int. j. aquat. biol. (2017) 5(6): 408-412 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology short communication length-weight relationship of black sea urchin (stomopneustes variolaris) in sri lanka heethaka krishantha sameera de zoysa*1, 2, bedigama kankanamge kolitha kamal jinadasa3, edirisinghe mudiyanselage ranjith keerthi bandara edirisinghe2, gabadage dona thilini madurangika jayasinghe3 1department of food technology, faculty of technology, rajarata university of sri lanka, mihintale, sri lanka. 2department of physical sciences, faculty of applied sciences, rajarata university of sri lanka, mihintale, sri lanka. 3institute of post-harvest technology (ipht), national aquatic resources research and development agency (nara), colombo, sri lanka. article history: received 6 june 2017 accepted 24 december 2017 available online 2 5 december 2017 keywords: indian ocean echinodermata biology sea urchin abstract: this study attempted to describe the length and weight frequency, and length-weight relationship in the black sea urchin, stomopneustes variolaris, in sri lanka. the sampling sites mount-lavinia (n=43), beruwala (n=99) and tangalle (n=55) were selected from south-west coast in sri lanka. the shell length and body weight were measured separately for three sampling sites. the mean length and weight of s. variolaris were 5.55±1.04 cm, 101.40±57.76 g; 6.54±0.86 cm, 147.90±50.40 g, and 6.41±1.05 cm, 150.50±59.45 for mount-lavinia, beruwala and tangalle, respectively. in addition, the length-weight relationship of s. variolaris were w=0.9953*l2.6472, w=0.9651*l2.6536 and w=1.4665*l2.4637 for mount-lavinia, beruwala and tangalle, respectively. introduction the black sea urchin (stomopneustes variolaris) (fig. 1) is one of the warm water species found in the indian ocean. they belong to the family stomopneustidae and are distributed in the tropical and subtropical regions of the indo-pacific from the east african coast to samoa and to the bonin islands to the north (giese et al., 1964). they are omnivores feeding mainly on algae and seaweeds. distribution of s. variolaris is limited to a depth up to 18 m (giese et al., 1964; james, 1982; kroh, 2014). of the 28 species of sea urchins found in sri lankan seas (jayakody, 2012), s. variolaris is highly restricted in distribution to the western and southern coastal areas (de zoysaa et al., 2016; jinadasa et al., 2016). in biological aspects, the growth rate of sea urchins and natural mortality mainly depend on temperature and food availability (reynolds and wilen, 2000). sea urchins accumulate nutrients in the gonads, which is a highly economically important delicacy in the world (james and siikavuopio, 2011; salon, 1985; scheibling and mladenov, 1987). according to james (1983), this species is the most abundant edible *corresponding author: heethaka krishantha sameera de zoysa doi: https://doi.org/10.22034/ijab.v5i6.304 e-mail address: dezoysahks@yahoo.com species found in the indian ocean such as lakshadweep islands, andaman islands and sri lanka. apart from the few studies done on the diversity, abundance and their distribution (jayakody, 2012), very little is known about the biology of sri lankan sea urchins. therefore, this study was conducted to examine the length-weight relationships of the common sea urchin, s. variolaris in sri lanka. materials and methods a total of 197 individuals were collected from rocky reefs in mount-lavinia (n=43), beruwala (n=99) and tangalle (n=55) (fig. 2) in 2014. all collected individuals were transported to the institute of postharvest technology (ipht), national aquatic resources research and development agency (nara). after that, the total body weights of all specimens were weighed to the nearest 0.01 g. the length of the sea urchin body was determined by measuring horizontal test diameter twice in right angles to the nearest 0.02 mm in all the specimens using a vernier caliper and the two measurements were averaged to obtain the diameter. 409 int. j. aquat. biol. (2017) 5(6): 408-412 the length-weight relationship was calculated using the w=alb equation and after logarithmically transferred form log w=log a b log l. where w is the weight in g, l is the total length cm, ‘a’ is the intercept and b are the slope. both of a and b were estimated by using the linear regression analysis (le cren, 1951). all the data were analysed by using the minitab 16.0 version and the microsoft excel 2010 version. results weight and average shell length frequency distribution: the most common shell length in the sample from mount-lavinia reef was 5.4 cm with an average of 5.55±1.04 cm (fig. 3). the most frequent total body weight in the mount-lavinia reef sample was 80 g and the average weight of the sample was and 101.40±57.76 g (fig. 4). for the beruwala reef, the commonest shell length was 6.5 cm with the average of 6.54±0.86 cm (fig. 5) and most frequent weight was 160 g with the average of 147.90±50.40 g (fig. 6). the tangalle reef population 6.5 cm was most frequent shell length while the average was 6.41±1.05 cm (fig. 7) and the most frequent total body weight was 150g with an average of 150.50 ± 59.45 g (fig. 8). length-weight relationships (lwr): there was a significant correlation between s. variolaris shell length and total body weight (p<0.05) for all three sampling sites (figs. 9, 11, 13). there was significant linear correlation between logarithmic values of shell length and total body weight (p<0.05) for all three sampling sites for s. variolaris (figs. 10, 12, 14, table 1). discussion the frequency distribution of shell length and weight of s. variolaris reveals that the shell length ranges from 3.30 to 8.90 cm and weight from 30.15 to 346.56 g for all the studied populations. but according to the smith and kroh (2011), s. variolaris from visakhapatnam coast (india) has a maximum shell length of 11 cm. this is the first time a study has been conducted in sri lanka to determine the lwr of s. variolaris. in fisheries research, the lwr is used as a good indicator, because it gives an idea about well-being, figure 1. stomopneustes variolaris. figure 2. sampling sites of stomopneustes variolaris. 410 de zoysa et al. / length-weight relationship of black sea urchin in sri lanka maturity, the rate of feeding and the rate of growth of a particular species (le cren, 1951; rahman et al., 2012). the value of the exponent (b) determine whether the growth isn weight is isometric (b=3) or figure 3. shell length frequency distribution at mount-lavinia reef. figure 4. weight frequency distribution at mount-lavinia reef. figure 5. shell length frequency distribution at beruwala reef. figure 6. weight frequency distribution at distribution at beruwala reef. figure 7. shell length frequency distribution at tangalle reef. figure 8. weight frequency distribution at tangalle reef. table 1. length-weight relationships and other parameters of stomopneustes variolaris at selected sites locations length-weight relationships (log w = log a + b log l) a b w r2 mountlavinia y = 0.0047 + 2.6472x 0.9953 2.6472 0.9953*l(2.6472) 0.9360 beruwala y = 0.0355 + 2.6536x 0.9651 2.6536 0.9651*l(2.6536) 0.8600 tangalle y = + 0.3829 + 2.4637x 1.4665 2.4637 1.4665*l(2.4637) 0.9402 411 int. j. aquat. biol. (2017) 5(6): 408-412 not and if the b value differs from 3, it indicates the change of the body shape as they grow. normally allometric growth can be negative (if b<3) or positive (if b>3) (rahman et al., 2012). the present study revealed the b value for mount-lavinia (2.6472), beruwala (2.6536) and tangalle (2.4637) were less than 3, which concludes that s. variolaris from all selected sites were close to isometric growth in weight, because “b” exponent value usually lay between 2.5 to 4.0 and that depends on the age, sex or maturity of species (le cren, 1951; rahman et al., 2012). according to the above “b” value, s. variolaris have relatively negative allometric growth. the reasons for negative allometric growth should be further explored under the different environmental parameters and feeding conditions. these findings about length-weight relationship study will be helpful to get an idea about the growth of s. variolaris in sri lanka. acknowledgments the authors would like to acknowledge nara, crow figure 9. length-weight relationship for mount-lavinia population. figure 10. logarithmic scale length-weight relationship for mount-lavinia population. figure 11. length-weight relationship for beruwala population. figure 12. logarithmic scale length-weight relationship for beruwala population. figure 13. length-weight relationship for tangalle population. figure 14. logarithmic scale length-weight relationship for tangalle population. 412 de zoysa et al. / length-weight relationship of black sea urchin in sri lanka island, colombo 15, for provided grant to complete this study and we take this opportunity to thank k. ukuwela for his invaluable comments on manuscript. references de zoysa h.k.s., jinadasa b.k.k.k., edirisinghe e.m.r.k.b. (2016). black sea urchin (stomopneustes variolaris): nutritional composition and trace metals accumulation of edible sea urchin of sri lanka. germany: lap lambert academic publishing. 116 p. giese a.c., krishnaswamy s., vasu b.s., lawrence j. (1964). reproductive and biochemical studies on a sea urchin, stomopneustes variolaris from madras harbor. comparative biochemistry and physiology, 13(4): 367380. james d.b. (1982). ecology of intertidal echinoderms of the indian seas. journal of the marine biological association of india, 24(1&2): 124-129. james d.b. (1983). research on indian echinoderms-a review. journal of the marine biological association of india, 25(1&2): 91-108. james p., siikavuopio s. (2011). a guide to the sea urchin reproductive cycle and staging sea urchin gonad samples. nofima, 1-20 jayakody s. (2012). provisional checklist of sea urchins (echinodermata: echinoidea) of sri lanka. the national red list 2012 of sri lanka. 370 p. jinadasa b.k.k.k., de zoysa h.k.s., jayasinghe g.d.t.m., edirisinghe e.m.r.k.b. (2016). determination of the biometrical parameters, biochemical composition and essential trace metals of edible sea urchin (stomopneustes variolaris) in sri lanka. cogent food and agriculture, 2(1): 1-12. kroh a. (2014). stomopneustes variolaris (lamarck, 1816). in: a. kroh, r. mooi (2014). retrieved 12-06, 2014, from world echinoidea database at http://www. marinespecies.org/echinoidea/aphia.php?p=taxdetails& id=212440. le cren e. (1951). the length-weight relationship and seasonal cycle in gonad weight and condition in the perch (perca fluviatilis). the journal of animal ecology, 201-219. rahman m., amin s., yusoff f.m., arshad a., kuppan p., nor shamsudin m. (2012). length weight relationships and fecundity estimates of long-spined sea urchin, diadema setosum, from the pulau pangkor, peninsular malaysia. aquatic ecosystem health and management, 15(3): 311-315. reynolds j.a., wilen j.e. (2000). the sea urchin fishery: harvesting, processing and the market. marine resource economics, 15: 115-126. salon n.a. (1985). echinoderm fisheries of the world: a review. a balkema, rotterdam. pp: 109-124. scheibling r.e., mladenov p.v. (1987). the decline of the sea urchin, tripneustes ventricosus, fishery of barbados: a survey of fishermen and consumers. marine fisheries review, 49(3): 62-69. smith a.b., kroh a. (2011). the echinoid directory. retrieved 12-06, 2014, from http://www.nhm.ac.uk /research-curation/projects/echinoid-directory int. j. aquat. biol. (2022) 10(4): 299-302 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article new record of cypridopsis vidua (muller, 1776) (ostracoda, podocopida: cyprididae) from east al-hammar marshes, iraq mohammed g. khalifa*1, shaker g. ajeel2 1department of biology, college of science, university of basrah, basrah, iraq. 2department of marine biology, marine science centre, university of basrah, basrah, iraq. article history: received 7 june 2022 accepted 21 august 2022 available online 2 5 august 2022 keywords: new record, al-hammar marshes, ostracoda, cypridopsis vidua. abstract: in the present study, cypridopsis vidua (muller, 1776), was recorded for the first time from the east al-hammar marshes, south of iraq. this species was collected from three stations during january and december 2021. the morphological features of the collected specimens were described for further taxonomic studies. in addition, some water quality parameters viz. temperature, ph, salinity, dissolved oxygen, turbidity, conductivity, and total dissolved solids were measured of the sampling station during the study period are provided. introduction ostracoda are small crustaceans inhabiting aquatic environments characterized by having a body completely enclosed between two valves as calcified shells. cypridopsis vidua (muller, 1776) (podocopida: cyprididae) is found in a wide range of aquatic habitats as an active swimmer, which prefers both large and small permanent water bodies that are rich in vegetation, such as fish ponds (roca and danielopol, 1993), the littoral zone of lakes, and slow rivers. it prefers shady areas with rich vegetation (mbahinzireki et al., 1991). in addition, c. vidua is found in temporary ponds, springs, well, and the interstitial habitat. it feeds on the aufwuchs, developing on aquatic plants, especially char stems and leaves (roca and danielopol, 1991). al-hammar marsh extends from suq al-shuyoukh area in dhi qar to basra governorate, iraq, with a length of 90 km, a width of 30 km, and an average depth of 3 m (salman et al., 2014). this marsh is affected by the half-daily tides through the shatt alarab (hussain et al., 2007) (fig. 1). in a study on the ostracoda of east al-hammar marshes, located south of the euphrates river in iraq, we collected c. vidua *correspondence: mohammed g. khalifa doi: https://doi.org/10.22034/ijab.v10i4.1712 e-mail: mohammed.ghazi@uobasrah.edu.iq for the first time from this marsh and reported it here. materials and methods cypridopsis vidua was collected from east alhammar marshes from three sampling stations, including al-sallal, al-nakara, and al-meshab stations using zooplankton net (with 0.1 mm mesh and 43 cm ring diameter) the water surface and pulling net for 15 minutes by boat of under the surface of the water during january and december 2021. the samples were fixed into 4% buffered formalin in 500 ml bottles labeled by collection data and brought to the laboratory. then they were examined under a wildtype dissecting microscope (humascope type). in addition, some c. vidua samples were collected from sediment at a depth 0f 3 m from three sampling stations using a grab-bottom sampler. these samples were washed with station water using a zooplankton net and, then these waters were filtered into a 500 ml plastic bottle, fixed 4% buffered formalin, and transferred to the laboratory. the samples were washed and filtered by a zooplankton net (0.1 mm mesh) in the laboratory, and the organisms were separated from other debris. under pressurized tap 300 khalifa and ajeel / cypridopsis vidua in al-hammar marshes, iraq water, the valve of c. vidua was separated from the soft body, dissected, and identified based on both soft body parts and valve morphology (using proper keys (meisch, 2000; karanovic, 2012), then the samples were classified by compound microscope humascope type. furthermore, the morphological features of the collected specimens were described for further taxonomic studies. in addition, some water quality parameters viz. temperature, ph, salinity, dissolved oxygen, turbidity, conductivity, and total dissolved solids were measured in the field during the sampling period. results cypridopsis vidua (muller, 1776) (fig. 2) description: female, carapace oval shape in both lateral and dorsal views, left valve slightly longer than right one, and left valve overlaps right one ventrally. carapace in dorsal view: anterior end rounded to pointed, left valve slightly overlaps right valve anteriorly. both valves about equally long at posterior end. posteroventral marginal zone of left valve with double-folded inner list, in right valve, list runs close to self-age. anterior external marginal zone of right valve with a row of 17 tiny pustules (fig. 2). antennae nugatory setae extending beyond tips of terminal claws with 1/4 of total length; mandibular palp with setae thick and feathery; maxillular palp: terminal segment cylindrical, longer than broad; teeth bristles of third thoracic leg masticatory lobe smooth. respiratory plate of maxilliped usually with five filaments; walking leg short and strongly developed, while cleaning leg without special characters; size of female 0.5 mm. male: unknown. environmental parameters: al-hammar marshes’ depth ranges from 0-3 m. water temperature in the sampling stations was recorded at its highest value of 34.5°c in the summer and its lowest value of 12.2°c in the winter. ph was at its highest value of 6.7 and 9.06 in the summer and winter, respectively. the salinity was between 1.3 ppt in summer and 5.8 ppt in winter, and the turbidity was at its highest at 101 ntu in spring and 2.2 ntu in winter. the conductivity was at its highest value of 9.2 mi.c/cm in spring and its lowest value of 2.8 mi.c./cm in autumn. the highest value of the total dissolved solid was 5.7 mg/l in spring and 1.8 mg/l in autumn was the lowest value. figure 1. sampling stations in the east al-hammar marshes, south of iraq. 301 int. j. aquat. biol. (2022) 10(4): 299-302 discussions in the present study, c. vidua was recorded for the first time in the east al-hammar marshes. the east hammar marsh has encountered a rise in its salinity due to the incursion of the seawater through the shatt al-arab due to the decrease in the flows of the euphrates-tigris river system, which directly or indirectly affected its living organisms in general and ostracoda in particular. the biotic and abiotic factors affect the abundance and distribution of c. vidua in the eastern al-hammar marshes. the densest ostracoda presence was recorded in the eastern alhammar marsh in the sediments and surface water. cypridopsis vidua is found worldwide (meisch, 2000). in the lake, the animals were found at depth of 0-30 m (loffler, 1969) and can tolerate a slight increase in salinity up to 8 ppt (hiller, 1972). the species has a very low tolerance for poorly oxygenated waters. in the laboratory, the animal survives only a few days at an oxygen tension of 0.1 mg/l (danielopol, 1991). the remarkable phenotypic variation of c. vidua has led to the description of a number of distinct species differing in carapace shape, size, color pattern, and the number of filaments of the mxp respiratory plate. as animals with transitional characters have repeatedly been found in c. okeecho (furtos, 1936), described from north america might be the bisexual form of c. vidua, but this needs further investigation. the three species of pionocypris viz. p. assimilis (sars, 1895), p. intermedia (sars, 1924), and p. videos (sars, 1895) described by sars (1924) from south africa are probably junior synonyms of c. vidua (meisch, 2000). cypridopsis obesa, c. parva, and c. helvetica (brady and robertsons, 1869) are subspecies or varieties of c. vidua (sars, 1925; beyer and meisch, 1996). references baird w. (1845). arrangement of the british entomostraca, with a list of species, particularly noticing those which have as yet been discovered within the bounds of the club. trans berwicks nat club, 2: 145-158. baird w. (1850). description of several new species of entomostraca. annals of natural history, 10: 56-59. beyer g., meisch c. (1996). freshwater ostracods collected on la go era (canary islands) with description of cyprideis obesa. travis science national museum of natural history of luxembourg, 23: 29-56. brady g.s. (1867). a synopsis of the recent british ostracoda. intelligence observer, 12: 110-130. brady g.s. (1868). a monograph of the recent british ostracoda. transactions of the linnean society of london, 26: 353-495. brady g.s., robertson d. (1869). notes of a week's dredging in the west of ireland-annals and magazine of natural history, 3: 353-374. danielopol d.l. (1991). spatial distribution and dispersal of interstitial crustacea in alluvial sediments of a backwaters of the danube at vienna. stygologia, 6(2): 97-110. furtos n.c. (1933). the ostracoda of chio. bulletin of the chio biological survey, 29: 211-524 hiller d. (1972). untersuchungen zur biologie und vzur okologie liminscher ostracoden a us dear umgebung von hamburg. archiv fur hydrobiologia, 40(4): 400497 hussain n.a., abdullah s.s., al-mahdi a.a. (2007). some features of the physical oceanography in iraqi marine waters. mesopotamian journal of marine science, 22(2): 209-222. karanovic i. (2012). recent freshwater ostracods of the world. springer-verlag berlin heidelberg. 619 p. kaufmann a. (1900). cypriden und darwinuliden der schweiz revue suisse de zoologie, 8: 209-423. kaufmann a. (1900). neue ostracoden aus der schweiz. zoologischer anzeiger, 23: 131-133. loffler h. (1969). recent and subfossil distribution of cytheroidea lacustris, ostracoda, in lake constance figure 2. cypridopsis vidua collected from al-hammar marshes. 302 khalifa and ajeel / cypridopsis vidua in al-hammar marshes, iraq mitteilungenb dear international's vereinigung fur theoretische und angewandte. limnologie, 17: 240-251. mbahinzirekig., heinlein f., winter h. (1991). microhabitat selection of ostracods in relation to predation and food. hydrobiologia, 222: 115-119. meisch c. (2000). freshwater ostracoda of western and central europe. susswasserfauna von mitteleuropa 8/3 spectrum akad vlog, gustav fischer, heidelberg, berlin. 522 p. muller o.f. (1776). zoologie danicae prodromus, seu animalium danie et norvegie indigenarum characters, nomina, et synonymy imprimis popularium. i-xxxii: 198-199. roca j.r., danileopol d.l. (1991). exploration of interstitial habitats by the phytophilous ostracod cypridopsis vidua (o.f. muller): experimental evidence. annales de limnologie, 27: 243-252. roca j.r., baltanas a., uiblein f. (1993). adaptive responses in cypridopsis vidua (crustacea, ostracoda) to food and shelter offered by a macrophyte (chara fragilis). hydrobiologia, 262: 127-131. salman d., abbas m.f., ghazi a.h.h., ahmed h.k., akash a.n., doubly a.a., warner b.g., asada t. (2014). seasonal changes in zooplankton communities in the reflooded mesopotamia wetlands, iraq. journal of freshwater ecology, 29(3): 397-12. sars g.o. (1866). oversigt af borges marine ostracoden. forth vidensk-selsk christian, 2865: 1-130. sars g.o. (1896). on a freshwater ostracod stenocypris chevreuxi with notes on some other entomostraca raised from dried mud from algeria. archiv for mathematik oh naturvidenskab, 1(7): 1-27. sars g.o. (1924). the freshwater entomostraca of the cape province (union of south africa). part 2: ostracoda. annels of the south african museum, 20(3): 195-211. sar g.o. (1925). an account of the crustacea of norway with short descriptions and figures of all museum, bergen. 208 p. int. j. aquat. biol. (2023) 11(1): 30-33 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology short communication length-weight relationships of three endemic fish species of the arabian peninsula amir hassan masoumi1, saud m. al jufaili2, fereshteh pourhosseini1, hamid reza esmaeili1 1ichthyology and molecular systematics research laboratory, department of biology, school of science, shiraz university, shiraz, iran. 2department of marine science and fisheries, sultan qaboos university, muscat, oman. article history: received 30 october 2022 accepted 29 december 2022 available online 2 5 february 2023 keywords: growth coefficient, cube law, population dynamics, oman. abstract: length-weight relationships (lwrs) were evaluated for three endemic fish species collected from the north and south of inland waters of oman in 2022 including garra sindhae, g. smartae, and oxyurichthys omanensis by using foldable shrimp and crab fishing traps (mesh size of 3*3mm) and hand nets (mesh size of 3*3mm). the parameter of b for all three species was between the estimated ranges of 2.5-3.5 as proposed for different other fish species, and there was high and significant coefficient of determination for all three species (0.87-0.984). introduction parameters of length-weight relationships (lwrs) for marine and freshwater fishes have a historical background in ichthyology and fisheries science; hence, these relationships have been estimated for many commercial and noncommercial species (esmaeili et al., 2014, 2015; keivany et al., 2015; sadeghi and esmaeili, 2018). at the beginning of the 20th century, length-weight relationships were implemented to evaluate fish population conditions in fish farms (froese, 2006; jellyman et al., 2013; purrafee dizaj et al., 2020). lwrs play a significant role in fisheries science (esmaeili and ebrahimi, 2006; esmaeili et al., 2014, 2015; hossain and sultana, 2014; sadeghi and esmaeili, 2018; mouludi-saleh and eagderi, 2019; al-jufaili et al., 2021). lwrs can be used to estimate unmeasured length or weight. as the lwrs data are the most available information in fisheries sciences, therefore, these parameters make a backbone in population dynamic investigations such as sex ratio, age at first maturity, longevity, and fecundity (esmaeili et al., 2014, 2015; sadeghi and esmaeili, 2018; mouludisaleh and eagderi, 2019). to date, lwrs for many endemic fishes of the correspondence: hamid reza esmaeili doi: https://doi.org/10.22034/ijab.v11i1.1789 e-mail: hresmaeili@shirazu.ac.ir dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.4.4 middle east, including the arabian peninsula, have been reported; these parameters have not been estimated for several other endemics, especially those restricted to the arabian peninsula. however, biological information, especially lwrs for three endemic fishes: garra sindhae lyon, geiger and freyhof, 2016, garra smartae krupp and budd, 2009 (cyprinidae: labeoninae); and eyebrow goby oxyurichthys omanensis zarei, al jufaili and esmaeili, 2022 (gobiidae: gobionellinae). garra sindhae is found in the south of oman near salalah in the wadi andhur. garra smartae have been recorded from two localities, including the wadis hadhabram and laggashalyon. oxyurichthys omanensis is restricted to the yeti estuary, oman (esmaeili et al., 2022; zarei et al., 2022). this study aims to provide lwrs for these three species of the arabian peninsula. materials and methods specimens of g. sindhae, g. smartae, and o. omanensis were collected from the wadis andhur; hadhabram and laggashalyon, and yeti, respectively, using foldable shrimp and crab fishing traps (mesh size of 3*3 mm, 3 nets for overnight) 31 int. j. aquat. biol. (2023) 11(1): 30-33 (fig. 1). the sampling was conducted twice in winter and summer 2022. the total length (tl), standard length (sl), and fork length (fl) of the individuals were measured to the nearest 0.1 mm using digital calipers, and total weight (tw) to the nearest 0.01 g using a digital electronic balance. the parameters of length and weight relationships were computed with the formula w=alb and expressed by linear regression of the logtransformed length and weight which gives the linear equation (koutrakis and tsikliras, 2003): log w = log a + b log l, where w= total weight in grams, l= length (total, standard and fork) in cm, a = a constant being the initial growth index, and b= growth coefficient. prior to regression analysis, log– log plots of length and weight values were performed for visual inspection of outliers (froese, 2006). the significance of the regression between length-weight relationships of fishes was tested by anova. bailey's t-test was used to determine whether b value significantly deviated from the expected cube value of 3. data were analyzed statistically by using ibm spss (version 22) statistical software package. results the parameters of length-weight relationships are given in table 1. lwrs were significant for all three species (p<0.001) with a high coefficient of determination (r2≥0.870). the b values of 2.6273.285, 2.772-2.965, and 2.999-3.362 were estimated for garra sindhae, g. smartae, and o. omanensis respectively, based on standard length. bailey's t-test reveals that the b values significantly deviated from 3. discussion length and weight measurements are generally recorded in fisheries surveys as these parameters make a foundation for other research and management investigations in fisheries sciences. the b-value of lwr is an exponent commonly different among fish species being 2.5 to 4. the slope parameter, b, is used to describe the growth pattern of a fish: allometric growth (b ≠ 3) represents a fish that has less girth as length increases (b < 3) or has an increase in plumpness as length increases (b > 3) and occurs more commonly among fish species compared to isometric growth. isometric growth (b = 3) describes a fish that grows with an unchanging body form. therefore, the b value represents the well-being and general morphology of fishes (esmaeili et al., 2014, 2015; sadeghi and esmaeili, 2018; mouludi-saleh and eagderi, 2019). lwrs of some garra species in oman are available e.g. bfigure 1. collection sites of three endemic fish species in the arabian peninsula (oman). 32 masoumi et al./ length-weight relationships of three endemic fish species value of g. barreimiae fowler and steinitz, 1956, and g. longipinnis banister and clarke, 1977, estimated for tl, fl, and sl are 3.441, 3.470, 3.269, and 3.104, 3.098, and 3.253, respectively (al jufaili et al., 2021). compared with the studied g. sindhae, g. smartae in this paper, they have a higher b value. this can be due to the habitat characteristics of these two studied fishes being restricted to small ponds or streams with no co-existing species and low vegetation. for the genus oxyurichthys, the b values have been reported to be 2.677 and 3.057 for o. microlepis (bleeker, 1849) from the pulicat lagoon in india and o. petersi (klunzinger, 1871) from the eastern mediterranean coast of turkey respectively (taskavak and bilecenoglu, 2001; nallathambi et al., 2020). the b value of 2.999-3.362 reported here for o. omanensis is close to o. petersi. previous studies suggested that variation in bvalue in fishes might be because of several factors like season, species, habitat, sex, gonad maturity, diet, stomach fullness, health, preservation techniques, and locality (le cren, 1951; esmaeili, 2001; sadeghi and esmaeili, 2018). one or more of the above factors could be the reason for variation in the lwrs in studied species. in conclusion, this study provides basic information about the lwrs of three endemic fishes, including two garra species and a goby from the arabian peninsula (oman), which will be useful in their fisheries and future conservation management. acknowledgments this research results from the scientific collaboration between sultan qaboos and shiraz universities. references al jufaili s.m., sayyadzadeh g., jawad l., esmaeili h.r. (2021). length-weight relationships of five fish species from the inland waters of oman. iranian journal of ichthyology, 8(1): 63-67. esmaeili h.r., al jufaili s.m., masoumi a.h., zarei f. (2022). ichthyodiversity in southeastern arabian peninsula: annotated checklist, taxonomy, short description and distribution of inland fishes of oman. zootaxa, 5134(4): 451-503. esmaeili h.r., ebrahimi m. (2006). length-weight relationships of some freshwater fishes of iran. journal of applied ichthyology, 22(4): 328-329. esmaeili h.r., gholamifard a., vatandoust s., sayyadzadeh g., zare r., babaei s. (2014). lengthweight relationships for 37 freshwater fish species of iran. journal of applied ichthyology, 30(5): 10731076. esmaeili h.r., masoudi m., sayyadzadeh g., mehraban h.r., gholami z., teimori a. (2015). length–weight relationships for four aphanius species of iran (teleostei: cyprinodontidae). journal of applied ichthyology, 31(3): 578-579. esmaeili, h.r. (2001). biology of an exotic fish silver carp hypophthalmichthys molitrix valenciennes from gobindsagar reservoir himachal pradesh india. froese r. (2006). cube law, condition factor and weightlength relationships: history, meta‐analysis and recommendations. journal of applied ichthyology, 22(4): 241-253. hossain m., sultana n. (2014). morphometric characters and length-weight relationship of bele, (glossogobius giuris) from mithamoin haor, kissorgonj, species n characters tl/sl range (cm) tw range (g) a 95% ci of a b 95% ci of b r2 garra sindhae 30 tl 5.37-9.66 1.1-10.7 0.01288 0.00567-0.0293 2.886 2.454-3.318 0.87 30 sl 4.1-7.73 1.1-10.7 0.02182 0.01259-0.03793 2.956 2.627-3.285 0.924 30 fl 4.74-8.69 1.1-10.7 0.175 0.00837-0.03656 2.886 2.475-3.296 0.881 garra smartae 142 tl 2.79-8.05 0.29-5.445 0.01534 0.01285-0.01832 2.914 2.792-3.035 0.941 142 sl 2.27-6.65 0.29-5.445 0.03 0.02667-0.03396 2.868 2.772-2.965 0.961 142 fl 2.5-7.3 0.29-5.445 0.02037 0.01774-0.0234 2.911 2.81-3.012 0.958 oxyurichthys omanensis 23 tl 4.7-9.03 0.483-4.083 0.00387 0.00195-0.00769 3.128 2.767-3.49 0.939 23 sl 3.28-6.26 0.483-4.083 0.01096 0.0083-0.01449 3.18 2.999-3.362 0.984 n: number of specimens; tl: total length; fl: fork length; sl: standard length; tw: total weight; a: intercept; b: regression slope; ci: confidence interval; r2: coefficient of determination. table 1. descriptive statistics and parameters of lwrs for three fish species from oman. 33 int. j. aquat. biol. (2023) 11(1): 30-33 bangladesh. journal of the bangladesh agricultural university, 12(2): 389-395. jellyman p.g., booker d.j., crow s.k., bonnett m.l., jellyman d.j. (2013). does one size fit all? an evaluation of length–weight relationships for new zealand's freshwater fish species. new zealand journal of marine and freshwater research, 47(4): 450-468. keivany y., nezamoleslami a., dorafshan s., eagderi s. (2015). length-weight and length-length relationships in populations of garra rufa from different rivers and basins of iran. international journal of aquatic biology, 3(6): 409-413. koutrakis e., tsikliras a. (2003). length-weight relationships of fishes from three northern aegean estuarine systems (greece). journal of applied ichthyology, 19(4): 258-260. le cren e. (1951). the length-weight relationship and seasonal cycle in gonad weight and condition in the perch (perca fluviatilis). the journal of animal ecology, 20(2): 201-219. mouludi-saleh a., eagderi s. (2019). length-weight relationship and condition factor of ten fish species (cyprinidae, sisoridae, mugilidae, cichlidae, gobiidae and channidae) from iranian inland waters. journal of wildlife and biodiversity 3(4): 12-15. nallathambi m., arumugam u., jayasimhan p., chandran s., paramasivam k. (2020). length‐weight relationships of six tropical estuarine fish species from pulicat lagoon, india. journal of applied ichthyology, 36(1): 125-127. purrafee dizaj l.p., esmaeili h.r., abbasi k., valinassab t., salarpouri a. (2020). does lengthweight equation fit clupeid fishes? an evaluation of lwrs for six clupeids from iran (teleostei: clupeiformes). international journal of aquatic biology, 8(2): 126-131. sadeghi r., esmaeili h.r. (2018). length‐weight relationships of three gobiid species (perciformes: gobiidae) along the iranian intertidal coast of the persian gulf and makran sea. journal of applied ichthyology, 34(5): 1233-1234. taskavak e., bilecenoglu, m. (2001). length-weight relationships for 18 lessepsian (red sea) immigrant fish species from the eastern mediterranean coast of turkey. journal of the marine biological association of the united kingdom, 81(5): 895-896. zarei f., al jufaili s.m., esmaeili h.r. (2022). oxyurichthys omanensis sp. nov., a new eyebrow goby (teleostei: gobiidae) from oman. zootaxa, 5182(4): 361-376. int. j. aquat. biol. (2022) 10(5): 384-387 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology short communication observations on the ghost fishing in two species of marine heterobranchs (mollusca gastropoda): aplysia depilans gmelin, 1791 (aplysidae) and umbraculum umbraculum ([lightfoot], 1786) (umbraculidae) andrea lombardo, giuliana marletta*1 department of biological, geological and environmental sciences, university of catania, 95124 catania, italy. article history: received 14 july 2022 accepted 28 august 2022 available online 2 5 october 2022 keywords: a.l.d.f.g., ghost net, gillnet, marine heterobranchia. abstract: the present note reports for the first time the finding of two species of marine heterobranchia within a ghost net. the stretch of the net was repeatedly wrapped in itself and formed a large tangle of spherical shape. this created several "layers" of meshes wrapped in each other, where the following species of marine heterobranchs were found: aplysia depilans and umbraculum umbraculum. in particular, of the former, an alive specimen was found tied to the wire of the net, and a dead specimen floating within the ghost net. of the second species, only the shell was found. introduction the term “ghost fishing” indicates the capability of given fishing gear to continue fishing even after the fisherman has lost control of it (smolowitz, 1978). such equipment can be lost in the sea due to multiple factors (thomas and sandhya, 2019): bad weather/sea conditions, damaged equipment, attachment of the same with natural (e.g. reefs) or artificial (e.g. wrecks) parts of the seabed, contact with other fishing equipment or for intentional causes. lost fishing gears are indicated with the acronym a.l.d.f.g. (abandoned lost discarder fishing gear) or ghost nets. through the phenomenon of ghost fishing, they continue to cause extensive damage to marine biodiversity (smolowitz, 1978; thomas and sandhya, 2019). in the mediterranean, the fishing gears mainly used in small-scale fisheries (ssfs) are passive (i.e. gillnets and trammel nets) (lucchetti et al., 2020). a gillnet is a wall of netting that hangs in the water column (it can be fixed to the substrate or floating), typically made of monofilament or multifilament nylon (noaa, 2022). trammel nets are similar to the previous one, except that the wall is formed by three layers of the net *correspondence: giuliana marletta doi: https://doi.org/10.22034/ijab.v10i5.1670 e-mail: giuliana.marletta@phd.unict.it dor: 20.1001.1.23830956.2022.10.5.5.6 (seafish, 2022). ghost fishing is mainly caused by such passive fishing gears (brown et al., 2005), which continue to fish, even for years, a high number of target, non-target and threatened organisms after being lost at sea (nakashima and matspuka, 2004; thomas and sandhya, 2019). fortunately, these ghost nets have become damaged or colonized by encrusting organisms and have lost their effectiveness (nakashima and matspuka, 2004). the marine organisms that are mainly threatened by ghost nets are fishes and crustaceans (thomas and sandhya, 2019). in this regard, the information regarding ghost fishing related to marine heterobranchs (previously known as opisthobranchia), a group that includes the most colorful and structurally diversified gastropods (rudman and willan, 1998), are practically nonexistent. consequently, this note describes observations made on a stretch of abandoned gillnet found during a scuba dive and in which two species of these marine gastropods have been found. materials and methods on 25th july 2022, during a scuba dive carried out between 9-11 am in the site of acque fredde https://ij-aquaticbiology.com/index.php/ijab/article/view/1670 385 int. j. aquat. biol. (2022) 10(5): 384-387 (37°38'15.7"n, 15°10'52.1"e), a diving point located within the hamlet of santa tecla in the municipality of acireale (eastern coast of sicily, italy), a stretch of abandoned gillnet was found at a depth of about 4-5 meters. the stretch of the net was repeatedly wrapped on itself and formed a large tangle of spherical shape. this shape created several "layers" of meshes wrapped in each other. the ghost net covered, wrapping it, a small area (about one m2) of the seabed. the covered area was a flat boulder placed between larger rocks and a small rocky wall, nearby, there were also some pebbles. the entanglement contained many marine organisms trapped inside and, on its surface, both living and dead (see results). after releasing all the living organisms from the net, the latter was removed from the bottom and brought to the surface, where it was almost completely unrolled. when unrolled, the piece of the net had approximately a rectangular shape (about 142 cm long and 50 cm high) (fig. 1a). along most of the margins, there were some ropes. the side of each mesh had a length of 3 cm (fig. 1b). at some points of the net, the meshes formed small knots (fig. 1c). results and discussions in the found ghost net, three different groups of animals were found. the most numerous groups of trapped animals were that of crustaceans, with about ten live specimens, including 5 maja squinado (herbst, 1788); 3 herbstia condyliata (fabricius, 1787); 1 dromia personata (linnaeus, 1758); 1 carcinus sp. and portions of the exoskeleton of scyllarus arctus (linnaeus, 1758). in the net, there were also two specimens of stramonita haemastoma (linnaeus, 1767) (mollusca, gastropoda) and an individual of symphodus tinca (linnaeus, 1758) (chordata, actinopteri). two species of marine heterobranchs were also found in the net stretch: 2 specimens of aplysia depilans gmelin, 1791 and a shell of umbraculum umbraculum ([lightfoot], 1786). as for the 2 individuals of a. depilans, one was alive and quite active, while the other specimen was dead, and its body was in a state of decomposition (with a yellowish-gray body and unrecognizable extremities of the head). both individuals were about the same size (about 5 cm long). the live specimen (fig. 1d) had the final part of the foot tied to a small knot formed by the wire of the net. this knot created an obvious bottleneck between the animal's tail and the rest of the body. this specimen also had an evident oblique groove on the tegument of both sides of the visceral mass. in addition, the mantle, usually found between the parapodia (and therefore relatively hidden), was located higher and beyond the margin of the latter. during the attempts made to free the animal (i.e. cutting gently the knot that choked the tail), the latter, whenever it was slightly pressed by one of the authors, secreted the purple ink. instead, the deceased specimen was free (not tied or embedded) within the agglomeration formed by the net. inside the net was also found a shell of u. umbraculum (fig 1d-e) with some organic parts still attached to the margins of the ventral side. the observations reported here on the finding of 2 species of marine heterobranchs within a ghost net would seem to be, to our knowledge, the first concerning this group of animals regarding the issue of ghost fishing. in fact, in literature, the only studies that have reported the finding of marine heterobranchs in fishing nets exclusively concern cases of "bycatch" [generally defined as the incidental capture of nontarget species during fishing activities (alverson et al., 1994; kumar and deepthi, 2006)] in the course of trawling [aplysia brasiliana rang, 1828 (branco et al., 2015) and aplysia sp. (mendonça et al., 2019)] or fishing with nets placed parallel to the seabed [bulla ampulla linnaeus, 1758 (samuel et al., 2018)] and therefore not through ghost nets. interestingly, both specimens of a. depilans found inside the net had a diameter of the body that would have quietly allowed it to cross the net’s meshes (3 cm per side) (unlike all other organisms found in it, even u. umbraculum). nevertheless, both animals were found inside the agglomeration formed by the net. this probably happened because the piece of the net was wrapped several times on itself, forming a spherical agglomerate composed of several "layers". as a result, the two animals, after entering the net, 386 lombardo and marletta/ ghost fishing of aplysia depilans and umbraculum umbraculum found themselves in a real sensory labyrinth that made the orientation almost impossible for them. in fact, in marine heterobranchs the sense of sight is poorly developed, and they are oriented mainly through touch, chemical sensations and balance (kandel, 1979). the hypothesis that they were "lost" within the net is confirmed by the fact that one of the two individuals was found dead and free (not stuck in the wires) inside the network. therefore, it is likely that this individual had died of starvation, having been lost within the agglomeration for a long time. on the other hand, the other specimen of a. depilans was found with his tail tied to the knots of the net and, therefore, unable to escape. this same specimen had an evident groove in the tegument on both sides of the visceral mass and the mantle slightly outside parapodia. this groove and the strange position of the mantle may have been caused by the continuous unnatural rubbing and chokes that the animal's body had undergone to pass through the various layers of the net. the finding of the shell of u. umbraculum inside the net suggests that even this animal was lost and could no longer get out of it. the shell had on some of the margins of the ventral side small organic parts that indicated that this organism was previously alive and probably, after being trapped in the net (since the animal was too large to get out), it was preyed upon by one of the many crustaceans found in it. to conclude, the present observations confirm the results of matsuoka et al. (2005) that the abandoned gillnets intertwined/wedged on a three-dimensional seabed maintain a high capacity for ghost fishing. consequently, it is evident that the ghost nets, under certain conditions (particular entanglements of the same), in addition to representing a threat to the group of marine heterobranchs, can be real mortal traps even for invertebrates that potentially would be able to cross them for their small size. references alverson d.l., freebag m.h., murawski s.a., pope j.g. (1994). a global assessment of fisheries by-catch and discards. fao fisheries technical paper 339. rome. branco j.o., freitas júnior f., christoffersen m.l. (2015). figure 1. a: the unrolled ghost net (scale bar 22 cm); b: net meshes (scale bar 3 cm); c: small knots between the meshes; d: schematic picture of the alive aplysia depilans. the lower black arrow indicates the groove on tegument; the upper arrow shows the mantle extroflextion.; e: dorsal view of umbraculum umbraculum shell; f: ventral view of the shell (scale bar 4.2 cm). 387 int. j. aquat. biol. (2022) 10(5): 384-387 bycatch fauna of seabob shrimp trawl fisheries from santa catarina state, southern brazil. biota neotropica, 15(2): e20140143. brown j., macfadyen g., huntington t., magnus j., tumilty j. (2005). ghost fishing by lost fishing gear. final report to dg fisheries and maritime affairs of the european commission: fish/2004/20. kandel e.r. (1979). behavioral biology of aplysia. w.h. freeman and company. united states of america, 463 p. kumar a.b., deepthi g.r. (2006). trawling and by-catch: implications on marine ecosystem. current science, 90(7): 922-931. lucchetti a., virgili m., petetta a., sartor p. (2020). an overview of gill net and trammel net size selectivity in the mediterranean sea. fisheries research, 230: 105677. matsuoka t., nakashima t., nagasawa n. (2005). a review of ghost fishing: scientific approaches to evaluation and solutions. fisheries science, 71(4): 691-702. mendonça l.m., guimarães c.r., lima s.f. (2019). mollusk bycatch in trawl fisheries targeting the atlantic seabob shrimp xiphopenaeus kroyeri on the coast of sergipe, northeastern brazil. papéis avulsos de zoologia, 59(33): 1-12. nakashima t., matsuoka t. (2004). ghost-fishing ability decreasing over time for lost bottom-gillnet and estimation of total number of mortality. nippon suisan gakkaishi, 70(5): 728-737. noaa (2022). gillnets. available from: www.fisheries.no oaa.gov/national/bycatch/fishing-gear-gillnets. retrieved 8/05/2022. rudman w.b., willan r.c. (1998). introduction. in: p.l. beesley, g.j.b. ross, a. wells (eds.). mollusca: the southern synthesis. fauna of australia vol. 5, part b. csiro. melbourne. pp: 915-942. samuel d.v., krishnan p., abhilash k.r., sreeraj c.r., shesdev p., sankar r., margi p., purvaja r., ramesh r. (2018). diversity of marine molluscs in the bycatch from loster nets, erwadi, gulf of mannar. indian journal of geo marine sciences, 47 (01): 170-175. seafish (2022). trammel-nets. available from: www.seafish. org/responsible-sourcing/fishing-gear-database/gear/tram mel-nets/. retrieved 8/05/2022. smolowitz r.j. (1978). trap design and ghost fishing: an overview. marine fisheries review, 1306: 2-8. thomas s.n., sandhya k.m. (2019). ghost nets: invisible fishers in the seas. aqua international, 66: october. international journal of aquatic biology (2014) 2(5): 286-291 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article redescription of paracobitis rhadinaea (regan, 1906) from sistan basin, iran (teleostei: nemacheiliidae) hamed mousavi-sabet*1, ahmad gharaei2, akbar nasrollahzade1, abouzar habibi1, soheil eagderi3 1department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. 2international hamun wetland institute, university of zabol, iran. 3department of fisheries, faculty of natural resources, university of tehran, p.o. box 4314, karaj, iran. article history: received 2 june 2014 accepted 30 july 2014 available online 2 5 october 2014 keywords: crested loach, taxonomy, chahnime reservoirs, morphology. abstract: paracobitis rhadinaea, a member of the family nemacheiliidae, originally described by regan in 1906 from sistan basin, is poorely known by the rare materials. the inadequately studied species is redescribed on the base of freshly collected materials. the species differs from all other paracobitis species by combination of the following characters: stout and elongated body; large size, up to 288 mm; a fully squamated body; slit like posterior nare; and midlateral series of large, irregular set and shaped dark, brown blotches. p. rhadinaea is endemic to sistan, iran. introduction nemacheilid loaches with a high dorsal adipose crest have been placed in the genus paracobitis for many years, especially those from central asia (banarescu and nalbant, 1964), vietnam (nguyen, 2005), the middle east (prokofiev, 2009) and china (min et al., 2010). paracobitis is restricted to near east and middle asia, and the species of paracobitis from china should be assigned to the genera homatula and schistura (nalbant and bianco, 1998). species of the genus paracobitis (bleeker 1863), are comparatively large-sized loaches inhabiting freshwaters of western asia, from the eastern mediterranean sea drainages to the eastern middle asia in afghanistan where it occurs only in the river helmand (banarescu and nalbant, 1995; nalbant and bianco, 1998). there are 8 valid species in the world, with six valid species in iran and the two others in the adjusted countries (kottelat, 2012; coad, 2014). from these valid species, paracobitis iranica nalbant and bianco, 1998 is known from the * corresponding author: hamed mousavi-sabet e-mail address: mousavi-sabet@guilan.ac.ir namak lake basin, paracobitis longicauda kessler, 1872 is found in the tedzhen and murgab rivers of afghanistan and turkmenistan and in the aral sea basin, and in the tedzhen (= hari) river basin of iran, paracobitis malapterura valenciennes, 1846 is found in the namak lake basin or in the southern caspian sea basin, paracobitis rhadinaea regan, 1906 is probably restricted to the sistan basin of iran and presumably afghanistan, paracobitis smithi greenwood, 1976 is found only from a cave in lorestan province in the tigris river basin, and paracobitis vignai nalbant and bianco, 1998 is endemic to sistan basin (nalbant and bianco, 1998; coad, 2014). also paracobitis boutanensis mcclelland, 1842 and paracobitis ghazniensis bănărescu and nalbant, 1966 are described from the helmand river drainage of afghanistan, with no iranian record (coad, 2014). the taxonomic history of nemacheiline loaches from iranian part of sistan basin dates back to the early 19th century, when the fishes were collected by 287 mousavi-sabet et al/ redescription of paracobitis rhadinaea colonel sir a. henry mcmahon, the officer of the sistan arbitration commission of 1901-1904. the collected fishes had been examined by charles tate regan, former director of the british natural history museum, in 1906, who described the nemacheilus rhadinaeus [=paracobitis rhadinaea] (coad, 2014). from more than a hundred years ago, there is no taxonomic data about the fish. therefore, a detailed re-description on the species is interesting for taxonomic studies on the loach family. some large specimens of paracobitis were collected in 2012, in the chahnime reservoirs, sistan basin, 30°76′97.17n, 61°68′46.28e, which were identified as p. rhadinaea. the present study aims to give a detailed description of this poorly documented species, because the data of the fresh deposited material enables a more precise definition of the species. material and methods ten fish were collected by gillnetting from the chahnime reservoirs, sistan basin, 30°76′97.17n, 61°68′46.28e (fig. 1); all fishes were fixed in 5% buffered formaldehyde. measurements were made with a digital calliper and recorded to 0.1 mm. all measurements are made point to point, never by projections. methods for counts and measurements follow kottelat and freyhof (2007). standard length (sl) is measured from the tip of the snout to the end of the hypural complex. the length of the caudal peduncle is measured from behind the base of the last anal-fin ray to the end of the hypural complex, at mid-height of the caudal-fin base. the last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are noted as "1½". terminology of head canals and pores is followed kottelat (1990). abbreviations used: sl, standard length; hl, lateral head length; vmfc, vatandoust and mousavi-sabet fish collection, tehran. results paracobitis rhadinaea (regan, 1906) nemachilus rhadinaeus regan, 1906 (figs. 2-5) diagnosis: paracobitis rhadinaea is distinguished from all other species of paracobitis in iran by having a stout and elongated body; large size, up to figure 1. map of iranian part of the sistan basin, and the study area chahnime reservoirs. 288 international journal of aquatic biology (2014) 2(5): 286-291 288 mm; a fully squamated body; slit like posterior nare; mid-lateral series of large, irregular set and shaped dark, brown blotches. description: the snout is longer than the postorbital distance, body depth is 8-10 times in body length, head length 5.1-5.5 times in body length, the mouth cleft extends to below the nostrils, lower lip interrupted medially, outer rostral barbel as long as maxillary barbel reaching back to or beyond nostrils, dorsal fin origin nearer tip of snout than caudal fin base, caudal fin slightly forked, caudal peduncle 2.12.8 as long as deep, 4.9-5.1 in length of fish. dorsal fin with 2-4 unbranched (first two minute and not readily visible) and 7½, rarely 8½, branched rays, anal fin with 2-4 unbranched (first two minute) and 5½ branched rays, pectoral fin branched rays 9-10, usually 10, and pelvic fin branched rays 6-8, usually 7. the dorsal fin forked. there is a well-developed post-dorsal fin crest and a slight ventral crest on the caudal peduncle. the pelvic fin origin lies just in front of the mid-point of the dorsal fin base. the anterior nostril is a tube followed immediately by a figure 2. paracobitis rhadinaea, 166 mm tl (photo by sahel pakzad-touchaei, jun 2012). figure 3. paracobitis rhadinaea, vmfc pc510ag, 181.5 mm sl, dorsal views. figure 4. paracobitis rhadinaea, vmfc pc510ag, 193.8 mm sl, mouth and lips. figure 5. paracobitis rhadinaea, vmfc pc510ag, 193.8 mm sl, nostril and flap. 289 mousavi-sabet et al/ redescription of paracobitis rhadinaea horizontal slit. slightly developed axillary lobe. mouth well arched. maxillary barbels not reaching to eye origin. outer mandibular barbels reaching to nostril origin. inner mandibular barbels not reaching to maxillary barbels origin. lips well furrowed, interrupted in their middle and pigmented by dark pigmentations. mental lobes enlarged. processus dentiformis slightly developed (fig. 4). lateral line complete, reaching to base of caudal fin, with 83-89 pores. cephalic lateral line system with 8 supraorbital, 4+11 infraorbital, 12 preoperculomandibular and 3 supratemporal pores. slit nostril, flap completely covered and passes the nostril (fig. 5). small scales, especially behind of dorsal fin rear (fig. 6). morphometric characters of the specimens were measured (table 1). meristic values for the recently caught specimens are: dorsal fin unbranched rays 24, dorsal fin branched rays 7½ (2) or 8½ (8), anal fin unbranched ray 2, anal fin branched rays 5½ (3) or 6½ (7), pectoral fin branched rays 8(1), 9(4) or 10(5), pelvic fin branched rays 6(3), 7(4) or 8(3) , and caudal branched rays 9+9(6) or 10+9(4). the maximum size of the recent caught materials is 193.8 character mean ± sd range total length mm 162.18 ± 14·15 147.15 193.85 standard length mm 140.52 ± 12.97 126.97 168.38 in % standard length total length 115.46 ± 1.83 113.24 119.02 head length 24.36 ± 1.05 22.49 25.64 head depth 10.79 ± 0.75 9.23 11.60 pre dorsal distance 49.68 ± 1.14 48.37 50.92 post dorsal distance 57.64 ± 2.53 54.13 59.14 maximum body depth 12.97 ± 1.03 11.87 14.92 pre anal length 76.05 ± 1.33 73.30 77.51 caudal peduncle height 8.14 ± 0.35 7.53 8.74 caudal peduncle length 22.57 ± 3.23 19.3 – 28.99 caudal fin length 16.89 ± 1.15 15.59 18.63 dorsal fin length 18.80 ± 0.62 17.73 20.05 dorsal fin height 5.62 ± 0.81 4.20 6.98 anal fin length 15.12 ± 0.62 14.07 15.82 anal fin height 4.09 ± 0.65 2.90 5.08 pectoral fin length 15.86 ± 1.15 13.79 17.51 ventral fin length 14.43 ± 0.63 13.72 15.41 pectoventral distance 22.79 ± 2.56 18.86 24.07 ventral-anal distance 12.97 ± 1.03 11.87 14.92 pectoanal distance 31.48 ± 1.28 29.49 32.94 crest length 30.11 ± 2.85 28.26 34.06 crest depth 1.37 ± 0.59 0.86 2.76 head length % snout length 44.87 ± 1.04 43.15 46.19 eye diameter 9.57 ± 0.88 7.67 10.58 head depth 44.28 ± 1.91 41.07 46.51 post orbital distance 49.10 ± 3.43 44.29 54.67 inter orbital length 22.56 ± 1.35 20.99 25.38 inter nasal length 18.87 ± 2.40 16.68 20.81 first barbel length 18.35 ± 2.51 16.55 19.86 table 1. morphometric characters of paracobitis rhadinaea. 290 international journal of aquatic biology (2014) 2(5): 286-291 mm in tl. coloration: in preserved specimens body is yellowish-white with a row of dark-grey blotchs on middle of the back and on the midsides of the body. in live specimens the flanks are pale yellow with 915 dark grey blotches on dorsal and lateral, which are connected together by a row of dark streak (figs. 2 and 3). the blotches extend onto the adipose crest. blotches are irregularly arranged anteriorly but may line up in upper and mid flank rows posteriorly. the flank blotches extend onto the caudal peduncle crest. all over the head, opercula and snout covered by small dark grey blotches. the dorsal, caudal and pectoral fins are pigmented by dark spots on the rays. the anal and pelvic fins lack pigment. a single obvious black waved-shaped and relatively narrow bar at caudal-fin base. the belly is yellowish white. no distinct dark stripe from eye to snout. all fins are yellow to orange (in live specimens) or hyaline (in preserved specimens); dorsal, pectoral and caudal fins with 3-4 rows of dark spots on rays, except distally so the fin margin is white; pelvic and anal fins without dark pigmentation. when touching the dorsal margin of the crest, the reticulations make the crest there dark, otherwise the crest is a light creamy color along the margin. the lateral line is light, sometimes in marked contrast to the rest of the flank. the rostral barbels and their bases are dark pigmented, but the maxillary pair is almost immaculate. remarks: this species is restricted to the sistan basin of iran and presumably afghanistan. banarescu and nalbant (1995) and nalbant and bianco (1998) placed this species in paracobitis. banarescu and nalbant (1966) place this species in the atrak and safid rivers of the caspian sea basin, the abkhar river of central iran, probably most of iran, the helmand drainage and the tedzhen river, evidently confusing it with p. malapterura and p. iranica. abdoli (2000) lists as questionably from the bejestan, kerman-na'in and dasht-e lut basins, from the middle and lower halil and bampur rivers of the hamun-e jaz murian basin, and from the simish and the mashkid rivers to its north in the mashkid river basin. distribution: this species is restricted to the sistan basin of iran and presumably afghanistan. habitat: paracobitis rhadinaea was found from chahnime reserviours. the species was distributed in the hamoun wetland (sistan basin, in southeast of iran), but since making some dams on hirmand river (the water supply of the wetland) in afghanistan, the wetland was dried from one decade ago. after the wetland dried the fish has remained in related reservoirs to the wetland (mousavi-sabet et al., 2013). conservation: urgent actions such as habitat protection, education of local fishermen and fishing management are needed to preserve this important species (mousavi-sabet et al., 2013). examined materials: paracobitis iranica: vmfc pc116se, 6, 31.4 67.9 mm sl; iran: qom province, qom river, namak lake basin, 34°43'14"n; 50°26'96"e; vmfc pc122ka, 2, 35.4 65.1 mm sl; iran: qazvin province, khar-rud river, namak lake basin, 35°47'48.00"n 49°22'46.11"e; vmfc pc133ka, 3, 45.4 68.5 mm sl; iran: hamedan province, zehtaran stream, namak lake basin, 35°14'17.31" n; 49°06'45.09" e, 1771m altitude. paracobitis longicauda: vmfc pc213hm, 3, 31.4 88.6 mm sl; iran: khorasan-erazavi province, laiin stream, hari river basin, figure 6. paracobitis rhadinaea, vmfc pc510ag, 193.8 mm sl, scale. 291 mousavi-sabet et al/ redescription of paracobitis rhadinaea 36°59'38.72"n; 59°45'11.97"e. paracobitis malapterura: vmfc pc414sv, 4, 66.0 127.1 mm sl; iran: markazi province, mazlaghan-chay river, namak lake basin, 34°54'27"n; 50°11'09"e; vmfc pc4226sv, 26, 57.6 84.1 mm sl; iran: markazi province, ghareh-chay river, namak lake basin, 34°53'25"n; 50°02'25"e; vmfc pc4312se, 12, 34.3 84.1 mm sl; iran: alborz province, kordan river, namak lake basin, 35°56'51.30"n; 50°49'46.76"e; vmfc pc443hm, 3, 79.3 88.0 mm sl; iran: alborz province, kordan river, namak lake basin, 35°56'51.30"n; 50°49'46.76"e. paracobitis rhadinaea: vmfc pc510ag, 10, 147.8 193.8 mm sl; iran: sistan and balouchestan province, chahnime reservoirs, sistan basin, 30°52’19"n 061°39’33"e; paracobitis vignai: vmfc pc612ka, 2, 108.7 – 118.0 mm sl; iran, sistan and balouchestan province, chahnime reservoir, sistan basin, 30°53’47"n 061°40'31"e. references abdoli a. (2000). the inland water fishes of iran. iranian museum of nature and wildlife, tehran: 378 pp. (in persian). banarescu p., nalbant t. (1964). süßwasserfische der türkei. 2. teil cobitidae. mitteilungen aus dem hamburgischen zoologischen museum und institut, 61: 159-201. banarescu p., nalbant t. (1966). the 3rd danish expedition to central asia. zoological results, 34. cobitidae (pisces) from afghanistan and iran. videnskabelige meddelelser fra dansk naturhistorisk forening, 129: 149-186. banarescu p., nalbant t. (1995). a generical classification of nemacheilinae with description of two new genera (teleostei: cypriniformes: cobitidae). travaux du muséum d'histoire naturelle grigore antipa, bucurešti, 35: 429-496. bleeker p. (1863). sur les genres de la famille des cobitioïdes. nederlands tijdschrift van dierkunde, 1: 361-368. coad b.w. (2014). freshwater fishes of iran. available from: www.briancoad.com. retrieved 2/10/2004. kottelat m., freyhof j. (2007). handbook of european freshwater fishes. kottelat, cornol and freyhof, berlin, 646 p. kottelat m. (1990). indochinese nemacheilines. a revision of nemacheiline loaches (pisces: cypriniformes) of thailand, burma, laos, cambodia and southern viet nam. verlag dr. friedrich pfeil, münchen: 262 p. kottelat m. (2012). conspectus cobitidum: an inventory of the loaches of the world (teleostei: cypriniformes: cobitoidei). the raffles bulletin of zoology, 26: 1199. min r., chen x.-y., yang j.-x. (2010). paracobitis nanpanjiangensis, a new loach (balitoridae: nemacheilinae) from yunnan, china. environmental biology of fishes, 87: 199-204. mousavi-sabet h., gharaei a., ghaffari m. (2013) .threatened fishes of the world: paracobitis rhadinaeus (regan, 1906) (nemacheilidae). croatian journal of fisheries, 71: 87-89. nalbant t.t., bianco p.g. (1998). the loaches of iran and adjacent regions with description of six new species (cobitoidea). italian journal of zoology, 65: 109-123. nguyen v.h. (2005). freshwater fishes of vietnam. v. 2: 760 p. prokofiev a.m. (2009). problems of the classification and phylogeny of nemacheiline loaches of the group lacking the preethmoid i (cypriniformes: balitoridae: nemacheilinae). journal of ichthyology, 49 (10): 874898. int. j. aquat. biol. (2013) 1(2): 55-60 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology sublethal effect of nanosilver on the structure of gill of caspian roach (rutilus rutilus caspicus) fingerlings maryam sharifian1, fatemeh khani1, kheirollah khosravi2, mohsen khalili *1,3, aliakbar hedayati1 1department of fishery, faculty of fisheries and environment, gorgan university of agricultural science and natural resources, gorgan, iran. 2department of fisheries science, faculty of natural resources, university of tehran, iran. 3young researchers club, bandargaz branch, islamic azad university, bandargaz, iran. article history: received 17 april 2013 accepted 30 april 2013 available online 4 may 2013 keywords: aquatic ecosystem fish nanosilver gill histopathology abstract: widespread use of nanosilver can be led the contamination of aquatic environment and impact on living organisms such as fishes. we investigated histopathological changes in the gills tissue of caspian roach fingerlings after two weeks exposure to sublethal concentrations of nanosilver. following one and two weeks exposure, necrosis, shortening of secondary lamellae, edema, destruction of epithelial lamella, shortening of secondary lamellae, epithelial lifting and curling of secondary lamellae were observed in fingerlings’ gill tissues. this observation showed that exposure to sub-lethal concentrations of nanosilver is caused damages in the gill tissues of caspian roach. the results demonstrated direct correlation of gill tissue damage and toxin exposure i.e. increasing nanosilver concentration is caused more tissue damage. hence, histopathological changes of gill can considered as a proper indicator for nanosilver contamination of aquatic environments. introduction development of science and technology has been led to increase the production of substances that can cause welfare in some aspects of human life. unfortunately, the improper use of these chemicals can have adverse effect on humans and other organisms. the use of nanoparticles (nps) has been dramatically developed in many fields. nanosilver is one of important nps because of its bactericide effect (farkas et al., 2011). in addition, nanosilver is applied in odor resistant textiles, food packaging, cosmetics, household appliances and medical devices. like other chemicals, overuse of this substance may contaminate the environment and release ag particles (nps or aggregates) via sewage discharge into the aquatic environment (benn and westerhoff, 2008). also, waste nanosilver can contaminate groundwater through drainage and finally influence on non-target aquatic organisms * corresponding author: mohsen khalili e-mail address: mohsen.khalili369@gmail.com such as fishes and crab. fishes are very susceptible to environmental pollution which may cause significant damage on its vital organ such as gill. (banaee et al., 2011; john, 2007). despite of widespread use of this nps i.e. nanosilver, there is little information is available about its potential detrimental effects on the environments particularly on fishes (farkas et al., 2011; handy and shaw, 2007). few studies has discussed on the toxicity of nanosilver on human health particularly on respiratory exposure or from in vitro assays using mammalian cells (lovern et al., 2007). the evaluation of ecotoxicological risks resulted by pesticides was done to demonstrate the influence of toxicants on non-target organisms such as fish. the present study is a contribution to the evaluation of toxicity and effects of a nanosilver based toxic to fish gill tissue. histological changes in animal tissues 56 sharifian et al./ int. j. aquat. biol. (2013) 1(2): 55-60 provide a suitable and easy method to discern of chronic effect of contaminate, in different tissues and organs (bernet et al., 1999). also, histopathological studies of exposed fish to pollutants have been revealed that fish organs can be considered as an adequate indicators of water quality (velmurugan et al., 2007; 2009). the gill is vital organs in fish respiration, osmoregulation, acid base balance and ammonia excretion (heath, 1995). fish gill are also sensitive to water pollution because of their great surface area and external position. for this reason fish gills are considered to be most suitable indicators of water contamination levels. the caspian roach (rutilus rutilus caspicus), an anadromous species, is an economically valuable (coad, 1980; soleimani et al., 2011) and a main food sources for sturgeon species in the caspian sea (keyvanshokooh and kalbassi, 2006). this fish enter the rivers of the caspian sea that are in close proximity to municipal waste for reproduction, so there is a possibility of exposure to this substance. hence, this study was aimed to determine the histopathological effects of nanosilver on gill tissue of caspian roach fingerlings. material and methods experimental design: caspian roach fingerlings (with average weight of 1.66 ± 0.05 g; mean ± s.d.) figure 1. (a) control gill: gill filament or primary lamella, secondary lamellae. h&e, x400, (b) gill tissue exposed to 11.6 mg l-1 nanosilver for one week: a, epithelial lifting. h&e, x100, (c) gill tissue exposed to 23.2 mg l-1 nanosilver for one week: a, shortening of secondary lamellae; b, haemorrhage at primary lamella; c, epithelial lifting and d, odema. h&e, x100 and (d) gill tissue exposed to 34.8 mg l-1 nanosilver for two weeks: a, curling; b, necrosis. h&e, x100. 57 sharifian et al./ int. j. aquat. biol. (2013) 1(2): 55-60 of this experiment were obtained from the sijwal fish reproduction center (golestan province, iran). their lengths were in the range 5-7 cm. the specimens were acclimated to the laboratory conditions for two weeks prior to experiment in a 20l glass aquarium filled by dechlorinated tape water. during two week acclimatization period, fish were fed twice a day whit a commercial food (sari animal feed and aquatic factories). fish were randomly divided into fifteen tanks at a density of seven fish per tank. fish were exposed to 0 (control), 11.6, 23.2, 34.8 and 46.4 mg l-1 of nanosilver with three replicates provided from commercially available nanosilvere, (partonar co). this concentrations were determined based on 0, 25, 50, 75 and 100% of h lc50 value for caspian roach fingerling that reported by shaluei et al., (2012), (46.4 mg l-1). during experiment, water temperature, dissolved oxygen, ph and salinity were 27.3 °c, 7.07 mg l-1, 7.3 and 1.2 ppt, respectively. also, photoperiod regime was adjusted to 13l: 11d during experiment. tanks were aerated continuously and 10% of the water was replaced daily with water containing the experiment concentration of nanosilvere. preparation of tissue: at the end of one week (3 specimens per tank) and two weeks (3 specimens per tank), fish removed from tanks and anesthetized using clove oil (150ppm). fish gill dissected and fixed in 10% buffered formalin (roberts, 1989). tissues sections of 4 µm were prepared and stained with haematoxylin-eosin based on cruz and pitogo (1989). the mounted slides were observed and photographed using a nikon e 200 eclipse microscope. degree of tissue changes have been shown in table 1 as mild (+), moderate (++), severe (+++) and none (-). results no alterations were observed in the control gill tissue. the structural details of the control gill is shown in figure 1a. the histological changes affected by nanosilver on gill of treatments are shown in table 1. results revealed the epithelial lifting of fish gill in 11.6 mg l-1 treatment (fig. 1b). curling and necrosis was observed after two weeks of exposure to 34.8 mg l-1 nanosilver (fig. 1d). two weeks of exposure to 46.4 mg l-1 nanosilver was caused epithelial lifting, haemorrhage at primary lamella, collapsing of secondary lamellae and figure 2. (a) gill tissue exposed to 46.4 mg l-1 nanosilver for two weeks: a, epithelial lifting; b, haemorrhage at primary lamella and c, collapsed of secondary lamellae. h&e, x100 and (b) gill tissue exposed to 46.4 mg l-1 nanosilver for two weeks: a, haemorrhage at primary lamella, (b) shortening of secondary lamellae. h&e, x100. 57 58 sharifian et al./ int. j. aquat. biol. (2013) 1(2): 55-60 shortening of secondary lamellae in the gills of experimental fish (fig. 2a, b). discussion in most aquatic animals such as fish, gills are vital organs for their respiratory, osmoregulatory and secretory functions. in present study, the histopathological changes of gill tissues in caspian roach during one and two weeks exposure to nanosilver include necrosis, shortening of secondary lamellae, edema, destruction of epithelial lamella, shortening of secondary lamellae, epithelial lifting and curling of secondary lamellae. several other studies have shown similar effects of contaminates on gills of difference fish species (yildirim et al., 2006; velmurugan et al., 2007; xing et al., 2012). hyperemia, fusion of secondary lamellae and telangiectasis were histopathological alterations of gill in nile tilapia (oreochromis niloticus l.) exposed to deltamethrin (yildirim et al. 2006). khoshnood et al. (2011) have reported the lamellar epithelia and lamellae fusion after 48 h exposure to mercuric chloride in the gill of persian sturgeon (acipenser persicus). epithelial hyperplasia, aneurism, epithelial necrosis, desquamation, epithelial lifting, edema and lamellar fusion were observed in merigal (cirrhinus mrigala) exposed to different sublethal concentrations of lambda-cyhalothrin (velmurugan et al., 2007). histopathological changes in the gill tissues of labeo fish (labeo rohita) exposed to atrazine were epithelial hyperplasia, curling of secondary lamellae, changes in chloride cells and degradation of epithelial and pillar cells (jayachandran and pugazhendy, 2009). in a study that scown et al (2010) conducted, it was found exposure of rainbow trout to nps can have highly negative effect on different tissues such as gill, including the presence of high concentrations of nps in the gills. gill tissues injuries can be divided into two groups: direct and indirect (richmonds and dutta, 1989). for example, the observed epithelial damage of the gill is a direct responses induced by the action of pesticide. the defense responses are lifting up of the epithelium and lamellar fusion. former pathological change increases the distance to avoid the toxicant to reach the blood circulation and later one i.e. lamellar fusion a rejoinder that reduces the amount of sensitivity of gill surface area. also, gill hyperplasia may use as a defensive mechanism leading to diminish in the respiratory area and an enhancement in the toxicant-blood diffusion distance (cengiz, 2006). costs of defense response occur at the gills and the results are a respiratory disorder that itself caused is destruction of gills. all these damages can cause reduction of up taking concentratio n (mg l−1) terms (week) shortening of secondary lamellae necrosis of secondary lamella haemorrhage at primary lamella epitheli al lifting collapsed of secondary lamellae adhesion of secondary lamellae curling of secondary lamellae control 11.6 1 + ++ 2 ++ + ++ + + ++ 23.2 1 + + ++ + + 2 ++ + ++ ++ ++ + + 34.8 1 ++ + ++ +++ ++ ++ ++ 2 +++ ++ ++ +++ ++ ++ ++ 46.4 1 ++ +++ + +++ + + ++ 2 +++ +++ +++ +++ ++ ++ ++ table 1. summarized histopathological effects in the gills of r. rutilus caspicus exposed to nanosilver and control fish. 59 sharifian et al./ int. j. aquat. biol. (2013) 1(2): 55-60 oxygen via gill resulting decrease of fish activity particularly in larvae and juveniles. destruction of gill tissue may cause severe physiological problems and eventually leading to the loss of fish. the histopathological observation of this study showed that exposure to sub-lethal concentrations of nanosilver is caused damages in the gill tissues of caspian roach. as a conclusion, this study demonstrated direct correlation of gill tissue damage and toxin exposure i.e. increasing nanosilver concentration is caused more tissue damage. references banaee m., sureda a., mirvaghefi a., ahmadi k. (2011). effects of diazinon on biochemical parameters of blood in rainbow trout (oncorhynchus mykiss). pesticide biochemical and physiology, 99: 1-6. benn t.m., westerhoff p. (2008). nanoparticle silver released into water from commercially available sock fabrics. environmental science and technology, 42: 7025-7026. bernet d., schmidt h., meier w., burkhardt-holm p., wahli t. (1999). histopathology in fish: proposal for a protocol to assess aquatic pollution. journal of fish diseases, 22: 25–33. cengiz e.i. (2006). gill and kidney histopathology in the freshwater fish (cyprinus carpio) after acute exposure to deltamethrin. environmental toxicology and pharmacology, 22: 200-204. coad b.w. (1980) environmental change and its impact on the freshwater fishes of iran. biologycal conservation, 19: 51-80. farkas j., christian p., gallego-urrea j.a., roos n., hassellov m., tollefsen k.e. (2011). uptake and effects of manufactured silver nanoparticles in rainbow trout (oncorhynchus mykiss) gill cells. aquatic toxicology, 101: 117-125. handy r.d., shaw b.j. (2007). ecotoxicity of nanomaterials to fish: challenges for ecotoxicity testing. integrated environmental assessment and management, 3: 458-460. jayachandran k., pugazhendy k. (2009). histopathological changes in the gill of labeo rohita (hamilton) fingerlings exposed to atrazine. european journal of scientific research, 4: 219221. john p.j. (2007). alteration of certain blood parameters of freshwater teleost mystus vittatus after chronic exposure to metasystox and sevin. fish physiology and biochemistry, 33: 15-20. keyvanshokooh s., kalbassi m.r. (2006). genetic variation of rutilus rutilus caspicus (jakowlew 1870) populations in iran based on random amplified polymorphic dna markers: a preliminary study. aquaculture research, 37: 1437-1440. kiabi b.h., abdoli a., naderi m. (1999). status of the fish fauna in the south caspian basin of iranian. zoology in the middle east, 18: 57-65. khoshnood z., khodabandeh s., shahryari moghaddam m., mosafer khorjestan s. (2011). histopathological and pathomorphological effects of mercuric chloride on the gills of persian sturgeon, acipenser persicus, fry. international journal of natural resources and marine sciences, 1: 23-32. lovern s.b., strickler j.r., klaper r. (2007). behavioral and physiological changes in daphnia magna when exposed to nanoparticle suspensions (titanium dioxide, nano-c-60, and c (60) hxc (70) hx). environmental science and technology, 41: 4465–4470. mallatt j. (1985). fish gill structural changes induced by toxicants and other irritants: a statistical review. canadian journal of fisheries and aquatic sciences, 42: 630-648. richmonds c., dutta h. (1989). histopathological changes induced by malathion in the gills of blue gill lepomis macrochirus. bulletin of environmental contamination and toxicology, 43: 123-130. roberts r. (1989). fish pathology, 2nd edition. 467. scown t.m., santos e.m., johnston b.d., gaiser b., baalousha m., mitov s., lead j.r., stone v., fernandes t.f., jepson m. (2010). effects of aqueous exposure to silver nanoparticles of different sizes in rainbow trout. toxicological 59 60 sharifian et al./ int. j. aquat. biol. (2013) 1(2): 55-60 sciences, 115: 521-534. shaluei f., hedayati a., jahanbakhshi a., baghfalaki m. (2012). effects of nanometer-sized silver materials on survival response of caspian roach (rutilus rutilus caspicus). toxicology and industrial health, doi: 10.1177/0748233712457445. soleimani n., hoseinifar s.h., merrifield d.l., barati m., abadi z.h. (2012). dietary supplementation of fructooligosaccharide (fos) improves the innate immune response, stress resistance, digestive enzyme activities and growth performance of caspian roach (rutilus rutilus) fry. fish and shellfish immunology, 32: 316-321. velmurugan b., selvanayagam m., cengiz e.i., unlu e. (2007). histopathology of lambdacyhalothrin on tissues (gill, kidney, liver and intestine) of cirrhinus mrigala. environmental toxicology and pharmacology, 24: 286-291. velmurugan b., selvanayagam m., cengiz e.i., unlu e. (2009). histopathological changes in the gill and liver tissues of freshwater fish, cirrhinus mrigala exposed to dichlorvos. brazilian archives of biology and technology, 52: 12911296. wendelaar bonga s., lock r. (1991). toxicants and osmoregulation in fish. netherlands journal of zoology, 42: 2-3. xing h., li s., wang z., gao x., x.u. s., wang x. (2012). histopathological changes and antioxidant response in brain and kidney of common carp exposed to atrazine and chlorpyrifos. chemosphere, 88: 377-383. yildirim m.z., benlı a., selvı m., özkul a., erkoç f., koçak o. (2006). acute toxicity, behavioral changes, and histopathological effects of deltamethrin on tissues (gills, liver, brain, spleen, kidney, muscle, skin) of nile tilapia (oreochromis niloticus l.) fingerlings. environmental toxicology, 21: 614-620. int. j. aquat. biol. (2016) 4(5): 325-329: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article occurrence of orthetrum abbotti calvert (1892) (odonata, libellulidae) and intraguild predation on clarias gareipinus burchell, 1822 (suliformes, clariidae) and oreochromis niloticus l., 1758 (perciformes, cichlidae) fry in lagos fish farms abiodun akinpelu denloye*1, oyindamola azeezat alafia1, kafayat oluwakemi ajelara1, oluwaseun olusegun babalola1, olanrewaju abayomi dosumu2, fatai gbolahan owodeinde3, oluwaseun oluyomi solomon1 1department of zoology and environmental biology, faculty of science, lagos state university, ojo, lagos, nigeria. 2department of mathematical sciences, lagos state university, ojo, lagos, nigeria. 3department of fisheries, lagos state university, ojo, lagos, nigeria. article history: received 15 july 2016 accepted 8 october 2016 available online 2 5 october 2016 keywords: intraguild predation orthetrum abbotti fish farms dragonfly nymph abstract: intraguild predation occurs when species competing for the same resource prey upon or parasitize one another. this may result in economic losses under commercial circumstances. a survey of the insect species of fish farms in badagry and ojo areas of lagos state, nigeria was carried out followed by an evaluation of the predatory ability of orthetrum abbotti nymphs on fish fry. nymph predation was evaluated in the laboratory against fry of clarias gariepinus and oreochromis niloticus. samples of insects were randomly collected from 10 earthen ponds, 10 concrete ponds and the vegetation surrounding the ponds and identified over a period of 12 weeks from three study fish farms. six species of insects belonging to four orders namely notonecta unifasciata, gerris remigis, o. abbotti, aedes aegypti, dysticus marginalis and acentria ephemerella syn. niveus were collected from the ponds. studies on feeding preference of 5th nymphal instar of o. abbotti on fry of c. gariepinus and o. niloticus over other food types revealed that the dragonfly preferred to feed more on c. gariepinus fry than on o. niloticus although there was no significant difference in the number of o. niloticus and c. gariepinus fry preyed upon by o. abbotti nymphs. introduction insects play important roles in the aquatic ecosystem, apart from being tools to reveal information about the water quality and ecology of a particular water body; they are also crucial in balancing the aquatic ecosystem. more importantly, odonates are good indicators for monitoring anthropogenic impacts on freshwater ecosystems. in small water bodies such as ponds, myriads of insect such as dragonflies, caddisflies and mayflies spend most of their lives in the pond feeding on larvae of other insect, plant matter, decaying vegetation and algae (cardoba-aguilar, 2008). most aquatic insects are predacious feeders, and the predatory effect of dragonflies larvae on aquatic organisms, including catfish and tilapia fry are * corresponding author: abiodun akinpelu denloye e-mail address: bio_denloye@yahoo.com becoming alarming leading to annual colossal losses (adeyemo et al., 1997). the predatory habit of dragonfly larvae on other co-occurring species may be as a result of competition for limiting resources such as food and is referred to as intraguild predation (igp) (berg et al., 2012). vashini and ganagappan (2014) stated that odonate nymphs are very efficient predators in lowland streams and ponds. according to sulem and brummett (2006), about 9% of total clarias gariepinus larval mortality in earthen pond in cameroun was attributed to predation by aquatic insects. nguenga et al. (2000) identified predation as the major cause of low and viable survival of c. gariepinus in cameroun. basically, predation by birds, amphibians and other fishes are believed to be more than in aquatic insects although, rashed (2005) 326 denloye et al./ occurrence of orththrum abbotti and intraguild predation in lagos fish farms explained that aquatic insect’s predation may exceed avian predation in some localities. considering the importance of c. gariepinus and oreochromis niloticus to fish farming in african countries, it is imperative to understand the feeding preference of dragonfly (orthetrum abbotti) nymphs for fry of c. gariepinus and o. niloticus. the first part of this study reported here assessed the entomofauna of three fish farms and the second evaluated the feeding preference of the dragonfly, o. abbotti. materials and methods study sites: three farms were studied in ojo and badagry area of lagos state, namely lagos state university (lasu) farm (site a) (in ojo area), anetekhai farm (site b) and bombata farm (site c) (sites b and c are located in badagry area). the farms rear c. gariepinus and o. niloticus for research and commercial purposes. in all the three farms, ponds are in close proximity to vegetation growing on sandy loam soil. site a (lasu farm) is surrounded by few buildings and tall grasses with vegetable farm about 500 metres away from the fish ponds and hatching earthen ponds and hatchery. site b (anetekhai farm) and site c (bombata farm) were surrounded respectively with few buildings and sparse vegetation. fish is reared in earthen ponds in site b while in site c, fish is reared in concrete tanks. anetekhai farm uses rectangular earthen ponds, while bombata farm use only concrete ponds. all the farms are very close to the vegetation. at each site, sampling areas were demarcated with the use of measuring tapes, into zones a-d. each sampling zone is about 500 m apart and collection was done at different parts of the zones. insect collection and identification: collection of insect was done fortnightly for a period of six months. sampling was started in august 2005 which represent the period of “break” in rain in lagos state. light traps, pitfall traps and sweep nets were used to collect larvae as well as adult insects from 10 earthen ponds and 10 concrete ponds and the vegetation around the ponds, respectively. insect collection was done twice daily, in the early morning 0700-1000 hr and later in the afternoon 1400-1800 hr. insects were collected from the ponds and the surrounding vegetation. the collected insects were killed in hot boiling water and thereafter placed in 70% alcohol for preservation and identification was done by comparison with already identified specimens in insect collections at university of lagos and university of ibadan, respectively (denloye et al., 2014). feeding preference of o. abbotti: nymphs of o. abbotti were obtained from the lagos state university pond. only the 5th nymphal instars were used following suling et al. (2004). nymphs were acclimatized with aquatic weeds to simulate their natural habitat in pond water for 48 hours before commencement of the experiment. fry of c. gariepinus and o. niloticus weighing 0.1 g and mean length of 0.92±0.03 cm and 0.87±0.02 cm, respectively were obtained from the hatchery of lagos state university. the fry (6-8 days post hatching) were scooped with the pond water. fifteen nymphs of o. abbotti already starved for 24 hours were introduced into 20 l plastic tanks filled with pond water. fifty fry of c. gariepinus were subsequently introduced into each tank in three replicates. the same set up was used for fry of o. niloticus. a mixed culture of both c. gariepinus (25 fry) and o. niloticus (25 fry) was also set up. control experiment had no nymph of o. abbotti. all experimental and control set up were replicated three times each. the tanks were covered with mosquito net of mesh size 0.25 cm to prevent adult mosquitoes from depositing batches of eggs during the experiment. predation was counted hourly after 24 hours. the number of fry preyed by the nymphs was recorded. in addition, the feeding behaviours of the larvae on the fry were observed. data analyses: insect species collected from earthen pond or concrete ponds were identified, counted and expressed as percentage and means of the total from each pond type. the mean number of insect taxa collected from the pond types and those from 327 int. j. aquat. biol. (2016) 4(5): 325-329 surrounding vegetation as well as feeding preference treatments were compared by analysis of variance (anova) to determine significant differences between them (p≤0.05). results species occurrence in pond area: four insect orders and six species were collected from all the fish ponds (table 1). the result showed that notonecta unifasciata had the highest occurrence with 44.59% in earthen ponds and 54.31% in concrete ponds. the least represented were the dipterans and coleopterans. five species of insects were collected from all the farms, but not at every site, while crematogaster herculeanus had the highest occurrence (73.17%) in the surrounding vegetation at all the sites larvae and adult o. abbotti and dysticus sp. occurred both in the ponds and in the surrounding vegetation having widespread occurrence in all the farms. the mean number of taxa collected in the ponds was less than that of surrounding vegetation; although numerically more insect species were collected from the ponds in all the farms (table 1). bombata farm having concrete ponds had the least number of insects of the order lepidoptera and odonata. analysis of variance reveals a significant difference for all order of the insects collected at the farm. feeding preference of o. abbotti nymphs for fry of c. gariepinus and o. niloticus: the results showed that nymphs of o. abbotti feed highly on fish fry of the two fish species when introduced into the tanks. the number of fry reduced as the period of exposure increased. after a period of 24 hours, only 16% of the c. gariepinus and 32% of o. niloticus remained in the experimental tanks relative to the control. in the mixed culture, it was observed that the nymphs exhibited a preferential feeding, consuming more c. gariepinus than o. niloticus. at the end of 24 hours all the c. gariepinus introduced had been consumed by o. abbotti nymphs while 45% of the o. niloticus fry remained. comparison of feeding preference of o. abbotti on fry of o. niloticus and c. gariepinus and control shows that the feeding rate of o. abbotti was significantly higher (p≤0.05) in the experiment than in the control. discussion the objectives of this study were to evaluate the diversity of insect species found in the ponds and vegetation of three farms in ojo and badagry areas of lagos state and to test if o. abbotti actually select its prey while feeding on fish fry. the ponds and fish farm are breeding grounds for a wide diversity of insect species, while some insects lay their eggs on the water surface, others find the ponds as an excellent feeding ground, feeding on larvae of other insects and even fish fry. man-made ponds attracts a wide array of insect species, selection of the pond as insect orders species ponds surrounding vegetation (%) earthen (%) concrete (%) hemiptera notonecta unifasciata 44.59 54.31 gerris remigis 35.06 26.72 odonata orthetrum abbotti 12.12 12.09 2.79 coleoptera dysticus marginalis 4.76 2.59 4.58 diptera aedes aegypti 3.46 4.31 lepidoptera acentria niveus 4.88 hymenoptera crematogaster herculeanis 73.17 orthoptera catantops melanosticum 8.80 zonocerus variegetus 2.00 homoptera hermetia illucens 3.88 table 1. insects collected from ponds and surrounding vegetation. 328 denloye et al./ occurrence of orththrum abbotti and intraguild predation in lagos fish farms a preferred habitat by such insects cannot be unconnected with the fact that it provides for them a wide variety of food sources and effective breeding ground, so much that they can locate the ponds soon after their construction. the diversity of insects in a pond and the vegetation around the ponds is related to the number of available predators around. foltz and dodson (2009) reported that the abundance of notonecta undulate, was positively correlated with shallow, fishless ponds, while cooks and streams (1984), reasoned that fish predation is an important determinant of notonecta habitat utilization pattern. another factor that determines aquatic insects’ abundance is the fish species composition in the aquatic body. wittwer et al. (2010) reported that the major determinant of the dragonfly community in swedish lakes is its fish species composition, and that this in turn affects the lower trophic levels, although different fish will affect the dragonflies population differently. the presence of vegetation also affect the species richness of the aquatic insects, since the vegetation covers serve as a place of refuge, although these cannot change the level of risk among prey species significantly (cook and streams, 1984). the common belief that some aquatic insects such as odonates and notonectids are general feeders can be erroneous when considering the predatory behavior exhibited by o. abbotti in this study. zalom (1978) reported that notonectid populations exhibited cannibalistic tendencies in the laboratory with special preference to certain species of insects. in another study by tave et al. (2008), dragonflies fed selectively on black o. mossambicus as compared to gold coloured ones when presented with the two fishes in a 1: 10 ratio. earlier studies have also shown that dragonflies were more likely to attack smaller prey than larger ones (rashed, 2005). the results of this study support the position that dragonfly nymphs are predatory on fish species. in this study, c. gariepinus and o. niloticus fry which are generally considered to be one of the most important tropical catfish and tilapia species respectively were slightly larger than the o. abbotti larvae. although there was no significant difference between the number of c. gariepinus and o. niloticus fry fed upon by the dragonflies nymph, results show that o. abbotti nymphs exhibited selective and preferential feeding on c. gariepinus and o. niloticus relative to the control. the results indicate that dragonflies preyed upon fry of test fish species. farmers need to be equipped with this information which shows that fry missing in the ponds may be as a result of predation by dragonfly nymphs. moreover abowei and ukoroije (2012) reported that it was better to have the knowledge of the biodiversity, biology, behaviour, roles and so on of aquatic insects to allow for sustainable aquaculture management. the result of this study supports that assertion. references abowei j.f.n., ukoroije b.r. (2012). the identification, types, taxonomic orders, biodiversity and importance of aquatic insects. british journal of pharmacology and toxicology, 3(5): 218-229. adeyemo a.a., yakubu a.f., oladosu g.a., ayinla o.a. (1997). predation by aquatic insects on african catfish fry. aquaculture international, 5(1): 101-103. cardoba-aguilar a. (2008). dragonflies and damselflies; modern organisms for ecological and evolutionary research. oxford university press.295 p. cook w.l., streams f.a. (1984). fish predation on notonecta (hemiptera): relationship between prey risk and habitat utilization. oecologia (berlin), 64: 177-183. corbet p.s. (1999). dragonflies: behaivour and ecology of odonata. cornell university press, ithaca, new york, usa. 829 p. de-graaf g., jannsen h. (1996). artificial reproduction and pond rearing of the african catfish clarais gariepinus in sub-saharan africa. fisheries technical paper 362. fao. rome. italy. 73 p. denloye a.a., makinde o.s.c., ajelara k.o., alafia a.o., oiku e.a., dosunmu o.a., makanjuola w.a., olowu r.a. (2014). insects infesting selected vegetables in lagos and the control of infestation on celosia argentea (l.) with two plant oils. international journal of pure and applied zoology, 2(3): 187-195. foltz s.j., dodson s.i. (2009). aquatic hemiptera 329 int. j. aquat. biol. (2016) 4(5): 325-329 community structure in stormwater retention ponds: a watershed land cover approach. hydrobiologia, 621: 49-62. kadoya t., suda s., washitani i. (2007). dragonfly species richness on man-made ponds: effects of pond size and pond age on newly established assemblages. ecological research, 8:182-187. lasswell j., forrest m.l. 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(2006). relative importance of various predators in clarias gariepinus fry mortality in cameroun. naga world fish center quaterly, 29(3-4): 74-77. tave d., rezk m., smitherman r.o. (2008). effect of body colour of oreochromis mossambicus (peters) on predation by dragonfly nymphs. aquaculture research, 21(2):157-162. vershini r.a., kanagapan m. (2014). effect of quantity of water on the feeding efficiency of dragonfly nymph bradynopyga geminate (rambur). journal of entomology and zoology studies, 2(6): 249-252. weir j.b. (1972). diversity and abundance of aquatic insects reduced by introduction of the fish, clarias gareipinus to ponds in central africa. biological conservation, 4(1): 169-175. wittwer t., sahlen g., suhling f. (2010). does one community shape the other? dragonflies and fish in swedish lakes. insect conservation and diversity, 3(2): 124-133. int. j. aquat. biol. (2022) 11(1): 69-75 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article mapping of diatom indices by remote sensing to evaluate the um el-naaj marshes' water quality jinan s. al hassany1, neran a. al naqeeb2, g.h. al-rubaiee3, fouad k. mashee4 1department of biology, college of science for women, university of baghdad, baghdad, iraq 2department of biology, college of science, university of misan, maysan, iraq. 3department of biology, college of science, university of mustansiriya, baghdad, iraq. 4remote sensing unit, college of science, university of baghdad, baghdad, iraq. article history: received 7 december 2022 accepted 21 february 2023 available online 2 5 february 2023 keywords: wetland remote sensing techniques epiphytic algae water quality abstract: this study was conducted to evaluate the water quality of the um el-naaj marshes using the community of diatoms bioindicator and mapping them by remote sensing data. from november 2018 to april 2019, epiphytic diatoms were collected from host aquatic plants of zannichllia palustnia from six sites in the um el-naaj marshes. according to the results, the diatom index (di) ranged from 3.5 in autumn 2018 to 2.4 in summer 2019, i.e in sites 3 and 2, respectively. the index of trophic diatoms (tdi) for the majority of study sites showed the mesotrophic state ranged from 47.5 to 62.5 in all seasons. except for site 2, which had the lowest value (35) in the summer of 2019, and station 3 which had a greater value than 60 in the autumn of 2018 and spring of 2019, other sites had oligo-mesotrophic states. the general diatomic index (gdi) values showed the lowest value (9.6) at site 2 in the summer of 2019 and the greatest value (14.4) at site 3 in the spring of 2019. in addition, in the current study, maps were produced using remote sensing methods to visualize the values close to the studied sites to show the quality state of the whole marsh. introduction the mesopotamian marshlands are one of the middle east's most internationally significant wetlands due to their high biodiversity and role as a breeding place for numerous bird species that migrate from north europe. because freshwater discharge has decreased over the past three decades, the water quality of these marshes has deteriorated, and the hydrological regime has changed, causing substantial environmental impacts. this has significantly impacted the marshes' vegetation and fauna, reducing their ecological viability and importance (al-handal et al., 2016). epiphytic algae are residing on submerged aquatic vegetation, including freshwater angiosperms and macroalgae (dunn et al., 2008). due to the recurrent process of separating epiphyton from the host plant, these species are the main food supply for small fish and other invertebrates in the correspondence: neran a. al naqeeb doi: ttps://doi.org/10.22034/ijab.v11i1.1846 e-mail: neranecology@uomisan.edu.iq dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.9.9 littoral zone (al ramahi and al bahadly, 2017). recent research has demonstrated their significance as primary producers that remove carbon from the water column and absorb key nutrients, making these vital elements available to other species, such as tiny aquatic habitats in lakes (fawzy et al., 2016; neran et al., 2020). because of being sessile, having rapid production rates, and unique set of environmental tolerances and preferences, epiphytic algae serve as reliable indicators of water quality and environmental conditions (hassan and albdulameer, 2014; naqeeb et al., 2022). they play a significant role in the ecological equilibrium between various types of macrophytes and their associated water bodies in the lotic water system, where they are the dominating species (usgs, 2017). this study aimed to evaluate the water quality of the um el-naaj marshes based on bioindicators for the community of epiphytic diatoms collected 70 hassany et al./ mapping of diatom indices by remote sensing from host aquatic plants of zannichllia palustnia. materials and methods study area: um el-naaj lake is located in the kahala district having two entryways: one is through al kahla district in the direction of bani hashem, and the other one is through kahla-al mueel-abo khasaf village (table 1). the um el-naaj lake has a 25 km width and a 30 km width along the iran-iraq border. although there were some dried fields, it was a part of the partially dried wetlands. currently, this marsh receives water through its outlets from both inside and outside of iraq, including the al khala, the al mshrah, al-dwiridj and karkheh, nissan, and al-kfagia rivers (hassan et al., 2012). processing of remote sensing (rs): in our investigation, we employed four bands of landsat-8 oli terrestrial remote sensing data, which were produced from november 2018 to october 2019. images were obtained from the web at the same time as fieldwork data collection (muhsin and foud, 2012). in order to determine the study area's greatest visual clarity, data from bands 7, 4, and 3 were combined. they were also used to track any changes that resulted from the analysis of parameter samples from the six stations located throughout the study area (werner, 1977). arcgis (10.4) was used to produce prediction maps using the interpolation method (idw). these maps show the circumstances surrounding um el-naaj lake inside iraqi borders. inverse distance weighted (idw) (santos and ferragut, 2018) was the approach used in this study to spatially interpolate water reflectance during seasonal changes in um el-naaj marshes in 20182019. six sampling sites were selected in um el-naaj marshes (hor al huweiza) by gps (table 1, fig. 1). water samples were taken monthly from november sites (st) sites (e) x-axis coordinate (utm) y-axis coordinate (utm) 1 um bzazen 746945 3500950 2 aboathba 1 750024 3504050 3 aboathba 2 750046 3502900 4 alkhabta 743841 3500870 5 aboliefa 1 753309 3500520 6 aboliefa 2 753652 3497750 table 1. coordinates and names of sampling sites of um el naaj lake. figure 1. the study area of the, observation station name in study area al-naaj lake include six stations. 71 int. j. aquat. biol. (2023) 11(1): 69-75 2018 to october 2019. for qualitative study; 10g of host plants were cut into small parts of 2-3 cm and washed with 50-100 ml of environment water and then scrapped by a smooth brush or the blade. the samples were preserved in containers plastic with 1ml of lugol’s solution. after separating the diatom from the plant, the sample is placed in tubes (100 ml) for 10-15 days with 1ml of lugol's solution to precipitate, after which the precipitate is taken (20-30 ml) and stored in containers with a few drops of lugol's solution (hassany and al-bueajee, 2015; salman et al., 2017; prescott, 1964). marked samples in containers with the date and location of the collection. epiphytic diatoms are prepared on permanent slides for microscopic inspection. diatoms were identified using the references listed below (patrick, 1966; werner, 1977; hadi, 1984). trophic indices: diatomic index (di) was calculated according to descy’s list (descy, 1979) which consists of 155 species using the formula of di = aj × ij × vj/aj × vj aj, that it is the relative abundance of the species j present in the sample. diatom taxa were chosen for their indicator value (tolerance to inorganic nutrients) and ease of identification which are crucial to measuring of trophic diatom index (kelly and whitton, 1995) that was calculated using the formula of tdi = ∑(ajs jvj / ajvj x25)-25, where aj = the frequency or percentage of species j in the sample, sj = varies from 1 for sites with extremely low nutrient concentrations to 5 for sites with very high nutrient concentrations i.e. the species nutritional sensitivity of species j in the sample ranges from 1 to 5. (table 3), and vj = indicating the index's value (ranging 13) generic diatom index (gdi): this index is measured using equation of 𝐺𝐷𝐼 = (∑ ajsjvjajvj ) × 4 (lecointe et al., 2003), where aj = the number or percentage of different species in the sample, sj = species that are sensitive to nutrients (1–5) (table 4), and vj = value of index (1-3). results and discussion diatom index: the results of the di showed that the water quality of the el-naaj marshes ranged from 3.5 in autumn 2018 to 2.4 in summer 2019. the greatest value was 3.6 in site 3 in spring 2019 and interpretation values value of diatom index optimum biological quality, zero contamination 4.5 almost standard caliber (slight changes in the community, slight pollution) 4-4.5 substantial eutrophication, declines in sensitive species, mild pollution, or community-wide effects that are more significant 3-4 high pollution, dominant species resistance, sensitive species declines or extinctions (reduced biodiversity), and resistant species 2-3 a clear preference for a few numbers of resistant species (many species vanish), and extreme pollution 1-2 table 2. the description of the values of the diatom index (di) (descy, 1979). value of index pollution degree tdi< 35 tdi 35-50 tdi 50-60 tdi 60-75 tdi>75 oligotrophic state oligo-mesotrophic state mesotrophic state eutrophic state hypertrophic state table 3. the description of tdi values ranged from 0-100. (kelly and whitton, 1995). value of index water quality 17.5-20 high 14-17.5 good 10.5-14 moderate 7-10.5 poor < 7 bad table 4. the values of generic diatom index (gdi). 72 hassany et al./ mapping of diatom indices by remote sensing the lowest was 2.4 in site 2 in summer 2019 (fig. 2). according to descy (1979), the um el-naaj lake's water quality ranged from moderate contamination (more sensitive species have a moderate to good quality) to good quality. species of a. minutissima and nitzchia were prevalent in contaminated water. both species are tolerant to various pollutants (todd and mays, 2004). reductions of vulnerable species show low pollution or severe eutrophication. during the study time period, the water quality of um el-naaj lake ranged from moderate water (moderate pollution) to low pollution categories. the high diversity of some species in the um el-naaj lake may be attributable to the river's water quality, which is suited for high diversity due to the wide range of favorable development conditions and when there are challenging conditions, like a shortage of nutrients or an excess of them. it allows species that were only initially present in a few natural settings to grow while reducing the presence of many species (kusstatscher et al., 2019) the findings demonstrate that the shape of the host plant and seasonal water level variations influence the temporal variations of epiphytic algae on aquatic macrophyta (phragmites australis). the range and density of epiphytic algae are restricted by elements including temperature, light intensity, turbidity, and nutrient-rich plants. this study came to the conclusion that the water quality of um el-naaj lake, which has a large diversity of diatoms, is favorable for the survival of many species of creatures (salman and hadi, 2015). the nitzschia genus is regarded as a biological indicator that tracks the levels of high pollutant toxins and organic pollutants in sediments due to an abundance of organic materials brought to the river by agricultural runoff and human waste. trophic diatom index: epiphytic diatoms' tdi values indicated that the majority of studied sites in all seasons had mesotrophic states, with values ranging from 47.5 to 62.5. site 2 in the summer of 2019 recorded the lowest value (35), and site 6 the spring of 2019 had the highest value (62.5.5) showing a eutrophic state (fig. 3). tdi showed variations in spring and summer at some showing an oligo-mesotrophic state. tdi data showed the level of water quality in the al hawaiza marshes ranging from mesotrophic to eutrophic conditions. increased bacterial activity to decompose organic materials in the water and promotes the growth of algae can be the cause of the increase in nutrients (ccme, 2007; köbbing et al., 2013). the inverse distance weighted (idw) approach results of the current study to spatially interpolate water reflectance figure 2. the seasonal variations of diatom index. 73 int. j. aquat. biol. (2023) 11(1): 69-75 during seasonal changes in um el-naaj marshes in 2018-2019 are shown in figure 3. generic diatoms index: according to the results, gdi values were lowest at site 2 in the summer of 2019 and highest at site 3 in the spring of 2019 (fig. 4). the quality of the um el-naaj lake ranged from mediocre to good based on these values (hungary ministry of environment and water, 2005) (table 2). the greater the water quality and appropriateness for living creatures, the higher the gdi indicator figure 3. the seasonal variations of trophic diatom index. autumn 2018 tdi gdi di st1 60 13.66 3.4 st2 55.75 12.94 3.23 st3 62.5 14.04 3.5 st4 52.5 12.79 3.1 st5 60 13.83 3.4 st6 60 13.6 3.4 winter 2019 st1 60 13.66 3.4 st2 55.75 12.94 3.23 st3 62.5 14.04 3.5 st4 52.5 12.79 3.1 st5 60 13.83 3.4 st6 60 13.6 3.4 spring 2019 st1 50 12.02 3 st2 50 12.16 3 st3 65 14.4 3.6 st4 47.5 11.77 2.9 st5 52.25 12.36 3.09 st6 50 12 3 summer 2019 st1 42.5 11.1 2.7 st2 35 9.6 2.4 st3 45 11.2 2.8 st4 50.25 12.07 3.01 st5 49.5 11.94 2.98 st6 48.75 11.8 2.95 table 5. seasonal variations of tdi, gdi, di in the um-al naaj lake. 74 hassany et al./ mapping of diatom indices by remote sensing readings. additionally, there aren't many nutrients in the water, and poor water quality results from low index values. the abu-zirig marsh has a moderate pollution level, according to the gdi data (abu hadal and al hassany, 2020). references al-handal a., taffs k., abdullah d., zawadzki a. 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(2020). assessment of the water quality of um el-naaj marshes by diatoms. ecology, environment and conservation, 26(1): 449-464. hassan f.m., al-bdulameer s.h. (2014). qualitative and quantitative study of epipelic algae in tigris river within baghdad city, iraq. baghdad science journal, 11(3): 1074-1082. naqeeb a., neran a., al hassany j.s., mashee f.k. (2022). use remote sensing techniques to study epiphytic algae on phragmites australis in um elnaaj lake, mysan province, southern iraq. iop iop conference series: earth and environmental science, 1002 012012 usgs (united states geological survey) (2017). landsat-a global land-imaging mission. [online] available at: http://remotesensing.usgs.gov. hassan f.m., hadi r.a., kassim t.i., al-hassany j.s. (2012). systematic study of epiphytic algal after restoration of al-hawizah marshes, southern of iraq. international journal of aquatic science, 3(1): 37-57. muhsin i.j., foud k.m. (2012). improving spatial resolution of satellite image using data fusion method. iraqi journal of science 53(4): 943-949. imuhsin i.j., mahmood f.h., mashee f.k. (2012). multispectral scanner “mss” and panchromatic components difference of al-haditha dam region using gis and remote sensing techniques. almustansiriyah journal of science, 23(6): 233-242. hassany j.s., al-bueajee a.i.m. (2015). a qualitative study of epiphytic algae (diatom) on some aquatic plants in al-auda marshes within maysan province/southern iraq. baghdad science journal, figure 4. the generic diatoms index seasonal variations. 75 int. j. aquat. biol. (2023) 11(1): 69-75 12(4): 665-676. salman j.m., hassan f.m., baiee m.a. (2017). practical methods in environmental and pollution laboratory. environmental research and studies center, university of babylon. 144 p. prescott g.w. (1964). how to know the freshwater algae. how to know the freshwater algae. michigan state univ., east lansing. 272 p. werner d. (1977). the biology of diatom. botanical monographs, university of california press. berkeley and los angeles. 13 p. hadi r.a.m. (1984). diatoms of the shatt-al-arab river, iraq. nova hedwigia, 39(3-4): 513-558 patrick r. 1966. the diatoms of the united states (exclusive of alaska and hawaii). monographs of the academy of natural sciences of philadelphia, 13: 1688. descy j.p. (1979). a new approach to water quality estimation using diatoms. nova hedwingia, 64: 305323. kelly m.g., whitton b.a. (1995). the trophic diatom index: a new index for monitoring eutrophication in rivers. journal of applied phycology, 7(4): 433-444. lecointe c., coste m., prygiel j. (2003). omnidia 3.2. diatom index software including diatom database with taxonomic names, references and codes of 11645 diatom tax. todd d.k., mays l.w. (2004). groundwater hydrology. john wiley & sons. 336 p. kusstatscher p., zachow c., harms k., maier j., eigner h., berg g., cernava t. (2019). microbiome-driven identification of microbial indicators for postharvest diseases of sugar beets. microbiome, 7(1): 112. salman j.m., hadi s.j. (2015). environmental study of epiphytic algae on 86 some aquatic plants in alabasiya river, iraq. mesopotamia environmental journal, 1(3): 1-15. köbbing j.f., thevs n., zerbe s. (2013). the utilization of reed (phragmites australis): a review. mires peat, 13(1): 1-14. canadian council of ministers of the environment. (2007). canadian water quality guidelines for the protection of aquatic life: imidacloprid. in: canadian environmental quality guidelines, 1999, canadian council of ministers of the environment, winnipeg. 8 p. santos t.r., ferragut c. (2018). changes in the taxonomic structure of periphytic algae on a freefloating macrophyte (utricularia foliosa l.) in relation to macrophyte richness over seasons. acta botanica brasilica, 32(4): 595-601. abu-hadal l.s., al hassany j.s. 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(2016) 4(4): 233-238; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article neem leaf juice as bio-safe anaesthetic against two live fish, anabas testudineus and channa punctata mohammad kamrul ahsan1, mohammad manjurul alam*2,1selina parween1 1fisheries research laboratory, department of zoology, university of rajshahi, rajshahi 6205, bangladesh. 2department of fisheries, university of rajshahi, rajshahi 6205, bangladesh. article history: received 7 may 2016 accepted 1 july 2016 available online 2 5 august 2016 keywords: neem juice anaesthetic toxicity anabas channa abstract: activity of fresh neem leaf juice to immobilize, anaesthetize and fatal effect were studied against two live fish species, anabas testudineus and channa punctatus. the concentrations, 5, 10, 15 and 20% of neem leaf juice affected both the species, but a. testudineus was found to be comparatively more susceptible to the treatment than c. punctatus. the time required to immobilize were 4 to >6 hours for a. testudineus and 7 to 9 hours for c. punctatus; to anaesthetize were 6 to >12 hours and 8 to 10.15 hours for a. testudineus and c. punctatus, respectively; and death occurred 7 to >13 hours in a. testudineus and 9 to 10.45 hours in c. punctatus after treatment depending on the concentrations. the toxic effects were positively co-related with the concentrations and negatively co-related with the total weight of the fish, though the values of the coefficient of the correlations (r) were low. at the anaesthetic stage, the colour of skin, gills and eye were changed, the fishes showed irritated movement first (a. testudineus) and then became sluggish (both species), and instead of the normal fishy smell the fishes gave neem leaf like smell. introduction application of plant materials in the management of aquaculture ponds is gaining momentum because they are safe, effective, widely available and comparatively cost-effective (mousa et al., 2008). in countries like bangladesh, fish parasites and fish predators, which cause a great economic loss in productivity, are mainly controlled by using toxic chemicals. these chemicals are mostly applied indiscriminately and without training of the fish farmers. as a result, high level of chemical residues has accumulated not only in the fish but also in the pond environment. it is reported that most of these chemicals used in the aquaculture system interfere with the homeostasis of the fish and thus affect their life performances (barton and iwama, 1991; wendelaar bonga, 1997). to overcome these adverse effects of the traditional chemical treatments extensive research on the alternative materials for * corresponding author: mohammad manjurul alam e-mail address: mamillat@yahoo.com fish disease control are ongoing. among the plants, neem tree is a very significant one, because the plant has chemicals which are of medicinal, insecticidal and anti-bacterial values. the major chemical component of the neem plant (azadirachta indica) is ‘azadirachtin’, which is one of the most promising natural compound exhibiting antiviral, antibacterial and antifungal properties (harikrishnan et al., 2003), and has been used successfully in aquaculture system to control fish predators and parasites (dunkel and ricilards, 1998; winkaler et al., 2007) and pathogenic bacteria in culture water (das et al., 2002). neem extract has also been found to be potential in controlling bacteria isolated from marine fishes (dhayanithi et al., 2010). however, omoregie and okpanachi (1997) reported that aqueous extract of neem bark caused respiratory problems in tilapia zillii. saravanan et al. (2011) reported that as neem 234 ahsan et al./ effects of neem leaf juice on a. testudineus and c. punctata extracts affect the haematological, ionoregulatory, biochemical and enzymological parameters in fish (cirrhinus mrigala). therefore, there is a need for establishing safe limit of neem or any other biopesticides against the culture fishes, before introducing these bio-pesticides in culture ponds to eradicate insect pests or pathogens. the present study was designed to determine as a bio-safe anaesthetic, toxic and morphological effects of neem leaf juice against the two live fishes, anabas testudineus and channa punctata. materials and methods collection and acclimatization of experimental fish: in the present experiment, two live fish species viz. a. testudineus and c. punctata were used. live fishes of both species were collected from the fish landing centre, and carried to the laboratory in plastic containers containing water. in the laboratory, the total length and total weight of each specimen were recorded, and then released them in earthen pots containing pond water. the fishes were acclimatized to the lab condition for three days providing feed, changing the water after 24-hour. the dead fishes were removed immediately. preparation of neem leaf juice: freshly collected neem leaves were thoroughly washed. about 500 g of the leaf was taken with 1 l fresh water in an electrical blender. the juice was then filtered, and the filtrate liquid was considered as the stock solution. required quantity of the stock solution was mixed with water to prepare 5 l solution, for obtaining the concentrations of the neem leaf juice as 5, 10, 15 and 20%. experimentation: two sets of experiments were done, toxicity test and observation on morphological characters. in each experiment three fishes of each species of similar length and weight, were used separately for each of the concentrations. each fish was kept in separate plastic container, and the mouth of the container was covered with a piece of mosquito curtain tied with a rubber band to avoid the escaping of the fish. the experiments were carried at room temperature with three replications for each. the fishes under experiments were kept starved for two hours before they were released in the treatment. during the experiment no food was given to the fishes. parameters recorded: for toxicity test, the times when the fish became immobilize, anaesthetize and finally died were recorded. to study the effects of the treatment on the morphology of the fish, the skin, gill and eye colours were compared with that of the fishes kept in fresh water only as control. movement of the treated fish and change in smell were also observed in the treated and control fish. these data were taken when the fishes were anaesthetized due to the treatment. statistical analysis: the toxicity data were presented as the average time taken to product the different toxic stages of the fish. regression equation (y=a+ bx) was calculated to find out the correlations between the time required to immobilize and anaesthetize the fishes and the concentrations of the neem leaf juice, and that in between the time required to immobilize and anaesthetize the fishes and the total weight of fish. results and discussion toxic effect of neem juice: the concentrations of neem leaf juice used were found to be toxic against both species of the fish. channa punctata showed slightly increased tolerance to the treatment. anabas testudineus were immobilized at 5.17, 6.30, 5.30 and 4.20 hours after treatment with 5, 10, 15 and 20% neem leaf juice, respectively. channa punctata became immobilized at 8.45, 9.05, 8.15 and 7.45 hours after treatment, respectively (fig. 1). the fishes were anaesthetized or became senseless at 7.10, 12.10, 6.30 and 6.10 hours (a. testudineus), and 10.15, 10.05, 9.25 and 8.28 hours (c. punctata) after treatment with the mentioned concentrations, respectively (fig. 2). however, the time required for death, from anaesthetic stage was less in c. punctata than a. testudineus. the time required for death of a. testudineus were 9.10, 13.20, 8.00 and 7.30 hours; and that for death of c. punctata were 10.45, 10.35, 235 int. j. aquat. biol. (2016) 4(4): 233-238 10.25 and 9.05 hours after treatment with 5, 10, 15 and 20% of neem leaf juice, respectively (fig. 3). the time required for immobilize and anaesthetize the fish were found to be negatively correlated with the concentrations of neem leaf juice used, where the coefficient of correlation values were obtained as r=0.59 and r=0.41, respectively (a. testudineus), and r=0.76 and r=0.95, respectively (c. punctata) (figs. 4 and 5). total weight of the fish was an important factor for the efficacy of the neem leaf juice as anaesthetic. the relations between the total weight and the time required to immobilize and anaesthetize the fish were poorly but positively correlated with coefficient of correlation values as r=0.23 and r= 0.33, respectively (a. testudineus). whereas, the correlation were high in case of c. punctata, where the r values were obtained as 0.63 and 0.79, respectively (figs. 6 and 7). morphological effects: the change of morphological figure 1. immobilizing time of a. testudineus and c. punctata in different concentrations of neem leaf juice. figure 2. anaesthetizing time of a. testudineus and c. punctate in different concentrations of neem leaf juice. figure 3. dying time of a. testudineus and c. punctate in different concentrations of neem leaf juice. figure 4. relationships among concentrations of neem leaf juice with immobilizing and anaesthetizing time of a. testudineus. y = 9.25 0.078x (r = 0.76) y =11.035 0.1282x (r = 0.95) 6 7 8 9 10 11 12 2 5 8 11 14 17 20 concent rat ions (%) t im e ( h r) immobilizing time anaesthetizing time y = 10.1 0.176x (r = 0.41) y = 6.22 0.0782x (r = 0.59) 0 2 4 6 8 10 12 3 5 7 9 11 13 15 17 19 21 concent rat ions (%) t im e ( h r) immobilizing time anaesthetizing time figure 5. relationships among concentrations of neem leaf juice with immobilizing and anaesthetizing time of c. punctata. y = 3.3677 + 0.0435x (r = 0.23) y = 0.8743 + 0.2035x (r = 0.33) 0 2 4 6 8 10 12 35 37 39 41 43 45 47 t ot al weight (g) t im e ( h r) immobilizing time anaesthetizing time figure 6. relationships among total weight and immobilizing and anaesthetizing time of a. testudineus. 236 ahsan et al./ effects of neem leaf juice on a. testudineus and c. punctata features of the experimental fishes were assessed when they became senseless or anaesthetized due to the treatment with the aforementioned concentrations of neem leaf juice. the normal skin colour of a. testudineus was changed to yellowish and pale brown at different concentrations. the skin colour of c. punctata was changed to yellowish, but at higher concentrations it became greyish. eye of the anaesthetized fishes became cloudy, and the normal fishy odour was changed to a smell like neem leaf. gills of the fresh fishes were deep red in colour, which finally changed either to yellowish or to brownish. movement of a. testudineus was frequent showing some restlessness in the treated water at lower concentration, but the agility was decreased in the higher concentrations. whereas c. punctata showed very slow movement in the treated water at all concentrations. the above mentioned characters of the treated fishes are presented in table 1. the present results revealed that the neem leaf juice has anaesthetic effect against both fish species concentration (%) *exposure time (hr) morphological characters skin color eye gill movement odor a n a b a s te st u d in e u s control (only water) normal (grayish) transparent or clear deep red normal (frequent) normal 5 4 yellowish transparent deep red frequent smelled like neem leaf 8 pale brown cloudy yellowish rapid and restless smelled like neem leaf 10 4 brownish transparent deep red frequent smelled like neem leaf 8 yellowish transparent brownish slow smelled like neem leaf 12 pale brown cloudy yellowish very slow smelled like neem leaf 15 4 pale brown cloudy brownish very slow smelled like neem leaf 20 4 yellowish cloudy yellowish immobile smelled like neem leaf c h a n n a p u n c ta te control (only water) normal (blackish) transparent or clear deep red normal (frequent) normal 5 4 yellowish transparent pale red slow smelled like neem leaf 8 yellowish cloudy pale red slow smelled like neem leaf 10 4 yellowish black transparent pale red slow smelled like neem leaf 8 yellowish cloudy brownish very slow smelled like neem leaf 15 4 yellowish cloudy brownish slow smelled like neem leaf 8 grayish cloudy yellowish very slow smelled like neem leaf 20 4 grayish cloudy yellowish very slow smelled like neem leaf * the exposure time was measured from the time of release up to the time the fish were anaesthetized. table 1. effect of neem leaf juice on the morphological characters of a. testudineus and c. punctata with time. y = 2.4578 + 0.1607x (r = 0..63) y = 0.0873 + 0.2629x (r = 0.79) 6 7 8 9 10 11 12 32 33 34 35 36 37 38 39 t ot al weight (g) t im e ( h r) immobilizing time anaesthetizing time figure 7. relationships among total weight and immobilizing and anaesthetizing time of c. punctata. 237 int. j. aquat. biol. (2016) 4(4): 233-238 a. testudineus and c. punctata, and both these fishes are designated as hardy fish. the time calculated between the anaesthetic stages to death for a. testudineus ranged from 1.30 to 2 hours and that for c. punctata ranged from 0.35 to around 1 hour at different concentrations of the juice used. however, the time of effectiveness of the neem leaf juice was found to be dependent on the body weight of the fish, i.e., younger fish of both the species will need >5% of the juice for anaesthetization. though the fish farmers use neem extract to control fish parasites and fish fry predators, but it has also been reported that longer exposure can be fatal for the fish. winkaler et al. (2007) reported that lc50 of the aqueous extract of neem was estimated as 4.8 gl-1 at 24-hour exposure for the juveniles of prochilodus lineatus. the authors also reported that the extract at concentrations of 5 and 7.5 gl-1 damaged the gill and kidney tissues, but not influenced the osmoregulatory capacity of the fish. akinwande et al. (2007) estimated the mortality rate and opercular ventilation under laboratory conditions over 96-hour exposure to aqueous extract of the mesocarp of neem fruit against the hybrid heteroclarias sp. catfish. the lc50 value at 96-hour exposure was calculated as 81.28 mgl-1, and the treated fishes exhibited respiratory distress (as they were gasping air), loss of appetite, loss of balance and erratic swimming prior to death. the present results showed that the respiratory distress observed clearly in anaesthetized a. testudineus. the species showed somewhat erratic movement at lower concentrations but became lethargic at higher concentrations. channa punctata is normally a sluggish fish, and in treatment its movement became slower. the lc50 values of neem leaf extract were found as 4 and 11 gl-1 for the juvenile oreochromis niloticus and clarias gariepinus, respectively, when exposed for 24 hour (mousa et al., 2008). labeo rohita when exposed to sub-lethal concentrations of neem extract for 25 days, the lc50 value was obtained as 1.035 gl-1 (saravanan et al., 2010). neem based pesticides like nimbicidine and neem gold were reported to be toxic against the freshwater loach, lepidocephalichthys guntea. the 96-hour lc50 values of nimbicidine, neem gold and combination of two were determined as 0.0135, 0.0525 and 0.0396 mgl-1, respectively (mondal et al., 2007). the authors also reported that the fish under toxicity stress suffered several abnormalities like erratic and rapid movement, imbalance and surface floating, which were correlated with the concentrations of the pesticides used. kumar et al. (2010) observed severe effects of neem extract on the gills of heteropneustes fossilis exposed to a treatment with 0.2 mgl-1 for 28 days. the effects were over secretion of mucous, hyperplasia, fusion of gill lamellae, necrosis of gill epithelial cells, and epithelial uplifting. these changes were time and dose dependent. the result of mousa et al. (2008) revealed that neem extract is safe to use in fish farm, as it exhibited low toxicity to non-target aquatic life. the neem extract may have lethal effects but at low concentrations it could be a good anaesthetic if the recovery rate from effect is high and quick. the present results revealed that for handling live fish during vaccination or other purpose, 5-10% neem leaf juice will provide enough time to keep the fishes immobile. moreover, bathing the fish at these concentrations may provide treatment against the bacterial or fungal diseases, and the fish will not lose its normal morphological characters. preparation of neem leaf juice is easy, and of low cost. fish farmers can easily use it for their cultured fish. further works with neem leaf juice are needed to estimate the recovery rate and recovery time from the anaesthetic state of different species of fish. acknowledgments the authors are grateful to the chairman of the department of zoology and department of fisheries, rajshahi university for providing all sorts of laboratory facilities. references akinwande a.a., sogbesan a.o., moody f.o., ugwumba a.a.a. (2007). piscicidal potential of 238 ahsan et al./ effects of neem leaf juice on a. testudineus and c. punctata mesocarp of neem plant (azadirachta indica l.) fruit on hybrid heteroclarias. journal of environmental biology, 28(3): 533-536. barton b.a., iwama g.k. (1991). physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids. annual review of fish disease, 1: 3-26. das b.k., mukherjee s.c., murjani g. (2002). acute toxicity of neem azadirachta indica in indian major carps. journal of aquaculture in the tropics, 17(1): 23-33. dhayanithi n.b., kumar t.t.a., kathiresan k. (2010). effect of neem extract against the bacteria isolated from marine fish. journal of environmental biology, 31(4): 409-412. dunkel f.v., richards d.c. (1998). effect of an azadirachtin formulation on six nontarget aquatic macroinvertebrates. environmental entomology, 27(3): 667-674. harikrishnan r., rani m.n., balasundaram c. (2003). hematological and biochemical parameters in common carp, cyprinus carpio, following herbal treatment for aeromonas hydrophila infection. aquaculture, 221(1-4): 41-50. kumar a., prasad m.r., srivastava k., tripathi s., srivastav a.k. (2010). branchial histopathological study of catfish heteropneustes fossilis following exposure to purified neem extract, azadirachtin. world journal of zoology, 5(4): 239-243. mondal d., barat s., mukhopadhyay m.k. (2007). toxicity of neem pesticides on a fresh water loach, lepidocephalichthys guntea (hamilton buchanan) of darjeeling district in west bengal. journal of environmental biology, 28(1): 119-122. mousa m.a.a., el-ashram a.m.m., hamed m. (2008). effect of neem leaf extract on freshwater fishes and zooplankton community. in: 8th international symposium on tilapia in aquaculture. pp: 307-318. omoregie e., okpanachi m.a. (1997). acute toxicity of water extracts of bark of the neem plant, azadirachta indica (lodd) to the cichlid tilapia zillii (gervais). acta hydrobiologica, 39: 47-51. saravanan m., kumar d.v., malarvizhi a., ramesh m. (2010). biosafety of azadirachta indica (a. juss) leaves extracts on certain biochemical parameters of labeo rohita. journal of biopesticides, 3(1): 227-231. saravanan m., ramesh m., malarvizhi a., petkam r. (2011). toxicity of neem leaf extracts (azadirachta indica a. juss) on some haematological, ionoregulatory, biochemical and enzymological parameters of indian major carp, cirrhinus mrigala. journal of tropical forestry and environment, 1(1): 14-26. wendelaar bonga s.e. (1997). the stress response in fish. in: physiological reviews, 77(3): 591-625. winkaler e.u., santos t.r.m., machado-neto j.g., martinez c.b.r. (2007). acute lethal and sublethal effects of neem leaf extract on the neotropical freshwater fish prochilodus lineatus. comparatice biochemistry and physiology part c: toxicology and pharmacology, 145(2): 236-244. int. j. aquat. biol. (2016) 4(5): 318-324: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article the sagittal otolith morphology of four selected mugilid species from iranian waters of the persian gulf (teleostei: mugilidae) vahideh salehi1, majid askari hesni1, azad teimori*1, mohammad reza lashkari2 1department of biology, faculty of sciences, shahid bahonar university of kerman, 76169-14111 kerman, iran. 2department of biodiversity, institute of science and high technology and environmental sciences, graduate university of advanced technology, kerman, iran. article history: received 2 july 2016 accepted 24 september 2016 available online 2 5 october 2016 keywords: sagitta phylogeny taxonomy fisheries management abstract: the members of mugilid species are usually difficult to recognize because of the wellknown similarity observed in their external morphology. nevertheless, their identification is very important for local fisheries management and conservation action. therefore, in the present study we applied otolith morphology to evaluate its significance in identification of four selected mugilid species; chelon subviridis (valenciennes, 1836), liza klunzingeri (day, 1888), ellochelon vaigiensis (quoy & gaimard, 1825) and mugil cephalus linnaeus, 1758 occurring in the iranian waters of the persian gulf in southern iran. the results indicated several otolith features to be important for identification of the selected mugilid species as follow; the position and sulcus centrality, the curvature of the cauda, and the type of anterior and posterior regions. based on the total approach evidences, we conclude that otolith morphology in mugilid fishes can be evidently used for the species identification and probably estimation of their phylogeny. the findings are in agreement with the previous studies which documented taxonomic importance of otolith morphology. introduction the members of the family mugilidae with 17 genera and 72 species show a world-wide distribution in temperate to tropical coastal environments (nelson et al., 2016; eschmeyer and fong, 2016). the fishes inhabit various coastal environment, brackish waters, and lagoons with high salinity (golani et al., 2002). however, some species are even resident in freshwater ecosystems (nelson et al., 2016). until date, four genera i.e. liza jordan and swain, 1884; mugil linnaeus, 1758, chelon; and ellochelon whitley, 1930 with seven species, including abu mullet, liza abu (heckel, 1843), golden grey mullet, chelon aurata (risso, 1810), leaping mullet, chelon saliens (risso, 1810), klunzinger's mullet, liza klunzingeri (day, 1888), flathead grey mullet, mugil cephalus linnaeus, 1758, and greenback mullet, chelon subviridis (valenciennes, 1836), and squaretail mullet, * corresponding author: azad teimori e-mail address: a.teimori@uk.ac.ir ellochelon vaigiensis (quoy and gaimard, 1824) have been recorded from iranian waters, including brackish and sea waters (coad, 2015). in addition to the above mentioned species, some other taxa have also been reported from the iranian waters but there are no acceptable scientific references for their existence. therefore, we did not explain them here. since species of the mugilid fishes have valuable fisheries catch over the world, therefore, species distinction in this family is very important for the local fisheries management, and the trophic studies. however, owing to the wide variety and similarities seen in their external morphology and meristic characters, usually considerable doubt exists regarding of their systematic classification (whitfield et al., 2012). therefore, hypothesis is that using hard structures such as otolith may provide an appropriate tool to discriminate species in this family. by considering the above mentioned hypothesis, 319 int. j. aquat. biol. (2016) 4(5): 318-324 we selected four morphologically similar mugilid species (i.e. chelon subviridis, ellochelon vaigiensis, liza klunzingeri, and mugil cephalus) from the iranian waters of the persian gulf, and applied morphology of the saccular otolith (sagitta) to evaluate the possible application of the otolith in identification of these species. this is the first comparative study on otolith morphology of the mugilid species in this region. the results could contribute to correct management of mugilidae fisheries resource, and even for trophic and paleoecological studies in this poorly studied area. materials and methods sampling: the four selected mullet species chelon subviridis (zm-fisbuk 1-10, n=10), ellochelon vaigiensis (zm-fisbuk 21-24, n=4), and liza klunzingeri (zm-fisbuk 11-20, n=10) were collected from bandar-e-lengeh (46°02'50.1"n, 37°01'53.5"e), and the mugil cephalus (zmfisbuk 25-34, n=10) was obtained from bandar abbas (27°11'29.93"n, 56°20'32.01"e). both locations are located in the coastal waters of hormuzgan province in the persian gulf, southern iran (fig. 1). all the specimens are adult, with total length between 111 mm and 276.6 mm (table 1). the specimens were preserved in ethanol 70% as whole fish, and the otolith were kept dry in plastic boxes. the fish material and their otoliths are deposited in the collection of the zoological museum of shahid bahonar university of kerman, iran (zm-fisbuk, see above). otolith preparation, description and measurement: to extract the otoliths, we follow the procedure explained in reichenbacher et al. (2007). four skulls per species were opened ventrally, and right and left otoliths were removed. the otoliths were cleaned from tissue remains with 1% potassium hydroxide solution for 6 hrs and rinsed in distilled water for 12 hrs. they were dried and stored in plastic vials for later description. description of general morphology of the otoliths was based on the criteria proposed by tuset et al. (2008) and accordingly, the following definitions used to describe different parts of the otoliths (figs. 2a-b): heterosulcoid sulcus: sulcus with ostium and cauda clearly differentiated, but very different in shape (fig. 2a). supramedian sulcus: sulcus generally positioned above the longitudinal midline of the otolith and the ventral area is noticeably larger than the dorsal area (fig. 2a). ostial sulcus: sulcus with an ostium opens widely in figure 1. sampling locations (bandar abbas and bandar-e-lengeh) of the mugilid species along the persian gulf coast in southern iran, hormuzgan province, and the persian gulf. map modified from google map 2015. 320 salehi et al./ otolith morphology of mugilid species the anterior margin of the otolith, and with a cauda distinctly closed far away from the posterior margin (fig. 2a). tubular cauda: the cauda is rather long and its walls are usually straight or curved (fig. 2b). moreover, since the position of sulcus is particularly important for species identification, therefore, an index of sulcus centrality (sc) was calculated (fortunato et al., 2014) to compare the degree of sulcus centrality in each studied species. according to fortunato et al. (2014), the sc represents relative position of the sulcus in relation to the otolith's anterior-posterior axis. in this calculation, sc=sm (distance from the cauda superior margin to the dorsal margin) / ow (otolith total width). by considering the proposed index, a sulcus with an sc=0.50 has central position; a sulcus with an sc<0.50 shift towards the dorsal margin of otolith, and a sulcus with an sc>0.50 shift towards the ventral margin of otolith (fortunato et al., 2014). results the sagittal otoliths in the selected mugilids are rectangular to oblong in shape, laterally compressed, longer in length than width, and represent irregular margins with obvious protuberances irregularly arranged. their otoliths are longer characterized by heterosulcoid and ostial sulcus acusticus, which are, are usually supramedian. the sulcus has funnel-like ostium, and open to the anterior margin. there is usually a tubular cauda, which is closed towards the posterior one. the cauda is always longer than the ostium (two or three times). there is a very short and broad rostrum in anterior region, while, antirostrum absent or not well defined. excisura is relatively wide chelon subviridis: the sagitta is rectangular to trapezoid in shape. dorsal rim is straight with a clear dorsal tip in anterior region, ventral rim irregular to sinuate (fig. 3a-b). sulcus is heterosulcoid, ostial and supramedian. sulcus has a centrality (sc index) = 0.14, ostium funnel-like and slightly deep. cauda obviously tubular and sinuous distinctly bent towards the ventral region, ending towards the posterior region; cauda length is bigger than three times of ostium. anterior region angled, while posterior region is mostly rounded (fig. 3a-b). the distal face of sagitta is concave (fig. 3c). relative otolith height (oh)/otolith length (ol) is 39.0%. liza klunzingeri: the sagitta is rectangular in shape. dorsal rim is straight, ventral rim irregular and strongly serrated (fig. 4a-b). sulcus is heterosulcoid, ostial and supramedian. sulcus has a centrality (sc index) = 0.13. ostium funnel-like and in some individuals rectangular. cauda obviously tubular and straight bent towards the ventral region, ending towards the posterior region; cauda length is bigger than three times of ostium. anterior region is rounded to blunt, posterior region mostly rounded (fig. 4a-b). the distal face of sagitta is concave (fig. 4c). relative otolith height (oh)/otolith length (ol) is 50.0%. mugil cephalus: the sagitta is rectangular in shape. dorsal rim is straight, ventral rim irregular and strongly serrated (fig. 5a-b). sulcus is heterosulcoid, ostial and supramedian. sulcus has a centrality (sc index) = 0.16. ostium funnel-like. cauda obviously tubular and bent towards the ventral region, ending figure 2. the definitions, which are used to describe different parts of the otoliths general morphology. (a) represent heterosulcoid sulcus, supramedian sulcus and tubular cauda; and (b) show the ostial sulcus. the criteria modified from tuset et al. (2008). 321 int. j. aquat. biol. (2016) 4(5): 318-324 figure 3. left sagitta of chelon subviridis (21.5 cm sl, zm-fisbuk1). a-c. internal view; d. external view; e. dorsal view; f. ventral view; a. anterior part; p. posterior part. figure 4. left sagitta of chelon klunzingeri (12.6 cm sl, zm-fisbuk14). a-c. internal view; d. external view; e. dorsal view; f. ventral view; a. anterior part; p. posterior part. 322 salehi et al./ otolith morphology of mugilid species towards the posterior region; cauda length is bigger than three times of ostium. anterior region peaked, while posterior region is rounded (fig. 5a-b). the distal face of sagitta is concave (fig. 5c). relative otolith height (oh)/otolith length (ol) is 42.1%. ellochelon vaigiensis: the sagitta is clearly rectangular in shape. dorsal rim irregular with obvious dorsal tip in anterior end, ventral rim serrated (fig. 6a-b). sulcus is heterosulcoid, ostial and supramedian. sulcus has a centrality (sc index) = 0.12. ostium is funnel-like and tubular in some individuals. cauda obviously tubular and bent towards the ventral region, ending towards the posterior region; cauda length is bigger than two times of ostium. anterior region peaked, while posterior region is rounded with clear processes (fig. 6a-b). the distal face of otolith is concave (fig. 6c). relative otolith height (oh)/otolith length (ol) is 47.2%. sagittal otolith morphology key to identify four studied mugilid species: 1a tubular cauda distinctly bent towards the ventral region………………………………….……..…... 2 1b tubular cauda straight bent towards the ventral region………………...…………... liza klunzingeri 2a the relative otolith height (oh)/otolith length (ol) is bigger than 42%.……………………….… 3 2b the relative otolith height (oh)/otolith length (ol) is 39%…………………..… chelon subviridis 3a sagitta has sulcus with a centrality (sc) index of 0.16………………………………... mugil cephalus 3b sagitta has sulcus with a centrality (sc) index of 0.12…………………………. ellochelon vaigiensis discussion several studies have recently indicated that general morphology of otolith and its morphological features contain taxonomic and even genetic data, and therefore, are very useful tools for identification of fish species (e.g., esmaeili et al., 2014; teimori et al., 2012a, 2014; gholami et al., 2014;) and populations (e.g. reichenbacher et al., 2007; figure 5. left sagitta of mugil cephalus (24.0 cm sl, zm-fisbuk28). a-c. internal view; d. external view; e. dorsal view; f. ventral view; a. anterior part; p. posterior part. 323 int. j. aquat. biol. (2016) 4(5): 318-324 teimori et al., 2012b-c; annabi et al., 2013). the otolith morphology could even be more important for discrimination of closely related species where external morphology of fish specimens could not help. the potential power of otolith morphology in discriminate of closely related fish species has already been examined in several genetically close species of the genus aphanius within iranian inland waters (teimori et al., 2012a; esmaeili et al., 2014), in which they have separated several endemic aphanius species by using otolith morphology. the utility of otolith morphology for identification of mugilid species has already been examined in northeastern atlantic ocean and the mediterranean sea region (fortunato et al., 2014). they concluded that general shape of the saccular otolith, presence of an ostial sulcus acusticus, type of ostium and cauda and characteristics of dorsal and ventral margins could be sufficient to identify species in this family. our comparative study is in agreement with the founding of fortunato et al. (2014), in which several otolith features such as type of ostium and cauda, and characteristics of dorsal and ventral margins considered to be important for discrimination of the studied mugilid species from iranian waters of persian gulf. the further otolith features that could play role in separation of mugilid species in iranian waters of persian gulf are position of sulcus (sulcus centrality), curvature of cauda, and type of anterior and posterior regions. these features have also recognized in adult specimens of mugil liza and mugil curema from the west coast of southwestern atlantic ocean (fortunato et al., 2014). since members of mugilidae family form an important part of the feeding regime of the local people in coastal parts of the persian gulf and probably other parts of the world, therefore, this type of study are particularly relevant for determination of the species. it can also be important even in figure 6. left sagitta of ellochelon vaigiensis (27.6 cm sl, zm-fisbuk24). a-c. internal view; d. external view; e. dorsal view; f. ventral view; a. anterior part; p. posterior part. 324 salehi et al./ otolith morphology of mugilid species trophic ecology where otoliths present in stomach contents of ichtyophagus organisms (bustos et al., 2012; veen et al., 2012; riet-sapriza et al., 2013). acknowledgements this work was supported by the shahid bahonar university of kerman. references annabi a., said k., reichenbacher b. (2013). interpopulation differences in otolith morphology are genetically encoded in the killifish aphanius asciatus (cyprinodontiformes). scientia marina, 77(2): 269279. bustos r., daneri l.g.a., volpedo a.v., harrington a., varela e.a. (2012). the diet of the south american sea lion (otaria flavescens) at río negro, patagonia, argentina. iheringia, série zoologia, 102(4): 394-400. coad b.w. (1998). systematic biodiversity in the freshwater fishes of iran. italian journal of zoology, 65(1): 101-108. coad b.w. (2015). freshwater fishes of iran. available from: www.briancoad.com. retrieved 12/11/2015. eschmeyer w.n., fong j.d. (2016). species by families/subfamilies. in: catalog of fishes. availabe from: http://researcharchive.calacademy.org/research/ ichthyology/catalog/speciesbyfamily.asp. retrieved 02/03/2016. esmaeili h.r., teimori a., gholami z., reichenbacher b. (2014). two new species of the tooth-carp aphanius (teleostei: cyprinodontidae) and the evolutionary history of the iranian inland and inlandrelated aphanius species. zootaxa, 3786(3): 246-268. fortunato r.c., dura v.b., volpedo a. (2014). the morphology of saccular otoliths as a tool to identify different mugilid species from the northeastern atlantic and mediterranean sea. estuarine, coastal and marine science, 146(5): 95-101. gholami z., esmaeili h.r., erpenbeck d., reichenbacher b. (2014). phylogenetic analysis of aphanius from the endorheic kor river basin in the zagros mountains, southwestern iran (teleostei: cyprinodontiformes: cyprinodontidae). journal of zoological systematics and evolutionary research, 52(2): 130-141. golani d., orsi relini l., massutí e., quignard j.p. (2002). ciesm atlas of exotic species in the mediterranean. in: briand, f. (ed.). fishes, vol. 1. ciesm publishers, monaco. nelson j.s., grande t.c., wilson mvh. (2006). fishes of the world, 5th ed. hoboken john wiley and sons publishing. 756 p. teimori a., esmaeili h.r., gholami z., zarei n., reichenbacher b. (2012a). aphanius arakensis, a new species of tooth-carp (actinopterygii, cyprinodontidae) from the endorheic namak lake basin in iran. zookeys, 17(215): 55-76. teimori a., schulz-mirbach t., esmaeili h.r., reichenbacher b. (2012b). geographical differentiation of aphanius dispar (teleostei: cyprinodontidae) from southern iran. journal of zoological systematics and evolutionary research, 50(4): 289-304. teimori a., jawad j.l.a., al-kharusi l.h., al-mamry., j.m. reichenbacher b. (2012c).late pleistocene to holocene diversification and historical zoogeography of the arabian killifish (aphanius dispar) inferred from otolith morphology. scientia marina, 76(4): 637645. teimori a., esmaeili h.r., erpenbeck d., reichenbacher b. (2014). a new and unique species of the genus aphanius (teleostei: cyprinodontidae) from southern iran: a case of regressive evolution. zoologischer anzeiger a journal of comparative zoology, 253(4): 327-337. tuset v.m., lombarte a., assis a.c. (2008). otolith atlas for the western mediterranean, north and central eastern atlantic. scientia marina, 72(1): 7-198. veen j., mullie w., veen t. (2012). the diet of the whitebreasted csormorant phalacrocorax carbolucidus along the atlantic coast of west africa. ardea, 100(2): 137-148. whitfield, a., panfili j., durand j.d. (2012). a global review of the cosmopolitan flathead mullet mugil cephalus linnaeus1758 (teleostei: mugilidae), with emphasis on the biology, genetics, ecology and fisheries aspects. reviews in fish biology and fisheries, 22(3): 641-681 int. j. aquat. biol. (2016) 4(5): 318-324 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی فارس خلیج ایرانی هایآب از ماهی کفال گونه چهار در ساژیتا اتولیت شناسیریخت 2ریک، محمد رضا لش1*تیموری آزاد ،1حصنی عسکری مجید ،1صالحی وحیده .ایران ،کرمان ،کرمان باهنر شهید دانشگاه علوم، دانشکده شناسی،زیست گروه1 .ایران کرمان، پیشرفته، فناوری و صنعتی تکمیلی تحصیالت دانشگاه محیطی، علوم و پیشرفته تکنولوژی و علوم پژوهشگاه زیستی، تنوع گروه2 چکیده: این با. است دشوار اغلب ظاهری ریختی صفات بر تکیه با آنها شناسایی دارد، وجود ماهیان کفال اعضای بین در که زیادی ظاهری شباهت خاطرهب برای نواییش سنگریزه شناسیریخت حاضر مطالعه در بنابراین،. باشدمی مهم بسیار آنها حفاظت و شیالتی مدیریت برای آنها دقیق شناسایی وجود، mugil و chelon subviridis، liza klunzingeri، ellochelon vaigiensisشامل) فارس خلیج ماهیان کفال از گونه چهار شناسایی cephalus )باشند؛یم مهم مطالعه مورد هایگونه تفکیک برای شنوایی سنگریزه ریختی صفت چندین که داد نشان نتایج. گرفت قرار ارزیابی مورد دشومی گیرینتیجه مطالعه این هاییافته اساس بر. اتولیت عقبی و قدامی نواحی شکل و( cauda) کوادا شیار انحنای سولکوس، مرکزیت و موقعیت مطالعات اب منطبق مطالعه این هاییافته. باشدمی مهم آنها فیلوژنتیکی روابط احتماالً و گونه تشخیص در ماهیان کفال در اتولیت شناسیریخت که .دارند زیادی اهمیت ماهیان تاکسونومیکی جایگاه شناخت در اتولیت ریختی صفات که نمایدمی تایید و است گذشته .شیالتی مدیریت تاکسونومی، فیلوژنی، ساژیتا، :کلمات کلیدی int. j. aquat. biol. (2017) 5(4): 275-281 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article variation in the shell form of the swan mussel, anodonta cygnea (linea, 1876) in response to water current fateh moëzzi1, hadi poorbagher*1,1amir ghadermazi2, fatemeh parvizi3, saleh benam2 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. 3department of fisheries, university of hormozgan, bandar abbas, iran. article history: received 19 march 2017 accepted 6 august 2017 available online 2 5 august 2017 keywords: anodonta cygnea biometry population structure water condition abstract: a biometric study was conducted on the populations of swan mussel, anodonta cygnea, belonged to water bodies with different water current velocity (high current: hc; low current: lc). the shell length, width, height, weight and age of the collected mussels were measured. the two groups had different age-classes distributions. the hc mussels had larger mean and maximal values of the biometric parameters. a high and medium correlation coefficient (width-length, height-length, and weight-length) were found in the hc and lc mussels, respectively. the weight-length relationships showed negative allometric pattern (hc: weight=3.6121x0.8561; lc: weight= 3.1362x0.8508). the 1-2 years old mussels had the highest rates of increase in length, width and height in both groups. based on the results, water current velocity influences biometric features and population structure of a. cygnea. introduction swan mussel, anodonta cygnea, is a member of the family unionidae, having a wide distribution in the world (pourang et al., 2004). this species inhabits shallow and eutrophic waters and also slow-flowing streams. swan mussel lives in water bodies with depths ranging from 0.2 m to several meters (rosinska et al., 2008). this species is sensitive to inappropriate environmental conditions (moezzi et al., 2013), therefore, it is a bioindicator of unpolluted waters with well-oxygenated bed (zajac, 2004). swan mussel is an endemic species of northern iran, i.e. the caspian sea basin (parvaneh, 1994; pourang, 1996). morphometric studies of mollusks provide useful information about their growth rates (alunno-bruscia et al., 2001) to assess their habitats (devescovi, 2009). the size of the valves in bivalve molluscs is an index representing the trophic state of their environment (azzopardi et al., 2013). in addition, the shell size, per se, and its correlation with other biometric para-meters can be appropriate indices for monitoring their *corresponding author: hadi poorbagher doi: https://doi.org/10.22034/ijab.v5i4.296 e-mail address: poorbagher@ut.ac.ir population dynamics in natural and deteriorated environments (deidun et al., 2014; bayne and newell, 1983; palmer, 1990). growth in bivalves is mainly estimated using the shell dimensions or volume (hibbert, 1977; rodhouse et al., 1984; bailey and green, 1988; ross and lima, 1994; rueda and urban, 1998; ravera and sproocati, 1997; deval, 2001; mutvei and westermark, 2001). using allometric equations in bivalves, shell dimension may be used to calculate biomass (hibbert, 1977; ross and lima, 1994; thorarinsdottir and jahanesson, 1996; ravera and sprocati, 1997; deval, 2001). reproductive conditions (rueda and urban, 1998), population density (seed, 1968), physical and biological characteristics of habitat (thorarinsdottir and jahannesson, 1996) are among the factors that affect the growth of bivalves and allometric relationships between the shell size and weight. as yet, biometric and morphometric characteristics of the iranian populations of a. cygnea in different habitats have not been investigated. the present study 276 moezzi et al./ variation in the shell form of the swan mussel thus aimed to investigate biometric characteristics (shell length, width, height, weight and their relationships plus age structure) of the populations of swan mussel collected from different flowing water bodies. materials and methods study area: a total of 277 individuals of a. cygnea were collected from two fastand slow-flowing water streams (1.5-2 m s-1 and < 0.1 m s-1, being named as hc and lc hereafter, respectively) from the semeskandeh, mazandaran province, northern iran in the autumn 2013 (fig. 1). water characteristics were nearly similar in all sampling locations (table 1), except current velocity, where it was 1.5-2 m s-1 at hc environment, which was a water channel, but the lc sampling place was a lentic water habitat with no considerable water current. during the sampling, specimens were collected from the area of 1 m2 using a quadrat with three replicates. 124 and 153 specimens were collected from hc and lc locations, respectively. collected mussels were placed in fiberglass tanks and transferred to the laboratory for biometric measurements (tenjing sing et al., 2013). measurements: length (l), width (w), and height (h) of shells were measured to the nearest 1 mm using slide calipers. the total weight was measured to the nearest of 0.01 g. the age was determined by counting the annual rings on the shell surface. statistical analysis: measurements results were determined statistically as arithmetical mean, maximum, minimum, variation range, variance, and standard deviation. width-length and height-length relationships was established using linear equation, y=ax+b, where a: slope (relative growth rates of the variables), b: intercept (initial growth coefficient), and y: is width (mm) or height (mm) (tenjing sing et al., 2013). the bivalve weight-length relationships was derived by applying the equation y=axb (tenjing sing et al., 2013). relationships between length, width, height, and weight with age were also examined for the two groups of the specimens. all statistical analyses were performed using microsoft office excel (ver. 2013). results density of bivalves per unit of area in studied sites were 31.2 and 24.8 (individual/m2) in hc and lc places, respectively. age of mussels in hc or flowthrough water channel ranged from 1 to 6 years, but in lc from 2 to 5 years. mussels with age 4 years old was dominate specimens in both populations (fig. 2). table 2 presents the mean length, width, height and weight of swan mussels based on age groups in the studied sampling places. the mean values of the studied parameters in age groups of 2 and 3 years in figure 1. sampling region at semes-kandeh, mazandaran province, north of the iran. table 1. water characteristics at sampling points (hc: high current; lc: low current). t (°c) ph do (mg/l) sampling depth (m) transparency (sd depth(m)) hc 9±3 7.3±0.4 12.7±3.4 0.6±0.3 0.3 lc 12±2 6.9±0.3 9.2±2.1 0.75±0.2 1.5 277 int. j. aquat. biol. (2017) 5(4): 275-281 hc population were lower than those of lc, but in higher age classes, the values of these parameters were higher in the hc population compared with the lc population. the mean values of length, width, height, weight and age of mussels were higher in the hc group (table 3). the highest and lowest measured values of the parameters were observed in the hc group. range of all studied parameters were higher in the hc group compared to the lc group (table 2). bivariate relationships between width, height, and weight with length of mussels are presented in figure 3. linear and positive relationships were found for all bivariate plots. strong correlations were observed for mussels belongs to the hc group, but correlations between the measured parameters of lc mussel population specimens indicated medium strength. the scatter plot of the weight against the length showed negative allometric pattern for both populations (bhc =0.8561; blc=0.8508). figure 4 shows the relationship between length, width, and height with age for mussels of both populations. correlations of length, width, and height with age of mussels were strong in the hc population, but in the lc mussels, these relationships has showed moderate correlation for all parameters. discussion there was no specimen with the age older than 6 years old. the 4-year mussels were the dominant age-class table 2. mean values of length, width, height and weight in different age groups of swan mussel populations collected in studied water bodies. age n length (mm) width (mm) height (mm) weight (g) hc 1 8 46.75 16.625 25.25 110.6175 2 7 69.43 25.29 41.14 135.32 3 15 97.33 32.00 49.67 169.58 4 46 122.96 48.20 63.14 214.53 5 33 132.30 51.00 65.41 252.15 6 15 136.73 53.60 67.27 276.21 lc 2 6 85.83 26.17 38.67 126.69 3 39 103.62 34.67 49.36 163.03 4 74 108.93 38.19 51.85 172.47 5 34 118.56 42.59 57.35 178.41 table 3. mean, minimum (min), maximum (max), variation range, variance (var.), and standard deviation (sd) for length, width, height, weight and age of swan mussel specimens collected from water bodies. n mean min max variation range var. sd. hc length 124 116.07 36 152 116 649.15 25.47 width 44.30 12 61 49 128.92 11.35 height 58.92 21 72 51 145.73 12.07 weight 212.96 92.43 300.35 207.92 2868.744 53.56 age 4.08 1 6 5 1.68 1.298 lc length 153 108.81 76 126 50 75.70 8.70 width 37.79 24 52 28 29.03 5.38 height 51.92 34 67 33 50.30 7.09 weight 169.58 113.65 194 80.35 176.89 13.30 age 3.88 2 5 3 0.62 0.791 figure 2. frequency (%) of mussel individuals at different age groups collected from studied water bodies. 278 moezzi et al./ variation in the shell form of the swan mussel at both studied groups. similarly, the maximum observed age of this species in iranian waters in prior studies was 4 years (parvaneh et al., 1998; pourang et al., 2004), whereas specimens with 15 years old have been reported in european waters (ravera and sprocati, 1997). in the lc group, there were no specimen belonged to 1 and 6 years age-classes, but in the hc group, the specimens were in 1 to 6 years ageclasses. the lack of mussels in some of year classes may be due to rigorous population fluctuations (rosinska et al., 2008), that may stem from different factors such as environmental pollution, decrease in number of fish, which are the host of the parasitic larvae of this mussel, and temporal water quality changes in these waters (zajac, 2002). the maximal mean values of the length, width, height and weight of mussels were belonged to the hc group and also, the maximal values of these parameters were found in that group (length: 152 mm; width: 61 mm; height: 72 mm). the maximal length reported for swan mussel is 200 mm (wirebellose, 2001). higher variation in measured biometric parameters in the hc mussels compared to lc mussels may be attributed to dynamic water system in the hc sampling places and higher quality and quantity of nutrient sources in this water body (azzopardi et al., 2013). also, population density is reported as an important factor that can affect growth and shell morphometry through either food regulation, physical interface or their interaction (alunno-bruscia et al., 2001). the relationships between width, height, and weight with length showed significant correlations, but these correlations were high and at a medium rate in the hc and lc mussels, respectively. the lower correlation coefficients for the lc mussels maybe is a result of smaller age and size range of these mussels in this water body due to their population fluctuations (zajac, 2004). increase in length is happened with linear increase in width and height of mussels in both mussel groups. linear positive relationships between width-length and height-length of the bivalve’s shell are reported previously in different studies on bivalves (alunno-bruscia et al., 2001). the weight-length relationship of mussels in the present study showed negative allometric pattern, where b values were lower than unity for both groups. weight-length relationships have been isometric in most of the studied species (müller and patzner, 1996; tenjing singh et al., 2013), but positive and negative allometric patterns have also been reported for different species and within a species in different conditions (park and oh, 2002). the mean b values for figure 3. scatter plots of width-length (a), height-length (b), and weight-length (c) relationships of anodonta cygnea specimens collected from studied water bodies. 279 int. j. aquat. biol. (2017) 5(4): 275-281 swan mussel populations were between 2 and 4 (tesch, 1971), but in present study, b values were lower than those values. changes in b value can be due to different reasons, such as different growth and maturity levels, sex, geographical and local conditions, diseases, environmental pollution and parasitic infections (tesch, 1971). the length, width and height were increased along with increase in age, and the highest increases of these parameters were occurred during 1-2 years oldness. r2 values of biometric parameters at age relationships were high for hc mussels. shell growth increases with increase in age and bivalve species with thinner shells have faster growth than species with thicker shells (harmon and joy, 1990). maximal growth rate of a. cygnea occurs in the second year of the life (zajac, 2002) and sexual maturity happens in ages of 2-3 years (rosinska et al., 2008). these changes in growth rate with increase in age is a result of higher energy consumption for gamete production rather than body growth in multiple species after sexual maturity (zajac, 2004). generally, in bivalve populations, larger bivalves have higher ages than smaller ones, but it can be observed that mussels with similar size belong to different age-classes (ravera et al., 2007). such a situation was observed in the present study in both groups and may be attributed to better habitat conditions (i.e., higher nutrient levels in water) during the period with highest density at the second year of bivalve’s life (rosinska et al., 2008). based on the results, it can be concluded that different investigated mussel populations in different figure 4. plot of length (a and b), width (c and d), and height (e and f) at age of anodonta cygnea individuals collected from studied water bodies. 280 moezzi et al./ variation in the shell form of the swan mussel environments in terms of their water current, has different biometric and population structures, which probably can be due to environmental fluctuations in the water body with no significant water current. despite of mentioned differences, both populations have similar characteristics that could not be affected by such factors. acknowledgments this study was financially supported by university of tehran. references alunno-bruscia m., bourget e., fréchette m. 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(2017) 5(4): 275-281 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی در پاسخ به جریان آب anodonta cygnea (linea, 1876)ای تغییرات در فرم صدف دوکفه 2، صالح بنام3، فاطمه پرویزی2امیر قادرمزی ،1*هادی پورباقر، 1معزیفاتح .ایران ،کرج ،تهران دانشگاه ،طبیعیمنابع دانشکده، شیالت گروه1 .ایران گرگان، گرگان، طبیعی منابع و کشاورزی علوم دانشگاه زیست، محیط و شیالت دانشکده شیالت، گروه2 .ایران بندرعباس، هرمزگان، دانشگاه شیالت، گروه3 چکیده: گروه) بودند متعلق متفاوت جریان سرعت با محیط دو به که شد انجام( anodonta cygnea) آنودونت صدف از جوامعی روی بر بیومتریک مطالعه . شدند گیریاندازه هاصدف سن و وزن همچنین کفه، ارتفاع و عرض طول،(. lc نام با پایین سرعت در واقع گروه و hc نام با باال سرعت در موحود شده گیریاندازه پارامترهای در باالتری حداکثر و میانگین مقادیر hc هایصدف. داشتند تفاوت هم با گروه دو این هایصدف سنی رده توزیع الگوی طول-وزن روابط. آمد دستهب lc و hc هایصدف برای طول وزن و طول-ارتفاع طول، -وزن بین متوسطی و باال همبستگی ضرایب. داشتند دارای ساله 2 و 1 هایصدف(. وزن=lc: 0.8508x3.1362 هایصدف در وزن=hc: 0.8561x3.6121 هایصدف در) داد نشان را منفی آلومتریک جمعیتی ساختار و بیومتریک هایویژگی بر آب جریان سرعت نتایج، مبنای بر. داشتند گروه دو هر در را ارتفاع و پهنا طول، افزایش نرخ باالترین .گذاردمی تاثیر a. cygnea صدف ساختار جمعیت، شرایط آب.، ومترییب ، anodonta cygnea :کلمات کلیدی int. j. aquat. biol. (2013) 1(2): 61-67 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology histopathological effects of zinc (zn) on mantle, digestive gland and foot in freshwater mussel, anodonta cygnea (linea, 1876) fateh moëzzi *1, arash javanshir1, soheil eagderi1, hadi poorbagher1 1department of fisheries science, faculty of natural resources, university of tehran, iran. article history: received 12 april 2013 accepted 1 may 2013 available online 5 may 2013 keywords: zinc histopathology swan mussel mantle digestive gland foot abstract: heavy metals are the most important pollutants in aquatic ecosystems that may cause adverse effects on its biota. in this study, histopathological effects of zinc (zn) and their incidence time on swan mussel, anodonta cygnea, were studied. exposure to zn was done during 18 days and histopathological investigations were conducted in mantle, digestive gland and foot in days 0, 4, 9, and 18. histopathological changes observed were: damages of epithelium cells with increasing mucous cells (in mantle), atrophy of digestive tubules and haemocyte aggregation (in digestive gland), and hypoplasia, increasing mucous cells and myocyte swelling (in foot). moreover, granuloma and tissue rupture were found in all organs. primary histopathological changes were observed in fourth day of examination in all of studied organs. results showed that sensitivity of digestive gland is lesser than mantle and foot in exposure to zn. also the results indicated the histopathological alterations in the organs of swan mussel can be considered as reliable biomarkers in biomonitoring of heavy metal pollution in aquatic ecosystems. introduction heavy metal is a general name for a group of metallic elements that have toxic effects in concentrations higher than tolerable physiological levels of animals (forstner and wittman, 1983; rainbow, 2002; banfalvi, 2011). they have two categories: essential (with structural and biological functions) and nonessential (without any biological role) groups (simkiss, 1981; williams, 1981; banfalvi, 2011). these pollutants may have adverse impacts on biota of aquatic habitats. zinc (zn) is one of the essential heavy metals contaminants of freshwater ecosystems. the major anthropogenic sources of this element are processing of ores, electroplating, domestic and industrial effluents, combustion of fossil fuels and soil erosion (liobet et al., 1988; buhl and hamilton, 1990). the reported natural concentrations of zn were in the range of 0.1-50 * corresponding author: fateh moëzzi e-mail address: fmoezi.fateh@gmail.com tel: +989185177054 µg l-1 in freshwaters and 0.002-0.1 µg l-1 in marine ecosystems (who, 2001). in last decades, aquatic organisms are used as bioindicators for monitoring chemical pollution of freshwater and marine environments. bivalves are a group of molluscs that are appropriate for study of biological impacts of environmental pollutants in aquatic ecosystems (livingstone et al., 2000) and are widely used as bioindicators in biomonitoring (sanders et al., 1993). with high potential of accumulation of heavy metals in tissues and organs of bivalves, it is possible to detect and assess their occurrence using cellular and physiological responses (van duren et al., 2006). long term exposure to heavy metals can increase susceptibility to disease and development of histopathological malformations (zorita et al., 2006). broad varieties of histopathological alterations in fish and bivalves 62 moëzzi et al./ int. j. aquat. biol. (2013) 1(2): 61-67 have been developed and offered as biomarkers for monitoring of pollutants (lowe, 1988; cajaraville et al., 1992; yevich and yevich, 1994; syasina et al., 1997; au, 2004). this study aimed to investigate the histopathological alterations in different organs of swan mussel, anodonta cygnea, exposed to zn. swan mussel which belongs to family unionidae, has a wide dispersal area including north america, europe, northern africa and asia (pourang et al., 2010). in iran, this species is found in northern wetlands and rivers and in some coastal regions and due to its wide distribution area, can be considered as an appropriate bioindicator of heavy metal pollution in aquatic ecosystems. material and methods specimen collection and experimental condition: in october 2011, eighty specimens of a. cygnea were collected from the tajan river estuary (36°48΄46΄΄n, 53°6΄57΄΄e) (mazandaran province, iran). these specimens were transferred to 60l fiberglass tanks for adaption to laboratory conditions. these tanks have been equipped to a circulatory system and figure 1. light micrographs of transverse sections (h&e) of control mantle and metal-exposed specimens. (a, c and e) control and (b, d and f) exposed to zn. mc: mucous cell; ep: epithelium; ct: connective tissue; gr: granuloma; tr: tissue rupture; ha: haemocyte. 63 moëzzi et al./ int. j. aquat. biol. (2013) 1(2): 61-67 aeration. during the experiment, dechlorinated tap water was used. water temperature and ph were measured daily, which were 15-17°c and 6.8-7.4, respectively. specimens were not fed through the experiment. exposure plan: twenty four specimens (length: 12.7-13.3 cm) were introduced randomly into 3 experimental tanks (8 specimens per tank). there were 3 replicates in every sampling time and a control tank with 4 specimens. considering natural concentrations of zn in aquatic environments and based on previous studies (naimo, 1995), bivalves were exposed to 125 µg l-1 of zn for 18 days. at days figure 2. light micrographs of transverse sections (h&e) of digestive gland of control and metal-exposed specimens. (a and c) control and (b and d) exposed to zn. haa: haemocyte aggregation; gr: granuloma; ha: haemocyte. figure 3. light micrographs of transverse sections (h&e) of foot of control and metal-exposed specimens. (a and c) control and (b and d) exposed to zn. mc: mucous cell; c: cilia; s: swelling; tr: tissue rupture; ls: longitudinal section of myocyte; ps: transverse section of myocyte. 63 64 moëzzi et al./ int. j. aquat. biol. (2013) 1(2): 61-67 4, 9 and 18, specimens of each tank were removed to collect obtaining tissue samples. tissue preparation: the valves of specimens were opened by cutting anterior and posterior abductor mussels using a scalpel. samples of ≈ 0.5 cm3 from each target organ (mantle, digestive gland and foot) were removed and fixed into bouin’s solution (saturated picric acid: 75 parts, 40% formaldehyde: 25 parts and glacial acetic acid: 5 parts) for 48h and then stored in 70% ethanol. the histological sections (5-7 µm thickness) were prepared based on hewitson and darby (2010) and stained using haematoxylin and eosin. stained tissues were observed under a light microscope (leica ms5). results histopathological observations: mantle: histological sections of the mantle of control group had normal status with contiguous external epithelium with no sign of alteration in connective tissue of sub-epithelial (fig. 1a, c and e). mucous cells were seen sporadically in epithelium and sub-epithelium with equal size (fig. 1a). the muscular cells were with conjunct structure (fig. 1c) and the connective tissue was healthy (fig. 1e). histological observations of treatments showed specific signs of tissue damage in mantle (fig. 1b, d and f). the width of external epithelium was decreased and the number of the mucous cells in subepithelial layer were increased (fig. 1b). granuloma (pigmented cells with yellow to brown color) was found in sub-epithelial layers (fig. 1d). tissue rupture and dissociation of regular cellular structure in the muscular tissue observed (fig. 1b, d and e). in addition, the haemocyte infiltration and aggregation of the myocytes was occurred (fig. 1d). digestive gland: figures 2a and c, display histological status of digestive gland and their junctions in control group with healthy basophilic columnar epithelial cells and digestive (dingy) cells in tubule structure. also, connective tissue of interstitial space between tubules was normal with similar distribution of haemocytes (fig. 2c). digestive gland exposed specimens showed atrophy of digestive tubules and loss of digestive and basophile cells into the tubule (fig. 2b). intensive haemocyte aggregations in connective tissue and interstitial space (fig. 2b and d), as well as, granuloma was found in connective tissue observed (fig. 2d). foot: in the control group, external epithelium with integrated ciliary structure was observed (fig. 3.a). in subepithellial layer, the mucous cells had uniform distribution. in inner parts of foot, groups of myocytes were in various directions as their longitudinal and cross-sections were visible (fig. 3c). organ time day 4 day 9 day 18 mantle increase in mucous cells count haemocyte infiltration and aggregation in sub-epithelial connective tissues; granuloma; sub-epithelial hyperplasia; haemocyte infiltration and aggregation in connective and muscular tissues; tissue rupture digestive gland loss of digestive cells into tubules haemocyte aggregation in connective tissue; atrophy of tubules; granuloma in interstitial space between tubules. foot hypoplasia of external epithelium; increase in mucous cells count haemocyte infiltration and aggregation in sub-epithelial tissues; epithelium rupture myocyte inflammation (swelling); muscular tissue rupture. table 1. incidence time pattern of histopathological alterations in mantle, digestive gland and foot of zn-exposed mussels during study period. presented histopathological damages were observed in all 3 replicates of mussels in days 4, 9 and 18. 65 moëzzi et al./ int. j. aquat. biol. (2013) 1(2): 61-67 the foot of zn-exposed specimens represents an increase in the number of mucous cells and swelling of connective tissue of subepithellial layers (fig.3b). hypoplasia of external epithelium was occurred. there were tissue rupture in epithelium and layers below that (fig. 3b). also, inflammatory myocytes were observed (fig. 3d). incidence time pattern of changes: the chronological trends of histopathological effects of metal exposure are presented in table 1. in the fourth day, histological changes were found in all organs. intensity of damage in the mantle and foot was greater than the digestive gland. haemocyte aggregation in the connective tissues was a common change in all studied organs in the ninth day. in day 18, haemocyte aggregation in muscular tissue of mantle was found, while in other two organs, such damage was not occurred. in day 18, an intense histological damage was observed in mantle and foot. discussion exposure to zn is led to histopathological alterations in mantle, digestive gland and foot tissues. haemocytes and other lipopigmented cells (garanulocytomes) are related to sorption and storing of toxic chemicals (johansson and sӧderhäll, 1992). these cells were found in mantle and digestive gland of studied organism. occurrence of this type of cells due to metal exposure has been reported in previous studies (neff et al., 1987; wolfe, 1992; chakraborty et al., 2012). haemocyte infiltration and aggregation in damaged area of different tissues is a common defensive response of organism against toxic agents (oliver and fisher, 1999) which was found in this study in connective and muscular tissues of mantle and digestive gland. cilia of the external epithelial cells of mantle and foot are involved in dynamic activity of these organs. exposure to zn led to destruction of cilia. the loss of cilia (hypoplasia) of gill has been reported by alsubiai et al., (2011) as a result of cu exposure. damaging of ciliary structures of epithelial cells weakens the moving performance of these organs. increase in number of mucous cells in mantle and foot was occurred in subepithellial regions after loss of cilia. likely, increase in number of these cells was occurred to compensate attenuation of dynamic operation. our results showed apparent histopathological signs in different parts of digestive gland as result of exposure to zn. morphology of digestive tubules changed. atrophy of digestive tubules in digestive gland in several studies reported in metal exposed organisms as a histopathological damage (chakraborty et al., 2010; sheir and handy, 2010; sheir et al., 2010; al-subiai et al., 2011). digestive cells of tubules spilled into internal space of tubules. usheva et al. (2006) reported such a histological damage in crenomytilus grayanus in exposure to ddt. as the results of this study, the regions where damages were greater, tissue rupture and disintegration including connective and muscular tissues (in mantle and foot) were found. tissue rupture has been reported by al-subiai et al. (2011) in mytilus edulis, due to cu exposure. besides, it has been reported that heavy metals can be stored in haemocytes and interstitial storage tissues in bivalve organs (regoli and orlando, 1994; soto et al., 1996). thus, tissue rupture can be assigned to zn storage in connective tissue of the mantle. results showed swelling and inflammation of myocytes and mucous cells in foot in treatments. previous studies reported swelling of myocytes in abductor muscles of mytilus edulis (al-subiai et al., 2010) and swelling of pore cells in littorina litorea (watermann et al., 2008) due to exposure of heavy metals. first signs of histopathological damages during experiment were appeared in mantle, digestive gland and foot in fourth day after exposure. foot and mantle are directly contacted with pollutant, but exposure route of digestive gland is indirect. during experiment, by approaching to day 18, the extent of damages increased. in organs such as foot in which tissue mass intensity is greater, damages have been extended to inner parts at the end of experiment. 65 66 moëzzi et al./ int. j. aquat. biol. (2013) 1(2): 61-67 based on finding of this study, exposure of a. cygnea to chronic concentrations of zn is led to histopathological changes in their mantle, digestive gland and foot. sensitivity of the mantle and foot are greater than digestive gland to zn exposure. as a general conclusion, histopathological alterations in mantle, digestive gland and foot in a. cygnea are suggested as biomarkers for biomonitoring of heavy metal pollution in aquatic ecosystems. references al-subiai sh.n., moody a.j., mustafa s.a., jha a.n. (2011). a multiple biomarker approach to investigate the effects of copper on the marine bivalve mollusc, mytilus edulis. ecotoxicology and environmental safety, 74: 1913-1920. au d.w.t. (2004). the application of histocytopathological biomarkers in marine pollution monitoring: a review. marine pollution bulletin, 18: 817-834. banfalvi g. (2011). cellular effects of heavy metals. springer. 348p. buhl k.j., hamilton s.j. 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(2017) 5(5): 342-347 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article effects of dietary vitamin c supplementation on some oxidative status biomarkers in erythrocytes of common carp (cyprinus carpio) hamideh ghodrati azadi*1, davar shahsavani2, hasan baghshani1 1department of basic sciences, school of veterinary medicine, ferdowsi university of mashhad, mashhad, iran. 2department of food hygiene and aquaculture, school of veterinary medicine, ferdowsi university of mashhad, mashhad, iran. article history: received 17 aug 2017 accepted 20 october 2017 available online 2 5 october 2017 keywords: vitamin c antioxidant enzymes lipid peroxidation cyprinus carpio abstract: regarding to the high content of polyunsaturated fatty acids in fish tissues, improving the fish antioxidant status seems to be necessary and may be associated with beneficial effects on fish health. the present study aimed to investigate the effects of dietary vitamin c supplementation (20 mg/kg body weight, 4 weeks) on some oxidative status biomarkers in rbc of common carp (cyprinus carpio). the results showed that the activities of antioxidant enzymes including catalase (cat), glutathione peroxidase (gpx) and superoxide dismutase (sod) were not changed significantly following dietary vitamin c supplementation in comparison to the control group. moreover, dietary vitamin c supplementation for 28 days significantly lowered malondialdehyde (mda) concentration in erythrocyte haemolysate by approximately 26% compared to that of the control group. in conclusion, dietary vitamin c supplementation appears to be able to protect carp erythrocytes against oxidative stress by decreasing lipid peroxidation. introduction a variety of reactive oxygen species (ros), derived from reduction of oxygen, ionizing radiations, reactive metals, and other environmental initiators, permanently threatens living organisms (birben et al., 2012). oxidative stress is a consequence of an increased generation of free radicals and/or reduced physiological activity of antioxidant defenses against them. oxidative stress impairs dna, causes enzymes and membrane disorders, changes the activity of the immune system, and alters the structure of basic biopolymers, which, cause various disorders (abd ellah, 2010). in both mammals and fish, insufficient dietary antioxidants have been followed by a decrease in antioxidative defense and increased susceptibility to oxidative stress (sies et al., 2005; welker and congleton, 2009). the antioxidant capacity of fish may be insufficient in captivity (mohebbi et al., 2011). regarding to increased environmental pollutants and thus increased risk of oxidative stress, administration of dietary antioxidants would be beneficial for cultured fish. moreover, in view of the high content of *corresponding author: hamideh ghodrati azadi doi: https://doi.org/10.22034/ijab.v5i5.314 e-mail address: ghodrati@ferdowsi.um.ac.ir polyunsaturated fatty acids in aquaculture feeds and fish tissues, improving antioxidant capacity of fish seems to be necessary and may be associated with many beneficial effects on fish health (mohebbi et al., 2011). fish tissues are characterized by high concentrations of polyunsaturated fatty acids and may therefore be particularly susceptible to lipid peroxidation. both oxidative responses and antioxidant potential of fish differ according to species habitat and feeding behavior (yonar and sakin, 2011). dietary antioxidant vitamins may play an important role in protecting fish against oxidant damages. vitamin c is important for many enzymatic reactions and also acts as a freeradical scavenger. vitamin c deficiency has been shown to retard growth and impair wound healing in o. mykiss and oncorhynchus kisutch (zhou et al., 2003). vitamin c supplementation in diet promoted growth in sea bass (lates calcarifer) and common carp (gouillou-coustans et al., 1998). on the other hand, it has been documented that vitamin c affects metabolism of lipids and carnitine, which is essential 343 int. j. aquat. biol. (2017) 5(5): 342-347 for the β-oxidation of long-chain fatty acids (feller and rudman, 1988). potential functions of vitamin c in rbc include maintenance of plasma ascorbate concentrations by ascorbate or dehydroascorbic acid efflux from red blood cells, transmembrane electron transfer from erythrocyte ascorbate, and antioxidant functions to protect erythrocytes from oxidative damage or to recycle membrane tocopherol (li et al., 2012). it has been reported that dietary vitamin c supplementation could significantly increase growth performance and antioxidant status in some aquatic animals (asaikkutti et al., 2016; gouillou-coustans et al., 1998). common carp (cyprinus carpio) has great commercial importance because it is widely consumed all over the world. although cyprinids appear to be able to synthesize vitamin c at rates sufficient to meet its physiological needs, dietary supplementation with vitamin c might be beneficial when metabolic demand exceeds endogenous supply (combs, 1998). therefore, the present study was conducted to assess the effects of dietary vitamin c supplementation on some oxidative status biomarkers in rbc of common carp. materials and methods vitamin c was purchased from nature made (california, usa). commercial enzyme kits for superoxide dismutase (ransod, randox/sd-125) and glutathione peroxidase (ransel, randox/rs505) were obtained from randox laboratories (crumlin, uk). ms-222 (ethyl 3-aminobenzoate methanesulfonate, tricaine) and 2-thiobarbituric acid (tba) was purchased from sigma chemical co. (st. louis, mo, usa). the rest of the utilized chemicals were of analytical grade and were supplied by sigma (st lewis, mo, usa) or merck (darmstadt, germany). common carp (total n=60), weighing 60-80 g, were obtained from a local commercial farm. they were held in four glass aquaria, each containing 250 l freshwater. the fish were acclimatized for 7 days (naeiji et al., 2013) before the commencement of the experiment and were daily fed with commercial fish feed at 3% of total body weight at a fixed time. the aquaria were aerated continuously. physicochemical characteristics of the water during the experimental period were: dissolved oxygen, 5.5-6 ppm; temperature, 25±1°c; ph, 7±0.5; photoperiod, 12:12 light–dark. the aquaria water was renewed every 48 h (naeiji et al., 2013). the fish were divided randomly into two groups of 30 each. group 1 fish were held in two aquaria (each containing 15 fish) and fed with basal diet; served as control. group 2 fish were held in two aquaria (each containing 15 fish) and fed the basal diet supplemented with vitamin c (20 mg/kg body weight, daily (nrc, 1993)) for 4 weeks. the vitaminsupplemented diet was prepared by spraying vitamin c solution onto the pellet (treves-brown, 2000). at the end of the experimental period, twenty fish were sampled randomly from each aquarium and anesthetized in diluted ms-222. blood samples were taken by cardiac puncture using heparinized syringes and tubes. after plasma separation by centrifugation at 750×g for 20 min (nazifi et al., 2010), erythrocyte pellet was washed three times with normal saline solution. the washed centrifuged erythrocytes were hemolyzed by the addition of an equal volume of icecold redistilled water (nazifi et al., 2010) and prepared hemolysate aliquots were stored at -70°c until analysis. glutathione peroxidase (gpx) activity was measured using randox-ransel enzyme kit. in this method, gpx catalyzes the oxidation of gsh by cumene hydroperoxide. in the presence of glutathione reductase (gr) and nadph, the oxidized glutathione (gssg) is immediately converted to the reduced form, with a concomitant oxidation of nadph to nadp+. the decrease in absorbance at 340 nm was measured spectrophotometrically, and the results were expressed as units per gram hemoglobin. hemoglobin (hb) concentration was measured by cyanmethemoglobin method (prakash and banerji, 1972). superoxide dismutase (sod) activity was determined by a modified method of iodophenyl nitrophenol phenyltetrazolium chloride using the randox-ransod enzyme kit. this method employs xanthine and xanthine oxidase to generate superoxide https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/antioxidant https://www.sciencedirect.com/science/article/pii/s235251341630028x#%21 https://www.ncbi.nlm.nih.gov/pubmed/?term=prakash%20n%5bauthor%5d&cauthor=true&cauthor_uid=4635482 https://www.ncbi.nlm.nih.gov/pubmed/?term=banerji%20hn%5bauthor%5d&cauthor=true&cauthor_uid=4635482 344 ghodrati azadi et al./ effects of vitamin c on oxidative status in common carp radicals which react with 2-(4-iodophenyl)-3-(4nitrophenol)-5-phenyltetrazolium chloride (int) to form a red formazan dye. the sod activity was then measured by the degree of inhibition of this reaction. one unit of sod was considered a 50% inhibition of reduction of int under the condition of the assay. the results were expressed as units per gram hemoglobin. catalase (cat) activity was measured in the rbc hemolysate by the method described by claiborne (1986) and expressed as units per gram hemoglobin. the decomposition of h2o2 can be directly followed by the decrease in absorbance at 240 nm. the difference in absorbance at 240 nm per time unit allows determining the cat activity. 50 µl of the diluted hemolysate was mixed with 1.9 ml of the phosphate buffer (0.05 m, ph 7) and 1 ml of 30 mm h2o2. the decrease in absorption was measured at 240 nm for 1 min. activity of catalase was calculated using the extinction coefficient of 43.6 m−1cm−1. one unit of catalase activity is equal to the amount of enzyme that will decompose 1 µmol h2o2 per minute. lipid peroxidation was assayed by measurement of malondialdehyde (mda). determination of mda concentration was based on spectrophotometry of the pink-colored product of thiobarbituric acid reactive substances, as described by latha and pari (2003). the concentration of mda was calculated using a molar extinction coefficient value of 156,000 m−1 cm−1. the results were expressed as nanomoles of mda per gram hemoglobin. statistical analysis: all experimental values have been represented as mean ±standard error of mean (sem). the obtained data were analyzed using student's t-test. the level of significance was set at p<0.05. all calculations were performed using spss/pc software, version 18. results the values (mean±sem) of the measured erythrocyte oxidative status biomarkers in experimental groups are presented in table 1. the activities of antioxidant enzymes including cat, gpx and sod were not changed significantly following dietary vitamin c supplementation in comparison to the control group. as shown in table 1, dietary vitamin c supplementation for 28 days significantly lowered the mda concentration in erythrocyte hemolysate by approximately 26% compared to concentration in the control group. discussion measurement of circulatory biomarkers of oxidative stress has emerged as a reliable method for screening putative antioxidative agents (balasenthil et al., 2000). the extent of lipid peroxidation is most frequently measured by estimating mda levels (latha and pari, 2003). fish erythrocytes have been proposed as a useful model to investigate oxidative stress, since their membranes are rich in long chain n-3 polyunsaturated fatty acids, which are oxidized under oxidative stress conditions (roche and boge, 1993; gabryelak et al., 2000; nagasaka et al., 2004). moreover, repeatedly exposure to high concentration of oxygen or presence of iron renders erythrocytes highly susceptible to peroxidative damage (clemens and waller, 1987). the effect of vitamin c in decreasing mda levels in the present study suggest that this vitamin may provide an effective protection against lipid peroxidation. similar to this finding, it has been reported that the level of thiobarbituric acid reactivesubstances (tbars) in the hepatopancreas and muscle of common carp was decreased following vitamin c supplementation (hwang and lin, 2002). parameter control vitamin c catalase (u/g hb) 273.41±30.95 310.25±24.74 superoxide dismutase (u/g hb) 1573.67±68.53 1557.90±53.67 glutathione peroxidase (u/g hb) 744.96±38.37 836.4±38.86 malondialdehyde (nmol/g hb) 129.29 ±10.37 a 95.25±7.40 b a, b mean±sem in each row with no common superscript differ significantly (p<0.05). table 1. mean±sem of measured oxidative status biomarkers in experimental groups (n= 20 in each group). 345 int. j. aquat. biol. (2017) 5(5): 342-347 moreover, chien and hwang (2001) indicated that vitamin c can prevent the increase in liver lipid peroxidation due to high water temperature in thorn fish terapon jarbua. similarly, it has been reported that dietary vitamin c supplementation caused a significant decrease in the levels of tissue lipid peroxides in chicken erythrocytes (aydemir et al., 2000). vitamin c as a strong reducing reagent has a direct reactive oxygen scavenger action. additionally, depletion of tissue gsh is one of the primary factors that permit lipid peroxidation and vitamin c has been shown to increase intracellular glutathione concentrations (hwang and lin, 2002; johnston, 1993). another criterion, which might contribute to the lowering effect of vitamin c on peroxidation of hydrophobic regions of the cells may be its ability to reduce the semi-stable β-tocopheroxyl radical (vitamin e radical form after performing the antioxidant role), thus regenerating the metabolically active form of the lipid antioxidant vitamin e (kontush et al., 1996). the detoxification of ros involves the cooperative action of the intracellular antioxidant enzymes sod, cat, and gpx. gpx contributes to the oxidative defense of animal tissues by catalyzing the reduction of hydrogen peroxide and lipid peroxides (harvey, 1997). cat has an equal importance to in the defense of human erythrocytes against h2o2 generating reactions (harvey, 1997). superoxide dismutase is also important in the antioxidant defense mechanism and protects against lipid peroxidation (miller et al., 1993). animal studies have shown that antioxidant enzymes’ levels are variable depending on the availability of antioxidants in food (meydani, 1993; puangkaew et al., 2005). it has been reported that sod and gpx activities increased but cat activity decreased with an increase of plasma vitamin c levels (monget et al., 1996). in chicken the activities of erythrocyte sod and gpx increased following vitamin c supplementation although no significant variations were observed in the cat activity (aydemir et al., 2000). similarly, the present results indicate no significant increment of the measured antioxidant enzymes following vitamin c supplementation in carp erythrocytes. the results of the present work suggest that dietary supplementation of vitamin c appears to be able to protect erythrocytes of carp against oxidative stress by decreasing lipid peroxidation. however, more investigations are required to elucidate the pharmacokinetic effects of this vitamin and also precise molecular basis of the beneficial effects of vitamin c in fish species. acknowledgments this study was supported by grant from ferdowsi university of mashhad, mashhad, iran. references abd ellah m.r. 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(2017) 5(4): 342-347 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی معمولی کپور قرمز هایگلبول در اکسیداتیو وضعیت بیومارکرهای برخی بر c ویتامین غذایی مکمل تاثیر (cyprinus carpio) 1باغشنی حسن ،2شاهسونی داور ، *1آزادی قدرتی حمیده 1گروه علوم پایه، دانشکده دامپزشکی، دانشگاه فردوسی مشهد، مشهد، ایران. .ایران مشهد، مشهد، فردوسی دانشگاه دامپزشکی دانشکده آبزیان، و غذایی مواد بهداشت گروه2 چکیده: اثرات تواندمی که رسدمی نظر به الزم ماهی در اکسیدانی آنتی وضعیت بهبود ماهی، هایبافت در اشباع غیر چرب اسیدهای باالی مقادیر به توجه با بر( هفته 4 بدن، وزن کیلوگرم/گرممیلی 20) c ویتامین غذایی مکمل اثرات بررسی منظوربه حاضر تحقیق. باشد داشته ماهی سالمت در مفیدی آنتی های آنزیم فعالیت که داد نشان نتایج. شد انجام( cyprinus carpio) معمولی کپور ماهی خون در اکسیداتیو وضعیت هایفراسنجه برخی گروه با مقایسه در c ویتامین مکمل حضور در( sod) دیسموتاز سوپراکسید و( gpx) پراکسیداز گلوتاتیون ،(cat) کاتاالز جمله از اکسیدان هایگلبول( mda) آلدهید دی مالون غلظت در داریمعنی کاهش باعث c ویتامین مکمل دیگر، سوی از. است نداشته داریمعنی تفاوت شاهد از محافظت به قادر c ویتامین غذایی مکمل رسدمی نظربه طرح این نتایج اساس بر. گردید شاهد گروه با مقایسه در درصد 26 حدود تا قرمز .باشدمی چربی پراکسیداسیون کاهش با اکسیداتیو استرس برابر در کپور ماهی قرمز هایگلبول .کپور ماهی چربی، پراکسیداسیون اکسیدان، آنتی های آنزیم ،c ویتامین :کلمات کلیدی int. j. aquat. biol. (2013) 1(2): 48-54 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology reproductive season, maturation size (lm50) and sex ratio of metapenaeus affinis (decapoda: penaeidae) in hormozgan shrimp fishing grounds, south of iran mohammad hasan gerami*1, rasool ghorbani2, seyed yousof paighmabari2, mohammad momeni3 1deptartment of fishery and forestry, gonbad-e kavous university, gonbad-e kavous, iran. 2deptartment of fisheries, gorgan university of agricultural and natural resources, gorgan, iran. 3persian gulf and oman sea ecology research institute, bandar-e abbas, iran. article history: received 12 april 2013 accepted 30 april 2013 available online 5 may 2013 keywords: reproductive biology metapenaeus affinis length at first maturity persian gulf abstract: this study aimed to investigate the spawning season, length at first maturity (lm50) and sex ratio of metapenaeus affinis in the shrimp fishing grounds of hormozgan province, west of the persian gulf, iran. samples were taken by the swept area method and trawl net with 2 cm mesh size in the cod end from january 2010 to february 2011. results showed that the sex ratio deviated from 1:1 and female’s number were significantly higher than males. metapenaeus affinis females had continuous spawning in all seasons but the peak spawning season was found in spring, and stage 3 of maturity was observed in all seasons. length at maturity (lm50) for females was estimated at 27.12 mm based on carapace length. our finding showed that lm50 of m. affinis do not change with sex ratio deviates from equal ratio. introduction metapenaeus affinis is considered to be one of the important commercial species which is caught by fishermen in shrimp fishing season in the coastal waters of the hormozgan province on the persian gulf, iran (from early october to december) (kamrani et al., 2003). the maximum width of the persian gulf is 640 km and its average depth is 35m (reynolds, 1993; valinassab et al., 2006). therefore, this gulf provides an important habitant for a number of penaeid shrimp species. metapenaeus affinis can be found in hormozgan fishing grounds from towla in the west to the jask area in the east (safaei, 2001). in recent years, several studies have been conducted on this species in iran. between 2000 and 2002, safaie and kamrani (2003) studied the population structure of m. affinis in the coastal waters of the hormozgan province. safaie (2009) investigated ovarian tissue sections of m. affinis in the coastal waters of the hormozgan province. furthermore, * corresponding author: mohammad hasan gerami e-mail address: m.h.gerami@gmail.com several studies have been carried out on m. affinis spawning periods in other locations. ayub and ahmed (1992) investigated the population structure, length at maturity (lm50; the length at which 50% of specimens reach the stage 3 or 4 of maturation and can be inseminated), sex ratio and spawning season of some penaeid shrimps penaeus pencillatus, p. merguiensis and metapenaeus affinis in the waters of pakistan. mathews (1989) studied the biology and management of the m. affinis stock in kuwait. pinheiro and fransozo (2002) stated that the breeding season in decapods may be restricted to a few months (discontinuous pattern) or may be yearround (continuous pattern) due to thermal conditions in winter (sastry, 1983). the spawning cycle periodicity is affected by endogenous and environmental factors (wenner et al., 1974; batoy et al., 1987), like temperature (heasman et al., 1985; campbell and fielder, 1986) and photoperiod 49 gerami et al./ int. j. aquat. biol. (2013) 1(2): 48-54 (knudsen, 1964; little, 1968; saigusa, 1992; flores and negreiros-fransozo, 1998). the present study aimed to present an update of information on sex ratio, lm50 and reproductive season of m. affinis in the waters of hormozgan province, west of the persian gulf, iran. the present study, particularly, the length at first maturity, may help the sustainable fishery of the species through saving the juveniles from the catch (paighambari et al., 2004). furthermore, information on the species spawning season is necessary for setting the fishing seasons, and determining the effectiveness of the environmental factors (momeni and kamrani, 2006). material and methods samples were collected from bandar-e-abbas 27° 07' n in the west of hormozgan province and 56° 06' e to bandar-e-jask 26° 25' n and 57° 07' e in the east, and covered most of the fishing grounds of m. affinis. the sampling were performed in three depths: >5, 5-10 and 10-30 m using a trawl with a 2 cm mesh size at the cod end with a 450 hp vessel. the swept area method was used for sampling (gerami, 2011). the netting operation was 5 days a month, 6 to 8 times a day. each netting operation lasted one hour. the samples were treated by 10% formalin and transferred to the laboratory. in the laboratory, species were separated by gender. the following biometric data were obtained: total length and carapace length in cm and mm, respectively (to the nearest to 0.1 cm and mm), total weight in g (nearest to 0.01 g) and the ovarian developmental stage of females, which was classified into four stages (lim et al.,1987; primavera, 1985; ausbon brown and patalan, 1974): stage one, undeveloped (transparent, small ovaries); stage two, developing (larger, yellowish. with increasing pigmentation); stage three, early ripe (light green color); stage four, ripe stages (intense green color with a further increase in size). in addition, stage five is the spawning stage and similar to stage one but the ovary is more clear than stage one. all reproduction stages were identified based on visual selection. to determine lm50, length classes was specified and the abundance of each length classes was extracted from all data and the frequency of matured stages (stages 3 and 4) was compared to the total frequency of all maturation stages. then the shrimp size at maturity was calculated by the following equation and using the least square method in excel 2007 with data analysis solver (king, 1995) where p is the predicted mature proportion, a and b the estimated coefficients of the logistic equation, and cl the carapace length. size at sexual maturity (lm50), corresponding to a proportion of 0.5 sexually mature, was estimated as the negative ratio of the two coefficients [lm50= (a/b)] (cha et al., 2004). the frequency of the reproductive stages was used to determine the spawning season of females (biswas, 1993). the chi-square examination was used to test the sex ratio in seasons. the spss 19 software was used to calculate the data with a confidence level of 5%. results the weight and length characteristics of m. affinis are represented in table 1. the maximum length frequency belonged to the 20 and 22 cm length class for males and females, respectively (fig. 1 and 2). also maximum frequency of carapace length in ripped stage belonged to 25-28 carapace length classes. the sex ratio of male and female deviated significantly from 1:1 (p<0.05) except in winter (table 2). 49 figure 1. males carapace length frequency of m. affinis (n=966). 50 gerami et al./ int. j. aquat. biol. (2013) 1(2): 48-54 according to figure 3, number of m. affinis are in stage three of maturity in all months; but the major spawning season of this species is from late winter and last to the early spring (march and april) due to high rate of stages three and four. the most immature shrimps were found in june, july and august. lm50 of the m. affinis in the hormozgan province shrimp fishing grounds was 27.12 cm. discussion in late winter and early spring, there was an increase in the number of mature females. gerami (2011) stated that with increase of carapace length, the frequency of mature shrimps increased. this may be related to the presence of adult shrimps in the stock and the consequent increase of the average carapace length (safaie and kamrani, 2003). in the present study, there was significant deviation from 1:1 sex ratios in most seasons of the year (table 2). our study is consistent with safaie (2004) who found that there was a significant difference between sex ratio in penaeus merguiensis in the hormozgan province. such deviation has been reported in other studies. for example, studies on the sex ratio of shrimp by kamrani et al. (2005). kim (2005) suggested that differences in sex ratio may be due to differences in mortality rates between the two sexes or because of differences in behavioral characteristics such as migration. gerami et al. (2012) suggested that the higher natural mortality of males may deviate in favor of females in most years, which agrees with cha et al. (2002) and kim (1977). da costa et al. (2010) suggested that the sex ratio of females may be related to the greater vulnerability of females to fishing due to their size. a complex interaction between endogenous and exogenous factors influences breeding periods causing intra and interspecific variation in the duration of the reproductive season (sastry, 1983; sexes max total length (cm) max total weight mean carapace length (mm) max min max min male 5.5 13.7 1 19.3 21.04 ± 3.5 female 3.5 17.8 1.3 45.3 24.86 ± 5.8 figure 2. females carapace length frequency of m. affinis (n=1445). figure 3. frequency stages of sexual maturity of m. affinis in 2010-2011 (n=1445). table 1. weight and length characteristic of m. affinis in hormozgan coasts (2010-2011). figure 4. percentage of ripped stages of m. affinis in different carapace length (n=2411). 51 gerami et al./ int. j. aquat. biol. (2013) 1(2): 48-54 henmi and koga, 2009). many biotic and abiotic factors such as photoperiod, food availability, temperature, and salinity have been identified as major modulators of reproduction in crustaceans (aiken, 1969; pillay and nair, 1971; snowden et al., 1991; henmi, 1993; emmerson, 1994; company and sarda, 1997; litulo, 2005) and genotypic response to these environmental variations may maximize reproductive success under favourable conditions (henmi and koga, 2009). for example, higher temperature in late winter and early spring makes environmental factors well-defined for breeding seasons for m. affinis (mathews, 1989; safaie, 2008). our results show that the peak spawning season of m. affinis occurs in late winter and early spring when reproduction stages 3 and 4 are the dominant stages. notably, the stage 3 was found over the year. this indicates that m. affinis has continuous spawning over the year as stage 1 and 2 were observed in the study duration. metapenaeus affinis have resilient ovarian and is capable to prepare and fertilize itself for re-spawning (safaie, 2008). in this study, the highest frequency of stage 1 was found in december. this is due to the spawning of shrimp species from august to november. after spawning, shrimps are in stage 1. from february to april, with the beginning of spring, stage 3 and 4 reach their peaks. mathews (1989) reported two peaks of spawning seasons in spring and autumn for this species in kuwaiti waters and pillary and nair (1971) found that the period of the breeding of this species of shrimp along the south-west coast of india were from august to april, with major and minor peaks in december in april, respectively. he explained that juveniles from the spring spawning season mature and ripen during the summer and spawn in autumn, and their descendants spawn in the upcoming spring. according to our results, stages 1 and 2 were mostly observed in summer indicating that m. affinis spawn in autumn. cha et al. (2002) found that penaeus chinensis recruited in august continued to grow in summer and spawn in april–june. it has been found that species of penaeidae are able to have one or two peaks of spawning their its life (garcia, 1977, 1985; o’connor, 1979) and that m. affinis had at least two spawning seasons in life (treece, 2001). studies on mean carapace length, when 50% of female shrimps were matured, indicated that the ovaries of m. affinis reach stage 3 and 4 of sexual maturation in the carapace length of 20-35 mm (mathews, 1989). garcia (1985) noted that the percentage of mature females is a biased index of population reproduction. kamrani et al. (2005) reported the mean carapace length of m. affinis females, when 50% had stage 4 fertility, to be 27.16 mm carapace length in hormozgan coastal waters. also, asadi (2000) observed that the lm50 of m. affinis occurs in 25.4 mm carapace length in the north of the persian gulf. in conclusion, according to previous studies; lm50 of this species did not change significantly while sex ratio tends to predominance by females in this region. kamrani et al. 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(2016) 4(4): 269-276; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article community structure of macrobenthos in ponnani estuary, south india with reference to occurrence of invasive alien species ranjeet kutty*1, muhsina chakkayil2, shahul hameed pentam veli pura3 1department of fishery hydrography, kerala university of fisheries and ocean studies, panangad p.o., 682 506, kerala, india. 2department of aquaculture and fishery microbiology mes ponnani college, ponnani south p.o., 679 586 malappuram dist., kerala, india 3department of aquaculture and fishery microbiology mes ponnani college, ponnani south p.o., 679 586, malappuram dist., kerala, india. article history: received 28 march 2016 accepted 9 august 2016 available online 2 5 august 2016 keywords: amphipod melita zeylanica benthic community open estuary climatic variability abstract: benthic organisms have been studied in past to assess the health of an aquatic ecosystem. moreover, being sedentary forms they have been used as indicator organisms. in the present study, an attempt has been made to study the distribution and assemblage of major macrobenthic forms occurring in ponnani estuary. a total of 23 genera of benthic invertebrate fauna belonging to 6 groups viz., polychaetes, bivalve, gastropod, amphipod, isopod and tanaidacian were recorded during the study. faunistic study revealed that in high saline regions of the estuary (veliyancode), a very good macrobenthic biodiversity was present. bivalves were the major group enumerated from veliyancode and ponnani (39% and 35%, respectively), while in biyyam predominance of gastropods (37%) were observed. among the bivalve groups, presence of maculista senhousia and pholas candida, both considered as invasive species was noteworthy. another interesting observation was the presence of amphipod melita zeylanica, another alien species evenly represented in all the three stations, indicating that the species had well established in ponnani estuary. the present study is the first description of benthic organism assemblages inhabiting in the region. results indicate that salinity is a major parameter that governs the diversity, occurrence, abundance and distribution of macrobenthos in ponnani estuary. it also elucidates that any perturbance in the physico-chemical nature of the estuary favours the invasiveness of alien macrobenthic species in ponnani backwaters. introduction estuary is one of the most important coastal life supporting system and an ideal tryst of various economically important marine and fresh water organisms. however, owing to the high primary productivity, suitable physic-chemical conditions and less hydrographical perturbations, they also are favourable grounds for establishment of new alien species. usually the establishment of a new group in estuary is facilitated with sharp fluctuation in the physico-chemical parameters as part of climate change in the region (occhipinti-ambrogi, 2007). climate change could alter the structure and composition of native communities and, as a consequence, the way an ecosystem functions, increasing the risk of biological invasion. as * corresponding author: ranjeet kutty e-mail address: ranjeet.mes@gmail.com invasive species and climate change are considered two of the three main threats to biodiversity, the two operating together could be expected to produce extreme outcomes (masters and norgrove, 2010). it is also likely to increase the potential distribution and abundance of invasive species, further enlarging areas at risk of invasion, and threatening even the survival of less prolific groups such as the benthic forms. temperature is a major factor influencing the settlement of tropical alien species (ben rais lasram et al., 2008). warming of seawater influences the spatial and temporal distribution of many marine organisms, ranging from phytoplankton and zoobenthos to higher trophic levels (beaugrand et al., 2008). this warming not only stresses the native 270 kutty et al./ macrobenthic fauna of ponnani estuary dwellers, but also facilitates the arrival of alien ones, adding extra pressure on the ecosystem (harris and tyrell, 2001). macrobenthos are an important component of estuarine ecosystem and play an important role in the system dynamics (herman et al., 1999). they are a central element of estuarine food webs, being an important food resource for large crustaceans, fish and birds (day et al., 1989). macroinvertebrates cycle nutrients throughout the water column and provide a food source for many economically important fish species (silva et al., 2006). the spatial heterogeneity of macrobenthos along the estuarine gradient is traditionally described in relation to salinity and sediment composition (ysebaert et al., 1993). studies have shown a complex interaction between hydrodynamics, sediment dynamics, and benthic biology in structuring distribution patterns of benthos (herman et al., 2001). benthic communities are sensitive to many environmental stresses. benthic invertebrates are extremely important indicators of environmental changes. the study of the alien species is necessary to understand the long term consequences of marine ecosystems, their goods and services. since estuarine environments are continuously dominated by species that can thrive well under harsh physico-chemical conditions, the possibility of invasive species establishing in the new environment cannot be ruled out. the impact of these species on native communities has been evaluated in many localities all over the world leading to the concept of biotic pollution. once invasive alien species become established in large numbers, their consequences are often irreversible. although there has been fairly good research on the ichthyofaunal diversity of ponnani estuary, an open estuary of south india, very little works pertaining to the diversity of macrobenthic community in the region has been published. moreover, the influence of hydrographical parameters on the resident community and occurrence of any invasive alien species in the region has yet to be reported. under the above pretext, a study was conducted to understand the distribution of macrobenthic community in ponnani backwaters with a view to develop an in depth knowledge on the environmental dynamics prevailing in the region and also to investigate the establishment of any alien/invasive groups in the region. the major objectives included to make community structure analysis of benthic fauna in ponnani estuary, analyze spatial distribution of benthos based on salinity regime prevailing in the region and to identify the presence of any alien/invasive macrobenthic group in the region. materials and methods study area: the ponnani estuarine system is located between 10°46' and 10°48' n and 75°54' to 75°56' e. it is an open estuary drained by a tributary of the bharathappuzha river, the largest river of south india, and drains into the arabian sea at this region. the estuarine system is exposed to tides from the arabian sea and hence water is brackish almost throughout the year. the region receives an annual average rainfall of 180 mm. in the present study, three stations viz., veliyancode, ponnani and biyyam that are 2 km apart from the barmouth were selected as sampling stations. this ensured that a judicious representation of the varying hydrographical parameters of ponnani estuary was represented. sampling: all sampling for the collection of water and sediment samples in the estuary was made during the early morning hours. the water quality parameters recorded on site were temperature, dissolved oxygen, salinity, ph, turbidity and total suspended solids (ei-model 191). nutrients such as nitrate and phosphate were analyzed in lab based on strickland and parsons (1972) and measured on systronics uv-vis spectrophotometer. the data were pooled in to three separate seasons (monsoon, post monsoon and pre-monsoon) for analysis. macro benthos samples were collected using a grab. two replications were made at each of the two stations. these samples were washed through sieves of mesh size 500 micron for separating the macro fauna from sediments and preserved in 5% formalin and stained 271 int. j. aquat. biol. (2016) 4(4): 269-276 in rose bengal (1:500) to facilitate segregation of the organism from other components of the soil. the samples were then analyzed for macro fauna by hand picking and microscopic analysis. in the laboratory, the samples were sorted into different groups and the actual numbers of organisms counted were converted to nos. ind. /m2. the fauna were identified to the lowest taxonomic level (species) wherever possible following standard references (gosner, 1971). data analysis: the enumerated samples were pooled for different stations and analysis on the incidence of occurrence, percentage of abundance and species diversity were worked out following gosner (1971). the species identified were cross referred with the national database on benthic forms under the website for world register of marine species (worms) for identifying alien species. results the physico-chemical parameters in the three stations of the ponnani estuary were influenced by the tidal regime, freshwater inflow and other biological processes. the mean values of different environmental variables recorded in the present study are depicted in table 1. the results indicate a significant difference (p<0.01) in all the parameters studied during the three seasons and across the three stations. a detailed checklist of macrobenthic communities recorded from ponnani backwater is depicted in table 2. a total of 23 species of benthic invertebrate fauna belonging to 6 groups and 23 genera were collected from the ponnani backwaters during the study. the benthic macrofauna at ponnani backwater was represented by 6 groups viz. polychaetes, bivalve, gastropod, amphipod, isopod and tanaidacian. more number of species were represented under polychaetes (10) followed by bivalves (5). among the three stations, veliyancode was comparatively more saline due to its proximity to the arabian sea, and likewise corroborated to the high diversity of polychaetes. the three stations also showed diverse pattern in the abundance of bivalves and other macrobenthic groups. among the bivalve group, presence of maculista senhousia and pholas candida, both considered as invasive species was noteworthy. another interesting observation was the presence of amphipod, melita zeylanica, considered as an alien species evenly represented in all the three stations, indicating that the species had very well established in ponnani backwaters. figures 1, 2, and table 1. mean values of environmental variables during different seasons in the three stations of ponnani backwaters. figure 1. macrobenthic representation in veliyancode station. 272 kutty et al./ macrobenthic fauna of ponnani estuary 3 depict the percentage composition of the benthic macro invertebrate fauna in veliyancode, ponnani and biyyam, respectively. bivalves were the major group enumerated from veliyancode and ponnani (39% and 35%, respectively), while in biyyam it was gastropods (37%). polychaete population was high in ponnani macrobenthos biyyam ponnani veliyancode polychaete ancistrosyllis constricta + branchiocapitella singularis + capitella capitata + + dendronereis aestuarina + eunice tubifex + lumbrinereis impatiens + nephthys dibranchis + + notomastus aberans + perinereis cavifrons + prionospio polybranchiata + gastropod baccinum sp. + dentalium sp. + littorilla littorea + + + bivalve masculista senhousia + meretrix casta + paphia malabarica + + perna viridis + pholas candida + isopod cirolana fluviatilis + + + amphipod melita zeylanica + + + eriopisa chilkensis + caprillids + tanaidacian apseudes sp + + + present, absent table 2. distribution of macrobenthos in different studied stations. figure 2. macrobenthic representation in ponnani station. figure 3. macrobenthic representation in biyyam station. 273 int. j. aquat. biol. (2016) 4(4): 269-276 (24%) compared to veliyancode (17%). gastropod population was rather evenly distributed ranging from 20% in ponnani to 37% in biyyam. nearly 25% of the total species present in veliyancode was contributed by gastropods. isopods, amphipods and tanaidacians together contributed 19% of total biodiversity of station veliyancode. in ponnani, the bivalves were found to be dominant group representing 35% of the total population followed by gastropods and polychaetes. the other groups’ viz. amphipod, tanaidacian and isopods were meagerly represented in both the stations. among the three stations, polychaetes were more abundant in ponnani. in biyyam, the diversity is very less compared to other two stations. here only four groups are identified in the present study. here, the gastropod contributed 37% of total biodiversity. the half of the total biodiversity in biyyam was contributed by the bivalve (25%) and isopod (25%). figures 4, 5 and 6 shows the abundance of macrobenthic fauna in the three stations i.e., veliyancode, ponnani and biyyam, respectively. the species abundance was very high in veliyancode. the polychaetes were the most diverse taxon followed by bivalve and gastropod. the polychaetes are lesser in number and higher in diversity. veliyancode provides a good environmental condition and habitat for a variety of polychaetes and bivalves. the presence of juveniles of m. senhousia shows that veliyancode also provides breeding ground for invasive species. abundance of macrobenthic fauna in ponnani station is shown in figure 5. in ponnani however, bivalves such as paphia malabarica was predominant followed by the gastropod, littorina littorea. polychaetes, capitella capitata is also seen figure 4. abundance of macrobenthic fauna in veliyancode station. figure 5. abundance of macrobenthic fauna in ponnani station. 274 kutty et al./ macrobenthic fauna of ponnani estuary abundantly in ponnani. the number of c. capitata was comparably higher in less saline ponnani than in high saline veliyancode. the number of juveniles of mertrix casta and baccinum spp. was also higher in this region. it shows that the ponnani is also provides a nursery ground for these molluscs. figure 6 shows the macrobenthic abundance in biyyam. the macrobenthic abundance and diversity are very less in biyyam. only four types of species are found in the present study. in biyyam, the isopod cirolana fluviatilis is seen abundantly followed by the bivalve littorina littorea. the bivalve, p. malabarica and amphipod, m. zeylanica are also found in lesser number i.e., 1 nos./m2. while studying the species richness it could be seen that polychaetes were more prolifically distributed in ponnani estuary than any other group. among them capitella capitata and nephthys dibranchis were recorded in two stations. bivalves are another dominant group observed in the present study, while, gastropod, amphipod, isopod and tanaidacian were sporadically represented. the present study clearly indicates that physico-chemical parameters in ponnani estuary is responsible for the distribution pattern of macrobenthic groups in the region. among them salinity had an inverse relationship with species richness. discussion the present study is the first description of benthic organism assemblages inhabiting in the ponnani estuary. among the 23 species reported from the study area only one species, m. zeylanica, alone was found to be both alien and invasive in nature as it was distributed in all the three stations. among the three stations, biyyam was less saline compared to ponnani and veliyancode, therefore species diversity in biyyam was comparatively less than the other two regions. the present study shows the higher abundance of m. zeylanica in veliyancode (3.80 nos.m-2), followed by ponnani (2.35 nos.m-2). population in biyyam was rather meagre (0.52 nos.m-2). this indicates that the distribution of m. zeylanica is dependent on the salinity, similar to the results of the earlier study by ysebaert et al. (2003). melita zeylanica is found in the bottom of the estuary. it is a slow moving community. therefore, its distribution depends on many factors like temperature, salinity and most importantly sediment characteristics since the distribution of benthos is closely related to the sediment characteristics. the different types of benthos require different sediment characteristics and salinity. the sediment characteristics include ph, nitrates, phosphates and organic carbon etc. sediment characteristics and salinity vary for different species. according to the present study, the sediment characteristics of veliyancode and ponnani was rather same, however in biyyam the sediment were more of silt. the heterogeneity in the distribution also depends on the sediment composition (ysebaert et al., 1993). this may be the reason for greater abundance and heterogeneity in macrobenthos community in veliyancode. the benthic community in ponnani estuary was characterized by low species richness but with better abundance of individuals, especially bivalves of p. viridis and p. malabarica, and gastropod of l.littorea. the great abundance of juveniles of both the bivalve and gastropod observed from certain pockets of the back water also indicated that this region provides good conditions as nursery grounds for these species. the results showed the higher macrobenthic abundance and diversity in veliyancode. it designates that the macrobenthos distribution is dependent on the salinity regime. this may be the reason for better species abundance and heterogeneity for macrobenthos in veliyancode. figure 6. abundance of macrobenthic fauna in biyyam station. 275 int. j. aquat. biol. (2016) 4(4): 269-276 the distribution and assemblage of macrobenthos depends on many factors like tide, fresh water inflow and environmental factors etc. environmental parameters such as temperature, salinity and do have major role in the availability and abundance of macrobenthos as reported earlier by racchor (1990). in the present study, macrobenthic distribution is different in studied stations. the fresh water inflow has greater importance in the spatial and temporal distribution of macrobenthic species (zajac and whittach, 1982). fresh water inflow decreases the salinity and likewise the species dwelling in the region. in veliyancode, the species richness and diversity is high for both polychaetes and bivalves. in the present study, species richness was found to decreases gradually in ponnani and biyyam. this corroborates with earlier studies of mannino and montagna (1997), describing that the pattern of species richness and diversity decline with decreasing salinity. similar results from other parts of india have been reported (kathiresan, 2000; saravanakumar et al., 2007). although the present study was not intended to delineate the influence of alien species and their influence to native community, the impact has very well been defined by earlier authors. still it would be worth investigating their influence on native biodiversity and ecosystem process and their competition for food and or space. this is particularly important for benthic ecosystems that are not able to adjust as fast as alien species. the results emerging from the present study indicate that salinity is the most important driving force determining the distribution of macrobenthos in ponnani backwaters. presence of alien species is yet another important information which implies to certain bivalves and amphipod that have established well in the region. with better adaptability, these alien species have better chance of coping with the stressful environment such as changing salinity profile, surface temperature, siltation and other anthropogenic alterations. hence, there is a possibility that these species would displace native species altogether from the region, owing to the harsh physico-chemical factors prevailing in the region. presence of specific alien species such as m. zeylanica which have established in these waters is also alarming and whether or not they have turned into invasive species needs further investigations. conclusion the present study was carried out to understand the biodiversity of macrobenthos in ponnani backwaters and thereby determine its health. presence of invasive benthic species was also another area of interest. the results showed a direct correlation between the physico-chemical parameters and distribution of macrobenthos in three stations of ponnani backwaters. the abundance of macrobenthos depends on the parameters like temperature, salinity, do and ph. the abundance and distribution are high in more saline station i.e., veliyancode. ponnani backwater is a good water body for bivalves because from three stations bivalves are found profoundly. the three species that have potential to be invasive were seen from the veliyancode, i.e. m. senhausia, m. zeylanica and p. candida. presence of invasive species has not yet posed any threat to other macrobenthic groups however, under harsh environmental conditions in future, these groups have all possibilities to distribute and establish in the region thereby causing competition among natives groups for food and space and ultimately changing the community structure in ponnani backwaters. the present study is particularly important and once again establishes the fact that the benthic organisms are important because they are biological indicators of environmental changes in aquatic ecosystem and also help to assess the health of an aquatic ecosystem. acknowledgments the authors are grateful to the fishermen of ponnani for their cooperation during sample collection. this work was done as part of a doecc, govt. of kerala ad hoc project and the financial received from the directorate of environment and climate change, 276 kutty et al./ macrobenthic fauna of ponnani estuary thiruvananthapuram is greatly acknowledged. references beaugrand g., edwards m., brander k., luczak c., ibanez f. 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(2017) 5(1): 40-46; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article construction of earthen structure as a sexual signals in the fiddler crabs noor us saher*1, naureen aziz qureshi2 1centre of excellence in marine biology, karachi university, karachi, pakistan. 2faculty of science and technology, government college, university, faisalabad, pakistan. article history: received 27 october 2016 accepted 28 january 2017 available online 2 5 february 2017 keywords: uca earthen structure habitat sexual attraction abstract: we studied the reproductive behavior and the construction of an earthen structure in the four species of fiddler crab with reference to their habitat. males of the uca sindensis and u. iranica inhibit on open mudflats, construct the pillar and semidome structures at or near to the burrows openings. these structures perform the function to attract females, who wandering on the surface between male burrows for mating as well as provide protection or hiding object against the avian predator to mates. there were no earthen structures were observed in u. urvillie and u. annulipes, which inhibit among the vegetation. the well-marked intersexual different arrangement of mudballs was observed in u. annulipes. the earthen structures (pillar and semidome) and mudballs arrangements help courting females to assess the quality of mate as well as internal conditions of the burrow. introduction it is a well-known phenomenon that adult males of some species of fiddler crabs belonging to the genus uca construct an earthen structure at the entrance of the burrow (crane, 1975). von-hagen (1968) listed twelve different species that construct such structures. studies have shown that there are specific differences in size and shape of the structures. crane (1975) reviewed these structures in fiddler crabs of the world. the males of 17 species have been reported to build an earthen structure as semidomes (shelter like structure), rims (a more delicate, vertical structure, which ranges from a tall, wide hood to a tall, narrow column or pillar) and chimneys next to their burrows during reproductive season (christy, 1988a; christy et al., 2001). chimneys were found in some subgenera: australuca (u. elegans), amphiuca (u. iranica), boboruca (u. thayeri), deltuca (u. dussumieri, u. arcuata, u. forcipata, u. coarctata, and u. urvillei), celuca (u. cumulanta and u. stenodactylus), minuca (u. vocator, and u. subcylindrica) and thalassuca (u. tetragonon) * corresponding author: noor us saher e-mail address: noorusaher@yahoo.com (crane, 1975; george and jones, 1982; thurman, 1984). the constructions of earthen structures have been shown to function for sexual attraction that build as pillars (christy, 1988a, b), hoods (zucker, 1974, 1981; christy et al., 2002, 2003), mud balls (oliveira et al., 1998) and chimneys (shih et al., 2005). these earthen structures are useful to protect the fiddler crabs in different conflicting behavior such as fighting, burrow occupation and disturbance caused by neighbours (zucker, 1974; salmon, 1987; oliveira et al., 1998; wada and murata, 2000; shih et al., 2005), while some other studies have indicated usefulness of these structures in environmental regulation through hoods (powers and cole, 1976) and chimneys (crane, 1975; thurman, 1984) as well as in parental care (incubation of eggs) and protection from enemies through chimney (shih et al., 2005). in many species, only reproductively active males build earthen structures and they have been supportive to reduce aggression behavior between neighbouring males (zucker, 1974; 41 int. j. aquat. biol. (2017) 5(1): 40-46 clayton, 1988; wada et al., 1994; oliveira et al., 1998), attract females for mating (crane, 1975; christy, 1988a, b, 1995; oliveira et al., 1998; christy et al., 2001, 2002), and protection of gravid females after mating (shih et al., 2005). in addition kim and choe (2003) and kim et al. (2004) considered semidome to be, an indication of courtship activity. kim et al. (2004) suggested that the semidome of u. lactea perform the function of reducing aggression or to attract females. the objectives of this study were to study the appearance and the construction of earthen structure of fiddler species and the correlation of these structures with the breeding season. materials and methods study site: two sites, sandspit and korangi creeks, were selected for the fiddler crabs collection due to the presence of marked distinct distribution patches of fiddler crabs. the sandspit backwaters mangrove area is located at south west of karachi (24o50'n, 66o56'e). the backwater is connected to the arabian sea through the manora channel. the sandspit beach is bifurcated by a dry strip of land, with mudflats and mangrove vegetation found on northern side and the sandy coast on its south. the dense vegetation is comprised of the monospecific stand of mangrove species, avicennia marina. the second study site was located (24o79'n, 67o20'e) in the korangi creek mangrove area near the salts works located in fishing village of ibrahim hyedri. the north most creeks of the indus delta are the korangi and phitti creeks of which korangi creek is 12 km from karachi harbour and 9 km from quaidabad. korangi creek is connected at its northeastern end with phitti creek and kadiro creek, while at its southwestern end, it connects with open sea and with gizri creek, and is bounded on its sides by extensive mangrove vegetation of a. marina. study fiddler crab species: the reproductive behaviour of four commonly distributed species of fiddler crabs was studied. uca urvillei is the only narrow fronted species found in the shadow or canopy of the mangrove trees; u. iranica is the most frequently found species mostly distributed from low tide level to high tide level of sandy cum muddy areas; u. annulipes is mostly associated with fringing mangrove or among pneumatophores; u. sindensis is mostly found at the high tide level of muddy cum sandy area. uca iranica and u. sindensis were usually distributed on the open mudflat areas adjacent to the mangrove vegetation. earthen structure construction behavior: initially the data on the size of male crab and burrow diameter relationship were collected for the male crabs of the species. the construction behavior and appearance of the earthen structures in the species of fiddler crabs were studied. data were collected from the visual field observations (figs. 1 and 4). figure 1. earthen structures constructed by males of uca species during breeding season. (a) pillar constructed by male crab of uca sindensis by bring the mud from inside the burrows and (b) semidome structure constructed by the male crab of uca iranica by scrapping the mud from opposite side. (b) (a) 42 saher and qureshi/ construction of earthen structure in fiddler crabs results like most of the species of fiddler crabs, construction of earthen structures have been associated with reproductive season. the appearance of earthen structures starts with the initiation of breeding season. uca sindensis: the structures of this species start to appear from the end of february or beginning of march with increasing temperature and were observed up to july. the male crabs began to build a cylindrical structure, a pillar (with mean height of 90.0±16.0 mm) for an average burrow diameter (11.9±1.2 mm), with height range of 50-125 mm, next to the burrow opening south wards in the direction of low tidal level (table 1). these pillars were composed of large wet soil pellets or mudballs (fig. 1). the sediment material of mudballs is darker in color than the substratum and indicates it has been excavated and was carried up from inside the burrow. a linear relationship (r2=0.694) between burrow diameter and pillar height (mm) was observed (fig. 2). in addition, male crabs were observed to make definite waving display and waving increase (double than usual) in the presence of females. the underground mating was observed as during excavation of burrow, the presence of male and female crabs suggested the mating takes place within the burrow. uca iranica: the courting males of u. iranica build semidomes (or shelter like) structures on their burrows openings. these structures were observed from the month of may to september at the collection site of this species. to build the structure bits of mud from the substratum surface was scraped with the help of their walking and staked the similar material on the opposite side of burrow entrance with the help of his major claw (fig. 1b). the size of burrows and the height and width of these semidomes structures showed the mean burrow diameter as 16.6 ± 2.5 mm with the mean height of 60.0±6.0 mm (table 1). in addition, male crabs were observed to increase waving display of enlarge cheliped in the presence of females. the surface mating was also never observed and during excavation of burrows both, the presence of male and female crabs confirmed the underground mating. a good corresponding positive linear relationship was observed between burrow diameter (bd) and the width of semidome (r2=0.57) and between bd and the height of semidome (r2=0.64; fig. 3). uca annulipes: there were no special structures around the burrow, but markedly different arrangements of mudball sequence were observed during the month of september to november. the table 1. summary of descriptive statistics for different parameters of the earthen structure constructed by three species of fiddler crab. species variable n mean ± sd min max uca sindensis burrow diameter (mm) 45 11.9 +1.2 10.0 14.0 pillar height (mm) 45 90.0+16.0 50.0 125.0 uca iranica burrow diameter (mm) 50 16.6+2.5 12.0 20.0 semidome height (mm) 50 60.0+6.0 50.0 75.0 semidome width (mm) 50 75.0+7.7 62.5 87.5 uca annulipes burrow diameter (mm) 50 14.0+3.1 8.0 19.0 number of mudballs 50 24.0+5.9 13.0 38.0 farthest mudball distance (mm) 50 130.0+26.0 87.5 200.0 figure 2. linear relationship between burrow diameter and pillar height (mm) for uca sindensis. 43 int. j. aquat. biol. (2017) 5(1): 40-46 mudball placement behavior was observed during the reproductive period for the male and female crabs. apparently intersexual difference in the arrangement and number of mudballs were observed (fig. 4). the male crabs placed more number of larger mudballs forming an arc (north wards mostly towards the high tidal level) around its burrow. the mean numbers of mudballs was 24.6±5.9 that were found and at the mean distance of 130.0±26.0 mm from the burrow opening with an average diameter of 14.0±3.1 mm (table 1). positive correlation was observed between bd and the number of mudballs adjacent to the burrows and the bd with the farthest placement of mudballs (r2=0.52 and r2=0.35) was observed (fig. 5). there is no evidence for surface mating, thus the mating was underground or within burrows. uca urvillei: there were no special structure around the burrow nor were any marked arrangements of mudballs sequence observed. during field observation the surface mating was observed. in u. urvillei, male crabs do not construct the burrows and usually wandering on the surface within vegetation and at the time of the threat they escape in to the nearest burrow of other ocypodid or grapsid crabs. discussion in present study, u. sindensis build a cylindrical pillar next to the burrow opening south wards in the direction of low tidal level these pillars were composed of large wet soil-pellets or mudballs and the courting males of u. iranica build semidomes (or shelter like) structures on the mouth of burrows openings by scraping the mud from the surface substratum towards the high tidal level. like most of the species of fiddler crabs construction of earthen structures have been associated with reproductive behaviour. the appearance of earthen structures clearly coincides with the presence of ovigerous females and initiation of the breeding season. these structures likely increase the attractiveness of the mate for the sexual selection. kim et al. (2004) figure 3. linear relationship between (a) burrow diameter (mm) and semidome width (mm) and (b) burrow diameter (mm) and semidome height (mm) for uca iranica. (b) (a) figure 4. the mouthball arrangement of uca annulipes around the burrows. (a) male and (b) female. 44 saher and qureshi/ construction of earthen structure in fiddler crabs reported during mating season male crabs of u. lactea built semidomes at their burrow entrances to attract females for mating in the upper intertidal zone of mudflats, which were beyond the reaches of neap tides. zucker (1981) suggested that male of u. musica build hoods to increase the time during mating and to reduce territorial overlap and aggressive interaction between neighbouring male crabs. the pillar and hood building are condition dependent behaviours (blackwell et al., 1995; christy et al., 2001). the primary functions of these structures are related to courtship signaling but not to aggression between males (christy, 1988; kim et al., 2004). male crabs of u. beebei also built pillars next to their burrow entrances for attracting mate searching female crabs (christy et al., 2003). christy et al. (2003) observed that females attracted to structure building males likely mate with superior males that may end in fitness benefits and reduce predation risks and thus ecological factors shape evolutionary patterns in different species. in u. formosensis chimney is built by male crabs after successfully luring the female into his burrow for underground mating (shih et al., 2005). shih et al. (2005) hypothesized chimney building of male u. formosensis before neap tides as a byproduct of excavation of the burrow that widens the shaft and deepens the burrow so that it reaches the water table and keeps the chamber, moist for female to incubate her eggs. in addition, the mudballs are piled to make chimney at burrow openings also serve as a refuge from enemies (shih et al., 2005). the association of the claw-waving display with a defended territory and with earthen structures serves as a territorial advertisement signal to both receptive females and rival males (brown, 1975; crane, 1975; weygoldt, 1977; von-hagen, 1993). mudball formation is a common characteristic feature of genus uca. oliveria et al. (1998) observed european fiddler crab u. tangeri both male and female crabs placed their mudballs around their burrow openings with major difference in mudballing behavior. they suggested that mudballs of females were the byproduct of burrow excavation and mudballs placement by males may exhibit dual function, to reduce the number and intensity of aggressive interactions between neighboring male crabs and to attract females. this type of mudballing behaviour has been observed in u. annulipes and was more evident during the reproductive season. there was no intersexual difference in the number of mudballs, but the placement and arrangement of mudballs was evidently different between sexes, this difference was more obvious during the breeding season and this difference indicated that mudball function in males spacing by making territories for attracting and copulatory activities. the size of territory and number of mudballs helps the female to assess male quality as well as internal condition of male burrow. some authors have noted that males of some species apparently increase their rate of waving when approached by either males or females (doherty, 1982; crane, 1975) although salmon, stout (1962) demonstrated that male u. pugilator increased their waving rate when presented with females, but not with males. the male crabs of u. urvillei usually does not construct their burrows (data unpublished, observed figure 5. linear relationship between (a) burrow diameter (mm) and the number of mudballs, and (b) burrow diameter (mm) and distance of farthest mudballs (mm) for uca annulipes. 45 int. j. aquat. biol. 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(2016) 4(6): 378-386: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article composition and seasonal variation of phytoplankton community in lake hlan, republic of bénin arsène mathieu houssou*,1, 2, hyppolite agadjihouédé1,2, clément a. bonou3, elie montchowui1, 2 1laboratory of hydrobiology and aquaculture, faculty of agricultural sciences, university of abomey-calavi, republic of bénin. 2laboratory of research in aquaculture and aquatics biology and ecology, school of aquaculture of vallée, national university of agriculture, republic of bénin. 3research laboratory in applied biology, polytechnic school of abomey-calavi, university of abomey-calavi, republic of bénin. article history: received 12 september 2016 accepted 7 december 2016 available online 2 5 december 2016 keywords: cyanobacteria diatoms dinoflagellate hydrological season phytoplankton diversity abstract: knowledge of biodiversity of aquatic ecosystems is nowadays a challenge for global research. phytoplankton being very important in the sustainability of ecosystems, its mastery allows the development of early monitoring and evaluation tools of the health status of aquatic environments. the study aims to make an initial inventory of phytoplankton of the lake hlan and to evaluate the influence of hydrologic season on its dynamics. plankton samples were collected monthly between may and december 2012 using plankton net of 30 µm size. they were then treated and species identified using light microscopy. 39 species in 7 classes (bacillariophyceae, 18 species in 10 genera), (cyanophyceae, 5 species in 5 genera), (chlorophyceae, 5 species in 3 genera), (zygnematophyceae, 3 species in 2 genera), (trebouxiophyceae, 2 species in 2 genera) (euglenophyceae, 4 species in 3 genera) and (dinophyceae, 2 species in 2 genera) have been identified. the shannon index varied between 4.8 and 5.1 bit cell-1. this shows that the ecosystem is balanced. nevertheless, the presence of potentially toxic species requires a monitoring program for lake hlan. introduction highly diversified phytoplankton is the basis of food webs in aquatic ecosystems. despite its importance, phytoplankton is very sensitive to environmental changes. hence, environmental variations often cause major damage which may be irreversible on the phytoplankton population. the final consequence is the loss of diversity in aquatic impacted environments. that at the higher level could affect the well-being of human. due to its high sensitivity, phytoplankton is now considered effective ecosystem indicator. therefore, it is widely used to assess or predict the state of aquatic ecosystems (gao and song, 2005; barinova et al., 2005; barinova et al., 2006; barinova and nevo, 2012; madhu et al., 2007). knowledge of biodiversity of inland water phytoplankton is therefore an essential basis for the use of this biological compartment for the purpose of * corresponding author: arsène mathieu houssou e-mail address: arsnehous@yahoo.fr ecosystems biomonitoring (houssou et al., 2015). hence, this study aims to be a first evaluation of phytoplankton of the lake hlan in the south of benin republic. based on its location and topography, the lake hlan is roughly difficult to access. it is an isolated ecosystem with little direct human impact. indirect inputs received may be drained by its tributaries (mainly hlan river). so this is an interesting ecosystem with very little known biodiversity (only its ichthyofauna was assessed (montchowui et al., 2008). no data exist on its phytoplankton diversity. the purpose of this study is to provide the first data on the diversity of phytoplankton. such information will provide the basis for further studies. materials and methods study area and sampling site: the lake hlan is localized at toffo town in the south of benin 379 int. j. aquat. biol. (2016) 4(6): 378-386 republic. it is a widening of the hlan river at kpomè (6°56.88'n, 2°19.48'e). its area covers 165 ha. in the flood times, its water is influenced by that of the rivers samion, da, hoho and hlan. those rivers flow into the lake starting from the swamp of adogbé and hon village. two other great rivers influences the lake at the flood time. it is about zou and ouémé rivers through respectively zounga and agbagbé tributary. three stations with different levels of anthropogenic activities and composed different habitats were chosen as representative of the water map for sampling plankton (fig. 1). so from upstream to downstream, the sampling stations were: houvènou (06°57'49.6" n, 002°19'04.4" e, 53 m), awayamey (06°56'29.2" n, 02°19'57.4" e, 25 m) and dezinmey (06°55'57.3''n, 02°20'17.3"e, 35 m). phytoplankton sampling and analysis: monthly phytoplankton sampling was performed from may to december 2012. sampling was done according to the hydrological seasons of benin republic. the period from august to november represented the flood time while may to july represented the recession period. the december samples are used to see the changes just after the flood. samples were collected using plankton net of 30 µm mesh. vertical sampling procedure (full depth) was used at three different points of each station. a composite sample resulting from the mixture of the three sub-samples is immediately preserved with formaldehyde 5%. it is then transported to lab and stored in darkness condition for future analysis. the recorded environmental parameters are presented in table 1. samples were settled and precipitated to a final same volume of 100 ml. species were identified under a light microscope (olympus b102). the species determination was based on prescott (1954), compère (1974, 1975), vanlandingham (1982), nogueira and correia (2000), tsukii (2005), kinross (2007), bellinger and sigee (2010), and oyadomari (2011). counting of cells/colonies of different species identified was done with a hematimeter (burker turk four grids). four hundred (400) cells/colonies were counted for each species. high abundant species which density per milliliter of sample was greater than 400 were counted in three consecutive 1 ml aliquots (regularity is then searched). the very rare species have been counted throughout the sample volume (milliliter per milliliter). the abundance of each species was calculated using the following formula: 𝐷 = 1 2 ( 𝑁 𝑇𝑑 ∗ 100) where d is the density per liter of the species, n is the number of cells/colonies counted and td is the sample rate corresponding to n. data analysis: all biological data were transformed by using log (x+1) according to frontier (1973). the principal component analysis (pca) and the cluster analysis were used to discriminate the typology of species distribution. the monthly variations of phytoplankton abundance was studied with the anova test, followed by the lsd of ficher post-hoc test. pca and one way anova tests were performed in statistica v7, while the cluster analysis was done with past program. the biological diversity was also study using the shannon diversity index, calculated by: h′ = −∑[( 𝑛𝑖 𝑁𝑖 ) ∗ 𝑙𝑜𝑔2( 𝑛𝑖 𝑁𝑖 )] figure 1. geographical map of lake hlan. 380 houssou et al./ phytoplankton diversity assessment in lake hlan where h' is the index of diversity expressed in bit/individual, ni the number of the species, n the total number of individual constituting all the species, log2 the logarithm on base 2. results seasonal spread of phytoplankton species: a total of 39 species of phytoplankton were recorded during the study belonging to 7 classes (table 2). the bacillariophyceae was represented by 18 species in 8 genera. chlorophyceae was composed of 5 species in 3 genera, while zygnematophyceae and trebouxiophyceae were represented by 3 species in 2 genera and 2 species in 2 genera, respectively. cyanophyceae was composed of 5 species in 5 genera. euglenophyceae and dinophyceae were respectively composed of 4 species in 3 genera and 2 species in 2 genera. during low water season, all species were identified, while urosolenia eriensis, peridiniopsis quadridens, stigeoclonium aestivale, rhizosolenia setigera, navicula sp., urosolenia sp., closterium parvulum and coelastrum sp. had disappeared with the increase in the water depth. the relative abundance showed absence of net dominant species. however, aulacoseira sp. nitzschia sigma, synedra acus, s. splendens, closterium sp. and closteriopsis sp. are most abundant species. phytoplankton abundance in relation with diversity index: the total phytoplankton abundance and the shannon diversity index variations throughout the study period are showed on figure 2. phytoplankton abundance had same profile with the diversity index. the high diversity and abundance was observed during low water season. the observed phytoplankton community had a very good diversity index, ranged between 4.8 bit cell-1 and 5.1 bit cell-1. significant variation was observed both in total phytoplankton abundance and the diversity index seasonality. typology of phytoplankton community: principal components analysis (pca) of phytoplankton species is presented in the factorial design (1 and 2) on figure 3. the cumulated eigenvalues was 75.25% and 13.95% respectively for first and second axis. the first axis was composed by both low water and flood seasons (may and august to december), while the second axis particularly and positively selected the month of july (first coming of flood waters). the first axis selected negatively both of two hydrologic seasons. the species as melosira sp., nitzschia sigma, s. acus, s. splendens, closterium sp. and closteriopsis sp. were not affected by hydrological season, while gomphonema vibrio, r. setigera, u. eriensis, urosolenia sp., c. parvulum, stigeoclonium sp., s. aestivale and coelastrum sp. were affected the flood. as to the month of july, gomphonema sp. highly correlated is opposed to g. ventricosum and stigeoclonium sp. in general, principal component analysis showed two groups of species. one is composed by species not affected by seasons and the second is affected by the flood event. these two groups of species are also showed on the cluster analysis (fig. 4). relationship between total phytoplankton and environmental parameters: the linear relationship table 1. monthly variation of abiotic factor in hlan lake during the study. may june july august september october november december temperature 29.8±0.1a 27.8±0.8b 27.3±0.5c 27.2±0.3d 27.4±0.2e 28.1±0.2f 28.1±0.4g 28.6±0.01h dissolved oxygen (mg.l-1) 5.0±0.9a 4.5±0.4b 4.2±0.8c 3.5±0.2d 4.0±0.3e 4.3±0.2f 4.2 ±0.1g 4.6±0.5h ph 6.9±0.1a 6.8±0.2b 6.8±0.1c 6.3±0.2d 6.4±0.3e 6.9±0.2f 6.8±0.4g 7.0±0.2h tds (ppm) 37.2±4.2a 39.8±1.9b 48.5±4.5abc 42.2±3.3cd 45.0±2.7ae 36.2±3.1cdef 42.3±0.8cfg 46.3±1.9abfh conductivity (µs.cm-1) 71.7±6.7a 83.2±11.6b 92.5±6.8ac 82.0±6.0d 87.0±5.5ae 72.5±6.0cef 82.2±2.3g 90.2±3.4afh transparency (cm) 95.7±25.4a 76.0±5.3b 67.0±33.8c 65.0±5.0d 76.0±1.7e 64.0±32.3f 81.0±13.9g 93.5±14.3h depth (m) 2.4±0.4a 2.6±0.2b 3.0±0.4c 3.9±0.5d 5.9±1.3abcde 4.3±0.4af 3.5±0.8eg 2.8±0.3eh the values of the same line with common letter as power are significantly different one way anova, post-hoc: lsd of fisher, p<0.05. 381 int. j. aquat. biol. (2016) 4(6): 378-386 between the total phytoplankton abundance and environmental parameter with significant variation throughout the study period is presented on figure 5. the total dissolved solid (tds) and conductivity had low positive effect on the total phytoplankton abundance. the respective correlation coefficients are r=0.21 and r=0.32. regarding the water volume (lake body depth), a negative impair is observed on table 2. distribution of phytoplankton species according to hydrologic seasons; the values in square bracket are the percentage of dominance (%). phytoplankton species hydrodynamic code low depth flood after flood bacillariophyceae aulacoseira granulata simonsen, 1979 d1 + (2.7) + (2.6) + (3.3) aulacoseira sp. thwaites, 1848 d2 + (4.8) + (5.9) + (5.9) bacillaria paxillifera, marsson, 1901 d3 + (3.1) + (2.9) + (2.8) nitzschia reversa smith, 1853 d4 + (3.7) + (3.7) + (3.3) nitzschia sigma smith, 1853 d5 + (4.3) + (5.3) + (5.3) synedra acus heurck, 1885 d6 + (4.0) + (4.7) + (4.8) synedra splendens kützing, 1844 d7 + (4.0) + (4.6) + (4.5) synedra sp ehrenberg, 1830 d8 + (3.2) + (3.8) + (3.7) diatoma tenuis agardh, 1812 d9 + (3.4) + (4.1) + (4.1) gomphonema ventricosum gregory, 1856 d10 + (1.0) + (1.0) + (2.2) gomphonema vibrio ehrenb, 1843 d11 + (0.8) + (0.3) + (1.1) gomphonema sp. ehrenberg, 1832 d12 + (1.1) + (1.1) + (1.0) navicula sp. bory 1822 d13 + (2.7) + (0.9) surirella sp. turpin, 1828 d14 + (3.0) + (3.3) + (3.5) surirella capronii kitton, 1869 d15 + (3.5) + (3.2) + (4.1) rhizosolenia setigera brightwell, 1858 d16 + (1.5) + (0.2) urosolenia eriensis .smith, 1872 d17 + (0.4) urosolenia sp. round & crawford, 1990 d18 + (1.8) + (0.7) cyanophyceae spirulina sp. turpin, 1892 cy1 + (2.0) + (1.0) + (1.1) oscillatoria sp. vaucher, 1893 cy2 + (3.5) + (3.8) + (4.1) raphidiopsis curvata fritsch and rich, 1929 cy3 + (2.6) + (3.0) + (3.2) microcystis flos-aquae kirchner, 1898 cy4 + (3.0) + (3.8) + (3.6) stigonema sp agardh, 1886 cy5 + (2.7) + (3.2) + (3.3) chlorophyceae tetraedron incus smith, 1926 ch1 + (2.6) + (2.4) + (2.6) tetraedron sp. kützing, 1845 ch2 + (2.3) + (2.3) + (2.5) stigeoclonium sp. kützing, 1843 ch3 + (0.2) + (1.0) + (1.9) stigeoclonium aestivale collins, 1909 ch4 + (1.9) coelastrum sp. nageli, 1849 ch5 + (1.2) + (1.0) zygnematophyceae closterium parvulum nägeli, 1849 zy1 + (0.5) + (0.9) closterium sp. nitzsch, 1848 zy2 + (3.9) + (4.0) + (4.9) gonatozygon sp. de bary, 1858 zy3 + (3.5) + (3.5) + (3.2) trebouxiophyceae actinastrum sp. lagerheim, 1882 tr1 + (1.3) + (0.9) + (1.0) closteriopsis sp. lemmermann, 1899 tr2 + (3.8) + (4.5) + (4.5) euglenophyceae strombomona sp. ehrenberg, 1835 e1 + (2.8) + (2.8) + (2.9) euglena sp. ehrenberg, 1830 e2 + (3.4) + (3.9) + (3.2) phacus longicauda dujardin, 1841 e3 + (2.8) + (2.4) + (2.4) phacus caudatus hübner, 1886 e4 + (2.2) + (2.5) + (2.6) dinophyceae peridiniopsis quadridens bourrelly, 1968 d1 + (1.1) peridinium bipes stein, 1883 d2 + (3.6) + (4.3) + (3.4) + present; absent; numeric italic scores in bracket: relative abundance (%). 382 houssou et al./ phytoplankton diversity assessment in lake hlan the phytoplankton abundance (r=-0.59). discussion this study constituting the first inventory of phytoplankton species in the hlan lake. it shows in addition to those identified species their assigned behaviors vis-à-vis to the hydrological seasons. a total of 39 phytoplankton species were identified. the structure of the assembly appeared dominated by diatoms species, showing a sufficient presence of mineralized organic matter (maestrini and robert, 1981; bennouna et al., 2000; kemka et al., 2004; atanle et al., 2012, 2013; houssou et al., 2015). this obtained structure also shows that the lake hlan has an apparent ecosystem health (houssou et al., 2016), diatomic species being potentially more sensitive to pollution. the shannon diversity index ranging between 4.8 and 5.1 bit cell-1 confirms the figure 2. compared variation of shannon index (h’) and total recorded phytoplankton abundance. histogram with different letter are significantly different (one way anova, lsd post-hoc of fisher, p<0.05). figure 3. principal component analysis (pca) of 39 recorded phytoplankton species. 383 int. j. aquat. biol. (2016) 4(6): 378-386 apparent good health of the environment (simboura and zenetos, 2002). species richness obtained appeared lower than that of lake azili (51 species) (a lake located in the same basin of ouémé) with the same method of study (houssou et al., 2015). the identified species are cosmopolitan with wide distribution. the typology allowed to see that species such as: melosira sp., n. sigma, s. acus, s. splendens, closterium sp. and closteriopsis sp. are characteristic of the ecosystem and are not affected by the season. most of the taxa in genus nitzschia including n. sigma are α-mesopolysaprobe. they are known to proliferate in eutrophic and hypertrophic environments (leland and porter, 2000). in this study, density of species of genus nitzschia including the characteristic (n. sigma) is still low. this is justified by the low accessibility of the lake, limiting exogenous inputs from anthropogenic sources (houssou et al., 2016). similarly, diatoms generally proliferate in favor of mineralized organic matter. the relatively low abundance of diatoms characteristics of the lake therefore reflects a low mineralization in the ecosystem. the presence of a number of potentially toxic or toxin producing species in the lake could draw attention to the monitoring of the ecosystem. an increase in pollutant may lead to lake hlan figure 4. cluster analysis of 39 recorded phytoplankton species. 384 houssou et al./ phytoplankton diversity assessment in lake hlan degradation in favor of a proliferation of harmful species. cyanobacteria, oscillatoria species are known as toxin-producing as microcystins, anatoxin and aplysiatoxines and lipopolysaccharide (chorus and bartrum, 1999). the genus microcystis is a producer of microcystins and lipopolysaccharide. these toxins in aquatic environments can cause several physiological damages on aquatic life. these effects can be direct or indirect with acute or chronic toxicity depending on the toxin. according djediat et al. (2010), microcystins are responsible for cell lysis of the liver, hepatocyte vacuolation and loss of reserves (glycogen and glycoproteins) in fish. on human health, cyanotoxins may have effects of different levels (body irritation to food poisoning) according to the type of exposure. similarly, the identified dinoflagellate species are potentially harmful. thus appear the importance of monitoring hlan lake ecosystem not only for maintaining biodiversity but also for preserving the health of surrounding human populations. the environmental factors effects on the abundance of the total phytoplankton were not very important due to low mineralization of the ecosystem (houssou et al., 2016). illustrated by the low correlation of phytoplankton with tds (r=0.21) and conductivity (r=0.32). the conductivity values recorded during the study were below of 200 μs cm-1. these values showed low impacts due to human activities (crel, 2009-2011). similarly tds values are relatively low due to the strong bond between the two parameters. in view of the link between phytoplankton and water depth, the effect of the flood on the population is thus justified. in summary, the phytoplankton species identified in the lake hlan during this study are generally cosmopolitan. some of them are producing toxin and therefore predispose the lake to a degradation of its ecosystem in case of significant input of organic matter. it is therefore essential to control the exogenous inputs into the lake. based on the observed diversity, hlan lake is a balanced ecosystem. a thorough study of the phytoplankton compartment will therefore allow appreciating the real level of the ecosystem health. acknowledgements we are grateful to all colleagues who have figure 5. linear regression between total phytoplankton abundance and environmental parameters that varied significantly across the study period. 385 int. j. aquat. biol. 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(2023) 11(2): 124-130 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article evaluation of imidacloprid-induced toxicity and lipid peroxidation in the freshwater bivalve, lamellidens marginalis poonam b. ahire, vinaykamal d. dethe department of zoology, mvp’s arts, science and commerce college, ozar (mig). nashik, india. s article history: received 26 january 2023 accepted 14 april 2023 available online 2 5 april 2023 keywords: pesticide bivalve hepatopancreas lamellae toxicity abstract: the toxicity of the neonicotinoid pesticide imidacloprid was tested on lamellidens marginalis by 96-hours lc50 test and histopathology. after acute exposure to imidacloprid, histopathological changes were noted in the gill, mantle, and digestive gland. exposed gills showed deformed interfilamental space, fused lamellae, disrupted chitenous rod, and vacuolated cytoplasm. in the mantle, damage was observed in the inner and outer mantle epithelium and vacuole in connective tissue. the digestive gland has ruptured the digestive tubule, and basement membrane, the lumen deteriorates, and the cytoplasm appears vacuolated. lipid peroxidation was also observed after the exposure. these findings suggest that acute exposure to imidacloprid caused significant histological alterations in vital organs and can affect the non-targeted freshwater bivalve lamellidens marginalis. introduction pesticides are chemical compounds used to control pests and enhance crop yields. there are various types of pesticides, including organochlorines, organophosphates, carbamates, pyrethroids, and neonicotinoids (sparks et al., 2020). among these, neonicotinoids, such as imidacloprid, have gained significant attention due to their widespread use and potential ecological impacts. imidacloprid is a neonicotinoid insecticide that targets the nicotinic acetylcholine receptor (nachr), leading to the prolonged opening of sodium channels in neurons and ultimately resulting in their depolarization and death (mencke and jeschke, 2002; selvam and srinivasan, 2019). this systemic pesticide is effective against a range of pests, including jassid, aphid, thrips, termites, and mites, and is used on crops such as cotton and ladyfinger, as well as in veterinary applications (stanneck et al., 2012; krämer and mencke, 2012). however, the extensive use of imidacloprid in agriculture has resulted in spray drifts and runoff, conveying the pesticide to nearby water bodies (morrissey et al., 2015; klarich correspondence: vinaykamal d. dethe doi: http//doi.org/10.22034/ijab.v11i2.1902 e-mail: mevdethe@gmail.com dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.6.8 et al., 2017). consequently, neonicotinoids have been commonly found in water bodies, potentially harming non-targeted organisms (borsuah et al., 2020). natarajan (2016) emphasizes the vital role of water quality in supporting aquatic life. unfortunately, the presence of pesticides in water bodies can negatively impact water quality. pesticides are a significant contributor to water pollution and can cause direct or indirect harm to economically and ecologically important species by producing toxic stress (köprücü et al., 2010). these substances can infiltrate water resources and accumulate in water columns, causing changes to the physical, chemical, and biological processes of water (agrawal et al., 2010). moreover, pesticides can lead to physiological, biochemical, and histological changes in mussels due to their bioaccumulation and biomagnification in tissues (yancheva et al., 2017; zhang et al., 2020). additionally, pesticide exposure can affect gene expression (kuchovská et al., 2021) and cause immunotoxicity, genotoxicity (kayis et al., 2019), as well as histopathological changes by 125 int. j. aquat. biol. (2023) 11(2): 124-130 damaging tissue cells (shaikh et al., 2010; sula et al., 2020). bivalves, including freshwater mussels, are ecologically important since they are part of the food web (strayer, 2008) and possess medicinal and nutritional value (laxmilatha, 2013; charaborty et al., 2016). unfortunately, pesticide pollution in water bodies can negatively impact the normal metabolism, development, growth, behaviour, reproductive cycle, and life span of organisms (amoatey and baawain, 2019). lamellidens marginalis, a benthic organism, is distributed extensively and exhibits feeding habits that increase its exposure to diverse environmental pollutants. this freshwater bivalve is frequently used as a model organism for toxicological investigations (yusufzai et al., 2010). the objective of this investigation is to evaluate the toxicity of the neonicotinoid pesticide imidacloprid on l. marginalis with respect to histopathology and lipid peroxidation. material and methods lamellidens marginalis were collected from the darna river, chehadi (19°55’54.02"n, 73°55’ 30.42"e), and brought to the laboratory where they were acclimatized in aged tap water for 6 days in plastic troughs. the bivalves were fed spirulina algae daily to ensure proper nutrition. overcrowding was avoided by keeping 10 bivalves in separate plastic troughs, and the water was changed every day with dechlorinated tap water. the bivalves were exposed to a 12:12 light-dark cycle during the experiment. healthy bivalves with a shell length of 9-10 cm and weight of 70-75 g, regardless of their sex, were selected for the toxicity assay. the pesticide imidacloprid (bayer confider super, 30.5m/m sc) was obtained from the market. to study the effect of imidacloprid, l. marginalis were exposed to the lc50 concentration (40 ppm) for 96 hrs. the gill, digestive gland, and mantle tissues were fixed in bouin's fluid, followed by treatment with alcohol (30, 50, 70, 80, 90, and 100%) for 2 hrs each. the tissues were cleared in xylene and embedded in paraffin wax. sections of 5-8 µ were obtained using a leica microtome rm 2235, and the sections were stained with delafield's hematoxylineosin. the stained sections were observed and microphotographed using hl-22/coslab. statistical analysis was performed using the microsoft excel data analysis program. in the tbars assay, the end product of lipid peroxidation, mda (malondialdehyde), was measured based on ohkawa et al. (1989). absorbance was recorded spectrophotometrically at 532 nm, and lipid peroxidation was expressed in nmol/mg protein. the bivalves were exposed to 0.7 ppm for studying lipid peroxidation. results acute toxicity studies: the acute toxicity test reported that the lc50 value of imidacloprid was 40 ppm for 96 hrs. behavioural changes: behavioural changes like secretion of mucous after 24 hrs of imidacloprid exposure were observed during the experiment. histopathological observation: normal gill lamellae of control bivalve consists of a large number of closely set, thin, vertical, gill filaments perforated by minute opening bound by filaments. the gill filament is connected by horizontal bars. gill filaments are composed of connective tissue. the margin of the gills is covered by ciliated epithelium and supported by chitanous rods. the gill lamellae are joined together by intrallamellar junction so that space is present between gill lamellae. lamellae are divided into water tubes. gills are covered by three types of cilia, lateral, latero-frontal, and frontal cilia. the prominent nucleus is present in each epithelium cell (fig. 1i). after the exposure of 40 ppm of imidacloprid, the gill structure showed anomalous changes. necrosis was noticed in the gill lamellae. the damaged nucleus was observed at the base of the gill. at certain places, fused gill lamellae were observed with degenerative changes like vacuolated cytoplasm, deformed inter filamental space, and disrupted chitenous rods (fig. 1ii). the mantle consists of two lobes that lie inner 126 ahire and dethe / evaluation of imidacloprid-induced toxicity and lipid peroxidation in the freshwater bivalve surface of the shell and encloses the animal inside the shell. the mantle consists of connective tissue, blood vessel, nerves, and muscles. below the columnar epithelium is a layer of connective tissue. nuclei were prominent (fig. 1iii). treated mantle epithelium shows necrosis and a gap was observed in the inner mantle fold and outer mantle fold. the inner mantle epithelium layer is separated from connective tissue. dissolved nuclei were observed in the middle of the epithelium (fig. 1iv). the digestive gland or hepatopancreas is the main site of all metabolic activities in l. marginalis. it is composed of tubules and ducts. digestive tubules are internally lined with columnar epithelial cells resting on a thin fibrous connective tissue layer and externally covered with a basement membrane. the epithelium of digestive tubules consists of basophilic cells (secretary in function) and acidophilic cells figure 1. (i) microphotograph of l.s (h & e) of control gill (g.f: gill filament, r.e: respiratory epithelium, ils: interlamellaer space, n: nucleus, and c: cilia), (ii) imidacloprid exposed gill (ils: interlamellar space, fgl: fused gill filament, vc: vaculated cytoplasm, fgl: and fused gill lamellae), (iii.) t.s of mantle of control (fm: mantle fold, omel: outer mantle epithelial layer, imel: inner mantle epithelial layer, ct: connective tissue, and sp: space), (iv) exposed mantle (fm: mantle fold, domel: outer mantle epithelial layer, dimel: inner mantle epithelial layer, ct: connective tissue, and sp: space), (v) t.s of digestive gland of control (c: cytoplasm, l: lumen, bm: basement membrane and imidacloprid exposed specimen, and (vi) t.s of exposed digestive gland, rbm: ruptured basement membrane, dl: degenerated lumen, and vc: vaculated cytoplasm). 127 int. j. aquat. biol. (2023) 11(2): 124-130 (digestive cells). the digestive cells are columnar in shape with a spherical nucleus situated in the basal region (fig. 1v). after the exposure to imidacloprid, the complete destruction of the epithelial lining and irregular placement of necrotic cells was observed along with vacuolated cytoplasm (fig. 1vi). tbars (thiobarbituric acid reactive substance assay): levels of tbars were evaluated in the control and experimental animals (table 1). in the treated group, a significant (p<0.05) increment of tbars in gill (237.47%), adductor muscle (194.28%), hpt (188.12%), mantle (175.81%), and foot (145.81%) were found. increased lipid peroxidation in gills might be because of its importance as the primary site of absorption. discussion the lc50 value for imidacloprid after 96 hours of exposure was found to be 40 ppm. interestingly, the lc50 value for l. marginalis was higher than that for the freshwater bivalves u. mancus and c. fluminea when exposed to imidacloprid (yoloğlu, 2019; shan et al., 2020). this suggests that l. marginalis is more resistant and able to tolerate higher concentrations of imidacloprid. lc50 values of 14.21 and 12.89 ppm were found for the freshwater bivalve p. cylindrical and l. marginalis, respectively, after exposure to the neonicotinoid thiamethoxam for acute treatment (rane and mahajan, 2013; patil, 2019). there is considerable variation in the lc50 values of imidacloprid reported by various investigators in both freshwater and marine water mussels and fish (iturburu et al., 2017; iturburu et al., 2018; vieira et al., 2018; alvim and dos reis martinez, 2019; shan et al., 2020). histology is a useful tool to study the toxicological effect of environmental stressors on animals (yavaşoğlu et al., 2016). gills play a crucial role in respiration, food capturing, and maintaining the acid-base balance (kumar et al., 2012; ray et al., 2020). as the gills come directly into contact with water, they are often the first target organ of contaminants found in water (giarratano et al., 2014), and hence, act as an indicator of environmental stress (cappello et al., 2013). therefore, the study of gills after exposure to imidacloprid is crucial to assess its toxicity. the gills of l. marginalis showed degenerative changes in the nucleus, reduced interlamellar space, broken chitenous rods, and fused gill filaments. similar degenerative changes in gills were observed in the freshwater clam c. fluminea and the marine water mussel m.galloprovincialis after exposure to imidacloprid (shan et al., 2020; pagano et al., 2020). moreover, exposure to thiamethoxam led to the degeneration of epithelial cells and swollen gill filaments in l. marginalis (rane et al., 2019), while exposure to chloropyrifos and dimethoate led to damaged epithelial cells, irregular gill lamellae, hypertrophic nuclei, and dilated sinus filaments in l. marginalis (stalin et al., 2011; kumar et al., 2012). in p. cylindrica, the shape of the gill was lost due to chitenous rods, and the connective tissue core was damaged after exposure to endosulfan (bhalchandra, 2010). in marine bivalve p. radi, necrosis of lamellar cells and fused filaments were observed after exposure to dimethoate (hassan and sheiko, 2013). similarly, exposure of the marine water mussel m. galloprovincialis to the neonicotinoid calypso 480 sc (cal) resulted in epithelial alteration and vacuolization (stara et al., 2020). as reported by patil (2019), exposure to thiamethoxam at 14.21 ppm caused necrosis along with hypertrophy in p. cylindrica. similarly, kumar et al. (2011) observed epithelial cell disruption, hypertrophy, and hyperplasia in l. marginalis after exposure to dimethoate at 27.3 ppm. kamble et al. (2015) reported necrosis, hyperplasia, damage to the table 1. treated groups exposed to 0.07 ppm imidacloprid (a. there are significant differences (p<0.05) between the control and treated groups). tbars (nmol/mg protein) tissue exposed gill foot mantle muscle hpt control 1.82±0.10 2.51±0.62 2.44±0.58 1.75±0.20 1.60±0.06 treated group 15.11±0.84a 19.56±1.90a 11.44±1.17a 13.89±1.34a 8.66±0.88a 128 ahire and dethe / evaluation of imidacloprid-induced toxicity and lipid peroxidation in the freshwater bivalve basement membrane, and vacuolization in l. corrianus during three different seasons after exposure to thiodan. in the present study, histopathological alterations were observed in the hepatopancreas of the test organism following exposure to imidacloprid. even at lower concentrations, imidacloprid is capable of inducing lipid peroxidation. mundhe and pandit (2014) observed an increase in lipid peroxidation in gills compared to other tissues upon exposure to monocrotophos. this finding is consistent with lackner's (1998) assertion that increased lipid peroxidation after pesticide exposure suggests the involvement of free radical-induced oxidative cell injury, indicating the toxicity of pesticides. similarly, köpriicü et al. (2010) reported increased lipid peroxidation in mussels after exposure to pesticides, which further supports the notion that pesticide exposure induces oxidative stress in organisms. our study reveals that imidacloprid induces significant histopathological changes in the gill, mantle, and digestive glands of l. marginalis, indicating its toxicity towards these vital organs. imidacloprid, a neonicotinoid pesticide, is extensively utilized in agriculture to combat pests. nonetheless, the indiscriminate use of this pesticide has led to environmental pollution, which could have adverse effects on non-targeted organisms. hence, it is crucial to take into account the ecological consequences of pesticide usage when devising agricultural practices acknowledgment the author is thankful to p.r. bhamre, and a.e. desai, for their encouragement and providing laboratory facilities. references agrawal a., ravi s.p., bechan s. 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(2014) 2(1): 1-8 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article growth, reproduction and feeding biology of an endemic sucker catfish, glyptothorax silviae (coad, 1981) (actinopterygii: sisoridae), in the maroon river, iran mohammad amini chermahini*,1samad bahrami, farideh bakhshi, amir hossein tahmasebi, fatemeh shahrani department of fisheries, college of natural resources, behbahan khatam alanbia university of technology, behbahan, khuzestan. article history: received 30 october 2013 accepted 31 january 2014 available online 2 5 february 2014 keywords: length-weight relationship fecundity gonadosomatic index diet maroon river abstract: this study was carried out to provide some information on the biological features of the endemic sucker catfish, glyptothorax silviae, from the maroon river in the tigris basin, iran. samples were collected at monthly intervals throughout the year and 277 individuals of g. silviae were caught. the mean total length and weight were 8.1 ± 1.7 (±sd) cm and 5.8 ± 3.5 (±sd) g and maximum length and weight were 11.9 cm and 17.4 g, respectively. length-weight relationship of the total specimens was calculated as w = 0.006 tl3.205, (r2 = 0.956) indicating allometric growth pattern and was not significantly different between males and females. the overall condition factor was 0.62. the sex-ratio (1.23m:1f) was not significantly different from the expected 1:1 ratio. glyptothorax silviae reproduces during june-september. absolute and relative fecundity was 1129 eggs/fish and 105 eggs/g body weight, respectively. the egg size ranged from 0.994 to 1.76 mm with a mean value of 1.29 ± 0.171 (±sd) mm. the highest values of gonadosomatic index were observed in june-july. the gut content analysis revealed that g. silviae feeds only on aquatic insects. introduction two species of the genus glyptothorax are reported from iran (coad, 2013): glyptothorax silviae coad, 1981 and g. kurdistanicus (berg, 1931). they are rheophilic fish in fast flowing streams, being able to attach themselves on hard substrates to resist strong currents (ng and rachmatika, 2005). glyptothorax silviae is only found in rivers draining to the persian gulf in south western iran (coad, 2013). abdoli (2000) has also reported this species in the karun and middle to lower khersan, and middle to lower dez rivers in the tigris basin and in the mond and shur rivers of the bushehr basin. glyptothorax silviae is an endemic species in south western iran and, therefore, conservation of gene pool of this fish is very important. for this purpose, basic biological information on the fish is necessary. * corresponding author: mohammad amini chermahini e-mail address: mamini57@yahoo.com tel: +989132078479 some information on the biology of g. silviae is already available (coad, 1981; esmaeili and ebrahimi, 2006; esmaeili et al., 2009; coad, 2013), but comprehensive data for this species are still missing. the principal aim of this paper was to provide an update of data on some biological features of this endemic catfish, including growth (length frequency distribution, length-weight relationship, fulton condition factor), reproduction (sex ratio, fecundity, oocytes diameter, gonadosomatic index), and feeding (relative length of gut, gastrosomatic and hepatosomatic indices). materials and methods study area and sampling: the present study was carried out in the maroon river, which is situated mainly in khuzestan province, and passes through 2 amini chermahini et al./ int. j. aquat. biol. (2014) 2(1): 1-8 north of behbahan city (fig. 1). the length of the maroon river is about 421 km, with an average slope of 2% in mountain area and less than 1% in plain area. the mean discharge of the river is about 48 m3/s. because of damming, its discharge is almost constant, but it slightly increases in early autumn. sampling were performed in two locations adjacent to behbahan city, emamreza village (30º40'11.12"n, 50º18'13.27"e) and kharestan (30º39'21.32"n, 50º11'39.56"e). sampling were carried out once per month in the late mornings using a small seine (4-6 m long and 1.5 m depth) with a mesh size of about 2 mm, between october 2010 and september 2011. this species is a weak swimmer and swept away when detached from gravels. the seine was placed across the flood-ways and narrow parts of river, to close entire width of water and fish were driven away into the net by disturbing the substrate. sometimes, in shallow and clear waters, larger fish were caught by small scoop net or by hand, when attached to coarse gravels. biometry and data analysis: a total of 277 specimens were collected, transported to laboratory freshly, and all measurements were performed at the same day. the recorded biometric characteristics and measurements were total length (tl), fork length (fl), standard length (sl), gut length (gl), body weight (w), gonad weight (gw), hepatic weight (hw), and gut weight (guw) with its contents. all lengths were measured to the nearest 0.05 mm using digital calipers and were rounded to the nearest 0.1 cm, whereas weights were recorded with an electronic balance to the nearest 0.01 g. in addition, gut contents were identified. geographical locations of sampling areas were registered to the nearest 5 m using a gps (model garmin). the relationships between tl-fl and tl-sl were calculated. the total length-weight relationship (lwr) was established by the exponential regression equation, w = alb, where w is the weight in g, l is the total length (tl) in cm, a and b are the parameters to be established (esmaeili and ebrahimi, 2006); a is a coefficient related to body form and b is an exponent indicating isometric growth when equal to 3 and indicating allometric growth when significantly different from 3. fulton condition factor (cf) was calculated using cf = (w / tlx) × 100 (johari et al., 2009), where x = 3.2, because exponent b in lwr equation was significantly different from 3. sex was determined by visual observation of the gonads (n = 241). to estimate absolute fecundity (af), ovaries of 16 ripe females caught in june and july were removed and after weighing, placed in gilson’s fluid for 3-4 days to harden eggs and facilitate the separation and counting the eggs. the number of eggs was estimated using the gravimetric method. pieces of approximately 0.01 g were removed from the anterior, middle and posterior parts of each ovarian lobe. the eggs in each piece were counted under a binocular microscope. the af was calculated as the proportion of eggs in the sample to the weight of whole ovary. relative fecundity (rf) was calculated as rf = af / w, where w is the body weight. three samples of each ovary (about 20 ova from each sample) from 16 ripe females caught in june and july were chosen randomly to measure the egg diameter. the gonadosomatic index, gsi = gonad weight × 100 / body weight, was calculated for each fish and all values averaged for each month. guts were stored in 4% formaldehyde and gut contents were examined monthly. the primary food items were determined using a binocular figure 1. sampling location of glyptothorax silviae in iran (1 = emamreza and 2 = kharestan). 3 amini chermahini et al./ int. j. aquat. biol. (2014) 2(1): 1-8 microscope. the formula for relative length of gut, rlg = gl / tl, where gl is the gut length and tl the total fish length, was used to indicate the feeding habits. the gastrosomatic index was calculated using gi = guw × 100 / w, where guw is the gut weight and w the body weight for each specimen and averaged for each month to determine the degree of feeding intensity (biswas, 1993). the hepatosomatic index was calculated as hsi = hw × 100 / w, where hw is the hepatic weight. the significance of lwr regression was assessed using anova (esmaeili and ebrahimi, 2006). to examine significant differences in b values of lwr between males and females, ancova was performed (abdoli et al., 2008; patimar et al., 2010). t-test (ts = b – 3 / se), where se = standard error, was used to examine significant differences between b exponent and 3 (morey et al., 2003; sangun et al., 2007). comparison of cf and gsi of males and females was carried out using a paired t-test. the overall ratio of males and females was evaluated using chi-square test (patimar et al., 2010). statistical analysis of data was carried out using microsoft excel 2003 and spss 16.0 software package at a significance level of 0.05. results size, growth and sex ratio: biometric results of the 277 g. silviae specimens (133 male, 108 female) are summarized in table 1. length frequency distributions of males and females are shown in figure 2. the specimens in the 8.5-9 cm length class had the highest frequency. the relationship between tl-fl and tl-sl were calculated as fl = 0.888 tl + 0.094 (r2 = 0.98) and sl = 0.823 tl – 0.06 (r2 = 0.97), respectively. the lwr was w = 0.006 tl3.205 (r2 = 0.956) (fig. 3) and w = 0.008 fl3.206 (r2 = 0.948). based on the anova, weight and length were significantly correlated (p≤0.05). the lwr of males and females were wm = 0.005 tl3.240 (r² = 0.947) and wf = 0.005 tl3.265 (r² = 0.947), respectively. there was no significant difference between lwr of males and females (ancova, p>0.05). however, based on the t-test analyses, exponent b was significantly different from 3 (p≤0.05). the condition factor parameter sex min max average ± sd total length (cm) total 3.4 11.9 8.1 ± 1.73 male 4.1 10.9 8.3 ± 1.52 female 3.8 11.9 8.3 ± 1.65 fork length (cm) total 3.1 10.6 7.3 ± 1.55 male 3.7 9.9 7.4 ± 1.36 female 3.6 10.6 7.5 ± 1.48 standard length (cm) total 2.7 9.6 6.6 ± 1.45 male 3.2 9.3 6.7 ± 1.28 female 3.3 9.6 6.8 ± 1.38 weight (g) total 0.31 17.39 5.83 ± 3.505 male 0.65 16.06 5.99 ± 3.191 female 0.53 17.39 6.35 ± 3.782 condition factor total 0.43 0.95 0.62 ± 0.104 male 0.44 0.93 0.62 ± 0.101 female 0.43 0.95 0.62 ± 0.107 table 1. length, weight, and cf of the 277 glyptothorax silviae specimens captured in the maroon river figure 2. length frequency distribution of glyptothorax silviae from the maroon river, iran. 4 amini chermahini et al./ int. j. aquat. biol. (2014) 2(1): 1-8 (table 1, fig. 4), was not significantly different in males and females (p>0.05). sex determination was carried out for 241 specimens (including 133 males, 108 females and 36 immature). the overall ratio of males to females was 1.2m:1f, but was not significantly different (p>0.05) from the expected 1:1 ratio (table 2). reproduction: the maximum value of 1165 eggs was observed in the largest fish weighing 17.39 g and the minimum value of 1053 eggs was recorded in the fish weighing 7.45 g (1129 ± 35.6 eggs/fish). relative fecundity fluctuated from 67 to 141 eggs/g (105 ± 32.25 eggs/g). the egg size ranged from 0.99 to 1.76 mm with a mean value of 1.29 ± 0.171 (±sd) mm. gsi varied from 0.54 to 3.15 (with a mean value of 1.64 ± 0.858) and from 0.75 to 13.17 (6.14 ± 4.772) for males and females, respectively (fig. 5). the highest values of gsi were observed in junejuly. gsi values of males were lower than for females (p>0.05). all ova of each female were at the same developmental stage and had uniform size. feeding: the primary food items in all months were aquatic insects such as plecoptera (perlodidae) and ephemeroptera (baetidae and potamanthidae). in addition, other orders were also identified in the river substrate such as: plecoptera (luctridae), ephemeroptera (caenidae and ephemerellidae), diptera (chironomidae, simuliidae, tabanidae and tipulidae), hemiptera (veliidae and notonectidae), odonata (cordulegastridae) and trichoptera (hydropsychidae). the remains of crustaceans and molluscs (snail) observed in river, were not identified in guts. contents of the alimentary canal of small, medium and large specimens were compared, and there were not significant differences between them. furthermore, different developmental figure 4. fulton condition factor of males and females of glyptothorax silviae from the maroon river, iran. figure 3. length–weight relation of glyptothorax silviae from the maroon river, iran. month no. male no. female season male:female ratio χ2 value apr 7 3 spring 1.4:1 0.862 may 7 5 jun 3 4 jul 7 3 summer 1.7:1 3.449 aug 10 8 sep 14 7 oct 3 5 autumn 1.3:1 0.714 nov 5 3 dec 12 7 jan 31 25 winter 1.0:1 0.031 feb 30 27 mar 4 11 total 133 108 1.2:1 2.593 table 2. sex ratio in glyptothorax silviae during different seasons 5 amini chermahini et al./ int. j. aquat. biol. (2014) 2(1): 1-8 stages (pupa and nymph) of aquatic insects were found in different seasons. relative length of gut (rlg) was 0.66 ± 0.123 in average. the severity of feeding based on the gi in different months is shown in figure 6. based on the gi, the greatest intensity of feeding was in january-february and august-september, while the least intensity was in april-may and october. the hsi for g. silviae was calculated over a period of 12 months and is shown in figure 7. the lowest values of hsi were observed in novemberdecember, and the highest in may-june. discussion size, growth and sex ratio: the highest tl observed in g. silviae from the maroon river was 11.9 cm. coad (2013) reported the maximum of sl as 13.5 cm (16.47 cm tl, employing sl-tl relationships). esmaeili and ebrahimi (2006) indicated the maximum tl of g. silviae as 11.85cm (n = 10). their size distribution pattern is very close to the results obtained in this study. however, they did not present any information about the location and physicochemical properties of the sampling area. the variations in maximum size (i.e., lengths or weights) among different populations of the same species could be due to different exploitation patterns and/or ecological condition. in this respect, as far as the fish is not either a commercially important species or a sport fish, the ecological conditions seem to be the most important factor affecting the size and growth of this species. the most frequent range of lengths for all specimens was 8-9.5 cm and specimens were more frequent in 8.519 length class. determining the sex of the fish smaller than about 4.5 cm was impossible using visual method. as a result, distribution of length classes of total specimens was different from distribution of length classes of males and females. in terms of growth type, the results showed that g. silviae had an allometric growth. therefore, this species become more rotund as their length increase. esmaeili and ebrahimi (2006) demonstrated a positive b value for total length and negative b value for fork length (b = 3.102 and 2.975, respectively, n = 10). but in the present study, b value was positive for both tl and fl. according to strong relationship between tl-fl (r2 = 0.98), positive b value for both tl and fl is more expectable. variation in b exponent could be attributable to fish response to different ecological condition (sangun et al., 2007; figure 6. monthly changes of gastrosomatic index of glyptothorax silviae from the maroon river, iran. figure 5. monthly gonadosomatic index of males and females of glyptothorax silviae from the maroon river, iran. figure 7. monthly changes of hepatosomatic index of glyptothorax silviae from the maroon river, iran. 6 amini chermahini et al./ int. j. aquat. biol. (2014) 2(1): 1-8 patimar, 2008). as mentioned previously, esmaeili and ebrahimi (2006) did not present any information about the location and physicochemical properties of sampling area, and furthermore they have calculated lwr using limited number of samples (n = 10). in the present study, there was no significant difference between males and females in lwr. in addition, cf was similar for both sexes. therefore they had similar growth type and fish condition. however, cf of females was higher than those of males in reproduction season. it suggests that females are more rotund in this period because of accumulating yolk in ovaries. in the present study, overall sex ratio and sex ratio in various seasons showed different values, but they were not significantly different from 1:1 (p>0.05). however, male:female ratio was at the maximum value in reproduction season (summer) and minimum value in winter. the overall sex ratio was different compared to g. madraspatanum in india (1m:1.24f) (dobriyal and singh, 1993). in g. silviae, ratio of males was higher than females in all seasons, but in g. madraspatanum ratio of females was higher than males. reproduction: this is the first study concerning reproductive biology of g. silviae. the study of reproductive biology is useful in various applied aspects of fisheries. the process of accumulating yolk in the ovaries of the females can be determined partly by tracing the changes in the gonadosomatic index. in species which spawn in late spring and in summer such as sisorid catfish, the index remains low in winter and then rises sharply just before the spawn (wootton, 1979; rinchard and kestemont, 1996; mousavi-sabet, 2011). a rapid increase in the weight of ovaries takes place when the temperature rises and increasing amounts of food are consumed (wootton, 1979). males had a lower gsi, because egg is much larger than sperm and it includes energy for further development of embryo. in addition, hsi and cf of males were higher than females before reproduction season. but as stated earlier, growth parameters of both sexes were not significantly different. based on gsi, reproduction of the endemic sucker catfish starts in june and lasts to september. a similar time of spawning was observed for g. madraspatanum (dobriyal and singh, 1993) and g. poonaensis (dahanukar et al., 2011) from india. glyptothorax silviae has a relatively protracted spawning period. in this study, histological examination was not performed. in addition, measuring the diameter of ova was carried out only in the middle of reproductive season, and in that period the ova have approximately the same size. according to rinchard and kestemont (1996), this fish probably is a batch spawner. however, dobriyal and singh (1993) indicated that g. madraspatanum spawns for a limited period (july-august) with a single batch of developing eggs showing a single spawning frequency. considering the climate of this region, it is expected that g. silviae to have a spawning pattern such as g. madraspatanum. however, damming as well as irregular release of dam water could influence mean temperature and sexual maturation of this species in the maroon river. it seems necessary to compare biological characteristics of g. silviae in different rivers of the region, for example kheyrabad river, in the vicinity of behbahan city or other hillstreams of karun basin. fecundity was relatively low (af = 1129 eggs/fish, rf = 105 eggs/g), maybe due to relatively large egg size of this species (1.29 ± 0.171 (±sd) mm). there is no any information about fecundity of g. silviae. however, dobriyal and singh (1993) reported a total length ranging from 12.1 to 15.2 cm and fecundity ranging from 1640 to 6830 eggs/fish for g. madraspatanum. these results are significantly different from those obtained in the present study. according to their study, g. madraspatanum with similar size to g. silviae has higher af. but they indicated that average relative fecundity was 122.5 eggs/g. in this particular case their results are close to ours. dobriyal and singh (1993) found that fecundity correlates closely with size. but further investigation is required with more sample size, which makes it possible to analyze the correlations between the fecundity and length and weight. feeding: investigating gut contents revealed that this 7 amini chermahini et al./ int. j. aquat. biol. (2014) 2(1): 1-8 species is carnivorous. results of rlg confirmed this hypothesis. rlg was 0.66, indicating that g. silviae feeds only on animal materials. there were no significant differences between gut contents of different fish sizes. guts of the smallest and biggest specimens were examined, and the only food items were aquatic insects in different sizes and developmental stages such as pupa and nymph. it is probable that in addition to plecoptera and ephemeroptera, some other insects have been consumed. dahanukar et al. (2011) reported that g. poonaensis feeds on benthic microinvertebrates such as freshwater prawns, maxillopod crustaceans (branchiura) and odonata nymph. changes in gi showed that feeding was the highest in winter and summer and the lowest in spring, late summer and autumn. it seems that increasing and decreasing of this index is related to water temperature and reproduction cycle. it was increased immediately after reproduction season to recover energy lost during this season (johari et al., 2009) and in the winter due to low temperature, foods remains longer in the gut. changes in hsi approved results of gi. hsi decreased in cold season due to decrease of metabolism and increase in spring and summer, coincided with increasing water temperature. likewise, hsi variations followed gsi variations. in fact, an increase in hsi is highly correlated with energy mobilization for reproduction. in conclusion, the present study shows that g. silviae has an allometric growth, and is a carnivorous species that feeds only on aquatic insects. this species reproduces between june-september. changes in gi and hsi show that feeding is correlated with water temperature and reproduction cycle. more research is needed to determine age structure of populations of this species and further examinations to investigate correlation between fecundity and size. this information will be useful for conservation of this endemic sucker catfish. acknowledgements we wish to thank dr. brian w. coad, dr. rasool ghorbani, dr. hamed mousavi-sabet, mrs. sara nikoo and samira nazemroaya, mr. saeed asadollah and hossein madadi. references abdoli a. 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(2022) 10(4): 273-279 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article evaluation of polyethylene microplastic bio-accumulation in hepatopancreas, intestine and hemolymph of freshwater crayfish, astacus leptodactylus amir zeidi1, mohammad reza rezaei*1, mohammad hossein sayadi1, amin gholamhosseini2, mahdi banaee3 1department of environmental engineering, faculty of natural resources and environment, university of birjand, birjand, iran. 2division of aquatic animal health and diseases, department of clinical sciences, school of veterinary medicine, shiraz university, shiraz, iran. 3aquaculture department, faculty of natural resources and the environment, behbahan khatam alanbia university of technology, behbahan, iran. s article history: received 8 may 2022 accepted 11 august 2022 available online 2 5 august 2022 keywords: crustaceans microplastics ftir bio-concentration abstract: microplastics (mps) are one of the biggest environmental problems threatening aquatic life. the accumulation of mps in the body of aquatic animals can play a role in transferring these pollutants into the food chain. these pollutants can significantly affect the physiology of aquatic animals. in this study, the bioaccumulation capability of mps in the body of freshwater crayfish, astacus leptodactylus has been evaluated. for this purpose, crayfish were exposed to 0, 500, and 1000 µg l-1 of polyethylene mps (pe-mps) for 28 days. then, the accumulation of mps in hemolymph, hepatopancreas, and intestine of crabs was investigated by fourier transform infrared spectroscopy (ftir). bioaccumulation of pe-mps in the hemolymph, hepatopancreas, and intestines was observed in the crayfish exposed to pe-mps. this study showed that ftir is a suitable method for identifying and measuring mps in aquatic organisms. introduction the increase in the global production of plastics has caused a large amount of plastic waste to be released into the environment every day. wastes and plastic wastes eventually enter aquatic ecosystems through washing or sewage. therefore, aquatic ecosystems, especially seas and oceans, are the centers of the accumulation of plastic waste in the world. plastic waste's physical and chemical decomposition causes the larger pieces to be broken into smaller pieces (chamas et al., 2020). from this point of view, microplastics (mps) of different sizes are one of the most obvious plastic pollutants in aquatic ecosystems (guzzetti et al., 2018; alimba and faggio, 2019; strungaru et al., 2019; prokić et al., 2021). mps can affect biological dynamics and biodiversity in aquatic ecosystems. the change in physical and chemical characteristics and the release of plasticizers from mps have caused an increase in their risk compared to virgin plastic polymers (karami et al., 2016; liu et al., 2020). in recent years, the various types of mps, including polystyrene (ps), polypropylene (pp), *correspondence: mohammad reza rezaei doi: https://doi.org/10.22034/ijab.v10i4.1661 e-mail: mrrezaei@birjand.ac.ir polyethylene (pe), polyethylene terephthalate (pet), polyvinyl chloride (pvc), and polyamide (pa) in different forms, have been reported in aquatic ecosystems (erni-cassola et al., 2019; schwarz et al., 2019). polyethylene (pe) has been classified as a major source of mps in aquatic and terrestrial environments (wang et al., 2018; sun et al., 2021). despite the variety of available polymers, about 45% of global production is made with polyethylene. mps can enter the body of aquatic animals through the digestive or respiratory system and are distributed in the body through the blood (banaee et al., 2021; dey et al., 2021). the exposure of aquatic organisms to plastic waste can have various consequences (paulpont et al., 2016; chen et al., 2020; banihashemi et al., 2022). metabolic damage, oxidative stress, dysfunction of the immune system (espinosa et al., 2017, 2019; tang et al., 2018; banaee et al., 2019b;) and change in intestinal microbial diversity (lu et al., 2018), growth inhibition (besseling et al., 2014; au et al., 2015), reduced growth (della torre et al., 2014), reduced feeding activity (de sá et al., 2015) and 274 zeidi et al./ polyethylene microplastic bio-accumulation in astacus leptodactylus abnormal behavior (rist et al., 2016), physical damage in the digestive system (scratching, perforation, and obstruction), hepatotoxicity (nematdoost haghi and banaee, 2017; huang et al., 2021) and bioaccumulation of mps in various tissues (dey et al., 2021) have been reported in aquatic animals exposed to mps. crustaceans, as aquatic scavenger organisms, may expose to mps. thus, these animals are an excellent indicator to assay mps effects. due to its feeding habits, the freshwater crayfish is one of the best biological indicators for monitoring the pollution of aquatic ecosystems (hong et al., 2018; banaee et al., 2019a, 2020). therefore, the biological response of these crustaceans to environmental pollution can reflect the health status of aquatic ecosystems. narrow-clawed crayfish, astacus leptodactylus (astacidae: malacostraca), is a freshwater crustacean found naturally and widely in some lakes, pools, and rivers in northwestern iran. in recent decades, this species has been introduced to many dams and internal lakes of iran. therefore, a. leptodactylus was selected as a model organism in this study and exposed to different concentrations of mps to understand the effect of mps on it. materials and methods this study was conducted from september to december 2021 at the department of aquatic health and diseases, faculty of veterinary medicine, shiraz university, iran. the birjand and shiraz universities' animal ethics committee approved all experimental procedures. freshwater crayfish, a. leptodactylus of both sexes weighing 42.11±0.31 cm and length 10±6.23 g, were caught from local waters (heft brom, shiraz) and transported to the laboratory. before starting the experiment, the crabs were adapted to the laboratory conditions (15±2°c, ph 7.2±0.3, with dissolved oxygen 8.2±0.6 mg/l, and under a photoperiod (14 light: 10 dark) cycle, electrical conductivity 693.85±174 μs cm and salinity 0.3±0.102 g/l) for two weeks. during the acclimatization period, the crayfish were fed formulated shrimp feed (beyza feed company, shiraz, iran: 45-55% protein, 10-11% lipid, 20-30% carbohydrate, 1.5-2% fiber), twice per day. the surplus food and fecal matter were removed from each aquarium, and water was renewed daily by adding fresh water. ninety adult crayfish were randomly introduced to nine aquariums (10 crayfish per aquarium) to carry out three experimental treatments (with three independent replicas). the crayfish were divided into three experimental groups and exposed to 0.0, 500, and 1000 µg.l-1 pe-mps for 28 days. during the experimental periods, crayfish were fed two times daily, while crayfish were starved for one day before taking the sample. after the experimental procedure, 12 crayfish from each treatment were sampled and anesthetized on ice. hemolymph was acquired from the sternal artery by a sterile syringe containing alsever's solution adjusted for a. leptodactylus as the anticoagulant (banaee et al., 2019). after the autopsy, the hepatopancreas of the crayfish was separated and placed in liquid nitrogen. next, the hemolymph and hepatopancreas samples were then dried in a freezer dryer. subsequently, polyethylene (mps) was detected in the tissues by ftir spectrometer (bruker). ftir could detect the high density of polyethylene in the 1445-1650 wavenumber (cm-1) (li et al., 2022). results ftir results are presented in figures 1-3. the ftir spectrum showed the presence of polyethylene mps in the hemolymph and hepatopancreas of crayfish. the change in ftir peaks in the wavenumber range of 717, 1600, 1680, and 2928 (cm-1) indicated the presence of polyethylene mps. furthermore, these results prove that mps could penetrate other vital organs such as the hepatopancreas after entering the hemolymph. the difference between the ftir peaks in the experimental groups treated with different amounts of mps and the standard may indicate the existence of differences in the concentration of mps in hemolymph and hepatopancreas. ftir results are presented in figures 1-3. the ftir spectrum showed the presence of polyethylene 275 int. j. aquat. biol. (2022) 10(4): 273-279 mps in the hemolymph and hepatopancreas of crayfish. the change in ftir peaks in the wavenumber range of 717, 1600, 1680, and 2928 (cm1) indicated the presence of polyethylene mps. furthermore, these results prove that mps could penetrate other vital organs such as the hepatopancreas after entering the hemolymph. the difference between the ftir peaks in the experimental groups treated with different amounts of mps and the standard may indicate the existence of differences in the concentration of mps in hemolymph and hepatopancreas. discussions the spectra of the polyethylene obtained in disk form are shown in figures 1-3. the results of this study showed that exposure of crayfish to mps for 28 days led to the accumulation of mps in the hemolymph, intestine and hepatopancreas (wang et al., 2020; kim et al., 2021) fourier-transform infrared spectroscopy (ftir) is a well-known analytical tool that can detect organic substances' functional groups and molecular structure (rytwo et al., 2015; lohumi et al., 2017; litvak et al., 2018). li et al. (2022) characterized the ftir bands as the functional groups related to polyethylene (table 1). the band i and ii at 721-993 cm-1, indicated the existence of aromatics derivatives. the band iii and iv at 950-1300 cm-1 demonstrated phenols, alcohols, and ethers groups. the results showed a significant transmittance pattern in the region of 1455-1646 cm -1 (band v and vi) (gulmine et al., 2002; xu et al., 2019; daniel et al., 2020; daniel et al., 2021). these peaks belong to the light aromatics and aromatic ring (aryl). the band vii at 2919 cm-1 showed methylene c-h asymmetric stretch groups. different chemical bonds and structures of various plastics show unique spectra through ftir spectroscopy. since mps are composed of carbonbased functional groups linked by covalent bonds, these analytical tools can identify all types of plastics (mecozzi et al., 2016). figure 1. fourier infrared spectroscopic spectrum related to hemolymph (a) samples to detect pe-mps. figure 2. fourier infrared spectroscopic spectrum related to hepatopancreas (b) samples to detect pe-mps. figure 3. fourier infrared spectroscopic spectrum related to intestine (c) samples to detect pe-mps. 276 zeidi et al./ polyethylene microplastic bio-accumulation in astacus leptodactylus the bio-accumulation of mps was reported in the visceral mass and haemolymph of the bivalve (amarilladesma mactroides) and mussels (brachidontes. rodriguezii) (truchet et al., 2021). also, fibre of mps was detected in the whole body of crangon crangon (devriese et al., 2015), pandalus borealis (fang et al., 2018), paratya australiensis (nan et al., 2020), fenneropenaeus indicus (daniel et al., 2020), and in the stomach and foregut of plesionika narval (bordbar et al., 2018), c. crangon (mcgoran et al., 2018), and macrobrachium rosenbergii (li et al., 2021). bio-accumulation of mps was reported in the digestive system of crustaceans (mcgoran et al., 2018; bordbar et al., 2018; carrerascolom et al., 2018), the whole body (daniel et al., 2020), visceral organs (carreras-colom et al., 2020), soft tissue (nakao et al., 2020; daniel et al., 2021), gills (zhang et al., 2019; not et al., 2020), hepatopancreas (martinelli et al., 2021; zhang et al., 2022), and hemolymph (zhang et al, 2022). as conclusion, polyethylene mps can be absorbed in the gills and intestines and enter the hemolymph. then mps are distributed through hemolymph in different body tissues and may accumulate. therefore, the measurement of pe-mps by the fourier transform infrared spectroscopy method of chemical imaging, i.e., ftir, is useful for mps to be automatically analyzed. references alimba c.g., faggio c. 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(2022). accumulation of polyethylene microplastics induces oxidative stress, microbiome dysbiosis and immunoregulation in crayfish. fish and shellfish immunology, 125: 276-284. international journal of aquatic biology (2013) 1(6): 273-280 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article effects of the aromatase inhibitore letrozole on serum immunoglobulin and lysozyme levels in immunized rainbow trout (oncorhynchus mykiss walbaum) females paria akbary1, alireza mirvaghefi*1, mostafa akhlaghi2, bagher majazi amiri1, mohammad saeid fereidouni2 1department of fisheries and environmental sciences, faculty of natural resources, college of agriculture and natural resources, university of tehran, karaj, iran. 2department of aquatic animal health unit, school of veterinary medicine, shiraz university, shiraz, iran. article history: received 1 november 2013 accepted 12 december 2013 available online 2 5 december 2013 keywords: letrozole lysozyme 17-βestradiol (e2) immunoglobulin oncorhynchus mykiss abstract: letrozole is a synthetic aromatase inhibitor and interfere in the committed step in the synthesis of endogenous estrogens from androgens. also estrogens regulate the immune system in teleost. changes of 17βesrtradiol (e2), serum immunoglobulin and lysozyme levels were measured using a method based on the ability of lysozyme to lyse the bacterium micrococcus lysodeikticus, enzyme-linked immunosorbent assay (elisa) and elisa respectively. twelve broodstocks were injected weekly with 2.5 mg kg-1 letrozole (an endocrine disrupter component) two months before spawning season and vaccinated intraperitoneally (i.p) with a bacterin (inactivated l. garviae) one month before spawning. twelve broodstocks for vaccination and twelve female rainbow trout as control group were also immiunised (i.p) with the bacterin and injected (i.p) with pbs, respectively. in the group received 2.5 mg ai kg-1 per week, serum e2 levels were significantly lower than that of other groups. total immunoglobulin level and lysozyme activity were significantly higher in the parents received 2.5 mg kg-1 per week and were immunized with 10-9 cells ml-1 lactococcus garvieae compared to the group which immunized with l. garvieae and the control (nonimmunized). the present study, suggests that aromatase inhibitors such as letrozole may be a potential tool to regulate the synthesis of e2, is involved in the hormoneimmune system interaction in rainbow trout. introduction letrozole (cgs 20264), with commonly used brand name femara, is a non-steroidal trizole derivation and one of the most potent aromatase inhibitors yet developed (smith, 1999). as such, aromatase inhibitors (ais) have been used to treat metastatic breast cancer for over 25 years (howell and buzdar, 2005). letrozole has a potential to prevent the conversion of androgenic steroids to estrogens (haynes et al., 2003; smith, 1999). letrozole is capable of inhibiting aromatase 98-99% and reducing serum concentrations of estrone and e2 beyond the limit of detection in patients (smith, 1999). the potential of aromatase inhibitors to adversely affect sexual differentiation and * corresponding author: alireza mirvaghefi e-mail address: avaghefi@ut.ac.ir reproduction in fish was demonstrated in a study by piferrer et al. (1994), and similar results have been reported by afonso et al. (1999, 2000), ankley et al. (2002) and panter et al. (2004). during the last decade, a number of studies have shown that in addition to their classically described reproductive functions, estrogens and androgens also regulate the immune system in teleosts (swain and nayak, 2009). in aquatic environments, fishes are often used as bioindicators for environmental contaminants. many environmental contaminants (for example letrozole), interfere with hormonal regulation in fish and are therefore known as endocrine disruptors (shilling et al., 1999). the studies on reproductive mailto:avaghefi@ut.ac.ir 274 akbary et al/ international journal of aquatic biology (2013) 1(6): 273-280 immune interactions in fishes indicate that sexual maturation (i.e., gonadal maturity and reproductive activities) can potentially influence both the innate and adaptive immune responses (harris and bird, 2000; milla et al., 2011). the reproductive cycle of fishes is marked by seasonal variation in plasma sex-steroids and changes in immune parameters which render them more sensitive to the presence of pathogens (hou, 1998). furthermore, immune biomarkers may prove useful for the identification of contamination in the wild (milla et al., 2011) some studies, have shown that the increase of 17-βestradiol during the spawning season affects the lymphocyte proliferation and igm level, and antibodyproducing cells in several fish species like oncorhynchus mykiss, salmo trutta, chinook salmon, oncorhynchus tshawytscha, gold fish, carassius auratus (hou, 1998; slater and schreck, 1993; suzuki et al., 1997; thilagam et al., 2009). for example, in o. mykiss (hou et al., 1999; lei et al., 2010) and gilthead seabream (cuasco et al., 2008), the rise of sex hormones can suppress the plasma igm and igm secreting cells during spawning period leading to immunosuppressive condition. however the suppressive effects of e2 on igm production are not observed in carp (saha et al., 2004) and zebra fish (danio rerio) (lei et al., 2010). elevation of sex steroids can make fish more vulnerable to several microbial infections during the spawning season and subsequent mortality following the spawning phase. therefore the overall health and immune status of brood fish is very important not only for breeding performances but also for the health condition of offspring (lei et al., 2010). in this paper, we investigated the effects of endocrine disrupter component (edc) letrozole on lysozyme and igm levels after female immunization of rainbow trout. materials and methods letrozole: the non-steroidal aromatase inhibitor letrozole (cgs 20267) [1h]-4-androstron-3,17 dione, obtained from iran hormone venture pharmaceutical technology development co., ltd., iran and was dissolved in the vehicle ethanol (shilling et al., 1999) stock solution containing 2.5 mg of ai ml-1 was prepared. bacteria and bacterin (vaccine) preparation: lactococcus garvieae (eu727199) was isolated from diseased fish showing symptoms of lactococcosis which were collected from rainbow trout farms in fars province (obtained from the shiraz university, shiraz, iran). this strain was selected on the basis of its antigenic characteristics that were determined in a previous work (sharifiyazdi et al., 2010). the strain was routinely grown onto columbia sheep blood agar (oxid, madrid, spain) plates at 25 °c for 24-48 h. stock cultures were maintained fozen at -80 °c in tryptone soy broth (difco, madrid, spain) with 15% glycerol. a formaline-killed vaccine was prepared as previously described (toranzo et al., 1995; romalde et al., 2004). the selected strain of l. garvieae was grown in trypticase soy broth (tsb, difco) for 48 h. bacterial cells were killed by addition of formalin to achieve a final concentration of 0.3% and incubated for 3h at 25 °c and then 4 °c overnight. bacterial cells were collected by centrifugation at 6500 ×g for 30 min at 4 °c and washed three times with phosphate buffered saline (pbs: ph 7.4) and then were re-suspended in pbs at a final concentration of 1.2 ×108 cells ml-1. fish and rearing conditions: thirty six adult rainbow trout (o. mykiss) females were obtained in mid september 2011 from dalkhan rainbow trout hatchery farm (sepidan, west of shiraz, iran) and held outdoor in a 10 m2 concrete pond in a flowthrough water system. the specimens were kept in well-aerated water at 15.8 ± 0.5 °c, dissolved oxygen 5.5 ± 0.1 ppm and ph 7.8 ± 0.18 (mean ± sd). fish were initially weighed (854 ± 0.1 g) under anesthesia (150 ppm clove oil). after 7 days of acclimation to the condition, they were randomly divided in three groups and each group was kept in a 2 m2 concrete pond with a water depth of 50 cm in 275 akbary et al/ international journal of aquatic biology (2013) 1(6): 273-280 the same flow-through water system. during the experiment, the specimens were fed with commercial salmon food (beyza 121 feed mill (bfm) co., ltd., iran). letrozole injection of brood fish: these procedures were carried out at least 3 days after females had been transferred to two m2 concrete ponds. in midaugust 2011, twelve specimens were weekly injected as itraperitonially injection (i.p) with 2.5 mg kg-1 letrozole at the base of the right ventral fin using individual 4 ml syringes fitted with an 18.5 gauge needle. twenty four fish were injected with the vehicle ethanol only (1.0 ml kg-1 body weight). vaccination of brood fish: in mid-september, letrozole injected parents, were immunized with 1.0 ml of formalin (3%) inactivated l. garvieae (109 cells ml-1) (n=12). also twenty four fish injected only with ethanol were divided into two groups. control group (n=12) was immunized (i.p) with 1.0 ml sterile pbs (phosphatebuffered saline 0.1 m, ph 7.2) and group immunized with 1.0 ml of formalin (3%) inactivated l. garvieae (109 cells ml1. before any handling procedure, fish were anaesthetized using a solution of freshly powdered clove oil with concentration of 150 ppm. serum sampling: after injection of letrozole, blood samples (3 ml) were collected from the caudal vein at 1, 2, 4, 6, 8, 12, 16, 20, 22 days after injection with ai. serum samples were obtained from six fish per pound. after immunization blood was collected from the caudal vein of fish prior to immunization and every ten day post-injection, random serum samples were obtained from six fish per pond (non-lethal bleeding) of the immunized (the group only immunized and the group injected with 2.5 mg kg-1 letrozole and immunized) and control fish (non immunized), and allowed to clot at room temperature for 1-2 h and then at 4 °c overnight. serum was collected and divided into ependroff tubes and stored at -20 °c (salamat et al., 2012). blood was collected from the caudal vein of fish prior to immunization and every ten day post injection, random serum samples were obtained from six fish per pond (non-lethal bleeding) of the immunized and control fish, and allowed to clot at room temperature for 1-2 h and then at 4 °c overnight. sera were collected and stored at -20 °c. serum e2 levels: for steroid analysis, serum samples were extracted with alcohol ice-cold methanol, added to the serum (6:1 v/v), shaken and centrifuged (3000 g, 15 min, 4 °c). the pellet was re-extracted twice with 200 µl of methanol. supernatants were pooled, dried and reconstituted in 120 µl of potassium phosphate buffer (0.1 m, ph 7.4), then stored at -20 °c for analysis. serum e2, levels were measured by enzyme linked imunosorbent assays described by navas and segner (2000) and guzmán et al. (2008) with slight modification. in the steroid elisa, the microtiter plate provided in this kit has been pre coated with a goat anti-rabbit antibody (cusabio biotech co., ltd). fifty µl standards or samples were then added to the appropriate microtiter plate wells with a hrp-conjugated (e2) and antibody preparation specific for steroid and incubated. then substrate solutions were added to each well. the enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution and the color density was measured spectrophotometrically at a wavelength of 450 ± 2 nm. the concentration of steroid in the samples was then determined using comparison of the optical density (o.d) of the samples to the standard curve. all samples were placed in triplicate on the plates. data were expressed as ng ml-1 for serum steroid levels (navas and segner, 2000; guzmán et al., 2008). lysozyme activity: the lysozyme activity of samples was measured using a method based on the ability of lysozyme to lyse the bacterium micrococcus lysodeikticus (ellis, 1990). in a 96 well microplate, 250 μl of samples in four twofold serial dilutions in pbs (0.05m, ph 7.2) were mixed with 250 μl of 0.7 mg ml-1 suspension of m. lysodeikticus (sigma) in phosphate buffer (175 ml). the microplate was incubated at 24 °с and o.d. was read at 450 nm at 15 and 30 min. for a 275 276 akbary et al/ international journal of aquatic biology (2013) 1(6): 273-280 positive and a control, serum was replaced by the hen egg white lysozyme (serial dilutions starting at 1.6 μg ml-1) and buffer, respectively. a unit of lysozyme activity was defined as the amount of serum causing a decrease in the o.d. reading of 0.001 min. total igm: the serum samples from weekly letrozole injected immunized, only immunized and non-immunized were assessed by elisa to measure the total igm using the method of hanif et al. (2004) with slight modifications. samples were washed three times with pbs containing 0.05% tween 20 (pbs-tween) between steps. each well of microplate (falcon, usa), which was coated with 50 μl of rabbit anti-rainbow trout igm (6.6 μg ml-1) (sigma, st. louis, u.s.a), was incubated for 2 h at 37 °с. after washing, 50 μl of serum samples, serially diluted standards from 800 to 3.2 ng ml-1 and were added to the wells, and the plate was incubated overnight at 4 °с. after washing, 50 μl of goat anti-trout antibody which was conjugated with horse radish peroxidase (sigma, st. louis, mo), and diluted in pbs-tween, was added to the plate, and incubated for 2 h at 37 °с. after washing, peroxidase activity was measured by adding 50 μl of substrate solution which contained оphenylenediamine dihydrochloride (1 mg ml-1, brl, usa) and 0.04 % h2o2 in 0.1 m citrate/ 0.2 m phosphate buffer (ph 5.5). after incubation for 30 min at room temperature, the enzyme-substrate reaction was stopped by adding 25 μl of 2m h2so4 to each well and the color change was measured spectrophotometrically at a wavelength of 450 ± 2 nm. all samples were placed in triplicate on the plates and the mean and standard error (se) were calculated for each sample igm concentration. data were expressed either as mg ml-1. statistical analysis: anova was used to detect variations in igm and lysozyme parameters and all data were shown as mean ± standard error (se). unpaired t-test was also used to compare the group injected with letrozole and the control, and data were shown as mean ± standard error (se). a significant difference among the different treatments was detected by anova following by all pairwise multiple comparisons using the duncan test. statistically significant differences were determined at α=0.05 using spss for windows version 11.5 (spss, chicago, usa). results the injection procedure of letrozole was started in mid-september 2011 while all females were in prepubertal stage (two months before spawning). in the control group injected with the ethanol vehicle, serum e2 levels 22 days after injection increased (2.12 ± 0.08 ng ml-1) significantly in relation to the first blood collection. in the group injected weekly with 2.5 mg kg-1 letrozole, declined significantly one day after injection, and remained low throughout the experimental period (0.07 ± 0 ng ml-1) (fig. 1). the lysozyme activity (μg ml-1) in sera of the injected with letrozole and immunized group was higher (293.31 ± 2.78 μg ml-1) than the immunized (288.27 ± 2.86 μg ml-1) and the non-immunized (182.27 ± 1.07 μg ml-1) broodstocks (fig. 2). there was a significant difference among the groups. the immunized groups expressed more lysozyme activity than the non-immunized group. the total immunoglobulin level in the sera of the injected with letrozole and immunized broodstocks (11.06 ± 0.03mg ml-1) was higher the immunized (10.47 ± 0.29 mg ml-1) and the non-immunized (7.91 ± 0.23 mg ml-1) broodstocks (fig. 3). the figure 1. serum concentration of 17βestradiol in female rainbow trout injected or not with letrozole (ai). each data point represents the mean (± se) of six fish (n=6). 277 akbary et al/ international journal of aquatic biology (2013) 1(6): 273-280 difference was significant among the groups. the immunized groups expressed more igm level than the nonimmunized group. discussion the interactions between hypothalamic-pituitary adrenal axis and immune system in fishes are yet to be fully understood (lutton and callard, 2006). also aquaculture industry is recently facing a serious setback due to infectious diseases leading to sever economic loss (sugita et al., 2002; swain et al., 2006). lactococcosis is a bacterial disease, caused by l. garvieae that occurs in both fresh water and seawater and has been a source of major economic losses since the early seventies for the rainbow trout industry (vendrell et al., 2006; hosseini et al., 2011). the reproductive–immune interactions studied in fishes indicates that sexual maturation (i.e., gonadal maturity and reproductive activities) can potentially affect both the innate and adaptive immune responses (milla et al., 2011). therefore, reliable methods to control the onset of puberty are required (afonso et al., 1999). the present study aimed to investigate as an approach to control e2 level and gonadal maturity by a sharp decrease in aromatase activity and effects of letrozole on serum lysozyme and immunoglobulin levels in rainbow trout. in the present study, basal e2 levels of were twice as high non-injected females as injected females from 1 to 2 days after injection with letrozole and serum e2 levels were significantly lower in females injected weekly with 2.5 mg kg-1 letrozole. this suggests that letrozole, non-steroidal inhibitor, inhibits aromatase which catalyzes the conversion of androgens, androstendione and testosterone via three hydroxylation steps to estrone and estradiol (sun et al., 2007). similar results were obtained by kelloff et al. (1998) who showed a dose responsive increase in blood serum steroid in trout injected with 50 mg fadrozole kg-1 per day. shilling et al (1999) used letrozole (cgs20267) and aminoglotethimide (ag) as non-steroid and examined in vitro for activity in trout ovarian microsomes. they showed that letrozole reduced aromatase activity a maximum of 90% in dose-dependent manner. but letrozole and clorimazole fed to juvenile rainbow trout at doses up to 1000 ppm for 2 weeks were not effective in suppressing 17 β-estradiol levels (shilling et al., 1999). our in vivo data showed that the mechanism and efficiency of inhibition of letrozole are different. also the study of afonso et al. (1999) provided the first evidence that injection of the fadrozole was effective in vivo in lowering plasma e2 levels (afonso et al., 1999). sun et al. (2007) showed that letrozole for 21 days affected the reproductive, gonadal development and vitellogenin production of japanese medaka (oryzias latipes) females (sun et al., 2007). the total immunoglobulin level in the sera of the injected with letrozole and immunized broodstocks (11.06 ± 0.03mg ml-1) was higher than those of the immunized (10.47 ± 0.29 mg ml-1) and the nonfigure 2. total lysozyme level changes after immunization with l. garvieae (10-9 cells ml-1) of the rainbow trout broodstock serum (μg ml-1) .each data point represents the mean (± se.) with three replications. a significant difference among the groups was identified by the different superscript letter (p>0.05). figure 3. total igm level changes after immunization with l. garvieae (10-9 cells ml-1) of the rainbow trout broodstock serum (μg ml-1) .each data point represents the mean (± se.) with three replications. a significant difference among the groups was identified by the symbols * and ** (p<0.05). 277 278 akbary et al/ international journal of aquatic biology (2013) 1(6): 273-280 immunized (7.91 ± 0.23 mg ml-1) broodstocks. this finding is in agreement with observation in rainbow trout (hou et al., 1999), gilthead seabream (cuesta et al., 2008). sex hormones are usually elevated during spawning season of fishes and the rise of sex hormones can severely affect the immunity of fishes (harris and bird, 2000; slater and schreck, 1993). the rise of estradiol-17 β is reported to affect igm level, and antibodyproducing cells in several fish species like oncorhynchus mykiss, salmo trutta, chinook salmon, oncorhynchus tshawytscha, gold fish, carassius auratus (hou, 1998; slater and schreck, 1993; suzuki et al., 1997; thilagam et al., 2009). for example, in o. mykiss (lei et al., 2010; hou. y. et al., 1999) and gilthead seabream (cuasco et al., 2008), the rise of sex hormones can suppress the plasma igm and igm secreting cells during the spawning period leading to immunosuppressive condition. the elevation of sex steroids can prone fishes more vulnerable to several microbial infections during the spawning season and subsequent mortality following the spawning phase. we used the endocrine disrupter component (edc) letrozole for rising lysozyme and igm levels in immunized females. the significant rise in lysozyme igm levels of the broodstocks injected with letrozole and immunized and only immunized, one month before breeding was found. a set of genes related to innate immunity in vertebrates, including chemotoxin, poly saccharidebinding protein1, and antimicrobial peptide hepcidin were downregulated after exposure to estrogen (williams et al., 2007; wang et al., 2009; roberson et al., 2009). sin et al. (1994) reported antibody against ichthyophinus multifilis in oreochromis aureus and hanif et al (2004) in sparus aurata against photobacterium damsella by immunizing one moth prior to spawning. finally, the injection of letrozole with the vaccination of brood fish through bacteria (l. garvieae) has not only enhanced the specific immunity (igm) but also the non-specific factor (lysozyme). further studies are required to elucidate the modulation of different nonspecific factors following injection of aromatase inhibitors and specific immunization. hence, strategies should be taken to inject with letrozole and immunized broodstocks of rainbow trout to breeding for better health management of females. acknowledgments the authors thank the ministry of science, research and technology of iran, for financial support. references afonso l.o.b., iwama g.k., smith j., donaldson e.m. 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(2009). cloning and expression of a hepcidin gene from a marine fish (pseudosciaena crocea) and the antimicrobial activity of its synthetic peptide. peptides, 30: 638-646. williams t.d., amer m.d., george s.g., sabine v., chipman j.k. (2007). gene expression responses of european flounder (platichtys flesus) to 17beta estradiol. toxicology letters, 168: 236-248. international journal of aquatic biology (2013) 1(6): 258-265 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article risk assessment of the total mercury in golden gray mullet (liza aurata) from caspian sea seyed mehdi hosseini *1, noorollah mirghaffari1, nasrollah mahbubi sufiani2, seyed vali hosseini3, amir faraz ghasemi4 1department of environmental science, faculty of natural resources, isfahan university of technology, isfahan, iran, p.o. box 8415683111, isfahan, iran. 2department of fisheries, faculty of natural resources, isfahan university of technology, isfahan, iran, p.o. box 8415683111, isfahan, iran. 3department of fisheries, faculty of natural resources, university of tehran, karaj, iran, p.o. box: 31585-3314, karaj, iran. 4department of marine biology, faculty of marine science and oceanography, khorramshahr university of marine science and technology, p.o. box 64199-669, khorramshahr, khozestan, iran. article history: received 30 october 2013 accepted 24 november 2013 available online 2 5 december 2013 keywords: mercury liza aurata risk assessment safety caspian sea ptwi abstract: mercury is the most toxic heavy metal in the aquatic ecosystems which originates both from natural and industrial resources and is ultimately deposited in sediments as methyl mercury. this metal is quickly transferred through the food chain and accumulated in organisms. in this study, the human health risk due to consumption of golden grey mullet (liza aurata) in the caspian sea, were evaluated by measuring the concentration of mercury in muscle samples using atomic absorption spectrophotometer (perkin elmer fias-100) and cold vapor technique. a total of 60 fresh mullet samples were collected by local fisherman from 12 stations on the southern coast of caspian sea in mazandaran province situated in the north of iran. the average concentration of mercury in mullet muscle was 0.137 µg/g of fresh weight (0.432 µg/g dry weight) which was less than the allowable amount for human consumption determined by the international organizations such as united states environmental protection agency, world health organization, food and agriculture organizations and the food and drug administration. the calculations indicated that daily and weekly mercury uptake for iranian consumers is lower than the guide values (acceptable daily intake and provisional tolerable weekly intake) provided by international organizations. also, hazard quotient index was below 1 (0.35). therefore, the consumption of the mullet is not a serious threat to the consumer’s health and a consumption permitted rate of 51 g per day is recommended. introduction mercury is the most toxic heavy metal in the aquatic ecosystems which originates both from natural sources and human activities. the mercury cycle in aquatic environments has been receiving considerable attention because of the high toxicity of its compounds, accumulation of both the organic and inorganic forms of the element in organisms and their biotransformation and bio-magnification in the aquatic food chains (houserova et al., 2006). methylmercury is the main form of organic mercury found in the environment and due to its chemical persistence and lipophilicity, have a tendency to accumulate up the food chain. therefore, human * corresponding author: seyed mehdi hosseini e-mail address: hosseini.sayedmehdi@gmail.com exposure to these pollutants occurs mainly from eating seafood. in this regard, fish consumption is often considered to be a major source of intake of mercury for humans (ruelas-inzunza et al., 2008). communities that rely on fish intake for daily nutrient sustenance may be at risk from chronic, high exposure to methylmercury as well as other persistent organic pollutants (burger et al., 2007). united states environmental protection agency (usepa) classifies methylmercury as group “c” based on inadequate data in humans and increased incidence of kidney tumors in a single species and sex (usepa, 2005). methylmercury has also been shown to be a developmental toxicant, causing subtle 259 hoseini et al/ international journal of aquatic biology (2013) 1(6): 258-265 to severe neurological effects. epa considers there is sufficient evidence for methylmercury to be considered a developmental toxicant, and to be of concern for potential human germ cell mutagenicity (usepa, 2001). the caspian sea is the biggest land-locked body of water and it has five major inlet rivers but no outlets and acts as a watershed reservoir for the region (hosseini et al., 2008). the most widespread pollutants of surface waters are petroleum compounds, phenols, heavy metals and etc. from anthropogenic activities, including both land-based and offshore pollution (unep, 2008). many potentially toxic contaminants such as heavy metals released into the caspian sea are lipophilic and insoluble in water. golden mullets are an important and commercial fish in caspian sea particularly in iran. previous investigations have demonstrated the occurrence of hg in fishes from the caspian sea. however, no studies have been conducted on contamination status of mercury in marine fish mid its risk assessment on human health in caspian sea. in the present study, contamination status of hg was assessed in muscle of golden gray mullet (liza aurata) collected in the south coastal waters of the caspian sea (mazandaran, iran) and was compared with other studies and food guideline values to evaluate potential human hazard (food safety and hygiene) from fish consumption. materials and methods study area: the investigated area (35˚47΄ 36˚35΄n, 50˚34΄e) is located in the southern coastal of caspian sea, and stretch of sampling area is about 340 km. twelve sampling sites were selected according to the localization of principal sources of pollution (waste from the main urban and sewage discharge points) (fig. 1). sampling: liza aurata were caught using beach seines from february through march 2010. after biometric measurements (weight determined 500600 gr), the fish were immediately transported to the laboratory in ice box. samples of muscle (the middorsal muscles) were dissected, washed with deionized water, packed in polyethylene bags and stored at -80 ˚c until chemical analysis. chemical analysis: the samples were thawed and a weighted sample (0.500 g) of homogenized tissue was taken from each specimen. each sample was placed in a teflon digestion vessel with 5 ml mixture of nitric acid and perchloric acid (3:1 v/v). the mixture was heated to 100-150 ˚c for 45 minutes until the tissue was dissolved, and was filtered through a membrane filter. after cooling, the solution was diluted to 50 ml with deionized water. mercury was analyzed by the cold vapor technique using the perkin elmer fims-100 mercury analyzer (usepa, 1998). hg concentrations are expressed in µg/g (ppm or mg/kg) on a wet weight basis. a dorm-2 certified dogfish tissue was used as the calibration verification standard. recoveries between 90% and 110% were accepted to validate the calibration. the detection limit for hg analysis was 0.005 µg/g. exposure assessment: fish constitutes the main source of dietary exposure to mercury, which can cause adverse health effects in humans at sufficiently high exposures. the exposure assessment evaluates the potential exposure to methylmercury from the consumption of fish. potential exposure is a function of (a) the amount of fish that is consumed on a figure 1. sampling area and stations. 259 260 hoseini et al/ international journal of aquatic biology (2013) 1(6): 258-265 regular basis, and (b) the amount of methylmercury that is present in fish (bureau of nutritional sciences of canada, 2007). ratio of methylmercury to total mercury and mercury toxicity assessment: from a human health perspective, it is the amount of methylmercury rather than total mercury that is of most interest, since methylmercury is much more readily absorbed into the human bloodstream. as a result, in the absence of detailed information on mercury speciation, it is simply assumed for the purposes of health risk assessments, that 100% of total mercury is in the methylated form as methylmercury (bureau of nutritional sciences of canada, 2007). several studies have measured the actual portion of total mercury that is present in fish as methylmercury (bloom, 1991; lansens et al., 1991; bureau of nutritional sciences of canada, 2007). when making quantitative estimates of non-cancer hazards from mercury exposure, the methylmercury reference dose (rfd) (developed by usepa is used (rfd = 0.0001 mg/kg day or µg/g/day). specifically, the rfd for methylmercury is used because the sampling program was not designed to differentiate between elemental, organic, and inorganic mercury. this approach is consistent with observations that most (>95%) of the total mercury content of fresh and saltwater fish is methylmercury (usepa, 1997). therefore it was assumed that all mercury present was methylmercury. this assumption will tend to overestimate the toxicity of mercury. by using the rfd for methylmercury, the toxicity assessment takes a conservative approach to estimating the potential health hazard from exposure. risk characterization (estimation of human exposure to methylmercury in fish) daily and weekly intake: to evaluate the potential health risk to people through consumption of golden grey mullets, hg intake rates (estimated daily intake (edi) and estimated weekly intake (ewi)) were estimated for the general adult population (µg/day/adult) on the basis of the mean hg levels in fish muscle (wet weight basis) multiply daily and weekly fish consumption (kojadinovic et al., 2006; hajeb et al., 2009). hazard quotient (hq): a hazard quotient (hq) is the ratio of the estimated exposure dose of a contaminant (a single substance exposure level) to its rfd or mrl. the hq can be calculated with the following formula: hq = ((mcc x cr) / bw) / rfd where: mcc: mean contaminant concentrations in fish cr: consumption rate rfd = reference dose (hg = 0.0001 mg/kg/day) bw = body weight (70 kg for adults) station hg (w/w) hg (d/w) ramsar mean 0.205 0.645 min 0.098 0.309 max 0.316 0.997 tonekabon mean 0.205 0.646 min 0.116 0.366 max 0.401 1.265 chaloos mean 0.221 0.696 min 0.141 0.445 max 0.298 0.940 noshahr mean 0.222 0.701 min 0.113 0.356 max 0.401 1.265 noor mean 0.150 0.474 min 0.064 0.202 max 0.377 1.189 mahmudabad mean 0.164 0.516 min 0.067 0.211 max 0.312 0.984 fereidunkenar mean 0.112 0.353 min 0.014 0.044 max 0.319 1.006 babolsar mean 0.129 0.408 min 0.025 0.079 max 0.331 1.044 juibar mean 0.102 0.321 min 0.038 0.120 max 0.301 0.950 sari mean 0.079 0.248 min 0.032 0.101 max 0.299 0.943 neka mean 0.098 0.309 min 0.021 0.066 max 0.351 1.107 behshahr mean 0.077 0.243 min 0.019 0.060 max 0.286 0.902 total 0.137 0.432 ¹geomean ²the mean moisture content of tissue in liza aurata was 68.3. table 1. total hg concentration (µg/g)1 in muscles (wet weight² and dry weight) of liza aurata from study area. 261 hoseini et al/ international journal of aquatic biology (2013) 1(6): 258-265 an hq exceeding one, suggests the potential of health effects (castilhos et al., 2006). consumption limits (crlim): the maximum allowable fish consumption rate for a noncarcinogen can be calculated with the following formula (usepa, 2000): crlim = (rfd or mrl x bw) / mcc where: crlim = maximum allowable fish consumption (kg/day) rfd = reference dose (hg = 0.0001 mg/kg/day) bw = body weight (70 kg for adults) mcc: mean contaminant concentrations in fish statistical analysis: analysis of variance and means comparison (duncan’s multiple range test) were performed using spss (chicago, il) software. results and discussion mercury level: levels of the total mercury in the muscle of l. aurata from coastal waters of caspian sea are shown in table 1. no significant difference (p>0.05) in hg concentration between various stations was found. the total mercury levels in the samples of this study were comparable with mercury concentrations of fish muscle in similar or related studies of the caspian sea fishes. average of mercury concentrations have been reported 0.190 µg/g dry weight in muscle of rutillus frisii kutum (anan et al., 2005). yazdaninasab et al. (2004) reported the mercury concentrations of 0.259 and 0.262 µg/g dry weight in abdominal muscles and tail muscles of l. aurata (yazdaninasab et al., 2004). our study indicates that accumulation of mercury in the muscle of mullet is higher comparing to the before mentioned studies. in a study carried out by agusa et al. (2004), the accumulation of mercury in muscle of five species of caspian sea sturgeon (huso huso, acipenser persicus, acipenser guldenstadti, acipenser nudiventris and acipenser stellatus), was reported as 0.33, 0.07, 0.08, 0.16 and 0.015 ʮg/g wet weight, respectively, (or 1.40, 0.330, 0.320, 0.670 and 0.06 µg/g dry weight, respectively) (agusa et al., 2008). nozari (2011) reported the average concentration of mercury in muscle of pike (esox lucius) 0/322 µg/g dry weight (nozari et al., 2011). comparison of mean concentration of mercury in muscle of golden grey mullet with the studies listed above indicate that although these fishes are bottom feeders and almost are at the top of trophic chains in the caspian sea ecosystem, but the accumulation of mercury in their muscles has been relatively lower than those of reported by united states environmental protection agency, the world health organization, food and agriculture organization and the food and drug administration which is 0.3, 0.5, 0.5 and 1 µg/g wet weight of fish, respectively. these higher levels of mercury are considered as dangerous levels for human body (shi et al., 2005). risk assessment for fish consumption: in general, mercury levels increase with the size and age of the fish. however, this is not always the case (stafford and haines, 2001). we have used marketable and equality sized fish (553 ± 56 g) to elimination of the effect of size on mercury bioaccumulation. on the other hand, because of popularity of marketable size for consumption, investigation of risk assessment was carried out for this size. risk to the food chain: accumulation of mercury (methylmercury) in the fish poses a risk both to the fishes themselves, and to their predators. in the fishes themselves, levels of 5–20 μg/g in the muscle are associated with toxicity (wiener et al., 2003). hq di (μg hg day−1 adult−1) wi (μg hg week−1 adult−1) cr (g/day) number of meals per week based on usepa2 number of meals per week based on jecfa2 0.35 2.402 16.814 51 1 3 1these estimations do not apply for pregnant woman and children. 2based on an adult standard portion size of 230 g. table 2. results of risk assessment of mercury in liza aurata from study area1. 261 262 hoseini et al/ international journal of aquatic biology (2013) 1(6): 258-265 the mean mercury level of 0.137 μg/g in l. aurata in this study was well below these levels. mercury accumulates in larger fish, so it magnifies as it moves up the food chain (to humans or other toplevel predators). however, mercury concentrations in muscle are available to predators. the critical effects levels for consumption by piscivorous mammals are 0.1 μg/g, and for birds are 0.02 μg/g (yeardley et al., 1998), although seabirds are generally less sensitive (furness, 1996). the mean mercury levels in the l. aurata from caspian sea (0.137 μg/g in muscle) are clearly higher than the levels known to pose a problem for sensitive birds or mammals that scavenge them along the shore, or for sensitive marine mammals especially caspian seal (phoca caspica). the caspian seal, which is endemic to the caspian sea, is the only mammal within the aquatic fauna of the region. it is an ichthyophagous predator and is at the top of trophic chains in the caspian sea ecosystem. crab, shrimps and mullet are consumed by caspian seal to a different extent (badamshin, 1966). however, the ability to detoxify (demethylate) and store mercury in the form of less toxic (divalent) may not be present in newborn and young seals following exposure to the mother’s burden in utero and while nursing, thus, these young and developing seals may be at risk for mercury-related neurotoxicity and other effects (wagemann et al., 2000). risk to human: the average of iranian fish consumption is 6400 g (6.4 kg/year) per capita (fao, 2009); therefore, the 17.5 g/day (122.5 g/week) of consumption rate is used in our healthrisk assessment. based on the mean of hg concentrations in l. aurata (table 1) and seafood consumption rate of an iranian, dietary exposure to hg via fish consumption was estimated for iranian people. edi and ewi were obtained 2.402 and 16.814 μg day−1, respectively. different acceptable daily intake (adi) limits have been established by national and international instances. the adi set by the world health organization (who) for t-hg is 0.71 μg day−1 kg−1 body weight, and restricted to 0.35 μg day−1 kg−1 body weight for pregnant women because foetus are more sensitive to hg toxicity, as well as nursing mothers and children less than 10 years (dhhs and epa, 2004). the french (french agency for food safety (afssa) ) and the canadian health agencies follow the same guidelines as the who, whereas the us fda and us epa have set more restrictive adi limits for mehg (0.4 and 0.1 μg day−1 kg−1 body weight, respectively for all the population) (hirsch, 2002). considering an average adult body weight of 70 kg (usepa, 1994), the t-hg who adi, mehg us fda adi and mehg usepa adi can be approximated as 50 (hence 350 μg hg week−1), 28 (196 μg hg week−1) and 7 (49 μg hg week−1) μg day−1 adult−1, respectively (kojadinovic et al., 2006; goldblum et al., 2006). on the other hand, a provisional tolerable weekly intake (ptwi) of 1.6 mg mehg/kg body weight/ week was established in the 61st meeting of the joint fao/who expert committee on food additives (jecfa) (jecfa, 2003). considering an average adult body weight of 70 kg, the guideline value calculated from ptwi of jecfa was 16 μg /day and 112 μg/week, respectively. these health risk limit were compared to the estimated daily and weekly intake of hg in this study. the results indicated that the edi and ewi of total mercury by a 70 kg adult consuming 17.5 g fish/day and 122.5 g fish/week is below the respective adi and ptwi. the resulting hq is a unitless number that represents the ratio of the estimated exposure dose from hg at the site to its rfd, which is assumed to be without adverse health impacts. in this study hq index was below 1 (0.35). since the hq is < 1, adverse health effects are not expected from the exposure described in the assessment. therefore, the consumption of golden gray mullet from the given location is not a serious threat. based on the above mentioned levels, consumption of l. aurata is safe, although pregnant women and infants should take into account some considerations 263 hoseini et al/ international journal of aquatic biology (2013) 1(6): 258-265 for consumption of these fishes. because fetuses, infants and children under 10 years old are the most sensitive group to mercury toxicity (unep, 1999). in addition, according to world health organization, every levels of mercury can be harmful, and no specific level for the health effects of mercury can be identified (who, 1990) and guidance or standards for hg in fish tissues are not always uniform (burger and gochfeld, 2005), it is recommended that the population restrains from consuming, on a regular basis, species exceeding these values. therefore, it is essential to determine the allowable fish consumption (daily or weekly). based on the measured concentration and body weight, a consumption permitted rate of 51 g/day (357 g/week) is recommended. the crlim is the maximum consumption rate allowable without human health effects. in order to better appreciate the safe amounts of fish for consumption, exposure limits can be expressed as the number of meals that an adult can eat per day, or per week. in risk assessment, the standard portion size of uncooked fish eaten by an average adult is estimated to be 230 g (usepa, 1994). safety limits, expressed as the frequency of meals for which fish is the main element. because almost all hg are present as mehg in the edible portions of fish (bloom, 1991), we assumed that concentration of total hg is equal to that of mehg. also, the body weight of an iranian was assumed to be 70 kg. based on these assumptions, the number of meals for safe consumption calculated from adi of us epa and ptwi of jecfa for mehg was 1 and 3 meals per week, respectively (table 2). for individuals weighing more or less than 70 kg, it is assumed that their consumption rates and number of meals will be proportionally higher or lower, respectively. conclusion major source of methylmercury for fish is from mercury that has been methylated after atmospheric transport and precipitation or runoff, followed by food chain bio-magnification. fish consumption is the only significant source of methylmercury exposure for the public. therefore, fish consumption is a matter of risk balancing. the average concentration of mercury in mullet muscle from southern coast of caspian sea in the mazandaran province in iran, was 0.137 µg/g of fresh weight (0.432 µg/g dry weight) which was less than the allowable amount for human consumption but more than the allowable amount for piscivorous mammals and birds determined by the international organizations. the calculations indicated that daily and weekly mercury uptake for iranian consumers, according to fao (the amount consumed per capita) is lower than the guide values (adi and ptwi). also, hq index was below 1 (0.35). therefore, the consumption of the golden grey mullet is not a serious threat to the consumer’s health and a consumption permitted rate of 51g is recommended. acknowledgement we sincerely thank to mr fereidoon aflaki, from department of nuclear science research school, nuclear science and technology research institute, tehran, iran for making a number of helpful suggestions. we are also grateful to ms fatemeh monsefrad for her assistance in the preparation of the manuscript. references anan y., kunito t., tanabe s., mitrofanov i., aubrey d. 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(1998). elemental fish tissue concentration in northeastern us lakes: evaluation of an approach to regional assessment. environmental toxicology and chemistry, 17: 1875–1884. 265 international journal of aquatic biology (2015) 3(4): 263-273 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article fishes of the dasht-e kavir basin of iran: an updated checklist arash jouladeh roudbar1, soheil eagderi*2,1hamid reza esmaeili3 1department of fisheries, faculty of natural resources, sari university of agricultural sciences and natural resources, sari, iran. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 3department of biology, college of sciences, shiraz university, shiraz, 71454–iran. article history: received 2 may 2015 accepted 7 july 2015 available online 2 5 august 2015 keywords: checklist biodiversity freshwater fishes dasht-e kavir basin abstract: this study provide a new and updated checklist of the freshwater fishes of the dasht-e kavir basin of iran. the list is based on historical literature records and taxon occurrence data obtained as a result of extensive field expeditions, examination of ichthyological collections and literature review. the total confirmed freshwater fish species of the dasht-e kavir basin comprise 22 species in 17 genera, 6 families, 4 orders and one class. the most diverse order is the cypriniformes with 18 species (81.82%) followed by cyprinodontiformes (2 species, 9.09%), salmoniformes and gasterosteiformes each with 1 species (1 species, 4.55%). the most diverse family is the cyprinidae with 18 species (72.73%), nemacheilidae (2 species, 9.09%) followed by salmonidae, cyprinodontidae, poeciliidae and gasterosteidae each with only one species (4.55%). new species are supposed to be discovered, the taxonomic status of some species has been changed, some are being resurrected from synonymy, and some taxonomic problems remain and are commented on briefly. four endemic species (18.18%) in 4 genera and 2 families; and 12 exotic species (54.55%) in 10 genera and 4 families are listed here. introduction the iran is located in the palearctic region bordering the oriental and african zones (coad and vilenkin, 2004) and its north-west, west and south-west are parts of irano-anatolian hot spot with high biodiversity especially freshwater fish (esmaeili et al., 2010, 2014a, b). iran is divided into nineteen drainage basins (fig. 1) both exorheic where the rivers and lakes drain to the sea and endorheic, where rivers drain to an internal basin such as a lake, or are lost in the desert, and have no connection with the sea (coad, 2015; esmaeili et al., 2015). dasht-e kavir is a large desert lying in the middle of the iranian plateau and is one of the endorheic basins of iran that occupies about 230,400 sq km in the rain shadow of the alborz mountains (fig. 2) (afshin, 1994; coad, 2015). the main streams entering this basin, drain the alborz mountains and their eastern extensions in * corresponding author: soheil eagderi e-mail address: soheil.eagderi@ut.ac.ir khorasan. intermittent streams drain to several kavirs viz the damghan kavir in the north, the sabzevar or kalshur kavir in the north-east and the great kavir which are grouped together under dasht-e kavir basin. the great kavir is the largest one that its rivers except hableh (fig. 3) and golrudbar rivers, are temporary streams (afshin, 1994). the great kavir also receive waters exiting from other kavirs (afshin, 1994; coad, 2015). the damghan kavir receives two major streams including the damghan and hasanabad rivers, and other streams dry up in early summer. the sabzevar kavir has numerous small and temporary streams which feed it as well as two major streams, the mureh and kalshur rivers. the kalshur drains the kuh-e binalud and flows west to meet the south flowing mureh. in addition, qanats support fishes in this area (fig. 4). qanat discharges in this area were 20-50 l/sec (coad, 2015). mahdavi and anderson 264 international journal of aquatic biology (2015) 3(4): 263-273 (1983) detailed the qanat water supply of the margins of this basin. it can be expected that endorheic and exorheic basins of iran represent the higher diversity of freshwater fish species i.e. approximately 222 species or more (esmaeili et al., 2014a, b). therefore, the present study provides an updated checklist including natives, endemics, exotics and transplanted fish species, from the dasht-e kavir basin which is considerably higher than that given in the last checklist provided by coad (1998) and esmaeili et al. (2010). materials and methods this checklist has been resulted from the works listed in the references (see the references) and also by examination or accessing available data in ichthyological collections in iran (e.g., zm-cbsu, zoological museum of shiraz university, collection of biology department, shiraz; cmnfi, canadian museum of nature, ottawa, canada; bmnh, natural history museum, london, uk; personal fish collection of soheil eagderi, fisheries department, university of tehran) and extensive figure 1. map of iran showing different drainage basins of iran (l.m: lake maharlu basin). figure 2. map of the daesht-e kavir basin of iran. 265 juladeh roudbar et al/ fishes of the dasht-e kavir basin of iran field expeditions till june 2015 from different river systems of the dasht-e kavir basin of iran (fig. 2). results the total fish species of the dasht-e kavir basin comprise 22 species in 18 genera, 6 families, 4 orders and one class. the most diverse order is the cypriniformes with 18 species (81.82%) followed by cyprinodontiformes (2 species, 9.09%), salmoniformes and gasterosteiformes each with one species (1 species, 4.55%). the most diverse family is the cyprinidae with 18 species (72.73%), nemacheilidae (2 species, 9.09%) followed by salmonidae, cyprinodontidae, poeciliidae and gasterosteidae each with only one species (4.55%). the dasht-e kavir basin comprises four confirmed endemic (18.18%) and 12 exotic species (54.55%) (table 1). checklist * = endemic to iran, ** = exotic. class actinopterygii order cypriniformes (2 families, 14 genera and 18 species) family cyprinidae (12 genera and, 16 species) genus alburnoides jeitteles, 1861 1. alburnoides sp. * comment: the members of the genus alburnoides is under revision in iran. the recent studies suggest that the members of this genus from the dasht-e kavir is different from that of a. namaki in terms of molecular and morphological characteristics and probably belongs to a separate taxa (fig. 5). genus alburnus rafinesque, 1820 2. alburnus hohenackeri kessler, 1877 ** comments: alburnus hohenackeri was originally described from karabakh, azerbaijan, on the kura river. previously the wide-ranging species alburnus alburnus (linnaeus, 1758) was identified as the taxon in iran. alburnus charusini herzenstein, 1889 is a synonym. introduced to the dasht-e kavir basin. genus barbus cuvier, 1816 3. barbus sp. * comments: barbus miliaris de filippi, 1863 was described from a "fiumicelli presso teheran" (= a stream near tehran). karaman (1971) considers barbus miliaris from the namak lake basin of iran figure 3. nam river, a tributary of hableh river, kavir basin, iran. figure 4. qanat pir salman (qanats are important habitat for fishes of the dasht-e kavir basin) figure 5. alburnoides sp. figure 6. barbus sp. 266 international journal of aquatic biology (2015) 3(4): 263-273 to be a subspecies of the caspian sea basin type subspecies, differentiated by larger scales (78-92 versus 85-103 of luciobarbus mursa), less fleshy lips, an undeveloped lower lip lobe, feebly ossified last dorsal fin spine, and shorter pectoral fins. berg (1948-1949) recognised miliaris as distinct from mursa on the basis of a shorter snout, somewhat larger scales, fewer scale rows above the lateral line, smaller dimensions and different colour. according to our morphological studied, it seems that population of barbus in the dasht-e kavir basin belongs to barbus miliaris, but more studies are required (fig. 6). genus capoeta valenciennes, 1842 4. capoeta aculeata (valenciennes, 1844) comment: chondrostoma aculeatum was originally described from "eaux douces de la perse" (=iran freshwater). scaphiodon macrolepis heckel, 1847 was described from probably the pulvar (= sivan) river, fars near persepolis and varicorhinus bergi derzhavin, 1929 was described from karaj river near tehran. however, both now are synonyms (fig. 7). 5. capoeta buhsei kessler, 1877 * comment: type locality is not clear, probably karaj river near tehran, iran. varicorhinus nikolskii derzhavin, 1929 was described in latin from the "keredsh flumen" (= karaj river near tehran) is a synonym (fig. 8). 6. capoeta fusca nikol’skii, 1897 comment: type locality is mondechi and kuss, iran. capoeta nudiventris nikol’skii, 1897 is a synonym (fig. 9). genus carassius jarocki, 1822 7. carassius auratus (linnaeus, 1758) ** table 1. list of fish taxa of dasht-e kavir basin in different ecological group. family species ecological group cyprinidae alburnoides sp. fluvial alburnus hohenackeri fluvial barbus sp. fluvial capoeta aculeata fluvial capoeta buhsei fluvial capoeta fusca fluvial carassius auratus fluvial carassius gibelio fluvial ctenopharyngodon idella fluvial cyprinus carpio fluvial-semi anadromous hemiculter leucisculus fluvial hypophthalmichthys molitrix fluvial hypophthalmichthys nobilis fluvial pseudorasbora parva fluvial schizothorax pelzami fluvial squalius cf. orientalis fluvial nemacheilidae paracobitis malapterura fluvial paraschistura turcmenica fluvial salmonidae oncorhynchus mykiss fluvial cyprinodontidae aphanius kavirensis fluvial? poeciliidae gambusia holbrooki fluvial gasterosteidae gasterosteus aculeatus fluvial figure 7. capoeta aculeata. figure 8. capoeta buhsei. 267 juladeh roudbar et al/ fishes of the dasht-e kavir basin of iran comment: cyprinus auratus was originally described from china and japanese rivers. introduced to the caspian sea and sistan basins. 8. carassius gibelio (bloch, 1782) ** comment: cyprinus gibelio was originally described from odra river system, silesia, czech republic. kottelat and freyhof (2007); bogutskaya et al. (2008, with question); esmaeili et al. (2010); kalous et al. (2012) considered it as distinct species. genus ctenopharyngodon steindachner, 1866 9. ctenopharyngodon idella (valenciennes, 1844) ** comment: type locality in china. no types known. genus cyprinus linnaeus, 1758 10. cyprinus carpio linnaeus, 1758 ** comment: type locality in europe. native populations in the caspian sea basin; also introduced to the dasht-e kavin basin and elsewhere in iran. cyprinion cypris heckel, 1843 is a synonym. genus hemiculter bleeker, 1859 11. hemiculter leucisculus (basilewsky, 1855) ** comment: culter leucisculus was originally described from peking, china. hemiculter eigenmanni (jordan and metz, 1913) is a synonym. introduced to the dasht-e kavir basin. genus hypophthalmichthys bleeker, 1859 12. hypophthalmichthys molitrix (valenciennes, 1844) ** comment: introduced to the dasht-e kavir basin. leuciscus molitrix was originally described from china. 13. hypophthalmichthys nobilis (richardson, 1844) ** comment: introduced to the dasht-e kavir basin. leuciscus nobilis was originally described from canton, china. genus pseudorasbora bleeker, 1859 14. pseudorasbora parva (temminck and schlegel, 1846) ** comment: introduced to the dasht-e kavir basin. leuciscus parvus temminck and schlegel, 1846 was originally described from japan. genus schizothorax heckel, 1838 15. schizothorax pelzami kessler, 1870 comment: type locality is cabul river at jullalabad. tarnuck river in the indus river basin. schizothorax schumacheri fowler and steinitz, 1956 is an iranian synonym (fig. 10) genus squalius bonaparte, 1837 16. squalius cf. orientalis (nordmann, 1840) comment: leusciscus orientalis was originally described from abkhazia georgia, no types known. it has been considered as valid species (doadrio and carmona, 2004; bogutskaya and zupančič, 2010; turan et al., 2009; perea et al., 2010; esmaeili et al., 2014a) (fig. 11). family nemacheilidae (1 genus and 1 species) comment: formerly included in the family cobitidae or the family was named balitoridae (see tang et al. (2006) and kottelat and freyhof (2007)). this species were placed in the genera nemacheilus, adiposia, barbatula, orthrias and schistura in earlier literature. genus paracobitis bleeker, 1863 17. paracobitis malapterura (valenciennes, 1846) comment: cobitis malapterura was described from the namak lake basin of iran (freyhof et al., 2015). based on vatandoust et al. (2014) and freyhof et al. (2015), the mitochondrial gene tree showed the figure 9. capoeta fusca. figure 10. schizothorax pelzami. figure 11. squalius cf. orientalis. 268 international journal of aquatic biology (2015) 3(4): 263-273 dasht-e kavir basin population belongs to p. malapterura (fig. 12). 18. paraschistura turcmenica (berg, 1932) comment: nemachilus turcmenicus was originally described from keltechinar river [cherokh river] near gyaurs (37°47'n, 58°44'e), turkmenistan. order salmoniformes (1 family, 1 genus and 1 species) family salmonidae (1 genus and 1 species) genus oncorhynchus suckley, 1861 19. oncorhynchus mykiss (walbaum, 1792) ** comment: salmo mykiss was originally described from kamchatka, russia. introduced to the dasht-e kavir basin. order cyprinodontiformes (1 families, 1 genus and 1 species) family cyprinodontidae (1 genus and 1 species) genus aphanius nardo, 1827 20. aphanius kavirensis esmaeili, teimori, gholami and reichenbacher, 2014 * comment: type locality in semnan, damghan, cheshmeh ali spring, kavir basin, 36°16'45.6"n, 54°05'01.6"e, iran, altitude 1569 meters. holotype: zm-cbsu 9587a (figs. 13-14). family poeciliidae (1 genus and 1 species) genus gambusia poey, 1854 21. gambusia holbrooki girard, 1859 ** comment: type locality in palatka, eastern florida; charleston, south carolina, u.s.a. introduced to the dasht-e kavir basin. order gasterosteiformes (1 families, 1 genus and 1 species) family gasterosteidae (1 genus and 1 species) genus gasterosteus linnaeus, 1758 22. gasterosteus aculeatus linnaeus, 1758 ** comment: gasterosteus aculeatus was originally described from europe. introduced to the dasht-e kavir basin. discussion anthropogenic activities have played a significant role in changing distribution pattern of iranian freshwater fishes especially in the past few decades (esmaeili et al., 2010a, 2012, 2015) which can be well-understood regarding the dasht-e kavir basin due to presence of 12 exotic species (54.55%). ctenopharyngodon idella, cyprinus carpio, hypophthalmichthys molitrix, h. nobilis and ocorynchus mykiss are commercially valuable exotic species recorded from this basin and probably introduced to the natural aquatic ecosystems of the dasht-e kavir basin by fish farmers. in addition, a. hohenackeri, c. auratus, c. gibelio, h. leucisculus, p. parva, and g. aculeatus have been figure 12. paracobitis malapterura. figure 13. female (above) and male (below) of aphanius kavirensis. figure 14. cheshmeh ali spring at damghan city (dasht-e kavir basin), natural habitat of aphanius kavirensis. 269 juladeh roudbar et al/ fishes of the dasht-e kavir basin of iran probably introduced to this basin along with commercially important cyprinds from the caspian sea basin as accidental introduction. furthermore, g. holbrooki has been released as a control agent for anopheles controlling (malaria) (tabibzadeh et al., 1970). some of them (e.g., cyprinus carpio, carassius auratus, pseudorasbora parva and gambusia holbrooki) have been established in natural water bodies acting as invasion species. the introduction of a non-native species in an ecosystem is likely present an ecological risk if the species is able to integrate itself successfully into the ecosystem (gozlan and newton, 2009), resulting in possible detrimental interactions with native species or even on ecosystem functioning (gozlan et al., 2010). freshwater fishes provide relatively conservative system for examining zoogeographical patterns. since, they are limited to drainage systems and cannot disperse without connections of the freshwater systems (berra, 2001). iran occupies a significant portion of the middle east and its freshwater fish fauna stands out from its neighboring counterpart in terms of species richness and level of endemism (see esmaeili et al., 2010, 2014a-b; coad, 2015). geographical isolation, together with the climatic conditions and watershed fragmentation experienced by iranian plateau over geological time, has led to differentiation of the freshwater ichthyofauna into several independent and isolated populations promoting speciation. iran contains elements of both ethiopian and oriental ichthyofauna, although it is predominantly a part of the palearctic realm. this zoogeographical situation, coupled with the past geological history, vicariance events and recent anthropogenic effects have played a significant role on the ichthyodiversity of iran (coad, 1982; kosswig, 1951; naseka, 2010). iranian current inland basins were present since the early tertiary (about 70 mya) before entrance of fish species. from the pliocene period (about 6 to 2 mya) along with originating of the middle east ichthyofauna, the inland basins of iran had arid climate that prevents permanent settlement of the ichthyofauna due to lacking proper habitats (por, 1975; coad 1995). a vast desert belt is extended from the west of africa toward saudi arabia, syria, iran and india possessing almost similar animal fauna that climatic features is considered as main factor operating in their distribution. across this belt, there were higher speciation events of fishes with a basic ichthyofauna formed by the members of the families, including cyprinidae, nemacheilidae, mastacembelidae, cyprinodontidae and cichlidae that characterised by a relatively high resistance to salt waters. hence, they can find from africa to india across this belt and the migrations could be occurred along this belt toward the east and vice versa (berra, 1981). the dasht-e kavir basin, a part of this desert belt has arid climate characterised by less than 200 mm annual rainfall. therefore, its native limited freshwater fish species, including capoeta, schizothorax and some members of the family nemacheilidae have an origin of this desert belt. the diversity of ichthyofauna in india, southeast asia and west africa is much more than that of its center i.e. the middle east despite having opportunities to produce a higher diversity that may limited due to arid climate (por, 1975; coad, 1995). north of this desert belt along the middle east is confined by the taurus mountains in turkey, alborz mountains in iran and hindu kush-himalayan mountains in afghanistan and india. these mountain chains are topographic barrier to reach precipitation from the north and create drought conditions in the southern regions (abell et al., 2008). in addition, these mountain chains have separated the fish fauna of the southern drought regions from palearctic fish fauna acting as a filter because some exchanges have occurred between them particularly at the western parts of this high mountains in iran and turkey, where the mountain barrier is not continuous (gleick, 1993). in some regions, the rivers including hari and murghab rivers (in east of iran), sefid river (in north of iran) and aras river (in west of iran and east of turkey) cross the mountains and therefore provide the passages for exchanges (abell et al., 2008; coad, 270 international journal of aquatic biology (2015) 3(4): 263-273 2015). three taxa of freshwater fishes with the origin of the desert belt, including capoeta, barbus and members of the family nemacheilidae are found in the north of above mentioned mountains i.e. the palearctic ecozone (nümann, 1966). furthermore, some members of the subfamily leuciscinae such as alburnus and squalius with the origin from the palearctic are found in the southern part of this mountains (naseka, 2010). these distribution patterns of fish species in both sides of the mountain chains shows the occurrence of exchange between them that have provides opportunity for further movement of some palearctic fish species toward south. the occurrence of the genera alburnoides, alburnus and squalius in the dasht-e kavir basin could be as result of such an exchange (coad, 2015). ichthyofauna of iran except remains of marine species e.g. a. kavirensis, could not reached iranian inland water until the pliocene and pleistocene periods that main exchange occurred (frenkel, 1995). after to the pliocene and pleistocene periods, the most of iranian freshwater fishes were settled except cyprinion and some other isolated genera (menon, 1964; 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(2014). first record of the loach fish paracobitis cf. malapterura in the kavir basin, northern iran. international journal of aquatic biology, 2(1): 27-28. international journal of aquatic biology (2015) 3(4): 263-273 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved چکیده فارسی ایران کویر دشت آبریز حوضه ماهیان شده روزهب فهرست 3اسماعیلی حمیدرضا ،2ایگدری سهیل ،1رودبار جوالده آرش .ایران ساری، ساری، طبیعی منابع و کشاورزی دانشگاه شیالت، گروه1 .ایران کرج، تهران، دانشگاه ، دانشکده منابع طبیعی،طبیعی منابع و کشاورزی پردیس شیالت، گروه2 .ایران شیراز، شیراز، دانشگاهانشکده علوم، د شناسی،زیست گروه3 چکیده: ین،پیش مطالعات اساس بر فهرست این. نمایدمی ارائه را ایران کویردشت آبریز حوضه شیرینآب ماهیانروز شده به وجدید فهرست حاضر مطالعه جنس، 11 به متعلق ماهی گونه 22 مجموع در. است تهیه شده و مرور منابع ایموزه هاینمونه بررسی ،گسترده هایبردارینمونه میدانی، مشاهدات با cypriniformes شکالن کپورماهی به مربوط راسته ترینفراوان. گردید گزارشتایید و کویر دشت آبریز حوضه از رده 1 و راسته 4 خانواده، 6 شکالن آزادماهی راسته ،(درصد 90/0) گونه 2با cyprinodontiformes داردندان کپورماهیان راستهدنبال آن و به( درصد 12/11) گونه 11 salmoniformes شکالن ماهیخارهسه و gasterosteiformes خانواده ترینفراوان. ندشتدا قرار( درصد 55/4 گونه، 1) گونه 1 با کدام هر ،salmonidae آزادماهیان هایخانواده آندنبال و به( درصد 90/0 گونه، 2) جویباری ماهیانسگ ،(درصد 13/12) گونه 11 با کپورماهیان به مربوط 55/4 گونه، 1) گونه یک با کدام هر gasterosteidae ماهیان خارهسه و poeciliidae ماهیان پشه ،cyprinodontidae داردندان کپورماهیان بر های معتها تغییر کرده، برخی گونهشناختی برخی گونههای جدید وجود داد، وضعیت آرایهاحتمال کشف گونه در این حوضه آبریز .بودند (درصد بر مشتملدرصد( 11/11) بومزاد گونهاند. چهار صورت توضیح کوتاه بیان شدهشناختی هنوز وجود دارد که بهشده و همچنین برخی مشکالت آرایه .گرددمی فهرست مقاله این در خانواده 4 و جنس 19 بر مشتملدرصد( 55/54) مهاجم گونه 12 و خانواده 2 و جنس 4 .دشت کویر آبریز حوضه شیرین،آب ماهیانتنوع زیستی، فهرست، :کلمات کلیدی int. j. aquat. biol. (2016) 4(6): 360-369; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article diversity of phytoplankton of a sub-tropical reservoir of mizoram, northeast india bhushan kumar sharma*,1 lalthlamuana pachuau freshwater biology laboratory, department of zoology, north-eastern hill university, permanent campus, shillong-793 022, meghalaya, india. article history: received 4 august 2016 accepted 29 october 2016 available online 2 5 december 2016 keywords: abundance composition richness diversity indices ecology abstract: phytoplankton of khawiva reservoir of mizoram, northeast india (nei) revealed a total of 55 species; nearly concurrent mean monthly richness and high community similarities (vide sørensen index) during two years affirmed homogeneity in its species composition. phytoplankton comprised dominant component (61.1±14.3%) of net plankton and recorded wider density variations. chlorophyta influenced phytoplankton abundance with quantitative importance of staurastrum spp. > xanthidium spp. > cosmarium spp. in particular. bacillariophyta formed subdominant group; cryptophyta and cyanophyta showed limited importance; and euglenophyta and dinophyta recorded poor densities. phytoplankton is characterized by moderate species diversity, high evenness and low dominance but with wide variations. richness, abundance and species diversity followed no definite patterns of monthly variations during two years. insignificant influence of individual abiotic factors on phytoplankton assemblages coupled with low cumulative influence of fifteen abiotic parameters (vide cca) yielded little insight on overall role of abiotic parameters. introduction limnological studies in india began nearly one century ago and culminated in several investigations on reservoir ecology from different regions (jana, 1998) and dealing with phytoplankton diversity. there is yet paucity of information on phytoplankton of sub-tropical environs of this country in general and from northeast india (nei) in particular (sharma, 2015). the related studies from nei are limited to analysis of these primary producers and fish-food organisms of certain reservoirs of meghalaya (sharma, 1995; sharma and lyngdoh, 2003; sharma and lyngskor, 2003), while other notable contributions related to certain floodplain lakes of this region (sharma, 2004, 2010, 2012, 2015). the present study forms a part of first limnological endeavor undertaken by the authors from mizoram state of nei; the results on zooplankton diversity are published earlier by sharma and pachuau (2013). nevertheless, this communication on phytoplankton diversity of * corresponding author: bhushan kumar sharma e-mail address: profbksharma@gmail.com khawiva reservoir merits ecological value in view of the stated lacunae. the observations are made on monthly variations of phytoplankton assemblages of the sampled reservoir for a period of two years with emphasis on composition, richness, community similarities, abundance, species diversity, dominance and evenness. in addition, individual and cumulative influence of abiotic factors is analyzed to assess their limnological value vis-à-vis phytoplankton diversity. materials and methods our observations are based on limnology study (november, 2005-october, 2007) of khawiva reservoir (22o35'n, 93o 47'e; area: about 10 ha; maximum depth: 40 m), lunglei district, mizoram state (fig. 1a-c), nei; located nearly 12 km from lunglei town, it was commissioned in 1988 on khawiva river with a capacity of generating 1050 kw power. this reservoir is surrounded by phyllanthus sp., cyperus sp., eupatorium sp., 361 int. j. aquat. biol. (2016) 4(6): 360-369 farmaria sp., and centella sp., and itself lacked any aquatic vegetation. water as well as qualitative and quantitative plankton samples was collected at monthly intervals at two sampling stations for a period of two years as per details of sharma and pachuau (2013). water temperature, ph and specific conductivity were recorded using the field probes; rainfall data was collected from lunglei meteorological station; winkler’s method was used for estimation of dissolved oxygen and apha (1992) was followed to analyze the rest of the abiotic factors. the phytoplankton was identified following the works of islam and haroon (1980), adoni et al. (1985) and fritter and manuel (1986). quantitative enumeration of phytoplankton and its constituent taxa was done with a sedgewick-rafter counting cell and abundance was expressed as n/l. phytoplankton community similarities, species diversity, dominance and evenness were calculated vide sørensen, shannon, berger-parker and pileou indices, respectively (ludwig and reynolds, 1988; magurran, 1988). anova (two-way) was used to ascertain significance of temporal variations of different parameters; and spss (version 20.0) was used for the hierarchical cluster analysis. pearson’s correlation coefficients (r) were used to analyze ecological correlations amongst abiotic-vis-biotic attributes; p-values were calculated and significance was ascertained following bonferroni corrections. xlstat (2012) was used for the canonical correspondence analysis to assess cumulative influence of fifteen abiotic factors namely rainfall, water temperature, specific conductivity, ph, dissolved oxygen, free carbon dioxide, total alkalinity, total hardness, chloride, sulphate, phosphate, nitrate, silicate, dissolved organic matter and total dissolved solids on phytoplankton assemblages. results the variations in abiotic parameters (annual ranges and mean ±sd) of khawiva reservoir observed during two years as well as during the study period are indicated in table 1. water temperature ranged between 22.1±4.0oc and total monthly rainfall ranged between 270.5±302.6 mm during the study period; specific conductivity, ph, dissolved oxygen, free carbon dioxide, total alkalinity, total hardness, chloride and sulphate varied between 40.5±11.0 µs cm-1, 6.64±0.31 mg l-1, 7.7±1.6 mg l-1, 11.5±3.0 mg l-1, 31.6±4.7 mg l-1, 30.0±8.0 mg l-1, 7.9±3.5 mg l-1 and 1.890±1.478 mg l-1, while phosphate, nitrate, silicate and dissolved organic matter varied figure 1. (a) map of india showing mizoram state (red color), (b) district map of mizoram showing lunglei district with location of khawiva reservoir and (c) khawiva reservoir (google photo) with sampling station marked by dots. 362 sharma and pachuau/ phytoplankton diversity of khawiva reservoir of mizoram, northeast india between 0.089±0.093 mg l-1, 0.185±0.088 mg l-1, 0.417± 0.364 mg l-1, and 0.302±0.582 mg l-1 during the study period, respectively. anova registered significant annual variations of ph (f1, 23=7.553, p=0.019) and sulphate (f1, 23=9.465, p=0.011), while specific conductivity (f11, 23=3.589, p=0.022) and hardness (f11, 23=3.238, p=0.032) recorded significant monthly variations. the richness and density variations (ranges and mean ±sd) of phytoplankton are presented in table 2. we observed 55 species of phytoplankton; the monthly richness (fig. 2) ranged between 24-38 and 20-41 species, and community similarities varied between 51.7-84.4% and 50.0-95.8%. chlorophyta, the diverse group, is represented by 22±4 and 23±6 species during two years, respectively. the hierarchical cluster analyses, based on sørensen’s community similarities, are presented in figures 3-4. the phytoplankton density varied between 132-1199 (369±253 n/l) and it comprised 61.1±14.3% of net plankton; chlorophyta exhibited wide density variations (57-1087, 239±238 n/l) and it formed between 59.5±21.9% of total phytoplankton during the study period; bacillariophyta (71±62 n/l), cyanophyta (24±52 n/l) and cryptophyta (26±44 n/l) comprised 21.2±15.2%, 8.1±17.7% and 6.1±6.9% of phytoplankton abundance, respectively in this study while euglenophyta (9±19 n/l) and dinophyta ((1±1 n/l) depicted poor abundance. the species diversity (1.693-3.399, 2.607±0.451), dominance (0.080-0.649, 0.265±0.151) and evenness (0.472-0.921, 0.760±0.119) recorded wide variations. the monthly variations of abundance of phytoplankton, chlorophyta and bacillariophyta, and that of species diversity are presented in figures 5-8, respectively. this study indicated quantitative importance of staurastrum spp. (100±142 n/l), xanthidium spp. (73±83 n/l) and cosmarium spp. (32±30 n/l); staurastrum inflexum (36±42 n/l), s. nonanum (23±57 n/l), s. kurzianum (13±16 n/l), s. chaetoceras (11±20 n/l), oscillatoria sp. (23±52 n/l), frustulia sp. (22±17 n/l) and navicula sp. (19±23 n/l) are important individual taxa. our results lacked significant influence of any individual abiotic parameter on richness and abundance of phytoplankton as well as on abundance of the chlorophyta, bacillariophyta and cyanophyta table 1. temporal variations of abiotic parameters (modified after sharma and pachuau, 2013). parameters first year second year study period range, mean±sd range, mean±sd range, mean±sd rainfall (mm) 0-890.0, 268.7±283.4 0-901.8, 272.4±320.7 0-901.8, 270.5±302.6 water temperature (°c) 14.5-28.0, 22.4±4.0 14.0-27.0, 21.7±3.9 14.0-28.0, 22.1±4.0 specific conductivity (µs cm-1) 20.0-62.0, 42.8±13.5 28.0-50.0, 38.3±7.2 20.0-62.0, 40.5±11.0 ph 6.34-7.18, 6.81±0.24 5.86-6.83, 6.48±0.29 5.86-7.18, 6.64±0.31 dissolved oxygen (mg l-1) 5.6-10.4, 8.1±1.5 4.8-9.6, 7.2±1.6 4.8-10.4, 7.7±1.6 free carbon dioxide (mg l-1) 8.0-16.0, 12.8±2.6 6.0-14.0, 10.2±2.8 6.0-16.0, 11.5±3.0 total alkalinity (mg l-1) 24.0-40.0, 32.3±5.5 26.0-34.0, 30.8±3.6 24.0-40.0, 31.6±4.7 total hardness (mg l-1) 18.0-46.0, 30.3±9.5 22.0-38.0, 29.7±6.2 18.0-38.0, 30.0±8.0 chloride (mg l-1) 1.0-12.0, 9.2±3.6 4.0-11.0, 6.6±2.8 1.0-11.0, 7.9±3.5 sulphate (mg l-1) 0.714-2.584, 1.055±0.640 0.285-4.638, 2.725±1.601 0.285-4.638, 1.890±1.478 phosphate (mg l-1) 0.017-0.445, 0.079±0.115 0.017-0.221, 0.100±0.061 0.017-0.445, 0.089±0.093 nitrate (mg l-1) 0.074-0.392, 0.199±0.105 0.074-0.238, 0.171±0.065 0.074-0.392, 0.185±0.088 silicate (mg l-1) 0.037-1.384, 0.482±0.456 0.037-0.664, 0.353±0.221 0.037-1.384, 0.417±0.364 dissolved organic matter (mg l-1) 0.016-2.236, 0.413±0.797 0.025-0.452, 0.192±0.138 0.016-2.236, 0.302±0.582 total dissolved solids (mg l-1) 0.018-0.296, 0.191±0.221 0.075-0.347, 0.183±0.081 0.018-0.347, 0.187±0.166 figure 2. monthly variations in species richness of phytoplankton. 363 int. j. aquat. biol. (2016) 4(6): 360-369 table 2. variations (range, average and sd) of phytoplankton. nov. 2005-oct. 2006 nov. 2006-oct. 2007 study period richness phytoplankton > zooplankton phytoplankton 55 species: 24-38 32±4 55 species: 20-41 34±7 55 species: 20-41 33±6 % similarity 51.7-84.4 50.0-95.8 chlorophyta 40 species: 15-27 22±4 40 species: 11-30 23±6 40 species: 22-30 22±5 abundance phytoplankton > zooplankton net plankton n/l 254-1344 589±296 276-1077 550±234 254-1344 570±267 phytoplankton n/l 132-1199 430±288 138-836 308±265 132-1199 369±253 % composition 41.3-89.2 68.8±12.7 39.1-77.6 53.4±11.3 39.1-89.3 61.1±14.3 species diversity 1.922-3.086 2.534±0.314 1.693-3.399 2.680±0.546 1.693-3.399 2.607±0.451 dominance 0.144-0.590 0.285±0.127 0.080-0.649 0.245±0.127 0.080-0.649 0.265±0.151 evenness 0.545-0.919 0.756±0.107 0.472-0.921 0.764±0.130 0.472-0.921 0.760±0.119 different groups chlorophyta > bacillariophyta > cyanophyta > cryptophyta > euglenophyta > dinophyta chlorophyta n/l 83-1087 306±265 57-739 171±184 57-1087 239±238 % composition 37.7-93.3 67.5±19.7 17.3-88.3 51.5±21.1 17.3-93.3 59.5±21.9 bacillariophyta n/l 6-313 83±83 27-101 58±24 6-313 71±62 % composition 2.1-59.8 20.1±19.4 8.8-38.5 22.4±9.1 2.1-59.8 21.2±15.2 cyanophyta n/l 0-54 6±15 2-192 42±68 0-192 24±52 % composition 0-23.8 2.5±6.4 0-73.5 13.7±22.9 0-73.5 8.1±17.7 cryptophyta n/l 1-192 31±51 1-130 21±34 1-192 26±44 % composition 0.7-25.8 6.2±6.9 0.7-23.5 6.1±6.9 0.7-25.8 6.1±6.9 euglenophyta n/l 0-21 2±6 3-96 16±25 0-96 9±19 % composition 0-9.2 1.0±2.5 0.3-33.9 7.0±9.0 0-33.9 4.0±7.8 dinophyta n/l 0-6 1±2 0-2 0±1 0 6 1±1 % composition 0-2.6 0.5±0.9 0-0.9 0.2±0.3 0-2.6 0.4±0.7 important genera (n/l) staurastrum spp. 11-663 108±175 27-367 92±97 11-663 100±142 xanthidium spp. 1-290 12±88 0-215 35±56 0-290 73±83 cosmarium spp. 14-106 43±31 3-88 20±25 3-106 32±30 important species (n/l) staurastrum inflexum 2-186 45±49 1-100 26±32 1-192 36±42 s. nonanum 0-257 27±71 3-96 16±25 0-257 23±57 s. kurzianum 0-65 10±18 2-45 16 ±13 0-65 13±16 s. chaetoceras 0-7 2±2 2-80 20±24 0-80 11±20 oscillatoria sp. 0-44 5±12 0-192 41±68 0-192 23±52 frustulia sp. 1-76 21±21 9-47 23±11 1-76 22±17 navicula sp. 1-100 23±30 4-30 15±8 1-100 19±23 figure 3. hierarchical cluster analysis of phytoplankton (first year). 364 sharma and pachuau/ phytoplankton diversity of khawiva reservoir of mizoram, northeast india during the study period. the canonical correspondence analysis (cca) with fifteen abiotic factors explained 57.6% of cumulative variance of phytoplankton assemblages along axis 1 and 2 (fig. 9). discussion the sub-tropical khawiva reservoir is characterized by ‘soft, slightly acidic-circum neutral and welloxygenated waters with distinctly low specific conductivity, low free co2 throughout the study period, low chloride content, and low concentration of nutrients and other abiotic factors (sharma and pachuau, 2013). of these, ph and sulphate recorded significant annual variations (vide anova) while specific conductivity and hardness recorded significant monthly variations thus indicating limited temporal variations during this study. fifty-five species of phytoplankton, belonging to six groups, observed from khawiva reservoir figure 4. hierarchical cluster analysis of phytoplankton (second year). figure 5. monthly variations in abundance (n/l) of phytoplankton. figure 6. monthly variations in abundance (n/l) of chlorophyta. figure 7. monthly variations in abundance (n/l) of bacillariophyta. figure 8. monthly variations in species diversity of phytoplankton. 365 int. j. aquat. biol. (2016) 4(6): 360-369 exhibited fairly diverse nature of these primary producers compared with the richness known from sub-tropical reservoirs of meghalaya (sharma, 1995; sharma and lyngdoh, 2003; sharma and lyngskor, 2003) and with the reports from kashmir (zutshi et al., 1980), bihar (baruah et al., 1993; sanjer and sharma, 1995) and assam (sharma, 2004). on the other hand, it broadly compared with the results from two floodplain lakes of nei (sharma, 2012, 2015). the richness indicated no definite pattern of annual variations but exhibited peaks during april (first year) and september (second year) while lowest richness is observed during october (first year) and april (second year). the occurrence of all phytoplankton species during both years asserted homogeneity in their overall composition; this generalization is also supported by high community similarities during two years with values between 60-80% in majority of instances (88.0%) in the matrix during first year while 58% instances affirmed the stated range in the following year. peak phytoplankton similarity is observed concurrently between january-february communities in both years. the hierarchical cluster analysis showed differences in clusters groupings during two years with high associations during january-february and most divergence in december during first year. high affinities are observed between january-february, august-september and december-july phytoplankton while divergence is noted in april in the following year. the most diverse chlorophyta largely contributed to phytoplankton richness and thus mainly influenced phytoplankton similarities; these features figure 9. cca ordination biplot of phytoplankton and abiotic factors of khawiva reservoir. abbreviations: abiotic: alk (alkalinity), co2 (free carbon dioxide), cl (chloride), cond (conductivity), do (dissolved oxygen), dom (dissolved oxygen matter), ph (hydrogen-ion concentration), no3 (nitrate), po4 (phosphate), rain (rainfall), sio2 (silicate), so4 (sulphate), tds (total dissolved solids), trans (transparency) and wt (water temperature). biotic: an.con (anthrodesmus convergens), cr (chlorophyta richness), pr (phytoplankton richness), bac (bacillariophyta), chl (chlorophyta), cryp (cryptophyta), cry.sp (cryptomonas sp.), (cyan (cyanophyta), eugl (euglenophyta), phy (phytoplankton), stuar (staurastrum), st.gl (s. gladiosum ), st.inf (s. inflexum ), st.ku. (s. kurzianum), st.non (s. nonanum), cos. (cosmarium), na.sp (navicula sp), fr.sp. (frustulia sp), xan (xanthidium), xan sp (xanthidium sp), osc.sp (oscillatoria sp). 366 sharma and pachuau/ phytoplankton diversity of khawiva reservoir of mizoram, northeast india agreed with the reports from nei (sharma, 1995, 2009, 2010, 2012, 2015; sharma and lyngdoh, 2003). the slightly acidic-circum neutral soft waters with low ionic concentrations exhibited high desmid richness (payne, 1986) while karikal (1995) observed that soft waters with low nutrients favored the growth of desmids. these remarks hold true for de-mineralized soft waters of khawiva reservoir with staurastrum (11 species) > cosmarium (10 species) > micrasterias (4 species). the importance of desmid richness concurred with those from reservoirs of meghalaya indicating identical characteristics (sharma, 1995; sharma and lyngdoh, 2003; sharma and lyngskor, 2003) and also with the reports of sharma (2009, 2010, 2012, 2015) from nei. phytoplankton recorded wider density variations with more difference during first year; it registered insignificant monthly and annual differences (vide anova). it formed main component of net plankton and significantly influenced their density (r = 0.946) during the study with >50.0% of abundance of the former throughout first year except in december while their contribution is >50.0% in the months of november, february and august through october during second year. in general, quantitative importance of phytoplankton in khawiva reservoir concurred with the results (sharma, 1995; das et al., 1996, sharma and lyngdoh, 2003; sharma and lyngskor, 2003) from certain reservoirs of meghalaya state of nei and also with the reports from the floodplains of kashmir (kaul and pandit, 1982), bihar (rai and dutta-munshi, 1982; sinha et al., 1994; sanjer and sharma, 1995), assam (yadava et al., 1987) and kerala (krishnan et al., 1999). on the contrary, our results differed from phytoplankton sub-dominance reported by (sharma, 2004, 2009, 2010). the present study showed no definite annual pattern of abundance while peak density is noticed in may (summer) and november (autumn) during two years, respectively; the former concurred with the report of sharma (2015) but differed from winter peaks reported by yadava et al. (1987), wanganeo and wanganeo (1991), sharma and lyngdoh (2003) and sharma (2009). the chlorophyta, dominant component, significantly influenced phytoplankton abundance (r=0.935) and contributed to its peaks during two years. the green algae registered insignificant monthly and annual density differences during the study; they exhibited annual peaks during march and november during two years, respectively. the average densities of this group are high than the reports from sub-tropical environs of meghalaya (sharma, 1995; das et al., 1996; sharma and lyngdoh, 2003; sharma and lyngskor, 2003) as well as than the results of yadava et al. (1987) and sharma (2004, 2009). the chlorophyta is characterized by high abundance of staurastrum spp. >xanthidium spp. >cosmarium spp.; the first two genera in particular largely influenced density variations of the former and contributed to chlorophyta peak abundance. in general, quantitative significance of desmids agreed with the reports of sharma (1995, 2009, 2010) and sharma and lyngdoh (2003). rao and govind (1964) recorded desmid maxima in summer and winter while doddagauder (1989), karikal (1995) and hulyal and kaliwal (2009) reported their winter peaks. our results did not affirm any such generalizations but indicated peak in autumn (november) during the second year. staurastrum spp. and cosmarium spp. registered concurrent peaks in march but xanthidium spp. showed peak density in september during first year. staurastrum inflexum > s. nonanum > s. kurzianum > s. chaetoceras are notable desmid species. the bacillariophyta comprised 21.2±15.2% of phytoplankton abundance and recorded relatively high abundance during first year. the diatoms followed oscillating but different annual patterns and peak densities during october and march during two years, respectively not concurrent with phytoplankton peaks. our results recorded higher diatom abundance than the results of sharma (1995, 2009, 2010), das et al. (1996), sharma and lyngdoh (2003) and sharma and lyngskor (2003). frustulia sp. and navicula sp. are important diatoms in this 367 int. j. aquat. biol. (2016) 4(6): 360-369 study. the cyanophyta > cryptophyta showed importance during certain months. the former recorded relatively high abundance during second year and january through march in particular with peak density in march attributed to oscillatoria sp. cryptophyta occurred in low densities except for peaks during april and february during two years, respectively attributed to cryptomonas sp. euglenophyta > dinophyta recorded poor densities; the former showed peak density in march during second year. the phytoplankton species diversity (1.6933.399; 2.607±0.451) recorded relatively wider variations during second year. high values (>3.0) are observed only during july during first year, and during december and august during second year while low diversity (below 2.0) is observed in august (first year) and during january and march in the following year. the diversity followed multimodal and bimodal patterns during two years respectively, and anova registered its insignificant annual and monthly variations. khawiva reservoir indicated higher phytoplankton diversity than the reservoirs of meghalaya (sharma, 1995; sharma and lyngdoh, 2003; sharma and lyngskor, 2003). an equitable abundance of majority of species resulted in general higher phytoplankton evenness (0.760±0.119) during the study except in august-september during first year and in january-march during second year. it is positively correlated with species diversity (r=0.843, p<0.0001), thereby, indicating that periods of high diversity correspond with high equitability. the evenness followed oscillating annual variations. this study indicated relatively wide variations in dominance (0.080-0.649) indicating period of very low dominance to certain months. on the other hand, xanthidium sp. mainly resulted in high dominance in august during first year while its peak value in january during second year is attributed to abundance of oscillatoria sp. and xanthidium sp. the dominance is inversely corrected with species diversity (r=-0.839, p<0.0001) and evenness (r=0.868, p<0.0001). in general, high evenness and low dominance affirmed the previous reports from subtropical reservoirs of meghalaya (sharma, 1995; sharma and lyngdoh, 2003; sharma and lyngskor, 2003) and also concurred with reports from various ecosystems of nei (sharma, 1995, 2004, 2009, 2010, 2012, 2015). this study lacked significant influence of any individual abiotic parameter on phytoplankton richness and abundance as well as on abundance of chlorophyta, bacillariophyta and cyanophyta. this interesting feature is concurrent with the report of sharma (2009) but differed from importance of various abiotic factors reported by sharma and lyngskor (2003), sharma and lyngdoh (2003) and sharma (2010). our results explained lower (57.6%) cumulative variance of fifteen abiotic factors along first two axes (vide the canonical correspondence analysis) on phytoplankton assemblages; and recorded importance of hardness, dom, rainfall, chloride and phosphate. staurastrum, s. gladiosum, s. kurzianum, s. nonanum, and chrysophyta abundance are influenced by hardness and dissolved organic matter. s. inflexum density is influenced by conductivity and nitrate; ph influenced cosmarium abundance; richness of phytoplankton and chlorophyta and phytoplankton abundance is influenced by phosphate; high rainfall influence abundance of chlorophyta, frustulia sp. and navicula sp. while high rainfall and chloride influenced bacillariophyta abundance. in general, this study thus yielded little insight on overall importance of abiotic parameters on variations phytoplankton taxa. conclusions the fairly rich phytoplankton of khawiva reservoir indicated homogeneity in its composition. high richness of chlorophyta and diverse nature of desmids with staurastrum > cosmarium > micrasterias are interesting. phytoplankton and its dominant component chlorophyta indicated wider density variations; bacillariophyta showed subdominance; and cryptophyta and cyanophyta exhibited limited importance; staurastrum > 368 sharma and pachuau/ phytoplankton diversity of khawiva reservoir of mizoram, northeast india xanthidium are quantitatively important genera while s. inflexum > s. nonanum > s. kurzianum > s. chaetoceras> oscillatoria sp. > frustulia sp. > navicula sp. are notable individual taxa. richness and abundance followed no definite pattern of monthly variations. phytoplankton indicated moderate species diversity, high equitability and low dominance. with lack of significant influence of abiotic factors and cca with 15 abiotic factors explaining low cumulative phytoplankton variance, this study yielded little insight on overall role of abiotic parameters. the results thus suggested importance of biotic factors associated with microhabitat variations. acknowledgments the authors are thankful to the head, department of zoology, north-eastern hill university, shillong for laboratory facilities. we thank s. sharma, shillong for valuable suggestions on the draft of this paper. the authors have no conflict of interests. references adoni a.d., ghosh g., chourasia s.k., vaishya a.k., yadav m., verma h.g. 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(2015) 3(5): 290-300 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article osteological characteristics of turkmenian stone loach, paraschistura cristata (cypriniformes: nemacheilidae) hoda azimi1, hamed mousavi-sabet*1, soheil eagderi2, saber vatandoust3 1department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, p.o. box 1144, guilan, iran. 2department of fisheries, faculty of natural resources, university of tehran, p.o. box 4314, karaj, iran. 3department of fisheries, babol branch, islamic azad university, babol, iran. article history: received 10 june 2015 accepted 27 september 2015 available online 2 5 october 2015 keywords: osteology metaschistura skeleton hari river basin iran abstract: formerly the turkmenian stone loach was the only member of the genus metaschistura based on osteological characters. but recently, it is placed in the genus paraschistura based on mtdna coi data. to provide a detailed description of the osteological characteristics of paraschistura cristata (berg 1898), ten specimens of p. cristata were collected from the hari river basin in iran and their osteological characteristics were examined. according to the results, p. cristata is osteologically characterized by a foramen in the ventral part of the exoccipital, two extra urohyals, sesamoid ossifications, trapezoid-shaped prevomer, three basibranchials, five hypural, lack of bony bridge between the parietal and pterotic, having over 20 procurrent rays supporting the adipose crest. the detailed skeletal description of p. cristata showed that this species can be easily distinguished from the related genera. despite the mtdna coi result, the osteological data of this species showed some features to describe it as a distinctive genus, but this needs the osteological data of the all other paraschistura species to be compared. introduction stone loaches, family nemacheilidae, are mostly small fishes occurring in freshwaters of asia and its islands (including the greater sunda islands), europe, and northeast africa (coad, 2015). the members of this family has a great diversity in iranian inland waters after the cyprinidae (nelson, 2006; azimi et al., 2014). because of little systematic and phylogenetic studies, classification of this taxa is complex and many experts are trying to determine their phylogenic status (prokofiev, 2010). recent classifications of this taxa have been mostly based on external morphological features and to a small extent on osteological and molecular data (bănărescu and nalbant, 1995; prokofiev, 2009, 201; freyhof et al., 2015; azimi et al., 2015). hence, classification and phylogenetic status of this group is still a subject to debate and osteological features can * corresponding author: hamed mousavi-sabet e-mail address: mousavi-sabet@guilan.ac.ir play an important role in this regard. the first osteological studies on the family nemacheilidae were performed by reagan (1911), who separated subfamily nemacheilinae from the family cobitidae. recent taxonomic studies on the loaches of iran are available based on osteological features (jalili and eagderi, 2015; jalili et al., 2014; jalili et al., 2015a; jalili et al., 2015b; mafakheri et al., 2014; mafakheri et al., 2015). the only comprehensive phylogenic study on the members of family nemacheilidae was done by prokofiev (2010), who described the genus metaschistura and considered the turkmenian crested loach as the only member of this genus. among the members of nemacheilids, paraschistura prokofiev, 2009 is a newly described genus, and therefore, not all of its species have been fully examined and ascribed to it or related genera (coad, 2015). recently vatandoust and eagderi (2015) 291 azimi et al./ osteology of paraschistura cristata described p. ilamensis from the tigris river drainage as the first species of the genus of paraschistura from iranian part of this basin. thereafter, freyhof et al. (2015) reviewed the genus paraschistura from iran and described six new species, including p. abdolii (from the sirjan basin and the western tributaries of the hamun-e jaz murian basin), p. aredvii (from the zohreh drainage), p. hormuzensis (from the minab drainage), p. naumanni (from the kol drainage, the mond drainage and the lake maharlo basin), p. pasatigris (from the karun and karkheh drainages) and p. susiani (from the jarahi drainage) based on the morphological and molecular (the mtdna coi barcode region) data set. based on the described characteristics for p. pasatigris, it is suggested that p. pasatigris is a synonym of p. ilamensis. freyhof et al. (2015) provided the diagnostic characters for all eleven recognized species and treated metaschistura prokofiev, 2009 as a synonym of paraschistura prokofiev, 2009. paraschistura cristata, known as turkmenian stone loach, is distinguished from the other crested loaches in the north east of iran by a unique color pattern and a short and thick crest. this species is found in rivers of north slope of the kopet dag mountain ranges in turkmenistan and its appearance in iranian waters is due to the connection of these rivers with iran’s borders (coad, 2015). as the basal classification of the family nemacheilidae has been done based on osteological characteristics, the present study was conducted to provide a detailed osteological characteristic of p. cristata. materials and methods ten specimens of p. cristata (55.1-74.6 mm in standard length) were collected by electrofishing device from the kalat stream, in the hari river basin, located in khorasan-e-razavi province, northeast of iran. after catching, samples were anesthetized with clove solution and fixed in 10% buffered formalin. for osteological examination, the specimens were cleared and stained using alcian blue and alizarn red based on taylor and van dyke (1985). the skeletal structures were dissected and photographed using scanner (epson v600) equipped with a glycerol bath. the skeletal structures of the cleared and stained specimens were observed and studied by ms5 leica stereomicroscope. the skeletal elements were drawn based on digital pictures using coreldrawx6 software. the terminology and abbreviation of the skeletal elements follows prokofiev (2009, 2010). results neurocranum: the neurocranium is almost wide and flat posteriorly, forming the maximum width of the skull at the level of the pterotic. the posterior part of the neurocranum is oval-shaped and flattened, while its anterior part is narrow. the ethmoid region consists of the paired lateral ethmoid and unpaired prevomer and supraethmoid-ethmoid bones (fig. 2a). the supraethmoid-ethmoid is narrower in the middle. this bone is vertically fused to the prevomer figure 1. lateral view of turkmenian stone loach paraschistura cristata. 292 int. j. aquat. biol. (2015) 3(5): 290-300 figure 2. the neurocranium of paraschistura cristata (from the dorsal (a), lateral (b), and ventral (c) sides): pr-bo: basioccipital process; bo: basioccipital; epo: epiotic; exo: exoccipital ; fon: fontanelle; fr: frontal; fr-exo: foramen exoccipital; let: lateral ethmoid; orb: orbitosphenoid; pa: parietal; pe: prevomer; pro: prootic; ps: parasphenoid; pto: pterotic; pts: pterosphenoid; se: supraethmoid-ethmoid; soc: supraoccipital; spo: sphenotic. 293 azimi et al./ osteology of paraschistura cristata and firmly connected to the frontal by a zigzagshaped suture posteriorly. in the middle of the narrow part of the neurocranium, the paired lshaped lateral ethmoids are situated. the anterior part of this bone is not completely ossified and joined laterally to the orbitosphenoid. the prevomer is almost trapezoid in shape having two processes with rounded edges antero-laterally. furthermore, this bone is connected to the orbitosphenoid and parasphenoid posteriorly (fig. 2b). there is a small bone series anterior to the ethmoid region, including paired bones of the preethmoid-ii, sesamoid and prepalatine, and unpaired kinethmoid (fig. 3a-c). the preethmoid-ii is small and rod-like, connecting to the prepalatine laterally (fig. 3b). it is also connected to the prevomer and maxilla anteriorly. the kinethmoid is a small and free bone, positioning between the maxillae. two small sesamoid bones are present in the ethmoid region (fig. 3a). the orbital region includes the frontal, orbitosphenoid, ptersphenoid, parasphenoid, and sclerotic bones (fig. 2a). the frontals are the largest bones of the skull roof. they involve about half of the length of the neurocranium separating by the fontanel posteriorly. the frontals are connected to the orbitosphenoid, pterosphenoid and sphenotic laterally, and parietal posteriorly (fig. 2a, b). the orbitosphenoid is connected to the parasphenoid ventrally and to the ptersphenoid postero-dorsally. all these bones together create a large olfactory foramen (fig. 2b). the ptersphenoid is connected to the frontal and sphenoid dorsally and posterolaterally, respectively. also, the posterior margin of the ptersphenoid is curved, creating a cavity along with the prootic and parasphenoid (fig. 2c). the parasphenoid is a large bony element at the base of the neurocranium, and it is extended from the prevomer to the basioccipital. the parasphenoid is wider in the middle part forming the alar and bifurcated at its two ends (fig. 2c). the otic region comprises of the parietal, sphenotic, pterotic, prootic, and epiotic (fig. 2). the parietal is connected to the supraoccipital and epiotic posteriorly and to the pterotic and sphenotic laterally. these paired bone is separated by the fontanel (fig. 2a). the pterotic is quarter-circle in shape and connected to the epiotic and sphenotic posteriorly and to the prootic and exoccipital ventrally. the sphenotic positions at the rear of the orbit, creating a part of the lateral wall of the skull (fig. 2b). the sphenotic is connected to the pterotic ventrally and to the parietal postero-dorsally. in the anterior part of the basioccipital, the paired prootics are the largest bones of the ventral face of the skull (fig. 2c). these paired bones are connected to each other latero-posteriorly. there is a depression in the antero-lateral part of these paired bones. the anterior and postero-lateral parts of this depression are connected to the parasphenoid and pterotic, respectively. the epiotic is the most posterior element of the otic region (fig. 2c). the occipital region comprises of the exoccipital, supraoccipital, and basioccipital bones. the supraoccipital is a pentagon-shaped bone and connected to the exoccipital dorsally and to the fontanel anteriorly. the fontanel accounts a length equal to approximately one-fourth of the neurocranium. the paired exoccipitals form the foramen exoccipitalis. the basioccipital is positioned between the two exoccipitals and connected to the prootic anteriorly. also, this bone has a ring-like process posteriorly (process basioccipital) (fig. 2b). the neurocranium has two articulatory facets for the articulation with the heads of the hyomandibular. the anterior facet is formed figure 3. bones of the ethmoid region in paraschistura cristata. ke: kinethmoid; ses: sesamoid (a); peth-ii: preethmoid-ii (b); ppl: prepalatine (c). 294 int. j. aquat. biol. (2015) 3(5): 290-300 by the ptersphenoid, sphenotic, and prootic, and the posterior one by the pterotic and sphenotic (fig. 2b). jaws: the upper jaw includes the maxilla and premaxilla (fig. 4a). the premaxilla is a l-shaped bone with two processes, i.e. ascendes and alveoral premaxilla processes. the horizontal part of the premaxilla is arc-shaped while, the vertical part is narrow and longer. the maxilla is a large laminar bone with a broken contour and slightly twisted along its longitudinal axis. this bone is wider in the middle and connected to the prepalatine and preethmoid-ii. the lower jaw is composed of the dental, articular, retroarticular, and cronomeckelian (fig. 4b). the dental is the largest element of the lower jaw and has two parts, including the anterior ramus dentalis and postero-dorsal coronoid process. this bone is connected to the articular postero-dorsally and to the retroarticular dorsally. the articular is connected to the dental anteriorly, to the retroarticular ventrally, and to the quadrate posteriorly. the coronomeckelian is a small and triangular bone which is situated dorso-medial to the articular. suspensorium: the suspensorium is composed of the autopalatine, endopterygoid, ectopterygoid, metapterygoid, hyomandibular, quadrate and symplectic bones (fig. 5). the suspensorium is inclined forward. the autopalatine possesses a blade-like process in the middle which is connected to the prevomer laterally. furthermore, this bone is connected to the prepalatine anteriorly, and to the preethmoid-ii and endopterygoid posteriorly. the endoptrygoid is wide and elongated and connected to the metaptrygoid and ectoptrygoid ventrally. the metaptrygoid is roughly rectangular in shape which is situated between the hyomandibular and quadrate. the anterior part of the metaptrygoid is wider. the hyomandibular is a large bone which stretched longitudinally and its dorsal part is wider. the hyomandibular is connected to the interhyal and symplectic ventrally, and to the metaptrygoid anteriorly. in addition, the hyomandibular possesses two condyles on its dorsal rim (anterior and posterior hyomandibular condyles) for articulation to the neurocranium. the quadrate has a pointed and stretched ventral process which is inclined posteriorly. there is also a smaller process at the anterior margin of the quadrate inclining anteriorly (fig. 5). this bone is connected to the endoptrygoid dorsally and to the metaptrygoid posteriorly. the symplectic is almost triangular in shape and positioned beneath the endoptrygoid and the posterior to the quadrate. the ectopterygoid bears a process anteriorly and a downward semicircular process ventrally. the metapterygoid, hyomandibular, symplectic and interhyal are connected by a ligamentous sheet covering their medial faces. opercular series: the operclar apparatus includes the opercle, preopercle, subopercle and interopercle (fig. 5). the opercle is the largest bone in this complex which has a rod-shaped process anterofigure 4. upper jaw (a) and lower jaw (b) bones in paraschistura cristata. art: articular; cm: coronomeckelian; den: dental; mx: maxilla; pmx: premaxilla; rar: retroarticular. figure 5. lateral view of suspensorium in paraschistura cristata. apl: autopalatine; ect: ectopterygoid; end: endopterygoid; hm: hyomandibular; io: interopercle; mtp: metapterygoid; op: opercle; po: preopercle; q: quadrate; so: subopercle; sym: symplectic. 295 azimi et al./ osteology of paraschistura cristata dorsally for connection to the operculi levator muscle. in addition, the ventral margin of this bone is connected to the subopercle. the subopercle is a stretched bone and connected to the interopercle anteriorly. the interopercle is a narrow and long bone and wider in the middle part. branchial arches: the branchial arch consists of the basibranchial, hypobranchial, ceratobranchial, epibranchial, and infrapharyngobranchials (fig. 6). there are three unpaired basibranchials. five paired ceratobranchials are the largest elements of the branchial arch and the fifth one is modified as pharyngeal teeth. three paired hypobranchials are situated between the ceratobranchial and basibranchial bones. also, there are four paired epibranchials positioning between the ceratobra nchials and two pairs of the infrapharyngobranchials. hyoid arch: the hyoid arch is composed of the basihyal, hypohyal, ceratohyal, epihyal, interhyal, urohyal, extra urohyal, and branchiostegal rays (fig. 7). the unpaired basihyal is a t-shaped bone and its anterior part is wider. the paired hypohyal are consisted of the dorsal and ventral parts. the ceratohyal is the largest bone of the hyoid arch. the epihyal is a triangular-shaped bone with a pointed process posteriorly. the interhyal is a small and cylindrical bone and connected to the epihyal ventrally, and to the hyomandibular and symplectic dorsally. the unpaired urohyal is narrow and triangular in shape and bears two ventral and dorsal parts. the dorsal part is blade-like and perpendicular to the ventral part. there are two extra urohyals between the hypohyals and urohyal. the branchiostegal rays are extended to the dorsal margin of the subopercle. the first and second branchiostegal rays are attached to the middle of the ceratohyal and at the junction of the ceratohyal and epihyal, respectively. the third one is connected to the middle of the epihyal. pectoral girdle: the pectoral girdle consists of the cleithrum, supracleithrum, coracoid, mesocoracoid, scapula, posttemporal, supratemporal, and radials (fig. 8). the supracleithrum is a wide bone and its anterior part has a projected process dorsally. this bone is connected to the cleithrum dorso-ventrally. the posttemporal is a thin and long bone which is connected to the epiotic posteriorly. the posttemporal is positioned between the supracleithrum and supratemporal. the supratemporal is small bone locating in the anterior part of the figure 6. branchial apparatus of paraschistura cristata. bbr: basibranchial; cbr: ceratobranchial; ebr: epibranchial; hbr: hypobranchial; pbr: infrapharyngobranchials. figure 7. hyoid arch of paraschistura cristata: bhy: basihyal; br: branchiostegale; chy: ceratohyal; dhy and vhy: dorsal and ventral hypohyal; ehy: epihyal; ihy: interhyal; uhy: urohyal; uhye: urohyal extra. 296 int. j. aquat. biol. (2015) 3(5): 290-300 posttemporal. the cleithrum is the largest element of the pectoral girdle and is connected to the supracleithrum dorsally and to the coracoid through mesocoracoid latero-medially. the anterior part of the coracoid is narrow and inclined downward, whereas its posterior part is wider. the scapula is almost trapezoidal in shape and positioned between the cleithrum and coracoid. this bone has a dent at its posterior part and is connected to the first ray of the pectoral fin (fig. 8). the pectoral girdle possesses four cylindrical radials. pelvic girdle: the pelvic girdle consists of the paired pelvic bones and radials (fig. 9c). it is not connected to another skeletal element and enclosed by muscles. three radials of the pelvic girdle are small and round in shape and positioned between the pelvic bones and rays. the anterior part of the pelvic bone is narrower. the pelvic bones have three processes including the anterior pubic process that has some depression laterally, the postero-lateral iliacus process that the rays are connected on it, and the posterior ischiadicus process. in addition, there is a developed bone in this collection viz. styloid positioned outside the rays. dorsal fin skeleton: the dorsal fin skeleton consists of 10 pterygiophores and one stay (fig. 9a). the first pterygiophore is positioned in front of the ninth centrum and there are 4 unbranched and 7½ branched rays (fig. 9a). anal fin: the anal fin is composed of 7 pterygiophores and one stay. in addition, this fin bears 3 unbranched and 5½ radiating rays (fig. 9b). the first pterygiophore of the anal fin is positioned in the front of the twenty-first centrum. caudal skeleton: there are 5 hypurals in the caudal skeleton. the hypural-1 is wide with a pointed process posteriorly (fig. 10). the hypural-1 is also connected to the parahypural ventrally. the figure 8. pectoral girdle of paraschistura cristata. cl: cleithrum; cor: coracoid; mcor: mesocoracoid; pect-r: ray of the pectoral fin; rad: ossified pectoral radial; sc: scapula. figure 9. (a) dorsal fin, (b) anal fin and (c) pelvic girdle of paraschistura cristata. adp: anal distal pterygiophore; dfr: dorsal fin rays; dfs: dorsal fin spin; dr: distal radial; mp: mesial pterygiophore; mr: medial radial; pp: pterygiophore; sty: stay. 297 azimi et al./ osteology of paraschistura cristata parahypural is flat with a triangular process midventrally and its posterior end is connected to the centrum. the hypurals-3, 4, and 5 are situated between the pleurostile and hyporal-2. the hypural1 and hypural-2 are fused medially. the epural bone is situated between the pleurostyle and neural process from the second pleural of the centrum (fig. 10). in addition, the haemal spine of the second pleural is wider compared with the previous one. weberian apparatus and swim bladder capsule: the weberian apparatus consists of the claustrum, scaphium, intercalarium, and tripus (fig. 11a, b). the claustrum is round in shape and situated ventral to the scaphium. in addition, the claustrum is connected to the supraneural-2 dorsally. the intercalarium is small and positioned between the scaphium and y-shaped tripus. the first centrum has two lateral processes which are ligamentously connected to the pectoral girdle. the second, third, and fourth centra participate in the formation of bony capsule. the second and third centra are fused. the third centrum is situated between the supraneural-2 and fourth centrum. the parapophysis of the forth centrum is modified and has processes that forms the posterior part of the bony capsule. in the lateral of the bony capsule, two openings are observed that posterior one is larger. the surface of the bony capsule is alveolar. the right and left lobes of the bony capsule are symmetrical and divided by the manubrium (fig. 11b). discussion the present study provided a detailed skeletal description of paraschistura cristata. the lateral ethmoid, supraethmoid-ethmoid and prevomer of this species were fused as other loaches, with the exception of lefua spp., oreonectes platycephalus, yunnanilus pleurotaenia, triplophysa microphthalma, t. tenuis (prokofiev, 2010) and schistura fasciolata (sawada, 1982). in p. cristata, the supraethmoid-ethmoid is tightly connected to the frontals, as other loaches with the exception of indoreonectes evezardi (sawada, 1982). the junction of the lateral ethmoid to the neurocranium in p. cristata is at the level of the anterior margin of the orbitosphenoid similar to oxynoemacheilus kiabii (mafakheri et al., 2014; mafakheri et al., 2015) and o. bergianus (jalili and eagderi, 2015), but according to prokofiev (2010), the lateral ethmoid of loaches is stationarily joined with the supraethmoid-ethmoid. similar to o. kiabii (mafakheri et al., 2014) and o. bergianus (jalili and eagderi, 2015), there is no preethmoid-i in p. cristata, while in species of the genera lefua, oreonectes, yunnanilus, eonemachilus, micronoemacheilus, hedinichthys, orthrias and triplophysa, a paired preethmoids-i joined to the lateral edge of the antero-lateral processes of the prevomer is present (prokofiev, 2010). in p. cristata similar to o. kiabii (mafakheri figure 10. the caudal skeleton of paraschistura cristata. epu: epural; hp: hypural; npu2: neural processes of the second preural centrum; hpu2: hemal processes of the second preural centrum; ph: parhypural; pst: pleurostyle. figure 11. the swim bladder capsule of paraschistura cristata. cla: claustrum; dpr-2 -4: descending processes of the second and fourth centra; hpr-2-4: horizontal processes of the second and fourth centra; na4: neural arch 4; sca: scaphium; sn2: supraneural 2; sn3: supraneural 3. 298 int. j. aquat. biol. (2015) 3(5): 290-300 et al., 2014) and p. nielseni (azimi et al., 2015), there are sesamoid ossifications. the presence of the sesamoid ossifications in nemacheilids has been rejected by sawada (1982); however, this bone is found in paracobitis malapterura, p. longicauda, dzihunia amudarjensis and oxynoemacheilus angorae (prokofiev, 2004, 2009). the prevomer of p. cristata is trapezoid in shape, while this bone is square-shaped in o. kiabii (mafakheri et al., 2014) and p. nielseni (azimi et al., 2015) and rectangularshaped in o. bergianus (jalili and eagderi, 2015). in addition, the supraethmoid-ethmoid of p. cristata is thin and longer compared to o. kiabii (mafakheri et al., 2014). in p. cristata similar to p. sargadensis, there is no bony bridge between the parietal and pterotic (prokofiev, 2009). the occipital part of the neurocranium is less than one-third of its length in p. cristata, similar to the majority of the genera (prokofiev, 2010). there is a foramen in the ventral part of the exoccipitals in p. cristata and p. nielseni (azimi et al., 2015), whereas such a state is not observed in o. kiabii and o. bergianus (mafakheri et al., 2014; jalili and eagderi, 2015). prokofiev (2010) pointed out that the coronomeckelian is connected to the base and dorsal edge of the coronoid process in loaches but this bone is connected to the medial face of the articular in p. cristata and o. kiabii (mafakheri et al., 2014) and to the dorso-medial part of the dental in p. nielseni (azimi et al., 2015). the anterior facet of the hyomandibular in p. cristata, p. nielseni (azimi et al., 2015), o. kiabii (mafakheri et al., 2014) and o. bergianus (jalili and eagderi, 2015) are formed by the prootic, pterosphenoid and sphenotic, and posterior facet formed by the pterotic and sphenotic. while, these observations were inconsistent with the report of prokofiev (2010) regarding nemacheilids. there are three unpaired basibranchials in p. cristata, whereas in p. nielseni, o. kiabii and o. bergianus four basibranchials are present. in p. cristata, two extra urohyals similar to o. kiabii and o. bergianus are exist, whereas this two extra urohyals were not found in p. nielseni (azimi et al., 2015). the number of the infrapharyngobranchials is two pairs in p. cristata, but this bone is three pairs in o. kiabii and o. bergianus. in the caudal skeletal of p. cristata, five hypurals are present, while in o. bergianus, the hypural number is six (jalili and eagderi, 2015). in addition, the number of the procurrent rays are less than 15 and densely grouped at the base of caudal fin in p. sargadensis and p. nielseni (azimi et al., 2015), whereas in p. cristata, over 20 procurrent rays support the adipose crest (prokofiev, 2009). the surface of the bony capsule is alveolar in p. cristata, whereas a non-alveolar bony swim bladder capsule is found in p. nielseni (azimi et al., 2015), based on the observed skeletal features, p. cristata can be distinguish from the other members of family nemacheilidae by some features e.g. bearing a foramen in the ventral part of the exoccipital, two extra urohyals, sesamoid ossifications, trapezoidshaped prevomer, three basibranchials, five hypurals, lack of bony bridge between the parietal and pterotic, and having over 20 procurrent rays supporting the adipose crest. since identification of species and populations of this family using meristic characteristics and color pattern is difficult, uncertain, and sometimes unfeasible; therefore, application of the osteological characteristics can be considered as an appropriate method for taxonomic studies of this taxa. finally, the results showed that this species has some unique osteological characters compared to the related taxa. hence, its osteological characters can help to better understanding of its taxonomic position as a distinct genus, if more details data about the skeletal structure of the other paraschistura species are available. however, despite the mtdna coi results, validation status of the genus metaschistura needs more taxonomic examinations. therefore, further taxonomic study is suggested to clarify the taxonomic status of the turkmenian stone loach. references azimi h., mousavi-sabet h., eagderi s. (2015). osteological characteristics of paraschistura nielseni 299 azimi et al./ osteology of paraschistura cristata (nalbant & bianco, 1998) (cypriniformes: nemacheilidae). iranian journal of ichthyology, 2(3): 155-164. bănărescu p.m., nalbant t. (1995). a generical classification of nemacheilinae with description of two new genera (teleostei: cypriniformes: cobitidae). travaux du museum d'histoire naturelle grigore antipa, 35: 429-496. coad b. (2015). fresh water fishes of iran. retrieved from http://www.briancoad.com. freyhof j., sayyadzadeh g., esmaeili h.r., geiger m. (2015). review of the genus paraschistura from iran with description of six new species (teleostei: nemacheilidae). ichthyological exploration freshwaters, 26(1): 1-48. jalili p., eagderi s. (2015). cephalic osteology of safidrud stone loach oxynoemacheilus bergianus. journal of animal researches, 28(1): 21-34. jalili p., eagderi s., mousavi-sabet h. (2014). description of skeletal structure and cranial myology of cobitis keyvani (cypriniformes: cobitidae). international journal of aquatic biology, 2(6): 337345. jalili p., eagderi s., mousavi-sabet h. (2015a). descriptive osteology of a spined loach, cobitis avicennae from iranian part of the tigris basin. iranian journal of ichthyology, 2(1): 53-60. jalili p., eagderi s., mousavi-sabet h. (2015b). descriptive osteology of the endemic spined loach cobitis linea from iran. aacl bioflux, 8(4): 526-534. mafakheri p., eagderi s., farahmand h., mosavii-sabet h. (2015). descriptive osteology of oxynoemacheilus kermanshahensis (bănărescu and nalbant, 1966) (cypriniformes, nemacheilidae). croatian journal of fisheries, 73(3): 115-123. mafakheri p., eagderi s., farahmand h., mousavi-sabet h. (2014). osteological structure of kiabi loach, oxynoemacheilus kiabii (actinopterygii: nemacheilidae). iranian journal of ichthyology, 1(3): 197-205. nelson j.s. (2006). fishes of the world. john wiley and sons, inc. new york. 523 p. prokofiev a.m. (2004). osteology and relationships between loaches of the genus dzihunia (osteichthyes, balitoridae). zoologicheskii zhurnal, 83: 826-838. prokofiev a.m. (2009). problems of the classification and phylogeny of nemacheiline loaches of the group lacking the preethmoid i (cypriniformes: balitoridae: nemacheilinae). journal of ichthyology, 49: 874-898. prokofiev a.m. (2010). morphological classification of loaches (nemacheilinae). journal of ichthyology, 50: 827-913. regan c.t. (1911). the classification of teleostean fishes of the order ostariophysi. i. cyprinoidea. annals and magazine of natural history, 8: 13-32. sawada y. (1982). phylogeny and zoogeography of the superfamily cobitoidea (cyprinoidei, cypriniformes). memoirs of the faculty of fisheries of hokkaido university, 28: 65-223. taylor w.r., van dyke g.c. (1985). revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. cybium, 9: 107-119. int. j. aquat. biol. (2015) 3(5): 290-300 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی paraschistura cristataماهی جويباری ترکمنی شناسی سگهای استخوانيژگیو (cypriniformes: nemacheilidae) 3صابر وطن دوست ،2ايگدری سهیل، *1حامد موسوی ثابتسید، 1هدی عظیمی .ایران، گیالن، 1111صندوق پستی ،صومعه سرا ،گیالن دانشگاهدانشکده منابع طبیعی، شیالت، گروه1 .ایران ،1111صندوق پستی کرج، تهران، دانشگاه طبیعی منابع و کشاورزی پردیس دانشکده منابع طبیعی، شیالت، گروه2 .ایران ،بابل، واحد بابل دانشگاه آزاد اسالمی ،شیالت گروه1 چکیده: توصیف شده بود. اما اخیراً این گونه metaschistura عنوان تنها گونه جنسهای استخوانی قبالً بهماهی جویباری ترکمنی براساس ویژگیسگ شناسیهای استخوانکردن و توصیف ویژگی منظور فراهمقرار گرفت. به paraschisturaدر جنس mtdna coi مولکولیهای براساس داده ایران جمع آوری گردید و رودحوضه آبریز هریاز p. cristataگونه از ، تعداد ده نمونه paraschistura cristata (berg 1898) گونه در فورامن یک داشتن شامل p. cristata شناسیاستخوان هایویژگی براساس نتایج، شناسی آن مورد بررسی قرار گرفت.های استخوانویژگی ایقاعده استخوان سه شکل، ایذوزنقه ومر پیش ،یسزاموئید هایاستخوان ،یاضاف اوروهیال استخوان دو خارجی، سریپساستخوان شکمی بخش . باشدمی چربی بالچه کننده حمایت شعاع 22 از بیش داشتن پرواتیک، و آهیانه بین استخوانی ارتباط فقدان المی،تحت استخوان پنج آبششی، مرغعلی. شود داده تشخیص های مرتبطجنس هایگونه دیگر از آسانی به تواندمی گونه این که داد نشان p. cristata گونه اسکلت جزئیات توصیف جنس متمایز یک عنوانهب آن صیفتو برای را هاویژگی برخی گونه این شناسیاستخوان هایداده ،coi میتوکندریایی ژن اساس بر مولکولی نتایج .باشدمی paraschisturaجنس هایگونه تمام شناسیاستخوان حاصل از هایداده مقایسه نیازمند امر این اما ،نشان داد .هریرود، ایران آبریز حوضه ،اسکلت، metaschistura ،شناسیاستخوان :کلمات کلیدی international journal of aquatic biology (2015) 3(4): 245-257 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article monsoon effects on the copepod community structure in the chabahar bay, oman sea neda fazeli*1, rasool zare2, seyed mohammad bagher nabavi3, saeed sanjani3 1guilan university, department of marine biology, rasht, iran. 2department of fisheries, faculty of natural resources university of tarbiat modarres, noor, iran. 3khorramshahr university of marine science and technology, department of biology, khorramshahr, iran. article history: received 6 july 2014 accepted 24 january 2015 available online 2 5 august 2015 keywords: abundance copepods diversity monsoon oman sea abstract: calanoid, cyclopoid, harpacticoid and poecilostomatoid copepods were investigated over the year at five stations in the chabahar bay, oman sea. this area is under the influence of the indian ocean seasonal monsoons. the samples were collected using vertical plankton tows with 100 µm mesh nets. copepods were identified into 20 genera and 59 species. calanoid formed about 15% to 62% and cyclopoid 26% to 39% of total copepod abundance. harpacticoid constituted about 6% in south west (sw)-monsoon and flourished well in pre (sw)-monsoon, formed 46% of copepod abundance. poecilostomatoid accounted for approximately 5% to 13% of the total copepods. the most dominant species were temora turbinata, paracalanus elegans, oithona nana and euterpina acutifrons. the results showed that the species composition and distribution of copepods differed between the monsoon seasons, due to changes in hydrographic conditions. furthermore, high abundance of small-sized copepods observed in offshore stations. introduction the chabahar bay is a small semi-enclosed bay on the southeastern coasts of iran (25°17'45"n, 60°37'45"e). this bay is connected to the indian ocean via oman sea being influenced by indian monsoonal winds (fazeli and zare, 2011). the asian monsoon in the oman sea is characterized by two distinct seasons separated by two transitional (intermonsoon) periods: the southwest (sw) monsoon from june through september, the northeast monsoon (ne) from december through march, the spring transition (pre-monsoon) in april and may and the fall transition (post-monsoon) in october and november (caulfield, 1990). the chabahar bay is one of the five major regions of the oman sea providing an ideal breeding ground for many fishes and shell fishes (wilson, 2000). the bay is located between chabahar and konarak. it has 14 km wide and a surface area of 290 km2. the average depth of the bay is 12 m (ranging from 8 to 22 m) (fazeli and * corresponding author: neda fazeli e-mail address: neda_fazeli200@yahoo.com zare, 2011; fazeli et al., 2013). the monsoon has remarkable effects in the region. a peak in chlorophyll-α biomass dominates in the indian ocean at sw monsoon (yoder et al., 1993). lower salinity water reaches the sea surface in coastal upwelling that occurs during sw monsoon along the coast of oman (morrison et al., 1998). water temperatures decrease from 27-29°c at oman sea (caulfield, 1990). during the fall transition, water temperatures cool slowly. average sea temperatures cool from 28-29°c in october to 27°c in november and oligotrophic conditions slowly return (caulfield, 1990). the cool, dry northeaster winds that characterize ne monsoon result in a typical winter time convection/nutrient enrichment scenario in the northern arabian sea (banse and mcclain, 1986; madhupratap et al., 1996). in this season, the monsoon results in a surface water mass with salinity between 35.5-36.5 and temperatures greater than 22°c as arabian sea. water 246 international journal of aquatic biology (2015) 3(4): 245-257 temperatures range from 25°c in april to 29°c in may (caulfield, 1990). the responses of the zooplankton community structure to seasonal changes in the physical environment have not been analyzed fully yet during 1990s. there is one trend, however, which has been noted in every seasonal study so far: the ne monsoon is characterized by increased abundance of cyclopoid copepods, such as the genera oithona and oncaea, compared with the sw monsoon. small calanoid copepods predominate during the sw monsoon (madhupratap et al., 1996). in coastal waters of the region, undinula vulgaris and paracalanus aculeatus were reproductive in both the ne and sw monsoons, along with cosmocalanus darwini and c. plumulosus in the sw monsoon (smith et al., 1998). many studies have described copepod community structure in many parts of indian ocean, arabian sea and the persian gulf (madhupratap, 1987; smith, 1995; savari et al., 2004) but little is known about the copepods of the chabahar bay (fazeli et al., 2010; fazeli and zare, 2011; fazeli et al., 2012; fazeli and zare, 2012; fazeli et al., 2013). therefore, the main objective of this study is to investigate the spatial and temporal variability in abundance and diversity of copepods and effect of monsoon on species composition of the chabahar bay. materials and methods sampling was conducted during four oceanography cruises, including august 2007 (sw monsoon), november 2007 (post-monsoon), february 2008 (ne monsoon) and may 2008 (pre-monsoon). five stations were investigated through the bay that have been provided in detailed by fazeli et al. (2013). four plankton samples were collected vertically at each station twice for counting, using a simple net with a mouth diameter of 30 cm and a mesh size of 100 μm and with a hydrobios flow meter mounted in the center of the net opening. samples were preserved immediately in 4-5% formalin, buffered to a ph of 8 with sodium tetra borate (borax), and identified to the lowest taxa. zooplankton abundance was expressed as ind. m-³ (somoue et al., 2005). at each station, prior to sampling, the environmental parameters were recorded using a ctd profiler lowered from the sea surface to near the bottom. seasonal changes of the environmental parameters viz. temperature, salinity and chlorophyll-α during studied period have been provided in detailed by fazeli et al. (2013). zooplankton was identified to species level using chen and zhang (1974), nishida (1985) and conway et al. (2003). a two-way anova was used to examine a significant of difference in abundance of zooplanktons amongst periods and locations. the pearson correlation were performed to determine the significance between environmental parameters and copepod genera abundance. species diversity was calculated using shannon-weaver diversity index (shannon and weaver, 1963) and species richness (margalef, 1968). the data were further subjected to hierarchical clustering analyze to identify the similarity between the stations based on the composition. this was calculated as a bray-curtis similarity index with log10 (x+1) data using primer version 5.2.8 (clarke and warwick, 1994). results abundance and composition: sixty six copepod species belonged to calanoid, cyclopoid, harpacticoid and poecilostomatoid were identified. copepod density was significantly higher during pre-monsoon (with a total abundance of 3276.81 ind. figure 1. total abundance of calanoid, cyclopoid, harpacticoid and poecilostomatoid by season in the chabahar bay. 247 fazeli et al/ monsoon effects on the copepod community structure in the chabahar bay sw m. post-m. ne m. pre-m. calanoid acartiidae acartia sp. 0.24 0.37 0.15 0.30 a. longiremis 0.27 0.54 0.32 centropagidae centropages tenuremis 0.91 0.52 12.81 1.14 clausocalanidae clausocalanus sp. 0.06 0.38 eucalanidae eucalanus subcrassus 0.29 0.57 1.36 e. sp. 0.11 0.00 0.04 e. crassus 0.30 0.18 0.04 e. attenuates 0.05 0.05 e. monachus 0.11 0.07 paracalanidae acrocalanus longicornis 1.63 0.31 0.30 a. gracilis 2.86 0.08 0.85 a. gibber 3.29 0.25 0.33 a. monachus 1.82 0.12 0.42 0.35 a. sp 1.62 0.73 0.71 0.56 calocalanus plumulosus 0.08 0.04 paracalanus crassirostris 1.49 2.81 0.34 0.79 p. elegans 4.70 5.60 2.11 0.85 p. aculeatus 2.23 2.29 0.21 0.57 p. denudatus 0.77 0.73 0.22 p. parvus 0.25 0.19 0.16 0.67 p. sp 0.40 4.95 1.45 pontellidae labidocera sp. 36.60 0.07 0.50 pseudodiaptomidae pseudodiptomus sp. 0.18 6.77 0.90 temoridae temora desicaudata 0.11 2.13 0.05 t. turbinate 1.02 7.66 12.35 7.94 t. stylifera 0.40 0.23 cyclopoid oithonidae oithona aculata 2.59 3.34 2.11 2.21 o. attenuate 4.78 3.71 2.56 1.31 o. brevicornis 0.44 3.36 5.60 1.69 o. plumifera 0.06 1.42 1.80 1.22 o. rigida 4.63 2.65 1.74 1.51 o. simplex 1.61 7.56 1.42 1.04 o. nana 4.72 8.66 13.84 8.63 o. ssp. 7.01 8.95 7.78 9.78 poecilostomatoid corycaeidae corycaeidae corycaeus andrewsi 1.33 0.89 2.07 7.01 c. asiaticus 0.42 0.38 0.32 2.61 c. erythraeus 0.11 0.42 0.36 c. pacificus 0.96 0.52 0.39 c. affinis 0.33 0.16 0.45 c. dalhi 0.52 0.49 c. speciosus 0.09 0.29 table 1. copepod relative abundance (%) in the chabahar bay. 248 international journal of aquatic biology (2015) 3(4): 245-257 m-³) and sw monsoon (with a mean of 2790.55 ind. m-³) than other seasons (fig. 1). among calanoids, the species belonged to the genera of acartia, acrocalanus, calocalanus, centropages, clausocalanus, eucalanus, labidocera, pseudodiaptomus, paracalanus and temora were the major components of copepod community during sw monsoon with an average of 1604.13 ± 1318.53 ind. m-³. in post–monsoon, calanoid copepods represented 354.30 ± 43.15 ind. m-³ showing the lowest abundance. thirty-four calanoid species representing 8 families and 12 genera were identified during four periods in the chabahar bay. some species were observed only in one season in very low abundance (table 1). acartia pacifica, a. erythraea, eucalanus pileatus and e. hyalinus were present only in sw monsoon. clausocalanus gracilis, e. miscanthus, sapphirina gastrica, s. nigromaculata, calocalanus styliremis, lucicutia flavicormis and l. gaussae were observed during post-monsoon and disappeared in other seasons. centropages furcatus, c. furcatus, pseudodiptomus marinus and c. minor were observed only in ne monsoon. the dominant calanoid throughout the year were temora turbinate and paracalanus elegans. cyclopoids were presented all year in large numbers with a density of 701.30 ± 75.05 ind. m-³ during postmonsoon. all identified species of cyclopoid belonged to the genus oithona. the lowest abundance was found during pre-monsoon with an average of 503.30 ± 293.95 ind. m-³. oithona fallax appeared only in ne monsoon. the dominant cyclopoid throughout the year was o. nana. harpacticoids had four genera, including clytemnestra, microsetella, macrosetella and euterpina. the maximum abundance was observed in pre-monsoon (with an average of 2122.76 ± 994.42 ind. m-³) which comprised 45.81% of total copepod abundance. harpacticoids had the lowest abundance in post-monsoon with an average of 352.80 ± 22.21 ind. m-³. clytemnestra scutellata showed the lowest abundance through the year and disappeared in sw monsoon. the dominant harpacticoids throughout the year were represented by e. acutifrons and macrosetella gracilis. the species of poecilostomatoid belonged to the genera corycaeus, oncaea and sapphirina. the highest abundance was found in pre-monsoon with a density of 276.64 ± 75.87 ind. m-³. corycaeus was present throughout the year but in large numbers in pre-monsoon. the next common poecilostomatoid was oncaea which it showed maximum abundance during post-monsoon. a small abundance of sapphirina was found during post-monsoon and ne monsoon and disappeared entirely during sw monsoon and pre-monsoon. the dominant sw m. post-m. ne m. pre-m. oncaeidae oncaea media 0.80 4.88 3.94 o. venusta 0.93 2.50 0.86 0.35 o. clevei 0.55 1.58 0.21 o. minuta 0.05 0.90 0.52 sapphirinidae sapphirina sp. 0.06 0.44 harpacticoid clytemnestridae clytemnestra scutellata 0.17 1.66 0.96 ectinostomatidae microsetella rosea 0.23 0.38 0.49 11.08 miraciidae macrosetella gracilis 0.40 3.19 1.02 19.51 euterpinidae euterpina acutifrons 5.39 11.82 6.55 14.26 table 1. continued. 249 fazeli et al/ monsoon effects on the copepod community structure in the chabahar bay poecilostomatoid throughout the year were corycaeus andrewsi and oncaea media. temporal and spatial variation of copepods: the variation of copepod genera showed a fluctuation figure 2. average copepod genera abundance in the chabahar bay during each sampling station. 250 international journal of aquatic biology (2015) 3(4): 245-257 seasonally and spatially (fig. 2). a significant increase in acartia was found during sw monsoon (with an average of 104.73 ± 62.83 ind. m-³) and post-monsoon (88.78 ± 52.33 ind. m-³) compared to other seasons. acrocalanus (with an average of 355.83 ± 116.06 ind. m-³) and labidocera (with an average of 5503.76 ± 5492.75 ind. m-³) also showed highest abundance during sw monsoon. in post-monsoon, paracalanus (499.01 ± 88.73 ind. m-³), oncaea (366.55 ± 211.09 ind. m-³) and eucalanus (65.64 ± 18.84 ind. m-³) showed the highest abundance. a significant increase of euterpina was found during post-monsoon and ne monsoon. ne monsoon showed the maximum abundance of temora (1866.26 ± 485.84 ind. m-³), pseudodiaptomus (933.15 ± 292.28 ind. m-³), clausocalanus (98.83 ± 40.20 ind. m-³) calocalanus (86.09 ± 151.79 ind. m-³), centropages (1888.40 ± 795.48 ind. m-³) and sapphirina (66.27 ± 46.29 ind. m-³). macrosetella gracilis and m. rosea showed a high abundance during ne monsoon compared to other periods. in pre-monsoon, the maximum abundance of corycaeus (382.64 ± 124.77 ind. m-³) was found. clytemnestra scutellata showed a higher abundance during pre-monsoon and ne monsoon. spatial variations in abundance of copepod are shown in figure 2. among calanoids, we observed the maximum average abundance of eucalanus and paracalanus at station 1, centropages and clausocalanus at station 2, pseudodiaptomus at station 3, temora, acartia and acrocalanus at station 4. in pontellidae, labidocera showed a higher abundance in station 5. calocalanus was observed only at stations 2 and 5. among cyclopoids, the maximum abundance of oithona was found at station 2. among poecilostomatoids, oncaea showed a higher abundance at stations 1 and 2, whereas sapphira showed the maximum abundance at station 3. corycaeus showed the highest abundance at stations 3 and 4. among harpacticoids, a low abundance of e. acutifrons was found at station 2, while m. gracilis and m. rosea showed a higher average abundance at station 1. a higher abundance of c. scutellata was found in stations 3 and 4 (fig. 3). figure 4 shows cluster analysis for calanoid, cyclopoid, harpacticoid and poecilostomatoid to investigate similarities between stations based on density data. the results indicated the presence of two groups; station(s) in group i separated from other stations in group ii. in calanoids, the highest similarity of stations was observed in stations 2 and 3. stations 1 and 4 showed the maximum similarity of cyclopoids density. in harpacticoids, the highest similarity of stations was observed in stations 2 and 4. in poecilostomatoids, the highest similarity of stations was presented in stations 1 and 2. species diversity and richness: in sw monsoon, calanoid formed by the relatively high diversity figure 3. average density of dominate copepod species in the chabahar bay. 251 fazeli et al/ monsoon effects on the copepod community structure in the chabahar bay index and species richness (2.28/2.60), while a low value was observed in pre-monsoon (1.01/0.44) (table 2). the highest and lowest cyclopoid species diversity and richness were observed in ne monsoon and pre-monsoon, respectively. in poecilostomatoids, the highest and lowest species diversity and richness were observed during postmonsoon and pre-monsoon, respectively. harpacticoid species diversity and richness were relatively low during four periods. the highest species diversity and richness of harpacticoid was recorded in pre-monsoon (0.97/0.28) and the lowest in sw monsoon (0.36/0.15). environmental parameters and zooplankton: a pearson product correlation was calculated to examine whether any relationship existed between copepod genera and environmental parameters in the chabahar bay. the results are presented in table 3. discussion ne monsoon consists of the moderate wind-driven mixing, a net flux of heat from the ocean to the figure 4. cluster analyses showing similarity of stations during monsoonal seasons based on calanoid, cyclopoid, harpacticoid and poecilostomatoid density in the chabahar bay. 252 international journal of aquatic biology (2015) 3(4): 245-257 atmosphere, and elevated evaporation (wiggert et al., 2000). apparently, nutrients transported via this wintertime mixing fuel the subsequent spring phytoplankton bloom as defined by the appearance of the chlorophyll-α in the chabahar bay which confirmed the record of kumar and prasad (1999) in arabian sea. high temperature was observed in sw monsoon result in high sunlight. in post-monsoon and ne monsoon, temperature decreased throughout the bay by decreasing sunlight. salinity did not vary significantly during warm and cold season as evaporation is high during cold season (wiggert et al., 2000). water temperature, chlorophyll-α and salinity in the chabahar bay were similar to those of caulfield (l990) in oman sea and arabian sea. the variation of species among the four sampling seasons is apparently influenced by monsoon (chen, 1992). in summer, by the prevailing sw monsoon some species e.g., a. gibber, a. gracilis, labidocera sp., o. rigida and o. attenuate were dominant. labidocera sp. was the most common species (36%) in this season but showed a low abundance during post-monsoon and ne monsoon, and disappeared during pre-monsoon. acrocalanus gibber and a. gracilis were abundant during sw monsoon but decreased during other seasons which confirmed the record of vengadesh et al. (2009). trophic resources may play an important role in controlling of acrocalanus in tropical environments (gusma and mckinnon, 2009). it seems that temperature play a major role in abundance of o. rigida and o. attenuate as they were positively related to temperature. in autumn, the prevailing post-monsoon declined, the copepod abundance was observed that it can be explained due to decrease in chlorophyll-α concentrations. the chabahar bay seems to have an sw m.(h/d) postm.(h/d) ne m. (h/d) pre-m.(h/d) calanoid station 1 2.73(3.83) 2.36(2.46) 2.51(2.61) 1.33(0.46) station 2 2.68(3.01) 2.82(2.50) 1.60(0.97) 1.85(0.92) station 3 2.22(1.89) 1.61(0.65) 1.56(0.56) station 4 2.66(2.66) 2.39(1.84) 1.66(1.04) 1.89(0.83) station 5 1.13(1.63) 0.59(0.86) 1.71(1.23) mean 2.28(2.60) 1.95(1.66) 1.80(1.28) 1.01(0.44) cyclopoid station 1 1.62(0.75) 1.70(0.83) 1.77(0.80) 0.83(0.22) station 2 1.89(0.93) 2.02(0.77) 1.78(0.77) 1.89(0.74) station 3 1.68(0.59) 1.59(0.69) 1.64(0.73) 1.38(0.50) station 4 1.58(0.70) 1.62(0.70) 0.91(0.44) 0.69(0.14) station 5 1.50(0.48) 1.26(0.49) 1.50(0.75) mean 1.64(0.69) 1.63(0.69) 1.52(0.69) 0.95(0.32) harpacticoid station 1 0.84(0.29) 0.97(0.38) 0.35(0.12) 0.89(0.20) station 2 0.86(0.34) 0.91(0.26) 1.20(0.37) 0.74(0.22) station 3 0.10(0.12) 1.18(0.37) station 4 0.11(0.12) 0.47(0.25) 0.54(0.14) 1.21(0.36) station 5 0.44(0.13) 1(0.37) 0.86(0.25) mean 0.36(0.15) 0.57(0.22) 0.61(0.20) 0.97(0.28) poecilostomatoid station 1 1.67(0.85) 2.16(1.43) 1.80(0.99) 0.63(0.16) station 2 1.39(0.58) 1.66(1.27) 0.69(0.19) 0.98(0.40) station 3 2.07(1.15) 1.55(0.63) 0.91 (0.24) station 4 1.37(0.58) 2.01(1.39) 0.98(0.27) station 5 0.18(0.28) 0.63(0.18) 0.91(0.63) 0.37(0.13) mean 0.92(0.45) 1.70(1.08) 0.99(0.48) 0.77(0.24) table 2. diversity indices (h) and species richness (d) of calanoid, cyclopoid, harpacticoid and poecilostomatoid. 253 fazeli et al/ monsoon effects on the copepod community structure in the chabahar bay oligotrophic condition during this season similar to arabian sea (baars et al., 1998). temora turbinate, o. simplex, o. media and m. gracilis were the most common species in this season. centropages tenuremis, t. turbinate, o. media and pseudodiptomus sp. were dominant during winter by the prevailing ne monsoon. entrainment of nutrients into the upper layer by winter cooling was caused producing phytoplankton blooms in this season similar to those of the gulf of aden (baars, 1998) and arabian sea (kumar and prasad, 1999). on the other hand, abundance of c. tenuremis, t. turbinate and pseudodiaptomus sp. was negatively related to temperature. it was stated that there is a negative relationship between nutrients and temperatures below 22°c. high concentrations of the phosphates and low temperatures allow development of different plankton compartments (roy, 1992). the joint effect of high nutrients and low temperature can result in high abundance of some opportunistic species such as t. turbinate (madhupratap, 1987) which is the major species of temora in the chabahar bay. they almost flourish in high chlorophyll-𝛼 concentration (madhupratap, 1987). this observation confirmed those of hsieh and chiu (2002), hwang and wong (2005) and shih and chiu (1998) along coast of taiwan. in spring, by the prevailing pre-monsoon c. andrewsi, microsetella rosea and m. gracilis were dominant, while they decreased significantly during other seasons. a high abundance of harpacticoid was remarkable during this season. it can be explained by their adaptive strategy to overcome the harsh mechanical disturbances which occur in premonsoon. these copepods are always reproductively active, and tolerant to harsh climatic regime (mantha et al., 2012). paracalanus elegans, t. turbinate, o. nana, e. acutifrons were occurred in all sampling seasons. the high abundance of these species during a year might be due to high tolerance to temperature and salinity variation (e.g., o. nana, nishida, 1985), reproductive adaptive natures (e.g. e. acutifrons, mantha et al., 2012) and opportunistic behavior (e.g., t. turbinate, madhupratap, 1987). similar to the present result, paracalanus spp. and t. turbinate were the most dominant copepods species in mida creek (mwaluma et al., 2003). also paracalanus, oithona, microsetella and oncaea were dominant in malaysia (nakajima et al., 2008). in this study, the high abundance of small-sized copepod such as o. media, p. elegans, t. turbinate and o. nana were observed in offshore samples which confirmed those of rezai et al. (2004) in the strait of malacca. these species showed positive relationship with depth. the results showed a chl(a) salinity temperatu re depth p. elegans 0.15 -0.42* -0.14 0.49* a. gibber a.gracilis -0.27 -0.25 -0.23 -0.37 -0.46* 0.29 -0.01 0.51* t. turbinate 0.31 0.01 -0.62** 0.47* pseudodiaptomus sp. 0.23 -0.19 -0.61** -0.22 centropages tenuremis 0.19 -0.16 -0.46* -0.10 labidocera sp. -0.13 -0.01 0.31 -0.21 o. nana 0.21 -0.31 -0.63** 0.65** o. attenuate 0.23 0.23 0.67** 0.23 o. rigida -0.28 0.23 0.53* 0.23 corycaeus andrewsi 0.28 0.53* 0.29 -0.39 oncaea media 0.05 -0.39 -0.32 0.55* euterpina acutifrons -0.07 0.37 -0.28 -0.55* macrosetella gracilis -0.05 -0.25 -0.52* 0.30 microsetella rosea -0.17 -0.21 -0.46* 0.24 table 3. pearson correlation of major environmental parameters and major species density (‘*’ significant at 0.05 level; ‘**’ significant at 0.01 level) 254 international journal of aquatic biology (2015) 3(4): 245-257 negative correlation between e. acutifrons and depth. this species is a near-shore species (jitchum and wongrat, 2009; russell et al., 1996; somoue et al., 2005) which showed higher abundances in the chabahar bay when salinity was the highest similar to those of moreira (1975). from sw monsoon to pre-monsoon, the calanoid and cyclopoid species diversity and richness tended to be lower, reflecting lower structured communities. the highest richness of harpacticoid was observed in pre-monsoon suggesting their high population density in this season. during post-monsoon, species richness of poecilostomatoid increased along with increasing population density. spatially, copepods showed higher species diversity and richness in offshore stations. a higher diversity in offshore stations was due to stable environmental conditions prevailing which permitted plankton community to increase diversify which confirmed those of sivasamy (1990) and shanthi and ramanibai (2011) in northern arabian sea. in conclusion, the species composition and distribution of copepods differed between the monsoon seasons, resulted in changes of hydrographic conditions. community structure of copepod changed by the prevailing monsoon in each season and caused some species flourished or disappeared. however, p. elegans, t. turbinate, o. nana and e. acutifrons were dominant through the year. the present information would form a useful tool for further ecological assessment in the chabahar bay. acknowledgments we wish to thank irina prusova for guide and iranian national center of oceanography (inco) for the encouragement and support. references baars m., schalk p., veldhuis m. 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(1993). annual cycles of phytoplankton chlorophyll concentrations in the global ocean: a satellite view. journal of global biogeochemical cycles, 7: 181-194. international journal of aquatic biology (2015) 3(4): 245-257 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved چکیده فارسی عمان دریایچابهار، خلیج پاروپایان جوامع ساختار بر مونسون اثرات 3سنجانی سعید ،3نبوی باقر محمد ،سید 2زارع رسول ،1فاضلی ندا .ایران رشت، گیالن، دانشگاه یا،شناسی درزیست گروه1 .ایران نور، مدرس، تربیت دانشگاه طبیعی، منابع دانشکده شیالت، گروه2 .ایران خرمشهر، خرمشهر، دریایی فنون و علوم دانشگاه ،شناسیزیست گروه3 چکیده: مدت طی عمان ریاید، چابهار خلیج ایستگاه پنج در هاپوسیلوستوماتوئید و هارپکتیکوئید سیکلوئید، کاالنوئید،شامل پاروپایان مطالعه، این در با النکتونیپ تور از استفاده با هانمونه. باشدمی هند اقیانوس مونسونفصلی بادهای تاثیر تحت منطقه این. گرفتند قرار بررسی مورد سال یک و درصد 22 تا 19 کاالنوئیدها. شدند شناسایی گونه 95 و جنس 21 از پاروپایان. شدند آوریجمع عمودی طور به و میکرون 111 چشمه فروانی. بود درصد 2 حدود تابستانه مونسون در هارپکتیکوئیدها فراوانی. دادندمی تشکیل را پاروپایان کل فراوانی درصد 35 تا 22 سیکلوئیدها درصد 13 تا 9 تقریباً پوسیلوستوماتوئیدها فراوانی. داد اختصاص خود به را پاروپایان کل فروانی درصد 62 و یافت افزایش بهاره مونسون در آنها euterpina و temora turbinata ، paracalanus elegans ، oithona nana شامل غالب هایگونه. بود پاروپایان کل acutifrons رایطش تغییر از ناشی که باشدمی متفاوت مونسون مختلف فصول در پاروپایان پراکنش و ترکیب که داد نشان نتایج. بودند .بود لساح نزدیک های ایستگاه از بیشتر ساحل از دور هایایستگاه در کوچک پاروپای هایگونه فراوانی این بر عالوه. باشدمی هیدروگرافیک .عمان دریای مونسون، تنوع، پاروپایان، فراوانی، :کلمات کلیدی int. j. aquat. biol. (2017) 5(1): 12-21; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article diatom community structure along physicochemical gradients in upland river segments of tamiraparani river system, south india meenakshisundaram amutha, murugan muralidharan*1 sri paramakalyani centre for environmental sciences, manonmaniam sundaranar university, alwarkurichi, 627 412, tamilnadu, india. article history: received 25 september 2016 accepted 9 january 2017 available online 2 5 february 2017 keywords: benthic diatoms water quality streams tamiraparani river abstract: this study examines the diversity and distributional patterns of benthic diatom assemblages in the upland streams of tamiraparani river system, southern part of western ghats of peninsular india. a total of 168 benthic diatoms representing 16 orders, 32 families, and 47 genera were enumerated. structuring of diatom community was dependent on the environmental conditions of the respective habitats. canonical correspondence analysis described both physical habitat quality and water parameter gradient and factors that correlated significantly were substrate type, water temperature, dissolved oxygen and nitrate content. gomphonema gandhii, cocconeis placentula and navicula cuspidata were strongly associated with sites with embedded substrates and clear water. diatoms could be effective in assessing physical habitat alterations in streams. introduction aquatic systems are complex with heterogeneous habitats to support a variety of biota having unique features and occupying specific niche (dudgeon et al., 2006). structural characteristics in running waters are important as they define the organisms that are associated with. unfortunately the increased dependability on freshwater resources for various human needs has immensely impacted the systems and organisms. biodiversity loss has also been enhanced by the policies that permit infrastructure development in various sectors that rely on lotic systems (andres et al., 2012). though these impacts are increasingly recognized, it has become essential to quantify the biotic responses to the altered conditions and propose better management options (bunn and davies, 2000). considerable progress has been made over the past four decades in designing and developing tools to monitor the contamination and potential sources of pollution of water bodies, however all such attempts are regarded incomplete without biological assessment to estimate the ecological damage of the systems (barbour et al., * corresponding author: murugan muralidharan e-mail address: muralistream@gmail.com 1999; karr, 2006). periphytons are among the few ecologically sensitive organisms that serve as indicators of system health and troves of biodiversity (taylor et al., 2007; larned, 2010; bere and mangadze, 2014). diatoms constitute integral part in most stream food webs (wehr and sheath, 2003) serving as primary food source and habitat for many invertebrates and juvenile fishes (lowe and pan, 1996, stevenson and pan, 1999). furthermore, they are regarded as good indicators to predict the ecological health of lotic systems because they occupy variety of habitats (cantonati and lowe, 2001; potapova and charles, 2005), sensitive to even subtle changes in the environmental conditions and also for their being easier to sample. streams and rivers are dynamic systems with inherent variability leading to temporal and spatial variation in water quality (palmer and poff, 1997; grubbs et al., 2007). the quality of a stream is generally determined by the factors influencing it, which also reflects the biological diversity depending on it. this forms the basis for predicting 13 int. j. aquat. biol. (2017) 5(1): 12-21 the quality of water by the type of biota that it supports (camargo and jimenez, 2007). diatoms are responsive to changes in the water chemistry and hence their distribution and abundance are influenced by environmental conditions (kelly, 1998; hering et al., 2006; urrea and sabatar, 2009). the other factors to influence diatom composition are the flow pattern and substrate type, characteristic of lotic ecosystems, wherein high and low flows have the potential to alter ecosystem structure (francoeur and biggs, 2006). habitats of diatoms are diverse (kelly, 1998) and unique for certain species (tang et al., 2006) as the reason all species found are not available everywhere but occur in different micro habitats (stevenson and pan, 1999; rimet, 2012). traits such as preferential colonization and ability to react to changing nutrient levels make diatoms effective candidates indicating water quality (hering et al., 2006; neustupa et al., 2013; fidlerová and hlúbiková, 2016). diatoms like fishes and other aquatic insects can be utilized for a basin-wide monitoring which further could be an essential part of comprehensive monitoring programme. head water reaches of river systems are critical, the disturbances that occur in this region could be detrimental to the ecological health of downstream. survey and studies on diatoms of indian peninsula is limited to few river systems and water bodies of some selected states with varied physiography (nandan and patel, 1984; mishra and saksena, 1993; trivedy and khatavkar, 1996; nautiyal et al., 2004; sharma et al., 2007; nautiyal and verma, 2009; ramanujam and siangbood, 2009; sah and hema, 2010; baba et al., 2011; siangbood and ramanujam, 2014; dwivedi and misra, 2015). despite the reason that sufficient works on periphyton are available and are being undertaken in waterbodies of southern india, most of them are related to diversity of algal community (suresh et al., 2011; selvin-samuel et al., 2012), except for a few that also discuss the ecological aspects of the species and the systems (venkatachalapathy and karthikeyan, 2012; venkatachalapathy et al., 2013). yet a lacuna remains on the ecology of diatoms and their ability to predict habitat integrity. data on the distribution of aquatic diversity at spatial and temporal scales and changes in the communities in relation to the perturbances would enable effective understanding of the consequent biological integrity. thus, the objective of this study was to explore and document the community composition of the benthic diatoms and to determine their relationship with environmental variables at various spatial reaches of the upland part along the main river and the tributaries of the tamiraparani river system of southern part of india. materials and methods study area: tamiraparani, medium sized perennial river basin in peninsular india, but a major river system in southern tamil nadu originates from the agasthiamalai or pothigai hills in the southern western ghats, flows eastwardly to an extent of 120 km to drain into the bay of bengal. the study sites include small rivers forming sub-basins (manimuthar, gadananathi, karupanathi, gundar and chittar) and the main river system (servalar and karaiyar). epilithic and epiphytic diatoms were sampled from eleven sites of tamiraparani river over a period of eight months during august 2013-april 2014. site selection was aimed at having comparison of upstream river sections of tributaries and the main river of the system (fig. 1). diatom sample collection: sampling was carried out in completely open to partially open canopy segments of the river. sampling area in a particular river stretch was selected in such a way that it had rapids and care was taken to collect samples away from the bank to avoid aerophilous diatoms. samples were collected from five or more cobbles (>64 and <256 mm diameter) or small boulders (>256 mm) from a reach of at least 10 m in the river. substrata were placed in a tray, along with approximately 50 ml of river water. diatoms were removed by vigorously scrubbing the upper surface (the side exposed to flowing water) of the substratum with a brush to dislodge the diatoms. collection of epiphytic desmids was made by vigorously shaking 14 amutha and muralidharan / diatom assemblage in relation to environmental variables macropytes followed by plant squeezing or careful brushing in a plastic bag with 50 ml of stream water and the resulting brown suspension poured into a sample bottle. samples were resuspended by thoroughly mixing and 20 ml of stock sample was transferred into a clean 150 ml beaker. approximately 10 ml of concentrated hno3 was added (a pinch of k2cr2o7 was added, if the sample was rich in organic content). the beaker was placed onto a hot plate, covered with watch glass and digested. samples were boiled until the total volume of the treated sample was reduced to 10-15 ml. the samples were further transferred to a 15 ml centrifuge tube. the volume of the samples was adjusted to 15 ml by adding distilled water. it was further centrifuged at 1700 ppm for 10 min. the supernatant was discarded. the washing and centrifuging processes were repeated for 5-6 times so as to get pellet, free of acid. clean coverslip was placed on a slide-drying table with diatoms allowing the coverslip to dry after which the diatom-coated coverslips were allowed to cool and then one or two drops of mountant were placed onto each by means of a glass rod or pipette. mountant on the coverslips were heated gently for 30 seconds to 1 min. a previously cleaned glass slide was then lowered onto the coverslip, inverted, and then heated at 90-120°c on a hot plate until the mounting medium ‘boils’ and all the solvent evaporates within two to five minutes. after mounting, the hot slide was quickly removed from the hot plate, and laid on the work bench, the coverslip adjusted into position, the slide is ready for microscopic examination. the permanent slide of diatom samples was viewed at a 100x with an oil immersion. per sample 200, 300, 400 valves were counted and their relative abundance calculated diatom cells were counted at random. identification of diatoms to the species level was done based on gandhi (1955), barber (198), beaver (1981) krammer (1980) krammer and lange-bertalot (1985) and karthick et al. (2013). physico-chemical variables: water samples were collected (in 2l pvc container) at each site at a depth of 20-30 cm below the surface of the water during the day. physical and chemical variables, including ph, temperature, turbidity, conductivity, alkalinity, dissolved oxygen, biological oxygen demand, chemical oxygen demand, total solids, phosphate, nitrate and chloride of the stream water at each pre-selected sampling sites were determined based on apha (2005). other environmental determinants observed and noted were water velocity, mean depth, embeddedness and substrate composition (%fines and sand, %gravel, %cobble, %boulder). data analysis: detrended correspondence analysis (dca) was used to determine gradient lengths of the biological data. canonical correspondence analysis (cca) was employed to establish the relationship between algal assemblages and environmental parameters. prior to the analysis, the parameters were log transformed and diatom taxa were square root transformed to meet the assumptions of homoscedasicity. the statistical mean of each variable was tested with a monte carlo permutation test (500 permutations). analyses were performed figure 1. detailed map of the site distribution in the upland part of tamiraparani river system. 15 int. j. aquat. biol. (2017) 5(1): 12-21 using past (2.15) (hammer et al., 2001). results diatom assemblages: the samplings made during this study yielded 168 species representing 47 genera, 32 families and 16 orders comprising both epilithic and epiphytic diatoms from 11 upland river stretches that included the main river and the tributaries of river tamiraparani. diversity varied with the stream habitats which ranged from 57 to 92 species. sample collections were species-rich, mostly containing a minimum of 40 species to a maximum of 90 species. the greatest number of species per site was from mottaiar (s11) with 92 species and least species count of 57 was from karupanathi (s9). the most speciose genera were cymbella and navicula each representing 14 species followed by nitzschia and amphipora with 12 and 11 species, respectively. taxa prevalent irrespective of stream habitat conditions were achnanthes exigua, a. inflata, achnanthidium saprophilum, amphora coffeaeformis, a. sabiniana, cocconeis placentula v. euglypta, c. placentula v. lineata, cyclotella meneghiniana, cymbella parva, c. pusilla, c. turgidula, gomphonema angutastum, g. gandhii, g. insigne, g. olivaceum, g. pseubsphaerophorum, g. pseudoaugur, navicula dicephala, n. gallica, n. heimansioodes, n. angusta, n. cuspidata, n. lanceolata, n. rostellata, nitzschia obtusa var. kurzii and n. scalpeliformis. diatoma vulgaris was sensitive to fine sediment whereas achnanthidium minutissimum, c. placentula, c. meneghiniana, c. tumida, gomphonema parvulum, n. palea, and rhopalodia gibba also occupied sites influenced of some activities. diatom diversity in most of the sites studied was equably high due to heterogeneity of microhabitats except few. water quality and environmental variables: relative abundance data of diatoms were used for dca, eigen values for first two dca axes (λ1=0.43, λ2=0.19) accounted for 35% variation (fig. 2, table 1). the cca detected patterns within species and sites based on the influencing environmental variables. the species–environment variance was explained by the first axis 29.4% and second axis 23.9% with eigen values 0.32 and 0.26, respectively. proportion of substrate type and the presence of fine substrate were influential in the structuring of diatom community. the first axis described both physical habitat quality and water parameter gradient distinguishing between sites based on the proportion of sand among the substrates and hardness of the water. the diversity and abundance of diatoms corresponded to water temperature and increasing proportion of boulder and cobble substrates, and decreasing proportions of sand. further, the increasing abundance of diatoms increased with the proportion of habitat dominated by embedded cobbles and gravels. the placement of a species in the cca ordination plane represented the environmental optima for the particular species relative to the other taxa. water temperature (0.75, p<0.05), %sandy substrate (0.69, p<0.05), dissolved oxygen (0.66) and chloride (0.60) were highly loaded variables in the first two components, other variables were not significant. the second cca axis described the water quality gradient. sites and species were placed in the ordination plane according to the loadings of the factors. sites s9 and s11 were characterized by higher values for temperature, %sandy substrate and chloride and other chemical parameters alkalinity, hardness, conductivity and ph (fig. 3). predominant diatoms that occurred in these streams were c. parva, g. pseudoaugur, n. rostellata, c. pediculus and g. parvulam. anthropogenic disturbance observed in the sites were diversion of stream for washing, bathing and agricultural activities. nitrate levels determined the placement of sites and clustering of species in the right side of the cca plot accordingly manimuthar (s1), ramanathi (s6), and jambunathi (s7) are placed closer. aulacoseira granulata, a. ambigua, a. fogediana, c. meneghiniana, eunotia valida, fragilaria ungeriana, n. dicephala, pinnularia acrosphaeria were species prevalent in sites with higher levels of nitrate (fig. 2). water temperature was low in sampling stations s2 and s3, perhaps at a higher altitude than other 16 amutha and muralidharan / diatom assemblage in relation to environmental variables table 1. relative abundance data of diatoms used for detrended canonical analysis (* 1-5%, ** 6-10%, *** 11-20%). species/sites s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 s11 achnanthes exigua grun (ae) * * * * * * * ** * achnanthes inflata (kutzing) grunow (ai) * * * * * * * *** achnanthes microcphala kutzing (am) * *** *** * achnanthidium minutissimum (kutzing) czarnecki (amt) * * * * *** * achnanthidium saprophilum round and bukhtiyarova (as) * * * ** * * ** * * * aulacoseira ambigua (grunow) simonsen (aam) *** * ** ** * * * aulacoseira granulata (ehrenberg) simonsen (agr) ** * * ** * * * brachysira microcephala (grunow) compere (bmi) *** ** * * * brachysira wygaschii lange-bertalot (bwy) * ** *** * * * cocconeis pediculus ehrenberg (cpd) * * ** * * * * * * cocconeis placentula ehrenberg (cpl) * * *** * * * * * * cyclotella meneghiniana kutz. (cme) * * * * ** * * * eunotia minor (kutzing) grunow (emi) * * * ** fragilaria crotonensis kitton (fcr) * ** * * * * * fragilaria ungeriana grunow (fun) * * ** * * * * gomphonema affine kutzing (gaf) * ** * * gomphonema gandhii karthick and kociolek (gga) * * *** * * ** * * gomphonema parvulam kutz. (gpa) * ** * *** ** * * gomphonema pseudoaugur lang. (gps) * * * * * * ** ** ** * gomphonema pseudosphaerophorum (gpp) * * ** * * * * * * navicula rostellata kutzing (nro) * * * * * ** * * planothidium rostratum lange-bertalot (pro) * * *** * pseudostaurosira tenuis morales and edlund (pte) * *** ulnaria acus (kutzing) aboal (uac) * * * * ** * * figure 2. detrended correspondence analysis (dca) showing the distribution of species in sites. aam: aulacoseira ambigua, ae: achnanthes exigua, ai: achnanthes inflata, agr: aulacoseira granulata, am: achnanthes microcphala, amt: achnanthidium minutissimum, as: achnanthidium saprophilum, bmi: brachysira microcephala bwy: brachysira wygaschii, cme: cyclotella meneghiniana, cpl: cocconeis placentula, cpd: cocconeis pediculus, emi: eunotia minor, fcr: fragilaria crotonensis, fun: fragilaria ungeriana, gaf: gomphonema affine, gga: gomphonema gandhii, gpa: gomphonema parvulam, gps: gomphonema pseudoaugur, gpp: gomphonema pseudosphaerophorum, nro: navicula rostellata, pro: planothidium rostratum, pte: pseudostaurosira tenuis and uac: ulnaria acus. ◼ anthropogenic impact □ agricultural impact ▲ no notable influence 17 int. j. aquat. biol. (2017) 5(1): 12-21 sites, the quality was also soft marked by the lower values for alkalinity and hardness. encyonema silessiacum, frustulia rhomboids, halamphora veneta, pinnularia streptoraphe and ulnaria acus were abundant in these sites. prevalence of g. gandhii, c. placentula and n. cuspidata could be associated with the higher proportion of embedded substrates and lower proportion of fine substrates. these species are known to prefer water enriched with oxygen which is related to the high values for dissolved oxygen in the respective sites (fig. 3). gomphonema parvulam kutz., g. pseudoaugur lang. nitzschia palea were abundant in s9 and s11. similarity across streams with respect to diatom diversity was not evident, even though the sites were spatially closer, but the diversity of diatom community differed relative to the environmental factors that characterize that particular site (figs. 2, 3). discussion distribution of diatoms in lotic systems, constantly under influence by flow modifications, is intriguing. streams and small river networks and the topography of the regions they flow through determine the quality of the river system and the biota they support (brown, 2000). the presence and distribution of diatom taxa collected during this study was in relation to the in-steam habitat features, accordingly epilithon abundance was observed in cobbles and boulders dominated river segments and epiphyton representation was high in sites with submerged plants. apart from rocky surfaces and macrophytes, fine and coarse sand also influenced the prevalence of species and hence the composition of species varied corresponding to the proportion of sandy surface. achnanthidium minutissimum was more abundant in s2 where the sandy substrate is periodically influenced by water flow from reservoir, similar such observation has been reported in unstable sandy substrates (passy and bode, 2004). diatoma vulgaris is sensitive to sediment and was found only in sites that had least proportion of find sand (s5). the preferential pattern in diatom distribution with regard to the quality of water has been specific, whereby the sensitive species occupy only the clear undisturbed areas and the tolerant group of species survives even in disturbed segments (potapova and charles, 2005). cymbella parva, g. pseudoaugur, n. rostellata, c. pediculus and g. parvulam were abundantly colonized in karuppanathi (s9) and periyar (s10) with comparatively high ionic strength, high conductivity, high alkalinity and figure 3. canonical correspondence analysis (cca) biplot of sites showing their relationships with environmental gradients. 18 amutha and muralidharan / diatom assemblage in relation to environmental variables hardness. richness of diatom communities has been observed to be correlated with ionic composition (leland et al., 2001) and distribution pattern differed along different tds gradients (tudor et al., 1991, stevenson and pan, 1999). human mediated disturbances could alter both the water quality as well as the habitat of the diatoms. removal of gravel and sand, channelization of streams, embankments across flow direction were some reasons observed in the sites that could severely impact on diatom colonization. localities s1, s6 and s7 were characterized by the influence of agricultural activities in the vicinity of the sites and the frequent draining of run-off in the river stretches as evident from the grouping in dca. interestingly, illupaiyar (s4) stream though flows through cultivated land had minimal influence on the water quality but for the slight increase in tss. cyclotella meneghiniana, g. parvulum and n. palea regarded as tolerant species were present in both the disturbed sites and sites that had minimum influence, however with variation in the abundance this could be an indication that the streams that presently seems undisturbed could have some been subjected to anthropogenic impact in the recent past. n. palea occurs in organically polluted waters (fore and grafe, 2002) and colonizes in greatest proportions in the deforested streams (bellinger et al., 2006). the quality and quantity of fresh water resources are critically affected by alterations in land use and land cover which could substantially alter algal diversity (li et al., 2008; walsh and wepener, 2009). stream diatom assemblages generally are associated to physical habitat conditions, much accounted for variables like riparian conditions, instream habitat and channel morphology (pan et al., 2006; veselá and johansen, 2009). however, there are studies that show nutrients and salinity as the influencing factors than physical conditions (leland et al., 2001). physiochemical parameters such as dissolved oxygen, conductivity and dissolved solids fluctuate between sites are dependent on flow and the organic matter content at the particular site, thus could vary between sites and rivers (zheng and stevenson, 2006; walker and pan, 2006; shetty et al., 2015). this was true with respect to the sites sampled and variations observed in the parameters were related to the in-stream structure. the rapids, characteristic of upland rivers, frequently agitate along their course enriching with rich quotient of dissolved oxygen, the problem is that they are also bound to carry organic load in the form of drift and dissolved solids. similar condition was observed in illupaiyar (s4) that had higher values for dissolved oxygen and dissolved solids. during the course of establishing the relationship between environmental conditions and diatom diversity, we indented to verify whether diatom community composition in each site could explain the alterations to the habitat. dca effectively segregated the sites into three divisions accordingly a group comprised habitats under anthropogenic influence (s9, s10, s11), the next group had sites influenced by agricultural activities (s1, s6, s7, s8) and the other uncategorised (s2, s3, s4, s5) with respect to the influence based on physico-chemical quality of the respective streams and tributaries of the river system. dynamic streams without human mediated disturbances could better maintain complexity and integrity among lotic systems (palmer et al., 2005). however, disturbed sites tend to show more diatom diversity than less influenced sites which is due to the presence and abundance of both moderately sensitive and tolerant species (yu and lin, 2009). apart from variations in environmental features, the diversity of macrophytes define the diversity of epiphytons which was beyond the scope of this study. references andres s.m., mir l.c, van den bergh j.c.j.m, ring i., verburg p.h. 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(2015) 3(6): 362-371 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article amelioration of cadmium-induced changes in biochemical parameters of the muscle of common carp (cyprinus carpio) by vitamin c and chitosan mahdi banaee*1, milad mehrpak1, behzad nematdoost haghi1, ahmad noori2 1department of aquaculture, faculty of natural resources and environment, behbahan khatam alanbia university of technology, iran. 2department of aquaculture, faculty of marine science and technology, hormozgan university, bandar abbas, iran. article history: received 29 august 2015 accepted 8 n o v e m b e r 2015 available online 2 5 d e c e m b e r 2015 keywords: antioxidants cadmium biomarkers oxidative stress edible tissue abstract: the aim of this study was to investigate the effects of administering antioxidants, including vitamin c and chitosan on oxidative stress markers in muscle as edible tissues of cyprinus carpio exposed to cadmium chloride. in this experiment, by exposing to 0.2 mg/l cadmium chloride for 21 days, fish were fed a normal diet, diet containing chitosan (1000 mg/kg diet), vitamin c (1000 mg/kg diet) or both vitamin c and chitosan. oxidative stress markers, including the activity of catalase, total antioxidant and malondialdehyde (mda) as well as biochemical parameters, including the activity of aspartate aminotransferase (ast), alanine aminotransferase (alt), creatine phosphokinase (cpk), lactate dehydrogenase (ldh), and acetylcholinesterase (ache) were measured. fish exposure to cadmium chloride significantly increased ast, ldh, cpk, catalase, and mda activity, while it significantly decreased ast and ache activity, and levels of total antioxidant in muscle cells. administration of chitosan or vitamin c alone or in combination with each other to fish exposed to cadmium chloride was effective in regulating alt, cpk, and catalase activity. although administration of vitamin c and chitosan caused a significant decrease in mda, ast and ldh, these enzymes were still significantly higher than those in the control group. administration of vitamin c and chitosan had no significant effects on the activity of ache and levels of total antioxidant. although, chitosan alone could not prevent oxidative stress damages in muscle tissues of cadmiumtreated fish, administration of vitamin c combined with chitosan may increase the efficiency of antioxidant defense system and improve the detoxification system in the muscles of fish exposed to cadmium chloride. introduction with increasing pollution of aquatic ecosystems with heavy metals such as cadmium, fish exposure to these compounds and their consequent biological effects on the health of species inhabiting polluted waters are inevitable (choi et al., 2013; banaee et al., 2015a). when cadmium exists in water, it may enter the fish body through the gills, skin or the digestive system and then distribute in varied tissues (choi et al., 2013; banaee et al., 2015a). cadmium can affect the physiological activity of cells in different ways. it can disrupt the metabolic processes in cell mitochondria, disrupt the electron transport chain, and cause lipid peroxidation of cell membranes and * corresponding author: mahdi banaee e-mail address: banaee@bkatu.ac.ir affect the permeability of cell membrane, inhibit oxidative phosphorylation and protein synthesis, and disturb ion transfer (wang et al., 2004; gonzalez et al., 2006). cadmium binds to thiol groups of proteins, causes oxidant-antioxidant imbalance and destroys cell membrane proteins. therefore, the first stage in the oxidative stress caused by cadmium toxicity can be attributed to cadmium binding to glutathione sulfhydryl and oxidation of cell membrane proteins. reactive oxygen species produced during fish exposure to cadmium can be omitted by cellular antioxidant defense system. cadmium is known as an exacerbations sarcopenia disease, the degenerative loss of muscle mass, in 363 banaee et al./ amelioration of cadmium-induced changes in biochemical parameters of common carp humans (papa et al., 2014). changes in biochemical markers of smooth muscle cells in vitro condition indicates the effect of cadmium on cell homeostasis (kaji et al., 1994). mould and dulhunty, (2000) observed that disorder in muscle contraction can be caused by changes in electrical voltage in the membranes of cells after exposure to cadmium. in recent years, extensive studies have been conducted to find out simple and appropriate strategies to reduce the effects of environmental pollutants on the health of organisms and to improve the consumers’ food safety. natural and synthetic antioxidants such as vitamins (ozturk et al., 2009; ibrahim and banaee, 2014; banaee et al., 2015b) and antioxidants like chitosan with unique biological properties (e.g. drug delivery systems) (sun et al., 2011; yoon et al., 2011; alishahi et al., 2011a, b), can be effective in this regard (mehrpak et al., 2015). physiologically, vitamin c is one of the most potent antioxidants which is soluble in water. furthermore, it has a key role in the regeneration of vitamin e and increasing cellular stores of glutathione. therefore, vitamin c is really effective in protecting protein thiol groups against oxidation (naziroğlu et al., 2010). vitamin c (ascorbic acid) can act as a natural antioxidant in negating free radicals and preventing lipid peroxidation of unsaturated fatty acids in cell membrane; both during the normal functioning of cells and when the organism is exposed to a toxic compound (ozturk et al., 2009). chitosan is another natural compound of great interest to researchers due to its pharmacological properties, including anti-cancer, anti-ulcer, antibacterial, immunostimulant and many other biological properties. as a drug and hormone carrier, chitosan is used as vaccine for peptide and protein antigens (chua et al., 2012), growth factors, anticancer drugs (wei et al., 2013), analgesics and antiinflammatory drugs (agrawal et al., 2010; grenha et al., 2010a, b), antibiotics (kavaz et al., 2010) and vitamins (alishahi et al., 2011a, b). recently, several researchers have become interested in the antioxidant activity of chitosan (santhosh et al., 2007; sun et al., 2011; yoon et al., 2011). regarding chitosan properties in vitamin transfer in biological systems and the antioxidant properties of vitamin c and chitosan, we hypothesize that administration of vitamin c and/or chitosan may reduce the toxic effects of cadmium and reinforce antioxidant defense system in skeletal muscle of fish which were exposed to cadmium chloride. therefore, this study aimed to investigate the protective effects of vitamin c and chitosan against the oxidative stress in skeletal cells in common carp exposed to cadmium. also, the activity of antioxidant defense system and biochemical properties of skeletal muscles in exposed fish, the effects of vitamin c and chitosan administration on causing a balance between lipid peroxidation rate and the antioxidant capacity of the cell, as well as the activity of intracellular enzymes in skeletal muscles were measured. materials and methods chmical materials: low weight chitosan (80% deacetylated) were purchased from aldrich chemical company inc. (usa). all other chemical materials were obtained from merk chemical company (germany). ascorbic acid (vitamin c) was purchased from rooyan daroe company (iran). fish treatment: a total of 180 juvenile common carp, cyprinus carpio, weighting 37.65±4.40 g were obtained from a private fish farm in behbahan, khuzestan province, iran (december 2014) and used according to national ethical framework for animal research in iran (mobasher et al., 2008). specimens were randomly introduced into 18 plastic tanks (80 liter) and acclimatized in aerated freshwater (24±2°c, ph=7.4±0.2, 16l/8d, and 40% water nutrients value gross energy (kcal/kg) 3500 crude protein (%) 35-37 crude lipid (%) 9-11 crude fiber (%) 5% moisture (%) <10 ash (%) <10 tvn (mg/100gr) <45 tvn: total volatile nitrogen table 1. composition of commercial diet. 364 int. j. aquat. biol. (2015) 3(6): 362-371 exchange rate/day) for two weeks before experiment. during the acclimatization period, fish were fed twice a day with commercial diet from beyza feed mill, shiraz, iran (table 1). fish were randomly assigned to six groups, including specimens (i) fed with a normal diet for 21 days considering as control group, (ii) exposed to 0.2 mg.l-1 cadmium chloride, (iii) fed a diet enriched with 1000 mg chitosan per 1 kg feed for 21 days, (iv) exposed to 0.2 mg.l-1 cadmium chloride and were fed a diet enriched with 1000 mg chitosan per 1 kg feed for 15 days, (v) exposed to 0.2 mg.l-1 cadmium chloride and were fed a diet enriched with 100 mg vitamin c per 1 kg feed for 21 days, and (vi) exposed to 0.2 mg.l-1 cadmium chloride and were fed 100 mg vitamin c combined with 1000 mg chitosan per 1 kg feed for 21 days. the water was exchanged daily to reduce the buildup of metabolic wastes and to keep concentrations of cadmium chloride near the nominal level. at the end of the experiment, specimens were euthanized by decapitation and muscles were carefully removed, washed repeatedly in ice-cold physiological saline and accurately weighed. tissue samples were homogenized for two minutes in ice cold phosphate buffer (ph 7.4; 1:10 w/v) using a glass homogenizer and then centrifuged for 15 min at 15000 g at 4°c in a refrigerated centrifuge. the resulting supernatants were immediately used to measure the biochemical parameters by using spectrophotometric assays (mehrpak et al., 2015). biochemical parameters analysis: during activity measurement, creatine phosphokinase (cpk) reacts with creatine phosphate and adp to form atp, which is coupled to the hexokinase/gdp reaction generating nadph. lactate dehydrogenase (ldh) activity was measured based on the conversion of pyruvate to l-lactate by monitoring the oxidation of nadh. aspartate aminotransferase (ast) was assayed in a coupled reaction with malate dehydrogenase in the presence of nadh. in alanine aminotransferase (alt) assay, the enzyme reacts with alanine and α-ketoglutarate to form glutamate and pyruvate. lactate dehydrogenase converts pyruvate to lactate and nad+. all these activities were monitored by measuring changes in absorbance at 340 nm (moss and henderson, 1999). acetylcholinsetrase (ache) activity was determined by adding an adequate volume of sample into a cuvette containing 0.1 m phosphate ph 8.0, and acetylcholine iodide (0.015 m) and dithiobis nitrobenzoic acid (0.01 m) as substrates. ache activity was recorded during 180 s at 405 nm (knedel and boetteger, 1967). protein levels in tissues were determined by standard procedures used in clinical biochemistry laboratories according to the biochemical kits user manuals (parsazemon co, iran) (johnson et al., 1999). catalase (cat) activity was determined according to góth (1991), although with some modifications. catalase activity was measured by hydrogen peroxidase assay based on the formation of its stable complex with ammonium molybdate. 200 µl of the supernatant was incubated in working solution including 1000 µl hydrogen peroxide and 500 µl phosphate buffer (ph: 7.4) at 25°c for 60 s. then 1000 µl of 32.4 mmol.l-1 ammonium molybdate was added to the reaction solution and the concentration of the yellow complex of molybdate and hydrogen peroxide was measured at 405 nm wavelengths. catalase activity (ku. l−1) = a(sample) − a(blank 1) a(blank2) − a(blank3) × 271 blank 1 contained 1.0 ml substrate, 1.0 ml molybdate and 0.2 ml distilled water; blank 2 contained 1.0 ml substrate, 1.0 ml molybdate and 0.2 ml buffer; blank 3 contained 1.0 ml buffer, 1.0 ml molybdate and 0.2 ml buffer. total antioxidant capacity was estimated according to the ferric reducing ability of plasma (frap). briefly, the frap reagent contained 5 ml of a (10 mmol/l) tptz (2,4,6tripyridylstriazine) solution in 40 mmol/l hcl plus 5 ml of fecl3 (20 mmol/l) and 50 ml of acetate buffer (0.3 mol/l, ph=3.6) that was prepared freshly. 100 µl aliquots of the supernatant were mixed with 3 ml frap reagent. the conversion rate of ferric tripyridyl-s365 banaee et al./ amelioration of cadmium-induced changes in biochemical parameters of common carp triazine (fe3+-tptz) complex to ferrous tripyridyls-triazine (fe2+-tptz) at ph 3.6 and 25°c is directly proportional to the concentration of total antioxidant in the sample. fe2+-tptz has an intense blue color that can be monitored for up to 5 min at 593 nm by a uv/vis spectrophotometer. calculations were performed using a calibration curve of feso4•7h2o (100 to 1000 µm/l) (benzie and strain, 1996). malondialdehyde (mda) content was assessed by modified thiobarbituric acid assay and was expressed as µmol/g tissue (placer et al., 1996). briefly, 500 µl of the supernatant was transferred to a pyrex tube and mixed with 2500 µl trichloroacetic acid (20%) and 1000 µml thiobarbituric acid (67%). then, the tubes were placed in boiling water (100°c) for 15 min. after cooling, the chromogenic substrate was extracted into the organic phase with 1000 µl of distilled water and 5000 µl n-butanol: pyridine (15: 1). the mixture was then centrifuged at 2000 g for 15 min at 4°c. the pink color produced by these reactions was measured spectrophotometrically at 532 nm to measure mda levels. mda concentration was calculated using mda standard. tetraethoxypropane and absolute ethanol were used to prepare the mda standards. concentrations of mda in muscle samples are expressed in µm per g tissue. all biochemical parameters were measured by uv/vis spectrophotometer (model unicco 2100). statistical analysis: all data were examined for normality (shapiro-wilk test). statistical tests were performed with spss (ibm, release 19) software by means of one way analysis of variance, followed by duncan multiple comparison test (p<0.01). data are presented as mean ± sd in each experimental group. significant differences between values were characterized by alphabetical symbols (p<0.05). results changes in biochemical parameters of muscle cells are presented in figures 1-6. during the experiment, no mortality was observed in different groups of the experiment. the activity of aspartate aminotransferase (ast) in muscle cells of fish exposed to cadmium chloride was significantly more than that of the control group. the administration of chitosan or vitamin c alone had a significant effect to reduce the activity of ast and restoring it to the normal level. administering both chitosan and vitamin c to the fish exposed to cadmium chloride had no significant effects on restoring this enzyme to the normal levels (fig. 1). no significant difference was found in the activity of alanine aminotransferase (alt) in muscles of the control or chitosan-treated group. however, fish exposure to cadmium chloride caused a significant decrease in the activity of alt. the results indicate that combined administration of chitosan and figure 1. protective effect of vitamin c and chitosan on the muscle ast activity of fish. figure 2. protective effect of vitamin c and chitosan on the muscle alt activity of fish. 366 int. j. aquat. biol. (2015) 3(6): 362-371 vitamin c to the fish treated with cadmium chloride restored the activity of alt (fig. 2). common carp exposure to cadmium chloride increased lactate dehydrogenase (ldh) activity in muscle cells, though the administration of vitamin c and/or chitosan to cadmium chloride-exposed fish regulated the enzyme’s activity. yet, ldh activity in treated fish was significantly higher than that of the control group (fig. 3). the increased activity of creatine phosphokinase (cpk) was observed in muscle cells of fish exposed to cadmium chloride. the administration of vitamin c and/or chitosan reduced the activity of this enzyme (fig. 4). the results showed that acetylcholinesterase (ache) activity in muscles of exposed fish to cadmium chloride was significantly lower than that of the control group. vitamin c and/or chitosan administration had no effects on the activity of this enzyme (fig. 5). compared with the control group, a significant elevation was found in catalase activity in fish which were exposed to cadmium chloride. this is while the activity of this enzyme reduced after the administration chitosan and/or vitamin c. however, catalase activity returned to normal only in edible tissues of fish which were treated with chitosan and cadmium chloride (fig. 6). we found that chitosan administration alone had no significant effects on the cellular total antioxidant figure 3. protective effect of vitamin c and chitosan on the muscle ldh activity of fish. figure 4. protective effect of vitamin c and chitosan on the muscle cpk activity of fish. figure 5. protective effect of vitamin c and chitosan on the muscle ache activity of fish. figure 6. protective effect of vitamin c and chitosan on the muscle cat activity of fish. 367 banaee et al./ amelioration of cadmium-induced changes in biochemical parameters of common carp capacity in muscle of fish compared to the control group. fish exposure to cadmium chloride is caused a significant decrease in total antioxidant level of muscle cells. also, administration of chitosan and/or vitamin c to the fish exposed to cadmium chloride had no significant effects on increasing total antioxidant level of muscle cells and their return to normal levels (fig. 7). a significant increase in malondialdehyde (mda) was observed in muscle tissues of fish which were exposed to cadmium chloride, compared to the control group. according to the results, the combined administration of chitosan and vitamin c reduced mda in exposed fish and returned the enzyme to the normal level. although administration of chitosan or vitamin c alone caused a significant reduction in mda in muscle tissues compared with that in fish which were just treated with cadmium chloride, mda level was still high in the former groups compared to that of the control group (fig. 8). discussion cadmium is one of the non-biological trace elements with very active chemical properties. pervious study indicate that one possible molecular mechanism involved cadmium toxicity is the disruption of delicate oxidant/antioxidant balance, which can cause histopathological alternations via oxidative damage (wang et al., 2004; gonzalez et al., 2006). the findings of this study show that fish exposure to cadmium chloride increases free radicals significantly (zahedi et al., 2013). therefore, the significant elevation in catalase activity in these fish may be a response to the increased hydrogen peroxide radicals. a significant increase in mda as well as the decrease in total antioxidant levels were observed in the present study that may be related to high production of reactive oxygen species during the detoxification of cadmium chloride. due to the presence of long-chain polyunsaturated fatty acids, phospholipids, glycolipids, glycerides and sterols in the biochemical structure of cell membrane, cells are too sensitive to lipid peroxidation. therefore, damage to the cell membrane can affect the activity of intracellular enzymes. pervious authors demonstrated that cadmium can increase lipid peroxidation and cause oxidative stress (defo et al., 2015), inducing the generation of free radicals (jin et al., 2015). reduced levels of catalase may signify the influence of vitamin c and chitosan administration in reducing hydrogen peroxide. however, administration of vitamin c and chitosan had no significant effects on total antioxidant capacity of cells or preventing lipid peroxidation of muscles cell membrane in fish exposed to cadmium chloride. the increased activity of ast may be a physiological response to the use of amino acids in figure 7. protective effect of vitamin c and chitosan on the muscle total antioxidant capacity of fish. figure 8. protective effect of vitamin c and chitosan on the muscle mda levels of fish. 368 int. j. aquat. biol. (2015) 3(6): 362-371 oxidation or glycogen process in cells to provide them energy to cope with the toxic effects of cadmium (banaee et al., 2011). fish exposure to cadmium may inhibit alt synthesis or activity. the elevated ldh in muscles of exposed fish may indicate impairment of oxidative phosphorylation in mitochondria and development of cellular hypoxia, providing energy in the absence of oxygen and reoxidation of nadph by lactate dehydrogenase (murray et al., 2003). a significant decreases in muscle energy sources, and increases in muscle lactate were reported in silver carp (hypophthalmichthys molitrix) exposed to cadmium (zhang et al., 2013). a significant increase in cpk in fish muscles may indicate the cell response to the increasing energy need to cope with cadmium chloride toxicity. the transfer of phosphate group from creatine phosphate to atp in order to regenerate atp is done by cpk (murray et al., 2003). vitamin c can regulate the activity of cellular enzymes as a radical scavenger and due to its effect on the cellular glutathione level. vitamin c efficiency improves by chitosan which acts as its carrier. therefore, simultaneous administration of vitamin c and chitosan to the fish treated with cadmium chloride regulated the activity of alt, alp and cpk. although chitosan and/or vitamin c affected ldh activity, its activity is still high compared with that in the control group. vitamin c and chitosan administration had no effects on returning ldh activity to the normal level in fish exposed to paraquat (sharifinasab et al., 2016). although chitosan has antioxidant properties (yan et al., 2006; ramasamy et al., 2014) and acts as a radical scavenger (samarakoon et al., 2013; ozcelik et al., 2014), the results of our study show that chitosan alone could not prevent oxidative stress damages in muscle tissues of cadmium-treated fish. oxidative stress has an important role in changing levels of ache (frasco et al., 2005; rodríguezfuentes et al., 2008). activation or deactivation of acetylcholinesterase (ache) activity is attributed to the effect of hydrogen peroxide on this enzyme (schallreuter et al., 2004). reduced ache activity may be caused by the inhibitory effect of cadmium and the increased level of free radicals on cadmium chloride-treated fish. the administration of vitamin c and chitosan did not return this enzyme to its original state. in conclusion, although administration of antioxidants such as vitamin c and chitosan may reduce cadmium toxicity by increasing the efficiency of antioxidant defense system and detoxifying muscles of fish exposed to cadmium chloride. administration of antioxidants may not always have a protective effect on the muscles of fish exposed to contaminants. therefore, regarding the great importance of muscle tissues for the consumers, an appropriate strategy must be found to maintain the quality and health of edible tissues of fish which are exposed to environmental pollutants. acknowledgments this study was supported by grant from behbahan khatam alanbia university of technology. also, the authors are grateful to maryam banaee, our english editor, for proofreading the manuscript. references agrawal p., strijkers g.j., nicolay k. 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(2015) 3(6): 362-371 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی کپورمعمولی ماهی عضله در اکسیداتیو استرس زیستی هایشاخص بر کیتوزان و c ویتامین حفاظتی تاثیر (cyprinus carpio )کادمیوم کلراید معرض در 2نوری احمد ،1حقی دوستنعمت بهزاد ،1مهرپاک میالد ،*1بنایی مهدی .ایران بهبهان،( ص) االنبیاء خاتم صنعتی دانشگاه زیست، محیط و طبیعی منابع دانشکده شیالت، گروه1 .ایران بندرعباس، هرمزگان، دانشگاه دریایی، فنون و علوم دانشکده شیالت، گروه2 چکیده: معرض در هایماهی عضله در اکسیداتیو استرس هایشاخص بر کیتوزان و c ویتامین شامل هااکسیدانآنتی تجویز تاثیر بررسی مطالعه این از هدف ویتامین ،(غذا کیلوگرم هر ازای به گرممیلی 1111) کیتوزان واجد جیره نرمال، جیره با روز 21 مدت به هاماهی آزمایش این در. است کادمیوم کلراید c (1111 غذا کیلوگرم هر ازای به گرممیلی )ویتامین و c کلراید لیتر بر گرممیلی 2/1 معرض در همزمان طور به و شدند تغذیه کیتوزان با همراه و( mda) آلدهید دیمالون و کل اکسیدانآنتی کاتاالز، آنزیم فعالیت شامل اکسیداتیو استرس هایشاخص آزمایش، این در. گرفتند قرار کادمیوم ،(cpk) فسفوکیناز کراتین ،(ast) آمینوترانسفراز آالنین ،(ast) آمینوترانسفراز آسپارتات هایآنزیم فعالیت نظیر سلولی بیوشیمیایی پارامترهای سطح در دارمعنی افزایش سبب کادمیوم کلراید با هاماهی تماس. شد گیریاندازه( ache) استراز کولیناستیل و( ldh) دهیدروژناز الکتات سلول tao سطحو alt، ache آنزیم فعالیت سطح دارمعنی کاهش و mda سطح همچنین و ast، ldh، cpk، cat آنزیم فعالیت آنزیم فعالیت سطح تنظیم در کادمیوم، کلراید تیمار تحت هایماهی به یکدیگر با توام یا و تنهایی به c ویتامین و کیتوزان تجویز. گردید عضله های alt، cpk و cat ویتامین تجویز اگرچه. بود مؤثر c سطح کاهش سبب کیتوزان و mda فعالیت سطح و ast و ldh ،اما گردید تاثیر کیتوزان و c ویتامین تجویز که حالی در. بود باال داریمعنی طور به کنترل گروه هایماهی با مقایسه در هاگروه این در آنها سطح همچنان بافت در اکسیداتیو استرس از ناشی هایآسیب از نتوانست تنهایی به کیتوزان اگرچه. نداشت tao سطحو ،ache فعالیت سطح بر داریمعنی آنتی دفاع سیستم کارایی است ممکن کیتوزان و c ویتامین نظیر هایاکسیدانآنتی تجویز اما کند، پیشگیری کادمیوم تیمار تحت ماهیان عضله .دهد افزایش را کادمیوم کلراید معرض در هایماهی عضله هایسلول زداییسم و اکسیدانی .خوراکی بافت اکسیداتیو، استرس زیستی، هایشاخص کادمیوم، ها،اکسیدانآنتی :کلمات کلیدی int. j. aquat. biol. (2021) 9(2): 105-114 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article life history parameters of yellowfin hind, cephalopholis hemistiktos (rüppell, 1830) in the coast of united arab emirates elsayed farrag*1, walid aly2, ahmed el-zaabi1 1marine environment research department, ministry of climate change and environment, united arab emirates. 2national institute of oceanography and fisheries, niof, egypt. s article history: received 5 january 2021 accepted 20 februry 2021 available online 2 5 april 2021 keywords: age growth mortality recruitment exploitation rate abstract: the life history parameters, including age, growth, mortality and recruitment of yellowfin hind, cephalopholis hemistiktos were studied in monthly collected samples from january to december 2018. otolith was used for age determination. mean size by the end of each year of life was estimated and showed that, the highest annual increment was identified at the end of the first year of life then gradually decreased with increase of fish age. the estimated von bertalanffy growth parameters were l∞=43.51 cm, k=0.26 per year, t0=-0.74 year. asymptotic weight w∞ was estimated as 1375.23 g. the length-weight relationship was w=0.0126 l3.0746 with r2=0.94 for both sexes. the instantaneous rates of total mortality and natural mortality were estimated as 0.77 and 0.49 per year, respectively. the gonado-somatic index showed increasing from april to august with a peak in june for both sexes. size at first capture (lc) was estimated as 24.30 cm, which was smaller than the mean size at first sexual maturity 25.31 cm. the value of fishing mortality (f=0.28y-1) was slightly higher than the optimum (fopt=0.25y-1) and smaller than the limit (flimit=0.33y-1) biological reference point, indicating that species was exploited within sustainable limit. estimated parameters and the relative yield-per-recruit analysis showed that this species is not over-exploited. introduction the fisheries of the united arab emirates are typically multi-species in nature with over 100 species being exploited (grandcourt et al., 2010). they provide a source of income, employment and recreation contributing to the cultural heritage and food security of the inhabitants of the littoral states (grandcourt, 2008). groupers are of great importance in the marine ecosystems of all of the subtropical and tropical seas, and they play a basic role in the food chain, since they are one of the largest carnivores of the ecosystems (grandcourt et al., 2009; erisman et al., 2010; craig et al., 2011). they are important to both commercial and recreational fisheries worldwide (heemstra and randall, 1999). grouper populations have been depleted by overfishing, destruction of both juvenile and adult habitats, ineffective management plans for their fisheries or lack of any management policies (sadovy et al., 2013). grouper ecology is well-known *correspondence: elsayed farrag doi: https://doi.org/10.22034/ijab.v9i2.1089 e-mail: eefarrag@moccae.gov.ae in general, but detailed information on biological characteristics is scarce for many species. cephalopholis, bloch & schneider is the most common genus of the family serranidae in the aquarium trade and some species of this genus feature colorful bodies (abied et al., 2014). the genus comprises 22 species (fao, 2002), of these, c. hemistiktos is one of the most abundant species that have a disjunctive distribution, being known with certainty only from the northern part of the red sea to the coast of the pakistan (randall, 1995). cephalopholis hemistiktos is primarily caught using traps, and to a lesser degree hand lines (hartmann, 2013). in the emirates fisheries, c. hemistiktos has a minor components of the demersal species and represents 3.30% of the secondary commercial species caught by traps (farrag et al., 2020). due to the scarcity of published information on c. hemistiktos, this study was carried out to shed the light on the its 106 farrag et al./ life history parameters of yellowfin hind life history and provide its status assessments as one of the subordinate species in the united arab emirates fisheries. materials and methods study area and samples collection: monthly size frequency data and biological samples were collected from four locations along the coast of the united arab emirates namely ras al-kheima, umm alqwain, ajman and sharjah from january to december 2018 (fig. 1). fishes were mainly captured by traps and selected random from landings. age and growth parameters: total length was recorded to the nearest mm by measuring board. whole wet weight was measured with an electronic balance and recorded to the nearest g. the sex was determined by macroscopic examination of the gonad, which was removed and weighed to 0.1 g with electronic balance. sagittal otoliths were extracted, cleaned, dried, weighed to 0.1 mg. one of each pair of sagittae embedded in epoxy resin and transverse sections through the nucleus were obtained using a twin blade saw. sections were mounted on glass slides and examined using a low power microscope and transmitted light. two different observers recorded the number of alternating opaque and translucent bands. the counts of the two observers were then compared, and any that differed by more than one zone were removed from further analysis. parameters of the length-weight relationship were estimated by fitting the power function to length and weight data according to the equation w=a*lb (ricker, 1975), where w is the wet weight, a is a constant, l is the total length and b is close to 3.0 for species with isometric growth. in order to verify if calculated b was significantly different from 3, the students t-test was employed (froese, 2006). fatness of fish was described by calculating the coefficient of condition (fulton, 1904) from the formula k=100*w/l3 where w is the total weight in g and l is the total length cm. growth was investigated by fitting the von bertalanffy growth function (von bertalanffy, 1938) as follows: lt=l∞*1-exp-k (t-to), where lt is length at age t, l∞ is the asymptotic length, k is the growth coefficient and to is the hypothetical age at which length is equal to 0. the parameters of von bertalanffy were estimated according to ford (1933) and walford (1946), and age at length 0 was calculated using empirical formula (pauly, 1984). growth performance index ø=logk+2logl∞ and ø=k+2/3logw∞ for length and weight, respectively was calculated according to pauly and munro (1984). the potential longevity tmax was estimated based on the formula of beverton (1992) as tmax=3/k. reproductive biology: gonado-somatic index (gsi) was calculated using the following equation according to anderson and gutreuter (1983): gsi=100* wt/wg, where wt is the gonad weight and wg is the gutted weight g. sex ratio and its percentage was estimated as the number of females to the number of males in the catch, the significant differences from the theoretical ratio (1:1) were tested by chi-squared test x2. the mean size (lm) at first sexual maturity was estimated by fitting the logistic function to the proportion of mature fish in 10.0 mm size categories (king, 1995). the corresponding age at first sexual maturity was estimated based on froese and binohlan (2000). juvenile retention was calculated as the proportion of fish in landings that were below the mean size at first maturity. fisheries assessment and per-recruit analysis: the annual instantaneous rate of total mortality (z) was determined using methods of length converted catch curve (pauly, 1983) and linearized catch curve figure 1. map showing the sampling sites. 107 int. j. aquat. biol. (2021) 9(2): 105-114 (ricker, 1975) and the mean rate of the total mortality was used for further analysis. backwards extrapolation of length converted catch curve were used to determine probability of capture (lc) and the corresponding age at first capture (tc) was estimated based on beverton and holt (1957). the mean annual natural mortality (m) was calculated by three different methods according to rikhter and efanov (1976), pauly (1980) and hoening (1983). fishing mortality was estimated from the equation f=z–m. the biological reference point brp was estimated by the formula of patterson (1992) as fopt=0.5*m and flimit=2/3*m. the exploitation rate e was calculated as the proportion of the fishing mortality relative to total mortality e=f/z (gulland, 1971). the exploitation rate producing maximum yield emax, the exploitation rate at which the marginal increase of y'/r is 10% of its virgin stock e0.1 and the exploitation rate which the stock is reduced to 50% of its unexploited biomass (e0.5) were estimated. the relative yield per recruit y’/r and relative biomass per recruit b’/r were calculated using the model of (beverton and holt, 1966) and modified by pauly et al. (1996). results age and growth: a total of 1504 length frequency samples were collected ranging 16.0-42.0 cm and the length groups of 25, 26, 27 and 28 cm were the most frequent in the catch while the terminal length groups of 16, 17 and 41 cm were the least frequent ones (fig. 2). a total of 578 c. hemistiktos were used to estimate the length weight relationship (511 females and 67 males). the relationship between fish total length and total weight of male, female and sexes combined shows in table 1. the t-test analysis showed a nonsignificant difference (t<tcritical at ci 95%), which reflect that growth is isometric. the high values of regression coefficient (r2) indicate that the correlation of total length is best described by linear regression equations. the maximum values of condition factor for males (1.68) and females (1.79) were observed in march i.e. before the maturation of gonads. while the minimum values (males: 1.50, females: 1.51) were recorded in december and november, respectively i.e. after the spawning season. in general, the average condition table 1. length-weight relationship of cephalopholis hemistiktos for males, females and sexes combined. sex no. tl* range (cm) tw* range (g) a b r2 growth type average k* male 67 18.0-41.0 92.0-999.00 0.0095 3.1568 0.9635 isometric 1.58 female 511 16.0-42.0 69.0-1251.0 0.0132 3.0621 0.9413 isometric 1.61 combined 578 16.0-42.0 69.0-1251.0 0.0126 3.0746 0.9444 isometric 1.60 * tl: total length, tw: total weight, k: condition factor figure 2. length frequency distribution of cephalopholis hemistiktos. figure 3. monthly changes in values of condition factor for cephalopholis hemistiktos. 108 farrag et al./ life history parameters of yellowfin hind factor of females was higher than males (fig. 3). a subsamples of 468 c. hemistiktos were aged using sagittal otolith using transmitted light under low power magnification (fig. 4). two independent readers agreed on the age of 89.0% (418/468). otolith radii were found to be directly proportional and correlated with total length of c. hemistiktos l=0.0089or+9.581 (r=0.94). the back calculated size at age showed that this species attains length at the end of each year from 1st to 7th year as follows: 16.60, 22.84, 27.73, 31.43, 34.07, 36.14 and 38.05 cm, respectively (table 2). the catch was characterized by lacking of juvenile stage (age group 0). the annual growth rate was rapid in the first year (37.59%), then gradually decrease with increasing in to reach 1.91% in 7th year. the most dominant age group in the catch was the 3rd year forming 36.36%, while the last age group (7th year) represent 1.67%. parameters of the von bertalanffy growth function were estimated as: k=0.26, l∞=43.51 cm and to=-0.74 years for combined sex. the value of w∞ was obtained by applying the length weight relationship as 1375.23 g. the growth performance index ø was found to be 2.70 for growth in length and 2.36 for growth in weight. the theoretical growth in length of the whole population of c. hemistiktos can be expressed as: lt= 43.51*[1-exp(-0.26*(t+0.74))]. the theoretical growth in weight (the combination between von bertalanffy and length-weight relationship equations) of the whole population of c. hemistiktos was as: wt = 1375.23*[1exp(-0.26*(t+0.74))]3.0746. the longevity of c. hemistiktos was estimated to be 11.5 years. reproductive biology: the monthly gsi of male and female are given in figure 5. the mean monthly gsi fluctuated 0.09-0.68 and 0.11-1.10 in novembers and june for male and female, respectively. the gsi increased from april to august with a peak in june for both sexes. the ratio of males to females was 1.0: 8.0 and the x2 goodness of fit tests indicated that c. hemistiktos had significantly female biased sex table 2. back calculated lengths at the end of each year of life as well as the annual increment of cephalopholis hemistiktos. age group no. observed length back calculated lengths at the end of each year of life i ii iii iv v vi vii i 42 17.3 18.03 ii 89 24.21 17.43 25.02 iii 152 28.12 17.02 24.60 29.82 iv 76 32.26 16.47 23.13 28.02 32.22 v 36 35.19 16.12 22.26 27.89 32.04 35.04 vi 16 37.91 15.90 21.23 26.80 31.16 34.12 37.16 vii 7 39.98 15.23 20.80 26.13 30.29 33.06 35.12 38.05 mean 16.60 22.84 27.73 31.43 34.07 36.14 38.05 increment 16.60 6.24 4.89 3.70 2.65 2.07 1.91 increment% 37.59 29.47 22.26 15.94 12.45 11.51 figure 4. sectioned sagittal otoliths of cephalopholis hemistiktos ((4 annual rings) 32.2 cm tl). mi is the marginal increment). figure 5. mean monthly gonado-somatic index for cephalopholis hemistiktos. 109 int. j. aquat. biol. (2021) 9(2): 105-114 ratio (x2=52.62, p<0.05). size at first sexual maturity was estimated as 25.31 cm and the corresponding age was 2.55 years (fig. 6). the proportion of immature fish in aggregated size frequency samples that were below the mean size at first sexual maturity (juvenile retention rate) was 32%. the recruitment pattern of c. hemistiktos shows defined peaks in recruitment to fishery in may and august (14.27 and 12.84%, respectively). mortality estimation and fishery assessment: the length converted catch curve (fig. 7) gave the total mortality coefficient (z=0.76; ci 95% of z=0.680.83y-1) close to that estimated with linearized catch curve (z=0.77y-1). thus the mean value of total mortality 0.765y-1 was chosen for further analysis. the mean value of the natural mortality coefficient (m) estimated based on the three applied methods was 0.49y-1 and the survival rate was 0.47y-1. the length at first capture (length at 50% capture) was estimated as 24.30 cm, which was smaller than the mean size at first sexual maturity. the corresponding age at first capture was calculated as 2.35 years. the length and age at first recruitment (lr & tr) were estimated as 16.0 cm and 0.99 years. the values of z and m gave a value of fishing mortality f=0.28y-1, slightly higher than the optimum (fopt=0.25y-1) and smaller than the limit (flimit=0.33y-1) biological reference point. the exploitation rate is very important to estimate the state of the stock whether optimum, underexploited or overexploited. the exploitation rate for c. hemistiktos was 0.36 which is less than the optimum (e=0.5) given by gulland (1971). figure 8 shows the yield per recruit and biomass per recruit of c. hemistiktos. at the current level of fishing mortality and age at first capture, the yield per recruit and biomass per recruit were estimated as 79.66 and 284.50 g, respectively. based on the results, the increasing of fishing mortality at the current level of age at first capture will increase the ypr to reach the maximum while the bpr will decrease. the relative spawner biomass per recruit was estimated as 52.35% of the theoretical unexploited level at the existing fishing mortality rate, indicating species was exploited within sustainable figure 6. length at first sexual maturity (lm) for cephalopholis hemistiktos. figure 7. length converted catch curve of cephalopholis hemistiktos k=0.26y-1, l∞= 43.51 cm. figure 8. yield per recruit and biomass per recruit curves of cephalopholis hemistiktos (f=0.28year-1; m=0.49 and tc=2.35years. 110 farrag et al./ life history parameters of yellowfin hind limit. estimates of relative yield per recruit and relative biomass per recruit as graphically represented in figure 9. the values of e0.1, e0.5 were 0.76 and 0.38, respectively, higher than the current level of exploitation rate (e=0.36). discussions in all studies performed on species of the genus cephalopholis, it is necessary to use otolith for ageing (chan and sadovy, 2002; araùjo and martins, 2009). sagittal otolith of c. hemistiktos grow proportionally through the life of fish as seen in other cephalopholis species (trott, 2006; araùjo and martins, 2006, 2009). almost all tropical epinephelids and all cephalopholis species lay down increments annually (craig et al., 1999; potts and manooch, 1999; chan and sadovy, 2002; bustos et al., 2009; choat et al., 2009). the oldest fish found in this study was 7.0 years old similar to c. boenak and c. miniata (9 years) in gulf of aqaba, jordan (wahbeh, 2005); c. urodeta (10 years); c. spiloparaea (7 years); c. sexmaculata (8 years) and c. argus (5 years) in papua new guinea (fry et al., 2006); c. argus (6 years) in red sea coast (mehanna et al., 2019). however, the maximum age observed differs substantially from what was described for c. hemistiktos in jamaica where specimens of up to 26 years were found (mathews and samuel, 1987); c. panamensis in mexico (14 years) (craig et al., 1999); c. fulva in brazil (25 years) (araùjo and martins, 2006, 2009); c. taeniops (20 years) in cape verde archipelago. this study indicated that c. hemistiktos in the united arab emirates is slightly fast growing species (k=0.26 year-1). this is different from that reported by grandcourt et al. (2013) in emirate of abu-dhabi (k=0.14), while similar to the result of mehanna et al. (2019) (k=0.26 for c. argus). negative to is common in species that grow rapidly in the first year, and slowly later in life (sadovy et al., 1992; craig et al., 1997). in the present study, the to was negative, as other works on this genus (craig et al., 1999; potts and manooch, 1999; araùjo and martins, 2009). asymptotic length (l∞) was estimated as 43.51 cm similar to c. fulva (potts and manooch, 1999) and c. argus (mehanna et al., 2019). table 3 shows a comparison between the von bertalanffy growth parameters estimated by other authors for cephalopholis species and this study. the variation between growth coefficient may be due to different localities, methods used, environmental condition, fish maximum length and number of samples. table 3. biogeographic comparison of vbgf parameters (cephalopholis species). author location species l∞ k to potts & manooch, 1999) south-eastern coast of usa c. cruentata 38.50 0.32 0.49 c. fulva 44.60 0.13 -1.15 wahbeh, 2005 gulf of aqaba, jordan c.miniata 58.65 0.08 -1.84 araùjo & martins, 2006 central coast of brazil c. fulva 31.60 0.14 -5.74 mohammad, 2007 red sea c.argus 52.60 0.16 -1.41 grandcourt el al., 2013 united arab emirates (abu-dhabi) c. hemistiktos 26.20 0.14 -10.9 tarich et al., 2015 cape verde archipelago c.taeniops 54.26 0.14 -0.85 mehanna et al., 2019 hurghada red sea coast c.argus 44.22 0.26 1.33 present study united arab emirates c.hemistiktos 43.51 0.26 -0.74 figure 9. relative yield per recruit and relative biomass per recruit of cephalopholis hemistiktos (m/k = 1.88; lc/l∞=0.56; emax= 0.88; e 0.1 = 0.76; e 0.5 = 0.38). 111 int. j. aquat. biol. (2021) 9(2): 105-114 (wahbeh, 2005; araùjo and martins, 2006 and tarich et al., 2015). in the present work, ø of c. hemistiktos was similar to c. argus in red sea (mehanna et al., 2019) and lower than cephalopholis species in red sea (2.93) (mohammad, 2007). the growth performance index estimated by us was higher than that estimated by wahbeh (2005) in gulf of aqaba (2.27). comparison with other species of groupers, ø falls within the range 2.11-2.65 as reported for species of cephalopholis (matheson and huntsman, 1984; mathews and samuel, 1987; chan and sadvoy, 2002). in the present study, the b-value was estimated as 3.0746 which is similar to c. hemistiktos (3.042) and c. argus (3.038) (grandcourt et al., 2013; mehanna et al., 2019). wahbeh (2005) stated the length-weight relationship showed allometric growth in females and isometric in males of c. miniata. according to bennet (1970), fulton’s condition factor ≥0.56 considered as well-being bench mark values of a fish, hence fishes with condition factor values above the well-being bench mark were considered to be in good condition. spawning in groupers tends to be restricted to less than half the year, with many species spawning primarily during 1 to 2 months (shapiro, 1987). in the present study, the gsi and maturity stage data suggest the spawning period in the spring-summer seasons, starting in april and ending in august. grandcourt et al. (2013) declared two peaks of gsi of c. hemistiktos, during the may to august and october to november. the present study agrees with spawning seasons reported for groupers from elsewhere (manickandheilman and philips, 2000; mackie, 2000; chan and sadvoy, 2002). the ratio of males to females was 1.0: 8.0 and the x2 goodness of fit tests indicated that c. hemistiktos has significantly female biased sex ratio. in the present work, the total mortality was 0.77year-1 compared to the double value of z reported by grandcourt et al. (2013). the annual instantaneous rate of z may have been overestimated if larger fish were less vulnerable to the fishing gear or if adult fish underwent migrations for example. for c. argus, the total mortality was estimated as 1.88 and 1.31year-1 in red sea (mohammad, 2007; mehanna et al., 2019, respectively). our estimate of the natural mortality rate derived from different methods was 0.49 year-1 which is higher than that estimated by mohammad (2007) and grandcourt et al. (2013) where the values were 0.44 and 0.21year-1 for c. argus and c. hemistiktos, respectively. while the current m was lower than that estimated by mehanna et al. (2019) (0.56year-1). as the size at first capture (24.30 cm) was smaller than the size at first sexual maturity (25.31 cm) and the size at which yield per recruit would be optimum (lopt=26.91 cm), an increase in the mesh size for the trap fishery should be considered by management authorities. this is particularly important given that 32% of the yield in numbers consisted of fish that were below the size at first sexual maturity. if fish were retained by traps at the size at which yield per recruit would be optimum (26.91 cm), they would have a chance to spawn at least one time before reaching the mean size at first capture. the use of yield per recruit models may be particularly restrictive for fast growing tropical species with high rates of natural mortality as the curves may not reach a maximum within a reasonable range of fishing mortality values (gayanilo and pauly, 1997). the result of the present study indicated that, with the increasing of fishing mortality and at the current level of age at first capture, the ypr increased to reach the maximum while the bpr will decrease. the relative yield per recruit analyses indicated that an increase in the size at first capture to that which would maximise yield per recruit would be associated with an increase in yield at the existing fishing mortality rate. the specified precautionary target (fopt=0.5*m) and limit (flimit=2/3*m) values are more appropriate biological reference points in light of the constraints of the yield per recruit model. in the present work, the value of fishing mortality i.e. f=0.28y-1 was close to the optimum (fopt=0.25y-1) and smaller than the limit (flimit=0.33y-1) biological reference point, indicating that species was exploited within sustainable limit. in conclusion, the present study gave the life history parameters (age, growth and mortality) of 112 farrag et al./ life history parameters of yellowfin hind subordinate species c. hemistiktos and provide more details about the impact of fishing gear on the demersal species, and suggest that, both reduction in fishing effort and mesh size regulations will be required for the demersal trap fishery. references abied a.m., abu el regal m.a., khalil a.h. 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(2017) 5(4): 236-245; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article responses of beluga (huso huso) to salinity exposure: a laboratory evaluation of the effect of field-based salinity levels on osmoregulatory characteristics and growth performance ali jalali1, 2, 3*, mohammad sudagar1, seyed mostafa aghilinejhad1, 3, hamed kolangi miandare1 1faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. 2center for integrative ecology, school of life and environmental sciences, deakin university, victoria, australia. 3sturgeon affairs management of golestan province, gorgan, iran. article history: received 6 march 2017 accepted 24 july 2017 available online 2 5 august 2017 keywords: blood biochemistry juvenile sturgeon osmoregulation acclimation sturgeon rearing abstract: there is a need for a better understanding of how sturgeon, especially hatchery reared juveniles, respond to salinity challenges. therefore, here we examined the effects of different fieldbased salinities (freshwater [fw] (0.5), 3, 6, 9 and 12 ppt) on osmoregulatory characteristics and growth performance of juvenile beluga sturgeon, huso huso, (22.1±1.1 g body weight) over a 60day period. survival rate was relatively high in all treatments although there was a sign of adverse effects of salinity on the survival as fish at 12 ppt salinity. growth performance was better in fish reared at 3 ppt, followed by 6, 9 and 12 ppt. overall, an increase in plasma sodium, potassium, calcium, magnesium and glucose levels was found in association with the increase of salinity, while the fw control group maintained basal levels. haematocrit levels were also affected by the salinity and the observed levels in fw, 3 and 6 ppt salinities were lower than other salinity concentrations. the results indicated that the beluga sturgeon juveniles are able to survive and acclimate to moderate salinities. here, we also discussed the importance of evaluating and comparing specific mechanisms of acclimation in populations across brackish waters of the southern caspian sea as such investigations may aid and improve aquaculture strategies. introduction sturgeons are migratory species that occur in major river systems of the northern hemisphere (bemis and kynard, 1997; grande and bemis, 1991), especially in the caspian sea basin. most sturgeon species have been listed on the iucn red list of endangered species due to the drastic declines in their wild populations (birstein, 1993; birstein et al., 1997; iucn, 2012), and many are unfortunately at the brink of extinction. overfishing, poaching, pollution and habitat degradation are the major threats to sturgeon longevity and sustainability (pourkazemi, 2006; ruban and khodorevskaya, 2011). many efforts have been made, especially in the past decade, to protect sturgeons, and aquaculture plans for restoration, conservation and commercial purposes have received the most attempt and attention. in iran, fisheries organization has widely been involved in breeding programs to develop *corresponding author: ali jalali doi: https://doi.org/10.22034/ijab.v5i4.285 e-mail address: jalalifc@gmail.com sturgeon aquaculture and restock sturgeon populations, so that thousands of sturgeon fingerlings are released into the caspian sea annually or kept for sturgeon rearing to meet meat and caviar demands. acipenseriformes are similar to teleosts with respect to the main features of their osmoregulation (potts and rudy, 1972; krayushkina et al., 1995). fish challenged with an altered environmental salinity must maintain their body osmolality and ionic balance. therefore, it is necessary for both sturgeons and teleosts to maintain rather tight control of serum water and ion concentration for efficient physiological function when they move between fresh water and salt water (natochin et al., 1985; krayushkina et al., 1995). this is mainly accomplished by profound morphological and physiological changes, such as drinking rate (tytler and blaxter, 1988; ura et al., 1996; miyazaki et al., 1998), stress hormone levels, 237 int. j. aquat. biol. (2017) 5(4): 236-245 which can disturb hydromineral balance and blood parameters such as haematocrit (woo and chung, 1995; wendelaar bonga, 1997; brown et al., 2001) and functions of the osmoregulatory surfaces (hwang and hirano, 1985; hwang et al., 1989; arai et al., 1997; perry, 1998; kelly and woo, 1999). there are a variety of studies assessing osmoregulatory mechanisms and salinity tolerance in sturgeon species, and it has been documented that many sturgeons species are able to adapt different salinity ranges (mcenroe and cech, 1985; krayushkina, 1996; krayushkina, 1998; altinok et al., 1998; martinz alvarez et al., 2002; jarvis and ballantyne, 2003; allen and cech, 2007; he et al., 2009; zhao et al., 2010). however, exposure length, fish size and age appear to play key roles in adaptation capacity as salinity tolerance may decrease especially when fish in smaller sizes are subjected to high salinities. it has previously been suggested that salinity can also affect fish growth, and an interaction between growth and salinity has been demonstrated in several fish species (altinok and grizzle, 2001; wada et al., 2004; martinez-palácios et al., 2004; tibblin et al., 2012). in tilapia, oreochromis mossambicus, its rearing in sea water is resulted in an improved growth performance compared to freshwater (kuwaye et al., 1993; riley et al., 2003). improved growth at intermediate salinity may be explained by a reduction of the metabolic cost for osmoregulation, whereas appetite and/or the endocrine system may also play a role (boeuf and payan, 2001). sardella and kultz (2009) demonstrated that green sturgeon, acipenser medirostris, with a mean weight of 121 g were able to survive and acclimate following a salinity transfer and they observed minimal osmotic stress in the exposed fish. juvenile atlantic sturgeon, a. oxyrinchus, with a mean weight of 440 g were reared under three salinity conditions (0, 10, or 33 ppt) for 6 months and it was found that fish in 0 and 10 ppt grew more than those of 33 ppt (allen et al., 2014). in contrast, after 30 days rearing, zhao et al. (2010) did not observe significant effect of salinity exposure (up to 25 ppt) on the growth of 5-month-old amur sturgeon, a. schrenckii, with a mean initial body weight of 106.8 g. however, growth performance in sturgeons under different saline environments appears to be species dependent and can also be impacted by the size of fish (allen and cech, 2007). there are few studies on the effects of salinity on the caspian sea sturgeons, especially beluga sturgeon. although previous studies have shown that sturgeons are capable of adopting different salinities, however, it has also been indicated that adaptation capacity in small juveniles reduces as they may not be able to tolerate even brackish waters (jalali et al., 2008). thus, detailed information regarding the salinity effects and during a various exposure times can provide a better understanding of sturgeon responses to environmental challenges. it can also be a useful method for aiding aquaculture programs especially when the fish are kept for coastal-based-rearing and aquaculture purposes. sea-cage-based and pen culture of sturgeons have recently been considered in the iranian part of the caspian sea as it can provide an alternative to wild stocks and for supply of sturgeon meat and caviar. thus, in this study we selected filedbased salinity doses of freshwater (fw), 3, 6, 9 and 12 ppt representing salinity ranges that the beluga are likely to encounter in the iranian part of the caspian sea. we then aimed to assess osmoregulatory characteristics (including sodium, potassium, calcium and magnesium concentrations), survival and growth performance of beluga (huso huso) juveniles to these salinities over two months, a relatively long experimental period. materials and methods fish rearing conditions: five months old beluga with a mean body weight of 22.1±1.1 g (mean±sd) were obtained from the shahid marjani sturgeon propagation center (aq qala, golestan province, iran). fish were transferred to the aquaculture research centre at the university of gorgan, then stocked in five groups with triplicate per group (200 l tanks with a stocking density of 15 fish per tank), and cultured in fw (0.5), 3, 6, 9 and 12 ppt for 60 days. acclimation to salinity was performed by increasing water salinity at an approximate rate of 3 ppt per day 238 jalali et al./ growth performance and physiological parameters of beluga in response to salinity until reaching 12 ppt. salinity levels were obtained by mixing dechlorinated tap water with salt, and measured by water checker (horiba u-10, japan). temperature was kept at 21°c, and supplemental aeration was also provided to maintain dissolved oxygen levels near saturation. the photoperiod was maintained at 13 h light /11 h dark. during the experiment, the fish were fed three times a day with the same commercial pellet diet (approximately 3% of body weight/day; 54% protein, 18% lipid, 11% ash and 0.3% fiber; biomar company). the amount of feed offered was daily recorded for food conversion ratio calculation. the tanks were siphoned daily to remove uneaten feed and feces. in each tank, half the water volume was renewed every day to assure water quality. it was tried to minimize any other stress during the entire period of the experiment. sampling and data analysis: at the end of the experiment, feeding was discontinued 24 hrs prior to measurements and all fish were weighed and growth parameters were calculated: specific growth rate ({[ln final body weight-ln initial body weight] × 100}/total days), feed conversion ratio (dry feed fed/body weight gain) and condition factor ({[body weight/body length (cm3)] × 100}) (lugert et al., 2014; shalaby et al., 2006). percent of survival were also calculated. in order to evaluate the haematocrit (% pcv), ions (sodium [na+], potassium [k +], calcium [ca 2+] and magnesium [mg2+]) and glucose concentrations, the blood was collected from the caudal vein of individual fish employing heparinized syringes. blood samples were centrifuged at 16000 g for 5 min in a clinical centrifuge (hettich-d7200, tuttlingen, germany) for haematocrit evaluation. the blood plasma was decanted and pipetted into eppendorf tubes and preserved at -20°c. in order to evaluate how different salinities impact plasma ion concentrations, sodium (na+) and potassium (k+) concentrations were measured with flame photometer (corning 405c: iri). magnesium (mg2+), calcium (ca2+) and glucose concentrations were measured with an absorption spectrophotometer (unico 3115233: usa) (hoseini et al., 2011). statistical analysis: data were initially checked for normality and homogeneity of variance (using bartlett and kolmogorov-smirnov tests) and then salinity was considered as the independent variable and fish haematological, biochemical and growth parameters as the dependent variables. data were analyzed by one-way analysis of variance (anova) with duncan’s new multiple range tests (spss software version 18). statistical values are expressed as mean±sd. the values of p<0.05 were considered significantly different. results biochemical and haematological variables: the blood plasma electrolytes, including sodium, potassium, calcium, magnesium and glucose concentrations were affected by increasing the salinity, and significant differences were observed among treatments (p<0.05). plasma sodium concentration was higher in the fish reared at 9 and 12 ppt salinities and there was a decline in the levels of this parameter with decrease in salinity (fig. 1a). plasma potassium was also higher in the fish exposed to 9 and 12 ppt salinities (fig. 1b). plasma calcium and magnesium levels showed a relatively similar patterns, with high concentrations in the fish reared at 6, 9 and 12 ppt salinities (fig. 1c-d). glucose level in fish exposed to fw (0.5 ppt) , and 3 and 6 ppt salinities was lower compared to other treatments, and it raised at higher salinities (fig. 1e). haematocrit levels of fish reared at salinities of 0.5, 3 and 6 ppt were lower than those of 9 and 12 ppt salinities (fig. 1f). survival and growth performance: although survival was relatively high in all groups, there were some significant differences among salinity treatments in term of survival rate (fig. 2), and fish subjected to 12 ppt salinity had lower survival compared to the fish exposed to other salinity levels (p<0.05). there was no significant difference in survival rate between salinities of 3, 6 ppt and 9 ppt. growth parameters were significantly different between treatments (p<0.05; table 1). fish reared at lower salinities (fw, 3 and 6 ppt) showed higher final weight, final length and specific growth rates compared to the fish reared at higher salinities (table 1, p<0.05). condition factor 239 int. j. aquat. biol. (2017) 5(4): 236-245 figure 1. trends in the levels of (a) sodium [na+], (b) potassium [k+], (c) calcium [ca2+], (d) magnesium [mg2+], (e) glucose and (f) haematocrit in the blood samples of beluga sturgeon (huso huso) on the 0th day and 60th day of the experiment. data are presented as mean (±sd). groups having different letters are significantly different (p<0.05). table 1. growth parameters of juvenile beluga sturgeon (huso huso) after a 60-day rearing period at various salinities. parameters treatments freshwater (0.5) salinity 3 salinity 6 salinity 9 salinity 12 initial weight (g/fish) 22.3±1.0a 22.0±1.0a 21.9±1.0 a 22.2±1.2 a 22.2±1.3a final weight (g/fish) 102.2±3.7a 105.5±2.6b 103.3±3.6a 97.8±3.1c 97.0±2.7c initial length (cm) 19.7±0.4a 19.8±0.3a 19.8±0.2a 19.9±0.2a 19.8±0.1a final length (cm) 31.0±1.1a 32.3±1.1b 30.9±1.7a 28.8±1.7c 28.5±1.4 c sgr1 2.53±0.01a 2.61±0.02b 2.57±0.01b 2.46±0.03c 2.45±0.01c fcr2 1.7±0.10a 1.61±0.07a 1.71±0.12a 1.88±0.07b 1.95±0.05b cf3 0.33±0.005a 0.30±0.01b 0.34±0.005a 0.40±0.01c 0.41±0.01c note: 1specific growth rate, 2food conversion ratio and 3condition factor; means in the same row with different superscripts are significantly different (p<0.05); data are presented as mean ±sd 240 jalali et al./ growth performance and physiological parameters of beluga in response to salinity in the fish exposed to 9 and 12 ppt salinities was higher than those of lower salinities (p<0.05). in addition, food conversion ratio increased when fish were subjected to 9 and 12 ppt (p<0.05). discussion the results of the present study showed how juvenile beluga sturgeon respond when exposed to brackish water in terms of osmoregulatory ion concentrations, survival, and growth. fish were acclimated to several salinity ranges (fw (0.5) to 12 ppt) by increasing water salinity at a rate of 3 ppt per day. these doses and rate of increase were chosen on the basis of conditions that juvenile beluga sturgeon may encounter in the iranian part of the caspian sea especially if they are kept for penand cage-based culture. although fish at low salinities showed a greater survival rate, the difference in survival between the lowest (fw) and the highest (12 ppt) salinities was 6.3% after 60 days. thus, it appears that five-month-old juvenile beluga sturgeon is able to acclimate to brackish water with salinity doses close to the concentrations observed in the southern caspian sea. in this regard, previous studies also indicated that salinity tolerance in sturgeons is improved after acclimation to different salinity levels (mcenroe and cech, 1985; altinok et al., 1998; mckenzie et al., 2001). such acclimation is probably needed to initiate enzymatic and cellular osmoregulatory changes necessary for withstanding osmotic challenge (morgan et al., 1997; morgan and iwama, 1999). nonetheless, fish body size appear to largely influence the process of successful acclimation to salinity changes. several studies have indicated that osmoregulatory abilities are positively correlated with fish size and larger fish show less sensitivity due to the structural and physiological developments during their ontogeny (mcenroe and cech, 1987; altinok et al., 1998; lebreton and beamish 1998; cataldi et al., 1999; allen and cech, 2007; jalali et al., 2010; allen et al., 2011). these mechanisms, however, relatively differ amongst sturgeon species due mainly to the species specific osmoregulatory characteristics and life history. for example, juvenile green sturgeon, a. medirostris, are capable of surviving seawater (34 ppt) and near brackish water (15 ppt) concentrations even following an immediate transfer (sardella and kultz, 2009; allen et al. 2011). but according to the previous investigation, abrupt transfer to such concentrations would lead a serious osmotic shock and catastrophic mortality in juvenile beluga sturgeon especially at small and fingerling range sizes (jalali et al., 2010). this is probably related to the species evolutionary history. green sturgeons move between oceanic waters and freshwater encountering an extended salinity ranges (0 to 35 ppt) within their habitats. in contrast, average salinity recorded in the northern and southern caspian sea respectively fall around 9 and 13 ppt (near an isosmotic medium), about a third salinity of most seawater. therefore, compared to other sturgeon species rearing at higher salinities or migrating to oceans, caspian sea sturgeon including beluga sturgeon habitats occur within relatively limited salinity ranges and this is likely to affect caspian sturgeon’s salinity tolerance. however, it has generally been said that salt tolerance is poor for early life history stages of sturgeon. therefore, more detailed assessment of how tolerance to a salinity gradient evolves in sturgeons could provide a better understanding of species and environmental associations. in addition, this would help successful aquaculture plans in producing sturgeon meat considering that high potential exist along the caspian sea to develop sturgeon aquaculture. figure 2. the beluga sturgeon (huso huso) survival (%) exposed to different salinities over a 60-day experimental period. data are presented as mean (±sd). groups having different letters are significantly different (p<0.05). 241 int. j. aquat. biol. (2017) 5(4): 236-245 there are numerous similarities in osmoregulatory mechanisms between sturgeon and teleosts such as the composition of the blood parameters (i.e. plasma osmolality and electrolyte concentrations) in both fresh water and sea water. they are hyperosmotic with respect to fresh water and hyposmotic with respect to salt water (holmes and donaldson, 1969). in this experiment, we detected an increase in electrolyte concentrations with enhance in salinity. our results indicated an elevation in haematocrit levels after 60 days exposure especially to 12 ppt salinity whereas these levels were low in the fish reared at lower salinities. a previous study indicated that haematocrit levels rose in juvenile shortnose sturgeon, a. brevirostrum, after 10 weeks in hyperosmotic conditions (jarvis and ballantyne, 2003). on the other hand, the haematocrit levels were not different between long-term fresh water and salt wateracclimated juvenile adriatic sturgeon, a. naccarii, and gulf sturgeons, a. oxyrinchus (altinok et al., 1998; martinez-alvarez et al., 2002). it has been shown that stressful conditions can lead to alterations in haematocrit levels and an elevated haematocrit level can reflect a stress response in fish (soivio and nikinmaa, 1981; maxime et al., 1990; franklin et al., 1992). it may also reflect haemo-concentration or demand of fish to oxygen because of the increased respiratory demand caused by salinity exposure, although there is no evidence for this theory as this study did not evaluate oxygen demands in the exposed fish and such demand could also be species-dependent (ern et al., 2014). the results indicated that plasma sodium, potassium, calcium and magnesium contents increased when the fish were reared in brackish water, and ion concentrations were higher at 9 and 12 ppt treatments. these alternations can be due to changes in the water content in the blood, resulted from the change in environmental salinity as indicated in other salt water-exposed sturgeons (plaut, 1998; krayushkina, 1998; mckenzie et al., 1999; martınezalvarez et al., 2002; he et al., 2009). sturgeons hatch and grow at least initially in fresh water and thus, they regulate at elevated ion homeostasis levels in hyperosmotic salinities (mcenroe and cech, 1987; altinok et al., 1998; krayushkina, 1998; rodriguez et al., 2002). changes in blood and osmotic parameters have been shown to be time-dependent as the levels return to steady state during the acclimation period when the fish remain at a constant salinity and reach homeostasis. when fish is exposed to the concentrated environment, it loses water and the content of the elements in the blood increases. fish would consequently tend to ingest more water to dilute the level of the blood parameters (he et al., 2009; allen and cech, 2007). at the end, the contents of these parameters reach the constant levels as a consequence of the rest of the osmoregulatory mechanisms, which act to re-establish the extracellular volume salinity (martınez-alvarez et al., 2002; he et al., 2009). glucose is an essential fuel for various tissues and its level in plasma was also higher in the fish-kept at 9 and 12 ppt treatments. it has previously been indicated that glucose showed both a rise (bashamohideen and parvatheswararao, 1972; assem and hanke, 1979) and a fall (soengas et al., 1991; krumschnabel and lackner, 1993; allen and cech, 2007) during seawater adaptation. the plasma glucose proliferation can be affected by cortisol changes though cortisol level was not evaluated here. nonetheless, there appears to be a high glucose demand to supply the energy by osmoregulatory mechanisms (krumschnabel and lackner, 1993; plaut, 1998), where upon glyconeogenesis even increases (jürss and bittorf, 1990). growth performance and food conversion ratio in the fish reared at low salinities (fw, 3 and 6 ppt) were significantly higher than those of fish reared at higher salinities. in the case of marine teleost fish, several authors reported better performance at intermediate salinities. atlantic cod, gadus morhua, larvae reared at 7, 14 and 28 ppt, showed higher growth rates at the intermediate salinity (14 ppt), possibly due to a more efficient conversion ratio (lambert et al., 1994). whitefish larvae, chirostoma estor estor, exhibited greater specific growth rates at 10 and 15 ppt, compared to those at 0 and 5 ppt, but net production, based on survival and growth, was clearly superior at 242 jalali et al./ growth performance and physiological parameters of beluga in response to salinity 10 ppt, with a very low response at 0 ppt (martinezpalácios et al., 2004). wada et al. (2004) found that growth rates in spotted halibut, verasper variegatus, juveniles kept at 8 and 16 ppt were higher than fish kept at 32 ppt (control) and at 4 ppt. specific growth rates in gulf sturgeon, a. oxyrinchus, were higher at 3 and 9 ppt compared to those of fresh water (altinok and grizzle, 2001). jarvis et al. (2001) found that weight gain and feed conversion efficiency in juvenile shortnose sturgeon was the highest in fresh water, and the lowest at 20 ppt, the highest salinity tested. juvenile adriatic sturgeon, a. naccarii, had lower specific growth rates and food conversion efficiencies at 20 than 0 ppt (mckenzie et al., 1999) and lower specific growth rates at 11 than 0 ppt (mckenzie et al., 2001). a recent study by allen et al. (2014) indicated that growth rate in atlantic sturgeon was greater at 0 and 10 ppt than in seawater (33 ppt). the beluga sturgeon in this study also grew better in near freshwater and lower salinity medium. physiological and structural changes in fish following exposure to higher salinities require energy which may negatively impact the growth due to an increased osmoregulatory costs. although juveniles of many fish species grow optimally in intermediate salinities (boeuf and payan, 2001), the results obtained from sturgeon species vary probably due to life history, age/body size differences, species-specific morphophysiological mechanisms and osmoregulatory ability. it can be concluded that juvenile beluga sturgeon are capable to acclimate, grow and survive in brackish water. this capability can lead the selection of beluga sturgeon as a good candidate for pen and cage culture in brackish water of the iranian area of the caspian sea. nonetheless, it should be noted that initial salinity adaptation plays a key role in survivorship and growth rates in saline environments. acknowledgments the authors wish to thank shahid marjani sturgeon center for their assistance in providing specimens. we are also grateful to the aquaculture research center and the laboratory of biology at gorgan university of agricultural sciences and natural resources (guasnr) for accessing their equipment and facilities. the project was supported by financial supported by guasnr. references allen p.j., cech, j.j.jr. 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(2017) 5(4): 236-245 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی روی طبیعی محیط هایغلظت بر مبتنی شوری اثرات آزمایشگاهی ارزیابی: شوری به (huso huso) ماهیفیل پاسخ رشد کارایی و اسمزی تنطیم 1میاندره کلنگی حامد ،3،1نژاد عقیلی مصطفی سید ،1سوداگر محمد ،3 ،2 ،1*جاللی علی .ایران گرگان، گرگان، طبیعی منابع و کشاورزی علوم دانشگاه زیست، محیط و شیالت دانشکده1 .استرالیا ویکتوریا، دیکین، دانشگاه محیطی، و زیستی علوم دانشکده2 .ایران گلستان، استان خاویاری ماهیان امور مدیریت3 چکیده: شوری اثرات مطالعه، این در. است ضروری پرورشی جوان تاسماهیان در ویژهبه شوری محیطی چالش به خاویاری ماهیان پاسخ چگونگی بهتر درک وزن گرم huso huso( )22/1) ماهیفیل بچه کارایی و اسمزی تنظیم روی( لیتر در گرم 12 و 9 ،6 ،3 ،5/0) طبیعی محیط هایغلظت بر مبتنی شوری جانبی عوارض از ای نشانه اگرچه ،بود باال نسبتاً آزمایش دوره تمام در ماندگاری نرخ. گرفت قرار بررسی مورد روزه 60 دوره یک طی در( اولیه هاگروه سایر از بهتر لیتر در گرم 3 شوری در یافته پرورش ماهیان در رشد عملکرد. گردید مشاهده لیتر در گرم 12 شوری در ماهیان بقای میزان بر و زیممنی کلسیم، پتاسیم، سدیم، سطح افزایش کلی،طوربه. داشتند را رشد بیشترین ترتیببه 12 و 9 ،6 هایشوری در ماهیان آن از پس و بود، اتوکریتهم سطح. داشت را پارامترها این اولیه سطوح( شیرین بآ) کنترل گروه که حالی در شد، مشاهده شوری افزایش با ارتباط در پالسما در گلوکز نشان نتایج. بود هاغلظت سایر از ترپایین 6 و 3 شوری شیرین، آب گروه ماهیان در هماتوکریت تغییرات و گرفت قرار تاثیر تحت شوری توسط نیز آب رد شوری به سازگاری خاص هایمکانیسم مقایسه و ارزیابی اهمیت. بودند متوسط شوری با انطباق و ماندن زنده به قادر ماهیانفیل بچه که داد ایهاستراتژی بهبود به تواندمی تحقیقاتی چنین انجام از حاصل نتایج. گرفت قرار بحث مورد مطالعه این در خزر دریای جنوبی منطقه شورلب .نماید کمک تاسماهیان پروریآبزی .تاسماهیان پرورش ،سازگاری ،اسمزی تنظیم ،جوان خاویاری ماهی ،خون بیوشیمی :کلمات کلیدی int. j. aquat. biol. (2017) 5(1): 7-11; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology short communication seasonal changes in length-weight relationship and condition factor of nile tilapia, oreochromis niloticus (linnaeus, 1758) (cichlidae) in lake naivasha, kenya james last keyombe*1, john o. malala1, edna waithaka2, ruth m. lewo3, benson ojowi obwanga4 1kenya marine and fisheries research institute, turkana research station, p.o. box 205-30500, lodwar, kenya. 2kenya marine and fisheries research institute, naivasha research station, p.o. box 837-20117, naivasha, kenya. 3nakuru county fisheries department, naivasha sub-county, p.o. box 135-20117, naivasha, kenya. 4laikipia university, department of biological and biomedical sciences, p.o. box 1100-20300, nyahururu, kenya. article history: received 13 october 2016 accepted 22 january 2017 available online 2 5 february 2017 keywords: allometry condition factor length-weight relationship oreochromis niloticus abstract: the study compared the length-weight relationship and condition factor (k) of nile tilapia (oreochromis niloticus) in lake naivasha, kenya, between the wet and dry seasons. fish samples were collected monthly by gill netting from february to december 2015. a total of 372 samples of o. niloticus were analysed. the b values in the length-weight relationships were observed as 3.077 and 3.366 in the wet and dry seasons, respectively. the values of b exhibited positive allometric growth which were the important indication that the species was growing faster in weight than length. the k values of the fish ranged 1.18-4.1 during the wet season and 0.8-3.0 during the dry season. all mean monthly k values were found to be greater than 1, the highest being in june (2.33±0.05) and lowest in february (1.31±0.28), an indication of a healthy status and general well-being of the o. niloticus population in lake naivasha. the study concluded that seasonal variation has no great influence in the length-weight relationship and condition factor of o. niloticus in lake naivasha. introduction the fishery of lake naivasha mainly depends on six non-native fish species: oreochromis niloticus (nile tilapia), o. leucostictus (blue spotted tilapia), tilapia zilli (gervais), micropterus salmoides (black bass), cyprinus carpio (common carp) and clarias gariepinus (african catfish). the riverine barbus amphigramma and procambarus clarkii (red louisiana crayfish) have been supporting the fishery of lake naivasha significantly since 1980s (oyugi et al., 2011). oreochromis niloticus (nile tilapia) was first introduced in lake naivasha around 1967 but disappeared by 1971 for reasons unclear to date (hickley et al., 2008). anthropogenic disturbance of the littoral vegetation, particularly through clearance and burning could have previously reduced its spawning areas as reported by hickley et al. (2008) resulting to its disappearance in the 1970s. the government of kenya through the economic * corresponding author: james last keyombe e-mail address: katalitsa@yahoo.com stimulus package (esp) reintroduced the nile tilapia between 2010 and 2013 to boost the local fisheries in lake naivasha. the fingerlings were sourced locally in accredited government fish hatcheries. since its reintroduction, kenya marine and fisheries research institute (kmfri) data shows a constant increase in landings by fishermen in all the fish landing beaches around lake naivasha. the same has been witnessed during the monthly fish stock assessment exercises on the lake. length-weight relationship (lwr) is a useful tool in a wide range of applications such as estimation of biomass from length data, estimation of a species condition factor and comparisons among life history and morphologic differentiations of the same species in other aquatic systems (binohlan and pauly, 2000). lwr measurement is also a useful tool that provides important information concerning the structure and function of fish populations in any aquatic systems 8 keyombe et al./ seasonal changes in length-weight relationship and condition factor of nile tilapia (anderson and neumann, 1996). when the b value is less than 3, the fish has a negative allometric growth but when it is greater than 3, it has a positive allometric growth and when it is equal to 3, the fish has isometric growth (khairenizam and normarashid, 2002). change of b values depend primarily on the shape and fatness of the fish species as well as physical, chemical and biological factors such as temperature, salinity, food, stomach fullness, sex and stage of maturity (sparre and venema, 1998; sarkar et al., 2013). fish condition factor is an important concept in fisheries management and can be used to assess the health and potential of any fishery to support the fishing pressure. the condition factor often referred to as k provides information on the general wellbeing of a fish and health condition of a population. condition factor is estimated by comparing individual fish weight of a given length to a standard weight. fish body condition is known to vary seasonally depending on changes in gonadal development, food availability, and other environmental factors (pope and willis, 1996). it is usually influenced by the type of fish species, sex, season, maturity stage among other factors (anyanwu et al., 2007). the role of the condition indices as stated by stevenson and woods (2006) is to quantify the health of individuals in a population or to tell whether a population is healthy relative to other populations. when fish of a given length exhibits higher weight it means they are in better condition (anwa-udondiah and pepple, 2011). the determination of seasonal variations of b and k values of o. niloticus, a reintroduced fish species in lake naivasha, is therefore useful in assessing the well-being, growth performance and feed utilization of the fish in the lake. the main focus of this paper is to assess the seasonal variations in lwr and condition factor of o. niloticus in lake naivasha between february and december 2015. materials and methods the study was carried out at random sampling sites in lake naivasha, an equatorial rift valley lake with an annual atmospheric temperature that rarely falls below 20°c (hickley, 2008). the lake is a shallow freshwater body, situated in the eastern rift valley of kenya (0°46's, 36°20'e) at an altitude of about 1890 m above sea level. it covers a surface area varying between 120 km2 and 160 km2 depending on the wet and dry seasons respectively (hickley et al., 2008). the lake’s mean depth varies 4-6 m (hickley et al., 2008). the lake is the major source of fish for the surrounding community and fresh water for the numerous horticultural industries in the area. apart from transient streams, the lake is fed by the perennial malewa and gilgil rivers with the former being the main one (kitaka et al., 2002). there are 2 wet seasons in the months of march-may (long rains) and october-november (short rains) characterized by high lake water levels (oyugi et al., 2011). sampling procedure comprised setting of gill nets of variable mesh size ranging from 25-70 mm at dusk, with lifting after approximately 12 hours of fishing. fish caught in the nets were identified at the species level, length and weight of individual fish were measured in situ and recorded for each sampling occasion. the total length (tl in cm) of each fish was measured using a meter rule. weight of each fish was measured using the sf-400 digital weighing balance. the length-weight relationship was calculated using the formula by wooton (1990) as: w= alb where w is the body weight of fish in grams, l the total length in centimeters, a, the intercept and b the slope of the regression line. condition factor (k) was estimated following fulton (1902) and le cren (1951): k=w/l3x100 where k is the condition factor, w is the body weight of fish in grams, l the total length in centimeters. results and discussion a total of 372 specimens of o. niloticus (191 during wet and 181 during dry season) were used in this 9 int. j. aquat. biol. (2017) 5(1): 7-11 study. allometry coefficient values b obtained were 3.077 and 3.366 in the wet and dry seasons, respectively (figs. 1, 2). the relative condition factor of o. niloticus in lake naivasha ranged from 1.18 to 4.1 during the wet season and 0.8 to 3.0 during the dry season. the wet and dry seasons had fish with mean k values of 1.92±0.09 and 1.89±0.49, respectively. the highest and lowest k values recorded during the sampling period were 2.33±0.05 and 1.31±0.28 in june and february 2015 respectively, both falling within the dry season (fig. 3). the results showed that o. niloticus had positive allometric growth patterns (b>3) in both the dry and wet seasons indicating that the fish is growing faster in weight than length. this could be linked to the high productivity of lake naivasha in both the wet and dry seasons (kitaka et al., 2002). however, these results differed with a study by outa et al. (2014) done between november 2013 and january 2014 in the same lake who recorded the b value as 2.3. differences in b values can be ascribed to one or a combination of factors including differences in the number of specimens examined, location and season effects and distinctions in the observed length ranges of the specimens caught and the duration of sample collection (moutopoulos and stregiou, 2002). the marked difference in the b value obtained by outa et al. (2014) could also be attributed to the short period spent by the fish in lake naivasha since its introduction in 2011. the current study showed no marked differences in a and b values between o. niloticus in lake naivasha and populations elsewhere. this is supported by a study of o. niloticus in lake baringo, a rift valley lake in kenya, by kembenya et al. (2014) where b values were 3.08 and 3.04 in the wet and dry seasons, respectively. based on these results, o. niloticus in lake naivasha had mean monthly k values of greater than 1 in all the months sampled an indication of general well-being and stable physiological status of the fish in the lake. however, the condition factors were relatively higher than the values (1.97-2.63) documented by outa et al. (2014) in an earlier study of the same fish species in lake naivasha. the present higher k values might be attributed to adaptation of feeding habits of o. niloticus in lake naivasha to the different trophic levels in the lake enabling it to comfortably obtain food all year round. according to canonico and arthington (2005), o. niloticus is known to adapt readily to a range of figure 1. length-weight relationship of oreochromis niloticus in lake naivasha during the wet season in 2015. figure 2. length-weight relationship of oreochromis niloticus in lake naivasha during the dry season in 2015. figure 3. mean monthly condition factor of oreochromis niloticus in lake naivasha in both wet and dry seasons of 2015. 10 keyombe et al./ seasonal changes in length-weight relationship and condition factor of nile tilapia environmental factors such as salinity, low oxygen levels and can feed at different tropic levels when need arises. deekae et al. (2010) noted that several factors affect the condition factor of fishes. these range from feeding, spawning, food nutrient composition and fat accumulation. the variations of condition factor (k) in fish according to king (1995) may be due to food abundance, adaptation to the environment and gonadal development. the mean condition factor in the wet and dry seasons in this study varied slightly with those obtained by kembenya et al. (2014) in lake baringo, where o. niloticus were observed to have k values ranging from 0.59 to 1.89 during the dry season and 0.80 to 3.70 during the wet season. conclusions this study revealed that seasonal variation has no great influence in the length-weight relationship and condition factor of o. niloticus in lake naivasha. the faster growth of o. niloticus in weight than length favours the fishermen and fish traders around lake naivasha as it enhances its profitability. the k values obtained for the fish showed that the population was in good condition, an indication of the healthy status of the population. it is equally an indication of the ability of lake naivasha to sustain a healthy population of o. niloticus. references anderson o.r., neumann r.m. (1996). length, weight and associated structural indices. in: l.a. nielsen, d.l. johnson (eds.). fisheries techniques. bethesda, american fish society. pp: 447-482. anwa–udondiah e.p., pepple p.c.g. (2011). lengthweight relationship and condition factor of black chin tilapia (sarotherodon melanotheron) cultured in sheltered outdoor tanks. in: r.j. kolo, a.m. orire (eds.). proceedings of the 26th annual conference of the fisheries society of nigeria (fison), minna. 28th november-2nd december. pp: 98-102. anyanwu p.e., okoro b.c., anyanwu a.o., matanmi m.a., ebonwu b.i., ayabu-cookey i.k., hamzat m.b., ihumekpen f., afolabi s.e. (2007). lengthweight relationship, condition factor and sex ratio of african mud catfish (clarias gariepinus) reared in indoor water recirculation system tanks. research journal of biological sciences, 2(7): 780-783. binohlan c., pauly d. (2000). the length-weight table. in: fishbase 2000: concepts, design and data sources, r. froese, d. pauly, (ed). iclarm, isbn 971-870999-1. manila, philippines. pp: 121-123 canonico g.c., arthington a. 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(2011). management implications of the response of two tilapiine cichlids to long-term changes in lake level, allodiversity and exploitation in an equatorial lake. ambio, 40(5): 469-478. pope k.l., willis d.w. (1996). seasonal influences on freshwater fisheries sampling data. revised fish science, 4(1): 57-73. sarkar u.k., khan g.e., dabas a., pathak a.k., mir j.i., rebello s.c., pal a., singh s.p. (2013). length weight relationship and condition factor of selected fresh water fish species found in river ganga, gomti and rapti, india. journal of environmental biology, 34: 951-956. sparre p., venema s.c. (1998). introduction to tropical fish stock assessment. manual part 1. fao fisheries technical paper no 306. wooton j. (1990). ecology of teleost fishes. chapman and hall, new york. 404 p. int. j. aquat. biol. (2013) (1): 28-32 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology sulfuric acid treatment for artemia cyst decapsulation seyyed morteza hoseini *1, mohammad hadi abolhassani1, rasool ghorbani1 1 department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 2 march 2013 accepted 12 april 2013 available online 14 april 2013 keywords: acipenseridae decapsulation artemia sulfuric acid hypochlorite solution abstract: in the present study, sulfuric acid was used for artemia cysts decapsulation. cysts of artemia franciscana were hatched out in regular manner or following hypochlorite or acid decapsulation. two acid concentrations (1 and 5%), three acid immersion times (10, 30 and 50 min) were used and hatching rates were recorded after 15, 18 and 24 h incubation. hatching rates increased but hatching time decreased in line with acid concentration and acid immersion time increment. hypochlorite-treated cysts had significantly higher hatching rate (97%) compared to other groups. however, among the acidtreated cysts, the best hatching rate (92.4%) was achieved in cysts treated with 1% acid over 50 min. acid treatment could be used as a decapsulation method which saves cost and labor because of increasing the hatching rate and speed. introduction live food is necessary for fish larvae, especially in marine species. undeveloped digestive system of larvae, makes them completely dependent on live food. live foods have other advantages over the formulated feed including appropriate size, palatability due to high water content, immersion in water column and stimulation of larval predatory behavior (bengston, 2003). nauplius of artemia sp. is one of the important live foods with the widespread use in aquaculture. it contains 41-62% protein and 12-23% lipid which is a good source of linolenic acid and eicosapentaenoic acid (dhot and van stappen, 2003). small size, acceptable nutritional values, easy to produce and suitability for use in bio-encapsulation process are the advantages of artemia nauplii. the common technique to produce artemia nauplii is to collect and hatch the cysts under artificial condition. the cysts have a chorion shell constituted of lipoproteins, chitin and haematin (garcía-ortega et al., 1998). this layer is completely indigestible by all known cultured species and may cause gut obstruction * corresponding author: seyyed morteza hoseini e-mail address: seyyedmorteza.hoseini@gmail.com (dhot and van stappen, 2003). during nauplii production, some cysts might not hatch. consumption of these cysts may cause gut obstruction in fish larvae, which should be considered. decapsulation is a process in which the chorion layer is dissolved and removed. decapsulated cysts could be used as a food source for fish larvae, although its application is limited compared to the nauplii. decapsulated cysts have been used to rear larvae of freshwater catfish (clarias gariepinus), common carp (cyprinus carpio), and marine shrimp (penaeus indicus and penaeus monodon) and milkfish (chanos chanos) (verreth et al., 1987; vanhaecke et al., 1990; stael et al., 1995; ribeiro and jones, 1998; sui, 2000). the decapsulated cysts offer a number of advantages over nauplii and non-decapsulated cysts, in larval production (dhot and van stappen, 2003): 1. use of decapsulated cysts instead of nauplii eliminates the need for labor and additional hatching-related facilities. 2. cyst shells are not introduced into the culture tanks. non-decapsulated cysts introduce shell to the 29 hoseini et al./ int. j. aquat. biol. (2013) (1) 28-32 rearing tanks. it is not easy to separate all shells. in addition, hatching rate is not always 100% and the remaining unhatched cysts may cause gut obstruction in larvae. 3. decapsulated nauplii have higher energy content and individual weight (30–55% depending on strain) compared to nauplii hatched out of non-decapsulated cysts. where cysts have relatively a low energy content, the hatchability could be improved by decapsulation as the larvae need a lower energy to break out of a decapsulated cyst. 4. cysts would be completely disinfected by decapsulation. 5. cysts with poor hatching quality or even nonhatching cysts can still be used as a food source. decapsulation is normally performed by hypochlorite solution. the detailed protocol was described by dhot and van stappen (2003). it would be of interest to investigate other solvent for decapsulation. although cysts are washed following hypochlorite exposure, the risk of hypochlorite residual is not eliminated. hypochlorite releases chlorine, which is highly toxic to fish (brungs, 1973). the present study aimed to investigate the efficiency of sulfuric acid on decapsulation of artemia cysts. the residual of sulfuric acid is sulfate which is a natural water anion. materials and methods hatching technique was based on dhont and van stappen (2003). briefly, cysts of a. franciscana were artificially hatched by adding 1 g cyst to 1 liter water (33 ppt). cysts were incubated for 24 h. temperature and light intensity was maintained at 28 ºc and 2000 lux, respectively. continuous aeration was provided to ensure dissolved oxygen up to 5 ppm and suitable turbulence for cysts. hatching rate was determined by counting and averaging the nauplii in three samples (0.1 ml) under stereoscopic loupe (olympus, szx7, japan). control cysts were hatched following the aforementioned method. the cysts were decapsulated using the method by dhont and van stappen, 2003), cysts were allowed to hydrate for 1 h in clean aerating water. then, cysts were added to hypochlorite (liquid bleach, naocl, 12%) or acid solution (1 and 5% sulfuric acid, mihanjaz co., iran). cysts were removed from hypochlorite solution after 5 min. the cysts were washed with clean water 5 times prior to incubation. in acid treatments, cysts remained 10, 30 and 50 min in both 1 and 5% acid solution. thereafter, cysts were washed 5 times prior to incubation. hatching rates were recorded at 15, 18 and 24 h after incubation. to compare hatching rates of acid treated cysts, data were analyzed using a split-plot design, which incubation time considered as the whole plot and acid immersion time and concentration as split plots table 1. analyses of variance of effect of acid concentration (%), acid immersion time (min) and incubation time (h) on hatching rate of artemia cysts. source type iii sum of squares df mean square f p value it1 24298.7 2 12149.3 830 <0.0001 error 87.7 6 14.6 354.8 at2 4250.5 2 2125.2 35.2 <0.0001 it × at 844.35 4 211.1 10.3 <0.0001 con3 61.65 1 61.65 0.25 0.003 it × con 2.96 2 1.48 59.5 0.486 at × con 712.65 2 356.32 2.24 <0.0001 it × con × at 53.61 4 13.4 0.09 error 179.69 30 5.99 corrected total 30491.84 53 1. incubation time, 2. acid immersion time, 3. acid concentration. 30 hoseini et al./ int. j. aquat. biol. (2013) (1) 28-32 on the incubation time. to compare hatching rate of acid-treated groups, control group and hypochlorite treated group, data of 24 h incubation were analyzed using a one-way anova. duncan test was used as post hoc to determine significant difference among the treatments. p<0.05 was considered as significant difference. data are presented as mean ± se. results results showed that acid concentration, acid immersion time, incubation time, interaction between acid concentration × acid immersion time as well as acid immersion time × incubation time had significant effect on hatching rate (table 1). hatching rates of acid-treated cysts are shown in table 2. there was significant difference in hatching rate among 15, 18 and 24 h incubated cysts as hatching rate increased with increase of the incubation time (fig. 1). hatching rates increased with increase of the acid immersion time from 10 to 50 min (fig. 2). hatching rate of the cysts exposed to 5% acid was significantly higher than those exposed to 1% acid (fig. 3). comparison of hatching rates among acid treated, hypochlorite-treated and control cysts are shown in fig. 4. the hatching rates were in following order: 1% acid over 10 min < 5% acid over 10 min < control < 1% acid over 30 min and 5% acid over 30 min < 5% acid over 50 min < 1% acid over 50 min < hypochlorite. table 2. hatching rate (mean and se) in different acid concentration × acid immersion time × incubation time combinations. 95% confidence interval con (%) at (min) it (h) mean se lower bound upper bound 1 10 15 12.16 a 1.499218 9.126112 15.20722 18 25.90 bc 1.499218 22.85945 28.94055 24 51.23 f 1.499218 48.19278 54.27389 30 15 29.46 c 1.499218 26.42611 32.50722 18 43.43 e 1.499218 40.39278 46.47389 24 80.10 h 1.499218 77.05945 83.14055 50 15 34.16 d 1.499218 31.12611 37.20722 18 50.93 f 1.499218 47.89278 53.97389 24 92.40 j 1.499218 89.35945 95.44055 5 10 15 23.36 b 1.499218 20.32611 26.40722 18 29.90 cd 1.499218 26.85945 32.94055 24 59.36 g 1.499218 56.32611 62.40722 30 15 23.60 b 1.499218 20.55945 26.64055 18 40.16 e 1.499218 37.12611 43.20722 24 77.53 h 1.499218 74.49278 80.57389 50 15 22.56 b 1.499218 19.52611 25.60722 18 41.96 e 1.499218 38.92611 45.00722 24 84.76 i 1.499218 81.72611 87.80722 a b c 0 10 20 30 40 50 60 70 80 h a tc h in g r a te ( % ) incubation time (h) figure 1. effect of incubation time on hatching rate of acid-treated cysts. different letters above the bars show significant difference. p<0.05. se=3.63.combinations. 31 hoseini et al./ int. j. aquat. biol. (2013) (1) 28-32 discussion hypochlorite oxidation is the common method for artemia cysts decapsulation. however, if hypochlorite is not neutralized appropriately, it may damage the fish larvae. the present study showed that acid digestion could be used instead of hypochlorite oxidation for artemia cyst decapsulation. this method would be safe, since residual of acid, if any, may only change the ph slightly, depending on the water alkalinity. however, 3-5 times washing would completely eliminate the risk of ph change. previous studies showed that hatching rate of a. franciscana cysts were about 70 % (bruggeman et al., 1980; triantaphyllidis et al., 1994). in the present study, similar hatching rate was also obtained in nondecapsulated cysts. data of this study showed that decapsulation has a significant impact on hatching rate of artemia cysts. on the other hand, the present results showed that increase in acid immersion could speed up hatching process. this feature is important because this could be cost and labor saving in practice. increase in acid concentration and acid immersion time cause increase in hatching rate. this could be due to increased shell digestion allowing nauplii to hatch out spending lees energy and over a shorter time. similar results were reported in artemia parthenogenetica decapsulated with hypochlorite solution over 2-5 min (hosseini najd gerami and agh, 2008). the best hatching rate (97%) was observed in hypochlorite-treated cysts. although, hatching rate of the cysts treated with 1% acid over 50 min was significantly lower than hypochlorite-treated ones, it was still high (92.4%). however, all acid-treated cysts showed significantly higher hatching rate compared with control, except those exposed to acid over 10 min. this suggests that 10 min acid exposure is not suitable for decapsulation. more studies are needed to illustrate effects of acid-treatment on cysts in term of ph, shell thickness and embryo condition. it is concluded that acid-treatment could be a useful method for cysts decapsulation. there is no risk of hypochlorite residuals in this method. decapsulation with acid cause higher hatching rate and shorter hatching time compared to control. acid concentration and acid immersion time are important figure 2. effect of acid immersion time on hatching rate of acidtreated cysts. different letters above the bars show significant difference. p<0.05. se = 7.48. a b c 0 10 20 30 40 50 60 h a tc h in g r a te ( % ) acid immersion time (min) a b 40 41 42 43 44 45 46 47 48 49 50 h a tc h in g r a te ( % ) acid concentration (%) figure 3. effects of acid concentration on hatching rate of artemia cysts. different letters above the bars show significant difference. p<0.05. se = 0.50 c g a d f b d e 0 20 40 60 80 100 120 c o n tro l h p c a -1 -1 0 a -1 -3 0 a -1 -5 0 a -5 -1 0 a -5 -3 0 a -5 -5 0 h a tc h in g r a te ( % ) treatments figure 4. twenty four hours hatching rate of control, hypochloritetreated (hpc) and acid-treated (a-1-10 = 1% acid over 10 min, a1-30 = 1% acid over 30 min, a-1-50 = 1% acid over 50 min, a-510 = 5% acid over 10 min, a-5-30 = 5% acid over 30 min, a-5-50 = 5% acid over 50 min) cysts. different letters above the bars show significant difference. p < 0.05. 32 hoseini et al./ int. j. aquat. biol. (2013) (1) 28-32 factors affecting hatching rate and time. the best hatching rate (92.4%) in acid-treated cysts was achieved when cysts were exposed to 1% acid over 50 min. references bengston d. a. (2003). status of marine aquaculture in relation to live prey: past, present and future. in: j. g. støttrup, and l. a. mcevoy (eds.) live feeds in marine aquaculture, wiley-blackwell. pp: 1-16. bruggeman e., sorgeloos p., vanhaecke p. (1980). improvements in the decapsulation technique of artemia cysts. in: the brine shrimp artemia, vol. 3. (eds: persoone, g., sorgeloos, p., roels, o., jaspers, e.). university press, western belgium. 456 p. brungs w.a. (1973). effects of residual chlorine on aquatic life. journal (water pollution control federation), 2180-2193. dhot j., van stappen g. (2003). biology, tank production and nutritional value of artemia. in: j.g. støttrup and l.a. mcevoy (eds.) live feeds in marine aquaculture, wiley-blackwell. pp: 65112. garcía-ortega a., verreth j. a. j., coutteau p., segner h., huisman e. a., sorgeloos p. (1998). biochemical and enzymatic characterization of decapsulated cysts and nauplii of the brine shrimp artemia at different developmental stages. aquaculture, 161: 501–514. hosseini najd gerami e., agh n. (2008). optimization of decapsulation method for parthenogenetic artemia, artemia parthenogenetica originated from urmia lake. pajouhesh and sazandegi, 77: 42-47. ribeiro f.a., jones d.a. (1998). the potential of dried, low-hatch, decapsulated artemia cysts for feeding prawn post-larvae. aquaculture international, 6: 421-440. stael m., sanggontanagit t., van ballaer e., puwapanich n., tunsutapanich a., lavens p. (1995). decapsulated cysts and artemia flakes as alternative food sources for the culture of penaeus monodon postlarvae. in: p. lavens, e. jaspers, i. roelants (eds.) larvi’ 95, book of abstracts. european aquaculture society, special publication no. 24, ghent. pp: 342–345 sui l. (2000). use of artemia biomass in practical diets and decapsulated cysts as food source for common carp (cyprinus carpio l.). msc thesis, ghent university, ghent. triantaphyllidis g.v., pilla e.j., thomas k.m., abatzopoulos t.j., beardmore j.a., sorgeloos p. (1994). international study on artemia l ii. incubation of artemia cyst samples at high temperature reveals mixed nature with artemia franciscana cysts. journal of experimental marine biology and ecology, 183: 273-282. vanhaecke p., de vrieze l., tackaert w., sorgeloos p. (1990). the use of decapsulated cysts of the brine shrimp artemia as direct food for carp cyprinus carpio l. larvae. journal of world aquaculture society, 21: 257–262. verreth j., segner h., storch v. (1987). a comparative study on the nutritional quality of decap-sulated artemia cysts, micro-encapsulated egg diets and enriched dry feeds for clarias gariepinus (burchell) larvae. aquaculture, 63: 269–282. int. j. aquat. biol. (2023) 11(2): 115-118 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article the leucosiid crabs of the iraqi coast with a new record of nursia plicata (herbst, 1803) amaal gh. yasser1, 2, murtada d. naser1,2 1marine science centre, university of basrah, basrah, iraq. 2school of environment and science, griffith university, 170 kessels road, nathan, queensland, 4111, australia. article history: received 20 february 2023 accepted 24 march 2023 available online 2 5 april 2023 keywords: iraqi marine waters, morphological traits, leucosiidae. abstract: the leucosiid crabs are common species that can be found on soft sediments ranging in depth from the intertidal flat to the shelf and slop. the leucosiid crab, nursia plicata (herbst, 1803), is reported in this study based on the collection of two specimens in april 2019 from the iraqi marin waters. photographs and illustrations of the species and its external morphological traits and the first male gonapod g1 are given. introduction there are many records of decapod crabs from the iraqi coast of the persian gulf (naser, 2009, 2011, 2018, 2019; ng et al., 2012; naser et al., 2010; naser, 2011; naser et al., 2012; naser et al., 2013; yasser and naser, 2019a, b; yasser et al., 2021). there are three subfamilies within the leucosiidae viz. leucosiinae samouelle, 1819, cryptocneminae stimpson, 1907, and ebaliinae stimpson, 1871 (ng et al., 2008; emmerson, 2016). the leucosiid taxonomic statute is not, however, well-defined. there are 7 species of leucosiid crabs in the iraqi coast, including arcania erinacea fabricius, 1787, ixa holthuisi tirmizi, 1970, seulocia anahita galil, 2005 (yasser and naser, 2019), hiplyra sagitta galil, 2009 (al-khafaji et al., 2017), h. elegans gravier, 1920 (al-maliky et al., 2020), lyphira perplexa galil, 2009 (al-maliky, 2020), and l. heterograna (ortmann, 1892) (al-maliky and almaliky, 2021). the genus nursia leach, 1817 consists of three species in the persian gulf, namely n. blandfordi alcock, 1896, n. persica alcock, 1896 and n. plicata (herbst, 1803). nursia plicata is already recorded from the kuwaiti coast (apel, 2001). this work aimed to record n. plicata as a new correspondence: murtada d. naser doi: https://doi.org/10.22034/ijab.v11i2.1861 e-mail: murtada.naser@uobasrah.edu.iq dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.4.6 record family leucosiidae from the northwest of the persian gulf on the iraqi coast. materials and methods nursia plicata was collected in april 2019 from two locations along the iraqi shore of the northwest persian gulf (fig. 1). the samples were preserved in 95% ethanol and deposited at the marine science center (msc), university of basrah, iraq, with a collection voucher of msc401. the specimens were photographed with a digital camera (nikon d7100). the abbreviations “cl” and “cw” are used for the length of the carapace from the front to the posterior dorsal margin, and the width of the carapace at its widest point, respectively. the measurements of the species are reported to the closest millimeter and were taken with an electronic caliper. the systematics of this species was adapted from naderloo and sari (2005) and naderloo (2017). results nursia plicata (herbst, 1803) (figs. 2a-b) cancer plicatus herbst 1803: 2, 3, pl 59, fig 2, nursia plicata — alcock 1896: 179 (in key), 180, 116 yasser et al./ the leucosiid crabs of the iraqi coast with a new record of nursia plicata 181; stephensen 1946: 70, fig 6c; titgen 1982: 248 (in list); sakai 1999: 16; apel 2001: 54; naderloo and sari 2005a: 35, fig 5, 2007a: 342, tab 1; ng et al. 2008: 92 (in list). nursia plicata plicata — campbell and stephenson 1970: 249 (in key). type locality: “ostindien”. east india or indomalayan archipelago. material examined: two specimens, 1♂ cl. 19.0 mm, cw. 13.8 mm; 1♂ cl. 21.9 mm, cw. 16mm. carapace nearly pentagonal, slightly wider than long, with two semicircular lobes on posterior margin (fig. 2a). front barely visible beyond eyes, with a thickened granular edge. third maxillipeds, pterygostomian regions, thoracic sterna, and proximal part of male abdomen having granular surfaces (fig. 2b). the manus is longer than the finger, the carpus has an upper and inner surface covered in very minute granules, and chelipeds long figure 1. sampling sites (red dots). figure 2. nursia plicata (herbst, 1804): a: carapace, b: abdomen of male; cw. 13.8 mm. (photographs by m.d. naser). figure 3. nursia plicata (herbst, 1804): first male gonopod g1. 117 int. j. aquat. biol. (2023) 11(2): 115-118 and stout. first gonopod sinuous, proximally setose, and distally bent (fig. 3). the previous records of the leucosiid crabs from northwest of the persian gulf from iraqi coasts are shown in table 1. discussion the leucosiidae family is widespread in the red sea, the gulf of oman, and the persian gulf. of all the brachyuran families, this one is the most diversified and widely distributed (stephensen, 1946; titgen, 1982; apel, 2001; naderloo and türkay, 2012). about 16.6% of the brachyuran crab species in the persian gulf belong to the leucosiidae family (apel, 2001). the reported leucosiid species in the persian gulf has increased to 37 according to naderloo and apel (2012) and naderloo and türkay (2012). recently, al-maliky and al-maliky (2021) listed lyphira heterograna (ortmann, 1892) for the first time from iraqi marine waters, which may raise the number of this family to 38 species in the persian gulf. however, we can’t confirm the identity of l. heterograna reported by al-maliky and almaliky (2021) due to the poor quality of the picture. most leucosiid crabs from the iraqi coast belong to the subfamily ebaliinae, except the leucosiid seulocia anahita galil, 2005 which belongs to the subfamily leucosiinae. the indo-west pacific genus nursia leach, 1817, contains 20 species (ng et al., 2008). most of its species are distinguished by their plate-like appearance with distinct ridges on the dorsal surface of the carapace. this genus is extremely diverse and needs further revision. there are three species of the genus nursia in the persian gulf, with only one species recorded from the northwest of the persian gulf. nursia plicata is widely distributed in the persian gulf: iran (naderloo and sari 2005; naderloo and sari 2007), and kuwait (apel, 2001). alcock (1896) recorded this species from an unknown locality. there is no record from the gulf of oman. the distribution of this species in the world is indo-west pacific: persian gulf, india, palk straits, mumbai, china, hong kong, and japan. this species is found at the subtidal 3-20 m, living on a sandy substrate with clay and stones. acknowledgements we thank r. naderloo (school of biology, university of tehran, iran), for his valuable comments. references al-maliky t.h. (2020). new records of leucosiid crabs lyphira perplexa galil, 2009 (crustacea; decapoda; leucosiidae) in the northwest of the gulf–iraq. indo pacific journal of ocean life, 4(1): 1-3. al-maliky t.h., al-maliky a.m. (2021). first record of lyphira heterograna (ortmann, 1892) from the north west of the gulf. jordan journal of biological sciences, 14(1): 7-9. al-maliky t.h., ukash a.n., al-maliky a.m., zeini a., carnin r.l.p. (2020). first record of hiplyra elegans (gravier, 1920) (crustacea; decapoda; leucosiidae) in the north-west gulf–iraq. revista meio ambiente e sustentabilidade, 9(19): 91-98. al-khafaji k.k., abdul-sahib i.m., ajeel s.g. (2017). first records of leucosiid crabs: hiplyra sagitta (galil, 2009) from iraqi coast, nw gulf. international journal of aquaculture, 7: 139-142. apel m. (2001). taxonomie und zoogeographie der brachyura, paguridea und porcellanidae (crustacea: table 1. species and subfamilies of the family leucosiidae from the iraqi coast. species family subfamily arcania erinacea fabricius, 1787 leucosiidae ebaliinae ixa holthuisi tirmizi, 1970 leucosiidae ebaliinae seulocia anahita galil, 2005 leucosiidae leucosiinae hiplyra sagitta galil, 2009* leucosiidae ebaliinae hiplyra elegans (gravier, 1920) leucosiidae ebaliinae lyphira heterograna (ortmann, 1892) leucosiidae ebaliinae lyphira perplexa galil, 2009 leucosiidae ebaliinae nursia plicata (herbst, 1803) leucosiidae ebaliinae * revised by al-maliky et al. (2020) as hiplyra elegans (gravier, 1920) 118 yasser et al./ the leucosiid crabs of the iraqi coast with a new record of nursia plicata decapoda) des persisch golfes. fachbereich biologie, frankfurt am main. emmerson w.d. (2016). a guide to, and checklist for, the decapoda of namibia, south africa and mozambique. cambridge scholars publishing, cambridge, u.k. 2: 1-645. naderloo r. (2017). atlas of crabs of the persian gulf. atlas of crabs of the persian gulf. 1st ed. 20 p. naderloo r., sari a. (2005). iranian subtidal leucosiid crabs (crustacea: deacapoda: brachyura) of the persian gulf: taxonomy and zoogeography. iranian journal of animal biosystematics, 1(1): 31-46. naderloo r., türkay m. (2012). decapod crustaceans of littoral and shallow sublittoral habitats along the eastern (iranian) coast of the persian gulf: faunistics, biodiversity and zoogeography, zootaxa, 3374: 1-67 naser m. (2011). the sesarmid crab parasesarma persicum naderloo and schubart, 2010 (crustacea: decapoda: brachyura: sesarmidae), new to the iraqi coastal waters of khor alzubair and shatt albasrah canal, basrah, iraq. jordan journal of biological sciences, 4: 185-190. naser m. (2019). a new record of eurycarcinus orientalis a. milne-edwards, 1867 (decapoda, brachyura, pilumnidae) from the northwestern part of the persian gulf. journal of biological studies, 4: 160164. naser m. (2009). first record of the freshwater crab, potamon mesopotamicum brandis, storch & türkay, 1998 (decapoda, brachyura, potamidae) from the alhuwaizah marshes, iraq. crustaceana, 82: 1599-1602. naser m. (2018). a new record of eurycarcinus integrifrons de man, 1879 (decapoda, brachyura, pilumnidae) from nw of the persian gulf, iraq. journal of biological studies, 1: 9-13. naser m., ali m., yasser a. (2010). new record of the fiddler crab uca (paraleptuca) sindensis (crustacea: brachyura: ocypodidae) from khor al-zubair, basrah, iraq. marine biodiversity records, 3: 1-3. naser m., page t., ng n., yasser a., bishop j.m., ng p.k.l., clark p.f. 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(1983). the systematics and ecology of the decapods of dubai, and their zoogeographic relationships to the persian gulf and the western indian ocean, dissertation, texas a & m university, usa. yasser a.g., alkhafaji k.s., darweesh h.s., naser m.d. (2020). a new record of the hairy crab pilumnus savignyi heller, 1861 (pilumnidae samouelle, 1819) from the northwest of the persian gulf. eurasian journal of biosciences, 14: 7575-7577. yasser a.g., naser m.d., jabbar i.m. (2020). far from the persian gulf, ilyograpsus rhizophorae barnard, 1955 (crustacea: decapoda: brachyura: macrophthalmidae) as a new record of khur alzubair, basra, iraq. eurasian journal of biosciences, 14: 7645-7649. yasser a.g., abdulsahib i.m., naser m.d., al-khafaji k.k., darweesh h.s. (2013). two records of macrophthalmus desmarest, 1823 (decapoda: brachyura: thoracotremata) from the nw of the gulf. arthropods, 2(3): 105-110. yasser a., naser m. 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(2022) 10(3): 187-200 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article reproductive cycle of laevistrombus canarium (littorinimorpha: strombidae) in krabi province, andaman sea, thailand lamai thongboon, saree niyomdecha, jintamas suwanjarat, chutchawan muenpo*1 division of biological science, faculty of science, prince of songkla university, songkhla 90110, thailand. s article history: received 24 december 2021 accepted 22 june 2022 available online 2 5 june 2022 keywords: dog conch spawning period gonadal histology gastropoda abstract: the mesogastropod laevistrombus canarium, or dog conch, is a commercially valuable species in the indo-pacific region. however, there is no information on its reproductive cycle in thailand. from december 2012 to january 2014, specimens of dog conch were sampled monthly from ao thung beach, on the andaman sea, southern thailand. seawater ph, temperature and salinity during the sampling period ranged 6.97-8.1, 28-30.8°c, and 27-34 ppt, respectively. spawning times were investigated through standard gonadal histology and calculation of the gonad index (gi). the population’s sex ratio departed from 1:1 and females dominated. five stages of gonad development were found in both sexes: resting, developing, mature, spawning and spent. monthly variations in these stages and gi showed that this species exhibited two major spawning periods: december to april and august to september, and synchronicity existed between the sexes. however, spawning in females was less frequent and of shorter duration than in males. the results also revealed that spawning times of s. canarium were not related to the environmental conditions recorded at the study site. the present study classified this population of l. canarium that spawn in two clear pulses. the collected data provide baseline information to manage the resources and conservation of l. canarium in the andaman sea. introduction the dog conch, laevistrombus canarium l., 1758, of the family strombidae is an economically significant mesogastropod species. indigenous to the indo-pacific region, this species occurs from southern india eastward to melanesia, from japan in the north to australia in the south (abbott, 1960; poutiers, 1998). in many parts of southeast asia, l. canarium is found within seagrass beds and lives on muddy and sandy bottoms (cob et al., 2008a, 2009c; uneputty et al., 2021). the dog conch is a delicacy and popular seafood in southeast asia. it is also traditionally picked for its shell, which has decorative value (poutiers, 1998; said et al., 2013). due to the commercial value of l. canarium, many aspects of its biology have been extensively determined, including age, growth, survival, distribution, mortality, sexual maturity, sex determination, population structure, species *correspondence: chutchawan muenpo doi: https://doi.org/10.22034/ijab.v10i3.1466 e-mail: chutchawan.m@psu.ac.th description, imposex, sex ratio, food consumption, polyculture, and metal accumulation (cob et al., 2008b, 2009a, b, c, 2011; hassan et al., 2019; ramses et al., 2019; maxwell et al., 2020; chang et al., 2021; kobkeatthawin et al., 2021). however, studies of its reproductive cycle are relatively scare. most information in this field has generally been found elsewhere in the strombidae family, particularly in the economically important caribbean species. data for strombus gracilior from conception bay, baja california, and mexico show two main spawning periods (aranda et al., 2003a). strombus pugilis from seybaplaya to campeche city, mexico exhibited continuous spawning (aranda et al., 2003a; cardenas et al., 2005). aliger gigas from the alacranes reef and banco chinchorro, quintana roo, mexico (aranda et al., 2003a, b) and the caribbean region (boman et al., 2018) displayed one short spawning, whereas a. gigas from the archipelago of san andres, providencia and 188 thongboon et al./ reproductive cycle of laevistrombus canarium in thailand santa catalina, colombia displayed two main spawning periods (avila-poveda and baqueirocárdenas, 2009). by comparison, data on the reproductive cycle of l. canarium in the indo-pacific region is limited despite the fact that it is a commercially exploited species. indeed, only the reproductive cycle of l. canarium in waters of the philippines has been investigated, where continuous spawning behavior was identified (libutaque, 2000). the current work aims to describe the reproductive cycle of l. canarium off ao thung beach, andaman sea, thailand. the information from the current study will be crucial to managing the fishing and conservation of dog conch in the region. environmental factors, sex ratio, gonad developmental stages, and spawning periods were investigated. materials and methods study site and sampling: specimens of dog conch were collected from ao thung beach, krabi province on the andaman coast of southern thailand (8°02’n, 98°45’e) (fig. 1). at monthly intervals during a 14month study period (december 2012 – january 2014), thirty to forty adult individuals were collected randomly by hand among seagrass beds during low tide. using a thermometer, manual optical refractometer, and ph meter, respectively, sea temperature, salinity, and ph within each sampling site were measured at a 2 m depth during specimen collection. in the laboratory, the total weight (tw) of whole individuals was measured with a digital balance (with a precision of 0.001 g), and shell length (sl) and shell width (sw) were measured using a vernier caliper to the nearest 0.05 mm. after breaking the shell in a vice, the soft part of the specimens was separated, the flesh weight (fw) was measured and individuals were sexed. males were recognized by the presence of a penis and a prostate gland, and females were recognized by the presence of a vagina and a capsule gland. reproductive cycle and gonad index: for histological investigations of the gonads, small pieces of the tissue were removed and fixed in bouin’s solution. after 24 hours, the collected tissues were rinsed with tap water and preserved in 70% ethyl alcohol. the gonad samples were dehydrated using a sequence of ascending alcohol series (70-100%), cleared with xylene, and embedded in paraplast. sections of 6 μm thick were cut with a rotary microtome and counterstained with harris’s haematoxylin-eosin. all sections were observed under a light microscope. identifying the stages of gonadal development was based on the categories defined for a. gigas by aranda et al. (2003b) and s. pugilis by cardenas et al. (2005). these categorizations comprised five stages: resting, developing, mature, spawning, and spent. mean gonad indices (gi) were calculated based on the methods described by king et al. (1989) using the following formula: gi = sum [number of specimens in each stage x numerical ranking of that stage] / [total number of specimens in the sample]. the gonad index would vary from 1 (minimum) if all specimens were resting and/or spent to 3 (maximum) if all specimens were mature. statistical analysis: the difference in the sex ratio was evaluated monthly using the chi-square test in spss program version 28.0.1 for windows. figure 1. location of sampling site of laevistrombus canarium off ao thung beach, krabi province, thailand. 189 int. j. aquat. biol. (2022) 10(3): 187-200 results environmental parameters: the ph of the seawater in the study area fluctuated between a high of 8.10 in april 2013 and a low of 6.97 in october 2013 (fig. 2). from december 2012 to february 2013, water temperatures ranged from 28.20 to 29.30ºc, then steadily increased during march (30ºc) to reach the maximum temperature of 30.8ºc in may (fig. 3). from june (29.87ºc), temperature declined to the minimum of 28ºc, recorded in august. the temperature increased to 28.7ºc in september and reached a steady level of 30.1ºc during october and november. in december 2013 and january 2014, the last two months of the study, sea temperature dropped to 28.6 and 28.8ºc, respectively. in december 2012, the salinity of the seawater was 31 ppt. there was a slight decrease to 30 ppt in january 2013 (fig. 3). salinity increased in february (32 ppt). it continued through march and april to reach the maximum level of 34 ppt. salinity declined steeply in a single month from 34 ppt in april to 30 ppt in may but rapidly rose again to reach a steady level of 33.00 ppt through june, july and august. in september, salinity slightly decreased to 32 ppt and fell abruptly in october to the minimum of 27 ppt. during november and december 2013 and january 2014, the last three months of the study, salinity returned to levels similar to those of the previous year, ranging 30-31 ppt. gonad developmental stages: gonad development in both sexes could be classified at the microscopic level into five distinct stages according to the number of different germ cells and the percentage of connective tissue between testicular tubules and/or ovarian follicles. testis: the testis is composed of testicular tubules. the five histological stages of the testis are described as following: resting stage: in this phase, testes comprised mostly reticular connective tissue, which occupied 90% of the testes. few testicular tubules were found, and they were relatively small and irregularly shaped (fig. 4a). they were mainly composed of the spermatogonia, which lined the thickened tubular wall and had started to fill with primary and secondary spermatocytes. developing stage: in this stage, about 80% of the testes were occupied by connective tissue. the testes had abundant testicular tubules which had increased in diameter and were filled with maturing cells in all stages of development (fig. 4b). spermatogonia were present in smaller numbers and, having grown, separated from the tubular walls and appeared in the lumen. male germ cells within the testicular tubules were mostly primary spermatocytes, secondary spermatocytes, and dark-colored spermatids near the middle of the lumen. spermatozoa were occasionally observed in the lumen but were not well arranged. mature stage: the testes contain numerous expanded testicular tubules. connective tissue occupied the testicular tubules walls and markedly decreased in size (fig. 4c, d). the testicular tubules were typically figure 2. monthly variation in the ph level of seawater at the sampling site off ao thung beach, krabi province from december 2012 to january 2014. figure 3. monthly seawater temperature (°c) and salinity (ppt) at the sampling site off ao thung beach, krabi province from december 2012 to january 2014. 190 thongboon et al./ reproductive cycle of laevistrombus canarium in thailand fused, and apparent cells were spermatids and mature spermatozoa, which were more numerous than spermatids and formed a compact mass with the tails close to the middle of the lumen. in contrast, spermatogonia were scarce and close to the tubular walls, and spermatocytes were also less abundant than spermatozoa. spawning stage: the testicular tubules continued to fuse. a dense band of spermatozoa at the center of tubular lumens was still apparent but spermatozoa were less than in the mature stage because some had been discharged and were located in the testis duct figure 4. photomicrographs of testicular phases of collected male dog conchs stained with h&e: (a) resting stage; (b) developing stage; (c and d) mature stage; (e) spawning stage; (f) spent stage. scale bars = 50 µm (a, b, c, e, and f) and 20 µm (d). 191 int. j. aquat. biol. (2022) 10(3): 187-200 (fig. 4e). the release of these spermatozoa caused empty spaces in the testicular tubules. at the same time, the walls of the testicular tubules erupted, resulting in the formation of large irregular lumens containing cells at almost all stages. most of the lumen was empty, and although some spermatids remained, they were fewer in number than at the previous stage. spent stage: the testes had collapsed and degenerated. the testicular tissue was inhabited by reticular connective tissue. this stage was distinguished by broken and degenerated testicular tubules with mostly empty lumens that contained some reproductive cells at different stages of development (fig. 4f). very few spermatogonia were found close to tubular walls, and remaining spermatozoa were visible in the lumen. abundant phagocytes were observed among the rest of the testicular tubules and residual reproductive cells. ovary: the ovary is composed of ovarian follicles. the five histological stages of the ovary are described in the following. resting stage: ovaries presented mostly reticular connective tissue that occupied 90% of the ovaries. it was hard to distinguish this tissue from digestive gland tissue. low numbers of irregularly shaped follicles were found in this stage (fig. 5a). follicles mainly were lined with oogonia and a few small, developing pre-vitellogenic oocyte buds from the follicular wall. developing stage: ovaries became thicker and occupied 30% of the ovarian section. the follicles were well-developed and contained reproductive cells in all stages of development: oogonia, pre-vitellogenic oocyte, vitellogenic oocyte and mature oocyte (fig. 5b). oogonia were found in clusters of four to five cells and they had a spherical nucleus with a clear nucleolus. in the early stage, small ovaries and thickening follicles were evident. these follicles were mainly filled with pre-vitellogenic oocytes. in the late stage, the ovary was larger, and vitellogenic oocytes and mature oocytes were usually present in the follicles. mature stage: as the ovary ripened, occupying 4050% of the section, ovarian follicles were spherical, and their thin walls exhibited no germinal cells. the connective tissue between follicles had almost disappeared (fig. 5c, d). in this stage, most of the follicles were in contact with other follicles, and some had fused to form a large lumen. most follicles were filled with vitellogenic and mature oocytes. previtellogenic oocytes were also seen in the follicles but were less than in the developing stage. mature oocytes or eggs exhibited granular cytoplasm containing many yolk or vitelline globules stained red to pink with eosin. spawning stage: the irregularly shaped follicles contained partially empty spaces in the lumen, and follicular walls were thin and degenerated. vitellogenic oocytes were usually detected close to the follicle walls and were less abundant than in the mature stage (fig. 5e). connective tissue reappeared, starting from the external wall of the ovary and in the middle of the ovary and digestive gland. mature oocytes packed with vitelline in the cytoplasm were still present in the follicular lumen, but their numbers were less than in the previous stage. sometimes the walls of these cells were broken or burst, and phagocytes could be identified among them. reabsorption of residual oocytes was common. spent stage: ovaries had collapsed and contained more reticular connective tissue. this stage showed broken and degenerated follicles, and their lumens usually contained remaining gametes and cellular debris but were sometimes empty (fig. 5f). the residual female gametes within the follicles were mostly mature oocytes exhibiting autolysis and broken and wrinkled walls. a great number of phagocytes were found among the rest of the ovarian follicles and unspawned gametes. new oogonia production occurred in the follicles' periphery in the late stage. reproductive cycle: in males, resting, developing and spent testes were observed during eleven to twelve months of the study period. in contrast, the testes in the mature stage were only noted for four months. resting testes were present throughout the year. low percentages of resting testes were detected during january and february 2013 (5.0-14.3%), with an increase from march (25.0%) to june (89.3%) (fig. 6a). a steep decline occurred during july (12.0%). 192 thongboon et al./ reproductive cycle of laevistrombus canarium in thailand the resting testes frequency started to recover in september (31.0%) and was at the maximum level in october (90.0%). developing testes were also observed throughout the year but presented low proportions, with a maximum in july 2013 (65.5%) and a minimum in january 2014 (5.0%). mature testes were registered in two periods which were december 2012 (23.1%), february 2013 (5.0%), and august 2013 (45.7%, maximum). spawning testes were found with variable figure 5. photomicrographs of ovarian phases of collected female dog conchs stained with h&e: (a) resting stage; (b) developing stage; (c and d) mature stage; (e) spawning stage; (f) spent stage. scale bars = 50 µm (a, b, d, and f) and 100 µm (c, and e). 193 int. j. aquat. biol. (2022) 10(3): 187-200 frequencies during nine months of the study period. two main spawning periods were noted, the first from december 2012 to april 2013 (ranging from 9.1% in april 75.0% in january 2013), and the second from august (28.6%) to september 2013 (52.0%). after that, the dog conch started a new phase of spawning in december 2013 (38.9%) and the frequency of spawning testes was highest in january 2014 (90.0%). testes in the spent stage were first encountered in december 2012 (7.7%) and percentages of spent testes increased to a maximum level in march 2013 (43.8%). after that, during the remaining months spent testes presented at low percentages, ranging from 3.4% in july to 38.9% in december. in females, the resting and developing ovaries were observed during the twelve months of the study period. mature and spawning ovaries were noted during six months. resting ovaries were present throughout the year. in the four months of april, june, july, and december 2013, resting ovaries ranged from 50.2 to 58.3%. in the four months of february, may, october, and november, ovaries in this stage were observed, ranging from 80.1 to 96.0% (fig. 6b). in the remaining months, the resting ovaries were found in figure 6. percentage frequency of five gonad developmental stages in (a) male and (b) female laevistrombus canarium specimens collected monthly from december 2012 to january 2014. 194 thongboon et al./ reproductive cycle of laevistrombus canarium in thailand 8.3-46.2% of the specimens. developing ovaries were also marked throughout the year but represented small proportions with a maximum in august (60.0%) and a minimum in january 2014 (5.0%). mature ovaries were found in two periods: december 2012 (15.0%) to january 2013 (50.0%), and august (20.0%) to september (33.3%). whereas spawning testes were present in nine months of the study phase, spawning ovaries were only present in six months. similar to testes, the spawning ovaries were found in two main periods. the first period was from december 2012 (25.9%) to january 2013 (33.3%), while the second time of spawning was in september (13.3%). after that, a new spawning phase started in december 2013 (15.0%) and peaked in january 2014 (35.0%). spent ovaries were noted in nine months of the year but occurred at low percentages, with a maximum in march (46.2%) and a minimum in october (4.0%). gi: the gi of both males and females displayed a similar pattern with two major peaks annually. the first peak was in december 2012 and january 2013, while the second one was in august. in males, gi values ranged from a minimum of 1.0 in october to a maximum of 2.3 in august. in females, the highest gi value occurred in december 2012 (2.5), while the lowest value (1.0) was observed in may and october 2013. gi values ≥ 2.0 were only noted over short periods of three and four months in males and females, respectively (fig. 7). the male gi decreased slightly from december 2012 (2.2) to january 2013 (1.8). female gi was higher during these months (2.4-2.5). in february, the male gi remained fairly stable, whereas the female gi decreased sharply to 1.1 and was stable from march to may, ranging from 1.0 to 1.2. during these three months and in june, the male gi decreased to the same level as the female gi (ranging from 1.1-1.3). the female gi increased to a steady level during june and july (1.4) and then rose steeply to a high value in august (2.0), while the male gi increased sharply from july (1.7) to an annual high in august (2.3). after that, a similar trend between males and females was observed in which there were steep declines in gi values of both sexes between september (1.8 in males and 1.9 in females) and october (1.0, both sexes), followed by a steady increase in gi values in both sexes between november 2013 and january 2014 (ranging from 1.4-2.1 in males and 1.2-2.0 in females). sex ratio: a total of 551 adult specimens (238 males and 313 females) were observed between december 2012 and january 2014. the male/female ratio of the population collected over 14 months was 0.76, which differed significantly from a 1:1 ratio (x2 = 11.022, p<0.05) and revealed that females were dominant (table 1). there were no differences in sex ratios from january 2013 and february, april and may, september, and between december and january 2014 (x2-test, p>0.05). the sex ratios showed more males than females in july and august (x2-test, p<0.05), whereas females outnumbered males in december 2012, and in march, june, october and november 2013 (x2-test, p<0.05). discussions the sex ratio of the l. canarium population from the ao thung beach, andaman sea, thailand was unequal, with an obvious dominance of females. this female-biased sex ratio is in agreement with previous studies conducted on this species along the johor straits, malaysia (cob et al., 2009b), on the reefs in far north queensland, australia (maxwell et al., 2017) and in the waters of kota batam, indonesia (ramses et al., 2019). the findings of the present figure 7. monthly gonad index (gi) of laevistrombus canarium males (squares) and females (circles) from a population sampled off ao thung beach, krabi province, southern thailand from december 2012 to january 2014. 195 int. j. aquat. biol. (2022) 10(3): 187-200 study is also in agreement with a study on s. pugilis collected from seybaplaya to campeche city, mexico (cardenas et al., 2005). in contrast, the studies on s. gracilior from playa panamá, costa rica (jiménezarce, 1993) and canarium labiatum population on green island, located near cairns, queensland, australia (maxwell et al., 2020) found an equal sex ratio (1:1). although most monthly samples in the present study showed a balanced sex ratio, females dominated in december 2012, march, june, and in october and november and males dominated in july and august. the monthly alteration in the sex-ratio can be related to the species’ behavior during the reproductive cycle. for example, the high proportions of females observed during the major spawning seasons of bolinus brandaris (vasconcelos et al., 2012; elhasni et al., 2013) was accompanied by the establishment of packed collections of females to lay down collective spawns (martín et al., 1995; vasconcelos et al., 2008a). the congregation of females for this purpose is absolutely accountable for the female-biased sex ratios found in late spring and early summer. recently, it is evident that crepipatella dilatata has a high reproductive potential, considering a large number of months that individuals are actively reproducing and the number of brooding events per female each year (chaparro et al., 2019), whereas in haliotis gigantea, the female proportion (f/f+m) of the 290 specimens was 36.6%, indicating a distinctly higher proportion of males (shin et al., 2020). in hexaplex trunculus (elhasni et al., 2010), high numbers of males were discovered mostly during maturation and copulation and agreed with the peak of copulative activity in b. brandaris (vasconcelos et al., 2012). however, this correlation was not the case in the present study. based on known spawning intensity and duration, all three types of gastropod spawning patterns are found among species in the strombus group: one very extended spawning period, with or without a dominant peak, two or more clear peaks or spawning pulses, and one short defined pulse as proposed by aranda et al. (2003a). the population of l. canarium in the present study showed two main spawning periods with simultaneous gametogenesis and spawning between sexes: one period from december 2012 to april 2013 and another from august to september 2013. this synchrony between males and females produced coincident gametic releases in both sexes. in the andaman sea, thailand, however, male dog conchs spawned for 9 months (december 2012 – april 2013, august – september 2013, and december 2013 – january 2014) for a total of 14 months, whereas females spawned only for 6 months: from december month total males females sex ratio (m:f) p-value december 2012 40 12 28 0.43 : 1 0.011* january 2013 40 24 16 1.50 : 1 0.160 february 40 22 18 1.22 : 1 0.527 march 34 11 23 0.48 : 1 0.040* april 40 14 26 0.54 : 1 0.058 may 40 14 26 0.54 : 1 0.058 june 40 9 31 0.29 : 1 0.001* july 40 30 10 3.00 : 1 0.002* august 40 28 12 2.33 : 1 0.011* september 40 20 20 1.00 : 1 1.000 october 40 6 34 0.18 : 1 0.000* november 40 11 29 0.38 : 1 0.004* december 37 15 22 0.68 : 1 0.250 january 2014 40 20 20 1.00 : 1 1.000 total 551 238 313 0.76 : 1 0.001* *indicates samples with statistically unbalanced sex ratios (x2 – test) (p<0.05) table 1. monthly variation in the sex ratios of laevistrombus canarium collected from ao thung beach, krabi province during december 2012 to january 2014. 196 thongboon et al./ reproductive cycle of laevistrombus canarium in thailand 2012 to january 2013, in april and september 2013, and from december 2013 to january 2014, indicating that periods of maximum spawning were less regular in females and lasted for shorter periods than males. in the oyster, crassostrea rhizophorae, males also recovered and ripened faster than females. it is believed that oogenesis needs more time and more energy than spermatogenesis, owing to the necessity for yolk creation (nascimento and lunetta, 1978; eckelbarger and hodgson, 2021). this difference would explain the variations between sexes in the frequency and span of spawning observed in the present work. the annual dual spawning periods observed in the present study of l. canarium differ from the populations of miag-ao, iloilo, the philippines (libutaque, 2000), which exhibited continuous or constant spawning, in which males and females spawned monthly during the thirteen-month study period. spawning peaks in that study occurred in january, april and may for males and in april, august, september and october for females. this pattern of constant spawning of l. canarium along the philippine coast was also observed in populations of s. pugilis from seybaplaya to campeche city, mexico, where spawning organisms were found in june 1990, from june to september 1996, in february 1997 and from may to july 1997 (aranda et al., 2003a). also, in the same location (from july 1996 to july 1997), spawn was present throughout the year in over 70% of the male population, with a maximum of 100% in august and 2 periods of low intensity in july (15%) and october (40%), while spawning in females was discontinuous, with 4 pulses of low intensity in february and june 10%, and august and october 20%. (cardenas et al., 2005). similar behavior to the present population of l. canarium was exhibited by a population of s. gracilior from conception bay, baja california, mexico. two spawning pulses were observed, the first at the beginning of both years from february to march 1979 and 1980, and the second from august to november 1979 (aranda et al., 2003a). when compared to spawning patterns of other species, the spawning pattern observed in l. canarium and s. gracilior is different from that reported for a. gigas. most studies have suggested that a. gigas displayed one short, defined spawning pulse. a population from seybaplaya to campeche city, mexico exhibited this type of spawning from june to august with a peak during july and another population on the alacranes reef, quintana roo, mexico (aranda et al., 2003a) spawned from june to october. a similar observation in the banco chinchorro, quintana roo, mexico also found that the male a. gigas produced a short spawning period during june and july, with a maximum of 60% in july and a spawning period in females limited to the months of july and august in 20 and 33% of the population, respectively (aranda et al., 2003b). in addition, a. gigas collected from the caribbean region, also had a significantly shorter reproductive season (boman et al., 2018). in the case of a. gigas, only a study in the archipelago of san andres, providencia and santa catalina, colombia demonstrated a different spawning pattern, in which spawning occurred twice, once from march to april (6%) and again in september (6%) in males and from march to april (20%) and in september (43%) in females (avila-poveda and baqueiro-cárdenas, 2009). the gonadal development, gametogenesis, and spawning of molluscs are regulated by endogenous factors, including the endocrine system (euler and heller, 1963: horiguchi et al., 2021), genetics and nutrition (horiguchi et al., 2021), and exogenous factors, including seawater temperature (morriconi, 1999; vélez-arellano et al., 2009; elhasni et al., 2010; vasconcelos et al., 2012; elhasni et al., 2013; boman et al., 2018; panchenko and balanov, 2020; horiguchi et al., 2021; popović et al., 2021), food supply (jaramillo and navarro, 1995), day length (photoperiod) (elhasni et al., 2010, 2013; horiguchi et al., 2021), salinity (cain, 1974; stephner, 1981), storms (cronin, 2000), currents (davis and chanley, 1955; ino, 1970; guallart et al., 2020; horiguchi et al., 2021), specific gravity of seawater (taki, 1949), presence of microalgae (breese and robinson, 1981), onshore wind (horiguchi et al., 2021) and geographical location (cronin, 2000). among 197 int. j. aquat. biol. (2022) 10(3): 187-200 environmental factors, day length, seawater temperature, and food availability establish in many marine gastropods the most significant environmental cues for the regulators of reproduction such as maturation and spawning (sternberg et al., 2010). in the present study at ao thung beach, the recorded environmental parameters of ph, temperature, and salinity ranged in 2013 from 6.97 (october) to 8.1 (april), 28 (august) to 30.8ºc (may), and 27 (october) to 34 ppt (march and april), respectively. as mentioned earlier, the dog conch in ao thung beach, andaman sea, thailand produced two main spawning periods. however, there was apparently no association between these factors (ph, temperature and salinity) and the reproductive cycle of the dog conch. the findings of the present study are in agreement with the population of s. pugilis from seybaplaya to campeche city, mexico where there was no correlation between environmental factors (temperature and salinity) and the reproductive cycle of s. pugilis displaying continuous spawning (baqueiro-cardenas et al., 2005). the small amplitude of annual fluctuations in temperature and salinity (range = 33.5-35.0 ppt) in tropical regions may also explain why dog conchs occupy the inshore waters of miag-ao, iloilo, philippines spawning continuously throughout the year (libutaque, 2000). continuous spawning in the limpet acmaea scabra (gould) had a direct connection with food abundance (sutherland, 1970), whereas in endangered freshwater snail heleobia atacamensis would reproduce continuously throughout the year; it is not known what ecological parameters (e.g., feeding, microhabitats, abundance) influence its reproduction (collado et al., 2021). in ao thung beach, the temperature is around 29.29°c with an annual thermal amplitude of around 2 to 3°c and salinity is around 31.5 ppt with an annual amplitude of around 7 ppt. these values are approximately steady and hence, they might not affect the gametogenesis and spawning periods in l. canarium. information about the mechanisms that control the annual reproductive cycles in gastropods is limited but most studies have concluded that these cycles are induced by temperature. for example, the spawning season of nacella (p.) deaurata in lapataia bay, argentina was synchronous with a rise in water temperature (morriconi, 1999). similarly, in conception bay, baja california, mexico, the periods of highest maturation and spawning in tegula eiseni concurred with the seasonal temperature rise from july to november (vélez-arellano et al., 2009) and in bolinus brandaris sampled from the ria formosa lagoon (southern portugal), spawning coincided with rising seawater temperatures (vasconcelos et al., 2012). furthermore, a. gigas collected from the caribbean region found that locations with a relatively high variation in water temperature had a significantly shorter reproductive season (boman et al., 2018) and in the green ormer (haliotis tuberculata l.) from the northern adriatic sea, off western istria (croatia) showed that the cycles of gametogenesis were directly correlated with the seasonal condition of the temperature change (popović et al., 2021). acting together, in some gastropod species, day length and seawater temperature regulate the annual reproductive cycle, such as in hexaplex trunculus taken from the gulf of gabès (tunisia), whose main spawning period (april–may), strictly coincided with increasing day length (from 13.2 to 14.2 h) and seawater temperature (from 19.3 to 23.4ºc) during this period (elhasni et al., 2010) and in b. brandaris collected from the gulf of gabès (tunisia) whose spawning season (april and july) seems to be regulated by the annual cycle in day length and seawater temperature (elhasni et al., 2013). in nacella magellanica from punta ninfas, chubut, argentina, spawning period extended over all months except june in males, while females spawned from late winter to spring, with an increase from august to november; this prolonged period of gamete release of n. magellanica could be related to more favourable temperature and photoperiod (vilela et al., 2019). in addition to seawater temperature and day length/photoperiod, a recent study of gastropods has suggested that there are four exogenous factors that control the reproductive cycle (including spawning behavior) of thais spp. i.e., water temperature, illumination/photoperiod, tidal cycles, and onshore winds (horiguchi et al., 2021). 198 thongboon et al./ reproductive cycle of laevistrombus canarium in thailand conclusions this study of the reproductive cycle of l. canarium from the andaman sea, thailand, identified this gastropod population in tropical waters as a species that spawns in two major pulses. the information gathered from this study is crucial to specific ecosystem management plans, guaranteeing the conservation of the species along the coast of the andaman sea in thailand. these plans should establish, for example, closed seasons during the spawning peaks, from december to april and august to september and limit the collection of small-scale snails throughout the year. acknowledgments this research was supported by a grant from prince of songkla university (contract no. sci550425s). we are grateful to p. soonsun and her staff at the krabi coastal fisheries research and development center, coastal fisheries research and development bureau, and department of fisheries for their assistance in specimen collection and t. coyne for his assistance and proofreading the manuscript. references aranda d.a., cardenas e.b., morales i.m., baez r.i.o., brule t. 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(2014) 2(1): 29-35 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article phytoremediation of heavy metals (cd, pb and v) in gas refinery wastewater using common reed (phragmitis australis) amir hossein hamidian*,1majid atashgahi, nematollah khorasani department of environment, faculty of natural resources, university of tehran, p.o. box 31585-4314, karaj, iran. article history: received 11 november 2013 accepted 28 january 2014 available online 2 5 february 2014 keywords: gas refinery heavy metal phytoremediation phragmitis australis wastewater abstract: industrial wastewaters are of the major sources of heavy metal pollution in the environment. in the middle east, gas and oil industry is the major source of heavy metal pollution and releases significant amounts of metals into the terrestrial and aquatic environment. in this research the capability of the common reed (phragmitis australis) in absorbing heavy metals cd, pb and v from the wastewater of a gas refinery plant in iran was investigated. the plant samples were collected from the vicinity of the bidboland gas refinery plant in iran and were used for the treatment of wastewaters collected from the outflow of the refinery plant. the metal concentrations were measured in the roots of the plant species before treatment and after 2, 6 and 10 days of treatment procedure. the heavy metal concentrations were measured after acid digestion and using an icp-oes instrument. after 10 days of treatment, the concentrations of the metals in plant tissues increased by 2.5, 6.9 and 2.7 times for cd, pb and v, respectively. the best treatment time was suggested to be 6 days. the common reed was observed to have a very good capability in removing heavy metals from the gas refinery wastewater. introduction human beings around the globe release huge amount of wastewater into the environment. in particular, significant concentrations of heavy and hazardous metals, are released in industrial wastewater and these can cause numerous environmental problems (atashgahi et al., 2011). these adverse effects associated with metals can include the alteration of the structure of crops and functions of organisms and their effects can be observed in food chains. high metal concentrations can be toxic to some organisms and metal imbalance in plant tissues can be critical for different kinds of diseases (ha et al., 2010). the capability of some plant species to absorb and accumulate high levels of metals in their organs can be used for the purification of polluted environments; in a process known as * corresponding author: amir hossein hamidian e-mail address: a.hamidian@ut.ac.ir phytoremediation. aquatic macrophytes exhibit a high potential for heavy metal phytoremediation due to their rapid growth and high biomass (carranzaalvarez et al., 2008). among the different possible methods of wastewater treatment, phytoremediation is an environmental friendly and economic method. one of the plant species which has been widely used for treatment of heavy metals is common reed (phragmeties australis), which can tolerate different environmental conditions, including high concentrations of toxic heavy metals (ye et al., 1997; stoltz and greger 2002; kropfelova et al., 2006; sardar khan, 2009). in the middle east, one of the major industries is oil and gas production and the resulting wastewater contains elevated levels of heavy metals such as lead, cadmium and vanadium. in this study, the potential 30 hamidian et al./ int. j. aquat. biol. (2014) 2(1): 29-35 of common reed was investigated for the phytoremediation of cd, pb and v in wastewater from the bidboland gas refinery in iran. material and methods study area: bidboland gas refinery is located 32 km north east of behbahan in the province of khozestan, iran (fig.1). samples of wastewater were collected from the outflow, 300 m south of the refinery. wastewater discharge was 60 to 70 m3 per hour. sixty samples of the common reed (phragmeties australis) were collected from the surrounding area; their roots were placed in plastic bags and immediately transferred to the laboratory. the roots of thirteen of these samples were washed with water, dried and prepared for chemical analysis. three plastic containers were used in order to plant 30 other plant samples. these 30 plant samples were nourished in these containers within 15 days to reach a height of approximately 40 cm. wastewater samples were transferred into another plastic container (100×150×40 cm) and the 40 cm plant samples were transferred and planted in this container. samples were taken from the roots (using acid washed plastic knives) of the plants during three phases, namely after 2, 6 and 10 days from transferring wastewater into the containers. during each phase, 13 plant samples were selected by random and taken out from the container. the roots of these samples were washed with water, dried in a fume hood and stored in clean plastic bags for chemical analysis. preparation of plant samples: all plastic and glassware were washed with soap and hot water, rinsed three times with distilled water and stored in a solution of 10% hno3 and hcl for 2 weeks; and rinsed three times with dionized water afterwards. the plant tissues were dried in an oven for 48 to 72 hours in 110°c and ashed in a furnace in 550°c for 48 hours. wet and dry weights of the samples were recorded. ashed samples were digested using concentrated analar grade hno3 (merck, germany) and diluted by %1 hno3. the concentrations of the metals were measured using an icp-oes instrument. (perkinelmer, usa). for certifying the analysis method, for each 20 root samples 2 blanks and 2 homogenised and spiked samples were analyzed under the same laboratory conditions. statistical analysis: statistical analyses were performed using spss 16. the normalization of data was checked using kolmogorov smirnov test. figure 1. location of bidboland gas refinery. 31 hamidian et al./ int. j. aquat. biol. (2014) 2(1): 29-35 anova and duncan tests were used to compare the average concentrations of the metals in different treatment times (0, 2, 6 and 10 days). pearson correlation coefficients were used for determination of any correlation between concentrations of the metals in the common reed tissues. results and discussion heavy metal concentrations in plant tissues: percentage recoveries were between 90 to 105 percent for all metals measured. the detection limits (equal to 3×sd of blanks) were 2, 10 and 100 ppb for cd, pb and v, respectively. the mean and standard deviation of cd, pb and v concentrations in plant tissues for different treatment times (0, 2, 6 and 10 days after treatment) are shown in table 1. figure 2. cd concentrations in common reed samples (µg g-1 dry weight). figure 3. pb concentrations in common reed samples (µg g-1 dry weight). figure 4. v concentrations in common reed samples (µg g-1 dry weight) 32 hamidian et al./ int. j. aquat. biol. (2014) 2(1): 29-35 concentrations of the metals in plant tissues are showed in figures 2 to 4. the average concentrations of pb in common reed tissues were 1.78, 13.25, 14.28 and 9.38 (µg g-1 dry weight) for 0, 2, 6 and 10 days of the treatment, respectively. the average concentrations of cd in plant tissues were 0.61, 1.53, 1.95 and 1.21 (µg g-1 dry weight) for 0, 2, 6 and 10 days after the treatment, respectively. the concentration of v in plant tissues was 11.54 (µg g-1 dry weight); which increased to 29.24, 40.83 and 24.75 (µg g-1 dry weight) after 2, 6 and 10 days of treatment, respectively. based on statistical analysis, significant differences (p<0.05) were found between the concentrations of heavy metals in common reed tissues; and between the concentrations of each metal in samples taken from different retention times of 0, 2, 6 and 10 days. the increase in the concentration of heavy metals in the plant roots: lead in the roots increased 6.87 times, the highest increase related to the beginning of the treatment, followed by 2.73 times for v and 2.54 times for cd, as showed in table 2. the average concentrations of heavy metals in roots of the common reed samples before and after 10 days of treatment followed the order of cd < pb < v. cadmium: among the metals, cadmium showed the lowest concentrations in roots of common reed, as was observed before in other studies (bonanno and lo giudice, 2010; nirmal kumar et al., 2008). this metal is a non-essential element with high toxic effects on growth and metabolism of the plant species. this metal releases free radicals and reactive oxygen species; which cause oxidative stress and consequently the death of plants with damaging membrane proteins, lipids, nucleic acid and pigments. however, the common reed (phragmeties australis) is one of the species with high tolerance to cd, as a result of high activity of antioxidant enzymes (bonanno and lo giudice 2010). common reed (phragmites australis) accumulates cd in its roots and hardly transfers it to the shoots. this might be a mechanism to avoid the potential toxic effects on photosynthetic organs (dhir et al., 2009). the average increase in the concentration of cd in roots of common reed in this study was 1.56 µg g-1 dry weight, which is comparable with 1.4 µg g-1 dry weight in roots of carex rostrata and 4.6 and 4.3 µg g-1 dry weight in roots of phragmites australis and eriophorom ansustifolium, respectively (soltoz and greger, 2002). sardar khan (2009) found cd uptake of 2.9 and 3.6 µg g-1 dry weight in roots of phragmites australis and thypha latifolia, respectively. lead: lead can be naturally found in plant tissues; however it is not recognized as an essential element. broyer et al. (1972) stated that if pb is necessary for plants, its concentration at the level of 2–6 μg/kg should be sufficient. transportation and industrial activities release huge amount of lead into the environment. rühling and tyler (1968) reported an increase of pb treatment time metal concentration (µg g-1 dry weight) 0 2 6 10 mean sd mean sd mean sd mean sd cd 0.61 0.25 1.53 0.84 1.95 0.74 1.21 0.59 pb 1.78 1.20 13.25 5.03 14.28 7.46 9.38 3.96 v 11.54 6.39 29.24 8.47 40.83 7.65 24.75 7.18 table 1. means and standard deviations of cd, pb and v concentrations in common reed tissues in different retention times cd (µg g-1 dry weight) pb (µg g-1 dry weight) v (µg g-1 dry weight) mean in the beginning 0.61 1.78 11.54 mean after treatment 1.56 12.30 31.61 increase 2.54 time 6.87 time 2.73 time table 2. the increase in the concentration of cd, pb and v by common reed roots of the a. braschnicowi populations 33 hamidian et al./ int. j. aquat. biol. (2014) 2(1): 29-35 concentration in hypnum cuperssiforme tissues within a 100-year period. various lead uptake rates have been reported for plant species. this might be because of several environmental factors including geochemical anomalies, type of pollutants, the plant species and seasonal changes (kabata-pendias and pendias, 2010). in this study, a lead uptake rate of 12.307 µg g-1 dry weight was observed, which was higher than the rates sardar khan (2009) reported for phragmites australis 2/9 (µg g-1 dry weight) and typha latifolia 3/6 (µg g-1 dry weight). vanadium: plants can easily uptake vanadium by their roots, especially in acidic soil. a linear relationship between the concentration of v in soil and plant tissues indicates that plant species can uptake vo2+ in acidic conditions more rapidly than v3− and hv42− species, which are dominated in neutral and alkaline solutions. it seems that cationic v might be more available to plant species than anionic v (welch, 1979). vanadium is an essential element for algae and some bacterial species. it is not proved to be an essential metal for higher plant species, however some evidences imply it might be a catalyst in n2 fixation (kabata-pendias and pendias, 2010). there is no evidence of deterioration effects of v on plants, while welch and cray (1975) expressed that v is an essential elements for plant species and a concentration of less than 2 μg/kg is needed for plant growth. shacklette and connor (1973) determined concentration between 50–180 mg/kg v in spanish moss samples collected from areas affected by crude oil discharges. gough and severson (1976), reported v concentrations as high as 700 mg/kg in sagebrush found in vicinity of a plant producing phosphorus fertilizer. in neighborhood of crude oil refinery plant, pawlak (1976) reported v concentrations of 13 and 8 mg/kg in clover and grass tissues, respectively. in this study a v uptake rate of 31.610 µg g-1 dry weight was found for phragmites australis. there is not much information available on the removal of v using phragmites australis, therefore, it is not possible to make comparisons. proper treatment time for metal removal using phytoremediation: this study showed that a treatment time of 6 days is the best for the removal of cd, pb and v from wastewater using common reed. this is in agreement with a treatment time of 7 days that hadad et al. (2006) found for typha domingensis. he suggested this treatment time for treatment of wastewater with high ec, ph and heavy metal content. after 10 days of treatment, the concentrations of metal, in plant tissues were decreased, which might be due to metal release and that uptake rate, might have gone higher that tolerant threshold of the plant. ye et al. (2003) suggested a retention time of 12 days as the proper time for the removed of nutrient and metals. correlation between cd, p and v concentrations in plants roots: balancing heavy metals in plant organs is very important and necessary; and affect their physiology and consequently their growth. in this study, significant correlations (p<0.01) were found between the concentrations of pb and cd; pb and v; and cd and v in plant tissues with correlation coefficients of 0.40, 0.51 and 0.41, respectively. the interaction between metals is one of the major causes of their toxicity. for example, hernandez et al. (1998) reported that uptake of mn by plant can be reduced in presence of cd. when plants uptake metals to the concentrations higher than their tolerance or toxic threshold, there might be interactions between the metals and their metabolic functions might be interfered. for instance, excessive uptake of zn by plant might affect fe metabolic function, and even in normal concentrations of fe, the plant might suffer from chlorosis (market, 1987). conclusions phytoremediation is a natural method for wastewater treatment with numerous benefits such as its easy processing, energy saving, environmental conservation; e.g. improving local biodiversity, 34 hamidian et al./ int. j. aquat. biol. (2014) 2(1): 29-35 enhancing the quality of local weather, less emission of co2, using natural resources of energy (such as solar radiation and wind) and soil, plants and animals in wastewater treatment, control of soil erosion and increasing available water supply (schröder et al., 2007). due to our observed uptake rates of pb, v and cd by common reed plant during wastewater treatment (compared to other studies) and because it is a native plant species in khozestan province (due to appropriate climate conditions), and because this plant species has a very high growth rate in this area, it is suggested to use common reed for the removal of heavy metals from this specific wastewater. however, in similar situations when common reed is regarded to be used for the treatment of wastewater, the source and type of pollutants should be investigated and a pilot study is needed before any further investment. acknowledgement we would like to thank professor barrie m. peake from the university of otago, new zealand, for reading the paper and giving the comments for the improvement of the paper. references agunbiade f.o., olu-owolabi b.i. adebowale k.o. 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(2022) 10(5): 364-369 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article new distribution record of crassostreine oyster magallana gryphoides (schlotheim, 1820) in kerala, india vineetha vijayan santhi, smrithi sreekanth, mugdha sukumaran, mano mohan antony department of zoology, (research centre, university of kerala), university college, palayam, thiruvananthapuram, kerala, india. s article history: received 12 june 2022 accepted 14 august 2022 available online 2 5 october 2022 keywords: morphology dharmadom estuary magallana bilineata gene sequencing phylogeny abstract: the crassostreine oyster magallana gryphoides (bivalvia: ostreidae) has been recorded for the first time on the kerala coasts from dharmadom estuary, kannur, kerala, india. the report indicates the range extension of m. gryphoides on the south-west coast of india. the external morphological characters were phenotypic and insufficient for species identification as it resembles magallana bilineata. however, internal shell characters gave important information, especially the adductor muscle scar. the accurate species determination was achieved from the mitochondrial coi and 16s gene sequencing, followed by molecular phylogenetic analysis. the native oyster m. bilineata and m. gryphoides were found to co-exist in the same habitat sharing similar ecological conditions, sharing a sister group relationship. introduction the true oysters of the family ostreidae rafinesque, 1851 have a cosmopolitan distribution and encompass species with high economic importance. they are considered the most efficient ‘keystone niches’ among all the keystone species known for making unique micro-ecosystems (sanjeeva, 2008). in india, oysters are widely distributed in estuaries, bays, harbours, and backwaters. however, comprehensive data on the distribution of oysters dated way back to 1987 by rao (1987), who reported crassostrea (magallana) gryphoides from gujarat, maharashtra and goa. subsequently, m. gryphoides had been reported from different regions on indian coasts, from konkan coast (sawant, 1997), maharashtra (tibile and singh, 2003), sunderbans (trivedi et al., 2015), goa (reece et al., 2008; nagi et al., 2010) and from nearby coasts of karachi (siddiqui and ahmed, 2002), myanmar (li et al., 2017) and bangladesh (chowdury et al., 2021). initially, the indian oysters were assigned to the genus ostrea by awati and rai (1931), later revised correspondence: mano mohan antony doi: https://doi.org/10.22034/ijab.v10i5.1672 e-mail: manomohanantony@universitycollege.ac.in dor: 20.1001.1.23830956.2022.10.5.2.3 and re-assigned under the genus crassostrea by rao (1956, 1958) and durve (1967). recently, salvi et al. (2014) and salvi and mariottini (2017) proposed a new genus, magallana salvi & mariottini, 2016, for the asian-pacific clade of true oysters of the subfamily crassostreinae, thereby, asian-pacific species of crassostrea were re-assigned into a new genus magallana gen. nov (salvi et al., 2014). the genus magallana was valid based on the 2016 description and all the species in the genus were included in worms in 2017, which makes m. gryphoides (schlotheim, 1820), the accepted name for the asian-pacific species of c. gryphoides (schlotheim, 1820) and m. bilineata (roding, 1798) for the native indian backwater oyster, c. madrasensis, preston 1916. however, it is noteworthy to observe the molecular data deficiency of oysters from the indian sub-continent in the above-mentioned papers. therefore, in the current study, we attempted to establish the new distribution record of m. gryphoides in the south-west coast of india https://ij-aquaticbiology.com/index.php/ijab/article/view/1672 364 santhi et al./ new record of crassostreine oyster, magallana gryphoides based on the mitochondrial coi and 16s gene sequencing and phylogenetic analysis along with ecological and morphological information. materials and methods oysters were collected from dharmadom estuary (11.796918n, 75.462153e), kannur, kerala, india, by hand-picking from shallow coastal waters. tissue from the adductor muscle was used for molecular analysis. the ecological factors such as depth, nature of substrate, salinity, ph, hardness, dissolved oxygen, temperature, and population density were recorded (apha, 1989; peters et al., 2017). the collected species were primarily identified based on published papers (siddiqui and ahmed, 2002; li et al., 2017; chowdhury et al., 2021). the classification and scientific names followed worms (www.marinespecies.org). for species confirmation, the mitochondrial gene sequencing method was employed using mitochondrial cytochrome oxidase i (coi) and 16s ribosomal rna (16s rrna). the genomic dna was isolated from muscle tissue using nucleospin® tissue kit (macherey-nagel). partial coi and 16s rrna was amplified via polymerase chain reaction using the primers of lco1490 and hco2198 (folmer et al., 1994) for coi and 16sar and 16sbr (palumbi et al., 1991) for 16s rrna. all the retrieved sequences were deposited in ncbi-genbank. for both 16s and coi datasets, multiple sequence alignment (msa) was done in mega 7 (kumar et al., 2016) and the phylogenetic tree (bayesian inference tree) was constructed in mrbayes 3.2.7 (ronquist et al., 2012). bayesian analysis (gtr+g+i model as best fitting model for both 16s and coi dataset) was done and two independent runs were performed for 2×106 generations sampling per 1000 generations. the first 25% of the trees acquired were discarded as burn-in, and a 50% majority-rule consensus tree with posterior probability (pp) values were generated from the leftover trees. the phylogenetic tree was constructed using figtree v1.4.2. talonostrea salpinx was taken as the outgroup. results the studied site showed a depth of 0.8-1.2 m with a muddy substratum. individuals of m. gryphoides were collected from the beds of m. bilineata. solitary individuals along with gregarious forms with m. bilineata were observed. compared to m. bilineata (11-18/m2), the population density of m. gryphoides was very fewer ranges of 2-5/m2. the ph of the studied site was 7.55, surface water temperature 32°c, salinity 30.14 ppt, hardness-284 mg/l and dissolved oxygen 7.33 mg/l. all the external shell characters were similar for both m. gryphoides and m. bilineata since they are phenoplastic and share a common habitat. however, characteristic differences were observed in the internal shell characters, especially in the adductor muscle scar. magallana gryphoides exhibited characteristic pearly white colouration, reniform or crescent-shaped adductor muscle scar, nearly straight dorsally and close to the posterodorsal margin and the shell interior was whitish and shiny devoid of any purplish or blackish markings. for m. bilineata, the adductor muscle scar was characteristically dark purplish-black in both valves. the white umbo cavity always had purple markings around the whole or some parts of the margin. in the ventral margin, deeper colouration was observed facing the posterior portion of the gill, especially in the lower valve (fig. 1). the mean and standard deviation of shell length (sl), shell height (sh), shell width (sd), and total weight (tw) in m. gryphoides was 3.64±4.751, 79.68±6.215, 32.86±2.572, and 128.19±2.08, and for m. bilineata as 74.4±13.682, 100.74±14.504, 35.026±10.607, and 191.56±31.735, respectively. a total of eight gene sequences were obtained from m. gryphoides and m. bilineata and submitted to the ncbi-genbank and acquired accession numbers (table 1). the phylogenetic relationship of m. gryphoides with the other asian-pacific species was inferred from coi and 16s gene datasets. the bi tree was constructed for each gene dataset, using four original sequences and other sequences of the asian-pacific species retrieved from ncbi365 int. j. aquat. biol. (2022) 10(5): 364-369 genbank (figs. 2 and 3). the topology of both the trees was similar with m. belcheri in the upper clade, m. dianbeinsis, m. bilineata and m. gryphoides placed in the middle clade and m. gigas, m. sikamea, m. hongonenesis, m. nippona, m. ariakensis and m. angulata in the bottom clade. the original sequences of the m. gryphoides shared the same clade with the other 3 sequences of m. gryphoides in the coi tree and showed more similarity with the sequences from goa, india (eu007488 and figure 1. shell characteristics of magallana gryphoides and m. bilineata. (a) lower valve of m. gryphoides (b) lower valve of m. bilineata (c) m. gryphoides among the cluster of m. bilineata (d) adductor muscle scar of m. gryphoides (e) adductor muscle scar of m. bilineata (f) upper valve of m. gryphoides (g) upper valve of m. bilineata. species species id genbank accession numbers 16s coi magallana gryphoides db1 on926958 on920920 db2 on926959 on921053 magallana bilineata du1 on926953 on912074 du2 on926954 on920843 table 1. gene sequence data of collected oysters. 366 santhi et al./ new record of crassostreine oyster, magallana gryphoides figure 2. phylogenetic tree based on bi analysis of coi sequences. bayesian posterior probability is shown at the nodes. original sequences are marked in red. figure 3. phylogenetic tree based on bi analysis of 16s rrna sequences. bayesian posterior probability is shown at the nodes. original sequences are marked in red. 367 int. j. aquat. biol. (2022) 10(5): 364-369 eu007492) (reece et al., 2008). the clade of m. gryphoides exhibited a sister group relationship with m. bilineata and m. dianbaiensis in coi and 16s trees. however, 16s sequences were scarce in ncbi-genbank. discussion the new report of m. gryphoides from dharmadom estuary represents the range extension of the species on the south-west coast as it was earlier reported from the coasts of gujarat, maharashtra and goa (rao, 1987; tibile and singh, 2003; reece et al., 2008). since, the indian oyster taxonomy is mostly dependent on the morphological shell traits, which are highly plastic, the presence of m. gryphoides on the south-west coast might be masked earlier. therefore, the presence of more oyster species and their distribution is highly anticipated on the indian coasts. however, molecular identification methods should be encouraged and given priority as they can provide decisive outcomes in oyster taxonomy. all the ecological conditions observed were optimum for the growth and distribution of oysters, highlighting the scope of oyster culture in the dharmadom estuary. being benthic, shells cope with the external environmental conditions that result in ecophenotypic plasticity (lam and morton, 2004; huber, 2010; liu et al., 2011; santhi et al., 2021). therefore, species identification could not be achieved based on external shell characteristics. however, it is the adductor muscle scar that gave important information for species identification. a similar finding was also made by siddiqui and ahmed (2002) in c. madrasensis (=m. bilineata) and c. gryphoides (=m. gryphoides) with respect to the white colour of the adductor muscle scar in c. gryphoides (=m. gryphoides) and purpleness in c. madrasensis (=m. bilineata). however, molecular markers had proved to be efficient markers for species delineation. nevertheless, the molecular database of indian oysters is still lacking and only a few sequences are available in ncbigenbank. though, accurate identification was achieved by mitochondrial coi gene sequencing and thereby blast analysis. the previous name of crassostrea gryphoides was also used for a european fossil species as crasssostrea gryphoides von schlotheim, 1813 that existed during the miocene and pliocene, and became extinct over three million years ago (harzhauser et al., 2016). however, it has been considered as not conspecific with the extant m. gryphoides (harzhauser et al., 2016) differing with respect to the shape of adductor muscle scar (durve, 1974), shell outline (durve and bal, 1961), and size (chatterji et al., 1985; nagi et al., 2011). therefore, according to huber (2010), harzhauser et al. (2016), and li et al. (2017), the same name should not be used also for an extant species. after the recent revision of asia-pacific oysters into the newly assigned genus magallana salvi and mariottinni, 2016, the extant species of c. gryphoides from the india and pakistan region were re-assigned as m. gryphoides (schlotheim, 1820). the current study also provides molecular phylogenetic data that agrees with the recent revision in the crassostreinae subfamily by salvi and mariottini (2017). the bi tree showed the monophyletic origin of asia-pacific true oysters (subfamily: crassostreinae) recently assigned to the genus magallana in coi and 16s trees. moreover, m. bilineata and m. gryphoides exhibited a sister group relationship with m. dianbaiensis in both trees with a high bayesian probability of 1. this is in accordance with melo et al. (2010), wu et al. (2013), xia et al. (2014), li et al. (2017), chowdhury et al. (2021), and ghaffari et al. (2022). the sister group relationship of m. bilineata and m. dianbeiensis was also demonstrated by salvi and mariottini (2017, 2021), al-kandari et al. (2021), and willan et al. (2021). however, the gene data of m. gryphoides is still scarce in ncbi-genbank. therefore, more molecular studies are required on this species and other magallana species of the indian waters to resolve their taxonomic uncertainties. since, the systematics of true oysters is critical to developing the sustainable use of species and understanding the diversity of oysters worldwide 368 santhi et al./ new record of crassostreine oyster, magallana gryphoides (sigwart et al., 2021), the gap areas should be immediately dealt with to document and conserve the biodiversity of indian oysters. the aid of molecular taxonomy and evolutionary analyses via a phylogenetic approach would unmask the biodiversity and evolutionary reactions of indian oysters that remained unnoticed and unknown to the world. being the keystone species, any conservation measures to this taxon will ultimately benefit the ecosystem. as the oyster population of india is radically decreasing (laxmilatha, 2022), it has become an emergency issue to be dealt with. references al-kandari m., oliver p.g., salvi, d. 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(2017) 5(2): 108-113; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article using kriging and co-kriging to predict distributional areas of kilka species (clupeonella spp.) in the southern caspian sea kaveh amiri1, nader shabanipour*1, 2, soheil eagderi3 1department of biology, faculty of science, university of guilan, rasht, iran. 2department of marine science, caspian sea basin research centre, university of guilan, rasht, iran. 3department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 22 december 2016 accepted 14 march 2017 available online 2 5 april 2017 keywords: modeling predict catch abundance kilka caspian sea abstract: understanding ecological and anthropogenic drivers of fish population dynamics and achieving a sustainable yield requires detailed studies on habitat selection and spatial distribution. the objective of this study was to predict spatial density and distribution of kilka species in the southern caspian sea in relation to satellite-derived sea surface temperature, chlorophyll-a concentration, turbidity and water depths using ordinary kriging and co-kriging geostatistical methods and introduction an appropriate potential fishing area according to the present fishing points. three hundred and fifty fishing surveys were done in two main kilka fishing ports in the southern caspian sea (anzali and babolsar ports) from 2015 to 2016. the geostatistical analysis showed that the co-kriging spatial interpolation method provided the best prediction of fish abundance when chlorophyll-a content was included in model. introduction the caspian sea is the largest inland water body in the world, occupying a deep depression on the boundary of europe and asia with a level of approximately 27 m below the world’s sea level (cep, 2002). three small valuable clupeid fishes known as “kilka” including common kilka, clupeonella cultriventris bordin, 1904, anchovy, c. engrauliformis svetovidov, 1941, and bigeye, c. grimmi kessler, 1877 are among the most abundant fishes of the caspian sea (svetovidov, 1963). kilka fishing was an important source of income and protein for people of the southern caspian sea. in addition, kilka species are important food reserve for sturgeons and the caspian seal (prikhod'ko, 1979) showing their ecological importance. annual catches of kilka fishes in the caspian sea reached to the highest level i.e. 423, 0000 t in 1970 (ivanov, 2000), constituting about 70% of the total fish catch in the caspian sea (sedov et al., 1997). during the past 30 years, the environmental status of the caspian sea has significantly changed * corresponding author: nader shabanipour doi: https://doi.org/10.22034/ijab.v5i2.309 e-mail address: shabani@guilan.ac.ir due to fluctuations of the sea level, water pollution (ivanov, 2000), invasive species and overfishing (fazli et al., 2009). the relationship between fish abundance and biotic and abiotic features define their habitat suitability (laevastu and hayes, 1981). these factors also influence feeding, reproduction, predator avoidance and migration of fish species and, therefore, are considered as spatial characteristics governing the biomass distribution (horne et al., 1999; freon et al., 2005). the relationship between fish distribution and environmental factors is supposed to be a non-linear or chaotic i.e. spatial fish biomass structure is stochastic in most observation (webster and oliver, 2001). geostatistical analysis of pelagic fish catch data has been recognized as the best method for modeling of spatial distribution of biomass to understand the relationship between spatial pattern of biomass and environmental features (simard et al., 2002). the physical and biological characteristics of marine 109 int. j. aquat. biol. (2017) 5(2): 108-113 ecosystems can be represented by sea surface temperature (sst), chlorophyll-a (chl-a), turbidity and water depth (solanki et al., 2005a). chl-a is known as an important oceanographic parameter of productivity (solanki et al., 2001) that could be related to fish production (bertrand et al., 2002). sst is assumed to be an index of the physical environment, which controls the physiology of the living organisms (solanki et al., 2005b; tang et al., 2003). turbidity is a fundamental index used to assess coastal and estuarine water quality conditions affecting light attenuation and the plankton productivity (pennock and sharp, 1994). according to positive phototropism of kilka species, these factors can affect their fishing yield. physical and biological features can be measured using sensors of satellites. this technology is able to provide reliable global ocean coverage of sst and chl-a and turbidity at a relatively high spatial and temporal resolution, which can be measured from space. the satellite remote sensing is an effective and efficient way compared with field sampling that requires time, cost and limited coverage areas. meanwhile, geographic information systems (gis) techniques are widely used in processing satellite images (castillo et al., 1996). it integrates theoretical aspects of oceanography and ecology with spatial database and statistical functions. some studies investigated potential fishing ground of fishes using stepwise regression models in relation to satellite derived environmental factors e.g. sst and chl-a (nurdin et al., 2015), whereas others used geostatistical methods e.g. kriging and co-kriging (rueda and defeo, 2001; georgakarakos and kitsiou, 2008; aidoo et al., 2015; pierre et al., 2016; woillez et al., 2016). kriging is an interpolation technique that minimizes the estimated variance measured from a prior model for a covariance. it calculates weights that result in optimal and unbiased estimates. within a probabilistic framework, kriging attempts to minimize the error variance and systematically sets the mean of the prediction errors to zero. however, a data set will often contain not only the primary variable of interest but also one or more secondary variables. these secondary variables where spatially cross correlated with the primary variable can contain useful information about the primary variable. this information can be included within the estimation process via co-kriging. it seems reasonable to add the cross correlated information contained in the secondary variable to help further decrease in the variance of the estimation error (david, 1977). cokriging uses a secondary variable (covariate) that is cross correlated with the primary or sample variable of interest. this can aid to minimize the error variance of the estimation (isaaks, 1992). many aspects of kilka stocks in the southern caspian sea such as biology, population dynamic (karimzadeh et al., 2010) and reproductive cycle (amiri et al., 2012) have been studied. however, there is no data on spatial distribution of kilka species or their potential fishing grounds. hence, this study aimed to determine their potential fishing grounds in the southern caspian sea using geostatistical methods to produce choropleth maps. a decline has been occurred in the fishing of kilka fishes in the caspian sea due to invitation of the warty comb jelly, mnemiopsis leidyi, during last decade and therefore the results of this study can help to management of its fishing by introducing proper fishing grounds. materials and methods data collection: the catch points of 350 fishing surveys in anzali (37°28' n, 49°25'e) and babolsar) (36°42′n, 52°39′ e) ports as two main fishing regions of kilka in iranian waters of the caspian sea were recorded using a gps (fig. 1). spring (98 points), summer (89 points), autumn (106 points) and winter (57 points) fishing done from 2015 to 2016. a density index (catch per unit of effort, cpue) was calculated as the catch of 72 vessels (28 vessels in anzali and 43 vessels in babolsar ports) per night. the tracked fishing points were standardize using arc map 10.3 (gis) and recorded on georeferenced map of the caspian sea as a shape file. remotely sensed environmental data: the primary satellite data set used in this study were sst, chl-a and turbidity data derived from modis measurement. the 110 amiri et al./ prediction of kilka abundance distribution sst (°c) and chl-a (mg/m3) level 3 (4 km) monthly standard mapped image data from 2015 to 2016 were downloaded from the ocean color website (http://oceancolor.gsfc.nasa.gov/). the bottom topography data of the caspian sea is constructed using the etopo1 dataset (amante and eakins, 2009). the wavelength 645 nm was used to measure turbidity (ntu) (chen et al., 2007). according to schooling of kilka fishes, the geographical latitudes (lat) and longitudes (lon) considered as environmental parameters (petitgas et al., 2001). seawifs data analysis system (seadas) version 7.3 was used to extract and process the data (o’reilly et al. 1998). the data were subset to the study area with geographical arc map (gis) 10.3 version software. data analysis: multivariate linear regression analysis was used to identify a main environmental factor or factors influencing spatial changes of cpue. kriging and co-kriging methods were applied to estimate the parameters including the cpue abundance of kilka species. for this reason, arcgis 10.3 and gs+ 7.2 software were used. after applying kriging and cokriging methods, to make an assessment: firstly, an empirical variogram was drawn from data and a theory variogram was fitted. each time a measured value is omitted in a point and another amount is estimated for it from the neighboring points. then, the real value is returned to the previous position and this was repeated for all measurement points. the assessment was carried out using determination coefficient (r2) and the root mean square error (rmse) and average standard error (ase) (goovaerts, 1997). data variogram was analyzed to examine the spatial correlation and the spatial structure of variables. to make analysis on the data variogram of the cpue and other co-factors after normalization, initially, the variogram and cross variogram of variables was drawn by using gs+ and, then, an appropriate model selected. results statistical parameters such as mean and standard deviation of variation are presented in table 1 for the environmental factors. for data analysis, a histogram was drawn for each study variable after normalization. geostatistical analysis: since some parameters are affected by environmental factors, they can be involved in the estimation of the main variable by using the co-kriging estimator, if a correlation exists. by the examination of the stepwise correlation among the studied variables, it was observed that there was a figure 1. kilka fishing points (in green) in southern part of the caspian sea from 2015 to 2016 (red triangles indicate fishing ports). 111 int. j. aquat. biol. (2017) 5(2): 108-113 correlation between the cpue and chl-a concentration (table 2). therefore, the estimation of the cpue spatial changes and the through chl-a by using the cokriging estimator will be entirely reasonable. after evaluating different models, it was demonstrated that the exponential and pentaspherical models were best suited for the variables using kriging and co-kriging, respectively and therefore, it was selected as a best fitted model on the data (table 3). evaluation of geostatistical methods: using rmse, as presented depicted in table 3, the estimation of cpue ratio by co-kriging with a rmse=1175.4 was obviously more precise than kriging method, though the two methods were reasonably accurate. to estimate the cpue rate, the accuracy of co-kriging was higher than that of kriging (rmse=1187.7) (table 3). discussion the results showed that kriging and co-kriging methods can be applied as a tool to estimate the abundance of kilka fishes in areas with data restriction. chl-a has impacts on created map using co-kriging and have positive linear correlation with cpue. the positive impact of chl-a on some fishes density distribution has been proven (gower, 1972; sachoemar et al., 2012). visual comparison of modis images showed that the chl-a concentration has spatial changes more than sst and turbidity in south part of the caspian sea region. these changes table 1. statistical parameters of studied variables. parameters no of data mean std. deviatio n lat 350 37.30 0.39 lon 350 50.65 1.4 chl-a (mg/m3) 350 0.4 0.2 depth (m) 350 63 12.43 sst (°c) 350 20.4 6.8 turbidity (ntu) 350 0.4 0.2 cpue (kg) 350 2682 1711.2 table 2. stepwise regression result. survey years dependent variable no of data independent variable model r2 f sig. 2015 and 2016 cpue (kg) 350 chl-a & sst4 & tur& lon & lat & depth 0.26 3.534 0.03 2015 and 2016 cpue (kg) 350 chl-a 0.30 4.334 0.01 table 3. compare indicator of kriging and co-kriging prediction accuracy. model variogram type range (km) rmse ase r2 cpue kriging exponential 1.03 1187.797 1381.042 0.11 cpue and chl-a co-kriging pentaspherical 6.3 1175.457 1355.567 0.15 figure 2. predicted densities (kg) for kilka fishes, 2015 and 2016, ordinary kriging and co-kriging (black circles = fishing points). 112 amiri et al./ prediction of kilka abundance distribution can increase predictive power of co-kriging method and influence fishing distribution of kilka fishes as planktovorous fishes. according to the choropleth maps (fig. 2), the cpue of kilka is more likely in eastern and western region of the anzali and babolsar ports than those of the other parts, respectively. satellite imagery analysis shows that the western region of the babolsar port has relatively more phytoplankton concentration throughout the year than the other pars. sefid river, as the largest river in the north of iran entering the caspian sea at the eastern part of the anzali port, and its runoff may have an influence on enhancement of kilka cpue. in addition, it is determined than shore line area between the rudsar (37°07' 43n; 50°18'51e) and chalus (36°41'28n; 51°18'15e) in the southern caspian sea may has great potential for kilka fishing as a new area (fig. 2). acknowledgments we would like to thanks mr. sheikhtabar, nikbakht, hadifar, zakariaei, mahboob, hamedanian, ghorbani, and dehghan, the fishermen and fishery managers of the anzali and babolsar ports for helps during fishing surveys. we also appreciate dr. poorbagher (university of tehran), dale best (university of california), dr. mirzaei (university of shiraz) and dr. kabiri (iranian national institute for oceanography and atmospheric science) for their scientific guidance. references aidooa e.n., mueller u., goovaerts p., hyndes g.a. 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(2017) 5(2): 108-113 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی در (.clupeonella spp) ماهیان کیلکا تراکم پراکنش بینیپیش در کوکریجینگ و کریجینگ هایمدل از استفاده خزر دریای جنوبی ناحیه 3ایگدری سهیل ،2،1*پور شعبانی نادر ،1امیری کاوه رشت، ایران. گیالن، دانشگاه پایه، علوم دانشکده، شناسی زیست گروه1 رشت، ایران. گیالن، دانشگاه ،دریایی علوم گروه خزر، دریای حوزه تحقیقات مرکز2 کرج، ایران. تهران، دانشگاه طبیعی، منابع دانشکده، شیالت گروه3 چکیده: با مرتبط تحقیقات انجام نیازمند هاآن منابع پایدار استحصال به دستیابی و ماهیان جمعیت پویایی بر موثر شناختیبوم و انسانی عوامل از آگاهی خزر، دریای جنوب ماهیان کیلکا فضایی تراکم بینیپیش و بررسی هدف با حاضر تحقیق. است ها آن مکانی پراکنش چگونگی و گزینی زیستگاه هایمدل از استفاده با ایماهواره تصاویر از حاصل محیطی های متغیر با آن ارتباط بررسی و صید کنونی نقاط اساس بر صید مناسب نواحی پیشنهاد بآ عمق همچنین و آب سطح کدورت و آ-کلروفیل تراکم آب، دمای شامل بررسی مورد محیطی عوامل. رسید انجام به کریجینگ-کو و کریجینگ دریای جنوبی ناحیه در ماهیان کیلکا صید اصلی منطقه دو عنوانبه بابلسر و انزلی بنادر ساحلی های آب در صید سفر 350 تعداد. بود صید مکان در عنوانبه آ-کلروفیل تراکم گرفتن نظر در با و کریجینگ-کو روش از استفاده با نتایج اساس بر. رسید انجام به 1395 و 1394 های سال در خزر، .شد خواهد حاصل تریدقیق نتایج کمکی فاکتور .خزر دریای ماهیان، کیلکا صید کمی بینی،پیش مدلسازی :کلمات کلیدی int. j. aquat. biol. (2017) 5(4): 268-274oi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article caspian sea’s navicula salinicola hustedt 1939 and effect of the prolonged culture on its fatty acid profile ehsan etesami1, farkhondeh saba1, mostafa noroozi*2,1mohammad ali amoozegar1, 3, gholamreza bakhshi khaniki4, seyed abolhassan shahzadeh fazeli1, 5 1microorganisms bank, iranian biological resource centre (ibrc), tehran, iran. 2department of biotechnology, faculty of biological sciences, alzahra university, tehran, iran. 3department of microbiology, faculty of biology and centre of excellence in phylogeny of living organisms, college of science, university of tehran, tehran, iran. 4department of biotechnology, faculty of sciences, payam-e noor tehran-shargh university, tehran, iran. 5departments of molecular and cellular biology, faculty of basic sciences and advanced technologies in biology, university of science and culture, tehran, iran. article history: received 3 june 2017 accepted 24 august 2017 available online 2 5 august 2017 keywords: algae omega3 eicosapentaenoic acid docosahexaenoic acid abstract: diatoms are a potent source of polyunsaturated fatty acids. this study was conducted for screening a naviculoid diatom strains from the southern caspian sea with analyzing its lipid production and accumulation potentials. the isolate was identified as navicula salinicola strain ibrc-m 5083 based on micro-morphological characterization and analysis of 18s rrna genomic region. navicula salinicola were cultured in the f/2 medium under both normal and prolonged culture (21 days) conditions. total lipid percentages of this strain were found to be 31.83% under normal condition and 43.72±1.4% in prolonged culture respectively on the basis of their dry cell weight (dcw). also, the oil droplets were detected in 21 days’ cells as shown by sudan black b staining experiments. furthermore, the main fatty acids were found by gas chromatography analyses of this strain under prolonged condition to be eicosapentaenoic acid (25.58%tfa). such oil accumulation capabilities seem to be promising for performing further studies on this strain as a source of omega3 in aquafeed, pharmaceutical and biofuel industries. introduction microalgae have many applications in different fields such as pharmaceutical, food industries, aquafeed, phytoremediation, sewage treatment and biofuel. there are several microalgae species which are able to accumulate lipids such as diatoms having a high potential of lipid production (dunahay et al., 1998; hildebrand et al., 2012). therefore, they are potent and proper candidates for many biotechnological applications like biofuels (merz and main, 2014). among them, the marine diatoms have been investigated more than others (griffiths and harrison, 2009) except the freshwater genus navicula (sheehan et al., 1998; graham et al., 2012). lipid in diatoms could be accumulated as intracellular droplets included tags (triacylglycerol) (yoneda et al., 2016; *corresponding author: mostafa noroozi doi: https://doi.org/10.22034/ijab.v5i4.271 e-mail address: noroozi.mostafa@alzahra.ac.ir yongmanitchai and ward, 1991). the 16 or 18 carbon tags could be used as biodiesel sources because of its chemical similarity with aliphatic hydrocarbons in petrol (yongmanitchai and ward, 1991; sivakumar et al., 2010). furthermore, over 18 carbons such as c20:5ω3 (epa) and c22:6ω3 (dha) have also been found in diatoms which could be valuable sources for omega3 (brett and muller‐navarra, 1997). strong evidence of beneficial properties of omega3 for human nutrition led to increase of interest in omega3 as nutritional supplements and aquafeed (merz and main, 2014). previous studies about navicula sp. showed its proper level of c16:0, c16:1, c17:01, c18:0 (matsumoto et al., 2010; thajuddin et al., 2015), but many environmental variations such as nutrient 269 int. j. aquat. biol. (2017) 5(4): 268-274 deficiency can change its fatty acid composition (lison, 1934; mekhalfi et al., 2010). for instance, under nitrogen and silicon deficiencies in n. salinicola, the lipid content was induced up to 58% and 22-49%, respectively (nagle and lemke, 1990). nonetheless, prior to any lipid analysis, the target cells should be identified. identification of diatoms is done using morphological characters such as the siliceous wall (frustules) ornamentations and protoplast features (preisig and andersen, 2005). for precise morphological identification of diatoms, it would be better to observe the frustules by scanning electron microscope (sem) (crawford et al., 2001). due to the presence of organic material on the frustule and cell contents, the characteristics of the valve are obscured thus it is necessary to clean diatom frustules from organic matter to provide a permanent slide and observe the frustule ornamentations under sem. recently molecular tools such as 18s rrna gene sequence are used to confirm the morphological identifications (dawson and pace, 2002). the objective of this research was to identify the isolated cell and determine the effect of a prolonged culture period (21 days) on the fatty acid profile of navicula salinicola, collected from the caspian sea, iran. materials and methods isolation and culture conditions: the samples were collected from the gohar-baran port, the southern caspian sea, iran (36°49'51.1''n, 53°11'38.4''e). the samples were left for 24 hrs to settle the organisms in ibrc microalgae laboratory and then cultivated in filter sterilized seawater enriched with guillard f/2 medium (nano3 8.82104 m; kh2po4 3.62105 m; fecl3.6h2o 1.17105 m; na2edta.2h2o 1.17105 m; cuso4.5h2o 3.93108 m; na2moo4.2h2o 2.60108 m; znso4.7h2o 7.65108 m; cocl2.6h2o 4.20108 m; mncl2.4h2o 9.10 107 m; thiamine hcl 2.96107 m; biotin 2.05 109 m; cyanocobalamin 3.691010 m, 100mg.l-1 imipenem all chemicals purchased from sigma aldrich inc., st louis, mo, usa) (guillard, 1975; presieg and andersen, 2005). the ph of the medium was set at 7 and the sample was grown at 18±1°c under a light/dark cycle (12h: 12h) in f/2 medium that prepared from seawater under a low intensity of 50 µmol m–2 s–1 (preisig and andersen, 2005). first, the axenic colonies of diatom on the plate were transferred to the triple 150 ml flask after 7 days, subsequently they were inoculated with 10% (v/v) into triple 500 ml fresh f/2 medium over another 7 days and kept to grow for 7 days at the same condition (prolonged culture). then, one of the triple sample of normal and prolonged culture were randomly chosen and centrifuged at a speed of 3,000 rpm for 30 min, then the supernatant were removed. the pellets were washed twice with distilled water and dried at 60°c to obtain their constant weight. then, they were weighted and stored at 4ºc for lyophilizing step. finally, before starting a lipid extraction process, the pellets were lyophilized and weighted. morphological identification: morphological identification was carried out through micromorphology under a light microscope (lm microscope, olympus, cx31) and sem (vega 3, tescan). for precise morphological identification, the permanent slide was used with h2o2 treatment. following cox and mann’s method (cox, 1990), the permanent slide was made with naphrax and pictures were captured with a dic and phase contrast microscope (nikon eclipse 80i). molecular identification: total genomic dna from the cultured sample was isolated based on liu method (liu et al., 2000) and stored at -20°c for further analysis. the primers ssu1 5'-aacctggttgatc ctgccagt-3' (kociolek, 2013) and its1dr 5' ccttgttacgacttcaccttcc-3' (kociolek, 2013; edgar and theriot, 2004) and amplification of the target gene (18s rrna) were done according to kociolek protocol (kociolek, 2013) by pcr (pcr bio-rad my cycler personal thermal cycler, usa). the genomic dna and pcr product were separated by agarose gel electrophoresis (bio-rad power pac universal power supply electrophoresis, usa). pcr product was sequenced based on the sanger method and edited by chromaspro and compared with registered sequences at ncbi by using basic local alignment search tool (blast). lipid analysis: lipid of the normal and prolonged sample was qualitatively analyzed with modified 270 etesami et al./ effect of prolonged culture on fatty acid profile of navicula salinicola sudan black b staining method (jape, 2014; thakur et al., 1989). the procedure initiated by fixing the air dried slides and flooding the slide in sudan black b stain for 20 min at room temperature (prepared about 0.3% sudan black b in 70% ethanol). finally, the slide was stained by safranin (0.5% aqueous solution) for 5 to 8 seconds (thakur et al., 1989). the lipid of the lyophilized samples (normal and prolonged) which were in late stationary growth phase were extracted via chci3: meoh (2:1 v/v) solvent mix method (folch et al., 1957; axelsson and gentili, 2014). the samples were homogenized and the residues were removed by filtration after homogenization. 0.73% nacl solution was added which led to the production of final solvent (2:1:0.8 chloroform: methanol: water (v/v/v)). solvents were removed by evaporation and the dry extracts were weighted for determination of total lipid percentages in normal and prolonged condition. as a final process, the dry extract of prolonged condition sample dissolved in dichloromethane for further quantitative analysis by gas chromatography (gc). in the following, the extracted oil was transesterified by two-step procedure (2-te) (cavonius, 2014) and the fatty acid profile was determined by gcyoung lin 6000 with an oven temperature 145°c isothermal, the injector temperature 280°c, detector temperature 300°c, capillary column-length: 50 m, internal diameter: 0.25 mm, varian-cp-sil 88, 60 m. the gc analysis was performed at the department of food science, iranian national standards organization (inso) karaj, iran, based on the national standard 4090 and 4091. results morphological and molecular analysis: identification of the isolated n. salinicola from the gohar-baran port in the caspian sea were confirmed according to the morphological and molecular characters explained in stoermer et al. (1999) and kociolek (2005). in n. salinicola, the valves were linear-lanceolate, rounded ends with the length-width 7-20, 2-4.5 μm, respectively. filiform raphe and striae features were slightly paralleled to radiate, convergent at the ends (fig. 1b, d, e, f). the amplified sequence of small subunit 18s rdna which blasted in ncbi revealed 99% similarity to n. salinicola (fr865499) identified from, san francisco, california, usa. as a result, this strain was identified as n. salinicola strain ibrc-m 5083 in ibrc (iranian biological research centre) microalgae collection. lipid analysis: determination of total lipids percentages of this strain were obtained 31.83% (dcw) in normal condition and 43.72±1.4% under prolonged culture. in addition, primary screening of lipid vacuoles of n. salinicola under prolonged condition by staining with sudan black b indicated blue–black color droplets (fig. 1c). finally, the fatty acid composition of prolonged cultivation of n. salinicola was demonstrated by gc analysis (table 1). the result revealed the high level production of epa (25.58% tfa) under prolonged culture in this strain. discussion the diatoms could be an affordable and appropriate source for biotechnological applications, however, studies on diatoms are rare in iran and need more attention. for this purpose, the isolated sample should be identified in the first step. taxonomic of diatoms are based upon morphology characteristics such as frustules structure (round et al., 1990) as well as ribosomal dna sequencing (kociolek et al., 2013). it needs to clarify that the ornamentations of frustule have enough information for diatom identification, however, the gene sequences of diatoms in databases table 1. the fatty acid profile of navicula salinicola extracts under prolonged culture (21 d) by gc strain fatty acid composition (% of total fatty acids) navicula salinicola c16:0 c16:1 c18:1 c18:2 c18:3 (n-6) c20:5 4.79 9.28 2.25 0.48 0.07 25.58 271 int. j. aquat. biol. (2017) 5(4): 268-274 are not complete yet and in many cases, it is not feasible to compare the sequence of an isolate with the sequence information in ncbi. the results of this study revealed that the isolate algae is n. salinicola based on morphological and dna data. algae produce carbohydrates, lipids and protein in photosynthesis process that depend on environmental factors (juneja et al., 2013). environmental conditions like nutrient availability are closely effective on quantity and content of carbon fixation and they lead to change of fatty acids composition as well (juneja et al., 2013; sharma et al., 2012). a study about evaluation of the nutrients in culture medium on lipid metabolism has shown that limitation of macronutrients such as nitrogen, silicon, phosphate can effect on lipid metabolism and contents of neutral lipids (tags) (hu et al., 2008). it should be noted that determining the algae products under variable figure 1. (a) navicula salinicola under lm, (b) frustule features in p ermanent slide, (c) appearance of lipid droplets via sudan black b staining, (d) striae slightly parallel, (e) a filiform raphe in n. salinicola., and (f) linear-lanceolate and rounded ends valves. 272 etesami et al./ effect of prolonged culture on fatty acid profile of navicula salinicola environmental factors could be important for understanding how influence these factors on algae. under prolonged incubation time of microalgae like diatoms, environmental factors could be altered and these changes effect on fatty acid composition and enhance tags during stationary phase in many algal species (hu et al., 2008); for instance, the total lipid content of nacicula sp. under normal condition was determined as 41.10 (% dcw) which could be improved to 60.28±4.92 (% dcw) under nitrogen starvation (thajuddin et al., 2015). according to previous study, lipid content of n. salinicola was commonly induced up to 58% under nitrogen deficiency and to 22-49% under silicon deficiency (nagle and lemke, 1990). based on the results, the total lipid content of isolated n. salinicola (ibrc-m 5083) was 43.72±1.4 (% dcw) which was higher than the total lipid content of nacicula sp. under normal condition but it was lower than under nitrogen limitation conditions. the content of lipids e.g. neutral lipids in the biomass like tags are important for biotechnological applications (d’ippolito et al., 2015) such as aquafeed, omega3 production and biofuel sources. for example in biofuel sources, linolenic acid and four double bonds in fatty acid methyl esters should not increase to 12% and 1%, respectively according to biodiesel standard en14214 methods (juneja et al., 2013). the gc analysis data in our work showed that the amount of linolenic acid in n. salinicola (ibrc-m 5083) is lower than 12%, which encounters the requirements of the european standard en 14214 for biodiesel. the above reports and absence of c18:3 demonstrated that n. salinicola (ibrc-m 5083) could be an appropriate source of biodiesel (matsumoto et al., 2010; thajuddin et al., 2015). in addition, nacicula sp. has suitable level of the other fatty acids such as c16:0, c16:1, c17:01 and c18:0 (matsumoto et al., 2010; thajuddin et al., 2015). the pervious works showed c16:0 (21.4% tfa) and c16:1 (25.1% tfa) under 200 hrs incubation in nacicula sp. (matsumoto et al., 2010) and c17:01 (29.36% tfa) and c18:0 (26.48% tfa) in naviculoid for 336 hrs (14 days) incubation period under normal condition (thajuddin et al., 2015). the valuable polyunsaturated fatty acids, especially omega 3 is one of the valuable products of microalgae. studies revealed that omega3 such as eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) have proper effect on human health (simopoulos, 1991). the results of this research demonstrated that the prolonged culture of n. salinicola produces a high level of epa (25.58% tfa) after 21 days which agrees with the previous studies (kitano et al., 1997; khatoon, 2006; zheng et al., 2007). however, the level of omega3 were lower than the results of this study (kitano et al., 2007; khatoon, 2006; zheng et al., 2007). it was reported c20:5 (8% tfa), c22:6 (2% tfa) (khatoon, 2006), c20:5 (21.52% tfa) and c22:6 (3.64% tfa) in navicula sp. (zheng et al., 2007). our findings indicated that n. salinicola contains considerable lipid vacuole and it seems not only suitable for biofuel but also it is an appropriate source of omega3 in prolonged culture conditions. by further study such as inducing genetic and physiologic variations on this strain for improving the efficiency, it is hoped to accelerate commercialization of such investigations. acknowledgments authors greatly acknowledge the iranian biological research center, karaj, iran (ibrc) for supporting this project. we also thank to a. afsharian, m. papizadeh and ibrc microalgae lab staff for their valuable cooperation. references axelsson m., gentili f. (2014). a single-step method for rapid extraction of total lipids from green microalgae. plos one, 9(2): e89643. brett m., muller-navarra d.o. (1997). the role of highly unsaturated fatty acids in aquatic food web processes. freshwater biology, 38(3): 483-499. burdon k.l. 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(2017) 5(4): 268-274 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی تاثیر کشت طوالنی مدت بر محتوای از دریای خزر و بررسی navicula salinicola (hustedt 1939) جداسازی اسید چرب آن 5،1، سید ابوالحسن شاهزاده فاضلی4، غالمرضا بخشی خانیکی3،1محمد علی آموزگار ،2*مصطفی نوروزی ،1فرخنده صبا ،1اعتصامی ناحسا .ایران ،مرکز ملی ذخایر ژنتیکی و زیستی ایران، تهران ها،بانک میکروارگانیسم 1 .ایران ،تهران ،س الزهرا دانشگاه ،علوم زیستی دانشکده، بیوتکنولوژی گروه2 .دانشگاه تهران، ایرانعلوم، سیپرد ،موجودات زنده ییقطب تبارزاروبیولوژی، دانشکده زیست شناسی، گروه میک 3 .ایران ،تهران ،پیام نور تهران شرق دانشگاه ،علوم دانشکده، بیوتکنولوژی گروه4 .ایران ،تهران ،علم و فرهنگ دانشگاه علوم پایه و دانشکدهبیولوژی سلولی و ملکولی، گروه5 چکیده: در این تحقیق دیاتوم ناویکلوئید از بخش جنوبی . شوندمی محسوب طبیعی اشباع غیر چرب هایاسید تولیددارای پتانسیل منابع از یکی هادیاتوم و شناسیریخت مشاهده صفات کمک به شده جداسازی آرایهو توانایی تولید و تجمع لیپید در آن مورد ارزیابی قرار گرفت. دریای خزر جداسازی در ibrc-m 5083 دسترسی شماره باو گرفت قرار شناسایی مورد navicula salinicola عنوانهب 18s rrna ژنومی طقهمن مولکولی آنالیز در سویه این( روز 21) مدت طوالنی کشت و نرمال کشت تیمارهای اعمال و کشت از پس. نگهداری شد ذخایر ژنتیکی و زیستی ایرانمرکز ملی رنگ توسط لیپیدی ذرات تولید همچنین. آمد بدست سلول خشک وزن درصد 43.72 و 31.83 ترتیب به کل چربی تولید میزان ،f/2 کشت محیط گازی، کروماتوگرافی روش از استفاده با سویه این مدت طوالنی کشت چرب اسید محتوای سنجش. شد مشخص سویه این در bبلک سودان آمیزی در 3 امگا مناسب محتوای و لیپید تجمع توانایی به توجه با. داد نشان را اسید ایکوزاپنتانوئیک( چرب های اسید کل درصد) 58/25 میزان n. salinicola، زیستی سوخت همچنین و دارویی آبزیان، خوراک صنایع در سویه این از استفاده جهت مناسبی بستر بیشتر تحقیقات با توانمی .نمود فراهم .اسید، دوکوزاهگزانوئیک اسید، ایکوزاپنتانوئیک 3جلبک، امگا :کلمات کلیدی int. j. aquat. biol. (2023) 11(2): 159-169 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article effects of different physical shocks and sampling time on lipid content and fatty acids composition of nannochloropsis oculata farzaneh noori, saeid vahdat artemia and aquaculture research institute, urmia university, urmia, iran. s article history: received 15 december 2022 accepted 11 march 2023 available online 2 5 april 2023 keywords: sampling time fatty acid physical shocks biochemical composition abstract: the microalga nannochloropsis oculata is marine widely used in aquaculture systems as an essential source of protein, lipid, and polyunsaturated fatty acids. day/night ph fluctuations driven by photosynthesis and respiration create an environment that exhibits changing ph ranges. the aim of this study was to find whether physical shocks could change the complete profile of nutrients (lipid, fatty acid, carbohydrate, chlorophylls, and proteins) in n. oculata. the algae were cultivated in 32 flasks of ten-liter for biomass production for 12 days using guillard medium (f/2). the cells were reared at 0.5 molar salinity (29 ppt), under 3500 lux light intensity with a 12l:12d photoperiod and 21ºc temperature. after 12 days, when the cell density reached its stationary phase, they were centrifuged. the pellets were then re-suspended in fresh seawater thoroughly and transferred into thirty-two containers with 10-liter volumes, including eight treatments with four replicates. the algae in treatments 3, 4, and 8 were subjected to salinity (88 ppt), starvation, and ph (11) shocks, respectively and treatments 2, 5, 6, and 7 were subjected to salinity + ph + starvation, ph + starvation, salinity + ph and salinity + starvation shocks, respectively. the biochemical composition of n. oculata demonstrated that t3 at the end of the dark period, and t1 at the end of the light period, possessed significantly higher (51.51%), and lower (24.9%) lipid content, respectively. according to the results, epa under ph shock, linoleic acid under ph + salinity + starvation shock, and dha and omega-3 under ph + salinity shock at the end of the dark period revealed significant differences with the control group. the saturated fatty acids showed significantly higher value in the control group during the dark period. the monounsaturated fatty acids increased significantly under ph shock at the end of the light period on day 18. based on the results, the best treatment to obtain more lipid production in n. oculata was using six-day salinity shock and harvesting algae at the end of the dark period, and for more epa synthesis in n. oculata, ph shock for six days and harvesting the algae at the end of dark period is recommended. introduction some species of microalgae synthesize very longchain fatty acids (carbon chains 20+ in length), including eicosapentaenoic acid (epa, c20:5n-3) and docosahexaenoic acid (dha, c22:6n-3) (bigogno et al., 2002; guiheneuf et al., 2013). these omega-3 (ω-3) fatty acids are essential components of high-quality fish diets (muhlroth et al., 2013). fatty acids are the building blocks of lipids, but they are not distributed equally amongst different lipid classes. algal polar lipids are located in structural and functional cell membranes, while neutral lipid triacylglycerols (tags) function as storage correspondence: farzaneh noori doi: http//doi.org/10.22034/ijab.v11i2.1809 e-mail: f.noori@urmia.ac.ir dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.10.2 molecules (hu et al., 2008). the microalga nannochloropsis sp. is a eustigmatophyte widely used in marine aquaculture systems as an important source of vitamins, pigments, and pufas (sukenik, 1991; sukenik et al., 1993; pal et al., 2011). this microalga has been widely studied due to its high pufa content. as found in many other species of algae, nutrient depletion significantly increased fa accumulation in nannochloropsis. other studies have reported that nannochloropsis salina reaches a total fa accumulation of 37.5% lipid/dw when arriving at the stationary phase and could grow well at 34 ppt 160 noori and vahdat/ effects of different physical shocks and sampling time on lipid content and fatty acids salinity (bartley et al., 2013). similarly, nannochloropsis gadiata grows at low irradiance demonstrating accumulation of up to 50% lipid/dw after a nutrient deprivation phase (bartley et al., 2013). doan and obbard (2011, 2012) reported a total lipid production of 50 and 55% of dw in nannochloropsis sp. using cell sorting and ethyl methanesulfonate, respectively. nannochloropsis is a genus of robust, oleaginous microalgae that synthesizes epa during balanced growth and is a promising candidate for commercial applications (kilian et al., 2011; radakovits et al., 2012; li et al., 2014; sharma et al., 2015). research demonstrated that subjecting the culture to a temporary change of environmental factors such as ph can decrease unwanted organisms and improve final culture densities. day/night ph fluctuations driven by photosynthesis and respiration create an environment that exhibits changing ph ranges (richmond and becker, 1986). further, ph may affect the lipid composition (griffiths et al., 2009, rodolfi et al., 2009, moheimani, 2013). nitrogen and phosphorus starvation shifts the lipid metabolism from membrane lipid synthesis to neutral lipid storage. this, in turn, increases the total lipid content of green algae (hu, 2004). higher salinity increases the algae's lipid content (fabregas et al., 1984; zhila et al., 2011). besides the pigment contents, other algal cell components such as carbohydrates and proteins can also be influenced by the light regime (renaud et al., 1991). the aim of this study was to find out how different physical shocks, alone or in combination, can influence the synthesis of different nutrients such as lipids, fatty acids, carbohydrates, chlorophylls, and proteins by nannochloropsis oculata. materials and methods nannochloropsis oculata was obtained from artemia and aquaculture research institute (aari), urmia university, iran. the algae were cultivated in ten-liter conical flasks for biomass production for 12 days using guillard medium (f/2)1 (guillard, 1973). the cells were reared at 0.5 molar salinity (29 ppt), 3500 lux light intensity with a 12l: 12d photoperiod and 21ºc temperature. after 12 days when the cell density reached its stationary phase, they were centrifuged (3500 rpm, 5 min). the pellets were collected and then re-suspended thoroughly, equally divided (0.2713 g.l-1 dw), and transferred into thirty-two 10-liter containers, including eight treatments and four replicates. then the nannochloropsis algae were exposed to different shocks for six days. treatments 3, 4, and 8 were subjected to salinity of 88 ppt, nutrient starvation, and ph (11) shocks, respectively and treatments 2, 5, 6, and 7 were subjected to salinity + ph + starvation, ph + starvation, salinity + ph, and salinity + starvation shocks, respectively. however, the control group (t1) was cultured under normal conditions. each container was aerated continuously at a rate of 758 ml.min-1. the samples were collected every three days and at the end of light and dark periods. chlorophyll a, b and total carotene analysis: fifty ml algae were frozen under -20°c and then defrost (for breaking down of cell wall), filtered with s&x filter (1 µm), and weighed. the pigments were extracted with 96% methanol (50 ml.g-1 algae). the samples were homogenized for one minute at 1000 rpm. the homogeneous mixture was filtered and centrifuged for 10 min at 2500 rpm. supernatants were separated and the absorbance at 666, 653 and 470 nm was recorded using the microplate reader synergy ht. the following formula was used to calculate chlorophyll and total carotene: ca= 15.65a666 – 7.340a653 cb= 27.05a653 – 11.21a666 cc= 1000a470 – 2.860ca – 129.2cb /245 where ca = chlorophyll a, cb= chlorophyll b, and cc= total carotene in terms of micrograms per gram of wet weight (dereet al., 1998). lipid and ash contents analysis: laboratory methods were used to measure lipid content by ether extraction (aoac, 2005). for this purpose, a certain amount of samples was weighted and held for 7 hours on soxhlet within diethyl ether (98%). to calculate the ash content samples were weighed and placed in an electric furnace for 6 hours at 550°c 161 int. j. aquat. biol. (2023) 11(2): 159-169 (aoac, 2005). fatty acid profiles: the fatty acid composition of nannochloropsis algae was determined based on miquel and browse (1992). in brief, 200 mg of algae was heated to 80°c in 1 ml of a mixed solution of h2so4 2.5% and methanol 98% (1:40, v/v) for one hour in a teflon lined screw cap glass tube. the mixture was cooled at room temperature and then 500 µl of hexane and 1.5 ml of nacl 0.9% (w/v) were mixed and added to the samples. the samples were centrifuged for 10 min at 4000 rpm and supernatant (1 µl) was injected to gas chromatograph (gc) to determine the fatty acid profiles (miquel and browse,1992). protein content: the protein content was measured according to slocombe et al. (2013). five mg of dried algae was mixed with 0.2 ml trichloroacetic acid (tca) 24% (w/v). the mixture was incubated for 15 min at 95°c and cooled at room temperature. the homogenate was centrifuged at 15000 g for 20 min at 4°c and the supernatant was discarded. the pellets were re-suspended in 0.5 ml lowry reagent and were incubated for 3 hours at 55°c. the samples were cooled at room temperature and centrifuged at 15000 g for 20 min. the supernatant was placed for 30 min in lowry reagent and absorption was read at 600 nm (slocombe et al., 2013). the standard curve was prepared using bovine serum albumin (bsa) as standard. total carbohydrate content: the carbohydrate content of the samples was measured by adding 5 ml of 2.5 n hcl to 100 mg of algae. the samples were hydrolyzed to simple sugars by keeping them in a boiling water bath for three hours and then cooled at room temperature. the sample was neutralized by solid na2co3 until the effervescence ceases and then the volume was made to 100 ml with distilled water and centrifuged at 5000 rpm for 5 minutes. to 0.5 ml of supernatant, 0.5 ml of distilled water and 4 ml of 0.2% anthrone reagent were added and the mixture was heated for 8 min in boiling water bath. the sample cooled rapidly and the absorbance was read at 630 nm. the glucose standard solution was prepared at a concentration of 100 µ.ml-1 (hedge and hofreiter, 1962). statistical analysis: the normality of all data was checked by the kolmogorov-smirnov test. mean (±sd) and factorial multivariate were used. oneway anova, ancova (bonferroni test) and post-hoc tukey analysis were used at 5% (p<0.05). calculated data and drawing charts were done by spss (version 22) and excel (2013 version), respectively. results biochemical compositions and some fatty acid profiles of nannochloropsis cultivated in f/2 medium on day 12 under laboratory conditions are shown in table 1. the results (fig. 1) showed that the protein content was affected by salinity and ph shock (t6) at the end of the dark period showing the lowest value, while the highest protein content was found in t4 at the end of the dark period on day 18 (p<0.05). lipid content was significantly influenced by the dark period and significantly higher values were observed in t7 and t3 (p<0.05) and the lowest value was achieved in t1 (4.68%) at the end of the dark period on day 18 (p<0.05). the result of ash reveals a significantly higher value at the end of the light period in starvation + salinity shock (t7) on day 15 (p<0.05). carbohydrate content was influenced significantly by starvation + salinity (t7) and salinity (t3) shocks at the end of the dark period and exhibited the highest and lowest values, respectively (p<0.05). the protein content was significantly decreased at the end of the light and dark period in t6 on day 18 and increased in t4 at the end of the dark period on day 18 (p<0.05). moreover, at the end of the dark period on day 18, t3 showed a significantly higher lipid content (51.51%) (p<0.05). carbohydrate content in t7 at the end of the dark period on day 18 showed the highest values (p<0.05). the chlorophyll a and b on day 18 and at the end of the dark photoperiod under ph + starvation + salinity shock revealed highly significant (p<0.05). the ph + starvation + salinity shock at the end of the dark period on day 18 increased total carotene 162 noori and vahdat/ effects of different physical shocks and sampling time on lipid content and fatty acids level significantly (p<0.05) (fig. 2). the results on day 18 showed ph + starvation + salinity shock at the end of dark period made chlorophyll a and b and total carotene to increased significantly (p<0.05) (fig. 2). based on the results, epa in ph, dha, and omega-3 in ph + salinity, and mufa under starvation + salinity treatments at the end of the dark period on day 15 demonstrated significant day 12 protein (%) lipid (%) carbohydrate (%) ash (%) chlorophyll a (µg.g-1 fw) chlorophyll b (µg.g-1 fw) total carotenoid (µg.g-1 fw) end of light 33.24±0.25 18.62±0.15 35.65±0.24 12.01±0.20 35.62±0.14 8.15±0.31 126.24±1.01 end of dark 30.12±0.08 19.31±1.11 37.99±1.01 12.08±0.35 34.21±0.14 7.16±0.44 124.21±0.14 day 12 ara dha epa linoleic acid linolenic acid sfa mufa pufa sum of ω3 sum of ω6 end of light nd nd 10.35±0.62 8.55±0.65 3.68±0.54 41.35±0.35 37.15±0.31 21.35±1.21 14.03±1.24 8.55±0.65 end of dark nd nd 10.95±0.35 8.84±0.24 3.71±0.84 41.95±1.21 37.45±1.32 21.42±1.67 14.66±0.71 8.84±0.24 table 1. bio-chemical and fatty acid compositions of nannochloropsis oculata cultivated in f/2 medium on day 12 under standard conditions. a a ab d bc a a a b a a a c c b a cd ab ab f b a cd cdd ab e c b a d c 0 5 10 15 20 25 30 35 p ro te in ( % ) l15 d15 d18 b f g c c c g ee bc bc a b d a cc d b e e e b d a f h c f e g c 0 10 20 30 40 50 60 li p id ( % ) d15 l15 l18 d18 d c bc b a a b c d b a f e d c f cd d cd d c e e ef c c a f c b g c 0 5 10 15 20 25 30 35 40 45 50 t1 t2 t3 t4 t5 t6 t7 t8 before shock c a rb o h y d ra te ( % ) l15 d15 l18 d18 c d a bc b b ab a c bc d a bc c e a b c bc a c d ab c a a a d bc b a c 0 2 4 6 8 10 12 14 16 18 20 a sh ( % ) d15 l15 l18 d18 figure 1. mean (±se) lipid, carbohydrate, ash and protein contents of nannochloropsis oculata cultivated in f/2 medium on day 15 and 18 under different shocks (p<0.05). a, b, c and … indicate the significance differences on day 15 (dark and light) and a, b, c and … indicate the significance differences on day 18 (dark and light). 163 int. j. aquat. biol. (2023) 11(2): 159-169 differences compared to other treatments (p<0.05). furthermore, linoleic acid (or omega-6) in t2 (ph + salinity + starvation) at the end of the dark period showed a significantly higher value compared to other treatments (p<0.05). at the end of the dark period, in t1, sfas were increased significantly (p<0.05). moreover, the pufas values under t8 and at the end of the dark period on day 15 increased significantly (p<0.05). linolenic acid in ph + salinity at the end of the light period on day 15 showed a significant difference between all groups (p<0.05) (table 2). according to the result obtained omega-6 and linoleic acid in t4 (starvation), epa in t4, and sfas in t1 at the end of the dark period revealed significantly higher values than other treatments (p<0.05). the dha in t2, pufas in t7, and linolenic acid in t3 at the end of the light period on day 18 showed significantly higher amounts (p<0.05). the mufas in t8 at the end of the light period exhibited significantly higher than other treatments (p<0.05). moreover, the total omega-3 at the end of the dark period on day 18, in t8 was significantly higher than other treatments (p<0.05) (table 3). discussion microalgae can accumulate considerable quantities of carbohydrates, proteins, and/or lipids (ho et al., 2012, 2013). changes in microalgae biochemical composition are likely to occur as a result of variations in ph (khalil et al., 2010), temperature (roleda et al., 2013), light, salinity (ruangsomboon et al., 2013), and metal contents (sun et al., 2014). ab ab a bc bc c ab d ab b a b c b ab c a cd ab a e cd ab d b c ab a f d a e 0 1 2 3 4 5 6 7 8 c h lo ro p h y ll b l15 d15 l18 d18 a ab a c e e ab g ab d a d f d ab f b c b a f d a e a c a a e c a d 0 5 10 15 20 25 c h lo ro p h y ll a l15 d15 d18 l18 b bc a e f g c i d f a f h f b h a b a a d b a b a b a a e b a c 0 500 1000 1500 2000 t1 t2 t3 t4 t5 t6 t7 t8 before shock t o ta l c a ro te n e l15 d15 l18 d18 figure 2. mean (±se) chlorophyll a, b and total carotenoids contents of nannochloropsis oculata cultivated in f/2 medium on day 15 and 18 under different shocks (p<0.05). a, b, c and … indicate the significance differences on day 15 (dark and light) and a,b,c and … indicate the significance differences on day 18 (dark and light). 1 6 4 n o o ri a n d v ah d at / e ff ec ts o f d if fe re n t p h y si ca l sh o ck s an d s am p li n g t im e o n l ip id c o n te n t an d f at ty a ci d s s a m p li n g t im e c o n d it io n s (s h o c k ) a r a d h a e p a l in o le ic a c id l in o le n ic a c id s f a m u f a p u f a s u m o f ω 3 s u m o f ω 6 e n d o f d a rk (d a y 1 5 ) t 1 ( c o n tr o l) n d 0 .2 2 ± 0 .0 3 a 7 .0 8 ± 0 .3 5 a n d 2 5 .8 5 ± 1 .7 0 g h 3 4 .6 9 ± 0 .6 5 f 1 3 .4 9 ± 3 .6 9 a 3 3 .1 5 ± 1 .7 5 a 3 3 .1 5 ± 1 .7 5 a n d t 2 ( p h + s a li n it y + s ta rv a ti o n ) n d n d 3 3 .4 7 ± 1 .4 8 h 6 .5 5 ± 1 .4 1 4 .1 3 ± 0 .2 6 a 2 2 .6 6 ± 0 .1 3 c d e 1 6 .7 9 ± 1 .8 8 a b 4 4 .1 4 ± 3 .1 6 b c 3 7 .6 0 ± 1 .7 4 a b 6 .5 5 ± 1 .4 1 t 3 ( s a li n it y ) n d n d 3 1 .4 4 ± 0 .6 8 g h n d 7 .1 3 ± 0 .8 2 b 1 9 .8 2 ± 0 .3 2 b c d 3 4 .9 2 ± 3 .4 3 fg 3 8 .5 7 ± 1 .5 0 a b 3 8 .5 7 ± 1 .5 0 a b n d t 4 ( s ta rv a ti o n ) n d n d 1 2 .2 6 ± 1 .7 8 a b c n d 2 4 .8 9 ± 1 .4 4 fg h 2 3 .7 8 ± 1 .3 6 c d e f 2 5 .6 8 ± 2 .7 5 c d e 3 7 .1 5 ± 0 .3 3 a b 3 7 .1 5 ± 0 .3 3 a b n d t 5 ( p h + s ta rv a ti o n ) n d n d 2 4 .2 9 ± 1 .0 8 e fg n d 1 3 .2 5 ± 0 .5 3 c 2 4 .0 1 ± 0 .3 9 c d e f 3 1 .8 0 ± 0 .3 2 e fg 3 7 .5 4 ± 1 .6 2 a b 3 7 .5 4 ± 1 .6 2 a b n d t 6 ( s ta rv a ti o n + s a li n it y ) n d n d 2 0 .8 3 ± 1 .0 2 d e f n d 1 6 .4 9 ± 1 .3 2 c d 1 7 .7 2 ± 0 .5 5 a b c 3 8 .8 2 ± 1 .0 2 g 3 7 .3 2 ± 0 .3 0 a b 3 7 .3 2 ± 0 .3 0 a b n d t 7 ( p h + s a li n it y ) n d 4 .7 5 ± 0 .2 3 b 9 .7 4 ± 3 .5 6 a b n d 1 9 .9 2 ± 1 .7 6 d e f 2 5 .5 0 ± 1 .1 2 d e f 3 0 .8 1 ± 0 .0 9 d e f 3 4 .4 2 ± 1 .5 6 a b 3 4 .4 2 ± 1 .5 6 a b n d t 8 (p h ) n d n d 4 7 .2 0 ± 0 .5 0 i n d 1 5 .8 9 ± 0 .8 4 c d 1 4 .8 9 ± 1 .6 3 a b 2 0 .9 4 ± 0 .9 4 a b c 6 3 .0 9 ± 0 .3 3 e 6 3 .0 9 ± 0 .3 3 e n d e n d o f li g h t (d a y 1 5 ) t 1 ( c o n tr o l) n d n d 1 6 .2 9 ± 0 .5 7 b c d n d 1 8 .4 9 ± 0 .5 0 c d e 2 7 .5 2 ± 3 .0 8 g 2 2 .6 4 ± 0 .2 2 b c d 3 4 .7 8 ± 0 .0 7 a b 3 4 .7 8 ± 0 .0 7 a b n d t 2 ( p h + s a li n it y + s ta rv a ti o n ) n d n d 4 3 .4 7 ± 4 .2 1 i n d 5 .3 7 ± 1 .4 8 a b 2 1 .1 9 ± 0 .3 0 c d e 2 2 .7 9 ± 2 .7 3 b c d 4 8 .8 3 ± 2 .7 3 c d 4 8 .8 3 ± 2 .7 3 c d n d t 3 ( s a li n it y ) n d n d 5 5 .3 4 ± 3 .0 9 k n d n d 2 0 .5 6 ± 3 .6 8 b c d 1 7 .1 5 ± 2 .3 9 a b 5 5 .3 4 ± 3 .0 9 d e 5 5 .3 4 ± 3 .0 9 d e n d t 4 ( s ta rv a ti o n ) n d n d 1 5 .4 8 ± 1 .5 1 b c d n d 2 0 .8 2 ± 2 .0 3 d e fg 3 0 .4 4 ± 2 .9 7 e f 3 2 .1 8 ± 3 .1 4 e fg 3 6 .2 9 ± 3 .5 4 a b 3 6 .2 9 ± 3 .5 4 a b n d t 5 ( p h + s ta rv a ti o n ) n d n d 2 7 .4 4 ± 2 .1 2 fg h n d 1 6 .0 9 ± 3 .1 8 c d 2 3 .0 1 ± 0 .7 7 c d e 2 6 .1 1 ± 1 .2 2 c d e 4 3 .5 2 ± 5 .3 0 b c 4 3 .5 2 ± 5 .3 0 b c n d t 6 ( s ta rv a ti o n + s a li n it y ) n d n d 1 5 .0 0 ± 0 .2 4 b c d n d 2 3 .3 6 ± 0 .1 4 e fg 2 2 .8 5 ± 0 .8 5 c d e 2 5 .5 7 ± 0 .4 4 c d e 3 8 .3 7 ± 0 .3 9 a b 3 8 .3 7 ± 0 .3 9 a b n d t 7 ( p h + s a li n it y ) n d n d 2 1 .6 9 ± 1 .7 8 d e f n d 2 8 .2 8 ± 2 .3 2 h 1 9 .2 7 ± 1 .5 8 b c d 2 9 .0 0 ± 2 .3 8 c d e f 4 9 .9 7 ± 4 .1 1 c d 4 9 .9 7 ± 4 .1 1 c d n d t 8 (p h ) n d n d 1 7 .4 8 ± 0 .1 0 c d e n d 1 9 .1 7 ± 0 .3 9 d e f 1 3 .0 1 ± 2 .2 4 a 3 0 .0 2 ± 0 .5 4 d e f 3 6 .6 5 ± 0 .2 8 a b 3 6 .6 5 ± 0 .2 8 a b n d t ab le 2 . m ea n ( ± s e ) so m e fa tt y a ci d c o m p o si ti o n s o f n a n n o ch lo ro p si s o cu la ta c u lt iv at ed i n f /2 m ed iu m o n d ay 1 5 u n d er d if fe re n t sh o ck s. t h e d at a sh o w n r ep re se n t th e m ea n v al u es o f th re e re p et it io n s. s a m p li n g t im e c o n d it io n s (s h o c k ) a r a d h a e p a l in o le ic a c id l in o le n ic a c id s f a m u f a p u f a s u m o f ω 3 s u m o f ω 6 e n d o f d a rk (d a y 1 8 ) t 1 ( c o n tr o l) n d 5 .7 4 ± 0 .7 2 b 1 7 .1 5 ± 2 .3 2 a b n d 6 .1 9 ± 1 .1 2 c d 3 7 .4 4 ± 4 .0 2 d e 3 0 .9 7 ± 0 .4 7 b 2 2 .5 8 ± 1 .6 6 b c d 3 0 .9 7 ± 0 .4 7 b n d t 2 ( p h + s a li n it y + s ta rv a ti o n ) n d n d 1 7 .8 8 ± 5 .0 5 a b n d 1 5 .0 9 ± 3 .5 4 g h 1 0 .8 2 ± 2 .4 6 a 3 2 .9 7 ± 1 .5 0 b 2 7 .7 6 ± 3 .1 9 c d e 3 2 .9 7 ± 1 .5 0 b c n d t 3 ( s a li n it y ) n d n d 3 0 .8 1 ± 0 .3 8 d e n d 4 .1 2 ± 0 .6 5 b c 4 3 .4 9 ± 2 .3 3 e 3 4 .9 4 ± 0 .2 7 b c 1 5 .1 4 ± 0 .8 5 a 3 4 .9 4 ± 0 .2 7 b c d n d t 4 ( s ta rv a ti o n ) n d n d 4 4 .3 6 ± 1 .1 8 f 4 .7 3 ± 0 .9 2 c n d 1 2 .4 4 ± 0 .3 5 a 4 9 .0 8 ± 2 .1 0 d e 2 7 .8 6 ± 0 .7 2 c d e 4 4 .3 6 ± 1 .1 8 e f 4 .7 3 ± 0 .9 3 c t 5 ( p h + s ta rv a ti o n ) n d n d 3 9 .3 7 ± 0 .8 9 e f n d 9 .2 2 ± 0 .2 5 d e 1 2 .4 2 ± 1 .0 8 a 4 8 .5 9 ± 0 .6 3 d 2 7 .4 3 ± 0 .4 9 c d e 4 8 .5 9 ± 0 .6 3 f n d t 6 ( s ta rv a ti o n + s a li n it y ) n d n d 2 8 .3 1 ± 1 .4 2 c d n d 7 .6 3 ± 0 .6 8 c d 2 3 .4 8 ± 1 .7 5 b c 3 9 .0 4 ± 2 .6 5 b c 2 1 .0 5 ± 3 .5 3 b c d 3 9 .0 4 ± 2 .6 5 c d e n d t 7 ( p h + s a li n it y ) n d n d 1 8 .9 6 ± 0 .4 3 a b c n d 1 2 .4 6 ± 0 .8 0 fg 2 0 .5 8 ± 1 .2 9 b 3 1 .4 2 ± 0 .3 6 b 4 4 .4 0 ± 2 .0 2 f 3 1 .4 2 ± 0 .3 6 b c n d t 8 (p h ) n d n d 6 3 .4 6 ± 1 .2 4 h n d 1 .6 4 ± 0 .7 3 a b 1 1 .5 7 ± 0 .0 9 a 6 5 .0 9 ± 0 .5 1 f 1 7 .9 5 ± 0 .0 8 a b 6 5 .0 9 ± 0 .5 1 h n d e n d o f li g h t (d a y 1 8 ) t 1 ( c o n tr o l) n d 3 .8 2 ± 0 .9 8 a 1 5 .7 0 ± 1 .6 1 a n d 2 .8 2 ± 0 .0 8 a b 5 2 .0 7 ± 0 .1 4 f 2 2 .3 4 ± 2 .5 1 a 1 4 .8 8 ± 1 .2 1 a 2 2 .3 4 ± 2 .5 1 a n d t 2 ( p h + s a li n it y + s ta rv a ti o n ) n d 7 .6 3 ± 0 .5 8 c 3 4 .0 9 ± 2 .7 7 d e n d 2 .5 2 ± 0 .4 9 a b 3 6 .1 9 ± 1 .0 9 d 4 2 .3 5 ± 2 .6 8 c d 1 5 .0 3 ± 0 .2 8 a 4 2 .3 5 ± 2 .6 8 d e f n d t 3 ( s a li n it y ) n d n d 1 7 .6 6 ± 1 .4 0 g h n d 1 7 .6 4 ± 1 .4 9 h 2 6 .6 0 ± 0 .2 1 b c 3 5 .3 1 ± 0 .0 9 b c 2 7 .8 0 ± 0 .0 7 c d e 3 5 .3 1 ± 0 .0 9 b c d n d t 4 ( s ta rv a ti o n ) n d n d 5 4 .3 2 ± 5 .4 8 g n d 2 .7 9 ± 0 .0 1 a b 2 0 .7 8 ± 1 .9 6 b 5 7 .1 0 ± 5 .4 8 e f 2 1 .2 5 ± 3 .5 8 b c d 5 7 .1 0 ± 5 .4 8 g n d t 5 ( p h + s ta rv a ti o n ) n d n d 1 9 .0 5 ± 2 .6 2 a b c n d 1 3 .5 2 ± 1 .3 2 fg 2 7 .1 0 ± 0 .5 0 b c 3 2 .5 6 ± 1 .2 9 b 2 8 .4 7 ± 3 .0 4 d e 3 2 .5 6 ± 1 .2 9 b c n d t 6 ( s ta rv a ti o n + s a li n it y ) n d n d 4 8 .2 3 ± 0 .0 4 fg 2 .6 6 ± 0 .1 6 b 8 .7 7 ± 0 .3 7 d e 8 .9 2 ± 1 .2 0 a 5 9 .7 2 ± 0 .4 8 f 2 9 .0 0 ± 0 .9 5 d e 5 7 .0 5 ± 0 .3 2 g 2 .6 6 ± 0 .1 6 b t 7 ( p h + s a li n it y ) n d n d 2 5 .2 2 ± 0 .6 0 b c d n d n d 2 8 .1 1 ± 0 .5 9 c 3 2 .1 7 ± 0 .4 6 b 3 5 .7 4 ± 6 .5 8 e f 3 2 .1 7 ± 0 .4 6 b c n d t 8 (p h ) n d n d 3 0 .7 4 ± 1 .7 0 d e 1 .0 9 ± 0 .0 4 a 1 .6 0 ± 0 .5 4 a 4 0 .1 3 ± 1 .5 2 d e 3 3 .4 4 ± 2 .2 9 b 1 8 .7 8 ± 1 .8 6 b c 3 2 .3 4 ± 2 .2 5 b c 1 .0 9 ± 0 .0 4 a t ab le 3 . m ea n ( ± s e ) so m e fa tt y a ci d c o m p o si ti o n s o f n a n n o ch lo ro p si s o cu la ta c u lt iv at ed i n f /2 m ed iu m o n d ay 1 8 u n d er d if fe re n t sh o ck s. t h e d at a sh o w n r ep re se n t th e m ea n v al u es o f th re e re p et it io n s. 165 int. j. aquat. biol. (2023) 11(2): 159-169 regarding the effects of starvation, studies demonstrated that n and/or p limitation in growth media causes metabolism alterations that induce lipid accumulation (liang et al., 2013; li et al., 2014). despite several studies made in an attempt to understand the potential effects of n and/or p limitations on microalgae biochemical composition changes, the phenomena seem to be speciesdependent (shifrin and chisholm, 1981). nitrogen and phosphorus starvation shifts the lipid metabolism from membrane lipid synthesis to neutral lipid storage. this, in turn, increases the total lipid content of green algae (hu, 2004). major effects of nitrogen deficiency in algal culture include the enhanced biosynthesis and accumulation of lipids (thompson, 1996, shifrin and chisholm, 1981; converti et al., 2009; demirbas, 2010) and triglycerides (takagi et al., 1999; stephenson et al., 2010) with a concomitant reduction in protein content (fogg, 1956; morris et al., 1974; kilham et al., 1997; heraud et al., 2005), resulting in a higher lipid/protein ratio (converti et al., 2009). in this study, a high alkaline ph + starvation + high salinity increased carbohydrate and decreased lipid and protein contents of algae, however high salinity in combination with any other shock caused higher lipid production, especially on day 18. this could be explained that under salinity conditions the cell would release nitrogen from the photosynthetic pigments and utilize the same for the metabolic processes. higher salinity increases the algae lipid content (fabregas et al., 1984; zhila et al., 2011). dunaliella, a marine alga, exhibited an increase in saturated and monounsaturated fatty acids with an increase in nacl concentration from 0.4 to 4 m (xu and beardall, 1997). in another study with dunaliella tertiolecta, an increase in intracellular lipids (60 to 67%) and triglyceride concentration (40 to 56%) with an increase in nacl concentration from 0.5 (freshwater concentration) to 1.0 m was observed (takagi and karseno, 2006). in this work, when the n. oculate biomass was cultured under normal conditions and transferred to high salinity, they showed different levels of lipid accumulation in the cell. among all conditions and sampling time, the most favorable treatment to achieve high lipid content per cell was at the end of the dark period on day 18. nannochloropsis oculata, showed a profound increase in lipid content under salinity shock at the end of the dark period on day 18 (51.51%). the lipid accumulation is enhanced by a combination of salinity shock. this inference is achieved on the bases of the results obtained from treatment 3 (end of light, day 15, and end of dark, day 18), where a significant increase in the cellular lipid content of n. oculate was observed under salinity shock individually. in a study conducted by ben-amotz, the lipid content of botryococcus braunii cultured in 0.50 m nacl was higher compared to media without the addition of nacl, but protein, carbohydrates, and pigments levels were lower (ben-amotz et al., 1985). another study with the same alga reported a decrease in protein content with unchanged carbohydrate and lipid content with an increase in salinity (vazquez-duhalt et al., 1991). research on tetraselmis suecica also reported a reduction in protein content per cell of up to 20% with an increase in salinity (fabregas et al., 1984), but protein in all shocks decreased to less than 25%. variations in salinity also influence several biochemical and physiological mechanisms such as lipid production and growth which are essential in marine organisms (fava and martnini, 1988). in the current study, the treatments of 2 (salinity + ph + starvation), 7 (salinity + starvation), and 8 (ph) on day 15 at the end of the light period showed the highest lipid level, while the other treatments revealed different action, that it would be due to different physiological mechanisms in a specific algal. lipids in general (as a percentage of particulate organic matter or as absolute amounts) and particularly fatty acids have been targeted as important variables determining the food quality of algae (ahlgren et al., 1990; coutteau and sorgeloos, 1997; weers and gulati, 1997a). the characteristic types of fatty acids produced by diatoms, green algae and/or cryptomonads have been studied (piorreck et al., 1984; cranwell et al., 166 noori and vahdat/ effects of different physical shocks and sampling time on lipid content and fatty acids 1989; ahlgren et al., 1990). the general pattern observed is that green algae rarely produce fatty acids in excess of eighteen carbons, while diatoms and cryptophytes make many long-chained (18c) polyunsaturated fatty acids (pufas). ahlgren et al. (1990) showed that the long-chained pufas produced by cryptophytes enhance reproduction in zooplankton, including daphnia. many studies of algal biochemistry have been undertaken in the past on cells that were either growing at the maximal rate (µmax) or at the stationary phase. growth rates of algae measured in lakes are frequently found to be at some intermediate growth rate (lehman and sandgren, 1985; sommer, 1989), rather than at stationary phase or µmax. algae grown in continuous or semi-continuous culture have reduced variation in physiological conditions, can be easily replicated, and are more representative of the in-situ condition. immediate effects of phosphorus limitation include a reduction in the synthesis and regeneration of substrates in the calvin-benson cycle and a consequential reduction in the rate of light utilization required for carbon fixation (reitan barsanti and gualtieri, 2005). phosphate limitation also reduces the synthesis of n-3 pufa (reitan barsanti and gualtieri, 2005). salinity and ph (t6) shocks together on day 15 and combination of ph and salinity and starvation (t2) shocks on day 18 induced dha production in n. oculata at the end of dark period, however, ph shock alone did influence the cell epa level to more than 47% of fame on day 15 and 63% of fame on day 18 at the end of the dark period. conclusion in conclusion based on the results for high lipid production (more than 51% of whole-body weight), it is recommended to keep the n. oculate in salinity shock for 6 days (day 18) and harvest at the end of dark period. our data indicate that high dha (7.63% of fame) is obtained at the end of the dark period on day 18 and epa level (up to 63% of fame) is improved under ph shock at the end of light period on day 18 (incubate under shock condition for 6 days). while to obtain high level of total carotene (more than 1825 µg/g fw), it is recommended to apply ph + starvation + salinity shock and harvest the algae at the end of the dark period on day 18 (6 days shock). finally, in this research, the highest value of carbohydrates was produced under ph + salinity + starvation shock at the end of the dark period on day 18 (6 days shock). references ahlgren g., lundstedt l., brett m., forsberg c. 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(2017) 5(1): 33-39; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article bioaccumulation of heavy metals in eight fish species of the khur-e khuran international wetland in the persian gulf, iran mohsen dehghani1 department of environmental science, islamic azad university bandar abbas branch, bandar abbas, iran. article history: received 5 august 2016 accepted 11 january 2017 available online 2 5 february 2017 keywords: bioaccumulation persian gulf wetland heavy metals khur-e khuran abstract: heavy metals are among the most toxic pollutants in aquatic ecosystems. this study was conducted to evaluate the concentration of heavy metals viz. lead, cadmium, chromium, zinc, cobalt, and copper in eight commercial fishes of khur-e khuran international wetland (kiw), iran. the results showed that the highest concentration of heavy metals attributed to zn as 176.5 µg g-1 dry weight in platycephalus indicus and the lowest to pb as 0.12 µg g-1 dry weight in sillago sihama. average concentrations of heavy metals in eight examined fish species were 29.15, 49.73, 1.39, 0.45, 1.43 and 1.56 µg g-1 dry weight for cu, zn, cd, pb, co and cr, respectively. the results also show that measured values of most heavy metals in some examined fishes of kiw were higher than those maximum permissible limit (mpl) according to international standards. the high concentrations of some metals in some examined fishes of kiw may be due to industrial and residential activities in adjacent coast i.e. in mainland and qeshm island, and marine traffic introduction the importance of aquatic ecosystems is significant due to providing irreplaceable habitats for maintaining the biodiversity (macfarlane and burchettt, 2000). therefore, knowledge on disturbances of these ecosystems is crucial for their protection (storelli et al., 2005). in this regard, understanding the sources and concentrations of pollutants in aquatic environments and their effects requires their monitoring, and evaluation of contamination rate by analysis of water, sediment and organisms (ochieng et al., 2007). heavy metals are persistent pollutants and due to the biological toxicity, are not biodegradable (de mora et al., 2004). these elements enter into the aquatic environment from different sources such as municipal and industrial sewage, leaching and carrying toxic chemicals by water from lands, agricultural activities and atmospheric deposition (sekhar et al., 2003). the amount of these pollutants is much higher in aquatic organisms than the * corresponding author: mohsen dehghani e-mail address: dehghani933@gmail.com surrounding environment due to accumulation and bio-magnification. since, many marine species are consumed by human, therefor, knowing the normal levels of metals, or at least their regular concentrations in the marine environment is necessary to identify and evaluate heavy metals’ pollution (ruelas-inzunza and paez-osuna, 2000; castro-gonzalez et al., 2008). aquatic organisms, especially fishes, are in the food basket of human as valuable protein source and it is estimated providing 15-20% of animal protein (fao, 2010). however, aquatic organisms can be contained high levels of some heavy metals that may be risky for human consumption. fish are relatively situated at the top of the aquatic food chain; therefore, they can accumulate heavy metals (yilmaz et al., 2007; vinodhini and narayanan, 2008; zhao et al., 2012); i.e. they generally have a large potential to accumulate biological environmental pollutants (türkmen et al., 2009). age, length, weight, sex, nutritional habits, ecological requirements, and 34 dehghani/ bioaccumulation of heavy metals in some fishes of kiw seasonal changes in chemical properties of water such as salinity, hardness, temperature, concentration of heavy metals in water and sediment are among the effective factors in accumulation of heavy metals in different organs of fishes (demirak et al., 2006). therefore fishes are also used as a biological indicator to determine the effect of heavy metal pollution in aquatic ecosystems (fernandes et al., 2008). in addition, fish species can be used to assess the health of aquatic ecosystems (begüm et al., 2005). wetlands are ecologically important due to their hydrologic attributes and their role as an ecotone between terrestrial and aquatic ecosystems (pascoe, 1993). there are three main sources of risk that are commonly considered in wetlands, including heavy metals and non-metal elements, such as cd, cr, cu, hg, ni, pb, zn, as, bo, and se (pollard et al., 2007; nabulo et al., 2008; bai et al., 2010); organic pollutants, such as oil compounds and pesticides (ji et al., 2007; rumbold et al., 2008; gao et al., 2009), and natural parameters such as water availability and salinity (speelmans et al., 2007; sun et al., 2009; xie et al., 2011). khur-e khuran international wetland (kiw) is a narrow strait separating the qeshm island from the iranian mainland and it is one of the valuable coastal marine areas in the persian gulf. many residents live on the coast of this wetland. the residents of the region are highly dependent on the marine environment and its resources, especially fishes. this wetland has high ecological importance as a unique aquatic ecosystems due to its shallow water and muddy bed that is suitable for spawning and rearing larva of many marine fishes. therefore, this study aimed to evaluate the concentration of heavy metals in valuable commercial fishes of kiw. materials and methods study area: kiw is located between 27-00 and 2640 north latitude, 52-55 and 55-21 east longitude and between the khamir port and qeshm island in the southern coast of iran (fig. 1). the area now is protected as "hara protected area" managed by the department of environmental protection of hormozgan province. a part of this coastal-marine region, the hara protected area was registered in ramsar convention (1975) as khur-e khuran international wetland including an area of 100000 hectares (hde, 2001). sampling and measurement: three area in the southern, northern and central parts of kiw was determined on 2010 tm satellite images as sampling stations. then, samplings were performed by a motorized boats equipped to trawl, on january 2012. among the 21 caught fish species, 8 species including lutjanus johni, pomadasys furcatus, platycephalus indicus, anodontostoma chacunda, epinephelus coioides, sillago sihama, plectorhinchus pictus and pampus argentus were selected as valuable food fishes for further examinations. in total 96 fishes, 12 specimens of each selected species, were collected and transferred to the laboratory, where the samples from their muscle were removed and dried in a freeze dryer (alam et al., 2002). then 8 ml nitric acid (65%) was added to1 gram of the dried muscle tissue of each sample and left in room temperature for initial digestion for 12 hours. for the final digestion, 3 ml of perchloric acid (70%) was added to each sample and put on a sand bath at 160°c based on storelli and marcotrigiano (2005). the obtained samples within each flask was then cooled in room temperature and diluted up to 25 ml with double distilled water. the diluted sample was filtered using 0.45 micrometers filter paper. the figure 1. location of the khur-e khuran international wetland. 35 int. j. aquat. biol. (2017) 5(1): 33-39 concentration of heavy metals, including pb, cd, cr, co, cu and zn were analyzed using graphite furnace atomic absorption spectrometer (elmer perkin) model was used (moopam, 1983). standard reference material (dorm-2 national research council, canada) was employed to control experiment quality and data validation, and measurements revealed 95% retrieval of all standard samples. metal concentrations were expressed as μg g-1 of dry weight. statistical analysis: data analysis was performed using spss 17. the mean of the heavy metals in fishes were compared using one-way anova with significance level of 0.05. all data were tested for the homogeneity of variances and normality; the data which were not normally distributed or not homogeneous were transformed. results and discussion the concentration of heavy metals in examined species are presented in table 1. the lowest and highest concentration of cr were observed in p. pictus (1.06 µg g-1 dry weight) and a. chacunda (1.91 µg g-1 dry weight), respectively. the highest concentration of cu was 136.00 µg g-1 dry weight in p. indicus, and the lowest as 2.13 µg g-1 dry weight in e. coioides. maximum concentration of zn was found in p. indicus (176.5 µg g-1 dry weight) and its minimum in a. chacunda (18.35 µg g-1 dry weight). the maximum and minimum concentration of co was 3.68 and 0.56 µg g-1 dry weight in p. indicus, and s. sihama, respectively. the highest and lowest concentrations of cd were found in p. indicus (2.58 µg g-1 dry weight) and l. johni (0.50 µg g-1 dry weight), respectively. in addition, pb had the highest concentration in e. coioides (0.91 µg g-1 dry weight). accumulation patterns of all metals were significantly different (p<0.05) between the different species (table 2). the correlation coefficients between heavy metals are shown in table 3. a strong correlations were find between cu/cr, cd/cr, cd/co, pb/cr and pb/cd. order of the accumulated heavy metals in the muscle of studied fishes was as pb < cd < cr < co < cu < zn. the highest combined concentration of zn, cu, cr, co, cd and pb was recorded in p. indicus (176.50), p. indicus (136.00), a. chacunda (1.91), p. indicus (3.68), p. indicus (2.58) and e. coioides (0.91) µg g-1 dry weight, respectively. the combined average concentration of heavy metals in examined species table 1. average (±sd) concentrations of heavy metals in muscle of examined fish species (µg g-1 dry weight) species cr cu zn co cd pb pampus argentus 1.80±0.27 15.23±2.35 30.00±4.32 1.38±0.33 1.87±0.25 0.08±0.1 sillago sihama 1.65±0.24 60.00±8.45 67.50±9.12 0.56±0.14 1.99±0.28 0.12±0.05 pomadasys furcatus 1.15±0.19 4.88±0.98 24.42±5.17 1.01±0.19 0.75±0.16 0.45±0.09 lutjanus johni 1.69±0.23 3.63±1.22 20.50±4.28 0.75±0.17 0.50±0.16 0.35±0.09 epinephelus coioides 1.44±0.20 2.13±0.87 40.00±8.82 1.01±0.24 1.96±0.31 0.91±0.18 plectorhinchus pictus 1.06±0.14 5.63±1.75 20.55±5.25 2.01±0.19 0.60±0.17 0.48±0.07 anodontostoma chacunda 1.91±0.54 5.75±1.32) 18.35±4.93 1.05±0.20 0.58±0.13 0.76±0.12 platycephalus indicus 1.83±0.47 136.00±26.55 176.5±37.71 3.68±0.89 2.58±0.75 0.44±0.08 table 2. the results of one-way anova showing differences in heavy metals’ concentrations between the examined species. metal df f sig. cr 7 48 462.784 0.000 cu 62.288 0.000 zn 124.312 0.000 co 901.115 0.000 cd 540.084 0.000 pb 440.096 0.000 table 3. pearson correlation between the heavy metals values of the examined fishes. cr cu zn co cd pb cr 1 cu 0.74 1 zn 0.47 -0.11 1 co 0.07 0.35 0.29 1 cd 0.73 0.47 0.38 0.82 1 pb 0.65 0.34 0.09 0.38 0.87 1 36 dehghani/ bioaccumulation of heavy metals in some fishes of kiw are shown in figure 2. table 4 shows heavy metal concentrations in the muscle tissues from different regions of the world. for example, oriyan et al. (2011) reported the concentration of pb in muscle tissues of p. argentus to be 0.3±0.29 ppm which is lower than the result of this study. nabizadeh and pourkhabbaz (2011) reported cr concentration in p. indicus and s. sihama as 0.6 and 0.72 µg g-1, whereas its concentration were 1.83 and 1.65 µg g-1 in the present study, respectively. khazaei et al. (2013) reported the amount of cu in muscles of p. argentus (3.30 ppm) that was more than that one in e. coioides (2.13 ppm) (khazaei et al., 2013). in addition all reported values in de mora et al. (2004) are lower than those obtained in the present study. accumulation of heavy metals in living organisms is effected by absorption rate of metals and metabolism rate of the living organism. difference in concentrations of heavy metals in fishes depends on a variety of factors such as feeding behavior (obasohan and oronsaye, 2004), distance to the pollution source (barlas, 1999), age, length table 4. heavy metal concentrations in the muscle tissues from different regions of the world. reference cr cu zn co cd pb µg g-1 dry weight de mora et al. (2004), qatar coast 0.03 0.56 8.2 <0.005 0.013 0.113 de mora et al. (2004),uae coast 0.01 0.37 13.5 0.014 <0.001 0.025 de mora et al. (2004), bahrin coast 0.013 0.235 15.9 <0.01 0.001 0.028 de mora et al. (2004), oman coast 0.072 0.513 13.4 <0.05 <0.005 0.025 oriyan et al. (2009) (pampus argentus) 0.05 0.29 askari sari et al. (2011) (otolithes ruber) 121.4 0.98 askari sari et al. (2011) (scomberomorus guttatus) 33.7 0.41 nabizadeh and pourkhabbaz (2011) (sillago sihama) 0.72 0.5 0.76 nabizadeh and pourkhabbaz (2011) (platycephalus indicus) 0.6 0.39 0.73 gorur et al. (2012) (thunnus albacares) 4.00 42.00 1.20 4.70 khazaei et al. (2013) (pampus argentus) 3.30 14.44 0.22 elnabris et al. (2013) (8 commercially fishes) 2.62 2.90 2.73 2.78 el-moselhy et al. (2014) (epinephelus sp.) 0.29 2.42 0.12 0.88 el-moselhy et al. (2014) (thunnus albacares) 0.35 1.99 0.06 0.32 khaled (2004) (siganus rivulatus) 2.70 43.90 2.80 1.20 the present study 1.56 29.15 49.73 1.43 1.39 0.45 figure 2. average heavy metals concentrations in 8 examined species fish khur-e khuran international wetland. 37 int. j. aquat. biol. (2017) 5(1): 33-39 and weight (benson et al., 2007) and type of habitat (saei-dehkordi and fallah, 2011). based on the results, the average concentration of pb in the examined fishes (0.45 µg g-1 dry weight) was lower than standards of who (cheung et al., 2008) and fda (2001) and higher than those of iaea-407, unep (1985) and tfc (2002) and for cd with an average of 1.39 µg g-1 dry weight was more than the standards given in table 5. iaea-407 and who standards for cr are 0.73 and 0.15 µg g-1 dry weight, respectively and its values in the examined fishes were less than these standards. in addition, cu values obtained in the current study (29.15 µg g-1 dry weight) were less than of fao/who standards. finally, that of zn was more than maximum permissible limit based in fao/who (1989) and maff (2000) standards. the results show that measured values of heavy metals in this study are higher than those maximum permissible limit (mpl) according to international standards (table 5). the high concentrations of some metals in examined fishes of kiw may be due to industrial and residential activities in adjacent coast i.e. in the mainland and qeshm island, and marine traffic that directly influence coastal waters by releasing pollutants. furthermore, benthic species have higher rate of heavy metal concentrations due to absorbing pollutants from sediments in addition to water column and food as seen in p. indicus in the present study. references alam m.g.m., tanaka a., allinson g., laurenson l.j.b., stagnitti s. and snow e.t. 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(2017) 5(1): 33-39 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی تجمع فلزات سنگین در برخی از ماهیان تاالب بین المللی خورخوران در خلیج فارس، ایران محسن دهقانی .ایران ،بندرعباس ،بندرعباسآزاد اسالمی واحد دانشگاه ،علوم دریایی و طبیعی منابع ، دانشکدهمحیط زیست گروه چکیده: سرب، کادمیوم، این مطالعه به منظور ارزیابی غلظت فلزات سنگین .آبی هستند يهااکوسیستم در سمی يهاآالینده ترینمهم سنگین جزو فلزات ن در بین فلزات سنگین بیشتریکه نتایج نشان داد خوران ایران انجام شد. گونه از ماهیان تجاري تاالب بین المللی خور 8 در کبالت و مس روي، کرم، 12/0و کمترین غلظت مربوط به فلز سرب با platycephalus indicus میکروگرم در گرم وزن خشک در گونه 5/176غلظت مربوط به فلز روي با غلظت فلزات سنگین در هشت گونه مورد مطالعه براي مس، روي، باشد. میانگین می sillago sihamaمیکروگرم در گرم وزن خشک در گونه دهد نتایج همچنین نشان میمیکروگرم در گرم وزن خشک بود. 56/1و 43/1 ،45/0، 39/1، 73/49، 15/29ترتیب کادمیوم، سرب، کبالت و کرم به االب بین المللی خورخوران با توجه به استانداردهاي هاي مورد مطالعه در تماهیبرخی گیري شده در اغلب فلزات سنگین در که مقادیر اندازه هاي صنعتی و مسکونی ها ممکن است ناشی از فعالیت( بود. غلظت باالي برخی از فلزات در ماهیmplالمللی باالتر از حداکثر حد مجاز )بین هاي دریایی در سواحل مجاور در سرزمین اصلی و جزیره قشم باشد.همچنین تردد .خورانتجمع زیستی، خلیج فارس، تاالب، فلزات سنگین، خور :کلیدیکلمات int. j. aquat. biol. (2013) 1(2): 76-81 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology effect of dietary tryptophan and betaine on tolerance of caspian roach (rutilus rutilus caspicus) to copper toxicity sajjad fatahi1, seyyed morteza hoseini *1 1 department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 10 april 2013 accepted 1 may 2013 available online 6 may 2013 keywords: tryptophan betaine copper nutrition toxicity abstract: the present study investigated effects of dietary tryptophan (trp) and betaine (bet) on copper (cu) toxicity tolerance in the caspian roach (rutilus rutilus caspicus). the caspian roach fingerlings were fed diets containing 0, 0.25 and 0.5% trp or 0, 0.5 and 1 % bet and combination of trp and bet. specimens were exposed to cu (0.8 ppm) after either 30 or 60 days feeding. mortality was recorded in each treatment 48 h after the cu exposure. trp decreased significantly fish mortality. however, bet had no significant effect on fish mortality. specimens fed on the diet supplemented with 0.25% trp had the lowest mortality among the treatments. it is concluded that 0.25% trp reduces mortality of caspian roach during cu exposure. introduction copper (cu) is an essential microelement for living organism; however, its higher concentrations are very toxic to aquatic life. cu naturally exists in all aquatic environments, however, multiple anthropogenic activities may result in elevated concentrations, increased exposure, and potential toxicity to aquatic organisms (grosell et al., 2007). cu may be introduced into water bodies from industrial waste or through the use of copper sulfate as therapeutic or algaecide. cu toxic effects in fish are related to gill damage and hydromineral imbalance, gas exchange disturbance (adhikari, 2003; evans et al., 2005, grosell et al., 2007), stress response (flik et al., 2002; fırat et al., 2011; hoseini and hosseini, 2012) and oxidative damages (roméo et al., 2000). previous studies revealed the cu contamination of the caspian sea (agah et al., 2006; abtahi et al., 2007; taghipour and azizi, 2011). caspian roach is one of the valuable species inhabits in caspian sea. * corresponding author: seyyed morteza hoseini e-mail address: seyyedmorteza.hoseini@gmail.com tel: +989112750713 recently, the roach population decreased in the caspian sea due to over fishing, degraded habitat and pollution (hoseini and jafar nodeh, 2012). for restocking purpose, the caspian roach fries and fingerlings (0.5-3 g in weight) are released into the rivers associated with the caspian sea. caspian roach may challenge with cu toxicity, when it is released into the caspian sea. thus it would be of interest to increase its resistance to cu intoxication. nutritional manipulation is a useful means for increasing fish tolerance to cu toxicity (hoseini and hosseini, 2012). dietary tryptophan (trp) can suppress the toxic effects of cu (hoseini and hosseini, 2012). trp is precursor of serotonin (5hydroxytryptamine, 5-ht) with a stress-relieving effect (lepage et al., 2002; hseu et al., 2003; hosseini and hoseini, 2013). lepage et al. (2003) showed that the brain levels of 5-ht is accompanied with an increase in trp intake whereas de boeck et al. (1995) found that the brain levels of 5-ht decreased as a result of cu exposure in common carp 77 fatahi and hoseini/ int. j. aquat. biol. (2013) 1(2): 76-81 (cyprinus carpio) suggesting the involvement of 5ht in cu toxicity. on the other hand, previous studies (lepage et al., 2002; tejpal et al., 2009; hoseini and hosseini, 2010) showed stress mitigation as a result of dietary trp in different fish species. christen et al. (1990), weiss et al. (2002) and reyes-gonzales et al. (2009) reported antioxidant properties of trp. betaine (bet) is an important compound in living organisms with two main biological roles: methyl donation and osmoprotection. it protects intracellular enzymes against osmoticallyor temperature-induced inactivation (yancey et al., 1982). also, it has been shown that bet may mitigate stress response related to endosulfan exposure in labeo ruhia (kumar et al., 2012). considering the aforementioned roles of trp and bet, these two compounds can increase the tolerance to cu intoxication in the caspian roach. thus, the aim of the present study was to investigate effect of dietary trp and bet on survival of caspian roach exposed to water cu. materials and methods fish and maintenance conditions: a total of 1080 fish (1.91± 0.01 g) were randomly distributed among 18 tanks (400 l). fish were fed control diet (table 1) for 2 weeks to acclimatize the experimental conditions. thereafter, the experimental tanks were assigned to nine treatments (each had two tanks). the treatments were fed diets supplemented with trp and bet as follow (amount in percent of diet): trp = 0-bet= 0 (control), trp = 0-bet = 0.5 (trp0-bet0.5), trp = 0-bet = 1 (trp0-bet1), trp = 0.25 –bet = 0 (trp0.25-bet0), trp = 0.25-bet = 0.5 (trp0.25-bet0.5), trp = 0.25bet = 1 (trp0.25-bet1), trp = 0.5-bet = 0 (trp0.5-bet0), trp = 0.5-bet = 0.5 (trp0.5bet0.5) and trp = 0.5-bet = 1 (trp0.5-bet1). the specimens were fed three times a day, based on 4% of fish wet weight. all tanks were continuously aerated throughout the trial. water was replaced at a rate of 60%, every day. water temperature, ph, dissolved oxygen and total hardness were measured weekly while were 23±1 ºc, 7.7±0.1, > 6 ppm and 180 ppm (caco3), respectively. dissolved oxygen and ph were measured using portable multitable 1. control diet composition. ingredients % fish meal 29 soybean meal 32.9 wheat meal 14 barely meal 14 fish oil 5 phytase 0.1 mineral mix 1 vitamin mix 1 methionine 1 lysine 1 d.c.p 1 proximate composition % moisture 14 crude protein 33.9 crude lipid 7 ash 4.99 figure 1. copper-induced mortality in the caspian roach fed with diet containing different levels of tryptophan, over 30 d. different letters show significant difference among the treatments. figure 2. copper-induced mortality in the caspian roach fed with diet containing different levels of tryptophan, over 60 d. different letters show significant difference among the treatments. 77 78 fatahi and hoseini/ int. j. aquat. biol. (2013) 1(2): 76-81 parameter meter (sension 156, usa). water total hardness was measured using portable photometer with commercial kits provided by the manufacturer (wagtecch portable photometer 7100, berkshire, uk). copper toxicity test: after 30 and 60 days of feeding, fish were exposed to 0.8 ppm cu (as copper sulfate) and survival rate was recorded after 48 h. to expose fish to cu, 30 fish were removed from each treatment and placed in three plastic tanks (20 l). all tanks were aerated continuously. cu was added to the tanks after a stock solution preparation. fish were not fed during the cu exposure. mortality was checked every 6 h until 48 h. dead fish were removed immediately. statistical analyses: data were analyzed using two way anova and duncan test to examine the effects of trp and bet on survival, at each time, separately. data are presented as mean ± se. p<0.05 considered as significant difference. all analyses were performed using statistical software spss 18. results there was no mortality during the feeding trials. results showed that fish survival during cu exposure was significantly (p<0.0001) affected by dietary trp, in both 30 (table 2) and 60 d (table 3) tests. mortality of fish fed different diets is presented in table 4 and figures 1 and 2. generally, fish fed with 0.25 trp had lower mortality during cu exposure, compared to other treatments, after both feeding trials. after 30 d feeding, fish fed with 0.25% trp had significantly lower mortality compared to the other treatments, however, there was no significant difference between the control group and fish fed on the diet supplemented with 0.5% trp (fig. 1). source type iii sum of squares df mean square f sig. corrected model 114 8 14.25 12.41 < 0.0001 intercept 385.3 1 385.33 335.6 < 0.0001 trp 96.8 2 48.44 42.19 < 0.0001 bet 4.66 2 2.33 2.03 0.16 trp × bet 12.44 4 3.11 2.70 0.063 error 20.66 18 1.14 total 520 27 corrected total 134.6 26 r squared = 0.847 (adjusted r squared = 0.778) table 2. two-way anova for survival of the caspian roach exposed to water copper and fed with tryptophanand betaine-supplemented diet, over 30 d. source type iii sum of squares df mean square f sig. corrected model 7496.2 8 937.07 4.68 0.003 intercept 23703.7 1 23703.7 118.5 < 0.0001 trp 6607.4 2 3303.7 16.51 < 0.0001 bet 474.0 2 237.0 1.18 0.32 trp × bet 414.8 4 103.70 0.51 0.72 error 3600 18 200 total 34800 27 corrected total 11096.3 26 r squared = 0.676 (adjusted r squared = 0.531) table 3: two-way anova for survival of the caspian roach exposed to water copper and fed with tryptophanand betaine-supplemented diet, over 60 d. 79 fatahi and hoseini/ int. j. aquat. biol. (2013) 1(2): 76-81 after 60 d feeding, fish fed with the 0.25% trp diet had significantly lower mortality than the control fish and those fed with the diet containing 0.5% trp treatments (fig. 2). also, fish in 0.5% trp group had significantly lower mortality compared to the control group (fig. 2). discussion the present study showed that the caspian roach is susceptible to cu intoxication. considering the cu contamination of southern coast of the caspian sea, using dietary trp can be a useful way to increase caspian roach survival during stock rehabilitation. the reported level of cu in the southern coast of the caspian sea and its tributaries is near 10% of lc50 96 h for the caspian roach (de mora et al., 2004; khakpour et al., 2009; hoseini and jafar nodeh, 2012), however, it could cause chronic toxicity and threatens fish health (hoseini and jafar nodeh, 2012). the present study showed that dietary trp could increase tolerance to cu intoxication in caspian roach. the result is in line with the previous study on common carp (hoseini and hoseini, 2012). there are several potential reasons explaining how trp enhanced the tolerance to cu intoxication. it was reported that cu exposure is stressful to fish (flik et al., 2002; firat et al., 2011; hoseini and hosseini, 2012). trp have been reported to mitigate stress response in several fish species, via serotonergic system activation (aldegunde et al., 1998, 2000; winberg et al., 2001; lepage et al., 2002; hseu et al., 2003; hoseini and hosseini, 2010; hoseini and hosseini, 2012). thus, one of the potential reasons for higher tolerance to cu intoxication in trp-fed groups might be mitigation of stress induced by cu. on the other hand, de boeck et al. (1995) reported that cu exposure decreased significantly the brain serotonin level and serotonergic system disruption. trp may counteract this phenomenon to increase fish survival during cu exposure, as winberg et al. (2001), lepage et al. (2002) and hseu et al. (2003) found an increase in brain serotonin content in trpfed fish. likewise, cu causes oxidative stress in fish (roméo et al., 2000), thus, some protective effect of trp during cu toxicity might be related to its antioxidant properties, as previously reported by other authors (christen et al., 1990; weiss et al., 2002; keithahn and lerchl, 2005; reyes-gonzales et al., 2009). in the present study, increase of dietary trp from 0.25 to 0.5% influenced the fish tolerance to cu toxicity adversely, which may be related to dietary amino acid imbalance and nutritional stress. previous studies showed that high dietary trp caused poor growth and physiological condition in other fish species (papoutsoglou and koustas, 2005; papoutsoglou et al., 2005). dietary requirement for trp in caspian roach is unknown which make it difficult to discuss about amino acid imbalance in 30 d 60 d trp bet mean pooled s.e. trp bet mean pooled s.e. 0 0 63.3 c 6.18 0 0 46.6 cd 7.93 0.5 50.0 bc 6.18 0.5 56.6 d 7.93 1 46.6 bc 6.18 1 36.6 bcd 7.93 0.25 0 3.33 a 6.18 0.25 0 6.66 a 7.93 0.5 16.6 a 6.18 0.5 10.0 ab 7.93 1 13.3 a 6.18 1 10.0 ab 7.93 0.5 0 63.3 c 6.18 0.5 0 23.3 bc 7.93 0.5 43.3 bc 6.18 0.5 40.0 cd 7.93 1 40.0 b 6.18 1 33.3 bcd 7.93 table 4. mortality (%) of the caspian roach exposed to water cu and fed with tryptophanand betaine-supplemented diet, over 30 and 60 d. different letters show significant difference among the treatments, after 30 and 60 d, separately. 79 80 fatahi and hoseini/ int. j. aquat. biol. (2013) 1(2): 76-81 fish fed with 0.5% trp. in the present study, bet had no benefit for fish exposed to cu. to our knowledge, there is no study in this topic to be compared with the present results. kumar et al. (2012) found benefit of bet in endosulfan-exposed l. ruhita. the author suggested that bet could suppress stress and energy expenditure in endosulfan-exposed fish. difference in fish species and experimental condition could potentially explain such contradictories between the two studies. in conclusion, dietary trp, especially 0.25%, can elevate tolerance to cu intoxication in caspian roach. trp supplementation may have benefit in stock rehabilitation program of the caspian roach, where fish may face cu in the sea, rivers and rearing ponds. references abtahi b., ghodrati shojaii m., esmaili sari a., rahnema m., sharif pour i., bahmni m., kazemi r., hallajian a. 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(2017) 5(4): 246-251; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article quality evaluation of the invader species, artemia franciscana from covelong salt works, kelambakkam, south india krishnakumar velayudhannair1, 3*, k.r. divya2, natesan munuswamy3 1department of life sciences, christ university, hosur road, bangaluru – 560 029, india. 2the key laboratory of aquatic biodiversity and conservation, institute of hydrobiology, chinese academy of sciences, wuhan430072, china. 3unit of aquaculture and cryobiology, department of zoology, university of madras, guindy campus, chennai – 600 025, india. article history: received 6 march 2017 accepted 24 july 2017 available online 2 5 august 2017 keywords: artemia fransciscana biometrics hatching characteristics nutritional composition abstract: the quality of the invader brine shrimp, artemia franciscana, colonized in covelong salt works, kelambakkam (south india) was evaluated based on biometrics, hatching characteristics and nutritional composition of cyst and freshly hatched nauplii. hydrated cysts measured 238.31±10.42 μm in diameter, while the freshly hatched nauplii measured about 429.00±12.19 μm in length. a hatching of 76.80±5.68% was obtained with hydrated cysts. time required to obtain 90% hatching was 22.31 hrs for this strain. the hatching synchronization time was of 4.41 hrs. the proximate composition of the decapsulated cysts and newly hatched nauplii showed high level of protein, energy content and low ash content. fatty acids analysis of the freshly hatched nauplii indicated low level of epa and absence (or undetectable) of dha. introduction providing nutritionally adequate diet is a major problem in finfish and shellfish industries, especially at hatchery level. the main thrust of basic fish and crustacean research, with regard to nutrition, is focused on ensuring efficient utilization of feed for their growth (rao, 1994). in the natural environment, the young ones of marine fishes have less yolk reserves, compared to freshwater species, which are depleted rapidly. also, the mouth of marine fish fry is relatively small (lavens and sorgeloos, 1996) and they can only feed on the pray of appropriate size. many commercially important marine fish species have a reproductive strategy that results in larvae of altricial type. altricial larvae require good amount of nutrients, the functional capacity and the developmental status of the larval digestive system is sufficient to support growth with live diets from first feeding onwards (govoni et al., 1986). altricial larvae having rudimentary digestive system (conceicao et al., 2010; wold et al., 2007) which makes them dependent on live food, such as artemia and rotifers, *corresponding author: krishnakumar velayudhannair doi: https://doi.org/10.22034/ijab.v5i4.316 e-mail address: krishnakumar.v@christuniversity.in during their early stages of development (rehberghass et al., 2015). because of essential growth factors present in natural prays, they promotes growth, high survival, conversion efficiency and enhanced immune response in predators in natural environment (awaiss et al., 1992). although there are several live food organisms such as tubifex sp., brachionus sp., daphnia sp., artemia sp., and moina sp. used in shellfish and finfish hatcheries, the brine shrimp, artemia nauplius is universally accepted live feed in both finfish and shellfish hatcheries, because of their ‘on demand’ character, nutritional efficacy and mainly on the knowledge accumulated to improve the hatching quality of the harvested cysts (sorgeloos et al., 1986). due to increasing demand and various climatic phenomenons such as el-nino, the global availability of artemia cyst reduced drastically and the price rose exponentially (lavens and sorgeloos, 2000). this condition leads to the diversification of artemia resources worldwide. however, the cyst quality in terms of hatching rate and nutritional values proved to 247 int. j. aquat. biol. (2017) 5(4): 246-251 be highly variables, among, not only various strains and species of artemia sp., but also different groups from the same location (vanhaecke and sorgeloos, 1982). efforts were made for the mass production of artemia cyst and biomass in man-made saltpans at covelong salt works, kelambakkam, tamilnadu, india for hatchery operations. a total of 80 kg of cysts were harvested successfully in one hector of artemia culture pond (krishnakumar, 2012). the present study was undertaken to evaluate the quality of harvested cysts and nauplii in terms of its size, hatching characteristics and nutritional composition of artemia franciscana (krishnakumar, 2012, sivagnanam et al, 2011), colonized in covelong salt works, kelambakkam. materials and methods sampling site and cyst collection: the brine shrimp cysts were collected from covelong salt works, kelambakkam, southeast coast of india. the covelong salt works, kelambakkam is one of the major salt manufacturers in tamilnadu. it covers an area of 1307 acre. this saltpan is divided in to series of salt ponds, such as reservoir, evaporation and crystallizers. this salt pan is sourced sea water from bay of bengal through backwater opened near muttukadu. salt is produced by evaporating the seawater using sunlight. the distribution of artemia was restricted in evaporation only and no artemia population was recorded in reservoir ponds (krishnakumar, 2012). an evaporation pond was chosen for mass production of artemia biomass and cyst. the pond was modified and care was taken to prevent the entry of predators in to the pond. the pond was filled with 80 ppt of salinity, rich with algae. locally available cysts were used for artemia inoculation in the culture pond. once the biomass increased in the culture pond, salinity was increased by pumping high salinity water (>180 ppt) in to the culture to induce cyst production. immediate next cysts were found floating near the shore. the floating cysts were harvested using 200 μm pore sized scoop net from the culture pond. the collected cyst were cleaned free from other debris by sieving them using different pore sized meshes (300, 250 and 200 μm) and soaked in brine solution (of >180 ppt of salinity for about 1 hr) and in freshwater (0 ppt salinity), and then treated as described by sorgeloos et al. (1986). the cysts were layered and dried in a thermostatically controlled oven at around 35°c. biometry and cyst hatching: the diameter of one hundred fully hydrated (soaked in seawater for 1 hr) and decapsulated cysts were measured using an ocular micrometer (erma) calibrated with stage micrometer fitted in compound microscopes. the hydrated cysts were decapsulated according to sorgeloos and kulasekarapandian (1984). for hatching, the cysts were incubated in filtered seawater of 35 ppt salinity at 30°c under continuous illumination (~1000 lux). hatching characteristics such as hatching percentage, hatching efficiency and hatching rate were determined as described by sorgeloos and kulasekarapandian (1984) and vanhaecke and sorgeloos (1982). hatching was performed in 100 ml conical glass tubes, with three replicates. proximate analysis: proximate analysis of decapsulated cyst and nauplii was carried out following the standard methods of aoac (2000). nitrogen content of the sample was analysed using kjeltec system (2100 kjeltec auto distillation, foss tecator, sweden) and crude protein (cp) was calculated by multiplying nitrogen percentage by 6.25. ether extract (ee) was determined using soxtec system (1045 soxtec extraction unit, foss tecator, höganäs, sweden) using diethyl ether (boiling point, 92°c for 90 min) as a solvent and ash content was estimated by incinerating the sample in a muffle furnace at 600°c for 12 hrs. total carbohydrate (tc) was calculated by differences as tc=100-(cp+ee +ash). fatty acid profile was determined as fatty acid methyl esters (fames). the fames were prepared according to the method of aoac (2000). the prepared methyl ester was injected to the gas chromatography (gc-ms, gc: agilent technologies; model 6890n; germany and ms: agilent technologies; model l 5973; usa) equipped with the flame 248 velayudhannair et al./ quality evaluation of artemia from kelambakkam, south india ionisation detector (fid) at a split ratio of 1:20. a fused silica capillary column (30.0 m x 250 µm x 0.25 µm), coated with bonded polyglycol liquid phase, was used. the analytical conditions were: injection port temperature of 250°c and detector temperature of 250°c. the oven was programmed from 170°c to 225°c at a rate of 1°c/min. retention times of fame standards were used to identify chromatographic peaks of the samples. fatty acid content was calculated, based on the peak area ratio and expressed as mg fatty acid/ g oil. statistical analysis: all the data were subjected to statistical analysis (one-way anova and duncan) using spss statistical software (version 11.5 for windows, spss, chicago, il, usa). limits of significance for all critical ranges were set at p<0.05. results mean diameter of hydrated and decapsulated cysts were measured 238.31±10.42 μm and 224.07±12.77 μm, respectively, while the nauplii had a mean length of 429.00±12.19 μm (table 1). as much as 76.80% of hatching was obtained prior to decapsulation and the percentage of hatching increased significantly, after decapsulation, up to 88.96% with the difference of 5.16%. the best hatching efficiency obtained was 221078.60±12829.33 nauplii g-1. hatching rate revealed that the first appearance of nauplii (t0) was observed on 15.35 hrs of incubation and 90% of hatching was obtained (t90) after 21.31 hrs after incubation under normal conditions (30 ppt and 30°c) (table 2). proximate composition of decapsulated cysts showed high level of protein, lipid, carbohydrate, energy content and low ash content compared to freshly hatched nauplii. the protein and carbohydrate amounted to 54.13 and 18.85% respectively in cysts and 52.86 and 11.01% respectively in nauplii. lipid content registered in nauplii was high (15.46%) compared to cyst (14.23%). high level of carbohydrate and energy was recorded in cyst as 18.85±0.25 and 351.80±1.91 kcal 100g-1, respectively (table 3). a total of 18 fatty acids were recorded in freshly hatched artemia nauplii, 8 saturated and 10 unsaturated fatty acids. among the saturated fatty acids, palmitic acid and stearic acid were recorded high of 14.31 and 5.66 mg.g-1, respectively. lignoceric acid and lauric acid registered with minimum content of 0.07 and 0.11 mg.g-1, respectively. among the 10 unsaturated fatty acids, α linolenic acid and oleic acid amounted to be 33.36 and 31.01 mg.g-1, respectively and erucic acid was recorded with low amount of 0.05 mg.g-1 (table 4). discussion the brine shrimp artemia is a micro-crustacean, with a wide distribution over all continents except antarctica well-adapted to the harsh conditions that severely hypersaline environments impose on survival and reproduction (vanhaecke and sorgeloos, 1982). artemia is well-known as an invaluable live food for aquaculture (sorgeloos et al., 2001; john et al., 2004; ben naceur et al., 2012). due to the increasing aquaculture practices worldwide, demand for quality artemia cyst has been increased and price rose exponentially (lavens and sorgeloos, 2000). during 1980’s, parthenogenetic species of artemia was dominated in kelambakkam salt pans and the size of table 1. biometry of cyst and freshly hatched nauplii. measured parameters diameter/length hydrated cysts (μm) 238.31±10.42 decapsulated cysts (μm) 224.07±12.77 nauplii (μm) 429.00±12.19 (* mean±sd of 100 observations) table 2. hatching characteristics of artemia cysts from covelong salt works. characters kbm strain of artemia* hatching efficiency (no. nauplii/g) 221078.60±12829.33 hatching rate (hrs) t0 15.35±0.07 t10 17.91±0.51 t90 22.31±0.19 ts 4.41±0.44 hatching percentage (%) 76.80±5.68 t0: time until appearance of the first nauplii; t10: time until 10% hatching is attained; t90: time until 90% hatching is attained; ts (t90-t10): determination of hatching synchrony (* mean±sd of three observations) 249 int. j. aquat. biol. (2017) 5(4): 246-251 the nauplii was fairly larger to feed the developing shrimps larvae at hatchery level. but at present bisexual strain of artemia was a colonized in kelambakkam saltpans (sivagnanam et al., 2011). present study reveals that the diameter of cyst and the nauplii length are smaller and comparable to the san francisco bay strain. the cysts hatching characters such as hatching percentage, hatching rate were seemed to be depends on salinity, temperature and ph (krishnakumar, 2012). earlier experiments on the hatching characteristics revealed that maximum hatching percentage was obtained with 30 ppt salinity (data not shown here). the hatching efficacy of kelambakkam artemia population yielded about 221078 nauplii/g of cyst; which means that 4.52 g of cysts needed to get 1 million of artemia nauplii. kara et al. (2004) observed obtained 1 million nauplii from 5.5 g of cysts chott marouane (northeast algeria) strain of artemia. artemia cysts of kelambakkam begin to hatch after 15 hrs of incubation whereas sorgeloos et al. (1986) reported the time limits from 17.91 to 22.31 hrs. the hatching synchronization time was relatively small (4.41 hrs) and is still within the limits of 4.4–17.3 h as reported by sorgeloos et al. (1986). this synchronized condition; it is possible to collect most of the nauplli in early stage (i.e. first instar stage) before stage ii conversion. if the size of nauplii size and mobility increased, they lose 27% of their energetic value (lavens and sorgeloos, 1996), and become less accessible to predators. the proximate composition of artemia differed species to species and even location to location in the same area. protein content averaged 54.13% in kelambakkam population and was higher compared to 53.25% in san francisco bay strain of artemia (barrata et al., 1996), 58.4% in artemia from texococo lake in mexico (barrara et al., 1994), 52.974.04% in artemia from natural habitats in spain (gonzalbo et al., 1987), and in artemia from the great salt lake (utah) had 58.39% (gonzalbo et al., 1987). rich proximate composition was observed in artemia nauplii may because of the naturally existing algae of rich protein sources such as fresh spirulina (castro, 1993; leger et al., 1986). although wild artemia lacks an external protein source, their natural protein content might be originated from feed sources that thrive in their natural environment. the average lipid content of 14.23% was recorded with kelambakkam strain which was higher compared to pichilingue strain (7.78%) (naegel et al., 2002) and the lipid content varied from 6.45 to 14% for various populations of artemia determined (gonzalbo and table 3. proximate composition of the cyst and nauplii of kbm strain. proximate composition cyst nauplii total protein (dry wt. %) 54.13±3.16 52.86±2.56 total lipid (dry wt. %) 14.23±0.21 15.46±0.36 total carbohydrate (dry wt. %) 18.85±0.25 11.01±0.75 ash (dry wt. %) 5.98±0.14 9.79±0.43 energy (kcal/100g) 351.80±1.91 330.34±1.50 (mean±sd of five observations) table 4. : fatty acid content in freshly hatched artemia nauplii. fatty acids mg.g-1 c12:0 0.11 c14:0 0.98 c15:0 0.23 c16:0 14.31 c17:0 0.92 c18:0 5.66 c22:0 0.18 c22:0 0 c23:0 0 c24:0 0.07 c16:1n-7 3.16 c18:1n-9 31.09 c22:1n-9 0.05 c18:2n-6 7.71 c18:3n-3 33.36 c20:2 0.27 c20:3n-6 0.09 c20:3n-3 0.87 c20:4n-6 0.37 c20:5n-3 1.46 250 velayudhannair et al./ quality evaluation of artemia from kelambakkam, south india amat, 1988). barrera et al. (1994) obtained lipid contents of 7.29 and 3.37%, respectively, from texcoco lake artemia and a. franciscana from san francisco. the ash content of artemia cyst and nauplii amounted to 5.98 and 9.79%, respectively. in case of ash content due to the feeding on organic matter that cause ash accumulation in the telopodites and the digestive tube, thus increasing the ash proportion and lowering that of other nutritional elements (gonzalbo et al., 1987). nevertheless, the ash content of the natural populations of the artemia nauplii and decapsulated cyst in this study was not substantially different from that reported for other artemia grown also under controlled conditions with different feed supplements (barrera et al., 1994; leger et al., 1986; rosinvalli et al., 1987). sakamoto et al. (1982) have reasoned that the biochemical composition of the artemia reflects the diet administered. eicosapentaenoic (epa) and decosahexanoic (dha) fatty acids are considered essential components of the diet of marine organisms (kanazawa et al., 1979; watanabe, 1993). eicosanoid production from arachidonic acid (n-6 fatty acid) is modulated by epa, and failure to supply these two essential fatty acid in the appropriate balance may result in adverse biochemical responses when fed to the predator organisms (sorgeloos and kulasekarapandian, 1984). in the present study, epa reached a value of only 1.46 mg.g-1 and no dha was detected. lavens and sorgeloos (1991) found that when artemia was cultivated with agricultural sub-products, the harvest contained small amounts of epa and dha acid. therefore, it is reasonably to assume that the low amounts of epa and dha found in the present study is likely to be due to the mixing of agricultural products in the diet. compilation of results on the hatching characteristics and nutritional composition of a. franciscana, confined to kelambakkam is very much suitable for shellfish and finfish aquaculture industries at hatchery level. hence, mass propagation of the brine shrimp artemia in kelambakkam saltpan is proposed to reduce the feed cost at hatchery level and production of more artemia cyst using the species available locally. acknowledgments financial assistance from the ministry of earth sciences (moes/11-mrdf/1/8/p/07), government of india and thanks are due to salt commissioner, jaipur, deputy salt commissioner, chennai and saltpan lessee of cove long salt works (skms salt works), kelambakkam are gratefully acknowledged for their kind permission for salt pans. references aoac (association of official analytical chemists) (2000). official methods of analysis, 17th ed., aoac, gaithersburg, maryland, usa. awaiss a., kestemont p., micha j.c. 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(2014) 2(1): 36-42 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article a swot analysis of aquaculture development in rural areas of iran, an application to rainbow trout (oncorhynchus mykiss) roxana moogouei*1 department of environmental planning, management and education, north tehran branch, islamic azad university, tehran, iran. article history: received 12 december 2013 accepted 7 january 2014 available online 2 5 february 2014 keywords: aquaculture rainbow trout rural areas iran abstract: in this study various important indices were selected to assess the sustainable aquaculture strategies in rural areas of iran. in addition the government officials, consultants and managers were surveyed to assess the indices of aquaculture development. the strengths, weaknesses, opportunities and threats analyses were used to make a comprehensive evaluation on internal and external factors, participating the development of aquaculture strategies. the sum of the attractiveness scores from the internal factor evaluation matrix was approximately 2.55, being larger than 2.5, indicating that the strengths exceed the weaknesses. the sum of the external factor evaluation matrix scores was 3.49, indicating that opportunities were higher than threats. this analysis showed that the development of aquaculture, promotion of new cold-water species production, productivity enhancement, establishment of hatchery facilities and formation of an effective support organization are the most important strategies that should be considered in the studied area. results obtained on this research help decision makers on work of the aquaculture sector in rural areas of iran. introduction in many areas rapidly growing populations have increased demand for expanded food production capabilities which, in turn, places pressure on environmental resources. both terrestrial and aquatic environments have experienced severe degradation. various post-1996 studies have suggested that fish species are threatened with extinction (perman et al., 1996; gilvear et al., 2002; keith et al., 2004; united nations environment programme, 2010). despite threats to marine fish populations, capture fisheries continue to supply a large quantity of food to local and world markets. aquaculture has increasingly become important in supplying fish and shellfish, and dominates all other animal food-production sectors in terms of growth rate. with an annual average growth rate of 8.9% since 1970, aquaculture had exceeded capture fisheries (1.4%) and terrestrial * corresponding author: roxana moogouei e-mail address: r_moogoui@iau-tnb.ac.ir farmed meat production operations (2.8%) over the same period (gopal paul, 2013). numerous fish species are used in small and large aquaculture operations. some salmonids, such as rainbow trout (oncorhynchus mykiss), are an oft-cultured fish because of their popularity as a food item and knowledge appropriate rearing methods (johansen and overturf, 2005; larsen et al., 2012). even though iran, with a population of more than 70 million, is one of world’s most populous countries, its aquatic meat consumption is only 7.32 kg per capita (iran fisheries organization, 2012) which is very low compared to the rest of the world mean consumption rate of 17.2 kg (per capita food fish supply) food and agriculture organization (2012). the primary objective of this research was to evaluate the current status of aquaculture farms in terms of its strengths, weaknesses, opportunities and 37 moogouei/ int. j. aquat. biol. (2014) 2(1): 36-42 threats, (swot). in many organizations the internal strengths and weaknesses as well as the external opportunities and threats have been evaluated (sarter et al., 2010). this study develops strategic planning for culturing of rainbow trout in a developing country such as iran. in fact swot is a planning tool that is used to identify the major factors affecting the competitiveness and viability before creating a production strategy (british columbia ministry of agriculture, food and fisheries, 2004). the swot analysis aimed to method was to identify the strengths and weaknesses of an organization and the opportunities and threats in the environment (dyson, 2004; chang and huang, 2006). it is believed that swot is important for strategy formulation and development. material and methods study area: the area of study is one of the most unstable tectonic plates in iran, and is located between 46°50' to 50°01'n and 32°40' to 34°32'e (fig. 1). the soil porosity is relatively high due to the presence of variously-sized sand (national geoscience database of iran, 2007). the annual average maximum and minimum temperatures are 20.4°c and 7.6°c, respectively; annual average temperature is 14°c. annual precipitation is 612.7 mm and the (annual) average number of sunny hours per month is 268 (fig. 2). the number of cold (near or below freezing) days per year is 111 (iran management and planning organization, 2009). methods: in this research various important indices were selected to assess sustainable aquaculture strategies in rural areas of iran. in addition, government officials, consultants and managers were surveyed to assess the indices of aquaculture development. this paper used swot analysis to make a comprehensive evaluation on strengths, weaknesses, opportunities and threats in the figure 1. sarab gerdu area in aligudarz city in iran. figure 2. raceways used for aquaculture in the area of sarab gerdu, iran. 38 moogouei/ int. j. aquat. biol. (2014) 2(1): 36-42 development of aquaculture strategies. the procedure is described in the following sections (nikolaou and evangelinos, 2010; manteghi and zohrabi, 2011). internal factors evaluation (ife) matrix: internal factors are consisted of strengths and weaknesses (gallego-ayala and juízo, 2011; zhao and yan, 2012). ten to twenty indices (strengths and weaknesses) that affect the productivity of management of rainbow trout raceways were selected. these indices were extracted from the review literature and interview with experts (delphi method). all these factors were weighed using questionnaires and one variable statistical analysis (using spss software) so that the sum of the weights was equal to one. a score was calculated to each factor. these scores ranged from 1 to 4, with the following orders: 1-severe weakness, 2common weakness, 3-common strength and 4-important strength. the chosen score was mode of the data obtained from questionnaires for each variable. the calculated weight of each factor was multiplied by the calculated score of the same factor to produce an attractiveness value, and all of the attractiveness values were added to each other. if the sum of all attractiveness values was less than 2.5, (mean of 1and 4) then it was concluded that weaknesses are more critical than strengths. the sums more than 2.5 indicated that strengths were the dominating weaknesses. external factors evaluation (efe) matrix: external factors consist of opportunities and threats. the steps used to complete the efe matrix were parallel to those used to develop the ife matrix. table 1. swot analysis of aquaculture in the sarab gerdu area strengths opportunities 1. emphasis on aquatic protein consumption in the 4th five year economic, social and cultural development plans of the country. 2. rural area development along with employment and economic prosperity. 3. appropriate conditions for aquaculture practices. 4. fish consumption effects on human health due to valuable foodstuff. 5. availability of production factors such as labors, fish food production in the country, controlled and uncontrolled water resources. 6. integrated agriculture aquaculture. 7. food security; decrease of stress on the other food production resources like pastures. 8. decrease of stress on the marine resources. 1. standardization of cultured fish quality. 2. promotion of productivity. 3. support of culturing new species. 4. decreasing food poverty due to valuable high caloric food production resources. 5. availability of internal and external marketing 6. marine ecosystem conservation through promotion of aquaculture. 7. use of biotechnology with safety assessment such as risk assessment. 8. possibility of aquaculture by-product processing industries establishment. 9. presence of experts for aquatics hatchery and culture. 10. possibility of endangered species culture. 11. hatching possibility. weaknesses threats 1. unavailability of waste water treatment system in the area of study. 2. absence of electricity line in the area. 3. damage to aquaculture race ways due to mud floods. 4. unfavorable insurance coverage. 5. absence of supportive rules and regulations. 6. absence of hygienic supervising and quality control of foods production in relative industries. 7. bad road conditions during snow fall periods. 8. weak economic conditions prevail over aquaculture race ways. 9. high expenditures for transport of fish associated with cars equipped with refrigerator. 10. abuse by middle-men for proper distribution. 11. inconsistent fish price due to lack of storage facilities during different seasons. 12. insufficient propaganda for consumption aquatics' foods. 1. farms instability due to lack of ecological capability evaluation and environmental assessment. 2. location of study area in earthquake zone. 3. increase of fish food costs in total costs. 4. incidence of fungi and bacterial diseases. 5. native people misery in others' economical activities. 6. aquaculture farmer have no information about new breeding and culturing methods. 7. no quality classification for food supplies that are used in fish feeding. 39 moogouei/ int. j. aquat. biol. (2014) 2(1): 36-42 theory: swot, is an acronym presenting main components: strengths, weaknesses, opportunities, and threats (glaister and falshaw, 1999). in fact, swot is not an analysis but, like a tool, it can assist in efficiently performing an extensive analysis (duarte et al., 2006). data supporting the analysis are obtained from several channels including governmental reports, related laws and regulations, group meetings and literature reviews (yuan, 2013). results the ife and efe matrices for aquaculture in the sarab gerdu are shown in table 1. based on the governmental reports, related laws and regulations and questionnaire for each of the four analysis variables (strengths, weaknesses, opportunities and threats) several factors are listed, with weaknesses having the highest (12) and threats having the lowest (7). scores for strengths were all 4, except #6, “integrated agriculture aquaculture,” which was scored as 3 (table 2). weakness scores were either 1 or 2. scores for opportunities and threats had higher fluctuations (table 3). opportunity scores ranged from 2 to 4 while threat scores ranged from 1 to 4. discussion the sum of the attractiveness scores in the ife matrix was approximately 2.55, which was larger than 2.5 and showed strengths are more than threats. the efe matrix sum of 3.49 was also more than 2.5, indicating that opportunities exceed possible threats. consequently before establishing aquaculture facilities for commercial production it is appropriate to conduct an ecological impact assessment. to this end, an accurate estimation of available water resources and environmental impacts is very factor weight score attractiveness strengths 2. rural area development along with employment and economic prosperity. 0.02 4 0.08 3. appropriate conditions for aquaculture practices. 0.07 4 0.28 4. fish consumption effects on human health due to valuable foodstuff. 0.07 4 0.28 5. availability of production factors such as labors, fish food production in the country, controlled and uncontrolled water resources. 0.06 4 0.24 6. integrated agriculture aquaculture. 0.02 3 0.06 7. food security; decrease of stress on the other food production resources like pastures. 0.04 4 0.16 8. decrease of stress on the marine resources. 0.02 4 0.08 weaknesses 1. unavailability of waste water treatment system in the area of study. 0.03 2 0.06 2. absence of electricity line in the area. 0.03 2 0.06 3. damage to aquaculture race ways due to mud floods. 0.10 1 0.10 4. unfavorable insurance coverage. 0.02 2 0.04 5. absence of supportive rules and regulations. 0.08 2 0.16 6. absence of hygienic supervising and quality control of foods production in relative industries. 0.05 2 0.10 7. bad road conditions during snow fall periods. 0.02 2 0.04 8. weak economic conditions prevail over aquaculture race ways. 0.07 1 0.07 9. high expenditures for transport of fish associated with cars equipped with refrigerator. 0.06 2 0.12 10. abuse by middle-men for proper distribution. 0.06 2 0.12 11. inconsistent fish price due to lack of storage facilities during different seasons. 0.07 2 0.14 12. insufficient propaganda for consumption aquatics foods. 0.04 2 0.08 sum 2.55 table 2. internal factors evaluation matrix of aquaculture in the area of sarab gerdu 40 moogouei/ int. j. aquat. biol. (2014) 2(1): 36-42 important (smith and liou, 2010). it is also vital to develop appropriate quality standards to promote purchase and use of aquaculture products by consumers. establishment of an effective supportive organization can be useful to support aquaculture farmers, propose corrective measures towards more effective rules and regulations, furnish standard aquaculture raceways with a certification of quality, conduct educational training courses, reflect and verbalize to legal authorities and the difficulties faced by farmers, support the culturing of new species, and decrease shortages of food in rural areas. it is feasible and economically appropriate to use rural resources, including land and labor, to set up and run aquaculture facilities. development of an aquaculture industry, using controlled and uncontrolled water resources and employing local expert residents, can decrease the stress placed on other resources. integrating aquaculture with other agricultural practices in an area could potentially increase regional productivity although environmental impact assessment is necessary (murshed-e-jahan and pemsl, 2011; efole ewoukem et al., 2012). advanced biotechnology can be employed to develop species that are resistant to pathogens (sabasinghe et al., 2007), thus decreasing the possibility of a significant stock that may destroy the production. this is in agreement with studies by other researchers (johansen and overturf, 2005). establishing a waste water treatment system on the basis of environmental impact assessment is necessary (lefrançois et al., 2010; burridge et al., 2010). a steady and reliable supply of electricity is critical in order to maintain homeostasis within the culture system and thus prevent damage to stock. it is also necessary to prevent damage from mud floods through appropriate watershed management. since roads are often blocked during the winter due to heavy snow fall, it is necessary to have an ample supply of drugs and food on hand. an important factor that would help makes aquaculture a commercial success would be the development of local markets, which would lower transportation costs for producers and factor weight score attractiveness opportunities 1. standardization of cultured fish quality. 0.07 4 0.28 2. promotion of productivity. 0.04 4 0.16 3. support of culturing new species. 0.04 3 0.12 4. decreasing food poverty due to valuable high caloric food production resources. 0.10 4 0.40 5. availability of internal and external marketing. 0.08 4 0.32 6. marine ecosystem conservation through promotion of aquaculture. 0.07 2 0.14 7. use of biotechnology with safety assessment such as risk assessment. 0.07 4 0.28 8. possibility of aquaculture by-product processing industries establishment. 0.05 3 0.15 9. presence of experts for aquatics hatchery and culture. 0.05 4 0.20 10. possibility of endangered species culture. 0.01 2 0.02 11. hatching possibility. 0.10 4 0.40 threats 1. farms instability due to lack of ecological capability evaluation and environmental assessment. 0.10 4 0.40 2. location of study area in earthquake zone. 0.01 1 0.01 3. increase of fish food costs in total costs. 0.04 3 0.12 4. incidence of fungi and bacterial diseases. 0.05 3 0.15 5. native people misery in others' economical activities. 0.03 1 0.03 6. aquaculture farmer have no information about new breeding and culturing methods. 0.04 4 0.16 7. no quality classification for food supplies that are used in fish feeding. 0.05 3 0.15 sum 3.49 table 3. external factors evaluation matrix of aquaculture in the area of sarab gerdu 41 moogouei/ int. j. aquat. biol. (2014) 2(1): 36-42 decrease costs. direct sales to consumers would also eliminate the need for a separate distributor or “middle man” who would help keep costs low. once local markets are developed, the need for refrigerator storage will also be minimized. production of a new aquaculture species or variety and maintenance of an adequate and sustainable supply can encourage consumption of aquaculture products (bc ministry of agriculture, food and fisheries, 2004, larsen et al., 2012) and also lead to the conservation of wild populations. it is not necessary to restrict aquaculture practices to rainbow trout many other species may be appropriate for culture and enhance the variety of products available to consumers. generally, it is important to lower the costs of aquaculture foods and also to develop appropriate hygienic and quality control mechanisms. according to philcox et al. (2010) many quantitative and qualitative methods are used to analyze management options related to aquaculture development. in the present study, to achieve the above goals, important strategies may be suggested. strategies that must be considered within the study area in iran are the development of an aquaculture industry, increasing production of a new cold water species (or variety of existing species) and conservation of endangered species, increasing productivity (vilhelm skov et al., 2011), establishment of hatchery facilities and establishment of an effective support organization. conclusion the data used to evaluate the status of aquaculture in the area of study resulted showed in order to exploit its current status and maximize future strengths, the aquaculture system must use existing capabilities to utilize internal opportunities whenever possible, remove internal weaknesses, and avoid external threats. acknowledgment spatial thanks to ms fereshteh heydari, mr. siavash moogouei, mrs. shahla teymouri, mr. morteza heydari and mr. arash bahmanpoor for their invaluable technical assistance. references british columbia ministry of agriculture, food and fisheries. 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(2022) 10(6): 451-459 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article isolation of symbiont bacteria of stylissa massa as potential candidates for producing antimicrobial compounds from the waters of rote island, east nusa tenggara, indonesia jefry presson1, lukas pardosi2, jendri mamangkey3, dicky frengky hanas2 1chemistry study program, faculty of agriculture, university of timor, kefamenanu, indonesia. 2biology study program, faculty of agriculture, university of timor, kefamenanu, indonesia. 3department of biology education, faculty of education and teacher training, universitas kristen indonesia, jakarta, indonesia. s article history: received 12 august 2022 accepted 3 december 2022 available online 2 5 december 2022 keywords: isolation characterization antimicrobial sponge abstract: current work aimed to isolate and characterize symbiont bacteria of stylissa massa inhabiting rote island of east nusa tenggara, indonesia. the symbiont bacteria are potential candidates for producing antimicrobial bioactive compounds. a total of 22 bacteria were isolated from s. massa with different characteristics. these isolates were screened to understand their capability to inhibit the growth of escherichia coli and staphylococcus aureus. further, the secondary metabolites of the sm10 isolate, with the best inhibition results. this isolate could inhibit pathogenic bacteria of e. coli (11.24 mm) and s. aureus (12.11 mm). the molecular identification using 16 s rrna, revealed that the sm10 isolate is pseudomonas aeruginosa. based on the gc-ms results, the sm10 isolate had produced two alkaloid compounds, pyrrole alkaloid (hymenialdisine) and bromopyrrole alkaloid (agelongine and spongiacidin d), and 2 unknown compounds. this finding showed that s. massa has symbiont bacteria isolates that can be used in the biochemical industry. introduction east nusa tenggara (ntt) is an archipelagic province in indonesia with approximately 200,000 km² of sea area. the wide open waters of ntt are rich in marine resources, including fisheries, seaweed, coral reefs, and sponges (indonesia statistics, 2018). rote island is one of the most advanced islands in the southern part of east nusa tenggara waters, the southern fence of the republic of indonesia, and its sponge symbiont bacteria diversity has not yet been explored. rote island waters have ecological conditions with specific characteristics that can be used as the main indicator to find new sponge symbiont bacteria that can produce antimicrobial compounds. stylissa massa is widely distributed in tropical indonesian seas, including the banda sea, sulawesi sea, mergui islands, and papua new guinea (van soest, 2017). several compounds that are categorized as potential medicinal ingredients have correspondence: lukas pardosi doi: https://doi.org/10.22034/ijab.v10i6.1692 e-mail: lukaspardosi51@unimor.ac.id dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.2.5 been isolated from sponges of this species, such as stylissatin a (cyclic peptide) as an antiinflammatory agent (kita et al., 2013), stylissatin b as an antitumor agent (sun et al., 2015), spongiacidin c as a usp7 inhibitor (yamaguchi et al., 2013), and aldisine alkaloid as a mek-1 inhibitor (tasdemir et al., 2002). currently, research concerning s. massa sponge symbiont bacteria is rare. stylissa sponges are abundant in rote island waters, and they are promising as an indicator of finding symbiotic bacteria. bacteria are frequently found living symbiotically in various marine organisms, including sponges. sponges are known as the producers of bioactive compounds, and some of them are also assumed to be produced by bacteria that live symbiotically with them. such condition allows sponge symbiont bacteria to play a major role in producing bioactive compounds that have antimicrobial activity. therefore, research on sponge 452 presson et al./ isolation of symbiont bacteria of stylissa massa symbiont bacteria through their isolation and characterization is crucial. therefore, this study was conducted to identify the symbiotic bacteria of the s. massa sponge that produces antimicrobial compounds in the waters of rote island, east nusa tenggara. materials and methods isolation of symbiont bacteria: the bacterial symbionts were isolated from the sponge as follows: 1 g of the sponge sample was ground finely using the pestle and mortar. the ground sample was diluted by adding 1 g of sponge into a test tube containing 9 ml sterile nacl. afterward, serial dilutions were performed from 10-1 to 10-4. 0.1 ml of the dilution results were spread on a petri dish containing na media and incubated at a temperature of 270°c for 24 hours. characterization of bacterial isolates: the morphological characteristics (macroscopic) of the bacterial isolates were investigated based on the colony's shape, color, elevation, size, and edge. biochemically, they were analyzed using citrate and tsia tests. antagonistic test of bacterial isolates against pathogenic bacteria: antagonistic test of the isolates was performed by testing against two pathogens of escherichia coli and staphylococcus aureus on muller hinton agar (mha) media based on the agar diffusion method. bacteria were taken using an ose needle and added into a test tube. furthermore, 10 microns of the suspension results were taken using a micropipette, dripped on paper discs, and incubated for 24 hours. the bacteria with the best results in terms of antimicrobial activity were used for the growth curve test, extraction of bioactive compounds, and molecular identification. growth curve of the potential bacterial isolate: the isolate’s growth curve was measured by calculating the number of living bacterial populations based on the spread plate method. this method spread 0.1 ml of the bacteria on plant count agar (pca) media evenly. the isolates were grown on nutrient broth media and incubated in a shaker, and bacterial colonies were counted every 4 hours using a colony counter for 48 hours. extraction of bioactive compounds of the potential bacterial isolates: 100 ml of the nutrient broth (nb) media was used to culture the bacterial isolates. furthermore, these isolates were incubated for 3 x 24 hours. the grown isolates were then dissolved in 100 ml of petroleum ether and extracted using a funnel. the extraction results were stored in bottles. antibacterial activity of bioactive compounds extracted from potential symbiont bacterial isolates: the diffusion method was applied to conduct the antibacterial test (brooks et al., 2007). this test was done first by making wells on the mha media that had been inoculated previously using pathogenic bacteria. furthermore, 0.1 ml of the selected symbiont bacterial extract was added to the well and incubated for 24 hours. molecular identification of potential bacterial isolates: dna isolation (for sm10 isolate) was done using the pcr method. for this purpose, 1.5 ml of pure bacterial culture aged 24 hours on nb media was taken and centrifuged for 10 minutes. the supernatant was discarded and added with 100 l of distilled water under aseptic conditions. the cell suspension was frozen at -10°c until the solution crystallized and then thawed at 90°c for 10 minutes. this cycle was repeated 5-10 times so that the cell broke down efficiently. dna amplification of the bacterial genome against the 16s rrna gene was further carried out using a forward primer of 27f (5'aga gtt tga tca ctg gct cag-3') and reverse primer of 1492r (5'-tac ggc tta cct tgt tac ga-3') (elavazhagan et al., 2009). gas chromatography-mass spectrometry analysis: gc-ms analysis was done by a fraction of sm10 using thermo trace 1300gc coupled with thermo tsq 800 triple quadrupole ms equipped with column bp 5ms 30 m x 0.25 mm, 0.25 mm. the oven was initially set at 60°c and further increased by a rate of 5°c/min, until reaching 230°c. the carrier gas was helium at the flow rate of 1 ml/min. the injection volume was 2.0 ml (split ratio of 5:1) while its temperature was maintained at 453 int. j. aquat. biol. (2022) 10(6): 451-459 250°c and the ion source temperature was 230°c (ashraf et al., 2017). results the results of the isolated bacteria associated with s. massa showed the presence of 22 isolates. the isolated bacteria had different morphological characters, as shown in table 1. among the 22 isolates of symbiotic bacteria, 19 isolates had irregular colony shapes and 3 rhizoid colony shapes. the edges of colonies were lobate, entire, serrate, and undulate. the colony elevation was flat, raised, and umbonate. in addition, the color of the colonies was white and yellow and gram-negative staining. five bacterial isolates, including isolates coded sm2, sm3, sm5, sm8 and sm10 were able to inhibit the pathogenic bacteria of both e. coli and s. aureus. sm10 had the best antimicrobial activity and was selected for further analysis. antagonist test of the isolates against e. coli and s. aureus: the antagonist results of bacterial isolates against pathogenic bacteria are shown in table 2. out of 22 bacterial isolates, 17 were able to inhibit e. coli growth, while 15 isolates inhibited s. aureus growth. curve of sm10 isolate growth: based on the results of the sm10 growth curve (fig. 1), the number of colonies increased in each phase. the adaptation phase (lag) had a relatively short time of growth increase in 0-4 hours. the logarithmic phase had a significant increase in growth at 8-20 hours, while the growth increase in the stationary phase occurred at 28-36 hours, and this phase was characterized by constant growth. the growth experienced a death phase after 42 hours, in line with the decrease in bacterial colonies. antibacterial test of the extract of sm10 isolate bioactive compounds: the results of sm10 isolate extract using petroleum ether are presented in table 3. based on the results of sm10 bacteria extract against e. coli and s. aureus, sm10 isolate could inhibit e. coli by an average of 11.24 mm, and s. aureus by an average of 12.11 mm (fig. 2). meanwhile, in the positive control test (+) using the antibiotic chloramphenicol, the inhibition against e. coli had a zone of 18.24 mm, and for s. aureus, isolate code colony shape colony edge elevation size color sm1 irregular entire flat medium white sm2 irregular lobate flat medium white sm3 irregular lobate flat medium white sm4 irregular undulate flat large yellow sm5 irregular serate flat large white sm6 irregular serate flat large white sm7 irregular entire convex small white sm8 irregular entire raised medium white sm9 irregular undulate umbonate medium white sm10 irregular undulate flat small yellow sm11 irregular lobate flat small white sm12 irregular entire flat medium white sm13 irregular undulate raised large white sm14 rhizoid serate flat medium white sm15 irregular undulate flat small white sm16 irregular undulate raised small white sm17 irregular undulate raised medium white sm18 irregular lobate flat large white sm19 rhizoid serate umbonate medium white sm20 rhizoid filamentous flat small white sm21 irregular undulate flat medium white sm22 irregular entire raised small yellow table 1. morphological character of stylissa massa sponge symbiont bacteria. 454 presson et al./ isolation of symbiont bacteria of stylissa massa 18.86 mm. furthermore, in the negative control test (-) using petroleum ether, the isolates could not inhibit e. coli and s. aureus. identification of isolates based on the 16s rrna gene: the results of molecular identification based on 16s rrna gene revealed that isolate sm10 was pseudomonas aeruginosa bacteria with 100% similarity to a bacterium with the accession number of cp012001 (fig. 3). identification of compounds produced from gcms: the identification of compounds extracted from sm10 was made by comparing the data spectrum with other literatures. the compound at the retention time of 43.525 (fig. 4) had a fragmentation pattern of m/z 281 [m-isopropyl]+, and m/z 325 [m+h]+, indicating that the mass was 324 sma. the compound with a mass of 324 sma was assumed to be 2hymenialdisine (fig. 5) that has been isolated from s. massa sponge from guam island (rohde et al., 2012), and stylissa carteri sponge (hamed et al., 2018), axinella verrucosa sponge, and acantella figure 2. antagonistic test of the sm10 isolate against the pathogenic bacteria; (a) e. coli, (b) aureus. table 2. testing of the bacteria isolate activity against e. coli and s. aureus bacteria isolate code e. coli s. aureus sm1 ₊₊ ₊ sm2 ₊₊₊ ₊ sm3 ₊₊ ₊₊ sm4 ₊₊ ₊₊ sm5 ₊₊ ₊₊ sm6 sm7 ₊ ₊ sm8 ₊₊ ₊ sm9 ₊ sm10 ₊₊ ₊₊ sm11 sm12 sm13 ₊ ₊₊ sm14 ₊ sm15 sm16 ₊ ₊ sm17 ₊ ₊ sm18 ₊ ₊₊ sm19 ₊ sm20 ₊ ₊ sm21 ₊ ₊₊ sm22 ₊ ₊₊ figure 1. diagram of curve of sm10 isolate growth. c e ll n u m b e r time (hour) 455 int. j. aquat. biol. (2022) 10(6): 451-459 aurantiaca (sharma et al., 2004). this compound has the inhibitory activity of interleukin-2 (ic50 = 2.4 µm) (sharma et al., 2004). the compound at the retention time of 43.683 (fig. 6) had a fragmentation pattern of m/z 249 [mmethyl]+, m/z 207 [m-isobutyl]+, m/z 193 [mtertpentyl]+, and m/z 265 [m+h]+, and the compound had mass of 264 sma. however, the compound with a mass of 264 sma has not been reported from stylissa sponge, so it is a new compound. the compound with the retention time of 43.725 (fig. 7) had a fragmentation pattern of m/z 327 [mmetyl]+, m/z 283 [m-isobutyl]+, and m/z 341 [m]+. the compound of the mass of 341 sma has been isolated from s. carteri sponge from read sea – table 3. the antibacterial activities of sm10 isolate bioactive compounds. e. coli s. aureus inhibition zone diameter (mm) inhibition zone diameter (mm) repetition 1 repetition 2 repetition 3 average repetition 1 repetition 2 repetition 3 average 9.22 12.58 11.92 11.24 15.50 8.44 12.40 12.11 figure 3. phylogenic tree of sm10 isolates. figure 4. mass spectrum results at retention time of 43.525. figure 5. hymenialdisine. sm10 456 presson et al./ isolation of symbiont bacteria of stylissa massa mesir (hamed et al., 2018). this compound was agelongine (fig. 8), categorized as pyrrole alkaloid group. the compound at the retention time of 44.067 (fig. 9) had fragmentation patterns of m/z 283 [misopropyl]+, m/z 267 [m-isobutyl]+, m/z 254 [mtertpentyl]+, and m/z 325 [m]+. the compound at the mass of 325 sma has been reported from s. carteri from the red sea – egypt (hamed et al., 2018), and hymeniacidon sp. sponge from ishigaki island – japan (inaba et al., 1998). this compound was categorized as bromopyrrole group as spongiacidin d (fig. 10). the compounds at the retention time of 45.175 (fig. 11) had fragmentation patterns of m/z 156 [m-cl]+, m/z 135 [m-isobutyl]+, m/z 191 [m]+, and m/z 193 [m+2]+, and the compound had mass of 191 sma. the spectrum of this mass indicated that the compound contained cl atom, seen at the peak of [m]+ and [m+2]+ with the ratio of 3:1. with the amount of odd mass (191 sma), this compound also indicated to have nitrogen atom. however, a compound with 191 sma has never been reported from stylissa sponge, and it is assumed as a new compound. discussion based on the results, 22 bacterial isolates were identified that 17 isolates inhibited the growth of e. coli, and 15 inhibited the growth of s. aureus. five bacterial isolates, including isolates coded sm2, sm3, sm5, sm8 and sm10 were able to inhibit the pathogenic bacteria of both e. coli and s. aureus. nineteen isolates had irregular colony shapes and three rhizoid colony shapes. the morphological characters of the edge in the colony were lobate, entire, serrate, and undulate. the colony elevation was flat, raised, and umbonate. there were two bacterial colonies colors, white and yellow. this result is in line with the findings of kandio's (2021) that reported white and orange colony bacteria isolated from stylissa sp. sponge. one isolate produced yellow pigment and inhibited both e. coli and s. aureus. bacteria that produce pigment generally have different physiological and biochemical properties than others. in addition, such ability was affected by environmental factors, and in figure 6. mass spectrum at retention time of 43.683. figure 7. spectrum of mass at retention time of 43.725. figure 8. agelongine. 457 int. j. aquat. biol. (2022) 10(6): 451-459 this case, the bacteria could produce various pigments with unique characteristics that can be correlated to the symbiosis between bacteria and their ecosystem (celedón and díaz, 2021). the sm10 isolate had optimal growth after 28 hours of incubation. the incubation time can be used as an indicator of producing secondary compounds by bacteria and the final logarithmic phase until entering the initial stationary phase, has the optimal condition to produce useful secondary compounds. antimicrobial compounds can be applied as drug candidates to prevent the growth of pathogenic bacteria. based on our findings, crude extract of sm10 isolate inhibits the growth of e. coli by 11.24 mm and s. aureus by 12.11 mm. krishnan et al. (2013) reported s8 isolate, symbiont with stylissa sp., can inhibit the growth of klebsiella pneumoniae. pardosi et al. (2022) reported that sm4 isolate from s. massa could inhibit s. aureus and e. coli. molecular identification of sm10 isolate identified it as p. aeruginosa. several studies have reported pseudomonas spp. symbiosis with sea sponges. pseudomonas stutzeri lives in symbiosis with the sponges of hyrtios erectus, clathria, and callyspongia sp. (marzuki et al., 2021), and pseudomonas putida was symbiosis with the mycale microsigmatosa sponge (marinho et al., 2009). pseudomonas putida isolated from the mycale microsigmatosa sponge showed strong antimicrobial activity, which was active against multidrugresistant bacteria (s. aureus, s. epidermidis, and e. coli). in our study, p. aeruginosa could produce antimicrobial compounds. interpretation of spectra using gc-ms found two groups of alkaloid compounds, including pyrrole alkaloids (hymenialdisine) and bromopyrrole alkaloids (agelongine and spongiacidin d), and two other unknown compounds. figure 9. spectrum of mass at the retention time of 44.067. figure 10. spongiacidin d. figure 11. spectrum of mass at the retention time of 45.175. 458 presson et al./ isolation of symbiont bacteria of stylissa massa sponges are one of the most prolific marine organisms for producing new bioactive compounds (thomas et al., 2010; joseph and sujatha, 2011). the pigmentation produced by the sponge symbiont bacteria indicates the characteristics of their habitat (sponges) that produce various bioactive compounds. sponges of the genus stylissa are unique because of having bioactive compounds, including dimeric alkaloids such as hymenialdisine (fouad et al., 2012; ebada et al., 2015; presson et al., 2021; win et al., 2021), agelongine (hamed et al., 2018), and pongiacidin (yamaguchi et al., 2013; hamed et al., 2018; wang et al., 2022). the agelongine and pongiacidin d compounds are members of the bromopyrrole alkaloid family (scala et al., 2010; aktas et al., 2011). bromopyrrole alkaloids have several pharmacological activities that chemists tied to their synthesis in the last two decades because of having antiproliferative, antimicrobial, and immunosuppressive activities (ebada et al., 2015). furthermore, the hymenialdisine compound is a member of the pyrrole alkaloid family (hamed et al., 2018). sm10 isolate is not one type of bioactive compound to prevent the growth of e. coli and s. aureus, but it also produces other bioactive compounds. in conclusion, sm10 isolate, a symbiont of the stylissa sponge from the waters of rote island, east nusa tenggara showed antimicrobial activity. the sm10 isolate was identified as p. aeruginosa and the application of its crude extract can inhibit the growth of e. coli by 11.24 mm and s. aureus by 12.11 mm. the crude extract contains two alkaloid compounds: pyrrole alkaloids (hymenialdisine) and bromopyrrole alkaloids (agelongine and spongiacidin d). in addition, there were two unknown compounds in the crude extract of sm10. acknowledgments gratitude was expressed to the department of research and community service institute, lembaga penelitian dan pengabdian masyarakat (lppm) of the university of timor for funding this research. references aktas n., gözcelioğlu b., konuklugil b. 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(2017) 5(3): 208-217; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article histomorphological and mucin histochemical study of the alimentary canal of pangas catfish, pangasius pangasius (hamilton 1822) javd sadeghinezhad1, hooman rahmati-holasoo*2,1ali reza mirzaei2, hosseinali ebrahimzadeh mousavi2, narges zadsar1 1department of basic sciences, faculty of veterinary medicine, university of tehran, tehran, iran. 2department of aquatic animal health, faculty of veterinary medicine, university of tehran, tehran, iran. article history: received 27 february 2017 accepted 18 june 2017 available online 2 5 june 2017 keywords: pangas catfish oesophagus stomach intestine morphology abstract: the present study describes the histological and mucin histochemical properties of the alimentary canal (ac) of the pangas catfish, pangasius pangasius. the results revealed that the mucosa of the oesophagus was lined by a stratified epithelium containing chloride cells and taste buds which suggested mechanic, gustatory and physiologic roles of the oesophagus in this species. the stomach mucosa was lined by a simple columnar epithelium. the lamina propria-submucosa in cardiac and fundic stomach contained gastric glands. the pyloric stomach had the thickest muscularis layer among all the parts of the ac. the villi showed the maximum height and width in the middle intestine. the tunica muscularis and serosa showed the thinnest thickness among all parts of ac. the mucin histochemistry showed that the goblet cells of oesophagus and intestine contained both neutral and acidic with carboxylated and sulfated mucins and there was not acidic mucins in epithelial cells of the stomach. introduction the alimentary canal (ac) in fishes, includes oesophagus, stomach and intestine which enable transportation, storage, digestion and absorption of the food (wilson and castro, 2011; dos santos et al., 2015). there are several reports available regarding histological study of the ac in numerous fish species (albrecht et al., 2001; diaz et al., 2003; suicmez and ulus, 2005; chatchavalvanch et al., 2006; cao and wang, 2009; hopperdietzel et al., 2014; dos santos et al., 2015) that show differences among species which are related to taxonomy, feeding habits, food, age, body shape and size. the mucin histochemistry of digestive tract has also been studied in different fish species which showed the diversity among species and along the fishes’ ac (pedini et al., 2001; diaz et al., 2008; faccioli et al., 2014). the presence of mucosubstances in the fishes’ ac is correlated with lubrication, protection against proteolytic degradation, inhibition of microorganisms and osmotic function (loretz, 1995; diaz et al., 2008). * corresponding author: hooman rahmati-holasoo doi: https://doi.org/10.22034/ijab.v5i3.280 e-mail address: rahmatih@ut.ac.ir pangas catfish, pangasius pangasius (hamilton 1822) is a carnivorous species belong to the family pangasiidae. it mostly feeds on mollusks, fishes, insects and crustaceans (gupta, 2016). pangasius pangasius is widely distributed in india, bangladesh, pakistan, myanmar, malaya-peninsula, indonesia, vietnam, java and thailand (tripathi, 1996). however, over exploitation, habitat degradation, water pollution, destruction of the breeding grounds are major threats of this species in its natural habitats (sarkar et al., 2006; gupta, 2016). it is an important freshwater food due to its good taste and deliciousness, and also a popular game fish and recently is being kept as ornamental fish (mohindra et al., 2015; gupta, 2016). there are no data is available about the morphology of the ac in p. pangasius, therefore, such a study can contribute to the digestive physiology, feeding habit, formulation of diet, diagnosis of disease and promote knowledge in zoological and phylogenetic field (carrason et al., 2006; chatchavalvanich et al., 2006; 209 int. j. aquat. biol. (2017) 5(3): 208-217 xiong et al., 2011). considering that the literature is poor on digestive tract of aquarium fish species, the ac morphological investigation in this species is important (hale, 1965; caceci, 1984; onal et al., 2010; hopperdietzel et al., 2014). hence, the present study was aimed to determine the detailed histological and mucin histochemical feature of the ac in p. pangasius. materials and methods tissue sampling and histology: seven healthy p. pangasius with total length of 50-55 cm were obtained from a fish ornamental shop. the fishes were anesthetized with 250 mg l-1 tricaine methanesulfonate (ms-222, argent chemical laboratories redmond, wa, usa) based on the ethics and animalcare approvals (faculty of veterinary medicine, university of tehran, tehran, iran. n. 3867672). an abdominal wall incision was made and their ac were gently dissected. the entire ac was divided into the oesophagus, the cardiac, fundic and pyloric stomach and the anterior, middle and posterior intestine based on morphological differences. the body length, the length of ac and the lengths of each part of ac were measured in each specimen. for histological investigation, the sections of each part of ac was cleaned of its contents using 0.01 mol l-1 phosphate buffer saline (pbs) and fixed in 10% neutral-buffered formalin. the histological sections were prepared based on sadeghinezhad et al. (2015) and stained using hematoxylin and eosin (h&e) and masson’s trichrome for general histological examination. in addition, the following methods were used for the mucin histochemical study: periodic acidschiff (pas) staining to demonstrate neutral mucins (schumacher et al., 2004), alcian blue (ab) staining at ph 1.0 and 2.5 for acidic mucins (bancroft and cook, 1994), ab at ph 2.5 staining followed by pas to detect neutral and acidic mucins (mowry, 1956) and aldehyde fuchsin (af) with ab (ph 2.5) staining was also used to assess the nature of the acidic mucins (spicer and meyer 1960). the mounted slides were observed under an axioplan microscope equipped with zeiss axiocam mrm and the axiovision software (carl zeiss, oberkochen, germany). the height and width of the intestinal villi, the thickness of the tunica muscularis, the thickness of tunica serosa and the number of goblet cells were measured in various parts of the ac. for each specimen, all measurements were made with ten replications in each section. the evaluation of the goblet cells was made on randomly selected 100 µm length of the mucosal epithelium. statistical analysis: comparisons were performed using kruskal-wallis test. if it was significant, mannwhitney was used to determine the significant differences between the pairs of means. p<0.05 was considered statistically significant. results histomorphological characteristics: the ac of p. pangasius was 41±3 cm in length and consisted of the oesophagus, stomach (with cardiac, fundic and pyloric parts) and intestine (including anterior, middle and posterior parts). the wall of ac was divided into the mucosa, lamina propria-submucosa, muscularis and serosa. the results of the histomorphometrical study of various parts of the ac is summarized in figure 1. the oesophagus was a short tubular organ (2.7±0.5 mm in length) which connects the oropharynx to the stomach. its mucosa was lined by a stratified epithelium containing surface epithelial, goblet and undifferentiated basal club cells (fig. 2a). the chloride cells, contained the eosinophilic cytoplasm with a large central nucleus, were present in the epithelium. the taste buds were sparsely observed in the epithelium of the anterior part of the oesophagus (fig. 2a). the muscularis mucosa was not observed and the lamina propria-submucosa was consisted of the dens connective tissue with abundant collagen fibers. the striated tunica muscularis was composed of an inner and outer muscle layers. the tunica serosa was the outermost layer and made up mesothelium with underlying connective tissue (fig. 2b). the stomach was a small blind sac consisting of three well-defined cardiac, fundic and pyloric parts. their mucosa layers were lined by a simple columnar 210 sadeghinezhad et al./ pangas catfish alimentary canal histology epithelium. many regular mucosal folds with secondary branches were seen in the pyloric stomach. the lamina propria-submucosa in the cardiac and fundic parts contained the gastric glands. these tubular glands were surrounded by a layer of connective tissue and opened into the bottom of the gastric pits. the limania propria-submucosa of the cardia and fundus contained collagen fibers with few thin strands of smooth muscles while in pyloric stomach the dense collagen fibers were predominant. the tunica muscularis is consisted of the smooth muscle which was formed an inner circular muscle layer (cml) and an outer longitudinal muscle layer (lml). the pyloric stomach had the thickest muscularis (1783±139.6 µm) among all the parts of the ac (p<0.05). the histological features of serosa was similar to that of the oesophagus (fig. 3a-c). the intestine was 33±2 mm in length and consisted of the anterior, middle and posterior parts showing similar histological structure. the pyloric caeca were not observed. the villi showed the maximum height (418±103.8 µm) and width (118.3±26 µm) in the middle intestine. the mucosa was lined by a simple epithelium, in which the enterocytes and goblet cells were present. the apical border of the enterocytes possessed many microvilli arranged as a striated border. the goblet cells were scattered between the enterocytes. the number of goblet cells significantly figure 1. the histometric characteristics of the alimenray canal in pangasius pangasius (different superscript letters indicate a significant difference, p<0.05; o: oesophagus, cs: cardiac stomach, fs: fundic stomach, ps: pyloric stomach, ai: anterior intestine, mi: middle intestine and pi: posterior intestine). techniques employed mucous secreting cells remarks goblet cells epithelial cells oesophagus intestine of stomach pas +++ + +++ r ab (ph 1) +++ ++ b ab (ph 2.5) +++ ++ b pas-ab (ph 2.5) +++ +++ +++ rp, bp af-ab (ph 2.5) +++ +++ b, p r red, b blue; rp, reddish purple; bp, bluish purple; p, purple; pas, periodic acid-schiff; ab, alcian blue; af, aldehyde fuchsin, = negative staining; + = weak staining; ++ = moderate staining; +++ = intense staining. table 1. the histochemical characteristics of the mucous-secreting cells in the alimentary canal of pangasius pangasius. 211 int. j. aquat. biol. (2017) 5(3): 208-217 increased toward the end of the intestine (p<0.05) with the highest average number in the posterior intestine (106±2.4 cells in 100 µm). the muscularis was composed of the smooth cml and lml which showed the least thickness (267±51, 239.3±34.4 and 310.5±140.8 µm in anterior, middle and posterior intestine, respectively) part of the ac (p<0.05). the serosa of intestine had similar structure to other parts of the ac but without a distinct connective tissue and thinnest serosa layer (10.7±3.8, 15.8±4.7 and 18.2±3.1 µm in anterior, middle and posterior intestine, respectively) (p<0.05) (fig. 4a, b). mucin histochemical profile: the characteristics of the mucins secreted by the mucous secreting cells in the ac of p. pangasius are summarized in table 1. histochemical analysis showed that goblet cells in the oesophagus and intestine, and epithelial cells in the stomach as secretory unites. the neutral mucins were present in both mucous secreting unites that were pas-positive. the apical cytoplasm of epithelial cell in the stomach showed a strong reaction. the intensity of the red color of the goblet cells was more in the oesophagus than intestine (figs. 5a, 6a, 7a). the acidic sulfated and carboxylated mucins were observed in the goblet cells which were blue owing to the ab staining at ph 1.0 and 2.5, respectively. there was no acidic mucin in the epithelial cells of the stomach. the reaction in goblet cells of the oesophagus was stronger than those of intestine (figs. 5b, 6b, 7b). the investigation of pas-ab (ph 2.5) staining showed that the goblet cells stained bluish purple were more than the reddish purple stained cells, implying that the secreted mixed mucins were more acidic than neutral. however, the stomach epithelial cells were labeled red due to loss of acidic mucins (figs. 5c, 6c, 7c). the results of the af–ab (ph 2.5) staining indicated that both types of carbonic and sulfated acidic mucins were present in the goblet cells owing to blue and purple appearances, respectively (figs. 5d, 6d, 7d). regarding the histochemical reactions all observations mentioned above were seen in a similar way in different parts of stomach and also intestine. discussion in the present study, the histomorphology and mucin histochemistry of the p. pangasius ac were examined for the first time. the results showed that the general morphological features were in accordance with those described for fishes, with some differences. the oesophagus was lined with stratified squamous epithelium in this species as seen in most fishes. however, the stratified columnar epithelium was reported in some fishes like pelteobagrus fulvidraco figure 2. the photomicrographs of pangasius pangasius oesophagus. (a) the mucosa of the oesophagus. the epithelium was lined by a stratified epithelium containing surface epithelial cells (sc), goblet cells (gc) and undifferentiated basal club cells (cc). note the taste bud (arrowhead) and chloride cells (arrows) within the epithelium. the lamina propria (lp) beneath the epithelium is labeled (h&e, scale bar=50 µm). (b) the tunica muscularis and tunica serosa of the oesophagus. note the collagenous fibers (arrows) around striated muscle fibers (sm) of the tunica muscularis (tm). the tunica serosa (ts) is the outermost layer (masson’s trichrome staining, scale bar=200 µm). 212 sadeghinezhad et al./ pangas catfish alimentary canal histology (richardson 1846) (cao and wang, 2009), himantura signifer (compagno and roberts, 1982) (chatchavalvanich et al., 2006) and raja clavata (l. 1758) (holmgren and nilsson, 1999) in the oesophagus. the two different anterior and posterior regions in oesophagus mucosa with outer most layers of squamous and columnar cells were observed in some fishes e.g. engraulis anchoita (hubbs and marini, figure 3. the photomicrographs of pangasius pangasius stomach. (a) the cardiac stomach wall. gastric glands (g) within the lamina propria are visible. the strands of the muscularis mucosae (arrows), tunica submucosa (tsm), tunica muscularis (tm) with the circular muscle layer (cml) and longitudinal muscle layer (lml), and tunica serosa (ts) are visible (masson’s trichrome staining, scale bar=200 µm). (b) the mucosal fold of the pyloric stomach. the secondary branches of a mucosal fold with simple columnar epithelium (e) and underlying lamina propria (lp) are visible (masson’s trichrome staining, scale bar=50 µm). (c): the gastric glands of the fundic stomach. note the layer of connective tissue (arrows) surrounded the glands (g) (masson’s trichrome staining, scale bar=100 µm). figure 4. the photomicrographs of pangasius pangasius intestine. (a) the intestine villus. the mucosa is lined with a simple columnar epithelium with enterocytes (e) and goblet cells (g). on the apical border of the enterocytes, microvilli are arranged as a striated border (sb). note the presence of connective tissue fibers inside the lamina propria (lp) of the villus (masson’s trichrome staining, scale bar=50 µm). (b) the intestinal wall. the intestinal villi (stars), tunica muscularis (tm) with the circular muscle layer (cml) and longitudinal muscle layer (lml), and tunica serosa (ts) are visible (h&e, scale bar=200 µm). 213 int. j. aquat. biol. (2017) 5(3): 208-217 1935) (diaz et al., 2003), anguilla anguilla (l. 1758) (abaurrea-equisoain and ostos-garrido, 1996), scomberomorus maculatus (mitchill 1815), (motaalves, 1969) and seriola dumerili (risso 1810) (grau et al., 1992). albrecht et al. (2001) suggested the figure 5. histochemical characteristics of the goblet cells in the oesophagus of pangasius pangasius. (a) the representative photomicrograph of pas-stained goblet cells. (b) the representative photomicrograph of the alcian blue (ab) (ph 1.0) staining. (c) the representative photomicrograph of the pas–ab (ph 2.5) stained goblet cells. note that most goblet cells stained bluish purple and were secreting mixed mucins that were more acidic than neutral (arrows) and that the cells stained reddish purple secreted mixed mucins that were more neutral than acidic (arrowheads). (e) the representative photomicrograph of the af– ab (ph 2.5) stained goblet cells. the sulfated mucin content (arrows) and the carbonic mucin content (arrowheads) are visible (scale bars=50 µm). figure 6. histochemical characteristics of the mucous-secreting cells on the apical cytoplasm of epithelial cell of stomach in pangasius pangasius. (a) the representative photomicrograph of the pas staining. (b) the representative photomicrograph of the ab (ph 2.5) staining. note that there is not any ab reaction within epithelial cells. (c) the representative photomicrograph of the pas–ab (ph 2.5) staining. note that all of the secreting units stained red, indicating only neutral mucin production. (d) the representative photomicrograph of the af– ab (ph 2.5) staining indicating no reactions in epithelial cells (scale bars=50 µm). 214 sadeghinezhad et al./ pangas catfish alimentary canal histology protective role of the stratified epithelium against abrasion in oesophagus. the longitudinal folds, which allow distention during swallowing, were present like other species (holmgren and nilsson, 1999). goblet cells were scattered all over the esophageal epithelium of p. pangasius. the abundant goblet cells in the oesophagus of fishes may help the food transit to the stomach (grau et al., 1992; chatchavalvanich et al., 2006). the distribution of mucins secreted by these cells, detected by the series of histochemical tests, revealed that the goblet cells contained neutral, acidic carboxylated, and acidic sulfated mucins. the neutral and acidic mucosubstances in esophageal goblet cells were described previously with different intensity (pedini et al., 2001; zdravko et al., 2005; cao and wang, 2009; faccioli et al., 2014). different amount of the acidic and neutral mucins might be related to their different functions. the secretions of acidic mucins are used to increase mucous viscosity, lubricate epithelium and protect against pathogens (cao and wang, 2009; faccioli et al., 2014). the neutral mucins enable pre-gastric digestion in oesophagus with enzymatic digestion of food and its transformation into chyme (cao and wang, 2009). chloride cells were observed in the esophageal epithelium of p. pangasius. these cells which have osmoregulatory function are reported in euryhaline species like a. anguilla (genten et al., 2009). the presence of taste buds on the esophageal epithelium figure 7. histochemical characteristics of the goblet cells in the intestine of pangasius pangasius. (a) the representative photomicrograph of the pas staining indicating weak staining in intestinal goblet cells. (b) the representative photomicrograph of the ab (ph 1) staining. (c) the representative photomicrograph of the pas–ab (ph 2.5) staining. the goblet cells secreting mixed mucins that were more acidic than neutral (arrows) and neutral than acidic (arrowhead) are visible. (d) photomicrograph of the af–ab (ph 2.5) stained goblet cells. the sulfated mucin content (arrows) and the carbonic mucin content (arrowheads) are visible (scale bars=50 µm). 215 int. j. aquat. biol. (2017) 5(3): 208-217 indicates the gustatory role of the oesophagus in this species. it has been suggested that taste buds indicate final acceptance or rejection of food items passing in oesophagus towards the stomach (oliveira-ribeiro and fanta, 2000). the presence of well-developed two layers of striated muscle in the oesophagus of p. pangasius facilitating antiperistalsis might suggest potential assessment of ingested food quality in this segment of ac. however, absence of taste buds in some species leporinus friderici (bloch 1794), leporinus taenifasciatus, britski 1997 (albercht et al., 2001), orthrias angorae, (steindachner 1897) (suicmez and ulus, 2005), s. dumerili (grau et al., 1992) and e. anchoita (diaz et al., 2003) with different diets suggested that feeding habits was not related to the taste buds in the oesophagus. the stomach of p. pangasius lined with single layer of the columnar epithelium was similar to that of other species. the gastric glands were absent in pyloric stomach that were similar with those of most species e.g. l. friderici and l. taeniofasciatus (albercht et al., 2001), h. signifier (chatchavalvanich et al., 2006), p. fulvidraco (cao and wang, 2009), hemisorubim platyrhynchos (valenciennes 1840) (faccioli et al., 2014) and schizodon knerii (steindachner 1875) (dos santos et al., 2015). however, the tubular glands in pyloric stomach have been reported in e. anchoita (diaz et al., 2003) and dentex dentex (l. 1758) (carrasson et al., 2006). the gastric glands were found in initial and terminal regions in oreochromis niloticus (l. 1758) (caceci et al., 1997) and only in the fundic stomach in oncorhynchus mykiss (walbaum 1792) (arias and garrido, 1994). the mucin histochemistry demonstrated extensive neutral mucins on the apical surface of the columnar cells in different regions of the p. pangasius stomach while the gastric glands were unreactive to histochemical tests. the neutral mucins in stomach with a role in absorption of easy digested substances (murray et al., 1996) and buffering effects on acidic content of the stomach (zdravko et al., 2005) have been also reported in h. signifer (chatchavalvanich et al., 2006) and p. fulvidraco (cao and wang, 2009) on the gastric epithelial cells. the surface epithelium of the stomach in h. platyrhynchos (faccioli et al., 2014), s. knerii (dos santos et al, 2015), d. dentex (carrasson et al., 2006), cynoscion guatucupa (cuvier 1830) (diaz et al., 2008) and e. anchoita (diaz et al., 2003) contained both neutral and acidic mucins. in umbrina cirrosa (l. 1758) both epithelial surface and gastric glands show histochemical reaction with neutral and acidic profile (pedini et al., 2001). in contrast, the secretory unites of the stomach in solea solea (l. 1758) revealed no reaction with histochemical tests (veggetti et al., 1999). these variations have been suggested to be related to different feeding habits or species specific (gargiulo et al., 1997). the mucosa of cardiac and fundic stomach contained few smooth muscle fibers which were replaced by abundant collagen fibers in pyloric stomach. the same feature has been reported for descending and ascending stomach of h. signifer (chatchavalvanich et al., 2006). the collagenous fibers in lamina propria-submucosa in some species may have a role in increase in elasticity for carrying food in stomach (cao and wang, 2009). the stratum compactum which protect the wall of ac has been reported in the stomach and intestine of some fishes (diaz et al., 2003; carrasson et al., 2006; sadeghinezhad et al., 2015), however, it was not observed in p. pangasius. the muscular layers that cause motility in stomach and its thickening in pyloric stomach enhance the movement for passing food into the intestine (dos santos et al., 2015). histometrical results showed the significant dimension of the villi in the middle intestine. similar results was reported in the initial parts of the intestine in esox lucius (l. 1758) (sadeghinezhad et al., 2015), amatitlania nigrofasciata (günther 1867) (hopperdietzel et al., 2014) and p. fulvidraco (cao and wang, 2009) and have introduced this part of the intestine as a major site for digestive processes. goblet cells were scattered within epithelium of the intestine and their number were increased in the posterior part due to need for more mucosal lubrication and protection in this part of intestine (pedini et al., 2001; carrasson et al., 2006). goblet cells were stained positive for both neutral and acidic mucins in p. pangasius intestine as found generally in the intestine of fishes with role in 216 sadeghinezhad et al./ pangas catfish alimentary canal histology lubrication of the mucosa and absorption of food (pedini et al., 2001; carrasson et al., 2006; cao and wang, 2009; hopperdietzel et al., 2014; dos santos et al., 2015). cao and wang (2009) reported abundant neutral content with few acidic mucin in intestine of p. fulvidraco while contrariwise results in p. pangasius may be related to the different feeding habit. the thickness of tunica muscularis was consistent along the length of the intestine in p. pangasius similar to a. nigrofasciata (hopperdietzel et al., 2014). in contrast, the thickness of tunica muscularis varies in the intestine of carnivorous fishes with irregular large food items to increase intestinal motility (grau et al., 1992). based on the results, the histomorphological and mucin histochemical features described in p. pangasius can be considered as adaptations to feeding behavior of this species. specific structures of the oesophagus in this species were indicative of mechanic, gustatory and physiologic roles in the oesophagus. the stomach and intestine presented the typical features of that of most fishes with some variations. references abaurrea-equisoain m.a., ostos-garrido m.v. 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(2005). mucosubstances of the digestive tract mucosa in northern pike (esox lucius l) and european catfish (silurus glanis l). veterinarski arhiv, 75: 317-327. http://www.ncbi.nlm.nih.gov/pubmed/?term=mohindra%20v%5bauthor%5d&cauthor=true&cauthor_uid=24409876 http://www.ncbi.nlm.nih.gov/pubmed/?term=kumar%20r%5bauthor%5d&cauthor=true&cauthor_uid=24409876 http://www.ncbi.nlm.nih.gov/pubmed/?term=sah%20rs%5bauthor%5d&cauthor=true&cauthor_uid=24409876 http://www.ncbi.nlm.nih.gov/pubmed/?term=lal%20kk%5bauthor%5d&cauthor=true&cauthor_uid=24409876 https://www.ncbi.nlm.nih.gov/pubmed/?term=pedini%20v%5bauthor%5d&cauthor=true&cauthor_uid=11820403 https://www.ncbi.nlm.nih.gov/pubmed/?term=scocco%20p%5bauthor%5d&cauthor=true&cauthor_uid=11820403 https://www.ncbi.nlm.nih.gov/pubmed/?term=fagioli%20o%5bauthor%5d&cauthor=true&cauthor_uid=11820403 https://www.ncbi.nlm.nih.gov/pubmed/?term=ceccarelli%20p%5bauthor%5d&cauthor=true&cauthor_uid=11820403 https://www.ncbi.nlm.nih.gov/pubmed/?term=su%c3%ad%c3%a7mez%20m%5bauthor%5d&cauthor=true&cauthor_uid=16212116 https://www.ncbi.nlm.nih.gov/pubmed/?term=a+study+of+the+anatomy%2c+histology+and+ultrastructure+of+the+digestive+tract+of+orthrias https://www.ncbi.nlm.nih.gov/pubmed/?term=a+study+of+the+anatomy%2c+histology+and+ultrastructure+of+the+digestive+tract+of+orthrias int. j. aquat. biol. (2017) 5(3): 208-217 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی (pangasius pangasius) پنگوسی گربه ماهی گوارشی لوله موسین هیستوشیمی و هیستومورفولوژی مطالعه 1زادسر نرگس ،2موسوی زاده ابراهیم علیحسین ،2میرزایی رضا علی ،2*هوالسو رحمتی هومن ،1نژاد صادقی جواد ، تهران، ایران.تهران دانشگاه ،دامپزشکی دانشکده ،پایه علوم گروه1 ، تهران، ایران.تهران دانشگاه ،دامپزشکی دانشکده، آبزیان های بیماری و بهداشت گروه2 چکیده: مخاط که داد نشان نماید. نتایجرا توصیف می( pangasius pangasius) پنگوسی ماهی گوارش لوله هیستوشیمی و بافتی خصوصیات حاضر مطالعه در را ریم فیزیولوژیک و چشایی مکانیکی، هاینقش که بود شده پوشیده چشایی هایجوانه و کلراید هایسلول حاوی مطبق اپیتلیوم بوسیله مری ددغ شامل معده فوندوس و کاردیا در مخاط زیر-پارین الیه. بود شده پوشیده ساده ایاستوانه اپیتلیوم وسیلههب معده مخاط. داد نشان گونه این اهدهمش میانی روده در پرزها عرض و ارتفاع بیشترین. داشت گوارش لوله هایبخش همه میان در را عضالنی الیه ترینضخیم معده پیلور. بود معدی یمر جامی هایسلول که داد نشان موسین هیستوشیمی. داشت گوارش لوله هایبخش همه میان در را ضخامت تریننازک سروز و عضالنی الیه. شد . داشتن وجود معده اپیتلیال هایسلول در اسیدی هایموسین و هستند سولفاتی و کربوکسیلی اسیدی و خنثی هایموسین نوع دو هر دارای روده و .شناسیریخت روده، معده، مری، پنگوسی، گربه ماهی :کلمات کلیدی int. j. aquat. biol. (2014) 2(1): 27-28 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology short communication first record of the loach fish paracobitis cf. malapterura in the kavir basin, northern iran saber vatandoust1, arash joladeh roudbar2, hamed mousavi-sabet*31 1 department of fisheries, babol branch, islamic azad university, mazandarn, iran. 2department of animal science and fisheries, sari university of agriculture sciences and natural resources, mazandran, iran. 3department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. abstract: the loach fish paracobitis cf. malapterura valenciennes, 1846 (nemacheilidae), formerly believed to be an endemic species in the caspian sea basin, was recorded for the first time in the upper kavir basin. this extension of its recorded range makes it likely that it has been overlooked in other parts of the kavir basin. keywords: nam river, iran, zoogeography, nemacheilidae article history: received 2 january 2014 accepted 15 february 2014 available online 25 february 2014 accepted 14 april 2013 available online 2 5 april 2014 introduction there are six valid species of the genus paracobitis bleeker, 1863 in iran (kottelat, 2012; coad, 2014). from these valid species, paracobitis iranica nalbant and bianco, 1998 is known from the namak lake basin, paracobitis longicauda kessler, 1872 is found in the tedzhen and murgab rivers of afghanistan and turkmenistan and in the aral sea basin, and in the tedzhen (= hari) river basin of iran, paracobitis malapterura valenciennes, 1846 is found in the southern caspian sea basin, paracobitis rhadinaea regan, 1906 is restricted to the sistan basin, paracobitis smithi greenwood, 1976 is found only from a cave in lorestan province in the tigris river basin, and paracobitis vignai nalbant and bianco, 1998 is endemic to sistan basin (nalbant and bianco, 1998; coad, 2014). and now the present study confirms the presence of the crested loach paracobitis cf. malapterura for the first time in nam river, kavir basin. materials and methods the study was conducted in nam river (35°44’44"n 052°34'23"e; 1975 m altitude), in tehran province. * corresponding author: hamed mousavi-sabet e-mail address: mousavi-sabet@guilan.ac.ir the river flows into the kavir basin. the specimens were collected by electrofishing and were preserved into 4% formaldehyde. results fourteen specimens of paracobitis cf. malapterura (fig. 1) were found on 16 august 2012 in the nam river near the city firouzkuh in the province of tehran, northern kavir basin, iran (35°44’44"n 052°34'23"e). the body proportions and meristic counts of the preserved specimens agree well with the available keys of paracobitis malapterura (coad, 2014). the reported species was not previously recorded from the kavir basin, and this constitutes new record. discussion the new record of the genus paracobitis and the species p. malapterura is the first in the basin of kavir for the genus and the first outside the basin of caspian sea for the species. considering the large distance between all the reported sampling localities of the genus paracobitis, the fishes can be expected 28 vatandoust et al. / int. j. aquat. biol. (2014) 2(1): 27-28 to occur more widely in iranian basins than is known today. acknowledgements we would like to express our sincere thanks to hamid reza bagherpour for his help in specimen collection. references coad b.w. (2014). freshwater fishes of iran (available at http://www.briancoad.com) (accessed on 10 december 2013). kottelat m. (2012). conspectus cobitidum: an inventory of the loaches of the world (teleostei: cypriniformes: cobitoidei). the raffles bulletin of zoology, 26(suppl.): 1-199. nalbant t., bianco p. (1998). the loaches of iran and adjacent regions with description of six new species (cobitoidea). italian journal of zoology, 65: 109-123. figure 1. paracobitis cf. malapterura from nam river in the kavir basin, northern iran. int. j. aquat. biol. (2017) 5(1): 47-51; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article first record and distribution of alburnus qalilus krupp, 1992 (teleostei: cyprinidae) in turkey sevil sungur birecikligil*1, ronald fricke2, erdoğan çiçek1 1department department of biology, faculty of art and sciences, nevşehir hacı bektaş veli university, turkey. 2im ramstal 76, 97922 lauda-königshofen, germany. article history: received 17 november 2016 accepted 2 january 2017 available online 2 5 february 2017 keywords: bleak orontes river gaziantep kilis abstract: the syrian spotted bleak, alburnus qalilus krupp, 1992 was described from the nahr alhawaiz, the mediterranean coastal drainage, syria. the species differs from all other members of the genus alburnus in the following combination of characters: 8 branched dorsal-fin rays, 9-11 branched anal-fin rays, 43-47 scales in the lateral line and 9-11 gill rakers on the lower limb of the first gill arch. in the present study, the occurrence of this species is reported for the first time from the turkish parts of the orontes river basin in kilis, gaziantep and hatay provinces. our new data indicate that the home range of this species has been increased towards the turkish parts of the orontes river basin. introduction the genus alburnus rafinesque, 1820, a member of the family cyprinidae, are widely distributed in europe and the northern parts of southwest asia (kottelat and freyhof, 2007). despite their wide distribution, the taxonomy and actual distribution of the members of this genus are still not well-known (buj et al., 2010). this genus comprises 41 valid species and also all species of chalcalburnus berg, 1933, which was proved to be a synonym of alburnus using molecular studies (zardoya and doadrio, 1999; durand et al., 2002; eschmeyer et al., 2017). in addition, this genus is an excellent example of high diversity and endemism in the western palaearctic freshwater fishes (mohammadian-kalat et al., 2015). the genus alburnus has a rich diversity in turkey (özulug and freyhof, 2007) and, still, there are some uncertainties about alburnus species living in anatolia (elp et al., 2015). by now, a total of 26 species of the genus alburnus have been reported from turkey, among which 15 are endemic. alburnus orontis sauvage, 1882, has been originally described from the orontes river drainage in syria * corresponding author: sungur birecikligil e-mail address: sevilsungur@nevsehir.edu.tr and is the only species that has been hitherto reported from the orontes basin in turkey (geldiay and balık, 2007; kuru, 2004, fricke et al., 2007; kuru et al., 2014; çiçek et al., 2015). the syrian spotted bleak, alburnus qalilus krupp, 1992, was originally described from the nahr al-hawaiz, syria (35°22'n, 35°58'e) based on 12 specimens collected at the mediterranean coastal drainage. the holotype and 21 paratypes are deposited in the senckenberg museum, frankfurt am main (holotype: smf 24480). this species is found in the turkish parts of the orontes basin [asi nehri] indicating that its distribution has increased. materials and methods the specimens of a. qalilus were collected from the orontes river basin, in the gaziantep, kilis and hatay provinces (turkey) during july-september 2013 using an electrofishing device (fig. 1). the collected fish were anesthetized with 1% clove solution, preserved in 10% formaldehyde and transferred to the laboratory for further study. the fish were identified according to krupp (1992). several characteristics were measured and the 48 sungur bırecıklıgıl et al./ first record of alburnus qalilus from turkey terminology of morphological characteristics were based on krupp (1992). the characteristics were measured to the nearest 0.1 mm using digital calipers. results and discussion the general body shape of the collected specimens is displayed in figure 2. fifty-six specimens of a. qalilus were collected from the orontes river basin with a total length ranging from 41.1 to 90.3 mm (table 1) being deposited in nevsehir hacı bektaş veli university (nhvuic-2013-6-1;4). the morphological characteristics of the specimens are presented in table 1. all morphological measurements of the specimens collected from turkey overlapped with those of the type specimens (krupp, 1992) (table 1). the syrian spotted bleak was diagnosed from all other members of this genus by a combination of characteristics including 8 branched dorsal-fin rays, 9-11 branched anal-fin rays, 43-47 scales in the lateral line and 9-11 gill rakers on the lower limb of figure 1. habitat of alburnus qalilus in the orontes river basin, turkey. figure 2. alburnus qalilus from the afrin river, the kilis province. 49 int. j. aquat. biol. (2017) 5(1): 47-51 the first gill arch (krupp, 1992). in addition, this species is characterized by moderately compressed head and body; greatest depth of body about halfway between head and dorsal fin origin. dorsal fin with 3 unbranched and 8 branched rays; anal fin with 3 unbranched and 10 branched rays; one unbranched and 13 branched pectoral fin rays complete lateral line with 45 scales, 10 scales between lateral line and dorsal fin origin, and 3 between lateral line and anal fin short gill rakers, 10 on lower limb of first gill arch, pharyngeal teeth in two rows: 2.5-5.2. origin of pelvic fins slightly in advance of dorsal fin origin, distal margin of dorsal fin oblique and straight, distal margin of anal fin slightly concave, caudal fin deeply forked, lateral line strongly curved ventrally (krupp, 1992). alburnus qalilus was previously known only from the nahr al-hawaiz, a mediterranean coastal drainage of syria (krupp, 1992). in addition, this species has not been reported in the previous checklists of the freshwater fishes of turkey (geldiay and balık, 2007; kuru, 2004), fricke et al., 2007; kuru et al., 2014; çiçek et al., 2015). the present study is the first record on the occurrence of this species and its geographical distribution in turkish parts of orontes river basin (fig. 3). alburnus qalilus, like its syrian population, inhabits in the rivers having quite clear water with substrate consisting silt and gravel (fig. 1). salaria fluviatilis, capoeta barroisi, planiliza abu, oxynoemacheilus insignis and oxynoemacheilus namiri co-exist with this species in its natural habitat in the orontes and afrin rivers. the global distribution of species diversity and richness are interesting for naturalists for centuries being important research topics in ecology (gaston, 2000). the knowledge of natural distribution and zoogeographic features of freshwater fishes help to identify hotspots of biodiversity, endemism, possible population connectivity and phylogeography, which are useful for conservation (jouladeh-roudbar, 2015; mohammadian-kalat, 2015). moreover, table 1. morphometric and meristic characteristics of alburnus qalilus (sd: standard deviation). characters holotype paratypes (n=11) orontes river basin (n=56) mean sd ranges mean sd ranges standard length (mm) 35.4 29.8-38.4 66.2 1.1 41.1-90.3 percent of standard length predorsal length 57.1 55.8 0.8 54.4-57.1 56.0 3.2 41.0-60.7 prepelvic length 52.0 50.6 1.1 49.2-52.9 48.9 2.9 34.9-53.3 preanal length 69.2 67.7 0.9 66.2-69.1 68.1 4.0 49.4-75.0 head length 29.1 28.6 0.4 27.9-29.5 26.3 1.8 19.3-31.3 body depth 27.1 26.2 0.7 25.0-27.3 25.1 2.2 16.9-29.0 caudal peduncle depth 12.1 11.6 0.3 11.1-12.1 10.6 0.9 8.3-12.5 dorsal fin base length 14.1 13.3 0.5 12.5-14.3 12.1 1.1 8.8-14.3 anal fin base length 15.0 14.5 0.9 12.9-16.1 13.2 1.4 10.8-18.1 longest dorsal ray 24.0 22.8 1.4 20.8-24.9 18.6 1.8 14.6-22.7 longest anal ray 19.2 18.5 0.9 17.1-20.2 15.5 1.7 12.0-18.4 longest pelvic ray 16.4 16.4 0.5 15.7-17.2 14.5 1.4 10.8-17.6 longest pectoral ray 23.2 21.7 0.8 20.3-23.1 22.1 0.9 18.3-27.4 eye diameter 9.6 9.8 0.5 9.1-10.4 9.2 1.0 4.8-9.4 interorbital width 7.4 7.1 0.6 6.4-8.0 7.4 1.1 4.8-10.0 meristic characters dorsal fin rays iii 8 iii 8 anal fin rays iii 9-11 iii 9-11 pectoral fin rays i 12-13 i 12-14 scales in lateral line 43-47 42-52 scales between lateral line and dorsal fin origin 9-11 9-11 scales between lateral line and anal fin origin 3 4-5 gill rakers 9-11 9-11 pharyngeal teeth 2.5-5.2 2.5-5.2 50 sungur bırecıklıgıl et al./ first record of alburnus qalilus from turkey historical biogeographical analyses of freshwater fishes provide a natural link between the geological and biotic evolution of a region (jouladeh-roudbar, 2015). however, the freshwater fishes of turkey are still poorly known and their ecology and habitat requirements would need further study for conservation. materials examined: all materials from turkey. alburnus qalilus: — nhvuic 2013-6-1, 37, 41-85 mm sl; gaziantep prov.: karasu river near islahiye, orontes basin, 37°01'14''n 36°52'57''e; e. çiçek & s. sungur, july-september 2013. — nhvuic 20136-2. 6, 49-87 mm sl; gaziantep prov.: karasu river near yesemek, orontes basin, 36°54'21''n 36°44'44''e; e. çiçek & s. sungur, july-september 2013. — nhvuic 2013-6-3.10, 51-65 mm sl; kilis prov.: afrin river near musabeyli, orontes basin , 37°52'24''n, 36°51'40''e; e. çiçek & s. sungur, july-september 2013. — nhvuic 2013-6-4. 3, 6372 mm sl; hatay prov.: afrin river near kırıkhan, orontes basin , 36°48'44''n, 36°59'09''e; e. çiçek & s. sungur, july-september 2013. acknowledgments the samples were collected during bap 13f34 project (scientific research projects) and was financially supported by nevsehir hacı bektas veli university. references buj i., vukić j., šanda r., perea s., ćaleta m., marčić z., bogut i., povž m., mrakovčić m. (2010). morphological comparison of bleaks (alburnus, cyprinidae) from the adriatic basin with the description of a new species. folia zoologica, 59 (2): 129-141. çiçek e., sungur birecikligil s., fricke r. (2015). freshwater fishes of turkey: a revised and updated annotated checklist. biharean biologist, 9 (2): 141figure 3. distribution map and sampling points (●). 51 int. j. aquat. biol. (2017) 5(1): 47-51 157. durand j.d., tsigenopoulos c.s., ünlü e., berrebi p. (2002). phylogeny and biogeography of the family cyprinidae in the middle east infeered from cytochrome b dna-evolutionary significance of this region. molecular phylogenetics and evolution, 22(1): 91-100. elp m., şen f., özuluğ m. (2015). alburnus selcuklui, a new species of cyprinid fish from east anatolia, turkey (teleostei: cyprinidae). turkish journal of fisheries and aquatic sciences, 15: 181-186. eschmeyer w.n., fricke r., van der laan r. 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(2007). alburnus demiri, a new species of bleak from western anatolia, turkey (teleostei: cyprinidae). ichthyological exploration of freshwaters, 18: 307-312. van der laan r. (2016). freshwater fish list, 19th edition, october 2016. richard van der laan, almere, the netherlands. 962 p. zardoya r., doadrio i. (1999). molecular evidence on the evolutionary and biogeographical patterns of european cyprinids. journal of molecular evolution, 49: 227. int. j. aquat. biol. (2017) 5(1): 47-51 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی ترکیه درکپورماهیان( )ماهیان استخوانی عالی: alburnus qalilus krupp, 1992نخستین گزارش حضور و پراکنش 2اردوغان چیچک ،1 رونالد فریک ،1*لیگیلکیریجیب سویل سونگر .ترکیه ،ولی نویشهر حاجی بکتاش دانشگاه ،علوم هنر و دانشکده، زیست شناسیگروه1 .آلمان ،کونیگسهوفن-الودا ،، 9792، 76ام رامستال آی1 چکیده: از . این گونه سوریه توصیف شده است مدیترانه احلوس ، از نهرالهویز، حوضهalburnus qalilus krupp, 1992 ،دارای خالماهی کولی سوریه -47مخرجی، باله شعاع منشعب 9-11، باله پشتی شعاع منشعب 8شامل: های زیرای از ویژگیواسطه مجموعهبه alburnusتمامی اعضای جنس در مطالعه حاضر، حضور این گونه برای .باشدمیخیص قابل تش بخش پایینی نخستین کمان آبششی خار آبششی در 9-11فلس در خط جانبی و 43 دهد که داده های جدید ما نشان میشود. های کیلیس، گازیانتپ و هاتای گزارش میدر استانرنتس اورودخانه ای حوضه اولین بار در بخش ترکیه یافته است گسترش رنتساورودخانه حوضه ایترکیهبخش طرف دامنه پراکنش این گونه به .، کلیسگازیانتپ ،رنتساورودخانه کولی، :کلمات کلیدی int. j. aquat. biol. (2021) 9(5): 344-349 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article anticoagulant effectiveness of glycosaminoglycan extracted from the scale of binni, mesopotamichthys sharpeyi (cyprinidae) hanaa a. abdulameer*, 1alyaa i. aldebs, neeran f. hassan college of pharmacy, department of clinical science, university of al-qadisiyah, al diwaniyah, iraq. s article history: received 12 july 2021 accepted 7 september 2021 available online 2 5 october 2021 keywords: fish scales glycosaminoglycan prothrombin thrombin physiology parameters abstract: fishery wastes are one of natural resources to extract bioactive substances such as collagen and glycosaminoglycan (gag). the anticoagulant activity of glycosaminoglycans extracted from binni fish, mesopotamichthys sharpeyi scales was the aim of this study. the cationic salt of cetyl pyridinium chloride was used to extract the glycosaminoglycan. the structure of the isolated glycosaminoglycan was identified by elise glycosaminoglycan kit and compared to that of heparin. prothrombin time (pt), and thrombin time (tt) on plasma of male mice at three concentrations of 20, 40, and 100 g/ml were used to determine the coagulant property of the extracted substance. the extracted glycosaminoglycan was calculated to be around 27.7 mg/g of dry tissue. the presence of heparin-like molecules in the glycosaminoglycan isolated from fish scales was confirmed by elise gag kit. when the concentration of isolated glycosaminoglycan was increased, the time to coagulate rose. the pt and tt coagulation times were 4:1 and 2:1. times faster than the control at 100 g/ml. when compared to synthetic anticoagulant substances like heparin, the glycosaminoglycan isolated from fish scales displayed good anticoagulation qualities. introduction glycosaminoglycans are polysaccharides with a negative charge (capila and linhardt, 2002), involving in variety of biological functions, including angiogenesis, host defense against viral infection, and blood coagulation (zhao et al., 2013). they are divided into four families based on their disaccharide composition viz. chondroitin sulfate cs, keratan sulfate ks, hyaluronic acid ha, and heparin sulfate hs (yamada and sugahara, 2008). heparin is a sulphated gags with anticoagulant properties utilizing in the treatment of viral infections, inflammation, and cellular proliferation in wounds and cancer, as well as in the acute coronary syndrome, lung embolism, vascular fibrillation, and deep pulmonary thumbs (linhardt, 2003). glycosaminoglycans are special type of glycoprotein (neil et al., 2005) that are prevalent in cartilages, vasculature, cell surfaces, intracellular granules, and plasma and other vertebrate’s organs (clarke, 2004; im et al., 2013). all of these are by *correspondence: hanaa a. abdulameer doi: https://doi.org/10.22034/ijab.v9i5. 1448 e-mail: hanaa.abdulameer@qu.edu.iq products of the meat and animal industries (volpi and maccari, 2002) from guts and lung of cows and pigs (linhardt and claude, 2003). as the demand for glycosaminoglycan’s rises, alternative marine resources are being used more frequently as a result, aquatic vertebrates and invertebrates have recently become increasingly important suppliers of glycosaminoglycan’s (rajapakse et al., 2005). several researches are concentrating on the extraction of glycosaminoglycans using marine sources (goldberg and buckwalter, 2005). bluefin tuna bone, jellyfish cartilaginous, and fishery wastes as potential glycosaminoglycan’s sources (maccari et al., 2015). reason of mortality in the poor countries, are disorders resulting from defective clotting in the circulatory system (zhang et al., 2014). to inhibit platelet aggregation and restore blood circulation via capillaries, medicines having anti-thrombotic, anticoagulant, and antiplatelet characteristics are utilized (vazquez et al., 2013). the existence of heparin and heparin derivatives in marine creatures, 345 int. j. aquat. biol. (2021) 9(5): 344-349 particularly squids, has been discovered (akbulut and akgul, 2014; pan et al., 2017). one of significant economic fish in iraq is the binni, mesopotamichthys sharpeyi that has substantial wastes during its process especially scales, and the scales might be a promising bioactive substances resource (tingbø et al., 2005). therefore, in this study scales were used to extract glycol-saminoglycan (gag) and assess their anticoagulant effectiveness in vitro utilizing traditional coagulation assays (world health organization 2008). materials and methods sampling and preparation: scales of the binni were obtained from a fish market in al-diwaniyah, iraq. to produce the dry powder, the fish scales were cleaned thoroughly with tap water, freeze-dried, then ground in a mixer. a total of 5 g scale powder was incubated for 1 hr at 80°c in 100 ml of mgso4 8.0 mm, then nacl was used to raise the ph of the solution to 10. the ph was then reduced to 7 using ammonium sulfate (2s04nh4) and heated at 50°c for 120 minutes. after filtering, the solution has been subjected to cinh 38 c21 / benzydamin (cpc). the filtrate was then treated with 20 ml of cpc (3: 0.8 m nacl). the solution was kept at 37°c for 1 day, resulting in a white color precipitate. 5 ml nacl before heated to 50°c was applied to extract n5h5c salt from of the precipitation. the sulfonated polysaccharide was precipitated with 75% ethanol and afterward the methanol was used to wash the precipitate multiple times before freeze-drying for an hour (najjam et al., 1997; tomatsu et al., 2005; gui et al., 2015) (fig. 1). glycolsaminoglycans (gags) detection: gags are extensively dispersed and, based on the particular gag, are linked to physiological and pathological functions. as a result, the development of precise, quick, sensitive, and specific assays of certain gags is critical. gags are used not only for diagnosis and therapeutic efficiency, but for many other abnormalities in which are down or up regulated, such as mucolipidoses (tomatsu et al., 2005), cancer (yip et al., 2006), osteoarthritis (plaas et al., 1998), rheumatoid arthritis (wang and roehrl, 2002), diabetes (olczyk et al., 1997), infectious diseases (iaci et al., 2007), and spinal cord damage (aydin, 2015; engvall, 2010). in this study, glycolsaminoglycans assay kit (colorimetric method) we used and for this purpose enzyme-linked immunosorbent assay (480 nm) bought from chondrex company (woodinville, wa 98072, usa) and using the company protocol, the antigen is bound to an antibody that is coupled to an enzyme, and antigen is detected by hydrolysis of a substrate by the linked enzymes (mucci et al., 2000; shute, 2012). coagulant test: the anticoagulation test was performed using citrated icr mice platelet deficient plasma. the plasma was taken from 36 male mice (with 20±2 g weight) after dividing into 3 groups as control, glycolsaminoglycans and heparin treatments each containing 12 mice. the plasma was mixed with 0.5% na3c6h5o7 solution as 99:1 blood to sodium citrate. the plasma was prepared by centrifuging at 5400 g for 25 min and then filtered and kept at -50°c until to use (krylov et al., 2011). standard clotting assays were used to determine the anticoagulant action of the isolated prothrombin time (pt), and thrombin time (tt). sodium hydroxide was used to dissolve the sample glycolsaminoglycans. all of the procedures had been done three times (majdoub et al., 2009). evaluating prothrombin time (pt): 70 pl of mice plasma was mixed with 30 pl heparin, 30 g/ml nacl, and different concentrations of isolated gags (20, 50, figure 1. mesopotamichthys sharpeyi (1), fish scales (2) demineralization of scales (3) and filtration and extraction glycolsaminoglycans (4). 346 abdulameer et al./ extraction of glycosaminoglycan from scale of mesopotamichthys sharpeyi and 100 pg/l) to measure the pt. for 15 minutes, the mixes were incubated at 50°c (chondrex, usa). then 100 pl of thromboplastin reagent before incubating at 50°c pt was added, and the clotting time was measured using a coagulometer (garnjanagoonchorn et al., 2007). evaluating thrombin time (tt): 70 pl of mice plasma were mixed 10 s of various gag levels (20, 40, and 100 pg/ml) and incubated at 50°c for 5 minutes. the tt was then recorded when 100 pl of thrombin was added. heparin and nacl were used as control sample, consequently, in place of the 10 pl of glycolsaminoglycans (pomin and mulloy, 2015). data analysis: due to the normal distribution, twoway analysis of variance anova was employed to compare the groups. in addition, as a post hoc test, the duncan multiple-range test was used. the mean and standard deviation were used to express all of the data (se). p<0.05 was used to determine the significance of the difference. spss software version 20.0 was used for data analysis. results the anticoagulant properties of the extracted glycolsaminoglycans are shown in table 1. the mean of pt and tt for mice plasmas were 20.4±0.4, 16.0±0.3 seconds, respectively. there was a significant difference in pt and tt between concentrations of the extracted glycolsaminoglycans (p<0.05). the anticoagulant indices of glycolsaminoglycans of fish scale increased as the concentrations of glycolsaminoglycans elevated. the prolonged coagulation times were observed in the presence of the extracted glycolsaminoglycans at the same concentrations, compared to control. at a concentration of 3 100 g/ml, the pt and tt glycolsaminoglycans of were 20.4±0.4 and 19.4±0.6 seconds, respectively which was 4 and 2 times slower than the control group. although, in the pt (p<0.05), there was significant variance between glycolsaminoglycans and heparin compared with control group. discussions anticoagulant properties are found in heparin and heparin-like substances such as glycolsaminoglycans and keratan sulphate in marine fishes (athukorala et al., 2006). the quantity of glycolsaminoglycans found in fish scale of the binni fish was 27.7mg/g of tissue. salmo salar, somniosus microcephalus, galeus melastomus, deania calcea, amblyraja hyperborea, and acipenser sinensis had 10.31, 13.96, 7.53, 6.66, and 15.51g of the sulphated glycolsaminoglycans from their cartilage in pervious researches, respectively (plaas et al., 1998; tomatsu et al., 2005; maccari et al., 2015). furthermore, the amount of glycolsaminoglycans extracted from 100 g of squid shell was estimated to be around 2 g /100 g dry weight (yang et al., 2015). coagulation analyzes of pt, and tt were performed in this work to assess anticoagulant activity. intrinsic and extrinsic coagulation cascades are assessed using the pt test and the tt test assesses the fibrin polymerization process. the findings of the table 1. relationship between protheombin time (pt) and thrombin (tt) with glycosaminoglycan as pt at various concentrations (/ml). pt 20 40 100 p glycolsaminoglycans 20.4±0.4 <0.05 heparin 23.6±0.7 <0.05 control 10.2±0.3 10.2±0.3 10.2±0.3 control 12.7±0.5 12.7±0.5 12.7±0.5 glycolsaminoglycans 20.5±0.4 23.5±1.2 24.7±0.2 <0.05 the results were expressed as means ±se (n = 12/group) figure 1. stander curve of glycolsaminoglycans assay kit from normal values. 347 int. j. aquat. biol. (2021) 9(5): 344-349 tt showed that scale glycolsaminoglycans can extend the coagulation time by 4:1 to 2: 1 times. there were significant variations in pt in the influence of glycolsaminoglycans. anticoagulant action of chondroitin sulphate isolated from various fish species, i.e. shark and sturgeon cartilage, has also been observed (pan et al., 2017). the glycolsaminoglycans extracted from sea cucumber produced similar results, with longer coagulation times as the content of glycolsaminoglycans elevated (yip et al., 2006). as a result, scale glycolsaminoglycans seem to have effect on the activity of extrinsic clotting factors. the increased tt levels imply that scale glycolsaminoglycans can influence the action of intrinsic coagulation factors and thrombin (engvall, 2010). heparin, for example, is a similar glycolsaminoglycans molecule with a wide range of biological functions. several factors, including species, structural composition, and sulphating patterns of glucose amines, might alter the anticoagulation function of glycolsaminoglycans substances (goldberg and buckwalter, 2005; tomatsu et al., 2005; maccari et al., 2015). the size of the scales’ mono-sulfated disaccharides, which are found in glycolsaminoglycans, play an important role in anticoagulation and anti-thrombin activities. the interaction of glycolsaminoglycans with the factors involved in the coagulation pathway results in a decrease in factor activity and an increase in clotting times (majdoub et al., 2009; pomin and mulloy, 2015). when compared to heparin, glycolsaminoglycans compounds isolated from fish scales demonstrated reduced coagulant activity. it is probably the amount of pollutants in the extracted glycolsaminoglycans molecules is to blame (plaas et al., 1998; iaci et al., 2007). the cpc method could extract a large amount of glycolsaminoglycans compound from fish scales. the coagulation tests pt, and tt revealed that the glycolsaminoglycans molecules might delay coagulation periods. finally, non-usable scales of fish can be a useful source of biological active chemicals (engvall, 2010; aydin, 2015). as conclusion, glycolsaminoglycans from fish scales were found to have coagulant properties by delayed prothrombin and thrombin times in the plasma of male mice and it can be used as a heparin alternative, at least in experimental studies. acknowledgements we would like to express our sincere thanks to people who took part in the experiments. the author also expresses gratitude to the research clinical science laboratory staff in pharmacy collage, university of alqadisiyah, for supporting and completion of this work. references akbulut m.d., akgül e. 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(2013). extraction, purification and characterisation of chondroitin sulfate in chinese sturgeon cartilage. journal of the science of food and agriculture, 93(7): 1633-1640. international journal of aquatic biology (2013) 1(6): 254-257 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article acute toxicity of diazinon to the caspian vimba, vimba vimba persa (cypriniformes: cyprinidae) mohammad mansouri chorehi, hamed ghaffari farsani, seyede amene hossaini*,1elahe hassan nataje niazie, mohammad forouhar vajargah, aliakbar hedayati 1 department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran article history: received 23 june 2013 accepted 16 october 2013 available online 2 5 december 2013 keywords: insecticide diazinon vimba lc50 abstract: the present research was performed to determine lethal concentrations of diazinon for the caspian vimba, vimba vimba persa. fish samples (50 ± 5 g) collected from sefidroud river were acclimatized for 5 days and exposed to 5 concentrations of diazinon, 0.07, 0.08, 0.1, 0.13 and 0.16 mg/l (with three replicates) and lethal concentrations (lc) lc1, lc10, lc30, lc50, lc70, lc90 and lc99 for 24, 48, 72 and 96 h were determined using a probit analysis. the results indicated that the 96 h lc50 value of diazinon for caspian vimba was 0.08 mg/l. introduction organophosphate pesticides are a major group of chemical insecticides being used widely throughout the world (garfitt et al., 2002) in agriculture, gardening, pest control, residential areas for protection of public health, veterinary and industry (bonilla et al., 2008). these insecticides frequently enter into surface water and groundwater through drainage from agricultural fields (sohrabi et al., 2001). diazinon is one of the most frequently used organophosphate insecticides for control of insects in agriculture. it becomes degraded rapidly in the aquatic environment. its half-life, in aerobic mineral soils, may be more than one month (garfitt et al., 2002; ahmadi et al., 2011). although diazinon is rapidly degraded, it may remain biologically active in the soil up to six month and even more under certain conditions such as low temperature, low humidity, high alkalinity and lack of active microbial decomposers (eisler, 1986). diazinon enters into * corresponding author: seyede amene hossaini e-mail address: amenehoseini@rocketmail.com tel: +981712220320 aquatic ecosystem in large quantities and affects non-target organisms (burkepile, 2000; maxwell and dutta, 2005). diazinon has been detected in significant amount in many coastal, deltaic and surface waters and also in municipal wastewater treatment plants around the world, including iran (shayeghi et al., 2001; u.s. epa, 2005). it has been detected in some drainage of rice fields in north of iran (nouri et al., 2000; tavakol, 2007) and mahabad, simine (honarpajouh, 2003), nahand (tarahi tabrizi, 2001), kor, sivand, shahpoor, mand, dalki (shayeghi et al., 2007), gorgan, gharesoo (bagheri, 2007), karaj river and most of mazandaran rivers (shayeghi et al., 2007; arjmandi et al., 2010). once entered into surface waters, even low doses of diazinon can cause several adverse effects in fish such as neurological impairment and abnormalities in gills, impairment in immune system, olfactory system and reproductive behavior, damage to ovary and testis and delay in 255 mansouri chorehi et al/ international journal of aquatic biology (2013) 1(6): 254-257 sexual maturity (eisler, 1986; moore and waring, 1996; dutta et al., 1997, dutta and maxwell, 2003; dutta and meijer, 2003, dutta et al., 2006). lc50 of diazinon is highly variable, and depends on the age, weight, and genus of fish and the environmental conditions. it affects sexual hormones, increases lh and fsh level and decreases testosterone levels significantly in mice (fattahi et al., 2009). vimba vimba is valuable species of cyprinid family (schweyer et al., 1991) and lives in the caspian sea, black sea and eastern part of the north sea. in iran, caspian vimba is distributed in the southern caspian sea from the anzali lagoon to the gorgan river. based on iucn classification the caspian vimba (vimba vimba persa) is a threatened species in the caspian sea (kiabi et al., 1999) and today the fish needs protection because of significant decline in stock, over fishing and destruction of habitat (jolodar and abdoli, 2004). the purpose of this research was to determine acute toxicity of diazinon to vimba vimba persa. materials and methods experiments were performed according to the oecd standard method (oecd, 1989), to determine 96 h lc50 of diazinon to caspian vimba. for these experiments 90 fish with an average weight of 50 ± 5 g were collected from sefidroud river and were acclimatized for 5 days, then divided into five treatments and one control (with three replications) in same aquaria (30 × 40 × 70 cm). physicochemical properties of water used for these experiments were as follows: 23 ± 1°c temperature, 7 to 9.5 mg/l dissolved oxygen, 6.5 to 8 ph and 220 mg/l total hardness. during the experiment, water was not exchanged. before the test, fish were fed twice daily with biomar feed at 2% body weight. five concentrations of diazinon used were 0.07, 0.08, 0.1, 0.13 and 0.16 mg/l. the nominal concentration of diazinon causing 50% mortality of caspian vimba within 24 h (24 h lc50), 48 h, 72 h and 96 h was determined using probit analysis in the software spss 16. results values of different lethal concentrations of diazinon for 24-96 h to caspian vimba have been presented in table 1. the results show that 96h lc50 value of diazinon for caspian vimba is 0.08 mg/l. discussion contrasting results are available on acute toxicity of diazinon to fish. tumer (2002) observed 96 h lc50 values for some freshwater teleost greater than 90 mg/l. on the contrary 96 h lc50 values of diazinon for acipenser persicus, acipenser stellatus and acipenser nudiventris were determined as 4.38, 2.54 and 0.36 mg/l, respectively (pazhand, 1999; shamooshaki, 2005) and those for rutilus frisii kutum, hypophthalmichthys molitrix and abramis brama were determined respectively as 0.34, 1.9 and 1.8 mg/l (nasri tajan, 1997). compared to these researches, vimba vimba is considered as highly sensitive and vulnerable to diazinon. 96 h lc50 value of diazinon to caspian vimba is even much lower than the sub lethal levels of diazinon (0.3 to 3.2 concentration (mg/l) points 24 h 48 h 72 h 96 h 1lc 0.01 0.06 0.04 0.007 10lc 0.16 0.1 0.07 0.04 30lc 0.28 0.12 0/09 0.06 50lc 0.36 0.14 0.1 0.08 70lc 0.44 0.16 0.12 0.1 90lc 0.55 0.19 0.14 0.12 99lc 0.71 0.23 0.17 0.16 table 1. lethal concentrations of diazinon for caspian vimba. 255 256 mansouri chorehi et al/ international journal of aquatic biology (2013) 1(6): 254-257 mg/l) that reduce emergence of stream insects cause potential mutagenicity in freshwater fish and spinal deformities in fish (eisler, 1986). references ahmadi s., jafari m., asgari a.r., salehi m. 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(2003). sub lethal effects of diazinon on the structure of the testis of bluegill, lepomis macrochirus: a microscopic analysis. environmental pollution, 125: 355–360. dutta h.m., maxwell l. (2003). histological examination of sublethal effects of diazinon on ovary of bluegill, lepomis macrochirus. environmental pollution journal, 121: 95-102. dutta h.m., munshi j.s.d., roy p.k., singh n.k., adhikari s., killius j. (1996). ultra structural changes in the respiratory lamellae of the catfish, heteropneustes fossils, after sub lethal exposure to malathion. environmental pollution journal, 92: 329–341. dutta h.m., qadri n., ojha j., singh n.k., adhikari s., datta munshi j.s., roy p.k. (1997). effect of diazinon on macrophages of bluegill sunfish, lepomis macrochirus: a cytochemical evaluation. bulletin of environmental contamination and toxicology, 58: 134-141. fattahi f., parivar k., jorsaraei s.g.a., moghadamnia a.a. (2009). the effect of diazinon on testosterone fsh and lh levels and testicular tissue in mice. iranian journal report medical sciences, 7(2): 59-64. garfitt s.j., jones k., mason h.j, cocker j. (2002). exposure to the organophosphate diazinon: datagram human volunteer study with oral and dermal doses. toxicology letters, 134(1-3): 105113. honarpajouh k. (2003). study and identification of op pesticides residues (azinphosmethyl and diazinon) in the mahabad and siminerood rivers, m.sc. thesis, tehran university of medical science. tehran, iran. jolodar m.n., abdoli a. (2004). fish species atlas of south caspian basin (iranian waters). iranian fisheries research organization, tehran. kiabi b.h., abdoli a., naderi m. (1999). status of the fish fauna in the south caspian basin of iran. journal of zoology in the middle east, 18: 57-65. maxwell b.l., dutta h.m. 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(cas registry number 33341-5). 257 international journal of aquatic biology (2015) 2(4): 274-281 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article evaluation of 5.8s rrna to identify penaeus semisulcatus and its subspecies, penaeus semisulcatus persicus (penaeidae) and some decapoda species zahra noroozi1, seyed javad hosseini*1, 21 1cell and molecular biology department, faculty of science, persian gulf university, bushehr, iran. 2biotechnology group of persian gulf institute, persian gulf university, bushehr, iran. article history: received 1 july 2014 accepted 2 june 2015 available online 2 5 august 2015 keywords: genetic variation nuclear marker crustacean decapoda abstract: the green tiger prawn, penaeus semisulcatus is one of the most important members of the family penaeidae in the persian gulf. based on the morphological characteristics, two groups, including p. semisulcatus and its subspecies viz. p. s. persicus are recognized. this study was conducted to investigate the genetic distance between p. semisulcatus and p. s. persicus by analyzing partial sequence of 5.8s rrna. another objective of this study is to evaluate the ability of 5.8s rrna to identify the species of decapoda. the results indicated that the 5.8s rrna gene of both p. semisulcatus and p. s. persicus were exactly identical, and sequence variation was not observed. the results also indicated that 5.8s rrna sequences between species of the same genus of analysed species of decapoda are conserved, and no genetic distance was observed in species level. the low evolutionary rate and efficient conservation of the 5.8s rrna can be attributed to its role in the translation process. introduction penaeid shrimps are the most important economic resource in the world’s crustacean fishery industry (voloch et al., 2005; holthuis, 1980; dall et al., 1990). the genus penaeus has 27 species (holthuis, 1980) and among them, the green tiger shrimp (penaeus semisulcatus) is included more than 90% of shrimp fishing in the persian gulf (hosseini et al., 2004). based on the morphological characteristics, two groups of p. semisulcatus are distinguished in the persian gulf. the first group (i) is characterized by a reddish body color with deep red or brown transverse bands, and cream and brown striped color of the whip antenna. second group (ii) i.e. subspecies of p. s. persicus, is characterized by a creamy pink body color without distinct transverse stripes, and its whip antenna has a cream color without stripes (rahnama et al., 2010). the group i is the main species in the coast of hormozgan * corresponding author: seyed javad hosseini e-mail address: sjhosseini@pgu.ac.ir province but group ii is found in the coast of bushehr province. the subspecies of p. s. persicus has been described based on the carapace morphology and protein electrophoresis patterns (matinfar, 1999). the identification of shrimps traditionally are relied on morphometric analysis; however, it is wellknown that such characteristics are influenced by environmental conditions (bowman et al., 1982). to overcome this problem, molecular markers e.g. nuclear and mitochondrial dna have been developed in the past two decades for study of the phylogenetic relationship and genetic diversity of such an aquatic organism (ferguson and danzmann, 1998; liu and cordes, 2004; chauhan and rajiv, 2010; askary et al., 2013). both nuclear and mitochondrial sequences are used for species identification and genetic diversity evaluation. the mitochondrial genes of 16s rrna and subunit i of cytochrome oxidase (coi) were extensively used for 275 noroozi and hosseini/ evaluation of 5.8s rrna to identify decapod species molecular study of the crustaceans especially shrimps of the family penaeidae (lavery et al., 2004; chan et al., 2008; nayak and umadevi, 2012). of the nuclear genes, 28s ribosomal rna, 18s rrna, 5.8s rrna, phosphoenolpyruvate carboxy kinase and sodium-potassium atpase α-subunit have been considered for the study of the phylogenetic relationships among shrimp (porter et al., 2005; calomata et al., 2009; ma et al., 2009). the molecular comparison of p. semisulcatus in persian gulf with its subspecies, p. s. persicus using mitochondrial 16s rrna showed a significant difference (rahnema et al., 2010). the genetic distance between them, based on a 561 bp section of the mitochondrial 16s rrna was calculated as 3.3% (rahnema et al., 2010). the high mutation rate of the mitochondrial dna limits its utility in the phylogenetics of deep divergences. furthermore, the highly a/t-biased mitochondrial dna, especially at the third codon position of the protein coding genes, suffers from high levels of homoplasy and thus exhibits strong negative effects in phylogenetic analyses (chu et al., 2009). therefore, it is necessary to evaluate the genetic distance between p. semisulcatus and the subspecies of p. s. persicus based on a proper and robust molecular marker. of the nuclear marker, the 5.8s rrna is considered for the study of the phylogenetic relationships among shrimps (calomata et al., 2009) and other organisms (gulling and voglers, 1998). hence, in the present study, we investigated the genetic distance between p. semisulcatus and p. s. persicus by analyzing partial sequence of 5.8s rrna. another objective of this study is to evaluate the ability of 5.8s rrna to identify the different species of decapoda. materials and methods sample collection and genomic dna extraction: 10 and 8 specimens of p. semisulcatus and p. s. persicus were collected from bushehr and dayyer (bushehr province, south of iran), respectively. total genomic dna was extracted from 100-150 mg muscle tissue from the ethanol-preserved samples according to brandfass and karlovsky (2008). dna amplification: the partial sequence of 5.8s rrna and complete sequence of internal transcribed spacer-ii were amplified using its2f (5’ gatcacttggctcgtgcgtc 3’) and its2r (5’ gctcttcccgtttcggtcgc 3’) primers. these primers have been designed based on 5.8s rrna sequence of p. merguiensis (ay331590) and p. vannamei (af 124597) and 28s rrna of p. semisulcatus (dq079809) and p. vannamei (af 124597), respectively. in fact, the its2f was designed to amplify of a 150 bp (93%) fragment of 3’ end of 5.8s rrna. in the first stage, pcr conditions were optimized using dmso (0, 2, 4 and 6%) and annealing temperature gradient. polymerase chain reactions (pcrs) were performed in 50 μl volume, containing 5 μl mg2+ free-pcr buffer (10x), 3 mm mgcl2, 0.4 mm of dntp mix, 20 pico mole of each primer, 2.5 u taq dna polymerase (fermentas), 200-400 nanogram dna, and ddh2o. the pcr reaction was carried out according to the following thermal program: 4 min at 94°c for initial denaturation, followed by 30 cycle with 94°c for 1 min, 59°c for 45 sec and 72°c for 1.5 min. the final extension was at 72ºc for 5 min. the negative control reactions were also used. the size and quality of pcr products were visualized on 1% agarose gel. dna sequencing and analysis: three samples of p. semisulcatus (sem3, sem4 and sem6) and three samples of p. s. persicus (per3, per4 and per6) were selected for sequencing. pcr products were sequenced in both strands, using the same primer pairs for pcr. the sequencing was performed using abi 370 automated sequencer (seq/teqh/california, usa). chromatograms of each of the forward and reverse sequences were checked using chromaspro and chromas lite softwares (technelysium pty ltd, australia). the sequences confirmation and homologies were searched using blast (ncbi). the sequences were aligned using the multiple– alignment program clustalw2 (larkin et al., 2007). base composition was calculated using mega6 software (tamura et al., 2013). the sequences distance matrix was calculated using kimura 2parameter (k2p) (kimura, 1980) and subjected to 276 international journal of aquatic biology (2015) 3(4): 274-281 the construction of neighbor-joining tree with 1000 bootstrap replicates (tamura et al., 2013). other conditions, for calculation of the genetic distance and tree construction include: transition and transversion substitutions, uniform rate among sites, homogeneous (same) pattern among lineages and complete deletion. for phylogenetic evaluation, the 5.8s rrna of other available species of the decapoda were retrieved from genbank database following blast search. at least two species of each genus were selected (table 1). then, their 114 bp of 5.8s rrna 3’ end were selected for phylogenetic reconstruction. all molecular analyses include sequence alignment, nucleotide composition, the pattern of nucleotide substitution, pairwise sequence distance and phylogenetic tree were conducted in mega6 (tamura et al., 2013). results and discussion the nuclear dna marker has been widely recruited for studies of phylogenetic relationship of crustacean (ahyong et al., 2007; liu and cordes, 2004; porter et al. 2005). the dna-based nuclear molecular markers can be classified into two types, the nuclear ribosomal rna (rrna or rdna) genes and protein –coding genes (ma et al., 2009; tsang et al., 2008; blanck et al., 2013). the nuclear ribosomal dna has three rrna genes (5.8s, 18s and 28s rrna) and two internal transcribed spacers (its-i and its-ii). the its-i and its-ii are located between 18s and 5.8s rrna and 28s rrna, respectively (gillespie et al., 2006). analysis of 5.8s rrna between p. semisulcatus and p. semisulcatus persicus: the 5.8s rrna section of pcr products were well-sequenced using its2f primer pairs, and reverse sequencing was failed. therefore, only 114 bp section of the 3’ end of 5.8s rrna, corresponds to more than 70% of the 5.8s rrna, was obtained from 6 studied samples. the base composition of the 5.8s rrna fragment of p. semisulcatus and p. s. persicus samples was as taxonomic designation abbreviation accession number region used penaeus vannamei p.vann af124597 853-967 macrbrchium rosenbergii m.ros hm804252 1180-1294 macrbrchium nipponense m.nipp gq369796 1519-1633 exopalaemon carinicauda e.car gq369794 469-583 exopalaemon cf. modestus e.cfmod gq369793 685-799 pandalus goniurus pa.gon ef035129 450-564 pandalus hypsinotus pa.hip ab193480 970-1021 pandalus eous pa.eou ab193477 790-904 eriocheir japonica e.jap af316381 382-496 eriocheir leptognathus e.lept af316385 385-499 eriocheir formosa e.for af316375 389-503 epilobocera sinuatifrons ep.sin fn395447 616-779 sesarma meridies s.mer fn396099 457-571 sesarma dolphinum s.dol fn396039 468-582 chionoecetes japonicus ch.jap hq909101 866-980 chionoecetes opili ch.opi hq909100 942-1056 table 1. accession numbers of the materials of decapod species retrieved from genbank database. table 2. maximum composite likelihood estimation of the pattern of nucleotide substitution. rates of different transitional substitutions are shown in bold and those of transversionsal substitutions are shown in italics. 277 noroozi and hosseini/ evaluation of 5.8s rrna to identify decapod species follows: a:17.5, t:24.6, g:26.3 and c:31.6. the multiple sequence alignment (fig. 1) indicated that the 5.8s rrna gene of both studied taxa were exactly identical, and no variation was observed. analysis of 5.8s rrna variation among decapoda: the pattern of nucleotide substitution between analyzed decapoda is shown in table 2. the rate of substitution of thymine by cytosine was 18.41%. the nucleotide frequencies were 23.16% (a), 21.08% (t/u), 29.00% (c), and 26.75% (g). the transition/transversion rate ratios were k1 = 1.273 (purines) and k2 = 2.453 (pyrimidines). the overall transition/transversion bias is r = 0.915, where r = [a*g*k1 + t*c*k2]/[(a+g)*(t+c)]. the alignment of 5.8s rrna resulted 129 sites. the aligned sequences showed that the gc content was more than at content (50.4% to 57.9% in pandalus and penaeus, respectively) and gc content average was calculated as 55.7%. the 129 sites of the 5.8s rrna gene were containing 74 conserved and 55 variable and parsimony informative sites. the average distance between all taxa was 0.339 and ranged from 0.00 between the species of one genus to 0.707 between penaeus and exopalaemon (table 3). the phylogenetic tree was inferred using the neighbor-joining method (saitou and nei, 1987) in table 3. the 5.8s rrna gene distance among some decapoda species analyzed by pairwise distance calculation using kimura two-parameter model. standard error estimate(s) are shown above the diagonal and were obtained by a bootstrap procedure (1000 replicates). figure 1. alignment of penaeus senisulcatus, and its subspecies based on a 114 pb fragment of 5.8s rrna (sem3, 4 and 6= p. senisulcatus and per3, 4 and 6= p. s. persicus). 278 international journal of aquatic biology (2015) 3(4): 274-281 mega6 software (tamura et al., 2013) based on k2p (fig. 2). the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (fleckenstein, 1985). the same tree topology was obtained by minimum evolution, upgma and maximum likelihood (tamura et al., 2013). the results of molecular analyses indicated that except for chionoecetes, no genetic variation observed between species of same genus among the studied members of decapoda. the constructed neighbor-joining tree separated taxa into three major clades, including clade a consisting sesarma (sesramidea), epilobocera (pseudothelphusidae), eriocheir (varunidae) and chionocetes (oregoniidae), clade b consists of pandalus (pandalidae), macrobrachium and exopalaemon (palaemonidae) and clade c consists of penaeus (penaeidae). the results indicated that clades a and b are closer to each other than to clade c. this result predictable, because, the members of the clades a and b taxa belong to suborder pleocyemata and clades c to suborder dendrobranchiata. the divergences within clades a, b, and c were 0.00.039, 0.0-0.136 and 0.0, respectively. the highest genetic diversity was estimated between the genus penaeus and exopalaemon (table 3), and the average distance between all taxa was 0.339. the maximum genetic distance (0.515) between the genus penaeus and exopalaemon, and minimum genetic distance (0.00) between the genus eriocheir and epilobacera were calculated, and the average genetic distance was calculated to be 0.185. when the full length of 5.8s rrna was used, compared with 114 bp section of the 3’ end of 5.8s rrna gene, genetic diversity is decreased. this suggests that 5.8s rrna 5’ end, compared with 3’ end, is the most conserved. similar to other nuclear genes, the 5.8s, 18s and 28s rrna genes evolve relatively slowly and are useful in addressing broad phylogenetic hypotheses involving a broad range of organisms i.e. a high level taxonomy (gulling and voglar, 1998). molecular studies using nuclear protein-coding genes indicated that they are highly informative for phylogeny estimation across all taxonomic levels of decapoda (chu et al., 2009). in addition, this study suggests that evolutionary rate of protein-coding genes are more than rrna genes. this phenomenon could be due to the fundamental role played by rrna in translation. the 5.8s rrna plays an important role in mrna translation (elela and nazar, 1997; graifer et al., 2005). studies on the inhibition of protein synthesis by specific anti 5.8s rrna oligonucleotides have suggested that 5.8s rrna plays an important role in eukaryotic ribosome function (elela and nazar, 1997). references askari g., shabani a., kolangi miandare h. 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(2005). model-based multi-locus estimation of decapod phylogeny and divergence times. molecular phylogenetics and evolution, 37: 355-369. porter m.l., perez-losada m., crandall k.a. (2005). model-based multi-locus estimation of decapod phylogeny and divergence times. molecular phylogenetics and evolution, 37: 355-369. rahnama r., hosseini s.j., qasemi s.a., yavari v., zolgharnein h., matinfar a. (2010). preiminary molecular comparison of p.(penaeus) semisulcatus of persion gulf and its subspecies penaeus semisulcatus persicus using 16s rrna. modern genetics, 25: 2331. saitou n., nei m. (1987). the neighbor-joining method: a new method for reconstructing phylogenetic trees. molecular biology and evolution, 4: 406-425. tamura k., stecher g., peterson d., filipski a., kumar s. (2013). mega6: molecular evolutionary genetics analysis version 6.0. molecular biology and evolution, 30: 2725-2729. tsang l.m., ma k.y., ahyong s.t. chan t.y., chu k.h. (2008). phylogeny of decapoda using two nuclear protein-coding genes: origin and evolution of the reptantia. molecular phylogenetic evolution, 48(1):359-68 voloch c.m., freir p.r., russo c.a.m. (2005). molecular phylogeny of penaeid shrimps inferred from two mitochondrial markers. genetics and molecular research, 4: 668-674. international journal of aquatic biology (2015) 3(4): 274-281 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved چکیده فارسی ،و زیرگونه آن penaeus semisulcatus میگوی سبز ببری در تشخیص 5.8s rrnaارزیابی ژن penaeus semisulcatus persicus (penaeidae) ای هو برخی گونهdecapoda 2، 1*، سید جواد حسینی1زهرا نوروزی شناسی سلولی و مولکولی، دانشکده علوم، دانشگاه خلیج فارس، بوشهر، ایران.زیست گروه1 ، دانشگاه خلیج فارس، بوشهر، ایران.خلیج فارس انستیتوبیوتکنولوژی گروه2 چکیده: این مطالعه برای بررسی در خلیج فارس است. penaeidaeخانواده مهم بسیار یکی از اعضای penaeus semisulcatusمیگوی سبز ببری، دیگر این هدف .به اجرا درآمد 5.8s rrnaوسیله آنالیز توالی بخشی از ژن هب p. s. persicus و p. semisulcatusفاصله ژنتیکی بین .pهر دو گروه 5.8s rrna ژن کهپایان است. نتایج نشان داد های دهبرای تشخیص گونه 5.8s rrnaتحقیق ارزیابی قابلیت ژن semisulcatus و p. s. persicus .ژنهای توالی که کهنشان داد همچنین نتایج کامالً مشابه هستند و تنوعی در توالی آنها مشاهده نشد 5.8s rrna اهده ای مشو هیچ فاصله ژنتیکی در سطح گونه بودهحفاظت شده پایان های آنالیز شده دههای مشابه گونههای جنسبین گونه .نسبت داده شود ترجمهتواند به نقش آن در فرایند می 5.8s rrnaو حفاظت موثر ژن نرخ پایین تکاملی شد.مین .پایانده، پوستان سخت، ایتهسه نشانگر، تنوع ژنتیکی: کلمات کلیدی int. j. aquat. biol. (2017) 5(1): 29-32; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology short communication a new record of the dwarf snakehead, channa ornatipinnis britz 2007 (perciformes: channidae) from india sivaramapillai muthukumar*1, muthukumarasamy arunachalam2, uthandakalaipandiyan ramesh1, murugiah umamaheswari2, alagappan vanarajan2 1department of molecular biology, school of biological sciences, madurai kamaraj university, madurai, india. 2sri paramakalyani centre for environmental sciences, manonmaniam sundaranar university, alwarkurichi, india. article history: received 7 january 2017 accepted 3 february 2017 available online 2 5 february 2017 keywords: channa ornatipinnis, new record, tuivawl, india. abstract: the snakehead of the family are represented 33 species from asia of which two species channa ornatipinnis and c. pulchra were recently described from myanmar. the recent record of c. ornatipinnis from tuivawl river, tuivawl village in champhai district, mizoram, india is of ichthyological interest introduction the snakeheads of the family channidae comprises of two genera, one in asia (channa) and the other in africa (parachanna). the genus channa comprises of 33 species in asia (courtenay and williams, 2004; serrao, 2014). in india, 12 species including channa amphibeus (mcclelland 1845), c. aurantimaculata (musikasinthorn 2000), c. barca (hamilton 1822), c. bleheri (vierke 1991), c. diplogramma (day 1865), c. gachua (hamilton 1822), c. marulius (hamilton 1822), c. melanostigma (geethakumari & vishwanath 2011), c. orientalis (bloch & schneider 1801), c. punctata (bloch 1793), c. stewartii (playfair 1867), c. striata (bloch 1793) are reported. fishes within this genus are characterized by an elongated cylindrical body, long and entirely soft-rayed dorsal and anal fins, a large mouth with well-developed teeth on both upper and lower jaws, and an accessory air-breathing apparatus known as the suprabranchial organ (musikasinthorn, 1998, 2003). snakeheads are of considerable interest as food fish and in aquarium trade and also as predators (courtenay and williams, 2004). * corresponding author: sivaramapillai muthukumar e-mail address: smk2882@gmail.com the dwarf snakehead, channa ornatipinnis, was described from waloun chaung in rakhine state of central myanmar by britz (2007). due to lack of information on its distribution, biology and population trends, this species was assessed as data deficient (britz, 2010). during recent field trips in tuivawl stream of champhai district, mizoram state in india, we collected 10 specimens of a snakehead that matched c. ornatipinnis, in its morphometric and meristic characters. therefore, this paper reports extension of its range of occurrence to india. materials and methods fish samples were collected from tuivawl stream in champhai district of mizoram state during may 2012 using gill nets and cast nets, and preserved in 10% formalin. voucher specimens are currently deposited in the manonmaniam sundaranar university museum of natural history (msumnh), alwarkurichi, tamil nadu, india, and specimens were preserved in collections of m. arunachalam, (cma). morphometric measurements and meristic counts generally follow hubbs and lagler (1964) and musikasinthorn (1998). a total of 10 individuals 30 muthukumar et al./ channa ornatipinnis, a new record of dwarf snakehead from india in the range of 98.44 to 161.77 mm sl were measured. abbreviations: d, dorsal fin; p, pectoral fin; c, caudal fin; hl, head length; sl, standard length. examined materials: msumnh 60, 8, 98.44-161.17 mm sl. cma 20, 2, 112.45-120.72 mm sl, india, mizoram, tuivawl river, tuivawl village, champhai district (23°48'16.4''n, 92°054'44''e), m. arunachalam, m. raja, c. vijayakumar and s. nandagopal. 06 may 2012. results and discussion description : body elongate, round in cross section anteriorly, laterally compressed at caudal peduncle, head large and wider in the opercular region with 1020 black spots on each side of cheeks and several irregular spots scattered on opercular region to caudal base. mouth large, maxilla extending posteriorly beyond jaw angle, lips fleshy, jaws with multiple rows of sharp, pointed minute teeth. dorsal fin rays 34(8)-36(2), anal fin rays 23(6) 24(4), pectoral fin rays 16, pelvic fin rays 6, principal caudal fin ray's 10-11, cheek scales 4(4)-5(6), lateral line scales 46(1)-48(9), scale rows above lateral line 4.5, below lateral line 7.5, circumpeduncular scales 28, circumferential scales 31(4)-32(6), pre-dorsal scales 14(2)-15(8), transverse breast row scales 9(3)10(7), anal scale rows 1, pre-anal scales 20(8)-21(2), lateral line scales dropping one row following 1516th anterior most scales. a pair of maxillary barbel. morphometric data of the examined specimens are listed in table 1. coloration: alcohol preserved specimens: dorsal surface of body dark grayish and ventral surface white grey, several small black irregular spots scattered on opercular region, cheek and body. pectoral fins with 6-7 semicircular white bands alternating with black bands. dorsal, anal and caudal figure 1. lateral view of channa ornatipinnis, msumnh 60, 132.52 mm sl), tuivawl river, mizoram, india. figure 2. natural habitat of channa ornatipinnis in tuivawl river, tuivawl village, champhai district, mizoram, india. 31 int. j. aquat. biol. (2017) 5(1): 29-32 fins with dark grey to blackish with few white spots, pelvic fin reddish white with black marks. live coloration: (fig. 1) dorsal side of the head greenish with golden orange, cheeks bluish grey with 15-20 bluish black spots, pectoral fin with red base and 6-7 semicircular white bands narrower than black bands, pelvic fin grayish brown with whitish margins. dorsal fin origin with 3 black blotches. dorsal, anal and caudal fins with reddish rays and bluish grey membranes. habitat: the tuivawl is a clear water third order stream with thick vegetation on its right and left banks (fig. 2) and substrate comprised of boulders, pebbles and small proportion of sand. the microhabitat from where the fish were collected was mostly run with low flow with a side pool in the 100 m thalweg length. two habitats were identified in the 100 m length with the density of c. ornatipinnis table 1. morphometric data of channa ornatipinnis from tuivawl river, mizoram, india (n=10). min max mean standard length (mm) 98.44 161.17 121.92 % of standard length pre dorsal length 34.16 37.95 35.63 pre anal length 47.49 50.34 48.99 pre pelvic length 28.93 31.02 30.10 pre pectoral length 25.69 29.98 28.85 pectoral fin length 19.66 23.73 21.10 pelvic fin length 9.03 12.50 11.04 dorsal fin base length 52.47 56.63 54.95 anal fin base length 39.53 41.62 40.73 caudal fin length 18.19 21.79 20.73 peduncle length 7.71 10.64 9.61 peduncle depth 8.90 11.85 10.40 occiput to dorsal origin 7.79 12.64 10.59 occiput to pectoral insertion 11.35 16.51 14.26 occiput to pelvic insertion 13.56 18.92 17.40 body depth 13.79 18.87 16.75 dorsal origin to pelvic insert. 14.03 18.06 16.10 dorsal insert to pelvic insert. 53.60 59.77 57.24 dorsal origin to pectoral insert. 12.44 18.54 14.97 dorsal origin to anal origin 18.92 26.47 22.99 dorsal insert to caudal base 6.64 8.91 7.73 dorsal insertion to anal origin 35.12 45.07 41.92 dorsal insert to anal insert. 9.56 19.39 12.63 pectoral insert to pelvic insert. 5.09 9.62 7.68 pectoral insert to anal origin 18.64 23.17 20.65 pelvic insert to anal origin 16.19 20.16 17.48 distance b/w pectoral fin to vent 17.26 23.88 21.81 distance b/w pelvic fin to vent 17.68 21.60 18.80 head length 34.36 37.53 36.17 % of head length pre occipital length 83.96 89.93 86.65 snout to opercle 88.16 94.84 91.71 upper jaw length 34.13 44.90 39.02 snout length 20.73 25.74 23.62 pre nasal length 14.39 21.19 16.70 orbit width 11.22 14.15 13.20 inter orbital width 28.07 35.23 30.13 inter nasal width 14.40 20.12 17.78 head width 57.74 61.16 58.96 head depth 37.68 45.36 42.02 head depth at nostril 12.82 19.35 16.54 head depth at pupil 27.26 32.49 29.93 head depth at occiput 39.09 43.53 40.56 32 muthukumar et al./ channa ornatipinnis, a new record of dwarf snakehead from india occurred in pool and in the inflow and outflow of the pool. velocity of the water was between 15 to 20 cm/s and the depth between 0.4-1.2 m. collection of pebbles and gravels and sands was a major disturbance in the channel. remarks: till date, there has been no documented record of the occurrence of c. ornatipinnis outside of the rakhine state in central myanmar and therefore the present record of the species from a stream in mizoram state, india is of special interest. acknowledgements the author muthukumarasamy arunachalam was supported by manonmaniam sundaranar university under one time grant by university grants commission, new delhi for faculty/professors produced 15 ph.d.s in ugc-bsr. {no.1988/2013(bsr) dt..21, nov., 2013}. this research was also possible with grants to r.l. mayden under saint louis university and the usa national science foundation grants ef0431326, deb1021840 and dbi-0956370 for the taxonomy and systematics of cypriniformes. the two initiatives, cypriniformes tree of life and all cypriniformes global biodiversity initiative (www.cypriniformes. org) have aided in this mission. references britz r. (2007). channa ornatipinnis and c. pulchra, two new species of dwarf snakeheads from myanmar (teleostei: channidae). ichthyological exploration of freshwaters, 18(4): 335-344. courtenay w.r., williams j.d. (2004). snakeheads (pisces, channidae): a biological synopsis and risk assessment. us geological survey circular, 1251. 143 p. hubbs c.l., lagler k.f. (1964). fishes of the great lake region. ann arbor, university of michigan press. 213 p. iucn. (2012). iucn red list of threatened species. version 2012.1. www.iucnredlist.org. downloaded on 21 august 2012. mcclelland j. (1845). description of four species of fishes from the rivers at the foot of boutan mountains. journal natural history, calcutta, 5(18): 274-282. musikasinthorn p. (1998). channa panaw, a new channid fish from the irrawaddy and sittang river basins, myanmar. ichthyological research, 45: 355-362. musikasinthorn p. (2000). channa aurantimaculata, a new channid fish from assam (the brahmaputra river basin), india, with designation of a neotype for c. amphibeus (mcclelland, 1845). ichthyological research, 47: 27-37. musikasinthorn p. (2003). channoidei (snakeheads) in: m. hutchins, a. thoney, p.v. loiselle, n. schlager (eds.). grzimek’s animal life encyclopedia, 2nd ed. vols 4, 5. fishes i–ii. gale group, farmington hills. pp: 437-447. viswanath w. (2000). fish fauna of manipur. manipur association for science and society, 137 p. viswanath w. (2002). fishes of north east india, a field guide to species identification. manipur university and natp. 198 p. vishwanath w., geetakumari kh. (2009). diagnosis and interrelationships of fishes of the genus channa scopoli (teleostei: channidae) of northeastern india. journal of threatened taxa, 1(2): 97-105. serrao n.r., steinke d., hanner r.h. (2014). calibrating snakehead diversity with dna barcodes: expanding taxonomic coverage to enable identification of potential and established invasive species. plos one, dx.doi.org/10.1371/journal.pone.0099546. int. j. aquat. biol. (2017) 5(1): 29-32 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی :ن اانواد ماهیا:ن ماهی شکال)سوف channa ornatipinnis britz 2007ش ماهی سرماری دارف، رنخستین گزا سرماری( از هندوستا:ن 2، االگاپپا:ن واناراجا:ن2، موروگیا اماماهسواری1اوتانداکاالی پاندییا:ن رامش ،2موتوکوماراسامی آرونچاالم ،1*سیواراناپیالی موتوکومار .مادورای کاماراج، مادورای، هندوستان دانشگاه ،مدرسه علوم زیستی لی،ولکوشناسی مگروه زیست1 وستان.د، دانشگاه مانونمانیام سوندارانار، الوارکوریچی، هنسری پاراماکاالیانیمرکز علوم محیطی 2 چکید از میانمار توصیف شده است. این c. pulchraو channa ornatipinnis دو گونهاخیراً وباشد آسیا میگونه در 33ن سرماری دارای خانواده ماهیا ب لیافته جاعنوان به قه چامپهای، میزورام هندوستان رامنطل ول، روستای تویواوودخانه تویوار از c. ornatipinnis مقاله نخستین گزارش حضور نماید.شناسی گزارش میماهی .هند ،لوتویوا ،شنخستین گزار ،channa ornatipinnis کلمات کلیدی int. j. aquat. biol. (2021) 9(1): 66-70 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology short communication anatomical, histoarchitectural and topological studies on the olfactory organ of freshwater garfish, xenentodon cancila (hamilton, 1822) saroj kumar ghosh* 1 department of zoology, bejoy narayan mahavidyalaya, itachuna, hooghly-712 147, west bengal, india. article history: received 7 june 2020 accepted 20 february 2021 available online 2 5 february 2021 keywords: needlefish olfactory epithelium cellular constituent microarchitecture chemoattraction abstract: the olfactory structure of xenentodon cancila (hamilton, 1822) were explored by advancement in microtomy, staining and ultrastructural practices. the unique feature of the olfactory system was that the olfactory cavity, an open groove with an obtruding sole lamella, no rosette like organization. the lamella was constituted of the central core, lined on both sides by well-organized epithelium. the central core usually consisted of connective tissue fibres and blood capillaries. the epithelium exhibited compact cellular distribution and made up of receptor cells, supporting cells, lymphatic cells, inner most basal cells and almost never mucous cells. morphologically specific two types of receptor neurons were recognizable: ciliated and microvillous, comprising sensory terminals. the cellular constitution of olfactory mucosa was explained with olfactory sensitivity of the fish necessitated. introduction olfaction is the crucial chemosensory channel, playing essential role in the behaviour of teleosts. it is involved in food finding, enemies’ recognition, mate selection, parental behaviour, shoaling, migration and in many more approaches (song, 1987). olfaction is accomplished by olfactory receptor cells on the mucosal surface which remains directly exposure to surrounding aquatic environment (singh et al., 1995). among vertebrates, fishes exhibit considerable variations in the anatomical and morphological features of the olfactory organ as depicted by kasumyan (2004), hansen and zielinski (2005), ghosh (2012), kuciel et al. (2013), and oliver and oliver (2019). olfactory mucosa consists of a mosaic of sensory cells, classified as ciliar, microvillar, rod and crypt receptor neurons which have high particularity and sensitivity to chemical stimulation. in teleostean fishes, the structural specialization and function of olfactory organ is related to their ecological niche (hara, 1994). the structural peculiarity of the olfactory organ in needlefishes has been attracted by researchers (singh, 1972, 1977; *correspondence: saroj kumar ghosh doi: https://doi.org/10.22034/ijab.v9i1.1041 e-mail: saroj.fisherylab@gmail.com theisen et al., 1980). xenentodon cancila (hamilton, 1822) is a carnivorous silver needlefish, feeds preferably on insects, crustaceans and small fishes (gupta and banerjee, 2017). there is no report about the cellular details in the olfactory mucosa of asian garfish. thus an attempt has been taken to investigate the olfactory structure in x. cancila (beloniformes: belonidae) at the light and scanning electron microscopic level. materials and methods collection of sample: a total fourteen mature specimens of x. cancila (18.5-24.5 cm) were collected from damodar river, nearby jamalpur (23.061089°n, 87.992584°e) of purba bardhaman, west bengal. the specimens were identified based on misra (2003). fishes were anaesthetized with 0.01% ethyl 3aminobenzoate methanesulfonate (ms-222; merck) solution and sacrificed following the protocol of the institutional animal ethics committee. the olfactory organs were dissected out precisely to display the position under a stereoscopic binocular microscope (magnus ms24) and rapidly processed for histology 67 int. j. aquat. biol. (2021) 9(1): 66-70 and scanning electron microscopy. histological preparation: olfactory tissues were immersed in bouin’s fluid for 18 h. after fixation, the tissues were washed in 70% ethanol, dewatered in an ascending series of ethanol, cleared in dimethylbenzene and embedded in paraffin (56-58˚c, sigmaaldrich). serial sections of tissue block were cut at 4 µm thickness using a rotary microtome (weswox mt1090a). tissue sections were stained with delafield’s haematoxylin-eosin (romies, 1968) and mallory’s triple (mallory, 1936). the staining slides were viewed and photographed under light microscope (carl zeiss primo star) equipped with microscope camera (tucsen 5.0 mp). scanning electron microscopic preparation: ahead removal of the olfactory organs, they were rinsed with 2.5% glutaraldehyde in 0.1 m phosphate buffer (ph 7.4) for 10 min. then the olfactory organs were dissected out and washed with 1% polyoxyethylenesorbitan monopalmitate solution (tween 40; merck) to remove the adhering mucus and debris from the surface. the samples were fixed in glutaraldehyde of same concentration for overnight at 4˚c and additionally fixed in 0.1 m phosphate buffered (ph 7.4) 1% solution of osmium tetroxide (oso4; sigmaaldrich) for 1 h at room temperature. after dehydration through a graded acetone series followed by isoamyl acetate, samples were dried by co2 critical point apparatus. after sputtered with platinum (using bt-150 sputter coater, hind high vacuum co. pvt. ltd.), the olfactory organs were observed and photographed in the scanning electron microscope (zeiss evo 18). results and discussion anatomy: paired olfactory organs of x. cancila are located in the depression of ethmoidal region of the skull (fig. 1a). they are placed on the dorso-lateral side of the head, near the eyes in the shape of simple open pits. the usual olfactory rosette is absent, replaced by a protruding solid lamella (fig. 1b). this laminate rosette has stubby base which is lodged in the ground of the groove. the thin elongated olfactory tracts which are composed of a bundle of olfactory fibres, connect the olfactory organ and olfactory lobes of forebrain (fig. 1c). histology: the olfactory lamella is made up of central core, enclosed by pseudostratified olfactory figure 1. the olfactory organ of xenentodon cancila. (a) lateral aspect of x. cancila showing the olfactory pit (arrow). (b) enlarged view of head showing the position of olfactory organ (arrow) in front of eye (e). (c) dissected dorsal view of head region shows olfactory organ (o) along with elongated olfactory tracts (arrows), olfactory lobe (ol), cerebrum (cb), optic lobe (opl), cerebellum (cl) and medulla oblongata (mo). 68 ghosh / olfactory structure of xenentodon cancila epithelium on both sides (fig. 2a-b). the basement membrane separates the olfactory epithelium from the central core (fig. 2b). the central core is composed of connective tissue, nerve fibres, fibroblasts and pigment cells. blood capillaries are encountered in this region. the olfactory epithelium is comprised of supporting cells, basal cells, rarely occurring mucous cells, lymphatic cells and notably two types receptor neurons: ciliated and microvillous cells. the cell types are evenly differentiated by the morphology, contour, staining property and their position in the mucosa. the ciliated receptor cells are compactly arranged in proximal zones, characterized with spindle shaped body having basally located dark nuclei and extension of dendrites bearing apical swelling over the epithelial surface (fig. 2a-b). relatively smaller microvillous cells are closer to epithelial lining; contain gentle stained rounded nuclei (fig. 2b). the non-ciliated supporting cells are intermingled in the epithelium, provided with intensely stained nuclei and faintly discernible chromatin material. the ciliated supporting cells are columnar in shape, having acentally located nuclei and granular cytoplasm (fig. 2a). the mucous cells are rarely observed in the surface zone. they are empty in nature due to release of muciferous contents. oval shaped lymphatic cells are scattered throughout the mucosa, contain profoundly stained nuclei and discreet cytoplasm (fig. 2a-b). the nuclei hold maximum of cell size. the rounded basal cells with large central nuclei are buried in the distal zone of mucosa, over the basement membrane. scanning electron microscopy: the olfactory organ is rapheless and contains one lamella. the surface of lamella contains structurally distinct ciliated receptor cells, microvillous receptor cells along with ciliated supporting cells and stratified epithelial cells. sporadically occurring ciliated receptor cells have cylindrical projected sensory dendrites on the free surface (fig. 3a, c). microvillous cells bear murky carpet of tiny microvilli, display sculpt surfacing (fig. 3a). the microvilli are stubbier in comparison to cilia. the sensory receptor cells are overlapped in the mucosa. mass of the ciliated supporting cells show squashy appearance. they bear nonsensory ciliary endings emerge from cell bodies (fig. 3b-c). stratified epithelial cells are arranged like epidermal outside pattern, marking with microridges (fig. 3cd). a very few number of mucous cells along with secreted mucin are observed in between stratified epithelial cells. chemical stimuli are exposed by apical parts of figure 2. histology of the olfactory lamella of xenentodon cancila stained with delafield’s haematoxylin-eosin (he) and mallory’s triple (mt). (a) olfactory epithelium (oep) exhibiting ciliated receptor cells (c) with apical protrusions (solid arrows), ciliated supporting cells (csc), mucous cell (broken arrow), lymphatic cells (asterisks) and basal cells (bc). central core (cc) contains blood vessels (bv). (b) pseudostratified olfactory epithelium (oe) shows ciliated receptor cells (c), microvillous receptor cells (mv), non-ciliated supporting cells (sc), lymphatic cells (asterisks) and basal cells (arrow heads). cc consists of connective tissue (ct), blood vessels (bv) and pigment cells (pc). note the presence of basement membrane (bm) in between oep and cc. 69 int. j. aquat. biol. (2021) 9(1): 66-70 sensory cells bounded to the olfactory mucosa and admissible behaviours expressed by fish species. in x. cancila, the olfactory cues are detected by unilamellar olfactory organs and conveyed precisely to the central nervous system by olfactory tract. xenentodon cancila have a pair of unilamellar olfactory organ, appearing as an open pit, without nostrils, which is related to their behavioural adaptations. the results showed that x. cancila belongs to teichmann’s (1954) second group of eye fishes i.e., sight feeder and pol gerard (1954) first category i.e., anosmic in which olfactory organ is regressed and seek their food by vision. morphologically distinct ciliated and microvillous neurons on the mucosa are responsible for appropriate reception of olfactory sensation. stratified epithelial cells bearing microridges are thought to safeguard for supporting tissues while disclosed to water forces and help in mechanical dissociation (uehara et al., 1991). numerous ciliated supporting cells are able to generate a gentle water flow over the epithelial surface and perform as guides allowing chemicals to contact with the receptor sites of sensory neurons. same finding was cited by singh and singh (1989) in the olfactory organ of heteropneustes fossilis. occurrence of lymphatic cells in the epithelial layer are function as part of cell immunity (lieschke and trede, 2009; kim et al., 2019). mucous cell discharges mucus which shields the epithelium from mechanical rubbing. graziadei and metcalf (1971) mentioned that the basal cells in the deeper layer of mucosa are capable to differentiate into receptor cells while ojha and kapoor (1973) reported their conversion into supporting cells. acknowledgements the author is obliged to the authority of sophisticated analytical instrumentation facility (saif), figure 3. scanning electron micrographs of olfactory lamella of xenentodon cancila (a) surface of lamella shows ciliated receptor cells (c), microvillous receptor cells (mv) and stratified epithelial cells (sc). arrows mark mucin masses over sc. (b) mucosal surface shows thick ciliated supporting cells (csc) and secreted mucus clumps (arrows). (c) part of epithelium displays ciliated receptor cells (c) with prolonged dendrites, ciliated supporting cells (csc), stratified epithelial cells (sc) and opening of mucous cells (arrows). (d) olfactory epithelium furnishes stratified epithelial cells (sc) with microridges (arrow heads) and aperture of mucous cells (omc). solid arrows mark lump of mucin on top of sc. 70 ghosh / olfactory structure of xenentodon cancila department of anatomy, all india institute of medical sciences (aiims), new delhi-110 029, india for kind assistance in scanning electron microscopy. references ghosh s.k. (2012). histoarchitecture, surface ultrastructure and histochemical studies of the olfactory organ of labeo bata (hamilton), rita rita (hamilton) and etroplus suratensis (bloch): a comparative study. ph.d. thesis, department of zoology, the university of burdwan. 117 p. graziadei p.p.c., metcalf j.f. (1971). autoradiographic and ultrastructural observations on the frog’s olfactory mucosa. zeitschrift fur zellforschung und mikroskopische anatomie, 116: 305-318. gupta s., banerjee s. (2017). food, feeding habit and reproductive biology of freshwater garfish, xenentodon cancila: a short review. international journal of fisheries and aquatic studies, 5: 423-427. hansen a., zielinski b.s. (2005). diversity in the olfactory epithelium of bony fishes: development, lamellar arrangement, sensory neuron cell types and transduction components. journal of neurocytology, 34: 183-208. hara t.j. (1994). the diversity of chemical stimulation in fish olfaction and gestation. reviews in fish biology and fisheries, 4: 1-35. kasumyan a.o. (2004). the olfactory system in fish: structure, function, and role in behavior. journal of ichthyology, 44: 180-223. kim h.t., yun s.w., park j.y. (2019). anatomy, ultrastructure and histology of the olfactory organ of the largemouth bass micropterus salmoides, centrarchidae. applied microscopy, 49: 1-6. kuciel m., zuwała k., satapoomin u. (2013). comparative morphology (sem) of the peripheral olfactory organ in the oxudercinae subfamily (gobiidae, perciformes). zoologischer anzeiger-a journal of comparative zoology, 252: 424-430. lieschke g.j., trede n.s. (2009). fish immunology. current biology, 19: 678-682. mallory f.b. (1936). the aniline blue collagen stain. stain technology, 11: 101. misra k.s. (2003). an aid to the identification of the common commercial fishes of india and pakistan. narendra publishing house. delhi. 320 p. ojha p.p., kapoor a.s. (1973). histology of the olfactory epithelium of the fish, labeo rohita ham. buch. archives de biologie, 44: 425-441. oliver j., oliver s. (2019). an update on anatomy and function of the teleost olfactory system. peerj, 7: e7808. pol gerard (1954). organe olfactif. traité de zoologie, 12: 522-533. romeis b. (1968). mikroskopische technik. oldenbourg verlag, münchen, wien. 757 p. singh c.p. (1972). a comparative study of the olfactory organ of some indian freshwater teleostean fishes. anatomischer anzeiger, 131: 225-233. singh s.p. (1977). functional anatomy of olfactory organs in some marine teleosts. zoologischer anzeiger, 5b: 441-444. singh s.p., singh s.b. (1989). a sem study of the olfactory lamellae of the catfish heteropneustes fossilis (bl.). folia morphologica, 37: 407-409. singh n., bhatt k.c., bahuguna m.k., kumar d. (1995). fine structure of olfactory epithelium in schizothoraichthys richardsonii gray (cyprinidae: teleostei) from garhwal himalaya (india). journal of biosciences, 20: 385-396. song t.f. (1987). chemical communication of fish. journal of fisheries of china, 11: 359-371. teichmann h. (1954). vergleichende untersuchungen an der nase der fische. zeitschrift für morphologie und ökologie der tiere, 43: 171-212. theisen b., breucker h., zeiske e., melinkat r. (1980). structure and development of the olfactory organ in the garfish belone belone (l.) (teleostei, atheriniformes). acta zoologica, 61: 161-170. uehara k., miyoshi m., miyoshi s. (1991). cytoskeleton in microridges of the oral mucosal epithelium in the carp, (cyprinus carpio). the anatomical record, 230: 164-168. int. j. aquat. biol. (2017) 5(2): 71-78; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article histopathological evaluation of the zebrafish (danio rerio) testis following exposure to methyl paraben nasrin hassanzadeh*1 department of environmental sciences, faculty of environment and natural resource, malayer university, malayer, p.o. box. 65719-9581863, iran. article history: received 5 december 2016 accepted 2 march 2017 available online 2 5 april 2017 keywords: histopathology parabens reproductive toxicity abstract: methyl paraben (mp) is widely used as a preservative in various products. it frequently enters into aquatic environment and renders potential threat to fish. the aim of this study was to evaluate reproductive toxicity of mp on zebrafish (danio rerio) under laboratory conditions. male zebrafish were exposed to four concentrations of mp (0.001, 0.01, 1, and 10 mg l-1) for 21 days in semi-static condition. changes in mean length, mean weight, gonadosomatic index (gsi) and histology of testis were studied. treatment at 0.001 to 10 mg l-1 mp had no significant effect on the survival, mean length and mean weight of fish. but, gsi decreased in a dose dependent manner and the decrease was significant in the group that received the highest dose. histological alteration of testis consisted of general testicular atrophy, multi-nucleated gonocytes (mngs), impaired germ cell, spermatogonial proliferation, leydig cell hyperplasia, interstitial fibrosis and apoptosis of sertoli cells. it was concluded that sub-chronic exposures of mp could adversely affect gsi, disrupt the histology of testis and produce estrogenic and antispermatogenic activity in male zebrafish. introduction the endocrine disruption compounds (ecds) are a large group of chemicals which enter into the aquatic environment from manufacture of various industrial and consumer products, agriculture and food/drug processing, waste water treatment plants and human wastes. this group includes certain polychlorinated biphenyls, polyaromatic hydrocarbons, dioxins, furans, pesticides, alkylphenols, synthetic steroids, plant sterols and parabens (hamilton et al., 2008; jasinska et al., 2015). some of the most potent endocrine disruptors share p-substituted phenol as a molecular structure, which includes parabens. parabens (esters of 4-hydroxybenzoic acid, also known as alkyl p-hydroxybenzoates), are used as preservatives in cosmetics, toiletries, pharmaceuticals and foods, and are antibacterial agents in certain toothpastes (fig. 1). four main parabens are in use: methyl, ethyl, propyl and butyl parabens; many products will have two or more of these chemicals as * corresponding author: nasrin hassanzadeh doi: https://doi.org/10.22034/ijab.v5i2.245 e-mail address: nasrinhassanzadeh@gmail.com part of a preservative system. in a survey of 215 cosmetic products, methyl paraben was detected in 98% of products (barse et al., 2010; oishi, 2002). approximately 8000 tons of parabens are consumed annually around the world, and most are continuously released into the environment during their production, use, and disposal (gao et al., 2016). methyl paraben is one of a homologous series of parabens, used singly or in combination to exert the intended antimicrobial effects and it has been used as an antimicrobial preservative in foods, drugs and cosmetics for over 50 years (taylor et al., 2002). exposure to synthetic estrogens (such as parabens) may adversely affect human health, particularly with regard to the reproductive cycle and reproductive function. ethyl, propyl and butyl parabens have previously been shown to possess estrogen-mimicking properties in fish (barse et al., 2010; taylor et al., 2002; bjerregaard et al., 2003). recently, parabens have been shown to act as xenoestrogens, a class of 72 hassanzadeh/ histopathological effect of methyl paraben on the zebrafish testis endocrine disruptors (eds), whose chemical structures can be closely associated with differences in their estrogenicity. like other xenoestrogens, parabens can also mimic the effects of physiological estrogens. they may bind to estrogen receptors (ers), stimulate the er-dependent response, and/or influence the expression of estrogen-responsive genes, including erα, the progesterone receptor (pr) and ps2. the potency of parabens as estrogens appears to depend on the lengths of their alkyl side chains(vo et al., 2010). in addition, some recent studies have reported adverse reproductive effects of parabens (routledge et al., 1998; boberg et al., 2010; gonzalezdoncel et al., 2014). the zebrafish (danio rerio) is increasingly used as an ecological model species for studies of stress physiology including the effects of the exposure to different environmental chemicals (segner, 2009). in fact, zebrafish has been found useful in edc screening, in edc effects assessment and in studying targets and mechanisms of edc action. since many of the environmental edcs interfere with the sex steroid system of vertebrates, most edc studies with zebrafish addressed disruption of sexual differentiation and reproduction (yang et al., 2009). numerous studies have evaluated susceptibility of aquatic organisms such as fish to toxic effects of exposure to different parabens (barse et al., 2010; vo et al., 2010; bjerregaard et al., 2003), in vivo studies of methyl paraben with zebrafish, which are lacking in the literature. gonad histopathology is also a valuable tool for the assessment of endocrine-disrupting effects on fish. the molecular events evoked by hormonally active agents have an effect on the levels of cell, tissue, and organ organization and morphology, and the nature of the evoked effects is specific and depends on the hormonal system that has been disrupted. in addition, histopathological changes, specifically those in the gonads, can be predictive of the (reproductive) fitness of the specimen under study and thus for the fitness of the population (en et al., 2003). therefore, the aim of this study was to investigate the effects of sub-chronic exposure of methyl paraben on gsi and testis histopathology in zebrafish under laboratory conditions. materials and methods test organisms: adult male zebrafish, with an age of about 120 day, were obtained from a local dealer and acclimated to laboratory conditions for 2 weeks before the experiment. during the acclimation period and experiment phases, fish were given daily supplies of standard fish pellets (1% body weight / day) and inspections were conducted twice a day to discard wounded, diseased and dead fish. fish were kept at a maximal density of 5 fish l-1 in 10l tanks (27±1°c) on a 12-h light:12-h dark photoperiod. ion concentrations in the synthetic water were (mg l-1): 6.26 k+, 11.5 na+, 4.74 mg2+, 11.6 ca2+, 32.4cl−, 31.0 no3 −, 9.61 so4 2− and 0.45 co3 2. experimental design: methyl paraben (cas no: 9976-3) (fluka chemika, germany), hplc grade ethanol (merck), were purchased. stock solution of the methyl paraben was prepared in ethanol at concentration 100 mg l-1. four groups of fish were subjected to serial dilutions of the stock solution of methyl paraben of 0.001, 0.01, 1.0 and 10.0 mg l-1. in addition to natural controls (unexposed males), solvent control (ethanol) and a positive control group (17-β estradiol; 10.0 ng l-1) were employed in the study. each treatment was performed in two different tanks (20 fish each). the test was performed by the semistatic (renewal) method in which the exposure medium was exchanged every 24 hrs to maintain toxicant strength and level of dissolved oxygen as well as minimizing the level of ammonia excretion during this experiment. the exposure period lasted 3 weeks. length and weight of the fish were recorded after the experiment. on the last day of experiment, fish were anesthetized on ice and testis were excised and figure 1. chemical structure of paraben. 73 int. j. aquat. biol. (2017) 5(2): 71-78 weighed for gonadosomatic index (gsi) determination. the gsi was expressed in grams per 100 g body weight. histological preparation: histological examinations were performed as described by (khodabandeh and abtahi, 2006; eagderi et al., 2013). for histological studies, gonads were immediately fixed in bouin’s fluid for 24 hrs. fixed testes were dehydrated through a graded series of ethanol and embedded in paraffin. serial sections were cut at 4 µm and collected onto glass slides, stained by haematoxylin and eosin, and then examined under a light microscope. gonads from six males were examined from each treatment. statistical analysis: to compare the mean length, mean weight and gsi of fish between controls and mean values of different concentrations of methy paraben, one-way analysis of variance (anova) was used. the data were tested for homogeneity of variances before anova and significance was tested at a level of p<0.05. significant difference between means were tested by duncan’s multiple range test (p<0.05). the statistical package spss, version 16, (chicago, il, usa) were used for data analysis. ethical considerations and animal rights in this paper were considered and the study was approved by the international agency for protection of experimental animals and by the in house animal welfare committee. results there was no mortality during the experimental period in control and treatments. the mean length, mean weight and gonadosomatic index (gsi) of fish from different controls and methyl parabenare treated groups have been presented in table 1. the results indicated that the mean length and mean weight of male zebrafish were not significantly different between controls and methyl paraben treatments. but gsi was significantly different between methyl paraben treatments and control groups. changes in gsi in fish exposed to different doses of methyl paraben (0.001, 0.01, 1 and 10 mg l-1) as compared to positive control (17-β estradiol; 10.0 ng l-1) have been shown in figure 2. the results showed a significant difference in gsi between control and two higher doses of methyl paraben i.e. 1.0 and 10.0 mg l-1. these two doses significantly reduced gsi suggesting that methyl paraben inhibited the development of the zebrafish testes. the histopathological results of control and methyl paraben exposed fish testis are displayed in figure 3. testicular sections from the control group had no visible histopathological alterations and were composed of normal, well-organized and uniform seminiferous tubules with complete spermatogenesis and normal interstitial connective tissue (fig. 3a). the arrangement of the seminiferous tubules was regular and the tubular walls were smooth. the sertoli cells were sparsely distributed, among the spermatogenic cells (i.e. spermatogonia, spermatocytes, and spermatids) at different stages of differentiation. table 1. mean (±sd) values of length, weight and gsi following 21 days exposure of male zebrafish to control and methyl paraben treatments. control solvent control 17-β estradiol methyl paraben (mean of all doses) length (mm) 37.81±4.21a* 38±1.10a 37.94±1.34a 37.40±6.06a weight (g) 36.02± 8.12a 35.45±6.04a 35.02±2.71a 36.10±1.21a gsi (%) 5.32±0.88a 5.02±1.06b 4.12±0.94c 3.16±0.81d *values in a column followed by different letters are significantly (p<0. 05) different duncan’s multiple range test). figure 2. changes in gonadosomatic index (gsi) in adult male zebrafish exposed to 17-β estradiol (10.0 ng l-1, positive control) and different concentrations of methyl paraben. data are given as mean (n=5) ± standard deviation (sd) 74 hassanzadeh/ histopathological effect of methyl paraben on the zebrafish testis there was no remarkable histopathological differences between the control and low dose (0.001 mg l-1) of methyl paraben. exposure to 0.001–10 mg l-1 methyl paraben did not induce ova-testes condition in the male zebrafish. however, different degrees of seminiferous tubular changes were observed in higher doses of methyl paraben. these damages to fish testis increased with increasing concentrations of methyl paraben. slight distortion of seminiferous tubules was recorded at the 1.0 mg l-1 methyl paraben treatment group, and these changes were characterized by slight figure 3. testicular histopathology in zebrafish exposed to methyl paraben for 21 days (h&e, 40x). the panels include a: control group, b-c: 0.01 mg l-1, d-f: 1 mg l-1 and g-i: 10 mg l-1. the testis of control fish indicated normal histology, while other treatments show injuries, including general atrophy (ga), multi-nucleated gonocytes (mngs), impaired germ cell (ig), leydig cell hyperplasia (lch), opoptosis (opt), spermatogonial proliferation (sg p), interstitial fibrosis (fb) (sg: spermatogonia, sc: spermatocyte, st: spermatic, sz: spermatozoa, le: leydig cell and se: sertoli cell). 75 int. j. aquat. biol. (2017) 5(2): 71-78 sloughing (atrophy) of germcells and vacuolar degeneration of spermatogenic, sertoli cells, as well as, the reduction in number of spermatids in the testicular parenchyma (fig. 3d-f). at higher dose (10 mg l-1) of methyl paraben, the general atrophy of testis was higher as compared to control and lower doses of methyl paraben (fig. 3g, h, i). the number of gonocytes appeared to be increased, and they were located centrally in the sseminiferous tubules due to vacuolization of sertoli cell cytoplasm. leydig cells were found in clusters and had small cytoplasm and small irregular nuclei. treatments with 1.0 and 10.0 mg l-1 of methyl paraben induced a significant proliferation of the spermatogonia (p≤0.05). a corresponding significant decrease in the proportion of the spermatozoa in the 1.0 and10.0 mg l-1 methyl paraben treated fish was noted (p≤0.05). atrophic tubules with a massive reduction of germ cells layers was also observed. the small scrotal testis in the highest dose also displayed multifocal leydig cell hyperplasia and presence of enlarged cells with enlarged or multinucleated nuclei. the leydig cells in the areas of hyperplasia were smaller in size with less cytoplasm relative to controls and were grouped in large aggregates versus smaller clusters of cells in the control testes. however, the most severe injuries in zebrafish testis were observed in 10 mg l-1. discussion gonads are frequently used in the evaluation of estrogenic potential of different endocrine disruptors in fish. impairment of spermatogenesis by estrogenic compounds has been already reported (taxvig et al., 2008; boberg et al., 2010). in the present study, methyl paraben at environmentally relevant concentrations was found to interfere with the reproductive performance of adult male zebrafish. the gonadosomatic index is a useful criterion to quantify sperm production, since spermatozoa form the majority of cells in the mature testis. the lower gsi induced by methyl paraben is probably a result of reduction in the testis size due to the loss of mature germ cells. gsi decreases have been reported in adult male of other fish species exposed to parabens (taxvig et al., 2008; boberg et al., 2010). the resuts of the present study also showed that in the methyl paraben treated fish histopathological changes in testes such as multi-nucleated gonocytes (mngs) and leydig cell hyperplasia occurred at different exposure concentrations. the cause of mngs is unknown, but it is not due to abnormal/ incomplete cell division because it occurs at ages when the germ cells are quiescent and not dividing. it seems likely that the mechanisms underlying the induction of mngs are separate from those involved in the formation of focal dysgenetic areas, because mngs occur throughout the testis and are equally evident in normally formed seminiferous cords as in areas destined to form dysgenetic areas (soni et al., 2005). multinucleated germs cells (mngs) are of particular interest because of their theoretical potential to undergo mutations (given their abnormal dna content) and thus the potential to give rise to testis germ cell cancer and the mngs that are formed typically have 2-4 nuclei, but may have as many as 13 nuclei all contained within a common cytoplasm. theoretically, mngs could arise either from nuclear division without cytoplasmic division, or from the collapse of intercellular bridges. given the ability of parabens to induce mngs by a single exposure during a time when the germ cells are not proliferating, the most logical conclusion is that mngs form the opening of intercellular bridges (barse et al., 2010) the abnormal clustering of leydig cells in the testes of the methyl paraben-exposed fish appears to be due to abnormal cell migration, which also trapped sertoli cells, and peritubular myoid cells among the leydig cell aggregates (taxvig et al., 2008). these data would suggest that the observation of multinuclear gonocytes in methyl paraben treated testes is due to an inappropriate initation of cell division, whilst the observed increase in leydig cell number may be due to a compensatory mechanism as a results of lowered testicular testosterone levels. however, at higher exposure concentrations (1.0 and 10.0 mg l-1) there was a general increase in general atrophy, fibrosis, impaired germ cell. unlike 76 hassanzadeh/ histopathological effect of methyl paraben on the zebrafish testis control fish, where the seminiferous lobule lumen was filled with spermatozoa, testis of fish exposed to mp showed altered testicular structure including atrophy, reduced spermatozoa cells and fibrotic changes. the interstitium became narrow and there were lees spermatozoa. other studies have suggested that leydig cell hyperplasia is associated with disturbances in paracrine relationships between sertoli and leydig cells due to germ cell loss and testicular atrophy. adverse effects on testis structure of mature male common carp (cyprinus carpio) exposed to 901,000 4-tertpentyl phenol (p-alkylphenol) have been reported (gimeno et al., 1998). these include inhibition of spermato-genesis, disappearance of spermatozoa and spermato-genic cyst and higher incidence of pathological alterations such as fibrosis, vacuolation, and atrophy of germinal epithelium. in the present study, the reduced gonadosomatic index (gsi) and testicular damages were observed in fish exposed to methyl paraben. several in vitro and in vivo studies have confirmed that parabens are weak estrogenic compounds (routledge et al., 1998; soni et al., 2005; tayloret al., 2002). estrogens may act either directly on the mature testis to regulate androgen production, reducing the basal testosterone and 11-ketotestosterone levels as in e2 treated goldfish (carassius auratus)(gimeno et al., 1998), or indirectly on gonadotropin secretion. little is known on how (pseudo-)estrogens, including methyl parabens induce the regression of the testes. based on the data available from fish models, it is suggested that exposure of fish to pseudo-estrogens decreases the ability of the testestosteron to synthesize androgens by inhibiting genes involved in steroidogenesis (star and cyp17α1) and suppressing gnrhregulated fsh and lh levels (scholz and mayer, 2008; van der oost et al., 2003; silva et al., 2012). steroid hormone levels were not assessed in the present study. but, it is known that estradiol regulates the proliferation of spermatogonia in teleosts (bhatia et al., 2014). we suggest that slower transformation of spermatogonia to the spermatocyte, spermatid and spermatozoa stage after treatment with methyl paraben is a multicausal condition involving hormonal imbalance and testicular damages. the interstitial fibrosis observed in the testes of parabens-exposed fish is in line with previous studies reporting testicular fibrosis after exposure to estrogens in male comon carp (barse et al., 2010). the localisation or expression levels of the gene for erα in the interstitial tissue of the testes was not measured in the present study. we suggest that an increase in estrogenicity in the micro-environment of the testicular cells after treatment of fish with parabens could have caused these anomalies. in addition, we observed apoptotic cells in the testes of the parabentreated fish. this is in agreement with the mammalian studies using parabens (scialli, 2011). however, the intracellular mechanisms of testicular histopathological changes with the possible involvement of (edcs) are not known and further investigation is needed. as demonstrated in mammalian studies, the development of abnormal germ cells could also be due to abnormal interactions between the germ cells and the sertoli cells resulting in multinucleated gonocytes (clewell et al., 2013; gray et al., 2001). the multinucleated cells could have resulted decreasing in the proportion of spermatocytes (fisher et al., 2003). it is concluded that methyl paraben can cause reproductive toxicity to male zebrafish. exposure to subacute concentrations of methyl paraben for 3 weeks can cause irrepairable damages on testes. estrogenic and antispermatogenic activity of methyl paraben on fish, as observed in the present study, is of great concern because parabens are widely used as preservatives in topical cosmetic preparations and find their ways to aquatic ecosystem. acknowledgments we sincerely acknowledge professor h. heidari vala from the avicenna research institute, acecr, tehran, iran for making a number of helpful suggestions references barse a.v., chakrabarti t., ghosh t.k., pal a.k., kumar n., raman r.p., jadhao s.b. 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(2017) 5(2): 71-78 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی پس از مواجهه با متیل پارابن (danio rerio) گورخریبیضه ماهی آسیب شناسی بافتارزیابی *زادهنسرین حسن 65719-9581863، کد پستی: ایران ،دانشگاه مالیر، طبیعی منابعمحیط زیست و دانشکده ،محیط زیستگروه چکیده: برای آبزیان ایهای آبی، تهدید بالقوهعنوان نگهدارنده کاربرد وسیعی در تولید محصوالت مختلف دارد. ورود مکرر این آالینده به محیطمتیل پارابن به در (danio rerio) گورخریاین مطالعه بررسی سمیت تولید مثلی متیل پارابن بر تغییرات هیستوپاتولوژی بیضه ماهی از د. هدفگردمحسوب می هایغلظت با روزه 21 مواجهه از پس گورخری ماهی در گنادی شاخص بیضه و هیستوپاتولوژی تغییرات مطالعه این شرایط آزمایشگاهی است. در از یکهیچ در متیل پارابن با مواجهه که داد نشان شد. نتایج بررسی استاتیکنیمه شرایط در پارابن متیل لیتر بر گرممیلی 10 و 1 ،01/0 ،001/0 داری با افزایش غلظت متیل پارابن کاهش یافت. طور معنینشد. شاخص گنادی به k فاکتور و مانیزنده در داریمعنی تاثیر بروز به منجر تیمارها ون های زایشی، پرولیفراسیای، تخریب سلولهای چند هستهشناسی بیضه شامل ایجاد آتروفی عمومی بیضه، تشکیل گونوسیتتغییرات بافت مه های سرتولی بود. این نتایج نشان داد که مواجهه نیریزی شده سلولهای لیدیگ، فیبروز بینابینی و مرگ برنامهاسپرماتوگونیا، هایپرپالزی سلول ماهی نر تولید مثلی رین اندامتارابن می تواند تاثیرات مضری بر کاهش شاخص گنادی و تخریب بافت شناسی بیضه به عنوان مهممزمن با متیل پ شود. داشته باشد. متیل پارابن با خاصیت استروژنی منجر به بروز تاثیرات غیر قابل برگشتی در سیستم تولید مثلی ماهی نر می . مثلی تولید سمیت ها،پارابن بافتی، هایآسیب ،گورخری ماهی :کلمات کلیدی int. j. aquat. biol. (2016) 4(4): 239-255; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article promising anti-oxidative therapeutic potentials of edible freshwater snail bellamya bengalensis extract against arsenic-induced rat hepatic tissue and dna damage sk. sajed ali1, nirmallya acharyya1, smarajit maiti*1, 21 1department of biochemistry and biotechnology, cell and molecular therapeutics laboratory, oriental institute of science and technology, vidyasagar university, midnapore-721102, west bengal, india. 2founder and secretary, agricure biotech research society, epidemiology and human health division, midnapore-721101, india. article history: received 26 may 2016 accepted 10 august 2016 available online 2 5 august 2016 keywords: arsenic toxicity tissue necrosis dna damage carcinogenesis ethnomedicinal effects abstract: epidemiological data suggest that arsenic ultimately results in cancer in different parts of the body. several synthetic therapeutic agents manifest inadequate potency with severe side effects against arsenic toxicity. the flesh of b. bengalensis, has long been used as an ethno-medicine in case of arthritis, blood-impurities, impaired immune system, conjunctivitis and liver anomalies. this potent organism might be a natural choice against arsenic and several other toxicities. our earlier studies on arsenic-exposed human can correlate carcinogenesis with dna-damage. in an attempt to investigate the possible protective and therapeutic effect against arsenic induced hepatotoxicity, the extract of b. bengalensis was tested in arsenic intoxicated rat model. the timeand dose-dependent effect of arsenic toxicity was also tested in b. bengalensis. sodium-meta-arsenite naaso2 (0.6 ppm/100g bw/day for 28 days, as earlier reported) was treated alone or in combination with the b. bengalensis water extract (bbe, 100 mg/100g bw) to rat and compared with vehicle treated control. in a separate experiment, the b. bengalensis was exposed to high concentration of naaso2 contaminated water (5 to 20 ppm for 1 to 9 days) in laboratory condition and their dna quality was evaluated in relation to its possible oxidative threat. any concentration of arsenic was incapable to initiate a significant dna damage in b. bengalensis. lipid peroxidation was increased in arsenic exposed b. bengalensis after longer duration of its exposure. increase in reduced antioxidant like non-protein-soluble thiol (npsh) is concordant with the decrease in lipid peroxidation and dna stability in this organism. in rat experiment, the bbe supplementation strongly prevented arsenicinduced oxidative, necrotic and apoptotic damages to liver tissue/dna by strengthening antioxidant systems, which has been shown in hepatic dna-fragmentation, comet-assay, histo-architecture (hematoxylin/eosin), alkaline-phosphatase, serum-glutamate-pyruvate-transaminase and lactatedehydrogenase (tissue-degeneration-marker) results. only arsenic exposure decreased hepatic superoxide-dismutase (sod) in-vivo and in-vitro (h2o2/arsenite redox-stress to dialyzed and concentrated, 6-8kd cutoff-millipore liver cytosolic sod), catalase, xanthine-oxidase, lactoperoxidase activities and the level of npsh with a concomitant increase in malondialdehyde resulting in mutagenic dna-breakage and apoptotic liver damage which has been decisively restrained in b. bengalensis extract. the present investigation offers strong evidence on the hepatoprotective and medicinal efficiencies of bbe against oxidative stress induced by arsenic. introduction clinical manifestations of the chronic arsenicosis are the skin lesions, keratosis, different metabolic disorders and finally cancer in several organs including liver, lung, and skin, of the exposed individuals. the liver is a major target tissue of * corresponding author: smarajit maiti e-mail address: maitism@rediffmail.com arsenic toxicity. a few drugs like british anti lewsite (bal) and dimercaptosuccinic acid (dmsa) are available and mundane in the market as arsenic chelating agents, but all these have severe side effects (inns et al., 1990). several components of herbal origin like quercetin (a flavonol, iupac 240 ali et al./ antioxidative pharmacological efficacies of b. bengalensis against arsenic-toxicity name: 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4 h-chromen-4-one), combination of monoisoamyl dimercaptosuccinic acid (miadmsa) and moringa oleifera lam (family moringaceae), hippophae rhamnoides l., curcumin ((a diarylheptanoid, iupac name: (1e,6e)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)), emblica officinalis gaertn (a deciduous tree of the family phyllanthaceae and synonym of phyllanthus emblica l.) and some other phytochemicals have been demonstrated to show varied extent of protection against arsenic-induced oxidative stress, dna breakage, hepatic damage, fibrosis and carcinogenesis (ghosh et al., 2010; chattopadhyay et al., 2011; flora et al., 2012; maiti et al., 2014, srivastava et al., 2014). recent studies in our laboratory elucidate the protective role of green tea flavonoids (camellia sinensis, family theaceae) and vitamins (b12; cyanocobalamin + folate) against arsenicinduced mutagenic dna-breakage/ intestinal damages in experimental rat model (acharyya et al., 2015a, acharyya et al., 2015b). though several researches investigated on phyto-chemicals, synthetic/natural micronutrients, but focus has not been made on tissue extracts from some small organisms/animals of certain ethno-medicinal importance. some edible shell fishes are well-known to provide the economically important nutraceuticals, which are of great medicinal values. these are consumed by the indigenous people of the rural and urban areas of the different parts of the world (deb and emdad haque, 2011; kim and pallela, 2012). different species of these organisms are biologically diverse in nature and belongs to the genera of the paratelphusa, macrobrachium, bellamya, pila, achatina, lamellidens, novaculina and parreysia (mahata, 2002; westneat and alfaro, 2005; ma et al., 2010; arul prakash et al., 2011; venugopal and siva kumar, 2014; shabelnikov and kiselev, 2015; ma et al., 2016). those are long been used as the traditional medicines and potent micronutrient supplier in a number of ailments such as rheumatism, cardiac diseases, hypertension, asthma, rickets, hypocalcemia and nervousness (fenical, 1997; mahata, 2002; deb and emdad haque, 2011; kim and pallela, 2012). the foot of pila sp., bellamya sp., lamellidens sp. and parreysia sp. are large muscular and proteins/ micronutrient rich fat free part of these organisms (prabhakar and roy, 2009; ma et al., 2010; kim and pallela, 2012; venugopal and siva kumar, 2014). secretion extract of bellamya bengalensis constitute potent anti-neoplastic/ antiproliferative agent which is cytotoxic and apoptogenic against human myeloid leukemia cells (besra et al., 2013). the hepatoprotective activity of b. bengalensis flesh extract has been shown in carbon tetrachloride-induced liver damages in rat model (gomes et al., 2011). in this background, the present study was designed to elucidate the therapeutic effects of the water extract of b. bengalensis for the management of arsenic induced hepatic toxicity and its possible mechanistic approach in the experimental rat model. we also made effort to find a cheap non-invasive medicinal-protective source against arsenicassociated toxic health hazards. materials and methods chemicals and reagents: sodium arsenite, bovine serum albumin (bsa), thiobarbituric acid (tba), reduced glutathione (gsh), 5-5´-dithiobis-2-nitro benzoic acid (dtnb), ammonium molybdate, ascorbic acid, nitro blue tetrazolium (nbt), agarose (low melting point), acrylamide, n,n'-methylenebis-acrylamide, ammonium per sulphate, xanthine, and tetramethylethylenediamine (temed) were purchased from sigma chemicals (st. louis, mo). sodium dihydrogen phosphate (nah2po4), disodium hydrogen phosphate (na2hpo4), ethylenediamine tetraacetic acid (edta), and eosin and hematoxylin were supplied from srl, india or merck, india. for urea, total protein, serum glutamate pyruvate transaminase (sgpt), alkaline phosphatase (alp), lactate dehydrogenase (ldh), and uric acid assay kits were purchased from either from ranbaxy laboratories limited, india or merck india, worli, mumbai 400018. 241 int. j. aquat. biol. (2016) 4(4): 239-255 bellamya bengalensis extract (bbe) preparation: mature b. bengalensis were collected from local control ponds at midnapore (22.424°n, 87.319°e) region (animal sp. are nurtured in a standard condition) and carefully washed to remove clay particle then blotted on a soak paper to remove excess moisture. the flesh (edible part) of the organism was homogenized by electric homogenizer to prepare 40% b. bengalensis water extract (bbe). the total homogenate tissue was collected in cold condition and centrifuged at 4°c temperature for 10 min at 10,000 rpm. the supernatant was collected and used freshly or stored for couple of days at -4°c. estimation of non-protein soluble thiol from bbe: the nonprotein soluble thiol (npsh) in b. bengalensis tissue extract was determined by standard 5, 5’-dithiobis-2-nitrobenzoic acid (dtnb) method with a slight modification (forman, 2009). in brief, the protein was precipitated by trichloroacetic acid and clear cytosol was added to 0.1 m sodium phosphate buffer containing 5 mm dtnb. the level of npsh was determined against a glutathione (gsh) standard curve. estimation of ascorbic acid, phosphorus and glycogen from bbe: the reaction of ascorbic acid with ammonium molybdate was initiated to generate molybdenum blue which was estimated to evaluate this vitamin in the extract. the method is appreciably sensitive (2 µg/ml) and specific for having no interference from common reducing sugars, antioxidants and degradation products of the vitamin (sayed elnenaey and soliman, 1979; barrows et al., 1985). total phosphorus was estimated spectrophotometrically by the method of chen et al. (1956) with some modifications (motomizu et al., 1984; barrows et al., 1985). the glycogen content in b. bengalensis tissues was determined by the method of seifter et al. (1950) with slight modifications (muriel and deheza, 2003). estimation of total protein, lipid and amino acids in bbe: the total protein was estimated from b. bengalensis tissue extract by the method of lowry et al. (1951). total lipid was estimated by an established (barnes and blackstock, 1973; rodríguez-gonzález et al., 2006) method. the total amino acid was estimated by ninhydrin method (moore and stein, 1954; bergstrom et al., 1974). determination of calcium from bbe: quantitative determination of calcium from bbe by arsenazo iii method was done by accucare assay kit. calcium with arsenazo iii (1, 8dihydroxy-3, 6-disulpho-2, 7-naphthalene-bis (azo)-dibenzenearsonic acid) yielded a blue colored complex (hazari et al., 2012). absolute values were calculated from the calcium standard curve. time and dose dependant exposure of sodium arsenite (naaso2) to b. bengalensis: live adult b. bengalensis were separated in 6 groups and housed in laboratory environment. those were exposed to naaso2 with different doses (0, 10, 12.5, 15, 17.5 and 20 ppm). the animals were collected in 48, 72 and 96 hrs interval and tissue extract was prepared by standard laboratory protocol and used for following biochemical experiments. in another experiment, organisms were exposed to 15 ppm of naaso2 for nine days and in regular interval their tissues were collected for dna stability study. estimation of non-protein soluble thiol (npsh) and malondialdehyde (mda) level in arsenic exposed b. bengalensis: the npsh assay has been described previous section (forman, 2009; maiti et al., 2012). the mda assay was conducted following the protocol of buege and aust (1978) and maiti et al. (2014) with a slight modification from control and arsenic exposed bellemaya tissue extract. dna fragmentation in arsenic exposed b. bengalensis: dna fragmentation assay was performed in the tissues of b. bengalensis exposed to arsenic in a dose and time-dependent manner (beltran et al., 2008). this assay is described in the preceding section. testing of protective potential of b. bengalensis in arsenic–induced hepatotoxic rat model: animal selection and arsenic treatment: female albino rats weighing 150-160 g were acclimatized for 10 days at 12-h light-dark cycle, 32±2°c temperature and 5070% humidity in the institutional animal resource facility. those were fed with a standard pellet diet 242 ali et al./ antioxidative pharmacological efficacies of b. bengalensis against arsenic-toxicity (hindustan lever, mumbai, india) and water ad libitum. studies were carried out in accordance with the national institutes of health, usa guidelines and the institutional ethical concerns were maintained throughout the investigation. rats were randomly distributed in three groups having six in each. animals of group-ii and group-iii were fed with 0.5 ml drinking water containing sodium arsenite at a concentration of 0.6 ppm/100 g bw/day for 28 days. initially, several dose response studies of arsenic were conducted on rat model. the present dose range usually does not cause animal mortality but exposure for a moderate time period (≥ 3weeks) increased the level of liver and kidney toxicity marker and other clinical marker suggesting significant level of cellular toxicity (maiti et al., 2014). the group-i, designated as control was supplied with same amount of drinking water for stipulated duration. the group-iii animals were supplemented with b. bengalensis water extract by gavages at a concentration of 100 mg tissue of b. bengalensis /100 g body weight/ day for 28 days. on the day 29, animals were exposed to light anesthesia (by ether), blood was collected using a disposable syringe (21-gauge needle) and serum was separated and organs required for biochemical and histological examinations were dissected out, stored at -20ºc. evaluation of general toxicity: liver and kidney function tests: serum glutamate pyruvate transaminase (sgpt), alkaline phosphatase (alp), urea, uric acid and lactate dehydrogenase (ldh) were measured from the rats by standard protocol with the assay kits (ranbaxy, india or other reputed company). total protein (serum) was measured following biuret method using standard kit from ranbaxy diagnostic india limited, mumbai, india. determination of catalase activities: the catalase activity was measured spectrophotometrically in hepatic cytosolic sample observing the rate of degradation of h2o2, the substrate of this enzyme (aebi, 1984; maiti and chatterjee, 2001). estimation of liver xanthine oxidase activities: xanthine oxidase (xo) activity was measured spectrophotometrically from liver tissue homogenate by following the oxidation of xanthine to uric acid according to the method of terada et al. (1990) with some modifications. the reaction was started by adding 0.15 mm xanthine in 100 mm phosphate buffer (ph=7.5). liver homogenate was used as the enzyme source. the rate of uric acid production was recorded for 5 min at 290 nm (ε=12,200 m-1cm-1). the results were expressed as units of xo/mg protein, whereby 1 unit of xo was defined as the amount of enzyme converting 1.0 μmol xanthine to uric acid at 25°c. determination of lactoperoxidase activities: lactoperoxidase (lpo) activity was measured in 0.1 m phosphate buffer (ph=7.4), containing 0.6 mm 2, 2´azionbis (3-ethylbenezthiazoline-6sulfonic acid (abts) and 0.1 mm h2o2 as described by (barrett et al., 1999). estimation of malondialdehyde (mda) level: the mda assay was conducted following the protocol of buege and aust (1978) with a slight modification. to chelate iron and reduce its interference in peroxidation reaction of unsaturated fatty acid, 1 mm edta was used in the reaction mixture. to reduce the interference caused by a yellow-orange color produced by some carbohydrates, the reaction mixture was heated at 80°c instead of 100°c. finally, the mda was measured and calculated using the molar extinction coefficient of mda (ε=1.56×105 cm2/mmol) (maiti and chatterjee, 2000; maiti et al., 2014). assay of super oxide dismutase activities: a tablet of nitro blue tetrazolium (nbt) was dissolved in 30 ml water and the non-denaturing (10%) acrylamide gel was soaked in it for 30 min with shaking. the gel was shaken in 40 ml superoxide dismutase (sod) solution [0.028 m tetra methyl ethylene di amine (temed), 2.8×10-5 m riboflavin, and 0.036 m potassium phosphate at ph=7.8] for 15 min. the soaked gel was placed on a clean acetate sheet and illuminated for 5-15 min. the gel became purple except at the position containing sod (ismail, 2006). the gel was scanned when the maximum contrast between the band and background has been 243 int. j. aquat. biol. (2016) 4(4): 239-255 achieved. an identical gel was stained with coomassie brilliant blue to verify the liver protein of the corresponding sod protein bands in different group of animals (acharyya et al., 2014). estimation of non-protein soluble thiol: the nonprotein soluble thiol (npsh) in liver tissue homogenate and b. bengalensis extract (prepared in 0.1 m phosphate buffer, ph=7.4) was determined by standard 5, 5’-dithiobis-2-nitrobenzoic acid (dtnb) method with a slight modification (forman, 2009). in brief, the protein was precipitated by trichloroacetic acid and clear cytosol was added to 0.1 m sodium phosphate buffer containing 5 mm dtnb. npsh was measured and calculated using a gsh standard curve. histology and dna fragmentation analysis: liver tissue was embedded in paraffin, serially sectioned at 5 mm, stained with eosin and hematoxylin (harris) and observed under a microscope (nikon, eclipse lv100, magnification x400) to study the histoarchitechture. the liver cells were used for dna preparation, tissue was treated with 500 ml of lysis buffer (50 mm tris ph 8.0, 20 mm edta, 10 mm nacl, 1% sds, 0.5 mg/ml proteinase k) for 20 min on ice (4°c) and centrifuged in cold at 12,000 g for 30 min. the supernatant was extracted with 1:1 mixture of phenol: chloroform with gentle agitation for 5 min followed by centrifugation and precipitated in two equivalence of cold ethanol and one tenth equivalence of sodium acetate. after spinning down and decanting, the precipitate was re-suspended in 30 ml of deionized water-rnase solution (0.4 ml water15 ml of rnase) and 5 ml of loading buffer for 30 min at 37°c. the 0.8% agarose gel with ethidium bromide was run in bio-rad submarine electrophoresis system at 5v for 5 min before increasing to 100 v and the band image was captured in a gel documentation system. the band intensity of dna at its different relative positions on the agarose gel was evaluated by imagej software (garciamartinez et al., 1993), expressed in relative/ normalized values and plotted in a graph. comet assay: the alkaline comet assay was conducted for the confirmation of dna damage following the guidelines proposed by singh et al. (1988) with slight modifications. the microscope slides were coated with 0.75% normal melting point agarose in pbs, 25 µl of liver cell suspension was mixed with 110 µl of 0.5% low-melting-point agarose in phosphate buffered saline (pbs) and applied to slides. after electrophoresis, the slides were neutralized with neutralizing buffer (tris 0.4 m, ph=7.5) and then stained with ethidium bromide. detection of dna breakage was measured by using imagej software. slides were read using a fluorescence microscope (nikon, eclipse lv100 pol), with the viscomet (impuls bildanalyse) software. a total of 100 comets/ slide were read for each experiment. in vitro regulation of sod activity: post-centrifuged (12,000×g) cytosolic fraction was prepared from control rat hepatic tissue homogenate, and that was cleaned by dialysis membrane (spectrum lab, usa) and concentrated with the amicon centrifugal filter units (millipore, usa, 6-8 kd mwco) to negate small molecules interferences. the concentrated fraction was incubated with different concentration of arsenite-h2o2 combination or with bbe and b. bengalensis venom, bbv (which is reported to be enriched with thiol containing protein) in kreb’s buffer. the sod activity was tested by nbt test in polyacrylamide gel as described earlier (acharyya et al., 2014). results arsenic toxicity in b. bengalensis tissue: a dose and time dependant increase in mda production is noticed in this tissue which has been ceased and further decreased in longer time at higher dose (fig. 1). this suggests the possible acclimatization by the organism in the present toxic environment. this result is found to be related by the protecting antioxidant agent i.e. npsh. a dose and time dependant increase of npsh is evident (fig. 1). no significant dna-fragmentation is noticed in dose (520 ppm naaso2) and time (1-9 days) dependent experiment (fig. 2). components in b. bengalensis tissue extract: the 244 ali et al./ antioxidative pharmacological efficacies of b. bengalensis against arsenic-toxicity level of nutrients and micronutrients are presented in the table 1. the protein content in the flesh of b. bengalensis was found to be 54.59 mg/g tissue. flesh of b. bengalensis contains moderate amount of carbohydrate, 25.75 mg/g and lipid, 242 µg/g. similarly, the micronutrient and non-protein soluble thiol (free -sh) levels are also described in table 1. status of oxidative stress markers: the mda content in liver homogenates significantly increased in the sodium arsenite-exposed rats (p<0.05) which is restored in the bbe supplemented group and this result is significantly reciprocated by the tissue npsh level (fig. 3). the strong antioxidant enzymes i.e. lactoperoxidase (p<0.05) and xanthine oxidase (p<0.01) and the oxidant-neutralizing molecule uric acid (p<0.05) have been found to be decreased significantly in arsenic treated group and restrained in the bbe supplemented group (fig. 3). general toxicity: liver and kidney function test: figure 4 suggests that the serum sgpt and alp enzymatic activities are significantly increased (p<0.05) in arsenic intoxicated rat suggesting necrotic tissue damage which has been ceased and restored by the bbe supplementation. similarly, the increase in the kidney function marker, urea and general tissue degeneration marker like ldh are also nullified by the bbe supplemented group in arsenic exposed rats (fig. 4). hepatic tissue architecture, dna fragmentation and figure 1. non protein soluble thiol (npsh) and malondialdehyde (mda) level in bellamya bengalensis tissues after its dose and time dependent exposure to arsenic (naaso2). bars in the figure represent as follows; s1 control, s2 10 ppm, s3 12.5 ppm, s4 15 ppm, s5 17.5 ppm, and s6 20 ppm naaso2 exposed group and 1 denotes 48 hrs, 2 is 72 hrs and 3 is 96 hrs. figure 2. the effect of arsenic exposure on the quality and stability of dna of bellamya bengalensis tissue. lane distribution; (panel a) lane 148 hrs control, 248 hrs control, 3 48 hrs 10 ppm, 448 hrs 12.5 ppm, 548 hrs 15 ppm, 648 hrs 17.5 ppm, 748 hrs 20 ppm, 896 hrs control, 996 hrs control, 1096 hrs 10 ppm, 1196 hrs 12.5 ppm, 1296 hrs 15 ppm, 13 96 hrs 17.5 ppm and 1496 hrs 20 ppm of naaso2 in the water where the animals were kept. (panel b) the 15 ppm of naaso2 was treated to the b. bengalensis in a time dependant manner. lane distribution; lane 124 hrs control, 224 hrs of naaso2 exposed, 348 hrs control, 448 hrs of naaso2 exposed 572 hrs control, 672 hrs of naaso2 exposed 7120 hrs control, 8120 hrs of naaso2 exposed, 9144 hrs control, 10144 hrs of naaso2 exposed, 11168 hrs control, 12168 hrs of naaso2 exposed, 13192 hrs control, 14192 hrs of naaso2 exposed, 15216 hrs control, 16216 hrs of naaso2 exposed. at every 48 hrs, the animal incubation-water was refreshed maintaining the stipulated concentration of naaso2. 245 int. j. aquat. biol. (2016) 4(4): 239-255 comet assay result: arsenic ingestion with the present dose and duration decisively resulted in appreciable amount of “ladder” of dna fragments (lane 4, 5) in liver with comparison to the control rats (lane 1, 2, 3), whereas, the dna is found to be partially but significantly protected from fragmentation in bbe co-administered group (fig. 5). the dna/ladder density (normalized value) is calculated for the different migrated locations on the lane in the gel and the mean values are plotted against the relative migrated location which clearly reveals the outcome noticed in the ladder image. the single cell dna status (comet assay) basically supports the dna fragmentation results. the number of comet forming cells is found to be higher in arsenic exposed rat liver whereas, in bbe supplemented group the genetic materials of the cells are noticed to be highly protected (fig. 6). parameters concentration in bbe phosphorous (µg/g) 52.24±3.89 vitamin c (µg/g) 71.85±4.74 calcium (mg/g) 0.36±0.023 protein (mg/g) 54.59±4.75 total lipid (µg/g) 242±23.54 total carbohydrate (mg/g) 25.75±2.94 total amino acid (mg/g) 0.96±0.056 npsh (µg/g) 50.8±3.7 table 1. composition of nutrients and micro-nutrients in the bellamya bengalensis tissue. figure 3. the effects of arsenic toxicity (0.6 ppm/100g bw/day for 28 days) and its possible preventive/ therapeutic effects of bbe (100 mg/100g bw) on different antioxidant enzymes and cellular components in female rat liver. the malondialdehyde (mda) is regarded as the oxidative stress marker representing the deleterious effects of reactive oxygen species (ros) on cellular lipid components. all the parameters were measured in the hepatic tissues except the antioxidant uric acid, which was measured in serum sample. results are mean ±se (n=6 in each group). data of treated group or supplemented group is compared to the corresponding vehicle treated group (student ‘t’ test). levels of significances are denoted as; *=p<0.05; **=p<0.01. 246 ali et al./ antioxidative pharmacological efficacies of b. bengalensis against arsenic-toxicity figure 4. the effects of arsenic toxicity (0.6 ppm/100g bw/day for 28 days) and its possible preventive/ therapeutic effects of bbe (100 mg/100g bw) on different functional markers of liver and kidney, metabolic inflammatory, and tissue necrotic markers. the parameters were measured with the standard assay kit (refer to the materials and methods section). result shows that arsenic induced impairment of liver and kidney functions are significantly restored by the bbe supplementation. results are mean ±se (n=6 in each group). data of treated group or supplemented group is compared to the corresponding vehicle treated group (student ‘t’ test). levels of significances are denoted as; *=p<0.05; **=p<0.01. figure 5. dna fragmentation result is shown in liver of female rats treated with arsenic. lane distribution; lanes 1, 2, 3control; lanes 4, 5 naaso2 exposed and lanes 6, 7, 8naaso2 + bbe (left panel). densitometry analysis of the different bands was done in imagej software and the mean normalized values are plotted at the position of their relative migration. 247 int. j. aquat. biol. (2016) 4(4): 239-255 the results on the dna stability in the figures 5 and 6 are concordant with the histo-architecture picture (fig. 7). we noticed a significant alteration in the liver functions of the experimental animal. this is supported by our present hepatic histological study. arsenic caused hepatic injury due to the necrotic tissue lesions and disruption of the central canal and associated convergent lobular structure of the hepatic cells. in vivo and in vitro sod regulation: the result suggests that in the whole animal study, the sod activity did not alter significantly in arsenic treated group (lane 4-7) with comparison to the control (fig. 8a). but in the bbe supplemented group, this activity augmented significantly suggesting its protective role. in the in vitro study, a significant decrease in arsenic and h2o2 treated group (lane 3, fig. 8b) is noticed which is markedly reversed bbe and bbv treated group (lane 4 and 5, respectively, fig. 8b). lane 6 and 7 served the control to test the possible sod activity in the only bbe and bbv, respectively. similarly, only h2o2 was used in oxidant-stress associated sod inactivation and its reversal and even activation by the b. bengalensis extract and b. bengalensis venom (fig. 8c). discussion in the present study, we demonstrate that arsenicinduced hepatic toxicity with present treatment schedule made a significant alteration in the liver functions of the experimental animal. this is supported by our present hepatic histological study. arsenic caused hepatic injury due to the necrotic tissue lesions and disruption of the central canal and figure 6. arsenic induced hepatic dna breakage in single cell apoptotic damage which is markedly prevented by the bbe administration. panels are represented as control rat (a) naaso2 exposed (b) naaso2+bbe (c). the alkaline comet assay is done following the lyses of cells then electrophoresis and staining with ethidium bromide. (please refer to the method section). figure 7. the hepatic histoarchitecture is shown by h&e staining (10x) of female rat treated with arsenic. control rat (a) or treated with sodium arsenite (b) or sodium arsenite + bbe. the lumen lining of the central vein of liver is present. the hepatic lobules are comprised mostly of plates of hepatocytes. tissue degeneration is clearly visible in slide b as the formation of a mesh like structure losing the eccentric feature of hepatocyte organization. the present histoarchitectural results are justified by the serum markers of liver and kidney functions and tissue degenerative marker i.e. ldh activity. 248 ali et al./ antioxidative pharmacological efficacies of b. bengalensis against arsenic-toxicity associated convergent lobular structure of the hepatic cells. report reveals that ingestion of arsenic-contaminated drinking water caused infiltration of inflammatory cells in the periportal area in the liver (liu et al., 1999). during the investigation on arsenic speciation and their mechanism, calatayud (2013) suggests that as (iii) and its organic derivatives induce tissue death mainly by necrosis, apoptosis, cancers, and other abnormalities. it is evident that arsenic-induced liver damages could be the result from oxidative stress which is substantiated by the histological and biochemical manifestations i.e. an elevated level of serum transaminases (karimov et al., 2002; li et al., 2007) and lactate dehydrogenase. not only in the experimental animals, has a similar increase of serum transaminases been reported in arsenicexposed workers (zaldivar et al., 1981). the present result on the bbe protection of liver and kidney functions indicates that the extract of this organism which has some traditional ethno-pharmacological effect, also possess a strong therapeutic potentials against arsenic-related toxicities. bellamya bengalensis is a freshwater organism. therapeutic potentials and drug development provisions from marine natural products have been extensively reviewed by molinski (2009). in the present study, a potent self-protective/ preventive ability are shown in b. bengalensis itself against very high arsenic concentration. this is interesting that, a very low dose of arsenic (0.6 ppm) become highly toxic and damaging to the rat tissues and several of its macromolecular structures nevertheless, the dna of b. bengalensis manifested a strong stress withstanding ability against the continuous exposure to 10-20 ppm of arsenic for several days. the inability of arsenic to damage b. bengalensis dna is paralleled with the higher increase of npsh and decrease of mda in this organism. this suggests that increase in npsh result in minimizing the generation of lipid peroxidation products (mda) and other free radicals. as a result, the organism’s dna remained highly protected. arsenic produces reactive oxygen species (ros) when react with h2o2 and/or metal cataions and produces high level of lipid peroxides and conjugated di-enes (messarah et al., 2013; acharyya et al., 2014). arsenic exposure also exhibits oxidative stress through a significant reduction of gsh in liver, cultured lung epithelial cells and brain tissues (li et al., 2002). the npsh, an antioxidant itself and a precursor of gsh is significantly declined by arsenic but that is restored by the bbe supplementation in the present investigation. the impairment of the antioxidant enzymes such as sod figure 8. cytosolic sod (cu-zn sod or sod1) activity of hepatic cells is shown on a polyacrylamide gel. upper panel a (in vivo experiment)lane distribution; 1, 2, 3 control; 4, 5, 6, 7 arsenic treated, and 8, 9, 10 arsenic + bbe treated. middle panel bin vitro h2o2 inactivation of rat cytosolic sod1 and its protection by bbe and bbv. lane distribution: lane 1control, 2control (2 hrs), 3h2o2 (100 mm) (2 hrs), 4h2o2 (100 mm) + bbe 15 µl (25%) (2 hrs), 5h2o2 (100 mm) + bbv 15 µl (2 hrs), 6bbe 15 µl (25%) (2 hrs) and 7bbv 15 µl (25%) (2 hrs). lower panel cin vitro h2o2 inactivation of rat cytosolic sod1 and its protection by bbe and bbv. lane 1control., 2con incubated 2 hrs., 3h2o2 (1 m) 2h rs., 4bbe 18 µl (25%) + h2o2 (1 m) 2 hrs., 5bbe 18 µl (25%) + h2o2 (1m) 2 hrs., 6bbv 18 µl + h2o2(1m) 2 hrs. and 7bbv 18 µl + h2o2 (1m) 2 hrs. 249 int. j. aquat. biol. (2016) 4(4): 239-255 and catalase activity has been reported to link with oxidative stress and dna damage (sinha et al., 2007; maiti et al., 2012; calatayud et al., 2013), which is demonstrated in the present investigation. the present protection of bbe has been demonstrated at the level of antioxidative efficacy of several enzymes that results in a protection in macromolecular structure like dna and proteins. the possible anti-apoptotic role of this medicinally potent organism has been demonstrated in the comet assay results. anti-inflammatory role of this organism extracts has been pointed out. anti-cancer activity of some natural products from several marine organisms has been demonstrated. commercial forms of the drugs prepared from these products are used in different physiological ailments (antitumor, analgesia, antiinflammatory, immunomodulation, allergy, and anti-viral) (singh et al., 2008; montaser and luesch, 2011). arsenic is reported to induce cellular inflammatory responses by increasing the tumor necrosis factor α (tnfα), interferon γ (ifn-γ) and interleukin 6 (il-6) (dutta et al., 2015). arsenic induced ros aggravate the situation by furthering the nf-ƙβ action (kaul et al., 2014). the bbe has been shown to decrease the lps-induced inflammatory and oedematous responses like tnfα and macrophage activation (bhattacharya et al., 2014). bbe has also been shown to effectively nullify the lps induced nuclear translocation of nfƙβ and p65 (bhattacharya et al., 2014). possible anti-inflammatory action of bbe might be occurring in the protection mechanism in the present study, where a remarkable amount of ros is produced by the arsenic. further investigation is required for more conclusive remark. ramanathan et al. (2005) evaluated the molecular changes during arsenic exposure and possible therapeutic efficacy of antioxidants like vitamin c and vitamin e on arsenic induced apoptosis in rats. they reported that administration of vitamin c and vitamin e along with arsenic significantly reduced the apoptosis. vitamin c was found in b. bengalensis at a satisfactory level i.e. 71.85±4.74 µg/g tissue. vitamin c acts as a scavenger of free radicals and plays an important role in the regeneration of α-tocopherol (young and woodside, 2001). moreover, supplementation of ascorbic acid and α-tocopherol has been known to minimize the dna damage by reducing tnf-α level and disfavoring the activation of caspase cascade in arsenic intoxicated animals (ramanathan et al., 2005). anti-carcinogenic and anti-leukemic activity of bbe has been demonstrated. selective degradation of cancerous cells by apoptogenic pathway by bbe-induced mitochondrial caspase cascade has been associated to these anti-cancer effects (besra et al., 2013). mitochondrial death signal is linked to the influences of the p38, p53, and c-myc regulations and dna damages (maiti, 2015). arsenic-induced mitochondrial deregulation is evident. in the present study, arsenic induced dna fragmentation has been restored by the bbe extract which has been demonstrated in ladder assay results and single cell dna damage picture (comet assay). this data suggest the apoptotic cell death by arsenic is circumvented by the bbe extract. the b. bengalensis extract has a higher amount of phosphate i.e. 52.24±3.89 µg/g tissue, which possibly offers a better protective effect against arsenic toxicity. arsenic in the form of arsenate is chemically similar to phosphate. it uncouples oxidative phosphorylation by substituting the phosphate molecule in atp synthesis (mitchell et al., 1971; gresser, 1981). it has been reported that arsenate and phosphate share the same transport mechanism and compete for the same sites with a moderate preference for arsenate adsorption (maiti, 2015). the intestinal absorption of arsenic is significantly decreased with the phosphate infusion in rat. this transportation is an active carriermediated system depending on na+ and h+ gradient (gonzalez et al., 1995; dixon, 1996). furthermore, even in complex systems, simulating natural conditions like in ground water, phosphate showed dominance in regulating the arsenic concentrations (stachowicz et al., 2008). thus higher phosphate in bbe might play a better protective role more 250 ali et al./ antioxidative pharmacological efficacies of b. bengalensis against arsenic-toxicity efficiently than that what only nutrient dose. specific functional groups within enzymes, receptors or coenzymes, such as thiols have shown a major influence in the activity of these molecules. a significant amount of non-protein soluble thiols has been demonstrated in the b. bengalensis extract (50.8±3.7 µg/g). the trivalent arsenicals readily react with thiol-containing molecules such as gsh and cysteine in in vitro experimental condition (scott et al., 1993; delnomdedieu et al., 1994). the in vitro binding of mmaiii and dmaiii to proteins occurs at a greater extent than with their corresponding pentavalent forms (styblo et al., 1995). arsenite has a higher affinity for dithiols than monothiols, as shown by the highly favored transfer of arsenite from a (gsh)3-arsenic complex to the dithiol 2, 3dimercaptosuccinic acid (delnomdedieu et al., 1993). the binding of trivalent arsenic to a critical thiol group may inhibit important biochemical events which could lead to toxicity. however, binding of arsenite at nonessential sites in proteins may offer a detoxication mechanism (aposhian, 1989). the inhibition of hepatic sod in arsenic intoxicated rats and its protection by bbe support the role of thiol. this finding is further justified by our in vitro study, where naaso2+h2o2-mediated sod inactivation is circumvented by bbe or bbv (b. bengalensis venom). these fractions is reported and found in the present study to have high thiol content. role of important cys residues in the catalytic/substrate-binding domain of sod is evident (acharyya et al., 2014). in most of the cases, the metal-binding site contains a cys or a his residue (kojima et al., 1999; cobbett and goldsbrough, 2002). proteins and peptides functioning in the uptake, distribution and detoxification of metal ions possess one or several metal-binding sites. the -cys-x-x-cysand -cyscysmotifs of various proteins are well-known for their heavy metal binding properties (kojima et al., 1999; cobbett and goldsbrough, 2002). it has long been acknowledged that sulfhydryl-containing compounds have the ability to chelate metals. the sulfur-containing amino acids methionine and cysteine, n-acetylcysteine, an acetylated analogue of cysteine, methionine metabolite s-adenosylmethionine, α-lipoic acid, and tripeptide glutathione (gsh), all contribute to the chelation and excretion of metals from the human body. gouri et al. (2011) reported an anti-microbial peptide of 1676 da, purified from b. bengalensis venom (collected carefully beneath the lid and inside the mantle part of the organism) having, three cysteine residues (cys3, cys5 and cys16) and may possess heavy metal binding/ chelating properties. we also noticed a high level of thiol containing substances in the b. bengalensis extract in our study. cysteine-rich secretory protein from the snail achatina achatina has been reported (shabelnikov and kiselev, 2015). bbe has been shown to be effective against the hepato-toxicity model in animal. in the light of the information regarding the ability of arsenic to develop hepato-carcinogenesis, the bbe potency is very significant. anti-cancer potentials of marine organisms are reported (singh et al., 2008). in the present study, we demonstrate for the first time, its strong hepato-protective role against a natural toxic contaminant like arsenic. further exploration of this work might be helpful for the optimization of the protective/therapeutic effects of bbe against arsenic and other toxic heavy metals. the aquatic edible snail b. bengalensis resides at the water-soil (muddy) interface. notwithstanding, this aquatic environment is highly exposed to the toxic deposit that is naturally occurring in that habitat or accumulates during sewage, drainage and during natural/artificial water circulatory processes (dutta et al., 2014). possibly, due to the implication of a strong natural selection pressure generated from the sustained environmental exposure of this organism to different toxic deposits/ metals increase its adaptation and confronting ability against stressassociated tissue and macromolecular damages. it is clearly reflected in the present study. not only against the arsenic-induced oxidative stress, b. bengalensis demonstrated a remarkable protection against heat-induced oxidant stress by buttressing the sod, catalase activity and heat shock 251 int. j. aquat. biol. (2016) 4(4): 239-255 protein 70 (hsp70) expression (dutta et al., 2014). arsenic is also reported to influence different hsps (hsp70) in exposed individuals as well as in experimental laboratory animals (maiti, 2015). here, we report for the first time on the effective medicinal role of any animal/ organism extract against the arsenic associated disorder and carcinogenesis. the clinical implications of this study focus on the immense therapeutic possibility of this organism against arsenic-induced organ toxicity/carcinogenesis. more investigations are necessary to explore the active constituent of b. bengalensis extract and its hepato-protective, antineoplastic-antioxidative activities. conclusion our study established that apparently banal, notwithstanding valuable this common edible snail (b. bengalensis) may provide a beneficial role against liver injury by arsenic toxicity. not only as a nutrient material, several of its components (i.e. thiols, phosphate, calcium, vitamins) can perform via cellular signal-transduction, nuclear factors/ receptors regulations, sod activity alteration, tnfα,/caspase modification, direct dna and cytoskeleton protection and possibly many more yet to be explored mechanistic approaches. a concept of folk medicine regarding the b. bengalensis is found here for the first time, unambiguously to be potent against the hepato-toxicity induced by arsenic which needs more detail study. acknowledgments we are indebted to a.k. mishra, dept. of chemistry, vidyasagar university for providing us several instrumentation in the usic facilities of the university. sa and na are the ph.d. students working in the post graduate department of biochemistry. we sincerely acknowledge d. mandal, technical officer of the usic dept. vidyasagar university. references acharyya n., chattopadhyay s., maiti s. 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(2016) 4(5): 308-317: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article histological characteristic of interrenal and chromaffin cells in relation to ovarian activities in mystus vittatus (bloch) during growth, maturation, spawning and post-spawning phases padmanabha chakrabarti*,1mainak banerjee fisheries laboratory, department of zoology, the university of burdwan, burdwan-713104, west bengal, india. article history: received 1 july 2016 accepted 25 september 2016 available online 2 5 october 2016 keywords: interrenal cells chromaffin cells ovary reproductive phases abstract: the histological status of adrenocortical tissues and the correlated seasonal changes in ovarian activities in mystus vittatus was performed. the tubules and nests of interrenal and chromaffin cells were located in cephalic kidney around the main branches of posterior cardinal vein. various female germ line cells were identified in the ovary based on size, distinctive features and histoarchitechture of the cells. however, on the basis of relative abundance and size of the different oocytes, the event of oogenesis has been found to occur in four distinct phases, including growth, maturation, spawning and post-spawning. the cytoplasmic features and the architecture of the interrenal and chromaffin cells varied during different phases of the reproductive cycle. during growth and maturation phases, the amount of cytoplasmic granules of interrenal cells increased than chromaffin cells that was in coincidence with the increase of early and late perinucleolar oocytes followed by highest frequency percentage of oocyte at stages iv and v. the cytoplasmic mass of interrenal cells was gradually elevated along with hypertrophied nuclei from the end of maturation and spawning phases also correlated with the increased frequency of mature oocytes. therefore, gradual accumulation of cytoplasmic granules in the interrenal cells was noticed during postspawning phase. the cytological variations in the interrenal and chromaffin cells harmonized with constitution of different ovarian cells during different reproductive phase in m. vittatus. introduction the morphology and distributional pattern of teleostean interrenal or adrenocortical and chromaffin cells surrounding the main blood vessels localized in the head kidneys are extremely diverse. it has been intimated that the interrenal cells are homologous to the mammalian adrenal cortex, whereas the chromaffin cells are homologous to the mammalian medullary cells (gazola et al., 1995). in teleosts, cortisol is synthesized and secreted by steroidogenic cells which in addition to chromaffin cells constitute the adrenal gland (grassi milano et al., 1997). civinini et al. (1997) noticed that interrenal steroidogenic cells present a considerable variety of cytological aspects that characterize them in relation to a metabolic cell cycle and periods of the annual cycle. aminergic chromaffin and * corresponding author: padmanabha chakrabarti e-mail address: dr.pchakrabarti@yahoo.in interrenal steroidogenic cells are found to be mixed, adjacent or completely separated and also can line the endothelium of the venous blood vessels or may be located in close proximity (gallo and civinini, 2003). the structural pattern and distribution of interrenal and chromaffin cells in various teleosts have been studied by several workers (joshi and satyanesan, 1980; verma and misra, 1992; borella et al., 1999; civinini et al., 2001; sampour, 2008). however, few authors have been studied the relationships between changes in the interrenal and gonadal tissue in different teleosts (singh et al., 1974; chakrabarti, 2014; chakrabarti and ghosh, 2014). as the interrenal cells and gonads are the essential elements involve in steroidogenic along with other physiological processes the aim of the present work 309 int. j. aquat. biol. (2016) 4(5): 308-317 was to examine more precisely the correlative changes of the cytology of interrenal and chromaffin cells with ovarian cells during different reproductive phases in mystus vittatus. materials and methods adult living female specimens of m. vittatus (with length of 12-14 cm and weight of 40-55 gm) were procured from particular local freshwater body during the second week of every month from february 2014 to january 2015. they were housed in an aquarium and treated with 1% methylene blue for 15 mins. the fishes were acclimatized for 5 days by feeding finely chopped goat liver and tubifex. data on total body weight and after decapitation the total ovarian weight of ten fishes were taken to calculate the mean gonadosomatic index (gsi) using the following formula: gsi = total ovary weight body weight − weight of the ovary × 100 histological methods: for histological studies after decapitation of the fish, the head kidneys containing the adrenal homologue and ovaries were dissected out, cut into small pieces and were fixed in aqueous bouin’s fluid for 18 hrs. subsequent to dehydration properly through graded ethanol, the tissues were dehydrated in acetone and were cleared in benzene. tissues were embedded in paraffin wax (melting point, 56°c-58°c) and serial sections of tissues were cut at 4 µm thickness. the sections were stained with delafield’s haematoxylin-eosin (h&e) and mallory’s triple (mt) (mallory, 1936) stains, respectively. from the histological preparations the measurement of interrenal and chromaffin cells and the nuclei were measured with the help of reticulomicrometer and ocular micrometer, respectively. results histologically, the adrenal gland is composed of tubules and clusters of interrenal and chromaffin cells surrounding the posterior cardinal veins and their branches in the anterior-most part of the pronephric kidney (fig. 1). the interrenal cells are usually round in shape and arranged in the form of tubules varying in thickness. the tubules are closely arranged interspersed with blood vessels and haemopoetic tissues (figs. 1, 2). the round interrenal cells provided with granular homogeneous cytoplasm and a distinct uninucleated nucleus with prominent nucleolus (fig. 1). in the mallory’s triple stain, the cytoplasm of interrenal cells become light pink and the nuclei are stained light orange brown. the chromaffin cells are generally present in clusters in close proximity to the blood vessels and also figure 1. the section of interrenal and chromaffin cells in mystus vittatus. interrenal cells (ir) in form of tubules having prominent nucleus and cytoplasm during growth phase. note chromaffin cells (arrow heads) adjacent to blood capilleries (bc) (h&e, 400x). figure 2. the section of interrenal and chromaffin cells in mystus vittatus. enlargement of interrenal cells (ir) adjacent to blood capilleries (bc) at the end of growth phase. note prominent nucleus and scanty cytoplasm in chromaffin cells (arrow heads) (mt, 400x). 310 chakrabarti and banerjee/ changes in the interregnal and chromaffin cells in relation to ovarian activities in m. vittatus dispersed between haemopoietic tissues and interrenal cells (figs. 1, 2). the nucleus of chromaffin cells appear oval or round and cytoplasmic granules are sparse than interrenal cells (figs. 1, 2). the inner wall of the germinal epithelium of ovary projected into the ovarian cavity is termed as ovigerous lamellae where development of new crops of oogonia take place. the sequence of oocyte maturation in m. vittatus has been divided into six developmental stages viz., oogonia (stage i), early and late perinucleolar stage (stage ii and stage iii), yolk vesicle stage (stage iv), yolk granule stage (stage v) and mature follicle (stage vi). oogonia (stage i) (8×11 µm to 14×12 µm): oogonia are present either singly or in small nests within the lamellar epithelium. an oogonium is made up of a large nucleus (4-5 µm) with chromatin threads (fig. 3). early perinuclolus oocyte (stage ii) (23×28 µm to 36×40 µm): this stage is larger than the oogonium. figure 4. the section of ovary in mystus vittatus. increasing number of oocyte iii (oiii) at the end of growth phase along with oogonia (og) and primary oocytes (mt, 100x). figure 3. the section of ovary in mystus vittatus. oogonial cells (og), oocyte i (oi) and oocyte ii (oii) within the ovigerous lamella during growth phase (h&e, 150x). figure 5. the section of interrenal and chromaffin cells in mystus vittatus. hypertrophied interrenal cells within the tubules with prominent nucleus and cytoplasm encircling the blood vessels (bv) during maturation phase. arrows indicate enlarged chromaffin cells adjacent to bv (h&e, 400x). figure 6. the section of interrenal and chromaffin cells in mystus vittatus. acidophilic homogeneous cytoplasm of enlarged interrenal cells (solid arrows) and hypertrophied chromaffin cells (cc) (broken arrows) adjacent to blood vessels (bv) and blood capillaries (bc) during maturation phase (mt, 600x). 311 int. j. aquat. biol. (2016) 4(5): 308-317 the central nucleus (10-14 µm) is provided with a few nucleoli near the periphery of nucleus together with central chromatin mass (fig. 3). some oocytes at this stage increased in size and possess a yolk nucleus which lies close to the nuclear membrane in the ooplasm (fig. 3). late perinucleolus oocyte (stage iii) (68×72 µm to 80×88 µm): the stage is characterized by the appearance of cortical alveoli along the periphery of the ooplasm. it consists of centrally placed nucleus with an average diameter of 18-20 µm together with condensed chromatin materials (fig. 4). a thin layer enclosing the zona radiata is also noticed in this stage. yolk vesicles oocyte (stage iv) (110×120 µm to 145×154 µm): the yolk vesicle finally cover the entire ooplasm of stage iv oocyte. most of the vesicles are empty but some of them are filled with homogeneous materials (fig. 8). the nucleus is large spherical and centrally placed having condensed materials. the oocyte is enveloped with a zona figure 7. the section of interrenal and chromaffin cells in mystus vittatus. higher magnification of interrenal cells (solid arrows) and chromaffin cells (broken arrows) adjacent to the blood vessels (bv) at the end of maturation phase (mt, 1000x). figure 8. the section of ovary in mystus vittatus. oocyte iv (oiv) stage with prominent yolk vesicles within the ooplasm and germinal vesicle (broken arrow) at the centre. note oogonia (arrow head), oocyte iii (oiii) and atretic follicle (af) in between during maturation phase (mt, 400x). figure 9. the section of ovary in mystus vittatus. increased number of oocyte v (ov) having condensed yolk granules (yg) and ecentric germinal vesicle (solid arrow) at the end of maturation phase (h&e, 400x). figure 10. the section of ovary in mystus vittatus. oocyte v (ov) having prominent granulosa cells in zona granulosa and thick zona radiata (arrow heads) (h&e, 1000x). 312 chakrabarti and banerjee/ changes in the interregnal and chromaffin cells in relation to ovarian activities in m. vittatus radiata, middle zona granulosa and outermost theca (fig. 8). yolk granules stage (stage v) (230×250 µm to 280×320 µm): in this vitellogenic oocyte stage, migration of germinal vesicle from the centre of egg towards the periphery is started (fig. 9). the yolk granules are condensed and as a result the cell volume and diameter increased considerably. the theca is thin but the granulose cells are prominent with distinct nucleus (fig. 10). mature follicle (stage vi) (380×400 µm to 420×480 µm): the yolk granules coalesced and remain packed to form homogeneous yolk mass. the germinal vesicle move towards zona radiata. the thickness of theca and zona granulosa reduced considerably (figs. 13, 14). discharged follicle: after the discharge of mature ovum, the theca and follicular cells are left behimd. the granulosa layer shows definite changes in structure (figs. 13, 17). atretic oocyte: sometimes the developing oocytes figure 11. the section of interrenal cells in mystus vittatus. columnar or lobular interrenal cells (solid arrows) in the form of tubules encircling central blood vessels (broken arrow) during spawning phase. bv indicates blood vessels (mt, 1000x). figure 12. the section of chromaffin cells in mystus vittatus. enlarged chromaffin cells (solid arrows) close to the blood vessels (bv) (h&e, 1000x). figure 13. the section of ovary in mystus vittatus. mature follicles (mf) with full of yolk granules (yg), thick zona radiata (broken arrows) and ecentric germinal vesicle (arrow head) during spawning phase. note the presence of discharged follicle (df), atretic follicle (solid arrow) and few oocyte iii (oiii) (mt, 400x). figure 14. the section of ovary in mystus vittatus. higher magnification of mature follicles showing distorted zona granulosa cells (arrow heads) thick zona radiata (arrows) and condensed yolk granules (yg) (h&e, 1000x). 313 int. j. aquat. biol. (2016) 4(5): 308-317 failed to attain maturity and they are characterize by irreregular shaped, disintegrated nuclei and liquefied yolk granules. they are found to be more apparent during maturation and spawning phases (figs. 8, 13). sequential changes of interrenal and chromaffin cells in relation to ovarian cells during different reproductive phases: the activities of the interrenal and chromaffin cells undergo correlative changes during the different reproductive phases. changes in the activities of adrenocortical tissues have been studied in terms of their number, distributional patterns and cell size along with decreases or increases of cytoplasm. based on the histological characteristics of ovary and the gonadosomatic index (gsi) values and the frequency of occurrence of various oogenetic cells, the reproductive phases of m. vittatus is conveniently divided into four phases, ıncludıng growth (december to february), maturation (march to may), spawning (june to august) and the post-spawning (september to november) phases. growth phase (december to february): during the growth phase the interrenal cells are round or oval (4±0.12 to 5±0.06 µm) with prominent, centrally located nuclei and the cytoplasm is acidophilic. the cells are arranged two layers in tubules, separated from each other and from the parenchyma of haematopoietic cells, chromaffin cells (2±0.05 to 3±0.02 µm) are situated in groups or intermingled with interrenal cells and exhibit a pale cytoplasm and spherical nucleus (figs. 1, 2). the gonadosomatic index (gsi) is recorded from 0.98±0.02 in december, 1.10±0.87 in january and 1.56±0.53 in february, respectively. the oogonia are few in number and the dominant cell types in this period are early perinucleolar oocytes (fig. 3). however, the percentage of late perinucleolar oocytes are gradually increasing during the end of growth phase (fig. 4). maturation phase (march to may): during this phase, the clusters of interrenal and chromaffin cells are oriented encircling the sinusoids. the diameter of interrenal cells increased (6.0 to 6.50±0.18 µm) and undergo hypertrophy and are arranged in tubules (figs. 5, 6). when stained with mallory’s triple stain, the homogeneous cytoplasm is lightly acidophilic while the nucleus is basophilic and mostly rounded (fig. 6). the chromaffin cells also increased in diameter (4.0 to 4.5±0.11 µm) and undergo hypertrophy. in some areas, the interrenal and chromaffin cells are present in clusters along the wall of the blood vessels (fig. 5). during the onset of maturation phase in march figure 15. the section of interrenal and chromaffin cells in mystus vittatus. attenuated interrenal cells with the tubules (solid arrows) encircling central blood vessel (broken arrow) during post-spawning phase. note the presence of chromaffin cells (arrow heads) associated with blood capilleries (bc) (h&e, 400x). figure 16. the section of interrenal and chromaffin cells in mystus vittatus. interrenal cells (ir) with cytoplasmic granules and prominent nuclei arranged in tubules during post spawning phase. note vacuolated chromaffin cells (solid arrows) adjacent to blood capilleries (bc) (mt, 400x). 314 chakrabarti and banerjee/ changes in the interregnal and chromaffin cells in relation to ovarian activities in m. vittatus onwards when the ovary enter into the maturation, gsi gradually increase to 2.20±0.11 in march, 5.56±0.16 in april and 9.40±0.22 in may. the highest oogenetic activity is found to occur in this phase. majority of the developing oocytes are of stage iv and v, respectively. the immature oocytes are decrease in number (figs. 8, 9). from the month of april to may yolk filled oocytes v are appeared and are gradually increased in number very sharply (fig. 9). a few atretic follicles are also found in this phase (fig. 8). the zona granulosa and zona radiata have an average thickness of 2.25±0.07 µm and 3.75±0.15 µm, respectively (fig. 10). spawning phase (june to august): in the spawning phase, the interrenal and chromaffin cells undergo momentous changes. the interrenal cells are lobular or columnar cells with more acidophilic cytoplasm and hypertrophic nuclei encircling the central blood vessels (fig. 11). the diameter of interrenal cells varies from 7.25±0.10 to 7.75±0.16 µm. however, in the late spawning phase, the interrenal cells are depleted of their cytoplasmic contents. the chromaffin cells are almost round with prominent nuclei and the cytoplasm stains brightly with eosin in haematoxylin-eosin stain (fig. 12). the diameter of chromaffin cells ranges from 6.15±0.09 to 6.25±0.18 µm. both the interrenal and chromaffin cells are usually arranged surrounding the blood vessels. in june the ovary is full of mature follicles and the gsi is recorded to be 10.25±0.04. however, the gsi rises up to a peak value (12.20±0.17) in july but in august it shows a declining trend (9.52±0.08). the ovaries of this stage are full of ripe ova. the mature follicles become larger and irregular in shape with ecentric germinal vesicle and the yolk granules condensed (fig. 13). a few discharge follicles and atretic follicles are also found (fig. 13). at the end of this phase the zona granulosa of the mature follicles are thin and distorted in several places (fig. 14). post-spawning phase (september to november): during the post-spawning phase, the interrenal cells and chromaffin cells are attenuated in size with vacuolated cytoplasm and prominent nucleus. these cells are located around the blood vessels (figs. 15, 16), although some interrenal cells has a considerable amount of cytoplasmic mass (fig. 15). in post-spawning phase, diameter of interrenal cells reduced considerably (3.5±0.02 to 3.8±0.05 µm) and provided with cytoplasmic granules and prominent nuclei (fig. 16). the cytoplasm of chromaffin cells become vacuolated because of their degranulation (figs. 15, 16) and the diameter varies from 3.0±0.17 figure 18. the section of ovary in mystus vittatus. considerable number of primary oocytes (arrow heads), oogonia (broken arrow), distorted mature follicles (solid arrows) and oocyte v (ov) during post-spawning phase (h&e, 400x). figure 17. the section of ovary in mystus vittatus. distorted mature follicles along with oocyte iv (oiv), discharged follicle (df) and primary oocytes (arrows) during post-spawning phase (h&e, 400x). 315 int. j. aquat. biol. (2016) 4(5): 308-317 to 3.20±0.14 µm, respectively. in post-spawning period, the ovaries show a regression state. the gsi is recorded as 3.38±0.34 in september followed by 0.38±0.24 in october and 0.80±0.88 in november, respectively. during this phase the mature follicles are collapse and irregular in shape with distorted condition of yolk granules. oogonia appear in large numbers along with primary oocytes in between the distorted follicles (figs. 17, 18). discussion in fish, two tissues were found in the head kidney, i.e. the interrenal tissue (cortical portion) and chromaffin tissue (medullar portion). the interrenal tissue of teleost is homologous to the mammalian adrenal cortex and is the source of cortical steroids (jones and phillips, 1986). the amount of interrenal tissue observed in the head kidney of fishes vary among species. in the present investigation in m. vittatus, the cephalic portion of the kidney is bilateral where the interrenal gland is composed of two main types of cells chromaffin and steroidogenic and are mainly associated with the posterior cardinal veins and their tributaries. similar observations were also made by civinini et al. (2001) in gasterosteus aculeatus and sampour (2008) in carassius auratus. abdel-aziz et al. (2010) also considered that interrenal and chromaffin cells were associated with the cardinal veins to be typically teleostean. in cichlasoma dimerus, the interrenal gland components were found exclusively within the posterior portion of cephalic kidney arranged in a relatively diffuse manner. the steroidogenic and chromaffin cells were in close association with the wall of the posterior cardinal vein, its tributaries and sinusoids (morandini et al., 2014). in m. vittatus, the interrenal cells were comparatively larger than the chromaffin cells, and they were basophilic. the chromaffin cells contained pale cytoplasm and slightly basophilic nuclei. though the physiological role of adrenocortical tissue during sexual maturation and spawning was not clearly understood but significant hyper activity of interrenal tissues corresponds with the breeding phases of m. vittatus. ball (1960) observed interrenal cells to be active during the teleost reproductive phase. yadav et al. (1970) reported that epinephrine content is higher in heteropneustes fossilis during reproduction, but that the nor-epinephrine content does not fluctuate. according to them, the rise of epinephrine content might be associated with the increased active phosphorylase levels required for metabolism during the breeding period. nussdorfer (1986) opined that different cytological aspects of interrenal cells could be linked to steroidogenic cells undergoing different degrees of hormonal activity. in the present investigation in m. vittatus, the accumulation of cytoplasm contents of interrenal and chromaffin cells coincided with the transformation of various oogenetic cells and the oogenetic activities continued until spawning. there were also a few degranulated and vacuolated interrenal cells and also some chromaffin cells which could have possibly released their content for oocyte maturation. the hyperactivity of the interrenal cells could be attributed to the higher level of corticosteroid production required during maturation and spawning phases. in m. vittatus, the maximum and minimum ovarian weights were recorded in july and october respectively, which coincided with the maximum and minimum nuclear diameter of interrenal cells. therefore, the activity of these cells appeared to be closely associated with ovarian activity. the gonadosomatic index (gsi) in m. vittatus increased slightly during the growth period. the storage of cytoplasmic granules in the interrenal cells began at the end of this period, which was clearly reflected in tinctorial reactions. in channa punctatus, the interrenal cells exhibit clear seasonal changes becoming either hyperactive or inactive during the breeding and non-breeding periods of this fish species (verma and mishra, 1992). during the postspawning phase, however, only a few mature follicles and primary oocytes were noted, and the gonadosomatic index (gsi) gradually decreased. subsequent to the release of cytoplasmic contents, 316 chakrabarti and banerjee/ changes in the interregnal and chromaffin cells in relation to ovarian activities in m. vittatus the cortical cells became less efficient to gonadal stimulation and were soon transformed into quiescent phase. in the present study, the chromaffin cells in m. vittatus were more or less uniform in appearance except during maturation and spawning phase when they were hypertrophied. the occurrence of chromaffin cells close to the blood vessels indicated releasing their contents into the blood circulation. reid et al. (1998) opined that in teleosts, chromaffin tissues were associated with the synthesis and secretion of the catecholamines. sampour (2008) with the help of electron microscopic studies, observed numerous mitochondria in different shapes in cytoplasm of chromaffin cells probably produce energy for the activities of the cells during the synthesis of catecholamine hormones. further studies of electron microscopy, immunocytochemical examinations and quantitative estimations of corticosteroids and catecholamine levels would be useful in corroborating the present findings. acknowledgements the authors are grateful to the authorities of the university of burdwan, for providing necessary laboratory facilities. references aguilar c. (1997). chromosomal studies in south atlantic serranids (pisces, perciformes). cytobios, 89: 105-114. abdel-aziz el-s.h., el-sayed ali t., abd s.b.s., fouad, h.f. (2010). chromaffin cells and interrenal tissue in the head kidney of the grouper, epinephilus tauvina (teleostei, serranidae); a morphological (optical and ultrastructural) study. journal of applied ichthyology, 26: 522-527. ball j.n. (1980). reproduction in female bony fishes. symposium of zoological society of london, 1: 105135. borella m.i., morais c.r., gazalo r., rossana v., bernardino g. (1999). pituitary gland, gonads and interrenal gland of the immature pacu piaractus mesopotamicus holmberg, 1987 (teleost, characidae): morphological study. b. tec. cepta, pirassununga, 12: 57-70. chakrabarti p. (2014). histological features of interrenal and chromaffin cells in relation to seasonal testicular activities in notopterus notopterus (pallas). international journal of fisheries and aquatic studies, 1(3): 206-213. chakrabarti p., ghosh s.k. (2014). cyclical changes in interrenal and chromaffin cells in relation to testicular activity of olive barb, puntius sarana (hamilton). archives of polish fishery, 22: 151-158. civinini a., tallini m., gallo v.p. (1997). the steriodogenic possibilities of oavarian and interrenal tissues of the female stickleback (gasterosteus aculeatus) during the annual cycle: histochemical and ultrastructural observations. in: s.k. maitra (ed.). frontiers in environmental and metabolic endocrinology, burdwan university, india. pp: 77-90, civinini a., padula d., gallo v.p. (2001). ultrastructure and histochemical study on the interrenal cells of the male stickleback (gasterosteus aculeatus), in relation to the reproductive annual cycle. journal of anatomy, 199: 303-316. gallo v.p., civinini a. (2003). survey of the adrenal homolog in teleosts. international review of cytology, 230: 89-187. gazola r., borella m.i., val-sella m.v., fava de morases f., bernardino g. (1995). histophysiological aspects of the interrenal of the pacu female, piaractus mesopotamicus (teleostei, cypriniformes). b. tec. cepta, pirassununga. 8: 1-12. grassi milano e., basari f., chimenti c. (1997). adrenocortical and adrenomedullary homologs in eight species of adult and developing teleost: morphology, histology and immunohistochemistry. general and comparative endocrinology, 108(3): 483-496. hanke w., kloas w. (1995). comparative aspects of regulations and functions of the adrenal complex in different groups of vertebrates. hormone and metabolic research, 27: 389-397. jones i.c., phillips j.g. (1986). the adrenal and interrenal gland. in: p.k.t. pang, m.p. schreibman (eds.). vertebrate endocrinology, springer verlag, new york. pp: 319-350. joshi, b.n. sathyanesan a.g. (1980). a histochemical study on the adrenal components of the teleost cirrhinus mrigala (hamilton). zeitschrift fur mikroskopisch-anatomische forschung, 94: 327-336. 317 int. j. aquat. biol. (2016) 4(5): 308-317 mallory f.b. (1936). the aniline blue collagen stain. stain technology, 11: 101. morandini l., honji r.m., ramallo m.r., moreira r.g., pandolfi m. (2014) .the interrenal gland in males of the cichlid fish cichlasoma dimerus: relationship with stress and the establishment of social hierarchies. general and comparative endocrinology, 195: 88-98. nussdorfer g.g. (1986). cytophysiology of the adrenal cortex. international review of cytology, 98: 1-405. reid s.g., bernier n.j., perry s.f. (1998). the adrenergic stress response in fish: control of catecholamine storage and release. comparative biochemistry and physiology, c120: 1-27. sampour m. (2008). the study of adrenal chromaffin of fish, carassius auratus (teleostei). pakistan journal of biological science, 11: 1032-1036. singh b.r., thakur r.n., yadav b.n. (1974). the relationship between the changes in the interrenal, gonadal and thyroidal tissue of the air-breathing fish, heteropneustes fossilis (bloch) at different periods of the breeding cycle. journal of endocrinology, 61: 309316. verma g.p., misra s.k. (1992). morphological and histochemical aspects of interrenal tissue of teleost, channa gachua (hamilton). proceedings of national academy of science, india, 52: 147-154. yadav b.n., singh b.r., munshi j.s.d. (1970). histophysiology of the adrenocortical tissue of an airbreathing fish, heteropneustes fossilis (bloch.) mikroskopie, 26: 41-49. int. j. aquat. biol. (2017) 5(3): 218-227; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article reproductive biology of pseudotocinclus tietensis (siluriformes: loricariidae: hypoptopomatinae), a threatened fish species jandyr a. rodrigues-filho1, 4, renato m. honji2, paulo h. mello2, maria i. borella3, alexandre w.s. hilsdorf1, renata g. moreira*1, 21 1universidade de mogi das cruzes. av. dr. cândido xavier de almeida e souza, 08701-970, brasil. 2instituto de biociências, universidade de são paulo, rua do matão, trav. 14, 321, 05508-090, brasil 3instituto de ciências biomédicas, universidade de são paulo av. prof. lineu prestes, 1524, são paulo 05508-900, brasil. 4fundação instituto de pesca do estado do rio de janeiro, km 2, 28470-000, santo antônio de pádua, brasil. article history: received 9 january 2017 accepted 5 may 2017 available online 2 5 june 2017 keywords: reproductive cycle gonads pseudotocinclus tietensis fecundity cascudo abstract: pseudotocinclus tietensis is endemic to the upper tietê river basin and classified as vulnerable. the reproductive biology of this species is still unknown, therefore, we investigated its reproductive strategy and gonad development during its annual reproductive cycle. the fish were collected throughout one year, and histology of the gonads, fecundity and oocyte diameter was conducted. three phases of gonad maturation were found in males and females (immature, developing, and spawning capable), and the development stages of the gametes were identified within each stage. in the testes, four stages of gamete development were distinguished: spermatogonia, spermatocytes, spermatids and spermatozoa. during spermiation, the spermatozoa were released into the tubular lumen and then continued through the efferent ducts. in the ovaries, five stages of gamete development were identified: chromatin nuclear, perinucleolar, yolk vesicle formation, vitellogenic and ripe. the minimum diameter of ovulating oocytes was 297 µm, and the absolute fecundity was 64 to 306 oocytes. males with spermatozoa in the lobular lumen and females with vitellogenic and ripe oocytes were found throughout the year. pseudotocinclus tietensis has asynchronous ovarian development and gametes with fertilization capacity can be eliminated throughout the annual cycle. introduction reproduction is an important biological process and information on the cyclical development of the gonads, time and place of spawning and length in which individuals enter the reproductive process are critical for understanding the reproductive biology of a species. for teleosts, this information is particularly important for the development of fishery regulations, including the period of capture, the place and size that fish can be caught within a management program, the creation of conservation management measures as well as for the control of undesirable species (marques et al., 2000). the upper reaches of rivers such as the upper tietê river basin (utrb) are characterized by the prevalence of small fish species with high endemism, * corresponding author: renata g. moreira doi: https://doi.org/10.22034/ijab.v5i3.260 e-mail address: renatagm@ib.usp.br caused mostly by a low degree of spatial dispersion (menezes, 1994). the icthyofauna of the utrb is distinct from those of other upper reaches of drainage in the upper paraná basin (langeani-neto, 1989). this is mainly due to the presence of valleys and hills, which provide isolation of the animals, allowing a high rate of speciation and endemism (castro and casatti, 1997). this is the case for the pseudotocinclus tietensis (loricariidae: hypoptopomatinae), a species that is endemic to utrb (langeani-neto, 1989; wolf et al., 2005; marceniuk et al., 2011), listed as “endangered” in the national list of threatened species (brasil, 2016) and classified as "vulnerable" in the red list of threatened species (iucn, 2016). the subfamily hypoptopomatinae is a monophyletic group of loricariids composed of 19 genera 219 int. j. aquat. biol. (2017) 5(3): 218-227 and 139 species (eschmeyer and fong, 2016) that are widely distributed in continental waters of south america and have been studied in terms of molecular phylogeny (cramer et al., 2011). this subfamily is divided into two tribes, hypoptopomatini and otothyrini, and the genus pseudotocinclus is a member of the latter. until 2005, this genus included one species. i.e. p. tietensis (langeani-neto, 1989), but the genus was revised and two new species were described as p. juquiae from the ribeira do iguape basin and p. parahybae from paraíba do sul river basin (takako et al., 2005). the presence of a small naked area on the snout tip and transverse dark-brown bands on the dorsal region of the body distinguishes p. tietensis from the other species in the genus (takako et al., 2005). the main focus of our study, which took place at the upper reaches of the tietê river and tributaries, was to analyze the gonadal and oocyte development of p. tietensis during its annual cycle to gain some expertise on the reproductive biology of this species and to support decisions on conservation management of this threatened species. materials and methods the specimens of p. tietensis (ihering, 1907) (fig. 1) were sampled in two tributaries of the urtb, são paulo state, brazil (fig. 2) including the biritiba mirim river (23º42'01.81''s, 46º05'27.32''o) (biritiba mirim/sp) and the paraitinga river (23º31'36.09''s, 45º48'52.08''o) (salesópolis/sp). fish were captured every two months with sieves and electrofishing device (honda generator ex1000w, ca). specimens were identified based on takako et al. (2005). after sampling, fish were placed in plastic bags and transported to the laboratory of metabolism and reproduction of aquatic organisms (lameroa), where they were anesthetized with tricaine methanesulfonate (ms-222, ins sigma diagnostics, st. louis, mo) (1 g.l-1), neutralized with sodium bicarbonate and killed by decapitation (according to the institutional animal care protocols and approval. the total length and weight (wt) were recorded, and the ovaries were excised and weighed (wg) for the calculation of the gonadosomatic index (gsi) according to vazzoler (1996): gsi = (wg x100) /wt. the middle third of the gonads was fixed in bouin's solution for 18-20 hours and transferred to ethanol (700 gl). the fragments of gonads were dehydrated and embedded in paraplast, diaphanized (erv-plast; erviegas surgical instruments, são paulo, brazil), cut with a microtome (5 µm), stained with hematoxylineosin and examined under light microscopy (zeiss axioskop ii light microscope, zeiss mc80 dx camera and a computer image capture pixera professional). the different developmental stages of the gametes were identified according to west (1990) for the ovaries and according to grier and aranzábaluribe (2009) for the testes. based on the predominance of gamete development stages, females and males were divided into the different phases of figure 1. the collected pseudotocinclus tietensis from the river paraitinga located in the upper tietê basin region, state of são paulo, brazil. dorsal, lateral and ventral view displayed here. 220 rodrigues-filho et al./ reproductive biology of pseudotocinclus tietensis gonadal development, as described by brownpeterson et al. (2011). the remaining portion of the ovaries was weighed again for the fecundity analysis, immersed in a gilson solution (simpson, 1951) and stored for 30 days for dissociation of the tissue containing the oocytes. after this period, the oocytes were kept in ethanol (700 gl), ready to be evaluated for the fecundity and diameter analyses. an acrylic reticulated plate was used for counting and measuring oocyte diameter under a stereomicroscope (leica mz125) with micrometric ocular (vazzoler, 1981; romagosa et al., 2001; honji et al., 2009; gomes et al., 2015). the absolute fecundity was calculated in the developing and spawning capable phases. relative fecundity was calculated using the mean body mass, total length and gonad weight. the data will show in linear fit of the type y = 𝛼 + bx, where y = fecundity, 𝛼 = regression constant, b = regression coefficient and x = variable (body weight, ovaries weight and total length). analysis of data: all values were expressed as the mean ± standard deviation (m±sd). body weight, length, weight of ovaries, gsi, number of oocytes/females and number of oocytes/ovary mass were compared among the different maturation phases using analysis of variance (one-way anova), followed by either the student–newmann–keuls (snk) test for parametric analyses or dunn’s test for non-parametric analyses. fecundity correlations were calculated using linear regression applied to total length, ovary and body mass. in all analyses, the differences were considered to be significant when p<0.05. these analyses were performed using the statistical software sigmastat for windows ver. 3.10 (systat software, san jose, ca). results a total of 41 specimens of p. tietensis were collected during the annual cycle, every two months: 20 females (january, n=4; march, n=3; may, n=3; july, n=3; september, n=4; november, n=3) with a total length of 5.9-8.3 cm (7.1±0.6) and body weight of 1.5-3.6 g (2.7±0.4 g), 16 males (january, n=3; march, n=2; may, n=2; july, n=3; september, n=3; november, n=3) with total length of 4.5-7.8 cm (6.2±0.4 cm) and figure 2. hydrographic basin from the upper tietê river. (a) head of tietê river; (b) ponte nova reservoir; (c) tietê river; d) paraitinga river; (e) paraitinga reservoir; (f) claro river; (g) ribeirão do campo reservoir; (h) biritiba mirim reservoir; (i) biritiba mirim river; (j) jundiaí reservoir; (k) jundiaí river; (l) taiçupeba reservoir; (m) taiaçupeba river (marceniuk et al., 2011). sampling points: red arrow (23º31'36.09''s, 45º48'52.08''o; paraitinga river) and black arrow (23º42'01.81''s, 46º05'27.32''o; biritiba mirim river). 221 int. j. aquat. biol. (2017) 5(3): 218-227 body weight of 1.2-2.9 g (2.0±0.4 g). five specimens with total length below 4 cm (3.5±0.3 cm) and body weight of 1 g (1±0.2) that we were not able to remove their gonads, discarded. the testes were paired structures located along the bladder and connected to the dorsal wall of the coelom. the posterior end of the organs continued through a common duct that opened to the outside via the urogenital papilla. the testes were surrounded by the tunica albuginea, from which septa of connective tissue were directed towards the internal part of the organs. microscopically, the testes were classified as unrestricted spermatogonial testes, anastomosing tubular type, composed of numerous tubules containing the germinal compartment, separated by interstitial tissue (fig. 3a, c). the germinal compartment was surrounded by sertoli cells (fig. 3c) and contained seminiferous tubules and cysts containing germ cells (fig. 3d, e). it was possible to figure 3. histological sections showing the maturation phases and the stages of cellular development in the testes of pseudotocinclus tietensis. (a) immature, (b-c) developing, (d) spawning capable, and (e-f) spawning capable (sg=spermatogonial, it=interstitial tissue, s=sertoli cells; sc=spermatocytes; st.=spermatids; sz=sperm, lc=luminal cavity). 222 rodrigues-filho et al./ reproductive biology of pseudotocinclus tietensis establish three stages of testicular development during the annual cycle: immature, developing and spawning capable. these stages of testicular development were distinguished due to the following characteristics: immature: testes with a great amount of interstitial tissue and spermatogonia predominated within the germ cells (fig. 3a). spermatogonia were larger cells among the germline, with a well-developed central nucleus, prominent nucleoli and a clear but abundant cytoplasm (fig. 3a). developing: the interstitial tissue diminished and the number of spermatogonia was reduced (fig. 3b). spermatocytes and spermatids (fig. 3b, c) were figure 4. histological sections showing the maturation phases and stages of development of gametes in the ovary of psudotocinclus tietensis. immature: oocytes in stage i (chromatin nucleolar, a) and ii (perinucleolar, b-c); developing: oocytes in phase iii (cortical alveoli, c-d) and iv (vitellogenic, e); spawning capable: phase v oocytes (ripe, f) (cn=chromatin nucleolar, nog="nests" of oogonia, og=oogonia, pn=perinucleolar, c=cytoplasm, n=nucleus, n=nucleolus, ca=cortical alveolar, lv=lipid vacuoles, zp=zona pellucida, fc=follicular cells, pv=protein vitellogenesis). 223 int. j. aquat. biol. (2017) 5(3): 218-227 located within the intratubular cysts. spermatocytes were smaller than spermatogonial cells, contained a clear cytoplasm and a large central nucleus and were organized as groups of rounded cells (fig. 3b, c). the spermatids were smaller arising from the division of spermatocytes that were present in large numbers within the cysts (fig. 3b). these spermatids are completely spherical with little cytoplasm and a rounded nucleus with a very compact chromatin (fig. 3c). spawning capable: at this stage, the interstitial tissue was scarce and spermatid cysts were still present (fig. 3d, e), but in small numbers. the spermatocysts were opened to release the spermatozoa (fig. 3d, e) in the luminal cavity, the main locale of spermatozoa (fig. 3e, f). the microscopic classification of the ovaries was based on cytologic features of the developmental stages of the follicles and structures. the development resulted in changes of the germ cells that characterized the different phases of gonadal maturation. the ovaries were paired organs located laterally to the gas bladder and connected to the dorsal wall of the coelom. each ovary showed free ends, and the rear extremity continued through a common duct that was opened to the outside through the urogenital papilla. histological analysis allowed for identification of three phases of gonadal development in females throughout the annual cycle: immature, developing and spawning capable. these stages were observed in all seasons. the ovaries presented lamellae filled with oocytes and surrounded by follicular cells. it was possible to identify five stages of oocyte development, described below, together with the description of gonadal development phases: immature: this is the initial phase of gonadal development that includes the following stages of oocyte development: (1) chromatin nucleolar: these cells were grouped into nests (fig. 4a), embedded into the lamellae, and were either oogonia or oocytes in the initial phases of development. the cytoplasm of the cells was slightly stained, and each cell had a large nucleus that was spherical and intensely basophilic, with one nucleolus in the central position (fig. 4a). (2) perinucleolar. the oocytes detached themselves from their nests and began a growth phase; the basophilic cytoplasm was developed, and the nucleus contained numerous rounded nucleoli of different sizes, located on the periphery of the nucleus (fig. 4b). developing: during this phase, two stages of oocyte development are found: (3) yolk vesicle formation (cortical alveolar), in which oocyte size increased and the oocytes entered the phase in which endogenous vitellogenesis occurs, characterized by intense lipid deposition, which was represented by vacuolization of cytoplasm (empty spherical structures located close to the cell membrane resulting from the processing of histological analyses) (figs. 4c, d). the cytoplasm became acidophilic. zona pellucida became evident. (4) vitellogenic (yolk) characterized by the deposition of highly acidophilus protein granules (fig. 4 e). this deposition occurred in the form of platelets from the periphery of the oocyte cytoplasm, causing a further increase in size. spawning capable: in this phase, germ cells were present in the ovaries at most stages of development, as described above. additionally, at stage (5), rip (mature) oocytes were present, with larger and more numerous protein granules (fig. 4f). lipid vesicles on the periphery were still present but reduced. the end of this phase is characterized by the migration of the nucleus from the core to the periphery of the cytoplasm (fig. 4f), a process known as germinative vesicle breakdown (vgbd). table 1. ovarian parameters of pseudotocinclus tietensis during late maturation phases. maturation phase gonads weight (g) gsi absolute fecundity relative fecundity developing 0.040.07a 1.80.24a 586.167.36a 861.571.75a spawning capable 0.240.06b 7.41.80b 1795.3210,57b 2190.1233.27b values (mean ± standard deviation) followed by different letters are statistically different among maturation phases (p≤0.05). gsi: gonadosomatic index 224 rodrigues-filho et al./ reproductive biology of pseudotocinclus tietensis there was no relationship between body weight and stage of gonadal maturation; the animals at all phases of gonad development presented no difference in body weight. however, there was a relationship between the total length and the phase of gonadal development (p=0.024), showing that the animals in the developing phase were shorter. the data in table 1 show that females at the spawning capable phase presented with larger ovaries and gsi calculations than those in developing phase (p=0.006 and p=0.012, respectively). furthermore, the absolute number of oocytes per female (absolute fecundity) and the ovarian mass were also higher in the spawning capable females than the developing females (p<0.001 for both parameters). the frequency distribution of oocyte diameters, calculated based on the average value of females in each maturation stage is presented in figure 5. this pattern of oocyte diameter distribution defines p. tietensis ovaries as asynchronous. vitellogenic oocytes were defined as those with a diameter greater than or equal to 297 µm. below this diameter, all oocytes are still at the cortical alveolar or perinucleolar stages. based on these analyses, the absolute fecundity of p. tietensis was 64-306 oocytes, all which were eliminated every spawning. the relationships between fecundity and body weight, gonad weight and total length are presented in figure 6a, b, and c, respectively. table 2 presents the correlation between fecundity and body weight, total length and gonad weight. the linear correlation showed that fecundity in p. tietensis was more related to gonad weight than to body weight or total length. these data were corroborated the fact that body mass did not vary with the maturation phase. discussion the results showed that p. tietensis presents an asynchronous oocyte development, with females in the capable spawn phase found throughout the year. even considering the conservation status of the species and the difficulties in capturing the fish, we realize that the number of specimens captured in this study is reduced, but enough to understand the gonad development. according to grier et al. (1980), the testicular structure of teleosts can be divided into unrestricted spermatogonial (spermatogonia are distributed over the entire length of the seminiferous lobules) and restricted spermatogonial (spermatogonia are confined to the distal tubule terminal). in p. tietensis, testicular structure was identified as unrestricted spermatogonial type, with spermatogonia present table 2. linear correlation between absolute fecundity (fabs) with body mass (bm), total length and gonad weight of pseudotocinclus tietensis. relationship 𝛼 b r2 body weight fabs=33.99bm–18.96 18.96 33.99 0.361 total length fabs=46.43tl–253.27 253.27 46.43 0.429 gonad mass fabs=269.36gm+40.98 40.98 269.36 0.581 figure 5. frequency distribution of oocyte diameters in pseudotocinclus tietensis. (a) developing and (b) spawning capable. 225 int. j. aquat. biol. (2017) 5(3): 218-227 across the length of the seminiferous tubules, forming germinal cysts all over the walls of the tubules, according to grier (1996). the traditional method for classification of the reproductive stages establishes four basic stages: resting, maturing, mature and spent. the criteria for defining these stages and appropriate terminology vary widely between authors. menezes et al. (2000) classify pseudotothyris obtuse, a species from the same subfamily (hypoptopomatinae) of p. tietensis, as having seven microscopic stages of development of the testes. in the present study, the testes were classified into only three stages, according to the recent criteria established by brown-peterson et al. (2011) that standardizes the classification of the reproductive phases. most of the traditional studies that classify testicular maturation phases consider only four phases: resting, maturing, mature and regression/exhausted. this four-phase classification scheme is based on the principle that a smaller number of stages should decrease the chances of errors in classification. however, it is important to highlight that the regressing phase was not found in p. tietensis, suggesting that either the gametes can be successfully released in the regions that fish were sampled or that the regressing and regenerating phases were so fast that the number of samplings did not allow the capture of animals in these phases. the histological examination of the ovaries provides information about transitions and structural and morphological changes that occur during oocyte development, considering specific stages through which the oocytes pass during their maturation (dias et al., 1998). researchers studying the classification of maturation phases of p. obtusa females (menezes et al., 2000), as well as in males, identified seven phases of ovarian development. following the classification of maturation phases proposed by brown-peterson et al. (2011) for p. tietensis, we found three phases of maturation. however, because parental care is common in loricariids and it is believed that females could be burrowed by keeping the eggs, there is the potential that females are inaccessible for sampling at the regressing/spent phase. the fecundity, which was found to be between 64 and 306, was calculated using the oocytes that were ready to be released in the next spawning. these oocytes included those that were in the end of maturation phase and those that were already mature. a wide range in fecundity is common in asynchronous species, as observed in oreochromis niloticus (duponchelle et al., 2000), which was found to have a total fecundity ranging from 149 to 2797. the number figure 6. the relationship between fecundity and body weight (a), fecundity and total length (c), and fecundity and gonad mass (b) in pseudotocinclus tietensis by considering all animals sampled. 226 rodrigues-filho et al./ reproductive biology of pseudotocinclus tietensis of oocytes varied mainly in relation to the gonadal mass; for the length and body mass, there was no correlation with the number of oocytes to be ovulated. at all stages of maturation, there are a large number of residual oocytes (oogonia each with a small diameter that would be recruited in the next reproductive cycle. the fact that p. tietensis lacks one unique batch of oocytes with a large diameter but contains many small batches of oocytes above 297 µm that would be ovulated in the next ovulation cycle characterizes the species as a multiple spawner. these data were corroborated by the presence of gametes in various stages of development in the gonads throughout the annual cycle, with no predominance of one cell type. however, the calculation of fecundity in asynchronous species presents some problems regarding the choice of the batch of the smaller oocytes to be ovulated. according to narahara et al. (1989), the choice of this batch, assumes that an oocyte at this diameter will be successfully ovulated. however, many processes are known to trigger the reabsorption of oocytes, which can impair ovulation. in conclusion, even considering the threatened status of p. tietensis, the ability to follow the gonadal development of a representative number of males and females throughout the annual cycle allows for classification of the species as a multiple spawner, with a variable number of oocytes to be ovulated at each spawn. these data will help fill current gaps regarding the biology and reproductive strategy of this species, which as of yet is still not known (www.fishbase.org). we suggest that conservation attempts for this species could be successful using proper management practices, such as the establishment of pairs of fish in fish farms and ongoing supervision of reproductive behavior throughout the year. acknowledgements the authors would like to thank the employees of the ponte nova fish farm for providing the facilities for sampling in the upper tietê river basin and s. batlouni for his help with the testes analysis. this work was supported by an undergrad student fellowship (cnpq/pibic). references brasil. 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(1998). análise macroscópica dos ovários de teleósteos: problemas de classificação e recomendações de procedimentos. revista brasileira de biologia, 58: 55-69. duponchelle f., cecchi p., corbin d., nunes j., legendre m. (2000). variations in fecundity and egg size of female nile tilapia, oreochromis niloticus, from manmade lakes of côte d’ivoire. environmental biological fishes, 57: 155-170. eschmeyer w.n., fong j.d. (2016). catalog of fishes, species by family/subfamily. http://research.calacademy.org/research/ichthyology/catalog/speciesbyfa mily.asp/ [online version, updated 01 november 2016] grier h.j., linton j.r., leatherland j.f., vlaming v.l. (1980). structural evidence of two different testicular types in teleostei fishes. american journal of anatomy, 159: 331-45. gomes a.d., tolussi c.e., ribeiro c.s., honji r.m., moreira r.g. (2015). the role of ovarian steroids in reproductive plasticity in hoplias malabaricus (teleostei: characiformes: erythrinidae) in tropical reservoirs with different degrees of pollution. general and comparative endocrinology, 222: 1-10. grier h.j., uribe-aranzábal m.c. (2009). the testis and spermatogenesis in teleosts. in: b.g.m. jamieson, (ed.). reproductive biology and phylogeny of fishes http://lattes.cnpq.br/8819118069017566 227 int. j. aquat. biol. (2017) 5(3): 218-227 (agnathans and bony fishes), new hampshire: science publishers, endfield, 119-142. honji r.m., narcizo a.m., borella m.i., romagosa e., moreira r.g. (2009). patterns of oocyte development in natural habitat and captive salminus hilarii valenciennes, 1850 (teleostei: characidae). fish physiology and biochemistry, 35: 109-123. ihering r.v. 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(1994). importance of the conservation of the ichthyofauna of brazilian aquatic ecosystems. in: seminar on aquatic fauna and the brazilian electricity sector. caderno 3. conservação. comitê coordenador das atividades de meio ambiente do setor elétrico comase. rio de janeiro, eletrobrás. 7-13. menezes m.s., takeuti d.f., aranha j.m.r., verani j. r. (2000). males and females gonadal development of pseudotothyris obtuse (ribeiro, 1911). acta biológica paranaense, 29: 89-100. narahara m.y., godinho h.m., romagosa e. (1989). type of spawning and fecundity of the catfish, rhamdia hilarii (valenciennes, 1840) (siluriformes, pimelodidae). boletim do instituto de pesca, 16: 37-45. romagosa e., narahara m.y., borella m.i., fenerichverani n. (2001). selection and characterization of matrinxã, brycon cephalus females, induced reproduction. boletim do instituto de pesca, 27: 139147 simpson a.c. (1951). the fecundity of the plaice. fish invest, 5: 1-27. takano a.k., oliveira c., oyakawa o.t. (2005). revision of the genus pseudotocinclus (siluriformes: loricariidae: hypoptopomatinae), with descriptions of two new species. neotropical ichthyology, 3: 499-508. vazzoler a.e.a.m. (1996). biologia reprodutiva de teleósteos: teoria e pratica. eduem. 169 p. vazzoler a.e.a.m. (1981). manual de métodos para estudos biológicos de populações de peixes. cnpq, brasília. zaiden s.f. (2000). morfologia gonadal e metabolismo energético da piraputanga brycon hilarii (cuvier e valenciennes, 1849) (pisces, characidae), em cativeiro, durante o ciclo reprodutivo anual. unpublished master dissertation, centro de aquicultura da universidade estadual paulista, jaboticabal. 152 p. west g. (1990). methods of assessing ovarian development in fishes: a review. journal of marine freshwater research, 41: 199-222. http://lattes.cnpq.br/4569484016705542 http://lattes.cnpq.br/7494205396994252 http://lattes.cnpq.br/4352170261682357 http://www.iucnredlist.org/ int. j. aquat. biol. (2017) 5(3): 218-227 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی pseudotocinclus tietensis زیست شناسی تولید مثل گونه در معرض تهدید (siluriformes: loricariidae: hypoptopomatinae) *1،2 ج. موریرا ، رناتا1، الکساندر و.س. هیلسدروف3ی. بورالآ، ماریو 2ملو، پائولو ح. 2م. هونجی رناتو ،1،4فیلهو-. رودریگزآجاندیر ، برزیل.08701-970دانشگاه د موگی داس کروزس، آو. دکتر کاندیدو د آلمیدا ای سوزا، 1 .، برزیل05508-090، 321، 14انیستیتو د بیوسینکیاس، دانشگاه د سائوپائولو، روآ د ماتو، تراو، 2 برزیل. 05508-900، سائوپائولو 1524انیستیتو د سینکیاس بیو مدیکال، دانشگاه د سائوپائولو، آو. پوفسور لینئو پرستس، 3 ، سنتو آنتونیو د پائودا، برزیل.28470-000، 2فونداچائو انیستیتو د پسکا دو استادو دو ریو د جانیرو، کیلومتر 4 چکیده: .شودبندی میپذیر طبقهآسیبگونه عنوان یک بهحوضه رودخانه تیته بوده و ناحیه باالدست ساکن pseudotocinclus tietensisماهی بومزاد مورد نهاالس ی چرخه تولیدمثلگنادی آن را در ط توسعهمثل و اتژی تولیدربنابراین ما است ،ناشناخته استتاکنون مثل این گونه شناسی تولیدزیست ر د یگناد بلوغمرحله سهشد. طالعهمها آن اووسیتآوری و قطر شناسی گناد، هم، بافتندآوری شدسال جمعها در طی یکبرسی قرار دادیم. نمونه مرحله چهار، هادر بیضهمرحله مشخص شد. ها در هر توسعه گامتمراحل و شد یافتها )نابالغ، در حال توسعه و قادر به تخمریزی( نرها و ماده لوله ه درونب، اسپرماتوزوآ ریزیاسپرم مرحله . در طیشد تشخیص داده اسپرماتوگونیا، اسپرماتوسیت، اسپرماتید و اسپرماتوزوآشامل یگامت توسعه هسته کروماتینی، پیش شامل یمرحله توسعه گامت پنجها ، د. در تخمداننشوهدایت میله خارج ی وابران ارمج از طریقزاد شده و سپس آلومن بود. نرهای 306تا 64آوری مطلق میکرومتر و هم 297. حداقل قطر تخمک شد تشخیص داده زایی و رسیدگیهستک، تشکیل وزیکول زرده، زرده دارای تخمدان با p. tietensisماهی رسیده در طول سال یافت شدند. سازی شده وزرده هایبا تخمک هادارای اسپرماتوزوآ در لوله لومن و ماده شوند. خارجتوانند های با قابلیت لقاح در طول چرخه ساالنه میو گامت بوده توسعه غیر همزمان .آوری، کاسکودوهم ، pseudotocinclus tietensis،هاگناد ،مثلچرخه تولید :کلمات کلیدی int. j. aquat. biol. (2023) 11(2): 119-123 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article first record of favonigobius gymnauchen (bleeker, 1860) (teleostei: gobiidae) in the north-western indian ocean: evidence for potential taxonomic diversity fatah zarei1, hamid reza esmaeili1, seyed hassan hashemi2 1ichthyology and molecular systematics research laboratory, zoology section, department of biology, school of science, shiraz university, shiraz, iran. 2department of environment, bandar abbas, hormuzgan province, iran. article history: received 5 january 2023 accepted 1 april 2023 available online 2 5 april 2023 keywords: gobioidei, distribution, dna barcoding, persian gulf, sharp-nosed sand goby. abstract: a single specimen of favonigobius gymnauchen was captured from the iranian coast of the persian gulf, north-western indian ocean. species identification was performed based on sequence analysis of the mitochondrial coi barcode region. this significant record from the northern persian gulf, adds a new species to the gobiid fauna of the north-western indian ocean and represents the easternmost documentation of the species. the presence of a deep phylogeographic break between the indian ocean and the west pacific groups of haplotypes suggests the potential existence of hidden taxonomic diversity, hereby highlighting the necessity of a comprehensive morphological and molecular analysis of the sharp-nosed sand gobies from the two oceans. introduction gobiidae is the largest fish family with some 1974 recognized species (fricke et al., 2022). while gobiids are found both in marine and freshwater environments throughout the world (nelson et al., 2016), a few other fish families having more than a thousand valid species are strictly freshwater fishes. among fish, they are unparalleled for their ability to adapt to microhabitats and specialize in novel ecological niches, leading to rapid speciation and adaptive radiations (patzner et al., 2011). gobiids of the northern indian ocean have not been thoroughly studied yet, mainly due to their low economic importance, small sizes, cryptic appearance, poor geographic sampling, and distributions in difficultto-sample habitats. recent systematic observations in this region resulted in the description of a few new species (kovačić et al., 2020; zarei et al., 2022) and new records (e.g., sadeghi and esmaeili 2019; sadeghi et al., 2017, 2019; ghanbarifardi and lagzian, 2019; al jufaili et al., 2022), suggesting a rich but yet unexplored gobiid fauna for the region. correspondence: hamid reza esmaeili doi: http//doi.org/10.22034/ijab.v11i2.1879 e-mail: hresmaeili@shirazu.ac.ir dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.5.7 species of the gobiid genus favonigobius whitley, 1930, belonging to the gobiopsis-lineage sensu agorreta et al. (2013) of the subfamily gobiinae with nine currently recognized species, are found in the seas and freshwaters environments of the indo-pacific. the sharp-nosed sand goby, favonigobius gymnauchen (bleeker, 1860) is mainly distributed in the west pacific region (fricke et al., 2022), however, a few georeferenced coi barcodes deposited in the genbank confirm its presence in the coastal habitats of india, bangladesh, and malaysia in the bay of bengal (fig. 1). the present study for the first time records it from the iranian waters of the persian gulf, which adds a new species to the fish fauna of the north-western indian ocean. we investigate the new record’s phylogenetic placement based on the mitochondrial coi, and preliminary discuss the species’ phylogeographic pattern in the indo-west pacific. materials and methods on october 2017, one goby specimen was collected 120 zarei et al./ first record of favonigobius gymnauchen in the north-western indian ocean using a hand net from the iranian coast of the persian gulf (fig. 1). a sample of pectoral fin tissue was taken from this specimen and stored in 96% ethanol for genetic analysis. the voucher specimen was preserved in formaldehyde (10%), and deposited in the zoological museum of shiraz university, collection of biology department (zm-cbsu). total dna was extracted from the fin clip according to a salt method protocol described in bruford et al. (1992). following zarei et al. (2021) and using a bioer xp thermal cycler (bioer technology co. ltd., hangzhou, china), the mitochondrial cytochrome c oxidase subunit i (coi) barcode region was amplified using a primer pair fish-bcl and fish-bch (baldwin et al., 2009). following purification with the exosap-it® (usb) kit, the pcr product was sanger sequenced with bigdye® terminator v3.1 cycle sequencing kit (applied biosystems, foster city, ca) on an abi prism 3730xl dna analyzer (applied biosystems, foster city, ca) by the faghihi lab., shiraz, iran. bioedit 7.1 (hall, 1999) was used to read and edit the new dna chromatogram, while clustalw procedure in mega 7 (kumar et al., 2016) was used for sequence alignment. the new coi sequence is deposited in the genbank (oq892260). a total of 46 coi sequences representing 35 gobiid species from different lineages were obtained from genbank. after alignment with the new haplotype sequence from the persian gulf, the coi dataset was analyzed using the maximum likelihood (ml) method in mega. popart 1.7 (leigh and bryant, 2015) was used to investigate the phylogeographic depth. to delineate putative species, (i) a statistical parsimony (sp) network based on a 95% connection probability threshold was estimated in tcs 1.21 (clement et al., figure 1. left: phylogenetic placement (ml) of the favonigobius gymnauchen coi haplotype sampled from the persian gulf basin, iran (marked with a black arrow). right: point distribution map of f. gymnauchen: red circles indicate previous records (froese and pauly, 2022; gbif, 2022; genbank, 2022), whereas the blue circle (marked with a black arrow) represents the new record from the persian gulf, iran (arabian sea basin). the mj haplotype network on the map for the six coi haplotypes observed in 13 f. gymnauchen specimens shows a deep phylogeographic break (10 mutational steps) between the indian ocean (yellow circles) and the west pacific (green circles) groups of haplotypes. species figures: male (upper) and female (lower) f. gymnauchen (after masuda et al., 1984). 121 int. j. aquat. biol. (2023) 11(2): 119-123 2000), and (ii) an assemble species by automatic partitioning analysis (asap; puillandre et al., 2021) was performed through its web interface (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.ht ml) using kimura (k80) ts/tv (=0.2). furthermore, cutoff value of 2% k2p sequence divergence for coi was used as an indicator of distinct species (ward, 2009). results the favonigobius specimen recorded from the persian gulf, phylogenetically grouped with f. gymnauchen (fig. 1). the sequence analysis of a 615 bp fragment of the coi region detected 20 variable nucleotide sites, including 8 singletons and 12 parsimony informative sites between 13 examined f. gymnauchen specimens from the indowest pacific region, leading to the definition of 6 haplotypes. corroborating the ml phylogeny, the median-joining haplotype network shows that the three haplotypes from the indian ocean localities (i.e., persian gulf and bay of bengal) are separated from the three west pacific haplotypes from china and philippines by 10 mutational steps (fig. 1). the two lineage are separated by 2.4% k2p genetic distance in the sequence of coi barcode, i.e., larger than the conventional threshold proposed for coi in fish (i.e., 2%; ward, 2009) as an indicator of distinct species. the statistical parsimony (sp) and assemble species by automatic partitioning (asap; the partition with the highest asap-score) analyses found support for only one species in the f. gymnauchen clade. the partition with the secondbest asap-score however, found support for two putative species in the f. gymnauchen clade, corresponding to the indian ocean and west pacific subclades. discussion favonigobius gymnauchen is well known from the west pacific (fricke et al., 2022), and there are few unpublished genbank records from the bay of bengal, northeastern indian ocean. this significant record from the iranian waters of the persian gulf, adds a brand new species to the gobiid fauna of the northwestern indian ocean and represents the easternmost documentation of the species. our results validated two deeply diverged lineages within the range of f. gymnauchen: one lineage in the indian ocean and a second lineage in the west pacific. considering that the west pacific lineage includes sequences close to the type locality of f. gymnauchen (i.e., tokyo, japan; bleeker, 1860), it is possible that the indian ocean lineage may represent a yet undescribed species depending on the outcome of additional sampling, and an integrative morphological and molecular analysis. the geographic distribution of these two lineages suggests a significant phylogeographic break at the sunda shelf barrier. this phylogeographic break was also documented for several marine fish (rocha et al., 2007), including blenniids omobranchus punctatus (mehraban et al., 2021), o. ferox and o. elongates (gibbs et al., 2018), and the japanese threadfin bream, nemipterus japonicas (farivar et al., 2017). similar phylogeographic patterns have also been documented for macroalgae (wichachucherd et al., 2014), and bivalves (mahidol et al., 2007). this concordance in the geography of gene-tree partitions across multiple genes and codistributed taxa implicates a shared historical biogeographic factor—allopatric divergence driven by the effect of sunda shelf barrier—in shaping intraspecific genealogies. acknowledgment shiraz university and department of the environment are acknowledged for financial and logistic supports. references agorreta a., san mauro d., schliewen u., van tassell j.l., kovačić m., zardoya r., rüber l. 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(2016) 4(4): 224-232; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article allometric growth pattern, sexual dimorphism and size at the onset of sexual maturity in opusia indica (brachyura: ocypodoidea: camptandriidae) from mangrove areas of pakistan noor us saher*1, naureen aziz qureshi2, uroj aziz3 1centre of excellence in marine biology, university of karachi, karachi, pakistan. 2department of zoology, government women college university, faisalabad, pakistan. 3a.p.w.a govt. degree college for women, karimabad, karachi, pakistan. article history: received 7 july 2016 accepted 8 august 2016 available online 2 5 august 2016 keywords: sexual maturity somatic growth intertidal morphometry allometry abstract: size at sexual maturity and patterns of somatic growth are important aspects of reproductive history of crab. the main purpose of this study is to provide an estimate for the onset of morphological sexual maturity in mangrove crab, opusia indica from a population located in korangi creek intertidal mud flat (karachi, pakistan) based on relative growth. the crabs were monthly collected through quadrat method from march 2001 to february 2002. a total of 1702 crabs was obtained, of which 764 were males, 939 were female. the morphometric measurement of carapace, abdomen, cheliped and male gonopod was related to carapace width. based on carapace width males were significantly larger than female, indicating sexual dimorphism. the size at onset of sexual maturity in males was estimated as 5.51 mm carapace width and 5.3 mm carapace width in females. the positive allometric growth of female abdominal width were likely related to the incubation process. introduction during the course of growth and development in brachyuran crabs, certain dimensions of animal’s body increases at different rates in proportion to size, resulting in a phenomenon known as relative growth. a growth rate between male and female crabs as well as between juvenile and adult crabs shows great differences during ontogeny and can be used to determine morphological sexual maturity (hartnoll, 1978). the difference in relative growth patterns among male and female crabs for size of chelipeds, abdomen and pleopods indicates sexual dimorphism, and can be used to predict maturity in crab (hartnoll, 1978; vannini and gherardi, 1988; saher and qureshi, 2011b). estimation of size at sexual maturity is key to describe population biology and structure in brachyuran crabs (pinehiro and fransozo, 1998). it can be determined by different methodologies i.e. determining morphometric (allometric) changes at * corresponding author: noor us saher e-mail address: noorusaher@yahoo.com puberty molt (hartnoll, 1969), gonadal development after attaining maturity (viau et al., 2006) and functional maturity (viau et al., 2006). functional maturity in decapods can be estimated by the determination of ovigerous female in population and situation of actual participation of both in the reproductive process (viau et al., 2006). determining the size at sexual maturity in crabs is useful to understand population aspects concerned with abundance, structure and number of mature individuals in a given population as it is responsible by the future generation as well as for researches involves in conservation of biodiversity. opusia indica (alcock, 1900) is a small ocypodoid crab of the family camptandriidae (ng et al., 2008). these small detritivorous crabs are inhabitant of intertidal mud flat associated with tropical and subtropical mangrove forest (ng et al., 2009). crab dig burrows in soft sediment to tolerate harsh environmental conditions as well as refuges 225 int. j. aquat. biol. (2016) 4(4): 224-232 from predators. these burrowing activities help in penetrating air into the deeper sediment layer. deposit feeding crabs emerge from their burrows when tide descends, there by changing the quality and quantity of resources distributed along the coast in turn play a key role in ecological functioning and environmental management on the intertidal detrital food web (saher and qureshi, 2011a, b). the review of literature reveals that there is no previous work on the morphometric studies on these small detritivorous crabs. the aim of the current study is to identify the allometric relationship in male and female crabs of o. indica and also to determine the size at the onset of sexual maturity based on morphological and functional maturity. materials and methods study area: the pakistan coastal belt is about 1050 km long, with 350 km along sindh and 700 km along balochistan coast. karachi, pakistan is located on the northern border of arabian sea having a coastal belt of about 100 km having indus delta on its southeastern side and hub river in the west. the coast along the indus delta consists of numerous dendritic tidal creeks network, including korangi creek (24°79'n, 67°20'e) (fig. 1), located in the south east of karachi, entouched with a system of creeks in its northeastern side, and open waters on its southern end (qureshi and saher, 2012). mangrove forest mainly comprises of avicenna marina along creeks coastal zone provides a critical habitat for variety of brachyuran crabs. field procedures and sampling methodology: crabs were collected from tidal mud flats regularly from march 2001 to february 2002 through transect quadrat method. during low tide period sampling were done by digging square down to 30 cm. excavated sediment were sieved (1 mm mesh size), the crab samples were collected in labelled bags and freeze till analyses. morphometric analyses: for morphometry crabs were initially sorted and sexed (juvenile male (jm), juvenile female (jf), adult male (am) and adult female (af), weighed and measured. the carapace dimensions used in morphological analysis were carapace length (cl) (carapace length from anterior median part to posterior carapace margin) and carapace width (cw) (carapace width measured at its widest point). cheliped dimensions included chela propodus length (ch.l), chela propodus width (ch.w) and chela propodus height (ch.h). abdominal dimensions included abdominal width (width of fifth abdominal somites in male and female crab) and length of first male pleopod (p1) (fig. 2). all measurements were taken using vernier caliper to the nearest 0.1 mm. carapace width was used as independent variable which is denoted as x related to other body dimension which were considered as dependent variables y. the mean values, minimum, maximum and standard deviation were calculated for each variable of female and male and compared by independent (students) t-test. crab wet weight was estimated using analysis of variance (glm) to estimate variation among juvenile and adult of o. indica. the relative growth was described using the power function y=axb and linearized (log y = log a+b log x) (huxley, 1950) where “a” is the intercept and “b” is the slope or allometric growth constant. for size weight relationship, exponent ‘b’ represent the weight gain which can be isometric, b=3; positively allometric, b>3; and negatively allometric b<3. the pattern of allometric growth for each developmental phase was established by the ‘b’ figure 1. coastal areas of pakistan showing study site korangi creek mangrove areas of pakistan. 226 saher et al./ allometric growth pattern and sexual maturity in opusia indica values, considered as positive allometry when b>1, negative allometry b<1 and growth was considered isometric when 0.9<b<1.1 (hartnoll, 1982; castiglioni and negreiros-fransozo, 2004; saher and qureshi, 2011b). to analyze growth patterns between male and female and between developmental phase slopes were statistically compared by analysis of covariance (ancova). functional maturity analysis: functional maturity in crabs was determined by identifying smallest ovigerous female (of) carrying eggs found in the collected samples. male and female smaller than this size were classified as juvenile (saher and qureshi, 2011b). for identifying adult females, additional criteria, i.e. convex abdomen which form an incubatory chamber in adult females was also considered, for adult male reflexed pleopod 1 (p1) which become more robust after attaining maturity were considered (snowden and jones, 1995). in order to estimate the size at which 50% population got functional maturity (y), size classes of 2 mm carapace width were made. the relative frequency of adult in each size class was plotted and fitted in a sigmoid curve following the logarithmic equation. 𝑦 = 1 1 + 𝑒𝑟(cw−cw50) cw50 is the carapace width at which 50% population attains sexual maturity, r is the slope of the curve. the adjusted equation was fitted by the least square regression method (vazzoler, 1996; qureshi and saher, 2011b). results during study period, a total of 1703 crabs was collected, of which 764 were males (118 juvenile male, 646 adult male) and a total of 939 were females (204 were juvenile female, 735 adult females). descriptive statistics of each variable were shown in table 1 with carapace width (cw) size ranged 2.25figure 2. opusia indica (alcock, 1900). (a) dorsal view of male crab, (b) abdominal width of female, (c) chela of male, (d) abdominal length of male and (e) pleopod length of male. 227 int. j. aquat. biol. (2016) 4(4): 224-232 11 mm and 2-10 mm for male and female crab, respectively. intersexual difference in crab size (cw) was observed, as adult male crabs were significantly larger than females (f=7.41, p=0.000). wet weight relationship also showed significant difference (f=190.48, p=0.000) among adult male and female crabs of o. indica, juvenile of both sex did not differs significantly (f=0.63, p=0.43). relative growth relationship: growth relationship of length (cl) and size (cw) with weight (wt): the relationship between wet weight and length showed negative allometry and show higher value of the slope in male than in female crabs (table 2). the growth relationship between cw and wet weight showed negative allometry in both sexes (table 2). relative growth of carapace width (cw) with carapace length (cl): during ontogeny carapace width and length showed no significant difference, isometric relationship was observed in both male and female crabs (fig. 3). relative growth of carapace width (cw) with abdominal width (ab.w) and length (cl): the growth rate of abdomen width versus carapace width showed an isometric relationship in juvenile and adult female as well as in juvenile male while in adult male crabs it showed negative allometry. the student t-test for the slope values suggested that the growth variable sex juvenile crabs adult crabs mean±s.d t-test, p-value mean±s.d t-test, p-value carapace length (cl) f 3.08±1.06 ns 5.1±0.74 -7.61, 0.000 m 3.37±0.89 5.4±0.83 carapace width (cw) f 4.3±1.3 ns 7.0±1.02 -7.41, 0.000 m 4.7±1.1 7.5±1.1 abdominal length (ab.l) f 2.7±1.02 ns 4.9±0.75 -6.54, 0.000 m 4.9±0.75 5.2±0.8 abdominal width (ab.w) f 2.2±0.83 -8.21, 0.000 6.0±1.80 82.16, 0.000 m 1.5±0.50 2.2±0.47 cheliped length (ch.l) f 2.0±0.7 3.24, 0.001 3.2±0.46 -10.50, 0.000 m 2.3±0.62 3.5±0.58 cheliped width (ch.w) f 0.8±0.4 3.02, 0.003 1.4±0.35 -9.32, 0.000 m 0.96±0.39 1.90±0.38 cheliped height (ch.h) f 0.46±0.4 ns 0.7±0.28 -6.71, 0.000 m 0.46±0.28 0.8±0.28 (ns=not significant; s.d=standerd deviation) table 1. summary statistics for the biometric analysis of opusia indica collected from korangi creek, karachi, pakistan. figure 3. relationships between (a) abdominal width and carapace width in female and (b) pleopod length and carapace width of male of opusia indica (am = adult male; af = adult female; jm = juvenile male and jf = juvenile females). a b 228 saher et al./ allometric growth pattern and sexual maturity in opusia indica rate of abdominal width showed significant difference in juveniles as well as in adults (table 2). abdominal length against carapace width showed isometric growth in juvenile and adult female as well as in juvenile male, for adult male crabs it showed negative allometry (table 2). the relative growth of cheliped length, cheliped width and cheliped height: allometric relationship of carapace width with the chela propodus length was negative in juvenile female which become more pronounced in adult (table 2). in juvenile and adult male, growth is isometric showing higher growth rate in juvenile than adult. chela width relationship with carapace width in juvenile male and female showed positive allometry while isometry for adult in both sexes. positive allometric relationship exists between cheliped height and carapace width in male and female crabs. value of slope is higher in male than in female. relative growth of pleopod with carapace width: the allometric growth of pleopod showed a positive relationship with carapace width in juvenile, but isometric in adult male crab and significantly different (table 2). analyses of co-variance: by means of covariance analysis, results showed that relative growth pattern between juvenile and adult of both sex differs statistically in abdominal width versus carapace width, while, the growth pattern of chela length versus chela width relationship also showed variables sex/ phase n linearized equation log y = loga + b log x allometry determinaion coefficient “r” wet weight (wt.) vs. carapace width (cw) jf af jm am 184 753 117 644 logwt= 3.04 + 2.26cw logwt= 2.95 + 2.27cw logwt= 2.64 + 1.72cw logwt= 3.16+ 2.59cw negative negative negative negative 0.733 0.680 0.578 0.760 wet weight (wt.) vs. carapace length (cl) jf af jm am 184 753 117 644 logwt= 2.63 +2.14cl logwt= 2.58 + 2.19cl logwt= 2.27 + 1.50cl logwt= 2.81 + 2.60cl negative negative negative negative 0.761 0.674 0.560 0.764 carapace length (cl) vs. carapace width (cw) jf af jm am 184 753 117 644 logcl= 0.15 + 0.99cw logcl= 0.05+ 0.90cw logcl= 0.14 + 0.99cw logcl = 0.04+ 0.89cw isometry isometry isometry negative 0.928 0.864 0.905 0.887 abdominal width (ab.w) vs. carapace width (cw) jf af jm am 184 753 117 644 logab.w= -0.25+ 0.92cw logab.w =-0.02 + 0.94cw logab.w =-0.45+ 0.94cw logab.w =-0.32 + 0.77cw isometry isometry isometry negative 0.548 0.727 0.626 0.577 chela length (ch.l) vs. carapace width (cw) jf af jm am 183 756 177 644 logch.l= 0.26 + 0.89cw logch.l = 0.06 + 0.66cw logch.l = 0.29 + 0.96cw logch.l = 0.11 + 0.75 cw negative negative isometry negative 0.833 0.681 0.773 0.691 chela width (ch.w) vs. carapace width (cw) jf af jm am 184 753 116 664 logch.w=-1.22 + 1.71 cw logch.w=-0.64 + 0.92cw logch.w =-1.01 + 1.44 cw logch.w =-0.67 + 1.00 cw positive isometry positive isometry 0.835 0.506 0.666 0.616 chela height (ch.h) vs. carapace width (cw) jf af jm am 184 753 117 644 logch.h= 1.42+ 1.52cw logch.h = 1.31+ 1.37cw logch.h=-1.52 + 1.68cw logch.h=-1.15+1.23cw positive positive positive positive 0.367 0.477 0.529 0.49 first pleopod length (p1.l) vs. carapace width (cw) jm am 96 575 logpl.l= 0.64 + 1.26 cw logpl.l= 0.18 + 0.92 cw positive isometry 0.54 0.70 (note: n, specimen number; jf, juvenile female; af, adult female; jm , juvenile male; am, adult male). table 2. results of regression analysis for morphometric data of opusia indica from the mangrove area of korangi creek, pakistan. 229 int. j. aquat. biol. (2016) 4(4): 224-232 different pattern of growth for adult male and females showed in table 3. analysis for functional sexual maturity: the smallest ovigerous female (female having eggs on their pereopod) was 4 mm in size (cw). the size at which 50% population got sexual maturity was calculated as 5.32 mm in female crab and 5.51 mm in males (fig. 4). discussion the size at sexual maturity and patterns of somatic growth are important biological events occurs in reproductive cycle of crab which promotes morphological, physiological and behavioural changes (hartnoll, 1982). allometric analysis carried out in this study showed that male o. indica acquired greater averaged body sized than those of female. size variation between sex can be explained by differential reproductive needs as size dominance in male crab increase the opportunities of winning during intra-specific competition, courtship and handling female during copulation (castiglioni and negreiros-fransozo, 2004; saher and qureshi, 2011b). in brachyuran, female crabs showed slower growth after attaining sexual maturity, probably due to direct their large portion of the energy budget for reproductive strategies i.e. spawn and incubation of eggs production (hartnoll, 1982; diaz and conde, 1989; gregati and negreiros-fransozo, 2007) tend to mature with inferior sizes than males, who invest their energy resources in somatic growth, reaching larger sizes (shine, 1988). the logistic function also variable relationship n sex/ phase parameter ss f p-value carapace length (cl) vs. carapace width (cw) 302 jf vs jm slope 0.01 0.04 0.838 1398 af vs am slope 0.65 4.51 0.034* abdominal length (ab.l) vs. carapace width (cw) 301 jf vs jm slope 0.03 0.17 0.678 1398 af vs am slope 0.07 0.35 0.554 abdominal width (ab.w) vs. carapace width (cw) 301 jf vs jm slope 48.04 132.6 0.000* 1398 af vs am slope 5315.3 1174 0.000* cheliped length (ch.l) vs. carapace width (cw) 301 jf vs jm slope 0.51 3.36 0.068 1398 af vs am slope 8.08 55.48 0.000* cheliped width (ch.w) vs. carapace width (cw) 302 jf vs jm slope 0.187 2.53 0.113 1398 af vs am slope 55.64 2.58 0.109 cheliped height (ch.h) vs. carapace width (cw) 292 jf vs jm slope 0.148 1.17 0.280 1398 af vs am slope 1.686 28.02 0.000* first pleopod length (p1.l) vs. carapace width (cw) 671 jm vs am slope 0.02 0.075 0.112 table 3. results of analysis of covariance (ancova) for test of significant differences between phases and sex (ss=sum of squares, f=fstatistics, p= the significance, * =significant at p<0.05). figure 4. sigmoid curve showing 50% maturity in adult female and male crab of opusia indica. 230 saher et al./ allometric growth pattern and sexual maturity in opusia indica showed that the size in which male o. indica attained morphological sexual maturity was higher than the values for females, similar to the pattern proposed by shine (1988) for brachyurans. in brachyuran, male crabs can be considered morphologically mature when they are able to retain female during courtship and mating while female when they are able to copulate, carry and protect incubating eggs (castiglioni and negreiros-franssozo, 2004). sexual maturity study based on functionally maturity showed that female mature earlier than male crabs. the wet weight relationship with size showed that body weight was greater in males than females, this differences were associated with the males' faster growth and larger averaged body size which is the expected pattern to many brachyuran crabs which may result of the androgen gland secretion, improving the weight in the male crab after maturity (bliss, 1968; pinheiro and fiscarelli, 2009; marina et al., 2012). puberty moult in brachyuran is characterized by allometric changes in female abdomen and male cheliped and they are best representative secondary sexual characters. after puberty molt sexual dimorphism is well-pronounced in abdominal width which is related to the difference of function performed by pleopod in male and female crabs. the proficient size and shape of female abdomen facilitate developing eggs and act as an incubatory chamber (hartnoll, 1978). in male, however, abdomen only protect two pairs of pleopod that are responsible of transferring sperm during mating. relative growth relationship of abdominal width with body size showed positive allometry for both juvenile phase and adult female during entire life as predicted by hartnoll (1982) indicated that growth of abdomen might be incomplete when female attain functional maturity thus growth remain continuous to enlarge incubatory chamber for protecting egg mass. these facts are also observed in studies conducted by oh and hartnoll (1999). in present study, the higher growth rate for cheliped was observed in juvenile and adult male than female crabs but was not significantly differs, these observation indicated that no sexual dimorphism and allometric growth at certain level in size of cheliped for male and female crabs. as size of male crab’s increases, cheliped become stouter and larger in male o. indica presumably for a compensation of feeding and reproductive requirements. these results are similar to the observations made by snowden and clayton (1995) and schuwerack et al. (2006). another secondary sexual character used in brachyuran crabs is the length of first gonopod in male specimen. the level of allometric growth obtained for carapace width with pleopod width indicated positive growth during the juvenile phase that changes to isometric growth with the passage to the adult phase, differential growth has been related to the ability of male crabs to copulate successfully with wide range of females improving their reproductive output (hartnoll, 1974; bertini et al., 2007; saher and qureshi, 2011). pleopod in male crabs do not increase in same rate as female positive growth of gonopod has no reproductive advantage comparative to female. it has been confirmed from many studies that analysis of male pleopod and abdominal width in female are among the best estimator of size at sexual maturity than cheliped, similar to the findings of silva et al. (2007) and ribeiro et al. (2013). acknowledgments the research was supported by the pakistan science foundation project psf/res/s-ku/envr(51) and it is equally acknowledged. references alcock a. (1900). material for a carcinological fauna of india. no. 6. the brachyura catopmetopa or grapsoidea. journal of the asiatic society of bengal, 69: 279-486. bliss d.e. (1968). transition from water to land in decapod crustaceans. american zoologist, 8: 355392. bertini g., braga a.a., fransozo a., correa m.o.d., freire f.a.m. (2007). relative growth and sexual maturity of the stone crab menippe nodifrons stimpson, 1859 (brachyura, xanthoidea) in 231 int. j. aquat. biol. (2016) 4(4): 224-232 southeastern brazil. brazilian archives of biology and technolog, 50: 259-267. castiglioni d.s., negreiros-fransozo m.l. (2004). comparative analysis of the relative growth of uca rapax (smith, 1870) (crustacea, ocypodidae) from two mangroves in são paulo, brazil. revista brasileira de zoologia, 21: 137-144. diaz h., conde j.e. (1989). population dynamics and life history of the mangrove crab aratus pisonii (brachyura, grapsidae) in a marine environment. marine bulletin of science, 45: 148-163. felder d.l. (1971). the decapods crustacean of seven one-half fathom reef. m.s thesis, taxes a&i uni. pp. 103. gregati r.a., negreiros-fransozo m.l. (2007). relative growth and morphological sexual matrurity of chasmagnathus granulates (crustacea, varunidae) from a mangrove area in southeastern brazilian coast. iheringia série zoologia, 97: 268-272. hartnoll r.g. (1969). mating in brachyura. crustaceana, 16: 161-181. hartnoll r.g. (1974). variation in growth pattern between some secondary sexual characters in crabs (decapoda brachyura). crustaceana, 27: 131-136. hartnoll r.g. (1978). the determination of relative growth in crustacea. crustaceana, 34: 281–289. hartnoll r.g. (1982). growth. in: d.e. bliss, l.g. abele (eds). the biology of crustacea: embryology, morphology, and genetics. new york, academic press. 2: 111-196. huxley j.s. (1950). relative growth and form transmittion. proceedings of the royal society of london, 137: 465-469. ng p.k.l., guinot d., daive p.j.f. (2008). systema brachyurorun: part i. an annomated checklist of extant brachyuran crabs of the world. raffles bulletin of zoology, 17: 1–286. ng p.k.l., rahayu, d.l., naser m. (2009). the camptandriidae of iraq, with description of a new genus and notes on leptochryseus al-khayat & jones, 1996 (crustacea: decapoda: brachyura). zootaxa, 2312: 1-26. oh c.w., hartnoll r.g. (1990). size at sexual maturity, reproductive output, and seasonal reproduction of philocheras trispinsous (decapoda) in port erin bay, isle of man. journal of crustacean biology, 10: 608612. pinheiro m.a.a., fiscarelli a.g. (2009). length-weight relationship and condition factor of the mangrove crab ucides cordatus (linnaeus, 1763) (crustacea, brachyura, ucididae. brazilian archives of biology and technology, 52(2): 397-406. pinheiro m.a.a., fransozo a. (1998). sexual maturity of the speckled swimming crab arenaeus cribrarius (lamarck, 1818) (decapoda, brachyura, portunidae), in the ubatuba littoral, são paulo state. crustaceana, 71: 434-452. qureshi n.a., saher n.u. (2011). relative growth and morphological sexual maturity of macrophthalmus (venitus) dentipes lucas, in guerin meneville, 1836, from two mangrove area of karachi coast. biharean biologist, 5: 56-62. ribeiro f.b., cascon h.m., arruda-bezerra l.e. (2013). morphometric sexual maturity and allometric growth of the crab sesarma rectum. randall, 1840 (crustacea: sesarmidae) in an impacted tropical mangrove in northeast). latin american journal of aquatic research, 41: 361-368. saher n.u., qureshi n.a. (2011a). density, distribution and population structure of opusia indica (ocypodoidae: camptandriidae) in a coastral mangrove creek in pakistan. biologia, 61: 138-145. saher n.u., qureshi n.a. (2011b). relative growth and morphological sexual maturity of ilyoplax frater (brachyura: ocypodoidea: dotillidae) from mangrove area of korangi creek. pakistan journal of zoology, 43: 133-140. schuwerack p.m.m., barnes r.s.k., underwood g.j.c., jones p.w. (2006). gender and species differences in sentinel crabs (macrophthalmus) feeding on an indonesian mudflat. journal of crustacean biology, 26: 119-123. shine r. (1988). the evolution of the large body size in females; a critique of darwin fecundity advantage model. the american naturalist, 131: 124-131 silva s.m.j., hirose, g.l., negreiros-fransozo, m.l. 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(2022) 10(3): 234-241 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article bioaccumulation of hydrocarbon compounds in the muscle of three aquatic birds in um alnaaj marsh, iraq firas sabeeh idan, salih hassan jazza department of biology, college of science, university of misan, misan, iraq. s article history: received 5 march 2022 accepted 25 may 2022 available online 2 5 june 2022 keywords: bioaccumulation tphs pahs bcf abstract: the current study was conducted to determine the concentrations and origins of total petroleum hydrocarbon tphs and polycyclic aromatic hydrocarbons pahs in three bird species of anas platyrhynchos, a. crecca and a. acuta collected from november 2020 to april 2021 in um alnaaj marsh, iraq. based on the results, the tphs (g/g.dry weight) in muscle tissues were 13.79 and 16.74 in a. platyrhynchos, 40.84 and 43.0 in a. crecca, and 10.08 and 11.18 in a. acuta during winter and autumn, respectively. the pahs (ng/g.dw) in muscle tissues were 41.22 and 146.86 in a. platyrhynchos and 31.17 and 42.98 in a. crecca during winter and autumn, respectively, whereas it was 24.41 to 75.51 in a. acuta during autumn and winter, respectively. the lower molecular weight pahs was less than higher molecular weight pahs in all bird species throughout the period study. the origins of pahs in muscles tissues of the studied bird species were estimated according to the ratio of lpahs/hpahs, fluo/pyr, phe/ant, inpy/(inpy+bghip), ant/(ant+phe), and baa/(baa+chr) and based on the findings, they were mostly pyrogenic and few petrogenic. the results also showed that the bird species have the ability to accumulate these compounds in their muscles according to values of bcf which is between 2.29 and 5.81 in a. crecca and a. platyrhynchos, respectively. these bird species were contaminated with petroleum compounds and the consumption of their meat may pose public health hazards. introduction the marshes are unique ecosystems because of their biodiversity (unep, 2006; bedair et al., 2006; saleh et al., 2020). the mesopotamian marshes are important wetlands in the middle east as resting places and passages for many migratory birds (alhandal et al., 2016). pollution is one of the main problems threatening all living organisms, particularly in the marshes (nrc, 2003). crude oil spills are a major problem that often occurs during oil drilling and result in the pollution of water bodies (oshienemen et al., 2018; carpenter, 2019), leading to serious environmental concerns worldwide. crude oil spreads over the water to form a thin layer and prevents the access of atmospheric oxygen to aquatic biota. it also prevents photosynthesis and leads to disturbances in the food chain (frank and boisa, correspondence: salih hassan jazza doi: http//doi.org/10.22034/ijab.v10i3.1666 e-mail: salih-jazza@uomisan.edu.iq 2018). environmental disasters resulting from oil production and transportation are growing (jernelov, 2010; eckle et al., 2012) and their recovery takes 210 years, with long-term effects generally limited to changes in communities (mendelssohn et al., 2012). one of the oil pollution problems is polycyclic aromatic hydrocarbons (pahs), which impact water quality, sediments, and living organisms in the aquatic ecosystem (zhang et al., 2015; kuppusamy et al., 2020; jazza and khwadem, 2021). waterfowl are sensitive to petroleum pollution, especially pahs, which enter their tissues through water and food, having significant behavioral and physiological effects (shore et al., 1999). exposure to pahs leads to many carcinogenic effects in adult birds (malcolm and shore, 2003). albers (2006) and giese et al. (2000) pointed out that high molecular 234 idan and jazza / bioaccumulation of tphs and pahs in three aquatic birds in um alnaaj marsh weight pahs consisting of 4-6 rings are the most toxic to birds, especially to embryos and young birds, while in adult birds, they lead to a decrease in egg production and hatching and their effects on the production of sex cells; thus lead to a reduction in reproductive levels. pereiara et al. (2009) discovered that the pahs compounds accumulated in eggs of golden eagles in high concentrations. kwok et al. (2013) observed that eggs of waterfowl and egrets in jiangsu province (central china) contain high amounts of these compounds. birds, like other vertebrates, contain highly oxidized p450 enzymes and, therefore, can rapidly metabolize and excrete most pahs readily (verbrugge et al., 2001; troisi et al., 2006). the present study aimed to investigate the levels and origins of petroleum compounds in three bird species viz. anas platyrhynchos, a. crecca and a. acuta in um alnaaj marsh, iraq. materials and methods study area: um alnaaj marsh is one of the alhawizeh marshes, extending along the iraqi-iranian border, with a length of about 30 km and a width of about 25 km (fig. 1) (yuonis et al., 2011; hassan et al., 2012). it is fed with water from outside iraq, such as al-dwiridj, nissan, karkheh and al-kfagia rivers, and inside of iraq from al-mshrah and alkahlaa rivers (taylor et al., 2011). the climate in the marsh is dry and subtropical, with less than 200 mm annual rainfall and low humidity (al-khatib, 2008; hashim et al., 2019). collecting samples: the birds were collected from november 2020 to june 2021 using the netting (douche), and after anesthesia, they were placed in the icebox until reaching the laboratory. three bird species of a. platyrhynchos, a. crecca and a. acuta were sampled. the specimens were washed with distilled water and after removing their feathers, the muscles were cut into small parts, then dried, grounded, and sieved using the metal sieve of 63 μm and placed in clean glass to prepare for analysis. bioconcentration factor (bcf): bcf was calculated according to mccarty (1986) using the equation of bcf with pahs in water = cb / cwd, where cb is the concentration of pahs in the organism and cwd = concentration of pahs in water. measurement of pahs in muscle tissues: pahs were extracted from muscle tissues based on grimalt and oliver (1993). for this, a five g of sample was removed, and then a mixture of methanol: benzene (1:1) was added to it and the extraction process was carried out for 24-36 hours at 40°c. then saponification process was performed by adding an aqueous solution of koh for 2 hours at 40°c. the extract was left to cool and transferred to a separating funnel, and 50 ml of n-hexane was added. the sample was shaken vigorously, left to settle, and separated into two layers i.e. a soaped and an unsoaped layer containing hydrocarbons dissolved in hexane. the sample was passed through a chromatographic column consisting of glass wool at the bottom, topped with a layer of silica gel and a layer of alumina and a layer of anhydrous sodium sulfate (na2so4) to obtain the aliphatic fraction. then 30 ml benzene was added to extract the aromatic fractions and placed in a vial for reading by spectrofluorometer in the marine science center laboratory to determine tphs and gas chromatography at nihran aomr laboratories to measure pahs. origins of pahs: the following parameters were calculated to determine the origins of pahs. the ratio of lpahs/hpahs: if the value is more than one, the origin of pahs is considered petrogenic, and the values less than one show the pyrogenic origin (vrana et al., 2001). the ratio of phe/ant: if the value is greater than ten, the origin of pahs is considered petrogenic, whereas the value less than ten indicates its origin pyrogenic (doong and lin, 2004). fluo/pyr: if the ratio value is greater than one, the origin of pahs is considered pyrogenic. the values less than one show the origin of pahs petrogenic (zakaria et al., 2002). the ratio inpy/(inpy + bghip): if the ratio is less than 0.2, the origin of pahs would be petrogenic, and the ratio between 0.2-0.5 indicates mixed origins. in a ratio higher than 0.5, the origin of pahs is pyrogenic 235 int. j. aquat. biol. (2022) 10(3): 234-241 (tolosa et al., 2004; guo et al., 2007). the ratio (ant/(ant+phe): the ratio less than 0.1 indicates the sources of pahs from petroleum, while a ratio more than 0.1 indicates the sources of pahs pyrogenic (guo et al., 2007). the ratio baa/(baa+chr): a ratio less than 0.2 indicates petrogenic origins, 0.2 to 0.35 mixed origins, and greater than 0.35 indicates pyrogenic origins (guo et al., 2007). results and discussion the results showed that the concentrations tphs range 40.84-43 μg/g dw in a. crecca and 10.08 to 11.18 in a. acuta during autumn and winter, figure 1. the sampling stations in um alnaaj marsh. 236 idan and jazza / bioaccumulation of tphs and pahs in three aquatic birds in um alnaaj marsh respectively, whereas it ranged from 13.79 to 16.74 in a. platyrhynchos in winter and autumn, respectively (fig. 2). the ability of the birds to accumulate tphs varies and this may be due to different seasons, lipid content, feeding habits, age, and sex of birds. the highest levels of tphs in the average fat contents % seasons species 1.66 1.75 autumn a. platyrhynchos 1.58 winter 1.58 1.76 autumn a. crecca 1.4 winter 1.69 1.62 autumn a. acuta 1.76 winter table 1. seasonal variation in the fat content of the studied aquatic birds. a. acuta a. crecca a. platyrhynchos pahs individuals winter autumn winter autumn winter autumn nd nd nd nd nd nd ace nd nd nd nd nd 2.22 phe nd nd nd 1.98 nd 3.31 ant 7.89 3.13 nd 5.54 2.08 6.99 fluo 2.10 nd nd 7.87 2.50 8.12 pyr 16.30 nd 4.91 nd 9.31 69.41 chr 3.16 nd nd 1.66 nd 19.79 baa 1.35 nd nd 1.16 1.16 11.61 bbf 6.80 3.75 4.01 7.28 2.47 16.54 bkf 34.67 10.04 22.25 2.89 23.70 5.99 bap nd nd nd nd nd nd inp+daha 3.24 7.49 nd 14.60 nd 2.88 bghip 75.51 24.41 31.17 42.98 41.22 146.86 ∑pahs 1.98 5.53 lpahs 75.51 24.41 31.17 43 41.22 141.33 hpahs 0.04 0.03 lpahs/hpahs 0.67 phe/ant 3.75 0.70 0.83 0.86 fluo/pyr inpy/(inpy+bghip) 1 0.59 ant/(ant+phe) 0.44 1 0.22 baa/(baa+chr table 2. seasonal variations of pahs levels (ng/g dw) in muscles tissues (nd: not detected). figure 2. seasonal variation of tphs levels in the studied bird species. 237 int. j. aquat. biol. (2022) 10(3): 234-241 a. crecca, during autumn, maybe due to its feeding habits on aquatic organisms such as plant material and invertebrates (sterry et al., 2001), because the aquatic plants accumulate petroleum hydrocarbons in their tissues higher than in the aquatic environment (al-saad, 1995). in addition, different species of waterfowl feed different prey types, which accumulate petroleum hydrocarbons in their tissues because of missing mixed-function oxidase systems (lawrence and weber, 1984; jazza, 2015). the highest levels of pahs were recorded in a. platyrhynchos during winter since they feed plants and protein-rich invertebrates, increasing during spawning i.e. late spring (gammonley, 1995). if it is time for birds to migrate to other feeding sites, they increase the rates of nutrients and lipid reserves for spring migration, thus, the levels of lipophilic petroleum hydrocarbons can increase (tidwell, 2010). the lowest pahs were found in a. acuta in winter, the lipid reserves of wintering waterfowl naturally reach their lowest content in mid-late winter which was 1.62 g (table 1). this result was consistent with the findings of thompson and baldassarre (1990). in a. platyrhynchos, pahs had high molecular weight ranging from 41.22 ng/g dw in winter to 146.86 ng/g dw in autumn (table 3). the origins of pahs, according to the ratio of lpahs / hpahs was 0.03 less than one, which indicated its source pyrogenic (fernandes et al., 1997; vrana et al., 2001; al-khatib, 2008), while phe / ant ratio was 0.67, less than ten, therefore its source is pyrogenic (doong and lin, 2004; al-khion, 2012). flu/pyr ratio ranged from 0.86-0.83, less than one; thus its origin is petrogenic (zakaria et al., 2002; kafilzadeh et al., 2011). ant/(ant+phe) was 0.59, more than 0.1, indicating that its origin is pyrogenic (yunker et al., 2002; guo et al., 2007) baa /(baa+chr) was 0.22, indicating its sources are mixed (jazza and khwadem, 2021). in a. crecca, pahs in the muscle tissues ranged from 31.17 to 42.98 ng/g dw in winter and autumn, respectively (table 3). the highest levels recorded for the two seasons were 25.14 ng/g dw for b(a)p and the lowest 1.16 ng/g dw for b(b)f. the ratio of lpahs/hpahs was 0.04, indicating the source of pahs is pyrogenic (vrana et al., 2001). the flu/pyr ratio was 0.70 showing the origins of pahs as petrogenic (qui et al., 2009). ant/(ant+phe) was one indicating the origin of pahs as pyrogenic (yunker et al., 2002). the ratio of baa/(baa+chr) was 1 indicating the source of pahs pyrogenic (tolosa et al., 2004; guo et al., 2007). in a. acuta, the most frequent compounds in both seasons were fluoranthene, b(k)f, b(a)p, and b(g,h,i)p, and ∑pahs in the muscle tissues was 24.41 ng/g dw in autumn and 75.51 ng/g dw in winter(table 3). flu/pyr ratio was 3.75, indicating a. acuta a. crecca a.platyrhynchos indices pyrogenic pyrogenic lpahs/hpahs pyrogenic phe/ant pyrogenic petrogenic petrogenic flu/pyr pyrogenic inpy/(inpy+bghip) pyrogenic pyrogenic ant/(ant+phe) pyrogenic pyrogenic mixed baa/(baa+chr) table 2. origin of pahs in the studied aquatic bird species. bcf concentration of pahs in water concentrations of pahs in birds species 5.81 16.17 94.04 a. platyrhynchos 3.08 16.17 49.96 a. acuta 2.29 16.17 37.07 a. crecca table 3. bioconcentration factor (bcf) in the studied aquatic bird species. 238 idan and jazza / bioaccumulation of tphs and pahs in three aquatic birds in um alnaaj marsh the source of pahs is pyrogenic (qui et al., 2009; jazza and khwadem, 2021). the baa/(baa+chr) ratio was 0.44 in autumn, indicating the sources of pahs pyrogenic (guo et al., 2007). waterfowl are exposed to pahs via nutrition, water, inhalation, feather conditioning, and direct ingestion of oil (fernie et al., 2018). accumulating pahs in birds is associated with decreased metabolic capacity and a high-fat percentage (eisler, 2000). in general, the reason for the differences in the levels of total pahs in the studied birds during seasons may be due to their metabolism and detoxification capacity (custer et al., 2001; roscales et al., 2011) or the fat content that increases the accumulation of pahs in muscle tissues (bandowe et al., 2014; maisano et al., 2016). also, jardine et al. (2006) pointed out that pollutant levels can be affected by environmental factors and nutritional status. birds can eat insects containing different pah concentrations (mengelkoch et al., 2004). the results of the current study revealed that most of the aromatic compounds in all birds have high molecular weight, and few have a low molecular weight because they are high stability and resistant to microbial degradation (anyakora and coker, 2007). based on the results, the origins of pahs in the studied birds were pyrogenic from human activities and organic wastes (table 4). the results also revealed that the bcf values ranged from 2.29 to 5.81 in a. crecca and a. platyrhynchos, respectively (table 5). the variation of bcf values in different bird species may be attributed to exposure duration, age, sex, habits, fat contents, metabolism process, detoxification and ecological factors (roscales et al., 2011; sun et al., 2016). conclusion the results showed high concentrations of pahs during winter in the studied birds. according to the ratios of lmw-pahs/hmw-pahs, baa/(baa+ chr), inp/(inp+bghip), phe/ant, ant/(ant+phe), and fluo/pyr, the sources of pahs were mainly pyrogenic and few were petrogenic. the presence of hydrocarbon compounds in the meats of the birds can have risks to human health by their consumption. acknowledgments the authors are grateful to the labs in marine science center of the university of basrah, college of science, misan university, and nihran omer, basrah oil company for providing the facilities to conduct this research. references albers p.h. 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(2016) 4(5): 340-344: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article mortality rate and immune responses of rainbow trout (oncorhynchus mykiss) infected with yersinia ruckeri subsequent to feeding on diet supplemented with ducrosia anethifolia essential oil fatemeh dehghan1, arya vazirzadeh*1, siyavash soltanian2, akbar karami3, mostafa akhlaghi2 1department of natural resources and environmental engineering, school of agriculture, shiraz university, shiraz 71441-65186, iran. 2department of aquatics health and diseases, school of veterinary, shiraz university, shiraz, iran. 3department of horticultural science, school of agriculture, shiraz university, shiraz 71441-65186, iran. . article history: received 12 april 2016 accepted 3 october 2016 available online 2 5 october 2016 keywords: immunostimulant lysozyme bactericidal blood protein abstract: application of the immunostimulant is the most promising method for controlling diseases in aquaculture. in this study, the mortality rate and immune responses of rainbow trout (oncorhynchus mykiss) fed on diet supplemented with ducrosia anethifolia essential oil was investigated after challenging with yersinia ruckeri. the essential oil mixed with sunflower oil at different concentrations (0.001, 0.01 and 0.1%) and the commercial food was coated with this oil. fish were fed with diets for 8 weeks and infected with y. ruckeri at the ending of feeding trial. serum protein, albumin, globulin and lysozyme and bactericidal activity of challenged fish were evaluated one week after injection and mortality were counted till day 10. the results showed that albumin had not differed among treatments. the highest level of the protein and globulin were found in control group. serum lysozyme activity showed no difference between groups. the highest and lowest serum bactericidal activity was observed in 0.001% and control group, respectively. the mortality rates in infected fish were as 55% in control group, 40% in 0.001%, 70% in 0.01% and 70% in 0.1% treatment. lowest rate of mortality was observed in group 0.001%, while began two days earlier than other groups. introduction the increase in fish culture productivity is accompanied with stressful conditions that provide a susceptible environment for infectious diseases (soltani et al., 2010; sheikhzadeh et al., 2011). improving the immune system using immunestimulants seems to be the most promising method to control diseases in aquaculture (harikrishnan et al., 2011). yersinia ruckeri is an opportunistic gramnegative bacterium that causes enteric red mouth disease (erm). antibiotic treatments would be useless if they induce antibiotic resistance in pathogen (ispir et al., 2009). hence, administration of the herbal immunostimulants is considered as an alternative method to chemotherapy and vaccination * corresponding author: arya vazirzadeh e-mail address: vazirzadeh@shirazu.ac.ir (harikrishnan et al., 2011a, b). herbal products are safer both for consumer and environment, cheaper and more available (hajibeglou and sudagar, 2010; soltani et al., 2010). ducrosia anethifolia is a medicinal plant that belongs to the family apiaceae. it widely grows in afghanistan, pakistan, syria, lebanon, iraq and west and south of iran. in iran, it is known as moshgak, roshgak or moshkbu (hajhashemi et al., 2010; shahabipour et al., 2013). in previous studies, the antibacterial, antimicrobial and antianxiety effects of d. anethifolia were examined in laboratory or in homoeothermic animals like rat and the results were positive (hajhashemi et al., 2010; shahabipour et al., 2013), but the effects of this plant in a poikilothermic animals such as fish have not been studied yet. 341 int. j. aquat. biol. (2016) 4(5): 340-344 therefore, the objective of this study was to investigate the mortality rate and changes in immune parameters of rainbow trout (oncorhynchus mykiss) infected by y. ruckeri subsequent to fed on diet containing d. anethifolia to discover its immunestimulant potential. materials and methods examined fish: rainbow trout (with average weight of 32.6±8.1 g.) were obtained from a local fish farm (22 bahman farm, sepidan, iran), transported to the laboratory and acclimatized for 2 weeks in a recirculating aquaculture system (ras). the ras were included 12 700-l tanks. water quality parameters, including dissolved oxygen, temperature, ph, tss and ec were 5.91±1.08 ppm, 8.22±2.32°c, 8.31±0.26, 0.27±0.01 ppm and 0.53±0.02 μs during the experiment, respectively. preparation of immunostimulant diets: ducrosia anethifolia was collected from the jahrom city (southern iran), air dried in room temperature and its essential oil was extracted by clevenger based on hajhashemi et al. (2010). the essential oil mixed with sunflower oil in concentrations of 0.001, 0.01 and 0.1% and commercial rainbow trout diets (beyza feed mill, shiraz, iran) were coated with this suspension. test diets were stored in the freezer until use. in control group, sunflower oil without essential oil was used. culture of y. ruckeri: the source of bacterium was obtained from the fish health and diseases laboratory, shiraz university. the bacterium was injected to 4 fish intraperitoneally. after 3 days, new plates were prepared from the kidney of infected fish. the colonies of new culture were proliferated for challenge test. experimental design: fish were randomly divided into 12 groups (4 treatments in triplicates) and fed with the test diets for 8 weeks. after the experiment period, 20 fish of each treatment with average weight of 63.64±12.68 g. were intraperitoneally injected with 2.24±106 y. ruckeri. five fish of each treatment were bled a week after challenge test and sera were separated. serum lysozyme activity, bactericidal activity, total protein, albumin and globulin were evaluated and the mortality of fish was monitored for 10 days. serum lysozyme activity: the serum lysozyme activity was measured spectrophotometrically based on parry et al. (1965). micrococcus lysodeikticus (0.2 mg ml-1) dissolved in pbs (0.04 m, ph 5.75). two µl serum was added to the bacterium suspension for the final volume of 3 ml. the absorbance was read in 530 nm after 0.5 and 4.5 min in room temperature. the level of enzyme activity was defined as the amount of enzyme that caused a decrease in absorbance of 0.001 per min using following formula: 𝑈/𝑚𝑙 = 𝑂𝐷1 − 𝑂𝐷2 4 × 0.001 × 2 × 1000 serum bactericidal activity: serum bactericidal activity was determined as described by rao et al. (2006). aeromonas hydrophila bacterial culture was centrifuged and the pellet was washed and suspended in pbs. od of the suspension was adjusted to 0.5 at 546 nm. this suspension was serially diluted (1:10) with pbs 5 times and the least dilution was used for assay. ten µl suspension was added to 100 µl serum and incubated at 20°c for 1 hour. after incubation, the combination of bacteria and sera were cultured on the nutrient agar plates in triplicates and colonies were counted after 24 hours incubation at 25oc. pbs was used instead of serum for control samples. total protein, albumin and globulin: serum total protein and albumin were determined using a diagnostic kit (pars azmun, iran) following the protocol recommended by factory (hajibeglou and sudagar, 2010). serum globulin was determined by subtracting the amount of albumin from total protein (rao et al., 2006). statistical analysis: data were analyzed using sas 9.1.3 software. the normality of data and homogeneity of variance were performed by kolmogorov-smirnov and leven test, respectively. groups were compared by one-way analysis of variance (anova) and if there were any significant differences between treatments, the means were compared by tukey's test to determine differences 342 dehghan et al./ mortality rate and immunity of rainbow trout infected with y. ruckeri fed on d. anethifolia among treatments at a significant level of p<0.05. results serum lysozyme activity: the levels of serum lysozyme in different groups are shown in fig. 1. the serum lysozyme activity showed no significant differences among the groups (p≥0.05). serum bactericidal activity: the highest serum bactericidal activity was observed in fish fed diet containing 0.001% d. anethifolia essential oil and the least bactericidal activity was observed in fish in control group (fig. 2). total protein, albumin and globulin: the highest protein level was observed in control group which was significantly different from those in 0.001 and 0.1% treatments (fig. 3). no significant differences were observed in albumin levels among treatments after challenging with y. ruckeri (fig. 4). the figure 3. changes in serum total protein level of rainbow trout one week after challenge with yersinia ruckeri. fish were fed on diets containing different amount of ducrosia anethifolia essential oil for 8 weeks before challenge. data are expressed as mean±sd. bars with different letters are significantly different (p<0.05). figure 4. changes in serum total albumin level of rainbow trout one week after challenge with yersinia ruckeri. fish were fed on diets containing different amount of ducrosia anethifolia essential oil for 8 weeks before challenge. data are expressed as mean±sd. no significant differences were observed among treatments (p>0.05). figure 5. changes in serum globulin level of rainbow trout a week after challenge with yersinia ruckeri. fish were fed on diets containing different amount of ducrosia anethifolia essential oil for 8 weeks before challenge. data are expressed as mean ± sd. bars with different letters are significantly different (p<0.05). figure 1. changes in serum lysozyme activity of rainbow trout one week after challenging with yersinia ruckeri. fish were fed on diets containing different amount of ducrosia anethifolia essential oil for 8 weeks before challenge. data are expressed as mean±sd. no significant differences were observed among treatments (p>0.05). figure 2. changes in serum bactericidal activity level of rainbow trout one week after challenge with yersinia ruckeri. fish were fed on diets containing different amount of ducrosia anethifolia essential oil for 8 weeks before challenge. data are expressed as mean ± sd. bars with different letters are significantly different (p<0.05). 343 int. j. aquat. biol. (2016) 4(5): 340-344 highest level of globulin was observed in control group which was significantly different compared to other treatments (fig. 5). mortality rate: fish were monitored for 10 days after challenge test and mortality were counted. mortality in 0.001 treatment was started on day 6, but fish started to die on day 4 after challenge in other treatments. the mortality rates in infected fish were 55% in control group, 40% in 0.001%, 70% in 0.01% and 70% in 0.1% treatment. therefore, mortality started earlier than other treatments in 0.001% treatment but its rate was lowest (fig. 6). discussion the result showed no differences in lysozyme activity among all treatments. bowden et al. (2004) stated that lysozyme activity affected by seasonal changes; this means that lysozyme activity increases in summer and decreases in winter. since this study carried out in winter, lack of difference among groups might be due to seasonal changes. the results also showed increasing of the serum bactericidal activity in treatments fed on the diet supplemented with immunostimulant compared to the control group. increasing serum bactericidal activity represents enhancement of the protective proteins that is occurred after natural infections like outbreaks or artificial ones like vaccination or challenge tests (biller-takahashi et al., 2013). in the line of this study, an increase of serum bactericidal activity was observed after administration of aegle marmelos extract or combination of different herbal extracts (hajibeglou and sudagar, 2010; pratheepa et al., 2010). janssen et al. (1984) also stated that d. anethifolia essential oil has antibacterial effects against gram-positive bacteria. since y. ruckeri is a gram-negative bacterium (ispir et al., 2009), it is possible that d. anethifolia cannot induce all the immune responses as an immunostimulant. in this study, the levels of proteins viz. albumin and globulin were evaluated after challenge and the results showed that these indices did not enhance in groups that used the herbal essential oil as immunostimulant. in contrast, hajibeglou and sudagar (2010) reported the enhancement of the protein, albumin and globulin after challenge in common carp (cyprinus carpio) subsequent to using mixed herbal extracts as immunostimulant. although increasing of all these three indices is of surprising, because serum globulin is estimated by subtracting the amount of protein and albumin. mortality rate was lower in 0.001% group and figure 6. cumulative mortality rate (%) of rainbow trout monitored for 10 days after challenging with yersinia ruckeri. fish were fed on diets containing different amount of ducrosia anethifolia essential oil for 8 weeks before challenge. 344 dehghan et al./ mortality rate and immunity of rainbow trout infected with y. ruckeri fed on d. anethifolia higher in 0.01 and 0.1% treatments compared to that of the control group. similarly, harikrishnan et al. 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(2016) 4(3): 208-214; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article effect of milk thistle plant, vitis vinifera extract on immune system of rainbow trout (oncorhynchus mykiss) challenge by diazinon mina rabie*1, younes asri2, kamal ahmadi3 1department of natural resources and environmental engineering, payame noor university, tehran, iran. 2research institute of forests and rangelands, botany division, tehran, iran. 3agriculture department, payame noor university, alborz devision, karaj, iran. article history: received 21 march 2016 accepted 6 june 2016 available online 2 5 june 2016 keywords: pollutant diazinon milk thistle igm immune system abstract: the pollutants due to effect on the immune system of fish increase fish sensitivity to pathogens. diazinon is one of the most used organophosphates pesticide in many agricultural areas. this study aimed to evaluate the effect of diazinon on the immune system of rainbow trout (oncorhynchus mykiss) and application of milk thistle plant, vitis vinifera extract to reduce the adverse effects of this pesticide on its immune system. the reduction in the level of plasma peroxides, igm, total complement and lysozyme were observed in fish exposed to diazinon showing its effect on the fish’s immune system. no significant difference between control group and fish fed by milk thistle plant extract and exposed to diazinon can reflect protective impact of milk thistle plant extract on the immune system of rainbow trout by eliminating the free radicals and boosting the immune system. introduction the immune system of aquatic organisms such as fishes is continuously affected by unwanted environmental changes, threatening their health as an acute or chronic stressor. with overuse of pesticides, herbicides and other toxic compounds, the incidence of disorders and increase of irreparable damages are inevitable in aquatic organisms. therefore, they can influence the immune system of the fishes. diazinon is one of the most used agricultural organophosphate pesticides during past decade in iran (selsele, 2001; tartahi tbrizi, 2001; honarpajo, 2003; qomqisi, 2004; hosseini, 2005; babai, 2009). a significant reduction of white blood cells, particularly lymphocytes and increase in neutrophils and eosinophils were observed in acipenser stellatus (khoshbavar rostami et al., 2005), a. nudiventris (khoshbavar rostami and sultani, 2005), onchorhynchus mykiss (banaii et al., 2009) and cyprinus carpio (banaii et al., 2008) treated by * corresponding author: mina rabie e-mail address: minarabie@pnu.ac.ir diazinon. in addition, a change in the activity level of lysozyme were reported in ctenopharyngodon idella (pourgholam and sultani, 2007) and o. mykiss (banaii et al., 2009) under treat of diazinon. production of the free radicals by the diazinon’s metabolism has been proven in various tissues, especially in the liver of the treated fishes (üner et al., 2006) threatening their survival. animals’ immune system has a relatively high sensitivity to oxidative stress. glutathione (gsh) as the most important intracellular regenerative factor has various functions such as protection against oxidative stress, regulation of gene expression, regulation of apoptosis and activation and proliferation of t lymphocytes; however, it is observed that the low level of gsh is associated with the incidence of functional disorders in lymphocytes (droge et al., 2000; townsend et al., 2003). therefore, strengthening of fishes’ immune system by providing dietary supplements can be a suitable 209 int. j. aquat. biol. (2016) 4(3): 208-2014 solution for preventing their immune system weakness. many plants extracts directly reduce the effects of oxidative stress whereby the elimination of a variety of reactive oxygen species (ros) and can indirectly play their protective role through the activation of antioxidant defense system (lang et al., 1988; dietzmann et al., 2002; townsend et al., 2003). medicinal plants as antifungal (ebrahimzadeh mousavi et al., 2006), antibacterial (naghdi badi and makizadeh tafti, 2007) and immunostimulant compounds (ahmadi et al., 2008) have been used to increase the ability of fishes’ immune system. milk thistle, vitis vinifera is considered as a cell protective compound, especially liver cells. many studies have so far been conducted on the antioxidant properties, removing feature of oxy-radicals, the ability to chelate ion and addictive effects of intracellular glutathione content of v. vinifera showing its ability to enhance the antioxidant immune system (borsari et al., 2001; valenzuela and garrido, 1994). however, the mechanism of milk thistle plant extract has not been well-described as a stimulant of the immune system. therefore, this study aims to evaluate the effect of milk thistle plant extract on the immune system of o. mykiss in exposure to diazinon. materials and methods a total of 120 healthy rainbow trout (with mean weight of 85.5±15 g) were randomly distributed into 12 100l-tanks with continuous aeration. fish were adapted to in-vitro conditions for 15 days before experiments. during this period, fish were fed by a commercial trout grower diet (fft2, chineh, tehran, iran). for preparing experimental food, 400 mg milk thistle plant extract per 1 kg of commercial food powder (fft2, chineh, tehran, iran) were mixed and the mixture was mixed with water to yield a suitable mash. the prepared diet was made into pellet and dried at 40°c in drying cabinet. food was weekly prepared. toxicology test was performed according to oecd guideline. during the experiment, the physicochemical conditions of the water were regularly controlled. chronic toxicity test was designed for 28 days as completely randomized design. four treatment each with three replicates were considered for this study. in control group, fish only fed by commercial food; other treatments were fish fed by diet supplemented by milk thistle plant extract, fish treated by diazinon (0.1 mg l-1) and fish treated by diazinon (0.1 mg l-1) and fed by diet supplemented by milk thistle plant extract. the fish were reared in static water tanks with 100% water exchange on every other day. throughout the experiment, the rearing water was constantly supplied with oxygen to maintain the dissolved oxygen above 6 mg l-1. after each water change, the amount of pesticide was renewed. the blood were randomly sampled from 3 fish of each tank at 7th, 14th and 28th days of the experiment. plasma was separated after the centrifugation in 6000 rpm for 15 min at 4°c, then the samples were kept in -78°c for further analysis. to assess the level of peroxidase activity, 15 ml of plasma with 35 ml hbss buffer without magnesium and calcium was diluted. then 50 ml tmb solution and 5 mm hydrogen peroxide were added so that the solution turned blue. after 2 min, by adding 50 ml of 2 m sulfuric acid, color reaction was stopped and the color became bright yellow. the amount of optical absorption at 450 nm was measured to determine the peroxidase activity. measuring complement of ch50 was done by kit (bahar afshan company, tehran) based on radial immuno diffusion method. lysozyme activity level was measured by turbidity method using micrococcus lysodeikticus suspension and moramydaz enzyme. the turbidity was measured at a wavelength of 670 nm. igm levels in plasma were measured using inkjet tehran spring kit and hitachi auto analyzer. statistical analysis was performed using minitab software version 13 and diagrams were drawn by microsoft excel 2003. statistical analysis was performed by one-way anova and the significance of means was performed at 95% level of significance 210 rabie et al./ effect of milk thistle plant extract on immune system of rainbow trout applying tukey test. results during the experiment, no death were observed; however, fish treated by pesticide seemed to be nervous at the end of the trial period. some fish were swimming as unbalanced in the water surface which was due to the deterrent effect of diazinon. such behavior was not observed in other treatments. changes in the level of immunoglobulin igm and peroxidase, total complement and lysozyme were shown figures 1-4. at 14th and 28th days, the level of immunoglobulin was significantly decreased in fish treated by diazinon (p<0.05); however, at 14th, no significant change was observed in the concentration of igm in the treated fish. no significant difference (p<0.05) was observed in the level of peroxidase in the treated fish compared to the control group in all stages of sampling; whereas, a significant difference was observed between the level of peroxidase in fish treated by diazinon and fish fed with diet figure 1. change in the level of plasma immunoglobulin in oncorhynchus mykiss treated by pesticide, milk thistle plant extract and complement of milk thistle plant extract and pesticide. figure 2. change in the level of peroxidase in oncorhynchus mykiss treated by pesticide, milk thistle plant extract and complement of milk thistle plant extract and pesticide. 211 int. j. aquat. biol. (2016) 4(3): 208-2014 supplemented by milk thistle plant extract at the third phase of the sampling (p<0.05). the level of total complements of plasma was significantly decreased in fish exposed to diazinon compared to control group after 28 days (p<0.05). a significant increase (p<0.05) in the level of plasma complements was also observed in fish fed by milk thistle plant extract. the level of lysozyme significantly decreased at the third phase of sampling in fish treated by pesticide compared to that of control group (p<0.05); while in the other treatments, no significant differences were observed in the level of plasma lysozyme. discussion in recent years, numerous studies have been conducted on the therapeutic effects of milk thistle plant extract on various diseases and significant results have been achieved. in vitro studies have shown the therapeutic effects of plant extracts with similar combinations to milk thistle plant on figure 3. change in the level of total complement in oncorhynchus mykiss treated by pesticide, milk thistle plant extract and complement of milk thistle plant extract and pesticide. figure 4. change in the level of plasma lysosyme in oncorhynchus mykiss treated by pesticide, milk thistle plant extract and complement of milk thistle plant extract and pesticide. 212 rabie et al./ effect of milk thistle plant extract on immune system of rainbow trout hyperlipidemia, atherosclerosis and formation of atherosclerotic plaque (kreman et al., 1998), poisoning and kidney disorders (zima et al., 1998), drug toxicity (muriel and mourelle, 1990), kidney dysfunction (fiebrich and koch, 1979), food (desplaces et al., 1975) and chemical poisoning (janiak, 1974 ), viral diseases (lirussi and okolicsanyi, 1992), neurological disorders (zhang et al., 1993), regulatory of blood sugar (velussi et al., 1993), anti-cancer property (zi et al., 1998) and prevention of hemolysis of red (zou et al., 2001) and white blood cells (locher et al., 1998). peroxide enzymes play significant role in oxidation of many xenobiotics by hydrogen peroxide (saunders, 1973). in this study, the ability of antioxidant enzyme system to remove the free radicals produced through ditazinon metabolism was increased in accordance with increasing the level of peroxidases’ activity in the plasma of fish fed by complement of milk thistle plant extract. while reducing the level of this enzyme in fish exposed to diazinon led to imbalance between the antioxidant defense system and production of ros causing serious damages of their immune system. free radicals produced during the metabolism and decomposition of diazinon in the detoxification process in the liver of fishes can cause disorder in the normal function through oxidation of intracellular proteins. the integrity and stability of membrane is eliminated due to the lipid peroxidation of the cellular membranes. it causes disorder in the cell interactions which will underlie cell death (üner et al., 2006). because the membrane of the immune cells is rich by long-chain polyunsaturated fatty acids, they are more susceptible to lipid peroxidation by ros produced in active phagocytic cells (üner et al., 2006). immunoglobulins or antibodies a class of glycoproteins are found in serum and tissues fluids of all vertebrates. antibodies are produced by plasmocytes derived from b lymphocytes (stites, 1991). immunoglobulins play an important role in fight against bacterial infectious diseases. therefore, reducing the level of their activity can weaken the immune system of the fishes. in the present study, a significant reduction of immunoglobulin level indicates the effect of diazinon in reducing the level of plasma immunoglobulin in fish treated by diazinon. no significant difference among fish fed by milk thistle plant extract and exposed to pesticide also reveals the effect of milk thistle plant extract on reducing complications caused by diazinon. according to the results, reducing the levels of total plasma complements in treatments exposed to diazinon can represent the effect of pesticide in weakening the fish's immune system, while milk thistle plant extract can reduce the adverse impact of the free radicals on fish's immune system by the metabolism of diazinon through preventing cell death and increasing the regenerative capacity of tissues. lymphocytes capability for proliferative response and production of il-2 are deeply damaged in exposure to ros (flescher et al., 1994). decreasing the level of lysozyme in fish exposed to pesticide also clearly shows the impact of diazinon on weakening the immune system. also, no significant difference between lysozyme of fish in the control group and those fed by milk thistle plant extract can indicate the effect of milk thistle plant extract on boosting the immune system (pourgholam and soltani, 2007). as results, the extract of milk thistle plant, particularly antioxidant compounds can prevent harmful effects of diazinon on the immune system of fish by eliminating the free radicals and boosting the immune system (ditzmann et al., 2002; levin et al., 1996; heyborn and silver, 1996). according to the results, the impact of milk thistle plant extract as a medicinal extract was proved on improvement of the immune system. therefore, using milk thistle plant extract in fishes’ diet can be a suitable solution to prevent of possible adverse effects of entering toxic pollutants into fish farms and their effects on fishes’ immune system. acknowledgments the authors are grateful to saif, nehu, shillong for the use of aas. 213 int. j. aquat. biol. 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(2022) 10(3): 242-253 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article ichthyofauna of the iranian part of the sirvan river drainage with the first record of cobitis avicenna and oxynoemacheilus euphraticus atta mouludi-saleh, soheil eagderi*,1hadi poorbagher department of fisheries, faculty of natural resources, university of tehran, karaj, iran. s article history: received 17 march 2022 accepted 1 june 2022 available online 2 5 june 2022 keywords: cyprinidae kurdistan invasive loaches conservation endemic species abstract: this study investigated the fish diversity of the sirvan river drainage, tigris river basin. sampling was performed at 20 stations during 2020-2021 using an electrofishing device. a total of 32 species in 23 genera, 12 families and seven orders, including barbus lecerta, capoeta damascina, c. trutta, c. umbla, carassius gibelio, cyprinion macrostomum, c. kais, garra rufa, luciobarbus barbulus, cyprinus carpio (cyprinidae), ctenopharyngodon idella, hypophthalmichthys molitrix, h. nobilis, hemiculter leucisculus (xenocyprididae), cobitis avicennae (cobitidae), oxynoemacheilus euphraticus, o. zarzianus, o. kurdistanicus, o. parvinae, turcinoemacheilus kosswigi, paracobitis molavi (neomacheilidae), squalius berak, s. lepidus, alburnus sellal, a. hohenackeri (leuciscidae), pseudorasbora parva (gobionidae), rhinogobius lindbergi (gobiidae), esox lucius (esocidae), mastacembelus mastacembelus (mastacembelidae), glyptothorax pallens (sisoridae), oncorhynchus mykiss (salmonidae) and gambosia holbrooki (poeciliidae) were recorded. carassius gibelio, c. idella, c. carpio, h. molitrix and h. nobilis, a. hohenackeri p. parva, h. leucisculus, o. mykiss, r. lindbergi, e. lucius and g. holbrooki were exotic species introduced to this river system. this study confirms the new records of o. euphraticus and c. avicennae for the first time from the iranian part of the sirvan river drainage. introduction iran has a high diversity of freshwater fishes (esmaeili et al., 2018) distributed in 19 exorheic and endorheic basins. in recent years, many new species, and records were described and reported from remote areas of iran that had been investigated rarely (eagderi et al., 2019a; eagderi & mousavi-sabet, 2021; mousavisabet et al., 2021; esmaeili et al., 2022). sirvan river drainage with an area of 7500 km2 located in the mountainous region of the kurdistan province with a length of 213 km and a maximum flow of 250m3/s (jafari, 2000). due to the importance of this river system and the lack of a comprehensive study on its fish inventory, the current study was conducted to investigate the ichthyofauna of the iranian part of the sirvan river drainage with two new records from this drainage. materials and methods during 2020-2021, some 985 specimens from 20 *correspondence: soheil eagderi doi: https://doi.org/10.22034/ijab.v10i3.1639 e-mail: soheil.eagderi@ut.ac.ir stations were sampled in the sirvan river drainage (fig. 1). sampling was performed using an electrofishing device. then, using identification keys keivany et al. (2016), and esmaeili et al. (2018), the fishes were identified. some of the fishes were fixed into 10% fixed formalin after anesthesia and the rest were released to the river. results the collected samples represented 32 species in 23 genera, 12 families, and seven orders. dominant order was cypriniformes (26 species; 81%), followed by gobiiformes, esociformes, synbranchiformes, siluriformes, salmoniformes and cyprinodontiformes each having 1 species (figs. 2, 3). checklist species name (author)―[iucn], english name/figure. * = endemic, ** = exotic. 243 int. j. aquat. biol. (2022) 10(3): 242-253 class actinopterygii order cypriniformes (6 families, 17 genera, and 26 species) family cyprinidae (7 genera and 10 species) genus barbus cuvier, 1816 1barbus lecerta heckel, 1843―[least concern (lc)], kura barbell (fig. 4a) genus capoeta valenciennes, 1842 2capoeta damascina (valenciennes, 1842)―[least concern (lc)], mesopotamian barb (fig. 4b) figure 1. sampling stations of the sirvan river. figure 2. percent of fish species in different fish orders. 244 mouludi-saleh et al./ ichthyofauna of iranian part of the sirvan river drainage remark: this species is found as sympatric with c. umbla in sirvan river drainage. 3capoeta trutta (heckel, 1843)―[least concern (lc)], longspine scraper (fig. 4c) 4capoeta umbla (heckel, 1843)―[least concern (lc)], tigris scraper (fig. 4d) remark: it was reported from iran by esmaeili et al. (2016). it differs from c. damascina in its morphological (esmaeili et al., 2016) and osteological (jawad and alwan, 2020; çiçek et al., 2021) characteristics. genus carassius jarocki, 1822 5carassius gibelio (bloch, 1782)**―[potential pest], prussian carp (fig. 4e) genus cyprinion heckel, 1843 6cyprinion macrostomum heckel, 1843―[least concern (lc)], tigris kingfish (fig. 4f) 7cyprinion kais heckel, 1843―[least concern (lc)], kais kingfish (fig. 4g) remark: both c. macrostomum and c. kais are inhabited sympatrically in the sirvan river drainage. genus garra hamilton, 1822 8garra rufa (heckel, 1843)―[least concern (lc)], red garra (fig. 4h) genus luciobarbus heckel, 1843 9luciobarbus barbulus (heckel, 1849)―[not evaluated], qarah aqaj barbell (fig. 4i) genus cyprinus linnaeus, 1758 (1 species) 10cyprinus carpio linnaeus, 1758**―[ least concern (lc)], common carp family xenocyprididae (3 genera and 4 species) genus ctenopharyngodon steindachner, 1866 (1 species) 11ctenopharyngodon idella (valenciennes, 1844)** ―[not evaluated], grass carp genus hypophthalmichthys bleeker, 1859 (2 species) 12hypophthalmichthys molitrix (valenciennes, 1844)** ―[ least concern (lc)], silver carp 13hypophthalmichthys nobilis (richardson, 1844) **―[least concern (lc)], bighead carp genus hemiculter bleeker, 1859 (1 species) 14hemiculter leucisculus (basilewsky, 1855)** ―[least concern (lc)], sharpbelly (fig. 4j) family cobitidae (1 genus, 1 species) genus cobitis linnaeus, 1758 (1 species) 15cobitis avicennae mousavi-sabet, vatandoust, esmaeili, geiger & freyhof, 2015*―[not evaluated], figure 3. number of fish species in different families. 245 int. j. aquat. biol. (2022) 10(3): 242-253 zagros spined loach (fig. 4k) remark: cobitis avicennae has been described from the karkheh river drainage in the tigris river basin (mousavi-sabet et al., 2015). this species is reported from the sirvan river for the first time. family nemacheilidae (2 genera and 6 species) genus oxynoemacheilus bănăraescu & nalbant, 1967 (3 species) 16oxynoemacheilus euphraticus (bănărescu & nalbant, 1964)―[least concern (lc)] (fig. 4l) remark: it was already reported from karoun river drainage as o. freyhofi and later treated as a junior synonym of o. euphraticus (freyhof (2016). it is a widely distributed species in the sirvan river drainage. 17oxynoemacheilus zarzianus freyhof & geiger, 2017―[ least concern (lc)] (fig. 4m) its record has been confirmed in garan (marivan) and sirvan rivers by eagderi et al. (2022). 18oxynoemacheilus kurdistanicus kamangar, prokofiev, ghaderi & nalbant, 2014―[not evaluated], kurdistan stone loach (fig. 4n) remark: this species was described by kamangar et al. (2014), from lesser zab, which is reported from the sirvan river drainage in the current study. 19oxynoemacheilus parvinae sayyadzadeh, eagderi & esmaeili, 2016*―[not evaluated], parvin stone loach (fig. 4o) genus turcinoemacheilus bănărescu & nalbant, 1964 (1 species) 20turcinoemacheilus kosswigi bănărescu and nalbant, 1964―[least concern (lc)], kosswig’s loach (fig. 4p) remark: its molecular identification was confirmed by nikmehr et al. (2020) from gaveh river. genus paracobitis bleeker, 1863 (1 species) 21paracobitis molavii freyhof, esmaeili, sayyadzadeh & geiger, 2014*―[not evaluated], molavi’s crested loach (fig. 4q) family leuciscidae (2 genera and 4 species) genus squalius bonaparte, 1837 (2 species) 22squalius berak heckel, 1843―[least concern (lc)], mesopotamian chub (fig. 4r) 23squalius lepidus heckel, 1843―[least concern (lc)], mesopotamian pike chub (fig. 4s) genus alburnus rafinesque, 1820 (2 species) 24alburnus sellal heckel, 1843―[least concern (lc)], sellal bleak (fig. 4t) 25alburnus hohenackeri kessler, 1877**―[least concern (lc)], north caucasian bleak (fig. 4u) family gobionidae (1 genus and 1 species) genus pseudorasbora bleeker, 1859 (1 species) 26pseudorasbora parva (temminck & schlegel, 1846)**―[least concern (lc)], stone moroko (fig. 4v) order gobiiformes (1 family, 1 genus and 1 species) family gobiidae (1 genus and 1 species) genus rhinogobius gill, 1859 (1 species) 27rhinogobius lindbergi berg, 1933**―[not evaluated], amur goby (fig. 4w) remark: this species was first recorded by eagderi et al. (2018) and we collect it only in the gaveh river. order esociformes (1 family, 1 genus and 1species) family esocidae (1 genus and 1species) genus esox linnaeus, 1758 (1species) 28esox lucius linnaceus, 1758**―[least concern (lc)], northern pike (fig. 4x) order synbranchiformes (1 family, 1 genus and 1 species) family mastacembelidae (1 genus and 1 species) genus mastacembelus scopoli, 1777 (1 species) 29mastacembelus mastacembelus (banks & solander, 1794)―[least concern (lc)], euphrates spiny eel (fig. 4y) order siluriformes (1 family, 1 genus and 1 species) family sisoridae (1 genus and 1 species) genus glyptothorax blyth, 1860 (1 species) 30glyptothorax pallens (mousavi-sabet, eagderi, vatandoust & freyhof, 2021)*―[not evaluated], pallens catfish (fig. 4z) 246 mouludi-saleh et al./ ichthyofauna of iranian part of the sirvan river drainage 247 int. j. aquat. biol. (2022) 10(3): 242-253 248 mouludi-saleh et al./ ichthyofauna of iranian part of the sirvan river drainage 249 int. j. aquat. biol. (2022) 10(3): 242-253 250 mouludi-saleh et al./ ichthyofauna of iranian part of the sirvan river drainage figure 3. fish’s pictures of the sirvan river drainage (a) barbus lecerta, (b) capoeta damascina, (c) c. trutta, (d) c. umbla, (e) carassius gibelio, (f) cyprinion macrostomum, (g) c. kais, (h) garra rufa, (i) luciobarbus barbulus, (j) hemiculter leucisculus, (k) cobitis avicennae, (l) oxynoemacheilus euphraticus, (m) o. zarzianus, (n) o. kurdistanicus, (o) o.parvinae, (p) turcinoemacheilus kosswigi, (q) paracobitis molavii, (r) squalius berak, (s) s. lepidus, (t) alburnus sellal, (u) a. hohenackeri, (v) pseudorasbora parva, (4) rhinogobius lindbergi, (x) esox lucius, (y) mastacembelus mastacembelus and (z) glyptothorax pallens. 251 int. j. aquat. biol. (2022) 10(3): 242-253 order salmoniformes (1 family, 1 genus and 1 species) family salmonidae (1 genus and 1 species) genus oncorhynchus suckley, 1861(1 species) 31oncorhynchus mykiss (walbaum, 1792) **―[not evaluated], rainbow trout order cyprinodontiformes (1 family, 1 genus and 1 species) family poeciliidae (1 genus and 1 species) genus gambusia poey, 185 (1 species) 32gambusia holbrooki girard, 1859**―[least concern (lc)], eastern mosquitofish discussions iran occupies a significant part of the middle east, and its freshwater fish fauna shows a high level of species richness and endemism (esmaeili et al., 2018). the sirvan river drainage is part of the persian gulf basin, having elements of ethiopian, oriental and palearctic ichthyofauna. the isolation of fishes in the mountainous region of kurdistan province has led to promoting speciation e.g. p. molavii, o. parvinae and g. pallens. in addition, we can expect new species in this river drainage as described past year (freyhof et al., 2014; sayyadzadeh et al., 2016; mousavi-sabet et al., 2021). there were some studies regarding ichthyofauna of the sirvan river, but partially such as esmaeili et al. (2011) about ichthyofauna of zarivar lake (iran) with the first records of h. leucisculus and a. hohenackeri, and reporting 12 other species i.e. b. lacerta, c. barroisi, c. damascina, c. gibelio, c. idella, c. carpio, h. molitrix, h. nobilis, p. parva, s. lepidus, m. mastacembelus and g. holbrooki. however, c. barroisi is not found in iran (zareian et al., 2018) and is probably misidentified by c. trutta. rezaee and rafiee (2014) by sampling 8 stations in the oramanat region and paveh city, identified three families of cyprinidae, sisoridae and balitoridae. in this list, genus capoeta with two species of c. damascina and c. trutta; b. barbulus (barbus); leuciscus cephalus (leuciscus) and g. rufa. leuciscus cephalus (as s. berak or s. lepidus) and b. barbulus (as luciobarbus barbulus) have erroneously been identified. alizadeh-marzani et al. (2015) reported 12 species from the gaveh river, and hasankhani et al. (2019), in their study on the abundance and biodiversity of sirvan river have listed 17 fish species. however, these two works had focused on the limited area of this river drainage. there are some awkward reports regarding some fish species viz. rhinogobius cf. similis, oxynoemacheilus angorae, squalius cephalus, alburnus mossulensis, capoeta barroisi, barbus barbulus, leuciscus cephalus that probably are erroneously identified (esmaeili et al., 2011; rezaee and rafiee, 2014; hasankhani et al., 2019). anthropological activities have led to many invasive fishes, including c. gibelio, c. idella, c. carpio, h. molitrix, h. nobilis, a. hohenackeri, p. parva, h. leucisculus, o. mykiss and g. holbrooki in this drainage. these fishes are commercially valuable exotic species introduced to natural aquatic ecosystems of kurdistan province by fish farms or ornamental and aquarium fishes. the results revealed 12 exotic species had been introduced to the sirvan river drainage. based on our field observations, c. gibeli, a. hohenackeri, p. parva, h. leucisculus, r. lindbergi and g. holbrooki have been established and should be considered invasive. cyprinus carpio, c. idella, h. molitrix, h. nobilis and o. mykiss have entered this river due to stocking fishes in the dam lakes and releasing them from fish farms and seem not to be established yet. alburnus hohenackeri is a native species to western water bodies of the sothern caspian sea basin (esmaeili et al., 2018) and probably has been released to zrebar lake with other economic species and could be established. following increasing the abundance of c. gibeli and p. parva in the zrebar lake, e. lucius was probably introduced to control these exotics. introducing e. lucius to this lake led to crucial concerns regarding native fishes. in addition, this species could enter the main canal of sirvan river, which was now found in the lower part of the sirvan river drainage. it seems that the expansion and threats of exotic species are constantly increasing. although not all https://www.fishbase.se/summary/orderssummary.php?order=salmoniformes https://www.fishbase.se/summary/orderssummary.php?order=cyprinodontiformes 252 mouludi-saleh et al./ ichthyofauna of iranian part of the sirvan river drainage exotic species are endangered, their progress is irresponsible, and the predictive effect of exotic species is complex and there is a little proper method to evaluate their effects. therefore, to protect native fish in sensitive ecosystems such as the sirvan river, avoiding any development that could lead to the release of invasive species is recommended. our intensive work conducted whole iranian part of this river showed higher diversity than before. in addition, during the last decade, the taxonomic status of many iranian freshwater fishes (esmaeili et al., 2018) has been revised and some of their names have been changed. therefore, updating the fish checklist of the sirvan river drainage seems to be crucial. in addition, two new species are recorded from this river, viz. c. avicenna and o. euphraticus. the first record of the c. avicennae in the sirvan river shows a range expansion of this species toward the more western part of iran. cobitis avicennae was described from the karkheh river drainage, persian gulf basin (mosavisabet et al., 2015). the presence of this species shows its probable presence in other parts of the tigris river drainage. acknowledgments the authors would like to thank n. ahmadi, o. abdiani, n. mahmoudi, z. ghafouri and b. molodinia for their help in fish collections, m. gheysori for help to draw map, the university of tehran for financial support, and the environmental department of kurdistan province for their collaboration. references alizadeh a., shojaei kavan l., taghoyan h., shahriari r. 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(2016) 4(3): 215-223; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article descriptive osteology of squalius orientalis from urmia lake basin of iran saber vatandoust*1, sedat v. yerli2, parya jalili3 1department of fisheries, babol branch, islamic azad university, babol, iran. 2department of biology, faculty of science, hacettepe university, beytepe campus, ankara, turkey. 3department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 17 february 2016 accepted 30 may 2016 available online 2 5 june 2016 keywords: inland water chub squalius skeleton urmia abstract: osteological features are important to study the taxonomy and phylogenetic relationship of fishes. since there is no information is available about osteological features of squalius orientalis, therefore this study was aimed to provide a detailed description of the osteological features of this species from the urmia lake basin of iran and comparing it with population of s. orientalis from the caspian sea basin. for this purpose, the specimens were collected from zarineh river of the urmia lake basin and cleared and stained with alizarin red s and alcian blue for osteological examinations. finally, a detailed osteological features of this species was provided and compared with those of the caspian sea basin. based on the results, having a longer pre-vomer, dorsally oriented parasphenoid alar, longer ventral blade-shaped process of the orbitosphenoid, concaved masticatory plate, pointed ascending process of the palatine, concaved posterior margin of the opercle, and a small posterior process of the cleithrum can differentiate s. orientalis of the urmia lake basin from those of the caspian sea basin. in addition, the observed osteological difference suggest that both studied populations belong to same taxon. introduction the genus squalius bonaparte, 1837 comprises medium-sized midwater fishes widely distributed in europe and west asia. the species of this genus were placed in leuciscus cuvier, 1816 (coad, 2016; turan et al., 2013), until morphological and molecular data showed that leuciscus as earlier understood was paraphyletic (bogutskaya, 1994; zardoya and doadrio, 1999; coad, 2016). approximately 45 species are recognized in this genus (özuluğ and freyhof, 2011) with 3 species, including s. lepidus, s. orientalis and s. cephalus reported from iran (jouladeh roudbar et al., 2015a). the members of the genus squalius were characterised by numerous total vertebrae (commonly more than 40, up to 48); increased number of sensory cephalic pores (up to 12-20 in the supraorbital canal) in most species; often fused and very expanded fourth and fifth infraorbitals; and * corresponding author: saber vatandoust e-mail address: s.vatandoust@gmail.com depressed neurocranium with a reduced interorbital septum. other characters are a somewhat compressed body; moderate to large scales; a complete lateral line; no barbels; mouth terminal or subterminal; no notch in the upper jaw accommodating a tubercle on the lower jaw; thin lips with the lower one interrupted medially; a short dorsal fin without a thickened ray; a moderately long anal fin; long and hooked pharyngeal teeth in 2 rows (2,5-4,2, 2,5-5,2 or 3,5-5,3 modally) usually with hooked tips and spoon-shaped crowns; short gut; no keel on the belly; and short and relatively few gill rakers (bogutskaya, 2002; coad, 2016). the populations of the genus squalius in iranian part of the caspian sea basin was considered as s. orientalis (esmaeili et al., 2014; jouladeh roudbar et al., 2015a). in addition, this species was reported from the namak lake, urmia lake and dasht-e kavir basins of iran (jouladeh roudbar et 216 vatandoust et al./ osteology of squalius orientalis from urmia lake basin, iran al., 2015a, b; ghasemi et al., 2015). squalius orientalis described from abkhazia, georgia and s. turcicus described from aras river at erzurum, turkey (coad, 2016). both species were given as subspecies of s. cephalus in previous studies (kuru 1975a, 1975b; zengin et al., 2012). both species have been listed as valid species by turan et al. (2013). however, coad (2016) put forward that s. orientalis and s. turcicus are synonym of s. cephalus. therefore, this situation needs to be clarified. since, the osteological characters are important diagnostic traits for interpreting systematics and phylogenetic relationships within teleostei (nybelin, 1973; schultze and arratia, 1988; arratia, 1999; keivany and nelson, 2004, 2006). hence, this study was conducted to provide a detailed descriptive osteology of s. orientalis from the zarineh river, urmia lake basin and comparing it with those of s. orientalis from the caspian sea basin as well as to find out the taxonomic statue of those of the urmia lake basin. in addition, the results will provide a basis for further phylogenetic study of iranian members of this genus based on osteological data. materials and methods ten specimens of s. orientalis (fig. 1), with a mean standard length of 97.2±13.5 (mean±sd) mm, were collected from the zarineh river (near the shahindezh town, western azarbaijan province, iran) during summer 2013 by electrofishing device. the collected specimens were anaesthetized using 1% clove oil solution and fixed in 10% buffered formaldehyde. for osteological examination, the specimens were cleared and stained with alizarin red s and alcian blue according to taylor and van dyke (1985) (fig. 2). the cleared and stained specimens were studied under a stereomicroscope (leica mc5); and different skeletal elements were dissected and scanned by a scanner equipped to a glycerol bath (epson v600). drawings of the skeletal elements were performed from obtained images using coreldraw x6 software. nomenclature and abbreviation of the skeletal elements were followed rojo (1991) and jalili et al. (2014 a, b and c). the detailed osteological features of s. orientalis from the caspian sea basin was provided by jalili (2015). results the anterior half of the neurocarnium is narrower and shallow. in the lateral view, three and four foramens present at the anterior and posterior half of the neurocranium, respectively. six bony elements, including the supraethmoid-ethmoid, lateral ethmoid, pre-vomer, nasal, kine-ethmoid and preethmoid-i form the ethmoid region. the supraethmoid-ethmoid bears the horizontal and vertical parts; the horizontal part is wide with two developed posterior processes and the vertical poart has a triangular shape. the pre-vomer is v-shaped anteriorly and serrated posteriorly. the pre-ethmoidi can be bony or cartilaginous and is connected to the prevomer antero-laterally. two long and narrow nasal bones were positioned at the lateral side of the supraethmoid-ethmoid (fig. 3c). a small kineethmoid is situated at the anterior part of the pre figure 1. lateral view of squalius orientalis from the zarineh river of the urmia lake basin. 217 int. j. aquat. biol. (2016) 4(3): 215-223 figure 2. the lateral view of the cleared and stained squalius orientalis from the zarineh river of the urmia lake basin (scale bare=2 mm). figure 3. lateral (a), ventral (b) and dorsal (c) views of the neurocranium of squalius orientalis from the zarineh river of the urmia lake basin. abbreviations: boc: basioccipital; epo: epiotic; exo: exoccipital; fr: frontal; le: lateral ethmoid; mp: masticatory palate; nas: nasal; orb: orbitosphenoid; pa: parietal; pe: preethmoid i; pr-pp: posterior pharyngeal process; pro: prootic; ps: parasphenoid; pts: pterosphenoid; pto: pterotic; so-cr: supraoccipital crest; so: supraorbital; soc: supraoccipital; sp: sphenotic; se: supraethmoid; pv: pre-vomer (scale bar=3 mm). 218 vatandoust et al./ osteology of squalius orientalis from urmia lake basin, iran vomer that its posterior margin is wider. the medial part of the lateral-ethmoid is the widest part of this bone and is connected to the orbitosphenoid, frontal and supraethmoid-ethmoid. there is a cartilaginous protuberance at the abdominal level of the lateralethmoid (fig. 3). the orbital regin comprises the frontal, parasphenoid, ptersphenoid, orbitosphenoid and circumorbital series. the anterior margin of the frontal is thin and its posterior part bears a posterolateral processes. the supraorbital canal goes around the lateral margin of the frontal. two trapizoidshaped orbitosphenoids are connected and form a blade shape process connecting to the parasphenoid. there are two upward processes and two fossa in the middle portion of the parasphenoid; these two internal and external processes are attached to the ptersphenoid and pterotic, respectively. the anterior part of the parasphenoid is serrated and its posterior part is bifurcated (fig. 3b). the lateral edge of the ptersphenoid is concaved and its abdominal is triangular in shape; there are a dorsal fossa and two protuberances on the middle part of this bone extending ventrally. the otic region of the neurocranium includes the prootic, epiotic, sphenotic, parietal and pterotic bones. the posterior edge of the parietal encloses the supratemporal canal. the sphenotics is a small bone with a lateral process bended posteriorly. the pterotic is triangulare in shape on dorsal view. the epiotic has a process posteriorly. there is a rounded process with some pores at the posterior and anterior parts of the prootic. the prootic possess a blade shape process tilted ventrally connectiong to the parasphenoid (fig. 3). the occipital region consists of the supraoccipital, exoccipital and basioccipital. the posterior margin of the supraoccipital is pointed and its dorsal level has a developed crest. the exoccipital bears two dorsal and ventral processes. the basioccipital is trapizoid in shape and its posterior part is narrower. the posterior part of the basioccipital bears a pharyngeal process with a postero-ventral fossa; at the anterior portion of this process, there is a masticatory palate narrower than the width of the basioccipital (fig. 3b). in the upper jaw, the maxillae has a mid-dorsal ascending protuberance bending anteriorly; this bone also has an anterior descending process. the premaxillae is l-shaped and its horizontal part is larger than the ventral part (fig. 4a). the lower jaw includes the dentary, angular, retroarticular and coronomeckelian (fig. 4b). the coronoid process of the dentary is short and bended posteriorly; its posterior part is wider overlapping with the angular; at the postero-ventral edge of the angular, a triangular-shaped retroarticular is present. the tiny and long coronomeckelian presents at the medial face of the angular and dentary. the opercle, preopercle, subopercle and interopercle form the opercular series (fig. 5). the posterior margin of the opercle is concaved and bears a developed antero-dorsal process. the horizontal portion of the preopercle is longer than its vertical part. the subopercle has crescent shape with a wide anterior portion. the suspensorium includes the quadrate, symplectic, hyomandibular, endopterygoid, figure 4. upper (a) and lower (b) jaws of squalius orientalis from the zarineh river of the urmia lake basin. abbreviations: an: angular; crb: coronoid process; cm: coronomeckelian; dn: dentary; keth: kinethmoid; ra: retroarticular; mx: maxillary; pmx: premaxillary; mdip: maxillary descending process; mip: maxillary mid-lateral ascending process; msc: mandibular sensory canal; rap: rostral ascending process (scale bar=3 mm). 219 int. j. aquat. biol. (2016) 4(3): 215-223 ectopterygoid, metapterygoid and palatine (fig. 5). the anterior margin of the hyomandibular is thin and round. the symplectic is positioned ventral to the hyomandibular and posterior to the quadrate. this bone has the blade-shaped vertical and thickened horizontal portions, respectively. the endopterygoid has an anterior triangular-shaped process and some mid-lateral protuberances; and its posterior part is broader. the ventral part of the endopterygoid is thicker than its dorsal portion. the middle part of the palatine is narrow; there are three small process anteriorly. the long ectopterygoid is positioned ventral to the endopterygoid (fig. 5). the branchial apparatus comprises three unpaired basibranchial, three paired hypobranchial, five paired ceratobranchial, four paired epibrancchial and two paired infrapharyngobranchial (fig. 6a). the infrapharyngobranchials are semi-circular in shape and overlap each other. the epibranchial bears some pointed process posteriorly. the hyoid series possess the unpaired basihyal and urohyal, and paired epihyal, ceratohyal, hypohyal and interhyal and three paired branchiostegal rays (fig. 6a). the urohyal has a blade shape portion perpendicular to the ventral part; the posterior portion of its ventral part is wider (fig. 6b). the anterior margin of the urohyal has a narrow groove ventrally. the anterior edge of the basihyal is wider. there is a protuberance at the anterior part of this bone. the hypohyal bears two dorsal and ventral parts. the pectoral girdle consists of eight bones including cliethrum, supracleithum, postcliethrum, coracoid, mesocoracoid, scapula, posttemporal, supratemporal and four radials (fig. 7a). the cliethrum is l-shaped and its horizontal part has a lateral protuberance and its vertical part has a developed process. in addition, the dorsal margin of the vertical part is thin connecting to the supracleithum. there is a tiny supratemporal anterior to the posttemporal that is connected to the pterotic. the dorsal margin of the coracoid is broad with a protuberance connecting to the mesocoracoid. it is long and connected to the coracoid and scapula. the scapula bears a large foramen. the pectoral girdle possesses four radials. the paired pelvic bones, styloid and radials form the pelvic girdle (fig. 7b). the anterior margin of the pelvic bone is bifurcated deeply and its posterior part has mid-lateral and posterior processes; the latter is weakly developed. there are three radials at the dorsal side of the pelvic bones. two external radials are paired and the internal one is unpaired. two long and thin styloid bone is located on the lateral side of figure 5. lateral view of the suspensorioum and opercular series of squalius orientalis from the zarineh river of the urmia lake basin. abbreviations: ect: ectopterygoid; end: endopterygoid; hy: hyomandibulare; iop: interopercle; mtp: metapterygoid; op: opercle; opp: opercular prominent process; opj: opercular joint; p: palatine; pop: preopercle; q: quadrate; sop: subopercle; sym: symplectic (scale bar=3 mm). figure 6 dorsal view of the hyoid arch and branchial apparatus (a) and urohyal bone (b) of squalius orientalis from the zarineh river of the urmia lake basin. abbreviation: bhy: basihyal; chy: ceratohyal; epy: epihyal; hhy: dorsal and ventral hypohyal; ihy: interhyal; uhy: urohyal; bbr: basibranchial; cbr: ceratobranchial; ebr: epibranchial; hbr: hypobranchial; pbr: infrapharyngobranchial (scale bar=3 mm). 220 vatandoust et al./ osteology of squalius orientalis from urmia lake basin, iran the pelvic bone. there are 42 centra in the axial skeleton; the four anterior centra with tripus, intercalarium, scaphium and claustrum form the weberian apparatus (fig. figure 7. medial view of the pectoral (a) and pelvic (b) girdles squalius orientalis from the zarineh river of the urmia lake basin. abbreviations: cl: cleithrum; co: coracoid; mco: mesocoracoid; mlp: mid-lateral process; pb: pelvic bone; pcl: postcleithrum; pop: posterior process; ps: pelvic splint; r: radials; sc: scapula (scale bar=3 mm). figure 8. lateral view of the weberian apparatus (a), dorsal (b), anal (c) and caudal (d) fins in squalius orientalis from the zarineh river of the urmia lake basin. abbreviations: c14-21: centrum 14-21; dfs: dorsal fin spine; dpt: distal pterygiophore; epu: epural; fvc: first vertebra centrum; hp 1-6: hypural plates 1-6; hsp: hemal spine; mtp: medial pterygiophore; nua: neural arch; ns: neural spine; pr 4: pleural rib 4; ppt: proximal pterygiophore; ph: parhypurale; pls: pleurostyle; rna: rudimentary neural arch; sun: supraneural; sty: stay (scale bar=3 mm). 221 int. j. aquat. biol. (2016) 4(3): 215-223 8a). the dorsal fin has 3 unbranched and 8½ branched rays, 9 pterygoiphore and one small stay bones (fig. 8b). the dorsal portion of the stay bone is bifurcated and thinner. the anal fin has 3 unbranched, 9½ branched rays, 10 pterygoiphore and one stay bone (fig. 8c). the first pterygoiphore of the dorsal and anal fins are located at the 14th and 21st centra, respectively. the caudal fin is formed by 6 hyporals, unpaired parhyporal, pleurstyle and paired uroneurals. the caudal fin has 10 dorsal and 9 ventral procurrent rays and 19 caudal rays (fig. 8d). discussion intra-specific variation such as osteological characters are important in species delineation, as it is acknowledged that inadequate information on intra-specific geographic variation can lead to erroneous species descriptions (ishihara, 1987; irfan and suneetha gunawickrama, 2011). osteological characters may also provide a major tool in examining variability within a species (eastman, 1980), since different populations of the same species, which share external appearance, may vary in skeletal structures (hilton and bemis, 1999). the results of the present study revealed some differences in osteology among the two geographic populations of s. orientalis in iran. the observed differences were in the structures of the neurocranium, opercle, palatine and pectoral girdle. at the dorsal view of the neurocranium, the anterior portion of the pre-vomer is extended the supraethmoid-ethmoid in s. orientalis of the urmia lake basin population, whereas that of the caspian sea is almost at the level of the supraethmoidethmoid (jalili, 2015). the parasphenoid alar in the urmia lake basin population is banded dorsally versus laterally oriented one of the caspian sea specimens (jalili, 2015). also the ventral blade shape process of the orbitosphenoid in the urmia lake basin specimens is longer than that of the caspian sea specimens (jalili, 2015). the two later characters are led a deeper neurocranium in the urmia lake basin specimens. according to zhang (2005), the basioccipital is important character in cyprinids. in the examined population, the masticatory plate is concaved, whereas that of the caspian sea is flattened (jalili, 2015). the anterior ascending process of the palatine in the urmia lake basin specimens were pointed and well-developed versus short and weakly developed one of the caspian sea population (jalili, 2015). the posterior margin of the opercle is concaved in the urmia lake basin population versus straight one of the caspian sea population (jalili, 2015). structure of the pectoral and pelvic girdles are important to identify the members of the subfamily schizothoracine (chen et al., 2001). in the pectoral girdle of the urmia lake basin specimens, the posterior process of the cleithrum is triangular in shape and small versus wide and well-developed one of the caspian sea specimens (jalili, 2015). the results revealed that osteological characters including having a longer pre-vomer, dorsally oriented parasphenoid alar, longer ventral bladeshaped process of the orbitosphenoid, concaved masticatory plate, pointed ascending process of the palatine, concaved posterior margin of the opercle, and a small posterior process of the cleithrum can differentiate s. orientalis of the urmia lake basin from that of the caspian sea basin. in addition, there is no variation in the bones forming the skull roof, postcranial and caudal fin skeletons of both populations. as conclusion, the results showed that bone arrangement seems to be a character for population differentiation in this species that can be caused under the influence of different environmental conditions. additionally, the observed osteological differences suggest that both studied populations belong to same taxon. acknowledgments the senior author was financially supported by islamic azad university, babol branch for a sabbatical period in hacettepe university, turkey. we would like to express our sincere thanks to h. mousavi sabet and s. eagderi for providing specimens. 222 vatandoust et al./ osteology of squalius orientalis from urmia lake basin, iran references arratia g. 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(2015) 3(6): 372-378 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article reproductive biology, maturation size and sex ratio of black tiger shrimp (penaeus monodon fabricius, 1798) from fishing grounds of digha coast, west bengal, india. naser uddin sk1, shubhadeep ghosh1, joydev maity*21 1regional centre of central marine fisheries research institute (icar), visakhapatnam 530 003, andhra pradesh, india. 2department of aquaculture management and technology, vidyasagar university, midnapore 721 102, west bengal, india. article history: received 1 september 2015 accepted 27 n o v e m b e r 2015 available online 2 5 d e c e m b e r 2015 keywords: lm50 spawning season gsi abstract: the present paper studies the reproductive biology, maturation size and sex ratio of penaeus monodon collected from digha fishing grounds, india during 2011-2013. a total of 633 individual of p. monodon were examined and among them 242 were males and 391 were females. the overall yearly sex ratio was observed to be 1:1.6 (males: females). based on the results, the spawning season of p. monodon was mainly in january-february and was extended up to june. the first maturity was observed at 163.5 mm length. the estimated number of ova in the mature ovary ranged from 120155 to 961240 in p. monodon. introduction penaeus monodon is the largest species among the penaeids prawns commercially known as jumbo tiger prawn or black tiger prawn. it is widely distributed and commercially very important species in india and national market. rao (2000) studied the reproduction and the spawning of p. monodon and reported it to be heterosexual as is the case with all the penaeids. the females attain maturity at 196-200 mm and males at 166-170 mm tl. the five maturity stages distinguished in the ovary could be termed as immature, maturing, mature, ripe and spent. fecundity varies from 200000 to 1000000 eggs depending on the size of the females. spawning takes place in the sea at 40-80 m depth. knowledge of spawning seasons and distribution of spawning areas are important for the management of fisheries the spawning seasons of penaeid shrimps are most commonly determined by the percentage of mature females present in the catch or from the changes in gonadal indices (crocos and kerr, 1983; garcia, 1985; bauer and vega, 1992; crocos and coman, 1997; minagawa et al., 2000; crocos et al., * corresponding author: joydev maity e-mail address: jmaity@mail.vidyasagar.ac.in 2001; costa and fransozo, 2004; aragon-noriega and garcia-juarez, 2007). in the philippines, spawning is year round but there seems to be two peak seasons in a year. february-march or july and october-november, although it varies from year to year (motoh, 1981). rajyalakshmi et al. (1985) reported the peak season as october–april corresponding to the post-monsoon season coinciding with an increase in the salinity along the odisha cost, while it varies from place to place in philippine and taiwan (su and liao, 1986). su et al. (1990) observed september-december as the peak season. however, there is no published information available till date on the breeding season, size at sexual maturity and sex ratio of p. monodon from digha coast of west bengal, india. the present study is to provide information on the reproductive biology including the breeding season, size at sexual maturity and sex ratio of p. monodon in coastal waters of digha coast, india. materials and methods the samples of p. monodon were collected from 373 uddin sk et al./ reproductive biology of black tiger shrimp from west bengal, india commercial catches of digha mohana landing centre caught by trawlers from bay of bengal of west bengal coast during 2011-2013. shrimps with petasma and presence of spermatophores in the terminal ampoule were taken as matured males. in females, the ovaries were classified into immature, early maturing, late maturing, mature and spentrecovering stages based on the size, colour and yolk formation (primacera, 1989) specimens with immature, spent-recovering and early maturing ovaries were rated as immature while those with late maturing and mature ovaries were considered as matured ones. the gonads were cut open on the dorsal side and removed to observe its condition. the weight of gonad was taken by using electronic balance to the nearest 0.001 g. the gonads were preserved in 7% formalin for further analysis. sex ratio: the sex ratio is based on the monthly estimated number as to get an actual representation of males and females in the population. homogeneity of the sex ratio (based on observed numbers) over months in three years has been tested using chi-square test (snedecor and cochran, 1967). this is computed as follows: x2 = ∑ (o-e)2 / e where, o = observed number of males and females in each month/length group, and e= expected number of males and females in each month /length group. significance at probability level of p = 0.05 was carried out. homogeneity was tested for 1:1 ratio. length at first maturity (lm50): sex was determined and stages were classified as immature, early maturing, late maturing, mature and spent (dall et al., 1990). the maturity classification was made (farfante, 1969) where stages iii and above were considered to be mature for males and females, respectively. for both males and females, the size at first maturity was determined by calculating the proportion of mature individuals in each size class (carapace length and total length). the size at which 50% of individuals were mature was taken as the length at first maturity (lm50) (king, 1995). spawning season: to determine the spawning season, proportion of the gravid and ripe females (iv and v) in each month were observed and the highest percentage were taken to determine the spawning period. ganado somatic index (gsi): the spawning season of p. monodon was estimated from the mean index value of gonad in different months of the year. for gonado-somatic index (gsi) estimation, females were weighed individually after wiping it dry. the gonad was dissected out carefully and weighed by using an electronic balance. the gsi are calculated by using the formula: gsi = (weight of the gonad x 100)/ weight of the fish. fecundity: for studying the fecundity, the preserved ovary was washed and dried by placing it between two blotting papers. the ovary sub-sample was taken from the anterior, middle and posterior regions of the ovary. the weight of ovary was recorded, and all sub-samples of the ovary were weighed to the nearest 0.001 g. using an electronic balance. the mature ova present in the sub-samples were counted by using a counting slide. from the number of ova in the weighed sub-sample, fecundity was calculated using the formula: fecundity = (total weight of the ovary/weight of the sample) × number of ova in the sample the relationship between the fecundity to total length, total weight and ovary weight were found out by fitting regression on logarithms of observed values by least square method (snedecor and cochran, 1967). f= αxb where, f = fecundity, 𝛼 = constant, x = variable (total length, body weight or ovary weight) and b = correlation coefficient. the exponential relationship was transformed into a straight line logarithmic form based on the equation: log f = log 𝛼+ b log x. results sex ratio: during 2011-2013, total numbers of 631 specimens of p. monodon were examined, of them 242 were males, and 391 were females. the sex ratio is presented in tables 1 and 2. the overall yearly sex 374 int. j. aquat. biol. (2015) 3(6): 372-378 ratio was observed to be 1:1.6 (males: females). a chi-square test showed that the annual distribution of females and males is not significantly difference from 1:1 ratio at 0.05% level (p>0.05), although the sex ratio varied from month to month during the study period. spawning season: average gonadosomatic index of females of p. monodon is presented in figure 1. in the present study, gsi was observed to be higher between january and july. the maximum value (gsi=10.36) was observed in june and minimum value (gsi=7.8) in october. the gsi of females of p. monodon showed two peaks, in the months of january and june. the highest gsi of 10.36 in june, indicating that most females were mature. the percentage of mature females is presented in figure 2. the highest mature percentage was observed in the month of february (88.89%) and june (85.4%). the lowest percentage was observed in the month of september (65.2%). mature females in all months were observed to be >50% indicating that p. monodon spawns more or less throughout the year. lm 50: the length at first maturity is presented in figure 3. in the present study, 163.5 mm was 2011 2012 2013 month male female d2/e sig male female d2/e sig male female d2/e sig jan 10 9 0.026 s 8 13 0.595 s feb 14 9 0.543 s 9 11 0.1 s 11 16 0.463 s mar 6 7 0.038 s 6 15 1.929 s apr 0 0 0 s 5 5 0 jun 3 14 3.559 s 5 21 4.923 ns 13 13 0 s jul 4 11 1.633 s 5 10 0.833 s 19 9 1.7857 s aug 3 19 5.818 ns 8 18 1.923 s 4 17 4.0238 ns sep 5 20 4.5 ns 11 13 0.083 s 6 13 1.2895 s oct 4 23 6.685 ns 13 8 0.595 s 11 9 0.1 s nov 4 12 2 s 7 21 3.5 s 19 5 4.0833 ns dec 4 21 5.78 ns 14 15 0.017 s 11 14 0.18 s s=significant, ns=not significant table 1. annual sex ratio of p. monodon during fishing period of 2011-2013. month male female total chi squre value significant f/m jan 18 22 40 0.2 s 1.222222 feb 34 36 70 0.028571 s 1.058824 mar 12 22 34 1.470588 s 1.833333 apr 5 5 10 0 s 1 jun 21 48 69 5.282609 ns 2.285714 jul 28 30 58 0.034483 s 1.071429 aug 15 54 69 11.02174 ns 3.6 sep 22 46 68 4.235294 ns 2.090909 oct 28 40 68 1.058824 s 1.428571 nov 30 38 68 0.470588 s 1.266667 dec 29 50 79 2.791139 s 1.724138 total 242 391 633 17.53633 ns 1.615702 s=significant, ns=not significant table 2. month-wise sex ratio of p. monodon during fishing period of 2011-2013. figure 1. average gonadosomatic index (gsi) of p. monodon females from digha coast during 2011 – 2013. 375 uddin sk et al./ reproductive biology of black tiger shrimp from west bengal, india observed as the length at first maturity. fecundity: in the present study, length and weight of mature females of p. monodon ranged from 125250 mm and 47-261 g, respectively. the estimated number of ova in the mature ovary ranged from 120155 to 961240 in ovary weight ranging 5 to 40 g. the relationship between fecundity and length and fecundity and weight of p. monodon was log f= 1.295461+3.135046 logl (r=0.607847) and log f= 3.253808+1.022533 logw (r= 0.58288). there was significant variation (p<0.05) in the slope of regression relations of body length and weight with fecundity. discussion in the present study, the observed sex ratio was 1:1.6 (males: females). this could be attributed to changes in the fishing ground and fishing pattern of trawl nets and the pattern of migration during breeding seasons of both sexes. in general, sex ratio is known to be close to 1:1 (males: females) in nature (fisher, 1958). costa et al. (2010) suggested that the sex ratio of females may be related to the greater vulnerability of females to fishing due to their size. sarada (2010) observed from kozhikode, kerala, india on the annual sex ratio between male and female to be 1.07:1. two peak spawning seasons were observed with the highest mature percentage of females in february and june. the results of gsi indicated the spawning season to be during january-february and june. similar results were reported by khan et al. (2003) who observed the spawning season of p. monodon twice a year i.e. during winter (february) and summer (september). along the kakinada coast, the species spawns throughout the year with different peak periods in different years. rao (2000) observed a similar phenomenon in the spawning off visakhapatnam. amanat and qureshi (2011) observed the peak spawning activity from the coastal waters of pakistan during august to october with a secondary peak during february to march. shrimps exhibit more than one spawning period in a year. the process of ovarian maturation goes through different reproductive stages viz., undeveloped, developing, nearly ripe, ripe and spent. after completion of first spawning, shrimps rest in a developing stage before going through the maturation cycle again (jayawardane et al., 2003). the estimated number of ova in the mature ovary ranged from 120155 to 961240. fecundity depends on the size of females. babu (2014) observed the fecundity at 723251 for 270 g from bhairavapalem (a.p) india, which slightly varies from the results of the present study. this may be due to differences of environment and on the availability of food. fecundity increased with prawn size, suggesting that much of the available energy in larger prawns is devoted to egg production rather than growth. motoh (1981) established a positive correlation between figure 2. average maturity percentage of p. monodon females from digha coast during 2011-2013. figure 3. length at first maturity (lm50) of p. monodon females at digha coast, india. 376 int. j. aquat. biol. (2015) 3(6): 372-378 fecundity and female size of p. monodon in terms of carapace length. villegas et al. (1986) demonstrated a positive correlation between fecundity and spawner weight of p. monodon. primavera (1989) stated that fecundity or number of eggs of p. monodon in a complete spawning averages 300000 (range: 100000-800000) for ablated females and 500000 (range: 200000-1 million) for wild spawners. length at first maturity was observed at 163.5 mm in the present investigation. the results differ from rao (2000), who studied the reproduction and the spawning of p. monodon from kakinada, possibly because of differences in food, temperature and water quality, impacting maturity. according to rao (2000), the females attain maturity at 196-200 mm and males at 166-170 mm tl. amanat and qureshi (2011) estimated size at onset of sexual maturity at 13.33 cm from the lagoon water of pakistan. acknowledgments the authors are thankful to the director, cmfri and pi nicra for his support and encouragement and the author also acknowledge icar for the funding to carry out your study. references amanat z., qureshi n. (2011). ovarian maturation stages and size at sexual maturity of penaeus indicus (h. milne edwards, 1937) in the lagoon water of sonmiani bay, balochistan. pakistan journal of zoology, 43(3): 447-459, aragon-noriega e.a., garcia-juarez a.r. (2007). comparison of two methods to determine the maturity period in penaeid shrimps (decapoda, penaeidae). crustaceana, 80: 513 -521. bauer r.t., vega l.w.r. (1992). pattern of reproduction and recruitment in two sicyoniid shrimp species (decapoda: penaeoidea) from a tropical seagrass habitat. journal of experimental marine biology and ecology, 161: 223-240. crocos p.j., coman g.j. (1997). seasonal and age variability in the reproductive performance of penaeus semisulcatus broodstock: optimizing broodstock selection. aquaculture, 155: 55-67. crocos p.j., kerr j.d. (1983). maturation and spawning of the banana prawn penaeus merguiensis in albatross bay, gulf of carpentaria, australia. journal of experimemtal marine biology and ecology, 69: 3759. crocos p.j., park y.c., die d.j., warburton k., manson f. (2001). reproductive dynamics of endeavour prawns, metapenaeus endeavouri and m. ensis, in albatross bay, gulf of carpentaria. australia. marine biology, 138: 63-75. costa r.c., fransozo a. (2004). reproductive biology of the shrimp rimapenaeus constrictus (decapoda: penaeidae) in the ubatuba region of brazil. journal of crustacean biology, 24: 274-281. dall w., hill b.j., rothlisberg p.c., staples d.j. (1990). the biology of penaeidae. advances in marine biology, 27: 1-488. fisher r.a. (1958). the genetical theory of natural selection. dover, new york, 2nd edition. 304 p. da costa r.c., branco j.o., machado i.f (2010). population biology of shrimp artemesia longinaris (crustacea: decapoda: penaeidae) from the southern coast of brazil. journal of the marine biological association of the united kingdom, 90: 1-7. farfante i.p. (1969) western atlantic shrimp of the genus penaeus. fishery bulletin. united states fish and wildlife service, 67: 461-591. garcia s. (1985). reproduction, stock assessment models and population parameters in exploited penaeid shrimp populations. in: p.c. rothlisberg, b.j. hill, d.j. staples (eds.). proceedings of the second australian national prawn seminar, cooralbyn, 22-26 october, 1984. cleveland, queensland, australia. pp: 139–158 jayawardane p.a.a.t., mclusky d.s., tytler p. (2003). reproductive biology of metapenaeus dobsoni (miers, 1878) from the western coastal waters of sri lanka. asian fishery science, 16: 91-106 king m. (1995). fisheries biology assessment and management. blackwell science ltd. (fishing news books), osney mead, oxford. 341 p. khan m.a.a., sada n.u., chowdhury z.a. (2003). status of the demersal india fishery resources of bangladesh, in: g. silvestre, l. garces, i. stobutzki, c. luna, m. ahmed, r.a. valmonte-santos, l. lachica-alino, p. munro, v. christensen, d. pauly (eds.). assessment, management and future directions for coastal fisheries in asian countries. 377 uddin sk et al./ reproductive biology of black tiger shrimp from west bengal, india worldfish center conference proceeding. pp: 63-82. motoh h. (1981). studies on the fisheries biology of the giant tiger shrimp penaeus monodon in the philippines. technical report. no. 7. aquaculture department, seafdec. philippines. 128 p. minagawa m., yasumoto s., ariyoshi t., umemoto t., ueda t. (2000). inter-annual, seasonal, local and body size variations in reproduction of the prawn penaeus (marsupenaeus) japonicus (crustacea: decapoda: penaeidae) in the ariake sea and tachibana bay, japan. marine biology, 136: 223-231. primavera j.h. (1989). broodstock of sugpo, (penaeus monodon (fabricius). aquaculture extension manual, no. 7, tigbauan, iloilo, philippines, aquaculture department, southeast asian fisheries development center. pp: 1-26 subrahmanyam c.b. (1965). on the reproductive cycle of penaeus indicus (m. edw.). journal of marine biological association of india, 7: 284-290. sudhakara r.g. (2000). the indian tiger prawn penaeus monodon (fabricius). in: v.n. pillai, n.g. menon (eds.). marine fisheries research and management, central marine fishery research institute, kochi. pp: 511-524. ramesh babu k. (2014). fecundity variations of black tiger shrimp penaeus monodon from two different geographical locations, east coast of andhra pradesh, india. journal of global biosciences, 3(4): 725-730. rajyalakshimi t., pillai s.m., ravichandran p. (1985). the biology of penaeus monodon in the capture fisheries of orissa coast in the context of occurrence of natural brood stock. in: y. taki, j.h. primavera, j.a. liborera (eds.). first international confurence. culture of penaeid prawn and shrimp, 1984 december 4-7; iloilo city, philippines, seafdec aquaculture department.175 p. snedecor g.w., cochran m.g. (1967). statistical methods. oxford and ibh publishing co., new delhi, 6th edn. 539 p. su m.s., liao l.c. (1986). distribution and feeding ecology of penaeus monodon along the coast of tungkang, taiwan. in: j.l. maclean, l.b. dizon, l.v. hisollos (eds.). proceding of the first asian fisheries forum. pp: 207-210. su m.s., liao l.c., hirano r. (1990). life history of grass prawn, penaeus monodon, in the coastal water of southwest taiwan. in: r hirano, i. hanyu (eds.). the second asian fisheries forum. pp: 369-372. sarada p.t. (2010). fishery and biology of green tiger shrimp penaeus semisulcatus (de haan, 1844) landed at puthiappa, kozhikode, kerala coast. journal marine biological association of india, 52(1): 24-28. villegas c.t., trino a., travina r. (1986). spawner size and the biological components of the reproduction process in penaeus monodon (fabricius). in j.l. maclean, l.v. hosillos (eds.). proceedings of the first asian fisheries forum, 26-31 may 1986, manila, philippines. 701 p. int. j. aquat. biol. (2015) 3(6): 372-378 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی ,penaeus monodon fabriciusشناسی تولیدمثل، اندازه بلوغ و نسبت جنسی میگوی ببری سیاه )زیست طق صیادی سواحل دیقا، بنگال غربی، هندوستانادر من (1798 2جویدو مایتی ،2قش شوبهادیپ ،1ناصر الدین س.ک. .هندوستان ،آندراپرادش ،353335ویساخاپاتنام ،ای انستیتو تحقیقات شیالت دریاییمرکز ناحیه1 .هندوستان ،بنگال غربی ،221132میدناپور ویدیاساگار، دانشگاهپروری، مدیریت و تکنولوژی آبزی گروه2 چکیده: 2311طی سال هندوستان دیقا، صیادیمناطق صید شده از penaeus monodon، اندازه بلوغ و نسبت جنسی شناسی تولیدمثلزیست حاضر الهقم نسبت جنسی عدد ماده بودند. 591عدد نر و 242ها مورد بررسی قرار گرفت که از آن p. monodonنمونه 355کند. تعداد مطالعه می 2315الی بود و تا های ژانویه و فوریهطور عمده ماهبه p. monodonفصل تخمریزی ،اساس نتایجبر)نر به ماده( بود. 3/1به 1مشاهده شده در دوره مطالعه 123133 در محدوده های بالغدر تخمدان نمونهتخم متری مشاهده شد. تعداد تخمینی میلی 3/135ماه ژوئن نیز ادامه داشت. نخستین بلوغ در طول عدد بود. 931243تا .gsi، فصل تخمریزی ،50lc :کلمات کلیدی int. j. aquat. biol. (2016) 4(4): 285-294doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article influence of probiotic, lactobacillus plantarum on serum biochemical and immune parameters in vaccinated rainbow trout (oncorhynchus mykiss) against streptococcosis/lactococosis ali. m. kane1,2, mehdi soltani*1, hossein ali ebrahimzahe-mousavi1, komeil pakzad1 1department of aquatic animal health, faculty of veterinary medicine, university of tehran, tehran, iran. 2department of pathology, faculty of veterinary medicine, university of kufa, kufa, iraq. article history: received 2 may 2016 accepted 27 june 2016 available online 2 5 august 2016 keywords: trout streptococcosis lactococosis immune parameters abstract: this study evaluated the effects of probiotic, lactobacillus plantarum on serum biochemical and some immune parameters of immunized rainbow trout weighting 29.6±1.84 g, with streptococcosis/lactococosis vaccine at 16±1.5°c, for 60 days. a commercial diet was used as the control. fish in the first treatment were immunized with streptococcosis/lactococosis vaccine in bathing route for 1 min. in the second group, the vaccinated trout were also fed diet containing l. plantarum (108 cfu g-1). in the third treatment, fish were only fed the diet supplemented with l. plantarum (108 cfu g-1). the results showed that vaccinated trout with or without l. plantarum feeding diets significantly decreased heterophils. meanwhile it enhances serum lysozyme, alternative complement activities, antibody titer, total leukocytes, lymphocytes, and serum biochemical parameters, including alp, igm, and total protein levels compared to control groups. moreover, the highest levels of above mentioned parameters were found in vaccinated fish that fed l. plantarum. in addition, the vaccinated fish that fed l. plantarum showed significantly elevated cholesterol levels compared to the control group. the results showed that the dietary l. plantarum improved the immunity of immunized trout with streptococcosis/lactococosis vaccine. introduction streptococcosis and lactococcosis, gram-positive bacteria, are major systemic bacterial diseases occurring in both wild and cultured fishes (pridgeon and klesius, 2012; soltani et al., 2005). during the examinations of the 108 gram-positive isolates cultured from diseased trout in seven provinces of iran with major trout production from 2008 to 2009, 49 samples were identified as s. iniae, 37 samples were matched lactococcus garvieae, and 22 samples identified as streptooccus sp. (haghighi karsidani et al., 2010), suggesting that the trout farms in iran are severely affected by these diseases. due to increasing in the resistant bacteria, demands for an effective vaccine have prompted over the past decade (soltani et al., 2007; sun et al., 2010; pridgeon and klesius, 2011; sun et al., 2013). nonetheless, to date the initiative to control these * corresponding author: mehdi soltani e-mail address: msoltani@ut.ac.ir diseases have focused on the use of antibiotics, including enrofloxacin, oxytetracycline erythromycin and amoxicillin (austin and austin, 2007; evans et al., 2004). currently, several experimental streptococcosis and lactococcosis vaccines in the forms of formalinkilled (soltani et al., 2007), subunit vaccines (cheng et al., 2010; zou et al., 2011), dna vaccines (sun et al., 2013), and attenuated live vaccines (buchanan et al., 2005) are reported. however, the only licensed vaccines against streptococcosis and lactococcosis are bacterins consisting of inactivated whole-cell bacteria (sommerset et al., 2005). in iran, a bacterin vaccine is currently available to protect rainbow trout (oncorhynchus mykiss) from streptococcosis/ lactococcosis, which has given impressive results (soltani et al., 2007). bacterins have also been used to immunize farmed fish in korea, australia, spain 286 kane et al./ effect l. plantarum on vaccinated rainbow trout and chile (hastein et al., 2005; sommerset et al., 2005; austin and austin, 2007). however, the protectivity of these bacterins in some cases was not completely satisfied (bachrach et al., 2001; eyngor et al., 2008). recently, the use of probiotic, especially lactic acid bacteria (lab), as a preferable method that enhances the non-specific immune response of fish has been grown significantly. this improves the prevention and control of various diseases in aquaculture (cruz et al., 2012). lactobacillus plantarum is a rod-shaped gram-positive bacterium belongs to lab, and is known to produce plantaricin that is active against certain pathogens (cebeci and gurakan, 2003). in aquaculture, the administration of l. plantarum induced immune modulation, enhances the growth performance, and increases disease resistance in fishes (giri et al., 2013, 2014; son et al., 2009). nonetheless, there is a lack of information regarding l. plantarum effects on vaccinated fish. therefore, the present study was carried out to explore the influence of probiotic, l. plantarum on some serum biochemical and some immune parameters of vaccinated rainbow trout against streptococcosis/lactococosis. materials and methods probiotic: a commercial probiotic l. plantarum (kc426951) isolated from the intestinal tract of rainbow trout was used in this study. the fish fed with commercial diet (faradaneh, iran) supplemented with l. plantarum as the probiotic cells suspension that has added in the feed as 1 g (108 cells g-1)∕ kg feed. fish culture and experimental design: juveniles of rainbow trout weighting 29.6±1.84 g were obtained from a local hatchery in arak, iran. the fish were acclimatized for two weeks in the laboratory condition in a 1000-l tank with a flow-through system (7 l min-1) at 16±1.5°c and were fed a commercial diet (faradaneh, iran). after checking the health status, acclimatized fish were randomly distributed into twelve 1000-l tanks, four groups each in three replicate with 80 fish per replicate. two groups of fish were immunized with streptococcosis/lactococosis vaccine (soltani et al., 2007) in bathing route for 1 min under aeration. the other two groups were considered as unvaccinated fish. one of each vaccinated and unvaccinated group were fed with diet containing l. plantarum (1 g/kg food), while the other two groups were fed without l .plantarum. protocol of experimental groups studied is given in table 1. on 12, 24, 36, 48 and 60 days of the experiment, nine fish from each replication were anesthetized with clove powder solution (200 mg l-1), and blood was collected from caudal vein puncture. then, serum biochemical and some immune parameters analysis were carried out on blood samples. during the feeding trial, the fish were fed three times a day (09:00, 12:00 and 15:00) at rate of 2% of body weight. the feeding rate was corrected every 12 days following a 24 hrs starvation period and batch weighing. the experiment was carried out for 60 days. water quality parameters including water temperature, ph and dissolved oxygen were monitored daily and were maintained at 16±1.5ºc, 7.5±0.5 and 8±0.4 mg/l, during the experimental period, respectively. serum biochemical parameters assay: serum biochemical analysis, including alkaline phosphatase (alp), cholesterol, total protein, and albumin levels were measured by a commercial kit according to the manufacturer protocol (parsazmon trial immunization regime feeding regime feeding rate 1 streptococcosis/lactococosis vaccine l. plantarum (1 g/ kg food) 2% 2 streptococcosis/lactococosis vaccine commercial diet without probiotic 2% 3 unvaccinated l. plantarum (1 g/kg food) 2% 4 unvaccinated commercial diet without probiotic 2% table 1. protocol of experimental groups studied in the present study. 287 int. j. aquat. biol. (2016) 4(4): 285-294 co. iran). serum total igm levels were measured with an enzyme-linked immunosorbent assay (elisa) using a commercial kit (cusabio, wuhan, hubei, china), as described by sun et al. (2010). immune parameters leukocytes: to assess leukocytes, appropriate blood smears were prepared from blood samples which were obtained on the 12, 24, 36, 48 and 60th days of the experiment. smears were then air-dried, fixed in 96% ethanol for 30 min and stained with giemsa for 30 min. the smears were examined for total leukocyte counts, lymphocytes, and heterophils under a compound microscope (klontz, 1994). serum lysozyme activity: lysozyme activity was measured based on ellis (1990) with a slight modifications carried out. alternative complement activity (ach50): alternative complement activity assays was carried out following the procedure of yano (1992) using rabbit red blood cells (rarbc). the calculation of the complement activity was carried out using below equation: ach50 value (units m l -1) =1/k × (reciprocal of the serum dilution) × 0.5 where k is the amount of serum (ml) giving 50% lyses and 0.5 is the correction factor since this assay is performed on the half scale of the original method. agglutination antibody titer: agglutinating antibody titer to s. iniae were measured using the microagglutination test (roberson, 1990). statistical analysis: the mean and sem were determined for 27 fish per each treatment. all the parameter/treatment time elapsed (days) 12 24 36 48 60 alp (u dl-1) control 210.7 ± 46a 220.7 ± 32a 249.3 ± 35a 287.7 ± 33a 320.7±39a p 246 ± 38a 287.3 ± 32a 353 ± 29b 403.7 ± 37b 453.7 ± 31bc v 383.67±33b 491±48b 567±49c 490.67±54c 437.67±38b v+ p 384.33±43b 570±47c 607±42c 584.67±31d 514.33±47b total protein (g dl-1) control 2.58±0.3a 2.98±0.22a 3.07±0.26a 3.49±0.15a 3.25±0.2a p 2.81±0.23a 3.14±0.21a 3.27±0.21a 3.44±0.22a 3.51±0.27ab v 3.88±0.34b 4.26±0.17b 3.95±0.16b 3.84±0.18b 3.92±0.23b v+ p 3.93±0.28b 4.35±0.25b 4.21±0.22b 4.05±0.26b 4.1±0.21b albumin (g dl-1) control 1.5 ± 0.18a 1.61±0.16a 1.46±0.18a 1.5±0.09a 1.44±0.08a p 1.52 ± 0.23a 1.65±0.15a 1.4±0.1a 1.63±0.06a 1.79±0.1b v 1.47 ± 0.17a 1.75±0.11a 1.62±0.1a 1.58±0.08a 1.49±0.11a v+ p 1.56 ± 0.16a 1.54±0.26a 1.69±0.13a 1.62±0.11a 1.43±0.15a igm (mg ml-1) control 1.68±0.28a 1.95±0.46a 2.02±0.4a 2.29±0.37a 2.25±0.33a p 1.79±0.33a 1.96±0.3a 2±0.36a 2.21±0.46a 2.7±0.25ab v 2.77±0.57b 3.68±0.31b 4.08±0.36b 3.53±0.54b 3.31±0.46b v+ p 2.68±0.46b 3.96±0.47b 4.29±0.41b 3.8±0.29b 3.57±0.36b cholesterol (mg dl-1) control 215±40a 292±10a 262±14a 295±14a 279±20ab p 207±36a 286±17a 218±25a 237±13a 217±24a v 213±30a 295±16a 229±10a 268±31a 245±36a v+ p 233±22a 293±9a 220±19a 271±22a 311±11b p: fish fed with probiotic (l. plantarum), v: fish immunized with streptococcus/lactococosis vaccine, p+v: fish fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine, control: fish fed with commercial diet. values (mean±se, n=27) containing different superscripts in the same row denotes significant difference between the treatments (p<0.05). table 2. serum biochemical parameters in rainbow trout (oncorhynchus mykiss) fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine. 288 kane et al./ effect l. plantarum on vaccinated rainbow trout data were analyzed using one-way anova by spss package (spss 1998) at the 0.05 significance level. results no mortality was observed during the experiment. the water quality of each group did not change during the experiment due to daily renewing of water. serum biochemical parameters: the results of serum biochemical parameters are presented in table 2. the alkaline phosphatase (alp) levels of all treatments were significantly higher (p<0.05) than that of the control group after 36, 48 and 60 days. moreover, this value was significantly higher in vaccinated trout with/without l. plantarum after 12 and 24 days of the experiment. however, its levels changed differently only in vaccinated fish fed probiotic on day 12 (p<0.05). after 60 days of feeding, the vaccinated fish fed the diet supplemented with l. plantarum showed significantly higher cholesterol than the control group (p<0.05). the total protein and igm levels of vaccinated trout with/without l. plantarum were significantly higher than those of the control group on all sampling day of the experiment (p<0.05). however, no significant difference was found in albumin value in treatment groups compared to the control group (p>0.05). immune parameters leukocytes: total leukocyte counts (fig. 1) and lymphocyte values (fig. 2a) in immunized trout with streptococcosis/lactococosis vaccine with/ without feeding diets containing l. plantarum after 12, 24, 36, 48 and 60 days were significantly higher than the control group (p<0.05). in contrast, the level of heterophil (fig. 2b) was lower in the treated groups compared to the control o (p<0.05). however, no significant changes were found in total leukocyte counts, lymphocytes and heterophil levels of fish fed the diet supplementation of l. plantarum (p>0.05). lysozyme activity: the results of serum lysozyme figure 1. total leukocyte counts in rainbow trout fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine at 16±1.5°c, for 60 days (p: fish fed with probiotic (l. plantarum), v: fish immunized with streptococcus/lactococosis vaccine, p+v: fish fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine, control: fish fed with commercial diet). values (mean±se, n=27) containing different superscripts in the same row denotes significant difference between the treatments (p<0.05). 289 int. j. aquat. biol. (2016) 4(4): 285-294 figure 2. leukocyte differential count (a: lymphocyte, b: heterophil) in rainbow trout fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine at 16±1.5°c, for 60 days (p: fish fed with probiotic (l. plantarum), v: fish immunized with streptococcus/ lactococosis vaccine, p+v: fish fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine, control: fish fed with commercial diet). values (mean±se, n=27) containing different superscripts in the same row denotes significant difference between the treatments (p<0.05). 290 kane et al./ effect l. plantarum on vaccinated rainbow trout figure 3. lysozyme levels in rainbow trout fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine at 16±1.5°c, for 60 days (p: fish fed with probiotic (l. plantarum), v: fish immunized with streptococcus/lactococosis vaccine, p+v: fish fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine, control: fish fed with commercial diet). values (mean±se, n=27) containing different superscripts in the same row denotes significant difference between the treatments (p<0.05). figure 4. ach50 values in rainbow trout fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine at 16±1.5°c, for 60 days (p: fish fed with probiotic (l. plantarum), v: fish immunized with streptococcus / lactococosis vaccine, p+v: fish fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine, control: fish fed with commercial diet). values (mean±se, n=27) containing different superscripts in the same row denotes significant difference between the treatments (p<0.05). 291 int. j. aquat. biol. (2016) 4(4): 285-294 activity are shown in figure 3. lysozyme activity of vaccinated trout fed with l. plantarum was significantly higher (p<0.05) than the control group after 60 days of feeding. moreover, its value increased significantly for 12 and 24 days of the experiment in vaccinated trout without feeding dietary l. plantarum (p<0.05). nonetheless, no significance change was observed in unvaccinated fish that was only fed probiotic (p>0.05). alternative complement pathway activity (ach50): the results of ach50 are shown in figure 4. the serum ach50 of both vaccinated groups with/ without feeding l. plantarum diet was significantly higher (p<0.05) than the control group after 12, 24, 36, 48 and 60 days of feeding. however, no significant changes were found in ach50 in fish fed the diet supplementation of l. plantarum (p>0.05). antibody titer: the results of antibody titer are shown in figure 5. antibody titer to s. iniae in both of vaccinated groups with/without l. plantarum feeding was gradually decreased from day 12 to 60 days after the vaccination but it was significantly higher than control one up to 48 days of feeding. the highest antibody titer was found in vaccinated trout that fed diet l. plantarum with no significantly different with vaccinated fish fed with probiotic; the value was significantly different from the other groups (p<0.05). discussion the probiotics have been recognized to function as immune-modulators in finfish which is often through stimulation of innate and cellular immunity, including enhanced phagocytic, lysozyme, respiratory burst, cytotoxicity, complement activity, superoxide dismutase, increased numbers of leukocytes, erythrocytes, monocytes and lymphocytes, migration of neutrophils, neutrophil adherence, antiprotease and peroxidase activities, and plasma bactericidal activity (newaj-fyzul and austin, 2015). in addition, there may be increases in serum bacterial agglutination antibody titer (ridha and azad, 2012), albumin (humoral immunity) and total igm levels (sharifuzzaman and austin, 2010a, 2010b). nonetheless, various probiotics may show different type of immune response. in the present study, serum biochemical parameters, including alp, cholesterol, serum total figure 5. agglutination antibody titers in rainbow trout fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine at 16±1.5°c, for 60 days (p: fish fed with probiotic (l. plantarum), v: fish immunized with streptococcus/lactococosis vaccine, p+v: fish fed with probiotic (l. plantarum) and immunized with streptococcus/lactococosis vaccine, control: fish fed with commercial diet). values (mean±se, n=27) containing different superscripts in the same row denotes significant difference between the treatments (p<0.05). 292 kane et al./ effect l. plantarum on vaccinated rainbow trout protein and total igm values of vaccinated trout fed the probiotic were significantly higher compared to the control group. the alp is associated with the absorption of lipid, glucose, calcium and inorganic phosphate (eguchi, 1995). in this study, the alkaline phosphatase (alp) levels of all treatment groups were significantly higher than the control group. increased phosphatase activity indicates a higher breakdown of the energy reserve, which may be utilized for the enhancement of growth or immunity (ghosh et al., 2008), as the result of vaccination and/or fed l. plantarum diet. furthermore, higher levels of cholesterol were shown in vaccinated trout fed l. plantarum diet, after 60 days of feeding, which may be due to the increased activity of liver enzymes like the alp. this indicates the disorders of lipid and lipoprotein metabolism (allen et al., 2005). in the present study, the highest levels of serum igm were observed in vaccinated trout fed with l. plantarum after 36 days of feeding. in spite of the slight decrease, its levels increased significantly more than the control group after 60 days of feeding. nonetheless, serum total igm levels in unvaccinated trout fed l. plantarum diet did not show a significant change compared to control group. in contrast, dietary supplement-ation of bacillus in grouper until 30 days (sun et al., 2010), and in rainbow trout fed dietary l. rhamnosus (jcm 1136) up to 20 days (panigrahi et al., 2005) have shown an increase in igm levels trend of feeding, and thereafter a dropping pattern prevailed. the total protein levels of vaccinated trout with/without feeding diets containing l. plantarum were significantly higher than those of the control groups on all sampling days of the experiment. the increase in serum total protein content might be due to an increase in the leukocytes, which is a major source of serum protein production, including complement factors, lysozyme, and bactericidal peptides (misra et al., 2006). this is supported by increase in leukocytes value in the both vaccinated groups. probably, the increase in the leukocyte count might have resulted in the enhancement of the nonspecific immunity. besides, the total leukocyte count, lymphocytes increased in both mentioned groups. meanwhile, heterophil were significantly decreased compared to the control group, similar to pervious study by soltani et al. (2007) who evaluated these parameters in immunized trout with streptococcosis/lactococosis vaccine. significant higher serum lysozyme activity and alternative complement activity (acp50), and serum antibody titers were shown in both vaccinated groups with/without feeding dietary l. plantarum, compared to the control groups. while these parameters did not increase in fish that were fed only probiotic, l. plantarum, in lysozyme and ach50 activity simulation of l. plantarum has been previously showed in different studies (son et al., 2009; giri et al., 2013). the discrepancy of these findings may be attributable to the difference in probiotic doses and the feeding duration. beside, vaccines have been shown to increase serum lysozyme activities, alternative complement activity and antibody titers in rainbow trout (kim and austin, 2006; soltani et al., 2007), which suggests an enhancement of these parameters due to using vaccine. in conclusion, the present results demonstrated that the administration of dietary l. plantarum (1 g/ kg food) in vaccinated trout against streptococcosis /lactococosis can induce some of the specific and non-specific immune responses. this appears to be obtained by increasing antibody titer, lysozyme activity, alternative complement activity and some serum biochemical parameters such as igm levels. to our knowledge, this is the first study that investigated a positive probiotic dietary in vaccinated trout. acknowledgement this work was financially supported by a grant (no. 32.6.7508002) from research council of the university of tehran and center of excellence of aquatic animal health, university of tehran, and financial support no.9912 provided by the ministry of higher education and scientific research, university of kufa, najaf, iraq. http://scialert.net/fulltext/?doi=pjbs.2014.451.461#1119076_ja 293 int. j. aquat. biol. 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(2016) 4(5): 345-352: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article assessment of fish fauna in relation to biodiversity indices of chalan beel, bangladesh md. abu bakar siddique, muhammad afzal hussain, fawzia adib flowra, mohammad manjurul alam*1 department of fisheries, university of rajshahi, rajshahi 6205, bangladesh. article history: received 2 july 2016 accepted 21 september 2016 available online 2 5 october 2016 keywords: fish fauna shannon-weiner margalef’s richness pielou’s index abstract: the research was carried out to enlist the fish species diversity along with diversity indices of existing fish species of chalan beel, bangladesh. during the study, a total of 78 fish species including 69 native and 9 exotic fish species were recorded belonging 10 orders and 26 families. the values of shannon-weiner diversity index (h') indicated a good spread of fish population in chalan beel although the number of species was not found satisfactory. the degradation of water quality due to domestic discharges, jute rotting, excessive usage of agro-chemicals, indiscriminate fishing activity etc. were revealed as the causes of declining the fish diversity of this beel. the margalef’s richness index (d) expressed the species richness of chalan beel that started in july by joining new fish population with the existing fish species and reached gradually highest in november after breeding. pielou’s index (j) showed the equal distribution pattern of fish species throughout the sampling area indicating a stable but incompatible habitat for existing fish species. the assessment of the study revealed the number of species was not found in expected level in view of the overall fish biodiversity of the country. considering the observations, it is recommended for stopping water pollution, ensuring normal water flow and developing awareness of fishermen to retrieve the fish diversity of the study area. besides, an inclusive management and conservation scheme is crying need for the beel fishery to enrich the fish species diversity of the chalan beel as well as the country. introduction a beel is a term for a lake-like wetland with static water in the ganges-brahmaputra flood plains of the eastern indian states of west bengal and asam; and in the country of bangladesh. a number of beels is existing northwest regions of bangladesh. the chalan beel is one of the largest inland depressions of marshy character and also one of the richest wetland areas of this country. it was originated about 2000 years ago when the brahmaputra diverted its water into the new channel of the jamuna (banglapedia, 2006). several decades ago, this beel was abundant with variety of fishes but in recent years like other water bodies of bangladesh, aquatic resources from this water body is decreasing to a large extent due to uncontrolled fishing and highly destructive devices of fish capture in beel deplete fisheries resources and are followed by great * corresponding author: mohammad manjurul alam e-mail address: mamillat@yahoo.com economic distress (karim, 2003). a number of research works have been done on different aspects of fisheries species diversity of chalan beel. hoq (2006) recorded 121 small indigenous fish species (sis), including 41 riverine, 29 migratory and 51 flood plain fish species; galib et al. (2009) found a total of 81 fish species, including 72 indigenous fish species and 9 exotic fish species; mostafa et al. (2009) recorded a total of 114 fish species; gillespie (2011) found 129 fish species and kostori et al. (2011) recorded a total of 82 sis fish species from chalan beel. the variation in number of species in the previous works noticeably ranged from 81 to 129 during the period from 2006 to 2011. and diversity in the two research works in 2009, a far difference is observed in the species between galib et al. (2009) and mostafa et al. (2009). that is why the research work is aimed to enlist the fish species diversity 346 siddique et al./ assessment of fish fauna in relation to biodiversity indices of chalan beel, bangladesh along with diversity indices of existing fish species of chalan beel to make some recommendations for its proper management and conservation. materials and methods duration and location of the study: the study was conducted from january 2015 to december 2015 to enlist fish species diversity and to calculate fish species diversity indices based on four main sampling areas of chalan beel. the biodiversity indices were calculated based on the fishing duration and fishing activity of the beel from july to december. the geographical positions of the four sampling areas are mentioned here as sampling area-1 (24°28'n, 89°14'e), sampling area-2 (24°39'n, 88°49'e), sampling area-3 (24°10'n, 89°20'e) and sampling area-4 (24°36'n, 89°37'e) (fig. 1). sampling procedure: both field and fish landing center based survey was carried out for collecting and enlisting fish species diversity fortnightly. fish species were collected from fishermen who use traditional fishing gear, nets and traps such as seine net (length, width and mesh size was 12.20 m, 1.25 m and 0.5 cm), lift net (length, width and mesh size was 45 cm, 5 m and 0.5 cm), bitti (length, width and height was 45 cm, 15 cm and 35 cm) and dohair (length, width and height was 45 cm, 25 cm and 40 cm) for sampling the biodiversity indices. the fishing nets operation time was considered for the morning (4.00 to 6.30 am) where one attempt of fishing was counted one sampling for seine net and 10 hauls counted as a one sampling for lift net. traps (bitti and dohair) were set up for overnight which counted one sampling. fish species collection and identification: specimens of recorded fish were collected from study areas and then identified based on the morphometric and meristics characteristics following talwar and jhingran (1991) and rahman (2005). identified species were preserved in 10% buffered formalin solution in leveled plastic jars. the abundance (vc=very common, c=common, r=rare and vr= very rare) and seasonal availability status of fish species (m=monsoon, pm=post monsoon, w= winter and as=all season) were categorized based on the catch records and interview of 336 fishermen from studied areas. fish species diversity indices: in the present study, the biodeversity indices viz. diversity index (h') by shannon-weiner (1949), species richness index (d) by margalef (1968) and species evenness index (j') by pielou (1966) were calculated as follows: h' =-sum [pi × log(pi)], where, h'=shannon-weiner index, pi=ni/n, ni= no. of indivduals of a species, and n=total number of individuals. d = (s-1)/log(n) where, s = total species, n = total individuals. j = h(s)/h(max) where, h(s)=the shannon-weiner diversity index, and h(max)=the theoretical maximum value for h(s) if all species in the sample were equally abundant. figure 1. the four sampling areas of chalan beel. 347 int. j. aquat. biol. (2016) 4(5): 345-352 analysis of data: descriptive analysis and graphical presentation of data were carried out using microsoft excel (version 2010). biodiversity indices were calculated using past (paleontological statistics) and spss (statistical analysis for social sciences, version 20) software. results and discussion status of fish fauna in chalan beel: during the study period, a total of 78 fish species including 69 native fish species belonging 10 orders and 26 families and 9 exotic fish species belonging 2 orders and 2 families recorded from chalan beel. the order and family based abundance and seasonal availability of each species are shown in the table 1. the findings are very much similar to the findings of galib et al. (2009) and kostori et al. (2011) who recorded 81 and 82 fish species respectively from chalan beel. but the findings varied with hossain et al. (2009) who reported a total of 114 fish species from chalan beel. order based distribution of fish fauna: in the present study, the dominant order considering the species number found as cypriniformes which contribute 34.78% of the total orders and then followed by siluriformes (30.43%), perciformes (18.84%), channiformes (5.80%), oteoglosiformes (2.90%) and the rest of the orders are occupied 1.45% (fig. 2). among the recorded families, the cyprinidae was the most diversified with 16 species and followed by bagridae (7) and schilbeidae (5). this finding is strongly agreed with the findings of kostori et al. (2011) and galib (2015). therefore, the present findings indicate that the chalan beel area is suitable habitat for minnows, barbs and catfishes. table 1. status of order and family based fish species abundance in the chalan beel area including seasonal availability. order family species name abundance seasonal availability native fish species beloniformes belonidae xenentodon cancila (hamilton, 1822) c w clupeiformes clupeidae gudusia chapra (hamilton, 1822) c as cypriniformes cyprinidae amblypharyngodon mola (hamilton, 1822) vc m catla catla (hamilton, 1822) c as cirrhinus mrigala (day, 1878) vc as cirrhinus reba (day, 1878) r m labeo bata (hamilton, 1822) c as labeo boga (hamilton, 1822) r as labeo calbasu (hamilton, 1822) vr as labeo rohita (hamilton, 1822) c m puntius chola (hamilton, 1822) vc w puntius conchonius (hamilton, 1822) vr pm puntius phutunio (hamilton, 1822) vc as puntius sophore (hamilton, 1822) vc as puntius ticto (hamilton, 1822) c pm puntius sarana (hamilton, 1822) c pm rohtee cotio (hamilton, 1822) vr pm salmostoma bacaila (hamilton, 1822) c pm rasborinae danio devario (day, 1878) vc pm esomous danricus (hamilton, 1822) c pm cobitidae botia dario (hamilton, 1822) c m botia lohachata (chaudhuri, 1912) r as lepidocephalus berdmorei (blyth, 1860) vc as lepidocephalus guntia (hamilton, 1822) r m balitoridae acanthocobities botia (hamilton, 1822) r w somileptus gongota (hamilton, 1822) vr m cypridontiformes aplocheilidae aplocheilus panchax (hamilton, 1822) c pm 348 siddique et al./ assessment of fish fauna in relation to biodiversity indices of chalan beel, bangladesh table 1. to be continued. order family species name abundance seasonal availability perciformes ambassidae chanda lala (hamilton, 1822) vc pm chanda nama (hamilton, 1822) c pm chanda ranga (hamilton, 1822) c w anabantidae anabus testudineus (bloch, 1792) c m gobidae glossogobius giuris (hamilton, 1822) c w osphronemidae colisa fasciata (bloch and schneider, 1801) c w colisa lalia (hamilton, 1822) c w trichogaster chuna (hamilton, 1822) r w nandidae nandus nandus (hamilton, 1822) c m mastacembelidae mastacembelus armatus (lacepede, 1800) vc m mastacembelus pancalus (hamilton-buchanan, 1822) c m macrognathus aculeatus (bloch, 1786) r pm pristolepidae badis badis (hamilton, 1822) c w channiformes channidae channa orientalis (bloch and schneider, 1801) r w channa marulius (hamilton, 1822) vc w channa punctata (bloch, 1793) c w channa striata (bloch, 1793) c as siluriformes bagridae chandramara chandramara (hamilton, 1822) vr as mystus aor (hamilton, 1822) vr as mystus cavasius (hamilton, 1822) r as mystus menoda (hamilton, 1822) r as mystus seenghala (sykes, 1839) vc as mystus tengara (hamilton, 1822) c w rita rita (hamilton, 1822) c w claridae clarias batrachus (linnaeus, 1758) c m heteropneustidae heteropneustes fossilis (bloch, 1794) c m pangasiidae pangasius pangasius (hamilton, 1822) vr w schilbeidae ailia coila (hamilton, 1822) vr w ailia punctata (day, 1871) r m clupisoma garua (hamilton, 1822) vr m eutropiichthys vacha (hamilton, 1822) c w pseudeutropius atherinoides (bloch, 1794) vr w chacidae chaca chaca (hamilton, 1822) c as siluridae ompok pabda (hamilton, 1822) r as ompok bimaculatus (bloch, 1794) vc w wallago attu (bloch and schneider, 1801) r w sisoridae gagata cenia (hamilton, 1822) r pm glyptothorax telchitta (hamilton, 1822) vr pm synbranchiformes synbranchidae monopterus cuchia (hamilton, 1822) vr w osteoglossiformes notopteridae notopterus chitala (hamilton-buchanan, 1822) c w notopterus notopterus (pallas, 1769) vr p m tetradontiformes tetradontidae tetradon cutcutia (hamilton, 1822) c as exotic fish species cypriniformes cyprinidae aristichthys nobilis c as ctenopharyngodon idella c as cyprinus carpio var. communis c as cyprinus carpio var. specularis c as hypopthalmichthys molitrix c as oreochromis mossambicus c as oreochromis niloticus c as puntius goninotus c as siluriformes pangasiidae pangasius hypophthalmus c w 349 int. j. aquat. biol. (2016) 4(5): 345-352 abundance and seasonal availability status: the abundance of native fish species were categorized into four statuses which observed as very common (17.39%), common (43.48%), rare (20.29%) and very rare (18.84%) (fig. 3a). the study focusing the declining trend of the existing fish population considering the percentage of rare and very rare species. flowra et al. (2013) observed a total of 60 species in baral river which has direct linkage with chalan beel and also found 45% available, 33.33% less available, 13.33% rare and 8.33% very rare fish species from this river. but, herein between two findings the percentage of rare and very rare species differed noticeably. the findings indicated some probable factors or barriers that are responsible for this abundance status. during the study period the highest number (31.43%) of fish species were found in winter (november-december) season (fig. 3b), because success of beel fishery mainly depends on the water volume of the habitat and the fishermen can easily catch the fish by netting, trapping and even dewatering. the highest catch of chalan beel was found in december where kostori et al. (2011) observed in september-october and karim (2003) reported maximum abundance in monsoon period. fish diversity indices: the diversity index indicates correlation with overall species richness and figure 2. order based percentage distribution of native fish species of chalan beel. figure 3. abundance and seasonal availability status of indigenous fish species of chalan beel. 350 siddique et al./ assessment of fish fauna in relation to biodiversity indices of chalan beel, bangladesh evenness across the sampling areas and could be utilized by the biodiversity conservation managers for prioritization of study areas of conservation and habitat restoration. shannon-weiner diversity indices (h'): shannonweiner diversity (h') index considers both the number of species and the distribution of individuals among species of chalan beel. this index takes into account the number of individuals as well as number of taxa. the value of h' usually falls between the values 1.5 to 3.5 and it rarely surpasses the value of 4.5. a value near 4.6 would indicate that the numbers of individuals are evenly distributed between all the species. during the study period, the highest value of h' was found as 3.15 (december) in sampling area2 and the lowest value was found as 2.54 (july) in sampling area-4 (fig. 4). in each case of the highest shannon-weiner, diversity index is involved with high individuals and the lowest diversity involved with low number of individuals. the main causes of the findings occurring in the biodiversity indexes are seasonal variations of nutrients, effectiveness of breeding activities, affecting the coexistence of many fish species, atmospheric air currents, environmental conditions and seasonal fish migrations. the average highest h' value of chalan beel was found as 3.05 in december and lowest was found as 2.74 in july and august as a whole. this finding is closely related with the findings of iqbal et al. (2015) who recorded h' value from 2.90 to 3.12 in konoskhaihaor in sunamgonj. murugan and prabaharan (2012) recommended that low diversity (h') occurs in pre monsoon due to shrinkage of water spread of the water body and the highest diversity occurs in post monsoon due to sufficient water and ample food in resources. therefore, these findings strongly agreed with the present findings and also expressed that the recorded value of h' both monthly and every sampling area indicates a good spread of species diversity in chalan beel. but, biligrami (1988) recommended better condition of water body for fish diversity when h' index ranged from 3.0-4.5. this mean chalan beel is slightly degraded which has negative impact on decline the fish diversity. margalef’s richness index (d): margalef’s richness is the simplest measure of biodiversity and is simply a count of the number of different species in a given area. this measure is strongly dependent on sampling size and effort. in this study, the lowest and highest margalef richness index value was observed as 4.03 in july and 9.58 in december at sampling area-2 respectively (fig. 5). the average highest and lowest richness (d) value in the entire chalan beel was recorded as 8.39 in december and 4.53 in july respectively. most of fish species start their breed from july when the monsoon begin in bangladesh which may be the reason behind the lowest and highest richness value during july and december. as a result, numbers of new individuals joined the fish stocks increased the species richness up to december. galib et al. (2013) calculated fish species richness value in the choto jamuna river of bangladesh and found values varied from 6.973 (june) to 8.932 (november). the margalef’s index may deviate from actual diversity value to some extent because it does not confound the evenness and species richness value properly and it is depend on sample size (nair et al., 1989). this may occur as a result of reduced water depth due to lack of rainfall, which disturbed fishermen to employ their fishing gears more effectively (iqbal et al., 2015). in addition, ecological conditions also have an effect on the distribution of the fish species. equitability or pielou's evenness index (j): equitability is an evenness measure index which measures the evenness which individuals are divided among the taxa present. during the study period, the recorded highest evenness (j) value was found in july as 0.69 in sampling area-1 and the lowest as 0.25 (november) in sampling area-2 (fig. 6) whereas the average highest and lowest value was recorded as 0.62 in july and 0.29 in november in the whole chalan beel (fig. 7). therefore, the species equitability index among the sampling area and in the different months reveals that the distribution of species or fish population of chalan beel is equally distributed. the values are also close to those reported by emmanuel and modupe (2010) for river 351 int. j. aquat. biol. (2016) 4(5): 345-352 ore, hence, the category of water body is different. murugan and prabharan (2012) found highest evenness value (0.99) in late monsoon indicating on evenly distributed and rich fauna in the monsoon and post monsoon. conclusion: bangladesh is one of the diversified countries with respect to freshwater fish species (260) in the world. as only 69 native fish species were recorded from chalan beel which representing the overall fish biodiversity status of chalan beel indicating a question mark in respect of number of total species. the value of h' indicating good spread of fish population in entire chalan beel as well as poor water quality that may cause by the domestic discharge, excessive uses of insecticides and pesticides in cultivation of the beel and adjacent lands. on the other hand, indiscriminate and excessive fishing is one of the major causes for declining the native fish species from this beel. a good management and conservation scheme is badly recommended to enrich the species diversity of this prominent waterbody of the country. acknowledgements authors are grateful for all logistic support to the sub project cp-3557 of the department of fisheries, university of rajshahi under higher education quality enhancement project (heqep), university grant commission (ugc) funded by the world bank. references banglapedia. (2006). national encyclopedia of figure 4. sampling area based shannon-weiner indices. figure 5. sampling area based margalef's richness indices. figure 6. sampling area based pielus's evenness indices. figure 7. the average biodiversity indices of chalan beel. 352 siddique et al./ assessment of fish fauna in relation to biodiversity indices of chalan beel, bangladesh bangladesh, asiatic society of bangladesh, 1st edn. february 2006. dhaka, bangladesh. available from url: www.banglapedia.org. biligrami k.s. (1988). biological monitoring of rivers, problems and prospect in india. aquatic ecotoxicology, 245-250. emmanuel l.o., modupe o.o. (2010). fish diversity in three tributaries of river ore, south west, nigeria. world journal of fisheries and marine science, 2(6): 524-531. flowra f.a., islam m.a., jahan s.n., hussain m.a., alam m.m., bashir f.a., mazlan a.g., simon k.d. (2013). status and decline causes of fish diversity of baral river, natore, bangladesh. accl bioflux, 6(4): 352-357. galib s.m. (2015). fish fauna of the brahmaputra river, bangladesh: threats and conservation needs. international journal of biodiversity and conservation, 3(3): 285-292. galib s.m., naser s.m.a., mohsin a.b.m., chaki n., fahad m.f.h. (2013). fish diversity of the river choto jamuna, bangladesh. present status and conservation needs. international journal of biodiversity and conservation, 5(6): 389-395. galib s.m., samad m.a., mohsin a.b.m., flowra f.a., alam m.t. (2009). present status of fishes in chalan beel–the largest beel (wetland) of bangladesh. international journal of animal and fisheries science, 2(3): 214-218. gillespie p. (2011). climate change impacts, community perceptions and adaptation practices by rice-growing communities in bangladesh. sikkha sastha unnayon karzakram (education, health and development program), pesticide action network, 10850 penang, malaysia, pp: 4-15. hoq e. (2006). bangladesher choto mach. published by graphic sign, 8 gkmc shah road, choto bazar, mymensingh. 20 p. hossain m.a.r., nahiduzzaman m., sayeed m.a., azim m.e., wahab m.a., lin p.g. (2009). the chalan beel in bangladesh: habitat and biodiversity degradation and implication for future management. lakes and reservoirs: research and management, 14: 3-19. iqbal m.m., kanon m.h., hossain m.a., hossain a., nasreen s., islam m.j., rahman m.a. (2015). diversity of indigenous fish species in konoskhaihaor, northeast bangladesh. pujab university journal of zoology, 30 (2): 073-079. karim m.s. (2003). discussion on the causes of reduction of fisheries resources in chalan beel, matsha pakkha. pp: 95-96. (in bengali) kostori f.a., parween s, islam m.n. (2011). availability of small indigenous species (sis) of fish in the chalan beel the largest wetland of bangladesh. university journal of zoology, 30: 62-72. margalef r. (1968). perspectives in ecological theory. chicago ii. university of chicago press, chicago. 111 p mostafa a.r., hossain m., nahiduzzaman m., sayeed a., azim, m.e., wahab m.a., olin p.g. (2009). the chalan beel in bangladesh: habitat and biodiversity degradation and implications for future management. lakes and reservoirs: research and management, 14: 3-19. murugan a.s., prabaharan c. (2012). fish diversity in relation to physico-chemical characteristics of kamala basin of darbhanga district, bihar, india. international journal of pharmaceutical and biological archives, 3(1): 211-217. nair n.b., arunachalam m., nair m.k.c., suryanarayanan h. (1989). seasonal variation and species diversity of fishes in the neyyar river of the west ghats. tropical ecology, 30(1): 69-74. pielou e.c. (1966). species diversity and pattern diversity in the study of ecological succession. journal of theoretical biology, 13: 131-144. rahman a.k.a. (2005). freshwater fishes of bangladesh. 2nd edition, zoological society of bangladesh, department of zoology, university of dhaka, dhaka. 263 p. shannon c.e., weiner w. (1949). the mathematical theory of communication. urbana, il: university of illinois press, urbana. 117 p. talwar p.k., jhingran a.g. (1991). inland fishes of india and adjacent countries. vol. 1 and 2. oxford and ibh publishing co. pvt. new delhi, calcutta, india. 1158 p. int. j. aquat. biol. (2018) 6(1): 8-14 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article comparison of meristic traits in transcaucasian chub (squalius turcicus de filippi, 1865) from caspian sea basin atta mouludi-saleh, yazdan keivany*,1seyed amir hossein jalali department of natural resources (fisheries division), isfahan university of technology, isfahan 84156-83111, iran. article history: received 10 september 2017 accepted 2 january 2018 available online 2 5 february 2018 keywords: cluster analysis kolmogorov-smirnov meristic morphology abstract: for comparison of meristic characters of squalius turcicus, in 12 rivers of caspian sea basin, 535 specimens were captured. some 14 meristic characters were counted. classification of meristic characters showed that most specimens of all populations have 8 soft dorsal rays, 9 soft anal rays, 19 branched caudal fin rays, 15 soft pectoral rays, 9 soft pelvic rays, 41-47 lateral line scales, 7-9 scales above ll, 3-5 scales below ll, 18-21 predorsal scales and 14-16 circumpeduncle scales. the results showed significant differences (p<0.05) in means of all meristic characters except dorsal, pelvic and pectoral fin spins between the populations. the pca and cva showed overlapping among the populations, although some populations were separated from the others. also, cluster analysis divided divandareh river population in a separate group and it was distinct from other populations. generally, the results of meristic characters cannot well-separate the populations of this species from each other. introduction transcaucasian chub, squalius turcicus formerly known as s. cephalus in the iranian inland waters is a cyprinid species distributed in the caspian sea and urmia lake basins (keivany et al., 2016a; esmaeili et al., 2017). this species lives in the middle and upper parts of rivers with relatively cool water and cobblestone bottoms. turan et al. (2007) studied the morphological variation in s. cephalus, across turkish inland waters. mouludi-saleh et al. (2016b, c, 2017a) studied the morphology of s. namak populations in rivers of the namak lake basin. poria et al. (2014) studied morphometric and meristic characteristics of s. cephalus in the shohaday-e-songhor dam lake. gorjian arabi et al. (2011) surveyed morphological diversity of s. cephalus in the talar river, mazandaran province. babazadeh and vatandoost (2013) surveyed morphometric and meristic characteristics of s. cephalus in tajan river, mazandaran province. alizadeh et al. (2015) studied morphological variation of s. orientalis in the southern caspian sea basin. despite its wide distribution, there is no comprehensive work on the species. *corresponding author: yazdan keivany doi: https://doi.org/10.22034/ijab.v6i1.317 e-mail address: keivany@cc.iut.ac.ir recently, özuluğ and freyhof (2011) suggested that s. turcicus de filippi, 1865 might be a valid species occurring in the southern caspian sea basin and turan et al. (2013) supported this view and provided some morphological data distinguishing this species from s. orientalis. khaefi et al. (2016) suggest that s. orientalis and s. turcicus are very closely related and might represent just one species. squalius turcicus might be more widespread and squalius populations of the urmia lake and caspian sea basins might belong to this species (esmaeili et al., 2017). herein, we followed esmaeili et al. (2017) and consider the southern caspian sea species as s. turcicus. the aim of this study was to evaluate the diversity of meristic characters in populations of s. turcicus in the caspian sea basin for possibility of finding distinct populations. materials and methods during 2010-2011 from 16 rivers of the caspian sea basin, including babol, palangab, tajan, talvar, chalak, divandareh, zarin, zalkie, sefid, shafa ghezel-ozan, kasma, tonekabon, neka, noor and 9 int. j. aquat. biol. (2018) 6(1): 8-14 haraz rivers (fig. 1), 535 specimens were collected by a seine net. after anesthetizing with 1% clove oil solution and fixing in 10% neutralized formalin, specimens were transferred to the isfahan university of technology ichthyology museum (iut-im) for further studies. some 14 meristic characters were counted under a stereomicroscope (tables 1, 2, 3). the data were analysed for normality using kolmogorov-smirnov test and non-normal data were analyzed by kruskal-wallis test. these analyses were carried out using excel 2013 and spss 19 for windows at 95% confidence limit. significantly different data were used for principal component analysis (pca), canonical variate analysis (cva) and cluster analyses (ca) in past software. results according to the results, all data were not normal. except dorsal, pelvic and pectoral fin spins, all other 11 examined characters were significantly different among the populations. the significant characters were used for pca, cva and cluster analyses. dorsal spins were 2-3 in all specimens from different rivers and their soft rays ranged 7-10. there were significant differences between some populations (p<0.05) (table 1). anal spins were 2-3 in all specimens from different basins and their soft rays ranged 8-10. there were significant differences between some populations (p<0.05) (table 1). the principal caudal rays ranged 18-21 and a significant differences found between basins (p<0.05) (table 2). table 1. minimum-maximum, mean±sd and frequency of each count (%) of the dorsal and anal fin rays of squalius turcicus from different caspian sea basin tributaries in iran, collected during summer 2010-2011. dorsal soft rays frequency of each count (%) anal soft rays frequency of each count (%) rivers min-max mean±sd 7 8 9 10 min-max mean±sd 25 52 10 babol 8-10 9.31±0.71 0 16 37 47 8-10 8.97±0.70 18 32 23 palangab 8-10 9.14±0.59 0 10 64 26 8-10 9.32±0.77 22 56 50 tajan 8-10 8.83±0.71 0 34 48 18 8-10 9.00±0.67 7 79 22 talvar 8-9 8.50 ±0.52 0 50 50 0 8-10 9.07±0.47 0 56 14 tonekabon 8-10 8.81±0.75 0 38 44 18 9-10 9.44±0.51 0 88 44 chalak 8-9 8.11±0.32 0 88 12 0 9-10 8.11±0.32 25 65 12 divandareh 8-10 8.40±0.68 0 70 20 10 8-10 8.85±0.59 54 46 10 zarin 8 7.00±0.00 0 100 0 0 8 8.33±0.49 92 4 0 zalkie 8-9 8.11±0.32 0 11 89 0 8-10 8.12±0.44 67 33 4 sefied 7-9 7.89±0.47 17 78 5 0 8-9 8.33±0.49 62 38 0 shafa 8-9 8.23±0.44 0 38 62 0 8-9 8.23±0.44 43 67 0 gheze-ozan 8-9 8.63±0.49 0 37 63 0 8-9 8.57±0.50 54 46 0 kasma 8-9 8.03±0.19 0 3 97 0 8-9 8.47±0.51 59 31 0 neka 8 8.00±0.00 0 100 0 0 8-9 8.41±0.50 71 29 0 noor 8 8.00±0.00 0 100 0 0 8-9 8.29±0.46 59 31 0 haraz 8-9 8.00±0.00 0 97 3 0 8-9 8.23±0.44 42 46 0 total 7-10 8.47±0.69 1 61 30 8 8-10 8.69±0.66 25 52 12 figure 1. collection points of squalius turcicus in the caspian sea basin. 10 mouludi-saleh et al./ comparison of meristic traits in squalius turcicus the pectoral spiny rays were 0-1 in the examined specimens and their branched rays ranged 14-17. the pelvic spiny rays were 1-2 in the examined specimens and their rays ranged 7-9 in all populations. a significant differences was found between some of the rivers for the pectoral rays (p<0.05) (table 4). the scales below the lateral line ranged 3-5, on the lateral line 41-47, above the lateral line 7-9, predorsal 18-21 and circumpeduncle 12-16 in the examined specimens. there was a significant differences between some of the rivers (p<0.05) (table 4). according to pca, 52.83% of the variance were accounted for first two components positioned above the jolliffe line and circumpeduncle scales along the two main axes of pc1 and pc2 had the highest variation (fig. 2). according to the classification of the populations (fig. 3), the studied populations partially overlap and do not fully differentiate from each other. although some populations (ghezel-ozan, kasma, sefid and talvar) are separated. the results of table 2. minimum-maximum, mean±sd and frequency of each count (%) of the caudal fin rays of squalius cephalus from different caspian sea basin tributaries in iran, collected during summer 2010-2011. caudal rays frequency of each count (%) rivers min-max min-max 18 19 20 21 babol 18-21 18-21 2 51 35 13 palangab 18-20 18-20 7 79 14 0 tajan 18-20 18-20 10 78 12 0 talvar 18-20 18-20 14 64 22 0 tonekabon 18-20 18-20 6 82 12 0 chalak 19-20 19-20 0 74 26 0 divandareh 18-20 18-20 10 85 5 0 zarin 18-19 18-19 20 80 0 0 zalkie 18-19 18-19 12 88 0 0 sefied 18-20 18-20 6 88 6 0 shafa 18-20 18-20 7 61 32 0 ghezel-ozan 18-20 18-20 4 93 3 0 kasma 19-21 19-21 0 86 7 7 neka 18-19 18-19 2 98 0 0 noor 18-19 18-19 2 98 0 0 haraz 18-20 18-20 3 3 14 80 total 18-21 18-21 5 80 12 3 table 3. minimum-maximum and mean±sd of the scales in squalius turcicus from caspian sea basin during 2010-2011. scales below ll ll sclaes scales above ll predorsal sclaes circumpeduncle scales rivers minmax mean±sd minmax mean±sd minmax mean±sd minmax mean±sd minmax mean±sd babol 3-4 3.79±0.58 41-45 43.29±0.6 3 7-8 7.97±0.16 18-20 18.79±0.66 14-15 14.01±0.12 palangab 3-4 3.68±0.48 43-46 43.96±0.9 2 8-9 8.22±0.15 18-19 18.32±0.48 14-16 14.21±0.63 tajan 4-5 4.43±0.50 42-46 43.33±1.0 0 7-8 7.97±0.18 18-20 18.24±0.50 14 14.00±0.00 talvar 3-4 3.43±0.51 43-47 44.14±1.1 0 7-8 7.36±0.50 18-20 19.07±0.92 14-15 14.14±0.53 tonekabon 3-4 3.19±0.40 43-46 43.88±1.0 2 8-9 8.38±0.25 18-20 18.50±0.52 14-16 14.75±1.00 chalak 3-4 3.06±0.24 42-47 43.79±1.2 2 7-8 7.97±0.17 18-20 18.57±0.70 14-16 14.74±0.98 divandareh 3-4 3.35±0.75 43-45 43.90±0.9 1 7-8 7.30±0.47 18-21 19.05±0.94 14-15 14.05±0.60 zarin 4-5 4.13±0.35 43-46 43.80±0.7 7 7-8 7.60±0.51 18-19 18.53±0.52 14-16 14.53±0.92 zalkie 3-4 3.80±0.82 42-46 43.80±1.0 8 7-8 7.20±0.41 18-21 18.88±0.83 14-16 14.48±0.77 sefied 4-5 4.89±0.32 43-46 43.67±0.9 7 7-8 7.17±0.38 18-20 18.50±0.62 14-16 15.00±0.97 shafa 4-5 3.77±0.60 43-46 43.77±1.1 7 7-8 7.23±0.44 18-20 18.77±0.73 14 14.00±0.00 ghezel-ozan 4-5 4.69±0.47 43-45 44.02±0.8 5 7-8 7.55±0.85 18-20 18.76±0.52 14-16 15.10±1.01 kasma 3-4 3.97±0.18 44-47 45.87±0.9 7 7-8 7.27±0.45 18-21 18.90±0.76 14-16 15.20±1.00 neka 3-4 3.69±0.47 44-47 44.59±0.7 0 7-8 7.18±0.39 18-20 18.88±0.56 14-16 14.18±0.57 noor 4-5 4.29±0.51 43-47 44.81±0.8 7 7-8 7.02±0.14 18-20 18.69±0.55 14-15 14.73±0.74 haraz 3-4 3.07±0.26 44-46 44.59±0.5 7 7-8 7.14±0.35 18-19 18.31±0.47 14 14.00±0.00 total 3-5 3.60±0.71 41-47 44.03±1.1 0 7-9 7.55±0.59 18-21 18.65±0.66 14-16 14.42±0.78 11 int. j. aquat. biol. (2018) 6(1): 8-14 table 4. minimum-maximum, mean±sd and frequency of each count (%) of the pectoral and pelvic fin rays of squalius turcicus from different caspian sea basin tributaries in iran, collected during summer 2010-2011. pectoral fin rays frequency of each count (%) pelvic fin rays frequency of each count (%) rivers min-max mean±sd 14 15 16 17 min-max mean±sd 7 8 9 babol 14-16 15.12±0.77 24 40 36 0 9 9.00±0.00 0 0 100 plangab 14-16 15.07±0.90 32 32 36 0 9 9.00±0.00 0 0 100 tajan 14-17 15.25±0.97 26 35 28 11 8-9 8.65±0.48 0 39 61 talvar 14-16 15.29±0.61 7 57 36 0 9 9.00±0.00 0 0 100 tonekabon 14-15 14.69±0.48 5 95 0 0 8-9 8.56±0.51 0 43 67 chalak 14-16 14.49±0.61 57 37 6 0 8-9 8.37±0.49 0 66 34 divandareh 15-16 15.55±0.51 0 45 55 0 8-9 8.80±0.41 20 80 zarin 14-15 14.33±0.49 67 33 0 0 8 8.00±0.00 0 100 0 zalki 14-16 14.84±0.75 36 44 20 0 8-9 8.88±0.33 0 8 92 sefied 14-15 14.33±0.49 67 33 0 0 8-9 8.28±0.46 0 72 28 shafa 14-16 15.00±0.82 31 38 31 0 8-9 8.62±0.51 0 38 62 gheze-ozan 14-16 15.00±0.61 18 63 19 0 8-9 8.49±0.51 0 50 50 kasma 14-16 15.17±0.53 6 70 24 0 8-9 8.87±0.35 0 13 87 neka 14-15 14.51±0.51 49 51 0 0 8-9 8.43±0.50 0 57 43 noor 14-16 14.65±0.53 37 61 2 0 8-9 8.40±0.49 0 65 35 haraz 14-15 14.62±0.49 38 62 0 0 8-9 8.86±0.35 0 10 90 total 14-17 14.89±0.74 32 42 18 8 8-9 8.64±0.47 0 35 65 figure 2. the role of the first character along the two axes. 12 mouludi-saleh et al./ comparison of meristic traits in squalius turcicus cva showed significant differences amongst the populations and little overlap can be observed. although some populations (palangab, talvar, zari and zalkie) are well-separated from each other by great distances (fig. 4). in the cluster analysis, six major clusters were recognized (fig. 5). kasma population was the most distinct population, and other groups were tajan+palangab+babol+tonekabon, divandareh+talvar, shafa+haraz+noor+neka+ zalkie, chalak+zari, and sefid+ ghezel-ozan. discussion the meristic characters of fishes such as scales, fin rays, gill rakers and pharyngeal teeth are genetically controlled, while, morphometric characters such as lengths and their ratios are highly affected by environmental factors (keivany et al., 2016b). the final number of meristic characters on fish depends on prevailing environmental conditions during early development of the individuals. fish populations in habitats with different environmental conditions, will figure 3. the result of pca of meristic characters of the studied populations in the caspian sea basin. figure 4. the results of cva of meristic characters of the studied populations in caspian sea basin. 13 int. j. aquat. biol. (2018) 6(1): 8-14 show a high morphological diversity (keivany et al., 2016c). although there are significant differences in mean values of most meristic characters, but they are highly overlapping. it seems that genetic differentiations among the studied populations are not sufficient to fully separate the populations from each other (keivany et al., 2012). besides, these characters are affected by size and environmental conditions (khara et al., 2006; daneshvar et al., 2013). the results separated some studied populations (such as ghezel-ozan, kasma, sefid and talvar) based on meristic data using pca that this method used in other studies as well (keivany et al., 1997, 2015; patimar et al., 2010). also, in cva, there were overlaps, however, some populations were separated. in cluster analysis, divandareh river population was clustered in one group and was separated from other populations and had the highest distinction. in the current study the dorsal spiny and soft rays were 2-3, 7-10, anal spiny and soft rays 3, 8-10, caudal fin rays 18-21, pelvic soft rays 8-9, pectoral soft rays 14-17, circumpeduncle scales 12-16, predorsal scales, 18-21, lateral line sclaes41-47, scales above ll 7-9 and scales below ll 3-5. in other studies conducted in iran, the dorsal spines and soft rays of this species were 2-3, 7-9, anal spines anal soft rays 2-3, 7-10, caudal fin rays as 18-21, pelvic 6-9, pectoral soft rays 13-19, circumpeduncle scales 12-16, predorsal scales 18-21, lateral line scales 38-48, scales above ll 5-7 and scales below ll 7-8 (abdoli and naderi, 2009; dadashpour ahangari et al., 2011; alizadeh et al., 2015). for s. cephalus in europe, dorsal spines and soft rays were reported as 3, 8, anal soft rays 7-9, pectoral spine and soft rays 1, 14-17, pelvic spine and soft rays 2, 8, lateral line scales 42-48, scales above ll 7-9, scales below ll 3-5, (steindachner, 1895; karaman, 1924; drensky, 1951; libosvarsky, 1956; banarescu, 1964; dimovski and grupce, 1972; ivanovi, 1973; economidis, 1974; georgieve, 2000). in general, the figures in this study are consistent with previous studies for this species and for s. cephalus complex in other regions (e.g., mouludisaleh et al., 2017b). thus, the meristic characters do not well-defined characters in s. cephalus senso lato populations (in iran s. turcicus, s. orientalis and s. berak) due to their low variation and overlapping. acknowledgements we would like to thank s. dorafshan, m. nasri, s. asadollah, a. nezamoleslami and a. mirzaei for their help in field work and m. zamani-faradonbe for his help in laboratory. this research was financially supported by isfahan university of technology. references abdoli a., naderi m. 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(2018) 6(1): 8-14 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی خزر حوضه در( squalius turcicus de filippi, 1865) ایرودخانه سفید ماهی شمارشی هایویژگی مقایسه جاللی امیرحسین سید ،*کیوانی یزدان صالح، مولودی عطا علی .ایران ،۸۴۱۵۶۸۳۱۱۱ اصفهان اصفهان، صنعتی دانشگاه طبیعی منابع دانشکده شیالت گروه چکیده: ۱۴نمونه صید گردید. حدود ۵۳۵رودخانه حوضه خزر، ۱۲در squalius turcicus ایهای شمارشی ماهی سفید رودخانهبرای مقایسه ویژگی شعاع نرم مخرجی، ۹شعاع نرم پشتی، ۸ها دارای های جمعیتبندی صفات شمارشی نشان داد که اغلب نمونهویژگی شمارشی شمارش گردید. طبقه فلس زیر خط جانبی، ۳-۵فلس باالی خط جانبی، ۹-7فلس خط جانبی، ۴۱-۴7شعاع شکمی، ۹ای، شعاع نرم سینه ۱۵شعاع منشعب دمی، ۱۹ های شمارشی به در بین همه ویژگی( p >0۵/0) داریهای معنی. نتایج تفاوتهستندفلس دور ساقه دمی ۱۴-۱۶فلس جلوی باله پشتی و ۲۱-۱۸ ها همپوشانی نشان داد، گرچه برخی از جمعیت های اصلی و متغیرهای کانونی بینآنالیز مؤلفهای نشان داد. جز خارهای باله پشتی، مخرجی و سینه ها قرار داد. در ای جمعیت رودخانه دیواندره را در یک خوشه جداگانه و جدای از سایر جمعیتها از هم جدا بودند. همچنین، آنالیز خوشهجمعیت های این گونه را به خوبی از هم جدا نماید.تواند جمعیتمجموع، نتایج صفات شمارشی نمی .شناسی، ریختشمارشی هایویژگی اسمیرنو،-کولمولورو آزمون ای،خوشه آنالیز :کلمات کلیدی international journal of aquatic biology (2013) 1(6): 294-305 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article seasonal variation of zooplankton abundance, composition and biomass in the chabahar bay, oman sea neda fazeli *,1ahmad savari, seyed mohammad bagher nabavi, rasool zare khorramshahr university of marine science and technology. department of biology, khorramshahr, iran. article history: received 31 june 2013 accepted 10 december 2013 available online 2 5 december 2013 keywords: abundance biomass chabahar bay diversity monsoon zooplankton abstract: temporal and spatial variation of zooplankton abundance, composition and biomass were examined on the chabahar bay, oman sea. the chabahar bay, a subtropical and semi-enclosed bay, provides an ideal breeding ground for many fish and shellfish. five stations were investigated along the bay. this area is under the influence of the indian ocean seasonal monsoons. zooplankton was collected with vertical plankton tows using 100 µm mesh nets. copepods dominated the zooplankton community followed by larvacea, cladocera and chaetognatha. fifteen taxa of zooplankton were identified. oithona nana and euterpina acutifrons were dominated in the whole year and larvacea showed a bloom in northeast monsoon. a two-way anova indicated that there were differences in abundance and biomass between sampling periods and between stations were significant. the peak zooplankton abundance in ne monsoon could be due to winter cooling, with entrainment of nutrients into the upper layer producing phytoplankton blooms. the decline of zooplankton abundance and biomass in south west monsoon and post-monsoon could be explained by decrease in chlorophyll a concentrations. the present result showed the composition and distribution of zooplankton differed between the monsoon seasons, resulted from changes in hydrographic conditions. introduction the chabahar bay is a small semi-enclosed bay on the southeastern coasts of iran (25° 17' 45"n 60° 37' 45" e). the bay is connected to the indian ocean through oman sea. the effect of indian monsoonal winds on this area is remarkable (fazeli and zare, 2011). this bay is one of the five major ports in oman sea and provides an ideal breeding ground for many fish and shell fish (wilson, 2000). the bay is located between chabahar and konarak. it is 14 km wide and has a surface area of 290 km2.the average depth of the bay is 12 m (ranging from 8-22 m) (fazeli and zare, 2011). oman sea is located in the northwest of the arabian sea. the continental shelf of the region is widest off * corresponding author: neda fazeli e-mail address: neda_fazeli200@yahoo.com tel: +989172029800 the northwest coast of india, which also experiences wind-induced upwelling. the asia monsoon in oman sea is characterized by two distinct seasons separated by two transition (inter-monsoon) periods: the southwest (sw) monsoon from june through september and the northeast monsoon (ne) from december through march with the spring transition (pre-monsoon) occurs in april and may and the fall transition (post-monsoon) occurs in october and november, respectively. southwesterly flow has a direct effect on the arabian sea, coast of oman, oman sea, the arabian sea, coast of iran, southern pakistan, and india (caulfield, 1990). although the information of the chabahar bay is rare, the effect of monsoon has been studied well in 295 fazeli et al/ international journal of aquatic biology (2013) 1(6): 294-305 the region. in sw monsoon, a peak in chlorophyll a biomass dominates in the indian ocean (yoder et al., 1993). lower salinity water reach the sea surface in coastal upwelling that occurs in this season along the coast of oman (morrision et al., 1998). prominent features along the oman coast generated during the sw monsoon tend to linger in place during the fall transition. during the autumnal inter-monsoon, water temperatures decreases slowly. average sea temperatures decreases from 28 and 29 °c in october to 27 °c in november (caulfield, 1990). oligotrophic conditions return slowly, but during the northeast monsoon winter cooling may imply the entrainment of new nutrients into the upper layer during windy periods, resulting blooms of phytoplankton (baars, 1998). the cool, dry northeasterly winds that characterize the ne monsoon result in a typical winter time convection/nutrient enrichment scenario in the northern arabian sea (banse and mcclain, 1986; madhupratap et al., 1996). evaporative cooling increases the salinity of the surface waters, which further promotes the convective mixing (wiggert et al., 2000). donguy and meyers (1996) defined a surface water mass with a salinity between 35.5-36.5 ppt and temperatures greater than in the 22 °c in the arabian sea waters. the productivity observed during the ne monsoon was much higher than anticipated (mahdupratap et al., 1996b). convective mixing during the ne monsoon (weller et al., 1998; wiggert et al., 2000) turned out to be an important source of nutrients. water temperatures decreases from 25 °c in december to 21 °c in february, then rise to 23 °c in march (caulfield, 1990). after the termination of winter cooling and subsequent warming during the spring inter-monsoon, when primary production drops, bacteria and microzooplankton proliferate. this suggests that the organisms of the microbial loop may be an important food source that sustains mesozooplanktons throughout this period (madhupratap et al., 1996), when water temperatures ranges from 25 °c in april to 29 °c in may (caulfield, 1990). importance of zooplankton in marine pelagic food webs as food for larval fish (jitlang et al., 2007) and as a good indicator of changes in water quality (gannon and stemberger, 1978). many studies have described zooplankton and copepod community structure in many parts of indian ocean, arabian sea and the persian gulf (madhupratap, 1987; smith, 1995; baars, 1998; savari et al., 2004; madhu et al., 2007) but there is little published information on them in the chabahar bay (wilson, 2000). the propose of the present investigation is to describe the zooplankton community in the chabahar bay oman sea, and provide an estimation of abundance, composition, biomass and their distribution influenced by monsoons. materials and methods sampling was performed in august 2007 (south west monsoon), november 2007 (post-monsoon), figure 1. map of sampling stations in the chabahar bay. 295 296 fazeli et al/ international journal of aquatic biology (2013) 1(6): 294-305 february 2008 (north east monsoon), and may 2008 (pre-monsoon). five stations were selected in the chabahar bay (fig. 1): two stations (stations 1 and 2) were located far from shore waters (22 m depth), while the other two (stations 3 and 5) were near the shore (6 m depth), and the final station (station 4) was located in the middle of the bay (12 m depth). quadruple plankton samples were collected vertically at each station using a simple net (with a mouth diameter of 30 cm and a mesh size of 100 μm), with a hydrobios flow meter mounted in the center of the net opening to estimate volume of the water, from the quadruple samples, two were used for counting and the other two for biomass measurement. the samples were immediately preserved in 4-5% formalin, buffered to a ph of 8 with sodium tetra borate (borax), and identified to the lowest taxa possible. zooplankton abundance was expressed as individuals m-³ (somoue et al., 2005). at each station, prior to taking a plankton sample, the environmental parameters were recorded using ctd profiler lowered from the sea surface to near the bottom. zooplankton was identified to species level using the keys of chen et al. (1965), nishida (1985), krishnapillai (1986) and conway et al. (2003). dry weight was calculated after drying organisms for 20 hours at 60 °c (edmondson and winberg, 1971). weights were estimated using a microbalance mettler mt5. single large organisms > 1 cm (fish, large crustaceans and medusae) were weighed separately and were not included in the biomass data (böttger-schnack, 1990). the pearson correlation was performed to determine the significant relationship between environmental parameters and zooplankton abundance and biomass. species diversity was calculated using shannon–weaver diversity index (shannon and weaver, 1949) and species richness (margalef, 1968). the data were further subjected to hierarchical clustering analysis to identify the similarity between stations based on the composition of zooplankton. using bray-curtis similarity index with log10 (x+1) data transformation using primer version 5.2.8 (clarke and warwick, 1994). results environmental variables: seasonal changes in environmental parameters are shown in figure 2. the mean of water temperature varied from (20.52 ± 0.20 °c) in ne monsoon to (29.92 ± 0.05 °c in sw monsoon. the mean of salinity ranged from (36.70 ± 0.06) in sw monsoon figure 2. seasonal variation of major environmental parameters in the chabahar bay (x axis as seasons and y axis as environmental parameters) (sw-m=sw monsoon, post-m=post-monsoon, ne-m=ne monsoon, pre-m=pre-monsoon). 297 fazeli et al/ international journal of aquatic biology (2013) 1(6): 294-305 to (36.91) in pre-monsoon. the minimum and maximum value of chlorophyll α concentrations were noticed (0.77 ± 0.08 mg.m-3) to (1.84 ± 0.92 mg.m1-3) in sw monsoon and ne monsoon, respectively. zooplankton abundance and biomass: seasonally, mean abundance of zooplankton varied considerably (fig. 3). the highest abundance of zooplankton was in ne monsoon (5728 ± 337.20 individuals m-³) and the lowest was in post-monsoon (2453 ± 480.14 individuals m-³). dry weight biomass was observed highest in pre-monsoon (101.10 ± 13.11 mg dry wt. m-³) compared to other periods. post-monsoon was characterized by lowest dry weight biomass value of 30.80 ± 6.54 mg dry wt. m-³. near shore stations (3 and 5) showed maximum zooplankton abundance and dry weight biomass, while offshore stations (1 and 2) showed the lowest (fig. 3). taxa composition: figure 4 shows total identified zooplankton in the chabahar bay. copepods dominated the zooplankton community followed by larvacea, cladocera and chaetognatha. the other dominant groups comprised ostracoda, siphonophora, decapoda and thalliacea. other taxa were rarely encountered. copepods were the dominant group during four seasons, reaching 1254 ± 302.65 individuals m-3 during pre-monsoon, 613 ± 326.35 individuals m-3 during sw monsoon, 594 ± 54.11 individuals m-3 during post-monsoon and 890 ± 161.7 individuals m3 in ne monsoon. copepod species belonging to 18 families were recorded during the investigation period (table 1). some species were observed only in one season with lowest abundance (< 25 individuals m-3) such as sapphirina gastrica, sapphirina nigromoculata, figure 3. temporal and spatial variation of zooplankton abundance and dry-weight in the chabahar bay. 297 298 fazeli et al/ international journal of aquatic biology (2013) 1(6): 294-305 lucicutia flavicornis, lucicutia gaussae that only occurred during post-monsoon. oithona fallax, paracandacia truncata and euchaeta marina were observed only during ne monsoon. bestiolina similis and delibus nudus just appeared during premonsoon. euterpina acutifrons, macrosetella gracilis and microsetella rosea were observed in highest abundance during pre-monsoon. temora turbinata were greater during pre-monsoon and ne monsoon than other seasons. moreover, pseudodiaptomus marinus increased remarkably during ne monsoon but was rarely observed in other seasons. larvacea: a bloom of larvacea was noticed in ne monsoon while it completely disappeared during pre-monsoon. among them oikopleura longicauda was the most dominant species. their abundances ranged from 1136 to 16778 individuals m-3. cladocera: cladocera was present all year round but in large number (5104.11 individuals m-3) in ne monsoon. the highest abundance was found in station 3 (8471.80 individuals m-3). their abundances ranged from 166 to 15389 individuals m3. evadne sp. was dominant during four periods but showed highest abundance in ne monsoon at station 3. chaetognatha: the most dominant chaetognatha species was sagitta enflata and sagitta sp. and thrived best in post-monsoon. maximum number of chaetognatha was found at station 4 with a mean of 1349 individuals m-3. other dominant groups: siphonophora occurred throughout the year but were most abundant during pre-monsoon. they were represented by bassia bassensia. siphonophora abundance ranged from 1 figure 4. relative abundance (individuals m-³) of dominant groups of zooplankton in the chabahar bay. 299 fazeli et al/ international journal of aquatic biology (2013) 1(6): 294-305 to 2653 individuals m-3; the maximum value was found at station 5 with an average of 1124 individuals m-3. ctenophora was rare and present in low number ranging from 0 to 60 individuals m-3. they were completely absent in pre-monsoon. ctenophora was not observed at station 5. among thaliaceans, doliolum sp. was common in distribution. the maximum number of thaliaceans was found at station 5 (mean of 957 individuals m3). their abundances ranged from 45 to 2653 individuals m-3. polychaete larvae ranged from 1 to 3301 individuals m-3, and reached to the highest abundance in premonsoon (average of 1084 individuals m-3). ostracoda showed highest abundance (472 individuals m-3) in post-monsoon and disappeared in ne monsoon. the lowest abundance was observed at station 4 (25 individuals m-3) and completely absent at station 5. amongst mollusks, the most dominant gastropoda were atlanta helicinoides. pterotrachea hippocampus was oly observed in station 4 and only during pre-monsoon. crustacea (copepods, decapods, ostracods and euphausids) were highly encountered in premonsoon as compared to other periods. they comprised ~80% of the zooplankton community in this season. echinodermata were rare in distribution and consisted of auricularia larvae and ophiopluteus larvae. zooplankton diversity and composition: zooplankton species showed wide variations in diversity (0.49–1.87) and richness (0.49–1.27). highest diversity and richness values were observed figure 5. diversity indices (h') and species richness (d) of zooplankton (sw m=sw monsoon, post-m=post-monsoon, ne m=ne monsoon, prem=pre-monsoon). 299 301 fazeli et al/ international journal of aquatic biology (2013) 1(6): 294-305 siphonophora copepoda family proboscidactyla family paracalanidae proboscidactyla stellata parvocalanus crassirostris fmily diphyidae p.elegans diphyes spp. paracalanus parvus family abylidae acrocalanus longicornis enneagonum hyalinum a.gracilis bassia bassensia a.monachus ctenophora a.gibber family pleurobranchiidae paracalanus aculeatus pleurobranchia sp. bestiolina similis family bolinopsidae delibus nudus bolinopsis family acartiidae appendicularia acartia pacifica family fritillaridae a. erythraea .fritillaria sp a. longiremis family oikopleuridae family candaciidae oikopleura longicauda paracandacia truncata decapoda family clausocalanoidea family luciferidae clausocalanus furcatus lusifer typus c. gracilis l. sp. c. minor thaliacea family calocalanidae family salpidae calocalanus plumulosus salpa sp. family centropagidae family doliolidae centropages tenuiremis doliolum sp. family pseudodiaptomidae bivalvia pseudodiaptomus marinus brachiopod larvae family oncaeidae polychaeta oncaea media family tomoperidae o. venusta tomopteris sp. o. clevei family lopadorhynchidae family sapphirinidae maupasia gracilis sapphirina gastrica cladocera s.nigromoculata family podonidae family ectinosomatidae evadne sp. microsetella rosea podon sp. subeucalanus crassus echinodermata subeucalanus monachus auricularia larvae pareucalanus attenuatus ophioplutes larvae family lucicutiidae cephalochordata lucicutia flavicornis branchiostoma sp. l. gaussae family euphausiidae family euchaetidae nematoscelis sp. euchaeta marina euphausia sp. family oithonidae vertebrata dioithona aculata fish larvae oithona attenuata ostracoda o. nana family halocypridae o. brevicornis conchoecia sp. o. simplex chaetognatha o. plumifera family sagittidae o. fallax sagitta enflata family corycaeidae s. sp. corycaeus pacificus gastropoda c. andrewsi family atlantidae c. asiaticus atlanta helicinoides c. affinis munthaea sp. c. dahlia family euterpinidae euterpina acutifrons family miraciidae macrosetella gracilis family clytemnestridae clytemnestra scutellata table 1. list of zooplankton species collected in the chabahar bay. 300 300 fazeli et al/ international journal of aquatic biology (2013) 1(6): 294-305 at stations 1 and 2 (off shore stations) compared to the other stations. peak diversity occurred in postmonsoon and the lowest was in ne monsoon. the highest and lowest richness was observed in postmonsoon and pre-monsoon respectively (fig. 5). the cluster analyses indicated a relatively high degree of homogeneity in the zooplankton composition between stations 2 and 4 during a year (fig. 6). the similarity was lowest between stations 2-4 and station 5. environmental parameters and zooplankton: it seems chlorophyll α, temperature and depth are major factors controlling distribution of zooplankton in the chabahar bay. pearson correlation showed a positive relationship between chlorophyll α value and zooplankton and copepod abundance (table 2). total zooplankton abundance and copepod indicated a positive relation with dry weight biomass. among zooplankton groups, a negative relationship was observed between larvacea and temperature, and between ctenophora and biomass. siphonophora negatively related to depth. also, a positive relationship was observed between depth and abundance of ostracoda. discussion the first objective of this study was to document seasonal and spatial variation of marine condition in the chabahar bay. we found temperature and chlorophyll α in the bay were seasonally structured. ne monsoon generally consists of moderate winddriven mixing, a net flux of heat from the ocean to the atmosphere, and elevated evaporation (wiggert et al., 2000). thus, it appears nutrients transported via this wintertime mixing fuel the subsequent spring phytoplankton bloom as defined by the appearance chlorophyll α in chabahar bay which confirmed the record of kumar and prasad (1999) in the arabian sea. high temperature was observed in sw monsoon resulted in high sunlight. in post-monsoon and ne monsoon temperature showed decreasing trend through the bay by decreasing in sunlight. salinity did not vary significantly during warm and cold season as evaporation is high during cold season (wiggert et al., 2000). our findings in water temperature, chlorophyll α and salinity in the chabahar bay were similar to the record of caulfield (l990) in oman sea and arabian sea. the second objective of this study was to understand zooplankton link to the changing environmental parameters affected by monsoon in the chabahar bay. in ne monsoon, high zooplankton abundance occurred in the chabahar bay. positive relationship between chlorophyll α value and zooplankton abundance suggests that the abundance is regulated by food availability (e.g. phytoplankton) similar to other tropical waters (yoshida et al., 2006; mahdu, 2007). baars (1998) stated that entrainment of nutrients into the upper layer by winter cooling caused producing phytoplankton blooms. furthermore, temperature fluctuations are good indicators of the variability of nutrient input. several days after a decrease in temperature, the enrichment of water lead to intensive primary and secondary production (roy, 1992). thus, the population rises to a higher level in the winter as a result of favorable environmental conditions, including temperature, dissolved oxygen and the availability of abundant food in the form of bacteria, nanoplankton and suspended detritus (salve and hiware, 2010). surprisingly, biomass of zooplankton in this season figure 6. cluster analyses showing similarity of stations based on zooplankton composition during the whole year. 301 302 fazeli et al/ international journal of aquatic biology (2013) 1(6): 294-305 was not high as much as expected. a bloom of larvacea, which is not unexpected in indian ocean region (tsujimoto et al., 2006), noticed in this season and increased abundance of zooplankton but had insignificant effect on biomass. in pre-monsoon, abundance of zooplankton was lower than ne monsoon while biomass was higher. the reason could be explained by the high abundance of crustacea which comprised ~80% of the zooplankton community in this season. furthermore, madhupratap and haridas (1990) stated that biomass is elevated by the presence of siphonophorea and medusa in indian ocean. the decline of zooplankton abundance and biomass in sw monsoon and post-monsoon may be explained by decrease in chlorophyll α concentrations. similar to the present result, ashjiana et al. (2002) reported highest biomass during pre-monsoon and ne monsoon, and the lowest during the sw monsoon and post-monsoon in arabian sea. in the present study, near shore stations showed higher abundance and biomass of zooplankton which confirmed the record of madhupratap and haridas (1990) and padmavati et al. (998), who reported a sharp decline in zooplankton abundance and biomass with depth. however, station 1 (an offshore station) was different from other stations based on several characteristics. the offshore currents could be responsible for an unexpected high abundance and biomass as it has been reported in omani coastal region and northern somalia by smith and bottero (1977) and smith and codispoti (1980). the third objective of this study was to understand seasonal succession of zooplankton in the chabahar bay. the variation of dominant zooplankton among four sampling seasons is apparently influenced by monsoon (chen, 1992). in sw monsoon, the summer population of total zooplankton falls due to a dilution effect that confirmed the record of salve and hiware (2010). however, high abundance of larvacea and calanoid copepod was remarkable. during autumn, by the prevailing post-monsoon chaetognatha and ostracoda were observed dominant. in ne monsoon, many organisms were increased in number such as copepods (oithona nana, temora turbinata, centropages tenuiremis), larvacea and cladocera. similarity, the ne monsoon is characterized by increased number of oithona as smith et al. (1998) mentioned, temora turbinata as madhupratap (1987) observed and cladocera as sharma and cyril (2007) reported. in pre-monsoon, harpacticoid copepod, decapods, ostracods, euphausids, siphonophors, gastropoda variables chla (mg m-³) temperature (°c) salinity (ppt) depth (m) zoo.biomass (mg dry wt. m-³) zooplankton 0.44* -0.31 -0.12 -0.15 0.52* copepoda 0.46* -0.06 -0.00 -0.02 0.79* larvacea 0.03 -0.56* -0.24 -0.25 -0.14 cladocera 0.43 -0.17 0.17 -0.21 0.29 chaetognatha 0.15 -0.15 -0.15 -0.26 -0.20 siphonophora 0.07 0.02 0.26 -0.47* 0.13 thalliacea 0.20 0.02 0.17 -0.24 -0.07 polychaeta -0.23 -0.05 -0.13 0.18 0.10 ostracoda 0.02 0.13 -0.22 0.55* -0.08 ctenophora -0.15 0.18 0.20 -0.03 -0.47* gastropoda 0.04 0.30 0.10 -0.18 -0.29 decapoda -0.37 0.04 0.00 0.01 -0.23 table 2. pearson correlation of environmental parameters and abundance of major zooplankton and biomass. 303 fazeli et al/ international journal of aquatic biology (2013) 1(6): 294-305 and polychaeta larvae were dominant which was similar to the record of geetha et al. (1997). in post-monsoon, species richness of zooplankton increased with increasing population density. high diversity in this season reflected higher structured communities. the lowest richness was observed in ne monsoon that could be due to dominance of larvacea. spatially, mean zooplankton diversity and richness were found to be inversely related to zooplankton abundance. higher diversity value in offshore stations could be due to stable environmental conditions prevailing there which permitted plankton community to diversify (goswami et al., 1992). the swarming of zooplankton in coastal waters resulted in low diversity but high biomass community (madhupratap, 1987). acknowledgement we wish to thank dr. irina prusova for guidance and iranian national institute for oceanography for helpful comments. references ashjiana c.j., smith s.l., flaggc c.n. idrisi n. 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(2015) 3(5): 301-313 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article biodiversity of freshwater rotifers (rotifera: eurotatoria) of mizoram, northeast india: composition, new records and interesting features bhushan kumar sharma*,1sumita sharma freshwater biology laboratory, department of zoology, north-eastern hill university, shillong 793 022, meghalaya, india. article history: received 1 september 2015 accepted 5 october 2015 2015 available online 2 5 october 2015 keywords: composition biogeography interesting taxa richness sub-tropical waters abstract: the plankton and semi-plankton samples examined from mizoram state of northeast india (nei) revealed speciose and diverse rotifera assemblage including a total richness (s) of 162 species belonging to 19 families and 35 genera. the reports of six species new to india, four species new to nei and 76 new records to mizoram merit biodiversity interest. the occurrence of one australasian, one oriental, seven paleotropical, one holarctic, one cosmo (sub) tropical and five other interesting species imparts biogeographical value while several species indicate regional distribution importance. lecanidae > lepadellidae > brachionidae > trichocercidae collectively comprised 69.7% of total richness (s). lecane > lepadella > trichocerca are diverse genera (~52.0% of s) while brachionus spp. (~8.0%) deserve cautious mention. the rotifer diversity pattern is predominantly ‘tropical’ with a large component of cosmopolitans (~71.0% of s) while tropicopolitan and pantropical species contributed ~16.0%. this study indicated high richness of the littoral-periphytonic and relative paucity of planktonic taxa. analysis of periphytic, sessile, colonial and benthic taxa, and of cryptic diversity in certain species-groups merit attention for further biodiversity update and we estimate occurrence of 250+ rotifer species in mizoram. introduction mizoram, one of the hill-states of nei, has received little attention on aquatic metazoans diversity in general and on rotifera, an important group of fishfood organisms and an integral component of aquatic food-webs, in particular. initial reports of sharma (1987), sharma and sharma (1987a, 1987b), and sharma and sharma (1990) on the latter dealt with eleven species of lecanidae, three species of lepadella, one species of notommatidae and 10 species of brachionidae, respectively. recently, sharma and sharma (2014a) reported occurrence of 76 species and remarked on yet partially studied diversity of the taxon from this state while sharma and sharma (2014b, 2015) highlighted inadequately documented brachionidae and lepadellidae in particular, respectively. the present study, hence, attempts to provide a more exhaustive account of * corresponding author: bhushan kumar sharma e-mail address: profbksharma@gmail.com rotifera biodiversity of mizoram based on the recent sampling surveys. remarks are made on composition and richness of the observed diversity, various interesting elements and distribution of different taxa. materials and methods the present observations are based on water and plankton and semi-plankton samples collected, during four field surveys (january, march, july and october) in 2012 and one in march 2013, from all eight districts of mizoram (21°58'-24°35' n and 92°15'-93°29' e). a total of 330 samples were collected from varied aquatic ecosystems. water samples were examined for water temperature, specific conductivity and ph by the field probes; dissolved oxygen was estimated by winkler’s method while free co2, alkalinity, hardness and 302 int. j. aquat. biol. (2015) 3(5): 301-313 chloride were analyzed following apha (1992). the qualitative plankton samples were collected by towing a nylobolt plankton net (no. 25) and were preserved in 5% formalin. individual collections were screened with a wild stereoscopic binocular microscope; the rotifer taxa were isolated and mounted in polyvinyl alcohol–lactophenol, and observed with leica (dm 1000) stereoscopic phase contrast microscope fitted with an image analyzer. the measurements were given in micrometers (µm). various rotifera taxa were identified following the works of koste (1978), koste and shiel (1987, 1989, 1990), shiel and koste (1992, 1993), segers (1995), sharma (1983, 1987, 1998), sharma and sharma (1997, 1999, 2000, 2008, 2013). segers (2002) was followed for the system of rotifera classification and the remarks on distribution of various taxa were made following segers (2007) and jersabek and leitner (2013). the reference materials are in the holdings of freshwater biology laboratory, department of zoology, north-eastern hill university, shillong. results the variations (ranges, mean±sd) in the recorded abiotic parameters of the sampled water bodies are indicated in table 1. the collections from mizoram revealed 162 species of rotifera belonging to 19 families and 35 genera of eurotatoria (appendix i). of these, lecane aeganea (fig. 1a), testudinella walkeri (fig. 1b), trichocerca hollaerti (fig. 1c), t. maior (fig. 1d), t. siamensis (fig. 1e) and t. taurocephala (fig. 1f) are new records to india. brachionus leydigii (fig. 1g), lecane syngenes (fig. 1h), macrochaetus subquadratus (fig. 1i) and trichocerca edmondsoni (fig. 1j) are new records to northeast india (nei). in addition, 76 species are new additions to the rotifer fauna of mizoram. testudinella walkeri is an interesting australasian species; filinia camasecla (fig. 2a) is an oriental endemic. our collections included seven paleotropical species namely keratella javana (fig. 2b), lepadella discoidea (fig. 2c), l. vandenbrandei (fig. 2d), lecane lateralis (fig. 2e), l. unguitata (fig. 2f), testudinella greeni (fig. 2g), and trichocerca hollaerti; t. taurocephala is the holarctic species; and brachionus durgae (fig. 2h) is a cosmo (sub) tropical element. besides, lepadella elongata (fig. 2i) and testudinella amphora (fig. 2j) are other interesting species. discussion water temperature affirmed sub-tropical nature of the sampled ecosystems concurrent with their geographical location. the slightly acidic-circum neutral and ‘soft’ waters of mizoram are characterized by low ionic concentration as indicated by specific conductivity values; the latter warranted their inclusion under ‘class i’ category of trophic classification vide talling and talling (1965). the results exhibited well-oxygenated waters, low free co2 and low chloride content. various abiotic parameters concurred broadly with the report of sharma and pachuau (2013). one hundred and sixty-two species of rotifera, belonging to 35 genera and 19 families of eurotatoria, observed from mizoram revealed rich parameters ↓ range mean sd water temperature °c 12.0 28.0 17.4 4.2 specific conductivity µs cm-1 20.0 65.2 42.5 12.0 ph 5.60 6.98 6.70 0.24 dissolved oxygen mg l-1 4.0 10.2 6.7 2.1 free carbon dioxide mg l-1 5.0 17.6 12.1 3.2 alkalinity mg l-1 21.0 40.0 29.6 4.3 hardness mg l-1 19.0 38.6 31.2 6.8 chloride mg l-1 4.0 10.9 6.5 3.1 table 1. variations in some basic abiotic parameters. 303 sharma and sharma/ freshwater rotifers of mizoram (northeast india) figure 1. a, lecane aeganea harring (ventral view); b, testudinella walkeri koste & shiel (ventral view); c, trichocerca hollaerti de smet (lateral view); d, trichocerca siamensis segers & pholpunthin (lateral view); e, trichocerca maior hauer (lateral view); f, trichocerca taurocephala (hauer) (lateral view); g, brachionus leydigii cohn (ventral view); h, lecane syngenes (hauer) (ventral view); i, macrochaetus subquadratus (perty) (dorsal view); j, trichocerca edmondsoni (myers) (lateral view). 304 int. j. aquat. biol. (2015) 3(5): 301-313 figure 2. a, filinia camasecla myers (ventral view); b, keratella javana hauer (ventral view); c, lepadella discoidea segers (ventral view); d, lepadella vandenbrandei gillard (ventral view); e, lecane lateralis sharma (ventral view); f, lecane unguitata (fadeev) (ventral view); g, testudinella greeni koste (dorsal view); h, brachionus durgae dhanapathi (dorsal view); i, lepadella elongata koste (ventral view); j, testudinella amphora hauer (ventral view). 305 sharma and sharma/ freshwater rotifers of mizoram (northeast india) and diverse assemblage of the phylum. this salient feature is hypothesized to habitat diversity and environmental heterogeneity of the sampled aquatic ecosystems and merits biodiversity interest particularly in light of the scarcity of perennial lentic biotopes in sub-tropical environs of this hill-state of nei. six species namely lecane aeganea, testudinella walkeri, trichocerca hollaerti, t. maior, t. siamensis and t. taurocephala are new records to the indian rotifera. brachionus leydigii, lecane syngenes, macrochaetus subquadratus and trichocerca edmondsoni are new to nei and 76 species are new to the rotifer fauna of mizoram. interestingly, the documented species comprised ~ 22.0% and ~66.0% of the rotifer richness known from india (bks, unpublished) and nei (sharma and sharma, 2014a), respectively. this study marked more than two-fold increase in the species reported from mizoram (sharma and sharma, 2014a) and incidentally it represented the third richest rotifer diversity known till date from any state of india following the reports of 220 and 177 species from assam (sharma and sharma, 2014c) and tamil nadu (sharma and sharma, 2009), respectively. the stated features imparted special biodiversity value to the present study. the globally important species (~10% of s) included the australasian testudinella walkeri; the oriental endemic filinia camasecla; seven paleotropical species namely keratella javana, lepadella discoidea, l. vandenbrandei, lecane lateralis, l. unguitata, testudinella greeni and trichocerca hollaerti; the holarctic trichocerca taurocephala; cosmo (sub) tropical brachionus durgae and five other interesting species i.e., lepadella elongata, trichocerca edmondsoni, t. maior, t. siamensis and testudinella amphora; these assigned biogeographic importance to mizoram rotifera. of these, testudinella walkeri is an interesting addition of the first category to the fauna of nei (sharma and sharma, 2014a) and thus affirmed affinity of rotifera assemblage of this region as well as of mizoram with southeast asia and australia. this generalization supported earlier remarks of sharma (2005) and sharma and sharma (2005, 2008, 2013, 2014a, 2014c). lecane aeganea, a new record from india, is often confused with l. tensuiseta but differed from the latter in having shorter lorica and shorter toes. it is known from the oriental region from cambodia (meas and sanoamuang, 2010) and thailand (saardrit et al., 2013). testudinella walkeri is another addition to the indian rotifera. described from australia (koste and shiel, 1980), it is enlisted in several reports from thailand (sa-ardrit et al., 2013). the specimens from mizoram, however, agreed with those reported by segers and pholpunthin (1997). the present report thus extended the distribution of these two species to the indian sub-region. of the other new records to india, trichocerca hollaerti is characterized by a lateral keel over the entire body, by a head aperture with longitudinal folds and by having a single elongate right, and a sshaped, short left toe. this species resembled t. lophoessa f. carinata koste; the identity of latter cannot be ascertained (segers, 2003). we assign our specimens to t. hollaerti following segers and sarma (1993) and segers (2003). this paleotropical species is known (segers, 2007) from african, neotropical, pacific and oriental regions; the present report further extended its distribution within the last region. trichocerca maior (hauer), often considered t. porcellus f. maior, is characterized by distinct shape of its lorica and trophi, and was hence assigned the status of a distinct species by segers (2003); the latter treatment is followed in this account. this species is distributed in nearctic and palearctic regions (segers, 2007) while it is known from the oriental region by a number of workers from thailand (sa-ardrit et al., 2013); the present report extended distribution range of t. maior to the indian sub-continent. trichocerca siamensis, new addition to the indian rotifera, was described from thailand (segers and pholpunthin, 1997). considering the almost identical external morphology of the two species, it is likely 306 int. j. aquat. biol. (2015) 3(5): 301-313 to be confused with the holarctic t. uncinata while the tropical records are referred (segers, 2003) to belong to t. siamensis. sharma et al. (2015) recently reported t. uncinata from the floodplains of the majuli river island of the brahmaputra river basin; this report yet required examination in view of the stated distributional limits of the two species. trichocerca taurocephala (hauer), an interesting species new to india, deserved attention. koste and zhuge (1996) compared the specimens from china considered this species and identified the same as trichocerca pygocera (wiszniewski) while considering the former as its synonym. segers (2003) commented t. taurocephala after koste and zhuge (1996) to be possibly an unnamed taxon endemic to hainan, china (segers, 1998). the specimens from mizoram differed from trichocerca pygocera and the chinese material in shorter anterior spines and in the absence of characteristic posterior or caudal spike of lorica and are thus identified as t. taurocephala. the present study extended distribution ranges of brachionus leydigii, lecane syngenes, macrochaetus subquadratus and trichocerca edmondsoni to nei. the first species is validly known from delhi (arora and mehra, 2003) and panjab (sharma, 1980) while the reports from jammu and kashmir, maharashtra, tamil nadu and uttarakhand are unverifiable. lecane syngenes is reported till date from west bengal (sharma, 1979). macrochaetus subquadratus is known from kerala (kakkassery, 2003) while the report from andhra pradesh warranted validation. trichocerca edmondsoni is documented from kashmir (edmondson, 1938) as t. compressa (a synonym of the former) while segers (2003) remarked on the confirmation of this sole report outside the americas. the present report confirmed occurrence of this new world species in the indian sub-region and extended its distribution to eastern himalayas. the occurrence of eastern hemisphere species namely brachionus diversicornis, b. forficula, lecane lateralis, l. simonneae, l. unguitata, lepadella discoidea, l. vandenbrandei, and testudinella greeni is a noteworthy feature of mizoram rotifera. in addition, brachionus bennini, colurella adriatica, euchlanis meneta, lecane bifurca, l. doryssa, l. haliclysta, l. pusilla, l. tenuiseta, l. thienemanni, lepadella benjamini, l. costatoides, l. dactyliseta, l. elongata, l. quadricarinata, l. quinquecostata, macrochaetus longipes, mytilina acanthophora, platyias leloupi, testudinella amphora, t. tridentata, trichocerca bidens, t. scipio, t. tigris, t. weberi and wolga spinifera indicated regional distributional interest. of these, b. bennini, lepadella benjamini, l. elongata, l. vandenbrandei, trichocerca bidens, t. scipio, testudinella amphora and t. greeni are restricted till date to nei. testudinella amphora is reported from india from assam (sharma et al., 2015) while e. meneta and w. spinifera are recent additions to nei from the brahmaputra basin of assam state (sharma and sharma, 2014b). our collections are characterized by the stated order of importance of lecanidae > lepadellidae ≥ brachionidae > trichocercidae; these families comprised 69.7% of total rotifer richness (s) known from the state. their biodiversity importance concurred with the reports from the floodplains of thailand (sanoamuang, 1998), indian rotifera (sharma, 1996) and the oriental fauna (segers, 2008). their richness pattern primarily concurred with the rotifer fauna of nei (sharma and sharma, 2014a) but it differed in particular from assam rotifera (sharma and sharma, 2014b). euchlanidae = testudinellidae ≥ trichotriidae ≥ notommatidae are other notable families (15.5% of s). interestingly, the stated families, except brachionidae, included predominantly the littoralperiphytic taxa (segers, 2001). on the other hand, the general paucity of planktonic rotifers in mizoram collections is hypothesized to the scarcity of perennial lentic ecosystems in sub-tropical environs of this hill-state of nei, their usually shallow nature and to the lack of definite pelagic habitats (de manuel, 1994). incidentally, this study doubled richness of brachionidae than an earlier report of sharma and sharma (2014a); this feature as well as 307 sharma and sharma/ freshwater rotifers of mizoram (northeast india) brachionus spp. (~8.0%) desired cautious attention because of fewer planktonic species occurring in limnetic environs of certain fish ponds. lecane > lepadella > trichocerca are diverse genera (~52.7% of s) of mizoram rotifera. the relative importance of richness of these periphytic genera concurred with the report by sharma (2014) and endorsed the possibility of assemblage rules for the periphytic rotifer assemblages as hypothesized by green (2003). the importance of `tropic-centered’ lecane is distinct (24.8% of s), the richness of trichocerca (21 species, 13.0% of s) marked a significant increase than only eight species reported by sharma and sharma (2014a) while `tropiccentered’ brachionus (~8.0%) is rather limited. nevertheless, significance of lecane and to a lesser degree of brachionus affirmed the role of thermophiles in our collections concurrent with the rotifer fauna of southeast asia (segers, 2001). the said features along with occurrence of a large component of cosmopolitans (~71.0% of s) and of various tropicopolitan and pantropical species (~16.0%) impart a general ‘tropical character’ to the rotifer fauna of mizoram. these remarks are supported by low richness of `temperate-centered’ keratella and scarcity of ‘cold-water’ genera cephalodella and synchaeta. these generalizations are in conformity with the composition of the tropical faunas from different parts of the globe (green, 1972; pejler, 1977; fernando, 1980; de ridder, 1981; dussart et al., 1984; segers, 2001, 2008). to sum up, the rich and diverse mizoram rotifera is hypothesized to habitat diversity and environmental heterogeneity of the sampled ecosystems of this hillstate of nei which are characterized by the scarcity of perennial lentic biotopes and the lack of definite pelagic habitats in particular. various new records, globally interesting elements and eastern hemisphere species impart biodiversity and biogeographic value to this study. the rotifer fauna is predominantly of ‘tropical nature’ and is characterized by notable richness of the littoralperiphytic taxa and general paucity of planktonic rotifers. our collections are biased towards planktonic and semi-planktonic taxa and specific analysis of the periphytic, sessile, colonial and benthic taxa, and of cryptic diversity in certain species-groups merit interest for future biodiversity update. we estimate occurrence of 250+ rotifer species in mizoram state. acknowledgements the senior author (bks) is thankful to the ministry of environment and forests (govt. of india) for a research project no. 22018-09/2010-cs (tax) under its aicoptax program which facilitated the present sampling. thanks are due to the head, department of zoology, nehu, shillong for laboratory facilities and to mr. n noroh for the field collections. references a.p.h.a. 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(2015) 3(5): 301-313 family: brachionidae 1. anuraeopsis coelata de beauchamp, 1932 *** 2. a. fissa (gosse, 1851) 3. brachionus ahlstromi lindeman, 1939 *** 4. b. angularis gosse, 1851 5. b. bennini leissling, 1924 6. b. bidentatus anderson, 1889 *** 7. b. calyciflorus pallas, 1766 8. b. caudatus barrois & daday, 1894 s. lato 9. b. diversicornis (daday, 1883) *** 10. b. durgae dhanapathi, 1974 *** 11. b. falcatus zacharias, 1898 12. b. forficula wierzejski, 1891 *** 13. b. leydigii cohn, 1862** 14. b. rubens ehrenberg, 1838 *** 15. b. quadridentatus hermann, 1783 16. keratella cochlearis (gosse, 1851) 17. k. javana hauer, 1937 *** 18. k. tecta (gosse, 1851) *** 19. k. tropica (apstein, 1907) 20. platyias leloupi (gillard, 1967) *** 21. p. quadricornis (ehrenberg, 1832) 22. plationus patulus (o.f. muller, 1786) family: euchlanidae 23. beauchampiella eudactylota (gosse, 1886) 24. euchlanis. dilatata ehrenberg, 1832 25. e. incisa carlin, 1939 *** 26. e. meneta myers, 1930 *** 27. e. triquetra ehrenberg, 1838 28. dipleuchlanis propatula (gosse, 1886) 29. tripleuchlanis plicata (levander, 1894) *** family: mytilinidae 30. lophocharis salpina (ehrenberg, 1834) 31. mytilina acanthophora hauer, 1938 *** 32. m. bisulcata (lucks, 1912) *** 33. m. ventralis (ehrenberg, 1830) family: trichotriidae 34. macrochaetus collinsi (gosse, 1867) 35. m. subquadratus (perty, 1850) ** 36. m. longipes myers, 1934 *** 37. m. sericus (thorpe, 1893) 38. trichotria tetractis (ehrenberg, 1830) 39. wolga spinifera (western, 1894) *** family: lepadellidae 40. colurella adriatica ehrenberg, 1831 *** 41. c. colurus (ehrenberg, 1830) *** 42. c. obtusa (gosse, 1886) 43. c. sulcata (stenroos, 1898) 44. c. uncinata (o.f. muller, 1773) s. lato 45. lepadella acuminata (ehrenberg, 1834) 46. l. apsida harring, 1916 *** 47. l benjamini harring, 1916 *** 48. l. biloba hauer, 1958 *** 49. l. costatoides segers, 1992 50. l. cristata (rousselet, 1893) *** 51. l. dactyliseta (stenroos, 1898) *** 52. l. discoidea segers, 1993 53. l. elongata koste, 1992 *** 54. l. eurysterna myers, 1942 *** 55. l. latusinus (hilgendorf, 1889 *** 56. l. lindaui koste, 1981*** 57. l. minuta (weber & montet, 1918) *** 58. l. ovalis (o.f. muller, 1786) s. lato 59. l. patella (o.f. muller, 1773) s. lato 60. l. quadricarinata (stenroos, 1898) *** 61. l. quinquecostata (lucks, 1912) *** 62. l. rhomboides (gosse, 1886)s. lato 63. l. triba myers, 1934 *** 64. l. triptera ehrenberg 1832 *** 65. l. vandenbrandei gillard, 1952 *** 66. l. (heterolepadella) apsicora myers, 1934 67. l. (h.) ehrenbergi (perty, 1850) 68. l. (h.) heterostyla (murray, 1913) 69. squatinella lamellaris (o. f. müller, 1786) *** family: lecanidae 70. lecane aculeata (jakubski, 1912) appendix i: a detailed systematic list of the rotifer species examined from mizoram phylum: rotifera class: eurotatoria subclass: monogononta order: ploima 311 sharma and sharma/ freshwater rotifers of mizoram (northeast india) 71. l. arcula harring, 1914 72. l. aeganea harring, 1914 * 73. l. bifurca (bryce, 1892) *** 74. l. bulla (gosse, 1851) s. lato 75. l. closterocerca (schmarda, 1859) 76. l. crepida harring, 1914 77. l. curvicornis (murray, 1913) s. lato 78. l. decipiens (murray, 1913) *** 79. l. doryssa harring, 1914 *** 80. l. elegans harring, 1914 *** 81. l. flexilis (gosse, 1886) *** 82. l. furcata (murray, 1913)*** 83. l. haliclysta harring & myers, 1926 *** 84. l. hamata (stokes, 1896) s. lato 85. l. hornemanni (ehrenberg, 1834) *** 86. l. inermis (bryce, 1892) 87. l. inopinata harring & myers, 1926 88. l. lateralis sharma, 1978 *** 89. l. leontina (turner, 1892) s.lato 90. l. ludwigii (eckstein, 1883) 91. l. luna (müller, 1776) 92. l. lunaris (ehrenberg, 1832) s. lato *** 93. l. monostyla (daday, 1897) *** 94. l. nana (murray, 1913) *** 95. l. obtusa (murray, 1913) 96. l. ohioensis (herrick, 1885) 97. l. papuana (murray, 1913) 98. l. ploenensis (voigt, 1902) 99. l. pusilla harring, 1914 *** 100. l. pyriformis (daday, 1905) 101. l. quadridentata (ehrenberg,1830) s. lato 102. l. stenroosi (meissner, 1908) *** 103. l. styrax (harring & myers, 1926) 104. l. syngenes (hauer, 1938) ** 105. l. tenuiseta harring, 1914 *** 106. l. thienemanni (hauer, 1938) *** 107. l. undulata hauer, 1938 *** 108. l unguitata (fadeev, 1925) 109. l. ungulata (gosse, 1887) family : notommatidae 110. cephalodella forficula (ehrenberg, 1830) 111. c. gibba (ehrenberg, 1830) 112. c. mucronata myers, 1924 *** 113. monommata longiseta (o.f. müller, 1786) 114. monommata sp. family: scaridiidae 115. scaridium longicaudum (o.f. müller, 1786) family: trichocercidae 116. trichocerca bicristata (gosse, 1887) 117. t. cylindrica (imhof, 1891) *** 118. t. bidens (lucks, 1912) *** 119. t. edmondsoni (myers, 1936) ** 120. t. elongata (gosse, 1886) 121. t. hollaerti de smet, 1990 * 122. t. iernis (gosse, 1887) *** 123. t insignis (herrick, 1885) *** 124. t. insulana (hauer, 1937) *** 125. t. maior hauer, 1936 * 126. t. pusilla (jennings, 1903) 127. t. rattus (o.f. müller, 1776) 128. t. scipio (gosse, 1886) *** 129. t. siamensis segers & pholpunthin, 1997 * 130. t. similis (wierzejski, 1893) 131. t. stylata (gosse, 1851) *** 132. t. taurocephala (hauer, 1931) * 133. t. tigris (o.f. müller, 1786) *** 134. t. vernalis (hauer, 1936) *** 135. t. voluta (murray, 1913) *** 136. t. weberi (jennings, 1903) *** family: asplanchnidae 137. asplanchna priodonta gosse, 1850 family: synchaetidae 138. polyarthra euryptera wierzejski, 1891 *** 139. p. vulgaris carlin, 1943 140. ploesoma lenticulare herrick, 1885 *** 141. synchaeta pectinata ehrenberg, 1832 *** family: dicranophoridae 142. dicranophoroides caudatus (ehrenberg, 1834) *** 143. dicranophorus forcipatus (o.f. müller, 1786) order: flosculariaceae family: floscularidae 144. floscularia ringens (linnaeus, 1758) *** 145. sinantherina socialis (linne, 1758) 146. s. spinosa (thorpe, 1893) family: hexarthridae 147. hexarthra mira (hudson, 1871) family: testudinellidae 312 int. j. aquat. biol. (2015) 3(5): 301-313 148. testudinella amphora hauer, 1938 *** 149. t. emarginula (stenroos, 1898) s. lato 150. t. greeni koste, 1981 *** 151. t. parva (ternetz, 1892) *** 152. t. patina (hermann, 1783) s. lato 153. t. tridentata smirnov, 1931 *** 154. t. walkeri koste & shiel, 1980* 155. pompholyx sulcata hudson,1885 *** family: trochosphaeridae 156. filinia camasecla myers, 1938 157. f. longiseta (ehrenberg, 1834) s. lato 158. f. opoliensis (zacharias, 1898) 159. f. terminalis (plate, 1886) s. lato *** family: collothecidae 160. collotheca ornata (ehrenberg, 1832) *** sub-class: bdelloidea family: philodinidae 161. philodina roseola ehrenberg, 1832 *** 162. rotaria neptunia (ehrenberg, 1830) ----------------------------------------------------------------------------------------------------------------------------------------------- * new record from india, ** new record from northeast india, *** new record from mizoram int. j. aquat. biol. (2015) 3(5): 301-313 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: w www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی شرقی هندوستان:میزورما، شمال شیرین آب (rotifera: eurotatoriaتنوع روتفرهای ) های جالبای، ثبت جدید و ویژگیترکیب گونه سومیت شارما، *بوشان کومار شارما ، مقالالیا، هندوستان. 220377شرقی، شیلونگ گروه جانورشناسی، دانشگاه هیل شمال ،شیرینهایشناسی آبزمایشگاه زیستآ چکیده: گونه 267شامل غنایی با اجتماعی پرگونه و متنوع از روتیفرا وستانشرقی هندایالت میزورما در شمال پالنکتونهای پالنکتون و نیمهبررسی نمونه گونه جدید 26شرقی هند و شمالگونه جدید برای 4، وستانگونه جدید برای هند 6این مطالعه جنس را آشکار کرد. 00خانواده و 22متعلق به یک گونه گونه پالئوتروپیکال، 2رالیایی، یک گونه شرقی، استکند. وقوع یک گونه برای ایالت میزورما که از نظر تنوع زیستی جالب هستند را ثبت می که چندین گونه اهمیت د، در حالیسازمی آشکارجالب دیگر ارزش جغرافیای زیستی را گونه 5هولوآرکتیک، یک گونه جهانی )زیر( استوایی و درصد از 2/62در مجموع lecanidae>lepadellidae>brachionidae>trichocercidae هایخانواده د.ندهای را نشان میپراکنش منطقه که ای( در حالیدرصد از غنای گونه 57ها هستند )با حدود ترین جنسمتنوعlecane>lepadella> trichocercaگیرند. دربرمیرا ای غنای گونه غنای پریفیتونی و -ساحلی هایاز آرایه یدهند. این مطالعه غنای باالیدرصد را تشکیل می26حدود pantropicalو tropicopolitanهای گونه ن نیاز به توجه ای معیهای گونههای پریفیتیک، ساکن، کلونی و بنتیک و تنوع نهفته برخی گروهپالنکتونی را نشان داد. آنالیز آرایههای از آرایهکمتر .شودزده میمین گونه روتیفر در میزورما تخ 753و حضور بیش از دکنای را بیان میبروز رسانی تنوع گونه هبیشتر ب .استواییهای زیرآب، ایغنای گونه های جالب،آرایه ،جغرافیای زیستی ،ترکیب کلمات کلیدی: int. j. aquat. biol. (2013) 1(2): 82-92 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology habitat-associated morphological divergence in four shemaya, alburnus chalcoides (actinopterygii: cyprinidae) populations in the southern caspian sea using geometric morphometrics analysis mohammad mohadasi1, nader shabanipour *1, soheil eagderi2 1department of biology, faculty of science, guilan university, rasht, iran. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 2 april 2013 accepted 29 april 2013 available online 5 may 2013 keywords: phenotypic plasticity generalized procrustes analysis shape variation shemaya abstract: in this study, geometric morphometrics approach was used to explore body shape variations and growth trajectory among four population of shemaya (alburnus chalcoides). the shape of 114 individuals from three rivers (lisar, shiroud and babolroud) and one lagoon (anzali) from the south of caspian sea was extracted by recording the 2-d coordinates of 16 landmark points. we applied a gpa analysis to eliminate non-shape variations. pca, cva, manova and dfa analysis were used to examine shape differences among populations. the significant differences found among the shape of populations. since shemaya is an anaderemus fish and all their populations have a common origin, we concluded that differences between habitat features might create selective pressures resulting morphological divergence among conspecific populations. we suggest that high level of plasticity, particularly in the depth of body, head and caudal peduncle shape may reflect low costs of maintaining the plastic response even in relatively isolated populations. introduction study of phenotypic diversity between populations can help to better understanding of diversification of species within ecosystems and intraspecific diversification in fishes is well documented (reviewed in robinson and wilson, 1994; smith and skulason, 1996; taylor, 1999; jonsson and jonsson, 2001). the body shape differences of populations is considered as essential steps in process of speciation (balon, 1993; margurran, 1998). fish body shape can be the results of evolutionary adaptations to environmental pressures (gatz, 1979; watson and balon, 1984; winemiller, 1991), particularly, food collection and hydrodynamic conditions (matthews, 1998) making feasible more efficient utilization of available resources and improving fitness and performance (pianka, 1994). hence, morphological characters can provide information about the * corresponding author: nader shabanipour e-mail address: shabani@guilan.ac.ir tel: +981313227642 ecological niches of fishes (winemiller, 1991) allowing inferences about its distribution (watson and balon 1984), trophic patterns (hugueny and pouilly, 1999) and predicting its life habitats (keast and webb, 1966; karr and james, 1975). understanding general patterns and causes of diversification requires an examination of divergence in multiple species (endler, 1982; johnson and belk, 2001; jennions and telford, 2002; van buskirk, 2002) and an evaluation of potential constraints on divergence (endler, 1977; slatkin, 1987; losos, 1996; hendry et al., 2000). divergent selection can be led to phenotypic differences through either genetic differences or phenotypic plasticity (levins, 1968; west-eberhard, 1989; robinson and wilson, 1994; orr and smith, 1998; schluter, 2000). both sources of divergence can drive microevolutionary change within species 83 mohadasi et al./ int. j. aquat. biol. (2013) 1(2): 82-92 leading to speciation (west-eberhard, 1989; rice and hostert, 1993; losos et al., 2000; schluter, 2000; agrawal, 2001; kaneko, 2002). geometric morphometrics is defined as a statistical study of biological shapes and shape variations among different populations (bookstein, 1991) and it allows the characterization of growth trajectory and the visualization of allometric growth (alberch et al., 1979; klingenberg, 1996; loy et al., 1998). many reports on applications of geometric morphometrics method in different biological fields including fisheries are available (marcus et al., 1996). these method, which allow the study of shape and size, offering powerful analytical and graphical tools for the quantification and visualization of morphological variation within and among organisms. the shemaya (alburnus chalcoides) is widely distributed in the river systems of the black, caspian and aral seas (bogutskala, 1997). this benthoplagic and anadromous species lives in fresh and brackish water. the populations that live in lakes migrate upstream for spawning from the early may till late july (slastenenko, 1959). little information is available about the environmental biology of shemaya. since, the shemaya populations have a common genealogy population, therefore, its morphological variation may be considered a results of environmental expression. hence, this study conducted to compare the morphological space occupied by shemaya assemblages in three rivers (lisar, shiroud and babolroud) and one lagoon (anzali) along the southern caspian sea, for analyzing the hypothesis that morphological space changes among mentioned regions. for this purpose, a homologous landmark-based geometric morphometric technique was applied (coordinating of points located unambiguously on each specimen’s profile or structure) (bookstein, 1991; rohlf and marcus, 1993; marcus et al., 1996). this study tries to obtain the relationship between morphological characters and environmental conditions in the shemaya. materials and methods sampling: in total 114 specimens of the shemaya were collected from four regions of the south caspian sea (rivers of the lisar: n: 37"58, e: 48"56, shiroud: n: 36"49, e: 50"52, babolroud: n: 36"42, e: 52"39 and anzali lagoon: n: 37"28, e: 49"27) (fig. 1) using hand net, cast net, and electrofishing. after anaesthetizing in clove solution, they were fixed into 10% formalin solution and transformed to 70% ethanol for further examinations. all collected specimens were deposited in the zoological museum collection of guilan university. geometric morphometrics analysis of shape variations: the specimens were photographed using a digital camera (canon g12, 10 mp) and sixteen homologous landmark-points were digitized using tpsdig2 software version 2.16 (rohlf, 2004) on their figure 1. sampling stations in the south caspian sea. figure 2. used landmark points to extract shape of a. chalcoides. 1. tip of the premaxilla; 2. end of the mouth; 3. the lower beginning of operculum; 4. end of operculum; 5. beginning of the scales at the dorsal side; 6. front of the eye; 7. end of the eye; 8. base of the pectoral fin; 9. base of the pelvic fin; 10, 11. anterior and posterior insertion of the anal fin; 12. lower margin of caudal peduncle. 13. end of the medial region of caudal peduncle; 14. upper margin of caudal peduncle. 15, 16. anterior and posterior insertion of the dorsal fin. 83 84 mohadasi et al./ int. j. aquat. biol. (2013) 1(2): 82-92 left side (fig. 2). the landmark-points were chosen at the specific points, in which a proper model of fish body shape was extracted (bookstein, 1991). the digitization error was estimated according to adriaens (2013). the obtained error based on a subsample was about 12% that is low enough to be ignored. correlations between the procrustes and tangent shape distances were calculated using tpssmall software version 1.2 (rohlf, 2003) to certify that the amount of shape variation in the original data set is small adequate to allow statistical analyses to be performed in the linear tangent space, approximating the non-linear kendall shape space (rohlf, 1998a). as a measure of size variation of the shapes, the centroid size (bookstein, 1991) were calculated for each shape in studied populations using tpsrelw (rohlf, 2008) and tested for normality using the shapiro-wilk test. one-way anova analysis were performed to compare the population’s cs using their mean size. to explore allometry (how shapes vary with size; klingenberg, 1998), multivariate regression of partial warps and uniform component on centroid size was performed with tpsregr (rohlf, 1998b). within-species changes were investigated as linked with centroid size of the species and illustrated deformation in shape of the anatomical aspects related to centroid size in the smallest and the largest specimen. the landmarks were submitted to a generalized procrustes analysis (gpa). partial warp (shape variables) and relative warp scores (with α=0 which is a pca of shape variables) (rohlf 1993) were calculated using the software tpsrelw version 1.46 (rohlf, 2008). principal component analysis (pca) was performed to summarize the variation among the specimens as few dimensions as possible. canonical variant analysis (cva)/manova was accomplished to investigate power of distinction among the populations. for discrimination of the individuals of four populations using shape variety, laniary discriminate analysis (lda) by a cross-validation was performed for pair-wise of the populations. partial warp scores have been used in cva and discriminate analysis. to display the shape variation linked with the das in four body part aspects, thinplate spline interpolation was used to produce transformation grids that show the transformation from a grid which superimposed onto the average configuration i.e. consensus shape. the relationship between shape variables and centroid size (cs) was evaluated to compute the allometric growth patterns. therefore, a principal component analysis (pca) was performed for each new set of variables. the correlation test was used between cs and pca scores (rohlf, 1993). when significant correlation was found, the pc with the highest correlation plotted against cs representing the growth trajectory. the use of the thin-plate spline function allows the visualization of the shape change in the deformation grids (splines). size related shape changes were then visualized as splines relative to the extreme values of the relative warp axis. as a complement to discriminant analysis, morphometric distances between the individuals of two groups were inferred to cluster analysis (veasey et al., 2001) by adopting the euclidean square distance as a measure of dissimilarity and the upgma (unweighted pair group method with arithmetical average) method as the clustering algorithm (sneath and sokal, 1973). results the variety of the specimens in shape spaces were perfectly correlated (for all the shapes r=1), therefore, they allow the use of the tangent plane approximation in further statistical analyses and interpretation of the results. comparison of centroid size (cs) of studied populations showed that variations among populations were completely figure 3. deformation wireframe related to centroid size of a. chalcoides. darker wireframe represent smallest specimen and lightness wireframe shows largest one. 85 mohadasi et al./ int. j. aquat. biol. (2013) 1(2): 82-92 significant (f=6.10e04, p=0.0001). deformations in coordinate configurations related to cs have been showed in figure 3 and figure 7 representing the variation in cs of four populations (table 1). pca analysis for all specimens explained 44.4% of shape variations by the first two pc axes extracted from the variance-covariance matrix (pc1=33.3% and pc2=13.1%). for covering more than 90% of the shape variation, 11 axes were needed. anzali and lisar populations showed more separation than the other populations along the first and second axis, respectively (fig. 4). the manova/cva analysis showed that geographically separated populations significantly differ in body shape (table 2 and fig. 5). the population of lisar is separated from other groups, whereas babolroud population showed an overlap with others. according to the table 2, shape variation among all populations is highly significant (wilks’ lamba= 0.0276, f=9.197, p=1.055e-45). hotelling’s pair-wise comparison showed that all populations are significantly different (p<0.01). the results of mahalanobis distance confirmed the results of hotelling’s pair-wise comparisons (table 2). discriminant analysis (da) on relative warps classified 87.4% in origin data and 69.2 in crossvalidation of specimen into the correct groups (table figure 4. scatter plot of individual scores from the first two principal components of a. chalcoides. deformation wireframes show the most extreme positive (light wireframe) and negative (black wireframe). figure 5. scatter plot of individual scores from the first two canonical variant functions of a. chalcoides. deformation grids show the mean shape of each population in relation to consensus shape. sum of squares df mean square f sig between groups 3.88229 15 0.258819 6.10e04 2.337e-97 within groups .203485e-3 48 4.24e-06 total 3.88249 63 table 1. on-way anova test for centroid size of a. chalcoides. lisar anzali shiroud babolroud lisar 0 8.68953e-10 1.28294e-06 0.000351966 anzali 5.21372e-09 0 5.61428e-07 9.0279e-07 shiroud 7.69761e-06 3.36857e-06 0 0.000263703 babolroud 0.0021118 0.0021118 0.00158222 0 table 2. hotelling’s pair-wise comparisons and mahalanobis distance analysis for 4 populations of a. chalcoides. 85 86 mohadasi et al./ int. j. aquat. biol. (2013) 1(2): 82-92 3). histogram of discriminant functions for pairwise groups has been shown in figure 6. for distinguishing correlation between size and shape, the pearson product-moment correlation was used to find the highest correlation between the first three pc scores and cs. the scores of pc1 had the highest correlation (r=0.72; p<0.001). the growth trajectory related in pc1 clarifies high shape variability in small specimens followed by a better defined pattern of shape change in larger specimens. figure 6. histogram of discriminate analysis (da) functions for pair wise competitions’ between studied populations of a. chalcoides. lisar anzali shiroud babolroud total original (%) lisar 88.9 2.8 2.8 5.6 100 anzali .0 97.2 .0 2.8 100 shiroud 2.9 2.9 91.4 2.9 100 babolroud 11.1 11.1 5.6 72.2 100 cross-validate (%) lisar 58.3 2.8 16.7 22.2 100 anzali .0 88.9 5.6 5.6 100 shiroud 11.4 11.4 68.6 8.6 100 babolroud 13.9 11.1 13.9 61.1 100 table 3. classification matrix showing the number and percentage of individuals that were correctly classified. (bold values indicate correct classifications). 87 mohadasi et al./ int. j. aquat. biol. (2013) 1(2): 82-92 figure 8 shows the plot of pc1 versus cs and shapes related in the extreme values of axis, and it appears as a saturating curve. the major shape changes observed in fusiform shape of the fish. gradually, the shape of larger fish is more fusiform, the anterior region sharpens and the caudal peduncle is longer and slimmer as they grow. the upmga analysis for the studied populations showed that they divided into two major distinct groups. the first branch is included the anzali’s population and the second group is divided into lisar’s populations and another group including the babolroud and shiroud populations (fig. 9). discussion in the present study, landmark-based geometric morphometrics tool was applied to compare and visualize the body shape changes as well as to display growth trajectories among four wild populations of shemaya in the southern caspian sea. manova, cva and dfa showed a significant morphological difference in terms of body shape among populations. these discriminations between river populations and lagoon inhabitants is higher than those among river populations. this discriminations observed on three main morphological parts; (1) abdominal circumference, (2) caudal peduncle shape and (3) position of the mouth. comparison of the lagoon and river inhabitants specified that in similar ages, lagoon specimens have larger size, more fusiform body shape and slimmer caudal region. comparison among three rivers populatons revealed that the lisar population bear the bigger abdominal circumference, and upper position of mouth. many fish species show morphological differences among habitats (robinson and wilson, 1994; smith and skulason, 1996; taylor, 1999; jonsson and jonsson, 2001) and intraspecific polymorphism is typically believed to arise from divergent selection pressures among various environments (robinson and wilson, 1994; smith and skulason, 1996; schluter, 2000). it is common that morphological characteristics can show high plasticity in response to different environmental circumstances (wimberger, 1992). lagoons are fairly rich in terms of nutritional quality and quantity (whitfield, 1999; mariani et al., 2002) and their fishes live in a wide and deep water body with low flow water as seen in anzali lagoon, whereas the lisar is a river characterized with less depth, muddy bottom, high turbidity and fastrunning water and less nutritious. on the other side the shiroud and babolroud rivers characterized with low turbidity, deeper, and more nutritious showing a better conditions than the lisar. hence, tough environmental conditions of the lisar specimens may be led to smaller size of individuals. insatiable condition takes more energy and results in low growth (boily and magnan, 2002). it is commonly known that growth of lagoon fish is higher than that of river specimens (warburton, 1979; mariani et al., 2002). coban et al. (2008) reported that there is no significant shape variation between cultured fish (that are always fed well) and lagoon caught. also, the results of this study revealed that fish in a lagoon which is rich in terms of nutrition than rivers, are bigger in size. many fishes show distinct morphological differences between lotic and lentic habitats (robinson and wilson, 1994; taylor et al., 1997; figure 7. the 95% confidence intervals (ci) of centroid size (cs) vs. locations of a. chalcoides. 87 88 mohadasi et al./ int. j. aquat. biol. (2013) 1(2): 82-92 hendry et al., 2000; pakkasmaa and piironen, 2000; brinsmead and fox, 2002). hydrodynamic theory prove that a more fusiform body shape decrease drag, and hence reduces the energetic expenditure essential to maintain position in the flowing water (keast and webb, 1966; blake, 1983; webb, 1984; videler, 1993; vogel, 1994). but the scenario in this study can be applied when nutrition in whole station be similar and analogous in similar ages, specimens contains better feeding, indicate better fusiform discrete from velocity of water flow (flowing of water in the anzali lagoon is slow). because as indicated, by growing the size of fish body form becoming more fusiform (fig. 8). bagherian and rahmani (2007) studied two river inhabiting populations of shemaya in the south caspian sea, expressing that more intensity of water flowing cause to be more slender body in this fish. intra-specific trophic diversification is also well known in fishes (robinson and wilson, 1994; wimberger, 1994; robinson and wilson, 1995; smith and skulason, 1996; ruzzante et al., 1998; mittelbach et al., 1999; holtmeier, 2001). the observed differences in mouth position among habitats would show discriminations in feeding, such as foraging mode, orientation or diet composition (keast and webb, 1966; winemiller, 1992; moyle and cech, 2000). the results showed that lisar specimens have upturned mouths but other populations have terminal mouths. the depth of lisar river is low and maybe the fishes of this river are fed from surface. other population might be expected to forage more frequently on these midwater prey items in lagoon and rivers with enough depth. mid-water foragers naturally show terminal mouths, benthic feeders exhibit sub-terminal figure 8. relative warp 1 (rw1) analogous to principal components of shape variability versus centroid size (cs). figure 9. the upgma graph for four studied populations of a. chalcoides. shape differences on the extremities of each population are presented. 89 mohadasi et al./ int. j. aquat. biol. (2013) 1(2): 82-92 mouths, and surface feeders have upturned mouths (keast and webb, 1966; winemiller, 1992; moyle and cech, 2000). the upgma graph shows two main branches, including anzali’s population as firs group and the rest in the second one. further, the second branch is divided into two groups comprising (a) the lisar and (b) the babolroud and shiroud populations. as mentioned above, anzali is a lagoon with different environmental condition rom rivers and this have been probably caused different body shape (langerhans et al., 2003). also, lisar population is far from shiroud and babolroud ones and maybe little gene exchange between lisar and others river populations (via and lande, 1985; west-eberhard, 1989; robinson and wilson, 1994; orr and smith, 1998; schluter, 2000), whereas, geographically, the anzali and lisar specimens are very close to each other, but showing a high shape differences. hence, it seems that ecological pressures have more importance role in shape differences in shemaya and gene exchange has less rate in equalization of the shape in populations. the member of branch including shiroud and babolroud have similar body shape with morphological common features. these two sites have similar environmental conditions rather than two others (lisar and anzali). these results indicated that feeding habits (coban, 2008; langerhans, et al. 2003) and flow conditions (langerhans, et al. 2003) along with geographical distance play an effective role in body shape variation in studied shemaya populations and can be considered as main evolutionary drivers acting on aquatic biodiversity. acknowledgements this article dedicate to gholam reza and negareh eslami moghaddam for providing necessary facilities. we would like to thank roozbahan khaefi, saeed ghareebi, dr. behrouz haydari and dr. shahram abdolmalaki for their help in during study, and dr. ali bagherian, for the financial support. references adriaens d. 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(2022) 10(4): 303-309 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article stock assessment of the african moony, monodactylus sebae (cuvier, 1829) in the new calabar, nigeria olaniyi alaba olopade, henry eyina dienye, desire precious dike department of fisheries, faculty of agriculture, university of port harcourt, choba, east-west road, pmb 5323, port harcourt, rivers state, nigeria. s article history: received 14 june 2022 accepted 8 august 2022 available online 2 5 august 2022 keywords: growth mortality asymptotic length maximum sustainable yield abstract: the population biology and stock assessment of african moony, monodactylus sebae was studied based on monthly length frequency data collected from new calabar river, nigeria from february 2020 to march 2021. the estimated von bertalanffy growth parameters were growth performance index (2.91 per year), asymptotic length (l∞ = 36.54 cm) and growth curvature (k = 0.61yr-1). the estimated theoretical age at birth (t0) and longevity for the assessed fish species were 0.55 years and 2.91 years, respectively. the total mortality (z), natural mortality (m), and fishing mortality (f), were 1.87 year-1, 1.23 year-1 and 0.64 0.64 year-1, respectively. the length at first capture (lc) was 8.56 cm. the exploitation rate (e) and maximum exploitation rate (emax) were calculated as 0.34 and 0.36, respectively. the recruitment pattern occurs throughout the year, with only one major peak in may with 19.9% recruits. emax was 0.36, while e50 was 0.23 indicating that the current exploitation rate was below the maximum sustainable yield, showing that this stock was underexploited. introduction fish populations are subject to natural control processes that continually modify and adjust the structure and abundance of the population and their life cycle in response to a wide range of factors (milner et al., 2003), apart from those caused by human activities such as overfishing and habitat alteration, as well as pollution and lately climate change. stock assessment is the basis for understanding changing fishery patterns and issues such as habitat destruction, predation and optimal harvesting rates (olopade et al., 2019). regular stock assessment and reference points are required for monitoring and determining whether the stocks are subject to overfishing or overfished and developing fishery management plans (mohamed et al., 2021). kebtieneh et al. (2016) stated that the basic purpose of stock assessment is to provide decision-makers with the information necessary to make rational choices on the optimum level of exploitation of aquatic living resources such as fish. stock correspondence: olaniyi alaba olopade doi: https://doi.org/10.22034/ijab.v10i4.1695 e-mail: olaniyi.olopade@uniport.edu.ng assessment forms the basis for calculations leading to the knowledge of the growth, mortality, recruitment, and other fundamental parameters of their populations (olopade et al., 2019). the family monodactylidae contains six extant species in two genera, monodactylus and schuettea. monodactylidae is found in the eastern tropical atlantic ocean along the african coast from senegal to angola and the canary islands (desoutter, 1990). monodactylidae is primarily found in estuaries and coastal mangrove habitats but can live in freshwater and marine habitats (schneider, 1990). reproduction takes place in marshes and lower courses of rivers, sometimes ascending over long distances into freshwater (bauchot, 2003). they are laterally compressed with an approximately diamond shape body with a long anal, and dorsal fin extended distance that gives this fish a square-like. african moony, monodactylus sebae is a member of the family and the only species identified so far in nigerian fresh water (adesulu and sydenham, 304 olopade et al./ stock assessment of the african moony, monodactylus sebae 2007). even though it is marine species that can survive in fresh water for some times. this species lacks the yellowish coloration in the caudal fin seen in other species of monodactylus (monks, 2006). it is economically important as it can be found in the aquarium trade, and lately, this fish has assumed importance in the niger delta region of nigeria by its acceptance as a food fish in both fresh and dried conditions. according to the iucn (2021), the fish is assessed as at least concern (lc). however, there is massive fishing pressure on this species in the new calabar river and in other water bodies in the niger delta region. this could be attributed to the absence of commonly important fish species. in spite of the importance of the m. sebae in nigeria, there is no information on stock assessment in the country or elsewhere. the objective of the present study was to assess the growth parameters, mortality rates, probability of capture, recruitment pattern, yield per recruit, and virtual population analysis of m. sebae in the new calabar river, nigeria. materials and methods the new calabar river, nigeria is a partially mixed estuary system that lies between latitude 4º25′n and longitude 7º16′e (olopade et al., 2019) (fig. 1). the entire river course is situated in the coastal area of the niger delta and empties into the atlantic ocean. data collection: fish samples were collected from the new calabar river at two fishing landing sites, namely choba and ogbogoro from the local fishermen using different gears. these samples were taken twice monthly, starting from the month of february 2020 to march 2021. the species were identified to the species level using the identification keys by monks (2006). fish specimens were immediately iced and transported to the laboratory to measure the weight (to the nearest 0.5 g) and length (to the nearest 1.0 cm) of each specimen. data analysis: the length-frequency data for m. sebae were collected monthly from a number of different gears at all sites and then grouped into class intervals for analysis. the data were analyzed using fisat ii (fao-iclarm stock assessment tools) (gayanilo et al., 2005). the von bertalanffy (pauly, 1980) growth parameters, asymptotic length l∞ and annual growth coefficient k were computed by elefan i (electronic length frequency analysis) method (beverton and holt, 1966). the total mortality rate (z) was estimated by the lengthconverted catch curve (pauly, 1984). the natural mortality rate (m) was also calculated by using pauly’s empirical formula (pauly, 1980). the fishing mortality rates (f) were then calculated by the difference between (z) and (m). the rate of exploitation (e) was calculated by the quotient between fishing and total mortality: e= f/z (pauly, 1984). figure 1. map of new calabar river, nigeria. 305 int. j. aquat. biol. (2022) 10(4): 303-309 relative yield per recruit (y/r) was estimated using the model of beverton and holt (beverton and holt, 1966) as modified by pauly and soriano (1986) and incorporated into the fisat software. lengths at first capture (lc50) and first maturity (lm50): the left ascending part of the length converted catch curve was used to estimate the probabilities of length at 50, 75, and 95 capture which correlates with the cumulative probability at 50, 75 and 95 percent, respectively (pauly, 1984). the length at first maturity (lm50) was estimated using the expression: length at first maturity (lm50) = 2 * l∞ /3 (hoggarth et al., 2006). one-year recruitment pattern was obtained by projecting the length frequency data backward onto the time axis as described in the fisat routine. biological reference points: emsy which depicts the exploitation rate producing a maximum yield of a cohort and e0.5 implying the exploitation rate under which the population is reduced to half its virgin biomass were computed together with the corresponding fishing mortality rate (i.e. fmsy and f0.5). the length-based virtual population analysis (vpa) was performed on the pooled annual length frequencies from the fishery to estimate the mean number in the population and the overall fishing mortality by length group. results length-frequency: 390 specimens were collected for this study and the size-frequency distribution (fig. 2) shows a unimodal type. they were grouped into twenty classes of total length frequency with the collected samples falling in the length range of 7.7 to 34 cm and with a mean of 13.53±2.94. the 12.8 cm tl size group was numerically dominant, followed by 10.8 cm, and constituted 53.86% of the total population. estimation of growth parameters and growth performance index: figure 3 shows the growth curve of m. sebae. the asymptotic length (l∞) and growth rate (k) of m. sebae were 36.54 cm tl and 0.61 year-1, respectively (table 1). the growth performance index or phi prime (φ′) was 2.91 (fig. 4), and the estimated theoretical age at birth (t0) and longevity for the assessed fish species were 0.55 and 2.91, respectively (table 1). the length-converted catch curve to estimate the annual total mortality rate (z) is shown in figure 5. the natural mortality (m/year) as per pauly’s empirical formula was calculated as 1.23 year-1 and the total mortality (z) as 1.87 year-1 while the fishing mortality (f) was taken by subtraction of m from z figure 2. length-frequency distribution of monodactylus sebae from the new calabar river, nigeria. indicators unit value growth rate (k) year-1 0.61 asymptotic length (l∞) cm tl 36.54 age at birth (t0) years -0.55 longevity (tmax) years 4.37 growth performance index (phi) 2.91 natural mortality rate (m) year-1 1.23 total mortality rate (z) year-1 1.87 fishing mortality rate (f) year-1 0.64 exploitation rate (e) 0.34 m/k 2.02 e-10 0.26 e-50 0.23 e-max 0.36 l25 cm 7.06 l50 cm 8.56 l75 cm 10.20 lm cm 24.36 table 1. population parameters of monodactylus sebae in the new calabar river, nigeria. 306 olopade et al./ stock assessment of the african moony, monodactylus sebae and was 0.64 year-1 (table 1). the current exploitation rate (ecurrent) was computed as f/z= 0.34 and the m/k ratio found was 2.02 (table1). length at first capture (l50): the logistic of the probability of the capture routine of m. sebae is presented in figure 6. the length at which 50% of the stock biomass is vulnerable to capture estimated at l50 = 8.56 cm. the l25 was calculated as 7.06cm while l75 was found to be 10.20 cm. the length at first maturity (lm50) was estimated at 24.36 cm. the reproductive load ratio (l50/ l∞) (8.56/36.54) = 0.23 for m. sebae indicating the length at first capture is quite low for the population (table 1). recruitment pattern: as shown in figure 7, the annual recruitment pattern of m. sebae indicated that recruitment occurred throughout the year with only one prominent peak in may with 19.9% recruits. relative yield per recruit (y'/r) and biomass per recruit (b'/r) analyses: the beverton-holt relative yield per recruit (y’/r) and relative biomass per recruit (b’/r) estimated using the selective ogive procedure of fisat for the species is given in the exploitation rate which maximizes yield per recruit produced values of emax was 0.36 while e50 was 0.23 (fig. 8). length-structured virtual population analysis: figure 9 shows that natural mortality is the only cause of loss in m. sebae at lengths from 6.8 to 9.8 cm. this species is caught by fishing gear in sizes from 6.8 cm, with the highest quantities in lengths figure 3. growth curve of monodactylus sebae in the new calabar river, nigeria. figure 4. growth performance index (kscan routines) of monodactylus sebae in the new calabar river, nigeria. figure 5. length converted catch curves of monodactylus sebae in the new calabar river, nigeria (the goodness-of-fit was at rn= 0.344). 307 int. j. aquat. biol. (2022) 10(4): 303-309 from 10.8 to 12.8 cm. the fishing mortality was at its lowest 22.8 and 30.8 cm. the smallest length groups have lower catches (harvesting rates) than the largest ones (table 1), indicating that the fishing mortality rate is size specific. natural losses were highest among individuals within the length range of 6.8 to 9.8 cm and then decreased gradually to the length group of 30.8 cm. discussion length frequency distribution showed that smallsize fish were the most abundant in the catches. the 12.8 cm tl size group was numerically dominant, followed by 10.8 cm, and constituted 53.86% of the total population. the growth parameters in this study were as follows: asymptotic length (l∞) = 36.54 cm, growth curvature (k) = 0.61 per year, growth performance index = 2.91, and t0 (per year) = -0.55. the growth rate (k) of 0.61 year-1 signifies that m. sebae exhibited a fast growth rate, evinced by the low longevity of 4.37 years. the growth coefficient (k) of m. sebae was high (2.91). the greater of these values indicates that the fish growth rate to achieve the maximum size is faster. the total mortality (z), natural mortality (m/year), fishing mortality (f), and exploitation rate (e) of m. sebae were found to be 1.87, 1.23, 0.64, and 0.34, respectively. the natural mortality of fish becomes much higher in an unexploited population than in an exploited one. however, since natural figure 6. length at first capture l50 for monodactylus sebae in the new calabar river, nigeria. figure 7. recruitment pattern of monodactylus sebae in the new calabar river, nigeria. figure 8. relative yield per recruit (y'/r) and biomass per recruit (b'/r) analyses of monodactylus sebae in the new calabar river, nigeria. figure 9. length-structured virtual population analysis of monodactylus sebae in the new calabar river, nigeria. 308 olopade et al./ stock assessment of the african moony, monodactylus sebae mortality (1.23) exceeded fishing mortality (0.64), the stock is not over-exploited. according to macer (1977), the consistency of the estimated natural mortality rates (𝑀) was ascertained using the 𝑀/𝐾 ratio, which has been reported to be 1.12-2.5 for most fishes. the 𝑀/𝐾 ratio in this study was 2.02 which was within the normal range. the exploitation rate allows for determining whether a stock is overfished or not based on the assumption that the optimal value of e is 0.5 (gulland, 1971; pauly, 1983). based on the exploitation rate (e) of m. sebae in this study (0.34), it is clear that the stock is currently underexploited. in this study, the length at first maturity (lm50 = 24.36 cm) was higher than the length at which the species become vulnerable to the fishing gears (lc50 = 8.56 cm) indicating that this species is harvested before they mature, a characteristic feature of growth overfishing (fröese, 2004). furthermore, the critical length at capture which is the ratio of lc50 / l∞ (8.56/36.54 = 0.23), indicated that it was lower than 0.5. this signals the harvesting of more juvenile fish species (pauly and soriano, 1986). the presence of many small-sized fish species in the catches could be explained by the unselective use of small mesh-sized fishing gears. continuous exploitation of this at this level could result in growth overfishing and eventually lead to a possible collapse. in m. sebae population, one major peak was recorded as continuous recruitment began from may to october. this result was in line with pauly (1982) that observed a double recruitment pulse per year for tropical fish species and for short-lived species the recruitment pattern is related to the spawning time (fiorentino et al., 2008). the present study agrees with the spawning seasons reported for tropical fish species. the yield per recruit model is an efficient approach for fish stock assessment, consisting in an important tool to the management for fisheries (sparre and venema, 1997). the predicted emax of the selective ogive procedure for m. sebae (0.36) was higher than the current exploitation rate e (0.34) showing that m. sebae was lower than both target reference points. this is a further implication that the stock of the species is underexploited. virtual population analysis (vpa) data were utilised to make management decisions and provide more information about the status of fish stocks in terms of growth, recruitment, and overfishing (chen et al., 2008). according to vpa, the 12.8 cm length group was more vulnerable to fishing and more harvested. this implies that more individuals are caught before they reach length at first sexual maturity. this situation is also described by fröese (2004) as growth overfishing; when fishes are caught before they can realize their full potential. if this condition continues without any efforts to regulate m. sebae stock, the fish species will be threatened in the long term. the protection of juveniles through fish size stipulation and mesh size limitation is probably a key factor for the sustainability of this species. this can be achieved by compliance or enforcement of the mesh size (7.5 cm) recommended as the standard as the minimum mesh size for all inland water bodies in nigeria by a joint effort between resource users and the governing authority. conclusion the present study is the first effort to evaluate the growth parameters and some important information on the population biology and the stock assessment of m. sebae in the new calabar river, nigeria. the study revealed that the m. sebae stock was underexploited, and more individuals were caught before they are reached length at first sexual maturity. this study suggests that mesh size regulations will be required to protect m. sebae in the new calabar river in nigeria. references adesulu e.a., sydenham d.h.j. 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(2016) 4(4): 256-268: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology review article review of the lampreys of iran (family petromyzontidae) brian w. coad1 canadian museum of nature, ottawa, ontario, k1p 6p4 canada. article history: received 11 may 2016 accepted 14 july 2016 available online 2 5 august 2016 keywords: caspian sea, biology, caspiomyzon, morphology. abstract: the systematics, morphology, distribution, biology, economic importance and conservation of the lamprey of iran are described, the species is illustrated, and a bibliography on this fish in iran is provided. there is one native and endemic species in the caspian sea basin, caspiomyzon wagneri. the genus caspiomyzon is characterised by having 2 dorsal fins, an oral disc narrower than the body, teeth are generally low and blunt, the supraoral lamina is small, oval and sometimes has 2 tubercles and rarely 2 teeth, the infraoral lamina has 4-6, usually 5, teeth which may be bicuspid at their tips, there are about 8 small teeth of equal size in the transverse lingual lamina, the exolaterals, anterials and posterials are strong and close together, anterior and endolateral circumorals 9-11, usually 11, and 3 long, papillose velar tentacles are present. characters of the species are the same as the genus. trunk myomeres number of c. wagneri are 5368 in ammocoetes. introduction the freshwater ichthyofauna of iran comprises a diverse set of families and species. these form important elements of the aquatic ecosystem and a number of species are of commercial or other significance. the literature on these fishes is widely scattered, both in time and place. summaries of the morphology and biology of these species were given in a website (www.briancoad.com) which is updated here for one family, while the relevant section of that website is now closed down. family petromyzontidae lampreys in the family petromyzontidae are found in cooler waters of the northern hemisphere, with a few related species in other families in the southern hemisphere. their origins lie at least 300 million years in the past. there are about 44 lamprey species in 9 genera (eschmeyer and fong 2011; renaud, 2011; maitland et al., 2015) with only one recorded from iran. coad (1987, 1998) and coad and abdoli * corresponding author: brian w. coad e-mail address: bcoad@mus-nature.ca (1996) place this species in context with the iranian ichthyofauna. lampreys are jawless fishes, lacking bone in the skeleton and having 7 pairs of pore-like gill openings. the eel-like body has no pectoral or pelvic fins. there are one or two dorsal fins and a caudal fin. an anal fin-like fold develops in spawning females. the mouth is a suctorial disc armed with rows of horny teeth. there are also teeth on the tongue. the median nostril, or nasohypophyseal opening, is not connected to the mouth. there is a light-sensitive pineal organ or "third eye" behind the nostril. the skin is covered in mucus which is poisonous to fishes and humans. lampreys are edible if the mucus is cleaned off. their tooth arrangement is used in classification and identification along with the number of myomeres (muscle blocks along the body). both tooth counts and the number of cusps are used, in particular those on the supraoral lamina (bar above the "mouth", the oesophageal opening), the infraoral 257 int. j. aquat. biol. (2016) 4(4): 256-268 lamina (bar below the "mouth") and the row of teeth on both sides of the "mouth". there are various series of smaller teeth and of course teeth on the tongue. larval lampreys lack teeth and are particularly difficult to identify and their determination often requires specialist knowledge. characters for the larvae include counts of myomeres and pigmentation patterns. lampreys have an unusual life cycle. adults die after spawning and the eggs develop into a larva, known as an ammocoete, which lacks teeth, has an oral hood, eyes covered by skin, a light-sensitive area near the tail, and is a filter-feeder while buried in mud and silt. fleshy tentacles in the oral hood are used to extract minute organisms from the water, such as algae (desmids and diatoms) and protozoans. after several years (up to 19 but usually 7 or less), the ammocoete transforms into an adult with enlarged eyes, teeth, a different colour and pronounced dorsal fins. the body shrinks during this metamorphosis and adults are only larger than ammocoetes if they feed. the adult may be a parasite on other fishes and marine mammals, or nonfeeding. individuals of a species may or may not be parasitic and different species may be parasitic or non-parasitic. the non-parasitic species are believed to have evolved from a parasitic species so there tends to be closely related parasitic/non-parasitic species pairs. parasitic adults feed mostly on other fishes, attaching to their bodies by suction and using their toothed tongue to rasp through the skin and scales to take blood and tissue fragments. prey is detected by sight but some lampreys attach to hosts during the night. perhaps this reduces their own predation risks and enables them to approach their quiescent hosts more easily. lampreys tend to select larger fish as these survive longer and ensure a good food supply. the flow of blood is aided by an anti-coagulant in lamprey saliva called lamphedrin which also serves to break down muscle tissue. the attack may weaken or even kill the host. weakened fishes are more prone to diseases and the wound provides an easy path of entry for them. the fish (and marine mammal) species parasitised are varied and reflect availability in the habitat. marine lampreys enter fresh water to spawn and freshwater species may move into or up streams. the scientific name of the family means "stone sucker" and the adult mouth is used to hold or suck onto stones as well as on prey. this suction enables the lamprey to maintain position in fast-flowing streams when spawning and even to climb over rapids and small waterfalls. usually spawning occurs in shallow water with a moderate current, a bottom of gravel and nearby sand and silt for the ammocoetes to live in. either or both sexes build a nest by moving gravel around with their sucking mouths and by thrashing their bodies. a shallow depression is formed, about 0.5-1.0 metre long. spawning often occurs in groups and several males may attach to a female with the sucking disc. the process takes several days as only a few white to yellow eggs are laid at a time. the eggs are adhesive. adult lampreys are usually caught when attached to a host or when spawning. electro-shocking will force ammocoetes out of their u-shaped burrows to the surface and immobilize adults. they sometimes attach to boats and occasionally to human swimmers when their skin is cool but are easily removed, perhaps because nobody has left a lamprey on their skin long enough to see if the tongue starts rasping flesh! genus caspiomyzon berg, 1906 this genus is characterised by having 2 dorsal fins, an oral disc narrower than the body, teeth are generally low and blunt, the supraoral lamina is small, oval and sometimes has 2 tubercles and rarely 2 teeth, the infraoral lamina has 4-6, usually 5, teeth which may be bicuspid at their tips, there are about 8 small teeth of equal size in the transverse lingual lamina, the exolaterals, anterials and posterials are strong and close together, anterior and endolateral circumorals 9-11, usually 11, and 3 long, papillose velar tentacles are present. there is a single species in the genus found only in the caspian sea basin. agnathomyzon 258 coad/ lampreys of iran gratzianow, 1906 and its subgenus haploglossa gratzianow, 1906 are synonyms of caspiomyzon (eschmeyer et al. 1996). caspiomyzon wagneri (kessler, 1870) (figs. 1-6) common names: mar mahi (= snake fish), marmahiye dehangerd (= round mouth snake fish), mahi dehangerd, mahi dehangerd daryacheh-ye khazar or dahangerd-e-daryaye khazar (= caspian sea round mouth fish). [ilanbaligi or xazar ilanbaligi, djilanbalux or morma in azerbaijan; kaspiiskaya minoga or caspian lamprey in russian; volga lamprey]. systematics: the type locality of p. wagneri is from the mouth of the tvertsa to astrakhan; oka and kama rivers, and the 3 syntypes (29.0-33.0 cm) are in the zoological institute, st. petersburg (zisp 31) (holčík, 1986). the zoological museum of moscow university (zmmu) has one syntype from the kura river near evlakh (p-1393) and one from the moskva river (p-555) with p-569 from the volga river near kazan being lost (pavlinov and borissenko, 2001). the naturhistorisches museum wien in 1997 had one specimen listed as "? syntype, ?paratype" (sic) under nmw 61053. agnathomyzon (haploglossa) caspicus gratzianow, 1907 is a synonym. key characters: this is the only lamprey species in iran, easily recognised by the absence of pectoral and pelvic fins, a round, suctorial mouth containing blunt teeth, and 7 branchial openings. morphology: characters of the species are the same as the genus. trunk myomeres number 53-68 in ammocoetes, and 68(2) or 69(1) in adults from iran. ginzburg (1936) describes ammocoetes from iran. renaud et al. (2009) give details of the feeding apparatus and renaud (2011) of morphology. nazari et al. (2009) found significant differences for morphometric, but not meristic, characters, between fish from the shirud and talar river, although a principal components analysis showed relatively high overlap. vatandoust et al. (2015) examined fish from the babol and khey rivers and found morphological differences in such characters as predorsal length, interdorsal length, interorbital distance, tail length and first dorsal fin length, with high and moderate overlap between the two localities in males and females, respectively. sexual dimorphism: females reach larger sizes than males and have a smaller urogenital papilla. during the spawning migration, the lamprey undergoes certain morphological changes some of which have been linked to sex of the fish. the teeth become blunt, fin size increases, the dorsal fins become almost united at the base in males, and there is a change in colour. the urogenital papilla length in males increases from a mean of 1.1 mm to 4.9 mm and in females from a mean of 0.6 to 1.7 mm. figure 1. lateral view of adult caspiomyzon wagneri, shirud river, courtesy h. nazari. figure 2. dorsal view of adult caspiomyzon wagneri, courtesy of afshin afzali. 259 int. j. aquat. biol. (2016) 4(4): 256-268 colour: adults are dark grey with a silvery-white belly. spawning adults become black on the back and flanks with a grey belly covered with dark oval spots, or are an overall golden colour (hassan nazari, pers. comm. 28 july 2011, see photo above). ammocoetes are a pale grey to yellowish with a white belly. size: attains 57.5 cm total length and 205.5 g as the adult and 13.0 cm total length as the ammocoete. after metamorphosis of the ammocoete there is shrinkage in length, the difference between prespawning and spawning adults being on average 22.3% in iranian samples (renaud, 1982). there is also a small variety which measures 19-31 cm and can attain sexual maturity at 19.1 cm (forma praecox). distribution: found only in the caspian sea and rivers draining to it, in particular the volga where it had its largest distribution but is now known only as far as the volgograd reservoir; also in the ural, terek, and kura rivers. it is recorded in iran from the upper reaches of the aras river, and from the astara to the gorgan river along the whole caspian coast. specific localities include the aras river, anzali mordab and the gelroudkhan, nahang roga, pir bazar roga, pasikhan river and siah darvishan river in the anzali region, to kisom on the safid river (but see under conservation), and in the babol, chalus, cheshmkelya, golshan, gorgan, haraz, hevigh, kargan, kheyrod, pol-e rud, sardab, shafa, shirud, siardarvishan, tajan, talar and tonekabon figure 3. adult caspiomyzon wagneri, shirud river, courtesy h. nazari. figure 4. line drawing of an ammocoete of caspiomyzon wagneri by s. laurie-bourque. figure 5. line drawing of disc of caspiomyzon wagneri by p. i. voevodine (41.8 cm total length, russia, volga river, cmnfi 1980-0926). 260 coad/ lampreys of iran rivers, and in most large streams (derzhavin, 1934; holčík and oláh, 1992; hosseinpour, 1995; ramin, 1997; karimpour, 1998; abbasi et al., 1999, 2007; kiabi et al., 1999; abdoli, 2000; nazari, 2002; abbasi, 2006; banagar et al., 2008; abdoli and naderi, 2009; piri et al., 2009; esmaeili et al., 2014). migrations into the babol, gorgan and sardab rivers are reported by ghasempouri (1993), the chalus and sardab rivers by the annual report, 1994-1995, iranian fisheries research and training organization, tehran (1996), and the shirud (nazari and abdoli, 2010), for example. zoogeography: known only from the caspian sea, its relationships with other lamprey genera have been reviewed by potter et al. (2015). habitat: the habitat of this species in the southern caspian sea proper is unknown although some specimens have been caught in the caspian at 600700 m (jolodar and abdoli, 2004). larvae burrow 12 cm into the river bottom and favour areas where current is moderate at river bends. they can also be found in the centre of rivers or in backwaters. finegrained sand with some ooze and detritus is preferred at all stages of larval growth but larger larvae can also be found in a silt-sand bottom with much plant debris and macrophytes. the ammocoetes select and change habitat according to sediment size as they grow. they prefer depths greater than 3 m as protection against drying out, are mostly shallower than 11 m but as deep as 22 m (ginzburg, 1970), yet in different rivers or at different times will be concentrated in water of markedly different depths, e.g., 30-85 cm versus 6-8 m. metamorphosing lampreys are found in areas of faster current in deeper water and without macrophytes. spawning migrations up the volga river used to exceed 1500 km but construction of dams now prevents this. the lamprey migrates in schools with the smaller fish arriving in estuaries first. larger lampreys migrate more quickly and travel further. the speed varies from 1.9 to 15.9 km/day. the migration is triggered by decreasing water temperature and increasing water level. the strongest migration is reported at 6-11°c. movement upriver only occurs at night, near the surface when dark and on the bottom when the moon is out. during the day, the lampreys hide among stones. body fat in the volga delta was 34% but by the time the fish reached the spawning grounds upriver it had declined to 1-2%. in the kura river of azerbaijan, the lamprey migrates at the same time as the caspian salmon (salmo caspius) and often attaches to the opercular region of this species. the peak of this run is in december and january. the migration in the volga takes place from the middle of september to the end of december. migrating lampreys prefer a current velocity of 0.4-0.6 m/sec and stay close to banks and the bottom. prespawning adults overwinter among stones or in the substrate of rivers. during winter-spring several individuals may be found coiled in a ball under stones (askerov et al., 2001). they hardly respond to external stimuli such as noise or being handled. transformed lampreys migrate to the caspian sea. before breeding, males change colour, increase slightly in size, develop their fins, and become much more active (askerov et al., 2001). nazari and abdoli (2010) note a short fall migration in late september to october with the main figure 6. live adult caspiomyzon wagneri disc, courtesy h. nazari. 261 int. j. aquat. biol. (2016) 4(4): 256-268 migration being in spring (see below). movement was mostly at night and involved swimming and resting attached to the concrete of a bridge used as the observation post. farrokhnejad et al. (2014) examined migrating lampreys in the shirud from 20 march to 26 april 2012. during this period 833 lampreys started to migrate in the dark and 332 in the light. the dark migration started at 10.5°c and the sex ratio was 0.87:1 in favour of females. the most intensive migration (77%) was from 1800 hours (twilight) to 0200 hours. daylight migration occurred when temperature was more than 15°c and higher temperatures near the end of the migration altered the normal nocturnal activity and extended it into daylight hours. age and growth: the growth rates of metamorphosing lampreys and adults are almost unknown. length and weight decrease but coefficient of condition increases in spawning as opposed to pre-spawning adults. the shrinkage in mean total length is 18-26%. females are heavier than males up to about 43 cm but past this point, males weigh more. there are 3 age groups of larvae in the volga (ginzburg, 1970), with average lengths of 3.1 cm, 6.2 cm and 10.1 cm and 2-4 age groups in the kura. in their fourth year of life, they metamorphose to adults after a downstream migration into the caspian sea. adult life span is at least 1 year and 5 months. maximum age is 6 years. maturity is attained in may and the beginning of june in the volga, and from may to the end of july in the kura river. mature lampreys are mostly 3541 cm in the volga and 41-46 cm in the kura river. the female lamprey dies after spawning but the male may live longer until sperm production ceases. nazari et al. (2010) investigated growth parameters in fish from the shirud and talar river. most fish were in the 367-369 mm length group, length-weight relationship was positive, high and significant, growth was negatively allometric, the coefficient of condition was higher in females, sex ratio was nearly equal, and growth parameters were similar in the two rivers. rasta et al. (2015) found shirud fish had a total length range of 330.4-465.4 mm, mean 393.24 mm, weight range 82.34-179.94 g, mean 118.62 g, and female to male ratio was 1.2:1. farrokhnejad et al. (2014a) and nazari et al. (2015) found the migrating shirud lampreys lost body length (6.8 and 18.8%) and weight (13.6 and 26.16%) in males and females, respectively. food: abakumov (1959) maintains that this lamprey attacks caspian salmon (salmo caspius) based on nineteenth century observations by kessler (1870) and kavraiskii (1896-1897). lelek (1987) also considers it to be parasitic. the lampreys may only have been using caspian salmon for transport. certainly the teeth in this lamprey are blunt, unlike those in lamprey species known to parasitise fishes. in contrast, holčík (1986) states that it is nonparasitic and ghasempouri (1993) agrees. renaud (1982) supposes that adults feed on amphipods since juvenile acanthocephalans (corynosoma sp.) are found in prespawners. this worm has amphipods as the intermediate host. however, holčík (1986) thinks that the acanthocephalans are swallowed while the adult lampreys are feeding on the internal organs of dead fish they scavenge. certainly larvae of corynosoma strumosum (perhaps correctly c. caspicum: b. kiabi, in litt. 1994) are found only in the body cavity of fishes. renaud et al. (2009) list it as a carrion feeder but note the well-developed buccal glands which may compensate for the blunt teeth and it may well feed on fishes. the feeding habits of the adult of this species remain to be confirmed by direct observation. gut contents include aquatic vegetation in iran and in the volga delta. migratory, transforming and spawning lampreys do not feed. the gut diameter decreases from 2.7 mm in prespawners to 1.4 mm in spawners in iran (renaud, 1982). ammocoetes feed on detritus and diatoms. joorabian shooshtari et al. (2011) compared mercury concentrations in iranian caspian lampreys with ammocoetes of other jawless fishes and found a concentration 10 times less. this difference was attributed to the non-parasitic diet of a detritivore. benam et al. (2016) compared the gastrointestinal tract of mature and maturing male and female lampreys in the shirud and confirmed 262 coad/ lampreys of iran non-feeding on the migration by absence of significant gut contents and by liver degeneration (greater in females which produce eggs). reproduction: ginzburg (1969, 1970) examined the reproduction of this species below the volgograd dam on the volga river and similar conditions may obtain in iran. the dam has probably increased fecundity by reducing the length of the spawning migration so that the fish have more energy reserves for egg production. a spawning migration exists from december to may with a peak concentration in the second 10 days of february although the catches declined in april at least in part because of the opening of the spillway of the dam. before the dam was built the migration from the caspian sea passed through the delta from mid-october to middecember, with a peak in december. the fish migrated when water temperatures reached 10-11°c and moved through channels where the current was strongest. spawning begins at 15-16°c, usually in early june but sometimes at the end of march through to the beginning of july, and temperatures during spawning are usually 15-23°c. each female produces up to 60,000 turquoise, or blue-green, eggs and spawns once in her lifetime. eggs are ovate and diameter reaches 1.5 mm. the eggs are laid on coarseto fine-grained, turquoise sand at a water depth of 3.5-19.0 m, sometimes shallower. the egg colour is cryptic against the sand substrate. many eggs are carried downstream by the current. a redd is excavated in sand or gravel by the male or by the female (authors differ on this point) and the lamprey attaches to stones by their suctorial disc. the male attaches to the female's head with his disc and wraps his body around hers. the tails of both fish quiver and eggs and sperm are released at the same time. females release all their eggs and die but males may spawn again with other females. ammocoetes hatch after 8-10 days at 17-23°c at lengths of 0.33-0.42 cm. metamorphosis of ammocoetes occurs at 8.011.0 cm in october in iran. nazari and abdoli (2010) examined migration and reproduction in lampreys from the shirud in the southern caspian sea from 16 march to 2 may at 11.0-21.25°c. the most intensive migration was at night (peaking at 2100 and declining to 0300 hours) at 16°c (34.4% of the run) (fig. 7). about 75% of the run had passed by the time water temperature reached 16-17°c. migration stopped when temperature reached 21°c. numbers observed each night varied from 1 to 60, average 17, with peak migrations at 26 march to 10 april and 15 april to 25 april. sex ratio was 1.07:1 in favour of males but not significantly different. absolute fecundity was 31,758-51,198 eggs (mean 41,924 eggs) relative fecundity was 80.3-148.1 eggs/mm length (mean 107.2 eggs/mm length) and 260.8-677.4 eggs/g (mean 397.6 eggs/g). egg diameter was 0.78-1.15 mm (mean 0.92 mm). the gonadosomatic index of females was 5.83-31.44 (mean 11.22), the peak being in mid-april. downstream migrating lampreys were spent but no dead ones were noted so some may survive to spawn a second time. two ammocoetes, 20 and 22 mm long, were found near the mouth of the shirud river on 18 april 2006 (river bank in a substrate of the sand-mud, water depth <30 cm). they probably belong to the autumn migratory group (hassan nazari, pers. comm. 28 july 2011). ahmady et al. (2010a, 2010b), ahmadi et al. (2011, 2011), mojazi amiri et al. (2012) and ahmadi (2016) also examined fish from the shirud and found both fall and spring migrants ready for spawning with no differences in numbers of males and females, weight, length, absolute fecundity (17,778 eggs in figure 7. shirud migration, courtesy h. nazari. 263 int. j. aquat. biol. (2016) 4(4): 256-268 spring, 20,247 in fall see above study), egg diameter (800 μm in spring, 710 μm in fall) and sex ratio (close to 1:1). the gonadosomatic and hepatosomatic indices were higher in fall females. fall migratory fish had a lower condition factor. sexual steroids and gonad histology also confirmed spring and fall spawning. farrokhnejad et al. (2014) determined shirud fish had 208-470 maturing degree days from the start of the upstream migration to maturation, and males matured earlier than females. farrokhnejad et al. (2014a) found the migrating shirud lampreys had a maximum gonadosomatic index of 9.51 and 35.12 for maturing and mature females respectively and 4.84 and 13.23 for males. the hepatosomatic index of males and females in the maturing stage was 0.85 and 1.02 and in the mature stage 1.31 and 0.975. this suggests a starvation period and rapid production of gametes. parasites and predators: see above under food. nazari et al. (2010) also record corynosoma in their fish. caspian lampreys are eaten by silurus glanis (coad, 2015), lota lota, sander lucioperca, and huso huso. economic importance: this species was consumed and used for oil extraction in the former u.s.s.r. (thomas, 1961; ginzburg, 1969). their fat content is so high that they were once dried and used as candles (kottelat and freyhof, 2007) and the high fat level makes them tasty (askerov et al., 2001). the caloric value for the caspian lamprey is 3.4 kcal/g wet weight. the catch in the volga-caspian region was 3,420,000 kg or 33.4 million fish in 1913 but fishing by state organizations ceased after the volgograd reservoir was constructed. the mean annual catch in azerbaijan for 1930-1963 ranged from 10 to 269 tonnes. local fisheries continue but are of little significance. it is not commercially important in iran for religious reasons but catches of several hundred kilograms can be made in an hour in such rivers as the gorgan, babol and sardab (ghasempouri, 1993). this lamprey is ingested medicinally for treatment of haemorrhoids and besmi (sic, ?) by turkmen of the southeastern caspian (hassan nazari, pers. comm. 29 july 2011). robins et al. (1991) list this species as important to north americans. importance is based on its use as food, in textbooks and because it is reputedly ichthyosarcotoxic. intoxication results from eating the flesh, skin or surface mucus of raw or cooked caspian lamprey, the location of the toxin being uncertain. a biogenic amine is believed to be responsible. mucus may cause skin irritations. poisoning can be avoided by soaking the lamprey in brine as cooking alone is insufficient. symptoms develop in a few hours and include nausea, vomiting, dysenteric diarrhoea, urge to urinate or defecate without ability to do so, abdominal pain and weakness. recovery takes several days and treatment is symptomatic (coad, 1979). however, lampreys lack scales and are not eaten in iran. conservation: the caspian lamprey has been proposed for inclusion in the "red book of the u.s.s.r." which forms the basis for measures to protect species (pavlov et al., 1985) and is listed as "vulnerable" in europe by lelek (1987) and maitland (1991). it is vulnerable because it migrates into rivers which are polluted and dammed and because of its restricted and declining distribution. these conditions apply particularly in iran, although there is some evidence for spawning based on captures in the 1990s (holčík and oláh, 1992). joorabian shooshtari et al. (2011) note that it is extirpated in the safid river. çiçek and sungur birekcikligil (2016) state that it is extinct in the turkish aras river basin. kiabi et al. (1999) consider this species to be near threatened in the south caspian sea basin according to iucn criteria. criteria include medium numbers, habitat destruction, widespread range (75% of water bodies), absent in other water bodies in iran, and absent outside the caspian sea basin. mostafavi (2007) lists it as near threatened in the talar river, as do banagar et al. (2008) for the haraz river, both in mazandaran. the iucn (2015) lists it as near threatened. abdollahi and imanpoor (2011), imanpoor and 264 coad/ lampreys of iran abdollahi (2011) and abdollahi et al. (2013) determined blood serum biochemical parameters and their relationship with gonadal parameters for shirud fish, useful in management of reproduction and culture of this species. relationships were attributed to the lack of feeding on migration and the use of reserved protein and fat in muscles. joorabian shooshtari et al. (2011) found iranian lampreys had relative mercury concentration in their tissues as follows: muscle > ovaries > liver > skin > testes. the mean values of mercury in muscle and testes were 192.25±7.10 and 21.42±1.48 hg ng/g dry weight, respectively. there were no significant differences between the sexes in the hg level of most tissues except for gonads. they concluded that this species could be used as an indicator of aquatic pollution as it is a scavenger on the sea floor. nasrolah pourmoghadam et al. (2015) showed that manganese could seriously affect reproductive success as spermatozoid motility decreased with an increase in concentration of this heavy metal pollutant. sources: the main source of information on this species are the summaries by holčík (1986) and renaud (2011) which should be consulted for further details on morphology and biology. further details on collections examined can be found in the museum catalogues. iranian material: cmnfi 1970-0511, 7 ammocoetes, 30-82 mm total length, gilan, shafa river estuary (37º35'n, 49º09'e); cmnfi 1970-0514, 33 ammocoetes, 24-92 mm total length, gilan, shafa river estuary (37º35'n, 49º09'e); cmnfi 19700515, 23 ammocoetes, 25-98 mm total length, gilan, shafa river estuary (37º35'n, 49º09'e); cmnfi 1970-0534, 30 ammocoetes, 47-91 mm total length, gilan, shafa river estuary (37º35'n, 49º09'e); cmnfi 1970-0535, 14 ammocoetes, 78-102 mm total length, gilan, shafa river estuary (37º35'n, 49º09'e); cmnfi 1970-0546, 2 adults, 352-355 mm total length, gilan, safid river (no other locality data); cmnfi 1970-0585, 3 adults, 406-455 mm total length, gilan, nahang roga river (37º28'n, 49º28'e); cmnfi 1971-0327a, 1 adult (part of trunk), gilan, shafa river estuary (37º35'n, 49º09'e); cmnfi 1979-0787, 9 adults, 353-428 mm total length, gilan, nahang roga river (37º28'n, 49º28'e); cmnfi 1980-0118, 8 adults, 270-317 mm total length, gilan, gelroudkhan river, tributary of the anzali mordab (no other locality data); cmnfi 1980-0119, 10 adults, 290-348 mm total length, gilan, gelroudkhan river, tributary of the anzali mordab (no other locality data); cmnfi 1980-0139, 44 ammocoetes, 36-95 mm total length, gilan, golshan river estuary (37º26'n, 49º40'e); cmnfi 2001-0011, 4, 356-365 mm total length, mazandaran, shirud (36º51'n, 50º49'e). acknowledgements i am indebted to the department of biology, shiraz university and the canadian museum of nature, ottawa for funding of research. numerous colleagues and co-authors assisted in developing the website on iranian fishes, providing specimens, data and photographs and are listed at www.briancoad.com references abakumov v.a. 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(2022) 10(5): 388-397 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article individual and mixture effects of nano-tio2 and microplastics on antioxidant and immunological responses of nile tilapia juvenile, oreochromis niloticus (l.) behzad nematdoost haghi, alireza mirvaghefi, hamid farahmand department of fisheries, faculty of natural resources, university of tehran, karaj, iran. s article history: received 14 june 2022 accepted 24 august 2022 available online 2 5 october 2022 keywords: oxidative stress immunity environmental contaminant oreochromis niloticus abstract: this study aims to evaluate the effect of nano titanium dioxide and microplastics, alone and in combination, on immune system function and oxidative stress of juvenile nile tilapia, oreochromis niloticus. for this purpose, fish were randomly divided into 27 100l tanks using a 3×3 factorial design, including three concentrations of nano titanium dioxide (0, 1, and 5 mg/l) and three concentrations of microplastic (0, 0.5, and 1 mg/l) separately and together each in triplicate. the results showed that immunoglobulin, malondialdehyde (mda), and glutathione reductase (gr) had significant effects in all groups (p<0.05). lysozyme, total antioxidant capacity (tac), and glutathione peroxidase (gpx) showed significant differences between the combined groups and the control (p<0.05). no significant differences were observed in c3 and c4 between groups exposed to mps and nano-tio2 and the control (p>0.05). superoxide dismutase (sod) showed significant differences between the group exposed to mps and nano-tio2 alone and the control group (p<0.05). in conclusion, the results showed that mps could have a synergistic effect on the toxicity of nanotio2. introduction fishes are directly or indirectly exposed to chemical compounds; their effects depend on the concentration of these substances and how they are absorbed through the gills, skin, and gastrointestinal tract (christophe et al., 2015). pollutants have various effects on fish, including physiological, immune, and reproductive responses (lawrence and hemingway, 2008; banaee et al., 2015; derikvandy et al., 2020). furthermore, since pollutants can produce active oxygen radicals and damage these substances in living organisms (lackner, 1998), oxidative stress-related damage can be used to evaluate the effects of contaminants on organisms (sies, 1985; ibrahim et al., 2021). high-consumption compounds can harm aquatic organisms such as fish if their residues enter the water. nanoparticles are used in various industries, and their production and application volume is increasing. these materials also have many correspondence: alireza mirvaghefi doi: https://doi.org/10.22034/ijab.v10i5.1676 e-mail: avaghefi@ut.ac.ir dor: 20.1001.1.23830956.2022.10.5.6.7 applications in aquaculture, including vaccine transfer, food product transport, and water refinement (shah and marz, 2020). despite these applications, entering these materials into aquatic environments harms the environment and living organisms in them (wang et al., 2008). nano titanium dioxide (nano-tio2) is one of these materials with excellent environmental dispersion. tio2, because of its unique properties, has many applications, including sunscreen production, water purification, and medication delivery (chen and selloni, 2014). due to its wide use, it can enter the aquatic environment and influence aquatic organisms. microplastics (mps), generated from garbage and plastic waste breakdown, are one of aquatic ecosystems' most critical environmental pollutants (crawford and quinn, 2016). they can act as potential vectors of various biological and nonbiological pollutants (kinigopoulou et al., 2022; https://ij-aquaticbiology.com/index.php/ijab/article/view/1676 389 int. j. aquat. biol. (2022) 10(5): 388-397 banihashemi et al., 2022). mps can absorb environmental contaminants and enter them into organisms' bodies (verla et al., 2019). this process elevates pollutant bioavailability for organisms (nematdoost haghi and banaee, 2017; banaee et al., 2019a), and pollutant accumulation (thammatorn and palic, 2022). thus, mps help transfer xenobiotics through the food chain (carbery et al., 2018). therefore, investigation of the simultaneous effect of mps and titanium dioxide nanoparticles on nile tilapia, oreochromis niloticus, was considered as this study's aim. materials and methods the fish were purchased from a tilapia breeding farm in qom and kept in a laboratory until they reached about 24 g. two weeks of acclimatization were performed on these fish before the experiment. a factorial design (3×3) was chosen for the experiment, including 0, 0.5, and 1 mg/l microplastic and 0, 1, and 5 mg/l nano-tio2in triplicate. each group consisted of 10 fish with an average weight of 23.5±0.6 g in triplicate. 100% daily water exchange was done (50% every 12 hours) to maintain a constant concentration of nano tio2 (banaee et al., 2019). water chemical factors, including temperature (25±1°c), oxygen (6±1 mg/l), and ph (7.6±0.2), were kept constant during the experimental period. the photoperiod was 12l:12d during the experiment. nano-tio2 was purchased from iranian nanomaterials pioneers (table 1). stock preparation was done every day. after ultrasonication by an ultrasonic bath (10 min, 35 khz, 100/400 w), the stocks added up to 10 l of dechlorinated water in the tanks to reach nominal concentrations (1 and 5 mg/l). sampling: on the 30th day, six fish were randomly caught from each replication of the treatments. after anesthetizing with clove extract (1/5000), blood was drawn from the caudal vein by heparinized syringes. the samples were separated by centrifugation (6000 g for 10 minutes) and stored at -28°c until the measurement of the parameters. analysis of oxidative stress and immunological parameters: sod (mccord and feridovich, 1969), gpx (johnson et al., 1999), and gr (zanetti, 1979) were measured by kits of kiazist company. cat (góth, 1991), mda (placer et al., 1966), and total antioxidants capacity (tac) (benzie and strain, 1996) were assessed. total immunoglobulin (panigrahi et al., 2005), lysozymes (lange et al., 2001), total complement (ac50), and c3 and c4 (yano, 1992) were measured using pars azmun company kits. assessment of synergism and antagonism was carried out based on banaee et al. (2020). statistical analysis: the data was analyzed based on factorial design. statistical analysis was performed using spss22 software. a two-way analysis of variance was used for data analysis, and mean comparisons were performed using the tukey test. the data normality test was performed using the shapiro-wilks test. the confidence level used in this study was 95%. finally, the results were presented as mean ± standard deviation. results there was no mortality during the experimental tio2,80%anatase20%rutile titanium dioxide +99% purity 20 nm average primary particle size (d50) 10-45 m2 g-1 specific surface area (ssa) white color 0.46 g ml-1 bulk density 5.5-6.0 ph 0.48% loss of weight in drying 0.99% loss of weight on ignition table 1. properties of nano tio2 based on the iranian nanomaterials pioneer. 390 nematdoost haghi et al./ individual and mixture effects of nano-tio2 and microplastics period. the results of oxidative stress markers are shown in table 2. sod activity showed a significant impact on factor interaction (p<0.05) (table 2). nano tio2 and mps significantly impacted sod activity (p<0.05). furthermore, sod activity showed a significant difference between groups of nano tio2 0 mg/l + mps 0.5 mg/l, nano-tio2 0 mg/l + mps 1 mg/l, nanotio2 1 mg/l + mps 1 mg/l, nano tio2 5 mg/l + mps 0 mg/l and control (p<0.05) (fig. 1). sig treatment parameters sig treatment parameter 0.000 nano-tio2 0.000 2tio-nano 0.000 microplastic lysozyme 0.000 microplastic sod 0.000 interaction 0.000 interaction 0.000 nano-tio2 0.199 2tio-nano 0.001 microplastic ach50 0.000 microplastic cat 0.086 interaction 0.000 interaction 0.000 nano-tio2 0.000 2tio-nano 0.808 microplastic c3 0.000 microplastic mda 0.713 interaction 0.000 interaction 0.078 nano-tio2 0.000 2tio-nano 0.072 microplastic c4 0.000 microplastic gpx 0.005 interaction 0.000 interaction 0.000 nano-tio2 0.000 2tio-nano 0.000 microplastic immunoglobulin 0.000 microplastic gr 0.006 interaction 0.000 interaction 0.000 2tio-nano 0.000 microplastic tao 0.000 interaction table 2. the results of two-way anova analysis of immunological and oxidative stress parameters of nile tilapia juvenile. figure 1. the antioxidative parameters of juvenile nile tilapia expose to nano titanium dioxide and moicroplastics. different letters show significant differences. data are shown as mean±sd. 391 int. j. aquat. biol. (2022) 10(5): 388-397 the impact of the two factors' interaction and mps on cat activity was significant (p<0.05), but the effect of nano tio2 showed no significant difference (p>0.05) (table 2). cat activity indicated significant changes in all groups with control (p<0.05) except nanotio2 1 mg/l + mps 1 mg/l and nano-tio2 5 mg/l + mps 1 mg/l (fig. 1). mda activity revealed significant impacts of the interaction of two factors and each of them separately (p<0.05) (table 2). mda activity showed a significant increase in all groups (p<0.05) (fig. 1). the interaction of nano-tio2, and mps showed a significant effect on gpx activity (p<0.05) (table 2). gpx activity increased in all groups compared to control but only significant in nano-tio2 0 mg/l + mps 1 mg/l, nano-tio2 1 mg/l + mps 1 mg/l, nanotio2 5 mg/l +mps 0.5 mg/l and nano-tio2 5 mg/l + mps 1 mg/l in groups (p<0.05) (fig. 1). the effect of each parameter on gr activity was similar (p<0.05) (table 2). gr activity decreased significantly in all groups (p<0.05) (fig. 1). tac showed a significant impact of two factors and their interaction on the results (p<0.05) (table 2). tac showed a significant decrease in all groups (p<0.05) (fig. 1). table 3 shows that nano-tio2 and mps had a synergistic effect on sod, cat, mda, and gpx activities. the results of ach50 revealed no significant effects of factor interaction. the impact of nanotio2 and mps were also significant (p<0.05) (table 2). total complement’s results revealed a significant decrease in nano-tio2 1 mg/l + mps 0 mg/l, nanotio2 1 mg/l + mps 1 mg/l, nano-tio2 5 mg/l + mps 0.5mg/l, and nano-tio2 5 mg/l + mps 1m g/l with control (p<0.05) (fig. 2). no significant effect of interaction observed in the c3 (p>0.05). among the two factors, only nano-tio2 showed a significant impact on the results of c3 (p<0.05) (table 2). c4 showed a significant effect of nano-tio2 and mps interaction (p<0.05). none of the contaminants alone showed a significant impact on results (p>0.05) (table 2). c4 result showed no significant parameters groups predicted effect observed effect synergy ratio combined effects sod 1nano+0.5 microplastic 4.78 0.73 6.54 s 1nano+1 microplastic 4.24 0.58 7.31 s 5nano+0.5 microplastic 2.91 0.68 4.27 s 5nano+1 microplastic 2.6 0.62 4.19 s cat 1nano+0.5 microplastic 4.64 3.78 1.22 s 1nano+1 microplastic 7.82 0.67 11.67 s 5nano+0.5 microplastic 5.71 3.77 1.51 s 5nano+1 microplastic 9.62 0.58 16.58 s mda 1nano+0.5 microplastic 53.78 26.84 2 s 1nano+1 microplastic 60.91 36.66 1.66 s 5nano+0.5 microplastic 60.29 25.78 2.33 s 5nano+1 microplastic 68.27 55.61 1.22 s gpx 1nano+0.5 microplastic 1.47 1.2 1.22 s 1nano+1 microplastic 1.53 1.37 1.11 s 5nano+0.5 microplastic 1.36 1.3 1.04 s 5nano+1 microplastic 1.41 2.24 0.62 a gr 1nano+0.5 microplastic 0.14 0.21 0.66 a 1nano+1 microplastic 0.03 0.15 0.2 a 5nano+0.5 microplastic 0.09 0.24 0.37 a 5nano+1 microplastic 0.05 0.15 0.33 a tao 1nano+0.5 microplastic 0.37 0.55 0.67 a 1nano+1 microplastic 0.35 0.44 0.79 a 5nano+0.5 microplastic 0.34 0.42 0.8 a 5nano+1 microplastic 0.33 0.29 1.13 s table 3. the combined effects of contaminants on enzyme activity. 392 nematdoost haghi et al./ individual and mixture effects of nano-tio2 and microplastics differences (p<0.05) (fig. 2). lysozyme activity significantly affected nano tio2 and mps interaction (p<0.05). the impact of nano-tio2 and mps on lysozyme activity was significant (p<0.05) (table 2). lysozyme activity showed a significant decrease in nano tio2 1 mg/l + mps 1 mg/l and nano-tio2 5 mg/l + mps 1 mg/l group (p<0.05) (fig. 2). immunoglobulin showed a significant effect of each figure 2. immunological parameters of juvenile nile tilapia exposed to nano-tio2 and mps. different letters show significantly differences. data are shown as mean±sd. parameters groups predicted effect observed effect synergy ratio combined effects ach50 1nano+0.5 microplastic 0.85 0.93 0.91 a 1nano+1 microplastic 0.87 0.9 0.96 a 5nano+0.5 microplastic 0.86 0.92 0.93 a 5nano+1 microplastic 0.88 0.87 1.01 s c3 1nano+0.5 microplastic 0.88 0.78 1.13 s 1nano+1 microplastic 0.78 0.79 0.98 a 5nano+0.5 microplastic 0.79 0.76 1.03 s 5nano+1 microplastic 0.7 0.78 0.89 a c4 1nano+0.5 microplastic 1.09 1.12 0.97 a 1nano+1 microplastic 1.12 0.99 1.13 s 5nano+0.5 microplastic 1.12 0.97 1.15 s 5nano+1 microplastic 1.15 1 1.15 s lysozyme 1nano+0.5 microplastic 1.04 0.63 1.65 s 1nano+1 microplastic 1.18 0.4 2.95 s 5nano+0.5 microplastic 0.71 0.72 0.98 a 5nano+1 microplastic 0.81 0.45 1.8 s immunoglobulin 1nano+0.5 microplastic 0.26 0.2 1.3 s 1nano+1 microplastic 0.19 0.15 1.26 s 5nano+0.5 microplastic 0.16 0.18 0.89 a 5nano+1 microplastic 0.12 0.08 1.5 s table 4. the combined effects of contaminants on immunological parameters. 393 int. j. aquat. biol. (2022) 10(5): 388-397 contaminant and their interaction (p<0.05) (table 2). the result of immunoglobulin showed a significant decrease in all groups (p<0.05) (fig. 2). table 4 shows nano-tio2 and mps synergistic effects on c4, immunoglobulin, and lysozyme activity. discussion biomarkers of oxidative stress: simultaneous exposure of organisms to different contaminants in the aquatic environment is inevitable, but awareness of their co-exposure impact can help deal with their effects. one of the critical impacts of nano-titanium and mps on organisms is oxidative stress (kim et al., 2021; mahboob et al., 2017) and the generation of reactive oxygen species (ros). oxidative stress occurs when an imbalance of oxidants/antioxidants happens in the cells (birben et al., 2012). organisms can counteract ros's impact using an antioxidant defense system that includes enzymatic and nonenzymatic parts. the enzymatic part contains a set of enzymes that prevent the formation of free radicals (li et al., 2010). the prominent role of these enzymes is to neutralize active oxygen radicals. sod, cat, and gpx act as the primary line of this system (ighodaro and akinloye, 2018; derikvandy et al., 2020). the first part of these enzymes, when organisms are exposed to xenobiotics, is sod (ighodaro and akinloye, 2018). when sod is exposed to ros, it converts superoxide radicals to hydrogen peroxide (sakamoto and imai, 2017; ibrahim et al., 2021), and afterward, the other enzymes act. thus, higher sod activity can counteract the organism with higher production of ros. however, with the elevation of ros concentration, due to the inability of the body to respond to ros increase, the system collapses and loses the ability to return to normal conditions. sod declines after fish exposure to contaminants such as pesticides (kaur and jindal, 2017). catalase plays an essential role in the antioxidant defense system of organisms by breaking down hydrogen peroxide (catalan et al., 2018), preventing oxidative stress and protecting cells. catalase elevation after exposure to nano-tio2 and mps can be due to the body’s response to the increasing hydrogen radicals. also, the decrease in catalase activity in combination groups can be due to producing catalase to reduce the ros levels. le thu et al. (2021) reported catalase decline after exposure of nile tilapia to cadmium. similar results were presented by chitra and maiby (2014) and sayed et al. (2003). mda is one of the final products of lipid peroxidation that can form non-enzymatically by ros inducement or enzymatically by lipoxygenase activity (farmer and muller, 2013). the level of mda shows induced oxidative stress (karadag et al., 2014) and injuries to cells, tissues, and organs (ayala et al., 2014). elevation of lipid peroxidation is a sign of ros increase (faheem and lone, 2018) and probably the incapability of antioxidant defense to cope with ros generation. gpx is an enzyme that finds in mitochondria and peroxisome compartments and reduces h2o2 to h2o. this reaction inhibits the harmful impacts of hydrogen peroxide; therefore, the elevation of gpx activity leads to the diminishment of oxidative stress (lubos et al., 2011). gr is another member of the glutathione family that produces gsh from gssg through utilizing nadph and maintains the reduced glutathione (gsh) content (couto et al., 2016). it plays an essential role in cellular antioxidant protection and regulates metabolic pathways (cazenave et al., 2006). elevation of gpx can show the reaction of the antioxidant system of fish against oxidative stress, as reported by hanachi et al. (2020) in the exposure to pesticides and microplastic in zebrafish. cellular total antioxidant capacity includes the enzymatic and non-enzymatic antioxidants that regulate the balance between peroxidants and antioxidants in cells. therefore, a significant decrease in the total antioxidant levels may indicate the collapse of cellular antioxidant defense systems (fraga et al., 2014). a significant decrease in tac in the present study is similar to other studies (banaee and taheri, 2019; taheri et al., 2017). tac decrease can signify oxidative stress in the organism exposed to mps and nano-tio2 394 nematdoost haghi et al./ individual and mixture effects of nano-tio2 and microplastics (hamed et al., 2020). moreover, it can be due to the consumption of antioxidants to cope with stress. immunity parameters: magnadottir (2010) opined that the immune system of fish is diverse, and its response rate can differ in different fish species exposed to different conditions. immunoglobulins are used as a marker of the immune system function when the organism is exposed to environmental stressors. immunoglobulins are essential in maintaining mucosal homeostasis (angeles esteban, 2012). as mature b lymphocytes synthesize immunoglobulin, the factors that affect the number of lymphocytes and the tissues that generate them can affect immunoglobulin levels (banaee et al., 2019b). immunoglobulin reduction in this study indicates debilitation of the immune system, as reported by hong et al. (2018). immunoglobulin decline due to a decrease in total ig synthesis and destruction and apoptosis of liver cells exposed to contaminants reported previously by banaee (2013). lysozyme, a mucolytic enzyme, is one of the best indicators of the innate immune system (saurabh and sahoo, 2008; li et al., 2021). exposure to nanotio2 and mps decreased the lysozyme activity, which can signify immune system impairment (ahmadi et al., 2014). li et al. (2013) reported that a significant reduction of lysozyme activity after exposure to environmental contaminants could be due to a decrease in this enzyme biosynthesis by phagocytic cells and, or a reduction of monocyte and granulocyte counts. furthermore, its increase after exposure to low concentrations of xenobiotics can be due to the effort of the immune system to neutralize changes in body condition. the complement system, as a part of the fish defense system, comprises a group of proteins produced by different organ cells, including the liver and intestinal epithelium (boshra et al., 2006). a decrease in ach50 can be another sign of the immune system weakening. such a decline in ach50 can also be due to hepatocyte and mononuclear phagocyte damage (bitsayah et al., 2018; banaee et al., 2019a, b). c3 is a protein made by liver cells and monocytes. activation of this component of the complement system makes the classical and alternative systems active (janssen et al., 2005). c4 is a protein made by monocytes and macrophages. it plays a vital role in the immune response (mortensen et al., 2015), and its cooperation after identifying the infectious agents with other immune system proteins help to destroy them (pushpa et al., 2014). high levels of c3 and c4 in the serum indicate fish health (yano, 1992). a significant reduction in the c3 and c4 levels can signify stressors' impact on weakening the immune system. these proteins did not show significant changes when exposed to the selected concentration of nano-tio2 and mps alone and in combination. conclusion mps and nano-tio2 can cause an imbalance in reactive oxygen production and antioxidant capacity, leading to oxidative stress. they also can affect the immune response. co-exposure to these contaminants can cause synergistic and accumulative impacts on organisms. the results indicated that even low concentrations of mps and nano-tio2 could impact oxidative parameters in juvenile nile tilapia. simultaneously exposure to mps and nano-tio2 showed a more significant impact and synergistic effect on the assessed indicators. although some immunological parameters did not show significant changes (probably due to low concentration of contaminant or low experimental period), the results showed that mps and nano-tio2 could synergistically impact on fish health parameters. acknowledgments it is necessary to appreciate the fisheries department of the faculty of natural resources of tehran university for providing the conditions for conducting experiments and for financial support. references ahmadi k., mirvaghefei a. r., banaee m., vosoghei ar. 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(2022) 10(2): 119-130 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article aquaculture by-product meal as a fishmeal replacer in african catfish (clarias gariepinus) diet: effects on serum biochemistry, innate immune response, and oxidative stress markers wasiu adeyemi jimoh*,1ahmed ayodeji ayeloja, isioma emmanuel mowete, yusuf olatunji yusuf, musa idi-ogede abubakar department of aquaculture and fisheries, faculty of agriculture, university of ilorin, pmb 1515, ilorin, nigeria. s article history: received 7 june 2021 accepted 21 august 2022 available online 2 5 april 2022 keywords: antioxidants superoxide dismutase blood electrolytes blood glucose catalase abstract: the effect of feeding aquaculture by-product meal (abp) to african catfish (clarias gariepinus) was investigated in a 56-day feeding trial using serum biochemistry, innate immune response, and oxidative stress markers as indices of assessment. fishmeal protein in control diets was replaced at a rate of 15, 30, 45, and 60% by aquaculture by-product meal protein. each experimental diet was randomly distributed into triplicate tanks containing catfish fingerlings (n = 15 fingerlings/replicate, 5.58±0.05 g). the primary haematological parameters (haemoglobin, packed cell volume, red blood cell count) and secondary haematological parameters (mch, mcv, and mchc) were similar to the control. the white blood cell count and its differential of the fish group fed abp meal was numerically higher than control but not significant, except in fish fed d30t that had high lymphocyte count. the platelet count in all the dietary groups was similar. there was no significant variation in some of the serum biochemistry parameters: total protein, albumin, globulin and albumin/globulin ratio, urea, hdl-c and ldl-c. creatinine values of the d60t-fed group were significantly higher than all other dietary treatment groups including the control. triglyceride level was statistically similar with control up to 30% replacement level, while there were no significant variations in the cholesterol levels of the blood of c. gariepinus fed the different dietary treatments. except for catalase, there were no significant differences in other oxidative stress biomarkers under study, primarily sod, gsh, and gpx. catalase enzyme activities of the fish group fed d30t were statistically higher than other fed groups. some serum electrolytes, such as calcium and chloride ions of the differently fed fish groups, were not significantly different. lastly, serum potassium ions were significantly higher among d60t-fed group though statistically similar to d45t-fed group. no stress conditions were recorded among the dietary groups. these results showed that the health status and immunity of african catfish were not degraded by feeding aquaculture by-product meal to the fish. introduction the global human population increase will reach 9.7 billion by 2050 (united nations, 2019; united nations, 2015). this will lead to increased food demand and the need to increase food supply by up to 70% of the current supply capacity (hunter et al., 2017). increased animal production to meet man's growing protein demand has drawbacks, including, but not limited to, water scarcity, habitat destruction, and biodiversity loss (rivera‐ferre et al., 2016; benton et al., 2018; michalk et al., 2019). aquaculture production, being the fastest growing sector in food production, portends the ability to meet a larger *correspondence: wasiu adeyemi jimoh e-mail: jimoh.wa@unilorin.edu.ng percentage of animal-derived protein (fao, 2018). thus, the expansion of aquaculture production in recent years is central to sustained human nutrition (hua et al., 2019). however, the long-term expansion of aquaculture means a reduced reliance on forage fish, which is a key component of fishmeal. although the proportions of fish meal and fish oil from forage fish in aquafeeds have declined over the last 20 years, they remain key feed components for many carnivorous fish and crustaceans (turchini et al., 2019). increased demand for fishmeal and fish oil as raw materials for aquafeed manufacture has been identified as a likely contributor to overfishing and a 120 jimoh et al./ aquaculture by-product meal as a fishmeal replacer in african catfish global fisheries disaster (türkmen, 2019). many researchers have reported the viability of replacing fish meal with a variety of plant protein sources (see tacon et al., 2009), but antimetabolites, and limited expansion potential due to increased pressure on land and water resources, serve as a barrier to the long-term use of plant protein sources as fish feed ingredients (döös, 2002; malcorps et al., 2019). hua et al. (2019) projected that in some years (2025), extra 37.4 million tons of aquafeed would be needed to meet aquaculture production feed requirements. hence, researching alternative feed ingredients to fishmeal beyond plant protein sources becomes imperative. aquaculture by-product meal remains a veritable substitute for conventional fish meal, and more imprtly, it is an animal protein source that is economically viable and sustainable (gümüş and aydin, 2013; kim et al., 2018). the inedible portion of fish, mainly fish viscera, discarded as waste from the fish processing industry could be used as a replacer of fishmeal (irm et al., 2020). they are generally referred to as aquaculture by-products, i.e., remnants obtained after fish processing for human consumption (olsen et al., 2014; stevens et al., 2018). nearly 10% of globally-produced fishmeal is from aquaculture byproducts (ytrestøyl et al., 2015; fao, 2018). fagbenro et al. (2005) reported that 25-35% of waste products could be generated from catfish processing. these can be reduced to aquaculture by-product meal (abm) after cooking and drying the waste products. the chemical composition of fish visceral differs with processing, species, nutrient intake, sex, age, seasonality, etc. (huss, 1995). abm has inferior nutritional contents consisting mainly of reduced crude protein (52-67%), higher lipid (12-23%), and ash content (7-14%) (goddard et al., 2008; kim et al., 2014) compared to a fish meal made from wild catches (national research council, 2011; ween et al., 2017). this could restrict its complete replacement for conventional fishmeal. hua et al. (2019) indicated that a vast potential exists to increase the volume of meals generated from aquaculture by-products due to aquaculture production projected to reach 109 million tons in 2030. in nigeria, catfish is the most cultured fish species. farmers supplement their fish feeding in earthen ponds using fresh fish viscera from fish processing plants without aeration. this could lead to oxygen depletion and pathogenic organisms could also develop due to the decay of uneaten fish viscera. abm obtained from aquaculture has been successfully used in practical diets of many fish (goddard et al., 2008; tacon et al., 2009; kim et al., 2014; kim et al., 2018; irm et al., 2020). hernández et al. (2004) included fish viscera meal in the diet of shrimp (litopenaeus vannamei), while trout processing waste was also used in gilthead bream (sparus aurata) practical diets (kotzamanis et al., 2001). other scientists that have included fish visceral in the practical diets of asian catfish (clarias batrachus) are giri et al. (2000) and gupta and gupta (2013). as far as we know, a lack of information exists on the use of fish visceral meal in the diet of african catfish (clarias gariepinus burchell) (osho et al., 2019; jimoh et al., 2021). the effect of abm on serum biochemistry, innate immune response and oxidative stress markers, and the response of african catfish needs to be investigated. therefore, this study examines the effect of fishmeal substitution, abm, on serum biochemistry, innate immune response, and oxidative stress markers of african catfish. materials and methods preparation of aquaculture by-product meal and diet preparation: fresh offal of fish, consisting mainly viscera, was obtained from fish processing facilities of the university of ibadan fish farm (7°26’29.1”n, 3v53’59.1”e), and cooked for 25 minutes following procedures to reduce the fat content and eliminate possible pathogen (saleh et al., 2020). the cooked offal was blended and sieved to produce an abm after oven-drying it at 50°c for 72 hours. the resulting meal was analyzed for its proximate composition and kept frozen (-4°c) until used for diet formulation (table 1). proximate composition of the feed ingredients and feed preparation process were given in our previous study (jimoh et al., 2021). 121 int. j. aquat. biol. (2022) 10(2): 119-130 experimental fish and system: the healthy and actively swimming fingerlings of african catfish (5.58±0.05 g) were procured from a hatchery at yesha-yahu nigeria ltd., egbejila, ilorin, kwara state, nigeria and acclimated for 14 days in an intermediate bulk container (ibc tanks) in the laboratory of the university of ilorin, nigeria. the fingerlings were fed a commercial pelleted diet (1.8 mm skretting feed production co. inc) for african catfish. after acclimation, 225 fingerlings were randomly stocked into fifteen 60-l rectangular glass tanks (76x35x30 cm) and each tank was continuously aerated. fishmeal protein in control diets was replaced at a rate of 15, 30, 45, and 60% by aquaculture byproduct meal protein. each experimental diet was randomly distributed into triplicate tanks containing catfish fingerlings (n = 15 fingerlings/replicate). the fish were fed in two equal proportions at 0900 and 1700 h for eight weeks. the following water quality parameters, temperature (27.32±0.26oc); ph experimental diet ingredient composition (g kg-1) control d15t d30t d45t d60t fishmeal @ 68% crude protein 287.2 244.1 200.9 158.0 114.9 abm @ 47.83% crude protein 0.00 61.2 122.5 183.7 245.0 sbm @ 38% crude protein 450.0 450.0 450.0 450.0 450.0 maize @ 10% crude protein 100.0 100.0 100.0 100.0 100.0 fish premix* 40.0 40.0 40.0 40.0 40.0 fish oil 5.0 5.0 5.0 5.0 5.0 soybean oil 5.0 5.0 5.0 5.0 5.0 starch 112.8 946 76.6 58.3 40.1 proximate composition (g kg-1) moisture 86.7 82.1 77.5 72.9 67.9 crude protein 391.8 389.6 387.2 385.1 383.9 crude lipid 143.1 142.7 142.4 142.0 141.2 ash 71.8 69.4 67.1 64.8 62.4 crude fiber 20.2 21.5 22.8 24.1 25.5 nfe 286.5 294.7 303.0 311.1 319.2 energy (kcal/g) 48.2 48.4 48.7 48.9 49.2 calculated amino acid composition (g kg-1) arginine 30.3 28.8 27.3 25.9 24.3 histidine 9.7 9.6 9.5 9.4 9.3 isoleucine 18.6 17.7 16.8 15.8 14.8 leucine 30.7 29.3 27.9 26.5 25.0 lysine 28.7 27.4 26.0 24.6 23.2 methionine 8.9 8.4 7.9 7.4 6.9 methionine + cysteine 14.1 13.6 13.0 12.5 11.9 phenylalanine 17.8 17.1 16.4 15.6 14.9 phenylalanine + tyrosine 30.9 29.5 28.1 26.7 25.2 threonine 18.2 17.2 16.2 15.2 14.1 tryptophan 4.7 4.4 4.2 3.9 3.6 valine 20.2 19.3 18.4 17.5 16.6 *1 kg aero-mix® fish premix contains vitamin a 25,000,000 iu, vitamin d3 2,000,000 iu, vitamin e 200,000 iu, vitamin k 8000 mg, vitamin b2 20,000 mg, vitamin c 500,000 mg, niacin 150,000 mg, pantothenic acid 50,000 mg, vitamin b6 12,000 mg, vitamin b12 10 mg, folic acid 4000 mg, biotin 800 mg, choline chloride 600,000 mg, cobalt 2,000 mg, copper 4,000 mg, iodine 5,000 mg, iron 40,000 mg, manganese 50,000 mg, selenium 200 mg, zinc 40,000 mg, antioxidant 100,000 mg, lysine 100,000 mg, methionine 100,000 mg manufactured by aerobic integrated concept limited km 130, lagos ibadan expressway, hossanah bus stop, opposite islim filling station, p. o. box 22109 ui post office , oyo state, nigeria table 1. ingredients (g kg-1 as fed basis) and nutrient composition (g kg-1) of the experimental diets containing abm (jimoh et al., 2021). 122 jimoh et al./ aquaculture by-product meal as a fishmeal replacer in african catfish (6.57±0.42), and dissolved oxygen (6.87±0.26 mg/l) were recorded during the period of the experiment. blood sampling and assessment: six (n=6) fish per replicate tank were removed and mildly euthanized with clove oil (100 mg l-1) at the end of the feeding trial for blood sampling. 3.5 ml blood per replicate was obtained by caudal vein piercing using a 1 ml disposable syringe and 25g needle. approximately, 1 ml blood sample was collected in an ethylene diaminetetraacetic acid (edta) treated bottle for haematological examination using an automatic full blood cell counter. the remaining (above 2.5 ml) blood sample was collected in untreated or plain sampling bottles for serum biochemistry after it was allowed to clot at 4°c. after 30 minutes of clotting, the coagulated blood samples were centrifuged at 8,000 rpm for 6 min, and the serum was collected following the procedure of tomlinson et al. (2013) for onward serum biomarkers assay. the serum total protein was determined using a commercial kit (randox laboratories ltd, u.k), while the bromocresol green method was used to determine albumin value (doumas et al., 1971; doumas and peters jr, 1997). the differential between the serum total protein and serum albumin formed serum globulin value (colville, 2002). the method of toro and ackermann (1975) was used to determine blood glucose. the procedures of reitman and frankel (1957) were used to determine serum alanine aminotransferase (alt) and serum aspartate aminotransferase (ast). a commercial kit (randox laboratories ltd, u.k) was used to analyze serum creatinine and urea nitrogen by deproteinization and urease-berhelot colorimetric methods modified by berthelot-searcy (searcy, 1967; scott, 1979). lowdensity lipoproteins (ldl) and high-density lipoproteins (hdl) using kits (biomed-cholesterol, chem. for lab technology, cairo, egypt) and serum triglyceride were determined by the colourimetric method (chawla, 2014). oxidative stress biomarkers and serum electrolytes: serum antioxidants, such as catalase (cat), superoxide dismutase (sod), glutathione (gsh) and glutathione peroxidase (gpx), thiobarbituric acid and reactive substances (tbars), were determined spectrophotometrically using commercial kits (randoxtm, united kingdom), following standard manufacturer’s guidelines after homogenization and centrifugation of the serum. serum electrolytes (na+, k+, ca2+and cl-1) were obtained using atomic absorption spectrophotometer. these were done at central laboratory, tanke ilorin. proximate analysis: feed and feed ingredients’ proximate composition were assessed with the procedures of aoac (2010). dry matter was evaluated after the samples were subjected to 105°c for 24 h in an oven (dhg 9053a, axiom medical ltd, usa). crude protein content was measured after acid digestion using a protein auto-analyzer (foss tecator kjeltectm 8400) using a factor of 6.25 to convert the nitrogen into crude protein. crude lipid was analyzed by the soxhlet extraction method using foss tecator soxtectm 8000. the crude fiber was determined using foss tecator fibertec 2010 analyzer. the ash content was obtained by igniting the samples in a furnace (krupp widia-fabrik) at a temperature of 600°c for 4 h. the nfe was calculated using the formula of: nfe = 100(moisture + crude protein + crude lipid + ash = crude fibre) ethical statement: the ethics of animal research as contained in the university of ilorin’s research policy on the use and care of animals were strictly followed. statistical analysis: the data obtained from body indices were expressed as mean ± se and analyzed using one-way analysis of variance (anova) after the data had passed the levene test of homogeneity of variance. duncan multiple range tests were used to separate treatment means where the significant difference (p<0.05) was recorded. results haematology: the haematological parameters of c. gariepinus fed diets containing abm are presented in table 2. the primary haematological parameters (haemoglobin, packed cell volume, and red blood cell count) and secondary haematological parameters (mean corpuscular haemoglobin, mean corpuscular volume, and mean corpuscular haemoglobin 123 int. j. aquat. biol. (2022) 10(2): 119-130 concentration) were similar (p>0.05) with control. figure 1 shows the innate immune response parameters (wbc, its differential, and platelet count) of african catfish fed abm. the white blood cell count and its differential of the fish group fed abp meal were numerically higher than control but statistically not significant (p>0.05), except in fish fed d30t that had higher lymphocyte count. the platelet count in all the dietary groups was similar (p>0.05). serum biochemistry: there was no significant variation (p>0.05) in some of the serum biochemistry parameters: total protein, albumin, globulin, and albumin/globulin ratio, urea, high-density lipoprotein cholesterol (hdl-c) and low-density lipoprotein cholesterol (ldl-c) (table 3). creatinine values of the d60t-fed group were significantly higher (p<0.05) than all other dietary treatments, including the control. glucose level was significantly lower (p<0.05) among the abm-fed groups than in the control group. the cortisol was significantly lower (p<0.05) among the abp meal-fed groups than the control group except for d45t and d60t (fig. 2). lipid function indices (triglycerides and cholesterol levels) of c. gariepinus fed diets containing abm are given in figure 3. triglyceride level was similar (p>0.05) to control up to 30% replacement level, while there were no significant variations (p>0.05) in the cholesterol levels of the blood of c. gariepinus fed the different dietary treatments. oxidative stress biomarkers and serum electrolytes: except for catalase, there were no significant differences (p>0.05) in other oxidative table 2. haematological parameters of african catfish fed diets containing abm. ctr d15t d30t d45t d60t haemoglobin (g dl-1) 11.50±0.65a 12.50±1.20a 13.75±0.15a 11.65±1.25a 11.35±0.75a pcv (%) 39.85±2.05a 43.4±5.20a 53.60±4.40a 40.40±3.60a 34.35±0.15a rbc (106 l-1 ) 2.75±0.19a 3.00±0.40a 3.89±0.24a 2.62±0.29a 2.10±0.35a mch (ρg) 40.50±0.55a 42.90±0.40a 41.95±1.65a 44.45±0.05a 41.90±1.50a mcv (fl) 144.90±2.50a 146.55±0.05a 151.55±1.75a 154.85±2.85a 140.45±0.35a mchc (g l-1) 280.00±1.00a 293.00±3.00a 277.50±8.50a 154.85±5.00a 298.50±11.50a values (means with standard errors, n=3) within the same row with different superscripts are significantly different (duncan test; p<0.05) from each other. (tacon et al., 2009). table 3. serum biochemistry of african catfish fed diets containing abm. ctr d15t d30t d45t d60t total protein (mg ml-1) 16.18±2.03a 16.11±0.26a 12.75±1.60a 16.65±0.17a 17.18±0.70a albumin (mg ml-1) 13.79±1.66a 11.01±0.88a 9.87±0.24a 11.03±0.11a 10.82±0.41a globulin (mg ml-1) 2.32±0.37a 5.10±1.14a 2.88±1.37a 5.62±0.07a 6.36±0.29a a/g ratio 5.98±0.23a 2.31±0.69a 4.39±2.01a 1.96±0.01a 1.70±0.01a urea (mg dl-1) 7.55±0.25a 9.45±1.75a 11.65±1.65a 13.55±0.07a 9.90±0.28a creatinine (mg dl-1) 2.19±0.68b 2.56±0.10ab 1.26±0.17b 2.60±0.14ab 3.70±0.43a hdl-c (mg dl-1) 36.60±3.00a 36.10±2.00a 32.25±0.05a 36.70±2.20a 35.20±0.70a ldl-c (mg dl-1) 82.55±33.61a 83.86±4.81a 82.53±4.46a 106.77±16.75a 71.64±17.08a values (means with standard errors, n=3) within the same row with different superscripts are significantly different (duncan test; p<0.05) from each other. table 4. oxidative stress biomarkers in the blood of african catfish fed diets containing abm. ctr d15t d30t d45t d60t sod (u mg-1 protein) 118.07±19.03a 154.24±15.99a 147.63±2.24a 121.31±31.02a 153.63±1.61a gsh (u mg-1 protein) 156.55±6.28a 207.10±18.57a 277.77±9.47a 174.48±41.26a 209.81±6.93a gpx (u mg-1 protein) 24.82±9.29a 26.98±3.28a 24.33±0.59a 25.44±11.18a 22.65±0.39a cat (u mg-1 protein) 1932.10±103.48c 1666.90±62.33c 3541.4±265a 2059.5±266.99bc 2674.9±113.71b tbars (u mg-1 protein) 0.26±0.01a 0.34±0.03a 0.37±0.02a 0.29±0.07a 0.34±0.01a values (means with standard errors, n=3) within the same row with different superscripts are significantly different (p<0.05) from each other. sod: superoxide dismutase; cat: catalase; gsh: glutathione; gpxglutathione stransferase, thiobarbituric acid reactive substances (tbars). 124 jimoh et al./ aquaculture by-product meal as a fishmeal replacer in african catfish stress biomarkers under study, primarily, sod, gsh, and gpx (table 4). catalase enzyme activities of the fish group fed d30t was higher (p<0.05) than other groups. no significant variations (p>0.05) were recorded in the catalase enzyme of fish fed ctr, d15t, and d45t. t-bars of the different dietary treatment groups were similar (p>0.05). some serum electrolytes, such as calcium and chloride ions of the fish fed the different diets, were not significantly different (p>0.05) (table 5). serum potassium ions were significantly higher (p<0.05) among d60t-fed group though similar (p>0.05) to the d45t-fed group. serum sodium ions of the fish group fed d30t was higher (p<0.05) than other groups; the fish group fed d30t was higher (p<0.05) than other fed groups through similarity (p>0.05) exists in serum sodium ion of fish fed ctr, d45t and d60t. discussions this study recorded a lower stress condition occasioned by comparable haematology and the higher antioxidant levels among abm-fed groups. hematological parameters are important in evaluating the health status of fish (fazio et al., 2013; jimoh et table 5. serum electrolytes of african catfish fed diets containing abm. ctr d15t d30t d45t d60t calcium (mg dl-1) 17.65±2.57a 20.69±0.90a 19.62±0.49a 19.34±1.47a 20.38±0.25a chlorine (meq l-1) 17.67±2.05a 18.22±0.90a 13.37±0.09a ±18.81±0.95a 13.95±1.09a potassium (meq l-1) 4.01±0.75bc 3.33±0.41bc 2.29±0.08c 6.05±0.44ab 7.05±1.39a sodium (meq l-1) 32.18±0.38c 37.05±2.10b 52.05±1.20a 34.13±0.98bc 35.10±0.45bc values (means with standard errors, n=3) within the same row with different superscripts are significantly different (p<0.05) from each other. figure 1. wbc, wbc differential and platelet count of african catfish fed abm. bar values (means with standard errors, n = 3) with different letters are significantly different (duncan test; p<0.05) from each other. mid: combined values of the other types of wbc not grouped as lymphocytes or neutrophil (eosinophil and basophil and monocytes) 125 int. j. aquat. biol. (2022) 10(2): 119-130 al., 2015; jimoh et al., 2020a, b; sezgin and aydın, 2021). primary haematological parameters (rbc, wbc, hb, and pcv) recorded in this study show that the fish health was not compromised by feeding abm. improved health status of fish is represented by an increase in the level of pcv, rbc, and hb arising from blood cell component formation (hematopoiesis) and reduced rupture of rbc (hemolysis) in the body system (clauss et al., 2008; hoseini et al., 2020b). hoseini et al. (2020a) reported that haematological parameters, especially rbcs, enhance the oxygenation of tissues which promotes energy expenditure, good health, and growth improvement. wbc and their differentials, consisting of granulocytes (basophil, neutrophil, and eosinophil) and agranulocytes (lymphocytes and monocytes), are frontline defence systems and constitute innate immune response against invading pathogens in fish (taheri mirghaed et al., 2020). a statistically similar elevated level of wbc and their differentials recorded in this study among abp meal-fed group relative to the control group indicated that the use of abm in the feed did not have a negative effect on the immune system of the fish similar to what was reported by yousefi et al. (2012). thus potentiate a similar capacity to boost innate immunity as the fishmeal. platelet counts in all the dietary groups were similar, revealing that the fish fed different dietary treatments had a similar immune response. platelets play a significant role in innate immune and inflammation responses aside from their traditional role in blood clotting (holinstat, 2017). plasma globulin improved among abm-fed fish compared to control though no significant variation existed among the dietary treatment groups. plasma globulin also plays a diverse role in fish immunity and the antioxidant capacity of fish (gerwick et al., 2002). lymphocytes produce immunoglobin (ig) that helps detect the pathogen (taheri mirghaed et al., 2020). thus, the higher but non-significant level of wbc and their differentials among the fish fed with different dietary treatments potentiates higher but the comparable ability of the figure 2. physiological stress indices (blood glucose and cortisol levels) of clarias gariepinus fed diets containing abm. bar values (means with standard errors, n = 3) with different letters are significantly different (duncan test; p<0.05) from each other. figure 3. lipid function indices (triglycerides and cholesterol levels) of clarias gariepinus fed diets containing abm. bar values (means with standard errors, n = 3) with different letters are significantly different (duncan test; p<0.05) from each otherr. 126 jimoh et al./ aquaculture by-product meal as a fishmeal replacer in african catfish fish to fight against invading pathogen when compared to control. this improves fish health and immune function as evidenced by reduced glucose and cortisol level among the fish group fed abp meal in this study. elevated cortisol among the d45t and d60t-fed fish groups could lead to immunosuppression. cortisol is a stress hormone that activates energy expenditure and immunosupression, thus growth reduction (dawood et al., 2020; hoseini et al., 2021; taheri mirghaed et al., 2020). the non-significant levels of oxidative stress biomarkers, primarily antioxidant enzymes (sod. cat. gpx and gsh) and lipoperoxidation marker (tbars), among the fish, fed the various dietary treatments further showed no stress was recorded by feeding abm. sod, gpx and cat are important components of the antioxidant system (jimoh et al., 2021), but a higher level of these enzymes in the blood would overwhelm stress (yilmaz, 2019; hoseini et al., 2020a). t-bars being similar among the treatments indicated comparatively no change in the oxidative stress condition of fish fed the different diets. instead, the antioxidant system was stimulated because the lipid was protected from being oxidized as shown in the elevated levels of t-bars among the abm-fed fish. the increased level of t-bars was consequently counteracted by the increased antioxidant enzyme activities to reduce the negative effect of oxidative stress. this is consistent with the reports of hoseini et al. (2019) and rajabiesterabadi et al. (2020). antioxidants offer profound protection to rbc membrane lipid from being oxidized, guiding against oxidative stress conditions which cause lower rbc destruction and hemolysis (yang et al., 2013; gao et al., 2016; hoseini et al., 2020a). lipid function indices, such as triglycerides and cholesterol in this study, appeared to have a similar trend to the report of saleh et al. (2020) on european seabass (dicentrarchus labrax) fed bioconverted fish waste. the serum ionic composition of fish plays important role in the maintenance of osmotic potentials and the normal physiological functioning of blood cells (edori et al., 2013). hence, they could be used as indices of assessing stress conditions in fish because of their osmoregulatory functions (shui et al., 2018). mayer et al. (1992) reported serum na+, ca2+, k+, and clare important for the regulation of internal homeostasis because they are indispensable elements of cellular metabolism (karthikeyan et al., 2006). the statistical similarity that existed in serum ca2+and cl among dietary treatment groups further suggested that feeding abm did not impact negatively on the blood cells’ permeability property, hence no stress condition among the dietary groups. ca2+, at a significantly elevated level, could damage the permeability of the blood cell membrane (hoar, 1983; edori et al., 2013). thus, blood cell integrity was protected as the serum electrolytes were similar to control. to the best of our knowledge, work on the effect of dietary treatment on serum electrolytes is little investigated. this report constitutes the first mention of it on fed c. gariepinus. a lot of investigations abound on the impact of salinity gradient or toxic constituents on the serum or tissue electrolytes of fish (wood, 2011; stewart et al., 2016; shui et al., 2018). serum na+ and k+ moved between blood cells and plasma by active transport to maintain the blood volume in the body and enhance the body's physiological functioning. k+ was statistically similar to control up to 45% replacement level by abm in this study. so also, the serum na+ of the control group was the lowest but shared statistical similarity with the fish group fed d45t and d60t. fish attains equilibrium by the balance between na+ and k+ as they participate in the transport of k++ na+-atpase activities (towle and mangum, 1985; edori et al., 2013). the normal range of serum k+ maintains the integrity of the central nervous system (adedeji, 2010). significant reduction of serum na+ could negatively impact the iso-osmolarity condition of the blood cell resulting in stress (gabriel et al., 2009). hence, this could be a pointer to osmoregulatory difficulties (laiz-carrión et al., 2005a, b). since statistical similarity exists with control in the different constituents of serum electrolytes understudy, no stress condition among the dietary groups could be further deduced. references adedeji o. 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(2016) 4(2): 125-142; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article the role of coral in art and architecture: an overview zohreh moradi*1 department of crafts, university of hormozgan, bandar abbas, iran. article history: received 4 february 2016 accepted 27 march 2016 available online 2 5 april 2016 keywords: coral art architecture jewelry persian gulf abstract: coral is one of the richest ecosystems in the world and divided into two major groups. first group consist of soft corals living in tropical and semitropical oceans and seas. people in the past used these kinds of coral to make jewelry and decorative objects. second group contains hard coral which have been used as construction material in coastal zones. this paper presents the role of corals in art and architecture in four southern islands (qeshm, hormoz, hengam and larak) of the persian gulf by studying objects in some museums around the world and some ancient iranian jewelry manuscripts. the results showed that despite references made about corals, especially the red one, in jewelry and historical books and in persian literature, there have never been any traces of an object made by coral in the persian gulf. in scientific researchers, there is no report on red coral in the persian gulf and studies indicate that red coral as a jewelry and decorative objects were exported to these islands via india and africa. instead three groups of coral reefs, lumashell and coquina were used as construction materials in these islands mostly in historical, religious, hydraulic and offshore architectures. introduction coral reefs are among the most diverse underwater ecosystems held together by calcium carbonate structures (limestone) secreted by corals in shallow tropical seas over thousands of years (hickman et al., 2015). coral reefs from some of the world’s most productive ecosystems, providing complex and varied marine habitat that support a wide range of other organisms. reefs are home to a large variety of animals, including fish, seabirds, sponges, cnidarians, worms, crustaceans, mollusks, echinoderms, sea squirts, sea turtles and sea snakes. a few of these varied species feed directly on corals, while others graze on algae on the reef. reef biomass is positively related to species diversity. coral reefs provide essential services to humans. large human populations live on islands built solely by coral reefs or by coral reefs in conjunction with other marine sediments. to many coastal and island communities, particularly in the developing * corresponding author: zohreh moradi e-mail address: zo.moradi@hormozgan.ac.ir countries of central america, the caribbean, africa and asia, coral reef biota are important sources of food and of reef limestone, sands, rubble and blocks for use as building materials. the physical barriers provided by coral reefs protect coasts from erosion by storm waves. tourism associated with coral reefs provides many countries with significant foreign exchange earnings. beyond these perhaps obvious benefits, reefs have been effective tools in art and architecture of many nations. the case which has not been highlighted in the persian gulf region where harbors extensive coral growth in one of the highest latitude locations in the world. i therefore have the opportunity to provide an overview on the role of corals in art and architecture of propel especially those, which are living in coastal area and islands of the persian gulf. materials and methods this work has been compiled by examination of 126 moradi/ the role of coral in art and architecture available coral objects in the museums around the world including; victoria and albert museum, british museum, metropolitan new york museum, hermitage museum, coral museum of naples, coral museum of alghero, italy and pepoli trapani musuem of sicily, italy. also this paper is compiled from the works listed in the references and case study of different villages of four southern islands (qeshm, hormoz, hengam and larak) in the persian gulf. results and discussion coral in different beliefs and cultures: the origin of the name ‘coral’ is generally attributed to the greek word for pebble (‘korallion’), but hebrew (‘goral’, meaning small pebble), arabic (‘garal’, small stone) origins are also possible, maybe due to the names given to them by early traders travelling between europe and the middle east (hickson, 1924). also in persian, coral was called bessad, bousad, vasad, kameh, khoruhak and red ink and in india called munga and sometimes kameh (noorbakhsh, 1991). from archaic times, the coral in the persian gulf and other seas was known as a kind of plant. in 1700s, naturalists believed that coral is a kind of sea plant turning into stone once it comes out of the water. furthermore, different cultures describe coral as stone, plant, animal and a creature of halfplant half-human (noorbakhsh, 1991). in iranian writings left about jewelry like arayes al-jawahir and nafays al-atayb by al-kashani and gvhrnameh by mohammad ibnmansour, there are four species of coral; red, white, black and dark black. all of them can be found at sea in white, however they take different colors when they are extracted and petrified. nonetheless, a different viewpoint is also seen in these epistles regarding different species of coral with different colors in the sea (al -kashani, 2007; ibn-mansour, 1976). from old times, it was believed that corals were effective in curing dementia, increase of wisdom, disinfection, decrease of aggression and anxiety, increase of positive social activities, and increase of immunity gainst plague and dangers derived from it. they also believed that carrying coral causes travelers to cross the seas and rivers safely (figs. 1, 2). in different cultures of different tribes from the past up to the present, it is believed that corals protect their owners from evil eye and bad omen (figs. 3, 4); this is still common among children and young women to wear coral necklaces to protect themselves. in china, coral is the symbol of life span and it is considered as one of the eight treasures. coral is one of the three groups of japans tajaramono, where it is considered as a symbol that removes badness. protective powers were also figure 1. amulet, silver set with red coral, spain, 1800-1850, victoria and albert museum. figure 2. amulet, made by fossil of coral traunstein, germany, 1800-1850, victoria and albert museum. 127 int. j. aquat. biol. (2016) 4(2): 125-142 attributed to black coral. the name antipathes translated from latin means ‘against disease’, and albertus magnus (catholic saint, 1200) mentioned corals used as cures (tsounis et al., 2010). according to indonesian folklore, a black coral bracelet worn on the right arm increases virility, whereas on the left it cures rheumatism (wells, 1983). a coral necklace is the sign of africa, one of the four parts of the world. in the vedas (hindu scripture in sanskrit), part astrology, red coral is associated with the planet mars (hall, 2010). coral is one of the five sacred stones of tibetans and american indians and it represents energy, force and life (chkrn, 2001). coral necklaces or coral branches are an element in many works of spiritual or religious art, such as the works of the fifteenth century renaissance artists piero della francesca and andrea mantegna (tsounis. et al., 2010). in the treatise, arays al-jawahir and nafays alatayb, by kashani (2007) and gvhrnameh by mohammad ibn-mansour (1976), carrying a branch of coral around the neck will be useful for those suffering from epilepsy, gout and stomach ailments. they considered coral as a kind of medication for heart disease and treating shortness of breath and swelling of the spleen and stomach. review of literatures revealed that the coral has been used to improve hearing and relief of dental pain (by putting some erosion of coral on gum). coral was used to brush the teeth and recover the gums. coral can also stop the bleeding from the throat and the chest (alkashani, 2007; ibn-mansour, 1976). nowadays, most corals have commercial values. they are being used not only in the aquarium business, but also they are used in pharmaceutical industry, manufacturing ophthalmic lenses, bone marrow transplantation and many others (ellis and sharron, 1999). in medicine, chemical composition of coral is used in treating diseases such as cancer, aids, pain and other disease. for example, isididae coral skeletons are used in bone grafts in humans (ehrlich et al., 2006). in sanskrit, coral claw is figure 3. amulet white coral, bavaria, germany, 1800-1850, victoria and albert museum. figure 4. shoulder brooch, red coral, algeria 1800, victoria and albert museum. 128 moradi/ the role of coral in art and architecture called praval bhasma that is widely used in traditional indian medicine as an adjunct in the treatment of bone defects and disorders associated with calcium deficiency (reddy et al., 2003). today, the usage of corals as an amulet or in indigenous medicine is rare in the villages of the four studied islands (qeshm hormoz, hengam and larak) in the persian gulf, and other kinds of amulets are ingrained with them such as; the speech of holy quran, salt and pottery beads and so on. coral in the arts, including jewelry: use of ornaments and jewelry is as old as human history. most people were naked cavemen and they often used tattoos and painted some pictures on their bodies, women adorn themselves with necklaces and bracelets and earrings from the seeds of plants and birds, ivory, shell, or colorful marble (ravandi, 1977). it seems that stone age people like people of thousands of years later used decorative objects for two purposes of ornament and amulet. in the civilizations of egypt and mesopotamia, hanging jewelry was considered as a symbol to express power, a sign of the privileged class of people. this symbolic role, sometimes using jewelry for immunity from disease is linked to the growth of superstitious interests (ghirshman, 2005). in iran, from the start of its history, the use of ornaments was common. in addition to the existence of some iranian jewelry in museums around the world, different books including shahnameh of ferdowsi are replete with names of different jewelry and ornaments that our ancestors have invented and used. similarly, famous books written in different periods after prominent iranian kings are a proof for the same (wilson, 1987). coral is one of the several jewelries labeled as organic gemstone like pearl, shell, ivory and amber. the findings of perforated red coral beads with paleolithic human remains demonstrates that corals have been treasured by humans for at least 25,000 yr (tescione, 1973; skeates, 1993). probably red coral with amber was common currency for trade by paleolithic humans (grigg, 1993). the excavations carried out in the period neolithic, 8000 bc, the ancient artifacts including pieces of coral in the mediterranean city of çatalhöyük, turkey, (central anatolia) and amulets made of red coral from switzerland are found (grigg, 1993). in the ancient egypt, coral jewelry has been very popular for two thousand years (until ptolemy in the second century ad). also red coral developed a tremendous cultural importance, as its appearance in decorative arts of the minoan and mycenean civilization documents (tescione, 1973). almost every civilization that lived in the vicinity of shallow seas with warm water or has had an advanced business has eagerly used coral. for centuries italian commercial road, was the main place for transportation of corals to other parts of the figure 5. dorsal view of bracelet made with red coral, victorian school, 1850, italy, victoria and albert museum. figure 6. whole view of bracelet made with red coral, victorian school, 1850, italy, victoria and albert museum. 129 int. j. aquat. biol. (2016) 4(2): 125-142 world (tescione, 1973). in the nineteenth century, the victorian corals (figs. 5, 6) were very popular and they were engraved with elegant roses with jeweled and featured designs (wood engraving). since the late 1800s, american indians mixed red coral with many of their jewelry designs. today, coral has an equal value with other jewelry. precious and semi-precious corals: one of the new sciences in the gem and jewelry industry is gemology. this science mainly deals with classifying, identifying and evaluating parts and pearls used in jewelry. four gems like diamonds, rubies, sapphires and emeralds and other precious stones are considered ornamental stones and all other ornamental stones are considered semi-precious stones. features that make mineral or organic precious gems be placed in the category of precious gems are beauty (shining and nice luster), durability (strength), scarcity, color and portability. although corals are among the semi-precious stones, according to grigg (2002), they can be placed in two categories: precious and semi-precious corals. he describes; those corals precious that are rigid and can easily be polished and semi-precious term is applied to the coral with skeleton that has pores and filler must be used while producing the jewelry (grigg, 2002). precious corals belong primarily to three orders of the class anthozoa (phylum cnidaria), which are the gorgonacea, zoanthidea (subclass hexacorallia; cairns et al., 2002), and antipatharia (subclass ceriantipatharia; cairns et al., 2002). the most valuable species are red and pink corals of the genus corallium in the order gorgonacea, such as the legendary mediterranean red coral (corallium rubrum, fig. 7), as well as the pacific species paracorallium japonicum, corallium elatius, c. konojoi and c. secundum. another important group of precious corals are black corals in the order antipatharia (fig. 8). at least 10 species (mostly from the genus antipathes) from the 150 species known worldwide are used in the jewelry industry. other gorgonians used for jewelry are gold corals from the families gerardiidae and primnoidae (primnoa resedaeformis, p. willeyi, narella spp., callogorgia sp.), as well as bamboo corals in the family isididae (acanella spp., keratoisis spp., lepidisis olapa) (fig. 9). semi-precious species, allopora (now stylaster) figure 7. precious or red coral, corallium rubrum. figure 8. live black corals, antipatharia. figure 9. hawaiian-gold-coral. 130 moradi/ the role of coral in art and architecture in the class hydrozoa, blue corals (heliopora: anthozoa), the organ pipe coral, (tubipora: anthozoa) and several gorgonians (sea fans) in the family melithaeidae, are used for jewelry to some extent but are of low value due to their skeleton quality (fig. 10). reef-building stony corals (madreporaria or scleractinia) are also of commercial value, but generally not within the jewelry industry (due to their pores) and are thus not considered as precious corals. precious corals are non-reef-building deep corals. deep corals live mainly in continental shelves, slopes, canyons and seamounts, in depths of more than 50 m (although some species also extend into shallower water). deep corals are slow growing, long-lived organisms with low growth rates and low reproductive rates and their skeletons consist of a complex of proteins or calcium carbonate (baco et al., 2006). red coral is one of the most thoroughly studied gorgonians because it has been of interest to science since the controversies over whether it should be included in the plant or animal kingdom (marsili, 1707). lacaze-duthiers’s pioneering (1864) study on the biology of red coral started a series of studies on its reproduction, growth and population dynamics. in addition, because red coral has been used as a jewelry and amulets and of course for decoration by human for centuries around the world, is discussed in continuous section. red-pink coral or precious coral: the red coral are also known with different common names such as angel skin coral, midway coral, midway deep-sea coral, noble coral, red coral, pink color, red coral of commerce, and sardinia coral. the genus corallium along with the genus paracorallium (bayer and cairns, 2003) belong to the family corallidae and contain the most valuable precious corals due to their hard calcium-carbonate skeleton. thirty-one corallium species are known, of which seven species are currently used for jewelry (cairns, 2007): corallium secundum, c. regale, c. elatius, c. konojoi, c. sp., c. rubrum, and paracorallium japonicum (figs.11, 12). corallium species are found throughout the world in tropical, subtropical and temperate oceans (grigg, 1993; bayer and cairns, 2003), including five species in the atlantic, two from the indian ocean, three from the eastern pacific ocean, and 15 from the western pacific ocean (grigg, 1993; cairns, 2007). they have also been found on seamounts in the gulf of alaska (baco and shank, 2005; heifetz et al., 2005), the davidson seamount off the california figure 10. blue coral, heliopora coerulea figure 11. earrings made by red coral, 19th century, victoria and albert museum. 131 int. j. aquat. biol. (2016) 4(2): 125-142 coast (devogeleare et al., 2005) and the new england seamounts in the atlantic (morgan et al., 2006). red corals are found at depths ranging from 7 to 1,500 meters (anon, 2009). colonies of coralliidae are branched and fan-like or bush-shaped, giving them the appearance of small trees. colonies may reach a size of 50 cm across (anon, 2009; pedersen, 2004; dridi, 2009). the coralliidae have a solid axial skeleton that is made primarily of calcium carbonate (85 percent of the wet weight) in the form of calcite (fig. 13), plus a small amount of other elements in an organic matrix (anon, 2009; allemand and bénazet-tambutté, 1996; dridi, 2009; pedersen, 2004). the history of precious corals starts with the mediterranean red coral, the precious coral par excellence and it was used in the production of jewelry, religious objects and medical objects and some bowls and ornaments (magsaysay, 2013) (fig. 14). in ancient times, mediterranean red coral was collected when washed up on beaches after heavy storms had broken off branches in shallow water. these nations separated the healthy and good corals, eroded with a rasp, polished with sandpaper, submerged in steel and dipped with wax to create valuable jewelries (tsounis et al., 2010) (fig.15). for centuries; on one hand, corals were transferred via the italian commercial road, (a place where main workshops of coral has been built), to the center of europe, siberia and the other celtic countries and on the other hand they were transferred via the indian ocean in asia to tibet and mongolia. figure 12. necklace, central asia, 19th century, gold, silver, coral, pearls, turquoise, beryl and glass, hermitage museum. figure 13. corallium rubrum after polish. figure 14. carving on coral, alghero coral museum, italy. 132 moradi/ the role of coral in art and architecture marco polo reported, red coral has been found as far away from the mediterranean as ancient tibetan temples. it has also been used as a decoration in chinese clothes dating back several millennia (knuth, 1999). according to alkashani (2007) the best coral is red one and grown in the west (farangestan) sea. the importance and value of red coral has been nicely given in the poems too: لولو شد صاف رخش زان عمان بحر به (عنصری) مرجان شد سرخ جوش زآن مغرب بحر به (the sea of oman is smooth and white because of pearl and the sea of west is red because of coral, unsuri, 1986) (fig.16). red coral from indian ocean was the other famous source for making jewelry. pliny mentioned trade of red coral from india (tsounis et al., 2010). ibn-mansour (1976) noted that, coral was used in making handles for knife, which are very valuable good in india and china (figs. 17, 18). most of the adorned idols and beautified faces of these lands are made of corals. abu rayhan biruni (973 ad) in his figure 17. netsuke: seawids and branch of coral, japan, early 19thcentury, hermitage museum. figure 15. brooch, red coral, algeria, 1800-1850 victoria and albert museum. figure 16. pendant, made by silver, gold, pearl and red coral, 1500, germany, metropolitan museum. figure 18. snuff bottle with boy flying a bird, china, qing dynasty, metropolitan museum. 133 int. j. aquat. biol. (2016) 4(2): 125-142 book "al-jamahir fel marafet al-javahr” wrote about red coral and its fishing method; stone tree is a kind of tree which is called coral and grows in deep water. whenever the sun shines at it, it turns red (biruni, 1995). in the tenth and eleventh centuries, marsa’el karez on the northern african coast was the largest coral port and trade centre. in the fourteenth century, barcelona was famous for its coral art (lieonart and camarassa, 1987); later, the main activity shifted to lisbon and in the seventeenth century to marseille. in the treatise of gvhrnameh mohammad ibn mansour, red coral grows in sea of africa, where it is called mursi al-khor, as well as in the west sea in deep waters. he mentioned african people sent raw coral from africa to alexandria, and then in alexandria they were engraved and polished. after being polished, their price was specified based on their beauty, color, and size (ibn-mansour, 1976) (figs. 19, 20). few records on the use of corals in the jewelry industry in the coastal area and islands of the persian gulf have been documented in the gemology books e.g., "al-jamahir fel marafet by al-javahr rayhan biruni (973 ad), arays al-jawahir and nafays alatayb, by kashani (14th century) and gvhrnameh by mohammad ibnmansour (15th century) and in different poems in persian literature. these books show that corals especially red coral was the most famous one among people and was transported from india (indian ocean) and africa to different persian gulf islands. red coral is therefore a symbol of rebirth, dependence and altruism. greek mythology originally elevated red coral to magical status (fig. 21). in iranian beliefs, red coral protects against lightning and storms (kosuge, 1993), so red coral used as an amulets in the past. the attractive blood red color made it a symbol for the blood in different cultures, for example in persian literature; poets used the red color of red coral in their poems; نمک جان بی بدخواه کتر تن )فردوسی) نمک مرجان سنگ دل زخونش (i am killing malevolent enemy and made hard coral with his blood, ferdowsi, 2013). figure 19. clothes made by red coral, benin city, edo state, nigeria, ca. 1898 or earlier british museum. figure 20. hat made by red coral. figure 21. bird and rabbit, made by francesco degni, 1933, coral museum, naples, italy. 134 moradi/ the role of coral in art and architecture زمرد چون باشد سبز مورد تا )فرخی) مرجان چون باشد سرخ الله تا (till the green mort (scientific name: myrtus) is like emerald and till the red tulip is like coral, farokhi, 2015). بسد چون شد خنده از رستم لب )فردوسی) رسد یزدان ز یکنی گفت چنین (the lips of rostam are like besad due to smile and said goodness come from god in this way, ferdowsi, 2013). according to noorbakhsh (1991) local fishers sometimes cut little parts of corals collected from the persian gulf and sale them in the local markets. people use them just for decoration (without polish or carving) in shops or exported to big cities like tehran (noorbakhsh, 1991) (fig. 22). from 1100 to 1600, torre del greco established a firm position in the coral-fishing business and remains the main centre of corallium rubrum jewelry manufacture (tescione, 1973). sunny torre del greco, small town on the coast of naples, italy, is known as the capital of precious red coral since the year 1400 ad. moreover, italy is the main production location objects made of red coral and until 1980; it was the largest storage and harvest of the coral (figs. 23, 24). in the 1950s, coral beads were still widely used as ornaments in different countries (liverino, 1983) (fig. 25). pink coral is also highly paid attention by jewelry industry as it has relatively high density and hardness. this coral is able to cover a wide range of corals from bright pink to red coral. in addition, the figure 24. detail of the museum of coral, naples, italy. figure 22. spoon, holland, 1600-1650, victoria and albert museum. figure 23. the museum of coral, naples, italy. figure 25. necklace, red coral, 20th century, algeria, british museum. 135 int. j. aquat. biol. (2016) 4(2): 125-142 live coral of coralloid whose fossil is called sciacca, also, is widely used in jewelry and harvested in the mediterranean (cicogna and cattaneo-vietti, 1993; tsounis et al., 2010). corals grow very slowly so that the growth of red coral in the mediterranean is 1 mm and pacific species grow between 3 and 8 mm per year. they also have relatively long life so that some of them are over 75 years and one meter tall. mediterranean red coral grow at depths of 10 to 280 meters, often in caves and crevices and gaps, while red and pink corals grow at depths of 350 to 1,500 meters in the west pacific. they prefer areas with moderate to strong water flow (pedersen, 2004). because of its slow growth, high commercial value and uncontrolled exploitation in the jewelry industry, coralliidae is very vulnerable and the population of both mediterranean and pacific corals in the last 20 years has declined about 66 percent (pedersen, 2004). coral and its role in architecture: in the preceding era, essential construction materials were obtained locally. mud and rough coral rag in costal places are used as a source of building material. in many towns houses were abutted or backed onto each other and remained single-storied and rectangular, though usually there was more use of dressed coral blocks and limestone mortar. plaster made from coral lime was used frequently to cover walls and pave floors (pouwels, 2002). also ancient (fossil) coral limestone, notably including the coral rag formation of the hills around oxford (england), was once used as a building stone, and can be seen in some of the oldest buildings in that city including the saxon tower of st michael at the northgate, st. george's tower of oxford castle, and the mediaeval walls of the city (horsfield, 2011). based on the field studies carried out in 2015 and 2016, coral was virtually one of the important construction materials available in the four islands of qeshm, hormoz, hengam and larak and corals have been mined in these islands for decades. coral mining are widespread in different places like holor village in dargan, near qeshm city, in salakh, laft and other villages. coral blocks have been historically used for more important constructions such as historical, religious, hydraulic and offshore (artificial maritime) in the qeshm and hormoz islands. therefore, the role of coral in the four island’s architecture can be divided into three groups in terms of hardness and raw materials and type of materials used in different structures. 1. coral reefs in architecture: corals habitats of the persian gulf can withstand temperatures much higher than coral reef ecosystems from many other similar zones (harrington, 1986), therefore it is considered that the persian gulf is the hottest coral sea in the world. it means some corals in the persian gulf can stand summer temperatures up to 10°c higher than corals elsewhere (coles and riegl, 2013). the coral reefs ecosystems of the persian gulf, iranian waters are not very diverse compared to other tropical areas such as red sea, due to fluctuation of ecological condition and environmental stress (ropme, 1999). only about 10% of the species that occur in the indo-pacific are found in the persian gulf, and community species compositions substantially differ from assemblages that normally dominate indo-pacific reefs (coles and riegl, 2013). the use of coral rock in the construction of stone buildings goes back many centuries, notably in houses along the red sea, the east african coast, maldives and so on (spalding et al., 2001). in addition, residents of the four islands (qeshm, hormoz, hengam and larak) use coral stone in architecture. as pedro tiksara from portugal in his book "print ties" in 1610 wrote about the coral reefs of the waters of the hormoz strait and building material: “buildings have been built well. construction material include a kind of stone that is not very hard and is found in the island as well as malarial extracted from seabed. he also says: in pars sea near the island of hormoz under the sea there are a few quarry rocks from which some stone is extracted. because this stone is very malleable, 136 moradi/ the role of coral in art and architecture people use it and it is called sangmay. its distinctive feature is that it is found in seabed and is light and it grows in the places it was removed or extracted” (noorbakhsh, 1991). base on observation in 2015-2016, the residents of the four islands of the persian gulf have used coral reefs not only due to their lightness and protection from earthquake but also due to the coral reefs power to treat water. in the historical architectures; such as portuguese castle in qheshm and hormoz islands (fig. 26) and naderi castle in laft (fig. 27), in the religious architectures like; bibi maryam shrine (pir e-tom seniti, bibi maryam) (figs. 28, 29) in tom seniti village and saydan tomb in laft, and also in hydraulic constructions like round cisterns (berkeh) in all four islands (figs. 30a-d); coral reefs used as a construction material. coral reefs only used in dome and arch parts of these architectures due to their lightness and steadiness. unfortunately, these days not only, these valuable works of architecture is being misused but also we are witnessing the destruction of the species due to human activities, temperature rise and water pollution. excessive water turbulence, sudden changes in water temperature due to indiscriminate visiting of tourists will intensify the destruction of these vital ecosystems. 2lumashell: today earth has many limestoneforming environments. most of them are found in shallow water areas between 30 degrees north latitude and 30 degrees south latitude. limestone is forming in the caribbean sea, indian ocean, persian gulf, gulf of mexico, around pacific ocean islands and within the indonesian archipelago. limestone lumashell is an important source of figure 29. coral reef in the dome of bibi maryam shrine (photographed by zohreh moradi). figure 26. figure 26. basement of portuguese castle in hormoz island (photographed by: morteza golzari). figure 27. coral reefs in arch, naderi castle, laft (photographed by zohreh moradi). figure 28. bibi maryam shrine, tom seniti village (photographed by zohreh moradi). 137 int. j. aquat. biol. (2016) 4(2): 125-142 stone for construction of some important structures such as sea breakwaters and other structures in the southern ports of iran. lumashell is a sedimentary biodegradable rock and it includes materials that are extracted with ease and great abundance in stone equerry located in the southern coast of iran and has been developed in miocene to pliocene formations (tlkhablv et al., 2007). this type of rock, unlike other limestone that has chemical origin, has a destructive source. destructive particles forming this rock was formed from the remains of crushed shells of marine organisms which often are combined with calcite or aragonite that have been connected to each other with sea cement lime (hosseini et al., 2006). lumashell or the limpet is one of 64 types of minerals found in iranian mines. the mineral is composed of the fossilized shells of marine organisms and is rich in calcium, therefore it is used as a dietary supplement in livestock and poultry breeding industry. in fact, the limpet mines have been accumulated from the remains of marine organisms in the course of life, in this way, they are first developed from fault layers and long and short hills in the deep sea and over millions of years that had been buried under the sand, they reached the land with the retreat of the sea into dry land. the hills affected by natural disasters such as wind and rain were eroded over time and provided the opportunity to use this valuable resource. in lumashell, there is a wide variety of animal species such as corals, clams, snails and gastropods that can help separate the mines. for example, some mines consist only of coral and some others are a mixture of clams and snails. also, in other mines of lumashell, the same amount of oysters snails and corals are found. of course, there are mines, the biggest constituent of them is mussels and snails and corals constitute a small percentage of them. the high percentage of calcium and nutrients in the mines that have been mostly comprised of shells and snails has given them an advantage over coral mines. if lumashell is highly purified and micronized, it can be used in pigment, petrochemical, pharmaceutical and other industries. also, if it has low rate of limestone in the shell, it can be used in the manufacture of white cement. lumashell, which is a sedimentary biodegradable figure 30. (a) berkeh (cistern), department of the environment, hormoz island, ghazal esmaeili, (b) coral reefs, (c) fan coral reefs and (d) brain coral reef. 138 moradi/ the role of coral in art and architecture rock, is a kind of material that is easily extracted and can be found in abundance in stone equerries located in the southern cost of iran in mishan and aghajari formations. some local formations in qeshm, larak and khamir are stretched along the coasts of persian gulf (fig. 31). also lumashell is used in some historical buildings like portuguese castle in the qeshm island (fig. 32) and some tombstones in cemeteries (fig. 33). 3-coquina: coquina is mainly composed of the mineral calcite, often including some phosphate, in the form of seashells or coral. coquinas dating from the devonian period through to the much more recent pleistocene are a common find all over the world. the stone makes a very good material for forts, particularly those built during the period of heavy cannon use. because of coquina's softness, cannonballs would sink into, rather than shatter or puncture, the walls of the portuguese castle of qeshm and hormoz islands were built of coquina. given the potential of the quarries in the coastal areas of the country and particularly the four islands, it is usually impossible to find high quality stone. therefore, given the lack of high quality stone material, coquina is used a lot, examples of that are seen in the local houses in four islands, stones of the figure 33. wall of portuguese castle, qeshm island (photographed by zohreh moradi). figure 31. lumashell, khamir port breakwater (photographed by zohreh moradi). figure 32. lumashell in tombston, ramkan, qeshm island village cemetery (photographed by zohreh moradi). figure 34. coquina in tombstone, tomseneyti village, qeshm (photographed by zohreh moradi). 139 int. j. aquat. biol. (2016) 4(2): 125-142 graveyards of villages of gorbehehdan, kosheh, turian, towla and other villages in qeshm and the other island’s cemeteries (figs. 34-36) and historical (figs. 37-39), religious and hydraulic constructions. due to solidity in the buildings, people in these islands used coquina in diagonal lines and then cover them with coral lime. lime, cement or concrete are used to bond the coral pieces together to form walls and other building structures. in the four islands, coral and coral debris collected from the reefs crushed by manual labor into irregular and smaller pieces and then burnt in a pit in the ground with locally available firewood. corals are converted to lime by this high heat treatment and are used to bond coral pieces to build houses and other constructions. figure 35. details of tombstone. ramkan, village, qeshm (photographed by zohreh moradi). figure 36. details of tombstone. ramkan, village, qeshm (photographed by zohreh moradi). figure 37. building made by indian people in safavid era, basaidu village, qeshm island (photographed by zohreh moradi). figure 38. using coquina in diagonal line, bassidu village, qeshm island (photographed by zohreh moradi). figure 39. coral and shells in coquina, basaidu, village qeshm island (photographed by zohreh moradi). 140 moradi/ the role of coral in art and architecture acknowledgments i would like to thank h.r. esmaeili, a. moradi, a.r. dashtizadeh, ghorbani, gholamaliyan, ranjbar and safari for their valuable comments. references al-kashani a.a. 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(2022) 10(6): 489-503 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article zno nanoparticles and cu2+ enhanced toxicity in acute rather than chronic exposure of the freshwater rotifer lecane papuana iliana e. medina-ramírez1, adriana daniela rodríguez-padilla1, catalina ester haro pérez2, mario alberto arzate-cárdenas1,3 1departamento de química, universidad autónoma de aguascalientes. av. universidad 940, ciudad universitaria, aguascalientes, ags., 20134. mexico. 2área de física de procesos irreversibles, departamento de ciencias básicas, universidad autónoma metropolitana – azcapotzalco, av. san pablo no. 180, col. reynosa tamaulipas, mexico city, 02200, mexico. 3investigadores por méxico conacyt. universidad autónoma de aguascalientes. av. universidad 940, ciudad universitaria, aguascalientes, ags., 20134. mexico. s article history: received 7 september 2022 accepted 1 december 2022 available online 2 5 december 2022 keywords: concentration-addition model dissolved cations metal oxide nanoparticles mixture toxicity nanotoxicology abstract: zinc oxide nanoparticles (zno nps) are currently used in several fields, including removing dissolved metals and organic contaminants from wastewater by adsorption and/or photocatalytic mechanisms. thereafter, zno nps can be released into the environment and reach aquatic ecosystems, where their interaction with dissolved trace metals can alter their solubility and toxicity. for these reasons, the present study aimed to assess the enhanced toxicity of zno nps and dissolved copper (cu2+), using the littoral rotifer, lecane papuana as a test organism. the zno nps synthesized in this research are colloidally stable at high concentrations in either distilled water or test media according to their z-potential and hydrodynamic diameter. thus, they remain as colloids along the duration of the exposure experiments. acute toxicity tests showed median lethal concentrations (lc50) of 28.24×10-3 mg cu2+/l, 21.34 mg zn2+/l, and 78.74 mg zno np/l. enhanced acute toxicity was elicited at low concentrations of cu2+ (1.42 to 5.68×10-3 mg/l) and zno nps (0.841 mg/l). the interaction of dissolved ions, cu2+ and zn2+, was discarded as the main source of toxicity towards l. papuana as the mixtures tested followed a concentration-addition pattern that produced ~50% mortality (1 toxicity unit [tu] = lc50). thus, we considered that the enhanced toxicity was mainly caused by the synergistic interaction of zno nps and cu2+. in contrast, chronic exposure to cu2+ and zno nps significantly inhibited the rotifers rate of population growth only in the groups exposed to the higher concentrations tested, representing about 10-20% of their respective lc50 values. therefore, reports based only on acute tests might bias the ecological significance of the results as long-term and more complex matrices are required for a better understanding of the potential risks that nanoparticles and co-contaminants represent to the aquatic biota. introduction nanostructured materials are defined based on their particle dimensions (1-100 nm), conferring physical and chemical properties completely different from the bulk or dissolved components (khan et al., 2019). for example, nanoparticles (nps) show high contact surface and mechanical strength, making them very attractive for several industrial applications due to their high efficiency. nevertheless, these same properties are related to their increased toxicity to living organisms. thus, several international legislative bodies in the european union and the united states have focused correspondence: mario alberto arzate-cárdenas doi: https://doi.org/10.22034/ijab.v10i6.1708 e-mail: marzate@conacyt.mx dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.6.9 on the risk assessment of nps to human and environmental health (bondarenko et al., 2013). zinc oxide nanoparticles (zno nps) are among the most used engineered nanomaterials (enms), with an annual production estimated at over 550 tons (piccinno et al., 2012). these enms have been used in the rubber industry (qin et al., 2020), in antifouling paints (kaiser et al., 2013), in textiles as antimicrobial agents (el-nahhal et al., 2017), for uv protection (alebeid and zhao, 2017), and also in personal care products like sunscreen because of the uv scavenging properties of the zno (subramaniam et al., 2019). 490 medina-ramírez et al./ zno nanoparticles and cu2+ enhanced toxicity in acute rather than chronic due to the increase in the use of enms, it is estimated that more than 50% of their total production enters the environment through landfills (keller et al., 2013) and water systems (gottschalk et al., 2013), posing a significant threat to aquatic ecosystems (vijayakumar et al., 2017). despite the efforts to study their (eco) toxicological characteristics, there is still a gap in aspects like the nps fate and distribution in natural environments, their effects on key species in aquatic food webs, and their potential for biomagnification, among others. environmental toxicologists have selected different model organisms to assess the effects of nps in aquatic ecosystems; among such organisms, the most commonly used are zooplankters because of their high sensitivity to environmental perturbations, their ease of handling, their short life cycles, and they represent a key component in aquatic food webs as secondary production (biomass production); besides, they serve as prey for fish and larger invertebrates. the use of rotifers as test organisms is described in the iso guideline 19827 (iso, 2016), and several authors have demonstrated their suitability for the standard assessment of toxic effects of chemical substances, effluents, and treated or untreated water from industrial or municipal sources (rico-martínez et al., 2016). the most widely used rotifers are limnetic species like brachionus calyciflours (freshwater) and b. plicatilis (brakish and marine water), but also littoral species of the genus lecane have shown high accuracy and reliability in assessing the impact of metals (klimek et al., 2013; hernández-ruiz et al., 2016). they also showed higher sensitivity than the conventional test cladoceran, daphnia magna, when both species were exposed to the nanocomposite cu@zno (medina-ramírez et al., 2019). when nps reach aquatic environments, they can co-exist with other chemicals, dissolved inorganic and organic compounds, ionic forms of metals, and other nanomaterials. thus, the co-occurrence of dissolved copper (cu2+) and zno nps is likely because cu2+ is a trace element that can be found at low concentrations in natural water systems (rader et al., 2019), and zno nps can enter aquatic environments in any part of their life cycle because of their extensive use. synergistic effects have been described for diverse nanomaterials and toxic compounds. some authors have studied the interaction of nano plastics and persistent organic pollutants, which exhibited a synergistic model effect on the rotifer brachionus koreanus (jeong et al., 2018). for zno nps, a similar model was described in the presence of cadmium, which promoted the lixiviation of zinc ions and enhanced toxicity toward phytolacca americana (xiao et al., 2022). the cladoceran d. magna exposed to ionic silver (ag+) and zno nps exhibited different response patterns (antagonism or synergism) depending on the toxicity unit’s ratio, with synergistic toxicity when zno nps were dominant in the mixture (baek et al., 2020). some studies considered that toxicity is related to ionic forms or lixiviated ions; thus, the antibacterial effect (toxicity) of zno nps as the result of lixiviated zn2+ rather than particles, finding zn2+ concentrations of up to 4.5 mg/l after 96 hours of incubation (wang et al., 2016). it is worth pointing out that the initial concentration (nominal) was 5.0 zno nps mg/l. nevertheless, other studies showed that the dissolution of zno nps in a culture medium for vibrio fischeri was about 15% (zhang et al., 2020), which was higher than the dissolution of cuo nps, but still lower lixiviation of zn2+ than that in previous reports (wang et al., 2016). the composite cu@zno was described to exert antibacterial effects against different clinical bacterial and fungal isolates but also toxic effects on zooplanktonic species, which might be due to the lixiviation of copper ions (medina-ramírez et al., 2019). for these reasons, the present work is aimed to assess the toxic effects due to the interaction of dissolved copper (cu2+) and zinc oxide nanoparticles (zno nps) in the rotifer, lecane papuana. different toxicity assays were implemented: (a) acute toxicity tests (without food supplementation), (b) chronic toxicity tests, which are supplemented with food (microalgae) and might pose a factor that modifies 491 int. j. aquat. biol. (2022) 10(6): 489-503 the toxicity of the mixture zno nps-cu2+ and (c) hatching of amictic eggs, to demonstrate that nps enhance the toxicity of dissolved metals towards the aquatic biota. materials and methods chemicals: the zno nps were synthesized by our research group using a microwave-activated solvothermal route. the materials have been fully characterized, exhibiting uniform size (~30 nm), mixed morphology (spheres and small rods), and high purity (medina-ramírez et al., 2019). nahco3, caso4·2h2o, mgso4·7h2o, kcl, cuso4·5h2o, and znso4·5h2o were purchased from j.t. baker and used as received. stock solutions and further dilutions were prepared in deionized water. work solutions were prepared in reconstituted mediumhard water, constituting the culture medium for rotifers (usepa, 2002). evaluation of the stability of zno nanomaterials in test media: the dynamic light scattering (dls) technique (zetasizer zs90, malvern panalytical) was used to determine the hydrodynamic diameter of zno nps. the hydrodynamic diameter value was obtained by performing a second-order cumulant fit to the field correlation function measured at a scattering angle of 90° (pecora, 2000). the zeta potential of the zno nps was obtained from electrophoretic mobility measurements using o’brien-white theory (delgado et al., 2005). for these analyses, zno nps were suspended in deionized water, epa medium, and epa medium with cu2+ (5 mg/l) at the highest particle concentration tested in the biological assays (100 mg zno nps /l). measurements were performed at different times (0, 24, and 48 h) to observe the size temporal evolution of the zno nps and to rule out the possible destabilization of the colloidal system. the mean value is obtained by averaging six independent measurements and the error by the sample standard deviation. culture conditions for rotifers: the strain of l. papuana has been cultured for at least five years in the laboratory of aquatic toxicology of the autonomous university of aguascalientes, mexico. lecane females were maintained in a bioclimatic chamber at 25±2ºc, photoperiod of 16 h light for 8 h dark, fed on the green algae nannochloropsis oculata (utex strain lb2164) at 106 cells/ml, and reconstituted medium-hard water according to the environmental protection agency o the united states (usepa) guidelines (nahco3 96 mg/l, caso4·2h2o 60 mg/l, mgso4·7h2o 60 mg/l, and kcl 4 mg/l; at ph 7.5) (usepa, 2002). acute toxicity test of single chemicals: twelve hours before the beginning of the test, the amictic eggs of l. papuana were separated and placed in a medium without food supplementation. then, neonates (<12 h) were randomly placed in a 24-well polystyrene microplate. five different concentrations of cu2+, zn2+, or zno nps were analyzed. the negative control consisted of neonates in epa medium without exposure to toxic substances. the conditions for the experiments were as follows: 1 ml test volume without food supplementation, at 25±2°c, for 48 h with a photoperiod of 16:8h (light/dark), and ten organisms per replica. all experiments were conducted in quadruplicate. at the end of the test, immobility and mortality were recorded, and these data were used to calculate the median lethal concentration (lc50) and lethal concentration at 10% (lc10). the lethal concentrations were estimated with the statistical package drc in r. all concentrations used in this study are expressed as nominal. chronic toxicity test of single chemical: five different nominal concentrations were selected for cu2+ (0.355, 0.710, 1.421, 2.841, and 5.683 × 10-3 mg/l) or zno nps (0.984, 1.968, 3.937, 7.874 and 15.747 mg/l), which correspond to 1/80, 1/40, 1/20, 1/10 and 1/5 of their respective lc50 values. the negative control consisted of neonates reared only in an epa medium (not exposed to any toxic substance). all controls and treatments (different concentrations of cu2+ or zno nps) consisted of four replicas (n = 4), and five neonates per well were placed in a 24-well polystyrene microplate with a test volume of 2 ml. the green alga n. oculata was 492 medina-ramírez et al./ zno nanoparticles and cu2+ enhanced toxicity in acute rather than chronic added as food supplement at 106 cells/ml. the temperature was controlled in a bioclimatic chamber at 25±2°c, with a photoperiod of 16 h light and 8 h dark. distilled water (100 μl) was added to the medium every other day to avoid desiccation. after five days of exposure, all living rotifers were counted. the total number of rotifers was used to estimate the intrinsic rate of natural increase (r) for all experimental groups (control and treatments), according to the formula of 𝑟 = 𝑙𝑛(𝑁𝑓 𝑁𝑖⁄ ) 𝑡 where ln is the natural logarithm, ni is the initial number of rotifers, nf is the number of organisms at the end of the test, and t corresponds to the exposure time (five days for these experiments). acute toxicity tests of binary mixtures: we assayed the following mixtures: cu2+ and zn2+, and cu2+ and zno nps. the mixtures for the immobilization test were prepared based on their respective lc50 values, which were considered one acute toxicity unit (1 tua). thus, 1 tua elicits the death of 50% of the exposed organisms. five combinations of cu2+ or zn2+ with zno nps were initially assayed (table 1). this experimental setup was changed for the interaction of cu2+ and zno nps because of the 100% mortality in all treatments. thus, different combinations were tested to find an interval that allowed comparisons (mortality <100%). the test conditions (final volume, no food supplementation, photoperiod, and temperature) were as aforementioned in the section on acute toxicity tests. chronic toxicity test of binary mixtures: five different ratios were selected to assess the interaction of cu2+ and zno nps (table 2). the control group consisted of organisms reared only in an epa medium without exposure to toxic compounds. the test conditions were described in the aforementioned chronic toxicity assays. at the end of the exposure period, all living organisms were counted. the total number of rotifers was used to estimate the intrinsic growth rate (r) with the formula previously described. lixiviation of zn2+ and toxicity towards l. papuana: to assess the effect of lixiviated zn2+ from zno nps, we experimented with the same conditions of the acute toxicity tests but without rotifers. thus, all test concentrations of zno nps were prepared in 2 ml of epa medium, incubated at 25°c for 24 h, and 48 h, with a photoperiod of 16 h light and 8 h dark. after incubation, test solutions were transferred to microtubes and centrifuged at 12,000 g for 20 min. supernatants were recovered and transferred to a new microplate of 24 wells, one test solution per well and four replicas per concentration. then, 10 neonates (<24-h old) of l. papuana were transferred to each well and incubated for 48 h. at the end of the test, dead animals were counted, and mortality rates were estimated. statistical analysis: the lc50 values were treatment zno nps + cu2+ zn2+ + cu2+ mg/l tua x10-3 mg/l tua mg/l tua x10-3 mg/l tua a 15.747 0.20 22.731 0.80 3.617 0.20 22.731 0.80 b 31.495 0.40 17.048 0.60 7.235 0.40 17.048 0.60 c 39.369 0.50 14.207 0.50 9.043 0.50 14.207 0.50 d 47.242 0.60 11.366 0.40 10.852 0.60 11.366 0.40 e 62.990 0.80 5.683 0.20 14.470 0.80 5.683 0.20 the expected effect is based on the postulate that lc50 = 1 tua. thus, exposure concentrations of 1 tua were expected to exert ~50% of immobilization/dead of exposed individuals. all treatments consisted of 1 tua (the sum of zno tua and cu2+ tua), except for the control, in which no mortality was neither expected nor observed. table 1. experimental design to assess the interaction of cu2+ and zno nps in acute toxicity tests with lecane papuana (rotifera: monogononta). 493 int. j. aquat. biol. (2022) 10(6): 489-503 obtained with the help of the statistical software r and the drc package. the results of the chronic toxicity experiments were compared using the oneway analysis of variance (anova) and tukey's multiple comparison tests with the help of statistical software r for windows and the agricolae and the ggplot2 packages. significant differences were established when p<0.05. results stability of zno nanomaterials in test media: table 3 presents the results of the stability of zno as suspended materials. the zeta potential of zno nps in deionized water took values from -40.5 to -35.6 mv, showing slight differences in deionized water with cu2+. in both epa media, the zeta potential showed values from -30.7 to -27.5 mv. in all cases, the zeta potential values are negative, indicating that the particles are negatively charged and higher than 25 mv in absolute values. this suggests aggregation is unlikely due to electrostatic repulsion among particles. acute toxicity test of single chemicals: the effective concentrations for cu2+, zn2+, and zno nps are shown in table 4, where lc50 represents the concentration that caused death to 50% of the treatment cu2+ zno nps ×10-3 mg/l acute loec ratio mg/l acute loec ratio control 0.000 0.00 0.000 0.00 a 2.814 1.00 0.00 0.00 b 2.131 0.75 3.95 0.25 c 1.421 0.50 7.90 0.50 d 0.711 0.25 11.85 0.75 e 0.00 0.00 15.80 1.00 the highest concentration tested for cu2+ (2.814 × 10-3 mg/l) and zno nps (15.80 mg/l) correspond to their respective loec (lowest observed effect concentration) in acute toxicity tests previously performed with l. papuana. table 2. assessment of the interaction of dissolved copper (cu2+) and zno nanomaterials in chronic toxicity tests with lecane papuana. hydrodynamic diameter nm zeta potential mv 0 h 24 h 48 h 0 h 24 h 48 h deionized water 230±22 225±13 222±8 -40.5±3.3 -38.2±1.0 -35.6±0.4 deionized water + cu2+ 235±17 221±11 218±8 -36.2±1.1 -42.0±1.6 -41.7±1.3 epa medium 245±13 227±11 237±16 -27.5±0.9 -29.5±1.3 -29.1±0.8 epa medium + cu2+ 247±9 227±8 242±18 -27.5±0.9 -30.2±0.7 -30.7±1.3 zno nps were prepared at 100 mg/l. epa medium represents a moderate hard reconstituted water used in the culture of rotifers (usepa, 2002). cu2+ was added at 5 mg/l. all data were obtained as the average of six replicas. table 3. hydrodynamic diameter and zeta potential values of zno nps at time zero (at the time the sample was prepared), 24 and 48 hours in different test media. cu2+ × 10-3 mg/l zn2+ mg/l zno nps mg/l lc1 5.574 (1.081) 0.902 (0.402) 1.873 (1.1074) lc10 13.041 (1.355) 4.312 (1.056) 13.177 (4.192) lc50 28.414 (1.328) 18.087 (2.142) 78.737 (8.362) lcx, lethal median concentration that affects determined percentage of the exposed population, where x takes values 1, 10 and 50 for the present study. data are presented as the mean value (standard error of the mean) (n = 4). table 4. results of the acute toxicity tests with lecane papuana exposed to cu2+, zn2+, or nanoparticles of zno. 494 medina-ramírez et al./ zno nanoparticles and cu2+ enhanced toxicity in acute rather than chronic total organisms. copper concentrations of some micrograms can significantly affect the survival of l. papuana (lc50 = 28.4×10-3 mg/l). chronic toxicity test of single chemical: the results of the chronic tests are presented with their respective confidence intervals (p<0.05) in figure 1. for these assays, we selected concentrations from 1/80 (0.0125) to 1/5 (0.20) of their respective lc50 values and found different patterns. in the case of cu2+, the population growth of l. papuana was significantly inhibited at concentrations above 0.1 lc50 (2.841×10-3 mg cu2+/l). however, zno nps elicited significant changes at 0.025 lc50 (1.968 mg zno nps /l) and higher concentrations. as the initial hypothesis revolved around the interaction of cu2+ and zno nps and the likely synergistic effect, we first assessed the interaction of dissolved cations. for the assays with dissolved cations (cu2+ and zn2+), it was not necessary to adjust the concentrations as the observed mortality was close to the expected mortality (~50%) (fig. 2). in this experimental design, all mixtures tested (0+21.34, 7.085+16.005, 14.170+10.670, 21.255+ 5.335, 28.340+0 mg/l, cu2+ at 10-3 mg/l and zn2+ at mg/l, respectively) were expected to cause ~50% (we considered 1 tua as equivalent to the lc50). thus, the cations’ effect was related to adding their respective toxicity units and summing ~1 tua. the criterion of the mixture toxicity index states that if the result of dividing the observed effect of the mixture by the observed effect of the single toxic compound is comprised between 0.8 and 1.2, then it can be stated that the effect corresponds to the concentration-addition concept (arreguin-rebolledo et al., 2021). therefore, the concentration-addition model can describe the effect of the mixture of dissolved cu2+ and zn2+. it was impossible to follow the same experimental design to assess the combined effect of cu2+ and zno nps as their mixtures elicited 100% mortality; thus, different sets of concentrations were tested to achieve those reported in table 2 finally. these results showed that the combination of only 0.025 tua of cu2+ and 0.011 tua of zno nps produced the same effect that either 0.028 mg cu2+/l or 78.74 mg zno nps/l (the respective lc50 values). the mortality of rotifers increased according to the increment of cu2+ concentration, reaching the highest mortality (~100%) at 0.840 mg/l of zno nps and 5.682×10-3 mg of cu2+/l. thus, the concentrations in these tests correspond to approximately 1/100 of the lc50 for zno nps, and from 1/40 to 1/5 of the lc50 for cu2+. then, the combination of these metal forms expressed as tu (0.036-0.212) was expected to cause mortality between 1 and 10%, but mortality occurred at rates from 46 to 95%, which are, on average, 10-fold higher than the expected response (table 5). based on the aforementioned criterion (arreguinrebolledo et al., 2021), the results of such a division figure 1. results of the chronic toxicity tests with lecane papuana exposed to cu2+ (left) and zno nps (right). different letters represent significant differences among treatments, which were compared through one way anova and multiple comparison test of bonferroni (p<0.05). 495 int. j. aquat. biol. (2022) 10(6): 489-503 are higher than 1.2, which corresponds to more than that of the additive model (synergistic effects). lixiviation of ions from zno nps: incubation of freshwater media with nanoparticles at the same concentrations used in the acute toxicity tests and their posterior removal through centrifugation were performed to assess the effect of ions lixiviation. rotifers exposed to free-zno nps media showed mortality rates of up to 10% at the highest zno nps concentration (fig. 3). chronic toxicity test of binary mixtures: figure 4 displays the results of the chronic toxicity tests with both cu2+ and zno nps, showing that mixtures of cu2+ and zno nps elicited no significant changes in the intrinsic rate of population increase in l. papuana. the concentration of either cu2+ and zno nps were fractions of the respective lc50 values, which were expected to follow the same synergistic pattern as in the acute toxicity tests. nonetheless, the combination of cu2+ and zno nps in tests with food supply (algae) showed no significant changes compared to the control group. figure 5 presents the population growth rates of rotifers born from amictic eggs exposed to mixtures of cu2+ and zno nps. here, significant changes were only observed for the two highest concentrations, corresponding to 0.162 tua (0.840 mg zno nps/l and 4.26×10-3 mg cu2+/l) and 0.212 treatment zno np cu2+ total tu mg/l tu x10-3 mg/l tu mortality sd control 0 0 0.000 0.00 0.000 n.o. n.a. a 0.036 0.840 0.011 0.71 0.025 0.463 0.169 b 0.061 0.840 0.011 1.42 0.050 0.700 0.200 c 0.111 0.840 0.011 2.84 0.100 0.875 0.139 d 0.162 0.840 0.011 4.26 0.151 0.713 0.136 e 0.212 0.840 0.011 5.68 0.201 0.950 0.053 lc50 = 1 tu. n.a., non-applicable; n.o., no observed. tu = toxicity units; all combinations were expected to cause mortality ≤10% in accordance to the total tu. table 5. results of the acute toxicity tests with lecana papuana exposed to mixtures of cu2+ and zno nps for 48 h. figure 2. results of the acute toxicity test of the mixture cu2+ + zn2+. neonates (<24 h) of lecane papuana were exposed to: 0 × 10-3 mg cu2+ /l + 21.34 mg zn2+/l (0+1 tua), 7.085 × 10-3 mg cu2+ /l + 16.005 mg zn2+/l (0.25 + 0.75 tua), 14.170 × 10-3 mg cu2+ /l + 10.670 mg zn2+/l (0.5 + 0.5 tua), 21.255 × 10-3 mg cu2+ /l + 5.335 mg zn2+/l (0.75+0.25 tua), 28.340 × 10-3 mg cu2+ /l+0 mg zn2+/l (1 + 0 tua). 1 tua = lc50. control group consisted of non-exposed organisms. different letters above indicate significant differences according to one-way anova and multiple comparison test of bonferroni (p<0.05). 496 medina-ramírez et al./ zno nanoparticles and cu2+ enhanced toxicity in acute rather than chronic tua (0.840 zno nps/l and 4.68×10-3 mg cu2+/l). discussion stability of zno nanomaterials in test media: the stability of zno nps was further confirmed by measuring the hydrodynamic size of the nanoparticles and following its time evolution during 48 h, the duration of the acute toxicity tests. the mean hydrodynamic diameter of the zno nps in deionized water was around 220-230 nm, with no significant differences when suspended in deionized water with cu2+ (5 mg/l). similar values were obtained for the dispersion of zno nps in epa medium and epa medium + cu2+. moreover, these values remain almost constant after 48 h of sample preparation, confirming the colloidal stability of all figure 3. results of the acute toxicity test with lecane papuana exposed to media without nanoparticles. test media consisted of reconstituted medium hard water with zno nps, incubated for 24 h (left) and 48 h (right). after incubation in the same environmental conditions than rotifers culture, test media were centrifuged at 12,000×g and supernatants were recovered. neonates of l. papuana (<24-h old) were exposed to supernatants. mortality was recorded after 48 h. different letters represent significant differences among treatments, which were compared through one way anova and multiple comparison test of bonferroni (p<0.05). figure 4. results of the chronic toxicity test with the rotifer lecane papuana exposed to mixtures of cu2+ and zno nps. rotifers were exposed to fractions of the acute loec (lowest observed effect concentration). thus, mixtures of cu2+ and zno correspond to: 1 + 0 (2.814 × 10-3 mg cu2+/l + 0 mg zno nps /l), 0.75 + 0.25 (2.131 × 10-3 mg cu2+/l + 3.950 mg zno nps /l), 0.5 + 0.5 (1.421 × 10-3 mg cu2+/l + 7.90 mg zno nps /l), 0.25 + 0.75 (0.711 × 10-3 mg cu2+/l + 11.850 mg zno nps /l), and 0 + 1 (0 mg cu2+/l + 15.80 mg zno nps /l). different letters represent significant differences among treatments, which were compared through one way anova and multiple comparison test of bonferroni (p<0.05). 497 int. j. aquat. biol. (2022) 10(6): 489-503 the nanoparticle dispersions. the colloidal stability study was carried out using suspensions prepared with the highest concentration of both zno nps and cu2+. thus, if nanomaterials remain stable as a colloidal suspension during the exposure time under the most unfavorable conditions (salt and nanoparticle concentrations), then it can be assumed that zno nps suspensions remain stable at lower concentrations of either cu2+ or zno nps. acute toxicity test of single chemicals: the lc50 values for this rotifer were very similar to data previously reported for other organisms; thus, l. papuana is the most sensitive to cu2+ within the family lecanidae, while their sensitivity is similar to other genera like brachionus and also to some cladoceran species which are commonly used in aquatic toxicology studies (usepa, 2017). for zn2+, l. papuana exhibits the highest lc50 (21.330 mg/l) compared to other zooplanktonic species. literature reports the lc50 of zno nps for freshwater invertebrates from 1.25 to 7.1 mg/l with d. magna (cladoceran) (heinlaan et al., 2008, zhu et al., 2009, naddafi et al., 2011) and about 0.165 mg/l for thamnocephalus platyurus (heinlaan et al., 2008). within freshwater test organisms, l. papuana exhibited one of the highest lc50 values, which are comparable to the results of artemia salina (lc50 = 58.3 mg/l, at 96 h) (schiavo et al., 2018). thereafter, l. papuana seems e very tolerant to zinc whereas the sensitivity towards copper is similar to other freshwater species used as test organisms. it was generally assumed that the nanomaterials of zno could be considered innocuous; nevertheless, nanostructured materials negatively affect the exposed organisms (jarvis et al., 2013). the lc50 values found that d. magna is more sensitive (lc50 = 1.511 mg/l; (zhu et al., 2009) than l. papuana (lc50 = 78.74 mg/l). in addition, all individuals of ceriodaphnia cornuta died when exposed to zno nps at 0.160 mg/l (vijayakumar et al., 2016), which is nearly 500-fold lower than the lc50 of l. papuana. it is evident that c. cornuta is more sensitive than l. papuana to zno nps, but this one is among the most susceptible organisms within rotifera, which consists of species important for aquaculture and ecotoxicology. chronic toxicity test of single chemicals: despite reports on the chronic toxicity of cu2+ and zno nps, only some authors reported the respective ec50 values (usepa, 2017). in the present study, the figure 5. reproductive performance of the rotifers hatched from amictic eggs that were exposed to cu2+ (0.71, 1.42, 2.84, 4.26 or 5.68 × 10-3 mg/l) and zno nps (0.840 mg/l), which correspond to 0.037, 0.062, 0.112, 0.162, and 0.212 tua, respectively. amicitic eggs were exposed until hatching and rotifers were transferred to clean epa medium supplemented with n. oculata at 106 cells/ml. then, the rate of population increase was estimated at the end of 120 h through the formula r = (lnnf-lnni)/t. different letters above the boxes refer significant differences among the treatments, which were compared through one-way anova and multiple comparison test of bonferroni (p<0.05). 498 medina-ramírez et al./ zno nanoparticles and cu2+ enhanced toxicity in acute rather than chronic ec50 for either cu2+ or zno nps was not estimated because the population growth rate inhibition was below 50% at the concentrations tested. higher concentrations could be required (hernández-flores et al., 2020) found a similar pattern with the rotifer euchlanis dilatata and reported an ec50 higher than the lc50 for cu2+, stating that the alga nannochloropsis oculata (the same algal species used in this study) might interfere with the bioavailability of cu2+ and diminished its toxicity due to quelation or absorption of the micronutrient (because its concentration was much lower than that in the culture media for algae). in the case of zno nps, it was reported that algal exudates and the interaction with their cell wall lessened the lixiviation of zn2+ from the zno nps and their toxicity by aggregation or adsorption (chen et al., 2012). acute toxicity of binary mixtures: the increased toxicity of cu2+ and zno nps as mixtures could find a possible explanation for the capacity of zno nps to remove metal ions, like cu2+, from the water (le et al., 2019) and its further release within the rotifers to cause their death. metal ions from zno nps are released within the digestive tract of zooplankters and facilitate the bioconcentration of zn, which might not generate significant effects until metals exceed threshold concentrations (li and wang, 2013). baek et al. (2020) reported the enhanced toxicity of ag+ and zno nps, when zno is dominant in the mixture and suggested the formation of ag+zno nps complexes as promoters of trojan horselike effects releasing cations within the gut of d. magna. similarly, zno nps (0.840 mg/l) enhanced the toxicity of cu2+ towards l. papuana, diminishing the lc50 of cu2+ from 0.028 mg/l to 0.71×10-3 mg/l, representing a 40-fold increase of toxicity. these results confirm that environmental concentrations of soluble copper and low concentrations of zno nps could exert deleterious effects on rotifers. lixiviation of ions from zno nps: since it is of high relevance to elucidate if enhanced toxicity is due to released soluble ions (zn2+) and cu2+, the interaction of zno nps and cu2+, or the participation of both mechanisms, we performed bioassays in which organisms were exposed to epa medium in which zno nps were previously dispersed and incubated for up to 48 h to allow zinc lixiviation from nanomaterials, and then, such nanoparticles were removed through centrifugation (12,000× g, 20 min). according to our results, mortality was low and zn2+ (lixiviated from nps) might not represent the main source of toxicity toward l. papuana. however, these results point out that it is certainly important to consider that zn2+ can interact with the other components of the mixture and modify their toxicity. the interaction of cu2+ and zn2+ in assays with l. papuana showed that their interaction complies with the criterion of the concentration-addition model. the stability studies, zeta potential and hydrodynamic size of zno nps in pure water and the media used in this study show that nanoparticles remain dispersed during the assays; thus, rotifers are exposed and might be able to consume the zno nps (zhang et al., 2020). the estimated solubility of zno nps was around 15% of the initial powder concentration, which might correspond to 0.126 mg zn2+/l in the present study. in case that zno nps could be completely dissolved into the medium, zn2+ concentration would correspond to 0.676 mg zn2+/l, which is 31 times lower than the respective lc50. in addition, when rotifers were exposed to mixtures of dissolved cations, cu2+ (7.06 to 21.18×10-3 mg/l) and zn2+ (5 to 16 mg/l) concentrations were higher than the result of complete solubilization of zno nps. moreover, the concentration of cu2+ in the mixture zno np + cu2+ was set from 1 to 5×10-3 mg/l, representing concentrations 5 to 28 times lower than the cu2+ lc50 value. therefore, the high mortality observed due to zno np+cu2+ exposure cannot be related to the lixiviation of zn2+ but to the interaction of cu2+ and zno nps. furthermore, lixiviation from zno nps has been tested in a medium of moderately hard water (the same used in the present study) and showed that zn2+ has low solubility due to carbonate precipitation and 499 int. j. aquat. biol. (2022) 10(6): 489-503 its likely adsorption onto caco3, thus reducing the zn2+ bioavailability and its toxicity (reed et al., 2012). therefore, zn2+ might not have an important contribution to the enhanced toxicity of the mixture zno nps+cu2+. hence, we hypothesized that the most relevant interaction could be the adsorption of cu2+ on the surface of zno nps, ingestion, and posterior release of cu2+ within the rotifers in accordance to previous reports about the synergy of nanoparticles and dissolved ions, at least in the acute toxicity tests, similarly to those effects observed in d. magna (yan et al., 2018, park et al., 2019). chronic toxicity test of binary mixtures: in chronic tests, it is important to consider that microalgae can modify the solubility and bioavailability of metals and nps. chen et al. (2012) described that chlorella vulgaris (chlorophyceae) depleted the zn2+ released from zno nps (62 nm, zno nps coated with 3-aminopropyl triethoxysilane) because their aggregation onto the algal cell wall, which was accomplished through algal exudates that bind the solubilized zn2+, depleting the bioavailability of cu2+ and zn2+. ionic zinc toxicity and its release from zno nps can be reduced by algae due to either the aggregation of zno nps or their interaction with algal exudates (mcintyre and guéguen, 2013). moreover, the concentration of both cu2+ and zn2+ and zno nps used in this study were very low and did not exceed those concentrations in the bold’s basal medium (0.4 mg/l and 2 mg/l for cu2+ and zn2+, respectively) (nichols and bold, 1965); thus, microalgae might take advantage of these metals and uptake the mineral nutrients (cu2+ and zn2+) from the medium, depleting their concentration, and as a consequence, their toxicity, which resulted in the inhibition of the population growth of l. papuana in about 30% with respect to the controls. higher algal densities ameliorate the effect of toxicants and allow zooplankton species to deal with the toxicant-induced stress (xue et al., 2021, sun et al., 2022). the algal density was selected in the present study according to the literature. however, such a concentration can be the best for rotifers culture, but it might not be representative of natural environments unless attempting to simulate eutrophic conditions (high algal density). therefore, although it could be convenient to assess the interaction of cu2+, zn2+, zno nps, their mixtures, and the food supply, that goal is beyond the aim of this manuscript. the intrinsic rate of population increase has been used for several years as a sensitive and environmental-relevant endpoint, which is modified by different sorts of toxic compounds. however, when population growth is diminished, it is important to consider that the exposure to toxicants can alter either hatching or fecundity. in the present study, hatching was not significantly affected by the exposure to cu2+ and zno nps, but the population growth suffered slight alterations. embryonic malformations, hatching delay, and larval hyperactivity are induced in danio rerio by exposition to zno nps (zhao et al., 2013, chen et al., 2014). it is worth noting that this experimental design assessed the effects of the mixture on the rotifer eggs without food supplementation, exposed for 24 h, and then transferred to toxicant-free medium, in which altered organisms developed lower intrinsic growth rates than the control group. therefore, the mixture of zno nps and cu2+ elicited pre-hatching alterations in l. papuana that conditioned their survival and fecundity, compromising their population growth. biased conclusions can be achieved by only reporting effects related to acute toxicity tests as they do not consider various factors intervening in chronic toxicity tests. as aforementioned, algal cells modify the bioavailability of copper and zinc cations, probably due to mechanisms like precipitation or absorption as mineral nutrients, mainly because the concentrations tested were lower than those commonly used in algae culture medium. higher concentrations of either cu2+ or zno nps could have been tested, but toxicological studies aimed to establish the potential risks of chemicals at environmentally relevant concentrations. some authors have described a phenomenon 500 medina-ramírez et al./ zno nanoparticles and cu2+ enhanced toxicity in acute rather than chronic called trojan-horse effect, in which the nanoparticles act as carriers for environmental contaminants, which are then released within the exposed organisms. this route has been accepted in general terms as a possible explanation for the enhanced toxicity of nanomaterials and dissolved cations, either found in the media or released from the nanomaterials (baek et al., 2020). nevertheless, such estimations were based on acute toxicity tests, which do not require feeding organisms. on the contrary, chronic assays with zooplankters imply microalgae supplementation as a food source for zooplankton species. thus, microalgae play a significant role because negative nanoparticleassociated effects depend on algal density, algal species, and phenomena of absorption and release of dissolved cations. thereafter, the inclusion of more complex experiments will lead to a better understanding of the nanoparticles’ fate and effects in aquatic systems. as observed in this study, conclusions based on acute assays overestimate the toxicity of dissolved cu2+ and zno nps, but chronic assays show that higher concentrations of cu2+ and zno nps are required to cause significant changes in the reproductive performance of l. papuana. in conclusion, the interaction between cu2+ and zno nps in the acute toxicity tests followed a synergistic pattern, increasing their toxicity about 40 times compared to the individual chemicals. release of zn2+ from zno nps was not the main toxicity mechanism on l. papuana, as demonstrated by the lixiviation assays that consisted in incubating suspensions of zno nps (same concentration used in the acute toxicity tests and the same environmental conditions), removed them from the media through centrifugation, and found that mortality was low among the rotifers exposed to zno nps-free media. in addition, the mixture of cu2+ and zn2+ caused mortality of l. papuana following a pattern that complies with the concentration-addition model, while the combination of cu2+ and zno nps followed a synergistic pattern. biased conclusions are based only on the results of acute toxicity tests since chronic exposure to cu2+ and zno nps required higher concentrations to cause significant alterations in the population growth rate of l. papuana. amictic eggs of l. papuana exposed for 24 h to relatively high concentrations of cu2+ and zno nps (those used in the acute toxicity tests) produced neonates that showed lower population growth rates than the controls; thus, those mixtures can negatively affect the population dynamics of rotifers. although it is worth pointing out that exposure was in media without algal cells, which can modify the toxicity of metal compounds. in general, reports of the interaction of metal ions and metal oxide nanoparticles are based on acute toxicity tests rather than chronic assays, which involve factors like the interaction of food supply (algal cells) that can alter the stability of metal oxide nanomaterials and the bioavailability of metal ions, and likely reduce their toxicity as it is herein reported. thus, the interaction of cu2+ and zno nps followed a different pattern than that observed in the acute toxicity tests. it might require higher concentrations or varying the ratios of both chemical species to cause significant changes in the population growth rate of l. papuana. finally, we consider that the results of acute toxicity 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(2013). acute zno nanoparticles exposure induces developmental toxicity, oxidative stress and dna damage in embryolarval zebrafish. aquatic toxicology, 136-137: 49-59. zhu x., zhu l., chen y., tian s. (2009). acute toxicities of six manufactured nanomaterial suspensions to daphnia magna. journal of nanoparticle research, 11: 67-75. international journal of aquatic biology (2015) 3(4): 258-262 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved short communication selected population characteristics of the non-native catfish hoplosternum littorale (hancock, 1828) in central florida, usa alexander benjamin orfinger1 department of biology, university of central florida, 4000 central florida boulevard, orlando, florida 32825, usa. article history: received 2 march 2015 accepted 14 april 2015 available online 2 5 august 2015 keywords: hoplosternum length-weight relationship fulton's condition factor sex ratio abstract: this study reports the length-weight relationship (lwr), fulton's condition factor (k), and sex ratio of the non-native catfish hoplosternum littorale (hancock, 1828) in florida for the first time. sampling was conducted from november 2013 to april 2014 in tosohatchee wildlife management area in christmas, florida. a total of 477 specimens were caught (6.4-13.5 cm tl). the allometric coefficient b of the lwr was greater than the isometric value (b=3.11), suggesting positive allometric growth. the average value for fulton's condition factor (k) was 1.396, with no significant differences between juvenile and adult size classes. the results present certain disparities from data recorded from the fishes' native range, suggesting that this freshwater invasive may be flourishing in florida. in addition, a new maximum total length of the species is reported. introduction in 1995, an established population of hoplosternum littorale (hancock, 1828) (siluriformes, callichthyidae) was detected in the indian river lagoon in central florida (nico et al., 1996). this neotropical species is a microphagous scavenger commonly found throughout much of tropical south america and trinidad (winemiller, 1987). the species has since spread over much of peninsular florida, possibly constituting an invasive threat to the freshwater ecosystem (duxbury et al., 2010) and creating a new small-scale commercial fishery (gestring et al., 2009). the present work provides the first data on the length-weight relationships (lwr), fulton's condition factor (k), and sex ratio of this species from florida. lwrs and fulton's condition factor are biological parameters commonly used to measure the physiological welfare of fish populations and are an effective means to assess the general productivity of a population (froese, 2006). * corresponding author: alexander b. orfinger e-mail address: aorfinger@knights.ucf.edu the information herein may be applied for population dynamic models in comparing native and introduced populations as well as population structure across the state of florida. materials and methods from november 2013 to april 2014, the samples were collected from sites throughout tosohatchee wildlife management area (c. 28°29'56"n, 80°55'1"w), a protected area of the middle st. johns river basin spanning 12,424.25 hectares. sampling efforts focused on tosohatchee creek, the st. johns river, and large ditches connecting the former two water bodies, all rich with various aquatic macrophytes. fish were captured using a 1.524 m radius cast net with 0.9525 cm nylon mesh. species identification was confirmed according to reis (1997). samples were transported on ice to the laboratory where they were fixed in 10% buffered formalin and counted and measured for total length (tl) and weight (g). lengths were measured to the 259 orfinger/ population characteristics of hoplosternum littorale nearest 0.1 cm using a digital caliper and weights to the nearest 0.1 g with a digital balance. lwr was calculated using the formula according le cren (1951) as follows: w = 𝛼lb where, w is the total body weight (g), l is the total length (cm), 𝛼 is the intercept of the regression and b is the regression coefficient (i.e. growth rate). the parameters 𝛼 and b were estimated using the leastsquare linear regression method (froese, 2006) after log-transforming the above equation as follows: logw = 𝛼 + b logl the regression coefficient provides information on fish growth. growth is isometric if b = 3 and allometric if b ≠ 3; negative allometry (fish becomes relatively slender as it grows) is noted if b<3 and positive allometry (fish becomes relatively fatter or deeper-bodied as it grows) is noted if b>3 (froese, 2006). the 95% confidence intervals for b were calculated to determine if the predicted value of isometry (3.0) fitted between these intervals (froese, 2006). fulton's condition factor was derived using the relationship: k = 100w/l3, where, w = weight of the fish in grams, l = total length of the fish in centimeters, and 3 is the length coefficient to allow the k values to tend towards one. condition factor was evaluated by size classes (6.09.9 cm and 10.0-13.9 cm) to evaluate physiological differences through ontogeny) and by overall population. size classes divided juvenile and adult individuals as suggested by hahn et al. (1997) and hostache and mol (1998). sex ratio was determined from 100 haphazardly subsampled individuals. those individuals were dissected and their gonads inspected to determine their sex. results and discussion a total of 477 h. littorale individuals were caught ranging in size from 6.4-13.5 cm and weight from 3.6-37 g. the results of lwr are summarized in table 1. the value of b was significantly different than the isometric value of 3 (b = 3.11 ± 0.075, p<0.05, student's t-test) suggesting slight positive allometric growth. the average condition factor for the population was k=1.396 and did not vary significantly between length classes (p<0.05). finally, of the 100 individuals evaluated for sex, 51 were males and 49 were females, emulating an approximately normal (i.e. 1:1) ratio. the b parameter of the lwr from this study is significantly larger than those provided in former studies (b=2.84 (oliva-paterna et al., 2009) and b=2.72 (do nascimento et al., 2013)). also, the maximum condition factor calculated by do nascimento et al. (2013) was significantly lower than in this study (k=1.15 versus k=1.396, respectively; p<0.05). these data suggest that h. littorale populations are obtaining greater relative weights in florida in comparison to their native range (e.g. brazil in the case of oliva-paterna et al., 2009 and do nascimento et al., 2013). such success could be contributed to access to more or novel resources, parasite or predator release, reduced competition than is found in the native range of h. littorale, or a combination thereof. supporting the above notion, the central florida population demonstrated a pattern of positive allometric growth whereas the former two studies noted negative allometry. the calculated lwr and condition factor for florida will be useful for understanding the population dynamics of this invasive fish and further investigations on its growth in the region. the 1:1 sex ratio is congruent with that in wild populations of h. littorale in trinidad (singh, 1978), but differs from the ratio (1.2:0.8) noted in northeastern brazil by do nascimento et al. (2013). the former study consisted of multi-year field n total length (cm) total weight (g) regression parameters min max min max log(a) b 95% ci (a) 95% ci (b) r2 477 6.4 13.5 3.6 37 -1.98 3.11 (-2.05)-(-1.90) 3.03-3.19 0.93 table 1. minimum and maximum length range, regression parameters, and 95% confidence intervals for hoplosternum littorale in central florida. 260 international journal of aquatic biology (2015) 3(4): 258-262 sampling of hundreds of fishes. the 2013 study by do nascimento et al. (2013) inspected only 70 fishes and was conducted solely during breeding season. this sampling regime may explain the male bias given that males aggressively protect their nests, making them much easier to catch. the present study sampled fish during the entirety of the nonreproductive season for h. littorale in florida, ensuring that the sex ratio is not biased by guarding males (nico et al., 2004). as an additional note, while searching for specimens of h. littorale in the university of central florida department of biology ichthyology collection, the author found two notably large male specimens of 25 cm tl and 26.3 cm tl, respectively. the latter length represents the new maximum length of the species; the former maximum length was 25.4 cm tl as reported by antonetti et al. (2013). the two specimens noted here were collected in march 2001 in the st. johns river in central florida at approximately 29°10'3"n, 81°31'23"w by an unknown collector and are currently stored in 70% ethanol. to date, the lwr, condition factor, and sex ratio of this species have not been reported from florida. thus, this work provides the first data available for the region. the population characteristics noted here may be applied in population dynamic modeling to aid in conservation efforts. likewise, the disparity of lwr and condition factor in native and non-native ranges provide incentive to investigate deeper the mechanisms of success for this species in florida. ultimately, the data provided here are a useful metric in measuring the success of a freshwater non-native species in florida. the baseline information herein could help to influence necessary management strategies of h. littorale in the state. acknowledgments the author wishes to thank aileen perilla for assistance during field work and the personnel of tosohatchee wildlife management area. juan bogota and julia behler reviewed the manuscript, providing helpful feedback. references antonetti d.a., leal m.e., schulz u.h. (2014). length‐ weight relationships for 19 fish species from the jacuí delta, rs, brazil. journal of applied ichthyology, 30: 259-260. do nascimento chaves f.d., sánchez-botero j.i., sequeira garce d., cavalcanti dos reis v. (2013). population features of hoplosternum littorale (hancock, 1828) (siluriformes, callichthyidae) at santo anastacio reservoir, brazil. revista mvz córdoba, 18: 3767-3772. duxbury c., holland j., marianne p. (2010). experimental evaluation of the impacts of the invasive catfish hoplosternum littorale (hancock, 1828) on aquatic macroinvertebrates. aquatic invasions, 5(1): 97-102. froese r. (2006). cube law, condition factor and weight– length relationships: history, meta-analysis and recommendations. journal of applied ichthyology, 22: 241-253. gestring k.b., shafland p.l., stanford, m.s, eisenhauer r.l. (2009). status and selected life history attibutes of the illegally introduced brown hoplo (hoplosternum littorale) in florida. florida scientist, 72: 37-47. hahn n.s., almeida v.l.l.d., luz k.d.g.d. (1997). feeding and diel cycle of hoplosternum uttorale (hancock)(siluriformes, callichthyidae) in the lagoons guaraná and patos of the upper paraná river, brazil. revista brasileira de zoologia, 14(1), 57-64. hostache g., mol j.h. (1998). reproductive biology of the neotropical armoured catfish hoplosternum littorale (siluriformes-callichthyidae): a synthesis stressing the role of the floating bubble nest. aquatic living resources, 11(03): 173-185. le cren e.d. (1951). the length-weight relationship and seasonal cycle in gonad weight and condition in the perch (perca fluviatilis). journal of animal ecology, 20(2): 201-219. nico l.g., walsh s.j., robins r.h. (1996). an introduced population of the south american callichthyid catfish hoplosternum littorale in the indian river lagoon system, florida. florida scientist, 59: 189-200. nico l.g., muench a.m. (2004). nests and nest habitats of the invasive catfish hoplosternum littorale in lake tohopekaliga, florida: a novel association with nonnative hydrilla verticillata. southeastern naturalist, 261 orfinger/ population characteristics of hoplosternum littorale 3(3): 451-466. oliva-paterna f.j., torralva m., carvalho e.d. (2009). length-weight relationship for 20 species collected in the jurumirim reservoir (paranapanema basin, brazil). journal of applied ichthyology, 25: 360-361. reis, r.e. (1997). revision of the neotropical catfish genus hoplosternum (ostariophysi: siluriformes: callichthyidae), with the description of two new genera and three new species. ichthyological exploration of freshwaters, 7: 299-326. singh t.b. (1978). the biology of the cascadura, hoplosternum littorale with reference to its reproductive biology and population dynamics. ph.d. thesis. university of west indies, st.-augustine, trinidad, 298 p. winemiller k.o. (1987). feeding and reproductive biology of the currito, hoplosternum littorale, in the venezuelan llanos with comments on the possible function of the enlarged male pectoral spines. environmental biology of fishes, 20(3): 219-227. international journal of aquatic biology (2015) 3(4): 258-262 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved چکیده فارسی از فلوریدا hoplosternum littorale (hancock, 1828) بومیماهی غیرهای جمعیت انتخاب شده گربهویژگی مرکزی، ایالت متحده آمریکا اورفینگر نجامینب الکساندر .آمریکا متحده ایالت فلوریدا،، 0444دانشگاه مرکزی فلوریدا، بلوار مرکزی فلوریدا ،شناسیزیست گروه چکیده: hoplosternum littorale( و نسبت جنسی گربه ماهی غیر بومی kفاکتور وضعیت فولتون ) ،(lwrوزن )-این مطالعه رابطه طول (hancock, 1828) ناحیه مدیریت در 3400الی آوریل 3402از نوامبر سال برداری. نمونهدکنبرای اولین بار گزارش می آمریکا را در فلوریدا . ندمتر صید شدیتسان 0/6-5/02نمونه با دامنه طول کل 044در کریسمس فلوریدا انجام شد. در مجموع tosohatchee حیات وحش ( که بیانگر الگوی رشد آلومتریک مثبت b=00/2ار برای رشد ایزومتریک بود )انتظوزن بیشتر از مقدار مورد -ه طولطراب b تریکمضریب آلو های طولی نوجوان و بالغ وجود نداشت. داری بین کالسبود و تفاوت معنی 296/0( kوضعیت فولتون ) باشد. مقدار میانگین فاکتورمیاین گونه اشد که تواند بدر محدوده بومی پراکنش آن نشان داد که بیانگر این موضوع می های ثبت شده این گونهاختالف بارزی را نسبت به دادهنتایج عالوه یک ثبت جدید از حداکثر طول کل این گونه نیز در این مطالعه گزارش . بهاستاین گونه مهاجم آب شیرین در حال گسترش در فلوریدا شود.می .نسبت جنسی، فاکتور وضعیت فولتون، وزن-رابطه طول، hoplosternum:کلمات کلیدی int. j. aquat. biol. (2013) 1(2): 42-47 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology effects of dietary free l-lysine on growth performance and muscle composition of beluga huso huso (linnaeus 1785) juveniles seyyed morteza hoseini *1, seyed abbas hosseini1, mohammad soudagar1 1 department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran article history: received 2 march 2013 accepted 14 april 2013 available online 2 5 april 2013 keywords: l-lysine huso huso food intake muscle composition abstract: effect of dietary free l-lysine on growth, food intake, and muscle composition of beluga juveniles were investigated over 6 weeks. control diet lysine content was 2.1% of dry matter (4.4% of dietary protein). three experimental diets were prepared by adding lysine (0.5, 1 and 1.5%) to control diet to obtain diets containing 2.6, 3.1 and 3.6% of dry matter lysine (corresponding to 5.5, 6.6 and 7.6 of dietary protein). fish were fed 2.6% of dry matter lysine showed significantly higher final weight, weight gain and sgr and lower fcr compared to other treatments. there was no significant change in food intake and survival between treatments. lysine supplementation resulted significantly in increase and decrease in muscle protein and lipid, respectively. dietary lysine has no effect on muscle ash and moisture content. results showed that lysine supplementation had no significant effect on food intake in beluga juveniles. it seems that dietary lysine level of 2.6% of dry matter (corresponding to 5.5% of dietary protein) is suitable for growth of beluga juveniles. introduction sturgeon populations are under pressure due to overfishing, pollution and habitat degradation (birstein, 1993). beluga is one of the most important species inhabits in caspian sea. this species is included as endangered species in iucn red list and in 1997 in the ciies appendices. aquaculture is a critical technique to decrease pressure on beluga wild population and increase caviar and meat production. fishes require the same ten indispensable amino acids as other animals for growth (wilson, 1985). indispensable amino acids deficiency will result in reduced growth performance and feed utilization (wilson and halver, 1986). it is important to fulfill the indispensable amino acid requirements of fish by formulating balanced nutrients in fish feeds. lysine is one of the indispensable amino acids (wilson, 2002) which is often the limiting amino acid in commercial feeds (harris, 1980; forster and ogata, * corresponding author: seyyed morteza hoseini e-mail address: seyyedmorteza.hoseini@gmail.com tel: +989112750713 1998; small and soares, 2000; tantikitti and chimsung, 2001). an important function of lysine is to serve as the precursor of carnitine which carries long chain fatty acids into the mitochondria for βoxidation of lipids (walton et al., 1984). researchers had reported the lysine requirements of commonly cultured fish species to range from 3.2 to 6.2% dietary protein (wilson, 2002). lysine deficiency causes poor growth performance in number fish species (zhou et al., 2007; mai et al., 2006 and wang et al., 2005). but no information is available about effect of different levels of dietary lysine on beluga juveniles' growth performance. amino acids are one of the major classes of feeding stimulants (jones, 1992) which can maximize food consumption and reduce food waste by improving initial feeding and food palatability (lee and 43 hoseini et al./ int. j. aquat. biol. (2013) 1(2): 42-47 meyers, 1996). sturgeons are virtually blind and olfaction is one of the fundamental senses for feeding behavior of them (kasumyan, 1999). when the food odor (like as chironomid larvae extract) is released in water body, sturgeons will receive it and move along circular and s-shaped trajectories or loops around food place (kasumyan, 1999). therefore, the main objective of this study was to investigate the effects of adding free lysine to beluga juveniles' diet, on food intake, growth performance, survival and muscle composition. materials and methods fish and maintenance condition: 240 beluga juveniles (17.71±2.68 g) were randomly distributed in 12 tanks (500 l.) and were allowed to acclimate for one week. all tanks were supplied by dechlorinated tap water. all tanks were aerated continuously over the experiment. water exchange was 50% daily. during acclimation week, all fish were fed control diet until apparent satiation once a day. thereafter, a six-week trial carried out with four triplicate treatments (one control and three lysine treatments). during whole experiment, fecal and food waste were siphoned every day. dissolved oxygen, ph and water temperature were measured daily (by digital dissolved oxygen meter + ph meter apparatus) over the six weeks, which ranged 5.5±0.5 mg/l, 8.04 ± 0.05 and 24 ± 1 (mean ± sd) °c, respectively. diet preparation: the diet ingredients and approximate composition of the control group are shown in table 1. it contained 2.1% of diet (correspond to 4.4% of dietary protein) lysine. other experimental diets were prepared by adding crystalline l-lysine at levels of 0.5, 1 and 1.5% of diet. thus, we reached four treatments contained 2.1, 2.6, 3.1 and 3.6% of diet total lysine. free and protein banded amino acid profiles is shown in table 2. all diets were pelleted 3 mm in diameter. experimental protocol: each treatment was fed corresponding diet until 6th week. feeding was performed by hand, once a day. in each meal, exceed food was offered to fish and allowed to eat until table 1. basal diet ingredient. ingredients % fish meal 49 soybean meal 15.5 wheat meal 4 corn meal 3.2 wheat gluten 2.2 barely meal 2.5 meat meal 7.5 cottonseed 2.5 antioxidant 0.1 salt 0.5 mineral mix 1 vitamin mix 1 binder 1 fish oil 5 soybean oil 5 crude protein 46.8 crude lipid 12.4 carbohydrate 17.1 ash 4 moisture 15 amino acid band free trp 0.9 0 asp 31.7 1.3 glu 74.2 0.3 ser 14.8 0.8 gly 28 0.6 his 8.4 1.2 arg 26.2 6.1 thr 13.4 0.6 ala 22.1 1.5 pro 25.7 1 tyr 6.7 0.3 val 22.1 0.7 met 7.1 0.3 cys 1.9 0.7 ileu 17.4 1.2 leu 28.7 0.9 phe 15.8 0.3 lys 20 1 table 2. basal diet amino acids profile. 43 44 hoseini et al./ int. j. aquat. biol. (2013) 1(2): 42-47 satiation and then remaining pellets were counted and food intake was calculated. at the end of trial all fish were weighed and growth factors were calculated. for determination of approximate muscle composition, dorsal muscle of 15 fish in each treatment were collected and stored in -20 °c until further analyses. analyses: at the end of the trial, all fish were weighed and percent weight gain (wg%), feed conversion ratio (fcr), specific growth rate (sgr), average food intake (fi) and percent survival were calculated using the following equations: wg% = final weight × 100/ initial weight fcr = dry feed fed / body weight gain sgr = (ln final weight ln initial weight) × 100/ days fi = total consumed food / days muscles and diet dry matter (at 105 ˚c for 24 h), crude protein (kjeldahl apparatus, gerhardt, königswinter, germany. nitrogen* 6.25), crude fat (extraction with petroleum ether by soxhlet apparatus, behr, düsseldorf, germany) and ash (incineration at 600 1c for 6 h) were determined (aoac, 1995). diet carbohydrate was measured using the method by lane – eynon method (aoac, 1978). amino acid profile of diet with exception of protein banded tryptophan was determined using hplc as described by ovissipour et al. (2009). for protein banded tryptophan determination, protein was extracted according to the method of concon (1975) and tryptophan was estimated by the method described by szakacs and perl (1990). statistical analysis: data were analyzed by one-way anova using the statistical software spss version 9.0. subsequent significance among groups was delineated by lsd test. data are presented as treatment mean ± sd. the values of p<0.05 were considered significantly different. results 2.6% lysine treatment exhibited the best wg, sgr and fcr than other groups (table 3). there was no significant difference in food intake and survival among the treatments (table 3). there were no differences in moisture and ash content of muscle dietary lysine iw fw wg fcr sgr fi sur 2.1 16.3±1.0a 43.0±1.8a 263±5.3a 1.0±0.02a 2.3±0.04a 2.8±0.2a 91.6±10a 2.6 17.5±3.2a 57.0±7.1b 328±24b 0.8±0.06b 2.8±0.20b 3.0±0.5a 98.3±2.9a 3.1 18.5±1.5a 47.6±6.6a 256±18a 1.0±0.04a 2.2±0.10a 2.8±0.2a 98.3±2.9a 3.6 18.1±3.1a 40.2±2.9a 224±32a 1.1±0.16a 1.9±0.30a 2.4±0.3a 98.3±2.9a pvalue 0.71 0.017 0.003 0.016 0.006 0.11 0.44 iw = initial weight, fw = final weight, wg = weight gain, fcr = food conservation ratio, sgr = specific growth rate, fi = foo d intake, sur = survival table 3. effects of dietary l-lysine (%) on weight gain (%), fcr, sgr, food intake (% of body weight per day) and survival (%) of h. huso juveniles. dietary lysine moisture protein lipid ash 2.1 77.96±0.62a 14.45±0.53a 6.27±0.19a 1.13±0.04a 2.6 78.35±0.55a 15.83±0.37b 4.58±0.15b 1.08±0.02a 3.1 78.21±0.28a 15.98±0.14b 4.58±0.23b 1.08±0.09a 3.6 78.06±0.33a 16.64±0.26bc 4.03±0.1c 1.13±0.04a pvalue 0.76 < 0.001 < 0.001 0.62 table 4. effects of dietary l-lysine (%) on muscle composition (%) of h. huso juveniles. 45 hoseini et al./ int. j. aquat. biol. (2013) 1(2): 42-47 samples among the groups. increasing of dietary lysine resulted in increase and decrease in muscles protein and lipid, respectively (table 4; p< 0.05). discussion one of the major classes of olfactory stimulants are amino acids that cause exhibition of food searching behavior in a variety of fishes (jones, 1992). thus, diet supplementation with free amino acid can improve food intake and growth. our results showed that lysine failed to stimulate food intake in beluga juveniles. based on our knowledge, there is no study on stimulatory characteristic of amino acids on beluga juveniles. previous study on other sturgeon species have shown stimulatory effect of amino acids is species specific (kasumyan, 1999; kuzmin et al., 1999). however, present results demonstrated lysine, at least in our experimental conditions, was not a stimulator amino acid in beluga juveniles. high growth performance in 2.6% lysine group seems to be as a result of optimization of dietary total lysine (5.5% of dietary protein) that is approximately similar to the data published for cobia, rachycentron canadum (5.3%, zhou et al., 2007) white sturgeon, acipenser transmontanus (5.5%, ng and hung, 1995), grass carp, ctenopharyngodon idella (5.89%, wang et al., 2005) and japanese sea bass, lateolabrax japonicus (5.8%, mai et al., 2006) but higher than those reported for rainbow trout, 3.7% (kim et al., 1992), and coho salmon, 3.8% (arai and ogata, 1991), and lower than that for catla, 6.2% (ravi and devaraj, 1991), or rainbow trout, 6.1% (ketola, 1983). the wide variation observed in the requirements for lysine among fish species may be due to the differences in dietary protein sources, the reference protein which amino acid pattern is being imitated (forster and ogata, 1998), diet formulation, size and age of fish, genetic differences, feeding practices and rearing conditions (ruchimat et al., 1997). digestibility, amino acid profile and energy content may also bring variable effects in amino acid requirement studies (simmons et al., 1999; de silva et al., 2000). in present study, fish which fed higher lysine than 2.6 (3.1 and 3.6% group) showed slightly lower sgr, wg and final weight. it was suggested that decrease of growth of indian carp fed high amount of lysine (2.78% dry diet) may be due to the negative effects (lysine–arginine interaction) of excessive amount of free lysine at this level (ahmed and khan, 2004). dietary lysine–arginine antagonism has been well known in poultry and rats (jones, 1964; harper et al., 1970; fico et al., 1982), but there are few studies on fishes. kaushik and fauconneau (1984) have offered some biochemistry evidences indicating that some metabolic antagonism may exist between lysine and arginine in rainbow trout. the increasing dietary lysine intake affected plasma arginine and urea levels, and ammonia excretion. similarly, some studies showed that some metabolic interactions occur between arginine and lysine when elevated levels of either were fed to atlantic salmon (berge et al., 1997, 1998). mai et al. (2006) also reported lower sgr of japanese sea bass fed high amount of lysine (3.66% and 4.25%). muscle protein content increased while diet lysine increased that was reported by mai et al., (2006). this can be attributed to an increase of net protein synthesis with the increase in dietary lysine level (ruchimat et al., 1997) or optimum nutritional status which corresponded to the optimum dietary lysine level. muscle lipid decreased while dietary lysine increased that is partially in agreement with other studies (kim et al., 1992; zarate and lovell, 1997; ahmed and khan, 2004; luo et al., 2006). in conclusion, lysine supplementation has no significant effect on food intake in beluga juveniles. it seems that dietary lysine level of 2.6% of dry matter (corresponding to 5.5% of dietary protein) is suitable for growth of beluga juveniles. acknowledgements the authors wish to thank aquaculture research center of gorgan university or agricultural sciences and natural resources for providing necessary facilities for the study and shahid marjani sturgeon propagation and culture complex for sturgeon fish. 45 46 hoseini et al./ int. j. aquat. biol. 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(2014) 2(1): 14-19 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article morphometric characters and condition factors of five freshwater fishes from pagla river of bangladesh m. manjurul alam*1, md. tohidur rahman1, selina parween2 1department of fisheries, university of rajshahi, rajshahi-6205, bangladesh. 2department of zoology, university of rajshahi, rajshahi-6205, bangladesh. article history: received 23 december 2013 accepted 27 january 2014 available online 2 5 february 2014 keywords: amblypharyngodon aspidoparia puntius cirrhinus chanda abstract: the research was aimed to carry out the length-length relationships, length-weight relationship and condition factor of five freshwater small indigenous fish species i.e. amblypharyngodon mola, puntius ticto, cirrhinus reba, chanda nama and aspidoparia morar from the pagla river bangladesh. the relationships equations among different body length parameters of each species were found highly significant. the length-weight relationship equation were found as tw=0.0351 tl2.86 for a. mola, tw = 0.0104 tl3.10 for p. ticto, tw = 0.0261 tl2.96 for c. reba, tw = 0.0175 tl2.845 for c. nama tw= 0.0101 tl3.05 for a. morar. the “b” values ranged from 2.8453.10 which remained on the expected range of optimum growth. introduction the statistical analysis of morphometric characters is one of the vital factors for the proper management of a species. in recent years, significance of the statistical relationship of morphometric characters has also been recognized in all taxonomic and systematic studies to solve various problems concerned with the life history of fish. morphometric measurements and statistical relationships of fishes are therefore imperative for both fishery biology (sparre et al., 1989; mustafa and brooks, 2008) and taxonomic studies (tandon et al., 1993; simon et al., 2010). these relationships also give information on the condition and growth patterns of fish (bagenal and tesch, 1978; oscoz et al., 2005). in addition, condition factors may be used to detect seasonal variations in the growth of fish, which may vary with food abundance and average reproductive stage of the stock (king, 1995). for proper management and conservation of the wild population of fish species morphometric study is necessary. * corresponding author: m. manjurul alam e-mail address: mamillat@yahoo.com in bangladesh, a total of 260 indigenous freshwater fish species (rahman, 2005) of which small indigenous species of fishes are important target species for the small-scale fishermen of bangladesh (craig et al., 2004; mustafa and brooks, 2008) and serve as a major source of protein and vitamin for the rural community (rubbi et al., 1978). the present study aimed to find out the present status of length-length and length-weight relationship and condition factor of five species i.e. amblypharyngodon mola, puntius ticto, cirrhinus reba, chanda nama, aspidoparia morar. findings of the work will play an important role for the successful management of these species to conserve from probable depletion of their wild stock in future. materials and methods the specimens were collected from the fishers of pagla river, chapai nawabgonj district (lies between 24°40'n and 24°42'e latitude and between 88°08'n and 88.11°'e longitudes) north-west end of 15 alam et al./ int. j. aquat. biol. (2014) 2(1): 14-19 bangladesh during day time from october, 2011 to january, 2012. during the period a total of 260 specimens (each of 52) were collected and identified to the species level according to rahman (2005) and preserved by date in plastic jars with 5% formalin to save from spoilage. for each individual different body lengths e.g. total length (tl), standard length (sl), dorsal length (dl), pectoral length (pl), pelvic length (pvl), anal length (al), head length (hl) were measured using a digital slide calipurse and the total weight (tw) was measured using a digital balance model: kd-300kc with 0.01g accuracy according to simon and mazlan (2008) and alam et al. (2012). the relationships among all body length parameters were determined usiny the method of least squares to fit a linear regression as: y = a + bx. where, y = various body lengths, x = total length, a = proportionality constant and b = regression coefficient (alam et al., 2012). the length-weight relationships were determined by the general equation of tw = atlb (lecren, 1951), where, tw is the total weight (expressed in g), tl is the total length (expressed in cm), “a” is a coefficient related to body form and “b” is an exponent indicating isometric growth when equal to 3 and indicating allometric growth when significantly different from 3 (simon and mazlan, 2008; simon et al., 2009). the parameters “a” and “b” of the exponential curve were estimated by linear regression analysis over log-transformed data expressed as: logtw=loga + blog tl. the values of the constant “a” and “b” of the linear regression was determined by following rounsefell and everhart (1953) and lagler (1966). the fulton’s condition factor, k was calculated by using the following formulak= (tw/tl3) × 100, where, k = fulton’s condition factor, tw= total weight, tl = total length. here, factor 100 is used to bring k close unity. the data were analyzed using software spss, version 15.0. results and discussion the length-length relationships with total length among standard length, dorsal length, pectoral length, pelvic length, anal length, head length and the coefficient of correlation of five fish species are presented in table 1. the body lengths were frond highly significant with all “r” values being >0.900 and positively correlated with tl. the obtained regression equations clearly revealed that the lengths of the body parts are proportional to the total length. such finding were also observed by tandon et al. (1993) while working with the morphometry of cirhinus reba of kanjli wetland of india. these relationships were also observed in puntius chola (bhuiyan and biswas, 1982), mystus vittatus (hoque and hossin, 1992; hossain et al., 2006), parastromateus niger, (dadzie et al., 2008) and puntius sophore (alam et al., 2012). the findings are more similar to the findings of hossain et al. (2009) on the length-weight and length-length relationship of 10 small fish species from the ganges, bangladesh; and alam et al. (2013) on length-length relationship, length-weight relationship and condition factor of freshwater fish species of bangladesh. the range of length and weight parameters of the length-weight equations and values of fulton’s condition factors were shown in table 2. the values of slope “b” of length-weight equations were obtained as 2.860 for a. mola, 3.10 for p. ticto, 2.960 for c. reba, 2.845 for c. nama and 3.051 for a. morar. the length-weight relationships were found highly significant with all “r” values being >0.900 where the parameter “b” remained mostly within the expected range of 2.5-3.5. therefore, all the species seemed to be followed the cube law. the reason behind may be the observed specimens were the inhabitants of quite good environment and gravid females were more in the samples (lecren, 1951). the equations are therefore applicable for the total population as a whole. while working with different morphometric characters of other fish species bagenal and tesch (1978), hoque and hossain (1992), kiran et al. (2004), oscoz et al. (2005), froese (2006), britton and devies (2007), aguirre et al. (2008), arshad et al. (2008), hossain et al. (2009) and alam et al. (2013) observed similar results. the obtained values of fulton’s condition factor were 16 alam et al./ int. j. aquat. biol. (2014) 2(1): 14-19 found >1 for p. ticto and a. morar which indicate good growth performance of these species whereas for the rest species the values were being <1 but very closer to 1. this fluctuation may occur due to age and stage of maturity of the species as well as environmental conditions of habitat such as temperature, salinity and seasonality. such findings were also observed in p. chola (bhuiyan and biswas, 1982), h. fossilis (mia, 1984), a. mola (afroze et al., 1992), m. vittaus (hoque and hossain, 1992), p. stigma (islam and hossain, 1992), a. coila (alam et al., 1994), b. canis (mir, 1996), chanda nama and species (ordinate tl) abscissa meanse of abscissa regression equation r a. mola (tl = 5.87±0.09) sl 4.53±0.07 sl = 0.4719+ 0.6918tl 0.972** dl 2.43±0.03 dl = 0.3203+ 0.3604tl 0.936** pl 1.24±0.04 pl = 0.1847+ 0.1799tl 0.934** pvl 2.23±0.04 pvl = 0.3323+ 0.4375tl 0.979** al 3.07±0.05 al = 0.0981+ 0.5392tl 0.964** hl 1.18±0.01 hl = 0.2949+ 0.1513tl 0.948** p. ticto (tl = 6.98±0.08) sl 5.39±0.06 sl = 0.276+ 0.7323tl 0.971** dl 2.72±0.02 dl = 1.0787+ 0.2345tl 0.918** pl 1.32±0.02 pl = 0.0176+ 0.1865tl 0.964** pvl 2.53±0.04 pvl = 0.3683+ 0.4154tl 0.938** al 3.87±0.06 al = 0.4028+ 0.6124tl 0.966** hl 1.51±0.02 hl = 0.0079+ 0.2149tl 0.952** c. reba (tl = 9.64±0.16) sl 7.39±0.11 sl = 0.6881+ 0.6949tl 0.986** dl 3.42±0.05 dl = 0.7073+ 0.2816tl 0.946** pl 1.67±0.03 pl = 0.1877+ 0.1536tl 0.963** pvl 3.59±0.05 pvl = 0.5637+ 0.3136tl 0.946** al 5.73±0.09 al = 0.1178+ 0.5816tl 0.987** hl 1.69±0.02 hl = 0.5561+ 0.1172tl 0.937** c. nama (tl = 5.59±0.10) sl 4.29±0.09 sl = 0.4553+ 0.8487tl 0.992** dl 1.68±0.03 dl = 0.2454+ 0.2577tl 0.954** pl 1.34±0.01 pl = 0.7222+ 0.1100tl 0.918** pvl 1.42±0.03 pvl = 0.1017+ 0.2356tl 0.931** al 2.40±0.05 al = 0.0827+ 0.4439tl 0.984** hl 1.30±0.02 hl = 0.1955+ 0.1981tl 0.956** a. morar (tl = 6.04±0.06) sl 4.84±0.05 sl = 0.3242+ 0.8541tl 0.976** dl 3.25±0.04 dl = 0.2049+ 0.5721tl 0.955** pl 1.05±0.02 pl = 0.6239+ 0.2773tl 0.922** pvl 2.37±0.02 pvl = 0.1262+ 0.3706tl 0.933** al 3.26±0.03 al = 0.3886+ 0.4748tl 0.932** hl 0.91±0.01 hl = 0.1073+ 0.1683tl 0.903** table 1. relationships with total length among different body lengths of five species species total length characteristics total weight characteristics parameters of the relationship fulton’s condition factor range (cm) mean±se range (cm) mean±se a b r (k) a. mola 4.50-6.70 5.87±0.09 0.88-3.26 1.97±0.10 0.0351 2.860 0.935 0.97 p. ticto 5.10-8.00 6.98±0.08 1.50-8.11 5.36±0.20 0.0104 3.10 0.939 1.36 c. reba 7.00-12.00 9.64±0.16 3.76-13.20 8.69±0.35 0.0261 2.960 0.976 0.99 c. nama 4.50-6.80 5.59±0.10 1.40-3.60 2.39±0.07 0.0175 2.845 0.933 0.95 a. morar 4.90-6.80 6.04±0.06 0.68-1.78 1.24±0.04 0.0101 3.051 0.928 1.28 table 2. length-weight relationships and fulton’s condition factors of five species 17 alam et al./ int. j. aquat. biol. (2014) 2(1): 14-19 c. ranga (iqbal et al., 1995-1996), b. lohachata (mortuza and mokarrama, 2000), t. mediterraneus (santic et al., 2006), p. niger (dadzie et al., 2008) and p. ticto (hossain et al., 2012). findings of the present study has provided some new and updated information on the morphometric characters of five freshwater fish species of pagla river representing the status of other small indigenous fish species of bangladesh. outcome of the present investigation will play important role for the management and conservation of these species as well as other small indigenous fish species of bangladesh. acknowledgement we are grateful to the chairman, department of fisheries and department of zoology, university of rajshahi for providing all sorts of lab facilities during the study period. references afroze s., hossain m.a., parween s. 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(2016) 4(2): 117-124; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article the complete description of the skeletal structure of hafez loach, turcinoemacheilus hafezi (cypriniformes, nemacheilidae) nasrin nikmehr, soheil eagderi*,1hadi poorbagher, hamid farahmand department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 21 january 2016 accepted 17 april 2016 available online 2 5 april 2016 keywords: nemacheilidae osteology loach turcinoemacheilus iran abstract: the osteological characteristics is an important tool for clarification of the phylogenetic statue of the family nemechelidae. since any information is available about osteological features of the genus turcinoemacheilus, hence the present study provides a detailed osteological characteristics of the turcinoemacheilus hafezi as representative of this genus. ten specimens of t. hafezi were collected from the shalamzar stream, tigris basin, iran and cleared and stained for osteological examination. according to the results, t. hafezi is osteologicaly characterized by absence of the preethmoideum-i and postcleithrum, having four basibranchials, presence of the sesamoid ossification, free and short epural with the reduced neural process and pleurostyle, connection of the hypural-1 to the parahypural, no connection of the hypurals 3, 4, and 5 to the pleurostyle, and no bony bridge between the parietal. introduction nemacheilids are small and benthic species that found in flowing waters of europe, asia, and northeast of africa (coad, 2016). loaches of the genus turcinoemacheilus, a member of the family nemacheilidae, is easily distinguished from other members of this family by a more anterior position of the anus (bânârescu and nalbant, 1964; freyhof et al., 2011). turcinoemacheilus was a monotypic genus for 47 years (conway et al., 2011; golzarianpour et al., 2013). turcinoemacheilus kosswigi bănărescu and nalbant, 1964 was recorded for the first time in iran from the karoun river drainage (golzarianpour et al., 2009). turcinoemacheilus hafezi, a new species from karoun and dez rivers drainages of iran (golzarianpour et al., 2013), was second species of this genus from iran. also, esmaeili et al. (2014) described two new species of the genus turcinoemacheilus from iran, including t. bahaii from the zayandeh river and t. saadii from the karoun river drainage in iran. turcinoemacheilus hafezi golzarianpour et al. * corresponding author: soheil eagderi e-mail address: soheil.eagderi@ut.ac.ir 2013 is distinguished from the other species of this genus by the more posterior position of the anus, which is closer to the anal-fin origin than to the pelvic-fin origin (distance from anus to anal-fin origin 0.2-0.4 times in distance from pelvic-fin origin to anal-fin origin), a mottled colour pattern or a prominent irregular dark brown or black midlateral stripe disconnect from blotches and saddles on back, a slightly emarginated caudal fin and a completely scaleless body (golzarianpour et al., 2013). due to small size and low economic value, the members of the family nemacheilidae are less known (kottelat, 1990) and their classification is still complicated; therefore, ichthyologists are trying to reveal their phylogenic status (prokofiev, 2010). hence, the osteological characteristics can play an important role in this regard (sawada, 1982; mafakheri et al., 2015). the only comprehensive osteology and phylogenetic studies on nemacheilids performed by prokofiev (2009, 2010), but in these studies, the turcinoemacheilus did not include. 118 nikmehr et al./ osteology of turcinoemacheilus hafezi since, there is no information is available about the osteological features of the genus turcinoemacheilus, therefore, this study aimed to provide a detailed osteological description of hafez loach, t. hafezi from the karoun river drainage. materials and methods ten specimens of t. hafezi with average standard length of 38.22(±0.64) (mean±sd) were collected by electrofishing device from the shalamzar stream (charmahal-e bakhtiari province, iran) (fig. 1). then, the specimens were anesthetized in 1% clove solution and fixed in 10% buffered formaldehyde. for osteological examination, the specimens were cleared and stained using alcian blue and alizarn red based on taylor and van dyke (1985). images of the stained skeletal structure obtained by a scanner (epson v600) equipped to a glycerin bath. skeletal structures were observed and studied by a stereomicroscope (leica ms5). drawings of the skeletal elements were performed using coreldraw x6 software. nomenclature and abbreviation of the skeletal elements were based on prokofiev (2009). results the neurocranium is wider posteriorly, forming the maximum width of the skull at the level of the pterotic. the ethmoid region is comprised of a pair of the lateral ethmoid, and unpaired prevomer and supraethmoid-ethmoid bones (fig. 2a). the paired l-shaped lateral ethmoids posteriorly form the anterior part of the orbit. the prevomer is relatively flat having two processes with rounded edges anterolaterally. this bone is connected to the orbitosphenoid and parasphenoid posteriorly (fig. 2b). the supraethmoid-ethmoid is narrower in the middle part. the kinethmoid is a small and free bone situated between two maxillae. this bone has two pointed lateral processes in the middle part, forming its maximum width. the orbital region includes the frontal, orbitosphenoid, ptersphenoid, parasphenoid, lachrymal, and sclerotic bones. the paired large frontals are asymmetric and have two processes in latero-middle part; its anterior part is narrow. these bones include about half of the length of the neurocranium separating by the fontanel posteriorly. the frontal is connected to the orbitosphenoid antero-laterally. the posterior margins of the frontals contribute in formation of the fontanel. the ptersphenoid is connected to the frontal dorsally and to the sphenoid posteriorly, forming the posterior part of the orbit. the parasphenoid is bifurcated at two ends and its middle part is connected to the pterosphenoids. it has two small lateral and one large middle pores. the orbitosphenoid is connected to the lateral ethmoid ventrally, to the parasphenoid anterolaterally, and to the pterosphenoid posteriorly (fig. 2). the otic region includes five bones viz. the parietal, epiotic, sphenotic, pterotic, and prootic. the parietal is posteriorly connected to the supraoccipital and epiotic and ventrally to the prootic. in addition, no bony bridge present between the parietal and pterotic. the parietal is roughly rectangular. the pterotic is almost triangular in shape. there is no connection between the parietals due to a long fontanel. the sphenotic bears a lateral process and a figure 1. turcinoemacheilus hafezi from the shalamzari stream river. 119 int. j. aquat. biol. (2016) 4(2): 117-124 (a) (c) (b) latero-ventral facet. the occipital region is composed of the supraoccipital, exoccipitals, and basioccipital. the supraoccipital has a pentagon shape; it is connected to the exoccipitals ventrally. the exoccipital encloses the foramen magnum and bears a small foramen laterally. the basioccipital bears a posterior process with a large cavity centrally. there is an occipital condyle at the posterior part of the basioccipital for connecting to the vertebrae. in the branchiocranium, the upper jaw consists of the maxilla and premaxila (fig. 3a). the maxilla is a figure 2. the neurocranium of turcinoemacheilus hafezi. (a) dorsal, (b) lateral, and (c) ventral views. apl: autopalatine, fon: fontanel, fr-exo: foramen exoccipital, pr-bo: basioccipital process, epo: epiotic, exo: exoccipital, fr: frontal, let: lateral ethmoid, orb: orbitosphenoid, pa: parietal, pe: prevomer, pro: prootic, ps: parasphenoid, pto: pterotic, pts: pterosphenoid, se: supraethmoid, soc: supraoccipital, and s po: sphenotic. 120 nikmehr et al./ osteology of turcinoemacheilus hafezi large laminar bone and its middle part is the wider; its anterior part is narrow with an antero-ventral process tilting ventrally. the premaxila is l-shaped comprising two horizontal and vertical parts. the horizontal part is arc-shaped and slightly wider in its middle part and the vertical part is narrower and longer. there is two small triangular process at the middle portion of the vertical part. the lower jaw is composed of four bones, including the dental, angular, retroarticular and coronomeckelian (fig. 3b). the dental is the largest element of the mandible, including a narrow anteroventral branch and a deep section (process coronoideus) postero-dorsally; it is connected to the articular postero-dorsally and to the retroarticular dorsally. the coronomeckelian is triangular in shape and situated in the medial side of the dental beneath the process coronoideus. the suspensorium is formed by the hyomandibular, ectopterygoid, endopterygoid, metapterygoid, symplectic, quadrate, and autopalatine (fig. 4). the hyomandibular is a relatively large bone extending longitudinally that its dorsal part is wider. in addition, there are two anterior and posterior hyomandibular condyles on its dorsal margin. the quadrate bears a condyle anteroventrally where it is articulated to the dental. the ectopterygoid, endopterygoid, metapterygoid, symplectic, and quadrate form a complex connecting to the anterior part of the neurocranium via autopalatine. the autopalatine is wider and posteriorly connected to the endopterygoid and anteriorly to the prevomer. the endopterygoid is pointed posteriorly connecting to the ectopterygoid figure 3. the upper (a) and lower (b) jaws of turcinoemacheilus hafezi. mx: maxilla, pmx: premaxilla, art: articular, cm: coronomeckelian, den: dental, and ret: retroarticular. figure 4. the suspensorium, palatine, and opercular series of turcinoemacheilus hafezi. apl: atutopalatine; ect: ectopterygoid, end: endopterygoid, hm: hyomandibular, io: interopercle, mtp: metapterygoid, op: opercle, q: quadrate, so: subopercle, and sym: symplectic. 121 int. j. aquat. biol. (2016) 4(2): 117-124 antero-ventrally and metapterygoid posteriorly. the metapterygoid bears several pores on its lateral face. the ectopterygoid is situated anterior to the quadrate. the opercular series consists of the opercle, peropercle, subopercle, and interopercle (fig. 4). the opercle is the largest element of this series and possesses a condyle anteriorly. the paddle-shaped subopercular is located ventral part to the opercle. the narrow preopercular is pointed anteriorly and bears a notch at its posterior rim. the interopercular is a curved bone with pointed anterior part. the branchial apparatus includes five pairs of the ceratobranchial, four pairs of the epibranchiasls, three pairs of the hypobranchials and pharyngobranchials, and four unpaired basibranchials (fig. 5a). the anterior part of the first and second basibranchials are the wider. two first hypobranchials are similar in shape and the last one is the smallest. four pairs of the ceratobranchials are situated between the hypobranchials and epibranchials and the last ceratobranchials are modified as the tooth plats. the last epibranchial curves dorsally and its anterior part is narrower. the hyoid arch consists of the paired interhyal, epihyals, ceratohyals, dorsal and ventral hypohyals, and the unpaired urohyal and basihyal, and three pairs of the branchiostegals (fig. 5b). the urohyal is t-shaped and anteriorly bifurcated. the basihyal is also t-shaped with a deep notch on its anterior end. the dorsal and ventral hypohyals are connected firmly. the ceratohyals are the largest elements of the hyoid arch. there are three curved long branchiostegals; the first one is attached to the ceratohyal, the second one connected between the ceratohyal and epihyal, and the third one is attached to the epihyal. the interhyal is small with pointed posterior end (fig. 5b). in the postcranial skeleton, the pectoral girdle consists of the cleithrum, supracleithrum, coracoid, mesocoracoid, scapula, posttemporal, supratemporal and radials (fig. 6a). the largest element of this complex is the cleithrum that bears a longer vertical and a horizontal sections. the dorsal part of the supracleithrum is wider. the coracoid is connected to the cleihtrum posteriorly and middle-ventrally. the mesocoracoid is narrow and its ventral part ia wider and connected to the coracoid. the mesocoracoid is firmly connected to the cleithrum. the scapula is trapezoid in shape and connected to the coracoid ventrally. the pectoral girdle bears four radials that overlap together and first one is the widest. the pectoral fin has one unbranched and 9 branched rays. figure 5. dorsal view of the branchial apparatus (a) and hyoid arch (b) of turcinoemacheilus hafezi. bbr: basibranchial, cbr: ceratobranchial, ebr: epibranchial, hbr: hypobranchial, pbr: pharyngobranchial, bhy: basihyal, chy: ceratohyal, dhy and vhy: dorsal and ventral hypohyal, epi: epihyal, ihy: interhyal, and uhy: urohyal. 122 nikmehr et al./ osteology of turcinoemacheilus hafezi the pelvic girdle includes the paired pelvic bones, pelvic splint and radials (fig. 6b). the pelvic bone is horizontally positioned in the middle part of the belly. it has two long processes anteriorly and its posterior part is wider. the pelvic bone has a round process latero-posteriorly. the pelvic girdle possesses three radials that the medial one is the smallest one. the styloid is situated lateral to the radials. the pelvic fin bears one unbranched and 7 branched rays. the dorsal fin bears 4 unbranched and 6 branched rays, 8 pterygiophores and one stay bone. the first pterygiophor is next to the 17th or 18th vertebra. the largest bone of the dorsal fin is the first pterygiophores that supports 4 unbranched rays. the first and last petrygiophores are bifurcated ventrally. a triangular stay supports the last branched ray (fig. 7a). the anal fine originates at 26th centrum; it has 4 figure 6. medial view of pectoral girdle (a) and pelvic girdle (b) of turcinoemacheilus hafezi. cl: cleitherum, cor: coracoid, mcor: mesocoracoid, pb: pelvic bone, ps: pelvic splint, r: radials, and sc: scapula. figure 7. dorsal (a), anal (b) and caudal (d) fin skeleton of turcinoemacheilus hafezi. adp: anal distal pterygiophore; c 18-26: 18th and 26th centrum; dfs: dorsal fin spine; mp: mesial pterygiophore; ph; parhyporal; pp: pterygiophore; sty: stay; epu: epural; hp1–5: hypural plates; npu 2: neural processes of the second preural centrum; hpu 2: hemal processes of the second preural centrum; ust: pleurostyle. 123 int. j. aquat. biol. (2016) 4(2): 117-124 unbranched and 5 branched rays and is supported by 6 pterygiophores and one small stays bone (fig. 7b). the caudal skeleton is made up of four centra along with the epural, parhypural, pleurostyle, uroneural and 5 hypurals (fig. 7c). the anterior part of the hypural-1 is wider and its anterior end reaches to the last centrum. the hypural-1 is also connected to the parahypural ventrally. the parahypural is relatively flat, and its posterior end is connected to the last centrum. the hypurals 3, 4, and 5 are positioned between the pleurostyle and hypural-2. the neural process of the second pleural is long and narrow. the caudal fin bears 18 branched rays. the number of the dorsal procurrent is 5 and thar of the ventral procurrent is 6. discussion the present study provided a detailed skeletal description of the t. hafezi. based on the results, t. hafezi showed various differences with other members of the family nemacheilidae. in the neurocranium, the lateral ethmoid, supraethmoidethmoid and prevomer are fused, as other loaches, with the exception of oreonectes platycephalus, lefua spp., yunnanilus pleurotaenia, triplophysa microphthalma, t. tenuis and schistura fasciolata (sawada, 1982). in contrast to t. kosswigi, the lshaped lateral ethmoid of t. hafezi has a process posteriorly. turcinoemacheilus hafezi bears a sesamoid ossification similar to t. kosswigi (azimi, 2014), paracobitis malapterurus (azimi et al., 2015a), p. longicauda, dzihunia amudarjensis and oxynoemacheilus angorae (prokofiev, 2004, 2009). however, the presence of the sesamoid ossifications in loaches had been rejected previously (sawada, 1982). in t. hafezi similar to t. kosswigi, (azimi, 2014), the supraethmoid-ethmoid is tightly attached to the frontals, as other loaches with the exception of indoreonectes evezardi (sawada, 1982). there are two opening on the ventral part of the exoccipital in t. hafezi similar to t. kosswigi (azimi, 2014), o. platycephalus, l. costata, orthrias barbatulus, t. stoliczkae, t. alticeps, t. strauchii, d. amudarjensis, o. angorae, micronoemacheilus pulcher, iskandaria kuschakewitschi and afronemacheilus abyssinicus, with the exception of p. cristata (prokofiev, 2010; azimi et al., 2015b). in branchial apparatus of t. hafezi, there are four basibranchials similar to t. kosswigi (azimi, 2014), o. angorae and o. brandti (mafakheri et al., 2015) that fourth one is very small; prokofiev (2010) are reported three basibranchials in lefua spp., o. platycephalus, y. pleurotaenia, hedinicthys, in the majority of species of the genus orthrias (with the exception of o. dgebuadzei), in i. kuschakewitschi, paracobitis longicauda, p. malapterurus, schistura spp. paraschistura, nemacheilus masyae, nun, seminemacheilus, acanthocobitis botia, and i. evezardi (prokofiev, 2010; azimi et al., 2015a, b). in addition, the basihyal of t. hafezi has a deep notch compared to that of t. kosswigi (azimi, 2014; azimi et al., 2015a, b). in the caudal skeleton, the hypoural-1 of t. kosswigi attaches to the terminal centrum similar to o. brandti (prokofiev, 2010; mafakheri et al., 2015). the hypural-1 is connected to the parahypural ventrally in t. hafezi similar to t. kosswigi (azimi, 2014), and unlike majority of loaches with the exception of o. angorae, triplophysa orientalis, and t. scleroptera (prokofiev, 2010). the epural of t. hafezi is long and not connected to the reduced neural process of the terminal centrum similar to p. cristata (prokofiev, 2010; azimi et al., 2015b), whereas in t. kosswigi, it is fused to the urostyle (azimi, 2014). according to prokofiev (2010), the most stable and important diagnostic and phylogenic feature of the pectoral girdle in loaches is the number and shape of bony radials in the pectoral fin; unlike o. platycephalus and lefua spp., which have two radials (sawada, 1982), there are three pelvic radials in t. hafezi and t. kosswigi (azimi, 2014) like other loaches. in loaches, the caudal skeleton has 5-6 hypurals (prokofiev, 2010), which five hypurals were observed in t. hafezi similar to t. kosswigi (azimi, 2014). finally, since the identification of the species of the genus turcinoemacheilus is difficult based on morphometric characteristics and color 124 nikmehr et al./ osteology of turcinoemacheilus hafezi pattern, the present study provides a useful distinguishing osteological features of this genus from the other members of the nemacheilid family. acknowledgments we would like to express our sincere thanks to a. jouladeh-roudbar for his help during sampling, h. mousavi-sabet for providing some materials and p. jalili for her help during clearing and staining of specimens. this study was financially supported by the university of tehran. references azimi h., mousavi-sabet h., eagderi s. 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(2022) 10(3): 254-261 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article bioconcentration factor of heavy metals in some aquatic plants in the shatt al-arab river and the possibility of using them as bioindicators of heavy metal pollution rajaa abdul-kadhim hanaf college of marine sciences, department of natural marine science, university of basrah, basrah, iraq. s article history: received 11 march 2022 accepted 22 may 2022 available online 2 5 august 2022 keywords: pollutant aquatic plants bioconcentration factor river abstract: this study was conducted to measure the concentration of heavy metals (manganese, copper, zinc, iron, and cadmium) in water and aquatic plants of ceratophullum demersum as a submerged, phragmites australis as an emergent, and lemna minor as a floating aquatic plant to calculate their bioconcentration factor and compare them in aquatic plants to find out proper one as heavy metal bioindicator. three stations were selected for sampling from the shatt al-arab river, based on the differences in their characteristics in terms of human activities and distribution of aquatic plants. the results showed that c. demersum can absorb most of the heavy metals, followed by the ph. australis. the manganese element was the distinguished element with the highest accumulation rate. based on the results c. demersum can be suggested as a bioindicator of heavy metal pollution in the shatt al-arab river. introduction heavy metals have a specific density of more than 5 g/cm3 (polle and schutzendubel, 2002). their concentration in the environment is low, known as trace elements (minkoff and baker, 2001). heavy metal pollution leads to the destruction of living organisms if they are found in high concentrations (acute), cause congenital malformations, skin infections, reducing fertility, sterility, stunting, or reducing the number of species, at concentrations higher than their normal levels for long-term exposure periods (chronic) (taobi et al., 2000). increasing the concentration of heavy metals can destroy cells by replacing some active groups of enzymes, inhibiting their effectiveness, and releasing effective oxygen radicals (ros) (scheid et al., 2017). heavy metals enter the aquatic environment from natural and industrial sources (singh et al., 2020). they enter the water due to ion exchange or adsorption on the outer surfaces of granules of clay and organic materials or through joint precipitation through rock weathering, as some elements are precipitated and associated with correspondence: rajaa abdul-kadhim hanaf doi: https://doi.org/10.22034/ijab.v10i3.1668 e-mail: rajaa.hanif@uobasrah.edu.iq sediments (karak et al., 2010). the assessment of heavy metals in the aquatic environment is important in controlling pollution because of their ability to move through the food chain or accumulate in the tissues of aquatic organisms (rekasi et al., 2006; arkadiusz et al., 2007). the use of different aquatic organisms as bioindicators of pollution for different types of pollutants, such as heavy metals, is popular, and these organisms are used in many environmental monitoring programs (duruibe et al., 2007). it also gives a clear picture of the environment that is exposed to stress, such as chemical stress by accumulating some pollutants such as heavy metals, and they help to understand the effects of these elements on their vital activities (kakar et al., 2010). the accumulation of heavy elements in plant tissues varies according to the types of plants, soil and water's physical and chemical properties, and the specificity of absorption, transfer, and accumulation of elements. increasing the level of heavy metals within plant tissues leads to their aggregation in different parts of the plants, or 255 int. j. aquat. biol. (2022) 10(3): 254-261 converting them to other non-toxic forms that can be distributed and used again in metabolic processes (memon et al., 2001). many studies used aquatic plants as bioindicators of pollution (matache et al., 2013; burada et al., 2014; hanaf, 2016; al-edani et al., 2019). this study aimed to measure the concentration of heavy metals of manganese, copper, zinc, iron, and cadmium and calculate their bioconcentration factor in ceratophullum demersum as a submerged, phragmites australis as an emergent, and lemna minor as floating aquatic plants and to compare the concentrations of the heavy metals in these aquatic plants to find out their efficiency to remove heavy metals as an indicator of pollution. materials and methods sample collection: three stations were selected to collect samples from the shatt al-arab river based on the differences in their characteristics in terms of human activities and their distinction by the presence of aquatic plants, their diversity, and densities. the first station was al-sharsh, about 20 km south of qurna. the second one was on sindibad island, about 7-8 km north of basrah, 36 km away from the first station. the third station was in the al-amiya area, near the former fertilizer plant in abu al-khasib located 22.29 km from the second station. monthly samples of water and aquatic plants were collected from the study stations. for water sample collection, we used 10 l pre-washed plastic bottles. for the aquatic plants, samples were collected by hand, washed with river water to remove suspended substances, and kept in nylon bags until reaching the laboratory. extraction of heavy metals from water samples: according to apha (2005), a 100 ml sample was taken after shaking the bottle and mixing them well, and 5 ml nitric acid was added. then, it was heated on a hot plate leaving it to near drying, and 5 ml of nitric acid was added to ensure the sample was digested entirely and left to cool and kept in special containers after being diluted with ion-free distilled water to a specific volume until measurement with a flame atomic absorption spectrophotometer (unicam sp9 air acetylene pye). the results were expressed in µg/l. measurement of heavy metal in aquatic plants: the aquatic plants were washed with tap and distilled water in the lab, respectively. then, they were dried in an electric oven at 85°c for 24 hours, ground and passed through a laboratory sieve with a mesh of 40, and prepared to measure the heavy metals according to apha (2005). one g of aquatic plant powder was weighed and placed in25 ml pyrex flasks, then nitric acid (hno3) and perchloric acid (hclo3) were added at a ratio of 1:3 and its nozzle was closed with a glass lid. the samples were shaken out, then left for 24 hours under a vacuum, and the flasks were placed in a water bath for an hour to speed up digestion. 2-3 ml of distilled water were added to the samples, then put the beakers on a hot plate at 70°c and left till the volume reached 2 ml. the centrifuge was used to eliminate the sediment, and the volume of the filtrate was completed to 50 ml with distilled water and kept in bottles until examination. heavy metals were measured using a flame atomic absorption spectrophotometer, and the result was expressed as µg/g dry weight. bioconcentration factor (b.c.f): the bioconcentration factor was calculated according to kumar et al. (2009) by dividing the total concentration rate of each element in the used tissue (a) by its concentration in water (b), i.e. b.c.f = a/ b. according to the us environmental protection agency (usepa, 2012), the substance was considered as not bioaccumulative when it is less than 1000, bioaccumulative in less than 1000, and hyper accumulative bioaccumulation when greater than 5000, results and discussions the use of water for industrial and agricultural purposes is led to its contamination with heavy metals (al-asadi et al., 2020). also, the environmental impact of heavy elements in the water system is related to their distribution between the liquid and solid phases in the water body (aldoghachi and altamimi, 2021). aquatic plants provide proper 256 hanaf / bioconcentration factor of heavy metals in some aquatic plants in the shatt al-arab river evidence of water pollution with heavy metals due to their ability to accumulate these elements in their tissues (coleman et al., 2001). in addition, they grow rapidly and adapt to living in different environments with simple environmental requirements (dirilgen, 2001). also, the concentrations of heavy metals in the bodies of plants may differ according to the plant species (forstner and wittman, 1981). the current study showed the concentrations of some heavy metals in three aquatic plants in the shatt al-arab, which may give a clear picture of the degree of pollution of the river with heavy metals. memon et al. (2001) pointed out that the aquatic plants clearly show the river's pollution with heavy metals better than the water and sediments. because aquatic plants are found submerged in the water, prominently or on the cliff, they obtain these elements either by absorbing them from the sediment through their root system or by directly absorbing them (forstner and wittmann, 1981; ekval and greger, 2002). the high concentration of heavy metals during the summer and spring is due to the rise in temperatures and increasing the evaporation rates or agricultural activity that increase the load of the fluxes of salts and fertilizers (alqam, 2002). the concentration of the heavy metals decreased during the autumn and winter, which can be due to the occurrence of rain and increasing water discharge (hanaf, 2016; lateef, 2020). the fe and mn, followed by zn had higher concentrations (figs. 1, 2, 3), and these results agreed with the study of al-khuzaie et al. (2020). this may be due to the exposure of the study area to human and agricultural wastewater (al-asadi et al., 2019). in addition, the low concentration of heavy metals in river water may be due to the tendency of these elements to accumulate in the bodies of phytoplankton, aquatic plants and other aquatic organisms (vardanyan et al., 2008; aldoghachi and altamimi, 2021), or to adsorb on sediment surfaces (dhir and kumar, 2010). many heavy metals react chemically and physically with other natural substances in the aquatic environment, which changes their oxidative state; thus, they are concentrated, figure 2. concentration of heavy elements in water of station 2. figure 1. the concentration of heavy elements in water of station 1. figure 3. concentration of heavy elements in water of station 3. 257 int. j. aquat. biol. (2022) 10(3): 254-261 precipitated, or adsorbed on the surfaces of particulate matter (dube et al., 2001). the results did not reveal a significant variation in the concentrations of the heavy metals in the different stations, and the reason for this may be due to the similar geological nature of the river (essa, 1995). the river passes through agricultural lands, and the adjunct lands lead to the addition of these elements into the river water (al-khuzaie et al., 2020). the increase in the movement of boats and ships can increase river pollution (al-amara, 2001). the loaded oil spilled through the tidal water bodies from the downstream waters of the shatt al-arab during the whole year (aljabri et al., 2016; al-asadi et al., 2019). based on the results, the order of concentration and abundance of heavy metals in the shatt al-arab waters during the study period was fe > mn > zn > cu > cd. iron is one of the basic elements for most living organisms, and it is usually more abundant in the aquatic environment (hassan et al., 2008), also the shatt al-arab sediments contain a high percentage of dark minerals rich in iron, which adds a large proportion of this element to the water during decomposition and dissolution (albdran et al., 1996). the results showed that the submerged aquatic plant, c. demersum possesses the highest concentration of cu, pb, mn, zn and cd, as its accumulation rates were higher than other plants, and this is consistent with findings of jamnicka et al. (2007). the results also revealed a high concentration of mn and zn, indicating that these plants tolerate high levels of these elements. this may be due to the accumulation and storage of these elements within the plant tissues in non-toxic forms, or it has a special mechanism to tolerate high concentrations of elements (memon et al., 1980), or it absorbs elements in high concentrations and converts them to inert forms in the vacuoles (peverly, 1988). aquatic plants that grow near bridges and population areas with heavy traffic may have a major role in increasing the concentrations of some heavy elements in the tissues of aquatic plants, especially zinc (el-gamal, 2000). dirilgen (2001) indicated that the elements in natural systems are not prepared for free uptake by the plant but rather in the form of soluble complexes, and this depends on the physical and chemical conditions of the surrounding medium, which makes a strong impact on the processes related to the absorption of elemental ions. the order of heavy elements in aquatic plants according to their concentration was mn > fe > zn > cu > cd. the order of aquatic plants in terms of their concentration of heavy elements was c. demersum > p. australis > l. minor (figs. 4, 5, 6). the concentrations of heavy metals in aquatic plants have varied, and this may be due to the species of aquatic plants and may be attributed to their tolerance to high concentrations of heavy metals (memon et al., 2001). the bioconcentration factor was adopted as a measure to infer the possibility of using aquatic plants and phytoplankton as evidence of pollution. as a result, the rates of bioconcentration in the figure 4. concentrations of heavy metals in the studied aquatic plants in station 1. 258 hanaf / bioconcentration factor of heavy metals in some aquatic plants in the shatt al-arab river c. demersum were higher than p. australis and l. minor for all the studied elements (figs. 7, 8, 9), therefore, c. demursum can be considered as a biological index for heavy elements in the aquatic environment (hanaf, 2016). the concentration factor increases when there is an increase in the figure 5. concentrations of heavy metals in the studied aquatic plants in station 2. figure 6. concentrations of heavy metals in the studied aquatic plants in station 3. figure 7. bioconcentration factor of heavy metals in the studied aquatic plants in station 1. 259 int. j. aquat. biol. (2022) 10(3): 254-261 concentrations and decreases when the concentrations of the elements decline. the c. demursum can accumulate heavy metals inside its bodies in larger quantities athbi et al. (2018). in line with our findings, al-khafaji and al-awady (2014) showed the possibility of c. demursum and p. australis removing some heavy metals from the aquatic environment. references al-asadi s.a.r., al-hawash a.b., alkhlifa n.a.h., ghalib h.b. (2019). factors affecting the levels of toxic metals in the shatt al-arab river, southern iraq. earth systems and environment, 3: 313-325. al-asadi s.a.r., al-qurnawi w.s., al hawash a.b., ghalib h.b., alkhlifa n.a.h. (2020). water quality and impacting factors on heavy metals levels in shatt alarab river, basra, iraq. applied water science, 10: 103. albadran b., al-beyati f., abdullah y. 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lee et al., 2004). there are many pathways by which chlorpyrifos distribute throughout the aquatic ecosystems. the major route of chlorpyrifos to aquatic ecosystems is through rainfall runoff and air-drift (xing et al., 2012). different concentrations of chlorpyrifos were detected in the world's groundwater and surface waters (turner, 2003). moreover, chlorpyrifos is moderately persistent in aquatic environments, especially in freshwater and estuaries (turner, * corresponding author: mahdi banaee e-mail address: banaee@bkatu.ac.ir tel: +986712221230 2003). so, during and after treatment with chlorpyrifos to control pest, non-target animals are probably exposed to the pesticide. the fishes living in aquatic ecosystems close to agricultural fields are the most important non-target organisms that can be affected by pesticides. in surface waters chlorpyrifos may be absorbed through the gill, skin and digestive system of fish and distributed in different tissues via the blood. due to the lipophilic property of this pesticide, it accumulates mainly in fatty tissues. bioaccumulation and detoxification of chlorpyrifos in the liver may effect on the physiological function of cells (wheelock et al., 2005; oruç, 2010; xing et al., 2012; ural, 2013). since dysfunction in the 282 banaee et al/ international journal of aquatic biology (2013) 1(6): 281-288 physiology of a cell exposed to toxic substances can change the levels of biochemical parameters in cytoplasm and extracellular fluids such as blood (banaee et al., 2011; saravanan et al., 2011). monitoring the alterations of the blood biochemical parameters could be a useful tool to diagnose toxicity effects in target organs to determine the physiological status in fish exposed to pesticides (banaee et al., 2008; banaee et al., 2011; banaee, 2012; banaee, 2013). common carp (cyprinus carpio) is one of the most important cultured fish in iran. common carp and other species belonging to the family cyprinidae are found in most rivers in iran, making it a proper model species to study eco-toxicity of pesticides. therefore, the aim of this study was to identify the changes in the blood biochemical parameters of carp exposed to sub-lethal concentrations of commercial formulation of chlorpyrifos. materials and methods experimental animals: healthy common carp (c. carpio) (body weight: 95 ± 15 g (mean ± sd)), were purchased from a local fish farm and acclimated to the laboratory conditions for 2 weeks before the experiments. fishes were randomly introduced into 12 closed 200-l recirculating tanks supplied with oxygenated water maintaining constant dissolved oxygen at 6.5 ± 0.5 mg/l, temperature at 24 ± 2 °c, ph at 7.4 ± 0.2, water hardness 150 ± 5 mg/l caco3 and natural photoperiod. during acclimation and all experimental tests, specimens were fed with commercial carp pellets (beyza fedd mill co. iran) at the manufacturer’s recommended rate (2% of their body weight twice a day). fishes were starved for 24 h before sacrifice. acute toxicity experiments: 180 immature common carp were used in acute toxicity tests. the acute toxicity test was conducted following the oecd guideline no. 23 under static-renewal test conditions (oecd, 2000). test solutions of chlorpyrifos were prepared from a commercial chlorpyrifos, emulsifiable concentrate (ec) 40.8%, with the active molecule chlorpyrifos [0, 0-diethyl 0(3, 5, 6-trichloro-2-pyridinyl)-phosphorothioate], were purchased from national agrochemical co. iran. nominal concentrations of chlorpyrifos were 0.0 (control), 50, 150, 250, 500 and 1000 µg/l. thus, the acute toxicity was conducted with six different concentrations of pesticides and three replicates for each concentration in 18 fiberglass tanks (200 l). during the 96 h acute toxicity experiment, water was aerated and had the same conditions as the acclimation period. the static-renewal tests exposed the specimens for 96 h to test solution. after 24 h test solution was replaced and all stock solutions were made immediately prior to use. water was changed daily to reduce the build-up of metabolic wastes and to keep concentrations of chlorpyrifos close to the nominal level. fish mortality was recorded 0, 24, 48, 72, and 96 h after exposure to chlorpyrifos. lc50 values were calculated using the probit analysis (hahn and soyer (date unknown)). sub-lethal toxicity experiments: ninety common carp were randomly distributed in nine 200-l fiberglass tanks (3 treatments with three replicates) the experiment was run for 30 day period sub-lethal toxicity tests. every tank had 10 specimens were exposed to test solutions with the following concentrations of chlorpyrifos: 0.0 (control), 20 (10% 96 h lc50) and 40 µg/l (20% 96 h lc50). sublethal concentrations were selected according the previous acute toxicity test. the water was changed daily to reduce the build-up of metabolic wastes and to keep concentrations of chlorpyrifos near the nominal level. nine fishes per each exposure concentration were captured and anesthetized with extract of clove powder (200 mg/l) after 10, 20 and 30 days of exposure. blood samples of specimens from each experimental group were collected from the dorsal aorta and stored in sterilized glass vials at 4 °c containing the anticoagulant 1% edta. the blood was centrifuged for 15 min at 4000 rpm and the plasma was recovered. samples were maintained at -21°c until biochemical analyses. biochemical analyses: glucose, total protein, albumin, globulin, triglyceride and cholesterol of 283 banaee et al/ international journal of aquatic biology (2013) 1(6): 281-288 plasma were determined by standard procedures used in clinical biochemistry laboratories based on manual biochemical kits (pars azemon co, iran) (thomas, 1998; johnson et al., 1999). in measurement of creatine kinase (ck) activity, the enzyme reacts with creatine phosphate and adp to form atp, which is coupled to the hexokinase/gdp reaction generating nadph. lactate dehydrogenase (ldh) activity was measured based on the conversion of pyruvate to l-lactate by monitoring the oxidation of nadh. aspartate aminotransferase (ast) was assayed in a coupled reaction with malate dehydrogenase in the presence of nadh. in alanine aminotransferase (alt) assay, the enzyme reacts with alanine and α-ketoglutarate to form glutamate and pyruvate. pyruvate is converted by lactate dehydrogenase to make lactate and nad+. all these activities were monitored by measuring the change in absorbance at 340 nm. alkaline phosphatase (alp) assay is based on the enzyme-mediated conversion of p-nitrophenol phosphate to nitrophenol in an alkaline buffer at 405 nm (moss and henderson, 1999). plasma acetylcholinsetrase (ache) activity was determined by adding and adequate volume of sample into a cuvette containing 0.1 m phosphate ph 8.0, and acetylcholine iodide (0.015 m) and dithiobis nitrobenzoic acid (0.01 m) as substrates. ache activity was recorded during 180 s at 405 nm (knedel and boetteger, 1967). all biochemical parameters were measured by uv/vis spectrophotometer (model unicco 2100). statistical analysis: a significant difference in biochemical characteristics of specimens statistical exposed to the different concentrations of chlorpyrifos was examined using one-way anova. all the data were examined for normality (kolmogorov-smirnov test). the significant means were compared by duncan’s test and a p < 0.05 was considered statistically significant. statistical analyses were performed using spss (ibm, release 19) software. data are presented as mean ± sd. results lc50 of chlorpyrifos at 24, 48, 72 and 96 hours are presented in table 1. fish mortality elevated with increasing the concentration of chlorpyrifos. sublethal concentrations of chlorpyrifos were determined according to 96 h lc50. no mortality was observed during the experiment. increased mucus secretion, color changes, behavioral changes such as tremors, lethargy, unbalanced swimming, swimming in the surface water and extreme irritability were important changes found in the individuals exposed to chloryprifos. alterations in enzymatic activities in plasma are presented in table 2. levels of ast activity in fish exposed to 40 µg/l chlorpyrifos were significantly higher than control group from day 10 onwards (p<0.05). at the end of experiment, alt activity in lc 24h 48h 72h 96h lc99 2149.55±309.61 1205.68±126.67 774.58±88.90 596.29±66.88 lc90 1570.78±210.25 867.27±84.11 551.41±57.52 419.56±42.75 lc80 1327.08±169.61 724.78±67.42 457.43±45.26 345.14±33.51 lc70 1151.36±141.27 622.03±56.38 389.67±37.24 291.48±27.66 lc60 1001.21±118.20 534.24±48.13 331.77±31.41 245.63±23.66 lc50 860.87±98.27 452.18±42.01 277.65±27.38 202.78±21.23 lc40 720.53±81.15 370.12±38.17 223.53±25.39 159.92±20.55 lc30 570.38±68.35 282.33±37.41 165.63±26.06 114.07±21.93 lc20 394.66±65.07 179.58±41.09 97.87±30.21 60.41±25.81 lc10 150.96±82.75 37.09±52.21 3.90±39.74 0.00±33.65 table 1. lethal concentrations of chlorpyrifos at different times. 283 284 banaee et al/ international journal of aquatic biology (2013) 1(6): 281-288 plasma of the individuals exposed to 40 µg/l chlorpyrifos increased significantly compared with the control group (p<0.05). ldh activity increased significantly on the 20th and 30th day, in both concentrations. no significant changes were detected in alp activity. ck activity was significantly higher than the control group on the 30th day. the specimens exposed to 40 µg/l chlorpyrifos indicated a reduced ache activity on the 10th day and 20th day of the experiment. changes in plasma biochemical parameters of fish exposed to different concentrations of chlorpyrifos are presented in table 3. although no significant changes were found in plasma total protein and globulin on the 10th day (p>0.05), both decreased significantly on the 20th and 30th day of trial in the two concentrations. however, a significant reduction was found in plasma albumin of the specimens exposed to 20 µg/l chlorpyrifos on the 30th day (p<0.05). there were no significant changes in albumin levels in plasma of fish exposed to chlorpyrifos on the 10th and 20th days of experiment (p>0.05). glucose and triglyceride increased significantly at 40 µg/l. cholesterol increased at 40 µg/l on the 20th day. discussion the present results show that chlorpyrifos is highly toxic to common carp. the observed 96 h lc50 was 203 ± 21 µg/l. halappa and david (2009) found a 96 h lc50 values for chlorpyrifos in common carp of 160 µg/l, which is highly comparable to our results. the 96 h lc50 values of chlorpyrifos in oreochromis mossambicus, o. niloticus and gambusia affinis were 154, 25.9 µg/l and 297 µg/l, respectively (oruç, 2010; rao et al., 2003; rao et al., 2005). the main breakdown product of chlorpyrifos in the environment and in organisms is the chloropyrifosoxon, which disrupts ache activity (chawanrat et al., 2007; oruç, 2010). decreases in ache activity in the plasma of piava (leporinus obtusidens) exposed to glyphosate have been reported (glusczak et al., 2006). halappa and david (2009) believed that parameter (u/l) concentration (µg/l) duration of exposure (days) 10 20 30 ast 0.0 277±44a 218±59a 265±66a 20 293±42ab 324±51b 326±62a 40 330±36b 343±49b 415±76b alt 0.0 28±8a 27±8a 25±3a 20 27±6a 28±7a 30±5ab 40 28±6a 30±7a 31±7b ldh 0.0 529±116a 477±46a 496±20a 20 554±79a 598±83b 623±100b 40 570±87a 605±81b 643±109b alp 0.0 81±14a 102±23a 107±24a 20 81±12a 88±15a 94±16a 40 81±11a 88±13a 107±11a ache 0.0 844±84b 839±32c 860±52b 20 775±55b 556±104b 343±89a 40 560±104a 393±28a 288±51a cpk 0.0 792±79ab 816±41a 841±54a 20 838±40b 869±41a 937±25b 40 740±92a 865±118a 1049±87c table 2. changes in the level of enzyme activities in blood of the fish exposed to different concentrations of chlorpyrifos. 285 banaee et al/ international journal of aquatic biology (2013) 1(6): 281-288 chlorpyrifos is not only inhibiting ache activity, but also has negative effects on functioning of the respiratory and digestive systems of fish. aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ldh), alkaline phosphatase (alp) and creatine kinase (ck) are found in almost all cells of the body tissues, such as heart, kidneys, liver, skeletal muscle, brain, erythrocyte, intestine and gills. the release of intercellular enzymes into the blood and their increased activity in plasma are the most important clinical signs in the diagnosis of damage to cell membranes (banaee et al., 2011). so, increase in plasma ast, alt, ldh and ck activities of fish exposed to the chlorpyrifos may indicate damage to cell membranes, particularly liver cells. increased activities of ast and alt were observed in plasma of channa punctatus (agrahari et al., 2007), cyprinus carpio (banaee et al., 2008), and oncorhynchus mykiss (banaee et al., 2011) exposed to organophosphorus pesticides. the observed increase of ldh activity can be attributed to the conversion of accumulated pyruvate into lactate which is transported through muscle to hepatopancreas and regenerated glucose and glycogen to supply energy for fish exposed to insecticides. similar results have been reported by goel et al., 2006; lavanya et al., 2011; li et al., 2011; saravanan et al., 2011. the increased activity of ck on the 30 day of trial may be indicative of a disorder in muscle fibers. these results agree with a previous study carried out on carp that had been exposed to bifenthrin (velisek et al., 2008). increase in blood glucose of fish exposed to the chlorpyrifos may reflect an increased need for energy to counteract the effects of stress caused by chlorpyrifos toxicity. hyperglycemia or elevated blood glucose levels indicate impaired glucose and lipid metabolism and degradation of glycogen in liver (acker and nogueira, 2012). increases in blood glucose levels have been reported in heteropneustes fossilis (saha and kaviraj, 2009), c. carpio (banaee et al., 2008), and o. mykiss (banaee et al., 2011) after exposure to cypermethrin and diazinon, parameters concentration (µg/l) duration of exposure (days) 10 20 30 protein (g/dl) 0.0 3.51±0.28a 3.85±0.31b 3.93±0.18c 20 3.46±0.27a 3.26±0.19a 3.30±0.19b 40 3.26±0.41a 3.15±0.40a 3.04±0.20a albumin (g/dl) 0.0 2.07±0.13a 2.04±0.17a 2.15±0.10b 20 2.13±0.10a 2.04±0.09a 2.03±0.12a 40 2.08±0.12a 2.15±0.30a 2.19±0.11b globulin (g/dl) 0.0 1.43±0.18a 1.81±0.30b 1.77±0.21c 20 1.32±0.20a 1.21±0.18a 1.28±0.25b 40 1.18±0.36a 1.00±0.20a 0.85±0.26a glucose (mg/dl) 0.0 78.61±14.16a 81.33±15.53a 65.29±10.92a 20 93.60±16.30ab 95.67±23.23ab 91.92±12.30b 40 98.11±16.06b 103.01±18.05b 116.41±20.39c triglyceride (mg/dl) 0.0 180.81±18.13a 186.39±20.62a 185.53±29.84a 20 207.44±20.77b 198.11±23.73a 210.94±21.66a 40 208.56±12.35b 224.68±23.38b 245.45±26.89b cholesterol (mg/dl) 0.0 173.75±34.31a 165.35±26.85a 189.72±39.46a 20 200.43±37.70a 182.50±35.02ab 212.24±35.01a 40 196.38±30.87a 200.86±38.53b 207.99±44.07a table 3. changes in blood biochemical parameters of the specimens exposed to different concentrations of chlorpyrifos. 285 286 banaee et al/ international journal of aquatic biology (2013) 1(6): 281-288 respectively. in stressful situations, glucose is converted to pyruvate in the glycolytic pathway, and pyruvate is metabolized to acetyl-coa in aerobic tissues, which can be used as a precursor in the synthesis of cholesterol and fatty acids in the citric acid cycle (murray et al., 2003). the increase of blood triglyceride and cholesterol may be due to liver dysfunction. destruction of cell membranes can also lead to increased levels of cholesterol in plasma. disorder in triglyceride uptake by adipose tissue may also increase triglycerides. increase of stress hormones such as cortisol in blood of fish exposed to various insecticides, which stimulates lipid breakdown in adipose tissue, were also found in banaee (2013). cholesterol and glucose levels in blood of freshwater fish, mystus vittatus, increased after exposure to metasystox and sevin (john, 2007). similar changes have also been reported by ibrahim and el-gamal (2003), lasram et al. (2009) and acker and nogueira (2012). a reaction between chlorpyrifos and plasma proteins may decrease levels of plasma proteins. malnutrition, reduced efficiency of the liver in protein synthesis, and reduction of nutrient absorption, especially protein, in the digestive system may be important factors in decreasing plasma total protein. decrease in plasma albumin and globulin levels of fish exposed to the chlorpyrifos may be due to decreases in total protein levels in plasma. decreased globulin levels may reduce the resistance of fish to pathogens. banaee et al. (2011) showed that the levels of total protein, albumin and globulin decreased in fish exposed to diazinon. similar results have been reported by banaee et al. 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(2017) 5(1): 22-28; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article morphometric analysis of olfactory organ and telencephalon in maturing and mature migrants of caspian lamprey (caspiomyzon wagneri, kessler 1870) ashraf namdariyan rad1, bagher mojazi amiri*1, soheil eagderi1, allison kupsco2 1department of fisheries, faculty of natural resources, university of tehran, 31585-4314 karaj, iran. 2department of environmental sciences, university of california, riverside, ca 92521 usa. article history: received 4 september 2016 accepted 11 january 2017 available online 2 5 february 2017 keywords: caspian lamprey mature maturing morphometry olfactory organ abstract: this study was conducted to provide a detailed information about changes of the olfactory organ and telencephalon morphology in spring and fall spawning run maturing and mature caspian lamprey, caspiomyzon wagneri, in the shirud river, sothern caspian sea basin, iran. a total of 71 maturing and mature fish were collected during their spawning migration. the results showed that the thickness of the olfactory epithelium and the density of ciliated olfactory receptor cells (orc) were lower in mature migrants. in addition, the nasal cavity, relative weight of olfactory organ and relative telecephalon area in mature migrants were larger indicating its more sensitivity to external queues. based on the results, the olfactory organ and telencephalon of maturing migrants of caspian lamprey have not developed completely and needs a period of rest in the river to its full development for spawning. introduction the caspian lamprey, caspiomyzon wagneri, is an endemic species to the caspian sea basin and migrates into its northern, western and southern rivers (coad, 2016). its populations in the southern part of the caspian sea have dramatically decreased due to river pollution, damming, and loss of spawning grounds (kiabi et al., 1999). now with “near threatened” status (iucn, 1996), there is need for special protection of this species. caspian lampreys inhabit freshwater rivers during larval development and migrate to the caspian sea for feeding after metamorphosis. when sexual maturation approaches, adults migrate to the rivers for reproduction (holcik, 1986). spawning migrations to the southern caspian sea rivers (e.g. shirud river, northern iran) occur in the middle of march to late-april in spring, and mid-september to late october in fall (ahmadi et al., 2011) and these migrants can be classified into two sexual types: mature and maturing. * corresponding author: bagher mojazi amiri e-mail address: bmamiri@ut.ac.ir the olfactory organ of lampreys is located in a shallow nasal cavity on the dorsal surface of the head and its olfactory receptors are long having thin dendrites with rounded apical nodes (thornhill, 1967). during sexual maturation and particular season, the olfactory sensitivity increases (moore and scott, 1992), e.g. sea lampreys (petromyzon marinus) use a specific chemical or pheromone to migrate back to the river, also males secrete pheromones to attract females to spawning grounds (teeter, 1980; bjerselius et al., 2000). pheromones stimulate the olfactory receptors to send the appropriate message to the brain (weiming et al., 2007). lamprey has a small brain comprising the telencephalon, diencephalon, mesencephalon and metencephalon. their telencephalon, in particular, is very small. the shape of the brain does not change much through the adult period, but increase in size (scott, 1887). there is significant coupling between the olfactory system and brain controlling areas. the 23 int. j. aquat. biol. (2017) 5(1): 22-28 telencephalon is the olfactory center and mediates a variety of behaviors such as classical conditioning (overmier and hollis, 1983), processing of sensory information (davis and kassel, 1983; hofmann, 2001), mating and reproduction (demski and beaver, 2001), social behavior (huber et al., 1997; kotrschal et al., 1998; hofmann, 2001; pollen et al., 2007), aggression (davis and kassel, 1983), schooling behavior (davis and kassel, 1983; shinozuka and watanabe, 2004), and avoidance learning (portavella et al., 2003, 2004; vargas et al., 2009). in addition, the size of the telencephalon is associated with residence in structurally complex habitats (bauchot et al., 1977; huber et al., 1997), superior spatial learning (vargas et al., 2000, 2009), greater parental care (gonzalez-voyer et al., 2009), monogamy (pollen et al., 2007), and sociality (pollen et al., 2007). sexual maturation can trigger changes in brain morphology (jacobs et al., 1990; jacobs and spencer, 1994; clayton et al., 1997; sherry, 1998). little studies are available about the reproductive biology and physiology of the caspian lamprey (kiabi et al., 1999; nazari and abdoli, 2010; ahmadi et al., 2011). to the best of our knowledge, no information is available about the olfactory organ and its target regions in the brain i.e. telencephalon of the caspian lamprey. hence, this study aimed to analysis morphometric features of the olfactory organ and telencephalon in maturing and mature migrants of caspian lamprey. the findings of the present study can increases our understanding about the changes of the olfactory organ and telencephalon during reproductive spawning migration in mature and maturing caspian lamprey and provide valuable information on the spawning migration physiology in this endangered species. materials and methods study site: specimens were collected by hand at night in spring (middle of april) and fall (middle of october) 2013. a total of 71 maturing and mature fish were collected under the shirud river bridge (200 m upstream from the river mouth; 36°51′n, 50°47′e). the mean water temperature was 14±3°c and 11±2°c during sampling period in spring and fall, respectively. sampling and biometry: fish were anaesthetized using a solution of clove powder (250 ppm). a total of 40 specimens, including mature and maturing males and females were selected from the collected specimens in each sampling season (5 per each group). mature males were opted from maturing males by applying moderate pressure to their abdomen to stimulate sperm ejaculation. ejaculation was observed in mature males but not in maturing males. mature and maturing females were separated according to their ovary color i.e. mature female had ovaries in bluish-green color, whereas those of maturing were yellowish. in addition, mature migrants had empty intestines unlike those of maturing migrants which had green and full intestines (ahmadi et al., 2011). the body weight (g) and total length (mm) of fish were measured using a digital balance and measuring board, respectively (table 1). the nasal cavity opening was measure from 2d pictures using imagej software. then, the olfactory organ (without inlet tube and surrounding table 1. body weight (g) and total length (mm) of maturing and mature males or females of the caspian lamprey in spring and fall. each value represents the mean ±se. season sex total length (mm) weight (g) spring male mature maturing 3788±362 3917±110 79±9 93±8 female mature maturing 3876±40 4274±390 102±5 127±8 fall male mature maturing 3675±234 3745±388 83±6 90±7 female mature maturing 3723±168 3827±277 93±6 104±9 24 namdariyan rad et al./ morphometric analysis of olfactory organ and telencephalon in caspian lamprey cartilages) and brain of fish were removed (figs. 1, 2) and their weight were measured using a digital balance to the nearest 0. 01 g. the relative weight of olfactory organ was calculated using the following equation: nose weight/ body weight × 100. finally, the nasal and brain were fixed in bouin’s fixative. histological study: the olfactory organ of specimens was embedded in paraffin and histological sections were prepared at 4-6 µm thickness and stained with delafield’s hematoxylin and eosin based on eagderi et al. (2013). numbers of olfactory receptor cells (orc) per 100 µm of the olfactory epithelium, and olfactory epithelium width were measured using olympus bh-2 optical microscope equipped to a dino-capture camera and imagej software. orcs were distinguished by their ciliated olfactory knobs with basal bodies at the outer margins and analyzed according to van denbossche et al. (1995). the area of brain and telencephalon region (as µm2 in dorsal view) were examined under stereomicroscopy equipped to a dino-capture camera and imagej software. the relative size of telencephalon was calculated using the equation: telencephalon area/ brain area × 100. statistical analysis: differences between sex ratios were determined using student’s t-test. mean data were compared using analysis of variance (anova). t-test was used to compare data between maturing and mature fish. data are expressed as mean ±sd. p-value<0.05 was considered significant. results nasal cavity opening in mature caspian lampreys was larger than that of maturing fish in both spring and fall migrants (fig. 1). the relative weight of the olfactory organ was significantly greater in mature fish than those of maturing migrants (0.3223±0.002 vs. 0.2847±0.0011 and 0.3347±0.0023 vs. 0.3013± 0.0022 in spring and fall migrant males, respectively; 0.3067± 0.0019 vs. 0.2793±0.0017 and 0.324± 0.0023 vs. 0.295±0.0015 in spring and fall migrant females, respectively) (p<0.05) (fig. 3). the thickness of olfactory epithelium (µm) in mature fish were significantly lower than that of maturing migrants (393±1.2 vs. 249.67±3.65 and figure 1. nasal cavity (external feature, up and internal feature, down) in the caspian lamprey (a: maturing and b: mature migrants). 25 int. j. aquat. biol. (2017) 5(1): 22-28 389±1 vs. 256.6±2.2 in spring and fall migrant males, respectively; 354.9±1.1 vs. 276.63±3.31 and 364.2±1.6 vs. 279.3±3.5 in spring and fall migrant females, respectively) (p<0.05) (fig. 4). the numbers of orc/100 µm of olfactory epithelium in maturing migrants were closely spaced and greater, compared to those of matures (8.67±3.65 vs. 3.33±1.2 and 8.67±2.2 vs. 3.66±1 in spring and fall migrant males, respectively; 6.67±3.31 vs. 4.67±1.1 and 7.33±3.5 vs. 4.67±1.6 in spring and fall migrant females, respectively) (p<0.05) (fig. 4). telencephalon area/ brain area (µm2) (relative telecephalon area) was significantly greater in mature migrants than that of maturing fish (37.3±0.2 vs. 29.4±0.23 and 33.4.33±0.17 vs. 28.1±0.19 in spring and fall migrant males, respectively; 35.8 ±0.15 vs. 29.1±0.23 and 30.5±0.12 vs. 26.9±0.28 in spring and fall migrant females, respectively) (p<0.05). there was a high positive correlation (r2=0.98) between the weight of olfactory organ and telecephalon weight in maturing and mature migrants during spring or fall. discussion this study provided a detailed information about change of the olfactory organ and telencephalon region of maturing and mature migrants of c. wagneri during spring and fall migrations. the results showed that the nasal cavity and relative weight of the olfactory organ in mature migrants were larger. in sea lamprey, progressive enlargement of the nasal sac during metamorphosis and maturation has been reported (lowe et al., 1973; van denbossche et al., 1997). the thickness of olfactory epithelium and orc figure 2. nasal cavity (external feature, up and internal feature, down) in the caspian lamprey (a: maturing migrants b: mature migrants). figure 3. the relative weight of the olfactory organ in maturing and mature male and female migrants in spring or fall (a, b=significant, a, a= not significant, p<0.05). 26 namdariyan rad et al./ morphometric analysis of olfactory organ and telencephalon in caspian lamprey density were greater in non-mature specimens than in mature fish. thomhill (1967) reported that the olfactory epithelium in adult lampretra fluviatilis had ciliated orc and our results also extend this observation to the maturing and mature stages. the range of orc numbers in the olfactory epithelium of upstream migrant p. marinus was 2-12 (zielinski et al., 1994). the enlarged olfactory organ may enable fish to receive large quantities of water and becomes more efficient at chemoreception as more water enters in the cavity and more odors can be detected (kleerekoper, 1972; vandenbossche et al., 1995). maturing migrant lamprey must find food with a smaller nasal cavity, which may lead to an increase in orc. in contrast, mature migrants not eat and build the reproductive products, resulting in a lower body weight and larger nasal cavity, which may explain the deformed orc in current study. telencephalon area/ brain area was significantly greater in mature fish compared to maturing migrants. during the larval period, the telencephalon grows slowly, then doubles in size during metamorphosis, and increases linearly until adulthood (hardisty and potter, 1971). furthermore, olfactory sensitivity increases and changes are occurred in olfactory receptors during sexual maturation due to presence of pheromones in the water (moore and scott, 1992) maybe causing enlargement of the telencephalon region as observed in this study. nanomolar concentrations of a sexual pheromone in males can stimulate the olfactory epithelium of adult females (li et al., 2002). behavioral responses in vertebrates are closely related to the olfactory system and olfactory-brain regions (healey, 1972). olfactory input is relayed on the medial part of the olfactory bulb, the posterior tuberculum, the mesencephalic locomotor region, and finally reach reticulospinal cells in the hindbrain. activation of this olfactory-motor pathway generated rhythmic ventral root discharges and swimming movements (derjean et al., 2010). based on the findings of the present study, mature fish (males or females) show larger relative nasal sac and telencephalon in both migration seasons indicating more sensitivity to external queues compare to maturing fish. also, it can be concluded that the olfactory organ and telencephalon of maturing migrants of caspian lamprey have not developed completely and needs a period of rest in the river to its full development for spawning. acknowledgments this study was supported by fisheries department, university of tehran. references ahmadi m., mojazi amiri b., abdoli a., fakharzade s.m.e., hoseinifar s.h. 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(2017) 5(1): 22-28 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی خزری گرددهان مارماهی در مغز سفالونتلن و بویایی اندام سنجیریخت تحلیل (caspiomyzon wagneri, kessler 1870 )بلوغ حال در و بالغ 2کوپسکو آلیسون ،1ایگدری سهیل ،1*امیری باقرمجازی ،1رادنامداریان اشرف .ایرانکرج، ،31585-4314 ،تهران دانشگاه ،طبیعی منابع دانشکدهگروه شیالت، 1 .آمریکاایالت متحده ،92521ca ریورساید ،کالیفرنیا دانشگاه ،علوم محیط زیستگروه 1 چکیده: در بلوغ حال در و بالغ خزری گرددهان مارماهی مغز سفالونتلن و بویایی اندام ریختی تغییرات مورد در اطالعات نمودن فراهم هدف با مطالعه این ماهی 71 تعداد منظور این برای. درآمد اجرا به ایران خزر، دریای جنوب آبریز حوضه شیرود، رودخانه به پاییزه و بهاره تولیدمثلی هایمهاجرت زمان گیرنده هایسلول تراکم و بویایی اپیتلیوم ضخامت که داد نشان نتایج. شدند آوریجمع تولیدمثلی مهاجرت زمان در زمان در بلوغ حال در و بالغ بیشتر الغب مهاجر ماهیان در سفالونتلن نسبی مساحت و بویایی اندام نسبی وزن بویایی، حفره عالوهبه. بودند کمتر غبال ماهیان در بویایی دارمژک حال رد مهاجر گرددهان مارماهی مغز سفالونتلن و بویایی اندام نتایج براساس. باشدمی خارجی هایمحرک به آن بیشتر حساسیت بیانگر که بودند .است تخمریزی منظوربه توسه تکمیل برای رودخانه در استراحت دوره یک نیازمند و نیافته توسعه کامل طوربه هنوز بلوغ .بویایی اندام سنجی،ریخت بلوغ، حال در بالغ، خزری، گرددهان مارماهی :کلمات کلیدی int. j. aquat. biol. (2017) 5(3): 193-200; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article gonad morphology and histology of an endemic tooth-carp, aphanius sophiae (heckel, 1847) from iran shadi aminaghaie, hamid reza esmaeili*1 developmental biosystematics research laboratory, zoology section, department of biology, college of sciences, shiraz university, shiraz, iran. article history: received 4 february 2017 accepted 27 april 2017 available online 2 5 june 2017 keywords: reproduction gonad histology sexual maturation gonado-somatic index abstract: this study presents the first details on morphological and histological characteristics of gonads and gonadal development stages of an endemic tooth-carp, aphanius sophiae (heckel, 1847) from a spring-stream system (south of iran). the sampling was done from march 2012 to march 2013 using dip net, and a total of 223 individuals were collected. the gonads of specimens were removed, and then fixed in 10% formalin solution after checking their morphology and measuring their weights, lengths and widths. based on the size, shape and weight of the gonads, degree of occupation of the body cavity, presence or absence of ripe oocytes or milt, diameter of the oocytes in the ovary, and histological observations, five stages of sexual maturation in females and males were determined by macroscopic and microscopic criteria. the results of the gonadal stages indicated that a. sophiae spawns at the beginning of spring. introduction the species of the old-world tooth-carps, aphanius (teleostei: cyprinodontidae) typically thrive in costal and freshwater environments along the mediterranean sea, red sea, persian gulf and arabian sea (clavero et al., 2007; esmaeili et al., 2014, 2016). most species tolerate a wide range of temperature and salinity regimes, and their small size permits viable population to persist in restricted habitats (e.g., wildekamp, 1993). the genus aphanius is the only genus of cyprinodontidae available in iran which is represented by 14 extant and one fissile species (esmaeili et al., 2016, 2017). aphanius sophiae is an endemic of iran that inhabits in the kor river basin, fars province, southern iran. the reproductive biology and gonad histological changes of several species of cyprinodontid fishes had been studied by leonardos and sinis (1999), keyvani and soofiani (2004), esmaeili and shiva (2006), monsefi et al. (2009), güҫlü and küҫük (2008) and karsli and aral (2010). the genus is sexually dimorphic (berra, 2001; esmaeili et al., 2012; 2014; * corresponding author: hamid reza esmaeili doi: https://doi.org/10.22034/ijab.v5i3.303 e-mail address: hresmaeili@shirazu.ac.ir 2016; gholami et al., 2014, 2015a, b; teimori et al., 2014). as no study on the gonad histology of a. sophiae has been published, therefore this study presents the first detailed description of gonad histology of this tooth-carp fish. materials and methods a total of 223 a. sophiae specimens were monthly collected during one year from march 2012 to march 2013 using 1 mm mesh-size dip net from beiza springsystem in denjan village (beiza), kor river basin, fars province (29˚57ʹ47.5"n, 52˚24ʹ12"e, alt. 1832 m asl). all collected specimens were deposited in the zoological museum, collection of biology department, shiraz university (zm-cbsu). the biometry of specimens was carried out using a digital scale and a vernier caliper with accuracies of 0.001 g and 0.01 mm, respectively. for histological studies, the specimens were dissected and their ovary or testis were removed. the sexes and stages of sexual maturation were determined as possible as by naked eye examination and under a compound microscope 194 aminaghaie and esmaeili / gonad morphology and histology of aphanius sophiae (olympus). weight, length, width, color, and shape of each gonad were recorded and the maturity stage of them was recognized macroscopically based on nikolsky (1963). the histological sections of ovary or testis of each maturing stages were prepared by routine histology method (bancroft and stevens, 1991; mirghiyasi et al., 2016; eagderi et al., 2013) as follow: they dehydrated in alcohol, cleared in xylene, imbedded in paraffin wax at 56°c melting point, sectioned at 5-7 μm thickness, and then the sections were stained by hemotoxylin and eosin (h&e). the histological slides were studied under a light compound microscopy and their pictures were taken by a compound microscope equipped to a digital camera. results characteristics of ovaries: the ovary is composed of 2 sac shaped parts extending along the body cavity in a dorsal position above the intestine (fig. 1). in the mature individuals, eggs could easily be seen (beneath the ovarian membrane). ovaries were in white color during non-spawning periods, and dirty yellow or gold yellow during the spawning period. the ovary composed of follicles that derived from the germinal epithelium and contain oogonia that develop into oocytes and ultimately ova, and the surrounding follicular epithelium (guraya, 1988; selman wallace, 1989). based on the histological characteristic, ovaries are classified into five stages as follow: stage i (oogonia and chromatin nucleolar stage): in the chromatin nucleolus stage, oocytes were small spherical cells with a thin indistinct peripheral zone (primordial follicle) having strongly basophilic cytoplasm. the nucleus was spherical and large. the ratio of nucleus to cytoplasm was high, and a very thin layer of connective tissue originative from the ovarian, surrounded each oogonia (fig. 2). stage ii (perinuclear stage): in perinuclear stage, the growing follicles exhibited a weak basophilic cytoplasm. the ovary was composed of nests of oocytes of chromatin nucleolus stage and perinuclear stage of different sizes which increased both their cytoplasmic mass and nuclear volume. the nucleoli appeared randomly at various depths in the ooplasm. this stage was representing the oocytes with average diameters of 0.122 mm (fig. 2). stage iii (yolk vesicle (cortical alveoli) stage): the size of ovaries in this stage was enlarged. the cytoplasm became weekly basophilic and the nucleus still occupied a central position and it was contained a lot of nucleoli attached to the nuclear membrane; number and size of cortical alveoli increased arranging in two distinct layers; one close to the nuclear membrane and the other one located near the oocytes membrane. the yolk globules were formed in this stage with mostly oval or round in shape. in this stage, zona radiate was surrounded by zona granuloza and a thin external follicular layer of theca cells. the average diameter of oocytes was 0.202 mm (fig. 3). stage iv (vitellogenic stage): the most perennial phase of oogenesis was the vitellogenic phase. the ovary was filled mainly with previtellogenic and vitellogenic oocytes in different stage of yolk figure 1. position of the ovary in aphanius sophiae, from kor river basin, fars province, iran. 195 int. j. aquat. biol. (2017) 5(3): 193-200 deposition, and the nucleus was slightly displaced from the central position to the animal pole. at the end of this stage, cytoplasm started to fill-up with yolk granules (proteins) and formation of fat vacuoles that reached their maximum size. the zona radiate was more conspicuous. the oocyte average diameter was 0.405 mm (fig. 4). stage v (maturation stage): this stage was distinguished by migration of the nucleus to the animal pole where it remained, but the nucleus membrane was disintegrated. during the migration of nucleus, it began to liberate its substances into the cytoplasm. the hydrated oocytes were spherical in shape and contain single yolk mass and large lipid droplets. the development of the eggshell was completed with the completely thickness of the zona radiate, and the outer layer (follicular epithelium) became ruptured. the oocyte average diameter reached 0.526 mm which was figure 2. microphotographs of the ovaries in aphanius sophiae in different stages. (a and b) stage i (chromatin nucleolar phase), and (c and d) stage ii (perinucleolar phase). n: nucleus, nu: nucleoli, a: alveoli, tl: theca layer, fcl: follicular cell layer, zr: zona radiata. figure 3. microphotographs of the ovary in aphanius sophiae in stage iii (cortical alveolar phase). n: nucleus, nu: nucleoli, a: alveoli, tl: theca layer, fcl: follicular cell layer, zr: zona radiata. 196 aminaghaie and esmaeili / gonad morphology and histology of aphanius sophiae the maximum size of the oocytes during the oogenesis (fig. 4). characteristics of testes: the pair testes of a. sophiae are an elongated milky white organ that have two lobes composed of numerous lobules that connective tissue surrounding the testicular surface as capsule (fig. 6). the immature fish have a very thin, steak like organ which become steadily thicker and bigger as the mature. in mature fish, the organ becomes creamy in color. based on the histological characteristic, testes are classified into five stages. stage 1: spermatogonia and primary spermatocytes were the dominant cells. the number of primary spermatogonia had increased which was parallel to the testicular development. spermatogonia had a light cytoplasm and a large nucleus. primary spermatocytes were smaller than spermatogonia and had a dense nucleus covered with small and pale cytoplasm. the secondary spermatocyte was smaller than primary spermatocyte (fig. 7a). stage 2: spermatogonia, primary and secondary spermatocytes were more remarkable in the histological sections. secondary spermatocytes were figure 4. microphotographs of the ovary in aphanius sophiae. (a and b) stage iv (vitellogenic phase) and (c and d) stage v (maturation phase). n: nucleous, nu: nucleoli, a: alveoli, tl: theca layer, fcl: follicular cell layer, zr: zona radiata, yg: yolk globule, af: atretic follicle, hym: homogeneous yolk mass. figure 5. microphotographs of a single mature oocyte of aphanius sophiae. nucleus migrated to the animal pole and oocyte contained single yolk mass. ap: animal pole, hym: homogeneous yolk mass, n: nucleus. 197 int. j. aquat. biol. (2017) 5(3): 193-200 similar to the primary spermatocytes but smaller. few spermatids were also observed in some lobules. spermatid was the smallest cell in lobules appeared during the spermiogenesis (fig. 7b). stage 3: the lobule diameter increased in this stage and spermatocytes were predominate, but the clusters of spermatozoa and spermatid were present. spermatocytes were recognized by their smaller nucleus and darkly staining chromatin material (fig. 7c). stage 4: in this stage, the gonad had well-defined lobules with the large numbers of small spermatids and spermatozoa (fig. 7d). stage 5: tubules were characterized by presence of all developing germ cells. the predominant cells were spermatozoa with dark blue stain related to their nucleus. they were the smallest spermatogenic cells. a small number of spermatogonia were evident around the subcapsular lobules (fig. 7e). discussion histology can be powerful tool, especially when used in association with measurements of reproductive factors such as vitellogenin and morphological studies. classification keys of ovary development was gain with used of indices like color, size of egg, and degree of occupation of body cavity in teleost fishes (see mirghiyasi et al., 2016). based on interspecies similarities, maturity stages of ovary in fishes were divided into different steps between 5 and 8 stages (west, 1990; salem et al., 1999; ünver and ünversaraydin, 2004; monsefi et al., 2007; mirghiyasi et al., 2016). this study provided the details on gonad morphology and histology of a. sophiae, an endemic fish of fars province that was found in the kor river basin. based on diameter of the oocytes in the ovary and yolk vesicle oocytes, five stage of ovarian development were observed. similar five developmental stages have also been reported in the ovary of a. persicus (monsefi et al., 2007) which be similar to many species such as thunnus albacares, cyprinus carpio and lutjanus fulviflamma. zona radiata (zr) of a. sophiae was observed at the cortical alveolar stage and became thicker along with progress of oocyte development and yolk deposition. it can be concluded that zr has no functional features at early oocyte development. the reproductive cycle of male a. sophiae is characterized by relative testicular size and developmental stage. five stage of testicular development according to the most advanced type of germ cell were observed in lobular testis: spermatogonia (stage 1), spermatogonia and spermatocytes (stage 2), spermatogonia, spermatocytes and spermatids (stage 3), spermatogonia, sprrmatocytes, spermatids and spermatozoa (stages 4 and 5). the results of the present study showed that a. sophiae spawns in the spring. percentage of late gonad maturation stages (iv and v) and high frequency of large oocyte confirmed the spawning season. the season of spring reported as the spawning figure 6. position of testes in aphanius sophiae, from kor river basin. fars province, iran. 198 aminaghaie and esmaeili / gonad morphology and histology of aphanius sophiae season for some other aphanius such as, a. fasciatus (lenardos and sinis, 1998), a. mento (güclü and kücük, 2008), and a. disaper disaper (bibak et al., 2012). in the spring, the ecological factors, such as temperature, photoperiod and nutrition increased fat storage in oocyte and thus increase fish size and to increase the size of gonads. based on histological section in which oocytes are in different stage of development, a. sophiae is asynchronous. batch spawning of an individual in a spawning season is an advantage for this fish that lives in unstable and changeable environments, such as temporary lagoon or a very small pool. this strategy of spawning allows relatively large eggs to be laid, which have a greater chance of survival (wooton, 1990; leonardos and sinis, 1998). acknowledgments we are thankful to m. masoudi for his kind help in fish collection, and environment department and figure 7. microphotographd of testis of aphanius sophiae in different stages.(a) stage 1, (b) stage 2, (c) stage 3, (d) stage 4, and (e) stage 5. s: spermatogonia, ps: primary spermatocytes, ss: secondary spermatocytes, st: spermatids, and sz: spermatozoa. 199 int. j. aquat. biol. 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(2017) 5(3): 193-200 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی ایران در aphanius sophiae (heckel, 1847) کر ماهیگور جنسی غدد شناسیبافت و شناسیریخت *اسماعیلی حمیدرضا آقایی، امین شادیه .ايران شیراز، شیراز، دانشگاه علوم، دانشکده جانورشناسی، بخش شناسی،زيست گروه تکوينی، سیستماتیک و شناسیماهی آزمايشگاه چکیده: يک که ،aphanius sophiae (heckel, 1847) کر گورماهی ماده و نر گنادهاي جنسی بلوغ مراحل و بافتی ريختی، هايويژگی مطالعه، اين در ماهی قطعه 223 تعداد کل در وشد انجام دستی تور وسیلهبه ،2013 مارچ تا 2012 مارچ ماه از بردارينمونه. است شده ارائه است، ايران بومزاد گونه ها،آن عرض و طول وزن، گیرياندازه ريختی، بررسی از پس و خارج ماهی بدن از گنادها. گرديد آوري جمع کر رودخانه حوضه در جويباري چشمه از وجود عدم يا و وجود شکمی، حفره طول به نسبت گناد اندازه گنادها، وزن و شکل اندازه، اساس بر .گرديد تثبیت درصد 10 فرمالین محلول در و نر افراد براي جنسی بلوغ مرحله 5 شناسی،بافت مشاهدات و تخمدان در تخمک قطر نر، گناد در میلت رنگ شیري مايع يا و ماده گناد در تخمک .ندکمی تخمريزي بهار درآغاز ماهی اين که داد نشان کر گورماهی ماده و نر گنادهاي جنسی بلوغ مراحل بررسی نتايج. گرديد منظور ماده .بدنی-گنادي نمايه جنسی، بلوغ گناد، شناسیبافت تولیدمثل، :کلمات کلیدی int. j. aquat. biol. (2014) 2(1): 20-26 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article a study of genetic population of alosa braschnicowi (borodin, 1904) in sari and mahmodabad coasts in the caspian sea, using microsatellite loci omid jafari*1, ali shabany, hamed kolangi miandare 1 department of fisheries, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 2 november 2013 accepted 10 february 2014 available online 2 5 february 2014 keywords: alosa braschnicowi caspian sea microsatellites molecular marker polymorphism abstract: in this study, five microsatellite loci were used to evaluate genetic diversity of a. braschnicowi between two populations of the caspian sea. sixty samples were collected from the coasts of mahmodabad and sari (30 specimens for each population). five microsatellite loci were highly polymorphic among all samples. the number of alleles per locus observed ranged from 17 to 32 and averaged 14.1 alleles across two populations. the average observed heterozygosis in mahmodabad and sari were 57.1% to 60.1% and average observed heterozygosis between two populations was 58.9%. among 10 population-locus (5 loci × 2 populations) only two tests were in the hardy-weinberg equilibrium, so highly deviation from the hardy-weinberg equilibrium was found. the average values of fis and nm were 0.33 and 14.19, respectively. also amova on base fst index showed low genetic difference between two populations (2%), while the genetic diversity within population was 98%. due to allelic diversity and estimates of heterozygosity, these markers can be useful in the genus alosa for population level analysis in the caspian sea. introduction the caspian sea is the largest lake in over the world. there are several species of fishes from agnatha to teleostomi in the caspian sea. alosa braschnicowi (borodin, 1904) from order clupeiformes is one of important endemic fish in the southern caspian sea. all of the caspian subspecies of a. braschnicowi are widely distributed in the sea but are found in limited number in the south over the winter, moving to the north to spawn in the spring and in the winter moves into deeper water of the iranian coast (vetchanin, 1984). the shores of the caspian sea are very different based on ecological conditions such as temperature, salinity, morphology, hydrology, geography. these factors can influence the populations of fishes including a. braschnicowi. generally, the species and subspecies of alosa in the caspian are larger * corresponding author: omid jafari e-mail address: jaafari.omid@yahoo.com than their related species in the black sea basin. these observations are attributed to the variable environment of the caspian sea over time, with repeated changes in salinity and temperature (coad, 2013). there is no evidence of genetic diversity of this genus in the caspian sea. microsatellite dna markers or simple sequence repeats (ssrs) are tandem repeated motif of 1-6 bases found in all prokaryotic and eukaryotic genomes that have been utilized in the assessment of genetic variation and population differentiation studies for a variety of vertebrates (o’connell and wright, 1997; neff and gross, 2001). among the molecular markers available in population genetics, microsatellites are preferred due to their abundance in genomes, high levels of polymorphism, even distribution, small locus size facilitating polymerase chain reaction (pcr)-based genotyping and co21 jafari et al./ int. j. aquat. biol. (2014) 2(1): 20-26 dominant inheritance pattern (neff and gross, 2001; chen et al., 2008). genetic diversity of populations or species is a result of their evolutionary history and its decrease can reduce their adaptability and surviving potential in a changing environment (lucentini et al., 2009). also genetic diversity is an important factor for the conservation of endangered species (na-nakorn et al., 2006). alosa braschnicowi is a key species in ecological pyramid and food chain. over fishing, pollution, destroying habitats declined its population. thus studying its genetic diversity and genetic population as one of important endemic species of the caspian sea may help to understand how crucial the status of this species in the caspian sea is. materials and methods sample collection and dna extraction: a total of 60 specimens were collected from two regions in the coasts of mazandaran province, mahmodabad and sari (30 specimens from each region). fin tissues were obtained from each sample and preserved in 96% ethanol. dna was extracted using standard phenol/chloroform extraction protocol and then the dna extract was stored at −20 °c until use (hillis et al., 1996). dna was evaluated for quality and quantity on 1% agaros gels and spectrophotometry (king et al., 2001). polymerase chain reaction and electrophoresis: five microsatellite loci, asad030, asad042, asac249, asac051 and asac059 (table 1) were used (julian and bartron, 2007). pcr amplification was carried out in 0.2 ml pcr tubes with an eppendorf thermal cycler (bio-rad, mj mini thermal cycler). amplifications were performed with a reaction volume of 25 μl component (geng et al., 2006). the profile of thermal cycling was as follows: a predenaturation for 3 min at 94°c, 35 cycles of 30 s at 94°c, 30 s at the selected higher annealing temperature, 30 s at 72°c, and 5 min at 72°c. pcr products were run on 10% polyacrylamide gels stained with silver nitrate (rajora et al., 2000). a 50 bp molecular weight marker (fermentas) was used as the molecular weight standard. microsatellite allele lengths were estimated using gel-pro analyzer 3.9 software (gene, usa). data analysis: scoring errors, large allele dropout and null alleles were checked using the microchecker (oosterhout et al., 2004). deviation from hardy-weinberg expected proportions and linkage disequilibrium between pairwise loci were analyzed using genepop (version 3.4) (raymond and rousset, 2003). at this software markov chain method was used to estimate the probability of significant deviation setting the parameters, de memorization = 10000, batches = 100 and iterations per batch = 5000. additionally, to determine if deviations from hardyweinberg equilibrium (hwe) were in the direction of heterozygote excess or deficit, hwe tests were carried out for all loci in each population. the inbreeding coefficient (fis) and its significance were calculated using fstat 2.9.3 (goudet, 2001). all cases with multiple tests, significant levels were adjusted using the sequential bonferroni correction (rice, 1989). allele number (na), expected heterozygosity (he), observed heterozygosity (ho), locus genbank accession no. size (bps) primer sequence (5′→3′) no. of alleles annealing (°c) asad030 ef014998 104-144 f:ccacagcatcatctctttactg r: accttgaatttctccttggg 17 55 asad042 ef015000 128-192 f: actggtcaattgtaagacaccc r:caagatgaccaagggttaagac 25 50 asac249 ef014994 236-324 f:ttattacaacggtgaattgagtg r: taagtgcatgttgtgtgtgatg 37 53 asac051 ef014992 260-356 f: gtaagtcgctttggactaccag r: tctaaatgcccaggtaaagatg 32 53 asac059 ef014993 292-388 f: cttggacttacaatgcttttgg r: agcaagtgtggagtcagtcg 30 53 table 1. characteristics of microsatellite loci used in the present study 22 jafari et al./ int. j. aquat. biol. (2014) 2(1): 20-26 effective number of alleles (ne), gene flow (nm, = [(1/fst) − 1]/4) between populations were calculated using genalex 6.3 (peakall and smouse, 2006). the significance of differences among na and he values of populations were examined using a wilcoxon-mann-witney test performed in spss. an analysis of molecular variation (amova) on base fst index (weir and cockerham, 1984) was used to examine the regional structure of genetic variation within and among populations. amova was carried out using genalex 6.3 (peakall and smouse, 2006). unbiased genetic identity (i) and genetic distance (d) were calculated according to nei (1978) using popgene 1.0 (yeh et al., 1999). popgene was also used to construct an upgma phylogenetic tree based on the nei’ genetic distance data. significance of an excess or defect of heterozygosity was estimated using the wilcoxon test. results a five microsatellite loci analyzed had high polymorphism. micro-checker showed no evidence for large allele dropout or stutter-band scoring at any of the five loci but null alleles can exist in all loci. allele distribution and effective number of alleles as well as expected and observed heterozygosity are presented in table 2. across all loci, 141 different alleles were found within populations. the average alleles number of all loci was 14.1, ranging from the lowest 7 alleles for asad030 (in sari) to the highest 20 alleles for asac249 (in mahmodabad). the average number of effective alleles per locus (ne) in mahmodabad and sari were 14.4 and 14.1, respectively, showing no significant difference (p>0.05) between two populations (wilcoxonmann-whitney test). the expected heterozygosity (he) was high, ranging from 0.80 to 0.92. the average observed heterozygosity (ho) was the highest in sari (0.607), followed by mahmodabad (0.571). there was no significant difference in the average expected and observed heterozygosity between the populations (wilcoxon-mann-whitney test). the highest and lowest value of observed asac059 asac051 asac249 asad042 asad030 mahmodabad na 17 15 20 10 10 ne 9.333 10.248 12.645 7.000 7.293 ho 0.321 0.714 0.571 0.536 0.714 he 0.893 0.902 0.921 0.857 0.863 fis 0.651 0.226 0.395 0.391 0.190 phw *** *** ** *** ns sari na 13 17 17 15 7 ne 8.809 10.814 9.800 11.529 4.962 ho 0.393 0.714 0.500 0.571 0.857 he 0.886 0.908 0.898 0.913 0.798 fis 0.569 0.230 0.458 0.390 -0.055 phw *** *** *** *** ns na = number of alleles; ne = number of effective alleles; ho = observed heterozygosity; he =expected heterozygosity; fis = fixation index; phw = hardy-weinberg probability test: *p <0.05; **p <0.01; ***p <0.001; ns = not significant were corrected with the sequential test of bonferroni (rice, 1989); significant fis values in bold. table 2. genetic diversity parameters for ten microsatellite loci in a. braschnicowi figure 1. scatter plot for relationship between the spawning latency and body weight of snow trout (group 1). 23 jafari et al./ int. j. aquat. biol. (2014) 2(1): 20-26 heterozygosity (0.857, 0.321) were found in locus asad030 (for sari) and asac059 (for mahmodabad) populations, respectively. there were significant deviations from hardy-weinberg equilibrium at most of the loci in the two populations. after sequential bonferroni correction (rice, 1989), among the 10 population-locus tests (2 populations × 5 loci) only two tests followed the hardyweinberg equilibrium (table 2). the average content of fis index was 0.330 across all loci and ranged from -0.055 (asad030 loci in sari) to 0.651 (asac059 loci in mahmodabad). most fis values at the five loci estimated for two populations were significantly different (p<0.05) in heterozygosity deficiency condition except for asad030 locus in sari population. the result of genepop software did not show linkage disequilibrium after bonferroni correction. although analysis of pairwise genetic differentiation revealed that fst values were small (table 3). amova showed a low percentage of variation among the regions (2%). the content of variation within populations 98% explained the majority of the variation. in addition, the content of genetic identity (i) and genetic distance (d) were obtained 0.83 and 0.18, respectively. in accordance with low percentage of variation among population, results revealed high levels of gene flow, average nm values was 14.19. discussion biological population variation is an important foundation for evaluating species resources. in the population genetic studies, researchers use several parameters of diversity and one parameter will provide a correct judgment on genetic diversity of populations. na, ne, ho, he and pic are all parameters of population genetic variations. the value of these parameters varies with the abundance. results showed that the maximum and minimum number of alleles were in asac249 and asad030, respectively. in terms of observed heterozygosity, our results were higher than the average for freshwater fish (ho = 0.54 ± 0.25, dewoody and avise, 2000). also the mean number of alleles are slightly higher than that reported for fresh water fish (na = 9.1 ± 6.1, dewoody and avise, 2000) and anadromous fish (na = 10.8 ± 7.2, dewoody and avise, 2000). in the genetic population studies allele number is used to determine diversity and is preferred to heterozygosity, because allele number may be lost faster than genetic heterozygosity loss because low frequency alleles contribute little to overall heterozygosity (lind et al., 2009). the genetic bottleneck could be an important factor for reduction of the number of alleles without having an obvious effect on heterozygosity (lundrigan et al., 2005). the allelic diversity and heterozygosity are both indicative of genetic variation, but allele number is dependent on the effective population size much more than that of heterozygosity (nei et al., 1975). therefore, allele number can be a suitable estimate of genetic diversity in populations for enhancement, conservation or selection programmes (diz and presa, 2009), although there were no statistical differences between populations. at the present asac059 asac051 asac249 asad042 asad030 fst 0.015 0.021 0.012 0.024 0.020 nm 16.607 11.825 20.371 10.206 11.950 table 3. nm and fst index of five microsatellite loci in two populations for a. braschnicowi population mahmodabad sari mahmodabad 0.83 sari 0.18 table 4. nei (1978) genetic similarity (above diagonal) and genetic distance (under diagonal) of the a. braschnicowi populations 24 jafari et al./ int. j. aquat. biol. (2014) 2(1): 20-26 study, some diversity indices such as the average values of the allele number and observed heterozygosity had a higher value than those in the study of alosa fallax (μ=4.50, ho= 0.445) and a. alosa (μ=4.88, ho= 0.560 ) (faria et al., 2004). there are several reasons for the high diversity in a. braschnicowi such as difference between number of specimens, the type of marker used and the species. the results obtained from genalex 6.3 (peakall and smouse, 2006) indicated that the mean value of ho was lower than that of he (ho= 0.59, he= 0.88). a high gene flow, mistake in reading alleles and a low number of specimens are some reasons for a low ho (li et al., 2009, skalla et al., 2004). deviation from the hardy-weinberg expectations may be related to a small sample size, the presence of migration, genetic drift, inbreeding propagation and selection programs (lucentini et al., 2006, zhao et al., 2005, dahle et al., 2006, mcquown et al., 2003, liu et al., 2005). this species do not have any selection programs in iran, so the deviation from the hardy-weinberg equilibrium may be related to the reasons referred earlier. the values obtained of inbreeding index (fis) showed significant deficiency in heterozygosity at the all loci except asad030 in sari population. the biological reasons of these shortages are not well known and many factors may be involved including null alleles, inbreeding, natural features of markers and selection (raymond et al., 1997, xu et al., 2001). based on microchecker results, the appearance of null alleles could be the major reason for deficiency of heterozygosity at these loci. the presence of inbreeding increases the proportion of homozygot individuals. the amova showed weak evidence for differentiation between populations. the average fst were obtained 0.018. the fst value of 0 to 0.05 indicates a low level of genetic differentiation (wright, 1978). our results showed high nm between populations and may be an agent factor for low genetic diversity between populations. the genetic identity was 0.83. thorpe (1982) calculated the values of genetic identity for different phylogenetic levels in vertebrate taxa. populations that belong to the same species have a genetic identity (i) between 0.80 and 0.90, on the other hand, the species that are belonged to the same genus, genetic identity values are between 0.35 and 0.85. the identity values obtained at the present study falls within the ranges of conspecific organism. conclusion the present study is the first report on genetic population of the genus alosa (a. braschnicowi) from the order clupeiformes in the southern coasts of the caspian sea. microsatellites are appropriate instruments in genetic population studies. all five microsatellite loci used in this study were highly polymorphic (100%). hence, our primers are suggested for other researches on species of alosa in the caspian sea. our data showed that genetic diversity of a. braschnicowi was rather high in the mazandaran coasts of the caspian sea, but genetic variation between two populations was very low (2%). some reasons that may explain this low genetic variation (high gene flow) are: overfishing, oil extraction, habitats destruction, development of industries around the sea, wastewater and thermal pollution. references chen l., li q., yang j. 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(2016) 4(5): 353-359: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article transcription of adaptive-immune genes upon challenge with infectious pancreatic necrosis virus (ipnv) in dna vaccinated rainbow trout (oncorhynchus mykiss) mehdi soltani*1, sohrab ahmadivand1, mahdi behdani2, reza hassanzadeh3, hooman rahmati-holasoo1, ali taheri-mirghaed1 1department of aquatic animal health, faculty of veterinary medicine, university of tehran, p.o. box 14155-6453, tehran, iran. 2biotechnology research center, venom and biotherapeutics molecules laboratory, pasteur institute of iran, tehran, iran. 3iranian veterinary organization, tehran, iran. article history: received 2 july 2016 accepted 21 september 2016 available online 2 5 october 2016 keywords: dna vaccine ipnv igt igm vp4 gene abstract: in the present study, rainbow trout weighting 3±0.3 g were vaccinated with an oral dna vaccine encoding vp2 gene of a prevalent isolate of ipnv in iranian trout farms encapsulated in sodium alginate microspheres and chitosan tripolyphosphate (cs/tpp) nanoparticles. the vaccinated fish were then challenged with a virulent isolate of ipnv at 30 days post-vaccination. the transcriptional changes of adaptiveimmune genes (igm and igt), as well as the vp4 gene of ipnv, as an indicators of viral replication were studied 45 days post-challenge. analysis of rt-qpcr data showed lower levels of vp4 gene expression in the oral dna vaccinated trout after ipnv challenge compared with the control one. moreover, the constructed dna vaccine did not enhance the expression of igm and igt genes above the levels observed in the carrier control group but it showed a mimic of viral activity and contributes to maintaining them at appreciable levels in vaccinated group. introduction infectious pancreatic necrosis (ipn) is an important viral disease of both salmonid and non-salmonid fishes caused by infectious pancreatic necrosis virus (ipnv) belonging to the family birnaviridae, and genus aquabirnavirus (dobos, 1977; reno, 1999; rodriguez saint-jean et al., 1991). this virus contains a bisegmented genome (a and b) of doublestranded rna with segment a being larger (approximately 3.1 kbp) encoding vp2 that the most of viral neutralizing epitopes are mapped to it (tarrab et al., 1993; frost et al., 1995; hill and way, 1995; fridholm et al., 2007). ipnv infects both fry and juveniles of atlantic salmon, brook trout and rainbow trout resulting in high morbidity and mortality (evensen, 2008). the surviving fish may become asymptomatic carriers shedding virus via urine, feces and reproductive products (rodriguez saint-jean et al., 1991; johansen and sommer, 1995). * corresponding author: mehdi soltani e-mail address: msoltani@ut.ac.ir after many researches still need to an appropriate vaccine against ipnv with a long lasting protection especially at an early age and preventing the carrier formation is crucial. however, different antigen delivery systems, including live, inactivated whole virus, fusion protein, subunit, virus-like particles, and intramuscular dna vaccines have been investigated (munang’andu and evensen, 2015). oral dna vaccines are considered as a new strategy and ideal way to immunize large numbers of small fish against ipnv (hølvold et al., 2014). dna vaccines are plasmid dnas encoding specific proteins that can be expressed in cells of an inoculated host, inducing strong and long-lasting humoral and cellular immune responses. intramuscular injection is an effective common route of dna vaccination in fishes. also, some antigenencapsulation methods have been approved for oral delivery of such vaccines (bozkir and saka, 2004; de las heras et al., 2010). however, the mechanisms 354 soltani et al./ transcription of adaptive-immune genes in dna vaccinated trout against ipnv responsible for protection of this delivery system are not completely clarified. iran is one of the leading countries in trout farming which its production remarkably decreased due to viral disease outbreaks such as ipn (soltani et al., 2015). in the present study, we assessed transcriptional changes of adaptiveimmune genes (igm and igt), as well as the vp4 gene of ipnv in vaccinated rainbow trout with an oral dna vaccine encoding vp2 gene of a prevalent isolate of ipnv in iranian trout farms encapsulated in sodium alginate microspheres and chitosan tripolyphosphate (cs/ tpp) nanoparticles. hence, the trout were challenged with a virulent ipnv at 30 days postvaccination and then the transcript change of mentioned genes measured in the survived fish at 45 days after challenge. materials and methods construction and preparation of the dna vaccine: the vp2 gene of a prevalent isolate of ipnv in iranian trout farms, cultured in chse-214 cell line, was cloned into a eukaryotic expression vector pcdna3.1 (invitrogen, usa) under the control of the immediate-early cytomegalovirus (cmv) promoter, yielding pcdna3.1-vp2. the pcdna3.1vp2 was verified using hind iii and xho i endonuclease analysis and the recombinant plasmid was then amplified in escherichia coli (top10), and the cells were grown in lb broth with ampicillin (fig. 1). the plasmid dna was isolated with the endofree plasmid mega purification kit (qiagen, usa) according to the manufacturer’s instructions. the dna concentration was measured in a spectrophotometer (nanodrop 2000, thermo scientific, spain) before it was aliquoted, and stored at -20°c until use. encapsulation of dna vaccine (pcdna3.1-vp2): the pcdna3.1-vp2 and pcdna3.1 plasmids were encapsulated in sodium alginate microspheres and cs/tpp nanoparticles as described by bozkir and saka (2004) and de las heras et al. (2010), and lyophilized and stored at 4ºc. briefly, an equal volume of heated cs/tpp (prepared by ionic gelation process; vimal et al., 2012) and pcdna3.1vp2 solutions was quickly mixed together and vortexed at 2500 rpm for 30 s. the pcdna3.1-vp2 loaded cs/tpp particles were separated by centrifugation at 20,000 rpm for 30 min at 10°c. for preparation of microspheres, 1.5 ml of pcdna-vp2 (1 mg ml-1) were mixed with 2.5 ml of sodium alginate (3% w/v), then the mixture after stirrer (10 min at 500 rpm) was emulsified (30 min at 900 rpm) with span 80 (0.5 ml) and paraffin oil (100 ml). a volume of 2.5 ml of 0.15m cacl2 was then added to emulsion and stirred (900 rpm for 2 hrs). microspheres were collected by centrifugation (10 min at 1000 g) and washed with ethanol (70%). the unbound pcdna-vp2 content in the supernatant was quantified by uv spectrophotometer at 260 nm. the encapsulation efficiency (ee) was calculated using the following equation: ee = [(total amount of pcdna3.1-vp2 − free amount of pcdna3.1-vp2 in supernatant) / total amount of pcdna3.1-vp2] ×100 fish vaccination and ipnv challenge: rainbow trout wheiting 3 g were orally immunized with feed pellets at 5% of body weight containing alginate microspheres and cs/tpp nanoparticles loaded with pcdna3.1-vp2 (named alg–pcdnavp2 and cs/ tpp–pcdnavp2, respectivly) in separate trial groups (each group 90 fish) at temperature 15ºc for 90 days. two groups of rainbow trout were immunized with vaccine impregnated feed pellets (containing alginate microspheres loaded with 10 and 25 µg of pcdna3.1-vp2, and boosted 15 days later with the same amount of the plasmid. in control group, fish orally immunized with alginate microspheres containing 10 µg of the empty-plasmid (pcdna3.1) and boosted 15 days later with the same amount of empty-plasmid. furthermore, a same experiment was performed via cs/tpp nanoparticles loaded with pcdna3.1-vp2 in three separate groups. unvaccinated fish were used as negative controls. at 30 days post vaccination, two subgroups of 15 fish form each group were challenged by ip injection of 0.2 ml/ fish with ipnv at a concentration of 107 355 int. j. aquat. biol. (2016) 4(5): 353-359 tcid50 ml -1. at day 45 post-challenge when fish did not exhibit clinical symptoms, three trout from each group were sacrificed by overexposure to clove oil, spleen and head kidney were removed for assessing of transcript change of igt, igm and ipnv-vp4 genes. transcriptional changes of igm, igt and ipnv-vp4 genes: rna was extracted from 20 mg of the homogenized head kidney and spleen samples using exgenetm viral dna/rna kit (geneall, korea), and then cdna was synthesis was carried out in a total volume of 25 μl from 5 μl of extracted rna using hyperscripttm first strand synthesis kit (geneall, korea) according to the manufacturer instruction. relative expression of adaptiveimmune genes (igm and igt), as well as the vp4 gene of ipnv, as an indicator of viral replication, were quantified in duplicate for each cdna sample on the real-time pcr detection system (applied biosystems) using sybr green qpcr master mix. each reaction containing 12.5 μl 2xsybr green pcr master mix, 200 nm of each primer 100 ng cdna template and nuclease-free water up to final volume of 25 μl were incubated for 15 min at 95°c (1 cycle), followed by 40 cycles at 95°c for 15 s, 59°c for 1 min. the melting curve of each amplicon was examined, and the expression of the target genes was corrected based on the endogenous control expression (ef-1 α) and calculated as fold change in relative expression according to the 2-δδct method (livak and schmittgen, 2001). primers are listed in table 1. statistics analysis: data were statistically analyzed by one-way analysis of variance (anova) using spss package (spss 1998). differences were considered statistically significant at p<0.05. results the encapsulation efficiency of alginate sodium and cs/tpp to encapsulate pcdna3.1-vp2 were determined and the results revealed a high encapsulation efficiencies with 87.8% and 84.2% of dna binding with alginate sodium microspheres figure 1. (a) diagram of the pcdna3.1 vector encoding vp2 genomic fragment of ipnv, and (b) the agarose gel (1%) of pcdna-vp2 digested by hind iii and xho i enzymes. table 1. the primers used for the real-time quantitative rt-pcr. genes acc. no fw-primer (5’-3’) rev-primer (5’-3’) vp4 m18049.1 aggagatgacatgtgctacaccg ccagcgaatattttctccacca igm x65263.1 accttaaccagccgaaaggg tgtcccattgctccagtcc igt ay870265 agcaccagggtgaaacca gcggtgggttcagagtca ef-1 α af498320 gatccagaaggaggtcacca ttacgttcgaccttccatcc 356 soltani et al./ transcription of adaptive-immune genes in dna vaccinated trout against ipnv and cs/tpp nanoparticles, respectively. furthermore, distribution of dna vaccine in different tissues of vaccinated fish was determined by rt-pcr amplification of vp2 gene at 30 days post vaccination (data not shown). ipnv-vp4 gene transcription: the relative expression of vp4 gene was determined to detect the rate of ipnv replication or the viral load (fig. 2). vp4 expression in the virus control group showed stronger expression in the kidney as approximately 15 and 9 times greater than the infected fish that were vaccinated with 25 µg alg–pcdnavp2 and 25 µg cs/tpp–pcdnavp2, respectivly. morover, vp4 expression in vaccinated fish with 25 µg of vaccines was around 3 times greater than 10 µg fish treatments. the results showed a significantly lower levels of the virus in the vaccinated fish 45 days postchallenge (p<0.05). adaptive immune-related genes (igm and igt) transcription: in frothy-five days post challenging fish with ipnv, the levels of igm and igt genes expression were examined in the kidney and spleen of the vaccinated fish (10 and 25 µg dose of pcdna3.1-vp2) and the results are showed in figure 3. transcript of igm and igt genes in the virus control group was greater than vaccinated fish (around 2 times). however, the expression patterns were similar. hence, the oral dna vaccine did not increase the transcript of igt and igm genes above the levels showed in the virus control, but it showed a mimic of viral activity and was kept them at appreciable levels in all vaccinated groups especially that of igt gene in vaccinated fish with 25 µg cs/tpp–pcdnavp2. discussion ipnv is the causal agent of an acute contagious disease particularly in young salmonids, with high morbidity and mortality, and establishes an asymptomatic carrier state in the survivors (evensen, 2008). in the present study, the relative expression of igm and igt genes, as well as the vp4 gene of ipnv were assessed in orally vaccinated rainbow trout with different dose of encapsulated pcdna3.1-vp2 dna vaccine with cs/tpp nanoparticles and alginate sodium microsphere, 45 days post-challenge with ipnv. the results showed that fish of all treatments survived and displayed no clinical figure 2. relative expression of the ipnv-vp4 gene in the kidney and spleen of vaccinated and ipnv infected trout. rainbow trout (n=90) orally immunized with alginate microspheres (a) and cs-tpp nanoparticles (b) containing 10 and 25 µg of pcdna3.1-vp2 and boosted 15 days later with the same amount of vaccine. fish orally immunized with alginate microspheres (a) or cs-tpp nanoparticles (b) containing 10 µg of the empty-plasmid and boosted 15 days later with the same amount of empty-plasmid, and unvaccinated fish were used as negative controls. at 30 days post-vaccination two subgroups of 15 fish form each group were challenged by ip injection 0.2 ml/fish with ipnv at a concentration of 107 tcid50 ml-1. transcription of the vp4 gene was recorded in the kidney and spleen of both vaccinated and virus control fish at 45 days postchallenge. data are represented relative to ef1-a expression (2-δδct method; n=3). different letters indicate significant differences between groups at p<0.05. 357 int. j. aquat. biol. (2016) 4(5): 353-359 symptoms of disease. the transcript of ipnv-vp4 gene in the control group was significantly higher than vaccinated fish suggesting that oral vaccination has decreased the viral load. similarly in the other studies, oral dna vaccination of fishes has reduced replication of different viral agents such as ipnv and ihnv (marjara et al., 2011; ballesteros et al., 2014; ballesteros et al., 2015), suggesting different immune modulatory effects of the vaccine may result in the inhibition of the viral replication. the results showed that cs/tpp nanoparticles and alginate sodium microsphere effectively protected the constructed ipn dna vaccine from degradation in the fish stomach. the results also showed that igt and igm genes are express-able in both vaccinated and virus control fish, but with more intensely in the control carrier fish. however, vaccination did not enhance the expression of igm and igt above the levels observed in the control carrier group, but it showed mimiced viral activity and contributes to maintain at the appreciable levels in all vaccinated groups suggesting the presence of ig secreting cells, and confirming the vaccine activity and the gene response to oral stimulation of vaccine administration. however, it remains unclear what immune activity is being induced by the virus that persists in surviving fish or by the exogenous vp2 gene. in conclusion, the results showed that cs/tpp nanoparticles and alginate sodium microspheres loaded with pcdna3.1-vp2 vaccine are able to reduce viral replication in the surviving fish after challenging with ipnv. moreover, the constructed dna vaccine did not enhance the expression of igm figure 3. expression of adaptive immune-related genes of igm and igt in the kidney and spleen of rainbow trout orally vaccinated with alg– pcdnavp2 (a and c) and cs/tpp–pcdnavp2 (b and d) after challenging with ipnv as described in legend figure 2. different letters indicate significant differences between groups at p<0.05. 358 soltani et al./ transcription of adaptive-immune genes in dna vaccinated trout against ipnv and igt genes above the levels observed in the virus control carrier group but it shows mimics viral activity and contribute to keep them at appreciable levels in all vaccinated groups. further works are in progress to assess the correlation between these genes and clinical efficacy of the vaccine in the same fish groups. acknowledgements this study was funded by the grant of research council of the university of tehran and the centre of excellence of aquatic animal health. authors thank e. alirahimi and m. ahmadpoor for their technical assistance. conflict of interest: authors declare that they do not have any conflict of interest. authors’ note: the subject matter of this publication is included in pending patent applications. references ballesteros n.a., alonso m., saint-jean s.r., perezprieto s.i. 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(2023) 11(2): 151-158 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article effect of antibiotic residues on some health parameters of nile tilapia, oreochromis niloticus l. collected from shatt al-arab mayada h. ahmed1, khalidah s. al-niaeem2, amjed k. resen2 1department of vertebrates, marine sciences center, university of basrah, basrah, iraq. 2department of fisheries and marine resources, college of agriculture, university of basrah, basrah, iraq. s article history: received 7 december 2022 accepted 27 february 2023 available online 2 5 april 2023 keywords: pollution antibiotics fish hematological parameters abstract: the presence of antibiotics in the aquatic environment poses great concerns due to their impact on water quality, aquatic organisms, and human health. the current study aims to detect the antibiotic residues (amoxicillin (amo), ciprofloxacin (cip), and levofloxacin (lev)) seasonally in the muscles and liver of nile tilapia, oreochromis niloticus, collected from shatt al-arab and to know their effect on some health aspects of fish during november 2020 to august 2021 by selecting two stations in the river. the samples were analyzed using high-performance liquid chromatography (hplc) for measuring antibiotics. the study revealed high concentrations of antibiotics in the muscles and liver of fish, and the concentrations were higher in the second station. cip had the highest concentration (7.4 mg/kg) in muscles in the spring, and the amo showed the highest concentration (4.1 mg/kg) in the liver during the spring. the study also showed that the accumulation of antibiotics in the liver and muscles of fish had negative effects on the health standards of fish. the presence of antibiotic residues in fish samples in high concentrations is a source of great concern as it is a major source of human food. introduction in recent years, antibiotics have been recognized as serious and active environmental pollutants due to their presence everywhere in high concentrations in surface waters, ground waters, soils, sediments, and animals in almost all parts of the world (kovalakova et al., 2020; lu et al., 2020). pollution with antibiotics is attributed to many factors, including the release of antibiotics that are not absorbed by humans and animals into the water bodies. in addition, most of the remaining unused antibiotics from laboratories, pharmaceutical factories, residential and commercial areas, and hospitals are disposed of in the water systems (qiao et al., 2018; ngigi et al., 2020) there are no local studies on antibiotic contamination in fish in iraq, and there are few international studies that dealt with antibiotic contamination in fish, including the study of fang et al. (2021) which dealt with the accumulation of correspondence: khalidah s. al-niaeem doi: http//doi.org/10.22034/ijab.v11i2.1934 e-mail: kalidah_salim@yahoo.com dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.9.1 antibiotics in oreochromis niloticus. their results showed that the accumulation of antibiotics in the muscles of the fish was positively related to its dose in the diet. this study also showed that the antibiotic sul led to the inhibition of growth in nile tilapia fish, emphasizing the need for rational and regulated use of antibiotics in aquaculture. han et al. (2021) studied the spatial distribution and risk assessment of 14 antibiotics in typical mariculture farms surrounding the bohai sea in china and antibiotics were detected in seawater, sediments, some marine fish, mollusks, and sea cucumbers. li et al. (2021) detected 12 antibiotics were detected in the muscle tissue of cultured aquatic organisms and in water and sediments in eastern china. this study also showed that the potential risks from ingesting these aquatic organisms are few and limited, except for the antibiotics belonging to the group of fluoroquinolones. bojarski and witeska (2020) showed the presence 152 ahmed et al./ effect of antibiotic residues on some health parameters of nile tilapia of antibiotics in the aquatic environment and their toxic effects on fish species revealing chronic exposure cause physiological disturbances such as hematological changes, oxidative stress, histopathological changes, weak immunity, metabolic disorders, and general stress. also, this study showed that low concentrations of antibiotics can affect the reproductive process. low concentrations of antibiotics can also affect aquatic bacterial communities, causing changes in the microorganisms that live symbiotically with fish. kondera et al. (2020) studied the effect of adding oxytetracycline and gentamicin in the diets on the hematological parameters of juvenile cyprinus carpio. their results showed no significant hematological or hepatotoxicity of therapeutic doses of otc and gen on juveniles, and this study also confirmed no significant changes in the values of hematological and biochemical parameters after taking otc and gen antibiotics. nibamureke et al. (2019) studied the effect of the antibiotic nevirapine on the liver of o. mossambicus in african surface waters under controlled conditions for 30 days concluding that nvp causes histological changes to liver cells and causes fibrosis around some veins and bile ducts and confirmed that long-term exposure to the antibiotic nvp causes negative effects on fish health. the current study aims to identify the effect of antibiotic residues accumulated in the liver and muscles of nile tilapia on some health parameters collected from shatt al-arab. materials and methods sampling: the shatt al-arab is the confluence of the tigris and euphrates rivers at the city of qurna, north of basrah, and then extends in the southeast direction for a distance of approximately 195 km to drain into the persian gulf. the width of the river ranges from 400 m in the basra to about 1500 m near ras al-bishah after its confluence with the karun river. its depth ranges from 8-15 m (al-mahmood et al., 2011). the southern part of the shatt al-arab river suffers from tidal phenomenon as a result of the entry of the marine waters, so the quality of the downstream water becomes mixed with marine water (abdullah et al., 2015). in this study, two stations were selected from shatt al-arab to detect antibiotics in water, sediments, and fish. the first station is located in the center of basra city near al-sadr teaching hospital (30°30'33"n, 47°51'03"e) located near a dock for commercial ships, and the movement of recreational boats and fishing boats. in addition to the presence of many tourist restaurants that throw their waste into the river, it is considered a source of great pollution to the river. the second station was near the salhiya river (30°30'24"n, 47°51'27"e). the movement of recreational boats, transport, and fishing boats is also active at this site, and the area is affected by the water coming from the salhia river, which contributes to increasing pollution. fish samples were collected from the two stations seasonally from november 2020 to august 2021. two fishing methods were used to collect fish samples, which are the gill net, 120 m long with a mesh size of 15 mm, and the cast net with a diameter of 9 m with a mesh size of 15 mm. the fish were kept in a cork container containing crushed ice until returning to the laboratory. detection of antibiotics preparation of standard solutions: standard solutions were prepared at a concentration of 20 mg/l of amoxicillin and 10 mg/l of ciprofloxacin, and levofloxacin by dissolving the pure substances in d.d.w (hamscher et al., 2002; gros et al., 2006). standard solutions were injected into the hplc device to draw the standard curve, which is used to compare with the curve of the sample to estimate the value of antibiotics. solid-phase extraction (spe): to measure the quantitative of amoxicillin, ciprofloxacin, and levofloxacin in the sample, 10 g of the sample was taken and placed in a volumetric vial with a capacity of 250 ml and then 100 ml methanol: distilled water (1:1) was added and mixed for one hour on a magnetic stirrer. then it was placed in a sonic boom device for 30 min, afterward which the sample was filtered through a 0.45 µm filter. the final volume 153 int. j. aquat. biol. (2023) 11(2): 151-158 was completed to 250 ml with distilled water. the sample was stored in the refrigerator for analysis by hplc. analytical methods amoxicillin: the examination was done using a high-performance liquid chromate-graphy device (hplc) (sykamn, german) (p1500 pump, uv2000 detector, as3000 automatic sampling device). the carrier phase consisting of acetonitrile: methanol: phosphite buffer was used according to the ratio of 10:30:60 (v/v/v), and a separation column (c18-ods 25 cm x) was applied. (4.6 mm) using an ultraviolet detector (uv-230 nm) at a flow rate of 1 ml/min (unutkan et al., 2018) . ciprofloxacin and levofloxacin: the measurements were done in the laboratories of the ministry of science and technology, department of environment and water using a high-performance liquid chromate-graphy device (hplc) (sykamn, german). the carrier phase consists of methanol: distilled water according to the ratios of 70: 30 (v/v), and a separation column (c18-ods-25 cm x 4.6 mm) using a radiation detector was used with an ultraviolet (uv-294 nm) device at a flow rate of 1 ml/min (naveed et al., 2014). hematological parameters: blood samples were collected by drawing blood from the heart in a test tube containing 2.5% edta (ethylenediaminetetraacetic acid). the total protein of the blood plasma was measured by a ready-made laboratory kit (randox, usa) using a spectrophotometer at a wavelength of 546 nm, according to the equation of total protein concentration (mg/ 100ml) = (sample reading/standard reading) x 6. the concentration of albumin in the blood plasma was measured by a ready-made kit (randox, usa) using a spectrophotometer at a wavelength of 630 nm according to the equation of albumin concentration (mg/ 100ml) = (sample reading/standard reading) x 4.5. globulin concentration was measured by subtracting the albumin value from the total protein value for all samples (wolf and darlington, 1971) i.e. globulin concentration (mg/ 100ml) = total protein concentration albumin concentration. the hemoglobin of the blood plasma was measured by a ready-made kit (randox, usa) using a spectrophotometer at a wavelength of 570 nm, according to the equation of hemoglobin concentration (mg/ 100ml) = (sample reading standard reading). statistical analysis: the statistical program statistical package for social science (spss) was used to conduct the statistical analysis under the significance level of 0.05. results in the current study, two groups of antibiotics, i.e. fluoroquinolone (levofloxacin (lev), and mol. wt (g mol-) chemical structures formula antibiotic class antibiotics 365.40 c16h19n3o5s β-lactam amoxicillin 331.34 c17h18fn3o3 fluoroquinolone ciprofloxacin 361.37 c18h20fn3o4, fluoroquinolone levofloxacin table 1. the aggregates, the chemical structure and the molecular formula of the antibiotics (amoxicillin, ciprofloxacin, levofloxacin) to be detected using the hplc device. 154 ahmed et al./ effect of antibiotic residues on some health parameters of nile tilapia amoxicillin (amo)) and β-lactam (ciprofloxacin, cip) were detected (table 1). the seasonal and local changes of the antibiotics (levofloxacin, amoxicillin, and ciprofloxacin) in the muscles of nile tilapia during the study period are shown in figure 1. the lowest values were recorded at the first station during autumn for lev (1.3 mg/kg), and the highest value recorded at the second station during spring was the cip (7.4 mg/kg). figure 2 shows the seasonal and local changes in the values of antibiotics (levofloxacin, amoxicillin, and ciprofloxacin,) in the liver of nile tilapia during the study period. the lowest values were recorded at the first station during summer for cip (0.38 mg/kg), and the highest values in the second station during spring for amo (4.1 mg/kg). tables 2 show the seasonal and local changes in the total protein, albumin, globulin, and hemoglobin in nile tilapia during the study period. the lowest values for total protein were recorded in the second station during the spring season (2.333 mg/100ml), and the highest values in the first station during autumn (3.88 mg/100 ml). the lowest values of albumin were recorded in the second station during summer (1.13 mg/100 ml), and the highest values in the first station during autumn (2.593 mg /100 ml). the lowest values for globulin were recorded in the first station during the winter (0.953 mg/100 ml), and the highest values in the second station during the winter season (1.49 mg/100 ml). the lowest values of hemoglobin were found in the second station in spring (28.833%), and the highest values in the first station during the winter (53.1%). discussion fish were used as bioindicators to determine the extent of the organism's response to environmental variables and its resistance to pollution. the figure 1. seasonal and local changes in the values of antibiotics (levofloxacin, amoxicillin, and ciprofloxacin,) in the muscles of nile tilapia fish during the study period (different letters in the stations are significantly different (p<0.05). 155 int. j. aquat. biol. (2023) 11(2): 151-158 concentrations of antibiotics were higher in the second station than in the first station, and this may be due to its proximity to the salhia river, which is polluted with sewage water. the results of the current study showed a large variation in the values of antibiotics in the muscles and liver of fish showing that most of the concentrations of antibiotics are very high and considered having high risk. these values have direct health risks to humans in different age groups, especially cip, and its low concentrations are toxic to children of one to three months (cui et al., 2018). hu et al. (2010) and kemper (2008) pointed out that the presence of antibiotics in the aquatic environment even at very low table 2. the concentrations of albumin, globulin, total protein and hemoglobin in the blood plasma of nile tilapia fish in the two study stations. hematological parameters seasons (mean ± standard deviation) autumn winter spring summer st.1 st.2 st.1 st.2 st.1 st.2 st.1 st.2 total protein )mg/ 100 ml ( 3.88±0.144a 3.566±0.351a 3.233 ±0.236a 2.57±0.320a 3.166±0.208a 2.333±0.251a 3.39±0.441a 3.1±0.173b albumin )mg/ 100 ml ( 2.593 ±0.061a 1.52±0.096b 2.286±0.155a 1.32±0.108b 2.14±0.069a 1.213±0.085b 1.656 ±0.228a 1.13±0.017b globulin )mg/ 100 ml ( 1.28±0.105a 1.366±0.309b 0.953±0.055a 1.49±0.351b 1.26±0.208a 1.143±0.005b 1.166±0.06a 1.153±0.049a hemoglobin (%) 42.833±1.755a 38.56±1.115b 53.1±2.007a 41.833±3.329b 41.333±2.516a 28.833±1.04b 38.733±0.461a 35.47±2.2a different letters in the same column (station 1 and station 2 for each adjective) mean that there are significant differences (p<0.05(. figure 2. seasonal and local changes in the values of antibiotics (levofloxacin, amoxicillin, and ciprofloxacin,) in livers of nile tilapia fish during the study period (different letters in the stations are significantly different (p<0.05). 156 ahmed et al./ effect of antibiotic residues on some health parameters of nile tilapia concentrations, causes chronic and hidden effects on fish behavior and the feminization of males in some fish species. the results also showed that the accumulation of antibiotics was higher in the muscles of fish than in the liver, and this may the muscles are the last part in which the absorption or accumulation of pollutants occurs, since muscles are inactive tissues (ben salem et al., 2014). high concentrations of cip were recorded in fish muscles, and this is probably due to its high stability and resistance to decomposition, which leads to its wider spread and its accumulation for a higher and longer period. the highest values of the amo were recorded in the liver of fish, and this may be due to its high ability to accumulate and its high presence in the environment as a result of its wide use in the treatment of humans and animals. it is a broad-spectrum antibiotic used to treat many infections (bielen et al., 2017). for lev, it was recorded in low concentrations in the liver and muscles of fish, and this may be due to its rapid chemical breakdown. hematological parameters: analysis of blood parameters is an important tool to assess the toxic effects of antibiotics on fish (serezli et al., 2005; burgos-aceves et al., 2019; bojarski and witeska, 2020). little is known about the effect of antibiotics on plasma or serum biochemical indicators in fish (reda et al., 2013; nakano et al., 2015; hoseini and yousefi, 2018). changes in the blood of fish indicate the morphological and physiological changes that can occur due to the negative effects in the aquatic environment resulting from pollution (javed and usmani, 2014). hoseinifar et al. (2011) and carbone and faggio (2016) mentioned that hematological parameters are useful tools for examining fish health and physiological responses and sensing ambient conditions affecting fish. changes in biochemical parameters (total protein, albumin, and globulin) can be attributed to changes in the liver. the results of the current study showed a slight variation in the total protein, albumin, and globulin between the two stations and fish. the highest values were recorded in the first station in tilapia. the rise in the level of these proteins may indicate an increase in their manufacture in the body and a decrease in the catabolism process, and the low level of both total protein and albumin in fish, it may be due to damage to blood vessels, and the exit of blood cells from the bloodstream into the tissues, causing hypoproteinemia (bly et al., 1994). kitancharoen and hatai (1996) also mentioned that the low level of albumin in the blood plasma is due to the low efficiency of the liver in the formation of this protein and that its decrease is another reason for the decrease in the percentage of total protein because albumin constitutes 80% of it i.e. globulintype proteins increase. in the current results, the level of globulin increased in the fish in the second station and decreased in the first station. hemoglobin is an important component of red blood cells and plays an important role in transporting oxygen in the blood of fish in general. poor formation of red blood cells shows affecting their formation centers (kidney/spleen) and destroys red blood cells due to a change in the permeability of the cell membrane and an increase in mechanical fragility leads to a decrease in hemoglobin in the blood plasma of fish ) kondera et al., 2020). the results of the current study showed a variation in hemoglobin concentrations, and high concentrations of hemoglobin were recorded in tilapia fish during the winter. this may be due to the low concentrations of oxygen in the tissues as a result of exposure to pollution, and thus there is a need to produce red blood cells (serezli et al., 2005). in addition, this increase may be due to the efficiency of the hormone thyroxine, which is one of the hormones that regulate metabolism that this hormone causes an increase in metabolic reactions in tissues, which leads to an increase in the need for oxygen increasing hemoglobin (thanikachalam et al., 2010). conclusions the study recorded a significant increase in the level of antibiotic contamination in nile tilapia. the second station recorded high concentrations of 157 int. j. aquat. biol. (2023) 11(2): 151-158 antibiotics compared to the first station. the results of the study also showed that the concentrations of the two antibiotics amoxicillin and ciprofloxacin were high during the study period with higher concentrations in spring. acknowledgments the authors would like to thank the ministry of science and technology, department of environment and water, university of baghdad, for facilitating the analysis of samples. references abdullah a.d., masih i., van der zaag p., karim u.f.a., popescu i., al suhail q. (2015). shatt al arab river system under escalating pressure: a preliminary exploration of the issues and options for mitigation. international journal of river basin management, 13(2): 215-227. al-mahmood h.k.h., al-sayaab h.a., al-miahi d.s.b., mahmoud a.b., mutasher w.r. 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(2017) 5(2): 63-70; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article histopathological study of common carp (cyprinus carpio) fed aflatoxin-contaminated diets shima shahafve, mahdi banaee*,1behzad nematdoost haghi, mohammad mohiseni aquaculture department, natural resources and environmental faculty, behbahan khatam alanbia university of technology, behbahan, iran. article history: received 14 december 2016 accepted 7 march 2017 available online 2 5 april 2017 keywords: aflatoxicosis feed poisoning common carp tissue damage abstract: this study aimed to evaluate the effects of aflatoxin-contaminated diet on histopathological alterations of the gill, liver, kidney and intestine tissues in common carp. fish were randomly distributed into 15 tanks, i.e. in five experimental groups; (i) control fed with normal diet without solvent and aflatoxin, (ii) positive control received feed with only solvent, and (iii-v) fed on diets containing 0.5, 0.7 and 1.4 mg kg-1 of aflatoxin, respectively. after 21-days, 12 fish per treatment were randomly caught, anesthetized and euthanized. then, histological sections of the tissues were prepared. the main aflatoxicosis symptoms in the gills were fusion and disorganisation of the secondary gill lamellae, shortening of the secondary lamellae, inflammation of mucous membranes, and exfoliation of the gill epithelium. liver of the infected fish indicated cloudy swelling of hepatocytes, cellular hypertrophy, formation of vacuoles in the cytoplasm, and necrosis of liver parenchyma. expansion of bowman’s space, necrosis of urinary tract, exfoliation and degeneration of the urinary tract epithelium, expansion of the urinary lumen and dilation of the urinary space were observed symptoms in the kidney. changes in the intestine of the aflatoxin-treated fish were; expansion of goblet cells, necrosis of mucous layers, exfoliation of the mucous epithelium, and bleeding in the intestinal wall. the results indicates that feeding common carp with diets contaminated with aflatoxin, even in low concentrations (≤ 1.4 mg kg-1 feed) can cause histopathological damages and disturb their physiological balance. introduction aflatoxin produced by aspergillus flavus was discovered about 50 years ago after an outbreak of turkey x disease in england (kensler et al., 2011). aflatoxins are recognized as the most toxic and carcinogenic compounds among mycotoxins. it is the secondary metabolite of a. parasiticus and a. flavus that are found in corns, peanuts, oilseed crops, almonds, and pistachios in tropical and subtropical regions (taheri et al., 2012; almeida et al., 2011; ruby et al., 2013). cereals, including corns, are the main materials in making feed in aquaculture and its contamination with aflatoxin can increase chances of toxicity in fish. aflatoxin can be produced in different phases of food production, during the harvesting stage, when crops are stored in warehouses or even after the aquatic feed production. feeding fish with * corresponding author: mahdi banaee doi: https://doi.org/10.22034/ijab.v5i2.264 e-mail address: mahdibanaee@yahoo.com aflatoxin-contaminated diet also increases the risk of aflatoxin-induced effects to consumers of the aquatic food (huang et al., 2011). four main groups of aflatoxins are b1, b2, g1 and g2 which contaminate agricultural products and have a potential risk for humans and farmed species (kensler et al., 2011; wacoo et al., 2014). aflatoxins, specifically aflatoxin b1, converts to exo-8,9-epoxide (afb1-8,9-epoxide) by cyp3a4 in the liver of fish and other organisms (deng et al., 2010; chawanthayatham et al., 2015). exo-8, 9-epoxide is very reactive and acts as the main mediator of cell damage. moreover, they have an important role in dna-adducts formation and hepatocarcinogenesis (kensler et al., 2011). although other enzymes of the cytochrome p450 are involved in degradation of aflatoxins and their metabolites (mary et al., 2012), 64 shahafve et al./ tissue damage induced by aflatoxin in common carp hydrogen peroxide and hydroxyl radicals produced during aflatoxin process (matur et al., 2011; adedara et al., 2010) may lead to peroxidation of cell membrane and cytological alterations (mary et al., 2012; bernabucci et al., 2011). alflatoxin b1 and its metabolites hinder dna and rna synthesis and reduce protein synthesis (mckean et al., 2006). aflatoxin is reported to cause immune-suppression (sahoo and mukherjee, 2001; almeida et al., 2011), growth disorder (tuan et al., 2002; almeida et al., 2011), gene expression alterations (mahfouz, 2015), changes in hematological parameters (mohapatra et al., 2011; vaziryan et al., 2017), tissue damage (mckean et al., 2006; mohapatra et al., 2011; raghavan et al., 2011), and mortality in fish (russo and yanong, 2010; spring and fegan, 2010; raghavan et al., 2011). also, aflatoxins are capable of disturbing dna replication, inhibiting rna polymerase and disturbing mrna transcription, hindering amino acid transport and protein synthesis (denli et al., 2009). therefore, its toxicity may prevent renewal of damaged tissues. that is why reversible and irreversible damage to tissues and cells can cause severe changes in biochemical parameters (el-sayed and khalil, 2009; arafa et al., 2014; vaziryan et al., 2017), cause oxidative stress and finally disturb biochemical and physiological balances of fish. pathological methods are quick, sensitive, relatively reliable and inexpensive tools to evaluate damages in vital tissues of fish exposed to toxic compounds. therefore, studying alterations and histopathological changes may provide direct evidence of the toxic effects of aflatoxins on fish. hence, this study investigated pathological alterations in gills, liver, kidney, and intestines of common carp, cyprinus carpio, that were treated with sublethal concentrations of aflatoxin in a 21-day period. materials and methods this study was performed in aquaculture and biology laboratory of behbahn khatam alanbia university of technology, iran in 2015. aspergillus flavus (ptcc 5006) purchased from persian type culture collection (iranian research organization for science and technology), and was cultured on potato dextrose agar (pda), and placed all the test tubes in incubator at 37°c for 17 day (shotwell et al., 1996). then, the fungal spores were transferred from inoculated test tubes on 200 gr-dried bread soaked in 30 ml distilled water. the material was shifted in eight 500 ml sterilized conical flasks and put in an orbital shaker at 28°c and 150 rpm for a period of 30 days. then, the aflatoxins were extracted from culture media with methanol, acetone (70:30 ratios) and diluted water and used for aflatoxin analysis by hplc method (varior, 2003). all the ingredients of commercial feed were powdered, sieved, blend and extruded through a kitchen noodle maker with a 3 mm die, dried at 55°c overnight and stored in freezer. the experiment diet had the same composition as that the control diet to which varying concentrations of the aflatoxin was added from the stock solution. for the feeding, aflatoxin solution (aflatoxin dissolved in ethanol and acetone as solvent) were added into the oil portion of the diet before blending. since acetone and ethanol mixture is toxic to fish if used as in diet, therefore it was allowed to evaporate. then the ingredients were mixed with water, extruded and then dried. healthy common carp were used in the present study according to the national ethical framework for animal research in iran (mobasher et al., 2008). the common carp were obtained from commercial suppliers (ahvaz, iran) and transported to the laboratory facilities. before the assay, fish were acclimated for two weeks in fiberglass 1000 l tank with de-chlorinated tap water, at a ph of 7.4±0.2, temperature 24±2°c, photoperiod: 16 h light: 8 h dark and with continues aeration enough for keeping the dissolved oxygen always higher than 6 mg l-1. after acclimation period, fish (n: 180; 30±5 g) were randomly distributed into 80 l polystyrene tanks, in five experimental groups (12 fish per tank); group i as control group fed with normal diet without solvent and aflatoxin. group ii received feed with solvent but without any aflatoxin. this group served as a positive control for the solvent in which the aflatoxin was dissolved. group iii-v was fed on diets containing 65 int. j. aquat. biol. (2017) 5(2): 63-70 0.5, 0.7 and 1.4 mg kg-1 of aflatoxin for 21 days, respectively. the water was renewed (50% rate) every 24 hrs and experiment was performed for 21 days. fishes were deprived of food 24 hrs before sampling. after 21 days, 12 fish from each treatment were randomly captured. then, fish were euthanized after being anesthetized with a solution of clove powder (1: 5000) (banaee et al., 2013). following autopsy, the liver, kidney, intestine and gill tissue of the control and aflatoxin-treated fish were removed to evaluate lesions and tissue damages. then, the tissues were fixed in buin's solution. then 5 µm histological slides of the tissues were prepared based on banaee et al. (2013) and eagderi et al. (2013). results histopathological alternations observed in the gills, liver, kidney, and intestine of the exposed fish to different concentrations of aflatoxin are illustrated in figures 1-4. bleeding and necrosis of gills, curling and clubbing of the lamellae, shortening of the secondary lamellae and their fusion, inflammation of mucous cells and gill epithelium necrosis are the major histopathological alterations found in the gill of fish treated with different concentrations of aflatoxin (fig. 1). liver in the control group and the positive control which received no aflatoxin had normal structures. hexagonal hepatocytes had round nuclei and a uniform cytoplasm. obvious histological changes were not found in fish treated with the extract (ethanol and acetone mixture) (fig. 2). the relative increase of bleeding and liver discoloration were the main apparent changes in aflatoxin-treated fish. deformation of hepatocytes, as well as disarrangement of cells, cloudy swelling of hepatocytes, cells hypertrophy, vacuole formation in cytoplasm (fig. 2: b2, b3, b4, b5), necrosis of liver parenchyma and histopathological alterations in pancreas were detected in treated fish. degenerated cells had big and dark nuclei (fig. 2: b3, b4, b5). great changes were observed in fat surrounding degenerated cells (fig. 2). destruction of the renal tract, glomerular atrophy and necrosis, enlargement of bowman’s space (fig. 3: c3, c4), urinary tract necrosis, exfoliation and degeneration of the urinary tract epithelium (fig. 3: c3, c5), increased urinary lumen space, dilation of urinary space (fig. 3: c3-5) and increase in melanomacrophage centers (fig. 3: c3-4) were figure 1. histopathology of gills in fish treated with different concentrations of aflatoxin. (a1) control group, (a2) positive control, (a3) 0.5 mg aflatoxin, (a4) 0.7 mg aflatoxin and (a5) 1.4 mg aflatoxin. primary lamellae (pl), secondary lamellae (sl), hypertrophy (h), and cartilage tissue (ct). 66 shahafve et al./ tissue damage induced by aflatoxin in common carp figure 2. liver histopathology of fish treated with different levels of aflatoxin; (b1) the control, (b2) positive control, (b3) 0.5 mg aflatoxin, (b4) 0.7 mg aflatoxin and (b5) 1.4 mg aflatoxin. kupffer cells (kc), liver sinusoid (s), hepatocytes (h), pancreas (p), portal veins (pv), cloudy swelling (cs), degeneration and formation of cytoplasmic vacuoles (v). figure 3. kidney histopathology of fish treated with different levels of aflatoxin; (c1) the control, (c2) positive control, (c3) 0.5 mg aflatoxin, (c4) 0.7 mg aflatoxin, and (c5) 1.4 mg aflatoxin; glomeruli (gl), bowman’s capsule (bc), collecting tubule (ct), distal convoluted tubule (dct), proximal convoluted tubule (pct), tubules with widened lumen (twl), degenerated epithelium (de), desquamation (d), glomerular shrinkage (gs), urinary tract dilation (td), pyknotic nuclei (pn), melano-macrophage centers (mmc), necrosis of distal convoluted tubule (npt). 67 int. j. aquat. biol. (2017) 5(2): 63-70 observed in aflatoxin-treated fish. in control and positive control fish, the intestine was normal. necrosis of mucous cells and mucous layer epithelium (fig. 4: d3-5), and bleeding in the intestinal wall were observed in the intestine of fish treated with aflatoxin. discussion aflatoxicosis can have an economically significant effect on the production of farmed fish. liver, gills, intestine and kidney are the most important target organs in fish that are susceptible to aflatoxicosis (zychowski et al., 2013). therefore, we evaluated their damages in common carp that were fed sublethal levels of aflatoxin (0.5, 0.7 and 1.4 mg kg-1 feed) for 21 days. bleeding in tissues indicates aflatoxins effects on the endothelial cells of the circulatory system. endothelial cells are apparently sensitive to aflatoxin and most pathologic damages were observed in these cells (mehrim et al., 2006). similar changes were observed in rohu (labeo rohita) (sahoo et al., 2001). gills are sensitive organs of teleost when exposed to toxic compounds (robert, 2001). epithelium hyperplasia, mucous cells of gill filaments, fusion of gill lamellae and bleeding were observed in fish fed aflatoxin-contaminated diets. damage to gills in aflatoxin-treated fish can disturb osmotic balance, gas exchange and ammonia excretion in the long term (mehrim et al., 2006). cell necrosis and bleeding are reported in gill lamellae of rohu with aflatoxicosis (sahoo et al., 2001, 2003). in the present study, fat changes in hepatocytes, cloudy swelling, formation of cytoplasmic vacuoles, necrosis of liver cells, tissue fibrosis of portal veins, excessive proliferation of bile duct cells and necrosis of pancreatic cells were found in liver of aflatoxintreated fish. hepatocytes necrosis in fish fed aflatoxincontaminated diet indicates aflatoxin’s effect in destruction of cell membranes and necrosis of tissues. nucleus hypertrophy, hyper chromosome, widespread biliary hyperplasia, liver focal necrosis and cellular inflammation are reported in hybrid sturgeons treated with aflatoxin-contaminated feed (raghavan et al., figure 4. intestine histopathology of fish treated with different levels of aflatoxin; (d1) the control, (d2) positive control, (d3) 0.5 mg aflatoxin, (d4) 0.7 mg aflatoxin, and (d5-1, 2) 1.4 mg aflatoxin; serous layer (sl), muscle layer (ml), mucosal epithelium (me), lamina propria (lp), submucosal layer (sml). 68 shahafve et al./ tissue damage induced by aflatoxin in common carp 2011). aflatoxin-induced damage to liver can disturb homeostasis and physiological balance of fish. similar histopathological damages are reported in liver of oreochromis niloticus (mahfouz and sherif, 2015; chávez-sánchez et al., 1994), rainbow trout, oncorhynchus mykiss, (arana et al., 2014) and sciaenops ocellatu (zychowski et al., 2013) fed aflatoxin-contaminated diets. in this study, microscopic examinations showed that aflatoxin has a significant effect on the kidney’s structure. increased urinary space, loss of renal tract epithelium, atrophy and necrosis of glomeruli and enlargement of the bowman’s space in fish treated with aflatoxin could be attributed to lipid peroxidation of cell membranes in kidney (mehrim et al., 2006). necrosis and atrophy of glomeruli, expansion of bowman’s capsule and increased urinary space lead to increased kidney filtration and disposal of large quantities of amino acids, proteins, glucose, electrolytes and water from the body of fish (singh, 2012; el-greisy and el-gamal, 2015; taheri et al., 2017). degenerated urinary tracts and necrosis of urinary tract epithelial cells are reported in aflatoxintreated rohu (labeo rohita) (sahoo et al., 2001, 2003). blood clots, necrosis and atrophy of glomeruli, as well as melanosis coli are other alterations found in oreochromis niloticus fed aflatoxin-contaminated diet (chávez-sánchez et al., 1994). major pathologic intestinal changes in fish fed diets contaminated with different levels of aflatoxin were atrophy and necrosis of mucous cells, exfoliation of the mucous layer epithelium, as well as bleeding and rupture of intestinal capillaries. damage to the intestine can negatively affect absorption of nutrients and lead to malnutrition, reduced growth rate and reduced physiological regeneration in aflatoxintreated fish (mehrim et al., 2006). exfoliation of the mucous layer epithelium and hemorrhage in the intestine were reported in rohu treated with aflatoxin (sahoo et al., 2001). pathological changes, reduced digestion and absorption of food in the intestine, decreased enzyme activity and malnutrition are all aflatoxins’ effects on the digestive system of fish (applegate et al., 2009). although the median lethal dose (ld50) of aflatoxin for warm water fish is 12.6 mg kg-1 (sahoo et al., 2001), the results of this study indicates the sensitivity of common carp to doses lower than ld50 (≤ 1.4 mg kg-1 feed) of aflatoxin in the long term. also, feeding fish aflatoxin-contaminated diets can provide the grounds for aflatoxicosis. however, the severity of tissue damage in fish treated with different levels of aflatoxin rises with an increase in dosage. tissue damage in fish treated with different levels of aflatoxin can be used as a clinical device in diagnosing aflatoxicosis. acknowledgments the authors gratefully acknowledge the support offered by the behbahan khatam alanbia university of technology. we also thank our english editor, m. banaee for proofreading the manuscript. references adedara i.a., owumi s.e., uwaifo a.o., farombi e.o. 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(2017) 5(2): 63-70 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی آلوده غذایی جیره با شده تغذیه( cyprinus carpio) معمولی کپور ماهی در مختلف هایبافت شناسیآسیب مطالعه آفالتوکسین به محیسنی محمد حقی، دوستنعمت بهزاد ،*بنایی مهدی عفو،شاه شیما 63616-47189: کدپستی ایران، بهبهان، شیالت، گروه زیست، محیط و طبیعی منابع دانشکده بهبهان،( ص) االنبیاء خاتم صنعتی دانشگاه چکیده: این در. است معمولی کپور ماهی مختلف هایبافت شناسیآسیب تغییرات بر غذایی جیره در موجود آفالتوکسین اثر ارزیابی مطالعه این از هدف کنترل عنوانبه ii گروه و نرمال جیره با کنترل گروه عنوانبه ،i گروه. شد توزیع آزمایشی گروه 5 در مخزن 15 در تصادفی بطورها ماهی مطالعه غذا کیلوگرم بر آفالتوکسین گرممیلی 4/1 و 7/0 ،5/0 حاوی جیره با ترتیببه iii-v هایگروه. شدند تغذیه گیریعصاره حالل حاوی جیره با مثبت هایبافت از سپس. شد کشیآسان و بیهوش و صید تصادفی صورتهب تیمار هر از ماهی 12 آزمایش، آغاز از روز 21 گذشت از پس. شدند تغذیه وکسیوزیسآفالت بالینی عالئم مهمترین. شد آماده میکروسکوپی بررسی جهت و تثبیت نظر مورد هایبافت و بردارینمونه روده و کلیه کبد، آبشش، ایهسلول تورم ثانویه، المالی شدن کوتاه ثانویه، المالهای آرایش ریختنبهم آنها، چسبیدگی بهم و المالها انتهای شدن گرزی شامل آبشش در نکروز ها،سلول مسیتوپالس در واکوئل پیدایش ها،سلول هیپرتروفی ها،هپاتوسیت ابری تورم مبتال، ماهیان کبد. بود آبششی اپیتلیوم نکروز و موکوسی و نکروز ه،هست پیکنوز ادراری، مجاری نکروز بومن، فضای افزایش این، بر عالوه. داد نشان پانکراس در هیستوپاتولوژیک تغییرات کبدی، پارانشیم اندازه افزایش به توانمی تغییرات دیگر از. بود کلیه در شده مشاهده عالئم از ادراری لومن فضای افزایش ادراری، مجاری اپیتلیوم شدن دژنره مطالعه این نتایج. است ماهیان روده دیواره در ریزیخون مخاطی، الیه اپیتیلیوم شدن پوسته پوسته مخاطی، هایسلول نکروز جامی، هایسلول نیز( غذا کیلوگرم هر ازای به گرممیلی 4/1≥) پایین هایغلظت در آفالتوکسین به آلوده غذایی جیره با معمولی کپور ماهی تغذیه که داد نشان .گردد آنها فیزیولوژیکی تعادل خوردن برهم و شناسیبافت هایآسیب بروز موجب تواندمی .بافتی آسیب کپور، ماهی غذایی، مسمومیت آفالتوکسیوزیس، :کلمات کلیدی int. j. aquat. biol. (2016) 4(4): 277-284doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article influence of dietary prebiotic mixture (α-mune) on growth performance, haematology and innate immunity of beluga sturgeon (huso huso) juvenile ahmad gharaei*1, mahdiye fadaei rayeni2, mostafa ghaffari3, reza akrami4, ehsan ahmadifar5 1department of fisheries, faculty of natural resources and international hamoun wetland research institute, university of zabol, zabol, iran. 2higher educational complex of saravan, saravan, iran. 3department of fisheries, chabahar maritime university, chabahar, iran. 4department of fisheries, azadshahr branch, islamic azad university, azadshahr, iran. 5department of fisheries, faculty of natural resources, university of zabol, zabol, iran. article history: received 14 november 2015 accepted 24 july 2016 available online 2 5 august 2016 keywords: prebiotic α-mune growth blood variables immune response abstract: the present study investigated the effects of prebiotic (α-mune), a mixture of mannan oligosaccharide, β-1,3 and β-1,6 glucan, on the growth performance, haematology and innate immunity of beluga sturgeon (huso huso) juvenile. fish (46±3 g) were allocated into 12 tanks (15 fish per tank) and triplicate groups were fed a control diet or diets containing 1.5, 3.0 and 4.5 g kg-1 prebiotic for 46 days. fish fed 1.5 g kg-1 prebiotic displayed significantly higher final weight, specific growth rate and feed conversion ratio. wbc, rbc, mcv, mch, haemoglobin, haematocrit and lymphocyte levels were also significantly higher in the fish fed 1.5 g kg-1 prebiotic. furthermore, the highest haematocrit content and lymphocyte level were found in the fish fed a diet containing 1.5 g kg-1 prebiotic. alternative complement activity (ach50), lysozyme activity and ig concentration were significantly higher in the fed 1.5 g kg-1 prebiotic. these results indicate that α-mune can be considered as a beneficial dietary supplement for improving the growth performance, haematological and immunological parameters of beluga sturgeon juvenile. introduction beluga sturgeon, huso huso, has been classified as a critically endangered species in the caspian sea (mohseni et al., 2006). along with declining the natural populations of this species, its farming has been developed across the world (abdolhay and tahori, 2006; mohseni et al., 2006). this developmental trend has been attributed to excellent characters of this species such as fast growth, easy adaptation to farm condition, and specifically, its expensive caviar (mohseni et al., 2006). since, little information is available about the nutritional requirements of h. huso in aquaculture (hoseinifar et al., 2011b), hence, the introduction of prebiotics in sturgeon nutrition could be an interesting alternative to improve their feed efficiency and health. prebiotics are non-digestible food ingredients which beneficially affect the host by selectively * corresponding author: ahmad gharaei e-mail address: agharaei551@uoz.ac.ir stimulating the growth and/or activity of healthpromoting bacteria in the intestinal tract (gibson, 2004). in this regard, the beneficial effects of dietary mannan oligosaccharides and β-glucan on fish and crustacean species have been reported. these prebiotics can positively influence the haematological parameters and non-specific immune responses (staykov et al., 2007; andrews et al., 2009; ta'ati et al., 2011; ye et al., 2011; soleimani et al., 2012) as well as growth performance (mahious et al., 2006; torrecillas et al., 2007; staykov et al., 2007; yilmaz et al., 2007; grisdale-helland et al., 2008; piaget et al., 2007; gultepe et al., 2010; ye et al., 2011; gultepe et al., 2012) of fish species. since, analysis of growth performance, haematological parameters and innate immunity are proper indicators for evaluating the conditions of aquatic animals (bahmani et al., 2001). hence, the 278 gharaei et al./ influence of dietary prebiotic mixture on h. huso present study was conducted to evaluate the effects of different levels of the prebiotic mixture (α-mune, a mixture of mannan oligosaccharide, β-1,3 and β1,6 glucan) on growth performance, haematology and innate immunity of the juvenile beluga sturgeon. materials and methods diet preparation: to prepare the experimental diets, a commercial pellet diet (skretting) supplemented with 0 (control), 1.5, 3.0 and 4.5 g kg-1 prebiotic αmune (alfarma, belgium) in triplicate group. for this purpose, the commercial pellet was crushed, mixed with the appropriate prebiotic concentrations and water, and pelleted; then were allowed to be dried for 18 hrs at 45°c by air circulation and stored at 4°c. the control diet was prepared by adding only water (cerezuela et al., 2008) (table 1). feeding trial: juveniles were obtained from the shahid marjani sturgeon hatchery center (gorgan, golstan province, iran). prior to experiment, fish were acclimated to experimental conditions for one week and fed by commercial diet without prebiotic to acclimatize for experimental diet. at the beginning of the experiment, the juveniles with an initial average weight of 46±0.8 g were randomly distributed into 12 fibreglass tanks (2×2×0.5 m) at a stocking density of 15 fish per tank. water temperature, dissolved oxygen, ph and salinity were monitored daily and maintained at 26.12±0.58°c, 6.55±0.49 mg l-1, 8.04±0.08 and 1.26±0.28, respectively. during the trial, fish were hand-fed 13% of the body weight 3 times a day at 07:00, 13:00 and 19:00 h for 46 days. growth performance: total fish weight in each tank was measured every 15 days to adjust the feeding rate and estimate growth performance. at the end of the experiment, the fish were fasted for 24 hrs before harvest and weighing. the total numbers were counted and the mean body weights of fish were measured. then weight gain (wg), specific growth rate (sgr), feed conversion ratio (fcr) and condition factor (cf) of each tank were calculated using the following equations: weight gain (wg, g) = final weight (g) initial weight (g) specific growth rate (sgr% / day) = 100 [ln final weight ln initial weight)] / days of feeding feed conversion ratio (fcr) = dry feed intake (g) / wet weight gain (g) condition factor (cf) = final weight (g) / (fork length (cm))3 ×100 survival rate (%) = 100 [initial number of fish final number of fish)]/ initial number of fish. chemical analyses: chemical analysis of formulated diet was performed according to aoac (2005) (table 1). crude protein content (n×6.25) using a kjeldahl system (buchi, switzerland), crude lipid content by a soxhlet system (buchi, switzerland), ash content by weighting after incinerating at 550ºc for 6 hrs in a hotspot furnace (gallenkamp, england), and crude fiber content by an automatic analyzer (fibertec, sweden) were measured. blood sample collection and haematological parameters: at the end of the experiment, 3 fish were randomly sampled from each tank. after anesthetizing with clove solution, about 2 ml of blood was sampled from the caudal vein, using a non-heparinized syringe. then, the blood samples were introduced to both heparinized (edta) and non-heparinized tubes to perform haematological and immunological analysis, respectively. serum samples were attained after centrifugation (4,500×g for 10 min) and stored at -20°c until analysis for immunological studies. red blood cells (rbc) and white blood cells table 1. proximate composition of the commercial diet (skretting). ingredients % dry weight crude protein 50 crude lipid 20 ash 7.5 fiber 7 nfe 1 15.5 gross energy (mj/kg) 2 22.33 1 nitrogen free extract (nfe) = 100 (% crude protein + % crude lipid +% ash +% fiber) 2 gross energy (ge) (mj/kg) = (% crude protein × 23.6 + % crude lipid × 39.5 + % nfe × 17) 279 int. j. aquat. biol. (2016) 4(4): 277-284 (wbc) were counted using a neubaur haemocytometer according to martins et al. (2004). haemoglobin (hb) according to collier (1944); haematocrit (hct) according to goldenfarb et al. (1971); mean corpuscular volume (mcv) and mean corpuscular haemoglobin (mch) according to wintrobe (1934), and differential white blood cell counts based on klontz (1994) were other measured haematological parameters. immunological assays: alternative complement activity (ach50): the serum complement activity was determined by assaying the alternative complement activity (ach50) (yano et al., 1988). rabbit blood was mixed with an equal volume of alsevers’s solution and stored at 4°c. subsequently, the cells were centrifuged at 400 g for 5 min; the pellet of rarbc was washed twice in 10 mm ethylene glycolbis tetraacetic acid (egta)-mg-gelatin veronol buffer (gvb) and suspended in the same buffer at a concentration of 2×108 cells ml-1. alternative complement pathway activity was assayed according to yano et al. (1988). briefly, 0.5 ml of serially 10fold diluted carp serum in egta–mg–gvb was placed in a set of test tubes and 0.2 ml of sheep red blood cells suspension (2×106 cells ml-1) was added. this mixture was incubated at 15°c for 90 min. addition of 2.8 ml of 10 mm edta-gvb buffer stopped the hemolytic reaction. after centrifugation, the value y (percent haemolysis /100) was calculated from the optical density at 414 nm of the supernatant. the value y/ (1-y) and the reciprocal of the serum dilution were plotted on log-log graph paper and the ach50 (u ml-1), the reciprocal dilution giving 50% haemolysis [y (1-y) =1], was read from the graph (sakai et al., 2001). lysozyme activity: lysozyme level was determined by turbidity assay according to the method of ellis (1990) with slight modifications. aliquots (1.75 ml1) of micrococcus lysodeikticus suspension (sigma) (0.375 mg ml-1, 0.05 m pbs, ph=6.2) were mixed with 250 μl-1 of each sample and the optical density was measured after 15 and 180 s by spectrophotometer (biophotometer eppendorf) at 670 nm. pbs was used as a blank and results were expressed according to amounts of lysozyme (μg) per 1 mg of sample calibrated using the standard curve determined with egg white lysozyme (sigma) in sterile sodium phosphate buffer. serum total immunoglobulin (ig): total immunoglobulin (ig) were determined according to siwicki and anderson (1993). briefly, serum total protein content was measured using a micro protein determination method (c-690; sigma), before and after precipitating down the immunoglobulin molecules, using a 12% solution of polyethylene glycol (sigma). the difference in protein content represents the ig content. statistical analysis: the normality and homogeneity of data were explored by examining the residual plots. the data were subjected to one-way analysis of variance (anova), and if significant (p<0.05) differences were found, duncan’s multiple range test (duncan, 1995) was used to rank the groups using the spss (version 15). results no mortality was recorded throughout the experiment. effects of different dietary prebiotic αmune levels on the growth performance and feed utilization are shown in table 2. at the end of the experiment, fish fed 1.5 g kg-1 prebiotic displayed significantly improved growth performance and feed utilization, including wg, sgr and fcr than other treatments (p<0.05). also, a non-significant elevation of cf was found in the fish fed diet 1.5 g kg-1 prebiotic (p>0.05). effects of different levels of dietary prebiotic αmune on the haematological parameters are presented in table 3. wbc, rbc, mcv, mch, haemoglobin, haematocrit and lymphocyte levels were significantly higher in the beluga fed 1.5 g kg-1 prebiotic compared to other groups (p<0.05). the highest monocyte level was observed in beluga juveniles fed control diet (p>0.05). immunological parameters of beluga sturgeon juvenile fed different levels of the dietary prebiotic α-mune are shown in figures 1-3. after the 46 days 280 gharaei et al./ influence of dietary prebiotic mixture on h. huso feeding trial, alternative complement activity (ach50) and ig concentration were significantly higher in the fish fed 1.5 g kg-1 prebiotic than other groups (p<0.05). furthermore, lysozyme activity was significantly higher in fish fed 1.5 g kg-1 prebiotic compared to fish fed control and 4.5 g kg-1 prebiotic treatments (p<0.05). discussion during the last decade, the use of dietary compounds with potential prebiotic effects is being considered as a possible tool for improving gut health and growth performance of farmed animals (ortiz et al., 2012). the present study showed that the highest growth levels of prebiotic control 1.5 g kg -1 3.0 g kg -1 4.5 g kg -1 parameters wg (g) 161.0± 4.9b 173.24± 4.5a 154.78± 3.7b 158.17± 1.6b sgr (%/day) 3.27± 0.05b 3.39± 0.05a 3.19± 0.04b 3.23± 0.00b fcr 1.37± 0.01ab 1.27± 0.11b 1.41± 0.02a 1.38± 0.05ab cf (%) 0.48± 0.02a 0.52± 0.05a 0.47± 0.00a 0.48± 0.02a survival (%) 100 100 100 100 data assigned with different superscripts indicate significant differences (p<0.05). table 2. growth performance of beluga juvenile fed the diets containing various prebiotic α-mune levels for 46 days. levels of prebiotic control 1.5 g kg -1 3.0 g kg -1 4.5 g kg -1 parameters wbc (per/mm3) 6850± 132.28b 7350± 150.00a 6616.7± 246.64b 6600± 200.00b rbc (per/mm6) 0.81± 0.00b 0.86± 0.01a 0.82± 0.00b 0.82± 0.00b mcv (fl) 227.60± 1.15c 236.74± 2.23a 230.60± 2.34bc 231.35± 1.07b mch (pg) 75.97± 0.94b 81.33± 1.01a 77.13± 1.01b 77.38± 0.57b haemoglobin (g/dl) 6.20± 0.10b 6.83± 0.05a 6.36± 0.15b 6.40± 0.10b haematocrit (%) 18.60± 0.30b 19.66± 0.25a 19.03± 0.40b 19.13± 0.25ab monocyte (%) 4.33± 1.15a 3.00± 1.00a 4.00± 1.00a 3.66± 0.57a lymphocyte (%) 74.33± 3.78ab 79.00± 1.00a 75.33± 2.51b 73.00± 2.00c basophil (%) 0.66± 0.57a 0.66± 0.57a 0.66± 0.57a 0.66± 0.57a data assigned with different superscripts indicate significant differences (p<0.05). table 3. haematological parameters of beluga juvenile fed the diets containing various prebiotic α-mune levels for 46 days figure 2. serum lysozyme activity of beluga juvenile fed the diets containing various prebiotic α-mune levels for 46 days. data assigned with different superscripts indicate significant differences (p<0.05). figure 1. serum alternative complement activity (ach50) of beluga juvenile fed the diets containing various prebiotic α-mune levels for 46 days. data assigned with different superscripts indicate significant differences (p<0.05). 281 int. j. aquat. biol. (2016) 4(4): 277-284 performance and other measured parameters including wg, sgr and fcr 1.5 g kg-1 prebiotic treatment. mannan oligosaccharide promotes the growth of beneficial lactic acid bacteria in the intestine and these bacteria can inhibit the growth of pathogens by producing bacteriosins (andrews et al., 2009). although previous studies have showed that dietary oligofructose and mannan oligosaccharide have no effect on growth performance of beluga (hoseinifar et al., 2011b; razeghi mansour et al., 2012). stimulation of growth by prebiotic has previously been reported in other aquatic animals such as turbot (psetta maxima) (mahious et al., 2006), rainbow trout (oncorhynchus mykiss) (staykov et al., 2007; yilmaz et al., 2007), sea bass (dicentrarchus labrax) (torrecillas et al., 2007), atlantic salmon (salmo salar) (grisdale-helland et al., 2008), flounder (paralichthys adspersus) (piaget et al., 2007), sea bream (sparus aurata) (gultepe et al., 2010, 2012) and japanese flounder (paralichthys olivaceus) (ye et al., 2011) due to promoting the synthesis of vitamins and enzymatic activity (lin et al., 2012). however, there are reports about negative effects of dietary prebiotic on other fish species (pryor et al., 2003; genc et al., 2006; genc et al., 2007; welker et al., 2007; sado et al., 2008; dimitroglou et al., 2010; akrami et al., 2009; akrami et al., 2010). haematological parameters are indices of fish health as well as the physiological status of an organism (yousefi et al., 2012). the present study indicated that haematological parameters were significantly higher in the beluga fed 1.5 g kg-1 prebiotic. white blood cells are used as indicators of health status in fish because of involving in the regulation of immunologic function (ballarin et al., 2004). in this study, white blood cells showed enhanced levels in the 1.5 g kg-1 prebiotic diet; also, lymphocyte and haematocrit cell counts were higher. ebrahimi et al. (2012) reported that increase in wbc count might be due to stress as a result of daily feeding on β-glucan. ahmadifar et al. (2010) and hoseinifar et al. (2011a) were studied the effects of dietary inulin and fructooligosaccharide on the beluga sturgeon juvenile, respectively, and pointed out that wbc levels, lymphocyte and haematocrit cell counts were significantly higher in the fish fed prebiotic. in addition, andrews et al. (2009) observed a significant improvement in wbc, rbc and hb, in labeo rohita fed on diets supplemented with mannan oligosaccharide. in contrast, razeghi mansour et al. (2012) reported that different levels of mannan oligosaccharide has no effect on haematological parameters of beluga sturgeon juvenile. also similar results have been reported in mannan oligosaccharide fed channel catfish (ictalurus punctatus) (welker et al., 2007), and nile tilapia (oreochromis niloticus) (hisano et al., 2007; sado et al., 2008). it appears that fluctuations in hematological and biochemical variables may be species specific, inclusion rates of prebiotic, ingredients of diets, rearing period, etc (ta'ati et al., 2011). probiotics and prebiotics stimulate the host immune system (ye et al., 2011). stimulation of the immune response of fish through dietary supplements is of high interest for commercial aquaculture (staykov et al., 2007). the present study showed that dietary prebiotic can modulate the innate immune responses of beluga sturgeon juvenile. based on the results, beluga sturgeon fed 1.5 g kg-1 prebiotic has significantly higher ach50 figure 3. serum total immunoglobulin (ig) levels of beluga juvenile fed the diets containing various prebiotic α-mune levels for 46 days. data assigned with different superscripts indicate significant differences (p<0.05). 282 gharaei et al./ influence of dietary prebiotic mixture on h. huso and ig concentration. these data are in agreement with the increased ach50 and ig level recorded in roach (rutilus caspicus) fed with the different levels of fructooligosaccharide (fos) (soleimani et al., 2012). serum lysozyme activity was significantly higher in 1.5 g kg-1 treatment than that of 4.5 g kg-1 and such a result was found by staykov et al. (2007), soleimani et al. (2012) and akrami et al. (2013). the increased serum lysozyme activity indicates that immune system is enhanced in treated fish. in contrast, lysozyme activity was lower in fish fed a mos diet in atlantic salmon (grisdale-helland et al., 2008). furthermore, ye et al. (2011) was evaluated the effects of different levels of dietary fos, mannan oligosaccharides and bacillus clausii on the japanese flounder (paralichthys olivaceus) and explained that the fos, mos and fos+mos dietary supplementations did not alter lysozyme activity. this contradictory result could be referred to the duration of prebiotic administration, the age and type of the treated fish (ibrahem et al., 2010). in conclusion, the results of present study showed that dietary administration of α-mune as a prebiotic at the level of 1.5 g kg-1 can positively influence on growth, haematology and innate immunity of beluga juvenile. references akinwande a.a., sogbesan a.o., moody f.o., ugwumba a.a.a. 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(2023) 11(2): 141-150 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article assessing the ecological quality status of arid mangroves in the gulf of oman, iran, using benthic indices of ambi, m-ambi, and bentix mohadeseh miri1, jafar seyfabadi1, mehdi ghodrati shojaei1, hassan rahimian2 1department of marine biology, faculty of natural resources and marine sciences, tarbiat modares university, noor, iran. 2school of biology, college of science, university of tehran, tehran, iran. s article history: received 2 january 2023 accepted 25 april 2023 available online 2 5 april 2023 keywords: mangrove bioindicator ecological quality status benthic communities abstract: polychaetes are suitable indicators to evaluate the benthic ecological status and respond to natural and anthropogenic. we evaluated the ecosystem health of the mangroves of azini and gwadar based on benthic indices including ambi, m-ambi, and bentix using polychaete communities. the results showed that in both regions, ecoq classifications ranged from "high" to "moderate" in bentix, "good" to "excellent" in ambi, and "good" in m-ambi. the m-ambi was significantly correlated with sediment variables, including total organic matter (tom), total organic carbon (toc), and silt/clay. the result revealed a significant correlation between the biotic indices and the toc content of sediments. according to the results, toc can be used as a descriptor and indicator to evaluate the health of mangrove ecosystems in relation to benthic indices. in addition, it is necessary to combine several indices to assess the status of ecosystems. introduction the mangrove forests grow in estuaries and intertidal zones of the tropics and subtropics (field et al. 1998). the mangrove ecosystem is a biologically active ecosystem with numerous ecological functions. mangrove forests provide many benefits, including breeding grounds for fish and shellfish, birds, and other wildlife, preventing shoreline erosion, and protection during hurricanes and tidal waves (quarto, 2005). in addition, they are responsible for providing many ecosystem goods and services, including natural barriers, carbon sequestration, and biodiversity (duke et al. 2007). however, it has been threatened by urbanization, pollution, and overexploitation over the past decades (alongi, 2002). the spatial distribution patterns of polychaetes have been extensively investigated with respect to environmental variables since they constitute a dominant element of benthic communities (tyler and kowalewski, 2018). the abundance of polychaetes on estuarine macrofauna and their correspondence: jafar seyfabadi doi: http//doi.org/10.22034/ijab.v11i2.1837 e-mail: jseyfabadi@gmail.com dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.8.0 occurrence under a variety of environmental conditions make them an excellent biological model for studying estuarine ecosystems (schüller et al., 2009). the assemblages of polychaetes display changes in standing stock as a response to variables in the environment, with the increase in organic sediment content being one of the most significant impacts of anthropogenic activity (dauvin et al., 2016; alvarez-aguilar et al., 2017). polychaetes can be used as a bioindicator of organic pollution due to their high diversity, abundance, and functional significance. they are frequently used as ecological groups to determine ecosystem quality as part of biotic indices (borja et al., 2014). several indices based on benthic invertebrate communities have been proposed to evaluate environmental health status, including the azti marine biotic index (ambi) (borja et al. 2000), the multivariate ambi (m-ambi) (muxika et al., 2007), and the bentix index (simboura and zenetos 2002). ambi is based on species assigned to one of five levels of sensitivity, ranging from very mailto:jseyfabadi@gmail.com 142 miri et al./ assessing the ecological quality status of arid mangroves in the gulf of oman sensitive to opportunistic species (borja et al., 2000). m-ambi is a multivariate analysis that combines shannon-wiener diversity, species richness, and ambi (muxika et al., 2007). bentix index calculates the relative contributions of tolerant and sensitive taxa based on their occurrence ratios in the benthic fauna by weighting them according to the concept of indicator groups (simboura and zenetos, 2002). ambi, m-ambi, and bentix indices are designed to evaluate the impact resulting from general stress factors and can not distinguish between natural and anthropogenic disturbances (borja et al., 2003). in order to detect and develop proper strategies for mitigating the effects of the discharge of anthropogenic chemical contaminants into marine ecosystems, we must assess and understand the ecological effects that affect habitat alteration and benthic marine communities. hence, our study assessed the ecological quality of the azini and gwadar mangrove ecosystems in the gulf of oman using benthic quality indices including ambi, mambi, and bentix based on polychaete communities and determining the correlation of these indices with environmental variables was investigated. materials and methods in the iranian coastal zone, mangrove forests are distributed along 1250 km. mangrove forests in iran consist of two species of avicennia marina and rhizophora mucronata, that both species are found in the azini estuary, but there is only a. marina in the gwadar estuary. in the summer and winter of 2019, sediment samples were taken from mangrove forests in gwadar and azini estuaries (fig. 1). sediment samples were collected using a metal quadrat (25*25*25 cm) from 10 stations i.e. 5 stations for each estuary. the sediments were collected in three independent replicates for sediment variables and polychaetes. polychaetes were sorted under a stereomicroscope and identified at the lowest taxonomic level possible. measurements of total organic matter (tom), total organic carbon (toc), and the grain size composition of the sediment surface were performed using the ignition method (heiri et al., 2001), the walkley-black (walkley and black, 1934) method, figure 1. positions of the sampling sites of polychaetes in the hara biosphere reserve, gulf of oman (the gwadar and azini estuaries). 143 int. j. aquat. biol. (2023) 11(2): 141-150 and the hydrometer technique (bouyoucos, 1962), respectively. the ambi and m-ambi indices were used to assess the benthic ecological status of the study areas and calculated using the ambi program (version 6.0; available at http://ambi.azti.es). the software divides polychaetes into five ecological groups (gi + gv) based on their sensitivity to environmental stress gradients. following the newest ambi species list (december 2020), most of the collected species were classified into different ecological groups (egs). the assignment of some species, including some native species, was based on expert opinion or on the assignment of other species in the same genus (borja et al., 2008). to improve the reliability of the results, ambi values with more than 20% unassigned individuals were removed from the ambi analyses but included in the m-ambi analyses (borja and muxika, 2005). m-ambi values were calculated using factor analysis of ambi, shannon diversity (h’), and species richness (s) (muxika et al., 2007). in order to assess ecological status, it was crucial to set an appropriate m-ambi reference condition. therefore, in the present study, the reference conditions for the m-ambi index proposed by borja and tunberg (2011) and forchino et al. (2011), which increased the highest species diversity and richness values by 15% and decreased ambi to half of the lowest value. bentix simplified the five ecological groups into two, the sensitive species group (gs), which includes gi and gii of the ambi method, and the tolerant species group (gt), which includes giii, giv, and gv (add-in v.1.0 version: (simboura and zenetos, 2002). table 1 shows the different ecological quality statuses determined by ambi, mambi, and bentix. to assess differences in benthic indices between regions, and seasons, we used a repeated measure permanova model (permutational multivariate analysis of variance), where ‘region’ and ‘season’ was fixed factors. permutations of row data were unlimited and 9999 permutations were applied. spearman correlation was done to examine the relationship between sediment parameters and benthic indices. the univariate and multivariate analyses were conducted using primer ver. 6 with the permanova add-on package and spss 21.0 software. 0 20 40 60 80 100 winter summer winter summer gwadar azini e c o lo g ic a l g ro u p s % i ii iii iv v figure 2. ambi ecological groups (eg) for the polychaete communities in mangrove ecosystems in the gulf of oman (the gwadar and azini estuaries). biotic index ecological quality status (ecoqs) high good moderate poor bad ambi 0-1.2 1.2-3.3 3.3-4.3 4.3-5.5 5.5-7 m-ambi 0.77-1 0.53-0.77 0.39-0.53 0.2-0.39 0.0-0.2 bentix 4.5-6 3.5-4.5 2.5-3.5 2-2.5 0.0-2 note: the thresholds of ambi and m-ambi are referred to borja et al. (2000), borja and muxika (2005), muxika et al. (2007), li et al. (2017), and the thresholds of bentix is referred to simboura and zenetos 92002) table 1. the threshold levels of three indices for benthic ecological quality status assessment. 144 miri et al./ assessing the ecological quality status of arid mangroves in the gulf of oman results the collected species were categorized into five ecological groups to calculate the ambi and mambi indices (fig. 2, table 2). the highest number of species was in the ecological group (i) in azini (84.9% of the total species in azini). the lowest number of species were ecological groups (v) in the gwadar. during two sampling seasons, ecological groups (i), (iii), and (iv) included the most dominant species in the study regions. the lowest and highest values of the ambi index in all three regions were 0.8±0.09 and 3.2±0.5, respectively, at azini and gwadar in summer. according to ambi, gwadar, and azini were of good to high status (fig. 3), and the results showed a significant difference between regions (p<0.05). the ambi index revealed that summer has better ecological quality, but there was no significant difference between seasons in both areas (p>0.05) (table 3). m-ambi showed that the ecological status of the study regions was good status and it ranged from 0.59±0.08 to 0.52±0.13 (fig. 4). no significant difference was found between the two regions and seasons (p>0.05) (table 3). two sensitive and tolerant ecological groups were used to calculate the bentix index (fig. 5). the most sensitive species were found in azini during summer (gs, 86.6%). the most tolerant species were observed in gwadar during winter (gt, 67%). species ambi groups bentix groups aricidea fragilis i i armandia intermedia i i armandia sp. i i capitella aberranta v ii ctenodrilus sp. na na glycinde sp. ii i heteromastus sp.1 iv ii heteromastus sp.2 iv ii heteromastus sp.3 iv ii heteromastus sp.4 iv ii leonnates indicus iii ii lepidonotus purpureus ii i lepidonotus sp. ii i levinsenia gracilis iii ii linopherus hirsuta iv ii marphysa sanguinea ii i marphysa sp. ii i mediomastus warrenae na na melinna monoceroides iii ii namalycastis sp. na na neanthes glandicincta i i paleaequor sp. i i perinereis horsti iii ii perinereis nuntia iii ii phyllodoce sp. ii i prionospio sp. na na questa riseri ii i scolelepis sp. iii ii scolelepis squamata iii ii sigambra sundarbanensis iv ii simplisetia erythraeensis iii ii syllis gracilis ii i tylonereis bogoyawlensky iii ii *na: not assigned table 2. list of polychaete species and their assigned ecological groups (eg) in mangrove ecosystems in the gulf of oman (the gwadar and azini estuaries). 145 int. j. aquat. biol. (2023) 11(2): 141-150 ambi df ms pseudo-f p region 2 8638.4 2.0915 0.048 season 1 6560.5 1.8017 0.259 region × season 1 5128.5 1.1307 0.438 residual 62 2304.8 total 71 m-ambi df ms pseudo-f p region 2 1055.4 0.81601 0.567 season 1 309.17 0.41735 0.142 region × season 1 150.01 0.2025 0.795 residual 62 803 total 71 bentix df ms pseudo-f p region 2 2825.3 1.3381 0.037 season 1 787.02 0.60774 0.561 region × season 1 834.85 0.64468 0.519 residual 62 582.58 total 71 table 3. the results of permanova for comparing ambi, m-ambi and bentix across regions and seasons in mangrove ecosystems in the gulf of oman (the gwadar and azini estuaries). factors: region (levels: gwadar and azini) and season (levels: winter and summer). 0 1 2 3 4 5 6 7 gwadar azini a m b i winter summer bad poor moderate good high figure 3. the ambi values of polychaete communities in mangrove ecosystems in the gulf of oman (the gwadar and azini estuaries). 0 0.2 0.4 0.6 0.8 1 gwadar azini m -a m b i winter summer bad poor moderate good high figure 4. the m-ambi values of polychaete communities in mangrove ecosystems in the gulf of oman (the gwadar and azini estuaries). 146 miri et al./ assessing the ecological quality status of arid mangroves in the gulf of oman bentix index values ranged from 2.8±0.32 to 5±0.67 and gwadar and azini had the lowest and the highest values in summer, respectively. using the bentix index, two regions were moderate to high status (fig. 6). there was a significant difference in ecological quality between gwadar and azini (p<0.05). the mean values of bentix did not differ significantly between winter and summer (p>0.05) (table 3). table 4 shows the physicochemical characteristics of the sediment during winter and gwadar winter summer tom 0.51±0.09 0.71±0.06 toc 0.91±0.3 1.23±0.4 silt/clay (%) 1.13±0.54 1.93±1.5 azini winter summer tom 0.78±0.1 0.57±0.08 toc 0.99±0.2 0.84±0.3 silt/clay (%) 1.96±0.91 1.62±0.88 table 4. variations in environmental data (mean ± sd) between regions and seasons in mangrove ecosystems in the gulf of oman (the gwadar and azini estuaries). (tom = total organic matter; toc = total organic carbon). 0 20 40 60 80 100 winter summer winter summer gwadar azini e c o lo g ic l g ro u p % sensetive group tolerant group figure 5. bentix ecological groups (eg) for the polychaete communities in mangrove ecosystems in the gulf of oman (the gwadar and azini estuaries). 0 1 2 3 4 5 6 gwadar azini b e n t ix winter summer bad poor moderate good high figure 6. the bentix values of polychaete communities in mangrove ecosystems in the gulf of oman (the gwadar and azini estuaries). 147 int. j. aquat. biol. (2023) 11(2): 141-150 summer from the gwadar and azini estuaries. the values of m-ambi showed the best response to increasing concentrations of tom (r = -0.91, p<0.01) and toc (r = -0.95, p<0.01). the ambi and bentix values showed a significant correlation with toc (r = -0.61, p<0.05, and r = -0.73, p<0.05 respectively). conversely, ambi did not show significant correlations with tom, and silt/clay (table 5). discussion ambi, m-ambi, and bentix were used to assess the ecological quality of gwadar and azini mangroves in the gulf of oman. there were noteworthy differences between the results of the three biological indices. in comparison to ambi and m-ambi, the bentix showed a much lower ecological status. the indices showed different ecoq classifications in the two regions, including "high" to "moderate" conditions in bentix and "high" to "good" conditions in ambi, and "good" conditions in m-ambi. some studies report inconsistent classifications of ecoqs based on different benthic indices for the same study area (simboura et al., 2014; pelletier et al., 2018; maghsoudlou et al., 2020; yan et al., 2020; xu et al., 2021; dong et al., 2021). these differences could be due to (1) these indicators are designed to evaluate european coastal waters' ecosystems (qiu et al., 2018; xu et al., 2021), (2) there are different boundary limits (simboura and reizopoulou, 2008; borja and tunberg, 2011; gamito et al., 2012; sun et al., 2018; ni et al., 2019), (3) in the various ecological groups, each index input is weighted differently (simboura and reizopoulou, 2008; sun et al., 2018), and (4) worldwide, macrobenthos is composed differently in marine ecosystems, and they may respond differently to disturbances (borja et al., 2000; pelletier et al., 2018; mulik et al., 2020; dong et al., 2021). the bentix classification method yields a wider high-status range (4.5-6) than the ambi (0-1.2), which often classifies good-status sites as high-status. in the current study, the toc content of sediment was significantly correlated with ambi, m-ambi, and bentix indices. according to the results, the bentix index showed a significant negative correlation with the toc content of the sediment. although organic matter provides food for benthic fauna in surface sediments, excessive amounts of organic enrichment can lead to oxygen depletion as well as toxic by-products, resulting in reduced species richness, abundance, and biomass of benthic fauna that are closely associated with bottom sediments (hyland et al., 2005). the toc content of sediment was higher in gwadar mangroves compared to azini mangroves. the bentix index was classified the gwadar mangroves in "moderate" status, while the azini mangroves classified as "good" to "high" status. therefore, the toc content of sediment can be an important descriptor and proxy for evaluating the benthic status of mangrove ecosystems in relation to ecological indices. the m-ambi index showed the highest correlation with tom and toc content of sediments. therefore, according to the results, the ambi, m-ambi, and bentix indices can be considered suitable indicators for enriching organic matter in sediments. some studies observed correlations between organic matter and ambi, mambi and bentix indices (borja et al., 2008; ambi m-ambi bentix silt/clay toc ambi m-ambi -0.59 bentix -0.97 0.66 silt/clay -0.26 -0.51* 0.07 toc 0.61* -0.95** -0.73* 0.58 tom 0.46 -0.91** -0.46 0.36 0.75 table 5. spearman correlation between biotic indices and sediment parameters in mangrove ecosystems in the gulf of oman (the gwadar and azini estuaries). (*, p<0.05; **, p<0.01). 148 miri et al./ assessing the ecological quality status of arid mangroves in the gulf of oman caglar and albayrak 2012; umehara et al. 2019; medeiros et al. 2021; munari et al. 2022; sarathy et al. 2022). however, according to hu et al. (2018), benthic indices are not correlated with organic matter, which may explain its overestimation of benthic quality. the azini mangroves were dominated by sensitive species (eg i), while the gwadar mangroves were dominated by tolerant species (eg iii) and second opportunistic species (iv). apart from the absence of industries in the coastline region, the presence of two types of trees, including a. marina and r. mucronata, may have contributed to the "high" ecological status in azini mangroves. according to delfan et al. (2021), the low species richness of the macrofauna may be explained by a low diversity of mangrove trees. avicennia marina is the only mangrove species in gwadar. in addition, the wastewater from shrimp farming centers may have disturbed the gwadar mangroves. in the current study, because of a lack of knowledge, some of the collected species, especially some native species, could not be assigned to the ambi species list, resulting in partly affecting index evaluation results. lastly, it is important not to ignore the characteristics of the local ecosystem when assessing its status of the local ecosystem. hence, no one biotic index is the most suitable, and when doing ecological assessments, it is more reliable to combine the results from several indices. conclusion the indices showed different ecoq classifications in the two regions studied, including "high" to "moderate" status in bentix and "high" to "good" status in ambi, and "good" status in m-ambi. sensitive species were dominant in the azini mangroves, while resistant species were dominant in the gwadar mangroves. there were significant correlations between benthic indices and the toc content of sediments. therefore, toc can be a suitable descriptor and indicator to evaluate the quality of mangrove ecosystems in relation to benthic indices. also, the highest correlation was observed between m-ambi and sediment parameters. our results showed that several benthic indices are needed for assessing the benthic quality of ecosystems; as well as suggest useful insights on identifying the key drivers of polychaete communities and benthic indices in mangrove ecosystems. acknowledgments we gratefully acknowledge the financial support provided by department of marine biology, faculty of natural resources and marine sciences, tarbiat modares university, noor, iran. references alongi d.m. 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(2016) 4(5): 301-307: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article karyosystematics of kol tooth-carp, aphanius darabensis (teleostei: cyprinodontidae) azam mansoori1, mehregan ebrahimi1, 2, ali gholamhosseini1, hamid reza esmaeili*1 1ichthyology and molecular systematics research lab., department of biology, college of sciences, shiraz university, shiraz, iran. 2school of biological sciences, flinders university, adelaide, sa, australia. article history: received 13 july 2016 accepted 22 september 2016 available online 2 5 october 2016 keywords: cyprinodontiformes chromosome cytogenetical analysis ideogram abstract: the karyological and cytological characteristics of an endemic cyprinodont fish of iran, aphanius darabensis esmaeili, teimori, gholami & reichenbacher, 2014 have been investigated for the first time by examining metaphase chromosomes spreads obtained from gill epithelial and kidney cells. the diploid chromosome number of a. darabensis is 48. the karyotype consisted of five submetacentric and 19 subtelocentric pairs of chromosomes (5sm+19st). the fundamental number (fn) is 58. sex chromosomes were cytologically indistinguishable in this tooth-carp. according to this study and previous karyological reports from other cyprinodont species, it can be suggested that the diploid number (2n=48) is common amongst cyprinodont fishes. these results can be used as basic informations in population studies and management and conservation programs. introduction fishes represent more than half of all extant vertebrates with more than 33,984 recognized species (eschmeyer and fong, 2016). cyprinidontiformes is a small fish order comprising about 1323 of mostly small species in 10 families (eschmeyer and fong, 2016). they live in fresh or brackish waters and some extreme environments, such as saline or very warm waters, or isolated water bodies where no other types of fishes occur (gholami et al., 2014; esmaeili et al., 2016). the cyprinodontidae with 135 species worldwide (eschmeyer and fong, 2016) are represented in iran by only one genus aphanius nardo, 1827. from a total of 32 aphanius species which have been described around the world, one fossil record, aphanius persicus and 14 alive species have been reported from iranian drainages including a. darabensis or kapour-e-dandandar-e-darab (farsi); kol tooth-carp (english) and darab zahnkärpfling (german) is an endemic species found in the uppermost reaches of the kol river tributary which drains to the persian gulf (esmaeili * corresponding author: hamid reza esmaeili e-mail address: hresmaeili@shirazu.ac.ir et al., 2014). aphanius darabensis is closely related to a. shirini from which it is distinguished by higher number of flank bars in males (9–18 in a. darabensis vs. 7-10 in a. shirini), small irregular vertical patches of brown color on the flank of females (vs. prominent dark brown blotches of round or irregular shape), and symmetrically shaped triangular to trapezoid otoliths with a rostrum clearly longer than the antirostrum (vs. quadrangular to trapezoid otoliths with short and equally sized rostrum and antirostrum). it is distinguished from the other aphanius species by the combination of four characters in both sexes: longer anal fin (15.5% sl in males, 12.1% sl in females), larger pelvic fin (8.1-12.5% sl in males, 7.04-10.3% sl in females), greater scale width (4.1-6.0% sl), and otolith characters. in addition, males can be distinguished by greater scale length (3.0-4.8% sl) and small caudal peduncle (0.9-1.5% minimum body depth); and females can be separated additionally by a short caudal fin length (12.7-19.2% sl) (esmaeili et al., 2014). tooth-carps of iran have been studied mainly 302 mansoori et al./ karyosystematics of aphanius darabensis based on their morphology but species identification on this basis is not always possible. the application of non-morphological methods such as cytogenetic studies may provide a complementary data source for more accurate and precise identification of fishes. fish karyosystematics is a branch of systematics that links systematics, cytology and genetics to find out structure and evolution of karyotypes and to reconstruct phylogenetic relationship of fish taxa (yu et al., 1987). a considerable attention has been paid to this type of studies in recent years (galetti jr et al., 2000; esmaeili and shiva, 2006; harrison et al., 2007). fish chromosome data have great importance in studies concerning evolutionary systematics, aquaculture, mutagenesis, genetic control and the rapid production of inbred lines (alsabti, 1991). the study of chromosomes in fishes has been expanding significantly due to the development of refined techniques of cell and tissue culture originally developed for mammals, but later adapted to the fish physiology (clem et al., 1961; booke, 1968; wolf and quimby, 1969; denton, 1973) and the development of less expensive in vivo direct methods (ozouf-costaz and foresti, 1992). due to the particular phylogenetic position of the ray-finned fishes among vertebrates, studies on their chromosomes have provided valuable information for understanding mechanisms of sex determination, evolution of sex chromosomes, distribution of the nucleolus organizers regions (nor), existence of supernumerary chromosomes and the role of polyploidy in evolution (pisano et al., 2007; nirchio et al., 2014). in this study we examined cytogenetical characteristics (i.e., diploid chromosome numbers, description of karyotypes, ideograms) of kol toothcarp, a. darabensis from the persian gulf basin in order to help future taxonomical and genetic studies. materials and methods twelve’s adult specimens of a. darabensis specimens were collected from the golabi spring figure 1. collection site of aphanius darabensis in the upper reaches of kol river drainage. 303 int. j. aquat. biol. (2016) 4(5): 301-307 located in the uppermost reaches of kol river tributary, darab city, fars, iran, 28°47′15˝ n 54°22′19˝ e, (fig. 1) using a dip net. the fishes were transported alive to the laboratory, and kept in a well-aerated aquarium at 20-25°c before analysis. for karyological studies the modified method of uwa (1986) was used. vinblastine solution was prepared with 0.005 g in 20 ml of physiological serum. the fish were injected intraperitoneally with 0.02 ml of vinblastine per gram of body weight using an insulin syringe, and then were put back in the aquarium for 3-4 hours. the gill filaments and kidneys of those specimens were then removed and placed in hypotonic 0.36% kci solution for 45 min at room temperature. thereafter, the solutions were centrifuged for 10 min at 1000 rpm, adding 2-3 drops of fresh and cold carnoy's fixative (1:3, acetic acid: methanol) before centrifugation. the supernatants were then discarded and 5 ml of fresh and cold fixative was added to the sediments, which were mixed thoroughly and then left for 1 hour. the fixation and centrifugation were repeated twice. the suspensions were then trickled onto cold slides. these slides were stained with 20% giemsa for 20 min. chromosomes were observed, selected and photographed by nikon light microscope with a camera mounted on it. karyotypes were prepared by arranging chromosomes in pairs by size and shape. for each chromosome, the average lengths of the short and long arms and arm ratio (the ratio of the long arm length to the short arm length of chromosomes) were calculated and then the chromosomes were classified according to the criteria given by levan et al. (1964). fundamental number (fn) was expressed as twice the number of atelocentric chromosomes plus the number of telocentric chromosomes. the ideogram was prepared in harvard graphics 2.0 software. results metaphase spread of this species is given in figure 2. the diploid chromosome number was 2n=48 (fig. 3). the quantitative data of the different measurements used to classify chromosomes and the ch. no. la sa tl ar ct 1 2.99 0.91 3.91 3.25 st 2 2.98 0.82 3.81 3.62 st 3 2.96 0.72 3.69 4.08 st 4 2.98 0.64 3.63 4.60 st 5 2.83 0.72 3.55 3.89 st 6 2.76 0.75 3.52 3.67 st 7 2.78 0.69 3.48 4.01 st 8 2.67 0.77 3.45 3.42 st 9 2.61 0.74 3.35 3.53 st 10 2.66 0.67 3.34 3.93 st 11 2.51 0.80 3.32 3.13 st 12 2.62 0.65 3.28 3.98 st 13 2.58 0.64 3.23 3.97 st 14 2.45 0.73 3.18 3.33 st 15 2.47 0.68 3.16 3.58 st 16 2.29 0.81 3.10 2.82 sm 17 2.28 0.79 3.07 2.87 sm 18 2.33 0.71 3.04 3.28 st 19 2.28 0.69 2.97 3.29 st 20 2.27 0.57 2.85 3.95 st 21 2.18 0.62 2.80 3.46 st 22 1.96 0.71 2.67 2.76 sm 23 1.85 0.65 2.51 2.83 sm 24 1.65 0.80 2.45 2.05 sm table 1. chromosome measurements (in µm) and classification of aphanius darabensis chromosomes (ch. no.: chromosome number; la: long arm; sa: short arm; tl: total length; ar: arm ratio; ct: chromosome type; sm: submetacentric; st: subtelocentric). 304 mansoori et al./ karyosystematics of aphanius darabensis ideogram are given in table 1 and figure 4, respectively. the karyotype consisted of five submetacentric and 19 subtelocentric pairs of chromosomes (5sm+19st). the chromosome arm number (fn) was 58. discussion according to our observations, the diploid chromosome number of a. darabensis (2n=48) is in confirmation with a. sophiae, a. farsicus, a. asquamatus, a. dispar, a. fasciatus, a. iberus and a. mento. hence, it can be concluded that the chromosome number in this genus is conserved. the number of chromosomes in this tooth-carp is also similar to that of other species of cyprinodontidae such as cyprinodon alvarezi, c. atrorus and c. beltrani (stevenson, 1981). in the order cyrinodontiformes, the most common fish species which have so far been cytologically investigated, such as gambusia affinis, g. holbrooki, g. gaigei, g. nobilis, girardinus metallicus, poecilia vivipara (poecillidae), fundulus diaphanus (fundulidae), figure 2. giemsa stained chromosome spread of aphanius darabensis. figure 3. giemsa stained karyotype of aphanius darabensis. figure 4. haploid ideogram of aphanius darabensis. 305 int. j. aquat. biol. (2016) 4(5): 301-307 allotoca maculata, goodea luitpoldi, g. atripinnis, g. gracilis, hubbsina turneri, ilyodon furcidens, i. lennoni, skiffia francesae, s. bilineata, xenoophorus captivus, xenotaenia resolanae, xenotoca eiseni, x. melanosoma, x. variata (goodeidae), have the diploid chromosome number of 2n=48 (arai, 2011). yet in a few species of the order such as aphyosemion bivittatum, a. bualanum, a. calliurum, fundulopanchax sjostedti, f. mirabilis (aplocheilidae); allotoca dugesi, allodontichthys hubbsi and ameca splendens (goodeidae), the diploid chromosome number is reported to vary from 2n=26 to 2n=42 (arai, 2011). it can be noted that the diploid number (2n=48) is modal in cyprinodont fish. in interpretation of karyotypic evolution it is often assumed that the primitive fish karyotype consists of 48 rods from which the karyotypes of all existing fish forms have been derived (khuda-bukhsh et al., 1986) but the issue seems yet to be resolved. the discovery of 48 rather large acrocentric chromosomes in the pacific hagfish, eptatretus stoutii, belonging to the order myxiniformes (taylor, 1967; vasil'yev, 1980) and the occurrence of 48 rods in the majority of fishes studied prior to 1967 led to the idea that the primitive karyotype of ancestral vertebrate freshly evolved from chordate might consist of 48 rods (khuda-bukhsh et al., 1986). therefore, most of the subsequent workers assumed the karyotypic evolution in different groups of fishes based on this basic assumption of 48 rods as the primitive number (khuda-bukhsh et al., 1986). but the discovery of 2n=24 rods in two species of freshwater eels (kitada and tagawa, 1973; rishi and haobam, 1984), 2n=36 rods in two species of myxine, low diploid numbers ranging between 14 and 42 in a large number of fish families showing fn less than 36 in some cases (khudabukhsh et al., 1986) would possibly call for a more cautious prediction on the primitive karyotype of fish. according to nirchio et al. (2014) in freshwater fishes both the average number of chromosomes and the fn are higher than in marine fishes, and a general higher degree of cytogenetic diversification and karyotype variation is observed, compared to a more conserved cytogenetic pattern in marine fishes. few decades ago the difference between karyotypes of freshwater and marine fishes was already observed and considered related to a more stable environment at sea as compared to inland waters, with some exceptions (nikolsky, 1976; nirchio et al., 2014). in the present study, no cytological evidence was found for sex chromosome dimorphism which agrees with reports on many fish species such as serranidae and mugilidae (aguilar, 1997; rossi et al., 1997). the karyotype formula of this tooth-carp was consisted of 5 submetacentric and 19 subtelocentric pairs of chromosomes (5sm+19st) and the chromosome arm number was 58. chromosome formula of 16sm+32st was reported for a. dispar and a. farsicus; 14sm+34st for a. ginaonis; 12sm+34st in a. isfahanensis and 8sm+40st for a. sophiae and a. vladykovi. the arm number of fn=32 was reported for a. dispar and a. farsicus and fn=28 for a. sophiae and a. vladykovi. the arm number in a. ginaonis and a. isfahanensis were reported to be 31 and 30, respectively (esmaeili et al., 2007; esmaeili et al., 2008a ; esmaeili et al., 2008b ; esmaeili et al., 2009) (table 2). though chromosome numbers of aphanius species are conserved despite of different geographical locations, the fundamental arm numbers are different. these differences within aphanius species of different geographical locations, suggest that structural rearrangement in chromosome complements, as a consequence changes in chromosome morphology without change in chromosome number. this divergence may be attributed to differences in the karyotype macrostructure, reflecting a real geographical variation common to widespread species or may be the result of differences in the scoring of submetacentric or metacentric chromosomes as different degrees of chromosome condensation, leads to differences in chromosome classification. based on nirchio et al. (2002), species with high arm number would be more recently appeared in 306 mansoori et al./ karyosystematics of aphanius darabensis evolutionary history of the lineage. in other word, low fn should be a plesiomorphy and high fn might be considered as apomorphy which suggested to be assessed for aphanius species using molecular data set. the data presented contributes with first knowledge on the karyotypes of a. darabensis. camparing to pervoius reported diploid chromosome number for other species of the genus, it can be concluded that the chromosome number in this genus is conserved despite variation in fundamental arm numbers. acknowledgements the authors give special thanks to a. marashi and g. sayyadzadeh for their help during field and labratoary works. the research work was funded by shiraz university and was approved by ethics committee of biology department (ecbd-su 9331011). references aguilar c. 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(2016) 4(5): 301-307 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی (داردندان کپورماهيان: عالي استخواني ماهيان) aphanius darabensis کل، گورماهي کاريوسيستماتيک ، **اسماعيلي حميدرضا غالمحسيني، علي ابراهيمي، مهرگان منصوري، اعظم .ايران شیراز، شیراز، دانشگاه علوم، دانشکده شناسی،زيست بخش مولکولی، سیستماتیک و شناسیماهی تحقیقاتی آزمايشگاه چکيده: ,aphanius darabensis esmaeili, teimori ايران، بومزاد داردندان کپور يک سیتولوژيکی و کاريولوژيکی هایويژگی بار اولین برای gholami & reichenbacher, 2014 کلیه و آبشش پوششی هایسلول از آمده دست به متافازی کروموزومی هایگسترش بررسی وسیله به جفت 19 و متاسنتريک ساب کروموزوم جفت پنج شامل آن کاريوتايپ و 48n=2 گونه اين ديپلوئید کروموزومی عدد. گرفت قرار مطالعه مورد نظر از جنسی هایکروموزوم. استfn =58 هاکروموزوم بازوی تعداد. باشدمی( sm19+st5) کاريوتايپی فرمول با تلوسنتريک ساب کروموزوم پیشنهاد توانمی داردندان کپور هایگونه ديگر از پیشین کاريولوژيکی هایگزارش و کنونی مطالعه اساس بر. است تشخیص قابلغیر سیتولوژيکی مطالعات در ایپايه اطالعات عنوان به توانندمی نتايج اين. باشدمی داردندان کپورماهیان بین در معمول ديپلوئید کروموزومی عدد ،48n=2 که کرد .شوند استفاده حفاظت و مديريت هایبرنامه جمعیتی، .ايدئوگرام سیتولوژيکی، آنالیز کروموزوم، دار،دندان شکالن کپورماهی :کلمات کليدي int. j. aquat. biol. (2021) 9(3): 148-158 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article reproduction and embryonic development in the african freshwater prawn macrobrachium macrobrachion (herklots, 1851) guillaume koussovi *1,2, dogbè clément adjahouinou1, farokh niass2, papa madiallacké diédhiou2, clément agossou bonou3, elie montchowui1 1school of aquaculture, national university of agriculture, bp 55, porto novo, republic of benin. 2complex systems modeling and biological & agronomic sciences laboratory (labam), gaston berger university, bp 234, saint-louis, senegal. 3applied-biology research laboratory, abomey-calavi polytechnic school, university of abomey-calavi, bp 2009, cotonou, republic of benin s article history: received 24 december 2020 accepted 19 february 2021 available online 2 5 june 2021 keywords: embryonic development larvae crustacean aquaculture food protein abstract: the current study aimed to determine parameters and conditions for successful reproduction of macrobrachium macrobrachion (herklots, 1851) in a controlled medium and describing its embryonic development. a total of 122 adult specimens were collected from the delta of ouémé river and stored in polyethylene tanks with 1:2 male-female sex ratio. this broodstock was fed on pelleted food (biomar efico) once a day. for the embryogeny monitoring, eggs were sampled each hour through the first two days after spawning and then every 2 hours till hatching. spawning happened at a mean temperature, dissolved oxygen concentration and ph of 27.80±0.56°c, 5.83±0.45 mg/l and 7.41±0.34, respectively. the eggs incubation meantime was 12±1 days with nine main embryonic development stages. hatching lasted on the average 21.00±1.94 h and led to larvae with a mean size of 2.30±0.90 mm. mean fecundity was 13062.4±5489.93 eggs and 14715.2±6108 eggs, respectively for the first and second seasons with a highly significant difference between them. the best hatching rates were obtained with salinities equal to 2 and 4‰. the results constitute the first database for larval breeding of m. macrobrachion species. introduction in benin, the total production of freshwater (palaemonidae) and marine (penaeidae) prawns is estimated at 4048.108 tons in 2018 (dph, 2019). freshwater prawns of the palaemonidae family distributed throughout the tropical and subtropical area (holthuis, 1980). there are more than 220 species of freshwater prawns in the genus macrobrachium (new, 2002). macrobrachium macrobrachion is an important fishery resource along the west coast of africa, from senegal (latitude 20°n) to angola (latitude 16°s) (holthuis, 1980). it is widely distributed in waterbodies and streams and, especially in the catchments of ouémé and mono rivers (kouton, 2004; agadjihouèdé, 2006; adite et al., 2013). macrobrachium macrobrachion and m. vollenhoveni are the most valued and their demand is high for local consumption as well as exportation to european union and other western africa’s countries (nigeria, *correspondence: guillaume koussovi doi: https://doi.org/10.22034/ijab.v9i3.1079 e-mail: guillaume.koussovi@yahoo.fr togo, ghana and ivory coast) (houngbo et al., 2015; ollabodé, 2019). therefore, m. macrobrachion is subjected to intensive fishing by artisanal fishermen especially during reproduction periods (koussovi et al., 2019) to satisfy the increasing demand. for example, in southern benin, these species are caught by up to 75 and 83% of fishermen, respectively (ollabodé, 2019). in this context, it is very important to develop some breeding techniques of m. macrobrachion in a controlled area to satisfy human consumption. that will contribute to the development of aquaculture and consequently to the preservation of the species in its natural environment (poncin and phillipart, 2002; rakaj et al., 2019). on the other hand, the development and mastery of the breeding techniques in african freshwater prawn species such as m. macrobrachion are also important since none of them is involved in commercial breeding of 149 int. j. aquat. biol. (2021) 9(3): 148-158 crustacean species worldwide despite its rapid development with a global production reaching 7.9 million tons in 2016 (fao, 2018). moreover, given m. macrobrachion is a native species, and its production in aquaculture may provide good yields when grown in their place of origin with no effect on habitat conservation (yamasaki-granados et al., 2013). successful breeding of prawn species depends on the mastery of the reproduction process and the availability of its juveniles for a continuous supply of production farms (vargas-ceballos et al., 2018). the mastery of artificially induced reproduction of m. macrobrachion constitutes the first step of its breeding process. in fact, this is a key step for improving reproduction yield and juveniles production of fish and crustacean in aquaculture (philippart, 1995; montchowui et al., 2011; rakaj et al., 2019). but, no basic knowledge exists on how the reproductive performance and embryonic development of m. macrobrachion respond to hatchery conditions. it is therefore important to work on these beginning stages of the development cycle of m. macrobrachion to understand the different phases of their process. also, such knowledge will be helpful to make easy the management of its broodstock and embryos during the breeding process. these pieces of knowledge may provide an insight into the process of changes, particularly of salinity, needed during these early stages of development. this is the first time such detail embryonic study with all stages, is done on the species. thus, the current study aims to (1) determine the reproduction parameters (mating time, spawning and fecundity, incubation time, hatching rate and hatching salinity) to evaluate the reproduction performances of m. macrobrachion in experimental conditions and (2) describe its embryonic development. materials and methods broodstock management and reproduction conditions: the broodstock (both sexes) of m. macrobrachion were collected from the ouémé river delta (6°30' and 10°n, 0°52' and 3°05'e) among specimens caught by artisanal fishermen using fishtrap during two successive reproduction seasons (july 2018 and july 2019). mean total length of the collected specimens was 8.27±0.41 cm for females and 7.98±0.52 cm for males. the sampled prawns were transported in plastic buckets (15 l) in the evening or early in the morning and stored in the hatchery at the school of aquaculture of the national university of agriculture (porto-novo, benin). before the trial, the broodstock was acclimated and stored in 1 m3 polyethylene tanks at a density of 60 individuals in 600 l tap water prior chloride-freed and filtered. a 1:2 male-female sex ratio was applied (koussovi et al., 2019). each tank was provided with two bubbles blowers connected to an aerator (resun, 1100 w; 1800 l/min) for permanent water oxygenation. the refuge (hiding-place) was provided to the prawns using punched pvc pipe (10 cm diameter and 30 cm length) put at the bottom of tanks to favour spawning. photoperiod was settled to 12h light and 12h darkness by the mean of akt fluorescent lamps with 40 w power in the hatchery. prawns were fed on pelleted food biomar efico (53% crude proteins, 8% lipids) once a day. remaining food, dejections and other wastes were daily removed by siphoning. the water temperature and ph were monitored using a multi-parameter (hanna, hi 99130), while the dissolved oxygen concentration was assessed with digital oximeter lutron version do-5509. the total length (from rostrum till the end of telson) and the weight of female specimens were taken using a sliding calliper draper with 0.05 mm precision and an electronic scale (kern 440-33n) with 0.01 g precision, respectively. determination of reproduction parameters in captivity: reproduction parameters of m. macrobrachion in captivity were determined for two reproduction seasons. during the trial, control fishing was carried out every 12 hours. ovigerous females were separated and put in a small floating cage (18x15x10 cm; 2 mm mesh size) to prevent cannibalism and monitor eggs development. the date and time of the spawning were recorded in each 150 koussovi et al./ reproduction and embryonic development of macrobrachium macrobrachion ovigerous female. floating cages containing ovigerous females were put in other tank (1 m3) filled with 600l water with continuous aeration till prehatching stage (fig. 1). each of these females was then transferred at hatching to a 30l bowl containing 10l of water to collect whole larvae and unhatched eggs. parameters such as incubation and hatching times, fecundity and hatching rate were determined in each ovigerous female. the eggs incubation time was noticed as the time interval between fertilization and hatching and hatching time was calculated as the time interval between the beginning and the end of this process (willführ-nast et al., 1993). absolute fecundity (af) is total number of the laid eggs by the female (willführ-nast et al., 1993; nhan, 2009). hatching rate (hr) was calculated as following: hr = (number of hatched eggs / non-hatched eggs) × 100 (nhan, 2009; vargas-ceballos et al., 2018). besides, to determine the optimal salinity for better eggs hatching in captivity, ovigerous females that eggs are near to hatch were selected and transferred to different bowls containing water with different salinity level. thus, six salinity concentrations (0, 2, 4, 6, 8 and 10‰) were tested in triplicate as previously documented for the macrobrachium species (bauer and delahoussaye, 2008; anger, 2013) especially in m. macrobrachion (koussovi et al., 2019). these salinity levels were obtained by mixing marine water (prior filtered by 10 μm mesh plankton net) with tap water. salinity was measured by portable atc refractometer and readjusted in need case. monitoring of the embryonic development: for the description of the different embryonic development stages in m. macrobrachion, 21 ovigerous females were monitored in the same above-mentioned conditions. the first sample was the freshly spawned eggs. then, eggs were hourly sampled during the first two days after spawning and every two hours during the following days till hatching. a cluster of eggs was taken from each female put in a 20 ml plastic recipient containing 5 ml of water. the eggs from the taken cluster were then separated using a sterile pincer, counted and observed under stereo zoom microscope (kern, ozm 554) at 45× magnification to describe their morphological characteristics and identify their development stage. microscopic observations were made on about 40 eggs for each sampling and some photos were taken with a digital photo device sony type ilce-5100 24 megapixels at 45× magnification. morphological features of embryos were described according to müller et al. (2003), garcía-guerrero and hendrickx (2009) and habashy et al. (2012). in addition, the different embryonic development stages were illustrated by drawings realized using adobe photoshop cs6 and adobe illustrator cc2017. the length (l) and size (h) of the observed eggs were immediately measured to evaluate their volume and follow up the progression from a development stage to another. these measurements were carried out on 30 eggs randomly chosen during each embryonic development stage. for that, eggs were put in petri dish set on millimetre paper and projected on the computer by mean of a "digital micro capture microscope" vetus model esd-50 (koussovi et al., 2019). then, the amcap version 4.9 software was used for images capture from which the direct measurement of eggs length and height were made by the "camera measure" software version 1.0. eggs volume at each stage was calculated from the formula v=πlh2/6 (odinetz-collart and rabelo, 1996; vadrucci et al., 2007) with l: egg length (long figure 1. stocking device of ovigerous females of macrobrachium macrobrachion separated for embryonic development monitoring. 151 int. j. aquat. biol. (2021) 9(3): 148-158 axis) in mm; h: egg height (short axis) in mm; π = 3.14 and v: egg volume in mm3. data analysis: the effect of salinity on hatching and survival rate, eggs volume at each development stage and fecundity were compared among females and seasons using a one-way analysis of variance (anova). the tukey post-hoc test was used to appreciate differences for each parameter in case of significance (p<0.05) (zar, 1999; vargas-ceballos et al., 2018). linear regression was carried out to determine the relationship between fecundity and hatching times. statistical analyses were realized using r software (version 1.1.4456). results fecundity and eggs characteristics: the mean total length and weight of females collected during the first season were 8.05±0.67 cm and 14.31±3.09 g, respectively while during the second season they were 8.75±0.48 cm and 16.10±1.00 g, respectively. therefore, the mean length (p<0.001) and weight (p<0.0001) of females from the second survey were quite significantly higher than those of the first survey. in hatchery conditions (27.8±0.56°c; 5.83±0.45 mg/l and 7.41±0.34 respectively for temperature, dissolved oxygen and ph), a total of 76 females (33 for the first survey and 43 for the second one) of m. macrobrachion spawned and incubated eggs in their pleopods till hatching. the mean incubation time was 12±1 days without no significant difference between seasons (p>0.05). hatching was started at night and lasted on average 21.46±1.94 h for both of seasons with significant variation from a female to another (p<0.05). mean absolute fecundity was 13062.4±5489.93 eggs and 14715.2±6108 eggs, respectively for the first and second seasons with a highly significant difference between them (p<0.001). eggs coming from each female underwent synchronic development and hatched a few minutes after the end of the embryonic process. concerning the colour of the eggs, two noticeable changes were observed during the incubation period similarly in both of study seasons. during spawning, the fertilized eggs were dark olive coloured and then remained changeless for seven days (fig. 2a). from the eighth day after spawning till hatching, eggs colour changed to dark grey (fig. 2b). hatching rates: hatching rate varied significantly (p<0.05) with salinity (fig. 3). thus, females incubated in salinity 4‰ presented the highest hatching rate (82%). nonetheless, this hatching rate did not significantly differ (p>0.05) from that of 2‰ salinity medium (71%). in return, the lowest hatching rates (19, 16.9 and 16%) were obtained in 8, 6 and 10‰ salinity, respectively with no significant difference (p>0.05). the medium with 0‰ salinity led figure 2. eggs colour change in macrobrachium macrobrachion during embryogenic: (a) 7 first days after spawning; (b) from the 8th day after spawning till hatching. figure 3. eggs hatching rate in macrobrachium macrobrachion in relation to salinity levels. letters show significant differences among treatments (p<0.05). 152 koussovi et al./ reproduction and embryonic development of macrobrachium macrobrachion to a hatching rate (31%) significantly different from others. embryonic development in m. macrobrachion: eggs volume varied significantly from 0.002±0.001 to 0.081±0.031 mm3 (p<0.05) throughout all the incubation period (fig. 4). the embryonic development of m. macrobrachion is divided into nine different stages characterized by important morphological changes (figs. 5, 6). stage i: fertilization (00-03 hours): starting from eggs fertilization, lasted about 3 hours and ended just before the first cell division. fertilized eggs were almost spherical in shape and include mainly a granulous mass uniform dark olive coloured surrounded with a transparent chorion (figs. 5a, 6a). stage ii: cleavage (03-07 hours): several cleavage furrows appear in the egg mass, pointing up the formation of the first embryonic cells. furthermore, a translucid area (germinal disc) set up at one pole of the egg shrinking slightly the egg’s inside mass (figs. 5b, 6b). these changes pointed out the beginning of embryonic development with a slight increase in eggs volume. stage iii: blastula (07-27 hours): the translucid area widened progressively without noteworthy morphological changes in the eggs (figs. 5c, d, 6c, d). therefore, two parts were noticed inside the eggs namely a light region (translucid area) representing the abdominal part of the developing embryo and a dark olive coloured part corresponding to its cephalic part. stage iv: gastrula (27-126 hours): 27 hours after the fertilization, the light region still increased by contracting the internal mass of the egg mostly in the peripheric part (figs. 5e, f, 6e, f). this enabled a perfect differentiation of the abdominal region with the appearance of some abdominal segments and the cephalic region taking a «v» form (figs. 5g, 6g). this stage ended 5 days after fertilization. stage v: nauplius (126-155 hours): a broad black spot appeared in the internal part of the embryo’s cephalic region which still decreased because of the abdominal part extension (figs. 5h, 6h). at 136 hours after fertilization, this black spot, representing a sketch of the embryo’s ocular region became more clear (figs. 5i, 6i). furthermore, some vitellin reserve vesicles appeared in the peripheric part of the cephalic region. stage vi: post-nauplius with a heartbeat (155-179 hours): the optic region, previously set up, enlarged with a more marked pigmentation (figs. 5j, 6j). at the abdominal region, the caudal papilla clearly appeared with a rudimentary telson and folded in the direction of the optic region. furthermore, heartbeats started with 89±18 beats/min on the average. at that time, the remaining content of the egg including mainly the vitellin reserve narrowed because of the development of embryonic structures and took a dark grey colour. stage vii: post-nauplius with eyes individualization (179-216 hours): eyes individualized from the optic region, enlarged, took an oval form and parted from the cephalic region but still stuck on it at their basis (figs. 5k, l, 6k, l). the embryo rolled itself up and took a marked «c» form due to the complete making up of figure 4. progression of eggs volume during incubation period in macrobrachium macrobrachion. letters show significant differences (p<0.05). 153 int. j. aquat. biol. (2021) 9(3): 148-158 the caudal papilla with a telson quite close to maxillaries. the heart beats increased reaching in average 97±12 beats/min. stage viii: final post-nauplius with eye condensation (216-264 hours): eyes diameter increased with an intensification of their colour. above each eye, some eyelashes appeared (figs. 5m, 6m). maxillipeds were well-developed, segmented and overlapped the abdomen. the embryo occupied whole egg space. the vitellin vesicles intensified and were more visible in the cephalic region of the embryo. besides, heartbeats frequency increased and reached 127±21 beats/min. stage ix: pre-hatching (264-288 hours): the dark grey part located in the cephalothorax (vitellin reserve) was much more reduced (figs. 5n, 6n). the heart was completely distinct from the vitellin mass and its contractions were more active than during the previous stages. moreover, some irregular movements such as abdomen contractions were noticed. the uropod (an abdomen part) was clearly segmented with its last segment provided with bristles. the edge of the telson overlapped the rostrum that spread over the head. embryonic development was completed at this figure 5. embryonic development stages in the freshwater prawn macrobrachium macrobrachion (45× magnification). scale bar = 0.1 mm. a: fertilization; b: cleavage; c: blastula; d: blastula; e: gastrula, making up of blastopores; f: gastrula with increase light region; g: gastrula with distinction of the abdomen; h: nauplius with black spot; i: nauplius with dark black spot; j: post-nauplius with heart beats; k: post-nauplius with eyes pigmentation; l: post-nauplius with oval eyes; m: post-nauplius with eyes condensation; n: pre-hatching; o: freshly hatched larva. ab: abdomen; abs: abdominal segment; an: antennae; ar: abdominal region; bey: base of the eye; bl: blastomeres; cf: cleavage sillon; ch: chorion; dey: developed eye; ey: eye; gd: germinal disc; hg: heart growth; hr: head region; ht: heart; lar: larva; ld: lipid droplet; ol: optical lobe; op: ocular pigment; or: ocular region; per: pereiopods; pes: primitive embryonic structure; ps: perivitellin space; sh: shell; str: streaks; ts: telson; vr: vitellin reserve; ym: yolk mass. 154 koussovi et al./ reproduction and embryonic development of macrobrachium macrobrachion stage (12 days after fertilization) leading to the hatching of the egg releasing a new larva (zoe i) (figs. 5o, 6o). discussions the results of the present study constitute the first data on the reproductive parameters and embryonic development of the freshwater prawn m. macrobrachion in captivity. a high fecundity proportional to the size of the female specimen is noticed in m. macrobrachion. this fecundity variation can be tied to the age and the reproduction capacity of females (graziani et al., 1993). however, the fecundity recorded in the females of m. macrobrachion was lower than that of most species belonging to macrobrachium genus (willführ-nast et al., 1993; makombu et al., 2014). this is probably due to the small size of m. macrobrachion allowing less space in their cephalothoracic cavity to enable the ovocysts growth during ovarian maturation (koussovi et al., 2019). besides, the narrowness of the abdomen of m. macrobrachion’s specimens does not allow the development of a large mass of egg (koussovi et al., 2019). the eggs incubation mean time in m. macrobrachion (12±1 days at 27.8°c) was shorter than that recorded in other macrobrachium species such as m. vollenhoveni (13 to 14 days at 28.15 and 30°c) (willführ-nast et al., 1993; sintondji et al., 2020), m. rosenbergii (20 days at 28.5°c) (habashy et al., 2012), m. olfersi (14 days at 26°c) (müller et al., 2003) and m. lar (29 days at 28°c) (lal, 2012). this is due to the small size of their eggs compared to figure 3. drawings of the embryonic development stages of macrobrachium macrobrachion (for abbreviations see fig. 5). 155 int. j. aquat. biol. (2021) 9(3): 148-158 those of other macrobrachium species, for instance, m. lar (0.072 to 0.133 mm3) (lal, 2012), m. mammillodactylus (0.044 to 0.099 mm3) (cuvinaralar, 2014) and m. vollenhoveni (0.0455±0.003 to 0.0857±0.002 mm3) (sintondji et al., 2020), given the embryonic development duration depends among others on eggs height (müller et al., 2004). furthermore, this difference in the eggs incubation time among macrobrachium species can also be due to the temperature of the incubation medium (habashy et al., 2012; cuvin-aralar, 2014). during the embryonic process, the eggs increased in size leading to their volume increasing as embryos developing. this increasing of eggs volume could be tied to the osmotic absorption of water to ensure cells’ mobility (kobayashi and matsuura, 1995), the structural organization (müller et al., 2003) and the biochemical composition of eggs (cuvin-aralar, 2014). as for hatching rates, the best ones were obtained in low-salinity media with 2 and 4‰. these salinities seem to be suitable for embryogeny process in m. macrobrachion. this justifies the migration of adult specimens of this species from rivers to estuaries or mouths (koussovi et al., 2019) as do others macrobrachium species during the reproduction period (anger, 2013; bauer, 2013). in return, the lowest hatching rates were noticed in media with pure freshwater (0‰) and at salinities higher than 4‰. this is because these ranges of salinity can stop embryogenic process by causing some physiological damages (ituarte et al., 2005) leading to their death and/or abortion (fukuda et al., 2017). concerning embryogeny, the results revealed the embryonic development in m. macrobrachion includes nine main stages almost based on the same principles as most of macrobrachium species (müller et al., 2003; müller et al., 2004; garcía-guerrero and hendrickx, 2009; habashy et al., 2012; sintondji et al., 2020). first of all, it is important to notice that eggs of m. macrobrachion are filled with yolk which could represent energy source and necessary food for embryo development (ma et al., 2019). the embryogenesis started with external fertilization eggs (stages i), previously laid and stocked on females’ pleopods. three to twenty-seven hours after fertilization, some furrows started to be established from the outside of eggs. the depression noticed at stage ii to iii highlighted the differentiation of blastomeres. however, this seemed to represent a holoblastic cleavage mode, in which blastomeres are separated by furrows. but these early cleavage furrows are superficially noticed as in most decapod crustaceans (anderson, 1973). during the embryonic development of m. macrobrachion, the first four embryonic development stages (stage i to iv) leading to gastrula, lasted approximately five days whereas the last five ones (stages v to ix) focusing on the nauplius development and leading to the zoe, lasted up to seven days. this distribution of time is expected because the more complex structures are mostly formed during the stages after gastrulation (chen et al., 2012; ma et al., 2019). the development of the nauplius, happening from stage v to stage viii, was much more focused on the abdominal segments formation and the eyes organization. at stage vi, the egg’s content decreased and this can be due to metabolic processes using lipidic and proteinic reserves for the formation of some embryonic structures, including especially the retina differentiation leading to a marked pigmentation of eyes at this stage (müller et al., 2004; cuvin-aralar, 2014). the progressive transformation of the embryo at this level of the embryogeny (stages vi) in m. macrobrachion is similar to that happening in most of the decapods (müller et al., 2000; müller et al., 2004; manush et al., 2006). this stage ended by the embryonic heart contraction showing the embryo of m. macrobrachion acquired the heart regulation capacity. the heartbeats were more and more frequent during the following stages (vii, viii and ix) also characterized by an intensification of the motor activity of post-nauplius appendixes as well as a marked development of eyes. once the embryonic development completed, the egg hatched releasing a zoe similarly to the majority of decapods crustaceans (yamaguchi, 2001; pinheiro and hattori, 2002; lal, 2012; sintondji et al., 2020). 156 koussovi et al./ reproduction and embryonic development of macrobrachium macrobrachion conclusion in m. macrobrachion, the mating, fertilization and embryonic development are happened in freshwater whereas the eggs hatched in low-salinity water. the embryonic development involved nine stages similar to that happening in most of the species belonging to macrobrachium genus with nonetheless some specificities among others eggs colour changes. the salinities for a better hatching rate were 2 and 4‰. the present study reports the results of the first successful reproduction of m. macrobrachion in hatchery conditions. these results are therefore useful for further surveys intended to develop techniques for the breeding of m. macrobrachion in captivity. acknowledgements this work received financial support from the benin national fund for scientific research and technological innovation (fnrsit, grant no. 011/mesrs/fnrsit/ ac/sse/ sai/sa) and the international foundation for science (ifs, grant n° i2-a-6247-1). the authors thank these two bodies for their support, as they do the anonymous referees for their constructive review of this manuscript. ethics approval the research protocol was approved by the animal ethics committee of the school of aquaculture, national university of agriculture/porto-novo in benin. thus, data collection was carried out in accordance with the ethical standards of this institution, and guidelines for the care and use of animals were followed during this study. references adite a., abou y., sossoukpe e.m.h.a., gbaguidi g., fiogbe e.d. 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(2014) 2(1): 43-52 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article the reproductive biology of shirbot (barbus grypus heckel, 1843) in the maroon river, iran mahdi banaee*1, mehdi naderi2 1department of aquaculture, natural resource and environment faculty, behbahan khatam alanbia university of technology, iran. 2department of aquaculture, natural resource, urmia university, iran. article history: received 5 september 2013 accepted 25 december 2014 available online 2 5 february 2014 keywords: shirbot reproductive cycle maroon river gonad histology abstract: shirbot (barbus grypus) is one of the species in south and southwest of iran which is greatly favorable to residents of the region. unfavorable ecological conditions in habitat of this species and overfishing have led to the reduction of the population of shirbot. therefore, to restore the natural stock of this species, identifying its reproductive cycle associated with its habitat is of a great importance. in this study, the reproductive status of shirbot in the maroon river in khuzestan province was studied in six sampling steps during four seasons. also, morphological indicators, sex ratio, age of fish, gonadosomatic and hepatosomatic indices, histological changes in the testis and ovary of the fish were studied. the ratio of male fish to female was 2.35 to 1. the maximum value of gonadosomatic index (gsi) is among the specimens aged 3 to 5 years and in march and april. an increase of hepatosomatic index (his) during march may indicate the increased activity of liver during vitellogenesis and vitellogenin synthesis which is well verified by histological results of ovarian tissue. based on our findings we recommend that the maximum reproductive activity of shirbot in the maroon river starts around the end of march and continues to middle of july. introduction sustainable use of aquatic ecosystem resources depends on reproduction, and the availability of proper conditions for reproduction and growth of aquatic organisms. thus, monitoring aquatic ecosystems is of a great importance. one of the main aspects of monitoring aquatic ecosystems is studying the reproductive cycle of varied aquatic organisms especially fishes, so that fishing without a thorough comprehension of the fish's breeding and evaluation of their resources may lead to complete disappearance of a species (hosseinzadeh sahafi et al., 2001; khorashadizadeh et al., 2006). in other words, biology and ecology of different species of fish in aquatic ecosystems can play a significant role in developing a pattern for conserving and restoring indigenous and commercial fish stocks (gharaei et al., 2011; aliasghari and parafkandeh haghighi, * corresponding author: mahdi banaee e-mail address: banaee@bkatu.ac.ir 2013). therefore, due to recent issues in breeding and population rehabilitation of many indigenous and commercial species, researchers in the field of fisheries have studied varied biological aspects of fish especially reproductive characteristics of fish (ronnback et al., 2002; orlando et al., 2003; sinsneros et al., 2004; guerriero et al., 2005; guerriero, 2007). the studies on reproductive indices such as gonadosomatic index, morphology of gonads, histological changes of gonads in different seasons in yellowfin tuna, thunnus albacares, (oryan et al., 2004), javelin grunt, pomadasys kaakan (valinasab et al., 2007), klunzinger mullet, liza klunzingeri (valinasab et al., 2004a, b), abudefduf sexfasciatus (hosseinzadeh sahafi et al., 2002), leaping mullet, liza saliens, (yousefian et al., 2003; balik et al., 2011), orange-spotted grouper, epinephelus coioides, (abbasi et al., 2005), 44 banaee and naderi/ int. j. aquat. biol. (2014) 2(1): 43-52 barbus esocinus (eskandari et al., 2004), kutum, rutilus frisii kutum, (najar lashgari et al., 2007; aminian at al., 2008), silver sillago, sillago sihama, (hosseinzadeh sahafi et al., 2001) as a key method in determining the spawning season and studying the life cycle of the fish verifies this issue. therefore, studying the changes in levels of sex steroid hormones, histology of gonads, and determining the biological indicators of the final maturation of wild fish along with constant monitoring of ecological and environmental conditions of aquatic ecosystems of iran can help to have access to reproduction biotechnology and restoration of commercial and indigenous fish stock. shirbot (barbus grypus) is one of the species of the genus barbus and carps being usually found in the southern and west-southern river basins of iran. this species can easily adapt to environmental conditions. however prefers slow flowing waters at 22°c. although this species usually live in shallower waters than five meters depth, chooses very shallow waters with a gravel bed for spawning (abdoli, 2000). this species is favorable among indigenous residents of south western provinces of iran, especially khuzestan province. the maroon river is one of the habitats of this species. this river originates in the dameh mountain in dehdasht region in kohkilouyeh and boyer ahmad province and after passing through behbahan and omidieh, joins the jarahi river. the discharge of this river in highlands is over 119 m3 per second and reaches 20 m3 per second in estuary regions. temperature of this river in different seasons of the year varies from 15 to 30°c. this river has a depth of 1 to 15 m and serves as a good habitat for different species of fish including shirbot (barbus grypus), hemri (b. luteus), berzem (b. barbulus), common carp (cyprinus carpio), siahmahi (capoeta trutta), gel-cheragh (garra rufa), lotak (cyprinion tenuiradius), mesopotamian spiny eel (mastacembelus mastacembelus), shah kuli (chalcalburnus mossulensis), nazok (chondrostoma regium), arteshi catfish (glyptothorax silviae), and loach (nemacheilus sp.). recently, many efforts have been done on the reproduction and breeding of indigenous species especially on the barbus in the khuzestan province and other areas of iran. however, further research could be helpful in overcoming problems and achieving its reproduction and breeding techniques. as previously mentioned, studying the life cycle of these fish in nature and with regard to the climatic conditions of each region may be a key to this problem. therefore, this study aimed to investigate the life cycle of shirbot in the maroon river with regard to the climatic conditions of the behbahan district and hydrologic conditions of the maroon river. materials and methods the data collected from local fishermen in three stations were used for sampling from the maroon river. these stations were located at the lake behind the maroon dam (30°42´n and 50°22´e), kheirabad (30°21´n and 50°19´e), and shohada regulatory dam (30°39´n and 50°18´e). sampling was done at 6 stages and during summer, fall, winter and spring via a gill net. samples were freshly transferred to the laboratory and the species intended for the purpose of this study was separated after identifying the fish. then, using a digital scale, biometric board/scale, and calipers, the weight, morphometric characteristics of the fish including the total length, standard length, head length, snout length, and meristic characteristics such as number of whiskers, number of lateral line scales and scales up and down the lateral line were measured to assure the initial identification of the species. their age was determined using the scales on different parts of their body. in this method, the age of fish was estimated by counting the annuli on the scale (adeli, 2008). then, the specimens were autopsied and after sex identification, their gonad and liver was weighed using a digital scale to determine gsi and hsi. at this stage, fecundity of the fish which had reached sexual maturity was estimated based on the gravimetric method. for histological study of the gonads and determining different stages of sexual maturation, small pieces of the gonad from initial, 45 banaee and naderi/ int. j. aquat. biol. (2014) 2(1): 43-52 middle, and final parts were sampled and fixed into bouin's solution for 48 hours. after preparation, dehydration, and bleaching in alcohol and xylol solution, the tissues were embedded in paraffin and sectioned with a microtome. after preparing the slides, they were stained with hematoxylin and eosin stains. statistical analysis: data were analyzed using oneway analysis of variance (anova) by spss 15. normality of the data was checked using the kolmogorov-smirnov test. the results were shown in mean ±s.e. the correlation between different variables was determined using the pearson correlation method. results shirbot, barbus grypus, is cylindrical in appearance. this fish is dark olive which looks lighter at the abdomen area (fig. 1). shirbot has 4 whiskers, protruding plump lips and the captured fish had the average length of 36.61 ± 7.95 cm and were 1-6. the sex ratio in the captured fish was 70.15 male to 29.85 female fish during the experiment. however, in some cases, sex identification was not possible. there is a significant relationship (p<0.01) between the total length of the captured fish in different seasons with their sexual maturity and gonadosomatic index. the results indicate a significant relationship (p<0.01) between total weight and gonadosomatic index and between the age and sexual maturity. there was a significant relationship between changes in hepatosomatic index and that of gonadosomatic index in fish captured in different seasons (p<0.01) (table 1). based on what is presented at table 2, the male fish captured in different seasons are in a higher ratio compared with the female fish (2.35 to 1). the higher value of gonadosomatic index was found in fish aged 3-5. the significant increase in hepatosomatic index (his) in march may indicate increased activity of liver during vitellogenesis and vitellogenis synthesis. the maximum value of gonadosomatic index (gsi) in the captured fish was found in march and april (table 3). figure 2 illustrates the testis histological structure of shirbot. in this figure, most spermatogonia have pale vesicular nuclei and granular cytoplasm. these cells are about 5-10 micrometers. spermatogonia ii are normally smaller than spermatogonia i and are usually visible as a cell mass in testis tissue. spermatocytes usually have a relatively dense nucleus and average cytoplasm content. spermatocytes are about 4-6 micrometers. primary spermatocytes are usually bigger than secondary spermatocytes. a significant proportion of the cells in the testis of the fish were primary and secondary spermatocytes. spermatid cells have a dense nucleus, which is surrounded by a ribbon-like cytoplasm and eosinophilic cytoplasm. these are the smallest cells that are found in generative epithelium (with an approximate size of 2-3 micrometers) and during spermatogenesis, their cytoplasmic connection figure 1. shirbot, barbus grypus, (a); shirbot testis (b); and shirbot ovary (c). studying the gut content of these fish shows that they are omnivore, so that it is hard to accurately determine the food items these fish consume. however, these fish usually feed on plants. 46 banaee and naderi/ int. j. aquat. biol. (2014) 2(1): 43-52 gradually breaks. spermatozoa cells have a dark round nucleus and very little cytoplasm which make cytoplasm identification in these cells very hard. identifying the flagella/cilium-like tail was/is not possible in the tissue samples. these cells are about two micrometers and are found in the lumen of the testis. sertoli cells have an elongated and triangular nucleus. cytoplasm of these cells is not usually clear and it is hard to identify them. cytoplasmic arms of a sertoli cell contain spermatogenic cells and form spermatocysts. compared with germ cells, sertoli cells are less and usually found as single cells adjacent to testicular lobules walls. in some cases, sertoli hypertrophic cells are much like spermatogonia in appearance. interstitial cells (leyding cells) have a round or oval nucleus, dense and vacuolated cytoplasm. compared total length total weight age sex hsi gsi total weight 0.786** sig. 0.00 age 0.513** 0.450** sig. 0.00 0.00 sex -0.311** -0.362** -0.319** sig. 0.003 0.001 0.003 his -0.102 -0.164 -0.011 -0.030 sig. 0.345 0.129 0.992 0.782 gsi -0.477** -0.437** 0.048 0.104 0.230* sig. 0.00 0.00 0.656 0.336 0.032 maturity -0.280 -0.169 -0.433 0.061 0.045 -0.063 sig. 0.009 0.118 0.00 0.573 0.682 0.564 table 1. the correlation between different biological factors in the captured shirbot (barbus grypus) during sampling age sex total length (cm) total weight (g) hepatosomaticindex gonadosomaticindex male female unknown 1 0 0 2 29.80±1.06 200.00±4.05 1.15±0.05 0.75±0.01 2 12 4 2 31.37±4.04 390.22±80.47 0.54±0.23 3.23±1.64 3 36 6 2 31.90±3.28 374.76±125.28 0.68±0.29 4.55±3.01 4 48 26 0 38.18±7.35 509.38±296.22 0.98±0.91 4.45±2.43 5 26 8 0 35.76±3.53 458.41±261.19 1.09±0.98 5.99±2.42 6 0 8 0 58.38±5.75 1800.05±476.1 0.68±0.14 1.64±0.95 table 2. total length, hepatosomatic index, and gonadosomatic index of fish in male and female fish. sampling time frequency total length (cm) total weight (g) hepatic-somatic index gonodo-somatic index september 26 38.04±9.87 518.93±359.05 0.68±0.15 2.84±0.47 november 24 37.07±5.84 376.18±76.80 0.82±0.25 3.61±1.07 february 42 39.92±9.65 664.46±589.14 0.69±0.68 4.19±1.93 march 28 31.98±2.35 315.34±61.38 1.83±1.32* 7.56±3.18* april 18 32.88±4.23 521.05±366.46 1.06±0.50 7.58±2.65* july 36 36.32±7.46 572.72±304.39 0.46±0.12 2.78±1.48 table 3. total length, total weight, hepatosomatic and gonadosomatic index of fish according to the sampling time. 47 banaee and naderi/ int. j. aquat. biol. (2014) 2(1): 43-52 with germ cells, these cells are usually single or as small cell communities in interstitial space and testis lobules. however, in appearance, they are more like spermatocytes, with interstitial cells just in the space between lobules. spermatocysts are functional units of testis and are figure 2. histology of testes in shirbot (barbus grypus), different parts of testis including spermatogonia, spermatocytes i and ii, spermatid, spermatocyst, spermatozoa, intestinal cells and sertoli cells are illustrated in figure a and b. figure 3. histology of shirbot (barbus grypus) ovary, different parts of ovary tissue are presented in figures a to d including oogonia (og), oocytes at chromatin nucleus stage (cno), oocytes at pronucleus stage (pno), oocytes at cortical alveolar stage (cao), oocytes at early (evo) and late stages of vitellogenesis (lvo), final maturation of oocytes (mo), yolk granules (yg), cortical vesicles (cv), and nucleus (n) in oocytes. 48 banaee and naderi/ int. j. aquat. biol. (2014) 2(1): 43-52 composed of spermatogenic cellular communities including spermatogonia, spermatocytes and spermatids which are surrounded by sertoli cells' cytoplasm. in spermatocycts, cells look like a cell mass and the cells are connected to each other by cellular connections and remain like this up to the final maturation and the release of spermatozoa (fig. 2). figure 3 illustrates the histological structure of shirbot ovary. in this figure, the oogonia are the smallest oocyte cells. the main characteristics of these cells are a big nucleus, which is sometimes indistinguishable, and a little cytoplasm. at chromatin nucleus stage, oocytes look a bit bigger than oogonia. at this stage, the oogonia are surrounded by pre-follicular cells (which are probably granulose cells) and this is when follicular layers begin to develop. as oocytes growth, size and volume of cytoplasm increases, which looks more dense and granular compared with the oogonia. now, oocytes have a relatively big nucleus containing a big and single nucleolus. the size of the nucleolus (germinative vesicle) increases along with oocytes growth, and several nucleoli appear which are usually around the nucleus. therefore, at this stage the oocytes are called pronuclear stage. irregular dark spots can be seen in cytoplasm, although pronuclear oocytes may have clear or amphophilic vacuoles in their cytoplasm. these cells are abundantly found in ovary tissue of mature fish (fig. 3). oocytes are usually bigger at cortical alveolar stage compared with pronucleus oocytes. at this stage, cortical alveolar appear in ooplasm. there is no doubt that alveolar cortical is different from yolk and has no role in feeding the fetus/embryo. chorionic layers appear at this stage and pre-follicle cells are easily visible. at the early stage of vitellogenesis, oocytes are bigger than those in cortical alveolar stage. at this stage, the most striking features of oocytes are their spherical shape and eosinophilic characteristics, as well as the presence of granules or yolk globules in their cytoplasm. in some cases, accumulation of yolk in the central region of the cell, especially in slides stained with hematoxylin and eosin may be mistakenly seen as nucleus. at the late stages of vitellogenesis, yolk granules accumulate in oocytes and it increases the cell size. the increase of yolk in oocyte cells marginalizes the corticals alveolar content. gradually, the nucleus moves to the cell margin. at the final maturation, oocytes reach their maximum volume due to absorbing the yolk and water intake/dehydration. the nucleus migrates toward the micropyle and the cell margin and germinal vesicle breakdown (gvbd) occurs. the loss of nucleus does not help identifying the features of this stage of oocytes that much. anyway, in most big oocytes, the nucleus is indistinguishable. due to the fragile nature of cells at this stage, it is hard to make histological slides. therefore, in most cases it was hard to see cells at the final maturation (fig. 3). discussion shirbot is one of the main edible species in the maroon river which is of a great importance to local people, so that due to local fishermen's great interest to this species and over fishing, and also river pollution, and loss of habitats caused by drought in recent years, the population of this fish has decreased dramatically and these fish are facing the risk of extinction. therefore, obtaining information on the reproductive physiology and biology of this fish may help restoring fish stock, reproducing, and cultivating them in cultural ponds. the findings of this study suggest that female shirbots reach sexual maturity at the age 3-4 years old, while males reach sexual maturity at the age 2-3 years old. furthermore, these fish start spawning as the weather warms since the beginning of april up to late july. changes in gonadosomatic index during this period verify these results. in this study, the male fish had a higher ratio compared with the female ones among the captured fish. the sex ratio in the shirbot that captured approximately was 2 male to 1 female fish during the experiment. sex ratio of b. barbulus and b. esocinus was 1:1 to 1:4 (female to male), respectively (mortezavizadeh et al., 2010; 49 banaee and naderi/ int. j. aquat. biol. (2014) 2(1): 43-52 eskandary et al., 2001). there was a significant relationship between total length and age of the fish captured in different seasons and their sexual maturity. a significant correlation between the gonadosomatic index with length and weight of fish was observed. patterns in the monthly gsi fluctuations that shows similar pattern to the reproductive cycles are common among other barbus fish (eskandary et al., 2001; mortezavizadeh et al., 2010; ghafari and jamili, 2010). the significant relationship between the numerical changes in hepatosomatic index with those of gonadosomatic index in the fish captured in different seasons suggests a relationship between the liver activity in different stages of sexual maturation and the growth of gonads. this relationship is evident in female fish during vitellogenesis. according to histological studies, most fish captured in september and even november are at the second, and some are at the first, stage of sexual maturity. at this stage, testis and ovary look like a dark red semitransparent ribbon. the ovary is only recognizable under the microscope. these fish are usually at their second sexual cycle. at this stage of sampling, a few of the fish are experiencing their first sexual maturity; and in some cases it is hard to distinguish their gender. for instance, in some mature fish captured in november, it was hard to identify their gender by the gonad appearance due to absence of sexual differentiation. in these fish, primary germ cells were developing. the main characteristics of these cells are their relative big size, lower ratio of nucleus to cytoplasm, and the presence of one or two nucleolus in them. stromal tissue is a dense and filament connective tissue with many collagen fibers which are seen red in hematoxylin and eosin staining. moreover, in this group of fish and another group of immature fish in which sex identification was difficult, only primary oogonia and oocytes were detectable in female fish, and primary spermatocytes and spermatogonia. in other words, in a few cases, besides oogonia, pronuclear oocytes were detectable in the ovary of juvenile fish. in the ovary of fish which were at the first phase of maturity, more than 90% of oocytes were at previtellogenesis, and often at pronuclear and cortical alveolar stage. however, various oogonia were found among larger ovarian follicles in the sexual gland of mature fish. at this stage, only spermatogonia were detectable in the testis of male fish. most of the fish captured in november or even some of the fish captured in february were at the second stage of sexual maturity; at this stage, oocytes were visible as polygons and dispersed granular particles in cytoplasm. most primary oocytes are morphologically spherical, oval, or polygon. these cells have a fairly large nucleus which occupies a great part of ovule/ovum and cytoplasm surrounds it as a thick layer. at this point, oocytes are normally at the first or second phase of vitellogenesis. by cytoplasm development, the diameter of the oocyte increases too. follicular layers surrounding developing oocytes are easily detectable. now, not only the spermatogonia, but also the primary and secondary spermatocytes, and in some cases the spermatozoa are observable in the testis of male fish. at this stage, size of the ovary and the testis has increased and they are full of capillaries. the oocytes are easily detectable and sex identification is easy. in most fish captured in march and a few of those captured in february, the ovary was at the final stage of vitellogenesis; at this stage, the cells were round and cytoplasm was full of yolk granules. lipid particles looked like intercellular hollow cavities. primary and secondary spermatocytes and spermatids were seen in the testis of male fish. seconday spermatocytes and spermatids were abundantly found in the testis tissue. the testes were white, but the ovaries were still red. the increased rate of secondary oocytes was evident compared with the previous stage. due to vitellogenesis, the volume of oocytes is increasing. the numerical value of hepatosomatic index (his) reaches its peak. this pattern is similar to that reported in b. capito by shajiei et al. (2002). however, the testes of many fish captured in march, and april was completely white and contained some 50 banaee and naderi/ int. j. aquat. biol. (2014) 2(1): 43-52 seminal fluid. increased spermatids and the appearance of spermatozoa are the most notable histological changes of the testes. at this stage, vitellogenesis is done and a few of oocytes have reached their final maturation. oocytes are generally round and big. at this point, the ovary is at the fourth stage of sexual maturation. in the ovary of mature fish, oocytes are at the final phase of vitellogenesis and mature oocytes are easily detectable. follicules reach their biggest size, especially after water intake. after ovulation of the oocytes, the fish start to spawn. this is evident in some of the fish captured in april and most of those captured in july. since due to the fragile nature of oocytes, preparing histological samples was very difficult, some of the samples were useless. at this stage, sex cells in sexual glands had reached their final maturity and the oocytes and sperm liquid were easily secreted with a slight pressure. gonadosomatic index (gsi) was in its highest peak. in a few of fishes captured in september, the ovaries and testes have shrunk and are full of capillaries. in the ovary of these fish, only the remains of follicular layers, including theca and granulose cells are visible. these histological changes in the ovarian and testis can be typical in many species of freshwater fish (eagderi et al., 2006; eagderi et al., 2013). therefore, since estimating the sexual maturation time and the fish's spawning season is done by several authors with regard to histological changes of gonads in breeding and pre-breeding fish in various aquatic ecosystems and is proposed as a reliable method (eagderi et al., 2006; eagderi et al., 2013). our results showed that the spawning season of shirbot (b. grypus) in maroon river normally starts early in march and continues till the middle of july. spawning season of b. xanthopterus in april (eskandary, 1999), b. esocinus in may (eskandary et al., 2001), b. grypus and b. sharpeyi (nikpey, 1996), b. barbulus (mortezavizadeh et al., 2010) and b. pectoralis (ghafari and jamili, 2010) between march to april in karoon river were reported. therefore, capturing the broodstock for the purpose of artificial breeding of these fish by experts and applying restrictive laws of fishing during this period could help restoring the stock of these important fish. acknowledgements we greatly appreciate the efforts of mr. ebrahimpour, the expert at histological laboratory in veterinary faculty of tehran university, in making histological slides. reference abbasi f., oryan sh., matinfar a. 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(2017) 5(3): 201-207; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article morphological development and allometric growth patterns of acipenser persicus borodin, 1897 (actinopterygii, acipenseridae) during early development soheil eagderi, hadi poorbagher*,1fatemeh moshayedi, seyed vali hosseini department of fisheries, faculty of natural resources, university of tehran, karaj po box 4314, iran. article history: received 11 november 2016 accepted 3 may 2017 available online 2 5 june 2017 keywords: ontogeny morphogenesis, morphometrics caspian sea abstract: morphological development and allometric growth patterns of reared persian sturgeon, acipenser persicus, were studied from hatching to 50 days post-hatching (dph). the larvae were sampled, their left sides photographed and seven morphometric characters, including total length, head length, tail length, trunk length, snout length, caudal peduncle and predorsal length were measured. allometric growth patterns were calculated as a power function of total length and described using the growth coefficient to find important steps in early life history. the total length of the newly hatched larvae and fry were 10.59±0.8 and 38.8±2.9 mm at 1 and 50 dph, respectively. morphogenesis and differentiation were the highest rates during the first 11 days of early development, i.e. endogenous feeding period. there were higher growth rate of head, snout and tail regions compared with those of other organs from the hatch up to yolk sac absorption, followed by positive or almost isometric patterns, after the begin of exogenous feeding, showing priority to enhance the feeding and swimming capabilities. this study confirmed that most of morphological changes of this species are occurred from hatching until the onset of exogenous feeding i.e. during the lecithotrophic phase. introduction fish larvae experience complex processes of morphogenesis and differentiation during early ontogeny by changing their morphology, metabolism rate, swimming ability and behaviour (osse and van den boogart, 1995; van snik et al., 1997; gisbert, 1999, 2014; koumoudouros et al., 1999). the alterations of body shape with increasing size (osse and van den boogart, 1995; van snik et al., 1997) as a result of different relative growth patterns of the body parts has been defined as allometry (fuiman, 1983). allometry is a common feature during early larval development, ensuring the development of the most essential organs for primary functions, followed by the development of organs with lower priority for survival (osse and van den boogart, 1995; hasanpour et al., 2015a). during the short period of early development, fish larvae are extremely vulnerable and subjected to * corresponding author: hadi poorbagher doi: https://doi.org/10.22034/ijab.v5i3.312 e-mail address: poorbagher@ut.ac.ir various environmental factors (claramunt and wahl, 2000; hardy and litvak, 2004). since the alteration of body shape is occured in response to environmental conditions (dettlaff et al., 1993; eagderi et al., 2015) and poor environmental conditions may cause deformities during early ontogeny resulting negative effects on growth performance and survival of reared fish larvae. therefore, information about the early morphological development and allometric growth patterns in the farmed fishes is crucial for their proper managment in aquaculture industry (chatain, 1994; koumoundoura et al., 1994, 1999; bengtson, 1999; hasanpour et al., 2015b; asgari et al., 2013a, b, 2014). furthermore, understanding normal growth pattern during early development will help to identify abnormal deformities and improving the quality of produced fish by improving environmental conditions (segner et al., 1995; koumoundoura et al., 1994, 1999; bengtson, 1999). 202 eagderi et al./ early ontogeny of persian sturgeon persian sturgeon, acipenser persicus, is an anadromous sturgeon distributed along the iranian coastal of the caspian sea (birstein et al., 1997). the artificial propagation and rearing of this species has been developed since 1971 for restocking the natural populations or production of marketable-size fish (chebanov and billard, 2001). hence, information about the growth patterns of this species fish may allow better understanding the biological process in the early life stages and their priorities during early growth. such information provides insight into fish biology, behaviour, ecology and especially aquaculture to promote the development of optimal rearing protocols and improve the quality of juveniles (koumoundouros et al., 1999; çoban et al., 2009) and evaluate the appropriateness of produced fish for restocking or further rearing (gisbert et al., 2002). therefore, in the present study, persian sturgeon, a. persicus, larvae and juvenile specimens were studied from hatching up to 50 days post hatching (dph) to describe its early morphological development and allometric growth pattern. materials and methods the larvae were obtained from artificial propagation of a hormonally-induced (lhrha2) female persian sturgeon fertilized with the milt of three males at the shahid rajaei propagation facility (sari, mazandaran province, iran) in 2015. eggs were incubated in the yushchenko incubators at 11-12°c (750 gram egg per unit) and hatched after about 1800 degree/day (6 day). the newly-hatched larvae were allocated to three 500-l circular fiberglass tanks connected to a flowthrough freshwater system. during the larval rearing period, water temperature, dissolved oxygen and ph were 15.9±0.6°c, 7.1±0.6 mg.l-1 and 7.2±0.1, respectively. the larvae were reared under natural photoperiod. the larvae were fed with a mixture of artemia nauplii and cladocerans (daphnia sp.) from 8-25 days post hatching (dph) (500 larvae/day–6 times a day). then, a short co-feeding phase based on cladocerans and an inert diet (biomar, denmark; d1 particle size=0.5) was conducted from 25 to 30 dph, at the rate of 20% of stocked fish biomass 6 times a day. the food particle size were progressively adjusted according to the fish size. all tanks were cleaned and the bottom of the tank siphoned to remove uneaten feed and faces three times a day. specimens were sampled from hatching till 10 dph, every day, up to 20 dph, every two days and afterwards, at 25, 30, 40 and 50 dph. ten specimens were randomly sampled for different stages using a scoop net, anaesthetized using 1% clove oil and weighed (bw, to the nearest 0.01 mg) using an analytical microbalance. the left sides of the specimens were photographed using a stereomicroscope equipped with a digital cannon camera with a 5 mp resolution. then the larvae were preserved in 5% buffered formalin and stored in 70% ethanol after 24 hours. seven morphometric characteristics including total length (tl), head length (hl), tail length (tal), trunk length (trl), snout length (snl), caudal peduncle (cp) and predorsal length (pdl) were measured using the software imagej (version 1.240). all characteristics were measured along the lines parallel or perpendicular to the horizontal axis of the body. the allometric growth patterns were calculated as a power function of total length using non-transformed data: y= axb, where y is the independent variable; x, the dependent variable, a, the intercept and b, the growth coefficient. isometric growth, positive and negative allometric growth are indicated by b=1, b>1, b<1, respectively (van snik et al., 1997). robustness of the regression was examined using r2 and its significance level. the inflexion points of growth curves were determined according to fuiman (1983) and van snik et al. (1997). graphs were drawn and data were analysed using excel 2013 (microsoft corporation) and past (version 2.17). results morphological development: at hatching, total length and weight of the larvae were 10.59±0.8 mm and 26.7±0.5 mg, respectively, and increased to 38.8±2.9 mm and 2.1±0.8 g at 50 dph. at hatching, the mouth and gill slits were closed and tail was as proterocercal 203 int. j. aquat. biol. (2017) 5(3): 201-207 and bordered by a wide primordial fin-fold. at this time, the notochord was visible, eyes were pigmented and olfactory organ was observed as an external opening. at 2 dph, the pectoral fin appeared, mouth was open with few taste buds around it, gill slit was open and its filaments were visible inside the branchial cavity (fig. 1a). at 3 dph, the eyes were darkly pigmented, size of the pectoral fin increased, melanin plug was appeared, yolk sac was divided into two parts, upper and lower lips were appeared as skin fold around the mouth silt, and a small barble was observed. at 4 dph, the length of barble increased and fin folds became wider in their base with clear pigmentation. at 5-6 dph, the ventral fin appeared and rays of the dorsal and anal fins were observed. in addition, the body pigmentations increased with dense pigmentation on the head and tail regions. larvae possessed about 10 canine teeth on the upper and lower jaws at 7-8 dph. at this time, the feeding apparatus for grabbing food items along with related sense organs e.g. taste buds and eye were developed for exogenous feeding. in addition, the dorsal and anal fins were clearly distinguishable from surrounding fin folds and rays of the paired fins were observed. at 910 dph, external yolk sac was almost depleted and food items were observed inside the digestive system of the most larvae and gill arches were completely covered with opercle. the rate of changes in external morphology was decreased after 11 dph. the first figure 1. early life stages of acipenser persicus. (a) 2 dph, (b) 11 dph, (c) 17 dph, (d) 28 dph and (e) 50 dph (the scale bars indicate 10 mm). 204 eagderi et al./ early ontogeny of persian sturgeon scutes were dorsal and ventral scutes that appeared at 11 dph. between 18-19 dph, lateral and ventral scutes were developed as some rows of small spines with their differentiation were completed during 0-40 dph in an anterior-posterior direction (fig. 1d, e). at 35-40 dph, remaining of the fin folds were disappeared completely and larvae were transformed as miniature shape of adults in the external morphology. allometric growth pattern: based on morphological development and growth coefficients, life stages of persian sturgeon from hatching (10.59±0.08 mm tl) up to 50 dph (38.8±2.8 mm tl), could be divided into two distinct phases, including larval and post-larval stages. the larval stage starts from hatching up to onset of the exogenous feeding at 11 dph (11.6-18.7 mm in tl) and the post-larval stage continues till metamorphosis (18.9-38.8 mm in tl). the growth pattern of all body segments can be divided into two phases (fig. 2 and table 1). the hl and snl had strongly-positive allometric growth in relation to tl during whole early developmental stage with inflection points at 13 dph (b=1.9 and 1.23) and 12 dph (b=1.7 and 1.41), respectively. the growth patterns of the tail region including cp and tal were positive allometric prior to their inflection points at 5 dph (b=1.18; 15.7 mm in tl) and 11 dph (b=1.43; tl=18.6 mm in tl), respectively, whereas tal showed a relatively-isometric (b=0.97) and cp, negative (b=0.68) allometric growth patterns during the flexion stage. there were negative allometric patterns for trl and pdl during the flexion stage at 11 dph (b=0.23; 18.6 mm in tl) and 4 dph (b=0.20; 14.1 mm in tl), respectively. there were relatively negative (b=0.85 ) and positve (b=1.36) growth patterns for trl and pdl, respectively, during the post-flexion period (fig. 2, table 1). discussion the organs developed and morpho-anatomical characteristics changes in a stepwise fashion, which is regulated by gene expression and influenced by environmental parameters (gilbert and bolker, 2003; osse and van den boogart, 1995). at hatching, most of the functional systems in persian sturgeon larvae were incomplete. thus, these larvae require to develop quickly the most essential organs for primary functions during the early development to survive (dettlaff et al., 1993; gisbert, 1999). this changes are accompanied with morphological alternations that is led to allometric growth patterns (gisbert and doroshov, 2006). based on the results, the growth pattern of the head in a. persicus was positive during early ontogeny, whereas it was divided into two distinct phases in a. baeri with different growth patterns (gisbert, 1999) i.e. during first phase, endogenous feeding was positive followed by isometric pattern during exogenous feeding (gisbert and doroshov, 2006). however, this difference may be due to their snout form and swimming hydrodynamics (gisbert and doroshov, 2006). positive allometric growth of the head is a common phenomenon during early ontogeny of many fish species, e.g. sturgeons (van snik et al., 1997). the positive growth pattern of head can show the developmental priority of this organ during early developmental stage because it is related to vital functions such as branchial respiration, brine development and feeding apparatus (splachnotable 1. a, the intercept; b, the growth coefficient and r2 correlated coefficient of different body segments before and after inflexion point of acipenser persicus (*p<0.01). characters before inflection point after inflection point b a r2 pvalue b a r2 pvalue head length 1.90 0.13 0.92 * 1.85 0.22 0.91 * caudal peduncle 1.18 0.07 0.63 * 0.68 0.09 0.71 * trunk length 0.23 0.57 0.25 * 0.85 0.37 0.85 * snout length 1.7 0.05 0.8 * 1.38 0.07 0.86 * tail length 1.43 0.31 0.95 * 0.97 0.40 0.89 * pre-dorsal length 0.20 0.22 0.01 * 1.36 0.15 0.91 * 205 int. j. aquat. biol. (2017) 5(3): 201-207 cranium) (van sink et al., 1997; gisbert, 1999; gisbert et al., 2002; osse and van den boogart, 2004). in addition, rapid growth of the head length leads larvae to have an efficient feeding system to capture the higher food particles which have more energetic than smaller one (osse et al., 1997; van sink et al., 1997; mathias and li, 1982). differentiation of the sensory structures i.e. barbles equipped with taste buds and eyes make larvae to be independent from yolk sac as an endogenous feeding (gisbert, 1999). the growth pattern of the snout was positive during all period with a flexion point at 12 dph similar to that of a. baeri (gisbert, 1999) and this growth pattern has been reported for many fishes (strauss, 1995; schmidt, 2001; geerinckx et al., 2008). the developed sensory systems such as taste buds and sensory chanells on the ventral face of the sturgeon’s snout can play a vital role for feeding (geerinckx et al., 2008). the negative growth pattern of the trunk from hatching up to 12 dph was similar to those of other fishes (osse and van den boogart, 2004), whereas it changed from 12-50 dph to relatively isometric pattern. similar result reported for a. medirostris (gisbert and doroshov, 2006), whereas in a. baeri, it was negative during whole developmental stages (gisbert, 1999). the positive allometric growth pattern of the tail in persian sturgeon from hatching up 13 dph may reflect its priority after development of the head that is related to vital functions such as feeding and swimming ability (osse and van den boogaart, 2004) and figure 2. growth allometries of the different body segments in acipenser persicus (r2=correlated coefficient). 206 eagderi et al./ early ontogeny of persian sturgeon changing swimming mode that can have an important role for locomotion (osse et al., 1997). similar results have been reported in other sturgeons e.g. a. baeri and a. medirostris (gisbert, 1999; gisbert and doroshov, 2006). since predation and starvation are main causes of mortality in fish larvae, the early development of feeding apparatus and swimming organs appear to have priorities during the early ontogeny (osse et al., 1997). there was positive growth pattern in the caudal peduncle of a. persicus up to 5 dph and then altered to negative. this pattern was positive in a. medirostris and isometric in a. baeri (gisbert, 1999; gisbert and doroshov, 2006). positive allometric growth pattern in anterior and posterior section of larvae body before complete development may reflect the larvae adaptation to decrease costs of transport (van snik et al., 1997). therefore, faster growth patterns of the head and tail regions would be a favorable condition for decreasing the stop pressure on the larvae body (osse and van den boogart, 1995) as seen in a. persicus. this study showed that important morphological and morphometric modifications occurred during early life stages of persian sturgeon holding valuable information on changes in functional demands throughout ontogeny. based on our results, most of morphological changes of this species, during the lecithotrophic phase, are occurred from hatching until the onset of exogenous feeding, i.e. 12 dph and after onset of exogenous feeding until 50 dph. acknowledgments authors wish to thank m. arabshahi for providing specimens. this study was financially supported by the university of tehran. references asgari r, eagderi s., rafiee g, poorbagher h., agh n., eshaghzadeh h. 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(2017) 5(3): 201-207 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی asipenser persicus borodin, 1897 در تاسماهی ایرانی، آلومتری رشد الگویو ریختی توسعه (actinopterygii, acipenseridaeدر مراحل اولیه تکوین ) ولی حسینی ، سیدمشیدی فاطمه ،*پورباقر هادی ،ایگدری سهیل .ايران كرج، تهران، دانشگاه طبیعی، منابع و كشاورزي پرديس طبیعی، منابع دانشکده ،شیالت گروه چکیده: روز پنجاه تا گشايیتخم زمان پرورش داده شده از asipenser persicus ايرانی، تاسماهی در آلومتري رشد الگوي و ريختی توسعه تحقیق، اين در طول سر، طول كل، طول شامل ريختی ويژگی 7 و شد برداريعکس آنها چپ نیمرخ از الروها، برداينمونه از پس. گرفت قرار مطالعه مورد آن از پس لك طول توانی تابع صورتهب آلومتري رشد يهاالگو. گرفت قرار سنجش مورد آنها در پشتی پیش طول و دمی ساقه طول پوزه، طول تنه، طول دم، كل طول گشايیتخم از پس 50 و 1 روزهاي در. شد توصیف چرخه حیات، اولیه فرآيند طی در مهم مراحل كردن پیدا براي رشد، ضريبساس ارب و گشايی،تخم از پس اول روز 11 طی در تمايز و زايیريخت. بود مترمیلی 59/10±8/0 و 8/38 ±9/2 ترتیببه ماهیان بچهالروهاي تازه تفريخ شده و اب مقايسه در دم و پوزه سر، هايبخش ،زرده كیسه كامل جذب تا گشايیتخم زمان از. بود بااليی نرخ داراي داخلی، تغذيه دوره به عبارت ديگر در فزايشا دنبال شد كه اولويت خارجی تغذيه شروع از پس ايزومتري يا مثبت آلومتري رشد الگويند كه با بود بیشتر رشد نرخ داراي هابخش ساير يعنی دوره ارجیخ تغذيه اولین تا گشايیتخم از زمان گونهاين ريختی تغییرات بیشتر كه كرد تايید مطالعه اين. دهدمی نشانرا شنا و تغذيه توانايی .پیونددجذب كیسه زرده به وقوع می .خزر درياي سنجی،ريخت زايی،ريخت نتوژنی،ا :کلمات کلیدی int. j. aquat. biol. (2021) 9(5): 326-332 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article assessment of flocculation induced by ph increase for harvesting microalgae cyanothece sp. zahra aminikhoei* 1, elnaz erfani far1, saaedeh taherpanah2, mahsa naderi samani3 1agricultural research education and extension organization (areeo), iranian fisheries science research institute (ifsri), offshore fisheries research center, chabahar, iran. 2iranian biological resource center, tehran, iran. 3iran fisheries organization, tehran, iran. s article history: received 7 september 2021 accepted 14 october 2021 available online 2 5 october 2021 keywords: microalgae harvesting flocculation induction and ph increase abstract: one of the most important challenges lies in the microalgae mass production is the high cost of harvesting process which is the separation of a low amount of biomass consisting of small individual cells from a large volume of culture medium. therefore, finding an efficient and costeffective technique for harvesting microalgae is important issue. in the current study, ph-induced flocculation method was tested for microalgae cyanothece sp. harvesting. the halophilic microalgae were cultured and grown in laboratory with hypersaline water in f/2 medium. after reaching the stationary phase, the impact of ph induction (from natural medium culture ph:8.2 to ph:11) on flocculation efficiency, chlorophyll a, chlorophyll b, total carotenoid, β-carotene and phycocyanin component and the possibility of reuse flocculated medium of microalgae cyanothece sp. were evaluated. the results indicated that the increasing the medium ph value by adding naoh from ph natural at 8.2 to 9.4 increased flocculation efficiency significantly from 10 up to 90% (p<0.05), but after that remained stable up to ph: 11. regarding the pigment content, the increase in ph value from natural ph: 8.2 to ph: 9.1 had a relatively a medium effect on pigment components, including chlorophyll a, chlorophyll b, total carotenoid, β-carotene and phycocyanin amount of the harvested biomass of cyanothece sp, but after that from ph: 9.4 to 11 the reduction was severe. the medium culture from ph: 8.5 to ph: 11 was reusable for new culture of microalgae. thus, the flocculation induced by ph increase up to ph: 9.1 is a suitable method for harvesting microalgae cyanothece sp. with no serious adverse effect on pigment component. introduction the commercial culture of microalgae is now over 60 years old with the main culture in large shallow openair ponds. in recent years too much attention has been paid in research and development for increasing the yield of microalga biomass production through open pond raceway and photo bioreactor design (helical tubular and flat panels systems), strain screening and selection, water quality, nutrients composition and climate condition (ben-amotz, 1999; borowitzka and borowitzka, 1988; borowitzka, 2013b; moheimani, 2013). but, much less work has been made on research and innovation in downstream processing such as harvesting and drying processes which is essential to reduce the cost of the production process. in order to moving microalgae production from laboratory to *correspondence: zahra aminikhoei doi: https://doi.org/10.22034/ijab.v9i5.1393 e-mail: zamini.41@gmail.com pilot scale and commercial-scale, we need to find cost and energy efficient downstream processing technologies. the separation and recovery of small microalgae biomass (about 0.5 g/l in open pond and 5 g/l in photobioreactors) from their large volume of culture medium is a challenging area and the costs related to harvesting, thickening and dewatering of microalgae biomass are about 20-30% of the total production cost (horiuchi et al., 2003; kwon et al., 2014). several separation processes are currently used, including sedimentation, centrifugation, filtration and flocculation of microalgae in their water culture medium. centrifugation and filtration methods are usually used for high-value products which is too expensive and energy-intensive. the flocculation is 327 int. j. aquat. biol. (2021) 9(5): 326-332 seen a low cost and convenient harvesting method. flocculation refers to the aggregation of unstable and small cells through surface charge neutralization, electrostatic patching and/or bridging after addition of flocculants to spate cells from their medium culture (spolaore et al., 2006; wu et al., 2012; ahmed et al., 2017). usually flocculation is proposed for the first step to concentrate a dilute suspension of 0.5 g/l dry matter 20-100 times to a slurry of 10-50 g/l (branyikova et al., 2018). the current study was designed to study flocculation method by ph inducing for unicellular cyanobacterium cyanothece sp. harvesting. cyanothece sp. is an aerobic, diazotrophic and halophyte cyanobacterium species that can grow in a wide range of salinities, light intensities, temperatures and nutrient concentrations. recent decade studies on different genera of cyanothece have shown that this genus has very important role in nitrogen cycle in aquatic and terrestrial environments. (bandyopadhyay et al., 2011). based on biotechnology research, there are several studies showed that cyanothece strains isolated from saline environments can produce high amount of extracellular polymeric substances which has characteristics suitable for food and pharmaceutical applications (chi et al., 2007; ohki et al., 2014). however, despite the great importance of this algae in the production of beneficial compounds, studies on suitable harvesting methods are still limited. therefore, this work was designed for evaluating the effect of ph-induced on harvesting efficiency, total carotenoid and β-carotene content of hypersaline microalgae, cyanothece sp. materials and methods microalga strain and culture conditions: the strain of cyanothece sp. was isolated from lipar lagoon in chabhar, iran. the halophilic microalgae were cultured and grown in laboratory with medium prepared with hypersaline water in f/2 medium (guillard and ryther, 1962) .the cultures were incubated in 500 ml erlenmeyer flasks with 300 ml of culture media at 29±1°c, and illuminated by fluorescent lamps for 24 h in 4300 lux (chi et al., 2007). the cultures were scaled up in 10 l and continuously aerated and mixed by the gentle bubbling filtered air. the seawater ph is typically limited to a range between 7.9 and 8.2. the growth was monitored by measuring the absorbance at 680 nm (od680) by a spectrophotometer (evolution™ 300 uv-vis spectrophotometer). dry weight biomass was determined by filtering a fixed volume of the algae suspension through a pre-weighed filter with a 0.45 μm porous membrane. then the filter and algal cells were dried at 105°c for 48 h (zhu and lee, 1997). determination of flocculation efficiency: flocculation tests were performed after reaching the stationary phase. flocculation experiments were carried out in cylindrical laboratory glass beakers of 1 l volume. 0.1-1 m sodium hydroxide (naoh) and hydrochloric acid (hcl) were used to adjust the ph value (from natural medium culture ph:8.2 to ph:11). the microalgae suspensions were mixed quickly just after ph adjustment and then allowed to settle at room temperature observe the behavior of flocs. an aliquot of the suspension at a height of two-thirds of the glass beakers was collected and flocculation efficiency percentage was calculated from the absorbance measured at 680 nm by a spectrophotometer (evolution™ 300 uv-vis spectrophotometer). a control biomass was collected by centrifuging as a reference (spilling et al. 2011). flocculating efficiency (%) = (1-b/a) * 100 where a represents the od680 of the control treatment and b is the od680 of the ph-adjusted treatment. the efficiency, in terms of biomass recovery percentage was determined for different samples at different time intervals 1, 3, 5 and 7 hours. floc visualization: after the sedimentation, flocs of the different treatments were observed under the optical microscope (eclipse e200, nikon). reuse of flocculated medium: after flocculation, the flocs and the growth medium were separated. the growth medium was return to refresh new alga culture and adjusted to the original ph value by adding the necessary amount of hcl and nutrients. pigment extraction and analysis: after flocculation, the microalgae biomass was collected and pigments 328 aminikhoei et al./ assessment of flocculation induced by ph increase for harvesting microalgae content including, chlorophyll a, chlorophyll b, carotenoid, β-carotene and phycocyanin concentrations were measured. the algal biomass harvested was washed with 0.5m ammonium bicarbonate until total reduction of the salinity. after washing, the biomass was lyophilized (jalteb, iran). a sample of 0.1 g of lyophilized microalgae was suspended in 5 ml of the acetone 100%. the suspension was sonicated for 3 minutes in an ultrasound apparatus (elma ultrasonic) and stored for 24 h at 4°c. the extract was separated from the pellet and recovered by centrifugation, immediately filtered through a 0.22 μm filter (cardoso et al., 2012). the absorbance of supernatant was noted at 660, 645, 470, 460, 431 and 412 nm using uv-visible spectrophotometer (evolution™ 300 uv-vis spectrophotometer). the amount of total carotenoids was determined using following equations (lichtenthaler, 1987; kumar et al., 2013): chlorophyll a (µg ml-1) = (12.25 a 663) – (2.79 a 646) chlorophyll b (µg ml-1) = (20.50 a 646) – (5.10 a 663) total carotenoids (µg ml-1) = (1000 a 470 – 1.82 chl a – 85.02 chl b) / 198 β-carotene (μg/ ml) = -0.430 (a412) + 0.251(a431) 4.376a (460) + 13.216a (480) phycocyanin = (mg/ ml) = (a615 a 720) -0.474× (a652a720))/5.34 where a663, a646, a470 and a412 represent absorbance at 663, 646 nm; 470 nm; 412 nm, respectively. the obtained extract was processed on the high-performance liquid chromatography (hplc) system for the separation and identification of β-carotene by comparison of their retention times with those of the commercial standards. statistical analysis: one-way analysis of variance was applied to analyze the experimental data using spss 16.0 software (spss, usa). the differences were considered significant at p<0.05. all the treatments were repeated three times, and data are reported as the mean±sd values. results effect of ph inducing on flocculation efficiency of microalgae cyanothece sp.: the effect of ph on the flocculation efficiency of marine microalgae cyanothece sp. was studied using 0.1-1 m naoh and hcl when the culture was in stationary phase (day 10). the results indicated that no significant automatic precipitation occurs in the culture medium without use of flocculating agent over time. the microalgae cells began to agglomerate when naoh was added and induced destabilization that led to the progressive formation of algal flocs. as, ph increased from ph: 8.2 in natural medium culture to 9.4, the flocculation efficiency was greatly raised to around 90% and reached plateaus. after that, with adding larger quantities of naoh, the solution behaved like a gel and no more sedimentation was observed from ph 10 to 11 (fig. 1). the method of adding naoh, either at low flow rate or at once, has no significant effect on recovery efficiency. reuse of growth medium for cultivation: it has been observed that the biomass of microalgae grown in a reused medium from ph: 8.5 to ph: 11 is close to that of fresh medium culture. effect of flocculants on pigment component including chlorophyll a, chlorophyll b, total carotenoid, β-carotene and phycocyanin of harvested biomass of cyanothece sp.: chlorophyll a, chlorophyll b, total carotenoid, β-carotene and phycocyanin of microalgae cyanothece sp. biomass which was collected from different ph induced treatment (figs. 2-5). chlorophyll a, chlorophyll b, figure 1. harvesting efficiency of ph-induce flocculation in microalga cyanothece sp. 329 int. j. aquat. biol. (2021) 9(5): 326-332 total carotenoid, β-carotene and phycocyanin content of the different ph induced treatment were significantly different (p<0.05). the chlorophyll a content experienced a relatively small decrease from 32.6 in ph: 8.2 to 20 μg/ml at ph: 9.1 after that sever reduction was observed up to ph: 14 and reached 3 μg/ml. the chlorophyll b also decreased very strongly from 10.3 to 2.6 μg/ml. the total carotenoid content was decreased from ph: 8.2 to 8.8. and there was no significant difference between ph; 8.8 and 9.1 however, a fast downward trend from ph 9.1 to 11 was observed from 3.3 to 0.2 μg/ml. β-carotene content of microalgae cyanothece sp. was very sensitive to adding naoh and ph increase specially in high ph value and it was reached to 0.02 μg/ml at ph: 10.6 and non-detected at ph: 11. changes in phycocyanin amount of microalgae cyanothece sp. biomass were also very evident with increasing ph of the culture medium and decreased from 13 to 1 μg/ml from ph: 8.2 to ph:11 discussions microalgae have been considered as the third generation sources of biomass production for biofuels. the algal biomass and extracted pigment have wide range of industrial applications, including animal feed and aquaculture, food supplements, nutrients and medicine. however, in the present, several important challenges related to algae cultivation, such as small size and negative surface of algae cells, as well as the dilution of the culture medium have led to their highcost commercial production. therefore, the method of figure 1. chlorophyll a, b of cyanothece sp. harvested with ph-induce flocculation figure 3. total carotenoid of cyanothece sp. harvested with phinduce flocculation. figure 4. β-carotene of cyanothece sp. harvested with ph-induce flocculation. 330 aminikhoei et al./ assessment of flocculation induced by ph increase for harvesting microalgae harvesting of microalgae is an important bottleneck for the large-scale industrial production process (wu et al., 2012). the results of this study showed that increasing the ph from 8.2 to 9.4 cause cells to coagulate and precipitate to the bottom in the culture medium and increases the harvest efficiency from 18 to 92%; however, an increase in ph to 11 did not have a significant effect on increasing harvesting efficiency. similarly, wu et al. (2012) showed that increasing the ph to 8-9 in saline aquatic species e.g. nannochloropsis oculataa and phaeodactylum tricornutum increased flocculation efficiency up to 90%. also, ph-induced flocculation method was successfully applied to harvest marine chlorella sp. and the flocculation efficiencies were up to 90% within 10 min (yang et al., 2016). additionally, based on perez et al. (2017), acid ph values (2 to 6) and basic ph values (8 to12) have been tasted for skeletonema costatum and chaetoceros gracilis microalgae and greater flocculation efficiency was achieved at high ph values whereas the lowest ph values reached a maximum algal biomass separation around 60% (pérez et al., 2017). the flocculation mechanism is related to the presence of metal cations in the culture medium, which are hydrolyzed by increasing ph and produce the hydroxide of metal cations. it has been proposed that this enhancement may be attributed mainly to the co-precipitation of ca2+ and mg2+ ions dissolved in the medium with algal cells. also, the algae cells with a negative charge become unstable with the formation of hydroxide of metal ions with a large adsorptive surface area and a positive superficial charge in basic ph (liu et al., 2013). in our experiment, adding sodium hydroxide to the culture medium naturally increased the ph from 8.2 to 9.4, but after that high amounts of sodium hydroxide were required to achieve ph 10 to 11, indicating a buffer state in this area. by increasing the larger quantities of naoh, the algae culture medium behaved like a gel and slow separation became due to the gelification. therefore, no significant difference was observed in the flocculation efficiency from ph 9.4 to 11. the reuse of water and viable algae cells fraction remaining after flocculation is an important issue for researchers and operators since, the recycle of flocculated medium could minimize the cost of nutrients and the demand for water (maji et al., 2018). in the present study, in ph-induced treatments, the culture medium was divided in to two parts, the upper part of which was clear and completely separate water, and the lower part of which was a sedimentary cell. the upper phase cultivated in the reused growth medium was close to that cultivated in the fresh solution, indicating the culture solution could be recycled, but the settling phase could not be reused due to the presence of non-living and cells lysis during the flocculating by ph increase. in term of inoculating cell culture to the new cell culture medium, this capability was eliminated in high ph value more than 9.4. the flocculated medium, after neutralizing the ph, could be reused for cultivating the microalgae. aluminum and ferric salts are of the most popular inorganic flocculants which have been used for long time in wastewater clarification. recently these multivalent inorganic chemicals used for algal biomass recovery of several species such as, tetraselmis sp., chlorella sp. and scenedesmus sp. with respectively 86, 100 and 90% flocculation efficiency (sanyano et al., 2013; gerde et al., 2014; kwon et al., 2014). the harvesting efficiency of inorganic flocculants depends largely on their physicochemical properties such as solubility and electronegativity. however economically, the cost of using aluminum salt is relatively low, which could be figure 5. phycocyanin of cyanothece sp. harvested with ph-induce flocculation. 331 int. j. aquat. biol. (2021) 9(5): 326-332 a potential choice for biomass recovery, but the main problem for using them is due to contamination and discoloration of the culture medium the release of such chemicals into the environment is a critical environmental and health concern (vandamme et al., 2015). effect of ph inducing flocculation method on pigment component of cyanothece sp.: due to the fact that the most important commercial and desired composition in microalgae cyanothece sp. algae are its pigments, especially carotenoid, β-carotene and phycocyanin. determining the effect of harvesting technique on this pigment is important for final use. the results of microalga biomass analysis after harvesting indicated that total carotenoid, β-carotene and phycocyanin content of cyanothece sp. were affected by changes in ph medium culture, but the rate of change was different and analyzable at different treatments. the increase in ph value from 8.2 to 9.1 had 80% harvesting efficiency with a reasonable decrease in total carotenoid, β-carotene and phycocyanin, however, the more increases in ph up to 9.4 improved harvesting efficiency till 90% with serious negative effect on total carotenoid, β-carotene and phycocyanin. adding high amounts of sodium hydroxide in ph 10 to 11 appear to damage pigment microalgae cells and it can have serious negative impacts on the down-stream processing and quality of products. there are some cases has been studied lipid component of microalgae after ph inducing harvesting. for instant, ph induced flocculation was used for harvesting chlorella sp. 725 and a high lipid extraction yield was obtained for the naoh flocculated cells, which was similar to the yield of the cells harvested by centrifugation, indicating that the method was suited for harvesting marine chlorella sp., for biofuel production (yang et al., 2016). therefore, it can be concluded that depending on the purpose of collecting algae, the harvesting method should be chosen. as conclusion, the results of this study showed that flocculation method induced by ph increase up to 9.1 is effective for harvesting microalgae cyanothece sp. with relatively low effect on its chlorophyll, total carotenoid, βcarotene and phycocyanin pigments content. acknowledgements this study was sponsored by agricultural research, education and extension organization (areeo), iranian fisheries science research institute. references ahmed r.a., he m., aftab r.a., zheng s., nagi m., bakri r., wang c. 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(cyanobacteriaceae: cyanophyceae) isolated from the eastern indian ocean. acta oceanologica sinica, 37(10): 4-10. international journal of aquatic biology (2013) 1(5): 266-272 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article morphology and ultrastructure of cysts in different species of the brine shrimp, artemia from southern india shanmugam sivagnanam1, velayudhannair krishnakumar*2,1natesan munuswamy2 1central institute of brackishwater aquaculture, santhome high road, chennai – 600 028, india. 2unit of aquaculture and cryobiology, department of zoology, university of madras, life science building, guindy campus, chennai – 600 025, india. article history: received 26 july 2013 accepted 24 november 2013 available online 2 5 december2013 keywords: cyst shell structure kelambakkam saltpan artemia abstract: surface topography and ultrastructure of dried cysts of artemia parthenogenetica (vedaranyam population) and bisexual artemia sp. (kelambakkam population) were studied electron microscopically in order to identify the bisexual strain, inhabiting covelong salterns, kelambakkam, south india. the scope of this study is to provide substantial information for further characterization of these artemia strains by the comparison of the cyst morphology and ultrastructure. a cyst of artemia franciscana was used as a reference cyst for comparison. scanning electron microscopic studies on cyst morphology revealed that the surface is smooth with no significant variation among the three artemia strains studied. transmission electron microscopic observations on the ultrastructure of the cyst both parthenogenetic and bisexual forms showed an apparent variation in thickness of the cortical layer. in kelambakkam population of artemia, the architecture of alveolar meshes is tightly arranged and is similar in shape. but they show minor variations in the arrangement of pores in the matrix and in the length of the cortical layer, compared to a. franciscana. in a. parthenogenetica, they are loosely arranged and are oval in shape. the present study clearly documents that the artemia species, colonized in kelambakkam saltpan is a. franciscana; thus, the variation in the cyst ultrastructure is much pronounced and a taxonomically important parameter for the genus artemia. introduction representatives of the branchiopod crustacean, the brine shrimp artemia spp. inhabit hypersaline habitats (van stappan et al., 2001). the genus artemia consists of several bisexual and parthenogenetic forms which probably diverged five to six million years ago from a common ancestor living in the mediterranean area (abatzopoulos et al., 2002; munoz et al., 2010) and the latter with a variety of ploidies (sun et al., 1999). it is well known that the reproductive pattern of artemia switches from ovoviviparity (browne, 1980) to oviparity (clegg et al., 1996) depending on the environmental factors in the habitat. brine shrimps can produce dormant eggs, covered by a characteristic shell or * corresponding author: velayudhannair krishnakumar e-mail address: kv.krishnakumar@hotmail.com tel: +919894304073 chorion under adverse environmental conditions such as high salinity, low dissolved oxygen and nitrate content (krishnakumar et al., in press). the shell around the egg is secreted by a cluster of shell glands. the shell itself plays an important role in the protection of the embryo (anderson et al., 1970) in both, parthenogenetic and bisexual species. encystment (cyst formation) protects the embryo from various extreme environmental conditions and during dormant stage (clegg, 1974; versichele and sorgeloos, 1980). embryo resumes its development, when the dehydrated cysts are hydrated at suitable environmental conditions, such as suitable osmotic pressure and sufficient oxygen (dutrieu, 1960). the cyst of artemia consists of several layers (lee et al., 267 sivagnanam et al/ international journal of aquatic biology (2013) 1(6): 266-272 1994; rosowski, 1997). outside the embryonic cuticle, and forming the outer surface of the cyst, there is a thick nonchitinous layer referred to as a ‘chorion’ (morris and afzelius, 1967). in india, the distribution of artemia has been well documented in the saltpans of various states like gujarat, rajasthan, tamilnadu and maharashtra (kulasekarapandian et al., 1992). it was reported that a. parthenogenetica was dominated in all the saltpans of south india until the late 1990s. the occurrence and even dominance of invasive bisexual species was reported in a few saltpans in tamilnadu, including kelambakkam (kulasekarapandian et al., 1992; sivagnanam et al., 2011; vikas et al., 2012) and tuticurin (sugumar and munuswamy, 2006) due to the improper disposal of artemia cysts used at various shrimp hatcheries to feed the shrimp larvae. earlier interest in the study of anostracan cysts led to the description of its structure and hypothecated the importance of cyst ultrastructure during diapausing period and development during the hydration process of the embryo. later, it became an integrated part of species or strain description (belk and sissom, 1992) and species identification keys were developed (thiery and gasc, 1991; brendonck and coomans, 1994). the structure of cyst and cyst membrane became an important character in species identification for several species (wurdak et al., 1978; gilbert et al., 1979; munuswamy et al., 1996). the present study was undertaken to identify, further characterize and to understand extend of invasion and naturalization of invader a. franciscana using the cyst morphology and ultrastructure of different strains of artemia inhabiting various salterns in south india. materials and methods sources of artemia: cysts of parthenogenetic and bisexual species of artemia were collected from the saltpans of vedaranyam (vrm) (08º59' n; 78°50' e) and kelambakkam (kbm) (12°08’n, 80°02’e), south india, respectively, during 2008 to 2011 during available period. collected cysts were transported to the laboratory in 2 or 3 l of screwfigure 1. scanning electron micrographs of the hydrated cyst of different strains of artemia, showing smooth and granular surface. bar = 30 µm; (a) a. franciscana (sfb population), (b) artemia sp. (kbm population) and (c) a. parthenogenetica (vrm population). 267 268 sivagnanam et al/ international journal of aquatic biology (2013) 1(6): 266-272 capped plastic containers having brine solution (>250 ppt) and washed with freshwater for 2 minutes. washed cysts were dried in an oven at 37ºc for 24 h and sieved through a 300 µm mesh and packed at vacuum (sevana’s quick seal, hyderabad, india) (sorgeloos and kulasekarapandian, 1984). vacuum packed cysts were stored at room temperature after proper labeling for further analysis. commercially available cysts of a. franciscana, (osi brand artemia cysts, purchased from m/s. southern india pvt. ltd., chennai, south india) were used as a reference. scanning electron microscopy: for scanning electron microscopy, cysts of all the three artemia samples, collected from the salterns of kelambakkam, vedaranyam and a. franciscana were rinsed with distilled water and immersed in 10% formalin for 30 min to remove the microorganisms and debris attached to the surface of the shell. the cysts were then fixed in 2.5% glutaraldehyde, prepared in cacodylate (sodium phosphate) buffer adjusted to ph 7.4 for several hours. the cysts were washed and post-fixed in 2% osmium tetroxide and then dehydrated in a graded alcohol series. thereafter, the cysts were air dried for a few minutes at room temperature, critical point dried and glued onto standard mica squares (1 cm2), mounted on metal stubs, subsequently coated with gold (sem leo 435 up) and viewed and photographed under a scanning electron microscope (leo stereo scan, 440). transmission electron microscopy: for transmission electron microscopy, dried cysts were fixed in 2% glutaraldehyde prepared in cacodylate (sodium phosphate) buffer (ph 7.4). the cysts were then post-fixed in 1% osmium tetraoxide and processed for electron microscopy. the tissue was embedded in epoxy resin and sectioned using ultra microtome. ultra-thin sections of the cysts were taken and stained with uranyl acetate and viewed using transmission electron microscope (philips, 20ic, netherlands). results scanning electron micrographs on the hydrated cyst of parthenogenetic and bisexual and exotic (bisexual) species of artemia showed smooth granular surface with no significant difference on the surface topography (fig. 1). transmission electron microscopic observations of the cysts showed a general pattern of outer cortex, inner alveolar layer and embryonic cuticle. the outer region is dark pigmented cortical layer followed by large alveolar layer which constitutes the tertiary cyst membrane. figure 2. transmission electron micrographs of a. parthenogenetica cyst (vrm population). (a) ultra-thick section showing the architecture of cyst membranes of an encysted gastrula, (b-d) transmission electron micrographs of tertiary cyst membranes, (c) ultrastructure of outer cortical layer and inner alveolar layer at higher magnification; note the thickness of the cortical layer and (d) alveolar membrane showing alveolar mesh with spongy nature. om outer membrane; clcortical layer; alalveolar layer; ocmouter cuticular membrane; flfibrous layer; icminner cuticular membrane; scssub cuticular space. emb-embryo. 269 sivagnanam et al/ international journal of aquatic biology (2013) 1(6): 266-272 the thickness of the outer membrane showed considerable variations between parthenogenetic and bisexual species. ultrastructure of outer cortical layer showed dense matrix with small pores arranged as a line in the centre and extended the width of the cortex. these pores are restricted in central matrix of the other cortical layer in a. parthenogenetica and it is distributed throughout the width of the matrix in artemia sp. (kbm population). a significant variation in the thickness of cyst membranes is found between bisexual and parthenogenetic species. the cortical region is not tightly bound to the alveolar region in a. parthenogenetica and is closely arranged in artemia sp. of both kbm and sfb populations. the outer protective cortex layer is followed by a broad spongy alveolar layer which is characterized by the presence of interconnecting chambers in all the strains. alveolar layer is much larger in size and tightly packed in a. parthenogenetica compared to kbm and sfb populations. in parthenogenetic cysts, the alveolar chambers are oval in shape and are loosely arranged; but in bisexual species, these are tightly attached each other and the chambers are round in shape and conjugated form and seems similar to that of sfb populations (figs. 2, 3 and 4). the alveolar layer is followed by a fibrous layer, located between the inner and outer cuticular layer. the outer and inner cuticular membranes are thin and appeared as triple layered biological membrane and not much variation noticed among the strains. embryonic mass is located safe beneath the inner cuticular membrane (figs. 2, 3 and 4). discussion the cysts of the two different populations (vrm parthenogenetic and kbm bisexual populations) of artemia have been thoroughly examined for morphological and ultrastructural variations. the surface topography of artemia cyst, distributed worldwide, has an externally smooth surface (anderson et al., 1970; trotman et al., 1987) except in a. monica. in the present study it was observed that the cysts of all the species studied exhibit smooth surface and no considerable variation was observed concerning the cyst surface topography. on the other hand, sugumar and munuswamy (2006) observed a button shaped structure on the cyst of puthalam artemia population apart from its normal smooth surface. tem studies showed different layers such cortical layer, alveolar layer, outer cuticular membrane, fibrous layer and inner cuticular membrane. the cross sections showed some difference in the arrangement of pores in cortical layer between figure 3. transmission electron micrographs of artemia sp. cyst (kbm population). (a) ultra thick section showing the architecture of cyst membranes of an encysted gastrula, (b-d) transmission electron micrographs of tertiary cyst membranes, (c) ultrastructure of outer cortical layer and inner alveolar layer at higher magnification; note the thickness of the cortical layer and (d) alveolar membrane showing alveolar mesh with spongy nature.omouter membrane; clcortical layer; alalveolar layer; ocmouter cuticular membrane; flfibrous layer; icminner cuticular membrane; scssub cuticular space; embembryo. 269 270 sivagnanam et al/ international journal of aquatic biology (2013) 1(6): 266-272 parthenogenetic and bisexual species. the consistently spherical shape of the alveoli suggests that they filled with or formed by gas bubbles, which may assist the cyst to float (morris and afzelius, 1967). the outer cover of the artemia cyst consists of an outer surface lamella, alveolar lamellae and a tertiary envelope (lee et al., 1994) followed by the embryonic cuticle and outer cuticle of the embryo. it is suggested that the function of the tertiary layer might be rather physical than chemical since many components can pass through this layer (hajirostamloo, 2008). because of hygroscopic nature, water must be omitted from the cysts if the shell is to act as a flotation device (hajirostamloo, 2008). the inner network space connection is restricted to the surface of the cyst shell and helps to reduce the water loss from the cysts when exposed to a dry environment. it may also help respiration to gastrula, like plastron respiration to keep it for a long period in live encysted conditions (clegg, 1974). similar results on the variation of the alveolar layer in different strains was reported by abatzopolous et al. (2006) and sugumar and munuswamy (2006). munuswamy et al. (1996) suggested that the difference in alveolar layer and alveolar mesh in rotifers provides a species specific pattern. emergence of nauplius from the hydrated cyst is induced by the light and the interpretation of light is based on the thickness of the cyst membrane. kulasekarapandian reported that parthenogenetic strain had low hatching percentage compared to bisexual strains, this may be due to the variations in the thickness (abatzopoulos et al., 2006). in the present study, parthenogenetic and bisexual species of artemia showed significant differences in their chorion thickness and such variations may because of the prevailing environmental conditions (such as salinity and ions) at kelambakkam saltpan. the studies on the cyst topography and ultrastructure of the three artemia populations in the present study demonstrate to a certain extent, that the variations in the alveolar and fibrous layers are more ‘suitable’ for characterizing artemia populations than whole homogenates of decapsulated cysts. our previous study on adult morphology, cyst and naupliiar biometry (krishnakumar, unpublished data) and protein profiles (sivagnanam, 2005) suggested that the artemia inhabiting kelambakkam saltern is like that of a. franciscana. the findings of the present study helps to further characterize this strain. further, the present study suggested that the cyst membrane thickness and difference in alveolar mesh can be considered to be taxonomic tools to characterize artemia strains as in rotifers (munuswamy et al., 1996) and fairy shrimps (walsche et al., 1991). this conclusion can also be figure 4. transmission electron micrographs of a. franciscana cyst (sfb population). (a) ultra thick section showing the architecture of cyst membranes of an encysted gastrula, (b-d) transmission electron micrographs of tertiary cyst membranes, (c) ultrastructure of outer cortical layer and inner alveolar layer at higher magnification note the thickness of the cortical layer and (d) alveolar membrane showing alveolar mesh with spongy nature. omouter membrane; clcortical layer; alalveolar layer; ocm outer cuticular membrane; flfibrous layer; icminner cuticular membrane; scssub cuticular space; embembryos. 271 sivagnanam et al/ international journal of aquatic biology (2013) 1(6): 266-272 better supported by close study of the results obtained with different artemia populations. acknowledgements financial assistance from the ministry of earth sciences (moes/11-mrdf/1/8/p/07), government of india is gratefully acknowledged. thanks are due to m/s. covelong salt works (skms), kelambakkam and salt commissioner, government of india for extending the saltpan facility for brine shrimp culture project. we are very much thankful to the anonymous reviewers and dr. hans dhams, sangmyung university, green life science department for their pertinent comments which improved the manuscript. references abatzopoulos t.j., beardmore j.a., clegg j.s., sorgeloos p. (2002). artemia: basic and applied biology, kluwer academic publishers, dordrecht. abatzopoulos t.j., baxevanis a.d., triantaphyllidis g.v., criel g., pador e.l., van stappen, g., razavi rouhani s.m., sorgeloos, p. (2006). quality evaluation of artemia urmiana gunther (urmia lake, iran) with special emphasis on its particular cyst characteristics (international study on artemia lxix), aquaculture, 254: 442-454. anderson e., lochhead j.h., lochhead m.s., huebner, e. (1970). the origin and structure of the tertiary envelope in thick-shelled eggs of the brine shrimp artemia. journal of ultrastructure research, 32(5-6): 497-525. belk d. sissom s.l. (1992). new branchinella (anostraca) from taxas, usa and the problem of antenna like processes. journal of crustacean biology, 12(2): 312-316. brendonck l., coomans a. 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(1987). abnormal development in artemia. defective emergence of the prenauplius with bicarbonate deficiency. journal of experimental zoology, 243(2): 225-232. van stappan g., fayazi g., sorgeloos p. (2001). international study on artemia lxiii. field study of artemia urmiana (gunther, 1890) population in lake urmiah, iran. saline lakes developments in hydrobiology, 162: 133-143 versichele d., sorgeloos p. (1980). controlled production of artemia cysts in batch cultures. in: g. personne, p. sorgeloos, o. roel, e. jaspers, (ed), the brine shrimp artemia, ecology, culturing and use in aquaculture, vol. 3, wettern, belgium, universa press, pp. 231-246 vikas p.a., sajeshkumar n.k., thomas p.c., kajal chakraborty, vijayan k.k. (2012). aquaculture related invasion of the exotic artemia franciscana and displacement of the autochthonous artemia populations from the hypersaline habitats of india. hydrobiologia, 684(1): 129-142. walsche c.d., munuswamy n., dumont h.j. (1991). structural differences between the cyst walls of streptocephalus dichotomus (baird), s. towicornis (waga), and thamnocephalus platyurus (packard) (crustacea: anostraca), and a comparison with other genera and species. hydrobiologia, 212(1): 195-202 wang s., sun s. (2007). comparative observations on the cyst shells of seven artemia strains from china. microscopic research and techniques, 70(8): 663–670. wurdak e.s., gilbert j.j., jagels r. (1978). fine structure of the resting eggs of the rotifers branchionus calyciflorus and asplanchna sieboldi. transactions of the american microscopical society, 97(1): 49-72. int. j. aquat. biol. (2023) 11(2): 104-114 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article effects of cultural conditions on life history characteristics of the freshwater rotifer brachionus calyciflorus trinh-dang mau1,2, duong quang-hung2 1the university of da nang-university of science and education, 459 ton duc thang street, lien chieu district, da nang, vietnam. 2danang environmental and biology resources teaching research team (dn-ebr), the university of da nang, 41 le duan street, hai chau district, da nang, vietnam. s article history: received 31 july 2022 accepted 23 april 2023 available online 2 5 april 2023 keywords: biological characteristics lifespan temperature algal density ph abstract: the effects of temperature, food concentration, and ph conditions on the life history characteristics of the freshwater rotifer, brachionus calyciflorus, were investigated. the culture temperature (20, 25, and 30°c) had a significant relationship with life history parameters. at lower temperatures, there was a prolongation of the mean lifespan and juvenile period. the optimal temperature for the fecundity of this species was 25°c with an average quantity of 23.67±6.99 offspring female-1 across the entire lifespan of 6.7 days. no significant difference was found between the mean lifespan at different algae densities but the maximum fecundity (25.75±6.99 offspring female-1) was obtained at an algae density of 10x106 cells.ml-1. brachionus calyciflorus could tolerate a broad range of ph (4-10) but preferred ph from 6-10. these results are critical for potential applications of this species in ecotoxicology, biomonitoring as well as in mass culture as live food for larval rearing in aquaculture. introduction rotifers are aquatic invertebrates that occur in almost all types of water bodies worldwide. rotifers are critical linkages between phytoplankton and planktivorous fish (wallace et al., 2006). they are a valuable live food source for the larvae culture of many fish species (lubzens, 1987). the rotifers were represented by many species of brachionus and others, indicating the trophic status of water bodies (baruah et al., 1996; nogueira, 2001; ismail and adnan, 2016). among freshwater monogonont rotifers, brachionus calyciflorus pallas, 1766 is widely studied in many fields, including ecology (gilbert, 1985; guo et al., 2011), evolutionary biology (becks and agrawal, 2012; scheuerl and stelzer, 2013; declerck et al., 2015), ecotoxicology (snell and moffat, 1992; cruciani et al., 2016; han et al., 2018), and aquaculture (lim and wong, 1997). brachionus calyciflorus was also the first monogonont rotifer correspondence: trinh-dang mau doi: https://doi.org/10.22034/ijab.v11i2.1659 e-mail: tdmau@ued.udn.vn dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.3.5 that its genome was characterized (kim et al., 2018) and suggested to be a species complex with at least four cryptic species (papakostas et al., 2016). in vietnam, most studies on rotifers focused on their biodiversity and distribution (shirota, 1966; dang and ho, 2002; zhdanova, 2011; phan and le, 2012; trinh-dang et al., 2015, 2019; duong-quang et al., 2020). some species of the genus brachionus such as b. plicatilis, b. rotundiformis, b. angularis, and b. have been investigated to be used as live food for aquaculture in laboratory conditions (le et al., 2017; quy et al., 2018; cong et al., 2019; vu et al., 2021). accurate estimation of the biological characteristics of rotifers in the field is quite difficult. meanwhile, laboratory culture of organisms under similar natural ranges of environmental parameters is one of the methods to determine growth and reproductive parameters. in addition, life history characteristics such as lifespan and fecundity are important approaches to studying the life history mailto:tdmau@ued.udn.vn 105 int. j. aquat. biol. (2023) 11(2): 104-114 strategy and population dynamics of zooplankton under continuously changing environmental conditions (wallace et al., 2006). in the present study, b. calyciflorus was collected from a freshwater body in danang city, central vietnam, an area with a tropical monsoon climate, and cultured under laboratory conditions. then, its juvenile period, embryonic development, spawning interval, fecundity, and mean lifespan were determined under different culture conditions of temperature, food concentration, and ph. materials and methods sample collection and stock culture: brachionus calyciflorus individuals were isolated in december 2020 from lake cong vien 29/3 (16°03'51.8"n 108°12'15.6"e) in danang city, vietnam, and then cultured under laboratory conditions at the laboratory of algal technology, faculty of biology and environmental science, the university of danang, vietnam. stock cultures were kept under static-renewal conditions for approximately 3 months with fluorescent light at 25±1°c in an epa medium. the epa medium is prepared by dissolving 96 mg of nahco3, 60 mg of caso4, 60 mg of mgso4, and 4 mg of kcl in 1 l of distilled water (peltier and weber, 1985). rotifers were fed every 2 days with the green algae of chlorella vulgaris at a density of 1.0-2.0x106 cells.ml-1, which was semicontinuously cultured in bbm medium with 16: 8 light: dark photoperiod of 3000 lx fluorescent light. before feeding the rotifers, the algae were precipitated by centrifugation (3000 rpm for 5 minutes), resuspended in an epa medium, and then stored at 4°c. the density of the stock algae was estimated using a hemocytometer (tiefe, 0.1 mm, 1/400 qmm, germany). life history assays: life history experiments were conducted in 96-well tissue cultures plates and started by introducing amictic neonate (< 2 h old) into each well containing 0.3 ml of epa medium. a total of 52 individuals were included and set up at three temperature levels, six ph levels, and four food concentrations. four replicates were made for each setup of the experiment. specifically, to examine the effects of different temperatures on the life cycle of b. calyciflorus, the solutions were maintained at different temperatures (20, 25, and 30°c) and fed on the algae c. vulgaris at 10x106 cells.ml-1. for the impact of different food concentrations, the experiments were implemented with solutions at different algae densities, including c. vulgaris at 1.5, 5, 10, and 15x106 cells.ml-1 at 25°c. for ph experiments, the solutions with the required ph (3.5, 4, 6, 7, 8, and 10) were maintained at 25°c and an algae density of 10x106 cells. ml-1. the ph of the culture medium was measured with a ph meter (hach hq411d) and adjusted by adding naoh (0.1 mol. l-1) and hcl (0.1 mol.l-1) into the medium (mitchell, 1992). the rotifers were observed every 1.5 h with a stereo microscope. the time of the first egg, the number of eggs and neonates produced, and the number of original individuals alive were recorded, respectively, and the neonates were removed. before each individual of every cohort died in the life history experiments, the original rotifers were transferred into a freshly prepared test solution every 24 h. based on the data collected, five life history parameters were calculated based on previously published methods, including the juvenile period (the time between a neonate and that laying the first egg), embryonic development (the time between an adult laying an egg and the egg hatching), spawning interval (the time between two spawnings), fecundity (the number of offspring produced per female), mean lifespan (the average surviving time of all females) (paez, 1991; walz, 1983). data analysis: differences between means for each life history parameter were compared using a oneway analysis of variance (anova). significant differences (p<0.05) were further analyzed with tukey's test. all statistics were calculated using the r program (r core team, 2020). results morphological characteristics: some morphologyical characteristics at different stages of the life cycle 106 mau and quang-hung./ effects of cultural conditions on life history characteristics of the freshwater rotifer of b. calyciflorus isolated from danang, central vietnam are shown in figure 1. the average body length and width (w) of adult females (mean±standard deviation, n=3) were 298.00±23.07 and 192.67±11.68 µm, respectively. newborn males and females produced by adult females had smaller body lengths, which were 165.67±4.04 and 219.33±10.07 µm, respectively. in addition, females of b. calyciflorus produced three different types of egg viz. diploid, haploid, and resting with average lengths of 112.67±5.69, 86.67±2.08, and 182.67± 6.43 µm, respectively (table 1). effects of temperature on life-history parameters of b. calyciflorus: the environmental temperature had a major effect on the life-history parameters of b. calyciflorus. the juvenile period, embryonic development, spawning internal, and mean lifespan steadily decreased with the increase of the environmental temperature (table 2, fig. 2). in particular, the mean juvenile period of b. calyciflorus was 33±1, 19.5±1.29, and 9±1 h at 20, 25, and 30°c, respectively (p<0.001). at the higher temperature, the mean lifespan was also significantly reduced (p<0.05). nevertheless, the optimal temperature for the fecundity of this species was at 25°c with an average quantity of 23.67±6.99 offspring female-1 across the entire lifespan of 6.7 days. at this temperature, the highest quantity of 35 figure 1. morphological characteristics of life-cycle stages of brachionus calyciflorus isolated from danang, central vietnam. (a) amictic female carrying three diploid eggs developing parthenogenetically into females; (b) newborn female; (c) mictic female carrying five haploid eggs developing parthenogenetically into haploid males; (d) newborn male; (e) fertilized mictic female carrying one encysted diapausing embryo, or resting egg. table 1. morphological characteristics of life-cycle stages of b. calyciflorus isolated from danang, central vietnam. life-cycle stages of b. calyciflorus length (µm) width (µm) posterior lateral spines (µm) anterior lateral spines (µm) anterior median spines (µm) adult female 298.00±23.07 192.67±11.68 60.00±2.65 43.00±2.00 52.33±2.52 newborn female 219.33±10.07 119.67±10.02 44.67±1.53 28.33±1.53 32.67±2.08 newborn male 165.67±4.04 60.67±2.08 diploid egg 112.67±5.69 102.00±6.24 haploid egg 86.67±2.08 62.33±2.52 resting egg 182.67±6.43 120.33±5.51 table 2. juvenile period (jp), embryonic development (ed), spawning internal (si), fecundity (f), mean lifespan (ml) of brachionus calyciflorus at different temperatures (mean±sd). within a column, means with the same superscript letter are not significantly different (p>0.05). temperatures (°c) jp (h) ed (h) si (h) f (offspring. female-1) ml (h) 20 33.00±1.00a 21.00±6.00a 6.11±0.51a 11.00±8.19a 218.67±14.43a 25 19.50±1.29b 11.50±0.58b 3.96±0.93b 23.67±6.99b 161.25±29.61b 30 9.00±1.00c 9.33±0.58b 2.22±0.38c 13.67±1.53ab 64.33±10.97c 107 int. j. aquat. biol. (2023) 11(2): 104-114 offspring female-1 was also reached. at 30°c, the mean fecundity of individuals was slightly higher than those maintained at 20°c with values of 13.67±1.53 and 11.00±8.19 offspring female-1, respectively. effects of algae density on life history parameters of b. calyciflorus: the life history parameters of b. calyciflorus at different densities of c. vulgaris as live food for the rotifers are shown in table 3. the juvenile period and spawning internal of this species figure 2. life history characteristics of brachionus calyciflorus at different temperatures. table 3. juvenile period (jp), embryonic development (ed), spawning internal (si), fecundity (f), mean lifespan (ml) of brachionus calyciflorus at different algae densities (mean±sd). within a column, means with the same superscript letter are not significantly different (p>0.05). densities of c. vulgaris (cells.ml-1) jp (h) ed (h) si (h) f (offsprings. female-1) ml (h) 1.5 x 106 25.33±6.03a 11.00±1.00a 15.78±5.83a 7.67±2.31a 177.33±61.01a 5 x 106 21.33±4.04a 11.00±1.73a 5.81±1.89b 14.33±6.81ab 155.33±24.01a 10 x 106 19.5±1.29a 11.50±0.58a 3.96±0.93b 25.75±6.99b 161.25±29.61a 15 x 106 18.50±0.58a 12.00±0.82a 3.25±0.69b 23.25±4.65b 176.75±25.75a figure 3. life history characteristics of brachionus calyciflorus at different densities of algae c. vulgaris. 108 mau and quang-hung./ effects of cultural conditions on life history characteristics of the freshwater rotifer were decreased with increasing food concentration (fig. 3). the mean juvenile period decreased from 25.33±6.03 h at an algae density of 1.5x106 cells.ml1 to 18.50±0.58 h at a density of 15x106 cells.ml-1. besides, a significant difference was also observed in the spawning internal between the algae density of 1.5x106 cells.ml-1 and the higher experimental algae densities (p<0.01). the maximum fecundity of b. calyciflorus was 25.75±6.99 offspring female-1 at a density of 10x10 6 cells.ml-1 and the minimum fecundity of 7.67±2.31 offspring female-1 was found at a density of 1.5x10 6 cells.ml-1 (3.4 times smaller). no significant difference was found between the fecundity at densities of 10x106 and 15x106 cells.ml-1, which showed that the number of offspring by females could reach a limited value in the experiment. for lifespan, no significant difference was found between different algae densities. the lowest mean lifespan (155.33±24.01 h) was observed at a density of 5x106 cells.ml-1. the highest mean lifespan (177.33±61.01 h) was found at a density of 1.5x106 cells.ml-1, however, the values of individuals were unstable. at densities of 10x106 and 15x106 cells.ml1, the mean lifespans were 161.25±29.61 and 176.75±25.75 h, respectively. effects of ph level on life history parameters of b. calyciflorus: brachionus calyciflorus could tolerate a broad range of ph from 4 to 10 in the experimental condition (table 4, fig. 3). however, this species prefers a ph of 6-8 and might adapt to alkaline environments rather than acidic environments. this is demonstrated by a better performance of most of the life history parameters at ph from 6 to 10. in contrast, the instability of these parameters was observed at ph 4, and all individuals of this species could not survive to 24h at ph 3.5. the embryonic development and spawning internal table 4. juvenile period (jp), embryonic development (ed), spawning internal (si), fecundity (f), mean lifespan (ml) of brachionus calyciflorus at different ph levels (mean±sd). within a column, means with the same superscript letter are not significantly different (p>0.05). ph jp (h) ed (h) si (h) f (offsprings. female-1) ml (h) 3.5 all individuals could not survive to 24h 4 22.50±4.12a 10.25±1.50a 8.00±2.76a 12.50±4.73a 158.00±34.13a 6 20.00±1.00a 12.00±1.00ab 3.22±0.38b 23.00±6.00a 168.00±0.00a 7 18.25±0.5a 13.75±0.50b 3.29±1.39b 24.50±6.45a 179.00±0.00a 8 19.33±1.53a 11.67±1.15ab 4.17±0.83ab 23.67±2.52a 183.00±17.35a 10 19.50±1.91a 12.00±0.82ab 4.71±1.69ab 20.75±5.91a 173.25±22.02a figure 4. life history characteristics of brachionus calyciflorus at different ph levels. 109 int. j. aquat. biol. (2023) 11(2): 104-114 of this species had a significant difference between ph 7 and ph 4 (p<0.05). discussion differences in morphological characteristics between b. calyciflorus strains: in this study, b. calyciflorus was found in both male and female forms, and also resting eggs were observed. most planktonic rotifers have a cyclical parthenogenetic life cycle where asexual reproduction predominates, but there are periods where both asexual and sexual reproduction occur simultaneously (snell and carmona, 1995). in monogonent rotifers, asexual reproduction in the absence of males (amictic phase) is mixed with occasional bouts of sexual reproduction (mictic phase). asexual (amictic) females are diploid, they produce eggs mitotically that develop into females (birky jr and gilbert, 1971). asexual females after receiving the mimic stimulus can produce both sexual (mictic) and asexual daughters. sexual female production is then followed by male production, fertilization, and resting egg formation. sexual females produce haploid eggs which can develop into haploid males if unfertilized. in contrast, if fertilization happens, mictic eggs become diploid and develop into resting eggs (gilbert, 1974). the resting eggs after a period of dormancy will hatch into asexual females when receiving specific cues and enter into the asexual phase again (pourriot and snell, 1983). there were significant differences in morphological characteristics and life table parameters between rotifer species or strains (wang et al., 2014). in this study, the morphological characteristics of the life-cycle stages of b. calyciflorus isolated from danang, central vietnam were investigated and the body size of adult females and diploid egg size were measured and compared with other strains from various water bodies (table 5). in general, the adult females of b. calyciflorus had body length ranges from 176 to 298 µm and body width ranges from 121 to 193 µm. brachionus calyciflorus isolated from danang is one of the largest known body lengths of adult females and is much larger than other strains from several water bodies around the world (rico-martínez et al., table 5. morphological characteristics of brachionus calyciflorus strains from water bodies. life-cycle stages of b. calyciflorus length (µm) width (µm) posterior lateral spines (µm) anterior lateral spines (µm) anterior median spines (µm) origin references adult female 298.0±23.1 192.7±11.6 60.0±2.7 43.0±2.0 52.3±2.5 da nang, vietnam this study 291±24 193±16 florida, usa lim et al. (1997) 244.0±21.1 160.7±18.6 gainesville, usa rico-martínez et al. (1992) 277.3±23.0 164.3±16.8 tampa, usa 195.3±16.8 121.3±18.1 mcfarland, usa 266.0±21.9 163.0±25.2 madison, usa 249.6±1.2 158.5±0.7 44.3±1.3 35.4±0.5 46.3±0.5 lake fengming, china xue et al. (2017) 239.8±1.6 153.2±0.9 33.0±1.2 33.0±0.5 48.2±0.7 lake hui, china 255.1±1.4 160.6±0.8 34.5±1.0 36.0±0.5 51.1±0.6 lake tingtang, china 196.9±14.6 a pond in guangzhou, china xi et al. (2002) 176.1±23.5 a pond in wuhu, china 196 ± 12 156 ± 8 a pond in beijing, china yin et al. (2008) diploid egg 112.7±5.7 102.0±6.2 da nang, vietnam this study 122.6±0.7 87.1±0.3 lake fengming, china xue et al. (2017) 116.7±0.9 84.8±0.5 lake hui, china 116.3±0.8 82.6±0.5 lake tingtang, china 110 mau and quang-hung./ effects of cultural conditions on life history characteristics of the freshwater rotifer 1992; xi et al., 2002; yin et al., 2008). some variation exists in size and growth rate among different strains of b. calyciflorus are similar to the variation found for b. plicatilis (snell and carrillo, 1984). consequently, the variation among strains gives this species an advantage over other species in its use as food for larval fish because different size strains will be suitable for different-sized larval fish (rico-martínez and dodson, 1992). in contrast, there were not many differences in the diploid egg size of b. calyciflorus among strains, which range from 112.67 to 122.6 µm in length and from 82 to 102 in width. effects of temperature, food concentration, and ph conditions on life history parameters of b. calyciflorus: various factors, including temperature, food concentration, and ph conditions affect the survival and reproduction of rotifers. temperature is one of the factors that most influence the population growth of rotifers. previous works indicated that the responses in life-history traits to increasing temperature differed not only for different species of rotifers but also for different strains of the same species (awaïss et al., 1992; kauler et al., 2011; wang et al., 2014; xiang et al., 2016). in general, the developmental rate of poikilothermic animals depends on the metabolic rate, which increases with temperature. therefore, increasing temperature will lead to a decrease in life expectancy at hatching, average lifespan, and generation time of rotifers (xiang et al., 2010). in the present study, we found that the juvenile period, embryonic development, spawning internal, and mean lifespan were steadily decreased with increasing environmental temperature, but the optimal temperature for the fecundity of this species was table 6. the effects of temperature conditions on life history characteristics of brachionus calyciflorus strains from water bodies. temperat ures (°c) jp (h) ed (h) f (offspring. female-1) ml (h) origin references 15-16 39.8±6.2 14.1±4.7 270.9±71.5 a pond in gainesville, florida kauler et al. (2011). 45.7±1.2 29.4±1.5 334.2±8.3 lake baixiang, china wang et al. (2014) 43.4±1.2 25.1±1.1 212.6±10.3 lake kongque, china 20-22 33.0±1.0 21.0±6.0 11.0±8.2 218.7±14.4 da nang, vietnam this study 24.6±2.2 13.4±1.7 ghent, belgium awaïss et al. (1992) 31.8±1.1 17.1±1.0 13.0 ± 0.29 154.4 ± 16.1 lake dianchi, china xiang et al. (2016) 31.9±0.8 16.5±0.2 11.3 ± 0.23 167.2 ± 1.6 xishuangbanna, china 18 ± 4.3 14.7±5.1 153.1±44.4 a pond in gainesville, florida kauler et al. (2011) 36.2±0.9 21.4±1.1 340.8±8.5 lake baixiang, china wang et al. (2014) 28.4±0.1 19.7±0.8 176.4±4.9 lake kongque, china 24-25 19.5± 1.3 11.5± 0.6 23.7± 6.9 161.3±29.6 da nang, vietnam this study 17.9±0.8 10.1±1.2 ghent, belgium awaïss et al. (1992) 32.4±0.7 17.7 ± 0.8 13.2 ± 0.6 146.4 ± 7.7 lake dianchi, china xiang et al. (2016) 17.9±1.1 17.9 ± 0.3 14.6 ± 0.9 166.4 ± 8.1 xishuangbanna, china 25.7±0.6 12.6±0.4 208.9±7.7 lake baixiang, china wang et al. (2014) 17.8±0.3 15.6±0.5 95.6±2.6 lake kongque, china 28-32 9.0±1.0 9.3±0.6 13.7±1.5 64.3±10.9 lake cong vien 29/3, vietnam this study 10.8±1.2 7.0±0.9 ghent, belgium awaïss et al. (1992) 16.3±1.6 11.3±0.6 11.5 ± 0.6 48.8 ± 5.8 lake dianchi, china xiang et al. (2016) 16.5±0.7 10.0±0.4 13.0±1.3 73.6±3.5 xishuangbanna, china 7.7±2.4 17.3±6.1 98.9±27.1 a pond in gainesville, florida kauler et al. (2011). 15.8±0.5 12.0±0.5 161.2±4.6 lake baixiang, china wang et al. (2014) 13.9±0.3 9.3±0.4 79.3±2.0 lake kongque, chin 111 int. j. aquat. biol. (2023) 11(2): 104-114 25°c. the effects of temperature on the net reproductive rate and fecundity differ not only among different rotifer strains but also among distant morphotypes (xiang et al., 2010; wang et al., 2014). xiang et al. (2010) suggested that the net reproductive rate of the two-spined b. calyciflorus was higher at 30°c than at the other temperatures, but the net reproductive rate of the unspined rotifer was not affected by temperatures. algae concentration is one of the important factors to influence the growth, movement, and reproduction of rotifers. in theory, a high algae concentration in the environment will increase the opportunity for rotifers to be fed enough, thus, might grow well (liang et al., 2017). aside from algal concentration, algal diets and algal food quality were also reported to affect the life history of amictic rotifers (ruttner-kolisko, 1984; jensen and verschoor, 2004). according to xi et al. (2001), there were no significant effects of algal food type on the duration of the reproductive period of the three types of females, but a significant effect on the duration of the juvenile period of amictic females and unfertilized mictic females was recorded. the duration of the juvenile period of amictic females fed c. pyrenoidosa was shorter than that of those fed s. obliquus or a mixture of both algae species, and that of unfertilized mictic females fed c. pyrenoidosa was longer than that of those fed s. obliquus. thus, these two types of females appear to respond differently to these algae. rico-martínez and dodson (1992) suggested that the optimum conditions for raising the rotifer b. calyciflorus were a temperature of 30°c and a food concentration of 107 cells.ml-1 of the algae c. vulgaris. this is confirmed by our results as we explored the maximum fecundity of b. calyciflorus at the algae concentration of 10x106 cells.ml-1 and no significant difference was found between the fecundity of this species at concentrations of 10x106 cells.ml-1 and 15x106 cells.ml-1. nevertheless, the appropriate algae concentration for raising the rotifer in general also depends on temperature. at a temperature of 20°c and high food concentration of 5x107 cells.ml-1, negative growth rates were found. this result was in line with other studies (halbach and halbach-keup, 1974; neimeroth, 1980), which explain the negative growth as being a consequence of high respiratory costs. the response of b. calyciflorus to ph also received many concerns (mitchell and joubert, 1986; mitchell, 1992; yin and niu, 2008). the distribution and abundance of rotifers are confirmed to be affected by ph (wallace and snell, 2001). species in the genus brachionus such as b. angularis, b. calyciflorus, and b. quadridentatus are believed to be common alkaline species. b. calyciflorus, b. quadridentatus, b. urceolaris, and b. patulus table 7. the effects of food concentrations on life history characteristics of brachionus calyciflorus strains from water bodies. concentrations of algae (cells.ml-1) jp (h) ed (h) f (offsprings. female-1) ml (h) origin references chlorella vulgaris 1.5x106 25.3±6.0 11.0±1.0 7.7±2.3 177.3±61.0 da nang, vietnam this study 5x106 21.3±4.0 11.0±1.7 14.3±6.8 155.3±24.0 10x106 19.5±1.3 11.5±0.6 25.75±7 161.3±29.6 15x106 18.5±0.6 12.0±0.8 23.3±4.7 176.8±25.8 scenedesmus obliquus 0.5x106 37.1±1.2 14.5±0.9 6.7±0.4 201.0±4.2 lake baixiang, china wang et al. (2014) 1.0x106 30.4±1.3 13.3±0.6 6.4±0.5 203.3±5.8 2x106 28.5±0.8 13.4±0.9 7.7±0.5 203.1±6.1 4x106 25.7±0.6 12.6±0.4 10.7±1.11 208.9±7.7 scenedesmus obliquus 2x106 32.4±0.7 17.7±0.8 13.2±0.6 146.4±7.7 lake dianchi, china xiang et al. (2016) 2x106 17.9±1.1 17.9±0.3 14.6±0.9 166.4±8.1 xishuangbanna, china scenedesmus obliquus 3x106 6.4±0.4 91.6±7.8 lake liantang, china xiang et al. (2010) chlorella pyrenoidosa 5x106 20.4±2.3 5.8±1.4 72.2±10.4 lake donghu, china xi et al. (2001) 112 mau and quang-hung./ effects of cultural conditions on life history characteristics of the freshwater rotifer showed a higher fecundity at ph from 6 to 8 (yin and niu, 2008). mitchell (1992) found that the organisms were unable to survive for 24 h at a ph of 11.5, and the lethal concentration of the organisms was less than 2 days at a ph of 2.5. similar results were obtained in this study as we recorded b. calyciflorus prefers a ph of 6-10 and might adapt to alkaline environments rather than acidic environments. this is demonstrated by a better performance of most of the life history parameters at ph from 6 to 10. in contrast, the instability of these parameters was observed at ph 4. all individuals of this species could not survive for 24 hours at ph 3.5. conclusion ambient temperature, ph, and food concentration are important factors to influence the rotifer b. calyciflorus isolated from the freshwater body in vietnam. the optimal temperature for the fecundity of this species was 25°c and a food concentration of 10x106 cells.ml-1 of c. vulgaris. similar to other species and strains in the genus brachionus, the species b. calyciflorus in our study also prefer alkaline environments than acidic environments, which ph ranges from 6 to 10. acknowledgements we would like to thank the faculty of biology and environmental science, university of science and education udn for providing research facilities. references awaïss a., kestemont p (1992). an investigation into the mass production of the freshwater rotifer brachionus calyciflorus pallas. 2. influence of temperature on the population dynamics. aquaculture, 105: 337-344. baruah b.k., baruah d., das m. 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(2017) 5(2): 95-107; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article assessment of suitability of macrobenthic mollusc diversity to monitor water quality and shallow sediment quality in a tropical rehabilitated and non-rehabilitated wetland system w.m. dimuthu nilmini wijeyaratne*,1bellanthudawage kushan aravinda bellanthudawa department of zoology and environmental management, faculty of science, university of kelaniya, sri lanka. article history: received 12 january 2017 accepted 9 april 2017 available online 2 5 april 2017 keywords: biomonitoring principal component analysis diversity indices sri lanka abstract: six sampling sites were selected to represent different land use types in the rehabilitated and non-rehabilitated areas of a recreational wetland in sri lanka to study the suitability of macrobenthic mollusc diversity to monitor spatial and temporal variation in physico-chemical parameters of water and shallow sediments. individuals belonging to six families and eight species were recorded during the study. the significantly highest mean abundance (individuals) of bithynia tentaculata and pila globosa were recorded in sites from the rehabilitated area and there was no significant temporal variation of mollusc abundance during the study. the abundance and diversity of mollusc community showed significant spatial variations and this study identified that b. tentaculata and p. globosa can be used as possible bioindicators to detect changes in water and shallow sediment quality in tropical wetland ecosystems introduction molluscs are a very diverse and species rich phylum that is widely distributed in many types of ecosystems throughout the world. different species of molluscs perform key functions in ecosystems maintaining the ecosystem balance and material cycling. also, they serve as important components in the food webs of ecosystems as they have representatives in almost all the trophic levels. in addition, molluscs have been identified as excellent bioindicators due to their relatively immobility or sedentary adult stages, close contact with both bottom sediments and the water column, and ability to respond to many types of anthropogenic impacts, such as nutrient enrichment, oxygen availability, and changes in habitat structure (bonada et al., 2006; feio et al., 2007; mereta et al., 2013). adult stages of mollusc life cycle mostly occupy the bottom habitats in a wetland and therefore they are always in contact with shallow soft sediments and overlying water column. this allows them to respond quickly to any physical or chemical change in these environments. therefore, the temporal and * corresponding author: w.m. dimuthu nilmini wijeyaratne doi: https://doi.org/10.22034/ijab.v5i2.288 e-mail address: dimuthu.wijeyaratne@kln.ac.lk spatial variation of abundance, composition, and distribution of molluscs provide an index of the ecosystem (barbour et al., 1999). the wide distribution, ecological and ecotoxicological importance of the molluscs in ecosystem monitoring have attracted a lot of attention. several biomonitoring studies have indicated that river bed compaction due to agricultural practices, eutrophiccation and different types of pollutants as causal factors for decline of freshwater molluscs species (hartmut and gerstmann, 2007). the adult and juvenile stages of freshwater mussel populations (villosa iris and anodonta grandis) were recorded to be declining due to activities such as dredging, agricultural runoff, industrial pollution and sedimentation (jacobson et al., 1993). the accumulation of chromium (cr) and cadmium (cd) in soft parts of bivalve caelatura companyoi and the gastropod snail cleopatra bulimoides have showed the suitability of these species as suitable bioindicators of heavy metal pollution (moloukhia and sleem, 2011). in addition, juvenile stages of lampsilis cardium has 96 wijeyaratne and bellanthudawa/ use of macrobenthic diversity as bioindicator in a tropical wetland system been used for assessment of pore water ammonia in several regions of usa (bartsch et al., 2003). heavy metal contamination of wetland ecosystems has also been assessed using bivalves such as mussels and oysters in temperate waters (paez-osuna et al., 1995; riget et al., 1997). also some recent ecological studies have used the presence-absence and abundance data of the gastropod patella caerulea to assess heavy metal contamination in the iskenderun bay (turkmen et al., 2005; yuzereroglu et al., 2010). gastropod and pelecypod species were identified as ideal bio-indicators for trophic stages classification of lentic and lotic environments (clarke, 1979; choubisa, 1992). the gastropod corbicula fluminea has been used in taiwan to study arsenic (as) pollution via a biological early warning system in wetland health assessment. in malaysia, melonides tuberculata a freshwater mollusc has been utilized to assess the acute toxicity of cu, cd, zn, pb, ni, fe, al, and mn (shuhaimi-othman et al., 2012). although the molluscs are considered as an important bioindicator in most different ecosystems in the world, in sri lanka, mollusc species are less popular as bioindicator organisms compared to fish and other bioindicator species. there are very few published research on use of mollusks to trace ecotoxicity in the aquatic systems in sri lanka. in a study in bolgoda canal and waga stream, paludomus loricatus, p. zeylanicus, planorbella trivolvis, promacea bridgesi, and cerithiidae sp. have shown significant relationships with the variation of physicochemical parameters (idroos and manage, 2012). a comprehensive study done in the stretch in the dutch canal, sri lanka, highlighted the importance of melanoides tuberculata, thiara acanthica, faunas ater, and neritina perottetiana as bioindicators (gamlath and wijeyaratne, 1997). also another study conducted in the negombo estuary to evaluate the diversity and distribution of microbenthic community with special reference to changing environmental variables indicated that the molluscs species such as cerithidea cingulate, dentalium sp., hydrobiid sp., terebralia palustris, faunus ater, thiarid sp., and several venerid sp., tellind sp., mytilid sp., are applicable as a good bioindicators in wetland health assessments (dahanayaka and wijeyaratne, 2010). the present study was conducted in the diyawwannawa wetland system which is a very popular recreational wetland located in the commercial capital of sri lanka. this wetland system is located in a very urbanized environment and part of the wetland is maintained under pristine conditions, while other part is a rehabilitated area surrounding the parliament of sri lanka. the rehabilitated part of the wetland is dedicated for recreational purposes and effects of human disturbances in the rehabilitated area is higher compared to the natural wetland area. due to the human interferences, there can be changes in the water and sediment quality of the rehabilitated area of the wetland system compared to the natural area and these changes may have significant effects on the associated sediment community structure. however, no previous research have been conducted to compare the changes of water and sediment quality and benthic community structure of these two areas of the dyawannawa wetland system. therefore, the present study was conducted to examine differences of water and shallow sediment quality parameters in the rehabilitated and natural areas of the diyawannawa wetland system. in addition, as diversity of molluscs is closely related to the sediment and water quality of their associated habitats, hence, this study focused on effects of differences in water and sediment quality parameters on the diversity and abundance of macrobenthic molluscs in the diyawannawa wetland system. materials and methods study area: diyawannawa wetland is one of the most popular urban wetlands in colombo district sri lanka. this wetland is composed of rehabilitated and nonrehabilitated areas. in the rehabilitated areas of the wetland, some parts are dredged, banks are restored and the aquatic vegetation is removed using mechanical removal methods to improve the water retention capacity of the wetland. in the non– rehabilitated area of the wetland, the human 97 int. j. aquat. biol. (2017) 5(2): 95-107 interventions on the wetland ecosystem are minimum and this area is rich in biodiversity and provides habitats for several threatened and endemic species in sri lanka. the present study was carried out from april to december 2016. six sampling sites were selected from the study area. the location of the sampling sites in the diyawannawa wetland is shown in figure 1. site a (06°54'.585''n, 079°54'.722''e), site b (06°54' .664''n, 079°54'.633''e) and site c (06°54'.609''n, 079°54'.604''e) were located in the non-rehabilitated area of the wetland. site d (06°54'.68''n, 079° 54'.610''e), site e (06°54'.751''n, 079°54'.735''e) and site e (06°54'.741''n, 079°54'.525''e) were located in the rehabilitated area. sites a is characterized by thick vegetation cover in the wetland banks as well as presence of high density of floating aquatic macrophytes. sites b and c are characterized by having a thick vegetation cover in the wetland banks and site d was characterized by absence of vegetation cover in the banks and presence of thick growth of aquatic macrophytes. site e was receiving runoff from small scale poultry farms located within 100 meters of the wetland. site f was located in a highly urbanized area. water and sediment quality parameters: from each site, water samples and shallow sediments samples (00.5 m depth) were collected. sampling was carried out once in 6 weeks for a period of 7 months from april to december in 2016. at each sampling sites, water ph, temperature, conductivity, total dissolved solids (tds) and salinity were measured in-situ using a calibrated digital multi parameter (ysi environmental model-556 mps). dissolved oxygen concentration (do) was measured using do meter (hq 40b modelhach). visibility was recorded using a secchi disk. the biological oxygen demand 5 days after incubation (bod5), chemical oxygen demand (cod), nitrate concentration, chlorophyll 𝛼 concentration and total and dissolved phosphorus concentrations were measured based on apha (1992). at each sampling site, sediment ph was measured in-situ using the calibrated digital multiparameter (ysi environmental model-556 mps). sediment organic matter content was measured in the laboratory using the loss on ignition method and the percentage sand, silt and clay content of the sediments were measured using the sedimentation jar. water and sediment quality parameters were measured in triplicate within a 1 m2 area and averaged for each sampling site. benthic mollusc diversity: benthic mollusc sampling was carried out using the peterson grab sampling method (dahanayake and wijeyaratne, 2006). the collected samples were preserved in 5% rose bengal solution on site and were transported to the laboratory. in the laboratory at the university of kelaniya, sri lanka, the samples were subjected to wet sieving through 4 mm, 2 mm and 1 mm mesh sieves to separate benthic molluscs. the organisms retained in each sieve were collected and preserved in 10% formalin and after a week they were transferred into 10% ethyl alcohol to prevent dehydration as described figure 1. the study sites in the diyawannawa wetland, sri lanka. sites a, b and c are located in the non-rehabilitated area and sites d, e and f are in the rehabilitated area. 98 wijeyaratne and bellanthudawa/ use of macrobenthic diversity as bioindicator in a tropical wetland system by dahanayaka and wijeyaratne (2006). molluscs were identified to family level using keys given by fernando and weerawardhena (2002). the number of individuals of each taxon and the number of families at each sampling site were counted. using the species abundance data, species richness, taxa richness (tr), species density, simpson’s diversity index (d), shannon-wiener diversity index (h’), pielou's evenness index (j), modified hilsenhoff biotic index and percent contribution of dominant family were calculated for each sampling site at each sampling occasion and data were averaged over time for six sampling sites. statistical analysis: after confirming the normality using anderson darling test, the data were analyzed using one way anova followed by tukey’s pairwise comparison to determine the significance of the spatial and temporal variation of selected water quality and sediment quality parameters in the rehabilitated and non-rehabilitated sites of the diyawannawa wetland. a principal component analysis (pca) was used to determine water and sediment quality parameters and diversity and biotic indices that describes the distribution of benthic mollusks in the diyawannawa wetland. minitab 14 statistical software package was used in the statistical analysis. results spatial variation of water quality parameters in sampling sites is given in table 1. temporal variation of shallow bottom water quality parameters is given in table 2. there was no spatial variation in temperature, salinity and total phosphorous (p>0.05). site a of the non-rehabilitated area showed significantly higher water ph, conductivity and tds. however, this site showed significantly lower cod, do and bod5 (p<0.05). the site b of the non-rehabilitated area and all the sites in the rehabilitated area showed significantly higher chlorophyll 𝛼 concentration throughout the study period (p<0.05). there was no significant temporal variation in temperature, do and salinity (p>0.05). however, the other physicochemical parameters showed significant temporal fluctuations throughout the study period (table 2). the spatial variation of shallow sediment quality parameters in each sampling site is given in table 3. the temporal variation of shallow sediment quality parameters is given in table 4. the ph, percentage total organic carbon (toc) and the percentage silt content of the shallow sediments did not show significant spatial variations (p>0.05), but the percentage sand content at sites e and f of the rehabilitated area were significantly lower and table 1. spatial variation of water quality parameters at the sampling sites of the diyawannawa wetland during the study period. the results are presented as mean±standard error of mean. different superscripts in each row indicate statistically significant differences (p<0.05). parameter non-rehabilitated area rehabilitated area site a site b site c site d site e site f ph 5.98±0.3b 7.54±0.2a 7.63±0.3a 7.78±0.2a 7.38±0.2a 8.05±0.3a visibility (cm) 42.7±4.0a 40.2±4.4a 37.7±1.5a 43.3±2.6b 30.8±1.7bc 23.76±1.2c temperature (oc) 30.74±0.4a 31.34±0.3a 31.44±0.3a 31.54±0.4a 31.75±0.5a 32.14±0.3a conductivity (µs/cm) 345.5±10.5a 253.7±9.7b 271.6±14.6b 252.4±8.3b 248.69±4.8b 245.23±7.4b depth (cm) 85.5±3.2bc 118.6±9.5a 98.9±5.9ab 119.4±2.5a 85.7±6.2bc 62.7±6.8c tds (mg.l-1) 166.25±5.0a 121.68±4.7b 129.98±7.1b 120.47±4.0b 109.90±5.3b 116.84±3.6b do (mg.l-1) 2.82±0.09a 6.84±0.4b 7.68±0.4b 7.81±0.4b 10.61±0.2c 10.28±1.0c salinity (o/oo) 0.16±0.004 a 0.12±0.004a 0.12±0.006a 0.12±0.004a 0.11±0.006a 0.12±0.003a bod5 (mg.l -1) 1.20±0.5c 2.13±0.5c 3.85±0.3b 3.79±0.3b 6.56±0.3a 5.13±0.3ab total phosphate (mg.l-1) 0.02±0.004a 0.02±0.003a 0.02±0.004a 0.03±0.005a 0.03±0.006a 0.04±0.008a nitrate (mg.l-1) 0.01±0.001a 0.04±0.001b 0.02±0.002a 0.02±0.003a 0.04±0.004b 0.04±0.002b chlorophyll-a (mg/dm3) 2.05±0.2a 11.45±1.7b 2.40±0.6a 10.61±0.5b 12.13±2.6b 12.42±1.9b cod (mg.l-1) 173.1±36.4a 305.3±44.7b 285.5±46.9b 384.9±38.8b 387.5±38.2b 454.8±30.1b 99 int. j. aquat. biol. (2017) 5(2): 95-107 percentage clay content of these sites were significantly higher compared to the other sites (table 3). the sites a and b of the non-rehabilitated area showed significantly lower sediment conductivity compared to other sites (p<0.05). there was no significant temporal variation of the percentage toc, conductivity and ph of the shallow sediments of the study sites (p>0.05). however, the percentage sand and silt contents showed significant decrease and percentage clay content of the sediments showed a significant increase during september and october compared to other months (p<0.05). the spatial variation of mean abundance of macrobenthic mollusk species in each sampling site is given in table 5. the temporal variation of mean abundance of macrobenthic mollusc species during the study period is given in table 6. in the present study, a total of 8 freshwater macrobenthic molluscs, table 2. temporal variation of water quality parameters at the sampling sites of the diyawannawa wetland during the study period. the results are presented as mean±standard error of mean. different superscripts in each row indicate statistically significant differences (p<0.05). parameter april may june september october ph 7.48±0.2a 6.13±0.2b 6.37±0.18b 8.73±0.12c 8.24±0.14c visibility (cm) 31.7±3.2a 48.0±3.4b 34.3±3.1a 36.0±1.3a 32.0±1.9a temperature (oc) 33.82±0.16a 30.78±0.19a 29.91±0.18a 30.59±0.11a 32.37±0.24a conductivity (µs/cm) 278.11±4.8ab 221.28±8.6c 254.3±12.7bc 289.4±11.8ab 304.50±9.8c depth (cm) 116.2±5.8a 114.4±6.5b 79.9±5.9b 81.6±6.1b 83.5±6.7b tds (mg.l-1) 133.57±2.52a 98.20±4.75b 121.49±6.13a 138.88±6.05a 145.45±4.72a dissolved oxygen (mg.l-1) 8.05±0.8a 6.90±0.7a 8.14±0.8a 7.26±0.5a 9.00±0.7a salinity (o/oo) 0.13±0.003 a 0.09±0.005a 0.12±0.006a 0.13±0.006a 0.14±0.004a bod5 (mg.l -1) 2.29±0.4a 3.57±0.5ab 3.94±0.4ab 3.72±0.5ab 5.37±0.4b total phosphate (mg.l-1) 0.009±0.0005a 0.011±0.001a 0.009±0.0009a 0.055±0.004b 0.044±0.004b nitrate (mg.l-1) 0.035±0.003a 0.031±0.004ab 0.027±0.003ab 0.024±0.003ab 0.021±0.002a chlorophyll-a (mg/dm3) 8.29±1.0a 5.961±0.8a 4.578±0.7a 14.83±2.8b 4.571±0.6a cod (mg.l-1) 504.6±16.9a 445.6±42.3a 288±20.1b 253.2±37.6b 167.7±19.5c table 3. spatial variation of shallow sediment quality parameters at the sampling sites of the diyawannawa wetland during the study period. the results are presented as mean±standard error of mean. different superscripts in each row indicate statistically significant differences (p<0.05). parameter non-rehabilitated area rehabilitated area site a site b site c site d site e site f percentage sand content 44.6±9.7a 54.3±11.9a 48.7±10.6a 42.7± 9.4a 7.3±1.6b 5.5±1.2b percentage silt content 9.9±2.1a 3.9±1.2a 8.1±1.7a 13.6± 3.1a 11.9±2.6a 13.1±2.8a percentage clay content 45.5±11.9a 41.8±12.7a 43.3±12.4a 43.8± 12.3a 80.76±4.20a 81.50±4.04a percentage toc 12.42±0.01ab 12.34±0.02b 12.36±0.03b 12.35± 0.02b 12.48±0.03a 12.48±0.03a conductivity (µs/cm) 47.98±0.6c 43.80±0.7c 70.87±2.5b 74.49± 2.4b 77.05±1.5b 91.44±1.2a ph 6.19±0.08a 5.76±0.24a 6.17±0.11a 6.12± 0.12a 6.23±0.13a 6.31±0.10a table 4. temporal variation of shallow sediment quality parameters at the sampling sites of the diyawannawa wetland during the study period. the results are presented as mean±standard error of mean. different superscripts in each row indicate statistically significant differences (p<0.05). parameter april may june september october percentage sand content 55.0±7.6a 57.2±8.2a 56.8±8.2a 3.41±0.01b 3.59±0.02b percentage silt content 18.4±1.3a 16.2±1.5a 15.9±1.5a 6.32±0.01b 5.86±0.02b sediment clay % 26.74±7.25b 26.49±7.15b 27.24±7.14b 90.3±0.01a 90.6±0.02a shallow sediment toc% 12.35±0.02a 12.43±0.03a 12.455±0.03a 12.42±0.03a 12.37±0.03a shallow sediment conductivity (µs/cm) 68.24±4.4a 68.39±4.4a 68.22±4.2a 68.01±4.04a 65.17±4.5a shallow sediment ph 6.13±0.23bc 5.75±0.02c 5.83±0.05c 6.32±0.06ab 6.60±0.05a 100 wijeyaratne and bellanthudawa/ use of macrobenthic diversity as bioindicator in a tropical wetland system species belonging to 6 families were recorded. the mean abundance of bithynia tentaculata was significantly higher in site f in the rehabilitated area (p<0.05, table 5). only b. tentaculata and melanoides turbeculata were recorded from site a of the non-rehabilitated area and the recorded numbers of these two species in this site were significantly lower compared to other study sites (table 5). pila globosa was recorded only at sites e and f of the rehabilitated area (table 5). there was no significant temporal variation of the mean abundance of freshwater macrobenthic molluscs during the study period (table 6). the shannon wiener diversity index, simpsons index, modified biotic index and pielou’s evenness index for each sampling site are given in table 7. the lowest values for simpsons diversity index, shannon wiener diversity index and pielou’s evenness index were recorded from site f in the rehabilitated area (table 7). the highest values for simpsons diversity index, shannon wiener diversity index and modified biotic index were recorded from the site d in the table 5. spatial variation of the mean abundance of freshwater macrobenthic molluscs species in each sampling sites of the diyawannawa wetland during the study period. different superscripts in each row indicate statistically significant differences (p<0.05). species name non-rehabilitated area rehabilitated area site 1 site 2 site 3 site 4 site 5 site 6 bithynia tentaculata 3d 3d 8cd 15bc 17b 35a melanoides turbeculata 0b 0b 1b 4a 1b 5a melanoides turriculus 1b 2ab 4ab 5a 2ab 3ab thiara scabra 0b 1b 2b 10a 1b 3ab lamellidens marginalis 0c 1c 1c 2b 0c 3a pila globosa 0b 0b 0b 0b 1b 2a gyraulus saigonensis 1b 1b 1b 1b 3a 2ab lymnaea stagnalis 1b 0b 0b 1b 3a 1b table 6. temporal variation of the mean abundance of freshwater macrobenthic molluscs species in each sampling sites in the diyawannawa wetland during the study period. different superscripts in each row indicate statistically significant differences (p<0.05). species name april may june september october bithynia tentaculata 8a 12a 18a 15a 15a melanoides turbeculata 3a 2a 3a 2a 1a melanoides turriculus 4a 3a 3a 3a 2a thiara scabra 7a 2a 2a 2a 1a lamellidens marginalis 1a 1a 1a 1a 1a pila globosa 1a 1a 1a 1a 1a gyraulus saigonensis 2a 1a 1a 2a 2a lymnaea stagnalis 1a 1a 1a 1a 1a table 7. spatial variation of diversity and biotic indices of macrobenthic molluscs in the diyawannawa wetland during the study period. site simpson’s diversity index (d) shannon-wiener diversity index (h’) modified biotic index pielou's evenness index a 0.613 1.1376 7.371 1.890 b 0.635 1.2033 7.160 1.721 c 0.660 1.3116 7.093 1.686 d 0.708 1.4177 6.873 1.678 e 0.506 1.1167 7.619 1.321 f 0.472 1.0978 7.548 1.216 table 8. the percentage dominance of dominant macrobenthic mollusc family in each study site during the study period. site. dominant family % dominance a bithyniidae 55.71 b bithyniidae 50.62 c bithyniidae 50.93 d thiaridae 53.50 e bithyniidae 68.66 f bithyniidae 71.56 101 int. j. aquat. biol. (2017) 5(2): 95-107 rehabilitated area (table 7). the diversity and biotic index values of the sites in the non-rehabilitated area showed similar values to each other (table 7). the percentage dominance of dominant macrobenthic mollusc family in each study site during the study period is given in table 8. all the study sites except site d were dominated by the members of the family bithyniidae and site d was dominated by the family thiaridae (table 8). pca showed the categorization six sampling sites figure 2. ordination of the study sites based on pc1 and pc2 scores of pca of the physico-chemical parameters of over lying water and sediments of the study sites in the diyawannawa wetland (sites a, b and c=non-rehabilitated area and sites d, e and f=rehabilitated area). figure 3. ordination of the study sites based on pc1 and pc2 scores of pca of the abundance of macrobenthic molluscs of the study sites in the diyawannawa wetland. (sites a, b and c=non-rehabilitated area and sites d, e and f=rehabilitated area). 102 wijeyaratne and bellanthudawa/ use of macrobenthic diversity as bioindicator in a tropical wetland system according to the water and sediment quality parameters. two principal components displaying a cumulative variance of 85.8% were obtained after applying pca on water and sediment quality parameters. pca score plot for variation of water quality and sediment quality parameters among the study sites in the diyawannawa wetland is given in figure 2. the eigenvalues of the first two principal components, eigenvectors of the water and sediment quality variables and the principal component scores for the study sites are given in table 9. according to the results of the pca on water quality and sediment quality parameters, the sites c and b of the nonrehabilitated area and site d of the rehabilitated area grouped together. sites e and f of the rehabilitated area were grouped together and site a of the nonrehabilitated area was separated from the other groups (fig. 2). two principal components displaying a cumulative variance of 99.3% were obtained after applying pca on total abundance of macrobenthic molluscs. pca score plot for variation of total abundance of macrobenthic molluscs among the study sites in the diyawannawa wetland is given in figure 3. the eigenvalues of the first two principal components, eigenvectors of the total abundance of macrobenthic molluscs and the principal component scores for the study sites are given in table 10. the results of the pca on total abundance of macrobenthic molluscs also categorized six sampling sites into three groups. the site f of the rehabilitated area was separated from other study sites. the three sampling sites of the nonrehabilitated area were grouped together and sites e and f of the rehabilitated area formed a separate group table 9. summary of the pca of physico-chemical parameters of water and shallow sediments of the study sites at the diyawannawa wetland. cumulative % variation of only the pc1 and pc2 are shown. a high cumulative percentage as high as 85.8% of the total variation among physicochemical parameters are explained by pc1 and pc2 axis. eigenvalues pc eigenvalues %variation cum.%variation 1 11.93 62.8 62.8 2 4.38 23.1 85.8 eigenvectors (coefficients in the linear combinations of variables making up pc's) variable pc1 pc2 pc3 pc4 pc5 water ph -0.214 0.263 -0.204 0.145 -0.388 temperature -0.281 0.068 0.055 0.069 -0.252 ec 0.229 -0.290 -0.057 -0.026 0.021 tds 0.240 -0.258 -0.018 -0.044 -0.187 do -0.277 0.104 0.026 0.186 0.144 bod5 -0.264 -0.023 0.080 0.241 0.470 cod -0.274 0.100 0.123 -0.197 -0.143 nitrate -0.213 0.184 -0.459 -0.046 -0.029 chlorophyll a -0.216 0.189 -0.180 -0.575 0.075 total phosphorus -0.255 -0.117 0.116 -0.384 -0.289 visibility 0.249 0.137 0.253 -0.225 0.303 depth 0.135 0.380 0.225 -0.187 0.322 %toc -0.177 -0.337 -0.274 -0.046 0.191 sediment ph -0.128 -0.386 -0.290 -0.192 -0.058 %sand 0.249 0.218 0.141 0.088 -0.177 % silt -0.157 -0.257 0.453 -0.373 0.179 %clay -0.248 0.194 -0.241 -0.028 0.160 sediment conductivity -0.251 -0.115 0.334 0.147 -0.134 principal component scores sample score1 score2 score3 score4 score5 site a(non rehabilitated site) 4.871 -2.879 -0.373 -0.363 0.033 site b(non rehabilitated site) 1.797 3.130 -1.451 -0.261 -0.250 site c (non rehabilitated site) 1.207 0.448 0.839 1.573 -0.170 site d (rehabilitated site) -0.071 1.198 1.957 -0.915 0.169 site e (rehabilitated site) -3.286 -0.513 -0.683 0.156 1.208 site f (rehabilitated site) -4.519 -1.384 -0.289 -0.181 -0.990 103 int. j. aquat. biol. (2017) 5(2): 95-107 on the pca plot (fig. 3). the results of the pearsons’ correlation analysis on the total abundance of macrobenthic molluscs with selected water quality parameters is given in table 11. the abundance of b. tentaculata, lamellidens marginalis, m. turbeculata and p. globosa showed significant positive correlations with sediment conductivity. in addition, the abundance b. tentaculata showed significant positive correlations with percentage clay content and significant negative table 10. summary of the pca of total abundance of macrobenthic molluscs of the study sites at the diyawannawa wetland. cumulative % variation of only the pc1 and pc2 are shown. a high cumulative percentage as high as 99.3% of the total variation among physico-chemical parameters are explained by pc1 and pc2 axis. eigenvalues pc eigenvalues %variation cum.%variation 1 7.58 94.7 94.7 2 0.36 4.6 99.3 eigenvectors (coefficients in the linear combinations of variables making up pc's) variable pc1 pc2 pc3 pc4 pc5 bithynia tentaculata -0.326 0.731 -0.037 -0.153 0.531 melanoides turbeculata -0.360 -0.199 0.176 0.282 0.288 melanoides turriculus -0.351 0.372 0.545 0.138 -0.650 thiara scabra -0.362 0.002 -0.316 -0.666 -0.209 lamellidens marginalis -0.361 -0.096 -0.374 0.267 -0.116 pila globosa -0.357 -0.298 -0.185 -0.209 -0.178 gyraulus saigonensis -0.362 -0.016 -0.320 0.526 0.030 lymnaea stagnalis -0.348 -0.435 0.543 -0.206 -0.350 principal component scores sample score1 score2 score3 score4 score5 site a(non rehabilitated site) 2.590 -0.059 -0.051 0.040 0.039 site b(non rehabilitated site) 2.550 -0.016 0.012 0.056 -0.036 site c (non rehabilitated site) 2.269 0.373 0.065 -0.095 -0.003 site d (rehabilitated site) -2.098 -0.751 0.327 -0.005 0.002 site e (rehabilitated site) -1.935 -0.486 -0.387 -0.022 -0.005 site f (rehabilitated site) -3.375 -0.939 0.033 0.026 0.002 table 11. pearsons’ correlation coefficients between the abundance of fresh water molluscs species and shallow sediment quality parameters (* indicates significant correlations at 95% level of significance). species shallow sediment toc shallow sediment ph shallow sediment conductivity sand % clay % silt % bithynia tentaculata .327 .134 .645* -.445* .365* .133 gyraulus saigonensis .120 .282 .310 -0.341 .314 -.050 lamellidens marginalis .216* .126 .497* -.267 .231 .023 lymnaea stagnalis .665* .154 .109 -.226 .189 .048 melanoides turbeculata 0.195 0.063 0.511* .005 -.092 .392 melanoides turriculus -.062 .057 .148 .156 -.187 .221 pila globosa .465* .144 .468* -.332 .285 .042 thiara scabra -.028 .140 .166 .122 -.178 .310 table 12. criteria for evaluation of water quality using the family-level biotic index (hilsenhoff, 1988). biotic index value water quality degree of organic pollution 0.00-3.50 excellent no apparent organic pollution 3.51-4.50 very good possible slight organic pollution 4.51-5.50 good some organic pollution 5.51-6.50 fair fairly significant organic pollution 6.51-7.50 fairly poor significant organic pollution 7.51-8.50 poor very significant organic pollution 8.50-10.0 very poor severe organic pollution 104 wijeyaratne and bellanthudawa/ use of macrobenthic diversity as bioindicator in a tropical wetland system correlation with percentage sand content of the sediments. pila globosa and lymnaea stagnalis showed significant correlations with percentage total organic carbon content of the shallow sediments (table 11). discussion the distribution of moluscs in aquatic ecosystems are influenced by various ecological features of the sediments an overlying water column. sediment characteristics are an important aspect of physical habitat in aquatic ecosystems. sediments are the primary component of the substrate upon which macrobenthic molluscs move, rest, shelter, and feed. therefore changes in the sediment characteristics can directly influence the mollusk community structure of the ecosystem. molluscs are commonly used for biological monitoring of freshwater ecosystems in many parts of the world as they have limited mobility and are quite easy to collect using standard sampling techniques (hellawell, 1986; abel, 1989). begon et al. (2006) stated that in a healthy aquatic community, the evenness index ranges between 0.0 and 2.0, and the shannon-wiener’s diversity index ranges between 1.5 and 3.5. during the present study in each site, shonnon-wiener diversity index values were ranged in between 1.091.42 and pioulos’ eveness indices values ranged between 1.2-1.9 indicating less variation of diversity and a healthy aquatic community in both non rehabilitated and rehabilitated areas of the diyawanawa wetland system. further, the significantly high value for pielou’s evenness index in sampling sites in nonrehabilitated area compared to the rehabilitated area reflect that there is an evenly distributed, less diverse freshwater macrobenthic molluscs community in rehabilitated area compared to the non-rehabilitated area. however, among the species of molluscs recorded during the study, the number of individuals of b. tentaculata collected from the rehabilitated sites were significantly higher compared to that of the nonrehabilitated sites during the study period. the reason may be that the environmental parameters of the rehabilitated area may be significantly favorable for the survival and distribution of b. tentaculata. according to the pearsons’ correlation analysis the abundance of b. tentaculata showed significant positive correlations with sediment clay content and sediment conductivity. the pca analysis based on water and shallow sediment quality parameters of the present study categorized sites e and f of the rehabilitated area together and these sites were characterized by high sediment clay content and high sediment conductivity. according to the pca on abundance of macrobenthic molluscs, site f of the rehabilitated area was separated from the other study sites and b. tentaculata was the responsible species for the separation of site f. therefore, the results of the present study agrees with studies conducted by kołodziejczyk (1984) and savage and gazey (1987) indicating that there is a statistical significant relationship with bottom sediments clay percentage and organic matter with the abundance of b. tentaculata in lentic ecosystems. in the present study, sites d and e of the rehabilitated area were grouped together and was characterized by p. globosa and l. stagnalis. further, p. globosa and l. stagnalis showed significant correlations with percentage total organic carbon content of the shallow sediments. this finding agrees with a study conducted in a tropical aquatic system in bangladesh indicating tolerance of these species to organic enrichment and poor water quality (badruzzaman et al., 2007). in addition, according to the pca on water and sediment quality parameters the sites a and b of the non-rehabilitated area and site d of the rehabilitated area were grouped together and were characterized by high visibility and high percentage sand content in the sediments. the sites a, b and d were located in close proximity to each other and this may have caused these sites to share common physical parameters. in the pca of abundance of macrobenthic molluscs, the sites (a, b and c) in the non-rehabilitated area were grouped together and were characterized by abundance of m. turriculus. this shows that m. turriculus is a pollution sensitive species and therefore restricted to non-rehabilitated area. also 105 int. j. aquat. biol. (2017) 5(2): 95-107 previous studies in tropical wetland systems has not identified the mussel species m. turriculus as a biomonitoring indicator that can be used in assessing the wetland health. plafkin et al. (1989) and bode et al. (1991, 1996) stated that modified biotic index is highly applicable and modified to incorporate non-arthropod species including molluscs to characterize the level of organic pollution input in the aquatic ecosystems. the input of organic pollutants and the water quality of the receiving aquatic system can be categorized based on the values of mbi and the categorization is given in table 12 (hilsenhoff, 1988). significantly, high modified biotic index (mbi) was recorded in sites e and f indicating a very significant level of input of organic pollutants and poor water quality in those sites. in addition, according to the variation of mbi sites, a, b and c in the non-rehabilitated area and site d of the rehabilitated area also indicated a significant organic pollution and fairly poor water quality. in a study conducted to assess the water quality in the diyawannawa wetland system for a five year period have recorded that the water nitrate concentration varied within a range 0.01-0.8 mg.l-1 (dharmasoma and piyadasa, 2010). in the present study, a similar variation of nitrate concentration from 0.02 to 0.09 mg.l-1 was recorded and the values recorded in the study are below the maximum standard ambient nitrate concentration (5 mg.l-1) to maintain a healthy aquatic life established by the central environmental authority of sri lanka (cea, 2001). based on the results, there was no statistically significant spatial variation in the total phosphate concentration. but compared to the study sites in the non-rehabilitated area, the sites in the rehabilitated area showed higher total phosphate concentrations. the total phosphate concentration of the study sites ranged from 0.0009 to 0.066 mg.l-1and it was also less than the maximum standard ambient nitrate concentration (0.4 mg.l-1) to maintain a healthy aquatic life (cea, 2001). do is identified to be negatively correlated with the organic pollution status of a wetland (kazi et al., 2009). as recorded in a study conducted by dharmasoma and piyadasa (2014), the mean dissolved oxygen concentration of the selected sites of the diyawannawa wetland ranged from 4.69-5.86 mg.l-1. in the present study, mean dissolved oxygen concentration showed a wider range of variation with a range from 2.40 to 14.91 mg.l-1. the dissolved oxygen concentration showed a significant spatial variation indicating a significantly lower dissolved oxygen concentration at site a of the non-rehabilitated area compared to other sites. during the field work, it was noted that this site had lot of floating macrophytes compared to other sites, thus disturbing the contact of aquatic and atmospheric interphase. this may have reduced the atmospheric input of oxygen of this site. the mean oxygen concentration recorded in site a of the non-rehabilitated area was less than the standard minimum ambient dissolved oxygen concentration (3 mg.l-1) established by the cea, but the other sites showed higher values than the ambient minimum standard. in the present study, there is a significant increment in bod5 and cod in the rehabilitated area compared to those of non-rehabilitated area. cod is a measurement used to detect the input of domestic sewage, agricultural and industrial waste into aquatic systems and high bod and cod can be recorded in aquatic systems where extensive domestic waste dumping is pracised (kazi et al., 2009; triest et al., 2001). in the present study, improper solid waste dumping and discharging of waste from nearby residents was observed in sites e and f of the rehabilitated area, which may have contributed to significantly high levels of bod and cod. however, the bod values recorded during the study were less than the maximum ambient bod (4 mg.l-1) and the cod values were higher than the maximum ambient cod (15 mg.l-1) values established by the cea of sri lanka. the results of the present study showed a significant spatial and temporal variation of physicochemical parameters of water and shallow sediment quality in the rehabilitated and nonrehabilitated areas of the diyawannawa wetland system affecting the abundance and diversity of 106 wijeyaratne and bellanthudawa/ use of macrobenthic diversity as bioindicator in a tropical wetland system macrobenthic molluscs. therefore, it highlights the importance of continuous monitoring of water and sediment quality, and fresh water macrobenthic mollusc diversity as reflect the health of this wetland system. the results also showed that abundance of b. tentaculata and p. globosa can be used as a biomonitoring tool to assess sediment quality in relation to their positive interaction with sediment quality and their abundance. references abel p.d. 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(2013) 1(1): 6-13 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology effects of pre-sampling fasting on serum characteristics of common carp (cyprinus carpio l.) seyyed morteza hoseini1*, melika ghelichpour1 1department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 2 march 2013 accepted 4 april 2013 available online 5 april 2013 keywords: biochemistry cyprinus carpio fasting metabolite sampling feeding abstract: common carp, cyprinus carpio (l.) were blood-sampled after 0, 2, 6, 12, 24, 48 and 72 h fasting to find serum baseline levels. there was a significant difference in serum glucose, lactate, triglyceride and total protein but not cholesterol, albumin and calcium levels among the treatments. glucose levels increased with fasting tine and reached to a peak after 6 h. then, the glucose level decreased to the lowest level after 12 h. changes of glucose and lactate had reverse trends, as lactate levels decreased with fasting time and reached to the dip point at 6 h and, thereafter, the levels increased to peak point at 12 h. serum glucose and lactate levels showed stable values during 24-72 h of fasting, compared with the values measured at 0 h. triglyceride levels showed an increasing trend parallel to that of the fasting period and reached to a peak point after 6 h. the levels reached 0 h values at 24 h and showed further decrement at 48 and 72h. total protein showed elevation while fasting progressed and reached the peak point at 6 h and remained stable during 24-48 h fasting; however, it decreased after 72 h fasting. according to the results, cholesterol, calcium and albumin baseline levels were not affected by 0-72 h fasting. glucose and lactate baseline could be determined after 24-72 h fasting. total protein baseline could be determined after 24-48 h fasting. triglyceride levels are significantly affected by fasting period which should be taken into account when it is measured. possible mechanisms involving in common carp serum fluctuation over 0-72 h fasting period are discussed. introduction blood sampling is necessary for monitoring of fish physiological condition in aquaculture and researches. period of pre-sampling fasting may alter the serum characteristics, thus choosing the correct sampling procedure guarantees precise scientific outputs. several researchers (schurmann and steffensen, 1997; stillwell and benfey, 1997; wagner et al., 2003) starved the fish prior to sampling to ensure the fish are in post absorptive state. during fasting period, the level of metabolites changes under the modulation of the hormones, mainly pancreatics (blasco et al., 1992). on the other hand, the metabolite levels are indicator of some other phenomenon like as stress and nutritional condition. thus, when the results of different studies * corresponding author: seyyed morteza hoseini e-mail address: seyyedmorteza.hoseini@gmail.com tel: +989112750713 are compared, the fasting period might mask or magnify the differences. to date, there are no studies on the effect of presampling fating period on the fish blood biochemistry exception of the study by shi et al. (2010) on amur sturgeon, acipenser schrenckii. researchers chose different fasting periods prior to blood collection (iversen et al., 2003; holloway et al., 2004; roubach et al., 2005; bystriansky et al., 2006; hoseini, 2010; hoseini and hosseini, 2010, hoseini et al., 2010). most studies have used a 24-h fasting period (cho and heath, 2000; roubach et al., 2005; bystriansky et al., 2006; hoseini, 2010; hoseini and hosseini, 2010, hoseini et al., 2010). however, cataldi et al. (1998) and wagner et al. (2003) chose 48-h pre-sampling fasting to study the mailto:seyyedmorteza.hoseini@gmail.com 7 hoseini and ghelichpour/ int. j. aquat. biol. (2013) 1(1): 6-13 stress response in adriatic sturgeon, acipenser naccarii and atlantic salmon, salmo salar (l). the others chose other periods, for example, 72 h or no fasting period (iversen et al., 2003; hyvarinen et al., 2004). on the other hand, other researchers investigated the effect of different factors on serum biochemistry of fish without mentioning the fasting period (ortuno et al., 2002a, b; holloway et al., 2004). common carp, c. carpio (l.) is a species with long term fasting tolerance (créach, 1972) during which the muscle protein mobilizes substantially. blasco et al. (1992) found decrease in common carp hepatosomatic index after 8 days fasting, mainly due to glycogen mobilization. blasco et al. (1992) reported short (2, 5 and 10 days) and long (20 and 50 days) term change in carp serum metabolites, insulin and glucagon levels. however the changes in serum glucose, lactate, triglyceride, cholesterol and total protein was not monitored during the first three days (the period which the researchers used as presampling fasting). thus it might be of interest to determine the effect of fasting period on serum biochemistry in common carp. in this case, there is not a common procedure. for example, while ruane et al. (2002a, b) and velisek et al. (2009) did not have a pre-sampling fasting period in their studies, ruane et al. (2001), sudova et al. (2009), hoseini (2010), hoseini and hosseini (2010) and hoseini et al. (2010) starved their specimens for 24 h prior to blood collection. thus, the aim of the present study was to investigate the changes in serum biochemistry in common carp starved for 0, 2, 6, 12, 24, 48 and 72 h. materials and methods fish were obtained from the artificial propagation of wild-caught brood stocks from caspian sea. the larvae were reared to juvenile (120 g) in the earthen ponds over 10 months (april 2010 january 2011). fish fed on natural food of the ponds as well as on artificial diet (34% protein and 8.5% lipid). total of 150 fish were transported to the laboratory using fish transporting tanks equipped to a pure oxygen supplier. a total of 90 fish were stocked in 15 rectangular glass tanks (0.8×0.55×0.55 m). all tanks were filled with 170 l dechlorinated tap water. continuous aeration was provided to all tanks and fish were fed on artificial diet (34% protein and 8.5% lipid) based on 1.4% of fish body weight, daily. fish were fed twice (08:00 am and 20:00 pm) a day. water exchange was performed daily corresponding to 85% of the tanks' water volume. water quality was as follow: temperature= 20.2 ± 1.1 ºc, dissolved oxygen = 6.74 ± 0.61 ppm, ph = 7.5 ± 0.18 and n-nitrite = 0.001 ppm. after 15 days acclimation, fish were blood-sampled at 0, 2, 6, 12, 24, 48 and 72 h after the last feeding (treatments 0, 2, 6, 12, 24, 48 and 72 h). blood sampling was conducted at 08:00; thus, to avoid the potential effect of daily rhythm on fish physiological status, fasting was begun at different time. accordingly, feeding was ceased at 08:00 in treatments 0, 24, 48 and 72 h, while the last meal of the treatment 12, 6 and 2 was on 20:00, 02:00 and 06:00, respectively. each treatment was constituted of three tanks. two fish were sampled from each tank to attain 6 samples per treatment. to avoid catch-born stress, fish were netted by a large dip net to allow 2 fish capture in a single effort. fish were immediately placed in anesthetic bath (eugenol 100 ppm) over 40 s, prior to caudal puncture. after blood-sampling, the specimens were placed in a freshwater tank to recover and their condition was monitored over a 3-day period. blood samples were collected in non-heparinized 1.5-ml tubes and centrifuged for 7 min at 5000 rpm to separate serum. serum samples were stored at 80 °c until further analyses. serum lactate levels were determined enzymatically using a commercial kit (pars azmun co. ltd, tehran, iran). serum levels of glucose, cholesterol, triglyceride, albumin and calcium were determined by glucose-oxidase, cholesterol oxidase, glycerol-3-phosphate oxidase, bromocresol green and cresolphthalein complexone methods, respectively (pars azmun co. ltd, tehran, iran). serum total protein was determined using the biuret reagent (pars azmun co. ltd, tehran, iran). 8 hoseini and ghelichpour/ int. j. aquat. biol. (2013) 1(1): 6-13 samples were analyzed three times and the averages over times were used for statistical analyses. normality of data and homogeneity of variance were examined using kolmogorove-smirnov and levene's tests, respectively. data were analyzed using a one-way anova followed by lsd test, using the statistical software spss version 16. all data are presented as mean ± sd. results serum levels of glucose and lactate are shown in figures 1 and 2, respectively. glucose and lactate levels were significantly affected by fasting period (p<0.05). there was a reverse trend in glucose and lactate levels as high levels of glucose were accompanied with low levels of lactate and vice versa. glucose levels increased with increase of fasting duration and reached to a peak at 6 h, when it decreased until the hour 12. then it increased again and, at 24-72 h, it reached to the concentrations recorded at the 0 h levels. in the contrary, lactate levels decreased in line with fasting period and reached its dip point at 6 h, thereafter it showed an increase and reached its peak at 12 h. lactate levels at 24, 48 and 72 h decreased again and reached the level of 0 h. figure 1. common carp serum glucose levels during fasting. different letters show significant difference (p < 0.05). serum levels of triglyceride are shown in figures 3. serum triglyceride levels were significantly different among the fasting points (p<0.05). the levels showed increase and reached the peak at 6 h. the levels, then, started to fall and reached the 0 h level at 24 h. the levels continued the decreasing pattern and reached the lowest levels at 72 h. figure 2. common carp serum lactate levels during fasting. different letters show significant difference (p < 0.05). n = 6. figure 3. common carp serum triglyceride levels during fasting. different letters show significant difference (p < 0.05). n = 6. figure 4. common carp serum total protein levels during fasting. different letters show significant difference (p < 0.05). n = 6. changes in serum total protein are shown in figure 4. total protein values were significantly affected by fasting period (p<0.05). the levels increased and reached the peak point at 6 h which was followed by a sharp decrease at 12 h. the levels remained c b a d c c c 0 1 2 3 4 5 6 7 8 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 g lu co se ( m m o l/ l ) time (h) b c c a b b b 0 1 2 3 4 5 6 7 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 l a ct a te ( m m o l/ l ) time (h) c b a c cd de e 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 t ri g ly ce ri d e ( m m o l/ l ) time (h) c b ab c c c d 0 5 10 15 20 25 30 35 40 45 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 t o ta l p ro te in ( g / l ) time (h) 9 hoseini and ghelichpour/ int. j. aquat. biol. (2013) 1(1): 6-13 unchanged until 48 h, however, it showed a significant decrease at 72 h. table 1. serum cholesterol (mmol l-1), calcium (mmol l-1) and albumin (g l-1) levels during fasting period in common carp. no significant difference was detected. n = 6. serum cholesterol, calcium and albumin levels are shown in table 1. there was no significant difference among the fasting points. discussion homeostasis of energy in fish during food deprivation is directly related to mobilization of energy reserves such as lipids, activation of hepatic gluconeogenesis and reduction in the rate of glucose utilization (sheridan and mommsen, 1991; navarro and gutierrez, 1995). protein catabolism (andenen et al., 1991), glycogenolysis (vijayan and moon, 1992), and tissue storage release and uptake are the factors affecting metabolite level of serum. on the other hand, serum levels of metabolite are affected by stressors (reviewed by wendelaar bonga, 1997). however, as the fish in this study were from the same origin and sampled following a constant protocol, any changes in the serum biochemistry are supposed to be as a result of fasting period. there was no significant difference in the tested parameters between 0 and 24 h fasting. the reason seems be due to short time between feeding and blood collection at 0 h. in fact, fish of 0 h groups were sampled immediately after last meal, which did not allow nutrient absorption from the gut. there is only one study (shi et al., 2010) focusing on the effect of short-term fasting on fish serum biochemistry, nevertheless, there are some studies on the effect of long term starvation or feeding rate on carp serum biochemistry (blasco et al., 1992; shimeno et al., 1997; ruane et al., 2002). however, results of these studies could not be used to determine the effect of pre-sampling fasting period of carp serum biochemistry, due to methodology and fasting period. glucose is one of the most important metabolites in fish serum which its level fluctuates over the fasting period (blasco et al., 1992; sala-rabanal et al., 2003). at the present study, a high level of glucose at 2 and 6 h stems from absorption of glucose from gut, introduction to blood stream and decrease at 12 h which is a result of uptake by tissues and energy production. recovery of glucose levels at 24-72 h suggests that glucose was produced via gluconeogenic and glycogenolysis (blasco et al., 1992). serum lactate levels showed reverse trend compared to glucose. lactate is back transported to hepatic cells for glucose production under hypoglycemic condition. this explains reverse trends in glucose and lactate over fasting periods, where the highest levels of one item was accompanied by the lowest levels of the other one, which was reported by other researchers (figueroa et al., 2000). increase in lactate levels at 12 h might be as a result of activation of gluconeogenesis pathway due to fall in glucose circulating levels. further decrement in lactate levels might be related to its utilization for glucose production and/or serum glucose elevation as a result of gluconeogenesis. at the present study, serum glucose level was statistically stable during 24 -72 h fasting, which is in agreement with the results on rainbow trout, o. mykiss (figueroa et al., 2000; congleton and wagner, 2006) and common carp, c. carpio (blasco et al., 1992). thus it is suggested that, for common carp serum glucose and lactate analyses, fish should be fasted 24-72 h to allow serum glucose and lactate to reach the baseline level. on the other hand, shi et al. (2010) found no change in serum glucose levels after 0, 12 and 48 h fasting and significant decrease at 24 and 72 h fasting in amur sturgeon, a. schrenckii. difference between this study and the present one, might be due to species differences, as common carp is different in some aspects of fasting duration (h) cholesterol calcium albumin 0 3.92 ± 0.60 1.75 ± 0.12 9.00 ± 1.15 2 4.73 ± 0.60 1.96 ± 0.11 9.92 ± 1.62 6 4.99 ± 0.40 1.90 ± 1.14 9.80 ± 1.51 12 4.00 ± 0.77 1.82 ± 0.19 8.00 ± 0.60 24 3.87 ± 0.63 1.81 ± 0.14 9.11 ± 0.90 48 4.38 ± 1.08 1.75 ± 0.18 9.35 ± 1.75 72 4.21 ± 1.19 1.88 ± 0.17 8.17 ± 2.16 10 hoseini and ghelichpour/ int. j. aquat. biol. (2013) 1(1): 6-13 metabolism compared to the other species, i.e. glycogen and lipid metabolism over food deprivation (navarro and gutierrez, 1995). on the other hand, navarro and gutierrez (1995) suggested that the sluggish (like carp) and active (like sturgeon) species are difference in serum glucose maintenance. lipids are stored as triglycerides in different tissues. lipid mobilization occurs during fasting, with or after carbohydrate mobilization (navarro and gutierrez, 1995). in our study, the increase of serum triglyceride at early fasting might be as a result of absorption from the gut. previous study by ruane et al. (2002b) showed a decrease in triglyceride and no changes in cholesterol due to the low ration in common carp. however, the decrease of triglyceride might be as a result of supplying energy demand together with carbohydrate mobilization, in common carp (navarro and gutierrez, 1995). also, decrease of triglyceride levels may come from low food intake. lack of significant changes in cholesterol levels might be due to short term of fasting, as mentioned above. shimeno et al. (1997) showed decrease in the both triglyceride and cholesterol levels as a result of low ration or fasting in common carp, after 30 d. congleton and wagner (2006) failed to detect any significant change in cholesterol levels in rainbow trout, o. mykiss and chinook salmon, oncorhynchus tshawytscha after 1 and 3 d fasting. there is no study on the effect of fasting on serum triglyceride and cholesterol levels in common carp, to make a precise comparison. however, shi et al. (2010) found no significant changes in triglyceride and cholesterol levels in amur sturgeon, a. schrenckii, after 0, 0.5, 1 and 2 d fasting; however, significant increase occurred at the third day. the difference in triglyceride and cholesterol patterns between the two studies might be due to species differences (sluggish carp vs. active sturgeon), diet (low lipid for carp vs. high lipid for sturgeon) and different metabolic pathways, as there are wide differences in basal levels of triglyceride (3.06 vs. 4.97 mmol l-1), cholesterol (4.71 vs. 1.99 mmol l-1) between the two species. based on the results, common carp serum levels of cholesterol are not related to pre sampling fasting period, however, serum triglyceride level is significantly affected by fasting which should be taken into account when it is measured and compared with the other data. total protein of serum is known as an indicator of the nutritional status which provides information on fish metabolism. there is a clear tendency for plasma proteins to decrease in fasting fish (navarro and gutierrez, 1995). increase in serum total protein might be due to absorption from the last meal and increase of liver protein synthesis. at the present study, total protein of serum remained stable during the 24-48 h fasting. thus to determine total protein baseline level in common carp, fish should be fasted for 24-48 h. significant decrease after 72 h fasting comes from decrease in hepatic protein synthesis as well as protein catabolism for energy production. serum albumin did not change along with the decrease of serum total protein, suggesting the physiological importance of this protein. measurement of the different serum proteins can help better understanding of protein catabolism over the fasting period. change in serum total protein levels are related to the change of serum globulin levels. according to the results, common carp serum albumin baseline level is not affected by 0-72 h fasting. in this study serum calcium were determined over the fasting period, because its linkage with serum proteins (bjornsson et al., 1989). about half of total plasma calcium is ionized and the rest is bound to serum proteins (andreasen, 1985; bjornsson et al., 1989), thus decline in proteins in the fasted fish might also lower plasma calcium concentrations. however, such results were not observed at the present study. similarly, andreasen (1985) failed to detect any correlation between plasma protein and total calcium concentrations. congleton and wagner (2006) did not find any changes in plasma calcium after 1 and 3 d fasting and even after extended periods of fasting (14 and 24 d, when plasma total protein decreased) in rainbow trout, o. mykiss and chinook salmon, o. tshawytscha. results of the 11 hoseini and ghelichpour/ int. j. aquat. biol. (2013) 1(1): 6-13 present study showed that serum calcium level is not sensitive to ore sampling fasting period. it is concluded that pre-sampling fasting period affects serum levels of glucose, lactate, triglyceride and total protein, in common carp. accordingly, serum glucose and lactate levels could be determined after 24-72 h fasting, while serum baseline level of total protein should be determined after 24-48 h fasting. the results, also, show serum triglyceride fluctuates over the fasting period which should be taken into account when it is measured. likewise, common carp serum cholesterol, albumin and calcium levels are not sensitive to fasting. references andenen d.e., reid s.d., moon t.w., perry s.f. (1991). metabolic effects associated with chronically elevated cortisol in rainbow trout (oncorhynchus mykiss). canadian journal of fisheries and aquatic sciences, 48:1811–1817. andreasen p. 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(2016) 4(3): 202-207; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology short communication range extension of moenkhausia oligolepis (günther, 1864) to the pindaré river drainage, of mearim river basin, and itapecuru river basin of northeastern brazil (characiformes: characidae) erick cristofore guimarães*1, felipe polivanov ottoni1, 2, axel makay katz2, pâmella silva de brito1 1laboratório de sistemática e ecologia de organismos aquáticos, centro de ciências agrárias e ambientais, universidade federal do maranhão. campus universitário, ccaa, br-222, km 04, s/n, boa vista, cep 65500-000, chapadinha, ma, brasil. 2laboratório de sistemática e evolução de peixes teleósteos, departamento de zoologia, instituto de biologia, universidade federal do rio de janeiro. cidade universitária, cep 21994-970. rio de janeiro, rj, brasil. article history: received 16 april 2016 accepted 15 june 2016 available online 2 5 june 2016 keywords: ichthyology, incertae sedis, maranhão state, taxonomy, teleostei. abstract: the present study reports range extension of moenkhausia oligolepis to the pindaré river drainage, of the mearim river basin, and itapecuru river basin, maranhão state, northeastern brazil. this species was previously known only from venezuela, guianas, and the amazon river basins. in addition, we present some meristic and morphometric data of the specimens herein examined and discuss on its diagnostic characters. introduction moenkhausia eigenmann, 1903 is one of the most specious characid genera, currently comprising about 80 valid species (eschmeyer and fricke, 2016). the genus is widely distributed in the cisandineans river basins, not occurring only in patagonia (lima et al., 2003; benine, 2009). the genus moenkhausia was firstly included in the subfamily tetragonopterinae (eigenmann, 1903, 1917), being recently located as incertae sedis in characidae (lima et al., 2003). according to géry (1977), benine (2004), benine et al. (2009) and petrolli and benine (2015), this genus is divided into some species complex; e.g.: moenkhausia chrysargyrea, moenkhausia dichroura, moenkhausia eigenmanni, moenkhausia grandisquanis, moenkhausia lepidura, moenkhausia jamesi and moenkhausia oligolepis species complexes. moenkhausia oligolepis complex is composed of species which share a set of diagnostic features, including presence of a conspicuous blotch in the caudal peduncle extending to the bases of caudal fin rays, preceded by a whitish brown area; scales of the side of body with a dark pigmentation on their * corresponding author: erick cristofore guimarães e-mail address: erick.ictio@yahoo.com.br posterior margin, forming a reticulated pattern; four to five series of scales above lateral line; and two to four series of scales between lateral line and pelvicfin origin (eigenmann, 1903; eigenmann, 1917; géry, 1977; costa 1994; lima et al., 2007; lima and toledo-piza, 2001; benine, 2009) distinguishing from all the other congeners. moenkhausia oligolepis was originally described as tetragonopterus oligolepis, based on specimens collected in guyana (günther, 1864). the geographical distribution of m. oligolepis is the river drainages of venezuela, in the guianas and amazon river basin, occurring in the following countries, including brazil, french guiana, guyana, peru, suriname, and venezuela (lima et al., 2003; eschmeyer and fricke, 2016; froese and pauly, 2016). this species has been now found in the pindaré river drainage, of the mearim river basin, and saco river of the itapecuru river basin of northeastern brazil showing the extension of its distribution further to west. materials and methods the specimens of m. oligolepis were collected from 203 int. j. aquat. biol. (2016) 4(3): 202-207 the pindaré river drainage, of the mearim river basin, northeastern brazil. the pindaré river origins from the serra do gurupi, with maximum altitude of about 300 m, and discharges into the mearim river, near its mouth in the são marcos bay, after going through 575.59 km (lima, 2013). the itapecuru river origins from the serras crueiras, in the south of maranão, with maximum altitude of about 530 m, and discharges into the arraial bay, in the southeastern of são luis island (lima, 2013). specimens of m. oligolepis were collected in seven sampling sites of the pindaré river drainage and one sampling station in the itapecuru river basin (fig. 1). the collected specimens were fixed in 10% formaldehyde, and then transferred to 70% ethanol for preservation, after 15 days. measurements and counts were made according to fink and weitzman (1974), menezes and weitzman (1990), weitzman and malabarba (1999) and bragança et al. (2015). counts and data related to the fin rays, branchiostegal rays, teeth, supraneurals, vertebrae and ribs were conducted in cleared and stained specimens based on to taylor and van dyke (1985). the weberian apparatus were not included in the vertebrae count and the fused pu1+u1 was considered as a single element. information about other congeners were based on the literatures, including günther (1864), eigenmann (1903), eigenmann (1917), géry (1977), costa, (1994), lima and toledo-piza (2001), lima et al. (2007), benine et al. (2009), sousa et al. (2010), lima et al. (2013), dagosta et al. (2015), and ohara and marinho (2016). the collected materials were deposited in the coleção ictiológica do centro de ciencias agrárias ambientais, da universidade federal do maranhão (cicca). collections were made under the “autorização de captura e transporte de material biológico, processo ibama nº 02001.007241/ 2004-37”. results and discussion the examined specimens present all character states cited above for the m. oligolepis complex (fig. 2) and were identified as m. oligolepis because they differ from the other congeners of the species complex by a set of features cited below: presence of a complete lateral line, while m. forestii benine, figure 1. distribution of moenkhausia oligolepis in pindaré river drainage, of the mearim river basin (red lozenge), and itapecuru river basin (red circle), maranhão state, northeastern brazil. figure 2. (a) life specimens of m. oligolepis: ciccaa 00094, 48.5 sl; codó municipality, itapecuru river basin, maranhão state, northeastern brazil, and (b) preserved specimen of m. oligolepis: ciccaa 00062, 50.0 mm sl; alto alegre municipality, pindaré river drainage, of the mearim river basin, maranhão state, northeastern brazil. 204 guimarães et al./ distribution extension of moenkhausia oligolepis. mariguela & oliveira, 2009, m. pyrophthalma costa, 1994, m. cosmops lima, britski & machado 2007, m. diktyota lima & toledo-piza 2001 and m. sanctaefilomenae (steindachener, 1907) have an incomplete or interrupted lateral line; presence of five series of scales between lateral line and dorsalfin origin and four series of scales between lateral line and pelvic-fin origin, while m. australe, (eigenmann, 1908), m. sanctaefilomenae and m. cosmops have four and two to three, respectively; presence of 28-30 perforated scales in the lateral line, while m. sanctaefilomenae and m. forestii have 2224 and 23-26, respectively; absence of longitudinal series of dark dots on body, while m. lineomaculata dagosta, marinho & benine, 2015, m. cotinho eigenmann, 1908, m. parecis ohara & marinho 2016, m. petymbuaba lima and birindelli, 2006 and m. plumbea sousa, netto-ferreira and birindelli, 2010 possess these series; and presence a dorsally and ventrally extended caudal peduncle blotch occupying its entire height, while m. pumblea, m. lineomaculata and m. parecis have a caudal peduncle spot centralized, not extended dorsally and ventrally. the present study extends the distribution of m. oligolepis for the pindaré river drainage, of the mearin river basin, and itapecuru river basin, northeastern brazil. in addition, our results confirm the brief descriptions and diagnostic features of the species previously proposed by günther (1864), planquette et al. (1996) and lima et al. (2013). some morphometric and meristic information of the specimens herein examined are presented in the (tables 1 and 2). examined material: brazil: estado do maranhão: município de alto alegre do pindaré: cicca 00057, 1, c&s, 41.1 mm sl; igarapé arapapá, 3°42'22.66''s 46°0'20.92''w; e. guimarães and c. characters range mean standard length 34.5-58.8 41.5 standard length % body length 37.2-44.3 41.3 head length 22.8-27.9 26.1 head depth 18.3-27.7 21.8 predorsal distance 31.0-56.7 52.1 prepectoral distance 24.1-31.5 28,3 prepelvic distance 40.0-52.0 48.7 preanal distance 16.8-24.6 21 caudal peduncle depth 10.5-14.1 12.1 dorsal-fin base length 9.3-17.0 12.9 anal-fin base length 21-33.3 28.8 pectoral-fin length 20.6-31.4 24.8 pelvic-fin length 16.0-32.2 21.3 dorsal-fin length 29.6-37.4 32.4 anal-fin length 13.6-25.7 20.7 caudal peduncle length 6.9-12.2 9,4 distance between dorsal and adipose fin 34.4-38.8 34.6 distance between orbit and dorsal fin 23.8-53.4 37.1 distance between dorsal fin and caudal peduncle 48-55.5 51.8 head length % horizontal eye diameter 21-47.1 34.5 snout length 20.1-38.7 26.2 least interorbital width 16.9-30.9 23.5 upper jaw length 15.9-35.4 25.8 table 1. morphometric characteristics of moenkhausia oligolepis specimens herein examined, (n = 30). http://en.wikipedia.org/wiki/carl_h._eigenmann http://researcharchive.calacademy.org/research/ichthyology/catalog/getref.asp?id=1221 205 int. j. aquat. biol. (2016) 4(3): 202-207 costa, 03 dec. 2015. cicca 00058, 1, c&s, 49.8 mm sl; igarapé araparizal, 3°54'31.68''s 46°12' 3.04'' w, e. guimarães and c. costa, 03 dec. 2015. cicca 00059, 1, c&s, 39.1 mm sl; igarapé araparizal; 3°54'31.68''s 46°12'3.04''w; e. guimarães and c. costa, 03 dec. 2015. cicca 00060, 1, c&s, 38.9 mm sl; igarapé do fausto, 3°42'47.45''s 46°3'26.26''w, e. guimarães and c. costa, 03 dec 2015. cicca 00061, 1, c&s, 47.1 mm sl; igarapé do fausto, 3°42'47.45''s 46°3'26.26''w, e. guimarães and c. costa, 03 dec 2015. cicca 00062, 4, 41.2-58.8 mm sl; igarapé do fausto, 3°42'47.45''s 46°3'26.26''w, e. guimarães and c. costa, 03 dec 2015. cicca 00063, 1, 46.3 mm sl; igarapé do fausto, 3°42'47.45''s 46°3'26.26''w, e. guimarães and c.costa, 03 dec 2015. cicca 00064, 3, 37.9-44.8 mm sl; igarapé do fausto, 3°42'47.45''s 46°3'26.26''w, e. guimarães and c. costa, 03 dec 2015. cicca 00065, 2, 39.8-44.3 mm sl; igarapé jenipapo, 3°51'15.77''s 46°11'6.19''w, e. guimarães and c. costa, 03 dec 2015. cicca 00066, 4, 38.640.1 mm sl; igarapé arapapa, 3°42' 22.66''s 46°0' 20.92'' w, e. guimarães and c. costa, 03 dec 2015. cicca 00067, 3, 38.0-52.3 mm sl; igarapé igarapá, bacia do rio mearim; 3°45'46.38''s 46°8' 11.67''w, e. guimarães and c. costa, 03 dec 2015. cicca 00068, 1, 55.5 mm sl; igarapé brejinho, 3°42' 27.54''s 46°1'17.00''w, e. guimarães and c.costa, 03 dec 2015. cicca 00069, 4, 34.5-44.6 mm sl; igarapé brejinho, 3°42'27.54''s 46°1'17.00''w, e. guimarães and c. costa, 03 dec 2015. cicca 00070, 3, 39.6-41.2 mm sl; igarapé mineirão, 3°42'26.96''s 45°56'15.12''w, e. guimarães and c. costa, 03 dec 2015. município de codó: cicca0094, 48.5 sl; rio saco, 04º31'48.2''s 43º56'09.5''w, e. guimarães and f. ottoni. acknowledgments this study was supported by cnpq (conselho nacional de desenvolvimento científico e tecnológico – ministério da ciência e tecnologia). references benine r.c. (2004). análise filogenética do gênero moenkhausia (characiformes: characidae) com uma characters n range number of dorsal-fin rays 5 ii+9 number of anal-fin rays 5 iv+24 – v25 number of pectoral-fin rays 5 i+12 number of pelvic-fin rays 5 i+7 number of perforated scales of upper lateral line 29 28 30 number of scales serie above lateral line 30 5 number of scales serie between lateral line and pelvic fin origin 30 4 number of predorsal scales 5 11 number of maxillary teeth 5 2 number of teeth in the outer row of premaxilla 5 4 number of teeth in the inner row of premaxilla 5 4 number of teeth in the dentary 5 13 -14 number of branchiostegal rays 5 4 number of supraneurals 5 4 number of principal caudal-fin rays 5 ii+19 number of dorsal procurrent rays 5 7 number of ventral procurrent rays 5 8 number of vertebrae (precaudal + caudal) 5 27–28 (12+15–16) number of rib pairs 5 11–12 (9–10 larger s+ 2 smallers) table 2. meristic data of moenkhausia oligolepis specimens herein examined. 206 guimarães et al./ distribution extension of moenkhausia oligolepis. revisão dos táxons do alto rio paraná. [unpublished report]., são paulo, universidade estadual paulista, são paulo, 358 p. benine r.c., mariguela t.c., oliveira c. (2009). new species of moenkhausia eigenmann, 1903 (characiformes: characidae) with comments on the moenkhausia oligolepis species complex. neotropical ichthyology, 7(2): 161-168. bragança p.h.n., ottoni f.p., rangel-pereira f.s. (2015). hyphessobrycon ellisae, a new species from northeastern brazil (teleostei: characidae). ichthyological exploration of freshwaters, 26(3): 255-262. costa w.j.e.m. (1994). description of two new species of the genus moenkhausia (characiformes: characidae) from the central brazil. zoologischer anzeiger, 232(1-2): 21-29. dagosta f.c.p., marinho m.m.f., benine r. (2015). a new species of moenkhausia eigenmann (characiformes: characidae) from the upper rio juruena basin, central brazil. zootaxa, 4032: 417425. eigenmann c.h. (1903). new genera of south american fresh-water fishes, and new names for old genera. smithsonian miscellaneous collections, 45: 144-148. eigenmann c.h. (1917). the american characidae (part 1). memoirs museum of comparative zoology, 43: 1 102. eschmeyer w.n., fong j.d. (2016). species of fishes by family/subfamily. available from: http://research. calacademy.org/research/ichthyology/catalog/species byfamily.asp. retrieved 14/04/2016. fink w.l., weitzman s.h. (1974). the so-called cheirodontin fishes of central america with descriptions of two new species (pisces: characidae). smithsonia contributions to zoology, 172: 1-46. froese r., pauly d. (2016). fishbase. world wide web electronic publication. available from: http://www.fishbase.org. retrieved 19/04/16. günther a. (1864). catalogue of the fishes in the british museum. catalogue of the physostomi, containing the families siluridae, characinidae, haplochitonidae, sternoptychidae, scopelidae, stomiatidae in the collection of the british museum. cat. fishes. v.5, 455p. lima f.c.t., malabarba l.r., buckup p.a., da silva j.f.p., vari r.p., harold a., benine r.c., oyakawa o.t., pavanelli c.s., menezes n.a., lucena c.a.s., reis r.e., langeani f., casatti l., bertaco v.a., moreira c.r., lucinda p.h.f. (2003). genera incertae sedis in characidae. pp. 106-169. in: r.e. reis, s.o. kullander, c.j. ferraris jr. (eds.). check list of the freshwater fishes of south and central america. edipucrs, porto alegre, 729 p. lima f.c.t., britski h.a., machado f.a. (2007). a new moenkhausia (characiformes: characidae) from central brazil, with comments on the area relationship between the upper rio tapajós and upper rio paraguai systems. aqua, international journal of ichthyology, 13(2): 45-54. lima f.c.t., toledo-piza m. (2001). new species of moenkhausia (characiformes: characidae) from the rio negro of brazil. copeia, 4: 1058-1063. lima a.s. (2013). análise geomorfológica da bacia hidrográfica do rio mearim-ma a partir do quadro geológico regional. tese de doutorado não publicada, universidade federal de minas gerais,belo horizonte. 142 p. lima f.c.t., pires t.h.s., ohara w.m., jerep f.c., carvalho f.r., marinho m.m.f., zuanon j. (2013). characidae. in: l.j.g. torrente-vilara, w.m. ohara, t.h.s. pires, j. zuanon, c.r.c. doria (eds.). peixes do rio madeira. vol. i, são paulo, dialeto. pp: 213395. menezes n.a., weitzman s.h. (1990). two new species of mimagoniates (teleostei: characidae: glandulocaudinae), their phylogeny and biogeography and a key to the glandulocaudin fishes of brazil and paraguay. proceedings of the biological society of washington, 103: 380-426. ohara w.m., marinho m.m.f. (2016). a new species of moenkhausia eigenmann (characiformes: characidae) from the upper rio machado at chapada dos parecis, rio madeira basin, brazil. neotropical ichthyology, 14(1): e150041. petrolli m.g., benine r.c. (2015). description of three new species of moenkhausia (teleostei, characiformes, characidae) with the definition of the moenkhausia jamesi species complex. zootaxa, 3986(4): 401–420. planquette p., keith p., le bail p.y. (1996). atlas des poissons d'eau douce de guyane (tome 1). muséum national d'historie naturelle, ministère de l'environnement, paris. 1-429. sousa l.m., netto-ferreira a.l., birindelli j.l.o. (2010). two new species of moenkhausia eigenmann 207 int. j. aquat. biol. (2016) 4(3): 202-207 (characiformes: characidae) from serra do cachimbo, pará, northern brazil. neotropical ichthyology, 8: 255-264. taylor w.r., vandyke g.c. (1985). revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. cybium, 9: 107-119. weitzman s.h., malabarba l.r. (1999). systematics of spintherobolus (teleostei: characidae: cheirodontinae) from eastern brazil. ichthyological exploration of freshwaters, 10(1): 1-43. int. j. aquat. biol. (2017) 5(2): 79-94; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article rotifers (rotifera: eurotatoria) from floodplain lakes of the dibru saikhowa biosphere reserve, upper assam, northeast india: ecosystem diversity and biogeography bhushan kumar sharma*,1nogen noroh, sumita sharma freshwater biology laboratory, department of zoology, north-eastern hill university, shillong 793 022, meghalaya, india. article history: received 7 february 2017 accepted 5 march 2017 available online 2 5 april 2017 keywords: conservation area composition distribution interesting taxa richness wetlands abstract: this study aims to assess ecosystem diversity of rotifera of the floodplain lakes (beels) of the brahmaputra river basin with reference to faunal diversity of the taxon in wetlands of conservation areas of india. we observed 141 rotifer species, belonging to 31 genera and 17 families, from three beels of the dibru-saikhowa biosphere reserve (dsbr) of assam, northeast india (nei) with high total richness (117±2 species) in individual beels. one, two and three species are new to the oriental region, india and assam state, respectively and 21 species are globally interesting. the diverse lecanidae > lepadellidae > trichocercidae; the paucity and scarceness of brachionidae and brachionus spp. in particular; and rare nature of keratella, filinia, asplanchna, polyarthra, and conochilus are salient. the monthly richness and community similarities affirmed heterogeneity in species composition in individual beels while this study exhibited overall rotifer homogeneity amongst beels. the richness followed monthly oscillations in the three beels and lacked significant variations amongst beels. the peak richness of 76 species during summer (may, 2014) from no. 11 beel is one of the richest rotifer assemblages known in single date collection from an aquatic ecosystem of south asia. our results explained little influence of individual abiotic factors while canonical correspondence analysis endorsed high cumulative influence of 17 abiotic factors on richness in all beels. introduction rotifera form an important group of freshwater metazoans and of fish-food-organisms, and an integral link of freshwater food-webs. segers et al. (1993) hypothesized tropical and subtropical floodplain lakes to be globally important rotifer habitats. the importance of the rotifer diversity of the indian floodplains in light of segers’s hypothesis have been affirmed for the floodplains of assam (sharma and sharma, 2014a; sharma et al., 2017) and manipur (sharma et al., 2016) states of northeastern india (nei). to augment our hypothesis on biodiverse nature of the floodplain wetlands of upper brahmaputra river basin with regard to ecosystem diversity, we analyzed the rotifer communities of three beels of the dibru-saikhowa biosphere reserve (dsbr) of assam, northeast india. this study * corresponding author: bhushan kumar sharma doi: https://doi.org/10.22034/ijab.v5i2.281 e-mail address: profbksharma@gmail.com deserves attention in view of limited studies till date on the rotifer diversity from wetlands of conservations areas of india. the notable related works included the reports from the majuli floodplains (sharma, 2014; sharma et al., 2015) and two ramsar sites of india namely deepor beel (sharma and sharma, 2015a) and loktak lake (sharma et al., 2016), and nokrek biosphere reserve of meghalaya (sharma and sharma, 2011). this study thus merits ecosystem diversity and biogeography importance for the indian rotifera and for biodiversity of the taxon in wetlands of conservation areas of india. rotifera of three dibru-saikhowa biosphere reserve beels of the upper brahmaputra river basin of nei are analyzed for a period of two years. we provide an inventory of the documented species and several interesting taxa are illustrated to warrant 80 sharma et al./ rotifers from floodplain lakes of the dibru saikhowa biosphere reserve, india validation. the nature and composition of the rotifer fauna is discussed with emphasis on new records, taxa of global and regional biogeography interest and distribution of various taxa. remarks are made on the rotifer diversity of the sampled beels with reference to monthly variations in richness, community similarities and the influence of abiotic factors. materials and methods this study is part of a limnological survey (october 2013–september 2015) undertaken in three floodplain lakes namely maghuri (27°34'19.2''-27°34'25.2''n; 95°22'04.5''-95°22'35.2''e), khamti guali (27°34' 23.4''-27°34'26.0''n; 95°20'27.4''-95°20'53.8''e) and no. 11 (27°34'04.8''-27°34'11.5''n; 95°20'21.8''-95° 20'25.8''e) beels located in the ‘buffer zone’ of the dibru-saikhowa biosphere reserve (dsbr), upper assam, nei (fig. 1). these beels are influenced by intensive fish harvesting and by floodwaters during south-west monsoon. aquatic vegetation of maghuri beel was comprised of eichhornia crassipes (mart.) solms, pistia stratiotes linnaeus, lemna sp., azolla sp., ludwigia sp., rumex sp. and cabomba caroliniana; and khamti guali and no. 11 beels showed aquatic vegetation composed of eichhornia crassipes (mart.) solms, pistia stratiotes linnaeus, hygroryza aristata (retz.) nees, trapa natans linnaeus, eleocharis sp., lemna sp., and nymphaea sp. water samples collected monthly from the three beels were analyzed for 17 environmental parameters. of these, water temperature, specific conductivity and ph were recorded with field probes, and rainfall data was obtained from local meteorological centre. dissolved oxygen was estimated by winkler’s method, and free carbon dioxide, total alkalinity, total figure 1. (a) map of india showing assam state of northeast india; (b) map of assam state indicating tinsukia district; (c) map showing sampled floodplain lakes (google map). a c b 81 int. j. aquat. biol. (2017) 5(2): 79-90 hardness, calcium, magnesium, chloride, dissolved organic matter, total dissolved solids, phosphate, nitrate, sulphate and silicate were analyzed following apha (1992). the qualitative plankton and semiplankton samples were collected monthly from the selected beels by towing a nylobolt plankton net (# 50 μm) and were preserved in 5% formalin. the rotifers were mounted in polyvinyl alcohol–lactophenol and were examined with leica (dm 1000) stereoscopic phase contrast microscope fitted with an image analyzer. we followed koste (1978), segers (1995), sharma (1998), sharma and sharma (2008, 2013) for identification of the rotifers. sørensen’s index was followed to calculate rotifer community similarities. two-way anova was used to investigate significance of the richness variations. the relationships between abiotic factors and rotifer richness were determined by pearson’s correlation coefficients (r); p values were computed and their significance was examined after applying bonferroni correction. the canonical correspondence analysis was done using xlstat (2015) to analyze influence of the stated 17 environmental variables on the rotifer richness in the three beels. results the variations (mean±sd) in abiotic factors of the three beels of the dibru-saikhowa biosphere reserve are indicated in table 1. a total of 141 species of rotifera belonging to 31 genera and 17 families are observed in our collections (appendix 1). squatinella bifurca (fig. 2 a-b) is a new record from the oriental region; lecane isanensis (fig. 2c) and l. shieli (fig. 2d) are new records from india; and lecane aeganea (fig. 2e), l. dorysimilis (fig. 2f), trichocerca maior (fig. 2g) and t. siamensis (fig. 2h) are new records from assam state. brachionus dichotomus reductus (fig. 3a), filinia camasecla (fig. 3b), keratella edmondsoni (fig. 3c), lecane blachei berzins (fig. 3d), l. batillifer (fig. 3e), l. bulla diabolica (fig. 3f), l. lateralis (fig. 3g), l. niwati (fig. 3h), l. simonneae (fig. 3i), l. superaculeata (fig. 3j), lepadella discoidea (fig. 4a), l. vandenbrandei (fig. 4b), notommata spinata (fig. 4c), testudinella amphora (fig. 4d), t. brevicaudata (fig. 4e), t. dendradena (fig. 4f), trichocerca edmondsoni (fig. 4g) and t. hollaerti (fig. 4h) are interesting species with regards to regional distribution. total rotifer richness (s) in three beels is observed to vary between 116-120 (117±2) species with no. 11 > khamti guali > maghuri beels and recorded 84.699.1% community similarities (sørensen’s index) amongst them. this study indicated annual richness of 107-112, 93-106 and 80-101 species and recorded 87.7%, 78.4% and 68.5% similarities of the rotifer assemblages during the study period; the similarities ranged between 30.0-70.4% and 28.9-71.7%; 27.569.0 and 28.9-76.1%: and 29.1-65.0 and 28.6-75.0% table 1. variations (mean±sd) in abiotic factors (october 2013–september 2015). parameters maghuri beel khamti guali beel no.11 beel range mean±sd range mean±sd range mean±sd water temp (o c) 15.0-30.8 25.2±5.2 14.0-32.6 25.9±5.7 15.5-30.7 25.6±5.2 rainfall (mm) 0.0 615.0 188.4-193.6 0.0-615.0 188.4-193.6 0.0-615.0 188.4-193.6 ph 6.75-8.09 7.37±0.49 6.84-8.04 7.38±0.46 6.92-7.94 7.28±0.36 conductivity (µs/cm) 69.0-140.0 104.8±21.2 74.0-150.0 109.7±23.9 46.0-139.0 82.5±26.2 dissolved oxygen (mg/l) 4.0-8.0 5.8±1.5 4.0-8.8 5.9±1.3 4.0-8.0 5.5±1.1 free carbon dioxide (mg/l) 10.0-28.0 15.8±5.3 10.0-24.0 16.3±4.2 8.0-24.0 15.8±4.8 total alkalinity (mg/l) 40.0-80.0 65.2±12.9 40.0-84.0 59.0±13.8 40.0-80.0 53.8±10.5 total hardness (mg/l) 54.0-86.0 70.5±11.0 50.0-80.0 67.7±9.9 40.0-80.0 64.2±11.5 calcium (mg/l) 16.8-25.2 20.5±2.6 14.7-27.3 20.8±3.6 12.7-23.1 18.6±2.8 magnesium (mg/l) 7.0-16.3 12.2±3.1 7.99-15.4 11.4±2.5 8.1-15.4 11.9±2.5 chloride (mg/l) 7.99-20.97 13.48±3.20 9.90-19.90 14.69±3.86 10.98-23.97 16.90±4.27 dissolved organic matter (mg/l) 0.041-0.131 0.095±0.033 0.048-0.131 0.104±0.029 0.045-0.120 0.090±0.026 total dissolved solids (mg/l) 0.080-0.200 0.131±0.051 0.040-0.240 0.123±0.053 0.040-0.280 0.147±0.078 phosphate (mg/l) 0.140-0.322 0.212±0.064 0.138-0.351 0.225±0.069 0.142-0.371 0.222±0.075 nitrate (mg/l) 0.352-1.881 0.862±0.456 0.440-1.702 0.852±0.378 0.369-1.550 0.839±0.374 sulphate (mg/l) 6.14-25.05 13.49±7.04 6.72-23.99 15.08±6.91 5.77-22.91 13.34±6.931 silica (mg/l) 0.657-1.361 0.890±0.209 0.678-1.372 0.896±0.212 0.681-1.379 0.893±0.213 82 sharma et al./ rotifers from floodplain lakes of the dibru saikhowa biosphere reserve, india during two years in the three beels, respectively. the monthly richness (figs. 5-7) ranged between 1561(30±10) species in maghuri beel, 15-68(34±14) species in khamti guali beel and between 1376(30±10) species in no. 11 beel. lecanidae and lepadellidae included 48 and 24 species, respectively and showed higher richness throughout the study (figs. 5-7). anova registered insignificant richness variations amongst beels but recorded significant monthly variations (f2, 46=2.636, p=0.0025) between them. it registered significant annual richness variations in maghuri beel (f1, 23=7.604, p=0.0186) and significant monthly variations in khamti guali beel (f11, 23=3.112, p=0.0363) but no. 11 beel recorded insignificant monthly and annual variations. no significant correlation between environmental parameters and species richness could be found in maghuri beel; brachionidae richness (r=0.592; p=0.0000) was positively influenced by water temperature in khamti guali beel; and lecanidae (r= 0.703; p=0.0001) and testudinellidae (r=0.569; p=0.000) richness in no. 11 beel were positively influenced by rainfall. the canonical correspondence analysis (fig. 8-10) registered 79.2%, 80.9% and 68.1% influence along first two axes on richness of rotifer assemblages in maghuri, khamti guali and no. 11 beels, respectively. discussion all three beels of the dibru-saikhowa biosphere reserve (dsbr) are characterized by tropical, circumneutral-alkaline, marginally hard, and moderately oxygenated calcium poor waters. low ionic concentrations warranted inclusion of these figure 2. new records. (a) squatinella bifurca (bolton), lateral view; (b) squatinella bifurca (bolton), dorsal view; (c) lecane isanensis sanoamuang & savatenalinton, ventral view; (d); lecane shieli segers & sanoamuang, dorsal view; (e) lecane aeganea harring, ventral view; (f) lecane dorysimilis trinh dang, segers & sanoamuang, ventral view; (g) trichocerca maior hauer, lateral view; (h) trichocerca siamensis segers & pholpunthin, lateral view. 83 int. j. aquat. biol. (2017) 5(2): 79-90 beels under class i category of trophic classification following talling and talling (1965). occurrence of free carbon dioxide throughout the study, total alkalinity attributed to bicarbonate ions, lack of human influence indicated by low chloride concentration and lower nutrients are notable. the relatively higher magnesium content and wide variations in sulphate content are interesting in comparison to various floodplain lakes of nei (sharma and sharma, 2014a; sharma et al., 2015, 2016). figure 3. interesting rotifers. (a) brachionus dichotomus reductus koste & shiel, ventral view; (b) filinia camasecla myers, dorsal view; (c) keratella edmondsoni ahlstrom, ventral view; (d); lecane blachei berzins, ventral view; (e) lecane batillifer (murray), dorsal view; (f) lecane bulla diabolica (hauer), lateral view; (g) lecane lateralis sharma, ventral view; (h) lecane niwati segers, kothetip & sanoamuang, ventral view; (i) lecane simonneae segers, dorsal view; (j) lecane superaculeata sanoamuang & segers, ventral view. 84 sharma et al./ rotifers from floodplain lakes of the dibru saikhowa biosphere reserve, india figure 4. interesting rotifers. (a)lepadella discoidea segers; (b) lepadella vandenbrandei gillard, dorsal view; (c) notommata spinata koste & shiel, dorsal view (partially compressed); (d); testudinella amphora hauer, dorsal view; (e) testudinella brevicaudata yamamoto, dorsal view; (f) testudinella dendradena de beauchamp, ventral view; (g) trichocerca edmondsoni (myers), lateral view; (h) t. hollaerti de smet, lateral view. figure 5. monthly variations in richness of rotifera and important families in maghuri beel (october 2013–september 2015). 85 int. j. aquat. biol. (2017) 5(2): 79-90 our collections from dsbr beels located in the limited study area of upper assam revealed total richness (s) of 141 species. the diversity accounts for ~34.0% and ~50.0% of species of the phylum known from india and nei, respectively and thus affirmed biodiversity importance. the report of 31 genera and 17 families endorsed rich higher diversity of the taxon in light of 50 genera and 23 families known from nei (bks, unpublished). the rich and diverse nature of the rotifer biocoenosis indicated high habitat and ecosystem diversity of the three beels in spite of influence of intensive fishing and floods. our results supported hypothesis of segers et al. (1993) on the biodiverse nature of tropical and subtropical floodplains and of sharma (2005), sharma and sharma (2014a, 2014b) and sharma et al. (2017) on wetlands of the brahmaputra basin of nei vis-a-vis rotifera diversity. squatinella bifurca is an interesting addition to the species list of the oriental rotifera; known to be a figure 6. monthly variations in richness of rotifera and important families in khamti guali beel (october 2013–september 2015). figure 7. monthly variations in richness of rotifera and important families in no. 11 beel (october 2013–september 2015). 86 sharma et al./ rotifers from floodplain lakes of the dibru saikhowa biosphere reserve, india figure 8. cca coordination biplot of rotifer richness and abiotic factors of maghuri beel. abbreviations: abiotic: ca (calcium), cl (chloride), dom (dissolved organic matter), do (dissolved oxygen), fco2 (free carbon dioxide), rain (rainfall), no3 (nitrate), po4 (phosphate), sio2 (silicate), sp cond (specific conductivity), so4 (sulphate), ta (total alkalinity), tds (total dissolved solids), th (total hardness), ph (hydrogen-ion concentration), wt (water temperature). biotic: bra (brachionidae richness), lec (lecanidae richness), lep (lepadellidae richness), test (testudinellidae) tri (trichocercidae richness), rr (rotifera richness). figure 9. cca coordination biplot of rotifer richness and abiotic factors of khamti guali beel. abbreviations: abiotic: ca (calcium), cl (chloride), dom (dissolved organic matter), do (dissolved oxygen), fco2 (free carbon dioxide), rain (rainfall), no3 (nitrate), po4 (phosphate), sio2 (silicate), sp cond (specific conductivity), so4 (sulphate), ta (total alkalinity), tds (total dissolved solids), th (total hardness), ph (hydrogen-ion concentration), wt (water temperature). biotic: bra (brachionidae richness), lec (lecanidae richness), lep (lepadellidae richness), test (testudinellidae) tri (trichocercidae richness), rr (rotifera richness). 87 int. j. aquat. biol. (2017) 5(2): 79-90 palearctic species (segers, 2007), our report from upper assam, nei extended its distribution to the oriental region. the tropical-latitude population of this cold-water taxon is likely to represent glacial relicts as hypothesized by segers (1996) while the report of this species at foot hills in eastern himalayas may be attributed to extension of the himalayan mountain ranges as hypothesized by sharma and sharma (2014c). lecane isanensis and l. shieli are new records to the indian rotifera. the former was described from northeast thailand (sanoamuang and savatenalinton, 2001) and was designated as a thai endemic (segers and savatenalinton, 2010). the report from upper assam extended its distribution to the indian subregion and we thus categorize this lecanid to be an oriental endemic. the australasian lecane shieli was described from nam pung reservoir, thailand (segers and sanoamuang, 1994) and was subsequently recorded from several localities in thailand (sa-ardrit et al., 2013). kobayashi et al. (2007) recorded it from australia and altındağ et al. (2009) examined specimens from turkey while it is known as an alien species in different parts of the world (pociecha et al., 2016). we observed 21 biogeographically interesting rotifers (~15.0% of s); these included (a) four australasian endemics brachionus dichotomus reductus, lecane batillifer, l. shieli and notommata spinata; (b) seven oriental endemics namely keratella edmondsoni, lecane blachei, lecane bulla diabolica, l. isanensis, l. niwati, l. superaculeata and filinia camasecla; and (c) seven paleotropical species i.e., lepadella discoidea, l. vandenbrandei, lecane lateralis, l. simonneae, l. unguitata, testudinella brevicaudata and trichocerca hollaerti; (d) the palaearctic cephalodella trigona and squatinella bifurca; and (e) the indo-chinese lecane dorysimilis. the first two categories in particular affirmed affinity of our rotifer inventory with those of southeast asia and australia. lecane aeganea, trichocerca maior and t. siamensis are new records to the rotifer fauna of figure 10. cca coordination biplot of rotifer richness and abiotic factors of no.11 beel. abbreviations: abiotic: ca (calcium), cl (chloride), dom (dissolved organic matter), do (dissolved oxygen), fco2 (free carbon dioxide), rain (rainfall), no3 (nitrate), po4 (phosphate), sio2 (silicate), sp cond (specific conductivity), so4 (sulphate), ta (total alkalinity), tds (total dissolved solids), th (total hardness), ph (hydrogen-ion concentration), wt (water temperature). biotic: bra (brachionidae richness), lec (lecanidae richness), lep (lepadellidae richness), test (testudinellidae) tri (trichocercidae richness), rr (rotifera richness). 88 sharma et al./ rotifers from floodplain lakes of the dibru saikhowa biosphere reserve, india assam. the first two species were added to the indian rotifera based on the reports from mizoram (sharma and sharma, 2015b) while t. siamensis was known till date from india from mizoram and meghalaya states of nei. the present report from upper assam further extended distribution of three species within nei. our samples from dsbr beels contained several species of regional biogeography interest in the indian sub-region; these included brachionus mirabilis, keratella lenzi, platyias leloupi, tripleuchlanis plicata, lophocharis salpina, macrochaetus longipes, mytilina acanthophora, m. bisulcata, m. michelangellii, lecane doryssa, l. elegans, l. flexilis, l. haliclysta, lepadella benjamini, l. costatoides, l. dactyliseta, l. lindaui, l. quinquecostata, l. hornemanni, l. inermis, l. monostyla, l. nitida, l. obtusa, l. pusilla, l. rhenana, l. rhytida, l. thienemanni, l. undulata, monommata grandis, notommata glyphura, testudinella amphora, t. dendradena, t. parva, t. tridentata, trichocerca bidens, t. edmondsoni, t. flagellata, t. insignis, t. scipio, t. tigris and t. weberi. amongst the taxa of global and regional interest, a notable fraction (~10% of s) namely brachionus dichotomus reductus, cephalodella trigona, lecane batillifer, l. blachei, l. dorysimilis, l. niwati, l. rhenana, l. superaculeata, lepadella benjamini, l. vandenbrandei, mytilina michelangellii, notommata spinata, trichocerca hollaerti and t. maior are known from india with distribution exclusively restricted till date to nei. total rotifer richness (s) of dsbr beels was reasonably comparable with 161 species (sharma et al., 2016) known from loktak lake (a ramsar site), manipur and 160 species reported from four beels of lower assam (sharma et al., 2017). the richness broadly concurred with 144 species known from 12 beels of the majuli river island, upper assam (sharma, 2014; sharma et al., 2015) and it is more diverse than the reports of 124 species from seven beels of lower assam (sharma and sharma, 2001) and 110 species (arora and mehra, 2003) from the yamuna floodplains at delhi. further, dsbr rotifera is distinctly rich than 38 species recorded from southwest west bengal floodplains (khan, 2003); this comparison deserved caution because of likely overlooking identification of smaller taxa culminating in incomplete species list. a more critical caution is focussed on routine ‘ad-hoc’ reports of underestimated richness of 16 (kar and kar, 2013), two (gupta and devi, 2014), 17 (das and kar, 2016) and 21 (kar and kar (2016) species from beels and wetlands of south assam. the diverse nature of lecanidae (34.0% of s); and the collective importance (27.6% of s) of lepadellidae > trichocercidae, and of lecane > lepadella > trichocerca (17.7% of s) impart the littoral-periphytic nature to the rotifer assemblages. the relative importance of the stated taxa endorsed hypothesis of green (2003) on the possibility of assemblage rules for the periphytic rotifers. this generalization also corroborated with the reports from indian floodplain wetlands from nei (sharma and sharma, 2015a; sharma et al., 2015, 2016, 2017). the richness of brachionidae (13 species) deserved cautious mention because of its notable scarceness in the three beels. the paucity and scarceness of brachionus species (5 species) and rare occurrence of keratella, filinia, asplanchna, polyarthra, and conochilus are salient. these interesting features are hypothesized to lack of limnetic conditions in dbsr beels and even to certain factors limiting distribution of these taxa; the latter yet need to be investigated. in general, the brachionid paucity concurred with the reports (sharma, 2014; sharma et al., 2015, 2016) from certain nei floodplain wetlands. the rich nature and common occurrence of ‘tropic-centered’ lecane and cosmopolitan species (~63.0% of s), and collective importance (~19.3 % of s) of cosmotropical and pantropical species imparted ‘tropical character’ to rotifera of dsbr beels. these features are concurrent with the remarks on tropical rotifer faunas (green, 1972; fernando, 1980; segers, 1996). our collections revealed high total richness and limited variations (117±2 species) with no. 11 > khamti guali > maghuri beels. this generalization supported by high community similarities due to common occurrence of large fraction (~68.% of s) of species in the three beels affirmed overall rotifer 89 int. j. aquat. biol. (2017) 5(2): 79-90 homogeneity amongst beels. the stated features, in turn, corroborated with the rotifer assemblages of four beels of lower brahmaputra river basin (sharma et al., 2017). the richness broadly concurred with the reports of 114 species (jose de paggi, 2001) from the floodplains of argentina; it is marginally more biodiverse than 106 taxa known from thale-noi lake, thailand (segers and pholpunthin, 1997); and 104 species from laguana bufeos, bolivia (segers et al., 1998). dbsr rotifers are rather diverse than 69-93 and 60-100 species documented from various beels of assam by sharma and sharma (2008) and sharma et al. (2015), respectively. the wider monthly variations with lowest and maximum richness between 13-15 species and 61-76 species, respectively; low mean values and lower community similarities with inter-annual variations and maximum instances of below 60% similarities affirmed heterogeneity in the rotifer composition within individual beels. the monthly variations and oscillating patterns of the richness in the three beels may be attributed to habitat disturbances caused due to fishing and floodwaters as hypothesized by sharma et al. (2017). it may also be attributed to invasion of eichhornia crassipes as indicated by sharma et al. (2015). anova registered insignificant richness variations amongst beels but recorded significant monthly variations between them. further, it registered significant annual richness variations in maghuri beel and significant monthly variations in khamti guali beel but no. 11 beel recorded insignificant monthly and annual variations. the peak richness of 76 species in may, 2014 (summer) single date collection from no. 11 beel indicated ecosystem diversity importance. it broadly concurred with our highest records from the indian sub-region: 79 species from deepor beel (sharma and sharma, 2013) in july, 2010; 79 species each reported from loktak lake – a ramsar site in december 2002 (sharma, 2009) and december, 2011 (sharma et al., 2016). incidentally, it is one of the richest rotifer assemblages known in single date collection from any freshwater ecosystem of south asia. nevertheless, it merits certain interest than the highest global report of 102+ rotifer species in broa reservoir, brazil (segers and dumont, 1995) in august, 1994 but not withstanding 135 species reported in august, 1999 (segers and sanoamuang, 2007) otherwise originating from two wetlands i.e., a laotian rice field and an adjacent pond. sharma et al. (2017) proposed l/b quotient based on lecane: brachionus species ratios to characterize habitat variations of the beels of lower brahmaputra floodplains of assam. we are unable to use this quotient in the beels of upper assam because of characteristic feature of paucity of brachionus spp.. sladecek’s b/t quotient based on brachionus: trichocerca species (sladecek, 1983) also holds no validity to assess trophic status of dsbr beels for the said paucity of the brachionid taxon. our results explained little influence of abiotic parameters on the rotifer richness in the three beels; this remark is supported by lack of significance influence of any individual abiotic factor in maghuri beel while temperature exerted positive influence on brachionidae richness in khamti guali beel, and rainfall positively influenced lecanidae and testudinellidae richness in no. 11 beel. the canonical correspondence analysis, however, indicated high influence of 17 environmental parameters along first two axes on richness of rotifer assemblages in the first two beels and moderately high influence in no. 11 beel, thereby, supporting collective role of abiotic attributes. the importance of different factors vides cca on richness of various rotifer taxa in the three beels affirmed habitat diversity amongst the sampled wetlands. in general, our report on the collective influence of environmental parameters rather than limited influence of individual factors corroborated with the rotifer assemblages of four beels of lower brahmaputra river basin (sharma et al., 2017). the species rich and diverse rotifera of dsbr beels, interesting new records and the report of a sizable fraction of species of global and regional distribution interest are features of biodiversity interest for indian rotifera and for meta-analysis of diversity of the taxon in the indian as well as tropical and subtropical floodplains. the littoral-periphytic 90 sharma et al./ rotifers from floodplain lakes of the dibru saikhowa biosphere reserve, india rotifer assemblages with scarceness and paucity of various taxa; high total richness and homogeneity amongst the three beels in spite of influence of fishing and floods vis-a vis lower monthly richness and the rotifer heterogeneity in individual beels; oscillating monthly richness variations; cumulative influence of 17 abiotic factors on the richness rather than individual role provide useful inputs on ecosystem diversity. acknowledgments the senior author (bks) is thankful to the ministry of environment and forests (govt. of india) for sanction of a research grant no. 22018-09/2010-cs (tax) under which this study was initiated. the subsequent samples collected by nn and additional collections by ss were analyzed for this study. we thank the head, department of zoology, north-eastern hill university, shillong for laboratory facilities. the authors have no conflict of research interests. references altındağ a., segers h., kaya m. 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(2017) 5(2): 79-90 order: ploima family: brachionidae 1. anuraeopsis fissa (gosse, 1851) 2. brachionus dichotomus reductus koste & shiel, 1980 3. b. diversicornis (daday, 1883) 4. b. falcatus zacharias, 1898 5. b. mirabilis daday, 1897 6. b. quadridentatus hermann, 1783 7. keratella cochlearis (gosse, 1851) 8. k. edmondsoni ahlstrom, 1943 9. k. lenzi hauer, 1953 10. k. tropica (apstein, 1907) 11. plationus patulus (o.f. muller, 1786) 12. platyias leloupi (gillard, 1967) 13. p. quadricornis (ehrenberg, 1832) family: euchlanidae 14. beauchampiella eudactylota (gosse, 1886) 15. dipleuchlanis propatula (gosse, 1886) 16. euchlanis dilatata ehrenberg, 1832 17. e. incisa carlin, 1939 18. tripleuchlanis plicata (levander, 1894) family: mytilinidae 19. lophocharis salpina (ehrenberg, 1834) 20. mytilina acanthophora hauer, 1938 21. mytilina bisulcata (lucks, 1912) 22. m. brevispina (ehrenberg, 1830) 23. m. michelangellii reid & turner, 1988 24. m. ventralis (ehrenberg, 1830) family: trichotriidae 25. macrochaetus longipes myers, 1934 26. m. sericus (thorpe, 1893) 27. trichotria tetractis (ehrenberg, 1830) family: lepadellidae 28. colurella obtusa (gosse, 1886) 29. c. sulcata (stenroos, 1898) 30. c. uncinata (o.f. muller, 1773) 31. lepadella acuminata (ehrenberg, 1834) 32. l. apsida harring, 1916 33. l benjamini harring, 1916 34. l. biloba hauer, 1958 35. l. costatoides segers, 1992 36. l. dactyliseta (stenroos, 1898) 37. l. discoidea segers, 1993 38. l. eurysterna myers, 1942 39. l. lindaui koste, 1981 40. l. minuta (weber & montet, 1918) 41. l. ovalis (o.f. muller, 1786) 42. l. patella (o.f. muller, 1773) 43. l. quinquecostata (lucks, 1912) 44. l. rhomboides (gosse, 1886) 45. l. triptera ehrenberg, 1832 46. l. vandenbrandei gillard, 1952 47. l. (h.) apsicora myers, 1934 48. l. (h.) ehrenbergi (perty, 1850) 49. l. (h.) heterostyla (murray, 1913) 50. squatinella bifurca (bolton, 1884) * 51. s. lamellaris (o. f. müller, 1786) family: lecanidae 52. lecane aculeata (jakubski, 1912) 53. l. aeganea harring, 1914 *** 54. l. arcuata (bryce, 1891) 55. l. arcula harring, 1914 56. l. batillifer (murray, 1913) 57. l. bifurca (bryce, 1892) 58. l. blachei berzins, 1973 59. l. bulla bulla (gosse, 1851) l. bulla diabolica (hauer, 1936) 60. l. closterocerca (schmarda, 1859) 61. l. crepida harring, 1914 62. l. curvicornis (murray, 1913) 63. l. decipiens (murray, 1913) 64. l. dorysimilis trinh dang, segers & sanoamuang, 2015 65. l. doryssa harring, 1914 66. l. elegans harring, 1914 67. l. flexilis (gosse, 1886) 68. l. furcata (murray, 1913) 69. l. haliclysta harring & myers, 1926 70. l. hamata (stokes, 1896) 71. l. hornemanni (ehrenberg, 1834) 72. l. inermis (bryce, 1892) 73. l. inopinata harring & myers, 1926 74. l. isanensis sanoamuang & savatenalinton, 2001 ** 75. l. lateralis sharma, 1978 76. l. leontina (turner, 1892) 77. l. ludwigii (eckstein, 1883) 78. l. luna (müller, 1776) 79. l. lunaris (ehrenberg, 1832) 80. l. monostyla (daday, 1897) 81. l. nitida (murray, 1913) 82. l. niwati segers, kothetip & sanoamuang, 2004 83. l. obtusa (murray, 1913) 84. l. ohioensis (herrick, 1885) 85. l. papuana (murray, 1913) 86. l. ploenensis (voigt, 1902) 87. l. pusilla harring, 1914 88. l. pyriformis (daday, 1905) 89. l. quadridentata (ehrenberg,1830) 90. l. rhenana hauer, 1929 91. l. rhytida harring & myers, 1926 92. l. shieli segers & sanoamuang, 1994 ** 93. l. signifera (jennings, 1896) 94. l. simonneae segers, 1993 95. l. stenroosi (meissner, 1908) appendix 1: systematic list of rotifera known from the dibru-saikhowa biosphere reserve beels. phylum: rotifera class: eurotatoria subclass: monogononta 94 sharma et al./ rotifers from floodplain lakes of the dibru saikhowa biosphere reserve, india 96. l. superaculeata sanoamuang & segers, 1997 97. l. thienemanni (hauer, 1938) 98. l. undulata hauer, 1938 99. l unguitata (fadeev, 1925) 100. l. ungulata (gosse, 1887) family: notommatidae 101. cephalodella gibba (ehrenberg, 1830) 102. c. mucronata myers, 1924 103. cephalodella trigona (rousselet, 1895) 104. monommata. grandis tessin, 1890 105. m. longiseta (o.f. müller, 1786) 106. notommata glyphura wulfert, 1935 107. n. spinata koste & shiel, 1991 family: scaridiidae 108. scaridium longicaudum (o.f. müller, 1786) family: trichocercidae 109. trichocerca bicristata (gosse, 1887) 110. t. bidens (lucks, 1912) 111. t. edmondsoni (myers, 1936) *** 112. t. elongata (gosse, 1886) 113. t. flagellata hauer, 1937 114. t. hollaerti de smet, 1990 115. t insignis (herrick, 1885) 116. t. maior hauer, 1936 117. t. pusilla (jennings, 1903) 118. t. rattus (o.f. müller, 1776) 119. t. scipio (gosse, 1886) 120. t. siamensis segers & pholpunthin, 1997 *** 121. t. similis (wierzejski, 1893) 122. t. tigris (o.f. müller, 1786) 123. t. weberi (jennings, 1903) family: asplanchnidae 124. asplanchna brightwelli gosse, 1850 125. a. priodonta gosse, 1850 family: synchaetidae 126. polyarthra vulgaris carlin, 1943 family: dicranophoridae 127. dicranophorus forcipatus (o.f. müller, 1786) order: flosculariaceae family: floscularidae 128. sinantherina socialis (linne, 1758) 129. s. spinosa (thorpe, 1893) family: conochilidae 130. conochilus unicornis rousselet, 1892 family: testudinellidae 131. testudinella amphora hauer, 1938 132. t. brevicaudata yamamoto, 1951 133. t. dendradena de beauchamp, 1955 134. t. emarginula (stenroos, 1898) 135. t. parva (ternetz, 1892) 136. t. patina (hermann, 1783) 137. t. tridentata smirnov, 1931 family: trochosphaeridae 138. filinia camasecla myers, 1938 139. f. longiseta (ehrenberg, 1834) 140. f. opoliensis (zacharias, 1898) sub-class: digononta order: bdelloidea family: philodinidae 141. rotaria neptunia (ehrenberg, 1830) ---------------------------------------------------------------------------------------------------------------------------------------------------- * new record from the oriental region; ** new record from india; *** new record from assam int. j. aquat. biol. (2023) 11(1): 86-97 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article discovery and dna analysis of the invasive freshwater mussel sinanodonta lauta (unionidae) in south iran kazem alwanzadegan1, hadise kashiri1, ivan n. bolotov2, ertan ercan3, ainaz shirangi4, edris ghaderi1 1department of aquatic ecology, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. 2n. laverov federal center for integrated arctic research of the ural branch of the russian academy of sciences, northern dvina emb. 23, 163000 arkhangelsk, russia. 3fisheries faculty, mugla sitki koçman university, mugla, turkey. 4basic sciences faculty, gonbad kavous university, gonbad, iran. article history: received 7 december 2022 accepted 13 january 2023 available online 2 5 april 2023 keywords: alien population coi haplotype dez river khuzestan abstract: some members of the genus sinanodonta (modell, 1944) are known as successful invaders of freshwater habitats. here we report the first record of sinanodonta lauta (martens, 1877) from iran and provide morphological and molecular data on this alien population. this species was observed in the dez river and a fish farm in khuzestan, south of iran. the s. lauta introduction seems to be closely related to the introduction of asian carps from east asia. the individuals collected from the river exhibited two coi haplotypes, probably due to several introduction events of host fish. based on our haplotype network, the alien individuals found in iran is closer to the native ones from south korea (two-three substitutions) compared to other native individuals reported from japan and russian far east. the presence of 10-11 years old specimens in dez river shows that the species can survive well in the natural environment of southern iran. further expansion and colonization of s. lauta in south iran or beyond it are not unexpected due to some human-mediated dispersal events as well as waterways in the region. introduction freshwater bivalves of the family unionidae are key ecological members of aquatic ecosystems due to contributing in purifying water, bioturbating, nutrient cycling and provisioning habitat (vaughn, 2018). unionid populations have been considerably declined, with many critically endangered (lydeard et al., 2004; böhm et al., 2020). however, some members of the genus sinanodonta are found to be hyper-successful invaders of freshwater habitats (sousa et al., 2014). sinanodonta comprises several species native to east asia (lopes-lima et al., 2020), but some have been widely introduced across many regions, including europe, north america, and parts of asia outside the native range (lopes-lima et al., 2016; bespalaya et al., 2018; bauer et al., 2021). among the species of this genus, s. woodiana is regarded as one of the most invasive aquatic macroinvertebrates (beran, 2020). recent molecular correspondence: hadise kashiri doi: https://doi.org/10.22034/ijab.v11i2.1796 e-mail: hadiskashiri@gmail.com dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.1.3 studies have shown that s. woodiana is a closely related species complex that includes several separate species-level lineages (kondakov et al., 2018). two species-level lineages of the complex (temperate and tropical) are known to be successful invaders sharing thoroughly allopatric non-native areas (bolotov et al., 2016). however, a molecular study on s. woodiana complex revealed that s. lauta is one more invasive lineage of this complex (kondakov et al., 2020a). the native distribution of s. lauta ranges across japan, south korea, and russian far east (primorye region) (lopes-lima et al., 2020), but its non-native populations have been reported outside the native range, including eastern siberia (yenisei river) (bespalaya et al., 2018), european russia (volga river), western siberia (ob river) (kondakov et al., 2020b), kazakhstan (ili river) (kondakov et al., 2020a) and borneo (zieritz et al., 2020). the species https://link.springer.com/article/10.1007/s10750-020-04385-w#auth-monika-b_hm 87 int. j. aquat. biol. (2022) 11(1): 86-97 appears to have been indirectly introduced in nonnative areas through fish hosts carrying the glochidia or directly by aquarists (bespalaya et al., 2018; zieritz et al., 2020). the spread of such filter-feeding nonindigenous mussels represents a significant threat to freshwater ecosystems (douda et al., 2017) and may cause serious ecological and economic consequences in invaded regions (sousa et al., 2014). due to the capability to cope with a broad range of environmental situations (douda et al., 2012), including hypoxia and pollution (sárkánykiss et al., 2000), fast growth, high reproduction rate (huber and geist, 2019), high filtration rate (douda et al., 2018) and host-generalist glochidia (douda et al., 2012; huber and geist, 2019), invasive sinanodonta populations can outcompete indigenous mussels (urbańska et al., 2021). these mussels can modify the biological and physical properties of freshwater ecosystems (douda et al., 2012). the glochidia of sinanodonta can also adversely affect the growth and physiological conditions of fish hosts (douda et al., 2017). in the late spring of 2021, we received a report from a fish farm in the south of iran about the existence of some giant mussels that caused trouble in the aquaculture activity. based on the information and photos we received, these large mussels seemed to be sinanodonta. finally, in july 2021, during a field trip to khuzestan province in the south of iran, we found the mussels not only on the fish farm but also in nature as well. here, we report the first record of invasive s. lauta in iran, describing its morphological traits, taxonomic status and possible affinities of the alien individuals in iran according to coi gene sequencing data and briefly discuss the possible vectors and expansion in the region. materials and methods study area and sampling: the study area is the dez river in khuzestan province, south of iran. the fieldwork was done on 8th and 9th july 2021. sinanodonta samples were collected from dez river, dezful )32°13'56.87"n; 48°18'43.96"e) (fig. 1), by a benthic hand net. no other unionid mussels were found in the sampling site. with a length of 400 km, the dez river is the main tributary of the largest river in iran, i.e. karun. the river originates from the mid-zagros mountains in lorestan province, flows to the khuzestan, and drains into the karun river. the minimum water temperature of the river is about 14.5°c in winter, and the maximum water temperature also does not exceed 32 in summer. the river bottom substrates range from muddy to stony in different areas (mohammadi ruzbahani et al., 2014). the bottom of the sampling site was dominated by silt substrate, with some smallmedium-sized stones scattered in some parts. in addition to the river, sinanodonta samples were also collected from a fish farm located in dezful, khuzestan province (32°13'49.85n; 48°19'2.24"e) (fig. 1a). for molecular studies, a piece of foot tissue was cut from live mussels and immediately preserved in 96% ethanol. mussel shells of the samples (n = 21 and 11 for dez and fish farm, respectively) were also collected for morphological studies. morphology and age estimation: the biometric variables of each mussel, including shell length (l), height (h) and width (w) were measured to the nearest 0.1 mm by an aaco caliper. the morphological indices of shell convexity (ci = w/l ratio × 100) and elongation (ei = h/l ratio × 100) were also calculated. the age of each sinanodonta individual was determined by counting the growth rings visible on the shell. dna extraction, polymerase chain reaction and sequencing: total genomic dna was extracted from the foot tissue of each specimen (n = 7 and 5 for dez and fish farm, respectively) via a high-salt procedure (sambrook et al., 1989) with a slight modification. dna quality and quantity were examined via agarose gel (1%) electrophoresis and a biophotometer spectrophotometer (eppendorf, hamburg, germany), respectively. we used the primers lco22me2 (5'-ggt caa caa ayc ata arg ata ttgg-3') and hco700dy2 (5'-tca ggg tga cca aaa aayca-3') (walker et al., 2006, 2007) to amplify the partial sequences of 88 alwanzadegan et al./ discovery and dna analysis of the invasive freshwater mussel cytochrome c oxidase subunit i (coi) gene. pcr was run on a thermal cycler (bio-rad mj mini thermal cycler, hercules, ca, usa) in 25 μl reaction mix containing 1 μl dna (20-160 ng/μl), 15 μl taq 2x master mix red (amplicon, denmark), 1 μl of each primer and 7 μl pcr grade water. the pcr condition was set as follows: 4 min at 94°c, 40 cycles at 94°c (30 s), 50°c (40 s) and 72°c (60 s), followed by 10 min at 72°c. the products were checked through agarose gel (1.5%) electrophoresis in tbe buffer (1x). finally, sequencing was carried out through an abi 3730xl automatic sequencer (applied biosystems, 3730/3730xl dna analyzers sequencing, bioneer, korea) using the same primers. molecular analysis: the obtained sequences were manually edited in bioedit 7.0.1 (hall, 1999). we used 136 coi sequences described in the previous figure 1. sinanodonta lauta; (a) the study area’s location. 1 = sampling site in dez river, khuzestan, 2 = fish farm in which the samples were collected. (b) habitat view of non-indigenous s. lauta in a branch of dez river, dezful, khuzestan (9th july 2021). 89 int. j. aquat. biol. (2022) 11(1): 86-97 studies (bolotov et al., 2016; kondakov et al., 2020b). furthermore, 24 more coi sequences were extracted from ncb i's genbank (table 1). multiple sequence alignment using clustalw also was implemented in bioedit. after trimming the sequences, a 616-bp coi fragment was left. similar sequences were removed through the online tool fabox 1.41 (villesen, 2007). finally, the phylogenetic tree was constructed based on 36 unique sequences. we also used two taxa, including margaritifera dahurica (kj161516) and m. laosensis (jx497731), as outgroups. phylogenetic relationships were reconstructed based on bayesian inference using mrbayes v3.2.2 taxon region population lineage accession number reference sinanodonta lauta iran alien c op048111 this study s. lauta iran alien c op048112 this study s. lauta iran alien c op048113 this study s. lauta iran alien c op048114 this study s. lauta iran alien c op048115 this study s. lauta iran alien c op048116 this study s. lauta iran alien c op048117 this study s. lauta iran alien c op048118 this study s. lauta iran alien c op048119 this study s. lauta iran alien c op048120 this study s. lauta iran alien c op048121 this study s. lauta iran alien c op048122 this study s. woodiana germany alien a mh319868 stelbrink et al. (2019) s. woodiana germany alien e ou070149 genbank s. woodiana iraq alien a lc656037 genbank s. woodiana philipines alien a kx424967 fornillos et al. (2020) s. woodiana philipines alien a kx424968 fornillos et al. (2020) s. woodiana philipines alien a kx424969 fornillos et al. (2020) s. woodiana philipines alien a kx424970 fornillos et al. (2020) s. woodiana philipines alien a kx424976 fornillos et al. (2020) s. woodiana philipines alien a kx424977 fornillos et al. (2020) s. woodiana philipines alien a kx424978 fornillos et al. (2020) s. woodiana philipines alien a kx424979 fornillos et al. (2020) s. woodiana philipines alien a kx424971 fornillos et al. (2020) s. woodiana philipines alien a kx424972 fornillos et al. (2020) s. woodiana philipines alien a kx424973 fornillos et al. (2020) s. woodiana philipines alien a kx424974 fornillos et al. (2020) s. woodiana philipines alien a kx424975 fornillos et al. (2020) s. schrenkii russia indigenous g ku853266 sayenko et al. (2017) s. schrenkii russia indigenous g ku853267 sayenko et al. (2017) s. schrenkii russia indigenous g ku853268 sayenko et al. (2017) s. schrenkii russia indigenous g ku853269 sayenko et al. (2017) s. lucida china indigenous f kx822667 genbank anodonta arcaeformis china indigenous kj434481 genbank a. arcaeformis china indigenous kj434479 genbank a. arcaeformis china indigenous kj434480 genbank table 1. coi sequences used in the present study (for other 136 sequences, see bolotov et al., 2016 and kondakov et al., 2020b). 90 alwanzadegan et al./ discovery and dna analysis of the invasive freshwater mussel (huelsenbeck and ronquist, 2001). the best-fitting models of nucleotide substitution on the basis of the akaike information criterion (akaike, 1973) were estimated through mrmodeltest v3.7 (posada and crandall, 1998) in paup v4.0 (swofford, 2003). two parallel runs were independently conducted. each included one cold and three heated metropolis coupled mcmc chains. the program was run for 10 million generations and sampled once every 10,000 generations with 20% burn-in fraction. the resulting tree was visualized through figtree v1.4.2 (rambaut, 2008). the genetic divergence based on p-distance was assessed in mega 6.0 (tamura et al., 2013). the median-joining network was also constructed through popart v1.7 (leigh and bryant, 2015) to study the relationships among haplotypes. results we detected a well-established viable population of the non-indigenous mussel sinanodonta in south of iran. the shell pattern of our samples was slightly elongated with brown/olive-green periostracum. as some sinanodonta species are morphologically too similar, we used coi sequencing to identify the species. according to the molecular data, s. lauta was recorded from khuzestan, iran (fig. 2). morphometric data: morphometric features of the s. lauta specimens collected from dez river (dr) and fish farm (ff) are shown in table 2. the youngest and oldest individual was 2 and 11 years old, respectively. the largest mussel was 152.96 mm (length) at 11 years old. the convexity index was figure 2. sinanodonta lauta; (a) and (b) represent shell interior and exterior view of the samples collected from fish farm and dez river, respectively. length height width convexity index elongation index age (year) fish farm min-max 125.92-146.58 72.62-91.28 47.70-55.64 37.13-40.4 57.67-64.4 6-10 mean±sd 137.23±7.17 85.02±5.36 52.80±3.26 38.46±0.94 61.93±1.68 8.45±1.44 dez river min-max 48.09-152.96 30.16-93.16 13.01-55.92 21.6-38.18 49.01-68.44 2-11 mean±sd 103.03±39.62 62.66±23.48 33.36±18.02 30.90±5.91 61.46±5.2 6.71±2.7 abbreviations: sd (standard deviation); min (minimum); max (maximum) table 2. morphometric features and age of sinanodonta lauta from iran. 91 int. j. aquat. biol. (2022) 11(1): 86-97 21.6-38.18 for dr samples and 37.13-40.4 for the samples collected from ff. the elongation index also ranged 49.01-68.44 and 57.6764.4 for dr and ff samples, respectively (table 2). molecular data: the coi sequences analysis confirmed the existence of the invasive species of s. lauta in iran. eight 664-bp and one 621-bp long fragments of the coi gene were acquired from the s. lauta specimens and deposited to the ncbi's genbank (table 1). we reconstructed the phylogenetic tree under tim+i+g model (fig. 3). the specimens from the fish farm belonged to a single haplotype, while the samples collected from dez river belonged to two haplotypes. our haplotypes were placed in the same clade with those from russian far east, south korea, kazakhstan, russia (siberia and volga), and japan (lineage c) with strong bootstrap support (99.98%), and there are seven more mitochondrial lineages (fig. 3). the mean coi p-distances among the sinanodonta spp. lineages are presented in table 3. this distance ranged from 2 (between lineages a and b) to 13.9 (between lineages a and f). the distance among lineage c comprising our samples and other lineages ranged from 2.8 to 13.6%. the mean p-distance within lineage c was also 1.1%. we constructed the median-joining network based on the coi sequences of lineage c (fig. 4). consistent with our phylogenetic data, the haplotype network recovered two haplotypes for the iranian samples. the ff and dr sequences were clustered into a single haplotype separated from the other dr figure 3. the bayesian phylogenetic tree on the basis of 36 unique coi sequences of sinanodonta spp. and related taxa; two sequences of margaritifera laosensis and m. dahurica are the outgroups. the numbers above branches represents the bootstrap support values. the scale bar shows the branch lengths. 92 alwanzadegan et al./ discovery and dna analysis of the invasive freshwater mussel haplotype by only one substitution. the iranian samples weakly differentiated from the respective ancestral haplotype. the samples diverged by only 1 and 2 substitutions from non-native individuals of kazakhstan and russia (volga, eastern and western siberia). the haplotype from russian far east was the most divergent, separated by 8-9 mutation sites from iranian haplotypes (8-9 substitutions). a haplotype from south korea was also the closest native haplotype to the alien ones from iran (2-3 mutation sites). discussion until now, no species of the genus sinanodonta have been recorded from iran. the present study documents the first record of the invasive freshwater mussel s. lauta from iran. there is no record of s. lauta in other middle east countries, but its closely related species s. woodiana was previously reported in some regions, including iraq (bogan et al., 2021) and turkey (ercan et al., 2012). however, in middle asia, a well-established population of the alien s. lauta was recorded from kazakhstan (kondakov et al., 2020a). the species was also recorded from siberia, european russia (bespalaya et al., 2018; kondakov et al., 2020b), and borneo (zieritz et al., 2020). our finding, together with the previous observations by bespalaya et al. (2018) and lineage a lineage b lineage c* lineage d lineage e lineage f lineage g lineage b 2.0 lineage c 3.6 2.8 lineage d 5.4 3.5 4.8 lineage e 5.8 5.5 4.9 4.9 lineage f 13.9 13.4 13.6 12.9 12.3 lineage g 9.8 10.4 10.6 9.5 8.1 12.7 lineage h 6 4.6 4.9 4.0 4.1 12.9 8.5 *the lineage that includes sinanodonta lauta. table 3. genetic divergences (mean uncorrected p-distance %) among the sinanodonta spp. lineages. figure 4. median joining network for coi sequences of sinanodonta lauta (n=55). short lines between the haplotypes indicate the number of mutation sites. 93 int. j. aquat. biol. (2022) 11(1): 86-97 kondakov et al. (2020a, b) suggest that this lineage of sinanodonta species complex may have high invasive capability to produce viable populations beyond its native ranges in japan, south korea and russian far east (primorye region) (lopes-lima et al., 2020). the mussels collected from ff had 100% identical coi sequence to some from dr. the farmers of this facility declared that the mussels have been observed since two years ago, after a flood in dez river. taking into account the farmers' information, and waterway between this facility and dez river and the maximum age of the specimens observed in ff (10 years old), it seems that s. lauta in this facility has originated from dez. however, the individuals collected from both the natural environment and fish farm were large, with numerous annual growth rings on the shells and the maximum age of samples was 11 years old, suggesting a rather recent introduction, at least in 2010. host fish carrying the glochidia is considered as the main vector for introducing s. lauta to nonnative areas (bespalaya et al., 2018; zieritz et al., 2020; kondakov et al., 2020a). during our survey in khuzestan, we found that besides the fish farm, we collected the samples, the mussel is also present in at least 2 other aquaculture facilities rearing native (e.g., common carp) and non-indigenous commercial fish species (e.g., chinese carps). the farmers in one facility declared that they saw the mussels in 2012 for the first time, one year after setting the facility up. unfortunately, we could not get sufficient information from the other facility about the exact origin and introduction time of the non-indigenous fish species. however, the farmers of both facilities stated that they imported the fish, including h. molitrix, c. idella and h. nobilis from east asia. asian carps have been introduced to serve as hosts for glochidia (von proschwitz, 2008) and the introduction of s. lauta to iran seems to be closely related to the introduction of carps from east asia to iran. the dr samples also exhibited two haplotypes that may be probably due to several introduction events of fish hosts by different aquaculture facilities in khuzestan. according to our haplotype network, the alien individuals found in iran is closer to the native ones from south korea (two-three substitutions) compared to other native individuals reported from japan and russian far east. however, due to the lack of enough information available, it is not possible to determine the exact origin and time the mussel was introduced to iran. currently, the alien h. molitrix inhabits the dez river. the introduction of h. molitrix to this river became possible due to the migration from hatcheries and fish farms (eskandari et al., 2005). besides that, cyprinids species, including c. carp and alien h. molitrix produced in aquaculture facilities in khuzestan, have been directly released into the dez river by the khuzestan department of fisheries during the fish stocking program. we, therefore, suggest that this activity by the fisheries department could facilitate the further introduction of s. lauta into nature. additionally, the species, together with anodonta anatina are newly being sold by aquarists in khuzestan as water purificators. the aquarium trade has been considered a possible means of aquatic invertebrates’ introduction into non-native areas (von proschwitz, 2008; duggan, 2010). when spoke with the aquarists, they were not aware that they are offering an alien species, and it is being unintentionally sold as a. anatina. however, marketing s. lauta may accelerate the spread of this alien species in the country. water temperature is regarded as a main environmental parameter that may have a main role in the growth and survival rate of sinanodonta spp. (kraszewski and zdanowski, 2001; liu et al., 2014). the thermophilic feature of the closely related species s. woodiana was previously suggested by various evidences but less has been studied about s. lauta. sinanodonta lauta originates from east asia with cold winters, and it is suggested that this species may be more cold-resistant than s. woodiana. however, it is possible that lower temperatures limit the s. lauta to produce viable populations (kondakov et al., 2020b). liu et al. 94 alwanzadegan et al./ discovery and dna analysis of the invasive freshwater mussel (2014) reported that s. lauta from japan has higher feeding and growth rate in higher temperatures during summer compared to the spring, and temperature is one of the main factors for this species’ growth. our record of 10-11 years old specimens in dez river shows that the species can well survive in the natural environment of south iran with the warm weather conditions. currently, we found a well-established population of s. lauta in the dez river but we suggest that s. lauta may be able to produce new populations in other areas of the tigris river drainage. considering multiple introductions of chinese carps from east asia, stocking dez river with such species and aquarium trade together with waterways in the region, rapid expansion and colonization of this invasive mussel in karun or even other parts are not unexpected in the future since some water transport projects from this river to other freshwater basins including zayandeh river in isfahan are under construction. the possible expansion of alien s. lauta may create important adverse ecological outcomes. no study has yet been done on the fauna of native freshwater bivalves in this basin. however, dez and karun support populations of more than 30 native fish species (eskandari et al., 2000; zare et al., 2019; eagderi et al., 2019, 2022; abbasi et al., 2020), with some impacted by river dragging, damming, industrial and agricultural pollution and also some invasive species, including oreochromis aureus. we assume that establishing viable populations of s. lauta may cause worse outcomes for the region. theoretically, sinanodonta may be able to produce positive impacts on the aquatic environment, including an increase of water clarification via water filtration and particles deposition or serve as a food resource for vertebrates (sousa et al., 2014), but these may cause some changes in the whole ecosystem including aquatics composition and structure, as observed in the case of dreissena polymorpha. douda et al. (2017) also reported that sinanodonta glochidia can adversely impact the growth and physiological properties of the fish host. however, less is known about the host and ecological preferences of s. lauta compared with the widely spread s. woodiana. as the spatial niche of sinanodonta may be changed based on the environmental situation, sampling in other seasons and different parts of the river is necessary for a better estimate of the actual distribution structure and habitat preference of s. lauta in dez river. the taxonomic status of sinanodonta species should be exactly determined since the current status is still under discussion due to the deficiency of molecular information available for indigenous and non-indigenous populations and high morphological similarity among the cryptic taxa (bolotov et al., 2016; sayenko et al., 2017; bespalaya et al., 2018; kondakov et al., 2018, 2020a). there is high morphological diversity which complicates the relationships among sinanodonta species and may confuse identifying them (sano et al., 2017). here, as it is obvious in our phylogenetic tree, the previously reported sequences for s. woodiana in south korea (gq451867 and gq451868) are s. schrenkii (fig. 3, lineage g). our s. lauta haplotypes, together with those from kazakhstan and russia were also clustered in the same clade with sequences extracted from genbank for a. arcaeformis (south korea, gq451870 and gq451869) and s. woodiana (japan, ab055627) (lineage c), representing misidentification of these species. bespalaya et al. (2018) also declared that with respect to the broad range of the species, s. lauta (noted under its junior name s. ovata) may indeed represent a junior synonym for some older nominal species reported from south korea and japan (sano et al., 2017). kondakov et al. (2020a) also stated that the previously recorded species s. puerorum and s. gibba based on morphological features from kazakhstan, refer to s. lauta and/or s. woodiana. based on the coi distance observed in the present study, each clade in our phylogenetic tree could correspond to a separate cryptic species of the genus sinanodonta. therefore, there are at least eight species-level lineages in this species complex, but it is possible that more species-level lineages be detected based on an increased molecular data set. 95 int. j. aquat. biol. (2022) 11(1): 86-97 furthermore, later studies indicate high intraspecific variation within different unionid taxa (maybe more than 3% barcoding threshold) that is usually applied to species delimitation (källersjö et al., 2005; soroka, 2010; prié and puillandre, 2014). here, we do not have enough data to discuss the complex systematics, and more information is needed on the basis of both molecular data and morphological characteristics. altogether, morphospecies of the unionidae needs complete revising on the basis of the integrative taxonomic method, examining topotypes and type series and also studying the inter and intraspecific diversity in genetic and morphological features. in this regard, valuable steps have been taken in the right course by bolotov et al. (2015), klishko et al. (2017), and sayenko et al. 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(2021) 9(1): 23-32 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article population structure and dynamics of the invasive procambarus clarkii (girard, 1852) in a tiber river ramsar site, central italy maxim veroli* 1, marco martinoli1, riccardo caprioli2, christian angelici3, domitilla pulcini1, fabrizio capoccioni1 1council for agricultural research and economics (crea), research centre for animal production and acquaculture via salaria, 31, 00015 monterotondo, rome, italy. 2arpa lazio, regional agency for environmental protection via giuseppe saredo, 52, 00173, rome, italy. 3regional nature reserve “nazzano tevere-farfa”, s.p. tiberina km 28,100, località meana, 00060 nazzano, rome, italy. s article history: received 18 september 2020 accepted 17 january 2020 available online 2 5 february 2021 keywords: crayfish freshwater invasive alien species population dynamics abstract: procambarus clarkii is a native species of central america, but strongly invasive in many regions of the world. an investigation on the red swamp crayfish was carried out to obtain more information about its population dynamics in the tiber river, in central italy. a total of 900 individuals, both males and females, were sampled within two different campaigns (2017 and 2019) aimed at collecting biometric data. a strong fishing effort was deployed (more than 100 nets set), to guarantee a large and randomized number of samples. the crayfish populations were grouped into seven different cohorts, according to bhattacharya’s method. the population showed a balanced sex ratio, the average cephalothorax length was 42.52 mm, with the most represented size class between 40-50 mm. k and l∞, as well as the growth parameter index (ø), the mortality rate (z), and longevity value (tmax), were calculated. k and ø values resulted very high, showing an impressive growth rate in the study area; tmax ranged from 4 to 5 years, l∞ values were lower compared with other studies (58.0-59.0 mm), while z was very high for this population (4.2-4.5 year). the results revealed that crayfish population dynamics can be complex and vary depending on habitat type, available trophic resource and competition. introduction the red swamp crayfish, procambarus clarkii girard, 1852, is a native species of northeastern mexico and southcentral usa. it is an r-selected species, which shows several invasive characteristics, such as rapid life cycle, strong dispersal capacity and high population densities. therefore, it is able to colonize and disrupt a great variety of habitats, constituting a serious threat to the natural environment worldwide (anastácio and marques, 1997; rodríguez et al., 2003, souty-grosset et al., 2016). procambarus clarkii is considered among the 100 worst invasive species (daisie, 2011) and it is listed in the “union list” or “black list” of invasive alien species (ias), an important management tool included in the eu regulation 1143/2014 (eu, 2014). the main introduction agent of freshwater crayfish was farming activity and the hobby stand bait industry *correspondence: maxim veroli doi: https://doi.org/10.22034/ijab.v9i1.1006 e-mail: maxim_veroli@hotmail.it (hänfling et al., 2011). procambarus clarkii has been reported in several continents all over the world, and it represents so far as the second freshwater crayfish species more commercially farmed and more captured globally (holdich, 1993; fao 2020). in europe, crayfish was legally imported from louisiana for commercial purposes to seville and badajoz in 1973 (habsburgo-lorena, 1979). later, the economic success of crayfish sale led to its illegal introductions in several european countries, even after the eu regulation 1143/2014, which banned its farming and sale (chucholl, 2013). due to the high dispersal capacity of this species, individuals started a rapid colonization throughout the mediterranean basin and central europe (anastácio and marques, 1997; barbaresi and gherardi, 2000). nowadays, p. clarkii is widespread and abundant all over europe, including central and northern european countries 24 veroli et al./ population structure and dynamics of procambarus clarkii (louriero et al., 2015). this species represents a strong competitor and affects native freshwater species via direct predation on eggs, larvae, and juveniles and by competing with them for both resources and habitats (gherardi, 2006). moreover, digging activities of crayfish populations were demonstrated to affect river banks stability and to increase water turbidity (rodriguez, 2005). several studies highlighted the significant negative impacts of red swamp crayfish on natural ecosystems, which can be quantified in terms of ecosystem services (es) loss. in particular, provisioning, regulatory and supporting services could be lost in freshwater habitats as a direct consequence of red swamp crayfish presence (lodge et al., 2012). for instance, in italy, where red swamp crayfish spread out in the 1990s, likely as a consequence of accidental release by some aquaculture farms in piedmont (del mastro et al., 1992), p. clarkii threatened the native fauna (barbaresi and gherardi et al., 2001; renai and gherardi, 2004), altering the ecological community structure and reducing the food web complexity (casellato and masiero, 2011). these invasive ecological skills make p. clarkii an impressive competitor and a habitat destroyer, threatening local biodiversity through a direct impact and a decrease in the environmental quality parameters (gherardi, 2006; souty-grosset, 2016). crayfish activities affected both habitats used by fish for shelter or spawning and the whole aquatic ecosystem (lodge et al., 2012; souty-grosset, 2016). plant communities are altered both by direct consumption of macrophytes and by burrowing activities, responsible for shifts from clear water macrophyte-dominated areas to phytoplankton dominated areas (rodríguez et al., 2003; geiger et al., 2005; matsuzaki et al., 2009). procambarus clarkii activities are also believed to induce cyanobacteria blooms (yamamoto, 2010). threats and damages caused by red swamp crayfish are not only limited to freshwater communities but also extend to the coastal area, i.e. this species can invade the estuarine and brackish environments of the adriatic coast as already reported for some tyrrhenian areas (scalici et al., 2010). in addition, groundwater native communities may be impaired by the presence of red swamp crayfish in the caves (mazza et al., 2014). here we provide a first study on the population structure of p. clarkii in the regional nature reserve “nazzano tevere-farfa”, an important wetland area of the tiber river basin as one of italian protected sites under the international ramsar convention (carp, 1972). the site hosts fragile environments inhabited by species included in both european directives and the iucn red list (rondinini et al., 2013). this area needs, therefore, to be highly preserved and it is necessary to identify and characterize possible threats. hence, the aims of the present study were to: (i) investigate the presence of crayfish in the study area, (ii) gather biological data about the local crayfish population and (iii) preliminary assess the red swamp crayfish population structure and dynamics in an important conservation site. this study will provide a major baseline for the future management and control of this invasive species. materials and methods this study was carried out along a stretch of the middle course of the tiber river, within the nature regional reserve of “nazzano tevere-farfa” (nazzano, rome). the reserve is 700 ha wide locating near the confluence of the farfa stream, 40 km northern rome (42°12'n, 12°37'e), at an altitude about 30 m a.s.l. since 1979, the site is protected according to the ramsar convention, the birds directive (european commission, 1979) and the habitat directive (eu, 1992). the site is also included in a special protection area (spa) and extends upstream of hydroelectric power station of “nazzano”, including a section of the tiber river up to the poggio mirteto mountain and a stretch of farfa river up to granica bridge. the area is composed by lotic faces, associated with the natural flow of the stream, and lentic faces, due to a dam construction. the core zone (fig. 1) is located near the reserve center, within an area of about 15 ha with a maximum wet riverbed length of 420 m. along the river shores, there are several small coves and islets characterized by reduced hydrodynamism and low 25 int. j. aquat. biol. (2021) 9(1): 23-32 water depth (<1 m). two sampling campaigns (22 samplings) were carried out in 2017 and 2019 (april-october). sampling frequency was once a week and fixed traps (collapsible cylindrical traps, consisting of nylon threads of 1 cm mesh size with a length of 1 m and a 0.3 m mouth width) were used (fig. 1). each trap was baited with pork liver or with “caperlan” boiles. ten series of fifteen linked-traps were prepared, randomly set in the area, and regularly moved to test the overall catchability in different zones inside the study area. traps were placed in small coves, characterized by shallow, lentic and turbid water, and in running water, slightly deeper and cleaner, mostly near vegetated banks. lentic water had a maximum depth of about 50 cm, whereas lotic water ranged from 40 to 100 cm. water temperature was measured weekly. a subsample from total catches (about 50-60 individuals) was randomly selected to record biometric data. cephalothorax length (cl; from the rostrum to the middle margin, to nearest 1 mm), weight (w; to nearest 1 g) and sex (s) were recorded on each specimen. cl was measured using a caliper, and the weight using a precision balance. gender and female’s reproductive status (i.e. without eggs, eggscarrying, and larvae-carrying) were recorded. statistical analysis was performed in the r-project environment (2.2.0 version), using “tropfishr” (mildenberger et al., 2017) and “flife” packages (kell et al., 2016). individuals were pooled in groups according to the month of capture, to reach a minimum number of observations (n>150; france et al., 1991) and to apply the batthacharya method (bhattacharya, 1967). fishing selectivity on sex, for each year separately and for the overall fishing period, was determined by chi-squared test. difference in sex ratio for each sampling group was tested using t-test. von bertalanffy equation (von bertalanffy, 1938) was used to assess crayfish growth, growth parameter index (ø) and growth coefficient (k). cl data were used to generate 10 mm frequency distributions, which were analyzed through the bhattacharya’s method, resulting in a yearly plot. the total mortality (z) was estimated with the powell-wetherall plot equation (powell et al., 1979; wetherall et al., 1986) by determination of l∞ (asymptotic cl) and z/k, figure 1. map of the study area located within natural regional reserve “nazzano tevere-farfa”. each point indicates the exact positions of crayfish traps used during both 2017 and 2019 sampling campaigns. 26 veroli et al./ population structure and dynamics of procambarus clarkii using the length-frequency plot. the parameter k was also used to estimation of z and calculated through the electronic length frequency analysis (pauly and david, 1981; taylor, mildenberger, 2017) using an optimized approach based on simulated annealing (elefan_sa) instead of the classical method based on the k-value scan method. von bertalanffy growth curve is described by the equation of lt=l∞(1-e-k(t-t0)) (von bertalanffy, 1938); where k is the curvature of the function and it is needed to estimate the relative animal growth; t0 is the time 0, theoretical time at which individuals hatch while t is the time at this moment and l∞ is the asymptotic cl. in order to have an overview of the crayfish growth, also the growth parameter index (ø) was estimated through the formula of ø=ln k+2ln l∞ (pauly and munro, 1984). finally, the value of age at time 0 (when crayfish have cl=0 mm) was assessed by the formula of ln(−t0)=− 0.3922–0.2752ln l∞−1.308ln k (jin et al., 2019). while the expected longevity (tmax), derived from k value and t0, has been calculated as tmax=k(3+t0)-1 (huang et al., 2012). results during the study period (april-october), water temperature was 21-25°c and water depth of the sampling sites varied 40-180 cm. we analyzed a total of 900 crayfish, 441 males and 452 females, over a 2year period. in 2017, we collected a total of 421 specimens (158 males and 263 females), and in 2019 472 (283 males and 189 females). chi-squared test showed for the whole sample a 1:1 sex ratio (χ2 = 0.05, df=1, p>0.05), while in 2017 the ratio was in favor of females (χ2=26.188, df=1, p<0.05) compared to 2019, when we observed an opposite trend (χ2= 20.878, df=1, p<0.05). the average cl was 42.52 mm for the entire sample (42.52±0.96 mm), whereas in 2017 the mean value was 41.36 mm (41.36±0.93 mm; fig. 2a) and in 2019, 43.69 mm (43.69±1.00; fig. 2b). the most represented size class was 40-50 mm cl (n=255) for 2019 (n=154), while for 2017, it was 30-40 mm (n=105). comparison between sexes of logtransformed cl data per sampling date did not show any significant difference (p>0.05). according to bhattacharya’s procedure, both sex and years were separately analyzed (fig. 3) to classify the collected crayfish into several age classes: for all year and sex, we found seven cohorts, except for september 2019 (6 cohorts) and also for females on 2017 (6 cohorts). comparing the two sampling years, most represented classes showed different patterns: most crayfish of both sexes belonged to 30-40 mm class in july 2017, while in september males and females fell into 40-50 mm class. in april, males’ abundance was found in the sixth cohort (50-60 mm) while most of females fit in 40-50 mm length class. concerning 2019, july and september listed in and, for both sexes, frequency peaks 40-50 mm in each month we recorded. table 1. growth parameters (k, ø, l∞) and mortality rate (z) values in different locations. site sex type habitat origin ø k l∞ z nazzano tevere-farfa reserve (this study) m f lotic river creeks main stream natural 3.34 3.42 0.76 0.65 59.00 58.00 4.24 4.54 river nile m f lotic river delta natural 3.20 2.70 3.65 5.60 delta del pò m f lotic pond natural 0.54 0.60 58.80 63.00 2.10 2.48 qianjiang m f lentic swamp artificial 8.01 7.97 0.81 0.86 60.93 58.12 2.32 1.93 torre flavia swamp m f lentic oligohalin natural 0.32 0.33 68.30 74.60 2.99 4.71 preola lake reserve m f lentic lakes natural 3.19 3.19 0.34 0.35 68.25 67.20 3.43 3.83 trasimeno lake m f lentic shallow lake natural 0.59 0.58 69.35 73.71 5.50 5.10 circeo national park m f lentic coastal lake natural 0.66 0.70 64.30 63.30 3.43 4.07 27 int. j. aquat. biol. (2021) 9(1): 23-32 figure 2. mean and standard deviation of the cephalothorax length (cl) recorded during field samplings grouped by year (2017, 2019) and gender (females and males). figure 3. length-frequency distributions calculated by elf (electronic length frequency) analysis that uses an optimized approach based on simulated annealing (elefan_sa). 28 veroli et al./ population structure and dynamics of procambarus clarkii table 1 reported growth parameters (k and l∞), growth parameter index (ø), and mortality rate (z) for both years. values in table 1 were calculated for both sexes and years. longevity values (tmax) ranged from 4 to 5 years: the highest longevity was recorded in 2019 for males (4.9 years), while the lowest value was recorded in 2019 for females (4.3 years old). in 2017, similar values, of about 4.5 years old, were recorded. discussions this study investigated the characteristics of the invasive red swamp crayfish in the regional nature reserve of “nazzano tevere-farfa”, an important biodiversity hotspot of central italy. first reports of the presence of the red swamp crayfish in the tiber river in latium date back to 1997 at the castel giubileo dam (giucca, 1997), about 40 km below the nature reserve. this alien species may be very dangerous for the maintenance of ecosystem balances of vulnerable habitats such as freshwater ones. we analyzed a total of 900 specimens in order to gather information about population structure and dynamics, that could be useful for future management actions on p. clarkii population of the reserve. the majority of analyzed crayfish were adult (cl>20 mm), likely as a consequence of the fishing gear selectivity (10 mm wide), that only allows the capture of larger individuals. the population was composed of crayfish aged at least 2+ (20 mm<cl<30 mm) for both years, while smaller size classes (the 0+ cohort, cl < 10 mm) were largely missing from length-frequency diagrams, confirming the poor capabilities of fishing equipment on juveniles. according to other studies (scalici et al., 2007; mistri et al., 2019), sex ratio is balanced considering both sampling campaigns. lengthfrequency class distribution evidenced the presence of 7 different cohorts: there are no individuals in the first two classes, while for the others, different patterns related to year and sex were found. according to other studies (maccarone et al. 2016; jin et al., 2019) a greater number of crayfish belonging to the fifth cohort (40-50 mm) in both july and september can be observed in 2019. during the warm season, crayfish is more active and its catchability is higher, (reynolds and souty-grosset, 2011). in this study, the highest number of individuals belonging to the oldest age classes has been recorded in july as reported in previous works (e.g. gherardi, 2007b). the growth coefficient (k) estimated in this study was considerably high, second only to that of the qianjiang population, that is considered one of the most important production zone of red swamp crayfish in china (jin et al., 2019). growth parameter index (ø) is comparable to several studies shown in table 1, except for the aforementioned research (jin et al., 2019). red swamp crayfish growth rate is influenced by several ecological factors and the most important factors are temperature and food availability (chucholl, 2011; dörr and scalici, 2013). consequently, the high growth parameters recorded by jin et al. (2019) are mainly due to their direct feeding by humans and the higher temperature (jin et al., 2019). hence, growth coefficient recorded in “nazzano tevere-farfa” natural reserve represents the highest ever recorded in natural conditions. the l∞ value in the current study was lower respect to other studies. a possible explanation could be the higher population density that leads to a decline in trophic resources availability (svedäng and hornborg, 2014). the habitat investigated is characterized by a large amount of debris, particularly consistent near vegetated islets, although limited space availability may result in a strong competition for the resources. the highest l∞ in previous studies were recorded in typical lentic and very wide habitat, such as the trasimeno lake (dörr and scalici, 2013), characterized by a great amount of trophic and spatial resources. the estimated mortality rate in this study (z; >4 years) is one of the highest ever recorded. the population studied is located in the middle stretch of the tiber river, a lotic environment characterized by slow, shallow and productive waters. hence, the study area could be ecologically compared to those where crayfish showed high z values, such as the nile river (saadet al., 2015) and trasimeno lake (dorr and scalici, 2013). the river confluence area of the 29 int. j. aquat. biol. (2021) 9(1): 23-32 “nazzano tevere-farfa” nature reserve is highly productive due to the large amount of terrigenous contributions and low water flow (nijboe and verdonschot, 2004). in addition, the river forms some islets that increase habitat diversity and shelters. this environment was created in the 1950s, immediately after the construction of a hydroelectric dam (unpublished data, 1950). landscape transformation brought to a deep alteration of physical characteristics, such as oscillation of water level, increase of external inputs and the consequent increase of trophic conditions (baxter, 1977), influencing biological communities. local crayfish population found in this area the ideal conditions that allow high density, fast growth rate and longer life span (momot et al., 1978; jackson et al., 2017). our findings may infer some considerations about crayfish spawning period. since water temperature plays an important role in crustacean reproduction (huner, 2002; alcorlo et al., 2008; jin et al., 2019), in southern mediterranean permanent water bodies, spawning may take place even twice a year, in spring and autumn (scalici and gherardi, 2007; alcorlo et al., 2008; dörr and scalici, 2013). this phase usually takes place in late summer and presents a variable duration, mostly dependent on water temperature, food resources and habitat (huner, 2002; alcorlo et al., 2008; jin et al. 2019). during samplings, we observed ovigerous females only in the autumnal period (september/october), as already observed in similar studies (dörr et al., 2006; jin et al., 2019; mistri et al., 2019). as aforementioned, the number of spawning crayfish events is mostly related to water temperature and therefore it is linked to local climatic and environmental factors (jin et al., 2019). therefore, we can speculate that the peculiar characteristics of the study area allow the crayfish population to have only one autumnal spawning period. conclusion in this study, we found a consistent presence of procambarus clarkii, which colonizes the whole study area. the good health of the studied population, characterized by balanced sex ratio and rapid growth, suggests the need of an effective action plan aimed at rapidly mitigating and remediating the negative impacts of this invasive species. acknowledgements this study was part of a larger research project supported by ager foundation (project sushin n°0112-2016). the authors are grateful to the "nazzano tevere-farfa" regional nature reserve, which permitted to carry out scientific samplings. references alcorlo p., geiger w., otero m. 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(2018) 6(6): 340-342 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology short communication length‐weight relationship for two flounder species from iranian waters of the persian gulf azad teimori*1 department of biology, faculty of sciences, shahid bahonar university of kerman, 76169-14111 kerman, iran. article history: received 1 november 2018 accepted 20 december 2018 available online 2 5 december 2018 keywords: lwr, flounder species, persian gulf, pseudorhombus, solea. abstract: the length-weight (lwr) relationship was estimated for the first time for two flounder species viz. pseudorhombus elevatus ogilby, 1912 and solea elongata day, 1877 from iranian waters of the persian gulf. fishes were collected using fishing boat during august to december 2017. no information is available in fishbase regarding the lwrs of these species. the b values of the lwr was 2.98 for p. elevatus and 3.10 for c. elongate, and the r2 values are 0.996 and 0.989, respectively. these estimates can be used for estimation of biomass of the studied fishes employing length frequency distributions. introduction among the fish body parameters, the length is often more easily measured factor in field studies; therefore, it can be used to estimate biomass. by taking account of this issue, to date, numerous studies have addressed length‐weight relationships (lwrs) as useful tool for conservation and stock assessment of freshwater and marine fishes (hossain et al., 2014; mousavi-sabet et al., 2014; esmaeili et al., 2015; zamani-faradonbe et al., 2015a, b; keivany et al., 2016; akhtar and khan, 2018), studying comparative growth of fishes in fisheries management (panase and mengumphan, 2015; rábago-quiroz et al., 2017), and in environmental monitoring programs (froese, 2006). this study provides length–weight data for two flounder species collected from iranian waters of the persian gulf, which have not lwr information available in fishbase (http://www.fishbase.org). materials and methods fish species were collected using fishing boat during august to december 2017 from the iranian waters of the persian gulf, southern iran. the sampling locations lie between 27°03’16.98"n 56°11'24.05"e to 27°03'32.98"n 56°20'40.06"e. specimens were *corresponding author: azad teimori doi: https://doi.org/10.22034/ijab.v6i6.578 e-mail address: a.teimori@uk.ac.ir measured to the nearest 0.05 mm total length (tl) using a vernier caliper and weighed to the nearest 0.001 g (total weight, tw). reference specimens from each fish species were preserved in ethanol (after fixing in formalin) in the zoological museum of shahid bahonar university of kerman (zm-sbuk). the parameters of the length-weight relationship w = atlb were estimated by linear regression of the logtransformed weight and length (koutrakis and tsikliras, 2003), where w is the total weight (g), tl is the total length (cm), a is the intercept, and b is the slope. prior to regression analysis, log-log plots of the length–weight pairs were performed for visual inspection of outliers (froese et al., 2011). extreme outliers were excluded from the analysis. the 95% confidence limits for b were calculated to see if the hypothetical value of isometry (i.e., 3) fell between these limits. all statistical analyses were performed in excel 2018. results totally, one hundred and eleven specimens were used to estimate the length weight relationships (lwrs) of the studied species. the results of the estimated values of lwr including; length range, weight range, a, b, 341 int. j. aquat. biol. (2018) 6(6): 303-306 95% cl of a, 95% cl of b and r2 for two species are presented in table 1. the obtained intercept (a) and growth coefficients (b) were 0.008 and 2.98 for p. elevatus, 0.004 and 3.10 for c. elongate, respectively. the regression values were highly significant (p<0.005). discussion our results indicated that “b” values of the lwr were 2.98 and 3.10, for p. elevatus and c. elongate, respectively. the estimated b values of the regression for these species were within the expected range for teleost fishes between 2.5 and 3.5 (froese, 2006; carlander, 1969). like any other lwr data, the presented estimates can be used for estimation of biomass of the studied fishes employing length frequency distributions. the results of present study are useful for common fisheries investigations. acknowledgments shahid bahonar university of kerman (sbuk) is acknowledged for its financial support. we also wish to thank local people in bandar abbas for their help during fish sampling. references akhtar n., khan m.f. 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(2017). length–weight relation for eleven demersal fish species in the artisanal shrimp fishery areas from the bahia magdalena almejas lagoon system, mexico. acta ichthyologica et piscatoria, 47(3): 303-305. zamani-faradonbe m., eagderi s., shahbazi-naserabad s. table 1. the length–weight relationship (lwr) for two flounder species from iranian waters of the persian gulf. species n tl range (cm) w range (g) a 95% ci of a b 95% ci of b r2 p. elevates 29 9.90–30.90 11.40–372.0 0.008 0.006–0.005 2.98 3.89–3.99 0.996 s. elongata 42 7.40–19.10 3.50–25.10 0.004 0.004–0.006 3.10 2.24–2.87 0.989 n, sample size; tl, total length; w, body weight; a, intercept; b, slope of the linear regression; ci, confidence intervals; r2, coefficient of determination. 342 teimori / length‐weight relationship for two flounder species from the persian gulf (2015a). length-weight relationships and condition factor of three fish species from taleghan river (alborz province, iran). journal of advanced botany and zoology, 2(3): 1-3 zamani faradonbeh m., eagderi s., ghojoghi f. (2015b). length-weight relationship and condition factor of seven fish species of totkabon river (southern caspian sea basin), guilan, iran. international journal of aquatic biology, 3(3): 172-176 international journal of aquatic biology (2013) 1(6): 289-293 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article effect of testosterone and fluoxetine on aggressive behaviors of fighting fish, betta splendens mohammad navid forsatkar, maedeh abedi, mohammad ali nematollahi*,1elahe rahbari department of fisheries, faculty of natural resources, university of tehran, karaj, iran, p. o. box: 31587-77871. article history: received 21 july 2013 accepted 14 november 2013 available online 2 5 december 2013 keywords: fighting fish testosterone fluoxetine exposure aggressive behavior abstract: effects of oral administration of testosterone and fluoxetine exposure on aggressive behavior of the fighting fish, betta splendens, were investigated. testosterone diluted in ethanol and sprayed on pre-weighted pellet to achieve concentrations of 0, 1, 2 and 4 mg/kg of hormone in food. two main behaviors were recorded: the time in front of mirror and duration of the gill flaring using a mirror 8 and 15 days after the start of the experiment. then, half of the specimens in each treatment subjected to waterborne fluoxetine at a concentration of 100 µg/l for 24 hours and the behavior was recorded. after 8 days of feeding, the time in front of mirror and duration of gill flaring were not significantly different between the treatments. duration of the gill flaring increased significantly after 15 days; however there was no significant difference for the behavior in front of the mirror. over time the aggressive behaviors were reduced significantly after fluoxetine exposure. this study indicated that fluoxetine in the aquatic environment alters the aggressive behaviors of the fighting fish. introduction the role of sex steroids especially androgens in fish is very clear. in males, this type of hormones are involved in gonadal development, defense of spawning site, mating behavior performance and in some cases parental care (dey et al., 2010). the hypothalamus-pituitary-gonad (hpg) axis controls the circulating levels of androgens. many factors such as environmental conditions (season, temperature, photoperiod, etc) and social status (being dominant or subordinate) can be effective on synthesis and secretion of hormones. androgens are also important in aggression and spawning behaviors (dey et al., 2010). change in androgen levels, alters behavioral consistency of aggression and courtship. dzieweczynski et al. (2006) reported that male 11ketotestosterone level increases as a result of being encountered in siamese fighting fish, betta splendens. sex reversal is one the main uses of * corresponding author: mohammad ali nematollahi e-mail address: malahi@ut.ac.ir tel: +982632223044 androgens in aquaculture. however, there is little information on aggressive behavior after artificial increase of androgens in fishes. natural resource pollution has always been a subject of great interest. the drugs used by humans, may enters to aquatic systems through waste water treatment plant at levels of ng/l to μg/l (fent et al., 2006). fluoxetine is a selective serotonin reuptake inhibitor (ssri) drug with 11% of it excreted from body as parent compound (de vane, 2000) and in natural system is biologically active because of its resistance to hydrolysis and photolysis (kwon and armbrust, 2006). exposure to fluoxetine disrupt reproductive axis of goldfish, carassius auratus (mennigen et al., 2010). fluoxetine at a concentration of 54 μg/l reduced significantly serum testosterone levels after 7 days of exposure. also, there are some studies on reduction effects of fluoxetine on aggression behaviors in different 290 forsatkar et al/ international journal of aquatic biology (2013) 1(6): 289-293 animal taxa such as mammals (fuller, 1996), birds (sperry et al., 2003), reptiles (deckel, 1996) and fish (lynn et al., 2007). the present study aimed to first increase the plasma levels of androgens by diets containing different amounts of testosterone and then the fish were exposed to waterborne fluoxetine. finally, the effect of both experiments on aggression behaviors of betta fish was investigated. results of this study can be used for better understanding of relationship between physiology and behavior in fish and determine the adverse effect of pollutant in nontarget species. material and methods fish: forty mature male b. splendens with a mean weight of 1.64 ± 0.46 g were obtained from a local distributor. the specimens were transported to laboratory and kept individually in a 1 lit opaque container. water temperature was 26 °c and photoperiod set as 12d:12l. the specimens were fed one or two times daily for two weeks using 0.9 mm commercial pellet (biomar, norway). food and stock solution preparation: we used four concentrations: 0, 1, 2 and 4 mg/kg of hormone in food. these levels selected because it was found that 3 or 4 mg/kg of 17α-methyltestosterone (via inert food) resulted in 100% masculinization of fighting fish fry (kipouros et al., 2011). for this, different amount of testosterone (andriol testocapstm) dissolved in ethanol and sprayed on pre-weighted pellet. fish were fed daily 3% of body mass for 15 days and then half of them exposed for 24 h in waterborne fluoxetine at a concentration of 100 µg/l. experimental protocol and behaviors: male specimens were divided into four groups. on days 8, 15 and 16 of the experiment, fish were transported to 25 lit experiment tanks. after 15 min acclimatization, a 16 × 24.5 cm mirror was placed in one side of the tank and male behavior was recorded in 10 min. two main behaviors, i.e. duration of gill flaring and time spent in front of the mirror was measured. the gill flaring is known to be associated with fight outcome (clotfelter et al., 2007) being fish reflected in opercular activity. also, response to the mirror show the importance of fighting for the male specimens. statistics: the data were statistically analyzed using one-way anova in spss v. 19.0. duncan’s multiple range test was used to identify significant differences between treatments (α<0.05). results androgen administration: after 15 days, fishes were tested two times on day 8 and 15 of the experiment. on the 8th day of the experiment, the time spent in front of the mirror (f= 1.220, p= 0.317; fig. 1a) and duration of gill flaring (f= 1.205, p= 0.322; fig. 1b) had no significant difference between the treatments. there was no significant differences in time spent in front of the mirror on day 15 (f= 0.508, p= 0.679; figure 1. the aggressive behavior of b. splendens (a) mean (± sd) of time spent in front of the mirror and (b) duration of gill flaring administered by oral testosterone after 8 days of feeding experiment. 291 forsatkar et al/ international journal of aquatic biology (2013) 1(6): 289-293 fig. 2a) but there was a significant difference for duration of the gill flaring (f= 3.414, p= 0.028; fig. 2b). fluoxetine exposure: following 24 h exposure, five fish from each treatment was examined for aggressive behaviors. there was significant difference in the time spent in front of the mirror among treatments (f= 4.181, p=0.023; fig. 3a). also, fluoxetine significantly affected the duration of gill flaring behavior (f= 11.326, p=0.000, fig. 3b). discussion increasing entrance of pharmaceuticals into the aquatic environment has attracted the attention of workers on their adverse effect. these compounds disrupt endocrine system as well as behaviors. result of this study showed that orally administration of testosterone increases aggression behavior of fighting fish after 15 days feeding trial. this result is in agreement with the same previous studies (e.g. cardwell and liley, 1991; pankhurst and barnett, 1993), which have correlated the aggressive behavior with circulating androgens. in an african cichlid fish, astatotilapia burtoni, the dominant male had more circulating levels of testosterone in comparison to sub-ordinate fish (parikh et al., 2006). also, it is showed that at the time of social contrast, androgens concentrations increase (dzieweczynski et al., 2006). these researchers reported that circulating levels of 11-ketotestosterone increased after observing each other in fighting fish. therefore, a high initial concentration of testosterone in plasma and increased after the introduction of the mirror, makes a significant difference in the incidence of aggressive behaviors after 15 days feeding trial. on 8th day of the experiment showed that there was not a significant differences between treatments in any of the measured behaviors. this showed that figure 2. the aggressive behavior of b. splendens (a) mean (± sd) of time spent in front of the mirror and (b) duration of the gill flaring administered by oral testosterone after 15 days feeding experiment. figure 3. the aggressive behavior of b. splendens (a) mean (± sd) of time spent in front of the mirror and (b) duration of the gill flaring following 24 h fluoxetine exposure at 100 µg/l. 291 292 forsatkar et al/ international journal of aquatic biology (2013) 1(6): 289-293 short term intake of androgen could not influence on the fish behaviors. this is noted that until the end of the experiment, there was no significant difference between treatments for the time spent in front of the mirror. this showed the importance of the conspecifics that expressed aggressive behavior in male fighting fish (personal observation); so, all the males were similar in exhibiting of this behavior. after fluoxetine exposure, the aggressive behavior has been reduced in compared to the previous measurements. this is consistent with other studies. perreault et al. (2003) reported that territorial aggression in a coral reef fish (thalassoma bifasciatum) reduced after long-term and acute fluoxetine administration. short-term exposure of male fighting fish to fluoxetine concentration at 3 µg/ml reduced the expression of specific aggressive behaviors (lynn et al., 2007). at the present study, there is a significant difference between treatments after exposure to fluoxetine and this was very obvious at 1 mg/kg of testosterone in food for both behaviors. fluoxetine in both injected (clotfelter et al., 2007) and exposure (gaworecki and klaine, 2008) forms caused reduction in serotonin and its metabolite, 5-hydroxyindoleacetic acid levels within the brain. therefore, serotonergic pathway reduction and its interaction with increase testosterone levels, maybe the main reason of significant reduction of aggressive behaviors of fighting fish in 1 mg/kg treatment. further physiological studies are needed to clarify the relationship between testosterone and fluoxetine interactions. in conclusion, the present study indicates that exogenous testosterone resulted in increase of aggression in fighting fish. however, waterborne fluoxetine cause reduction in these behaviors. therefore, due to the increasing prescribing different drugs, their consequences in the environment and finally established methods should be investigated. references cardwell j.r., liley n.r. (1991). androgen control of status in males of a wild population of stoplight parrotfish sparisoma viride. hormones and behavior, 25: 1-18. clotfelter e.d., o’hare e.p., mcnitt m.m., carpenter r.e., summers c.h. (2007). serotonin decreases aggression via 5-ht1a receptors in the fighting fish betta splendens. pharmacology biochemistry and behavior, 87: 222-231. de vane c.l. (2000). metabolism and pharmacokinetics of selective serotonin reuptake inhibitors. cellular molecular neurobiology, 19: 443-466. deckel a.w. (1996). behavioral changes in anolis carolinensis following injection with fluoxetine. behavioural brain research, 78(2): 175-182. dey c.j., o'connor c.m., gilmour k.m., van der kraak g., cooke s.j. (2010). behavioral and physiological responses of a wild teleost fish to cortisol and androgen manipulation during parental care. hormones and behavior, 58: 599-605. dzieweczynski t.l., eklund a.c., rowland w.j. (2006). male 11-ketotestosterone levels change as a result of being watched in siamese fighting fish, betta splendens. general and comparative endocrinology, 147: 184-189. fent k., weston a.a., caminada d. (2006). ecotoxicology of human pharmaceuticals. aquatic toxicology, 76: 122-159. fuller r.w. (1996). the influence of fluoxetine on aggressive behavior. neuropsychopharmacology, 14: 77-81. gaworecki k.m., klain s.j. (2008). behavioral and biochemical responses of hybrid striped bass during and after fluoxetine exposure. aquatic toxicology, 80: 207-13. kipouros k., paschos i., gouva e., ergolavou a., perdikaris c. (2011). masculinization of the ornamental siamese fighting fish with oral hormonal administration. science asia, 37: 277-280. kwon j.w., armbrust, k.l. (2006). laboratory persistence and fate of fluoxetine in aquatic environments. environmental toxicology and chemistry, 25: 2561-2568. lynn s.e., egar j.m., walker b.g., sperry t.s., ramenofsky m. (2007). fish on prozac: a simple, 293 forsatkar et al/ international journal of aquatic biology (2013) 1(6): 289-293 noninvasive physiology laboratory investigating the mechanisms of aggressive behavior in betta splendens. advances in physiology education, 31: 353-63. mennigen j.a., sassine j., trudeau v.l., moon t.w. (2010). waterborne fluoxetine disrupts feeding and energy metabolism in the goldfish carassius auratus. aquatic toxicology, 100: 128-137. pankhurst n.w., barnett c.w. (1993). relationship of population density, territorial interaction and plasma levels of gonadal steroids in spawning male demoiselles chromis dispilus (pisces: pomacentridae). general and comparative endocrinology, 90(2): 168-76. parikh v.n., clement t.s., fernald r.d. (2006). androgen level and male social status in the african cichlid, astatotilapia burtoni. behavioural brain research, 166: 291-295. perreault h.a.n., semsar k., godwin j. (2003). fluoxetine treatment decreases territorial aggression in a coral reef fish. physiology and behavior, 70: 719-24. sperry t.s., thompsony c.k., wingfield j.c. (2003). effects of acute treatment with 8-ohdpat and fluoxetine on aggressive behaviour in male song sparrows (melospiza melodia morphna). journal of neuroendocrinology, 15: 150-160. 293 int. j. aquat. biol. (2017) 5(1): 1-6; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article acute toxicity of nonylphenol ethoxylate-6 to whiteleg shrimp, penaeus vannamei (boone, 1931) (decapoda, penaeidae) azin ahmadi1, ahmad noori1*, mahdi banaee2 1department of fisheries science, faculty of marine science and technology, university of hormozgan, bandar abbas, iran. 2department of aquaculture, faculty of natural resources and environment, behbahan khatam al-anbia university of technology, iran. article history: received 24 april 2016 accepted 15 december 2016 available online 2 5 february 2017 keywords: nonylphenol ethoxylate-6 shrimp penaeus vannamei acute toxicity abstract: nonylphenol ethoxylate-6 (np6eo) is widely used in industrial and domestic products and easily detected in the environment. the toxicity and estrogenic potency of alkylphenols have been investigated in several studies. however, to the best of our knowledge available, acute toxicity data about the effects of np6eo on decapod and aquatic ecosystem in literature is yet scarce. therefore, in this study the adult male and female whiteleg shrimp, penaeus vannamei, were exposed to various concentrations of np6eo (0.04, 1, 5, 25, 125, 625 µl l-1) for four days. acute toxicity potential of np6eo on adult p. vannamei was assessed by calculating lc50 for different times. median lethal concentration (lc50) of np6eo at 96 hours was 7.017 μl l-1. the lc50 of this compound revealed a positive correlation between shrimp mortality and exposure periods. the data exhibited that np6eo was considered as "toxic" to p. vannamei and further toxicity assessment to other species is strongly recommended. introduction man-made chemicals are an important part of the modern life. human beings as well as wildlife populations cannot avoid coming into contact with many of chemicals employed in variety of industries like food production (plants and meat), pathogen control (insecticides), production of modern materials (plastics), or in the built environment (insulations and fire retardants) (bergman et al., 2012). considering the importance of these compounds and their widespread presence in the environment, it is important that comprehensive strategies are developed to preclude widespread environmental contamination with endocrine disruptors (eds) and protect environment (david et al., 2009; bergman et al., 2012). nonylphenol ethoxylate-6 (np6eo) is used in countless number of applications and because of its extensive use, discharged to the sewer system and make their way into wastewater and aquatic systems * corresponding author: ahmad noori e-mail address: nooryahmad@gmail.com (ying et al., 2002). np6eo is a nonionic surfactant that is used in a wide range of industrial applications and consumer products, such as laundry detergents, dust-control agents and deicers, industrial liquid soaps and cleaners, cosmetics, paints, and as the dispersing agents in pesticides and herbicides (jobling and sumpter, 1993). concern has recently increased about the use of alkylphenol ethoxylates (apes) because of the relative stability of their metabolites such as nonylphenol (np), octylphenol (op) and nonylphenol ethoxylate-1-3 (np1-3eo) in the environment (giger et al., 1984) and their estrogenic effects on organisms (ying et al., 2002) which is considered as eds. testing strategies employed acute toxicity studies to evaluate and measure the effect(s) of one or more pollutants on one or more species. this implies that tests at high doses will inform us about low-dose exposures (reish and oshida, 1986; bergman et al., 2012). the lethality of the eds was used as the 2 ahmadi et al./ acute toxicity of nonylphenol ethoxylate-6 to whiteleg shrimp endpoint in an aquatic acute toxicity testing system (faheem and lone, 2013). in general, determination of lethal concentrations, such as the median lethal concentration (lc50), is recognized as the first step for risk assessment of synthetic and natural chemicals (johnson and finley, 1980; ura et al., 2002). these data assist in the development and application of water quality criteria for the protection of the aquatic environment. in spite of evidence that proved the toxicity of npneo on many aquatic animals (dorn et al., 1993; lussier et al., 2000; hirano et al., 2004; oliveirafilho et al., 2005; ricciardi et al., 2008; liu et al., 2011), there is no attempts have been made to determine the impacts of np6eo on penaeus vannamei. hence, this study aims to determine and compare the acute toxicity of np6eo to p. vannamei upon modification of the exposure conditions. materials and methods chemicals: np6eo (cas no.9016-45-9) was obtained from kimiagaran emroz company (tehran, iran). stock solution of the np6eo was prepared by dissolving appropriate concentration in 96% ethanol as solvent. required concentrations were obtained by serial dilution and stored in dark at 4°c until usage. solvent concentration was kept at 0.01% (v/v) for all treatments. animal maintenance and exposure to np6eo: adult p. vannamei (both sexes; body weight: 25.89±0.79 g, total length: 14.26±0.16 cm) were obtained from shrimp farms located at southern coast lines of iran and were transferred to kolahi aquatic restock center (karc). adult shrimps were acclimated in 300l fiberglass tanks containing ultraviolet-filtered recirculating water (ph=7.77±0.017) for 2 weeks prior to the experiment. feeding was done on daily basis in four times at the rate of 2.5% of the body weight by commercial feed until 24 hrs prior to the initiation of the test. after the acclimation period, 240 adult shrimp were randomly distributed among 24 tanks, comprising control, vehicle control (ethanol with a final concentration of 1:1,000 v/v water), 0.04, 1, 5, 25, 125, 625 µl l-1 of np6eo. the experiment was run in triplicate, without feeding and water exchange during the experiment. the mortality of shrimps in each treatments were counted and recorded over the exposure period at 24, 48, 72 and 96 hrs. dead shrimps were removed from treatments immediately. the study was approved by the iranian society for the animal welfare. data analysis and statistics: data are expressed as mean with the corresponding standard error (se). lc values and 95% confidence intervals (95% ci) were calculated using probit analysis. shapiro-wilk and levene’s tests were used to check the normality of data distribution and the homogeneity of variances, respectively (zar, 2010). if data support the prerequisites for parametric analysis, one-way analysis of variance (anova) followed by tukey’s multiple range test was applied. otherwise, kruskalwallis and mann-whitney u test were applied to determine the statistical significance (zar, 2010). all the analysis was performed by spss 16.0. the significant level in all analysis was set at p≤0.05. results the percent mortality of p. vannamei after exposure to various concentrations of np6eo for 24, 48, 72 and 96 hrs has been depicted in figure 1. mortality increased with increasing concentrations and exposure time (fig. 1). the lc50 values were 437.052 ± 326.250 µl l-1 for 24 hrs, 33.627±15.443 µl l-1 for 48 hrs, 10.816±3.936 µl l-1 for 72 hrs and 7.017±2.391 µl l-1 for 96 hrs. the lc values, their upper and lower confidence limits and slope functions for np6eo have been given in table 1. discussion acute toxicity tests provide a measure of the toxicity of the given compounds to experimental species under specific environmental conditions (reish and oshida, 1986). they also reflect the severe and rapid damage caused by sudden exposure to lethal concentrations of contaminants (alam and maughan, 1993). in the present study, calculated lc50-96 hrs value https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahukewiystvgg4rnahvhbrqkhriecpeqfggkmae&url=https%3a%2f%2fen.wikipedia.org%2fwiki%2fultraviolet_germicidal_irradiation&usg=afqjcnhp4ceji4utfbnfuppqesvaxlwjqw&sig2=kgvhttnjloxvohxtaomr3q 3 int. j. aquat. biol. (2017) 5(1): 1-6 of np6eo for p. vannamei was assessed as 7.017 μl l-1, which was in agreement with the results reported for other species have been tested with np and npneo (dorn et al., 1993; mann and bidwell, 2000; oliveira-filho et al., 2005; ricciardi et al., 2008). the acute and chronic toxicity of alkylphenol ethoxylates and their metabolites have been investigated for several freshwater and marine species (servos, 1999; staples et al., 2004). previous studies showed that species sensitivity varies from 17 μg l-1 of para-nonylphenol (pnp) for winter flounder (pleuronectes americanus) (lussier et al., 2000) to 9.2 mg l-1 of np8eo for litoria adelaidensis (mann and bidwell, 2000). median lethal concentrations of 4 to 6.6 mg l-1 of np9eo was reported for fathead minnow, pimephales promelas (dorn et al., 1993; staples et al., 1998). also in the other similar studies, lc50-96 hrs for np, np1eo and np2eo for fathead minnow were 136, 218, and 323 μg l-1, respectively (teneyck and markee, 2007). the varying data of available toxicity tests resulted as a function of ethoxy chain table 1. effective dose, confidence limits, and slope function for nonylphenol ethoxylate-6 (np6eo) at different intervals for the whiteleg shrimp, penaeus vannamei. exposure periods effective dose (µl/l) se limits slope function 't' ratio heterogeneity lcl ucl 24 hrs lc1=0.006 lc5=0.161 lc10=0.922 lc20=7.642 lc50=437.052 lc80=* lc90=* lc95=* lc99=* 0.009 0.150 0.647 3.976 326.250 * * * * 0.000 0.012 0.144 2.346 131.656 3340.90 * * * 0.052 0.683 2.895 20.537 3241.25 * * * * 0.479±0.081 5.920 1.135 48 hrs lc1=0.001 lc5=0.026 lc10=0.128 lc20=0.866 lc50=33.627 lc80=1305.031 lc90=8834.585 lc95=* lc99=* 0.002 0.023 0.089 0.447 15.443 1032.11 8962.67 * * 0.000 0.003 0.022 0.255 14.568 362.622 1755.602 6344.154 * 0.010 0.107 0.400 2.124 95.011 9758.932 * * * 0.530±0.070 7.548 1.540 72 hrs lc1=0.002 lc5=0.028 lc10=0.104 lc20=0.512 lc50=10.816 lc80=228.489 lc90=1125.541 lc95=4199.877 lc99=* 0.002 0.020 0.063 0.237 3.936 127.492 802.962 3578.53 * 0.000 0.000 0.001 0.022 2.243 39.623 133.766 348.322 1984.653 0.042 0.247 0.664 2.438 74.971 * * * * 0.635±0.075 8.436 2.903 96 hrs lc1=0.003 lc5=0.027 lc10=0.091 lc20= 0.406 lc50=7.017 lc80=121.319 lc90=538.201 lc95=1841.874 lc99=* 0.002 0.018 0.052 0.182 2.391 59.905 338.229 1382.53 * 0.000 0.000 0.002 0.028 1.708 26.836 87.163 219.975 1182.462 0.035 0.193 0.495 1.673 33.301 2593.424 * * * 0.680±0.078 8.755 2.538 * values more than 10000 are not shown 4 ahmadi et al./ acute toxicity of nonylphenol ethoxylate-6 to whiteleg shrimp length, the type of test used, and the species tested. considering the relative toxicity values, or np toxic equivalency factors (tefs) which calculated for nonylphenol compounds, toxic concentrations for different nonylphenolic compounds such as npneo with various eo chain length could be matched with similar endpoints for np for the same species. if tef for np be considered as 1, for example, toxic equivalency factors for npneo (1<n<8) will be considered as 0.5 (environment canada, 2002). based on the present results, it can be proposed that in p. vannamei sensitivity to np toxicity is approximately the same as other studied species. according to the usepa toxicity categories (u.s. epa., 2015), chemicals with lc50 values ranging from 1 to 10 mg l-1 are considered as "toxic to aquatic species with long lasting effects". therefore, our result strongly indicate that np6eo is toxic for p. vannamei. this study assessed the acute toxicity of np6eo on p. vannamei, which is the first report of lc50 values of this chemical on this species. the work has revealed that np6eo has a potentially harmful impact on p. vannamei and probably other aquatic crustaceans and invertebrates. the response of various species to same chemicals are different at the same time of exposure. moreover, the potential of np6eo for endocrine disruption at even lower concentrations may cause further concerns for aquatic fauna. additional long term studies are needed to study other impacts of np6eo on aquatics. the findings of this research suggest that the policy makers must take necessary measures to prevent the crucial damage of this compound on aquatic organisms and human beings. acknowledgments the authors wish to thank m. darvishi for providing shrimp. also the authors thank mr. sirpoor for providing the facilities and karc staffs for their helps. references alam m.k., maughan o.e. 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(2017) 5(1): 1-6 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی غربی پاسفید میگوی روی بر اتوکسیالت-6 فنل نونیل حاد سمیت تعیین تاثیر penaeus vannamei (boone, 1931) (decapoda, penaeidae) 2بنایی مهدی، *1، احمد نوری1آذین احمدی .ایران بندرعباس، هرمزگان، دانشگاه دریایی، فنون و علوم دانشکده شیالت، گروه1 63616-47189: کدپستی ایران، بهبهان، ،بهبهان( ص) االنبیاء خاتم صنعتی دانشگاه زیست، محیط و طبیعی منابع شیالت، دانشکده گروه2 چکیده: می جشسن قابل زیست محیط در راحتی به دلیل همین به و داشته صنعتی و خانگی محصوالت تولید در وسیعی کاربرد اتوکسیالت-6 فنل نونیل در طالعاتا کمبود وجود این با. است گرفته قرار ارزیابی مورد مطالعات از بسیاری در فنل آلکیل ترکیبات استروژنی شبه خاصیت و سمیت. باشد منظور، همین به. گرددمی احساس خوبی به آبی هایاکوسیستم همچنین و پایانده روی بر اتوکسیالت-6 فنل نونیل حاد سمیت تاثیرات خصوص مختلف هایغلظت معرض در روزه چهار دوره یک در( penaeus vannamei) غربی پاسفید میگوی بالغ ماده و نر جنس دو هر حاضر مطالعه در غلظت مقدار به توجه با اتوکسیالت-6 فنل نونیل حاد سمیت. گرفت قرار( لیتر بر میکرولیتر 625 و 125 ،25 ،5 ،1 ،04/0) اتوکسیالت-6 فنل نونیل 50lc 50 غلظت مقدار حاصل، نتایج به توجه با. شد ارزیابی مختلف هایزمان درlc 017/7 با برابر ساعت 96 برای اتوکسیالت-6 فنل نونیل رتاثی زمان مدت و میگوها در میر و مرگ بروز بین مستقیم رابطه بیانگر تحقیق این در شده محاسبه 50lc غلظت. شد محاسبه لیتر بر میکرولیتر سمیت ارزیابی دلیل همین به و رودمی شمار به سمی ترکیبی غربی پاسفید میگوی برای اتوکسیالت-6 فنل نونیل که داد نشان نتایج. بود گذاری . شودمی توصیه و بوده ضروری آبزی هایگونه دیگر برای ترکیب این حاد. سمیت ،غربی پاسفید ، میگو،اتوکسیالت-6 فنل نونیل :کلمات کلیدی int. j. aquat. biol. (2017) 5(2): 52-62; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article an account on the assemblage of fish larvae in ponnani estuary, south india ranjeet kutty*1, shahana shamsudheen2, shafeena ottathaikkal2, jamsheeravallikkattil2 1department of fishery hydrography, kerala university of fisheries and ocean studies, panangad p.o., kerala, india. 2department of aquaculture and fishery microbiology, mes ponnani college, ponnani south p.o., kerala, india. . article history: received 28 april 2016 accepted 27 february 2017 available online 2 5 april 2017 keywords: ichthyoplankton assemblage estuary climate change ponnani abstract: estuarine environments are one of the most dynamic aquatic ecosystems and serve many important functions in coastal waters. larval fish dynamics contribute significantly to understanding the ecology of fish populations as they can indicate the spawning-stock biomass and recruitment in adult fish stocks. initial development stages of fishes are particularly vulnerable and are influenced by physical and biological processes. hence the present study was aimed to characterize ichthyoplankton assemblages, to evaluate environmental influence in its structure. ponnani backwater fish larvae assemblages displayed a clear seasonal pattern presenting higher abundances and diversities during warmer months. throughout the year there is a wide fluctuation in salinity, temperature and primary productivity in these backwaters enabling it to be classified under stressful environment for larval forms of certain economically important marine fishes. a detailed analysis made to study the interaction of selected environmental parameters with ichthyofaunal diversity in ponnani backwater provided a clear understanding on the influence of these variables on the distribution of marine fish larvae in the region. the results of the present analysis provided a model for the prediction of larval diversity from the prevailing environmental parameters. introduction fish communities inhabiting estuaries represent a combination of freshwater, resident and marine species, including many larvae and juveniles. habitat selection and non-random use of space occupied by these groups are often influenced by abiotic and biotic factors (whitfield, 1999). the importance of these two factors in driving the spatial and temporal patterns of occurrence of fish in estuaries is still not been investigated in depth. but a variety of abiotic factors have been shown to influence the utilization of estuaries by fishes (cabral et al., 2001; martinho et al., 2007). salinity and turbidity on a spatial scale and temperature on a temporal scale have been regarded as best predictors for estuarine species abundance and spatio-temporal community structure (bruno et al., 2013). nonetheless, it has also been suggested that biotic processes, such as food availability, competition and * corresponding author: ranjeet kutty doi: https://doi.org/10.22034/ijab.v5i2.170 e-mail address: ranjeet.mes@gmail.com predation, may be important in driving the occurrence of spatial and temporal patterns of fish in estuaries (akin et al., 2005). up to now, only few studies have analysed simultaneously the effect of both biotic and abiotic factors on estuarine fish structures and assemblages (beck et al., 2001; martino and able, 2003; trevor and whitfield, 2006; selleslah and amara, 2008; castro et al., 2009). many authors have also shown that they exhibit strong short and long-term variability, due to environmental fluctuations (day et al., 1989). both spatial and temporal studies of fish assemblages are therefore needed to understand fully the functioning of environmental variables in any dynamic ecosystem such as the estuary. tropical lagoons and estuaries are the subject of an increasing scientific interest because of the expansion of human activities in these areas and the awareness of their economic and ecological importance. 53 int. j. aquat. biol. (2017) 5(2): 52-62 estuaries have been the subject of considerable ichthyological research, some of which have focused on the key environmental factors affecting estuarine fish community structure (elliott et al., 2007). estuarine fish assemblages have been studied since the late seventies (whitfield et al., 1994; blaber, 2000). estuaries are the focus of attention because of the concentration of large components of the human population in these watersheds, the resulting increasing urbanization of coastal watersheds and concomitantly the alteration and loss of habitats. investigating climatic effects and climate change trends is an urgent issue all over the world, but even more so in tropical regions where alterations in temperature and precipitation are expected to be more accelerated than the global mean alteration rate (intergovernmental panel on climate change, 2001). alterations in temperature and precipitation have the potential to strongly affect the hydrological features that affect fish larvae, such as temperature and currents, and the magnitude of river plumes. it is thus very important to understand how climate and hydrology affect fish larvae movement towards estuarine nurseries. since each species has different environmental ranges and requirements, different species will be influenced differentially by different variables in the process of estuarine immigration (vinagre, 2009). ramya (2010) documented unusual occurrence of larvae and juveniles of economically important marine fish in the ponnani backwaters. also prevalence of species formerly rare in the area had increased. these changes in fish distribution correlate to the temperature and salinity preference of the species. larvae and juveniles of fishes that spawn near the coast prefer slightly lesser temperature and salinity than that in open ocean (kimura et al., 2010). however, in pursuit for better rearing environment they may migrate to estuaries (albe, 2005). the relationship between environmental factors and the distribution of organisms within estuaries has received considerable attention. because fishes are one of the dominant macrofaunal components of estuarine biota, many studies have focused on their distribution patterns. central to these studies are the determinations as to what factors, and what levels of those factors, are relevant in defining fish habitat within estuaries (able, 2005). the knowledge of juvenile fish growth in extreme environmental conditions is a key to the understanding of adaptive responses and to the relevant management of natural populations. under the above pretext, a detailed analysis on the impact of different environmental variables on the occurrence and distribution of larvae of commercially important marine fishes was studied. the results would provide an in depth knowledge on the environmental dynamics prevailing in the ponnani backwaters and help fishery biologist to develop models for postulating the impact of the climate change to the ichthyofauna of the region. materials and methods study area: the ponnani estuarine system is located between 10°46' and 10°48'n and 75°54' to 75°56'e. it is an open estuary that is drained by a tributary of the bharathapuzha river and drains in to the arabian sea at this estuary. the estuarine systems exposed to tides from the arabian sea and hence water is brackish almost throughout the year. sampling: sampling was carried out once every month during daylight at high tide, from may to april at three separate stations along the ponnani backwaters, viz., veliyancode, ponnani and biyyam (fig.1). samples were collected by horizontal subsurface tows using plankton net (mesh size 500 mm; mouth diameter: 0.5 m). the samples were then preserved in a 4% buffered formaldehyde seawater solution. in the laboratory, individuals were identified and counted. larval identification was done following leis and carson-ewart (2000) and the key provided in www.larvalbase.org. relative position of the dorsal and anal fin-ray, vertebrae counts, pigmentation patterns shape, number and arrangement of myomeres, and other prominent physical features were used to identify fish larvae (leis and carson-ewart, 2000). fish larvae were 54 kutty et al./ distribution of ichthyoplankton in ponnani estuary identified under stereomicroscope to family level by using the descriptions of related taxa. after identification, the larvae were photographed and preserved in 70% alcohol. unidentified larvae were placed in “unknown” category due to the samples were too small to identify and damaged larvae were placed in “incomplete” category. water samples from the three stations were collected on a monthly basis from may to april. the water quality parameters recorded on site were temperature, dissolved oxygen, salinity, ph, total suspended solids, and turbidity using portable water and soil analysis kit (ei-model 191), while nutrients such as nitrate and phosphate were analyzed in lab based on strickland and parsons (1972) and measured on systronics uv-vis spectrophotometer. subsequently from each site phytoplankton and zooplankton samples were also enumerated using sedgwick rafter counter or heamocytometer (apha, 2005). statistical analysis: the software programmes viz., spss (statistical product and service solutions version17.0) and primer 6 (plymouth routines in multivariate ecological research, version 6.1.9) were used for univariate and multivariate analyses of data. on the base data, descriptive statistics for the biological and hydrographical parameters of each sample were worked out and averaged over the stations. one-way anova, and duncuns multiple range tests (dmrt) were performed to test the significant differences between means of various parameters recorded from different stations during the three seasons. the results were used to correlate the environmental parameters with biological parameter by backward regression method, in which most statistically insignificant variables were avoided for further analysis. in the next step, two parametric multivariate analyses (factor analysis and regression analysis) were employed to examine the possible influence of environmental variables on the diversity of marine ichthyofauna. factor analysis was particularly useful for considering several related random environmental variables simultaneously to identify a new smaller set of uncorrelated variables that accounted for a large proportion of total variants in original variables (lau and lane, 2002). in factor analysis, the extracting factors that account for variance is called figure 1. map of the ponnani backwaters showing the stations. 55 int. j. aquat. biol. (2017) 5(2): 52-62 the eigen value. therefore higher eigen value means, higher percentage of variance exhibited by the factor (wang et al., 2007). hence the first factors with higher eigen value was considered for further analysis which will cumulatively contribute much to the total variance. to establish the relationship between the environmental variables and ichthyofaunal diversity, a backward multivariate regression analysis was employed using diversity indices as dependent variable and the factors obtained by factor analysis as independent variables using spss v17.0. principal component analysis was adopted for deriving the factors following varimax rotation with kaiser normalization. this was followed by construction of a factor loading matrix to delineate the most prominent environmental factor influencing the distribution of fish fauna in the ponnani estuary. community structure: primer 6 for windows was used for the analysis of community structure and following diversity indices i.e. number of individuals (abundance; n), total number of species (s), shannon-weiner diversity index (h’) and simpson’s evenness index (d). results during the present study, 754 fish larvae belonging to 14 different families were identified. however, four larvae could not be identified. the mean values of different environmental variables recorded in the present study are depicted in table 1. the results indicate a significant difference (p<0.01) in all the parameters studied during the three seasons and across the three stations. table 2 depicts the analysis of variance in the number of individuals and species, mean diversity and evenness of the population. the results indicate that all these parameters varied significantly (p<0.01) among the three stations. this implies that the assemblage of fish larvae in veliyancode, ponnani and biyyam were different table 1. mean values of environmental variables during different seasons in the three stations of the ponnani backwaters. table 2. analysis of fish larval assemblage, diversity and evenness in different stations of the ponnani backwaters. parameters sum of squares df mean square f number of individuals between groups 5130.667 2 2565.333 26.069** within groups 3247.333 33 98.404 total 8378.000 35 number of species between groups 262.889 2 131.444 24.449** within groups 177.417 33 5.376 total 440.306 35 shannon diversity index between groups 3.236 2 1.618 14.711** within groups 3.629 33 .110 total 6.865 35 simpson evenness between groups 0.084 2 0.042 7.785** within groups 0.177 33 0.005 total 0.261 35 **significant at 0.01% level. 56 kutty et al./ distribution of ichthyoplankton in ponnani estuary and while correlating this result with that of environmental variables, it could be ascertained that the cause of variation should be due to change in the physico-chemical characteristics of the region. the results also showed that mean values of number of individuals were significantly high (p<0.01) for veliyancode indicating a rich density of marine fish larvae compared to other two stations. similarly, the number of species in veliyancode showed significantly higher representation. veliyancode had highest diversity of fish larvae followed by ponnani and biyyam. in contrast, the simpson’s evenness index was least in veliyancode which indicate that the probability of a single being sample continuously is less. this also indicate that the larval diversity in veliyancode is significantly better than the other two stations thereby showing that this part of the estuary is most probable to have more marine fish larvae than any other part of the ponnani backwater. the results of dmrt indicated that in terms of number of individuals, shannon’s diversity and simpson’s evenness indices, ponnani and biyyam, were grouped together since they showed similar characteristics (table 3). however, veliyancode, which was more prone to salinity incursion distinctly stood out from rest of the stations in all the parameters analysed. this means that the physicchemical characteristics prevailing in this region is unique and favourable for better diversity and number of larvae of marine fishes. in the second set of analysis, seasons (premonsoon, monsoon and post monsoon) was kept as the variable and data was pooled accordingly for distinguishing variations in larval assemblage. the results also indicated that even season-wise has been a significant (p<0.01) variation in the number of individual, species, diversity and evenness in ponnani backwaters (table 4). hence, based on the above results, it can be confirmed that the environmental parameters correlates have a direct bearing on the distribution of ichthyofauna in backwaters of ponnani. figures 2 and 3 denotes the mean values of number of individuals and species respectively of marine fish parameters sum of squares df mean square f number of individuals between groups 6135.500 8 766.938 9.234** within groups 2242.500 27 83.056 total 8378.000 35 number of species between groups 340.056 8 42.507 11.448** within groups 100.250 27 3.713 total 440.306 35 shannon’s diversity index between groups 4.866 8 0.608 8.218** within groups 1.998 27 0.074 total 6.865 35 simpson’s evenness index between groups 0.145 8 0.018 4.246** within groups 0.115 27 0.004 total 0.261 35 **significant at 0.01% level. table 3. duncun’s multiple regression test on fish larval assemblage, diversity and evenness in different stations of the ponnani backwaters. variables veliancode ponnani biyyam number of individuals 37.67a 15.00b 10.33b number of species 11.75a 7.42b 5.25c shannon diversity index 2.27a 1.80b 1.54b simpson’s evenness index 0.12a 0.20b 0.24b means with similar sets of superscripts are homogenous. table 4. analysis of fish larval assemblage, diversity and evenness in different seasons in the ponnani backwaters. 57 int. j. aquat. biol. (2017) 5(2): 52-62 larvae encountered during different seasons in the three stations. the results show a clear dominance of number of individual during post monsoon season in all the stations. while analysing the pattern of distribution of fish species during different seasons, it could be seen that maximum representation in veliyancode was during premonsoon periods and in quite contrary the numbers were comparatively high during post monsoon season in ponnani and biyyam. the shannon’s diversity index also followed a similar trend as seen for number of species (fig. 4). however, an interesting observation was reduced values of marine fish diversity during premonsoon season in ponnani. in spite of being a comparatively lesser saline environment, ponnani estuary showed good representation of marine fish larvae similar to that seen in veliyancode during monsoon and post monsoon seasons. simpson evenness index on the figure 2. variation in the number of individuals during different seasons. season figure 3. variation in the number of species in different seasons. season 58 kutty et al./ distribution of ichthyoplankton in ponnani estuary contrary was high during premonsoon in ponnani (fig. 5). the results undoubtedly emphasized that chances of finding different families of marine fish larvae was high in veliyancode station during all seasons. similar results were noticed for monsoon and post monsoon periods in ponnani as well. this indicates that except for biyyam the other two stations had fairly good population of marine ichthyofauna throughout the year. in biyyam, the number of individuals (n), number of species (s), shannon’s diversity (h’) and simpson evenness remained rather same during three seasons (table 5). this means that the environmental perturbation in this station is comparatively less than the other two stations, thereby reflecting a similar kind of population throughout study period. factor analysis of environmental variables collected from the three stations of ponnani backwater pooled together during the three seasons showed that the eigen values for the first four factors figure 4. seasonal-wise variations in the shannons’s diversity indices. season figure 5. seasonal-wise variations in the simpson’s evenness. season 59 int. j. aquat. biol. (2017) 5(2): 52-62 were 3.119, 2.280, 1.774 and 1.034 (table 6). f1, f2, f3 and f4 together accounted for 74.61% of the total variance. f1 which contributed to 28.35% of the total variance was related to surface water temperature, dissolved oxygen and abundance of zooplankton, f2 explained 20.73% of the total variance and was related to bottom water temperature and salinity, f3 accounted 16.13% of the total variance and was related to ph and total suspended solids (tss), and, f4 supported only 9.40% of the total variance related to turbidity (table 7). the relationship of marine fish larval diversity indices to the factors, worked out for different seasons, was established using stepwise regression analysis. since the environmental parameters and diversity indices showed significant (p<0.01) variation with respect to season and region, regression equations were developed to correlate each ichthyofaunal diversity indices and related environmental variables for the study area by pooling the three stations and seasons. abundance of marine fish larvae (n): n=21.00-7.512×f2+5.035×f3-4.961×f1 the results suggest that f2 and f1 showed significant negative relationship with abundance of marine fish larval communities in ponnani table 5. duncun’s multiple regression test on fish larval assemblage, diversity and evenness in different seasons of the ponnani backwaters. means with similar sets of superscripts are homogenous. table 6. environmental variables based on eigen value, percentage of variance and cumulative percentage. component extraction sums of squared loadings eigen value percentage of variance cumulative percentage 1 3.119 28.352 28.352 2 2.280 20.728 49.080 3 1.774 16.128 65.207 4 1.034 9.400 74.608 table 7. factor loading matrix for different environmental variables observed in the ponnani backwaters. environmental/ biological parameters component 1 2 3 4 surface water temperature -0.744 0.122 0.474 0.017 bottom water temperature -0.319 0.83 -0.015 0.162 dissolved oxygen 0.758 0.398 -0.287 0.199 salinity 0.271 0.667 -0.494 0.331 ph -0.319 0.308 0.634 0.104 total suspended solids 0.476 -0.046 0.626 0.333 turbidity 0.415 -0.498 0.144 0.616 nitrite 0.61 -0.227 0.031 -0.381 phosphate 0.167 0.586 0.407 -0.033 phytoplankton abundance 0.577 0.48 0.194 -0.452 zooplankton abundance 0.763 -0.047 0.452 -0.077 60 kutty et al./ distribution of ichthyoplankton in ponnani estuary backwaters, while f3 had a positive bearing on the abundance of marine ichthyoplanktons. hence environmental variables positively favouring better abundance of fish larvae were ph and tss, while temperature, do and salinity negatively influenced this variable. species abundance (s): s=8.139-1.872×f2+1.317×f3+0.979×f4 species abundance was instead positively favoured by factors f3, f4 and negatively influenced by f2. a significant finding of the study was that both bottom water temperature and salinity were the deciding factors for larval assemblage in ponnani backwaters. shannon-wiener diversity index (h’): h'=2.442-0.314×f2+0.270×f3 similar to earlier results, f2 was the chief contributing factor for this variable. here only two factors (f2 and f3) contributed to the diversity indices. hence f2 could be considered as a limiting factor responsible for not only the population size but also the diversity of marine fish larvae inhabiting backwaters of ponnani. simpson’s evenness (1-λ): 1-λ= 0.186+0.039×f2 evenness was influence by only factor f2. hence there is a greater role of climatic variables such as temperature and salinity in an estuarine environment in determining the evenness of population available at a particular locality at a specific period of time. discussions the present investigation was conducted to explore the interactions between environmental factors with abundance and diversity of marine fish larvae in three regions of the ponnani backwaters. the results underline the fact that there is both spatial and temporal distribution of marine fish larvae in ponnani backwaters, the environmental parameters have a direct role. hence delineating the most salient environment factor influencing this distribution would throw more light on to the dynamics of larval assemblage. it could also be a combination of factors that influence the larval dispersal. hence, an in depth analysis based on stations and seasons was attempted to elucidate whether a specific set of parameters are responsible for the larval diversity in ponnani backwaters. among the three stations, veliyancode throughout the study period showed high proportions of marine ichthyofana both adult and larval forms. both in terms of number of individuals and families represented this station stood apart from other two. being in close proximity to the arabian sea and with salinity gradient much narrow, this harbours the richest diversity of marine ichthyofauna in the region. although in terms of shannon’s diversity and simpson’s evenness scales, the place shared a unique position with ponnani estuary, on closer observation it could be seen that typical marine fauna were more concentrated in veliyancode whereas ponnani was dominated by marine and estuarine fish fauna. hence in a nutshell it could be consolidated that although the three station were in close proximity to each other, the environmental regime featuring in the region was quite distinct, which ultimately reflected in the diverse pattern of fish fauna prevalent in the three stations. impact of monsoon was more pronounced in biyyam which has been receiving greater drainage of freshwater from bharathapuzha river. this is why lesser marine species were documented from the region. this also means that salinity does play a major role in the distribution of fish in ponnani backwaters. the statistical evidence suggested that there exist high spatial heterogeny among the relationship between environmental variables and fish larval diversity. although an array of environmental factors indulge on the biotic component of an aquatic system, very few affect them regularly. however, if any such limiting factor is observed, they either act singly or in combinations with other minor entities to produce a change in the ecological structure. in the present study, although there were eight factors that influence the faunal assemblage in the estuary only two variables were responsible for most of the changes. hence it can reasonably be concluded that the temporal and spatial distribution of marine fish larvae in ponnani backwaters is profoundly 61 int. j. aquat. biol. (2017) 5(2): 52-62 influenced by the physico-chemical conditions prevailing in a particular area. this makes biodiversity assessment more challenging in a dynamic environment such as an estuary where there are more than a dozen factors that continuously interact with the organisms. marine fish larvae hence are easy target of even a slightest change in these environmental parameters and which could deleteriously affect their recruitment and further survival. understanding the influence of environmental predictors would in turn be used as an effective tool for developing appropriate policies in the conservation of estuaries and fish fauna available in it. conclusion the present study suggests that there exist a high spatial heterogeneous relationship between environmental variables and ichthyofaunal diversity. in the present study although there were eight factors that influence the faunal assemblage in the estuary, however only temperature and salinity were the variables responsible for most of the changes. hence the major limiting factor for all the diversity indicators was temperature and salinity. the degree to which they contribute any variation depends on the influence of other factors either acting synergistically or antagonistically. whatever may be the case the present study clearly revealed the influence of environmental variables on the fish fauna of the ponnani backwaters. it would thus confirm the earlier finding of spatial and temporal variations that salinity along with bottom water temperature play a vital role in the survival and distribution of marine fish larvae in the ponnani backwaters. the present study paves way for an in depth analysis of all available environment parameters acting upon the fish fauna of ponnani estuary to ascertain their role in determining the availability and distribution of commercially important marine fish in the backwaters of ponnani. only then shall it be clearer how each environmental correlates contribute to the fish diversity of the region. references able k.w. 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(2000). the larvae of indopacific coastal fishes. an identification guide to marine fish larvae, brill, boston. 850 p. martinho f., leitão r., neto j.m., cabral h., marques j.c., pardal m.a. (2007). the use of nursery areas by juvenile fish in a temperate estuary, portugal. hydrobiologia, 587: 281-290. martino e.j., able k.w. (2003). fish assemblages across the marine to low salinity transition zone of a temperate estuary. estuarine and coastal shelf science, 56: 969-987. ramya k. (2010). fish occurrence and biodiversity in beeyam kayal. msc thesis, department of aquaculture and fishery microbiology, mes ponnani college. 35 p. trevor d.h., whitfield a.k. (2006). estuarine typology and the structuring of fish communities in south africa. environmental biology and fisheries, 75: 269293. selleslagh j., amara r. (2008). environmental factors structuring fish composition and assemblages in a small macrotidal estuary (eastern english channel). estuarine and coastal shelf science, 79(3): 507-517. strickland j.d.h., parsons t.r. (1972). a practical handbook of sea water analysis, 2nd ed. fisheries research board canada bulletin, 167: 310 p. whitfield a.k. (1999). ichthyofaunal assemblages in estuaries: a south african case study. reviews in fish biology and fisheries, 9: 151-186. whitfield a.k., paterson a.w., bok a.h., kok h.m. (1994). a comparison of the ichthyofaunas in two permanently open eastern cape estuaries. south african journal of zoology, 29: 175-185. int. j. aquat. biol. (2022) 10(4): 285-298 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article insight into the reproductive biology of euryhaline cyclopoid copepods apocyclops dengizicus and apocyclops royi muthupriya palanichamy1, sivakumar kandhasamy1, altaff kareem2 1school of aquaculture, department of biotechnology, karpaga vinyaga college of engineering and technology, chengalpattu-603308, tamilnadu, india. 2department of marine biotechnology, amet university, kanathur-603112, tamilnadu, india. s article history: received 7 june 2022 accepted 21 august 2022 available online 2 5 august 2022 keywords: apocyclops reproductive biology mating behavior spermatophore abstract: the present research work aimed to investigate the unexplored information about mating strategies and reproductive potential of two euryhaline cyclopoid species apocyclops dengizicus and a. royi. laboratory experiments on mating strategy and reproductive aspects such as the efficacy of females under different mating conditions, the reproductive potential of males, mating frequency, and the effect of starvation on survival and reproductive potential, were conducted and documented. the once-mated female and females pairing continuously with males favored higher reproductive potential. in the present study, the mate-pursuing males prefers to copulate with mature virgin females. the older female was unable to produce eggs even after the sperm discharge. the lifespan of both the apocyclops species under different states of mating and unmated conditions showed considerable variation. apocyclops dengizicus produced 7.92±0.57 pairs of spermatophores, while a. royi extruded 13.32±0.99 pairs of spermatophores during its lifespan. the results were analyzed and inferred; thus high throughput was applied to reveal the understudied topics of these apocyclops species to add considerable knowledge about its reproductive biology. introduction copepods are dominant group of zooplankton enjoying diversified distribution in various aquatic habitats, and play a pivotal role in the energy transfer of the aquatic ecosystem. copepods contribute more to global ecology by controlling the microbial food web and biogeochemical cycle and are also used as a test organism in ecotoxicological studies (burris and dam, 2014). despite a large number of outstanding research on copepods, much biological information remains obscure. copepods transfer spermatozoa to the female through the spermatophore, a highly organized structure with well-stored spermatozoa and a variety of secretary substances (muthupriya et al., 2004). successful spermatophores placement by males on a female genital system is a complicated process involving correspondence: sivakumar kandhasamy doi: https://doi.org/10.22034/ijab.v10i4.1689 e-mail: ksivakumar76@gmail.com behavioral and morphological compatibility (maier, 1992; lonsdale et al., 1998). misplacement of spermatophores outside the genital field has been reported for centropagid calanoid copepods by lee (1972). in copepods, the successful mating and transfer of spermatophore to female is the basic requirement to develop its population growth. but the success of reproduction is not only dependent on female fecundity but also equally shared by male copepods through mating frequency and it is energetic (kiorboe, 2006). according to burris and dam (2015), lifetime spermatophore production can provide valuable information about male energy and mating which is not known for many copepod species. ianora et al. (1989) opine that though it is important to study much about the potential of males in population 286 palanichamy et al./ reproductive biology of apocyclops dengizicus and a. royi dynamics, very less knowledge is gathered about its reproductive investment. the fecundity rate of the female is extensively studied while information about the spermatophore production rate is lacking (marshall and orr, 1958). the spermatophore production and fecundity may also be limited by factors like food and age (burris and dam, 2015) and it is also evident that even sperm quality is affected by these factors (sichlau and kiorboe, 2011). the genus apocyclops includes species found in continental brackish and hypersaline waters worldwide (cheng et al., 1999). apocyclops royi and a. dengizicus are the common dominant species in the adyar estuary, chennai, tamilnadu. both the species were experimentally studied to reveal the secrets of male mating biology and to explore whether males' reproductive investment limits females' fecundity rate. thus, the current study was conducted to assess male energetics by conducting experiments on the rate of spermatophore production and reproduction in females related to mating. further laboratory studies on reproduction in starvation and the status of unmated females were also conducted. the present study is highlighted to address the unanswered questions such as the mating preference of males, determine the frequency of mating, sperm storage, and usage, measuring spermatophore production, factors affecting mating and reproduction, rate of fecundity concerning spermatophore size, and whether remating is necessary to improve reproduction. as little is known about these species, this study will explore more information about their basic biology and energetics. materials and methods zooplankton samples were collected from the adyar estuary using a plankton net made up of bolten silk of 50 µm mesh size. the collections of zooplankton samples were carried out during the early hours of the day. the collected samples were preserved in 5% buffered formalin. apocyclops dengizicus and a. royi were identified based on the minute morphological details and taxonomic key characters provided by botelho (1999) and reid et al. (2002). live zooplankton samples were transported to the laboratory in a 5 l insulated polyethylene container within an hour. apocyclops dengizicus and a. royi were sorted out and maintained in pretreated water (seawater double filtered in 50 µm bolten silk and allowed to settle in large aquaria at room temperature for 48 hours and aerated for 12-24 hours before use). they were fed baker’s yeast (saccharomyces cerevisiae) on alternate days. both apocyclops species were reared in the laboratory, and male and female copepodid v (c v) were used for various experimental studies. reproduction potential of a. dengizicus and a. royi: the reproductive potential of female a. dengizicus and a. royi were carried out with different mating conditions in the laboratory, including trail i male mated with a female once in a lifetime, trail ii male mated in regular intervals with female and trail iii male continuously presents with female in the culture medium. mating behavior: each species of virgin male (c v) and female (c v) were transferred to the petri dish separately, and mating behavior was observed under an inverted microscope. the duration of mating, hatching, number of clutches, clutch interval, number of eggs, egg diameter, and clutch size produced by the mated female during its entire lifespan was recorded. reproduction potential of the unmated female of a. dengizicus and a. royi: both the species of females in the c v stages were maintained separately in unmated conditions and their reproductive variables were studied. spermatophore size variation in males: to study the reproductive potential of male a. royi and a. dengizicus, experiments were conducted for six hours and six days duration vice versa. in the first experiment: the number of females mated by a single male for six hours duration, and in the second experiment, males were allowed to mate with a single virgin female, once a day regularly. in both experiments, the size of the spermatophore transferred was micro-metrically measured and 287 int. j. aquat. biol. (2022) 10(4): 285-298 photomicrographed. effect of starvation on reproduction and survival: to study the influence of starvation on the reproduction and survival of a. dengizicus and a. royi, laboratory-reared females and males of c vi stages were exposed to three days of starvation. using these starved virgin males and females, three types of experiments were conducted, including trail a starved male with starved female, trail b starved male with well-fed female and trail c starved female with well-fed male. the experiment was conducted in a controlled condition with a temperature of 28±1ºc and ph of 7.5. the reproductive potential and survival were monitored. the experiments were maintained with the above conditions in 20 ml filtered seawater with ad libitum feed. the production of spermatophore number, the total number of clutches produced, interclutch period, number of eggs, egg diameter clutch size, and survival duration were recorded. the well-fed males and females were maintained separately in a yeast-fertilized medium. the number of clutches produced during their lifespan was monitored regularly. statistical analysis: the data of the number of clutches, clutch interval, number of eggs, egg diameter and clutch size, and survival of a. dengizicus and a. royi were subjected to statistical analysis. analysis of variance was used to test significant differences between the variable at p<0.05. the mean difference (p<0.05) of the parameters was analyzed by duncan multiple range's test. results mating behavior of a. dengizicus and a. royi: mating and transferring spermatophore in a. dengizicus and a. royi occur soon after the transfer from copepodid v (c v) to copepodid vi (c vi). before mating, the male and female show normal leaping, spiraling, and helical swimming movements. the male and female might get approximation through mechano and chemical sensations. when closer to the female, the male shows the higher activity as a preliminary step for mating; the male captures the caudal rami of the female for mate recognition (fig. 1). immediate wriggling movement of the male puts forth both the antennules towards the swimming legs of the female (fig. 2). in this condition, males actively perceive the female until it becomes passive. then the male makes a characteristic movement bending its body towards the genital segment of the female and sweeps the genital area with its caudal rami (fig. 3). after this act, it resumes its mating behavior to bring the genital segment of both sexes together. at this juncture, male and female faces were ventrally. the prehensile distal part of the male antennules firmly holds the female's third and fourth swimming legs. this is happening to be the proper copulatory position, and once the proper copulatory grasp is achieved male continues to show higher activity while the female becomes more and more passive. at this mating posture, the male’s urosome shows fluxion, leading to the extrusion of a pair of spermatophores. during the final phase of spermatophore extrusion, the genital segment fluxes anteriorly to the figure 1. male capture the female caudal rami–apocyclops royi. 288 palanichamy et al./ reproductive biology of apocyclops dengizicus and a. royi maximum extent, which swiftly transfers the spermatophores onto the setae of swimming legs of the male, which in turn directs forwards and in a quick succession place and attaches the spermatophore closer to the genital pore on the ventral surface of the female genital segment. the whole of this spermatophore extrusion, transfer, and attachment to the genital area is a rapid activity and takes about 15-30 seconds. during the spermatophore transfer and its attachment, females remain passive. however, the male holds the female’s swimming legs either in an upright position or facing the legs more towards the anterior side. such a copulatory position is maintained for about five minutes. during this male ensures that the genital area of the female where spermatophores are freshly attached is undisturbed either by the male's appendages or the female's swimming legs. finally, the male loosens its antennular holds and releases the female. subsequently, the male lies passively, fully stretching its antennules for one or two minutes, and then resumes its normal swimming. after mating and transfer of spermatophores, the female also lies passively. it remains passive for a longer duration compared to males. in the present study, the mate-pursuing males prefer to copulate with mature virgin females than with mated females carrying or not carrying fertilized eggs. mating was also observed in ovigerous females but was not always successful (fig. 4). many males attempted to mate with a female and mating with c v females were also observed (fig. 5). misplacement of spermatophores around the gonophore, in the urosomal segments, and caudal ramus were also observed in the a. royi (figs, 6, 7). in both species, the entire mating process figure 2. male antennules capture the female pleopods v– apocyclops royi. figure 3. male animal bending its body into female genital segment–apocyclops royi. figure 4. male mating with ovigerous female–apocyclops royi. 289 int. j. aquat. biol. (2022) 10(4): 285-298 takes place in about 15-30 minutes. after mating, a pair of spermatophores is attached to the ventral surface of the female genital segment. discharge of spermatophores content into the seminal receptacle and removal of exuviae of the spermatophores takes about 2 hours. the females of both species produced a pair of ovisacs containing fertilized eggs about one hour after mating (fig. 8). however, the duration taken for ovisac formation varies depending upon the maturation of oocytes. embryonic development is completed in 24 hours and nauplii are hatched thereafter. hatching takes about 20-30 minutes. single nauplii take about five minutes to release from the ovisac (fig. 9). in spent females, the spermatophore remained attached to the genital pore for even five days. if mature oocytes were available in the oviduct, the female readily produces a new clutch within one-hour duration after hatching. when recently mated females with or without spermatophores were allowed to mate immediately with a virgin male, the females could not mate. it escapes from the male's grip before the spermatophores were transferred. as the female aged, there was a gradual decrease in the egg-laying rate. in the final phase of reproduction, the female produced empty sacs without eggs. though the older female mates, it is unable to produce eggs even after the sperm discharge. in a few cases in the late phase of reproduction, a new ovisac emerges before the fall of the older empty sacs and this triovisac condition was observed many times (fig. 10). reproduction potential of a. dengizicus and a. royi: the reproductive potential of female a. dengizicus and a. royi in different mating conditions showed variations in the number of clutches, egg numbers, and ovisac length and width in the present study. the numbers of clutch were significantly (p<0.05) highest in females mated at regular intervals with males (trail iii) of 9.08±0.40 and 12.64±0.86, in a. dengizicus and a. royi, respectively. however, the number of eggs of 38.84±0.62 and 39.96±1.14 and ovisac length (308.80±1.50 and 288.12±1.86 µm) and width (210.04±2.07 and 146.24±1.61 µm) were recorded figure 5. two males attempting to mate with female–apocyclops royi. figures 6 and 7. misplacement of spermatophore around the female caudal rami and gonophores–apocyclops royi. (sph– spermatophore). 290 palanichamy et al./ reproductive biology of apocyclops dengizicus and a. royi significantly (p<0.05) high in once mated female (trail i), in a. dengizicus and a. royi, respectively. the interclutch period was found to be shorter in the females who were mated at a regular interval (trail ii) than once mated females (trail i) as well as females continuously paired with males (trai lii) in both the species (tables 1 and 2). anova for the number of clutches, ovisac length, ovisac width, number of eggs in the clutch, egg diameter, and clutch interval of both the species in different mating conditions showed significant differences (p<0.05). the gross observation indicated that once mated or pairing females continuously with males favored higher reproductive potential. in the once-mated female of both the species, during the final phase of reproduction, it extruded unfertilized eggs which were shredded or disintegrated. after extruding a few batches of unfertilized eggs, they released granular secretion (egg material) until death. unmated females: the female’s a. dengizicus and a. royi were maintained in unmated condition after the final molt extruded unfertilized eggs from the fifth day or one week onwards. it extruded very few infertile eggs (1-5) and rarely a complete batch of eggs. it was observed that there was oocyte production in the unmated females; from the early oocyte stage they transformed into germinal vesicle stage with a conspicuous nucleus and nucleolus. these oocytes remained in the oviduct without undergoing intensive vitellogenesis. though females without mating for 8 days showed normal production of oocytes, a greater abnormality was observed in the number and size of eggs. after this period, the oviducts of the unmated females contained eggs of normal texture and abnormal eggs in the form of disintegrated secretory material (fig. 11). further, advancement in the unmated condition leads to the production of mostly abnormal eggs in the form of figure 8. ovigerous female–apocyclops royi. figure 9. nauplii release from ovisac–apocyclops dengizicus. (na– nauplii; os–ovisac). figure 10. triovisac–apocyclops dengizicus. (eos–empty ovisac; os–ovisac). 291 int. j. aquat. biol. (2022) 10(4): 285-298 disintegrating secretory material. the female maintained around 40 days in unmated condition, when allowed to mate with a normal adult male there was no proper sequencing of the mating act, and due to the evasive activity of the female, there was no transfer of spermatophores. experiments on the reproductive potential of males: in the mating process, the males of a. dengizicus and a. royi transfer a pair of beanshaped spermatophores. apocyclops dengizicus produced 7.92±0.57 pairs of spermatophores, while a. royi extruded 13.32±0.99 pairs of spermatophores during its lifespan. in both the species during the peak reproductive period, after the extrusion of spermatophores from the spermatophore sac, a new pair was formed within 30 minutes (maximum 9 hours). laboratory experiments indicated that the size (15-62 μm in length and 6-30 μm in width) and shape of the spermatophore varied. the mating capacity was limited by the time taken by the animal to refill the spermatophore. depending on the duration of refilling the spermatophore, the size of the spermatophore varied. in some cases, instead of a pair, a spermatophore was also extruded. in the case of successive mating, the male was capable of transferring spermatophores to 3-4 females in 2-3 hours. during successive mating, the size (15-60 μm in length and 6-30 μm in width) of the spermatophore gradually decreased. further, variation in the shape was also observed. after transferring 3-4 pairs of spermatophores in a day, table 1. reproductive potential of apocyclops dengizicus in different mating conditions. no. of clutches ovisac length (µm) ovisac width (µm) no. of eggs egg diameters (µm) clutch interval trail i 8.04±0.45a 308.80±1.50a 210.04±2.07a 38.84±0.62a 84.08±0.64a 2.20±0.41a trail ii 7.04±0.35b 258.96±1.77b 179.92±2.12b 27.92±0.76b 82.00±0.71b 5.12±0.44b trail iii 9.08±0.40c 288.88±1.81c 182.92 2.12c 25.16±1.43c 83.96±0.68a 3.08±0.49c the values are represented as mean±sd (n=25). different superscripts on the column between the trails showed significant different (p<0.05) of either species. table 2. reproductive potential of apocyclops royi in different mating conditions. no. of clutches ovisac length (µm) ovisac width (µm) no. of eggs egg diameters (µm) clutch interval trail i 9.56±0.71c 288.12±1.86b 146.24±1.61b 39.96±1.14a 71.04±0.89b 8.00±0.50a trail ii 8.56±0.77b 210.72±1.59c 116.32±1.28c 29.96±1.24b 68.24±0.83a 1.12±0.33b trail iii 12.64±0.86a 231.96±1.79a 125.68±0.95a 21.12±1.33c 69.80±0.96c 3.04±0.35c the values are represented as mean±sd (n=25). different superscripts on the column between the trail showed significant different (p<0.05) of either species. table 3. survival of apocyclops dengizicus and a. royi under different mating conditions. parameters no. of days survived a. dengizicus a. royi male female male female once mated 21±2 35±1 38±1 36±1 intensively mated 11±1 27±1 15±1 16±2 unmated 24±1 63±2 50±2 64±1 figure 11. secretory materials formed in apocyclops dengizicus. 292 palanichamy et al./ reproductive biology of apocyclops dengizicus and a. royi even though the male initiated mating again by capturing the female, spermatophore transfer was not observed and it takes about 7-9 hours to refill its spermatophore sac. the size of the spermatophore transferred by a male of a. royi during daily mating (45-60 μm) was longer than the male which mated successively (1560 μm) on 6 hours duration with several females and transferred the spermatophores. the size difference in the spermatophores extruded during six hours duration showed a significant difference (p<0.05) while the spermatophore transferred after daily mating for six days did not show a significant difference. the male a. royi which was allowed to mate with a female every day extruded spermatophore in normal size (45-60 μm) and the mated females subsequently produced the normal number of clutches (10±2). in successive mating, depending on the size of the spermatophore, the clutches produced by the mated females varied. when the size of the spermatophore transferred was very small (20 μm) the mated female produced only two clutches during its lifespan. few males exhibit normal mating with the female and extruded normal size spermatophores. though females produced a smaller number of clutches and no case a complete egg batch was observed; only a few numbers of eggs were produced. this might be due to the poor viability of sperm. the lifespan of unmated males and females: the lifespan of apocyclops species under a different state of mating and unmated conditions showed considerable variation. unmated a. dengizicus and a. royi male and female survived longer than once mated animals. compared to these animals, individuals which were intensively mated showed survival for a short period (table 3). effect of starvation on survival and reproduction: the starved adult females of both species survived the longer duration of 10±1 days, while the males were 7±1 days. during starvation, both females and males were active for about two days, and subsequently, their locomotory activity reduced gradually. experimentation results on the reproduction of these animals indicated that the males could mate with the female and transfer spermatophores not more than twice under starved conditions. the size of the spermatophores ranged 35-50 μm. instead of a pair, the extrusion of a single spermatophore was also observed. the female fed to satiation when inseminated by a starved male could produce only one or two clutches during its lifespan. though the females appeared to be gravid with mature oocytes in oviducts (darkened oviducts), clutch production was not observed. instead, the oocytes from the oviduct were released to the medium through the reproductive pore. in the case of starved females which were inseminated by well-fed males, only two clutches were produced with the interval of two days. the development of eggs in the ovisac was arrested and eggs remained in the ovisac for about three days during this period. the eggs turned pale in appearance and the egg content disintegrated leading to its discharge from the sac to the medium (fig. 12). after the release of aborted eggs, the empty ovisac sometimes remained attached to the genital segment for about two days. the starved female thereafter did not produce normal eggs and ovisac. the oocytes of the oviduct mostly remained in the form of granular secretory material which oozed out of the reproductive pore. in the final phase of the starved period, there was no oocyte production in the female as evidenced by the transparent oviducts. the effect of starvation on the number of clutches produced, length and width of ovisac, number of eggs, and egg diameter of a. dengizicus and a. royi are presented in tables 4 and 5. the number of clutches (2.20±0.42 and 2.20±0.79), the number of eggs (29.40±0.84 and 29.40±0.52) and the length and width of the ovisac were significantly (p<0.05) higher in starved male with well-fed female (trail b), starved female with well-fed male (trail c), and starved male with starved female (trail a) in a. dengizicus and a. royi, respectively. dmrt’s test showed that the number of clutches between trail a and trail c and egg diameter between trail b and 293 int. j. aquat. biol. (2022) 10(4): 285-298 trial c were no significant (p>0.05) variation in both the species (tables 4 and 5). discussion it is well understood that the copepod taxa express an array of mating behavior depending on the life history and mating strategy of each species. in general, mating behavior involves a series of steps (foltz, 1995; vacquier et al., 1995). in the present study based on the observation, the sequential events of the mating process and spermatophore transfer was reconstructed and reported for the first time in both apocyclops species. it is interesting to note that in the current experimental studies males of both the apocyclops species prefer to mate with newly molted virgin females than mated females. similar to the present result the males of epischura lacustris (chow-fraser and maly, 1991) acartia hudsonica, and a. tonsa (burris and dam, 2015) also reported mating exclusively with virgin females of comparable size. uchima (1985) and uchima and murano (1988) suggested that a sex-attractant pheromone is present with mature virgin females and absent in immature or mated females, which enhances the males to prefer virgin females for copulation. leeuwen and maly (1991) opined that the chemical signal released by the female varies in strength with its reproductive status. although the general features of cyclopoid reproductive biology are known, many details, for example, the reproductive cycle or a male's mating capacity are still unclear (maier, 1992). defaye et al. (2003) stated that single mating is sufficient to fertilize several clutches in m. albidus as is usually observed in cyclopidae. muthupriya et al. (2004) observed that the freshwater cyclopoid species mesocyclops thermocyclopoides and thermocyclops decipines produced a maximum of five to nine clutches after a single mating. uchima (1985) found that in o. davisae one copulation is usually enough to fertilize all eggs produced by the female in its entire lifespan. similar to all these reports, in the present study, the reproduction of a. dengizicus and a. royi showed normal fecundity and clutch size after a single mating. table 4. reproductive potential of apocyclops dengizicus under starvation condition. no. of clutches ovisac length (µm) ovisac width (µm) no. of eggs egg diameters (µm) trail a 1.60±0.52a 258.80±5.94a 129.40±5.87a 24.40±0.84a 68.90±0.88b trail b 2.20±0.42b 231.40±6.72b 118.00±3.20b 28.40±0.84b 67.00±0.94a trail c 1.40±0.52a 241.60±7.01c 123.80±2.86c 29.40±0.84c 66.80±0.92a the values are represented as mean±sd (n=25). different superscripts on the column between the trail showed significant different (p<0.05) of either species. table 5. reproductive potential of apocyclops royi under starvation condition. no. of clutches ovisac length (µm) ovisac width (µm) no. of eggs egg diameters (µm) trail a 1.40±0.52a 253.80±5.94a 129.00± 4.85a 23.80±0.79a 68.20±0.79a trail b 2.20±0.79b 225.80±4.85b 117.80± 2.28b 28.00±0.67b 66.60±0.52b trail c 1.20±0.42a 235.80±5.34c 123.80± 3.36c 29.40±0.52c 66.40±0.52b the values are represented as mean ± sd (n=25). different superscripts on the column between the trail showed significant different (p<0.05) of either species. figure 12. discharge of egg from ovisac to medium–apocyclops dengizicus. (ae–aborted egg) 294 palanichamy et al./ reproductive biology of apocyclops dengizicus and a. royi the secretory material of the seminal epithelium would preserve the sperm and might constitute a sort of “nutritive medium” for the sperm giving the spermatozoa maximum chances for optimal fertilization efficiency (defaye et al., 2003). however, it remains unclear whether one mating is sufficient to permit the realization of a female's total reproductive potential (maier, 1992). supporting this statement, in the present research, in once-mated females the extrusion of unfertilized eggs and granular secretion was observed in the last phase of clutch production, which might be due to the insufficiency of sperms to fertilize more number of clutches. the investigations of maier (1992) on the reproductive biology of c. vicinus revealed that females that mate only once, show a similar reproductive pattern (clutch size and clutch succession) to those which remain combined with males and thus have the opportunity to remote but tend to produce fewer clutches. however, the extent of sperm storage and clutch formation after a single mate is not known in this group. under different mating conditions, in a. dengizicus and a. royi, the diameter of the egg remained similar, while variation was observed in the production of clutches, clutch size, and length and width of the ovisac. though the results indicated that once-mated females of both species could produce several clutches, there appears to be a wide variation concerning the interclutch period. apocyclops dengizicus and a. royi females are allowed to remain continuously with males showing a higher number of clutches and moderate interclutch period advocating higher reproductive performance with multiple mating. this suggests that there should be remating for optimizing the reproductive output in this species. it is interesting to note that a very short interclutch period was recorded in females mated at regular intervals. perhaps, regular mating may induce higher gonadal activity leading to increased reproductive potential. thus, even though the males and females of cyclopoids may mate more than once (wyngaard and chinnappa, 1982), the remating seems to be of little significance for egg production. willey et al. (1990) opined that remating is most likely to have no importance for egg production and hence for population growth. such a reason can be attributed to the less reproductive performance of females of a. dengizicus and a. royi which are mated at regular intervals; in addition to this it might be due to different qualities of insemination (maier, 1992). the data reported for egg number in successive broods of a. viridis suggested an initial increase followed by a decline during the last days (abdullahi, 1992). this report was similar to the present observation that in both the cyclopoids there was a gradual decline in eggs production during the last phase of reproduction. sciandra et al. (1990) opined that the synthesis of matter necessary for the maintenance of oocytes is a biochemical process whose efficiency decreases with age. the information provided by maier (1992) on the mating duration, spermatophore refilling time of c. vicinus is similar to the duration recorded in the present study. a pair of spermatophores discharged sperms into the female’s seminal receptacle and detached within two hours in both the apocyclops species. in the case of o. davisae, the discharge of spermatophore content takes 24 hours to 2 days (uchima and murano, 1988). in the present study, the long-time attachment of spermatophores to the female genital segment is observed in the spent females. however, in aged females, it took longer durations (up to 5 days) to completely discharge spermatophores' content to the seminal receptacles. spermatophore production is habitat related and in the marine media, they appear to have evolved to minimize sperm loss (subramoniam, 1993). spermatophore protects the delicate sperm cells from drying (schaller, 1980). well formed spermatophore layer and a variety of secretory substances enable spermatophore attachment and sperm expulsion after mating. maier (1992) stated that the mating capacity of males is possibly limited by the time needed to fill a new spermatophore. in the present study, males showed high reproductive potential by producing and 295 int. j. aquat. biol. (2022) 10(4): 285-298 transferring many pairs of spermatophores during their lifespan. compared to a. dengizicus higher number of spermatophores were produced by a. royi suggesting that more frequent remating is required in a. royi than a. dengizicus. the production of spermatophores depends on the sperm storage facility available in the female body. if the seminal receptacle can store a large quantity of seminal content, consequently large spermatophores are produced and transferred by males, and females can store the spermatozoa for a longer duration and hence require less frequent mating. whereas in females, storing seminal content for a shorter duration might require frequent mating with small spermatophores. thus, the spermatophore production in cyclopoid copepods might be based on the capacity for sperm storage in the female and their reproductive strategy. this strategy in the case of a. dengizicus is towards the adaptation of less frequent mating with a longer duration of sperm storage, while in a. royi frequent mating facility with a short duration of storage of spermatozoa. nevertheless, these strategies are highly suitable for maintaining the high-density population of these species in nature. the extrusion of a spermatophore in the present study was observed during successive mating and unfavorable condition such as starvation. laboratory experiments on the life cycle of unmated females suggest that mating and spermatophore transfer is necessary for the normal reproductive activity of the female. in the absence of mating and spermatophore transfer, the female is incapable of normal reproductive processes such as the production of previtellogenic oocytes, vitellogenic oocytes, secretory material meant for the formation of ovisac, and also normal embryonic development of fertilized eggs in the ovisac. there may be involvement of an intricate hormonal mechanism in regulating aspects such as oogenesis, vitellogenesis, fertilization, and embryogenesis. these processes may be regulated in a coordinated or well-directed direction. nevertheless, mating, spermatophore transfer, and discharge of spermatophore content into the seminal receptacle might act as the triggering factor for the successive female activity. inspite of the availability of ideal reproductive conditions, the absence of male stimulatory factors can be attributed as the most vital factor leading to the abnormal reproductive activity of the unmated female. uchima (1985) reported that the unmated females of o. davisae lay unfertilized eggs in the same mode as that of mated females. maier (1992) reported that unmated females of c. vicinus extruded a few (1-5) infertile eggs; in no case, a complete egg batch was observed. there are many reports on the longevity of many starved copepods, a. tonsa (dagg, 1977), paracalanus parvus (checkley, 1980), and calanus typicus (nival et al., 1990) which did not exceed more than a few days suggesting, the stored energy is rapidly spent. sciandra et al. (1990) showed that for periodicities more than 9 days of copepods die because their resources are insufficient for more than 5 days of starvation. similar to the previous reports, in the present study, both species survived for about 10 days under starvation conditions. in these animals, the depletion of food sources is well exhibited in their locomotor activity. in the first few days, the animals were active, while the locomotor activity declined progressively. during the last phase of survival under starved conditions either they became sedentary or showed feeble activity. the survival days in starved males are much shorter than those in starved females (marshall and orr, 1972; uchima and hirano, 1988). like previous reports, the present results also suggest that there is higher tolerance of starvation by females than the male. the experiments of preetha and altaff (1996) showed that starvation results in lower fecundity in freshwater calanoid copepods sinodiaptomus (rhinediaptomus) indicus. durbin et al. (1983) suggested that each species has the ability for accumulating large stores of energy (especially lipids) and the rate of egg-laying is strongly dependent on the amount of food ingested during the previous 6-48 hours. experiments on p. parvus (checkley, 1980) and c. typicus (nival et al., 1990) suggested that reproduction is stopped during starvation to reduce 296 palanichamy et al./ reproductive biology of apocyclops dengizicus and a. royi the energetic expenditures involved in the maturation of gametes and hence to permit copepods to survive for a longer time. thus, it is considered that the maturation of gametes is possible when the level of energetic storage remains higher than a given threshold (runge, 1985). such a reason can be attributed to the sharp decline in the reproductive potential of starved males and females of both a. dengizicus and a. royi. pandian (1994) stated that the interruptions of food supply lead to regression of ovaries. thus, the percentage of females producing eggs was greatly reduced (jayanthi, 2001) and substantially lowered the hatching success (ederington et al., 1995). the data of sciandra et al. (1990) reveals that the egglaying during starvation in t. stylifera originates from already matured oocytes whose vitellogenesis was nearly achieved after which due to starvation, the maturation is completely inhibited. such a process is observed concerning the egg and spermatophore production by the female and male of a. royi and a. dengizicus, respectively. during the first two days of starvation, the mature oocytes and spermatophores contents might have formed which was evident in the reproductive activity. as nutrition depleted under starved conditions, the reproductive activity was affected resulting either in the production of abnormal eggs or caesation of egg production. misplacement of spermatophores outside the genital field has been reported for many species of copepods (pandian, 1994). according to lonsdale et al. (1998) male spermatophore placement is likely to be guided at least partially by chemical signals on the surface of females. the present laboratory studies revealed that the misplacement of spermatophores occurred in a male biased population where more males compete for a single female, males copulate with inappropriate mate such as cv female and rarely due to the abnormal genital segment of females. thus, these incidences may result in improper placement of spermatophores. fleminger (1967) has reported that labidocera jollae, postulated that sexual swarming coupled with a high male and less female sex ratio could be the basis for a large number of misplaced spermatophores. cases of several males attempting to copulate at the same time with one female have been reported earlier for several copepods such as cyclops americanus and pseudodiaptomus coronatus (hill and coker, 1930; jacobs, 1961). thus, the slightly misplaced spermatophore may significantly reduce the number of sperm transferred and these mating mistakes would result in wastage of gametes and energy (blades-eckelbarger, 1991). the present study may add more important information for the first time about the reproduction of the apocyclops species which may provide a key for ecologists to understand the basic biology, copepod behavior, and life history. despite the fact that this study examines the reproductive strategies of both species with remarkable insight, much more research into its biology remains to be investigated. our finding has concluded that the successful copulation and reproduction in cyclopoids are influenced by various factors and reproductive potential of females of a. dengizicus and a. royi revealed remating is necessary for the continued reproduction of these species. the experiments on unmated females clearly indicated that the mating and spermatophore transfer is necessary for normal reproductive activity of female. the present attempt has documented that food and aging are the two important limiting factors of normal reproduction. the higher mating frequency of male with virgin females divulges the male mate-choice. the variation in reproduction between two species might be attributed to their genetic variation. thus, the investment of energetic male and utility of the sperm by female producing viable eggs disclose the equal contribution of both sexes are necessary for successful population growth. acknowledgement the authors are grateful to the principal, professor and head, postgraduate department of zoology, the new college, chennai, for providing the laboratory facilities to carry out the work. the authors are 297 int. j. aquat. biol. 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(2022) 10(2): 151-168 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article effect of bio-organic fertilizer and agro-industrial residue on the growth and reproduction of cyclopoid copepod, oithona rigida (giesbrecht, 1896) amirah yuslan1, hidayu suhaimi1, norhidayah m. taufek2, nadiah w. rasdi1,3 1faculty of fisheries and food science, universiti malaysia terengganu, 21030 kuala terengganu, terengganu, malaysia. 2aquanutri biotech research laboratory, institute of biological sciences, faculty of science, universiti malaya, 50603, kuala lumpur, malaysia. 3 institute of tropical biodiversity and sustainable development, universiti malaysia terengganu, terengganu, malaysia. s article history: received 5 november 2022 accepted 8 april 2022 available online 2 5 april 2022 keywords: copepod enrichment dietary composition fatty acids live feed abstract: production of live feed for larval development of aquatic species is crucial in the aquaculture industry. the cultivation of cyclopoid copepod, oithona rigida can enhance the growth performance and nutritional quality of fish and crustacean larvae. hence this study was conducted to evaluate the different dietary regimes containing swiftlet waste, soybean meal, rice bran and microalgae (nannochloropsis sp.) on the growth and productivity of o. rigida. the results showed that rice bran and nannochloropsis sp. additives produced the best outcomes in terms of specific growth rate (0.109±0.002 and 0.104±0.001 day -1) of o. rigida., protein content (66.83±2.25 and 72.08±2.02%), and lipid content (21.98±1.41 and 18.09±1.18%) respectively. a mixture of rice bran and nannochloropsis sp. (41.62%) as dietary additives also improved the polyunsaturated fatty acids (pufas) content of o. rigida as compared with mono diet applied such as rice bran (28.16%) and nannochloropsis sp. (31.35%). the use of rice bran as food additives for o. rigida has shown a comparable result with the nannochloropsis sp., in terms of the growth, survival, reproduction and dietary value. thus, rice bran and nannochloropsis sp. was marked as the best feeding regime for the cyclopoid copepod, o. rigida as potential characteristics for mass culture in aquaculture hatcheries. introduction primarily, zooplanktons are widely used as live food for crustacean larvae as they have a vital connection with the food system based on the larger targeted consumers (gürbüzer et al., 2017). live food organisms contain all required nutrients such as proteins, lipids, carbohydrates, vitamins, minerals, and amino acids, popularly regarded as ‘living nutritious capsules’ (manickam et al., 2020; radhakrishnan et al., 2020). copepods have a high rate of generative capacity and can survive under adverse conditions for a short period (loh et al., 2013; rasdi and qin, 2016). they are easier to ingest and digest, which is a crucial aspect of the growth and development of larval forms in aquaculture (maquirang et al., 2020; khoa et al., 2020). production of live feed is vital for exogenous feeding of commercially cultured larvae such as blue swimming crab, portunus pelagicus (affandi et al., correspondence: nadiah w. rasdi e-mail: nadiah.rasdi@umt.edu.my 2019), seabass, dicentrarchus labrax (el-sayed et al., 2021), common sole, solea solea (shawky et al., 2021), japanese flounder, paralichthys olivaceus (khoa et al., 2021), and tiger shrimp, penaeus monodon (jaseera et al., 2021). copepods are common live feed found in marine, freshwater and brackish water, representing up to 80% of the zooplankton biomass in the natural environment (mauchline, 1998; piasecki et al., 2004; kimmerer et al., 2018). cyclopoid copepod, oithona rigida has been reported to contain high content of hufas, comparable to artemia and rotifer. besides, copepods vary in sizes for efficient capture, consumption, and optimise ingestion of food consumed by fish and crustaceans’ larvae (genodepa et al., 2004; santhanam and perumal, 2012a). oithona rigida can swim freely in the water and is constantly accessible to newly hatched larvae due to their jerking movements in the water column that triggers the larvae's feeding 152 yuslan et al./ effect of bio-organic fertilizer and agro-industrial residue reaction (burbano et al., 2020). enrichment of live feed such as copepods and other zooplanktons has been widely applied using various media such as oatmeals and sunflower oil on freeliving nematode, panagrellus redivivus (affandi et al., 2019) and agro-waste from shochu distillery on brachionus plicatilis rotifer (khoa et al., 2021) as well as a waste by-product from defatted haematococcus pluvialis meal on moina macrocopa culture (li and liu, 2021; rasdi et al., 2021). although few studies are available on the utilization of agro-waste and byproducts for the enrichment media, it is still considered very limited compared to those enrichments from the microalgae like nanochloropsis sp. due to its high nutritional content (manisali et al., 2019; matsui et al., 2019; rasdi et al., 2021). live feed containing desirable nutrients such as lc-pufa is preferable since larvae require high levels of arachidonic acid (ara, 20:4n-6), omega-6 (dpan-6, 22:5n-6), eicosapentaenoic acid (epa 20:5n-3) and docosahexaenoic acid (dha, 22:6n-3) for their survival and growth (basford et al., 2020). enriching the live feed with high lipid sources ingredients, including lipid emulsions (dhert et al., 2014) and microalgae (ma and qin, 2014; rehberghaas et al., 2015) are common approaches in hatcheries. however, the form of neutral lipids and triacylglycerols present in the lipid emulsions is not as ideal as supplying polar lipids in the form of phospholipids contained in the microalgae (basford et al., 2020). the availability of different food sources for live feed enrichment is crucial in providing better quality live feed sources for aquatic larvae (radhakrishnan et al., 2020). various studies on the enrichment of live feed mainly focused on microalgae as the main and common food source (latib et al., 2020; ramlee et al., 2021). however, potential organic waste products and agro-industrial residue have been seen as possible food sources which could be explored to enhance the nutritional value of live feed culture in hatcheries (kamrunnahar et al., 2019; rasdi et al., 2020). hence this work aimed to evaluate the different dietary regimes containing swiftlet waste, soybean meal, rice bran and microalgae (nannochloropsis sp.) on the growth and productivity of o. rigida. material and methods sampling and experimental design of oithona rigida: the zooplanktons were collected and isolated from the setiu wetland of peninsular malaysia using horizontal and vertical plankton net (50 µm). during the collection, the lagoon temperature was 24-30°c, salinity 22-34 ppt, and 8.0-9.5 ph at noon (yuslan et al., 2021). upon collection, the live samples were washed with freshly filtered seawater (1 μm) to avoid contamination and transported directly to the laboratory. individual copepod was transferred to a new beaker containing filtered seawater for species identification following dussart and defaye (2001), lopes et al. (2001) and conway (2006). enrichment diets, including rice bran, soybean meal, swiftlet waste and nannochloropsis sp. were provided immediately after being transferred to the culture tank. oithona rigida (fig. 1) was isolated according to the morphological classification and gradually scaled up by standard protocols (kasturirangan, 1963; santhanam et al., 2015). all maintenance and feeding trials were conducted in fisheries and aquaculture sciences hatchery, universiti malaysia terengganu. oithona rigida stock culture was first cultivated in 1 l flask with an initial density of 8 ind/ml. the culture was upscale once the density increased where the cultures were transferred to 500 l tanks for stock culture. adult o. rigida was isolated from the stock culture and transferred to a 1000 l mass culture tanks and supplied with the specified food for one generation before data were collected to remove any consequences of previous feed regiments (kamrunnahar et al., 2019). oithona rigida were maintained under controlled laboratory conditions as specified in table 1. after six months, the new o. rigida were harvested and placed into the 100 l enrichment tanks for the feeding trial. the five enrichment tanks represent five experimental diets (one control) in triplicates for each treatment. enrichment of o. rigida with bio-organic fertilizer 153 int. j. aquat. biol. (2022) 10(2): 151-168 (swiftlet waste) and agro-industrial residue (rice bran band soybean meal): bio-organic fertilizer (swiftlet waste) was obtained from post-harvest facilities at universiti malaysia terengganu. the agro-industrial residue (rice bran and soybean meal) was purchased from a fertilizer supplier in kedah, malaysia. before swiftlet wastes were used as feed, they were first transformed from raw manure to bioorganic fertilizer by using mineralization technique to make the feed become more hygienic and safer to be used as feed for zooplanktons (nagavardhanam, 2017). these organic feeds were dried at 105°c for 24 hours to remove the moisture and stored in plastic jars for further use (war and altaff, 2011). prior to the feeding trial, all diets were grounded, and the micronized substances were reduced into powdered form (<0.1 mm) using a blender at maximum speed for approximately 3 minutes. the feeds were then weighed and dissolved in distilled water to achieve a heterogeneous suspension culture. next, the feed mixture was sieved using a net of 50 µm before being poured into the o. rigida culture tank (loh et al., 2013). the dietary suspensions were then used to fertilize the culture medium as the enrichment media for o. rigida. enrichment of o. rigida with microalgae (nannochloropsis sp.): nannochloropsis sp. pure culture stock strain was obtained from universiti malaysia terengganu hatchery and cultured prior to the experiment. nannochloropsis sp. was grown in conway medium at temperature 29oc, salinity 30 ppt with ratio of light: dark cycle of 12:12 h and continuous aeration (deville et al., 1995). weekly algae preparation using the optimization algal growth process was appropriate for copepod growth. population density and specific growth rate of figure 1. dorsal view of adult oithona rigida (left) and berried female oithona rigida (left) under a compound microscope. parameters references diets concentration swiftlet waste, rice bran and soybean meal = 500 mg/l paray and al-sadoon (2016) microalgae (nannochloropsis sp.) = 500 mg/l = 2 x 107 of algal cells/ml espinosa-rodríguez et al. (2012) and rasdi and qin (2015) duration of enrichment 6 hours payne and rippingale (2001) and estévez and giménez (2017) salinity 26±0.3 ppt santhanam and perumal (2012b) and santhanam et al. (2019) temperature 26 ± 0.3 °c milione and zeng (2008) and santhanam et al. (2019) table 1. the parameters for oithona rigida cultivation and enrichment. 154 yuslan et al./ effect of bio-organic fertilizer and agro-industrial residue o. rigida: a culture setup consisted of 100 l tank of o. rigida with a population consisting of mixtures of 20, 10, 6, and 4 ind ml-1 adult was cultured in the 500 ml beakers with triplicates for each dietary treatment. the measurements of life-history parameters in o. rigida were monitored, and the data were recorded. the 3 ml subsample of o. rigida was taken and calculated daily from the 500 ml culture medium under dissecting microscope. triplicate counting was performed from the subsamples. optimum water quality parameters were maintained daily using a ysi meter, and gentle aeration was provided. the experiment was performed for 16 days until the entire cohort dies. the specific population growth rate (r) (days-1) has been derived from population density data using the following equation by lee et al. (2013). the specific population growth rate (r) = ( ln 𝑁𝑒 − ln 𝑁𝑖 t ) where t = culture days, ni = initial density of copepods and ne = end density of copepods. hatching rate, hatching time, survival, and generation time of o. rigida: fifty berried females were randomly collected from each beaker and placed in a 30 ml petri dish with three replicates for each treatment. eggs were examined every 6 hours to count newly hatched nauplii over 24 hours. the nauplii were then transferred to a new petri dish with the same diet figure 2. the population density of nauplii copepod, oithona rigida fed with different diets. all values are mean ± standard deviation (n = 3). figure 3. the population density of copepodites oithona rigida fed with different diets. all values are mean ± standard deviation (n = 3). 155 int. j. aquat. biol. (2022) 10(2): 151-168 as the broodstock to test the following o. rigida survival from incubation to maturity. the number of unhatched eggs was counted after 48 hours, and the hatching rate was calculated (pan et al., 2014). hatching rate (%) = ( total number of the unhatched eggs total number of the eggs ) × 100 survival rate (%) = ( total number of the sample taken total number of initial density ) × 100 subsequently, the hatched nauplii were monitored daily for mortality in each treatment to assess the average lifespan from hatching to maturity. throughout the trial, food was applied regularly to each beaker using a plastic dropper to prevent overfeeding. the hatched o. rigida were then placed in a petri dish with three replicates for each dietary treatment to determine the developmental period from nauplii to the mature stage. the development period of o. rigida was monitored every 1 hour before the mature stage was reached, and the development time for each post-embryo stage was calculated from the mean triplicate for each treatment. the developmental stages were observed under a dissecting microscope to calculate the development time from nauplii i to vi, the generation time of copepodite i to adult and the generation time of nauplii i to gravid. offspring production, spawning, and lifespan: one mature female was taken from the preconditioning copepod stocks culture tank and placed into a 50 ml beaker, covered with aluminum foil with holes for ventilation for each diet treatment in triplicates. the females of o. rigida were fed with all the diets enrichment and the beakers were monitored daily for the occurrence of new eggs in the egg sacs. the individual products from the previous eggs were counted in the sedgewick-rafter chamber on a dissecting microscope using a 40 μm mesh. the daily development of offspring and offspring per egg sac was derived from the average of the triplicates of o. rigida for each diet. full spawning in a lifetime was recorded and the longevity of females was reached by an average of all life expectancy within 30 days before the entire cohort died. lipid and protein analysis of enrichment diets, unenriched and enriched o. rigida and artemia: the analyses have been done at the faculty of fisheries and food science laboratory in umt. enrichment diets and live feeds were analyzed using the association of official analytical chemists (aoac, 1995) standard protocol in triplicates. all prepared samples were finely crushed using mortar and pestle and further blend into powder form (<0.1 mm) using a blender at maximum speed for approximately 3 minutes (loh et al., 2013). the protein was analyzed using the kjeldahl method with a conversion factor of 6.25 to transform total nitrogen to crude protein (pearson, 1999). samples (200 mg) were approximately placed into glass tubes to conduct the digestion procedures, followed by the addition of the digestion mixture according to the treatments (silva et al., 2016). lipids were extracted from the soxhlet petroleum ether extractor. approximately 100 mg of samples were applied using a modified bligh and dyer method (branco-vieira, 2018; gorgich, 2020). after successful extraction, the residue was dried to a constant weight at 100°c. fatty acid methyl esters (fame) analysis of enrichment diets, unenriched and enriched o. rigida and artemia: plankton net (100 μm) was used to sieve the samples of live feeds. the samples were washed with distilled water and taken to the diets specific population growth rate (day− 1) swiftlet waste 0.067±0.005a soybean meal 0.082±0.001b rice bran 0.109±0.002b nannochloropsis sp. 0.104±0.001a unfed -0.084±0.015c table 2. the specific population growth rate of oithona rigida fed different enrichment diets. all values are mean ± standard deviation (n = 3). different letters on the same column indicate significant difference (p<0.05). 156 yuslan et al./ effect of bio-organic fertilizer and agro-industrial residue laboratory to be stored in a deep freezer below -80°c for 48 hours. the sample was freeze-dried for 48 h prior to the preparation of a fatty acid analysis. once each sample was thoroughly dried, it was finely ground prior to analysis. the combinations of the classical method (aoac, 2000) and a one-step method (atolani et al., 2015) have been used for preparing fatty acid methyl esters (fame). all the chemicals used (hexane, chloroform, methanol, and 14% bf3 in methanol) were analytical reagent grades (gc-2010, shimadzu, tokyo, japan). supelco tm 37 component fame mix, sigma was used as an internal standard. statistical analyses: the data were shown as mean±standard deviation. all data were tested for normality (shapiro-wilk test), homogeneity, and independence (levene’s test) to fulfill the anova assumptions (ibm spss version 25.0, 2017). the data were analyzed using one-way analysis of variance (anova) to see the effect of different diets on o. rigida population density, survival rate, and reproductive efficiency. the effects of selected diets on the nutritional compositions (protein, lipid and fatty acids) of o. rigida were compared with artemia. the post-hoc tukey’s test comparisons were carried out, and differences were considered statistically significant at probability levels p<0.05. results the total mean population density of o. rigida nauplii was highest when fed rice bran at 57.33±3.06 ind/ml in the final density (fig. 2). the growth pattern showed that the density of nauplii was higher in copepods-enriched microalgae, nannochloropsis sp. compared to other dietary treatments on day 0th until day 10. however, after 10 days of culture, the density of nauplii was drastically increased in copepods enriched-rice bran compared to other treatments. the abundance of o. rigida in copepodite stages was highest when fed rice bran (26.33±0.58 ind/ml; fig. 3). the growth pattern of copepodite density fed rice bran on day 0 until day 6th was comparable with treatments fed nannochloropsis sp. however, on the 7th day, the copepods enriched-rice bran diet showed drastically rising density compared to other treatments. the population density of adult o. rigida was higher with rice bran as diet (31.00±1.00 ind/ml; fig. 4). however, the concentration of unfed adult o. rigida reduced during the culture period and nearly died on 9th day. the highest total population of o. rigida occurred on day 16th when fed rice bran (77.46±27.93 ind/ml; fig. 5). in contrast, all developmental stages of unfed o. rigida were declined and died on day 9th of the culture period. the specific population growth rate of copepods varied with the different dietary sources in 16 days of the culture period (p<0.05; p = 0.001; table 2). oithona rigida cultured in rice bran diet treatments achieved the highest specific growth rates (0.109±0.002 day−1). diets swiftlet waste soybean meal rice bran nannochloropsis sp. unfed hatching time (days) 2.13±0.12a,b 1.90±0.02b 1.96±0.06b 1.84±0.07b 2.40±0.29 a generation time of nauplii i to vi (days) 4.17±0.03b 4.03±0.25a,b 4.15±0.05a,b 3.79±0.11c 5.35±0.12 c generation time of copepodite i to adult (days) 3.53±0.10a 3.21 ± 0.04a 3.57±0.40a 3.13±0.04a 4.32±0.31b generation time of nauplii i to gravid (days) 10.84±0.08b 9.57 ± 0.07d 9.81±0.03c 9.17±0.05b 13.32±0.14a mean lifespan of copepods (days) 13.00±1.00a 13.33 ± 1.53a 14.00±1.73a 14.67±2.31a 11.67±1.15a no. of spawning /lifespan 9.00±1.73a,b 10.67 ± 1.53a 9.33±0.58a,b 12.00±1.00 a 5.33±2.08b daily offspring/ female 5.60±0.69a,b 6.62 ±0.95a,b 7.53±0.50 b 8.67±0.70a 4.58±0.54b all values are mean ± standard deviation (n = 3). different letters on the same row indicate significant difference (p< 0.05). the parameters in bold letters have shown the best results achieved and fully described in the text. table 3. life table of oithona rigida fed with different dietary regimes. 157 int. j. aquat. biol. (2022) 10(2): 151-168 conversely, unfed o. rigida showed a negative specific population growth rate and almost died after 12 days (-0.084±0.015 day-1). reproductive performance of o. rigida: the hatching time of o. rigida depends on the enrichment diets (p<0.05; p = 0.011; table 3). oithona rigida fed with nannochloropsis sp. (1.84±0.07 days) took the shortest time to hatch their eggs. the diets also influenced the hatching rates of o. rigida (p<0.05; p = 0.001). the highest hatching rate was recorded when o. rigida fed with rice bran (82.00±7.21%) (fig. 6). generation time from nauplii i to nauplii vi in o. rigida fed nannochloropsis sp. had the shortest development time (3.79±0.11 days; table 3). the maturation of copepodite i until the adult phase of o. rigida fed nannochloropsis sp. (3.13±0.04 days) took a shorter time to develop. the generation time from newly hatched nauplii stage i to gravid o. rigida fed nannochloropsis sp. had the shortest development time (9.17±0.05 day). the survival rate of o. rigida from hatching to the adult stage was significantly stimulated by the varieties of nutrients during the cultivation period (p<0.05; p = 0.001; fig. 7). oithona rigida fed rice bran produced the highest survival rate from hatching to adult stage compared to other dietary treatments. the lifespan of o. rigida was not significantly affected by the types of food used (p>0.05; p = 0.098; figure 4. the population density of adults oithona rigida fed with different diets. all values are mean ± standard deviation (n = 3). composition (%) swiftlet waste soybean meal rice bran nannochloropsis sp. protein 5.65±0.99a 31.08±0.69b 24.63±0.51c 53.46±0.94d lipid 3.09±0.71a 11.90±0.45b 22.58±0.72c 17.10±0.79d all values are mean ± standard deviation (n = 3). different letters on the same row indicate significant difference (p<0.05). the nutritional composition in bold letters has shown the best results achieved and fully described in the text. table 4. protein and lipid composition of different diets used for the enrichment. composition (%) oithona rigida artemia swiftlet waste soybean meal rice bran nannochloropsis sp. unenriched unenriched protein 58.22± 1.30a 69.51± 2.7b 66.83±2.25b 72.08±2.02b 56.90±1.6a 50.34± 2.19c lipid 15.34±1.23a,c 16.86±0.63a,c 21.98±1.41b 18.09±1.18a,b 14.45±1.78a 18.90± 1.33bb,c all values are ± standard deviation (n = 3). different letters on the same row indicate significant difference (p<0.05). the nutritional composition in bold letters have shown the best results achieved and fully described in the text. table 5. protein and lipid composition of oithona rigida fed different diets. 158 yuslan et al./ effect of bio-organic fertilizer and agro-industrial residue table 3). a longer lifespan was observed in treatments with nannochloropsis sp. (14.67±0.53 days). oithona rigida had a high spawning rate when fed with nannochloropsis sp. (12.00±1.53), while the lowest spawning was recorded in swiftlet waste (9.00±1.73). the different types of diets significantly affected the number of spawns per lifetime and the total number of offspring per female produced in 16 days (p<0.05; p = 0.003; table 3). the protein and lipid compositions of different feeds used for enrichment: the protein and lipid content from the dietary treatments were recorded to be in the range of 6 to 54% and 4 to 22% of dry weight, respectively (p<0.05; p = 0.001; table 4). however, these variations are more likely reflect variations in the nutritional composition of different diets, such as bio-organic fertilizer, agro-industry residue, and microalgae e.g. nannochloropsis sp. (54.46±0.94%) had the highest protein content, whereas rice bran recorded the highest lipid composition (22.58±0.72%). the protein and lipid compositions of unenriched (o. rigida and artemia) and enriched o. rigida: the protein content in o. rigida was recorded at 56.90±1.61%, and it was higher than the commercial live feed, artemia (50.34±2.19%). the protein and figure 5. the total population density of oithona rigida fed with different diets. all values are mean ± standard deviation (n = 3). figure 6. the egg hatching rate of oithona rigida fed with different diets. all values are mean ± standard deviation (n = 3). different letters on the bar indicate significant difference (p<0.05). 159 int. j. aquat. biol. (2022) 10(2): 151-168 lipid content in o. rigida were influenced by the enrichment given (p<0.05; p = 0.001; table 5). oithona rigida yielded higher protein content when enriched with nannochloropsis sp. (72.08±2.02%) followed by soybean meal, rice bran and swiftlet waste yielded the lowest protein content (58.22±1.31%). the composition of lipid content in all the treatments gradually increased compared to unenriched o. rigida (14.45±1.79%), and artemia (18.90±1.33%). the lipid content was significantly fatty acid (%) oithona rigida artemia swiftlet waste soybean meal rice bran nannochloropsis sp. unenriched unenriched saturated c 12 0.18±0.02 0.54±0.06 1.46±0.55 1.05±0.43 0.16±0.05 0.00±0.00 c 14 6.90±0.08 8.80±0.09 9.44±0.12 9.76±0.15 5.47±0.09 1.63±0.34 c 16 20.24±0.08a 21.48±0.18b 26.44±0.27c 23.54±0.27d 19.40±0.15e 11.67±0.44f c 18 5.52±0.14 7.59±0.21 9.48±0.25 8.48±0.24 4.55±0.37 4.38±0.16 c 24 0.74±0.01 0.85±0.03 1.16±0.02 1.03±0.01 0.63±0.02 0.29±0.07 sum safas 33.5 39.25 47.98 43.86 30.22 18.98 monounsaturated c 16:1 4.87±0.65a 7.61±0.39b 9.89±0.09c 9.31±0.28c 4.61±0.39a 3.06±0.50d c 18:1n9c3 1.38±0.21 2.18±0.82 4.47±0.24 5.46±0.20 1.61±0.39 1.31±0.33 c 20:1n9 0.34±0.01 0.54±0.02 0.98±0.01 0.75±0.01 0.17±0.06 0.54±0.01 c 22:1n93 0.00 0.00 0.00 0.00 0.00 0.00 sum mufas 6.59 10.33 15.34 15.52 6.38 4.90 polyunsaturated c 18:2n6t2 0.44±0.03 0.64±0.03 0.85±0.02 0.54±0.01 0.00 0.00 c 18:2n6c2 0.36±0.01 0.52±0.04 2.54±0.30 1.37±0.12 0.58±0.17 0.00 c 18:3n62 0.39±0.01 0.66±0.01 0.91±0.07 0.75±0.01 0.32±0.00 0.20±0.01 c 18:3n3 (ala)1 0.35±0.06a,c 0.54±0.02a 0.79±0.01b 0.55±0.19a 0.13±0.03c 15.4±30.01d c 20:4n6 (ara)2 0.87±0.02a 1.75±0.05b 2.75±0.06c 3.86±0.08d 0.44±0.22e 1.04±0.03a c 20:5n3 (epa)1 5.52±0.15a 8.42±0.12b 10.53±0.19c 14.620.33d 6.61±0.39e 3.70±0.21f c 22:6n3 (dha)1 3.53±0.34a 6.61±0.39b 9.79±0.25c 7.65±0.11d 2.76±0.21e 2.31±0.22e sum pufas 13.46 19.18 28.16 31.35 10.84 22.68 total fatty acid 53.55 68.76 91.48 90.73 47.44 46.56 a value of 0 indicates that fa was not detected. different letters indicate significant differences between treatments: 1ω-3 fatty acids, 2ω6 fatty acids, and 3ω-9 fatty acids. ala: alpha-linolenic acid; ara: arachidonic acid; epa: eicosapentaenoic acid; dha: docosahexaenoic acid. the nutritional composition in bold letters is described in the results. table 6. the fame analysis of unenriched and enriched live feeds fed with different diets enrichment. all values are mean ± standard deviation (n = 3). different letters on the same row indicate a significant difference (p<0.05). figure 7. the survival rate of oithona rigida fed with different diets. all values are mean ± standard deviation (n = 3). different letters on the bar indicate significant difference (p<0.05). 160 yuslan et al./ effect of bio-organic fertilizer and agro-industrial residue higher in the o. rigida enhanced with rice bran (21.98±1.41%), than nannochloropsis sp. (18.09±1.18%), soybean meal (16.86±1.63%) and swiftlet waste (15.34±1.23%). the fatty acid composition of unenriched (o. rigida and artemia) and enriched o. rigida: fatty acid compositions such as safas, mufas and pufas of enriched o. rigida showed improvement after being supplemented with different dietary treatments (table 6). oithona rigida reached the higher fatty acid content after enriched with rice bran followed by nannochloropsis sp., soybean meal and lower fatty acid content when enriched with swiftlet waste at 91.48, 90.73, 68.76 and 53.55%, respectively. the ara content in o. rigida also varied between all dietary treatments (p<0.05). ara content was found highest in o. rigida enriched with nannochloropsis sp. (3.86±0.08%). on the other hand, epa and dha in o. rigida were significantly impacted by the different dietary treatments (p<0.05; table 6). the highest content of epa was observed in o. rigida enriched with nannochloropsis sp. (14.6±0.33%), whereas the highest dha was recorded in rice bran enrichment (9.79±0.25%). discussion the present study revealed significant variations in population and peak densities at different days of life stages in copepod (nauplii, copepodites and adults). these findings are in accordance with studies conducted by hyder et al. (2014) where a high density of thermocyclops decipiens and mesocyclops aspericornis was attained when chicken manure was used as diet. previous research has shown the intricacies of mass-cultivation of copepods due to the wide varieties of food quality and environmental conditions that are prerequisites for their development and reproduction (evjemo et al., 2003; punnarak et al., 2017). the production of copepods is highly influenced by the biochemical composition of food consumed, which will impact population growth and reproductive ability (khatoon et al., 2013). the highest population density and growth rate of o. rigida were observed in rice bran and microalgaeenriched treatments. the growth rate in both dietary treatments was higher than the previously documented growth rate of cyclopoids copepods such as apocyclops royi (0.096±0.013 day-1) (lee et al., 2006) and tigriopus japonicas (0.08±0.014 day− 1) (lee et al., 2013). this indicates that o. rigida can be grown at high densities as live food for fish and crustaceans’ larvae. furthermore, the use of rice bran as fertiliser has shown increasing the growth and development of copepods such as m. dussarti and t. neglectus and cladocerans such as moina micrura and diaphanosoma excisum (amian et al., 2018). the increase in these zooplanktons' production is closely linked to feeding decomposition, promoting the acceleration and maintenance of zooplankton abundance and survival (amian et al., 2018; rahmati et al., 2020). soybean meal also provides an outstanding result in sustaining copepod culture density and survival rate, owing to the good nutritional content digested by the copepods (paray and al-sadoon, 2016). according to el-khodary et al. (2020), the highest population density was observed in cyclops abyssorum divergens after feeding with soybean diet, and this may be attributable to the desirable food size. hyder et al. (2014) suggested that it is possible to maximise the viability of a mass culture of two freshwater cyclopoid copepods, t. decipiens and m. aspericornis, with a sufficient dosage of organic waste. animal waste such as swiftlet waste could be a potential enrichment diet for high-density mass cultivation of zooplankton culture. it could serve as an efficient, cheap and sustainable food supply to cultivate copepods (kabery et al., 2019). furthermore, manure contains numerous organic matters and bacteria that can be transformed into carbohydrates, proteins, pigments, oils, alcohols, and aldehydes, which could be filtered by zooplankton to promote their growth (ansa and jiya, 2002; a'tirah et al., 2016). the hatching rate is a critical factor for measuring copepod production as these were closely correlated with maternal nutrition as egg quality dictates the progress of hatching (rasdi and qin, 2018). the 161 int. j. aquat. biol. (2022) 10(2): 151-168 results indicated that efficient egg hatching and the quantity of spawns in copepods during a lifetime relied on different dietary treatments. rasdi and qin (2018) and milione and zeng (2007) have published comparable findings on cyclopina kasignete, acartia sinjiensis and bestiolina similis. however, contrary to parvocalanus crassirostris, there is no variation in egg hatching success under various algal treatments (alajmi and zeng, 2015). in short, the productivity achievement of copepods depends on the quality of foods and species cultured (species-specific). therefore, the need to evaluate each copepod species' dietary requirements was emphasized, especially for copepods with the capacity for aquaculture as a live feed. many copepod species are inadequate for largescale mariculture due to certain copepod species' long generation period and limited understanding of their nutritional requirements (conceição et al., 2010). besides, egg production and the maturity of live feed also depend on the consistent feed (ullimaz et al., 2020). the rice bran and nannochloropsis sp. produced more offspring, fast hatching rate, shortest developmental period and longer female lifespan than soybean meal and swiftlet waste. these results were consistent with previous studies, whereby rice bran suspension had accelerated the reproductive cycle by enhancing the m. macrocopa fecundity rate (mubarak et al., 2017; ullimaz et al., 2020). in addition, vitamin b, particularly thiamine and pyridoxine derived from rice bran, might contribute to the rising offspring production (mehdipour et al., 2011). the nutrient feasibility of copepods as feed for aquatic larvae can be manipulated by copepod-fed algal species (molejón and alvarez-lajonchere, 2003; jeyaraj and santhanam, 2013). rajkumar and rahman (2016) have shown that nannochloropsis oculata is a microalgae preferred for a. erythraea and oithona brevicornis, resulting in the best growth result. eltohamy et al. (2021) also declared that microalgae, isochrysis galbana proved to be the best algal diet for the best growth efficiency in the cultured calaniod copepod, gladioferens imparipes. these results were also proportional to the outcomes in microalgae diet treatments reported in the current study. santhanam and perumal (2012b) disclosed that o. rigida cultivated in four dietary algae species has a high reproductive ability. alajmi and zeng (2015) have suggested using microalgae tisochrysis lutea as an effective diet to achieve a greater number of progeny and a faster time of growth for the paracalanid copepod, p. crassirostris. isochrysis sp. and a. bilobata (lee et al., 2006; pan et al., 2014). tisochrysis lutea was also a superior food to increase cyclopina kasignete offspring production (rasdi and qin, 2018). however, as pointed out by payne and rippingale (2001), the hardness and indigestion of its cell wall was the key explanation for the weak success of nannochloropsis sp. in copepod cultivation compared to other species of microalgae, which was also observed when fed to artemia. the growth, fertilization, maturity and longevity may be enhanced by exploiting feed ingredients and their nutrients (wacker and creuzburg, 2007; mehdipour et al., 2011; nagaraju, 2011). our results showed that all the dietary treatments used could promote the growth and development of o. rigida. nevertheless, rice bran and nannochloropsis sp. were highlighted to contribute to a successful cyclopoid copepod-feeding regime for o. rigida. friedman (2013) explored rice bran as food to enhance the nutritional efficiency of aquatic animals as a live feed enhancement to improve fish and shellfish larvae development and survival, which indirectly contributed to the food quality management. faria et al. (2012), abbas et al. (2011), and mubarak et al. (2017) have concluded that rice bran is ideal for live feed cultivation that provides several nutrients and energy sources, including protein (12-13%) and lipid (16-20%). the results of nutritional properties of rice bran reported by choi et al. (2011) is approximately is in agreement with the amount obtained, which was a protein of 12-21% and lipid of 4-20%. the levels of protein and lipid in nannochloropsis sp. also recorded in the range of the previous research documented (5055%) protein and (24-27%) lipids (hulatt et al., 2017; tibbetts et al., 2017). the protein and lipid content in 162 yuslan et al./ effect of bio-organic fertilizer and agro-industrial residue unenriched o. rigida rise with the increasing nutrient elements in the dietary treatments. the quality of dietary enrichments, such as protein and lipid, and the amount of the diets, promote the production of copepod eggs and influence the population size with regards to the copepod diet manipulation (mitra and flynn, 2005; gusmão and mckinnon, 2009; jónasdóttir et al., 2009; finiguerra et al., 2013; manklinniam et al., 2018). a report by mubarak et al. (2017) resulted in a high rna/dna ratio and protein concentration in live feed culture with rice bran suspension, specifically in m. macrocopa. protein and lipid nutritional contents in rice bran were improved due to the catabolic bacterial interaction or break down of the feed's complex components such as complex proteins into amino acids to produce enzyme protease that utilized by microbes to increase the crude protein content indirectly and provide better digestion of zooplankton during cultivation (damayanti et al., 2020). therefore, rice bran is considered the best option for feed culture, which increased population fertility, broodstock percentage, and biomass of m. macrocopa (mubarak et al., 2017). el-khodary et al. (2020) found that the most remarkable differences in total protein and lipids in c. abyssorum divergens were obtained when enriched with tetraselmis chuii than soybean meal. the result was supported by the fact that protein providing the body with the energy required for the growth, enzyme metabolism, hormones and antibodies (el-khodary et al., 2020). the present works’ findings regarding the nutritional composition of the nannochloropsis sp. diet were in line with elkhodary et al. (2020) and macias-sancho et al. (2014), who stated that microalgae have high protein and amino acid profiles relative to other food proteins. a study by buttino et al. (2009) showed some of the critical issues in copepod mass cultivation where different copepod species have different dietary requirements in maintaining high populations. copepod typically relies on live microalgae and yeast in most mass culture conditions (zaleha et al., 2012; rasdi and qin, 2016). however, copepods fed yeast resulted in low pufas content relative to fresh algae, which indicates the unfavorable diet for copepods (hazel et al., 2011). in the current study, o. rigida was given different dietary ingredients from different sources equipped with sufficient protein, lipid, and fatty acids for the diet consistency evaluation. as a result, the total fatty acid content of o. rigida observed from the experiment was higher than artemia nauplii. similarly, the same findings were reported by santhanam and perumal's (2012b) and rasdi et al. (2015). different types of food used significantly impacted the concentrations of fatty acid in o. rigida pufas such as ala, ara, epa, and dha in copepod were essential and influenced by their feed (chen and chen, 2012). pufas are a vital nutrient implicated in many important marine copepod production and performance, such as growth and reproduction (nielsen et al., 2021). kabery et al. (2019) also revealed that safa and pufa content of live feed fed different diets such as poultry manure and food waste would result in variance outcomes. interestingly, the epa and dha in copepod can be synthesized from ala as documented in other copepod species such as tachidius discipes and tisbe sp. (caramujo et al., 2008). furthermore, rasdi and qin (2016) suggested that the cyclopoid copepod c. kasignete can convert ala to epa and dha when fed green marine microalgae, similar to the o. rigida in the current study. the results showed that o. rigida was better supplemented with rice bran and nannochloropsis sp. than other diets. nevertheless, the challenge of sustaining a sufficient supply due to the algal culture collapse is another problem (mostary et al., 2007). a good diet for copepods is an essential way that nutrient can be transferred to the larvae, which indirectly increases survival and development. therefore, there is a need to consider further use of these diet types in the cultivation of copepod. conclusions the findings of this work will contribute crucial information on copepods nutrition and valuable knowledge to guide management, technique and protocols for the effective development of copepods 163 int. j. aquat. biol. (2022) 10(2): 151-168 as live food for fish and crustacean larvae. the growth and life history parameters of o. rigida were pretentious by different feeding used in this study. oithona rigida fed or enriched with inadequate nutrients resulted in slower developmental performance. on the contrary, o. rigida provided good nutrient content such as high lipid and protein content successfully improving their growth and reproductive performance. the outcomes from this research indicated that all the diet types used could enhance the production of copepod. however, rice bran and nannochloropsis sp. were the recommended diet that facilitates the growth and reproduction in copepods either in a single or a combined diet. in addition, rice bran as a substitution for microalgae can maintain copepod growth and reproduction to overcome the shortage of microalgae supply in copepod culture acknowledgments the work was funded by long term research grant scheme (lrgs) issued under grant lrgs/1/2020/umt/01/1/1 (vote no. 56040) and strategic research grant (srg) with vote no. 55196 by the malaysian ministry of higher education to generate new ideas and methodologies for the adaptation of organism under adverse environmental condition. thank you to the universiti malaysia terengganu (umt), malaysia, for facilitation and accommodation providence. lastly, we would like to thank all umt’s staff and lab assistances that have contributed to helping us in this study. references abbas a., murtaza s., aslam f., khawar a., rafique s., naheed s. 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(2016) 4(2): 102-107doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology review article review of the freshwater eels of iran (family anguillidae) brian w. coad1 canadian museum of nature, ottawa, ontario, k1p 6p4 canada. article history: received 17 january 2016 accepted 1 april 2016 available online 2 5 april 2016 keywords: biology, morphology, anguilla. abstract: the systematics, morphology, distribution, biology, economic importance and conservation of the freshwater eel (anguilla anguilla) of iran are described, the species is illustrated, and a bibliography on this fish in iran is provided. the species is found in the caspian sea basin as an exotic. introduction the freshwater ichthyofauna of iran comprises a diverse set of families and species. these form important elements of the aquatic ecosystem and a number of species are of commercial or other significance. the literature on these fishes is widely scattered, both in time and place. summaries of the morphology and biology of these species were given in a website (www.briancoad.com) which is updated here, while the relevant section of that website is now closed down. family anguillidae freshwater eels are found world-wide in temperate to tropical waters except for the south atlantic ocean and the whole eastern pacific ocean. there are about 20 species in one genus (eschmeyer and fong, 2011) with one found in iran. the family is characterised by the elongate body, numerous vertebrae, small elliptical scales which are difficult to see casually, a small and elliptical gill opening just in front of the pectoral fin base, very long dorsal and anal fins confluent with a reduced caudal fin, a terminal mouth with the lower jaw projecting a little, small teeth in several rows on the jaws and palate, the dorsal fin origin well behind the pectoral fin level but in front of the anus level, no * corresponding author: brian w. coad e-mail address: bcoad@mus-nature.ca pelvic fins, and by a suite of osteological characters. the term eel-like is based on the body shape of freshwater eels and includes the muscular slipperiness associated with this fish and its mucusproducing skin. the life cycle of atlantic eels was unknown until johannes schmidt published his 1922 study based on years of collecting. where the adults went on their seaward migration and where the elvers ascending rivers came from were a mystery. these eels are catadromous, living in fresh water but migrating to the sea to spawn and die. in the north atlantic ocean spawning occurs in the sargasso sea. the young eels or leptocephali (= thin head larvae) are distinctive, being transparent and leaf-like. a newspaper can be read through the body of a leptocephalus. in this form they drift to the shores of america and europe, transform into elvers with the more familiar eelshape and move into rivers and lakes to feed and grow. some scientists believe that the european eel is not a distinct species but merely american eels (anguilla rostrata (le sueur, 1817)) which develop in cooler areas of the sargasso sea and are carried by different ocean currents to the shores of europe. differences between the american and european eels overlap and include such characters as vertebral number which is known to vary with development 103 int. j. aquat. biol. (2016) 4(2): 102-107 temperature. recent studies using mitochondrial dna (mtdna) showed no genetic divergence among samples of american eels along 4000 km of north american coastline reflecting a single spawning population. however, european eels had a distinct mtdna genotype and the conclusion to be drawn is that american and european eels have separate spawning sites such that larval dispersal ends up on different continents. the mtdna differences are marked but do not prove species distinction as this level of distinction is known to occur among fishes which are a single species (though some authorities would argue that these "single" species are themselves complexes of two or more species). however, icelandic eels seem to be hybrids between the two putative species. all other evidence (vertebral and other counts, body proportions, biology, and electrophoresis) suggests that the american and european eels are the same species but have different spawning sites. the biology of eels is based almost entirely on the freshwater phase of their life. adults in fresh water develop large eyes, the gut degenerates and coloration changes in preparation for the migration to the sargasso sea. adults were only caught in the deep ocean, at nearly 2000 m near the bahamas, in 1977. the sargasso spawning ground is deduced from collections of larvae across the atlantic ocean the smallest and youngest larvae are found around the sargasso sea. the spawning grounds are at about figure 1. line drawing of anguilla anguilla by s. laurie-bourque. figure 2. anguilla anguilla from wikimedia commons. 104 coad/ freshwater eels of iran 400 m, at a 17°c temperature and in saltier water than usual sea conditions according to some authors but since spawning adults have never been caught this remains dubious. the theory advanced by d.w. tucker in 1959 maintained that european eels lack the energy resources in their migratory, spawning phase to reach the sargasso sea 7000 km from europe. they are presumed to be following an instinct to head out to sea, dating from an earlier geological age when the atlantic ocean was narrower before the separation caused by continental drift. all european eels die at sea and europe is restocked by larvae drifting there spawned from american parents. the american populations are closer to the sargasso and can make the journey easily. differences between american and european eels are merely the consequence of different environmental regimes in different parts of the sargasso. this theory has not found general acceptance but, if true, means that all european eels can be harvested for food without depleting stocks. eels are valued as food, particularly in europe and japan. hochleithner (2010) gives a review of eel biology and aquaculture. genus anguilla schrank, 1798 characters of the family also serve for the genus. anguilla anguilla (linnaeus, 1758) (figs. 1-2) common names: marmahi-ye ma'muli (= common snake fish), marmahi mohajer, meaning migrating snakefish), marmahi-ye haghighi. [rechnoi ugor' or river eel in russian; european eel]. systematics: no major synonyms. muraena anguilla was originally described from europe. key characters: the eel shape is characteristic along with the long and spineless dorsal and anal fins and the absence of pelvic fins. the caspian lamprey, caspiomyzon wagneri, has a similar shape but lacks pelvic fins, has seven gill openings in a row behind the eye, and has a round suctorial mouth. morphology: the scales are small, elliptical in shape and embedded in the skin. the lateral line is distinct. some fish in any population may have a broad or a narrow head. fish approaching sexual maturity develop very large eyes, the olfactory organs atrophy, the lateral line becomes more conspicuous, a tougher and thicker skin develops, and the colour changes as detailed below. dorsal fin rays 243-275, anal fin rays 175-249 and pectoral fin rays 15-21. vertebrae number 110-120, usually 114-116. the chromosomes are 2n=38 (klinkhardt et al., 1995). the leptocephalus and elver stages are not found in iranian waters and are not described here (see below under reproduction). sexual dimorphism: at the silver eel stage males are 29-40 cm and females 38-130 cm long. male adults are smaller than females. colour: colour is variable but the back is usually grey-brown, olive-brown, brownish-green, yellowish or black and the belly is whitish to yellowish. the dorsal fin is dark, other fins are yellowish. the iris is yellow. this yellow or green eel stage changes to the silver or bronze eel at maturity. the mature fish is darker on the back, has silvery or bronze to coppery flanks and belly, a black pectoral fin and a clear contrasting black lateral line, as well as enlarged eyes. size: attains 2.0 m, but rarely, and 12.7 kg, possibly 14.0 kg. iranian specimens up to 1.0 m long have been caught near bandar-e anzali (firouz, 2005). distribution: common in europe including the mediterranean sea, and east to the black sea although few young eels migrate naturally as far as this. occasionally caught in iranian waters (p. walczak, pers. comm. 1978; holcík and razavi, 1992). holčík and oláh (1992) report single specimens from the anzali mordab (= talab) and its exit streams and near bandar-e anzali. also reported generally from the southeast caspian sea, southwest caspian sea and south-central caspian sea (kiabi et al., 1999). reported from the safid river and anzali talab by abbasi et al. (1999), and also from the sheikan river. berra (2001) does not show the iranian distribution because the fish are introduced. zoogeography: an exotic species in iranian waters, 105 int. j. aquat. biol. (2016) 4(2): 102-107 arriving there through the influence of mankind (esmaeili et al., 2014, 2014). this species established itself in the caspian sea after the opening of the volga-baltic waterway and the introduction of larvae from france and england and was recorded from fresh waters in azerbaijan by abdurakhmanov and kuliyev (1968). habitat: eels are caught by fishermen between bandar-e anzali and the mouth of the safid river in beach seines, in the anzali mordab and are probably present in rivers along the caspian coast. the catch appears to be increasing (holčík and razavi, 1992). about 10-40 specimens are caught annually weighing up to 3 kg (holčík and oláh, 1992). in europe freshwater populations show considerable migratory movements in summer and this helps explain their widening distribution in the caspian sea basin. however, abbasi (2005) states that the population has decreased. eels will live in almost any kind of water over a wide range of temperatures (0-30°c at least); warmer waters being preferred as long as oxygen is not low. they are found at depths of 0-1000 m. they are found in small streams, large rivers, lakes and coastal waters, all connected to the sea for natural populations. elvers flourish in sandy areas where grain size is 0.25 mm or in gravel areas where size is 2 mm or larger, the former for burrowing, the latter for insinuating between. adults also prefer a substrate that can be burrowed into during the day, emerging at night. the burrows are usually at a 45° angle and the eel sticks its head out at this angle too. eels show some migratory habits within fresh water, moving between summer and winter areas, over a distance of a few metres to tens of kilometres. in europe, eels in the silver eel stage begin to migrate to the sea in late summer and autumn on their journey to the sargasso sea where they arrive the following spring. they travel at about 2 km/hour, particularly at night when the moon is at or a few days after the last quarter and light levels are low. iranian fish cannot migrate, being constrained by distance and lack of ready access to the open ocean. age and growth: eels generally begin to mature only at sizes above 30 cm long. females grow much larger than males and usually begin to mature at 54 cm or longer. maturity is actually attained after leaving european waters en route to the sargasso sea. eel larvae do not all metamorphose at the same age (this can vary from 1 to 6 years), with subsequent effects on age at the same length. in addition, growth varies widely with the habitat and available food supply. fish of the same length often have very different weights. average life span is usually 15-20 years. life span is up to a reputed 88 years based on a captive specimen. food: eels are principally nocturnal but feed both at night and during the day. food includes almost any edible item and includes fish spawn, small fishes, and larger dead fish which have a mouthful of flesh torn off by a rapid rotation along the long axis of the eel body. food includes insect larvae and algae but fishes, worms, crustaceans and molluscs are the most important items in order. in the southern caspian, they have been reported to eat gobies (gobiidae) and rutilus sp. in november, suggesting that feeding continues late in the year in contrast to other waters where they dig into sand or silt and hibernate (abdurakhmanov and kuliyev, 1968). eels will lie buried in mud or gravel with just the head projecting, seizing by a sudden strike any food item passing by. eels will feed on commercially important species such as salmonids and crayfishes. they are reliably reported to even leave the water and enter fields, presumably to feed on slugs and worms. reproduction: this has not been observed in the wild but under artificial conditions eels are promiscuous and fertilisation is external. the eel is catadromous and is believed to spawn in the sargasso sea at 100200 m depths off the coast of america after a long migration from europe. adults die after spawning. spawning takes place at the beginning of march. mature females contain 3 million eggs per 1 kg body weight. the ovary is a rosy-pink because of numerous blood vessels. the pelagic eggs are 1.2 mm in diameter. the eggs develop into a distinctive leptocephalus larva which has a leaf-like shape quite unlike the adult eel. during its leptocephalus phase, 106 coad/ freshwater eels of iran the eel drifts on ocean currents and actively swims from the american side of the atlantic, arriving in europe in its third summer. it is now fully grown and 7.5 cm long. the larva gradually transforms into the elver at depths of 1000 m off the coast of europe. the elver is eel-shaped and transparent and reduces in length and weight during the autumn when it does not feed. the elvers begin to migrate into rivers and lakes in europe in winter. they are regarded as young eels once they begin to feed and are fully pigmented. the transition from a yellow to a silver eel stage lasts 6-12 years in males and 9-20 years in females. parasites and predators: there is a heavy toll on elvers which are taken on the migration into rivers and lakes by a wide variety of fish and birds. adults are eaten by large fishes including larger eels and by birds such as herons and cormorants. a large variety of parasites have been reported from eels. economic importance: not used in iran for food, probably because its minute scales make it appear scaleless, and in any case the annual catch is only about 40-60 specimens (holčík and razavi, 1992). it is of considerable economic importance in europe where annual catches have reached 22,000 tonnes. the 1981 catch in turkey, for example, was 374 tonnes. this species is also farmed quite extensively. the flesh has a high fat content and the eel is often smoked for sale. the blood of this fish is poisonous but the poison is destroyed by cooking. fresh eel blood should never be ingested. a dog injected with eel serum died within one minute. symptoms include diarrhoea, bloody stools, nausea, vomiting, frothing at the mouth, skin eruptions, cyanosis, apathy, irregular pulse, weakness, numbness, paralysis, respiratory distress, and death. severe inflammations will result if the blood touches the eye or tongue. robins et al. (1991) list this species as important to north americans. importance is based on its use in aquaculture and aquaria, as food, for sport and in textbooks conservation: the peculiar migratory behaviour of this species prevents spawning in iranian waters and all stocks must be replenished through migration from european waters or by artificial introductions. as a non-reproducing, exotic species, no conservation measures are required. critically endangered in turkey (fricke et al., 2007) and throughout its range (iucn, 2014) with a suite of reasons for this status, e.g., barriers to upstream and downstream migration caused by dams and mortality by hydropower turbines, pollution, etc. sources: there is little information on this species in iran because of its scarcity and general biology is taken from bertin (1956), tesch (1973), sinha and jones (1975), deelder (1984), hoestlandt (1991) and iucn (2014). these works should be consulted for the extensive data on biology and economic importance of this fish. comparative material: cmnfi 1983-0359, 3, 63.6180.9 mm total length, italy, rio porra at finale ligure (no other locality data); cmnfi 1986-0458, 2, 287.5-434.0 mm total length, germany, danube river at frengkofen (48º58'n, 12º18'e); cmnfi 1986-0462, 1, 209.9 mm total length, germany, vils river at mettenhausen (48º32'n, 12º50'e).. references abbasi k. (2005). studying alien fishes and macrocrustaceans distribution and their effects on rivers and wetlands of the iranian basin of caspian sea. borok ii, international workshop, invasions of alien species in holarctic, 27 september-1 october 2005, borok (abstract). abbasi k., valipour a., talebi haghighi d., sarpanah a., nezami s. (1999). atlas of iranian fishes. gilan inland waters. gilan fisheries research centre, rasht. 113 p. (in farsi) abdurakhmanov yu.i., kuliyev z.m. (1968). european eel in azerbaidzhan waters. problems of ichthyology, 8: 592-594. berra t.m. (2001). freshwater fish distribution. academic press, san diego. 604 p. bertin l. (1956). eels. a biological study. cleaver-hune, london. 192 p. deelder c.l. (1984). synopsis of biological data on the eel anguilla anguilla (linnaeus, 1758). food and agriculture organization, rome, fisheries synopsis 80, revision 1: 73 p. 107 int. j. aquat. biol. (2016) 4(2): 102-107 eschmeyer w.n., fong j.d. (2011). pisces. in: z.q. zhang (ed.). animal biodiversity: an outline of higher level classification and survey of taxonomic richness. zootaxa, 3148: 26-38. esmaeili h.r., coad b.w., mehraban h.r., masoudi m., khaefi r., abbasi k., mostafavi h., vatandsoust s. (2014). an updated checklist of fishes of the caspian sea basin of iran with a note on their zoogeography. iranian journal of ichthyology, 1(3): 152-184. esmaeili h.r., teimori a., owfi f., abbasi k., coad b.w. (2014). alien and invasive freshwater fish species in iran: diversity, environmental impacts and management. iranian journal of ichthyology, 1(2): 6172. firouz e. (2005). the complete fauna of iran. i.b. tauris, london. 322 p. fricke r., bilecenoglu m., sari h.m. (2007). annotated checklist of fish and lamprey species (gnathostomata and petromyzontomorphi) of turkey, including a red list of threatened and declining species. stuttgarter beiträge zur naturkunde, serie a (biologie), 706: 169 p. hochleithner m. (2010). aale (anguillidae) biologie und kultur. aquatech publications, kitzbühel. 156 p. hoestlandt h. (1991). the freshwater fishes of europe. volume 2. clupeidae, anguillidae. aula-verlag, wiesbaden. 448 p. holčík j., oláh j. (1992). fish, fisheries and water quality in anzali lagoon and its watershed. report prepared for the project anzali lagoon productivity and fish stock investigations. food and agriculture organization, rome, fi:undp/ira/88/001 field document. 109 p. holčík j., razavi b.a. (1992). on some new or little known freshwater fishes from the iranian coast of the caspian sea. folia zoologica, 41(3): 271-280. iucn. (2014). the iucn red list of threatened species. international union for the conservation of nature, gland, switzerland (http://www.iucnredlist.org/). kiabi b.h., abdoli a., naderi m. (1999). status of the fish fauna in the south caspian basin of iran. zoology in the middle east, 18: 57-65. klinkhardt m., tesche m., greven h. (1995). database of fish chromosomes. westarp wissenschaften, magdeburg. 237 p. robins c.r., bailey r.m., bond c.e., brooker j.r., lachner e.a., lea r.n., scott w.b. (1991). world fishes important to north americans exclusive of species from the continental waters of the united states and canada. american fisheries society special publication. 243 p. schmidt j. (1922). the breeding places of the eel. philosophical transactions of the royal society, b211: 179-208. sinha v.r.p., jones j.w. (1975). the european freshwater eel. liverpool university press, liverpool. 146 p. tesch f.-w. (1973). der aal. biologie und fischerei. verlag paul parey, hamburg. 306 p. tucker d.w. (1959). new solution to the atlantic eel problem. nature, 183(4660): 495-501. int. j. aquat. biol. (2016) 4(4): 295-300doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article histological and allometric growth analysis of eye in caspian kutum, rutilus kutum kamensky, 1901 (teleostei: cyprinidae) during early developmental stages shaghayegh hasanpour1, soheil eagderi*1, seyed valli hosseini1, mohamad hasan jafari sayadi2 1department of fisheries, faculty of natural resources, university of tehran, p.o. box: 4111, karaj, iran. 2department of agriculture and natural resources, university of payam noor, karaj, iran. article history: received 7 april 2016 accepted 22 june 2016 available online 2 5 august 2016 keywords: vision retina ontogeny growth pattern abstract: fish larvae have several sensory systems that are functional at or soon after hatching and then are developed further during larval and juvenile stages. this study was conducted to investigate development of the eye in rutilus kutum, based on histological and allometric growth analysis during early developmental stages up to 35 day post hatching with emphasis on retinal morphology. for this purpose, the histological sections were prepared and allometric growth pattern of the eye was calculated. the results showed that the most eye’s structures along with the retina of the newly hatched larvae, as the inner sensory (photosensitive) tissue were completely differentiated. allometric growth pattern of the eye diameter up to the inflexion point (7 dph) was somewhat positive and then it became negative. the results revealed that the caspian kutum is dependence on visual capability as visual feeder during their larval period which itself explains completion of eye structures and the high growth rate of eye before 3 dph i.e. beginning of mixed feeding. introduction fish larvae have several sensory systems that are functional at or soon after hatching and then are developed further during larval and juvenile stages (wahl et al., 1993; hall and wake, 1999; loosey et al., 2000). the feeding habits of fishes are reflected on the structure and size of the sense organs particularly the eyes (atta, 2013). eyes are among the major sensory organs in fishes for detecting photic stimuli and forming images of the environment (chai et al., 2006). fish visual capability is highly related to the eye structure, therefore study of the eye structure and its retinal morphology can provide insight to the visual capability of fish (lim et al., 2014). in addition, the development of the functional eye is correlated with the feeding habits of fishes for instance distinct changes in the retinal morphology occur concomitant with a shift from pelagic to a benthic habitat (hall and wake, 1999). different growth rate of various parts of the body * corresponding author: soheil eagderi e-mail address: soheil.eagderi@ut.ac.ir or allometric growth is a common phenomenon during early development of fishes (osse and van den boogart, 1995), which it is responsible for a progressive transformation of recently hatched specimen from a larval body shape to juvenile or adult form in a relatively short time (khemis et al., 2013). hence, understanding normal growth pattern and morphological changes are crucial to reduction of the hatchery losses (khmis et al., 2013; pena and dumas, 2009). caspian kutum, rutilus kutum is an important commercial and edible fish in the southern caspian sea distributed from the southwest (the atrak river) to northward (the volga river) (abdolhay et al., 2011). due to over fishing and deterioration of its spawning grounds and natural habitats, this species has experienced a dramatic reduction in its fishing yields. therefore, its artificial propagation in hatcheries was established to recruit its natural stocks by releasing its fingerlings into rivers that drain to the caspian sea basin (jafari et al., 2010). 296 hasanpour et al./ histological and allometric growth analysis of caspian kutum during early development since in restocking programs, providing basic biological information is crucial for breeding and rearing of larvae; therefore, this study was conducted to investigate the development of the eye in the caspian kutum based on histological and allometric growth analysis during early developmental stages from hatching up to 35 day post hatching (dph) with emphasis on retinal morphology. its ontogeny will provide insight on the visual capability of this species and can help to optimize its larval culture conditions. materials and methods specimens rearing and sampling: larval specimens were obtained from artificial propagation of 15 female and 30 male broodstocks in april-may 2012, in dr. yousef-pour fish hatchery center (siahkal, guilan, iran). the eggs were incubated in 10 l vase incubators with flow-through system at 22°c. after six days of incubation, eggs were hatched and transferred to a large larval collector tanks (200 l). after 3 days, at the beginning of exogenous feeding, 30,000 larvae were transferred to an earthen pond (0.1 ha) with a flow-through system with a mean temperature, ph and dissolved oxygen of 25±2.2°c, 8.1±0.5 and 7.4±1.1 mg l-1, respectively. the natural water flow provided some natural prey but additional artificial feed was supplied from 7 dph. the feed was a specialized feed for caspian kutum larvae and juveniles based on a mix of protein and cereal meals. larval specimens were randomly sampled from 1-20 dph every days and then every 5 days up to 35 dph (n=30) from the same larval batch prior to feeding, in the morning. allometric analysis: the samples were sacrificed by overdose of ms222 (sigma-aldrich), weighed to the nearest 0.0001 mg and fixed in 5% phosphate buffered formaldehyde solution. the left side of the fresh larvae, aged 1-12 dph, were photographed using a dissecting microscope equipped with a cannon camera (5mp resolution) and the older specimens were photographed using a copy-stand equipped to the camera. total length (tl: from tip of the snout to the end of the caudal fin) and eye diameter were measured from the digital images to the nearest 0.01 mm using the imagej software (version 1.240). tl was measured as the reference point in the description of the ontogeny because it is a proper measure of ontogenetic state than age (hasanpour et al., 2015, 2016; saka et al., 2008; sfakianakis et al., 2004, 2005). allometric growth pattern was calculated as a power function of tl using non-transformed data: y=αxb. where y is the dependent variable, x: the independent variable (tl), 𝛼 the intercept and b the growth coefficient. allometric growth pattern is considered as positive, when b is larger than the isometric value (b=1) and as negative when b>1 (gisbert, 1999). the inflexion point of growth curve was determined according to van sink et al (1997). both preparation of the plots and data analysis were performed using manitab (version 16), past and microsoft excel (version 2013) softwares. histological analysis: six fixed specimens per sampling day were randomly selected and subsequently dehydrated in a graded series of ethanol (70-100%) and cleared with xylene and finally embedded into paraffin. the histological sections were prepared with 6 µm thickness, mounted on the glass slides and stained with hematoxylin and eosin (hewitson and darby, 2010; eagderi et al., 2013). the sections were examined under a light microscope and photographed by a nicon camera (13mp resolution). results at 1 dph, the eye was developed and had almost similar structures as adults showing importance of this organ during eleuthero-embryonic stage. in addition, the retina was completely differentiated as inner sensory (photosensitive) tissue of eye at this stage. the pigment epithelium layer of the retina as a non-nervous area, was thin. the inner nervous area of the retina is composed of 9 layers, including (definitions are according to atta, 2013): (1) photoreceptor cell layer (ph), (2) outer or the external limiting membrane (om), (3) outer nuclear layer (on) representing the nuclei of the 297 int. j. aquat. biol. (2016) 4(4): 295-300 photoreceptor cells, (4) outer plexiform layer (op) i.e. the location of synaptic relationship between photoreceptor, bipolar, amacrine and horizontal cells as well as mullers cells, (5) inner nuclear layer (in) contains the nuclei of several types of neurons mainly of bipolar, amacrine and horizontal cells as well as mullers cells, (6) inner plexiform layer (ip), the location of synaptic relationship between the bipolar and ganglionic cells, (7) ganglionic layer (g) that is composed of a narrow chain of granular and spherical cells surrounded by a fine connective tissue network, (8) nerve fiber layer (n) represents axons of the ganglionic layer, and (9) inner limiting membrane (im) (fig. 1). in addition, the diameter of the inner nuclear layer was significant compared to the ganglionic layer. the median uveal layer had not been completely developed at 1 dph. the lens as an avascular spherical ball was made up of 4 tissue layers, including an extra cellular matrix (capsule), a monolayer of nucleated flattened or cuboidal cells capable of division and secretion, hyaline layer, and fourth layer consisting long, slender, transparent, non-nucleated fibrous cells that are arranged as parallel rows (fig. 1). at 2 dph, the blood vessels of the rete choroid was strongly developed to support the retina. there was no significant structural changes in the eye expect their size from 3 dph onward that is coincided with starting mixed feeding, i.e. development of the retina and choriocapillary layer is completed before exogenous feeding. after 3 dph, the only noticeable changes in the retina were increasing some layers’ diameter and the density of the rod, cone and figure 1. eye development of rutilus kutum. a-b: 1-dph, c: 2-dph, d-e: 3-dph, f: 4dph, g-h: 5-dph, j: 35-dph. n: nerve fiber layer, g: ganglionic layer, ip: inner plexiform layer, in: inner nucleus layer, op: outer plexiform layer, on: outer nucleus layer, om: outer membrane, ph: photoreceptor cell layer, pe: pigment epithelium, bm: brush membrane, cl: choriocapillar layer, l: lens, i: iris, and c: cornea (scale bar = 100 µm). 298 hasanpour et al./ histological and allometric growth analysis of caspian kutum during early development ganglion cells. during this period, the pigment epithelium was gradually thickened. the eye was externally unpigmented at hatching and pigmented at the beginning of the mixed feeding stage (3 dph). allometric growth pattern of the eye diameter up to the inflexion point (7 dph, tl=12.48±0.67) was somewhat positive (b = 1.05, r2=0.59), and it became negative after this point (b=0.79, r2=0.95) (fig. 2). discussion the eye determines the ability to feed, search, distinguish objects and orient in a three dimensional light environment (chai et al., 2006). therefore, major events in the functional ontogeny of the visual system are closely correlated with life history events where fish experiences changes in the photic environment due to a change in vertical or horizontal position or changes in behavioral repertoire (blaxter and stains, 1970; hall and wake, 1999). several studies found that many fishes possess only cone photoreceptor cells at the onset of exogenous feeding, as the larvae live near the surface of the water where sun light penetrates (blaxter and stains, 1970; hall and wake, 1999; lenkowski and raymond, 2014). the appearance of rod photoreceptor cells in the retina delay until the larvae move to deeper water (chai et al., 2006; ebbessen et al., 2007; hall and wake, 1999; lenkowski and raymond, 2014). at hatching, the retina of the caspian roach larvae had composed of welldifferentiated photoreceptor cells in contrast to many fish species (chai et al., 2006), showing their dependence on visual capability as visual feeder during early development. prior to initiation of exogenous feeding at 3-5 dph, whole layers of the retina were present in the caspian roach larvae, illustrating the importance of visual sense for its exogenous feeding. in addition, the complete development of the choriocapillar layer, which figure 2. the allometric growth pattern of the eye of rutilus kutum (the dashed line represents the inflexion points of growth). 299 int. j. aquat. biol. (2016) 4(4): 295-300 supply the high oxygen demand of the eye, up to 3 dph was in accordance with formation of other structures of the eye in this species. increasing of the rod photoreceptor cells of in older caspian roach specimens i.e. about 35 dph shows switching its habitat preference to deep water like many bottom feeders e.g. yellow perch (perca flavescens) (whal et al., 1993). allometric growth pattern of the eye diameter up to the inflexion point was somewhat positive, and it became negative after this point i.e. the most of the ontogenesis and differentiation of the eye structure had been completed before 3 dph. larger eye diameter commonly accommodates the larger eye lens. therefore, more light can be gathered and a higher resolution image can be generated in the brain. the greater eye size also provides the better visual sensitivity to the fish especially under dim light conditions with its better light gather feature (lim et al., 2014). in addition, changes in allometry of morphometric characters are hypothesized to be related to many functions such as predator avoidance and feeding (yúfera and darias, 2007). after exhaustion of the yolk-sac, larvae need to shift from endogenous to exogenous feeding, as a result, the positive allometry of those structures involved in exogenous feeding i.e. eye is predicted. positive allometry of eye diameter in early larvae (0-7 dph) confirmed this hypothesis and its critical role in feeding, prey detection, schooling behavior and predator avoidance (rodríguez and gisbert, 2001; 2002). our results shows that the caspian kutum is mainly eye-dependent during their larval period which itself explains completion of eye structure particularly the retina and the high growth rate of eye before starting mixed feeding. acknowledgments this study was financially supported by the university of tehran. references abdolhay h.a., daud s.k., rezvani ghilkolahi s., pourkazemi m., siraj s.s., abdul satar m.k. 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(2015) 3(5): 314-322 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article combined mitochondrial dna analysis of the mesopotamian spiny eel, mastacembelus mastacembelus (banks & solander 1794), and its phylogenetic position esen tutar*1 department of bioengineering and sciences, graduate school of natural and applied sciences, kahramanmaraş sütçü i̇mam university, kahramanmaraş, turkey. article history: received 7 april 2015 accepted 4 september 2015 available online 2 5 october 2015 keywords: mastacembelus 16s rrna 12s rrna trnaphe trnaval phylogenetic relationships abstract: nucleotide sequences of the 12s rrna, 16s rrna, trnaphe and trnaval genes of mtdna of mesopotamian spiny eel, m. mastacembelus, was determined for the first time. the comparison of the three populations of mesopotamian spiny eel from turkish part of the tigris basin based on the combined mitochondrial dna was performed. based on the results, no differences were determined and the identity found to be 100% among three populations. furthermore, the obtained results from molecular methods were compared with morphological findings to validate the position of the studied populations of m. mastacembelus. in addition, the phylogenetic position of the mesopotamian spiny eel was examined among the mastacembelidae and synbranchioformes based on 12s rrna and 16s rrna. the constructed phylogenetic relationship between m. mastacembelus and some other members of synbranchioformes order supported their taxonomic hierarchy. introduction the order synbranchioformes includes 120 species in three families, including chaudhuridae (10 species), synbranchidae (23 species) and mastacembelidae (87 species) (froose and pauly, 2014). the members of the family mastacembelidae, known as spiny eels, are found in freshwaters and distributed in tropical and subtropical africa, the middle east, south-east asia and north of china (coad, 2015). this family consists of three genera, including mastacembelus (64 species), macrognathus (22 species) and sinobdella (1 species) (vreven, 2005a; froose and pauly, 2014). nine species of the genus mastacembelus inhabit asian inland waters, whereas 52 species occur in african inland waters and all members of the genus macrognathus were recognized in asian inland waters (froose and pauly, 2014). mastacembelids can attain a maximum length of about 1 m. they are eel-like fishes having a long series of well-separated dorsal spines and a short * corresponding author: esen tutar e-mail address: esentutar@gmail.com series of anal spines. they have no pelvic girdle and fins (vreven, 2005b). more than 70 species of spiny eels are consumed as food fishes (britz, 2007). mastacembelus mastacembelus occurs in the river basins of the tigris and euphrates in the middle east; turkey, syria, iraq and iran (coad, 1996; froose and pauly, 2014) and is known as mesopotamian spiny eel referring to its inhabiting area. this taxon is a typical species of the mastacembelidae and contains all the characteristics of the family (coad, 2015). the phylogenetic structure of the family mastacembelidae is under debate and its classification has mainly been based on meristic and morphometric characters (travers, 1984; kottelat, 1991; johnson and patterson, 1993; britz, 1996; vreven and teugles, 1996; vreven, 2004; vreven, 2005a; vreven, 2005b; britz, 2007; çakmak and alp, 2010; plamoottil and abraham, 2013). although, the majority of the mastacembelids were morphologically described, a few species such as 315 tutar/ combined mitochondrial dna of the mesopotamian spiny eel mastacembelus aculeatus, m. erythrotenia, m. armatus, macrognathus aculeatus and m. pancalus were described based on molecular data (miya et al., 2001; chen et al., 2003; smith and wheeler, 2006). however, there is no molecular information for m. mastacembelus and many mastacembelid species. the mitochondrial dna (mtdna) is a circular and small molecule, self-replicating and usually about 15-18 kb in length. mitochondrial genome contains two ribosomal rna genes, which play primary role in protein synthesis (12s rrna and 16s rrna), 13 protein-coding genes (atpase 6, atpase 8, coi–iii, cytb, nd1-6 and 4l), 22 transfer rna genes and a non-coding control region (d-loop) in charge of its replication and transcription factor as other vertebrates (ishıguro et al., 2001; kartavtsev et al., 2007). the gene content and organization of complete vertebrate mtdna are quite conserved (boore, 1999). the mitochondrial dnas have been widely used as a marker for identification of species and phylogenetic researches, since a lot of characteristics are attributed to the maternal inheritance, high copy numbers in each cell, lack of recombination and high evolution rate (kartavtsev et al., 2007; cui et al., 2009; cawthorn et al., 2012). in addition, the complete mtdna has been widely used in the phylogenetic researches, partial gene fragments such as cytb, 12s rrna, 16s rrna and the control region has become also very useful molecular tools for mitochondrial analysis (cruzagüero et al., 2012). therefore, the mitochondrial dna has been considered a popular marker in many areas including fisheries biology, management and aquaculture, especially for population and evolutionary studies (avise, 1994; okumuş and çiftci, 2003; galtier et al., 2009; lin et al., 2014). the aim of this study is to determine nucleotide sequences of the 12s rrna, 16s rrna, trnaphe and trnaval genes of the spiny eel, m. mastacembelus, and to determine its phylogenetic position among the mastacembelids and the members of synbranchioformes. furthermore, it is aimed to validate the obtained results from molecular methods with morphological findings for identification of this species. to my best knowledge, there is no report on molecular taxonomy of m. mastacembelus. this is the first report on molecular identification of this species. these findings can contribute to understanding of the evolution and phylogenetic characterization of the m. mastacembelus based on mtdna. materials and methods total dna extraction: a total of 57 individuals (36 of karakaya reservoir, 7 of tohma stream and 14 of tigris river) of m. mastacembelus from three different locations at tigris and euphrates rivers were sampled. genomic dna samples were obtained from ethanol preserved caudal fin tissues. caudal fins of 20-30 mg were minced and 600 µl ten (100 mm tris, 10 mm edta and 250 mm nacl), 40 µl 20% sds (sodium dodecyl sulphate) and 10 µl proteinase k (10 mg/l) were added on the samples. they were incubated at 55°c for 24 hours. after the incubation, 10 µl rnase (5 mg/ml) was added and second incubation was applied at 55°c for 24 hours. the total dna was purified by standard phenol:chloroform extraction and ethanol precipitation (sambrook et al., 1989). isolated dna was inspected under uv light after 1% agarose gel electrophoresis. pcr and sequencing: pcr amplifications were performed in 50 µl tubes containing 5 µl 10x reaction buffer (50 mm kcl, 10 mm tris-hcl, 0.1% triton-x1-100), 0.5 µl 1mm dntp (250 μm from each of nucleotides), 1 µl each of 20 pmol forward and reverse primers, 1 u taq dna polymerase, 1 µl dna and 42 µl ddh2o. reaction mixtures were subjected to the following cycling protocol: initial denaturation (94°c: 4 min), 30 cycles (94°c: 1 min; primer denaturation annealing extension e1-e8 1 min in 94oc 30s in 59oc 2 min in 72oc e2 1 min in 94oc 30s in 55oc 2 min in 72oc e3-e6 1 min in 94oc 30s in 58oc 2 min in 72oc e4 1 min in 94oc 30s in 64oc 2 min in 72oc e5-e7 1 min in 94oc 30s in 62oc 2 min in 72oc table 1. denaturation, annealing and extension temperature and times in pcr. 316 int. j. aquat. biol. (2015) 3(5): 314-322 55°c: 30 s; 72°c: 2 min) and final extension (72°c: 5 min) (table 1). in order to amplify mitochondrial dna with standard pcr techniques, the new primers were designed because mtdna of m. mastacembelus has not been determined so far. in order to design pcr and sequencing primers for mtdna genes, sequence for each gene were retrieved from the mitochondrial genome data of m. favus (accession no. nc_003193). sequence length of 12s rrna gene of m. favus was 947 bp and 16s rrna gene was 1671 bp (http://www.ncbi.nlm.gov). the target dna fragment had 3036 bp and contains the region of dloop (last 100 bp), trnaphe, 12s rrna, trnaval, 16s rrna, trnaleu, and nd1 (initial 100 bp) (fig. 1). the primers were designed on the alignments of these sequences. dna fragment was divided into 8 sections because 400-600 bp were desired to sequencing. forward and reverse primers were designed for each section. the length and temperature of these primers were given in table 2. for sequence analyses, three samples from each population were used for sequence of mitochondrial 16s rrna, 12s rrna and trnas genes, which sequenced in iontek (http://www.iontek.com.tr). data analysis and phylogenetic relationships: the 16s rrna, 12s rrna, trnaphe and trnaval sequences of nine samples of m. mastacembelus figure 1. a diagram showing arrangement and position of all amplifying primers. nc 003193 location primer sequence ( 5′ → 3′ ) length start end tm (oc) e1-f ccggaaacaggaaaacctct 20 24 43 64.10 e1-r tagctttcgtggggtcagaa 20 565 582 64.56 e2-f ctacggcgtaaagagtggtt 20 453 472 60.71 e2-r ctttagaaccggtttcagca 20 976 995 61.72 e3-f caaacgtcaggtcgaggtgta 21 843 863 64.97 e3-r atcatgatgcaaaaggtacgag 22 1396 1417 62.69 e4-f tgcaagtcggatcaccctga 20 1172 1191 69.20 e4-r cgctttctattgtggtggctgc 22 1757 1778 69.61 e5-f atagctggttgcccgagaactg 20 1570 1591 68.31 e5-r ggtaaacaggcgaggcttataagg 20 2087 2110 66.31 e6-f gccaacctctctccaaacac 20 1894 1918 63.62 e6-r gtgtctaaagctccacaggg 20 2369 2388 61.27 e7-f ccccaaggaaaggctgaaag 21 2042 2061 66.76 e7-r cttgaaggggattgcgctg 22 2565 2584 67.93 e8-f cggggataactccataagac 20 2310 2329 64.20 e8-r ggatttgaacctctgtggtaaagg 22 2903 2926 65.43 table 2. specific primers used for pcr amplification of target genes of m. mastacembelus. 317 tutar/ combined mitochondrial dna of the mesopotamian spiny eel from three different locations were analyzed to determinate nucleotide composition by mega 5.2 software (tamura et al., 2011). a blast search was performed on ncbi to compare the sequences of m. mastacembelus populations from karakaya reservoir, tohma stream and tigris river, and its phylogenetic tree were constructed based on maximum likelihood model using mega 5.2 software (tamura et al., 2011). the 16s rrna and 12s rrna nucleotide sequences of seven species, including m. mastacembelus (in this study), m. armatus, m. erythrotaenia, m. favus, monopterus albus (synbranchidae) and synbranchus marmoratus (synbranchidae) from the order synbranchioformes registered to genbank as ingroup and acipenser stellatus as out-group was used to study the phylogenetic relationships of m. mastacembelus among the members of synbranchioformes (table 3). the 16s rrna and 12s rrna of these species were translated in different formats aligned using clustal x (thompson et al., 1997). the genes of trnaphe and trnaval were excluded from this step since there is no sequence knowledge found in public databases such as ncbi and embl. phylogenetic trees were constructed using maximum likelihood (ml) (felsenstein, 1981) and neighbor joining (nj) (saitou and nei, 1987) methods using mega 5.2 software (tamura et al., 2011). the robustness of the internal branches of trees was assessed by bootstrapping with 1000 replicates. phylogenetic trees including nucleotide sequences of m. mastacembelus individuals and m. favus were similarly constructed using ml and nj methods. results in the present study, i provided complete sequences of the mitochondrial 16s rrna, 12s rrna and trna genes of m. mastacembelus. the total length of the 12s rrna, 16s rrna, trnaphe and trnaval genes of m. mastacembelus were found to be 947 bp, 1667 bp, 69 and 73 bp, respectively. the 12s rrna, 16s rrna, trnaphe and trnaval gene sequences were deposited in genbank (with accession no gu174757, gu174759, km211690 and gu174758, respectively). the nucleotide composition of order family species common names* distribution* 12s rrna gene 16s rrna gene genbank accession no. genbank accession no. synbranchiformes mastacembelidae mastacembelus mastacembelus mesopotami an spiny eel asia: tigris and euphrates basin gu174757 gu174759 mastacembelidae mastacembelus armatus zig zag eel asia: pakistan to viet nam and indonesia af508066 dq532904 mastacembelidae mastacembelus erythrotaenia fire eel asia:thailand and cambodia to indonesia ay141349 ay141419 mastacembelidae mastacembelus favus tire track eel asia:thailand to the malay peninsula nc_003193 nc_003193 synbranchidae monopterus albus swamp eel asia:india to china, japan, malaysia and indonesia nc_003192 nc_003192 synbranchidae synbranchus marmoratus marbled swamp eel central and south america: mexica to northern argentine ap004439 ap004439 acipenseriformes acipenseridae acipenser stellatus starry sturgeon eurasia: caspain, balck, azov and aegean seas nc_005795 nc_005795 *common names and distribution were taken from www.fishbase.org. table 3. genbank accession numbers and location of examined species for phylogenetic relationships 318 int. j. aquat. biol. (2015) 3(5): 314-322 12s rrna is a: 32.4%, c: 26.7%, g: 19.6% and t: 21.2%. the content of a+t (53.6%) is higher than that of c+g (46.3%). the nucleotide composition of 16s rrna is a: 34.2%, c: 26.2%, g: 19.3% and t: 20.3%. the content of a+t (54.6%) is higher than that of c+g (45.5%). the base compositions of the 12s rrna, 16s rrna and trnas nucleotide sequences are given in table 4. the phylogenetic relationships of the studied m. mastacembelus populations i.e. the karakaya reservoir, tohma stream and tigris river populations were investigated using combined mitochondrial dna sequence. for this purpose, their combined identified sequences were compared using ncbi blast software and based on the results no differences were determined between populations and the identity found to be 100% among all three populations. furthermore, based on the combined target sequences i.e. 12s rrna, 16s rrna and trna genes of these populations and m. favus, the ml tree was constructed. in this tree, the members of three studied population were clustered together with m. favus in another branch (fig. 2). the phylogenetic position of m. mastacembelus among the mastacembelids and members of synbranchioformes that their 16s rrna and 12s rrna nucleotide sequences were available in genbank was constructed (figs. 3 and 4). both ml and nj phylogenetic trees showed two main branches viz. the members of synbranchiformes as in-group and a. stellatus as out-group showing monophyly of the synbranchiformes with the families mastacembelidae and synbranchidae in discrete clade as sister groups. mastacembelus favus, m. erythrotaenia and m. mastacembelus formed a monophyletic group with high bootstrap value. mastacembelus mastacembelus diverged with high bootstrap value from m. erythrotaenia in trees based on 12 rrna (fig. 3) whereas, phylogenetic trees of 16s rrna showed that m. mastacembelus and m. armatus are the closest (fig. 4). discussion identification of fish species is traditionally based on morphological methods i.e. morphometric, meristic and anatomical features. however, there are major fragment a (%) c (%) g (%) t (%) total (bp) trnaphe 39.1 23.2 20.3 17.4 69 12s rrna 32.4 26.7 19.6 21.2 947 trnaval 30.1 31.5 21.9 16.4 73 16s rrna 34.3 26.2 19.3 20.3 1667 average 33.7 26.4 19.5 20.4 565.2 table 4. base compositions (% of total number) of target genes of m. mastacembelus. figure 2. phylogenetic trees constructed from combined target nucleotide sequences (12s rrna, 16srrna and trnas genes) of different studied populations of m. mastacembelus and m. favus based on ml methods with bootstrap support values for each branch. figure 3. phylogenetic trees based on ml and nj methods of 12s rrna genes with bootstrap support values for each branch. acipenser stellatus was used as out-group. 319 tutar/ combined mitochondrial dna of the mesopotamian spiny eel problems to identify the fish species solely based on the morphological characters due to different ecological conditions, which are lead to morphological variations (lakra et al., 2009; teletchea, 2009; chen et al., 2012). therefore, molecular methods especially based on mtdna are used as alternative for their identifications. these methods are highly specific, sensitive and simple compared with morphological methods (comesana et al., 2003). based on the results, the sequences of 12s rrna, 16s rrna, trnaphe and trnaval genes in m. mastacembelus from different studied locations showed no differences. however, in a previous study significant differences among these populations were observed in terms of morphometric characters (çakmak and alp, 2010). based on çakmak and alp (2010), the karakaya reservoir population was morphologically different than two other populations. such condition was reported in seabass, dicentrarchus labrax by turan and erguven (2005). they noted that molecular techniques have great potential to support the detected phenotypic differentiation. furthermore, 12s rrna and 16s rrna genes are highly conserved in of animal kingdom (cawthorn et al., 2012). these genes have been proven to be the powerful phylogenetic tools (cruz-agüero et al., 2012). the 12s rrna has been used to higher categorical levels such as in phyla and 16s rrna often used for studies at middle categorical levels such as families or genera (arif and khan, 2009). therefore, the results also showed that this genes are not proper to study the genetic population of the genus mastacembelus as well. the 12s rrna and 16s rrna genes were respectively bordered by the trnaphe and trnaval genes and by the trnaval and trnaleu genes as in other vertebrates (nagase et al., 2005). the location of these genes are conserved in vertebrates (chang et al., 1994; cui et al., 2009). the results of this study were supported by location of these genes. the 12s rrna and 16s rrna genes of m. mastacembelus exhibit a+t rich-content like as other bony fishes (chang et al., 1994). phylogenetic trees based on ml and nj method using 12s rrna and 16s rrna gene sequences showed that members of the same genus have been clustered together confirming the current taxonomic classifications of the studied fish species (vreven, 2005a; froose and pauly, 2014). although little nucleotide sequences of the members of synbranchioformes were available in genbank. this study is the first attempt to identify phylogenetic position of mesopotamian spiny eel, m. mastacembelus based on mitochondrial sequences. the sequences of mesopotamian spiny eel, m. mastacembelus, generated that were previously unavailable in genbank. these sequences will be valuable for future molecular studies and phylogenetic researches in mastacembelid species and the order of synbranchioformes. references arif i.a., khan h.a. 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(2015) 3(5): 314-322 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی mastacembelus mastacembelusالنهرین،میتوکندیای مارماهی خاردار بین dnaرکیب ت و جایگاه تبارزایی آن *اسن توتار ترکیه.، کاهرامان ماراش، امام سوتچو کاهرامان ماراشدانشگاه مدرسه علوم طبیعی و کاربردی، ، مهندسی زیستی و علومگروه چکیده: ،m. mastacembelusالنهرین، مینوکندریایی مارماهی خاردار بین valtrna و rrna12s ،16s rrna ،phetrnaهای تیدی ژنوتوالی نوکلئ میتوکندریایی dnaحوضه تیگریس ترکیه براساس ترکیب بخش النهرینمارماهی خاردار بینمقایسه سه جمعیت برای اولین بار تعیین گردید. درصد یکسان 011طور ها بهیافت نشد و توالی آن m. mastacembelusهای مورد مطالعه حاصل انجام شد. براساس نتایج، تفاوتی بین جمعیت انجام m. mastacembelusهای مورد مطالعه جمعیت شناختیآرایه جایگاهاعتبارسنجی نتایج مولکولی و ریختی به منظور همچنین مقایسه. بود براساس synbranchioformesو راسته mastacembelidaeدر بین اعضای خانواده النهرینمارماهی خاردار بینبه عالوه جایگاه تبارزایی شد. 12s rrna 16 وs rrna مورد بررسی قرار گرفت. روابط تبارزایی حاصل بینm. mastacembelus راسته و برخی دیگر اعضای synbranchioformes شناختی آنها را مورد تایید قرار داد.جایگاه آرایه .، روابط تبارزاییmastacembelus، s rrna16، s rrna12، heptrna ،alvtrna کلمات کلیدی: int. j. aquat. biol. (2019) 7(4): 233-238 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article development of low-cost feeds for fattening of native catfish, clarias macrocephalus arlene l. avillanosa*1, jasper d. pacho1, christopher marlowe a. caipang2 1college of fisheries and aquatic sciences, western philippines university, puerto princesa campus, puerto princesa city 5300, palawan, philippines. 2college of liberal arts, sciences, and education and the center for chemical biology and biotechnology, university of san agustin, general luna st., iloilo city 5000, philippines. s article history: received 31 april 2019 accepted 10 august 2019 available online 2 5 august 2019 keywords: feed ingredients freshwater aquaculture recycling abstract: growth performance, survival, and feed efficiency in native catfish, clarias microcephalus, fed chicken entrails, earthworm meal, and low-value fish meal were investigated. a simple cost-benefit analysis using this fattening approach was done to evaluate the profitability of using these low-value feed ingredients. nine 30l aquaria were stocked with native catfish juveniles (about 20 cm in total length and 80 g in weight) at a density of 1 fish per liter. the catfish were fed cooked chicken entrails (treatment 1), earthworm meal (treatment 2) and low-value fish meal (treatment 3) at 3% body weight for 60 days. at the end of the feeding trial, the growth of the fish fed various low-cost feeds was not significantly different. survival was better in fish fed cooked chicken entrails than with either earthworm meal or low-value fish meal. feed conversion efficiency (fce) was relatively similar among the three types of feeds. a simple cost-benefit analysis using these low-cost feeds showed a return of investment (roi) of 68-79%, indicating the feasibility of using these feeds for fattening of catfish. these preliminary results show that utilizing low value feed ingredients or food wastes as sources of feeds during fattening of native catfish are feasible. in addition, food wastage is reduced by bringing these food sources back to the food chain during aquaculture operations. introduction the rapid global expansion of freshwater aquaculture in recent years has resulted increasing the industry and number of fish species that are being utilized (mather and de bruyn, 2003). fishing production has reached its limit, which calls for an increase of fish supply from aquaculture (fao, 2012). aquaculture currently produces about 50% of the world supply of fisheries products for direct human consumption (boyd, 2012). it is envisioned that aquaculture production will increase fish supply and bridge the gap between fish supply and demand. one potential area for aquaculture development is tapping the rich freshwater resources. in the philippines, there are at least 10,000 hectares of freshwater ponds and 250,000 hectares of inland freshwater resources that can be developed for freshwater aquaculture (psa, 2018). however, very few species are being utilized for aquaculture. the *correspondence: arlene l. avillanosa doi: https://doi.org/10.22034/ijab.v7i4.583 e-mail: arlavillanosa@yahoo.com freshwater asian catfish, clarias macrocephalus is one of the most important but declining fishes in the philippines (tan-fermin, 2003). world production of catfish had been dominated by the hybrid species of c. gariepinus x c. macrocephalus (fao, 2003). though native catfish i.e. c. macrocephalus has been overtaken by the african catfish, c. gariepinus as a preferred catfish species in aquaculture, there is still high demand for native catfish because of its superior meat quality and flavour (coniza et al., 2008). hence, there is a huge potential to develop native catfish as an aquaculture species. catfish culture requires the formulation of efficient food to meet the protein requirements of fish during grow-out period. protein is the most expensive macronutrient in the fish diet (pillay, 1990) and amount of the protein in the diet should be just enough for fish growth. although native catfish is an omnivorous species, fish meal is still considered as 234 avillanosa et al./ low-cost feeds for native catfish main animal protein source when developing formulated diets for this species. alternatively, the use of cheap and locally available resources could provide a way to reduce the total production costs (edwards and allan, 2004; munguti et al., 2012). one approach that can reduce the cost of animal feeds is the use of farm and food wastes, agro-industrial by-products or household wastes, either as direct feeds or incorporated as components of feeds along with other ingredients that will meet the nutritional requirements of native catfish (aletor, 1986; fagbenro and arowosoge, 1991). current estimates revealed that about 40–50% of catfish farmers still use wet farm-made feeds from locally available feedstuffs that have wide variations in their composition and nutritional value (edwards et al., 2004). the lack of efficient diets remains one of the bottlenecks in the development of native catfish culture in the philippines (coniza et al., 2003), therefore, there is a need to explore some of these alternative feed ingredients that are suitable for catfish culture. once these feed ingredients are identified, these can be further developed into commercial diets for native catfish. hence, this study aimed to assess the efficiency of selected locally available feed sources that can be used in the grow-out culture or fattening of native catfish. the feasibility of using these feed ingredients was also determined using a simple cost-benefit analysis. materials and methods experimental procedure: the study was conducted in aquatic science laboratory (asl) at western philippines university-puerto princesa campus, palawan, philippines. the native catfish were obtained from the local suppliers in the municipalities of quezon and roxas, palawan. a total of 27 fish with average body weight of 81.9±10.6 g were used as experimental animals. the fish were placed in glass aquaria and acclimated to experimental conditions for 2 weeks. a total of nine (9) rectangular glass aquaria (0.65x0.25x0.31 m) were used. the following lowcost feeds were assigned into three treatments, viz. cooked chicken’s entrails (treatment 1), earthworm meal (treatment 2), and low-value fish meal (treatment 3). the tank replicates were assigned using complete randomized design in triplicate. each aquarium was filled with 30l and aerated 24 hours before stocking. fish were stocked in each aquarium at a density of 3 fish per aquarium. all aquaria were covered with black plastic sheet to simulate the natural habitat. water quality was monitored for ph and temperature weekly at 09.00 a.m. using appropriate water kits. dissolved oxygen was monitored using an oxygen meter (hi 9143 microprocessor oxygen meter hanna, usa). debris at the bottom of the tanks was siphoned out daily while total water exchange was done every two weeks after sampling. feeding and sampling: all diets were divided into small portions and stored in the freezer. on the day of feeding, each portion was thawed and fish fed twice a day (09.00 and 17.00) at 3% body weight for 60 days. fish mortality was monitored daily. individual fish in each tank was weighed at the start and every 15 days to monitor growth and feed utilization. for the purpose of measuring growth parameters, all fish were taken from each aquarium replicate and measured for total length and weight. the length was measured using 30 cm ruler; while weight was measured using a weighing balance. after each sampling, survival (%), specific growth rate (sgr), feed conversion efficiency (fce) and growth rate (gr) was calculated using the formula given by aderolu et al. (2010). at the end of the study, a simple cost benefit analysis was done by computing the return of investment (roi) in a 500 m2 earthen pond using the data of the present study. statistical analysis: means and standard error (se) of each zootechnical parameter were computed. all statistical computations were performed at the 0.05 probability level. data for all zootechnical parameters of treatments were analysed using one-way analysis of variance (anova). when anova revealed significant differences, then duncan’s multiple-range test (zar, 1999) was applied to characterize and quantify the differences between treatments. analyses 235 int. j. aquat. biol. (2019) 7(4): 233-238 were performed using microsoft excel 2010. results and discussions the zootechnical parameters of native catfish fed different low-cost feeds are shown in table 1. after a 60-day fattening period, catfish fed three different types of feeds had increased in weight ranging 10 to 34 g. in terms of the weight gain and final weight, no significant differences were observed among treatments, demonstrating that these low-cost feed ingredients had similar effects in the growth of the fish. however, in terms of average final length, significant differences (p<0.05) were obtained among treatments, with catfish fed chicken entrails contributed to the highest average length of the fish. survival rates of catfish were significantly different (p<0.05) among the treatments, with highest survival in fish fed chicken entrails (100%). the groups fed earthworm meal and low-value fish meal had similar survival rates (77.78%). the mean feed conversion efficiency (fce) ranged from 12 to 20%; however, no significant differences (p>0.05) were observed between the treatments. proximate analyses of the feed ingredients were within the values in previous published studies. chicken entrails had moisture content of 76-83%; crude protein content of 56%; ash content of 11% and fat content of 2.5% (seong et al., 2015; kwikiriza et al., 2016). low-value fish meal had the following proximate composition: moisture content of 77-80%, crude protein content of 70%; ash content of 2.5-4% and fat content of 0.5-3% (emre et al., 2003; ramalingam et al., 2014; kwikiriza et al., 2016). earthworm meal contained 57-64% moisture; 18-24% crude protein; 0.9-1.2% ash and 0.5-1.8% fat (finke, 2002). a simple cost and return analysis is shown in table 2. the return of investment (roi) for rearing native catfish in a 500 m2 pond using the zootechnical data from the small-scale study were 68.37, 71.18 and 79.47% for low-value fish meal, chicken entrails, and earthworm meal, respectively. the native catfish is one of the important aquaculture commodities in the philippines. according to coniza et al. (2008), it is valued as a food fish in most southeast asian countries due to its tender and delicious flesh. small-scale fish farmers consider this species to have a good potential for aquaculture. due to the abundance of these low-value sources of feeds for catfish, the present study determined the growth, survival, feed efficiency and simple cost and return analysis of native catfish using these locally available feeds (chicken entrails, earthworm meal and low-value fish meal). based on the results, the growth performance of catfish in terms of weight gain was similar when fed either of the chicken entrails, earthworm meal or low-value fish meal. marketability of the fish is influenced by its weight and native catfish are usually sold in 80-200 g (seafdec/aqd, 2009). the catfish were of harvestable sizes during the table 1. zootechnical parameters of catfish fed various low-cost feeds. growth parameters chicken entrails (treatment 1) earthworm meal (treatment 2) low-value fish meal (treatment 3) initial weight (g) 86.81±7.93 82.12 76.92 final weight (g) 108.26±6.94 92.44 110.59 weight gain (g) 21.45±2.00 10.32 33.67 initial length (cm) 23.04±0.59 22.00 22.33 final length (cm) 24.5±0.67c 22.88 a 23.75b length gain (cm) 1.46±0.3c 0.88 a 1.42b specific growth rate %/day 0.37±0.03 0.20 0.61 feed conversion efficiency (%) 12.3±0.38 6.73 20.33 survival rate (%) 100±0.00a 77.78b 77.78 b values in each row with different superscripts indicate significant difference at p<0.05. n= 3 tank replicates per treatment. 236 avillanosa et al./ low-cost feeds for native catfish fattening experiment, but since the fish were thin, they were further reared in the aquaria for two months to increase their weight and selling price. in terms of growth performance, the locally available feed ingredients can be used as feeds for the fattening of native catfish, however, only chicken entrails resulted in 100% survival of the fish stock in this study. the survival of catfish fed chicken entrails was higher than those of nahar et al. (2000) and coniza et al. (2008), which was 83 and 81%, respectively. among the low-cost feeds for catfish culture, the use of chicken entrails is popular based on earlier studies of nahar et al. (2000) in african catfish fry and coniza et al. (2008) in native catfish fingerlings. in fact, use of the chicken entrails has been recommended by yaakob and ali (1994) for the canvass tank culture of hybrid catfish in malaysia. the authors stressed that catfish farmers still prefer to use this feed than commercial feed pellets because the former is cheap and readily available. the culture of catfish using earthworm as a replacement for fish meal was done by omeru et al. (2016) and demonstrated the potential of earthworm as an alternative ingredient in formulating feeds. the survival (75%) recorded by omeru et al. (2016) was comparable with our result (78%). based on literature review, most of the studies conducted on catfish are thai catfish and african catfish (nahar et al., 2000; akegbejo-samsons and fasakin, 2008; adewumi, 2015; tunde et al., 2016). further, these previous studies focused on fry and fingerlings using commercial and formulated feeds as well as natural feeds. the feed conversion efficiency (fce) of 6.73% for native catfish fed earthworm meal was lower than the fce of 12.83% that was obtained by omeru et al. (2016) in african catfish fed formulated feeds containing earthworm ingredients (50% earthworm and 50% fishmeal). the differences in the ages of the fish during the experiment could have contributed in disparity of the results. table 1. simple cost-benefit analysis of catfish fattening using low-cost feeds. technical basis chicken entrails (treatment 1) earthworm meal (treatment 2) low-value fish meal (treatment 3) stocking rate (fish/m 2 ) 3.00 3.00 3.00 total stock (fish/500 m² ) 1500.00 1500.00 1500.00 abw (g) @ stocking 82.00 82.00 82.00 culture period (days) 60.00 60.00 60.00 growth increment (g/month) 13.13 5.22 14.29 abw (g) @ harvest 108.26 92.44 110.59 survival (%) 100.00 78.00 78.00 yield (kg) 162.39 108.15 129.39 feed conversion rate 1.37 0.75 2.26 total feed consumed (kg) 53.90 9.16 75.60 operational cost (philippine peso, php) labor @ php 275.00/day 6000.00 6000.00 6000.00 native catfish @ php40/kl 731.71 731.71 731.71 feeds 4652.00 500.00 2490.00 total operational cost 11384.00 7232.00 9222.00 return (philippine peso, php) sales @ php 120 19487.00 12979.00 15527.00 profit 8103.00 5747.00 6305.00 return on investment (%) 71.18 79.47 68.37 n = 3 tank replicates per treatment. 237 int. j. aquat. biol. (2019) 7(4): 233-238 the economic potential of native catfish for fattening is promising because based on the simple cost analysis, the rois were in the range of 68-79%. the fish readily accept these locally available and cheap feed sources. small-scale farmers can easily adapt this technique of fattening catfish in portable canvass tanks (yaakob and ali, 1994) for 1-2 months resulting in better profits. in general, catfish aquaculture technology is simple and easily adaptable. the fish can be marketed live or profits can be enhanced through selling cooked products. in conclusion, the three types of feeds can be used for the culture of catfish because they have same growth and feed efficiency. survival is best attained when catfish fed chicken entrails and in terms of profitability, both chicken entrails and low-value fish meals are recommended for fattening or commercial production. future studies needs to be done to develop proper formulations for development of artificial feeds to enhance growth, nutrient absorption and minimize wastage of raw materials. acknowledgments the study was supported in part by the ched-nafes project “western philippines university learning environment-friendly advocacy farm and familybased education towards fisheries resource management and popularization of aquaculture and alternative agriculture in western philippines”. cma caipang is supported by the balik scientist program of the department of science and technology, philippines. the project staff of the ched-nafes project and some personnel of the college of fisheries and aquatic sciences of western philippines university-puerto princesa campus assisted in the conduct of the study. references aderolu a.z., seriki b.m., apatira a.l, ajaegbo c.u. 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(2021) 9(3): 167-176 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article evaluation of the population growth and fatty acid composition of copepoda, oithona nana, fed on different diets fawzy i. magouz1, mohamed a. essa2, mustafa matter2, mohamed ashour*2 1 1department of animal production, faculty of agriculture, kafrelsheikh university, egypt. 2national institute of oceanography and fisheries (niof), egypt. s article history: received 26 december 2020 accepted 9 february 2021 available online 2 5 august 2021 keywords: aquaculture corn starch fatty acid live food rice bran abstract: the marine copepoda species oithona nana, is considered as one of the most copepoda species that successfully mass cultured in marine hatcheries. this study investigated the effects of four feed diets (soybean, yeast, rice bran, and corn starch) on the population growth, growth rate, population composition, fecundity, and fatty acid composition of copepoda, o. nana. the experiment was continued for 15 days and the copepods were fed on four feed diets with concentration of 1 g/106 individual/day. the results found that o. nana fed on corn starch showed the highest significant population growth (9067 individual/l) and population growth rate (0.735). for nutritional value, copepods fed on rice bran were detected to have the highest content of monounsaturated fatty acid (mufa), polyunsaturated fatty acids (pufa), the lowest saturated fatty acids/unsaturated fatty acids ratio (sfa/ufa ratio) and the lowest sfa. more importantly, the rice bran diet was the only diet that showed eicosapentaenoic acid (epa; c20:5ω3). moreover, copepods fed on rice bran showed the highest significant female fecundity (8.33 egg/female), copepodite and nauplii percentages (33.27 and 32.65%, respectively). finally, regarding to the quantity, corn starch is the most suitable diet for mass culturing o. nana, while, regarding to the quality, rice bran enhances the nutritional value and fecundity of the calanoida copepoda o. nana. introduction livefoods are considered as one of the most key components of the production of marine fish and shrimp larvae in the marine hatcheries, still one of the most significant obstacles for the development of marine larvae production (abdel rahman et al., 2008, 2010; khairy and el-sayed, 2012; ashour and kamel, 2017). livefoods are divided into two types of microorganisms; phytoplankton (microalgae) and zooplankton (like rotifer, artemia, and copepods). microalgae are the basic food chain in aquatic environments (ashour 2015, 2020). sequentially, livefoods (phyto and/or zooplankton), are the main live-foods that consumed by marine fish and shrimp larvae. in general, marine algal-cells have many bioactive compounds like pufa, essential amino acids, carotene, flavonoid, and phenolic compounds, showing antimicrobial and antioxidant activities, nonspecific immunity, and growth promoting activities *correspondence: mohamed ashour doi: https://doi.org/10.22034/ijab.v9i3.1056 e-mail: microalgae_egypt@yahoo.com (hassan et al., 2017, ashour et al., 2020a, 2020b, elshobary et al., 2020). many livefoods species meet the complete nutritional requirements of marine fish and shrimp larvae, therefore, livefoods are considered “nutritional bags” for marine larvae and postlarvae (drillet et al., 2011; sharawy et al., 2020; abo-taleb et al., 2020a). the production of marine microalgae is more difficult than the production of marine zooplankton because of many consecrations. in marine hatcheries, despite the complexity of livefoods productions for marine larvae, there are many benefits and potential of consuming livefoods for aquatic animals (drillet et al., 2011). copepoda are one of the most nutritious livefoods used in marine hatcheries. despite rotifer and artemia were extensively used as preys in marine hatcheries (heneash et al., 2015; ashour et al., 2019), copepod species are considered the best live preys, due to their higher nutritional value than rotifers and artemia 168 magouz et al./ effects of diet on oithona nana (støttrup, 2000; olivotto et al., 2010; drillet et al., 2011; abate et al., 2016; øie et al., 2017). in wild, copepoda acting as trophic linkages between primary producers and secondary consumers in marine ecosystems (støttrup, 2000; ashour et al., 2018; abotaleb et al., 2020b). among copepoda, the order calanoida is considered the main organic matter consumer and energy transporter to higher trophic levels, including small fish, larvae, and juveniles of aquatic species in the marine ecosystem. thus, calanoida represents a major prey source for mesopelagic and bathypelagic fish (yamaguchi et al., 2015; abo-taleb et al., 2020b). calanoida copepoda, especially species oithona nana, is an excellent source of highly polyunsaturated fatty acids which makes copepods to be more nutritious and attractive food for larval and small fish (drillet et al., 2011). selecting a suitable feed diet for copepoda is a crucial key factor that extensively influences the quantity and quality of cultured copepods (kleppel et al., 2005; van der meeren et al., 2008; pan et al., 2018). microalgae are the basic live diet utilized in marine hatcheries for copepod cultures (vidhya et al., 2014; el-khodary et al., 2020). microalgal are more preferred for copepods due to many factors such as nutritional value, size, shape suitability, and digestibility (pan et al., 2018). many studies have focused on the evaluation of a convenient microalgal diet for copepoda species (payne and rippingale, 2000; milione and zeng, 2007; camus et al., 2009; ohs et al., 2010; pan et al., 2018). however, the high labor and high production cost of microalgae are increases the copepods price production. subsequently, the seeking of an optimal diet as microalgal alternatives diet for copepoda species is critical for sustainable marine aquaculture, in particular for cultured species that have commercial prospects. many feed regimes were referenced as microalgalalternative diets for different cultured copepods and for zooplankton species, in general, such as baker’s yeast saccharomyces cerevisiae (payne and rippingale, 2000; farhadian et al., 2008), fish diet (ribeiro et al., 2011), soybean (agbakimi et al., 2017; el-khodary et al., 2020), rice bran (depauw et al., 1981; mubarak et al., 2017; amian et al., 2018), starch and albumen (sulehria et al., 2010), and glucose (gyllenberg and lundqvist, 1978). according to references, the alternative feeding regimes for culturing either copepods or zooplankton species were resulted in adequate population growth and productivity, depending on cultured species, culture methods, and experimental conditions. therefore, studying the effects of feed types on copepods quality and quantity is need to find out their ideal prospect in marine aquaculture. to select the optimal food regime for calanoida copepoda, oithona nana, the current study was conducted to evaluate the population growth, population growth rates, population compositions, fecundity, and fatty acid compositions of o. nana fed on different diets of the commercial-grades of soybean, yeast, rice bran, and corn starch. materials and methods copepods stock culture: the marine copepods were isolated from an earthen pond at el-max research station, alexandria branch of national institute of oceanography and fisheries, (niof). during the copepod collections period in spring 2017, the earthen pond temperature (23±2°c), salinity (31±1 ppt), and ph (7.37±0.10) were recorded at noon. copepod samples were collected following the protocol described by abo-taleb et al. (2020a). isolated individuals were initially examined using a binocular stereomicroscope (optika microscopes, b190/b-290, italy), at 10x magnification. morphological identification and taxonomic characterization was conducted by the hydrobiology lab., marine environment division, niof, using the key references of bradford-grieve (1994), newell and newell (1933), and gonzalez and bowman (1965). after morphological classification, the isolated adult copepods were identified as copepoda calanoida: oithona nana (fig. 1). adult individuals of o. nana were cultured under laboratory-controlled conditions (27±1°c, 20 ppt, ph 7.7±0.15, and continuous gentle aeration) and enriched with microalga nanno169 int. j. aquat. biol. (2021) 9(3): 167-176 chloropsis oceanica niof15/001 (5×106 cells/ml). regime and experimental design: commercial grades of soybean, yeast (saccharomyces cervicates), rice bran, and corn starch were used as feed for copepods. yeast s. cervicates and corn starch was supplied by starch and yeast company, egypt, while soybean and rice barn was supplied from fish feed factory located in alexandria, egypt. the copepods were fed on four regimes (treatments), including soybean, yeast, rice bran, and corn starch, with concentration of 1 g/106 individual/24-hr of each (heneash et al., 2015). to prepare the concentration of the diets (three replicates for each treatment) of soybean, yeast, rice bran, and corn starch, 1 g of a commercial baker yeast and corn starch and a very finely grounded commercial soybean and rice bran were dissolved separately in 100 ml hot freshwater, shaken vigorously, and then blended using a kitchen mixer until fresh instant emulsion was formulated to be used as feeding regimes for copepods. the experiment was continued for 15 days, according to santhanam and perumal (2012). the density of copepods in each food regime was estimated as individual/ml and the needed concentration for each food regime was estimated depending on the previously accounted copepods individual/ml every three days (day-0, 3, 6, 9, 12, and 15). before experiment, the copepods were harvested from the stock culture tank and transferred to the new culture water for a 24 h gut evacuation to avoid the effects of resident soybean and algal diet (tseng et al., 2009; pan et al., 2018). at the beginning of the experiment, the adult copepods (with average size of 625 µm) were cultured in glass tanks field with 30 l of 1 µm bag-filtered, chlorine-disinfected of diluted seawater (20 ppt) with initial stock density of approximately 1 individual/ml (about 1,000 ind./l) with sex ratio 1:1. the culture conditions during the experiment were kept under controlled conditions of salinity 20 ppt, temperature 27±1˚c (using a digital thermometer), ph 7.7±0.15, and photoperiod 12:12. each tank was hand-fed three times per day (9.00 am, 12.00 pm and 3.00 pm), seven days a week. the tanks were conducted without water replacement and were supplied with gentile aeration to keep dissolved oxygen (do) over 4 mg/l (measured using oxymeter, china). ammonia (nh3) concentration (measured using digital multi-meter, italy) was <0.45±0.05 mg/l in all treatments, showed no negative effects of food regimes additions. tested parameters population growth, growth rate, composition, and fecundity: every three days, 25 ml of culture water from every replicate of each diet was taken to estimate the population growth of copepods, which estimated as increase in number (ind./ml). every 3 days, about one hundred individuals from each replicate were harvested, using a 38-μm mesh, and fixed with a 4% formalin solution to estimate the percentage of population composition and different developmental stages (nauplii, copepodite, male, and female) under a microscope (optika microscopes, b190/b-290, italy). twenty to thirty vigorous carrying-females from every replicate were sorted and placed on a petri dish to examine the fecundity. the population growth rate (r) was calculated according to yin et al. (2013), using the equation of r = (lnnt lnn0)/t, where n0 and nt are the initial and final population densities, and t is the incubation time in days. fatty acid analysis: at the end of the experiment (after day-15), all copepods of each replicate were harvested and preserved at −80°c for fatty acid figure 1. isolated adult copepoda (calanoida: oithona nana). 170 magouz et al./ effects of diet on oithona nana analysis. fatty acid profiles of o. nana fed different diets were extracted and estimated as described by elshenody et al. (2019). data analysis: statistical analyses were analyzed using spss version 16. the results are presented as the mean±standard error (n=3). all variables were evaluated in three replicates using one-way analysis of variance (anova) followed by duncan's multiple range tests to compare differences among individual means at a significance level of p≤0.05. results population growth, growth rate, composition, and fecundity: based on the results, the population growth (ind./ml), growth rate (r), population composition, and fecundity of o. nana were significantly affected by different diets (figs. 2-5). among all experimented diets, corn starch was exhibited the highest significant (p≤0.05) population growth and growth rate in all investigated days, including day-3 (2267 ind./l and 0.273, respectively), day-6 (3800 ind./l and 0.445, respectively), day-9 (6267 ind./l and 0.611, respectively), day-12 (7600 ind./l and 0.675, respectively), and day-15 (0967 ind./l and 0.735, respectively), followed by yeast, rice bran, while the lowest population growth and growth rate was observed in soybean treatment (figs. 2, 3). figure 4 presents the percentages of the population composition (male, female, copepodite, and nauplii) of o. nana fed different diets showing o. nana fed on rice bran having the highest significant copepodite and nauplii percentages (33.27 and 32.65%, respectively) and the lowest significant male and female percentages (16.50 and 17.58%, respectively) (fig. 4). figure 5 shows the fecundity (eggs/female) of o. nana fed on different diets with o. nana fed on rice bran diet having the highest significant fecundity (8.32±0.167 eggs/female), followed by the corn starch (7.67±0.159 eggs/female), while the lowest one found in the soybean (6.17±0.177 eggs/female) and yeast (6.50±0.289 eggs/female). fatty acid compositions: fatty acid compositions were significantly varied in o. nana fed on the different diets (table 1). among all diets, o. nana fed rice bran showed the highest significant (p≤0.05) percentages of mufa (34.87%) and pufa (1.45%), figure 2. effect of different diets on the population growth (individual/l) of calanoida copepoda oithona nana. data are presented as mean± standard errors. the letters (a, b, and c) above each bar indicate the significant differences (p≤0.05) between different diets in the same day. 171 int. j. aquat. biol. (2021) 9(3): 167-176 and the lowest significant percentage of sfa (63.87%) and the lowest sfa/ufa ratio (1.77), compared to the others. furthermore, o. nana fed on rice bran had a greater c18:1c (13.04%), c18:3ω3 figure 3. effect of different diets on the population growth rate (r) of calanoida copepoda oithona nana. data are presented as mean±standard errors. figure 4. effect of different diets on mean percentages of population compositions of oithona nana. data are presented as mean ± standard errors. the letters (a, ab, b, and c) above each bar indicate the significant differences (p≤0.05) between the developmental stages (adult males, adult females, nauplii, and copepodites) in different diets. 172 magouz et al./ effects of diet on oithona nana (1.20%). no recorded epa (c20:5ω3) in all o. nana fed on the different diets expect the diet of rice bran which was the only that revealed a small amount of (0.24%) (table 1). discussions the low yields, long generation time, seasonal variations of production, and high costs are the main problems limiting the success of the culture of copepods (støttrup, 2000; conceição et al., 2010; ajiboye et al., 2011). there are needs for cheap food to minimize the cost of productions copepod (drillet et al., 2011). the commercial-grades of soybean, yeast, rice bran, and corn starch did not show any negative effects on the environment water quality during the experiment. soybean meal is an attractive source of protein, which has high protein percentage, appealing smell, and low price (booman and jones, 2018). moreover, it also contains different classes of specific anti-nutrients, including oligosaccharides and allergic proteins (fao, 2011), while, the high concentration of soybean meal can be toxic (barrows and sealey, 2017). in marine hatcheries, baker’s yeast, s. cerevisiae, could be successively used as an algalsubstitute, moreover, it has obvious benefits, like the table 1. fatty acid profiles (% of total fatty acids) of copepods fed on different food regimes. fa soya bean yeast rice bran starch sfa 10:0 0.31±0.006d 0.46±0.17a 0.41±0.006b 0.373±0.003c 11:0 0.40±0.006c 0.81±0.075a 0.41±0.023c 0.56±0.006b 12:0 0.78±0.003ab 0.88±0.058a 0.70±0.020b 0.52±0.035c 13:0 2.17±0.040b 2.87±0.055a 2.39±0.205b 3.10±0.159a 14:0 10.51±0.309c 17.80±0.820a 14.15±0.471b 16.10±0.393a 15:0 0.91±0.147d 2.60±0.101b 1.99±0.012c 11.80±0.115a 16:0 38.54±0.12a 24.78±0.303c 30.48±0.029b 24.83±0.245c 17:0 0.55±0.003d 1.92±0.043a 1.16±0.015b 0.69±0.003c 18:0 21.88±0.245a 11.57±0.205b 9.76±0.064c 8.12±0.090d 20:4 0.83±0.046b 2.65±0.191a 2.43±0.300a 2.22±0.049a mufa 14:1 12.35±0.001b 15.16±0.823a 15.58±0.003a 13.93±0.543ab 15:1 0.77±0.015b 1.32±0.001b 1.16±0.479b 4.37±0.150a 16:1 1.89±0.020b 4.19±0.592a 2.44±0.032b 2.33±0.040b 18:1c 6.22±0.150c 9.07±0.297b 13.04±0.069a 9.02±0.433b 18:2c 1.04±0.104c 2.77±0.015a 2.46±0.006b 1.12±0.012c pufa 18:3ω3 0.83±0.038b 1.18±0.017a 1.20±0.061a 0.91±0.012b 20:5ω3 0.00±0.000b 0.00±0.000b 0.24±0.006a 0.00±0.000b ∑ sfa 76.89±0.297a 66.33±0.479c 63.87±0.647d 68.32±0.084b ∑ mufa 22.27±0.260d 32.49±0.514b 34.68±5.95a 30.77±0.095c ∑ pufa 0.83±0.037c 1.18±0.017b 1.45±0.055a 0.91±0.012c sfa/ufa 3.33±0.058a 1.97±0.043c 1.77±0.052d 2.16±0.009b values are means±se. means (n = 3) in the same row with different superscript are significantly different (p≤0.05). fa: fatty acids; sfa: saturated fatty acids; mufa: monounsaturated fatty acids; pufa: polyunsaturated fatty acids; ufa: unsaturated fatty acids. figure 5. effect of different diets on fecundity of oithona nana. data are presented as mean ± standard errors. the letters (a, b, and c) above each bar indicate the significant differences (p≤0.05) between different diets. 173 int. j. aquat. biol. (2021) 9(3): 167-176 reduction of algal production facilities and subsequently reduces the production cost. many authors cited that the low concentration of yeast did not influence water quality (payne and rippingale, 2000; farhadian et al., 2008; el-khodary et al., 2020). rice bran was successfully used as feed for copepods, daphnia, artemia, and moina cultures (depauw et al., 1981; mubarak et al., 2017). amian et al. (2018) stated that the diversity and abundance copepods, rotifera, and cladocera are enhanced when using rice bran in fish ponds during the rearing of tilapia. rice bran, as well as soybean (amian et al., 2018; el-khodary et al., 2020) must be processed into small particles suspension to fit the mouth of the cultured copepods. in current study, the highest significant population density and population growth rate were observed with copepods o. nana fed on corn starch diet. it may be due to its particle size, texture, energy content, and lower levels of anti-nutrients, or may be because of higher digestibility of corn starch. the result was in agreed with sulehria et al., (2010), who cited that rotifers cultured on diet mixed with corn starch. the starch grains are composed of two types of alphaglucan, amylopectin and amylose, which substitute approximately 98-99% of the dry weight of starch, moreover, starch contains relatively low quantities (0.4%) of minerals (tester et al., 2004). the findings of current study indicated that o. nana fed rice barn diet had a higher fecundity (8.33 eggs/female) compared with those fed on soybean, yeast, and corn starch (6.17, 6.50, and 7.67, respectively). carli et al. (1995) reported that the type of diets (algae monochrisis lutheri and yeast saccharomyces cerevisiae) are strongly affects the fecundity and survival of harpacticoid copepoda, tigriopus fulvus. previous studies have confirmed that the fecundity of copepods is probable to be linked to the content of pufa in their diets (støttrup and jensen, 1990; kleppel et al., 2005; pan et al., 2018). the importance of dietary pufa contents to the fecundity of o. nana was detected in the experimented diets. in current study, the fatty acids composition of copepods fed on rice bran diet may explain the increases in female fecundity and comparing to the fatty acid compositions of copepods fed soybean, yeast, and corn starch, the fatty acid composition of copepods fed rice bran resulted in the highest significant mufa and pufa, as well as, the lowest significant sfa and the lowest sfa/ufa ratio. these funding may be due to the nutritional value of rice bran (mubarak et al., 2017). najeeb et al. (2015) cited that the commercial grade of rice bran contains 50% carbohydrate, 15% protein, 20% fatty acids (linoleic and oleic acids), 5% vitamin e (tocatrienols, tocopherols, oryzanols, phytosterols), and a low amount of other micronutrients. many works cited that copepods have the ability to endogenously synthesize pufa from short-chain fatty acids (lee et al., 200; monroig et al., 2013). interestingly, in the current study, the copepods fed on rice bran are the only exhibited epa in their fatty acids profile. this funding may be attributed to the nutritional value of rice bran. moreover, the epa contents of copepod fed on rice bran may be explained the high fecundity, as well as, the high significant percentage of nauplii (32.65%) and copepodite (33.27%) in population composition of o. nana. the trend observed on fecundity was similar to the examination of population composition that rice bran provided the highest total percentage of nauplii and copepodite population. besides, the rice bran was markedly predominated by nauplii and copepodite, with relatively few adult males and/or female. our finding is agreed with the results of pan et al. (2018) who cited that the high pufa content of the diet positively affecting the fecundity, nauplii, and copepodite population of copepoda, apocyclops royi. many dry-diets have been utilizing in copepods production, however, there ideal prospects on copepods (the quality and the quantity) are still needed. overall, regarding to the high percentages of nauplii and copepodite, maximum fecundity, and improved fatty acid profiles, especially pufa content of copepoda, o. nana, the rice bran is the optimal diet for the culturing o. nana. while, regarding to improvement of the quantity of cultured o. nana, the corn starch diet is recommended to produce the maximum population density and the maximum 174 magouz et al./ effects of diet on oithona nana growth rate. references abate t.g., nielsen r., nielsen m., jepsen p.m., hansen b.w. 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(2022) 10(6): 460-473 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article influence of dietary fat sources on growth, bacterial resistance, and antioxidant ability of liver in common carp, cyprinus carpio thi mai nguyen1, thu hang nguyen2, thi ngoc anh do1, hung phuc nguyen3, nang thu tran thi1* 1department of aquaculture nutrition and feed, faculty of fisheries, vietnam national university of agriculture, hanoi, vietnam. 2pharmacology department, hanoi university of pharmacy, hanoi, vietnam. 3department of human and animal physiology, faculty of biology, hanoi national university of education, hanoi, vietnam. s article history: received 7 june 2022 accepted 21 august 2022 available online 2 5 december 2022 keywords: linseed oil sesame oil glutathione mda aeromonas veronii abstract: the current study was conducted to determine the effect of dietary fat sources on fish growth, resistance, and antioxidant ability in common carp. the experimental diets were based on various fat sources, including fish oil (fo), linseed oil (lo), sesame oil (so), and a mixture of lo and so (slo). the common carp (23.8±0.7 g) were distributed into a 100 l-tank system at a density of 25 fish per tank. fish were fed experimental feed to satiation for seven weeks, and the consumable feed amount was recorded daily. after a 7-week trial, fish were infected with aeromonas veronii at a dose of 0.43×106 cfu/ml and monitored for 14 days. the fish mortality was checked daily. fish livers were sampled after feeding and on the second day post-bacterial injection to analyze the antioxidant parameters. the results indicated that the fat sources did not affect the fish growth, feed conversion rate, survival, and cumulative mortality in the challenge test but modified the antioxidant ability in fish liver. the malondialdehyde activity in slo-fed fish was lower than that in fo group at the end of the feeding trial, while the glutathione activity in so-fed fish was higher than those in other plant oil-fed fish after the bacterial challenge. the highest values of superoxide dismutase activity were recorded in lo fish after the nutritional trial and fo ones after the challenge test. introduction lipid is one of the principal sources of nutrients in aquatic feeds. they have many roles, such as providing energy, participating in the cellular structure, dissolving vitamins, and joining the immune system (alabdulkarim et al., 2012; guo et al., 2020). among their structural components, fatty acids are important molecules that play many essential functions in living organisms. besides their nutritional and structural functions, fatty acids play important roles in fish immune responses and other physiological processes. they are classified into saturated fatty acids (sfa), mono-unsaturated fa (mufas), polyunsaturated fa (pufas), and highly unsaturated fa (hufa) (sargent et al., 2002; tocher, 2003; fahy et al., 2005). the n3 hufas, such as dha and epa induce antioxidant and antiinflammatory effects (anderson et al., 2014). therefore, the dietary lipid sources enriched in these correspondence: thi mai nguyen; nang thu tran thi doi: https://doi.org/10.22034/ijab.v10i6.1696 e-mail: ntmai.ntts@vnua.edu.vn; trannangthu@vnua.edu.vn dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.3.6 fas, such as fish oil, are ideal for fish health and physiology. besides fish oil, terrestrial vegetable oils are an abundant source of lipids used in aquafeeds, thanks to their great economic benefits. the plantderived oils are generally enriched in pufa, including linoleic acid, la (c18: 2n-6) and αlinolenic acid, ala (c18: 3n-3) (nguyen et al., 2021) that are the precursors for n3 and n6 hufa biosyntheses such as dha, epa, and ara. as their chain structure contains double bonds, these fatty acids are often highly oxidized but do not contain the trans-fats found in animal fat products. in addition, vegetable oils also contain several natural antioxidants, such as vitamin e (desai et al., 1988). some studies have previously shown that using different fat sources generally did not affect fish growth (monge-ortiz et al., 2018; nguyen et al., 2019b, 2020; sourabie et al., 2019) but influenced the tissue fa composition and fish health status. 461 int. j. aquat. biol. (2022) 10(6): 460-473 indeed, the modifications in innate immune activities such as lysozyme, complement, peroxidase, and phagocytosis were observed in fish fed on various fat sources (montero et al., 2010; xu et al., 2015; zuo et al., 2015; tan et al., 2016, 2017; conde-sieira et al., 2018). however, the effect of dietary oils on the antioxidant ability in fish tissues is still limited (peng et al., 2016; fu et al., 2017). oxidative stress is a general concept to describe a severe imbalance between the production of reactive oxygen species (ros) and the antioxidant defense mechanisms. under these conditions, ros can induce damage in membrane lipids and dna that affect the function of cellular proteins (buico et al., 2009). ros are free radicals, including superoxide anions, hydroxyl radicals, peroxyl radicals, and alkoxyl radicals. these radicals are increased during the bacterial infection or inflammatory response; and can inhibit the activity of pathogenic microorganisms that cause much damage to the host organism (chelombitko, 2018). in normal conditions, the over-production of free radicals is a pathological state (mittal et al., 2014). in these cases, the antioxidants could reduce free radical production and their negative effects on the body (sardesai, 1995; rani, 2017; wilson et al., 2017). common carp, cyprinus carpio linnaeus, 1758 is an omnivorous fish widely cultured worldwide (cai et al., 2019; nguyen et al., 2019b). this species can bioconvert the pufas, such as ala and la, to several important hufas (dha, epa, and ara) through the desaturation and chain elongation processes (nguyen et al., 2022). many studies have previously shown that the use of plant oils instead of fish oil, such as flaxseed/linseed oil, sesame oil, corn oil, canola oil, and sunflower oil, did not affect the growth performance and feed efficiency in common carp (ren et al., 2012; ljubojević et al., 2015; nguyen et al., 2019a, 2021). the effects were observed on the fa profile or fish health status, such as immune response and fish resistance; however, no studies have reported the antioxidant capacity of common carp fed the plant oil compared to fish oilfed ones. some studies were conducted on other fishes, such as rainbow trout oncorhynchus mykiss walbaum, 1792 (kutluyer et al., 2017), black carp mylopharyngodon piceus richardson, 1846 (sun et al., 2011), large yellow croaker larimichthys crocea richardson, 1846 (li et al., 2021), and nile tilapia oreochromis niloticus linnaeus, 1758 (larbi ayisi et al., 2018). therefore, the current research was carried out to evaluate the effects of dietary fat sources on growth, feed utilization, resistance, and antioxidant capacity of the liver in common carp. materials and method the protocol of the feeding trial and bacterial challenge was approved by the vietnam national university animal ethics committee (t2020-0303tđ). the experimental design is briefly described in figure 1. common carp juveniles were collected from a local hatchery and acclimatized for two weeks in the experimental tank system before beginning the experiment. during this period, fish were fed on a commercial feed (austfeed, 35% crude protein, 8% lipid). after two weeks, healthy fish were selected for the experiment. experimental diets: four isoproteic (crude protein = 30.7%) and isolipididic (10%) diets were formulated based on three fat sources: cod liver oil (enriched in hufa), linseed oil (enriched in αlinolenic acid, ala, c18: 3n-3), sesame oil (enriched in linoleic acid, la, c18: 2n-6), and a blend of linseed and sesame oil (v:v, 1:1). the diets are denoted as follows: fo (fish oil-based diet), lo (linseed oil-based diet), so (sesame oil-based diet), and slo (blend of sesame and linseed oil-based diet). the protein sources in experimental diets included fishmeal, wheat gluten, gelatin, and casein. in addition, the diets also contained other ingredients such as modified starch, mineral, and vitamin premix. the feed ingredients were chosen according to the nutritional requirements of common carp (table 1) (nrc, 2011). all the powdered ingredients were well-mixed; then oil was added to the mixture, followed by water to produce a stiff dough. the dough was pelleted with a laboratory pellet mill, then air-dried and stored at 4ºc until use. 462 nguyen et al./ influence of dietary fat sources on common carp feeding trial: seventy-five fish with an initial body weight (ibw) of 23.8±0.7 g were randomly allocated to each of 12 glass tanks (100 l holding capacity), resulting in triplicate tanks per dietary treatment. fish were fed to satiation twice a day with the experimental diets for seven weeks. the consumable feed amount of each tank was recorded daily to calculate the feed conversion ratio (fcr). the environmental parameters in the ras were maintained suitable for common carp, including temperature (from 22 to 25ºc); ph (7-7.5); dissolved oxygen (6-6.5 mg/l); and no2 (<0.05); nh3/nh4 + (<0.05). the rearing tank was siphoned daily to clean the fish faeces and continuously aerated. after a 7week trial, fish were weighed and counted to determine their growth rate and survival. bacterial challenge test: before the bacterial challenge, a median lethal dose (ld50) of aeromonas veronii bacteria was determined in another group of carp juveniles (size of 32.8±5.2 g). figure 1. brief diagram of experimental design. ingredient (g.kg-1 diet, dm) diet fo lo so slo fish meal1 300.0 300.0 300.0 300.0 wheat gluten2 120.0 120.0 120.0 120.0 gelatin3 10.0 10.0 10.0 10.0 casein4 25.0 25.0 25.0 25.0 modified starch5 415.0 415.0 415.0 415.0 cod liver oil (fo)6 100.0 --- linseed oil (lo)7 -100.0 -50.0 sesame oil (so)8 --100.0 50.0 vitamin and mineral premix9 30.0 30.0 30.0 30.0 total 1000.0 1000.0 1000.0 1000.0 estimated crude protein (%) 30.7 30.7 30.7 30.7 estimated crude lipid (%) 10.0 10.0 10.0 10.0 estimated gross energy (ge, mj.kg-1 dm) 18.3 18.3 18.3 18.3 note: fo, fish oil; lo, linseed oil; so, sesame oil; slo, mixture of so and lo. dm: dry matter. (1) phuc loc vung tau co. ltd., hoang a road, tan hai commune, phu my town, ba ria vung tau provinc. (2)(3)(4) sigma aldrich, st louis, mo, usa, (5) baaboo food, ho chi minh city, vietnam, (6) mollers tran, norway, (7)(8) naturgreen, spain, and (9) nova-premix for fish, anova, vietnam. table 1. ingredients and approximate composition of the experimental diets. 463 int. j. aquat. biol. (2022) 10(6): 460-473 briefly, the bacteria were isolated from diseased carp caused by a. veronii and stored in the laboratory of the department of environment and aquatic diseases, faculty of fisheries, vietnam national university of agriculture. the bacterial solution cultured at 30ºc for 24 hours was considered the stock solution (with od = 1.374 at 610 nm), corresponding to a concentration of 109 cfu.ml-1. the stock solution was diluted into concentrations of 108, 107, 106, 105, 104, and 103 cfu.ml-1. fish was injected with these bacterial concentrations at a volume of 0.1 ml per fish (ten fish for each concentration). fish were then monitored for 14 days, where the number of dead fish in each tank with the specific disease symptom caused by a. veronii was recorded daily. the ld50 dose was calculated based on the cumulative mortality rate in each bacterial concentration. at the end of the feeding trial, 30 fish in each experimental condition were intraperitoneally injected with a. veronii at a dose of ld50 and then randomly divided into a 120 l-tank system at a density of ten fish per tank. a group of ten fish from all experimental conditions was used as a control in which the fish were injected with a physiological saline solution instead of a bacterial one. the injected fish were then monitored for 14 days, and the number of dead fish in each tank were recorded daily. dead fish with pathological symptoms caused by a. veronii bacteria in common carp, as described by yu et al. (2010) were dissected and re-isolated to confirm the cause of death. sample collection and analysis: at the end of the nutritional trial and the second-day post-bacterial infection, fish were starved for 24 h, then anaesthetized with clove oil (50 mg/l) and used for sampling. fish livers (three samples per tank) were then collected to measure the antioxidant parameters such as malondialdehyde (mda), glutathione (gsh), and superoxide dismutase (sod) activities. the analysis protocols were detailed as follows: malondialdehyde activity (mda): the mda level in the liver homogenate was determined according to wasowicz et al. (1993). liver samples were first homogenized in phosphate buffer (100 mm, ph = 7.5) according to the ratio of 1 : 10 (w : v) at 4ºc. after cold centrifugation for 30 min, 150 µl of the solution was placed in a glass tube containing 1 ml of distilled water and 1 ml of 0.5 % thiobarbituric acid. the mixture was heated in a water bath at 99ºc for one hour. after cooling at room temperature, the mixture was added with 25 µl of 5 m hcl. the solution was then measured at 532 nm. in parallel, 150 µl of tetramethoxypropane solution was used as a standard. the mda content (nmol.ml-1 of homogenate) was calculated according to the linear regression equation with the tetramethoxypropane standard, and the mda content (nmol.mg-1 protein) of the fish liver was also considered. glutathione activity (gsh): gsh content in the liver was determined by ellman reagent (koh et al., 2012). the homogenate of the liver procedure was described as above. the homogenate was used for gsh quantification. the reaction mixture in each well of the 98-well plate was prepared as follows: 20 µl of homogenate solution, 160 µl of tris-hcl buffer ph 7.5, and 20 µl of 5.5′-dithiobis (2nitrobenzoic acid) (dtnb) 10 mm solution. the absorbance of the product was immediately recorded at 412 nm. the gsh content (µg.ml-1) in 1 ml of liver homogenate was determined based on a linear regression equation with the gsh standard. gsh content was expressed as μg reduced gsh.mg-1 protein liver. superoxide dismutase (sod): sod activity in liver homogenate was determined according to marklund's method with modifications (tung et al., 2017). the method is based on the ability of sod to inhibit the auto-oxidation reaction of pyrogallol. the reaction mixture includes tris-hcl buffer 100 mm, 1mm edta, ph 8.2; 10 µl homogenous, and 20 µl of 13 mm pyrogallol. the mixture solution was then measured at 420 nm. sod activity is determined by u.min-1 per mg protein, where the unit (u) of sod activity is defined as the total amount of enzyme that can inhibit the auto-oxidation of 50% of pyrogallol in the reaction. 464 nguyen et al./ influence of dietary fat sources on common carp data analyses: mean values were first checked for homogeneity by the univariate test. the data were then subjected to one-way anova followed by a lsd post-hoc test using the replicate tank as a statistical unit for husbandry variables (n = 3) and the number of samples for each experimental condition for antioxidant parameters (n = 9). differences between treatments were considered significant at p<0.05. all data were analyzed with the statistica 10.0 software (statsorf, inc., east 14 street, tulsa, usa). results growth, feed utilization, and survival rate: after a 7-week feeding trial, the husbandry parameters were shown in table 2, but no significant differences were observed between the experimental groups (p>0.05). the average fish weight increased from 23.84±0.73 to 33.70±0.69 g corresponding to 37.9±5.3 to 43.8±3.2% of the weight gain; the daily growth rate reached 0.2 g.fish-1 per day; the specific growth rate ranged from 0.7 to 0.8%.day-1. the feed conversion ratio (fcr) varied from 1.9 to 2.1. the fish survival reached 100% in all of the experimental groups. cumulative mortality rate: based on the cumulative mortality rate recorded in each tank, the ld50 dose of a. veronii in common carp juveniles was determined as 0.43x106 cfu.ml-1. after injection with ld50 dose, the fish mainly died from the first to the third day of the challenge experiment. the number of dead fish in each tank was daily recorded and the cumulative mortality results after a 14-day challenge are shown in figure 2. variable experimental groups fo lo so slo ibw (g.fish-1) 24.3±0.7 23.3±1.2 23.8±0.4 23.9±1.8 fbw (g.fish-1) 33.5±1.0 34.0±0.5 33.6±0.3 33.8±1.0 dwg (g.fish-1 per day) 0.2±0.0 0.2±0.0 0.2±0.0 0.2±0.0 sgr (%.day-1) 0.7±0.1 0.8±0.1 0.7±0.0 0.7±0.1 wg (%) 37.9±5.3 43.8±3.2 41.1±1.1 41.1±4.7 fi (g.fish-1 per day) 0.41±0.02 0.43±0.03 0.38±0.05 0.41±0.02 fcr 2.2±0.2 2.0±0.1 1.9±0.2 2.1±0.3 survival rate (%) 100.0 100.0 100.0 100.0 note: ibw: initial body weight; fbw: final body weight; dwg: daily weight gain; sgr: specific growth rate; wg: weight gain; fi: feed intake; fcr: feed conversion rate. values are represented as mean±sd. table 2. growth, feed utilization, and survival of common carp used the experimental diets based on the fish oil (fo), linseed oil (lo), sesame oil (so), and a mixture of so and lo (slo) after a 7-week feeding trial. figure 2. cumulative mortality rate of infected fish after bacterial challenge (note: fo, fish oil; lo, linseed oil; so, sesame oil; slo, mixture of so and lo; ctrl: the control without bacterial infection). 465 int. j. aquat. biol. (2022) 10(6): 460-473 accordingly, the cumulative mortality rate was not influenced by dietary fat sources (p>0.05) and ranged from 33.3±5.8 to 40.0±0.0 %. the signs of dead fish included abdominal distension, red swelling, and bleeding in the skin, base of the fins, and anus. the internal organs, such as the liver and spleen, were pale, while the intestines were hemorrhagic. the kidney tissues were then stained and used for bacterial re-isolation. the results showed that a. veronii belonged to the negative gram and rod-shaped bacterium. the colony form is round and creamy yellow (fig. 3). antioxidant capacity of liver: the activity of glutathione (gsh, fig. 4), malondialdehyde (mda, fig. 5), and superoxide dismutase (sod, fig. 6) were measured in the common carp liver samples at the end of the nutritional trial and after bacterial infection. the gsh contents in the liver of carp dissected at the end of the feeding trial (from 35.23 to 39.83 µg.mg-1 protein) were not affected by the dietary fat sources (p>0.05). the differences between experimental conditions were displayed in the liver samples collected after bacterial infection (p<0.05, fig. 4); accordingly, the gsh level in sofed fish (22.75±4.51 µg.mg-1 protein) was higher than those in other plant oil-fed groups and figure 3. images of dead fish after bacterial infection including external (a, b) and internal symptoms (c); comparison pictures between infected fish (above) and control fish (below) including external (d), anatomical (e), and intestinal symptoms (f); stained kidney tissue (g); bacterial colonies on nutrient agar medium (h); and gram-stained bacteria (i). 466 nguyen et al./ influence of dietary fat sources on common carp equivalent to the fo one (18.5±6.28 µg.mg-1 protein). furthermore, the gsh activities in the fish livers after bacterial infection in all of the treatments were reduced in comparison with those at the end of the nutritional experiment (p<0.05). similar to gsh activity, the malondialdehyde (mda) contents in the fish liver after bacterial infection were lower than those observed at the end of the feeding trial (p<0.05, fig. 5). the influence of dietary fat sources on mda level in carp livers was recorded at the end of the nutritional trial (p<0.05) while no differences were found after bacterial challenge (ranging from 3.25 to 3.80 nmol.mg-1 protein). accordingly, the highest value of mda activity was observed in the fo-fed fish (11.63±3.03 nmol.mg-1 protein) that was equivalent to lo-fed fish (9.40±3.80 nmol.mg-1 protein) and so (10.72±4.58 nmol.mg-1 protein); the lowest level of mda activity was found in slo-fed ones (7.21±3.10 nmol.mg-1 protein). sod levels in carp livers also tended to decrease after bacterial challenge compared to those at the end of the feeding trial in almost all of the experimental groups (fig. 6). the influence of dietary lipid figure 4. glutathione (gsh) content in liver of carp fed on the different fat sources including fish oil (fo), linseed oil (lo), sesame oil (so), and a mixture of so and lo (slo) for seven weeks and after infection with aeromonas veronii. data are represented as mean±sd. values with common superscript letter denote non-significant differences (p>0.05). figure 5. malondialdehyde (mda) content in liver of carp fed on the different fat sources including fish oil (fo), linseed oil (lo), sesame oil (so), and a mixture of so and lo (slo) for seven weeks and after infection with aeromonas veronii. data are represented as mean±sd. values with common superscript letter denote non-significant differences (p>0.05). 467 int. j. aquat. biol. (2022) 10(6): 460-473 sources on sod activity was displayed at the end of the nutritional trial as well as post-bacterial infection (p<0.05). specifically, after a 7-week feeding trial, the highest value of sod activity was found in lofed fish (218.92±52.15 u.mg-1 protein), while the lowest one was observed in the so-fed group (143.78±41.10 u.mg-1 protein). similarly, the lowest level of sod activity was still recorded in so-fed fish (71.97±31.40 u.mg-1 protein), whereas the highest value was observed in the fo-fed ones (120.32±43.09 u-1mg protein). furthermore, only the value of sod activity in fo-fed fish was not reduced after bacterial infection. discussions in the current experiment, plant oil instead of fish oil did not negatively affect fish growth and feed utilization (table 2). this result is consistent with previous findings in common carp when no adverse effects of dietary plant oil on growth and feed performances were reported in the common carp diet (ren et al., 2012; nguyen et al., 2019b, 2020). the same data were also previously documented in other omnivorous fish such as triangular bream, megalobrama terminalis richardson, 1846 (tian et al., 2018) and nile tilapia (teoh and ng, 2016). on the other hand, the reductions in growth, feed efficiency, or survival rate were usually found in carnivorous/marine fish such as pike-perch, eurasian perch linnaeus, 1758 (geay et al., 2015), turbot, scophthalmus maximus linnaeus, 1758 (benedito-palos et al., 2008; montero et al., 2010), european seabass, dicentrarchus labrax (geay et al., 2011; torrecillas et al., 2017), and rainbow trout, oncorhynchus mykiss (kutluyer et al., 2017; mellery et al., 2017) fed on plant oil. the differences in fish growth performance and feed efficiency were generally observed in fish fed different levels of lipid or protein sources (nguyen, 2020). in the current study, the experimental diets differed only in fat sources, while the lipid levels and protein ingredients were homogenous, leading to comparable results in fish growth and feed utilization for all of the conditions. furthermore, reductions in fish growth were observed when the fish ingredients, including fishmeal and fish oil were totally replaced with plant-based ingredients. conversely, no differences were found in the case of a low level of fish oil replacement and fish meal used as the main protein source (nguyen et al., 2019b). influence of fat sources on fish survival and resistance to a. veronii: after the feeding trial, the fish survival reached 100% in all of the treatments, indicating that the dietary fat sources in the feed formulation did not affect the fish survival rate under rearing conditions. in addition, the suitable figure 6. superoxide dismutase (sod) activity in liver of carp fed on the different fat sources including fish oil (fo), linseed oil (lo), sesame oil (so), and a mixture of so and lo (slo) for seven weeks and after infection with aeromonas veronii. data are represented as mean±sd. values with common superscript letter denote non-significant differences (p>0.05). 468 nguyen et al./ influence of dietary fat sources on common carp environmental parameters for common carp during the experiment were also a reason for the absolute value of fish survival rate. after bacterial infection, the fish mortality was not significantly different between experimental groups, suggesting that the dietary lipid sources did not affect the resistance to a. veronii. these results were similar to the previous studies on common carp fed on the linseed and sunflower oil-based diets (nguyen et al., 2019b), nile tilapia fed on coconut oil (apraku et al., 2017) and palm oil (larbi ayisi et al., 2018). contrarily, some other studies have shown that fish mortality increased in carnivorous/marine fish using vegetable oils such as large yellow croaker (tan et al., 2016) and japanese sea bass, lateolabrax japonicus (tan et al., 2017). this difference can be explained by the different capacities of hufa synthesis in these two fish groups. generally, omnivorous / freshwater fish have a good ability to synthesize hufas from the pufa precursors such as ala and la in plant oils, while this ability in carnivorous / marine fish is less efficient by the deficiency of several enzymes involved in this process (oliva-teles, 2012). in animals, some hufas such as ara, epa, and dha are the precursors synthesizing several lipid mediators in the immune response (nguyen et al., 2021). therefore, the resistance to a. veronii of common carp, an omnivorous fish that can convert the pufa precursors to hufa, was not affected by the tested plant oils. the symptoms of dead fish after bacterial infection, tissue histopathological, bacteria, and colony characteristics in this study were similar to those in the disease common carp caused by a. veronii described in previous studies (chen et al., 2019; hoai et al., 2019). effect of dietary fat sources on the antioxidant ability in fish liver: in the body, free radicals are formed by a number of exogenous and endogenous processes. their negative effects are generally neutralized by antioxidant defenses. oxidative stress occurs due to an imbalance between the production of free radicals and antioxidant defenses. when the free radicals are generated more than the threshold that antioxidants can keep in balance, the free radicals begin to damage the fat tissue, dna, and proteins in the body, consequently leading to numerous diseases, signs of aging, and reduced lifespan (buico et al., 2009; liguori et al., 2018). therefore, increasing antioxidants by food intake is a great idea to maintain a good health for animals in general and aquatic animals in particular. to evaluate the antioxidant capacity of tissues; some parameters such as gsh, mda, and sod were generally used. regarding the results of gsh content, no differences were observed between experimental groups at the end of the feeding trial, indicating that the replacement of fish oil with linseed oil, sesame oil, or their mixture did not induce any negative effect on gsh activity in carp liver. other studies have also previously shown that the use of dietary vegetable oils instead of fish oil did not reduce the antioxidant capacity in freshwater / omnivorous fish such as rockfish, sebastes schlegeli (aminikhoei et al., 2013), nile tilapia (larbi ayisi et al., 2018), and onychostoma macrolepis (gou et al., 2021). in contrast, several studies demonstrated the decreases in the antioxidant capacity in the liver of fish using plant oil-based diets in marine fish like japanese sea bass, l. japonicus (tan et al., 2017), large yellow croaker (mu et al., 2018, 2020; li et al., 2019), and red seabream, pagrus major (mzengereza et al., 2021). after bacterial infection, the gsh level in so-fed fish was higher than that measured in fish fed on the other plant oil diets (lo and slo), and comparable to the fo-fed group, indicating that the use of dietary sesame oil induced a higher antioxidant ability compared to those in other plant oil-fed carps. glutathione is an antioxidant compound found in plants, animals, fungi, and some bacteria that can prevent the important cellular components against the damage caused by reactive oxygen species such as free radicals, peroxides, and heavy metals (kerksick and willoughby, 2005; forman et al., 2009; silvagno et al., 2020). during the bacterial infection or inflammatory process, the host body generally produces a number of strong oxidants that could destruct and inhibit the activities 469 int. j. aquat. biol. (2022) 10(6): 460-473 of pathogens or other exogenous agents (abdulkhaleq et al., 2018). these processes endamage the exogenous agents and negatively affect the host body if no regulatory mechanisms appear. in the current study, the common carp can convert la, which is abundant in dietary sesame oil, to arachidonic acid (ara). ara contents in the liver (3.8 mg.g-1) and dorsal muscle (1.9 mg.g-1) were previously quantified (nguyen, 2020) that were even higher than those measured in fish oil-fed fish (0.9 mg.g-1). in the body, ara is a precursor to produce some lipid mediators involved in the inflammatory response and the recovery process after inflammation (nguyen et al., 2021), including antioxidant reaction. this argument may explain the highest antioxidant ability corresponding to the highest level of gsh was found in fish-fed sesame oil containing a high level of linoleic acid. sod is a special enzyme that could convert the destructive superoxides in the host body into nontoxic substances. regarding the results of sod activity, no influences of dietary fat sources were recorded at the end of the feeding period, suggesting that the dietary plant oil did not negatively affect the antioxidant ability in fish liver under normal conditions. however, this capacity in the lo-fed group was higher than that in so-fed fish. previous studies have also demonstrated the better antioxidant ability in fish fed linseed oil compared to other plant oil sources in rainbow trout (kutluyer et al., 2017), large yellow croaker (li et al., 2021), and o. macrolepis (gou et al., 2021). reported data have also demonstrated higher levels of dha and epa in fish-fed diet enriched in ala, an n3 pufa precursor. the supplementation of dha and epa in the fish diet has been previously documented to enhance the antioxidant ability in fish under basal conditions (engstrm et al., 2009; capó et al., 2015; othman et al., 2015). under the bacterial infection, the sod activity in so-fed fish was lower than that of fo-fed ones, while other plant oil-fed groups were comparable to fo-fed one, suggesting that sod activity decreased in the so group after bacterial infection. this result could be explained by the higher gsh content in so-fed fish post-bacterial challenge compared to other plant oil-fed groups, leading to the decrease of sod to keep the balance of the antioxidant process in the host body. contrary to gsh and sod, malondialdehyde (mda) is one of the products of lipid oxidation, known as an indicator of lipid oxidation degree in the body. the degree of oxidation in plasma and tissues is correlated with mda concentrations (ayala et al., 2014). the mda level in slo-fed fish at the end of the feeding trial was lower than that of fo-fed group, indicating that the lipid oxidation activity of the slo fish was lower than that of the fo group under normal conditions; i.e. the antioxidant ability in the liver of fish fed on the mixture of linseed and sesame oil was higher than that in fish oil-fed fish. this result was probably explained by the balance of pufa precursors in the slo diet, including la and ala, which are converted to an amount enough of important hufas involved in immunoregulatory processes such as the redox reactions. furthermore, the balance of the n3 / n6 fas ratio in slo probably positively affected the health status of experimental fish. as recommended, this ratio should be close to one (simopoulos, 1991; gómez candela et al., 2011; bhardwaj et al., 2016). although the n3 / n6 ratio in fish oil control is close to one, the levels of ala and la in fish oil were much lower than those of the slo, explaining why the antioxidant ability of slofed fish was higher than that of the fo-fed group. no difference in mda concentrations was observed after bacterial infection, indicating that terrestrial vegetable oils did not negatively affect the antioxidant ability of infected fish. conclusion the replacement of fish oil with linseed oil, sesame oil, and their blends did not affect the growth, feed efficiency, survival, and resistance to a. veronii bacteria in common carp. using these plant oil sources did not negatively affect the antioxidant ability in the liver of common carp. in addition, linseed oil and its mixture with sesame oil have improved the antioxidant capacity of common carp 470 nguyen et al./ influence of dietary fat sources on common carp under a basal condition, while sesame oil boosted this ability under a bacterial infection. acknowledgements this work was supported by the funding source for research activities from the vietnam national university of agriculture, project no. t2020-0303tđ. references abdulkhaleq l.a., assi m.a., abdullah r., zamri-saad m., taufiq-yap y.h., hezmee m.n.m. 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(2016) 4(6): 370-377: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article sublethal toxicity of tio2 nanoparticles to common carp (cyprinus carpio, linnaeus, 1758) under visible light and dark conditions mahdi banaee*,1shima shahafve, somaye tahery, behzad nematdoost haghi, maryam vaziriyan 1department of aquaculture, faculty of natural resources and environment, behbahan khatam al-anbia university of technology, iran. article history: received 12 november 2016 accepted 26 august 2016 available online 2 5 december 2016 keywords: tio2 nanoparticles common carp biochemical parameters photoperiod conditions abstract: the objective of this study was to determine the sublethal toxicity of tio2 nanoparticles (tio2-nps) on common carp (cyprinus carpio) under visible light and dark conditions. blood sampled was collected after 21 days and biochemical parameters, including glucose, total protein, albumin, globulin, creatinine, triglyceride and cholesterol levels, and aspartate aminotransferase (ast), alanine aminotransferase (alt), γ-glutamyl transferase (ggt), lactate dehydrogenase (ldh), creatine kinase (ck), and alkaline phosphatase (alp) activities were measured. the results showed that tio2-nps is caused a significant effect on blood biochemical parameters of c. carpio. by changing lighting conditions from darkness to light, significant differences were observed in certain blood biochemical parameters, including ast, alt, ldh, alp and ck activities, glucose, cholesterol and triglyceride levels in fish exposed to tio2-nps under light conditions as compared with fish exposed to tio2-nps under dark conditions. cholesterol and triglyceride levels in fish exposed to 0.0 mg l-1 tio2-nps under darkness conditions were significantly higher than the control. the results revealed that toxicity of tio2-nps under visible light conditions was more than darkness conditions. introduction the application of nanotechnology in industries such as biomedical sciences, pharmaceutical and cosmetics, wastewater treatments and electronic, and consequently the availability of nanoparticles into the environment are increasing. therefore, nanoparticle products are rapidly accumulating in the aquatic ecosystems (nowack and bucheli, 2007) and their potential for exhibiting environmental toxicity is growing. titanium dioxide (tio2) nanoparticles (tio2nps) have the most industrial application compared to other nanoparticles. ortlieb (2010) reported an annual amount of four million consumption of tio2 nanoparticle, used as pigments in a variety of products including food colorings, paper products, ink and plastic products (ortlieb, 2010), cosmetics (as a uv filter), shampoos, soaps, toothpastes, sunscreens (melquiades et al., 2008), building * corresponding author: mahdi banaee e-mail address: mahdibanaee@yahoo.com materials and paints (chen and poon, 2009) as well as many synthetic vitamin tablets, over-the-counter pain relievers, capsulated antidepressants and antibiotic products (luft et al., 2010). in addition, the nanoparticles of tio2 are used in drinking water treatment especially for arsenic removal and also used as a photocatalyst in wastewater treatment plants for removal of contaminants. therefore, tio2nps is one of the biggest environmental concern due to their easy transfer to aquatic ecosystems (kaegi et al., 2008). according to the reports, the amount of tio2-nps estimated to be 0.0007 to 0.0245 µg ml -1 is available in the environment (mueller and nowack, 2008; pérez et al., 2009). the widespread use of various nanoparticles in pharmaceutical and cosmetic industry, producing disinfectants, wastewater treatments and chemical removal from water have all contributed to increasing amount of nanoparticles to the surface 371 int. j. aquat. biol. (2016) 4(6): 370-377 water (zou et al., 2014; lei et al., 2016). therefore, aquatic organisms are constantly exposed to a variety of potentially toxic nanoparticles (li and lenhart, 2012; zou et al., 2014; koetsem et al., 2015; zhang et al., 2015; lei et al., 2016). exposure to tio2-nps significantly induced the antioxidant enzymatic activity and increased the lipid peroxidation levels most evidently in liver, and caused histopathological damages in different tissues of common carp (hao et al., 2009), zebrafish (xiong et al., 2011) and rainbow trout (federici et al., 2007). however, our knowledge on the potential toxic effects of tio2-nps on the health of these organisms in regard to biochemical parameters is really limited. many of the toxic effects of tio2-nps on the environment depend on the interactions between the physicochemical properties of tio2 and physical and chemical conditions of the water parameters. since light exposure can play an active role in the photocatalytic activity of tio2, we hypothesize that there is a significant difference between the toxicity of tio2-nps under visible light and darkness. therefore, the aim of this study was to evaluate the toxicity of nanoparticles of tio2 on blood biochemical parameters of common carp (cyprinus carpio) under light photoperiod (light: dark: 16: 8 h) and dark (no irradiation) conditions. materials and methods nanoparticles characterization: commercial tio2nps, with an average primary particle size of 20 nm in the powder form (table 1), were purchased from iranian nano-materials pioneers company. fish: juvenile common carp with average body weight of 30±5 g were maintained in the laboratory of the department of aquaculture, khatam al-anbia university of technology, iran. fish were maintained in 80 l tanks with de-chlorinated tap water under controlled environmental conditions at 24±2°c on a 16:8 h (light: dark) photoperiod during two weeks acclimatization period. fish were fed with commercial carp pellets (beyza fedd mill co. iran) at the manufacturer’s recommended rate. preparation of test nanoparticles: stock solutions of tio2-nps were prepared using distilled water and then ultrasonicated (10 min, 35 khz, 100/400w) using an ultrasound bath (elma, germany) for dissolution. then, solutions were added to 10 l of dechlorinated tap water in exposure tanks to obtain nominal concentrations of 0.125 mg l-1 tio2. sublethal toxicity: a total of 144 common carp were randomly distributed in twelve 80 l tanks (4 treatments with three replicates). the experiment was conducted for 21 day sublethal toxicity tests. every tank contained 12 fish which were exposed to test solutions with the following concentrations of tio2-nps: 0.0 mg l -1 tio2-nps, and 0.125 mg l -1 tio2-nps each under different dark and light conditions. sublethal concentrations were selected according to xiong et al. (2011), hao et al. (2009) and lee et al. (2012). the experiments were carried out according to a factorial scheme considering two factors (at different concentrations of tio2 and photoperiod conditions) in two parallel sets. visible light irradiation was provided by four white fluorescent lamps (600 lux, 40 w, pars-shahab, iran). in darkness experiments, the tanks were wrapped with opaque sheets and were kept in darkness without intervention of any visible light. the photoperiod (16:8 h) condition was considered as light condition and dark (no irradiation) as dark condition throughout the experiment. although continuous aeration of water may partly prevent deposition of nanoparticles on the bottom of tanks, nanoparticles tend to form agglomeration (hao et al., 2009). the actual amount of tio2 nps may decrease up to 50% after 3 days (hao et al., 2009). therefore, 50% of the water was exchanged every 12 hours and nanoparticles solution was added again to the tanks to maintain tio2-nps concentrations in constant (equivalent to 0.125 mg per liter). this is necessary to inhibit the agglomeration of nps and their absorption by fish feces and uneaten food. sampling and analysis of blood biochemical parameters: fish were starved for 24 hrs before sampling. after the 21-day exposure period, 12 fish from each treatment (4 fish from each tank) were 372 banaee et al./ toxicity of tio2 nanoparticles to common carp removed for sublethal toxicity studies. fish were quickly netted from tanks and placed in 4 liter buckets filled half with 200 mg l-1 of clove powder solution. blood sample was collected from the caudal vein using heparinized syringes, centrifuged at 6000×g for 10 min and stored at -25°c. all blood biochemical parameters were determined using a uv-visible spectrophotometer (unico 2100) and standard biochemical reagents (pars azmun company, tehran, iran). each blood biochemical parameter was measured by a certain method. total plasma protein was measured at 540 nm by the biuret reaction. albumin assay was based on the dye-binding properties of plasma albumin with bromocresol green. an increase in blue-green color was measured at 630 nm. the plasma globulin was estimated based on the ratio of albumin versus total protein (johnson et al., 1999). plasma glucose was measured by the glucose-oxidase method at 500 nm (sacks, 1999). plasma cholesterol levels were determined by the chod-pap method at 510 nm, triglyceride levels by gpo-pap method at 546 nm (rifai et al., 1999) and creatinine by jaffe method at 510 nm (foster-swanson et al., 1994). urea is hydrolyzed enzymatically by urease to yield ammonia and carbon dioxide. the ammonia and αoxoglutarate are converted to glutamate in a reaction catalyzed by l-glutamate dehydrogenase. simultaneously, one molar equivalent of reduced nadh is oxidized. two molecules of nadh are oxidized for each molecule of hydrolyzed urea. the rate of change in absorbance at 340 nm, due to the disappearance of nadh, is directly proportional to the blood urea concentration in the sample (lumeij and remple, 1991). the activities of aspartate aminotransferase (ast) and alanine aminotransferase (alt) in plasma were determined by nadph consumption and its conversion to nad+ at 340 nm. gamma-glutamyl transferase (ggt) activity is determined by a coupled enzyme assay in which ggt transfers the γ-glutamyl group from the substrate l-γ-glutamyl p-nitroanilide, liberating the chromogen p-nitroanilide at 418 nm proportional to ggt. lactate dehydrogenase (ldh) in plasma was determined based on the conversion of pyruvate to lactate at 340 nm, alkaline phosphatase (alp) based on converting nitrophenol phosphate into nitrophenol and phosphate at 405 nm, creatinine kinase (ck) based on the conversion of creatinine phosphate into creatinine at 340 nm and based on optical density (od) absorption and the formula presented in the kits' manual (moss and henderson, 1999). statistical analysis: all data were examined for normality (kolmogorov-smirnov test) and then analyzed for significance using one-way analysis of variance (anova). the significant means were compared by duncan’s test and a p<0.05 was table 1. physicochemical proprieties of tio2 nanoparticles according to the manufacturer. titanium oxide tio2, 80 vol% anatase + 20 vol% rutile purity +99% average primary particle size (d50) 20 nm specific surface area (ssa) 10-45 m2 g-1 color white bulk density 0.46 g ml-1 ph 5.5-6.0 tio2 ≥99% al ≤17 ppm mg ≤65 ppm si ≤120 ppm ca ≤75 ppm s ≤130 ppm nb ≤80 ppm loss of weight in drying 0.48% loss of weight on ignition 0.99% 373 int. j. aquat. biol. (2016) 4(6): 370-377 considered statistically significant. statistical analyses were performed using spss 19 (ibm corp.). data are presented as mean ±sd. results no significant mortality was observed in all treatments. during the assay, increased mucus secretion, color changes, a progressive loss of scales, behavioral changes such as tremors, lethargy, and erratic swimming in the surface water were important changes found in the individuals exposed to tio2-nps. the effects of sublethal concentration (0.125 mg l-1) of tio2-nps on the blood biochemical parameters of common carp under photoperiod (light: dark: 16: 8 h) and dark (no irradiation) conditions are shown in table 2. fish exposure to 0.0 mg l-1 tio2-nps under dark conditions showed no significant effects on the activity of ast, alt, ldh, alp, cpk and ggt and levels of glucose, creatinine, total protein, albumin and globulin. however, cholesterol and triglyceride levels in these fish were significantly higher than the control group i.e. under light condition (p<0.05). the plasma ast, alt, ldh, alp and ck activities showed a significant increase in fish exposed to tio2-nps compared to control group (p<0.05). the plasma ast, alt, ldh, alp and ck activities in fish exposed to tio2-nps under light conditions were significantly greater (p<0.05) than fish exposed to tio2-nps under dark conditions. ggt activity, total protein and globulin levels in the plasma of fish exposed to tio2-nps in both photoperiod conditions were significantly lower (p<0.05) compared to the respective control groups. hyperglycemic condition was significant (p<0.05) in both photoperiod conditions in fish exposed to tio2-nps. plasma glucose levels in fish exposed to 0.125 mg l-1 of tio2-nps under light photoperiod conditions were significantly lower than fish exposed to tio2-nps under dark conditions (p<0.05). there was a significant increase (p<0.05) in creatinine levels of fish exposed to tio2-nps compared to control group. however, no significant changes were observed in levels of plasma albumin in fish treated with tio2-nps compared to the control group (p>0.05). a significant decrease (p<0.05) was observed in cholesterol levels in fish exposed to 0.125 mg l-1 of tio2-nps under light photoperiod conditions compared with other groups. triglyceride levels in plasma of fish exposed to tio2-nps were significantly lower than control group, and a significant decrease (p<0.05) was observed in triglyceride levels in fish exposed to tio2-nps under light photoperiod conditions as compared to fish exposed to tio2-nps under dark table 2. alterations in the blood biochemical parameters of common carp, cprinus carpio exposed to tio2 nanoparticles (0.00 and 0.125 mg l-1) under photoperiod (light: dark: 16: 8 h) and dark (no irradiation) conditions. blood biochemical parameters 0.00 mg nano-tio2 (light) 0.00 mg nano-tio2 (dark) 0.125 mg nano-tio2 (light) 0.125 mg nano-tio2 (dark) ast (u l-1) 44.22±3.77a 54.35±6.60a 125.94±19.21c 70.95±6.65b alt (u l-1) 14.67±1.91a 11.67±0.47a 80.54±20.43c 26.84±4.29b ldh (u l-1) 146.94±11.34a 182.53±15.76a 305.76±67.74c 260.04±33.47b alp (u l-1) 59.33±5.80a 55.91±3.31a 113.95±10.40c 93.74±19.24b cpk (u l-1) 318.90±76.30a 324.25±14.16a 1421.37±250.52c 1126.75±258.81b ggt (u l-1) 14.15±1.06b 14.76±0.82b 4.93±1.15a 4.29±1.05a glucose (mg l-1) 58.32±9.76a 56.48±5.37a 79.09±8.58b 95.01±12.13c creatinine (mg l-1) 0.14±0.02a 0.17±0.02a 1.17±0.42b 1.08±0.24b total protein (g dl-1) 4.06±0.35b 4.29±0.25b 2.82±0.49a 2.94±0.54a albumin (g dl-1) 2.03±0.28a 2.15±0.37a 2.22±0.29ab 1.99±0.16a globulins (g dl-1) 2.03±0.53b 2.13±0.52b 0.60±0.44a 0.95±0.45a cholesterol (mg l-1) 145.03±16.86b 166.62±14.73c 90.31±17.03a 131.44±5.83b triglycerides (mg l1) 222.85±16.62c 251.51±30.27d 99.06±10.63a 139.91±19.46b statistically significant differences comparatively to controls if * (p<0.05). alphabet letters indicate significant differences between groups. 374 banaee et al./ toxicity of tio2 nanoparticles to common carp conditions (p<0.05). discussion the results have shown that damage to the skin and the consequent loss of fish scales may facilitate the penetration of tio2 into body of the fish. the attachment of tio2-nps to cells might impair the physiological function of cell membranes. since skin and gills are in direct contact with the aqueous pollutants, increased mucus may act as a barrier and defense mechanism in sensitive stratified gill epithelium and skin epithelium against tio2-nps (smith et al., 2007). biochemical data showed that tio2-nps at sublethal concentrations exerted a significant effect on some of the studied blood biochemical parameters. reeves et al. (2008) found that in addition to generation of reactive oxygen species (ros), possible mechanisms of toxicity to organisms induced the adhesion of tio2 to cells and physical disruption of the cell membranes. there is strong evidence that tio2-nps are able to generate ros such as hydroxyl (oh) radical (reeves et al., 2008), superoxide radical anions (o2 -) and hydrogen peroxide (h2o) (dodd and jha, 2009) not only in the presence of uv irradiation but also in the absence of photo-activation (armelao et al., 2007; gurr et al., 2005; reeves et al., 2008). when cells are attacked by ros, cell membrane happens to be the first target. in the present study, increased ast, alt, ldh, alp and ck activities were observed in fish exposed to tio2-nps, which might be due to increased cellular membrane damage and enzymes leaking out of cells which are damaged by tio2-nps. similar results have been reported by wang et al. (2007), chen et al. (2009), duan et al. (2009) and liu et al. (2009). however, the activity of these enzymes in the photoperiod condition was found to be more than dark condition. the results showed that damage to cell membrane in visible light conditions was more severe than that of darkness conditions. tio2 nanoparticles may react with oxygen and water molecules to produce reactive oxygen species under dark conditions (fenoglio et al., 2009). in addition to the direct interaction of tio2-nps with oxygen and water molecules under light conditions, tio2-nps can absorb the light to produce hydroxyl radicals, which can provide the basis for the production of superoxide anion, super hydroxide radicals and peroxide hydrogen (li et al., 2015). the results indicate that increased production of ros in an environment treated with nanoparticles of tio2 under light conditions can cause more damage to cell membranes. ros and its consequent oxidative stress may cause peroxidation of the phospholipid membranes and direct damage to proteins, and affect the physiological function of the cell membrane (reeves et al., 2008; dodd and jha, 2009). hao et al. (2009) confirmed that histopathological damage to various tissues of juvenile carps exposed to tio2-nps was related to oxidative stress. the hyperglycemic condition observed in the present study could be attributed to the mobilization of glucose or increased secretion of cortisol due to tio2 toxicity. hyperglycemia or elevated blood glucose levels indicated the impaired glucose and lipid metabolism and degradation of glycogen in liver (liu et al., 2009). glycogen depletion has also been reported by lourenço (2012) in gold fish (carassius auratus) exposed to concentrations of tio2-nps within a range from 0.01 to 800 mg l -1. a decrease in total protein and globulin levels in fish exposed to tio2-nps indicated reduced protein and globulin synthesis in hepatocytes. the decrease in protein levels may be related to malnutrition, the increased energy cost of homeostasis, tissue repair and the detoxification mechanism under stress conditions. griffitt et al. (2009) found that tio2-nps could affect the expression of genes involved in protein synthesis process. also, decreased globulin levels may reduce the resistance of fish to pathogens. increased photoperiod is reported to have a significant effect on the activity of enzymes involved in fatty acid synthesis and fat accumulation in tissues (faulconnier et al., 2001). moreover, the influence of photoperiod on the synthesis level and excretion of insulin, thyroid hormones and corticosteroids 375 int. j. aquat. biol. (2016) 4(6): 370-377 which play an important role in fat metabolism is interesting (marie et al., 2001). therefore, an increase in cholesterol and triglycerides in the blood of fish kept in dark condition may be due to the effect of darkness on fatty acid biosynthesis, reduced storage capability of blood lipid in adipose tissue and alterations in hormones involved in blood lipid homeostasis in fish. decreased cholesterol and triglyceride levels in the blood of fish exposed to tio2-nps under normal photoperiod conditions might be attributed to the influence of tio2-nps on the synthesis of cholesterol and triglycerides in liver or reduced intestinal uptake of lipids and fatty acids. using cholesterol and triglycerides to deal with the toxicity of tio2-nps under light conditions might be due to lower levels of cholesterol and triglycerides in blood. liu et al. (2009) demonstrated that tio2-nps have negative effects on metabolism of lipids in animals. prochazka et al. (2012) found that tio2nps have the potential to breakdown lipids, especially cholesterol, in biological systems. an increase in creatinine was observed in fish exposed to tio2-nps that might be due to kidney dysfunction and is considered as the physiological evidence of tio2-induced nephrotoxicity (wang et al., 2007; liu et al., 2009; wu et al., 2016). our results showed that alterations in blood biochemical parameters in fish exposed to tio2-nps under light photoperiod conditions were more acute than fish exposed to tio2-nps under dark conditions. in fact, the possibility of having reactive oxygen species in aquatic environments by tio2nps under visible light is more than that under darkness. acknowledgements the authors gratefully acknowledge the support offered from behbahan khatam al-anbia university of technology. also, the authors are grateful to m. banaee, our english editor, for proofreading the manuscript. references armelao l.b., bottaro g., gasparotto a., maccato c., maragno c. 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(2016) 4(6): 370-377 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی ( cyprinus carpio) معمولی کپور ماهی بر تیتانیوم اکسیددی نانوذرات زیرکشنده سمیت تاثیر تاریکی و مرئی نور شرایط تحت وزیریان مریم طاهری، سمیه عفو، شاه شیما حقی،دوست نعمت بهزاد ،*بنایی مهدی 63616-47189: کدپستی ایران، بهبهان، ،بهبهان( ص) االنبیاء خاتم صنعتی دانشگاه زیست، محیط و طبیعی منابع دانشکده شیالت، گروه چکیده: . است تاریک شرایط و مرئی نور زیر در( cyprinus carpio) معمولی کپور ماهی در 2tio نانوذرات کشنده تحت سمیت تعیین مطالعه این از هدف گلیسیرید ریت سطح کراتینین، گلوبولین، آلبومین، کل، پروتئین گلوکز، جمله از بیوشیمیایی پارامترهای و شده گیریخون ماهیان از روز 21 از پس ،(ldh) دهیدروژناز الکتات و( ggt) ترانسفراز گلوتامیل γ ،(alt) آمینوترانسفراز آالنین ،(ast) آمینوترانسفراز آسپارتات و خون کلسترول و پارامترهای بر داریمعنی اثر 2tio نانوذرات که داد نشان نتایج. شد گیریاندازه( alp) فسفاتاز آلکالین هایفعالیت و( ck) کیناز کراتین از ن،خو بیوشیمیایی پارامترهای از برخی در داریمعنی تفاوت روشنایی، به تاریکی از نوری شرایط تغییر با. داشت c. carpio خون بیوشیمیایی شرایط در 2tio نانوذرات معرض در ماهی در گلیسریدتری و کلسترول گلوکز، سطح و ،ck و ast، alt، ldh، alp هایآنزیم فعالیت جمله بیشتر روشنایی شرایط در 2tio نانوذرات سمیت که داد نشان نتایج. شد مشاهده تاریک شرایط در 2tio نانوذرات معرض در ماهی با مقایسه در نور . است تاریکی شرایط از .نوری دوره شرایط بیوشیمیایی، پارامترهای معمولی، کپور ،2tio نانوذرات :کلمات کلیدی int. j. aquat. biol. (2015) 3(5): 331-338 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article effect of density on some physiological responses to transportation stress in mesopotamichthys sharpeyi (günther 1874) fingerlings tayebe nazari1, vahid yavari1, amir parviz salati*1, abdolali movahedinia2 1department of fisheries, faculty of marine natural resources, khorramshahr university of marine science and technology, khorramshahr, iran. 2department of marine biology, faculty of marine science, khorramshahr university of marine science and technology, khorramshahr, iran. article history: received 14 june 2015 accepted 12 o c t o b e r 2015 available online 2 5 o c t o b e r 2015 keywords: stress cortisol lactate glucose mesopotamichthys sharpeyi abstract: in the present study, the effect of density on transportation stress in mesopotamichthys sharpey fingerlings was evaluated. for this purpose, four different densities, including 40, 80, 120 and 160 g/l were used as treatments each with 3 replicates. simulation of transport procedure was carried out for 4 hrs. the blood samples were collected from the fish prior to loading from the stocking tank (control), after 4 hrs of transportation and from released fish into recovery glass tanks at 6, 12, 24, 48 and 96 hrs after transportation during recovery period. for blood sampling, fish immediately anesthetized by adding 2% 2-phenoxy ethanol and the blood samples were prepared. the cortisol, glucose and lactate value of plasma were measured. the results showed a significant increase in cortisol and glucose levels (in highest density) after transportation in all treatments (p<0.05). lactate did not show a significant difference in experimental groups (p>0.05). the results showed that only cortisol level was significantly different with basal level at 96 hrs. our findings showed that this species can be transported at higher densities up to 120 g/l. introduction in aquaculture, fish is exposed to stressful stimuli during transport, handling, netting and stocking at high densities (dobsikova et al., 2009). transporting fish is inevitable in aquaculture (gbore et al., 2006), therefor, reduction of harmful effects of caused stress is a fundamental goal for its successful growth and production (ashly, 2007). hence, rearing programs of fishes in aquaculture industry usually involve subjecting fish to a variety of practices such as weighing, grading, transporting or increasing rearing density, all of which can be stressful to the fish. fish transportation is considered a traumatic procedure that exposes fish to a series of adverse stimuli responsible for several physiological responses. these stimuli include capture in ponds, handling, transportation, inappropriate stocking densities, physical handling, unfavorable water * corresponding author: amir parviz salati e-mail address: apsalati@kmsu.ac.ir quality and introducing fish to a new environment (urbinate et al., 2004; harmon, 2009). stocking density in fish transportation can be led mechanical abrasion due to their contact (urbinati et al., 2004). in addition, during transporting, fish is encountered a decrease in dissolved oxygen and an increase in carbon dioxide levels. excretion of nitrogenous wastes also increase the level of ammonia in the transport medium. the increase of co2 concentration decrease water ph (dobsikova et al., 2006). the poor water quality parameters are caused physiological stress affecting fish health (kayali et al., 2011). mesopotamoichthys sharpeyi (günther 1874) is a commercially valuable cyprinid species of karoun river drainage of tigris basin, which has been considered as an important candidate for the aquaculture industry of iran in recent years. due to the low number of the hatcheries to produce 332 int. j. aquat. biol. (2015) 3(5): 331-338 m. sharpeyi larvae, distribution and transport of produced larvae to rearing farms at long intervals is necessary. since suitable methods and conditions to reduce fish stress is favorable in aquaculture, therefore, this study was conducted to find a suitable density for the transportation of this species to rearing farms and characterize physiological responses of m. sharpeyi during this process. materials and methods a total of 180 m. sharpeyi (with a mean length and weight of 10±1 g and 9.8±1 cm, respectively) were obtained from shahid maleki farm (ahwaz, khuzestan province, iran) and transported to the laboratory of marine natural resources department, (khorramshahr university). the fish were acclimatized for one week in well-aerated 300 liters polyethylene tanks. during this period, they were fed twice a day with commercial pellet at 3% body weight. diet composition is presented in table 1. water temperature (25.6±0.84ºc), dissolved oxygen (7.11±0.36 mg/l) and ph (7.42±0.18) were recorded daily during the experimental period. eighty g/l is a routine density that used by practioniors for fish transportation, hence, densities of 40, 80, 120 and 160 g/l were considered as treatments in this study (each with 3 replicates). simulation of transportation procedure was performed for 4 hrs (urbinate et al., 2004), which represents the average transportation time. after examination, each group was introduced into 100 l glass aquarium for recovery and fed once a day with commercial fish pellet as 3% body weight, but they were not consumed the food. fish were sampled prior to loading from the stocking tank (control) in 3 replicates, immediately after transport (transportation effect) and 6, 12, 24, 48 and 96 hrs after transport (during the recovery period) based on urbinate et al. (2004). for prevention of handling stress, fish were anesthetized by 0.2% 2phenoxy ethanol to the glass aquarium and then blood samples were taken from the caudal vasculature using a heparinized syringe. survival was recorded in an experimental unit at the end of transportation and recovery. mortality rate determined by dividing the number of dead fish by number transported fish, multiplied by 100 (gbore et al., 2006). because of the small size of fish, each blood sample resulted from a pool of 3-4 fish (urbinate et al., 2004). the extracted blood was centrifuged at 3000 rpm for 10 min. the collected serum in eppendorf tubes was stored at -80°c for further assays (acerete et al., 2004). cortisol was measured in duplicate, using ria kit (immunotech, czech republic) with a sensitivity of 10 nm (ghaedi et al., 2013). glucose and lactate were determined by enzymatic colorimetric and by enzymatic method using commercial kits (pars axmoon, iran), respectively (salati et al., 2010). water temperature, ph, total ammonia and dissolved oxygen were monitored in the tank (before loading), immediately after the fish transportation and 6, 12, 24, 48 and 96 hrs after transport (table 2). water temperature, ph and dissolved oxygen were measured using hatch multimeter and total ammonia was determined by nessler method according to standard method (standard method, 1992). the data was analysed by spss 11.5 software. data are presented as mean ± se. one way anova and tukey’s post hoc were used to compare the parameters between treatments at each sampling time and different time at each density. p<0.05 was accepted for statistical significance. results blood cortisol was increased significantly in all treatments compared to the basal value after 4 hrs of ingredients diets (g/100g) fish meal 39 soybean meal 23 casein 15 wheat flour 7.5 rice flour 7.5 fish oilb 6 vitamin premix 1 mineral premix 1 table 1. composition of basal diet used in this study. 333 nazari et al./ effect of transportation density on stress response the transportation (p<0.05) (fig. 1). after 4 hrs transportation period, the blood cortisol levels of fish submitted to the highest loading density were higher than fish at lowest loading density, but their differences were not significant (p>0.05). a little increase in cortisol levels were observed in 6, 12 and 24 hrs compared to 0 hrs after transportation, but it was not significantly different. the reduction in cortisol levels continued after 48 hrs recovery. in the present experiment even after 96 hrs, the cortisol levels was remained elevated. after transportation, an increase in glucose was recorded compared to the base value, but these changes were only significant in 160 g/l treatment (p<0.05) (fig. 2). fish transported in the highest densities showed higher values of glucose, but no significant differences were verified in values of blood glucose level in all densities (p>0.05). in the recovery aquarium, the glucose value showed a little increase at 6 and 12 hrs after transportation compared to 0 hrs but was not significant. the glucose levels in all densities with the exception of lower density at 48 and 96 hrs after transport, returned to near baseline levels at the end of the recovery period. after transportation, no significant difference was found in the blood lactate between treatments (p>0.05) (fig. 3). in the present experiment, 24 hrs after transportation the blood lactate showed an increase and its concentration was decreased in 48 and 96 hrs. this alterations were not significant from that of the base value (p>0.05). parameters density before transport after transport empty aquarium 6 hrs 12 hrs 24 hrs 48 hrs 96 hrs ph 40 gl-1 7.16 7.31 7.16 7.20 7.30 7.25 7.25 7.24 80 gl-1 7.16 7.11 7.16 7.16 7.17 7.16 7.85 7.36 120 gl-1 7.16 6.80 7.60 7.83 7.64 7.37 7.78 7.36 160 gl-1 7.16 7.34 7.30 7.27 7.21 7.19 7.85 8.50 do (mgl-1) 40 gl-1 8.60 0.83 8.70 8.20 7.90 7.80 7.48 7.36 80 gl-1 8.60 0.78 8.60 8.08 8.01 8.10 7.64 7.83 120 gl-1 8.60 0.76 8.70 8.40 8.30 8.50 8.20 7.90 160 gl-1 8.60 0.85 8.40 8.37 8.33 8.11 8.06 7.81 nh3 (µgl-1) 40 gl-1 13.30 36.70 8.4 19.8 15.3 21.6 11.4 26.2 80 gl-1 13.30 52.70 7.2 8.0 7.4 12.9 13.2 14. 120 gl-1 13.30 63.40 10.7 23.5 19.2 15.5 16.5 24.2 160 gl-1 13.30 64.90 11.3 16.2 17.0 13.6 19.2 16.6 table 2. water quality parameters before and after transportation of mesopotamoichthys sharpeyi and during recovery period. figure 1. plasma cortisol (µg/dl) levels of mesopotamichthys sharpeyi in response to transportation stress in different densities. same capital letters indicates no differences among treatments within sampling time and lower case letters among same treatment in different sampling (p<0.05). figure 2. plasma glucose (mg/dl) level of mesopotamichthys sharpeyi in response to transportation stress in different densities. same capital letters indicate no differences among treatments within sampling time and lower case letters among same treatment in different sampling (p<0.05). 334 int. j. aquat. biol. (2015) 3(5): 331-338 the results showed increasing the mortality in plastic bags and aquariums in higher densities (table 3). discussion in the present study, the blood cortisol increased in all treatments compared to the basal values after 4 hrs of the transportation. furthermore, the blood cortisol levels of those fish submitted to the highest loading density were higher than those of the lowest loading density, but these differences were not significant. in response to stressful events, the hypothalamic portion of the brain stimulates the release of adrenocorticotropic hormone (acth). acth is circulated into the anterior kidney, where it stimulates the interrenal cells to produce cortisol and other corticosteroid hormones (kubilay and ulukoy, 2002). similar findings have been reported in other fish species. carneiro et al. (2009) in jundiã (rhaudia quelen), abreu et al. (2008) in matrinxã (brycon amazonicus), adamante et al. (2008) in salminu brasiliensis fingerlings, acerete et al. (2004) in perca fluviatilis. l, bolasina (2011) in juvenile paralichthys orbignyanus, hur et al. (2009) in paralichthys olivaceus, urbinati et al. (2004) in matrinxã (b. cephalus), dobsikova et al. (2009) in cyprinus carpio and grutter and pankurest (2000) in hemigymnus melapterus reported an increase in plasma cortisol level in response to transportation stress. whereas, dobsikova et al. (2009) and svobodova et al. (1999) reported a decrease in plasma cortisol in c. carpio during transportation. a little increase in cortisol levels were observed in 6, 12 and 24 hrs after transportation. furthermore, after 96 hrs, the cortisol levels was remained elevated that may be due to new environment and starvation stress during recovery period. new environment is a stressor that can alter the recovery procedure. many other factors including genetic, developmental, and environmental (e.g. temperature, nutrition and water quality) factors influence stress responses in fishes (barton, 2002). animals respond to challenges in their environment through several interacting mechanisms, including behavioral, hematochemical, physiological and neuro-hormonal parameters (fazio and ferlazzo, 2003). barcellose et al. (2001) observed similar result in r. quelen after transference to growing tank, which could explain the fast effect (adamante et al., 2008). the cortisol levels showed reducing after 48 hrs recovery. similar results were observed by carmichael (1983) who subjected largemouth bass (micropterus salmonides) to long distance transport. carneiro et al. (2009) in jundiã (r. quelen), abreu et al. (2008) in matrinxã (b. amazonicus), hur et al. (2009) in p. olivaceus and urbinati et al. (2004) in matrinxã (b. cephalus) reported that the blood cortisol values returned to normal value during the first 24 hrs after transportation. such rapid decreases in plasma cortisol levels could be related to the low intensity of stress (urbinati et al., 2004). adamante et al. (2008) in fingerlings of s. brasiliensis and sulikowski et al. (2006) in flounder, pseudopleuronectes americanus were reported that cortisol levels of lower densities, returned rapidly to near baseline levels. after transportation, an increase in glucose was recorded compared to base value, but this change was significant only in 160 g/l treatment. the high glucose level is characteristic of the secondary figure 3. plasma lactate (mg/dl) level of mesopotamichthys sharpeyi in response to transportation stress in different densities. same capital letters indicate no differences among treatments within sampling time and lower case letters among same treatment in different sampling (p<0.05). 40 gl-1 80 gl-1 120 gl-1 160 gl-1 polyethylene bag 0.79 5.55 6.34 11.90 aquarium 0.00 4.76 3.17 3.17 total 0.79 10.31 13.48 15.07 table 3. mortality rate (%) during the study. 335 nazari et al./ effect of transportation density on stress response response which is caused by an increase in catecholamines and cortisol levels and provides energy for the fish to respond to the demand generated by the behavioral response to stress stimuli (carneiro et al., 2009). urbinati et al. (2004) in matrinxã (b. cephalus), abreu et al. (2008) in matrinxã (b. amazonicus), carneiro et al. (2009) in jundiã (r. quelen), dobsikova et al. (2009) in (c. carpio), nomura et al. (2009) in (salmo salar) and hur et al. (2009) in (p. olivaceus) reported an increases in blood glucose level following encountered stress. in contradict, acerete et al. (2004) in p. fluviatilis did not record any change in blood glucose level. the low magnitude of glucose responses can indicate low activation of the brain-sympathetic-chromaffin cell axis, and hence a low release of catecholamines, which seems to occur at a higher extent of severe stresses (abreu et al., 2009). elevated cortisol secretion under stress increases the activation of plasma glucose by activity of the gluconeogenesis enzyme (hur et al., 2009). in the recovery aquarium, the glucose concentration showed a little increase at 6 and 12 hrs after transportation compared to that of 0 hrs but was not significant. this increase may be due to the new environment and demanding energy for behavioral responses to stress. in the present study, fish transported in high densities showed higher values of the glucose, but no significant differences were verified in values of blood glucose level in all densities. similar to our findings, abreu et al. (2008) in matrinxã (b. amazonicus) and carneiro et al. (2009) in jundiã (r. quelen) reported that glucose level is directly related to fish density. the glucose level was returned to near baseline levels at the end of the recovery. decrease in the glucose level appears to be a high glucose demand to supply the energy (martinez-alvarez et al., 2002). also, it may be as result of appetite decrease and starvation due to consumption of plasmatic glucose. glucose levels in all densities with the exception of lower density at 48 and 96 hrs after transport, returned to near baseline levels at the end of the recovery, which may be due to intensity of stress in different experimental groups. this difference can be related to low demand for breakdown of energy resource. after transportation no significant difference was verified in values of blood lactate in all densities. hur et al. (2009) in p. olivaceus, dobsikova et al. (2009) in c. carpio, inversen et al. (2005) in s. salar, nomura et al. (2009) in s. salar and inversen et al. (1998) in s. salar were reported lactate increased after transportation. blood lactate is considered as a secondary stress response that is released from white muscle following vigorous exercise (nomura et al., 2009). in the present study, 24 hrs after transportation, the lactate showed an increase and it was decreased in 48 and 96 hrs, which was not significantly different from base value. the lactate level in lower density showed a rapid decreases that could be related to low hypoxia. no significant difference in lactate levels were observed in this study that could be related to low severity of transportation stress for m. sharpeyi. in the present study, the mortality was observed in fish exposed to transport stress for 4 hrs. despite providing proper condition in the applied closed systems, the method can become a limiting factor, as well as an important stress factor due to the accumulation of metabolites in water such as carbon dioxide and total ammonia, and also due to the decreasing of dissolved oxygen (do) and changing ph (adamante et al., 2008). in our study, mortality in plastic bags and aquariums were increased in higher densities. the higher stocking densities overload with produced metabolites is led stress (conte, 2004). in addition, fish transported at high densities are submitted to mechanical abrasion due to contact between them, an important stress factor which can cause loss of fish scales and mucus facilitating the infection process and diseases (urbinati et al., 2004). transportation consists of several traumatic events (stressors), including capture, loading, transport, unloading and stocking (inversen et al., 2005). pavlidis et al. (2003) in red porgy (pagrus pagrus), without water renewal, reported an increased mortality with 336 int. j. aquat. biol. (2015) 3(5): 331-338 increasing stocking density. a high density associated with a very long time of transportation can stress fish, impair transportation efficiency, and cause mortality, as well as cause negative effects on animals performance. hence, an acute stress during transportation can predispose fish to pathologies after the stock-term due to immunosupression caused by stress (adamante et al., 2008). abreu et al. (2008) in matrinxã (b. amazonicus) no mortality was registered through the week following the transport. matrinxã demonstrated to be a crowding tolerant-species in transport. also urbinati et al. (2004) in matrinxã (b. cephalus) no mortality was observed among the fish during the whole experimental period. based on little changes in physiological indicators of stress after transportation and low mortality rate at different densities in the present study, it can be concluded that m. sharpeyi fingerlings could be transported at higher densities up to 120 g/l without significant changes in physiologic status. acknowledgments this work has been supported by khorramshahr university of marine science and technology. references abreu j., takahashi l., hoshiba m., urbinati e. (2009). biological indicators of stress in pacu (piaractus mesopotamicus) after capture. brazilian journal of biology, 69(2): 415-421. abreu j.s., sanabria-ochoa a.i., gonçalves f.d., urbinati e.c. 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(2015) 3(5): 231-338 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی بنی قد انگشت ماهیان در انتقال استرس به فیزیولوژیک هایپاسخ برخی روی بر تراکم اثر mesopotamoichthys sharpeyi (günther,1874) 2نیا موحدی عبدالعلی ،* 1سالطی پرویز امیر ،1یاوری وحید ،1نظری طیبه .ایران خرمشهر، خرمشهر، دریایی فنون و علوم دانشگاه دریا، طبیعی منابع دانشکده شیالت، گروه1 .ایران خرمشهر، خرمشهر، دریایی فنون و علوم دانشگاه دریایی، علوم دانشکده دریا، شناسیزیست گروه2 چکیده: برای این . گرفت قرار بررسی مورد mesopotamichthys sharpeyiبنی قد انگشت ماهیان در انتقال استرس بر تراکم اثر حاضر مطالعه در 0 مدت به انتقال فرایند سازی شبیه. گرفت قرار استفاده مورد مطالعه این در تکرار سه در لیتر بر گرم 164 و 124 ،04 ،04 تراکم چهارمنظور ریکاوری هایتانک در ماهیانو از انتقال ساعت 0 از پس ،(کنترل) سازی ذخیره های تانک در بارگیری از قبل ماهیان خون نمونه .شد انجام ساعت ماده با صید از پس بالفاصله ماهیانبرداری از خون، برای نمونه. شد آوریجمع انتقال پس ساعت 66 و 00 ،20 ،12 ،6 هایزمان در شده توزیع هاآن در الکتات و گلوکز کورتیزول، میزان پالسما جداسازی از پس .شد اخذ ها آن از خون نمونه و شدند بیهوش %2 اتانول فنوکسی -2 بیهوشی داریمعنی افزایش الکتات(. p>40/4) داد نشان ها تراکم همه در انتقال از پس گلوکز و کورتیزول میزان در داریمعنی افزایش نتایج. شد گیریاندازه ما هاییافته. داد نشان اختالف پایه سطح با انتقال از پس ساعت 66 زمان در کورتیزول سطح فقط(. p<40/4) نداد نشان آزمایشی های گروه در .شود جا جابه لیتر بر گرم 124 تا باال های تراکم در تواندمی گونه این دهدمی نشان .mesopotamichthys sharpeyi ،، الکتات، گلوکزکورتیزول استرس، :کلمات کلیدی int. j. aquat. biol. (2023) 11(2): 98-103 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article bioaccumulation of heavy metals in liver tissues of three anas species in al-hawizah marshes, southern iraq saja mahdi hussein1, salih hassan jazza1, safaa sabri najim2 1department of biology, college of science, university of misan, maysan, iraq. 2department of chemistry, college of science, university of misan, maysan, iraq. article history: received 11 october 2022 accepted 16 december 2022 available online 2 5 april 2023 keywords: pollution bioaccumulation heavy metals liver tissues abstract: bioaccumulation of cadmium, lead, copper, zinc, and iron was investigated in the liver tissues of three migratory bird species viz. anas platyrhynchos, a. crecca, and a. acuta of both sexes in autumn and winter. bird samples were collected from autumn 2021 to winter 2022 from alhawizeh marshes, misan province, iraq. the results showed that cd in liver tissues ranged from 0.73 to 8.174 ppm during winter and autumn, respectively in males of a. crecca. pb ranged from 0.076 to 0.922 ppm in females and males of a. platyrhynchos during winter and autumn, respectively. cu varied from 0.635 to 4.62 ppm in males of a. platyrhynchos during autumn and winter, respectively. zn ranged between 0.402 ppm in males of a. acuta and 2 ppm in females a. platyrhynchos and a. crecca during autumn and winter, respectively. fe ranged between 1.544 ppm in males of a. acuta and 15.85 ppm in females of a. platyrhynchos during autumn and winter, respectively. the concentrations of cd, cu, and fe were higher than the permissible limit, whereas zn and pb were within permissible fao. introduction al-hawizah marsh is one of the main and important marshes in the south of iraq, extending from the tigris river to iran. it provides a suitable environment for many living organisms, such as aquatic plants, invertebrates, fishes, birds, etc. in recent years, the marsh has suffered from pollutants such as pesticides, hydrocarbon compounds, and heavy metals (rushdi et al., 2006; hasab et al., 2020). aquatic ecosystems accumulate pollutants from natural and human activities, which pose adverse effects on aquatic biota. heavy metals are pollutants of concern attributed of their persistence, toxicity, and high ability to accumulate in the tissues of living organisms (kanwal et al., 2020). the main sources of heavy metals in the aquatic environment are anthropogenic activities and other industrial wastes or natural sources (behra et al., 2002; abdullah, 2013; bhardwaj et al., 2017). heavy metals are toxic with correspondence: salih hassan jazza doi: https://doi.org/10.22034/ijab.v11i2.1845 e-mail: salih-jazza@uomisan.edu.iq dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.2.4 chronic and acute toxicological impacts on living organisms through different trophic levels of the food chain (merian, 1991). some metals essential for living organisms, such as zinc, copper, manganese, and iron, have enzymatic functions in the metabolism of cells (kadhim et al., 2018). birds are susceptible to bioaccumulation of heavy metals through the consumption of contaminated water and food; therefore, they can provide interesting information to monitor the quality of the ecosystems (kim and oh, 2013; mansouri and majnoni, 2014; jazza et al., 2022). therefore, birds are a proper indicator for assessing heavy metals in the wetland because they feed on various levels of the food chains (burger and gochfeld, 2016). hence, the main objective of the present study was to investigate levels of some heavy metals of cadmium, lead, zinc, copper, and iron in the liver tissues of three migratory anas species (anas platyrhynchos, a. crecca, and a. acuta.) of both sexes in al99 int. j. aquat. biol. (2023) 11(2): 98-103 hawizah marsh. materials and methods study area: al-hawizeh marshes are located on the eastern side of the tigris river and extend between al-suwaib sub-district in the al-qurnah district in the north to al-uzayr district, maysan province to the villages of al-baydah, al-sawadah, and alturaba, which is a common marsh between iraq and iran. several subsidiary rivers run in the same direction due to the land’s natural slope as a result of the topographical situation of the area. some plants grow in saline soils, such as tahma and tarfa (hashim et al., 2019; idan and jazza, 2022). collection and prepare bird samples: bird samples were collected from al-hawizeh marshes from autumn 2021 to winter 2022 using a fishing net (douche). the birds’ samples were placed in a box containing crushed ice until they reached the laboratory. samples were three economic migratory bird species, namely a. crecca, a. platyrhynchos, and a. acuta, from both sexes (male and female). the liver tissues were removed and then wrapped in aluminum foil and stored in the freezer for further analysis. later, the liver samples were dried using a hot air oven at 80°c for 24 hours and then ground using an electric grinder, sieved using the metal sieve (0.3 mm), and placed in polyethylene tubes for digestion (jayakumar et al., 2013). all utensils and glassware were rinsed with tap water, soaked in nitric acid for 24 hours, rinsed with demineralized water, and dried under a laminar flow hood before use to minimize contamination with metals. digestion of liver samples: digestion of liver samples was done following the standard procedure suggested by jeffrey (2003) as follows: 0.5 g of the dried samples were weighted and transferred into teflon tubes, and then 6 ml concentrated mixture of hclo4 and nitric acid hno3 in a ratio of (1:1) was added. the samples are shaken well for 12-16 hours to complete the initial digestion process in the vacuum hood. then it was placed in a water bath at 70°c for 30 minutes and transferred to a hot plate to complete the digestion process until the mixture became clear. the solution was cooled at room temperature and transferred to a volumetric flask filled with deionized distilled water. heavy metals measurement: the concentrations of heavy metals of pb, cd, cu, fe, and zn were measured using a flame atomic absorption spectrometer, model ai 1200 aurora. results and discussion the results revealed that the highest cd in the liver of a. platyrhynchos was 7.579 and 8.139 ppm during autumn in males and females, respectively, and the lowest were 1.003 and 1.567 ppm during winter in females and males, respectively. accumulation of cd in the liver of a. crecca, and a. acuta in autumn and winter is shown in table 1. the variation in the concentrations of cd in different species may be attributed to their different feeding habits, age, weight, size, interaction with nutrients, and gender (peakall and burger, 2003; douterelo et al., 2004). sometimes they feed on agricultural lands contaminated with chemical fertilizers and pesticides, which increase the exposure of birds to heavy metals (degrnes, 2008; alloway, 2012). heavy metals in the organs of birds are affected by the level of contamination in food and water; the name of bird gender autumn winter a. platyrhynchos male 7.579 1.567 female 8.139 1.003 a. crecca male 8.174 0.73 female 7.782 1.44 a. acuta male 7.917 1.221 female 8.073 2.276 fao (1985) 0.2 table 1. seasonal variations of cd levels (ppm) in liver tissues of studied birds. 100 hussein et al./ bioaccumulation of heavy metals in liver tissues of three anas species liver tissue plays an important role in detoxification, reflecting long-term bioaccumulation (burger and gochfeld, 2016). metals are absorbed into the body and then enter the circulatory system. some interact with fats, whereas others dissolve and transfer to other tissues (burger et al., 2003; peakall and burger, 2003). the migration of birds increases cd accumulation during their flight (durkalec et al., 2022). this study showed that the levels of cd in the liver tissues for all studied species were higher than the permissible limit (0.2 ppm) of fao (1985). the results of pb accumulation in the liver of three studied birds from the al-hawizah marsh are shown in table 2. the ability of the birds to accumulate pb varies, which may be attributed to different factors such as weight, age, gender, and their feeding habits (idan and jazza, 2022; douterelo et al., 2004). lead is absorbed by birds with food and is associated with the uptake of ca+2, and accumulated in bone and liver tissues (scheuhammer, 1987). pb is toxic, and the bones are the main depot for lead. this may explain the reason for its low concentration in liver tissues in both sexes, i.e. it may be due to the deposition of pb in the skeleton compared to other tissues of birds (burger et al., 1992). birds feed on solid food materials to help grind larger food particles to make them digestible, hence pb will be consumed with such materials (best and stafford, 2002). pb concentrations in this study were within the permissible limits (5 ppm) recommended by fao (1985). the results of cu bioaccumulation in the liver of a. platyrhynchos, a. crecca, and a. acuta from the al-hawizah marsh are presented in table 3. the highest levels of cu were recorded during winter, whereas the lowest were during autumn. this seasonal variation may be due to dietary patterns impacting cu absorption (burger et al., 2003) or attributed to the benthic sediments spreading pollution back to the water body because of the changes that occur inside the water as a result of the difference in temperature during two seasons (fukue et al., 2006; lazim, 2019). cu in the aquatic ecosystem tends to accumulate in aquatic biota through food chains (edward et al., 2013). cu accumulates in the liver higher than in other organs due to its high ability to accumulate, which is attributed to its distinguished location within the circulatory system, thus receiving most elements through the blood (hamza-chaffai et al., 1997). cu is an important element in many physiological species gender autumn winter a. platyrhynchos male 0.922 0.077 female 0.113 0.076 a. crecca male 0.095 0.15 female 0.195 0.077 a. acuta male 0.35 0.077 female 0.002 0.15 fao (1985) 5 table 2. seasonal variations of lead levels (ppm) in liver tissues of studied birds. species gender autumn winter a. platyrhynchos male 0.635 4.62 female 0.84 3.9 a. crecca male 1.366 2.12 female 0.986 4.4 a. acuta male 0.807 1.67 female 1.816 2.03 fao (1985) 0.2 table 3. seasonal variations of copper levels (ppm) in liver tissues of studied birds. 101 int. j. aquat. biol. (2023) 11(2): 98-103 processes, but exposure to high concentrations of cu can cause damage to the endocrine gland, digestive, respiratory, and reproductive systems (abdullah et al., 2015). levels of cu in all studied birds were more than the permissible limit (0.2 ppm), according to fao (1985). the concentrations of zn in the liver tissues of a. platyrhynchos, a. crecca, and a. acuta are shown in table 4 and this low accumulation may be due to its tendency to accumulate in bones, feathers, and eggshells than liver tissues (stout and trust, 2002). zinc is an essential element in the formation of feathers, so will accumulate in large quantities in feathers (deng et al., 2007). birds regulate zn in their tissues which are associated with fat contents, gender, weight, and age (burger, 2007). the soil in al-hawizah marshes plays an important role in the formation of complexes with heavy elements because of having a high percentage of clay and silt, reducing the chance of birds being exposed to this element (bradl, 2004). zinc is essential, but its high levels cause renal toxicity (kaur and dhanju, 2013). our results revealed that the concentration of zn in all samples was less than the permissible limits (2 ppm) (fao, 1985). table 5 shows fe concentrations in the liver of a. platyrhynchos, a. crecca revealing seasonal variations. the higher levels may be due to the abundance of iron in the earth's crust, while the lower due to the tendency of fe to accumulate in aquatic organisms and sediment (akbar and khazali, 2012). increasing levels of fe in because of its high ability to accumulate iron (okati and rezaee, 2013). the results showed that the fe in most samples was more than the permissible limit (5 ppm) (fao, 1985). in the current study, seasonal variations in the accumulation of pb, cu, zn, and fe in the liver tissues of three studied bird species were observed showing their ability to accumulate heavy metals. the results also revealed that the levels of cd, cu, and fe in the liver tissues arere higher than the permissible limit, whereas the levels of zn and pb were within the permissible limits. references abdullah e.j. 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(2017) 5(3): 228-235doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article comparative assessment of diet and condition factor of cyprinus carpio and oreochromis leucostictus in lake naivasha, kenya james last keyombe*1, 2,1yasindi w. andrew2, oyugi o. dalmas3 1lake turkana research station, kenya marine and fisheries research institute, p.o. box 205 30500, lodwar, kenya. 2department of biological sciences, egerton university, p.o. box 536 20115, egerton, kenya. 3migori county, fisheries and livestock development, p.o. box 210 40400, suna-migori, kenya. article history: received 22 january 2017 accepted 30 m ay 2017 available online 2 5 june 2017 keywords: feeding detritus algae zooplankton phytoplankton plant materials abstract: the study compared and assessed the diet and condition factors of two fish species, oreochromis leucostictus and cyprinus carpio, in lake naivasha. fish samples were collected monthly using gill nets (35-70 mm mesh size) from july to december 2013. stomach contents of all the specimens were analysed using the point method. results indicated that detritus was the most abundant food item in the diet of both o. leucostictus and c. carpio accounting for 50% and 63%, respectively, while benthic macroinvertebrates contributed the least with each fish having 2%. rooting and digging behaviour of the carp probably led to both c. carpio and o. leucostictus ingesting the suspended detritus as their main source of food with c. carpio outcompeting o. leucostictus due to its prolific nature and better adaptability to benthic conditions. fulton’s condition factor of all the fish samples had values of >1. a comparison of the two fish species showed c. carpio had a condition factor of 1.51 while o. leucostictus had 1.32. the higher condition factor of c. carpio in lake naivasha is an indication that the fish have better tissue energy reserves, greater reproductive potential and higher survival rates compared to o. leucostictus with a lower condition factor. introduction the blue spotted tilapia, oreochromis leucostictus, is an exotic species of lake naivasha. it established itself quickly when it was first introduced, unintentionally, in the lake in 1956 from lake victoria basin and has been present to date (njiru et al., 2006). reason for its quick establishment after the introduction has not been exhaustively studied. currently, it is the most abundant tilapiine fish species in the lake (oyugi et al., 2011). other tilapiine species introduced in the 1950s were tilapia zillii, oreochromis niloticus and o. spirulus niger (gunther) (hickley et al., 2008). there was a hybrid produced between o. leucostictus and o.s. niger (gunther) which became abundant in the early 1960s but due to back crossing with o. leucostictus, it disappeared by 1972 (hickley et al., 2008). the purpose for the introduction of o. s. niger was to provide a forage fish * corresponding author: james last keyombe doi: https://doi.org/10.22034/ijab.v5i3.265 e-mail address: katalitsa@yahoo.com for the american largemouth bass, micropterus salmoides. o. leucostictus and t. zillii used to form an important fishery in the lake (muchiri and hickley, 991), with both species being commercially exploited using gill nets by fishermen. they have however been replaced from the commercial fishery by the invasive common carp, cyprinus carpio (oyugi et al., 2014). the common carp which was accidentally introduced in the year 1997 from a fish farm at the catchments of river gilgil has the largest populations and is the main component in the lake’s commercial fishery (oyugi et al., 2014). among the commercially important fish species of the lake, o. leucostictus is the most desired by the local community for consumption. this is because it has fewer bones in its flesh compared to c. carpio and is tastier (waithaka et al., 2015). however, its population has been on a decrease probably due to the invasion by the c. carpio 229 int. j. aquat. biol. (2017) 5(3): 228-235 in the lake (oyugi et al., 2014). the c. carpio interfered with the o. leucostictus breeding grounds through increasing turbidity of the water when feeding, and this could have reduced o. leucostictus spawning areas (oyugi et al., 2014). interactions between fish species and between them and planktonic organisms are frequently revealed through dietary studies. gut content analysis which provides evidence of whether the invading population has increased pressure on prey items or increased competition for resources (britton et al., 2007), is also lacking in this lake. similarly, common carp has had various impacts in lake naivasha ecosystem since its introduction (oyugi et al., 2012). however, knowledge of the effects of their interaction with o. leucostictus is limited. therefore, the aim of this study is to assess the diet and condition factor of o. leucostictus and c. carpio in lake naivasha, to facilitate informed decision making processes for effective management of the fisheries resources. materials and methods study area: lake naivasha is a shallow freshwater body, situated in the eastern arm of the great rift valley in kenya (0o46's, and 36o20'e). it lies at an altitude of about 1890 m above the sea level. the lake covers a surface area varying between 120 km2 and 160 km2 depending on the dry and wet seasons, respectively (harper and mavuti, 2004). the lake’s mean depth varies between 4-6 m (hickley et al., 2008). it lies in an endorheic basin but its freshness is mainly maintained by the inflows from the catchment area, biogeochemical sedimentation and the underground seepage (hickley et al., 2008). lake naivasha is a complex basin consisting of four lakes that include oloidien, crescent lake, the main lake naivasha and sonachi. rivers malewa and gilgil are the most important feeders of the lake (fig. 1). karati river flows into the lake intermittently. the lake is surrounded by a cyperus papyrus swamp which covers an area of 64 km2, but this can vary largely depending on rainfall intensity, livestock and prevailing wildlife populations in the riparian zone (harper and mavuti, 2004). the six sampling stations in both the main lake naivasha and crescent lake which were used in this study are indicated in figure 1. these stations are malewa (00o43'49.9"s 036o21'32.1"e), korongo (00o 44'22.6"s 036o19'28.9"e), hippo point (00o47'14.0"s 036o18'56.8"e), mid lake (00o46'30.8"s 036o20' 49.9"e), sher 00o49'19.1"s 036o21'49.4"e) and crescent (00o45'39.8"s 036o24'31.2"e). malewa and korongo sampling stations are located approximately 100 m from the shore and characterized by floating figure 1. a map of lake naivasha showing the sampling sites. 230 keyombe et al./ diet and condition factor of c. carpio and o. leucostictus in lake naivasha, kenya mats of water hyacinth (eichhornia crassipes), salvinia (salvinia molesta) and papyrus (c. papyrus) vegetation. they are characterized by muddy substrate, decayed plant materials and silt. the average depths of the stations are 3 and 3.5 m, respectively. sher bay and crescent lake are fairly sheltered from the wave action of the main lake and are characterized by calm waters occasionally invaded by the floating mats of e. crassipes and detached c. papyrus especially during strong winds at high water levels. the average depths in these stations are 2.5 and 3 m, respectively. the substrate is mainly silt and sand. sampling and data analyses: fish samples were collected monthly using gill nets (mesh size of 35, 40, 50, 60 and 70 mm) from malewa, korongo, hippo point, mid lake, sher and crescent sampling stations between july and december 2013. the variation in net mesh sizes allowed fish of different sizes to be caught. the nets were set at the first sampling station of the day at 7:00 am and hauled six hours later at 1:00 pm. the nets were then set at 1:30 pm and hauled at 7:30 for the second station of the day. two stations were sampled per day. six hours was the adequate time to ensure enough fish for sampling were caught in the nets during the day. immediately after retrieving the net, each fish caught was weighed in grams using an electronic weighing scale (digitron t745) to the nearest 0.1 grams. the total length of each fish was measured to the nearest centimeter using a measuring board. the sampled fish were then eviscerated and their sex determined according to witte and van densen (1995) as outlined in table 1. in the laboratory, fish stomachs were removed, fullness index determined using the modified method of hyslop (1980), and preserved in labeled plastic vials with 5% formalin for further analysis. condition factor (k) was estimated following le cren (1951): k =w/lb where k is the condition factor, w is the total body weight of fish in grams, l the total length in centimeters and b is the regression slope. data on gut contents was tested for normality and homogeneity of variance using ms excel 2010 and chi-square. the differences in the contribution of each food item were tested using quadratic fit. descriptive and inferential data analysis was conducted using ms excel 2010. in all the analyses, 95% level of significance was used as the critical point for rejection of the null hypotheses. results contribution of food items: during the period of july to december 2013 the gut contents of 153 o. leucostictus and 162 c. carpio were analysed. detritus, algae and zooplankton were the most abundant food types in the guts of the two fish species, table 1. the ovarian maturity stages of oreochromis leucostictus and cyprinus carpio (witte and van densen, 1995; bonneau, 1999; donkers, 2004). description maturity stage o. leucostictus c. carpio i cannot differentiate sex immature, gonad tissue developing ii small ovary, tube like. eggs not visible gonad non-vascularised iii ovary larger, occupies 1/3 of body cavity. eggs visible as yellow granules eggs/milt visible iv ovary dull grey. occupies ½ of body cavity. eggs visible as yellow granules mature, vascularised but not running v ovary large. greenish in colour. occupies almost entire body cavity running ripe golden green eggs extruded on applying pressure to abdomen vi red wrinkled ovary spent 231 int. j. aquat. biol. (2017) 5(3): 228-235 with fish of all sizes including the dietary items in their diet (fig. 2). detritus contributed the highest proportion in the diet of both o. leucostictus and c. carpio in all the sampling stations. despite o. leucostictus and c. carpio having diversified their feeding habit to include mainly detritus and phytoplankton, higher plant materials still contributed significant portion of food items consumed by the fish in lake naivasha. spatial variation in diet composition: there was minimal spatial variation in the composition of the food items consumed by both c. carpio and o. leucostictus in lake naivasha. detritus dominated c. carpio diet in both the inshore (crescent, korongo, malewa, sher, hippo point) and offshore (mid lake) sampling stations. the lowest composition of detritus was at korongo and the highest at sher sampling stations at 61% and 75%, respectively. the other important food items in the guts of c. carpio were zooplankton, benthic invertebrates and plant 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% c. carpio o. leucostictus p e r c e n ta g e c o m p o s it io n fish species benthic invertebrates plant materials zooplankton algae detritus figure 2. the dietary composition of (a) cyprinus carpio and (b) oreochromis leucostictus in lake naivasha from july to december 2013. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% p e r c e n ta g e c o m p o s it io n sampling site benthic invertebrates plant materials zooplankton algae detritus figure 3. the relative food contribution in the guts of cyprinus carpio at different sampling stations in lake naivasha from july to december 2013. 232 keyombe et al./ diet and condition factor of c. carpio and o. leucostictus in lake naivasha, kenya materials. zooplankton abundance ranged from 1-8% in all sampling stations. zooplankton in the guts of c. carpioat crescent, korongo and malewa sampling stations constituted 8%, 4% and 3%, respectively. carps from mid lake, sher and hippo point sampling stations had 2%, 1% and 2% of zooplankton as their gut contents (fig. 3). the results revealed no significant differences between the food items ingested by c. carpio in all the sampling stations (p<0.05). the important food items of o. leucostictusin all sampling stations was detritus at sher (52%) followed by algae at hippo point (35%) and zooplankton at mid lake (24%). benthic macroinvertebrates and higher plant materials constituted insignificant proportions of o. leucostictus food each at 4%. the results showed no significant differences between the food items 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% p e r c e n ta g e c o m p o s it io n sampling site benthic invertebrates plant materials zooplankton algae detritus figure 4. the relative contribution of food items in the guts of oreochromis leucostictus at different sampling stations in lake naivasha from july to december 2013. 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 c. carpio female c. carpio male c. carpio o. leucostictus female o. leucostictus male o. leucostictus c o n d it io n fa c to r ( s e ) fish species figure 5. the condition factor of oreochromis leucostictus and cyprinus carpio in lake naivasha from july to december 2013. 233 int. j. aquat. biol. (2017) 5(3): 228-235 ingested by o. leucostictus in all the sampling stations (p<0.005). no significant spatial variation was detected between the other food items. further analyses revealed that detritus was the most important food item for o. leucostictusin all the sampling stations (fig. 4). condition factor: all the fish samples collected had condition factor value of > 1. cyprinus carpio had a condition factor of 1.51 while that of o. leucostictus was 1.32. the females of c. carpio had a k-value of 1.53 while female o. leucostictus 1.35. similarly, male c. carpio had a k-value of 1.52 while males of o. leucostictus had 1.33 (fig. 5). discussion the highest proportion of phytoplankton was consumed in the open waters compared to the areas closer to the shore. this could be due to the openness and lack of free floating macrophytes in these off shore sampling stations allowing for more light penetration hence more phytoplankton biomass compared to areas closer to the shore where macrophytes shade the water from direct sunlight and inhibiting phytoplankton development. the higher percentages of plant materials consumed in the near shore areas (korongo and hippo point) than in the open deeper areas of the lake (sher bay, oserian and crescent island) could be due to the infestation and presence of water hyacinth and other macrophytes as compared to the open lake which only receives floating macrophytes occasionally especially during strong winds. there was no significant difference in the amount of detritus in the diet of both fish in all the sampling stations. however, slightly higher detritus amounts were recorded in the near shore areas. this could be attributed to the decaying of plant materials abundant at the littoral zone. occasionally, the decayed plant materials and other sediments are usually carried to the deeper waters through lake mixing particularly by wind thus distributing the detritus throughout the lake. this could explain the almost equal contribution of detritus in all the sampling station within the lake. an earlier study by njiru et al. (2006) found that c. carpio in lake naivasha had diversified its diet by feeding on plant materials (40%), plant seeds (17%), detritus (12%) and fish remains (11%). however, according to muchiri (1990), o. leucostictus feed on detritus as the principal component of their diet. detritus is the most abundant food material available to fish in the lake and its importance has been previously also noted by malvestuto (1974) and siddiqui (1977). of the other dietary constituents, algae, especially planktonic forms, were predominant. fish body condition varies seasonally depending on changes in gonadal development, food availability, and other environmental factors (pope and willis, 1996). the two fish species had k-values above 1. the results showed that both species were in good condition. according to braga (1986) and efitre et al. (2009), values of the condition factor vary according to seasons and are influenced by environmental conditions. the favourable physicochemical parameters in lake naivasha may therefore have been a catalyst for the good condition factor of the two fish species. the physico-chemical parameters of lake naivasha measured in this study were within the tolerable range for both o. leucostictus and c. carpio (edwards and twomey, 1982). in a study of o. leucostictus in lake naivasha, siddiqui (1977) found all stages of gonad maturation all year round and did not observe any seasonal fluctuation in relative condition factor, which he attributed to a constant proportion of fish with gonads in different stages of development. according to nathaniel et al. (1998), the condition factor of gravid females was within the range 1.6 and 2.0 irrespective of the location of sampling in the victoria reservoir (uganda). the condition factor of 1.52 for c. carpio in this study is in agreement with the results from victoria reservoir. based on the results of this study, it can be concluded that the habit of c. carpio of rooting or digging in the bottom has had a negative effect on the environmental condition of lake naivasha through increase in turbidity and decrease in oxygen in the water column. this has in turn led to both fish species in the study consuming the suspended detritus as their 234 keyombe et al./ diet and condition factor of c. carpio and o. leucostictus in lake naivasha, kenya main source of food with c. carpio outcompeting o. leucostictus due to its prolific nature and better adaptability to such conditions. the higher condition factor of c. carpio in lake naivasha is an indication that the fish have better tissue energy reserves, greater reproductive potential and higher survival compared to o. leucostictus with a lower condition factor. it can therefore be concluded that the feeding and reproductive ecology of c. carpio has disrupted the natural environmental conditions of lake naivasha causing a decline in the numbers of o. leucostictus through alteration of its feeding and reproductive strategies. since c. carpio is a better competitor than o. leucostictus, the fisheries stakeholders should look into cage culture of o. leucostictus as an alternative way of ensuring improved production of the fish species. acknowledgements the author gratefully acknowledges kenya marine and fisheries research institute (kmfri), management for co-funding this research work as part of his m.sc. thesis. special thanks to kmfri naivasha research team for technical and logistical support during data collection. the views expressed are those of the author and not necessarily those of their parent organizations. references bonneau j.l. 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(2017) 5(3): 228-235 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی در oreochromis leucostictus و cyprinus carpio چاقی ضریب و غذایی رژیم ایمقایسه ارزیابی توسعه کنیا نایواشا، دریاچه 3لماسا، اویوگی او. د2. آندرودبلیویاسیندی ، *1،2جیمز لست کیومبه .، لودوار، کنیا205-30500، صندوق پستی ی کنیاو شیالت اییدریایستگاه تحقیقاتی دریاچه تورکانا، انیستیتو تحقیقاتی علوم 1 .، اگرتون ، کنیا536-20115ی، دانشگاه اگرتون، صندوق پستی علوم زیست گروه2 .، کنیامیگوری-، سونا210-40400، صندوق پستی منطقه میگوری و دامداری توسعه شیالتاداره 3 چکیده: ارزیابی ودر دریاچه نایواشا کنیا oreochromis leucostictusو cyprinus carpio ماهی و ضریب چاقی دو گونه رژیم غذاییدر این مطالعه . ندصید شد 2013( از ماه جوالی تا دسامبر مترمیلی 35 -70)فاصله گره تا گره تور گوشکیر با استفاده ازهای ماهی ماهانه نمونهمقایسه شد. دو ررژیم غذایی هغذایی در ترین اقالم فراوان تریتیدکه . نتایج نشان داد ندتحلیل شد اینقطهبا استفاده از روش ها تمامی نمونهمحتویات معده شدند. شامل میبرا هر گونه درصد را 2مهرگان کفزی حداقل که بی، درحالیبوددرصد 63و 50به ترتیب با o. leucostictusو c. carpioگونه بهرا معلق هایتریتید از o. leucostictusو c. carpioگونه دو که هر شود میبه این منجر احتماالً کپور معمولیزنی رفتار ریشه کنی و نقب نسبت به بسترو سازگاری بهتر به شرایط توانایی ذاتیعلت به c. carpio در این رقابت با این وجودنمایند، استفاده عنوان غذای اصلی o. leucostictus مقدار آن در که گونه نشان داد دو ضریب چاقیمقایسه بود. 1از بیشتر هاماهی تمامی نمونه فولتونضریب چاقی .برتری دارد c. carpio 51/1 در وo. leucostictus 32/1 ضریب چاقی باالتر .باشدمیc. carpio ذخایر بافتی انرژیتیکی، دهندهدر دریاچه نایواشا نشان .استتر ضریب چاقی ایینپ مقداربا o. leucostictus در مقایسه با و نرخ بقای باالتر پتانسیل تولید مثلی .گیاهی مواد فیتوپالنکتون، زئوپالنکتون، جلبک، پوده، تغذیه، :کلمات کلیدی int. j. aquat. biol. (2019) 7(2): 65-70 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article plasma 17beta-estradiol and alkali-labile phosphoprotein levels in male and female tench (tinca tinca) in the anzali and amirkolayeh wetlands ali taheri mirghaed*1, marzieh abbasi2, seyyed morteza hoseini3, esmaeil pirali kheirabadi4 1department of aquatic animal health, faculty of veterinary medicine, university of tehran, tehran, iran. 2fisheries department, faculty of natural resources, university of guilan, sowmeh sara, iran. 3inland waters aquatics resources research center, iranian fisheries sciences research institute, agricultural research, education and extension organization, gorgan, iran. 4department of fisheries, faculty of natural sciences, shahrekord university, shahrekord, iran. s article history: received 2 january 2019 accepted 2 march 2019 available online 2 5 april 2019 keywords: alp anzali wetland steroid hormone tinca tinca abstract: environmental pollutants are potentiate to disturb biological processes such as metabolism, growth and reproduction of aquatic organisms. these compounds are able to cause gonadal abnormalities, biased sex ratios and alteration in reproductive physiology in fish. the aim of this study was to examine plasma 17β-estradiol (e2) and alkali-labile phosphoprotein (alp) levels in male and female tench (tinca tinca) from a polluted (the anzali wetland) and a non-polluted environments (the amirkolayeh wetland). samples were collected over the maturation season of tench between may and june 2017. the results revealed significant difference in mean alp and e2 between genders in the polluted environment. however, the mean plasma alp concentrations in male tench of the polluted environment (39.46±1.02 µg/ml) was 45% of the average recorded in female (86.18±2.25 µg/ml) and was two times higher than the amount measured in males in the nonpolluted environment (18.68±0.35 µg/ml). high concentrations of e2, were detected in the male samples from the anzali wetland. mean plasma e2 concentrations for male in the anzali wetland was almost two times higher than male in the amirkolayeh wetland. the results indicate that the reproductive physiology of tench was affected by contaminants found in the anzali wetland, a highly polluted area. introduction fish reproductive physiology is controlled by the endocrine system, which is affected by environmental factors such as light, temperature, etc. this system includes various glands, synthesizing and secreting hormones, which in turn regulate fish reproduction (pait and nelson, 2003), control and initiate steroidogenesis, vitellogenesis and gametogenesis process mainly through the activation of hypothalamic–pituitary–gonadal (hpg) axis (pankhurst and munday, 2011). in aquatic systems, there are varieties of environmental pollutants causing disturbance in the function of this system (ogundiran and fawole, 2018) by simulating or blocking the steroid hormones activity (mills and chichester, 2005) and binding to estrogen or androgen receptors (tapiero et al., 2002). these contaminants are called *correspondence: ali taheri mirghaed doi: https://doi.org/10.22034/ijab.v7i2.596 e-mail: mirghaed@ut.ac.ir endocrine disrupting chemicals (edcs) and include groups of natural and synthetic compounds such as phytoesrogens (burki et al., 2006), bisphenol-a (qiu et al., 2015), pcbs (walczak and reichert, 2016) and pahs (vignet et al., 2016). releasing of large quantities of municipal, agriculture and industrial wastewaters into the aquatic ecosystems cause serious impacts on the physiology of aquatic organisms. the anzali (sakizadeh et al., 2012) and amirkolayeh wetlands (zare khosh eghbal and sajadi nasab, 2015) are located in the north of iran and registered as two international wetlands based on 1975 ramsar convention. the main water source of the amirkolayeh wetland is underground water with no pollution from domestic and industrial wastes, and therefore, is considered as unpolluted and clean environment (zare khosh eghbal and sajadi 66 taheri mirghaed et al./ plasma 17beta-estradiol and alkali-labile phosphoprotein levels in tench nasab, 2015). in contrast, concentrations of edcs are considerably high in the anzali wetland (mortazavi et al., 2012, 2013) due to releasing large amounts of untreated municipal and industrial wastewater into the wetland; as a result, fish could be exposed to the chemical compounds directly and indirectly in this wetland. recent investigations have suggested that exposure of wild fish to edcs causes gonadal abnormalities and intersex (scholz and klüver, 2009), biased sex ratio (larsson and forlin, 2002) and induction of vitellogenin in males (tyler et al., 2002). vitellogenin is egg yolk protein precursor, which is synthesized in the liver of female fish (jobling et al., 1996). although there is vitellogenin gene in male, it is not expressed under normal conditions. however, when fish are exposed to edcs, they may respond to these compounds by increasing the level of plasma vitellogenin. a considerable increase in the level of vitellogenin has been reported in male squalius cephalus (randak et al., 2009) and carassius auratus (li et al., 2009) inhabited in aquatic ecosystems contaminated by edcs. vitellogenin is dominant in plasma after the onset of vitellogenesis and the protein is heavily phosphorylated. this enables indirect quantification through measurement of alkali-labile protein bound phosphate (alp) as alternative to the more expensive elisa assay (hallgren et al., 2009). in this study, tench (tinca tinca) was selected as target species, being an indicator of water quality, used as a model species for studies of physiology (martin et al., 1999), and lives in wetlands of iran north, including the anzali and amirkolayeh wetlands. the present study started with hypothesis that reproductive physiology of tench in the anzali wetland could be affected by environmental pollution, and the amirkolayeh wetland was considered as reference site. materials and methods sixty male and female tench were collected during the reproduction season (between may and june 2017) by fyke nets (mesh size, 34-54 mm) at two sites: the anzali wetland (pirbazar region), as a polluted environment and the amirkolayeh wetland as a nonpolluted environment or reference site. mean total length, sex, and number of collected samples are given in table 1. the fish were anesthetized in 150 mg/l clove powder solution and blood samples were immediately collected on site from the caudal vine using a heparinized 2.5 ml syringe. the collected blood from every fish was immediately placed in 2 ml plastic tubes, stored on ice, and transferred to the laboratory for further analyses. blood samples were centrifuged at 1500 g, 4°c for 3 min (scan speed, 1730 r, denmark). the separated plasma was stored at -80°c until analysis. total length of fish were measured to the nearest millimeter, and weighed to the nearest gram. gonads of each fish were observed macroscopically. plasma vitelloginine concentrations were measured indirectly by determination of plasma alp (negintaji et al., 2018; matthiessen et al., 2018). in this method, vitelloginine-related phosphate released into plasma and was measured by spectrophotometer with the wavelength at 630 nm according to hallgren et al. (2009). the levels of e2 were measured by estradiol ii kit (biomereux, france) and with enzyme linked fluorescent assay (elfa) method at 450 nm (diver, 1987). for calculating the effects of sex and location on measured parameters (alp and e2), two-way analysis of variance (anova) was applied. all data were tested for normality and homogeneity of variance using the kolmogrov–smirnov test and the levene test, respectively. statistical analyses were conducted table 1. total length and total weight (mean ±se) of tench captured from the anzali and amirkolayeh wetlands (n=15 fish for each gender in each wetlands). anzali wetland amirkolayeh wetland length (cm) weight (gr) length (cm) weight (gr) male 22.50±3.12 33.10±2.20 26.17±2.10 38.45±5.14 female 25.13±6.41 40.87±4.89 28.32±4.52 43.19±7.26 67 int. j. aquat. biol. (2019) 7(2): 65-70 using spss (version 16, inc., chicago, il, usa). all statistics were performed with a critical α of 0.05. results the males from the anzali wetland had significantly higher (~2 folds) alp and e2 compared to those of the amirkolaye wetland (figs. 1, 2). there were no significant differences in the female’s plasma alp levels between the sampling sites (fig. 1), but plasma e2 levels of the females from the anzali wetland were significantly higher than the females from the amirkolayeh wetland (fig. 2). in both wetlands, females had significantly higher plasma alp (fig. 1) and e2 compared to the males (fig. 2). discussions there are increasing concerns for the presence of varieties of environmental pollutants with endocrine systems disrupting ability in aquatic organisms. edc act by mimicking or interrupting hormone function, inducing vitellogenin synthesis in males, biased sex ratio and etc., which may affect natural reproductive and developmental processes (mills and chichester, 2005). in the present study, the plasma alp levels of the female fish from the anzali wetland were significantly different from males. vitellogenin is a significant source of nutrition for developing embryos and larvae that synthesized in female fish liver in response to circulating e2 hormone (monson et al., 2017). however, during the reproductive cycle of tench in the anzali wetland, synthesis vitellogenin was found in the male fish. there are vitellogenin genes in male fish and are not expressed under normal conditions, but might be induced when exposed to estrogen (juin et al., 2017) or estrogen like compounds in the aquatic environment (folmar et al., 2000). field studies on wild c. auratus (lu et al., 2010) and rhinichthys cataracte (jeffries et al., 2008) showed high concentrations of vitellogenin levels in the fish from edcs-contaminated environment. in this study, alp levels of the males from the anzali wetland were 45% of the females. fossi et al. (2002) studied xiphias gladius and thunnus thynnus from the mediterranean sea, and found plasma vitellogenin concentrations of the males were 28% and 11% of the females, respectively. plasma alp levels in the males from the anzali wetland were higher than the males from the amirkolayeh wetland, which is similar to previous reports by abbasi (2013) that suggested increased plasma alp levels in the male pike (esox lucius) from the anzali wetland more than from pike males captured from amirkolayeh wetland. other researchers also found similar results over different species and in different regions (hashimto et al., 2000; folmar et al., 2001; dick vethaak et al., 2002). mortazavi et al. (2012, 2013) stated that the levels of edcs (such as nonylphenol, octylphenol and bisphenol-a) at the anzali wetland were higher figure 1. concentrations of alp for male and female tench captured from the anzali and amirkolayeh wetlands (n = 15). data are presented as the mean ± standard error. different letters above the bars show significant differences. figure 2. concentrations of e2 for male and female of tench captured from the anzali and amirkolayeh wetlands (n = 15). data are presented as the mean ± standard error. different letters above the bars show significant differences. the bars show significant differences. 68 taheri mirghaed et al./ plasma 17beta-estradiol and alkali-labile phosphoprotein levels in tench compared to some other parts of the world such as rivers in china (jin et al., 2010) and japan (isobe et al., 2001). when xenoestrogens present in water, they may changes aromatase activity (the enzyme converts androgens to estrogens) in males by increasing plasma e2 concentrations (solé et al., 2003) and induction of vitellogenin. e2 is one of the most important sex steroids that are produced by ovarian follicles (monson et al., 2017) and regulates sexual processing and reproductive processes (martyniuk et al., 2006). our results demonstrated that the males captured from the anzali wetland had a doubled e2 levels compared to the males from the amirkolayeh wetland, indicating that edcs compounds at the anzali wetland interrupted reproduction system. conclusion these results suggest that edcs compounds in the anzali wetland might increase synthesis vitellogenin compared to the amirkolayeh wetland. the male tench from the anzali wetland have increased plasma alp and e2, suggesting disruption of reproduction system. however more studies will need to approve our results. acknowledgements we thank mr. ghorbanzade, ghane and darand for sampling. references abbasi m. 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(2019) 7(2): 65-70 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology چکیده فارسی و انزلی هایتاالب در( tinca tinca) ماهیالی ماده و نر در پالسما فسفوپروتئین-آلکالین و استرادیول-بتا17 سطوح امیرکالیه 4، اسماعیل پیرعلی خیرآبادی3، سید مرتضی حسینی2عباسی مرضیه ،1*میرقائد طاهری علی .ایران تهران، تهران، دانشگاه دامپزشکی، دانشکده آبزیان، هایبیماری و بهداشتگروه 1 .ایران سرا، صومعه گیالن، دانشگاه طبیعی، منابع دانشکده ،شیالت گروه2 .ایران ،گرگان ،سازمان تحقیقات کشاورزی، آموزش و توسعه ،سه تحقیقات علوم شیالتی ایرانسمو ،داخلی آبهای منابع تحقیقات مرکز گروه3 .ایران ،درشهرک ،شهرکرد دانشگاه طبیعی، منابع دانشکده شیالت، گروه4 چکیده: ثل یک متولیدهای متابولیسم، رشد و نمو و شوند توانایی مختل کردن فرایندهای آبی وارد میهای زیست محیطی که به درون اکوسیستمالیندهآ این از مثل ماهی شوند. هدفهای جنسی و تغییر فیزیولوژی تولیدهای گنادی، انحراف نسبتتوانند موجب ناهنجاریموجود آبزی را داشته و می االب آلوده )ترخون الی ماهی نر و ماده از یک محیط آلوده )تاالب انزلی( و یک محیط غی یمطالعه ارزیابی برخی پارامترهای بیوشیمیایی در پالسما آوری شدند. فسفوپروتئین متصل به آلکالین جمع 1396های اردیبهشت و خرداد ها در طول فصل بلوغ الی ماهی بین ماهباشد. نمونهامیرکالیه( می (alpبه )17گیری شد. همچنین مستقیم از مقدار ویتلوژنین پالسما اندازهعنوان یک شاخص غیر-( بتا استرادیولe2در نم )های پالسما اندازهونهدر الی ماهی alpهای نشان داد. با این حال غلظترا ها در محیط آلوده بین جنس e2و alpداری در میانگین گیری شد. نتایج، اختالف معنی برابر مقدار اندازه 2( و یترل میلی/ میکروگرم 18/86±25/2ها )میانگین ثبت شده در ماده %45/ میلی لیتر( میکروگرم 46/39±02/1نر محیط آلوده ) های نر تاالب انزلی در نمونه e2های باالیی از ( بود. غلظتلیتر میلی/ میکروگرم 68/18±35/0های نر محیط غیر آلوده )گیری شده در الی ماهی ثلی مد که فیزیولوژی تولیدادنتایج نشان دو برابر نرهای تاالب امیرکالیه بود. ر تاالب انزلی تقریباًدپالسما e2های تعیین شد. میانگین غلظت های تاالب انزلی، یک محیط به شدت آلوده، تحت تاثیر قرار گرفته است.ماهی توسط آالیندهالی .tinca tinca استروئیدی، هورمون انزلی، تاالب ،alp :کلمات کلیدی int. j. aquat. biol. (2015) 3(5): 282-289 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology review article review of the silversides of iran (family atherinidae) brian w. coad*1 canadian museum of nature, ottawa, ontario, k1p 6p4 canada. article history: received 2 may 2015 accepted 27 july 2015 available online 2 5 october 2015 keywords: atherina, biology, morphology, caspian sea. abstract: the silversides are found in coastal areas of temperate to tropical seas and in fresh water. there are about 14 genera and 68 species with only one native species in the caspian sea basin, atherina caspia. in this review, the systematics, morphology, distribution, biology, economic importance and conservation of the caspian silverside of iran are described, the species is illustrated, and a bibliography on this fish in iran is provided. introduction the freshwater ichthyofauna of iran comprises a diverse set of families and species. these form important elements of the aquatic ecosystem and a number of species are of commercial or other significance. the literature on these fishes is widely scattered, both in time and place. summaries of the morphology and biology of these species were given in a website (www.briancoad.com) which is updated here for one family, while the relevant section of that website is now closed down. other families will also being addressed in a similar fashion. family carcharhinidae the silversides or sand smelts are found in coastal areas of temperate to tropical seas and in fresh water. there are about 14 genera and 68 species (nelson, 2006; eschmeyer and fong, 2011) with only one reported from the caspian sea and iran. coad (1987, 1998) and coad and abdoli (1996) place this species in context with the iranian ichthyofauna. most are small fishes with a maximum length of 60 cm. these small, silvery fishes have a moderately elongate body, usually cycloid and moderately large scales, no lateral line but sometimes a pit on each * corresponding author: brian w. coad e-mail address: bcoad@mus-nature.ca scale, small teeth in the jaws and sometimes on the roof of the terminal, upwardly-directed mouth, wide gill openings with branchiostegal membranes free of the isthmus, 5-6 branchiostegal rays, gill rakers usually long and slender, two well-separated dorsal fins, the first with 3-10 unbranched but flexible rays, and the second with 1-2 unbranched and the rest branched rays, anal fin long, pectoral fins high on the flank, no pyloric caeca, egg membranes with filamentous outgrowths, back bluish to greenish with small melanophores but translucent, and a silvery stripe along the flank, often distinctively outlined with black. silversides can occur in vast schools in inshore waters and are an important item in the diet of other fishes. they have been used as bait but are too bony to be much used as food. their food is plankton. eggs are large and greenish. the sticky egg filaments entangle with plants, rocks or sand as anchors until hatching. genus atherina linnaeus, 1758 members of this genus are found in fresh and brackish waters with a single representative in iran. the record of atherina hepsetus linnaeus, 1758 in the caspian sea by quignard and pras in whitehead 283 coad/silversides of iran et al. (1984-1986) is an error (vasil'eva, 1994). the body is compressed with a rounded belly, the mouth large and terminal, jaws large, reaching back to the anterior eye level or beyond, sides of the upper jaw are straight and premaxillaries protractile, the dentary bone has a high central portion, a preopercular notch is absent, rows of setiform teeth on the jaws, vomer and palatines, cycloid scales extending onto the head, short pectoral fins, vent nearer the pelvic fin origin than the anal fin, numerous gill rakers, 5-10 flexible rays in the first dorsal fin, and the second dorsal fin is similar in length to the anal fin with its origin above the anal fin. atherina caspia eichwald, 1831 (figs. 1-2) common names: shisheh mahi (= glass fish), gol azin mahi (= flower decoration fish), atrinka (from the russian). [aterinka or xazar aterini in azerbaijan; kaspi aterinasy in turkmenian; kaspiiskaya aterinka or caspian silverside in russian; caspian silverside, caspian sand smelt, big-scale sand smelt]. systematics: formerly called atherina mochon pontica natio caspia eichwald, 1831 or atherina mochon caspia in caspian sea literature. atherina mochon cuvier, 1829 was described originally from ivasa and atherina presbyter pontica eichwald, 1831 from near odessa, ukraine. the caspian sea taxon was also known as atherina boyeri risso, 1810. atherina boyeri was originally described from the sea shore and lower course of rivers in the département du var, france. reshetnikov et al. (1997) give the date for this species as 1826. atherina presbyter var. caspia eichwald, 1831 described from "in caspii maris littore australiore, sinu balchanensi" (i.e., southern shore of the caspian sea, balkhan bay) was considered a synonym of atherina boyeri. naseka and bogutskaya (2009) consider caspia to be a full species and this is followed by esmaeili et al. (2014). syntypes of a. boyeri are in the muséum national d'histoire naturelle, paris under mnhn a-4342 (2) and mnhn b-860 (1) (eschmeyer's "catalog of fishes", downloaded 29 august 2007). no types are known for atherina presbyter var. caspia. bamber and henderson (1985) demonstrated how the morphology of atherina presbyter around britain varies according to temperature and salinity during embryonic development. isolation of populations results in local selection and random genetic drift and thus recognisable morphologies but these were not recognised as distinct. kottelat (1997) reviewed literature reports of variation in this species in the mediterranean and black seas and neighbouring fresh waters and concludes that lacustrine populations should be called a. boyeri while marine populations are a. mochon. the status of caspian populations was not commented on. these studies showed how variable these related silversides are and why the definition and limitation of species has been varied. the silverside in the caspian sea was referred to as atherina boyeri caspia by savenkova and asanov (1991) and by vasil'eva (1994, 1996) and this seemed a reasonable step as the population is isolated from other populations in the atlantic ocean and mediterranean sea. the subspecies was figure 1. line drawing of atherina caspia by s. laurie-bourque. 284 int. j. aquat. biol. (2015) 3(5): 282-289 characterised by a reduced number of infraorbital bones (3 bones in caspian sea fish as opposed to 4 in black sea fish; apparently infraorbital bones 2 and 3 fuse), a reduced number of gill rakers (generally 19-27 as opposed to 27-37), and the form of the maxilla (the inferior margin in caspian sea fish is always smooth while in the black sea and sea of azov a "wing" is usually developed). however, kiener and spillman (1972) allocated caspian populations to this subspecies on the basis of larger size, hardly an adequate criterion. vasil'eva (1994) makes the allocation based on a reduced number of gill rakers, reduction of the number of bones around the eye to three (preorbital, infraorbital and postorbital) and a smooth lower margin to the maxilla without a wing protuberance. dobrovolov and ivanova (1999) studied two nonenzymatic proteins and 11 enzymes for putative atherina boyeri and a. mochon pontica. they concluded that these are distinct species and indeed the black sea fish are a distinct species, a. pontica. they diverged 2.316 mya. these authors did not examine caspian sea material. miller in miller (2003) followed a conservative approach, regarding the various named and wide-ranging populations as representing a single polymorphic species. key characters: the two dorsal fins, cycloid scales, pectoral fin high on the flank and the vent remote from the anal fin are characteristic. morphology: first dorsal fin spines 5-10, second dorsal fin with 1-2 spines and 8-15 soft rays, anal fin with 1-2 spines and 9-18 soft rays, pectoral branched rays 10-17, usually 12-15, pelvic fin with 1 spine 57, usually 5, branched rays, lateral series scales 3753 (possibly to 61, see vasil'eva (1994)), total gill rakers 19-29 in the caspian sea, spinulose on the interior surface, and long and reaching about 7 rakers along the arch when appressed, and vertebrae 39-52. note narrower ranges for all preceding meristic characters for iranian material given below. the anus is 4-5 scales in advance of the anal fin. scales are higher than broad, with slight indentations on the otherwise straight dorsal and ventral margins, a rounded posterior margin, and a wavy to rounded anterior margin with a protuberant central point. there are no radii. circuli are restricted to the anterior third of the scale with a central and vertical roughened area posterior to the circuli, presumably made up of fragmented circuli. the focus is central. the lower jaw symphysis fits into a notch in the upper jaws. the haemal arches of the anterior 4-7 caudal vertebrae are expanded around the gas bladder. the gut is s-shaped. the chromosome number is 2n=48 (klinkhardt et al., 1995). meristic values for iranian specimens are: first dorsal fin spines 8(28), 9(5) or 10(1); second dorsal fin soft rays 11(10), 12(21), 13(2) or 14(1), anal fin soft rays 13(7), 14(15), 15(9) or 16(3), pectoral fin branched rays 12(1), 13(12), 14(14) or 15(6), pelvic fin branched rays 5 (34), caudal fin branched rays 13(1), 15(30), 16(2) or 17(1), lateral series scales 50(1), 51(10), 52(5), 53(3), 54(8), 55(3), 56(3) or 57(1), predorsal scales 17(1), 18(2), 19(12), 20(8), 21(3), 22(3), 23(1), 24(3) or 26(1), caudal peduncle scales 12(31), 13(2) or 14(1), transverse scales from anal fin antero-dorsally 10(2), 11(6), 12(23) or 13(3), total gill rakers 24(3), 25(4), 26(14), 27(8), 28(4) or 29(1), and total vertebrae 45(1), 46(19), 47(7) or figure 2. atherina caspia, anzali shore, december 2009, courtesy of k. abbasi. 285 coad/silversides of iran 48(7). sexual dimorphism: females tend to be larger than males and there are some differences in morphometrics and meristics (ghoorbanalidoost et al., 2003). colour: general colour is given in the family account. the lateral band is strongly developed and bright in caspian sea specimens. the belly is white, fins are pale to translucent grey. lagoon specimens are brownish or grey-brown on the back. the peritoneum is brown to black, eggs being encased in a black peritoneum while the abdomen wall is a light brown. size: reaches 11.8 cm and 17.6 g in the atrak river (savenkova and asanaov, 1991), to 14.5 cm standard length in the caspian sea basin generally (henderson and bamber, 1987). kiener and spillman (1972) found a maximum of 15.4 cm in their caspian sea sample and patimar et al. (2009) found 12.8 cm total length for their sample of 2256 fish in the gomishan wetland. distribution: found in the caspian sea and the uzboi valley of turkmenistan. in iran, it is reported along the whole caspian sea coast, and from the safid, tajan, tonekabon, havigh, karkan, chalus, farahabad, langarud, siahrud, sheikan, shafa, talar and atrak rivers, gomishan lagoon or wetland, boojagh wetland, anzali lagoon and the sowsar roga outlet of the lagoon seasonally, the caspian sea at bandar anzali breakwater and beach, hasan kiadeh, kazian beach and west of chalus, gorgan bay, alma-gol, adji-gol and ala-gol (savenkova and asanov, 1991; holčík and oláh, 1992; roshan tabari, 1997; karimpour, 1998; abbasi et al., 1999; kiabi et al., 1999; afraie and lalooie, 2000; khara et al., 2004; abbasi, 2006; patimar, 2008b; abdoli and naderi, 2009; patimar et al., 2009; esmaeili et al., 2014). zoogeography: this species probably entered the caspian sea from the black sea during khvalyn transgression (10-70,000 years b.p.) via the kumomanych depression (kosarev and yablonskaya, 1994). berg (1948-1949) contends that it entered the caspian from the black sea in post-glacial times while most other caspian fishes are relicts of earlier transgressions or migrants from northern waters. habitat: the caspian silverside is a schooling fish found at depths exceeding 100 m but is concentrated at 10-20 m. salinity tolerance is high, up to 77‰ (miller in miller, 2003), up to 60‰ in the atrak river (savenkova and asanov, 1991). reproduction can occur up to 42‰ while preferred levels are 312‰. a wide temperature range is tolerated, 0-31ºc (miller in miller, 2003). it is also found in lagoons and river mouths, and enters rivers to spawn, against currents up to 1.2 m/sec. it is the dominant species in the gomishan wetland in spring, summer and autumn (patimar et al., 2009). holčík and oláh (1992) report its apparent recent occurrence in the anzali lagoon in response to increased salinity there. it is also known from fresh water in lenkoran. it can rapidly adjust its life history to a range of environments, from fresh water to coastal water. age and growth: maturity is attained in the first year and life span varies from 1 to 5 years, 4-5 years usually in the caspian sea basin (henderson and bamber, 1987). ghoorbanalidoost et al. (2003) found populations in the south caspian sea to be 73.82 mm long on average and 3.15 g in weight, with age groups 1-3 years, average 1.7 years, total length and length-weight relationship w=0.00000615l3.02 and a sex ratio of male:female=0.47:0.53. heydarnejad (2009) gave the length-weight relationship for an iranian sample as w=0.0326tl3.033. patimar (1995) and patimar et al. (2009) found 2256 fish from the gomishan wetland had a 4-year life cycle (to age 4+ years). lengthweight relationships were w=0.0053tl3.0181 for males and w=0.0050tl3.063 for females, both allometrically positive. the von bertalanffy equation was lt=155.17[1-exp-0.28(t+0.738)] for males and lt=162.77[1-exp-0.27(t+0.727)] for females. the sex ratio was male:female 1:1.3 and females dominated in the older age classes. amri sahebi et al. (2015) examined 191 fish from the southeast coast of the caspian sea and found an average fork length of 7.7 cm, weight 3.64 g and age 2 years. 286 int. j. aquat. biol. (2015) 3(5): 282-289 food: savenkova and asanov (1991) report plankton, eggs and juvenile fishes to be food items in the atrak river of turkmenistan. some populations also eat benthic organisms such as amphipods, worms and molluscs. some iranian specimens contained encysted cladocerans and beetles in their guts. it has a trophic plasticity, adapting to whatever conditions obtain (miller in miller, 2003). amri sahebi et al. (2015) examined this fish in the southeast coastal waters of the caspian sea, finding it to be an opportunistic and carnivorous fish and a relatively voracious feeder. benthic gammarus and the zooplankton daphnia were favoured foods and various other cladocerans along with copepods, ostracods, nematodes and dipterans were taken. reproduction: spawning in this species is intermittent and occurs along the coast from may to july, peaking in mid-may to mid-june (savenkova and asanov, 1991) or from april to august (henderson and bamber, 1987). preserved iranian fish samples have relatively large eggs from april to september, e.g., 1.4 mm on 27 april, 1.6 mm on 14 may. savenkova and asanov (1991) also studied the annual spawning migration into the atrak river (lower reaches in turkmenistan, upper reaches shared with iran). fish are caught in a fish ladder 1819 km from the sea and in the river. the first schools appear in the atrek mouth as early as mid-february at water temperatures of 8-10°c but the mass migration takes place in mid to late march and the first half of april at 14-22°c. spawning occurs in march, april and may but most intensively in april at flood water temperatures of 13.7-23.0°c. there is a larger migration in high-water years. sex ratio is about 1:1. a female may be able to spawn 5-6 times in one season so that egg numbers and diameters vary within each individual for different generations of eggs. egg deposition in the caspian sea is associated with the alga cladophora to which the eggs are attached or entangled by long filaments (up to 15) and this plant is present in the atrak. egg diameters may reach up to 2.0 mm and the light yellow eggs number up to 5500 (caspian sea biodiversity database, www.caspianenvironment.o rg) presumably total seasonal fecundity. only one gonad develops. larvae are pelagic but may school close to shore. patimar et al. (2009) for their gomishan study found the reproductive season was march-july, peaking in march. average absolute and relative fecundities were 2976 eggs and 784 eggs g-1 of body weight respectively (874 in the abstract). maximum fecundity was 10,188 eggs for a 4+ fish. mean oocyte diameter was 0.68 mm. parasites and predators: the caspian seal, pusa caspica, is a predator on this species (krylov, 1984) as are the larger fishes such as stenodus leucichthys and alosa saposchnikowii (lönnberg, 1900) and the sturgeons acipenser persicus and a. stellatus (haddadi moghadam et al., 2009). economic importance: this species is food for a number of other fishes including such economic species as sturgeons, sander and the predacious shads. it has been caught as a by-catch in kilka seine nets and used in fishmeal production. in europe, the related a. boyeri has been sold fresh or canned, and in turkey has been investigated as a food in the form of fish chips (izci et al., 2011). conservation: kiabi et al. (1999) consider this species to be of least concern in the south caspian sea basin according to iucn criteria. criteria include abundance in numbers, widespread range (75% of water bodies), absent in other water bodies in iran, and present outside the caspian sea basin. the iucn (2014) has not assessed this species. sources: further details on collections examined can be found in the museum catalogues. iranian material: cmnfi 1970-0507, 11, 22.2-56.3 mm standard length, gilan, caspian sea at hasan kiadeh (37º24'n, 49º58'e); cmnfi 1970-0509, 5, 49.5-101.7 mm standard length, gilan, pond at hasan kiadeh (37º24'n, 49º58'e); cmnfi 19700543a, 7, 52.9-83.1 mm standard length, gilan, caspian sea at hasan kiadeh (37º24'n, 49º58'e); cmnfi 1970-0563, 18, 39.8-112.5 mm standard length, gilan, kazian beach (ca. 37º29'n, ca. 49º29'e); cmnfi 1970-0581, 5, 47.6-54.8 mm standard length, caspian sea near hasan kiadeh 287 coad/silversides of iran (37º24'n, 49º58'e); cmnfi 1970-0586, 7, 33.4-84.9 mm standard length, mazandaran, gorgan mordab at ashuradeh-ye kuchak (36º50’n, 53º56’e); cmnfi 1971-0343, 2, 65.9-72.5 mm standard length, gilan, langarud at chamkhaleh (37º13'n, 50º16'e); cmnfi 1979-0081, 3, 74.1-77.3 mm standard length, mazandaran, caspian sea, 3 km west of chalus (36º41'n, 51º24'e); cmnfi 1980-0127, 6, 39.7-44.4 mm standard length, gilan, caspian sea near hasan kiadeh (37º24’n, 49º58’e); cmnfi 1980-0146, 4, 92.1-103.6 mm standard length, mazandaran, gorgan mordab at ashuradeh-ye kuchak (36º50'n, 53º56'e); and cmnfi 1980-0160, 2, 49.2-73.8 mm standard length, iran, caspian sea basin (no other locality data); cmnfi 1993-0144, 1, 75.3 mm standard length, mazandaran, neka power plant (36º51’48”n, 53º23’24”e); cmnfi 20080105, 1, 87.6 mm standard length, gilan, bandar anzali breakwater (37º26’n, 49º29’e). acknowledgements i am indebted to the department of biology, shiraz university and the canadian museum of nature, ottawa for funding of research. numerous colleagues and co-authors assisted in developing the website on iranian fishes, providing specimens, data and photographs and are listed at www.briancoad.com. references abbasi k., valipour a., talebi haghighi d., sarpanah a., nezami s. 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(2015) 3(5): 282-289 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی (atherinidae)خانواده ایران گل آذین ماهیانمروری بر برایان کد نادا.، کاk1p 6p4انتاریو، ، موزه تاریخ طبیعی کانادا، اتاوا چکیده: گونه در این خانواده 86جنس و 41شوند. حدود معتدله تا گرمسیری و آبهای شیرین یافت میدر نواحی ساحلی دریاهای ماهیان آذین گل شناسی، یستماتیک، ریختشود. در این مقاله مروری، سدر دریای خزر یافت می atherina caspiaوجود دارد که تنها یک گونه بومی بنام هرستفشود. همچنین یک شده و به تصویر کشیده میایران توصیف گونه گل آذین خزری شناسی، اهمیت اقتصادی و حفاظت پراکنش، زیست گردد.در مورد این ماهی در ایران ارائه میمنابع .دریای خزرشناسی، شناسی، ریختزیست ،atherina :کلمات کلیدی int. j. aquat. biol. (2019) 7(4): 224-232 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article 1h nmr-based metabolomics approach to understanding the temperature-dependent pathogenicity of lactococcus garvieae nasim safari alighialo1, ruhollah rahimi*1, saeed hajirezaee2, farzaneh nikookhah1 1department of fisheries and environmental sciences, faculty of natural resources and earth sciences, shahrekord university, shahrekord, iran. 2department of fisheries and environmental sciences, faculty of natural resources, university of jiroft, jiroft, kerman, iran. s article history: received 24 april 2019 accepted 12 august 2019 available online 2 5 august 2019 keywords: metabolomics pathogenicity lactococcus garvieae fish abstract: lactococcus garvieae is known as main agent of the bacterial diseases, lactococcosis in trout farms. the present study was aimed to study the metabolic bases of the temperature-dependent pathogenicity of the l. garvieae using 1h nmr spectroscopy. the bacteria were grown at different temperatures, including 10, 14, 18 and 22˚c and then the metabolites extracted, identified and quantified. the results of pls-da analysis clearly separated the experimental treatments. the main metabolites responsible for this separation were acetate, acetoacetate, creatine phosphate, succinylacetone and trehalose. furthermore, the result of the analysis of variance indicated also significant differences in metabolome content between temperature treatments. the bacteria exposed to higher temperatures showed more concentration of acetate and acetoacetate compared to those grown at 10°c. the concentrations of trehalose were higher in the bacteria grown at 14 and 18°c compared to other temperature treatments. the higher levels of succinylacetone were found in the bacteria exposed to the temperature less than 14°c compared to those grown at 18 and 22°c. the creatine phosphate concentrations increased with temperature, however, a significant decline occurred at 22°c. the levels of isoeugenol, methionine and betaine significantly declined with increase of temperature from 10 to 22°c. also, the concentration of n-acetylglutamine significantly raised as the temperature increased from 10 to 22°c. in conclusion, the temperature altered the metabolome of l. garvie, which this may be linked to the pathogenicity. the temperature probably affects fermentation, homeostasis, energetic condition and metabolism of amino acids in l. garvieae. introduction the bacteria of genus lactococcus are well-known as agent of a wide spectrum of clinical lesions in aquatic animals, particularly in fish and shellfish (vendrell et al., 2006). in marine and freshwater aquaculture, the l. garvieae (a non-motile, anaerobic and grampositive species) has been well-recognized as the most important species of this genus, causing considerable economic losses (exceed approximately 50–80%) in fish farms throughout the world, especially in salmonid farms. in turkey, l. garvieae caused approximately 80% mortality in rainbow trout farms (diler et al., 2002). furthermore, this pathogen was identified as responsible for outbreaks (>50%) in rainbow trout production in australia, south africa, japan, portugal, france, taiwan, england and *correspondence: ruhollah rahimi doi: https://doi.org/10.22034/ijab.v7i4.609 e-mail: rrahimi6083@gmail.com countries of the mediterranean area (baeck et al., 2006; eyngor et al., 2004; pereira et al., 2004). lactococcus garvieae is the etiological agent of lactococcosis in fish with clinical signs, including anorexia, melanosis, lethargy, loss of orientation, erratic swimming, exophthalmia, accumulation of ascitic fluid in peritoneal cavity, surface (in the base of fins, opercula-bucal region) and internal hemorrhages (in the swim bladder, intestine, liver, spleen and kidney) and also necrosis in the liver and spleen (vendrell et al., 2006). based on epidemiological studies, the clinical signs of l. garvieae are temperature-dependent and usually emerged in fish when water temperature increases over 16°c in summer months (eldar and ghittino, 1999; soltani et al., 2008; vendrell et al., 2006). 225 int. j. aquat. biol. (2019) 7(4): 224-232 however, to our knowledge, there is no comprehensive information regarding the mechanisms underlying the temperature-dependent pathogenicity of this pathogen. in the present study, we used for the first time a 1hnmr based metabolic method to investigate the molecular bases of the temperature-dependent pathogenicity of the l. garvieae. metabolomics is known today as a new and growing approach to study low molecular mass metabolites (mts) (<500 da) within a cell, tissue, or biofluid of an organism (bundy et al., 2009; lankadurai et al., 2013; nicholson et al., 1999). the structure and function of almost all mts have been well-recognized, which this makes them easier to interpret compared to genomic and proteomic information. furthermore, mts are more stable during sample preparation, enhancing the accuracy of metabolic data in comparison with proteomic ones (ankley et al., 2006; lankadurai et al., 2013; van ravenzwaay et al., 2012). nmr based metabolic is one of the most important methods to analyse metabolome. nuclear magnetic resonance (nmr) is nondestructive, nonselective and laboratory reproducible (pan and raftery, 2007; robertson, 2005; viant et al., 2008). these advantages have made nmr as a good tool to assay metabolites within a biological sample (åslund et al., 2011; ritota et al., 2012; simpson and mckelvie, 2009; verpoorte et al., 2008). the results of the present study provide comprehensive data on temperature-dependent changes of metabolites in l. garvieae. the study also may help us to understand the molecular bases of the bacterial pathogenicity and subsequently to find solutions for reducing the pathogenicity using metabolic information. materials and methods bacterial strain and preparation: a previously proven strain of l. garvieae was provided from faculty of veterinary sciences, shahrekord university, iran. the bacteria were initially inoculated into trypticase soy agar (tsa) (merck, germany) and incubated at temperatures of 10, 14, 18 and 22°c for 24 h. after incubation, the bacteria were separately dissolved in distilled water to assay optical density (1-1.5) by spectrophotometer. 1.5 ml of the suspension was grown in 15 ml tryptic soy broth (tsb) for 24 h. the culture was regenerated using 4 ml of the suspension in tsb (96 ml) for 14 h. after that, the suspension was centrifuged (8000 rpm) at 8°c for 1.5 min and then 0.5 ml of deposit was separately inoculated in 45 ml tsb at 10, 14, 18 and 22°c for 24 h. after incubation, 15 ml of liquid nitrogen was added to each medium to reduce the metabolism in bacteria. the suspensions were then centrifuged (12000 rpm at 4°c) for two times, followed with washing by 10 ml and 1 ml nacl solution respectively. eventually, the bacterial was centrifuged (16100 rpm at 4°c for 1 min) and lyophilized overnight at -41°c and stored in liquid nitrogen until metabolite extraction (boroujerdi et al., 2009). metabolite extraction: the metabolites of bacteria were extracted using 2:1 (v/v) meoh:h2o at a constant cell mass/solvent ratio of 9 mg (dry weight) of lyophilized cells and 0.5 ml of meoh:h2o according to boroujerdi et al. (2009). the solutions were vortexed gently and then centrifuged at 11000 g for 6 min at 8°c to eliminate cell debris. finally, the supernatants containing metabolites were dried by a vacuum centrifuge drier (eppendorf vacufuge, westbury, ny) for 150 min at 30°c to obtain concentrated extracts. the extracts were then stored in liquid nitrogen (-196°c) until nuclear magnetic resonance (nmr) assay. 1h nmr assay: 1h nmr assay was carried out based on the method described by boroujerdi et al (2009) with some modifications. in brief, 500 mhz nmr spectrometer were used (brukerbiospin. 165 corporation, billerica, ma, usa). the bacterial extracts were resuspended in 600 µl nmr buffer [60 μl internal standard solution (5 mm sodium 2,2 dimethyl-2-silapentane-5-sulfonate (dss) (as internal reference) in 99% deuterium water (d2o) +540 μl of 0.2 m sodium phosphate buffer (ph 7.0) containing 0.018% sodium azide (nan3)] and then transferred into a 5-mm nmr tube (wilmad, buena, nj). the carr‐purcell‐meiboom‐gill (cpmg) technique was used to block signals related to proteins and other 226 safari alighialo et al./ metabolomic of lactococcus garvieae macromolecules (tian et al., 2013). free induction decays (fids) were obtained with 150 scans into 32k data points, using a spectral width of 8389.262 hz with a relaxation time of 1.5s, an acquisition time of 4s, and a mixing time of 400 ms. nmr spectra (nsps) were analyzed by chenomx nmr suite (chns) ver. 7.6 software (chenomx inc., 172 edmonton, ab, canada). the nsps were firstly phased and baselined by the processor program and then metabolites identified and quantified using profiler program. data analysis: principal component analysis (pca), followed by partial least squares discriminant analysis (pls-da), which converts multidimensional data into smaller model, was used for analysis of metabolome data (keun et al., 2003; xu and shao, 2004). before pca and pls-da analysis, nmr datasets were normalized using log transformation and autoscaling options. all above analyses were conducted using metaboanalyst, a comprehensive web-based tool designed for processing, analyzing, and interpreting metabolomic data (xia and wishart, 2011). the analysis of variance (anova) and comparison of means between temperature treatments was performed by spss software (spss 19.0, ibm software, inc., chicago, il, usa). before analysis, the normality of data was evaluated by kolmogorovesmirnov test. after calculating significant f-ratios by anova, the tukey test was used to identify which groups were different (p<0.05). all data were presented as mean and standard deviation (sd). the p<0.01 was used as significance level for data evaluation. results representative 1h nmr spectra of the bacterial metabolome are shown in figure 1. totally 23 metabolite were identified (fig. 1). the scores plot obtained from pls-da clearly confirmed the effects of different temperature treatments on endometabolites of the l. garvieae, because the bacteria incubated at 18 and 22°c showed significant overall separation from those grown at 10 and 14°c (fig. 2). the main metabolites responsible for this separation are indicated by corresponding loading plot (fig. 3). 12 metabolites contributed more in the separation of temperature treatments according to vip scores obtained from pls-da (fig. 3). in this regard, the more significant contributions were related to acetate, acetoacetate, creatine phosphate, succinylacetone, trehalose (fig. 3). the result showed also significant differences in metabolome content between temperature treatments (fig. 4a-e, p<0.05). the concentration of acetate and acetoacetate were lower in the bacteria exposed to 10°c compared to other temperature treatments (fig. figure 1. representative 500 mhz 1h nmr spectrum of the bacterial metabolome (lactococcus garvieae). 227 int. j. aquat. biol. (2019) 7(4): 224-232 4a, p<0.01). the levels of trehalose were higher in the bacteria treated with 14 and 18°c compared to other treatments (fig. 4a, p<0.01). the higher concentrations of succinylacetone were observed in the bacteria exposed to the temperatures less than 14°c compared to those treated with the temperatures of 18 and 22°c (fig. 4b, p<0.01). the creatine phosphate (cr) levels increased, as the temperature reached 18°c (fig. 4b, p<0.01). however, we found a considerable decline in cr concentrations at 22°c (fig. 4b, p<0.01). the concentration of isoeugenol (fig. 4b), methionine (fig. 4d) and betaine (fig. 4e) significantly declined with increase of temperature from 10 to 22°c (p<0.01). the n-acetylglutamine levels significantly elevated as the temperature increased from 10 to 22°c (fig. 4c, p<0.01). also, there were no significant differences between temperature treatments in terms of the levels of aconitate and n-acetylglutamine (fig. 4c, p<0.01). discussions it is well-recognized that the pathogenicity of l. garvieae increases with raises in water temperature, especially when fish are exposed to the temperature higher than 16°c (eldar and ghittino, 1999; soltani, et al., 2008; vendrell et al., 2006). the present study was carried out to investigate the temperaturedependent pathogenicity of l. garvieae from a metabolic point of view. the results of pls-da analysis showed significant separations between experimental groups, indicating the effects of temperature on the metabolome content of the bacteria. the metabolites, including acetate, acetoacetate, trehalose, succinylacetone, creatine phosphate, isoeugenol, betaine, glycine, nacetylglutamine, trans-aconitate, acetamide and methionine had more contribution in this separation based on vip scores. acetate and acetoacetate are known as ketone bodies in biological systems and produced during the oxidation of the lipids for meeting energetic demands of cells (newman and verdin, 2014). in anaerobic bacteria, it is recognized that acetate is produced as a result of the fermentation of organic matters including fatty acids (fujita et al., 2007; ljungdhal, 1986; mah et al., 1977). furthermore, acetate along with formaldehyde, butyrate, lactate and succinate were found to be the final product of lactic acid bacteria (gottschalk 2012). however, we could not found any data regarding the metabolism of fatty acids and figure 2. partial least-squares discriminant analysis (pls-da) of the bacterial metabolome (lactococcus garvieae) at different inoculation temperatures. figure 3. main metabolites identified by pls-da. the colored boxes on the right indicate the relative concentrations of the corresponding metabolite in each group under study. 228 safari alighialo et al./ metabolomic of lactococcus garvieae related metabolites such as acetate and acetoacetate in l. garvieae. in our study, lower levels of acetate and acetoacetate were observed in the bacteria treated with low temperature (i.e. 10°c) compared to other temperature treatments, probably indicating the retarding effects of low temperatures on the bacterial fermentation and subsequently its pathogenicity. trehalose, also known as mycose, is a 1-alpha disaccharide which plays important role in hydrobiosis, the ability of organism to withstand prolonged periods of dehydration. trehalose is found to form a gel phase as cells dehydrate, which prevents the disruption of internal cell organelles by effectively splinting them in position (filippov et al., 2015). in bacteria, trehalose is found to protect the bacteria against heat shocks and dehydration (argüelles, 2000; gomez zavaglia et al., 2003; leslie et al., 1995). in the l. garvieae, more accumulation of trehalose was observed in the bacteria exposed to temperatures of 14 and 18°c, which may be attributed to physiological response of the bacteria to high temperature. nevertheless, the levels of trehalose decreased in the bacteria exposed to 22°c, indicating the possible disrupting effects of high temperatures on the accumulation of trehalose. succinylacetone is an abnormal tyrosine metabolite that produced as a result of defect in the fumarylacetoacetase, an enzyme responsible for hydrolysis of fumarylacetoacetate into fumarate and acetoacetate (lerner, 2009). fumarylacetoacetase is involved in metabolism of phenylalanine and tyrosine (lerner, 2009). succinylacetone can also be generated by the oxidation of glycine (dindo et al., 2018). in the present study, the temperature less than 14°c induced figure 4. comparison of the metabolite levels (mean ±sd) between temperature treatments in lactococcus garvieae. significant differences are shown with different letters (p<0.01). 229 int. j. aquat. biol. (2019) 7(4): 224-232 succinylacetone production, which this may be associated with the disrupting effects of low temperature on metabolism of some amino acids such as tyrosine, glycine and phenylalanine. however, we found no information on metabolism of succinylacetone in bacteria. in biological systems, creatine phosphate (cr) is known as an energetic buffer, which reduced to atp and creatine to meet the immediate needs of cells to energy. cr also affects the metabolism of arginine and proline (pastoris et al., 1998). in our study, as the temperature reached the optimum temperature (18°c) of the bacteria, the intracellular concentration of cr also increased, suggesting an efficient regeneration for cr at temperatures in which the pathogenicity is high. nevertheless, at 22°c, we observed a considerable decline in cr concentrations, which may be related to the adverse effects of high temperature on the energetic homeostasis of the bacteria. the isoeugenol is known as a natural antimicrobial component and identified in essential oils extracted from many terrestrial plants (akhtar et al., 2012; dal pozzo et al., 2012; janssens et al., 1990; zemek et al., 1979; zemek et al., 1987). the metabolic conversion of phenolic compounds such as isoeugenol has been the focus of many studies in bacteria. for example, the metabolic conversion of phenolic compounds to vanillin has been well-reviewed by priefert et al. (2001). however, it is well-recognized that lactic acid bacteria are not able to produce vanillin from the components such as isoeugenol (bloem et al., 2007). in the present study, the intracellular concentration of isoeugenol decreased with temperature, which may be due to the direct destructive effect of temperature on metabolite or metabolic conversion of the isoeugenol to other metabolites at temperatures in which the pathogenicity is high. betaine is usually recognized as an osmoprotectant metabolite in organisms including bacteria (courtenay et al., 2000; csonka, 1989; csonka and hanson, 1991; roeßler and müller, 2001). in various bacteria, including lactic acid bacteria (van de guchte et al., 2002), the accumulation of betaine and other osmolytes have observed against high salinity shocks and other stressors, such as elevated temperature (caldas et al., 1999). most bacteria are not capable to synthesize betaine de novo and rely more on uptake from medium (culham et al., 1993; haardt et al., 1995; molenaar et al., 1993; racher et al., 1999). the results of present study were in contrast with above conclusions on betaine, since we observed declines in intracellular betaine with increases in temperature. these results may be attributed to the species-specific differences in the behavior of bacteria in response to the temperature shocks. in l. garvieae, the temperatures more than 16°c are recognized as optimum temperatures for bacterial growth and pathogenicity. therefore, the concept of temperature shock and its relationship with betaine accumulation may be different in l. garvieae in comparison with other bacteria. n-acetylglutamine is a modified stable amino acid that used as a source of glutamine (lópez-pedrosa et al., 2007; snowden et al., 2002). the production of nacetylglutamine is also reported in bacteria during fermentation (haran et al., 1983; kinoshita and tanaka, 1972; nakanishi, 1978; qu et al., 2002). however, we could not find evidences regarding the generation of this amino acid in l. garvieae. in our study, the n-acetylglutamine concentrations showed significant elevations with temperature, indicating the possible effect of temperature on the metabolism of glutamine. as an essential amino acid, methionine has a main role in the initiation of translation and following biosynthesis of the proteins. almost all bacterial species possess biosynthetic pathways for methionine (foster et al., 2005; mccutcheon and moran, 2012). in the present study, the temperature affected the metabolism of the methionine, because the concentration of this amino acid decreased as the temperature elevated. the reduction in intracellular methionine may be attributed to the metabolic consumption of this amino acid for synthesis of proteins, as the temperature approached the optimum temperatures for growth and pathogenicity of the l. garvieae. in conclusion, the temperature altered the 230 safari alighialo et al./ metabolomic of lactococcus garvieae metabolome of l. garvie, which this may be linked to the pathogenicity. the temperature probably affects fermentation, homeostasis, energetic condition and metabolism of amino acids in l. garvieae. references akhtar y., pages e., stevens a., bradbury r., da camara c.a., isman m.b. 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(2015) 3(6): 398-408 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article rodlet cells changes in oreochromis niloticus in response to organophosphate pesticide and their relevance as stress biomarker in teleost fishes natália de souza araujo*1, joão carlos shimada borges2,3 1universidade de são paulo, instituto de biociências usp, departamento de genética e biologia evolutiva, room 320. rua do matão, 321, cidade universitária, são paulo, sp, zip code: 05508-900, brazil. 2universidade paulista, vice reitoria de pós graduação e pesquisa unip. dr. bacelar, 1212 vila clementino são paulo – sp, zip code: 04026-002, brazil. 3faculdades metropolitanas unidas fmu, mestrado em saúde ambiental, rua ministro nelson hungria, 541, real parque, são paulo,sp, zip code: 05690-050, brazil. article history: received 24 september 2015 accepted 21 november 2015 available online 2 5 december 2015 keywords: organophosphate biomarkers nile tilapia methyl parathion abstract: rodlet cells are frequently found in teleost fishes and although their role in organisms is not completely understood. the occurrence of these cells are related to stress and may undergo changes in contaminated environments, thereby allowing their use as biomarkers. this hypothesis is tested in the present study. thirty specimens of oreochromis niloticus were divided into three groups, two groups were exposed to organophosphate pesticide methyl parathion at nominal concentrations of 4 mgl-1 and 8 mgl-1 and one group was kept as control. after ten days, the gills were removed for microscopic study and the number and area of the rodlet cells were analyzed and compared with a well-established method of assessing histological damages in fishes. no significant differences were found in the area of the cells, but there were significant differences in the number of rodlet cells among examined concentrations. the present study provides evidence for the use of this new biomarker in teleost fishes and discusses some of the potential confounding factors of this approach. introduction the nile tilapia (oreochromis niloticus linnaeus 1758) is a cichlid fish of great economic interest in pisciculture and aquaculture (borges et al., 2013). the species is originated from africa and is widely distributed in water bodies of many tropical countries because of its resistance to different trophic levels (agnèse et al., 1997; borges et al., 2013). the nile tilapia was reported to have a remarkable cell type called rodlet cell (rc) (borges et al., 2013) that have also been found within the epithelia of 36 teleost fish families (fishelson et al., 2011) from marine to freshwater habitats. the rcs are easy to distinguish when mature because of their elongated form, dense microfibrils capsule, basal nucleus and cytoplasmic corpuscles in the shape of rodlets or arrows that point to the apical area of the cell where they are discharged (bielek, 2005; schmachtenberg, 2007; depasquale, 2014). * corresponding author: natália de souza araujo e-mail address: na.araujo@usp.br for over 120 years, scientists have attempted to discover the origin and function of the enigmatic rc. thélohan first described these cells in 1892 and believing he had found a parasite the author named it rhabdospora thelohani (manera and dezfuli, 2004). plehn (1906) considered the rc to be defense cells with granules of endogenous action, thereby refuting thélohans’ hypothesis. both assumptions have continued to coexist with different interpretations about the function and origin of these cells until the last decade (schmachtenberg, 2007). currently it is mainly accepted that the rc are endogenous cells involved in defense (sfacteria et al., 2014). the rc presence is often associated with other elements of the fish innate immune system such as mast cells, eosinophils and neutrophils (bielek 2005). the rodlets are usually discharged at the external face of the epidermal layer and although 399 araujo and borges/ rodlet cells as biomarkers their composition material is still unknown there are evidences that the antibiotic peptide piscidin and dna reside in the rodlets (barber and mills westermann, 1986; silphaduang et al., 2006). thereby different works have suggested that the rc comprise part of the host inflammatory defense response (bielek, 2005; matisz et al., 2010; depasquale, 2014). regardless of its function, there are strong evidences for the potential use of the rc as biomarkers of exposure and effect as first observed by manera and dezfuli (2004). biomarkers are bodily fluids, cells, tissues and physiological or behavioral changes that indicate the presence of contaminants in exposed organisms (livingstone, 1993). biomarkers of exposure can be used to confirm and evaluate the exposure of an individual or group to a particular substance, thereby establishing a link between external exposure and the measurement of internal exposure. biomarkers of effect can be used to document pre-clinical changes or adverse health effects from the exposure to and absorption of chemicals (amorim, 2003; rüdiger, 1999; world health organization, 1993). thus, the rc may be potently important for monitoring environmental quality and the health of organisms that live in polluted or stressed environments. although there are many studies describing the occurrence of the rc in the presence of a variety of external stressors (dezfuli et al., 2003a; manera and dezfuli, 2004; giari et al., 2008; poltronieri et al., 2009; matisz, 2010; rebok et al., 2010). there is no previous work standardizing exactly how this information can be used for biomarkers proposes. rebok et al. (2010) attempted to address this issue analyzing the liver of barbus peloponnesius specimens from the river bregalnica in the republic of macedonia, but it is not clear if the place they have used as reference for comparisons was indeed free of stressors. as stated by the authors “the microscopy data, however, is only partially indicative of stress effects at site a” which is the supposed polluted environment. herein we use the pesticide methyl parathion as an environmental stressor in increasing concentrations to analyze the fish response in number and area of rc. the methyl parathion is an organophosphate pesticide that after degradation produces the compound methyl paraoxon through metabolic conversion, which is even more toxic (araujo et al., 2006; machado, 1999) inhibiting enzymes such as cholinesterase, carboxylases, mitochondrial oxidative phosphorylases, and acetylcholinesterase (machado, 1999). nevertheless, this compound is widely used in agriculture and aquaculture and also illegally as indoor pesticide (ruckart et al., 2004). thus to comprehend its effects in the environment is essential. herein we evaluate the toxic effect of the methyl parathion in the gill epithelium of fishes exposed to different concentrations and compare this response to changes in size and quantity of the rc in the tissue of this organ. materials and methods fish maintenance and experimental design: thirty juvenile specimens of the nile tilapia (with mean standard length of 8.79±0.82 cm, and mean weight of 17.9±4.69 g) were obtained from a local supplier. fishes were fasted for five days prior to and during the trial to avoid interference due to the permanence of food in the gut and water (lenon and walker, 1964). the specimens were randomly divided into three groups housed in three 16-liter aquaria for an additional five days period of acclimation. two groups were then exposed to nominal concentrations of 8 mgl-1 and 4 mgl-1 of methyl parathion [o,odimethyl-o-(-4-nitrophenyl) phosphorothioate, c8h10no5ps] (folisuper 600 br ® methyl parathion, 600 g/l, agripec) and one group was kept as control in non-contaminated water. the pesticide concentrations have being chosen based in two criteria, first in the lc50 dose for juveniles of piaractus mesopotamicus (9.89 mgl-1; machado, 1999) and second on small-scale experimental trail (unpublished data) designed to promote toxic effects without causing death within 5 days. throughout the periods of acclimation and exposure physiochemical parameters [nitrite, ammonia and ph (tetratest ®)] 400 int. j. aquat. biol. (2015) 3(6): 398-408 were monitored weekly and water temperature was maintained between 20-26(±0.5)°c using thermostats connected to heaters (40 w). cooling was provided by air conditioning. there were neither control of photoperiod nor maintenance of the pesticide after its initial dilution. after 10 days exposure, the animals were anesthetized (immersed in ethyl benzoate at 50 mgl-1) and euthanized with the disruption of the spinal cord proximal end (silva et al., 2005). the gills then were removed for histological analyses. all procedures involving animals were in accordance with the ethical standards of the animal care committee (019/09 cep/ics/unip). tissue processing for optical and electron microscopy: for optical microscopy, the gills were fixed in cold 2% glutaraldehyde solution buffered ph 7.2 with 0.1 m phosphate (mcdowell and trump, 1976) for 12 hrs. two middle gill arches (one on the left and one on the right) were selected for the preparation of the histological slides. after removal of the fixation solution, the gills filaments were submitted to dehydration in increasing alcohol and historesin (glycol methacrylate) solutions until their complete inclusion in historesin. the material was then embedded (resin + hardener) and placed to dry at 40ºc. histological sections 1 to 3 μm in thickness were obtained using a microtome and stained with fuchsine and toluidine blue. the slides were then photographed and analyzed under a light microscope (zeiss standard 25 ics). samples from each group have also been processed for electron microscopy. for this analysis the tissue was fixed at 0ºc in 2.5% glutaraldehyde in phosphate buffer (0.1 m, ph 7.2), postfixed in 1.0% oso4 (hayat, 1981) and embedded in spurr resin (spurr kit for electron microscopy, sigma chemical co., usa). ultra-thin sections (70 nm) were placed on copper grids and stained with 2% uranyl acetate in distilled water for 1 h, then washed in distilled water and stained with 0.5% lead citrate in distilled water. the ultrastructure was examined using a jeol 100 cx-ii electron microscope. gill analysis qualitative analysis: the method established by poleksic and mitrovic-tutundzic (1994) was used to evaluate tissue damage as a control for the effects of the pesticide. this method is a rating system based on changes that are frequently encountered in injured tissue and that gives an i number categorized in one of four groups. an i value between 0 to 10 denotes a functionally normal sample; 11 to 20 denotes mild to moderate alterations; 21 to 50 denotes moderate to severe alterations, and over 100 denotes severe alterations. in this method, each alteration reported in the tissue accounts for the overall score in a different way according to its severity, thus alterations from first stage include changes such as hypertrophy and hyperplasia while the third and last stages accounts for necrosis and fibrosis of the tissue. thus i values under 100 indicate that a greater number of reversible changes are present in the tissue but an i score higher than 100 evidences irreparable tissue damage. the observation of the sections was standardized in a way that the images could not be repeated. twenty-five fields (1000x magnification) in alterative sections when necessary were randomly chosen for analysis. however, when histological artifacts prevented the proper interpretation of the morphological features, the field was replaced by another. images observed in the electron microscope were used to improve the visualization of the rc and the histological alterations but not for counting them. quantification and measurement of rodlet cells: the same criteria for field observation used in the i value analyses were adopted for the rc tests in optical microscopy (25 random fields per fish 1000x magnification). the fields were always randomly selected and the total number of rc in the secondary lamella of each selected area was manually counted. to measure the area of rc alterative sections in different concentrations were checked to select 10 cells in each tested group (including control). these cells were then photographed and analyzed using the image j® program, which enabled the longitudinal measurement of the cell area. only clearly characterized rc were considered, i.e., cells 401 araujo and borges/ rodlet cells as biomarkers showing a given trait such as peripheral nucleus, thick cell-wall and presence of the cytoplasmatic rodlets (bielek, 2005). data analysis: all the statistic tests were performed in the r package software version 3.1.1 (r development core team, 2008). data were first checked for their normality with the shapiro-wilk normality test. when normal distribution was observed, the comparative analyses were performed with two-way anova. for non-normal data the kruskal-wallis non-parametric test was employed with post-hoc comparison made using mannwhitney test with bonferroni correction. the confidence intervals were calculated with 2000 bootstrap replicates. the results obtained with the i value of poleksic and mitrovic-tutundzic (1994) have being used to identify the occurrence and intensity of damage to the gill epithelium. once the injuries were detected the rc number and area have been compared with the damage verified to evaluate their efficiency as biomarkers. results qualitative analysis: the dose-response curve of the mean i values obtained in all nominal concentrations follows in figure 1. the results clearly indicate that the control fish were in healthy condition (mean i value: 6.8±9.6), whereas nominal concentrations of 4 and 8 mgl-1 caused irreparable damage to the gills (mean i value: 115.3±42.3 and 125.4±10.9, respectively). according to the shapiro-wilk the data is non-normal (p=0.001) with significant differences among the groups (kruskal-wallis p=0.0002). poleksic and mitrovic-tutundzic (1994) i value is based on the observation of a series of histologic information in the gill epithelium altered by contaminants. it indicates morphological changes such as hypertrophy and hyperplasia of the structural cells and the proliferation of mucous and chloride cells. these mechanisms may form a barrier between the irritating agent and the blood stream but will also hinder the gas exchange (mallat, 1985; perry, 1997). some examples of the histological alterations commonly observed in the samples exposed to the pesticide methyl parathion are displayed in figure 2. quantification and measurement of rodlet cells: figure 3 displays some of the rc observed at different concentrations. as indicated in the figure, rcs were found in all groups at different regions and stages of development. they were especially difficult to find in the gills of the control group and had been found usually close to blood vessels in fish, although in some cases, few cells were observed in the edge of the secondary lamella discharging the rodlets in the exterior (fig. 3d). however, this behavior were more frequent in the exposed fish in which most of the rcs were found in the base of the secondary lamella. central tendency measures from the rc analyses are summarized in table 1. the area of the rc follows a normal distribution but does not present any statistical significance difference among the groups, however the amount of rc shows a different pattern. the number of cells presented in the secondary lamella of each group is a non-normal dataset with a significant statistical difference (p=0.021) indicating that the amount of rc changes in the presence of the contaminant. post-hoc analyses have shown that the result is due to the difference between the control and the intermediated concentration (4 mgl-1 p=0.019* and 95% confidence interval of 0.500-6.996). but no significance is found when comparing the 8 mgl-1 group (p=0.368 for control x 8 mgl-1; p=0.697 for 4 mgl-1 x 8 mgl-1, 𝛼 = 95%). figure 1. i value observed in each tested concentrations (mgl-1). numbers refer to the mean observed in control (0 mgl-1), 4 mgl-1 and 8 mgl-1. * p <0.05 for α = 95%. 402 int. j. aquat. biol. (2015) 3(6): 398-408 discussion in the present study, the mean i values found in fish exposed to both nominal concentrations of the pesticide (4 and 8 mgl-1) indicate irreparable damage in the gills. the tested concentrations are close to the ones reported for regular use (machado, 1999) but figure 2. histological alterations commonly observed in gills during transmission electron microcopy; (a) necrosis in the tissue, indicated by the arrows (8 mgl-1, 1000x), (b) (δ) chloride cell (*) mucus cell (8 mgl-1, 3000x), (c) apoptosis in the cells indicated by the arrows (8 mgl-1, 2500x), and (d) (δ) monocytes and (*) erythrocytes in blood vessel (8 mgl-1, 3000x). rc count rc area control 4 mgl-1 8 mgl-1 control 4 mgl-1 8 mgl-1 median 0.00 2.00* 0.00 1 587.36 2 085.65 1 388.06 mean 0.10 ± 0.32 4.30* ± 6.80 3.00 ± 7.48 1 509.28 ± 503.50 1 986.23 ± 680.20 1 535.10 ± 450.57 * p=0.019 <0.05 for =95%. table 1. central tendency measures summarizing the rc data observed in all groups. area of the rc are in mm2 and the significant value is featured in bold. n = 10 fishes in each studied concentration. 403 araujo and borges/ rodlet cells as biomarkers nevertheless the results demonstrate that even at sublethal doses the organophosphate can harm fishes severely compromising the pulmonary function of the epithelium. when considering the rc data no significant difference was found among groups according to the area of the cells. it indicates that this aspect should not be used to measure the contamination of an environment by methyl parathion. however, a significant difference was found in the amount of rc. although this was not directly correlated to the intensity of the contamination, since the highest number of rc occurred in the intermediate concentration of 4 mgl-1. this result may be explained by a greater number of irreparable alterations (necrosis and fibrosis) at the higher concentration, indicating that increasing the dosage of the organophosphate agent might compromise the integrity of the rc. this hypothesis is supported by the microscopy analysis, where degenerated rc and other cell types have been observed at 8 mgl-1. previously the degradation of the rc have also been documented in a study where fishes were exposed to an herbicide (dezfuli et al., 2003b) thus it is known that very toxic compounds may harm these cells. in natural contaminated environments such as lakes related to urban areas, the correlation of the number of rc and the degree of contamination is more figure 3. rodlet cells observed in o. niloticus gills in transmission electron microscopy. indicated by the arrows: (a) immature rodlet cell (control group, 6000x), (b) rodlet cell degenerating (8 mgl-1, 7500x), (c) mature rodlet cells (4 mgl-1, 4000x), and (d) rodlet cell degranulating rodlets in the exterior (control group, 5000x). 404 int. j. aquat. biol. (2015) 3(6): 398-408 straightforward. borges et al. (2013) reported a correlation value of 0.98 between the number of rcs found in nile tilapias and the trophic state index of the studied water bodies. nevertheless in the cited work none of the i index found for the gills were over 100 corroborating the hypothesis that in the higher concentration of the pesticide the rcs were too compromised to be clearly identified and consequently indicate the toxic effect of the compound. that is because the capability to distinguish the rc in the tissue will vary according to the stage of development of these cells. opposed to their characteristic oval shape and rodlets, the rcs in the very early stage of development or when degenerating are not easy to distinguish from other cell types and are probably under recorded in literature (laurà et al., 2012). based on the results, it is possible to observe some of the changes in the rcs morphology due to increasing exposured concentration. in figure 3d, the image shows the mature and easy to distinguish rc, while in figure 3a and 3b follow the immature and degenerated rc, respectively. because the methods used to identify the rc only accounted for mature cells and degenerated cells have been observed in the highest exposure concentration due to the pesticide; it is reasonable to assume that the number of rcs were under estimated in the higher concentration. other variables to keep in mind when using the rcs as biomarkers are the species, used tissue and temporal factor. fishelson et al. (2011) encountered rcs that differed strongly from other fish in lizardfishes not only regarding its structure but also about their location. in turn, matisz et al. (2010) noticed a temporal change in the occurrence of the rcs in the optic lobe of pimephales promelas exposed to trematode cercariae. they have shown that the rc densities increased at 4 and 6 weeks after the infection followed by a decline in the ninth week. nevertheless in the present study and in many other cases in literatures, there are evidences of numerical changes in the amount of rc due to the influence of different stressors. such as parasites, herbicides, heavy metals, viral infections, tissue damage, differences in water depth and overcrowding (bielek, 2005; manera and dezfuli, 2004; dezfuli et al., 2006; giari et al., 2008; mazon et al., 2007; poltronieri et al., 2009). according to schultz et al. (2014), even unknown disturbing agent(s) in groundwater causes an increase in the number of rc in australian murrays. thus, even considering all the potentially confounding factors discussed before, the evidences indicating the use of rcs as biomarkers for most of the teleost fishes are overwhelming. therefore, the use of the rcs in the tissue as a biomarker is possible but when using this approach it is important to keep in mind the confounders, such as: tissue/ species used in the analyses (are rcs described in this tissue/ species); degree of contamination (is this environment/ compound too toxic for the rc) and time (is the temporal lap since contamination enough for the appearance and maturation of the rc). when these variables are considered during experimental design and interpretation of the results the amount of rcs in the tissue may be a very simple and fast way of detecting contamination. furthermore, although the exact function of the rcs has been debated, the current view is that they are involved in host non-specific immune response as discussed in sfacteria et al. (2014). this idea are supported by many studies connecting the rcs appearance to a number of stressors laurà et al. (2012) and in association to other immune system cells (kramer et al., 2005; reite and evensen, 2006). laurà et al. (2012) have registered in ultrastructural microscopy, the development of the rc from immature to complete mature stage in the intestinal epithelium of the sea bass (dicentrarchus labrax) and concluded that “the rcs may be considered to be a normal component of teleost tissues, probably with a secretory function” and also that it has a probable defensive role associated with secretory activity. thus, despite there are some important open questions about the rc such as: what is exactly the composition of the rodlets? what is the evolutionary origin of these cells? what are their exactly importance for the host response in teleost fishes? 405 araujo and borges/ rodlet cells as biomarkers this is the first time that their use as biomarkers has been tested in a standardized way in order to discuss the variables and confounding factors of this approach. the results described here allied to previous workers allow the conclusion that the number of rcs in tissues exposed to stressors (such as the gills for polluted water) is indeed a good biomarker of effect. but in order to avoid misinterpretation of the results, it is recommended to use species and tissues where the occurrence of the rc were well-documented and only when the tissue is not necrotic. we also indicate the use of the rc number altogether with other histological metrics if a more detailed analysis of the environment is needed, especially in highly contaminated habitats. conclusions we conclude that sublethal doses of the organophosphate methyl parathion can be harmful to the maintenance of animal life and that the number of rc is an efficient method to identify the stressor presence. this is the first time that these cells are systematically tested as biomarker and the evidences found in this work aligned to previous results indicate the usefulness of the rc as a biomarker of effect. however taking into account the higher amount of rc in the intermediate concentration and their temporal pattern of appearance (matisz et al., 2010), this technique should be used carefully and the possible confounding factors must be taken into consideration during experimental design. the count of rcs in the exposed tissues is a simple and fast way to identify contamination but in order to establish a more precise panorama regarding the intensity of the environmental contamination this parameter must be analyzed with other aspects of tissue alteration, such as those described by poleksic and mitrovictutundzic (1994). acknowledgments the authors are grateful to the brazilian fostering agency conselho nacional de desenvolvimento científico e tecnológico (cnpq) and to the universidade paulista (unip) for financial support; also to the universidade de são paulo and to josé roberto machado cunha da silva, phd for the laboratories, materials and the maintenance of the vivarium for the experiments; to agripec química e farmacêutica s/a for the donation of the organophosphate folisuper 600br® used in the analyses. declaration of interest the first author has received a fellowship from (cnpq/pibic process 116069/2008-2 and 117961/2009-4). the second author is employed at the universidade paulista (unip). the authors declare that they have no conflict of interest. references agnèse j.f., adépo-gourène b., abban e.f., fermon y. 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(2015) 3(6): 398-408 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی به آن رابطه و ارگانوفسفات ماهی سم به پاسخ در oreochromis niloticusدر ماهی rodlet هایسلول تغییرات استخوانی ماهیان در زیستیشاخص عنوان 2،3بورگس شیمادا کارلوس جوا ،*1آراوجو دسوزا ناتالیا .برزیل ،سائوپالو سائوپالو،، دانشگاه زیستی تکامل و ژنتیک گروه، uspزیستی انستیتو علوم 1 .برزیل سائوپالو، دانشگاه پاولیستا، ،unipتحصیالت تکمیلی و تحقیقات بخش2 .برزیل سائوپالو، ،fmu متحده متروپولیتنکالج ،محیط بهداشت ارشد کارشناس3 چکیده: این بروز ،باشدنمی مشخص کامل طوربه هنوز موجودات در آنها نقش چه اگر .شوندمی یافت استخوانی ماهیان در معمول طوربه rodlet هایسلول ینا. شوند استفاده زیستی شاخص عنوانبه توانندمی رواین از کنند، تغییر آلوده هایمحیط در است ممکن و باشدمی مربوط استرسبه هاسلول معرض در گروه دو. شدند تقسیم گروه سه به oreochromis niloticus ماهی قطعه 33 تعداد. گرفت قرار آزمایش مورد حاضر مطالعه در فرضیه ده از بعد. شدند داشته نگه شاهد عنوانبه نیز گروه یک و گرفتند قرار لیتر در گرممیلی 8 و 4 هایغلظت در پاراتیون دمتیل ارگانوفسفات ماهی سم ارزیابی برای شده ایجاد روش یک با و شدند آنالیز rodlet هایسلول مساحت و تعداد و شدند برداشته میکروسکوپی مطالعه برای ماهیان آبشش روز داریمعنی تفاوت اما نشد، مشاهده هاسلول مساحت در داریمعنی تفاوت هیچ . براساس نتایجگرفتند قرار مقایسه مورد ماهیان در بافتی هایآسیب فراهم جدید زیستیخصشا این از استفاده برای را شواهدی حاضر مطالعه. شد یافت آزمایش مورد هایغلظت بین در rodlet سلولهای تعداد در .کندمی بحث را روش این فاکتورهای هایپتانسیل از برخی و کندمی .پاراتیون متیل نیل، تیالپیا زیستی،شاخص فسفات، ارگان :کلمات کلیدی int. j. aquat. biol. (2018) 6(3): 122-125 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology short communication wild otter observation on gurney drive coast in penang island, malaysia ayaka yurimoto1, tatsuya yurimoto*2,1faizul mohd kassim3 1independent, nagasaki, nagasaki, japan 2japan international research center for agricultural sciences, tsukuba, ibaraki, japan 3penang office of japan international research center for agricultural sciences, penang, malaysia article history: received 10 may 2018 accepted 12 june 2018 available online 2 5 june 2018 keywords: otter lutrogale perspicillata feeding activity malaysia abstract: otter was irregularly observed on gurney drive coast in the northern part of georgetown, penang island from june 2015 to march 2016. authors examined relationship between the otters sighting date and time and the tide pattern. otter was observed a total of five times on 27th june, 17th august, 6th december 2015, 14th march, and 16th march 2016. these otters were identified same species which was the smooth-coated otter, lutrogale perspicillata, from the morphological characteristics. swimming direction of the otters were characterized two patterns that the first was the otters swam from inner part to east coast in a cove of gurney drive coast and the second was the otters swam from the east coast to inner part of the cove. from this result, it was suggested that the otters swam to drive fish toward tide flow and the otters catch the fish efficiently. introduction the 13 species of otter live in the world, and they are the top predator in waterside area. however, many of them have problems with reduction of habitat and population, and they are on the verge of extinction (iosf, 2018). eurasian otter (lutra lutra), shortclawed otter (aonyx cinerea), hairy-nosed otter (l. sumatrana) and smooth-coated otter (lutrogale perspicillata) live in the southeast asian including peninsular malaysia (esabii, 2009; iosf, 2018). among them, the smooth-coated otter was widely distributed in south and southeast asia and it was a common species in peninsular malaysia. however, this otter is also subject to the animal protection act in malaysia since 2010 (iosf, 2018). declining factors of the otters in the southeast asia are pointed out influence of hydroelectric power plant construction, pesticide contamination, poaching, habitat loss due to reclamation of wetlands (de silva, 2015), and the similar affects are suggested in malaysia (iosf, 2018). sighting of the smooth-coated otter in peninsular malaysia is reported many around penang island. *corresponding author: tatsuya yurimoto doi: https://doi.org/10.22034/ijab.v6i3.506 e-mail address: yurimoto@outlook.com according to recent reports, the otter is observed around coastal area of georgetown and seri tanjung pinang in penang island, and byram on the opposite coast of penang island (guy, 2013). on the other hand, the star online magazine, which is a major domestic newspaper in malaysia, sungai pinang river in penang island was concerned influence on the smooth-coated otter inhabits due to serious water pollution in around 2006. the penang drainage and irrigation department (did) established a cleanup infrastructure system in 2009, and they collected about 120 tons of garbage on every year and took measures to relocate residents (francis, 2015). thereafter, ten smooth-coated otters consisting 4 adults and 6 children were observed around the river in june 2015. it is suggested that fish, which is a food for otter, came back to the river, because the water quality was improved (francis, 2015). sighting report of the smooth-coated otter is increasing around penang island on recent. and then, authors observed the smooth-coated otters on gurney drive coast in penang island and got some findings about their feeding behavior from the observation results. 123 int. j. aquat. biol. (2018) 6(3): 122-125 materials and methods penang island is known the area is about 295 km2, length of east-west is about 12 km and length of northsouth is about 24 km (fig. 1b). there is a city called georgetown in the northeastern part of the island and the population is about 300,000 (wikipedia, 2018). there are many sighting of the smooth-coated otter around the city area. authors sighted otters on the gurney drive coast in georgetown and irregularly observed otters on the coast from june 2015 to march 2016. we recorded date and time and photographed with a digital camera when observed otters (fig. 1b). tide data around penang island was obtained from the tides 4 fishing (https://tides4fishing.com/as/malaysia/ pulau-pinang) to examine relationship between the sighting time and the tide level. results and discussion authors got a total of five times sighting of the otters on the gurney drive coast in 27th june, 17th august, 6th december 2015, 14th march and 16th march 2016. all otters were same species from the morphological characteristics. upper side of the body was dark brown color and the lower side was yellowish brown. both sides of the neck and the jaw were cream color and authors sometimes could observe no hair on the nasoscope. therefore, it was estimated to be the smooth coated otter (yasuma, 2013). also, the otters were observed a group with from 2 to 4 individuals on figure 1. location of penang island in the strait of malacca (a) and otters observation site (☆) on gurney drive coast in the island (b). figure 2. photographs of fish capture (a) and predation (b) by the otters on gurney drive coast in december 2015. 124 yurimoto et al./ wild otter observation on gurney drive coast in penang island, malaysia the coast and they were swimming while fish capture (sea catfish etc.) and the predation. then, this coastal area is considered a feeding area for the otters (fig. 2). also, there was two types of the otter’s swimming direction. the first type was they swam from east coast to inner part in a cove on gurney drive coast and they moved to the offshore (fig. 3a). and the second type was they swam from inner part to east coast in the cove (fig. 3b). therefore, authors compared relationship between sighting date and time of the otters and the day of tide data in penang island. when we sighted the otters, the tide was during neap tide and medium tide, and the sighting time was during tidal fluctuation (fig. 4). in addition, there was a relationship between the tidal change and swimming direction of the otters. they swam to inner part from east coast in the cove during ebb tide on 17th august and 14th march, and they swam to the east coast from the inner part during rising tide on 27th june and 6th december. from this result, it was suggested that the otters swam for fish capture and feeding while they were driving the fish toward tide flow. gurney drive coast is proceeding a coastal development which is a plan about 60 acres of coastal areas are reclaimed since june 2016 (onlypenang, 2016). the reclamation will be retail area of food and beverage, water garden, artificial beach and coastal forest land. therefore, coastal areas including the otters sighting area will be reclaimed by the coastal development. one of the main objectives on the development is to make that citizen access easier to the coastal areas and they have more opportunities to interact with nature. therefore, authors hope the coastal development will be harmonized well with inhabiting of wild animals such as the otters. references de silva p., khan w.a., kanchanasaka b., reza lubis i., feeroz m.m., al-sheikhly o.f. 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(2013). otters. animals of the rain forest. available from: www.doubutsu-no-kuni.net/?p= 12873. retrieved 4/20/2018. figure 4. tidal pattern of the sighting day on gurney drive coast in penang island. tide data from tides4fishing (https://tides4fishing.com/ as/malaysia/pulau-pinang). the otters were observed from neap tide (a, c, e) to mid-tide (b, d). arrow means sighting time of the otters. a: 27th june, b: 17th august, c: 6th december, d: 14th march, e: 16th march. int. j. aquat. biol. (2017) 5(2): 114-127; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology review article do alien species matter? impacts of invasions in indian freshwater systems and challenges in management murugan muralidharan*1 sri paramakalyani centre for environmental sciences, manonmaniam sundaranar university, alwarkurichi – 627 412, tamilnadu state, india. article history: received 1 september 2016 accepted 11 april 2017 available online 2 5 april 2017 keywords: aquaculture exotics freshwater invasive species management challenges abstract: alongside anthropogenic activities and habitat destruction, invasions are regarded as one of the most influential components of global change. india as a growing economy and rapidly developing nation has been constantly engaged in infrastructure development which consequently has led to depletion of natural resources and declining quality of habitats aquatic systems in particular. invasions that have established from the introductions in past during the colonial era and recently spread species are great challenges that hamper survival of aquatic resources. as of 2015, 20 plants, one mollusc and 38 fishes are known to have naturalized in the indian water bodies. awareness on the invasive species along with detailed information on the ecosystem-wide impacts is essential for management. introduction biological invasions are increasingly recognized as a primary threat to global biodiversity (wilcove et al., 1998; bax et al., 2001). invasive species are widely distributed in all kinds of ecosystems throughout the world, that include all categories of living organisms such as plants and animals, as competitors, predators, pathogens and parasites (dey, 2011). the spread of exotic species to regions without previous history of distribution is not a new phenomenon. this process has been happening naturally and there are enough evidences from the past that show organisms occupying new territories by crossing barriers. such invasions however, occurred at a very slow rate but presently the human activities have accelerated this movement. given the pre-adaptive ability of exotic species, the chances are always higher for them occupying the ecosystems which are constantly prone to habitat alterations as a result of urbanization and other man induced changes (dudgeon, 2002). success of invasion relies on the opportunity that leads the invader to a newer environment. international trade, travel, and transport are the major * corresponding author: murugan muralidharan doi: https://doi.org/10.22034/ijab.v5i2.211 e-mail address: muralistream@gmail.com drivers of biological invasion (mcneely et al., 2001). some species that become invasive are intentionally imported, and those that escape from captivity are carelessly released into the environment. accidental transports of invasive species are favoured through crates and containers (carriers of snails, slugs, molluscs, beetles) and military cargo. despite good intentions, developed countries occasionally facilitate the introduction of invasive species to other countries through development assistance programmes, military operations, famine relief projects and international financing (pallewatta et al., 2003). over the last few decades, technological advances have greatly increased the speed of transportation and stimulated by the expansion of the global transport of goods and people, the numbers and costs of invasive species are rising at an alarming rate (nisc, 2001). next to habitat loss and fragmentation, invasive species are currently the second greatest threat to biodiversity and aquatic systems. introduced species, freshwater fish in particular, are reported to thrive in degraded aquatic habitats in many areas of the world (kennard et al., 2005). invasives are thus the major 115 int. j. aquat. biol. (2017) 5(2): 114-127 focus of international conservation concern and the subject of cooperative international efforts, such as the global invasive species programme (gisp). however, the management and control of invasive species is one of the biggest challenges in conservation. the iucn guidelines on invasive alien species specifically emphasizes the following: (i) improving understanding and awareness, (ii) strengthening the management response (including prevention, eradication and control, (iii) providing appropriate legal and institutional mechanisms and (iv) enhancing knowledge and research efforts (iucn, 2000). of the many problems is the limited understanding of consistent and predictable impacts of non-native species on native diversity. the complex interactions of invasive species with native ecosystems make invasion ecology an interesting and important area of research. despite the growing worldwide awareness of alien species invasions, india still lacks specific legislation to regulate the introductions of potentially invasive species into the country (hiremath and sundaram, 2013). this review assesses the current knowledge of impacts of exotic species on aquatic systems with management strategy for effective management of invasives. list of species introduced either accidentally or deliberately occupying aquatic habitats in the indian subcontinent is provided. invasiveness and invasion success: a complete knowledge of the invasive species, traits and the distribution is essential prior to developing priorities for control. not all non-natives become ‘invasive’. some fail to thrive in their new environment and die off naturally. others survive, but without destroying or replacing native species, it is on this basis some ecologists decry that the term “invasive” is severely overused. however, exotics that do not affect residing species have appreciable effects on their new ecosystems, many exert significant ecological, evolutionary, and economic impacts. invasiveness is the most important trait in the invaders that makes it sustain against all odds. the role of other qualities put together would eventually lead to establishment. biological characteristics most often cited as associated with successful invasions are abundance and wide distribution in the native region, high physiological tolerance, genetic traits, r-selected reproductive strategy, generalist diet or habitat, rapid dispersal and invasion site characteristics (moyle and marchetti, 2006). first of all they are pre-adapted to harsh environments with ability to tolerate wide range of fluctuations in ecological parameters such as temperature, salinity, pollution etc., they are aggressive and out-compete native species and further in newer environments they are safe from natural predators and parasites. invasives also go through rapid genetic change due to newer environments. yet another notable trait as observed through various studies is the ‘invasion meltdown’ by which they facilitate the colonization and success of other exotics. a successful invasion happens when an invader’s symbiotic traits, biological characteristics, and invasion site (at multiple scales) are all favourable (moyle and marchetti, 2006). establishment of invasive species normally occurs in 3 stages (fig. 1). the dispersal is the first step to ensue which may be either in a weak/disturbed system when there are no other species or in a vacant niche when some species already exists. the second step is colonization, it includes all events related to improving a selfsustaining population much enough to invade nearby region. the last step is the succession stage where the new colony starts encroaching the surrounding areas the process during which the habitat space is fully gradually occupied. a successful invader eventually naturalizes and responds to local environmental conditions and to other members of the biotic community in ways apparently indistinguishable from those of native species (moyle and marchetti, 2006). the arrival of an exotic species with a high likelihood of becoming a significant invasive species should be regarded serious and prompted for urgent action, because this is the stage at which eradication is both feasible and easy to justify economically. of the known anthropogenic disturbances habitat degradation and pollution have triggered the invasion. at the same time there are also reports that show successful establishment of exotic species may not be 116 muralidharan/ invasive species in indian freshwater systems due to a single factor but could depend on multiple factor like e.g. complex interactions between the species and the target species (alpert et al., 2000). potential impacts of aquatic invasive species: species composition of a particular habitat is characterized by the environmental factors that govern it. the establishment of self-sustaining populations of alien species impact native communities at various levels and can alter fundamental ecological properties of the host ecosystems, even to the extent of diminishing ecosystem services (vincenzi et al., 2011). that invaders, through various activities, affect biodiversity leading to impaired ecosystems is well established (fig. 2). it is concerning to learn the fact that the impact of invasives on biodiversity is obviously greatest in the protected areas that are relatively undisturbed, which shows that habitat protection alone does not assure safer range for native biota (scott and helfman, 2001). further, the total impact of the invasive species on an ecosystem may be more than what we expect it would cause to the system where it is introduced, since the effect is actually the result of a combination of direct and indirect species impacts (gutiérrez et al., 2013). hence interactions between invasive species impacts and other anthropogenic influences can co-occur with possible factors like habitat degradation; other invasive species, pollution, altered climate, hydrology, or fire regimes (strayer, 2010; gutiérrez et al., 2013). aquatic ecosystems are more vulnerable than terrestrial systems in that they are the final recipient of variety of pollutants through multiple processes across a hierarchy of spatial and temporal scales (paukert et al., 2011). as the reason intact freshwater systems are becoming increasingly rare and many require protection from a range of threats (abell et al., 2007). invasive species are notorious for the impact they cause to the native organisms and the ecosystems. they are deemed as obnoxious for it out-competes native species for resources such as nutrient, light, physical space, and water. other well-known alterations they could directly or indirectly cause to the system include increased soil erosion, increased incidence of flooding in some situation, increased water use, reduction in water table, changes in soil chemistry, e.g., salt accumulation and loss in productivity. invasive alien species are as equally ancient as human civilization, and are ongoing chronologically indistinguishable by man. of late, biological invasions are among the major global issues of concern. lack of information related to introductions and their current distribution impedes management. though we know that invasives are capable of displacing, predating native species besides their ability to spread disease and alter habitats, a complete knowledge about the extent and variety of impacts in different regions worldwide is not available. as climate change is crucial in the future of the distribution of invasive species worldwide, it becomes essential to have data of current status of distribution and the dispersal rates (fig. 2). it would also be relevant on the basis that they are adaptive with broad environmental tolerances, short generation times and high rates of dispersal (hellmann et al., 2008). indian aquatic systems and species introductions: indian peninsula occupies a strategic position in southern asia, across the seas to arabia and africa on the west and to myanmar, malaysia and the indonesian archipelago on the east. the river systems in india support one of the richest fish germplasm resources (ca. 840 species) in the world including many rare and endemic species (vass et al., 2009, singh and lakra, 2011). aquatic systems and water in figure 1. establishment of invasive species. 117 int. j. aquat. biol. (2017) 5(2): 114-127 india are intricately intertwined with the cultural fabric of the country, and has both economic and social connotations (dudgeon, 1992; unicef, 2013). the total water potential of india, determined by mean annual river flows, is estimated to be 1,672,590 million m3. however, this could possibly not be sufficient to meet the needs of world’s second most populous country. as the reason several crucial issues prevail over the water sector in india notable of them are erratic distribution of rainfall, water use inefficiency; unregulated groundwater extraction; inter-state river disputes and growing financial crunch for management of resources. lack of relevant awareness on sustainable utilization in the part of citizens and the failure of decision makers to enforce stringent rules towards reckless activities leading to declining aquatic resources have made problems complex. the alterations in aquatic biogeochemistry and ecosystems are expected to have a profound impact on water quality and living resources. aquatic systems in the current state would only facilitate the establishment of invasive species (muralidaran et al., 2015). introduction of certain exotic species are said to have been deliberate during the pre-independent period and more prevalent during the colonial era to gratify the then rulers of their aesthetic interest (mcneely, 2001). the present day invasions in indian inland waters both of floral and faunal components could be attributed to the increasing dependence on aquaculture and flourishing aquarium trade. species from temperate and tropical regions of aquacultural and commercial value are being imported on regular basis that threaten native biodiversity (muralidharan et al., 2015). activities linked to such industries could be held responsible for the estimated occurrence of 300 alien fish species in india (singh and lakra, 2011). this practice could not be completely restricted given the revenue it yields in addition to the employment opportunities available for youth forming a considerable proportion of the total population at present. eradication of invasive and obnoxious species is globally accepted practice and has been adopted as a key management option in extenuating the impacts of biological invasions (genovesi and shine, 2003; genovesi, 2005). though invasion impacts had been experimentally quantified for non-native species, in all major freshwater and marine habitats, most are from the regions occupying temperate latitudes figure 2. impact of exotics on species and ecosystems. 118 muralidharan/ invasive species in indian freshwater systems (thomsen et al., 2014). in india, regulation of introduction of invasive alien species and their management has been covered by the national biodiversity action plan, in the absence of exclusive policy. nbfgr (national bureau of fish genetic resources) evaluated the impact of invasive fishes in india and a strategic plan for quarantine and exotic fish introductions has been prepared. action plans developed for non-native species management in india initiated under asia-pacific invasive species network, a cooperative alliance of 32 member countries, share information on the invasive species. despite the wellestablished quarantine system, which regulates the import and export of biological materials to check the entry of the undesirable species, there are registered cases of alien introductions (tripathi, 2015; bijukumar et al., 2015). studies on the impact of invasive species in indian waters are relatively poor as compared to other nations. species that have invaded the aquatic systems are not completely ascertained, however a provisional list of species based on literature their impacts on native community and habitats shows the occurrence of 20 plants, 1 mollusc and 38 fishes (appendix 1). most of the fish species were deliberately introduced, to augment aquaculture activities considering the social and economic importance of the fishery (ghosh et al., 2003; katiha et al., 2005; singh and lakra, 2011; singh et al., 2014). challenges: impact of invasions on the aquatic systems, as for as india, has not been perceived as a major issue however is likely to emerge as a serious problem because, the severity of the invasions on resources is not felt as of now. globalization has been the prevalent economic ideology, with prime objective of urbanization and infrastructure development which could seriously impact on the ecology of freshwater systems. carried by the marvels of urban growth we would have failed to realize the increasing thrust on the pristine habitats that harbor native diversity. also many alien invasives benefit from the reduced competition that follows habitat degradation. management plans to control invasions would not be effective when the level of awareness is inadequate to check the introduction and spread. hence understanding the factors related to invader abundance and impact is essential also the conservation measures have to be prioritized corresponding to the cause and the impact (kulhanek et al., 2011; tripathi, 2015). homogenization is a threat to indigenous species; it is commonly asserted that exotic species promote the homogenization of biological communities by influencing community composition (nentwig, 2007). freshwater fish fauna that are highly differentiated and isolated lose their uniqueness resulting in the loss of local and regional distinctiveness. introduction of exotic freshwater fishes, which is common worldwide mainly for aquaculture, is especially harmful in terms of biodiversity. even transfer of species from a different river system which already inhabits related congener could tremendously impact the system and the residential species. the morphological distinction of few species is completely chaotic and a very classic example of such a species, influenced by the impact of cultivable transferal species is the fish labeo most popular in inland aquaculture. this genus due to inbreeding and other reasons has become enigmatic with number of species with similar morphological features difficult to distinguish. invasives spread through pet trade are even worser with their hybrid origin, as the case of the armoured suckermouth cat fish pterygoplichthys exhibiting “hybrid superiority” through successive invasions (nico et al., 2012; bijukumar et al., 2015). interactions of the invasives with the native organisms after establishing in newer environments have modified and evolved significantly. nonindigenous species with morphological similarity to native residential species failed to establish due to the non-availability of niche space (azzurro et al., 2014). however there are contradictory findings against the widely popular hypothesis in invasion biology that species-rich communities are more resistant to invasion than species-poor communities. as expected the native communities are supposed to restrict and control the rate of invasion however, the rate of invasions will actually increase with time, because the disruption of native species promotes further 119 int. j. aquat. biol. (2017) 5(2): 114-127 invasions as some invaders are facilitative rather than being invasive (rooney et al. 2007). for example the invading dreissenid mussels by the provision of food in the form of fecal deposits favour further invaders such as the amphipod echinogammarus ischnus (ricciardi, 2001). oreochromis mossambica got introduced into tanks along with other fish fry during the transfer of commercial fish seeds from aquaculture farms. they are observed to co-exist with other residential native species. exotic species are known for their adaptive traits and in course of their evolution have developed strategies by sharing habitat and resources mutually with local residential species, which is referred as ‘invasive meltdown’. invasion of eiccornia crassipes (water hyacinth) due to eutrophication challenges the life supporting ability of aquatic systems remains the world’s most problematic water weed. purple swamphen (porphyrio porphyrio) a water bird that usually nests in mass of floating debris or amongst matted reeds slightly above water level utilizes invasive water hyacinth foliage in the absence of native fauna, it is also said to facilitate the proliferation of the weed. prosopis juliflora (mesquite) native to mexico, south america was introduced to india. however, it is a case of turn-about as it has been used as fodder for goat and other cattle and it also supports village dwellers for fuel and production of charcoal for industry. this species is hard and expensive to remove as the plant can regenerate from the roots. this is interesting case along similar lines is the alewife, a non-native fish that was first reported in the great lakes in 1873 which was considered a costly nuisance species in the mid-20th century. now it is considered a valuable (but still exotic) food source for salmon and lake trout, which supports billion dollars’ worth sport fishery. aquaculture is one of the fastest growing areas of food production that sustains the growing population by food and economy which has been the known sector to establish exotics in newer regions. as the reason the popularity and admiration gained by an exotic species is greater than the knowledge required to realize the long-term impacts it would cause. interestingly none of the invasive species has been declared as prohibited in any state or the country. transboundary rivers are ecologically important however are socially prone to dispute owing to problems arising from sharing of water between states. merger of rivers and construction of navigation canals between rivers develop a network of waterways that open long distance dispersal routes for aquatic species from several bio-geographic areas. this leads to homogenization of species. past and present governments have insisted in river linking projects by connecting major rivers of himalayan region to those in the southern part of india. the objective behind such scheme is water supply for irrigation, however with compromise on aquatic organisms. introduction of species, be it through any mode, either transplanting or translocating is said to be ecologically disastrous to major biodiversity nation like india, with more than 20 geographically distinct drainage basins (tripathi, 2015). further changing climate patterns will probably produce significant effects on the biodiversity of freshwater ecosystems throughout the world and possibly initiate varying adaptive responses. species might survive climate change by shifting their distributions or through evolution and become adapted to the new local climatic conditions. as the invasives are tolerant and capable of surviving in the harsh environments the spread of invasions are only facilitated by climate variations. removal of certain invasive species needs fundamental knowledge of population ecology which is lacking for many exotics. further control of parasitic infections in introduced culture species is another challenge as it could be spread to native forms (shomorendra et al., 2005; dash et al., 2008). the successful eradication of the parasites in aquaculture stations would be possible only after knowing the population biology sufficiently (kaur et al., 2012b). measures based on biological and chemical control are not sometimes advisable, which could have harmful impact on other native species and the habitat as a whole. implementation of similar such projects in india with objective of combating the impact of non-native species should be undertaken after due considerations related to safety aspects of the 120 muralidharan/ invasive species in indian freshwater systems environment as well as people. management and control strategies though, invasive species are considered as ecological threat throughout the world, the management methods adapted to control are not the same. however, certain aspects in general are applicable worldwide. careless behaviour leads to unintentional introductions: avoid using known invasive species. creating awareness among people would enable them to make informed choices among pets/ornamental species. reducing activities that alter landscape: invasive species thrive well in disturbed systems where the native community has been displaced. protection of healthy native species is the key to control invasive species. regular monitoring and assessment: all land use plans need to be monitored regularly and invasive species need to be checked for their removal. scouting at regular interval helps in preventing spread of invasive species. removal of invasive species when the population is low helps native species to occupy the empty niche. community awareness and perceptions: exotic species attract a range of opinion in country like india, based partly in terms of ecological impact but more on the human utility. promoting community participation through awareness and voluntary involvement in efforts to eradicate invasive species could well yield better results. development of database for species distribution: building species distribution database for the invasive alien species using spatial stochastic model with provisions for updation through region and species specific surveillance programme. adoption and applicability of control strategy: choosing the most appropriate control strategy is important. proper guidance is required in the choice of control measures. hence it is necessary to have rigorous comparisons of control success under field conditions to have a robust decision support tool. coordinating committee to control invasion: establish a coordinating committee consisting of members from various agencies preferably agriculture, irrigation, engineering, fisheries, environment and industry conclusion despite the growing concern for the impact of invasive species, exotics are constantly released as we have not completely halted activities promoting such introductions. the biology of invasive species and lack of site specific information on its ecology are major bottlenecks in developing effective tools for its management. as ecologists opine conservation of biodiversity needs good scientific information to inform our decisions on advocacy, public awarenessraising, and support to field and policy projects. conservation of indigenous species from the impact of invasive aliens could be successful through the following steps: (i) gathering complete information on the species and the ecosystem wide impact, (ii) innovative control and eradication methods developed after incorporating techniques found effective in successful invasive management projects and (iii) proper and periodic monitoring of restored sites to control against future invasions. strategies in effective management of the invaders are to be modified and developed in the rapidly changing trends of the climate. acknowledgements financial assistance through the major research project (ugc f. no. 39 – 332/2010) funded by university grants commission, new delhi is acknowledged. references abell r., allan j.d., lehner b. 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(2009). spatial and temporal dynamics of the weed community in a seashore paspalum turf. weed science, 57: 248-255. 1 2 5 m u ra li d h ar an / in v as iv e sp ec ie s in i n d ia n f re sh w at er s y st em s o rg a n is m /s p e c ie s n a ti v e r e g io n im p a c t o n i n v a d e d s y st e m l it e ra tu re p la n ts 1 a lt e rn a n th e ra p h il o x e ro id e s h e rb s o u th a m e ri c a in fe st in g r iv e rs , la k e s, p o n d s a n d i rr ig a ti o n c a n a ls ju li e n e t a l. ( 1 9 9 5 ); p ra m o d e t a l. ( 2 0 0 8 ); d e v i a n d s h a rm a ( 2 0 1 0 ); m a so o d i a n d k h a n ( 2 0 1 2 ) 2 a c a c ia n il o ti c a t re e a u st ra li a r e p la c e s n a ti v e n o n -t re e v e g e ta ti o n , su c h a s g ra ss la n d a n d sh ru b la n d p a n d e y e t a l. (1 9 9 9 ); b a rg a li a n d b a rg a li (2 0 0 9 ) 3 c a b o m b a c a ro li n ia n a b ra z il c lo g s d ra in a g e c a n a ls a n d f re sh w a te r st re a m s o rg a a rd ( 1 9 9 1 ); m a tt h e w s e t a l. ( 2 0 1 3 ) 4 e u p a to ri u m c a n n a b in u m l . h e rb b ri ti sh i sl e s a lt e r so il n u tr ie n ts a n d h y d ro lo g y p o te n ti a ll y r e d u c in g t h e s u it a b il it y o f th e a re a t o n a ti v e f lo ra is s g ( 2 0 1 1 ) 5 e ic h h o rn ia c ra ss ip e s (m a rt .) s o lm s f re e f lo a ti n g w e e d s o u th a m e ri c a im p e d e s fl o w a n d c lo g s p a te l (2 0 1 2 ) 6 g y m n o c o ro n is s p il a n th o id e s s o u th a m e ri c a r a p id ly c o v e r w a te r b o d ie s w it h a f lo a ti n g m a t, e x c lu d in g o th e r p la n ts a n d t h e a n im a ls t h a t re ly o n t h e m k u m a r (2 0 0 9 ) 7 ip o m o e a c o rn e a b ra z il d is ru p ts s u c c e ss io n a n d d e c re a si n g b io d iv e rs it y c o o k ( 1 9 8 7 ); c h a u d h u ri e t a l. ( 1 9 9 4 ); c h a n d ra se k a r (2 0 1 2 ) 8 l im n o c h a ri s fl a v a ( l .) ( b u c h e n a u , 1 9 0 3 ) a m e ri c a s e ri o u s w e e d i n r ic e f ie ld s, i rr ig a ti o n c a n a ls a n d w e tl a n d s a b h il a sh e t a l. ( 2 0 0 8 ) 9 l u d w ig ia p e ru v ia n a ( l .) h a ra s o u th a m e ri c a v ig o ro u sl y o p p o rt u n is ti c , c lo g g in g w a te rw a y s c h o w d h u ry e t a l. ( 2 0 1 3 ) 1 0 l a n ta n a c a m a ra l . c e n tr a l a n d n o rt h e rn s o u th a m e ri c a a n d t h e c a ri b b e a n d is ru p ts s u c c e ss io n a n d d e c re a si n g b io d iv e rs it y s h a rm a e t a l. ( 2 0 0 5 ); s a h u a n d s in g h , (2 0 0 8 ); p ri y a n k a a n d j o sh i (2 0 1 3 ) 1 1 m e la le u c a q u in q u e n e rv ia (c a v .) b la k e q u e e n sl a n d a n d n e w s o u th w a le s, a u st ra li a a lt e rs s o il c h e m is tr y a n d m o d if ie s e v e rg la d e s e c o sy st e m p ro c e ss e s o rw a e t a l. ( 2 0 0 9 ) 1 2 m ik a n ia m ic ra n th a ( l .) k u n th . c e n tr a l a n d s o u th a m e ri c a d a m a g e s o r k il ls o th e r p la n ts b y c u tt in g o u t th e l ig h t a n d sm o th e ri n g t h e m c h o u d h u ry ( 1 9 7 2 ); m u n ia p p a n a n d v ir a k ta m a th ( 1 9 9 3 ); r a m a c h a n d ra n a n d s o o sa ir a j (2 0 0 8 ) 1 3 p a sp a lu m v a g in a tu m s w . n o rt h a m e ri c a im p a c t o n f a u n a c o m m u n it ie s x ie e t a l. ( 2 0 0 9 ) 1 4 p h a la ri s a ru n d in a c e a l . e u ro p e a si a a n d n o rt h a m e ri c a f o rm s d e n se a n d i m p e n e tr a b le m a ts o f v e g e ta ti o n h o lm e t a l. (1 9 9 1 ) 1 5 p ro so p is s p p . p . ju li fl o ra a n d p . p a ll id a m e x ic o , s o u th a m e ri c a a n d th e c a ri b b e a n . s m o th e r n a ti v e v e g e ta ti o n h a rd a n d e x p e n si v e t o r e m o v e a s th e p la n t c a n r e g e n e ra te fr o m t h e r o o ts o rw a e t a l. ( 2 0 0 9 ); k a u r e t a l. ( 2 0 1 2 a ) 1 6 r u b u s m o lu c c a n u s l in n a e u s m a la y si a t o a u st ra li a , s o lo m o n is la n d s, n e w c a le d o n ia a n d f ij i th re a te n s n a ti v e p la n ts is s g ( 2 0 1 1 ) 1 7 s a lv in ia m o le st a so u th e a st e rn b ra z il a n d n o rt h e rn a rg e n ti n a f o rm d e n se v e g e ta ti o n m a ts t h a t re d u c e s w a te rfl o w a n d lo w e rs l ig h t a n d o x y g e n l e v e ls i n t h e w a te r k u m a r e t a l. ( 2 0 0 5 ) 1 8 s p a rt in a a lt e rn if lo ra l o is e l. n o rt h a n d s o u th a m e ri c a n e g a ti v e e ff e c t o n n a ti v e s p e c ie s in c lu d in g s o m e e n d a n g e re d is s g ( 2 0 1 1 ) 1 9 t ri a d ic a s e b if e ra ( l .) c h in a a n d j a p a n a g g re ss iv e ly d is p la c e s n a ti v e p la n ts a n d f o rm s m o n o sp e c if ic s ta n d s ja ry a n e t a l. ( 2 0 0 7 ) 2 0 u le x e u ro p a e u s l . e u ro p e a n d g re a t b ri ta in a n d ir e la n d d is p la c in g c u lt iv a te d a n d n a ti v e p la n ts r ic h a rd so n a n d h il l (1 9 9 8 ) m o ll u sc 2 1 a c h a ti n a f u li c a b o w d ic h , 1 8 2 2 e a st a fr ic a s e v e re d a m a g e i n i n fe st e d p la n ts , t ra n sm is si o n o f p a ra si te s r a u t a n d b a rk e r (2 0 0 2 ) a p p e n d ix 1 . l is t o f in v a si v e sp ec ie s re p o rt e d f ro m i n d ia n w at er s (b as ed o n l it er at u re ). 1 2 6 in t. j . a q u at . b io l. ( 2 0 1 7 ) 5 (2 ): 1 1 4 -1 2 7 o rg a n is m /s p e c ie s n a ti v e r e g io n im p a c t o n i n v a d e d s y st e m l it e ra tu re f is h e s 2 2 a m p h il o p h u s tr im a c u la tu m g u n th e r s o u th a m e ri c a p re d a ti o n k n ig h t (2 0 1 0 ) 2 3 a ri st ic h th y s n o b il is * r ic h a rd so n 1 8 4 5 s o u th e rn c h in a e n d a n g e rs n a ti v e s p e c ie s s in g h a n d l a k ra ( 2 0 1 1 ) 2 4 c a ra ss iu s a u ra tu s (l in n a e u s, 1 7 5 8 ) c e n tr a l a si a in c re a si n g t u rb id it y , p re d a ti o n u p o n n a ti v e f is h , a n d h e lp in g to f a c il it a te a lg a l b lo o m s r e m a d e v i (1 9 8 7 ); k n ig h t (2 0 1 0 ) 2 5 c ic h la so m a tr im a c u la tu m g u n th e r 1 8 6 7 e l s a lv a d o r, h o n d u ra s, n ic a ra g u a h ig h ly a g g re ss iv e k n ig h t (2 0 1 0 ) 2 6 c la ri a s g a ri e p in u s (b u rc h e ll , 1 8 2 2 ) a fr ic a t h re a t to e n d e m ic a q u a ti c f is h , p a rt ic u la rl y i n s o u th a fr ic a a n d i n d ia m is h ra e t a l. ( 2 0 0 0 ); g o p i a n d r a d a k ri sh n a n (2 0 0 2 ); k ri sh n a k u m a r e t a l. ( 2 0 1 1 ); s a rk a r e t a l. ( 2 0 1 2 ) 2 7 c te n o p h a ry n g o d o n i d e ll a ( v a le n c ie n n e s in c u v ie r a n d v a le n c ie n n e s, 1 8 4 4 ) c h in a a n d r u ss ia e li m in a te s p a w n in g s u b st ra te , d is tu rb s e d im e n t a n d m u d d y w a te rs , re d u c e w a te r q u a li ty , in c re a se n u tr ie n ts i n w a te rs a c c e le ra ti n g e u tr o p h ic a ti o n , d e c re a se o x y g e n l e v e ls , a n d p ro m o te a la g a l b lo o m . f is h b a se ( 2 0 0 4 ); s a rk a r e t a l. ( 2 0 1 2 ) 2 8 c y p ri n u s c a rp io c o m m u n is l in n a e u s 1 7 5 8 e u ro p e i n r iv e rs a ro u n d t h e b la c k s e a a n d t h e a e g e a n b a si n in c re a se d s il ta ti o n a n d b io tu rb id it y d a m a g e a q u a ti c m a c ro p h y te s d a n ie ls ( 2 0 0 6 ); k ri sh n a k u m a r e t a l. ( 2 0 1 1 ); s a rk a r e t a l. ( 2 0 1 2 ) 2 9 c y p ri n u s c a rp io s p e c u la ri s l a c e p e d e s o u th -e a st a si a d a m a g e a q u a ti c m a c ro p h y te s k ri sh n a k u m a r e t a l. ( 2 0 1 1 ); s in g h a n d l a k ra , (2 0 1 1 ) 3 0 c y p ri n u s c a rp io b lo c h * s o u th -e a st a si a c o m p e te w it h n a ti v e s p e c ie s s in g h a n d l a k ra ( 2 0 1 1 ) 3 1 g a m b u si a a ff in is ( b a ir d a n d g ir a rd , 1 8 5 3 )* s o u th e rn u s a a n d n o rt h e rn m e x ic o e x tr e m e ly a g g re ss iv e a n d a tt a c k o th e r fi sh , sh re d d in g f in s. h o st o f p a ra si te s in fe c ti n g n a ti v e f is h d a n ie ls ( 2 0 0 6 ) 3 2 g a m b u si a h o lb ro o k i (g ir a rd 1 8 5 9 ) n o rt h a m e ri c a p re d a te o n a m p h ib ia n e g g s; a n d p re d a te a n d c o m p e te w it h ta d p o le s s h a rm a ( 1 9 9 4 ); n a g d a li a n d g u p ta ( 2 0 0 2 ) 3 3 h y p o p h th a lm ic h th y s m o li tr ix v a l. ja p a n , a si a c o m p e te w it h n a ti v e s p e c ie s s in g h a n d l a k ra ( 2 0 1 1 ) 3 4 o n c h o rh y n c h u s m y k is s w a lb a u m e u ro p e in v a si v e , c a rr ie s p a ra si te s in g h a n d l a k ra ( 2 0 1 1 ) 3 5 o n c h o rh y n c h u s n e rk a w a lb a u m n o rt h a m e ri c a a g g re ss iv e , im p a c ts h a b it a t s in g h a n d l a k ra ( 2 0 1 1 ) 3 6 o re o c h ro m is m o ss a m b ic u s (p e te rs , 1 8 5 2 ) s o u th e rn a fr ic a t h re a t to n a ti v e s p e c ie s th ro u g h c o m p e ti ti o n f o r fo o d a n d n e st s p a c e s u g u n a n ( 1 9 9 5 ); l a k ra e t a l. ( 2 0 0 8 ); s a rk a r e t a l. ( 2 0 1 2 ) 3 7 o re o c h ro m is n il o ti c u s l in n . 1 7 5 8 s o u th e rn a fr ic a t h re a t to n a ti v e s p e c ie s th ro u g h c o m p e ti ti o n f o r fo o d a n d n e st s p a c e b h a k ta a n d b a n d y o p a d h y a y ( 2 0 0 7 ) 3 8 o sp h ro n e m u s g o ra m y l a c e p e d e ,1 8 0 1 s o u th e a st a si a r e so u rc e c o m p e ti ti o n , o p p o rt u n is ti c c a rn iv o re , c a rr y p a th o g e n s r a j (1 9 1 6 ); c h a n d ra se k h a r (2 0 0 4 ); r a g h a v a n e t a l. ( 2 0 0 8 ) 3 9 p a n g a si u s su tc h i s a u v a g e , 1 8 7 8 a fr ic a c a rn iv o ro u s d e v o u rs i n se c t, f is h f ry a n d f in g e rl in g s a n d ta d p o le s. b h a k ta a n d b a n d y o p a d h y a y ( 2 0 0 7 ) 4 0 p ia ra c tu s b ra c h y p o m u s c u v ie r 1 8 1 8 s o u th a m e ri c a o p p o rt u n is ti c s in g h a n d l a k ra ( 2 0 1 1 ) 4 1 p o e c il ia r e ti c u la te p e te rs , 1 8 5 9 s o u th a m e ri c a t h re a t to i n d ig e n o u s fa u n a k h a ra t e t a l. ( 2 0 0 3 ); d e a c o n e t a l. ( 2 0 1 1 ) 4 2 p te ry g o p li c h th y s a n is it si e ig e n m a n n & k e n n e d y , 1 9 0 0 s o u th a m e ri c a a lt e ra ti o n o f b a n k s tr u c tu re a n d e ro si o n , d is ru p ti o n o f a q u a ti c f o o d c h a in s s in h a e t a l. ( 2 0 1 0 ) 4 3 p te ry g o p li c h th y s d is ju n c ti v u s w e b e r, 1 8 9 1 s o u th a m e ri c a a lt e ra ti o n o f b a n k s tr u c tu re a n d e ro si o n , d is ru p ti o n o f a q u a ti c f o o d c h a in s s a rk a r e t a l. ( 2 0 1 2 ); s in g h ( 2 0 1 4 ) a p p e n d ix 1 . c o n ti n u e. 1 2 7 m u ra li d h ar an / in v as iv e sp ec ie s in i n d ia n f re sh w at er s y st em s a p p e n d ix 1 . c o n ti n u e. o rg a n is m /s p e c ie s n a ti v e r e g io n im p a c t o n i n v a d e d s y st e m l it e ra tu re 4 4 p te ry g o p li c h th y s m u lt ir a d ia tu s h a n c o c k , 1 8 2 8 s o u th a m e ri c a in c lu d e a lt e ra ti o n o f b a n k s tr u c tu re a n d e ro si o n , d is ru p ti o n o f a q u a ti c fo o d c h a in s, c o m p e ti ti o n w it h n a ti v e sp e c ie s, m o rt a li ty o f e n d a n g e re d s h o re b ir d s, c h a n g e s in a q u a ti c p la n ts h o o v e r e t a l. ( 2 0 0 4 ); d a n ie ls ( 2 0 0 6 ); k n ig h t (2 0 1 0 ) 4 5 p te ry g o p li c h th y s p a rd a li s c a st e ln a v , 1 8 5 5 s o u th a m e ri c a a lt e ra ti o n o f b a n k s tr u c tu re a n d e ro si o n , d is ru p ti o n o f a q u a ti c f o o d c h a in s, c o m p e ti ti o n w it h n a ti v e s p e c ie s, k n ig h t (2 0 1 0 ); m u ra li d h a ra n e t a l. ( 2 0 1 5 ) 4 6 p u n ti u s g o n io n o tu s b le e k e r s o u th e a st a si a n o t c o m p le te ly k n o w n , h o w e v e r d a m a g e s a q u a ti c h a b it a ts d u e t o i ts p o te n ti a l in w e e d c o n tr o l s in g h a n d l a k ra ( 2 0 1 1 ) 4 7 p y g o c e n tr u s n a tt e re ri k n e r, 1 8 5 5 s o u th a m e ri c a p re d a ti o n , th re a t to n a ti v e f is h e s k n ig h t (2 0 1 0 ) 4 8 s a lv e li n u s fo n ti n a li s m it c h il l 1 8 1 4 * n o rt h a m e ri c a c o m p e te w it h n a ti v e f is h e s, a q u a c u lt u re s in g h e t a l. ( 2 0 1 3 ) 4 9 s a lm o g a ir d n e ri i ri d e u s r a n a e e u ro p e c o m p e te w it h n a ti v e t ro u ts s in g h e t a l. ( 2 0 1 3 ) 5 0 s a lm o g a ir d n e ri s h a st a r a n a e e u ro p e c o m p e te w it h n a ti v e t ro u ts s in g h a n d l a k ra ( 2 0 1 1 ) 5 1 s a lm o t ru tt a f a ri o l .* g re a t b ri ta in n o t k n o w n s in g h a n d l a k ra ( 2 0 1 1 ) 5 2 s a lm o l e v e n si s p ic k e n s 1 9 2 8 g re a t b ri ta in y e t to e st a b li sh s in g h a n d l a k ra ( 2 0 1 1 ) 5 3 s a lv e li n u s n a m a y c u sh w a lb a u m 1 7 9 2 ja p a n n o t k n o w n s in g h a n d l a k ra ( 2 0 1 1 ) 5 4 s a lm o s a la r l in n a e u s n o rt h a m e ri c a im p a c t n o t e st a b li sh e d s in g h a n d l a k ra ( 2 0 1 1 ) 5 5 t in c a t in c a ( l in n a e u s, 1 7 5 8 )* e u ro p e a n d a c ro ss a si a t o c h in a c o n c e rn s o v e r c o m p e ti ti o n w it h n a ti v e f is h jo n e s a n d s a ro ji n i (1 9 5 2 ); f is h b a se ( 2 0 0 4 ) 5 6 t il a p ia z il li g e rv a is 1 8 4 8 s o u th e a st a si a n o t e x a c tl y k n o w n s in g h a n d l a k ra ( 2 0 1 1 ) 5 7 t ri c h o p o d u s tr ic h o p te ru s (p a ll a s, 1 7 7 0 ) m e k o n g b a si n i n c a m b o d ia , l a o s, t h a il a n d , v ie tn a m a n d y u n n a n r e so u rc e c o m p e ti ti o n d a n ie ls a n d r a ja g o p a l (2 0 0 4 ); d a n ie ls ( 2 0 0 6 ); k ri sh n a k u m a r e t a l. ( 2 0 0 9 ) 5 8 x ip h o p h o ru s h e ll e ri h e c k e l, 1 8 4 8 s o u th a m e ri c a r e so u rc e c o m p e ti ti o n , a g g re ss iv e n a tu re d a n ie ls ( 2 0 0 3 ); r a g h a v a n e t a l. ( 2 0 0 8 ) 5 9 x ip h o p h o ru s m a c u la tu s g u n th e r, 1 8 6 6 s o u th a m e ri c a r e so u rc e c o m p e ti ti o n , a g g re ss iv e n a tu re k h a ra t e t a l. ( 2 0 0 3 ); k ri sh n a k u m a r e t a l. (2 0 0 9 ) # 4 9 & 5 0 – s y n o n y m s o f o n co rh y n ch u s m y k is s jo rd a n ( ac c o rd in g t o f is h b as e) . int. j. aquat. biol. (2014) 2(1): 9-13 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article incidence of lernaea (crustacea: copepoda) parasitic in the mashkid river basin, southeast of iran mohammad hashem malekzehi1, hamid reza esmaeili2, halimeh zareian*2,1zahra farahani2, abdolrahim pazira1 1departement of fisheries, bushehr branch, islamic azad university, bushehr, iran. 2department of biology, college of sciences, shiraz university, shiraz, 71454. iran. article history: received 27 june 2013 accepted 28 january 2014 available online 2 5 february 2014 keywords: copepod mashkid river basin native fishes abstract: in the present investigation, lernaea parasite was reported in the examined fish species, collected from the mashkid river basin, southeast of iran in 2012 and 2013. lernaea parasites were isolated from the external surface of eye, fins, operculum and body of bangana dero, cyprinion microphthalmus, gonorhynchus diplocheilus (cyprinidae), aphanius dispar (cyprinodontidae), channa gachua (channidae) in different water bodies. the highest infection was found in native fish, b. dero with nine parasites in single specimen. the exotic fishes were not infected. introduction iran is located a region of major zoogeographical interchange having remarkable biodiversity attracting naturalists and scientists (esmaeili et al., 2010). based on field work, maps, fish distribution, previous studies (sadati,1977; armantrout, 1980; coad, 2013), and hydrography, 19 major basins have been recognized in iran (coad, 2013). the hamun-e mashkid (= mashkel) which lies within pakistan on the border with iran is one of these basins. the mashkid river starts from the east of the mountains draining into the hamun-e-jazmurian basin and flows east into pakistan. two tributaries of the mashkid within iran are the rutak river and the simish (= sunish river) which drain the lowlands between kuh-e-birag and the badamo range from the northwest to enter the mashkid river southeast of saravan (coad, 2013). this basin has been poorly investigated in terms of fish fauna and parasites. however, with increased attention on parasitism and disease as threats to biodiversity, there is a need to identify the pathogens and parasites, which pose significant risks (daszak et al., 2000; smith et al., * corresponding author: halimeh zareian e-mail address: h.zareian@gmail.com 2006) especially the globally distributed parasite lernaea. lernaea species, which commonly referred to anchor worms, are common pests in freshwater fishes particularly of cyprinids and amphibians (piasecki et al., 2004; kupferberg et al., 2009). pathogenicity of lernaeids depends largely on the size of their host and attachment site preferences. (fryer, 1968; paperna, 1996). lernaea spp. can cause severe fin damage (shariff and roberts, 1989). as a rule, lernaea has been the cause of great economic losses of fish in many parts of the world (shariff and roberts, 1989). parasite in fishes have been a great concern since they often create disease conditions in fish which will lead to an increase in fishes susceptibility to other disease causing nutritive evaluation of fish as well as fish loss (raissy et al., 2013). the present study reports the parasitic crustacean lernaea from freshwater fishes of mashkid river basin, iran. materials and methods fish specimens were collected during fieldwork in 2002 and 2013 in mashkid river basin (in dahak, 10 malekzehi et al./ int. j. aquat. biol. (2014) 2(1): 9-13 gavarnagan, roodtak and mashkid, saravan city), using electrofishing device and hand net. identification of fish specimen carried out based on coad (2013). external surface of all individuals were investigated macroand microscopically for detecting lernaeid parasites. the worm-like objects of lernaeid parasites were examined under light microscope for diagnosis of the infection. lernaea parasites were carefully detached from the infected parts of skin, fins, eyes, and musculature tissues. all the collected fish specimens were stored in the zoological museum-collection of biology department, shiraz university (zm-cbsu). results based on fieldwork conducted on the ichthyofauna of the mashkid river basin in 2012 and 2013, 698 fish specimens were collected belonging to 13 species, order family species native/ex otic cypriniformes cyprinidae aspidoparia morar native bangana dero native carassius auratus exotic ctenophryngodon idella exotic cyprinion microphthalmum native cyprinus carpio exotic garra rossica native gonorhynchus diplocheilus native pseudorasbora parva exotic nemacheilidae paraschistura bampurensis native cyprinodontiformes poecilidae aphanius dispar native cyprinodontodae gambusia holbrooki exotic perciformes channidae channa gachua native table 1. fish species collected from mashkid river basin species standard length (mm) weight (g) no. of collected specimen no. of infected specimen occurrence of parasite eyes fins muscles operculum a. dispar 23-28 0.4-0.6 63 3 * * a. morar 47-60 1.6-2.6 149 6 * * b. dero 58-204 3.88-141.33 6 4 * * * * c. gachua 131 42.67 52 1 * c. microphthalmum 81-109 13.3-35.44 174 7 * g. diplocheilus 32-94 0.62-18.3 95 13 * * table 2. infected fishes with lernaea parasite found in mashkid river basin of iran figure 1. aphanius dispar infected by lernea. figure 2. gonorhynchus diplocheilus infected by lernea parasite. 11 malekzehi et al./ int. j. aquat. biol. (2014) 2(1): 9-13 13 genera and five families (table 1). lernaeid parasite were separated from six species including aspidoparia morar, bangana dero, cyprinion microphthalmum, gonorhynchus diplocheilus (cyprinidae), aphanius dispar (cyprinodontidae) and channa gachua (channidae) in different water resource. parasites were detected in different body parts in variant species (table 2, figs. 1 and 2). the highest infection was found in b. dero with nine parasites in a single specimen. the other collected species in different localities were carassius auratus, ctenopharyngodoni idella, cyprinus carpio, garra rossica, pseudorasbora parva (cyprinidae), paraschistura bampurensis (nemacheilidae) and gambusia holbrooki (poecilidae). discussion lernaea is a copepod, which is parasitic on many species of freshwater fishes and is extremely common among the cyprinid fishes. the data reported here are concerned with the occurrence of lernaea parasites in six native species belonging to three orders collected from the mashkid river basin in iran. usually, lernaea is a common parasite of the cyprinid fishes. however, it has infected other distantly related group, a. dispar (cyprinodontiformes) and c. gachua (perciformes) in mashkid river basin. there are several reports, both historical and recent, of l. cyprinacea using amphibians as hosts in many countries (see kupferberg, 2009). although the parasite occurs on other fish species, bangana dero was the most heavily infected and the parasites were found in eyes, fins, body muscles and operculum of this native cyprinid causing lernaeasis. the lernaeasis is one of the most dangerous diseases appearing among different native and exotic fish species causing disastrous influence on the economically important fish species (jazebizadeh, 1983). here we present the first record of lernaeid parasite in southeast of iran and in the mashkid river (see pazooki and masoumian, 2012). at the present study, 34 out of 698 studied fish (≅5%) with different infection rate are infected with lerneaid parasite. interestingly all of the infected fishes are native species revealing sensitivity of the native fishes to this parasite. the difference in infection rate in studied fish species may be due to differences in biology, nutrition, behavior of fish and environmental conditions. lernaea sp., the most common copepod parasite in the freshwater aquaculture in iran, being highly pathogenic to fishes (barzegar et al., 2008). lernaea spp. can cause severe fin damage (shariff and roberts, 1989). however, pathogenicity of lernaeids largely depends on the size of their host and attachment site preferences (el-mansy, 2009). as a rule, large numbers of lernaea can cause serious problems resulted from severe wounds. the infected fishes are not eliminated directly by the parasite, however, it may open routes for secondary infection and finally, related growth retardation, behavioral changes and associated secondary invaders may lead to death of the infected individuals (robinson and avenat-oldewage, 1996). there are different views on the effect of length and weight of the fish on parasitic infection rate. some believe that smaller fishes had more parasitic infection rate (bazari moghadam, 2009), but other thought infection rate increases with increasing weight and length. we did not found any statistical relation between infection rate and biometric characteristics of the examined fish as we found parasites in both small (a. dispar) and large (b. dero) species. due to impacts of lernaea parasites on native fishes of the mashkid river basin, a long term monitoring of the parasites and fishes is highly recommended. acknowledgment the authors are thankful to shiraz university for financial supports. references el-mansy a.i. 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(2016) 4(6): 387-390; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology short communication length-weight and length-length relationships for six fish species from zohreh river, iran yazdan keivany*,1mazaher zamani-faradonbe department of natural resources (fisheries division), isfahan university of technology, isfahan, 84156-83111, iran. article history: received 17 august 2016 accepted 11 november 2016 available online 2 5 december 2016 keywords: cyprinidae cyprinodontidae sillaginidae sisoridae abstract: the present study reports length-weight and length-length parameters for six fish species belonging to four families from the zohreh river. the weight-length relationships were estimated using the equation w= 𝛼lb. the values of the slope parameter (b) varied between 2.72 and 3.72, with a mean±sd of 3.015±0.18. the values b parameter in the length-weight relationship equations were calculated as 2.72 for arabibarbus grypus (heckel, 1843), 2.96 for capoeta trutta (heckel, 1843), 2.72 for garra rufa (heckel, 1843), 3.25 for aphanius dispar (rüppell, 1829), 2.91 for sillago sihama (forsskål, 1775) and 3.15 for glyptothorax silviae coad, 1981. this study presents the first reference on lwr and llr for these species in zohreh river. introduction the length-weight relationships (lwrs) of fishes are commonly used to estimate the weight of a specimen from its length and vice versa (binohlan and pauly, 1998; keivany et al., 2015). in addition, lwrs of fishes are important in fisheries and biology (sarkar et al., 2008) and ecology of fishes (froes, 2006). lwrs are also applied for evaluation of fish stocks and give information on their growth and condition patterns (ricker, 1968), estimate biomass of the standing stock (martin-smith, 1996), condition indices, ontogenetic changes (sarafan, 1992) and growth studies (garcia et al., 1989; haimovici and velasco, 2000; moutopoulos and stergion, 2002). length-length relationships (llrs) are useful for standardization of length type when data are summarized (froes, 1998) and are also functional for comparative growth studies (moutopoulos and stergiou, 2002). length-weight relationships were reported for many freshwater fishes of iran (gerami et al., 2014; hasankhani et al., 2014; keivany et al., 2015; 2016; 2016; keivany and zamani faradonbe, 2016; tabatabaei et al., 2015; zamani faradonbe et al., 2015a, b; radkhah and eagderi, 2015), but such * corresponding author: yazdan keivany e-mail address: keivany@cc.iut.ac.ir a data for fishes of the zohreh river are limited. therefore, the present study aimed to reports lengthweight and length-length relationships’ parameters of six fish species from the zohreh river, a sub-basin of the tigris basin in the khuzestan provinces, southwestern iran. materials and methods a total of 114 specimens belonging to six species were collected by electrofishing device and net from the zohreh river, persian gulf basin, iran. the collected specimens were preserved in 10% buffered formalin and transferred to the laboratory for subsequent analyses. the total length (tl), fork length (fl) and standard length (sl) of specimens were measured with a digital caliper to the nearest 0.1 cm (length, l) and weight with a digital balance to the nearest 0.01 g (weight, w). length-weight relationships were determined by applying the equation w=𝛼lb, where w is the total body weight (g), l is the total body length (cm), 𝛼 is the intercept and b is the slope (le cren, 1951; ricker, 1975; froese, 2006; froese et al., 2011). the length-length relationship was estimated by linear regression analysis: tl=𝛼+b*sl and sl=𝛼+b*fl, where 𝛼 388 keivany and zamani-faradonbe/ lwrs and llrs for six fish species from zohreh river, iran parameter is the intercept and b parameter is the slope of the linear regression (cao et al., 2015). results and discussion this study increases the information related to lwrs and llrs for six fish species inhabit in the zohreh river. the number of samples, minimum and maximum of total length (cm), minimum and maximum of weight (g), length-weight relationships parameters (𝛼, 95%ci-𝛼, b and 95%ci-b) and the coefficient of determination (r2) are presented in table 1 and length-length relationships parameters (𝛼 and b) and the coefficient of determination (r2) are presented in table 2. the maximum value for b parameter in lwrs was 3.24 for aphanius dispar and minimum value was 2.72 for garra rufa. froese (2006) expressed that the parameter b should normally range between 2.5 to 3.5 and tesch (1971) reported values of b parameter varies usually between 2 and 4. in this study, the values b ranged from 2.72 for g. rufa to 3.24 for a. dispar, so the parameters can be used safely within the indicated length ranges. this study showing that linear regressions on data are highly significant (p<0.01) with all r2>0.905. the b values of lwrs that indicate allometric or isometric growth pattern and the 𝛼 parameter was close to 0.01, which is suggested as normal by froese (2006) for fusiform fish species. bibak et al. (2013) reported that for arabibarbus grypus in dalaki river and g. rufa in shahpur river, the b parameter were 2.93 and 3.242, respectively and the values of b parameter for g. rufa were 3.10 in tigris drainage, 3.02 in karkheh drainage, 3.06 in karun drainage, 3.06 in persis basin and 3.34 in hormuz basin, respectively (keivany et al., 2015), while our study b parameter for this species was 2.94 table 1. length-weight relationships parameters for six fish species collected from zohreh river, iran. family/species n tl (cm) min-max w (g) min-max parameters 𝛼 % ci-a (mean±se) b % ci-b (mean±se) r2 cyprinidae arabibarbus grypus 32 3.3-8.1 0.2-3.72 0.0066 0.0064±0. 0017 2.94 2.91±0.08 0.98 capoeta trutta 21 3.7-11.6 0.37-11.09 0.008 0.009±0.006 2.96 2.89±0.16 0.98 garra rufa 30 3.0-7.2 0.22-2.58 0.012 0.011±0.001 2.72 2.75±0.065 0.98 cyprinodontidae aphanius dispar 10 2.6-3.5 0.22-0.57 0.0105 0. 026±0. 58 3.24 3.22±0.16 0.97 sillaginidae sillago sihama 12 6.2-11.3 0.79-5.06 0.0026 0.0068±0.0033 3.15 2.72±0.12 0.96 sisoridae glyptothorax silviae 9 2.9-4.8 0.15-0.84 0.0058 0.006±0.0018 3.08 3.09±0.099 0.97 table 2. length–length relationship for six fish species from zohreh river, iran. species equation 𝛼 b r2 arabibarbus grypus tl = a + bsl 0.1373 1.256 0.99 sl = a + bfl -0.2696 0.93 0.99 capoeta trutta tl = a + bsl 0.2767 1.207 0.99 sl = a + bfl -0.2567 0.937 0.99 garra rufa tl = a + bsl 0.1078 1.206 0.99 sl = a + bfl -0.0889 0.914 0.99 aphanius dispar tl = a + bsl -0.055 1.209 0.99 sl = a + bfl sillago sihama tl = a + bsl 0.2175 1.13 0.99 sl = a + bfl 0.1306 0.865 0.99 glyptothorax silviae tl = a + bsl -0.0437 1.27 0.98 sl = a + bfl 0.1085 0.855 0.99 389 int. j. aquat. biol. (2016) 4(6): 387-390 and 2.72. the b parameter was 2.9475 and 2.9475 in male and female of c. trutta of shour river (taghavi niya et al., 2015) and were 30149 in male, 3.0003 in female and 3.0065 in overall of c. trutta in shour river downstream (baboli et al., 2012), whereas b parameter in this study was 2.93 for c. trutta. the b parameter for male, female and overall of a. dispar were 3.0745, 3.292 and 3.2053 from dalaki river, respectively (bibak et al., 2012), 3.03 and 2.98 for female and male in sillago sihama (mirzaei et al., 2013) and 3.045 for g. silviae (gerami et al., 2014); however in this study b parameter was 3.24 for a. dispar, 3.15 for s. sihama and 3.08 for g. silviae. the variances observed in values of the lengthweight relationship parameters in fishes is affected by a number of factors, including season, habitat, population, gonad maturity, sex, diet, stomach fullness, health, sample size, preservation techniques and locality (tesch, 1971; moutopoulos and stergiou, 2002; froese, 2006; hasankhani et al., 2014; zamani-faradonbe et al., 2015). this is the report of lwrs and llrs parameters for these species in the western iran, thus the data serve as a baseline for further studies of a possible alteration in the biometric parameters. with threats to fish communities such as damming and overexploitation, the data will be helpful in future species management and conservation. acknowledgements we would like to thank s. asadollah, m. pouladi, s.m.a. mousavi and a. nezamoleslami for their help in fish collection. this research was financially supported by isfahan university of technology. references baboli j.m., niya t.m., pazira a. (2012). length-weight relationship and condition factor of copoetta trutta in shour river downstream. advances in environmental biology, 1731-1735. binohlan c., pauly d. (1998). the length-weight table, in: r. froese, d. pauly (eds.). fishbase 1998: concepts, design and data sources. iclarm, manila. pp: 121-123. froese r. (1998). length-weight relationships for 18 less studied fish species. journal of applied ichthyology, 14: 117-118. froese r. (2006). cube law, condition factor and weightlength relationships: history, meta-analysis and recommendations. journal of applied ichthyology, 22: 241–253. froese r., tsikliras a.c., stergiou k.i. (2011). editorial note on weight-length relations of fishes. acta ichthyologica et piscatoria, 41: 261-263. garcia c.b., buarte j.o., sandoval n., von schiller d., mello n.p. (1989). length-weight relationships of demersal fishes from the gulf of salamanca, colombia. fishbyte, 21: 30-32. gerami m.h., abdollahi d., patimar r., abdolhahi m. (2014). length-weight relationship of two fish species from cholvar river, western iran: mastacembelus mastacembelus (banks & solander, 1794) and glyptothorax silviae coad, 1981. journal of applied ichthyology, 30(1): 214215. haimovici m., velasco g. (2000). length-weight relationship of marine fishes from southern brazil. the iclarm q, 23: 14-16. hasankhani m., keivany y., daliri m., pouladi m., soofiani n.m. (2014). length-weight and lengthlength relationships of four species (barbus lacerta heckel, 1843), oxynoemacheilus angorae (steindachner, 1897), squalius lepidus (heckel, 1843) and pseudorasbora parva (temminck & schlegel, 1846) from the sirwan river (western iran). journal of applied ichthyology, 30(1): 206207. keivany y., aalipour m., siami m., mortazavi s.s. (2015). length-weight relationships for three species from beheshtabad river, karun river basin, iran. iranian journal of ichthyology, 2(4): 296-298. keivany y., dopeikar h., ghorbani m., kiani f., paykanheyrati f. (2016). length-weight and length– length relationships of three cyprinid fish from the bibi-sayyedan river, western iran. journal of applied ichthyology, 32(3): 507-508. 390 keivany and zamani-faradonbe/ lwrs and llrs for six fish species from zohreh river, iran keivany y., nezamoleslami a., dorafshan s., eagderi s. (2016). length-weight and length-length relationships in populations of garra rufa from different rivers and basins of iran. international journal of aquatic biology, 3(6): 409-413. le cren e.d. (1951). the length-weight relationship and seasonal cycle in gonad weight and condition in the perch (perca fluviatilis). journal of animal ecology, 20: 201-219. martin-smith k.m. (1996). length/weight relationships of fishes in a diverse tropical fresh-water community, sabah, malaysia. journal of fish biology, 49: 731-734. mirzaei m.r., valinasab t., yasin z., hwai a.t.s. (2013). reproduction characteristics and length weight relationships of the sand whiting (sillago sihama) in the south coastal of iran (persian gulf and oman sea). annals of biological research, 4(5): 269-278. moutopoulos d.k., stergiou k.i. (2002). length-weight and length-length relationships of fish species from the aegean sea (greece). journal of applied ichthyology, 18: 200-203. radkhah a., eagderi s. (2015). length-weight and length-length relationships and condition factor of six cyprinid fish species of zarrineh river (urmia lake basin, iran). iranian journal of ichthyology, 2(1): 61-64. ricker w.e. (1975). computation and interpretation of biological statistics of fish populations. bulletin. fisheries research board of canada, 191: 382 p. ricker w.e. (1968(. methods for assessment of fish production in freshwaters. ibp handbook no. 3.blackwell scientific publications, oxford and edinburgh, uk. sarkar u.k., negi r.s., deepak p.k., lakra w.s., paul s.k. (2008). biological parameters of the endangered fish chitala chitala (osteoglossiforms: notopteridae) from some indian rivers. fisheries research, 90: 170-177. tabatabaei s.n., hashemzadeh segherloo i., eagderi s., zamani m. (2015). length-weight relationships of fish species in kordan river (namak lake basin), iran. journal of applied ichthyology, 31: 800-801. taghavi niya m., javaheri baboli m., roomiani l., pazira a., lakzaie f. (2015). study on the growth parameters of capoeta trutta (heckel, 1843) in shour river, iran. iranian journal of fisheries sciences, 14(1): 262-274. tesch f.w. (1971). age and growth. in: w.e. ricker (ed.). methods for assessment of fish production in fresh waters. blackwell scientific publications, oxford. pp: 98-130. zamani faradonbe m., eagderi s., naserabad s.s. (2015a). length-weight relationships and condition factor of three fish species from taleghan river (alborz province, iran). journal of advance botany and zoology, 2: 1-3. zamani faradonbeh m., eagderi s., ghojoghi f. (2015b). length-weight relationship and condition factor of seven fish species of totkabon river (southern caspian sea basin), guilan, iran. international journal of aquatic biology, 3(3): 172-176. int. j. aquat. biol. (2016) 4(6): 387-390 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی ایران زهره، رودخانه در ماهی گونه شش طول-طول و وزن-طول رابطه ارزیابی فرادنبه زمانی مظاهر ،*کیوانی یزدان .ایران ،84156-83111 اصفهان، اصفهان، صنعتی دانشگاه ،(شیالت گروه) طبیعی منابع دانشکده1 چکیده: -طول رابطه. است گرفته قرار بررسی مورد زهره رودخانه در خانواده چهار به متعلق ماهی گونه شش طول-طول و وزن-طول رابطه مطالعه این در معادله در b پارامتر مقادیر. دارد قرار 72/3 و 72/2 مقادیر بین( b) خط شیب به مربوط مقادیر. گردید محاسبه =bl𝛼w رابطه از استفاده با وزن گونه برای ،96/2 برابر capoeta trutta (heckel, 1843) گونه برای ،72/2 برابر arabibarbus grypus (heckel, 1843) گونه برای وزن-طول garra rufa (heckel, 1843) گونه برای ،72/2 برابر aphanius dispar (rüppell, 1829) گونه برای ،25/3 برابر sillago sihama (forsskål, 1775) گونه برای و 91/2 برابر glyptothorax silviae coad, 1981 شده ارائه اطالعات اولین از مطالعه این. آمد بدست 15/3 برابر .باشدمی زهره رودخانه از ماهی گونه شش این طول-طول و وزن-طول رابطه مورد در .ماهیانگربه ماهیان،شورت ماهیان،کپوردندان کپورماهیان، :کلمات کلیدی int. j. aquat. biol. (2021) 9(4): 248-253 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article assessing the stock status of atlantic bumper, chloroscombrus chrysurus, linnaeus 1766, from the coastal waters of ghana samuel k.k. amponsah1, nii amarquaye commey2, berchie asiedu1, hasan fazli*3 1 1department of fisheries and water resources, university of energy and natural resources, box 214, sunyani, ghana. 2esl consulting, accra, ghana. 3caspian sea ecology research center, iranian fisheries science research institute, agricultural research, education and extension organization, iran. s article history: received 3 march 2021 accepted 10 august 2021 available online 2 5 august 2021 keywords: growth parameters stock status mortality rates coastal waters abstract: the main objective of this study was to examine the growth, mortality, and exploitation rate of chloroscombrus chrysurus from the continental shelf of ghana, west africa between july 2018 and june 2019. this study provided results on fishery dynamics parameters to contribute to estimating the stock assessment of these fish species. monthly length-frequency data were collected from 697 samples and analyzed using fisheries models fitted in tropfish r. the von bertalanffy growth parameters were utilized to analyze the population dynamics of these species using elefan simulating annealing. the estimated asymptotic total length (l∞), coefficient of growth (k), and growth performance index (φ′) was 24.9 cm, 0.84 year-1, and 2.72, respectively, with a response surface (rn) value of 0.79. the total mortality (z), natural mortality (m), and fishing mortality (f) rates c. chrysurus from the continental shelf of ghana were 3.27 year-1, 1.31 year-1, and 1.96 year-1, respectively. the exploitation rate (e) estimated was above the optimum level of 0.5 which indicates that the species is overexploited. based on the emsy (0.69) value, analyses show that the exploitation rate has exceeded the sustainable limit and hence the need for proper fisheries management measures. introduction chloroscombrus chrysurus linnaeus 1766, known as the atlantic bumper or sapater, is an ecologically and economically important fish species of the family carangidae (smith-vaniz, 1990; edwards et al., 2001). this pelagic species is found in the tropical and subtropical western atlantic range, occuring from the coast of west africa from mauritania to angola. also, it is found in brackish and marine environments; usually at depths of 0-55 meters and inhabits soft bottoms, however, juveniles are often found in more oceanic waters (gabis et al., 2012). atlantic bumper is characterized by its readily distinguishable body shape, in which the lower profile is more arched than the upper part, and by having the small oblique mouth (edwards et al., 2001). it has a black spot on the upper part of the tail, is silvery, and many rainbow reflections when fresh (edwards et al., 2001; schwartz, 2013). this species primarily feeds on fish, cephalopods, and zooplankton and is one of *correspondence: hasan fazli doi: https://doi.org/10.22034/ijab.v9i4.1174 e-mail: h.fazli@areeo.ac.ir 12 species of the carangidae family landed in ghana (edwards et al., 2001; gabis et al., 2012). in ghana, c. chrysurus is caught from january to april (edwards et al., 2001). several researchers have documented the biology and stock status of c. chrysurus from several coastal countries, including growth and exploitation rate in benin (sossoukpe et al., 2017), life-history in tropical waters of the atlantic ocean (de queiroz et al., 2018), growth and mortality rates in the caribbean sea (garcia and duarte, 2006). however, there is limited information on the population dynamics of the commercially important c. chrysurus occurring in the waters of ghana. according to arra et al. (2020), population dynamics of fishes are undertaken with the major objective of sustainable management and conservation of fish species. therefore, this study aimed to examine the growth, mortality, and exploitation rate of c. chrysurus from the continental shelf of ghana, west africa, between july 2018 and 249 int. j. aquat. biol. (2021) 9(4): 248-253 june 2019. materials and methods study area: based on two stage-sampling, criteria included the level of fishing activity and geographical isolation, five fishing communities in the greater accra region of ghana were selected for the current study. these five fish landing sampling locations were sakumono, tema, nungua, kpone, and prampram (fig. 1). the primary form of livelihood for many of the inhabitants in the selected sampling sites is fishing and other activities related to the post-harvesting of fish from the wild. data collection: samples of fish species from the coastal waters of ghana were obtained from randomly selected fishermen who apply different fishing types and gears. samples were collected over 12 months (i.e. july 2018 to june 2019), preserved on ice, and taken to the laboratory. at the laboratory, identification of the species was done based on schneider's (1990) identification key. in all, 697 specimens of c. chrysurus were obtained during the study period. the total length (tl) of the specimens was measured to the nearest 1 cm. growth parameters: parameters for which the fish growth is assumed to follow von bertalanffy growth function (vbgf) were estimated using the elefan simulating annealing. the theoretical age at length zero (to) and longevity (tmax) were calculated according to the equations: log10 (-t0) = -0.3922 – 0.2752 log10 l∞ 1.038 log10 k (aleev, 1952) and tmax = 3/k + to (pauly, 1984), respectively. the growth performance index (φ′) was estimated using the formula: φ′ = 2 log l∞ + log k (munro and pauly, 1984). mortality: the total annual mortality rate, z, was estimated by constructing linearized length-converted catch curves (spare and venema, 1992). the natural (m) and fishing mortality (f), and exploitation rate (e) were calculated as m = 4.118k0.73 l∞−0.333 (then et al., 2015), f = z – m (qamar et al., 2016), and f/z (georgiev and kolarov, 1962), respectively. lengths at first capture (lc50) and first maturity (lm50): the left ascending part of the length converted catch curve was used to estimate the probabilities of length at 50, 75, and 95 capture which correlates with the cumulative probability at 50, 75 and 95 percent, respectively (pauly, 1984). the length at first maturity (lm50) was estimated by log 10 (lm50) = 0.8979 × log10 (l∞) 0.0782 (froese and figure 1. map showing the fish landing sampling locations. 250 amponsah et al./ stock status of chloroscombrus chrysurus in the coastal waters of ghana binohlan, 2000). biological reference points: emsy which depicts exploitation rate producing maximum yield and e0.5 implying exploitation rate under which the population is reduced to half its virgin biomass were computed together with the corresponding fishing mortality rate (i.e. fmsy and f0.5). data analyses: the length-frequency data were pooled into groups with 1 cm length intervals. then the data were analyzed using the tropfish r package in the r software (mildenberger et al., 2017). results figure 2 shows the restructured length-frequency with superimposed growth curves. the growth parameters of l∞, k, and t0 were estimated as 24.9 cm tl, 0.84 year-1, and -0.20 year with tmax of 3.37 years. the φ′ and rn were 2.72 and 0.79, respectively. the length at first capture at lc50, lc75, and lc95, was estimated at 11.3, 12.2, and 13.6 cm, respectively (fig. 3). the length at first maturity (lm50) was 15.0 cm. based on the length converted catch curve method, the z was estimated at 3.27±0.16 year-1 (fig. 4). the m, f, and e were 1.31 year-1, 1.96 year-1, and 0.60, respectively. the indices of the optimum sustainable yield (e0.5) and maximum sustainable yield (emax) were 00.44 and 0.69, respectively (fig. 5). the corresponding fishing mortality rates were 1.02 yr-1 for optimum sustainable yield (f0.5) and 2.92 yr-1 for the maximum sustainable yield (fmsy). discussion the information on growth parameters and the stock status gained from the present study will serve as a figure 2. reconstructed length-frequency distribution of chloroscombrus chrysurus from continental shelf of ghana. figure 3. probability of age at first capture for chloroscombrus chrysurus from continental shelf of ghana. figure 4. length converted catch for chloroscombrus chrysurus from continental shelf of ghana. 251 int. j. aquat. biol. (2021) 9(4): 248-253 springboard for the sustainable management of the assessed species in ghana. the asymptotic length (l∞) of c. chrysurus (24.9 cm tl) in this study varied from other studies, which de queiroz et al. (2018), sossoukpe et al. (2017) and garcia and duate (2006) reported higher l∞ values (25.6, 28.3 and 30.5 cm tl, respectively). the growth coefficient, k in the present study (k = 0.84 year-1) was higher than estimates by sossoupke et al. (2017), da costa et al. (2005), and de queiroz et al. (2018) who reported k at 0.49, 0.39 and 0.33 year-1, respectively. the changes in growth parameters as compared to other studies may be associated with climate type, and latitudinal differences (tarkan and vilizzi, 2015). also, the size ranges of individuals play a key role since growth parameters are very sensitive to sample size, which larger samples can increase the l∞ and decrease the k (espino barr et al., 2008). the growth performance index (ø’) of c. chrysurus (2.72) is more than that found by sossoupke et al. (2017) and da costa et al. (2005) (2.59 and 2.58, respectively). the critical length at capture which is the ratio of lc50/ l∞ (11.3/24.9 = 0.45), indicated that it was lower than 0.5 in the continental shelf of ghana. this signals the harvesting of more juvenile fish species (pauly and soriano, 1986). the indulgence in such practice for a long period of time could result in growth overfishing and consequentially, may render recruitment dysfunctional in the future leading to a possible collapse. furthermore, the length at which these species become vulnerable to the fishing gears (lc50 = 11.3 cm) used by ghanaian fishermen was lower than the lm50 (15.0 cm). this implies that species do not get the opportunity to spawn at least once before they are captured which could have some ramifications on the recruitment potential of these stocks in the future (gheshlaghi et al., 2012). it is therefore necessary to enforce length at 95% capture which is feasible when the mesh size is increased. the natural mortality rate (m) of c. chrysurus (1.31 year-1) was higher than that reported by sossoupke et al. (2017) and de queiroz et al. (2018) (1.17 and 0.92 year-1, respectively). according to macer (1977), the consistency of the estimated m was the ratio of m/k ratio, which has been ranged between 1.12-2.5 for most fishes. this ratio in the present study (1.63) fell within the acceptable demarcated range. according to the results, the total (z) and fishing (f) mortality were 3.27 and 1.96 year-1, which the f was higher than m (m = 1.31 year-1). similar results were reported by garcia and duarte (2006), with z, m, and f at 3.03 year-1, 1.29 year-1, and 1.71 year-1, respectively. in contrast, sossoupke et al. (2017) reported a lower z and f for the same species from benin (1.39 and 0.26 year-1) and the f was lower than m (m = 1.16 year-1). the relatively higher f than m indicates an imbalanced stock position (azim, 2017). the exploitation rate (e) of c. chrysurus was 0.60, showing that it is slightly overexploited because the exploitation rate surpasses the optimum level of 0.5 (gulland, 1971). also, this e was slightly lower than the exploitation rate at the msy (emax = 0.69) on the figure 5. thompson and bell model: (a) curves of yield and biomass per recruit. the black dot represents yield and biomass under current fishing pressure. the yellow and red dashed lines represent fishing mortality for maximum sustainable yield (fmsy) and fishing mortality to fish the stock at 50% of the virgin biomass (f0.5). (b) exploration of the impact of different exploitation rates and lc values on the relative yield per recruit. 252 amponsah et al./ stock status of chloroscombrus chrysurus in the coastal waters of ghana continental shelf of ghana. this observation may have accounted for the high exploitation rates estimated for this species, which calls for the management of these stocks through measures such as a reduction in fishing efforts, closed fishing season, creating marine protected areas (mpas) to help sustain these stocks. in conclusion, c. chrysurus encountered in ghana’s coastal waters is fast-growing fish species. the immature individuals are under high fishing pressure. this species appears to be overexploited in ghana’s marine waters. hence it needs to safeguard its stocks through proper management measures. such management measures may include mesh size regulations, closed fishing seasons, and compliance to fisheries policies. acknowledgment sincere gratitude goes to esl, ghana for permitting the authors to use the data from fieldwork. we would like to express our appreciation to the fishermen who assisted us with data collection during the study period. references aleev y.g. (1952). horse mackerel of the black sea, vniro press. 16 p. arra s., sylla s., zan-bi t.t., loukou a., ouattara m. (2020). stock assessment and population dynamics of senegal jack, caranx senegallus cuvier, 1833, from industrial fishery of cote d’ivoire (west africa). agronomie africaine, 32: 37-49. azim m.k., amin, s.m.n., romano n., arshad a., yusoff f.m. (2017). population dynamics of yellowtail scad, atule mate (cuvier 1833) in marudu bay, sabah, malaysia. sains malaysiana, 46 (12): 2263-2271. da costa m.r., albieri r.j., araújo f.g. (2005). size distribution of the jack chloroscombrus chrysurus (linnaeus) (actinopterygii, carangidae) in a tropical bay at southeastern brazil. revista brasileira de zoologia, 22(3): 580-586. de queiroz j.d.g.r., salvador n.l.a., sousa m.f., da silva v.e.l., fabré n.n., vandick s., batista v.s. (2018). life-history traits of chloroscombrus chrysurus (actinopterygii: perciformes: carangidae) in tropical waters of the atlantic ocean. acta ichthyologica et piscatoria, 48 (1): 1-8. edwards a.j., gill, c.a., abohwekyere p.o. (2001). a revision of irvine’s marine fishes of tropical west africa. 186 p. espino barr e., gallardo cabello m., cabral solís e.g., garcia boa a., puente gómez m. (2008). growth of the pacific jack caranxcaninus (pisces: carangidae) from the coast of colima, méxico. revista de biologia tropical, 56 (1): 171-179. froese r., binohlan c. (2000). empirical relationships to estimate asymptotic length, length at first maturity and length at maximum yield per recruit in fishes, with a simple method to evaluate length-frequency data. journal of fish biology, 56 (4): 758-773. gabis g., nichols e., kelpsaite e., parkins c., castro k., somers b. (2012). guide for the identification of commonly caught fish in the bottom set gillnet fishery in the gambia, coastal resources center, university of rhode island. 68 p. garcia c.b., duarte l.o. (2006). length-based estimates of growth parameters and mortality rates of fish populations of the caribbean sea. journal of applied ichthyology, 22: 193-200. gayanilo f.c., sparre p., pauly p. (2005). the fao‐ iclarm stock assessment tools ii (fisat ii) user’s guide. fao, rome. retrieved from: http://www.fao.org /fi/statist/fisoft/fisat/index.htm georgiev z.m., kolarov p. (1962). on the migration and distribution of horse mackerel (trachurusponticus aleev) in the westernpart of black sea. arbeiten des zentralen forschungsinstitutes fur fishzught und fisherei varna, 2: 148-172. gheshlaghi p., vahabnezhad a., taghavimotlagh s.a. (2012). growth parameters, mortality rates, yield per recruit, biomass, and msy of rutilus frisii kutum, using length-frequency analysis in the southern parts of the caspian sea. iranian journal of fisheries science, 11(1): 48-62. gulland j.a. (1971). the fish resources of the oceans. west by fleet survey. fishing news (books) ltd., for fao, west by fleet, england. 255 p. macer c.t. (1977). some aspects of the biology of the horse mackerel [trachurustrachurus(l.)] in waters around britain. journal of fish biology, 10(1): 51-62. munro j.l., pauly d. (1984). once more on the comparison of growth in fish and invertebrates, iclarm fishbyte, 2. 21 p. pauly d., soriano m.l. (1986). some practical extensions to beverton and holt's relative yieldper-recruit model. 253 int. j. aquat. biol. (2021) 9(4): 248-253 in: j.l. maclean, l.b. dizon, l.v. hosillos (eds.), first asianfisheries forum, asian fisheries society, manila, philippines. pp: l49-495. pauly d. (1984). fish population dynamics in tropical waters: a manual for use with programmable calculators. iclarm contribution, makati, metro, manila, philippines, 143. 325 p. qamar n., panhwar s.k., brouwer s. (2016). population characteristics and biological reference point estimates for two carangid fishes, megalaspiscordyla and scomberoidestol, in the northern arabian sea coast of pakistan. pakistan journal of zoology, 48(3): 869-874. schneider w. (1990). fao species identification sheets for fishery purposes. field guide to the commercial marine resources of the gulf of guinea. prepared and published with the support of the fao regional office for africa. rome: fao. 268 p. schwartz f. (2013). atlantic bumper (pisces, family carangidae) in north carolina. journal of the north carolina academy of science, 129(4): 184-185. smith-vaniz w.f. (1984). carangidae: relationships. in: h.g. moser et al. 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(2022) 10(4): 310-314 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article comparison of the traditional outdoor and recirculation indoor rearing systems on survival rate and growth performance of common carp (cyprinus carpio) larvae during early development nawras a. al-faiz*1, abdulkareem t. yesser2, amir a. jabir2, qusay h. alhamdany2 1natural science department, faculty of marine science, university of basrah, basrah, iraq. 2department of vertebrates, marine science centre, university of basrah, basrah, iraq. s article history: received 3 march 2022 accepted 6 july 2022 available online 2 5 august 2022 keywords: recirculatory system earthen ponds larval production survival rate abstract: the larvae of common carp (cyprinus carpio l.) were studied for 6 weeks in an indoor recirculatory system (ras) and an outdoor earthen pond to compare their effects on growth performance and survival rate. larvae reared in outdoor earthen ponds achieved significantly higher total length, weight, weight gain, and length increments than indoor groups (p≤0.05). however, the indoor recirculatory system had the highest survival rate (83±1.9%) than outdoor earthen ponds (42±3.6%) (p≤0.05). the results of the present study revealed that the raising system has a significant impact on the survival and growth performance of the larvae in common carp, and the best growth performance was in the outdoor earthen ponds, while the better survival rate was recorded in the indoor recirculatory system. therefore, to minimize mortality and maximize survival rate, it is suggested that the larvae were released after hatching into closed and controlled rearing systems before being reared into earthen ponds after starting the exogenous feeding. introduction larval rearing is vital to a successful aquaculture program (islam et al., 2004). larviculture in the common carp (cyprinus carpio) follows the usual practices in iraq, i.e., applying a semi-intensive system in the earthen ponds. in this system, larvae feeding during the exogenous stage is based on ingesting natural food items produced by fertilizing the ponds (portella et al., 2014). this approach causes losses during the initial phase i.e. up to grown to fry with a weight of 1 g (jelkić et al., 2012). these losses can be due to variations in the water quality of the earthen ponds. in nature, larval survival is determined by predator avoidance and feeding conditions (feldlite and milistein, 1999). large-scale mortality is a concern during the early developmental stages of some freshwater fish in the wild and even in some nursery ponds (jana and jana, 2003). dietary deficiencies have been reported to be the main reason for high larval mortality in hatcheries (ayyappan and jena, 2003; ghosh et al., 2004), along with low water quality (rice et al., 1987), predation and disease (smith and kernehan, 1981; ludwig, 1999; frimpong and lochmann, 2005). the greatest losses of fish larvae occur during the larvae to fry transition period, ranging from 50 to 90 % in earthen ponds (matic and jurakic, 2006; kumar et al., 2012; jelkić et al., 2012; gjurcevic et al., 2012). this could result in a severe shortage of common carp fingerlings in the developing aquaculture industry. modifications in larval rearing technology, like controlled production, can increase the survival rate of larvae from up to 80-90% (barr et al., 2007). in semiintensive production systems, larviculture is typically done in earthen ponds; however, rearing the larvae in an intensive system for a short period is more efficient than direct release into earthen ponds (jomori et al., 2003). indoor systems for larvae production are an alternative for improving survival rates (jomori et al., 2005; motta et al., 2019), especially during months in which climatic conditions are improper. nutrition is the main issue with raising common carp larvae in hatcheries under controlled conditions. however, outdoor earthen-pond-produced zooplankton is 310 al-faiz et al./ comparison of traditional outdoor and recirculation indoor rearing systems in common carp frequently irregular in quantity and quality, and there is a high risk of parasites in such hatcheries. artificial diets can be used in indoor hatcheries (charlon and bergot, 1986), but the development of common carp larvae using formulated diets are frequently reduced (kamler, 1992). in addition, feeding live food first can compensate for this deficiency and reduce the high mortality rates in common carp larvae fed formulated diets from the start of exogenous feeding. recent works on rearing common carp larvae using artificial feed instead of live-food have shown good results (regenda et al., 2003). although the survival rate of fish larvae is higher under controlled conditions due to food supply and the absence of predators, but mortality is high and varies depending on the species. hence, this study aimed to compare the growth performance and survival rate of common carp larvae during early development in an indoor recirculatory system fed artificial feed to those of traditional systems i.e. outdoor earthen pond system. materials and methods this experiment was conducted in the wet laboratory of the marine vertebrates department, marine science centre, the university of basrah in march-april of 2021. the experiment was conducted in an indoor recirculatory aquatic system including 12 plastic tanks of 30× 30× 40 cm, fitted with an underwater biofilter, solids settling tank, and oxygen pumps. the outdoor earthen ponds include three earthen ponds of 10×15×0.75 m. the recirculatory system was cleaned and filled with water before beginning the experiment. before the experiment, the earthen ponds were dewatered and sun-dried for about two weeks. the earthen ponds will be filled with water in february 2021. furthermore, extra water was added to each earthen pond 10 days before the experiment began to keep the water level stable. the healthy larvae with an average length of 0.45±0.001 cm and an average weight of 0.0029±0.0001 g were obtained from the marine science centre hatchery of the university of basrah. fish larvae were introduced into tanks of the recirculatory system and earthen ponds at random and reared for six weeks. the density of the tanks and earthen pond culture system were considered as five larvae per liter. the larvae were fed ad libitum four times per day during the rearing period. initially, the larvae were fed boiled egg yolk four times a day at four-hour intervals for the first three days. then, they were fed algae and mixed zooplankton for the next eleven days. the larvae were then fed commercially available feed pellets containing 25% crude protein, 2.5% crude fat, 7% fiber, and 10% moisture for the remaining study period. the feed pellets were crushed before feeding the larvae. some physio-chemical water quality parameters, including water temperature, ph, salinity, and dissolved oxygen, were measured daily during the rearing period (baird et al., 2012). the length, weight, and the total number of larvae were recorded at the end of the experiment (6 weeks). the following formula was used to calculate growth parameters, weight gain, average length increment, and survival rate. weight gain (g) = final fish weight (g) initial fish weight (g) length increment (cm) = final fish length (cm)-initial fish length (cm) survival rate (%) = harvested larvae / stocked larvae x 100 the statistical analysis was performed in spss (version 26) using the one-way analysis of variance (anova), followed by the lsd test. the differences were considered statistically significant at the level of p≤0.05. the results were all described as mean±sd. results all physical and chemical water quality parameters were within acceptable limits for larval rearing (table 1). based on the results, the larvae reared in outdoor earthen ponds achieved a higher average total length (6.54±0.13 cm) and weight (5.324±1.276 g) (fig. 2). those of the indoor recirculatory system had a lower average length (2.60±0.06 cm) and weight 311 int. j. aquat. biol. (2022) 10(4): 310-314 (0.267±0.088 g) (p≤0.05) (fig. 1). the larvae reared in outdoor earthen ponds had higher weight gain, and length increments, than those of reared indoor (p≤0.05) (table 2). the survival rates of larvae in two rearing systems are shown in table 2. the indoor recirculatory system had a higher survival rate (83±1.9%) than outdoor earthen ponds (42±3.6%) (p≤0.05). discussions the water quality parameters during the experiment were within the acceptable range for carp larvae cultivation (motta et al., 2019). the indoor tanks effectively maintained acceptable water quality parameters during the experiment. in addition, the used feeding protocol in this study, which was based on motta et al. (2019), showed proper results for the rearing of an indoor system. the adopted feeding frequency (4 times per day) was also effective for rearing larvae. fish larvae are transitional animals that require constant feeding (portella et al., 2014). in the current study, larvae reared in the outdoor earthen ponds gained higher total length and weight. under any management regime, larvae gained more weight in earthen ponds than indoor tanks (ayyappan and jena, 2003). similar findings have been reported parameters indoor recirculatory aquatic system range (mean) outdoor earthen ponds range (mean) temperature (ºc) 26.5-27.7 (27.1) 22.3-28.1 (25.2) ph 8.1-8.6 (8.3) 8.1-8.9 (8.5) do (mg/l) 7.2-8.9 (8.05) 5.4-7.8 (6.6) salinity (g/l) 1.23-2.52 (1.87) 2.45-3.27 (2.68) table 1. water physical and chemical properties in common carp larvae rearing systems. table 2. common carp larvae growth parameters in outdoor earthen ponds and indoor recirculatory aquatic systems. parameters indoor recirculatory system outdoor earthen ponds initial weight (g) a0.0029±.0001 a0029±.0001 final weight (g) 0.267±0.088b 5.324±1.276a weight gain (g) 0.257±0.088b 5.314±1.276a initial length (cm) 0.45±0.02a 0.45±0.02a final length (cm 2.60±0.06b 6.54±0.13a length increments (cm) 2.15±0.08b 6.09±0.17a survival rate (%) 831.9%a 42±3.6%b the data is presented as mean ± sd. the difference in means is represented by different letters in the rows (p≤0.05). figure 1. weekly increase in the weight of the common carp larvae. 312 al-faiz et al./ comparison of traditional outdoor and recirculation indoor rearing systems in common carp in studies with other fish species (summerfelt et al., 1996; jomori et al., 2003, malison, 2003). the increases in total length and weight of larvae in earthen ponds were significant, which attributed to the live food availability in earthen ponds as a result of fertilization and additional food (ayyappan and jena, 2003). whereas indoor tanks have small spaces limiting natural food production (fotedar, 2016). competition for space and food is the primary factor influencing larvae growth in indoor tanks (fotedar, 2016). even food for the larvae is sufficient; this did not guarantee that every larva would consume the estimated amount of food. differences in food availability were more directly related to growth, weight gain, and length increments. in contrast to our findings, ako et al. (2005) found that common carp were non-competitive feeders. despite laboratory success, tank culture of yellow perch (perca flavescens) fingerlings is not widely used, and commercial production is done in ponds with live food (malison, 2003). ponds are commonly used for walleye, stizostedion vitreum, and younger fry (up to 6.5 cm) because they grow faster (summerfelt et al., 1996). the high survival rate in the indoor recirculatory system was most likely due to the improved conditions promoted by larvae production, such as high-quality food supply and water quality control. the larval stage immediately the following hatching is the most critical developmental stage in fish life history, requiring a significant pause to maintain growth and survival until later life stages (jelkić et al., 2012). controlling environmental factors such as water quality, predators, diseases, and nutrition are crucial. common carp larvae were transferred to laboratory tanks while environmental factors were kept within acceptable limits, allowing for appropriate growth and survival rates, which is consistent with the findings of smagula and adelman (1982), and elliot (1982). the results of the present study showed that the raising system has an important effect on the survival rate and growth performance of common carp larvae. the best weight gain and length increments were found in the outdoor earthen ponds, while the better survival rate was recorded in indoor tanks. therefore, it is suggested that the larvae are released after hatching in a closed and controlled rearing system, and then released into earthen ponds to avoid mortality and achieve the highest growth performance. acknowledgments the authors would like to express their gratitude to the fish hatchery staff at the marine science center, the university of basrah, for their assistance and support in completing this study, as well as for providing the larvae and setting up the earthen ponds. references ako h., shimizu e., de lemos k., asano l., tamaru c. (2005). behavioral limitations of high-density fish grow figure 2. increase in the length of the common carp larvae. 313 int. j. aquat. biol. (2022) 10(4): 310-314 out. world aquaculture, 36: 25-29. fotedar r. (2016). water quality, growth and stress responses of juvenile barramudi (lates calcarifer bloch), reared at four different densities in integrated recirculating aquaculture systems. aquaculture, 458: 113-120. ayyappan s., jena j.k. (2003). grow-out production of carps in india. journal of applied aquaculture, 13: 251282. baird r.b., eaton a.d., clesceri l.s. (2012). standard methods for the examination of water and wastewater (vol. 10). in: e.w. rice (ed.). washington, dc: american public health association. barr y., gissis a., dulic z. 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(2013) 1(3): 93-99 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 npajournals. all rights reserved original article the skeletal deformity in response of dietary phosphorus and calcium level in the caspian roach (rutilus rutilus caspicus) larvae sohrab ahmadivand*1, soheil eagderi2, mohammad reza imanpour3 1department of aquatic animal health, faculty of veterinary medicine, university of tehran, p.o. box: 14155-6453, tehran, iran. 2fisheries, faculty of natural resources, university of tehran, p.o. box 4314, karaj, iran. 3fisheries department, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 14 april 2013 accepted 20 may 2013 available online 20 june 2013 keywords: skeletal deformity caspian roach phosphorus calcium abstract: skeletal deformities are a common problem in fish hatcheries and commercial farms that affect growth, development and survival as well as the market value of the final product. among the nutritional components, phosphorus (p) and calcium (ca) are of special interest as they are directly involved in the development and maintenance of the skeletal system. hence, the present study was carried out to investigate the effects of dietary p and ca on the skeletal deformity, growth and carcass composition the caspian roach (rutilus rutilus caspicus) larvae. in this study, six semi-purified diets were formulated. the diets a, b, c, d and e were supplemented with 0.0, 0.4, 0.8, 1.2 and 1.6% available p supplied as a 1:1mixture of nah2po4/kh2po4. these five diets were supplemented with 1% ca, supplied as caco3. diets f was ca-free and supplemented with 0.8% available p served as control level of p. each diet was randomly assigned to triplicate groups of fish, and each group was stocked with 30 larvae and fed three times a day for 60 days. at the end experiment, there was no significant effect of dietary p (0 to 1.6%) or ca (0 or 1%) supplementation on growth performance such as weight gain and fcr, carcass moisture, p and ca. however, a significant difference found between treatments in carcass ash. analysis of length, height and area of vertebrae in two regions of the vertebral column showed no significant difference between the dietary treatments. the skeletal abnormalities were highest incidence in the caspian roach fed with a low p. kyphosis placement of vertebrae was the most frequent abnormality. introduction main morphological change of the fish larvae is occurred during the early developmental stages which can be affected by xenobiotic, environmental, genetic, nutritional and physiological factors. during this period, abnormalities such as the skeletal deformities are frequently observed in reared fish larvae as a result of deficiencies in above mentioned factors (cahu et al., 2003; lall et al., 2007). the incidence of skeletal deformities in cultured fishes is led to financial losses (sadler et al., 2001). the most skeletal deformities in fishes include vertebral and spinal malformations such as kyphosis, lordosis, fusion of vertebrae and irregular column (lall et al., * corresponding author: sohrab ahmadivand e-mail address: s_ahmadivand@ut.ac.ir 2007). moreover, these deformities may be occurred as a combination of several types of deformities (lall et al., 2007). nutrition has been suggested having a key role in skeletogenesis (beattie and avenell, 1992; wallach, 2002). the effects of dietary factors related to nutrient deficiencies have received limited attention in fishes in despite many works on humans and terrestrial animals (beattie and avenell, 1992; wallach, 2002). it has been suggested that nutritional deficiencies, including some vitamins e.g. c, b, a or d and minerals e.g. zinc, magnesium, phosphorous (p), calcium (ca) and selenium, promote skeletal deformities in fish larvae under culture conditions 94 int. j. aquat. biol. (2013) 1(3): 93-99 (lall et al., 2007). p and ca are closely related to the development and maintenance of the skeletal system. fishes can absorb ca and p from water and their ca requirement is met by their ability to absorb directly this element from water (lall, 2002). ca deficiency, unlike p deficiency is related to very low concentration of this element in water and not common, however, ca deficiency induces a delay in the ontogeny of skeletal development without affecting final bone mineralization and leads to modifications in the shape and size of vertebrae (fontagné et al., 2009). p deficiency in most fish species is led to poor growth, poor bone mineralization, skeletal deformities, low ash and high lipid content in the whole body, while excess p affects survival (tacon, 1992; lall, 2002). information about the larval nutritional requirements such as ca and p is limited due to their fast-changing requirements during ontogeny and also; their requirements are slightly different from those of juveniles. hence, understanding of biological effects and function of these components is essential and can allow to improve the quality of hatchery-reared larvae (cahu et al., 2003). therefore, a special attention should be paid to the level and availability of p in formulation of diets for normal vertebral development during the early development of fish larvae. the caspian roach (rutilus rutilus caspicus jakowlew, 1870), a commercially important, has declined greatly in the iranian caspian sea due to overfishing and deterioration of its spawning grounds (eagderi and ghelichpour, 2012). therefore, its artificial propagation in hatcheries to restock its natural population is necessary. the iranian fisheries organization produces the caspian roach larvae in the earthen pond till 1-3 g and then releases them to the rivers ending to the caspian sea (kiabi et al., 1999). this study was conducted to investigate the effect of different levels of p and ca on the skeletal deformity, growth and carcass composition of the reared caspian roach larva with a view to determining the optimum p requirement for the growth of this fish. material and methods design of experiment: the caspian roach larvae were obtained (with a mean weight of 0.108 ± 0.01g) from the sijval fish hatchery (gorgan, iran). before the experiment, the larvae were acclimated to laboratory conditions in a single 1000 l tank for 10 days. during this period, water temperature (°c), ph, do (mg l-1) were 24 ± 2, 7.8 ± 0.2, 7 ± 1, respectively. after the acclimation period, the larvae were randomly divided into eighteen 100 l experimental tanks, each containing 30 larvae. diet a b c d e f casein-dextrin basisa 82.5 82.5 82.5 82.5 82.5 82.5 vitamin mixture 5 5 5 5 5 5 mineral mixture without p and cab 2.5 2.5 2.5 2.5 2.5 2.5 monobasic sodium and potassium phosphates (50/50) 0.00 1.68 3.36 5.04 6.72 3.36 α-cellulose 7.50 5.82 4.14 2.46 0.78 4.14 calcium carbonate 2.50 2.50 2.50 2.50 2.50 0.00 available p 0.0 0.4 0.8 1.2 1.6 0.8 available ca 1.0 1.0 1.0 1.0 1.0 0.0 acasein-dextrin basis (% diet): 52% casein); 0.65%, l-methionine; 0.85% , l-arginine; 8% soybean lecithin; 8% fish oil; 12% dextrin; 1% sodium alginate. bmineral mixture (g/kg mineral mix): kcl, 180; ki, 0.08; nacl, 80; cuso4·5h2o, 6; znso4·7h2o, 8; coso4, 0.04; feso4·7h2o, 40; mnso4·h2o, 6; mgoh, 248; na2seo3, 0.06; naf, 2. all ingredients were diluted with α-cellulose. table 1. formulation and composition of experimental diets (g/100 g dry weight). 95 ahmadivand et al./ skeletal deformity in response of p and ca in the caspian roach feeding and experimental diets: six semi-purified diets were prepared with graded levels of p and ca, based on fontagne et al. (2009) (table 1). the diets a, b, c, d and e were supplemented with 0.0, 0.4, 0.8, 1.2 and 1.6% available p supplied as a 1:1 mixture of nah2po4/kh2po4. these five diets were supplemented with 1% ca, supplied as caco3. diets f was ca-free and supplemented with 0.8% available p served as control level of p. each diet was provided to three replicate tanks. every two weeks, samples of fish were taken from each tank to weigh fish and calculate the required food for the following week. larvae were fed three times a day with experimental diets, 5% of their bw/d over a 60-days experiment period. sample collection: at the end of the experiment, the larvae were anaesthetized using the clove oil solution (1 ml diluted in 40 liters of water). mean weight of larvae was measured using the sum of individual weight divided by the number of fish in each tank. then, weight gain (gr) and fcr of treatment were calculated. fifteen larvae from each treatment (five larvae per tank) were sampled to analysis the composition of carcass (five larvae) and examination of skeletal deformity (10 larvae). chemical analysis: moisture determined by drying larvae in an oven at 105°c for 24 hrs. to determine the ash and mineral contents, dried samples were placed in a muffle furnace at 550°c for 24 hrs (aoac, 2000). minerals, including the p and ca were measured using an atomic emission spectrophotometer. examination of vertebrae: ten larvae were fixed in 5% formalin (from each treatment) and x-rayed using a mammography system (payamed, mamo xray, unit 100 khz, iran), with a manual exposure mode to give 10 mas and 20 kv. the films of radiographies were digitized using epson perfection v600 photo scanner and archived as jpeg images for subsequent analysis. three vertebrae in three anatomical regions were identified in the vertebral column of the caspian roach based on kacem et al. (2003) including, viz-cranial, trunco-cranial and caudal (fig.1). the length (l), height (h) of vertebrae and vertebral area (area = 2π × (h/2) × l) in trunco-cranial and caudal were measured using imagej (http://rsb.info.nih.gov/ij/). but viz-cranial was identified as the weberian apparatus were disregard. statistical analysis: one-way anova and duncan’s multiple range tests were performed using spss 20 to examine significance different between mean values of the dietary groups (duncan, 1955). results the weight gain (wg), fcr and carcass composition of the caspian roach larvae fed with different levels of p and ca are shown in table 2. dietary supplemented with different levels of available phosphorus (0, 0.4, 0.8, 1.2 and 1.6%) and calcium (0 and 1%) did not affect growth of the caspian roach larvae. although larvae in the group e with 1.2% available p had the highest wg and lowest fcr, but no significant differences in wg figure 1. x-ray image of caspian roach larva showing three distinct regions considered during measurement of the vertebral column; viz-cranial, trunco-cranial and caudal. 96 int. j. aquat. biol. (2013) 1(3): 93-99 and fcr were found between dietary treatments (p>0.05). also, no significant difference in carcass moisture, p and ca between the dietary groups was found (p>0.05). however, there was significant difference between the treatments in ash content of the carcass (p<0.05). analysis of the length, height and area of vertebrae in two identified regions in the vertebral column (table 3) revealed no significant figure 2. normal and abnormal skeletal structure in caspian roach larva fed the six diets supplemented with different levels of phosphorus (0, 0.4, 0.8, 1.2 and 1.6%) and calcium (0 and 1%) after 60 days of feeding trial. (a) normal vertebral column, (b) kyphosis, (c) irregular column and (d) twisted neural and haemal arches. table 2. weight gain, fcr and carcass composition of the caspian roach larvae fed diets with different levels of p and ca (% wet weight). a b c d e f ash 1.37 ± 0.07 a 2.08 ± 0.34 b 2.28 ± 0.30 b 2.40 ± 0.39 b 2.67 ± 0.20 b 2.23 ± 0.18 b moisture 74.4 ± 0.28 a 74.4 ± 0.39 a 74.3 ± 0.91 a 74.4 ± 0.76 a 74.3 ± 0.13 a 74.6 ± 0.9 a p 0. 23 ± 0.05 a 0.26 ± 0.08 a 0.31 ± 0.16 a 0.34 ± 0.03 a 0.32 ± 0.01 a 0.30 ± 0.12 a ca 0.19 ± 0.02 a 0.18 ± 0.04 a 0.25 ± 0.02 a 0.31 ± 0.04 a 0.22 ± 0.15 a 0.26 ± 0.1 a wg (g) 0.784 ± 0.07a 0.807 ±0.05a 0.805 ± 0.18a 0.867 ± 0.05a 0.806 ± 0.09 a 0.77 ± 0.11 a fcr 1.92 ± 0.17 a 1.86 ± 0.12 a 1.93 ± 0.49 a 1.74 ± 0.18 a 1.87 ± 0.22 a 1.97 ± 0.27 a means of triplicate values with similar superscript are not significantly different (p>0.05) 97 ahmadivand et al./ skeletal deformity in response of p and ca in the caspian roach difference between the dietary groups (p>0.05). skeletal abnormalities were grouped into three categories viz. twisted haemal and neural arches, kyphosis, irregular column. these abnormalities showed in figure 2. the frequency of each abnormality was calculated as a proportion of the total number of abnormalities per dietary group (n=10) (fig. 3). the highest incidence of internal abnormalities was observed in the treatment fed with a low p. the lowest abnormality was observed in group d (1.2% available p). kyphosis placement of vertebrae was the most frequent abnormality. discussion skeletal abnormality due to its impact on larva quality is led to financial losses; hence it is considered as an important issue for finfish industry and animal welfare (koumoundouros, 2001; sfakianakis et al., 2006). an approach to solve this problem is to determine accurately of larval nutritional requirements such as ca and p which are key parameters influencing the skeletogenesis during the early development of fish (negm et al., 2013). the present study investigated the effects of different dietary level of p and ca on the skeletal deformity, growth and carcass composition in the caspian roach larvae with a view to determine its optimal p requirement. the results revealed that the wholebody ash was significantly lower in the larvae fed a low p supplement (group a), whereas, the wholebody moisture not affected by any dietary groups indicating p is necessary for bone mineralization. the ash content of bone is considered to be the most sensitive criterion for evaluating dietary p utilization (ye et al., 2006). nwanna et al. (2008) determined that p deficiency produced lower ash in channel catfish (heterobranchus bidorsalis). similar results have also been reported in channel catfish (andrews et al., 1973), sunshine bass (brown et al., 1993) and haddock (roy and lall, 2003), which are in agreement with our results. the results also showed that growth parameters, including wg and fcr were not affected by different dietary p and ca levels; however, the results suggest that the best wg figure 3. percentage of the caspian roach larvae sampled on day 60 fed diets supplemented with different levels of available p (0, 0.4, 0.8, 1.2 and 1.6%) and ca (0 and 1%) which showed malformation include kyphosis, irregular column and twisted neural and haemal arches (n=10 for each dietary group). column region diet vertebral length vertebral height vertebral area trunco-cranial a 2.26 ± 3.4 a 26.0 ± 3.0 a 2132.4 ± 327.5 a b 25.3 ± 2.06 a 26.3 ± 2.2 a 2095.0 ± 220.3 a c 25.3 ± 2.7 a 25.3 ± 1.8 a 2024.2 ± 314.5 a d 27.07 ± 2.2 a 24.2 ± 1.9 a 2054.3 ± 157.3 a e 28.4 ± 1.2 a 27.3 ± 1.8 a 2438.3 ± 163.6 a f 25.3 ± 2.8 a 25.4 ± 2.0 a 2030.1 ± 352.1 a caudal a 28.0 ± 1.4a 24.1 ± 1.9 a 2118.0 ± 86.4 a b 27.3 ± 1.6 a 26.1 ± 1.6 a 2132.4 ± 327.5 a c 26.4 ± 2.5 a 26.1 ± 1.9 a 2174.2 ± 335.9 a d 26.5 ± 1.9 a 25.7 ± 1.5 a 2137.4 ± 189.2 a e 26.6 ± 1.3 a 26.3 ± 1.9 a 2201.5 ± 287.8 a f 27.4 ± 1.2 a 26.5 ± 1.0 a 2281.9 ± 190.6 a table 3. length, height and area(pixel) of 3 vertebrae in 2 regions identified in the vertebral column of the caspian roach sampled on day 60 and fed diets supplemented with different levels of available phosphorus ((0, 0.4, 0.8, 1.2 and 1.6%) and calcium (0 and 1%). 98 int. j. aquat. biol. (2013) 1(3): 93-99 were obtained with diet d containing 1.2% available p. it is well recognized that the need for p, to gain maximum bone strength and bone ash content, are higher than that to gain maximum wg (sauveur and perez, 1987). at the end of experiment, the mineral content of fish whole body, p and ca contents showed no significant different among p dietary groups. calcium supplement, also, had no significant effect on wg, fcr and carcass composition, including moisture, ash, phosphorus and calcium, which is in agreement with the generally accepted view that most fish can absorb ca directly from the aquatic environment or from food ingredients to meet their requirements (baeverfjord et al., 1998). the results of the present study showed that the dietary p deficiency induced skeletal deformities in the caspian roach larvae. in addition, sizes of vertebrae such as length, height and area of vertebrae were identified in two regions, i.e. the trunco-cranial and caudal regions showed no differences among treatments. however, some skeletal abnormalities such as twisted haemal and neural arches, kyphosis and irregular vertebral column were observed with dietary p deficiency that is in agreement with other studies (baeverfjord et al., 1998; vielma and lall, 1998; helland et al., 2005; uyan et al., 2007). visible external abnormalities are principally associated with the spine and differed in incidence between the fish fed with different diets (fontagné et al., 2009). in conclusion, the present study revealed that p deficiency affects bone calcification in the caspian roach larvae. thus, it is necessary to pay attention to the level and availability of p in the formulation of diets for normal vertebral development during the early development of caspian roach. references andrews j.w., murai t., campbell c. 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(2009). effects of dietary phosphrus and calcium level on growth and skeletal development in rainbow trout (oncorhynchus mykiss) fry. aquaculture, 297: 141150. helland s., refstie s., espmark a., hjelde k., baeverfjord g. (2005). mineral balance and bone formation in fast-growing atlantic salmon parr (salmo salar) in response to dissolved metabolic carbon dioxide and restricted dietary phosphorus supply. aquaculture, 250: 364-376. kiabi b.h., abdoli a., naderi m. (1999). status of the fish fauna in the south caspian basin of iran. zoology in middle east, 18: 57-65. koumoundouros g., divanach p., kentouri m. (2001). the effect of rearing conditions on development of saddleback syndrome and caudal fin deformities in dentex dentex (l.). aquaculture 200: 285-304 lall s.p. (2002). the minerals. in: halver, j.e., hardy, r.w. (eds.), fish nutrition, 3rd ed. academic press, san diego, ca, pp. 259-308. lall s.p., lewis-mccrea l.m. (2007). role of nutrients in skeletal metabolism and pathology in fish. aquaculture, 267: 3-19. nwanna l.c., adebayo i.a., omitoyin b. (2008). effect of different levels of phosphorus on growth and mineralization in african giant at fish heterobranchus bidorsalis (geoffrey saint hillarie, 1809). environmental management, 12: 25-32. 99 ahmadivand et al./ skeletal deformity in response of p and ca in the caspian roach roy p.k., lall s.p. (2003). dietary phosphorus requirement of juvenile haddock (melanogrammus aeglefinus l.). aquaculture, 221: 451-468. sadler j., pankhurst p.m., king h.r. (2001). high prevalence of skeletal deformity and reduced gill surface area in triploid atlantic salmon salmo salar l. aquaculture, 198: 369-386 sauveur b., perez j.m. 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(1998). phosphorus utilization by atlantic salmon (salmo salar) reared in freshwater is not influenced by higher dietary calcium intake. aquaculture, 160: 117-128. wallach s. (2002). disorders of skeleton and kidney stones. in: bedrdanier c.d. (ed.), handbook of nutrition and food. crc press, florida, usa, pp. 1275-1289. international journal of aquatic biology (2015) 3(4): 218-224 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved review article review of the freshwater sharks of iran (family carcharhinidae) brian w. coad*1 canadian museum of nature, ottawa, ontario, k1p 6p4 canada. article history: received 2 may 2015 accepted 27 july 2015 available online 2 5 august 2015 keywords: biology, morphology, carcharhinus. abstract: the systematics, morphology, distribution, biology, economic importance and conservation of the bull shark (carcharhinus leucas) in iran are described, the species is illustrated, and a bibliography on this fish in iran is provided. introduction the freshwater ichthyofauna of iran comprises a diverse set of families and species. these form important elements of the aquatic ecosystem and a number of species are of commercial or other significance. the literature on these fishes is widely scattered, both in time and place. summaries of the morphology and biology of these species were given in a website (www.briancoad.com) which is updated here for one family, while the relevant section of that website is now closed down. other families will also being addressed in a similar fashion. family carcharhinidae this family, the requiem or ground sharks, contains about 12 genera and about 58 species of large sharks found world-wide in tropical to warm-temperate waters (eschmeyer and fong, 2011). there is only one species found in iranian fresh waters. a second species is reported from an iranian river under special circumstances and is not regarded as a regular occurrence. this was carcharhinus dussumieri (müller and henle, 1839) found dead in the saline mehran river, hormozgan and kindly identified by l.j.v. compagno (see sources and below) (fig. 1). about 30 of these fish were found * corresponding author: brian w. coad e-mail address: bcoad@mus-nature.ca above shallow rapids and were probably trapped there, dying from an influx of fresher water, higher temperatures and/or low oxygen levels. this species is also recorded from the lower mand river at 28.1353ºn, 51.4309ºe, possibly under similar circumstances. requiem sharks are distinguished from other sharks by a complex of characters including having an anal fin, 5 gill slits, 2 dorsal fins, no fin spines, nictitating eyelids, and a scroll intestinal valve. the first dorsal fin base is in front of the pelvic bases, there is a wavy dorsal tail fin margin, well-developed, knife-like teeth with cutting edges, usually no spiracles, and precaudal pits. this is one of the largest and most economically important shark families. most members are voracious predators as their common name suggests (requiem = a mass said for the dead) and they are frequently dangerous to man. some of these species enter rivers and remain there for long periods causing human fatalities. these sharks are usually viviparous. food includes a variety of fishes, sharks, rays, squids, crustaceans, marine reptiles, birds and mammals, and carrion and garbage. shark flesh can be eaten and is religiously permissible in iran. 219 coad/freshwater sharks of iran genus carcharhinus blainville, 1816 there are about 31 species of gray sharks found world-wide but only one regularly enters fresh water in iran. a detailed definition of the genus is given by compagno (1988). carcharhinus leucas (müller & henle, 1839) (figs. 2-6) common names: kooseh, kuseh, kooseh-kuli, sag mahi (= dog fish). [kosetch or kossage, jarjur in arabic; bull shark]. systematics: carcharhinus leucas was originally described from the antilles. a number of shark species have been reported as entering rivers of the tigris-euphrates basin including iranian tributaries (günther, 1874; day, 1875-1878; sykes, 1902; kennedy, 1937; hunt, 1951; khalaf, 1961; mahdi, 1962; zorzi, 1995). the species appeared under such names as carcharhinus gangeticus (müller and henle, 1839), eulamia (= carcharhinus) lamia (blainville, 1820), and carcharhinus menisorrah (valenciennes in müller and henle, 1839). a revision of carcharhinid sharks by garrick (1982) cites only carcharhinus leucas from fresh waters of the tigris-euphrates basin and compagno (1984) concurs. coad and papahn (1988) and coad (2010) also list specimens and data which confirm this species to be present. key characters: this is the only shark species commonly encountered in iranian fresh waters and can be recognised by the 5 gill slits, upper caudal fin lobe larger than lower, and the arched mouth armed with teeth on the underside of the head. distinction from other sharks is given in compagno (1984). morphology: this is a heavily-built shark with a very short snout which is rounded and ends bluntly. snout length is less than the distance between the nostrils and much less than the mouth width. eyes are small. there are 12-14, usually 13, teeth on each side of a median tooth in the upper jaw and 12-13, usually 12, teeth on each side of a median tooth in the lower jaw. teeth are heavy, broad, almost triangular, erect near the jaw symphysis but becoming slightly oblique and more concave or notched nearer the mouth corners. the teeth are strongly serrated, more so near the base, and upper teeth more so than lower teeth. the first dorsal fin lies over or just behind the level of the axil of the pectoral fin. the apex of the first figure 1. carcharhinus dussumieri from the mehran river, hormozgan, 19 march 1978, cmnfi 1979-0410, brian w. coad. figure 2. line drawing of carcharhinus leucas by s. lauriebourque. figure 3. upper and lower tooth of carcharhinus leucas by s. laurie-bourque. figure 4. carcharhinus leucas, cochin, india, 69cm total length, courtesy of j. e. randall. 220 international journal of aquatic biology (2015) 3(4): 218-224 dorsal fin is pointed to somewhat rounded. the second dorsal fin is high, has a short posterior lobe and lies just over the level of the anal fin origin. pectoral fins are broad and their tips are narrow and pointed. there is no interdorsal ridge (the back is smooth between the dorsal fins). the upper precaudal pit is well-developed while the lower pit is weak. sexual dimorphism: males bear claspers. the pelvic fins are partially modified into grooved, rod-like structures which are held together to form a tube and are used in mating. they are not used to clasp the female but as an intromittent organ. females are larger than males. colour: the body is overall grey with a pale to white underside. fin tips are dusky to black, particularly in young, fading with age. there is no other obvious colour pattern although the back is darker than the belly, being bluish, grey or brown. fins are similar in colour to the neighbouring body. size: attains 3.24 m (garrick, 1982), 3.4 m (carpenter et al., 1997), rarely to 4.0 m and an estimated weight of over 600 kg (mccord and lamberth, 2009). fish in iranian fresh waters have been estimated up to 2.8 m in length but naturally circumstances were not always favourable for an objective and detached size judgment. distribution: this species is widespread in warmer marine waters world-wide but also entering rivers and lakes. sharks have long been known to enter fresh waters in the tigris-euphrates basin. zorzi (1995) records a book by pausanias, "guide to greece", written in the late second century a.d. which refers to sharks in "the euphrates...., which fatten monsters as man-eating as any in existence". one of the earliest distributional records is found in the arabic work "wonders of creation" by zakariya al-qazwini published in 1263 a.d. and later translated into persian. the sharks were found at basrah on the tigris river in what is now iraq and were cited as formidable because of their voracity and teeth like the points of spears. shark attacks still occur at basrah (coad and al-hassan, 1989). subsequently reported in the tigris river above figure 5. carcharhinus leucas, bm(nh)1924.10.1:1, tigris river at al karradah near baghdad, brian w. coad. figure 6. carcharhinus leucas, bm(nh) 1874.4.28:9, tigris river near baghdad, brian w. coad. 221 coad/freshwater sharks of iran baghdad about 850 km from the sea (günther, 1874; kennedy, 1937) before dams were built. a recent record for iraq is documented by hussain et al. (2012a, 20102b). sykes (1902) saw sharks in the ab-e gargar (karun river in iran) at shushtar 420 km from the sea, wilson (1942) reporting on events in 1908 records sharks from between shushtar and ahvaz and near shushtar, blegvad and løppenthin (1944) report them from khorramshahr, and hunt (1951) reported them from the karun river, khowr-e bahmanshir and shatt al arab (arvand river). coad and papahn (1988) report sharks at ahvaz on the karun river about 275 km from the sea as well as further up river at shushtar and down river in the khowr-e bahmanshir. zoogeography: this shark is found world-wide in warm temperate to tropical seas and is reported from fresh waters in africa, asia, australia and the americas. habitat: this is a shark of coastal waters such as harbours, bays and estuaries but unusually it will penetrate far up rivers, as far as 4000 km up the amazon river. it is said to be a sluggish bottom dweller except when attacking prey and in the sea may be found down to at least 150 m although usually at less than 30 m. it can cover great distances, up to 180 km in 24 hours. movements between fresh, brackish and marine waters are common and random. they are said to invade the khowr-e bahmanshir and karun river of iran from july to september when freshwater flow is at a minimum and tidal penetration of salt water is at its highest. however, they do travel well beyond tidal influence in iran. local people along the bahmanshir river near tangeh-ye seh (fig. 7) in khuzestan maintain that it is dangerous to swim there because of these sharks. they are occasionally trapped in nets set for the clupeid tenualosa ilisha and may be caught on hooks. they are not as common as in the past (n. najafpour, pers. comm. november 2000). mohamed et al. (2015) record them from the shatt al-arab near the iranian border in june and july. age and growth: maturity in males is attained at 1.60-2.25 m and in females at 1.80-2.30 m. mature fish are about 6 years old and life span is up to 32 years. food: food is a wide variety of fishes including tunas, small sharks, and rays, as well as crabs, shrimps, molluscs, cephalopods, sea snails, sea urchins, turtles, sea birds and mammals, and even garbage. diet in fresh water has not been investigated in southwest asia although blegvad and løppenthin (1944) reported that sharks station themselves under the date palms at khorramshahr to eat the falling dates! reproduction: birth size is about 56-81 cm and takes place in estuaries and river mouths. females may contain up to 13 embryos and the gestation period is 10-11 months. this species is known to breed in figure 7. habitat of carcharhinus leucas, khowr-e bahmanshir at tangeh-ye seh, 23 november 2000, brian w. coad. 222 international journal of aquatic biology (2015) 3(4): 218-224 fresh waters, such as lake nicaragua in central america, but there have been no reports of reproduction in the tigris-euphrates basin. parasites and predators: none are reported for iran. economic importance: this shark has a considerable impact on people using water directly in khuzestan. a number of severe injuries and fatalities have been reported in fresh waters through shark attacks. the first comprehensive report in modern times was by hunt (1951) although accounts date back to the thirteenth century (coad and papahn, 1988). the latter summarize recorded attacks and add new ones for a total of 34 in the period 1941-1985, of which about half were fatal. additionally, wilson (1942) reports a woman taken by a shark while drawing water between shushtar and ahvaz and a 2.8 m one near shushtar which killed two boys and a girl. these iranian records are a significant proportion of freshwater attacks worldwide, about 28%. a number of soldiers were apparently victims during the iraniraq war but no records have come to light. men, women and children are attacked as well as horses and sheep, only the massive water buffalo is said to be safe. many minor attacks and narrow misses are probably not reported. attacks are said to have declined in recent years since shark oil is no longer used to caulk boats but this is probably a local legend. people were attacked while swimming, paddling, bathing, washing vehicles or fishing. there were no apparent triggering factors for the attacks as victims were dressed in various colours and types of clothing, engaged in various activities and environmental conditions where known varied between attack sites. shark attacks at basrah, iraq are summarised in coad and al-hassan (1989). freshwater shark attacks have even appeared in a novel set in persia, "harem", by mossanen (2002). specimens from a fish shop in ahvaz have been investigated for gelatin and collagen yield, for their use in food and pharamaceutical industries (aberoumand, 2010, 2011). in other parts of the world, this species has been used for its flesh and fins, as leather, for its liver oil and for fishmeal. conservation : this shark appears to still be common in iranian fresh waters judging from the attacks reported over the past 50 years or more and no conservation measures are needed (or likely to be acceptable to the local population). sources: garrick (1982) and compagno (1984, 1988) for general anatomy and biology. further details on collections examined can be found in the museum catalogues. comparative material: bm(nh) 1874.4.28:9, 1, ca. 76.8 cm total length, iraq, tigris river near baghdad (ca. 33º21'n, ca. 44º25'e); bm(nh) 1924.10.1:1 1, (head only, recorded length 1.25 m), tigris river at al karradah near baghdad (33º17'n, 44º23'e). carcharhinus dussumieri: cmnfi 1979-0410, 2 (kept of ca. 30), 449-461 mm total length, hormozgan, mehran river (26º53’n, 55º17’e) acknowledgements i am indebted to the department of biology, shiraz university and the canadian museum of nature, ottawa for funding of research. numerous colleagues and co-authors assisted in developing the website on iranian fishes, providing specimens, data and photographs and are listed at www.briancoad.com. references aberoumand a. (2010). edible gelatin from some fishes skins as affected by chemical treatments. world journal of fish and marine sciences, 2(1): 59-61. aberoumand a. (2011). isolation of collagen from some fish skins in iran. journal of agricultural technology, 7(3): 783-788. blegvad h., løppenthin b. (1944). fishes of the iranian gulf. einar munksgaard, copenhagen. 247 p. (1999 translation into farsi by e. etemad and b. mokayyer with supplement, tehran university publications no. 1744: 26 + 416 pp.). carpenter k.e., krupp f., jones d.a., zajonz u. (1997). living marine resources of kuwait, eastern saudi arabia, bahrain, qatar, and the united arab emirates. fao species identification field guide for fishery purposes, food and agriculture organization, rome. 293 p. 223 coad/freshwater sharks of iran coad b.w. (2010). freshwater fishes of iraq. pensoft publishers, sofia-moscow. 294 p. coad b.w., al-hassan, l.a.j. (1989). freshwater shark attacks at basrah, iraq. zoology in the middle east, 3: 49-53. coad b.w., papahn f. (1988). shark attacks in the rivers of southern iran. environmental biology of fishes, 23(1-2): 131-134. compagno l.j.v. (1984). fao species catalogue. volume 4. sharks of the world. an annotated and illustrated catalogue of shark species known to date. part 1 hexanchiformes to lamniformes, part 2 carcharhiniformes. food and agriculture organization, rome, fisheries synopsis, 125(4), part 1: 1-249; part 2: 251-655. compagno l.j.v. (1988). sharks of the order carcharhiniformes. princeton university press, princeton, new jersey. 486 p. day f. (1875-1878). the fishes of india; being a natural history of the fishes known to inhabit the seas and fresh waters of india, burma and ceylon. vol. 1, london. 778 p. eschmeyer w.n., fong j.d. (2011). pisces. in: z.-q. zhang (ed.). animal biodiversity: an outline of higher level classification and survey of taxonomic richness. zootaxa, 3148: 26-38. garrick j.a.f. (1982). sharks of the genus carcharhinus. national oceanic and atmospheric administration technical report, national marine fisheries service circular 445. 194 p. günther a. (1874). a contribution to the fauna of the river tigris. the annals and magazine of natural history, 4(14): 36-38. hunt r.s. (1951). the sharks of ahwaz. journal of the royal army medical corps, 97(1): 79-85. hussain n.a., rasen a.k., al-kafiji b.y., coad b.w. (2012a). bull shark occurrence carcharhinus leucas (valenciennes, 1839) at the inland waters of southern iraq. the 1st scientific agricultural conference, faculty of agriculture and forestry university of duhok, april 10-12th 2012, duhok kurdistan region iraq, p. 25. hussain n.a., rasen a.k., al-kafiji, b.y., coad, b.w. (2012b). bull shark occurrence carcharhinus leucas (valenciennes, 1839) at the inland waters of southern iraq. journal of the university of duhok iraq, 15(special issue 1): 140-143. kennedy w.p. (1937). some additions to the fauna of iraq. journal of the bombay natural history society, 39: 745-749. khalaf k.t. (1961). the marine and freshwater fishes of iraq. ar-rabitta press, baghdad. 164 p. mahdi n. (1962). fishes of iraq. ministry of education, baghdad. 82 p. mccord m.e., lamberth s.j. (2009). catching and tracking the world's largest zambezi (bull) shark carcharhinus leucas in the breede estuary, south africa: the first 43 hours. african journal of marine science, 31(1): 107-111. mohamed a-r.m., hussein s.a., lazem l.f. (2015). spatiotemporal variability of fish assemblage in the shatt al-arab river, iraq. journal of coastal life medicine, 3(1): 27-34. mossanen d.l. (2002). harem. scribner paperback fiction, new york. 378 p. sykes p.m. (1902). ten thousand miles in persia; or, eight years in iran. john murray, london. 481 p. wilson a. (1942). s.w. persia. letters and diary of a young political officer 1907-1914. readers union limited and oxford university press, london. 305 p. zorzi g.d. (1995). the biology of freshwater elasmobranchs: an historical perspective. journal of aquaculture and aquatic sciences, 7: 10-31. international journal of aquatic biology (2015) 3(4): 218-224 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved چکیده فارسی (carcharhinidaeماهیان آب شیرین ایران )خانواده مروری بر کوسه برایان کد نادا.، کاk1p 6p4انتاریو، ، موزه تاریخ طبیعی کانادا، اتاوا چکیده: شده ( در ایران توصیف carcharhinus leucasکولی )شناسی، اهمیت اقتصادی و حفاظت کوسهزیستشناسی، پراکنش، سیستماتیک، ریخت گردد.در مورد این ماهی در ایران ارائه میمنابع فهرستشود. همچنین یک و به تصویر کشیده می .carcharhinusشناسی، شناسی، ریختزیست: کلمات کلیدی int. j. aquat. biol. (2013) 1(2): 68-75 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology relationships between nutrients and chlorophyll-a concentration in the international alma gol wetland, iran saeed balali*1, seyed abbas hoseini1, rasool ghorbani1, hamideh kordi2, elahe amooee khozani1 1fisheries department, gorgan university of agricultural sciences and natural resources, iran. 2ph. d. student, inland water aquaculture institute, bandar anzali, iran. article history: received 15 april 2013 accepted 1 may 2013 available online 5 may 2013 keywords: nutrients chlorophyll-a alma gol wetland abstract: this study investigated the relationships between nutrients and chlorophyll-a concentration in the international alma gol wetland. chlorophyll-a is the major photosynthetic pigment in lots of phytoplanktons and has been used as a trophy index in aquatic ecosystems. water samples were collected fortnightly from five stations in the wetland during summer and autumn. chlorophyll-a ranged between 4.38 to 156.55 mg/m3, sulfate ranged between 138 to 190 mg/l, total alkalinity ranged between 80 to 280 mg/l, silica ranged between 3.80 to 35.00 mg/l, phosphate ranged between 0.02 to 3.70 mg/l, ammonia ranged between 0.10 to 11.90 mg/l, nitrate ranged between 0.01 to 2.75 mg/l and nitrite ranged between 0.01 to 0.39 mg/l. there was a significant correlation between chlorophyll a and nitrate, nitrite and ammonia but there was no significant correlation between chlorophyll-a and silica, total alkalinity, sulfate and phosphorus. introduction wetlands are providing numerous useful services for people, fish and wildlife (environmental protection agency, 2001). they are one of the most important ecosystems on the earth and is considered unique because of their hydrology and their function as ecotones between terrestrial and aquatic ecosystems (mitsch and gosselink, 2007). also, wetlands are biodiversity shelters (junk et al., 2006). chlorophyll-a is the major photosynthetic pigment of phytoplanktons and a trophy index in aquatic ecosystems (vollenweider, 1969; dillon, 1975). chlorophyll-a (chl-a) is often used as an estimate of algal biomass, and in blooms concentration chl-a goes above 40 μgl-1 (stanley et al., 2003). so a method for the estimation of the growth and development of the phytoplankton community is the analysis of photosynthetic pigments, although the chlorophyll content in the cells changes with the * corresponding author: saeed balali e-mail address: saeed_balali85@yahoo.com tel: +989112777053 availability of light (wetzel, 2001), depth and trophic status (kasprzak et al., 2008). eutrophication is defined as the response of aquatic ecosystems to nutrient loading (edmondson, 1991). hence, identification of important factors and prediction of subsequent algal blooms using a chl-a equation can be a key in the management of lakes. both chemical and physical controls can be used to prevent or remove algae or algal byproducts from water (stanley et al., 2003). in particular, information about the form of nutrient-chlorophyll relationships has allowed lake managers to establish nutrient concentration (e.g. dillon and rigler, 1974; prairie et al., 1989). nitrogen and phosphorus are often identified as limiting nutrients for algal biomass and silicon is necessary for diatom growth (hecky and kilham, 1988). nitrogen occurs in fresh water in numerous forms: dissolved nitrogen, amino 69 balali et al./ int. j. aquat. biol. (2013) 1(2): 68-75 acids, amines, urea, ammonium (nh4+), nitrite (no2-), and nitrate (no3-) (wetzel, 2001). in aquatic ecosystems, phosphorus (p) can be found either in particulate form or soluble inorganic phosphorus, orthophosphate (po43-) (knud-hansen, 1997). a review of the biological abstract published from 1995 to 1997, indicates that, of 596 articles on estuaries and nutrients, 52% consider only nitrogen, 32% refer to both nitrogen and phosphorus, and 16% consider only phosphorus as significant factor for algal bloom, despite the preponderance of studies on n, the evidence for general n limitation of coastal systems is feeble compared to the data for general p limitation of freshwater systems (meeuwig et al., 1998). there is a small number of comprehensive analyses on the form of phosphorus-chlorophyll relationships. the phosphorus-chlorophyll relationship most probably originates from the dependence of algae on phosphorus availability (mccauley et al., 1989). nitrogen limitation of algal biomass seems to be usually prevalent in subtropical and tropical lakes (henry et al., 1985; davalos et al., 1989; hecky et al., 1993), while phosphorus appears to be the primary limiting nutrient in temperate lakes (reviewed by smith, 1990). other nutrients, for example iron and silicate, have been reported to be limiting in other regions (johnson et al., 1999; yolanda et al., 1997). the limiting nutrient is determined mainly by the mass equilibrium between elements such as c, n, p, and si, and their relationship to the growth requirements of the phytoplankton (wu and chou, 2003). this research was conducted to determine the relationships between chl-a and nutrients concentrations in the alma gol wetland to identify the important and effective nutrients on chlorophyll a concentration. materials and methods study area: this study was conducted on alma gol international wetland, which is situated on the turkmen steppes near the border with turkmenistan in the golestan province, in north of iran (fig.1). area of the wetland was 207 ha. field sampling: from summer to autumn of 2011, water samples were collected fortnightly from five sampling stations to determine nutrients and chl-a concentration. water samples were light-protected and transferred to laboratory at 4 ºc. chlorophyll a determination: to measure chl-a concentration, samples were shaken and a certain volume of water (based on water color) was filtered using a vacuum pump and gf/f filter. thereafter, filter was pulverized with 90% acetone in a mortar. the resulting mixture was centrifuged for 10 min. (3000 rpm) and supernatant was poured into a glass cuvette. the optical density was read at 630, 647, 664 and 750 nm. chlorophyll-a concentrations were calculated according to jeffrey and humphrey (jeffrey and humphrey, 1975). nutrient determination: ammonia, nitrite, nitrate, silica, sulfate, total alkalinity and dissolved phosphorus were measured using especial tablets and a photometer set (wagtech, berkshire, uk) with specific recipe. statistical analyses: data were analyzed by statistical software spss v. 18 and microsoft office excel 2007. data were examined using pearson correlation test to find significant relationship between chl-a and nutrient concentration. figure 1. location of alma gol wetland and situation of sampling stations. 69 70 balali et al./ int. j. aquat. biol. (2013) 1(2): 68-75 figure 2. log chl-a:no2, log chl-a:no3, log chl-a:nh3, log chl-a:so4, log chla:sio2, log chl-a:po4 and log chl-a:caco3 relations for alma gol wetland. chl -a log chl-a no2 no3 nh3 po4 sio2 caco3 so4 chl-a 1 log chl-a 0.889** 1 no2 -0.486 ** -0.654** 1 no3 -0.489 ** -0.701** 0.895** 1 nh3 -0.444 * -0.457* 0.482** 0.535** 1 po4 -0.038 0.005 -0.005 -0.120 -0.013 1 sio2 0.061 0.124 0.186 0.092 -0.043 -0.160 1 caco3 0.043 0.158 -0.220 -0.365 * -0.117 0.249 -0.312 1 so4 0.055 -0.105 0.227 0.283 0.027 -0.373* 0.159 -0.763** 1 ** correlation is significant at the 0.01 level (2-tailed) and * correlation is significant at the 0.05 level (2-tailed). table 1. nutrients and chl-a relationship in almagol wetland. 71 balali et al./ int. j. aquat. biol. (2013) 1(2): 68-75 results results illustrated that there was negative and significant relationships between chl-a and logarithm of chl-a with nitrate, nitrite (p<0.01) and ammonia (p<0.05) but there was no significant correlation between chl-a and logarithm of chl-a with silica, total alkalinity, sulfate and resolve phosphorus (p>0.05) (table 1). the average of various factors and significant differences between months are presented in table 2. regression lines for log chl-a:no2, log chla:no3, log chl-a:nh3, log chl-a:so4, log chla:sio2, log chl-a:po4 and log chl-a:caco3 relations were shown in figure 2 and also presented at below. y=-0.003x+2.118, r2=0.012, r=-0.105, log chla:so4 y=-1.888x+1.758, r2=0.161, r=-0.654, log chla:no2 y=0.004x+1.482, r2=0.002, r=0.124, log chla:sio2 y=-0.289x+1.706, r2=0.261, r=-0.701, log chla:no3 y=-0.046x+1.688, r2=0.110, r=-0.457, log chla:nh3 y=0.017x+1.519, r2=0.000, r=0.005, log chla:po4 y=0.001x+1.392, r2=0.014, r=0.158, log chla:caco3 discussion a significant correlation was found between chl-a and nitrite, nitrate and ammonia. when there was a high concentration of chl-a, a low concentration of nitrite, nitrate and ammonia were found. chl-a was not affected by silica, total alkalinity, sulfate and dissolved phosphorus. power relationships between phosphorus, chlorophyll, and water clarity have been observed for freshwater systems in the world (e.g., sakamoto 1966; brown et al., 2000). the strong relationship between chlorophyll and phosphorus established by sakamoto (1966) in numerous japanese lakes forms an appropriate testable hypothesis: chlorophyll is both a useful and an easy estimator of phytoplankton standing crop and is now more generally used than cell number or cell volume. smith (1982) achieved the most comprehensive analysis of phosphorus-chlorophyll relationships to date, and was the first to illustrate that information of both total phosphorus (tp) and total n (tn) concentrations could improve predictions of algal biomass. a hypothesis that fits more closely with the classic liebigian paradigm (von liebig, 1840; hutchinson, 1973; droop, 1974; tilman, 1982) would consider other nutrients that might limit algal growth at high levels of ambient phosphorus. it would predict that chlorophyll should rise linearly until some other factor (such as nitrogen, silicon, molybdenum and light) becomes limiting. despite the fact that nonlinear relationships between tp and chlorophyll are possible in lakes (forsberg and ryding 1980; canfield, 1983), there is few quantitative examinations on their shape or form. hoyer et al. (2002) suggested that phosphorus accounts for more variance in chlorophyll than that variables august september october november december no2 0.14±0.1 ab 0.18±0.1a 0.08±0.02b 0.11±0.03ab 0.06±0.02b no3 0.60±0.58 b 1.34±0.95a 0.18±0.03b 0.47±0.32b 0.33±0.08b nh3 4.72±3.50 a 4.78±3.88a 1.90±0.69a 5.00±2.32a 3.56±0.92a po4 0.50±1.13 a 0.14±0.06a 0.12±0.09a 0.11±0.07a 0.11±0.08a sio2 10.15±6.28 a 9.67±1.94a 9.36±1.24a 12.27±8.18a 7.16±1.51a caco3 153.00±67.59 a 88.50±5.80b 94.00±6.52b 108.50±13.75b 113.00±10.37b so4 160.30±16.53 b 184.00±3.16a 185.00±6.12a 176.50±3.37a 177.00±2.74a chl a 52.46±20.79b 23.03±14.59cd 136.83±15.80a 42.08±21.67bc 11.03±3.82d table 2. mean ± sd of nutrients during sampling in almagol wetland. different letters show significant difference between columns (p <0.05). 71 72 balali et al./ int. j. aquat. biol. (2013) 1(2): 68-75 of nitrogen in inshore coastal waters and fresh water of florida. while nitrogen had been shown to limit algal populations in both systems (elser et al., 1990; downing, 1997), the data presented suggested that phosphorus was the primary nutrient limiting algal populations amongst the 300 nearshore coastal locations sampled. they recognized, though, that some of those 300 stations may at some times be limited by nitrogen. canfield (1983) and brown et al. (2000) have formerly affirmed that phosphorus is the primary limiting nutrient in florida lakes, and when the total nitrogen to total phosphorus ratio decreased to < 10, nitrogen may become limiting. it is normally assumed, that nitrogen is the primary limiting nutrient for phytoplankton production in most coastal waters (downing, 1997). brown et al. (2000) explained that tp accounted for a significant amount of the variance (r2=0.76) of observed chlorophyll measurements and tn accounted for lower variance related to observed chlorophyll measurements (r2=0.46), but a multivariate model using both tp and tn also accounted for a significant amount of the observed variance (r2=0.78). the coefficient of determination values for tp-chlorophyll and the multivariate nutrient-chlorophyll, were similar (r2 =0.76 against 0.78), suggesting that chlorophyll concentrations can be predicted rationally well using tp alone. the coefficient of correlation for the relationship between tp and chlorophyll for both equations was positive and significant. redfield (1958), expressed that phosphorus (p) has been considered a key limiting nutrient in marine systems. furthermore, p controls phytoplankton biomass in numerous freshwater systems and similarities in phytoplankton physiology and nutrient requirements in coastal and freshwater systems (hecky and kilham, 1988). nevertheless, following ryther and dunstan's (1971) work, nitrogen is generally the limiting nutrient in coastal systems and has received the bulk of research interest. meeuwig et al. (1998) illustrated that the relation between chlorophyll and tn is marginally stronger than that between chlorophyll and tp suggesting that tn, rather than tp, limits estuarine chlorophyll. the average tn:tp ratio of 4.5 also supports the argument for tn as the key limiting nutrient in estuaries in prince edward island in canada (meeuwig et al., 1998). however, the relative strength of these patterns and, the tn:tp ratio cannot be used to conclude which nutrient is limiting phytoplankton biomass, the low yield of chlorophyll per unit of nutrient points to the importance of other factors such as herbivory and turbidity, and potentially to indirect control by iron, in determining phytoplankton biomass. canfield (1983) developed a chl-a equation using samples from 223 florida lakes, 27% of which were considered n-limited. because of the long growing season in florida, these samples were taken during august 1979 to september 1980. chl-a showed significant correlations with both tp (r=0.79, p<0.01) and tn (r=0.87, p<0.01). it has been shown that there is often a strong correlation between tp and algal biomass (sakamoto 1966; dillion and rigler 1974; jones and bachmann 1976; carlson, 1977). this suggests that p may be the element controlling algal growth. though, lakes surrounded by rich phosphate deposits and p-containing soils may be n-limited. wu and chou (2003) indicated that both the concentration of chlorophyll a and phytoplankton biomass have shorter euclidean distances to silicate, nitrate, biochemical oxygen demand, and temperature, than to phosphate, nitrite, ammonium, or physical factors such as conductivity, ph, and dissolved oxygen, suggesting that phytoplankton are associated with silicate, nitrate, biochemical oxygen demand and temperature. these results supported the hypothesis that nutrients such as silicate and nitrate play a more important role in regulating phytoplankton in subtropical eutrophic estuary of taiwan than do other factors. in conclusion, results illustrated that there was a negative and significant relationships between chl-a and logarithm chl-a with nitrate, nitrite and ammonia but there was no significant correlation between chla and logarithm chl-a with silica, total alkalinity, 73 balali et al./ int. j. aquat. biol. (2013) 1(2): 68-75 sulfate and dissolved phosphorus in this research. some research supported the result of this study and some of them were against. although we could not find any relationship between chl-a and p, it can be, because of measuring dissolved phosphorus instead of total phosphorus. acknowledgements thanks to a. jafar node, critic of shahid naser fazli barabadi aquaculture research station for helpful and constructive comments. this research was supported from grants from the department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources. references brown c.d., hoyer m.v., bachmann r.w., canfield d.e. (2000). nutrientflorida and northern temperate lake data. canadian journal of fisheries and aquatic sciences, 57: 1574-1583. canfield d.e. (1983). prediction of chlorophyll a concentrations in florida lakes: the importance of phosphorus and nitrogen. water resources bulletin, 19: 255-262. carlson r.e. 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(2016) 4(2): 96-101; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article the study of enrichment capability of adult artemia franciscana with singular or combined administration of pediococcus acidilactici and fructooligosaccharide mahmood azimirad*1, saeid meshkini2, nasrollah ahmadifard1, seyed hossein hoseinifar3 1fisheries department, faculty of natural resources, urmia university, iran. 2department of food hygiene and quality control, faculty of veterinary medicine, urmia university, iran. 3department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 9 february 2016 accepted 12 april 2016 available online 2 5 april 2016 keywords: probiotic synbiotic p. acidilactici enrichment artemia franciscana abstract: the present study investigates the possibility of enriching adult artemia franciscana with singular or combined administration of pediococcus acidilactici and fructooligosaccharide (fos). the experiment was conducted in a completely randomized design with four treatments, including synbiotic, p. acidilactici +fos (t1), probiotic, p. acidilactici (t2), prebiotic, fos (t3) and control (t4). to evaluate the enrichment of adult artemia with each treatment, sampling was performed at 2, 4 and 6 hrs post enrichment. the bacterial counts was measured using the microbial culture and expressed as log cfu per g artemia. a pre-experiment has been designed and probiotic was used in three levels (107, 108 and 109 cfu per litter of suspension) and prebiotic was used in three levels of 1, 2 and 5 g per litter of suspension. based on pre experiment results, 108 cfu per litter of probiotic and 5 g per litter of prebiotic was selected. the results of this experiment showed that over time, consumed bacteria increased by adult artemia and there was a significant difference between sampling in terms of ingested bacteria. the highest bacterial count (6.78±0.03 log cfu g-1) was observed 6 hrs after the start of enrichment. based on microbial culture, the number of bacteria p. acidilactici in t1 and t2 was significantly higher than those in t4 (control) and t3 (prebiotic). there was no significance difference between t2 (probiotic) and t1 (synbiotic). in conclusion, the results of this study showed that adult artemia in a short time (about 4 hrs) can retain a large amount of probiotic bacteria. introduction during the past decade, the use of probiotics in aquaculture is become prevalent and can overcome many of the problems associated with bacterial diseases. the use of probiotics as a food supplement for farmed animals goes back to the 1970s (fuller, 1989). various types of microalgae (tetraselmis), yeasts (phaffia and saccharomyces), gram-positive bacteria (bacillus, carnobacterium, enterococcus, lactobacillus, lactococcus, micrococcus, streptococcus and weissella) and gram-negative bacteria (aeromonas, alteromonas, photorhodobacterium, pseudomonas and vibrio) have been studied as probiotics (gatesoupe et al., 2010). the doubts in the use of probiotics such as the non-guaranteed viability * corresponding author: mahmood azimirad e-mail address: mahmoodazimirad@gmail.com of the probiotics in the gastrointestinal tract, necessity of competition autochthonous microbiota, the colonization ability and the long-term sustainability of the colony, caused the researchers to suggest the idea of prebiotic (gibson, 2004; mahious and ollevier, 2005). the prebiotics increase numbers and dominance of beneficial bacteria due to selectively fermentation (roberfroid, 2007). researches in this field have shown that non-digestible oligosaccharides such as inulin and oligofructose are the most important materials that have prebiotic function (flickinger et al., 2003). because of the inability of probiotic species to form stable masses and maintain dominance in the aquatic microbiota, simultaneous 97 int. j. aquat. biol. (2016) 4(2): 96-101 use of probiotics species with appropriate prebiotics (synbiotic) as a substrate to increase dominance and sustainable growth of probiotics bacteria has been suggested (hoseinifar et al., 2015). regarding the use of synbiotics in aquaculture, few studies have been performed and their positive effects on physiology and immunity have been reported (rodriguez-estrada et al., 2009; merrifield et al., 2010; montajami et al., 2012; abid et al., 2013; hosseinifar et al., 2015). however, the use of synbiotics during the early life stages of fish through the enrichment of live food and the effects on growth, physiology and immunity has not been considered. the use of synbiotic in artemia could be considered as a food for artemia, and also could affect the intestinal microbiota, immune system and increase resistance to pathogenic bacteria, enhance health and reduce the risk of disease outbreaks. artemia is among the live foods that widely used in the culture of ornamental fishes due to the high nutritional value, the proper size and the enrichment capability (sorgeloos et al., 2001). artemia can be used as the carrier of particles used in aquaculture such as nutrients (fatty acids, vitamins, etc.), antimicrobial substances, vaccines and probiotics (ziaei-nejad et al., 2006) application of live, useful and non-pathogenic bacteria to culture medium or artemia culture can positively affects cultured fish species by improving the intestinal microbial microbiota, eliminating harmful bacteria and improving the nutritional value of artemia (havennar et al., 1992; ringo et al., 1992). the number of bacteria in the artemia exponentially increases at the time of artemia hatching and enrichment processes by nutrients (ritar et al., 2004). it also has been observed that during the early stages of fish development, the increase in the number of bacteria in the intestinal microbiota of fish, is mainly associated with the bacteria in live food (makridis et al., 2000). it can be concluded that mortality increases in the intensive culture of early life stages of fish along with elevation of the number of opportunistic bacteria in the fish intestine. therefore, control of bacterial population in the live feed may lead to higher survival rates of fish larvae and profitability in hatcheries (olafsen, 2001). therefore, this study was conducted to study enrichment capability of adult artemia franciscana with singular or combined administration of pediococcus acidilactici and fructooligosaccharide (fos) as probiotic and prebiotic, respectively. materials and methods artemia culture conditions and bacterial strain: artemia cysts (a. franciscana) was obtained from great salt company, usa. chorionic layer of cysts were separated using sodium hypochlorite during decapsulation. hatching of the decapsulated cysts was performed by a cone-shaped container with a volume of 120 litters and sea water (with salinity of 30 g per litter). cysts were incubated with a density of 5 g per litter at 30°c with 2000 lux lighting conditions and vigorous aeration (sorgeloos et al., 1986). artemia naupliis were transferred to culture environment after hatching. the culture environment was a 150l cone-shaped plastic containers that were aerated by aeration pipes connected to the central pump. nauplii were fed during the first few days by spirulina algae (spirulina platensis), and then by a mixture of rice bran, baker's yeast and spirulina. feeding was performed three times a day with an interval of 4 hrs. stocking density was three nauplii per ml and culture period was 20 days to reach sexual maturity (teresita et al., 2005). during culture period, all physical and chemical parameters were measured and recorded daily. physical and chemical factors, including water temperature, salinity, dissolved oxygen, light and ph were 28.69°c, 32 g l-1, 7.75 mg l-1, 1500 lux and 7.88, respectively. the used commercial probiotic used in this experiment was obtained from tak gene company with pedi-guard brand name contains bacteria p. acidilactici to amount of 1×1010 cfu g-1. prebiotic, oligofructose (raftilose p95) was supplied from orafti company, belgium. enrichment of synbiotic to adult artemia: for 98 azimirad et al./ enrichment capability of artemia with of p. acidilactici and fructooligosaccharide enrichment of the adult artemia by synbiotic, combinations of the probiotics and prebiotics were used along with singular administration of the probiotic and prebiotic as described in table 1. for preparation of the synbiotic suspension, first a ratio of 0.1:10 lecithin and water at 40°c were poured into a clean and dry beaker and mixed using an electric mixer. then, the rapeseed oil was added to the solution and mixed very well by mixer. the ratio of lecithin, oil and water in suspension was 0.1, 1 and 10, respectively. to evaluate the diameter of oil particle, some samples were poured on slide and observed under light microscope. 150 ml was separated from the prepared suspension, 700 mg probiotic, p. acidilactici and 100 mg of prebiotic, fos were transferred to the beaker and were uniformed with an electric mixer, then mixed in 2 litters of seawater. the adult artemia with the number of 4000 was placed inside the culture container (agh and sorgeloos, 2005; daniels et al., 2013) (table 1). to determine the best level of the probiotic, p. acidilactici and prebiotic fos in enrichment suspension of artemia, a pre-experiment has been designed and probiotic was used in two levels with an amount of 107 cfu and 108 cfu per litter of suspension, and prebiotic was used in two levels of 2 and 5 g per litter of suspension. the results of this pre-experiment has been used as level of probiotic, prebiotic and synbiotic in this experiment. to examine the process of enrichment, sampling was performed from the all treatment at 2, 4 and 6 hrs (dhont and lavens, 1996). in each sampling time, 100 ml (containing 0.5 g of adult artemia) were collected using a sterile pipette and were transferred to a filter with a mesh size of 300 micrometer, then to elimination of the bacteria in the external surface of artemia body, were washed for 60 seconds in a salt solution, benzalkolium chloride (0.1%) and again were washed with sterile water and after that, water of samples was taken after a while (makridis et al., 2000). the sterile samples were weighted and transferred to sterile porcelain mortar. after the homogenization of samples using a sterile saline solution (0.87% w/v), dilutions of 10-1 to 10-7 were prepared. from prepared dilutions, under sterile conditions, 0.1 mm was removed and spread on surface of the mrs agar plates (for determine the number of lactic acid bacteria). the incubation of plates was conducted for 3-5 days in an incubator at a temperature of 30°c and under aerobic conditions. after the incubation period, the bacteria were counted, and recorded according to the logarithm of the colony unit (the number of bacterial colonies grown on culture medium × dilution coefficient -1) per g of artemia (rengpipat et al., 1998). pediococcus acidilactici was identified based on apparent characteristics, gram staining and also standard biochemical tests such as phenol red, citrate, indole, motion and methyl red (peter and sneath, 1986). statistical analysis: statistical analysis was performed using the spss software package (version 18). one-way anova was used for comparison between treatments and duncan's multiple range test was used for the comparison of means at confidence level of 0.05% (p<0.05). results the effects of different treatments and sampling time on the amount of bacteria in the artemia is shown in table 2. the results indicated that probiotic bacteria in each sampling time, were successfully enriched in artemia. the enrichment trend of a. franciscana was treatments rapeseed oil suspension (ml l-1) probiotic, p. acidilactici (mg l-1) prebiotic, fos (mg l-1) synbiotic (t1) 150 700 100 probiotic (t2) 150 700 0 prebiotic (t3) 150 0 100 control (t4) 150 0 0 table 1. the enrichment condition adult artemia in different treatments. 99 int. j. aquat. biol. (2016) 4(2): 96-101 different at different sampling times. in terms of the enrichment time, the results showed significant difference in the capability of artemia enrichment (p<0.05). regarding the synbiotic and probiotic treatments at 4 and 6 hrs enrichment, there was no significant difference in the number bacteria per g of artemia (p>0.05). the results of bacterial count in prebiotic and control treatments showed that the concentration of lactic acid bacteria in these treatments were lower than 20 cfu g-1 and no significant difference were observed between sampling times (p>0.05). the bacterial counts in treatments enriched by probiotic and synbiotic were almost at the same level, but increasing trend was observed along with enrichment period (fig. 1). however, no statistically significant differences was observed between bacterial level in adult artemia at 4 and 6 hrs of enrichment (p>0.05). discussion in this experiment, bacterial levels used in the enrichment solutions at all sampling times were at a level of 1010 cfu g-1. gomez-gil et al. (1998) were applied the concentrations of 107 cfu g-1 and 108 cfu g-1 of vibrio parahaemolyticus and v. alginolyticus, respectively, during enrichment experiment of a. franciscana and reported the same pattern in their changes at different sampling times. similar study were not observed regarding to enrichment of adult artemia with probiotic and synbiotic. based on the results, the concentration of bacteria in adult artemia showed a positive correlation with the duration of enrichment, similar to the results of parta et al. (2003) during the enrichment of a. franciscana nauplii with yeast (saccharamyces baulardii) which revealed accumulation of yeast in nauplii at a level of 3.5×103 cfu g-1. however, enrichment of a. franciscana hours treatments synbiotic (t1) probiotic (t2) prebiotic (t3) control (t4) 2 5.50±0.07b 5.58±0.04b 1.15±0.02a 1.09±0.05a 4 6.61±0.07b 6.67±0.03b 1.04±0.02a 1.23±0.04a 6 6.71±0.04b 6.78±0.03b 0.83±0.07a 1.15±0.04a table 2. the cultivable lactic acid bacteria levels (log cfu g-1 artemia) in a. fransiscana enriched in pre-, proand symbiotic. figure 1: the process of enrichment adult artemia enrichment at different times in different treatments. 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 2 hour 4 hour 6 hour l o g c f u /g synbiotic probiotic prebiotic control 100 azimirad et al./ enrichment capability of artemia with of p. acidilactici and fructooligosaccharide nauplii with two strains of vibrio sp. showed different patterns, so that, attached bacteria to artemia nauplii began to increase at first 30 minutes of enrichment, then suddenly declined at 8 hrs after enrichment and again a sharp rise occurred at 24 hrs at the levels of bacteria in nauplii which all naupliis died at the end of this time (gomez-gil et al., 1998). the a. urmiana showed a gradual trend in enrichment with mentioned probiotic increased over time. furthermore, campbell et al. (1993) enriched a. franciscana with the formalin-killed v. angualiurum and showed that when the concentration of bacteria in enrichment solution is 1.5×107 cfu g-1, the maximum accumulation of vibrio sp. in the artemia nauplii is happened at 60 min. moreover, in concentrations lower than 5×106 cfu g-1, the maximum accumulation is occurred at 120 min after the start of enrichment. changes in the number of bacteria in the a. franciscana is not limited by the number of bacteria in enrichment suspension and the same results reported by makridis et al. (2000) in the enrichment of a. franciscana nauplii with the probiotic bacteria. in conclusion, the results of this experiment indicated that adult artemia has high ability to be enriched with the probiotic bacteria, p. acidilactici and bacterial levels in artemia that is increased along with enrichment time. acknowledgments the authors express their thanks to a. esteghlalian for his assistance during experiment. references abid a., davies s.j., waines p., emery m., castex m., gioacchini g., carnevali o., brickerdike r., romero j., merrifield d.l. (2013). dietary symbiotic application modulates atlantic salmon (salmo salar) intestinal microbial communities and intestinal immunity. fish & shellfish immunology, 35(6): 19481956. agh n., sorgeloos p. (2005). handbook of protocols and guidelines for culture and enrichment of live food for use in larviculture. artemia and aquatic animals research center, urmia university, urmia, 60 p. campbell r., adams a., tatner m.f., chair m., sorgeloos p. (1993). uptake of vibrio anguillarum vaccine by artemia salina as a potential oral delivery system to fish fry. fish and shellfish immunology, 3: 451-459. daniels c.l., merrifield d.l., ringo e., davies s.j. (2013). probiotic, prebiotic, synbiotic applications for the improvement of larval european lobster (homarus gammarus) culture. aquaculture, 416: 396-406. dhont j., lavens p. (1996). tank production and use of ongrown artemia, in: p. lavens, p. soregeloos (eds.). manual on the production and use of live food for aquaculture. fao fisheries technical paper, rome, 361 p. flickinger e.a., van loo j., fahey g.c. (2003). nutritional responses to the presence of inulin and oligofructose in the diets of domesticated animals: a review. critical review food science nutrition, 43:19-60. fuller r. (1989). probiotics in man and animals. journal applied bacteriology, 66: 365-366. gatesoupe f.j., ronald ross w., victor r.p. (2010). probiotics and other microbial manipulations in fish feeds: prospective health benefits. bioactive foods in promoting health, boston, academic press, pp: 541-552. gibson g.r. (2004). fibre and effects on probiotics (the prebiotic concept). clinical nutrition supplements, 1: 25-31. gomez-gil b., herrera-vega m.a., aberu grobis f. a., roque a. (1998). bioencapsulation of two different vibrio species in nauplii of the brine shrimp (artemia fransiscana). applied environmental microbiology, 64: 2318-2322. havennar r., ten brink b., huisint j.h.j. (1992). selection of strains for probiotic use. in: r. fuller (ed.). probiotics, the scientific basis. chapman and hall, london, pp: 209-224. hoseinifar s.h., mirvaghefi a., amoozegar m.a., sharifian m., esteban m.a. (2015). modulation of innate immune response, mucosal parameters and disease resistance in rainbow trout (oncorhyncchus mykiss) upon symbiotic feeding. fish and shellfish immunology, 45: 27-32. mahious a.s., ollevier f. (2005). probiotics and prebiotics in aquaculture: a review. in: n. agh, p. sorgeloos (eds). 1st regional workshop on techniques for enrichment of live food for use in larviculture. 101 int. j. aquat. biol. (2016) 4(2): 96-101 urmia, iran, pp: 17-26. makridis p., fjellheim j.a., skjermo j., vadstein o. (2000). colonization of the gut in first feeding turbot by bacterial strains added to the water or bioencapsulated in rotifers. aquaculture international, 8: 367-380. merrifield d.l., dimitroglou a., foey a., davies s.j., baker r.t.m. (2010). the current status and future focus of probiotic and prebiotic applications for salmonids. aquaculture, 302: 1-18. montajami s., hajiahmadyan m., forouhar vajargah m., hosseini zarandeh a.s., shirood mirzaie f., hosseini s.a. (2012). effect of symbiotic (biomin imbo) on growth performance and survival rate of texas cichlid (herichthys cyanoguttatus) larvae. global vertebrate, 9(3): 358-361. olafsen j.a. (2001). interactions between fish larvae and bacteria in marine aquaculture. aquaculture, 200: 223-247. parta s.k., mohamed k.s. (2003). enrichment of artemia nauplii with the probiotic yeast sacharomyces boulardii and its resistance against a pathogenic vibrio. aquaculture international, 11: 505514. peter h., sneath a. (1986). bergeys manual of systematic bacteriology, 1104-1154. reitan k.i., rainuzzo j.r., oie g., olsen y. (1993). nutritional effects of algal addition in first feeding tanks of turbot scophthalmus maximus l. larvae. aquaculture, 118: 257-275. rengpipat s., phianphak w., piyatiratitivorakul s., menasveta p. (1998). effects of a probiotic bacterium on black tiger shrimp penaeus monodon survival and growth. aquaculture, 167: 301-313. ringo e., sinclair p.d., birkbeck h., barbour a. (1992). production of eicosapentaenoic acid 20:5 n-3 by vibrio pelagius isolated from turbot scophthalmus maximus l. larvae. applied environment microbiology, 58: 3777-3778. ritar a.j., dunstan g.a., nelson m.m., brown m.r., nichols p.d., thomas g.w., smith e.g., crear b.j., kolkovski s. (2004). nutritional and bacterial profiles of juvenile artemia fed different enrichments and during starvation. aquaculture, 239: 351-373. roberfroid m. (2007). prebiotics: the concept revisited. the journal of nutrition, 137:830s. rodriguez-estrada u., satoh s., haga y., fushimi h., sweetman j. (2009). effects of single and combined supplementation of enterococcus faecalis, mannanoligosaccharide and polyhydrobutyric acid on growth performance and immune response of rainbow trout oncorhynchus mykiss. aquaculture science, 57: 609-617. sorgeloos p., dhert p., candreva p. (2001). use of the brine shrimp, artemia spp., in marine fish larviculture. aquaculture, 200:147-159. sorgeloos p., lavens p., leger p., tackaert w., versichele d. (1986). manual for the culture and use of brine shrimp artemia in aquaculture. state university of ghent, belgium. 319 p. teresita d.n.j., maldonado-montiel leticia g. (2005). biomass production and nutritional value of artemia spp. (anostraca: artemiidae) in campeche. mexico revista de biological tropical, 53(3-4): 447-454. ziaei-nejad s., rezaei m.h., takami g.a., lovett d.l., mirvaghefi a.r., shakouri m. (2006). the effect of bacillus spp. bacteria used as probiotics on digestive enzyme activity, survival and growth in the indian white shrimp fenneropenaeus indicus. aquaculture, 252: 516-524. int. j. aquat. biol. (2016) 4(2): 96-101 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی pediococcus acidilacticiبا کاربرد منفرد یا همزمان artemia franciscanaسازی آرتمیا بالغ، مطالعه امکان غنی فروکتوالیگوساکارید و 3فر حسینی حسین سید ،1فرد احمدی نصراهلل ،2مشکینی سعید ،، 1*راد عظیمی محمود .ایران ،ارومیه ارومیه، دانشگاه طبیعی منابع دانشکده شیالت، گروه1 .ایران ،ارومیه ارومیه، دانشگاه طبیعی منابع دانشکده ،دامپزشکی دانشکده غذایی، مواد بهداشت گروه2 .ایران ،گرگان گرگان، طبیعی منابع و کشاورزی علوم دانشگاه ،زیست محیط و شیالت دانشکده آبزیان، پرورش و تکثیر گروه3 چکیده: pediococcus acidilactici از ترکیبی بیوتیکسین با بالغ (artemia franciscana) فرانسیسکانا آرتمیا سازی غنی امکان مطالعه این در p. acidilactici بیوتیک سین شامل تیمار چهار قالب در تصادفی کامالً طرح صورت به آزمایش این. گرفت قرار بررسی مورد فروکتوالیگوساکارید و جهت. گردید اجرا (t4) شاهد تیمار و (t3) ساکارید فروکتوالیگو بیوتیک پری ،p. acidilactici (t2) پروبیوتیک ،(t1) فروکتوالیگوساکارید و هایباکتری تعداد و انجام بردارینمونه سازی،غنی شروع از پس ساعت 6 و 4 ،2 هایزمان در تیمارها، از یک هر با بالغ آرتمیای سازیغنی ارزیابی که داد نشان نتایج. گردید شمارش آرتمیا گرم هر در cfu لگاریتم حسب بر هاباکتری تعداد میکروبی، کشت از پس آرتمیا بدن داخل در موجود نسبت الغب آرتمیای به شده الحاق باکتری تعداد و سازیغنی زمان بین و بوده بیشتر بالغ آرتمیای توسط شده مصرف هایباکتری زمان، گذشت با زمان با که گردید الحاق بالغ آرتمیای به( 2/0 × 615 ± 56/5) باکتری تعداد بیشترین سازی،غنی ساعت 6 از بعد(. p<50/5) دارد وجود یدارمعنی باکتری تعدادکه داد نشانهمچنین نتایج . نداد نشان داری معنی اختالف سازیغنی ساعت 4 زمان مدت با ولینشان داد دارمعنی اختالف ساعت 2 p. acidilactici باشدمی بیوتیک پری فقط واجد تیمار و شاهد تیمار از بیشتر داریمعنی میزان به 2 و 1 تیمارهای در (50/5p>.) اختالف اما ( ساعت 4 حدود) کوتاهی زمان مدت در بالغ آرتمیایداد که نشان همچنین نتایج. نگردید مشاهده بیوتیکسین و پروبیوتیک تیمار بین داریمعنی .نماید ذخیره خود در را پروبیوتیک باکتری از باالیی میزان تواندمی .فرانسیسکانا آرتمیا سازی، غنی فروکتوالیگوساکارید، ،pediococcus acidilactici بیوتیک،سین :کلمات کلیدی int. j. aquat. biol. (2013) 1(3): 116-118 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology technical note preliminary study on semi-closed incubator efficiency for hatching persian sturgeon (acipenser persicus) eggs maryam mardaneh khatooni1, seyed hossein hoseinifar*2,1bagher mojazi amiri1 1department of fisheries and environmental science, natural resources faculty, university of tehran, karaj, iran. 2department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 11 april 2013 accepted 14 may 2013 available online 2 0 june 2013 keywords: semi-closed incubator persian sturgeon eggs acipenser persicus hatching abstract: the present study investigated the efficiency of semi-closed incubator for hatching the persian sturgeon (acipenser persicus) eggs. the incubator was basically designed according to vase apparatus equipped with collector vessel, recirculation pump and aerator. 50% of water was changed every day. triplicate groups of persian sturgeon (50 g) fertilized eggs were stocked in the incubators. water flow rate was set not to harm the eggs and only circulating the eggs. the mortality of embryos and larvae at gasterula stage, formation of s-type heart stage, before hatching and hatching percent were recorded. our results showed that semi-closed incubator circulated eggs completely and the probability of fungal infections was lower than that of flow through incubators. since water used in semi-closed incubator is far less than that of flow through systems, thus semi-closed incubator may be a proper alternative for flow-through incubated systems in future. introduction sturgeons are of extremely great commercial value and widely distributed in the northern hemisphere (dettlaff et al., 1993). sturgeon populations have suffered from over fishing, loss of habitat and decrease of water quality (kynard, 1997). severe decline of sturgeon populations in the caspian sea resulted in artificial propagation, especially for huso huso and acipenser persicus, in iran (pourkazemi, 1997). incubation period is a critical stage in artificial propagation of sturgeons. before 1950, the eggs were incubated in river using seth–green incubators (yushchen, 1957). to resolve several limitation of this device, the first out of water incubator was built by yushchenko in 1952. after that other devices (e.g. osetr, mcdonald, azarakhsh) invented for incubation of sturgeon eggs but still the most popular * corresponding author: seyed hossein hoseinifar e-mail address: hoseinifar@ut.ac.ir tel: +989113706839 incubator in iran is the yushchenko (yushchen, 1957; azari takami and kohnehshahri, 1974). in most of mentioned incubators, constant water flow maintain oxygen for eggs and embryos which consumes large amount of water (farabi et al., 2007). development of incubators with a circulating system would be of tremendous benefit for hatcheries especially in areas with circulating water sources. hence, this study was conducted to investigate efficiency of semi-closed incubator with circulating water system for persian sturgeon (acipenser persicus) eggs. materials and methods eggs and sperm provided from migratory breeders of the southern caspian sea (golestan province). after fertilization and removing adhesiveness, 150 g eggs were stocked in three incubators with semi-closed 117 mardaneh khatooni et al./ int. j. aquat. biol. (2013) 1(3): 116-118 water system. the incubator was a 5l jar, equipped with collector vessel, recirculation pump and aerator (fig.1). circulative water was changed with freshwater by half every day. water flow set at the rate that caused not harm to the eggs but circulating them gently. the mortality of embryo and larvae at blastula and gastrula stage, total mortality percent of eggs during incubation period and survival rate of larvae during 15 day post hatch were recorded and compared with the other kind of incubators. results egg hatchability: hatching occurred four days after fertilization (120 h). the mortality of embryo and larvae at blastula and gastrula stages, and hatching percent of eggs were 64.06±3.85, 18.06±0.85, 88.22±3.81, respectively. mortality of larvae: cumulative mortality of larvae before and after exogenous feeding was 7.29% and 10.23%, respectively. survival percent of larvae up to 15 days post hatch was 88.62% (fig. 2). discussion our results showed that persian sturgeon eggs can successfully develop and hatch in semi-closed incubator with much lower need of water. as water circulated the eggs completely in this system, the probability of fungal infections would be lower than flow-through incubators and the unfertilized eggs were omitted automatically. circulation provided uniform water temperature and quality (the two major factors influences embryos mortality and abnormality) compared to other incubators. other incubators use river water that that should pass a boiler when the environment temperature becomes cold (abdolhay, 2006). since water used in semi-closed incubator is much lower than other incubators (e.g. osetr, yushchenko and azarakhsh), it is a good alternative where water is limited. in addition incubation in separate batch of eggs reduces the risk of disease transfer (azaritakami and kohnehshahri, 1974). although this incubation system shows some beneficial aspect, but it is mainly depend on electricity force. further studies are needed to address the use of this incubator for various sturgeon species. references azari takami g., koneshahri m. (1974). culture of sturgeon fishes. tehran university press, 30-66. abdolhay h.a., baradaran tahori h. (2006). fingerling production and release for stock enhancement of sturgeon in the southern caspian sea: an overview. journal of applied ichthyology, 22: 125-131. dettlaff t.a., ginsburg a.s., schmalhaulen o.t. (1993). sturgeon fishes developmental biology and aquaculture. berlin, heidelberg, newyork, springerverlag, 300 p. farabi s.m.v., taheri s.a. and nadri h. (2007). the figure 1. schematic design of the semi-closed incubator: a, collector vessel; b, aeration pump; c, water recirculation pump. figure 2. cumulative mortality of larvae during 15 days post hatch (the intonation of exogenous feeding is shown by an arrow). 117 118 mardaneh khatooni et al./ int. j. aquat. biol. (2013) 1(3): 116-118 comparison efficiency incubator of egg sturgeon fishes: yushchenko incubator of russian model with azarakhsh incubator of iranian model and emphasis on acipenser persicus (borodin, 1897). journal of pajouhesh and sazandegi, 74: 9-18. kynard b. (1997). life history, latitudinal patterns, and status of the shortnose sturgeon, acipenser brevirostrum. environmental biology of fishes, 48: 319-334. pourkazemi m. (1997). the survey status of sturgeon fishes and their conservation in the caspian sea. iranian journal of fisheries sciences, 3: 13-22. yushchen p.c. (1957). a device for incubation of the eggs of acipenserid fishes. ussr ministry of fisheries. moscow (in russian). int. j. aquat. biol. (2019) 7(2): 85-92 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article feeding habits of striped piggy, pomadasys stridens (forsskal, 1775) (haemulidae) in northern part of the persian gulf sajjad karimi*1, elham katiraei1, nasrollah mahboobi soofiani1, seyed aminollah taghavimotlagh2, amir vazirizadeh3 1fisheries division, department of natural resources, isfahan university of technology, isfahan, iran. 2iranian fisheries research organization, agricultural research education and extension organization, tehran, iran. 3persian gulf university, persian gulf research and study center, bushehr, iran. s article history: received 17 july 2018 accepted 15 april 2019 available online 2 5 april 2019 keywords: food diet composition feeding intensity abstract: feeding habits of pomadasys stridens were studied in northern part of the persian gulf. a total of 591 specimens were collected from the coastal water of bushehr province using trawl boats from may 2012 to april 2013 and diet composition, feeding intensity and season changes in diet composition were investigated. vacuity index was 81% during the study. rgl was 0.98±0.03 classifying this species as carnivorous to omnivorous fish. pomadasys stridens had consumed 48 different food categories in 7 taxonomic classes. crustacean, mollusca, foraminifera, nematodes, echinodermata, annelida and miscellaneous were the main observed food items. there was no significant difference between males and females regarding diet composition in different months. introduction haemulidae is one of the important tropical fishes in terms of ecology and economic point of views. this family is composed of 19 genera and 134 species (nelson et al., 2016). striped piggy, pomadasys stridens (forsskal, 1775), is one of the abundant haemulid fish in the persian gulf and oman sea (valinassab et al., 2006). this species has wide distribution in the pacific, atlantic and indian ocean inhabiting reefs, shelf area, muddy, sandy bottoms and in variety of inshore habitats (fehri-bedoui and gharbi, 2008). it is also important energy importer to reef communities and a major pray for many larger species such as snappers and groupers (darcy, 1983). despite economic and ecological importance of p. stridens, there is scarcity of information on its biological aspects and feeding habits. earlier studies were mainly focused on reproductive biology (atiqullah khan et al., 2013; karimi et al., 2014) and length-weight relationship (karimi et al., 2015; safi et al., 2014), and the only study on its feeding habits of this species was that of safi et al. (2013) from the karachi coast. therefore, many aspects of feeding *correspondence: sajjad karimi doi: https://doi.org/10.22034/ijab.v7i2.477 e-mail: sajjad.karimi@na.iut.ac.ir characteristics and habits of this species remain unknown. in general, maintenance of ecosystem balance and design of program for ecological management depends on availability of sufficient information about biology and life history of species. furthermore, knowledge about feeding features such as feeding habits and behavior, feeding intensity and diet composition usually provides useful tools for ecosystem management and biodiversity conservation. hence, this study was conducted to investigate the feeding habits of p. stridens to provide information about some feeding parameters, including diet composition and feeding behavior of the populations inhabiting the northern part of the persian gulf, bushehr coasts, for fisheries management and conservation purposes. materials and methods specimens of p. stridens were caught using trawl boat, from the bushehr province in the northern part of the persian gulf (28°55´19´´n 50°59´49´´e). a total of 591 specimens were collected at monthly intervals from may 2012 to april 2013 (except september). 86 karimi et al./ feeding habits of striped piggy samples were immediately preserved by placing in ice box and then transported to the laboratory, total length and weight of fish were measured using biometry ruler with accuracy of 1 mm and digital balance (andmodel fx300i) with 0.1 g precision, respectively. gender was determined visually by macroscopic examination of the specimens’ gonads. feeding habits: the intestine and stomachs of fish were removed and weighted in full and empty situation by digital balance with precision of 0.01 g. then, intestine was removed and detached from other tissues gently and its length measured using biometrical ruler to the nearest 1 mm. stomach and intestine contents were fixed in 4% formaldehyde. food items were identified and classified to the lowest possible taxonomic levels using various identification keys (fischer and bianchi, 1984; asadi and dehghani, 1996; carpenter et al., 1997; jereb and roper, 2005). for identification of food items, stereo zoom microscope was used and pray was identified by external morphological characteristics and hard parts of the body such tentacles, frustules, antenna, ornamentations, scales and etc. some food items which were digested to the extent that was difficult to identify, were considered as unidentifiable items. parameters which were used to evaluate the feeding habits were divided into two main categories viz. feeding intensity and food preference. feeding intensity: stomachs were divided into two groups; with food (designated as “full”) and without food (designated as “empty”). fullness or emptiness of stomach was determined visually. vacuity index (vi) measured using following equation: vi= number of empty stomachs number of examined stomachs × 100 according to vi, fish were grouped into 5 different levels from feeding intensity point of view, where 0≤vi≤20 represented for edacious, 20≤vi≤40 relatively edacious, 40≤vi≤60 moderate feeding, 60≤vi≤80 relatively abstemious and 80≤vi≤100 abstemious fish (euzen, 1987). gastrosomatic index (gasi) and stomach fullness index (sfi) were calculated by dividing total stomach weight and the weight of stomach content to fish body weight × 100, respectively (desai, 1970). food preferences: as a general index, relative gut length (rgl) was calculated by dividing intestine length to fish length. rgl values for carnivorous and omnivorous fish species is 0.6-0.8 and 0.8-1.0, respectively and rgl value about 2.5-16.4 will represented herbivorous fish species (kramer and bryant, 1995). after washing the collected food items from the stomach and intestine, they were identified and grouped into 8 different taxonomic groups (table 1). according to numerical index (ni) and frequency of occurrence (fo) values, fish preference for diet was recognized. equations (a) and (b) were used to measure ni and fo, respectively (berg, 1979; hyslop, 1980). ni= number of each pry item in all full stomachs total number of food items × 100 (a) fo= number of stomachs containing specific prey total number of full stomachs × 100 (b) if fo is <10: prey is considered randomly ingested and not preferred food item, if 10<fo<50: food is considered second preferred item and if fo>50: the item was considered as a main preferred food of fish (euzen, 1987). statistical analysis: all statistical analysis was done using spss (version 22). one-way anova was applied to examine the differences in frequency of occurrence and numerical index and also vacuity index values between various sampling months. where there was a significant difference (p<0.05), duncan analysis was employed for determination of significant differences source. sex ratio was examined by chi-square test for determination of the significant differences between frequencies of two sexes during various sampling months. results length-frequency distribution and sex ratio: minimum and maximum length of all samples were 11.7 and 23 cm. the females ranged 12.6-23 cm (18.55±2.2, mean± sd) and males 11.7-22 cm (17.57±2.1). weight ranged 27.4-144.8 g (88.92± 87 int. j. aquat. biol. (2019) 7(2): 85-92 table 1. numerical index and frequency of occurrence of different food items observed in pomadasys stridens stomachs. prey ni% fo% crustacea 39.60 84.53 decapoda 2.01 44.48 xanthidae actaea sp. 0.63 19.78 ocypodidae ocypode quadrata 0.87 26,92 portunidae portunus plagicus 0.27 10.44 grapsidae sesarma plicatum 0.08 1.92 calappidae matuta lunaris 0.06 2.20 penaidae 0.11 3.85 calanoida 1.89 6,91 amphipoda 14.60 56.63 gammaridae orchestia sp. 13.71 53.85 cummacea cyclaspis picta 17.56 46.96 ostracoda 3.54 35.64 mollusca 34.33 82.32 gastropoda 21.59 73.68 actenoidae pupa affinis 0.06 1.10 nassarididae 3.46 33.79 xenophoridae stellaria solaris 8.62 59.34 muricidae murex scolopax 0.02 0.55 cerithiidae 0.59 14.01 turritelidae 0.45 10.71 atyidae haminoea vitra 0.47 11.54 eulimidae eulima polita 0.35 9.89 epitonidae epitonium pallasii 0.51 11.54 trochidae umbonium vestiarium 1.00 21.43 pyramidelidae odostomia sp. 0.06 2.47 janthidelidae janthina janthina 0.64 11.26 haminoeaidae atys sp. 2.37 25.27 rissoina distans 1.60 22.80 columbelidae mitrella micera 0.09 0.82 marginilidae marginella sp. 1.29 24.18 bivalvia 11.96 67.68 tellinidae tellina inflate 1.08 15.93 veneridae bassina calophyla 0.96 18.96 veneridae callista sp. 5.38 22.8 paphia sp. 2.13 25.55 pteriidae pinctada radiate 1.01 2.47 cardiidae trachy cardium 0.27 7.14 solenidae solen brevis 0.09 1.65 schaphopodae 0.78 18.78 dentalidae dentalium longitrosum 0.08 3.02 dentalium octangulatum 0.70 18.41 nematode 2.92 35.36 foraminifera 4.30 46.96 nubeculariidae spiriloculina sp. 3.89 46.96 annelidae 0.48 7.46 polychaeta aciculata nephtyidae nephthys sp. 0.40 6.04 nereidae platynereis cultifera 0.04 0.55 pectinaridae pectinaria sp. 0.04 0.82 echinoderm 1.73 9.94 asterozoa ophiuroidea ophionereis sp. 1.73 8.25 miscellaneous 16.58 68.51 plant material 0.13 5.49 echinus 13.83 44.78 ctenoid scale 1.55 40.11 apanthura 0.16 3.85 gastropoda egg 0.08 3.47 fish bone 0.09 1.92 88 karimi et al./ feeding habits of striped piggy 26.2) and 21-124.9 g (77.41±25.9) in females and males, respectively. the females showed a slightly larger size than the males. the females were predominant sex in all sampling months. sex ratio (male: female) ranged 11-74% and chi-square test indicated that sex ratio values were significantly different (p<0.05) (fig. 1). according to the overall sex ratio of the specimens 1:3.3 (male: female), the population composition is in favor of female fish. feeding habits: in total 591 stomachs were examined and among them, 478 were empty, and 113 full (fig. 2). overall vacuity index (vi) was 81% and maximum number of the empty stomachs was observed in november and december (98 and 96%) and minimum number in february and may (55 and 53%). vi value was 84 and 80% for male and females, respectively and no significant differences were observed between sexes. in the present study, vi value was 81%. according to euzen (1987), p. stridens is considered as abstemious species. stomach fullness index (sfi) showed a significant differences between months (p<0.05) (fig. 3a). mean sfi increased from december and reached its maximum level in february (0.51) and may (0.52) and decreased from june, reaching its minimum level in december (0.16). gasi showed similar trend as sfi (fig. 4b). it maximum was observed in february (%93) and may (94%) and its minimum values were observed in november (26%) and december (23%) which was significantly different compared to other figure 1. female and male distribution of pomadasys stridens during various sampling months. figure 2. monthly proportion of full and empty stomachs in sampled pomadasys stridens. 89 int. j. aquat. biol. (2019) 7(2): 85-92 months (p<0.05). diet composition: based on the results, 48 categories of food items classified into 7 taxonomic classes, including crustacean, mollusca, foraminifera, nematodes, echinodermata, annelida and miscellaneous. figure 4 shows the general composition and proportion of each food items observed in the stomach and intestine of studied fish. the most abundant food items which was observed in 84.53% of gut with 39.6% numerical index (ni) value was crustacean followed by molluscs with fo=82.32% and ni=34.33%, miscellaneous with fo=68.51% and ni%=16.58%, foraminifera with fo=46.96% and ni=4.3%, nematode with fo=35.36% and ni%=2.92%, echinodermata with fo=9.94% and ni=1.73% and annelide was the least abundant food item with fo=7.46% and ni=0.48%. the results also showed that among crustaean, cummacea (fo=46.96% and ni=17.56%) and amphipoda (fo=56.63% and ni=14.6%) and in molluscs, gastropoda (fo=82.32% and ni=34.33%) and bivalvia (fo=67.68% and ni=11.96%) were most abundant food items (table 1). average calculated rgl value for all fish specimens was 0.98±0.03, indicating a relatively carnivorous to omnivorous feeding habits for p. stridens. no significant differences were found in comparing diet composition between male and female. discussions there is lack of data on feeding intensity, diet composition, feeding behavior and food preference regarding the feeding habits of p. stridens. the present study provides the first information from the northern (a) (b) figure 3. mean monthly variations in (a) stomach fullness index ± se and (b) gastrosomatic index ± se of pomadasys stridens. figure 4. seasonal variations in frequency of occurrence (%) of different food items in the diet of pomadasys stridens. 90 karimi et al./ feeding habits of striped piggy part of persian gulf. composition of the sampled fish was significantly in favor of females. predominance of the females p. stridens has been reported for various pomadasys species, such as p. argenteus (kulbicki et al., 2009), p. incises (pajuelo et al., 2003) and p. kaakan (falahatimarvast et al., 2012). high ratio of females may be due to different survival rates of females and males (sadovy et al., 1994), spatial segregation of size-classes in each sex (kulbicki et al., 2009) and also sampling strategy in various depths when males and females inhabit in different depths (falahatimarvast et al., 2012). considering the values of vi, sfi and gasi in different months of sampling, the feeding intensity of p. stridens can be related to its reproductive cycle. robillard et al. (2008) reported that there is an inverse relationship between spawning season and feeding intensity in fish. pomadasys stridens spawns in november and december (karimi et al., 2014), therefore by entering the fish to the last developmental stage of its maturation, the gonads especially ovaries occupy almost all abdominal cavity and therefore a limited space will be available for gastrointestinal tract. this issue is led to decrease feeding intensity by fish during this period. after spawning, a large amount of energy and nutrients is need to recruit resources for growth and preparation for next reproduction cycle (dadzie et al., 2000). similar feeding characteristic has been reported for p. kaakan, where lowest feeding intensity and highest value for vi found during spawning season i.e. spring (valinassab et al., 2011). whereas safi et al. (2013) found highest feeding intensity rate of p. stridens in october and march, concluding that feeding intensity is higher in colder seasons, followed by a decline in warm seasons. rgl is affected by different digestibility rate of plant, detritus and animal based diets that fish consumed as food (pogoreutz and ahnelt, 2014). rgl was 0.98 in p. stridens , hence, according to kramer and bryant (1995), p. stridens is an omnivorous fish, consuming a high protein content items as food. furthermore, the present study revealed that the crustacean and molluscs are the preferred and dominant food items, constituting about 39.6 and 34.33%, respectively, followed by miscellaneous items (16.58%), foraminifera (4.3%), nematode (2.92%) and annelida (0.48). foraminifera and nematodes were secondary food preferred items preferred. increasing the presence of the foraminifera in gut was observed in winter and spring, showing that with decreasing temperature in winter, fish prefer to stay in deeper waters. moghaddasi et al. (2009) reported similar finding by indicating a higher intensity of foraminifera in winter diet of p. stridens, when they live in deep waters of the persian gulf and oman sea. based on our findings, the crustacean and mollusks were main food for p. stridens in all year round and other food taxa intensity is dependent on their availability. the amount of food digestion is dependent on the type of feed stuffs, fish species and temperature. therefore, the digestion and absorption rate of consumed foods could be different (bond, 1996). the presence of identifiable remains from crabs, stomatopods and gastropods in the intestine indicates a relatively slow digestion rate of items having the chitinous exoskeletons, hence, overrepresented in the identified remains (bond, 1996). safi et al. (2013) found that semi digested items was the most dominant food items in the digestive tract and thereafter crustacean was the dominant food item followed by miscellaneous, molluscs, teleosts and polychaeta in digestive system of p. stridens as food. it seems that in both studies, crustacean was the preferred food item by fish, however, the observed differences in food preferences in both studies probably refer to geographical differences and food and prey availability (biswas, 1993). conclusion this study provided useful information about feeding habits of p. stridens to better understanding of the relationship between fish species and other living organisms in the persian gulf. further investigations is suggested to discover precise information about feeding behaviour and food preference by implementing digestion coefficient value for every food item consumed by p. stridens. 91 int. j. aquat. biol. (2019) 7(2): 85-92 acknowledgements financial and technical supports for the present study was provided by isfahan university of technology and iranian fisheries research organization. the authors would like to express their gratitude to s. asadollah, a. vahabnezhad and m.j. shabaani for their technical assistance and valuable discussions. references asadi h., dehghani r. (1996). fishes of the persian gulf and oman sea. iranian fisheries research organization, tehran: aieneh press. 226 p. atiqullah khan m., khan z., usman m., safi a. (2013). studies on gonadosomatic index and stages of gonadal development of striped piggy fish, pomadasys stridens (forsskal, 1775) (family; pomadasyidae) of karachi coast, pakistan. journal of entomology and zoology studies, 1(5): 28-31. berg j. (1979). discussion of methods of investigating the food of fishes, with reference to a preliminary-study of the prey of gobiusculus flavescens (gobiidae). marine biology, 50(3): 263-273. biswas s.p. (1993). manual of methods in fish biology. south asian publishers. 157 p. bond c.e. (1996). biology of fishes. saunders college pub. 750 p. dadzie s., abou-seedo f., al-qattan e. (2000). the food and feeding habits of the silver pomfret, pampus argenteus (euphrasen), in kuwait waters. journal of applied ichthyology-zeitschrift fur angewandte ichthyologie, 16(2): 61-67. darcy g.h. (1983). synopsis of biological data on the grunts: haemulon aurolineatum and h. plumieri (pisces: haemulidae): united states department of commerce, national oceanic and atmospheric administration, national marine fisheries service. no. 133. 448 p. falahatimarvast a., poorbagher h., lokman p.m. (2012). the reproductive biology of pomadasys kaakan (osteichthyes: haemulidae) in the northern persian gulf. cahiers de biologie marine, 53(1): 25-34. fehri-bedoui r., gharbi h. (2008). sex-ratio, reproduction and feeding habits of pomadasys incisus (haemulidae) in the gulf of tunis (tunisia). acta adriatica, 49(1): 518. hyslop e.j. (1980). stomach contents analysis a review of methods and their application. journal of fish biology, 17(4): 411-429. karimi s., katiraei e., soofiani n., paykanheirati f. (2015). investigating the length-weight relationship and growth parameters of stripped piggy pomadasys stridens (forrskål, 1775) in northern part of persian gulf (bushehr). gnbd-jair, 3(2): 97-106. karimi s., mahbobi soofiani n., paykanheirati f., katiraei e. (2014). reproductive biology of stripped piggy (pomadasys stridens forsskal, 1775) in northern part of persian gulf (bushehr). gnbd-jair, 2(3): 87-100. kramer d.l., bryant m.j. (1995). intestine length in the fishes of a tropical stream .2. relationships to diet the long and short of a convoluted issue. environmental biology of fishes, 42(2): 129-141. kulbicki m., morize e., wantiez l. (2009). synopsis of the biology and ecology of pomadasys argenteus (haemulidae) in new caledonia. cybium, 33(1): 45-59. moghaddasi b., nabavi s., vosoughi g., fatemi s.m.r., jamili s. (2009). abundance and distribution of benthic foraminifera in the northern oman sea (iranian side) continental shelf sediments. research journal of environmental sciences, 3(2):210-217 nelson j., grande t., wilson m. (2016). fishes of the world, fifth edition. 752 p. pajuelo j.g., lorenzo j.m., gregoire m. (2003). age and growth of the bastard grunt (pomadasys incisus: haemulidae) inhabiting the canarian archipelago, northwest africa. fishery bulletin, 101(4): 851-859. pogoreutz c., ahnelt h. (2014). gut morphology and relative gut length do not reliably reflect trophic level in gobiids: a comparison of four species from a tropical indo-pacific seagrass bed. journal of applied ichthyology, 30(2): 408-410 robillard e., reiss c.s., jones c.m. (2008). reproductive biology of bluefish (pomatomus saltatrix) along the east coast of the united states. fisheries research, 90(1-3): 198-208. sadovy y., rosario a., roman a. (1994). reproduction in an aggregating grouper, the red hind, epinephelus guttatus. environmental biology of fishes, 41(1-4): 269-286. safi a., atiqullah khan m., khan z. (2014). study of some morphometric and meristic characters of striped piggy fish, pomadasys stridens (forsskal, 1775) from karachi coast, pakistan. the journal of zoology studies, 1(4): 1-6. safi a., atiqullah khan m., khan z., usman m. (2013). observations on the food and feeding habits of striped 92 karimi et al./ feeding habits of striped piggy piggy, pomadasys stridens (forsskal, 1775) (family; pomadasyidae) from karachi coast, pakistan. international journal of fauna and biological studies, 1(1): 7-14. valinassab t., daryanabard r., dehghani r., pierce g.j. (2006). abundance of demersal fish resources in the persian gulf and oman sea. journal of the marine biological association of the united kingdom, 86(6): 1455-1462. valinassab t., jalali s., hafezieh m., zarshenas g.a. (2011). evaluation of some feeding indices of pomadasys kaakan in the northern persian gulf. iranian journal of fisheries sciences, 10(3): 497-504. int. j. aquat. biol. (2018) 6(4): 189-197 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article reproductive biology of two sympatric species of tooth-carps: aphanius hormuzensis and aphanius furcatus, from south of iran (teleostei: aphaniidae) mojtaba masoudi1, hamid reza esmaeili*1, mehrgan ebrahimi1,4, azad teimori2, mahvash seifali3 1developmental biosystematics research laboratory, zoology section, department of biology, college of sciences, shiraz university, shiraz, iran. 2department of biology, faculty of sciences, shahid-bahonar university of kerman, kerman, iran. 3department of plant sciences, faculty of biological sciences, alzahra university, tehran, iran. 4school of biological sciences, flinders university, gpo box 2100, adelaide, south australia 5001, australia. article history: received 5 september 2018 accepted 6 november 2018 available online 2 5 august 2018 keywords: reproductive indices fecundity spawning season sex ratio sexual dimorphism abstract: some aspects of the reproductive biology of two endemic tooth-carps, aphanius hormuzensis and a. furcatus, from southern iran, were studied by regular monthly collections throughout one year. significant differences were observed between the total number of females and males, females being more abundant. based on the pattern of reproductive indices including the gonado-somatic index and dobriyal index, it was concluded that these fishes spawn in april and may. the estimated absolute fecundity of a. hormuzensis ranged from 78 (tl = 32.2 mm) to 730 (tl = 51.1 mm), with a mean value of 219.78±66.50 oocytes per fish based on 15 females. the relative fecundity ranged from 68.45 to 518.54 oocytes/g body mass (mean±s.d: 237.67±96.87 oocytes/g). for a. furcatus, the estimated absolute fecundity ranged from 53 (tl = 26.9 mm) to 102 (tl = 32.04 mm), with a mean value of 93.73±45.37 oocytes per fish based on 15 females. the relative fecundity ranged from 22.41 to 123.65 oocytes/g body mass (mean±s.d: 64.98±23.37 oocytes/g). due to overlapping of spawning season in these two sympatric species, it seems that other preand post-zygotic factors are responsible for absence of natural hybrids in the studies tooth-carps in the mehran river. introduction the tooth-carps genus aphanius is the sole native representative of the family aphaniidae in the old world and represents a relic of the ancient ichthyofauna of the tethys (kosswig, 1955; hrbek and meyer, 2003; freyhof et al., 2017). they occur all over the southern part of the western palaearctic. several tooth-carp species are restricted to one or a few catchments, and some are even restricted to single spring fields (e.g., a. ginaonis and a. kavirensis), making them highly vulnerable to habitat alterations in their often semi-arid or arid environments (see freyhof et al., 2017). its present-day species diversity and distribution have largely been shaped by vicariance events since the early miocene, when the ancient tethys sea closed and uplift of the anatolian and iranian plateaus began (e.g., hrbek and meyer, 2003; teimori et al., 2014). *corresponding author: hamid reza esmaeili doi: https://doi.org/10.22034/ijab.v6i4.490 e-mail address: hresmaeili@shirazu.ac.ir as currently understood, there are four sympatric species, a. mesopotamicus and a. stoliczkanus in the shadegan wetland, tigris river, southwestern iran and also a. hormuzensis and a. furcatus in the mehran river, hormozgan basin (masoudi et al., 2016; esmaeili et al., 2018a, b; teimor et al., 2018). the hormuz tooth-carp, a. hormuzensis is endemic to the mehran river, hormuzgan basin in south of iran. it occurs mainly in two types of habitats, that is, brackish rivers of exorheic drainages and hot sulfur springs. in both habitat types, a. hormuzensis can be found sympatrically with a. furcatus and the endemic cichlid, iranocichla hormuzensis coad, 1982 (teimori et al., 2018). the scaleless tooth-carp, a. furcatus is found the shur river, kol river, mehran river, khurgu and faryab hot sulphuric springs in the hormuzgan drainage, southern iran (teimori et al., 2014). the 190 masoudi et al./ reproductive biology of two sympatric species of tooth-carps rivers and spring streams in southern iran are often seasonal and characterized by shallow water and white layers of salt around and within the river (teimori et al., 2014). aphanius furcatus individuals are often dominant on the riversides, where the water is shallow, warm and water flow is slow (teimori et al., 2014). species of aphanius are well-known for their remarkable capacity to adapt to adverse ecological conditions and evolve into new species when populations become isolated. this has made them particularly attractive as model species for biologists, and many researchers have studied their phenotypic variation, diversity, zoogeography, hybridization, sympatricity and phylogenetic relationships (see masoudi et al., 2016; teimori et al., 2018). the sympatricity increases the chance of natural hybridization between closely or distantly related species (masoudi et al., 2016). the natural hybridization has been reported for two sympatric species of a. mesopotamicus and a. stoliczkanus but there is not such record for a. hormuzensis and a. furcatus. as this phenomenon preliminary requires the same period of reproductive activity of two sympatric species (e.g., spawning season), hence, in this study some aspects of reproductive biology of a. hormuzensis and a. furcatus including gonadosomatic index, modified gonadosomatic index and dobriyal index which are indicators of spawning season which in turn causing or preventing natural hybridization, have been investigated. materials and methods study area and sampling: the fish individuals were collected from the mehran river (hormozgan basin) near bastak city, hormozgan province, south of iran (27°8'39.38"n., 54°15'42.38"e.). fishes were sampled regularly by hand net (mesh size, 5 mm) from august 2014 to july 2015. the specimens were immediately preserved in buffered formalin solution of 10% until they could be examined. data analysis: tooth-carp individuals were measured to the nearest 0.05 mm total length (tl) and standard length (sl) using a vernier caliper. the total body weight (tw) of all preserved fishes was measured using electronic balance to the nearest 0.001g. the sexes were determined based on the body color pattern (figs. 1). the parameters of length-weight relationships were estimated by linear regression of the log-transformed weight and length (koutrakis and tsikliras, 2003). prior to regression analysis, log–log plots of length and weight values were performed for visual inspection of outliers (froese, 2006) which is w=alb, where w is the total weight in gr, l the total length in mm, a a coefficient related to body form and b an exponent indicating allometric growth when unequal to 3. the fulton’s condition factor (k) was calculated monthly by k=w / l3 x 100 where w= whole body wet weight in grams and l= length in cm; the factor 100 is used to bring k close to unity (murphy and williams, 1996). we dissected the fishes and examined the ovary to determine stage of maturity. to examine the monthly changes in the gonads as a means for estimating the spawning season of theses tooth-carp, 3 indices were used: gonado-somatic index (gsi), modified gonadosomatic index (mgsi) and dobriyal index (di), which were calculated by gsi = (weight of gonads / weight of fish)×100 (nikolsky, 1963); mgsi = (weight of gonad / weight of fish weight of gonad)×100 (nikolsky, 1963); di =√𝐺𝑊 3 (dobrial et al., 1999), where gw is the average gonad weight. the gonads were examined and absolute fecundity was measured in terms of the number of oocytes [(f= 𝑛×𝐺 𝑔𝑤 ), f = absolute fecundity, n = number of oocytes, g = gonad weight, gw= gonad sample weight] with a diameter greater than 0.2 mm (leonards and sinis, 1998), using 15 female gonads for each species. the relative fecundity (number of ova per unit of body weight) [(r= 𝐹 𝑇𝑊 ) , r= relative fecundity, f= absolute fecundity, tw= total weight] was also estimated using the method suggested by bagenal (1967). to determine the ovum diameter, the ovaries were preserved in 10% formalin solution. the diameters of 100 ova from each female fish were measured using a zeiss light microscope which was 191 int. j. aquat. biol. (2018) 6(4): 189-197 fitted with an ocular micrometer. the ovum diameters which were measured in ocular divisions were transformed into mm. the chi square test was used to assess deviation from a 50:50 sex ratio (robards et al., 1999). all the collected specimens were deposited in zm-cbsu (zoological museum, collection of biology department, shiraz university). results descriptive information: we examined 427 specimens of a. hormuzensis (with ranging in total length from 253 to 521 mm, standard length 212 to 483 mm and total weight of 0.068-1.866 g) and 214 specimens of a. furcatus (with ranging in total length from 172 to 427 mm, standard length 130 to 381 mm and total weight of 0.061-0.701 g), from mehran river, hormozgan province in southern iran. sexual dimorphism: males of a. hormuzensis have 23 dark and light blue alternating bars on the caudal fins, the last bar being yellow. the bars are crescentshaped, with concave side posterior. males outside the breeding season are less brightly coloured with silvery on the flanks with a grey or black-brown back and irregular flank bars. females are brown-grey to silvery with 15-17 narrow dark brown flank bars (fig. 1). males of a. furcatus (fig. 2) usually have 7-11 vertical flank-bars, their dorsal, anal, caudal, pelvic and pectoral fins are white but with a dark pigmentation on the base of the 1st-4th dorsal rays. females (fig. 2) display 7-9 dark circular blotches on their flanks, starting behind the operculum and extending until the base of the caudal fin. similar to the males, their dorsal, anal, caudal, pelvic and pectoral fins are white. both males and females display a yellowish dorso-posterior part of the eyes. sex ratio: of 427 fish specimens of a. hormuzensis caught, 183 were males and 244 were females, giving an overall sex ratio of 1.34f:1m (chi square=17, p<0.05). in case of a. furcatus, out of 214 collected individuals, 118 were female and 96 were male with sex ratio of was 1.23f:1m (chi square= 18, p<0.05). length-weight relationship: descriptive statistics and estimated parameters of lwrs for the a. hormuzensis and a. furcatus species are given in table 1. the value of parameter b ranged from 2.643 for male a. furcatus to 3.385 for female a. hormuzensis, which remains within the expected range of 2–4 reported by tesch (1971) and almost to 2.5 < b < 3.5 by froese (2006). gonado-somatic and modified gonado-somatic indices: the results showed that female invest more in gonads than males (anova, p<0.001). significant differences were observed in female and male gsi and mgsi in different months (anova, p<0.05). the spawning of the a. hormuzensis was determined by figure 1. male (left) and female (right) aphanius hormuzensis collected from mehran river, south of iran. figure 2. male (left) and female (right) aphanius furcatus collected from mehran river, south of iran. 192 masoudi et al./ reproductive biology of two sympatric species of tooth-carps increased gsi during february to april showing two peaks in both sexes (fig. 3). the first peak was observed in february with maximum mean value of 2.984±1.603 and the second one in april with maximum mean value of 4.789±5.106 for male individuals and 8.915±0.916 and 15.824±5.126 for female individuals, respectively. spawning time of the a. furcatus was determined by increasing gsi value during february to may, showing two peaks in both sexes (fig. 4). the first peak was observed in february with maximum mean value of 1.375±1.143 and the second one was seen in may with maximum mean value of 3.913±1.226 for male specimens and 3.501±1.712 and 11.198±3.326 for female specimens, respectively. the analysis of mgsi of for both aphanius species (anova, p<0.05) are also consistent with the results of the gsi index (figs. 5, 6). dobriyal index (di): another indicator of spawning season is the dobriyal index (di). this index unlike the gsi index, does not consider the body weight, which is influenced by nutrition and environmental and physiological conditions. the results of the di were also consistent with the results of gsi and mgsi analysis. in a. hormuzensis, the mean of di for both sexes was maximum in april (0.338 for males and 0.558 for females) (fig. 7). respectively, for a. furcatus the mean of di for males and females was high as 0.303 in april and 0.341 in may (fig. 8). hsi index: an analysis of the values of the hsi index table 1. length-weight relationship of aphanius hormuzensis and a. furcatus from mehran river, bastak (in south of iran). species basin sex n tl range (cm) w range (g) a 95% ci of a b 95% ci of b r2 aphanius hormuzensis hormuz m f a 183 244 427 2.53-4.64 2.12-5.21 2.53-5.21 0.245-1.017 0.068-1.866 0.068-1.866 0.0095 0.0089 0.0090 0.0062–0.015 0.0079–0.0102 0.0081–0.0100 3.357 3.385 3.396 3.031–3.697 3.278–3.497 3.304–3.488 0.969 0.996 0.994 aphanius furcatus hormuz m f a 96 118 214 1.72-3.36 1.89-4.27 1.72-4.27 0.061-0.244 0.712-0.701 0.061-0.701 0.0126 0.0098 0.0010 0.0103-0.0158 0.0088-0.0109 0.0091-0.0111 2.643 3.035 2.981 2.353-2.921 2.882-3.154 2.845-3.091 0.964 0.987 0.972 m, male; f, female; a, sexes combined; n, sample size; tl, total length; w, weight; a, intercept of log-log relationship; b, regression slope; r2, coefficient of determination. table 2. mean of gonadosomatic index (gsi) of female (f) and male (m) aphanius hormuzensis and a. furcatus from the mehran river, bastak, south of iran. species sex no. mean±s.d. range sig. a. hormuzensis m f a 183 244 427 2.864±2.12 7.923±4.32 5.652±4.495 0.17-21.67 0.53-29.25 0.17-29.25 0.0001 a. furcatus m f a 96 118 214 1.614±2.12 2.229±3.17 1.921±3.12 0.11-17.26 0.15-20.71 0.11-20.71 figure 3. variation of mean gonadosomatic index (gsi) in aphanius hormuzensis in different months. figure 4. variation of mean gonadosomatic index (gsi) in aphanius furcatus in different months. 193 int. j. aquat. biol. (2018) 6(4): 189-197 for male and female individuals in both aphanius species are shown in the figures 9-10. in general, the mean values of this index, especially in the female specimens, showed a decreasing trend during the spawning period. condition factor: we calculated the condition factor separately for each sex in different months, and found monthly differences in this factor. in a. hormuzensis, the condition factor of females was highest in february and march, and reached its lowest value in september. from september to december it showed fluctuations. we observed an increasing trend in the condition factor from december to march. in male specimens, the condition factor was lowest in october, and increased until march. it showed a virtually decreasing trend from july to december (fig. 11). in a. furcatus the condition factor in both sexes, had a peak in march. in both males and females, the condition factor was the lowest in february, while increased up until march (fig. 12). fecundity: the estimated absolute fecundity of a. hormuzensis ranged from 78 (tl = 32.2 mm) to 730 (tl = 51.1 mm), with a mean value of 219.78±66.50 oocytes per fish based on 15 females. the relative fecundity ranged from 14 to 173 oocytes/cm body length (mean±s.d: 79.95±38.03 oocytes/cm) and 68.45 to 518.54 oocytes/g body mass figure 5. variation of mean modified gonadosomatic index (mgsi) in aphanius hormuzensis in different months. figure 6. variation of mean modified gonadosomatic index (mgsi) in aphanius furcatus in different months. . figure 7. variation of dobriyal index (d.i.) of aphanius hormuzensis in different months. figure 8. variation of dobriyal index (d.i.) of aphanius furcatus in different months figure 9. variation of hepatosomatic index (hsi) of aphanius hormuzensis in different months. 194 masoudi et al./ reproductive biology of two sympatric species of tooth-carps (mean±s.d: 237.67±96.87 oocytes/g). for a. furcatus the estimated absolute fecundity ranged from 53 (tl = 26.9 mm) to 102 (tl = 32.04 mm), with a mean value of 93.73±45.37 oocytes per fish based on 15 females. the relative fecundity ranged from 12 to 85 oocytes/cm body length (mean±s.d: 55.35±22.02 oocytes/cm) and from 22.41 to 123.65 oocytes/g body mass (mean±s.d: 64.98±23.37 oocytes/g). discussion this study provided the details on some aspects of reproductive biology of a. hormuzensis and a. furcatus, two endemic tooth-carps of iran. both species exhibit a clear external sexual dimorphism as found in the other aphaniid species and also cyprinodontid and poecilid fishes (esmaeili et al., 2014; teimori et al., 2014, 2018). sexual dimorphism includes color pattern differences as seen in aphaniid fishes or size differences and presences of gonopodium in poecilid fishes. it has been suggested/ shown that sexual selection promotes the evolution of costly secondary sexual traits in males of many animal species (darwin, 1871; sedlácek, et al., 2014). the evolutionary analysis suggests that both sexes of the common ancestor of killifish were relatively plainly coloured (sedlácek et al., 2014). during evolution of the group, changes in male and female colouration were correlated across most killifish lineages (sedlácek et al., 2014). this interspecific pattern suggests that intersexual genetic correlation is an important factor in the evolution of killifish colouration (sedlácek et al., 2014). in the presence of genetic correlation, females bear fully developed or rudimentary colour ornamentation and such female ornaments were more intensively developed in species with more variegated males as predicted by macroevolutionary scenario (sedlácek et al., 2014). the results of the present study showed that both tooth-carps spawn in april and may. the mean values of reproductive indices (gsi, mgsi and di) and high frequency of large oocytes confirmed the spawning season. spawning season for other aphanius species have already been reported. reproduction of a. dispar (now a. kruppi) occurs throughout the day and throughout the year in oman with 69% of females having ripe eggs (up to 41) in april-june. peak spawning in southern iraq in a. dispar (now a. stoliczkanus) is april to june when only a small proportion of eggs in the single ovary are fully developed eggs (al-daham et al., 1977). shafi and figure 10. variation of hepatosomatic index (hsi) of aphanius furcatus in different months. figure 11. variation of mean condition factor (k) of aphanius hormuzensis in different months. figure 12. variation of mean condition factor (k) of aphanius furcatus in different months. 195 int. j. aquat. biol. (2018) 6(4): 189-197 shalli (1986) record up to 73 mature eggs in southern iraq specimens with a diameter of 220 microns. breeding season was may-july. in saudi arabia mature and developing oocytes are observed in fish during the whole year. the egg cycle may be more than one year with spawning in march and april (elhawawi and al-imam, 1984). on the mediterranean coast of egypt, a. dispar spawning occurs from march to september with a peak in july and august. maximum egg diameter is 2.2 mm and size at maturity for females is 30 mm total length. iranian specimens of a. stoliczkanus with large eggs (2.0 mm) on 16 march but young fish (8.9 mm standard length) were caught on 26 november suggesting reproduction is almost year around (see coad, 2017). bibak et al. (2012) examined reproduction a. dispar (now a. stoliczkanus) in the dalaki river, bushehr and found females to predominate (390 fish versus 140), males had a maximum length of 49 mm and females 39 mm, and gonadosomatic indices indicated a spawning period of april to june, with peaks in each of these months of 6.449 and 6.632. according to zare et al. (2015) size composition analysis of oocyte diameter showed that females contained mature oocytes (>1.1 mm) throughout year. absolute fecundity of a. ginaonis ranges from 36 to 832 oocytes per individual (mean: 341±209 oocytes), varying considerably at given length and mass (zare et al., 2015). relative fecundity to total length of a. ginaonis fluctuates from 12 to 169 oocytes/mm (mean: 78±41 oocytes/mm) while relative fecundity to total mass varies from 61-526 oocytes/g (mean: 213±106 oocytes/g) (zare et al., 2015). aphanius ginaonis is an asynchronous, iteroparous spawner producing more than one oocyte clutch in a single reproductive season (zare et al., 2015). the absolute and relative fecundity in a. farsicus have been reported to be 115.7 and 90.01 respectively and it has been related to fish size (total length and total weight) and also to gonad weight (esmaeili and shiva, 2006). the relative fecundity of another aphaniid species a. fasciatus is reported to be136.2 for the mesolongi lagoon, 104.4 and 94.7 for alykes, both in greece (leonardos and sinis, 1998). the mean value of absolute fecundity in a. hormuzensis (219.78±66.50) is greater than a. ginaonis (zare et al., 2015) and lower than a. farsicus (esmaeili and shiva, 2006). the mean absolute fecundity of a. furcatus (93.73±45.37) is lower than a. ginaonis, a. hormuzensis and a. farsicus. fecundity is affected by several factors, such as the size and age of the female (thorpe et al., 1984), life history strategy (morita and takashima, 1998), food supply and temperature (fleming and gross, 1990). reduction of number of produced eggs (fecundity) in a. furcatus might be another case of regressive evolution in this fish. in a. furcatus, several phenotypic characters show reductions; most remarkable are the complete absence of scales and the lower degree of ossification (the reduced thickness of the neural and haemal spines of pu3, in the short length of the spines of the preceding caudal vertebrae and in the presence of thin and short ribs) (see teimori et al., 2014). the fecundity of the studied tooth-carp increases with fish size (total length and body weight). according to jonsson and jonsson (1999), fecundity increases with body size because the amount of energy available for egg production and the body cavity accommodating the eggs increases with fish size. conclusion aphanius hormuzensis and a. furcatus from southern iran demonstrate some reproductive strategies including sexual dimorphism, female biased sex ratio and a lengthy spawning period in response to its habitat. the small body size is another significant factor in the life history of these tooth-carps, which allows fish to colonise and exploit microenvironments. however, the distribution of these species is limited to the waters in southern iran. this limited distribution makes the species highly vulnerable and could result in significant loss or even extinction, if habitats are disturbed or destroyed. due to its restriction to the southern part of iran, the population of these small and beautiful fishes should be conserved because it is an important part of the natural heritage of this country. due to overlapping of 196 masoudi et al./ reproductive biology of two sympatric species of tooth-carps spawning season in these two sympatric species, it seems that other preand post-zygotic factors are responsible for absence of natural hybrids in these studies species in the mehran river. acknowledgments the research work was funded by shiraz university and alzahra university and it was approved by the ethics committee of biology department (su 909830). references al-daham n.k. 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(2023) 11(1): 41-49 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article hormonal failure and osmoregulatory disruption in laboratory food-deprived caspian kutum, rutilus frisii larvae during brackish water challenge mohammad mohiseni department of environmental science and fisheries, lorestan university, khorramabad, iran. article history: received 19 december 2021 accepted 10 december 2022 available online 2 5 february 2023 keywords: starvation osmoregulation caspian kutum salinity abstract: caspian kutum, rutilus frisii, is a valuable species in the caspian sea basin. with the aim of restocking, the iranian fisheries organization (ifo) annually released millions of caspian kutum larvae into the estuaries of the caspian sea. this study was conducted to evaluate the effects of starvation on fish during caspian seawater (csw) adaptation. caspian kutum larvae (0.5±0.1 g) were divided into two groups; one was considered a control fed ad libitum during the experiment and another group was left food deprived. both groups were continuously exposed to the csw challenge (13 ppt) for 7 days and sampling was done on the second, third, fourth and seventh days after initiation of the csw challenge. different physiological factors, including hormones (cortisol, t3, and t4), gill na+/k+-atpase activity, whole body glucose and protein, gill protein, body moisture, and seawater preferences, were analyzed in each sampling time. the results showed that although fed larvae can successfully overcome the physiological changes imposed by the csw challenge, the starved fish indicated significant failures in the most measured parameters and eventually demonstrated significantly lower salinity preferences. therefore, it can be concluded that starvation may negatively affect the success of csw adaptation. since physiological impairment during the csw adaptation period is directly related to the effectiveness of the restocking program, more studies about the feeding condition of caspian kutum larvae pre and post-releasee and the nutritional status of recipient rivers are suggested. introduction rutilus frisii (kamensky, 1901) is an important commercial fish in iran, with a wide distribution from north to south, and its main population is on the southern coast of the caspian sea (ebrahimi and ouraji, 2012; hasanpour et al., 2016; eagderi et al., 2022). having a migratory anadromous habit, caspian kutum migrates to the rivers and lagoons of the southern caspian sea for spawning. the spawning season of caspian kutum is from late march to mid-may. during reproduction, they spawn on aquatic weeds, gravel, and sandy substrates (bastami et al., 2012). because of the severe decline in the annual catch of this species during the 1970s and 1980s due to the demolition of their natural spawning substrates, overfishing, and other factors, ifo launched a restocking project in 1984 (salehi, 2008). in the restocking centers, larval rearing is performed in the earthen ponds until a releasing weight is around 1 g. then, correspondence: mohammad mohiseni doi: https://doi.org/10.22034/ijab.v11i1.1430 e-mail: mohiseni.m@lu.ac.ir dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.6.6 the fingerlings are released into the rivers that carry them toward the caspian sea (jafari et al., 2009). during downstream migration, changes in morphology and physiology will occur to successfully prepare the fish for seawater entry (lerner et al., 2007). this transformation is driven by a change in environmental factors and mediated by significant alteration in specific hormones, including thyroid hormones, cortisol, growth hormone, and insulin-like growth factor i (mccormick, 2001). cortisol has a critical role in hypoosmoregularoty capacity through the development and proliferation of gill chloride cells (mitochondria-rich cells) and upregulating of na+/k+atpase (nka) expression (madsen et al., 1995). the activation of these processes implies an increased energy requirement that eventually can alter gill energy metabolism and whole organism energy partitioning. integral in the seawater adaptation is a reduction in 42 mohiseni / hormonal failure and osmoregulatory disruption in food-deprived caspian kutum glycogen supply, change in body lipids, and depletion of energy stores. this will make the juveniles vulnerable to starvation during the early phase of seawater adaptation (stefansson et al., 2009). starvation is known to affect hypo-osmoregulatory ability in many fish species, including rainbow trout (jürss et al., 1983), mozambique tilapia (jürss et al., 1984), arctic charr (aas-hansen et al., 2003), sea bream (polakof et al., 2006) and atlantic salmon (stefansson et al., 2009). there are several reports which investigated and described the important factors affecting the reproduction of the caspian kutum broodstock (nikoo et al., 2010; shafiei sabet et al., 2010), growth and production of fingerlings in the hatcheries (afraei et al., 2010; ouraji et al., 2011; samarin et al., 2011). the effect of different salinities and fingerling sizes on the survival of the caspian kutum is also reported (enayat gholampoor et al., 2011; hosseini et al., 2012). despite the important role of normal and successful hypo-osmoregulation during the seawater adaptation in further survival and fitness of marine life stage, to the best of our knowledge, there is no information about the effects of starvation on salinity adaptation of caspian kutum fingerling. the present study investigated the behavioral and physiological changes in the starved caspian kutum larvae during caspian seawater csw challenges. material and methods the caspian kutum larvae (n=180, 0.5±0.1 g body weight) were provided from shahid rajaee, the center of fish reproduction (sari, iran) and transferred to the 300-l tank and maintained in normal condition (ph=7.3; temperature=18-20°c and oxygen around saturation level) for two weeks (mohiseni et al., 2017). the larvae were fed 3% of their body weight three times a day on the starter diet for rainbow trout (biomar, france). daily water exchange during adaptation and also experimental periods was 30%. after the acclimation period, larvae were divided into fed and feed-deprived groups (with three replications), and both groups were transferred to the brackish water (13 ppt) simultaneously. the first group was fed ad libitum during the csw challenge. the experimental salinity was made by mixing evaporated full-strength caspian seawater with dechlorinated tap water. the salinity challenge was done (randomly) for both groups for 7 days continuously, and sampling was done on the second, third, fourth, and seventh days after initiation of the csw challenge. the whole-body cortisol was measured according to peterson and booth (2010) with minor modifications. briefly, caspian kutum larvae were dried with a paper towel and weighed before extraction. the sample was then homogenized (buch and holm homogenizer, denmark) in pbs, and aliquots of ethyl ether were added and vortex for 1 min. the samples were then centrifuged and frozen immediately at -20°c, and an unfrozen portion was transferred to a fresh tube for ethyl ether evaporation under nitrogen. the remaining extract was stored at -20°c until ready for enzyme-linked immunosorbent assay (elisa). cortisol was measured with monobind, a cortisol assay kit (usa). thyroid hormone extraction was done based on mukhi et al. (2005). triiodothyronine (t3) and thyroxine (t4) were measured using pishtazteb, elisa assay kits (iran). gill arches were collected for gill na+/k+-atpase (nka) activity. it was determined following mccormick (1993) developed for microplates. the ouabain-sensitive hydrolysis of adenosine triphosphate is enzymatically coupled to the oxidation of nicotinamide adenine dinucleotide, which is directly measured in a microplate reader. glucose was determined by enzymaticcolorimetric test (moss and henderson, 1999). total protein concentrations were analyzed using the bradford method with bovine serum albumin as standard (bradford, 1976). to determine whole-body moisture, 5 larvae were dried with a paper towel and then weighed (based on mg) precisely. afterward, the larvae were dried until full dehydration at 60˚c (approximately 72 h) and then weighed. the difference between wet and dried mass was considered whole-body moisture and reported as a percentage (moustakas et al., 2004). the seawater (sw) preferences were evaluated based on lerner et al. (2007). briefly, the sw preference tank was constructed as two parallel chambers of styrofoam connected by a pvc bridge. two chambers were filled with freshwater (fw) and csw (13 ppt) separately. each chamber was filled just below the bridge. 10 fish from both groups were transferred into the fw chamber and allowed to acclimate for 2 h. the level of fw was then elevated until the connection between the two chambers was formed. afterward, the fish activity was videotaped from above the chambers for 1 h. the videotape was analyzed for the presence of fish in csw at 30 s intervals for 45 min and presented at the percent of fish in csw. trials were conducted in three replications from each experimental group. 43 int. j. aquat. biol. (2023) 11(1): 41-49 all datasets were statistically analyzed by independent sample t-test. pearson correlation was also used to determine correlated factors. all statistical analyses were performed using ibm spss statistics for windows (version 19) at the significance level of p<0.05. the results were reported as mean±se. results hormonal changes in both fed and starved groups are illustrated in figure 1. cortisol levels tend to increase over time and reach its maximum level at the end of the experiment in fed fish, while the pattern of the hormonal change in starved fish showed a significant reduction 7 days after the salinity challenge (p<0.05). similarly, t3 was also increased during the salinity challenge in fed fish and showed a significant difference with starved fish most of the time (p<0.05). the level of t4 in starved fish remained almost unchanged during the experiment and showed significant reduction at 2, 3, and 7 days after the challenge (p<0.05). t3/t4 ratio was consistently higher in fed treatment throughout the experiment, with significant differences on 3, 4, and 7 days after the challenge (p<0.05). gill nka activity was altered in response to the salinity challenge in both groups. the enzyme activity was consistently increased for fed fish over time, despite the transient elevation of enzyme level in starved fish until the 4th day. the enzyme activity dropped significantly at the end of the experiment (table 1). a similar trend was observed for glucose in both treatments, with a recorded significant decrease at 2, 4, and 7 days of salinity challenge for the starved group (p<0.05). although the whole-body protein was approximately unchanged in starved fish, the recorded values for the fed group indicated a slight increment and were higher than the starved group all the time, with a significant difference on the 7th day (p<0.05). the gill protein was elevated to the maximum level in both groups 3 days after the challenge, but the recorded value for fed was higher (p<0.05). the gill protein decreased in both groups, but the reduction rate for starved fish was significantly higher than for fed fish (p<0.05). the whole-body moisture in the starved group tended to decrease over time, but it remained largely stable in fed fish throughout the experiment, resulting in these fish having significantly higher levels on 3, 4, and 7 days after the challenge than those in starved fish (p<0.05). the starved fish were first observed in seawater immediately after the formation of the aqueous bridge, but with time advancement, the percentage of fish in seawater significantly decreased (about 92%) and a b c d figure 1. hormonal change (a: cortisol, b: t3, c: t4 and d: t3/t4 ratio) during seawater challenge in fed and starved caspian kutum. (*) and (**) show a significant difference between fed and starved fish at the same time at p<0.05 and p<0.01, respectively. dissimilar small letters show the differences among different times for fed and dissimilar capital letters show differences among different times for the starved groups (p<0.05). 44 mohiseni / hormonal failure and osmoregulatory disruption in food-deprived caspian kutum reached the minimum (5.22%) at the end of the experiment (fig. 2a, b). on the other hand, fed fish displayed obvious latency (about 12.5 min) to enter csw, but their csw preference behavior was increased until 30 min and remained stable toward 45 min. furthermore, the results exhibited that the most measured osmoregulatory parameters were significantly correlated (p<0.05) in fed fish, whereas a few correlated parameters were found for starved fish (table 2). discussion this study revealed that starvation may lead to endocrine disruption in the csw adaptation period in caspian kutum. cortisol is a mineralocorticoid hormone in teleost and has a direct role in osmoregulatory change and seawater adaptation in anadromous, downstream migratory juvenile fish (mccormick, 2001; nemova et al., 2021). cortisol is also participating in energyproviding through glycogenolysis for normal metabolism and stress response (laiz-carrion et al., 2002). in the current study, whole-body cortisol levels significantly increased over time in fed fish. a prolonged cortisol increase is reported during smolting and successful seawater adaptation in anadromous fish (mccormick, factor time (day) fed starved p-value gill na+k+atpase (nka) (µmol adp/mgpr/h) 2 26.97±3.02 a 27.99±3.42 0.834 3 43.33±3.55 a 33.32±1.51 0.06 4 43.41±4.84 a 41.94±5.72 0.854 7 70.88±10.85 b 38.61±2.06 0.043* whole body glucose (mg/dl) 2 72.95±4.75 56.93±2.26 b 0.048* 3 81.5±26.91 41.21±13.3 ab 0.15 4 105.01±16.06 57.9±9.98 ab 0.03* 7 108.88±0.71 39.89±17.31 a 0.025* whole body protein (mg/dl) 2 3.08±0.17 a 3.11±0.13 0.88 3 3.84±0.97 a 2.89±0.11 0.39 4 5.04±0.38 b 3.67±0.42 0.072 7 4.69±0.57 ab 3.03±0.11 0.047* gill protein (mg/dl) 2 2.19±0.21 1.61±0.08 a 0.062 3 3.34±0.24 2.17±0.17 b 0.018* 4 2.87±0.16 1.48±.08 a 0.002** 7 2.84±0.13 1.21±0.07 a 0.033* whole body moisture (%) 2 69.53±8.02 62.14±2.76 b 0.433 3 66.93±2.05 53.13±1.51 a 0.006** 4 73.28±2.65 60.99±1.85 b 0.046* 7 70.98±2.95 52.33±0.97 a 0.004** values are means±standard error; (*) shows a significant difference between two groups at p<0.05 and (**) shows a significant difference at p<0.01. dissimilar small letters show the differences among different times for fed and dissimilar capital letters show differences among different times for the starved groups (p<0.05). table 1. gill and whole-body parameters change after different times of salinity challenge in fed and starved caspian kutum. figure 2. seawater preferences quantity (a) and pattern (b) for fed and starved fish during seawater challenge. (*) indicate significant differences from the fed group at the same time. 45 int. j. aquat. biol. (2023) 11(1): 41-49 2001; mccormick et al., 2005; lerner et al., 2007; mancera and mccormick, 2019). cortisol also has a promotive effect on the development and proliferation of gill chloride cells, which is directly connected to an increase in gill nka activity (madsen et al., 1995; mccormick et al., 2008). the activation of this process involves an elevation in energy requirement that apparently could alter the gill energy metabolism (laizcarrion et al., 2005). there was a significantly positive correlation between cortisol levels and nka activity in the fed group. therefore, the significant decrease in cortisol levels due to starvation may explain this study's significant reduction in gill nka activity. thyroid hormones failed to increase during the csw challenge in the starved caspian kutum fish. thyroxin (t4) and triiodothyronine (t3) are the principal thyroid hormones involved in the development and growth of fishes (peter and peter, 2009). t4 is the main secreted prohormone by the thyroid gland in teleost. further enzymatic outer ring deiodination will transform the less potent t4 into the bioactive thyroid hormone (t3). in fishes, thyroid hormones have a fundamental role in different physiological processes, including somatic growth, metamorphosis, parr-smolt transformation, bioenergetics, and reproduction (arjona et al., 2010). several studies have shown that thyroid hormones regulate basal and active metabolic rates in different tissues of teleost (narayansingh and eales, 1975; pavlidis et al., 1997; aas-hansen et al., 2003; lópez-bojórquez et al., 2007; jarque and piña, 2014; tovo-neto et al., 2018; deal and volkoff, 2020). several studies have also reported the involvement of thyroid hormones during salinity acclimation. prolonged t4 treatment led to an increase in the number of chloride cells and gill nka activity in atlantic salmon (madsen and korsgaard, 1989). physiological levels of t3 and t4 have also increased chloride cell size and gill nka activity in mozambique tilapia (peter et al., 2000). accordingly, we found a significant positive correlation between nka activity and t3 levels only for the fed group. the liver metabolism may be enhanced during csw adaptation because of its direct involvement in glycogen/glucose turnover in fish. this process will make the glucose available to provide energy requirements for the osmoregulatory phenomenon in different tissues, especially the gill and kidney (sangiao-alvarellos et al., 2003). food deprivation resulted in changes in hepatic energy metabolism, as reported in several teleosts (sangiao-alvarellos et al., 2005; stefansson et al., 2009; costas et al., 2011). these included: (1) elevation of glycogenolysis and gluconeogenesis rate that can be attributed to the increased plasma cortisol concentration, cortisol t3 t4 t3/t4 na +k+ atpase glucose body protein gill protein body moisture salinity preference cortisol fed 1 .663* .177 .764** .675* .626* .319 -.330 .719** .178 starved 1 -.019 -.328 .105 -.186 -.514 -.004 -.354 .489 .327 t3 fed .663* 1 .101 .857** .673* .430 .764** .034 .696* .718* starved -.019 1 .317 .817** .052 -.155 -.194 .063 -.502 .100 t4 fed .177 .101 1 .043 .128 .213 .094 .819** .312 .436 starved -.328 .317 1 -.112 .223 .587* .223 -.147 -.179 -.187 t3/t4 fed .764** .857** .043 1 .839** .488 .487 .004 .685* .697* starved .105 .817** -.112 1 .085 -.336 -.270 -.070 -.421 .217 na+k+ atpase fed .675* .673* .128 .839** 1 .372 .366 .013 .477 .818** starved -.186 .052 .223 .085 1 .370 .510 -.108 .058 -.508 glucose fed .626* .430 .213 .488 .372 1 .131 -.032 .295 .004 starved -.514 -.155 .587* -.336 .370 1 .331 -.281 -.024 -.597 body protein fed .319 .764** .094 .487 .366 .131 1 .176 .486 .691* starved -.004 -.194 .223 -.270 .510 .331 1 -.321 .313 -.445 gill protein fed -.330 .034 .819** .004 .013 -.032 .176 1 -.182 .800** starved -.354 .063 -.147 -.070 -.108 -.281 -.321 1 -.249 .197 body moisture fed .719** .696* .312 .685* .477 .295 .486 -.182 1 .674* starved .489 -.502 -.179 -.421 .058 -.024 .313 -.249 1 -.313 salinity preference fed .178 .718* .436 .697* .818** .004 .691* .800** .674* 1 starved .327 .100 -.187 .217 -.508 -.597 -.445 .197 -.313 1 *. correlation is significant at the 0.05 level (2-tailed). **. correlation is significant at the 0.01 level (2-tailed). table 2. pearson correlation among different osmoregulatory factors during seawater challenge in fed and starved caspian kutum. 46 mohiseni / hormonal failure and osmoregulatory disruption in food-deprived caspian kutum (2) increased liver capacity for glucose transferring, and (3) decreased concentration of the plasma triglyceride and protein. cortisol is one of the most important factors during salinity adaptation and has a special role in chloride cell proliferation and development. chloride cells are the main site of nka that makes a driving force for monovalent ion secretion via gill arcs (madsen et al., 1995; evans et al., 2005). according to the results, the cortisol and nka levels were increased a time-dependent and correlated manner, whereas both factors finally failed to surge in the fasting group during the seawater challenge i.e. gluconeogenesis is the common role of cortisol action in the liver. cortisol is responsible for glycogen fraction and glucose production. accordingly, we found a significant correlation between body cortisol and glucose of caspian kutum fish only in the fed group. osmoregulatory adaptation during the csw challenge is one of the most energy-consuming processes in aquatic animals (madsen et al., 2015). various tissues prefer specific energy resources to overcome the major changes due to the csw challenges. the excess energy requirement of the liver and brain is mainly based on carbohydrates, while amino acids and lactate are more important in the gills and kidneys. therefore, a significant decrease in the whole body and gills protein content in food-deprived fish in the current study was in agreement with the previous studies (sangiao-alvarellos et al., 2005; polakof et al., 2006). on the other hand, the protein content of the body and gills of fed larvae remained approximately unchanged during different times after the csw challenge. during the first hours after csw challenges, the drinking rate is elevated constantly due to the water loss via gills epithelia. in this process, the water is absorbed from the intestine along with excess divalent ion secretions. excess monovalent ions will further be secreted via gills through the efficient actions of chloride cells. this process keeps the hydromineral balance and blood osmolality within the normal range (marshall and bryson, 1998; webb et al., 2001; grosell, 2010). it seems that fed larvae successfully kept their body moisture during the csw adapting periods while starved larvae, probably due to the failures of different osmoregulatory factors, would not be able to ameliorate water loss during the salinity challenge. the body moisture was, therefore, constantly decreased over time and reached the lowest level at the end of the experiment. voluntary movement into csw is a complete organism response dependent on the exact integration between physiological and developmental cues. the external environment can alter the timing and quality of response (lerner et al., 2007). based on the results, there was a behavioral instability in starved larvae, they were observed in csw immediately after the formation of the aqueous bridge, but their presence in csw was subsequently reduced and reached the minimum at the end of the recording. fed larvae showed more stability in csw entrance, and their presence in csw gradually increased and reached the maximum. disruption of csw preference is probably related to the physiological failure of the starved larvae in csw adaptation. several studies have also emphasized on negative effects of food deprivation on the seawater adaptation phase for different species (aas-hansen et al., 2003; taylor and grosell, 2006; stefansson et al., 2009; costas et al., 2011). conclusion this study showed that starving had potentially impaired effects on the osmoregulatory fitness of caspian kutum larvae. key factors related to the csw adaptation and cortisol, nka, and thyroid hormones failed due to starvation, and larvae could not overcome imposed changes during the csw challenge. since the caspian kutum larvae were released mainly in the small size (0.51 g) by ifo through an annual restocking program, the feeding condition of larvae should be considered and monitored before and after their release into the rivers. references aas-hansen ø., johnsen h.k., vijayan m.m., jørgensen e.h. 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(2022) 10(4): 336-343 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article evaluation of growth performance and feed utilization of oreochromis shiranus and coptodon rendalli fed diets combining moringa oleifera leaf meal and cajanus cajan meal jose uachisso savanguane*1, jeremiah kang’ombe2, daniel sikawa2, austin mtethiwa2 1faculty of agronomy science, catholic university of mozambique, p. o. box: 22, cuamba, niassa, mozambique. 2aquaculture and fisheries science department, aqua fish africa centre of excellence, bunda college, lilongwe university of agriculture and natural resources, p.o. box 219, lilongwe, malawi. s article history: received 7 june 2022 accepted 21 august 2022 available online 2 5 august 2022 keywords: growth performance feed utilization moringa cajanus aquaculture abstract: this study evaluated the growth performance and feed utilization of oreochromis shiranus and coptodon rendalli fed on diets combining moringa oleifera leaf and cajanus cajan. a factorial experiment (4×2) in a completely randomized design with three replicates was used. oreochromis shiranus and c. rendalli (22.20±2.03 g) were randomly selected, assigned to 24 experimental units and reared for 90 days. the fish were fed 32% protein diets formulated as follows: diet 1control (0 g/kg m. oleifera leaf and 0 g/kg c. cajan), diet 2 (100 g/kg m. oleifera leaf + 150 g/kg c. cajan), diet 3 (200 g/kg m. oleifera leaf + 300 g/kg c. cajan), and diet 4 (300 g/kg m. oleifera leaf + 450 g/kg c. cajan). the results revealed that there were significant differences in final body weight, body weight gain, and specific growth rate among dietary treatments for both species (p<0.05). coptodon rendalli performed better in final body weight, weight gain, and specific growth (60.94±0.54 g, 39.47±0.53 g and 1.13±0.02 %/d, respectively) than o. shiranus when fed diet 2. however, no significant difference was recorded in condition factor (cf) among the dietary treatments for both species (p>0.05). coptodon rendalli obtained the best apparent feed conversion ratio (2.75±0.06) and protein efficiency ratio (0.73±0.02) than o shiranus, when fed diet 2. it is therefore concluded that diet 2 (100g/kg m. oleifera leaf + 150g/kg c. cajan) recorded the best growth performance and feed utilization for both species and can replace soybean meal as a fish feed ingredient. introduction the increase in the world’s human population in the middle of the 21st century has brought enormous challenges in providing food to livelihoods. aquaculture and fisheries are key areas for food, nutrition and employment of millions of people (fao, 2018). however, studies have demonstrated that the future of the aquaculture industry will depend on the sustainable supply of plant proteins for feeds (hoseini et al., 2017). soybean meal is widely used as a source of protein for livestock and specifically for herbivorous and omnivorous fish in feed formulation (hoseini et al., 2018). however, the high cost of this raw material, associated with the competition of this ingredient for human and livestock consumption, has caused problems for fish farmers (karina et al., 2015). this situation calls for an urgent need to examine other *correspondence: jose uachisso savanguane doi: https://doi.org/10.22034/ijab.v10i4.1395 e-mail: jose.savanguane@gmail.com valuable and locally available resources as alternative protein sources for fish feed (egwui, 2013). several alternative plants have been investigated for their potential use in replacing soybean-based feed. moringa oleifera leaves and cajanus cajan seed have been identified as contributing to fish nutrition with the possibility of reducing th the possibility to reduce dependence on soybean meal (karina et al., 2015; salmon et al., 2017). additionally, given the availability of these plant ingredients in mozambique, m. oleifera leaves and c. cajan seed are considered cheaper than soybean meal. a study by nouman et al. (2014) showed that m. oleifera tolerates adverse conditions, is high yielding and adaptable to different agro ecological zones. their leaves contain 26.4% crude protein and are rich in vitamins and minerals. furthermore, m. oleifera leaves contain saponins and 336 savanguane et al./ moringa oleifera leaf and cajanus cajan in the diet of o. shiranus and c. rendalli phenols as anti-nutritional factors (egwui, 2013). on the other hand, c. cajan is an important leguminous crop that can be grown under rainfed conditions with the least inputs and is ideally suitable for production on small farms (walker, 2015). humans have less preference for c. cajan seed for food consumption (majili et al., 2020). these factors make it inexpensive to be considered as a feed ingredient (foidl et al., 2001). moringa oleifera leaves can replace soybean meal in tilapia diet at 10 to 30% (bello and nzeh, 2013; ganzon-naret, 2014; mehdi et al., 2016; el-elrahman et al., 2017). in the same way, researchers indicated that c. cajan seed can replace soybean meal at 10 to 45% (ndau and madalla, 2015). however, the combined effects of both herbal materials as soybean meal alternatives should be determined in oreochromis shirannus and coptodon rendalli. therefore, the present study aimed to evaluate the effects of combining dietary m. oleifera leaf and c. cajan seed on growth performance and feed utilization of o. shiranus and c. rendalli. materials and methods the study was conducted at bunda fish farm located at lilongwe university of agriculture and natural resources (luanar), in lilongwe, malawi (33°50’e, 14°35’s), from 15th august to 15th november 2019 for 90 days. feed formulation: the following ingredients were used to formulate different diets: m. oleifera leaf, c. cajan seed, fish (eugraliciprys sardella) meal, wheat bran, soybean meal, cassava root, yellow maize flour, additives (vitamin and mineral premixes and amino acids). the trial-and-error method was used to formulate the feed. each treatment contained 32% crude protein. the raw materials were sourced from the faculty of agriculture farm of the catholic university of mozambique (cum) and at 25 de setembro market in niassa province of mozambique. the m. oleifera leaves were dried under shade and processed using a hammer mill to reduce antinutritional contents (nsofor et al., 2012). while c. cajan seed was autoclaved at 121°c for one hour and processed using a hammer mill also to reduce antinutritional contents (salmon et al., 2017). the other ingredients were also milled as above to facilitate the production of feed. first, the ground ingredients were analyzed for chemical composition (table 1) following standard methods (aoac, 2011). the ingredients were used to formulate four diets to replace soybean meal with m. oleifera leaf and c. cajan seed meal at 0, 100, 200, and 300 g/kg, and 0, 150, 300, and 450 g/kg, respectively. these concentrations were selected based on the maximum of 300 g/kg of m. oleifera leaves that can replace soybean meal in the dietary for tilapia (bello and nzeh, 2013; ganzon-naret, 2014; karina et al., 2015; mehdi et al., 2016; el-el-rahman et al., 2017) and maximum of 450 g/kg of c. cajan that can replace soybean in the dietary for tilapia (ndau and madalla, 2015) without causing any negative effect on the fish performance. the feed were formulated locally and amino acids, minerals + vitamins were added to the experimental trial (table 2). all dry materials were mixed and stirred homogeneously and then water was ingredients crude protein (%) dry matter (%) ash (%) crude fat (%) crude fiber (%) gross energy (cal/g) soybean meal (roasted) 43.85±0.16 92.70±0.13 5.01±0.07 15.05±0.04 5.08±0.01 4507±11.55 c. cajan seed (roasted) 35.01±0.22 90.66±0.40 6.88±0.14 16.20±0.21 7.77±0.08 2381±21.93 m. oleifera leaf 27.89±0.18 89.81±0.43 8.89±0.89 6.72±0.03 8.89±0.11 2500±13.22 e.sardella (sundried) 63.50±0.27 92.62±0.84 8.20±0.35 11.86±0.18 0.22±0.01 5019±12.37 yellow maize 8.82±0.24 92.20±0.08 5.20±0.18 2.34±0.53 1.03±0.10 2297±10.40 rice bran 9.28±0.34 92.95±0.13 15.76±0.08 6.55±0.08 14.77±0.66 2326±8.74 wheat bran 14.96±0.28 93.95±0.18 4.34±0.02 3.85±0.91 11.21±0.45 2622±14.89 casava root 1.2±0.28 91.02±0.40 1.82±0.59 1.42±0.17 3.46±0.33 2335±9.70 values are the mean ± standard deviation; n=3 table 1. chemical composition (%) of feed ingredients on dry matter basis 337 int. j. aquat. biol. (2022) 10(4): 336-343 added gradually. the mixed ingredients were pressed into pellets of about 1.5 mm using a manual processing machine (paray et al., 2020). experimental fish: the fish fingerlings were obtained from the propagation of broodstocks in lilongwe university of agriculture and natural resources (luanar). juveniles of 22.20±2.03 g were selected and randomly placed in 24 experimental units. a 4 × 2 factorial experiment in completely randomized design with three replications was used. species had two levels (c. rendalli and o. shiranus) while diets had four levels: (1) control, (2) 100g/kg m. oleifera leaf + 150g/kg c. cajan, (3) 200g/kg m. oleifera leaf + 300 g/kg c. cajan and (4) 300 g/kg m. oleifera leaf + 450 g/kg c. cajan. a total of 24 hapas of 4 m2 each with stoking density of 5 fish/ m2 was used. the total number of 480 fish was used (240 o. shiranus and 240 c. rendalli). fish were hand-fed twice a day (9:00 am and 14:00 pm) at 5% body weight (saravanan et al., 2012). the experiment lasted for 90 days. calculation of fish growth performance: body weight gain, specific growth rate, condition factor and survival rate were determined using the following formulae (adineh et al., 2021): body weight gain (bwg) (g) = mean final weight (g) – mean initial weight (g) specific growth rate (sgr) (%/day) = 100 [(lnwf (g) – lnwi (g)]/t (days) condition factor (k) = [w(g)/l3(cm)]*100 survival rates (sr) (%) = (final number of fishes/ initial number of fishes)*100 where, w is weight in grams and l is standard length in cm. determination of feed utilization parameters: total amount of feed given to the fish during the study period were recorded and the apparent feed conversion ratio and the protein efficiency ratio were calculated using the following formula (hoseini et al., 2022): apparent feed conversion ratio (afcr) = total feed given (g)/weight gain (g) apparent protein efficiency ratio (aper) = weight diets1 soybean meal diet 1 diet 2 diet 3 diet 4 m. oleifera meal 59.65 44.65 29.65 14.65 c. cajan seed meal 0.00 5.65 11.65 17.65 fish meal 0.00 8.65 17.65 26.65 rice bran 3.65 7.65 11.65 14.65 yellow maize meal 8.65 4.65 7.65 3.65 cassava 11.65 11.65 9.65 11.65 wheat bran 4.65 4.65 4.65 4.65 mineral+vitamin2 9.65 10.65 5.65 4.65 monocalcium phosphate 0.50 0.50 0.50 0.50 salt 0.50 0.50 0.50 0.50 lysine% 0.50 0.50 0.50 0.50 methionine % 0.20 0.20 0.20 0.20 lime stone 0.20 0.20 0.20 0.20 analyzed proximate composition 0.20 0.20 0.20 0.20 crude protein % gross energy cal/g 32.59 32.78 32.93 32.76 moisture % 3768.89 3564.85 3346.59 3112.08 ash % 92.70 92.33 91.86 91.53 crude fat % 6.26 6.36 6.86 7.32 crude fiber % 10.83 10.65 10.52 10.39 soybean meal 5.00 5..58 5.92. 6.04 1diets: 1reference, 2test diet (100g/kg m. oleifera leaf+ 150g/kg c. cajan), 3 – test diet (200g/kg m. oleifera leaf + 300g/kg c. cajan) and 4test diet (300g/kg m. oleifera leaf + 450g/kg c. cajan). 2mineral+vitamins: calcium 26%, phosphorous 9 %, salt 4 %, selenium 0.2mg/kg, methionine 100 mg/kg vitamin a 8,000,000 iu, vitamin e 8,000 iu, vitamin d3 3,000,000 iu, vitamin 2,000 mg, pantothenic acid. table 2. dietary and nutrient compositions of the formulated diets. 338 savanguane et al./ moringa oleifera leaf and cajanus cajan in the diet of o. shiranus and c. rendalli gain (g)/protein intake (g) water quality parameters: water quality parameters such as temperature, ph and dissolved oxygen concentration (do) were measured at 20 cm below the water surface twice a day (morning 06: 00 h and afternoon 14:00 h). while ammonia (nh4), nitrate (no3), nitrites (no2) and phosphate (po4) were monitored biweekly using apha method. data analysis: data were analyzed using statistical analysis system (sisvar) v.13 and excel was used to plot graphs. shapiro-wilk and levene’s tests were used to check for normality and equality of variance. therefore, analysis of variance (two-way anova) was performed to test for significant variation in different parameters among the treatment combinations at 0.05 alpha level. turkey`s test was used to separate differences among individual treatments. results growth performance: the results presented in table 3 indicated that for weight gain, final body weight and specific growth rate (sgr) presented significant difference of diets and species as well as the interaction between factors (p<0.05). contrarily, for condition factor (cf), the isolated effect of diet, as well as the interaction between the factors, were not significantly different (p>0.05). means of final body weight, weight gain and sgr as a function of the effect of diets and species are presented in table 3. the results of final body weight and body weight gain, indicated that for species c. rendalli, diet 1 (control), diet 3 (200g/kg m. oleifera leaf + 300 g/kg c. cajan) and diet 4 (300 g/kg m. oleifera leaf + 450 g/kg c. cajan) were not significantly different. however, there were significantly different in diet 2 (100 g/kg m. oleifera leaf + 150 g/kg c. cajan). for o. shiranus, the final body weight and body weight gain were not significantly different in diets 1 and 2 but significant differences were observed in diets 3 and 4. the species c. rendalli had better performance than o. shiranus when fed diet 2 (100 g/kg m. oleifera leaf+ 150 g/kg c. cajan), resulting in an average of 60.94±0.54 g and 39.47±53 g for final body figure 1. biweekly increase in weight of o. shiranus and c. rendalli fed diets combining m. oleifera leaf and c. cajan meal. *diets: 1control, 2(100 g/kg m. oleifera leaf + 150 g/kg c. cajan), 3 – (200 g/kg m. oleifera leaf + 300 g/kg c. cajan) and 4(300 g/kg m. oleifera leaf + 450 g/kg c. cajan). data are presented as mean ± sd (n =3). 339 int. j. aquat. biol. (2022) 10(4): 336-343 weight and body weight gain respectively (table 3). the sgr results showed that c. rendalli differed significantly from o. shiranus when fed diet 3 (200g/kg m. oleifera leaf + 300 g/kg c. cajan) and diet 4 (300 g/kg m. oleifera leaf + 450 g/kg c. cajan). therefore, c. rendalli presented the highest sgr with 1.13±0.02 when fed diet 2. for condition factor (cf), the results indicated that the species c. rendalli and o. shiranus significantly differed among different treatment diets. contrary, diets and the interaction between factors did not present differences (table 3). the survival rate of o. shiranus and c. rendalli fed diets combining m. oleifera leaf and c. cajan were 100% in 6 treatments except for 2 treatments, namely diet 4 fed to o. shiranus and the same diet 4 fed to c. rendalli that were 95 and 98.35%, respectively. the biweekly growth of c. rendalli and o. shiranus indicated an exponential increase in weight with an increase in time (fig. 1). the c. rendalli, presented a higher average of 60.94±0.54 (g) when supplemented with died 2. while the o. shiranus specie showed weak growth in all experimental phases with the lowest final average weight of 47.85±0.54 (g) when supplemented with diet 4. feed utilization: the results presented in table 4 showed that for afcr and aper, the isolated effect of diet and species and the interaction between factors were significantly different (p<0.05). means (±se) of afcr and aper as a function of the effect of diets and species are presented in table 4. the afcr showed that c. rendalli had no significant difference from all diets (p<0.05) with a mean of 2.82±0.06. for o. shiranus, the afcr had no significant differences in diets 1, 2 and 3, with a mean of 2.85±0.06. however, o shiranus had significant differences in diet 4 with a mean of 3.19±0.06 kg of feed to produce 1 kg of fish. the two species performed well when fed diets 1, 2 and 3 but c. rendalli performed better than o. shiranus when fed diets 2 and 3 with the mean of 2.75 ±0.06 and 2.78±0.06, respectively (table 4). the aper results indicated significant differences among treatment diets and species (p<0.05), as well as evidencing the existence of significant interaction between the factors (table 4). the results from aper showed that c. rendalli and o. shiranus differed significantly when supplemented diets 2 and 4. for c. rendalli, all diets did not differ significantly from each other. for the o. shiranus, diets 1, 2 and 3 did not differ significantly except for diet 4. the best performance was obtained by c. rendalli compared to o. shiranus with an average of 0.73±0.02 when fed to diet 2 (table 4). water quality parameters: the results of water quality parameters, did not show significant differences in diets and species (p>0.05) nor the interaction between factors for all the analyzed variables. the means (±se) of water quality parameters (temperature, dissolved oxygen and ph) table 3. means (± se) of initial body weight, final body weight, weight gain, sgr and cf as a function of the effect of diets and species. diets1 initial weight (g) final weight (g) weight gain (g) sgr cf c. rendalli o. shiranus c. rendalli o. shiranus c. rendalli o. shiranus c. rendalli o. shiranus c. rendalli o. shiranus diet 1 22.28±0.16 22.36± 0.16 57.16±0.54 aa 60.88±0.54 ab 34.86±0.53 aa 38.08±0.53ab 1.07±0.02 aa 1.10±0.02 aa 1.81±0.05 aa 1.66±0.05ab diet 2 22.69±0.16 22.33±0.16 60.94±0.54ba 58.9±0.54ab 39.47±0.53 ba 36.50±0.53ab 1.13±0.02aa 1.09±0.02aba 1.83±0.05 aa 1.68±0.05ab diet 3 22.36±0.16 21.86±0.16 56.48±0.54 aa 53.55±0.54 bb 34.90±0.53 aa 31.48±0.53bb 1.05 ± 0.02 aa 1.00±0.02 ba 1.84±0.05 aa 1.69±0.05ab diet 4 22.21±0.16 22.10±0.16 55.34±0.54 aa 47.8±0.54 cb 33.81±0.53 aa 27.59±0.53cb 1.06 ± 0.02 aa 0.89±0.02 cb 1.88±0.05aa 1.71±0.05ab p-value diets 0.790n.s 0.000** 0.000** 0.000** 0.744ns species 0.060n.s 0.000** 0.000** 0.005** 0.001** diets x species 0.286n.s 0.000** 0.000** 0.007** 0.998ns 1pairs of means followed by the same lowercase letter in the vertical direction are not significantly different at 0.05 level (p>0.05) and, pairs of means followed by the same uppercase letter in the horizontal direction, do not present statistical differences between them by the test of tukey (p<0.05). 1diets: 1 control (0 g/kg m. oleifera leaf + 0 g/kg c. cajan), 2 (100 g/kg m. oleifera leaf + 150g/kg c. cajan), 3 (200 g/kg m. oleifera leaf + 300 g/kg c. cajan) and 4 (300 g/kg m. oleifera leaf + 450 g/kg c. cajan). data are presented as mean±se (n = 3). 340 savanguane et al./ moringa oleifera leaf and cajanus cajan in the diet of o. shiranus and c. rendalli were recorded with 19.91±0.09 am, 27.03±0.23 pm; 4.64±0.04 am 5.28±0.03 pm; 7.39±0.01am and 7.60±0.01 pm, respectively. additionally, the recorded results of ammonia (nh4), nitrate (no3), nitrite (no2) and phosphorous (po4) with the mean (±se) of 0.05±0.01, 5.56±0.13, 0.016±0.01 and 0.03±0.02, respectively. discussions in aquaculture, a proper diet with optimum nutrition have been recognized as a main factor in promoting a satisfactory growth performance (adewumi, 2018). the results indicated that c. rendalli had better performance in almost all the growth parameters, compared to o. shiranus when supplemented with diet 2 (100 g/kg m. oleifera leaf+ 150 g/kg cajanus cajan). from there, an increment on the level of inclusion of above 100 g/kg of m. oleifera leaf has decreased growth parameters. a similar result was reported by richter et al. (2003) and hussain et al. (2017) who found that there was a decrease in the growth performance at a level of inclusion of moringa leaf above 100 g/kg. the explanation for this may be attributed to what was reported by han et al. (2000), who justified a decrease in growth performance as a result of the presence of anti-nutritional factors such as saponins and phytate in the moringa leaves. according to chen et al. (2011), high levels of saponin in the feedstuff significantly depressed the feed intake and growth responses. the same scenario was observed by bello and nzeh (2013) when studying the effects of varying levels of m. oleifera leaf meal diet on the growth performance of clarias gariepinus. they found the better performance of nutrients utilization and survival parameters at 100 g/kg of inclusion without any negative effects on the growth and feed efficiency. previous studies observed that the specific growth of nile tilapia fed 15% of soaked c. cajanus, had a specific growth rate of 0.75±0.05% day-1 (ndau and madalla, 2015). in the current experiment, the specific growth overcomes for both c. rendalli and o. shiranus. when comparing the two fish species, the results indicated that c. rendalli had a positive response on growth performance parameters when fed by m. oleifera + c. cajanus combined diet than o. shiranus. this could have been because different fish species have different preferences for feed and their digestibility. accordingly, c. rendalli is a largely non-selective feeder and it eats a wide range of territorial plants when compared to o. shiranus (chikafumbwa et al., 1991). despite the low growth performance observed in o. shirannus, the m. oleifera + c. cajan based diets provided good health conditions for both c. rendalli and o. shiranus. the condition factor (general well-being of fish) was increasing as the level of inclusion of moringa and cajanus were increasing. this may be attributed to the table 4. means (±se) of afcr and aper as a function of the effect of diets and species. diets1 afcr2 aper3 c. rendallii o. shiranus c. rendalli o. shiranus diet 1 2.87±0.06aa 2.78±0.06aa 0.66±0.02aa 0.67±0.02aa diet 2 2.75±0.06aa 2.84±0.06aa 0.73±0.02aa 0.66±0.02ab diet 3 2.78±0.06aa 2.92±0.06aa 0.67±0.02aa 0.65±0.02aa diet 4 2.88±0.06aa 3.18±0.06bb 0.65.3±0.02aa 0.54±0.02bb p-value: p = 0.05 diets 0.005** 0.007** species 0.022** 0.002** diets x species 0.039** 0.008** pairs of means followed by the same lowercase letter in the vertical direction, do not present statistical differences between them by the tukey test (p<0.05) and, pairs of means followed by the same uppercase letter in the horizontal direction, do not present statistical differences between them by tukey test (p<0.05). 1diets: 1 control, 2 (100 g/kg m. oleifera leaf + 150 g/kg c. cajan), 3 – (200 g/kg m. oleifera leaf + 300 g/kg c. cajan) and 4(300 g/kg m. oleifera leaf + 450 g/kg c. cajan). 2afcrapparent feed conversion ratio, and 3 aper apparent protein efficiency ratio. data are presented as mean ± se (n =3) 341 int. j. aquat. biol. (2022) 10(4): 336-343 fact that m. oleifera leaves are rich in vitamins and minerals which can increase as we increase the inclusion in the diet (nouman et al., 2014). the study indicates that the highest cf was achieved in d4 when fed to c. rendalli with a mean of 1.88±0.05 and the lowest in diet 1 when fed to o. shiranus with a mean of 1.66±0.05. those figures are greater than 1 that indicated that the fish were in good condition (abobi, 2015). a condition factor of less than 1 indicates that fish are in bad health condition (rajkumar, 2006). previous studies conducted by odedeyi and ayegbusi (2018) found condition factors ranging from 1.07 to 1.08 when fishes were fed diets containing 150 and 300 g/kg m. oleifera leaf and justified this because of good utilization of the nutrients in the diet. the results in the present study are above these figures when soya bean is replaced at 25, 50 and 75% with a combined m. oleifera leaf and c. cajan. this could be because of combining m. oleifera leaf with c. cajanus is less harmful than soya bean (bello and nzeh, 2013). the authors argued that the anti-nutritional factors such as polyphenols, amylase and protease inhibitors, which are a known problem in most legumes, are less problematic in c. cajanus than in soybean. the results of the present study, clearly show that the combined m. oleifera leaf and c. cajanus can replace soya bean meal without affecting the fish health condition with a 100% survival rate up to 200 g/kg m. oleifera leaf + 300 g/kg c. cajan incorporated in the diets. feed utilization of o. shirannus and c. rendalli fed on combined m. oleifera and c. cajan revealed that once introduced the m. oleifera leaf and c. cajanus to diets 2 and 3, the afcr remained the same as the control for both fish species. however, with an increment in the levels of combined m. oleifera and c. cajanus in the diets, the afcr increased and inversely the aper decreased. the same trend was observed by richter et al. (2003) who concluded that a higher level of m. oleifera in diets suppress growth performance. the increase in afcr was attributed to anti-nutrients such as phenol, tannins, phytates and saponins (richter et al., 2003). anti-nutritional factors such as phytic acid can interfere with the nutritional value of feed, therefore, reducing mineral absorption, protein digestibility and resulting in toxicity and health problems when present in high concentrations in feedstuff (samtiya et al., 2020). although the decrease in feed utilization for the two species with high doses of moringa + c. cajanus in the feed, c. rendalli showed to use the feed compared to o. shiranus efficiently. this may be due to the poor digestibility and palatability of plant-based diets for o. shiranus (skelto, 2001). although the two species are omnivorous, c. rendalli is more of a plant feeder than o. shiranus (koekemoer and steyn, 2014). however, the feed conversion rate found by ndau and madalla (2015) was 2.77±0.79 and 2.70±0.56 in correa et al. (2020). these results are in the same range as observed in the present study indicating that c. cajan can replace the soya bean meal. the results of water quality parameters indicated no significant effects on c. rendalli and o. shiranus and had no significant differences across treatments and the levels were within the recommended concentration. the mean (±se) of water quality parameters (temperature, dissolved oxygen and ph) ranged from 19.91±0.09 am, 27.03±0.23 pm; 4.64±0.04 am 5.28±0.03 pm; 7.39±0.01am and 7.60±0.01 pm, respectively. additionally, ammonium (nh4), nitrate (no3 -), nitrite (no2 -) and phosphorous (po4 3-) ranged from mean (±se) of 0.05±0.01, 5.56±0.13, 0.016±0.01 and 0.03±0.02, respectively. the water quality parameters recorded during the experiment were within the recommended ranges as provided in bhatnagar, (2013); jaganmohan and kumari, (2018). conclusion it is concluded that the partial replacement of soya bean meal by the combination of m. oleifera leaves + c. cajan at levels of 100g/kg m. oleifera leaf + 150g/kg c. cajan improves growth performance, feed utilization and general health condition of fish. furthermore, the c. rendalli species performed better when fed the diets in question than o. shiranus. acknowledgement we would like to express our special gratitude to those 342 savanguane et al./ moringa oleifera leaf and cajanus cajan in the diet of o. shiranus and c. rendalli who supported this research in one way or the other at deutscher akademischer austauschdienst (daad) for funding the study, the host lilongwe university of agriculture and natural resources in malawi (luanar) and the sender catholic university of mozambique (cum) in mozambique. references abobi s.m. 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(2015). pigeon pea in mozambique: an emerging success story of crop expansion in smallholder agriculture. report. modernizing extension and advisory services project, university of illinois at urbana-champaign, illinois, usa. international journal of aquatic biology (2014) 2(6): 319-324 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2014 npajournals. all rights reserved original article concentration of heavy metals (pb, cd) in muscle and liver of perca fluviatilis and tinca tinca in anzali wetland, southwest of the caspian sea vahid eslami1, masoud sattari*1, javid imanpour namin1, seyed davood ashrafi2 1department of fisheries sciences, faculty of natural resources, university of guilan, sowmeh-sara, iran. 2department of environmental health, school of health, guilan university of medical sciences, rasht, iran. article history: received 10 july 2014 accepted 30 november 2014 available online 2 5 december 2014 keywords: anzali wetland heavy metals muscle liver abstract: anzali wetland is one of the most important aquatic ecosystems of iran located at southwest of the caspian sea. this wetland is a habitat for valuable fish with vital role in life cycle of this ecosystem. assessment of pollutants concentration is rational due to complications of determining biological effects in a habitat. the present study examined the concentration of lead (pb) and cadmium (cd) in muscles and livers of two fish species i.e. perca fluviatilis and tinca tinca collected from anzali wetland, and their relationships with fish size (length and weight). the results showed the highest concentration of metals in liver, and the lowest in muscle tissues of both species. highest concentrations of cd (0.09) and pb (3.66) were recorded in liver tissue of t. tinca. the results also showed significant negative correlation between metal concentrations and fish size. highly significant (p<0.01) negative relationships were observed between fish length and pb concentrations in liver of p. fluviatilis. cd and pb concentrations in liver of p. fluviatilis and cd concentrations in the liver of t. tinca showed significant negative relationships (p<0.05) with size factors. the concentrations of pb and cd were lower than the maximum acceptable concentrations for fish proposed by maff thus safe for human utilization. introduction awareness of heavy metal (hm) concentrations in fishes is essential in terms of management and human consumption (rauf et al., 2009). anthropogenic activities constantly enhance the amount of hms in environment, especially in aquatic ecosystems. pollution of hms in aquatic ecosystems is increasing at an alarming rate worldwide due to human activities (malik et al., 2010). hms enter water reservoirs via atmosphere, drainage and soil erosion. therefore, heavy metal pollutions are of great concern worldwide, and have a great ecological significance due to their toxicity and accumulative behavior. hence, hms can damage aquatic organisms (matta et al., 1999). researches have showed that fishes accumulate hms in their tissues and their concentrations depend on many factors * corresponding author: masoud sattari e-mail address: msattari@guilan.ac.ir such as concentration and duration of exposure, salinity, temperature, hardness of water and metabolic rate of organisms (pagenkopf, 1983; allen, 1995). in polluted waters, hms accumulate in organisms directly through skin and gill or indirectly via food chains (sinha et al., 2002; sure, 2003). hms cause mutation, disturb immune responses, change blood parameters, decrease organism’s adaptation qualities and increase aquatics susceptibility to diseases. also, hms have toxic effects, altering physiological activities and biochemical parameters both in tissues and blood (larsson et al., 1985; nemesok and huphes, 1988; abel et al., 1986). metals such as iron, copper, are essential due to their vital role in biological systems, whereas, lead and cadmium are non-essential and toxic. the essential metals can be toxic when their 320 international journal of aquatic biology (2014) 2(6): 319-324 concentration in aquatic environments increase beyond the tolerable level of organisms. since the toxic effects of metals have been recognized, hm levels in the tissues of aquatic animals are occasionally monitored. because the hms concentration in tissues reflects past exposure via water and/or food, hence it can demonstrate the current situation of animals before toxicity affects the ecological balance of populations in the aquatic environment and to assess their risk on human health (canli et al., 1998). therefore, this study was conducted to assess the concentrations of pb and cd, in muscle and liver of perca fluviatilis and tinca tinca collected from anzali wetland to estimate risk assessment of these fish species on consumer health. material and methods sixteen specimens of p. fluviatilis and twenty-three specimens of t. tinca were caught by local fishermen from anzali wetland, located at the southwest of the caspian sea, at 39°28'n, 49°25'w (fig. 1). the specimens were transported to the laboratory on ice without delay and kept frozen at -25°c until further analyses. the age of specimens were determined using scales according to bagenal and tesch (1978). in addition, total length and weight of specimens were measured to nearest 1 mm and 1 gr, respectively. following dissection, the liver and muscle tissues were removed, thawed (in an oven set at 90°c) and a weighted sample (0.5 g) of homogenized tissue was taken from each specimen. each sample was placed in a teflon digestion vessel with 12 ml mixture of nitric acid and perchloric acid (3:1 v/v) (merck) (canli and atli, 2003). the mixture was heated to 120˚c for 45 minutes until the tissue was dissolved. the digests were diluted by adding distilled water prepared from stock standard solution (merck). metal concentrations were measured using an inductive coupled plasma mass spectrometer (icp-ms-300d) and metal concentration in a tissue was presented as µg metal/g dry weight. data were plotted on graphs to show their distributions. the linear regression were applied on data to analyze the relationships between size of specimens and hms concentrations in the tissues. the concentration of hms in tissues were also compared using one-way anova test. all statistical analyses were carried out using spss statistical programs (version 16). results table 1 shows numbers, length and weight ranges and length-weight relationships of examined specimens. higher cd concentrations were observed in liver tissues of both species. pb concentrations in both tissues were higher than those of cadmium, especially in the liver (table 2). table 3 shows the relationships between metal concentrations and fish length and weight. significant negative relationship were found between length (and weight) of t. tinca and cadmium levels in its liver (p<0.01). furthermore, in the liver of p. fluviatilis negative relationships were observed between length and hms (cd and pb) levels (p<0.05) (table 3). discussion heavy metals are considered as the most important pollutants of aquatic environments because of their toxicity and accumulation. the toxic effects of heavy metals, particularly cadmium and lead have been figure 1. the map of anzali wetland at southern region of the caspian sea (north iran). 321 eslami et al/ heavy metals (pb, cd) in muscle and liver of perca fluviatilis and tinca tinca widely studied (inskip and piotrowsiki, 1985; kurieshy and de siliva, 1993; narvaes, 2002; nishihara et al., 1985; schoerder, 1965; venugopal and luckey, 1975). cadmium and lead have no known biological functions in human physiology and might potentially be toxic even at trace concentrations (robert, 1991). the symptoms of acute cadmium toxicity include high blood pressure, kidney damage, destruction of testicular tissue and destruction of red blood cells (gupta and mathur, 1983). hence information regarding levels of heavy metals content in fish species may have some advantageous for reducing their risk in public health (domingo et al., 2007). in this study, metal concentrations varied in both examined fish species. these difference may be related to differences in ecological requirements, swimming behaviors, metabolic activities between fish species, feeding habits (mormede and davies, 2001; romeoa et al., 1999; watanabe et al., 2003), species n age lenght rang (cm) weight rang (gr) equationa r value p. fluviatilis 16 1-4 17.6-23.4 87.5-156.3 y=0.0793x+10.897 0.961 t. tinca 23 1-3 13-21 35 -144 y=0.0708x+11.157 0.951 ay is total length (cm) and x is total weight (g). table 1. size ranges and the relationships between weight and total length of p. fluviatilis and t. tinca from anzali wetland. fish tissue cadmium lead mean ± sd mean ± sd p. fluviatilis muscle 0.003 ± 0.001 0.62 ± 0.15 t. tinca 0.03 ± 0.02 1.15 ± 0.42 p. fluviatilis liver 0.08 ± 0.02 1.2 ± 0.28 t. tinca 0.09 ± 0.03 3.66 ± 0.5 metal concentrations among the tissues from different fishes were compared statistically using one-way anova. all comparisons were statistically significant (p<0.05). table 2. the concentrations (µg /g d.w.) of heavy metals in the tissues of p. fluviatilis, and t. tinca collected from anzali wetland. fish tissue data cadmium lead p. fluviatilis muscle aequation y=18.47+(-422.44)x y=0.85+(-0.011)x p value bns ns liver equation y=24.09+(-48.15)x y=3.73+(-0.012)x p value *(*) *(*) t. tinca muscle equation y=16.52+(-11.71)x y=0.75+0.02x p value (ns) ns ns(ns) liver equation y=0.23+(-0.008)x y=1.545+(-0.009)x p value **(*) ns(ns) ain the equations, y is metal concentration (µg/g d.w.) and x is total length (cm) of fish. asterisks indicate significant results. bns, not significant, p>0.05. * p<0.05 ** p<0.01 table 3. the relationships between heavy metal concentrations and total lengths and weights of fish (p values are given in parenthesis) in the tissues of p. fluviatilis and t. tinca.a 322 international journal of aquatic biology (2014) 2(6): 319-324 age and size of fish (al-yousuf et al., 2000; linde et al., 1998) and their habitats (canli and atli, 2003). in addition, the results showed that metal concentrations in the liver was higher than muscle tissue of both fish species. the dissimilarity in metal concentrations in various tissues is due to the induction of metal-binding proteins i.e. metallothioneins in liver. it is well-known that large quantity of metallothionein induction occurs in liver tissue of fishes (heath, 1987; canli and furness, 1993a, b; roesijadi and robinson, 1994). yilmaz et al. (2007) reported highest accumulation of cadmium, cobalt and copper in the liver of leuciscus cephalus and lepornis gibbosus, whereas the lowest accumulation was observed in muscle tissues of these species. it is commonly known that muscle is not a tissue in which hms accumulate (legorburu et al., 1988). similar results in a number of fish species (karadede and unlo, 2000) show that muscle is not an active tissue in accumulating hms. canli and atli (2003) recorded the highest concentration of cadmium in liver and the lowest in muscle tissues of the fish. based on our results, the concentration of lead was higher than that of cadmium, particularly in the liver. the results showed a negative relationships between fish sizes viz. length and weight and hm levels. nussey et al. (2000) showed that accumulation of hms (cr, mn, ni, and pb) were reduced with increasing the length of labeo umbratus. widianarko et al. (2000), showed the relationship between hms (pb, zn, and cu) concentration and size of poecilia reticulata with a significant decline in lead concentrations with increasing fish size. metabolic activities play an essential role in hm accumulation in aquatic organisms (heath, 1987; langston, 1990; roesijadi and robinson, 1994). metabolic activity of a young individual is generally higher than that of older ones and therefore hm accumulation is higher in younger fish than elders (elder and collins, 1991; douben, 1989; canli and furness, 1993b; nussey et al., 2000; widianarko et al., 2000). a probable reason for observed negative relationships between hm concentrations and size may be related to differences in metabolic activities between younger and older fish. douben (1989) also showed that metal accumulation could reach a stable condition after a certain age. on the other hand, the dilution of tissue metal concentrations due to growth and/or lowered metabolic activity in old individuals may not be seen if metal concentration in water is higher than the capacity of these parameters. in this case, continuous accumulation of hms may occur and positive relationships may be observed among animals in different sizes. high concentration of hms in water can postpone fish growth causing variations in fish size (heath, 1987; weis and weis, 1989; friedmann et al., 1996). the concentrations of cd and pb in examined t. tinca and p. fluviatilis specimens were below the guidelines for food summarized by maff (cd, 0.2 μg/g wet wt.; pb, 2.0 μg/g wet wt.) 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(2014) 2(2): 53-57 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article penicillin improves the milt quality of persian sturgeon, acipenser persicus during short-term storage mostafa halimi*1, rouhallah norousta2 1department of fishery college, islamic azad university, babol-branch, iran. 2science and research branch, islamic azad university, tehran, iran. article history: received 2 march 2013 accepted 14 april 2013 available online 2 5 april 2014 keywords: penicillin sturgeon milt antibiotic abstract: this study was conducted to examine the effects of antibiotic (5000 units of penicillin) on sperm quality of persian sturgeon, acipenser persicus during 9 days in vitro storage of milt. for this purpose, the milt samples were stored in the presence and absence of 5000 units of penicillin. freshwater was used as sperm activator. the milt samples were stored at 4°c and the motility indices were measured 0, 3, 6 and 9 days after storage. the sperm duration and percentage of sperm motility decreased after 6 days of storage both in the presence and absence of antibiotic, although this decrease was more significant in the absence of antibiotic. after 9 days of storage, the lowest values of sperm motility indices was recorded for antibiotic receiving milt samples while no motile spermatozoa observed for antibiotic-free milt samples. in conclusion, our results demonstrated that 5000 units of penicillin improve the persian sturgeon milt quality during short-term storage. introduction the short and long term storage of fish spermatozoa are widely used to enhance the efficiency of artificial propagation of fish especially endangered species. short-term storage of fish milt is useful to reduce the risk of disease transmission, to decrease the costs of brood stock holding in the hatchery, to synchronize male and female gamete availability and to transport the milt samples between different regions (cloud et al., 1990; degraaf and berlincky, 2004). the milt quality during short-term storage is affected by temperature, oxygen supply, sterility, addition of antibiotics, and proper gas exchange (scott and baynes, 1980; babiak and dabrowsky, 2003; jensen and alderdice, 1984). some studies showed that the anaerobic conditions and associated microbial contaminations may reduce sperm motility and viability during short-term storage of fish milt. for example in channel catfish, ictalurus punctatus, the * corresponding author: mostafa halimi e-mail address: m_halimi82@yahoo.com milt quality decreased during 10 days storage at 4°c due to the increasing load of bacteria and subsequently the production of extracellular enzymes and consumption of oxygen (jenkins and tiersch, 1997). few studies have used antibiotics in order to the inhibition of bacterial activity during short-term storage of fish milt. in this respect, data is very rare about sturgeon fishes especially persian sturgeon, acipenser persicus. for instance, the milt quality of paddlefish, polyodon spathula in terms of sperm motility duration and percentage improved in the presence of 5000 units of penicillin + 5 mg streptomycin/ml (brown and mims, 1995). the sturgeons are ecologically and economically valuable fish species that have been considered in iucn red list due to significant decreases in their stocks in the nature. at now, the short-term storage of sturgeon milt is used widely in the hatcheries. the 54 halimi and norousta/ int. j. aquat. biol. (2014) 2(2): 53-57 increase of short-term storage efficiency can enhance the management of sturgeon propagation and aquaculture in hatchery. therefore, this study was aimed to examine the effects of antibiotic (5000 units of penicillin) on viability of persian sturgeon spermatozoa during short-term storage of milt. material and methods adult persian sturgeon were obtained from shahid beheshti artificial sturgeon propagation and rearing center (rasht, iran) during spawning season. after spermiation, the milt samples were collected by 50 ml syringe to prevent contamination with feces, urine, blood, or water, poured into separate sterilized plastic petri dishes. immediately after milt collection, 10 milt samples with good quality obtained from separate males were considered for the experiment. 5000 units of penicillin were added to 5 milt samples and 5 antibiotic-free milt samples were considered as control group. after addition of penicillin, the milt samples were stored in refrigerator at 4°c. the sperm motility indices were measured according to alavi et al. (2006) four times including 0, 3, 6 and 9 days after storage. to induce the initiation of sperm motility, a 50 µl drop of freshwater placed on a glass slide and then a drop of 1 µl fresh sperm was diluted using a microsampler. then, sperm motility was measured according to rurangwa et al. (2004) by a semi-quantitative method. in this regard, the motility was recorded by a video camera coupled with the optical lens of microscope. at the end, the video recordings were reviewed and the motility was presented as the percentage and duration of motility after the onset of motility until 100% of spermatozoa were immotile. only forward-moving sperm were considered motile, those simply vibrating or turning on their axes was considered immotile (aas et al., 1991). the spss software (version 16) was used for data analysis. the normality of data was investigated by kolmogorov-smirnov test but because of percentage data (percentage of motile spermatozoa) did not have a normal distribution, proportional data were converted by angular transformation (arcsin√p). one-way analysis of variance (anova) was employed to compare the means of sperm motility indices in different times of storage. when significant f-ratios were calculated by anova, the tukey test was applied to identify which groups were different. also, the independent samples t-test was used for the comparison of the means of sperm motility indices between antibiotic receiving and antibiotic-free milt samples. results in antibiotic receiving milt samples of persian sturgeon, the duration and percentage of sperm figure 1. changes of duration of persian sturgeon (acipenser persicus) sperm motility during short-term storage of antibiotic receiving milt samples. different letters indicate significant differences among samples (p<0.05). 55 halimi and norousta/ int. j. aquat. biol. (2014) 2(2): 53-57 motility were stable in 0 and 3 days (p>0.05) and then decreased after 6 and 9 days of storage (fig. 1, fig. 2) (p<0.05). in antibiotic-free group, the duration and percentage of sperm motility decreased significantly after 3 days of storage so that no motile spermatozoa was observed after 9 days of storage (fig. 3, fig. 4) (p<0.05). decreases in sperm motility was more significant during the storage days in the absence of antibiotic. the overall values of sperm motility duration and percentage in antibiotic receiving milt samples was significantly higher than that of antibiotic-free one (p<0.05) (table 1). discussion our results demonstrated that 5000 units of penicillin improve the milt quality of persian sturgeon in terms of sperm motility and percentage of motile spermatozoa during 9 days short-term storage at 4°c. studies on other fish species have confirmed that antibiotics such as penicillin, streptomycin and erythromycin can maintain the viability of spermatozoa during milt storage (stoss et al., 1978; stoss and refstie 1983; brown and mims, 1995; jenkins and tiersch, 1997; segovia et al., 2000). in rainbow trout, oncorhynchus mykiss decreasing of fertilization capacity during milt figure 3. changes of duration of persian sturgeon (acipenser persicus) sperm motility during short-term storage of antibiotic-free milt samples. different letters indicate significant differences among samples (p<0.05) figure 2. changes of percentage of persian sturgeon (acipenser persicus) sperm motility during short-term storage of antibiotic receiving milt samples. different letters indicate significant differences among samples (p<0.05). 56 halimi and norousta/ int. j. aquat. biol. (2014) 2(2): 53-57 storage was significant in absence of 125 units of penicillin and 125 μg streptomycin per ml sperm than antibiotic receiving milt samples. in paddlefish, an extender containing antibiotic provided better fertilization rate after 14 and 25 days of storage. anaerobic conditions and associated microbial contamination may reduce sperm motility and viability. channel catfish sperm maintains its motility at 4°c for 10 days, until the bacterial infection especially by genus pseudomonas (65%), decreased the percentage and duration of sperm motility by production of extracellular enzymes and consumption of oxygen (jenkins and tiersch, 1997). the same authors demonstrated that, in non-sterile solutions, motility was completely lost after three days. however, a higher concentrations of antibiotics (gentamicin, 750 µg ml−1; ampicillin, >250 µg ml−1) reduced sperm viability and mitochondrial function in tilapia, oreochromis niloticus (segovia et al., 2000). our result from antibiotic-free milt samples showed that persian sturgeon spermatozoa maintain their motility in appropriate level only during 3 days of storage. but in antibiotic receiving milt samples, the appropriate sperm motility was maintained for 6 days. therefore, the use of penicillin can enhances the viability of persian sturgeon spermatozoa in terms of motility percent and duration during in vitro short-term storage. references aas g.h., refstie t., gjerde b. (1991). evaluation of milt quality of atlantic salmon. aquaculture, 95: 125132. alavi s.m.h., cosson j. kazemi r. (2006). semen characteristics in acipenser persicusin relation to sequential stripping. journal of applied ichthyology, 22: 400-405. babiak i., dabrowski k. (2003). refrigeration of rainbow trout gametes and embryos. journal of experimental zoology, a. 300: 140-151. brown g.g., mims s.d. (1995). storage, transportation, and fertility of undiluted and diluted paddlefish milt. progresses in fish-culture, 57: 64-69. cloud j.g., miller w.h., levanduski m. j. (1990). cryopreservation of sperm as a means to store salmonid germ plasm and to transfer genes from wild fish to hatchery populations. progresses in fishculture, 52: 51-53. degraaf j.d., berlinsky d. l. (2004). cryogenic and refrigerated storage of atlantic cod (gadus morhua) and haddock (melanogrammus aeglefinus) spermatozoa. aquaculture, 234: 527-540. jenkins j.a., tiersch t.r. (1997). a preliminary bacteriological study of refrigerated channel catfish figure 4. changes of percentage of persian sturgeon sperm motility during short-term storage of antibiotic-free milt samples. different letters indicate significant differences among samples (p<0.05). 57 57 halimi and norousta/ int. j. aquat. biol. (2014) 2(2): 53-57 sperm. journal of world aquaculture society, 28: 282-288. jensen j.o.t., alderdice d.f. (1984). effect of temperature on short-term storage of eggs and sperm of chum salmon (oncorhynchus keta). aquaculture, 37: 251-265. rurangwa e., kime d.e., ollevier f., nash j.p. (2004). the measurement of sperm motility and factors affecting sperm quality in cultured fish. aquaculture, 234: 1-28. scott a.p., baynes s.m. (1980). a review of the biology, handling and storage of salmonid spermatozoa at low temperatures. progresses in fish-culture, 18: 99–403. segovia m., jenkins j.a., paniagua-chaves c., tiersch t.r. (2000). flow cytometric evaluation of antibiotic effects on viability and mitochondrial function of refrigerated spermatozoa of nile tilapia. theriogenology, 53: 489-499. stoss j., refstie t. (1983). short-term storage and cryopreservation of milt from atlantic salmon and sea trout. aquaculture, 30: 229-236. stoss j., bukhatipoglu s., holtz w. (1978). short-term and cryopreservation of rainbow trout (salmo gairdneri richardson) sperm. annales de biologie animale, biochimie, biophysique, 18: 1077-1082. int. j. aquat. biol. (2013) (1): 22-27 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology microsatellite variations and genetic structure of common carp (cyprinus carpio) populations in gomishan bay and gorganroud river (southeast of the caspian sea) melika ghelichpour*1, ali shabani1, bahare shabanpour1 1 department of fisheries, faculty of fisheries and environment, gorgan university of agriculture science and natural resources, gorgan, iran article history: received 3 march 2013 accepted 11 april 2013 available online 14 april 2013 keywords: population gorganroud river gomishan bay microsatellite genetic structure abstract: common carp (cyprinus carpio) population has been declined in the caspian sea in the recent years, mainly due to human manipulation. this valuable species needs to be protected in the caspian sea. considering the commercial value of common carp, its rehabilitation program has been established. in the present study, 8 microsatellite loci were used to assess genetic variation and population structure of common carp in gomishan bay (gb) and gorganroud river (gr). these two regions are the most important habitat of common carp. mean actual (na) and expected (ne) alleles numbers were 15.12 and 11.35 for gb and gr, respectively. mean observed (ho) and expected (he) heterozygocity were 0.99 and 0.90 for gb and gr, respectively. results, also, showed that all investigated loci were polymorphic. twelve out of 16 tested locus×region combinations showed significant deviation from hardy-weinberg equilibrium (hwe) which could be mainly due to increase in he. fst index was found to be 0.011. hence, amova showed that observed variation was related to within population (99%) as well as between populations (1%). according to the results, it is suggested that studied populations have a great allelic richness and gene flow. introduction nowadays, many fishes are under threat due to destruction of their habitats, over-exploitation, pollution and introduction of predator and competitor species. hence, they need to be protected via a restocking program (millennium ecosystem assessment, 2005). restocking programs are a managerial strategy, in which mature individuals are caught from wild and propagated under controlled conditions. then, fries are released to their natural habitats and this process is repeated next years (fiumera et al., 1999). species ability to survive in the nature is determined by genetic variation that affects their ability to adapt environmental changes. thus, genetic variation is necessary for the species survival and resistance (bataillon et al., 1996). genetic variation management needs the evaluation of genetic * corresponding author: melika ghelichpour e-mail address: ml.ghelichpour@gmail.com structure and separation rate of targeted species stocks (pujolar et al., 2009). hence, permanent monitoring of genetic status of species that subjected to rehabilitation program is necessary for their conservation and management. in the recent decades, little molecular studies were conducted on aquatic organism in compare to terrestrial species (shabani et al., 2006). a variety of molecular markers are used in population genetic studies, however, among them, the microsatellite markers are widely used. this is mainly due to high frequency in genome, mendelian inheritance, being semi-dominant, small loci size, ease to determine genotype by polymerase chain reaction (pcr) and great polymorphism (chen et al., 2008; dewoody and avise 2000). also, this marker has been widely used in fisheries and aquaculture studies, where 23 ghelichpour et al./ int. j. aquat. biol. (2013) (1) 22-27 interand intra-population variation may be limit (thai et al., 2007). cyprinids are one of the important families of fishes, containing more than 2000 species (kirpichnikov, 1972). among cyprinids, the common carp (c. carpio), a native fish of eurasia, is commercially valuable species that transferred to different regions of the world (kohlman et al., 2003). this species, also, inhabits in the caspian sea and considered as an important food resource for local people. although the common carp inhabits in all parts of southern part of the caspian sea and inter to its tributaries for reproduction, its population has been declined because of over-fishing and degradation of their spawning ground. therefore it needs to be conserved (abdoli and naderi, 2008). currently, stock rehabilitation program of common carp is conducted by releasing artificially propagated fries to the caspian sea. unfortunately, despite the commercial importance and huge market demand for this species, there is no comprehensive information about its population structure in different regions of the caspian sea. the available data are related to common carp population genetic in southern caspian sea, using mtdna (yousefian and laloei, 2011). therefore, in the present study, common carp population structure and genetic variation were studied in gorganroud river (gr) and gomishan bay (gb) (southeast caspian) using 8 microsatellite loci. materials and methods sampling: a total of 30 specimens were sampled from each study area. two grams of each fish caudal fin was sampled and preserved in 90% ethanol, separately. nuclear dna was extracted according to hillis et al., (1996). in this protocol, tissue samples were digested by k-proteinase in buffer [100 mm of acidic tris, 10 mm edta, 250 mm nacl and 1% sodium dodecyl sulfate (sds): ph=8] over 12h at 55 ˚c. the resulted product was purified using phenol-chloroform. dna was precipitated by adding table 1. characteristics and sequence of the primers used in microsatellite analyses in common carp. locus allele number allele size allele size sequence temperature (˚c) mfw2 22 200-228 200-228 f: cacaccgggctactgcagag r: gtgcagtgcaggcagtttgc 64 mfw7 22 160-272 160-272 f: tactttgctcaggacggatgc r: atcacctgcacatggccactc 62 mfw13 17 188-272 188-272 f: atgatgagaacattgtttacag r: tgagagaacaatgtggatgac 56 mfw16 18 128-204 128-204 f: gtccattgtgtcaagatagag r: tcttcatttcaggctgcaaag 57 mfw17 25 208-316 208-316 f: ctcaactacagagaaatttcatc r: gaaatggtacatgacctcaag 57 mfw20 19 208-304 208-304 f: cagtgagacgattaccttgg r: gtgagcagcccacattgaac 60 mfw26 16 108-172 108-172 f: ccctgagatagaaaccactg r: caccatgcttggatgcaaaag 60 cypg24 14 112-168 112-168 f: ctgccgcatcagagataaacactt r: tggcggtaagggtagaccac 58 table 2. cycle number, time (min) and temperature (˚c) used in pcr. cycle number stage time temperature 1 denaturation 5 94 denaturation 30 94 5 annealing 30 5 ˚c above the annealing temperature (marked by*) extension 30 72 denaturation 30 94 32 annealing 30 56-64* extension 30 72 1 final extension 10 72 locus allele number allele size allele size sequence temperature (˚c) 24 ghelichpour et al./ int. j. aquat. biol. (2013) (1) 22-27 cold ethanol (90%) and centrifugation. then extracted dna were dissolved in deionized water and kept at –20 ˚c. quality and quantity of extracted dna was determined by agarose gel (1%) and biophotometer. microsatellite analyses: eight microsatellite loci mfw2, mfw7, mfw13, mfw16, mfw17, mfw20, mfw26 and cypg24 were chosen based on the previous studies (crooijmans et al., 1997; baerwald, 2004) (table 1). pcr amplification was carried out in 0.2 ml pcr tubes with an eppendorf thermal cycler (table 2). fifteen μl pcr reactions contained, 0.5 u taq dna polymerase (fermentas), 1×pcr buffer, 0.2 mmdntp mix, 1.5 mm mgcl2, 1 μm of each primer set, and about 100 ng template dna. pcr temperature cycles were as follows: a predenaturation at 95 ◦c for 3 minute; followed by 35 cycles of denaturation at 95 ◦c for 30 s, annealing for 30 s and extension at 72 ◦c for 30 s and a final extension at 72 ◦c for 3 min. pcr products were separated on 10% polyacrylamid gels stained with silver nitrate (rajora et al., 2000). a 50 bp molecular weight marker (fermentas) was used as the molecular weight standard. microsatellite allele lengths were estimated using gel-pro analyzer 3.9 software (gene, usa). data analyses: na, ne, ho and he were estimated by arlequin 2.0 software (schneider et al., 2000). the reason of deviation from hwe and its significance was determined by fstat software. deviation distribution, genetic distance, genetic identity (nei, 1978), deviation from hwe and fst index (amova) were determined by statistical-geneticl software, genalex 6.5. difference in ho, he and allelic variation was determined by wilcoxon’s test using statistical software, spss v. 16. to adjust significance level for repetitive tests, sequential bonferroni correction was used (rice, 1989). results results showed that all studied loci were polymorphic. a total of 139 alleles with average of 17.37 alleles were observed in each locus. mfw17 with 23 alleles and cypg24 with 12 alleles had the most and least alleles, respectively. na and ne were 15.12 and 11.35 for gr, while they were 16 and 11.78 for gb, respectively (table 3). there was no significant difference in na and ne between two studied areas (p>0.05). mean ho was 0.99%, as ho was found to be 1.00 in all locus exception of mfw20 in gr which was 0.96. mean he was found to be 0.90 which highest and lowest values were observed in mfw20 (0.93) and cypg24 (0.82) in gr (table 3). there was no significant difference in ho and he values between the studied areas (p>0.05). fifteen out of 16 tests (8 locus ×2 areas) showed a significant deviation from hwe (p<0.05), however, only 12 out of 16 tests showed the significant deviation from hwe when sequential bonferroni correction was performed (p=0.005). fst index was found to be 0.011. hence, amova showed that observed variation was related to within table 3. na, ne, ho, he and phw of tested loci in gorganroud river and gomishan bay. area cypg24 mfw26 mfw20 mfw17 mfw16 mfw13 mfw7 mfw2 gr na 9 15 18 17 14 14 16 18 ne 5.64 12.74 14.38 10.96 10.66 10.31 13.17 12.95 ho 1.00 1.00 0.96 1.00 1.00 1.00 1.00 1.00 he 0.823 0.922 0.930 0.909 0.906 0.903 0.924 0.923 phw ** *** *** *** *** ns *** * gb na 12 16 18 21 16 14 11 20 ne 9.13 12.08 14.63 12.24 12.16 10.46 9.04 14.51 ho 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 he 0.891 0.917 0.932 0.918 0.918 0.904 0.889 0.931 phw *** *** ** *** *** *** *** *** gr=gorganroud river. gb=gomishan bay. phw = hardy-weinberg probability test after sequential bonferroni correction. ns = not significant. asterisks show significant difference: * p<0.05; ** p<0.01; *** p<0.001. 25 ghelichpour et al./ int. j. aquat. biol. (2013) (1) 22-27 population (99%) as well as between populations (1%) (table 4). fst (0.006 0.020) and gene flow (nm) (12.23-40.73) values for each tested locus are presented in table 5. results showed that genetic identity and genetic distance between the studied areas were 0.25 and 0.78, respectively (table 6). discussion many fishes have more than one stock which fisheries managements should conserve their genetic variation via their sustainable exploitation. thus, recognition of genetic information of different stocks is fundamental in fisheries managements (waldman et al., 1999). microsatellites markers have provided important information on genetic variation and factors affecting formation of population. these markers encompass a great polymorphism and inheritance (crooijmans et al., 1997). heterozygocity and allele number are of the important parameters in population genetic variation which determine the ability of organism to compete and survive in natural habitats (hakansson and jensen, 2005; frankharn, 2008). in genetic variation studies, allelic richness is more worthy than heterozygocity. in fact, higher allelic richness shows higher effective population size and use of allelic richness is suitable for populations which are treated by selection or conservation programs (diz and persa, 2009). in the case of allele number, ho and he, the present study is in line with the previous one on common carp with the similar primers (ghelichpour et al., 2010). however, the result of this study showed higher rates than that of crooijmans et al. (1997) on common carp using similar primers. such contradictories might be related to difference in sample size or higher genetic variation in studied populations compared to the previous one. crooijmans et al. (1997) stated that higher allele number and heterozygocity could be affected by sample size and studied area. hence, based on the results, there was no significant difference in allele number and heterozygocity between gb and gr, in the present study. twelve out of 16 tests showed a significant deviation from hwe after use of sequential bonferroni correction. this deviation is mainly due to increase in heterozygocity. as mentioned above, ho was greater than he. population combination or nonrandomized mating (liu et al., 2005) could cause deviation from hwe, in this study. generally, a single factor could not cause deviation from hwe, however, combination of some factors such as artificial propagation and stock rehabilitation programs could participate in increase of heterozygocity and deviation from hwe. environmental deterrents, life history and mating type could alter populations’ genetic structure (tiedemann et al., 2000). in addition, stock rehabilitation, due to selective propagation, could affect genetic structure. amova, as a statistical analyses, is a suitable means to determine population table 4. amova for fst. df ss ms est. var. % between populations 1 4.73 4.73 0.023 1 within populations 58 231.5 3.99 3.99 99  table 5. fst and nm for tested locus. mfw2 mfw7 mfw13 mfw16 mfw17 mfw20 mfw26 cypg24 fst 0.008 0.009 0.012 0.006 0.020 0.007 0.007 0.020 nm 29.17 27.58 20.97 40.71 12.23 33.57 36.04 12.43 table 6. genetic identity (regular font) and genetic distance (bolded font) of common carp originated from gorganroud river and gomishan bay. area gorganroud river gomishan bay gorganroud river 0.25 gomishan bay 0.78 26 ghelichpour et al./ int. j. aquat. biol. (2013) (1) 22-27 structure and between population variation (grassi et al., 2004). results of this study showed that observed variation was related to within population (99%) and between populations (1%) and were in line with the previous study on common carp populations which had 99% within population and 1% between populations’ variation in the southern caspian (ghelichpour et al., 2010). fst value (0.011), also, confirmed the little between populations’ variation. according to wright (1978), fst range of 0–0.05 means small variation. hence, genetic similarity and distance between the two studied areas were 0.78 and 0.25, respectively. according to value of genetic similarity in samespecies populations (0.8-0.9) and in same-genus populations (0.35-0.85), it could be stated that the studied populations belong to same-genus (thorpe, 1982). on the other hand, the present results showed a great gene flow between the studied areas, which could cause small variation in genetic structure of the areas. this high gene flow might be related to natural fish migration. also, stock rehabilitation programs could involve in this great gene flow; as produced fries of present restocking program of common carp in the southern caspian sea were released into the sea, without considering their parents origin, it could cause great gene flow. the results of this study suggest that despite of enclosed system of the caspian sea and artificial propagation, genetic variation of common carp is considerably high. however, as stock rehabilitation programs are running yet, management programs should be performed to conserve the genetic variation to avoid the problems caused by inbreeding. references abdoli a., naderi m. 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(2004). population structure and genetic variation within valeriana wallrothii kreyer in relation to different ecological locations. plant science, 166: 1437-1441. 27 ghelichpour et al./ int. j. aquat. biol. (2013) (1) 22-27 hakansson j., jensen p. (2005). behavioural and morphological variation between captive populations of red jungle fowl (gallus gallus) – possible implications for conservation. biological conservation, 122: 431-439. hillis d.m., mable b.k., larson a., davis s.k., zimmer e.a. (1996). nucleic acids iv: sequencing and cloning. in: molecular systematics. sinauer associates, sunderland, usa. kirpichnikov v.s. (1972). methods and effectiveness of ropsha carp breeding. communication i. breeding amis, original forms and cross system. russian journal of genetics, 8: 65-72. kohlmann, k., gross r., murakaeva a., kersten p. 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(1989). analyzing tables of statistical tests. evolution, 43: 223-225. schneider s., roessli d., excoffier l. (2000). arlequin ver. 2.000. a software for population genetics data analysis. genetics and biometry laboratory, university of geneva, switzerland. shabani a., pourkazemi m., rezvani s. (2006). study of mtdna variation of stellate sturgeon (acipenser stellatus) population from the north (volga river) and south (sefidrud river) caspian sea using rflp analysis of pcr amplified nd5/6 gene regions. journal of agricultural sciences and natural resources, 12: 195-204. thai t.b., christopher p.b., christopher m.a. (2007). genetic diversity of (cyprinus carpio l.) in vietnam using four microsatellite loci. aquaculture, 269: 174-186. thorpe j.p. (1982). the molecular clock hypothesis: biochemical evolution, genetic differentiation and systematic. annual review of ecology and systematics, 13: 139-168. tiedemann r., hardy o., vekemans x., milinkovitch m.c. 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(2015) 3(5): 339-345 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article determination of lethal concentrations of indoxacarb in fingerling cyprinus carpio at two temperatures ali taheri mirghaed, melika ghelichpour*1 department of aquatic animal health, faculty of veterinary medicine, university of tehran, tehran, iran. article history: received 8 september 2015 accepted 3 october 2015 available online 2 5 november 2015 keywords: common carp insecticide toxicity lc50 abstract: acute (24-96 hrs) toxicity of indoxacarb, a new insecticide generation, was determined in cyprinus carpio under the semi-static condition. fish (~5 g) were exposed to increasing concentrations of indoxacarb over 24-96 hrs at 17ºc, and over 24 hrs at 22ºc, and mortality was recorded every 24 hrs. indoxacarb-lc50 values at 17ºc were found to be 37.55, 20.92, 18.77 and 16.85 ppm after 24, 48, 72 and 96 hrs, respectively. lc50 after 24 hrs at 22ºc was 21.55 ppm, which was significantly lower than that obtained at 17ºc. the lowest observed effect concentration (loec) values at 17ºc were 14 ppm for 24, 48, 72 hrs, and 11 ppm for 96 hrs. no observed effect concentration (noec) values at 17ºc were 11 ppm for 24, 48, 72 hrs, and 8 ppm for 96 hrs. noec and loec values after 24 hrs at 22ºc were 8 and 11 ppm, respectively. the results indicated that indoxacarb is classified as "harmful" substance in common carp and, the higher temperature, the more toxicity of indoxacarb. introduction chemical pesticides are considered as an economic access to control pests; however, these chemicals may be highly toxic to non-target species in the environment. nowadays, the indiscriminate use of pesticides and their excretion to the aquatic ecosystem threaten the health of organisms (rao, 2006). indoxacarb is an oxadiazin pesticide used to control sucking pests of crops, especially acts against lepidopteran larvae. the chemical name of indoxacarb is (s)-methyl 7chloro-2, 5-dihydro-2 [[(methoxycarbonyl) [4-(trifluoromethoxy) phenyl] amino] carbonyl] indeno[1,2-e][1,3,4] oxadiazine4a(3h)-carboxylate (c22h17clf3n3o7). it is marketed under the names indoxacarb technical insecticide, steward insecticide and avaunt insecticide. avant is more common name for this substance in iran. this relatively new insecticide was registered in california, january 2001 (monkada, 2003). as soon as indoxacarb is absorbed or ingested * corresponding author: melika ghelichpour e-mail address: ml.ghelichpour@gmail.com by insects, feeding cessation occurs. it kills pests by binding to a site on sodium channels and blocking the flow of sodium ions into nerve cells. the result of exposure is impaired nerve function, feeding cessation, paralysis and death of pests. it may take days for insects to die (brugger, 1997). indoxacarb is used on a range of crops, which include fruits (apples, pears and tomatoes), vegetables (broccoli, brussels sprouts, cabbage, cauliflower, eggplant, potato, and lettuce), soybeans, alfalfa and peanuts. it is used to control or suppress many insects, including beet armyworm, cabbage looper, corn earworm, diamondback moth, fall armyworm, imported cabbageworm, southern armyworm, tomato pinworm and tomato fruitworm (dupont, 2002). this pesticide is somehow a hydrophobic pesticide and its penetration to aquatic system is slow, so it is considered as a safe insecticide in environmental standpoint. it provides a much safer alternative to some pesticides such as organophosphates and 340 int. j. aquat. biol. (2015) 3(5): 339-345 pyrethroids. the relatively low mammalian toxicity of this insecticide provides improved safety to workers, as well as terrestrial mammals and birds when compared to competitive organophosphates and carbamates. lack of cross resistance to existing insect control products, environmental suitability and its safety to non-target organisms makes indoxacarb an excellent candidate for controlling some pests (wing et al., 2000). since ph elevation and sunlight speed up indoxacarb hydrolysis and photolysis, it seems that this insecticide has low persistence in aquatic system. however, it is classified as moderately to very highly toxic to freshwater and estuarine/marine fish on an acute basis (moncada, 2003). there are very limited studies about the effect of this insecticide on fish. veeraiah et al. (2013) investigated the biochemical parameters of indian major carp labeo rohita exposed to lethal and sublethal concentrations of indoxacarb showing that indoxacarb is highly toxic to fish with 96 hrs lc50 of 0.053 ppm. also, indoxacarb exposure resulted in decrease in protein, glycogen and nucleic acids content in different organs. as, there is no information available about the toxicity of this pesticide to common carp, cyprinus carpio, hence, this study aimed to determine the lethal concentrations of indoxacarb in common carp at two different temperatures. materials and methods fish and maintenance conditions: in summer 2015, a total of 700 fingerlings were obtained from fishery research station of gharahsoo (bandar turkman, iran) and brought to the laboratory. fish were stocked in a 2000 l fiberglass tank for adaptation to the laboratory conditions. the fish were maintained under continuously-aerated condition in the tank for seven days. they were fed commercial pellets (energy co., 4ef3000) at 2% body weight per day, twice a day. water exchange was about fifty percent daily (ground water of gharahsoo). the fish total length and weight were 5.83 ± 0.87 cm and 5.64 ± 0.74 g, respectively. water quality parameters were as following: temperature = 17ºc, ph = 8.23 ± 0.02, dissolved oxygen = 8.68 ± 0.5 ppm, oxygen saturation = 97.05 ± 2.3 %, electro conductivity = 4.8 ± 0.15 μs m-1, salinity = 2.8 ± 0.08 ppt. these parameters were measured by hach hq40d portable ph, conductivity, dissolve oxygen and salinity meter (loveland, colorado, usa). total hardness 300 ± 17 ppm (as caco3), alkalinity 350.75 ± 20 ppm (as caco3), and calcium 110.8 ± 11 ppm were measured by a portable photometer (wagtech 7100, berkshire, uk). no mortality was observed during adaption period. toxicity test: lethal concentration was determined at two temperatures, 17 ± 1 and 22 ± 1°c, according to oecd (1992). based on the preliminary tests, the fish were exposed to concentrations of 0 (control), 8, 11, 14, 17, 20, 23, 26, 35, 45 and 60 ppm indoxacarb (hefiran co., 15%, tehran, iran). at 22°c, mortality was recorded after 24 hrs exposure, whereas, at 17°c mortality was recorded at 24, 48, 72 and 96 hrs. eleven tanks were assigned for each temperature. a 300 l, white and cylindrical tank was used for each concentration. each tank stocked with 30 fish (150 g biomass) and 160 l water. the fish were allowed to adapt to these conditions for seven days under aerated condition, during which they were fed (2% of body weight, twice a day) with commercial pellets (energy co., 4ef3000). tanks' water was exchanged fifty percent daily. water quality parameters were monitored during experiment and they were similar to those of adaptation period. no mortality was observed during this period. feeding was ceased 24 hrs before dosing. indoxacarb solution was added to each tank to set concentrations. seventy five percent of the exposed solution was renewed each day to maintain water quality and the appropriate concentration of indoxacarb. during the experiment, the fish behavior and the number of dead fish were recorded at 24 hrs intervals. statistical analysis: lc50 values were calculated using probit regression in spss v.22. the lowest observed effect concentration (loec) was 341 taheri mirghaed and ghelichpour/ effect of indoxacarb on common carp determined as the minimum concentration which caused mortalities at each time point (rand, 1995). no observed effect concentration (noec) was determined as maximum concentration at which no mortality was occurred (rand, 1995). difference between 24 hrs-lc50 of the temperatures was considered significance if lc50 of one temperature was outside the confidence interval of the other temperature (marking and bills, 1975; hoseini and jafar nodeh, 2011). significant difference between mortality percentages was investigated using t-test. results no mortality was observed in the control group during the exposure. the control and 8 ppm indoxacarb treatment showed normal swimming and natural body coloration during the experiment. abnormal behavioral changes such as dark coloration, unilateral or bilateral exophthalmia, loss of equilibrium, lateral swimming near the surface, motionless on the bottom of tank, hyper excitement and lethargy were observed in the fish exposed to higher indoxacarb concentrations (20-60 ppm) during 24 hrs of exposure period. these behaviors were observed in 11 ppm of indoxacarb 72 hrs after the start of the experiment, and in 14 ppm indoxacarb 48 hrs after exposure. lc5-95, noec and loec of indoxacarb are presented in tables 1-4. lc50 after 24, 48, 72 and 96 hrs at 17ºc were 37.55 (32.26-42.44), 20.92 (19.1223.08), 18.77 (17.87-19.70) and 16.85 (15.97-17.73) ppm, respectively. lc50 after 24 hrs at 22ºc was 21.55 (18.25-26.70). there was a significant difference between 24 hrs-lc50 at the tested 95% confidence limits time temperature lc5 lower upper slope ± s.e intercept ± s.e 24 hrs 22ºc 14.98 7.86 17.83 10.42 ± 1.25 -13.89 ± 1.65 24 hrs 17ºc 19.57 13.76 23.61 5.81 ± 0.68 -9.15 ± 0.96 48 hrs 17ºc 14.80 11.41 16.70 10.94 ± 1.37 -14.45 ± 1.80 72 hrs 17ºc 13.70 12.17 14.80 12.03 ± 1.50 -15.31 ± 1.93 96 hrs 17ºc 12.10 10.67 13.15 11.44 ± 1.39 -14.03 ± 1.73 table 1. lc5 (ppm) of indoxacarb after 24, 48, 72 and 96 hrs at 17 and 22ºc. 95% confidence limits time temperatu re lc50 lower upper slope±s.e intercept±s.e 24 hrs 22ºc 21.55* 18.25 26.70 10.42±1.25 -13.89±1.65 24 hrs 17ºc 37.55 32.26 46.44 5.81±0.68 -9.15±0.96 48 hrs 17ºc 20.92 19.12 23.08 10.94±1.37 -14.45±1.80 72 hrs 17ºc 18.77 17.87 19.70 12.03±1.50 -15.31±1.93 96 hrs 17ºc 16.85 15.97 17.73 11.44±1.39 -14.03±1.73 table 2. lc50 (ppm) of indoxacarb after 24, 48, 72 and 96 hrs at 17 and 22ºc. asterisk in front of 24 hrs lc50 at 22ºc means significant difference compared to 24 hrs lc50 at 17ºc. 95% confidence limits time temperature lc95 lower upper slope ± s.e intercept ± s.e 24 hrs 22ºc 31.00 55.33 124.9 10.42 ± 1.25 -13.89 ± 1.65 24 hrs 17ºc 72.05 25.54 66.26 5.81 ± 0.68 -9.15 ± 0.96 48 hrs 17ºc 29.57 25.95 39.38 10.94 ± 1.37 -14.45 ± 1.80 72 hrs 17ºc 25.72 23.90 28.87 12.03 ± 1.50 -15.31 ± 1.93 96 hrs 17ºc 23.48 21.79 26.22 11.44 ± 1.39 -14.03 ± 1.73 table 3. lc95 (ppm) of indoxacarb after 24, 48, 72 and 96 hrs at 17 and 22ºc. 342 int. j. aquat. biol. (2015) 3(5): 339-345 temperatures. noec after 24, 48, 72 and 96 hrs at 17ºc were 11, 11, 11 and 8 ppm, respectively. loec after 24, 48, 72 and 96 hrs at 17ºc were 14, 14, 14 and 11 ppm, respectively. noec and loec after 24 hrs at 22ºc were 8 and 11 ppm, respectively. mortality of fish exposed to different concentrations of indoxacarb, at 22ºc was significantly higher than 17ºc (fig. 1). discussion the results revealed that the severity of the behavioral responses was dependent to the indoxacarb concentration and exposure time. there were no studies about fish behavioral alterations which are exposed to indoxacarb. environmental pollutants such as metals, pesticides, and other organic pollutants cause serious risks to aquatic organisms. accordingly, there is a great deal of researches about physiological mechanisms in animals exposed to contaminants. behavioral signs of toxicity appear to be ideal tools to assess the effects of aquatic pollutants on fish, because behavioral indicators reflex the physiological states. toxicant exposure may completely alter behaviors that are essential for fish survival in natural ecosystems, particularly in toxicant concentrations lower than lethal concentration. behavioral changes are mainly related to cholinesterase inhibition, alternation of brain neurotransmitter levels, sensory deficiency, and impaired gonadal or thyroid hormone levels. scott and sloman (2004) investigated interrelationships between behavioral and physiological indicators of toxicity in fish. also, a positive correlation was observed between brain acetyl cholinesterase activity and swimming speed of mosquito fish, gambusia affinis after lethal exposure of an organophosphate pesticide, monocrotophos (kavitha and rao, 2007). according to 96 hrs lc50, indoxacarb is classified as "harmful" substance in common carp (commission directive, 2001). the results showed that lc50 of indoxacarb in the present study was higher than other studies which reported 96 hrs lc50 of 0.024-0.053 in different fish species (veeraiah et al., 2013; hoke, 1997). such differences may be species specific. also, water physico-chemical properties are the most important factors involved in the different results of indoxacarb toxicity. hydrolysis rates of indoxacarb elevate with increasing ph. the half-life at ph 5, 7 and 9 were considered to be approximately 500, 38 and 1 day, respectively (ferraro and mceuen, 1996). in this study, ph was 8.23 ± 0.02; therefore, it can cause faster hydrolysis of indoxacarb and continuously lower toxicity in comparison with other studies; although, those studies did not mention water ph. other water physic-chemical factors may affect toxicity of pesticide. for instance, increasing total alkalinity from 19 to 90-120 ppm is resulted in decreasing mortality of oncorhynchus mykiss exposed to methiocarb and endosulfan (altinok et al., 2006; capkin et al., 2006). water alkalinity in the present study was relatively high (~350 ppm) and this may be another reason for higher lc50 compared to the previous studies that needs a further study. cyprinus carpio is an ectothermic organism, hence, time temperature noec loe c 24 hrs 22ºc 8 11 24 hrs 17ºc 11 14 48 hrs 17ºc 11 14 72 hrs 17ºc 11 14 96 hrs 17ºc 8 11 table 4. noec and loec of indoxacarb after 24, 48, 72 and 96 hrs at 17 and 22ºc. figure 1. mortality percentages at the final time point of observation (17 and 22ºc: 24 hrs after exposure). columns represent means and standard deviation. gray bars show mortalities at 22ºc and black bars show mortalities at 17ºc. asterisks above the bars show significant difference in mortality percentages between the temperatures. * p<0.05; *** p<0.001. 343 taheri mirghaed and ghelichpour/ effect of indoxacarb on common carp temperature is a fundamental factor influencing all its physiological processes. many authors affirmed the assumption that temperature elevation increases toxicity of harmful substances in certain species (fisher and wadleigh, 1985; persoone et al., 1989; song et al., 1997; van wezel and jonker, 1998; heugens et al., 2001). exposure to a toxic substance would increase metabolism and oxygen demand of fish as an ectothermic organism; with elevated temperature, the oxygen solubility in water is decreased, therefore, temperature elevation may worsen the toxic effects of the toxic substance (osterauer and kohler, 2008). in addition, bioavailability of toxic materials like any substances is dependent on temperature. at high temperatures, the absorption of substances by aquatic animals is elevated due to better solubility of the substance and an intensified distribution or active uptake rate of the substance by gill or skin (heugens et al., 2001; osterauer and kohler, 2008). according to this, toxic effect of a substance on an aquatic organism may be potentiated with increasing temperature. similarly, altinok et al. 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(2015) 3(5): 339-345 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی مختلف دمای دو در قدانگشت کپورماهیان بر ایندوکساکارب سم کشندة هایغلظت تعیین *پور قلیچ ملیکا میرقائد، طاهری علی .ایران تهران، تهران، دانشگاه دامپزشکی، دانشکده آبزیان، بهداشت گروه چکیده: . شد تعیین ایستا نیمه شرایط در( cyprinus carpio) کپورمعمولی ماهی در( ساعت 69 -42) ایندوکساکارب جدید کشحشره حاد سمیت در ساعت 42 مدت به گرادسانتی درجة 44 حرارتدرجه در و ساعت 69 تا 42 مدت به گرادسانتی درجة 71 دمای در گرم 5 حدود هایماهی مقادیر گرادسانتی درجة 71 دمای در. شد گزارش ساعت 42 هر میرومرگ درصد و گرفتند قرار ایندوکساکارب مختلف هایغلظت معرض 50lc 45/79 و 11/74 ،55/64،71/42 ترتیببه ساعت 69 و 14 ،24 ،42 از پس ایندوکساکارب سم برای ppm درجه در. آمد دستبه در آن معادل مقدار از کمتر داریمعنی طوربه که شد محاسبه ppm 55/47 ساعت 42 از پس 50lc مقدار گراد،سانتی درجة 44 حرارت 14 و 24 ،42 هایزمان طی گرادسانتی درجة 71 ترحرادرجه در( loec) مؤثر غلظت کمترین مقدار. بود گرادسانتی درجة 71 دمای 24 ،42 از پس گرادسانتی درجة 71 دمای در سم این( noec) اثربی غلظت. شد محاسبه 77ppm ساعت 69 از پس و ppm 72 ساعت درجة 44 دمای در ایندوکساکارب سم مؤثر غلظت کمترین و اثربی غلظت. بود ppm 4 ساعت 69 گذشت از پس و ppm 77 ساعت 14 و دما افزایش و داشته قرار مضر مواد دستة در ایندوکساکارب سم که داد نشان نتایج. بود ppm 77 و 4 ترتیببه ساعت 42 از پس و گرادسانتی .گرددمی کشحشره این سمیت افزایش سبب .50lc سمیت، کش،حشره کپورمعمولی،: کلمات کلیدی int. j. aquat. biol. (2021) 9(5): 279-289 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article ecological status of a west african lagoon complex under anthropogenic pressure, tohotodougba complex, benin mardochée ephraïm achoh* 1, hyppolite agadjihouèdé1, luc gangbè2, arsène houssou1, romaric aïzonou1, simon ahouanssou montcho1, antoine chikou3 1unit of aquaculture research and fisheries management, laboratory of fisheries and animal sciences, national university of agriculture, po box 43 ketou, benin. 2national institute of agricultural research of benin, 01 box 884 cotonou, benin. 3laboratory of hydrobiology and aquaculture (lha), faculty of agronomic sciences, university of abomey-calavi, 01 po box: 526 cotonou, benin. s article history: received 24 january 2021 accepted 22 october 2021 available online 2 5 october 2021 keywords: eutrophication lagoon anthropogenic cage fish farming abstract: management and preservation of aquatic environments are essential for their productivity and the maintenance of aquatic life. this study aims to characterize the trophic state of tohotodougba lagoon complex which has been subject for several years, to an intense fish production in cages. for this purpose, physico-chemical parameters were measured from june 2019 to may 2020 as well as chlorophyll-α, phosphorus and nitrogen. the trophic characterization indices were calculated basedo n carlson (1977), burns and bryers (2000), neverova-dziopak and kowalewski (2018), primpas et al. (2010) and ccme-wqi (2001). it appears that the temperature (29°c), dissolved oxygen (3.19 mg.l-1 to 4.33 mg.l-1) and ph (6.66 to 7.31) are those characteristic of tropical lake environments. the production parameters revealed that the chlorophyll-α concentration varies from 0.19 to 37 mg.l-1 (stations and months combined). the concentration of phosphorus ranged from 0.02 to 0.42 mg.l-1 while nitrogen varied from 1.91 to 4.03 mg.l-1. only nitrogen is not in critical proportion for the ecosystem. it should be noted that with the exception of the carlson index, all other indices revealed that the ecosystem is in a state of advanced eutrophication with a tendency to hyper-trophication according to the eutrophication index of primpas et al. (2010). consequently, toho-todougba lagoon complex is eutrophic and requires adequate measures for its restoration despite its good health revealed by the physico-chemical parameters. introduction the food and agriculture organization of the united nations has revealed that fish contributes 50% of the average per capita animal protein intake in developing countries (fao, 2020). on the other hand, the projections made by delgado et al. (2001) revealed that, to make animal protein available to all, it will be necessary to reach a fish production of 98.6 million tons in 2020 compared to 62.7 million tons in 1997. consequently, the demand for fishery resources has reached a crucial level where the intensification of aquaculture in all its forms is required to meet the growing demand for food security especially in developing countries, including benin (fao, 2018). in the perspective of intensification of aquaculture production, republic of benin has opted for cage fish farming since 2011 as an objective production *correspondence: mardochée ephraïm achoh doi: https://doi.org/10.22034/ijab.v9i5.1033 e-mail: mardoachoh12345@gmail.com strategy. thus, the toho-todougba lagoon complex as other aquatic ecosystems in the country has experienced the installation of cages so that in 2019, 604 cages were counted (aïzonou et al., 2019). the total number of cages in this aquatic ecosystem allows for production of about 600 tons of fish per year and about 700 tons of feed used. the proximity of the toho-todougba lagoon complex in cotonou (a city that is a major market for fish farmers) has generated a strong interest among fish farming entrepreneurs to increase the number of production cages to meet demand. also, the territorial agency for agricultural development pole 7 has undertaken and signed agreements with the fish farmers' cooperatives that operate on the complex to develop fish farming infrastructure. thus, a floating aquaculture village made up 100 cages with dimensions of 5 m on each 280 achoh et al./ ecological statute of toho-todougba complex lagoon side and 3 m of net fall is being installed on the lagoon complex. despite the will of fish farmers and the public authorities to boost the production of caged fish in benin, concerns about ecological preservation arise because of the discharge of organic particles into the environment. indeed, schenone et al. (2011), degefu et al. (2011), gorlach-lira et al. (2013) and yoboue et al. (2018) have mentioned negative effects of the input of organic particles on aquatic ecological life. for organic particles discharged into the aquatic environment influence hydrobiological functioning both physically (change in water color, decrease in transparency, high water density, etc.) and chemically (high concentration of phosphorus, nitrogen, chlorophyll abundance etc.). they can constitute sources of ecological stress for living resources (vodougnon et al., 2018) and lead to eutrophication (mama et al., 2012) through a phytoplankton bloom. in spite of these risks incurred by the tohotodougba lagoon complex and in the face of the evident willingness of fish farmers to intensify production, very little scientific information giving rise to an environmental impact study exists. this crucial lack of scientific information hinders all initiatives aimed at managing the installation and production in this ecosystem. the present study aims to assess the current level of organic pollution of the complex based on production parameters (phosphorus, nitrogen, and chlorophyll-α), trophic status characterization index and other physicochemical water parameters to assist public institutions developers and aquaculture stakeholders in decisionmaking. materials and methods study area and sampling sites: the toho-todougba lagoon complex represents a significant portion of the ancient lagoons (known as the five-fingered lagoon) and is located between 6°23' and 6°27'n and 2°07' and 2°13'e in the heart of ramsar site 1017. the complex has a surface area of 995 ha in recession according to chippaux et al. (1990). for several decades, the communication of the toho-todougba lagoon with the sea has broken due to various developments (chippaux et al., 1990). the lagoon has become a closed ecosystem fed essentially by rainwater and runoff. the vegetation characteristic of this ecosystem is composed of grassy savannahs, meadows, swampy formations with raphia gigentea (capo-chichi et al., 2018). the climate of the area is characterized by two rainy seasons (a large season from april to july and a small one from september to november) and two dry seasons (a small one covering the month of august and a large one from december to march). the average rainfall in the area varies between 936 and 1.200 mm.yr-1. to conduct this study, the complex was subdivided into seven sites. the choice of stations throughout the complex was based on criteria, including the presence or absence of cages, the distribution of cages throughout the complex and accessibility. the geographic coordinates of the study sites are presented in table 1 and the location of each is shown in figure 1. at each study station, four points were selected for sampling. measurement of physico-chemical parameters and water sampling: from june 2019 to may 2020, the physico-chemical parameters (temperature in °c. dissolved oxygen in mg.l-1, ph, salinity in ‰, transparency in cm and depth in m) were measured in situ at each station once a month between 6:30 and 8:30 am. parallel to the in situ measurement of the parameters, water from the lagoon was taken from the euphotic zone (40-50 cm) using the van-dorn bottle, poured into dark bottles of 0.5 liter capacity and kept figure 1. map of the study area. 281 int. j. aquat. biol. (2021) 9(5): 279-289 under refrigeration temperature until it reached the laboratory for various analyses. chlorophyll-α, total phosphorus and total nitrogen concentrations were measured in the laboratory by spectrophotometry within 48 hours of sampling. measurement of ecological production parameters (chlorophyll-α, nitrogen, phosphorus): the determination of chlorophyll-α was carried out according to the monochromatic method (lorenzen, 1967) using 90% acetone as solvent. total nitrogen was determined by the peroxodisulphate mineralization method (rodier et al., 2009) and for total phosphorus, the persulphate digestion method in acidic medium was used (method no. 8190 of hach compagny, 2005). data processing and analysis: the averages the physico-chemical parameters were calculated per station for the entire period of the study and per month. the spatial variation of the data include means, standard deviations and minima-maxima. the temporal variation was presented as a figure by month. after verification of normality by the shapiro-wilk test and homogeneity by the levene test, the means of the different stations were compared to each other by the analysis of variance test (p<0.05). the r software was used for the different analyses. the spatiotemporal variation in chlorophyll-α, the total phosphorus and the total nitrogen concentrations was calculated and presented. the averages were compared with each other using the analysis of variance test (p<0.05). a reference value for the concentration of chlorophyll-α was calculated according to de bortoli and argillier (2008) as follows: reference[ch − α] =100.745-0.489 x log (medium depth) the characterization of the trophic status of the toho-todougba lagoon complex was calculated based on four different indices namely the trophic status index of carlson (1977), the trophic level index of burns and bryers (2000), the trophic status index of neverova-dziopak and kowalewski (2018) and the eutrophication index of primpas et al. (2010). these indeces are considered in the present study because they are specific to continental aquatic environments particularly lakes and lagoons and take into account most of the same parameters for their calculation. then, the water quality index for the protection of aquatic life proposed by the canadian council of ministers of the environment ccme-wqi (2001) was calculated and represented as a box-plot based on table 1. geographic coordinates and characteristics of study sites. sites latitude longitude site characteristics 1 adjadji 6.437893 2.174565 extreme zone of the right arm of the complex. in contact with the flood plain with low aquaculture production and receiving the water that has washed out the palm grove plantations 2 lokohoué 6.402386 2.184292 area of high aquaculture production 3 pont 6.392554 2.199051 exit area of the water circulation receiving all types of waste 4 tonon 6.391409 2.184055 zone housing the farm with more cages in operation on the ecosystem (300 cages in 2019). zone of high aquaculture production 5 tchiakpè 6.388619 2.170374 intersection zone between right and left arm. no aquaculture production 6 savi ii 6.422442 2.124627 average aquaculture production area receiving water that has washed out the palm plantations 7 savi i 6.404449 2.150161 extreme zone of the left arm in contact with the flood plain, receiving the water that has washed out the palm groves plantations. no aquaculture production table 2. quality criteria for the protection of freshwater aquatic life. environmental parameters quality criteria effect considered reference ph 6.5 9.0 chronic effect (usepa, 2006) o2 (mg.l-1) >4 chronic effect (squilbin and yourassowsky. 2005) pt (mg.l-1) <0.02 chronic effect (mddefp, 2013) nt (mg.l-1) <12 chronic effect (agrbc, 2011) pt=total phosphorus; nt=total nitrogen 282 achoh et al./ ecological statute of toho-todougba complex lagoon study stations. the quality criteria for the protection of aquatic life were selected in accordance with the literature (table 2). the principle for determining these indices is summarized in table 3. results physico-chemical parameters of the tohotodougba lagoon: the averages of the environmental parameters measured during the study are presented in table 4. the average depth varies from 3 to 3.37 m for all study stations. the transparency varies from 44.74 to 47.75 cm without being different between stations (p>0.05). the salinity varies between 2-2.33‰ with a significant difference between stations (p<0.05). the average temperature for all the stations is around 29°c for an average dissolved oxygen level that varies between 3.40-4.72 mg.l-1 and an average ph ranging from 6.73 to 7.21. the results revealed that with the exception of temperature, ph and dissolved oxygen vary significantly between stations (p<0.05). variation in production parameters (chlorophyllα, total phosphorus, total nitrogen): the spatial variation in production parameters, including chlorophyll-α, phosphorus and nitrogen are presented in table 5. the values for chlorophyll-α loading vary by station. the stations of adjadji, lokohoué and tchiakpè with respective means of 18.17±1.54, 14.38±0.72 and 19.80±1.6 µg.l-1 with a significantly higher chlorophyll-α load than the others (p<0.05). table 3. trophic status characterization indices. index classification scale trophic state index (tsi) of (carlson, 1977) iet between [0 40[ : oligotrophic area; iet between [40 50[ : mesotropic area; eit between [50 60[ : eutrophic area ; eit > 60 : hyper-eutrophic area the trophic level index (tli) of burns and bryers (2000) tli between 0 and 2, microtrophic area; tli between 2 and 3, oligotrophic area; tli between 3 and 4, mesotrophic area; tli between 4 and 5, eutrophic area; tli between 5 and 6, supertrophic area; tli between 6 and 7. hypertrophic area trophic status index of neverova-dziopak and kowalewski (2018) eit between 5.7 and 6, dystrophic area; eit between 6 and 6.66, ultraoligotrophic area; eit between 6.7-7.3, oligotrophic area; eit between 7.4-8, mesotrophic area; eit > 8; eutrophic area the eutrophication index (ei) of (primpas et al., 2010) ei between 0.04 and 0.38, oligotrophic area; ei between 0.37 and 0.87, mesotrophic area; ei between 0.83 and 1.51. eutrophic area water quality index for the protection of aquatic life (ccme-wqi, 2001) the index value between 0 and 100 is classified into five quality classes. poor quality: 0-44; poor quality: 45-64; moderate pollution: 65-79; good quality: 80 94; excellent quality: 95 and 100 table 4. annual average of physico-chemical parameters for each study station. sites t (°c) do (mg.l-1) ph transp (cm) sal (‰) dep (m) adjadji m±ecart type 29.22±1.45a 3.40±1.22c 7.03±0.83ab 44.74±0.32a 2.19±0.52ab 3.13±0.71bc min-max 27-32.6 1.2-6.60 5.0-9.0 40-70 1.0-3.0 2.0-4.98 lokohoue m±ecart type 29.3±1.34a 4.72±0.99ab 6.8±0.91bc 46.28±0.33a 2.19±0.57ab 3.35±0.61a min-max 26.8-32.6 1.60-6.90 5.10-9.14 41-80 1.0-3.0 2.20-4.79 pont m±ecart type 29.5±1.19a 4.17±1.11a 7.21±0.95a 47.06±0.35a 2.17±0.56b 3.18±0.56abc min-max 28.00-32.1 2.44-7.4 5.6-9.4 40-78 1.0-3.0 2.20-4.5 savi1 m±ecart type 29±3.34a 3.5±1.38bc 6.73±0.91c 44.83±0.32a 2±0.0c 3±0.68c min-max 29.10-32.2 1.20-7.7 5.0-8.7 24-85 2.0-2.0 1.80-4.8 savi2 m± ecart type 29.36±1.20a 3.99±1.34a 6.78±0.89bc 45.77±0.32a 2.33±0.47a 3.24±1ab min-max 27.1-31.80 2.5-8.10 5.0-8.93 40-78 2.0-3.0 2.0-6.65 tchiakpe m± ecart type 29.29±1.09a 4.06±1.25a 7.1±0.72a 47.75±0.34a 2.22±0.42ab 3.37±0.49a min-max 27.90-32.2 2.40-6.9 5.70-8.6 37-89 2.0-3.0 2.65-4.3 tonon m± ecart type 29.29±1.05a 4.16±1.22a 7.12±0.80a 46.7±0.34a 2.14±0.54bc 3.23±0.41ab min-max 27.50-31.7 2.5-7.3 5.29-9.1 40-98 1.0-3.0 2.10-4.3 p-value 0.741 0.00012 0.00157 0.998 0.00487 0.0133 averages with different letters on the vertical differ from each other p-value =0.05; m=mean; min=minimum; max=maximum; t=temperature; transp= transparency; sal= salinity; dep= depth. 283 int. j. aquat. biol. (2021) 9(5): 279-289 the savi ii station had the lowest chlorophyll-α load (5.68±0.49 µg.l-1) while the pont, tonon and savi i stations with respective averages of 7.41±0.61, 8.14±0.48 and 6.92±0.92 µg.l-1 recorded a moderate load. the reference value of chlorophyll-α for this ecosystem for the average depth of 3.20 m, is 13.781 µg.l-1. by comparing the chlorophyll-α load of each station to the calculated reference value, it appears that the sites adjadji and tchiakpè have an average significantly higher than the reference value while all the other sites have a value below it, except the station of lokohoué which has a value not different from the reference value. as for phosphorus concentration, the adjadji, pont and savi ii sites have significantly higher concentrations with respective values of 0.25±0.032, 0.18±0.02 and 0.22±0.02 mg.l-1, respectively (p<0.05). the tonon site has the lowest concentration (0.09±0.01 mg.l-1). the same trend was observed at the nitrogen level for the highest concentrations (adjadji: 3.01±0.96; pont: 3.7±0.16; savi ii: 3.7±0.35 mg.l-1). these concentrations are significantly different from those at the other stations i.e. the lowest nitrogen concentration was at the savi i station as 1.91±0.05 mg.l-1. the temporal variation in chlorophyll-α, phosphorus and nitrogen loading in toho-todougba lagoon is summarized in table 6. the chlorophyll-α load varied significantly between months. december, january, february and may are the months in which high chlorophyll-a concentrations are obtained (respectively 23.53±5.42, 37.88±6.81, 37.89±6.68, and 25.08±5.71 mg.l-1). march to april have a moderate load of 5.63±1.69 mg.l-1. the month of july recorded lower averages (0.19±0.09 mg.l-1). the concentration recorded for phosphorus has a tendency more or less opposite to that of chlorophyll-α so that the months of june, december, january, february, march, april and may had the lowest concentrations ranging from 0.09±0.01 to 0.16±0.02 mg.l-1. july (0.42±0.09 mg.l-1), august (0.42±0.09 mg.l-1) and september (0.29±0.07 mg.l-1) measured concentrations that are significantly higher than the other months (p<0.05). with regard to nitrogen, only table 5. spatial variation in chlorophyll-α, total phosphorus and total nitrogen concentration. sites of study adjadji lokohoue pont tonon tchiakpe savi i savi ii ch-a (µg.l-1) 18.17±1.54a 14.38±0.72a 7.41±0.61b 8.14±0.48b 19.80±1.6ac 6.92±0.92b 5.68±0.49c p (mg.l-1) 0.25±0.03a 0.10 ±0.07b 0.18±0.02a 0.09±0.01c 0.16±0.02b 0.13±0.07b 0.22±0.02a n (mg.l-1) 3.01±0.96a 2.10 ±0.02b 3.70±0.16a 2.47±0.04b 2.80±0.04b 1.91±0.05b 3.79±0.35a averages with different letters on the horizontal differ from each other p-value =0.05; ch-a = chlorophyll-a; p = phosphorus, n=nitrogen table 6. spatial variation in chlorophyll-α, total phosphorus and total nitrogen concentration. months organic pollution parameters chlorophyll-α (µg.l-1) phosphorus (mg.l-1) nitrogen (mg.l-1) june 0.33±0.10a 0.11±0.04a 2.58±0.89a july 0.19±0.09b 0.42±0.09b 2.56±0.88a august 0.28±0.12a 0.42±0.09b 2.57±0.89a september 0.26±0.14a 0.29±0.07c 2.58±0.89a october 0.73±0.16c 0.02±0.00d 3.24±0.99b november 0.73±0.2c 0.02±0.01d 3.24±0.99b december 23.53±5.42d 0.16±0.02ac 4.03±0.91b january 37.88±6.81e 0.09±0.01d 2.26±0.58a february 37.89±6.68e 0.09±0.01d 2.26±0.58a march 5.66±1.69f 0.10±0.01a 3.00±0.53a april 5.63±1.60f 0.10±0.01a 3.00±0.53a may 25.08±5.71d 0.13±0.01a 2.58±0.88a p-value <0.05 <0.04 <0.05 averages with different letters on the vertical differ from each other p-value =0.05 284 achoh et al./ ecological statute of toho-todougba complex lagoon the concentrations in october, november and december recorded significantly higher averages than the others, ranging from 3.24±0.99 to 4.03±0.91 mg.l1. concentrations in the other months ranged from 2.58±0.88 to 3.00±0.53 mg.l-1. trophic status characterization index: the trophic status of the toho-todougba lagoon complex has been characterized on the basis of four index. following the carlson (1977) scale, it appears from figure 2a that none of the stations recorded a carlson index that exceeds the limit of 40 representing the maximum limit for characterizing an oligotrophic environment i.e. the toho-todougba lagoon complex is in an oligotrophic state. the burns and bryers (2000) index showed in figure 2b indicating the opposite by revealing that all study stations recorded a value greater than 5 which is the critical threshold for a eutrophic environment. the values reached the threshold of 6 or even 7 indicating that the tohotodougba lagoon complex is in an enlarged state. the index of neverova-dziopak and kowalewski (2018) based on the relationship between ph and %o2 figure 2. (a) trophic status index of carlson (1977), (b) trophic level index burns and bryers (2000); (c) trophic status index of neverovadziopak and kowalewski (2018), (d) linear regression between ph and %o2; (e) eutrophication index of primpas et al. (2010); (f) water quality index for the protection of aquatic life. 285 int. j. aquat. biol. (2021) 9(5): 279-289 revealed that all the stations are eutrophied with the exception of savi i which recorded for this index a value lower than 8 and to some extent the station of lokohoue (fig. 2c). the sites of adjadji and savi ii recorded for this index a value higher than 10, which testifies to the level of pollution of these sites. the correlation coefficient r of the regression in ph and %o2 is higher than 0.6 (fig. 2d; r = 0.61155) and testifies that the criteria of use of this index is respected. this eutrophication trend of the tohotodougba lagoon complex is also confirmed by the eutrophication index of primpas et al. (2010) which recorded for all sites, a value above the critical threshold of eutrophication which is 1.51 (fig. 2e). in contrast to the index of neverova-dziopak and kowalewski (2018), the index of primpas et al. (2010) revealed that the savi ii site recorded the lowest eutrophication level compared to the other stations, but without guaranteeing an ecologically pleasing health of aquatic resources. water quality index for the protection of aquatic life (ccme-wqi): the water quality index for the protection of aquatic life was calculated for each station (fig. 2f). when considering the decision scale for this index, all sites recorded a value of less than 50. this bodes poor quality of life for the aquatic resources of the toho-todougba lagoon complex. considering each site, the site of adjadji and savi ii recorded the lowest values (35). discussions physico-chemical parameters and production parameters: physico-chemical parameters influence water quality and the health of aquatic ecosystems. in most developing countries, aquatic environments are subject to a variety of problems related to anthropogenic activities and the misuse of their resources (el-serehy et al., 2018). these activities are likely to influence the physical and chemical parameters of aquatic environments. thus, a state of permanent stress is increasingly observed in most aquatic ecosystems. in the present study, the parameters temperature and ph did not record average values likely to disturb aquatic life. the values obtained (29°c; 6.73 to 7.21) for temperature and ph are characteristic of tropical environments (houssou et al., 2017) and could not particularly affect the biotope of the ecosystem. however, transient decreases in ph below 6 in some locations are observed for some measurement sites indicating a slightly acidic ph. this transient and unevenly distributed acidity within the same study station may result from the decomposition of organic matter or the dissolution of atmospheric co2 at these sites (beaune et al., 2018). for dissolved oxygen, the average varies between 3.19 and 4.33 mg.l-1 exposes the quality of life at the water level. this concentration is vital for aquaculture species and in this case cichlids, the most dominant in benin's aquatic ecosystems (achoh et al., 2018). however, disparities are observed depending on the time of year for some stations, notably adjadji and savi ii, so that very low values of the order of 1.77 mg.l-1 are recorded for the months of june, january, february and march. this low concentration is characteristic of polluted environments with a reversible character (squilbin and yourassowsky, 2005). although having a reversible character, this low concentration of dissolved oxygen can indirectly contribute to global warming by promoting the oxidation of ammonia into nitrous oxide (n2o), which is a greenhouse gas (peng et al., 2015). regarding the production parameters (chlorophyllα, phosphorus and nitrogen), this study revealed that the sites of adjadji (18.17±7.54 µg.l-1) and tchiakpè (19.80±5.3 µg.l-1) and to a lesser extent that of lokohoue (14.38±2.52 µg.l-1) recorded values higher than the reference value of chlorophyll-α (13.781 µg.l-1) for this ecosystem. this indicates that there is increased phytoplankton production at these stations. for the adjadji site, agricultural activities and large livestock breeding in the large palm grove of gakpe are developing and runoff drains organic waste in this portion of the lagoon (koné et al., 2009). the tchiakpè site is a transit strait for the population of tchiakpecodji (a village in the district of the commune) on their way to the commercial city and market of pahou. this site receives waste of all kinds inciting phytoplankton production, hence its value as 286 achoh et al./ ecological statute of toho-todougba complex lagoon a polluted station. mama et al. (2012) and djihouessi et al. (2019) reported similar pollution on behalf of lake nokoué where fluvio-lagunar transport and human waste from the dantokpa market caused eutrophication of the lake. along with nitrogen, phosphorus is a limiting factor in the eutrophication of aquatic ecosystems (issola et al., 2008; dodds and smith, 2016; schindler et al., 2016). total phosphorus concentrations recorded in this study ranged from 0.02 to 0.42 mg.l-1 for all sites and months. these values exceed the phosphorus concentrations (0.005 to 0.1 mg.l-1) for continental aquatic ecosystems with hydrobiological functioning without exogenous influence (issola et al., 2008). consequently, the concentrations of phosphorus in the present study refer to anthropogenic input into the ecosystem (powers et al., 2016). dietary intake in the highly developed floating cage fish farming in the lagoon is also an important source of phosphorus for the ecosystem coupled with the organic load of runoff water. worse, a floating infrastructure dedicated to festive events is erected in the heart of this lagoon complex with all these corollaries of organic pollution. as phosphorus is a limiting factor in eutrophication, the risk will be increasingly high that fish farming will be further developed on the water body and that the discharge of organic matter by man will be increasingly significant. as for nitrogen, the averages range from 1.91 to 4.02 mg.l-1 considering the spatial and temporal variation. this concentration is below 12 mg.l-1 which is the threshold value for good water quality according to agrbc (2011). it appears from the values recorded that the nitrogen load in the tohotodougba lagoon ecosystem could not negatively influence the ecological balance of the environment. trophic status of the ecosystem: to characterize the trophic status of the toho-todougba lagoon complex, several indices were calculated and the recodef values were compared to the classification scale for each index. thus, carlson's (1977) trophic status index, based on transparency, phosphorus and chlorophyll-α, obtained values that are below the threshold value of an oligotrophic environment and shows that all the stations are in good ecological health and are not, at least for the moment, vulnerable to the pressure of organic pollution. in contrast, the trophic level index of burns and bryers (2000), the eutrophication index of primpas et al. (2010) and the eutrophication index of neverova-dziopak and kowalewski (2018) showed a pollution trend contrary to the carlson index. indeed, the toho-todougba lagoon complex is in a state of advanced eutrophication according to these indices. this level of pollution is all the more critical as the index of primpas et al. (2010) characterized the ecosystem in a hypertrophic state. the combination of chemical parameters to determine overall water quality in the ecosystem has shown that water quality is poor for the chronic protection of aquatic life based on the ccme-wqi water quality index. the values for this index at all stations is below 50 (minimum threshold for good quality of life). it therefore reflects that all stations are unfit for aquatic life. by grading the sites, adjadji and savi ii are the most polluted stations. this is in line with the results of the neverova-dziopak and kowalewski (2018) index. the level of pollution of these two stations exposed by these indices is related to the geographical position of the adjadji station which is presented as the receptacle of field leaching water on the one hand, and the receptacle of wastewater from the numerous hotels and rest homes that are installed all around and the fish farm on the other hand. it is the same for the savi ii site. it results from the pooling of the measurements of production parameters (chlorophyll-α, phosphorus and nitrogen) and calculated indices that the tohotodougba lagoon complex is in a eutrophic state. even if according to bourdin (2004), the eutrophication of a water body exists outside of any human action, activities such as fish farming and agriculture would have an important part in the eutrophic state of the toho-todougba lagoon. thus, the eutrophication of the lagoon will worsen over time as long as fish farming in cages is developed and significant input of organic matter from agriculture and dwellings is observed. in addition, another factor favoring the eutrophic evolution of this lagoon complex is the closure of the contact of the toho287 int. j. aquat. biol. (2021) 9(5): 279-289 todougba lagoon with the sea through the coastal lagoon of ouidah. as a result, there is no possibility of evacuation and renewal of water from the tohotodougba lagoon and all organic matter is concentrated there. as a result of this eutrophication, organisms in the toho-todougba lagoon complex live permanently under the stress of organic pollution. the long-term consequences are the decline of stocks, the rarefaction of certain species in the catches and the decrease in the size of adults. the consequence on fishing practice is the use of fine mesh nets for catches as a measure of resilience by fishermen and a progressive destruction of the ecosystem. continuous monitoring of the trophic level is necessary to mitigate or prevent serious negative impacts on aquatic resources and anthropogenic activities (napiórkowska -krzebietke and hutorowicz, 2014). conclusion the toho todougba lagoon complex is in a state of eutrophication considering the calculated trophic status indices. the paradox of this study is that the recorded physico-chemical parameters indicate that the environment is still conducive to an intensification of fish farming. what is the share of fish farming in this evolution of the trophic level of the tohotodougba lagoon? will we continue to intensify fish farming with regard to the physico-chemical parameters despite the organic pollution revealed by the trophic status indices? studies on the dynamics of chlorophyll production and carrying capacity are essential to further elucidate the decision-makers on management and environmental preservation measures to safeguard this ecosystem. acknowledgements the authors thank the laboratory of applied hydrology (lha) of the faculty of science and technology of the university of abomey-calavi for its contribution through laboratory analysis of production parameters. references achoh m.e., agadjihouèdé h., gangbé l., dougnon t.v., hounmanou y.m.g., baba-moussa l. 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(2021) 9(2): 88-96 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article food availability estimation of the blood cockle, anadara granosa (linnaeus, 1758), from the aquaculture grounds of the selangor coast, malaysia tatsuya yurimoto* 1, faizul mohd kassim1, reiko fuseya2, kazumi matsuoka3, alias man4 1japan international research center for agricultural sciences (jircas), tsukuba-shi, ibaraki, japan. 2fisheries technology institute, japan fisheries research and education agency, yokohama-shi, kanagawa, japan. 3institute for east china sea research, nagasaki university, nagasaki-shi, nagasaki, japan. 4fisheries research institute (fri), department of fisheries (dof), kampung acheh, sitiawan, perak, malaysia. s article history: received 29 january 2021 accepted 22 march 2021 available online 2 5 april 2021 keywords: food availability chlorophyll a phytopigment selangor abstract: blood cockles, anadara granosa (linnaeus, 1758), were collected from the aquaculture grounds (4 stations) of the selangor coast, malaysia, and the water quality (water temperature, salinity, dissolved oxygen, turbidity, and chlorophyll a) was measured from september 2011 to june 2013. at all stations, the water temperature fluctuated around 30℃. at station c, located at the mouth of the selangor river, the salinity was occasionally lower than 20 psu. however, the salinity of the other stations fluctuated around 30 psu. in addition, at all stations, the content of dissolved oxygen generally fluctuated around 3 mg.l-1 or above, and the turbidity changed irregularly, sometimes exceeding 300 ftu. the chlorophyll a content fluctuated mainly ranging 4-20 µg.l-1 at all stations, and values above 20 µg.l-1 were occasionally observed. the phytopigment content, a food availability indicator, in the digestive gland tissue of the blood cockles collected from all stations fluctuated ranging 30-770 µg.g-1. however, there was no proportional correlation between phytopigment content in the digestive gland and chlorophyll a content at all stations. therefore, even in a high chlorophyll a content (over 20 µg.l-1) environment, the accumulated phytopigment in the digestive gland was around 290 µg.g-1. in general, these results indicated the cockles were eating a sufficient amount of foods (organic materials including phytoplankton) all year round during the study period. and, the food availability environment in the aquaculture grounds of the selangor coast was estimated sufficient to grow the blood cockle. introduction the blood cockle, anadara granosa (linnaeus, 1758), inhabits a wide coastal area, ranging from tropical to temperate environments (narasimham, 1988; nakamura and shinotsuka, 2007; soegianto and supriyanto, 2008). in southeast asian countries, especially malaysia, thailand, and vietnam, the blood cockle is cultured in mudflats of the mangrove coastal areas (broom, 1985; tri and lin, 1999; chalermwat et al., 2003; phuong and minh, 2005; watanabe, 2009; yurimoto et al., 2013, 2014a, b, c, d), because it is an important protein source for the residents of these countries (pathansali and soong, 1958; broom, 1983; watanabe, 2009). the selangor coast, peninsular malaysia, has a broad coastline of approximately 100 km, with many inflowing rivers accompanied by huge *correspondence: tatsuya yurimoto doi: https://doi.org/10.22034/ijab.v9i2.1113 e-mail: yurimoto@outlook.com mudflats and mangrove areas around the river estuaries. in addition, along these shorelines, a total of 654 hectares of blood cockle farming plots area of approximately 50 ha per plot are arranged in a line (ramli et al., 2013; harith et al., 2016). moreover, this region is famous for supplying blood cockle spats to other regions in malaysia, because of the large number of cockle spats that occur on the mudflats every year (yurimoto, 2013; yurimoto et al., 2014c, d). production of blood cockle culture along the selangor coast has been decreasing since 2011, after peaking at approximately 40,000 tons in 2010 (ramli et al., 2013; shimoda et al., 2016). the temporary peak in production was caused by an increase in blood cockle farmers due to an incentive policy outlined by the malaysian government. however, it has been 89 int. j. aquat. biol. (2021) 9(2): 88-96 suggested that the decrease in production was due to mass mortalities (ramli et al., 2013; yurimoto et al., 2014d) and a reduction in suitable culture grounds caused by coastal erosion (jeyanny et al., 2009, 2012; asmawi and ibrahim, 2013). therefore, environmental evaluations around the estuary are important for the protection of suitable aquaculture grounds. in this study, four monitoring stations, located at the main areas for blood cockle aquaculture, were established along the selangor coast from september 2011 to june 2013. then, we sampled blood cockles to evaluate the food availability conditions (phytopigment content in the digestive gland). furthermore, the water quality (water temperature, salinity, dissolved oxygen, turbidity, and chlorophyll a) was monitored at all stations at the time of sampling. materials and methods from september 2011 to june 2013, sampling stations (st. a: bagan nakhoda omar, st. b: sungai besar, st. c: kuala selangor, and st. d: sungai buloh) were set in four sea areas along the selangor coast (fig. 1). these areas are known as main sites for blood cockle, a. granosa, aquaculture and the cockle landing. environmental survey and the blood cockle sampling were carried out with a fishing boat at the four stations mainly from medium to low tide period on almost every month. the cockles were periodically collected with a hand dredge (basket size: 60 cm wide, 15 cm high, 30 cm deep) and ten individuals for this study were randomly taken from the collected cockles. however, when enough the cockles could not be collected, possible number of the cockles were used for this study sample. the shell lengths (mean± standard deviation) of the collected cockles during the survey period were 27±3 (n=195), 28±5 (n=176), 40±9 (n=128), and 30±4 (n=191) mm for stations a, b, c, and d, respectively. additionally, the water quality (water temperature, salinity, dissolved oxygen, turbidity, and chlorophyll a content) of the bottom layer (from bottom surface to 50 cm above the bottom) of each station at the time of sampling was measured every 10 cm using a throw-in-type water quality sensor (aaq-rinko, jfe advantech co. ltd., japan), and the measured data were calculated as the average value±standard deviation. in addition, due to a failure in the turbidity sensor, the turbidity data were not collected after october 2012. the collected blood cockles were transported alive to a temporary laboratory, located near the monitoring site, where the shell lengths were measured, and the shells were opened to collect the soft tissues for phytopigment analysis. the phytopigment analysis was performed using a modified method of numaguchi et al. (1985, 2001). the soft tissue was stored in a freezer (lower than -20℃) until analysis, and a tissue block of the digestive gland was excised from the soft tissue at the time of analysis. the tissue block was immersed in 90% acetone solution under shading overnight for the extraction of the phytopigment. the solution was centrifuged (3,000 rpm, 10 min) to obtain the supernatant, and a small amount of 4n hydrochloric acid solution was added to completely convert the pigments to phytopigments. two wavelengths, 665 and 750 nm, were measured with a spectrophotometer to calculate the value of phytopigments (absorption wavelength 665 nm) figure 1. location of the selangor coast in peninsular malaysia (square in map a) and four sampling stations in the main aquaculture grounds of the area (map b). st. a: bagan nakhoda omar, st. b: sungai besar, st. c: kuala selangor, and st. d: sungai buloh. 90 yurimoto et al./ food availability of blood cockle minus the value of impurities (absorption wavelength 750 nm) to obtain an actual value of the phytopigments. finally, the phytopigment content in 1 g of the midgut gland tissue (wet weight) was calculated from the measured data using a modified formula from strickland and parsons (1968). results environment: annually, the water temperature fluctuated around 30℃ at all stations (fig. 2a, e, i, m). at station c, a low salinity of below 25 practical salinity units (psu) was observed in march, august, and january 2013, while the salinity in the other months was higher than 25 psu (fig. 2j). the salinity at the other stations fluctuated around 30 psu during the survey period (fig. 2b, f, n). at station d, the dissolved oxygen content decreased to 2.8 mg.l-1 in november 2012, whereas that of the stations a, b and c changed higher than 3 mg.l-1 during the monitoring (fig. 2c, g, k, o). the turbidity fluctuated irregularly at all stations, and frequently exceeded 300 forumajin turbidity units (ftu) (fig. 2d, h, l, p). notably, the highest turbidity reached 655 ftu, which was at station c in october 2011. the chlorophyll a content fluctuated mainly ranging from 4 to 20 µg.l-1 at stations a and b during the survey period. in addition, the content at the both stations exceeded 20 µg.l-1 in may and october 2012, which was also observed in january (st. b) and may (st. a) 2013 (fig. 3a, c). furthermore, at station c, the chlorophyll a content exceeding 20 µg.l-1 was observed on october, november in 2012 and april, may, july, september in 2013 (fig. 3e, g). in contrast, at station d, the chlorophyll a content fluctuated mainly ranging from 4 to 19 µg.l-1. and the high content exceeding 20 µg.l-1 observed only on august 2012. phytopigment: the phytopigment content in the midgut gland of the blood cockle, a. granosa, collected from stations a and b was less than 160 µg.l-1 from september to november 2011, and has been fluctuated ranging from 170 to 370 µg.l-1 figure 2. water quality monitoring in the bottom layer (bottom-50 cm) at the four stations (st. a-d) along the selangor coast. a, e, i, m: water temperature; b, f, j, n: salinity; c, g, k, o: dissolved oxygen and d, h, l, p: turbidity. a-d: st. a; e-h: st. b; i-l: st. c and m-p: st. d. mean±sd. 91 int. j. aquat. biol. (2021) 9(2): 88-96 (except 140 µg.l-1 at st. a in november 2012) since february 2012 (fig. 3b, d). and the content at station b has decreased to 200 µg.l-1 on february 2013. the phytopigment content at station c remained ranging from 150 to 370 µg.l-1 from september 2011 to october 2012, and then decreased to lower than 100 µg.l-1 from november to december 2012 (fig. 3f), after which it increased to over 500 µg.l-1 in february and may 2013. in contrast, the content at station d was less than 160 µg.l-1 in september-october 2011, and then increased ranging from 240 to 300 µg.l-1 from november 2011 to may 2012, before further increasing to over 500 µg.l-1 from september to october 2012 (fig. 3h). after that, the value decreased again to lower than 100 µg.l-1 in november 2012 and increased again to over 600 µg.l-1 in february 2013, and fluctuated ranging from 360 and 480 µg.l-1 from march to may 2013. additionally, to clarify the relationship between the chlorophyll a in the fishing ground and the phytopigment content in the digestive gland, we set the chlorophyll a on the x-axis and the phytopigment on the y-axis for both values obtained at each sampling station on each month and the mean value±sd was plotted to examine the both correlation (fig. 4). as the results, y=1.6887x+208.69, r2=0.0215, n=19 at station a, y=2.1762x+225.27, r2=0.0502, n=19 at station b, y=2.7869x+252.39, r2=0.0212, n=18 at station c, and y=3.3719x+267.58, r2=0.0132, n=20 at station d, and no clear correlation was observed at the all stations. discussions environment: at each station, the water temperature of the blood cockle aquaculture ground was almost stable at approximately 30℃ (fig. 2a, e, i, m). a low salinity, below 25 psu, was occasionally observed at station c, which is near the mouth of the selangor river (fig. 2j), while the salinity of the other stations fluctuated around 30 psu (fig. 2b, f, n). this result indicates that station c is the most susceptible to river water among the stations. in addition, the dissolved oxygen content was ordinally higher than 3 mg.l-1 at figure 3. changes in the chlorophyll a content of the bottom layer (bottom-50 cm) at the four stations (st. a-d) and phytopigment content in the blood cockle, anadara granosa, collected from the same stations along the selangor coast. a, c, e, g: chlorophyll a and b, d, f, h: phytopigment contents. a, b: st. a; c, d: st. b; e, f: st. c and g, h: st. d. the numbers in parentheses in the b, d, f, h graphs indicate the number of analysed samples from each plot, while plots with no parentheses indicate that 10 samples were analysed. mean±sd. 92 yurimoto et al./ food availability of blood cockle each station (fig. 2c, g, k, o). therefore, it is considered that the oxygen level was not serious hypoxic environment to the blood cockle, because it is known that the hypoxic level for many benthic organisms, including bivalves, is around 2 mg.l-1 or less, and organism survival is affected if low oxygen levels continue for several days (vaquer-sunyer and duarte, 2008). additionally, in case of the blood cockle, it inhabits in surface layer of sea bottom (sato, 2006) and it is known also an oxygen regulator adapted to hypoxic environment. davenport and wong (1986) investigated the blood cockles exposed in anoxia condition for 18 hours and they found the cockle could increase oxygen absorption ability to 2.8 times than the normal. on the other hand, in case of manila clam, ruditapes philippinarum, the feeding ability does not affect even if it is exposed under anoxic condition for 24 hours (nagasoe et al., 2011). therefore, the food availability of the blood cockle was suggested almost little or no affected by the dissolved oxygen condition, because serious low oxygen level was not observed on the all stations during the study period. on the other hand, high turbidity values were irregularly observed. the influence of high turbidity on the blood cockle is unknown. however, the blood cockle commonly inhabits muddy tidal flats around mangrove estuaries (lai et al., 2020). and, in case of the mangrove estuaries, the high turbidity contains high amount of organic matter as a food source for bivalve not only clay silt and it is a low negative affect on the cockle habitat such as their pseudo-feces production relating their debilitation (velasco and navarro, 2002; sroczyńska et al., 2012; de alencar leite et al., 2020). tanaka et al. (1982) highlighted that the resuspended mud in the mudflats of inner bays contain a large amount of particle matter, such as phytoplankton and organic particles, which are food sources for benthic organisms. in addition, yurimoto et al. (2008) conducted an indoor experiment to evaluate the effects of resuspended mud in a case of the pen shell, atrina pectinata, reporting resuspended mud from an inner bay contains organic matter and has a food source value for the bivalve in comparison with inorganic clay. normally, in mangrove areas large amounts of organic matter accumulate (sanders et al., 2010), especially suspension matter in the coastal waters, which also contains significant organic matter (williams and benson, 2010; saifullah et al., 2016). additionally, yurimoto et al. (2014b) reported that phytoplankton cells were mainly found in the digestive tube of the blood cockle in the matang mangrove estuary of malaysia, but various suspended particles were also observed such as mangrove litter. therefore, it is considered the suspension matter in the four stations had a food value and low negative effects on food availability and habitat of the blood cockle. the chlorophyll a content, which is the main food environment indicator for bivalves, generally fluctuated mainly ranging from 4 to 20 µg.l-1 at all stations, and the occasionally increased to over 20 µg.l-1 during the survey period. then, it was estimated figure 4. relationships between the chlorophyll a content of the bottom layer (bottom-50 cm) and phytopigment content in the blood cockle, anadara granosa, at the four stations (st. a-d) along the selangor coast. a: st. a; b: st. b; c: st. c and d: st. d. a thick red straight line represents a linear approximation. mean±sd. 93 int. j. aquat. biol. (2021) 9(2): 88-96 that the food environment had enough content levels for bivalve feeding at all stations. in many kinds of cockle, anadara (scapharca) kagoshimensis, a. inaequivalvis, cerastoderma edule and clinocardium nuttallii, those are known to grow well or to develop the gonad in environment of the chlorophyll a content around 3 µg.l-1 (yurimoto et al., 2007; acarli et al., 2012; martínez-castro and vázquez, 2012; dunham et al., 2013). moreover, the coastal waters around tropical mangroves commonly have a high chlorophyll a content of over 20 µg.l-1 in wet, dry or whole seasons depend on region (senthilkumar et al., 2008; rivera-monroy et al., 2011; teoh et al., 2016). especially, teoh et al. (2016) surveyed primary production including chlorophyll a content in water column of selangor coast and reported southern coast from kuala selangor to sungai buloh was high chlorophyll a content over 20 µg.l-1 in dry season (february-march 2015). therefore, the chlorophyll a content observed along the selangor coast in this study reconfirmed that the waters have a high basic productivity similarly with those around other tropical mangrove coasts. food availability: phytopigment is a generic name that encompasses decomposition products of pigments, such as chlorophyll and pheophytin (numaguchi, 1985). therefore, the pigments extracted from the digestive gland of a bivalve is a useful indicator for the food availability conditions of the individual. numaguchi (1985, 2001, 2002) focused on this index, evaluating the phytopigment (sum of chlorophyll a and pheophytin contents) content in the digestive gland of bivalves. the phytopigment content in the digestive gland of pearl oysters from the coastal waters of japan is as follows: the phytopigment content in the digestive gland was approximately 150 µg.g-1 when the chlorophyll a + pheophytin in the water column was increased to approximately 15 µg.l1 (numaguchi, 1985). in addition, the phytopigment content in the digestive gland of a. (s.) kagoshimensis, fluctuated around several tens of µg.g-1 during winter (yurimoto et al., 2007). on the contrary, when the water temperature rose to more than 25°c and the chlorophyll a content was detected at 10 µg.l-1 or more during summer, the phytopigment content increased to approximately 160 µg.g-1 (yurimoto et al., 2007). in contrast, the phytopigment content of the blood cockle along the selangor coast was rarely lower than 100 µg.g-1, but mostly fluctuated ranging from 150 to 350 µg.g-1. this value is almost twice that measured for other bivalves. therefore, it was considered that the food environment of the selangor coast was sufficient to grow the blood cockle. in addition, unlike blood cockles living in the temperate zone, this result showed that seasonal changes to the phytopigment content in the cockle were unclear. in the temperate zone, the water temperature shows huge variabilities depending upon the season, and the spawning period is short compared with that of the tropical zone. therefore, it is suggested that the food availability for the blood cockle in the temperate zone has clear seasonality compared with that in the tropical zone. additionally, there was no correlation between the phytopigment content in the blood cockle and the chlorophyll a content in the bottom layer of the coastal water, as the coefficient of determination (r2) was less than 0.06 at all stations. this suggests that the phytopigment content that can accumulate in the digestive gland of the blood cockle is around 290 µg.g1, even when the cockle is placed in an environment with an excessive chlorophyll a content. therefore, the above values were considered to be one of the indicator criteria to show that the cockles were feeding sufficiently. on the contrary, at stations c and d, in some cases when the chlorophyll a content in the sea bottom layer was approximately 12 µg.l-1, a high phytopigment content of above 500 µg.g-1 was detected in the digestive gland of the blood cockle. factors influencing the high accumulation of phytopigment by the blood cockle are currently unknown. however, these high levels were mainly detected in september, october, february, and may, which are periods when there is not necessarily a high chlorophyll a content in the sea bottom layer. according to yurimoto et al. (2014a, c), the sexual maturation of the blood cockle on the west coast of peninsular malaysia is less clear than that of a kind of blood cockle in the temperate zone. however, the 94 yurimoto et al./ food availability of blood cockle stages are roughly divided into development and mature stages in september-october (early rainy season), and spawning and spent stages in februarymay (post rainy season). therefore, this suggests that the cockles may engage in aggressive feeding to develop their gonads or to restore their own strength after spawning. the environmental conditions were suitable for such excessive feeding, as the water temperature was almost stable at stations c and d, while the chlorophyll a content in the bottom layer was mainly around 12 µg.l-1. thus, it was suggested that a chlorophyll a content around 12 µg.l-1 enhanced the feeding activity of the blood cockle. marescaux et al. (2016) found that rearing experiments with freshwater bivalves revealed a higher filtration rate at a chlorophyll a content of 10-20 µg.l-1. thus, it is likely that the blood cockle has the same favourable concentration. conclusion the chlorophyll a content in the bottom layer of the main blood cockle aquaculture ground of the selangor coast fluctuated mainly ranging from 4 to 20 µg.l-1, and occasionally increased to over 20 µg.l-1. at all stations, the phytopigment content in the digestive gland of the blood cockle fluctuated ranging from 30 to 770 µg.g-1. however, there was no correlation between the contents of phytopigment in the blood cockle and chlorophyll a in environmental water. therefore, even in a high chlorophyll a content (over 20 µg.l-1) environment, the accumulated phytopigment in the digestive gland was around 290 µg.g-1. in general, these results indicated the cockles were eating a sufficient amount of foods (organic materials including phytoplankton) all year round during the study period. and, the food availability environment in the aquaculture grounds of the selangor coast was estimated sufficient to grow the blood cockle. acknowledgement this research was conducted as part of the grant project of jircas. references acarli s., lok a., yigitkurt s. 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(2015) 3(5): 352-361 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article the effects of washing with tamarind (tamarindus indica l.) water solution on shelf life of silver carp (hypophthalmichthys molitrix) fillet during refrigerator storage eshagh zakipour rahimabadi*1, 2, mehri zolfaghari1, mahin rigi3 1department of fisheries, faculty of natural resources, university of zabol, 98615-538 zabol, iran. 2fisheries department, faculty of natural resources, university of guilan, sowmeh sara, 1144, guilan, iran. 3hamoon international wetland research institute, university of zabol, zabol, iran. article history: received 11 december 2014 accepted 8 november 2014 available online 2 5 november 2015 keywords: hypophthalmichthys molitrix, tamarindus indica shelf life cold storage abstract: this study evaluated the antibacterial and antioxidant effects of tamarind water solution on shelf life of silver carp (hypophthalmicthys molitrix) fillet during refrigerator storage. treatments of this study were unwashed samples (control), and samples washed with 1% and 2% tamarind water solution. microbial, physicochemical and sensory analysis including total viable count (tvc), peroxide value (pv), thiobarbituric acid (tba), total volatile base (tvb-n) and ph were measured during 15 day storage at refrigerator (with 3 days intervals). proximate analysis of samples also measured at day 0. tvc content was 0.93, 0.50 and 0.10 log cfu/g for control and treatments 1% and 2%, respectively and reached to 6.24, 5.82 and 5.21 log cfu/g at the end of storage period. at the end of storage period, the pv, tba and tvb-n content were 8.4, 4.3, and 3.0 meq o2/kg for control, 2.75, 1.35, and 0.50 mg/100g for 1% treatment, and 33.17, 23.90, and 22.10 mg n/100g for 2% treatment, respectively. this results showed the positive effect of tamarind to inhibit and delay fish fillet spoilage. according to sensory evaluation, the density of 1% tamarind was selected as the best density. introduction seafoods with high quality proteins, high content of unsaturated fat, vitamins and minerals are noticed as a useful food for human health (venugopal, 2006). many efforts have been done to supply fresh seafood rather than frozen or other processed products with high quality according to increasing consumer’s demands (hassan, 2002; fernández et al., 2009). fish are very perishable food due to large amounts of free amino acids, volatile nitrogen bases, highly unsaturated fatty acids and higher final ph (liston, 1980; razavi shirazi, 2001). therefore, they are very susceptible to bacterial spoilage and oxidative rancidity during storage (mexis et al., 2009). different methods have been presented to inhibit or delay of seafood rancidity and spoilage such as temperature control, vacuum packaging, modified atmosphere packaging (map), and supplying of * corresponding author: eshagh zakipour rahimabadi e-mail address: ezakipour@gmail.com antioxidants (lin and lin, 2005). various compounds such as chemical antimicrobials and antioxidants are used to prevent the spoilage of fresh fish (lu et al., 2010). as artificial antioxidants have some undesirable effects such as creating mutation, intoxication and carcinogenic; hence, application of natural antioxidants with the same inhibitory effect on oxidation is increasing (sakanaka et al., 2005). applying herbal compounds as natural preservatives in fish fillets can be effective especially on shelf life increasing during storage in refrigerator. tamarind (tamarinad indica l.) belongs to the family fabaceae and the subfamily of caesalpinioideae, which is the third extensive family of flowering plants (lewis and neelakantan, 1964). its chemical compositions includes 20.6% water, 3.1% protein, 0.4% lipid, 70.8% carbohydrate, 3% fiber and 1.2% ash (el-siddig et al., 1999). tamarind 353 zakipour rahimabadi et al./ effect of washing with tamarind solution on shelf life of silver carp fillet is used as an appropriate food preservative compound that inhibits food infectious bacteria growth. tamarind has also showed oxidation inhibitory activity due to high content of phenolic composition (el-siddig et al., 2006). although there are many studies regarding to usage of synthetic and natural antioxidant and antimicrobial compounds on shelf life extension of seafoods, information regarding to effects of tamarind in this area is rare (pezeshk et al., 2010). therefore, the present research was conducted to study the effect of tamarind water solution preservative effect on microbial, physicochemical and sensory characteristics of silver carp (hypophthalmichthys molitrix) fillet during the storage in refrigerator. materials and methods sample preparation: fifteen silver carp with the average weight and length of 1000 (±100) g and 40 (±5) cm, respectively, were purchased from a local fish market (zabol, iran) in january 2014. they were transferred in insulated boxes with ice to seafood processing laboratory. immediately, the fish were rinsed with drinking water and then eviscerated, headed and filleted by hand. the fillets were rinsed again and cut into 5×5×1 cm pieces. these pieces were soaked in tamarind water solution for 5 min based on mohsin et al. (1999) and then put on the strainer for 1 min to drip the water. three treatments, including unwashed samples (as control), washed with 1% and 2% tamarind water solution were considered in this study. after treating, all samples were battered, breaded, packaged and then kept in refrigerator for 15 days. preparation of tamarind water solution: 200 g thai tamarind was purchased from a retail shop (zabol, iran). water solution was prepared based on bekar and hamzeh (1997). thus, the paste was dissolved in boiled water, and its shell, seed and fiber were separated using a strainer. the solution was heated over a flame and the obtained paste was dissolved in distilled water (w/v) to get required concentration i.e. 1 and 2%. microbiological analysis: microbiological counts were determined by placing 10 g sample in 90 ml of 0.85% nacl solution, and homogenizing with a stomacher (moulinex, france) for 60 second. from this dilution, other decimal dilutions were prepared and plated in the appropriate media. meanwhile, the total viable counts (tvc) were determined using tryptic soy agar (tsa, merck) after incubation for 48 hrs at 25°c (aoac, 2005). the microbiological data were transformed into logarithms of the number of colony-forming units (cfu/g). chemical analysis: the moisture content of flesh was measured by drying to constant weight at 105°c for 24 hrs according to aoac (2005). the crude ash was determined after heating the sample overnight at 550°c (aoac, 2005). the crude protein content was measured by the kjeldahl method (aoac, 2005), employing the 6.25 conversion factor. the crude lipid was determined by ether extraction using a soxhelet method (aoac, 2002). pv value was determined based on aoac (2000) and expressed as meq o2/kg lipid sample. tba value was measured according to namaulema et al. (1999), and was expressed as mg malondialdehyde/kg sample. total volatile base nitrogen (tvb-n, mg n/100 g) was determined according to aoac (2005). sensory evaluation: sensory evaluation was performed by seven half-trained persons based on hedonic (astm, 1969). after deep frying the samples in sunflower oil (bahar, iran), properties of colour, odour, texture, taste and overall acceptability were evaluated by assessors. the samples were scored from 0-7: highest quality = 7, good quality = 5, fair quality = 3 and rejectable quality = 0. statistical analysis: statistical analysis were performed using the spss program, version 16. tukey’s test was performed to evaluate the significance of differences among mean values. a nonparametric anova of kruskal wallis test was used for sensory evaluation. results proximate analysis of the control and treated samples of silver carp fillet are shown in table 1. according to the results, silver carp is a fish with moderate fat 354 int. j. aquat. biol. (2015) 3(5): 352-361 content. microbial analysis: the content of total viable count (tvc) for control treatment, 1% tamarind and 2% tamarind was 0.93, 0.50 and 0.10 log cfu/g at day 0, respectively (table 2). increasing the values of tvc were found in all the treatments during storage, but the changes of tvc in control treatment was higher than those of treatments. the results showed that treating the silver carp fillets with tamarind water solution significantly decrease tvc (table 2). ph: the changes in ph of silver carp fillets as a function of treatments and storage time are presented in table 3. the ph of control samples at day 0 was 6.37 and significantly (p<0.05) lower ph values were found for treated samples with tamarind solution. for control samples, the highest ph was observed at day 15 and the least content of ph at day 3. for fillets treated with tamarind solution, the highest ph was observed at day 0 and least at day 15. peroxide value (pv): pv values of control and treated samples, and their changes during refrigerator storage are shown in table 4. the initial pv value of the control samples at day 0 was 1.34 meq o2/kg. this amount was higher as compared with raw samples of silver carp reported by zakipour rahimabadi and divband (2012). lower pv values were found in treated samples with 1 and 2% of tamarind solution at initial of storage period (table 4). pv values increased significantly (p<0.05) with time of storage at 4ºc for all treatments. pv value was significantly lower (p<0.05) for treated samples during the storage period compared with those of the chemical composition control group 1% tamarind treatment 2% tamarind treatment total protein 2.70a±17.80 0.90a±17.60 2.00a±17.77 total fat 1.20a±4.79 0.63a±4.69 0.50a±4.20 moisture 2.02a±75.80 1.00a±75.80 0.64a±76.80 ash 0.21a±1.61 0.45a±1.91 1.40a±1.23 values are means and s.d. of triplicate. means with the same capital letter in a row were not significantly different at p<0.05 level. table 1. chemical composition (%) of raw and treated samples of h. molitrix with tamarind solution. days of storage treatments control 1% tamarind 2% tamarind 0 0.93±0.08af 0.50±0.15be 0.10±0.02cd 3 1.50±0.04ae 0.70±0.20be 0.30±0.02bd 6 3.80±0.07ad 3.30±0.06bd 3.00±0.00cc 9 5.20±0.04ab 4.70±0.00bc 4.34±0.05cb 12 5.60±0.35ab 5.10±0.10bb 5.01±0.02ba 15 6.24±0.05aa 5.82±0.10ba 5.21±0.01ca values are means and s.d. of triplicate. means with the same capital letter in a row were not significantly different at p<0.05 level in different treatment. means with the same small letter in a column were not significantly different at different at p<0.05 level during storage at 4˚c. table 2. changes in tvc (log cfu ̸ g) in different treatments during refrigerator storage. days of storage treatments control 1% tamarind 2% tamarind 0 6.37±0.02ac 6.16±0.05ba 6.16±0.01ba 3 5.97±0.01ad 5.83±0.05ab 5.63±0.10bb 6 6.17±0.15ad 5.80±0.09bb 5.51±0.12bb 9 6.42±0.03ac 6.23±0.05ba 6.12±0.03ca 12 6.92±0.09aa 5.76±0.06bb 5.54±0.04cb 15 6.96±0.04aa 5.55±0.03bc 5.21±0.07cc values are means and s.d. of triplicate. means with the same capital letter in a row were not significantly different at p<0.05 level indifferent treatment. means with the same small letter in a column were not significantly difference at different at p<0.05 level during storage at 4˚c. table 3. changes in ph of control and treated samples of h. molitrix during refrigerator storage. 355 zakipour rahimabadi et al./ effect of washing with tamarind solution on shelf life of silver carp fillet control samples. there were no significant changes from day 3 until 9 but there were significant changes from day 9 afterward (table 4). thiobarbituric acid (tba) value: the tba values of control and treated samples were 0.053, 0.04 and 0.006 mg malondialdehyde/kg of lipid at initial of storage period, respectively. a continues increase in tba value was observed in control samples and other treatments throughout of 15 days storage period (p<0.05). the tba of treated samples with tamarind solution was lower than those that of control samples (p<0.05) (table 5). tvb-n: the initial tvb-n value in raw silver carp fillets was 10.33 mg/100 g. treatment with tamarind water solution did not produce a significant effect on the initial tvb-n values decrease. the tvb-n was significantly (p<0.05) decreased in the 1% samples than that of the control samples. tvb-n content in 2% samples was significantly lower than that of 1% treatment (p<0.05) (table 6). sensory evaluation: the results of sensory evaluation for control and treated fillets with tamarind solution are presented in table 7. at the beginning of the storage period, all treatments had a days of storage treatments control 1% tamarind 2% tamarind 0 1.34±1.20ac 0.73±0.50ab 0.26±0.06ac 3 2.98±0.23abc 2.87±0.80aa 2.90±0.10aa 6 0.92±0.09ac 0.72±0.26ab 0.68±0.28abc 9 5.13±1.01ab 3.17±0.80aa 2.73±1.40aab 12 3.90±0.70abc 3.70±0.60aa 2.50±0.50ab 15 8.40±1.90aa 4.30±0.70ba 3.00±0.27ba values are means and s.d. of triplicate. means with the same capital letter in a row were not significantly different at p<0.05 level in different treatment. means with the same small letter in a column were not significantly different at p<0.05 level during storage at 4˚c. table 4. changes in pv values in different treatments during storage at 4°c. days of storage treatments control 1% tamarind 2% tamarind 0 0.05±0.01ad 0.04±0.01ac 0.00±0.00bb 3 0.12±0.03ad 0.06±0.00bc 0.01±0.00cb 6 1.37±0.17ac 0.08±0.01bc 0.03±0.01bb 9 1.75±0.15ab 0.37±0.20bb 0.06±0.01bc 12 1.93±0.08ab 1.20±0.08ba 0.13±0.06cb 15 2.75±0.19aa 1.35±0.50ba 0.50±0.14ca values are means and s.d. of triplicate. means with the same capital letter in a row were not significantly different at p<0.05 level in different treatment. means with the same small letter in a column were not significantly different at p<0.05 level. table 5. changes in tba values in control and treated samples during refrigerator storage. days of storage treatments control 1% tamarind 2% tamarind 0 10.33±0.76af 9.03±0.71ae 9.10±0.80ad 3 14.40±1.00ae 12.27±0.71bd 11.06±0.44bd 6 20.23±1.03ad 14.37±0.60bc 13.33±1.00bc 9 22.87±0.81ac 15.84±0.40bc 13.70±0.90cc 12 27.74±0.65ab 20.21±0.92bb 19.10±0.86bb 15 33.17±1.15aa 23.91±1.08ba 22.11±0.60ba values are means and s.d. of triplicate. means with the same capital letter in a row were not significantly different at p<0.05 level in different treatment. means with the same small letter in a column were not significantly difference at different at p<0.05 level during storage at 4°c. table 6. changes in tvb-n content in different treatments during storage at 4°c. 356 int. j. aquat. biol. (2015) 3(5): 352-361 high quality of examined sensory indicators (texture, color, odor, taste, and overall acceptability). treated samples with 2% tamarind solution had lower sensory scores than those of 1% treatment and control one at day 0 in odor and taste indicators but this difference was not significant (p>0.05). in all treatments, the score of all sensory indicators decreased by passing the time in control treatment. as the score of texture and color at day 9 and the other indicators at day 6 reached to acceptable quality of minimum score. assessors expressed that the samples treated with 2% tamarind solution were unacceptable but the samples treated with 1% tamarind solution had a good quality score until day 15. discussion the results showed that washing silver carp fillets with tamarind solution has significant effects on all measured parameters. the highest growth of spoilage pathogens in control samples was occurred as reported in other works (samelis et al., 2001; schillinger et al., 1996). the number of total bacteria load in control samples was significantly higher than those treated fillets with tamarind solution showing inhibitory effects of tamarind on bacteria which it is in agreement with the results of parhusip adolf et al. (2011). the most outstanding characteristic of tamarind is its sweet acidic taste (el-sidding et al., 1999). phytochemical components such as tannins, flavonoids, alkaloids and several other aromatic compounds are secondary metabolites of plants that serve as defense mechanisms against microorganisms, insects and herbivores (lutterodt et al., 1999). phenolic compounds act on double membrane of phospholipid cells and causes increasing the penetration and leak of interacellular’s necessary elements (e.g. iron, atp, nucleic acid and amino acid) (yaldaei et al., 2013). in addition, it possibly harms the enzymatic system of bacteria causing bacteria’s death (hyytiä et al., 1999). the low initial muscle ph value reflects the good nutritional state of fish. the ph value in tamarind treatments decreased slowly during the storage period. this could be due to production of lactic acid produced during glycolysis (massa et al., 2005). furthermore, denaturation of proteins starts to produce some products such as amines (massa et al., 2005; woywoda et al., 1986). the degradation of nitrogen compositions is led to the increase of ph in meat during the storage, and also ph increases due to the production of alkaline compositions during factors treatments days of storage 0 3 6 9 12 15 texture control 0.00aa±7.00 0.00bb±5.00 0.22bc±3.50 0.00bc±3.00 0.00bd±1.00 0.00bd±1.00 1% 0.00aa±7.00 0.00aa±7.00 0.00ab±5.00 0.00ab±5.00 0.16ab±4.83 0.21ab±4.66 2% 0.00aa±7.00 0.00aa±7.00 0.00ab±5.00 0.00ab±5.00 0.00ab±5.00 0.33ab±4.70 color control 0.00aa±7.00 0.21ab±5.00 0.01bb±4.46 0.27bc±3.65 0.21bc±3.33 0.08bd±2.60 1% 0.00aa±7.00 0.16ab±4.83 0.16ab±4.83 0.21ab±4.66 0.21ab±4.66 0.21ac±4.30 2% 0.00aa±7.00 0.00ab±5.00 0.16ab±4.83 0.16ab±4.83 0.21ab±4.66 0.16ab±4.66 odor control 0.00aa±7.00 0.16ab±4.83 0.16bc±3.16 0.00bc±3.00 0.00bd±2.00 0.00be±1.00 1% 0.00aa±7.00 0.08ab±4.66 0.34ab±4.66 0.34ab±4.50 0.20ab±4.30 0.00ac±4.00 2% 0.00aa±5.90 0.20ab±4.30 0.00ab±4.00 0.21cc±2.60 0.00cd±1.00 0.00bd±1.00 taste control 0.00aa±7.00 0.02bb±4.34 0.02bc±3.16 0.00bd±1.00 0.00bd±1.00 0.00bd±1.00 1% 0.00aa±7.00 0.00ab±5.00 0.08ab±4.66 0.00ac±4.00 0.00ac±4.00 0.00ac±4.00 2% 0.00aa±5.90 0.20bb±3.16 0.21cc±2.60 0.00bd±1.00 0.00bd±1.00 0.00bd±1.00 overall acceptability control 0.00aa±7.00 0.00bb±4.00 0.20cc±3.16 0.21cd±2.60 0.00ce±1.00 0.00be±1.00 1% 0.00aa±7.00 0.00ab±5.00 0.08ab±4.66 0.08ab±4.66 0.00ac±4.00 0.00ac±4.00 2% 0.34ba±4.50 0.33aa±4.50 0.00ba±4.00 0.21bb±3.60 0.21bc±2.60 0.00bd ±1.00 values are means and s.d. (n= 7) means with the same capital letter in a row were not significantly different at p<0.05 level in different treatment. means with the same small letter in a column were not significantly different at p<0.05 level during storage at 4°c. table 7. the results of sensory evaluation for different treatments during storage at 4°c. 357 zakipour rahimabadi et al./ effect of washing with tamarind solution on shelf life of silver carp fillet this process. increasing ph can indicate the bacteria growth, reduction of quality and finally fish spoilage (gram and huss, 1996). low ph of the treated fillets with tamarind solution can be related to tamarind anti-bacterial characteristic (de et al., 1999). such process may contribute to decline muscle ph, by inhibiting growth of bacteria, as well as buffering the basic metabolites (calo-mata et al., 2008). it can be concluded that the lower ph values of 1 and 2% treated fillets may restrain microbial growth and inhibit the activity of the endogenous proteases, leading the extension of the preservation of silver carp fillets. the peroxide value (pv) is used to measure the primary lipid oxidation especially hydro-peroxides. these are mainly due to chemical changes in muscle tissue as a result of a wide range of factors particularly the nature of lipid, process involving oxidation of the unsaturated fatty acids or triglycerides in fish (ashie et al., 1996). factors that may influence lipid oxidation are microbial spoilage, biochemical substances and environmental conditions (erkan and özden, 2008). based on the results, pv of fillets was within the acceptable limit of 10-20 meq/kg of fat (connell, 1990). the increase of pv in fillets treated with tamarind solution indicated the development of spoilage and rancidity during fish storage. the increase of pv during the storage period was significant for all treatments which it is in agreement with the results of özogul et al. (2005) and pacherco-aguilar et al. (2000). the lipid oxidation value in fish commonly increases due to lipid oxidation by extending storage period. lipid oxidation is one of the basic factors to make an unpleasant taste in meat (guillén and ruiz, 2004). less peroxide value of fillets treated with tamarind solution is due to inhibiting the lipid oxidation by tamarind solution (el-siddig et al., 2006). tamarind absorbs free radicals because it has phenolic compositions with a convoluted basis, thus it inhibits spoilage, color change or lipid rancidity by impeding oxidation (el-siddig et al., 2006). in addition, it has an important role to inhibit lipid oxidation (el-siddig et al., 2006). there is a positive relationship between phenolic value and extracts, essences anti-oxidation property (tsai et al., 2008). according to the results, the treatment of fish fillets with 2% tamarind solution has higher anti-oxidation properties and therefore, the peroxide value in 2% treatment was significantly lower than those of control one and 1% treatment. in present study, tba value in control treatment was about 3 mg/kg at day 15 and the values of 1% and 2% tamarind treatments were 1.35 and 0.5 mg/kg, respectively. these results showed a positive effect of tamarind to inhibit and delay of fillet spoilage. increasing lipid oxidation during storage can be due to more free iron releasing and other per oxidants from the muscle with more analysis during the storage (chaijan et al., 2006). chaijan et al. (2006) pointed out that by increasing the storage period, hydrolysis and fish lipid oxidation increase and also hydroperoxids and paired dns is produced. therefore, time increases producing secondary reaction products of lipid oxidation that reacts with tba reactor (chaijan et al., 2006). the existence of such compositions in fish meat causes changes in its sensory characteristics such as taste and odor (dragoev et al., 1998; ladikos and lougovois, 1990). the decrease of the content of thiobarbitoric acid at some days of storage may be due to decreasing hydroperoxids and reaction between malonede aldehyde and proteins, amino acids and glycogen that decrease the content of malonede aldehyde (gomes et al., 2003; ojagh et al., 2010). the results showed that tamarind has anti-oxidant effect and it can delay fish meat spoilage which accords with the results of yaldaei et al. (2013). comparison of tvb-n value of the control sample with those of treated samples in different storage periods showed a significant difference from day 3 afterward. the increase of tvb-n value was presumably due to the bacteria activity during the storage period in refrigerator (ibrahim and desouky, 2009). the results also showed that tvb-n value in tamarind treatments was under the standard quantity (25 mg /100 g) at the end of the storage period. as it seems, tamarind causes less degradation of the 358 int. j. aquat. biol. (2015) 3(5): 352-361 proteins via microorganisms by decreasing ph and microbial load and it also effects on tvb-n (hebard et al., 1982; giménez et al., 2002; arashisar et al., 2004). the results of the present study was in agreement with the findings of rostamzad et al. (2010) who studied anti-oxidant effect of the citric acid on lipid spoilage in the frozen fillets of acipenser persicus during 6 months storage. sensory assessment is applied as one of the methods to evaluate fish quality during the storage period in many studies (fan et al., 2008; fan et al., 2009; mexis et al., 2009; ojagh et al., 2010). by storing the silver carp fillets in refrigerator, a considerable changes was found in its sensory attributes. the results of taste, odor, texture, color and total acceptability of silver carp fillets showed that all samples were in a very good condition at day 0 except 2% treatment that its odor and taste indicators were undesirable. by passing the time, tamarind treated samples had more desirable conditions of all factors than control samples. this showed the role of tamarind solution anti-oxidant attributes to keep tamarind samples quality (el-siddig et al., 2006). the total of evaluated sensory attributes expresses considerable prominence of 1% tamarind fillets in ratio with the other samples. fan et al. (2008) reported that sensory scores of silver carp fillets treated with tea polyphenol decrease by increasing storage period, and its sensory attributes receive a higher score. as conclusion, the chemical and microbial results of present study showed that using tamarind water solution as natural anti-oxidant and anti-bacterial compositions can decrease the intensity of bacteria activity which exists on the surface of fish meat. it can also delay the oxidation spoilage and therefore increases fish shelf life. the comparison between control samples, and 1 and 2% tamarind treatments showed a significant difference between them in terms of the examined chemical and bacterial parameters. the increases in the spoilage factor was significantly lower in the 1% samples than in the control samples and all the factors of 2% was significantly lower than 1%. according to the results, the concentration of 2% tamarind has more anti-oxidation properties and all the factors value in 2% treatment was significantly lower than those of control and 1% samples. this study showed the positive effect of tamarind to inhibit and delay fish fillet spoilage. according to sensory evaluation, the density of 1% tamarind was selected as the best concentration. acknowledgments authors thank rahdari, heidari, khosravanizadeh, and navidpour for their cooperation and technical assistances. references aoac. 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(2015) 3(5): 352-361 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی فیله ماندگاری زمان بر( .tamarindus indica l) هندی تمبر آبی محلول با شووشست اثر بررسی یخچال در نگهداری خالل در( hypophthalmichthys molitrix) اینقره کپور 3ریگی مهین ،1ذوالفقاری مهری ،*2،1آبادیرحیم پورزکی اسحق .ایران زابل، زابل، دانشگاه طبیعی، منابع دانشکده شیالت، گروه1 2گروه شیالت، دانشکده منابع طبیعی، دانشگاه گیالن، صومعه سرا، ایران. .ایران زابل، زابل، دانشگاه هامون، تاالب المللیبین پژوهشکده3 چکیده: hypophthalmichthys) اینقرهکپور فیله ماندگاری زمان بر هندیتمبر آبی محلول ضداکسیداسیونی و باکتریایی ضد اثرات حاضر مطالعه در molitrix )تیمار) هندیتمبر آبی محلول با شووشست بدون هایفیله شامل تحقیق تیمارهای. گرفت قرار بررسی مورد یخچال در نگهداری خالل در میکروبی، هایآزمایش. بودند درصد 2 هندی تمبر آبی محلول با شده شسته هایفیله و هندیتمبر درصد 1 آبی محلول با شده شسته فیله ،(شاهد نیتروژنی بازهای ،(tba) اسید تیوباربیتوریک شاخص ،(pv) پراکسید محتوای ،(tvc) کل باکتریایی بار شمارش شامل حسی و فیزیکوشیمیایی همچنین تقریبی ترکیب آنالیز. گرفتند قرار بررسی مورد( روزه 3 زمانی فواصل در) یخچال در نگهداری روزه 11 دوره یک در ph و( tvb-n) فرار به هندیتمبر آبی محلول با شووشست درصد 2 تیمار و درصد 1 تیمار شاهد، تیمار در صفر روز در کل باکتری میزان. پذیرفت انجام صفر روز در مقدار. یافت افزایش log cfu/g 21/1 و 22/1 ،22/6 میزان به ترتیب به دوره انتهای در که بود log cfu/g 10/0 و 10/0 ،33/0 با برابر ترتیب شووشست درصد 2 تیمار و درصد 1 تیمار شاهد، نمونه برای نگهداری دوره انتهای در فرار نیتروژنی بازهای مجموع و اسید تیوباربیتوریک پراکسید، گرممیلی 10/0 و 31/1 ،51/2 ماهی، چربی کیلوگرم در اکسیژن گرم واالناکی میلی 0/3 و 2/2،3/2 به برابر ترتیب به هندیتمبر آبی محلول با تاثیر دهنده نشان نتایج. رسید ماهی گوشت گرم 100 در نیتروژن گرم میلی 10/22 و 30/23 ،15/33 و ماهی گوشت گرم 100 در آلدئید مالون به هندیتمبر درصد 1 غلظت حسی، ارزیابی نتایج اساس بر. بود ماهیفیله فساد تاخیر و جلوگیری بر هندیتمبر بیآ محلول با شووشست مثبت . گردید انتخاب غلظت بهترین عنوان .یخچال در نگهداری ماندگاری، زمان هندی،-تمبر ای،نقرهکپور :کلمات کلیدی int. j. aquat. biol. (2014) 2(2): 91-98 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article comparative study on effects of different salinities on the reproduction rates of two parthenogenetic species of artemia (gaav khooni wetlands of isfahan and ponds around the urmia lake) from iran hamed aalamifar*1, naser agh2, reza malekzadeh2, mahbobe aalinezhad3 1department of fisheries, faculty of natural resources urmia university, iran. 2artemia and aquatic animals research center, urmia, iran. 3department of fisheries, faculty of marine natural resources, marine science and technology university, khoramshahr, iran. article history: received 28 october 2013 accepted 27 february 2014 available online 2 5 april 2014 keywords: artemia gaav khooni reproductive iran isfahan urmia abstract: this paper investigated effects of different salinities on reproductive of two parthenogenetic species of artemia (from gaav khooni wetlands of isfahan and ponds around lake urmia) in iran. nauplii from two iranian brine shrimp parthenogenetic populations, were grown up at three salinities (80, 120 and 150 ppt). the initial stocking density was 200 nauplii/litre at all the salinities. the shrimps were fed according to a standard feeding table. the results show that gaav khooni’s artemia has better reproductive than the brine shrimp from urmia ponds (except in reproductive characteristics such as number of offspring at each day of reproductive period, the number and the percent of offspring encysted). moreover, artemia from ponds around the urmia lake had a reduction in many characters with increasing salinity, but artemia from gaav khooni showed best results at optimal levelʼs of salinity (120 ppt). therefore the present study indicated that artemia from gaav khooni, unlike most species the decrease in salinity, (optimal level) is also vulnerable. introduction the brine shrimp artemia is a genus with a wide distribution on the five continents, inhabiting inland salt lakes, coastal lagoons and solar saltworks (vanhaecke et al., 1987). it is among the unique organisms that can adapt to very diverse living conditions that involve salinities as low as 10 g/l (abatzopoulos et al., 2006) to as high as 340 g/l (post and youssef, 1977). it comprises a complex of sibling species and superspecies defined by a criterion of reproductive isolation (browne and bowen, 1991). the gaav khooni lake is situated 140 km south-east of the isfahan province. it is one of the rare wetlands of central iran and, in this sense, plays a critical role for migratory and native birds. it is an internationally protected natural reserve. the gaav khooni lake is * corresponding author: hamed aalamifar e-mail address: hamed.aalamifar@gmail.com located on the center of the gaav khooni region, which extends over an area of 2800 km2. the soil is salty throughout the region and a permanent salt crust covers a large area around the lake. it is almost nearly permanent saline lake and its major water source is the river zayandeh roud. during the rainy season, many smaller lagoons and lakes with fluctuating salinity appear around the central lake. the average annual precipitation is 83 mm. the dry season lasts from late march until mid-october. the temperature in the region ranges from 6.6 to 37.48°c (asri et al., 2002). water salinity is usually above 120 g/l. agh et al. (2007) reported the presence of a possible parthenogenetic artemia population in the gaav khooni lake at isfahan (central of iran). agh and noori (1997) and agh et al. (2001) also reported the presence of a morphologically 92 aalamifar et al./ int. j. aquat. biol. (2014) 2(2): 91-98 distinctive parthenogenetic population in small lagoons in the vicinity of the urmia lake. the lagoons are scattered at the periphery of the lake both in west and easte azerbaijan. the size of the lagoons varies from a few square meters to maximum 10000 m2 surface area and their depth is always less than 0.7 m. therefore these lagoons are temporary small water catchments that are dried during the early summer and filled up again during the winter rainfall. water salinity in the lagoons ranges from 10-20 g/l in the early spring and gradually rises to saturation level within 10 weeks. parthenogenetic females were observed at high densities with rare males seen only at the ratio of one male to 100 females in these lagoons (agh and noori, 1997; abatzopoulos et al., 2006). there is a lot of information on survival and growth of many parthenogenetic artemia populations (vanhaecke et al., 1984; wear and haslett, 1986; browne et al., 1984, 1991; browne and wanigasekera, 2000; triantaphyllidis et al., 1995, 1997; baxevanis et al., 2004; el-bermawi et al., 2004; abatzopoulos et al., 2004, 2006; agh et al., 2007). but, the effect of salinity on artemia population in the gaav khooni lake at isfahan (central of iran) have been poorly examined or has not been studied at all therefore, the present study aimed to survey effects of salinity on reproduction rate of two parthenogenetic species of artemia (gaav khooni wetlands of isfahan, ponds around lake urmia) from iran. materials and methods culture procedure: the origins of the artemia cysts used in the experiment are as follows: (1) artemia parthenogenetica from the gaav khooni wetlands at isfahan, iran; (2) artemia parthenogenetica from the ponds around lake urmia, iran. in the laboratory, the artemia cysts were separated by floatation separation method and density separation in freshwater (gillbert, 1996). the cysts were maintained under conditions of continuous illumination and aeration with gaav khooni wetland-water collected in the sampling station and was hatched at a constant temperature of 23–25oc and salinity of 33–35 ppt, ph was adjusted to 8.0 throughout the hatching. after hatching, the nauplii were separated from their shells and from the unhatched cysts by the method described by amat (1985) then three replicates of 200 actively moving nauplii from each population were allocated to 500 ml conical containers filled with 400 ml of filtered water with different salinities. solutions of different salinities (80, 120 and 150 ppt) were prepared using synthetic gaav khooni wetlandwater and salt of gaav khooni wetland. the animals were cultured at 27 ± 1°c under constant aeration. the salinity in each cone was checked daily in order to maintain salinities according to the experimental set up. twenty four h after hatching, larvae were fed daily with yeast-based diet lansy pz, 1.66 gl-1, (inve aquaculture nv, belgium). density of artemia was adjusted to one animal per 2 ml at the beginning of the experiment, but the density was gradually decreased to one animal per 3 ml on day 8 and per 4 ml on the day 14. reproductive characteristics: as animals attained maturity, 30 adult parthenogenetic females from each population (from all salinities) were transferred into separate 50 ml falcon tubes in order to study their life cycle characteristics. the falcon tube were checked every day for the production of cysts or nauplii, which were counted and recorded separately. finally, the reproductive characteristics (offspring per brood, number of brood per female, offspring per female per day, during the reproductive period, brood intervals, percent offspring encysted, total offspring per female, average number of cysts and larvae) were determined for each population according to browne et al. (1984, 1988). statistical analysis: the results were statistically analyzed using spss (version 17). the normality of distribution of variables was examined using the kolmogorov–smirnov test. the homogeneity of variances was examined using the levene's f test. the differences in the variables among the treatments were tested using one-way anova at 93 aalamifar et al./ int. j. aquat. biol. (2014) 2(2): 91-98 regular sampling dates. post hoc comparisons between sample means performed using by tukey and lsd test. data were presented as mean ± standard error (s.e) and differences were considered statistically significant at p<0.05. results the number offspring per brood: the average number offspring per brood declined in the both populations when salinity increased. the brine shrimp ponds had significantly higher offspring per brood at salinities 120 ppt and 150 ppt compared to artemia from gaav khooni. the artemia from gaav khooni had significantly higher offspring per brood only at 80 ppt compared to the brine shrimp ponds. no significant differences were found in the offspring per brood of parthenogenetic strains at 80 ppt and 120 ppt, but significant difference was found at 80 and 150 ppt (p<0.05). the maximum number offspring per brood was found in artemia from gaav khooni at 80 ppt, significantly higher than those of the other salinities at intrapopulation level (p<0.05). also minimum number offspring per brood was observed in artemia from gaav khooni at 150 ppt (table 1). number of brood per female: the average number of brood per female declined in the both populations when salinity increased (except in artemia from gaav khooni at salinity 80 ppt). the artemia from gaav khooni had significantly higher number of brood per female at salinities 80 ppt and 120 ppt compared to brine shrimp ponds. the brine shrimp ponds had significantly higher number of brood per female only at 150 ppt compared to artemia from gaav khooni. the maximum number of brood per female was found in artemia from gaav khooni at 120 ppt, significantly higher than those of the other salinities at intrapopulation level (p<0.05). also the minimum number of brood per female was observed in artemia from gaav khooni at 150 ppt (table 2). offspring per female per day, during the reproductive period: different results obtained in the artemia species / salinity (ppt) 80 120 150 artemia gavkhouni a 16.785±2.04 abd 14.333±1.11 c 5.02±2.23 artemia ponds a 14.733±0.9 abd 14.863±1.52 ad 10.195±2.64 the rows that have no common letters are significantly different (p<0.05). table 1. comparison of two artemia populations in different levels of salinity in terms of average number offspring per brood artemia species / salinity (ppt) 80 120 150 artemia gavkhouni a 4.3±0.1 abe 5.6±0.23 c 1.3±0.11 artemia ponds b 3.0±0.45 ab 2.53±0.3 c 1.8±0.14 the rows that have no common letters are significantly different (p<0.05). table 2. comparison of two artemia populations in different levels of salinity in terms of average number of brood per female artemia species / salinity (ppt) 80 120 150 artemia gavkhouni a 2.423±0.1 ab 1.383±0.12 a 1.527±0.15 artemia ponds a 2.357±0.15 abd 3.877±0.17 a 2.813±0.11 the rows that have no common letters are significantly different (p<0.05). table 3. comparison of two artemia populations in different levels of salinity in terms of average number offspring per female per day, during the reproductive period artemia species / salinity (ppt) 80 120 150 artemia gavkhouni a 8.68±1.11 b 11.18±0.88 c 2.35±0.32 artemia ponds d 5.43±0.48 edc 3.83±0.71 c 2.83±0.11 the rows that have no common letters are significantly different (p<0.05). table 4. comparison of two artemia populations in different levels of salinity in terms of average number day of brood intervals 94 aalamifar et al./ int. j. aquat. biol. (2014) 2(2): 91-98 offspring per female per day, during the reproductive period for each one populations. therefore, the maximum number offspring per female per day, during the reproductive period was observed in brine shrimp ponds at 120 ppt, significantly higher than those of the other salinities at intrapopulation level (p<0.05), but the minimum number offspring per female per day, during the reproductive period was observed in artemia from gaav khooni at 120 ppt (table 3). brood intervals (in terms of day): the average number day of brood intervals declined in the both populations when salinity increased (except in artemia from gaav khooni at salinity 120 ppt). the artemia from gaav khooni had significantly higher number of day in the brood intervals at salinities 80 and 120 ppt compared to brine shrimp ponds. the brine shrimp ponds had significantly higher number of day in the brood intervals only at 150 ppt compared to artemia from gaav khooni. the maximum number day of brood intervals was observed in artemia from gaav khooni at 120 ppt, significantly higher compared to the values obtained at other salinities at intrapopulation level (p<0.05), also the minimum number of day in the brood intervals was observed in artemia from gaav khooni at 150 ppt (table 4). percent offspring encysted: no significant differences were found in the percent offspring encysted of parthenogenetic strains at all levels salinity (p<0.05) but the maximum percent offspring encysted was observed in both partinogenetic populations at 120 ppt (table 5). total offspring per female: total offspring per female declined in the both populations when salinity increased (except in artemia from gaav khooni at salinity 120 ppt). the artemia from gaav khooni had significantly higher total offspring per female at salinities 80 ppt and 120 ppt compared to brine shrimp ponds. the brine shrimp ponds had significantly higher total offspring per female only at 150 ppt compared to artemia from gaav khooni. artemia species / salinity (ppt) 80 120 150 artemia gavkhouni a 37.32±7.11 a 41.93±7.23 a 40.35±3.2 artemia ponds b 77.24±6.32 b 81.68±5.34 b 64.07±5.18 the rows that have no common letters are significantly different (p<0.05). table 5. comparison of two artemia populations in different levels of salinity in terms of percent offspring encysted artemia species / salinity (ppt) 80 120 150 artemia gavkhouni a 82.87±9.23 ab 91.53±8.76 c 11.27±1.97 artemia ponds d 54.13±10.3 edf 45.7±4.65 f 29.67±4.44 the rows that have no common letters are significantly different (p<0.05). table 6. comparison of two artemia populations in different levels of salinity in terms of total offspring per female artemia species / salinity (ppt) 80 120 150 artemia gavkhouni a 32.39±3.3 ab 42.05±5.93 c 6.46±0.76 artemia ponds d 47.56±6.67 abdf 41.92±8.41 f 28.56±2.19 the rows that have no common letters are significantly different (p<0.05). table 7. comparison of two artemia populations in different levels of salinity in terms of average number of cysts artemia species / salinity (ppt) 80 120 150 artemia gavkhouni a 50.48±6.55 ab 49.48±5.31 c 4.81±1.2 artemia ponds d 6.57±0.83 abdf 3.78±0.37 f 1.1±0.68 the rows that have no common letters are significantly different (p<0.05). table 8. comparison of two artemia populations in different levels of salinity in terms of average number of larvae 95 aalamifar et al./ int. j. aquat. biol. (2014) 2(2): 91-98 the maximum total offspring per female was observed in artemia from gaav khooni at 120 ppt, significantly higher than those of other salinities at intrapopulation level (p<0.05). also, the minimum total offspring per female was observed in artemia from gaav khooni at 150 ppt (table 6). average number of cysts: the average number of cysts declined in the both populations when salinity increased (except in artemia from gaav khooni at the salinity of 120 ppt). the brine shrimp ponds had significantly a higher average number of cysts at salinities 80 and 150 ppt compared that of the brine shrimp ponds. the artemia from gaav khooni had significantly a higher average number of cysts only at 120 ppt compared to that of the brine shrimp ponds. the maximum average number of cysts was observed in the brine shrimp ponds at 80 ppt, significantly higher compared to those of the other salinities at intrapopulation level (p<0.05), but the minimum average number of cysts was observed in artemia from gaav khooni at 150 ppt (table 7). average number of larvae: the average number of larvae declined in the both populations when salinity increased. the artemia from gaav khooni had significantly higher average number of larvae at all levels salinity compared to brine shrimp ponds. the maximum average number of larvae was observed in the artemia from gaav khooni at 80 ppt, significantly higher compared to the those of other salinities at intrapopulation level (p<0.05), but the minimum average number of larvae was observed in the brine shrimp ponds at 150 ppt (table 8). discussion reproductive characteristics: we aimed to investigate the effects of different salinities on reproductive characteristics of the two parthenogenetic species of artemia, one from gaav khooni wetlands of isfahan and the other, from ponds around urmia lake. our study showed that the average number of offspring per brood declines in both populations when salinity increases. the maximum number of offspring per brood is found in artemia from gaav khooni at 80 ppt, which is significantly higher than those of the other salinities at intrapopulation level. also minimum number of offspring per brood is observed in artemia from gaav khooni at 150 ppt. triantaphyllidis et al. (1995) did not find significant differences in offspring per brood in tanggu parthenogenetic artemia and a. franciscana from san francisco bay at interpopulation level, but there were significant differences at intrapopulation level at salinities below 100 g/l. abatzopoulos et al. (2003) studying an artemia clone from megalon embolon observed significant differences in all reproductive characteristics except for number of broods in all three salinities (50, 80, 120 g/l). browne and wanigasekera (2000) reported that all five populations in their study had highest reproduction period and peak production at 24°c at either 120 or 180 g/l. we can justify the results that the old world species including our two asexual populations from iran and the parthenogenetic population are more limited by temperature and salinity for reproduction whereas new world species are euryhaline and eurythermal being able to reproduce more successfully at more diverse salinity-temperature combinations. the average number of brood per female declines in both populations when salinity increases. the artemia from gaav khooni has significantly higher number of brood per female at salinities of 80 ppt and 120 ppt and the maximum number of brood per female is found at 120 ppt. also the minimum number of brood per female is observed in artemia from gaav khooni at 150 ppt whereas the brine shrimp ponds has the highest number of brood per female at 150 ppt. the present study supports findings by many studies on the negative impact of salinity values above 120–140 g/l on reproductive and life span characteristics in many other artemia species or strains (vanhaecke et al., 1984; wear and haslett, 1986; triantaphyllidis et al., 1995; browne and wanigasekera, 2000; baxevanis and abatzopoulos, 2004; baxevanis et al., 2004). in accordance with previous laboratory investigations 96 aalamifar et al./ int. j. aquat. biol. (2014) 2(2): 91-98 on several artemia species (browne et al., 1991; triantaphyllidis et al., 1995; baxevanis et al., 2004), it was found that the optimal range for reproduction of iranian asexual strains of artemia from region lies between 80 and 120 ppt (especially for artemia gaav khooni at salinity 120 ppt). since the salinity in gav khooni wetland equals 120 ppt it could be anticipated that the optimum salinity for artemia from gav khooni is 120 ppt as this is in accordance with our results. we found that the average number day of brood intervals declines in both populations when salinity increases. gilchrist, (1960), dana and lenz, (1986) and triantaphyllidis et al. (1995) who worked on a. salina, a. franciscana from mono lake and tanggu parthenogenetic artemia and a. franciscana from san francisco bay, respectively, reported that maturation is achieved fastest at salinities lower than 100 g/l, and much slower above 140 g/l. abatzopoulos et al. (2003) reported faster maturity at 50 and 80 g/l in comparison to 120 g/l for a parthenogenetic artemia from megalon embolon (greece). similarly baxevanis et al. (2004) reported early maturation at 35 g/l in three parthenogenetic populations and at 80 g/l in the bisexual a. salina from lake of wadi el-natrun, all from egypt. it was found that this bisexual artemia died before attaining maturity at 150 and 200 g/l. but browne and wanigasekera (2000) who performed the experiments at various combinations of temperature and salinity with five artemia populations (one parthenogenetic and four bisexual) reported parthenogenetic artemia from margherita di savoia (italy) as a niche specialist attaining maturity and reproducing only at salinities higher than 120 g/l at 24°c. the obtained results in the offspring per female per day were found to be different during the reproductive period for each population. therefore, the maximum number offspring per female per day, during the reproductive period is observed in brine shrimp ponds at 120 ppt but the minimum number offspring per female per day, during the reproductive period in artemia from gaav khooni is observed at 120 ppt. contrary to these, triantaphyllidis et al. (1995) in their study on tanggu parthenogenetic artemia and a. franciscana from san francisco bay found no significant differences between their study groups at interpopulation level. we found no significant differences in the percent of offspring encysted of parthenogenetic strains at all levels salinity and the maximum percent offspring encysted can be observed in both parthenogenetic populations at 120 ppt, similar to findings observed by triantaphyllidis et al. (1995). in conclusion our findings show that considering the number of offspring per brood, number of brood per female, brood intervals (in terms of day), percent offspring encysted, and average number of larvae, the artemia from gav khooni was significantly better than artemia from ponds; however, artemia from ponds had significantly better results considering offspring per female per day during the reproductive period, total offspring per female, average number of cysts. acknowlegments the authors would like to express their gratitude to reviewers of the present study for their nice and considerable comments on the study. refrences abatzopoulos t.j., baxevanis a.d., triantaphyllidis g.v., criel g., pador e.l., van stappen g., sorgeloos p. 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(2019) 7(3): 166-174 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article study on probiotic potential of bacillus species isolated from the intestine of farmed rainbow trout, oncorhynchus mykiss mina seifzadeh, mohammad rabbani khorasgani *,1rasoul shafiei department of biology, faculty of science, university of isfahan, isfahan, iran. s article history: received 22 april 2019 accepted 25 june 2019 available online 2 5 june 2019 keywords: bacillus farmed rainbow trout gastrointestinal tract probiotic abstract: the gastrointestinal tract of fishes is a complex ecosystem occupied by a large number of microorganisms, some of them could have potentially-valuable features. this research was conducted to study bacillus species in the intestine of farmed rainbow trout to examine their probiotic properties, and to provide a new source of probiotics. a total of 23 farmed rainbow trout were sampled and their intestine samples were cultured. following the morphological assay and biochemical analysis, isolated bacilli were amplified by polymerase chain reaction and universal primers 27f and 1492r. bacillus subtilis and b. amyloliquefaciens were isolated from 5 and 3 samples, respectively. bacillus tequilensis, b. cereus and b. licheniformis were isolated from 1 sample. probiotic properties of b. subtilis strain msm 24, b. amyloliquefaciens strain tmm 25 and b. licheniformis strain mr 78 were confirmed. since probiotic bacteria cause no foodborne diseases, their existence in farmed trout intestines, and their penetration into the fish tissues do not pose any risk to consumers’ health. introduction the intestinal microbiota is an integral part of the gastrointestinal tract of fishes. numerous internal and external factors regulate the microbial specifications of environment, thereby are affecting the fish intestinal microbiota (earl et al., 2008). bacteria are the main constituents of the intestinal microbiota of fish. the fish intestinal microbiota is characterized by high density and diversity of organisms with complex interactions (nithya and halami, 2013). diverse microbial populations exist in the intestinal contents. the numbers of bacteria are higher in the intestinal contents than in the surrounding water. this shows that the intestines provide favorable environments for microorganisms. this microbial population can prevent infection by interfering with pathogens and/or limiting occupying surface for them (huber et al., 2004). studies have indicated that bacillus species improve food consumption in fishes (kavitha et al., 2018). various studies reported that the intestinal microbiota of freshwater fishes encompasses of *correspondence: mohammad rabbani doi: https://doi.org/10.22034/ijab.v7i3.584 e-mail: m.rabbani@biol.ui.ac.ir different genera such as acinetobacter, aeromonas, bacillus, flavobacterium, pseudomonas, enterobacteriaceae, and obligate anaerobic bacteria of the genera bacteroides, fusobacterium, and clostridium (hovda et al., 2007; huber et al., 2004). bacilli bacteria are present in water and soil and they can easily reach the trout farms. given that rainbow trout are among high-quality and marketable fishes, its farming is considered as the largest aquaculture industry in iran (ghorbanzadeh and nazari, 2015). rainbow trout are sold as non-live form in many regions of iran, which may lead to bacilli bacteria penetrating to the fish flesh; as a result, it is necessary to determine the properties of these bacteria. since probiotics have health benefits, they may provide an alternative way to reduce the use of drugs in aquaculture and simultaneously may avoid the development of antibiotic-resistant bacteria (chen et al., 2016). bacilli are being explored for the production and preservation of food for many centuries. the inherent https://www.ncbi.nlm.nih.gov/pubmed/?term=earl%20am%5bauthor%5d&cauthor=true&cauthor_uid=18467096 https://www.sciencedirect.com/science/article/pii/s2352513418300176#! 167 int. j. aquat. biol. (2019) 7(3): 166-174 ability of production of the proteins, enzymes, antimicrobial compounds, vitamins, carotenoids and other specifications are importance of study of bacilli in food industry. additionally, bacillus spp. are gaining interest in human health related functional foods researches coupled with their tolerance and survivability under hostile environment of gastrointestinal tract. besides, bacilli are more stable during manufacturing process, food storage and pharmaceutical industry that making them more suitable candidate for health promoting (elshaghabee et al., 2017). therefore, providing a new source of probiotic bacilli is necessary. hence, this research was conducted to study bacillus species in the intestines of farmed rainbow trout, oncorhynchus mykiss, by examining their probiotic properties, and providing a new source of probiotics. materials and methods initial separation: a total of 23 farmed rainbow trout, 130-2230 g in weight, were collected from fishfarming ponds. the fish were sampled after euthanasia. first, the fish skin was washed using 70% alcohol. then, their intestine was removed and washed by sterile distilled water to remove fecal matter. next, the intestine was cut and rewashed by sterile distilled water to remove the remaining fecal matter. they were placed in sterile petri dishes. the intestinal wall was scraped with a plastic spatula. all steps of sampling were done on ice. undiluted mucus was used to explore the type of bacteria. mucus was cultured on the nutrient agar (cohen and laux, 1995). plates were incubated under aerobic conditions at 37°c for 5 days. after incubation, white colonies of gram-positive bacteria with 2-3 mm diameter were sampled. these bacteria were sub-cultured on nutrient agar under same conditions. bacterial identification was carried out by morphological assay, chemical tests, pcr and probiotic tests. morphological assay: gram staining and fluorescent microscopy were used for morphological study of the bacteria. bacterial identification by chemical methods: the bacteria were identified according to bergey manual of systemic bacteriology (holt et al., 1994). hence, they were identified by the mobility and catalase activity, followed by fermentation a set of carbohydrates, including arabinose, glucose, starch, mannose, mannitol, salicin, xylose, turanose and melibiose. the other biochemical tests were tween 20, o-nitrophenyl-β-d-galactopyranoside (onpg), ph, concentration of nacl range from 2-10%, temperature (5-65oc), citrate, vp and nitrate reduction. the ability to ferment lactose was utilized to differentiate bacillus subtilis from b. amyloliquefaciens. the sugars were sterilized by filtration (millipore filter, 0.45 μm pore size). bacterial identification by pcr: in order to identify the bacteria using the pcr (bioline, united kingdom), they were cultured on nutrient agar. the culture medium was incubated at 37°c for 18 hours. boiling method was used to extract dna from bacteria (dashti et al., 2009). the concentration and integrity of the extracted dna was determined by ethidium bromide stained 0.8% agarose gel electrophoresis and compared against known concentrations of lambda dna (chandrasekara bhagya et al., 2013). all chemicals used were molecular grade from sigma company. 16s ribosomal rna gene was used. the pcr primers were 27f (agagttt gatcctg gctcag) and 1492r (tacggytaccttgtta cg actt). master-mix of pcr was diluted to 10.30 μl reaction mixture contained 15 µl master, 1 µl each pcr primer and 8 µl sterile injectable water. the pcr program was as follow: initial denaturation at 94°c for 2 min followed by denaturation at 94°c for 1 min, annealing at 55°c for 1.5 min, 30 cycles of extension at 72°c for 1 min, and the final extension at 72°c for 3 min. the pcr product was visualized on a 1% agarose gel under uv light after ethidium bromide staining (silva et al., 2013). gel electrophoresis was carried out at 80 v. amplicons were purified from agarose gel using the gfx pcr dna and gel band purification kit (ge healthcare, united kingdom). the pcr products were sequenced using the sanger dna sequencing (teng et al., 2004). sequences were identified using blast. the results were analyzed 168 seifzadeh and rabbani / probiotic potential of bacillus species isolated from rainbow trout using the bioedit sequence editor. fasta sequences aligned using clustal x. pcr results aligned against bacterial dna database (ncbi) (http://blast.ncbi. nlm.nih.gov). a homology greater than 97% was acceptable for identifying the genus. these bacteria were registered in the ncbi and dgbi database. probiotic assay of the isolates: the isolates were screened for hemolytic activity, lecithinase, tolerance to acidic conditions, gastric juice and bile salt, and antibiotic resistance transferability. then, based on the results, the selected bacteria were examined for adhesiveness and invasiveness. microbial suspension was prepared for study of theses specifications (iranian national standard no. 19459. 2014). microbial suspension preparation: the identified bacteria were cultured in nutrient broth at 37ºc for 24 hours. culture media were centrifuged at 5000 rpm for 10 min. the supernatant was removed and pellets were washed in a phosphate-buffered saline (3 times). then, the pellets were dissolved in the same buffer. these suspensions were adjusted to a turbidity equivalent to the 0.5 mcfarland standard (iranian national standard no. 19459. 2014). acidic tolerance: in order to evaluate acidic tolerance, ph of mrs broth (10 ml) adjusted to 2.5 and 4 using hydrochloric acid. 100 µl of microbial suspensions were inoculated into mrs broth. they were incubated at 37ºc for 3 and 4 hours. after that, 1 ml of these media were inoculated into nutrient agar, followed by incubation at 37ºc for 24 hours to determine colony forming unit (iranian national standard no. 19459. 2014). gastric juice tolerance (pepsin and trypsin): in order to prepare the pepsin and trypsin media, 2 g of sodium chloride and 2.3 g of pepsin or trypsin were dissolved in 1 l of distilled water. the ph of these media were adjusted to 2-2.3. in order to evaluate gastric juice tolerance, the microbial suspension (2%) was inoculated into the pepsin and trypsin media, then, incubated at 37ºc for 24 hours. thereafter, a 103 dilution was prepared from the incubated media using saline solution supplanted by 0.1% peptone water. one ml of these dilutions was cultured in nutrient agar using pour-plate method. plates were incubated at 37ºc for 24 hours to determine colony forming unit (iranian national standard no. 19459. 2014). bile salt resistance: in order to assess bile salt resistance, 100 µl of the microbial suspension were inoculated into nutrient broth containing 0.3% of fish bile, followed by incubation at 37ºc for 8 hours. before and after the incubation period, absorbance of the suspension was measured at a wavelength of 600 nm using uv-vis spectrophotometer (eppendorf, germany). a medium without bile was used as a control sample for each bacterium (iranian national standard no. 19459. 2014). microbial adhesion and invasion: the adhesion assay was conducted according to nithya and halami (2013). the invasion assay was carried out as described by rowan et al. (2001). the hepg-2 cell line was used. the cell monolayers were seeded at 5×104 cells per well (in rpmi 1640 medium plus 10% fetal calf serum) in 96-well plate. the cells were infected with filter-sterilized (0.2 μm) supernatants from overnight cultures (bhi, 30°c) of bacteria. 100 μl of supernatant were added to the cultured cells immediately after heat treatment (95°c, 10 min). monolayers containing the bacterial supernatants were incubated overnight at 37°c in a 5% co2 atmosphere. after overnight incubation, the suspension from each well was discarded and 25 μl of fresh complete medium (rpmi+10% fetal calf serum) containing 0.004 g/ml of mtt reagent (sigma) was added. samples were incubated for 3 hours at 37°c in 5% co2 and the formazan product was solubilized by the addition of 100 ml of dimethyl sulfoxide. optical densities of the suspensions were measured at 540 nm using an elisa reader (biotek, usa) and cytotoxicity was calculated (mohkam et al. 2016). the results of microbial adhesion and invasion tests were analyzed by spss software. the samples were compared to each other by one way anova. t-test was used to compare the control with the test samples. antibiotic susceptibility examination: the susceptibility of the bacillus isolates to erythromycin (15 µg), penicillin (10 µg), gentamicin (10 µg), amoxicillin (25 µg), azithromycin (15 µg), chloramphenicol (30 µg), cefalotin (30 µg), clindamycin (2 µg), 169 int. j. aquat. biol. (2019) 7(3): 166-174 streptomycin (10 µ g) and tetracycline (30 µg) were determined using muller-hinton agar. after incubation, the formation of clear zone around the discs monitored (iranian national standard no. 19459. 2014). hemolytic activity test: this test was performed on all isolates. a loopful of overnight bacteria was cultured to spot form on blood agar plate. the plates were incubated for 24 hours at 37ºc and observed for clear zones around the colonies (iranian national standard no. 19459. 2014). lecithinase activity: this test was performed using mannitol-egg yolk-polymyxin agar. a loopful of overnight bacteria was cultured to spot form on this medium. after incubation for 24 hr at 37ºc, the plates were checked for clear zone around the bacterial colonies (iranian national standard no. 19459. 2014). abbreviations used: git, gastrointestinal tract; onpg, o-nitrophenyl-β-d-galactopyranoside; pcr, polymerase chain reaction. results morphological characteristics: the detected bacteria appeared to form of short or long chains of large gram positive bacilli. their spores had situations of ellipsoidal, central and sub terminal. chemical properties: eleven bacillus species were obtained from 23 examined fish. they showed catalase activity and could grow under both aerobic and anaerobic conditions. bacillus cereus was arabinose, glucose, mannose, mannitol, salicin, xylose, and starch negative. however, it was citrate, vp, and nitrate reduction positive. this bacterium was unable to grow in the presence of salt (2-10%). glucose, mannose, mannitol, salicin, xylose, arabinose, citrate and starch were used by b. subtilis, b. amyloliquefaciens, b. tequilensis and b. licheniformis. these bacteria were vp and nitrate reduction positive. these isolates were able to grow at the media containing 2-8% nacl. turanose and tween 20 were used by b. subtilis and b. amyloliquefaciens. bacillus amyloliquefaciens was melibiose and lactose positive; however, it was onpg negative. bacillus subtilis was lactose negative; however, it was tween 80 positive. according to table 1, 11 bacilli species were identified in rainbow trout. bacillus subtilis and b. amyloliquefaciens were isolated from 5 and 3 samples, respectively. bacillus tequilensis, b. cereus and b. licheniformis were isolated from one sample. all the isolated bacilli were motile. these bacteria were able to growth at ph 7; however, not at ph 10. bacillus cereus mr11 was not able to grow at ph at 8-9. bacillus cereus mr11 and b. licheniformis strain mr 78, but not the other bacilli, were not able to grow at temperatures 10, 50 and 60°c. the isolated bacilli were able to growth at temperatures of 20, 30 and 40°c, but not 5 and 65°c. bacillus amyloliquefaciens was found in the small and large fishes. bacillus subtilis was found in the small and medium fishes. bacillus licheni formis and b. cereus were observed in the medium fish. bacillus tequilensis was observed in the fish with 700 g weight. molecular characteristics: the isolated bacteria were table 1. the chemical identification results of bacillus species isolated from the intestines of farmed rainbow trout (n= 11). index bacteria weight motility ph capability at different temperatures 7 8 9 10 5 10 20 30 40 50 60 65 b. tequilensis strain ms21 700 + + + + + + + + b. cereus mr11 355 + + + + + b. subtilis strain mt 13 480 + + + + + + + + + + b. amyloliquefaciens ma 11 2040 + + + + + + + + + + b. subtilis strain msm 14 135 + + + + + + + + + + b. amyloliquefaciens strain tr 15 215 + + + + + + + + + + b. subtilis strain mr 20 247 + + + + + + + + + + b. subtilis strain tam 21 360 + + + + + + + + + + b. amyloliquefaciens strain tmm 25 2230 + + + + + + + + + + b. subtilis strain msm 24 247 + + + + + + + + + + b. licheniformis strain mr 78 350 + + + + + + + https://blast.ncbi.nlm.nih.gov/blast.cgi#alnhdr_948513421 170 seifzadeh and rabbani / probiotic potential of bacillus species isolated from rainbow trout 99% homologous to b. tequilensis, b. cereus, b. amyloliquefaciens and b. subtilis. probiotic potentials: from all the bacilli isolates, only b. licheniformis strain mr 78, b. amyloliquefaciens strain tmm 25, and b. subtilis strain msm 24 were able to grow in the presence of bile salts and gastric juice, and different acidity levels. however, these bacteria had no hemolytic capability and lecithinase activity. therefore, the other bacteria removed from cell toxicity and antibiotic resistance tests. as table 4 shows, there was no significant difference in adhesion among the samples (p<0.05), but, there was significant difference between test and positive probiotic control in this factor (p>0.05). adhesions of the test samples were significantly lower than the adhesion of the pathogenic control (l. monocytogenes) (p<0.05). no invasion was observed in b. subtilis strain msm 24 and b. amyloliquefaciens strain tmm 25, but these bacteria had no significant difference in invasion compared to negative control and the other bacilli. the highest invasion was related to the pathogenic control, which were significantly different compared to the other groups (p<0.05). as the table 5 shows, b. subtilis strain msm 24, b. amyloliquefaciens strain tmm 25 and b. licheniformis strain mr 78 have no antibiotic resistance. according to the tables 3, 4 and 5, b. licheniformis strain mr 78, table 2. accession numbers and similarity of bacillus species isolated from the intestine of farmed rainbow trout (n=11). bacteria ncbi dgbi similarity (%) b. tequilensis strain ms21 lc458434 99 b. cereus strain mr11 mk395545.1 b. subtilis strain mt 13 lc458433 99 b. amyloliquefaciens strain ma 11 mk392154.1 99 b. subtilis strain msm 14 mk400693.1 99 b. amyloliquefaciens strain tr 15 mk393391.1 99 b. subtilis strain mr 20 mk397791.1 99 b. subtilis strain tam 21 mk397798.1 99 b. amyloliquefaciens strain tmm 25 mk394994.1 99 b. subtilis strain msm 24 mk393445.1 99 b. licheniformis strain mr 78 mk395274.1 99 table 3. the culture results of bacillus species isolated from the intestine of the farmed rainbow trout in different conditions (n=11). growth at index bacteria ph 4 ph 2.5 bile 0.3% gastric juice tolerance lecithinase activity hemolytic activity 3h 4h 3h 4h pepsin trypsin b. tequilensis strain ms21 1×108±4.67 1×108±2.35 1×108±3.89 1×108±2.67 0.48±0.11 1×105±4.74 1×104±1.14 + + b. cereus strain mr11 1×108±4.13 1×108±3.56 1×108±2.87 1×108±3.78 0.56±0.16 1×104±4.71 1×106±3.91 + + b. subtilis strain mt 13 1×108±4.56 1×108±3.76 1×108±2.67 1×108±2.98 0.49±0.13 1×105±4.22 1×103±4.27 + + b. amyloliquefaciens strain ma 11 1×108±3.78 1×108±3.16 1×108±3.64 1×108±3.15 0.52±0.17 1×105±3.97 1×104±4.11 + + b. subtilis strain msm 14 1×108±3.21 1×108±3.13 1×108±3.13 1×108±3.24 0.57±0.19 1×103±2.89 1×104±3.91 + + b. amyloliquefaciens strain tr 15 1×108±3.45 1×108±2.58 1×108±4.43 1×108±3.74 0.61±0.18 1×103±2.95 1×105±3.47 + + b. subtilis strain mr 20 1×104±2.19 1×103±4.15 1×103±3.17 1×102±4.45 0.91±0.15 1×105±2.31 1×104±3.31 + + b. subtilis strain tam 21 1×108±4.15 1×108±3.18 1×108±3.78 1×108±2.54 0.06±0.02 1×108±4.17 1×108±3.42± _ _ b. amyloliquefaciens strain tmm 25 1×108±4.98 1×108±4.24 1×108±3.97 1×108±3.19 0.02±0.01 1×108±3.16 1×108±4.11 _ _ b. subtilis strain msm 24 1×108±2.78 1×108±4.19 1×108±3.65 1×108±4.21 0.4±0.14 1×108±2.23 1×108±4.61 _ _ b. licheniformis strain mr 78 1×108±4.19 1×108±4.25 1×108±2.12 1×107±4.18 0.4±0.12 1×108±2.59 1×108±3.90 _ _ http://getentry.ddbj.nig.ac.jp/getentry/na/lc458434/?filetype=html http://getentry.ddbj.nig.ac.jp/getentry/na/lc458433/?filetype=html 171 int. j. aquat. biol. (2019) 7(3): 166-174 b. amyloliquefaciens strain tmm 25, and b. subtilis strain msm 24 had probiotic potentiality. discussions bacilli are diverse bacterial species, found ubiquitously in nature. they are highly adaptable to different environmental conditions and capable to grow in various environments, including the gastrointestinal tract of animals, soil and plants. different strains of b. subtilis were isolated form different fishes, as probiotic for foods with aquatic origin viz. b. subtilis ab1 in rainbow trout (newajfyzul et al., 2007), b. subtilis in ornamental fish (lalloo et al., 2017), b. subtilis in labeo calbasu (kavitha et al., 2018), b. subtilis strain vitnj1 isolated from the gastrointestinal tract of oreochromis niloticus (efendi and usra, 2014) and b. subtilis strain g024 in paralichthys lethostigma (chen et al., 2016). they recommended using the bacillus isolates in the aquaculture industry for their probiotic potentiality. to the best of our knowledge, the present research is the first attempt to explore the isolation of b. subtilis from rainbow trout in iran. bacillus subtilis is found in the upper layers (1-3 cm) of various soils (mongkolthanaruk, 2012), therefore, this bacterium enters into trout body through water. hence, this species was observed in more fishes compared to the other bacillus species. the present study reported the isolation of probiotic b. amyloliquefaciens from trout. it has been isolated from aquatic animals and other sources in different countries. krishnan et al. (2014) isolated probiotic bacillus species from the gastrointestinal tract of anabas testudineus and labeo rohita. chen et al. (2016) isolated b. amyloliquefaciens strain n004 from the gastrointestinal tract of southern flounder and sediments of farming ponds of sea cucumber in china and recommended that it could be used as a probiotic strain. kavitha et al. (2018) isolated probiotic b. amyloliquefaciens from the gastrointestinal tract of l. calbasu. cao et al. (2010) isolated b. amyloliquefaciens from sediments in shanghai. sugita et al. (1998) isolated bacillus species strain nm 12 from the gastrointestinal tract of japanese coastal fishes. nevertheless, there is no report about isolation of this bacterium from trout in iran. some members of b. amyloliquefaciens are found in plants and their roots and provide benefits for plant growth. table 4. adhesion and invasion percentages of bacillus subtilis strain msm 24, b. amyloliquefaciens strain tmm 25 and b. licheniformis strain mr 78 in cell culture medium (n=3). invasion (%) adhesion (%) index experiment 0a 0a phosphate-buffered saline (as negative control) 0a 0.74±0.02b bacillus subtilis strain msm 24 0a 0.79±0.012b bacillus amyloliquefaciens strain tmm 25 0.01±0.0001a 0.59±0.014b bacillus licheniformis strain mr 78 0.09±0.003a 1.1±0.09b bacillus coagulans (ibrc-m 10807) (as positive probiotic control) 35.1±1.2b 2.3±0.11c l. monocytogenes (ibrc-m 10671) ( as pathogen control) the different letters in the same column indicate significant differences (p<0.05). table 5. the growth inhibition halo diameters of bacillus licheniformis, b. amyloliquefaciens and b. subtilis (mm). bacteria antibiotic disk b. subtilis strain msm 24 b. amyloliquefaciens strain tmm 25 b. licheniformis strain mr 78 erythromycin (15 µg) 32±3.19 1.14±29 32±2.28 penicillin (10 µg) 31±2.13 28±2.25 35±3.35 amoxicillin (25 µg) 33±3.21 32±3.23 33±2.43 gentamycin (10 µ g) 22±2.16 31±2.11 31±1.56 azithromycin (15 µg) 32±3.24 34±3.18 33±2.53 cefalotin (30 µg) 29±1.17 33±1.21 30±3.51 clindamycin (2 µg) 31±2.35 35±3.39 34±3.41 streptomycin (10 µ g) 29±3.41 30±3.59 32±2.65 tetracycline (30 µg) 34 ±2.31 29±2.42 34±2.54 chloramphenicol (30 µg) 33±2.56 33±2.34 30±1.52 172 seifzadeh and rabbani / probiotic potential of bacillus species isolated from rainbow trout bacillus amyloliquefaciens strains are widely used in various commercial formulations to boost plant growth. these bacteria are readily transmitted to aquatic animals from water flowing and through plants that harbor the bacteria (chen et al., 2007). bacillus tequilensis spp. was isolated from the intestines of the farmed rainbow trout. its isolation of aquatic animals has not been reported yet. there are a few reports of the presence of this bacterium in water. this indicates the effect of the physiochemical conditions of water or soil and/or antagonistic activity of other bacteria on the survival of this bacterium. bacillus licheniformis strain mr 78, b. amyloliquefaciens strain tmm 25, and b. subtilis strain msm 24 b. cereus spp. was isolated from the intestine of the farmed rainbow trout. bacillus cereus was isolated from other fishes by rasool et al. (2017) and lalloo et al. (2007). kavitha et al. (2018) also isolated b. cereus from the gastrointestinal tract of l. calbasu. nevertheless, its isolation of farmed rainbow trout has not been reported from iran. bacillus cereus might be as vegetative cells or spores, which can adapt to the environmental changes, thus, it resists in environment. then, penetrates to the rivers and trout farms (rasool et al., 2017). bacillus licheniformis spp. was isolated from the intestine of the farmed rainbow trout. lalloo et al. (2007) reported the isolation of b. licheniformis from common carp. pasmik et al. (2008) carried out a study on the isolation of b. licheniformis from atlantic menhaden (brevoortia tyrannus). bacillus licheniformis may be a common flora of some fish species. it is also a part of fish feed. in addition, b. licheniformis is ubiquitous in the environment and used in treatment plants often enter into rivers and trout farms (pasnik et al., 2008). based on the results, b. licheniformis strain mr 78, b. amyloliquefaciens strain tmm 25, and b. subtilis strain msm 24 have probiotic potentiality because they were resistant to bile salt, acid and gastric juice. furthermore, they had no antibiotic resistance, lecithinase and hemolytic activity and toxicity in cell culture. although, lactobacilli constitute the main population of probiotics; however, other bacteria such as b. coagulans and b. subtilis and so on are also among probiotics or are regarded as probiotic candidates for the development of probiotic products (elshaghabee et al., 2017). conclusion the present study demonstrated that, of the bacilli isolated from the trout intestine, b. licheniformis strain mr 78, b. amyloliquefaciens strain tmm 25, and b. subtilis strain msm 24 have probiotic properties. further studies are needed to determine the bacteria anti-micrbial properties and resistance to enable one for choosing the most suitable bacteria for use in food industry as probiotic. acknowledgements we are sincerely grateful to the experts at isfahan university and inland waters aquaculture research center for their unwavering support. references cao h., he s., wei r., diong m., lu l. 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(2017) 5(6): 393-400 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article anesthesia of juvenile persian sturgeon, acipenser persicus; borodin 1897, by peppermint, mentha piperita, extract – anesthetic efficacy, stress response and behavior mohammad mazandarani*1, seyyed morteza hoseini2 1department of fisheries, faculty of fisheries and environmental science, gorgan university of agricultural sciences and natural resources, gorgan, iran. 2inland waters aquatics resources research center, iranian fisheries sciences research institute, agricultural research, education and extension organization, gorgan, iran article history: received 2 september 2017 accepted 9 december 2017 available online 2 5 december 2017 keywords: anesthesia acipenser persicus mentha piperita stress cortisol abstract: anesthesia in large animals such as sturgeons is unavoidable, so in this regard it is important to choice a best anesthetic with lowest side effects in fish.in the present study anesthetic efficacy of mentha, mentha pipertia, extract was studied in persian sturgeon, acipenser persicus, to find if it is a suitable anesthetic for this species. in this regard, the fish were subjected to 300, 500, 750 and 1000 mg l-1 mentha extract, or 150 mg l-1 clove oil, and behavioral response, stress indices, induction and recovery time were recorded. according to the results, the fish exposed to mentha extract showed more severe exciting movements than those exposed to clove oil. no histopathological effects were recorded in gills and kidneys of the fish after anesthesia with both mentha extract and clove oil. exposure to either 150 mg l-1 clove oil or 750 mg l-1 mentha extract for 3 min resulted in the fish serum cortisol change. result showed a significant increase in serum cortisol at 6 hrs after anesthesia in both mentioned anesthetic. however, in the fish anesthetized by clove oil, serum cortisol level returned to the pre anesthesia value, at 24 hrs post anesthesia. in the fish anesthetized by mentha extract, a further significant increase in serum cortisol level was observed at 24 hrs after anesthesia. however, it returned to the pre anesthesia level at 72 hrs after anesthesia. at all sampling time, serum cortisol levels of the fish anesthetized by mentha extract were significantly higher than those anesthetized by clove oil. totally it is concluded that, in persian sturgeon, use of mentha extract as anesthetic results higher stress compared to clove oil, in the other word, it can be used as a good anesthetic agent but clove oil is better. introduction anesthetics are commonly used in fish handling, research, surgery, sorting and transportation to minimize stress (summerfelt and smith, 1990). herbal anesthetics have received attention in recent years because of their beneficial effects beside anesthesia, such as antioxidant property (chaieb et al., 2007; gressler et al., 2014). the results of the studies on anesthetic properties of different essential oils in fish are variable, as some authors reported efficiency of essential oil to induce anesthesia (gressler et al., 2014; toni et al., 2014) and others failed to reach similar conclusion (benovit et al., 2012). therefore, it is necessary to examine the anesthetic efficacy of each herbal essential oil in different fish species. mentha, mentha piperita, essential oil has antioxidant, antibacterial, antiviral and fungicidal *corresponding author: mohammad mazandarani doi: https://doi.org/10.22034/ijab.v5i6.391 e-mail address: mazandarani57@gmail.com activities (mckay and blumberg, 2006). the essential oil is mainly (33-60%) comprised of menthol (clark and menary, 1981; sang, 1982; pittler and ernst, 1998). menthol was found to induce anesthesia in some fish species (façanha and gomes, 2005; kasai et al., 2014). the anesthetic property of menthol was suggested to be related, at least in part, to the activation of gabaa receptors (kasai et al., 2014). to have appropriate anesthetics, knowledge on their side effects is necessary. early diagnosis is difficult because of the lack of clinical signs in the first stage of poisoning and in many cases, paraclinical tests are needed for determining toxicity levels of drugs and chemicals. histological analysis is a suitable tool to provide acceptable biomarker of toxicity of drugs. there are limited studies on histopathology as paraclinical test in anesthetic agents 394 mazandarani and hoseini/ anesthesia of persian sturgeon by mentha piperita extract (padiyoo et al., 2017; boijink et al., 2017; velisek et al., 2005; velisek et al., 2007). persian sturgeon, acipenser persicus, is an important fish species inhabiting the caspian sea. it is reared by iranian aquaculturists to produce meat and caviar. so use of anesthesia is unavoidable for handling, catheterization and sorting of this species on farms. at present, clove solution (mixture of powdered clove buds and water), clove oil and ms222 are used to anesthetize persian sturgeon. however, there is no data about the anesthetic efficacy of mentha extract in this species. therefore, it was tested if mentha extract is a suitable anesthetic for persian sturgeon, compared to clove oil. to determine its suitability, induction time, anesthetic dosage and stress response were compared between mentha extract and clove oil. materials and methods fish and maintenance conditions: persian sturgeons (76.55±2.17 g) were provided from a local farm and 120 fish were used in this experiment. the fish were stocked into two rectangular fiberglass tanks with 1000 l of water equipped with sponge filters. the fish were acclimatized to the experimental conditions for 20 days. during the acclimation period, the fish were fed daily with commercial feed (biomar co., france) at 1% of body weight. about 75% of the tanks’ water was replaced with fresh water every day. preparation of anesthetic solution: clove oil (purity= 99%) were purchased from sigma co. (st luis, usa), whereas, mentha extract was purchased from adonis gol darou co. (tehran, iran). the mentha extract was obtained by steam-distillation of mentha leaves and contained 50% menthol, according to the product fact sheet. both clove oil and mentha extract was dissolved in ethanol (1: 5 oil: ethanol) to facilitate solution in anesthetic bath water (hoseini et al., 2015). induction and recovery time record: ten fish were individually subjected to 300, 500, 750 and 1000 mg l-1 mentha extract, or 150 mg l-1 clove oil (ten fish per concentration), and induction (stage 5: decreased opercular beat) and recovery times and behavioral responses were recorded. for each concentration, five fish were tested from each 1000 l tanks, therefore, ten data was recorded for induction and recovery in each concentration (n=10). the fish were individually placed into an anesthetic bath (60 l plastic tank). the anesthetized fish were individually placed into fresh water (200 l tank) to recover. both anesthesia and recovery baths were aerated. the concentration of clove oil was chosen based on a preliminary trial to find the concentration inducing anesthesia (stage 5) within 3 min. each fish was only once exposed to anesthetic, and was excluded from experiment thereafter. fresh water was used for induction and recovery baths preparation. the water source has the following characteristics (mean±sd): oxygen 7.12± 0.12 mg l-1, ph=7.77±0.08, temperature= 20.45°c, unionized ammonia=0.001±0.0001 mg l-1 and nitrite =0.002±0.0003 mg l-1 and total alkalinity=243.8± 8.66 mg l-1 caco3. stress response study: to study the stress response induced by mentha extract and clove oil, a concentration of each anesthetic was chosen to induce anesthesia (stage 5) within 3 min. stage 5 anesthesia was chosen because fish can easily be handled for blood sampling at this stage. accordingly, 750 mg l1 mentha extract and 150 mg l-1 clove oil was chosen for the stress response study. a total number of 60 fish were randomly distributed into 6 fiberglass tanks filled with 250 l dechlorinated tap water. all tanks were provided by sponge filter and aerators. the fish were allowed to acclimatize to the experimental conditions for 30 days. during the acclimation period, the fish were fed daily with commercial feed (biomar co. france) based on 1% of body weight. about 75% of the water was replaced with fresh water every day. before the start of the study, the fish were fasted for 24 hrs. then, one fish was sampled from each tank. these samples were considered as pre anesthesia samples. then, the tanks were assigned into two treatments, each contains 3 tanks. one treatment was exposed to 750 mg l-1 mentha extract, and the other was subjected to 150 mg l-1 clove oil. exposure time for both anesthetics was 3 min. immediately after 3 min, the fish were returned to their tanks to recover. blood sampling were performed at 6, 24 and 72 hrs 395 int. j. aquat. biol. (2017) 5(6): 393-400 after anesthesia. at each sampling time, two fish were sampled from each tank, thus, sample size in all sampling time was six (n=6). the sampled fish was tagged by dorsal fin cutting and returned to their corresponding tank to avoid a change in fish density until 72 hrs and to ensure that the sampled fish were not sampled at the next sampling time. no anesthesia was applied in sampling time. blood samples were collected by caudal puncture without anticoagulant. capture and blood collection lasted less than 1.5 min. serum preparation and cortisol assay: immediately after blood collection, the blood samples were allowed to clot at room temperature for 2 hrs. then, the samples were centrifuged (7000 rpm, 10 min) to obtain the serum. the serums were kept at -20°c until analyses. serum cortisol was determined by chemiluminescence technique using liaison® cortisol assay kit (diasorin inc., usa) according to hoseini and hosseini (2010). the procedure was competitive luminometric assay based on a solid phase antigen-linked technique. in this procedure, cortisol is used for the coating of the solid phase (magnetic particles). the tracer consisted of a highly specific monoclonal antibody, which is labeled with an isolumonal derivative. histological analysis: for histopathological analysis, tissue samples were taken from gill and middle parts of kidney after anesthesia (before recovery test). tissues were fixed in 10% buffered formalin and after 24 hours the fixative were renewed. routine histological methods were followed and totally 5 μm tissue sections prepared (robert, 2012; eagderi et al., 2013), the sections were stained using harris’s hematoxylin and eosin (h&e) method and microscopically examined at magnifications of 40 to1,000 (nikon, japan). in the present study, fish histological analysis were done for mentha extract and clove oil in 750 mg l-1 and 150 mg l-1 concentrations, respectively. water physico-chemical parameters: water dissolved oxygen, ph, temperature, unionized ammonia and nitrite was recorded twice a week during the experiment using portable physico-chemical test kit (wagtech projects ltd, wagtech court, station road, thatcham, berkshire, rg19 4hz). statistical analyses: normality of the data was confirmed by shapiro-wilk test. the mean of induction (n=10) and recovery (n=10) time, and serum cortisol levels (n=6) were compared among the anesthetics’ concentrations using one-way anova and duncan test to find significant difference. p<0.05 was considered as significance. data are presented as mean±sd. all analyses were performed using spss v. 16. results behavioral observations of the fish exposed to mentha extract and clove oil are summarized in table 1. five anesthesia stages were identified in the fish anesthetized either by mentha extract or clove oil. generally, the fish exposed to mentha extract showed more severe exciting movements to anesthetic contact than those exposed to clove oil. the induction time of the fish exposed to 300 and 500 mg l-1 mentha extract was similar, being significantly longer than 750 and 1000 mg l-1 mentha extract and 150 mg l-1 clove oil (fig. 1). also, the recovery time of the fish exposed to 300 and 500 mg table 1. behavioral observation of different anesthesia stages in persian sturgeon subjected to mentha extract and clove oil. stage exhibited behavior mentha extract clove oil 0 normal normal 1 rapid movements, excitation and saltation slight excitation 2 calmness and operculum closure, no swimming calmness and no response to tactile touch, normal swimming 3 spiral and imbalance swimming, normal opercular pulse imbalanced swimming, normal opercular pulse 4 loss of equilibrium, pectoral fin vibrating and irregular opercular pulse loss of equilibrium and irregular opercular pulse 5 decreased opercular beat decreased opercular beat recovery fish regained its equilibrium fish regained its equilibrium 396 mazandarani and hoseini/ anesthesia of persian sturgeon by mentha piperita extract l-1 mentha extract was similar, being significantly shorter than 750 and 1000 mg l-1 mentha extract and 150 mg l-1 clove oil (fig. 1). increase in mentha extract concentration resulted in a significant decrease in induction time, and an increase in recovery time (fig. 2). anesthesia with both anesthetics resulted in a significant increase in serum cortisol at 6 hrs after anesthesia (fig. 3). however, in the fish anesthetized by clove oil, serum cortisol returned to the pre anesthesia value, at 24 and 72 hrs after stress. in the fish anesthetized with mentha extract, a further significant increase in serum cortisol was observed at 24 hrs after anesthesia, however, the cortisol returned to the pre anesthesia level at 72 hrs after anesthesia. in histological analysis, no pathological signs were observed in the fish anesthetized with both clove oil and mentha extract (fig. 4). discussion anesthesia of fish by essential oils can offer various health benefits such as antioxidant and antimicrobial effects (chaieb et al., 2007; gressler et al., 2014). the most well-known herbal essential oil to anesthetize fish is clove oil. its anesthetic efficacy was shown in many fish species (soto and burhanuddin, 1995; roubach et al., 2005; hoseini et al., 2015) and persian sturgeon (bagheri and imanpour, 2011; vali et al., 2016). mentha extract has frequently been less studied as an anesthetic in fish, and in this regard there are no data for its application in persian sturgeon. mentha extract caused more severe exciting movements than clove oil. early excitation due to anesthetic exposure of fish have been reported (hoseini et al., 2010), which may be due to sensation of chemical compound by the olfactory and gustatory systems. the more severe reactions to mentha extract exposure compared to clove oil may be as a result of activation of cold nociceptors (wei and seid, 1983; kasai et al., 2014). also, behavioral response at stage 2 anesthesia (operculum closure) seems to be related to gill cold nociceptor activation, which forced the fish to prevent gill exposure to mentha extract. kasai et al. (2014) reported rapid movements in fish anesthetized by menthol, which could be due to cold nociceptor activation. in the present study, 150 mg l-1 clove oil induced anesthesia stage 5 after about 173 s, which is longer figure 1. induction (open circles) and recovery (solid circles) time of persian sturgeon anesthetized by clove oil or mentha extract. different lowercase letters show significant difference among the induction time, whereas, different uppercase letters show significant difference among the recovery time. figure 2. correlation between induction or recovery time and mentha extract concentrations. figure 3. serum cortisol levels of persian sturgeons subjected to 750 mg l-1 mentha extract or 150 mg l-1 clove oil. different letters above the bars show significant difference among the treatments. 397 int. j. aquat. biol. (2017) 5(6): 393-400 than the time reported by hoseini et al. (2015) for 220 g iridescent shark, pangasius hypophthalmus (at 25°c) and waterstrat (1999) for 19 g channel catfish, ictalurus punctatus (at 23°c). the reason for longer induction time obtained in the present study compared to those reports may be due to the higher fish weight and lower water temperature in the present study compared to the previous ones. increase in fish weight affects induction time. for example, hoseini et al. (2015) found a decrease, and stehly and gingerich (1999) found no change in induction time due to fish weight increase. park et al. (2009) showed that temperature elevation significantly decreased induction and recovery time of clove oil in rock bream, oplegnathus fasciatus. there is little information about the induction and recovery time of mentha extract in fish. façanha and gomes (2005) found 105-358 s induction time for 250-50 mg l-1 menthol in tambaqui, colossoma macropomum, which is lower than the present concentrations. kasai et al. (2014) reported induction times of 50-300 s for 156-31 mg l-1 menthol in japanese medaka, oryzias latipes, goldfish, carassius auratus and zebrafish, danio rerio. it is suggested that mentha extract is not as efficacious as clove oil for persian sturgeon. the reason for the difference between the results of the present study and those conducted by façanha and gomes (2005) and kasai et al. (2014) is not exactly demonstrated. beside the environmental factors, which affect anesthetic figure 4. histological analysis of gill and kidney of persian sturgeon after deep anesthesia with eugenol and mentha extract (h&e). (a) normal gill structure in fish anesthetized with eugenol, secondary lamella (a), pillar cell (b), epithelial cell of secondary lamella (c), (bar=60 µm), (b) normal gill structure in fish anesthetized with mentha extract, secondary lamella (a), pillar cell (b), epithelial cell of secondary lamella (c), (bar=100 µm), (c) normal kidney structure in fish anesthetized with eugenol, kidney glomerulus (a), tubules (b), interstitial tissue (c), (bar=100 µm) and (d): normal kidney structure in fish anesthetized with mentha extract, kidney glomerulus (a), tubules (b), interstitial tissue (c), (bar=100 µm). 398 mazandarani and hoseini/ anesthesia of persian sturgeon by mentha piperita extract efficacy, species-specific traits are important factors affecting anesthetic efficacy. in the case of mentha extract, the present study suggests a potential pain generation in persian sturgeon. as suggested previously (wei and seid, 1983; kasai et al., 2014), menthol, the major component of mentha extract, activates cold nociceptors and generates a pain sensation. also, kasai et al. (2014) reported that rapid movement and pain sensation is induced by high menthol concentration (469 mg l-1), but not low ones (78.1-312.5 mg l-1). rapid movement and opercular closure may be evidence of pain generation in persian sturgeon, in this study. in the present study, also, it was observed that the recovered persian sturgeons were inactive for 0.5-6 hrs, particularly at the high mentha extract concentrations. they remained inactive in the tanks and initiated swimming, only after a tactile touch. there are many studies showing stress response as a result of anesthesia in fishes (auperin et al., 1997; holloway et al., 2002; park et al., 2009; hoseini et al., 2010). cortisol is the best indicator of stress in fish. the present results, generally, showed that mentha extract induced a greater stress response than clove oil. also, cortisol remained elevated until 24 hrs after anesthesia. the results of serum cortisol trend of different fish species after anesthesia are variable. park et al. (2008) showed no change in serum cortisol in kelp grouper, epinephelus bruneus 1-6 hrs after anesthesia with clove oil, but a significant increase at 12-48 hrs. on the other hand, park et al. (2009) found a significant increase in serum cortisol of rock bream, o. fasciatus 0-12 hrs after anesthesia with clove oil, which returned to normal level 24-72 hrs after anesthesia. there is no study monitoring serum cortisol in fish anesthetized by mentha extract. although no histopathologic effects was recorded in anesthetized fish with 750 mg l-1 peppermint extract, the higher cortisol response, longer period of cortisol elevation and behavioral responses (fish with minimum activity) suggest that the persian sturgeons anesthetized by mentha extract experienced more stress in compared to use of clove oil at dose of 150 mg l-1 in this fish. however, further studies are necessary to find the reason for these results. histological analysis is an applied method to diagnose tissue damage in medication toxicity (roberts, 2012). there are limited studies about histological effects of anesthetic agents in fish; for example anesthesia with 2-phenoxyethanol in sheatfish, silurus glanis l. 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(2017) 5(6): 393-400 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی نعنا عصاره کمک با جوان( acipenser persicus borodin, 1897) ایرانی تاسماهی بیهوشی ارزیابی (mentha piperita :)رفتاری و استرسی هایپاسخ بیهوشی، داروی اثرات 2حسینی مرتضی سید ،*1مازندرانی محمد .ایران ،گرگان ،گرگان طبیعی منابع و کشاورزی علوم دانشگاه، زیست محیط و شیالت دانشکده ،آبزیان پرورش و تکثیر گروه 1 ، گرگان، ایران.داخلی آبهای آبزیان ذخایر تحقیقات مرکز ایران، شیالتی علوم تحقیقات موسسه کشاورزی، ترویج و آموزش تحقیقات، سازمان2 چکیده: نندهک بیهوش ماده بهترین انتخاب راستا این در . بنابرایناست ناپذیر اجتناب امری خاویاری ماهیان جمله از بزرگ حیوانات در بیهوشی از استفاده ایرانی تاسماهی در( mentha pipertia) نعنا عصاره کنندگی بیهوش اثرات بررسی این در. است برخوردار باالیی اهمیت از جانبی عوارض کمترین با ،300 دوزهای با ماهیان منظور این به. است گرفته قرار بررسی مورد نه؟ یا باشد گونه این در مناسبی بیهوشی داروی تواندمی ماده این آیا کهاین و هایشاخص رفتاری، هایپاسخ و شده واقع مواجهه مورد میخک روغن لیتر در گرممیلی 150 و نعنا عصاره لیتر/گرممیلی 1000 و 750 ،500 میخک ارهعص با مقایسه در نعنا عصاره با شده بیهوش ماهیان نتایج اساس بر. گردید بررسی و ثبت ماهیان در بیهوشی از بازگشت و القاء استرسی یهوشب میخک اسانس و نعنا عصاره با که ماهیانی کلیه و آبشش در بافتی آسیب هیچ بررسی این در. دادند نشان خود از بیشتری هیجانی عالیم ساعت 6 در خون سرم کورتیزول افزایش به منجر لیتر/ گرممیلی 150 میخک روغن و لیتر/ گرممیلی 750 نعنا عصاره با مواجهه .نگردید ثبت شدند اما .برگشت بیهوشی از پیش قبلی سطح به سرم کورتیزول بیهوشی از پس ساعت 24 میخک روغن با شده بیهوش ماهیان در. گردید مواجهه از پس د،گردی ثبت بیهوشی از پیش زمان از باالتر داریمعنی طورهب سرم کورتیزول میزان بیهوشی از پس ساعت 24 نعنا عصاره با شده بیهوش ماهیان در کوتیزول سطح برداری نمونه های-زمان تمام در بررسی این در. برگشت بیهوشی از پیش سطح به ساعت 72 از پس کورتیزول میزان نیز گروه این در حاکی بررسی این گیرینتیجه. است بوده میخک روغن با شده بیهوش ماهیان از بیشتر نعنا عصاره با شده بیهوش ماهیان در داریمعنی طورهب سرم کندیم ایجاد باالتری استرس ایرانی تاسماهی در کننده بیهوش عنوان به میخک روغن با مقایسه در نعنا عصاره از استفاده که است بوده امر این از .دارد بهتری عملکرد میخک روغن اما گیرد قرار استفاده مورد ماهی این در کننده بیهوش یک عنوانبه تواندمی ترکیب این دیگر عبارت به .کورتیزول استرس، ،mentha piperita ،تاسماهی ایرانی بیهوشی، :کلمات کلیدی int. j. aquat. biol. (2019) 7(3): 123-131 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article seasonal heavy metal monitoring of water, sediment and common carp (cyprinus carpio) in aras dam lake of iran mehdi naderi farsani*1, rezvaneh jenabi haghparast2, saeid shahbazi naserabad3, fatemeh moghadas4, tahereh bagheri5, mohammad hasan gerami6 1young researchers and elite club, urmia branch, islamic azad university, urmia, iran. 2department of fisheries, faculty of natural resources, university of urmia, urmia, iran. 3young reasearchers and elite club, yasooj branch, islamic azad university, yasooj, iran. 4department of fisheries, islamic azad university of babol, babol, iran. 5offshore fisheries research center, iranian fisheries science research institute, agricultural research education and extension organization, chabahar, iran. 6young researchers and elite club, shiraz branch, islamic azad university, shiraz, iran. s article history: received 2 march 2019 accepted 29 ma7 2019 available online 2 5 june 2019 keywords: bioaccumulation pollution environment common carp abstract: heavy metal in aquatic ecosystems are monitored by measuring their concentrations in water, sediments and biota. in the presented study, cadmium, copper, lead, mercury, nickel, and zinc concentrations determined in water, sediment and tissues (liver and muscle) of cyprinus carpio in aras dam lake of iran, during spring, summer, autumn, and winter from 2016 to 2017. the concentration of pb, cd, zn, cu and ni of samples were measured using inductively coupled plasmaoptical emission spectroscopy. the concentration of hg was analyzed using atomic absorption equipped with mhs 15 cvaas. the trend in the metal mean concentrations of liver and muscle was zn>cu>pb≈ni>cd>hg, and water and sediment were cu>zn>pb>cd≈ni>hg. heavy metals concentration was more in sediment than fish tissues and water. this results revealed that heavy metals accumulation of muscle was the highest in summer showing the most contaminated season. whereas, winter had the lowest contamination in water (cd hg, cu, pb, ni, zn), sediments (hg, cu, zn, pb, ni), and the liver and muscle of fish (cd, zn, ni, cu). the amount of heavy metals was less or slightly higher than global standards (epa, who). introduction nowadays, global concern on metals contamination of aquatic ecosystems is growing (yi et al., 2017; jiang et al., 2018). technically, heavy metals concentration in various natural environments and ecosystems depends on local geology (fiorino et al., 2018; plhalova et al., 2018). however, anthropogenic release has been considered as the main source of heavy metals accumulation in water, sediment and aquatic organisms (savorelli et al., 2017; capillo et al., 2018; aliko et al., 2018). according to the guidelines of water quality of the european water framework directive (directive 2008/105/ec 2008), the list of metals in priority include hg, pb, cd and ni, while cu, cr, as and zn are on the list of other specific chemical dangerous substances. heavy metals are distributed in the forms of particulate and dissolved in water bodies and accumulated in the bottom (vukosav *correspondence: mehdi naderi farsani doi: https://doi.org/10.22034/ijab.v7i3.597 e-mail: m.naderi@urmia.ac.ir et al., 2014). therefore, potential source of heavy metals could be sediment which is releasing metals into the surrounding water through natural and anthropogenic processes (aliko et al., 2018). in addition, toxic metals can transferred via food chain to higher levels by fish grazing from benthos fauna (wang et al., 2010; zhang et al., 2015). aras dam-reservoir is important for many activities such as fisheries, agriculture, industry, domestic and drinking water supply (mohebbi et al., 2012). sewage and industrial effluents discharges daily into aras river through multifarious man-made activities. nasehi et al. (2013) stated that enhanced erosion of soil, dissolution of bed load, runoffs and excessive use of pesticides resulted in elevated metals’ concentration in the aras river, which led to heavy metals excessive concentration compare to the permissible values of who published. 124 naderi farsani et al./ seasonal heavy metal monitoring of water, sediment and common carp due to the heavy metals persistence in the environment, heavy metals monitoring in the environment would be a key role of ecosystem management (vukosav et al., 2014). no published papers dealing with the detailed study of trace metals concentrations in the water body, sediment and indicator fish species of the aras dam is available. therefore, this study was conducted on (1) estimation seasonal concentration of trace metals (cd, hg, cu, pb, ni and zn) in water, sediment and common carp liver and muscle in the aras dam and (2) evaluating the transfer factor and relationship between water/ sediment and fish body. the results could help us to understand status and seasonal heavy metals variation in aras dam and toxic metals accumulation rate in ecosystems with shallow lake. materials and methods sampling of fish, water and sediment: for each season, a total of 30 samples of fish, water and sediment were collected from regional stations in aras dam lake (fig. 1). before sampling from water and sediments, all sampling bottles were washed with 1:1 nitric acid and distilled water and dried with 60° c for 4 hours. three to five-liter water samples were taken at the depth of 20 cm using a plastic bottle. sediment samples were collected using a van veen grab device with 0.0256 m2 cross section and decanted to plastic bags. fish samples were collected using gillnet and after weight and length measurement, samples were placed into zipped plastic. all samples put in an iceberg and transferred to the laboratory for further analysis. sample preparation: for tissue preparing, at first stainless steel knife disinfected and cleaned with acetone and distilled water, then liver and muscle tissues were dissected. tissue and sediment samples were oven dried for 24 hours at 105°c. after cleaning teflon vessel and soaking in %5 hno3 for 1 day, it was rinsed with ultrapure water and dried. in order to analyze metal concentration in tissue and sediments, 0.5 g of samples were weighted and decanted to 50 ml close teflon vessels. tissue and sediment samples were treated with 5 ml 60% hclo3 and 8 ml 68% hno3 and 4 ml 30% h2o2. the solution was shaken well to complete tissues digestion. for determining the concentration of pb, cd, zn, cu and ni, the samples put within an inductively coupled plasma optical emission spectrometer (icp-oes, model 735-es, varian company, italy). the concentration of hg was analyzed with perkin elmer 4100zl atomic absorption equipped with mhs 15 cvaas. for determination of the heavy metals in water samples, 1 l of 60% hno3 were added to 250 ml of water samples. liquid-liquid extraction technique was performed according to mentasti et al. (1989). sample evaporation done on a low temperature reaching up to 20 ml. metal concentration in the final solution was determined using atomic absorption spectrometry (varian 710-es). transfer factor (tf): the transfer factor of samples, including muscle, liver, sediment and water were calculated according to kalfakakour and akridademertzi (2000) and rashed (2001) as follows: tf = m tissue / m sediment or water where m tissue is the metal concentration in liver and muscle and m sediment or water the metal concentration in sediment and water. geochemical index (igeo) and pollution load index (pli): the heavy metal contamination degrees in figure 1. sampling area of aras dam lake (39°33''82''n, 46°52'99''e). 125 int. j. aquat. biol. (2019) 7(3): 123-131 sediment were determined using measuring geochemical index (igeo) (muller, 1969; saleem et al., 2015) and pollution load index (pli). igeo is calculated as follows: igeo = log [cn /1.5 bn] where cn is the measured concentration of heavy metal n in the sediment and bn is the crustal shale background content of the metal. the plis defined as the nth root of the multiplications of the metal concentration (rajeshkumar et al., 2018): pli = (cf1×cf2×…×cfn)1/n where cf1 is concentration of the first metal, cf2 is concentration of the second metal and n is the total number of studied metal in the survey. classification of igoe contamination assessment model is as defined as follows: igoe<0 uncontaminated; 0≤igeo ≤1 moderately contaminated; 1≤igeo≤3 considerable contaminated; 3≤igeo≤5 high contaminated; 5≤igeo extremely contaminated (muller, 1969). zero value of pli shows excellence condition while a value of one or higher indicates baseline level of pollutants and progressive deterioration of the site and estuarine quality, respectively (tomlinson et al., 1980). results transfer factor: the results of transfer factor (tf) in water and sediments are given in table 1. for all the studied elements within muscle and liver, tfs from water were higher than sediments and exceeds 1 in all seasons. this confirmed intensive accumulation of studied metals from surrounding water within the evaluated species. the highest organ/water ratio was for zn in fish liver in summer while the lowest was for pb in muscle in spring. the tf from sediment for all measured heavy metals, fixed less than 1 within muscle and liver tissues throughout the seasons. it confirmed limited accumulation from sediment within fish liver and muscle tissues. water metals concentration: concentration of all heavy metal in surface water samples were higher in both summer and autumn than winter and spring (table 2). the mean concentration of metals in water followed the order of: cu> pb> zn> hg> ni> cd. the mean concentration of cu was 0.27, 0.37, 0.54 and 0.22 (mg/l) in spring, summer, autumn and winter, respectively, which were less than the who standard level for drinking water (table 2). however, the concentration of pb was 0.1, 0.17, 0.13 and 0.04 in spring, summer, autumn and winter, respectively, which were higher than the who for drinking water (table 2). metal concentration in sediment: seasonal concentrations of heavy metal in sediments are presented in table 3. the average concentration of table 1. seasonal transfer factor of metals (mg/kg) in different tissues of cyprinus carpio. ` cd hg cu pb ni zn m u sc le tf from water sp 16.83 5.04 30.38 1.90 18.52 278.17 tf from water su 8.18 5.30 41.89 3.92 13.27 323.58 tf from water au 5.85 6.22 26.74 7.08 5.26 227.56 tf from water wi 7.67 11.83 28.61 9.23 6.03 273.50 tf from sediment sp 0.11 0.07 0.09 0.03 0.07 0.20 tf from sediment su 0.06 0.11 0.15 0.05 0.08 0.22 tf from sediment au 0.07 0.10 0.10 0.09 0.06 0.15 tf from sediment wi 0.04 0.09 0.09 0.13 0.04 0.17 l iv e r tf from water sp 37.58 19.38 93.31 12.28 56.96 801.67 tf from water su 20.24 20.25 103.69 10.00 39.40 877.00 tf from water au 15.77 25.50 70.50 19.35 20.15 682.05 tf from water wi 28.33 31.33 82.04 33.85 20.29 623.10 tf from sediment sp 0.25 0.27 0.29 0.19 0.21 0.58 tf from sediment su 0.16 0.44 0.37 0.14 0.25 0.59 tf from sediment au 0.18 0.43 0.27 0.25 0.21 0.44 tf from sediment wi 0.14 0.25 0.25 0.48 0.14 0.39 *sp= spring, su= summer, au= autumn, wi= winter, cd: cadmium, hg: mercury, cu; copper, pb: lead, ni; nickel, zn; zinc 126 naderi farsani et al./ seasonal heavy metal monitoring of water, sediment and common carp studied metals in sediment is followed the order of: cu> zn>pb>ni>cd>hg. in the sediment, metals have higher concentrations in summer and autumn than winter and spring. according to epa (2001) in case of cu, sediment was heavily polluted and in case of zn, it was moderately polluted in summer and autumn. for other heavy metals, sediment were not polluted in this study (table 3). heavy metal content in fish liver and muscle tissue: seasonal variation of metal (cd, hg, cu, pb, ni and zn) accumulation in fish liver and muscle is shown in table 4. the decreased tendency of the mean heavy metal concentrations in fish tissues according to the elements was as zn>cu>pb>ni>cd. the values in summer and autumn were significantly more than winter and spring. accumulation of metals in the liver was obviously more than muscle. among the six studied metals, zn and cu concentration were high whereas hg and ni were low in fish organs. pollution load index (pli) and geoaccumulation index (igeo): figures 2 and 3 show the indexes of pli and igeo of heavy metals in sediment. based on the table 2. comparison of heavy metals in water (mg/l) with different international guideline and other studies in the world. different letters (a–d) in the same columns indicate significant differences (p<0.05). location cd hg cu pb ni zn this study sp 0.02±0.01a 0.04±0.01a 0.27±0.11ab 0.10±0.01a 0.02±0.01a 0.04±0.02a su 0.06±0.02b 0.07±0.03a 0.37±0.25abc 0.17±0.03b 0.05±0.02b 0.06±0.04ab au 0.07±0.01c 0.05±0.02a 0.54±0.10c 0.13±0.02b 0.08±0.0c 0.07±0.01b wi 0.02±0.02a 0.02±0.01b 0.22±0.12b 0.04±0.03c 0.02±0.01a 0.03±0.02a who 0.003 0.006 2 0.01 0.07 3 epa* 0.001 0.002 1.3 0.05 0.1 0.5 ataturk dam lake1 n.d. n.d. 0.02 n.d. 0.01 0.06 saline lake in karstic area2 sp 0.1×10-4 0.2×10-4 0.3×10-3 0.4×10-4 0.4×10-3 wi 0.2×10-4 0.1×10-5 0.3×10-3 0.5×10-4 0.3×10-3 enne and porsuk dam lakes3 0.0297 bdl 0.008 0.02 0.004 bdl buriganga reservior4 0.009 0.163 0.065 0.008 taihu lake5 sp 0.1×10-3 0.1×10-3 0.2×10-2 su 0.2×10-3 0.1×10-2 0.6×10-2 au 0.1×10-3 0.4×10-3 0.2×10-3 wi 0.7×10-3 0.3×10-3 0.5×10-3 1karadede and ünlü (2000), 2cuculic et al. (2009), 3cicek et al. (2009), 4ahmad et al. (2010), 5rajeshkumar et al. (2018), *guidelines (2002) for water, bdl= bellow detection limit. n.d.= not detected table 3. comparison of heavy metals in sediment (mg/kg) with different international guideline and other studies in the world. different letters (a–d) in the same columns indicate significant differences (p<0.05). location cd hg cu pb ni zn this study sp 2.98±0.06a 2.92±0.25a 89.5±5.77a 6.38±0.98a 5.23±0.45a 55.67±0.76a su 7.20±0.82b 3.10±0.90a 103.6±7.09b 12.2±1.12b 7.97±1b 94.00±4.58b au 5.55±1.13c 3.18±0.33a 142±8.19c 10.1±1.68c 7.55±1.63b 111±3.12c wi 3.93±1.14a 2.53±0.55a 73.33±7.64d 3.03±0.25d 3.23±1.17c 53.33±4.73a etueffont (france)1 su 1.67 177.21 37.29 45.61 371.17 au 0.51 63.13 42.12 38.73 132.12 saline lake in karstic area2 14.8 1.76 300 62.8 377 plitvice lakes3 10.9 1.22 115 11.7 258 epa guidelines (2001) for sediments4 not polluted / <25 <40 <20 <90 moderately polluted / 25-50 40-60 20-50 90-200 heavily polluted >60 >50 >60 >50 >200 sediment quality guidelinequotient (sqg-q)5 4.21 0.7 108 112 271 1ben salem (2014), 2cuculic et al. (2009), 3vukosav et al (2014), 4nvironmental protection agency, 5macdonald et al. (1996). 127 int. j. aquat. biol. (2019) 7(3): 123-131 results, the value of pli in autumn and summer significant higer (p<0.05). in the case of igeo index, hg and cu showed the highest (high pollution) and lowest (no pollution) igeo value in sediment, respectively. discussions aquatic environment easily destroyed as a consequences of human activities impairing water quality (zhang et al., 2015; rajeshkumar et al., 2018). the results revealed seasonal variation of heavy metals pollution within water, sediment and liver as well as muscle tissue of fish in aras lake, iran, throughout a year. heavy metal concentration results exhibited more density in summer and autumn than winter and spring. the transfer factor (tf) was measured for all of the heavy elements studied within fish liver and muscle table 4. comparison of heavy metals in liver and muscle of common carp (μg/g) with different international guideline and other studies in the world. different letters (a–d) in the same columns indicate significant differences (p<0.05). location cd hg cu pb ni zn this study muscle sp 0.33±0.12a 0.20±0.01a 8.36±0.40a 0.18±0.03a 0.36±0.04a 11.12±0.71a su 0.46±0.09a 0.35±0.04b 15.36±0.52b 0.65±0.08b 0.66±0.10b 20.49±0.86b au 0.38±0.06a 0.33±0.06b 14.48±1.29b 0.93±0.08c 0.42±0.08a 16.30±0.36c wi 0.15±0.03b 0.24±0.04a 6.39±0.78c 0.40±0.08d 0.14±0.05c 9.11±0.46d liver sp 0.75±0.12a 0.78±0.04a 25.66±0.77a 1.19±0.16a 1.09±0.18a 32.06±0.08a su 1.15±0.14b 1.35±0.17b 38.02±0.92b 1.67±0.24a 1.97±0.12b 55.54±1.44b au 1.03±0.27b 1.36±0.26b 38.19±1.30b 2.55±0.27b 1.60±0.21b 48.88±1.22c wi 0.57±0.06c 0.63±0.10c 18.32±1.21c 1.47±0.25a 0.46±0.12c 20.77±0.61d fao (1983) 0.05 30 0.5 30 fao/who limit 0.5 30 0.5 40 who 1989 1 30 2 100 nasser lake tilapia nilotica1 muscle 0.260 0.62 0.63 liver 7.5 0.13 2.28 lake kasumigaura cyprinus caripio2 muscle 0.009 0.24 0.03 0.04 5.43 liver 0.01 0.74 0.08 0.05 201 karoon river barbus sp3 muscle 0.84 0.73 1.75 1.15 liver 1.07 0.79 2.10 1.30 1rashed (2001), 2alam et al. (1980), 3mohammadi et al. (2011). figure 2. pollution load index (pli) value of heavy metals in sediment in aras dam lake, iran. different letters (a–c) in the same bars indicate significant differences (p<0.05). 128 naderi farsani et al./ seasonal heavy metal monitoring of water, sediment and common carp tissue for water and sediment. water tfs of heavy metals, which were higher than 1 in liver and muscle of all fish, were greater than sediments. the results show the fact that although the main source of heavy metals accumulation in aquatic environment is sediment, bioaccumulation of elements in fish mainly come from water. these results obtained from studies by ali and fishar (2005), abdel-baki et al. (2011) and salem et al. (2014). however, it must be noted that our findings are not essentially related to high transfer from water. heavy metals concentration in fish tissues are depends on other factors such as feeding behavior and size (vinodhini and narayanan, 2008) which were not considered in this study. according to the results, maximum tf found in summer. increased summer water temperature has favorable effect on biological activity of freshwater fish which led to higher metabolic activities of fish in this season, increased up taking and accumulation of heavy metal in tissues and finally increasing in tf (salem et al., 2014). based on table 1, overall heavy metal is more accumulated in liver than muscle. it is well-known that liver is the first organ in detoxification and excretion of heavy metals which is the target organ of metal absorption and considered as water pollution indicator in aquatic environments (kim and kang, 2004; farombi et al., 2007). nevertheless, heavy metals concentration in muscle is substantial because heavy metals accumulation in fish muscles effect on human through transferring high levels of accumulated heavy metals by fish consumption. whereas, the muscle is not potentially able to accumulate heavy metals, so the amount of heavy metals is less in this organ (visnjicjeftic et al., 2010). in comparison, the concentration of all studied metals was higher than other published literatures for water contamination (tables 2 and 3). however, concentration of cd, cu, pb and zn in the aras dam lake was significantly lower than those reported for saline lake in karstic area (cuculic et al., 2009) and plitvice lakes (vukosav et al., 2014). seasonal fluctuation of heavy metals in aquatic ecosystem might be affected through some water physical traits such as temperature and salinity as well as chemical traits of water including ph and dissolved oxygen levels (wong et al., 2001). in this survey, the highest heavy metals concentration in water was in summer. this could be due to more evaporation and less rainfall which resulted in higher metal concentrations as observed in the previous literatures (rajeshkumar et al., 2018). moreover, lower concentration of heavy metals in winter and spring than autumn and summer might be due to increasing in seasonal rainfall which could lead to alleviation of heavy metals throughout the rainy seasons as reported figure 3. geoaccumulation index of selected metals in the sediment in in aras dam lake, iran. 129 int. j. aquat. biol. (2019) 7(3): 123-131 by duman and kar (2012). these trends were also observed for sediments. the results on cu, showed the highest concentration between heavy metals in both water and sediment in summer. summer is the main seasons for agricultural activities and many cu based pesticides and fertilizers uses agricultural lands in aras reservoirs catchment area (nasehi et al., 2013). in addition, many industrial shutdowns through winter due to low temperatures and harsh weather, will cause relatively lower metal concentrations in. on the other hand, lower temperatures will reduce the affinity of metals to be dissolved in liquid phase (drever, 1997). the pollution load index (pli) values was significantly higher in autumn and summer. the pli values ranged from 17 in autumn and summer to 11 in spring and 7 in winter which indicates all seasons, sediment were contaminated. cu and zn were the more contributed factors in increasing pli in the sediments. researchers reported increasing in pli values in summer due to the effects of urban activities (suresh et al., 2012; ali et al., 2016). the igeo values showed the decreasing order as cu> zn> ni> pb> cd> hg. furthermore, small standard deviations indicated that variation between seasons is low and the heavy metal contamination in surface sediments is constant during a year. due to the muscle tissue of fish is the most important part of human cosnsumption, therefore, assessment heavy metals in this organ is more vital than other organs. according to table 4, concentration of all studied heavy metals in fish muscle was lower than guidelines. however, in comparison with c. carpio of lake kasumigaua (alam et al., 1980), heavy metals concentration of this study was much higher. in addition, compare to barbus sp. in karoon river (mohammadi et al., 2011), cd, hg, pb and ni was lower. the results indicated that the concentration of all heavy metals elevated more in summer and autumn than winter and spring. furthermore, increasing heavy metals in aras lake in summer and autumn could be contributed as high influx of metals as a result of pollution from the surrounding industries thereby increased bioavailability to the fish. based on the results, aras lake sediments are highly polluted. common carp is a sediment dependent species feed on benthic macro invertebrates. fish are considered as second trophic level in aquatic ecosystems with capacity of both essential and non-essential heavy metals accumulation (phillips, 1977). in addition, it is better to know the concentration of heavy metals in fish body in different seasons for fishing ban in high polluted times during a year. based on our findings, summer and autumn are the most contaminated seasons. winter had the lowest contamination in water (cd،hg, cu, pb, ni, zn), sediments (hg, cu, zn, pb, ni) and the liver and muscle of fish (cd, zn, ni, cu), and the amount of heavy metals in them was less or slightly higher than global standards (epa, who). references abdel-baki a.s., dkhil m.a., al-quraishy s. 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(2018) 6(3): 138-146 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article interaction of fish density and background color effects on growth performance, proximate body composition and skin color of common carp, cyprinus carpio ali marandi, mohammad harsij*,1hossein adineh, hojatollah jafaryan department of fisheries, faculty of agriculture and natural resources, gonbad kavous university, gonbad kavous, iran. article history: received 10 march 2018 accepted 24 june 2018 available online 2 5 june 2018 keywords: culture density tank color growth skin color abstract: this study was carried out to evaluate the combined effects of three stocking densities and two tank colors on growth, body composition and skin coloration of common carp (1.41±0.05 g). fish with low (ld: 20 specimens/tank or 0.70 g/l), medium (md: 40 specimens/tank or 1.41 g/l) and high (hd: 80 specimens/tank or 2.82 g/l) densities were reared in two tank colors (black and white) for 45 days. at the end of the experiment, density recorded 2.45 g/l and 7.00 g/l at low and high densities treatments, respectively. the final weight and specific growth rate of the fish at ld treatment were significantly higher than those of md and hd treatments. the highest weight (4.90±0.44 g) and the lowest feed conversion ratio (1.21±0.13) were obtained for the ld fish treatment reared in the black tanks. rearing density has a significant effect on the fish body total protein content, but the tank color had no effects on this factor. the fish body lipid content in the white tanks and high density was significantly higher than other treatments. significant interactions between tank color and rearing density were observed for the fish body protein, fiber and dry matter. the fish skin color was considered by three factors: l*, a* and b*. the results showed that black color had a negative effect on the fish skin color indices. brightness (l*) and yellowness (b*) values of the fish skin in the white tanks were higher than those of the black tanks. in the present study, tank color and rearing density significantly affected growth and feed performance of common carp, while no combined interaction was found between the two factors examined. introduction common carp, cyprinus carpio l. is one of the most important fish species currently being cultured. it generally inhabits freshwater environments, especially ponds, lakes and rivers, and rarely inhabits brackish-water environments (barus et al., 2001). in 2015, common carp accounted for the third highest production (4328083 t) among all freshwater species worldwide (fao, 2017). more than 90% of this production comes from asia, where c. carpio is cultured in earthen ponds, containing a variety of naturally abundant foods supplemented through nutrient fertilization and/or artificial feeds. it is being considered as a potential candidate for commercial aquaculture in asia and some european countries as it has a very high adaptive capability to both environment and food (soltani et al., 2010; manjappa et al., 2011). an important factor for successful fish production *corresponding author: mohammad harsij doi: https://doi.org/10.22034/ijab.v6i3.470 e-mail address: m_harsij80@yahoo.com is appropriate stocking density (rahman et al., 2008). stocking density directly depends on environmental conditions. it is a key factor in determining productivity and profitability of aquaculture systems production (north et al., 2006; braun et al., 2010). environmental stress caused by stocking density can affect digestion and absorption of food, growth rate and nutritional efficiency (north et al., 2006; abdeltawwab, 2012; abdel-tawwab et al., 2014). higher stocking densities were reported to cause chronic stress, which in turn reduced the growth rate due to the reallocation of energy towards activities aimed at restoring homeostasis such as respiration, locomotion, hydromineral regulation, and tissue repair (biswas et al., 2007; abdel-tawwab, 2012; abdel-tawwab et al., 2014). various studies have been carried out about the effects of stocking density on fish growth performance in different species such as nile tilapia, oreochromis niloticus (abdel-tawwab, 2012; abdel 139 int. j. aquat. biol. (2018) 6(3): 138-146 tawwab et al., 2014), c. carpio (nuwansi et al., 2017), rtilus frisii kutum (imanpoor and abdollahi, 2011), oncorhynchus mykiss (liu et al, 2016), paralichthys olivaceus (kang and kim, 2013) and anguilla marmorata (tan et al., 2018). in nature, light intensity and background color can affect feed detection, feed conversion rate and feeding success of cultured fish (strand et al., 2007). therefore, all these factors can affect the fish growth and mortality (jirsa et al., 2009). the tank color may affect fish growth and body composition (papoutsoglou et al., 2000; ginés et al., 2004; aly et al., 2017), stress reactions (rotllant et al., 2003) and their skin color (van der salm et al., 2004). in the fish body, the α-melanocyte-stimulating hormone and the melanin condensing hormones are the main components of pigmentation (clement et al., 2005). the pituitary gland releases the alpha-melanocyte hormone. this hormone disperses melanin granules into melanophores, which darkens the skin color. skin coloration in teleost is under multi-parametric control and a number of external or internal factors such as physical, nutritional, genetic, neuro-hormonal known as influential factors on chromatic state of fish (fujii, 1993). in addition, fish can alter their coloration in response to environmental conditions, physiological challenges, stressful stimuli (szisch et al., 2002) and rearing condition (in some fish such as red porgy) (rotllant et al., 2003). however, fish could adapt to the background color by changing the skin color (fernandez and bagnara, 1991; han et al., 2005). although the influence of tank color and rearing density on growth and metabolic activities of cultured fish has been reported in the literatures, the effect of both factors at once has not been studied. therefore, this study examined the combined effects of rearing density and background color on growth performance, feeding, body composition and skin color of common carp under experimental conditions. materials and methods fish and experimental design: common carp was obtained from a farm in gonbad-kavous, iran, and transferred to aquatic engineering laboratory of gonbad-kavous university, iran. the fish were acclimatized with laboratory conditions for 12 days. eight hundred and forty fish with mean initial body weight (±sd) of 1.41±0.05 g were randomly distributed, in six triplicated treatments according to a 2×3 factorial design for 45 days. the fish were reared in tanks with black and white color at three rearing densities of 20 (low density–ld), 40 (medium density–md) and 80 (high density–hd) fish per tank (the equivalent of 0.70, 1.41 and 2.82 g/l). the fish were fed thrice daily (8:00, 13:00 and 18:00 h) at a rate of 3% of body weight using commercial pellet (40% crude protein, 12% crude lipid, 4.5% crude fiber with a diameter of 1.5 mm) from animal feed and aquaculture of bezae company, iran. sample collection and analysis: at the end of the feeding experiment, the fish were fasted for 24 hrs, collected, counted, and then total length and final weights were measured in each tank. the fish were profoundly anesthetized with 500 mg/l of clove powder. the growth and feed utilization parameters were calculated as following: percentage of weight gain (wg%) = [(wt – w0)/(w0)]×100 specific growth rate (sgr; %/day) =100× [(ln wt – ln w0)/t] feed conversion ratio (fcr) = dry feed intake (g))/wet weight gain (g) protein retention efficiency (per) = weight gain (g)/protein fed (g) lipid efficiency ratio (ler) = weight gain (g)/lipid fed (g) condition factor (cf) =wt/l3 where wt (g) is fish body weight at day t and w0 at day 0, t (days) is the duration of experiment, l is total fish length (cm). body composition analysis: seven fish were randomly captured from each tank for measuring the proximate composition of the whole-fish body. contents of protein, lipid, moisture and ash were analyzed according to the standard methods (aoac, 1995) and crude protein by analysis of total nitrogen (tn× 6.25) content by the kjeldahl method (kjeltec 1030 auto analyzer, tector, sweden). crude lipid content was 140 marandi et al/ interaction of fish density and background color effects on common carp measured by petroleum ether extraction using the soxhlet method (model 1043 extraction unit; tecator, sweden). moisture content was measured by drying in oven at 105ºc to a constant weight and ash was measured by ignition at 600 ºc in a muffle furnace (heraeus, germany) for 24 hrs. fiber was analyzed using a fibertec system tector 1010. color analysis: fish skin color was measured in three fish in each tank using a chromameter wsc-s (spsic inc., shanghai, p.r. china) equipped with a d65 light source and a 108 observing angle calibrated to black and white standards. the value of l* represents lightness (0 for black and 100 for white), the a* value represents the red/green dimension with positive values for red and negative ones for green and the value of b* represents the yellow/blue dimension with positive values for yellow and negative ones for blue. colorimetric values of skin color were performed in two sides of each fish body (han et al., 2005). statistical analysis: normality of data was assessed using kolmogorov-smirnov test. data were analyzed by two-way analysis of variance (anova) with tank color and rearing density as factors using sas (statistical analysis system) program. each tank was considered as the experimental unit. where p values were significant (p< 0.05) multiple comparisons were carried out using the duncan test (karakatsouli et al., 2010). all values presented in the text and tables are means±sd. results comparing growth and feed indices of common carp reared in black and white tanks with three levels of density (low, moderate and high) are showed in table 1. the final weight was significantly different among the treatments (p<0.05), the highest was observed in ld (4.90±0.4 g) and lowest in hd (3.50±0.30), regardless of the tank color. at the end of 45 days of experiment, the wg showed a significant difference among the treatments from 136.55±20.88 to 252.76± 29.01. although the cf was not different among the treatments. the fish reared under black tank at ld had higher sgr compared to the other treatments (p<0.05). fcr was significantly (p<0.05) lower in the ld treatment (1.21±0.13) compared to the hd (2.37±0.33). statistically, per and ler was significantly different (p<0.05), so that the highest value was observed in the ld treatment in black tank and the lowest in the hd treatment in both black and white tanks. growth and feed parameters reduced in the hd treatment in both black and white tank. independently, tank color and rearing density significantly affected growth and feed performance of common carp, while no combined interaction was figure 1. values of l* in dark and white tanks at three rearing densities (bars assigned with different superscripts are significantly different; n=15; p =0.05). 141 int. j. aquat. biol. (2018) 6(3): 138-146 found between the two factors examined (table 1). table 2 shows the effects of tank color (black and white) and culture density on the fish body compositions. crude protein was significantly different among the treatments (p<0.05). the crude protein content ranged from 65.76±0.30 to 64.11±0.32%. fish under in the hd treatment and white tank showed the highest lipid content significantly different compared to the other treatments (p<0.05). experimental treatments show significant difference in dry matter, fiber and ash (p<0.05). statistical analysis showed that rearing density had significant effect on the fish body biochemical composition (p<0.05), while the tank color did not significantly affect the body composition (p>0.05). significant interaction between tank color figure 2. values of a* in dark and white tanks at three rearing densities (bars assigned with different superscripts are significantly different; n=15; p =0.05). figure 3. values of b* in dark and white tanks at three rearing densities (bars assigned with different superscripts are significantly different; n=15; p =0.05). 1 4 2 in t. j . a q u at . b io l. ( 2 0 1 8 ) 6 (3 ): 1 3 8 -1 4 6 t a n k c o lo r p v a lu e b la c k w h it e d e n si ty c o lo r d e n si ty c o lo r × d e n si ty l o w m e d iu m h ig h l o w m e d iu m h ig h f in a l w e ig h t (g ) 4 .9 0 ± 0 .4 0 a 3 .9 6 ± 0 .2 6 b c 3 .5 0 ± 0 .3 0 c 4 .4 6 ± 0 .3 1 a b 3 .9 1 ± 0 .2 5 b c 3 .5 9 ± 0 .3 0 c 0 .3 9 1 0 .0 0 1 0 .3 5 0 n s f in a l le n g th ( c m ) 6 .5 4 ± 0 .2 4 6 .1 0 ± 0 .1 1 6 .9 0 ± 0 .1 8 6 .4 7 ± 0 .1 9 6 .2 0 ± 0 .3 1 6 .0 7 ± 0 .3 0 0 .9 6 2 0 .0 1 8 0 .8 2 3 n s w g ( % ) 2 5 2 .7 6 ± 2 9 .0 1 a 1 9 3 .7 5 ± 1 9 .3 7 b c 1 3 6 .5 5 ± 2 0 .8 8 c 1 9 7 .9 6 ± 2 0 .7 7 a b 1 7 5 .5 7 ± 1 8 .1 4 b c 1 6 2 .4 4 ± 2 2 .2 9 b c 0 .3 0 8 0 .0 0 1 0 .1 6 2 n s s g r ( % g d a y − 1 ) 2 .7 9 ± 0 .1 7 a 2 .3 9 ± 0 .1 4 b c 1 .9 0 ± 0 .1 9 c 2 .4 2 ± 0 .1 5 a b 2 .2 4 ± 0 .1 4 b c 2 .1 3 ± 0 .1 8 b c 0 .2 5 6 0 .0 0 1 0 .0 2 7 s c f ( % ) 1 .7 5 ± 0 .0 5 1 .7 5 ± 0 .1 7 1 .5 4 ± 0 .0 6 1 .6 4 ± 0 .0 4 1 .6 5 ± 0 .1 8 1 .6 0 ± 0 .1 1 0 .4 1 7 0 .1 8 1 0 .4 3 3 n s f c r 1 .2 1 ± 0 .1 3 c 1 .8 9 ± 0 .1 9 a b 2 .3 7 ± 0 .3 3 a 1 .4 4 ± 0 .1 4 b c 1 .9 8 ± 0 .2 1 a b 2 .1 5 ± 0 .2 8 a 0 .7 8 0 0 .0 0 1 0 .2 6 4 n s p e r 0 .1 2 ± 0 .0 1 0 a 0 .0 9 ± 0 .0 0 6 b c 0 .0 8 ± 0 .0 0 7 c 0 .1 1 ± 0 .0 0 7 a b 0 .0 9 ± 0 .0 0 6 b c 0 .0 8 ± 0 .0 0 7 c 0 .3 9 1 0 .0 0 1 0 .3 5 0 n s l e r 0 .8 1 ± 0 .0 6 a 0 .6 6 ± 0 .0 4 b c 0 .5 8 ± 0 .0 5 c 0 .7 4 ± 0 .0 5 a b 0 .6 5 ± 0 .0 4 b c 0 .5 9 ± 0 .0 5 c 0 .3 9 1 0 .0 0 1 0 .3 5 0 n s v a lu e s in a r o w w it h d if fe re n t su p e rs c ri p ts d e n o te s ig n if ic a n t d if fe re n c e ( p < 0 .0 5 ). e a c h v a lu e r e p re se n ts t h e m e a n ± s d . t ab le 1 . g ro w th a n d f ee d in g p er fo rm a n ce o n c o m m o n c ar p r ea re d i n t w o t an k c o lo rs ( b la ck a n d w h it e) w it h d if fe re n t d en si ti es ( l o w l d ; m ed iu m m d ; h ig h d en si ty h d ) fo r 4 5 d ay s. t a n k c o lo r p v a lu e b la c k w h it e d e n si ty c o lo r d e n si ty c o lo r × d e n si ty l o w m e d iu m h ig h l o w m e d iu m h ig h p ro te in 6 5 .7 6 ± 0 .3 0 a 6 4 .1 1 ± 0 .3 2 b 6 5 .2 2 ± 0 .4 2 a 6 5 .6 2 ± 1 .0 5 a 6 5 .0 0 ± 0 .2 8 a b 6 4 .1 7 ± 0 .3 2 b 0 .7 0 1 0 .0 0 5 0 .0 2 5 s l ip id 2 3 .5 5 ± 0 .5 8 b 2 3 .8 2 ± 0 .4 5 b 2 3 .4 0 ± 0 .3 8 b 2 4 .0 4 ± 0 .1 6 a b 2 3 .6 2 ± 0 .3 7 b 2 4 .4 9 ± 0 .5 1 a 0 .0 4 3 0 .6 5 8 0 .0 6 9 n s f ib e r 0 .9 2 ± 0 .0 4 a 0 .9 3 ± 0 .0 7 a 0 .5 1 ± 0 .0 9 c 0 .7 1 ± 0 .0 9 a b c 0 .6 7 ± 0 .1 2 b c 0 .7 8 ± 0 .1 0 a b 0 .1 4 6 0 .0 1 3 0 .0 0 1 s a sh 9 .1 2 ± 0 .4 6 b 1 0 .4 2 ± 0 .6 0 a 1 0 .3 8 ± 0 .6 8 a 9 .1 9 ± 0 .3 1 b 1 0 .0 3 ± 0 .3 0 a b 9 .9 5 ± 0 .6 3 a b 0 .3 2 0 0 .0 0 6 0 .6 6 3 n s d ry m a tt e r 2 5 .1 4 ± 0 .2 4 b c 2 6 .9 5 ± 0 .3 3 a 2 7 .3 3 ± 0 .5 2 a 2 3 .7 6 ± 0 .4 2 c d 2 3 .1 0 ± 0 .3 6 d 2 5 .8 1 ± 0 .4 2 a b 0 .0 0 1 0 .0 0 1 0 .0 0 1 s c o m p u ta ti o n a l c a rb o h y d ra te s 0 .6 8 ± 0 .0 9 a 0 .6 1 ± 0 .0 5 a b 0 .4 8 ± 0 .0 4 b 0 .5 9 ± 0 .0 9 a b 0 .5 2 ± 0 .0 4 b 0 .6 1 ± 0 .0 2 a b 0 .6 0 8 0 .0 7 5 0 .0 1 7 n s v a lu e s in a r o w w it h d if fe re n t su p e rs c ri p ts d e n o te s ig n if ic a n t d if fe re n c e ( p < 0 .0 5 ). e a c h v a lu e r e p re se n ts t h e m e a n ± s d . t ab le 2 . t h e p ro x im at e co m p o si ti o n ( % , o n d ry m at te r b as is ) o f th e w h o le b o d y o f co m m o n c ar p r e ar ed i n t w o t an k c o lo rs ( b la ck a n d w h it e) w it h d if fe re n t d en si ti e s (l o w l d ; m ed iu m m d ; h ig h d en si ty h d ) fo r 4 5 d ay s. 142 marandi et al/ interaction of fish density and background color effects on common carp and density was detected for carcass protein, fiber and dry matter content (table 2). the tank color significantly affected all body color parameters (figs. 1, 2, 3). the values of l* in dark tank were significantly (p<0.05) lower than white tank. the values of a* in dark tanks were higher than white tanks. the value of b* in common carp skin in white color tanks was significantly higher than that in dark color tanks. the images produced at the end of the experiment indicated that the fish reared in dark tanks were darker than those fish reared in white tanks. skin color was affected only by tank color (fig. 4). discussion the results of the present study showed that growth performance was affected only by rearing density being lower in the hd treatment. no significant interaction was detected between tank color and rearing density for growth and feed factors. it is thought that fish growth is influenced by various environmental factors (i.e. density, nutrition and light) and behavioral (i.e. social interactions, life stage) (haga et al., 2002; ashley, 2007). the growth of some fish species is density dependent and there is an inverse relationship between stocking density and individual size of fish produced (yang et al 2011; abdel-tawwab, 2012, 2014). final weight and sgr were significantly lower in the hd treatment. in this regard, gang et al. (2010) and zhu et al. (2011) reported that growth, survival, and food utilization decreased along with increase in rearing density in scortum barcoo and acipenser schrenckii. using appropriate stocking densities and feeding strategies are two key factors for successful aquaculture management (kohinoor and rahman, 2014). there was a significant difference in fcr among different treatments. in this experiment, fcr was ranged from 1.21±0.13 (dark tank with low density) to 2.37±0.33 (dark tank with high density). in contrast to these results, oprea et al. (2015) reported that different stocking densities did not negatively influence the fish production (fcr value was 1.41 in density of 30 kg/ha and 1.33 in density of 15 kg/ha. during the present experiment, no significant interaction was observed between the two factors examined for cf, wg and fcr, revealing that when the fish were kept at low density, growth and feed utilization enhanced. density effect in intensive rearing of carp in tanks has been reported either to agree with the present results (jha and barat, 2005) or to suggest even much lower densities (biswas et al., 2006). different factors like feeding components (ali and al-asgah, 2001) and environmental color (brännäs et al., 2001) and stocking density (enache et al., 2011) may influence the growth performance of cyprinidae. furthermore, under culture conditions, tank color and light intensity can cause stress to fish (papoutsoglou et al., 2005), resulting in behavioral changes such as swimming performance, activity level and habitat utilization (mesa and schreck, 1989). in addition, the tank color can affect fish body composition (papoutsoglou et al., 2000; aly et al., 2017). in the present study, black and white tanks did not have any significant effects on the fish body biochemical composition. significant interaction between tank color and rearing density was detected for carcass protein, fiber and dry matter content. hassan akbarian et al. (2012) studied the effect of tank color on growth performance and body composition in common carp and reported that blue tank provided a better condition for growth factors and protein contact compered to white and black tanks. it may be because more physiological compatibility of fish with this color. contrary to the current research, karakatsouli et al. figure 4. pictures of common carp skin reared in dark and white tanks with different densities for 45 days. 143 143 int. j. aquat. biol. (2018) 6(3): 138-146 (2007) investigated combined effects of rearing density and tank color on the growth and welfare of juvenile white sea bream, diplodus sargus l. in a recirculating aquaculture system. the results of their research proved that white and blue tanks could increase the growth factors and protein content and reduces lipid content, because it caused stress reduction, increasing food intake and fish comfort in these tanks. the effect of tank and food color on growth and body composition of grey mullet (liza ramada) was tested. the highest wg and carcass protein content were observed in light tank and the lowest values were obtained in non-colored tank. therefore, it seems that the fish have different functions based on the conditions of life and adaptation to the specific color (el-sayed and elghobashy, 2010). the background color of the living media is an important factor in determining the skin color (papoutsoglou et al., 2000). body colors and patterns are determined by the distribution, density and aggregation state of different chromatophores in the integument. in fish, colors are produced by light absorption of pigments contained in the chromatosomes of melanophores, erythrophores and xanthophores and cyanophores, as well as by reflection from purine crystals, which, depending on their spatial organisation in refractosomes or leucosomes, produce the metallic iridescence of iridophores or the whitish hue of leucophores (goda and fujii, 1995; leclercq et al., 2010). fish can synthesize eumelanin from tyrosine and pteridine pigments from gtp (maan and sefc, 2013). in the present study, it was found that fish in white tank have suitable color status, and their light color makes them marketable. fish color changes are the result of adaptation to the environment, which is related to physiological changes (van der salm et al., 2004). acknowledgments this research was conducted with the support of gonbad-kavoush university. references abdel-tawwab m. 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(2017) 6(3): 138-146 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی معمولی کپور ماهی پوست رنگ و بدن ترکیبات تغذیه، رشد، عملکرد به مخزن رنگ و تراکم متقابل اثر (cyprinus carpio) جعفریان... ا حجت ،آدینه حسین ،*هرسیج محمد مرندی، علی .ایران گلستان، کاوس، گنبد دانشگاه طبیعی، منابع و کشاورزی دانشکده شیالت، گروه چکیده: عمولیم کپورماهی پوست رنگ تغییر و بدن ترکیباتبر رشد، مخزن رنگ دو سازی وذخیره تراکم سه ترکیبی اثر منظور بررسیمطالعه به این ماهی در هر مخزن یا md: 40)، متوسط گرم در لیتر( 70/0ماهی در هر مخزن یا ld: 20) کمماهیان با تراکم .انجام شد( گرم 41/1 05/0±) روز پرورش داده شد. 45مدت به (سفید و سیاه)رنگی مخزن دوگرم در لیتر( در 82/2ماهی در هر مخزن یا hd: 80) و زیادگرم در لیتر( 41/1 نرخ نهایی و وزنثبت رسید. گرم در لیتر به 00/7گرم در لیتر و 45/2ترتیب هدر تیمارهای تراکم کم و زیاد ب سازیپایان آزمایش، تراکم ذخیرهدر ضریب میزان کمترین و گرم( 90/4 ±44/0میزان وزن ) بیشترین .بود hd و md تیمار تراکم از بیشتر داریمعنی طوربه ldتیمار در ویژه رشد کل پروتئین محتوای بر توجهی قابل تاثیر تراکم .آمد دستهب ld در سیاه برای ماهیان پرورش یافته تحت مخازن( 21/1 ±13/0) تبدیل غذایی مارهاتی سایر از بیشتر داریمعنی طوربه باال تراکم با سفید مخزن در بدن چربی مقدار .دهد نمی تغییر را عامل این مقدار مخزن رنگ اما دارد، بدن سنجیده b*و l، *a* سه فاکتور توسط پوست رنگدست آمد. هب خشک ماده و فیبر الشه، پروتئین مخزن، در مقدار رنگ تراکم و بین متقابل اثر .بود از بیشتر سفید مخازن در ماهی پوست( b*) زردی و( l*) روشنایی دارد. مقدار منفی تأثیر رنگی هایشاخص بر سیاه رنگ که داد نشان نتایج. شد در ،است گذاشته تأثیر معمولی کپورو کارایی تغذیه ماهی رشد بر توجهی قابل طور به رشد تراکم و مخزن رنگ مطالعه، این در .بود سیاه مخازن .وجود ندارد عامل دواین میانی متقابل اثر هیچگونه حالیکه .پوست رنگ رشد، مخزن، رنگ پرورش، تراکم :کلمات کلیدی int. j. aquat. biol. (2021) 9(5): 297-308 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article evaluation of the fisheries and resource of sea cucumbers in the coastal waters of trincomalee district, eastern sri lanka kasun randika dalpathadu*1 marine biological resources division, national aquatic resource research and development agency, crow island, colombo, sri lanka. s article history: received 22 june 2021 accepted 4 october 2021 available online 2 5 october 2021 keywords: sea cucumber stock assessment depletion exploitation abstract: though sea cucumber is one of the key export-oriented fishery resources in sri lanka, there is some evidence for the population depletion of most of the sea cucumber species in the shallow coastal waters. the present study was aimed to study the status of the sea cucumber fishery in the irrakkakandi coastal area, eastern sri lanka, and carry out a stock assessment on the critical sea cucumber species presently harvested in this area. in order to achieve the objectives, logbook records based survey was conducted to cover the fishing season in 2019, from late february to the end of september. the harvest was collected by skin diving from shallow nearshore waters and scuba diving from distal coastal waters about 5 km away from the shore. thelenota anax was the dominant species in the catch from both fishing grounds, with a relative abundance of 86.82% in shallow waters and 91.30% in distal waters. the average catch per unit effort (cpue) during the fishing season in 2019 for scuba diving and skin diving was 50±8.59 individuals /boat/day and 8±1.12 individuals /diver/day, respectively. the dominant stock of the t. anax in the distal fishing ground was assessed using the depletion method. the initial stock size of t. anax at the onset of the fishing season was estimated at 112,067 individuals, and about 25% of the initial stock had been fished by the end of the fishing season in 2019. the estimated catchability coefficient (q) was 0.00046. the study revealed that the stock of t. anax along with other recorded threatened species in the irrakkakandi coastal waters might be led towards extinction if the fishery prevails without proper management. introduction sri lanka is an island nation located in between 5°55’ and 9°55’n and 72°42’ and 81°52’e, south of the indian subcontinent. it has a total land area of 65,000 km2 and a coastline of 1,770 km in length, containing several bays and shallow inlets (kumara et al., 2005; dissanayake et al., 2010). the continental shelf area is 30,000 km2, which is relatively narrow and small in the area when compared with other island nations (kumara et al., 2005). fishing activities are carried out all around the coast, but primarily within the continental shelf, which rarely extends more than 40 km and averages 25 km (dissanayake et al., 2010). though there are nearly 200 known species of sea cucumbers found in the waters around sri lanka, about 75 species inhabit shallow coastal waters, while nearly 50 species are abundant in intertidal areas (kumara et al., 2005). among them, 21 species are *correspondence: kasun randika dalpathadu doi: https://doi.org/10.22034/ijab.v9i5.1265 e-mail: kasun.randika@yahoo.com considered commercially important (dissanayake and athukoorala, 2010; dissanayake and stefanson, 2010). as in many asian countries, the sea cucumber fishery in sri lanka is an artisanal fishery confined to the shallow coastal waters (dissanayake et al., 2010; dissanayake and stefanson, 2012). at the present, the sea cucumber fishery is confined to the north-western (puttlam and mannar districts), eastern (trincomalee to ampara districts), northern (jaffna district) and north-eastern (mullaitivu district) coastal areas of the island. sea cucumbers were initially harvested by hand picking along the coast during the low tide period, and since the 1980’s fishers moved further offshore using snorkelling and at present by scuba diving as stock became depleted in shallow waters (kumara et al., 2005). fishing activities for sea cucumbers in sri lanka are highly seasonal which affected by the monsoon wind patterns. generally, the 298 dalpathadu / evaluation of the fisheries and resource of sea cucumbers in eastern sri lanka fishery for sea cucumbers in north-eastern and eastern coastal areas are carried out from the end of march to the end of september, while in north-western coastal areas, those prevail from the onset of october to onset march. as in the many parts of the asia region, all the harvest is exported as the ‘beche-de-mer’, mainly to singapore, hong kong and china (dissanayake et al., 2010; dissanayake and stefanson, 2010). holothurians are now considered valuable species worldwide (conand, 2008), and the recovery of depleted populations are slow and sporadic (kinch, 2002). even though there is a long history of sea cucumber fishery in sri lanka which dated back to about 1000 years back (dissanayake and wijeyaratne, 2007; dissanayake and stefanson, 2010), baseline data on the species composition, stock status, catch, effort etc. are very scarce. thus, the fishery for sea cucumbers in sri lanka has developed neither regulations nor precautionary approaches except diving and transportation licenses (dissanayake and wijeyaratne, 2007; dissanayake and athukoorala, 2010; dissanayake and stefanson, 2010, 2012). furthermore, some of the biological characteristics of the sea cucumbers, such as their lack of hard parts for ageing, difficulty in marking them, and plastic size and shape, may cause difficulties in conventional stock assessment methods (perry et al., 1999; uthicke, 2004). making the situation worse, due to the less availability of facilities for conducting the fisheryindependent surveys of resources, no one has attempted to carry out a stock assessment for sea cucumber resources in the trincomalee district, eastern sri lanka, via such method. based on the observations, it is evident that intense exploitation rates generally induce sharp depletions in abundance after the main recruitment within a year. the depletion model is designed to capture such within-season dynamics and can be used to track in real-time the depletions in abundance under fishing pressure, allowing in-season adaptations of management measures (pierce and guerra, 1994; agnew et al., 1998). thus depletion models (leslie and davis, 1939; de lury, 1947) are good candidates for in-season monitoring and management of invertebrate fisheries such as sea cucumbers (trianni, 2000; hoggarth et al., 2006; prescott et al., 2013). therefore, in this study, the present status of the sea cucumber fishery in the trincomalee district, eastern sri lanka, was assessed. further, the stock of dominant sea cumber species in the commercial catch was assessed by the depletion method. to my knowledge, this is the first effort to assess the sea cucumber fishery and the application of depletion methods for sea cucumber stock assessment in the trincomalee district, eastern sri lanka. the findings of this study will help to understand the sea cucumber fishery and the current status, and the exploitation rates of the sea cucumbers in the region. the results will be beneficial for the preparation and implementation of appropriate management strategies for a sustainable fishery for sea cucumbers in the region. materials and methods study area: a fisheries dependent survey of fisheries logbook records was carried out in 2019 in irrakkakandi coastal area, trincomalee district, eastern sri lanka. a single collector was responsible for carrying out the fishery for sea cucumbers in the irrakkakandi coastal area (fig. 1). data collection: the fishing season for sea cucumbers in the trincomalee district was initiated in late february till september 2019. thus the logbook data collection was carried out during this period. sea cucumber collectors typically maintain their logbooks in which they record daily species wise catch per boat. accordingly, data on the total landed catch per boat by species in terms of the number of individuals and the total number of boats operated each day was extracted from the daily logbook records of the collector. in addition, some of the fishers had involved in skin diving for collecting sea cucumbers in near shore shallow waters. the collector had recorded the daily species wise catch per each skin diver. those data were also available separately in the logbook records, which was used to assess the nearshore stocks of the dominant sea cucumber species. as the collector had recorded the species by their local names, scientific 299 int. j. aquat. biol. (2021) 9(5): 297-308 identification of the species was made in the field using available published literature and species identification guides (conand, 1998; dissanayake and athukoorala, 2010; purcell et al., 2012; dissanayake and nishanthan, 2016). further information was gathered via interviewing the divers and boat skippers. total monthly catch estimate by species for scuba diving: for the present study, a single fishing trip was considered as one unit of effort. therefore, for each sea cucumber species, the monthly mean catch per unit effort (cpue) in terms of catch in the number of individuals per boat per day was estimated based on the logbook data. accordingly, the total monthly collection of sea cucumber species by scuba diving was estimated by summing up the daily catch records of the particular month. for skin diving: the total effort of a skin diver in a single day was considered one unit of effort. for each sea cucumber species, the monthly mean cpue in terms of catch in the number of individuals per diver per day was estimated based on the logbook data. accordingly, the total monthly collection of sea cucumber species by skin diving was estimated by summing up the daily catch records of the particular month. stock assessment by depletion method: the depletion method consisted of modelling the depletion of stock during the main fishing season and analysing the influence of cumulative effort on an abundance index (royer et al., 2002). this method allows interpolation of the total initial stock size during each fishing season (leslie and davis, 1939; de lury, 1947). models were based on a biological understanding of the fishery. although not optimal, this was the best compromise between using the high resolution of the catch data and the lower resolution of the biological data (keller et al., 2015). the model estimates the following parameters: the initial population (n1) and the current stock size, the expected catches for each time step during the depletion event (all in numbers), the catchability coefficient (q) and the goodness of fit measure (rt). it was assumed that the population was a closed one because natural mortality would be low in the relatively short fishing season (gould and pollock, 1997). further, the catchability coefficient (q) was assumed to be constant during the study period. the assessments were conducted using the lesliedelury dms with the catch and effort data analysis (ceda) version 3.0 software package (kirkwood et al., 2001). the ceda software package assumes that the index (e.g. cpue) is simply proportional to the figure 1. sea cucumber landing site in irrakkakandi area, trincomalee district, eastern sri lanka (sri lankan map inset). 300 dalpathadu / evaluation of the fisheries and resource of sea cucumbers in eastern sri lanka stock size (hoggarth et al., 2006). for the analysis, ‘no recruitment’ model type was used as per availability of the data over a short period, certainly less than one year, and based on the assumption of the closed stock (trianni, 2000; kirkwood et al., 2001; hoggarth et al., 2006). thus it was assumed that there is no recruitment to the stock after the first data point but a constant natural mortality rate ‘m’ (kirkwood et al., 2001; hoggarth et al., 2006). for the analysis, ‘m’ was assumed to be not significantly different from zero over the period for which the model was fitted, which was generally of the order of about seven days (parkes et al., 1996; hoggarth et al., 2006; prescott et al., 2013). the depletion model is as follows: 𝑁𝑁𝑡𝑡+1 = 𝑁𝑁𝑡𝑡 𝑒𝑒−𝑀𝑀 − 𝐶𝐶𝑡𝑡 𝑒𝑒 −12𝑀𝑀 where nt is the abundance in terms of numbers of sea cucumber at the start of time t, c is total catch taken over time t, and m is natural mortality. the ‘no recruitment’ model type is as follows (kirkwood et al., 2001): 𝑁𝑁𝑡𝑡+1 = 𝑒𝑒−𝑀𝑀𝑁𝑁𝑡𝑡 − 𝑒𝑒 −12 𝑀𝑀 𝐶𝐶𝑡𝑡 𝑁𝑁 𝑡𝑡+12 = 𝑒𝑒− 1 2 𝑀𝑀 𝑁𝑁𝑡𝑡 − 1 2 𝐶𝐶𝑡𝑡 where nt is the abundance in terms of numbers of sea cucumber at the start of time t, c, is total catch taken over time t, and m is natural mortality. there are three error models in the ceda software package; least squares, gamma, log transform, deal with the measurement errors in the catch component of cpue, or, if a single abundance index is being used, in the abundance index itself (kirkwood et al., 2001), to achieve the best model fit (keller et al., 2015). considering the three error models in the ceda software package, a preliminary analysis was conducted to understand the effect of each error model on the results and determine the most suitable one. the best error model for the analysis with the available data set was decided after analysing the “residuals” graphs of the observed and expected values of catch and cpue (kirkwood et al., 2001; hoggarth et al., 2006). further within the selected error model, numerical measures of goodness of fit (r2) was used to decide how well the model fits (kirkwood et al., 2001; hoggarth et al., 2006; keller et al., 2015). results fishery, fishing season and fishing methods: the fishing season for the sea cucumbers in the trincomalee district was from february to the end of september 2019. after that, fishing for sea cucumbers could be carried out day and night. however, since a complete ban on night diving activities in sea cucumber fishery has been executed by the department of fisheries and aquatic resources (dfar) of sri lanka since april 2019, all fishing operations conducted targeting sea cucumber were confined to the daytime. fishing operations were usually carried out by 6 to 7 m long outboard motor fibre reinforced plastic (ofrp) boats. under the collector in the irrakkakandi area, there were 15 ofrp boats available for sea cucumber fishery though all of them had not operated in a single day. the collector used his boats interchangeably for the ease of service and repairs of the boats and engines. the catch had collected mainly by scuba divers by the method of hand-picking. according to the regulations implemented by the department of fisheries and aquatic resources (dfar), sri lanka, two scuba divers and the boat operator were allowed to participate in one fishing operation by scuba diving. furthermore, a boat could carry a maximum of 10 oxygen cylinders per single fishing operation. usually, boats left from the landing site around 7.00 am and reached the landing site with the catch by around 2.00 pm. the fishing ground for scuba diving was about 5 km away from the shore, about 20 m depth. in addition, few skin divers had engaged in sea cucumber collection in shallow waters on a more or less daily basis. there was no specific time for skin diving; thus, they had carried out the skin diving in search of sea cucumbers when the near shore waters got calm. those skin divers just swam towards the fishing ground, located within 1 km distance from the shore and collected the catch. then the catch was sold to the collector. the sea cucumber catch was processed at the collection centre to produce beach301 int. j. aquat. biol. (2021) 9(5): 297-308 de-mer. species composition in the present catch in irrakkakandi area: five species belonged to two families were recorded from the catch of the irrakkakandi waters on the eastern coast of sri lanka. considering the fishery by scuba diving, thelenota anax clark, 1921, was the dominant species with 91.30% relative abundance in terms of the number of individuals (table 1). holothuria atra jaeger, 1833 was recorded the lowest abundance with 0.14% representation in the catch in 2019. in addition, actinopyga miliaris (quoy & gaimard, 1834), bohadschia sp. and stichopus chloronotus brandt, 1835, had contributed to the rest of the catch (table 1). the catch of skin divers was composed of the same species as in the scuba diving but with different relative abundances. thelenota anax was the dominant species with 86.82% relative abundance in terms of the number of individuals and s. chloronotus recorded the lowest abundance with 0.17% relative abundance in 2019 (table 1). the fishing effort, cpue and production in the sea cucumber fishery scuba diving: the average effort during the fishing season in 2019 was estimated at 3.37±2.07 boat days. figure 1. location map of ashtamudi lake showing the study sites (station i-perumon lake, station ii-thekkumbhagam lake). table 1. species composition of the sea cucumber fishery based on the landed catch from in irrakkakandi area in 2019. family species common name local name % in the catch global conservation status scuba diving skin diving stichopodidae thelenota anax clark, 1921 amber fish poona attaya 91.30 86.82 dd (conand et al., 2013a) stichopus chloronotus brandt, 1835 greenfish dambala attaya 0.85 0.17 lc (conand et al., 2013c) holothuriidae actinopyga miliaris (quoy & gaimard, 1834) hairy blackfish kalu gal attaya 3.20 4.74 vu (conand et al., 2013b) bohadschia sp. sandfish nool attaya 4.51 7.79 holothuria atra jaeger, 1833 lollyfish nari attaya 0.14 0.48 lc (conand et al., 2013d) figure 2. the monthly variation of the average catch per unit effort (cpue) in 2019 in the sea cucumber fishery by scuba diving in the irrakkakandi area. 302 dalpathadu / evaluation of the fisheries and resource of sea cucumbers in eastern sri lanka the average cpue in the sea cucumber fishery was 50±8.59 individuals /boat/day. considering the cpue for the species caught by scuba diving, a gradual declining trend could be identified for t. anax from march towards the end of the season. a gradual incline till may and then after a declining trend towards the end of the season in the cpue was observed for a. miliaris and bohadschia sp. (fig. 2). the total production from the fishing ground for scuba diving in the irrakkakandi area was estimated at 31,014 individuals of sea cucumber species in 2019. among them, 28,317 individuals were represented by t. anax. considering monthly variations in the total production by scuba diving, a sharp increment in the catch till may then gradually decreased towards the end of the fishing season (fig. 3). the highest production was recorded in may 2019. the three most dominant species in the catch, t. anax, a. miliaris and bohadschia sp. showed a similar production trend, which increased rapidly till may and then decreased figure 3. the monthly production of sea cumber fishery by scuba diving in the irrakkakandi area in 2019. figure 4. the monthly variation of the average catch per unit effort (cpue) in 2019 in the sea cucumber fishery by skin diving in the irrakkakandi area. 303 int. j. aquat. biol. (2021) 9(5): 297-308 gradually to the end of the season in september. stichopus chloronotus was recorded only in may and june. skin diving: the average effort for skin diving fishery during the fishing season in 2019 was estimated at 4.00±2.29 diver days. the average cpue in the sea cucumber fishery by skin diving was estimated at 8±1.12 individuals /diver/ day. the highest average cpue was recorded in may, and a gradual decline in the cpue was observed then after towards the end of the fishing season for a. miliaris and bohadschia sp. the average cpue for t. anax remained almost constant from may to the end of the fishing season (fig. 4). the total production of sea cucumbers via skin diving in 2019 was estimated at 5,836 individuals, among which 5,067 individuals were represented by t. anax. considering the monthly variation in the total production by skin diving, the total production was boosted from february to march, and then a dropdown was observed in april. then, it gradually increased till july and recorded the highest production in 2019. after that, it decreased steeply in august. the most dominant species, t. anax, followed a trend similar to the total production. considering other species in the catch, the production of bohadschia sp. and a. miliaris exhibited an increasing trend till may and then decreased gradually towards the end of the season (fig. 5). stock assessment for thelenota anax in deep water fishing ground (scuba diving) in the irrakkakandi area: according to the results, the stock at the onset and end of the fishing season was estimated at n1 = 112,067 and n30 = 83,750 (r2 = 0.94). the estimated catchability coefficient (q) was 0.00046. thus, 25.27% of the t. anax stock thrived in the distal waters fishing ground was harvested by the end of the fishing season in 2019 (fig. 6). log transforms error model assumptions frequently showed minimisation failure in the stock assessment process for the other species recorded in the catch by the ceda software package. discussions harvesting and exporting coastal ecosystem associated organisms such as sea cucumbers have contributed substantially to the country's foreign exchange earnings while providing essential livelihood to the coastal fishing community (choo, 2008; kumara et al., 2008; dissanayake and stefansson, 2012). considering the fishery for sea cumbers in the irrakkakandi area, trincomalee district, eastern sri lanka, hand-picking by scuba diving was the main fishing method while ofrp boats were used fishing craft. those are the main fishing and figure 5. the monthly production of sea cumber fishery by skin diving in the irrakkakandi area in 2019. 304 dalpathadu / evaluation of the fisheries and resource of sea cucumbers in eastern sri lanka craft types for sea cucumber fishery in sri lanka (dissanayake and athukoorala, 2010; dissanayake and stefansson, 2012). in addition to the hand-picking by scuba divers, some fishers involved in the handpicking of sea cucumbers in nearshore shallow waters by skin diving. at the beginning of the sea cucumber fishery in sri lanka, hand-picking while wading or using snorkel gears were the main fishing methods (kumara et al., 2005). however, due to the overexploitation of the nearshore sea cucumber resources, fishers had to exploit resources located in far deeper areas by scuba diving (kumara et al., 2005; dissanayake and athukoorala, 2010). therefore, it was evident that the sea cucumber resource in nearshore shallow waters in the irrakkakandi area had not been over-exploited to date. though about 21 sea cucumber species are considered commercially important (dissanayake and stefanson, 2010) in sri lanka, only five species belonging to two families were recorded during this study. among them, the representation of holothuria atra in the catch from the deep distal waters was lower than that from the nearshore shallow waters. it may be attributed to the habitat preference of this species as it inhabits in aggregated populations in shallow waters where skin diving operated while it inhabits in more scatter in the deeper waters where the scuba diving operated (conand, 1998). further, this scattered nature of occupancy in the habitat may be the reason behind the lowest representation of h. atra in the catch from the scuba diving fishery. furthermore, the lowest representation of s. chloronotus in the catch from the skin diving may also be attributed to its habitat preference. this species generally occurs on back reef hollows (conand and mangion, 2002) and in areas with boulders mixed with live corals (choo, 2008), making it difficult to be spotted by the skin diver. however, due to the absence of scientific records of previous studies in sea cucumber fishery in trincomalee district, sri lanka, it is challenging to state whether the relatively low abundance of s. chloronotus, a. miliaris, h. atra and bohadschia figure 6. the results of the catch and effort data of thelenota anax with the 'log transform error model' in ceda software package version 3.0 for scuba diving fishery in the irrakkakandi coastal waters in 2019. 305 int. j. aquat. biol. (2021) 9(5): 297-308 sp. in the catch was due to depletion of the resources in the area or due to other factors such as the ecology of these species. the lowest production from both fishing methods in february may be attributed to the lowest total fishing effort in february, as the fishing season started in the last week of the month. the easter day terrorist attack in sri lanka on 21st april 2019 made a larger impact on sri lankan society, including the fishing community. it might be the reason behind the low production in april, as most of the fishing activities were seized for about two weeks due to security reasons. however, a continuous decline in the total monthly production was observed from may to september. the sharp decline in production could presumably be attributed to the depletion of the sea cucumber resource in the scuba fishing ground in the irrakkakandi area. when considering the fishery for sea cucumbers by skin diving, the sudden drop of the production from july to august and also the comparatively low production in september may be due to either the depletion of the resources or unfavourable weather conditions for skin diving activities in rocky shore area in irrakkakandi or due to the combination of both reasons. however, lacking historical data on the sea cucumber fishery in this area obstructed to conclude on either trend in the production or the fishing effort. according to the collector in the irrakkakandi area, the price paid for a diver per individual of different species in 2019 was 300-500 lkr (based on the size of the specimen) for t. anax; 400 lkr for a. miliaris and 200 lkr for bohadschia sp. therefore, when considering the species composition, production and cpue for both harvesting methods, t. anax was dominated while bohadschia sp. holds the second highest species in the catch. though a. miliaris has higher economic value than bohadschia sp, the higher percentage of bohadschia sp in the catch probably explained by the fishers' behaviour which aims to catch valuable species first, and when such species are not abundant or not found, they attempt to harvest low-value species (hasan, 2019). actinopyga miliaris was the third abundant species in the catch from both fishing methods in the irrakkakandi area. according to the global conservation status of this species, it has been categorised under the 'vulnerable' category, which is one of the 'threaten categories' in the iucn criteria (conand et al., 2013b). the price paid for an individual of a. miliaris was as same as for t. anax. furthermore, unlike t. anax, a. miliaris can be easily collected from their habitats (conand et al., 2013b). therefore, the very low abundance in the catch most probably attributed to the very low stock abundance in the habitat. thus prevailing fishing pressure may lead this species towards extinction from its local habitats. however, as there is a lack of historical data and unavailability of the stock assessment results from the present study for a. miliaris, it cannot be concluded on the present status of a. miliaris in the irrakkakandi area. some studies (purcell, 2010; koike, 2017) have proven that the decreasing trend in the cpue might indicate the decrease in the abundance of the target sea cucumber species in their habitat. however, the degree of confidence in cpue as an index of species abundance varies with behavioural interactions between the harvested sea cucumber species and the collectors (purcell, 2010; dissanayake and stefanson, 2012). the stock assessment results revealed that around 25% of the stock of t. anax had been removed at the end of the fishing season via scuba diving. though there might be other factors that affected the reduction of the stock, several studies have proven that the contribution of those factors on the decline in the abundance of sea cucumbers are probably negligible (conand, 1990; uthicke and benzie, 2000; hasan, 2019). therefore, the marked reduction in the size of the stock could result from the high fishing mortality. due to some of the biological traits of sea cucumbers such as late maturity, density-dependent reproduction, and low rate of recruitment of sea cucumbers (dissanayake and stefanson, 2012; hasan and elrady, 2012), some studies have suggested maintaining the exploitable level at a lower rate such as around 5% of the initial stock size to avoid the 306 dalpathadu / evaluation of the fisheries and resource of sea cucumbers in eastern sri lanka collapse of the fishery due to overexploitation (uthicke, 2004; purcell, 2010). if the fishery collapses due to over-exploitation of the resources in the particular area, it will take several decades to regain the stocks to their original condition (purcell, 2010; hasan and el-rady, 2012; hasan, 2019). as the current exploitation level is higher than the recommended level in the scuba fishing ground, there is a high risk of extinction of the t. anax in the habitat susceptible to scuba diving fishing if the current exploitation level remains unchanged in future. in this assessment, the depletion model used several assumptions. sea cucumber species exhibit minimal movements and a slow growth rate (conand, 1998; trianni, 2000). therefore, the t. anax population in the irrakkakandi coastal area would probably not have experienced any significant immigration or emigration during the seven-month harvest period, thus validates the assumption of a closed population. one of the fundamental assumptions in this method is constant catchability (q) over time. the constant catchability assumption would depend upon weather and the management unit fished (trianni, 2000). for sea cucumber fishing operations by scuba diving in irrakkakandi coastal area, the environmental conditions remained unchanged to some extent during the fishing season in 2019, and fishers used their fishing locations within the same fishing ground for the collection of sea cucumbers. moreover, the number of divers who were on-board for sea cucumber collection and the number of oxygen cylinders used for scuba diving per fishing operation also remained constant. therefore, the magnitude of change in catchability is expected to be low. however, it is imperative to formulate a proper management plan and implement appropriate management strategies to ascertain the sustainability of the sea cucumber fishery in the trincomalee district, eastern sri lanka. the results of this study could be use of when preparing such a management plan. further, it is highly recommended to implement a monitoring mechanism for the sea cucumber fishery in the trincomalee district to understand its existing trend. acknowledgements this study was undertaken using allocated funds to the national aquatic resources research & development agency (nara), sri lanka, by the ministry of finance, sri lanka. the authors are grateful to s.s.k. haputhantri (head of the division), c. karunarathne and u. prasad, and all other staff members of the marine biological resources division (mbrd) of nara who assisted in the fieldwork and office work. i would like to express my sincere thanks and gratitude to d.r. hearth of mbrd for her support in proofreading this paper. i would like to express my great appreciation for the divers and collector involved in the sea cucumber fisheries in trincomalee district, sri lanka, who supported in the field by providing with their logbook data. references agnew d.j., baranowski r., beddington j.r., des clers s., nolan c.p. 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(2019) 7(2): 71-84 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article identification of characteristic zooplankton species in the kinyankonge river basin, burundi simon buhungu*1,2, marcel donou3, gaspard ntakimazi1, clément agossou bonou4, elie montchowui2 1centre de recherche en sciences naturelles et de l’environnement, université du burundi, bp 2700 bujumbura, burundi. 2laboratoire de recherche en aquaculture et en biologie et ecologie aquatiques, ecole d’aquaculture, université nationale d’agriculture, benin. 3laboratoire de biomathématique et d’estimation forestière, université d’abomey calavi, benin. 4laboratoire de recherche en biologie appliquée, ecole polytechnique d’abomey-calavi, université d’abomey-calavi, bp 2009 cotonou, benin. s article history: received 28 august 2018 accepted 4 january 2019 available online 2 5 april 2019 keywords: zooplankton characteristic species indicator value kinyankonge abstract: the objective of this study is to determine the zooplankton species that characterize the kinyankonge river basin in burundi. thus, zooplankton was sampled monthly over a period of 18 months (from july 2015 to june 2016, then from january 2017 to june 2017) at seven stations. the indicator value (indval) of the identified zooplankton species and the coverage of stations were determined. the results showed that three species characterized significantly the most upstream station whereas the water of the irrigation channel was characterized by 4 species. the waters of the nyabagere tributary and the wastewater treatment plant are characterized by 1 and 5 species, respectively. furthermore, the dry season was characterized by 4 singletons and 13 pairs of species, while the rainy season was characterized by 11 pairs of species. moreover, the group of upstream stations was characterized by 5 species while 3 species characterized the group of downstream stations. these species highlighted by the indicator value method can be used to characterize stations in the kinyankonge river and provide information on seasonal changes. introduction zooplankton plays an important role in aquatic ecosystems (baloch et al., 2005). it is considered as one of the most important food sources to the aquatic organisms particularly to planktivorous. zooplankton community constitutes a way of energy flux transfer through aquatic food webs especially between phytoplankton and the high levels (santoswisniewski et al., 2006). zooplankton species are used as bioindicators of the quality of water in lakes and rivers (el-bassat and taylor, 2007; ahangar et al., 2012), because of their sensitivity to changes in the ecological and environmental conditions of their habitats (hanazato, 2001; carignan and villard, 2002; niemi and mcdonald, 2004; brito et al., 2011; güher et al., 2011; primo et al., 2015). their identification as characteristic species is a classical method often used in ecology (legendre and legendre, 2012). in fact, they early react to a large number of environmental changes. such species or groups of species are called bioindicators (parmesan, 2006; jakhar, 2013; primo et *correspondence: simon buhungu doi: https://doi.org/10.22034/ijab.v7i2.484 e-mail: buhusimon@gmail.com al., 2015) and are useful in predicting of the level or degree of pollution before the pollutants cause significant damage (pai, 2002; verma, 2002). their identification can provide an indication of ecosystem health. they can thus act as an early warning system allowing the implementation of intensive conservation strategy to anticipate ecologic catastrophe (chapin, 2000). in ecology, environmental bioindicators are identified by establishing a strong relationship with some environmental characteristics (kitching et al., 2000; davis, 2001). they are now one of tools used by water quality monitoring programs worldwide (furse et al., 2006; marchant et al., 2006; yagow et al., 2006; borja et al., 2008). especially, studies on the structure of zooplankton populations can be a tool for analyzing the environmental disturbances to which these organisms are subjected in aquatic environments (sampaio et al., 2002; eskinazi-santanna et al., 2013). therefore, through their indicator value of their community, the characteristic species can provide an 72 buhungu et al./ identification of characteristic zooplankton species in the kinyankonge river basin, burundi ecological significance to a classification of inspected stations and also highlight the functional characteristics of the studied system (touzin, 2008). studies conducted on the kinyankonge river have shown organic pollution coming from domestic discharges (buhungu et al., 2017, 2018) and a zooplankton community included rotifers, copepods and cladoceran species (buhungu et al., 2018). the current study aims to identify, using the method of indicator species analysis, the spatial and seasonal characteristic species of this river basin, based on a determination of their indicator values materials and methods study area and sampling stations: the kinyankonge river is approximately 6.5 km long. it crosses a nearly slightly populated locality and is characterized by arable land stretches. the soil is marshy and is therefore favorable mainly for rice and fodder cultivation. to conduct this study, seven sampling stations have been selected based on the types of discharges and activities occurring around the river (fig. 1). the first station s1 (3°20'22.765"s, 29°21'10.655"e, 774.5 m of altitude) is located upstream of the kinyankonge river. it has been chosen in the cibitoke district to investigate the river source which receives both wastewater and garbage. the second station s2 (3°20'30.527"s, 29°21'27. 655"e, 774.7 m of altitude) was chosen into the gikoma channel to assess the polluting load thrown out in kinyankonge river. the third station s3 (3°20' 43.598"s, 29°21'27.468"e, 774.8 m of altitude) is located on the nyabagere river, a tributary of the studied river. in fact, sand is extracted from nyabagere river for the construction of a new neighborhood located on its shores. sand removal operations cause a significant degradation of the substrate which is important for the aquatic organisms. on this station, the collected samples have also enabled the evaluation of the pollutants load discharged into the kinyankonge river. the fourth station s4 (3°20ˊ42.623"s, 29°21ˊ11.275"e, 771.3 m of altitude) is located on the kinyankonge river, downstream of the mouths of the nyabagere tributary and the gikoma canal. the fifth station s5 (3°21ˊ15.908"s, 29°20ˊ33.745"e, 765.6 m of altitude) is into the discharge channel of the wastewater treatment plant (wwtp) of buterere discharging their effluents into the kinyankonge river. the sixth station s6 (3°21ˊ16.657"s, 29°20ˊ32.535"e, 764.5 m of altitude) is positioned after the discharge point of the treatment plant. it receives the waters coming from the blending of wwtp effluents with the kinyankonge river water. as for the seventh station s7 (3°21'37,346"s, 29°20'22,794"e, 760.5 m of altitude), it is located near the mouth of the kinyankonge river and tanganyika lake. at this station, the river receives effluents from savonor soap factory that are discharged after a physical pretreatment. sampling: zooplankton samples were collected monthly over an 18-month period (from july 2015 to june 2016, then from january 2017 to june 2017). they were taken at morning between 7 am and 11 figure 1. geographic situation of sampling stations on kinyankonge river. 73 int. j. aquat. biol. (2019) 7(2): 71-84 am using a 50 μm-mesh plankton net. samples were taken vertically and over the entire water column. at each station, three different points were sampled to constitute a composite sample. the concentrated zooplankton was then recovered in a jar and immediately fixed with 5% formalin. observation, identification, and enumeration of zooplankton: in the laboratory, each zooplankton sample was concentrated to a volume of 100 ml. zooplankton species were identified by microscopic observation using n-120/ n-120a light microscope from ht-0205 hiprove. this species identification operation was based on the specific morphological characters observable using different determination keys (dussart, 1967; pourriot, 1968; rey and saint jean, 1968, 1969; dussart, 1982). then, individuals of identified species were also enumerated using a burker turk enumeration cell. the enumeration effort was set at 400 individuals for each inventoried species. thus, the count rate varied according to species abundance and reached 100% of sample for rare species. an extrapolation was then made on total volume of sample, on the one hand, and the volume of filtered water, on the other hand, to assess the densities per liter of river water. the density was calculated using the following relation: 𝐷 = 1000 ∗ (𝑛𝑖 ∗ 100 𝐴𝑅 ) 𝑉 where d is the density (expressed in individuals per liter); ni the number of individuals recorded for species i; ar sample analysis rate corresponding to ni; v volume of filtered river water (ml). data analysis: in order to identify characteristic species, the indicator value of species was calculated and the significance of this value was tested using the monte carlo permutation test. this test enables to verify whether the preference of a species for a type of habitat is significantly higher than it is suggested by a random distribution (dufrêne and legendre, 1997). the indicator value of species that measures its predictive value as indicator of the conditions prevailing in a station or a season (de cáceres and legendre, 2009) is given by the following relation according to dufrêne and legendre (1997): indvalij = aij × bij × 100 in this relation, aij = n individuals ij / n individuals i, and represents the specificity, while bij = n sites ij / n sites j, and corresponds to the fidelity. the indicspecies package of r (r core team, 2015) was used for testing singletons and species pairs, which provide better information on habitat ecology. in this study, analyses were limited to singletons and species pairs to limit the complexity of characteristic species identification. this option was done in order to avoid very large numbers of possibilities that could reduce the reliability of the analysis and making them too long (de cáceres and legendre, 2009). the coverage of stations, groups of stations and seasons was evaluated by the "strassoc", "coverage" and "plotcoverage" functions that were used for the calculation and graphical representation of the coverage according to the specificity (a) values. for this analysis, only species with fidelity values b > 0.1 were included for eliminating low fidelity species. a comparison was made between singleton coverage and species pairs. all the analyses were performed with the indicspecies package (de caceres and legendre, 2009) of the r software (r core team, 2015). results characteristic species of stations, group of stations and seasons: a total of 36 zooplankton species inventoried in the kinyankonge river basin (buhungu et al., 2018) were used for the identification of characteristic species. singletons and species pairs considered as characteristics of stations (table 1), groups of stations (table 2) and seasons (table 3) were the significant ones at 5% threshold with indicator value indval ≥ 0.50. thus, no species or pair of species characterized stations s6 and s7. the first station (s1) was characterized by 8 pairs of species and 3 singletons (lecane luna, l. bulla and alonella sp.), the second station (s2) by 70 pairs of species and 4 singletons (polyarthra vulgaris, brachionus quadridentatus, b. patulus and philodina sp.), the third station (s3) by one singleton (keratella tecta), the fourth station (s4) by 2 pairs of species and the fifth 74 buhungu et al./ identification of characteristic zooplankton species in the kinyankonge river basin, burundi table 1. indicator values (indval) of characteristic species of the stations. stations species a b indval p-value sig. station s1 singletons leca_lu 0.43 0.94 0.64 0.019 * leca_bul 0.30 1.00 0.55 0.040 * alon_sp(¥) 0.59 0.44 0.51 0.002 ** pairs alon_sp+rota_sp(¥) 0.67 0.44 0.55 0.001 *** leca_lu+rota_sp(¥) 0.34 0.83 0.53 0.008 ** leca_bul+leca_lu(¥) 0.30 0.94 0.53 0.005 ** leca_lu+naup(¥) 0.31 0.89 0.53 0.004 ** alonsp+brach pat(¥) 0.61 0.44 0.52 0.002 ** alon_sp+leca_bul(¥) 0.59 0.44 0.51 0.002 ** alon_sp+leca_lu(¥) 0.59 0.44 0.51 0.002 ** alon_sp+naup(¥) 0.59 0.44 0.51 0.002 ** alon_sp+brach_caly 0.52 0.44 0.48 0.002 ** alon_sp+lepa_pat 0.83 0.28 0.48 0.002 ** anur_fis+poly_vul 0.59 0.39 0.48 0.007 ** leca_bul+lepa_pat 0.37 0.61 0.47 0.069 ns alon_sp+brach_quad 0.67 0.33 0.47 0.003 ** alon_sp+brach_ang 0.57 0.39 0.47 0.002 ** alon_sp+poly_sp 0.97 0.22 0.47 0.003 ** brach_bid+leca_lu 0.27 0.78 0.46 0.038 * brach_ang+leca_lu 0.25 0.83 0.46 0.040 * alon_sp+moin_sp 0.46 0.44 0.45 0.001 *** anur_fis+leca_lu 0.44 0.44 0.44 0.025 * alon_sp+brach_bid 0.69 0.28 0.44 0.004 ** cepha_gib+lepa_pat 0.38 0.50 0.44 0.141 ns alon_sp+poly_vul 0.54 0.33 0.42 0.006 ** alon_sp+fili_ter 0.51 0.33 0.41 0.013 * cepha_gib+leca_bul 0.33 0.50 0.41 0.289 ns anur_fis+rota_sp 0.49 0.33 0.40 0.035 * alon_sp+aspl_sp 0.97 0.17 0.40 0.014 * alon_sp+aspl_pri 0.46 0.33 0.39 0.014 * anur_fis+brach_pat 0.39 0.39 0.39 0.037 * anur_fis+brachquad 0.45 0.33 0.39 0.026 * anur_fis+leca_bul 0.33 0.44 0.39 0.115 ns alon_sp+plat_quad 0.52 0.28 0.38 0.032 * alon_sp+micro_sp 0.61 0.22 0.37 0.019 * station s2 singletons poly_vul 0.80 0.88 0.84 0.003 ** brach_quad 0.90 0.75 0.82 0.006 *** brach_pat 0.59 1.00 0.77 0.001 *** phil_sp 0.42 0.81 0.58 0.010 ** rota_sp 0.35 0.94 0.58 0.065 ns aspl_sp 0.74 0.44 0.57 0.069 ns brach_bid 0.44 0.63 0.52 0.156 ns kera_trop 0.51 0.44 0.47 0.011 * moin_sp 0.31 0.69 0.46 0.367 ns pairs brachquad+poly vul(¥) 0.91 0.75 0.83 0.001 *** brach_quad+naup(¥) 0.84 0.75 0.79 0.003 ** brachquad+rota sp(¥) 0.83 0.75 0.79 0.002 ** brach_pat+naup(¥) 0.60 1.00 0.77 0.001 *** poly_vul+rota_sp(¥) 0.66 0.88 0.76 0.001 *** brach_pat+leca_bul 0.57 1.00 0.76 0.001 *** naup+poly_vul 0.65 0.88 0.76 0.002 ** brach_quad+phil_sp 0.77 0.69 0.73 0.001 *** 75 int. j. aquat. biol. (2019) 7(2): 71-84 table 1. continued. stations species a b indval p-value sig. station s2 pairs phil_sp+poly_vul 0.65 0.81 0.73 0.001 *** brach_pat+poly_vul 0.60 0.88 0.73 0.001 *** fili_ter+phil_sp 0.64 0.81 0.72 0.001 *** naup+rota_sp 0.56 0.94 0.72 0.002 ** brach_pat+fili_ter 0.59 0.88 0.72 0.001 *** brach_pat+phil_sp 0.59 0.81 0.70 0.001 *** brach_pat+rota_sp 0.52 0.94 0.70 0.001 *** brach_bid+brach_pat 0.75 0.63 0.69 0.001 *** brach_pat+brach quad 0.62 0.75 0.68 0.001 *** naup+phil_sp 0.56 0.81 0.68 0.001 *** brach_quad+fili_ter 0.60 0.75 0.67 0.004 ** aspl_pri+brach_pat 0.53 0.81 0.66 0.001 *** phil_sp+rota_sp 0.52 0.81 0.65 0.002 ** fili_ter+poly_vul 0.47 0.88 0.64 0.001 *** brach_pat+leca_lu 0.43 0.94 0.64 0.001 *** fili_ter+rota_sp 0.45 0.88 0.63 0.001 *** aspl_sp+brach_quad 0.89 0.44 0.62 0.006 ** aspl_pri+brach_quad 0.56 0.69 0.62 0.003 ** brach_ang+brach pat 0.44 0.88 0.62 0.001 *** aspl_sp+poly_vul 0.87 0.44 0.62 0.022 * brach_quad+lecabul 0.49 0.75 0.61 0.003 ** leca_lu+moin_sp 0.52 0.69 0.60 0.001 *** brach_pat+moin_sp 0.52 0.69 0.60 0.001 *** aspl_sp+naup 0.80 0.44 0.59 0.055 ns brach_bid+phil_sp 0.70 0.50 0.59 0.005 ** aspl_sp+rota_sp 0.79 0.44 0.59 0.044 * brach_ang+brach_qud 0.50 0.69 0.59 0.001 *** aspl_pri+poly_vul 0.42 0.81 0.59 0.001 *** aspl_pri+fili_ter 0.42 0.81 0.59 0.001 *** moin_sp+phil_sp 0.61 0.56 0.59 0.001 *** leca_bul+poly_vul 0.39 0.88 0.59 0.001 *** moin_sp+poly_vul 0.54 0.63 0.58 0.002 ** fili_ter+leca_bul 0.38 0.88 0.58 0.001 *** brach_quad+lepapat 0.60 0.56 0.58 0.001 *** leca_bul+moin_sp 0.48 0.69 0.58 0.001 *** brach_pat+lepa_pat 0.53 0.63 0.57 0.002 ** brach_bid+naup 0.52 0.63 0.57 0.023 * brach_pat+cephagib 0.58 0.56 0.57 0.001 *** brach_quad+leca_lu 0.43 0.75 0.57 0.001 *** aspl_sp+phil_sp 0.74 0.44 0.57 0.014 * fili_ter+leca_lu 0.37 0.88 0.57 0.001 *** brach_bid+rota_sp 0.56 0.56 0.56 0.015 * leca_lu+phil_sp 0.37 0.81 0.55 0.003 ** aspl_pri+rota_sp 0.37 0.81 0.55 0.002 ** cepha_gib+moin_sp 0.59 0.50 0.54 0.006 ** moin_sp+rota_sp 0.43 0.69 0.54 0.003 ** aspl_sp+brach_pat 0.67 0.44 0.54 0.008 ** leca_bul+phil_sp 0.36 0.81 0.54 0.003 ** aspl_pri+moin_sp 0.47 0.63 0.54 0.001 *** leca_lu+poly_vul 0.33 0.88 0.54 0.001 *** lepa_pat+phil_sp 0.51 0.56 0.54 0.010 ** brach_caly+brachpat 0.33 0.88 0.53 0.002 ** leca_bul+rota_sp 0.29 0.94 0.53 0.003 ** 76 buhungu et al./ identification of characteristic zooplankton species in the kinyankonge river basin, burundi table 1. continued. stations species a b indval p-value sig. station s2 pairs aspl_pri+phil_sp 0.37 0.75 0.52 0.016 * leca_bul+naup 0.27 1.00 0.52 0.005 ** brach_bid+leca_bul 0.43 0.63 0.52 0.024 * brach_bid+fili_ter 0.48 0.56 0.52 0.053 ns brach_quad+keratro 0.71 0.38 0.52 0.001 *** brach_quad+moinsp 0.47 0.56 0.51 0.001 *** aspl_pri+leca_lu 0.32 0.81 0.51 0.004 ** aspl_sp+lepa_pat 0.58 0.44 0.51 0.014 * brach_pat+kera_trop 0.58 0.44 0.50 0.003 ** brach_pat+micro_sp 0.57 0.44 0.50 0.013 * aspl_pri+lepa_pat 0.50 0.50 0.50 0.028 * brach_bid+kera_trop 0.77 0.31 0.49 0.003 ** cepha_gib+phil_sp 0.48 0.50 0.49 0.024 * aspl_pri+leca_bul 0.29 0.81 0.49 0.028 * brach_caly+leca_lu 0.27 0.88 0.49 0.010 ** kera_trop+naup 0.54 0.44 0.49 0.007 ** brach_ang+brachbid 0.41 0.56 0.48 0.037 * brach_quad+cephagi 0.46 0.50 0.48 0.039 * aspl_sp+fili_ter 0.53 0.44 0.48 0.044 * kera_trop+rota_sp 0.52 0.44 0.48 0.006 ** fili_ter+kera_trop 0.52 0.44 0.48 0.006 ** brach_pat+trop_sp 0.33 0.69 0.48 0.007 ** aspl_sp+aspl_pri 0.59 0.38 0.47 0.024 * fili_ter+lepa_pat 0.39 0.56 0.47 0.038 * brach_ang+poly_vul 0.27 0.81 0.47 0.040 * brach_quad+plat_qua 0.43 0.50 0.46 0.007 ** brach_ang+keratrop 0.49 0.44 0.46 0.013 * kera_trop+trop_sp 0.48 0.44 0.46 0.007 ** kera_trop+leca_lu 0.48 0.44 0.46 0.009 ** leca_lu+micro_sp 0.47 0.44 0.45 0.020 * kera_trop+poly_vul 0.46 0.44 0.45 0.010 ** poly_vul+trop_sp 0.30 0.63 0.43 0.046 * leca_lu+trop_sp 0.30 0.63 0.43 0.080 ns brach_quad+trop_sp 0.37 0.50 0.43 0.015 * kera_trop+leca_bul 0.42 0.44 0.43 0.020 * brach_caly+lepapat 0.31 0.56 0.41 0.063 ns moin_sp+plat_quad 0.39 0.44 0.41 0.024 * aspl_pri+kera_trop 0.39 0.44 0.41 0.024 * brach_bid+moin_sp 0.45 0.38 0.41 0.018 * lepa_pat+plat_quad 0.38 0.44 0.41 0.024 * aspl_sp+leca_lu 0.38 0.44 0.41 0.041 * brach_pat+méso_sp 0.88 0.19 0.41 0.048 * kera_trop+moin_sp 0.61 0.25 0.39 0.015 * phil_sp+trop_sp 0.27 0.56 0.39 0.289 ns brach_pli+poly_sp 0.80 0.19 0.39 0.045 * kera_trop+micro_sp 0.73 0.19 0.37 0.025 * station s3 singletons kera_tec 0.84 0.44 0.61 0.001 *** pairs kera_tec+rota_sp(¥) 0.85 0.44 0.61 0.001 *** aspl_pri+keratec(¥) 0.83 0.44 0.61 0.001 *** kera_tec+leca bul(¥) 0.78 0.44 0.59 0.001 *** kera_tec+phil_sp(¥) 0.88 0.39 0.59 0.001 *** brachang+keratec(¥) 0.78 0.39 0.55 0.001 *** cepha_gib+kera_tec 0.77 0.28 0.46 0.008 ** kera_tec+lepa_pat 0.76 0.28 0.46 0.005 ** 77 int. j. aquat. biol. (2019) 7(2): 71-84 station (s5) by 17 pairs of species and 5 singletons (b. calyciflorus, b. angularis, filina terminalis, microcyclops sp. and filina sp.) (table 1). the group of upstream stations (s1, s2, s3 and s4) was characterized by 14 pairs of species and 5 singletons (l. luna, l. bulla, p. vulgaris, b. quadridentatus and b. patulus), while 8 pairs of species and 3 singletons (b. angularis, b. calyciflorus and tropocyclops sp.) were characteristic of the group of downstream stations (s5, s6 and s7) (table 2). the table 1. continued. stations species a b indval p-value sig. station s4 pairs brach_caly+brach_qu 0.63 0.56 0.59 0.047 * brach_caly+poly_vul 0.40 0.83 0.58 0.021 * aspl_sp+brach_caly 0.53 0.39 0.46 0.122 ns brach_caly+cepha_gi 0.41 0.50 0.45 0.082 ns cepha_gib+fili_sp 0.63 0.22 0.38 0.038 * brach_ang+cephagib 0.31 0.44 0.37 0.350 ns aspl_sp+brach_fal 0.81 0.17 0.37 0.048 * station s5 singletons brach_caly 0.76 0.94 0.85 0.001 *** brach_ang 0.64 1.00 0.80 0.001 *** fili_ter 0.53 0.83 0.66 0.003 ** micro_sp 0.96 0.44 0.65 0.026 * naup 0.36 1.00 0.60 0.131 ns fili_sp 0.51 0.50 0.51 0.015 * trop_sp 0.33 0.72 0.49 0.029 * méso_sp 0.80 0.28 0.47 0.025 * pairs brachang+brachca(¥) 0.75 0.94 0.84 0.001 *** brach_caly+filiter(¥) 0.79 0.83 0.81 0.001 *** brach_caly+naup(¥) 0.61 0.94 0.76 0.001 *** brach_ang+filiter(¥) 0.65 0.83 0.74 0.002 ** brach_ang+naup(¥) 0.48 1.00 0.69 0.001 *** micro_sp+naup 0.86 0.44 0.62 0.012 * brach_caly+fili_sp 0.72 0.50 0.60 0.001 *** brach_caly+micro sp 0.79 0.44 0.59 0.007 ** fili_sp+naup 0.69 0.50 0.59 0.004 ** brach_ang+micro_sp 0.74 0.44 0.57 0.009 ** fili_sp+micro_sp 0.84 0.39 0.57 0.001 *** brach_ang+fili_sp 0.61 0.50 0.55 0.002 ** fili_ter+micro_sp 0.65 0.44 0.54 0.013 * brach_caly+trop_sp 0.42 0.67 0.53 0.004 ** fili_ter+naup 0.33 0.83 0.52 0.065 , brach_caly+méso_sp 0.92 0.28 0.51 0.010 ** brach_ang+méso_sp 0.88 0.28 0.50 0.007 ** naup+trop_sp 0.33 0.72 0.49 0.020 * brach_ang+trop_sp 0.32 0.72 0.48 0.016 * fili_ter+méso_sp 0.82 0.28 0.48 0.011 * aspl_pri+brach_caly 0.30 0.72 0.46 0.145 ns fili_sp+trop_sp 0.58 0.33 0.44 0.004 ** micro_sp+trop_sp 0.60 0.28 0.41 0.033 * station s6 pairs poly_sp+trop_sp 0.38 0.39 0.39 0.027 * brach_fal+fili_sal 1.00 0.11 0.33 0.157 ns aspl_pri+fili_sal 0.46 0.22 0.32 0.093 ns fili_sal+poly_vul 0.46 0.22 0.32 0.101 ns station s7 pairs anur_fis+phil_sp 0.27 0.33 0.30 0.350 ns cepha_gib+trop_sp 0.24 0.33 0.28 0.651 ns fili_sp+scar_lon 0.60 0.11 0.26 0.325 ns plat_quad+scar_lon 0.57 0.11 0.25 0.315 ns anur_fis+brach_fal 0.52 0.11 0.24 0.357 ns a=specificity, b=fidelity, p-value=probability, sig= significance level, code of significance: 0.001***; 0.01*; 0. 05*; ns: non significance, (¥): species more significantly characteristic of the station with highest indicator value. 78 buhungu et al./ identification of characteristic zooplankton species in the kinyankonge river basin, burundi dry season was characterized by 13 pairs of species and 4 singletons (l. bulla, asplanchna priodonta, brachionus bidentatus and anuraeopsis fissa) while the rainy season was characterized by 11 pairs (table 3). spatial coverage of characteristic species: station coverage by characteristic species is shown by figure 2. for each station, coverage of singletons and this of species pairs were compared. the coverage varied from a station to another according to characteristic species recorded. indeed, the coverage decreased as specificity increased for both singletons and pairs of characteristic species. therefore, when the selection of characteristic species were made more rigorously, the coverage of a station by the singletons or by the pairs of characteristic species decreased. at station s1, the coverage was total at specificity threshold for a=0.45 for singletons as well as characteristic pairs. at a higher specificity, it noticed that characteristic species number was no more sufficient to cover the entire station. this remark is more pronounced when considering only singletons. as for station s2, the coverage was total at up to a=0.6 for singletons and a=0.75 for species pairs. station s3 was the least covered. in this station, the coverage was total only a=0.18 for both singletons and species pairs. for stations s4, s6 and s7, singleton coverage decreased before pair coverage. stations s2 and s5 were covered by many species for both singletons and species pairs. table 2. species characteristic of groups of stations. group of stations species a b stat pvalue sig. upstream stations singletons leca_bul 0.73 1.00 0.86 0.001 *** leca_lu 0.75 0.84 0.80 0.005 ** poly_vul (¥) 0.93 0.66 0.78 0.036 * brach_quad 0.99 0.59 0.76 0.001 *** brach_pat 0.79 0.70 0.74 0.001 *** pairs leca_bul+rota_sp (¥) 0.72 0.87 0.79 0.001 *** leca_bul+leca_lu (¥) 0.74 0.84 0.79 0.001 *** leca_bul+naup (¥) 0.72 0.86 0.78 0.002 ** rota_sp 0.62 0.87 0.74 0.174 ns leca_lu+naup (¥) 0.79 0.74 0.77 0.001 *** aspl_pri+leca_bul 0.67 0.86 0.76 0.003 ** leca_lu+rota_sp 0.77 0.74 0.76 0.001 *** brach_pat+leca_bul 0.81 0.70 0.75 0.001 *** naup+rota_sp 0.76 0.74 0.75 0.105 ns brach_quad+naup 0.98 0.57 0.75 0.001 *** aspl_pri+leca_lu 0.77 0.73 0.75 0.001 *** brach_quad+leca_bul 0.94 0.59 0.74 0.001 *** brach_pat+naup 0.82 0.67 0.74 0.001 *** aspl_pri+rota_sp 0.73 0.74 0.74 0.001 *** naup+poly_vul 0.86 0.63 0.74 0.048 * poly_vul+rota_sp 0.85 0.63 0.73 0.019 * downstream stations singletons brach_ang 0.86 0.93 0.89 0.001 *** brach_caly 0.98 0.81 0.89 0.001 *** fili_ter 0.71 0.76 0.73 0.052 ns naup 0.54 0.94 0.71 0.649 ns trop_sp 0.65 0.74 0.70 0.013 * pairs brachang+brachcaly (¥) 0.95 0.80 0.87 0.001 *** brach_caly+fili_ter (¥) 0.94 0.72 0.82 0.001 *** brach_caly+naup (¥) 0.82 0.80 0.81 0.001 *** brach_ang+fili_ter (¥) 0.88 0.74 0.81 0.001 *** brach_ang+naup (¥) 0.69 0.91 0.79 0.001 *** brach_caly+trop_sp 0.79 0.65 0.71 0.001 *** naup+trop_sp 0.65 0.70 0.68 0.016 * brach_ang+trop_sp 0.65 0.70 0.68 0.019 * a=specificity, b=fidelity, p-value=probability, sig= significance level, code of significance: 0.001***; 0.01*; 0.05* 79 int. j. aquat. biol. (2019) 7(2): 71-84 table 3. seasonal characteristic species. seasons species a b stat p-value sig. rainy singletons moin_sp 0.71 0.51 0.60 0.126 ns pairs leca_bul+rota_sp(¥) 0.63 0.90 0.76 0.008 ** brach_ang+rota_sp 0.63 0.84 0.73 0.017 * brach_caly+leca_lu 0.65 0.66 0.65 0.043 * brach_pat+leca_lu 0.67 0.63 0.65 0.036 * rota_sp+trop_sp 0.69 0.58 0.64 0.03 * fili_ter+leca_bul 0.56 0.73 0.64 0.203 ns aspl_pri+rota_sp 0.52 0.74 0.62 0.371 ns leca_bul+moin_sp 0.78 0.48 0.61 0.028 * leca_lu+moin_sp 0.77 0.46 0.60 0.034 * brach_caly+brach_pat 0.62 0.57 0.60 0.078 ns brach_pat+moin_sp 0.83 0.42 0.59 0.012 * brach_caly+trop_sp 0.57 0.57 0.57 0.305 ns brach_pat+trop_sp 0.76 0.43 0.57 0.03 * moin_sp+phil_sp 0.86 0.37 0.57 0.029 * brach_ang+kera_trop 1.00 0.20 0.45 0.027 * brach_caly+kera_trop 1.00 0.20 0.45 0.019 * kera_trop+leca_bul 1.00 0.20 0.45 0.024 * kera_trop+leca_lu 1.00 0.20 0.45 0.02 * plat_quad+poly_vul 0.58 0.34 0.44 0.401 ns fili_ter+kera_trop 1.00 0.18 0.42 0.033 * kera_trop+naup 1.00 0.18 0.42 0.034 * kera_trop+rota_sp 1.00 0.18 0.42 0.032 * kera_trop+trop_sp 1.00 0.18 0.42 0.031 * micro_sp+trop_sp 1.00 0.18 0.42 0.024 * dry singletons brach_ang 0.79 0.85 0.82 0.152 ns leca_bul 0.71 0.91 0.81 0.025 * naup 0.71 0.91 0.80 0.14 ns aspl_pri 0.69 0.91 0.79 0.005 ** brach_bid 0.91 0.67 0.78 0.001 *** anur_fis 0.84 0.52 0.66 0.001 *** poly_vul 0.90 0.42 0.62 0.983 ns trop_sp 0.49 0.79 0.62 0.438 ns hexa_sp 0.75 0.24 0.43 0.013 * pairs aspl_pri+naup(¥) 0.68 0.82 0.75 0.011 * aspl_pri+brach_bid(¥) 0.84 0.64 0.73 0.001 *** aspl_pri+brach_ang(¥) 0.67 0.79 0.73 0.03 * aspl_pri+leca_bul(¥) 0.64 0.82 0.73 0.03 * brach_bid+naup(¥) 0.84 0.61 0.72 0.001 *** leca_bul+naup 0.62 0.82 0.71 0.204 ns brach_bid+leca_bul 0.77 0.64 0.70 0.001 *** brach_ang+naup 0.59 0.82 0.70 0.427 ns anur_fis+aspl_pri 0.85 0.52 0.66 0.001 *** brach_ang+brach_bid 0.79 0.55 0.66 0.001 *** brach_ang+brach_caly 0.83 0.52 0.65 0.776 ns anur_fis+leca_bul 0.84 0.48 0.64 0.001 *** leca_lu+naup 0.58 0.70 0.64 0.415 ns anur_fis+brach_bid 0.86 0.45 0.63 0.001 *** anur_fis+trop_sp 0.78 0.48 0.61 0.001 *** brach_ang+leca_lu 0.52 0.70 0.60 0.622 ns anur_fis+naup 0.77 0.45 0.59 0.002 ** brach_caly+naup 0.60 0.52 0.56 1 ns anur_fis+brach_ang 0.78 0.39 0.56 0.004 ** fili_ter+hexa_sp 0.75 0.24 0.43 0.011 * 80 buhungu et al./ identification of characteristic zooplankton species in the kinyankonge river basin, burundi coverage of characteristic species according to station groups: the coverage of the group of upstream and downstream stations is shown in figure 3. it remained maximal (100%) and decreased only beyond a specificity of 0.6 for both groups. in upstream group, this coverage is greater for pairs than for species singletons above 0.6. downstream station group covers were almost identical for characteristic species pairs and singletons. coverage of characteristic species according to season: seasonal coverage by characteristic species is presented in figure 4. for each season, it compares singletons and pairs of characteristic species. in fact, the cover is much higher during the rainy season than the dry season; it remained maximal (100%) and decreases only beyond a specificity threshold of 0.8. on the other hand, in the dry season, it decreased starting with a specificity of 0.5. the coverage rate was almost identical for both characteristic species pairs and the singletons. however, the coverage seemed to decrease faster in dry season (starting with a specificity of 0.8) than in rainy season. discussions this study on the identification of zooplankton species characteristic of the kinyankonge river basin provides a diversity of knowledge on the spatial and seasonal distribution of these species. the use of the indicator value for zooplankton species in the kinyankonge river basin has made it possible to develop a list of the most significant species for each station, group of stations and season. singletons and/or pairs of characteristic species were found mostly at stations located in the upstream part of kinyankonge river. thus, l. luna, l. bulla, and alonella sp. were identified as characteristic of the first station (s1) which receives domestic discharges. likewise, p. vulgaris, b. quadridentatus, b. patulus and philodina sp. were identified as characteristic of the second station (s2) located into an irrigation channel receiving both agricultural and domestic discharges. only k. tecta was characteristic of the third station (s3), enriched with suspended matter coming from sand operations. these aforementioned species table 3. continued. seasons species a b stat p-value sig. dry pairs brach_ang+hexa_sp 0.75 0.24 0.43 0.013 * brach_caly+hexa_sp 0.75 0.24 0.43 0.013 * hexa_sp+naup 0.75 0.24 0.43 0.013 * fili_sp+hexa_sp 0.85 0.21 0.42 0.005 ** brach_caly+brach_quad 0.85 0.21 0.42 0.934 ns brach_caly+cepha_gib 0.66 0.27 0.42 0.953 ns hexa_sp+moin_sp 0.96 0.18 0.42 0.001 *** brach_ang+brach_pli 0.82 0.21 0.42 0.026 * hexa_sp+leca_lu 0.96 0.18 0.42 0.007 ** hexa_sp+poly_vul 0.95 0.18 0.42 0.006 ** hexa_sp+leca_bul 0.93 0.18 0.41 0.012 * aspl_sp+naup 0.92 0.18 0.41 0.923 ns hexa_sp+rota_sp 0.91 0.18 0.41 0.012 * cepha_gib+hexa_sp 0.96 0.15 0.38 0.01 ** aspl_sp+brach_caly 0.79 0.18 0.38 0.845 ns hexa_sp+trop_sp 0.92 0.15 0.37 0.006 ** aspl_pri+micro_sp 0.80 0.15 0.35 0.74 ns kera_qua 1.00 0.12 0.35 0.008 ** brach_bid+kera_qua 1.00 0.12 0.35 0.008 ** kera_qua+leca_bul 1.00 0.12 0.35 0.008 ** kera_qua+naup 1.00 0.12 0.35 0.008 ** kera_qua+trop_sp 1.00 0.12 0.35 0.008 ** a= specificity, b=fidelity, p-value=probability, sig=level of significance, code of significance: 0.001***; 0.01*; 0.05* 81 int. j. aquat. biol. (2019) 7(2): 71-84 establish themselves in waters characterized by high dissolved oxygen level and high transparency (buhungu et al., 2018). for downstream stations, only station s5, which receives highly organic and mineralized effluents from wastewater treatment plant, was characterized by b. calyciflorus, b. angularis, f. terminalis, microcyclops sp. and filina sp. these species are characteristic of eutrophication environments (baloch et al., 2005). furthermore, the combination of stations revealed figure 2. coverage rates of characteristic species stations. figure 3. coverage rates of characteristic species according to station groups. 82 buhungu et al./ identification of characteristic zooplankton species in the kinyankonge river basin, burundi that 5 species (l. luna, l. bulla, p. vulgaris, b. quadridentatus and b. patulus) characterized the upstream stations, while 3 species (b. calyciflorus, b. angularis and tropocyclops sp.) characterized downstream stations which waters were polluted by organic matter (buhungu et al., 2017, 2018). in addition, the analysis of the indicator value for b. calyciflorus and b. angularis revealed that these species are pollutant-resistant. these results confirm those of starling (2000) which showed that zooplankton species richness decreases with eutrophication degree in rivers and lakes. similar results were found by pedrozo and rocha (2005) showing tolerance of b. calyciflorus and b. angularis to organic pollution and confirming several rotifers belonging to genera brachionus, keratella and fillina are characteristic of organic-enriched environments (isumbisho et al., 2006; moshood, 2009; ahmad et al., 2011). it is important to notice that rotifers were the most abundant zooplankton species identified in this study, in both rainy and dry season, in upstream as well as downstream stations. they were distributed according to downstream-upstream gradient since much more characteristic species were recorded at upstream. this abundance of rotifers species can be justified by their opportunistic nature, giving them the ability to better withstand changes of environmental conditions and of the availability of food resources (dumont, 1977; matsumura-tundisi et al., 1990; zébazé et al., 2004; bonecker et al., 2007). moreover, the river waters were characterized by singletons of rotifer species (l. bulla, a. priodonta, b. bidentatus and a. fissa) only during the dry season in which they were abundant. this may be due to the decreasing of water flow, creating thus favorable conditions for zooplankton egg-laying and hatching. in fact, in a river, the permanent renewal of the water does not favor the abundance of zooplankton (ouattara et al., 2001). a strong water current enhance turbidity which, by decreasing light penetration into the water, reduces the production of phytoplankton organisms and, thereby, limits the development of their predators which are zooplanktonic organisms (ouattara et al., 2001, 2007). on the other hand, season coverage seemed to decrease faster in the dry season than in the rainy season. this can be due to the fact that there are no other dry season characteristic species and the probability of finding it is low or even null (walther and moore, 2005). conclusion this study highlighted zooplankton species that significantly characterized the sampling stations in the kinyankonge river basin. the indicator species analysis method has identified the species that characterize each station, each group of stations, as well as seasons. it also pointed out the characteristic species favoured by dry season. their absence in the mentioned season could be due to the environment disturbance by human activities. this study provides therefore important information for future researches figure 4. seasonal coverage rates for characteristic species. 83 int. j. aquat. biol. 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(2019) 7(1): 45-55 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article how the body shape changes by the habitat hydrological factors in freshwater benthic fishes; case study on the genera cobitis (cobitidae) and ponticola (gobiidae) adeleh heidari1, meysam salehi2, hamed mousavi-sabet*1,3 1department of fisheries sciences, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. 2abzi-exir aquaculture co., agriculture section, kowsar economic organization, tehran, iran. 3the caspian sea basin research center, university of guilan, rasht, iran. s article history: received 7 november 2018 accepted 20 february 2019 available online 2 5 february 2019 keywords: environmental factors truss network system discrimination body shape abstract: benthic fish groups with low mobility are often restricted to a narrow range of a stream course, and their morphological characters tend to be affected substantially by the environmental conditions in their habitat. due to morphological affectability of fishes by many factors in lotic ecosystems, a landmark-based hypothesis was used to investigate the effects of habitat hydrological conditions on morphological characteristics in freshwater benthic fishes; a case study on the genera ponticola and cobitis. a total of 216 gobies and 128 spined loaches specimens were caught from six rivers with different hydrological conditions, along the southern caspian sea basin. in discriminant function analysis (dfa), the overall assignment of gobies and loaches into their original groups were 95.7% and 80.5%, respectively. discriminant analysis for pairwise groups shows a longer snout, shallow body/head, and elongated body for populations living in the large slope channel with faster water velocity versus relatively short snout and deep body/head for those living in small slope channel with slower water velocity. the results confirm the possibility of changes in the morphological characters of the benthic gobies and loaches, which should be considered in taxonomical and biological studies. introduction the environmental control of phenotypic expression combined with variability among local environments may result in populations being distinguishable from one another in morphology, behavior, physiology and life history (matthew, 1998; kawecki and ebert, 2004). rivers, in particular, are heterogeneous environments that are stochastic and highly variable in both space and time (matthews, 1998; fraser et al., 2011). fish living in such variable systems face multiple ecological constraints such as water temperature and flow regime. environmental heterogeneity across the geographical range of the species may exert local selective pressure actings to maximize individual fitness within specific environments (kawecki and ebert, 2004; fraser et al., 2011; jalili et al., 2015). fish can be classified into several functional groups (e.g., pelagic, slow water and benthic fishes) on the *correspondence author: hamed mousavi-sabet doi: https://doi.org/10.22034/ijab.v7i1.601 e-mail: mousavi-sabet@guilan.ac.ir basis of functional-morphological studies (sagnes et al., 1997; langerhans et al., 2003). benthic fishes are generally considered poor swimmers, which maintain their stream position on the substratum hydrodynamically. thus, their morphological characters should be affected by environmental conditions such as the water depth and current velocity of their habitat (sagnes et al., 1997; langerhans et al., 2003). therefore, the study of morphological variation in benthic fish populations in relation to environmental gradients may hold the key to evaluate the nature of natural selection. the analysis of morphometric characters has been widely accepted by fish biologists as a way to differentiate among different species and different populations within a species (yakubu and okunsebor, 2011). morphometric measurements are widely used to identify differences between fish populations and mainly important where the differences are mostly 46 heidari et al./ how the body shape changes by the habitat hydrological factors? attributed to environmental influences rather than genetic differentiation (bektas and belduz, 2009). geographical isolation of populations and interbreeding can lead to morphometric variations between populations, and this morphometric variation can provide a basis for population differentiation (bookstein, 1991; cadrin and friedland, 1999). the genera cobitis (family cobitidae) and ponticola (family gobiidae) are small benthic freshwater fishes with a wide distribution area covering large parts of the southern caspian sea basin and are of particular interest from a conservation point of view in many areas of its native range (coad, 2017; esmaeili et al., 2018). there are several reports, indicating the existence of morphological variability on benthic fish populations in the southern caspian sea basin (mousavi-sabet et al., 2011, 2012; mousavi-sabet and anvarifar, 2013; vasil′eva et al., 2015). despite to the recent morphology-based taxonomic studies on gobies and loaches species, there is no available data on habitat effects on morphological characters, which can help in better understanding of the real body shape differentiations between different taxa. therefore, it should be clear that; are the morphological variations between benthic fish species caused by (i) adaptive differences that reflect genetic differences, (ii) adaptive differences that reflect phenotypic plasticity or (iii) non-adaptive differences that reflect phenotypic plasticity. on the other hand, we want to clear that; how the environmental conditions and the river hydrology can affect the body shape in freshwater benthic fish species. therefore, the present study examines the relationship between body shape variation and environmental conditions in freshwater benthic fishes, the genera cobitis and ponticola, in the southern caspian sea basin. we predict that fish tend to have lower body depth and longer snout under environment with faster bottom current velocities. materials and methods for the study 216 goby and 128 loach specimens were collected from november 2015 to january 2016 by electrofishing device. fish specimens were sampled in the same month, out of the reproductive period from six rivers of the southern caspian sea basin, including the babol, sefid, tonekabon, siah, talar and gisum (fig. 1). the hydrologic and environmental factors, including altitude, temperature, water flow, substrate and slope were estimated in each studied river (table 1). the sampled fishes were photographed and fixed in 10% formaldehyde at the sampling site and transported to the laboratory for further studies. standard length (±1.0 mm) and body weight (±0.001 g) were recorded for each specimen. for goby specimens 105 distance measurements between 15 landmark points on lateral side and for loach specimens 78 distance measurements between 13 landmark points were measured using the truss network system according to bookstein (1991) with minor modifications (fig. 2). also, for gobies 44 and 28 distance measurements between 10 and 8 landmark points on ventral and dorsal views, respectively, were measured for investigation ventral and dorsal faces’ shape variation. the fishes were placed on a white board with dorsal and anal fins held erect by pins. the left body profile of each fish was photographed with a 300-dpi, 32-bit color digital camera (cybershot dscfigure 1. map of the iranian part of the southern caspian sea basin and location of sampling sites including guisum, sefid, tonekabon, babol, talar and siah rivers. 47 int. j. aquat. biol. (2019) 7(1): 45-55 f505; sony, japan). images were saved in jpg format and analyzed with tpsdig2 to digitize landmark points (mustafic et al., 2008). all measurements transformed into linear distances by computer for subsequent analysis. after image capture, the fish was dissected to identify the sex of the specimen by macroscopic examination of the gonads. gender was used as the class variable in anova to test for the significant differences in the morphometric characters if any, between male and female specimens. an allometric method (elliott et al., 1995) was used to remove size-dependent data in morphometric characters using following formula: madj = m (ls / l0)b table 1. the hydrologic data of the studied rivers in the southern caspian sea basin. environment conditions at sampling site river name altitude (m) water flow (m3/s) substrate slope (%) babol 158 2.1 sandy-stony 2.5 sefid 201 5.1 sandy-stony 5.4 talar 943 2.2 sandy-stony 2.0 guisum 24 0.5 sandy-muddy 1.7 tonekabon 179 1.6 sandy-stony 1.4 siah 117 1.2 sandy-muddy 1.3 figure 2. locations of the landmark points on lateral (a), ventral (b) and dorsal (c) sides of the goby and on lateral side of the loach (d). 48 heidari et al./ how the body shape changes by the habitat hydrological factors? where m is the original measurement, madj the size adjusted measurement, l0 the standard length of the fish, ls the overall mean of the standard length for all fish from all samples in each analysis, and b is estimated for each character from the observed data as the slope of the regression of log m on log l0 using all fish in any group. the results derived from the allometric method were confirmed by testing significance of the correlation between transformed variables and standard length (mustafic et al., 2008). univariate analysis of variance (anova) was performed for each morphometric character to evaluate the significant difference among the fish specimens (bookstein, 1991). to identify morphological differences of the loaches and gobies between rivers, discriminant function analysis (dfa) performed. this analysis will allow an identification of whether the differences between the rivers are significant and define the discriminant functions that account for these differences (bookstein, 1991; mustafic et al., 2008). the resultant discriminant function was used to calculate the percentage of correctly classified (pcc) fish. a cross-validation using the leaving one-out procedure was done to estimate the expected actual error rates of the classification functions. principal component analysis (pca) and canonical variates analysis (cva) were employed to discriminate these fishes from studied rivers. statistical analyses for morphometric data were performed using the spss version 16 software package, past ver. 1.36, morphoj and excel (microsoft office 2010). results descriptive data were examined for the mean and standard deviation (sd) of length and weight in the sampled specimens (table 2). although it is well known that the female and male specimens of the gobies and loaches have some morphological differences (mousavi-sabet, 2012; mousavi-sabet et al., 2012, 2015; vasil′eva et al., 2015), but the interaction between morphometric measurements used in this study by truss network system and sexes were not significant (p>0.05) demonstrating a negligible effect of sex on observed variations; hence, the data for both sexes were pooled for all subsequent analyses. there was no significant correlation between any of the transformed measured morphometric variables and standard length (p>0.05) indicating that size or allometric signature on the basic morphological data was accounted. statistically significant differences in goby specimens were observed in 79 morphometric characters out of 105 studied. of these 79 characters, 72 characters were found to be highly significant (p≤0.01) and used further for multivariate analysis. also, statistically significant differences for loach specimens were observed in 54 morphometric characters out of 78 studied which of these 54 characters, 41 characters were found to be highly significant (p≤0.01). those morphometric characters which showed highly significant variations (p≤0.01) table 2. descriptive data [mean ±sd standard length (mm) and body weight (g)] of goby and loach specimens collected from the studied rivers of the southern caspian sea basin. genus locality n standard length body weight min–max mean ± sd min–max mean ± sd tonekabon 32 35.71-112.87 70.55±21.85 1.15-34.90 10.25±9.30 ponticola babol 55 53.33-139.92 74.23±23.64 3.77-51.61 12.86±11.85 guisum 41 20.80-111.95 55.30±20.80 1.00-33.05 5.81±8.11 sefid 88 44.54-72.89 55.63±6.84 2.18-8.16 3.99±1.40 talar 39 22.66-99.15 55.41 ± 13.59 0.3-6.1 1.6±0.5 cobitis siah 40 22.47-98.80 53.61 ± 11.74 0.4-5.9 1.5±0.4 babol 49 17.65-95.73 50.01 ± 16.26 0.3-5.6 1.5±0.5 49 int. j. aquat. biol. (2019) 7(1): 45-55 were used to achieve the recommended ratio of the number of organisms measured (n) to the parameters included (p) in the analysis of at least 3-3.5 (bookstein, 1991), to obtain a stable outcome from multivariate analysis. in this study n: p ratio for goby and loach specimens were 3.00 (216/72) and 3.12 (128/41), respectively, that revealed samples size were adequate. the value of kaiser-meier-olkin coefficient (kmo) for overall matrix are 0.716 and 0.630 for goby and loach specimens, respectively and the bartlett’s test of sphericity is significant (p≤0.01). the results of kmo and bartlett’s suggest that the sampled data is appropriate to proceed with a factor analysis procedure. in order to determine which morphometric measurement affected populations differentiates mostly, the contributions of variables to principal components (pc) were examined. the pca of 72 morphometric measurements for goby specimens extracted 12 factors with eigenvalues >1, explaining 93.91% of the variance (table 3). of these, the first explained 28.37% and the second 16.35% of the variance. the most significant weightings on pc1 were from 1-5, 1-6, 5-15, 2-6, 2-5, 6-15, 6-9, 4-6, 4-5, 6-13, 6-14, 5-13, 1-14, 5-14, 1-4, 1-13, 1-11, 1-12, 12, 5-12, 1-10, 1-8, 1-3, 1-7, 1-9 and on pc2 were from 1-12, 12-15, 9-12, 12-13, 12-14, 3-12, 2-12, 10-12, 512, 4-12 and 3-14. the pca of 41 morphometric measurements for loach specimens extracted 9 factors with eigenvalues >1, explaining 89.65% of the variance (table 3). of these, the first explained 31.17%, the second 18.02% and the most significant weightings on pc1 were from 1-2, 1-4, 2-4, 4-11, 413 and on pc2 were from 1-10, 2-10, 3-10, 4-10, 1011, 10-12 and 10-13. canonical variates analysis confirmed the significant difference among the goby and loach specimens. the scores of the two canonical variables for each river revealed that goby specimens grouped into four distinct areas while there was a relativity low degree of overlap between tonekabon and babol populations and for loaches revealed that specimens grouped into three distinct groups while there was a relativity high degree of overlap among these populations (fig. 3). the wilks’ lambda tests indicated differences between fish when their morphometric measurements were compared by means of discriminant analysis. in table 3. eigen values, percentage of variance and percentage of cumulative variance of morphometric measurements for goby and loach specimens collected from the studied rivers in the southern caspian sea basin. genus factor eigenvalues percentage of variance percentage of cumulative variance 1 20.141 28.367 28.367 2 11.606 16.347 44.714 3 8.061 11.354 56.068 4 6.906 9.727 65.795 5 4.550 6.408 72.203 ponticola 6 4.242 5.974 78.178 7 2.754 3.879 82.056 8 2.447 3.447 85.503 9 2.097 2.953 88.456 10 1.478 2.082 90.538 11 1.350 1.901 92.439 12 1.041 1.466 93.905 1 12.780 31.170 31.170 2 7.389 18.021 49.191 3 5.715 13.938 63.129 4 2.930 7.146 70.275 cobitis 5 2.372 5.785 76.061 6 1.929 4.705 80.766 7 1.335 3.256 84.022 8 1.189 2.899 86.921 9 1.120 2.732 89.653 50 heidari et al./ how the body shape changes by the habitat hydrological factors? this test, all functions were highly significant (p≤0.01). for the discriminant analysis, the average of pcc for gobies and loaches morphometric characters were 95.7% and 80.5%, respectively, indicating a correct classification of specimens into their original populations. in this analysis, there was a high degree of separation in goby and a slight degree of separation in loach specimens in different rivers. the crossvalidation testing procedure was exactly the same as the pcc results (table 4). the histogram of discriminant functions for pairwise groups in goby specimens is shown in figure 4. body shape differences shows a longer snout, shallow body and head, and elongated body for sefid and babol groups versus relatively short snout, and deeper body and head for tonekabon and guisum table 4. percentage of specimens classified in each group and after cross validation for morphometric data of gobies and loaches specimens collected from the studied rivers along the southern caspian sea basin. genus rivers tonekabon babol guisum sefid total tonekabon 100.0 0.0 0.0 0.0 100.0 orginal babol 0.0 97.6 2.4 0.0 100.0 guisum 0.0 2.7 97.3 0.0 100.0 ponticola sefid 0.0 2.0 6.1 91.8 100.0 tonekabon 81.8 18.2 0.0 0.0 100.0 cross-validation babol 4.9 89.2 4.9 7.3 100.0 guisum 0.0 10.8 83.8 5.4 100.0 sefid 0.0 6.1 14.3 79.6 100.0 rivers babol siah talar total babol 79.6 14.3 6.1 100.0 orginal siah 7.5 75.0 17.5 100.0 cobitis talar 5.1 7.7 87.2 100.0 babol 75.5 16.3 8.2 100.0 cross-validation siah 7.5 75.0 17.5 100.0 talar 7.7 7.7 84.6 100.0 figure 3. scatterplot of standardized canonical variates functions 1 (cv1) and 2 (cv2) for morphometric characteristics of goby specimens (a) and loach specimens (b) collected from the studied rivers along the southern caspian sea basin. axis units for deformation grids are also shown. 51 int. j. aquat. biol. (2019) 7(1): 45-55 populations. also, the histogram of discriminant functions for pairwise groups in loach specimens shows a longer snout, shallow body and head, and elongated body for talar and babol populations versus relatively short snout, and high body and head depths siah river group (fig. 5). also, the ventral and dorsal shape variation analysis showed a longer snout and bigger disc for sefid and babol populations versus relatively short snout and smaller disc for tonekabon and guisum groups (fig. 6). discussions the results based indicated significant morphological differences in the studied benthic fishes between six different rivers system. which for gobies and loaches 95.7% and 80.5%, respectively, of individuals were classified correctly in each river. this study found the presence of the body shape variation of the benthic fishes at a macro spatial scale between different riverine systems along the southern caspian sea basin. the results also revealed no significant phenotypic variation between male and female specimens. the results showed that 79 out of 105 transformed morphometric measurements and 54 out of 78 transformed morphometric measurements were significantly different in these groups of goby and loach specimens living in the southern caspian sea basin, showing a high phenotypic variation among them. also, ddifferentiation between samples from adjacent rivers, such as those from tonekabon, babol, siah and talar and those from sefid and guisum may figure 4. histogram of discriminate analysis (da) functions for pair wise competitions’ of gobies (left). shape differences on the extremities of each river (right). 52 heidari et al./ how the body shape changes by the habitat hydrological factors? be due to the geographic isolation allowing morphological differentiation to proceed independently in each river. the tonkabon, siah and guisum are short rivers considering as cold rivers with high slope, whereas babol, talar and sefid are long and warm rivers with low slope. it is well-known that morphological characteristics can show high plasticity in response to differences in environmental conditions (mousavi-sabet and anvarifar, 2013; heidari et al., 2013, 2014). the pca showed a morphological segregation of the studied fishes based on the characters, i.e. head shape, pre-dorsal, pre-anal distances, pre-orbital and post-orbital distances, body length, body depth, caudal peduncle depth, caudal peduncle length, dorsal and anal fins origin and caudal fin origin positions for gobies and head length, pre-orbital and post-orbital distances, body length and caudal peduncle length for loaches. body shape differences for these benthic fishes shows a longer snout, shallow body and head depths, and elongated body for the sefid, talar and babol populations versus relatively short snout, and deep body and head for tonekabon, siah and guisum groups. these fishes tend to have a lower caudal peduncle depth in environments with faster water current in the sefid, talar and babol rivers. similar trends have been observed in other fish species inhabiting stream environments (mclaughlin and grant, 1994; pakkasmaa and piironen, 2000; paknejad et al., 2014). empirical study has indicated that the cost of deviating from the optimal current velocity for swimming increased markedly in such high-drag fishes with a bottom-dwelling body shape (pakkasmaa and piironen, 2000). gobies are typical benthic fishes in that they hold their large disc out ventrally when in a current and use them to hydrodynamically press onto the substratum (vasil'yeva and vasil'yev, 1995). for such benthic fishes, body shape with a lower caudal peduncle depth would be expected to reduce the influence of shear stress related to positioning energetics even under high water velocity (mclaughlin and grant, 1994; pakkasmaa and piironen, 2000). some empirical studies have indicated that the deterioration of body condition could influence the fitness-related attributes of individuals, such as fecundity, competitive ability and predator avoidance (e.g. hoey and mccormick, 2004). the most prominent differences in separate morphometric features were observed for four characters. the snout length, body and head depths and body length. snout and body lengths were smaller in short rivers as compared to longer rivers specimens and body and head depths were longer in short rivers specimens. in addition, these benthic fish have longer disc in rivers with faster water currents, including sefid, talar and babol in compare with tonekabon, siah and guisum rivers with slower water velocity. the influences of environmental parameters on morphometric characters are considered by several authors in the course of fish population segregation already (e.g. tura, 2004; yamamoto et al., 2006; pollar et al., 2007; eschmeyer and fong, 2011; figure 5. histogram of discriminate analysis (da) functions for pair wise competitions of loaches (left). shape differences on the extremities of each river (right). 53 int. j. aquat. biol. (2019) 7(1): 45-55 vatandoust et al., 2014a). different rivers in the same basin have various conditions that can change the feeding habits and food items, growth pattern and reproductive strategy of individual species (mousavisabet and anvarifar, 2013; kohestan-eskandari et al., 2014; vatandoust et al., 2014b, 2015; mousavi-sabet et al., 2016). the phenotypic plasticity of fish is very high and quickly adapt themselves by their physiology and behavior to environmental changes. these modifications ultimately change their morphology (turan et al., 2004). along the southern caspian sea basin there are probably very small environmental changes from place to place. therefore, each river system can possess a unique environment that differs from other rivers of along the southern caspian sea basin. the present study provides basic information about the body shape variation of these fishes in relation to different environmental condition in the southern caspian sea basin rivers. to accurately delineate units for conservation, multiple characters, such as ecological and genetic features, must be examined and the processes that influence those characters must be understood (yamamoto et al., 2006; eschmeyer and fong, 2011). therefore, to investigation of morphological variation between rivers of along the southern caspian sea basin, further exploration is necessary using dna techniques under different environmental conditions. references bektas y., belduz a.o. 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(2016) 4(1): 11-16 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article effects of pediococcus pentosaceus as a probiotic on intestinal microbiota and body composition of siberian sturgeon, acipenser baerii brandt, 1869 fateme moslehi1, masoud sattari*1, alireza shenavar masouleh2 1department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, iran. 2department of aquatic animal health, international sturgeon research institute, rasht, iran. article history: received 19 october 2015 accepted 14 december 2015 available online 2 5 february 2016 keywords: sturgeon probiotic intestine microflora body composition abstract: an eight-week experiment was carried out to determine the effects of dietary pediococcus pentosaceus as probiotic on the body composition and gut microbiota of siberian sturgeon, acipenser baerii. a total of 180 fish with mean body weight of 143±0.01g were randomly distributed into 12 200l fiberglass tanks as four treatments with three replicates, including groups fed with diet containing 2×107, 2×108 and 2×109 cfu g-1 of p. pentosaceus and probiotic-free as control group. body composition of treatment groups was not influenced by p. pentosaceus except for fat and moisture. the bacteria had a significant colonization in the intestine of fish fed with supplemented diet with p. pentosaceus. high level of acid lactic bacterial load was found in the treatment fed with highest amount of the probiotic i.e. 2×109 cfu g-1. the results showed that application of p. pentosaceus has positive effect on the body composition and intestinal microflora of the a. baerii. introduction probiotics are commonly defined as ‘live microbial feed supplements that beneficially affect the host animal by improving its intestinal microbial balance’ (fuller, 1989) and play a beneficial role on the health of the host if used in adequate amount (fao/who, 2002). the application of the probiotics in aquaculture is based on the concept that the balance of intestinal microorganisms in healthy animals increases resistance to diseases, and efficient digestion and maximum absorption of nutrients (fuller, 1992; verschuere, 2000). probiotics can also improve the immune response system, reduce mortality, increase growth parameters, improve water quality, enhance stress resistance, and increase reproduction efficiency (bairagi et al., 2002; wang and xu, 2004; gomez-gil et al., 2000; martınez cruz et al., 2012). the selection of suitable strain of a microorganism is a primary requirement for the use of probiotics. different types of microalgae, yeasts, gram-positive and gram-negative bacteria are * corresponding author: masoud sattari doi: http://dx.doi.org/10.7508/ijab.2016.01.002 e-mail address: msattari@guilan.ac.ir considered as probiotics. several bacterial strains which are common member of the non-pathogenic microflora are capable of inhabiting fish pathogenic bacteria in in-vitro assay (joborn et al., 1997; austin and zhang, 2006; gibson et al., 1998). this has been demonstrated for lactic acid bacteria (joborn et al., 1997), vibrio sp. (austin and zhang, 2006), and bacillus sp. (gibson et al., 1998) etc. pediococcus pentosaceus is a gram-positive, facultative anaerobic, non-motile, and non-spore forming bacterium. it is a member of the industrially important lactic acid bacteria that grows on lactobacilli mrs broth at 37°c. ferguson et al. (2010) stated that dietary supplementation of pediococcus acidilactici increases the intestinal microbial load of red tilapia, oreochromis niloticus. merrifield et al. (2011) also reported that this bacterium has a significant effect on intestinal microflora of rainbow trout, oncorhynchus mykiss. they reported that this probiotic has a proper colonization in the gut of treated fish species. acipenseridae are the most commercially 12 moslehi et al./ effects of p. pentosaceus on intestinal microbiota and body composition a. baerii important fish species in the caspian sea basin. they are endangered due to loss of habitat, overfishing and deterioration of water quality (bahmani et al., 2001). among sturgeon fishes, the siberian sturgeon, acipenser baerii, is a proper candidate for aquaculture because it can easily be adapted to hand feeding in captive condition. hence, in the present study, the effect of p. pentosaceus was examined as a probiotic on the body composition and intestinal microflora of the siberian sturgeon, a. baerii. materials and methods probiotic: pediococcus pentosaceus was isolated from the intestine of persian sturgeon, a. persicus identifying by 16s rrna gene based on merrifield et al. (2009). the probiotic powder was prepared in guilan science and technology park (rasht, guilan province, iran) with 1012 bacterial count. experimental diet: a commercial diet composed of 36% crude protein, 14% lipid, 1% phosphorus, 11% moisture, 10% ash and 4% fiber was used as a control diet (chineh commercial feed), and fish were acclimated to this diet for two weeks before beginning the experiment. the experimental diet was supplemented with p. pentosaceus. it was prepared by slowly spraying the mixture of 500 ml saline serum with 0.2, 2 and 20 g probiotic powder on 10 kg diet with 2×107, 2×108 and 2×109 cfu g-1 bacterial count, respectively, according to merrifield et al. (2009). experimental design: the specimens of the siberian sturgeons were obtained from beheshti sturgeon reproduction and restoration center (rasht, guilan province, iran). a total of 180 fish having average body weight of 143±0.01 g were randomly distributed into 12 fiberglass tanks (with 2000 l capacity) each containing15 fish. four treatments each with 3 replicates, including ta, tb, tc and c containing 2×107, 2×108, 2×109 cfu g-1 probiotic and probiotic free diet, respectively, were designed for this study. the treatments were fed on supplemented diet for 8 weeks at the rate of 2-2.5% of body weight 3 times a day (08:00, 14:00 and 20:00). the unconsumed feed was siphoned out six hrs after feeding. about 75% of water from each tank was changed daily with minimum disturbances to the fish. intestinal microbiology: at the end of experiment, after 24 hrs starvation, three fish from each tank were anesthetized by 200 ppm clove powder solution. the intestine of fish was removed in a sterile condition then washed thoroughly three times with saline serum (merrifield et al., 2009). samples were diluted with saline serum and then the specimens were added to tsa (tryptic soy agar) culture medium to count total number of intestinal bacteria (rawling et al., 2009) and mrs (man rogosa sharpe) culture medium to count slightly lactic acid bacteria (merrifield et al., 2009). chemical analysis: at the end of experiment, three fish per tank were taken to analyze carcass composition according to protocols of aoac (1990) for measuring moisture, ash, lipid and protein. amount of moisture was determined by drying the fish muscle in oven at 103-105°c for 18-24 hrs. thereafter, the dried samples were used to determine ash content by combusting them in a furnace. amount of lipid was estimated by ether extraction in soxhlet system. micro-kjeldahl system was used to determine the nitrogen content followed by multiplying the amount of nitrogen by 6.25. statistical analysis: data analysis were performed using spss program as installed by the university of guilan. differences between treatments were performed using one way anova and duncan tests. the results were considered significant at the 95% (p<0.05) level. results the results showed that p. pentosaceus has affected on fat and moisture of fish fed with probioticsupplemented diets. the highest level of moisture was found in the control group and a significant differences were found between probiotic-fed and control fish (p<0.05). the maximum level of fat was found in ta (fish fed with 2×107 cfu g-1 probiotic in the diet) and a significant difference was found between ta and the other groups in term of fat level. 13 int. j. aquat. biol. (2016) 4(1): 11-16 the lowest level of fat was observed in control group. there were no significant differences between probiotic-fed fish and control group in terms of protein and ash (table 1). tsa (tryptic soy agar) culture medium was used to calculate the total viable count (tvc). the results showed that tvc ranged from log 6.37 cfu g-1 in ta (fed with 2×107 cfu g-1 of the probiotic) to log 6.15 cfu g-1 in c (control group). however, no significant differences were found between treatments (fig. 1). the results of mrs (man rogosa sharpe) culture medium showed that the amounts of p. pentosaceus ranged from log 4.46 cfu g-1 in ta (fed with 2×107 cfu g-1 of the probiotic) to log 5.65 cfu g-1 in tc (fed with 2×109 cfu g-1 ) and the differences between the probiotic-fed fish were significant (p<0.05) (fig. 2). discussion in the present study, the isolated p. pentosaceus from the intestine of persian sturgeon was used as a probiotic in the diet of siberian sturgeon. based on the results, the supplementation of p. pentosaceus as probiotic affected the fat and moisture of body composition but it had no significant effect on protein and ash of treated fish. merrifield et al. (2010) used chlorogloeopsis cyanobacter as probiotic on the diet of o. niloticus and reported no significant effect on the body composition viz. crude protein, crude lipid and ash of the treated fish. hoseinifar et al. (2011) and merrifield et al. (2011) found similar results in h. huso and o. mykiss, by using saccharomyces cerevisiae and p. acidilactici, respectively, as probiotics. in contrast to our findings, bagheri et al. (2008) showed that the diet containing commercial bacillus probiotic has a significant effect on body composition of o. mykiss, but they did not observe significant difference between the groups consumed the lowest level of probiotic and control group. bagheri et al. (2008) also observed significant parameter treatment moisture (%) ash (%) fat (%) protein (%) c a76.383±0.12 a1.063±0.006 b6.53±0.33 a18.03±0.05 ta b74.52±0.28 a1.133±0.006 a8.59±0.26 a16.14±0.05 tb b75.073±0.12 a1.163±0.09 b7.28±0.21 a17.6±0.03 tc b74.99±0.25 a1.136±0.008 b7.01±0.31 a17.82±0.05 different superscript letters denote significant (p<0.05) difference. table 1. siberian sturgeon’s body composition at the end of the experiment. figure 1. effect of p. pentosaceus on siberian sturgeon intestinal total viable count of bacteria (log). figure 2. effect of p. pentosaceus on the number of siberian sturgeon intestinal p. pentosaceus bacterium (log). 14 moslehi et al./ effects of p. pentosaceus on intestinal microbiota and body composition a. baerii differences in carcass moisture and lipid content between the groups fed with the probiotic. el rhman et al. (2009) also showed that the body composition in o. niloticus, were significantly affected by the pseudomonas and micrococcus lateus as a probiotic, except for moisture. the difference between above mentioned findings and our results may refers to different bacteria and host. the results of the present study showed no significant differences in tvc among all treatments, but the highest number of p. pentosaceus was observed in group fed with 2×109 cfu g-1 probiotic. this means that probiotic has been colonized in the intestine of treated fish. an important action of probiotic is competitive exclusion by competition for adhesion sites, nutrient, oxygen, and by-product inhibitory compounds (irianto and austin, 2002). intestinal microbial flora change after application of probiotic and then pathogen bacteria can decrease in host animal. in addition, probiotic can reduce the pathogen by secretion of bacteriocins and then reduce intestinal ph (irianto and austin, 2002). similar to our results, ferguson et al. (2010) reported that the number of lactic acid bacteria (lab) is significantly higher in the intestine of the probiotic-fed o. niloticus. however there was no significant difference between the population of heterotrophic anaerobic and aerobic bacteria (ferguson et al., 2010). merrifield et al. (2011) stated that o. mykiss had a successful colonization of p. acidlactici in its digestive tract and the tvc of mucosal bacteria was significantly higher in the probiotic-fed fish. in another study, the tvc of heterotrophic bacteria in huso huso were not affected by saccharomyces cerevisia, but the levels of autochthonous lactic acid bacteria significantly increased in the fish fed with 2% yeast (hoseinifar et al., 2011). the effects of lactobasillus curvatus and leuconostoc mesenteroides on the gut microflora of h. huso and a. persicus were examined by askarian et al. (2011). they reported that the a. persicus fed with l. mesenteoides had higher levels of lab than fish fed with l. curvatus and lab mixture, but the highest level of lab in h. huso was observed when the fish were fed with l. curvatus. they reported that the levels of lab in h. huso and a. persicus fed with lab mixture, were similar to control group. as conclusion, the findings of this study showed that application of p. pentosaceus as a probiotic has positive effect on the body composition and intestinal microflora of the siberian sturgeon. references abd el-rhman a.m., khattab y.a.e., shalaby a.m.e. (2009). micrococcus luteus and pseudomonas species as probiotics for promoting the growth performance and health of nile tilapia, oreochromis niloticus. fish and shellfish immunology, 27: 175-180. aoac (1990). official methods of analysis of association of official analytical chemists (aoac), 15th ed, washington, usa. askarian f., kousha a., salma w., ringo e. 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(2002). enzyme producing bacterial flora isolated from fish digestive tracts. aquaculture international, 10: 109121. fao/who.2002. guidelines for the evaluation of probiotics in food. london ontario canada. 11 p. ferguson r.m.w., merrifield1 d.l., harper g.m., rawling m.d., mustafa1 s., picchietti s., balcazar j. l., davies s.j. (2010). the effect of pediococcus 15 int. j. aquat. biol. (2016) 4(1): 11-16 acidilactici on the gut microbiota and immune status of on-growing red tilapia (oreochromis niloticus). journal of applied microbiology 109(3): 51-62. fuller r. (1989). a review, probiotics in man and animals. journal of application in bacteriology, 66: 365-378. fuller r. 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(1997). colonization in the fish intestinal tract and production of inhibitory substances in intestinal mucus and faecal extract by carnobacterium sp. strain k. journal of fish diseases, 20: 383-392. martínez cruz p., ibáñez a.l., monroy hermosillo o.a., ramírez saad h.c. (2012). use of probiotics in aquaculture. isrn microbiology, id 916845: 1-13. merrifield d.l., dimitroglou a., bradley g., baker r.t.m., davies s.j. (2009). probiotic applications for rainbow trout (oncorhynchus mykiss walbaum) i. effects on growth performance, feed utilization, intestinal microbiota and related health criteria. aquaculture nutrition, 10: 1365-2095. merrifield d.l., güroy d., güroy b., emery m.j., llewellyn c.a., skill s., davies c.j. (2010). assessment of chlorogloeopsis as a novel microbial dietary supplement for red tilapia (oreochromis niloticus). aquaculture, 299: 128-133. merrifield d.l., bradley g., harper g.m., baker r.t.m., munn c.b., davies s.j. (2011). assessment of the effects of vegetative and lyophilized pediococcuc acidlactici on growth, feed utilization, intestinal colonization and health parameters of rainbow trout (oncorhynchus mykiss walbaum). aquaculture nutrition, 17: 73-79 verschuere l., rombaut g., verstraete w. (2000). probiotic bacteria as biological agents in aquaculture. microbiology and molecular biology, 64(4): 655-671. wang y.b., xu z.r. (2004). probiotic treatment as method of biocontrol in aquaculture. feed research, 12: 42-45. int. j. aquat. biol. (2016) 4(1): 11-16 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی و ترکیب بدن تاسماهی سیبری، روده عنوان پروبیوتیک بر فلور میکروبیبه pediococcus pentosaceusاثرات acipenser baerii brandt, 1869 2ماسوله ، علیرضا شناور1*، مسعود ستاری1فاطمه مصلحی سرا، ایران.صومعه گیالن، طبیعی، دانشگاه منابع دانشکده، شیالت گروه1 رشت، ایران.، المللی تحقیقات ماهیان خاویاریبهداشت آبزیان، انستیتو بین گروه2 چکیده: میکروبی فلوربر ترکیب بدن و عنوان پروبیوتیکبه pediococcus pentosaceusای ای به منظور تعیین اثرات تغذیهیک آزمایش هشت هفته یتری ل 200تانک فایبرگالس 12صورت تصادفی بین گرم به 143±01/0قطعه ماهی با وزن متوسط 180اجرا گردید. تعداد روده تاسماهی سیبری p. pentosaceusباکتری 910x2 1-cfu gو 710x2 ،810x2حاوی هایهای تغذیه شده با خوراکوهرشامل گ عنوان چهار تیمار با سه تکراربه ه باکتریوسیلرطوبت بههای تیمار به جز چربی و ترکیب بدن گروهبراساس نتایج .تقسیم شدندعنوان تیمار شاهد هو خوراک بدون پروبیوتیک ب p. pentosaceus داری در روده ماهیان تغذیه شده با خوراک حاوی پروبیوتیک شد. سطح سازی معنیرار نگرفت. باکتری سبب کلنیقتحت تاثیر یافت شد. نتایج نشان 910x2 1-cfu gتیمار عبارت دیگر ار پروبیوتیک بهدهای اسیدالکتیک در تیمار تغذیه شده با بیشترین مقباکتری باالی لود میکروفلور روده تاسماهی سیبری دارد. ومثبتی بر ترکیب بدن اتاثر p. pentosaceusباکتری داد که کاربرد .میکروفلور روده، ترکیب بدن ،خاویاری، پروبیوتیکماهی :کلمات کلیدی int. j. aquat. biol. (2017) 5(6): 375-386 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article implications of white spot syndrome virus disease on dna integrity, histology and biochemistry of procambarus clarkii in egypt haidan m. el-shorbagy*1, salwa abdelhamid hamdi department of zoology, faculty of science, cairo university, 12613, egypt. article history: received 11 september 2017 accepted 20 october 2017 available online 2 5 december 2017 keywords: comet assay dna damage white spot virus oxidative stress abstract: white spot syndrome virus (wssv) is a widespread and highly pathogenic virus; that infects shrimp, crayfish and other crustaceans. the objectives of the present study were to investigate wssv implications on some crayfish tissues, within light and sever stages of infections. several parameters have been investigated including dna integrity, oxidative stress, and histological changes in gills, muscles and hepatopancreas cells, using several techniques such as comet assay, dna fragmentation assay, oxidative stress biomarkers estimation and histopathological examination. specimens were divided into three groups according to the nested pcr results. group i included healthy specimens whose tissues were all negative two-step pcr; group ii involved lightly infected specimens with positive two-step pcr. group iii included heavily infected specimens whose tissues were tested mostly positive one-step pcr. wssv generates an increase in the different parameters of dna damage (p<0.05) with abnormal histological features and notable reduction (p<0.05) of the endogenous scavengers in the tested tissues of the infected crayfish in comparison with the normal healthy ones. furthermore, gills were found to be the most affected organ followed by muscle and finally hepatopancreas. these outcomes additionally demonstrated that comet test could profitably be utilized in genotoxicity evaluation protocols in aquatic invertebrates. introduction procambarus clarkii, the red swamp crayfish, is considered to be one of the largest invasive species in the egyptian aquatic environment that entered fresh water at the beginning of 1980'th (hamdi, 2011). crayfish exists in hardy warm freshwater that is found in reservoirs, marshes, rice fields, rivers and slow flowing water. it may become a keystone species, affecting many components of the ecosystem inhabits and altering the nature of native plant and animal communities. arthropods lack the highly advanced adaptive immune system of vertebrates. instead, they only depend on their innate immune system to fight the invading microorganisms (hoffmann et al., 1999). thus, their capability to live are at risk nowadays due to the infection with white spot syndrome virus (wssv). it is known to date that wssv can infect >90 species of deca-pod and non-decapod crustaceans *corresponding author: haidan mostafa el-shorbagy doi: https://doi.org/10.22034/ijab.v5i6.378 e-mail address: haidan@sci.cu.edu.eg from natural aquatic environments (sánchez‐martínez et al., 2007). for the past 16 yr, wssv has been identified as a severe and devastating pathogen of the farmed shrimp worldwide (ramos-carreño et al., 2014), as it is implicated in a serious economic impact on the shrimp aquaculture industry due to 80 to 100% mortality within 3 to 10 days post-infection (sablok et al., 2012). moreover, many reports indicated that wssv is capable of significant morbidity and mortality in crayfish as well (edgerton, 2004; jiravanichpaisal et al., 2004). this resulted in significant economic losses in many crayfishproducing farms. interestingly, researches in other crustacean species have demonstrated that many infected animals do not die, instead, they may act as reservoirs for the pathogen in ecosystems (sánchez‐ martínez et al., 2007), as wssv-infected crayfish caused severe disease in a feeder population when being used in the diets of zoo residents maintained at the national zoological park in washington, d.c., 376 el-shorbagy and hamdi / dna damage and ros in wssv-infected crayfish usa (richman et al., 1997). wssv is a double-stranded dna virus with a very steady genome, assigned to the new genus, whispovirus, in the family nimaviridae (vlak et al., 2005). wssv is 110–130×260–350 nm, trilaminar enveloped, and rod-shaped (van hulten et al., 2001). many symptoms can be distinguished after wssv infection, such as white spots in the exoskeleton and epidermis, loose cuticle, rapid reduction in food consumption, enlargement and yellowing of the hepatopancreas (hameed et al., 2001); swelling of bronchioles as a result of accumulation of fluid; thinning and delayed clotting of haemolymph (chakraborty and ghosh, 2014). in addition, tropism analysis of tissue from both wild-captured brooders and experimentally infected shrimp, showed that cuticular epithelium and subcuticular connective tissues, antennal gland and haematopoietic organ were the core object tissues for wssv. while samples from the gills, pleopods, abdominal muscle, haemolymph, still need further diagnostic testing (lo et al., 1997). although host-virus interaction has been one of the recent attentions in understanding wssv pathogenesis (rodríguez et al., 2012; leu et al., 2013), to our knowledge, there are no previous histopathological or molecular studies concerning wssv effects on different p. clarkii tissues. under these circumstances, an investigation of dna damage, biochemical and the pathological changes in wssv-infected crayfish are subjects worth studying to shed more lights on the most target organs for viral replication, host mortality, the potential anti-wssv applications and the viral risk assessments on the aquatic environment, which help controlling their prevalence. materials and methods chemicals: all chemicals used were of analytical grade and purchased from sigma-aldrich (st. louis, mo, usa). kits for all biochemical parameters were purchased from bio-diagnostic company (giza, egypt). other molecular kits are listed elsewhere. animals: fifty adult female crayfish, p. clarkii, approximately 20 g and 8 cm each, were captured from their natural environment al-ayat aquacurium, giza, egypt and maintained in the laboratory as previously described (zou et al., 2003). animals were collected according to the clinical signs to be: 20 wssv-free (no white spots) and considered as negative control and 30 wssv-infected crayfish (with white spots). the prevalence of wssv was further tested by wssv diagnostic two-step pcr using a piece of the walking legs as a pcr template source to distinguish healthy animals from infected one. all samples were put in crushed ice in insulated containers and brought to the laboratory for preservation prior to analysis. the exoskeleton was removed, and three parts excised from crayfish specimens (gills, muscles and hepatopancreas) were divided into three parts each. one part was homogenized in cold hanks buffer then stored at -20°c for comet analysis. the second part was used to extract dna and subjected to pcr assays to reconfirm wssv prevalence in the tested tissues and to carry out dna fragmentation and biochemical tests, while the third part was fixed in 10% formalin for further processing for histopathological examination. nested pcr analysis for wssv: tissues under test were used for dna extraction using dna mini-prep kit (qiagen, sigma) and dna quality was tested using decapod gene 143f: tgccttatcagctntcgatt gtag, 145r: ttcagntttgcaaccatacttcc c (848 bp) (oie, 2003). diagnosis of different wssv-infected stages was performed by nested pcr as described previously (lo et al.,1996) using outer nested primers 146f1: actactaacttcagccta tctag, 146r1: taatgcgggtgtaatgttctt acga (1447 bp), for the one-step pcr, and inner nested primers 146f2: gtaactgccccttccatc tcca, 146r2: tacggcagctgctgcaccttgt (941 bp) for the two-step pcr. positive and negative controls were also included to avoid any false positive pcr results. dna extracts from experimentally infected crayfish were used as a positive control where the wssv inoculum was prepared as previously described (wang et al., 2009), and was injected into the last abdominal segment of p. clarkii (2.6×107 virions per crayfish). one-step pcr was performed by one cycle of initially denaturation at 94°c for 4 min, 377 int. j. aquat. biol. (2017) 5(6): 375-386 55°c for 1 min, 72°c for 2 min, followed by 39 cycles at 94°c for 1 min, 55°c for 1 min and 72°c for 2 min and finally, final extension at 72°c for 5 min. then 10 µl of the pcr product from samples with negative results of one-step pcr were used as a template for the two-step pcr over the same 40 cycles described above. cycling was carried out using thermal cycler (ptc-100™ thermal cycler, watertown, ma, usa). the pcr products were separated by electrophoresis on 1% agarose gels, stained with 2% ethidium bromide and visualized by uv trans-illuminator (stratagene, usa). tested specimens were divided into three groups based on nested pcr results. group i comprised specimens whose tissues were all twostep pcr negative, group ii comprised lightly infected specimens (gp.ii) which had at least some tissues positive after re-amplification and group iii comprised heavily infected specimens (gp.iii) whose tissues tested one-step pcr positive. comet analysis: comet technique was carried out for 7 animals for each group, according to singh et al. (1998) with some modifications. cell suspension (10 µl), previously homogenized with hanks buffer was mixed with 75 µl of low melting agarose (0.5% in pbs). the mixture was spread on 1% agarose precoated slides. the slides were immersed in cold lysed buffer (2.5 m nacl, 100 mm edta, 10 mm tris, ph 10, to which 1% triton x-100 and 10% dmso were freshly added) for 24 hours at 4°c in dark. then they were placed in electrophoresis chamber containing the alkaline electrophoresis buffer for another 20 minutes. the electrophoresis was carried out for 20 minutes at 300 ma and 25 v. then neutralization was done using 0.4 m tris (ph7.5). the slides were stained with ethidium bromide 20 µg/ml. dna fragment migration patterns of 50 cells for each animal were examined using a fluorescent microscope (with excitation filter 420-490 nm (issue 510 nm) linked to a ccd camera. comet assay iv software (perceptive instruments, suffolk, uk), was used to assess tail moment, dna% in tail and tail length of dna damage, at 400x. dna-laddering assay: according to the standard protocol described by sriram et al. (2010), tested tissues were homogenized in cold lysis buffer containing 10 mm tris hcl, ph 7.4, 10 mm edta, and 0.2% tritonx-100. then incubated with 0.6 µg/ml proteinase k at 50°c overnight and consequently with rnase a to a final concentration 100 µg/ml at 37°c for one hour. extraction with phenol chloroform isoamyl was carried out, and dna in the aqueous phase was precipitated by 25 µl (5 m) sodium chloride and 100% isopropanol at -20°c. the concentration of the extracted dna was measured and 5 µg from each sample was detected by migration on a 1.5% ethidium bromide-treated agarose gel through electrophoresis at 80 volt (power supply biorad, hercules, ca, usa, model 200/2.0). finally, fragmented dna could be visualized under uv transillminator (stratagene, la jolla, ca, usa). oxidative stress markers: tissue samples of gills, muscle, and hepatopancreas were homogenized in a 10% (w/v) in cold tris hcl buffer 0.1 mm ph 7.4 at 4°c. the homogenates were centrifuged at 10000g for 15 min at 4°c and supernatants were immediately used for determining glutathione peroxidase (gpx), catalase (cat) and superoxide dismutase (sod) activities according to manufacturer’s instructions. glutathione peroxidase (gpx) activity: according to paglia and valentine (1967), gpx activity was measured indirectly upon the oxidation rate of nadph. the reaction depends on the reduction of peroxides in which oxidized glutathione (gssg) is produced and recycled to its reduced state by glutathione reductase. the oxidation of nadph to nadp+ is accompanied by a decrease in the absorbance at 340 nm (a340) providing a spectrophotometric means for monitoring gpx enzyme activity. the rate of the decrease in the a340 is directly proportional to the gpx activity in the sample. a blank without homogenate was used as a control for non-enzymatic oxidation of nadph upon addition of hydrogen peroxide in 0.1 m tris buffer, ph 8.0. enzyme activity were expressed as unit/g tissue. superoxide dismutase (sod) activity: sod activity was measured using nishikimi et al. (1972) method which is based on its ability to inhibit nitro blue tetrazolium (nbt) reduction by xanthine-xanthine oxidase after the addition of superoxide dismutase. 378 el-shorbagy and hamdi / dna damage and ros in wssv-infected crayfish sod activity was expressed as unit/g tissue. catalase (cat) activity: in aebi (1984) method , the catalase activity was measured by measuring the breakdown of h2o2 where cat reacts with a known quantity of h2o2 and stopped after exactly 1 min using cat inhibitor. in the presence of peroxidase, the remaining h2o2 reacts with 3, 5-dichloro-2hydroxybenzene sulfonic acid and 4aminophenazone forming a that can be measured spectrophotometrically at 510 nm. thus, the color intensity is inversely proportional to the amount of cat in the tested sample. results were expressed as u/min. histological examination: for pathological studies portions of gills, muscles and hepatopancreas tissues were fixed in neutral buffered formalin (10%) overnight, washed with tap water, dehydrated using grades of ethanol (70%, 90%, 95%, and 100%) and followed by clearing the samples in two changes of xylene. then impregnated with two changes of molten paraffin wax, embedded, and blocked out. samples were sliced to 3-4 μm thickness. the obtained tissue sections were collected on glass slides, dewaxed with xylene and rehydrated through a descending alcohol series and finally were stained with haematoxylin and eosin (h&e) (bancroft et al., 1996). the stained sections were evaluated using light microscopy (u-iii multi-point sensor system; nikon, tokyo, japan) and photographed using is_capture camera system. statistical analysis: the present data were analysed by statistical package for the social sciences (spss) version 15. two-ways anova was applied to test the effect of infection, type of tissue and their interaction on the studied parameters. duncan’s test of homogeneity was used to test the similarities between the studied groups. all the results were expressed as a mean ± standard error of mean (sem). results clinical signs: white spots or patches embedded in the exoskeleton and loose cuticle are the principle clinical signs of wssv-infected crayfish (fig. 1a, b). other signs may include whiteness of body and appendages. ssv nested pcr detection: samples with successful amplicons (1447 bp) from one-step pcr have been considered as gp.iii (heavily infected-crayfish), while those showed negative results have been subjected to two-step pcr, yielding two groups either contain positive amplicons that showed two bands (1447 and 941bp) which considered as gp.ii (lightly infectedcrayfish) or negative samples that considered to be (gp.i) healthy crayfish (fig. 2). comet assay: significant difference (p<0.05) in dna damage could be detected between lightly infected (gp.ii) and their control (gp.i). all the heavily infected tissues (gp.iii) (muscles (m), gills (g) and parameter tissue experimental group i ii iii tail length (µm) h 0.30±0.04cc 0.98±0.03bc 2.68±0.14ac m 0.32±0.03cb 1.97±0.05bb 3.06±0.21ab g 0.34±0.09ca 2.53±0.08ba 5.41±0.51aa %dna in tail h 1.16±0.13cc 1.98±0.21bc 2.51±0.11ac m 1.27±0.16cb 2.12±0.28bb 3.03±0.29ab g 1.61±0.13ca 2.98±0.15ba 3.65±0.29aa tail moment h 0.003±0.001cc 0.019±0.001bc 0.067±0.004ac m 0.004±0.001cb 0.063±0.001bb 0.094±0.014ab g 0.005 ± 0.002ca 0.089±0.001ba 0.198±0.023aa results are expressed as mean ±se. in each row, the mean values marked with the same superscript capital letters are similar (insignificant, p>0.05) whereas those with different ones are significantly differed (p<0.05). in each column, the mean values marked with the same superscript small letter are similar (insignificant, p>0.05) whereas those with different ones are significantly differed (p<0.05). p>0.05: insignificant effect; p<0.0001: significant effect at α=0.0001. table 1. dna damage represented as tail length, %dna in tail and tail moment in three tissues (hepatopancreas (h), muscles (m) and gills (g)) within control (gp.i), lightly infected (gp.ii) and heavily infected (gp.iii) crayfish. 379 int. j. aquat. biol. (2017) 5(6): 375-386 hepatopancreas (h)) showed statistically significant increase (p< 0.05) in all dna damage parameters (tail length, %dna in tail and tail moment) in comparison with their corresponding tissues in gp.i and gp.ii as shown in table 1. within gp.iii, gill showed the highest dna damage between all tested tissues while hepatopancreas revealed the lowest one at (p< 0.05) (fig. 3). dna–ladder assay: the present findings revealed slight smear in all tested tissues within gp.ii figure 1. external observations of procambarus clarkii (a) healthy p. clarkii, (b) wssvinfected p. clarkia showing white spots in the carapace. figure 2. representative photo showing pcr product of one-step pcr (1447bp) and two-step pcr (1447and 941bp), where (m): dna marker. lanes (1-2): gill (g), lanes (3-4) muscle (m), lanes (5-6) hepatopancreas (h) and lane (7): positive control. 380 el-shorbagy and hamdi / dna damage and ros in wssv-infected crayfish when compared to the negative control. wssv induced apoptotic dna fragmentation within gp.iii expressed as 200bp and 250bp laddering in gill tissue, while muscle and hepatopancreas show only one band at 200bp (fig. 4). histopathological examination: histopathological figure 3. representative photo of damaged dna in light wssv-infected procambarus clarkii (b), heavy wssv-infected p. clarkii (c) and compared with undamaged dna (a) (400x). figure 4. representative photo showing smeared and laddered dna laddering within lightly infected group (gp.ii) and heavily infected group (gp.iii) respectively, in the three tested tissues gill (g), muscles (m) and hepatopancreas (h) compared to the intact dna of the healthy control group (gp.i). parameter tissue experimental group effect of infection effect of tissue type effect of interaction i ii iii sod (u/g tissue) h 246.79±1.85ca 223.13±1.50ba 191.43±2.10aa f1,12=728.54, p<0.0001, f2,12=815.87, p<0.0001, f2,12=35.79, p<0.0001, --(-9.58%) p (-22.43%) p m 289.01±5.43cb 257.99±1.52bb 207.01±4.35ab --(-10.73%) p (-28.37%) p g 390.83±6.45cc 333.78±1.78bc 262.73±1.20ac --(-14.60%) p (-32.78%) p gpx (u/g tissue) h 1025.57±2.79ca 977.93±1.22ba 841.86±6.04aa f1,12=858.96, p<0.0001, f2,12=3028.61, p<0.0001, f2,12=139.93, p<0.0001, --(-4.65%) p (-17.91%) p m 1142.07±6.22cb 999.98±0.93bb 980.65±3.93ab --(-12.44%) p (-14.13%) g 1457.38±16.75cc 1233.23±1.59bc 1130.75±4.54ac --(-15.38%) p (-22.41%) p cat (u/min/g tissue) h 56.26±2.82cb 44.24±0.63bb 36.05±0.22ab f1,12=197.03, p<0.0001, f2,12=2895.32, p<0.0001, f2,12=41.28, p<0.0001, --(-21.38%) p (-35.93%) p m 32.14±0.89ca 26.47±0.33ba 23.83±0.49aa --(-17.62%) p (-25.85%) p g 118.67±1.64cc bc93.95±0.16 76.52±1.12ac --(-20.84%) p (-35.52%) p in each row, the mean values marked with the same superscript capital letters are similar (insignificant, p>0.05) whereas those with different ones are significantly differed (p<0.05). in each column, the mean values marked with the same superscript small letters are similar (insignificant, p>0.05) whereas those with different ones are significantly differed (p<0.05). p>0.05: insignificant effect; p<0.0001: significant effect at α=0.0001. p: percentage of change in comparison to the corresponding controls. 381 int. j. aquat. biol. (2017) 5(6): 375-386 figure 5. photomicrograph of crayfish muscles represents: (a) control muscles showing no histopathological changes. wssv-infected crayfish representing, (b) gp.ii showing intermuscular edema (indicated by arrow), (c) gp.iii showing: myolysis of focal myocytes, and (d) zenker’s necrosis of myocytes (indicated by arrows) (h&e). 382 el-shorbagy and hamdi / dna damage and ros in wssv-infected crayfish changes within control, gp.ii and gp.iii were evaluated. several abnormal architectures were observed in gill tissue within gp.ii and gp.iii showing necrosis and atrophy of gill lamellae respectively (fig. 5). alteration of muscle fibers including intermuscular edema within gp.ii, and myolysis of focal myocytes with zenker’s necrosis in gp.iii were clearly detected (fig. 6). moreover, necrotic cells of hepatopancreas tubules could be observed within gp.ii, while highly vacuolated cells and necrotic cells with inflammatory cell infiltration could be noticed only within gp.iii (fig. 7). oxidative stress markers: there was a significant reduction (p<0.05) in the levels of all measured endogenous enzymes sod, gpx1 and cat in gp ii and gp iii (table 2). the reduction of anti-oxidant enzymes was parallel to the severity of the infections. gills within gp.iii showed the most affected organ among all tested tissues for sod and gpx levels (32.78% and -22.41%) respectively, while the change in cat activity was equally higher in gills and hepatopancreas (-35.52% and -35.93%, respecttively) than in muscles tissues. discussion in the present study, wssv infected crayfishes have been randomly chosen according to the existence of clear white spots or patches embedded in their exoskeleton, and then tested to ensure wssv infection and classified using 1 or 2-step pcr assay. these clinical signs were previously reported in different crustacean species infected with wssv (lo et al., 1996; kasornchandra et al., 1998). the formation of white spot may be contribute to the accumulation of calcium salts within the cuticle as a result of the dysfunction of the integument results in the white spots formation(wang et al., 1999). figure 6. photomicrograph of crayfish hepatopancreas tubules represents: (a) control group showing apparent normal hepatopancreas tubules. wssv-infected crayfish representing, (b) gp.ii showing necrosis of cells of hepatopancreas tubules (indicated by arrow), (c) gp.iii showing: highly vacuolated cells and (d) inflammatory cells infiltration within necrotic cells (indicated by arrows) (h&e). figure 7. photomicrograph of crayfish hepatopancreas tubules represents: (a) control group showing no histopathological changes. wssv-infected crayfish representing, (b) gp.ii showing necrosis of gill lamellae (indicated by arrow) and (c) gp.iii showing: atrophy of gill lamellae (indicated by arrows) (h&e). 383 int. j. aquat. biol. (2017) 5(6): 375-386 white spot syndrome virus target the cells of ectodermal and mesodermal origin, including lymphoid organ, antennal gland, epidermis, gills, muscle and eye-stalk (kou et al., 1998). according to comet assay, the present data declare that wssv infection induced the highest dna damage in gill cells followed by muscle and finally hepatopancreas within both the gp.ii and gp.iii in comparison with the normal healthy group (gp.i). indeed, the percentage of dna in the tail (tail intensity) was found to be proportional to the frequency of dna strand breaks (olive et al., 1990). this is may be contributed to the increasing of the apoptotic rate in the late stage of infection due to the release of high viral copy numbers, that differ after post viral exposure from tissue to the other as indicated by tan et al. (2001) who revealed that viral copy numbers were below the detectable levels during the first 12 h post-exposure, and that viral dna was first detected 14 h post-exposure in gills and integument, while first detection was 22 h post-exposure in muscle tissue (tan et al., 2001). additionally, some reports investigated wssv replication in several infected specimens, and it was particularly prevalent in gills, followed by muscle and finally hepatopancreas, it is in these organs that the virus most frequently appears and replicates (kou et al., 1998, shi et al., 2005). these in contrary with sahtout et al. (2001) findings that revealed the highest number of apoptotic cells within gills then hepatopancreas and finally within muscle in black tiger shrimp, which may be due to the difference in crustaceans species (sahtout et al., 2001). however, double stranded viral dna can be distinguished from the host damaged dna using comet test and dna ladder assay by its heavy size (~300 kbp) which obligate viral dna to be close to the nucleus rather than moving by the electrophoretic current as fragmented dna in the damaged or lysed cells. anyway, the gp.ii revealed an extent of significant dna damage and smeared dna, this is may be contributed to the life-death struggle, in which virus intend to inhibit apoptosis in order to replicate in the early stage of infection, while the host defense system induce apoptosis to kill virus (leu et al., 2013). simultaneously, wssv in gp.iii showed signs of late stage apoptosis, as shown by two different laddered bands in the tested infected tissues indicating that the extend of apoptosis is proportional to the severity of the infection, these data are in a harmony with the study of sahtoot et al. (2001) on wssvinfected penaeus monodon who revealed that number of tunel positive cells present in gill, stomach, abdomen and muscle tissues increased with cumulative severity of infection, as determined by gross signs of white spots on the cuticle. however, this is a study of randomly captured crayfish, so it did not display whether the crayfish died rather due to wssv infection or survived for longer because of the reduction in the apoptotic level. as apoptosis is initiated through two major pathways: the extrinsic pathway and the intrinsic pathway (danial and korsmeyer, 2004), we can postulate that the intrinsic pathway is that main way of wssv induced apoptotic mechanism cause it initiated by signals arising within the cells such as survival factors depletion, dna damage, viral infection, and oxidative stress as indicated by the present study. recently, some studies have reported that the infection with wssv is accompanied with upregulation of zn-finger protein within crayfish haemocytes as previously reported by zeng et al. (2009). the zinc-finger antiviral protein (zap) is a kind of zinc-finger protein which considered as a host factor that inhibits the replication of many viruses by preventing the viral mrna accumulation in the cytoplasm. zap binds to the viral mrna and recruits the cellular rna degradation machinery to degrade the target rna (garcia and damonte, 2007; zhu and gao, 2008). du et al. (2012) reported that zn-finger protein induced dna double strand breaks, which may declare another possible mechanism of dnadamage induced by wssv in crayfish. knowing that crayfish being often considered as a biological indicator species of the heavy metal pollution existing in the aquatic environment and by considering the findings of most studies that observed the highest concentrations of each metal were inside the hepatopancreatic tissue (madigosky et al., 1991). 384 el-shorbagy and hamdi / dna damage and ros in wssv-infected crayfish we cannot conclude that the resultant dna damage could be due to metal accumulation as hepatopancreas reported the least dna damage among tested tissues. as wssv is a lytic virus that replicates and assembles in the nucleus, resulting in lysis of the infected nuclei/cells thus the cells of the infected tissues may become severely damaged in the late stages of infection; that may cause organ dysfunctions, necrosis and perhaps leads to death (kou et al., 1998, leu et al., 2013). these implications could be further proved in the present study using histopathological examinations that demonstrated several abnormal architecture recording ascending scores of damages according to the severity of infections. the observed histopathological changes including edema, zenker’s necrosis in muscle fibers and necrosis in gills and hepatopancreatic cells further confirmed wssv mediated apoptosis in the tested tissue. the observed necrotic cells with inflammatory cell infiltration have been reported as indicators of the oxidative stress triggered by depletion of glutathione in these cells (alarifi et al., 2013). the observed edema may attributed to the entry of solutes and water that resulted from the loss of membrane fluidity and function induced by oxidative stress (galli et al., 2003). wssv-induced oxidative stress could be confirmed by the significant reduction of the antioxidant defense systems represented by sod, gpx and cat activities in respect to the negative control. these three anti-oxidant enzymes provide the first line defense against oxidative stress. thus, the resultant reactive oxygen stress (ros), which overwhelmed the antioxidant defenses causing dna damage, is mostly caused by the hydroxyl radical (oh·) and superoxide anion radical (hayes and mclellan,1999). therefore, further investigations of the possible changes in mitochondrial genes expression levels or the expression of genes related to the antioxidant defense mechanism should be studied. the results presented here give some evidences to the prediction of the viral accommodation theory which states that when an active crayfish mechanism to accommodate fatal viral infection fails, they will show signs of cell death by apoptosis that explain the common persistence of viral infection in crayfish. of interest, in some insect baculoviruses, viral infections would usually cause apoptosis unless blocked by specific genes that involved in the production of the inhibitors of apoptosis proteins (iap) (clem et al., 1991), thus this might be the case. anyway, more studies are essential to determine (iap) genes in wssv other than p35 and iap which are able to control apoptosis in a variety of eukaryotes (clem et al., 1996), and whether it might activate apoptosis at some point during infection. conclusion: this is the first study that demonstrates the damaging effects of wssv on dna integrity, histopathological structure and endogenous defense antioxidant enzymes in different tested tissues of crayfish, especially gills which can be considered as a target organ for early detection of wssv. from the present work, we can speculate that apoptosis may be the reason of death in crayfish with mortal viral infections and that it may be considered as an essential part of the adaptive tolerance process against viruses in crustaceans. moreover, these findings declare that the comet assay had adequate sensitivity to detect the differences in the levels of dna damage among different levels of infection within aquatic animals. acknowledgments this work was supported by faculty of science-cairo university. authors would like to thank prof. dr. koukap abd_elazez ahmed for her great help in slicing, staining, photographing and examining the histological specimens. references aebi h. 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(2022) 10(6): 478-488 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article morphological and molecular analysis of the freshwater bivalve anodonta anatina in iran and finland hosein mohamadzadeh1, hadise kashiri1, ainaz shirangi2, jouni taskinen3, reza khaleghi1, amir ghadermarzi1 1department of aquatic ecology, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. 2department of biology, faculty of basic sciences, university of gonbad, gonbad kavus, iran. 3department of biological and environmental science, university of jyväskylä, jyväskylä, finland. s article history: received 4 august 2022 accepted 8 december 2022 available online 2 5 december 2022 keywords: anodonta haplogroup haplotype shell morphology abstract: duck mussel, anodonta anatina is a habitat generalist inhabiting both lentic and lotic aquatic ecosystems. due to high morphological similarity and phenotypic plasticity, a. anatina has sometimes been misidentified as a. cygnea. here, morphological and molecular studies were conducted on anodonta mussels inhabiting north iran and finland. the individuals were collected from anzali wetland, tajan river (north iran) and jyväsjärvi lake (finland). the coi sequence analysis showed the existence of a. anatina in the sampling areas. the iranian and finland specimens showed three and two haplotypes, respectively. the iranian haplotypes were placed in a single clade, while the finland haplotypes were clustered with those of central europe. the mean p-distance between these two clades was 2.4. the median-joining network showed that the iranian haplotypes were lumped into a single haplogroup, while the finland ones were in the same haplogroup as those from central europe. the mediterranean haplotypes were the most divergent haplogroup from both iranian and central european haplogroups. in morphological characteristics, the shell pattern of all individuals from both iranian and finland specimens was stretched and slightly compact with light/dark brown periostracum. the mean length of the specimens from anzali wetland was significantly higher than those of tajan and jyväsjärvi. no significant difference was observed in morphometric characteristics between tajan and jyväsjärvi populations. the results did not indicate significant variation in shell morphology in the studied groups. in this regard, the conventional linear measurements can be supplemented using more complex geometric morphology in further studies. introduction amon different groups of bivalvia, the family unionidae is highly distributed in freshand brackish-water ecosystems of the world. they are known for their significant ecological role (vaughn, 2018), specific life cycle, being parasite in the larval stage (modesto et al., 2018) and unusual doubly (paternal and maternal) mitochondrial inheritance (guerra et al., 2019). in biogeographical studies, the unionidae has great potential to study hydrological and geological events (zieritz et al., 2020). like many other freshwater fauna, bivalves are currently regarded as threatened groups and globally decreased because of anthropogenic activities (ferreira-rodrigues et al., 2019; riccardi et al., 2019), enhancing their conservation significance. in correspondence: hadise kashiri doi: https://doi.org/10.22034/ijab.v10i6.1771 e-mail: hadiskashiri@gmail.com dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.5.8 fact, conserving the unionidae populations is important due to their major ecological role in freshwater ecosystems (vaughn, 2018). among the unionidae, the duck mussel, anodonta anatina (linnaeus, 1758), known as the pan-european freshwater mussel, is an important species due to high distribution in the lakes and rivers of europe and asia below 65°n latitude down to sicily and portugal and expanding as far east as the siberian region. therefore, the duck mussel is a suitable model to assess hypotheses in biogeography about freshwater habitats (graf, 2007; froufe et al., 2014). as a habitat generalist, a. anatina inhabits both lotic and lentic aquatic ecosystems, from streams and rivers to reservoirs and lakes (hinzmann et al., 2013). this species has high ecological importance, 479 int. j. aquat. biol. (2022) 10(6): 478-488 including decreasing water turbidity and controlling suspended particles (vaghum, 2010). moreover, duck mussels can filter some parasitic nematodes and decrease their transition to fish hosts (gopko et al., 2017). it also has a high ability to filter and digest cyanobacterial colonies (bontes et al., 2007). despite its high ecological importance, there is little information on the conservation status of a. anatina worldwide. some regional studies have indicated a decrease in duck mussel populations in europe so that it is now regarded as near threatened or threatened species in austria, germany, irland, and romania. the global decrease in freshwater mussel populations has threatened biodiversity and some habitat services the mussel provides (hajisafarali et al., 2022). in this regard, some countries, including germany and luxemburg, have established conservation programs for a. anatina (byrne et al., 2009; binot-hafke et al., 2011). characteristics of bivalves’ shells, and calcified exoskeletons with protection functions, are considered common morphological markers in population studies (modestin, 2017). intraspecific variation in shell morphological characters in some bivalves has been previously studied (modestin, 2017; ghozzi et al., 2022; wu et al., 2022; mirzoeva and demchenko, 2022). however, classic morphometric measurements might be insufficient in distinguishing bivalve populations and/or species (morais et al., 2014). like other unionid mussels, a. anatina has high morphological plasticity that sometimes has resulted in some mistakes in identifying the species, so more than 400 synonyms are reported for this taxon (nagel et al., 1996; graf and cummings, 2019). the morphological differences between a. anatina populations from some aquatic basins in iberia resulted in the introduction of more than 20 synonyms (araujo et al., 2009). applying some morphological characteristics, especially those related to the shells, has caused some errors by taxonomists in characterizing the bivalve species and populations. it seems that morphological attributes traditionally used in identifying taxonomic units have some limitations since some characteristics have high morphological flexibility expressing different ecophenotypes (zieritz and aldridge, 2009; zieritz et al., 2010). however, due to their simplicity, low cost, and no need for complicated facilities, many scientists have widely used morphological markers. due to the high morphological similarity between a. anatina and a. cygnea and high phenotypic plasticity, these two species are sometimes misidentified (froufe et al., 2014). until now, the anodontini individuals inhabiting north iran have been misidentified as a. cygnea. the aim of the present study was to study the morphological traits and examine possible affinities of the anodontini populations in north iran and finland using coi gene data. materials and methods sampling was done during jun and july 2020 in anzali wetland, guilan province and tajan river, mazandaran province, iran. the samples were also collected from lake jyväsjärvi, jyväskylä, finlan) in november 2019. a small foot tissue was cut from live mussels and immediately preserved in 96% ethanol. mussel shells of the samples (n= 27, 28 and 12 for anzali wetland, tajan river, and jyvaskyla lake, respectively) were also collected for morphological studies. morphometry and age estimation: the biometric variables, including shell length (sl), shell height (sh), and shell width (sw), were measured for each specimen to the nearest 0.1 mm using an aaco caliper. the morphological indices, including shell convexity (ci = w/l ratio × 100) and elongation (ei = h/l ratio × 100), were calculated. the age of the samples was determined by counting the growth rings, which were visible on the shell. molecular studies: total genomic dna was extracted from the foot tissue of each mussel (n=6 and 4 for iranian and finland, respectively) using a high-salt procedure (sambrook et al., 1989) with slight modification. dna quality and quantity were examined via agarose gel (1%) electrophoresis and a 480 mohamadzadeh et al./ morphological and molecular analysis of anodonta anatina table 1. list of coi sequences used in the present study. taxon accession number lineage/haplotype reference anodonta anatina op905650 iran/i3 this study a. anatina op905651 iran/i3 this study a. anatina op905652 iran/i2 this study a. anatina op905653 iran/i3 this study a. anatina op905656 iran/i3 this study a. anatina op905661 iran/i2 this study a. anatina op905654 iran/i3 this study a. anatina op905655 iran/i1 this study a. anatina op905657 iran/i1 this study a. anatina op905658 iran/i3 this study a. anatina op905659 iran/i3 this study a. anatina op905660 iran/i3 this study a. anatina op906256 central europe/e8 this study a. anatina op906257 central europe/e9 this study a. anatina op906258 central europe/e8 this study a. anatina op906259 central europe/e8 this study a. anatina kc583482 central europe/e6 ncbi's genbank a. anatina kc583483 central europe/e3 ncbi's genbank a. anatina kc583484 central europe/e7 ncbi's genbank a. anatina kc583485 central europe/e2 ncbi's genbank a. anatina kc583487 central europe/e10 ncbi's genbank a. anatina kc583501 central europe/e4 ncbi's genbank a. anatina mf414222 central europe/e1 ncbi's genbank a. anatina ef440346 central europe/e11 ncbi's genbank a. anatina mf414221 central europe/e5 ncbi's genbank a. anatina ef571394 north iberia/ni3 ncbi's genbank a. anatina kc583507 north iberia/ni4 ncbi's genbank a. anatina kc583503 north iberia/ni2 ncbi's genbank a. anatina kc583462 north iberia/ni8 ncbi's genbank a. anatina kc583496 north iberia/ni10 ncbi's genbank a. anatina kc583459 north iberia/ni11 ncbi's genbank a. anatina kc583458 north iberia/ni9 ncbi's genbank a. anatina kc583456 north iberia/ni5 ncbi's genbank a. anatina kc583472 north iberia/ni1 ncbi's genbank a. anatina kc583470 north iberia/ni7 ncbi's genbank a. anatina kc583447 north iberia/ni6 ncbi's genbank a. anatina kc583450 south iberia/morocco/im10 ncbi's genbank a. anatina kc583451 south iberia/morocco/im9 ncbi's genbank a. anatina kc583452 south iberia/morocco/im8 ncbi's genbank a. anatina kc583464 south iberia/morocco/im5 ncbi's genbank a. anatina kc583476 south iberia/morocco/im3 ncbi's genbank a. anatina kj402054 south iberia/morocco/im4 ncbi's genbank a. anatina kc583479 south iberia/morocco/im1 ncbi's genbank a. anatina kc583481 south iberia/morocco/im2 ncbi's genbank a. anatina mk733420 south iberia/morocco/im11 ncbi's genbank a. anatina ef571396 south iberia/morocco/im6 ncbi's genbank a. anatina ef571397 south iberia/morocco/im7 ncbi's genbank a. anatina mf414241 mediterranean/m7 ncbi's genbank a. anatina mf414233 mediterranean/m4 ncbi's genbank a. anatina mf414230 mediterranean/m1 ncbi's genbank a. anatina kc583518 mediterranean/m6 ncbi's genbank a. anatina kc583512 mediterranean/m5 ncbi's genbank a. anatina mf414237 mediterranean/m3 ncbi's genbank a. anatina kc583475 mediterranean/m2 ncbi's genbank 481 int. j. aquat. biol. (2022) 10(6): 478-488 biophotometer spectrophotometer (eppendorf, hamburg, germany), respectively. we used the primers lco22me2 (5'-ggt caa caa ayc ata arg ata ttgg-3') and hco700dy2 (5'-tca ggg tga cca aaa aay ca-3') (walker et al., 2006, 2007) to amplify the partial sequences of cytochrome c oxidase subunit i (coi) gene. pcr was run on a thermal cycler (bio-rad mj mini thermal cycler, hercules, ca, usa) in 25 μl reaction mix containing 1 μl dna (20-160 ng/μl), 15 μl taq 2x master mix red (amplicon, denmark), 1 μl of each primer and 7 μl pcr grade water. the pcr condition was set as follows: 4 min at 94°c, 40 cycles at 94°c (30 s), 50°c (40 s) and 72°c (60 s), followed by 10 min at 72°c. the products were assessed using agarose gel (1.5%) electrophoresis in tbe buffer (1x). the high-quality products were sent to the genetic codon company (tehran, iran) for sanger sequencing using the same primers. data analysis: the obtained sequences were manually edited in bioedit 7.0.1 (hall, 1999). we extracted 334 coi sequences from ncb i's genbank. multiple sequence alignment using clustalw also was implemented in bioedit. after trimming the sequences, a 592-bp coi fragment was left. similar sequences were removed through the online tool fabox 1.41 (villesen, 2007). the phylogenetic tree was constructed based on 53 unique sequences (table 1); three and two of them were for iranian and finland specimens, respectively. we used sinanodonta woodiana (ky978735) and anemina arcaeformis (ky561633) as outgroups. the phylogenetic tree was reconstructed using bayesian inference in mrbayes v3.2.2 (huelsenbeck and ronquist, 2001). the best-fitting models of nucleotide substitution based on the akaike information criterion (akaike, 1973) were estimated using mrmodeltest v3.7 (posada and crandall, 1998) in paup v4.0 (swofford, 2003). two parallel runs were independently conducted. each included one cold and three heated metropolis coupled mcmc chains. the program was run for 10 million generations and sampled once every 10000 generations with 20% burn-in fraction. the obtained tree was visualized through figtree v1.4.2 (rambaut, 2008). genetic divergences based on pdistance were assessed in mega 6.0 (tamura et al., 2013). the median-joining network was also constructed using 43 sequences of a. anatina (table 1) through popart v1.7 (leigh and bryant, 2015) to study the relationships between haplotypes. results the shell pattern of iranian and finland samples were stretched and slightly compact with brown/olive-green periostracum. we used coi gene sequencing to identify the species. according to the molecular data, the studied bivalves were a. anatina (fig. 1). morphometric data: morphometric features of the a. anatina specimens collected from anzali taxon accession number lineage/haplotype reference a. cygnea mk034157 ncbi's genbank a. cygnea mk034159 ncbi's genbank a. cygnea mt027890 ncbi's genbank pseudanodonta complanata mk574186 ncbi's genbank p. complanata mk574187 ncbi's genbank p. complanata mk034156 ncbi's genbank p. complanata mk034155 ncbi's genbank a. exulcerata mf414313 ncbi's genbank a. exulcerata mf414306 ncbi's genbank a. exulcerata mf414301 ncbi's genbank anemina arcaeformis ky561633 ncbi's genbank sinanodonta woodiana ky978735 ncbi's genbank table 1. continued. 482 mohamadzadeh et al./ morphological and molecular analysis of anodonta anatina wetland, tajan river and jyväsjärvi lake are shown in table 2. the age of individuals from iran and finland ranged 1-8 and 4-8 years, respectively. the largest mussel was recorded from anzali with a length of 134.32 cm (8 years old). the mean length of the specimens from anzali wetland was significantly higher than those of tajan and jyväsjärvi (p≤0.05). no significant difference was observed in morphometric characteristics of the mussels from tajan and jyväsjärvi (p>0.05). in the length-age relationships, finland bivalves exhibited lower length at a given age than iranian specimens (table 3). the convexity index ranged between 27.49 and 3735.78 for tajen samples (mean ci: 32.57), 29.48 and 37.57 for anzali samples (mean ci: 32.81) and 29.96 and 38 for jyväsjärvi (mean ci: 33.39). the elongation index also ranged from 42.08 to 67.27 for tajan individuals (mean ei: 49.22), 41.63 to 59.57 for anzali individuals (mean ei: 50.65) and 47.96 to 56.16 for jyväsjärvi (mean ei: 51.31) (table 2). molecular data: the coi sequences analysis figure 1. anodonta anatina; a and b represent live duck mussel individuals and exterior/interior view of the mussel, respectively. length height width convexity index elongation index age (year) tajan river min-max 31.84-115.38 21.42-52.43 9.63-35.97 29.77-39.74 42.08-67.27 1-7 mean±sd 86.30±18.12 41.68±7.38 28.05±5.99 32.57±2.45 49.22±6.34 5±1.3 anzali wetland min-max 67.9-134.32 32.45-75.63 21.4-49.35 28.58-38.12 41.63-59.57 2-8 mean±sd 97.99±21.77 50.03±15.0 5 32.49±9.5 32.81±2.46 50.65±5.67 5.11±1.47 jyväsjärvi lake min-max 66.43-130.93 33.99-64.19 22.67-39.22 29.95-38 47.96-56.16 mean±sd 84.83±18.75 40.49±8.57 28.04±4.51 33.39±2.2 51.31±2.52 5.83±1.4 abbreviations: sd (standard deviation); min (minimum); max (maximum) table 2. morphometric features and age of sinanodonta lauta from iran. length age 1 2 3 4 5 6 7 8 tajan 31.84 40.28 62.08 74.76 88.7 98.91 97.09 anzali 68.04 73.4 77.76 92.88 115.58 126.11 133.06 jyväsjärvi 73.6 75.7 83.9 107.45 table 3. age-length relationships of anodonta anatina from iran and finland. 483 int. j. aquat. biol. (2022) 10(6): 478-488 confirmed the existence of a. anatina in the sampling areas. twelve 705-bp and four 652-bp long fragments of the coi gene were acquired from the iranian and finland duck mussel specimens and deposited to the ncbi's genbank (table 1). we reconstructed the phylogenetic tree under tim+i+g model (fig. 2). the specimens from the studied regions in iran belonged to three haplotypes, while the samples collected from finland belonged to two haplotypes. the finland haplotypes were placed in the same clade as those from central europe (lineage central europe). the iranian haplotypes were also placed in a single clade (lineage iran) with strong bootstrap support (100%). besides these two lineages, there are three more mitochondrial lineages of a. anatina (fig. 2). the mean coi p-distances between the a. anatina lineages are presented in table 4. this distance ranged from 1.7 (between the lineages north iberia and south iberia/morocco) to 3.5 (between iran and the mediterranean clades). the distance between the lineage comprising the iranian samples and other lineages ranged from 2.4 to 3.5. the haplotype network recovered three and two haplotypes for the iranian and finland samples (fig. 3). the haplotypes from iran were lumped into a single haplogroup separated by 1-2 substitutions. there are four more haplogroups. there was also one mutation site between two haplotypes from finland. the haplotypes from the mediterranean group were the most divergent haplogroup from the haplogroups comprising our samples from iran and finland. figure 2. the bayesian phylogenetic tree on the basis of 53 unique coi sequences of anodonta anatina and related taxa; two sequences of sinanodonta woodiana and anemina arcaeformis are the outgroups. the numbers above branches represents the bootstrap support values. the scale bar shows the branch lengths. 484 mohamadzadeh et al./ morphological and molecular analysis of anodonta anatina discussion anodontini has consistently been retrieved as a monophyletic group, comprising genera from north america, including pseuanodonta and anodonta spp. (williams et al., 2017; riccardi et al., 2019). the genus anodonta comprises a. californiensis, a. kennerlyi, a. nuttalliana, and a. oregonensis in north america (williams et al., 2017), and a. exulcerata, a. cygnea and a. anatina in europe and some parts in asia (graf, 2007; lopes-lima et al., 2017). however, the taxonomic status of unionid bivalves is under discussion due to the high morphological similarity between the cryptic taxa and deficient molecular information (bolotov et al., 2016; bespalaya et al., 2018; riccardi et al., 2019; kondakov et al., 2020). as reported by klishko et al. (2018), the difference in shell morphometric variables between a. anatina and other anodonta species is weak and cannot support the exact identification. therefore, due to great plasticity, the differences were even overlooked in most comprehensive classifications, considering s. iberia / morocco c. europe mediterranean n. iberia c. europe 2.1 mediterranean 2.8 3.1 n. iberia 1.7 2.5 3.4 iran 3.0 2.4 3.5 3.1 table 4. genetic divergences (mean uncorrected p-distance %) among anodonta anatina lineages. figure 3. median joining network for coi sequences of anodonta anatina (n=43). short lines between the haplotypes indicate the number of mutation sites. 485 int. j. aquat. biol. (2022) 10(6): 478-488 a. anatine and a. cygnea as a single species. that is why all nominal taxa in european countries had previously been regarded as a. cygnea (haas, 1969). furthermore, a recent molecular phylogenetic study has clustered a. anatina with a. cygnea and a. nuttalliana in one monophyletic clade (araujo et al., 2017). the anodontini mussel inhabiting the freshwater bodies in the north of iran has been considered as a. cygnea but based on our molecular data, they belong to a. anatina. according to the results, the iranian and finland specimens exhibited three and two haplotypes, respectively. the iranian specimens clustered in separate clade while the finland haplotypes were placed together with those of central europe. froufe et al. (2014) reported three mitochondrial clades for pan-european freshwater mussels, including iberia, europe, italy, and ebro, while riccardi et al. (2019) reported four clades, including n. iberian, w. iberian/moroccan, central european and mediterranean ones. tomilova et al. (2020) reported four intraspecific lineages comprising iberia, azov, europe and italy. in our study, there were five lineages and within each of them, some geographically related haplogroups. however, as low divergences between these lineages (1.7 and 3.5%) and a lack of samples from some areas, we do not decide on their taxonomy. froufe et al. (2014) reported the highest and lowest divergence between the iberian with italian and europe, respectively. they also demonstrated that the highest and lowest genetic divergence was between europe with italy and azov, respectively. here, we observed the lowest mean uncorrected coi p-distance between the north and south iberian/moroccan haplotypes, while the highest difference was between the iranian and mediterranean haplotypes. the lineage comprising the iranian group exhibited the highest and lowest affinity with the lineages central europe and mediterranean, respectively. based on the median-joining network, a. anatina could be divided into five haplogroups. consistent with our phylogenetic data, the haplotype network also recovered three and two haplotypes for iran and finland individuals, respectively. the specimens from finland and those from central europe are interrelated and comprise a coi haplotype cluster. the iranian haplotypes are placed in a single cluster. this cluster has a distinct source, likely around the caspian sea. however, the mediterranean was the most distant group from iran and finland, while the central european population was the closest to both. different taxa have shown plasticity in phenotype in response to environmental and landscape parameters (e.g., minton et al., 2008; inoue et al., 2013; modestin, 2017). slow changes in freshwater bivalve’s morphology have been reported from upstream to downstream of a river (graf, 1998; hornbach et al., 2010). in fact, the shell shape of bivalves can be influenced by their way of life (alyakrinskaya, 2005). according to selin (2007), a convexity index more than 0.5 shows the shell is convex, and the lower the elongation index (less than 0.9), the more the mussel is stretched; otherwise, it is truncated. the mean ci of our samples were 32.57, 32.81, and 33.39 for anzali, tajan and jyväsjärvi, respectively and none of the individuals had ci more than 0.5. the mean ei of our samples was low (49.23, 50.65, and 33.39 for anzali, tajan and jyväsjarvi, respectively). no significant ei and ci differences were observed among the iranian and finland specimens. the substrate characteristics can significantly influence the ci and ei indices (modestin, 2017). in both sampling sites in iran and lake jyväsjärvi in finland, the beds were dominated by fine sandy mud and the shells of the samples were stretched and slightly compact. this is in accordance with the results of modestin (2017), who reported that lucina pectinata was more stretched in fine sandy mud beds compared to the coarse sand and sandy mud hardened by the mangrove tree roots. although the a. anatina individuals did not display diversity in the shell shape and colour based on their region, the samples from finland exhibited lower shell length at a given age compared to the iranian ones. among the iranian samples, the anzali individuals were bigger than the tajan ones at a given age. similar to the present study, such a 486 mohamadzadeh et al./ morphological and molecular analysis of anodonta anatina different length growth at a given age had previously been reported by girgibo (2013) for a. anatina populations in different lakes, koijarvi and paijanne, in finland. this could be related to the suitability of the habitat, especially in terms of food availability (girgibo, 2013). although the morphology is usually applied to identify freshwater bivalves, this could be strongly variable based on habitat properties. the anodontini species had been considered morphologically as a. cygnea in iran and our data identified it as a. anatina based on molecular data. a combination of molecular and morphological data is applied to study the plasticity of freshwater bivalve’s phenotype (zieritz et al., 2010). however, our results did not indicate evidence for significant variation in shell morphology in the studied regions. in this regard, the conventional linear measurements can be supplemented using more complex geometric morphology in further studies. references akaike h. 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(2021) 9(5): 309-325 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article seasonal variation in the coastal water phytoplankton communities and their environmental responses at upstream and downstream of the steep naf river in the south-western bay of bengal md. jahangir sarker* 1, mehedi hasan tanmoy1, m shahanul islam2, khondokar mohammad shahriar nazrul3, shaharior hossen4, mir mohammad ali5 1department of fisheries and marine science, noakhali science and technology university, noakhali, bangladesh. 2college of food engineering and biotechnology, tianjin university of science and technology university, tianjin 300457, china. 3marine fisheries survey management unit, department of fisheries, ministry of fisheries and livestock, bangladesh. 4department of fisheries science, chonnam national university, yeosu 59626, korea. 5department of aquaculture, sher-e-bangla agricultural university, dhaka 1207, bangladesh. s article history: received 28 june 2021 accepted 10 september 2021 available online 2 5 october 2021 keywords: phytoplankton diversity nutriment content chlorophyll-α abstract: as a multinational river, the naf river flows into the bay of bengal in the indian ocean, between the cox's bazar district of bangladesh and the rakhine state of burma. in a multidisciplinary approach, several experiments were carried out to understand the seasonal diversity of the phytoplankton community structure. a total of four layers of water was sampled from four depths in the naf river during monsoon (september) and winter (december) of 2016. 41 species of phytoplankton were identified, and 3 different dominant groups (cyanobacteria, diatoms, and dinoflagellates) were found. diatoms and cyanobacteria alone were found to be most prevalent. higher species diversity was observed in the monsoon season, with synedra sp. (1.84×105 cells l-1, 18.76%) and winter with microcystis sp. (1.41×105 cells l-1, 17.74%), respectively. in monsoon, no3-n and po4-p were both higher than winter (450.9 and 34.4 µg l-1, respectively) especially, at downstream naf river. moreover, high diversity indexes (richness) of phytoplankton were recorded along with these estuarine stations. significant correlations (p<0.01) of nutrients with phytoplankton may liable behind these scenarios. an analysis of principal component analysis (pca) and linear regression supported this correspondence. in the monsoon season, the concentration of chlorophyllα reached the highest level (165 µg l-1) at a depth of 1.5 m, in station-d. cluster analysis based on the nutrient content of the naf river was found two (upstream and downstream) mentionable zones during the winter and monsoon seasons. the results of the present study indicate that estuarine downstream areas are more productive than upstream areas of the naf river at the southwest coastal zone of the bay of bengal. introduction phytoplankton is the most diverse group of photosynthetic microorganisms, and are adapted to live partly or continuously in open seas, lakes, reservoirs, ponds, and river waters, where they contribute part or most of the organic carbon available to pelagic food webs (reynolds, 2006; barsanti and gualtieri, 2006; klais et al., 2017; santana et al., 2017; lee et al., 2018; affe et al., 2019; beak et al., 2019; bhaskar et al., 2020). they are the agents of primary production and the key to the base of food chains and food webs, directly providing nutrients to *correspondence: md. jahangir sarker doi: https://doi.org/10.22034/ijab.v9i4.1275 e-mail: mjsarker@nstu.edu.bd zooplankton, fishes, and other aquatic animals (shubert, 1984; millman et al., 2005; moser et al., 2017; abonyi et al., 2018; kim et al., 2019a; bom and colling, 2020). changes in phytoplankton dominance can affect whole aquatic ecosystems because they are the main source of food for many small fish and invertebrates (dalelio et al., 2014; maranon, 2015; kim et al., 2019b; park et al., 2020). the rapid changes in the abundance and composition of phytoplankton species as a result of environmental changes make them useful as aquatic bio-indicators for water quality monitoring 310 sarker et al./ seasonal variation in phytoplankton communities of naf river (plongon et al., 2016; charalampous et al., 2018). specific annual biological features in tropical rivers and reservoirs are expressed in plankton density and species composition (palmer et al., 1977; shubert, 1984; washington, 1984; pongswat et al., 2000; nakajima et al., 2015; shanks et al., 2017; sai elangovan et al., 2019). the key limiting nutrients for the growth of marine phytoplankton are sometimes considered to be phosphorus (p) and nitrogen (n), with an increase in their availability resulting in increased primary production rates and changes in population abundance, species richness, and species composition (harvey, 1960; berdalet et. al., 1996; carter et al., 2005; hobday et al., 2006; choern et al., 2014; beak et al., 2015; spilling et al., 2015; gherardi et al., 2016; chan et al., 2020). chlorophyll-α is regarded as a well-accepted index in this case for phytoplankton abundance and the population of primary producers in an aquatic environment (camdevy'ren et al., 2005; yoshikawa et al., 2017; liu et al., 2020). the naf, a trans boundary river that forms bangladesh's border with myanmar, is one of the most important estuarine rivers in bangladesh. the only coral island in county saint martin, the most southerly point, sits at the mouth of the naf. the overall health of the bay of bengal ecosystem can also be affected by the discharge of nutrients from, and the productivity of, this river. which, in return, directly affects the development of fish and fisheries in and around the river. the estuarine environment plays many economically significant roles in many ways. though the estuarine area of bangladesh has high faunal diversity, it has given the least importance due to a lack of sufficient research. realizing the importance, several works pertaining to macrobenthos of coastal and estuarine waters of bangladesh has been carried out (hossain et al., 2009; abu hena et al., 2013; asadujjaman et al., 2012; noman et al., 2018). however, information on nutrients and primary producers, which act as a key platform of the naf river estuary is yet untouched despite this river provides many ecological and economic services to the people of bangladesh. therefore, our study aimed to (i) investigate the seasonal and spatial distribution of phytoplankton biomass and their communities and (ii) the relationship between shifts in river nutrient levels and phytoplankton communities, species composition, and productivity. materials and methods study area: the naf river flows through the apex of the bay of bengal, roughly between latitudes 20°47'n and 92°28'e. a solid hydrodynamic regime is present in the flow. the prevailing southerly winds and swells and the convergence of northbound currents influence oceanographic conditions. the river's bed is sandymuddy from the upper reaches to the lower reaches, where tidal water flows freely. sampling for the present study was undertaken throughout the length of the naf river, which runs from approximately 21°09'03"n 92°12'12"e to 20°44'32"n 92°21'40"e. it enters bangladesh near phalungkhali (ukhiya upazila) of cox's bazar district and joins the bay of bengal after shah-prior-dweep (teknaf upazila) (fig. 1). as bangladesh and myanmar are at odds over the naf river on the coast, only five sampling stations figure 1. location of the study area showing different stations in naf river (red-colored inset in the map of bangladesh indicates the position of naf river). 311 int. j. aquat. biol. (2021) 9(5): 309-325 from the mouth to the head of the naf river estuary stations were chosen. all of the stations were chosen from various estuary elevations. levels of the naf river estuary were named according to stations’ pattern (table 1). each station was sampled in triplicate fashion at four depths in the vertical water column (0, 1.5, 3, and 6 m) except in stations b and c (water depth was less than 6 m). sample collection and preservation: water samples were collected using a 1l kemmerer type water sampler (model: wildco-3-1200-e35) during monsoon and winter, 2016. sub-samples of water were then taken for phytoplankton, chlorophyll-α, and nutrient analysis. a country boat was used to collect the water samples throughout the study period. water samples collected for phytoplankton analysis were passed through plankton net (125 µm). filtrates were carefully decanted into plastic sampling bottles up to a standard volume of 50 ml. thereafter, phytoplankton samples were preserved in 5% formalin for the qualitative and quantitative analyses and species identification. 200 ml of the collected water sample was filtered using filter paper (whatman gf/f, 0.45 μm, diameter 47 mm) in situ for chlorophyll-α and nutrient content analysis. the filtrates and filtered water samples were then immediately brought back to the laboratory, kept frozen, and stored at -20°c until analysis. analysis of chlorophyll-α (µg l-1) and nutrient (no3-n and po4-p) content (µg l-1) were performed using hach (model dr 6600) at the laboratory of fisheries and marine science, noakhali science and technology university. sample extraction and analysis of chlorophyll-α: the filters were cut into small pieces and placed in a 50 ml centrifuge tube, then 15 ml of 90% acetone was added and allowed to stand overnight in a refrigerator. these were then centrifuged at room temperature for 10 min at 3,000 rpm. the supernatants were decanted into a 50 mm path length spectrophotometer cuvette. the methods employed for algal absorption measurements and calculations are described by parsons et al. (1984). phytoplankton enumeration, identification, and diversity indexes: for each sub-sample, 1 ml from every 50 ml the preserved sample was transferred to a 1 ml capacity sedgwick-rafter counter, and phytoplankton was counted using a luminous microscope (xsz21-05dn, china). phytoplankton was counted using the formula proposed by stirling (1985), with the resultant count expressed as the number of cells per l of water (cells l-1). the total number of phytoplankton present in the collected sample was calculated by the following formula: n = 𝑛𝑛×𝑣𝑣 𝑉𝑉 ×1000 where n = total number of phytoplankton cells per liter of water filtered, n = average number of phytoplankton cells in 1 ml of plankton sample, v = the volume of phytoplankton concentrates (ml), and v = the volume of total water filtered (l). phytoplankton was identified (mostly to species level) based on morphology, using taxonomic keys of prescott (1962) and referring to the monographs of yamazi (1979), hustedt (1985), and sahu et al. (2013). identification and measurement of the phytoplankton were carried out using a light microscope equipped with eyepiece graticules. shannon-wiener diversity index (shannon and weaver, 1949) was calculated by using the following respective formulae: h = -∑ 𝑃𝑃𝑃𝑃 𝐿𝐿𝐿𝐿 𝑃𝑃𝑃𝑃𝑠𝑠𝑖𝑖=1 where, s is total number of species in a sample, pi = proportion (pi=ni/n) of total sample individuals table 1. stations coordinates and estuary level in the study area. name of the station location of the station coordinates estuary level total length of the naf river in bangladesh along with the study area coverage n e a phalungkhali 21°8'60" 92°12'13" lower estuary 63 km (44 km; 70%) b silkhali 21°3'5" 92°15'23" lower-mid estuary c dhumdumia 20°57'26" 92°16'3" mid estuary d teknaf 20°53'23" 92°17'57" upper-mid estuary e dakhkhin para 20°46'8" 92°20'30" upper estuary 312 sarker et al./ seasonal variation in phytoplankton communities of naf river belonging to the ith species, n = total number of individuals of all the species and ni = number of individuals belonging to the ith species. margalef’s species richness (gleason, 1922) or margalef’s index was done by: d = 𝑆𝑆−1 ln𝑁𝑁 where, d = margalef’s index, s = number of species in the sample, ln = log-normal, and n = total number of individuals in the sample. and evenness index (pielou, 1967) of phytoplankton species evenness or equality was counted by j = h/ln s; where, j = evenness index, h = shannon-weiner index value, ln = log normal, s = total number of species in sample. statistical approaches and data demonstrations: a t-test was run using ibm (spss, version 20, 2011) to assess the change in chlorophyll-α concentrations between monsoon (september) and winter (december). statistical comparisons of different diversity indices were performed using paleontological statistics (past version 3.11) (hammer et al., 2001). surfer (version 12) was used for average surface demonstration of all tabulated and examined parameters. the concentration of different parameters was shown through a line graph by excel stats software and contour color map by ocean data view (odv 2018). cluster analysis (mca) was performed after using the pearson coefficient by multivariate statistical package software (kovach, 1998). pca (principal component analysis) plot was also deployed accordingly. linear regressions were performed by microsoft excel (v2016) software. spss (v25) was used for the pearson correlation and covariance. however, data were demonstrated in graphs based on significant relationships accordingly. results naf river hydrology: the naf river demonstrated high temperature at the mid and low across the upstream region (fig. 2a). however, dissolved oxygen (do; mg l-1), salinity (ppt), and ph were high at the downstream region near the naf river’s estuarine area. low do (fig. 2b) and salinity (fig. 2c) were observed at the upstream area and low ph was found in the mid of the river (fig. 2d). riverine nutrients patterns: vertical distributions of abiotic factors: the concentration of no3-n was approximately 10 times higher than po4-p, with both increasing towards the estuary (supplementary file 1). the highest concentration of no3-n (450.9 µg l-1) was recorded in monsoon at the surface of station-d (fig. 3c) while the lowest (66.4) was at the surface of station-a in winter (fig. 3g). monsoon and winter also showed temporal variations for all the variables, with a decrease in no3-n, po4-p, from 287.18 and 23.53 µg l-1 in monsoon to 223.90 and 20.60 µg l-1 in winter, respectively (table 1). the highest average concentration of no3-n was recorded (table 1) in monsoon at 6 m depth (374 µg l-1) whereas the lowest was in winter at the surface layer (209.32 µg l-1). the po4-p concentration ranged from 19.06 to 23.07 µg lfigure 2. environmental parameters of sampling stations in study area. 313 int. j. aquat. biol. (2021) 9(5): 309-325 1 in winter and 21.14 to 29.39 µg l-1 in monsoon (table 1). po4-p concentration was highest (34.4 µg l-1) in monsoon at 6 m (fig. 3d) and the lowest (14.7 µg l-1) was in winter at the surface of station-b (fig. 3h). horizontal distributions of abiotic factors: the average concentrations of no3-n and po4-p in monsoon were 287.18 and 23.53 µg l-1 while in winter they were 233.90 and 20.6 µg l-1, respectively. the highest average concentration of no3-n was recorded in monsoon at station-d (414.3 µg l-1) whereas the lowest was in winter at station-b (102.91 µg l-1). the average po4-p concentration ranged from 16.43 to 29.08 µg l-1 at station-a in winter and station-e in monsoon respectively. especially, station-d always showed the highest concentrations for no3-n compared to all depths during both seasons (fig. 3a, e). on the other hand, station-e demonstrated the highest concentrations for po4-p during both seasons accordingly (fig. 3b, e). distribution of biotic and abiotic factors: horizontal profile: the mean no3-n, po4-p, and chlorophyll-α concentrations in monsoon were 287.18, 23.53, and 0.85 µg l-1, respectively, while in winter they were 233.90, 20.6 and 0.52 µg l-1, respectively (table 1). mean phytoplankton cell density varied seasonally (table 1). spatial changes in the phytoplankton biomass (herein chlorophyll-α) were similar with phytoplankton cell density and yielded the highest value at station-d in both seasons (fig. 4a-b and e-f). vertical patterns: phytoplankton cell density decreased sharply with an increase in depth. the concentration of chlorophyll-α slightly increased for figure 3. average distributions of biotic and abiotic factors at naf the river. 314 sarker et al./ seasonal variation in phytoplankton communities of naf river both months when comparing the surface to the deepest sample (6 m), with a decrease in the middle layers (1.5 and 3 m). monsoon and winter also showed temporal variations for all the variables, with a decrease in chlorophyll-α and phytoplankton from 0.88 µg l-1 and 9.93×105 cells l-1 in monsoon to 0.53 µg l-1 and 7.03×105 cells l-1 in winter, respectively. the average chlorophyll-α concentration was the highest in monsoon at the depth of 6 m (0.91 µg l-1) whereas the lowest was in winter at the depth of 3 m (0.48 µg l-1) (table 1). the distribution of phytoplankton abundance was similar to that of biomass showing the highest and the lowest values in the northeastern and southwestern part of the naf river at station d and a, respectively, both in monsoon (fig. 4c-d) and winter (fig 4g-h). with an increase in profundity, phytoplankton cell density decreased dramatically. when comparing the surface to the deepest sample (6 m), the concentration of chlorophyll-α increased marginally for both months, with a decrease in the middle layers (1.5 and 3 m). with all the variables, monsoon and winter also showed temporal differences, with a reduction in chlorophyll-α and phytoplankton from 0.88 µg l-1 and 9.93×105 cells l-1 in monsoon to 0.53 µg l-1 and 7.03×105 cells l-1 in winter. the average concentration of chlorophyll-α was highest in monsoon at a depth of 6 m (0.91 µg l-1) while the lowest concentration was 3 m (0.48 µg l-1) in winter (table 1). species distribution: during the current analysis, a total of 41 phytoplankton taxa belonging to three separate classes (cyanobacteria, diatoms, and dinoflagellates) were found in which cyanobacteria figure 4. vertical distribution of biotic and abiotic factors at the naf river. 315 int. j. aquat. biol. (2021) 9(5): 309-325 belonged two species (microcystis sp. and trichodesmium erythraeum), dinoflagellates belonged seven and diatoms belonged the remaining 32 species. diatoms were the most common, followed by dinoflagellates and cyanobacteria, at all stations in both months. all of the 41 species reported were present in the monsoon, while 2 species of diatom (chaetoceros didymus and t. robertsianum) were absent in the winter (table 2). at the surface and 1.5 m depth at station-d, the highest 21 species were found in monsoon and winter, respectively. the lowest 7 species were found in both months at 3 m depth and 6 m depth at stationa as well as at station-a (6 m) and station-b (l0 m) in winter, respectively (fig. 5). synedra sp., (monsoon 18.76 and winter 13.66%; diatom), thalassiothrix fraunfeldii (monsoon 14.04 and winter 15.20%; diatom), and microcystis sp. were the organisms with the highest cell densities (monsoon 13.18%; cyanobacteria; winter 17.74%) (table 2). the single species with the highest cell density in monsoon was synedra sp. (1.84×105 cells l-1), while microcystis sp. table 2. group-wise lists of phytoplankton species identified from the naf river with average cell density (cells/l) of monsoon and winter, 2016 during the present study (bold=highest concentrations). groups species identified monsoon winter monsoon % winter % cyanobacteria microcystis sp. 1.3×105 1.41×105 13.18 17.74 trichodesmium erythraeum 3.25×104 2.96×104 3.31 3.72 diatoms amphora sp. 2×103 2.67×103 0.20 0.34 bacillaria paxillifer 2.08×104 1.18×104 2.11 1.49 bacteriastrum hyalinum 2×103 1.25×103 0.20 0.16 bellerochea malleus 8.25×103 9.67×103 0.84 1.22 cerataulina pelagica 1.5×104 2.03×104 1.53 2.55 chaetoceros didymus 1×103 0.10 0.00 coscinodiscus centralis 3.25×103 4.25×103 0.33 0.53 cyclotella sp. 1.33×104 1.38×104 1.36 1.74 diploneis weissflogii 9.67×103 5×103 0.98 0.63 eucampia zodiacus 6.25×103 5.25×103 0.64 0.66 grammatophora marina 2.67×104 2.06×104 2.71 2.59 lithodesmium undulatum 7.75×103 4.67×103 0.79 0.59 navicula sp. 3.47×104 3.18×104 3.53 3.99 paralia sp. 2.92×103 2.42×103 0.30 0.30 planktoniella sol 1.58×104 1.80×104 1.60 2.26 pleurosigma sp. 3.30×104 1.56×104 3.36 1.96 pseduonitzschia pungens 1.50×103 5.00×102 0.15 0.06 rhizosolenia alata 5.02×104 3.64×104 5.11 4.58 rhizosolenia castracanei 3.50×103 2.50×103 0.36 0.31 rhizosolenia crassispina 1×104 7×103 1.02 0.88 rhizosolenia imbricata 2.38×104 2.22×104 2.43 2.79 rhizosolenia setigera 1.88×104 4.08×103 1.92 0.51 skeletonema costatum 3.00×103 4.75×103 0.31 0.60 stephanopyxis turris 4.67×103 5.00×102 0.48 0.06 surirella sp. 3.50×103 7.50×102 0.36 0.09 synedra sp. 1.84×105 1.09×105 18.76 13.66 thalassionema nitzschioides 1.13×105 6.63×104 11.50 8.33 thalassiosira rotula 7.42×103 8.50×103 0.76 1.07 thalassiothrix fraunfeldii 1.38×105 1.21×105 14.04 15.20 triceratium favus 2.26×104 1.79×104 2.30 2.25 triceratium reticulatum 3.00×103 1.75×103 0.31 0.22 triceratium robertsianum 1.33×103 0.14 0.00 dinoflagellates ceratium macroceros 1×103 1.50×103 0.10 0.19 ceratium trichoceros 8.33×103 1.91×104 0.85 2.40 ceratium tripos 1.03×104 1.28×104 1.04 1.61 ornithocercus steinii 4.83×103 6.58×103 0.49 0.83 prorocentrum maximum 3.17×103 6.75×103 0.32 0.85 prorocentrum scutellum 1×103 4.50×103 0.10 0.57 protoperidinium steinii 1×103 3.75×103 0.10 0.47 total 41 8.90×105 7.23×105 100.00 100.00 316 sarker et al./ seasonal variation in phytoplankton communities of naf river obtained the highest position in winter (1.41×105 cells l-1). phytoplankton diversity: phytoplankton cell density showed a steady increase from station-a to station-d for both monsoon and winter, accompanied by a small decrease in station-e. the cell density in monsoon was higher than in winter, while the phytoplankton cell density during the study period was higher in the surface layer than the other river layers. a similar trend from station-a to station-e was also seen in the number of species described. the highest cell density (1.73×107 cells l-1) recorded was at the surface layer of station-d in monsoon and the lowest (3.6×105 cells l-1) was at a depth of 3 m in the month of winter at station-a. this showed a correlation with the concentration of no3-n that also figure 5. cluster analysis of sampling stations after considering nutrients as variables. figure 6. linear regressions among biotic factors and its correspondences with physical parameters of the naf river. 317 int. j. aquat. biol. (2021) 9(5): 309-325 showed a comparable trend with the stations (fig. 5). different indices of diversity were also tested on current data (table 3). in winter, the diversity of shannon-wiener (h) ranged from a maximum of 2.089 at station-d (1.5 m) to a minimum of 1.462 at station-a (6 m), while the minimum was 1.578 at station-a (3 m) at monsoon and a maximum of 1.972 at station-surface d's layer (table 3). the highest margalef (d) index in winter was 1.234 at station-d (1.5 m) and the lowest was 0.436 at station-b. (3 m). the maximum index of margalef (d) for monsoon was 1.21 for station-d (0 m), while the minimum was 0.4418 for station-a at 1.5 m depth (table 3). the maximum evenness or equitability (j) of the species observed was 0.613 at station-a (3 m) in winter, while the minimum was 0.3265 at station-d (0 m) in monsoon. during this study period, no consistent pattern of evenness was observed (table 3). environmental correlations among parameters: stations can be divided into ecological zones geographically, i.e., upstream (st. a, b and c) and downstream (st. d and e) of the river naf. cluster (fig. 5) analysis of both seasons reveals that after taking nutrients as a component, the sampled stations were divided into two sub-divisions, such as upstream (groups 1 and 3) and downstream (groups 2 and 4). in both seasons, on average, downstream stations displayed the highest concentration of all parameters (fig. 5). linear regressions in both seasons (fig. 6) showed a strong positive relationship between chlorophyll-α and phytoplankton. salinity also showed significant positive linearity with them, particularly during the monsoon with chlorophyll-α (fig. 6). besides, the n:p ratio demonstrated a significant positive relationship with the phytoplankton percentage (fig. 6). phytoplankton biomass showed strong positive linearity, especially in winter with phosphate and in monsoon with nitrate, however, cell density showed weak linear relationship with nutrients in both seasons (fig. 7). pca is an appropriate way to display a dataset's variety and clear patterns (pitchaikani and lipton, 2017). clustered nutrients and phytoplankton (green circle) showed close relationships between them (fig. table 3. station wise calculated phytoplankton diversity index (h= shannon, r= margalef, and j= evenness) of different layers (depth) during the study period. month indices depth (m) a b c d e m on so on h 0m 1.73 1.76 1.87 1.97 1.89 5m 1.58 1.58 1.83 1.83 1.89 10m 1.58 1.64 1.76 1.96 1.74 20m 1.63 1.81 1.64 r 0m 0.6 0.65 0.94 1.21 0.93 5m 0.49 0.54 0.84 0.99 0.77 10m 0.44 0.55 0.67 1.17 0.65 20m 0.5 0.95 0.6 j 0m 0.51 0.49 0.38 0.33 0.39 5m 0.54 0.49 0.42 0.35 0.47 10m 0.61 0.52 0.48 0.34 0.47 20m 0.57 0.36 0.47 w in te r h 0m 1.68 1.66 1.71 2.08 1.98 5m 1.6 1.57 1.79 2.09 2 10m 1.59 1.56 1.71 1.95 1.92 20m 1.46 1.86 1.86 r 0m 0.5 0.48 0.83 1.16 1.12 5m 0.56 0.48 0.73 1.23 1.07 10m 0.44 0.44 0.62 0.95 0.84 20m 0.44 0.78 0.84 j 0m 0.59 0.59 0.37 0.38 0.36 5m 0.5 0.53 0.46 0.37 0.39 10m 0.61 0.6 0.5 0.41 0.45 20m 0.54 0.46 0.43 318 sarker et al./ seasonal variation in phytoplankton communities of naf river 8). the extracted pca 1 showed a variance of 66.840% from the naf river (table 4). in monsoon, the pca plot showed a close relationship between nutrient concentration and plankton density from the naf river water depth of 0 m (fig. 8). the extracted pca 1 showed a variance of 66.84 % from the naf river (table 4). a paired sample t-test was used to test if there was a statistical difference between the mean chlorophyll-α concentrations during monsoon and winter. significant findings of the individual sample t-test {t (17) = -4.664, p = 0.0005} suggest a significant difference between monsoon (m = 0.8772, figure 7. linear regressions of biotic factors with nutrients of the naf river. figure 8. pca plot clarifies the highest plankton diversity with relation to nutrients (spo4; concentration of po4 in monsoon (monsoon), dpo4; concentration of po4 in winter (winter), sno3; concentration of no3 in monsoon, sno3; concentration of no3 in winter, schla; concentration of chlorophyll-α in monsoon, dchla; concentration of chlorophyll-α in winter, scell; plankton density in monsoon, dcell; plankton density in winter) in the naf river. 319 int. j. aquat. biol. (2021) 9(5): 309-325 sd = 0.39873, n = 18) and winter (m = 0.5250, sd = 0.09996, n = 18) in chlorophyll-α concentrations. the mean decrease was 0.35, with the gap between the mean of 0.51 to 0.19 having a 95% confidence interval. in different months, pearson's correlation (table 5) between water depth, plankton density, and nutrients showed positive important correspondences with each other (p>0.01). in both seasons, strong positive relationships (r = 0.86) between phytoplankton and chlorophyll-α showed the accuracy of phytoplankton data (table 5). nutrients have shown a more important seasonal association with chlorophyll-α than the corresponding mean density of phytoplankton. discussion environmental influences on phytoplankton diversity: the naf river showed similar zonal patterns of seasonally high phytoplankton and chlorophyll concentrations, particularly near the estuarine zone. a steady increase in all the variables was observed in both months as it shifted in a seaward direction (i.e., from station-a-e). the concentration of these variables may be diluted by constant freshwater inflows at the upstream stations. besides, domestic wastewater runoff (e.g., agricultural fertilizers) will effectively increase the concentration of nutrients (shen et al., 2008). also, salinity changes were observed from upstream to downstream of the river naf. it may also contribute to the development of phytoplankton (li et al., 2017). in both seasons, substantial linear regression between salinity and chlorophyll-α was also reported. it was found that coastal rivers compared to riverine waters are enriched with a high number of phytoplankton species in bangladesh (islam and aziz, 1977; hoque et al., 1999; sarker et al., 2018). these are common scenarios along the coast due to high salinity and nutritional enrichment (rahman et al., 2013). the substantial linearity of salinity with chlorophyll-α and phytoplankton in both seasons supported the present study. on the other hand, during both seasons, the naf river displayed medium phytoplankton diversity compared to other coastal table 4. eigen analyses of the significant principal components of different parameters. component pca 1 pca 2 eigen value 6.684 2.118 percentage of variance 66.84 33.16 factor loadings depth 0.269 0.922 station 0.901 -0.078 seasons monsoon winter monsoon winter no3-n 0.913 0.944 0.2 0.115 po4-p 0.883 0.929 0.466 0.22 chlorophyll-α 0.866 0.842 -0.255 -0.277 density 0.602 0.733 -0.711 -0.581 table 5. pearson correlation between nutrients and plankton diversity of the naf river. phytoplankton chlα no3-n po4 m on so on phytoplankton 1 chlα 0.86** 1 no3-n 0.66** 0.84** 1 po4 0.64** 0.77** 0.87** 1 phytoplankton chlα no3-n po4 w in te r phytoplankton 1 chlα 0.86** 1 no3-n 0.76** 0.79** 1 po4 0.77** 0.92** 0.84** 1 320 sarker et al./ seasonal variation in phytoplankton communities of naf river rivers. these differences can influence the bulk availability of nutrients, among other coastal areas (hossain et al., 2017). seasonal influences on biotic diversity: there are major variations in the diversity of phytoplankton and other environmental parameters in the naf river between two seasons. seasonal variances in the sampled data were evaluated and verified accordingly by pca and t-test. the extracted pca 1 showed a variance of 66.84% from the naf flow. similar findings were also recorded by mannar gulf, india (pitchaikani and lipton, 2017). in this study, high phytoplankton abundance was observed in the monsoon after winter, especially near the naf river estuary. the sampled nutrients are correspondingly strong during the monsoon as well. these were similar to the bangladesh eastern coast study (maheshkhali channel) (jewel et. al., 2002) and found a maximum no3-n concentration in monsoon, ranging from 0.8 to 3.0 mg l-1, when phytoplankton population cell density peaks were also found. the causes of elevated nitrate levels may be freshwater inflow and terrestrial run-off during the monsoon seasons, as well as the oxidation of ammonia from nitrogen to nitrite (hanninen et al., 2000; rajasegar, 2003). additionally, due to the bulk availability of necessary nutrients, post-monsoon was also recorded with high phytoplankton along coastal rivers (sharif et al., 2017). besides, during winter, the index of shannon and margalef (d) was also strong, associated with the highest heterogeneity or stability of this structure and maximum species richness of the phytoplankton group (pielou, 1967). it was comparable to the meghna river findings in the monsoon season because of the flow of ambient nutrients (hossain et al., 2017). cyanobacteria affect the food chain in aquatic table 6. reported phytoplankton diversity from other places. type reported rivers reported taxa references c oa st al r iv er s kirtankhola 5 sharif et al. 2017 meghna estuary 8 sharif et al. 2017 buragauranga 10 ahmed et al. 2010 karnafully 13 hosen et al. 2019 meghna 17 ahsan et al. 2012 meghna 23 sharif et al. 2017 reju khal 27 iqbal et al. 2017 shibsha 31 shah et al. 2008 meghna 40 flura et al. 2018 naf 41 this study meghna estuary 50 sarker et al. 2016 mathamuhuri 91 hoque et al. 1999 rupsha-pashur 97 rahman et al. 2013 bhola-baleswar 110 rahman et al. 2013 karnafully 111 islam and aziz 1977 khalpatua-arpangachia 122 rahman et al. 2013 in la nd r iv er s padma 17 ahsan et al. 2012 buriganga 27 ferdous et al. 2012 padma 29 ahmed and alfasane 2004 padma 35 rahman and huda 2012 turag 35 khatun and alam 2020 tetulia 39 ahsan et al. 2012 dhepa 52 ara et al. 2018 jamuna 54 alam et al. 2014 padma 54 haque et al. 2019 padma 60 flura et al. 2016 shitalakhya 62 islam and huda 2016 321 int. j. aquat. biol. (2021) 9(5): 309-325 environments by producing toxic blooms and are typically considered to be a hazard, with increases in their abundance commonly correlated with changes in nutrient levels (marshall, 2009). high cyanobacteria concentrations in both seasons and high nutrient concentrations, respectively, were also observed in the current research. phytoplankton succession is primarily affected by the interactions and seasonal cycling of physical, biological, and chemical factors such as temperature, grazing, and/or nutrients (sommer et al., 1986). overall, during both seasons, high substantial positive linearity and positive associations of phytoplankton and chlorophyll-α with naf nutrients assisted these phenomena. nutrients play a big proactive role in biotech development in this region because they are the most productive ecological system in bangladesh (noman et al., 2018). responses of phytoplankton to nutrients: nutrients are the main contributor to the water body's production of phytoplankton (sun et al., 2010). in both seasons, especially in winter, chlorophyll-α was found to correlate significantly with phosphate and then nitrate in the naf river. during these seasons, it can influence biotic development (yuan et al., 2014). the link between phytoplankton and nutrients was also confirmed by pearson. diatoms can thrive in turbulent water due to the nutrient richness of the water body (thomas et al., 1995). it may be responsible for the dominance of phytoplankton contributed by diatoms near the estuarine region of naf, especially at station d. potentially, estuarine vegetation often affects the diversity of planktons along the coastal zone (jiang et al., 2015). due to the input of nutrients, especially from the bottom, the diversity of phytoplankton and its concentration are high across the downstream of the naf river in each case. zonal impact on phytoplankton: phytoplankton densities increased with nutrient content, while salinity, temperature, and water depth decreased from place to place in coastal waters (jiang et al., 2015). these theoretically create an ecological zone in the body of water (li et al., 2017). therefore, it is possible to divide the interrelation between parameters into two ecological zones geographically, i.e. upstream and downstream of the naf river. cluster analysis was suggested as a useful method for marking ecological zonation within the study area after considering nutrients as variables (deng et al., 2008). there are two referential zones according to the latest cluster analysis: group 1 (upstream) and group 2 (downstream) during the monsoon, and group 3 (upstream) and group 4 (downstream) during the winter. in addition, the zone-specific pearson correlation with positive and negative correspondence also substantially differentiates the zonal features. upstream of naf river: the diversity of phytoplankton in the upstream of the naf river was found to be poor with less richness and evenness. this situation could be responsible for nutrient limitations (guo et al., 2014). except for the temperature, on average, do, salinity and ph were found very low across this area. in addition to a strong negative correlation of phosphate with phytoplankton in the monsoon and with chlorophyll-α an in the winter, corresponding correlations have been observed. significant portions of primary productivity may be restricted by phosphate, which can cause phytoplankton to decrease here (wang et al., 2003). in addition, a major limiting factor for the growth of diatoms was low nutritional levels (wei et al., 2017). the naf river phytoplankton is largely respectably dominated by diatoms and cyanobacteria. the combined effect of environmental scarcity (marshall, 2009) is responsible for low concentrations of phytoplankton across this area. downstream of naf river: the downstream naf river had a rather distinctive estuarine features, then the rest of the study region. high nutrient concentrations and other environmental parameters have been found in this area. nutrients, especially nitrate, can act as a precursor, i.e. diatom and cyanobacteria, for dominant phytoplankton (gong et al., 2003). this phenomenon was also confirmed in both seasons by the strong positive association of nitrate with phytoplankton. turbulence has been documented to provide phytoplankton nutrients (estrada and berdalet, 1997), and there is growing evidence that turbulence directly affects dinoflagellate physiology (juhl et al., 2000). 322 sarker et al./ seasonal variation in phytoplankton communities of naf river the brackish portion of the naf river (st. d and e) is a possible turbulent site due to the bay of bengal estuarine link. by enriching nutrients across this area, it can potentially increase the diversity of phytoplankton (juhl and latz, 2002). in summer and autumn, further sampling is recommended to consider the phytoplankton group structure in the naf river all year round. conclusions strong species richness and abundance have been shown by the naf river ecosystem. diatoms are dominant in the group, i.e. during the monsoon, synedra sp., and thalassiothrix fraunfeldii while cyanobacteria, i.e., microcystis sp. in winter. the density of phytoplankton cells increased from upstream to downstream, while also demonstrating an inverse association with the sampling depth. productive downstream has been found with high nutrient abundances that intrigue the diversity and richness of phytoplankton along the naf river estuarine region. therefore, this study has provided a framework on which future research can construct, promoting a deeper understanding of the naf river and its management. the limitations of the present analysis were, however, the sampling data in autumn and summer. in order to imagine the phytoplankton group structure of the naf river along the northwestern bay of bengal, more projects should be introduced covering certain seasonal data deficiencies with larger areas. 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(2021) 9(5): 309-325 average values of nutrients (no3-n and po4-p (µg/l)) and phytoplankton cell density (cells/l×105) along with chlorophyll-a concentration (µg/l) of the present study in the naf river. d ep th w is e seasons september no3-n po4-p chlorophyll-a cell density monsoon 0 263.57 21.14 0.90 12.49 5 274.96 22.28 0.88 10.98 10 307.52 25.08 0.83 8.43 20 374.00 29.37 0.91 7.82 average 305.01 24.47 0.88 9.93 winter 0 209.32 19.06 0.55 10.05 5 214.84 20.66 0.53 8.18 10 246.05 21.22 0.48 6.57 20 286.90 23.70 0.55 7.03 average 239.28 21.16 0.53 7.96 st at io n w is e monsoon a 238.33 20.58 0.51 5.53 b 157.85 19.60 0.53 8.50 c 233.47 20.30 0.74 11.68 d 414.30 28.08 1.51 14.31 e 391.95 29.08 0.97 10.76 average 287.18 23.53 0.85 10.16 winter a 161.75 16.43 0.42 4.20 b 102.91 16.77 0.43 6.80 c 163.30 19.43 0.53 7.77 d 356.63 23.88 0.64 11.50 e 334.90 26.50 0.58 9.65 average 223.90 20.60 0.52 7.98 supplementary file 1 int. j. aquat. biol. (2014) 2(2): 75-84 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 npajournals. all rights reserved original article biometric parameters of the redline torpedo fish puntius denisonii day 1865, an endemic barb in the western ghats hotspots of southern india sajan sajeevan*,1anna mercy thoranam varkey, malika vadakkedath college of fisheries, kerala university of fisheries and ocean studies (kufos), panangad, ernakulum, kerala -682 506, india. article history: received 26 december 2013 accepted 22 march 2014 available online 2 5 april 2014 keywords: growth condition length-weight relationship morphometric puntius denisonii western ghats abstract: studies on the biometric parameters of a species to have an important role in its population or stock management and conservation. present study investigated the morphometric, meristic, lengthweight relation (lwrs), length-length relation (llrs) and growth status (k and kn) of the redline torpedo barb, puntius denisonii collected during august 2012 to july 2013 from river valapattanam, kerala. the length-weight relationship was established for monthly to differentiate growth variation and also separately calculated for males, females pooled and indeterminate. length-length relationship, morphometric and meristic characteristics were also represented by the equation: y = a + bx. the length-weight relations established for males was: log w = -2.207 + 3.148 log l; for females: log w = -2.360 + 3.316 log l; for indeterminate: log w = -2.176 + 3.171 log l and for pooled was: log w = 2.076 + 3.030 log l. the regression coefficients between pooled, indeterminate, males and females of p. denisonii did not show any significant variation from isometric growth (p<0.05). all morphological characters were highly correlated with total length and correlation coefficient (r) was highest for standard length (0.9889) and pre anal length (0.9904) and lowest for pelvic fin length (0.6150). based on the study fin formula can be written as d ii-9, p i-16, v i-8, a i-6 and c 22-24. introduction biometric parameters have been commonly used to distinguish the species taxonomically, to identify stocks of fish and to separate different morphotypes (jayaprakash, 1974; lourie et al., 1999; doherty and mccarthy, 2004; tarkeshwar et al., 2012). lengthweight relationship (lwrs) of fishes are used for the estimation of average weight of the fish of a given length group (beyer, 1987) and also provided useful data for stock assessment and population dynamics studies (kolher et al., 1995). apart from this, the length-weight relationship can also be used for deriving comparisons between different stages in life history and between fish populations from regions or habitat groups (petrakis and stergiou, 1995; goncalves et al., 1997) and tracking seasonal variations in fish growth (richter et al., 2000). * corresponding author: sajan sajeevan e-mail address: sajanpolayil@gmail.com the western ghats in peninsular india supports over 48% of the fish biodiversity in india (ghosh and ponniah, 2001) and is one of the global biodiversity hotspots (myers et al., 2000). the western ghats are already known to have high levels of endemism (dahanukar et al., 2011) and this unique ecosystem should receive the highest priority for species conservation (marcus et al., 2012). puntius denisonii (day, 1865) an endemic ornamental barb of the western ghats of india belongs to the family cyprinidae and is distributed in twelve rivers of kerala (mercy et al., 2009). a large number of endemic species of the western ghats are known to be facing severe population decline due to indiscriminate collections for ornamental fish trade (raghavan et al., 2009). in order to manage endangered species effectively, it is 76 sajan et al./ int. j. aquat. biol. (2014) 2(2): 75-84 necessary to identify the reason for decline and a thorough knowledge of the ecology of the target species (leunda et al., 2007). over the last few decades wild population of p. denisonii has been declined due to various reasons and the species was categorized as endangered (ali et al., 2010). this work on the biometric measurements of p. denisonii was carried out as part of the study of population characteristics of this species from river valapattanam of the northern part of kerala, india. materials and methods monthly samples were collected during daytime from river valapattanam (latitude 11°93' n; longitude 73°73' e) during the period from august 2012 to july 2013. the fishes were caught by means of the traditional fishing gears veeshu vala (cast net) and koru vala (encircling net). the samples were immediately fixed in10% formalin and transferred to the laboratory. the length-weight relationships (lwrs) were established separately for male, female, indeterminate and pooled using the formula w= alb (le cren, 1951) and the relationship was expressed in the logarithmic form as: log w = log a + b log l. the constants ‘a’ and ‘b’ were estimated using least square method. length-length relationships (llrs) between lt vs lf; lt vs ls, ls vs lh, lh vs lt, lh vs lf and ls vs lf were also calculated by linear regressions. the ponderal index (k) was worked out to assess the well-being of the population with the assumption that the growth of fish in ideal condition maintains equilibrium in length and weight (hile, 1936). the general well-being of the fish (condition factor) was determined by the formula: k = w*100/l3. where, w = weight of the fish in (gm); l = total length of fish (cm). the relative condition factor (kn) is described as the ratio between the actual weight (observed weight) and the calculated weight based on the length weight equation i.e., kn = w/w^, (le cren, 1951), where, w = observed weight and w^= calculated weight. the data used for length-weight relationship were also utilized for calculating the condition factors. condition index (k and kn) was calculated separately for, males, females and pooled samples of different length class (1 cm class). eighteen morphometric and elven meristic characters as shown in figure 1 were studied by following the standard procedures described by dwivedi and menezes (1974). length of the specimen was measured with vernier callipers nearest to 0.1 cm and weighed with an electronic balance nearest to 0.1gm (sartorius gd-303). the length parameters were total length (lt), standard length (ls), fork length (lf), pre dorsal fin length (lprdf), pre-anal fin length (lpraf), pre-pelvic fin length (lprpvf), pre-pectoral fin length (lprprf), dorsal fin length (ldf), pelvic fin length (lpvf), dorsal fin base length (ldfb), anal fin base length (lafb), head length (lh), head height (hh), body depth (db), caudal peduncle depth (dcp), caudal peduncle length (lcp) snout length (lsn) and eye diameter (ed). all morphometric characters were expressed in percentage of total length (lt) and head length (lh). scatter gram of morphometric characters were plotted and the linear regression equation was fitted using least square method described by snedecor and cochran (1967). the relationships were represented by the equation: y = a + bx, where, y: a dependent variable, x= an independent variable, a= a constant (intercept) and b= the regression coefficient (slope). the correlation coefficient (r) is usually calculated to express the degree of linear association or interdependence of two variables. figure 1. schematic representation of puntius denisonii indicating morphometric measurements. 77 sajan et al./ int. j. aquat. biol. (2014) 2(2): 75-84 the significance of regression was tested by anova. the regression coefficients of the sexes and indeterminate were compared by analysis of covariance (anacova) (snedecor and cochran, 1967) to establish the variations in the ‘b’ values, if any, between them. bailey’s t-test (snedecor and cochran, 1967) was employed to find out whether ‘b’ value significantly deviated from the expected cube value of 3 [t= (b-3)/sb], where b = regression coefficient and sb = standard error of ‘b’. the coefficient of correlation ‘r’ was determined in order to know the relationship between the two variables. results and discussion the endemic and endangered status makes, p. denisonii an important freshwater species of study and the biometrics information is useful for more effective management of in-situ conservation. a total of 306 specimens comprising 111 males, 97 females and 98 indeterminate were used for the study. the smallest length of captured fish was 3.30 cm and highest was 14.00 cm in tl (table 1). when empirical values of lengths were plotted against their respective weight on a logarithmic scale, linear line were obtained (fig. 2 a, b, c and d). lwrs of males, females, indeterminate and pooled stock of p. denisonii were established as follows: males: log w = -2.207 + 3.148 log l females: log w = -2.360 + 3.316 log l indeterminate: log w = -2.176 + 3.171 log l pooled : log w = -2.076 + 3.030 log l the length-weight relationship for p. denisonii of pooled population was calculated as log w = -2.076 + 3.030 l log l (r2 = 0.97). the parameter b values of lwrs vary generally between 2.5 to 3.5 are more common (froese, 2006). normally, b is close to 3, indicating fish grow isometrically; values significantly different from 3.0 indicate allometric growth (tesch, 1971). the results of the present study shows that the regression coefficient does not significantly different from 3 (p<0.05) and thus follows the cubic law for isometric growth (grover and juliano, 1976). the calculated ‘b’ varied from a minimum of 2.68 (august) and to a maximum of 3.19 (january). le cren (1951) states that the lwrs in fishes may probably be related to the seasonal variations, as fishes do not retain the same shape or body contour throughout the year. all the earlier reports (brody, 1945; lagler, 1952; brown, 1957; balasunder reddy, 1981; narejo et al., 2003; rekha, 2007; choudhury et al., 2012) are in compliance with the present findings of the length-weight relationship in p. denisonii. the exponent of length-weight relationship b was higher than 3 (males 3.148; females 3.316, indeterminate 3.171 and pooled 3.303) and the 95% higher and lower confidence interval values were also above 3 indicating that the growth of p. denisonii in river valapattanam was isometric. the exponential value of 3.316 implies that the female gain weight at a faster rate in relation to the length than males (3.148). le cren (1951) reported table 1. logarithmic length-weight relationship (lwr) of puntius denisonii. length (cm) weight (gm) a b r2 min max min max august 2012 4.8 6.8 4.8 6.8 -1.81 2.68 0.75 september 2012 7.6 11.5 4.8 6.8 -1.96 2.91 0.96 october 2012 6.3 14 4.8 6.8 -2.01 3.01 0.91 november 2012 6.6 13 4.8 6.8 -2.09 3.10 0.99 december 2012 7.6 11.8 4.8 6.8 -1.96 2.92 0.94 january 2013 6.4 12.9 4.8 6.8 -2.24 3.19 0.97 february 2013 7.5 11.7 4.8 6.8 -1.96 2.87 0.85 march 2013 3.6 12.9 4.8 6.8 -2.01 2.96 0.99 april 2013 4.3 12.1 4.8 6.8 -1.93 2.95 0.99 may 2013 4.7 12.9 4.8 6.8 -2.01 2.95 0.97 june 2013 4.6 12.5 4.8 6.8 -2.07 3.08 0.99 july 2013 3.3 12.4 4.8 6.8 -2.01 2.99 0.99 78 sajan et al./ int. j. aquat. biol. (2014) 2(2): 75-84 that females are heavier than the males of the same lengths probably because of the difference in fatness and gonadal development. in addition, growth increment, differences in age and stage of maturity, food, as well as environmental conditions such as temperature, salinity and seasonality can also affect the value of ‘b’ for the same species (weatherley and gill, 1987; jaiswar and kulkarni, 2002). relationships between lt, lf, lh and ls of p. denisonii along with the estimated parameters of the length-length relationship and the coefficient of determination r2 are presented in table 2. all llrs were highly correlated to each other (0.92 to 0.98). condition factor can be used as an index to assess the status of the aquatic ecosystem in which fish live (anene, 2005). the mean values of condition factor (k) and relative condition factor (kn) worked out separately for male, female and pooled samples (figs. 3 and 4). in the present study, the condition factor (k) vary between 0.8858 to 1.0483 in the pooled sample, 0.8380 to 1.0865 in the male, 0.8282 to 1.004 in the female. the relative condition factor (kn) varied between 0.8180 and 1.9187 in the case of pooled, 0.0342 and 1.9638 in the male, 0.3577 and 3.0043 in the female. kn value of female is higher than that of male in p. denisonii, higher kn values in females may be due to the heavier gonadal development in females (shinkafi and ipinjolu, 2010). in p. denisonii, kn values showed gradual increment to higher length groups of males and females, which may be due to better foraging ability and conservation of stored food materials in adult than juveniles as suggested by ikomi and sikoki (2003) and shinkafi and ipinjolu (2010). table 2. logarithmic length-length relationships (llrs) of puntius denisonii. lt vs lf = y = 0.8671x -0.0563; r² = 0.9771 lt vs ls = y = 0.7892x -0.1385; r² = 0.9873 ls vs lf = y = 1.0832x +0.1855; r² = 0.9619 ls vs lh = y = 0.2199x +0.1947; r² = 0.9746 lh vs lt = y = 0.1737x +0.1632, r² = 0.9638 lh vs lf = y = 0.1939x +0.2159; r² =0.9239 figure 2. scatter plot of logarithmic length-weight relationships (lwrs) of puntius denisonii. 79 sajan et al./ int. j. aquat. biol. (2014) 2(2): 75-84 fluctuations in the condition of different length group may also be in relation to their reproductive cycle, feeding rhythms or physico-chemical factors of environment, age, physiological state of fish or some other unknown factors as suggested by many workers (le cren, 1951; kumar and kurup, 2010). morphometric and meristic features have been commonly used to distinguish the species taxonomically, to identify stocks of fish, and to separate different morphotypes (jayasankar et al., 2004; turan, 2004). in the present investigation, specimens ranging from 5.70-10.50 cm lt were used for the morphometric and meristic characteristics studies. measurements of various morphometric characters of p. denisonii, such as mean, median, standard deviation and coefficient of variation are presented in table 4 and 5. regarding proportional values between each character and lt, body depth (db) showed a maximum coefficient of variation (21.42%), while snout length (lsn) showed minimum variation (10.07%). body depth (db) of fishes mostly depends upon the stage of reproduction figure 3. condition factor (k) and relative condition (kn) of puntius denisonii (pooled). figure 4. condition factor (k) and relative condition (kn) of puntius denisonii. table 3. meristic characters of puntius denisonii. character number character number single fins: gill rakers (upper and lower) 26 dorsal fin (spines-rays) ii-9 number of barbel 02 anal fin (spines-rays) i-6 number of lateral line 01 caudal fin (rays) 22-24 scale along lateral line 24-26 adipose fin absent scale above lateral line 06 paired fins: scale below lateral line 04 pectoral fin (spines-rays) 1-16 total vertebrae 28 pelvic fin (spines-rays) i-8 pre anal vertebrae 17 80 sajan et al./ int. j. aquat. biol. (2014) 2(2): 75-84 (ripe, spawning, or spent) especially in females; it may be resulted by the variation of body height (bh) in related to growth (gunawickrama, 2008). the growth of the morphometric characters in relation to the total length was noted to be the least in the snout length (b=0.247) and the highest in the fork length (b=0.869). high degree of correlation of morphometric characters with total length is evident from r-values which ranged from 0.7527 to 0.9841 (tables 5 and 6). the morphometric characteristic such as lf, ls, db, lprdf, lpraf, lafb, dcp, lh, hh and ed were highly correlated with total length (r>0.990), as well as hh (0.965), ed (0.903) and lsn (0.834) were highly correlated with head length (lh) (fig. 6). the coefficient of correlation of head length (lh) against compared characters ranged from minimum of 0.6955 for snout length to maximum of 0.9325 for head height (fig. 5). in general, fishes table 4. statistical estimate of morphometric characters of puntius denisonii. table 6. regression analysis of morphometric characters of puntius denisonii as function of head length (lh) min= minimum, max= maximum; sd=standard deviation; c.v= coefficient of variation; lt (%) = percentage of total length; lh (%) = percentage of head length table 5. regression analysis of morphometric characters of puntius denisonii as function of total length (lt) 81 sajan et al./ int. j. aquat. biol. (2014) 2(2): 75-84 demonstrate greater variance in morphological traits both within and between populations, and are more susceptible to environmentally induced morphological variation (wimberger, 1992; swain and foote, 1999), which might reflect different feeding environment, prey types, food availability or other features. the results of various meristic characters were presented in table 3. based on the results from the present study, the fin formula for p. denisonii from river valapattanam could be written as: d ii-9, p i-16, v i-8, a i-6 and c 22-24. these results are in agreement with earlier studies (ali et al., 2010). the riverine ecosystem of valapattanam is affected by pollution, pesticides, habitat destruction and sand mining. swain and foote (1999) reported that, biometric characters of fishes may influence by environmental changes. during our study, we observe different types of malformation such as, vertebral deformity, semi-operculum and puff out belly in p. denisonii (day, 1865) from valapattanam river (unpublished results). similar reports of skeletal deformities in freshwater fish species in bhavani river (raj et al., 2004). these studies show rivers of kerala facing serious threats by these kinds of illegal and indiscriminate man-made alterations. these findings would give information to fishery biologists about morphometric, meristic characteristics, length-weight relation, length-length relation and condition index of redline torpedo barb, puntius denisonii (day, 1865) in the river valapattanam in southern india. acknowledgements we express our sincere thanks to college of fisheries, kerala university of fisheries and ocean studies (kufos), ernakulum, kerala, india for providing laboratory facility for this research work and express sincere gratitude to noushad, santhosh, ubaid, manoos and shaji for assistance during sample collection. the author was also thankful to the government of kerala for the financial assistance during 2010-2013. references anene a. 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(2019) 7(1): 38-44 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article species diversity of rotifers (rotifera: eurotatoria) of phu ninh lake with five new records from vietnam mau dang trinh *1, minh van vo1, anh nguyen quynh tran2, huyen thi ngoc le1, son ngoc tran1 1faculty of biology and environmental sciences, the university of danang university of education, danang, vietnam. 2faculty of chemistry, the university of danang university of education, danang, vietnam. s article history: received 29 august 2018 accepted 22 february 2019 available online 2 5 february 2019 keywords: biodiversity freshwater rotifera zooplankton abstract: a total of sixty-one taxa of rotifer, belonging to 15 families, 3 orders were identified from the phu ninh lake, vietnam. of these, five species were recorded new to vietnam rotifera fauna, including lecane acanthinula (hauer, 1938), l. sola (hauer, 1936), l. thailandensis segers & sanoamuang, 1994, l. pyriformis (daday, 1905), and mytilina bisulcata (lucks, 1912). the result raised the total number of rotifers known from vietnam to 227 species. moreover, the results showed that the number of species recorded increased with the increase of sample size, which followed the equation y=12.85+14.12log(x) (r2 = 0.99). based on the estimators, the α-diversity of rotifers in phu ninh lake might be up to 67±4 taxa (according to the bootstrap index), 71±8 taxa (according to the chao index), or even up to 79 taxa (according to jacknife 2 index). introduction rotifera is widely distributed in inland aquatic habitats playing an important role in freshwater ecosystem functioning. moreover, they can be used as indicators of water quality (sládeček, 1983), toxicology test organisms (arnold et al., 2011), and live feeds for fish larvae in aquaculture (lubzens, 1987; ogata and kurokura, 2011). although rotifer has such great roles, the knowledge of rotifer diversity in vietnam is still poor (trinh-dang et al., 2013, 2015). the first record of rotifer diversity in vietnam was reported by shirota (1966) with 63 species in fresh and saline water environments. dang et al. (1980) recorded 52 species of rotifer in freshwater bodies in south vietnam. the number of rotifer species recorded by a few studies after that were 65 species in central vietnam (zhdanova, 2011) and 49 species in south vietnam (phan and le, 2012). most notably, the recent studies conducted by trinh-dang et al. (2013, 2015) documented one hundred new record and four new species for vietnam rotifers fauna in freshwater bodies of the thua thien hue province indicating the high potential of rotifer diversity in *correspondence author: mau dang trinh doi: https://doi.org/10.22034/ijab.v7i1.581 e-mail: tdmau@ued.udn.vn vietnam. in order to contribute more knowledge on rotifers diversity for vietnam, we conducted the study on species diversity of rotifera of the phu ninh lake, the largest reservoir in the central of vietnam, which plays an important role for supplying water and regulating climate to the central of vietnam. materials and methods the phu ninh lake is a mesotrophic lake situated in northern quang nam province with a surface area of 23,409 ha (fig. 1). thirty plankton samples were collected in rainy season (december 2017) and dry season (april 2018). qualitative samples of rotifers were collected using a 50 μm mesh size cast-net and then preserved in 4% formaldehyde. rotifer specimens were sorted and examined using a hund (h600) compound microscope equipped with a camera. the trophi of rotifers was examined by adding a drop of commercial sodium-hypocloride (naocl) to dissolve and isolate the hard trophi parts. the species accumulator and species richness estimators were calculated using the vegan package (oksanen et al., 2013) in r program (r development 39 int. j. aquat. biol. (2019) 7(1): 38-44 core team, 2018). of these, jacknife 2 (eq. i) and chao 2 (eq. ii) estimators were selected to estimate the expected diversity of rotifer in phu ninh lake, as the following formulas: 𝐽𝑎𝑐𝑘2 = 𝑆0 + 𝑓1 2𝑁−3 𝑁 − 𝑓2 (𝑁−2)2 𝑁(𝑁−1) (equation i; smith and van belle, 1984) 𝐶ℎ𝑎𝑜2 = 𝑆0 + ( 𝑁−1 𝑁 )( 𝑓1(𝑓1−1) 2 2(𝑓2+1) ) (equation ii; chao, 1987) where: so = observed number of species in the pooled samples series, f1 = number of singletons; f2 = number of doubletons, and n = total number of samples. results species composition: a total of sixty-one taxa of rotifer, belonging to 15 families, and 3 orders were identified from the phu ninh lake (table 1). of these, five species were recorded new to vietnam rotifera fauna, including lecane acanthinula (hauer, 1938), l. sola (hauer, 1936), l. thailandensis segers & sanoamuang, 1994, l. pyriformis (daday, 1905), and mytilina bisulcata (lucks, 1912) (table 1; figs. 2-6). of the total 15 families recorded in the phu ninh lake, the most diverse family was lecanidae (18 taxa, 29.51 %), followed by brachionidae (14 taxa, 22.95%), trichocercidae (4 taxa, 6.56%) and synchaetidae (4 taxa, 6.56%). four families viz. collothecidae, hexarthridae, testudinellidae, and asplanchnidae were present in single species (fig. 7). species richness: the number of taxa recorded in the dry season (59 taxa) was higher than that of rainy season (37 taxa). the taxonomic species composition markedly differed between the two seasons. lecanidae was the most diverse family in the dry season with 17 taxa (28.81% of total taxa recorded in dry season), while brachionidae was the most diverse family in the rainy season with 11 taxa (29.73% of figure 1. schematic map of the study area at phu ninh lake. figure 2. lecane sola hauer, 1936, habitus dorsal view. 40 trinh-dang et al./ species diversity of rotifers from lake in central vietnam total taxa recorded in rainy season). the study also investigated that the average number of rotifer species per sample in the dry season (18±8 taxa) was higher than that in the rainy season (11±3 taxa) (fig. 8). to identify the species richness, we constructed species accumulation curves based on the rarefaction method, and fitted with a logarithmic model in each study site and in each season (figs. 9, 10). the result collothecaceae lecane acanthinula hauer, 1938 k* collothecidae lecane batillifer murray, 1913 k collotheca ornata (ehrenberg 1832) k m lecane bulla gosse 1851 k m flosculariaceae lecane closterocerca schmarda 1859 k m conochilidae lecane crepida harring 1914 k conochilus dossuarius (hudson 1885) k lecane curvicornis murray, 1913 k m conochilus unicornis (rousselet 1892) k m lecane doryssa harring, 1914 k trochosphaeridae lecane thailandensis segers & sanoamuang, 1994 k* filinia camasecla (myers 1938) k m lecane hamata stokes, 1896 k m finilia longiseta (ehrenberg 1834) k lecane hornemanni ehrenberg 1834 k m hexarthridae lecane luna müller 1776 k hexathra intermedia (wiszniewski) k m lecane lunaris ehrenberg, 1832 k m testudinellidae lecane obtusa murray, 1913 k testudinella patina (hermann 1783) k lecane rhenana hauer, 1929 k ploima lecane pyriformis (daday, 1905) k* asplanchnidae lecane ruttneri hauer, 1938 m asplanchna priodonta (gosse 1850) k m lecane sola hauer, 1936 k * brachionidae lecane thienemani hauer, 1938 k anuraeopsis navicula (rousselet 1911) k m lepadellidae brachionus angularis (gosse, 1851) k m colurella obtusa hauer, 1936 k brachionus calyciflorus (pallas, 1766) k m lepadella ovalis müller, 1786 k brachionus caudatus (barrois & daday 1894) k m lepadella patella müller 1773 k m brachionus dichotomus (shephard 1911)k m mytilinidae brachionus diversicornis (daday 1883) k mytilina ventralis ehrenberg, 1830 k brachionus donneri ( brehm, 1951) k m myttilina bisulcata lucks, 1912 k* brachionus falcatus zacharias, 1898 k m synchaetidae brachionus forficula wierzejski 1891 k m polyarthra dolichoptera idelson 1925 k m brachionus patulus müller, 1786 k polyarthra major burckhardt, 1900 k m brachionus quadridentatus hermann 1783 k m polyarthra vulgaris carlin 1943 k m keratella cochlearis gosse 1851 k m synchaeta stylata wierzejski 1893 k m keratella quadrata müller 1786 k trichocercidae keratella tropica apstein, 1907 k m trichocera longiseta alexander, 1927 k m euchlanidae trichocerca chattoni de beauchamp, 1907 k m euchlanis dilatata ehrenberg 1832 k m trichocerca cylindrica imhof 1891 k m euchlanis meneta myers,1930 k trichocerca similis wierzejski, 1893 k m gastropodidae trichotriidae ascomorpha ecaudis perty, 1850 k m macrochaetus collinsii gosse 1867 k ascomospha ovalis bergendal, 1892 k m macrouchaetus longipes myers, 1934 k ascomospha saltan bartsch, 1870 k m trichotria tetractis ehrenberg, 1830 k lecanidae table 1. list of rotifer fauna from phu ninh lake, quang nam province (* = new to vietnam, k = dry season, m = rainy season). 41 int. j. aquat. biol. (2019) 7(1): 38-44 showed that the average species number at each locality was 13 taxa, and the observed number of species in the pooled samples series was 61 taxa (fig. 9). besides, the number of species increased with the increase of sample size, following the equation y=12.85+14.12log(x) (r2=0.99). based on the estimators, the expected number of taxa in phu ninh lake could be 71±8 taxa (according to the chao index), 79 taxa (according to jacknife 2 index), and 67±4 taxa (according to the bootstrap index) (fig. 9). comparing the species richness recorded between two seasons, the number of species in the dry season (59 taxa) was higher. moreover, the number of recorded taxa also tended to increase with the increase of the sampling efforts in both seasons. the trend of increase was identified to follow the model as y=16.93+15.3log(x) (r2=9,998) in the dry season and y=10.11+10.02log(x) (r2=0.998) in the rainy season. this trend was also reflected in the cumulative curve model, in which the model slope in dry season (15.30) was higher than that in rainy season (10.02). in addition, the estimator, which estimated the maximum number of taxa could be present in the lake, again confirmed the higher species diversity in the dry figure 5. lecane pyriformis (daday, 1905), habitus 1: dorsal view and 2: ventral view. figure 6. mytilina bisulcata lucks, 1912, habitus lateral view. figure 7. percentage of species in each family of rotifera in phu ninh lake. figure 3. lecane acanthinula hauer, 1938, habitus 1: dorsal view and 2: ventral view. figure 4. lecane thailandensis segers & sanoamuang, 1994, habitus. 1: dorsal view and 2: ventral view. 42 trinh-dang et al./ species diversity of rotifers from lake in central vietnam season. in the detail, according to chao 2 estimator index, the number of taxa that could appear in the dry season was 78±13 taxa, and in rainy season was 39±3 taxa. according to the jackknife 2 index, these numbers were 85 taxa in the dry season, and 42 taxa in the rainy season. according to the bootstraps index, the numbers were 67±4 taxa and 41±3 taxa in dry season and rainy season, respectively (fig. 10). discussions this study reports 61 taxa of rotifer for phu ninh lake, quang nam province, increasing the number of rotifer fauna for vietnam to 227 taxa. of 61 taxa identified from phu ninh lake, l. acanthinula (hauer, 1938), l. sola (hauer, 1936), l. thailandensis segers & sanoamuang, 1994, l. pyriformis (daday, 1905) and m. bisulcata (lucks, 1912) are new to vietnam. these results confirmed the high potential of rotifer diversity in the country. the diversity of rotifer in phu ninh lake was lower than that in some other freshwater bodies in the central vietnam such as bau thiem (89 taxa) and thuy tien lakes (82 taxa) in thua thien hue province (trinh-dang et al., 2013, 2015). it was also lower than the rotifer diversity of the kud-thing lake, noing kai province, northern thailand (183 taxa). however, in compared to some lakes located in the southern vietnam (tra su=9 taxa, bung binh thien=24 taxa, figure 8. boxplot showing the average species number of each sample between dry and rainy season at phu ninh lake. figure 9. species accumulator of species richness at study sites, with the fitted curve and estimator curve. figure 10. species accumulator with fitted curve and estimator curve of species richness at study sites during dry (left) and rainy season (right). 43 int. j. aquat. biol. (2019) 7(1): 38-44 tri an=33 taxa (dang and nga, 2012), da ban=16 taxa, kam lam=19 taxa, suoi dau=20 taxa, and suoi trau=45 taxa (zhdanova, 2011)) and a lake in turkey (35 taxa) (yağci and ustaoğlu, 2012), the phu ninh lake showed a higher level of diversity. although there was a difference in the taxa number and rotifers species composition, the phu ninh lake was rather similar to the two lakes in thua thien hue province (thuy tien and bau thiem lakes) (trinhdang et al., 2013, 2015). in these three lakes, lecanidae was the most abundant family (18 species with 27% and 42 species with 40%, respectively), followed by brachionidae family (9 species with 13% and 11 species with 11%, respectively). this similarity might be due to the resemblance in the lakes environmental conditions. all these three lakes were the reservoirs with nutrients ranging from poor to medium (trinh-dang et al., 2013, 2015). this also explained the difference in the species structures between phu ninh lake and other reservoirs such as suoi dau, cam lam and da ban reservoirs, where the brachionidae was the most diverse family (zhdanova, 2011). conclusions with sixty-one taxa of rotifer and in which five new species for vietnam recorded in phu ninh lake, this study provided more evidence about the high potential of rotifers diversity in vietnam. it is should be noted that rotifer species number in the lake does not stop at the number sixty-one since the study found that the taxa number tended to increase with the collected sample number. it is suggested that more surveys should be conducted in this site and also in other areas with different environmental conditions to supply more information on this group in vietnam. acknowledgements we would like to thank the faculty of biology and environmental science, university of science and education udn for providing research facilities. this research is funded by funds for science and technology development of the university of danang under project number b2018-ddn03-26. references arnold w.r., diamond r.l., smith c.d. (2011). acute and chronic toxicity of copper to the euryhaline rotifer, brachionus plicatilis (“l” strain). archives of environmental contamination and toxicology, 60: 250-260. chao a. (1987). estimating the population size for capturerecapture data with unequal catchability. biometrics, 43: 783-791. dang p.d., nga l.t.n. (2012). diversity on rotifera species competitions in fresh inland waters of southern vietnam and some new records for zooplankton fauna of vietnam. journal of biology, 34 (3): 13-20. (in vietnamese) dang n.t., thai t.b., pham v.m. (1980). classification of fresh-water invertebrates in northern viet nam. science publishing house, ha noi. 573 p. (in vietnamese) lubzens e. (1987). raising rotifers for use in aquaculture. hydrobiologia, 147: 245-255. macêdo r.l., lopes v.g., kozlowskysuzuki b., branco c.w. (2018). zooplankton community attributes in an oligo-mesotrophic reservoir: a comparative study of two sampling strategies. anais da academia brasileira de ciências, 91(1). oksanen j., blanchet f.g., kindt r., legendre p., minchin p.r., o’hara r.b., simpson g.l., solymos p., stevens m.h.h., wagner h. (2013). the vegan package. community ecology package. ogata y., kurokura h. (2011). use of the freshwater rotifer brachionus angularis as the first food for larvae of the siamese fighting fish betta splendens. fisheries science, 78: 109-112. r development core team (2018). r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. available from: http://www.r-project.org. sanoamuang l., savatenalinton s. (2001). the rotifer fauna of lake kud-thing, a shallow lake in nong khai province, northeast thailand. hydrobiologia, 446/447: 297-304. segers h., sanoamuang l. (1994). two more new species of lecane (rotifera, monogononta) from thailand. belgian journal of zoology, 124: 39-46. segers h. (1995). guides to the identification of the microinvertebrates of the continental waters of the world: volume 2. rotifera: the lecanidae (monogononta). spb academic publishing. 226 p. 44 trinh-dang et al./ species diversity of rotifers from lake in central vietnam sharma b.k. (2004). rare and interesting monogonont rotifers (rotifera, eurotatoria) from north‐eastern india. zoosystematics and evolution, 80: 33-40. shirota a. (1966). the plankton of south viet nam: freshwater and marine plankton. overseas technical cooperation agency, tokyo. 463 p. sládeček v. (1983). rotifers as indicators of water quality. hydrobiologia, 100: 169-201. smith e.p., van belle g. (1984). nonparametric estimation of species richness. biometrics, 40: 119-129. zhdanova s.m. (2011). the species composition of rotifers in the water reservoirs of central vietnam. inland water biology, 4(4): 425-434. yağci m.a., ustaoğlu m.r. (2012). zooplankton fauna of lake i̇znik (bursa, turkey). turkish journal of zoology, 36(3): 341-350. int. j. aquat. biol. (2017) 5(5): 286-294; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article effects of dietary supplementation of zinc oxide nanoparticles on some biochemical biomarkers in common carp (cyprinus carpio) somayeh taheri, mahdi banaee*,1behzad nematdoost haghi, mohammad mohiseni aquaculture department, environment and natural resource faculty, behbahan khatam alanbia university of technology, iran. article history: received 23 august 2017 accepted 6 october 2017 available online 2 5 october 2017 keywords: zno-nps oxidative stress common carp biochemical parameters abstract: if the dose and duration of zinc oxide nanoparticle (zno-nps) supplementation optimize, low concentrations of zn nanoparticles can replace conventional zn sources in diets of different species of fish. since evaluating the cytotoxicity of any nutritional supplement is one of the requirements for optimizing the dose for a specified time, we conducted this study to investigate the effects of oral administration of zno-nps on oxidative stress and certain biochemical biomarkers in common carp, cyprinus carpio, as an experimental model. for this purpose, zno-nps were orally administered to fish for 21 days at 0 (control), 5, 10 and 15 mg kg-1 feed. administration of znonps (15 mg kg-1) significantly enhanced aspartate aminotransferase (ast), and lactate dehydrogenase (ldh) activities in liver, and alanine aminotransferase (alt), alkaline phosphatase (alp), and ldh activities in kidney. dietary zno-nps increased glucose-6-phosphate dehydrogenase (g6pdh) activity in liver of fish. the results indicated that administration of 10 mg kg-1 and 15 mg kg-1 zno-nps caused a significant increase in alt and catalase (cat) activities and malondialdehyde (mda) levels in liver, ast and cat activities and mda levels in kidney. znonps decreased the liver alp activity. administration of 5 mg kg-1 zno-nps significantly increased the cellular total antioxidant (ta) levels in various tissues. therefore, we suggest that oral administration of 10 and 15 mg kg-1 zno nps caused cytotoxicity and alterations in oxidative biomarkers, but 5 mg zno-nps per kg feed had no side effects on oxidative stress and biochemical biomarkers in fish. introduction zinc is an essential trace element for finfish and plays a critical role in biological processes and physiologyical functions such as biosynthesis of hormones, enzymatic activity, and metabolism of proteins and carbohydrates (wang and wang, 2015). the activity of more than 300 enzymes and around 2000 transcription factors in varied species of animals is closely related to zinc (chen et al., 2015; wang and wang, 2015; swain et al., 2016). zinc ions specifically bind to the receptors of cell membranes, carriers and channels and regulate their activity (swain et al., 2016). this element is vital in regulating the reception of cellular signals, metabolism of secondary messengers, the activity of protein kinases *corresponding author: mahdi banaee doi: https://doi.org/10.22034/ijab.v5i5.329 e-mail address: mahdibanaee@yahoo.com and phosphatases, as well as the binding of transcription factors to dna (chen et al., 2015). therefore, zinc deficiency can lead to a lower growth rate, increased mortality, cataracts, fins and skin erosion and dwarfism (wang and wang, 2015). the presence of tri-calcium phosphate in fish meal, phytate or phytic acid in soybean meal and other oil seeds and grains may reduce the bioavailability minerals, such as zinc and manganese in diet of freshwater fish, including carp and rainbow trout (hossain et al., 2003). therefore, using zinc supplement in foodstuff may prevent zinc deficiency (hossain et al., 2003). nevertheless, one of the consequences of zinc supplements in foodstuff is an increase in zn excretion from fish body and an increase in its concentration in 287 int. j. aquat. biol. (2017) 5(5): 286-294 the environment (swain et al., 2016). that is why researchers are looking for ways to decrease zinc content in food supplements and increase its bioavailability in diets (swain et al., 2016). with regard to the physiological, chemical and biological properties of zinc oxide nanoparticles (zno nps), their usage in food supplements could be an appropriate strategy for the aforementioned problem (swain et al., 2016). this has recently increased interests in using nano zinc oxide as a food supplement, a growth promoter, an antioxidant and antimicrobial compound and an immune-modulatory agent in diets of varied species of farmed animals (swain et al., 2015). moreover, using metal nanoparticles as a dietary and medical supplement is considered a new approach in pharmacology (bahrami et al., 2017). therefore, it is essential to study the side effects of these compounds on the health of experimental laboratory models (bahrami et al., 2017). dietary zno nps are so small that are easily absorbed by the digestive system (swain et al., 2016) and then distributed in different tissues, especially the liver (swain et al., 2016). this element can demonstrate its short-term effects on biochemical processes and physiological functions of cells (muthuraman and kim, 2015). regardless of nutritional and commercial aspects of aquaculture, fish can be used as a model in pharmaceutical toxicology (chen et al., 2017). data on the toxic effects of zinc in diets of different species of animals abound (vandebriel and de jong, 2012; pandurangan and kim, 2015a; pandurangan and kim, 2015b). also, there are several studies on the toxic effects of environmental zno nps on fish (hao and chen, 2012; hao et al., 2013; connolly et al., 2016; (xiong et al., 2011; cong et al., 2017; fernández, garcía-gómez and babín, 2013); however, there is not much information on the toxicological effects and potential risks of zno nps in high concentrations in diets of fish (swain et al., 2016). depending on the zno nps concentration and the exposure duration, their cellular toxicity is attributed to oxidative stress, lipid peroxidation, and damage to the cell membrane and oxidative damage to dna (najafzadeh et al., 2013; pandurangan and kim, 2015a; pandurangan and kim, 2015b). using zno nanoparticles may not sound cost-effective, but using metal nanoparticles can be used as a novel approach in treating many difficult-totreat diseases such as cancer (bahrami et al., 2017). thus, the purpose of this study was to investigate consequences of using zinc oxide nanoparticles and to determine the nontoxic dose in foodstuff of common carp. materials and methods fish: one hundred forty-four immature common carp, cyprinus carpio (mean weight 20.5±2.5 g) were obtained from a local fish farm (ahvaz, khuzestan province, iran) and were randomly distributed into twelve circular tanks of 80 l capacity (12 fish per each tank) at the department of aquaculture (khatam alanbia university of technology). prior to the experiment, fish were adapted in tap water (24±2°c; ph, 7.4±0.2; 50% water exchange rate/day) for two weeks. the fish were subjected to artificial light (16 l/8d). during the adaptation period, common carp were fed 2 times a day with commercial pelleted feed (3% of their body weight) according to the manufacturer’s recommendations (beyza feed mill, shiraz, iran). diet preparation: the formulated fish feed was enriched with nano-particles of zinc oxide (iranian nano-materials pioneers company, iran; table 1, figs. 1-3). nano-particles of zinc oxide were figure 1. tem micrographs of the nano-zno powders (adapted from iranian nano-materials pioneers company’s catalog). 288 taheri et al./ effects of dietary zno-nps on some biochemical biomarkers in common carp supplemented at 5, 10 and 15 mg per kg feed for a total of three treatments. zno nanoparticles were prepared using distilled water and then ultrasonicated (10 min, 35 khz, 100/400w) using an ultrasound bath (elma, germany) (banaee et al., 2016). then, solutions were added to powdered feed in order to obtain nominal concentrations of 5, 10 and 15 mg zno nps per kg. each supplemented diet was mixed in a mixer for 30 minutes and then homogenized into a paste by adding fish oil (20 ml kg-1) and distilled water into the food mixer. the amount of distilled water required for pelleting (20-40% of feed weight) was then added to the mixture and further homogenized. this mixture was passed through a meat grinder, producing string shapes, which were dried in an oven at 55°c for 12 h and then broken to produce 5 mm pellets. the pellets were packed and stored at -20°c in a freezer. the control diet was prepared by the same process, although no supplement was added. experimental design: during the experimental period, fish fed commercial pelleted feed with 0 (control), 5, 10 and 15 mg kg-1 zno nps supplement for 21 days. after 21 days, 12 fish per group were sampled randomly and then anesthetized with clove powder solution (200 mg l-1). fish were sacrificed by decapitation and dissected to remove the liver, and kidney. for enzymatic and biochemical analyses, tissue samples from the target organs were homogenized on ice in cold buffer 100 mm potassium phosphate (sigma-aldrich, germany) ph 7.0 containing 2 mm of edta (riedel-haën, germany). tissue homogenates were centrifuged at 12,000x g for 15 minutes at 4°c. the supernatant was removed and frozen at -25°c for further analysis. biochemical parameters analysis: aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ldh) and alkaline phosphatase (alp) were determined by the method of (moss and henderson, 1999). glucose-6-phosphate dehydrogenase (g6pdh) activity was measured by the method of gómez-milán and lozano (2007). protein levels in tissues were determined by standard procedures of (johnson et al., 1999). for determinations test kits from pars azemun co, iran, were used. cat activity was determined as the decrease of absorbance at 450 nm due to hydrogen peroxidase figure 2. sem micrographs of the nano-zno powders (adapted from iranian nano-materials pioneers company’s catalog). zinc oxide zno purity +99.9 % average primary particle size (d50) 10-30 nm specific surface area (ssa) 20-60 m2 g-1 color white bulk density 5.606 g cm-3 table 1. zinc oxide nanoparticles physicochemical proprieties (adapted from iranian nano-materials pioneers company’s catalog). figure 3. the x-ray powder diffraction (xrd) curves of nanocrystalline zno (adapted from iranian nano-materials pioneers company’s catalog). 289 int. j. aquat. biol. (2017) 5(5): 286-294 consummation as described by (góth, 1991), although with some modifications. total antioxidant capacity was estimated according to the ferric reducing ability of plasma (frap) as described by (benzie and strain, 1996) using tptz (2,4,6-tris(2-pyridyl)-s-triazine) as a substrate. malondialdehyde (mda) content was assessed by modified thiobarbituric acid assay according to (placer et al., 1996). all biochemical parameters were measured by uv/vis spectrophotometer (model biochrom libra s22). statistical analysis of the results was carried out by one-way anova; the data were checked for assumptions of normality and homogeneity (shapirowilk test) and when necessary, they were appropriately transformed. the duncan test was used to compare pairs of means and detect significant differences (p<0.05). the statistical analysis was performed at the significance level of 5%, using ibm spss 19. data are presented as mean ±sd. results during the experiment, mortality was not observed in the control group and fish fed zno nps supplement. hepatic biomarkers: the activities of hepatic marker enzymes are shown in table 2. activities of ast and ldh were found to be significantly increased in the 15 mg kg-1 zno nps-administrated group (p<0.05). moreover, alt, and cat activities in liver tissue was enhanced by 10 mg kg-1 and 15 mg kg-1 zno npssupplemented diets at day 21 compared to the control group (p<0.05). administration of zno nps caused a reduction in alp activities. in this study, dietary zno nps significantly increased g6pdh activity in liver of fish (p<0.05). compared with control diet, 10 and 15 mg kg-1 zno nps in the diet significantly enhanced malondialdehyde (mda) levels in liver of fish (p<0.05). supplementing 5 mg kg-1 zno nps in the diet increased the total antioxidant levels in liver of fish (table 2). biochemical parameters control 5 mg zno-nps per kg feed 10 mg zno-nps per kg feed 15 mg zno-nps per kg feed ast (u g-1 protein) 0.51±0.07a 0.53±0.21a 0.58±0.11a 1.27±0.33b alt (u g-1 protein) 0.17±0.03a 0.20±0.02a 0.23±0.03b 0.26±0.05b alp (u g-1 protein) 1.50±0.38a 0.99±0.08b 0.80±0.05b 0.82±0.17b ldh (u g-1 protein) 1.33±0.41a 1.34±0.15a 1.25±0.19a 1.87±0.32b g6pdh (u g-1 protein) 8.14±1.65a 18.36±2.32b 16.92±1.98b 16.49±2.77b cat (ku g-1 protein) 7.31±2.17a 9.10±1.36a 21.38±2.91b 23.15±2.79b ta (µm g-1 tissue) 10.97±2.37a 26.64±5.27b 11.79±3.02a 10.71±0.88a mda (µm g-1 tissue) 0.03±0.01a 0.03±0.01a 0.06±0.01b 0.19±0.05c different superscripts indicate the significant difference (p<0.05, duncan’s multiple comparison). data are expressed as means ±s.d. aspartate aminotransferase (ast); alanine aminotransferase (alt); alkaline phosphatase (alp); lactate dehydrogenase (ldh); glucose6-phosphate dehydrogenase (g6pdh); catalase (cat); malondialdehyde (mda); total antioxidant (ta). table 2. effects of dietary supplementation of zinc oxide nanoparticles (5, 10 and 15 mg kg-1 feed) on some biochemical biomarkers in liver of common carp (cyprinus carpio). biochemical parameters control 5 mg zno-nps per kg feed 10 mg zno-nps per kg feed 15 mg zno-nps per kg feed ast (u g-1 protein) 0.49±0.04a 0.46±0.05a 0.40±0.05b 0.33±0.06c alt (u g-1 protein) 0.08±0.01a 0.06±0.01a 0.07±0.01a 0.13±0.01b alp (u g-1 protein) 1.50±0.07a 1.54±0.29a 1.45±0.08a 2.12±0.60b ldh (u g-1 protein) 1.55±0.26a 1.65±0.14a 1.75±0.20a 2.02±0.41b cat (ku g-1 protein) 2.87±0.86a 3.64±0.77a 4.88±1.08b 5.96±0.61c ta (µm g-1 tissue) 4.09±0.98a 9.18±2.83c 8.06±3.75bc 6.14±1.29ab mda (µm g-1 tissue) 0.04±0.01a 0.03±0.01a 0.13±0.03b 0.26±0.06c different superscripts indicate the significant difference (p < 0.05, duncan’s multiple comparison). data are expressed as means ± s.d. -aspartate aminotransferase (ast); alanine aminotransferase (alt); alkaline phosphatase (alp); lactate dehydrogenase (ldh); catalase (cat); malondialdehyde (mda); total antioxidant (ta). table 3. effects of dietary supplementation of zinc oxide nanoparticles (5, 10 and 15 mg kg-1 feed) on some biochemical biomarkers in kidney of common carp (cyprinus carpio). 290 taheri et al./ effects of dietary zno-nps on some biochemical biomarkers in common carp kidney biomarkers: cat activity in kidney of fish fed with 10 and 15 mg kg-1 zno nps-supplemented diet was significantly higher than that found in fish fed 0.0 mg kg-1 zno nps-supplemented diet (p<0.05). ast activity was found to be elevated after the administration of 10 and 15 mg kg-1 zno nps. the findings demonstrated that alt, ldh and alp activities in kidney was enhanced in fish fed with 15 mg kg-1 zno nps as compared with control group (p<0.05). mda levels statistically increased in kidney of fish fed with 10 mg kg-1 and 15 mg kg-1 zno nps compared to the control group (p<0.05). the total antioxidant levels was significantly increased in kidney of fish fed with 5 mg kg-1 and 10 mg kg-1 zno npssupplemented diet on day 21 (table 3). discussion the required amount of zinc in diets of farmed common carp is between 15-30 mg kg-1 feed which is usually added as mineral salts, including zinc oxide or zinc sulphate (davis and gatlin, 1996). due to the use of oil seeds in the base diet, the bioavailability of zn may decrease for fish (gupta et al., 2015). since the physiological function of zinc is affected by its way of transfer and storage in the aquaculture (muralisankar et al., 2014), using zinc supplement in the form of nanoparticles may solve this issue. therefore, we evaluated the influence of zno nps in common carp on preventing zinc deficiency in the long term. we aimed at assessing oxidative stress biomarkers (as a general biomarker) in tissues of zno nps-treated carp. common carp were fed 5, 10, and 15 mg kg-1 zno nps in a 21-day experiment. ast and alt activities are important in cellular nitrogen metabolism, oxidation of amino acids, and liver gluconeogenesis (murray et al., 2003). the increased activity of ast and alt in tissues of zno nps-treated fish may indicate the increased rate of proteins metabolism in cells. a similar increase of ast and alt activities were previously reported by (fazilati, 2013) in the serum of zno nps-treated rats. in contrast, a decrease of plasma alt and ast activities was observed in chicken broilers that were fed with zno nps supplement dietary (fathi, 2016). alp plays a significant role in phosphate hydrolysis and in membrane transport and it also acts as a good biomarker of stress in biological systems (murray et al., 2003). the administration of zno nps for 21 days caused a significant decrease in alp activity in liver of fish. an increase in zinc level in liver can account for a reduced alp activity because high levels of zinc can have deterrent effects on alp activity (farah et al., 2012). increased alp activity in kidney may be due to the effects of zno nps on transphosphorylation activity as well as a metabolic dysfunction in cells. an increase in ldh activity in liver, and kidney may be caused by metabolic stress (muthuraman and kim, 2015). metabolic stress in hepatic and renal cells is dose-dependent. an increased ldh is reported in lung cells of rats and myoblast cell line of mice (muthuraman and kim, 2015; kao et al., 2012. muthuraman and kim (2015) found that zno nps increased ast, alt, alp and ldh activities and their mrna expression in c2c12 cells. an increase in ast, alt, ldh and alp activity was observed in plasma of common carp treated with high doses of zno nps (lee et al., 2014). zinc toxicity depends on the concentration of free ions (kool et al., 2011). increasing the concentration of zinc ions in the cytoplasm and influx of zn+2 from cytosol to mitochondria can affect permeability and stability of mitochondrial membrane, trigger caspase activation and cell apoptosis (pandurangan and kim, 2015b; kao et al., 2012). acidic lysozyme accelerates the release of zn+2 ions which accompanies the oxidative stress and damage to mitochondrial membrane (fröhlich and fröhlich, 2016). in high concentrations, zno nps may disturb the homeostasis of ion in cytoplasm (kao et al., 2012) and accordingly disturb the biochemical balance in cells. g6pdh is the rate-limiting enzyme in the pentose phosphate pathway and a key contributor to carbohydrate and fatty acid metabolism (murray et al., 2003). previous studies show that alterations in g6pdh activity are critical for cells (mehrpak et al., 2015). g6pdh plays an important role in cell growth by providing nadph and therefore leading to 291 int. j. aquat. biol. (2017) 5(5): 286-294 regulation of the redox activity (stanton, 2012). moreover, antioxidant enzyme activities are dependent on an adequate supply of nadph. thus, g6pdh activity should be increased to provide sufficient nadph (stanton, 2012). an increase in g6pdh activity following zno nps administration is a cellular physiological response to cope with reactive oxygen species (ros). although low levels of zno nps are nontoxic, increased g6pdh activity was still observed which indicates that g6pdh may act as a bio-sensor and response to very low levels of free radicals (sauer, 1998). an increase in the activity of g6pdh can eliminate ros by using nadph (sauer, 1998). however, a decrease of g6pdh activity was observed in the liver of white sucker, (catostomus commersonii) exposed to zinc oxide nanoparticle (dieni et al., 2014) zno nps have antioxidant properties (nagajyothia et al., 2014; nagajyothia et al., 2015). the results of this study show that administering low concentrations of zno nps may enhance total antioxidant capacity of the cell by increasing the activity level of enzymatic (sod and cat) and non-enzymatic (protein antioxidants and glutathione) antioxidant system, reducing the ros level and inhibiting the activity of nitric-oxide synthase and nadph oxidase (prasad, 2014). furthermore, zn inhibits the influence of lipid peroxidation products on the cellular antioxidant system (prasad, 2014). muthuraman et al. (2014) showed that zno nps increased antioxidant enzyme activities, and their mrna expression in the cocultured c2c12 (mouse myoblast cell line) and 3t3l1 cells. previous studies indicate that zno nps may remove free radicals, increase the efficiency of the cellular antioxidant defense system, increase the enzymatic activity of antioxidant defense system and reduce malondialdehyde level (dawei et al., 2010) and consequently protect cells against ros and oxidative damages (badkoobeh et al., 2013). zn, a cofactor of superoxide dismutase (sod), regulates the process of converting superoxide to hydrogen peroxide (prasad, 2014). therefore, an increase in cat activity in hepatic and renal cells of fish which were fed 10 and 15 mg kg-1 zno nps could be a response to increased h2o2 in these cells. saddick et al. (2015) found that oreochromis niloticus and tilapia zillii which were exposed to low concentrations of zno nps (500 μg l1) showed an increase in cat activity and gene expression of antioxidant enzymes in brain tissue. on the other hand, increased concentration of zno nps (2000 μg l-1) significantly decreased cat activity and gene expression of antioxidant enzymes. similarly, cat activity decreased in liver, and intestine of zebrafish which were exposed to zno nps (xiong et al., 2011). zn has antioxidant properties and a key role in inhibition and removal of free radicals; therefore, it can act as an antioxidant in low concentrations (swain et al., 2016). nonetheless, we found that the increased amount of zno nps in foodstuff increased lipid peroxidation in tissues. we suggest that an increase in mda level in liver, and kidney could be an appropriate biomarker of lipid peroxidation in common carp after oral exposure to zno nps. our findings correspond with those of (syama et al., 2013). they reported that zno nps are not toxic at low concentration, but at higher concentrations increase ros through increased mda levels (syama et al., 2013). muthuraman et al. (2014) found that zno nps increased reactive oxygen species (ros) and lipid peroxidation (mda) in 3t3-l1 adipocytes. an increase in mda is reported in c. commersonii (dieni et al., 2014), c. carpio (hao and chen, 2012; hao et al., 2013), oncorhynchus mykiss (connolly et al., 2016), and zebrafish (xiong et al., 2011) which were exposed to zno nps. cytotoxic effects of zno nps, depending on their concentration and exposure duration, may be caused by zno nps accumulation in the liver, the occurrence of oxidative stress, damage to dna and an increase in lipid peroxidation (najafzadeh et al., 2013). zno nps produce free radicals, cause cellular toxicity and therefore lead to oxidative damages, inflammation and programmed cell death (kumar et al., 2011; umrani and paknikar, 2014). the results obtained from the present study showed that zno nps given orally to fish could induce dose292 taheri et al./ effects of dietary zno-nps on some biochemical biomarkers in common carp dependent effects on oxidative stress biomarkers and biochemical parameters. on the basis of these results, it is deduced that supplementation of 5 mg kg−1 znonps had no side effect on biochemical parameters in liver and kidney tissue of common carp. nevertheless, to ensure the safety of using zno nps as a food supplement, future studies should consider effects of these nps in non-toxic concentrations on other physiological indicators such as growth, reproduction, the immune system. acknowledgments the authors gratefully acknowledge the support offered by the behbahan khatam al-anbia university of technology. we also thank our english editor, m. banaee for proofreading the manuscript. references badkoobeh p., parivar k., kalantar s.m., hosseini s.d., salabat a. 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(2017) 5(5): 286-294 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی بیوشیمیایی پارامترهای برخی بر روی اکسید نانوذرات خوراکی مکمل تاثیر (cyprinus carpio) معمولی کپور ماهی در محیسنی محمد حقی،دوستنعمت بهزاد ،*بنایی مهدی طاهری، سمیه .ایران بهبهان،( ص) االنبیاء خاتم صنعتی دانشگاه زیست، محیط و طبیعی منابع دانشکده شیالت، گروه چکیده: جایگزین تواندمی روی نانوذرات پایین هایغلظت شود، سازیبهینه( zno-nps) روی اکسید ذرات نانو مکمل تجویز زمانمدت و دوز کهدرصورتی بهینه ضروریات از یکی خوراکی افزودنی هرگونه سلولی سمیت ارزیابی کهآنجایی از. شود ماهیان مختلف هایگونه غذایی رژیم در روی متداول منابع بیوشیمیایی هایشاخص برخی بر zno-nps خوراکی مصرف تأثیر بررسی منظوربه مطالعه این ما است، مشخص زمانمدت در مصرفی دوز سازی نانوذرات هدف، این به نیل برای. ایمکرده طراحی آزمایشگاهی مدل یک عنوانبه( cyprinus carpio) معمولی کپور ماهی در اکسیداتیو استرس و خورانده ماهیان به روز 21 مدت به غذا کیلوگرم هر ازای به گرممیلی 15 و 10 ،5 ،(کنترل) صفر هایغلظت در خوراکی مکمل صورتبه روی اکسید کبد در را( ldh) دهیدروژنازالکتات و( ast) آمینوترانسفرازآسپارتات فعالیت داریمعنی طوربه( کیلوگرم بر گرممیلی 15) zno-nps تجویز. شد فسفات-6 گلوکز فعالیت zno nps خوراکی تجویز. داد افزایش کلیه در را ldh و( alp) فسفاتازآلکالین ،(alt) آمینوترانسفرازآالنین و افزایش به منجر zno-nps کیلوگرم بر گرممیلی 15 و 10 تجویز که دهدمی نشان نتایج. داد افزایش ماهیان کبد در را( g6pdh) دهیدروژناز سطح و cat و ast فعالیت افزایش نیز و کبد در( mda) آلدهیددیمالون سطح افزایش همچنین و( cat) کاتاالز و alt فعالیت دارمعنی mda شودمی کلیه در. zno nps فعالیت کاهش سبب alp گرممیلی 5 تجویز. شد کبد در zno-nps اکسیدانآنتی سطح دارمعنی افزایش سبب سمیت بروز به منجر zno-nps گرممیلی 15 و 10 خوراکی تجویز که کرد عنوان چنین توانمی بنابراین. گردید مختلف هایبافت در( ta) تام هایشاخص بر جانبی عوارض گونه هیچ zno-nps کیلوگرم بر گرممیلی 5 مصرف که حالی در شد، اکسیداتیو هایشاخص در تغییر و سلولی .نداشت ماهیان در اکسیداتیو استرس و بیوشیمیایی .بیوشیمیایی پارامترهای کپور، ماهی اکسیداتیو، استرس روی، نانواکسید :کلمات کلیدی int. j. aquat. biol. (2017) 5(4): 252-259; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article the effects of dietary myrtle (myrtus communis l.) supplementations on growth performance and some innate immune responses in rainbow trout (oncorhynchus mykiss) hadis mansouri taee1*, abdolmajid hajimoradloo1, seyed hossein hoseinifar1, hassan ahmadvand2 1department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. 2razi herbal researches center, lorestan university of medical sciences, khoram abad, iran. article history: received 10 may 2017 accepted 22 august 2017 available online 2 5 august 2017 keywords: medical herbs immunostimulant innate immune responses bioactive abstract: this study investigated the effect of dietary myrtle (myrtus communis l.) powder on the growth performance, immune responses and haematological parameters of rainbow trout fingerlings. twelve cages were assigned to four treatments in triplicate and thirty fish (6.50±0.55 g) were stocked. treatments were different levels 0 (control), 0.5 (m0.5), 1 (m1) and 1.5% (m1.5) of myrtle powder. the highest weight gain and specific growth rate and lowest feed conversion ratio were observed in fish fed with 1% myrtle. the rbc, wbc and haematocrit were higher in the myrtle fed treatments compared to the control group. the results showed increased total protein in m1.5 and m1 treatments compared to control and m0.5 treatment. also, the highest and lowest albumin were observed in m1.5 treatment and control group. furthermore, serum alp decreased along with the increasing myrtle levels in diet, and lowest level was observed in m1 treatment. lysozyme activity increased with increasing dietary myrtle inclusion levels, although no significant difference was noticed when compared with control. these results revealed the potential growth enhancing and health promoting effects of myrtle powder. introduction rainbow trout, oncorhynchus mykiss, is one of the most important fish species that commercially farmed in many countries (fao, 2009). this species is cultured in both semi-intensive or intensive systems. although these systems increase the production level, at the same time can cause stressful condition which negatively affect immune system and elevate the risk of disease outbreak (mona et al., 2015; hoseinifar et al., 2015; dawood and koshio, 2016). a practical approach for resolving the issue, especially in antibiotic free aquaculture, is by potentially stimulating the immune system of the host animal by the inclusion of natural immunostimulants (barman et al., 2013; wang et al., 2016). animal or plant originated immunostimulants contain various bioactive compounds which can elevate immune responses and disease resistance (bairwa et al., 2012). to date, many studies have focused on the administration of dietary *corresponding author: hadis mansouri taee doi: https://doi.org/10.22034/ijab.v5i4.331 e-mail address: mansouritaee@gmail.com medicinal herbs and their extracts as potential immunstimulants in aquaculture (asadi et al., 2012; haghighi et al., 2014; reverter et al., 2014; hai, 2015; adel et al., 2015; akrami et al., 2015; sonmez et al., 2015; safari et al., 2016; hoseinifar et al., 2016) and positive results reported on growth performance, feed utilization, immune response and disease resistance of various fish species. myrtle (myrtus communis l.) belongs to myrtaceae family and myrtales has been considered as beneficial medicinal plants in mediterranean regions and middle east (amensour et al., 2009; asgarpanah and ariamanesh, 2015). the foliage of this medicinal plant contains different types of biologically active compounds like flavonoids, tannins, saponins, vitamin c and essential oils (martin et al., 1990; yoshimura et al., 2008; habiballah et al., 2014). in two studies, the positive effects of myrtle powder on skin mucus immune parameters in rainbow 253 int. j. aquat. biol. (2017) 5(4): 252-259 trout and zebrafish (danio rerio) has been shown (mansouri taee et al., 2017; safari et al., 2017). in another experiment, the effect of savory and myrtle essential oils on growth performance, hematological and biochemical parameters in rainbow trout has been studied (mohamadi saei et al., 2016). there are no study to reveal the effects of powder myrtle on some innate immune parameters in fish serum. therefore, the present study was performed to investigate the effects of dietary administration of m. communis powder as immunostimulant on growth, hematological parameters and immune responses of rainbow trout fingerlings. materials and methods fish and experimental diets: three hundred and sixty healthy rainbow trout fingerling were purchased and transferred to a selected farm located in lorestan province, iran. the fishes were acclimatized to experimental condition for two weeks. after this period, fish (6.50±0.55 g) were randomly stocked into 12 square cages (65×65×65 cm) and assigned to their respective experimental treatments in triplicate. each replicate consisted of 30 fish and the cages were kept in a raceway pond (30×3×2 m) with the flow through water system. during the trial, the water temperature, dissolved oxygen and ph were 15±1.2°c, 6.4±0.1 mg.l-1 and 7.7±0.2, respectively. prior to preparation of the experimental diet, myrtle herb was supplied, dried and powdered as described by safari et al. (2016). then, a commercial diet (faradaneh, iran) formulated for trout fingerlings (table 1) was considered as basal diet and experimental diet were prepared by supplementation of basal diet with different levels (0.5, 1 and 1.5%) of myrtle powder after. the prepared diets were kept in plastic bags at -4°c until used. the feeding rate was 3.8% of body weight which corrected every 2 week after biometry. growth performance and survival rate: to measure growth performance, weight and length of 20 fish in each tank was monitored once every 15 days following a 12 hrs starvation until the end of experiment. growth performance and survival rate of the fingerlings were calculated using the following formula (mohamadi saei et al., 2016): fcr (feed conversion ratio) = f/(bt – b0) sgr% d-1 (specific growth rate) = (ln wt -ln w0) ×100/t cf or k (condition factor) = 100 × (wt/tl3) gbm (gain of body mass) = wt – w0 survival rate = 100 × (nt /n0). where: f: relative food intake (g), bt and b0: final and initial fish biomass (g); wt and w0: final and initial body weight (g); t: time of rearing (days); tl: total length; nt and n0: final and initial fish number. sample collection: five specimens were randomly selected from each cage and anesthetized with clove solution for blood sampling. the blood samples were obtained from the caudal vein and divided into two parts. for haematological parameters, the samples were transferred to heparinized tubes and the remainings to non-heparinized tubes for serum isolation. the samples were centrifuged (3000 g for 15 min) and the obtained serums were stored at -80°c until use (akrami et al., 2015). hematological parameters: red blood cells and white blood cells were counted using a neubauer haemocytometer according to martins et al. (2004). haemoglobin concentration (hb: g/dl) was measured spectrophotometrically at 540 nm using the cyanmethenoglobin method drobkin (1945). haematocrit (hct) was measured with microcentrifuge method, using standard heparinized microhaematocrit capillary tubes (75 mm at 7000 g for 10 min) (blaxhall and daisley, 1973). serum biochemical and non-specific immunity parameters: the serum total proteins were analysed using a biuret assay as described by doumas et al. (1981). the serum albumin level was determined according to the bromocresol green method table 1. analysis of the commercial feed for rainbow trout (faradaneh, iransft). analysis moisture 11% crude protein 46% total lipid 14% ash 10% phosphorus 1.2% fiber 3% 254 mansouri taee et al./ myrtle and rainbow trout physiological responses following the method of doumas et al. (1971), using a diagnostic kit (ziestchem, diagnostics co., iran). globulin content (subtracting albumin from the total protein) was calculated basedon kumar et al. (2005). alkaline phosphatase (alp) was estimated using the ziest chem diagnostics kit (tehran company, iran) and the absorbance was read at 405 nm in a spectrophotometer (sanchooli et al., 2012). serum lysozyme activity was determined according to ellis et al. (1990). data analysis: for statistical analysis, the normality of data were checked. then, the data were subjected to one-way analysis of variance (anova) followed by duncan’s multiple range tests. in case of all statistics analysis, the mean values were considered significantly different at p<0.05. the statistical were performed by spss software (version, 17) and the figures were drawn by microsoft excel. results the weight gain (wg), specific growth rate (sgr) and feed conversion ratio (fcr) of fish fed the different experimental diets are shown in table 2. regarding, wg and sgr a dose dependent increase was noticed following feeding on myrtle supplemented diets (p<0.05); with the highest increments in those fish fed 1% myrtle. feed conversion ratio (fcr) of fish fed 1% myrtle was lower than that of control group (p<0.05). the number of red blood cells (rbc) and haematocrit (hct) in myrtle (0.5, 1 and 1.5%) fed fish was higher than the control group. however, rbc and haematocrit was also significantly higher (p<0.05) in fish fed 1% compared to the control group (table 3). similarly, the haemoglobin level (hb) was significantly higher in all the groups fed myrtle enriched diets, but no significant difference was noticed between the treatments (p>0.05). also, feeding on the myrtle supplemented diets significantly increased (p<0.05) wbc compared with the control group. in addition, at the end of feeding trial, the highest wbc level was observed in the 1% myrtle group (table 3). a remarkable dose-dependent increase was observed in total protein and albumin, and the highest increments was recorded in those fish fed 1.5% myrtle enriched diets (p<0.05). also, the globulin levels was significantly higher in 1% and 1.5% myrtle treatments compared to the control group (p<0.05). feeding on myrtle supplemented diet (1 and 1.5%) decreased serum alp activity (p<0.05) respect to the control group. lysozyme activity increased with increasing dietary myrtle inclusion levels, although no statistically significant difference was noticed compared to control (table 4) (p>0.05). discussion the use of immunostimulants in aqua-feed is highly recommended in aquaculture (barman et al., 2013; wang et al., 2016). however, due to their high costs, table 2. growth parameters of rainbow trout fed diets enriched with different levels of myrtus communis (control, 0.5%, 1% and 1.5%) for 60 days. data are presented as mean ±s.d. values in each row with different superscripts shows significant difference (p<0.05). wg, weight gain; sgr, specific growth rate. control m0.5 m1 m1.5 wg (g) 42.23±7.02b 55.02±11.50ab 62.42±3.83a 53.08±4.37ab sgr (%) 3.34±0.25b 3.73±0.30ab 3.94±0.09a 3.69±0.13ab fcr 0.75±0.13a 0.60±0.11ab 0.55±0.03b 0.64±0.06ab table 3. hematological parameters of rainbow trout fed diets enriched with different levels of myrtus communis (control, 0.5%, 1% and 1.5%) for 60 days. values are presented as the mean±sd. values in a row with different superscripts denote significant difference (p<0.05). control m0.5 m1 m1.5 rbc (×106 mm-1) 0.7±0.03b 0.84±0.07ab 1.05±0.18a 0.91±0.25ab wbc (×103 mm-1) 13.86±0.22b 22.59±1.4a 23.65±3.43a 20.9±2.67a hb (gr dl-1) 6.77±0.67a 7.0±1.12a 7.15±0.5a 7.51±1.2a hct (%) 25.67±4.04b 28.33±1.53ab 33.67±2.08a 30.0±2.0ab https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&cad=rja&uact=8&ved=0ahukewjeylgk57jmahxlnpokhtk8bsyqfgglmae&url=http%3a%2f%2fparamed.ir%2f%25d8%25b2%25db%258c%25d8%25b3%25d8%25aa-%25d8%25b4%25db%258c%25d9%2585%25db%258c--ziest-chem-diagnostics_6440.html&usg=afqjcnfuxravjccfz5vjdscdgpa5e2b3ka&sig2=epscxx74p6job1lpgo7kog&bvm=bv.121070826,d.bgs 255 int. j. aquat. biol. (2017) 5(4): 252-259 a large number of these additives are not economical. there are numerous medicinal herbs that cheaper source can be considered as promising immunostimulants and growth enhancer in aquaculture (reverter et al., 2014; hai, 2015; gholipour kanani et al., 2014; caipang and lazado, 2015; jeney et al., 2015; newaj‐fyzul and austin, 2015). the present results revealed that m. communis powder as feed additive enhanced growth performance. similar to the present study, the increase of growth performance were recorded in rainbow trout fed savory (satureja khuzestanica), sage (salvia officinalis), thyme (thymus vulgaris) and ginger (zingiber officinale) (sonmez et al., 2015; mohamadi saei et al., 2016; nya and austin, 2009), nile tilapia (oreochromis niloticus) fed garlic (allium sativum) powder (metwally, 2009), caspian brown trout (salmo caspius) fed peppermint (mentha piperita) (adel et al., 2015), beluga (huso huso) fed onion (allium cepa) (akrami et al., 2015), common carp (cyprinus carpio) fed persian hogweed (heracleum persicum) (hoseinifar et al., 2016). regarding the mode of action of medicinal plants on growth promotion, it has been suggested that it may be due to appetite stimulation as well as elevation of digestive enzymes activity which per se increase protein synthesis and improve feed utilization (hai, 2015; adel et al., 2015). the results of previous studies revealed that evaluation of haematological parameters can be considered as means of monitoring fish health (banaee et al., 2008). in the present study, the haematological parameters such as haemoglobin was not affected by dietary myrtle inclusion. this finding is in agreement with previous findings where feeding with other herbal supplementary food in rainbow trout (haghighi et al., 2014; farahi et al., 2012). however, our results suggest that oral administration of 1% myrtle increased rbc (erythrocyte) count and haematocrit. this findings were in accordance with those of previous studies which suggested medicinal herbs could affect immune system through their effects on the blood cells (nya and austin, 2009; adel et al., 2015). there are several published works showing the medicinal herbs could act as immunostimulants and increase the total wbc (hai, 2015; newaj‐fyzul and austin, 2015; mohamadi saei et al., 2016). the increase in wbc, and other blood cells, following feeding of m. communis, may be due to positive effect of myrtle on health status. this immunomodulatory effect has been attributed to presence of biologically active substance such as tannins, flavonoids, saponins and vitamin c found in m. communis prevented fish from infection by triggering immune system (scalbert and williamson, 2000). in the present study, total protein in serum increased in fish fed 1% and 1.5% myrtle. similar results were reported in rainbow trout fed with a. sativum (nya, 2009), z. officinale (nya and austin, 2009), laurus nobilis (bilen and bulut, 2010), nasturtium nasturtium (asadi et al., 2012) and aloe vera (haghighi et al., 2014). it has been welldocumented that the increase of serum total protein is associated with enhanced non-specific immune response of fish (akrami et al., 2015). this is mainly due to the fact that serum proteins comprises various elements of the non-specific immune system. the results also revealed notable increase of globulin level in fish fed 1% and 1.5% myrtle. similar to the present study, the increase of globulin were table 3. the total protein level, albumin, globulin (gr dl-1), alp (u l-1) and lysozyme activity (u mg-1) in rainbow trout fed diets enriched with different levels of myrtus communis (control, 0.5%, 1% and 1.5%) for 60 days. values are presented as the mean±sd. values in a row with different superscripts denote significant difference (p<0.05). control m0.5 m1 m1.5 total protein level (gr dl-1) 4.63±0.25b 5.08±0.57b 5.75±0.86ab 7.02±0.85a albumin (gr dl-1) 2.09±0.03b 2.28±0.26b 2.46±0.59b 3.74±0.94a globulin (gr dl-1) 2.55±0.28b 2.79±0.59ab 3.29±0.28a 3.28±0.12a alp (u l-1) 982.50±102.53a 770±132.94ab 427±33.94c 617±70.71bc lysozyme activity (u mg-1) 13±4.24a 22±3.46a 25±7.07a 29±12.73a 256 mansouri taee et al./ myrtle and rainbow trout physiological responses recorded in rainbow trout after feeding n. nasturtium and a. vera (asadi et al., 2012; haghighi et al., 2014). likewise, gholipour kanani et al. (2014) and binaii et al. (2014) reported elevation of globulin in beluga (h. huso) fed ginger and nettle, respectively. indeed, the serum globulin level is associated with proteins involved in humoral immune responses (billertakahashi et al., 2013). therefore, the elevation of globulin level can be attributed to immunomulatory effects of myrtle powder inclusion in rainbow trout diet. also, regarding albumin level a dose-dependent increase was observed and the highest increments in those fish fed 1.5% myrtle. the activity of alp in blood serum showed significantly decrease in fish fed 1.5% and 1% myrtle. these results are in accordance with previous results reported by metwally (2009) and metwally et al. (2001). since anti-radical and anti-oxidant properties of the medicinal plants may prevent lipid peroxidation of cell membranes and the release of foresaid enzymes into the plasma (haghighi et al., 2014), myrtle might inhibit those diseases caused by oxidative stress. lysozyme is an important enzyme in non-specific immune defences which eliminates pathogenic bacteria through hydrolysis of their cell wall (saurabh and sahoo, 2008). in this study, an increasing trend in lysozyme activity observed following dietary administration of myrtle which is in agreement with several reports indicating the role of herbal immunostimulants in enhancing lysozyme activity (rao et al., 2006; choi et al., 2008; haghighi et al., 2014; adel et al., 2015). this increase can be attributed to modulation of non-specific immune system following dietary administration of medicinal plants. however, determination of the exact mechanisms underlies the effects of medicinal herbs on lysozyme activity merit future research. as conclusion, the present results showed beneficial effects of dietary myrtle as medicinal herb on growth performance, immune response and antioxidant enzymes activity in rainbow trout. this study encourages additional research on different aspect of myrtle application in aquaculture. acknowledgments the authors would like to thank s. masouri for providing the necessary facilities for the study. the authors are also grateful to m.h. mansouri, as laboratory technicians, for their cooperation and assistance throughout the research. references fao (food and agriculture organization). 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(2017) 5(4): 252-259 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی ایمنی پارامترهای برخی و رشد عملکرد بر جیره به( .myrtus communis l) مورد دارویی گیاه افزودن اثرات کمان رنگین آالی قزل ماهی غیراختصاصی 2احمدوند حسن ،1فر حسینی حسین سید ،1مرادلو حاجی عبدالمجید ،1*طایی منصوری حدیث .ایران گرگان، گرگان، طبیعی منابع و کشاورزی علوم دانشگاه شیالت، و زیست محیط دانشکده شیالت، گروه1 .ایران آباد، خرم لرستان، پزشکی علوم دانشگاه رازی، دارویی گیاهان تحقیقات مرکز2 چکیده: . پردازدمی کمان رنگین آالی قزل ماهی بچه شناسیخون پارامترهای و ایمنی پاسخ رشد، عملکرد روی مورد گیاه پودر اثرات بررسی به مطالعه این قفس هر در( گرم 50/6±55/0) ماهی عدد 30 و شد استفاده تکرار و تیمار 4 با( مترسانتی 65×65×65 ابعاد با) قفس 12 تعداد منظور این رایب و حاصله وزن بیشترین. بودند مورد گیاه پودر( درصد 5/1m) 5/1( و 1m) 1(، 5/0m) 5/0 ،(شاهد) صفر مختلف سطوح شامل تیمارها. گردید ذخیره تعداد(. >05/0p) گردید مشاهده ،بودند کرده دریافت مورد گیاه درصد 1 که هاییماهی در غذایی تبدیل ضریب میزان کمترین و ویژه رشد ضریب .(>05/0p) بود بیشتر شاهد گروه به نسبت بودند کرده دریافت مورد جیره در که تیمارهایی در هماتوکریت و خون سفید هایگلبول قرمز، هایگلبول در آلبومین میزان بیشترین همچنین .(p>05/0) داد نشان افزایش 5/0m تیمار و شاهد با مقایسه در 1m و 5/1m تیمارهای در کل پروتئین میزان کاهش جیره در مورد گیاه دوز افزایش با فسفاتاز آلکالین آنزیم این بر عالوه .(p>05/0) گردید مشاهده شاهد در آن میزان کمترین و 5/1m تیمار نشان افزایش جیره در مورد سطوح افزایش با لیزوزیم آنزیم فعالیت میزان .(p>05/0) گردید مشاهده 1m تیمار در آنزیم این میزان کمترین و یافت نشان مورد گیاه پودر سالمت و رشد تقویت اثرات نتایج این. (>05/0p) باشدنمی دارمعنی شاهد با تیمارها این اختالف که است حالی در این داد، .دهدمی .غیراختصاصی ایمنی پاسخ ایمنی، محرک کمان، رنگین آالی قزل ماهی مورد، :کلمات کلیدی int. j. aquat. biol. (2015) 3(5): 346-351 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article acute toxicity of euphorbia turcomanica on aphanius dispar homa zare1, ahmad noori*1, morteza yusefzadi2, mahdi banaee3 1department of fisheries science, faculty of marine science and technology, hormozgan university, bandar abbas, iran. 2department of marine biology, faculty of marine science and technology, hormozgan university, bandar abbas, iran. 3department of aquaculture, faculty of natural resources and environment, behbahan khatam alanbia university of technology, behbahan, iran. article history: received 10 july 2015 accepted 25 o c t o b e r 2015 available online 2 5 n o v e m b e r 2015 keywords: euphorbia turcomanica dried powder piscicidal killifish abstract: piscicidal and molluscicidal activities of aqueous extracts of many members of the family euphorbiaceae are well-known, but the toxicity potential of euphorbia turcomanica was not yet studied on any aquatic animals. an acute toxicity test was performed by using a four-day static renewal test to determine the lc50 value of dried powder of e. turcomanica for the euryhaline fish, aphanius dispar. the lc50 values at various exposure periods are 0.177±0.039 g/l for 24 hrs, 0.131±0.030 g/l for 48 hrs, 0.073±0.018 g/l for 72 hrs, and 0.052±0.013 g/l for 96 hrs. the toxicity of dried powder of e. turcomanica exhibits a positive correlation between fish mortality and exposure periods. as this is the first report about toxicity of e. turcomanica on a. dispar, the results could be only compared to that of other euphorbiaceae as well as other fishes. it is concluded that the toxicity potential of e. turcomanica is comparable and close to that of well-documented euphorbiaceae. it has been suggested that e. turcomanica products cannot be used directly in fish-inhabiting water reservoirs. introduction increased awareness of the negative effects caused by overexposure to synthetic organo-piscicides (reidinger and russell, 1976) has led to efforts for finding products from plant origin to substitute. being the products of biosynthesis, they are potentially biodegradable (marston and hostettmann, 1985). the euphorbiaceae is a large families with about 300 genera and 7500 species (vasas and hohmann, 2014). of the member of this family, the genus euphorbia with about 2000 species (frodin, 2004), is one of the five most species-rich genera of flowering plants (govaerts et al., 2000). they have a poisonous milky white latex-like sap and unique kind of floral structures. the chemical constituents of these plants, include triterpenoids and related compounds (sterols, alcohols and hydrocarbons), phenolic compounds (flavonoids, lignans, coumarins, tannins, phenanthrenes, * corresponding author: ahmad noori e-mail address: nooryahmad@gmail.com quinones, phenolic acids, etc.), alkaloids, cyanogenic glucosides, and glucosinolates (abdel‐ fattah, 1987; neuwinger, 2004; kumar et al., 2010) that are poisonous to target and non-target aquatic organisms (singh et al., 1996; ebenso, 2004; prasad et al., 2010). many of these plants are cosmopolitan distributing in subtropics and temperate regions (horn et al., 2012). in the flora of iran, this genus comprises 70 species, of which 17 species are endemic (mozaffarian, 1996). euphorbia turcomanica boiss, is an annual herb, which grows wild in plains of iran (mozaffarian, 1996; pahlevani and riina, 2011). different species of the genus euphorbia are used as insecticide, piscicide, and molluscicide (sastry and siddiqui, 1983; oliveira-filho and paumgartten, 2000; tiwari and singh, 2004; tiwari et al., 2004; singh et al., 2005; oliveira-filho et al., 2010; hassan et al., 2011). since, there is no information available 347 zare et al./ acute toxicity of e. turcomanica on a. dispar regarding the effect of e. turcomanica on fishes as piscicid. therefore, due to wide use of various parts of the members of the family euphorbiacea (bani et al., 1997; abdel-hamid, 2003; srivastava et al., 2004; tiwari et al., 2004), this study was conducted to assess the acute toxicity of the lethal concentrations of aqueous extracts of e. turcomanica’s aerial parts on aphanius dispar, an euryhaline fish of iranian inland water. materials and methods a total of 240 individuals of a. dispar (both sexes: with mean body weight and total length of 2.03±0.5 g, and 47.7±0.45 mm, respectively) were collected from seasonal rivers in bandar abbas and transported to the hormozgan university fisheries laboratory. in the laboratory, healthy fish were introduced into a 100 l tank with continuous aeration, where they were acclimatize for 14 days to the laboratory conditions. the fish were considered fully acclimatized when no death was observed for seven successive days. the fish were fed 2-3 times a day during this period with commercial pellets, containing protein >28%, lipid >3%, fiber <4% and moisture <10%. the stem, branches and leaves of e. turcomanica were dried in room temperature away from direct sunlight. then, they were powdered and mixed with water to obtain the required plant concentrations. for the determination of lc50 of e. turcomanica on a. dispar, the four-day static renewal acute toxicity test was performed based on clesceri et al. (1998). fish were exposed to 0.001, 0.01, 0.1, 1, 2, 4 and 6 g/l of the dried powder of e. turcomanica with three replicates each containing 10 fish (kept in 4 l plastic aquaria). in addition, control group with three replicates in similar stocking density and aquaria were considered for this experiment. the water was renewed every day and required dried plant was added after water renewal to keep experimental concentrations. the fish were not feed 24 hrs before and during the experiment. dead fish were counted and removed from the treatments immediately. a toxic effect was determined by a statistically significant decrease in the survival of fish exposed to the plant relative to the survival of figure 1. percent mortality of the fish aphanius dispar after 24, 48, 72 and 96 hrs exposures to different concentrations of euphorbia turcomanica powder (g/l). 348 int. j. aquat. biol. (2015) 3(5): 346-351 fish in a control. the physico-chemical parameters of the water during experiment, including temperature (23.5±1ºc), dissolved oxygen (4.17±0.1 mg/l), electrical conductivity (840.41±2.46 µs/cm), and ph (8.12±0.03) were measured daily. at different exposure periods (24, 48, 72 and 96 hrs), the mortality of the fish was subjected to probit analysis using minitab software (minitab®16.2.4) to calculate the lc values, their slope functions, and confidence limits. results the percent mortality of the exposed a. dispar to exposure periods effective dose (g/l) se limits slope function 't' ratio lcl ucl 24 hrs lc1=0.010 lc5=0.024 lc10=0.037 lc20=0.063 lc50=0.177 lc80=0.496 lc90=0.851 lc95=1.327 lc99=3.056 0.004 0.008 0.011 0.017 0.039 0.117 0.228 0.401 1.155 0.004 0.012 0.020 0.038 0.115 0.313 0.503 0.734 1.457 0.024 0.047 0.067 0.106 0.272 0.789 1.439 2.400 6.411 1.414±0.229 6.183 48 hrs lc1=0.005 lc5=0.014 lc10=0.023 lc20=0.041 lc50=0.131 lc80=0.414 lc90=0.757 lc95=1.246 lc99=3.172 0.002 0.005 0.007 0.011 0.030 0.104 0.219 0.409 1.317 0.002 0.007 0.012 0.024 0.084 0.253 0.430 0.654 1.406 0.013 0.028 0.043 0.071 0.204 0.678 1.334 2.373 7.156 1.485±0.226 6.556 72 hrs lc1=0.001 lc5=0.005 lc10=0.009 lc20=0.018 lc50=0.073 lc80=0.296 lc90=0.618 lc95=1.133 lc99=3.540 0.001 0.002 0.003 0.006 0.018 0.083 0.202 0.427 1.714 0.001 0.002 0.004 0.010 0.045 0.171 0.325 0.541 1.371 0.004 0.011 0.018 0.033 0.118 0.512 1.173 2.373 9.144 1.570±0.219 7.163 96 hrs lc1=0.001 lc5=0.003 lc10=0.005 lc20=0.012 lc50=0.052 lc80=0.230 lc90=0.500 lc95=0.949 lc99=3.155 0.000 0.001 0.002 0.004 0.013 0.067 0.172 0.378 1.621 0.000 0.001 0.003 0.006 0.032 0.130 0.255 0.435 1.153 0.003 0.007 0.012 0.023 0.087 0.407 0.981 2.070 8.635 1.675±0.228 7.332 table 1. effective dose, confidence limits, and slope function for aqueous extract of euphorbia turcomanica at different intervals on aphanius dispar. 349 zare et al./ acute toxicity of e. turcomanica on a. dispar various concentrations of the plant extract of e. turcomanica for 24, 48, 72 and 96 hrs are depicted in figure 1. the lc50 values at various exposure periods were 0.177 g/l for 24 hrs, 0.131 g/l for 48 hrs, 0.073 g/l for 72 hrs, and 0.052 g/l for 96 hrs. the lc values, their upper and lower confidence limits, and slope functions are given in table 1. the toxicity of dried powder of e. turcomanica was found to be time and dose-dependent (p<0.05). the regression coefficient demonstrated a significant positive correlation (p<0.05) between mortality percent and concentration of e. turcomanica. also a significant negative correlation (p<0.05) was found between the exposure time and different lc values. discussion the results revealed that the dried powder of e. turcomanica has a high piscicidal activity causing more mortality with increasing its concentration. the toxicity was both time and dose dependent. a significant negative correlation demonstrated between different lc values and exposure time. lc50 value of e. turcomanica decrease with increasing exposure time in a. dispar from 0.177 g/l after 24 hrs to 0.052 g/l after 96 hrs. to my best knowledge, there is no report on the toxicity effects of e. turcomanica on aquatic animals. although, some reports are present on the toxicity effects of the aqueous and latex extracts of other members of the family euphorbiaceae on some animals, including fish and molluscs (singh and singh, 2002; singh and singh, 2005; tiwari and singh, 2006; dos santos et al., 2007; oliveira-filho et al., 2010). the lc50 value for 24 hrs of dried powder of e. tirucalli stem bark latex for colisa fasciatus was 8.14 mg/l, whereas this value for channa punctatus was 9.01 mg/l (tiwari et al., 2003). in another experiment, the toxicity of four plants belonging to members of euphorbiaceae and apocynaceae on c. punctatus evaluated (singh and singh, 2005). in this study, 96 hrs lc50 values of e. royleana, nerium indicum, jatropha gossypifolia, and thevetia peruviana were 0.020, 0.041, 4.34, and 3.17 g/l, respectively (singh and singh, 2005). this value for e. turcomanica was 0.052 g/l on a. dispar. in general, juicy and latex-bearing euphorbiaceae are more potent in their toxic effects than rotenoneyielding plants (neuwinger, 2004). in the present study, the dried powder of whole plant is used directly and nevertheless, the lc50 values of e. turcomanica is comparable in potent toxicity to the other studies. also, it should be noted that the experimental fish in this study is very hardy comparing to other studies. therefore, it is suggested that e. turcomanica has more toxicity than other species such as j. gossypifolia and t. peruviana. it is concluded that dried powder of e. turcomanica has a potent piscicidal activity on a. dispar. therefore, enough precautions must be exercised when derivatives of e. turcomanica is being used near fish-inhabiting water reservoirs. references abdel-hamid h. 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(2015) 3(5): 346-351 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی ( euphorbia turcomanica) ترکمنی فرفیون گیاه کشنده های غلظت تعیین (aphanius dispar) گورخری آفانیوس ماهی روی بر 3بنایی مهدی ،2زادی یوسف مرتضی ،*1نوری احمد ،1زارع هما .ایران بندرعباس، هرمزگان، دانشگاه دریایی، فنون و علوم دانشکده شیالت، گروه1 .ایران بندرعباس، هرمزگان، دانشگاه دریایی، فنون و علوم دانشکده دریا، زیست گروه2 .ایران بهبهان، االنبیاء، خاتم دانشگاه طبیعی، منابع و زیست محیط دانشکده شیالت، گروه3 چکیده: مطالعه مورد تناننرم و ماهی کشنده سم عنوانبه euphorbiaceae خانواده به متعلق هایگونه از بسیاری از حاصل آبی هایعصاره تاثیر تاکنون در آبزی موجودات روی بر e. turcomanica ترکمنی فرفیون گیاه کشندگی و سمیت توان مورد در اطالعاتی وجود این با است، گرفته قرار هایغلظتمنظور تعیین به محصور محیط در روزه چهار آزمایش روش از استفاده با گیاه این کشنده هایغلظت تعیین آزمون. باشدمین دسترس 05lc گیاه شده خشک پودر e. turcomanica گورخری آفانیوس ماهی روی بر a. dispar، اجرایک گونه با دامنه تحمل باالی شوری، به لیتر بر گرم 131/5±535/5 ساعت، 22 برای لیتر بر گرم 111/5±530/5 ترتیب به در معرض قرارگیری مختلف هایزمان در 05lc مقادیر. درآمد شده خشک پودر سمیت. آمد دست به ساعت 09 برای لیتر بر گرم 502/5±513/5 و ساعت 12 برای لیتر بر گرم 513/5±514/5 ساعت، 24 برای گزارش یناول این تحقیق نتایج اینکه به توجه با. داد نشان تاثیرگذاری زمان مدت و ماهی تلفات بین را مثبتی همبستگی e. turcomanica گیاه euphorbiaceae های گونه سایر نتایج با مقایسه قابلتنها نتایج این باشد،می a. dispar ماهی روی بر e. turcomanica گیاه سمیت مورد در هایگونه سایر به نزدیک و مقایسه قابل e. turcomanica گیاه سمیت توان که نمود استنباط توانمی حاصل نتایج از. است دیگر ماهیان روی بر euphorbiaceae گیاه از تولیدی ترکیبات که شودمی پیشنهاد همچنین. اندگرفته قرار بررسی مورد تاکنون که است e. turcomanica توجه با .دگردن استفاده است، مختلف ماهیان زیست محل که آبی هایمحیط در مستقیم طوربه آن باالی سمیت به .ماهی گورخری ماهی، کشنده سم خشک، پودر ترکمنی، فرفیون :کلمات کلیدی int. j. aquat. biol. (2022) 10(5): 378-383 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article the efficiency of biosynthesized zinc oxide nanoparticles by fusarium sp. against saprolegnia parasitica isolated from common carp eggs in fish hatchery rana h.h. al-shammari1, majida hadi mahdi alsaady2, shaimaa satae m. ali3 1department of biology, college of sciences, mustansiriyah university, baghdad, iraq. 2collegef science, university of baghdad, baghdad, iraq. 3research center of environmental study, university of babylon, babylon, iraq. s article history: received 4 june 2022 accepted 17 august 2022 available online 2 5 october 2022 keywords: malachite green dieses aquaculture technique fish breeding abstract: saprolegnia spp. infect common carp (cyprinus carpio l.) eggs in hatcheries. therefore, this study aimed to evaluate the antifungal effect of biosynthesized zinc oxide nanoparticles (znnps) against saprolegnia spp. as an eco-friendly treatment. biosynthesized znnps were characterized using atomic force microscope (afm), size distributor and ultra violate-visible spectrometer (uvvis), and scanning electron microscope (sem). biosynthesized (znnps) had a spherical shape with diameters ranging 10-70 nm. antifungal activity was tested by fungal radial growth inhibition on corn meal agar. the highest concentration of 100 ppm of znnps showed a remarkable inhibition rate of 79% against saprolegnia spp., demonstrating similar efficiency as the positive control i.e. malachite green in the inhibition percentage rate of fungal growth. this study showed that biosynthesized zinc oxide nanoparticles had a significant antifungal effect (p<0.05) and can be used as an alternative option to control saprolegnia spp. in fish hatcheries. introduction one of the concerns in fish breeding is fungal infections with saprolegnia spp.. to control this, malachite green is used as an effective traditional method (west, 2006). this fungus attaches to the dead eggs and transfers to the live eggs; therefore, it is considered a crucial issue in aquaculture (tedesco et al., 2021). in aquaculture, malachite green and sodium chloride are traditionally used due to their fungicidal effects (hashimoto et al., 2011), but adverse effects of malachite green e.g. its carcinogenicity and negative environmental impacts, have limited its application in many countries (almahmood et al., 2017; al-mahmood et la., 2021). hence, it is important to replace malachite green with an effective antifungal agent with fewer side effects (fajardo et al., 2022). the rapid development of nanotechnology and its successful applications in the agri-food sector make correspondence: rana h.h. al-shammari doi: https://doi.org/10.22034/ijab.v10i5.1741 e-mail: dr.rana@uomustansiriyah.edu.iq dor : 20.1001.1.23830956.2022.10.5.4.5 it a promising candidate for this purpose (taha et al., 2021; al-ardi, 2022). nanotechnology has many applications in detecting and controlling pathogens, water treatment, and sterilizing ponds (bhattacharyya et al., 2015; luis et al., 2019). various studies have confirmed zinc oxide nanoparticles' antifungal and antibacterial effects (swain et al., 2014). aquaculture fungal infections can be controlled using nanoparticles with antibacterial and antifungal properties (shammari, 2016). studies have confirmed their effectiveness as an antifungal agent. zinc oxide nanoparticles (znnps) have antimicrobial activity (mendes et al., 2022). biosynthesized nanoparticles represent an eco-friendly alternative. in addition, a more effective synergetic effect is possible through a combination of nanoparticles and natural extracts. for these reasons, this study aimed to characterize and evaluate a biosynthesized znnps against https://ij-aquaticbiology.com/index.php/ijab/article/view/1741 379 int. j. aquat. biol. (2022) 10(5): 378-383 saprolegnia spp. by an in vitro assay. materials and methods isolation of saprolegnia: saprolegnia spp. was isolated from the infected eggs of common carp from al-wahda fish hatchery, baghdad, and identified based on their morphological characteristics. for the pure culture of saprolegnia parasitica. saprolegnia fragments from the eggs were removed and directly inoculated in corn meal agar (cma) medium with antibiotics (chloramphenicol). after the fungus (white, cotton-like mycelia), a disk of the colony (6 mm) was placed on a plate with sterilized distilled water and baits of 5-10 sesame seeds were distributed around it. the disk was kept at 20°c for 24 hours. the colonized seeds were placed on another plate with sterile distilled water and kept at 20°c for 5 days. subsequently, the baits were examined under a light microscope (lenovo, lx400) to verify identification by the hyphae, zoosporangium, zoospores, and sexual structures (sandoval-sierra and dieguez-uribeondo, 2015). biosynthesis of zn-nps by aqueous fusarium sp. extract: the fungus fusarium sp. was obtained from the biology department, college of science, mustansiriyah university. preparation of aqueous fusarium sp. extract cultured in liquid medium for 14 days at 25°c with a shaking rate of 150 rpm. then fungal biomass was homogenized and filtrated (100 ml). the extract was filtered using a millipore filter (0.2 μm), and stored at -4°c before use. the flask containing 5 g of zinc acetate with 50 ml of deionized and sterile distilled water mixed with 50 ml of the aqueous fusarium extract for 30 min under continuous stirring at 35°c and then allowed to keep at room temperature for 48 hrs. the initial ph of the solution was about 5.5, and the color of the aqueous solution changed to a dark brown solid. the product was collected by centrifugation at 10000 rpm for 10 min and careful washing with distilled water. the final products were obtained by drying overnight. the resulting dried sample was crushed into powder and stored in an airtight container for further analysis. preparation of zinc oxide nanoparticle and malachite green stock: one mm of zinc nitrate hexahydrate in a 1:2 ratio, zn(no3)26h2o was added to 50% fungal extract solution while stirring continuously. following complete dissolution, the mixture was stirred at 100°c for 5 hours, and the supernatant was discarded after cooling. after centrifuging twice at 6000 rpm for 15 minutes, washing, and drying at 85°c for 5 hours, the solid product (pale white) was collected. further studies were conducted using the dried powder, which was kept at room temperature until it changed color. in preparation for the malachite green stock, 10 parts per million were used (ahmad et al., 2019). characterization of biosynthesized zn-nps: nanoparticles were evaluated for their morphology and stability using a femto uv-vis photometer (ultraviolet-visible spectroscopy) within a week and a year after synthesis (800xi) at a wavelength of 400 to 800 nm (ghozali et al., 2015). for atomic force microscopy (afm), a thin sample layer was put on a glass slide by dropping 150 μl of the sample and drying for 10 mins. the slides were then scanned with the afm. scanning electron microscope (sem) photographs of the biosynthesized znnps were performed using a philips xl30 sem (netherlands). antifungal activity of biosynthesized zn-nps: the antifungal effect of the synthesized zn-nps was tested against saprolegnia spp. using cma medium prepared with different concentrations of nanoparticles (10, 25, 50 and 100 ppm) prepared by adding a volume of 0.5 ml of each 0.05, 0.10, 0.25, and 1 mg/ml aseptically into the plates loaded with 9.5 ml of cma medium compared with negative control of distilled water and positive control malachite green. all experiments were done in three replicates. petri dishes were inoculated in the center with 4 mm fungal plugs and incubated at 20±2°c for 10 days. the radial growth of the colonies was measured and the percentage of inhibition of mycelial growth was calculated using the equation of inhibition% = [(g1g2)/g1] x100; where g1 is the radius of normal growth in control plates and g2 is 380 al-shammari et al./ efficiency of biosynthesized zinc oxide nanoparticles by fusarium sp. the radius of inhibited growth. statistical analysis: the significant difference between values of the incubation conditions was determined using a two-way anova. in addition, the least significant difference (lsd) and correlation were performed to test whether group variance was significant or not (p≤0.05) in spss program version 26. results saprolegnia spp. was isolated from the infected eggs of common carp that were covered with white or grey threads of cotton-like mycelia (fig. 1). znnps were synthesized using fusarium extract, which changed the colour from colourless aqueous extract to red-brown as an indicator of the formation of znnps (fig. 2). biosynthesized (znnps) had a spherical shape with diameters ranging 10-70 nm. studies show that other metals where the colour change shows the synthesis of the respective nanoparticles; however, it was characterized by uv– visible spectroscopy using fusarium extracts at 360 nm that recorded an absorption peak, which is attributed to the formation of znnps. scanning electron microscopic images of znonps synthesis using fusarium extract are presented figure 1. infected common carp eggs labeled with arrows during incubation period after 48 hours, (10x). figure 2. color changes in aqueous solution (a) zinc oxide before adding fusarium sp. extract (b) after one hour of adding fungus extract at 25°c. figure 3. sem of biosynthesized znnps by fusarium sp. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc7175351/figure/biomolecules-10-00425-f002/ 381 int. j. aquat. biol. (2022) 10(5): 378-383 in figure 3, showing the accumulations of particles. the size and shape of synthesized znnps were reported by afm microscopy. the biosynthesized zno were spherical with a few cylindrical particles with variations in particle size ranging from 10-70 nm (fig. 4). biosynthesized znnps at 100 ppm showed a remarkable inhibition rate of 79% against s. parasitica followed by 75 and 47.2% for the lowest concentrations (table 1). there was no significant difference (p≤0.05) between inhibition rates at 100 ppm znnps with malachite green. mycelia growth of s. parasitica was inhibited in all the tested concentrations. discussion saprolegniaceae species such as achlya and saprolegnia can infect fish eggs of different species in aquaculture and nature (lone and manohar, 2018), considering crucial problems in their incubation (ali et al., 2020). malachite green is widely used as an antifungal agent, but it is teratogenic and mutagenic, causing abnormalities in eggs and fish, which since 1991, it was banned (jogaiah et al., 2019). therefore, other methods are used to treat saprolegniasis. it has been suggested application of nanoparticles in aquaculture. zinc oxide nanoparticle is an inorganic compound soluble in water and its antifungal properties are proven. it is connected to the membrane of microorganisms in the phase of the growth cycle, prolonged the time of germination of organisms (ahmad et al., 2018). the changes in color as an indicator of znnps formation approved the formation of the respective nanoparticles as reported in other works (jogaiah et al., 2019; mahmood et al., 2022). uv-vis spectroscopy is a suitable technique to detect the biological reaction compared to other methods, such as physical, chemical and hybrid methods, where an additional force is required, which may carry toxic bi-products that lose their stability (jeevanandam et al., 2018). the results showed that malachite green and zinc oxide nanoparticles have significant antifungal effects. based on the results, with an increase in the concentration of biosynthesized znnps, the antifungal activity was increased, similar to the results of (ahmad et al., 2018; johari et al., 2019) regarding the antifungal properties of silver oxide nanoparticles against saprolegnia. the antifungal activity of znnps was dose-dependent, and the best test fungi zn-nps concentrations (ppm) malachite green 10 (ppm) 10 25 50 100 inhibitory rate (%) 21.2±0.2c 47.2±1.2b 75±0.4b 79±0.5a 80.2±1.2a the inhibition rates in (%) mean± standard error (se). different letter(s) are significantly different. table 1. inhibitory rate (%) of saprolegnia parasitica fungi using different concentrations of zn nanoparticles compared with malachite green. figure 4. diameters, amount of biosynthesized znnps by fusarium. 382 al-shammari et al./ efficiency of biosynthesized zinc oxide nanoparticles by fusarium sp. inhibition concentration was 25 ppm in the current study. a significant increase in the inhibition of saprolegnia was recorded at higher concentrations of 50 and 100 ppm, respectively, which agrees with the findings of ahmad et al. 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(2018) 6(4): 208-220 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article the rotifers (rotifera: eurotatoria) from the kashmir himalayan floodplains and rotifera biodiversity of jammu and kashmir, north india bhushan kumar sharma*,1sumita sharma 1department of zoology, north-eastern hill university, shillong-793022, meghalaya, india. article history: received 11 july 2018 accepted 20 august 2018 available online 2 5 august 2018 keywords: composition distribution evaluation important taxa interesting species richness wetlands abstract: our plankton and semi-plankton collections from the floodplain wetlands of the kashmir valley reveal 140 rotifer species belonging to 43 genera and 22 families. the richness forms ~81% of 173 species, belonging to 51 genera and 23 families, recognized as validly known from jammu and kashmir (j&k) vide the present biodiversity evaluation. the rotifer assemblages of the kashmir himalayan wetlands and j&k comprise ~33% and ~41%, respectively of the indian rotifera; their biodiverse nature is hypothesized to habitat diversity and ecological heterogeneity of aquatic environs of this state of north india. one species is new to india and 25 species are new to northwest india. lecanidae > brachionidae form ~33% and trichocercidae = lepadellidae > notommatidae > euchlanidae comprise ~31% of the rotifer fauna of j&k which includes species of global and regional biogeographic interest and, a large component of cosmopolitan species (~81%). lecane > trichocerca > brachionus = lepadella collectively comprise ~39%; keratella > euchlanis > synchaeta are notable (~12%); and notholca and cephalodella species deserve attention from j&k. rotifera of the kashmir himalayan floodplains and j&k indicate the littoral-periphytic character, cold-water elements, smallsized species, and the relative paucity of brachionus, lepadella and lecane richness. we estimate 260+ rotifer species from j&k pending analysis of intensive latitudinal and longitudinal collections including samples from ramsar sites, high altitude lakes and other water bodies with emphasis on colonial, sessile and benthic taxa, and analysis of likely cryptic diversity of certain species. introduction rotifera has been studied from scattered localities of india ever since the first taxonomic survey by anderson (1889). nevertheless, the indian literature still lacks intensive biodiversity works from various states or regions; and it is loaded with anomalous, ambiguous and fuzzy reports, and ad-hoc species lists due to unverifiable taxa (sharma and sharma, 2014a, 2014b; 2017, 2018a). these lacunae impede effective biodiversity evaluation notwithstanding that the indian rotifera is yet by no means fully explored. the stated generalizations hold valid for the rotifer fauna of jammu and kashmir (j&k) state of north india in spite of a sizable number of limnological studies (shah et al., 2015) from the kashmir valley and jammu province invariably enlisting the rotifers. ironically, the compilation on the rotifers of kashmir himalayas by shah (loc. cit) deserves caution due to inclusion of *corresponding author: bhushan kumar sharma doi: https://doi.org/10.22034/ijab.v6i4.507 e-mail address: profbksharma@gmail.com duplicate species entries, invalid reports, misidentifications, and several un-validated species reports; the lapsi question the objectives of ‘documentation’ and `authentication’ of the stated work and its utility as a viable biodiversity reference. further, the recent report of litvinchuk (2018) lists only six rotifer species from the kashmir himalayas because of paucity of collections. the state of jammu and kashmir, a part of the himalayan biodiversity ‘hot-spot’, is dotted with ramsar sites, the floodplain wetlands, and lakes and water bodies located under varied ecological regimes which offer prime scope for exploration of aquatic metazoan biodiversity and that of phylum rotifera in particular. the latter taxon has received inadequate attention from j&k in spite of 35+ publications (shah et al., 2015) and several unpublished research dissertations listing the rotifers ever since the first 209 int. j. aquat. biol. (2018) 6(4): 208-220 report by edmondson and hutchinson (1934). additionally, the published works from this state are frequented with sloppy identifications and fuzzy records warranting validations, and incomplete species lists due to overlooking identification of the smaller rotifer taxa (sharma and sharma, 2017). the present endeavor is thus an attempt to provide an evaluation of rotifera biodiversity of j&k based on our collections from the floodplains of the kashmir himalayas as well as `critical assessment and validation’ of the viable published records. we provide a detailed inventory of the valid and authenticated rotifer taxa known till date from j&k while certain interesting taxa are illustrated to warrant validations. comments are made on nature and composition of the faunal diversity, anomalous records, distribution of several species, important taxa, and salient features of the rotifer assemblages of the kashmir himalayan floodplains and j&k. this study merits interest for biodiversity and biogeography of rotifera of india, the kashmir himalayas and the indian floodplains. materials and methods the present observations are based on analysis of our plankton and semi-plankton samples collected, during january-february 2014, july-august 2014, february 2015, june 2015 and november 2016 from various lakes and small wetlands from the floodplains of the kashmir valley (fig. 1a-c). the samples were collected by towing a nylobolt plankton net (50 μm) and were preserved in 5% formalin. all the samples were screened with wild-stereoscopic binocular, individual rotifers were isolated and mounted in polyvinyl alcohol-lactophenol mixture and were observed with a leica dm 1000 phase contrast microscope. the micro-photographs of various species were taken using an image analyzer. the rotifer taxa were identified following koste (1978), segers (1995, 2003), sharma (1983, 1998), sharma and sharma (1999, 2000, 2008, 2013, 2018b) and jersabek and leitner (2013). the voucher collections were in the holdings of department of zoology, north-eastern hill university, shillong. in addition, the viable published records from jammu and kashmir state were evaluated for the validity and inclusion in our rotifer species inventory. results a total of 140 rotifera species (marked as *: appendix i), spread over 43 genera and 22 families, are observed from our collections from the floodplain wetlands of the kashmir valley, while we present a detailed systematic list of 173 species, belonging to 51 genera and 23 families, considered as validly recorded from j&k (appendix i). lecane bulla diabolica (fig. 2a) and testudinella insinuata (fig. 2b) are the oriental endemics; and brachionus durgae (fig. 2c) is a cosmo (sub) tropical species. keratella hiemalis (fig. 2d), k. serrulata (fig. 2e), k. ticinensis (fig. 2f), notholca acuminata (fig. 2g), n. labis (fig. 2h), n. squamula (fig. 2j), testudinella mucronata (fig. 2k), trichocerca edmondsoni (fig. 2l) and t. maior (fig. 2m) are other interesting species observed in our collections. twenty-five species are new records from northwest india; these include b. durgae, b. urceolaris, beauchampiella eudactylota, dissotrocha aculeata, figure 1. a-c: (a) map of india indicating the state of jammu and kashmir, (b) map of jammu and kashmir indicating kashmir valley and (c) map of kashmir valley indicating the sampled districts. a b c 210 sharma and sharma / the rotifers from the kashmir himalayan floodplains keratella tecta, mytilina acanthophora (fig. 2n), m. michelangellii (fig. 2o), lepadella benjamini (fig. 2p), l. eurysterna, l. quadricarinata (fig. 2q), lecane arcula, l. bulla diabolica, l. hornemanni, l. inopinata, l. monostyla, l. paxiana (fig. 2r), l. obtusa, l. signifera, l. stenroosi, l. tenuiseta (fig. 2s), l. undulata, sinantherina socialis, testudinella emarginula, t. parva (fig. 2t) and figure 2. interesting rotifers from jammu and kashmir. (a) lecane bulla diabolica (hauer)(lateral view), (b) testudinella insinuata hauer (ventral view), after sharma and sharma (2018a), (c) brachionus durgae dhanapathi with parthenogenetic eggs (ventral view), (d) keratella hiemalis carlin (ventral view), (e) keratella serrulata (ehrenberg) (dorsal view), (f) keratella ticinensis (callerio) (ventral view), (g) notholca acuminata (ehrenberg) (ventral view), (h) notholca labis gosse (ventral view), (j) notholca squamula (o.f. muller) (ventral view) and (k) testudinella mucronata (gosse) (ventral view). 211 int. j. aquat. biol. (2018) 6(4): 208-220 trichocerca maior. fourteen species recorded from j&k warrant validations; these are designated as `un-verifiable, and thus are not included in our inventory: brachionus leydigii cohn, 1862 dicranophorus robustus harring & myers, 1928 lecane arcuata (bryce, 1891) l. arcuata (bryce, 1891) unconfirmed l. cornuta (müller, 1786) l. crenata (harring, 1913) figure 2. continued. (l) trichocerca edmondsoni (myers) (lateral view), (m) trichocerca maior hauer (lateral view), (n) mytilina acanthophora hauer (lateral view), (o) mytilina michelangellii reid & turner (lateral view), (p) lepadella benjamini harring (dorsal view), (q) lepadella quadricarinata (stenroos) (ventral view), (r) lecane paxiana hauer (ventral view), (s) lecane tenuiseta harring (ventral view) and (t) testudinella parva (ternetz) (ventral view). 212 sharma and sharma / the rotifers from the kashmir himalayan floodplains l. depressa (bryce, 1891) l. elasma harring & myers, 1926 notholca striata (müller, 1786) platyias polycanthus ehrenberg, 1834 polyarthra remata skorikov, 1896 resticula melandocus (gosse, 1887) sphyrias lofuana (rousselet, 1910) squatinella rostrum (schmarda, 1846) cephalodella wiszniewskii edmondson and hutchinson 1934, described from jammu and kashmir, is a species inquirendus while diplois sp. denotes genus inquirendus. keratella brehmi, colurella oblonga, l. angulata, l. periptera and t. tetractis are invalid species, while k. valga is a classic example of misidentification. a total of 83 species (marked as @: appendix i) are not listed in the compilation by shah et al. (2015). discussion our collections from the floodplain wetlands of the kashmir valley record 140 rotifer species belonging to 43 genera and 22 families, while we consider 173 species, belonging 51 genera and 23 families, as validly known as per the present evaluation of rotifera biodiversity of jammu and kashmir (j&k) state of north india. interstingly, the former represent a notable fraction (~81%) of the rotifer fauna of j&k and form ~33% of the indian rotifera. the richness recorded from j&k comprises ~40% of the rotifer species known from india (sharma and sharma, 2017), and this study records one of the richest higher level diversity of eurotatoria genera and families known from india (sharma and sharma, 2017). the rich and diverse rotifer assemblages of j&k and the kashmir himalayan floodplains are hypothesized to habitat diversity and ecological heterogeneity of aquatic environs of this state in general and the floodplain wetlands in particular. the rotifer richness known from j&k concurs with 172 species observed from the eastern himalayas (bks, unpublished) as well as broadly with mean richness (177±29 species) examined from seven states of nei (bks, unpublished), while it presents distinct variations than 152, 162 , 161, 150 and 189 species known from the states of tripura (sharma and sharma, 2000), mizoram (sharma and sharma, 2015), meghlaya (sharma et al., 2016), nagaland (sharma et al., 2017) and manipur (sharma and sharma, 2018a) of nei, respectively. the comparisons, howver, are not tenable with regards the detailed species composition of the rotifer assemblages of j&k and individual states of nei. the rotifer assemblages of the kashmir himalayan floodplains are biodiverse than the reports from certain global floodplains i.e., 114 species (jose de paggi, 2001) from the rio pilcomayo national park (a ramsar site) of argentina; 124 species (oguta lake) from the niger delta (segers et al., 1993) of africa; 130 species from lake guarana, brazil (bonecker et al., 1994); 106 taxa from thale-noi lake, a ramsar site in thailand (segers and pholpunthin, 1997); and 104 species from laguana bufeos of bolivia (segers et al., 1998) while it largely compares with the reports of 136 species (iyiefi lake) from the niger delta (segers et al., 1993) of africa and 151 (koste, 1974) and 148 species from rio tapajos and lago camaleao (koste and robertson, 1983) of brazil, respectively. the species-rich and diverse nature of the rotifers of the kashmir valley wetlands endorses our reports from the floodplains of nei (sharma and sharma, 2014a, 2014b, 2017, 2018a) while yet fragmentary information from other floodplains of india hinders any effective comparion. the observed richness compares with the reports of 144 species from the majuli river island floodplains, upper assam (sharma et al., 2015) and 141 species from the floodplain lakes of the dibrusaikhowa biosphere reserve of the brahmaputra river basin, upper assam (sharma et al., 2017), while it is lower than the reports of 164 species from 15 beels of lower assam (sharma, 2005); 162 species from loktak lake, manipur (sharma et al., 2016) and 160 species from four beels of barpeta district of lower assam (sharma et al., 2018). the kashmir valley wetlands, however, indicate higher rotifer richness than 110 species (arora and mehra, 2003) examined from the floodplains of the river yamuna at delhi. on the other hand, our rotifer inventory is distinctly biodiverse than the reports of 27 species from two floodplain lakes of kashmir (khan, 1987) and 38 species from four ox213 int. j. aquat. biol. (2018) 6(4): 208-220 bow lakes and nine floodplain lakes of south-eastern west bengal (khan, 2003); these comparisons deserve caution because of incomplete species lists due to lack of adequate sampling and taxonomic delineations. rotifera richness presently reported from j&k and from the kashmir valley floodplains marks a notable increase over 110 species enlisted from the kashmir himalayas by shah et al. (2015). this comparison even assumes more prominance in light of much lower effective richness known vide the latter work because of anomalous inclusions of duplicate enteries of 14 species, six invalid species, one lapsus and eight unvalidated species, while 22 taxa lack species determinations. our biodiversity evaluation marks yet another distinct departure for non-inclusion of 83 species, belonging to 18 families, in the compilation by shah et al. (2015). the stated aspects limit the objectives of `documentation‘ and `authentication‘ of the latter publication vis-a-vis our analysis of rotifera of the kashmir himalayan wetlands. this state-of-art is attributed to routine species lists without validations in various `ad-hoc‘ ecology works from j&k, lack of adequate sampling, overlooking identification of small rotifer taxa and even lack of taxonmic expertiese. our species inventory, however, excludes 14 species designated as `reports inquerenda` without validations in any published work; the actual richness is thus likely to increase subjected to the future conformation and validation of these reports. further, we categorize cephalodella wiszniewskii, described as a new species from jammu and kashmir by edmondson and hutchinson (1934), as species inquirendus and diplois sp. as genus inquirendus following segers (2007), jersabek and leitner (2013) and sharma and sharma (2017). in addition, we consider the records (vide shah et al., 2015) of k. brehmi, c. oblonga, l. angulata, l. periptera and t. tetractis as `invalid species’, while the reports of k. valga from j&k and elsewhere from india are examples of misidentification (sharma and sharma, 2014b). the oriental endemics t. insinuata and l. bulla diabolica; the cosmo (sub) tropical b. durgae; and k. hiemalis, k. serrulata, n. acuminata, n. labis, t. edmondsoni and t. maior are fewer species of global distribution interest observed from j&k vide our collections from the kashmir himalayas. the relative paucity of the members of this category is hypothesized to ‘geographic barrier’ nature of high northern and western himalayan mountains vis-à-vis rotifera biogeography. this salient feature marks a significant departure than a sizable fraction of species of global biogeographic importance observed from the rotifer assemblages of nei (sharma and sharma, 2005, 2014a, 2017, 2018a) and that of the eastern himalayas (bks, unpublished) in particular. the rotifer fauna of j&k is, however, notable for several examples of regional distribution interest for the indian rotifera namely: asplanchnopus multiceps, cephalodella catellina, c. panarista, conochilus hippocrepis, collotheca campanulata, cupelopagis vorax, dicranophorus myriophylli, eosphora najas, euchlanis alata, floscularia conifera, hexarthra bulgarica, horaella brehmi, itura aurita, lecane paxiana, l. tenuiseta, lepadella benjamini, l. quadricarinata, pompholyx complanata, proales decipiens mytilina acanthophora, m. bisulcata, m. michelangellii, m. mucronata, notommata aurita, n. copeus, n. tripus, synchaeta stylata, s. tremula, testudinella mucronata, trichocerca brachyura and t. cavia. of these, 14 species are reported from this state only by edmondson and hutchinson (1934) with c. panarista, d. myriophylli, h. bulgarica, i. aurita, n. aurita, t. brachyura and s. tremula even solely known from india vide the stated work. lecane paxiana, l. tenuiseta, lepadella benjamini and l. quadricarinata are exclusively observed in our collections from the kashmir valley while t. mucronata is validly known from india only from j&k (sharma and sharma, 2018b). testudinella insinuata is added as a new record to the indian rotifera by sharma and sharma (2018b). our collections from the wetlands of the kashmir himalayas reveal 25 species new to northwest india namely b. durgae b. urceolaris, beauchampiella eudactylota, dissotrocha aculeata, keratella tecta, mytilina acanthophora, m. michelangellii, lepadella benjamini, l. eurysterna, l. quadricarinata, lecane arcula, l. bulla diabolica, l. hornemanni, l. inopinata, 214 sharma and sharma / the rotifers from the kashmir himalayan floodplains l. monostyla, l. paxiana, l. obtusa, l. signifera, l. stenroosi, l. tenuiseta, l. undulata, sinantherina socialis, testudinella emarginula, t. parva and trichocerca maior. the report of the oriental endemic l. bulla diabolica from j&k indicates notable extension of distribution of this lecanid to north india with its disjunct populations known till date from nei and tamil nadu (sharma and sharma, 2014a, 2017). besides, this study marks extension of distribution ranges of dissotrocha aculeata, m.michelangellii, l. benjamini, l. tenuiseta, and t. maior to the kashmir himalayas as against earlier indian reports only from nei (sharma and sharma, 2017). eleven eurotatoria families comprise bulk (~83%) of species known from j&k thus depicting widespread nature of the rotifer species composition. of these, lecanidae ≥ brachionidae form ~33%; trichocercidae = lepadellidae > notommatidae > euchlanidae comprise ~31% while synchaetidae, testudinellidae, mytilinidae, gastropodidae and trochosphaeridae collectively represent ~19%. this trend is also observed in the collections from the kashmir himalayan floodplains with increased importance of the first five families (~70% of species known from wetlands) but with lepadellidae > trichocercidae while synchaetidae, testudinellidae and mytilinidae are other three notable families (13.6%). nevertheless, the relatively lower richness of lecanidae and lepadellidae marks a distinct contrast to composition of rotifera of nei and its floodplains (sharma and sharma, 2014a, 2018a) as well as elsewhere from india (sharma and sharma, 2017). the relatively rich brachionidae is due to occurrence of species of the record seven genera (sharma and sharma, 2014b). the importance pattern of the stated families in general imparts the littoral-periphytic character to the rotifer assemblages of j&k and to the floodplain wetland of the kashmir himalayas. the latter are characterized by several small-sized species of colurella, lecane, lepadella and trichocerca; this feature is hypothesized to influence of juvenile fish and invertebrate predation (baumgartner et al., 1997) concurrent with our remarks from nei floodplains (sharma, 2005, 2014; sharma and sharma, 2014a, 2018a). the ‘tropic-centred’ lecane is relatively speciose genus (~17% and ~21%); trichocerca > brachionus = lepadella collectively comprise ~22% and ~25% and species of the ‘temperate-centred’ keratella > cephalodella > synchaeta are noteworthy (~12%, ~10%) features of the rotifer assemblages of j&k and that of the kashmir himalayan wetlands, respectively. the ‘temperate-centred’ keratella, notholca and cephalodella also deserve attention while the report of eight keratella spp. marks a notable feature of the indian brachionidae (sharma and sharma, 2014b, 2017) which holds parallel to our exclusive report from the eastern himalayas (bks, unpublished). the rotifers from j&k and the kashmir valley floodplains highlight relative paucity of brachionus, lepadella and even of lecane richness than known from nei (sharma and sharma, 2014a, 2018a) and elsewhere from india (sharma and sharma, 2017). the paucity of brachionus spp., however, corresponds with our reports from the floodplains of the majuli river island (sharma, 2014) and the dibru-saikhowa biosphere reserve (sharma et al., 2017) of upper assam, and the loktak lake basin of manipur (sharma and sharma, 2018a), nei. the rotifer faunas of j&k and the kashmir wetlands thus shows a combination of ‘tropic’ and ‘cold-water’ elements concurrent with our report (bks, unpublished) from the eastern himalayas but this feature is in distinct departure to the reports from other states of nei (sharma and sharma, 2014a, 2015, 2018a). further, the cosmopolitan species form a large fraction (~81%), while tropicopolitan and pantropical species together comprise ~13% of the rotifer fauna of j&k; the former out-number the reports from nei (sharma and sharma, 2014a) and elsewhere from india (sharma and sharma, 2017). the collections from the kashmir valley floodplains are notable for morphological variations of brachionus angularis, b. bidentatus, b. calyciflorus, b. caudatus, b. quadridentatus, euchlanis dilatata, keratella cochlearis, lecane bulla, l. closterocerca, l. curvicornis, l. hamata, l. leontina, l. luna, l. lunaris, l. ludwigii, l. papuana, l. quadridentata, l. signifera, l. unguitata, l. ungulata, lepadella costatoides, l. ovalis, l. patella, plationus patulus, 215 int. j. aquat. biol. (2018) 6(4): 208-220 testudinella emarginula, t. patina and t. tridentata populations. molecular analysis of likely cryptic diversity of the stated species is desired in light of some interesting related works of schröder and walsh (2010) and mills et al. (2017). in addition, the concept of ‘reverse taxonomy’ by combining morphometric, morphological and phylogenetic analyses (michaloudi et al., 2018) merits future interest. to sum up, the present study highlights the speciesrich and diverse rotifera assemblages of j&k and the floodplain wetlands of the kashmir himalayas which, in turn, are hypothesized to habitat diversity and environmental heterogeneity of the sampled aquatic biotopes. various new records; species of global and regional biogeographic interest with relative paucity of the former; the littoral-periphytic nature of the two assemblages with reports of several small-sized species; the occurrence of ‘tropical vs. temperate’ taxa; high richness of brachionidae and the relatively low richness of lepadellidae and even of lecanidae are notable features. this study merits biodiversity and biogeographic importance as the first-ever critical evaluation and validation of rotifera biodiversity j&k and ecosystem diversity interest for the kashmir himalayan floodplains in particular. as our collections are biased to the planktonic and semi-planktonic taxa, specific analysis of sessile, colonial and benthic taxa are desired along with intensive latitudinal and longitudinal collections, including those from ramsar sites and high altitude lakes and other water bodies. analysis of likely cryptic diversity in certain speciesgroups following ‘molecular vs. reverse taxonomy’ marks future interest. we estimate 260+ rotifera species from j&k with more interesting elements from the kashmir himalayas and ladak plateau with the latter as yet being practically un-explored vis-a-vis the indian rotifera. acknowledgements the senior author (bks) thanks the head, department of zoology, north-eastern hill university, shillong for laboratory facilities. the collection for this study was initiated by a. perrey who joined research under the senior author but decided to discontinue without any response to the official contacts till date. this work would not have been completed without help of n. ahmad bhat, research scholar, centre for advanced studies in botany, north-eastern hill university, shillong and various friends for helping us in the subsequent collections from the kashmir valley. the authors have no conflict of interests. references anderson h.h. 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(2014a). northeast india an important region with a rich biodiversity of rotifera. in: b.k. sharma, h.j. dumont, r.l. wallace (eds.). rotifera xiii: rotifer biology a structural and functional approach. international review of hydrobiology, 99(12): 20-37. sharma b.k., sharma s. (2014b). the diversity of indian brachionidae (rotifera: eurotatoria: monogononta) and their distribution opuscula zoologica budapest, 45(2): 165-180. sharma b.k., sharma s. (2015). biodiversity of freshwater rotifers (rotifera: eurotatoria) of mizoram, northeast india: composition, new records and interesting features. international journal of aquatic biology, 3(5): 301-313. sharma b.k., sharma s. (2017). rotifera: eurotatoria (rotifers). in: k. chandra, k.c. gopi, d.v. rao, k. valarmathi, j.r.b alfred (eds.). current status of freshwater faunal diversity in india, zoological survey of india, kolkata. pp: 93-113. sharma b.k., sharma s. (2018a). loktak lake, manipur revisited: a ramsar site as the rotifer (rotifera: eurotatoria) biodiversity hot-spot of the indian subregion. bonn zoological bulletin, 67(1): 5-13. sharma b.k., sharma s. (2018b). the indian species of testudinella (rotifera: flosculariacea: testudinellidae) and their distribution. international journal of aquatic biology, 6(1): 15-20. sharma b.k., sharma s., hatimuria m.k. (2015). rotifer assemblages (rotifera: eurotatoria) of the floodplain lakes of majuli river island, the brahmaputra river basin, northeast india. international journal of aquatic biology, 3(1): 1-13. sharma s., sharma b.k. (2008). zooplankton diversity in floodplain lakes of assam. records of the zoological survey of india, occasional paper, 290: 1-307. sharma s., sharma b.k. (2013). faunal diversity of aquatic invertebrates of deepor beel (a ramsar site), assam, northeast india. wetland ecosystem series, 17: 1-226. 218 sharma and sharma / the rotifers from the kashmir himalayan floodplains family: brachionidae 1. anuraeopsis fissa (gosse, 1851) * 2. a. navicula rousselet, 1911* 3. brachionus angularis gosse, 1851 * 4. b. bidentatus anderson, 1889 * 5. b. calyciflorus pallas, 1766 * 6. b. caudatus barrois & daday, 1894 * 7. b. durgae dhanapathi, 1974 *#@ 8. b. falcatus zacharias, 1898 * 9. b. forficula wierzejski, 1891 * 10. b. plicatilis o.f. muller, 1786 11. b. quadridentatus hermann, 1783 * 12. b. rubens ehrenberg, 1838 * 13. b. urceolaris o. f. muller, 1773 *#@ 14. kellicottia longispina (kellicott) 15. keratella cochlearis (gosse, 1851) * 16. k. hiemalis carlin, 1943 * 17. k. procurva (thorpe, 1891) * 18. k. quadrata (o. f. muller, 1786) * 19. k. serrulata (ehrenberg, 1838) *@ 20. k. tecta (gosse, 1851) *#@ 21. k. ticinensis (callerio, 1921) *@ 22. k. tropica (apstein, 1907) * 23. notholca acuminata (ehrenberg, 1832) *@ 24. n. labis gosse, 1887 * 25. n. squamula (o.f. muller, 1786) *@ 26. plationus patulus (o.f. muller, 1786) * 27. platyias quadricornis (ehrenberg, 1832) * family: epiphanidae 28. epiphanes brachionus (ehrenberg, 1837) * @ 29. e. senta (o.f. muller, 1773) family: euchlanidae 30. beauchampiella eudactylota (gosse, 1886) *#@ 31. dipleuchlanis propatula (gosse, 1886) *@ 32. euchlanis alata voronkov, 1912 33. e. calpidia (myers, 1930) 34. e. deflexa gosse, 1851 35. e. dilatata ehrenberg, 1832 * 36. e. incisa carlin, 1939 *@ 37. e. meneta myers, 1930@ 38. e. oropha gosse, 1887 * @ 39. tripleuchlanis plicata (levander, 1894) *@ family: mytilinidae 40. lophocharis oxysternon (gosse, 1851) *@ 41. l. salpina (ehrenberg, 1834) *@ 42. mytilina acanthophora hauer, 1938 *#@ 43. m. bisulcata (lucks, 2012) *@ 44. m. michelangellii reid & turner, 1988 *#@ 45. m. mucronata (o.f. muller, 1773) * 46. m. ventralis (ehrenberg, 1830) * family: trichotriidae 47. macrochaetus collinsi (gosse, 1867) * 48. trichotria pocillum (o.f. muller, 1776) 49. t. tetractis (ehrenberg, 1830) * family: lepadellidae 50. colurella adriatica ehrenberg, 1831 * 51. c. colurus (ehrenberg, 1830) *@ 52. c. obtusa (gosse, 1886) * 53. c. uncinata (o.f. muller, 1773) * 54. lepadella acuminata (ehrenberg, 1834) * 55. l benjamini harring, 1916 *#@ 56. l. cristata (rousselet, 1893) * @ 57. l. ehrenbergi (perty, 1850) * 58. l. eurysterna myers, 1942*#@ 59. l. heterostyla (murray, 1913) * @ 60. l. ovalis (o.f. muller, 1786) * 61. l. patella (o.f. muller, 1773) * 62. l. quadricarinata (stenroos, 1898) *#@ 63. l. rhomboides (gosse, 1886) * 64. l. triptera ehrenberg, 1832*@ 65. squatinella lamellaris (o. f. müller, 1786) * family: lecanidae 66. lecane aculeata (jakubski, 1912) * 67. l. arcula harring, 1914 *#@ 68. l. bulla (gosse, 1851) * l. bulla diabolica (hauer, 1936) *@ 69. l. closterocerca (schmarda, 1859) * 70. l. crepida harring, 1914 * 71. l. curvicornis (murray, 1913) *@ 72. l. decipiens (murray, 1913) * 73. l. flexilis (gosse, 1886) * 74. l. furcata (murray, 1913) * 75. l. hamata (stokes, 1896) * 76. l. hornemanni (ehrenberg, 1834) *#@ 77. l. inermis (bryce, 1892) * #@ 78. l. inopinata harring & myers, 1926 *#@ 79. l. leontina (turner, 1892) *@ 80. l. ludwigii (eckstein, 1883) * 81. l. luna (müller, 1776) * 82. l. lunaris (ehrenberg, 1832) * 83. l. monostyla (daday, 1897) *# @ 84. l. nana (murray, 1913) * appendix i: systematic list of rotifera known from jammu and kashmir. phylum: rotifera class: eurotatoria subclass: monogononta order: ploima 219 int. j. aquat. biol. (2018) 6(4): 208-220 85. l. obtusa (murray, 1913) *#@ 86. l. papuana (murray, 1913) *@ 87. l. paxiana hauer, 1940 *#@ 88. l. pyriformis (daday, 1905) * 89. l. quadridentata (ehrenberg,1830) * 90. l. signifera (jennings, 1896) *#@ 91. l. stenroosi (meissner, 1908) *#@ 92. l. sympoda hauer, 1929 * 93. l. tensuiseta harring, 1914 *#@ 94. l. undulata hauer, 1938*#@ 95. l. ungulata (gosse, 1887) * family: proalidae 96. proales decipiens (ehrenberg, 1832) @ family: notommatidae 97. cephalodella auriculata (o.f. müller, 1773) @ 98. c. catellina (o.f. müller, 1786) *@ 99. c. exigua (gosse, 1886) * 100. c. gibba (ehrenberg, 1830) * 101. c. intuta myers, 1924@ 102. c. panarista myers, 1924 @ 103. eosphora najas ehrenberg, 1830@ 104. itura aurita (ehrenberg, 1830) @ 105. monommata grandis tessin, 1890 *@ 106. notommata aurita (müller, 1786) @ 107. n.copeus ehrenberg, 1834*@ 108. n. tripus ehrenberg, 1838*@ family: scaridiidae 109. scaridium longicaudum (o.f. müller, 1786) * family: gastropodidae 110. ascomorpha. ecaudis perty, 1850 111. a. saltans bartsch, 1870 * 112. a. ovalis (bergendal, 1892) *@ 113. ascomorphella volvocicola (plate, 1886) 114. gastropus hyptopus (ehrenberg, 1838) *@ 115. g. stylifer imhof, 1891@ family: trichocercidae 116. trichocerca bicristata (gosse, 1887) *@ 117. t. brachyura (gosse, 1851) *@ 118. t. cavia (gosse, 1886) @ 119. t. cylindrica (imhof, 1891) * 120. t. edmondsoni (myers, 1936) * 121. t. elongata (gosse, 1886) *@ 122. t. iernis (gosse, 1887) *@ 123. t insignis (herrick, 1885) * 124. t. longiseta (schrank, 1802 * 125. t. maior hauer, 1936 *# 126. t. porcellus (gosse, 1881) 127. t. rattus (o.f. müller, 1776) * 128. t. ruttneri donner, 1953 *@ 129. t. similis (wierzejski, 1893) *@ 130. t. tigris (o.f. müller, 1786) * 131. t. weberi (jennings, 1903) * family: asplanchnidae 132. asplanchna brightwelli gosse, 1850 * 133. a. priodonta gosse, 1850 * 134. a. sieboldi (leydig, 1854) 135. asplanchnopus multiceps (schrank, 1793) family: synchaetidae 136. ploesoma lenticulare herrick, 1885 * @ 137. polyarthra dolichoptera idelson, 1925 *@ 138. p. euryptera wierzejski, 1891 139. p. vulgaris carlin, 1943.* 140. synchaeta oblonga ehrenberg, 1832 *@ 141. s. pectinata ehrenberg, 1832 * 142. s. stylata wierzejski, 1893 * 143. s. tremula (o.f. müller, 1786) @ family: dicranophoridae 144. dicranophorus epicharis harring & myers, 1928 * 145. d. myriophylli (harring, 1913) @ 146. d. robustus harring & myers, 1928 order: flosculariaceae family: floscularidae 147. floscularia conifera (hudson, 1886) @ 148. sinantherina socialis (linne, 1758) *#@ family: conochilidae 149. conochilus dossuarius hudson, 1885@ 150. c. hippocrepis (schrank, 1803) @ 151. c. unicornis rousselet, 1892 * family: hexarthridae 152. hexarthra bulgarica (wiszniewski, 1933) @ 153. h. intermedia (wiszniewski, 1929) 154. h. mira (hudson, 1871) * family: testudinellidae 155. pompholyx complanata gosse, 1851 156. p. sulcata hudson,1885 *@ 157. testudinella emarginula (stenroos, 1898) *#@ 158. t. insinuata hauer, 1938 *@ 159. t. mucronata (gosse, 1886) *@ 160. t. parva (ternetz, 1892) *#@ 161. t. patina (hermann, 1783) *@ family: trochosphaeridae 162. filinia longiseta (ehrenberg, 1834) * 163. f. opoliensis (zacharias, 1898) * 164. f. terminalis (plate, 1886) * 165. horaella brehmi donner, 1949 166. trochosphaera solstitialis thorpe, 1893 * order: collothecaceae family: atrochidae 167. cupelopagis vorax (leidy, 1857) *@ family: collothecidae 168. collotheca campanulata (dobie, 1849) @ 169. c. ornata (ehrenberg, 1832) * sub-class: digononta order: bdelloidea family: philodinidae 220 sharma and sharma / the rotifers from the kashmir himalayan floodplains 170. *#@ 171. philodina roseola ehrenberg, 1832*@ 172. rotaria neptunia (ehrenberg, 1830) * 173. r. rotatoria (pallas, 1766) *@ ----------------------------------------------------------------------------------------------------------------------------------------------- * recorded in our collection from the kashmir himalayas; # new record from northwest india (nwi); @ not listed in the compilation by shah et al. (2015). int. j. aquat. biol. (2018) 6(3): 157-161 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article determination of mancozeb toxicity and biochemical effects in common carp (cyprinus carpio) pooria simakani1, mohammad hadi abolhasani*2,1seyyed morteza hoseini3 1department of environmental science, islamic azad university, isfahan (khorasgan) branch, isfahan, iran. 2waste and wastewater research center, department of environmental science, islamic azad university, isfahan (khorasgan) branch, isfahan, iran. 3inland waters aquatics resources research center, iranian fisheries sciences research institute, agricultural research, education and extension organization, gorgan, iran. article history: received 20 april 2018 accepted 22 june 2018 available online 2 5 june 2018 keywords: carp mancozeb blood toxicity pesticide abstract: the aim of this study was to investigate mancozeb toxicity and its effects on physiological characteristics of common carp. fish were reared for one week to acclimatize with the experimental conditions. for 96h-lc50 determination, the fish were stocked in 16 aquaria at the density of 10 fish per aquarium. the aquaria were exposed to 8 mancozeb concentrations (two aquaria per concentration) for 96 h (0, 0.94, 1.87, 3.75, 7.50, 15, 30 and 60 mg/l). 96h-lc50 was calculated based on the fish mortality, being 8.03 (4.95-13.2) mg/l. for sub-acute test, the fish were exposed to 0 (control), 1.6, 2.4 and 3.2 mg/l mancozeb (20, 30 and 40% of the 96h-lc50) for one week. blood samples were taken from each treatment for determination of plasma glucose, total protein, albumin, globulin, calcium, alanine aminotransferase (alt) and aspartate aminotransferase (ast). mancozeb exposure led to increase in glucose and ast, and decrease in plasma proteins and alt. in conclusion, mancozeb exposure causes stress response, health problem and tissue damage in common carp. introduction now a day, input and distribution of pollutants to ecosystems and their effects are of the main environmental concerns. industrial developments and population growth led to chemical pollutant accumulation in aquatic ecosystems (saghali et al., 2014). the pollutants behaviors may be assessed at three levels: water column, sediments and biomass of aquatic organisms (saghali et al., 2014). one of the serious threats for human is pollutant entry to waters, leading to accumulation in aquatic organisms’ body and moving towards higher levels of food chain (shaw and handy, 2011). agricultural pesticides are considered as one of the largest group of environmental pollutant, which are extensively studied in aquatic toxicology (wang et al., 2015). the effects of the pesticides on fishes are of important environmental concerns. mancozeb is a carbamate fungicide that is used for control of cucumber mildew. this pesticide has short half-life but may cause neural and humoral issues in human *corresponding author: mohammad hadi abolhasani doi: https://doi.org/10.22034/ijab.v6i3.494 e-mail address: hadi.mha2001@yahoo.com (wang et al., 2014). today, human consumes aquatic organisms, as the healthiest sources of food and protein. fish are one of the most important aquatic animals due to high economic value and susceptibility to pollutants, thus, are used for variety of biological studies (hedayati et al., 2016). different fish vary in susceptibility to pollutants, thus, it is necessary to conduct toxicological studies on different fish species (mazandarani and hoseini, 2017). mancozeb toxicity has been rarely studied in fish. 96h-lc50 of the pesticide has been 11.68 mg/l in oreochromis mossambicus, which caused behavioral changes in the fish (saha et al., 2016). mancozeb was found to induce oxidative stress in goldfish (carassius auratus) characterized by increased activities of antioxidant enzymes, and levels of protein carbonyle and lipid peroxides (kubrak et al., 2012). moreover, bisson and hontela (2002) found that mancozeb exposure interfere with cortisol secretion in response to stress. however, there are no data about mancozeb lethal concentration and biochemical effects in common 158 simakani et al. / determination of mancozeb toxicity and biochemical effects in common carp carp (cyprinus carpio). common carp is an economically important species and is reared in many parts of iran (hosseini and hoseini, 2012). it is a popular fish in iran north and its population in the caspian sea is supported by stock rehabilitation activities of iranian fisheries organization, because of declining the natural population in the sea. as mancozeb is used in agricultural fields of caspian region, it might enter surface water reaching the caspian sea and threatening the organisms. thus, the aim of this study was to assess acute toxicity and biochemical effects of mancozeb on common carp. materials and methods fish maintenance conditions: a total number of 280 common carp fingerlings (30 g) were purchased and transported to laboratory and allowed to acclimatize under the experimental conditions for one week. during the acclimation period, the fish were fed twice a day (1% of biomass) and uneaten feeds were removed from the aquaria to avoid water pollution. the fish were randomly distributed into 16 aquaria at the density of 10 fish per aquarium. the aquaria water was renewed by 50% per day. lc50 determination: the fish were exposed to 0, 0.94, 1.87, 3.75, 7.50, 15, 30 and 60 mg mancozeb per liter (as commercial product with 80% purity) and their mortality was recorded over a 96-h period. lc50 was determined according to hoseini and nodeh (2011) using probit analysis. two aquaria were assigned for each concentration and 10 fish were stocked in each aquarium. the fish were not fed during the experiment. the aquaria water were daily renewed by 50% and desired amount of mancozeb were added to the aquaria to maintain the pesticide concentration at constant levels. sub-acute experiment: according to lethal concentrations of mancozeb, three concentrations of 1.6, 2.4 and 3.2 mg/l (20, 30 and 40% of the 96hlc50) along with a control group (totally four treatments with three replicates). twelve aquaria (each containing 10 fish) were used for this experiment and the fish were allowed to acclimatize with the experimental conditions for one week. the fish were fed based on 1 % of biomass and the aquaria water was daily renewed at 50%. water dissolved oxygen, ph, temperature, hardness, nitrite and ammonia were determined during the experiment (hoseini and nodeh, 2011; hoseini et al., 2012; hoseini and jafar nodeh, 2012). the fish were bled after one-week exposure to mancozeb. sampling and blood processing: six fish were sampled form each treatment. the fish were immediately anesthetized in clove oil (100 mg/l) and bled using heparinized syringe. the blood samples were centrifuged for plasma separation. the obtained plasma samples were kept at -20°c until (taheri mirghaed et al., 2017; taheri mirghaed et al., 2018). plasma glucose (god method), total protein (biuret method), albumin (bromocresol green method), calcium (cresolphthalein complexone method), alanine aminotransferase (alt; conversion of alanine to lactate) and aspartate aminotransferase (ast; conversion of aspartate to malate) were determined using commercial kits (pars azmun). statistical analyses: mancozeb lc50 was determined by probit regression using probit analysis program v. 1.5. plasma data normality was checked and then the data were analyzed by one-way anova and duncan test in sas 9.4 software. p<0.05 was considered as significance level. results water quality data are presented in table 1 and were within acceptable range. fish mortality and mancozeb lc50: the fish mortality after 24-96 h exposure to different mancozeb concentrations are presented in table 2. the results showed that increase in the pesticide concentrations and/or exposure time led to increased mortality. probit analysis showed that mancozeb lc50 for common carp was 8.03 (4.95-13.2) mg/l. behavioral and morphological alterations: the results showed that the fish behaviors and morphology changed markedly. at the early exposure, the fish were anxious with fast swimming followed by anorexia. surface swimming, irregular and imbalance swimming were observed in the exposed fish. 159 int. j. aquat. biol. (2018) 6(3): 157-161 morphological changes were mucus hypersecretion, body and gill discoloration, scale loss and hemorrhage on body and around operculum. biochemical characteristics of fish exposed to mancozeb: table 3 shows biochemical data of the fish exposed to different concentrations of mancozeb. mancozeb concentrations had significant effects on plasma glucose and the highest glucose levels were observed ta the concentrations of 3.2 mg/l (p<0.05). mancozeb concentrations significantly affected plasma total protein and globulin levels and the highest and lowest levels were observed in the control and 3.2 mg/l mancozeb groups, respectively (p<0.05). mancozeb had significant effects on plasma albumin levels and 3.2 mg/l mancozeb led to significant decrease in plasma albumin compared to the other treatments. there was no significant difference in plasma calcium levels among the treatments. the highest and lowest plasma albumin levels were observed in the control and 3.2 mg/l mancozeb treatments, respectively (p<0.05). mancozeb exposure significantly affected alt activities and the highest and lowest activities were observed in the control and 3.2 mg/l mancozeb treatments, respectively (p<0.05). the lowest plasma ast were observed in the control group that was significantly different compared to the other groups. 1.6 and 2.4 mg/l mancozeb groups had similar enzyme activities and significantly lower than 3.2 mg/l mancozeb group (p<0.05). discussion in aquatic toxicology, lc50 lower than 1000 ppb says “very toxic” substance, between 1000 and 10000 ppb says “moderately toxic” substance, and higher than 10000 ppb says “less toxic” substance. accordingly, mancozeb is moderately toxic for common carp juveniles. the resulted lc50 in the present study was slightly lower than that reported in o. mossambicus (saha et al., 2016). fish behaviors under toxicant exposure are ideal tools to assess the effects of aquatic pollutants on fish, because behavioral indicators reflex the physiological states. toxicant exposure may induce a complete behavioral change that affects fish survival in natural ecosystems, particularly in toxicant concentrations lower than lethal concentration. table 1. water physicochemical parameters during the experiment. parameters hardness (mg/l caco3) temperature (°c) ph dissolved oxygen (mg/l) nitrite (mg/l) ammonia (mg/l) content 318 ± 21.12 18.25 ± 0.21 7.3 ± 0.35 7.3 ± 0.4 1.1 ± 0.12 0.67 ± 0.1 table 2. the fish mortality after 24-96 h exposure to different mancozeb concentrations. concentration (mg/l) number of fish 24h 48h 72h 96h 0.96 10 0 0 0 0 1.87 10 0 0 1 2 3.75 10 0 0 1 4 7.5 10 0 0 1 5 15 10 0 1 3 7 30 10 0 1 4 8 60 10 0 2 5 10 table 3. plasma biochemical characteristics of common carp exposed to different concentrations of mancozeb for one week (different letters show significant difference). parameters control 1.6 mg/l 2.4 mg/l 3.2 mg/l glucose (mg /dl) 67.3±5.43c 117±21.07b 140.45±23.12ab 153.44±12.34a total protein (g /dl) 4.33±0.21a 3.56±0.43b 2.43±0.44b 2.23±0.47b albumin (g /dl) 1.50±0.07a 1.45±0.06a 1.40±0.02a 1.32±0.03b globulin (g/dl) 2.80±0.11a 2.11±0.12b 1.02±0.08c 0.91±0.05c calcium (mg /dl) 7.32±0.5a 6.56±0.54a 6.95±0.32a 6.55±0.4a alt (u/l) 73.31±9.27a 66.23±11.26b 57.57±3.46c 54.13±3.21c ast (u/l) 23.11±3.66c 45.01±6.17b 46.69±5.83b 52.12±12.03a 160 simakani et al. / determination of mancozeb toxicity and biochemical effects in common carp behavioral changes are mainly related to cholinesterase inhibition, alternation of brain neurotransmitter levels, sensory deficiency, and impaired gonadal or thyroid hormone levels (taheri mirghaed and ghelichpour, 2015). scott and sloman (2004) monitored the relation between behavioral and physiological indicators of toxicity in fish. in addition, a positive correlation was observed between brain acetyl cholinesterase activity and swimming speed of gambusia affinis after lethal exposure to monocrotophos (kavitha and rao, 2007). plasma glucose increased in the mancozeb-treated fish suggesting stress induction due to the toxicant exposure. under stressful conditions and toxicant exposure, fish need more energy supply to cope with toxicant-induced adverse effects; thus, circulating levels of glucose increases (hoseini et al., 2018). previous studies on common carp have shown that exposure to toxicants led to hyperglycemia in the fish (hoseini and tarkhani, 2013; hoseini et al., 2014; hoseini et al., 2016a). plasma proteins are suitable indicators of fish health. they are responsible for many vital functions in body including immune response and antioxidant system (silva et al., 2010). decreased total protein, albumin and globulin may suggest liver damage as most of the proteins, including albumin, are synthesized in fish liver (hoseini and tarkhani, 2013). the present results are in agreement with those reported previously (shariff et al., 2001; hoseini and tarkhani, 2013; hoseini et al., 2016a). alt and ast are non-functional enzymes in circulation, but indicators of tissue damage. they are found at high concentrations in liver and kidney, thus damages to this organs lead to increased enzymes’ activity in blood (haschek et al., 2009). accordingly, the present results show that mancozeb had detrimental effects on carp liver and kidney. similar results was found in common carp exposed to other toxicants (hoseini et al., 2016b; hoseini et al., 2016a; ghelichpour et al., 2017; hoseini et al., 2018). in conclusion, mancozeb exposure causes stress response, health problem and tissue damage in common carp. such adverse effects may decrease the fish well-being and survivorship in natural environment. references bisson m., hontela a. 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(2018) 6(3): 157-161 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی (cyprinus carpio) معمولی کپور ماهی در مانکوزب بیوشیمیایی آثار و مسمومیت تعیین 3حسینی مرتضی سید، 2*ابوالحسنی هادی محمد ،1پوریا سیمکانی .ایران، اصفهان ،(خوراسگان) اصفهان واحد اسالمی آزاد دانشگاه زیست، محیط علوم گروه1 .ایران ،اصفهان ،(خوراسگان) اصفهان واحد اسالمی آزاد دانشگاه زیست،محیط علوم گروه پسآب، و پسماند تحقیقات مرکز2 .ایران ،ترویج کشاورزی، گرگانتو تحقیقات علوم شیالتی ایران، سازمان تحقیقات، آموزش و یهای داخلی، انستمرکز تحقیقات ذخایر آبزیان آب3 چکیده: مدت یک هفته با ها بهبود. ماهی ماهی کپور معمولیهای فیزیولوژیکی در هدف از این تحقیق بررسی مسمومیت مانکوزب و اثرات آن بر شاخص سازی شدند. ماهی در هر آکواریوم ذخیره 10آکواریوم با تراکم 16ساعته، ماهی ها در lc50 96شرایط آزمایشگاهی سازگار شدند. جهت تعیین گرم بر لیتر( قرار گرفتند میلی 60و 30، 18، 50/7، 75/3، 87/1، 94/0، 0غلظت مختلف مانکوزب ) 8ساعت در معرض 96مدت ها بهسپس آکواریوم گرم بر لیتر بود. برای انجام ( میلی95/4-2/13) 03/8ساعت محاسبه شد و برابر با 96بر اساس تلفات در خالل lc50آکواریوم برای هر غلظت(. 2) درصد 40و 30، 20گرم بر لیتر مانکوزب قرار گرفتند )میلی 2/3و 4/2، 6/1)شاهد(، 0مدت یک هفته در معرض ها بهآزمایش تحت حاد، ماهی lc50 ،گلبولین، کلسیم، آالنین آمینوترنسفراز و آسپارتات (. نمونه خون از همه تیمارها برای تعیین مقدار گلوکز، توتال پروتئین، آلبومین ز در پالسما ها و آالنین آمینوترنسفراآمینوترنسفراز در پالسما گرفته شد. مانکوزب باعث افزایش گلوکز و آسپارتات آمینوترنسفراز و کاهش پروتئین شود. آسیب بافتی در ماهی کپور معمولی می شود که مسمومیت با مانکوزب باعث بروز استرس، افت سالمت وگیری میشد. نتیجه .کش آفت مسمومیت، خون، مانکوزب، کپور، :کلمات کلیدی int. j. aquat. biol. (2015) 3(1): 52-59 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article simultaneous detection of aeromonas hydrophila, and escherichia coli in rainbow trout (oncorhynchus mykiss) by duplex pcr fatemeh fattahi1, alireza mirvaghefi*1, hamid farahmand1, gholamreza rafiee1, alireza abdollahi2 1department of fisheries, faculty of natural resource, university of tehran, karaj, iran. 2department of pathology, school of medicine, tehran university of medical sciences, tehran, iran. article history: received 14 october 2014 accepted 6 january 2015 available online 2 5 february 2015 keywords: duplex pcr rainbow trout simultaneous detection 16srrna gene abstract: rapid and accurate identification of microorganisms have a significant impact on strategies and fish health management programs. hence, in this study a duplex pcr assay based on the 16s rrna gene for simultaneous detection of aeromonas hydrophila rticc 1032 and escherichia coli rticc 2325 from pure cultures, and challenged fish tissues was performed and their results compared with the results of single pcr assays for each bacterium. for this purpose, an experiment with three treatments including artificially infected with a. hydrophila, e. coli and a mixture of them with a control group was designed. fish were injected intraperitoneally with 1 ml of sterile physiological saline containing 106 cfu/ml of the corresponding bacteria. samples were collected from liver, kidney and spleen 48 hrs post-injection. a duplex pcr based 16s rrna genes was developed for the simultaneous detection of a. hydrophila and e. coli. the pcr reaction conditions were optimized to permit detection of organisms from agar plates and fish tissues in less than 8 hrs. each of the two pairs of oligonucleotide primers exclusively targeted 16s rrna gene of the specific microorganism. when duplex pcr assay was used to simultaneous detection of the pathogens in asymptomatic fish, spleen and liver were negative for a. hydrophila, whereas kidney was positive for two bacteria. samples of control group with negative results of duplex pcr were also negative by the culture method. on the whole, the duplex pcr has advantages in terms of its accuracy, sensitivity, ease of use, time of length analysis and cost-effectiveness compared to the single pcr and traditional method. introduction fishes are often exposed to various microorganisms such as aeromonas hydrophila that is naturally present in the fresh water environments (belanco, 2000). this bacterium does not cause disease under optimal conditions, whereas under unfavorable environmental conditions, physiological stress or infection by other pathogens can cause motile aeromonad septicemia (mas), epizootic ulcerative syndrome (eus) and ulcerous dermatitis, which are led to economic losses in aquaculture industry (plumb et al., 1976; fang et al., 2000; laptera et al., 2010). in addition, the presence of escherichia coli in fishes is considered an indicator of the polluted or * corresponding author: alireza mirvaghefi e-mail address: avaghefi@ut.ac.ir stressful environments where fish inhabit (dhss, 1991; gelderich et al., 1966; sinderman, 1988). escherichia coli enters aquatic ecosystems via animal excreta, agricultural runoff and human consumed wastes (ferreira da silva et al., 2007; berier et al., 2008). therefore, significant numbers of e. coli on the skin and gut of the fishes can be led health risk to human (janssen 1974; ishii et al., 2007). rapid and accurate identification of the microorganisms, especially pathogen bacteria have a significant impact on fish health management programs (adams et al., 2006). traditionally, the diagnosis of the pathogenic bacteria is performed by 53 fattahi et al./ duplex pcr method detection of a. hydrophila, and e. coli in rainbow trout culturing bacteria on agar plates followed by phenotypic and serological properties (altinok et al., 2008). however, detections of some bacteria are difficult due to their morphological variations and unusual biochemical reactions. therefore, molecular diagnosis methods such as reverse transcription pcr, quantitative pcr, real-time pcr, aflp, rflp and rapd have been developed to detect specific nucleic acids without culture and isolation of the pathogens (tang et al., 2006; adams et al., 2008). in this regard, the individual pcr assay is an effective method for identification of the fish pathogens; however, a large number of individual pcr assays are necessary when a single primer set is used on a large number of the clinical samples, which can be a relatively costly and time-consuming process. hence, the simultaneous detection of several pathogens with a multiplex pcr (m pcr) approach would be a relatively rapid and cost effective method (mata et al., 2004). in this method, we seek to diagnose all possible pathogens, which can be occurred in each disease (belak, 2007). the multiplex pcr assay for the simultaneous detection of fish pathogenic bacteria has been recently described (del cerro et al., 2002; mata et al., 2004; altinok et al., 2008). hence, in this work, a duplex pcr assay based on the 16s rrna genes for the simultaneous detection of a. hydrophila and e. coli from pure cultures, and the challenged rainbow trout tissues (oncorhynchus mykiss) including liver, kidney and spleen were performed and the results compared with single pcr assays for each bacterium. materials and methods fish: fifty-two rainbow trout with a mean weight of 246 ± 20. 91 g (mean ± sd) and mean length of 27.04 ± 0.90 cm (mean ± sd) were obtained from a commercial fish farm in karaj (alborz province, iran). they were introduced at a rate of 13 fish per l000-liter to four tanks with proper aeration. fish were acclimatized to the laboratory conditions for 2 weeks prior to experiment. the water temperature during acclimatization period and experiment was 14.5°c (± 1.5). bacteria: bacterial isolates (a. hydrophila rticc 1032 and e. coli rticc 2325) were obtained from the razi vaccine and serum research institute (table 1). for the challenges, each bacterium strain was cultured on tsa (himedia-m 290) at 37°c for 24 hrs and harvested in a sterile physiological saline to 10-9 and diluted to an optical density of 1.0 at a wavelength of 640 nm. this corresponds to a bacterial concentration of 1 x 106 cfu/ml. challenge with a. hydrophila and e. coli: for bacterial challenge, 9 fish (three from every tank) were randomly selected, anaesthetized in 100 mg/ml of tricaine methane sulphonate (tms) and injected intraperitoneally with 1 ml of the bacterial suspension of a. hydrophila, e. coli and mixture of a. hydrophila and e. coli, respectively. then, injected fish were returned to treatment tanks and allowed to recover from the anesthetic. one of tanks was considered as controls group without injection. fish sampling: forty-eight hours after injection, three fish from each treatment were sampled and their body surface were swabbed using 70% ethyl alcohol after killing by overdosing using tms to prevent contamination from the rearing environment and normal external bacterial flora. to obtain similar size samples, 1 mm cubes of the liver, kidney and spleen were aseptically removed and put in the microcenterfuge tubes for detection of microorganism from fish tissue. then, the samples of liver, kidney and spleen were streaked on tryptic soy agar. following incubation, one typical colony was selected from each isolate and sub-cultured on bacteria species strain number donor escherichia coli rticc 2325 razi vaccine and serum research institute aeromonas hydrophila rticc 1032 razi vaccine and serum research institute table 1. bacteria strains used in the experiment. 54 int. j. aquat. biol. (2015) 3(1): 52-59 macconkey (merck-5465), emb (oxoid-cm69) and blood agar media to check purity of the isolates. all isolates were stored in a broth culture supplemented with 15% glycerol at -70°c. the isolates were classified as a. hydrophila or e. coli according to their reactions in the following conventional tests including catalase, motility, indole, voges-proskaues, urea, triple sugar iron (tsi), glucose, methyl red, h2s production and citrate utilization tests based on bergey’s manual of determinative bacteriology (holt, 2000). every substrate was incubated at 37°c and reactions read after 24 and 48 hrs. dna extraction: for dna extraction of the isolates (pure cultures), a boiled method was used based on sambrook et al. (2001) by phenol-chloroform– isoamyl alcohol. dna concentrations of samples were evaluated using a spectrophotometer. in addition, dna was extracted form liver, kidney and spleen of artificially infected fish based on altinok et al. (2008). the extracted dna quality was evaluated using electrophorese on a 0.8% agarose gel. primers and pcr conditions: the used primers in this study were based on nilsen et al. (2001) and sabat et al. (1991) to validate the duplex pcr assay for the simultaneous detection of a. hydrophila and e. coli in asymptomatic carrier fish. the pcr protocol was optimized by amplification reaction in a thermal cycler (astec, japan) using the ready–to– go pcr beads (cinagene, iran). reaction mixtures had 1 µl of each primer, 1 µl of the dna template, 17.5 µl of sterile distilled water, 1 µl of mgcl2, 0.5 µl of dntp, 2.5 µl of 10 x pcr buffer. pcr conditions consisted of an initial denaturation step at 94°c for 7 min followed by 30 cycles of amplification (denaturation at 94°c for 1 min, annealing at 58°c for 1 min, and extension at 72°c for 1 min) and a final 10 min elongation period at 72°c. controls received the pcr mixture containing (1) no template, (2) dna from control fish and (3) dna from e. coli and a. hydrophyla (positive control). after the pcr, the products were transferred to a 1.5% agarose gel, electrophoresed, and dna was visualized by ethidium bromide staining. table 2 shows the sequences of the two primer pairs used in this study. duplex pcr assay: a duplex pcr assay was developed for the simultaneous detection of a. hydrophila and e. coli in which rainbow trout were experimentally challenged with both bacteria. the specificity and sensitivity of this assay was evaluated by performing the duplex pcr to the detection of healthy carriers. samples were collected from kidney, liver and spleen and analyzed for the presence of these two pathogens by duplex pcr. to avoid contamination, each of the following steps were performed in a separate room: autopsy, dna extraction, pcr master mix preparation, dna quantification, addition to the pcr mixture, pcr reaction and electrophoresis. new disposable razor blades, forceps, and gloves were used for each fish to reduce potential contamination between fish. sequencing method: to verify that the specific primer-pair amplified a. hydrophila and e. coli dna, the pcr product was purified with a pcr purification kit (qiagen) and directly sequenced with an abi 3130 genetic analyzer (applied biosystems instrument) in avicenna research institute. the results of the sequencing were used for homology searches by the blast program available at the ncbi (national center for biotechnology information) website (http://www. ncbi.nlm.nih.gov). name gene sequence (5-3) described pathogen size (bp) fes res 16s rrna f:gaaaggttgatgcctaatacga r:cgtgctggcaacaaaggacag nielsen et al., 2006 a. hydrophila 700 fes res 16s rrna f:ggaagaagcttgcttctttgctg r:agcccggggatttcacatctga sabat et al., 1999 e. coli 544 table 1. primers used in this study. 5 5 f at ta h i et a l. / d u p le x p c r m et h o d d et ec ti o n o f a . h y d ro p h il a, a n d e . co li i n r ai n b o w t ro u t c h a r a c te r is ti c o f is o la te s id e n ti fi e d a s e . c o li a n d a .h y d ro p h y la b y b o th p c r a n d b io c h e m ic a l te st s b io c h e m ic a l te st s l it e r a tu r e ( m il le r sh ip , 1 9 9 6 ) a . h y d ro p h il a l it e r a tu r e ( f o r b e s e t a l. , 1 9 9 8 ) e . c o li m o le c u la r d ia g n o si s c it t s i g a s/ g lu c o se u c a t m r v p n it s im g a s/ g lu c o se u c a t m r v p n it s i m c it t s i g a s/ g lu c o se u c a t m r v p n it s i m c it t s i a h -1 + a/ a + + + + + + + + + + + + + + + + + a/ a 1 0 % + + + + + + a/ a a/ k 7 0 0 -b p e -1 a/ a + + + + + g a s/ g lu c o se u c a t m r v p n it s i m c it t s i 5 4 4 b p t y p e s tr a in r t ic c 1 0 3 2 a/ a + + + + + + + + 1 0 % + + + + + + a/ a a/ k 7 0 0 -b p t y p e s tr a in r t ic c 2 3 2 5 a/ a + + + + + g a s/ g lu c o se u c a t m r v p n it s i m c it t s i 5 4 4 b p u re a = u , c a ta la se = c a t , c it ra te = c it , g a s fr o m g lu c o se = g a s/ g , n it ra te = n it , s u lf id e , in d o le , m o ti li ty = s im , m r -v p = m e th y l re d , v o g e sp ro sk a u ; t s i= t ri p le s u g a r ir o n t ab le 3 . id e n ti fi ca ti o n a n d c h ar ac te ri za ti o n o f e . co li a n d a . h y d ro p h y la . f ig u re 1 . f is h i n je ct e d w it h a . h y d ro p h y la ( n o te p et ec h ia l at t h e fi n b as ed a n d t ai l ro t) . f ig u re 2 . f is h i n je ct ed w it h e . co li ( sh o w s n o c li n ic al s ig n s o f d is ea se ). 56 int. j. aquat. biol. (2015) 3(1): 52-59 results isolation and identification of a. hydrophyla and e. coli: pure cultures were obtained from all tissue samples and biochemical analysis were carried out on a. hydrophyla and e. coli isolates. all tissue samples were positive for microbiological and pcr identification (table 3). variation in citrate reaction was observed within the group identified as a. hydrophyla when compared to the type strain rticc 1032. occurrence of a. hydrophila and disease signs: mortality was observed two days post-injection in the group injected with a. hydrophila. aeromonas hydrophila clinical signs were observed in five days post-injection including anorexia, exophtalmus, petechiae and reddening due to haemorrhage of the skin, erosion of the tail and fins and swimming at the surface of the tank (fig. 1), and similar clinical signs were observed in the groups injected with both bacteria, while mortalities or clinical signs of disease were not observed in fish injected with e. coli (fig. 2). pcr identification of a. hydrophyla and e. coli: three annealing temperatures (58, 60 and 62°c) and two mgcl2 concentrations (1.5 and 2 mm) were examined for the optimal sensitivity of the duplex pcr assay. a good intensity of the amplicons for each target dna, as well as the absence of unspecific bands, was considered in selecting the optimal duplex pcr conditions. thus, the best results were obtained with an annealing temperature of 58°c and 2 mm mgcl2. each of the two pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganisms. positive pcr amplification of dna templates from a. hydrophila and e. coli were produced a single fragment of the expected, for each pathogen (700 bp and 544 bp, respectively) (fig. 3). the two bacterial pathogens were simultaneously amplified with relatively equal dna band intensities (fig. 3). escherichia coli and a. hydrophila were detected from cultures on agar plates and fish tissues (fig. 4). detection of the two bacterial pathogens within dna templates derived from liver, kidney and spleen were possible as early as 48 hrs after challenge in dead fish (fig. 4a). the two bacterial pathogens were simultaneously amplified in kidney (fig. 3); whereas, only e. coli was amplified in the liver and spleen tissues of fish injected with two bacteria. the size of pcr products from colonies were the same as tissues. representative examples of the product formation from each source are shown in figure 4. all tissue samples were positive for microbiological and pcr identification (table 3). the total procedure was accomplished in less than 8 hrs. no amplification products were obtained from control group (fig. 3). to confirm the positive pcr results, we sequenced the amplified dna products figure 3. representative pcr products from dead fish tissues, agar plate and positive control using the duplex pcr and single pcr assay under optimized condition. lane m, molecular size marker; lane 1, a. hydrophila rticc 1032 (700bp); lane 2, e. coli rticc 2325(544 bp); lane 3, mixture of the two bacteria; lanes 4 to 6, bacteria isolated from agar plates; lane 7, kidney tissue from fish injected with a. hydrophila; lane 8, kidney tissue from fish injected with e. coli; lane 9, kidney tissue from fish injected with two bacteria (asymptomatic fish); lane 10, liver tissue from fish injected with e. coli; lanes 11 to 12, liver and spleen tissue from fish were injected with two bacteria (asymptomatic fish), respectively; lanes 13 to15, negative control containing duplex pcr from fish tissues (kidney, liver and spleen, respectively); lane16, no dna. 57 fattahi et al./ duplex pcr method detection of a. hydrophila, and e. coli in rainbow trout from different pcr reactions and dna extractions. the blast analysis from sequencing of four randomly amplicons showed similar results, and found that the amplified fragment exactly matched the sequence of two bacteria. these findings suggest the high specificity of the primers to detect e. coli and a. hydrophila. discussion in aquaculture industry, diagnosis and treatment of the microbial diseases is crucial both from economic and sanitary point of views (blanco et al., 2000; stevenson, 1999). in recent years, there has been much interest in the development of multiplex pcr assay for the simultaneous detection of bacterial fish pathogens (del cerro et al., 2002; mata et al., 2004; altinok et al., 2008). mortality was observed in 48 hrs post-injection in injected fish with a. hydrophila, showing a hemorrhagic septicemia with hemorrhagic in internal organs and a red tinged ascetic fluid. the kidney, liver and spleen of challenged fish were processed for both microbiological and single pcr analysis. in the literature, a. hydrophila is usually reported to be citrate positive (millership, 1996). however, neil and nair (2004) reported that a. hydrophila to be citrate variable, which was confirmed in this study. this could be due to many biochemical identification schemes based on the analysis of human clinical isolates and that fish isolates may differ in several biochemical characters. different reaction patterns may be influenced by physical parameters, such as ph, temperature, and growth substrate concentrations (haenninen et al., 1994; janda et al., 2002; sautour et al., 2003). in this study, the characterization by biochemical identification methods was supported by the use of molecular technique. escherichia coli was detected 48 hrs after injection in the kidney and liver of the injected fish with this bacterium. buras et al. (1987) reported that the peaks in the concentration of fecal coliforms in water can be detected after 2 weeks in the kidney and liver. these fish showed no clinical signs of disease. all tissue samples were positive for microbiological and pcr identification. the results demonstrated that a. hydrophila and e. coli can be simultaneously detected in kidney from asymptomatic carrier fish and agar plates. in the duplex pcr assay, the amplification products corresponding to a. hydrophila (700 bp) and e. coli (544 bp) were obtained, which was supported by the sequencing results. the duplex pcr did not produce any non-specific amplification products. aeromonas hydrophila was not isolated from liver and spleen. the high specificity of this assay was verified by the absence of amplified a. hydrophila dna fragment in these samples. figure 4. specificity pcr products from dead fish tissues, agar plate and positive control using fas-ras primer set for detection of a. hydrophila (a). lane m, molecular size marker; lane 1, a. hydrophila rticc 1032 (700bp); lane 2, kidney; lane 3, spleen; lane 4, bacteria isolated from agar plate; lane 5, negative control (no dna). (b) specificity pcr products from fish tissues, agar plate and positive control using fes-res primer set for detection of e. coli. lane m, molecular size marker; lane 1, e. coli rticc 2325(544 bp); lane 2, kidney; lane 3, liver; lane 4, bacteria isolated from agar plate; lane 5, negative control (no dna). 58 int. j. aquat. biol. (2015) 3(1): 52-59 the duplex pcr method described in the present work provides improved capabilities to detect a. hydrophila and e. coli in healthy carriers and also decreases the time required to amplify the 16srrna gene. our results, based on the pcr suggested that the first target of a. hydrophila colonization is kidney of the rainbow trout showing the pathogen prior to clinical symptoms. furthermore, our findings suggest that the combination of sampling method and duplex pcr is suitable for a rapid detection and discrimination between apparently healthy and asymptomatic infected and uninfected fish. uninfected fish used as controls did not produce any amplification products with either method. our study has confirmed that the usage of an individual pcr reaction for each pathogen and laboratory culture based methods is costly, tedious and time consuming. but the duplex pcr method could identify two bacterial pathogens in less than 8 hrs. in conclusion, the duplex pcr presented in the current work is suitable tool for the detection of fish pathogen and at the same time, allows for rapid identification of bacterial causative agents of human diseases. these results, together with those obtained in dead fish and asymptomatic fish experiments, indicate that the kidney is suitable for epidemiological sampling. on the whole, the duplex pcr has advantages in terms of its accuracy, sensitivity, ease of use, time of length analysis and cost-effectiveness compared to the single pcr and traditional method. acknowledgement the authors acknowledge the aquatic animal laboratory and aquatic biotechnology laboratory staff of university of tehran for their assistance. references adams a., thompson k.d. 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(2017) 5(5): 321-327; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article the regeneration capacity of caudal fin in the common tooth-carp, aphanius dispar (rüppell, 1829) (teleostei: cyprinodontidae) faezeh zeinali, mina motamedi*1 department of biology, faculty of sciences, shahid bahonar university of kerman, 76169-14111 kerman, iran. article history: received 26 august 2017 accepted 22 october 2017 available online 2 5 october 2017 keywords: regeneration blastemal caudal fin cyprinodontids aphanius abstract: regeneration ability is known for several actinopterygii or ray-finned fishes. in order to assess the universality of regenerative potencies in this group of fishes, we have examined for the first time, caudal fin regeneration in tooth-carp, aphanius dispar (rüppell, 1829). the caudal fin is used because of its accessibility, simple structure and fast regeneration. the results revealed the regeneration ability in caudal fin of a. dispar. the initiation of the regenerative outgrowth was differing in three examined water temperatures. it is started approximately 5 days post amputation (5 dpa) at room temperature, and 2 dpa at 25°с and 28°с. our finding indicates that water temperature more than 25°с promotes procedure in caudal fin regeneration of a. dispar. by considering the high regeneration ability in a. dispar, and also the relatively short life span of the members of the genus aphanius, we concluded that these fishes could probably be used in regeneration researches. however, details of this mechanism in aphanius need further examinations. introduction the teleost fish together with some groups of amphibians among the vertebrates represent remarkable capability to regenerate their various organs (pfefferli and jazwinska, 2015). due to the simplicity in manipulation and macroscopic examination, most studies on regeneration potential focused on external appendages such as fins and limbs in abovementioned animal groups. the first report on fish regeneration was documented for the gold fish (broussonet, 1786). he discovered that in regeneration experiment of the fishes, caudal fin has advantage over other fin types, because it regenerates faster than pectoral, ventral and dorsal fin (pfefferli and jazwinska, 2015). among the teleost fishes, zebrafish danio rerio (hamilton, 1822) has been used as powerful model for regeneration studies (e.g., nechiporuk and keating, 2002; pfefferli and jazwinska, 2015). besides its simple architecture, its accessibility and its fast and robust regeneration, it is able to regenerate fins, scales, retina, spinal cord and heart among other internal organs (iovine, 2007; azevedo et al., 2011). in contrast to amphibians and fishes, other *corresponding author: mina motamedi doi: https://doi.org/10.22034/ijab.v5i5.375 e-mail address: m.motamedi@uk.ac.ir vertebrate such as reptiles, birds and mammals have a more limited capacity to regenerate complex body structures (brockes and kumar, 2008; galis et al., 2003; unguez, 2013). the limited capacity of humans to regenerate tissues and evolutionary loss of regenerative ability in animal groups attracts many biologists from medical perspective (bely and nyberg, 2010; unguez, 2013), and therefore researchers always attempt to elucidate the mechanisms responsible for the extensive regenerative capacities in specific groups of vertebrates. caudal fin is composed of segmented bony rays, mesenchymal tissue, blood vessels and nerve axons. each bony ray consists of two concave hemi-rays that define an inner space filled with intra-ray mesenchymal cells. the bifurcations are responsible for generating the characteristic shape of the caudal fin (poss et al., 2003). bony rays are produced and maintained by osteoblasts (also called scleroblasts), skeletogenic cells that secrete bone matrix (hall, 2005). based on the study of poss et al. (2003), an amputation of caudal fin triggers a regenerative 322 zeinali and motamedi/ regeneration in caudal fin of a. dispar program that occurs in three phases: (i) wound healing: the first phase after amputation started immediately after injury and continues within 12 hour-post-amputation (hpa). the injury is healed through migration of epidermal cells that cover and close the wound. (ii) blastema formation: the wound epithelium thickens forming an apical epidermal cap (aec). formation of blastema, is accomplished by the dedifferentiation and proliferation of cells in the disorganized mesenchymal tissue under aec. (iii) regenerative outgrowth: the onset of regenerative outgrowth starts after establishment of the blastema, at 48 hpa. the third phase is characterized by the extensive proliferation of cells. up to now, no information is available on the regeneration phenomenon in tooth-carp, genus aphanius nardo, 1827. to determine whether aphanius can regenerate its body parts, we used aphanius dispar from southern iran as model, and removed its caudal fin from the basal part. materials and methods study model: the common tooth-carp, a. dispar inhabits brackish and freshwater environments almost in the desert regions (wildekamp, 1993) (fig. 1). more especially, its principal habitats are coastal lagoons, but owing to its high tolerance to ecological changes, it can live in inland waters and hot sulphuric springs (wildekamp, 1993; teimori et al., 2012a, b). in this study, a. dispar specimens were captured from bandar abbas city in southern iran and transferred to the laboratory aquariums equipped with heater, thermometer and constantly aerated water. the animals were fed every day during experiment period with freeze-dried live food and also dry food that contain macronutrients, trace elements and vitamins necessary to keep captive fish in good health. the individuals were kept in three separate aquariums in ph 6.8-7, and each settled with specific temperature (room temperature (rt)=23°с, 25°с and 28°с). throughout the examination, temperature was recorded twice a day. fin amputation: in total, 12 fish individuals were examined for this experiment. they placed in 180 ppm of aqueous clove (eugenia caryophyllata) solution, and the anesthesia was induced after approximately 3 minutes. once fish asleep and immobile, it was moved to the dry lid of the petri dish, oriented so the head was positioned to the left, the caudal fin was fanned out with a clean razor blade. the fish was then transferred back to a tank of fresh water, observed for gill movement, and allowed to awaken. the caudal fins of four fish individuals (two males and two females) in each temperature were imaged 2, 5, and 7 days post amputation (dpa) (fig. 2). each captured image was used for measurement in figure 1. a fresh male common tooth-carp, aphanius dispar from southern iran, studied as model in this study (scale bar is about 1 cm; fish photo provided by h.r. esmaeili). figure 2. stages of the caudal fin including amputation: uncut (before cutting), amputation (immediately after cutting) and 2, 5, and 7 days after amputation (dpa). the dashed line indicates position of the cutting and the regeneration processes. the white arrows show the measurements from point of amputation. 323 int. j. aquat. biol. (2017) 5(5): 321-327 image-j software (schneider et al., 2012). the length of caudal fin regeneration from point of amputation was measured in three times (in millimeter, mm) to obtain an average values of the growth rate. eventually the obtained data was analyzed using ibm spss statistics 24. the t-test (p<0.05) was used to show if significant differences exist for the regeneration rates among different days at different water temperatures. results the results clearly indicate for the first time the regeneration ability in tooth-carp, a. dispar. the results reveal that a. dispar can obviously repair its caudal fin a short time (approximately 2 days) after traumatic injury as described below. there was little bleeding after caudal fin amputation. few hours after amputation, the wound is covered by epidermal layer which is called wound healing phase. next step in a. dispar fin regeneration is blastema formation which is occurred in 12-48 hours after amputation (2 dpa in fig. 3). during blastema formation, epidermis accumulates additional layers by cell migration. the hall mark of blastema is a proliferative mass of mesenchymal cells which sits atop each fin ray and gives rise to the new structures of the caudal fin. the last step in fin regeneration is switch from blastema formation to regenerative outgrowth. this transition occurs at approximately 5 dpa at rt, and 2 dpa at 25°с and 28°с (fig. 3). therefore, for quantitative analysis, we only compared regeneration rates for 2 and 7 days post amputation at 25°с and 28°с water temperature in males and females separately. descriptive analysis of regeneration rate in different days and water figure 3. the regeneration processes in caudal fin of aphanius dispar in different days at three water temperatures. 324 zeinali and motamedi/ regeneration in caudal fin of a. dispar temperatures is shown in table 1. in the 5 dpa, caudal fin regeneration was 0.77 mm at 25°с and 0.73 mm at 28°с in the males vs. 0.59 mm at 25°с and 0.83 mm at 28°с in the females. in the 7 dpa, at both 25°с and 28°с, the regeneration rate in the males was lower than the females (1.86 mm at 25°с and 2.05 mm at 28°с in the males vs 2.0 mm at 25°с and 2.90 mm at 28°с in the females) (table 1). in the males, there were not significant differences for the regeneration rates at different water temperatures in 2and 5 dpa, while the regeneration rates significantly differ between rt vs 25°c and rt vs. 28°c in 7 dpa (t-test, p<0.05) (table 2 and fig. 4). in the females, the regeneration rates differ significantly only between rt vs 28°c in both 5and 7 dpa (t-test, p<0.05) (table 2 and fig. 4). fin regeneration measurement revealed an overall increase in growth with increasing of water temperature. regeneration outgrowth was seen after 5 dpa in 25°с and 28°с. however, in rome temperature (rt) it is detected after 7 dpa (figs. 3-4). the increasing rate of regeneration was significant in sex t°c day 2 day 5 day 7 mean s.d. mean s.d. mean s.d. male rt 0.20 0.085 0.61 0.064 0.66 0.106 25 28 0.23 0.22 0.059 0.006 0.83 0.78 0.114 0.178 1.86 2.05 0.328 0.375 female rt 0.32 0.162 0.44 0.080 0.45 0.160 25 0.31 0.014 0.59 0.050 1.98 0.070 28 0.32 0.028 0.73 0.028 2.89 1.163 the rate of regeneration is shown for different days at three water temperatures. rt=room temperature and s.d. = standard deviation. table 1. descriptive analysis of the regeneration rates between males and females of the aphanius dispar. sex day t-test mean differences sig. female day 2 rt vs 25 0.0066 0.98 rt vs 28 0.0030 0.99 28 vs 25 0.0100 0.11 day 5 rt vs 25 0.1575 0.91 rt vs 28 0.2925 0.03 28 vs 25 0.1350 0.61 day 7 rt vs 25 1.5250 0.56 rt vs 28 2.4375 0.03 28 vs 25 0.9125 0.27 male day 2 rt vs 25 rt vs 28 0.0216 0.0183 0.89 0.92 28 vs 25 0.0033 0.99 day 5 rt vs 25 0.2200 0.97 rt vs 28 0.4625 0.24 28 vs 25 0.0575 0.81 day 7 rt vs 25 1.2009 0.00 rt vs 28 1.3895 0.00 28 vs 25 0.1885 0.48 table 2. t-test analysis to show significant differences (p<0.05) of the regeneration rates for different days at three water temperatures in the examined aphanius dispar. the analysis was carried out for the males and females separately. rt=room temperature and s.d.= standard deviation. figure 4. comparison of the rate of caudal fin regeneration in the male (above), and the female (below) specimens housed at three water temperatures over 7 days. 325 int. j. aquat. biol. (2017) 5(5): 321-327 5 and 7 days post amputation in comparison to the 2 dpa (t-test, p<0.05). also, our results indicate that no clear growths happened in the 2 dpa comparing the time of amputation (figs. 2-4). discussion regenerative capacity in different organs and organisms is interesting for biologists, and clinicians. there are many studies which attempt to discover various aspect of regeneration (e.g. poss et al., 2003; azevedo et al., 2011; rink, 2013; hoppe et al., 2015; kim et al., 2016). therefore, it is in the center of attention to reveal regenerative capacity in organisms from evolutionary point of view, the role of stem cells, signals which initiate regeneration and its controlling mechanisms. among teleost fishes, various degrees of regeneration abilities have been reported. good examples are cyprinid fish, d. rerio which can regenerate lost parts repeatedly (azevedo et al., 2011), and short-lived african turquoise killifish, nothobranchius furzeri (e.g. hoppe et al., 2015; kim et al., 2016). however, some teleost fishes exist with total absence of regeneration such as some members in the family blennidae (wagner and misof, 1992). in the present study, the possible occurrence of the regeneration ability in caudal fin of another relatively short-lived killifish, a. dispar is examined. the outcome of all these studied could help researchers to assess the universality of regenerative potencies in actinopterygii fishes. the studied taxon is a small tooth-carp with a maximum length up to 8 cm (usually 45-55 mm in total length). it is widely distributed throughout the coastal environments of the mediterranean sea, red sea and persian gulf regions (wildekamp et al., 1999; hrbek and meyer, 2003; reichenbacher et al., 2009; ferrito et al., 2013; teimori et al., 2016; esmaeili et al., 2017). most of the aphanius species tolerate euryhaline and eurythermal habitats as well as relatively low levels of oxygen and wide range of pollution. in addition, they are good candidates as biological control of malaria because they feed on mosquito larvae of anopheles (homski et al., 1994). based on the finding of the present study, the regeneration phenomenon is happening in a. dispar, and probably could be occurred also in the other members of the genus aphanius. the killifishes with special biological properties such as short life span and having regeneration ability are very interesting models for biomedical researchers. therefore, besides the already known killifish, nothobranchius furzeri (hoppe et al., 2015; kim et al., 2016), here we present genus aphanius as a new candidate model organism to be used to study the mechanisms of regeneration. according to the previous study, the regeneration procedure contains the wound healing, blastema formation and growth (pfefferli and jazwinska, 2015). blastema is a heterogeneous group of dedifferentiated/ proliferative cells, able to regrow and pattern a new complex organ (pfefferli and jazwinska, 2015). in this study, after the caudal fin amputation, the wound is healed in few hours. thereafter, blastema formation is occurred in 12-48 hours. it is documented that b-catenin is one of the earliest molecular markers for fin regeneration in wound epidermal cells, induced in the first few hours after amputation and maintained through regeneration (poss et al., 2000). initiation of regenerative outgrowth is differing in three examined water temperatures of this study. it is started approximately 5 dpa at rt, and 2 dpa at 25°с and 28°с. our finding indicates that temperature more than 25°с promotes procedure in caudal fin regeneration of a. dispar. as results, members of the genus aphanius can probably be a suitable candidate to study regeneration phenomenon. a noticeable biological characteristic of the aphanius is its mid-life span (12-24 months) in compare to the d. rerio (3-5 years), and this would probably make aphanius to be suitable for the aging researches, even though this assumption need further evaluation in future. acknowledgments this work was supported by shahid bahonar university of kerman (grant number, 1394). we would like to thank a. iranmanesh and s.f. sajjadi for their assistance during this work. we also thank h.r. 326 zeinali and motamedi/ regeneration in caudal fin of a. dispar esmaeili for providing the fish photo used in figure 1. references azevedo a.s., grotek b., jacinto a., weidinger g., saúde l. (2011). the regenerative capacity of the zebrafish caudal fin is not affected by repeated amputations. plos one, 6: e22820. azevedo a.s., sousa s., jacinto a. (2012). an amputation resets positional information to a proximal identity in the regenerating zebrafish caudal fin. bmc developmental biology, 12: 1-12. bely a.e., nyberg k.g. (2010). evolution of animal regeneration: re-emergence of a field. trends in ecology and evolution, 25: 161-170. brockes j.p., kumar a. (2008). comparative aspects of animal regeneration. annual review of cell and developmental biology, 24: 525-549. esmaeili h.r., mehraban h., abbasi k., keivany y., coad b.w. (2017). review and updated checklist of freshwater fishes of iran: taxonomy, distribution and conservation status. iranian journal of ichthyology, 4(suppl. 1): 1-114. ferrito v., pappalardo a.m., canapa a., barucca m., doadrio i., olmo e., tigano c. (2013). mitochondrial phylogeography of the killifish aphanius fasciatus (teleostei, cyprinodontidae) reveals highly divergent mediterranean populations. marine biology, 160: 31933208. galis f., wagner g.p., jockusch e.l. (2013). why is limb regeneration possible in amphibians but not in reptiles, birds, and mammals. evolutionary development, 5: 208-220. hall b.k. (2005). bones and cartilage: developmental and evolutionary skeletal biology. elsevier academic press, california., usa. 760 p. homski d., goren m., gasith a. (1994). comparative evaluation of the larvivorous fish gambusia affinis and aphanius dispar as mosquito control agents. hydrobiologia, 284: 137-146. hoppe b., pietsch s., franke m., engel s., groth m., platzer m., englert c. (2015). mir-21 is required for efficient kidney regeneration in fish. bmc developmental biology, 15: 2-10. hrbek t., meyer a. 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(2000). roles for fgf signaling during zebrafish fin regeneration. developmental biology, 222: 347-358. poss k.d., shen j., keating m.t. (2000). induction of lef1 during zebrafish fin regeneration. developmental dynamic, 219: 282-286. poss k.d., keating m.t., nechiporuk a. (2003). tales of regeneration in zebrafish. developmental dynamic, 226: 202-210. reichenbacher b., kamrani e., esmaeili h.r., teimori a. (2009). the endangered cyprinodont aphanius ginaonis (holly, 1929) from southern iran is a valid species: evidence from otolith morphology. environmental biology of fishes, 86: 507-521 rink j.c. (2013). stem cell systems and regeneration in planaria. development genes and evolution, 223: 6784. schneider c.a., rasband w., eliceiri k.w. (2012). nih image to image j: 25 years of image analysis. nature methods, 9: 671-675. teimori a., esmaeili h.r., gholami z., zarei n., reichenbacher b. (2012). aphanius arakensis, a new species of tooth-carp (actinopterygii, cyprinodontidae) from the endorheic namak lake basin in iran. zookeys, 215: 55-76. teimori a., motamedi m., askari hesni m. (2016). fish morphology and mitochondrial phylogeny reveal translocations of a native aphanius nardo, 1827 (teleostei: cyprinodontidae) in iran. iranian journal of ichthyology, 3: 181-189. unguez g.a. (2013). electric fish: new insights into 327 int. j. aquat. biol. (2017) 5(5): 321-327 conserved processes of adult tissue regeneration. journal of experimental biology, 216: 2478-2486. wagner g.p., misof b.y. (1992). evolutionary modification of regenerative capability in vertebrates: a comparative study on teleost pectoral fin regeneration. journal of experimental zoology. part a, ecological genetics and physiology, 261: 62-78. wildekamp r.h. (1993). a world of killies. atlas of the oviparous cyprinodontiform fishes of the world. the genera adamas, adinia, aphanius, aphyoplatys and aphyosemion. indiana, american killifish association, indiana, 311 p. int. j. aquat. biol. (2017) 5(4): 321-327 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی : عالی استخوانی ماهیان) aphanius dispar (rüppell, 1829) معمولی گورخری ماهی در دمی باله ترمیم توانایی (داردندان کپورماهیان *معتمدی مینا زینلی، فائزه .ایران کرمان، کرمان، باهنر شهید دانشگاه علوم، دانشکده شناسی،زیست بخش گروه چکیده: نخستین برای ماهیان، از گروه این در ترمیم هایتوانایی پذیریقابلیت ارزیابی برای. است شده شناخته باله شعاع ماهیان از تعدادی در ترمیم توانایی راحت، دسترسی دلیلهب دمی باله مطالعه، این در. گرفت قرار بررسی مورد aphanius disparمعمولی گورخری ماهی در دمی باله ترمیم بار، رشد ازآغ. است برخوردار دمی باله ترمیم توانایی از معمولی گورخری ماهی که داد نشان نتایج. گرفت قرار استفاده مورد سریع ترمیم و ساده ساختار 28 و 25 دمای در و( dpa5) برش اولین از پس روز 5 حدوداً ترمیم اتاق، دمای در. باشدمی متفاوت مطالعه، مورد آب حرارت درجه سه در ترمیمی موجب گرادسانتی درجه 25 از بیش آب دمای که دهدمی نشان مطالعه این هاییافته. شودمی شروع ترمیم برش از پس روز 2 گرادسانتی درجه اًنسبت عمر همچنین و معمولی گورخری ماهی در ترمیم باالی توانایی گرفتن نظر در با. گرددمی معمولی گورخری ماهی در ترمیم سرعت افزایش این با .گیرند قرار استفاده مورد ترمیم به مربوط مطالعات در توانندمی احتماال ماهیان این که نمود گیرینتیجه توانمی ماهیان، این اعضای کوتاه .باشدمی بیشتر مطالعه به نیاز ماهیان این در ترمیم مکانیسم جزئیات شناخت برای حال، .آفانیوس دار،دندان کپورماهیان دمی، باله بالستما، ترمیم، :کلمات کلیدی int. j. aquat. biol. (2021) 9(5): 290-296 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article evolution of ecophenotypic plasticity in indian oyster, crasssostrea madrasensis (preston) population from ashtamudi lake, kerala, india vineetha vijayan santhi, mano mohan antony*,1 leeanda lopez, lekshmi vasanthi, joelin joseph post graduate and research department of zoology, (research centre, university of kerala) university college, palayam, thiruvananthapuram, kerala, india. s article history: received 3 march 2021 accepted 12 october 2021 available online 2 5 october 2021 keywords: crassostrea madrasensis ashtamudi phenotypic plasticity gene sequencing abstract: the indian oyster, crassostrea madrasensis are abundant in the coastal waters of tamil nadu and kerala. since, they are benthic filter feeders, the external environmental conditions impose ceaseless effects on their shell affecting one or more of size, sculpture, anatomy pattern, shape and colour resulting in ecophenotypic plasticity. however, the identification of oyster species is still based on phenotypic characters that are highly plastic. therefore, this study aimed to investigate the phenotypic plasticity of the indian oyster, c. madrasensis population of ashtamudi lake with respect to certain ecological parameters. individuals were collected from the barmouth and upper reaches of ashtamudi lake, kerala and apparent variations were measured. significant variations were found in the shell colour and shell pattern. accordingly, two morphotypes of c. madrasensis were recorded. since, the two population exhibit remarkable differences in morphology, species confirmation was made possible using mitochondrial 16s rrna gene. even though 2.7% genetic distance observed between the two morphotypes of c. madrasensis is not enough to consider them as different species, it calls attention to the possibility of evolutionary divergence in the near future. introduction many speciation events and indeed whole adaptive radiations, result from the colonization of a new environment, and this entry into a new environment results in selection pressures favouring divergence from the ancestor, induce changes in an individual’s physiology, behaviour and morphology. such changes are collectively called as phenotypic plasticity. the indian oyster, crassostrea madrasensis are abundant in the coastal waters of tamil nadu and kerala. since, they are sessile and benthic, and the external environmental conditions such as salinity, temperature, substrate, diet, moisture, predators and pollution rate impose interminable effects on their shell traits resulting in ecophenotypic plasticity. ecophenotypic plasticity is referred to as the ability of a genotype to produce multiple phenotypes when exposed to different environmental conditions. the morphological variations which is an outcome of phenotypic plasticity had led to the description of at least 4000 freshwater mussel species, among which *correspondence: mano mohan antony doi: https://doi.org/10.22034/ijab.v9i5.1167 e-mail: manomohanantony@universitycollege.ac.in only ∼840 species have been recognised (haas, 1969; graf and cummings, 2007). this has been an interminable and a controversial issue that give rise to exaggerating the extant number of freshwater bivalves, as the species with high morphological plasticity variability on its shells tends to inflate the number of species, which is in reality, the ecophenotypes of the same species (klishko et al., 2018). ecophenotypic plasticity and cryptic morphology are far prevalent among relatively sessile or immobile animals as they have to persistently adapt themselves to cope up with the surrounding conditions (schlichting, 1986). moreover, the theory of genetic isolation by geographic distance predicts that increasing distance between populations results in the decreasing gene flow that will lead to the increasing phenotypic divergence (slatkin, 1987; hendry et al., 2001). this morphological variation has been the subject of many evolutionary studies. these have increased greatly due to increasing availability of dna sequences (bickford et al., 2007). 291 int. j. aquat. biol. (2021) 9(5): 290-296 as bivalve molluscs are closely related to their external environment, the changes in the allied habitat can be meticulously monitored using their shell morphology. therefore, this is an attempt to look over the phenotypic plasticity of the indian oyster, c. madrasensis population of ashtamudi lake with respect to certain ecological parameters. materials and methods live samples of c. madrasensis was collected randomly from the oyster beds of two ecologically different sites of ashtamudi lake, viz. site i, perumon (upper reach, lat-8.961508, long-76.607392) and site ii, thekkumbhagham (estuarine barmouth region, lat8.955707, long-76.549566) for a period of six months, from march 2019 to august 2019 (fig. 1). the study sites were surveyed every month at low tides and samples were collected by hand picking method from intertidal regions and shallow coastal waters. after collection, the live samples were brought to the laboratory for further investigations. samples for molecular analyses were immediately transferred to absolute alcohol. fifty individuals from each site was used for the morphological and morphometrical analysis. water samples were collected from the sampling sites during morning hours between 8 a.m. to 11 a.m. and used for the analyses. the ph of water samples was recorded using a calibrated digital ph pen. the surface water temperature was recorded at the sites using an accurate mercury thermometer. dissolved oxygen which was fixed in situ was estimated by winkler’s method (apha, 1989). salinity of the water sample was estimated by silver nitrate method (trivedy et al., 1987). total hardness and tds was estimated using standard procedures of apha (1989). samples were photographed and morphological characters were examined according to durve (1974). classification were referred to the world register of marine species (preston, 1916). thirty individuals were randomly selected and used for morphometric analysis. according to moore et al. (1971) and dillon and manzi (1989), 12 morphometric characters were selected and abbreviated as tw (total weight gm), sww (shell wet weight gm), sdw (shell dry weight gm), mw (meat weight gm), mc (meat content %), scv (shell cavity volume ml), sv (shell volume gm), sd (shell density sdw to sv), sh (shell height mm), sl (shell length mm), sd (shell width mm) and sh/sl (ratio of shell height to shell length). data analysis of the morphometric parameters were done by anova. the figure 1. location map of ashtamudi lake showing the study sites (station i-perumon lake, station ii-thekkumbhagam lake). 292 vijayan santhi et al./ evolution of ecophenotypic plasticity in crassostrea madrasensis differences in mean were tested using duncon analysis. significant level used was p≤0.05. all the statistical analysis was performed using the software spss 22.0 for windows. the uncertainity in species identification due to phenotypic plasticity was addressed by performing16s rrna gene sequencing proceeded by blast analysis. the genomic dna was isolated from the muscle tissue using nucleospin® tissue kit (macherey-nagel) following manufacturer’s instruction. dna amplification of partial mitochondrial 16s rrna was obtained using the primers 16sar 5’ cgcctgtttatcaaaaaacat 3’ and 16sbr 5’ccggtctgaactcagatcac gt3’ (palumbi et al., 1991). the pcr cycles were carried out in a mastercycler pcr system (eppendorf) under the following conditions: an initial denaturation for 3 min at 94°c, followed by 35 cycles of 40 s at 94°c, 40 s at 48°c annealing temperature, 40 s at 72°c, and with a final 5-min extension at 72°c. pcr products were purified using exosap-it (ge healthcare). sequencing reaction was done in a pcr thermal cycler (gene amp pcr system 9700, applied bio systems) using the bigdye terminator v3.1 cycle sequencing kit (applied bio systems, usa) following manufactures protocol. genetic distance between two morphotypes (‘p’ distance) was calculated, the estimates of evolutionary divergence between the sequences were inferred using mega 7. results morphological and morphometrical analysis: two different morphotypes of c. madrasensis were recorded from ashtamudi lake with respect to the two different ecological sites. the two morphotypes were clearly distinguishable from each other in terms of the shell type, shape, size, internal shell colour, colour and position of muscle scars (fig. 2): morphotype i from site i: greenish yellow shells with brittle lamellae, umbos not prominent with shallow cavity, smooth inner surface and having purple around whole or part with dark purple to black shell outline. adductor muscle scars reniform, deep purplish in colour and posterior in position. morphotype ii from site ii: whitish yellowish shells with solid lamellae, left valve (lower valve) thick, heavy, concave and deeply cupped, umbos prominent with deep and chalky cavity, inner surface white, outlines absent, adductor muscle scars dark purplish to black, d-shaped and sub-central in position. figure 2. morphotypes of crasssostrea madrasensis. (a) right valve dorsal (b) right valve ventral (c) left valve dorsal (d) left valve ventral. 293 int. j. aquat. biol. (2021) 9(5): 290-296 remarkable variations were also observed in the overall morphometry of two morphotypes. apart from mc, all the parameters show higher values for morphotype ii. the morphological parameters such as tw, sww, sdw and sv were two times greater for morphotype ii, whereas mw, scv and sh were doubled in morphotype ii. (table 1). molecular analysis: since the two morphotypes exhibited phenotypic plasticity, species confirmation was done through mitochondrial 16s rrna gene sequencing method. fasta sequences with 558 and 590 bp were interpreted from the chromatogram. the blast analysis of the 16s rrna gene sequences obtained from morphotypes 1 and ii showed 99.79 and 99.78% similarity with c. madrasensis (accession no. jf915515) in the ncbi genbank, respectively. these sequences were submitted in ncbi genbank with accession number of mn508435 (morphotype i) and mn508481 (morphotype ii). evolutionary divergence between the sequences were estimated. codon positions included were 1st+2nd+3rd+noncoding. there were a total of 487 positions in the final dataset with 13 base differences contributed to 2.7% genetic distance between both the morphotypes. water quality parameters: the water quality of perumon lake and thekkumbhagm lake exhibited a table 1. morphometric analysis of morphotype i and morphotype ii of crasssostrea madrasensis. morphotype i ( from site i) morphotype ii (from site ii) range mean sd range mean sd tw 51.94-72.14 60.38 6.95 149.14-268.1 183.5 29.9 sww 34.82-81 48.35 11.17 60.28-165.43 133.7 34.3 sdw 12.1-52.38 32.92 9.1 59.8-206.4 130.54 35.92 mw 4.9-8.7 6.3 1.32 7.76-19.07 11.33 2.9 mc 8.23-13.4 10.5 2.26 4.49-7.72 6.17 1.05 scv 7.00-31 16.93 7.2 21-48 34.2 8.17 sv 10.54-46.37 29.82 12.1 52.6-112.26 80.51 15.55 sdn 0.38-3.33 1.3 0.8 1.14-2.59 1.62 0.38 sh 48.2-107 69.2 13.43 79-125 103.21 12.9 sl 38.2-69 52.6 8.05 52-95 73.61 13.93 sd 21-46 31 7.71 22-61 37.62 10.91 sh/sl 1.02-1.55 1.32 0.14 1.04-1.75 1.43 0.22 figure 3. monthly variation of ph from the two sites of ashtamudi lake. figure 4. monthly variation of temperature from the two sites of ashtamudi lake. figure 5. monthly variation of salinity from two sites of ashtamudi lake. 294 vijayan santhi et al./ evolution of ecophenotypic plasticity in crassostrea madrasensis continuous change with respect to the environmental conditions. a seasonal pattern was observed in both the sites, where highest values were recorded from thekkumbhagam region (figs. 3-8). discussions the observed ecological parameters determine the morphological and physiological properties of c. madrasensis, so the variation in the external environment creates differences in the individuals of the same species between the different ecological sites. being ectosomatic organ, shell faster than the internal organs reacts to fluctuations in the external environment, which allows them to adapt to a wide range of environmental conditions (severtsov, 1939). several studies have showed morphological variations of the shell among populations in different habitats (mackie and topping, 1988; hornbach et al., 2010). a study reported that a single species could be light and laterally compressed in form in headwaters, but heavier and more laterally inflated in downstream waters (utterback, 1917). this agrees with the findings of the present study where the populations of c. madrasensis, exhibited remarkable variation in the external shell traits between the barmouth region of thekumbhagam lake and upper stream portions of perumon lake. as a result, two morphotypes of c. madrasensis were observed in the present study with respect to the changes in the morphology and morphometrical parameters. the uneven shell thickness and shorter forms of c. madrasensis from site i, was the result of crowding due to population density and increased depth gradient. therefore, the morphotype i showed an allometric growth of the shell and also resulted in the crooked forms of shells in site i (rao and nayar, 1956). therefore, crowding strongly affects shell shape. the shell size was found to be doubled, deeper and elongated in barmouth population (site ii), in order to counter involuntary dislodgement by turbulence and currents in the barmouth region and the protection offered by rocks or hard substrates in the site ii influences the lateral growth of morphotype ii (denny et al., 1985). the shell weight of c. madrasensis from sites i and ii varied greatly (48.348±2.883 gm and 133.662±8.854 gm, respectively). the comparative drop in salinity in the perumon region, cause the decreased caco3 supply thereby shell thickness and shell density in site i population, whereas a reciprocal reaction was observed in site ii with increased salinity ranges upto 32.08 ppt due to the influx of sea water. figure 6. monthly variation of hardness from two sites of ashtamudi lake. figure 7. monthly variation of dissolved oxygen from the two sites of ashtamudi lake. figure 8. monthly variation of total dissolved solids (tds) from the two sites of ashtamudi lake. 295 int. j. aquat. biol. (2021) 9(5): 290-296 this is in accordance with the findings of aguirre et al. (2006) and krapivka et al. (2007) in the mytilids, mytilus chilensis and brachidontes species, respectively, where the influence of salinity was observed on the shell shape along the clines. the parameters such as total weight, meat weight, shell weight, shell length, shell height, shell width etc. were also found to be doubled in the samples collected from thekkumbhagam region. being an estuarine portion, thekkumbhagam region possesses more nutrient sources than perumon region and the buffering activity of the estuary in turn have an influence on the fluctuations in water quality. the conventional identification thus failed to address the species identity due to phenotypic plasticity, where morphological and morphometrical data supports two distinct taxa, while it is the genetic data that provided information of phenotypic plasticity. therefore, combinations of genetic, biological, and morphological information are recommended for developing conservation strategies of these highly imperiled animals (inoue et al., 2013). ecophenotypic plasticity is controlled by one or two environmental factors that are subjected to the evolutionary change (schneider et al., 2010). even though 2.7% genetic distance observed between the two morphotypes of c. madrasensis is not enough to consider them as different species, in turn, it calls attention towards the emergence of evolutionary divergence of these two populations in the near future. stanley (1970) showed that much of this diversity is the result of evolutionary adaptation to the hydrology and sedimentology of a particular niche. therefore, the pattern of phenotypic integration can be modified by natural selection to enhance adaptation to the external environment or to maintain the coherence of the internal developmental system (hansen and houle, 2004), therefore ‘constraints’ are actually forms necessary substrate for natural selection to work (matthen and ariew, 2002). in this context, it is expected that if gene flow between distant localities is low, adaptation to local environments generates shell-shape variation via selection. therefore, variation between adjacent populations could be due to phenotypic plasticity, while between distant populations the restricted gene flow caused by physical and biological barriers which could promote adaptive divergence (marquez and molen, 2011). since bivalves are sessile, the habitat fragmentation and the restricted gene flow can give rise to reproductive isolation and genetic divergence. moreover, the given anthropogenic disturbance of habitats and human driven climate change accelerates this process, therefore, it is crucial to understand evolutionary responses to this vicariant phenomena and to develop proper conservation practices for taxa in peril. references aguirre m.l., perez s.i., sirch y.n. 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(2010. variation in freshwater mussel shell sculpture and shape along a river gradient. the american midland naturalist, 164(1): 22-36. inoue k., hayes d.m., harris j.l., christian a.d. (2013). phylogenetic and morphometric analyses reveal ecophenotypic plasticity in freshwater mussels obovaria jacksoniana and villosa arkansasensis (bivalvia: unionidae). ecology and evolution, 3(8): 2670-2683. klishko o.k., lopes-lima m., bogan a.e., matafonov d.v., froufe e. (2018). morphological and molecular analyses of anodontinae species (bivalvia, unionidae) of lake baikal and transbaikalia. plos one, 13(4): e0194944. krapivka s., toro j.e., alcapán a.c., astorga m., presa p., pérez m., guiñez r. (2007). shell‐shape variation along the latitudinal range of the chilean blue mussel mytilus chilensis (hupe 1854). aquaculture research, 38(16): 1770-1777. mackie g.l., topping j.m. 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(2016) 4(3): 171-178; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article bioaccumulation of heavy metals in anuran tadpoles: a study in barak valley, assam pammi singh*1, mithra dey1, sunkam narayana ramanujam2 1department of ecology and environmental science, assam university, silchar-788011, assam, india. 2department of zoology, north eastern hill university, shillong 793022, meghalaya, india. article history: received 22 february 2016 accepted 26 may 2016 available online 2 5 june 2016 keywords: amphibians accumulation heavy metals barak valley abstract: heavy metal pollution plays an important role in global biodiversity decline. however, there is paucity of information concerning the effects of metals on amphibians. in the present study, investigations were made on the accumulation of heavy metals, copper (cu) and lead (pb) in water, sediment and tadpoles inhabiting the water bodies of barak valley, assam. tadpoles of six different anuran species, hoplobatrachus tigerinus, leptobrachium smithi, clinotarsus alticola, fejarvarya sp., sylvirana leptoglossa and euphlyctis cyanophlyctis were selected for this purpose. heavy metal concentrations were determined in intestine, liver and tail of tadpole samples of these species. the results revealed that the copper concentration in water samples was within the maximum permissible limit of who (2 mg l-1), but the concentration of lead in water samples increased beyond the permissible limit of who (0.01 mg l-1) resulting in possibilities of higher accumulation of the metal in tadpoles and decline of amphibians’ population. total concentration of cu in the tadpoles of different species of amphibians followed the order: h. tigerinus > s. leptoglossa > e. cyanophlyctis > c. alticola > fejarvarya sp. > l. smithi, while concentration of lead followed the order: e. cyanophlyctis > c. alticola > s. leptoglossa > fejarvarya sp.> h. tigerinus > l. smithi. introduction amphibians are good bio-indicators of environmental pollution due to their susceptibility to chemicals and are well known for accumulating metals during their freshwater cycles (loumbourdis and wray, 1998). larval amphibians accumulate metals more readily than adults, possibly due to their semi permeable and highly vascularised skin which allows cutaneous respiration and high accumulation of environmental pollutants in the tissue from water and soil directly (hall and mullherm, 1984). their biology and habitat selection makes them candidates for heavy metal accumulation (hayes et al., 2010). decline in amphibian populations, throughout the world, in recent years have caused concern among the scientific community (ezemonye and enuneku, 2006). the reason of decline has been attributed to habitat destruction, introduction of invasive species, over-exploitation, emerging diseases, pathogen, * corresponding author: pammi singh e-mail address: singhpammi20@gmail.com climate change and environmental contamination (hayes et al., 2010). aquatic systems become polluted with heavy metals discharged from industries and agricultural sectors. among environmental pollutants heavy metals are of particular concern due to their potential toxic effects and ability to bioaccumulate in aquatic ecosystems (censi et al., 2006). bioaccumulation is the building up of a chemical to a toxic level in an organism. it is the net accumulation of a substance by an organism as a result of uptake directly from all environmental sources and from all routes of exposure (astm, 1998). as heavy metals cannot be degraded, they are deposited, assimilated or incorporated in water, sediment and aquatic animals causing heavy metal pollution in water bodies (mallick et al., 2010). toxicological research on amphibians is scarce compared to other vertebrates. metals have the tendency to accumulate in various organs of aquatic 172 singh et al./ heavy metals in tadpoles organisms especially in fish (karadede et al., 2004). frogs are opportunistic breeders and breed in wide variety of aquatic systems like ephemeral pools, small and large ponds, streams, ditch, drains, manmade tanks etc. barak valley region has ideal habitats for breeding of anurans. the objectives of this study were to assess susceptibility of anuran tadpoles to heavy metals, cu and pb in barak valley. accordingly, six different anuran species, hoplobatrachus tigerinus, leptobrachium smithi, clinotarsus alticola, fejarvarya sp., sylvirana leptoglossa and euphlyctis cyanophlyctis were selected from urban, rural and industrial area and the level of copper (cu) and lead (pb) in water, sediment and selected organs of tadpoles were determined. materials and methods barak valley is situated in the southern part of the assam, between 24º8'n and 25ºn latitudes and 92º15'e and 93º15'e longitudes. the region abounds in wetlands, streams, pools, marshes, ponds etc. of various shapes, sizes and have small hillocks. the valley has urban areas, brick kilns, industrial area like hpc/cachar paper mill at panchgram situated at a distance of 25 km from silchar town and large number of tea gardens. the study was carried out in different selected habitats of barak valley which included tea estates, urban and industrial area and brick kilns. for physicochemical analysis and determination of cu and pb of water, three replicates of water samples were collected from the sites where tadpoles were present. physicochemical parameters like ph, electrical conductivity (ec), dissolved oxygen (do), free carbon dioxide (fco2) and total alkalinity (ta) were analyzed using standard methods (trivedy and goel 1984; apha, 2005). tadpoles were collected by dip net and were washed properly with double distilled water in the laboratory. selected organs like intestine, tail and liver were removed from the tadpole samples and dried until reaching a constant weight. digestion of all tadpole samples was done according to fao/sida (1983). to each sample (0.1 g), 10 ml of perchloric acid: conc. hno3 (3:2 v/v) was added and the mixture was heated at 60ºc until a clear solution was formed. the resulting solutions were cooled, and the volumes were made up to 50 ml using double distilled water. the samples were then stored in plastic bottles till analysis to determine the amount of heavy metal bioaccumulated (ezemonye and enuneku, 2012). the water collected in sampling bottles were preconditioned with dilute nitric acid (hno3) and later rinsed thoroughly with double distilled water. precleaned polyethylene sampling bottles were immersed about 10 cm below the water surface and 1 liter of the water sample was taken. samples were acidified with concentrated nitric acid (hno3) for preservation. the samples were filtered through whatman filter paper no. 1 and kept in refrigerator until analysis. the sediment samples were oven dried at 45ºc followed by grinding and sieving using <2 mm sieve, 5 gm of dry sample was poured into a beaker and mixed with 2 ml of aqua regia (1 conc. hcl : 3 conc. hno3). the mixture was digested on a hot plate in open beakers at 95ºc for 1 hr and allowed to cool to room temperature. the supernatant was filtered and then diluted to 50 ml using distilled water. the heavy metal concentrations in the digested samples of tadpoles, water and sediment were determined in a graphite furnace-atomic absorption spectrophotometer (gf-aas), model analytik jena vario-6. abbreviation used: ht-hoplobatrachus tigerinus, ls-leptobrachium smithi, ca-clinotarsus alticola, fsp.-fejarvarya sp., sl-sylvirana leptoglossa and eceuphlyctis cyanophlyctis. results and discussion table 1 presents the distribution of different species of tadpoles in different habitats. the physicochemical parameters of water are an important indication of its quality. table 2 presents the physicochemical variables of the study sites. air temperature ranged between 27.1ºc to 31.1ºc. surface water temperature ranged from 22.7 to 26ºc. 173 int. j. aquat. biol. (2016) 4(3): 171-178 the ph varied between 5.4-7.1 and conductivity ranged between 24 μs cm-1 to 95 μs cm-1. dissolved o2 ranged between 1.2 mg l -1 to 3.2 mg l-1 and free co2 ranged between 6.2 to 12.1 mg l -1. total alkalinity ranged between 30.1 mg l-1 to 50.2 mg l1. ph in the water bodies inside the rosekandy tea estate were found to be acidic while the ponds in the industrial areas had ph ranging between 7-7.1. concentrations of cu and pb in water, sediment and tadpoles from different sites are presented in table 3. metal concentration in water depends in part on water quality and watershed influxes (mason, 2002). metal uptake and their toxicity in aquatic fauna are influenced by many factors such as ph, hardness, alkalinity and temperature of water (osman and kloas, 2010). a few studies have correlated aqueous concentrations of metals with other chemical parameters (stephenson and mackie, 1988, watras et al., 1998). for instance, aqueous cd, pb, and zn have been related to ph, dissolved oxygen, carbonate, and nutrients (prahalad and seenayya, 1989). correlation coefficients (r) computed between physicochemical properties and the concentration of cu and pb (table 4) showed that the metal concentrations were significantly and positively correlated with ph, conductivity, free co2 and water temperature. the concentration of cu and pb were significantly and negatively correlated with dissolved oxygen which is similar to the findings of singh et al. (2008) and insignificantly negatively correlated with alkalinity of water. therefore, water quality stations habitat types tadpole sp. month of collection in 2013 1. panchgram (ia) pond h. tigerinus may 2. rosekandy (tg) stream l. smithi march 3. rosekandy (tg) pond c. alticola september 4. rosekandy (tg) temporary water bodies fejervarya sp. march 5. rosekandy (tg) s. leptoglossa may 6. sildoobi (bk) e. cyanophlyctis september iaindustrial area, tgtea garden, bkbrick kiln table 1. habitat used by seven species of anuran tadpoles from barak valley, assam. sites air temp. (°c) surface water temp. (°c) ph cond. (µs cm-1) dissolved oxygen (mg l-1) free co2 (mg l-1) total alkalinity (mg l-1) 1.panchgram (ia) 31.1±1.9 25.1±1 7.1±0.6 95±1 1.2±0.1 12.1±0.5 35.9±1.3 2.rosekandy (tg) 28.5±0.4 23±0.9 5.4±0.3 24±1.5 3.2±0.7 6.2±0.4 45.4±3.1 3.rosekandy (tg) 27.1±0.3 24.1±0.3 6.5±0.3 46.6±1.5 3±0.1 10.5±0.3 30.6±1.5 4.rosekandy (tg) 28.2±0.9 22.7±0.9 5.4±0.3 31.3±1.5 2.9±0.4 6.3±0.5 50.2±2.3 5.rosekandy (tg) 29.5±0.7 24.2±0.3 5.6±0.1 24.3±1.5 2.3±0.3 6.8±0.2 30.1±1.6 6.sildoobi (bk) 28.2±0.3 26±0.2 7±0.1 69.6±0.5 1.2±0.1 11.1±0.2 42.6±2.1 table 2. physicochemical properties of water in different sampling sites. species sites heavy metal concentration tadpoles (µg gm-1) sediment (µg gm-1) water (mg l-l) copper lead copper lead copper lead 1. h. tigerinus ia 2.599 0.114 0.342 0.245 1.83 1.42 2. l. smithi tg 0.0052 0.084 0.0009 0.01 0.003 0.017 3. c. alticola tg 0.064 1.77 0.73 0.91 1.09 0.325 4. fejervarya sp. tg 0.037 0.288 0.0009 0.007 0.002 0.013 5. s. leptoglossa tg 2.456 1.476 3.135 0.445 0.815 0.341 6. e.cyanophlyctis bk 1.91 4.632 0.345 3.335 1.675 1.085 table 3. heavy metal concentrations in different species of tadpole, sediment and water in different sampling sites (iaindustrial area, tgtea garden, bkbrick kiln). 174 singh et al./ heavy metals in tadpoles parameters that affected metal concentration in water were ph, conductivity, free co2, do and water temperature. among different species of tadpoles, the total concentration of copper was in the order of h. tigerinus > s. leptoglossa > e. cyanophlyctis > c. alticola > fejarvarya sp. > l. smithi, while concentration of lead was in the order of e. cyanophlyctis > c. alticola > s. leptoglossa > fejarvayra sp. > h. tigerinus > l. smithi. in h. tigerinus and s. leptoglossa, copper concentration was more in comparison to lead concentration. in tadpoles of l. smithi, c. alticola and fejarvayra sp. and in sediment and water samples collected from the habitat, lead concentration was higher than copper concentration. in e. cyanophlyctis and in sediment sample lead concentration was more than the copper concentration but in water samples copper concentration was higher than the lead concentration. in the sampled tadpoles, concentration of copper in intestine was in the order of s. leptoglossa > h. tigerinus > c. alticola > fejarvayra sp. > l. smithi > e. cyanophlyctis and lead (pb) in the order of s. leptoglossa > e. cyanophlyctis > c. alticola > fejarvarya sp. > l. smithi > h. tigerinus. among different species of tadpoles e. cyanophlyctis accumulated highest concentration of copper in tail while least accumulation was recorded in the tail of l. smithi. sylvirana leptoglossa accumulated highest concentration of lead in tail. copper concentration was high in tail and liver in e. cyanophlyctis in comparison to other tadpoles. lead concentration was also high in liver of e. cyanophlyctis. (fig. 1 and 2). highest copper concentration was found in the sediment sample collected from the habitat of s. leptoglossa, while highest concentration of lead was recorded in the sediment sample collected from the habitat of e. cyanophlyctis. in water samples highest copper and lead concentrations were recorded in the habitat of h. tigerinus which is an industrial area (fig. 3). copper concentration in tadpoles were positively and significantly correlated with the concentration copper in water (r=0.725; fig. 4), while concentration of lead in tadpole was positively and significantly correlated with concentration of lead in water parameters copper (cu) lead (pb) ph 0.954** 0.911* conductivity 0.874* 0.951** dissolved oxygen -0.866* -0.949** free co2 0.935 ** 0.875* total alkalinity -0.515 -0.267 water temperature 0.943** 0.883* significant level at p values: * <0.05, ** <0.01 table 4. correlation coefficients between physico-chemical variables and concentration of heavy metals in water from sampling sites. figure 1. copper concentrations (µg gm-1) in intestine, tail and liver in six different species collected from six different sites. figure 2. lead concentrations (µg gm-1) in intestine, tail and liver in six different species collected from six different sites. 175 int. j. aquat. biol. (2016) 4(3): 171-178 sediment (r=0.944; fig. 5). different species of tadpoles exhibit different metals accumulation rates. all the tadpoles collected for heavy metal analysis belongs to the stage 26-35 (gosner, 1960). the result revealed that the concentration of lead in water samples was higher than the permissible limit of 0.01 mg l-1 but copper concentration was within the maximum permissible limit of 2 mg l-1 (who, 2011). the analysis for heavy metals (cu and pb) confirmed that the sediment concentrated more heavy metals than water. high concentration of heavy metal accumulation recorded in the study sites may be due to the excessive use of herbicides, chemicals, industrial effluents and other wastes in tea gardens. shaapera et al. (2013) studied the concentrations of seven heavy metals (pb, cu, zn, cr, fe, cd and mn) in the organs of adult rana esculenta obtained from river guma, benue state of nigeria and found that the trend of the heavy metals concentration in the organs were fe > mn > pb > zn > cu > cr > cd. the present study also reveals that in most of the samples pb was higher than the cu. sparling et al. (2000) reported that in amphibians, pb exposure resulted in a range of effects including decrease of erythrocytes and leucocytes, neutrophils and monocytes, sloughing of the skin, excessive bile figure 3. heavy metal concentrations in sediment (left) and water (right) collected from the habitat of tadpole species. figure 4. correlation between copper concentrations in tadpoles and the corresponding concentrations in sediment (left) and water (right). 176 singh et al./ heavy metals in tadpoles secretion, hypertrophy of liver, spleen and stomach, decreased muscle tone and loss of normal semi-erect posture, salivation, excitement and muscular twitching; and delayed metamorphosis. copper is an essential element that serves as a cofactor in a number of enzymes systems and necessary for the synthesis of hemoglobin (sivaperumal et al., 2007) but very high intake of cu can cause adverse health effect problems for most living organism. various researchers have reported strong positive correlations between pb concentrations in sediments and those in tadpoles (sparling and lowe, 1996). karasov et al. (2005) exposed tadpoles along a pollution gradient in the fox river and green bay ecosystem in wisconsin and found a positive correlation between levels in sediments and tadpoles for cd, cr, and pb. in the present study, positive correlations was found between pb and cu in tadpoles and the respective levels in the sediment and water. kelepertzis et al. (2012) reported that the significant correlation found between pb and cu in tadpoles and the respective levels in the sediments and water indicate that sediments provided the bulk of these metals for tadpoles. they also reported that differences in tadpole concentrations for the majority of analyzed metals were due to different metal levels in the sediments where they develop and breed. amphibian larvae accumulated the highest concentration of most trace elements, possibly due to their feeding ecology. since trace metals bioaccumulate in the food web, monitoring the quantities entering the trophic dynamics of the food chain can provide early warning systems on the potential environmental health consequences of current anthropogenic activities in the agricultural and industrial sectors. the edible frogs are an important link in the trophic transfer to higher vertebrates including birds, mammals, reptiles, predatory fish and man (tyokumbur and okorie, 2011). several problems, such as delayed development, morphological deformities, decreased growth and size at metamorphosis are related to heavy metals (sparling et al., 2006) and further investigation and monitoring of heavy metal residues in the environment is seriously necessary. it was concluded from this study that environment of barak valley is now becoming polluted by heavy metals and anuran tadpoles have become susceptible to this heavy metal pollution. acknowledgments the authors are grateful to saif, nehu, shillong for the use of aas. first and second authors express their gratitude to ugc, new delhi for providing figure 5. correlation between lead concentrations in tadpoles and the corresponding concentrations in sediment (left) and water (right). 177 int. j. aquat. biol. (2016) 4(3): 171-178 financial support. the authors are also grateful to the department of ecology and environmental science, assam university, silchar where the work was carried out. references apha. (2005). standard methods for the examination of water and waste water analysis, 21st ed. washington, dc. 964 p. astm. (1998)american society for testing of materials. standard practice for conducting toxicity tests with fishes, macroinvertebrates and amphibians. astm, philadelphia. publication no. e729-80. censi p., spoto s.e., saiano f., sprovieri m., mazzola s., nardone g., di geronimo s.i., puntu r., ottonello d. (2006). heavy metals in coastal water system. a case study from the north western gulf of thailand. chemosphere, 64: 1167-1176. ezemonye l.i., enuneku a.a. (2006). stage-dependent acute toxicity of exposure of bufo maculatus and ptychadena bibroni tadpoles to cadmium (cd2+). journal of applied science and technology, 11(1&2): 78-82. ezemonye l.i., enuneku a.a. 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(2019) 7(4): 202-210 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article effects of culture conditions on growth and survival of poecilia sphenops and poecilia reticulata sithara rasanjalee sirimanna*,1 chamari dissanayake department of zoology, university of sri jayewardenepura, gangodawila, nugegoda, sri lanka. s article history: received 2 march 2019 accepted 30 june 2019 available online 2 5 august 2019 keywords: culture conditions growth poeciliidae survival abstract: poecilia sphenops and p. reticulata are considered as two most popular and high demanding freshwater ornamental fish species globally. the effects of feeding frequency, feed protein level, photoperiod and salinity on growth and survival of these species and the effect of sex ratio on fecundity were studied under laboratory conditions from january to december 2016. although there was no any significant impact of feeding frequency on growth and survival of these species, feed protein content affected significantly on their growth. fry fed with newly hatched artemia, commercial larval feeds and chlorella sp. showed no significant impact on growth. poecilia sphenops and p. reticulata could tolerate salinity up to 10 and 25 ppt, respectively. fish exposed to 8 hrs photoperiod reported significantly lower growth than those who were exposed to 12 and 24 hrs photoperiods. the ideal female: male sex ratio for commercial stocking of p. sphenops and p. reticulata was 3:2. introduction ornamental fish culture is one of the high income generating practices in the world. millions of fresh, brackish and salt water fishes belonging to 2,000 species worth around 250-400 million us$ are annually traded as ornamental fish throughout the world (fao, 2015). the united states is the single largest importer of ornamental fish in the world (fao, 2015; dey, 2016) while singapore, hong kong, malaysia, thailand, the philippines and sri lanka are considered as the top exporters. favorable climatic conditions, natural water resources and ever growing market demand are some driving forces that promote the ornamental fish industry in sri lanka. around 35 ornamental fish species have been exported from sri lanka in 2016 reporting an average income of 13 million us$ (edb, 2016). both guppy (poecilia reticualta) and molly (p. sphenops) belonging to the family poeciliidae contribute more than 75% of ornamental fish exports from sri lanka (edb, 2016; perera, 2016). similar to other aquaculture practices, feed management, health management and water quality *correspondence: sithara rasanjalee sirimanna doi: https://doi.org/10.22034/ijab.v7i4.570 e-mail: sitha9234tcc@gmail.com management are the major concerns in ornamental fish culture. feed is the most potent exogenous factor that affects growth and other physiological mechanisms in fish. supplementary feeding is typically practiced in aquaculture to enhance growth of organisms to marketable size within a short period whereas limited feeding negatively affects survival, food intake and growth. ration size and feeding frequency varies with the age, season and species of fish (johnston et al., 2003). excess feeding not only pollutes the water but also increases production costs (james and sampath, 2003). nutrient requirements of fish differ with the age and species but protein is considered as the most important nutrient in fish growth (chong et al., 2000). levels beyond the optimum protein requirement result in excess deamination and catabolism of protein and the lower protein levels result poor growth (kruger et al., 2000). environmental factors such as temperature, photoperiod, salinity and dissolved oxygen (do) have a significant impact on growth, survival and reproduction of fish (boeuf and le bail, 1999). according to previous literature, 23-27°c temperature 203 int. j. aquat. biol. (2019) 7(4): 202-210 (dzikowski et al., 2001), do level higher than 6 mgl-1 (parameshwaran et al., 2001), photoperiod of 12 hours (boeuf and le bail, 1999) and 0 ppm salinity (rosen and bailey, 1963) are considered ideal for the optimum growth of freshwater ornamental fishes. considering all these factors, this study was carried out to determine the effects of feeding frequency, feed protein level, photoperiod and salinity on growth and survival of two highly exported freshwater ornamental fish species, p. sphenops and p. reticulata. an attempt was also made to identify the relationship between sex ratio and fecundity of these species. as ornamental fish industry is rapidly growing in the country with the support of government, this study would be a support to ornamental fish farmers in sri lanka to identify the most appropriate breeding and culture conditions for p. sphenops and p. reticulata to maximize the export revenue while enhancing the production and livelihood opportunities. materials and methods brood stock collection and maintenance: thirty brooders from each species, p. sphenops (1.2±0.2 g) and p. reticulata (1.0±0.2 g) were collected, from a commercial fish farm, packed in double layered polythene bags and immediately transported to the aquatic science laboratory of the department of zoology, university of sri jayewardenepura. upon arrival to the laboratory, brooders were quarantined with 2 ppt kmno4 for 30 minutes and transferred to the brood stock rearing tanks (glass tank with 55×30× 30 cm) where the stocking density was 1 fish per liter. brood stock rearing tanks were well-aerated and brooders were fed with commercial fish feed (prima fish feed, produced by prima ceylon pvt ltd, sri lanka) twice a day based on 10% of their body weight (harpaz et al., 2005). the bottom of each tank was siphoned out daily and the water quality parameters were maintained at ph, 7.2±0.2; temperature, 29±1°c; dissolved oxygen, 6.0±0.3 mg/l; ammonia, 0 ppm. experimental setup: the common experimental setup (fig. 1) was used to study the effects of different culture conditions, i.e. feeding frequency, feed protein level, salinity and photoperiod on growth and survival of p. sphenops and p. reticulata under the laboratory conditions. each experiment was replicated thrice and 30 laboratory bred fry (2 day old) were used in each replicate tank. initial length of fry both in terms of total length and standard length was measured before introducing them to the experimental tanks. experimental fish were fed with commercial fish feed pellets (prima fish feed) twice a day except for the feed frequency experiment. the tanks were provided with continuous aeration throughout the experimental period, tank bottoms were siphoned out daily and the water quality parameters were monitored twice a week. fish were reared under natural day light condition except for those who were subjected to the photoperiod experiment and the water quality parameters in experimental tanks were maintained at ph, 7.0±0.2; temperature, 29°c±1; dissolved oxygen, 6.2±0.3 mg/l; ammonia, 0 ppm. in all growth experiments, the standard length (cm) and wet weight (g) of each experimental fish was measured weekly. length measurements were taken since the beginning of all experiments but the weight measurements were taken 8 weeks after the experiments as the individual weight of each fry was difficult to measure accurately. effect of feeding frequency: poecilia sphenops and p. reticulata fish were fed with commercial fish feed (prima fish feed) subjecting them into three different figure 1. common experimental setup used to study the effects of culture conditions on growth and survival of poecilia reticulata and p. sphenops under laboratory conditions. 204 sirimanna and dissanayake / culture conditions of poecilia sphenops and p. reticulata feed frequency treatments; once (8.30 a.m), twice (8.30 a.m and 4.30 p.m) and thrice (8.30 a.m, 12.30 p.m and 4.30 p.m) a day. the amount of feed given at a time was assigned based on 10% of mean body weight of experimental fish. this experiment was carried out for 32 weeks and growth and survival of each species were estimated with respect to three different treatments. effect of feed protein content: three fish feed types each containing 20, 40 and 60% of protein were prepared following the method described by chong et al. (2000) (table 1) under the laboratory conditions and p. sphenops and p. reticulata were fed with experimental feed twice a day (8.30 a.m and 4.30 p.m) for a period of 24 weeks. the water ammonia level was measured weekly throughout the experimental period and at the end of the experiments, growth and survival of two species were estimated with respect to three different feed types. micro-kjeldahl method with acid digestion was used to determine the crude protein content in prepared fish feeds and conversion factor 6.25 was used to convert total nitrogen to crude protein (haider et al., 2015). effect of larval feeds: effect of three different larval feed types; newly hatched artemia nauplii, chlorella sp. and commercial larval feed (prima powder fish feed produced by prima ceylon pvt ltd, sri lanka) on the growth and survival of p. sphenops and p. reticulata fry was compared for a period of 4 weeks. artemia nauplii were hatched in 32 ppt saline water and the artemia concentration was maintained at 2 nauplii ml-1 under laboratory conditions as described by tolga (2012). mass culture of chlorella sp. was prepared using a stock solution and the green water (chlorella sp.) with 1.5×106 cells/ml cell concentration was maintained and daily cell counts were taken using sedgewick rafter counting chambers (raehanah et al., 2009). effect of salinity: three days old fry (n=20) and six months old adults (n=20) of p. sphenops and p. reticulata were exposed to six different salinity treatments; 5, 10, 15, 20, 25 and 30 ppt. at the beginning of this experiment, all the experimental fish were kept in freshwater (0 ppt) for two days and then they were directly subjected to each saline treatment (shikano and fujio, 1997). the survival of table 1. main ingredients used to prepare experimental feeds with three different protein levels. diets ingredients 20% protein 40% protein 60% protein danish fish meal (70%)1 125 125 125 soya 16.3 126 235 gelatin 15 15 15 cod liver oil 15 15 15 wheat flour 296.5 189 78 vitamin and mineral mix1 25 25 25 carboxy methyl cellulose 7.5 7.5 7.5 proximate composition crude protein 20.5 39.3 59.3 crude lipid 9.5 9.4 9.5 ash (%) 4.3 4.5 4.3 fibre (%) 4.3 4.6 3.9 gross energy (kj/g)3 16.5 16.5 16.5 1source: (moisture 42.3 g kg-1, crude protein 706.80 g kg-1, crude lipid 74.70 g kg-1, ash 92.50 g kg-1 and fibre 1.60 g kg-1). 2premix; as per kg, vitamin a 2.0×106 iu, vitamin d3 4.0×105 iu, vitamin e 5.0 g, vitamin c 20 g, vitamin b1 0.8 g, vitamin b2 1.0 g, vitamin b6 2.4 g, vitamin b12 40 mg, l-lysine 3 g, dl methionine 2 g, choline chloride 5 g, niacinamide 10 g, magnesium sulphate 24 g, coboltsulphate 80 mg, sodium selinate 20 mg, pottassium iodide 240 mg, biotin 150 mg, ferrous sulphate 28 g, copper sulphate 24 g, zinc sulphate 24 g, manganese sulphate 6.8 g, inositol 5 g, calcium carbonate as carrier. 3gross energy: calculated based on 0.17, 0.40 and 0.24 kj/g for carbohydrate, lipid and protein, respectively. 205 int. j. aquat. biol. (2019) 7(4): 202-210 experimental fish after one week of stocking in each salinity treatment was recorded. effect of photoperiod: the effect of photoperiod on growth and survival of p. sphenops and p. reticulata was estimated subjecting experimental fish into three different photoperiods, 8, 12 and 24 hours per day (boeuf and le bail, 1998; biswas et al., 2006). this experiment was carried out for 24 weeks and at the end of the experiment, the growth and survival of fish were estimated with respect to each treatment. effect of sex ratio on fecundity: three different female: male sex ratios; 2:1, 3:1 and 3:2 of p. sphenops and p. reticulata species were maintained for a period of 40 weeks. aquarium bred brooders of similar age (6 months old) and size (1.2±0.2 g) were reared in 55×30×30 cm glass tanks under laboratory conditions. the number of offspring produced under each sex ratio was counted and recorded. data analysis: in growth experiments, percentage body weight gain (bwg%), percentage length gain (lg%) and specific growth rate (sgr) were calculated following the formula described by anka et al. (2016). percentage body weight gain (bwg%) = 𝑊𝑡 − 𝑊0 𝑊𝑡 × 100% percentage length gain (lg%) = 𝐿𝑡 − 𝐿0 𝐿𝑡 × 100% specific growth rate (sgr) (%/day) = (𝑙𝑛𝑊𝑡 – 𝑙𝑛 𝑊0) × 100 𝑡 −1 where, wt and w0 were mean final and initial fish weight (g), lt and l0 were mean final and initial length (cm), respectively and t was the experimental duration in days. the survival was calculated according to the following equation, survival (%) = 𝑁𝑡 × 100 𝑁0 −1 where, nt and n0 were final and initial numbers of fry in each treatment, respectively. variations in the average growth parameters of fish subjected to different treatments of feeding frequency, feed protein level, larval feed, salinity and photoperiod were compared using one way analysis of variance (anova) followed by tukey’s multiple comparison tests. the effect of sex ratio on fecundity of p. sphenops and p. reticulata was also assessed using anova. differences were considered to be table 2. growth performance of poecilia reticulata and p. sphenops in feeding frequency, feed protein content and photoperiod experiments. p. reticulata mean initial length (cm) mean final length (cm) lg (%) mean initial weight (g) mean final weight (g) bwg (%) sgr (%day-1) feeding frequency 1 0.6±0.0 2.2 ±0.3 72.7 0.2 ±0.0 0.7 ±0.1 71.4 0.54 2 0.6±0.0 2.7±0.3 77.7 0.2 ±0.1 1.0 ±0.1 80 0.56 3 0.6±0.0 2.7±0.2 77.7 0.2 ±0.0 1.2 ±0.1 83.3 0.81 feed protein content 20% 0.6±0.0 1.4±0.1 57.1 0.1±0.0 0.4±0.1 75 0.82 40% 0.6±0.1 2.1±0.1 71.4 0.1±0.0 0.7±0.1 85.7 1.16 60% 0.6±0.0 2.5±0.3 76 0.1±0.0 0.9±0.1 88.8 1.31 photoperiod 8 hours 0.6±0.0 1.7±0.1 64.7 0.1± 0.0 0.6±0.1 83.3 0.82 12 hours 0.6±0.0 2.5±0.2 76 0.1±0.0 0.9±0.2 88.8 1.31 24 hours 0.6±0.0 2.6±0.3 76.9 0.2±0.1 0.8±0.1 75 1.06 p. sphenops feeding frequency 1 0.6±0.0 2.3 ±0.2 73.9 0.2 ±0.0 0.7 ±0.1 71.4 0.55 2 0.6±0.0 3.0 ±0.1 80 0.2 ±0.0 1.1 ±0.1 81.8 0.75 3 0.6±0.0 3.2 ±0.1 81.25 0.2 ±0.0 1.2 ±0.1 83.3 0.79 feed protein content 20% 0.6±0.0 1.7±0.1 64.7 0.1±0.0 0.5±0.1 80 0.96 40% 0.6±0.0 2.5±0.1 76 0.1±0.0 0.8±0.1 87.5 0.96 60% 0.6±0.1 2.7±0.1 77.7 0.1±0.0 1.0±0.1 90 1.24 photoperiod 8 hours 0.6±0.1 1.5±0.1 60 0.1±0.0 0.4±0.1 75 0.82 12 hours 0.6±0.1 2.7±0.2 77.7 0.1±0.0 1.0±0.1 90 1.16 24 hours 0.6±0.0 2.1±0.1 71.4 0.2±0.1 0.7±0.1 71.4 1.37 206 sirimanna and dissanayake / culture conditions of poecilia sphenops and p. reticulata significant when p<0.05. all the statistical tests were performed in minitab 14 for windows statistical package. results effect of feeding frequency: variations in weight (mean±sd g) of p. spenops and p. reticulata and reared in three different feeding frequency treatments were compared (fig. 2). in both these species, experimental fish fed thrice a day reported the highest average weight as well as the highest specific growth rate (sgr) while the fish fed with one time per day reported the lowest values (fig. 2, table 2). however, there was no significant impact of feeding frequency on weight gain of these species (p>0.05, anova, df =2). at the end of this experiment, fish showed 100% survival in all the experimental tanks. effect of feed protein content: the feed protein level has a significant impact on growth of p. reticulata and p. sphenops (fig. 3). fish fed with 20% protein feed showed significantly lower weight gain than the 40% and 60% protein feeds (p<0.05, anova, df=2). the lowest specific growth rate was also evident in the fish fed with 20% protein feed (table 2). however, experimental fish showed 100% survival at the end of this experiment. effect of larval feeds: fry of p. reticulata and p. sphenops fed with newly hatched artemia showed a higher mean weight than the other two larval feeds (fig. 4). however, there was no significant impact of feed types on growth and survival of early life stages of p. reticulata and p. sphenops (p>0.05, anova, df =2). effect of salinity: p. reticulata fry showed 100% survival in 5 and 10 ppt saline water but they showed only 40% survival in 15 and 20 ppt saline water. however, adult p. reticulata reported 100% survival up to 20 ppt salinity and 60% survival in 25 ppt salinity. they survived only 2 days in 30 ppt salinity. both fry and adults of p. sphenops could tolerate salinity up to 10 ppt. although 100% survival of adult p. sphenops was recorded in 10 ppt saline water, only 80% of fry were survived. both adults and fry figure 2. variations in mean weight (±sd g) of poecilia reticulata (above) and p. sphenops (below) reared under three different feeding frequencies. figure 3. variations in mean weight (±sd g) of poecilia reticulata (above) and p. sphenops (below) reared under three different feed protein levels. 207 int. j. aquat. biol. (2019) 7(4): 202-210 survived less than 24 hours in 15 ppt saline water. effect of photoperiod: a significant impact on photoperiod on growth of p. sphenops and p. reticulata was reported (fig. 5). experimental fish exposed for 8 hours photoperiod showed significantly lower weight gain than the fish exposed for 12 and 24 hours photoperiods (p<0.05, anova, df=2). both species reared under12 hours photoperiod showed the highest sgr (table 2). a survival rate of 100% was recorded in all photoperiod experiments. effect of sex ratio on fecundity: a total of 834 p. reticulata and 611 p. sphenops offspring were produced during the study period. in both these species, a significantly higher number of offspring were reported when (female:male) sex ratio was 3:2 (p<0.05, anova, df=2 ). (table 3). discussions although sri lanka is considered as one of the pioneers in ornamental fish industry in the world, very few scientific studies have been conducted to identify the appropriate culture conditions for ornamental fish species. therefore, this study was carried out to identify the most suitable conditions for the growth and survival of p. reticulata and p. sphenops under aquarium conditions. in feeding frequency experiments, the observed higher specific growth rate of fish fed 2 and 3 times per day compared to the fish fed once a day, could be probably due to fish reaching to the level of satisfaction or to the level of satiation when they were table 3. number of offspring produced by poecilia reticulata and p. sphenops under different sex ratios during the experimental period. sex ratio (female : male) number of offspring produced p. reticulata p. sphenops 2:1 130 97 3:1 178 162 3:2 526 352 figure 4. variations in mean length (±sd g) of poecilia reticulata (above) and p. sphenops (below) fry reared under three different larval feeds. figure 5. variations in mean weight (±sd g) of poecilia reticulata (above) and p. sphenops (below) reared in different photoperiods. 208 sirimanna and dissanayake / culture conditions of poecilia sphenops and p. reticulata fed 2 or 3 times a day (grayton and beamish, 1977). though, feeding fish thrice a day ensures the highest sgr of both species, a comparatively high accumulation of waste materials, including both excretory products of fish and uneaten feed particles and discolored turbid water was observed in all experimental tanks. therefore, more time and labor was required and the wastage of food was high in feeding frequency 3 treatment. as the feeding of fish considered to occupy 60-70% of total cost of production in ornamental aquaculture (rathnayake et al., 2016), feeding fish 2 times a day can be considered as the most economically feasible feeding frequency which ensures the optimum growth of p. reticulata and p. sphenops under aquarium conditions. as the survival rate of fish in all treatments was 100%, it confirms that there is no any effect of feeding frequency on the survival of p. reticulata and p. sphenops. feed protein plays a major role in the growth of fish and it has been reported that fish requires two to four times more dietary protein compared to warm-blooded animals, as they need relatively higher level of essential amino acids (sumithra et al., 2015). on dry weight basis, protein contributes about 65-75% of the total weight of fish tissue (shim and chua, 1986). it is reported that decrease of body weight can occur when fish are fed with low dietary protein levels (dabrowski, 1977). in feed protein experiment, 20% feed protein level showed a significantly lower growth in p. reticulata and p. sphenops compared to 40 and 60% feed protein levels. the reduced growth in fish fed with 20% or less protein levels also have observed by shim and chua (1986). the dietary protein is considered to be just enough for growth and repair as the protein sources are more expensive than carbohydrates and fat sources (sumithra et al., 2015). as the fish fed with feed containing 40 and 60% dietary protein levels showed no significant difference in growth of p. reticulata and p. sphenops, 40%, dietary protein level can be proposed as the ideal protein level in commercial fish feed due to cost effectiveness. although there was no any significant impact of feed types on growth and survival of p. reticulata and p. sphenops fry, the results revealed that fry fed with newly hatched artemia attained the highest mean weight followed by the fry fed with commercial larval feed and chlorella sp.. it was reported that newly hatched artemia contain high level of nutrients; especially they are rich with proteins (leger et al., 1991). further, nutrient loss from live feeds like artemia is much less or negligible when compared with formulated artificial diets (goldblatt et al., 1980). furthermore, artemia does not deteriorate the water quality by accumulating in the bottom when compared to commercial fish feed (harpaz et al., 2005). because of these favorable factors, when artemia are given to fry they can easily detect their food, get comparatively long feeding time and receive high protein diet, which finally enhance their growth. the observed reduced mean weight in fry fed with chlorella sp. can be due to the lack of animal protein in this feed (dussault and kramer, 1980). poeciliids are generally active fish during daylight (meffe and snelson, 1989). therefore, they have a clear connection with the photoperiod. the observed significant reduction in growth of p. reticulata and p. sphenops subjected to 8 hours photoperiod per day could be mainly due to their reduced feed intake. as a diurnally active fish species, poeciliids actively feed during daylight. when photoperiod is reduced, the amount of feed intake by these species is also reduced causing their growth retardation (biswas et al., 2006). high accumulation of excess feed in the experimental tanks which were subjected to 8 hours photoperiod than 12 and 24 hours photoperiods may be a result of reduced feeding rate during dark. but the survival of these species was not affected by the photoperiods. some fish are highly euryhaline but they do not use their ability to tolerate seawater in the natural environment. similarly, some species belonging to family poeciliidae are euryhaline though they live mostly in freshwater environments (rosen and bailey, 1963). the experiment recorded survival of p. reticulata in 25 ppt saline waters which also has been observed previously by shikano and fujio (1998). according to pisam et al. (1987), acclimation 209 int. j. aquat. biol. (2019) 7(4): 202-210 to the seawater can be induced by developing osmoregulatory functions such as branchial chloride cells in euryhaline fish including poeciliids. but due to unknown reasons p. sphenops showed comparatively lesser salinity tolerance compared to p. reticulata in this experiment. in both experiments a significant high fecundity was observed when female to male sex ratio was maintained as 3:2 than 2:1 and 3:1. the exact reason for this result is unknown but it might be the high number of males in the replicates which results a higher sperm production (evens and magurran, 1999). conclusion the study recommends several culture conditions for p. reticulata and p. sphenops namely, maintaining a feeding frequency of twice per day, dietary protein level of 40%, 12 hours photoperiod and female: male sex ratio of 3:2. the impact of the type of fry feed on the growth and survival was not significant. poecilia sphenops and p. reticulata could tolerate salinity up to 10 and 25 ppt, respectively. references anka i.z., jothi j.s., sarker j., talukder a., islam, m.s. 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(2021) 9(1): 33-40 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article capoeta baliki turan, kottelat, ekmekçi & imamoglu, 2006 a junior synonym of capoeta tinca (heckel, 1843) (teleostei: cyprinidae) erdoğan çiçek* 1, soheil eagderi2, sevil sungur3, burak secer1 1department of biology, faculty of art and sciences, nevşehir hacı bektaş veli university, nevşehir, turkey. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 3health services vocational school, nevşehir hacı bektaş veli university, 50300, nevşehir, turkey. s article history: received 6 october 2020 accepted 27 december 2020 available online 2 5 february 2021 keywords: sakarya basin kızılırmak basin susurluk basin interspecific variation abstract: capoeta baliki was described from sakarya basin, turkey. it was distinguished from its nearest congener i.e. c. tinca based on a combination of characters, including fewer serrae along posterior margin of last simple dorsal-fin ray, modally fewer scale rows between lateral line and dorsal-fin origin, fewer vertebrae, deeper and shorter head and slenderer caudal peduncle. we examined the synonymy hypothesis of c. baliki and c. tinca by comparing their morphometric, meristic and molecular characters. based on the results, their morphometric and meristic characters largely overlapped and no character was found to distinguish them. in addition, a low k2p mean genetic divergence of 0.37% c. baliki and c. tinca based on cytb gene and clustering in same clad showed that they are identical in molecular characters. as no character could be found to clearly distinguish these species, we treat c. baliki as a junior synonym of c. tinca. introduction capoeta tinca heckel (1843), described from nilüfer river, bursa province, susurluk basin of turkey, is found throughout the black sea watersheds, as well as sakarya and kizilirmak basins in the central anatolia (geldiay and balık, 2007; çiçek at al., 2020). turan et al. (2006) described the c. tinca populations of the central anatolian basins i.e. sakarya and kizilirmak as a distinct species of c. baliki based on some morphological characters. however, further works (özdemir, 2013, 2015; kaya, 2019) rejected distinguishing characters of c. baliki from c. tinca suggesting c. baliki as a junior synonym of c. tinca. additionally, pronounced discriminative differences was not found in the osteological characteristics of c. baliki and c. tinca (küçük et al., 2008). in recent years, molecular studies (özdemir, 2013; bektaş et al., 2017, 2019; ghanavi et al., 2016; levin, 2012; zareian and esmaeili, 2017; zareian et al., 2018) based on both coi and cytb genes of mitochondrion indicated clustering of c. tinca and c. baliki in the same clade supporting c. baliki as *correspondence: erdoğan çiçek doi: https://doi.org/10.22034/ijab.v9i1.1118 e-mail: erdogancicek@nevsehir.edu.tr junior synonym of c. tinca. hence in the present study, we decided to examine the synonymy hypothesis of c. baliki and c. tinca by comparing their morphometric, meristic and molecular (mtdna cytb gene) characters. materials and methods specimens of c. tinca were collected from its type locality (susurluk basin) and c. baliki from sakarya basins. all specimens caught by electrofishing, and after anaesthesia by ms222, they were fixed into 5% buffered formaldehyde and stored in 70% ethanol after two weeks. methods for counts and measurements followed armbruster (2012). measurements were made with a dial calliper to the nearest 0.1 mm. gill rakers were counted on the outer margin of the anterior gill arch. the posterior pair of the branched rays articulating on a single pterygiophore in the dorsal and anal fins were noted as “1½”. twenty morphometric characters were measured (table 1) and then standardized using an allometric method to remove size-dependent variation using madj 34 çiçek et al./ capoeta baliki a junior synonym of capoeta tinca = m (ls / l0)b, where m is the original measurement, madj the size adjusted measurement, l0 the standard length of the fish, ls the overall mean of the standard length for all fish from all samples in each analysis, and b was estimated for each character from the observed data as the slope of the regression of log m on log l0 using all fish in any group (elliot, 1999). the results derived from the allometric method were confirmed by testing significance of the correlation between transformed variables and standard length (buj et al., 2008). the morphometric data of the two species were analyzed using multivariate analyzes of table 1. cytochrome b sequences downloaded from ncbi genbank with information on basin, country of origin and reference. species drainage country published by genbank acc. no. capoeta baliki black sea basin, sakarya turkey bektas et al. 2017 gq424019 capoeta baliki black sea basin, sakarya turkey bektas et al. 2017 gq424016 capoeta baliki black sea basin, sakarya turkey bektas et al. 2017 gq424015 capoeta baliki black sea basin, sakarya turkey bektas et al. 2017 gq424014 capoeta baliki black sea basin, sakarya turkey bektas et al. 2017 gq424013 capoeta baliki black sea basin, sakarya turkey bektas et al. 2017 gq424012 capoeta baliki black sea basin, sakarya turkey bektas et al. 2017 gq424011 capoeta baliki lake iznik basin, çakirca stream turkey levin et al. 2012 jf798275 capoeta baliki sakarya basin, kurtbogazı dam lake turkey levin et al. 2012 jf798274 capoeta baliki sakarya basin, kurtbogazı dam lake turkey levin et al. 2012 jf798273 capoeta baliki black sea basin, kelkit river turkey levin et al. 2012 jf798272 capoeta baliki black sea basin, kızılırmak river turkey levin et al. 2012 jf798271 capoeta tinca eber lake basin, afyon turkey bektas et al. 2017 gq424010 capoeta tinca eber lake basin, afyon turkey bektas et al. 2017 gq424009 capoeta tinca marmara basin, susurluk turkey bektas et al. 2017 gq424008 capoeta tinca marmara basin, susurluk turkey bektas et al. 2017 gq424007 capoeta tinca marmara basin, susurluk turkey bektas et al. 2017 gq424006 capoeta tinca marmara basin, susurluk turkey bektas et al. 2017 gq424005 capoeta tinca marmara basin, susurluk turkey bektas et al. 2017 gq424004 capoeta damascina mediterranean sea basin, orontes river turkey levin et al. 2012 jf798306 capoeta damascina mediterranean sea basin, orontes river turkey levin et al. 2012 jf798305 capoeta aydinensis aegean sea basin, b. menderes turkey bektas et al. 2017 ky065274 capoeta aydinensis aegean sea basin, b. menderes turkey bektas et al. 2017 ky065275 capoeta caelestis mediterranean sea basin, kargi stream turkey levin et al. 2012 jf798287 capoeta caelestis mediterranean sea basin, kargi stream turkey levin et al. 2012 jf798288 capoeta antalyensis mediterranean sea basin, aksu at gokdere turkey bektas et al. 2017 gq424021 capoeta antalyensis mediterranean sea basin, aksu at gokdere turkey bektas et al. 2017 gq424023 capoeta sieboldii black sea basin, kelkit river turkey levin et al. 2012 jf798330 capoeta sieboldii black sea basin, kızılırmak river turkey levin et al. 2012 jf798329 capoeta sieboldii black sea basin, kizilirmak turkey bektas et al. 2017 ky065259 capoeta sieboldii black sea basin, kizilirmak turkey bektas et al. 2017 ky065258 capoeta sieboldii black sea basin, yesilirmak turkey bektas et al. 2017 ky065256 capoeta bergamae marmara basin, bakacak stream turkey levin et al. 2012 jf798282 capoeta bergamae bakırçay river turkey levin et al. 2012 jf798280 capoeta banarescui black sea basin, coruh turkey bektas et al. 2017 gq423988 capoeta banarescui black sea basin, coruh turkey bektas et al. 2017 gq423984 capoeta banarescui black sea basin, coruh turkey bektas et al. 2017 gq423992 capoeta banarescui black sea basin, coruh turkey bektas et al. 2017 gq423991 capoeta banarescui black sea basin, coruh turkey bektas et al. 2017 gq423990 capoeta banarescui black sea basin, coruh turkey bektas et al. 2017 gq423989 capoeta capoeta caspian sea basin, aras river iran ghanavi et al. 2016 ku167938 capoeta trutta karoun river drainage, lordegan iran ghanavi et al. 2016 km459673 luciobarbus esocinus tigris river turkey yang et al. 2015 kp712264 35 int. j. aquat. biol. (2021) 9(1): 33-40 principal component analysis (pca) and p-value obtained from permutation test of one-way npmanova. all outliers were removed from further analysis. all analyses were performed using past software. molecular data analysis: for this study, we retrieved 43 cytb sequences of the published capoeta from genbank using the (blastn) basic local alignment search tool (altschul et al., 1990) (table 1). for phylogenetic reconstruction, the datasets were analysed by bayesian inference (bi) using mrbayes 3.1.2 (ronquist et al., 2011) and the maximum likelihood (ml) method in iq-tree 1.6.0 (nguyen et al., 2015). we determined the best-fit model of molecular evolution for the genomic dataset using the bayesian information criterion (bic) in iqtree 1.6.0 (kalyaanamoorthy et al., 2017). mrbayes was run with 6 substitution types (nst=6) and considered the gamma-distributed rate variation across sites plus a proportion of invariable sites (gtr) for the coi datasets. for bi, bayesian inference was calculated with mrbayes v.3.2.6 (ronquist et al., 2011). two simultaneous analyses were run with each 2,000,000 generations and four mcmc chains sampling every 10,000 generations. convergence was checked on tracer 1.6 (rambaut and drummond, 2013). after discarding the first 10% of generations as burn-in, we obtained the 50% majority rule consensus tree and the posterior probabilities. for ml analyses, we conducted heuristic searches (1,000 runs) under a tn+f+g4 model. uncorrected pairwise genetic distances (p-distances) were investigated based on kimura two-parameter (k2p) distances (tamura et al., 2013). capoeta capoeta (ku167938), capoeta trutta (km459673) and luciobarbus esocinus (kp712264) were used as outgroups. abbreviations used. hl, head length; sl, standard length; k2p, kimura 2-parameter. collection codes: nuic, ichthyological collection of the nevsehir haci bektas veli university. results general appearances of c. tinca and c. baliki are presented in figure 1 showing their body shapes and colour patterns similarity. tables 2 and 3 represent their morphometric measurements and meristic counts, respectively. all morphometric and meristic features of c. baliki are largely overlapped with those of c. tinca. we failed to find any non-overlapping morphological differences between the c. tinca and figure 1. lateral view of (a) capoeta baliki, nuic-1816, 106.3 mm sl; derecik stream, ankara province, and (b) c. tinca, nuic-1717, 119.2 mm sl değirmen stream, balıkesir province (all from turkey). 36 çiçek et al./ capoeta baliki a junior synonym of capoeta tinca table 2. morphometric data of capoeta tinca (n=20) and c. baliki populations (n=20). morphometric characters capoeta tinca (nuic-1717) capoeta baliki (nuic-1816) min-max mean±sd min-max mean±sd standard length (mm) 90.6-140.7 117.0±15.2 90.8-227.3 127.4±37.2 in percent of standard length head length 23.4-24.9 24.3±0.5 21.4-26.4 24.2±1.3 body depth at dorsal fin origin 22.9-26.3 24.5±1.1 20.2-24.8 23.1±1.4 predorsal length 47.0-53.2 49.6±2.1 46.7-53.8 50.9±1.9 prepelvic length 51.2-54.4 52.5±1.1 49.8-54.9 52.8±1.5 preanal length 72.9-76.0 74.7±1.1 73.5-78.1 75.0±1.6 distance between pectoral-fin origin to anal fin 51.1-55.7 53.9±1.5 43.3-58.1 52.7±3.5 distance between pectoral-fin origin to pelvic fin 29.1-32.9 31.1±1.2 29.7-34.2 31.1±1.3 distance between pelvic-fin origin to anal fin 21.1-23.9 22.6±0.9 20.2-24.2 22.3±1.0 dorsal-fin height 19.3-22.8 21.4±1.1 18.1-22.7 20.4±1.5 anal-fin length 16.0-18.4 16.9±0.9 16.0-22.2 18.3±2.1 pectoral-fin length 17.6-19.7 18.7±0.7 17.3-19.6 18.6±0.8 pelvic-fin length 15.0-16.6 15.8±0.6 14.7-17.1 15.7±0.7 upper caudal-fin lobe 16.9-25.3 22.7±2.2 18.1-23.6 21.6±1.4 length of caudal peduncle 17.6-19.3 18.6±0.7 16.3-20.3 18.0±1.4 depth of caudal peduncle 11.5-12.8 12.1±0.4 10.9-12.9 12.2±0.6 in percent of head length head depth at eye 45.2-51.0 48.4±1.7 48.0-55.2 50.9±2.2 snout length 32.4-38.2 35.2±1.4 35.3-40.4 37.5±1.6 eye horizontal diameter 17.1-22.5 19.8±1.5 16.6-22.1 19.0±1.8 interorbital width 37.5-42.0 39.9±1.7 37.4-44.2 41.1±2.0 postorbital distance 46.2-51.2 48.8±1.8 46.2-52.3 49.1±1.7 maximum head width 60.2-68.3 62.9±2.5 60.5-69.2 63.7±2.8 table 3. meristic data of capoeta tinca (nuic-1717) and c. baliki (nuic-1816) (n=20 in each populations). gill raker examined materials 19 20 21 22 23 24 capoeta tinca 3 6 10 1 capoeta baliki 2 5 3 8 1 1 lateral line scales examined materials 75 76 77 78 79 80 capoeta tinca 8 4 3 capoeta baliki 12 5 3 scales above lateral line scales below lateral line examined materials 14 15 16 17 18 7 8 9 capoeta tinca 1 15 4 1 14 5 capoeta baliki 1 12 6 1 18 2 branched dorsal-fin rays examined materials 7½ 8½ 9½ mode capoeta tinca 4 16 9 capoeta baliki 2 18 9 branched anal-fin rays examined materials 5 6 7 mode capoeta tinca 2 18 6 capoeta baliki 4 16 6 pelvic-fin rays examined materials 7 8 9 mode capoeta tinca 2 18 8 capoeta baliki 20 8 pectoral-fin rays examined materials 17 18 19 20 mode capoeta tinca 2 16 2 18 capoeta baliki 18 2 18 37 int. j. aquat. biol. (2021) 9(1): 33-40 c. baliki. in pca, the first three pcs accounted a total of 84.49% of the variances (pc1=41.02, pc2=20.90 and pc3=12.857) (jolliffe cut-off=1.119). by plotting the first two pcs, the distribution of the studied species based on their morphometrics are presented in figure 2, showing overlapping the specimens of both c. tinca and c. baliki. the result of multivariate one-way npmanova showed no different between two species (p=0.1003, f=1.913). based on figure 3, capoeta species, including c. tinca, c. baliki, c. banarescui and c. antalyensis were clustered in the same clade with those c. baliki and c. tinca merged in the same clade. a low k2p mean genetic divergence of 0.37% was calculated between c. baliki and c. tinca (özdemir, 2013; bektaş et al., 2019) (table 4). discussions according to turan et al. (2006), c. baliki is distinguished from c. tinca by having fewer serrae along posterior margin of last simple dorsal-fin ray (17-23 vs. 24-28), modally fewer scale rows between lateral line and dorsal-fin origin (14 vs. 16), fewer vertebrae (43-44 vs. 44-46), shorter head (21.8-24.5 vs. 23.3-26.7% sl), deeper head (55.6-63.5 vs. 49.356.5% hl) and lower caudal peduncle (9.5-12.2 vs. 10.8-13.4% sl). based on the examined materials, our data in line with pervious findings (özdemir, 2013, 2015; kaya, 2019) showed overlapping of all above-mentioned distinguishing characters as well as others (tables 2 and 3). therefore, there is no morphological diagnostic characters to distinguish c. baliki from c. tinca. capoeta tinca and c. baliki were clustered in the figure 2. the pca graph of morphometric characters in capoeta tinca (nuic-1717, n=10) and c. baliki (nuic-1816, n=12). table 4. estimates of average k2p genetic divergence over sequence pairs between the studied capoeta species. species no 1 2 3 4 5 6 7 8 c. baliki 1 c. tinca 2 0.37 c. damascina 3 3.63 4.19 c. aydinensis 3 4.29 4.84 3.19 c. caelestis 4 4.11 4.47 1.43 3.93 c. antalyensis 5 1.81 2.02 3.77 4.71 4.04 c. sieboldi 6 4.49 5.07 2.99 4.15 3.58 4.74 c. bergamae 7 4.74 5.31 3.74 2.61 4.39 5.18 4.83 c. banarescu 8 4.30 4.33 3.51 5.05 4.18 3.56 5.40 5.53 38 çiçek et al./ capoeta baliki a junior synonym of capoeta tinca same clade and cannot be considered as distinct species based on phylogenetic species concept (psc). in addition, mean k2p genetic divergence of 0.37% between c. tinca and c. baliki is low for species delimitation criteria suggested by geiger et al. (2014) for freshwater fishes of the mediterranean region and also this genetic distance stands within intraspecific range in the genus capoeta based on the pervious figure 3. bayesian inference of the phylogenetic relationships based on the mitochondrial cytb barcode region (values at nodes correspond to bi posterior probability/ ml bootstrap). 39 int. j. aquat. biol. (2021) 9(1): 33-40 studies (bektaş et al., 2017, 2019; ghanavi et al., 2016; levin, 2012; zareian and esmaeili, 2017; zareian et al., 2018). furthermore, bektaş et al. (2017) reported that the haplotypes of c. baliki (from sakarya river drainage and lake eber) and c. tinca (from susurluk drainage) are closely related. the basins drain to black sea and the sea of marmara are not fully isolate from each other and exchanges routes are still available e.g. via river capture (yıldırım and emre, 2004). therefore, c. tinca and c. baliki have perhaps recently isolated. moreover, even existence of minor molecular and morphological differences between populations of widespread freshwater fish species, is a well-studied phenomenon (marcil et al., 2006). since no morphological diagnostic characters to distinguish c. baliki from c. tinca is available and they are identical in molecular characters i.e. cytb gene, therefore we treat c. baliki as a junior synonym of c. tinca. material examined. all from turkey. capoeta baliki, nuic-1816, 20, 90.8-227.3 mm sl; ankara prov.: derecik stream, sakarya basin, 40°30'44''n 32°19'30''e; 15 may 2018. ⸺ esfmpisi/2004-74 (holotype), 202 mm sl; ankara prov.: sakarya river: kizilcahamam stream, 60 km west of ankara, 40°29'n 32°39'e; 15 april 2004. ⸺ esfmpisi/2004-75, 4, 140-190 mm sl; same data as holotype. capoeta tinca, nuic-1717, 20, 90.6-140.7 mm sl; balıkesir prov.: değirmen stream, susurluk basin, 39°54'50''n 27°33'50''e. zoobank registration: urn:lsid:zoobank.org:pub:72 9c2166-8359-401d-b3ca-102a9cbacb4b acknowledgments the authors would like to thank nevsehir hacı bektas veli and tehran universities for financial supports. we also thank d. turan, c. kaya and e. bayçelebi for helping us to examine the types of c. baliki. references altschul s.f., gish w., miller w., myers e.w., lipman d.j. 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(2016) 4(5): 330-339: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article genotoxic evaluation of the ergene river, turkey, on mosquito fish, gambussia affinis (baird and girard, 1853) using the piscine micronucleus assay martin orlinov kanev*1, kezban ozdemir1, fulya dilek gökalp muranli2 1department of biotechnology and genetic, institute of science, trakya university, edirne, turkey. 2department of biology, faculty of science, trakya university, edirne, turkey. . article history: received 5 may 2016 accepted 11 october 2016 available online 2 5 october 2016 keywords: genotoxicity river in-vivo micronucleus test apoptosis nuclear abnormalities abstract: the ergene river is located in the thrace region of turkey and is polluted by industrial and municipal waste. in the present study, we investigated the genotoxic and cytotoxic effects of water samples on gambussia affinis in vivo using the piscine micronucleus (mn) test. fish were exposed to 50, 100, 150 and 300 ml l-1 of water samples for 24 hrs, and mn, nuclear abnormalities (na), polychromatic-normachromatic erythrocytes (pces/nces), and apoptotic erythrocytes were evaluated. in addition, water samples were analysed to determine the concentrations of the heavy metals. the results showed that mn, na, and apoptosis frequencies significantly increase at all concentrations compared to the control. a significant correlation was found between genotoxicity endpoints and the concentration of water samples. the pce-nce ratio was significantly decreased at all treatments. the metal content of river water was not associated with the increase in the seasonal frequency of genotoxicity endpoints. the results indicated that the ergene river has genotoxic and cytotoxic effects on erythrocytes of g. affinis in an in-vivo piscine mn test that could be due to organic and inorganic effluents. introduction one of the main problems of the industrial areas is pollution of water resources. if the industrial regions are near agricultural areas and cities, industrial effluents, agricultural runoff and urban contaminants are released into the environment and contaminate water sources. hence, these water bodies need to be evaluated for cytotoxic and genotoxic effects of pollutants on aquatic organisms (valko et al., 2005). genotoxic contaminants may interact with dna and induce mutations, chromosomal alterations, birth defects, and cancer in vertebrates (nigro et al., 2002; frenzilli et al., 2004). genetic alteration and mutations may be induced by many contaminants in the aquatic ecosystems (de flora et al., 1993). for monitoring the effects of aquatic ecosystems’ pollution, fish are often suitable models because of their ability to metabolize xenobiotics and bioaccumulate pollutants (al-sabti and metcalfe, *corresponding author: martin orlinov kanev e-mail address: martinkanev@trakya.edu.tr 1995). the observed genotoxic effects in fishes’ tissues are related to industrial, agricultural, and domestic activities (rajaguru et al., 2003; ergene et al., 2007; summak et al., 2010; osman et al., 2011; radic et al., 2013). the piscine micronucleus (mn) test is a common method to assess of the impact of pollution on aquatic ecosystems. mn, nuclear lesions, and binuclei are the most frequent parameters as bioindicators of the genotoxic effects in fish species (osman and kloas, 2010), and the mn test is considered one of the most useful methods for evaluating genotoxicity in aquatic systems (de lemos et al., 2001; andrade et al., 2004; da silva souza and fontanetti, 2006). micronuclei are chromosome fragments or whole chromosomes that lag at cell division due to the lack of a centromere, damage, or a defect in cytokinesis that are formed by both clastogenic and aneugenic compounds (heddle 331 int. j. aquat. biol. (2016) 4(5): 330-339 et al., 1991). binucleated cells, blebbed, lobed, and notched nuclei are different forms of morphological nuclear abnormalities (nas) described in fish erythrocytes (carrasco et al., 1990). these abnormalities are considered to be indicators of the genotoxic damage (ergene et al., 2007). it is important to assess cytotoxic effect of pollutants beside genotoxic effects on living organisms. the cytotoxic effect of chemicals are assessed using the polychromatic erythrocytenormachromatic erythrocyte (pce-nce) ratio, which reflects inhibition of cell division and maturation of nucleated erythropoietic cells (kirkland, 1990). measuring the pce-nce ratio is an approved method in erythrocytes of fishes (kirkland, 1990; randalli and farrell, 1992; cavas, 2008). also, it has been employed to visualize dna degradation due to apoptosis (olive et al., 1993; singh, 2000; osman et al., 2012), which is a form of nuclear destruction in which the nucleus disintegrates and nuclear fragments are formed (lawrence and hemingway, 2003). if the damage produced reaches a high level, it can lead to cell apoptosis (hao et al., 2009; osman et al., 2012). the water of the ergene river, in the thrace region of turkey, is mostly used for agricultural purposes although it runs in an industrial area. pollution of the river ergene continues as a consequence of increasing agricultural, industrial, and domestic effluents. for this reason, continuous monitoring of its water quality is necessary, and therefore, many data have been regularly recorded on the status of this river. however, there are no known data available regarding the genotoxic and cytotoxic effects of pollutants on its aquatic organisms using the in-vivo piscine mn assay or other genotoxicity techniques. therefore, we investigated the cytotoxic and genotoxic effects of the water of the ergene river on mosquito fish, gambussia affinis under laboratory conditions using the piscine mn test. mn, nas, apoptosis, and the pce-nce ratio are determined in erythrocytes after exposure of g. affinis to the ergene river water sampled from two stations. materials and methods study area and water samples: the ergene river is located in the middle of the thrace region of turkey. industrial, agricultural, and domestic regions are located along the river, and consequently a large volume of the industrial, agricultural, and domestic wastes contaminate this river. the ergene river origins from the istranca mountain in tekirdag city. it drians to the saroz gulf and then to the aegean sea after conneting to the meric river. its length is 283 km with a basin of 11,000 km2, and its water potential is 1.71 billion m3 per year (domestic waste water, 230,000 m3/day; industrial waste water, 330,000 m3/day). two stations, including station 1, cerkezkoy, 41°17'00''n, 28°00´00''e and station 2, muratli, 41°10'27''n-27°30'31''e, were selected along the ergene river. both sampling stations were located near the industrial and residential areas. the water samples were collected in september, december, march, and june, 2014. chemical analyses of water samples: water samples were taken manually with a horizontal water bottle and kept in dark containers at low temperature before transporting to the laboratory. measurements were done within one day. for measuring dissolved metals, the samples were filtered through a 0.45 μm membrane filter and acidified with hno3 (ph<2). element quantification was achieved by inductively coupled plasma mass spectrometry (icp-ms) according to epa 2008. average values of three replicates were taken for each measurement. test organisms: gambusia affinis (cyprinodontiformes; poeciliidae) were collected from the balkan campus (41°38'45''n, 26°37'21''e) in edirne, turkey, by a fish trap. the fish were transferred to the laboratory and kept in continuously aerated glass aquaria (100 l) for 2 weeks before the experiment. dissolved oxygen, ph and temperature of the aquarium water were monitored daily. the weight and length of the specimens were 0.14±0.1 (mean±sd) g and 23.9±3.5 (mean±sd) mm, respectively. experimental design: before the experiment, fish 332 kanev et al./ genotoxic evaluation of the ergene river, turkey, on gambussia affinis were kept in an aquarium (100 l) at 20-21°c. fish were then introduced into aquaria containing dechlorinated tap water (as control) and four different concentrations of ergene river’s water samples (50, 100, 150 and 300 ml l-1) for 24 hours exposure periods. five specimens were tested at each treatment with three replicates. analysis of micronuclei, nuclear abnormalities and apoptosis: fish erythrocytes were analysed for micronuclei, nas (notched, lobed, and blebbed nuclei), pce-nce ratio, microand bi-nucleated cells and apoptosis. slides were prepared according to ueda et al. (1992). briefly, peripheral blood samples were obtained from the caudal vein of the specimens and smeared on clean slides. cells were dried overnight, fixed with absolute methanol for 510 min, and stained with acridine orange (0.01 g/100 ml) in sorensen’s phosphate buffer. three slides were prepared from each fish, and 2000 cells were observed from different slides for each fish. the frequency of the abnormalities was determined in erythrocytes under 100x magnification in a fluorescence bx51 microscope according to carrasco et al. (1990). the slides were coded and randomized prior to scoring for mn, na, apoptosis, and pcence ratios. nas were classified according to carrasco et al. (1990). erythrocytes that had a small evagination of the nuclear membrane were classified as blebbed nuclei. erythrocytes with evaginations larger than those of blebbed nuclei and with several lobes were classified as lobed nuclei. nuclei with vacuoles and an appreciable depth into a nucleus that did not contain nuclear material were recorded as notched nuclei. micronuclei were considered as circular, non-refractive, small chromatin bodies showing the same staining pattern as the main nucleus (al-sabti and metcalfe, 1995). micronuclei with one-third or one-fifth’s the diameter of the main nucleus with same colour, refraction, and texture of the main nucleus, were counted as micronuclei. unmature erythrocytes containing ribosomes in their cytoplasm were considered pces. mature erythrocytes without ribosomes in their cytoplasm were considered nces. pces stain differently from nces because of the rna in the cytoplasm that reflects acridine orange with a reddish colour. decreases in the proportion of pce to nce were considered as indicators of induced cytotoxicity (suzuki et al., 1989). pce frequency was calculated based on pacheo and santos (2002) as: pce frequency (%)=[no.pces/(no.pces+nces)]x100. the condensed or fragmented erythrocytes were considered apoptotic cells. statistical analysis: student’s t-test was used to compare differences between two groups. multiple comparisions were performed using one-way analiysis of variance. correlations between total heavy metal content and frequencies of mn, pce/nce, and nas were analysed using linear regression. pearson correlation coefficients were calculated to compare concentration increases with genotoxicity endpoints and the relation amoung genotoxicity endpoints; mn, na, pce/nce, apoptosis. in all cases, p≤0.05 was considered as the accepted significance level. statistic analysis were performed using spss software (version 22). results mean values of genotoxicity and cytotoxicity endpoints at both stations and the significance of comparisions with the negative control (p≤0.01) are shown in table 1. the results showed that mn and nas were significantly increased due to a genotoxic effect. the frequencies of the observed parameters among seasons are not included in table 1 as the endpoints showed no correlation. fish in treatments of the station 1 were alive after 24 hrs exposure period, whereas all fish of the tratments at station 2 died during the spring and summer. the frequencies of genotoxicity endpoints were not significantly different among seasons. the results of the polychromatic and normachromatic cell and apoptotic cells are shown in figure 1. the correlation between concentrations of tested river water samples and mn, na, and apoptosis are shown in figure 2. water samples taken from station 1 had a significant correlation between concentration 333 int. j. aquat. biol. (2016) 4(5): 330-339 and mn (r=0.898*, p=0.038), na (r=0.909*, p=0.032), and apoptosis (0.899*, p=0.038) in g. affinis erythrocytes under laboratory conditions. these significant correlations were also obtained with the water samples of station 2 between concentration and mn (r=0.987**, p=0.002) and apoptosis (r=0.945*, p=0.015). apoptosis was showed a significant correlation with na (r=0.906, group station parameters mn pce/nce total na apoptosis (-) control 0.38±0.09 2.18±0.08 11.36±2.34 24.53±3.36 (+) control 5.46±0.4*** 0.16±0.02*** 26.47±1.38*** 242.01±4.37*** 50 ml/l 1 1.20±0.68 0.65±0.11*** 10.47±2.34 115.92±30.34* 2 0.98±0.26* 0.72±0.08*** 10.58±1.38 79.855±30.19* 100 ml/l 1 3.41±0.87** 0.45±0.09*** 17.73±1.86 173.59±30.12** 2 2.26±0.99* 0.46±0.11*** 25.20±4.08* 141.07±45.27* 150 ml/l 1 4.66±0.61*** 0.30±0.09*** 19.74±2.09* 216.80±30.26*** 2 3.64±6.06*** 0.30±0.08*** 19.61±1.44 183.97±36.37** 300 ml/l 1 5.32±0.51*** 0.25±0.03*** 23.67±0.86** 259.34±15.04*** 2 5.56±1.04** 0.18±0.07*** 24.83±0.33* 251.42±38.22*** values marked with *, ** and *** represent p values of p≤0.05, p≤0.01 and p≤0.001, respectively, in relation to the negative control using t-test. values presented as mean±se; mn: micronucleus; pce/nce: polychromatic erythrocyte/normachromatic erythrocyte; na: nuclear abnormalities (notched, lobed and budding nuclei); cyclophosphamide (5 mg l-1): positive control. table 1. frequencies of mn, pce/nce, total na and apoptosis after exposure to 50,100,150 and 300 ml l-1 concentrations of the ergene river water in erythrocytes of gambusia affinis. figure 1. apoptotic cells, polychromatic and normachromatic erythrocytes of gambusia affinis stained with acridine orange (black arrow: normal erythrocyte; datted arrow: apoptotic erythrocyte; white arrow: polychromatic erythrocyte; arrowhead: normachromatic erythrocyte). 334 kanev et al./ genotoxic evaluation of the ergene river, turkey, on gambussia affinis p=0.034), mn (r=0.97, p=0.006), and pce/nce (r=-0.89, p=0.043) for samples from station 1 and mn (r=0.972, p=0.006) and pce/nce (r=-0.874, p=0.05) for samples of station 2. the results of the metal and non-metal analysis of water samples in both stations are shown in table 2. the concentration of metals was not significantly different between stations except cr (p=0.03) and rb (p=0.03). table 2 also shows the limit values of pollutants established by the turkish water pollution control regulation (2004). the physical and chemical properties of the water samples were measured parameters station 1 station 2 limit values ph 7.9 8.1 6.5-8.5 temperature (°c) 22 24 25 al (µg 1-1) 204.583 347.474 300 cr (µg l-1) 10.034 15.330 20 mn (µg l-1) 210.662 304.774 100 fe (µg l-1) 187.383 232.163 300 co (µg l-1) 1.539 1.656 10 ni (µg l-1) 8.991 13.531 20 cu (µg l-1) 25.707 30.857 1000 zn (µg l-1) 57.168 28.272 200 as (µg l-1) 14.502 18.486 20 cd (µg l-1) 0.804 1.136 3 ba (µg l-1) 55.854 74.861 1000 pb (µg l-1) 3.192 2.248 10 table 2. physical and chemical properties of mean values of water samples collected at different seasons from the ergene river and their limit values (class i) according to turkish water pollution control regulation (2008). figure 2. frequencies of mn, na, pce/nce, and apoptosis after exposure of the ergene river water on gambussia affinis. 335 int. j. aquat. biol. (2016) 4(5): 330-339 under or near class i limit values according to the turkish water pollution control regulation (2004) (ministry of environmental and urban planning (meup), 2004). there were no correlations between stations for metal concentration and abnormalities. pearson’s correlation showed no significant correlation concentrations and na frequencies or mn. discussion the results showed a higher incidence of mn and nas in erythrocytes of g. affinis exposed to different concentrations of the ergene river water under laboratory conditions. similar results were obtained in environmental toxicology studies with such frequencies being significantly elevated with an increasing pollution gradient (osman et al., 2011). the researchers observed that micronuclei are induced in the fishes inhabiting polluted sites compared to those of clean river systems (al-sabti and hardig, 1990; pietripiana et al., 2002; bagdonas et al., 2003; rodriguez-cea et al., 2003; bombail et al., 2001; cavas and ergene-gozukara, 2005). it is known that mn formation as well as the induction of na are considered to be the consequence of genotoxic events in fish (metcalfe, 1988; pacheo and santos, 2002). mn and na frequencies observed in the present study indicate that the ergene river water has a potential genotoxic effect on its fish fauna. the ergene river basin is located near an industrial region including textile chemistry, domestic metal and mine industries and factory effluents and may contain metals, such as cu, cr, ni, cd, pb and zn (chino et al., 1991; aonghusa and gray, 2002; cavas and ergene-gozukara, 2003; manzoor et al., 2006). it is known that metals found in polluted water have a potential genotoxic effect on aquatic organisms (hei and filipic, 2004; barbosa et al., 2010; summak et al., 2010). toxic metals and their interactions with other compounds present in effluents may have genotoxic effects on living organisms, and metals in a mixture may have additive chronic toxicity compared to their individual effects (enserink et al., 2003). guner and muranli (2011) showed that nuclear abnormalities were significantly induced in erythrocytes of g. affinis when fish were exposed to cu and cd in combination. in the present study, there was no significant correlation between metal concentrations in water samples and mn or na frequencies. as shown by the metal analyses of water for both stations 1 and 2 (class i quality water, according to turkish water pollution control regulation (2004)), metal concentrations were found under limit concentrations meup, 2004). in addition, we found no correlation between metal concentration and mn, pce/nce, or nas. the results are in agreement with ergene et al. (2007) that found no significant correlations between metal concentrations and genotoxicity endpoints in erythrocytes of nile tilapia (oreochromis niloticus) exposed to berdan river water for different time periods (ergene et al., 2007). their result also revealed that mn and na frequencies were significantly increased, although the chemicals properties of the berdan river within the limit values according to the turkish water pollution control regulation (ergene et al., 2007). an increase of mn, pce/nce, and nas may indicate the presence of large quantities of organic and inorganic pollutants in river water due to industrial, domestic and agricultural runoff (osman and kloas, 2010; summak et al., 2010). it is known that genetic aberrations cannot be attributed to a single agent due to the constitution of a complex mixture (donbak et al., 2005). municipal effluents are recognized as a major source of many environmental contaminants, including polyaromatic hydrocarbons, pesticides, steroids, and metals (gagne et al., 2006). white and rasmussen (1998) noted that despite the noteworthy genotoxicity of some industrial wastewater, domestic wastewater constitutes a greater genotoxic hazard to aquatic systems and their associated biota (white and rasmussen, 1998). an ecological risk assessment of polluted waters is difficult due to antagonistic or synergistic effects of a mixture of contaminants. our 336 kanev et al./ genotoxic evaluation of the ergene river, turkey, on gambussia affinis results are in accordance with the other investigations that mn and na frequencies increased as the river water concentrations increased; this may have been due to other genotoxic chemicals and organic pollutants present in the ergene river water. the results showed a significant negative correlation between pce/nce ratio and apoptotic cell frequency; the pce-nce ratio significantly decreased at all treatments. homeostasis is provided by erythropoiesis and destruction of erythrocytes. new erythrocytes are continuously entering the circulatory system, and defeat erythrocytes are destroyed at the same rate (randalli and farrell, 1992). assessment of the pce-nce ratio can provide evidence of exposure to toxic substances. it is known that pces are immature erythrocytes that have ribosomes in the cytoplasm, and that nces are mature erythrocytes that lack ribosomes in the cytoplasm (kirkland, 1990). inhibition of maturation of nucleated erythropoietic cells causes a decrease in the pce-nce ratio. reductions in the proportion of pce/nce are considered to be indicators of mutagen-induced cytotoxicity (suzuki et al., 1989). the results of the present study indicated that ergene river water has cytotoxic effects due to a significant reduction in the pce-nce ratio, and this reduction is strongly correlated with the induction of apoptosis. although water quality monitoring studies involves measurements of physical-chemical parameters, biological monitoring has become essential as it reveals the harmful effects of noxious chemicals and can indicate risk to the environment and human health (ternjej et al., 2013). in the present study, we did not perform detailed chemical analyses of the ergene river water, since the ministry of environmental and urban planning (meup) investigates the ergene river on a regular basis. based on the criteria of dissolved oxygen, chemical oxygen demand, biological oxygen demand, sulphate, chlorine, sulphur, and sodium, class iv water quality for this river is considered (meup, 2014). a high concentration of sulphate as reported for this river, strongly interferes with the biogeochemical cycling of iron and phosphorus and can lead to eutrophication, which further enhances toxicity (lamers et al., 2002; geurts et al., 2009). it is known that sulphates present an ecotoxicological risk (mihaljevic et al., 2011; stankovic et al., 2011). in addition to the potential mutational effect related to the complex mixtures, there are other possible problems for resident organisms that could produce alarming indices of organic wastes and result in the death of various fish species (batzias and siontorou, 2006). in the present study, death of fish after exposure to river water taken during spring and summer may be due to low concentrations of chemical and biological oxygen demand, sulphur, and sulphate concentrations that has been indicated in class iv water quality (meup, 2014). in the present study, the polynucleated erythrocytes of g. affinis were the most frequent abnormalities with exposure to higher concentrations of riverine water. polynucleated cells were found to cause apoptosis of the cells due to dna degradation. apoptosis is a form of nuclear destruction in which the nucleus disintegrates and nuclear fragments are formed; apoptosis occurs both naturally and in response to chemically-induced cellular damage (lawrence and hemingway, 2003). the results also showed a positive correlation between apoptosis and concentration and between apoptosis and the other genotoxicity endpoints i.e. mn and na. the apoptotic effect of water pollution on fish erythrocytes has been previously investigated (omar et al., 2012; osman et al., 2012; walia et al., 2013). metals, such as iron, copper, and zinc lead to dna fragmentation (razzaque, 2007), and fragmentation patterns may involve more than one mechanism leading to cell death and inducing apoptosis and/or necrotic cell death (razzaque, 2007; omar et al., 2012). similarly, in the present study the high incidence of mn, na, and polynucleated cells that could cause cell death showed that the ergene river has a genotoxic effect, leading to cytotoxicity and apoptosis. the effects of chemical interactions and the influence of complex matrices on toxicity cannot be 337 int. j. aquat. biol. 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(2021) 9(6): 350-359 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article effect of garlic extract supplementation on growth performance, nonspecific immunity, and antibacterial activity of skin mucus in goldfish, carassius auratus hamidreza ahmadniaye motlagh*1, omid safari1, sajjad pourmozaffar⁕2 1department of fisheries, faculty of natural resources and environment, ferdowsi university of mashhad, mashhad, iran. 2persian gulf mollusks research station, persian gulf and oman sea ecology research center, iranian fisheries sciences research institute, agricultural research education and extension organization (areeo), bandar-e-lengeh, iran. s article history: received 24 august 2021 accepted 5 october 2021 available online 2 5 december 2021 keywords: antimicrobial activity garlic extract growth immune parameters skin mucus abstract: in the present study, the effects of dietary supplementation of garlic extract on growth performance, skin mucus immunological parameters and antibacterial activity of carassius auratus were examined. fish were stocked in 100 l glass tanks (6 fish per tank) in triplicate and fed diets containing different garlic extracts (0 (control), 5, 10, and 15 ml/kg] for eight weeks. at the end of feeding period, the fish skin mucus was collected for evaluating the components of non-specific immune system (including lysozyme, complement, total immunoglobulin, dissolved protein, and alkaline phosphatase). additionally, antimicrobial activity of the skin mucus against aeromonas hydrophila, yersinia ruckeri, micrococcus luteus, streptococcus faecium, and s. iniae was assessed. after the feeding trial, the fish fed diets containing garlic extract showed no significant difference in growth parameters. significantly higher skin mucus lysozyme, complement, alkaline phosphatase activities, and total immunoglobulin and dissolved protein concentration were observed in the fish fed garlic extract-supplemented diets (p<0.0001). the antimicrobial activity of the skin mucus increased along with the increase in the dietary garlic extract levels (p<0.0001). moreover, garlic extract exhibited the antimicrobial activity against pathogenic bacterial species. the highest level of dietary garlic extract (15 ml/kg) led to significantly higher inhibition zones against pathogenic bacterial species compared to the other garlic extract levels (p<0.0001). the optimal administration of garlic extract at 15 ml/kg enhance skin mucus immune parameters and antimicrobial activity in goldfish. introduction diseases are a notable restriction to the productivity of the aquaculture industry, imposing substantial losses. antibiotics and chemical disinfectants have been routinely used to control diseases in aquatic animals (li et al., 2019). nowadays, their application has been limited or banned in many countries due to bacterial resistance and persistence in the fish tissues and aquatic environment (fierro-coronado et al., 2018). moreover, antibiotics have negative effects on the host animal, including oxidative stress and immunosuppression, making the animal susceptible to non-bacterial infections (hoseini and yousefi, 2019; zargar et al., 2020). natural products such as prebiotics (chen et al., 2016), probiotics (daniels et al., 2013), medicinal plants (harikrishnan, et al., *correspondence: hamidreza ahmadniaye motlagh, sajjad pourmozaffar doi: https://doi.org/10.22034/ijab.v9i6.1306 e-mail: ahmadnia@um.ac.ir, sajjad5550@gmail.com 2011), phytochemicals (hoseini et al., 2018) seaweeds (tamadoni jahromi et al., 2021), and acidifiers (pourmozaffar et al., 2019) have been identified as promising alternatives to chemical drugs. in recent years, the use of medicinal plants in the aquaculture industry has increased and these herbal materials has been found to improve the immune system (yin et al., 2014), antioxidant system (hoseini et al., 2021), stress response (paray et al., 2020; yousefi et al., 2020a), growth performance (yu et al., 2009), gut function (ahmadniaye motlagh et al., 2019), and resistance to pathogens (yogeeswaran et al., 2012) of fish and crustaceans. garlic (allium sativum) is used as alternative medicine for various illnesses in many different cultures (tanekhy and fall, 2015). it contains many 351 int. j. aquat. biol. (2021) 9(6): 350-359 valuable compounds such as vitamins (a, c, and b complex), iodine salts, linoleic acid (labrador et al., 2016), organosulfur compounds (ajoene, allicin, diallyl disulfide, s-methylcysteine, s-allylcysteine, smethylcysteine sulfoxide, and s-allylcysteine) (lee et al., 2014) and flavonoid (sharma et al., 2010). allicin is the main biochemically and biologically active component of freshly crushed garlic (guo et al., 2012). it improves protection against pathogenic agents through stimulation of immunological functions including antibody responses, lysozyme and complement activities, and phagocytic activity (breyer et al., 2015). for example, the non-specific immune system of nile tilapia (oreochromis niloticus) has been improved, when garlic was incorporated into the diet (aly and mohamed, 2010). nya et al. (2010) have reported that the addition of allicin to rainbow trout (oncorhynchus mykiss diets enhances the red blood cells, serum lysozyme activity, phagocytic activity, and biochemical parameters such as total protein and globulin ratio. in another study, rainbow trout fed diets containing commercial garlic extract has shown a significant reduced mortality after challenge with aeromonas hydrophila (nya and austin 2009). several investigations have demonstrated the positive effects of dietary supplementation of garlic on the growth performance of aquatic animals such as caspian roach (rutilus caspicus) (ghehdarijani et al., 2016), beluga (huso huso) (gholipour kanani et al., 2014), grouper (epinephelus coioides) (guo et al., 2012), common carp (cyprinus carpio) (yousefi et al., 2020b), caspian trout (salmo caspius) (zaefarian et al., 2017), and asian sea bass (lates calcarifer) (talpur and ikhwanuddin, 2012). the skin mucus in fish contains many innate immune components such as lysozyme, alkaline phosphatase, lectins, proteolytic enzymes, and proteases, which play important roles against pathogens (wang et al., 2020). immune components in skin mucus are modulated by a number of abiotic and biotic factors (lallès, 2019). according to earlier reports, diets enriched with myo-inositol (wang et al., 2020), myrtus communis (mansouri taee et al., 2017), aloysia citrodora (hoseinifar et al., 2020), and zingiber officinale (sukumaran et al., 2016) were found to improve skin mucus immunological parameters in various fish species. however, little information is available concerning the effects of diet supplementations with herbal additives on the skin mucus immunological parameters in goldfish, carassius auratus. mimeault et al. (2005) reported that goldfish can be used as a model in nutritional and physiological studies. due to the high stocking density and stressful conditions in rearing and transportation systems, goldfish will be susceptible to the invasion of opportunistic pathogens such as a. hydrophila (harikrishnan et al., 2009) and yersinia ruckeri (wang et al., 2020). therefore, the present study was designed to investigate the effect of different levels of dietary garlic extracts on growth performance, skin mucus immunological parameters and antibacterial activity of goldfish. materials and methods fish: goldfish with average weight (17.12±2.44 g) were obtained from a commercial fish farm (topazland, mashhad, iran) and acclimated for 14 days into twelve 100 l glass tanks (6 fish/tank). during the acclimation period, the fish were fed a commercial feed (energy® with a proximate composition of 40.92±1.37 crude protein, 3.72±0.70 crude lipid, 2.72±0.59 crude ash, 3.15±0.95 crude fiber, and 19.23±1.12 nitrogen free extract) three times a day at 8, 12, and 16, at a rate of 2.5% of their body weight. the water was exchanged daily at the rate of 30% for removing the uneaten food and feces. the water was maintained at a temperature of 29±2.5οc, ph of 7.30±0.6, and dissolved oxygen of 7.90±0.14 ppm. all experiments were performed according to the ethical standards of working with laboratory animals issued by ferdowsi university of mashhad (number d/700/1028). preparation of garlic extract: fresh garlics have been obtained from a local farm in birjand, iran. the garlic was extracted based on ahmadniaye motlagh et al. (2020). one hundred grams of fresh garlic pieces 352 ahmadniaye motlagh et al./ effect of garlic extract on goldfish were added to 200 ml of distilled water and blended for 3 min. the homogenate was centrifuged at 10,000 rpm for 5 min. the supernatant was removed and then the extract was filtered with whatman filter paper. the prepared extract was stored at -20οc until being used. experimental design: goldfish (n= 72) were divided into four experimental groups in triplicate. four experimental diets were prepared by supplementing the commercial diet with different levels (5, 10 and 15 ml/kg) of garlic extract. garlic extract was sprayed over a basal diet. the gelatin powder (4 g/l) was used as a binder. finally, the diets were dried for 3 h at room temperature. the fish were fed the experimental diets at 2.5% of whole body weight twice daily for 57 days. growth performance: after the feeding trial, all fish were fasted for 24 h and then the growth parameters were calculated according to the following equations: weight gain = w1–w0 specific growth rate (sgr) = [(ln w1-ln w0) ÷ (t)] ×100 feed conversion ratio (fcr) = [(fi) ÷ (w1w0)] where, w0 is initial weight; w1 = final weight, t = experimental days and fi = feed intake. skin mucus biochemical analyses: at the end of the experiment, the fish were fasted for 24 h and three fish from each aquarium were randomly chosen. the method of skin mucus collection was performed according to earlier study (mansouri taee et al., 2017). fish (3 fish per tank) were anesthetized with clove oil (0.2 ml/l). individual fish were transferred into polyethylene bags containing 10 ml of nacl (50 mmol) and shaken gently to extract the skin mucus in the nacl buffer. after 2 min extraction, the mucus samples were transferred to sterile tubes and centrifuged at 1500 g for 10 min at 4°c. finally, the supernatant was stored at −80°c. protein concentrations were measured according to lowry et al. (1951). alkaline phosphatase (alp) was assayed by detection kits (co, pars azmon kit, iran) (tamadoni jahromi et al., 2020). each sample (0.1 ml) was mixed with 12% polyethylene glycol solution for assessment of total immunoglobulin (ig). ig molecules were precipitated down by centrifugation and total protein levels were re-determined. the difference in protein content was considered the total ig content of skin mucus (hoseinifar et al., 2016). lysozyme activity was determined according to the method described by zheng et al. (2009). briefly, 100 μl of mucus sample was added to a 3 ml suspension of micrococcus lysodeikticus (sigma-aldrich, germany) was prepared in 0.05 m sodium phosphate buffer (ph= 6.2). the reaction was carried out at 25±1°c, and absorbance at 540 nm was measured after 0.5 and 4.5 min. alternative hemolytic complement of skin mucus was determined using rabbit red blood cells. rabbit's red blood cells were added to mucus samples and the solution was incubated for 90 min at room temperature. 3.15 ml of nacl solution (0.85%) was added to the samples and the tubes were centrifuged for 10 min at 1600 rpm. the absorbance of the supernatants was measured at 412 nm. the values of maximum (100%) and minimum haemolysis were obtained by adding 100 µl of distilled water or buffer to 100 µl samples of rrbc, respectively. the volume of serum yielding 50% hemolysis was used to determine complement activity. the volume of the mucus, which causes 50% hemolysis, is the complement activity (stolen et al., 1994). skin mucus antibacterial activity: the skin mucus antibacterial activity was assessed with the disc diffusion method. the following bacterial strains were used as substrates: gram-negative bacteria, a. hydrophila atcc 7966 and y. ruckeri ptcc 1888. gram-positive bacteria, micrococcus luteus ptcc 1169, streptococcus faecium atcc 19434, and streptococcus iniae ptcc 1887 (magarinos et al., 1995). the bacterial species were grown in nutrient broth medium for 24 h at 37˚c, and then 0.1 ml of each broth culture medium (contain 1×105 cfu ml/1; od 600) was cultured on nutrient agar. 6 mm diameter paper discs (four discs per plate) were impregnated with 150 µl of the mucus samples. the agar plates were incubated at 37°c for 24 h. the antibacterial activity of mucus was defined as the diameter of the clear inhibitory zone (mm) formed around the paper discs (ahmadniaye motlagh et al., 2019). 353 int. j. aquat. biol. (2021) 9(6): 350-359 statistical analysis: the data were expressed as average ± standard error. statistical analysis involved one-way analysis of variance (anova) followed by a post hoc least significant difference (lsd) test. normality was tested using the kolmogorov–smirnov test. data homoscedasticity was checked by bartlett's test. a statistically significant difference was required at p<0.05. statistical analyses were conducted using spss software version 17.0 (spss inc., chicago il, usa). results the growth performance of goldfish fed diets containing different levels of garlic extract are presented in table 1. at the end of 57-day experimental periods, the final weight, weight gain, sgr, and fcr did not significantly differ among the treatments. non-specific immune parameters of goldfish fed dietary garlic extracts are shown in figure 1a-e. alp activity was significantly higher in the garlic extracttreated fish than the control fish (fig 1a) (p<0.0001). significantly lower lysozyme activity in goldfish mucus was observed in the control group than that in the others (fig. 1b) (p<0.0001). complement activity in the skin mucus increased significantly by 65, 75, and 100% for fish fed with 5, 10, and 15 ml/kg garlic extract supplemented diets, respectively (fig. 1c) table 1. growth performance of goldfish (mean ± standard deviation) after 56 days feeding with diets containing 0-15 ml/kg garlic extract (n = 3). parameters control 5 ml/kg 10 ml/kg 15 ml/kg p-value initial weight (g) final weight (g) weight gain (g) sgr fcr 17.10±0.11 31.56±0.84 14.46±0.86 1.07±0.05 1.83±0.09 17.09±0.11 30.22±0.02 13.13±0.08 1±0.01 1.96±.011 17.18±0.16 31.15±0.22 13.98±0.07 1.04±0.00 1.91±0.00 17.13±0.21 30.94±1.32 13.81±1.39 1.03±0.08 1.93±0.25 p = 0.89 p = 0.29 p = 0.33 p = 0.38 p = 0.65 figure 1. non-specific immune parameters of goldfish skin mucus following 56 days feeding with diets supplemented with 0-1.5 ml/kg garlic extract. a: alkaline phosphatase; b: lysozyme; c: complement; d: dissolved protein; e: total ig. values are presented as the mean±sd (n=9 in each group). different lowercase letters above the bars indicate significant differences among different experimental groups (p<0.05). 354 ahmadniaye motlagh et al./ effect of garlic extract on goldfish (p<0.0001). the total ig was significantly increased when garlic extract was supplemented in the diet (fig. 1d) (p<0.0001). fish fed diets containing garlic extract showed significantly higher dissolved protein concentration than those fed with the control diet (fig. 1e) (p<0.0001). experimental groups exhibited a different degree of inhibition against bacterial species (fig. 2a-e). anti-bacterial activity of the fish skin mucus significantly increased along with dietary garlic extract elevation. fish fed with diet supplemented with 15 ml/kg garlic extract exhibited the highest antimicrobial activity against a. hydrophila (1.57±0.02 cm) (fig. 2a), s. iniaee (1.16±0.04 cm) (fig. 2b), s. faecium (0.88±0.01 cm) (fig. 2c), y. ruckerii (0.89±0.02) (fig. 2e) and m. luteus (0.32±0.006 cm) (fig. 2d) (p<0.0001). discussions based on the present results, the growth parameters of goldfish were not affected by the supplementation of garlic extract. similarly, mahmoud et al. (2019) found that nile tilapia fed a diet containing 15 g/kg garlic powder for 60 days showed similar final weight. similarly, labrador et al. (2016) reported that diets enriched with garlic powder at 5, 10, and 15 g/kg did not have any significant effect on the growth performance of white leg shrimp, peneaus vannamei. in another study, diets containing garlic extract at up to 30 g/kg inclusion had no notable effect on the final weight of caspian trout (zaefarian et al., 2017). growth-boosting effects of herbal supplements are mediated by increased digestive enzymes’ activity and/or improved nutrient absorption and utilization; therefore, garlic extract may have no such effects in goldfish (hoseini et al., 2021) under the conditions of this experiment. fish mucus indicated valuable information for scientists to assess the fish health status (talpur and ikhwanuddin, 2012). the results showed the potentials of garlic extract in enhancing the skin mucus immune parameters alp acts as antimicrobial enzyme against water pathogens (lallès, 2019). in the present study, alp activity was significantly higher in figure 2. antimicrobial activities of skin mucus of carassius auratus fed diets containing different levels of garlic extract for 57 days (a-e). values are presented as the mean±sd (n=9 in each group). different lowercase letters indicate statistically significant differences between different experimental groups (p<0.05). 355 int. j. aquat. biol. (2021) 9(6): 350-359 the garlic fed groups. ghehdarijani et al. (2016) found that caspian roach fed with 5 and 10 g/kg garlic showed a significant increase in the skin mucus alp. change in skin mucus alp due to the inclusion of plant components has been previously reported. diets enriched with m. communis (0-15 g/kg), z. officinal (0-10 g/kg), and phoenix dactylifera water-soluble extracts (0-200 ml/kg) enhanced the skin mucus alp activity in rainbow trout, ruho carp (labeo rohita), and common carp, respectively (hoseinifar et al., 2015; sukumaran et al., 2016; mansouri taee et al., 2017). the enhancement of alp in goldfish might be due to the increased mucus secretion and mucosal immune response (wang et al., 2020). the goldfish fed diets enriched with garlic extract showed increased dissolved protein level, lysozyme, and complement activities in the skin mucus. ahmadniaye motlagh et al. (2019) found similar results in poecilia reticulate fed with different levels of a. sativum. rainbow trout and ruho carp fed diets supplemented with different levels of garlic powder showed increased lysozyme activity and total protein in the plasma (adineh et al., 2020; sahu et al., 2007). furthermore, diets enriched with garlic at 1-20 g/kg showed significantly enhanced plasma protein concentration in asian sea bass, caspian roach, and rainbow trout (nya and austin, 2009; talpur and ikhwanuddin, 2012 ghehdarijani et al., 2016). lectin is the most abundant protein in garlic, which involved in activation of the mannose-binding lectin. this protein is considered to bind to bacterial cells, trigger the complement cascade, and subsequently phagocytosis by macrophages (nya and austin, 2009). igs play a vital role in the adaptive immune system through the production of antibodies against various antigens (hoseinifar et al., 2020). in the present study, we found that dietary garlic extract significantly enhanced the total ig level, which is in agreement with ahmadniaye motlagh et al. (2019) who reported increase in total ig levels in p. reticulate fed diets containing garlic extract for 80 days. meanwhile, the highest serum complement activity and skin mucus total ig were observed in common carp fed 0.5 and 1.5 g/kg artemisia annua extract, respectively (sarhadi et al., 2020). it seems that sulfur compounds (such as allicin and s-allyl cysteine) in garlic contribute to activating the immune responses (ndong and fall, 2011). in addition, garlic can modulate cytokine production and activate immune response by stimulating antibody secretion and immune cells (arreola et al., 2015). in the present study, antibacterial activity against a. hydrophila, s. iniaee, s. faecium, y. ruckerii, and m. luteus was observed in goldfish fed garlic extract. wang et al. (2020) found similar results when different levels of myo-inositol were added to the diet of hucho taimen fry. additionally, the diets containing fermented saccharomyces cerevisiae exhibited the strongest antimicrobial activity of rainbow trout skin mucus against y. ruckeri (sheikhzadeh et al., 2012). antibacterial activity of garlic against vibrio parahemolyticus, v. harveyi and a. hydrophila was also reported by vuddhakul et al. (2007), vaseeharan et al. (2011) and natasya-ain et al. (2018), respectively. allicin is responsible for antimicrobial activity of garlic against pathogenic bacteria. when the garlic is crushed or cut, alliinase (c–s lyase enzymatic system) converts alliin (s (+)allyll-cysteine sulphoxide) into allicin (vaseeharan et al., 2011). in addition, allicin may react with sulfhydryl groups of the bacterial proteins. the ability of allicin to inhibit bacterial sulfhydryl enzymes such as urease, papain, amylase, and alcohol dehydrogenase reported by bhatwalkar et al. (2021). the smallest inhibition was observed against m. luteus while the largest was against a. hydrophila. ushimaru et al. (2007) reported that the efficacy of garlic against gram-positive bacteria is less than that of gramnegative bacteria. the enhancement of defense components with bactericidal properties such as lysozyme, complement, ig, and lectin could improve the skin mucus antibacterial activity against pathogens (wang et al., 2020). in conclusion, the present study demonstrates positive effects of garlic extract on the skin mucus immune parameters. dietary garlic supplementation at a dose of 15 ml/kg exhibits antibacterial properties against pathogenic bacteria. 356 ahmadniaye motlagh et al./ effect of garlic extract on goldfish references adineh h., harsij m., jafaryan h., asadi m. 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(2019) 7(1): 1-8 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article temporal and spatial phytoplankton biomass dynamics in southern gulf of lake tana, northwestern ethiopia dilnessa gashaye*1, goraw goshu2, berhanu abraha1 1biology department, bahir dar university, bahir dar, ethiopia. 2fisheries and aquatic sciences department, bahir dar university, bahir dar, ethiopia. s article history: received 29 august 2018 accepted 22 february 2019 available online 2 5 february 2019 keywords: chlorophyll-a cluster analysis human impacts sustainable use abstract: southern gulf of lake tana is southern part of lake tana in northwestern ethiopia. water samples were obtained from eight sites of the gulf for six months during november to april 2010/11 twice a month to determine temporal and spatial variation in phytoplankton biomass of the gulf. phytoplankton biomass data was estimated following total chlorophyll-a concentrations determination methods. there was no significant difference in sampling sites and interactions between sampling sites and months (p>0.05). however, there is a significant difference in sampling months (p<0.05). the absence of significant difference in sampling sites and the interactions might be due to the similarity in human or natural impacts, and phytoplankton growth and decline. but, the presence of significant difference among sampling months might be due to the difference in growth/decline periods of the phytoplankton. southern gulf of lake tana subparts are similarly impacted naturally or by humans who passed their time around the gulf. the ethiopian government and concerned non-governmental organizations in general and local communities, in particular, have better cooperate their efforts for conservation and sustainable use of the gulf. similarly, they have to play in awareness creation for stakeholders to reduce natural/human impacts to the lake gulf. introduction a common symptom of increasing nutrient enrichment (eutrophication) is an increase in phytoplankton biomass (nixon and buckley, 2002). there is a long history of the application of total chlorophyll-a as an index of the productivity and trophic condition of estuaries, coastal and oceanic waters (boyer et al., 2009). it is also very important parameters to understand the structure and tropic level of aquatic systems, the quality of water and environments (fakioglu, 2013) and for forecasting the extent of global change impact on aquatic ecosystem functioning (winder and cloern, 2017). phytoplankton biomass is affected by different biological, physical and chemical factors such as the grazing effect of zooplankton and benthic filterfeeding animals (levine et al., 1999), light conditions (berger et al., 2006), flow velocity (feipeng et al., 2013), temperature and nutrients (lin-lin et al., 2012; cao et al., 2016). according to goericke (2002), *correspondence author: dilnessa gashaye doi: https://doi.org/10.22034/ijab.v7i1.488 e-mail: dilugm.gashaye@gmail.com planktonic populations are interacted by both resource limitation (bottom up) and predation (top-down). lake tana, which is the largest lake in ethiopia (barker, 2004; gordon et al., 2007), is surrounded by agricultural lands (erkie, 2016) and fed from many small seasonal to large permanent rivers to the lake (nyssen et al., 2015). it is recently losing its natural conditions (vijverberg et al., 2009). however, significant inflow comes from three major rivers in the south, namely the gilgel abai, rib, and gumara, which carry a large amount of silt resulting from severe erosion, thereby increasing the turbidity of the water in the bahir dar gulf (berhanu et al., 2001). assessing phytoplankton biomass, could be beneficial for environmental managers and other stakeholders to understand the lake conditions and hereby alleviate human and natural hazards to the lake. the objective of this research was to assess spatial and temporal phytoplankton biomass variability in southern gulf of lake tana, 2 dilnessa et al./ temporal and spatial phytoplankton biomass lake tana, ethiopia southwestern ethiopia. materials and methods lake tana is located between 10°58`00''–12°47'00''n and 36°45'00''-38°14'00''e (dagnew et al., 2014) on the basaltic plateau of the north-western highlands of ethiopia at an altitude of 1,830 m above sea level (vijverberg et al., 2009). it is characterized by low nutrient concentrations, high silt and a low primary production (ayalew et al., 2007). the lake is the largest and third largest in ethiopia and the nile basin (abeyou, 2008), respectively. it is also the source of the blue nile river, which is the longest river in the world (vijverberg et al., 2009). morphometric and physicochemical characteristics of the lake are shown in table 1. climate around the study area is unimodal pattern or "seasonal rains" characterized as rainy in summer (june-august) and dry in the winter (january-march). the absolute, mean minimum and maximum temperature in an area, where is nearby to southern gulf of lake tana, are 5.9, 12 and 31°c during the study year. similarly, the rainfall pattern ranges from 0 ml (decebber, february and march) to 417.2 ml (august) (fig. 1). fieldworks and site selection: sites were selected based on the presence/absence of human-induced pressures along the southern gulf of lake tana. site 1 (debre mariam area) is characterized by impacts from agriculture. site 2 (west gojjam prison station) is characterized by human wastes. site 3 (shum abo recration) is characterized by transportation and recreation around the site. site 4 (titu recreation center and port for tana transport organization) is characterized by both transportation and hotel construction on the shoreline of the lake. site 5 (bahir dar city, legislative area) is characterized by swimming, washing clothes and bathing. site 6, sites 7 and 8 were are characterized by hotel construction (the blue nile resort relatively undisturbed and was control site for the data analysis), wastes from hospitals (flege hiwot referral hospital) and runoff from agricultural lands (tana medhanie alem integrated development association), respectively. sampling procedures and programs: sampling of table 1. morphometric and physicochemical characteristics of lake tana. morphometric characteristics physicochemical characteristics maximum depth (m) 14 lake temperature (°c) 22.8±1.19 mean depth (m) 8 conductivity (µscm-l) 84-230 lake area (km2) 3156 total alkalinity (mg/l) 85-159 catchment area (km2) 16500 na+(mg/l) 7-9 lake volume (km3) 28.4 k+ (mg/l) 1 water residence time (year) 3 ca++ (mg/l) 14 evaporation loss (mm) 1248 mg++ (mg/l) 12 altitude (m) 1830 hco-3 +co 3 (mg/l) 91 latitude (0n) 10058’-12047’ cl(mg/l) 1.25 longitude (0n) 36048-38014’ tds (mg/l) 50-138 runoff coefficient (k) 0.22 δ d 34.6 ‰ rainfall (mm/year) 1500 turbidity (ntu) 20-29 max. length (km) 78 no-3 (mg/l) 0.2 to 2 max. width (km) 67 po-4 (mg/l) 0.05-2.82 annual precipitation (mm) 1375 dissolved oxygen (mg/l) 3.5-8.5 major water use hydropower, navigation bod (mg/l) 3.2-23.7 kebede et al. (2006), shimelis et al. (2008) and dagnew et al. (2014). figure 1. climate diagram of the study area. 3 int. j. aquat. biol. (2019) 7(1): 1-8 water was done using mesh net (pore size 55 µm) starting from site 1 to site 8 and adding sampled water into 8 pure sample bottles, for phytoplankton biomass determination in the morning from 5 am to 7 am. water samples were put into a sampling bottle filled with ice to prevent chlorophyll degradation due to light and temperature during transportations to the laboratory. the samples were transported to the laboratory for analysis within a maximum of two hours after collection. sampling was done every two weeks in eight sites for six months from november 2010 to april 2011. a total of 96 (8 sites*2 times*6 months) samples of water were analyzed for phytoplankton biomass following chlorophyll-a concentration determination methods as proposed by lorenzen (1966). phytoplankton biomass determination: the water samples in the botany laboratory at bahir dar university were filtered through millipore microfilters (47 mm diameter; 45 μm pores) using a vacuum pump (ts8002). the filtrate was fourfold and placed in the tissue grinder and 3 ml of 90% acetone was added to the filtrate. it was ground until the filter fibers are separated and another 4 ml of acetone was added and put into the refrigerator (haier or hycd215) at -29°c for 24 hours. the ground filter was put into centrifuge tubes and added into the centrifuge (iec model cl centrifuge) to remove suspended particles. the centrifuge was revolved for 10 minutes in 3000 rpm to clarify samples. one cuvette was filled with 90% acetone for calibration and the rest cuvettes with their respective samples. the spectrophotometer (nv-203) was put on for at least five minutes before being used for measuring light absorption into the sample. the cuvette with acetone was put into spectrophotometer and the reading was adjusted to read zero. the respective samples were put to the spectrophotometer and the absorbencies were recorded at wavelengths of 750, 665, and 630 µm. the recorded samples were drawn out and two drops of 0.1n of hcl acid was added to samples. again the absorbance at 750, 665 and 630 µm was recorded. the content of total chlorophyll-a was calculated using the formula provided by jeffrey and humphrey (1975). other parameters such as temperature, rainfall, wind speed and humidity data were obtained from the nearby meteorological station (national metrological agency bahir dar branch). statistical data analysis: the data was compared for spatial and temporal phytoplankton biomass variation, correlation of phytoplankton biomass with temperature, rainfall, and wind speed and humidity conditions. similarity and difference between and among sites and months were determined by cluster analysis and one-way analysis of variance (anova) using r (r core team, 2016). bar graphs and table were used to present the summary of the result and presented using microsoft excel (2013) and r (r core team, 2016) to compare the relative phytoplankton biomass variations among sites and months. results and discussions spatial phytoplankton biomass in southern gulf of lake tana: phytoplankton biomass distribution in the southern gulf of lake tana was all most equal in all sites. mean phytoplankton biomass was calculated and presented in the bar graph (fig. 2). sites 6 and 8 had relatively high and same while the remaining sites low mean values (fig. 2). cluster analysis among sites showed that sites 2 and 4 are very similar. however, site 6 is very different from other sites. in a practical situation, sites 2 and 4 were areas where boats accumulated for transportation and it was amazing that they were similar in cluster analysis. similarly, site 6 was relatively undisturbed area and it was placed in figure 2. spatial phytoplankton biomass distributions in the southern gulf of lake tana. 4 dilnessa et al./ temporal and spatial phytoplankton biomass lake tana, ethiopia different positions by the analysis (fig. 3). the correlation analysis indicated that phytoplankton biomass is positively associated with environmental humidity and rainfall (r = 0.37 and 0.39, respectively) in one way and negatively correlated with temperature and wind speed (r = -0.1 and -0.16, respectively). in other words, increasing humidity and rainfall increase phytoplankton biomass, whereas elevation of temperature and wind speed decrease increase phytoplankton (table 2). the result also showed that there is a very strong relationship between temperature and wind speed around the study area i.e. the southern gulf of lake tana (r = 0.94). all sites except site 3 (impacted by transportation) were eutrophied in november. in the rest months, all sites were oligotrophic (polovina et al., 2001) (table 3). the gulf was also classified as high, good, moderate, poor and bad conditions (karydis, 2009). hence, all sites except site 8 (moderate) were bad in november 2010 (table 4). the highest and the lowest phytoplankton biomass values of the southern gulf of lake tana were recorded in november (21 mg/m3) and march (0.001 mg/m3), respectively. the monthly variation in total phytoplankton biomass was characterized by the lowest (0.001 mg /m3) in february and march 2011 and peaked (21 mg /m3) in november 2010 and the annual mean biomass of total phytoplankton in this gulf was 0.90 mg/m3 (fig. 4). annual phytoplankton biomass growth and decline was estimated by finding monthly average phytoplankton biomass. it was found that peak mean phytoplankton biomass in november (8 mg /m3) and lowest in march (0.04 mg /m3). the growth trend was decrement toward march from november and again increased toward a peak in april (fig. 3). the temporal cluster analysis showed that november (leveled as 1), december (leveled as 2) and april (leveled as 6) have high similarity. this means phytoplankton biomass returned back to its original state (november) after three months (january, february, and march). in other months (january, february, and march), it was nearly the same when compared to the first cluster (fig. 5). table 2. biomass correlation with temperature (°c), wind speed (m/s) and humidity (%). n = 96 temperature wind speed rainfall humidity temperature wind speed 0.94 rainfall -0.1 -0.11 humidity -0.71 -0.69 0.58 phytoplankton biomass -0.1 -0.16 0.37 0.39 table 3. site conditions in the southern gulf of lake tana from november 2010 to april 2011 (where e=eutrophic site and o=oligotrophic site) (polovina et al., 2001). months sites site 1 site 2 site 3 site 4 site 5 site 6 site 7 site 8 november e e o e e o e o december o o o o o o o o january o o o o o o o o february o o o o o o o o march o o o o o o o o april o o o o o o o o table 4. one way anova for phytoplankton biomass (mg/m3) in southern gulf of lake tana among months (november-april) of the year 2010/11 and sampling sites (where *** = very significant and--= not significant). source (n=96) df ss ms lambda f-value p-vale time (month = t) 5 251.062 50.212 44.879 1.000 <0.001*** space (site = s) 7 32.126 4.589 5.743 0.310 0.5752-- interactions (t+s) 35 181.204 5177 32.391 0.889 0.5902-- residual 48 268.524 5.594 5 int. j. aquat. biol. (2019) 7(1): 1-8 anthropogenic impacts on phytoplankton biomass significance test: statistically, there was a significant difference in phytoplankton biomass concentration in the southern gulf of lake tana among the six months (f = 9.4, p< .05 and r2 = 0.34) (table 4), but there was no spatial and interaction significance difference in the study area. phytoplankton biomass did not vary widely among all sites in southern gulf of lake tana. according to the trophic classification of polovina et al. (2001), the gulf can be classified as oligotrophic gulf although the gulf is eutrophic in november. the peak condition of total chlorophyll-a in this month is table 5. water quality of sites (where h=high, g = good, m = moderate, p =poor & b = bad). months site 1 site 2 site 3 site 4 site 5 site 6 site 7 site 8 november b b b b b m b b december g g h g g h h b january g g h h h h h h february h h h h h h h h march h h h h h h h g april m g p m p m g g figure 3. phytoplankton biomass spatial cluster analysis. figure 4. temporal algal biomass in lake tana southern gulf (where numbers are sample sites). 6 dilnessa et al./ temporal and spatial phytoplankton biomass lake tana, ethiopia in line with the study by ayalew et al. (2007) who found that highest production rates in the post-rainy season (oct.–nov.), which coincided with algal blooms (microcystis) and higher chlorophyll levels in the lake. this may be attributed to the low discharge of nutrients to the gulf because of low rainfall and hence low runoff which probably could carry nutrientrich soil to the lake from surrounding agricultural areas in the remaining studied months (except november). a similar study by tarekgne (2012) from july 2009 to may 2010 on the eastern side of lake tana showed that there was no significant difference of nutrients on spatial base but showed temporal significance variation. this study was in line with tarekgne (2012) results and agrees with phytoplankton biomass data analysis for the southern gulf of lake tana. conclusions and recommendations the results showed that all study sites range between oligotrophic to eutrophic though the gulf was most of the time oligotrophic. thus, this gulf might be most of the time nutrient limited. the absence of significant difference in sampling sites of the gulf indicated that there are similarity in phytoplankton grazers, anthropogenic and and/or natural impacts and phytoplankton growth conditions of southern gulf of lake tana. however, the presence of significant variations among monitoring months in terms of phytoplankton biomass showed that there is a dissimilarity in anthropogenic/natural impacts and phytoplankton growth on the gulf due upon month basis. there is a need for more work to be done at the southern gulf of lake tana in general and felege hiowt referal hospital and west gojam prison stations areas in particular for removing inputs pressures from surrounding area to the gulf ecosystem. such works can be policy, decision, and management and thus bring sustainable growth and protection of the aquatic biodiversity and resources of the gulf for future generations. since the gulf receives nutrients from the rivers during flood season, runoffs from various sources like for example hospitals and prison stations, there is a need to balance, at least minimize the hydrological changes and prevent such inputs to the gulf. acknowledgments this research was supported by bahir dar university figure 5. cluster analysis showing similarity and differences among months (where 1=november, 2=december, 6 april in the first cluster and december=3, january=4, february=5). 7 int. j. aquat. biol. 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(2018) 6(1): 1-7 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article effects of safflower (carthamus tinctorius) extract on serum antibacterial activity of rainbow trout (oncorhynchus mykiss) against aeromonas hydrophila, streptococcus iniae and yersinia ruckeri ashkan zargari1, mohammad mazandarani*2, seyyed morteza hoseini3 1department of fisheries, faculty of natural resources and marin sciences, tarbiat modares university, noor, iran. 2department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. 3inland waters aquatics resources research center, iranian fisheries sciences research institute, agricultural research, education and extension organization, gorgan, iran abstract: in the present study, the effects of safflower (carthamus tinctorius) extract on serum antibacterial activity of rainbow trout (oncorhynchus mykiss) against aeromonas hydrophila, streptococcus iniae and yersinia ruckeri was studied. in this regard, 450 fish with average weight of 100±10 g were stocked into fifteen fiberglass tanks. this experiment consisted of 3 treatment groups (received 50, 100 and 200 mg/kg bw safflower extract via ip injection), one positive control group (just received normal saline) and one negative control group (with no injection). blood samples were taken at the 3th, 7th and 10th days after the injections, and antibacterial activity of serum were determined in vitro using cfu method. the results showed that safflower extract injection had no significant effects on serum anti-bacterial activity against a. hydrophyla and y. rukeri during 10 days post injection. however, in the fish receiving 100 mg/kg safflower extract, serum bactericidal activity against s. iniae was significantly higher than the other groups. this study demonstrated that safflower extract at the doses of 50-200 mg/kg via ip injection did not cause significant changes in serum antibacterial activity against a. hydrophila and y. ruckeri, but injection of 100 mg/kg extract led to an increase in the serum antibacterial activity against s. iniae, in rainbow trout. article history: received 5 october 2017 accepted 23 january 2018 available online 2 5 february 2018 keywords: antibacterial activity safflower serum injection introduction nowadays rainbow trout is one of the most important species in cold-water aquaculture of the world. an increasing trend towards the breeding and reproduction of this fish has raised the incidences of various diseases, including the bacterial ones. bacterial pathogens are the most common pathogens in aquaculture, causing many problems and damages. improving the level of immunity along with the use of immunostimulants is a useful and cost-effective method to increase fish resistance to pathogens and to prevent the formation of drug resistance bacteria. every year, bacterial diseases are responsible for the loss in many trout farms. treatment with antibiotics is one of the common methods to control these diseases, while vaccinations would be also useful in some cases. due to the pathogens’ resistance to antibiotics, some limitations have been established against their application; besides, antibiotics are also responsible for killing the beneficial bacteria in the host's digestive tract (aoki, 1990). prophylactic methods are superior to therapeutic methods from the economic aspects, thus methods for improving fish immunity and resistance to diseases are more suitable than antibiotic application. due to the presence of effective natural compounds in medicinal plants, the use of medicinal plants and their natural derivatives has increased recently. most of these compounds have no side effects on fish and consumers, resulting in remarkable advantages compared to chemical drugs (velag and studlla, 2005). on the other hand, some fish bacterial diseases are important in human health and hygiene. an example is the infection with a. hydrophila, found in association with fish and crustacean. human infections with aeromonas spp. are often observed in *corresponding author: mohammad mazandarani doi: https://doi.org/10.22034/ijab.v6i1.392 e-mail address: mazandarani@gau.ac.ir http://www.ij-aquaticbiology.com/ mailto:mazandarani@gau.ac.ir 2 zargari et al./ effects of safflower extract on serum antibacterial activity of rainbow trout people with a weak immune system, and localized wounds, swelling and gastroenteritis are some of the most common signs of the infection (moyer, 1987; lowry and smith, 2007). streptococcus iniae is another zoonotic bacterium that can cause serious problems in humans, with the most clinical signs in the patients being endocarditis, cellulitis, meningitis and systemic arthritis (weinstein, 1997). to achieve sustainable development in aquaculture industry, it is necessary to find methods to reduce high costs of disease treatments in ponds. in this regard, it is essential to follow quarantined principles and prevent the spread of the pathogens. in addition, improving the fish resistance against pathogens and environmental stress is effective method with low cost. for this purpose, use of oral or injectable herbal extracts with antioxidant or antimicrobial properties would be beneficial; besides, some plants increase growth performance, immunological parameters and environmental stress resistance in fish (velag and studlla, 2005; sahu et al., 2008). safflower (carthamus tinctorius), a member of the compositae family (villa, 2017), has been of particular interest to humans for the past 2500 years for its therapeutic effects (li, 2013). nowadays more than 200 compounds have been extracted from this plant. many therapeutic effects on humans and laboratory animals, such as anti-hypoxia, liver anti fibrosis, antioxidant, stimulate the immune system and anti-tumor and anti-inflammatory effects have been reported for this extract (zhou, 2014; fan, 2009). however, in this regard, there are some researches about the effects of this extract on fish (dadras et al., 2016; choi et al., 2010). in the present study, antibacterial effects of safflower extract injection have been studied against some fish pathogenic bacteria (a. hydrophila, y. ruckeri and s. iniae) in rainbow trout. materials and methods a total numbers of 450 rainbow trout with an average weight of 100±10 g were transferred to the fisheries research station of gharehsou (bandar-turkmen, golestan province, iran). the fish were distributed into fifteen 500-l fiberglass tanks containing 200 l water at a flow rate of 2 l/min. acclimation was carried out for one week, during which the fish were fed with a commercial diet (3% of mass weight daily). this experiment was consisted of 5 treatments: negative and positive controls, 50, 100 and 200 mg/kg extract groups. to prepare the injectable extract, ethanolic extract was made from dried petals of safflower. briefly, the petals were washed (with cold sterile water), dried and grounded. five liters of 96% ethanol were added to 500 g of the dried powder as a solvent in a ratio of 1:10. later, the mixture was transferred to a dark container and was mixed well. after that, the container was tightly closed and shaken for seven days at room temperature to ensure well mixing. after seven days, the supernatant was passed through a filter paper to remove any insoluble suspended particles; after which the filtered supernatant was dried using an oven and finally the residual material was packed in sterile containers as ethanolic extract (harikrishnan, 2009). this extract was dissolved in 0.9% sodium chloride at 40°c to prepare the injectable solution for intraperitoneal injection at the dosages of 50, 100 and 200 mg/kg of fish weight. each dosage was injected in a 0.1 ml solution to the fish of three tanks. positive control was injected by the 0.9% sodium chloride solution. a negative control group, consistent of three tanks, was assigned with no injection. for injecting, the fish were netted and anesthetized in 100 ppm eugenol. the extract was injected intraperitoneally using an insulin syringe. the injected fish were also transferred to separate 500-l tanks filled with 200-l of water with constant flow rate. blood samples were taken three, seven and ten days after injection. six fish were sampled for treatment at each sampling time. for blood sampling, the fish were netted and anesthetized with 100 ppm eugenol. the blood samples were collected from the caudal vein using 2-ml syringes. sera was obtained by centrifugation at 6000 rpm for 7 min and stored at 80°c for analysis. three pathogenic bacteria including a. hydrophila, y. ruckeri and s. iniae were used to assess the serum int. j. aquat. biol. (2018) 6(1): 1-7 3 bactericidal activity. the bacteria were first cultured on nutrient agar medium. after 48 hrs of incubation at 25°c, the bacteria were collected from the second passage culture according to sahu (2008) and suspended in 0.9% sodium chloride solution with one optical density (od) at 640 nm. each of the bacterial suspensions was diluted three times and used as a main sample of bacteria. 100 μl of serum was mixed with 100 μl of the bacterial suspensions and then incubated at 37°c for 4 hrs after that 6-fold serial dilutions were prepared from the mixture (according to the national iranian standard, no. 3-8923, 2006). 100 μl of all dilutions for each treatment were inoculated in nutrient agar plates and after 48 hrs resting at room temperature, the viable total count calculated for each treatment based on colony forming unit (cfu). for estimating the total count of bacterial suspension, 100 μl of bacteria was added with 100 μl sterilized 9% sodium chloride solution and after 4 hrs at 37°c, a viable total count of each bacterium was determined. statistical analyses were implemented using the spss version 22.0 for windows (ibm spss inc., chicago, il, usa). the normality of the data and homogeneity of variances were confirmed by shapiro wilk and levene tests. data of the serum bactericidal activities were analyzed by one-way analyses of variance (anova) followed by duncan’s test. a statistical significance was accepted at p<0.05 levels. results data of bactericidal activity against a. hydrophyla are presented in the figures 1-3. the bacterial load has significantly decreased in serum treatments compared to that of the bacterial control (figs. 1-3). however, there was no significant difference among negative and positive controls and the extract treated sera at any sampling times. data of the bactericidal activity against y. rukeri are presented in figures 4-6. negative and positive controls had significantly lower bactericidal activity compared to the bacteria control at the third day (fig. 4). however, the extract-treated fish bactericidal activities were similar to the bacteria control but not figure 1. serum bactericidal levels of treatments compared to control plate containing aeromonas hydrophila on the third day. different letters above the bars show significant difference. figure 2. serum bactericidal levels of treatments compared to control plate containing aeromonas hydrophila on the seventh day. different letters above the bars show significant difference. figure 3. serum bactericidal levels of treatments compared to control plate containing aeromonas hydrophila on the tenth day. different letters above the bars show significant difference. significantly different compared to negative and positive controls (fig. 4). after, seven days, the serum treatments excluding the 50 and 200 dosages had a lower bacterial load compared to the bacteria control, however, there was no significant difference among the serum treatments (fig. 5). at the tenth day, the bacterial load significantly decreased in serum treatments compared to that of the bacterial control (fig. 6). however, there was no significant difference among negative and positive controls and the extract treated sera. 4 zargari et al./ effects of safflower extract on serum antibacterial activity of rainbow trout figure 4. serum bactericidal levels of treatments compared to control plate containing yersinia ruckeri on the third day. different letters above the bars show significant difference. figure 5. serum bactericidal levels of treatments compared to control plate containing yersinia ruckeri on the seventh day. different letters above the bars show significant difference. figure 6. serum bactericidal levels of treatments compared to control plate containing yersinia ruckeri on the tenth day. different letters above the bars show significant difference. data of bactericidal activity against s. iniae are presented in the figures 7-9. at the third day, the bacterial load of the serum treatments was significantly lower than the bacteria control; however, there was no significant difference among the serum treatments (fig. 7). after, seven days, the serum treatments excluding the dosage of 200 had lower bacterial loads compared to the bacteria control while the bacterial load of the dosage 100 was significantly lower than the other serum treatments. (fig. 8). at the figure 7. serum bactericidal levels of treatments compared to control plate containing streptococcus iniae on the third day. different letters above the bars show significant difference. figure 8. serum bactericidal levels of treatments compared to control plate containing streptococcus iniae on the seventh day. different letters above the bars show significant difference. figure 9. serum bactericidal levels of treatments compared to control plate containing streptococcus iniae on the tenth day. different letters above the bars show significant difference. tenth day, the bacterial loads of dosages 50 and 100 were significantly lower than the bacteria control, while the other treatments had bacterial loads similar to that of bacteria control (fig. 9). discussion under stressful conditions, microbial pathogens, especially bacteria can make problems in fish farms (zorrilla, 2003). this situation leads to a reduction in int. j. aquat. biol. (2018) 6(1): 1-7 5 the fish growth as well as economic losses. on the other hand, some fish diseases can also be transmitted to humans in the form of zoonotic diseases. therefore, the control of fish pathogens in farms is necessary and cost effective. in the present study, safflower extract effects on serum antibacterial activities against three fish pathogens, a. hydrophila, s. iniae (zoonotic bacteria) and y. ruckeri have been investigated. streptococcus iniae, is one of the important zoonotic bacteria because of its serious problems and clinical effects on humans. aged persons are more susceptible to the infection, and are infected via an external wound or ulcer; cooking can lead to the elimination of the bacterium (agnew, 2007; sun, 2007; weinstein, 1997). in addition, s. iniae the cause of streptococcusis, is one of the main diseases in aquatic industry in cold/warm and fresh/salt water species. consequently, due to economic losses in recent years, this disease has been recognized as a one of the most dangerous bacterial diseases among the fishes, resulting to significant losses in many cultivating species (dadson, 1999; agnew, 2007; austin, 2007). red mouth disease (rmd) or yersiniosis, is another common bacterial disease that is caused by y. ruckeri resulting to high annual losses in cold water fish farms throughout the world (davies, 1989; zorriehzahra, 2012). this bacterium may cause a large mortality in a wide variety of fish species, also it may cause health problems in humans (xia, 2004; poobalane, 2010; zhou, 2010). yersiniosis has been also reported in rainbow trout farms of iran for the first time in 1999, where six bacterial strains were isolated from the infected fish (soltani, 2014). in many cases, drug therapy for this disease was not successful due to antibiotic resistance (khushiramani, 2007); however, no human infections have been reported for this bacterium. aeromonas hydrophila is a gram-negative bacterium, which may cause problems in humans especially in the patients with a weak immune system. the patients were diagnosed with external swelling wounds and gastrointestinal problems (moyer, 1987; lowry and smith, 2007), therefore, the transmission of such bacterium to humans should always be taken into consideration. in aquaculture, this bacterium may cause high mortality in a wide variety of fish such as common carp, catfish, tilapia, eel, goldfish etc. (xia, 2004; poobalane, 2010; zhou, 2010). this disease causes different abnormal symptoms in fish such as hemorrhagic septicemia, ascites, ulcers, and exophthalmia (khushiramani, 2007). after some disease incidences and in some cases, antibiotic therapy is not easy and always successful, therefore, anti-aeromonas vaccines are administered in some cases (khushiramani, 2007; poobalane, 2010; sahoo, 2011). in order to maintain hygiene, the reduction of antibiotics consumption and the production of safe foods, the use of certain medical herbs that have numerous antibiotic properties was suggested that also elevate body immune activities. in some cases, it can also be used as a vaccine adjuvant, to reduce cost of vaccinations and increase effectiveness of vaccines by injection (reverter, 2014). there are several studies on effects of safflower on laboratory animals such as mice. hepatoprotective effects of this extract have been previously recorded in laboratory mice (asgari, 2012; rahimi 2009). these studies showed that intraperitoneal injection of the safflower extract in mice led to significant reductions of hepatocyte damage in exposure with alloxan poisoning. despite the reports about improvement of the immune system by safflower extract, there are some reports showing impaired immune systems as a result of safflower extract administration. for instance, louei monfared and salati (2012) reported that intraperitoneal injections of safflower extract at the doses of 1.4 and 2.8 mg/kg induced toxic changes in the placental structure, weight, and thickness of the placenta in the mice during pregnancy, resulting in a decrease in survival rate of neonates during 42 days after birth. ip administration of 50-450 mg/kg.day of safflower extract for 6-8 days decreased serum lysozyme concentration and phagocytic functions of both peritoneal macrophages and peripheral leukocytes in mice (al-snafi, 2015, 2016). in fish, dadras et al. 6 zargari et al./ effects of safflower extract on serum antibacterial activity of rainbow trout (2016) demonstrated that dietary administration of 1 2% safflower resulted in a significant increasing in immune function and led to an increased activity of lysozyme, serum alternative complement (ach50) activities and the number of white blood cells (wbc) in beluga (huso huso). such aforementioned contradictions suggest that the effects of safflower extract on immune components depend on experimental conditions, such as experimental animal and rout of administration. according to the present study, injection of the safflower extract does not lead to increased serum bactericidal activity against a. hydrophila and y. ruckeri in rainbow trout, but it improves serum antibacterial activity against s. iniae at the doses of 50 and 100 mg/kg during 7 and 10 days. these results may be related to difference in cell wall structures of bacteria. in fact, s. iniae is a gram-positive bacterium while the other two bacteria are gram-negative. therefore, further studies are needed to address the potential effects of bacteria type and rout of the extract administration on different immune components and bactericidal activity. references agnew w., barnes a.c. 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(2018) 6(1): 1-7 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی کمانرنگین آالی قزل در سرم باکتریایی ضد خاصیت بر( carthamus tinctorius) گلرنگ عصاره اثرات (oncorhynchus mykiss )هایباکتری علیه بر aeromonas hydrophila ، streptococcus iniae و yersinia ruckeri 3حسینی مرتضی سید ،2*مازندرانی محمد ،1زرگری اشکان ، ایران.نور مازندران، مدرس، تربیت دانشگاهدانشکده منابع طبیعی، ،شیالت گروه1 ، ایران.گرگان گلستان، گرگان، طبیعی منابع و شاورزیک علوم دانشگاه آبزیان، پرورش و تکثیر گروه2 .ایران گرگان، داخلی، هایآب آبزیان ذخایر تحقیقات مرکز ایران، شیالتی علوم تحقیقات موسسه کشاورزی، ترویج و آموزش تحقیقات، سازمان3 چکیده: های باکتریآالی رنگین کمان بر علیه ( بر خاصیت ضد باکتریایی سرم خون قزلcarthamus tinctoriusدر بررسی حاضر اثرات عصاره گلرنگ ) aeromonas hydrophila ، streptococcus iniaeو yersinia ruckeri نمونه ماهی با 450این منظور تعداد رایقرار گرفت. ب مطالعهمورد گرم/کیلوگرم میلی 200و 100، 50ترتیب به میزان . در این راستا سه گروه تیمار )بهشدندتانک فایبرگالس تقسیم 15گرم در 100±10میانگین وزنی اقع وعصاره گلرنگ از طریق تزریق داخل صفاقی دریافت نمودند(، یک گروه کنترل مثبت )تنها با سرم فیزیولوژی از طریق داخل صفاقی مورد تزریق پس از تزریق اخذ گردید و خاصیت ضد 10و 7، 3های خون در روزهای شدند( و یک گروه کنترل منفی )بدون تزریق( در نظر گرفته شد. نمونه ارزیابی گردید. بر اساس نتایج تزریق عصاره گلرنگ هیچ cfuهای یاد شده بر روی محیط کشت بر اساس روش کتریایی سرم آنها برای باکتریبا گرم/ کیلوگرم میلی 100نداشت. در ماهیان که با دوز y. ruckeri و a. hydrophila هایبرای باکتری تاثیری بر خاصیت ضد باکتریایی سرم خون داری در طور معنیبه s. iniaeپس از تزریق خاصیت ضد باکتریایی سرم برای باکتری 10و 7در روزهای ،ره گلرنگ مورد تزریق واقع شدندعصا گرم/کیلوگرم میلی 200تا 50های داشت. در نهایت بر اساس بررسی حاضر تزریق داخل صفافی عصاره گلرنگ در غلظت ها افزایشمقایسه با سایر گروه ،نگردید y. ruckeri و a. hydrophilaهای کمان برای علیه باکتریآالی رنگیندار در خاصیت ضد باکتریایی سرم خون قزلمنجر به تغییر معنی s. iniaeبر علیه باکتری کمانآالی رنگینگرم/کیلوگرم عصاره گلرنگ منجر به افزایش خاصیت ضد باکتریایی سرم خون قزلمیلی 100اما تزریق گردید. .تزریق سرم، گلرنگ، باکتریایی، ضد خاصیت :کلمات کلیدی int. j. aquat. biol. (2017) 5(5): 310-320; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article occurrence and intensity of parasites in chelon aurata (risso, 1810) and neogobius caspius (eichwald, 1831) (teleostei: perciformes) from southern caspian sea seyedeh bahereh mirnategh1, nader shabanipour1, 2, masoud sattari*2, 31 1department of biology, faculty of science, university of guilan, rasht, iran. 2department of marine science, caspian sea basin research centre, university of guilan, rasht, iran. 3fisheries department, faculty of natural resources, university of guilan, sowmeh sara, iran. 3 article history: received 16 june 2017 accepted 12 september 2017 available online 2 5 october 2017 keywords: ectoparasite endoparasite mugilidae gobiidae abstract: this study was performed to investigate ecto-and endo-parasites of golden grey mullet, chelon aurata (risso, 1810) (n=331) and caspian goby, neogobius caspius (eichwald, 1831) (n=170) from the southern caspian sea. the sampling was carried out for one year in three stations, including chamkhaleh (st1), kiashahr (st2) and anzali (st3) coastal areas, guilan province, iran. biometric characteristics were recorded, sexes were determined and specimens examined for ecto and endoparasites. a total of 158 specimens (58.31%) out of 331 grey mullets, and 61 (35.88%) out of 170 caspian gobies, were found to be infected. 1453 parasites belonging to 5 species were found consisting of trichodina reticulata, unknown protozoan, nematode larvae (in golden grey mullet) and a cestode, eustrongylides excisus larvae (in caspian goby). the occurrence of unknown protozoan and nematode larvae in c. aurata and cestode larvae in n. caspius are reported for the first time in iran. seasonal variations, the effects of host length, weight and localities on parasite prevalence and mean intensity have been examined during present investigation. introduction the mugilids are found in freshwater, brackish and marine regions of the world (özer and kirka, 2013). they are known to be euryhaline, therefore are mostly coastal marine fishes and fresh water ones are less in number. golden grey mullet, chelon aurata (risso, 1810) feeds on zooplankton, molluscs larvae, other invertebrates, debris, algae and some small aquatics (fazli et al., 2008). there have been several studies on the parasitic fauna of c. aurata reporting some parasites, including saccocoelium obesum, capillaria sp., contracaecum sp., neoechinorhynchus agilis (in mitras lagoon, sardinia), trichodina lepsii (at the black sea coast of sinop, turkey), ligophorus spp. (in eratino lagoon of greece) (merella and garippa, 2001; özer and öztürk, 2004; ragias et al., 2005). however, there are no reports on parasites of this fish species in the caspian sea. important species of the family gobiidae in the *corresponding author: masoud sattari doi: https://doi.org/10.22034/ijab.v5i5.323 e-mail address: msattari@guilan.ac.ir caspian sea are monkey goby (neogobius fluviatilis), caspian goby (neogobius caspicus), bighead goby (ponticola gorlap) and round goby (neogobius melanostomus) (daghigh-roohi and sattari, 2004). neogobius caspicus has wide distribution inhabiting close to the beach in shallow waters. they are benthic feeder so that feed mainly on benthic crustaceans such as cumaceae, gammaridae and molluscs (daghighroohi and sattari, 2004). parasites of some gobiids in the southern caspian sea, including eustrongylides excisus, dichelyne minutus and corynosomas strumosum, have been reported in recent works (pazooki and aghlmandi, 1999; daghigh-roohi and sattari, 2004; pazooki et al., 2011). as gobiids are valuable food items for some commercially important fishes such as sturgeons, then they can transmit parasitic infections. this study aimed to investigate the prevalence, intensity and composition of parasitic fauna in 311 int. j. aquat. biol. (2017) 5(5): 310-320 c. aurata and n. caspicus in the southern caspian sea and to determine the influence of seasonal differences, locality, sex, weight and length of host on the prevalence, intensity and composition. materials and methods this study was carried out for a time period of one year from october 2015 to september 2016. three sampling sites in the guilan province including chamkhaleh (station 1: st1; 37°13'44.8"n 50°18' 58.4"e), kiashahr (station 2: st2; 37°28'8.6"n 49°59' 34.4"e) and anzali (station 3: st3; 37°29'16.5"n 49°28'35.2"e) were selected. fish were caught by local fishermen using beach seine net, transported partly alive in tanks and partly as perished specimens in an ice-cooled box. all specimens were left to expire naturally before body dissection. fish examination and parasite collection: total length and body weight of fishes were measured and their gender was determined after dissection. a total of 331 specimens of c. aurata and 170 specimens of n. caspius were examined for ectoand endo-parasites using light microscope and stereoscope. the skin, fins, heart, gills, body cavity and visceral organs, including stomach, intestine, liver, swim bladder and gonads were examined. parasites counting and also methods of fixation, preservation, slide preparation were performed according to jalali (1999) and palm and caira (2008). all specimens were fixed in 10% formalin solution, stained with aqueous acetocarmine, dehydrated and mounted in premount. the worms were identified using parasites identification keys (yamaguti, 1961; bykhovskaya-pavlovskaya et al., 1962; moravec, 1994; jalali, 1999). the parasitological parameters i.e. prevalence, mean intensity and abundance were calculated according to bush et al. (1997). the prevalence (%) was calculated as number of hosts infected / number of hosts examined. the mean intensity was calculated as total number of individuals of a particular parasite / number of infected hosts. the mean abundance is total number of individuals of a particular parasite species in a host species / the total number of hosts examined. the dominance of a parasite species was calculated as n/n sum (where n=abundance of a parasite species and n sum=sum of the abundance of all parasite species found) and expressed as a percentage based on leong and holmes (1981). data analysis: the dominance values were used for classification of parasites as: eudominant (>10%), dominant (5.1-10%), subdominant (2.1-5%), recedent (1.1-2%) and subrecedent (<1.0%) of given species (niedbala and kasparzak, 1993). mean intensity of infection and abundances of parasite species (with prevalence >10%) among seasons and sexes were tested by kruskal-wallis test (kw, multiple comparisons) and mann-whitney u test (mw, pairwise comparisons). z test was carried out for prevalence comparison between sexes. the results were considered significant at 95% level (p<0.05). statistical analysis was carried out using spss 22 software. results the examined golden grey mullets were 118.73 g (±25.14, range=38.2-310 g) in weight and 24.38 cm (±1.11, range=15.2-38.5 cm) in total length. the examined caspian goby were 19.69 g (±1.34, range=10.22-65.21 g) in weight and 10.41 cm (±0.26, range=6.3-18.2 cm) in total length. 1453 individuals of 5 parasite species consisting of two ectoparasites: trichodina reticulata (fig. 1) and unknown protozoa (fig. 2) and three endoparasites: one nematode larvae (figs. 3, 4) in c. aurata, eustrongylides excisus larvae (figs. 5, 6) and one figure 1. trichodina reticulata (100x). 312 mirnategh et al./ ectoand endo-parasites in chelon aurata and neogobius caspius cestode larvae (fig. 7) in n. caspius were found. the occurrence of unknown protozoans and nematodes larvae in c. aurata and also cestode larvae in n. caspius were reported for the first time. prevalence, mean intensity and abundance of the parasites in two fish species are summarized in tables 1-8. trichodina reticulata and unknown protozoa had the highest prevalence, mean intensity, abundance and dominance. the eudominant parasites of c. aurata (table 1) were t. reticulata and unknown protozoa (d=61.3% and 38.04%, respectively). the subrecedent parasites were nematodes larvae (d=0.59%). the maximum prevalence, mean intensity and abundance of t. reticulata were determined to be in autumn (72.18%, 5.42±1.42, 3.91±1.08) and spring (70.59%, 4.38±1.03, 3.09±0.84) (table 2). the unknown protozoan parasites were observed only in autumn. there was no significance difference between seasons for parasites occurrence (kw, p>0.05). the abundance of t. reticulata in st2 was higher than other stations, but differences were not significant (kw test, p>0.05), while mean intensity showed a significant relationship between st1 and the other two stations (kw, χ2=8.499, df=2, p<0.05) figure 2. (a) unknown protozoa (400x) and (b) unknown protozoa (1000x). figure 3. (a) nematode larvae (40x), (b) the anterior of nematode larvae (400x) and (c) the posterior of nematode larvae (100x). figure 4. line drawing of the nematode larvae showing cephalic end in chelon aurata. parasite prevalence (%) mean±sd range abundance±sd dominance trichodina reticulate (n1=833) 67.05 4.71±1.31 0-14 3.16±0.94 61.3 unknown protozoa (n1= 517) 3.78 51.7±26.7 9-170 1.96±26.7 38.04 nematodes larvae (n1= 8) 2 1.6±0.82 1-2 0.02±0.02 0.59 n1=number of parasite, sd=standard deviation table 1. the prevalence, mean intensity, range, abundance and dominance of some parasites in chelon aurata (n=331) 313 int. j. aquat. biol. (2017) 5(5): 310-320 (table 3). there was no significant difference between abundance, mean intensity of the unknown protozoa and nematode larvae among three regions (kw test, p>0.05). the prevalence of t. reticulata in females was significantly higher than males, while the prevalence of the unknown protozoa in males was significantly higher than females (z test, p<0.05) (table 4). the differences between the prevalence of nematodes larvae in females and males were significant (z test, p<0.05). there was no significant difference between all parasites in terms of mean intensity and abundance (mann whitney u test, p>0.05). a total of 95 individuals of 2 parasite species were found in n. caspicus. of those, e. excisus had the highest prevalence, mean intensity, dominance and abundance (table 5). the eudominant parasites of n. caspicus were e. excisus (d=96.88%) and the parasite/ season trichodina reticulata prevalence (%) mean±sd range abundance±sd unknown protozoa prevalence (%) mean±sd range abundance±sd nematodes larvae prevalence (%) mean±sd range abundance±sd autumn n=133 72.18 5.42±1.42 0-14 3.91±1.08 7.52 51.7±26.7 9-170 11.24±26.7 0 winter n=97 58.76 3.59±1.48 0-10 2.14±1.13 0 0.03 1.33±0.68 1-2 0.04±0.05 spring n=51 70.59 4.38±1.03 0-9 3.09±0.84 0 0.02 2±1.03 2 0.04±0.07 summer n=50 _ _ 0.02 2±1.03 2 0.04±0.07 sd=standard deviation table 2. the prevalence, mean intensity, range and abundance of some parasites of chelon aurata (n=331) in different seasons. parasite locality trichodina reticulata prevalence (%) mean±sd range abundance±sd unknown protozoa prevalence (%) mean±sd range abundance±sd nematode larvae prevalence (%) mean±sd range abundance±sd chamkhaleh n=116 66 3.1±1.5 0-10 2.03±1.4 11 51.7±17.66 9-170 5.6±17.66 0 kiashahr n=107 77 5.3± 1.3 0-14 4.1±1.4 0 0.02 1.5±0.61 1-2 0.03±0.1 anzali n=108 58 5.9±1.6 0-11 3.4±1.3 0 0.03 1.67±0.67 1-2 0.05±0.11 sd=standard deviation table 3. the prevalence, mean intensity, range and abundance of some parasites of chelon aurata (n= 331) in different localities. 314 mirnategh et al./ ectoand endo-parasites in chelon aurata and neogobius caspius recedent parasite was cestode larvae (d=1.05%) (table 5). the cestode larvae was observed only in spring. the abundance of e. excisus in summer was significantly higher than other seasons (kw, χ2=8.586, df=3, p<0.05) (table 6). the cestode larvae was observed only in st1 (table 7). the prevalence and abundance of e. excisus in st1 were higher than other stations, but differences were not significant (kw test, p>0.05). the prevalence of cestode larvae and e. excisus larvae in females were significantly higher than males (z test, p<0.05). mean intensity and parasite sex trichodina reticulata prevalence (%) mean±sd range abundance±sd unknown protozoa prevalence (%) mean±sd range abundance±sd nematode larvae prevalence (%) mean±sd range abundance±sd male n=225 65.32 4.17±0.97 0-11 2.68±0.42 3.47 6.3± 13-170 0.22±0.11 1.33 1.33±0.68 1-2 0.02±0.02 female n=106 70.33 5.78±1.82 0-14 3.66±1.91 1.1 5± 9-101 0.05±0.03 1.89 2±1.03 2 0.04±0.05 sd=standard deviation table 4. the prevalence, mean intensity, range and abundance of parasites of males and females chelon aurata. parasite prevalence (%) mean±sd range abundance±sd dominance cestode larvae (n1=2) 1.43 2±1.03 2 0.01±0.005 1.05 eustrongylides excisus (n1= 93) 35.29 1.55±0.35 1-4 0.55±0.14 96.88 n1=number of parasite, sd=standard deviation. table 5. the prevalence, mean intensity, range, abundance and dominance of some parasites in neogobius caspicus (n=170). table 6. the prevalence, mean intensity, range and abundance of some parasites of neogobius caspicus (n=170) in different seasons. parasite/ season cestode larvae prevalence (%) mean±sd range abundance±sd eustrongylides excisus prevalence (%) mean±sd range abundance±sd autumn n=26 0 11.54 1.33±0.69 1-2 0.15±0.08 winter n=30 0 3.3 4±2.07 2 0.13±0.07 spring n=54 1.85 1 ± 0.52 1 0.02 ± 0.01 18.52 3.6±1.86 1-3 0.67±0.35 summer n=60 0 21.67 3.92±2.02 1-4 0.85±0.44 sd=standard deviation 315 int. j. aquat. biol. (2017) 5(5): 310-320 abundance of these parasites in females were higher than males, but differences were not significant (mann whitney u test, p>0.05) (table 8). the results showed a significant positive relation between length and weight of c. aurata and the prevalence of t. reticulata (fig. 8). there was also a significant positive relation between weight of table 7. the prevalence, mean intensity, range and abundance of some parasites of neogobius caspicus (n=170) in different localities. parasite locality cestode larvae prevalence (%) mean±sd range abundance±sd eustrongylides excisus prevalence (%) mean±sd range abundance±sd chamkhaleh n=63 2 1±0.34 1 0.02±0.09 37 1.96±0.9 1-4 0.71±0.6 kiashahr n=51 0 31 1.44±0.6 1-2 0.45±0.3 anzali n=56 0 38 1.2±0.3 1-2 0.45±0.2 sd=standard deviation table 8. the prevalence, mean intensity, range and abundance of some parasites of neogobius caspicus (n=170) in males and females. parasite/ sex cestode larvae prevalence (%) mean±sd range abundance±sd eustrongylides excisus prevalence (%) mean±sd range abundance±sd male n= 100 0 21 1.14±0.1 1-2 0.24±0.07 female n= 70 4 1± 1 0.05±0.03 54 1.79±0.6 1-4 0.97±0.28 sd=standard deviation figure 5. (a) the anterior of eustrongylides excisus (400x) and (b) the posterior of e. excisus (100x). figure 6. line drawings of eustrongylides excisus from neogobius caspicus. (a) cephalic end and (b) caudal end. 316 mirnategh et al./ ectoand endo-parasites in chelon aurata and neogobius caspius figure 7. (a) the anterior of cestode larvae (100x) and (b) the posterior of cestode larvae (100x). figure 8. (a) relationship between weight and prevalence of trichodina reticulata in chelon aurata and (b) relationship between length and prevalence of trichodina reticulata in chelon aurata. figure 9. (a) relationship between weight and prevalence of unknown protozoa in chelon aurata and (b) relationship between length and prevalence of unknown protozoa in chelon aurata. figure 10. (a) relationship between weight and infection of nematodes larvae in chelon aurata and (b) relationship between length and infection of nematodes larvae in chelon aurata. 317 int. j. aquat. biol. (2017) 5(5): 310-320 c. aurata and the prevalence of unknown protozoa but no significant relationship was observed by length (fig. 9). there was also significant relationship between weight of c. aurata and the prevalence of nematode larvae but no relation was found between length and the prevalence of these parasites (fig. 10). the results also showed a significant relationship between length and weight of n. caspius and the prevalence of cestode larvae (fig. 11). there were no relationships between length and weight of n. caspius and the prevalence of e. excisus (fig. 12). discussion in the present study, t. reticulata, unknown protozoa and nematode larvae were identified in c. aurata. there is one report concerning to infection of c. aurata by parasites in iran (taghavi et al., 2012) where four parasites, including ichthyobodo necator, ichthyophthirius multifiliis, trichodina sp. and s. obseum have been reported. the occurrence of unknown protozoa and nematodes larvae in c. aurata is reported for the first time from iran. the occurrence of trichodina sp. has been reported from other fish species in the southern caspian sea, including cyprinus carpio, blicca bjoerkna and tinca tinca from anzali wetland (sattari, 1996). there were also some reports about occurrence of t. reticulata in iran (mokhayer, 1972; adel et al., 2015; moghaddam, 2015). ectoparasites are much affected by water conditions. in contrast, endoparasites need intermediate hosts to complete their life cycle and as a result, their infection process is relatively stable compared to fish ectoparasites (özer and krica, 2013). trichodinids are ciliates belonging to the family trichodinidae. these ciliates infest many of marine and freshwater fishes in many parts of the world. the genus trichodina could be found on the gills and body surface (baker, 2007). in the present study, the prevalence of t. reticulata was highest in kiashahr (st2, 77%) and lowest in anzali (st3, 58%). such variations could be related to the differences in environmental parameters. female fishes have shown more infestations than the males. this may be related to specific physiological conditions of females. in addition, their increased food intake as a result of eggs development process might have exposed them to more contact with the parasites (omeji et al., 2011). similar results were figure 11. (a) relationship between weight and infection of cestode larvae in neogobius caspicus and (b) relationship between length and infection of cestode larvae in neogobius caspicus. figure 12. (a) relationship between weight and infection of eustrongylides excisus in neogobius caspicus and (b) relationship between length and infection of eustrongylides excisus in neogobius caspicus. 318 mirnategh et al./ ectoand endo-parasites in chelon aurata and neogobius caspius obtained by holden and reed (1972), adebanjo (1979) and emere and egbe (2006). there was a positive significant relationship between weight and length of c. aurata and prevalence of t. reticulata. thus, the larger fishes were more sensitive compared to the smaller ones. there is significant relation between weight of c. aurata and the prevalence of nematodes larvae. this could be a result of covering wider areas in search of food by larger fishes (omeji et al., 2011). as a result, they susceptible to higher contamination through food rather than the smaller ones. parasites infection in different seasons showed contradictory results. in the present study, there was no significant difference between infections caused by parasites and different seasons. similar result was obtained by jerônimo et al. (2011). on the other hand, chanda et al. (2011) reported that the mortality rate was 100% in fish infected by ichthyophthiriasis in low temperature, whilst the number was reduced when temperature increased. majumdar et al. (2013) and hossain et al. (2008) reported dependence of the protozoan parasites to seasonal changes in temperature. daghigh-roohi and sattari (2004) reported three parasite species from caspian goby consisting of 2 nematodes (e. excisus and dichelyne minutus) and one acanthocephalan (c. strumosum). in the present study, two parasites, including one nematode larvae (e. excisus) and one cestode larvae were found in n. caspius, of those, cestode larvae is reported for the first time from this fish in iran. the genus eustrongylides (jägerskiöld, 1909) include three species of e. tubifex, e. ignotus and e. excisus, which are found in the gut of aquatic birds and uses oligochaetes as first intermediate hosts, fish as second intermediate hosts and likely amphibians and reptiles are second intermediate/paratenic hosts (moravec, 1994; friend and franson, 1999; lezama and sarabia, 2002). karmanova (1968) reported the roach (rutilus rutilus), bighead goby (ponticola gorlap) and round goby (n. melanostomus) as obligatory second intermediate hosts for e. excisus in estuary of the volga river. daghigh-roohi and sattari (2004) reported monkey goby (n. fluviatilis) and caspian goby (n. caspicus) as obligatory second intermediate hosts for e. excisus. this parasite was also reported from fish families such as acipenseridae, cyprinidae and certain gobiidae (sattari et al., 2003; pazooki et al., 2011). dogiel and bykhovskiy (1939) stated that transmission of e. excisus larvae to acipenserids can lead to severe damages to their muscles. according to dubinin (1952), e. excisus larvae are very pathogenic for fishes, as a result, gobiidae are important as their intermediate host. noteworthy, these larvae are found in the abdominal cavity, ovary, testis, under the skin and between the muscles. in the present study, e. excisus was found in n. caspius with high prevalence, mean intensity and abundance. the occurrence of cestode larvae and e. excisus larvae in females were higher than males which may be related to physiological conditions of females as mentioned in c. aurata. there is a significant difference between the abundance of e. excisus and different seasons which might be due to the ecological and nutritional conditions in different seasons (reimchen and nosil, 2001). furthermore, seasonal pattern varies with water temperature and dissolved oxygen, so that high prevalence of e. excisus may be related to high temperature and low dissolved oxygen. these results are in agreement with findings of banu et al. 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(2017) 5(5): 310-320 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی و( chelon aurata) طالیی کفال ماهی انگلی آلودگی شدت و شیوع میزان خزر دریای جنوبی بخش در( neogobius caspius) خزری گاوماهی 3، 2*ستاری مسعود ،2، 1پور شعبانی نادر ،1میرناطق باهره سیده .ایران رشت، گیالن، دانشگاه علوم، دانشکده شناسی،زیست وهگر1 .ایران رشت، گیالن، دانشگاه خزر دریای آبی حوضه پژوهشکده دریایی، علوم گروه2 .ایران سرا، صومعه گیالن، دانشگاه طبیعی، منابع دانشکده شیالت، گروه3 چکیده: جنوب در( neogobius caspius) خزری گاوماهی و (chelon aurata) طالیی کفال ماهی داخلی و خارجی هایانگل بررسی برای مطالعه این کیاشهر چمخاله، شامل( ایران گیالن، استان) خزر دریای جنوب ساحلی ی منطقه سه در سال یک مدت به بردارینمونه. گرفت صورت خزر دریای قرار آزمایش مورد داخلی و خارجی های انگل بررسی برای سپس شدند، تعیین ماهیان جنس و ثبت سنجیزیست هایویژگی. شد انجام انزلی و . داشتند انگلی آلودگی خزری گاوماهی عدد 170 از( % 35/ 88) نمونه 61 و طالیی کفال ماهی عدد 331 مجموع از( % 31/58) نمونه 158. گرفتند نوزاد و سستود نوزاد و( طالیی کفال در) نماتد نوزاد ناشناخته، پروتوزوآی یک رتیکوالتا، تریکودینا شامل گونه 5 به متعلق انگل 1453 کلی طوربه در ودسست نوزاد و طالیی کفال ماهی در نماتد نوزاد و ناشناخته پروتوزوآی های انگل. شدند یافت( خزری گاوماهی در) اکسیسوس استرونژیلیدس یانگینم و شیوع روی هاایستگاه نیز و میزبان وزن و طول اثرات فصلی، تنوع مطالعه، این در. شوند می گزارش ایران در بار اولین برای خزری گاوماهی .است گرفته قرار بررسی مورد انگل شدت .گاوماهیان ماهیان، کفال داخلی، انگل خارجی، انگل :کلمات کلیدی int. j. aquat. biol. (2018) 6(2): 75-87 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article histological description of the larval development of brycon gouldingi lima, 2004 (teleostei, characidae) francine faustino*1, lilian cristina makino2, erika neumann3, laura satiko okada nakaghi1 1centro de aquicultura da universidade estadual paulista (caunesp), jaboticabal, são paulo, 14.884-900, brasil. 2campus experimental de registro, universidade estadual paulista (unesp), registro, são paulo, 11.900-000, brasil. 3piscicultura buriti, nova mutum, mato grosso, 78.450-000, brasil. article history: received 3 october 2017 accepted 15 april 2018 available online 2 5 april 2018 keywords: ontogeny larval development organic systems light microscopy abstract: piabanha, brycon gouldingi, is an endemic species in the tocantins-araguaia basin. it has aroused the interest of both fish farmers, who started its creation in confinement, and riverine people who appreciate it as a food source. in order to provide information about organic systems of b. gouldingi larvae, a histological description was performed after capturing adult specimens in the rio das mortes (mato grosso, brazil), adapted to captivity and induced to spawn at buriti fisheries (nova mutum, mt, brazil). the collection of samples took place at pre-defined moments after hatching, and the processes relating to morphological differentiation of digestive, excretory, cardiorespiratory, nervous/sensory systems and gas bladder were characterized. at the hatching were found: undifferentiated digestive system; pronephros (primitive kidney), rudimentary heart, central nervous system characterized by primary vesicles, optic vesicle forming the optic cup and crystalline lens. in the course of ontogeny, these organic systems were developed and at the time of the total absorption of the yolk at 55 hours post-hatching were found: the digestive system consisting of the head gut, foregut, midgut and hindgut; two heart chambers and branching of gill arches; three regions of the brain (forebrain, midbrain and hindbrain), neuromasts, olfactory cavity, taste buds; eye consisting of well-defined layers; presence of gas bladder. the results of this study may be useful in providing support for the captive breeding of b. gouldingi during the larval stage. introduction the genus brycon, a member of the family characidae, is widely distributed in south and central america (howes, 1982). brycon species are of great importance for brazilian fish farming, because they have excellent characteristics such as high growth rates and high flavor and quality of the meat for intensive production (zaniboni-filho et al., 2006). piabanha, b. gouldingi, is an endemic species of the tocantins basin araguaia, the largest river basin lying entirely within brazil (lima, 2004). this species has a diet based on fruits and insects, living in benthopelagic freshwater environments of tropical climate. they differ from other brycons for having narrow winding longitudinal stripes (not straight) along the body, darkened pectoral and pelvic fins, distinct v-shaped mark on the caudal fin and caudal *corresponding author: francine faustino doi: https://doi.org/10.22034/ijab.v6i2.361 e-mail address: francine.unesp@gmail.com peduncle, and about 66-82 scales in a lateral line (lima, 2004). studies on b. gouldingi is rare; in the literature, only the reports of faustino et al. (2011) which analysed the fertilization and embryonic development of this species based on light and scanning electron microscopy, and faustino et al. (20015) which conducted the first experiments on the larval stage of the species, with a focus on morphological and morphometric aspects under stereomicroscope can be found. therefore, research on early development b. gouldingi is crucial since it helps fish farmers to improve its reproduction in captivity. in addition, such investigations are urgent because b. gouldingi was included in the ordinance of the ministry of environment (mma) no. 445 (17 december 2014) as endangered species classified in endangered category 76 faustino et al. / organic systems of brycon gouldingi larvae (en) in the list "brazilian fauna of extinction threatened" (brasil, 2014). the larval stage is a limiting factor for brycon farming, considering that the nutritious reserves contained in the yolk sac run out approximately 36 hours post-hatching and larvae start exogenous feeding and cannibalism practices (oliveira et al., 2004). success in farming/breeding depends on factors such as quality and quantity of available zooplankton, stocking density of larvae, water quality and homogeneity in the size of the larvae (ceccarelli and senhorini, 1996). the early development of fish is also a critical period in which the differentiation of organ systems occur (brown and nuñez, 1994). nutrition is endogenous during early post-hatching days and once it is depleted, their feeding becomes exogenous; generally, the gastrointestinal tract is not completely developed at this time, leading to morphological constraints, such as mouth size which restricts the number and size of available prey; and physiological constraints, such as incomplete development of digestive glands or incipient enzyme activity, leading to high mortality rate (govoni et al., 1986). according to zavala-camin (1996), fish larvae generally have similar rudimentary digestive tracts, a single tube format, which undergoes transformations until it reaches the characteristic of adult form. according to bértin (1958), the digestive tract assumes the following morphological divisions: gut head, which corresponds to the oral cavity and pharynx; foregut, which is equivalent to the segment involving the esophagus and stomach (when present); mid-gut (intestine itself); hindgut, which corresponds to the rectum segment (when present); anus. this terminology corresponds to the morphology of the digestive tract of adults, because fish larvae do not have well-defined segments in their digestive tract during early developmental stages. studies related to the ontogenetic development of systems and organs that especially address the digestive system allow identifying the morphological structures related to sorting, capture, digestion and absorption of food, which may assist in evaluating the factors involved in the mortality of larvae (maciel, 2006). hence, this study aimed to provide detailed information regarding larval morphological development of organic systems in piabanha b. gouldingi from the hatching upto the total yolk absorption. materials and methods animals: adult piabanha, b. gouldingi breeders from rio das mortes, mato grosso (mt), brazil were adapted for cultivation in buriti fish farming, nova mutum mt, brazil, for about seven months, and selected for induced reproduction according to woynarovich and hórvath (1983) between december 2007 and january 2008. females and males received common carp, cyprinus carpio pituitary extract to induce spawning, applied to the base of the pectoral fin. in females, the first dose was 0.5 mg.kg-1 and the second one 5.0 mg.kg-1 after 10 hours. a single dose of 1.0 mg.kg-1 was applied to males at the time of the second injection to the females. after stripping, the oocytes were placed in plastic containers and then received the semen, which was lightly homogenized. then in a few seconds, water was added to the mixture, for activation of gametes and hydration of the eggs, and washed with water to remove excess semen. the eggs were transported to 200l fiberglass conic incubators with water exchange of 6 l.s-1. the water temperature of the incubators was 25.5±0.35ºc during the early development of b. gouldingi. as an exogenous food source, larvae of the foraging leporinus piau fish was supplied to the b. gouldingi larvae at 24 hours post-hatching (hph). the experiment used a randomized design due to the descriptive nature of the research, which aimed to characterize b. gouldingi larvae through microscopic examination. 10 specimens per sampling time were collected: from the hatching larvae (time zero); at every hour up to nine hph; at every 2 hours until 33 hph; and at every 3 hours until the total yolk absorption (55 hph). this research was approved by the ethics committee on animal use (0015738-08) and 77 int. j. aquat. biol. (2018) 6(2): 75-87 conducted according to the ethical principles in animal experimentation adopted by the brazilian college of experimentation. light microscopy: samples were fixed in the modified karnovsky's solution (2.5% glutaraldehyde and 2.5% paraformaldehyde) for 24 hours and then washed and transferred to 0.1 m sodium cacodylate ph=7.4 buffer, and stored under low temperature, being selected and processed for analysis by light microscopy (historesin embedding). sample processing was performed in the histology laboratory of the morphology and animal physiology department at fcav-unesp, jaboticabal – sp, brazil. for historesin embedding (historesin plus, leica, heidelberg, germany), the samples were dehydrated for 24 hours in 80% ethanol and then washed twice in 95% and 100% alcohol for 30 minutes each. afterwards, the samples were stored for 4 hours in a pre-embedding solution consisting of gma (glycol methacrylate) + ethanol (1:1) and 16 hours in the embedding stage (gma), for further inclusion in histomold. the samples were placed in a drier at 50°c for 24 hours. 2.0 mm semi-seriated histological sections (five sections were discarded) were obtained in a leica rm2255 microtome using a tungsten razor. section staining was performed with hematoxylin-phloxine (tolosa et al., 2003). slide analysis and photo documentation were obtained using a leica dm 5000 b light microscope with leica application suite (las) software. results brycon gouldingi larvae hatched 14 hours postfertilization and total absorption of the yolk happened at 55 hph. by monitoring the larval development, we were able to observe the morphological differentiation processes of the digestive, excretory, cardiorespiratory, nervous/sensory systems and gas bladder. digestive system: at the hatching, the digestive tract was undifferentiated with an identical epithelium continuous to the integument directly penetrating in the front of yolk sac, marking the future site of the oropharyngeal cavity (fig. 1a). this epithelium was sometimes observed as a single layer of cells extending over the yolk sac, and other times as several layers of undifferentiated cells where the intestine differentiates later (fig. 1b). the primordia of an oropharyngeal cavity and observation of an oral cleft, marking the location where upper and lower lips would separate later at 1 hph (fig. 1c). a small lumen was appeared in the digestive tube, at the intestinal region at 2 hph (fig. 1d), however its posterior part was closed (fig. 1e). the oropharyngeal cavity was formed at 5 hph, but the lips remained attached and the anterior part of the digestive tube was also closed by a connective tissue (fig. 1f). the separation of the upper and lower lips and opening of the oropharyngeal cavity were occurred at 9 hph, but the initial portion of the digestive tract remained closed (fig. 2b), with the first dental alveoli in the pre-maxilla (upper lip) and dental bone (lower lip) being visible (fig. 2c). the teeth were more elongated at 11 hph (fig. 2d). at 13 hph, the digestive tract consisted of the oropharyngeal cavity, esophagus (as a short and narrow tube) and intestine; however connection between gut head and foregut could not yet be identified (fig. 2e), and the posterior part of the digestive tube was opened (fig. 2f). it was also possible to identify a portion of the pancreas (fig. 2e). at 23 hph, the teeth in the lips were more protruding, covered by a simple undefined epithelium (fig. 3c). at this time, the pleating of the bowel was observed (fig. 3d). the pharynx was observed at 29 hph, formed by epithelial mucus cells, while the esophagus covered by a simple cylindrical epithelium (fig. 3e). a small portion of the liver and pancreas was also observed (fig. 3e). at this time, the digestive tube opened along its entire length. the intestine is dilated with villi and lined by simple cylindrical epithelium (fig. 3f). the presence of foraged larvae (supplied l. piau at 24 hph) in the gut of b. gouldingi larvae was detected at 32 hph, almost occurring from this point onwards, marking the functionality of the digestive tract. at 33 hph, the superficial cells of the pharynx presented cytoplasmic processes protruding inwards and 78 faustino et al. / organic systems of brycon gouldingi larvae pharyngeal teeth were also observed (fig. 4c). the pancreas presented an acinar aspect with basophilic nucleus and acidophilus apical cytoplasm with zymogen granules (fig. 4d), while liver hepatocytes presented vacuolar cytoplasm presumably due to containing high lipid and glycogen concentrations resulting from the yolk or consumed larvae (fig. 4e). the intestinal epithelial presented brush borders and the presence of digested material noticed in the intestine (fig. 4f). at this time, the bowel wall was composed of a simple cylindrical epithelium including goblet cells (fig. 4f). at 36 hph, the dental alveoli were visible on the ceiling of the pharynx that in the figure 1. photomicrographs of brycon gouldingi larvae. hatching: (a) significant presence of yolk sac (ys); epithelial tegument penetrating and defining the location of the oropharyngeal cavity (asterisk); developing heart (circle) developing eyes with the presence of optic cup (oc) and crystalline lens (cl), (b) pronephro (pro) and intestine (in) being defined, 1 hph: (c) primitive oropharyngeal cavity (arrow); oral cleft (ocf) marking the site of separation of upper and lower lips; developing central nervous system (cns) and developing eyes with the presence of optic cup (oc) and crystalline lens (cl), 2 hph: (d) pronephro (pro) and intestine (in) with a small visible light (lumen), (e) final portion of pronephro (pro) and intestine (in) and 5 hph: (f) oropharyngeal cavity developed (arrow); anterior region of the digestive tract closed (square); two chamber heart (circle); developing central nervous system (cns) and developing eyes with the presence of optic cup (oc) and crystalline lens (cl). 79 int. j. aquat. biol. (2018) 6(2): 75-87 future will originate pharyngeal teeth (fig. 5b). figure 5c shows an ingested forage larva at 39 hph, portraying mixed feeding, since the larvae ingestion occurred before the endogenous reserve (yolk sac) was depleted. many dental alveoli were present and incisor teeth formed protrusions, indicating that their externalization could happen shortly (fig. 5d). at 48 hph, it was possible to identify the septum separating the (actual) midgut and the hindgut (fig. 6a), digested material (probably consumed forage larva) was found in the midgut leaving it widely dilated (fig. 6a). at this point, the yolk was almost disappeared. at 55 hph, the yolk sac had already been fully absorbed, and we observed some b. gouldingi larvae having more than one foraged larvae within their gut (fig. 6b). at this moment the digestive system consisted of the head gut (buccal cavity and pharynx), foregut figure 2. photomicrographs of brycon gouldingi larvae. 7 hph: (a) anterior region of the digestive tract closed (square); developing central nervous system (cns) and developing eyes with the presence of optic cup (oc), optic vesicle (ov), hyaloid artery (ha) and pigment layer emerging (pi). 9 hph: (b) separation of upper and lower lips (asterisk); anterior region of the digestive tract closed (square); developing central nervous system (cns), (c) dental alveoli (da), 11 hph: (d) primitive teeth (t), 13 hph: (e) pharynx (pha), esophagus (es), intestine (in) and pancreas (p) and significant presence of the yolk sack (ys) and (f) pronephro (pro); end portion of the digestive tract open (circle). 80 faustino et al. / organic systems of brycon gouldingi larvae (represented by the esophagus), midgut (gut itself) and hindgut. the esophagus showed no significant changes in its structure throughout this study. most teeth remained covered by the epithelium, while many dental alveoli and some exteriorized teeth were observed. during the study period, there was also no differentiation of the stomach or gastric glands which probably occur later. differentiation of the rectal portion and the anus was also not verified. excretory system: the pronephros (primitive kidney) observed from the hatching, just above the gut, following the entire length thereof and consisting of a simple cylindrical epithelium (fig. 1b). a small lumen appears in the pronephros at 2 hph (fig. 1d, e). with larval development at 39 hph, the cranial portion figure 3. photomicrographs of brycon gouldingi larvae. 17 hph: (a) branching of gill arches (arrow); developing central nervous system (cns); otic vesicle (ot) with neuromasts (arrowhead); developing eyes with the presence of well-formed crystalline lens (cl), pigment layer (pl) and plexiform (ple) emerging, 21 hph: (b) esophagus (es); intestine (in); pancreas (p); superficial neuromast (arrowhead); gas bladder (gb); yolk sac (ys), 23 hph: (c) primary teeth (t); neuromast (arrowhead) near the eyes (e); presence of olfactory cavity (olf), (d) pronephro (pro); intestine circumvolutions (in), 29 hph: (e) pharynx (pha); esophagus (es); intestine (in); liver (l); pancreas (p); gas bladder (gb) and (f) middle portion of the intestine dilated (in). 81 int. j. aquat. biol. (2018) 6(2): 75-87 of the pronephros located in the dorsal part of the body cavity, above the gas bladder, becomes coiled and its lumen is more apparent (fig. 5e), while the caudal region remains straight (fig. 5c). cardiorespiratory system: a rudimentary heart is observed in the pericardial cavity, anterior to the yolk sac and the abdominal cavity at hatching (fig. 1a). two heart chambers (atrium and ventricle) were observed at 5 hph (fig. 1f). the branching of gill arches was detected at 17 hph (fig. 3a). nervous/sensory system: development of the central nervous system was characterized by initially being presented as primary vesicles at hatching (fig. 1a). at this moment, the optic vesicle (first embryonic stage of the eye) had already invaginated, forming the optic cup, as well as the crystalline lens (fig. 1a). figure 4. photomicrographs of brycon gouldingi larvae. 31 hph: (a) neuromasts (arrowhead); well-formed crystalline lens (l); pigment layer (pl) and photoreceptor layer (two asterisks) developing; evident plexiform layer (ple); olfactory cavity (olf), (b) olfactory cavity with cilia (olf), 33 hph: (c) pharynx (pha) with cytoplasmic processes (star), esophagus (es); liver (l); gill arches (arrow); primary pharyngeal teeth (dotted arrow), (d) gas bladder (gb); pancreas (p); cranial portion of pronephro coiling (pro), (e) liver (l) and (f) digested material (asterisk) in the intestine (in). 82 faustino et al. / organic systems of brycon gouldingi larvae at 7 hph, the hyaloid artery and pigment layer appeared in the peripheral region of the optic cup (fig. 2a). at 9 hph, development of the central nervous system was evidenced by observing the cells of the primitive vesicles filling out (fig. 2b). the eye lens was well-formed at 17 hph, and the plexiform layer began to emerge (fig. 3a). larvae showed free neuromasts inside the otic vesicle (fig. 3a) and also throughout the body surface in the cephalic region (fig. 3b). at 23 hph, several neuromasts were observed near the orbital optical vesicles (fig. 3c). at 31 hph, the plexiform layer of the eye was better visualized, and the double layer of photoreceptors could also be distinguished, with surface neuromasts (fig. 4a). the olfactory cavity was composed of the ciliated pseudostratified cylindrical epithelium (fig. 4b). the central nervous system was well-developed at 36 hph, figure 5. photomicrographs of brycon gouldingi larvae. 36 hph: (a) well-defined prosencephalon (pro), mesencephalon (mes) and rhombencephalon (rom); neuromasts (arrowhead); yolk sac (ys); pancreas (p), (b) presence of dental alveolus (dotted arrow) on the roof of the pharynx (pha); yolk sac (ys), 39 hph: (c) ingestion of forage larva (circle); notochord evident (no); pronephro (pro); intestine (in); yolk sac (ys), (d) tooth (t); taste buds (tb) and (e) partially inflated gas bladder (gb); cranial portion of the pronefro coiled (pro). 83 int. j. aquat. biol. (2018) 6(2): 75-87 when the three regions of the brain prosencephalon (forebrain), mesencephalon (midbrain) and rhombencephalon (hindbrain) were distinct (fig. 5a). the notochord was well characterized at 39 hph, (fig. 5c and 5e) extending throughout the body length of the larvae. the cells showed vacuolar cytoplasm. the presence of taste buds was also noticed (fig. 5d). the eye was well-developed at 55 hph, consisting of well-defined lens, pigment layers (outer) and well prominent plexiform, in addition to the dual layer of photoreceptors (fig. 6c). several neuromasts were seen around the eye orbital (fig. 6d). gas bladder: at 21 hph, the formation of the gas bladder began, and connected to the esophagus by a figure 6. photomicrographs of brycon gouldingi larvae. 45 hph: (a) digested material (asterisk) in the intestine (in); notochord evident (no); small remaining portion of the yolk sac (ys). 55 hph: (b) presence of forage larvae (circles) in the intestine; septum between middle and hindgut (square). (c) well-developed eyes with well-formed crystalline lens (l); pigment layer (pl), plexiform layer (ple), photoreceptor layer (two asterisks) and evident optic nerve (opn). (d) olfactory cavity (olf); neuromasts (arrowhead) around the eyes (e). 84 faustino et al. / organic systems of brycon gouldingi larvae connector duct (fig. 3b). it was formed and covered by a stratified squamous epithelium at 33 hph (fig. 4d), located below the notochord and above the digestive tract. at 39 hph, it was partially inflated (fig. 5e). discussion in most fish species, newly hatched larvae have simple, undifferentiated digestive tracts (govoni et al., 1986). indeed at the time of hatching, b. gouldingi presented a rudimentary digestive tract, while at the time of total yolk absorption the digestive tract divided into gut head, foregut, midgut and hindgut, the same classification adopted by bértin (1958). rodrigues et al. (2006) have stated that the mouth, oral cavity and pharynx, which constitute the head gut, are associated to the capture, guidance, and predigestive preparation of food. they also suggest that the thicker lips and oral teeth help to hold food, which in omnivorous species are used for pre-digestive preparation of plant food material, and to capture and hold animal prey. the pharyngeal teeth observed in b. gouldingi assist in capturing and macerating softbodied organisms. when exogenous feeding was initiated, the mucus cells were also found in the pharynx of b. gouldingi, and according to galvão et al. (1997), these cells participate in gliding prey. the presence of mucous cells was also observed in the esophagus, indicating that along with the development of the folds in the esophagus, which precede the differentiation of the stomach, these cells facilitate the rapid transition of food to the posterior segment (gonzáles et al., 2002; chen et al., 2006; yang et al., 2010). until the total yolk absorption, the esophagus in b. gouldingi showed no significant differentiation. according to zavala-camin (1996), the esophagus in fish is a tubular body that serves as a passageway between the oral-pharyngeal cavity and the stomach; and in the case of physostomous species and similarly in this study, with the pneumatic duct opening of the gas bladder in the esophagus. the formation of the stomach and gastric glands was not observed in b. gouldingi until the total yolk absorption. in that period, according to watanabe and kiron (1994), fish depend on the ability to select food, mechanical digestion, and pancreatic and intestinal enzymes which act in an alkaline medium to compensate for the absence of gastric enzymes. the efficiency of digestion and absorption of food is due to the presence of folds and microvilli in the intestines, considering that a mature liver and pancreas help in the digestion of food for producing digestive secretions which digest proteins, fats and sugars (gonzáles et al., 2002). a differentiated liver and pancreas are usually observed in larvae that had already begun exogenous feeding soon after hatching and which are functional before the yolk is completely absorbed; a fact confirmed in paralabrax maculofasciatus (peña et al., 2003), seriola lalandi (chen at al., 2006), pelteobagrus fulvidraco (yang et al., 2010), and also found in this study. according to gonzález et al. (2002), the presence of folds in the intestine, as observed in b. gouldingi, indicate an increase in efficiency of food digestion and absorption. seixas-filho et al. (2000) have suggested that mucosal transverse folds slow food passage, allowing for a greater digestive period and better utilization of nutrients in the midgut, as this type of arrangement does not occur in the hindgut. goblet cells were also observed in the midgut portion and, as reported by george et al. (1998), these cells secrete basic mucus, with the function to neutralize stomach acid and prepare the food bolus for the action of pancreatic and intestinal enzymes that act in a basic medium. regarding the development of the excretory system, the pronephros was present from the hatching of b. gouldingi larvae, and according to kimmel et al. (1995), it developed in the early stage of somitogenesis. according to drummond et al. (1999), pronephros only differentiate into mesonephro in youth/adult individuals. the heart is the first organ to develop and become functional during embryogenesis (hu et al., 2000). in b. gouldingi, the heart was rudimentary at the hatching; it was located in the pericardial cavity anterior to the yolk sac and the abdominal cavity, 85 int. j. aquat. biol. (2018) 6(2): 75-87 similarly to what has been reported in newly hatched larvae from other studies (hu et al., 2000; falkpetersen, 2005). according to bone et al. (1995), most teleost larvae hatch performing gas exchanges through the skin; this type of breathing is appropriate since many of these larvae are transparent and inhabit pelagic regions, where oxygen is plentiful and the required hemoglobin could make them visible to predators. however, with the growth of the larvae, the cutaneous respiration becomes inefficient, being performed through the gills. the protuberance from which the gill arches will emerge was visualized in b. gouldingi soon after hatching, and became well-developed after the total yolk absorption. leonardo et al. (2001) reported that gills are vital structures for fish health, since they are the main site for gas exchanges, as well as also being involved in osmoregulation processes, acid-base balance and excretion of nitrogenous compounds. the first sensory structures to be visualized in b. gouldingi larvae were the eyes, olfactory cavity, then the otic vesicle and neuromasts, while taste buds were the last to be identified, as reported by uyan et al. (2006) for eugraulis japonicus larvae. during larval development, the eye is the sensory organ that plays a key role in detection of prey and escape from predators (moorman, 2001). the eye development in b. gouldingi happened gradually. in the larvae when yolk was absorbed, the eye was developed in the last moment of observation and had its main layers formed and the nuclei of the rod and cone cells were starting to form two layers, similarly to larvae of oreochromis niloticus (morrison et al., 2001). the hyialoid artery found in this study irrigated the optic cup and the crystalline lens under development. vision is critical to fish survival, especially after the start of exogenous feeding, since most species are considered visual consumers, as well as to avoid predators (carvalho et al., 2004). nascimento et al. (2015) showed that feeding behavior of betta splendens larvae was particularly dependent visual ontogeny, since the larvae were able to capture artemia more efficiently with the progress of development, when there was a greater eye movement of larvae in the presence of food. the olfactory plate was present in b. gouldingi from the hatching, corroborating reports by hansen and zeiske (1993), which stated that the differentiation of the olfactory plate in embryos and fish larvae is fast; however, the process is slow until complete formation. the olfactory organs act as chemoreceptors to capture the smell of the saturated substances in the water and consequently, the food supply (matsuoka, 2001). the neuromasts are mechanoreceptors superficially located in the skin, which can be presented as free neuromasts and/or grooves and channels, forming the lateral line system. in b. goulgindi, neuromasts are observed in the lateral line, but they were also observed close to the eye sockets. according to gilbert (2010), the anterior portion of the neural tube undergoes dramatic changes and expands into three primary vesicles: forebrain (prosencephalon), midbrain (mesencephalon) and hindbrain (rhombencephalon). in larvae of b. gouldingi, the central nervous system has been completely filled observed for cells and delimited in the three portions at 36 hph. the development of the central nervous system is related to the onset of aggressive behavior in fish larvae such as cannibalism (sakakura and tsukamoto, 2002). the gas bladder, observed as an outline in b. gouldingi located in the dorsal part of the body, as other fishes above the center of gravity of the fish allowing them to maintain posture without relying on muscular effort (hidelbrand, 1995). godinho et al. (2003) reported that the rise of gas bladder (along with the emergence of the pectoral fins) are remarkable events of larval ontogeny, since they facilitate the balance and direction in the water column. based on the results, it can be concluded that the development of b. gouldingi was quick, a common characteristic among species of freshwater teleost, with simultaneous differentiation of structures that allow capturing of food early in the development before endogenous energy reserves run out, and also ensuring they can escape predators, increasing the 86 faustino et al. / organic systems of brycon gouldingi larvae chance of survival during the larval stage. this information is unprecedented for the b. gouldingi species and indispensable for farmers who already produce or intend to start farming of b. gouldingi; thus contributing to better performance and production in captivity. acknowledgments the authors thank buriti fisheries (nova mutum, mt, brazil) for providing the eggs and fish and to o. mateus, histo-technician from the histology and embryology laboratory of fcav/unesp, jaboticabal-sp, brazil, for his help in material processing; f.f. acknowledges fapesp for the award of a masters scholarship (2007/57826-7) and cnpq (473712-2007-5) for financial assistance. references bértin l. 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(2019) 7(3): 140-145 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article sub-lethal effects of potassium dichromate on hematological and histological parameters in climbing perch, anabas testudineus (anabantidae) liya vazhamattom benjamin1,2, ranjeet kutty *1 1department of aquatic environment management, kufos, kochi, india. 2nicra project, central marine fisheries research institute, kochi, india. s article history: received 5 march 2019 accepted 24 april 2019 available online 2 5 april 2019 keywords: heavy metal hematology histopathology chromium abstract: chromium, which enters the river through anthropogenic sources, is one of the potent heavy metals. the present study is an attempt to determine the lc50 of potassium dichromate for the climbing perch, anabas testudineus and to study the impact of two sub-lethal concentrations (6 and 12 mg/l) of potassium dichromate the toxic hexavalent cr(vi) form of chromium on this fish through investigating hematological and histopathological parameters. experimental set up included quadruplicate treatments for each dosage, and the results were compared with control treatments. the results showed that the lc50 value at 96 hr was 59.92 mg/l. the fishes exposed to sub-lethal concentrations showed severe abnormalities such as; degeneration of hepatocytes, necrosis of hepatic tissue and extensive haemorrhage in gills and renal tissue. the present study brings out the harmful impact of cr(vi) in the aquatic environment and necessitates regulations of its inflow to natural water bodies as a management plan to curb its contamination. introduction water pollution has been regarded as one of the most important threats globally impacting human health, environment and sustainable development (world water assessment programme, 2018). although there has been a much understanding on the impact of the addition of anthropogenic contaminants to the environment, the natural aquatic bodies have extensively been polluted with heavy metals released from domestic, industrial and other man-made activities (conacher et al., 1993; velez and montoro, 1998). according to the united states environmental protection agency (usepa), and the international agency for research on cancer (iarc), these metals are also classified as human carcinogens based on the epidemiological and experimental studies (tchounwou et al., 2012). among the various metals, chromium (cr) has turned out to be a vital pollutant, which has the potential to be toxic to living organisms due to their bioaccumulation and non-biodegradable properties (velma, 2009). with increasing industrialization and fewer means for safe disposal, *correspondence: ranjeet kutty doi: https://doi.org/10.22034/ijab.v7i3.604 e-mail: ranjeet@kufos.ac.in contaminations of cr in the aquatic environment is often being regarded as a menace in india. the situation gets even worse since its availability in nature either as dichromate in acidic environments or as chromate in alkaline environments has rampantly infiltrated into the drinking water system (risikesh et al., 2007). the permissible levels of cr for drinking water recommended by the indian drinking water quality standard (is: 10500:2012) is 50 µg/l. hexavalent chromium, cr(vi) is highly carcinogenic and may cause death to animals and humans if ingested in large doses (zyed and terry, 2003). this form of chromium rarely occur naturally but is produced from anthropogenic sources and profoundly used in industry for metal planting, cooling tower, water treatment, tanning, and wood preservation (palmer and wittbrod, 1991). among the various compounds, the dichromate compounds, especially k2cr2o7, is the most profoundly found form of chromate in india, mainly due to its wide industrial application. fishes are more prone to such pollution due to their 141 int. j. aquat. biol. (2019) 7(3): 140-145 continuous exposure to toxicants as well as bioaccumulation potential, which makes them good biological indicators of heavy metal toxicity (hedayati et al., 2010). the effects of toxicant in fish as studied from a haematological and histological perspective have been used as successful biomarkers for assessing the toxic effects of xenobiotics (yancheva et al., 2015). histological alterations in the tissues of liver, kidney, and gills of fish are sensitive biomarkers for metal pollution that provide for a better evaluation of the effects of pollution (poleksic et al., 2010). under the above background, a study was conducted to assess the sub-lethal concentration of chromium (k2cr2o7) on the selected tissues of climbing perch, anabas testudineus (bloch, 1972), to bring out the immediate impact of cr(vi) toxicity on this freshwater fish. materials and methods about 100 healthy adult a, testudineus (bloch, 1972) weighing between 40-45 gr and length 10-15 cm were procured from pulimugham hatchery, thakazhi, kerala. they were transported to the laboratory in anaesthetized condition, examined for any pathological symptoms, treated with 0.1% kmno4 solution to avoid any dermal infection and acclimatized in dechlorinated tap water for 30 days. fishes were raised in wide-mouthed glass tanks of 600 l capacity. stocking density was maintained at a rate of one fish per 30 l. adequate aeration was given without vigorous bubbling of the water. water was replaced with filtered dechlorinated water every 2 to 3 days, depending on the water quality. fishes were adequately fed with commercially available crumble fish feed (cp feeds), and any feed waste was siphoned out. at the end of the acclimatization phase, healthy fishes were selected and maintained in separate aquaria before actual experimentation. water was maintained at ph 7.4±0.3; d.o. 6.6±1.2 mg/l; total alkalinity 310±52 mg/l and temperature of 28±1ºc. a stock solution of k2cr2o7 was prepared, and their dilution was made according to standard guidelines (apha, 2012). to determine the appropriate range of toxicity, a series of different concentrations was prepared (in triplicate), and the sublethal toxicity of the heavy metal was determined following brungs et al. (1977). the fishes were not fed during the period of exposure. the water in the aquaria was replaced every 24 hr. the mortality data were used to calculate the 96 hr lc50 value. the lc50 value at 96 hr was statistically evaluated using probit analysis method (finney, 1952). this was followed by the analysis of the effect of sub-lethal concentration of k2cr2o7 on the selected tissues of a. testudineus. for this based the results of lc50, two sub-lethal concentrations of k2cr2o7 approximately 1/5th and 1/10th the value of the lethal concentration, i.e., 12 and 6 mg/l, respectively were selected. the experimental set-up included quadruplicate tanks for each treatment. t1 was maintained as control tanks devoid of any k2cr2o7, while t2 and t3 were treatments with 12 and 6 mg/l of k2cr2o7, respectively. the fishes in these treatments were exposed to k2cr2o7 for 15 days. the fishes were moderately fed during the period of exposure. the water in the aquaria was replaced every 24 hr. at the end of the 15-day exposure period, the hematological and histological changes in the gill, liver, and kidney of the a. testudineus were analyzed. haemoglobin (hb%) was measured by sahli's acid haemoglobin method (hesser, 1960). rbc counts were taken in neubauer's hemocytometer using hendrick's solution as diluting fluids. packed cell volume (pcv) was determined by the microhaematocrit method. differential leucocyte count (dlc) was carried out by preparing a thin blood smear and staining it with leishman’s stain. calculation of mean corpuscular haemoglobin (mch), mean corpuscular haemoglobin concentration (mchc) and mean cell volume (mcv) was done using standard formulae (dacie and lewis, 1982). mean values were compared using one-way anova and post hoc analysis (duncan multiple range tests) following ranjeet et al. (2013) using statistical software (spss 17.0 for windows). results the calculated 96 hr lc50 value of potassium 142 benjamin and kutty / effects of k2cr2o7on hematological and histological parameters in climbing perch dichromate for a. testudineus was found to be 59.92 mg/l in a static bioassay with aeration (table 1). acute toxicity tests of k2cr2o7 showed a direct relationship between mortality and concentration of the chemical, with no mortality recorded from the control groups. table 2 shows the impact of two sub-lethal concentrations of k2cr2o7 on the haematological parameters. inversely to the increasing concentration of k2cr2o7, a decrease in the haematological parameters such as total rbc, hb, and pcv could be seen, while that of total wbc count increased proportionately with concentration. duncan’s multiple range test (dmrt) analysis revealed the differences between the three treatments. the results showed that most of the haematological variables were significantly (p<0.01) different within the two sub-lethal concentrations indicating that even a slight change in the sub-lethal concentration had a direct bearing on the haematological parameters. studies on the morphology of a mature erythrocytes showed that healthy a. testudineus had an elliptical erythrocyte and nucleus, which was centrally located (fig. 1a). however, under sub-lethal concentrations of k2cr2o7 the cell wall become crenate and the cells club together. presence of hypochromic red cells (hrc) in fishes exposed to sub-lethal concentration indicates a disproportionate reduction of red cell hemoglobin, which ultimately table 1. probit analysis for anabas testudineus during a 96 hr exposure to various concentration of potassium dichromate exposure period (hr) lc50 (mg/l) 95% fiducial limits (mg/l) slope function (b) intercept (a) r value lower upper 96 59.92 47.59 75.44 3.657 -1.501 0.924 table 2. effect of potassium dichromate on hematological indices of anabas testudineus (mean ± sd) parameter t1 t2 t3 heamoglobin (gr%) 11.77±0.85 a 6.70±0.10 b 10.17±0.25 c tbc (x 106/cmm) 8400.00±360.56 a 11000.00±1000 b 9700.00±200 c pvc (%) 32.63±2.14 a 20.93±0.45 b 32.00±1.45 a mcv(fl) 123.70±10.52 a 73.27±0.47 b 100.93±1.23 c mch (pg) 42.53±3.01 a 23.33±0.38 b 33.03±1.61 c mchc (%) 35.10±1.25 a 31.67±0.15 b 33.23±1.03 a,b rbc 3.30±0.26 a 2.47±0.40 b 2.82 ±0.32 a,b neutrophils (%) 17.68±0.58 a 12.00±1.00 b 21.67±0.58 c lymphocytes (%) 75.00±1.00 a 81.00±1.00 b 72.00±2.65 a eosinophils (%) 3.00±0.00 a 1.00±0.00 b 4.67±0.58 c monocyte (%) 3.00±0.00 a 4.66±0.57 b 3.00±0.00 a figure 1. morphology in the blood corpuscles of control fish (1a) and those exposed to sublethal concentration k2cr2o7 (1b). presence of crenated red cell (crc), white cell (wc) and hypochromic red cells (hrc) in exposed fishes. 143 int. j. aquat. biol. (2019) 7(3): 140-145 affects the fish physiology (fig. 1b). the results of histopathological studies suggested that once exposed to 1/5th of the sublethal concentration of potassium dichromate, remarkable morphological alterations in the ultrastructure of gill, liver and kidney tissues could be noticed. the histological analysis in the treated fish showed abnormalities in secondary gill lamellae (sgl); lamellar fusion (lf), epithelial lifting (el) and haemorrhage (he) (fig. 2a, b). microscopic examination of the treated liver showed degenerated hepatocytes (fig. 3). the hepatic tissue of fish exhibited additional structural alteration such as dilated sinusoids, a modest collection of blood in the liver parenchyma (he), partial necrosis (nc). similarly, the renal tissue also showed marked alteration such as edema, interstitial haemorrhage, and degeneration of renal tubules (drt) (fig. 4). therefore, the results illustrate the severe impact of sub-lethal concentration of k2cr2o7 on the blood and vital organs of a. testudineus clearly. discussions it is evident from the present study that the lethal effect of k2cr2o7 in a. testudineus could be directly attributed to reduced hb levels. the reduction of figure 2. histological section showing gill of exposed fish to sublethal concentration of k2cr2o7. hyperplasia in lamellar epithelium leading to lamellar fusion (lf), epithelial lifting (el) and curling of secondary lamellae (sgl) due to exposure to dichromate. figure 3. histological section showing hepatic tissue of exposed sublethal concentration of k2cr2o7. presence of hepatic haemorrhage (he) necrosis (nc) and degenerated hepatocyte (dh) in hepatic tissues. figure 4. histological section showing renal tissue of exposed sublethal concentration of k2cr2o7. exposed fishes with degeneration of cortical tubules epithelial cell (ed), renal corpuscles (rc), renal haemorrhage (he) and degeneration of renal tubules (drt). 144 benjamin and kutty / effects of k2cr2o7on hematological and histological parameters in climbing perch haemoglobin affects the oxygen binding capacity (mini, 2014) and also manifest anaemic condition in fish, which may be due to the stress related hemolysis (mallesh et al., 2004). cr(vi) is highly water soluble, easily penetrable through the biological membranes and causes cellular damage by inducing oxidative stress (irwin et al., 1997; begum et al., 2006). the study also shows various abnormalities in the morphology of erythrocytes which is related to the capacity of cr(vi) to disintegrate erythrocytes and leaving the nuclei free in the blood film, as well as cell fragments (smith, 1968). the reduction in hematocrit values is an indication of anaemia or oligohemia (wepener et al., 1992). the lowered mcv and mch values were observed among the 12 mg/l concentration treatments (t2) indicating the possibility of microcytic anaemia. changes in mch may be due to increased lysis of rbcs and reduction in cellular blood iron, which reflects a decline in hb content due to metal toxicity (sharma and langer, 2014). in the present study, leucocytes count in fish of t2 was high compared to control (t1), and this indicates protective response during cr(vi) exposure (bhatkar, 2011). the changes observed in the blood cells were corresponding to the concentration of the toxicant and duration of the exposure time. the cr(vi) exposure exhibited marked degenerative changes in the histology of gills, kidney and liver tissues. in the present findings, fusion of gill lamellae was observed, and it seems to be resulting from changing and/or coagulation of mucus through altering the composition of glycoprotein as reported by khalesi et al. (2016). the present study also revealed that there is a strong link between liver damage and chromium toxicity, which results in extensive hepatocyte necrosis with chronic inflammation. the kidney is yet another organ susceptible to sub-lethal doses of k2cr2o7. in the present study, degeneration of tubular epithelial cells and tubular necrosis was observed at higher concentrations of cr(vi) exposures, which this may be due to the accumulation of inflammatory cells associated with cr toxicity (kurtović et al., 2008). conclusion the present study is an in situ analysis of the effect of cr(vi) that enter the aquatic ecosystem through effluents discharged from various industries. fishes are highly prone to chromium toxicity as they assimilate the metal by ingestion or through gill uptake and accumulate it in hepatic and renal tissues. the overall toxic impact on organs like gill, kidney, and liver may seriously affect the metabolic, physiologic activities and could impair the growth and behaviour of fish. the present study brings out the impact of potassium dichromate in freshwater airbreathing fish a. testudineus. exposure to the sublethal concentrations of this metal resulted in significant changes in hematological and histopathological parameters. acknowledgements the authors would like to thank n.n. raman, head, department of aquatic environment management for providing the necessary facilities, a. gopinath and m. sebastian for their technical support and kufos for providing the financial support. references agrawal a., ravi s.p., bechan s. 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(2013) 1(3): 100-108 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article can the pamolare 1-layer model predict eutrophication in hypertrophic lakes? a case study: the zaribar lake, iran amir hossein hamidian*,1mansooreh hasanzadeh department of environment, faculty of natural resources, university of tehran, p.o. box: 31585-4314, karaj, iran. article history: received 30 april 2013 accepted 23 may 2013 available online 2 0 june 2013 keywords: eutrophication hypertrophic pamolare zaribar lake abstract: eutrophication is known as the most common problem in water bodies, caused by high concentrations of different nutrients leading to unbalanced growth of aquatic plants, among other symptoms. hence, the possibility of eutrophication prediction can be beneficial to the sustainable management of these natural resources and create an opportunity to control their trophic conditions over time. a software package applied for generating these predictions is pamolare with its different models (layers). the 1-layer model of this method was selected to investigate the trophic condition of hypertrophic zaribar lake. prior to 2012, water samples were collected from six stations over a seven-year period. during the last year of this period, sediment samples were also collected. the concentrations of n and p were measured in the samples. the initial results showed that the zaribar lake is a hypertrophic water body. applying the pamolare 1-layer model showed that this model was not powerful enough to predict the trophic changes in this hypertrophic water body and suggested that other models should be examined and modified for use in these ecosystems. alternatively, it is necessary to improve the software for the prediction of eutrophication in hypertrophic water bodies. introduction water bodies such as lakes and wetlands provide facilities and services for the human beings, as a result of their high levels of production and their rich and unique biodiversity (herath, 2004; punt et al., 2010). in more than half of these ecosystems, the quality of these ecosystem services has been reduced due to environmental degradation (zedler and kercher, 2005). human activities have doubled the annual rate of nitrogen fixation and caused an increase of 75% in phosphorus accumulation in lakes and wetlands (vitousek et al., 1997; bennett et al., 2001). due to the acceleration of this trend in past decades, the water resources have faced a severe environmental problem, i.e. eutrophication (mitsch and jorgensen, 2003). among the numerous factors causing eutrophication in the water resources, the * corresponding author: amir hossein hamidian e-mail address: a.hamidian@ut.ac.ir accumulation of nutrients such as nitrogen and phosphorus is the most important factor. the response of these water bodies to the accumulation of nutrients results in a decrease in light penetration into the water column, increase in water temperature, and changes in biological, chemical and physical processes (zha et al., 2010). eutrophication is characterized by rapid reproduction of phytoplankton and other microorganisms and decreased water quality, both of which are harmful to the ecological conditions of lakes and wetlands and affect their natural functions (oecd, 1982). kautsky (1998) used salinity to investigate eutrophication and environmental pollution in lakes and wetlands. he found that there was an inverse relationship between salinity and the concentration of organic matter in these water 101 hamidian and hasanzadeh/ int. j. aquat. biol. (2013) 1(3): 100-108 resources. to investigate eutrophication in coastal areas, dale (2009) studied dinoflagellates populations in surface sediments. he found that climate change affects the eutrophication trend. reducing the various forms of nitrogen and phosphorus compounds is the most effective way of restoring eutrophic water bodies, which have been affected by agricultural pollutants (ardo’n et al., 2010; duff et al., 2009; bruland and richardson, 2005). total nitrogen and phosphorus concentration in the water column have been reported to be the best indicators of eutrophication trends and prediction (bobbink et al. 1998; gunnarsson and rydin, 2000; gunnarsson et al., 2004; koshikawa et al., 2005; tsujian et al., 2010). kokfelt et al. (2010) showed that in a lake in northern sudan concentrations of carbon and nitrogen increased during several years. in a similar study, larsen and hervey (2010) found interactions between plant growth, hydrology and sediment transport. hydrologic and biogeochemical conditions, plant and animal community status and the concentrations of nutrients were found to be a proper base for identification of trophic level of wetlands (richardson, 2010). the nutrient loadings have considerable effects on plant growth and consequently on eutrophication trends (tsujian et al., 2010). other studies showed that the concentrations of nitrogen and phosphorus in sediments are appropriate indicators of eutrophication (olander and vitousek, 2000; vicente et al., 2010; wang et al., 2010). due to the critical role of eutrophication in the limnology of lakes and wetlands, an evaluation of trophic status is the first step in their conservation (vicente et al., 2010). therefore various models have been introduced for the management of eutrophic wetlands, which are based on the physical, chemical and biological properties of lakes and wetlands. pamolare was first introduced at the “3rd international workshop on regional approaches for the development and management of reservoirs in the la plata basin,” in 2001. this methodology was produced at kyoto university at the request of unep-ietc (the united nations environment programme international environmental technology centre) and iucn (the international union for conservation of nature) (jørgensen et al., 2003). this methodology has been used and improved for management and control of eutrophication in eutrophic lakes and water resources (gurkan et al., 2006). structurally dynamic modeling has also been applied to explore the biological patterns related to system responses to forcing functions, such as changes in nutrient loads (jørgensen and de bernardi, 1998; zhang et al., 2003). the pamolare model has been successfully applied in 18 case studies for parameter estimation, calibration, validation and generation of prognoses (zhang, 2004). this type of model may be important for qualitatively predicting the outcomes of lake management strategies by generating prognoses although examining more case studies is required to support the possible generalizations relating to their use for such a purpose (jørgensen and de bernardi, 1998). it includes a watershed model, which works together with a simple lake model or a structurally dynamic lake model with a medium to high degree of complexity in order to generate outputs based on different restoration measures (jorgensen et al., 2003). hence, it is easy to apply the results from the watershed model to assess nutrient loadings (the most crucial forcing functions) using the lake model (jørgensen, 2010). testing pamolare using different lakes and water reservoirs has calibrated and validated the models to give better results (jørgensen, 2008). pamolare has been used in a study in iran for the anzali wetland (ramin, 2004). in this study, the trophic levels of the zaribar lake were identified, then, pamolare was used to predict the concentrations of nutrients and other chemical and biological responses in the lake to validate its use for assessing the various management alternatives. based on previous studies the concentrations of nitrogen and phosphorus were used for the trophic level identification (vollenweider and kerekes, 1980; carlson and simpson, 1996 and robert, 1996). 102 hamidian and hasanzadeh/ int. j. aquat. biol. (2013) 1(3): 100-108 material and methods study area: the zaribar lake is located on the west side of the zagros mountain, extending from 35o31’30” to 35o37’06”e latitude and from 46o03’52” to 46o10’47”n longitude at an elevation of 1278 m above sea level (fig. 1). it has a total area of 720 ha. this ecosystem consists of a unique plant and animal diversity, which in recent years resulted in a considerable increase in the numbers of tourists. however, the wastewater discharges from adjacent populated areas, chemical fertilizers and pesticides from farmlands, improper disposal of solid waste and the pressures caused by the increasing numbers of tourists are devastating its ecological and environmental quality (asarab consultanting co., 2007). determination of tropic level: six sampling sites were selected based on their spatial distribution in the wetland (fig. 1) and water samples were collected in pvc bottles during a seven-year period (2004-2010) during different seasons. one of the sites was located in the entrance of the lake. in 2010, sediment grab samples were collected from three randomly selected sites, stored in plastic bags and transported to the laboratory. these samples were dried and fixed at ambient temperatures prior to analysis. a pallin test photometer (model 8000) was used for the measurement of nitrogen and phosphorus concentrations in the water column. the water samples were analyzed for nitrite, nitrate, ammonium, and phosphate. total phosphorus concentrations were measured based on murphy and riley (1962) using spectrophotometer (cintra 40). kjeldahl nitrogen and total nitrogen concentrations were measured using the ascorbic acid method of inorg et al. (2005). a kolmogorov-smirnov test was used to investigate the normality of the distribution of total nitrogen and phosphorus in water column and a one way anova test was applied to examine a significance difference between total nitrogen and phosphorus concentrations among sampling sites. the results were compared to existing standards (table 1; vollenweider and kerekes, 1980; carlson and simpson, 1996; robert, 1996). using the 1-layer model for the prediction of trophic levels in zaribar lake over the next 20 years: pamolare contains four different eutrophication models; a vollenweider plot, a 1-layer model with four state variables and the possibility of simulating nutrient concentrations both in the water column and in the sediment (vollenweider, 1975); a two-layer model with 21 state variables, and a structurally dynamic model with 15 state variables using energy as the function for describing structural changes (zhang, 2004). the 1-layer model of pamolare is the verified version of vollenweider model and forecasts nitrogen and phosphorus concentrations in sediments and the water column. the empirical part of the model consists of simple regressions between input data and physico-chemical properties based on responses observed in similar water bodies. for executing the 1-layer model in pamolare software, information on wetland morphology and the concentrations of nitrogen and phosphorus are required. this model is developed as an ecological model that could account for structural changes in the water body affecting the prediction of trophic level. some of the required information is measured directly or estimated using other factors. the average length, width and depth and nutrient concentrations are measured in the field or in collected samples. other required factors, including a wide range of trophic indicators which have direct or indirect figure 1. the study area and sampling stations. 103 hamidian and hasanzadeh/ int. j. aquat. biol. (2013) 1(3): 100-108 effects on the trophic level of water bodies, are calculated. calculating the required input data: some of the required information is measured directly or estimated using other factors. calculated factors are (jørgensen et al., 2003): 1. water flow speed: s=√g.dm, where g=32.3 (foot/s2), dm = depth (foot). 2. nitrogen and phosphorus loadings: loading (g×m2×year) = percentage of element (concentration) × (annual volume of water inlet/ wetland area). 3. sediment bound nitrogen or phosphorus: bound = load – release, or 15-25% × loading from sediment. 4. denitrification rate: denit = n load – (0.34 × wres) – (0.16× z ×0.17), where denit= rate of denitrification, z= depth and wres= resting time (water residence time). all calculated data were put into the software and all pamolare indicators were predicted. the estimated results (outputs) were compared with field observations. results the average concentrations of no2, no3-, nh3 and po3-2 at different times and sites are presented in table 2. the comparison between total nitrogen and total phosphorus concentrations at the sampling sites is presented in figure 2. the kolmogorov smirnov test showed that the resultant data have a normal distribution (p>0.05). no significant difference was found among the five sampling sites in total nitrogen concentrations (df = 5, f = 1.101, p = 0.362) and total phosphorus concentrations (df = 5, f = 0.521, p = 0.761). the results of the chemical analysis of the sediment samples were processed using the 1-layer model. all required information required for this method are shown in tables 3, 4 and 5. the results of the simulation are presented in table 6 and figures 3, 4, 5 and 6. comparison of the calculated results with those measured in the zaribar lake showed that there was no compatibility between the pamolare predictions and those observed in the zaribar lake. figure 2. the comparison between total phosphorus and nitrogen concentrations. figure 3. the comparison between total phosphorus and nitrogen concentrations in sediment. figure 4. the predicted trend of nutrient changes in water. 104 hamidian and hasanzadeh/ int. j. aquat. biol. (2013) 1(3): 100-108 discussion lakes are valuable water resources, which are under pressures of increasing populations and the consequent increases in the different type of pollutants. pollutants, especially nutrients such as phosphorus, cause the eutrophication of these water bodies. therefore, the identification of their trophic status and prediction of eutrophication trends in these natural resources are necessary for selecting the best management and engineering methods for their conservation or restoration. in this study, no difference was found between the concentrations of nutrients at the different sampling sites (the sites were distributed evenly in the lake). all sites showed similar concentrations of nutrients in their water columns. this might be because of the geomorphology of the lake and the existence of springs on its bottom. the geomorphological and hydrological conditions, especially the high sedimentation rates, might have accelerated the eutrophication of the lake, as previously reported by zha et al. (2010). kokfelt et al. (2010) showed that the nutrient cycles in water resources are affected by the rate of sedimentation, similar to what was found in this study. nutrient compounds attach to sediments and can create more stability in lakes but cause future pollution if the nutrients are released back into the water column. hence, there could be a direct relationship between sedimentation rate, the presence of the nutrients and the eutrophication level. the effects of hydrobiological factors on plant vegetation in lakes were previously reported (larsen reference parameter oligotroph mesotroph euotroph hypertroph robert (1996) (mg/lit) chlorophyll a -0.002 0.002-0.006 +0.006 carlson (1996) (mg/lit )tp -0.008 0.008-0.0267 +0.0267 96-384 vollenweider and kerekes (1980) (mg/lit) tn -0.661 0.661-0.753 0. 753 table 1. the classification of trophic levels using different standards. figure 6. the predicted trend of changes in primary production and fish production (biomass). figure 5. the predicted trend of nutrient changes in sediment. stations 1 2 3 4 5 6 mean sd n u tr ie n ts ( m g /l it ) 22no 0.0012 0.0023 0.0048 0.0042 0.0058 0.0057 0.004 0.00187 3no 8.44 7.97 9.01 9.69 8.82 9.48 8.90 0.64147 3nh 0.18 0.25 0.21 0.47 0.22 0.28 0.23 0.10338 tn 8.63 8.23 9.23 10.17 9.04 9.77 9.18 0.71492 tp 0.57 0.51 0.57 0.60 0.59 0.96 0.63 0.163254 table 2. the average concentrations of nutrients in different sampling stations. 105 hamidian and hasanzadeh/ int. j. aquat. biol. (2013) 1(3): 100-108 and harvey, 2010; richardson, 2010). in the zaribar lake the hydrologic conditions and the high rates of sedimentation cause development of reed communities in different parts of the wetland. high concentrations of total phosphorus and nitrogen in the lake were found to be the main causes of eutrophication in this ecosystem, which is similar to what has been found in other studies (ardo’n et al., 2010; duff et al., 2009; bruland and richardson, 2005). the concentration of nitrogen in zaribar lake is more significant than that of phosphorus, so it support greatly the dense vegetation cover which traps additional nutrients and this reciprocal relationship propels this lake to hypertrophic levels. among all chemical and hydrobiological factors investigated in this research, the concentrations of total phosphorus and total nitrogen in water column were found to be the best indicators of the trophic level of the wetland, which supports previous research in other areas (tsujian et al., 2010; stations 1 2 3 mean sd average of nutrients tp 24.55 33.55 29.05 29.05 4.5 tn 0.7182 0.9576 0.830 0.8352 0.11 table 3. the concentrations of total phosphorus and nitrogen in the sediment samples. reduction of nutrient outflow due to thermocline (a) sedimentation constant (m/year) water residence time (year) length (m) water speed (foot/s) depth (m) initial factors 1 0.0077 0.0000267 5000 19.50 3.6 amount table 4. other factors required for pamolare 1-layer model. nutrient initial value in water (mg/lit) initial value in (3gr/m) sediment loading value (/year2gr/m) sediment release value (gr/m2/year) fraction bound in sediment (year) stabilized amount (year) phosp 0.63 29.05 2.58 2.064 7.26 0 nitrog 9.18 0.83 7.37 2.26 1.47 0 table 5. phosphorus and nitrogen required data for 1-layer model. table 6: the results of software (pamolare) simulation for a 20-year period. t im e p e ri o d (0 .0 2 y r) n it ro g e n o f w a te r ( m g /l it / ) n it ro g e n o f se d im e n t ( 2 g /m ) p h o sp h o ru s o f w a te r ( m g /l it / ) p h o sp h o ru s o f se d im e n t ( 2 g /m ) l im it in g n u tr ie n t c h lo ro p h y ll a ( m g /l it / ) s e c c h i d e p th ( m ) z o o p la n k to n (m g /l it ) ( m g /l it / ) f is h a v e ra g e p ri m a ry ( m g /l it / ) p ro d u c ti o n a v e ra g e m a x im u m ( m g /l it / ) p ro d u c ti o n a v e ra g e fi sh y ie ld 0.2 0.01 1.00 0.00 18.27 p 0.00 18.34 0.04 0.81 -0.07 -0.06 0.01 0.4 0.01 1.00 0.00 11.49 p 0.00 18.34 0.04 0.81 -0.07 -0.06 0.01 0.7 0.01 1.00 0.00 7.23 p 0.00 18.34 0.04 0.81 -0.07 -0.06 0.01 0.9 0.01 1.00 0.00 4.55 p 0.00 18.34 0.04 0.81 -0.07 -0.06 0.01 1.1 0.01 1.00 0.00 2.86 p 0.00 18.34 0.04 0.81 -0.07 -0.06 0.01 1.3 0.01 1.00 0.00 1.80 p 0.00 18.34 0.04 0.81 -0.07 -0.06 0.01 1.5 0.01 1.00 0.00 1.13 p 0.00 18.34 0.04 0.81 -0.07 -0.06 0.01 1.8 0.01 1.00 0.00 1.00 p 0.00 18.34 0.04 0.81 -0.07 -0.06 0.01 2.0 0.01 1.00 0.00 1.00 p 0.00 18.34 0.04 0.81 -0.07 -0.06 0.01 .. 20 0.01 … 1.00 … 0.00 … 1.00 … p … 0.00 … 18.34 … 0.04 … 0.81 … -0.07 … -0.06 … 0.01 … 106 hamidian and hasanzadeh/ int. j. aquat. biol. (2013) 1(3): 100-108 koshikawa et al., 2005; gunnarsson et al., 2004; gunnarsson and rydin, 2000; bobbink et al., 1998). this indicates that those pollutants which have the highest loading rates have the main effect on increasing trophic level in the zaribar lake, transforming it to an ecosystem of low value to humans. the application of the 1-layer model to this lake was unsuccessful in predicting of the trend changes in nutrient concentrations, fish and biomass production and primary production. this is in contrast to what ramin (2004) found in the anzali wetland. in the case of zaribar lake, these factors do not appear to reflect the real changes, especially related to our observations in recent years. therefore, the application of this model to hypertrophic lakes is inappropriate. to apply in hypertrophic conditions, the software needs to be improved to be able to simulate the trophic changes in lakes with higher trophic levels. gurkan et al. (2006) also reported the inability of pamolare software in modeling and simulation of lakes with special or critical conditions. pamolare failed to generate prognoses of the effects of various pollutants and did not predict the changes in some trophic indicators such as fish and biomass production, primary production and nutrient concentrations in our hypertrophic lakes. therefore, this model has limited utility for the management of hypertrophic lakes. in conclusion, the present study supports the idea of using observed total nitrogen and total phosphorus concentrations in the water column as the best method for the identification of the trophic levels of hypertrophic water resources. it is necessary to improve and develop pamolare and its software to predict the eutrophication factors in wetlands at high trophic levels. this necessity was highlighted as the purpose of the software is to be used as a management tool for the prediction of the effectiveness of restoration plans and programs. the zaribar lake, as a hypertrophic wetland being impaired by pollutants from different sources, requires a range of environmental and engineering methods for its restoration. however the effectiveness of these methods cannot be simulated or predicted using the existing software. therefore, it is strongly suggested that pamolare, as widely available software, be further developed and improved for better simulation and modeling of extreme conditions. references ardo’n m., morse j.l., doyle m.w., bernhardt e.s. 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(2018) 6(6): 330-339 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article how the selective breeding in aquaculture programs can change the body shape of cyprinids; a case study on the native cyprinus carpio and a cultured stock hamed mousavi-sabet*1,2, adeleh heidari1, meysam salehi3 1department of fisheries sciences, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. 2the caspian sea basin research center, university of guilan, rasht, iran. 3abzi-exir aquaculture co., agriculture section, kowsar economic organization, tehran, iran. article history: received 11 september 2018 accepted 19 december 2018 available online 2 5 december 2018 keywords: morphology stock structure caspian sea restocking program abstract: decades since restocking program of the vulnerable native cyprinus carpio in the southern caspian sea, the cultured stocks in hatcheries have created new challenge to protect the native population. releasing the cultured common carp in natural water-bodies caused an uncertainty about originality of the carp broodstocks within the restocking program. to clarify that how the selective breeding with aquaculture purpose could change the body shape with aiming to prepare an identification key for the indigenous and cultured stocks, a landmark based morphological characteristics of these stocks from the anzali wetland and a hatchery were analyzed. univariate analysis of variance of 100 adult specimens collected during the non-reproductive season were observed in 62 morphometric characters out of 78 (p<0.05). principle component analyze (pca) of morphometric characteristic showed a high differentiation between these stocks. in morphometric traits, linear discriminate function analysis (dfa), the overall assignments of individuals into their original groups between stocks were 100%. the pca and dfa showed a morphological segregation of the studied stocks based on the characters head shape, pre-dorsal, pre-pelvic and pre-anal distances, caudal peduncle depth, dorsal fin and ventral fin origins, body depth and caudal fin origin. the results showed stocks represent two distinct morphological forms of c. carpio that had high morphometric differentiation. the results can be useful as baseline information on the native stock for conservational policy. to protect the vulnerable population, using wild native broodstocks in the restocking program is strongly recommended. introduction information on the biology and population structure of any fish species is a prerequisite for developing management and conservation strategies (tudela, 1999) and maybe applicable for studying short-term and environmentally induced variations. morphological studies on fishes are important from various viewpoints, including evolution, ecology, behavior, conservation, water resource management and stock assessment (mousavi-sabet and anvarifar, 2013; kohestan-eskandari et al., 2013; heidari et al., 2013; natsumeda et al., 2014; jalili et al., 2015; vatandoust et al., 2015). the study of morphological characters with the aim of defining or characterizing fish stock units has been of a strong interest in ichthyology (tudela, 1999). traditional and truss morphometrics are often used *corresponding author: hamed mousavi-sabet doi: https://doi.org/10.22034/ijab.v6i6.599 e-mail address: mousavi-sabet@guilan.ac.ir to describe morphological variations between different species. the study of morphometrics using truss network system is a landmark-based on geometric morphometrics, which poses no restriction on the directions of variation and localization of shape changes, and much effective in capturing information on the shape of an organism (bookstein, 1991; cardin and fried, 1999; kocovsky 2009; bagherian and rahmani, 2009). it covers the entire fish body shape in a uniform network, and theoretically, it increases the likelihood of extracting morphometric differences between specimens (cardin and fried, 1999; turan, 2004a; kocovsky, 2009). the common carp (cyprinus carpio) is native to the caspian sea, which is a widespread of eutrophic waters in lakes and large rivers in europe and asia (esmaeili et al., 2018). the wild stocks are considered 331 int. j. aquat. biol. (2018) 6(6): 330-339 vulnerable to extinction, but the species has also been domesticated and introduced into environments worldwide, and often considered a very destructive invasive species, being included in the list of the world's 100 worst invasive species (radkhah et al., 2016). cyprinus carpio is one of the most important fishes for fisheries and stocking programs in south of the caspian sea. native bony fishes to the caspian sea are economically high value species, which are mainly distributed along the southern coasts of the caspian sea (kohestan-eskandari et al., 2013). due to limited natural reproduction of these species, caused by destruction of natural spawning areas and other factors (kohestan-eskandari et al., 2013), iranian fisheries organization has launched its restocking program from last decade (ebrahimi and ouraji, 2012). despite the biodiversity and commercial importance of c. carpio, there is no any available study on stock differentiation of the fish in the southern caspian sea. considering the abovementioned facts, the present study was aimed to obtain information about morphological differentiation between two wild and cultured stocks, from the anzali wetland and a hatchery in the southern caspian sea, respectively, which can be employed in the future enhancement programs of this species. the present study deals with the stock structure of c. carpio from a phenotypical point of view to determine the morphometric difference between the stocks. materials and methods during september to october 2014, 100 c. carpio were collected from 2 sources: the anzali wetland (50 specimens) in the southern caspian sea basin (fig. 1) and a hatchery (50 specimens). in order to investigate the body shape of the specimens, 13 homologous landmark-points were used. a total of 78 distance measurements between these 13 landmark-points were extracted using the truss network system according to strauss and bookstein (1982) with minor modifications (fig. 2). measurements of specimens were made by collecting x–y coordinate data for relevant morphological features, followed the threestep process as described below (turan, 2004a). the fish were placed on a white board with dorsal and anal fins held erect by pins. the left body profile of each fish was photographed with a 300-dpi, 32-bit color digital camera (cybershot dsc-f505; sony, japan). images were saved in jpg format and digitized with tpsdig to coordinates of 13 landmark points. a box truss of 26 lines connecting these landmark points was generated for each fish to represent the basic shape of the fish (cardin and fried, 1999). all measurements were transferred to a spreadsheet file (excel 2010), and the x–y coordinate data transformed into linear distances by computer for subsequent analysis (turan, 2004a). the extracted landmark-points were submitted to a generalized procrustes analysis (gpa) to remove nonshape data in past software for visualization purpose. in respect of truss network measurements, as variation should be attributable to body shape differences and not related to the relative size of the fish, an allometric method was used to remove size figure 1. map of iranian parts of the caspian sea basin showing sampling point. 332 mousavi-sabet et al./ effect of selective breeding on the body shape of cyprinus carpio dependent variation using following formula: madj = m (ls / l0)b where, m is original measurement, madj is the size adjusted measurement, l0 is the standard length of the fish, ls is the overall mean of standard length for all fish from all samples in each analysis, and b was estimated for each character from the observed data as the slope of the regression of log m on log l0 using all fish from both the groups. the results derived from the allometric method were confirmed by testing significance of the correlation between transformed variables and standard length (turan, 2004a; kocovsky, 2009). univariate analysis of variance (anova) was performed for each morphometric character to evaluate the significant difference between the two stocks. discriminant function analyses (dfa) and principal component analysis (pca) were employed to discriminate the two stocks. the analysis of variance revealed significant phenotypic variation between the two stocks. in order to determine which morphometric measurement made most effectively differentiates between the stocks, the contributions of variables to principal components (pc) were examined. to examine the suitability of the data for pca, bartlett’s test of sphericity and the kaiser– meyer–olkin (kmo) measure was performed. the bartlett’s test of sphericity tests the hypothesis that the values of the correlation matrix equal zero and the kmo measure of sampling adequacy tests whether the partial correlation among variables is sufficiently high (nimalathasan, 2009). a cross-validation using percentage of correctly classified (pcc) was done to estimate the expected actual error rates of the classification functions. a dendrogram of the stocks based on the morphometric and landmark distance data was drawn using the unweighted pair group method analysis (upgma). statistical analyses for morphometric data were performed using the spss version 16 software package, past software ver. 1.36, taxonomy and multivariate analysis system (ntsyspc), morphoj and excel (microsoft office, 2010). results the correlation between transformed morphometric variables and standard length was non-significant (p>0.05) confirming size or allometric signature on the basic morphological data was accounted. statistically significant differences between the wild and hatchery stocks were found in 62 morphometric characters out of 78 characters (table 1). of these 62 characters, 54 characters were found significantly different (p<0.01) and were used for multivariate analysis. this traits include 1-3, 1-4, 1-5, 1-6, 1-7, 18, 1-9, 1-10, 1-11, 1-12, 2-4, 2-6, 2-11, 3-4, 3-6, 3-7, figure 2. position of 13 digitized landmark points for constructing the truss network on cyprinus carpio.1. tip of snout; 2. center of the eye; 3. forehead (end of frontal bone); 4. end of the operculum; 5 ventral edge of the body appendicular to origin of the pectoral fin; 6 origin of the dorsal fin; 7 origin of the pelvic fin; 8 insertion of the dorsal fin; 9 origin of the anal fin; 10 insertion of the anal fin; 11 dorsal side of caudal peduncle, at the nadir; 12 ventral side of caudal peduncle, at the nadir; 13 end of the lateral line. 333 int. j. aquat. biol. (2018) 6(6): 330-339 3-8, 3-9, 3-11, 4-6, 4-8, 4-10, 4-12, 4-13, 5-7, 5-8, 59, 5-10, 5-12, 5-13, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 613, 7-10, 7-12, 7-13, 8-9, 8-10, 8-11, 8-12, 8-13, 9-10, 9-11, 9-12, 9-13, 10-11, 10-12, 11-12, 11-13 and 1213. the morphometric characters between two sexes (out of reproductive season) did not differ significantly (p>0.05) (table 2); hence, the data for both sexes were pooled for all subsequent analyses. the value of kmo for overall matrix was 0.773 and the bartlett’s test of sphericity is significant (p<0.01). the kmo statistics varies between 0 and 1. kaiser recommends that values greater than 0.5 are acceptable, the results of kmo and bartlett’s suggest that the sampled data is appropriate to proceed with a factor analysis procedure. principal component analysis of 54 morphometric measurements extracted 8 factors with eigenvalues >1, explaining 93.925 % of the variance (table 3). the first principal component (pc1) accounted for 46.92% of the variation and the second principal component (pc2) for 12.67%, respectively (table 3). the most significant loadings on pc1 were 1-3, 1-4, 1-6, 1-7, 1-8, 1-10, 1-12, 2-4, 2-6, 3-4, 3-6, 3-7, 4-6, 4-8, 4-10, 4-12, 5-7, 5-8, 5-10, 5-12, 6-9, 6-11, 6-13, 7-9, 7-10, 8-9, 8-13, 11-12 and table 1. the results of anova for morphometric measurements of cyprinus carpio stocks from the anzali wetland and a hatchery. characters f p characters f p characters f p 1-2 1.510 0.201 3-7 52.417 0.000 6-9 975.587 0.000 1-3 103.450 0.000 3-8 41.105 0.000 6-10 45.755 0.000 1-4 105.441 0.000 3-9 77.673 0.000 6-11 66.571 0.000 1-5 135.297 0.000 3-10 5.219 0.042 6-12 47.067 0.000 1-6 54.318 0.000 3-11 9.091 0.005 6-13 162.295 0.000 1-7 21.285 0.000 3-12 1.625 0.206 7-8 1.552 0.188 1-8 109.632 0.000 3-13 1.115 0.295 7-9 4.899 0.029 1-9 1.417 0.227 4-5 3.158 0.078 7-10 231.981 0.000 1-10 96.384 0.000 4-6 85.726 0.000 7-11 0.630 0.421 1-11 34.453 0.000 4-7 3.221 0.075 7-12 35.524 0.000 1-12 76.432 0.000 4-8 237.274 0.000 7-13 22.779 0.000 1-13 33.453 0.000 4-9 5.789 0.017 8-9 53.055 0.000 2-3 0.905 0.334 4-10 438.500 0.000 8-10 23.320 0.000 2-4 85.638 0.000 4-11 3.688 0.058 8-11 46.074 0.000 2-5 0.224 0.634 4-12 414.887 0.000 8-12 17.327 0.000 2-6 24.456 0.000 4-13 33.226 0.000 8-13 34.216 0.000 2-7 4.973 0.020 5-6 4.422 0.065 9-10 8.891 0.002 2-8 0.047 0.858 5-7 599.238 0.000 9-11 145.985 0.000 2-9 0.318 0.567 5-8 61.678 0.000 9-12 56.852 0.000 2-10 0.402 0.526 5-9 10.312 0.003 9-13 97.355 0.000 2-11 87.337 0.000 5-10 70.619 0.000 10-11 122.384 0.000 2-12 3.755 0.054 5-11 6.008 0.015 10-12 64.940 0.000 2-13 6.386 0.013 5-12 98.391 0.000 10-13 41.320 0.000 3-4 222.642 0.000 5-13 37.424 0.000 11-12 257.389 0.000 3-5 6.313 0.021 6-7 779.005 0.000 11-13 247.291 0.000 3-6 146.901 0.000 6-8 110.912 0.000 12-13 32.757 0.000 figure 2. plot of the factor scores for pc1 and pc2 of all morphometric measurements between cyprinus carpio stocks from the anzali wetland and a hatchery. 334 mousavi-sabet et al./ effect of selective breeding on the body shape of cyprinus carpio 11-13. the plot of pc1 and pc2 scores revealed that the 100 specimens grouped into two stocks (fig. 3). the wilks’  tests of discriminant analysis indicated significant differences in morphometric characters of the two stocks. in this test, one function was highly significant (p<0.01) (table 4). the histogram of discriminant functions for pairwise groups is shown in figure 4. there was a slight degree of separation between two stocks. the linear discriminant analysis gave an average pcc (percentage of specimens classified) of 100% for morphometric characters indicating a high rate of correct classification of individuals into their original stocks (table 5). clustering analysis based on euclidean square distances between the groups of centroids using an upgma resulted two main clusters, including the anzali wetland and hatchery groups in separate clades (fig. 5). discussion the aim of the present study was to investigate the hypothesis; morphological differentiation between native wild and cultured c. carpio stocks using table 2. the results of anova for morphometric measurements between two sexes of cyprinus carpio in the studied stocks from the anzali wetland and a hatchery. hatchery stock wild stock characters f p characters f p characters f p characters f p 1-3 1.003 0.322 5-10 0.986 0.326 1-3 1.357 0.250 5-10 0.421 0.518 1-4 0.069 0.794 5-12 0.380 0.541 1-4 0.744 0.393 5-12 0.226 0.636 1-5 0.814 0.371 5-13 3.598 0.064 1-5 0.525 0.472 5-13 0.144 0.706 1-6 3.374 0.072 6-7 0.000 0.989 1-6 0.865 0.357 6-7 1.384 0.197 1-7 0.362 0.550 6-8 0.005 0.947 1-7 1.585 0.214 6-8 2.981 0.087 1-8 3.100 0.085 6-9 1.174 0.284 1-8 0.665 0.419 6-9 0.881 0.351 1-9 1.247 0.265 6-10 0.928 0.340 1-9 3.113 0.084 6-10 1.545 0.217 1-10 3.636 0.063 6-11 2.853 0.098 1-10 0.713 0.403 6-11 1.952 0.166 1-11 2.810 0.100 6-12 1.123 0.294 1-11 3.191 0.080 6-12 2.031 0.157 1-12 0.500 0.483 6-13 0.029 0.865 1-12 2.874 0.097 6-13 0.840 0.361 2-4 0.003 0.955 7-10 1.054 0.310 2-4 0.691 0.410 7-10 0.299 0.586 2-6 0.649 0.424 7-12 3.297 0.076 2-6 0.137 0.713 7-12 3.569 0.062 2-11 0.738 0.394 7-13 0.023 0.881 2-11 0.628 0.432 7-13 0.186 0.663 3-4 2.397 0.128 8-9 0.230 0.633 3-4 1.224 0.274 8-9 0.518 0.475 3-6 2.344 0.132 8-10 0.015 0.903 3-6 0.605 0.440 8-10 2.081 0.155 3-7 2.216 0.143 8-11 2.978 0.088 3-7 2.833 0.092 8-11 0.781 0.385 3-8 0.449 0.506 8-12 0.786 0.388 3-8 0.217 0.644 8-12 2.341 0.135 3-9 0.062 0.805 8-13 0.034 0.855 3-9 3.466 0.069 8-13 2.345 0.133 3-11 0.001 0.974 9-10 2.086 0.152 3-11 3.103 0.085 9-10 0.692 0.411 4-6 3.715 0.060 9-11 1.426 0.235 4-6 1.605 0.203 9-11 0.851 0.365 4-8 0.001 0.974 9-12 1.786 0.377 4-8 0.722 0.400 9-12 0.695 0.415 4-10 0.247 0.622 9-13 0.043 0.837 4-10 0.517 0.476 9-13 0.288 0.578 4-12 0.028 0.867 10-11 2.426 0.123 4-12 2.603 0.108 10-11 2.067 0.105 4-13 3.204 0.080 10-12 0.518 0.473 4-13 0.004 0.951 10-12 1.611 0.204 5-7 0.426 0.517 11-12 0.187 0.666 5-7 0.998 0.320 11-12 0.035 0.851 5-8 0.690 0.410 11-13 3.579 0.361 5-8 0.016 0.901 11-13 3.106 0.089 5-9 0.849 0.361 12-13 0.299 0.586 5-9 0.230 0.370 12-13 1.425 0.234 table 3. eigenvalues, percentage of variance and percentage of cumulative variance for cyprinus carpio stocks from the anzali wetland and a hatchery. factors eigenvalues percentage of variance percentage of cumulative variance pc1 25.401 46.921 46.921 pc2 6.851 12.671 59.592 pc3 6.267 11.639 71.231 pc4 4.679 8.676 79.907 pc5 3.010 5.498 85.405 pc6 2.181 4.036 89.441 pc7 1.411 2.591 92.032 pc8 1.029 1.893 93.925 335 int. j. aquat. biol. (2018) 6(6): 330-339 landmark-based methods. the landmark-based morphometric method is recently used to investigate various hypothesis in freshwater and marine species in the region (kohestan-eskandari, 2013; heidari et al., 2013, 2014; paknejad et al., 2014; vatandoust, 2014a, b, 2015; mohadasi et al., 2014). the result of the present study also demonstrate there is significant phenotypic variation between the two studied stocks. many natural populations of fish species have decreased drastically in number, mainly because of the effects of over-exploitation, habitat alterations, including physiography, abiotic, and biotic features, the release and introduction of exotic fish species, etc. over fishing, especially when directed against a specific size or class age, can reduce the size of the population to a level where inbreeding and loss of genetic diversity may be a serious problem or may lead to extinction of local fishes (mostafa, 2010). discriminant function analysis could be a useful method to distinguish different stocks of a same species (karakousis, 1991). in the present study, achieved high classification of individuals that were correctly classified in to their respective groups by dfa, confirmed by pca. mostafa et al. (2010) compared morphometric characteristics among three groups of labeo calbasu, from stocks of two isolated rivers and a hatchery and reported high isolation of these stocks. the pca and dfa showed a table 4. result of wilks’ test for verifying difference between two stocks when morphometric measurements are separately compared using dfa. test of functions wilks’ x2 df significance 1 0.006 467.966 11 0.000 table 5. percentage of specimens classified in each group and after cross validation for morphometric data between cyprinus carpio stocks from the anzali wetland and a hatchery. stocks hatchery anzali wetland total o ri g in a l c o u n t hatchery 50 0 50 anzali wetland 0 50 50 % hatchery 100.0 0.0 100.0 anzali wetland 0.0 100.0 100.0 c ro ss v a li d a te d c o u n t hatchery 50 0 50 anzali wetland 0 50 50 % hatchery 100.0 0.0 100.0 anzali wetland 0.0 100.0 100.0 figure 4. histogram of discriminate analysis (da) functions for pairwise competition’ between cyprinus carpio stocks of the anzali wetland and a hatchery (left). shape differences on the extremities of each stock (right). 336 mousavi-sabet et al./ effect of selective breeding on the body shape of cyprinus carpio morphological segregation of the studied stocks based on the characters, including head shape, pre-dorsal, pre-pelvic and pre-anal distances, caudal peduncle depth, dorsal fin and ventral fins origins, body depth and caudal fin origin position (fig. 6). these morphological differences are solely related to body shape variation and not to size effect which was successfully accounted by allometric transformation. on the other hand, size related traits play a predominant role in morphometric analysis and the results may be erroneous if not removed for statistical analyses (tzeng, 2004). in the present study, the size effect had been removed successfully by allometric transformation, and the significant differences between the body shape of stocks are due to the body shape variation. the causes of morphological differences between stocks are often quite difficult to explain (poulet, 2004). it has been suggested that the figure 5. dendrogram derived from cluster analyses of morphometric characteristics for cyprinus carpio stocks from the anzali wetland and a hatchery. mean shape of species in relation of consensus shape of the populations are also represented. figure 6. cyprinus carpio from the anzali wetland (above) and a hatchery (below), in the southern caspian sea basin. 337 int. j. aquat. biol. (2018) 6(6): 330-339 morphological characteristics of fish are determined by genetic, environment and the interaction between them (swain and foote, 1999; poulet, 2004; vatandoust et al., 2015). the environmental factors prevailing during the early development stages, when individual’s phenotype is more amenable to environmental influence (pinheiro, 2005). the influences of environmental parameters on morphometric characters are well-discussed by several authors in the course of fish segregation (e.g., swain and foote, 1999). in general, fish demonstrate greater variances in morphological traits both within and between populations than any other vertebrates and are more susceptible to environmentally induced morphological variations (wimberger, 1992; mostafa, 2010; mohadasi et al., 2014; vatandoust et al., 2015). in this study, pca of morphometric characteristic showed a high differentiation between the stocks of c. carpio. the dendrogram resulted in two distinct clade i.e. the anzali wetland wild and hatchery reared stocks. the differences between the hatchery and wild stocks may have been due to environmental conditions as well as genetic variations (which needs further studies to confirm). the phenotypic plasticity of fish is very high. they adapt quickly by modifying their physiology and behavior to environmental changes. these modifications ultimately change their morphology (wimberger, 1992). the present study showed that each stock represents different body shape. the results also provide useful baseline information of c. carpio stocks for further studies and conservation programs. in both aquaculture and open-water management, it is essential to select genetically superior stocks along with better features. more research especially genetic studies and impacts of environmental factors are need for conservation and mass seed production of selected stocks to pave the way to protect this vulnerable species. the results also present a key to identify the native and cultured stocks, which can be useful for selecting broodstocks to the restocking program. to protect the vulnerable native population, using wild native broodstocks in the restocking program is strongly recommended. acknowledgements we express our sincere thanks to a. habibi for his help in specimen collection, and thank to h. mojdekanlou for helping us in fixing and taking the photograph. references bagherian a., rahmani h. 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(2022) 10(5): 406-416 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article study of pollution in shatt al-arab river using histological alternations and some other biochemical parameters of gill in nile tilapia (oreochromis niloticus) as water quality biomarkers nowfel hammadi jasim1, abdul jabbar rasmi huwait2, raghad issa abed3, majdy faisal majeed2 1department of physiology and pharmacology and chemistrycollege of veterinary medicineuniversity of basra, basrah, iraq. 2department of histology and anatomy, college of veterinary medicine, university of basrah, basrah, iraq. 3department of clinical laboratory sciencesfaculty of pharmacy, university of basrah, basrah, iraq. s article history: received 3 june 2022 accepted 11 august 2022 available online 2 5 october 2022 keywords: aquatic pollution gills enzymes activity micronucleus abstract: this study aims to assess the pollution of shatt al-arab river using histology and some other biochemical parameters of gill in nile tilapia (oreochromis niloticus) caught in winter 2021 at four locations as water pollution biomarkers. some water quality parameters were determined in these sites, and the results showed that sites 2 and 3 are polluted at levels above the world health organization's guideline. the enzymatic and metabolism activity, histological status, and cytogenetic mutation over time in the gills were assessed. biotransformation enzymes level showed total cytochrome p450 and ethoxyresurofin–o– demethylase (erod) activities are significantly increased in gills of tilapia in site 1, 2 and 3. the antioxidant enzymes activities were recorded significantly high in fish gills of catalase (cat) and glutathione -s-transferase (gst) in polluted sites of 1, 2, and 3, while superoxide dismutase (sod) were significantly higher only in site 3 compare with reference site of 4. the metallothionein-like protein (mtlp) was significantly higher in gills at site 3. also, lipid peroxidation (lop) and micronucleus analysis showed that sites 2 and 3 samples site the most affected. gill tissue index was appeared severe alterations levels at sites 2 and 3, followed by site 1 with a relatively lower level of damage, while site 4 (reference one) showed minor or invisible changes in gill tissue. the histological alterations in the gills of nile tilapia fish at sites 2 and 3 showed atrophy of cellular, hemorrhage, congestions, hyperplasia, and hypertrophy of the filament lamellae epithelium. introduction approximately 90% of anthropogenic discharges end up in aquatic systems resulting from industrial, agricultural, and urban activities (siraj basha and usha rani, 2003). the aquatic environment around industrial units is at high risk of contamination by different pollutants, such as heavy metals (vinodhini and narayanan, 2008). the histological alterations may help to detect the most affected target organs and reveal the organism's sensitivity to levels of pollutants (wester and canton, 1991). histological indicators are frequent practices in fish health research that provides information on the sub-lethal and chronic effects of xenobiotics on each biological correspondence: majdy faisal majeed doi: https://doi.org/10.22034/ijab.v10i5.1755 e-mail: majdy.majeed@uobasrah.edu.iq do r: 20.1001.1.23830956.2022.10.5.8.9 response (schwaiger et al., 1997; van der oost et al., 2003). they have been used in contaminated ecosystems as proper bio-indicators (camargo and martinez, 2007). in a biological system, the toxicant induces changes that are considered a biomarker, providing information about the state of the ecosystem’s health (maria et al., 2009; monteiro et al., 2010). the potential of xenobiotics to boost cellular levels of reactive oxygen species (ros), which can occur either through increased production or an imbalance in antioxidant cell defenses, is widely used to measure their toxicity (gravato et al., 2006). it may cause dna changes and oxidation of https://ij-aquaticbiology.com/index.php/ijab/article/view/1755 407 int. j. aquat. biol. (2022) 10(5): 406-416 membrane lipids initiating the process of cellular degeneration (sanchez et al., 2005). some physiological ways to remove free radicals (ros) toxicity are cellular defense systems that could be considered bio-indicators of healthy life in the surrounding water body. defenses against free radicles include scavenger compounds like (cat) catalase, (gst) glutathione stransferase, (gsh) glutathione, (sod) superoxide dismutase, (gpx) glutathione peroxidase, and (gr) glutathione reductase and (mt) metallothionein, (storey, 1996) as well as lipid peroxidation (lop) (ahamad et al., 2005). this imbalance may also cause dna damage, which has been recommended as a bioindicator for determining the level of genetic toxicants. the most prominent example of genotoxicity in a test of the micronucleus (mn) (rabello-gay, 1991) is connected with the identification of nuclear changes in fish erythrocytes, representing an important analysis tool (carrasco et al., 1990). the gills have important roles because, being the major position of gas exchange and taste (hughes, 1982), function in the osmoregulation process (verbost et al., 1994), nitrogen excretion (sayer and davenport, 1987), and acid-base equilibrium (goss et al., 1992). several studies were performed on the fish gills by microscopical examination that revealed an increase in the tissue damage degrees is correlated to the water quality (wael et al., 2013). in gills, the morphological alterations represent the adaptation strategies to maintain physiological functions and evaluate chronic or acute exposure to pollutants in the aquatic ecosystem (morina et al., 2013). therefore, this work aimed to study the pollution of the shatt al-arab river using histology and some other biochemical parameters of gill in nile tilapia (oreochromis niloticus) as water quality biomarkers. materials and methods specimens of nile tilapia with body weight and length of 200±50 g and 17±3 cm, respectively, (n =10, each site) were captured from four different sites in the shatt al arab river, including (1) electric power of the haratha, (2) electric power of the najebia, (3) kandac canal and (4) south part of the river in basrah, iraq, using a fishing-net in december 2021 (fig. 1). the sampling sites were located along the river, having various types of contamination sources (sites 1, 2, and 3), and a clean site with relatively well preserved, be free of industrial, agricultural, and human domestic effluents was selected as site 4 (fig. 1). the physical and chemical properties of the river water were determined using the methods described in (apha, 1998). in the field, a water quality checker was used to measure temperature, ph, hardness, dissolved oxygen (do), conductivity (ec), and biochemical oxygen demand (bod). the nitrite, po4, so4, and chlorides (cl-) were determined after filtering the water (mackereth et al., 1978). the concentration of heavy metals in the column water was determined by aas flam of atomic absorption spectrophotometry, using the srm 3114 nist (usa) as reference standards for quality control and assurance. in brief, the water samples were fixed with a solution of nitric acid, filtered through a glass filter (0.45 lm pore diameter), and the heavy metals concentration was determined by a flamer spectrophotometer. biochemical analyses: the gill tissue (1 g tissue/15 ml buffer) was homogenized in phosphate buffer (0.1 m, ph 7.4). this homogenate was divided into aliquots for erod, cat, sod, gsh, and gts activity enzymes. the ethoxyresorufin-odemethylase (erod) enzyme level was assayed in fish gills by fluorometric methods (galgani and payne, 1991). glutathione– s–transferase (gst) antioxidant enzyme activity was determined in cytosolic fraction by 1-chloro2, 4–dinitrobenzene as a substrate (habig et al., 1974). superoxide dismutase (sod) enzyme level was measured according to mccord and fridovich (1969). cytosolic catalase activity (cat) was measured at 240 nm (aebi, 1984). the metallothionein-like protein (mtlp) was determined in fish gills, according to thompson and cosson (1984). per oxidative and cytogenetic damage evaluation: ohkawa et al. (1979) procedure with some 408 jasim et al./ study of pollution in shatt al-arab river using histological alternations modifications was used to determine lpo in tissue homogenates. blood samples were taken from caudal vasculature with a heparinized syringe, the account of erythrocytes micronuclear by preparing a blood smear on a glass slide and stained with giemsa dye (al sabti, 1985). histological examinations: the mid-section of the second gill arch from the left side was sampled and fixed in 10% buffered formalin. after that, the samples were decalcified, dehydrated in ethanol in a graded series, cleared in xylene, and embedded in paraffin blocks (luna, 1968). sections of 5 µm thickness were cut, and three sections were stained with the standard hematoxylin and eosin (h&e) staining technique. histological examinations assessed the distribution of lesions in the analyzed organ and the severity of the alterations. however, depending on the analysis method developed and modified by tavares et al. (2019), the lesions were distributed in scores (sc) and an important factor (fi). the sc was distributed as (0 the absence of lesions or the presence of lesions on up to 10% of the total analyzed tissue), 1 low-frequency occurrence of lesions ranging from 11-25% for the total analyzed tissue, 2 moderate frequency lesions occur on 26 to 50% of the total analyzed tissue, 3 frequently contain apparition of lesions on 51-75% of analyzed tissue, whereas 4 frequently contain apparition of lesions on 76-100% of analyzed tissue. however, fi indicates how such a change would affect organ function as well as the likelihood of the fish surviving and receiving the following values: (1) lesions that are easily reversible and of minor pathological significance; (2) reversible lesions when the stressor is moderate and has been neutralized histo-pathologically; and (3) lesions that are generally irreversible and have a high histopathological prominence. the lesion index was calculated: i lesion = (sc x fi), and the lesion organ index is represented by i lorgan = i lesion. statistical analysis: the results were expressed as means±sd (standard deviation) for experimental groups of ten fish. statistical software was performed in spss program. anova was used to determine statistical differences between groups (zar, 1999). the significance of the results was ascertained at a p≤0.05. results the physical and chemical parameters of the shatt al-arab river water from the studied sites are presented in table 1. the chemical and physical profile and contamination status of sites 1, 2, and 3 show a significant polluting load, contain high-level of bod, nitrite, chloride (cl), po4, and so4, trace metals concentration (pb, cu, cd, zn, and cr), a high level of conductivity (ec) and a low do. also, the chemical analysis recorded low concentrations but dangers of chlorpyrifos insecticide in sites 1, 2, and 3. however, at site 3, the nitrite, heavy metals, and chlorpyrifos insecticide levels exceeded the water figure 1. map of shatt al-arab river showing sampling sites. 409 int. j. aquat. biol. (2022) 10(5): 406-416 quality guidelines for freshwater life protection established by national and international legislation. our results acquired after assessing the contamination indices provided supplementary evidence of the low water quality at sites 1, 2, and 3, where the fish were captured, compared with site 4. biotransformation enzymes as biomarkers: the fish gills of sites 1, 2, and 3 showed an increase in their biotransformation enzyme activities compared to site 4, which indicates a point of reference. parameter site 1 site 2 site 3 site 4 physical and chemical factors temperature (c0 ) 17.33±1.22 18.44±1.42 12.56±1.11 10.32±1.01 ph 7.8±0.056 8.01±0.087 8.13±0.045 7.54±0.055 hardness (mm ) 445±44.6 687±63.6 651±105.54 213±30.5 do (mg o2 /l) 4.62±0.98 4.54±0.97 4.19± 0.88 4.83±0.76 conductivity (ls cm/1) 4.21±0.003 4.43±0.87 16.54±1.32 2.11±0.00021a bod (mg/l) 2.15±0.22 2.41±0.34 3.2±0.36 1.5±0.21 nitrites (mg / l) 0.58±0.055 1.25±0.067 1.99±0.087 0.464±0.099 chlorides (mg/ l) 550±38.6 801±35.6 822±44.8 312±30.8 po4 (mg/l) 0.43±0.02 0.61±0.031 1.02±0.053 0.33±0.012 so4 (mg /l) 550±31.7 780±47.8 830±50.3 467±33.1 tds (mg/l) 2400±87.9 2030±80.00 2105±78.99 1340±68.97 heavy metals (ug /l) pb 0.143±0.004 0.342±0.010 0.87±0.021 0.054±0.002 cu 0.057±0.005 0.078±0.003 0.104±0.003 0.055±0.0013 cd 0.0051±0.00 0.017±0.002 0.031±0.0021 ns zn 0.29±0.003 0.33±0.004 0.39±0.002 ns cr 0.008±0.001 0.007±0.001 0.014±0.002 ns insecticides (ug/l) chlorpyrifos insecticide ns ns 0.018± 0.007 ns other organophosphates ns ns ns ns other organ chlorines ns ns ns ns table 1. shows the physicochemical profiles of shatt al-arab river water samples collected from sites 1, 2, 3, and 4. figure 2. effect of sampling sites on biochemical parameters in gills of o. niloticus (a) total cyt p450 activity, (b) erod activity, (c) cat activity, (d) gst activity, (e) sod activity, (f) mtlp level, and (g) lpo concentration level. the different letters denote indicate significance (p≤0.05). values are mean±sd (n=40). 410 jasim et al./ study of pollution in shatt al-arab river using histological alternations figure 3. microphotographs of the blood smear of o. niloticus fish, note micronucleus mutation (arrows) in sites 1, 2, and 3 (a, b, and c). giemsa stain (1000x). cytogenetic mutation site1 site2 site3 site4 micronucleus (rbc) (%) 7.88±0.51d 10.51±2.11c 18.8±3.32b 2.61±0.87a values are expressed as mean ±sd (n = 40), the significant differences were obtained (p≤0.05) when compared to control group values. table 2. micronucleus (%) in o. niloticus rbc from various sites of shatt al-arab river. fish species pathological signs site 1 site 2 site 3 site 4 o. niloticus hypertrophy of the epithelium + +++ hyperplasia of the epithelium ++ ++ +++ hyperplasia of the mucous cell + + hyperplasia of the chloride cell + ++ hypertrophy of the chloride cell + + secondary lamellae fusion ++ ++ +++ + edema + +++ lifting of the epithelium + + +++ necrosis + ++ hyperemia + + ++ hemorrhage + ++ telangiectasia + + inflammatory infiltration + ++ (–) no histological alterations or mild histopathological alterations; (+) moderate histological alterations; (++) severe histological alterations; and (+++) very severe histological alterations in the gill surface architecture. table 3. distribution of the detected histopathological damage in the gills of the o. niloticus collected from different sites along the whole course of the river shatt al-arab river. 411 int. j. aquat. biol. (2022) 10(5): 406-416 however, in sites 1, 2, and 3 the t-cyp450 and erod showed a significantly higher level (p≤0.05) than site 4. however, the erod activity was more noticeable and affected by the low water quality compared to the t-cyp450 enzyme (figs. 4, 5). antioxidant enzymes as biomarkers: the fish gills figure 3. a cross-section of a fish's gills containing the following: (3-a-site4) description of histological structure of semi-normal gills of o. niloticus fish gills contain primary and secondary respiratory lamellae (pl and sl), covered by epithelial layer (epi), and blood sinuses respectively (bs). (3-b – site1) fish's gills contain hyperplasia (hp), slightly fused secondary lamellae (sf), hyperemia (he), and epithelial lifting (el). fish's gills contain club shape (csh), hypertrophy of secondary lamellae cells (hyc), and hemorrhage (hem) (3-c and dsite2). section of fish gills contains many club shapes (mcsh), a hyper fusion of secondary lamellae (hf), edema (ed), acute hyperplasia (ahyp), inflammation cells (inf) and hypertrophy (ht) as well as severe bleeding (sb) and necrosis of secondary filaments (n), ( 3-e and fsite3). (h and e 400x). histopathological signs gill lesion index (i lesion%) site 1 site 2 site 3 site 4 fi sc fi sc fi sc fi sc epithelial hypertrophy 1 15.6 2 28.9 3 70.9 0 2.22 epithelial hyperplasia 1 11.7 2 25.5 4 86.5 1 3.11 mucous cell hyperplasia 1 11.8 2 25.8 3 75.4 0 0.00 chloride cell hyperplasia 1 13.8 1 12.8 3 66.8 0 0.00 chloride cell hypertrophy 1 11.1 1 13.9 3 60.7 0 0.00 fusion of secondary lamellae 1 14.6 1 12.7 4 90.8 1 5.35 edema 0 7.42 1 11.9 3 55.9 0 0.00 epithelial lifting 1 11.7 1 14.9 3 74.9 0 0.00 necrosis 0 6.11 1 13.8 4 80.6 0 0.00 hyperemia 1 12.9 2 28.9 3 67.7 1 0.00 hemorrhage 1 11.9 2 26.7 4 80.3 0 0.00 telangiectasia 1 11.3 1 15.8 3 65.4 0 0.00 inflammatory infiltration 0 7.71 1 12.1 3 60.7 0 0.00 table 4. gill alteration index (i lesion %) of o. niloticus from sites selected on the shatt al-arab river. fi important factor. 412 jasim et al./ study of pollution in shatt al-arab river using histological alternations of sites 1 2, and 3 showed an increase in their antioxidant enzyme activities compared to site 4. however, in sites 2 and 3, the cat showed a significant increase compared to sites 1 and 4. the increase in gst and sod activities was significant only in site 3, compared with other regions. this reveals that low water quality in sites 2 and 3 had a greater impact on cat than gst and sod enzymes (figs. 1, 2, 3). metallothionein protein (mltp): one of the goals of this study was to evaluate the relationship between low water quality on the gill quantitative expression of metallothionein as a defense response against heavy metals pollution, which shows significantly greater values (p≤0.05) in sites 1, 2, and 3 compared to site 4. however, site 3 recorded the highest value. lipid peroxidation: sits 1, 2, and 3 show an increase in lpo of fish gills compared to site 4; however, site 3 recorded the highest lpo (p≤0.05) than other locations. sites 1 and 3 also showed a significant increase in fish gills lpo (p≤0.05) than site 4. histological structure of gills: the micronucleus– rbc mutation is shown in table 2. our results confirmed that micronucleus–rbc mutation was higher in all sites compared with reference site 4 and site 3 had the highest value (figs. 1, 2, 3). the fish from sites 1, 2, and 3 revealed several histological changes in the gills (table 1). the gills in the reference region had normal structure (fig. 3a). the individuals from site 1 had a low prevalence of lesions. an increase in the histological lesion of fish gills was recorded in site 2, and the acute histological lesion in site 3. the most relevant histological changes were hyperplasia, which is accompanied by lamellar fusion and rupture of the epithelial layer (fig. 3b). the damage was more pronounced in site 2 due to the vacuole formation and necrosis in the interfilament region (fig. 3c). vasodilation, slight necrosis and epithelial lifting in site 2 (fig. 3d) and hypertrophy, spiked secondary lamellae, and end filaments in the shape of a club in site 3 (fig. 3e) were main observed histopathological alternations. in some cases, in site 3, the damage is exacerbated by the complete loss of gills structure at several points and prominent hemorrhage and necrosis of secondary lamellae (fig. 3f). the significant changes in the i lesion % index occurred in fish gills coinciding with the low water quality in sites 1, 2, and 3 compared with the reference i.e. site 4. however, site 3 recorded significant alterations in primary and secondary filaments represented by hypertrophy, hyperplasia, and the fusion of secondary lamellae, edema, epithelial lifting, necrosis, hyperemia, hemorrhage, telangiectasia, and inflammatory infiltration (table 3). discussion in the present article, o. niloticus was chosen as a bio-model because its fish is a pollution-resistant species ideal for a biomarker of water pollution. generally, the quality of physical and chemical factors provides vital data regarding water health. sites 2 and 3 had poor water quality containing higher concentrations of hardness, do, ec, bod, nitrate, chloride po4, so3, and tds; also recorded higher concentrations of trace metals and the chlorpyrifos insecticide. most physical and chemical standards in site 3 do not contain the limits permitted by the world health organization (who) for freshwater quality (al-asadi, 2014). however, the high concentrations of pollutants mentioned above were due to hartha and najebbia power plants and human activities, which discharge organic, inorganic compounds, heavy metals, ions, and insecticides into the river. most of the physical and chemical compounds in the shatt al arab river were not within the limits of acceptability according to who and iraq standards (al-hejuje, 2015). therefore, the fish inhabiting the shatt al-arab river were exposed to many mixtures and complex pollutants, whether chemical compounds or physical factors and our findings showed the ecotoxicological impact of multiple contaminants on o. niloticus. oliveira et al. (2010a) mention that low levels of do interfere with the fish population, causing 413 int. j. aquat. biol. (2022) 10(5): 406-416 abnormalities in breeding and death. disturbing the balance of oxygen supply/demand influences oxygen levels in tissues, interfering with antioxidant defenses. generally, the catalytic efficiency and binding capacity of enzymes were affected by ph and temperature. ph alteration may also affect the bioavailability of severely contaminated with special reference to metals (carvalho et al., 2004). because biological and chemical processes cannot degrade trace metals, they tend to accumulate in aquatic sediments and soils with water; therefore, they have the potential to enter the food chain, posing a health risk to aquatic animals such as fish (castiglione et al., 2009; eagderi et al., 2022). contamination substances such as heavy metals, pesticides, and other chemical compounds may immediately affect aquatic organisms by generating free oxygen radicals (ros), resulting in primary degeneration and genotoxicity (halliwell and gutteridge, 1985). our study revealed that the biotransformation enzymes, total cyp 450, and erod enzymes are good indicators for organic and inorganic pollutants in the water. the erod enzyme is considered the best biological marker for water quality. van der oost et al. (2003) pointed out that total cyp 450 and erod activities appeared in many fish species and seemed to be sensitive biomarkers for the pollution of aquatic systems. in freshwater fish, the cytochrome p450 has been studied as a biomarker indicating environmental contaminations due to agricultural sewage or industrial wastes. therefore, it is used for water pollution at low levels, or the pollutants are no longer dissolved in water but persist in the living organism, such as residues of biocidal agents (machala et al., 1997). however, in fish, the class of cyp p450 isozymes and erod are in charge of the biotransformation of a wide range of chemical compounds such as pahs, pcbs, and dioxins, etc.. erod enzyme is the most sensitive catalytic probe for determining fish's cyt p450 system's inductive response (goksøyr and forlin, 1992; whyte et al., 2000). the total cyp 450 is responsible for a wide range of chemical material biotransformation whose catalytic activity (siroka and drastichova 2004). erod is a highly sensitive indicator of contaminant uptake in fish (arellanoaguilar et al., 2009). the results of the current study confirmed that the antioxidant enzymes (cat and gst) are proper indicators for water quality monitoring in the studied sites 1, 2, and 3, while the sod was less sensitive to water pollutants. gst, cat, and sod are the primary line of defense compared to oxygen toxicity induced by the different contamination compounds such as heavy metals, insecticides, and organic and inorganic compounds. gst, cat, and sod are metalloenzymes that play an important role in ros defense by converting superoxide anions to hydrogen peroxide, which is detoxified by both cat and gpx activities (olsen et al., 2001). an increase in cat enzyme activity indicates the presence of a higher peroxide level, for the increased activity of this enzyme at sites 1, 2, and 3 indicated that oxidant defenses were insufficient to prevent the formation of lpo. hence, the removal strategy of free radicals by cat activity is a functional response to oxidative stress (monteiro et al., 2009). gst enzyme significantly prevents oxidative damage by conjugating gsh to lipid peroxide breakdown products (fernandes et al., 2007). gst induction indicates the presence of a variety of chemical pollutants (ahmad et al., 2005). monteiro et al., (2010) explain that gst enzyme induction occurs in various tissues at various times after exposure to inducers. our results showed that metallothionein levels were significantly higher in fish gills collected from the shatt al-arab river (sites 3) compared to the reference sites, i.e. site 4 and sites 1 and 2. metallothionein proteins (mt) have a physiological role in protecting cells from the poisonous effects of free oxygen radicals, which must be considered before elevated mt levels can be interpreted as a marker of metal exposure in aquatic organisms (oliveira et al., 2010b). our study found higher lpo levels in the fish gills, indicating oxidative stress in sites 2 and 3 compared to reference site 4. however, lpo compounds are typically formed due to 414 jasim et al./ study of pollution in shatt al-arab river using histological alternations complex free radical reactions in cell membranes that produce lipid hydroperoxides that decompose double bonds of unsaturated fatty acids and degrade membrane lipids. after being absorbed, pollutants have the potential to react with endogenous substances, creating a state of biological harm that may destroy the life quality (cerqueira and fernandes, 2002). the results confirmed that lpo is an excellent biomarker for sensing lower water quality. our results showed a wide range of gill alteration index (i lesion %) in sites of 1, 2, and 3 compared to the reference site 4. our results agreed with santos et al. (2014), who found several histological changes, such as hyperplasia and hypertrophy of epithelium lamellae, lifting of respiratory epithelium, edema of epithelial cells, and clubbing of secondary filaments. the fish gills account for the top 50% of the surface area of a body; therefore, understanding the level of tissue damage produced can help determine the toxicity of the environment, making fish highly suitable for assessing the health of aquatic systems (ogundiran et al., 2009). conclusions our study confirmed that the enzyme activities, micronucleus mutation of rbc, and histological alterations of the gill of o. niloticus could be used as a biomarker to monitor the water quality of the aquatic ecosystem and its effects on organisms that live in it. our results contribute to the knowledge of the linkage between biological responses and environmental variations and the importance of these biomarkers in environmental monitoring. further, it is proved that o. niloticus could be used in further environmental pollution studies in freshwater ecosystems. references aebi h. 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(2021) 9(3): 207-213 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article isolation and identification of bacteria degrading 2,2-dichloropropionic acid in water sample wafaa hassan muslem* 1, hassan muslem abdulhussein2, mohamed faraj edbeib3, roswanira abdul wahab4, fahrul huyop5 1department of biology, college of science, mustansiriyah university, baghdad, iraq. 2department of dentistry, dijlah university college, baghdad, iraq. 3department of animal production, faculty of agriculture, baniwalid university, libya. 4department of chemistry, faculty of science, universiti teknologi malaysia, 81310 utm johor bahru, malaysia. 5department of biosciences, faculty of science, universiti teknologi malaysia, 81310 utm, johor bahru, malaysia. s article history: received 17 april 2021 accepted 23 june 2021 available online 2 5 june 2021 keywords: enterobacter bioremediation biodegradation dehalogenase haloalkanoic acid abstract: the widespread use of herbicides containing 2,2-dichloropropionic acid (2,2-dcp) as a recalcitrant halogen compound poses significant environmental risks and can be harmful for human. consequently, it is important that the bio-based detoxification method is developed in an environmental manner. this study was aimed to isolate and identify a possible degradation 2,2-dcp bacterial strain as the sole source of carbon. a bacterial dehalogenase producing 2,2-dcp was isolated named as wm. the wm strain was shown to have 98% sequence identity and characteristics similar to enterobacter sp. based on 16s rrna analysis, biochemical and morphological tests. phylogenic analysis showed that the wm strain is enterobacter sp.. in media with 20 mm 3cp, the bacteria were well growing at 37°c, although an optimal chloride ion release was 0.48 μmol cl/ml. our finding is first report of an enterobacter sp. strain which can use 2, 2-dcp as sole carbon source in a competent manner. introduction our environments specially in aquatic ecosystems are seriously affected by increased levels of industrial chemicals, wastes and biocides. this is led important research programs to examine the concentraion of these substances in the nature (ejlertsson et al., 1996; baumann et al., 2005). classifications of chemical compounds in the environment are based on natural and biosphere-free products according to the groups (hutzinger and veerkamp, 1981), which have not natural origin but from human activities defined as xenobiotics. temporary or permanent xenobiotic contamination may affect biosphere's normal functions. modern agriculture lead to applications of herbicides and pesticides (atlas, 1996). in addition, numerous routes could introduce pollutants in the environment such as illegal dumping, leaked effluents, liquidation and chemical evaporation. the full extent of harm to the environment should be evaluated by effects such as toxicity, carcinogenicity, bio *correspondence: wafaa hassan muslem doi: https://doi.org/10.22034/ijab.v9i3.1269 e-mail: wafaahassan2017@uomustansiriyah.edu.iq agrandissement and persistence. halo-aliphatic compounds such as 2,2dichloropropionic acid and trichloroacetic acid are common herbicides (edbeib et al., 2020). 2,4 dichloroxyacetate (2,4-d) and 2,4,5-trichloro-trichlorophenoxyacetate (2,4,5-t) are of great interest for their degradation and therefore many researchers were initiated to investigate their environmental fate (ghosal et al., 1985; farhana et al., 1987). haloorganic compounds (e.g. methoxychloride, lindane, aldrin and 1,1,1 trichloro-2,2-bis (ddt)) contributed to the contamination of the environment (ritter et al., 1995). however, naturally occurring halogenated compounds are usual. in soil and fresh water ecosystems, halogens such as inorganic salts or minerals are relatively abundant (fetzner, 1998). more than 2000 halo-organic compounds are released by various marine organisms, plants, fern, insects, bacteria and fungi into the biosphere as natural products (gribble, 1994). furthermore, analogs of 208 muslem et al./ bacteria degrading 2,2-dichloropropionic acid in water bromins and iodine chloralkans in natural conditions have been identified (gschwend et al., 1985) and can be seen as biodegradation models for more halogenated alkanes, in view of their chemical and analytical implication. the primary agents of biological recycling are microorganisms, which have evolved a broad range of enzymes, pathways and control mechanisms to degrade and use of the pollutants as energy sources (madigan et al., 2000; talaro and talaro, 2002). there is no evidence from laboratory studies shows the involvement of microorganisms in transforming halogenated xenobiotics as an important factor in determining their environmental fate (talaro and talaro, 2002; muslem et al., 2018). as many other organisms participates, but to a lesser extent, microorganisms are not exclusively responsible for degradation of organic compounds. photochemical decomposition was proposed as a major way of degrading certain compounds (zabik et al., 1976). all existing organic compounds are considered thermodynamically unstable in varying degrees, and co2 and microbial growth energy can be generated in principle. but most organic compounds are completely stable in the kinetic sense, and they are not degraded or mineralized at significant rates under physiological conditions without catalysis. such enzyme-based catalysis is common in aerobic microorganisms seeking growth energy. it is a difficult chemical and biological activity relationships and may help to the recalcitrance of some halogenated compounds. the removal, especially from organic molecules of halogens, fluorines and chlorine has long been fascinating for chemical and microbiology professionals because these mechanisms ease the inhibitory effect and provide alternative carbon and energy sources for growth (slater et al., 1995). in general, the number of halogens per organic molecule is more difficult to degrade alone or microbially (commandeur and parsons, 1990). halogenated compounds are ideal pesticides. leasure (1964) observed that chlorinated substrates are the most suitable herbicidal activity over brominated, fluorinated and iodinated compounds. but an αchlorine result in the highest herbicidal activity in propionic acid and homologous butyric acid; dca (dichloroacetate) and trichloromethane (mca) do not result in activity, and with α-chlorinated materials, the proper effect can be weakened. also, the increase in the chain length seems a weakly active 2,2dichloropentanoic and 2,2-dichlorohexanoic inactive acid to decrease the activity even of very biocidal compounds. the acidic properties of halogen when connected with the parent molecule may be another factor that can make haloorgan compounds desirable for biocidal action. acid dissociation constant shows the relative strength of an acid (ka). the stronger the acid, the greater the dissociation; logarithmically, ka is often expressed because of a broad range of spans. stronger acids would therefore have lower pka values (solomons, 1994; wade, 2003). the more highly replaced aliphatic and aromatic compounds, the more strongly electron-negative. the acidic nature of the halo-organ compounds is also affected by the position of substitution, and the more the halogen replacement is separated from the –cooh group, the less acid is indicated by the pka values, known as "inductive effect" phenomenon. the inductive effect is obvious when one or more groups strongly draw electron from the carbon. the extent of the substitution effect is determined by its distance from carboxylic acid, while distant substitution agents have less acidity and a rapid decline in distance induction effects (wade, 2003). halogenized compounds may be used in crops as an herbicide to excessively damage the human and natural environment because of the persistent, biologically accumulative and toxic halogenated compositions. such biocides are used for plant protection directly and deliberately in the environment. underground water sources as well as plants could pollute by these non-degraded synthetic chemicals. halogenated analogs of intermediate metabolites were also shown to be toxic because they inhibited a major reaction in organisms' central metabolism (slater et al., 1995). for certain important pollutants, dehalogenation is the only known biodegradation system. it is usually less toxic and 209 int. j. aquat. biol. (2021) 9(3): 207-213 easier to degrade xenobiotic compounds. the significance of the study has been derived from little available information regarding the bacteria isolates degrading 2,2-dcp compounds. therefore, this study was aimed the isolation and identification of a possible degradation 2,2-dcp bacterial strain as the sole source of carbon. materials and methods isolation of bacteria strain: in an agriculture area that haloacids have been utilized i.e. previously exposure to pesticide, a soil sample were removed and packed into sterile jars, sealed, and stored at 4°c. sample processing and purification: in 30 ml of the sterile distilled water, a 10 g soil sample is suspended, and the mixing was continued until the particles’ settlement in the base. then the pipettes are spread over plates with 20 mm of 2,2-dcp on 0.1 ml of the mix of suspension soil. the plate was incubated at 37°c for 6 days so that bacterial growth is permitted. one plate is incubated as a control along with the other with blank inoculation. after the incubation, the colonies are carefully isolated and streamed through the streak-plating method into the same type of minimal medium plate. the method of streak plate is continued to achieve a pure colony. media for growth: based on miller (1972), the yeast extract containing nacl (10 g/l) and tryptone (5.0 g/l) was used as luria broth (lb) media. the 10x basal salts were prepared using nah2po4.2h2o (10.0 g/l), k2hpo4.3h2o (42.5 g/l) and nh4.2so4 (25.0 g/l) for minimal concentrations of pjc chloride-free. the solution for trace metal salts were included 10x of feso4.7h2o (120.0 mg/l), mgso4 (2.0 g/l), mnso4.4h2o (30.0 mg/l) and nitriloacetic acid (c6h9no6) (1.0 g/l) as well as disposed water cocl2.6h2o (10 mg/l) and znso4.h2o (30 mg/l) (hareland et al., 1975). minimal media for bacterial growth has been autoclaved (121°c for 15 min, 15 psi) containing 10 ml basal salts per 100 ml distilled water and 10 ml trace metal salts. sources of carbon i.e. 1m 2,2-dcp were separately sterilized, then added to the desired final concentration aseptically to the media. 0.05% (w/v) final concentration was added to the liquid minimal cultures by yeast extract. bacteriological oxoid agar (1.5% w/v) was added before sterilization to prepare a solid medium. organism culture was prepared by adding 0.3 ml 50% sterile glycerol to 0.7 ml culture and then mixed with the sample thoroughly and frozen in dry ice. cultivation of stocks was stored at -80°c. microbial growth measurement: the absorption was measured at 680 nm for microbial growth (a680 nm). for this, a sample of 2 ml culture was pipetted and absorption was measured in 680 nm in a spectrophotometer (t60 uv/vis). absorption readings have been taken at appropriate intervals for broth medium turbidity. a graph of log a680 nm against exponential phase time was determined to measure specific growth rate. μ was determined from the slope of the graph (stanbury and whitaker, 1984) and td equation time was calculated to double = 0.301/μ (shuler and kargi, 2002). he bacteria cultures were incubated using temperature-adjustable rotary shaker at 30±1oc, t150 rpm. test media optical density (od) was measured using a600 nm at every 6 hrs. growth profile: the wm isolate was first cultured in 20 mm of 2,2-dcp, and the culture was transferred to the test media. the test media were prepared in triplicates of liquid minimal media that consisted of three different concentrations of 2,2-dcp (10, 20 and 30 mm) and were based on substrate concentration and culturing condition as suggested (mesri et al., 2009). pcr and sequencing: following the pcr, the products were sent to the 1st base® company for dna sequencing in both direct and reverse directions. then, bioedit software was used to observe the sequence. finally, blast was used to find the similar bacteria species based on 16s rrna gene (https://blast.ncbi.nlm.nih.gov). 16s rrna gene phylogenetic analysis: the obtained 16s rrna sequences were compared using the blast with other sequences (table 1). the first ten similar sequences were retrieved and aligned using the culstalw. the phylogenetic tree was reconstructed with mega (version v6) based on neighbor210 muslem et al./ bacteria degrading 2,2-dichloropropionic acid in water joining algorithm (saitou and nei, 1987). halide ion assays: during the dehalogenation reaction, the free halides were measured by the adapting the bergman and sanik method (bergmann and sanik, 1957). sample (1 ml) was added and carefully mixed in 9 m nitric acid (reagent a) to 100 μl of 0.25 m ferric sulphate ammonium. to this, 100 μl of mercuric ethanol saturated with thiocyanates was added (reagent b), and the solution mixed by vortex. the color was measured at a460 nm for 10 min. by comparing the absorbance of the samples to a standard curve of known halide concentrations, the halide concentration was determined. the standard halide ion assay curve was built on the basis of the chloride concentration of sodium chloride (nacl) 0.01 m (10 mm). in each 0.8 ml of standard solutions in a clean cup, 0.1 ml of reagent a and 0.1 ml of reagent b were added and left for 10 min for color development in the cup (orange). the mix was measured at a460 nm. approximately 1.5 ml of culture sample liquid was pipetted and centrifuged for cell removal at 10.000×g for determining the production of chloride by bacteria. 0.8 ml of the supernatant was transferred to a clean cup and a reagent of 0.1 ml plus 0.1 ml of the reagent b, then left to fill for 10 min. finally, its absorption was read at a460 nm. results and discussions isolation and characterization of 2,2-dcp degrading bacteria: the bacterial colonies were grown on a 20 mm 2,2-dcp minimal medium as the only carbon source 6 days after its culture (fig. 1). one type of bacterial colony was identified as degrading bacteria after a series of sub-cultures of randomly selected primary plating colonies. this sole carbon source user at 37oc was named as "wm strain" that grown on a minimum medium of 20 mm 2,2dcp. analysis of bacteria's cellular morphology under a light microscope showed isolates of 20 mm 2,2-dcp minimum media agar which were usually formed in circular, white (pale brown through suntans) and creamy colonies. gram staining showed that this strain i.e. wm, is a gram negative one. minimum liquid medium growth different 2,2dcp concentration supplemented: the wm isolate growth was monitored as the sole source of carbon in liquid minimum media with differing concentrations accession number description max. score total score query coverage max. identity eu139848 enterobacter sp. gj1-11 2518 2518 98% %99 ef059865 enterobacter cloacae strain e717 2529 2529 98% 99% kf478236 enterobacter cloacae strain ipri 2525 2525 98% 99% he978272 enterobacter cloacae subsp. 2523 2523 98% 99% nr028912 enterobacter cloacae strain 279-56 2525 2525 98% 99% fj888607 enterobacter sp. bsra3 2523 2523 98% 99% kf523893 enterobacter cloacae strain gw-3 2521 2521 98% 99% kf984470 enterobacter sp. bab-3361 2518 2518 98% 99% jn986806 enterobacter sp. wy2-d9 2518 2518 98% 99% ab644526 enterobacter cloacae strain 56_2_1 2518 2518 98% 99% table 1. the retrieved sequences with them accession number from genbank. figure 1. growth on spread plate minimal media contain 20 mm 2,2-dcp observed after 6 days (incubated at 37oc). 211 int. j. aquat. biol. (2021) 9(3): 207-213 of 2,2-dcp. bacterial growth on these media showed that the 2,2-dcp carbon atoms are suitable for use. growth profile: the growth profile of isolated bacteria strain wm in different substrate concentrations are presented in figure 2. assay of halide ion: ion liberation was controlled by halide ion to confirm the ability of wm bacteria to degrade 2,2-dcp. the chloride ion concentration was determined using mmol/l as standard measurements of soluble chloride by converting the absorption value into the concentration of sodium chloride (bergmann and sanik, 1957) showing the results in figure 3. dna extraction and pcr amplification of 16s rrna gene: to extract genomic dan, the dna extraction and purification kits (promega, wizard® genomic dna kits) were used and after nano-drop analysis, and the concentration of the isolated genetic material was approximately 300 ng/μl. the 16s rrna gene has been amplified using universal primers of 16s-f and 16s-r. using gel electrophoresis, the pcr product was visualised, showing a gene with almost 1500 bp. a total of 1500 bases were aligned with other retrieved sequences, indicating that wm is a member figure 2. wm strain growth profile on minimum media of three different 2,2 dcp concentrations. figure 3. correlation of 20 mm 2,2-dcp of wm growth with chloride ion released. 212 muslem et al./ bacteria degrading 2,2-dichloropropionic acid in water of the genus enterobacter and its partial 16s rrna gene sequence was submitted to ncbi genbank (fig. 4). the 16s rrna, is a universal target of highest precision for identification of bacteria (ventura and zink, 2002). based on the results, bacterium wm is identical to enterobacter sp., which corresponds to a sequence identity of 98%. the reconstructed phylogeny tree of the extracted 16s rrna and those retrieved from ncbi with a bootstrap value based on nj algorithm are shown in figure 5. as finding of this study, a soil-isolated bacterium has a great potential in degrading 2,2-dcp, identified as enterobacter sp. that was well-growing in the minimum medium of 10, 20 and 30 mm. it produces the dehalogenase enzyme and as an environmentally friendly bacterial strain, can be useful as a bioremediation agent of detoxifying xenobiotic compounds through 2,2-dcp degradation. references abel s.e.r. 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(2017) 5(4): 260-262; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology short communication helminth parasites fauna of the green toad, bufo variabilis, laurenti, 1768 (anura: bufonidae) from the fars province, iran ehsan rakhshandehroo1, amin ahmadi1*, zahra asadollahi2 1department of pathobiology, school of veterinary medicine, shiraz university, shiraz, iran. 2department of parasitology, school of veterinary medicine, university of tehran, tehran, iran. article history: received 13 june 2017 accepted 24 august 2017 available online 2 5 august 2017 keywords: helminth infection polystoma nematotaenia rhabdias ranae abstract: the green toad, bufo variabilis has been a common species of toads in iran with a wide distribution in most provinces. the main goal of this study was to determine the helminth parasite fauna of b. variabilis in southwest of iran. among 30 toads inspected for helminth infection from different sites, 100% harbored one or more parasite species. some toads were afflicted by intestinal obstruction, due to the infection with a large number of cestodes. twenty-eight cases (93%) had mixed infection with cestodes (in intestine) and nematodes (in lung). also, a toad was infected with a single monogenean parasite in its urinary bladder. the identified helminth parasites were monogenean, polystoma viridis; cestode, nematotaenia dispar and a species of nematode, rhabdias ranae. this study reports for the first time the presence of nematode species r. ranae in b. variabilis in iran. introduction the green toad, bufo variabilis laurenti, 1768 is one of the most widespread old world amphibian species. it belongs to the family bufonidae, a widely distributed amphibian families (degani et al., 2013). bufo viridis is widely distributed in much of europe, russia, mongolia, mediterranean countries, and central and southwest asia (stock et al., 2001). the green toad is an important component of local ecosystems (vashetko and siddikov, 1999). in iran, its populations are widely distributed in most provinces and observed from below sea level to 4600 m above (baloutch and kami, 2007; derakhshan and nokhbatolfoghahai, 2015). in recent years, several species of helminth parasites has been reported from different organs of toads from iran. mashaiiet al. (2008) reported one monogenean (polystoma viridis), one cyclophyllid cestode (nematotaenia dispar), and two nematodes (rhabdias bufonis and cosmocerca sp.) in green toads collected from the north and northeast of iran. they also recorded monogenea, p. viridis; digenean, *corresponding author: amin ahmadi doi: https://doi.org/10.22034/ijab.v5i4.333 e-mail address: ahmadi.a63@gmail.com haplometra cylindracea; cyclophyllid cestode, n. dispar and nematoda, cosmocerca ornata, c. commutata, r. bufonis and aplectana sp. from toads in different parts of iran (mashaii, 1999; mashaii et al., 2000; mashaii, 2005; massoud and farahnak, 1994). these studies seem not enough and thus more precise investigations are still required to identify the complete fauna of helminthes in toads (dusen, 2011; mohammad, et al., 2010). therefore, the present study was conducted to determine the parasitic fauna of b. variabilis in the fars province, southwest of iran. materials and methods a total of thirty adult green toads were collected by hand from different locations in the fars province (29.1044°n, 53.0459°e), southwest of iran. the frog carcasses transferred to the laboratory and dissected. the body cavity was opened by a longitudinal ventral incision and the alimentary canal was excised. then, the contents of each organ (lungs, liver, gall bladder, kidneys and urinary bladder, and intestine) were 261 int. j. aquat. biol. (2017) 5(4): 260-262 mixed with 0.5% saline solution, poured into petridishes and examined under a stereomicroscope. the lungs were cut and checked out for infection. the worms were cleaned in saline and fixed in 70% ethanol. monogeneans and cestodes were fixed in 70% alcohol while being slightly pressed between two glass slides, stained with acetocarmine, dehydrated in a series of alcohol concentrations of 80%, 90% and 100%, cleared in xylene and mounted in canada balsam. nematodes were cleared in lactophenol for examination. all the specimens were investigated morphologically by microscopic observations and identified based on khalil et al. (1996) and yamaguti (1959). results and discussion despite its importance, the parasitic fauna in iranian toads has received little attention from researchers (mashaii, 1999, 2008; massoud and farahnak, 1994; combes and knoepffler, 1972). all collected toads were infected with at least one parasite. helminth parasites of b. viridis were the monogenean, b. variabilis in the urinary bladder, and the cyclophyllid cestode, n. dispar and the nematodes, r. ranae (fig. 1) in the intestine. one of the toads was infected with only one monogenea, p. viridi. twenty-eight out of thirty toads (93.3%) had mixed infection with cestodes and nematodes. except the nematode r. ranae, p. viridis and n. dispar has been previously reported as parasite of b. variabilis in various regions of iran (mashaii, 2005; mashaii et al., 2008); it is also noteworthy that n. dispar has a wide distribution in amphibians and reptiles all over the world except nearctic region (prudhoe, 1982). in this study, the nematode r. ranae was found in b. variabilis for the first time in iran; however previous studies have reported one different species, r. bufonis (mashaii, 2005; mashaii et al., 2008). the nematode genus rhabdias, stiles et hassall, 1905 is a world-wide distributed group of parasitic nematodes. most species of the genus inhabit the lungs of amphibians (kuzmin, 2005). rhabdias species have a direct life cycle (toad to toad infection) and also a very short life span. they, therefore, represent a potential model for searching the mechanisms involved in a number of phenomena of biological matters, such as trans-mission, genetics and ageing processes (saeed, 2007).the results of this works could contribute for increasing the current knowledge on geographic distribution of parasite species and also their relationships with their hosts. figure 1. rhabdias ranae walton, 1929 (a, b): posterior end of male, and (c) female. 262 rakhshanderoo et al./ helminths of the green toad from iran acknowledgments we would like to thank dr. mobedi, m. ahoo and a.m. alavi for their kind assistances. references baloutch m., kami h.g. (2007). amphibians of iran. tehran university publications, tehran. 177 p. combes c., knoepffler l.p. (1972). helminthes parasites de rana ridibunda ridibunda pallas, 1771 sur les rives iraniennes de la mercaspienne. vie et milieu / life and environment, 2: 329-334. degani d., goldberg t., gasith a., elron e., nevo e. (2013). dna variations of the green toad pseudepidalea viridis (syn. b. variabilis) from various habitats. zoological studies, 52: 1-15. derakhshan z., nokhbatolfoghahai m. (2015). thermal tolerance limits and effects of temperature on the growth and development of the green toad, bufotes viridis. salamandra, 51:129-136. dusen s. (2011). the helminth parasites of the two bufonid toads, european common toad, bufo bufo (linnaeus, 1758) and european green toad, bufo (pseudepidalea) viridis laurenti, 1768 (anura: bufonidae), collected from denizli province, inner-west anatolia region, turkey. helminthologia, 48:101-107. khalil l.f., jones a., bray r.a. (1994). keys to the cestode parasites of vertebrates. wallingford, oxon, cab international. kuzmin y. (2005). the description of rhabdias globocephala (nematoda, rhabdiasidae) from the new host buergeria pollicaris (amphibia, rhacophoridae). vestnik zoologii, 39: 9-14. mashaii n., balouch m., mobedi i. (2008). a report about helminth parasites of some amphibians (anura: ranidae, bufonidae) from the north and northeast of iran. journal of science and technology, 33: 9-13. mashaii n. (2005). helminth parasites of green toad, b. variabilis (anura: bufonidae); tree frog, hylaarborea savignyi (anura: hylidae) and marsh frog, rana ridibunda ridibunda (anura: ranidae) from southwest of iran. iranian journal of veterinary research, 6: 6773. mashaii n., balouch m., mobedi i. (2000). new records about helminth parasites of the marsh frog, rana ridibunda ridibunda (anura: ranidae), from the north of iran. journal of fisheries sciences, 2: 77-88. mashaii n. (1999). new records of trematode parasites (digenea) in the banded frog, rana camerani and marsh frog, rana ridibunda ridibunda (anura: ranidae), from southwest of iran. journal of fisheries sciences, 1: 4147. massoud j., farahnak a. (1994). study on heterophid trematodes in khuzestan, southwest of iran. abs. 8th inter. cong. parasi., izmir, turkey. 2: 363. mohammad k.m., azhar a.a.m., suhad y.j. (2010). helminth parasites of the green toad b. variabilis laurenti, 1768 in baghdad area, central iraq. egyptian academic journal of biological sciences, 2: 17-25. prudhoe b.y.s., bray r.a. (1982).platyhelminth parasites of the amphibia. oxford university press, oxford, and british museum (natural history), london. 217 p. saeed i., al-barwari s., al-harmni k.i. (2007). a metazoan parasitological research of some iraqi amphibians. turkish journal of parasitology, 31: 337345. stock m., frynta d., grosse w.r., steinlein c., schmid m. (2001). a review of the distribution of the diploid, triploid and tetraploid green toads (bufo viridis complex) in asia including new data from iran and pakistan. asian herpetological research, 9: 77-100. vashetko e.v., siddikov b.h. (1999). the effect of the ecology of toads on the distribution of helminths. zoological studies, 23: 107-110. yamaguti s. (1959). systema helminthum. vol ii. the cestodes of vertebrates. new york & london, interscience publishers. 860 p. https://zoologicalstudies.springeropen.com/ https://www.degruyter.com/view/j/vzoo int. j. aquat. biol. (2017) 5(4): 260-262 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی استان فارس، ایران در bufo variabilis, laurenti, 1768 (anura: bufonidae)سبز وزغ کرمی هایانگلفون 2زهرا اسداللهی ،1*، امین احمدی1رودهنرخش ناحسا .ایران ،شیراز ،شیراز دانشگاه ،دامپزشکی دانشکدهپاتوبیولوژی، گروه1 .ایران ،تهران ،تهران دانشگاه ،دامپزشکی دانشکدهپاتوبیولوژی، گروه2 چکیده: این هدف. دارد ای پراکنش وسیعی هااستان از بسیاری در که است ایران هایوزغ از شایع ایگونه( bufo variabilis laurenti, 1768) سبز وزغ 100 شد، بررسی مختلف نواحی از که وزغ 30 بین از. باشدمی ایران غربی جنوب در b. variabilis کرمی هایانگل لودگیآ میزان تعیین مطالعه ( درصد 93) مورد 28. بودند شده روده انسداد دچار سستودها با شدید لودگیآ علت به آنها از برخی. بودند انگلی گونه چند یا یک دارای هاآن درصد هایکرم. بود لودهآ مونوژن انگل یک به مثانه کیسه در هاوزغ از یکی همچنین. بودند( ریه) نماتودها و( روده) سستودها به همزمان عفونت دارای مطالعه این. بودند rhabdias ranae نماتود گونه یک تنها و nematotaenia dispar: سستود ،polystoma viridis: مونوژن داده، تشخیص انگلی .دهدمی گزارش را ایران در bufo variabilis غزو در r. ranae نماتود حضور بار اولین برای .polystoma ،nematotaenia ،rhabdias ranae ،کرمی آلودگی :کلمات کلیدی int. j. aquat. biol. (2018) 6(4): 235-241 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article investigation of different levels of glycerol on cyst hatching percentage, total length and survival of phallocryptus spinosa and artemia franciscana pooria gholamzadeh1, kamran rezaei tavabe*1, gholamreza rafiee1, masoud seidgar2 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2national artemia research center, iranian fisheries science research institute, agricultural research, education and extension organization, urmia, iran.. article history: received 1 june 2018 accepted 23 august 2018 available online 2 5 august 2018 keywords: live food phallocryptus spinosa artemia franciscana glycerol abstract: artemia and fairy shrimps due to propitious size and wide distribution throughout the world, purvey ample feed resources with relatively favorable nutritional value to profitable aquatic species. ambient water physical-chemical properties affect the biological function of zooplanctonic cysts. glycerol is an alcoholic compound which is soluble in water and it has three hydroxyl groups that are responsible for solubility in water. there is a relationship between the presence of free glycerol in water and cysts metabolic rate and dormancy duration. the present study aimed to investigate the effects of different levels of free glycerol in the hatchery water on hatching percentage, total length of nauplius and mortality rate of phallocryptus spinosa and artemia franciscana cysts. in this experiment, four triplicate treatments including 0% (control), 0.1%, 1% and 10% of glycerol were used on a. franciscana and p. spinosa hatchery water for 48 hours and 72 hours, respectively. the results revealed that 0.1% glycerol was the most efficient level for hatching percentage of p. spinosa cysts with 28.86±1.6%. also, the size of total length of newly hatched nauplii in this treatment was 0.75±0.08 mm that was significantly greater than the other treatments (p≤0.05); there was no significant difference in the mortality percentage between this treatment and the control treatment. in a. franciscana, the highest hatching rate (68.33±4.71%) and nauplius length (0.90±0.08 mm) were recorded in the 1% glycerol treatment. according to the results, glycerol at 0.1% level for p. spinosa and 1% level for a. franciscana are suitable in the cysts hatching media to increase hatching rate and nauplii performance. our work could contribute to a better understanding of the hatching biology of dormant life stages in zooplanctonic crustaceans. introduction along with the development of aquaculture in recent decades, usages of another class of freshwater crustaceans belong to anostraca such as artemia and fairy shrimp have been of great interest. these organisms with their unique biological characteristics can live in a wide range of environmental conditions and have a high potential to produce high biomass, rapid growth and cysts production. anostracas have a wide distribution in the world and since the beginning of 1910; nine families, 27 genera and 266 species of them have been recorded (brtek and mura, 2000). there is still no artificial feed formulation substitute for artemia. artemia is the most widely used live feed in larviculture due to its high nutritional quality (sorgeloos et al., 1986). in fact, artemia remains corresponding author: kamran rezaei tavabe doi: https://doi.org/10.22034/ijab.v6i4.472 e-mail address: krtavabe@ut.ac.ir essential food item in most marine finfish and shellfish hatchery operations especially during the earliest life stages (kolkovski et al., 2004). several studies examined temperature, salinity, soluble oxygen content and presence of few substances as influential factors on growth and reproduction of different artemia and fairy shrimps species in hatching condition (triantaphyllidis et al., 1995; brown and wanigasekera, 2000; lotfi et al., 2003; abatzopoulos et al., 2003; hafezieh and hosseinpour, 2009; agh et al., 2008). salinity, water temperature and nutrition are the most important factors affecting the growth and reproductive performance of artemia and fairy shrimp in hatching conditions or natural ecosystems or artificial environment (wear and hustler, 1987; 236 gholamzadeh et al./ effect of glycerol on phallocryptus spinosa and artemia franciscana hatching triantaphyllidis et al.,1995; zmora and shpigel, 2006; atashbar et al., 2014). the dehydrated encysted embryos resume development upon rehydration and aerobic incubation with an adequate salinity, coinciding with a decrease in the trehalose level of the cysts accompanied by a corresponding increase in the contents of glycogen and glycerol (nambu et al., 1997). when the nauplius is fully developed, the shell splits, which is the result of free glycerol production and causes a considerable uptake of water (clegg, 1964). glycerol or glycerin is an alcoholic compound which is soluble in water and it has three hydroxyl groups that are responsible for solubility in water. since, glycerol forms fats via its combination with fatty acids and glycolysis pathway in the studied organisms cysts, they absorb the glycerol as a precursor to provide energy for cellular metabolism. study of the glycerol content in extracts of artemia salina cysts revealed an absolute influence of glycerol on the artemia cysts metabolism (james and clegg, 1962). the main objective of the present study was to investigate on the effects of different free glycerol contents on hatching rate and quality of the larvae of phallocryptus spinosa and artemia franciscana as two important zooplanctonic crustaceans as live feed. materials and methods the p. spinosa cysts were collected from the eastern azarbaijan province water recources body and and a. franciscana cysts were obtained frim ornamental fish center market. the experiment was conducted in the laboratory of fisheries department in the university of tehran. experiment procedure was designed by triplicate in four treatments including 0% (control), 0.1%, 1% and 10% of glycerol. eech treatment had 200 cysts of to include p. spinosa and a. franciscana in 100-ml cylindrical containers kept under 2000-lux light in incubators. the treatments varies solely in temperature of aeration being 20°c and 30°c for the p. spinosa and a. franciscana treatments respectively. also water salinity for a. franciscana and p. spinosa treatments were adjusted in 30 ppt and 1 ppt, respectivly. water salinity was made by cacl2 that prepared from the merck company. also, ph in the treatments were 7.1±0.2 during the research period. in order to remove diapause, the cyst samples of both species were prepared according to levanse and sorgeloos (1996) chilling method. thus, the cysts were stored at -20°c during a month in the freezer and then exposed to room temperature throughout a week leading to hatch stage (levanse and sorgeloos, 1996). artemia franciscana and p. spinosa cystes were shaken 48 and 72 hours after incubation, respectively to provide homogenized fairy shrimp and artemia naupili. the samples (10 ml) were fixed in 4% formalin and the hatching percentage was calculated by the below formula (gholamzadeh et al., 2018). hatching percentage= 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 ℎ𝑎𝑡𝑐ℎ𝑒𝑑 𝑐𝑦𝑐𝑡𝑠 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑦𝑐𝑡𝑠+𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑛𝑎𝑢𝑝𝑙𝑖𝑖 × 100 to calculate the percentage of mortality, the number of dead nauplii in the 10 ml samples were divided into number of hatched nauply as below formula. mortality percentage= 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑒𝑎𝑑 𝑛𝑎𝑢𝑝𝑙𝑖𝑖 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 ℎ𝑎𝑡𝑐ℎ𝑒𝑑 𝑛𝑎𝑢𝑝𝑙𝑦 × 100 to achieve the desired amount of glycerol ratio in the treatments, the ratio of 0% (control), 0.1%, 1% and 10% liquid glycerol was diluted to the incubation containers. for obsevation of hatching fairy shrimp and artemia cysts procces and nummerating their mortality, we took samples from the treatments and studied them under the steromicroscop and to measure their length and then were moved on the lam under optical microscopy. also we used from image j software to analysis of the images (drewes, 2006). before analyzing the variance, the data normal distribution was confirmed by the shapiro-wilk test then the quality parameters were analyzed by one-way anova and significant differences among the means were found (p<0.05) by duncan's test in spss software version 21 (ibm co. ny, usa). results various glycerol concentrations in hatching media caused different records of mortality and hatching percentage in the treatments. the results showed that the highest hatching rate were recorded in p. spinosa 237 int. j. aquat. biol. (2018) 6(4): 235-241 (28.86±1.6%), a. franciscana uppermost hatching percentage (68.33±4.71%) in the 0.1% and 1% glycerol treatments, respectively. also, the results showed no hatching in the cysts of the both p. spinosa and a. franciscana in the 10% glycerol treatment (table 1). the results of nauplius’ total length have been presented in the table 2. the results confirmed that in the p. spinosa nauplius, the highest total length was recorded in the 0.1% glycerol treatment and this treayment was signifilantly different with the control treatment. total length of the a. franciscana nauplius among the treatments were similar and there were no significant differences among the glycerol treatments (table 2). the results showed that the highest mortality percentage of p. spinosa nauplius (27.66±4.49%) was recorded in the 1% tretament but in the a. franciscana was recorded (24±1.41%) in the 0.1% percentage. in the p. spinosa, there was sinificantly different between the 0.1% and the control treatment but in the a. franciscana these treatments had no significant different (table 3). in general, results of the present study about p. spinosa implies that the best hatching percentage, total length, and the mortality rate occurs in the 0.1% glycerol treatment. although, the mortality rate of this treatment was greater than the control, this difference was not statistically significant. also, the effects of various glycerol levels on the a. franciscana larval quality suggested that 1% glycyrol level is the best experimental treatment. nevertheless, increasing the amount of glycerol did not induce significant differences in total length of a. franciscana and treatments with 0.1% glycerol significantly increased the mortality rate in comparison to the control treatment while 1% level of glycerol did not show any significant difference. discussion the effects of external osmotic pressure on growth and hatching, respiration with changes in glycogen conctent, glycerol and trehaloses in different artemia species cysts have been investigated. the column at the right attempts to account for the decrease in trehalose by increases in glycogen, glycerol, and glucose equivalents of the amount of oxygen consumed (clegg, 1964). the severity of this action is directly related to external osmotic pressure factors. glycerol exists in two parts of the cyst, the space treatments species control 0.1% glycerol 1% glycerol 10% glycerol p. spinosa 14.4±1.5a 28.86±1.6b 25.5±20ab 0±0c a. franciscana 55±4.08a 63.33±2.35ab 68.33±4.71b 0±0c table 1. hatching percentage of phallocryptus spinosa and artemia franciscana exposed to different glycerol level (values are represented as means±sd (n = 3). means in the same row with different superscript show significant differences (p<0.05)). table 2. total length (mm) of phallocryptus spinosa and artemia franciscana nauplii in different glycerol level (values are represented as means±sd (n = 3). means in the same row with different superscript show significant differences ((p<0.05)). treatments species control 0.1% glycerol 1% glycerol 10% glycerol p. spinosa 0.61±0.04a 0.75±0.08b 0.65±0.02ab a. franciscana 0.87±0.01a 0.89±0.02a 0.90±0.08a table 3. mortality percentage of phallocryptus spinosa and artemia franciscana nauplii in different glycerol level (values are represented as means±sd (n = 3). means in the same row with different superscript show significant differences ((p<0.05)). treatments species control 0.1% glycerol 1% glycerol 10% glycerol p. spinosa 15.49±10.8a 22.33±15.92ab 27.66±4.49b a. franciscana 17.33±2.05a 24±1.41b 20±1.63ab 238 gholamzadeh et al./ effect of glycerol on phallocryptus spinosa and artemia franciscana hatching filling between embryonic layer and the shell from where glycerol is emitted to the environment as the shell is discharged and embryo produces embryonic glycerol immediately after hatching (jmaes and clegg, 1964). if free glycerol in artrmia cyst is functioning osmotically during the lag period, and thereby enabling the embryo to emerge, then the osmotic pressure exerted by this glycerol can be expected to bear some relation to the external osmotic pressure (clegg, 1964). the present study investigated how different levels of glycerol can affect on the hatching percentage of two zooplanctonic crustacean species i.e. p. spinosa and a. franciscana and to what extent help to improve the larval quality of the larva. the level of growth and survival of artemia in nature and cultivation conditions mainly depends on temperature, salinity, quantity, and quality of food (sorgeloos, 1986; saravanakumar and soundarapandian, 2009). according to the present study, glycerol factor also can affect the quality of p. spinosa and a. franciscana nauplii. the results indicated that the presence of specific amounts of glycerol in the incubation system of p. spinosa and a. franciscana cysts increased hatching percentage. glycerol, an osmotically active metabolite produced by the embryo, is responsible for mechanically breaking the tertiary shell in artemia cysts (murugan and dumont, 1995). it seems that the presence of glycerol in the cysts incubation system caused a change in the osmotic pressures on both sides of the cyst shell brought about changes in the time and hatching percentage in the studied species. the differences between the best treatment of glycerol level in p. spinosa and a. franciscana hatching percentage could be due to the presence of different glycerol amounts and variation in the pressure between the hatching media and the cysts internal condition. it seemed possible that glycerol might be involved in shell-rupture because of its presence in high concentration, its relatively low molecular weight, and its well-known hygroscopic properties (clegg, 1964). nowadays, the use of materials, compounds and resources improving the probability of growth, survival rate and reproductive performance of live feeds in breeding conditions are of particular importance regarding the mass production. when the nauplius is fully developed, the shell splits, which is the result of free glycerol production and causes a considerable uptake of water (wheeler et al., 1979). researches has examined the important factors such as temperature, salinity, oxygen concentration and alkalinity on growth conditions and reproductive performance of artemia and fairy shrimp (triantaphyllidis et al., 1995; brawn and wanigasekera, 2000; lotfi et al., 1382; abatzopoulos et al., 2003; hafezieh and hosseinpour, 2009; agh et al., 2008). in addition, results of current study suggested that raising the amount of glycerol to certain levels boosts the hatching percentage of both spiecies, a. franciscana and p. spinosa. low concentration of glycerol (0.5 and 0.75 mol/l) enhance hatching percentage of the a. parthenogenetica cysts (royan et al., 1987). in contrary to a previous study, advocating the downregulating effects of glycerol content increase on a. parthenogenetica hatching percentage (royan et al., 1987). the results of current study corroborate a concordance between increment of glycerol and augmented hatching rate and larva quality for both tested spiecies when other influencial factors except for glycerol level remains unwavering. a lowered hatching at higher concentrations of glycerol, conversely, might be due to an increase in the viscosity of the medium and perhaps to the rapid development of fungi and baeteria (murugan and dumont, 1995). another study showed that hatchability was significantly increased (23%) in 0.0125% glycerol and concentration above 0.025% glycerol had no or a negative influence on hatching of the thamnocephalus platyurus (murugan and dumont, 1995). carbohydrate metabolism, which involves the conversion of trehalose to glycerol and is required for hatching, responds to increasing salinity as reported in other artemia species: increasing amounts of glycerol must be synthesized as salinity is raised (drinkwater and crowe, 1991). despite the rising trend of hatching percentage as hatching medium was enriched by more glycerol, 239 int. j. aquat. biol. (2018) 6(4): 235-241 hatching was impeded in treatments containing highest glycerol amount (10%). this observation might be induced by several reasons. for instance, osmotic pressure could be the reason due to the fact that adding superfluous amounts of glycerol is likely to alter the osmotic pressure between cyst internal condition and hatching environment dramatically resulting in reversed or ceased water flow. the accumulating glycerol in the egg becomes the osmotic gradient and eventually the inelastic alveolar layer is fractured, rupturing the shell and propelling the instar from the shell (trotman 1991). from other point of view, adding excess amounts of glycerol afflicts the water quality and hatching medium ending up without any hatching for both examined species. the influence of different factors such as the level of water hardness and various ratios of water hardness factors such as calcium and magnesium on the hatching percentage and quality of larvae p. spinosa and a. franciscana were investigated. the results indicated that increasing the water hardness up to 500 mg/l can affect the amount of hatching rate of p. spinosa and a. franciscana and improve the nauplii quality (gholamzadeh et al., 2018). though glycerol, accumulated inside the cysts, aids the embryo to rupture the shell, its role in hatching when added externally is indeed not known (murugan and dumont, 1995). the stimulation of net glycerol synthesis by increased osmotic pressure indicated that free glycerol might play a role in overcoming the osmotic pressure difference between the interior of the artemia cyst and its environment (clegg, 1964). several researches studied the combined effects of photoperiod, temperature, and salinity, each with three levels, on the hatching percentage of artemia cysts. photoperiod and temperature, as well as temperaturesalinity interaction, were found to significantly affect the hatching percentage of artemia (arun et al, 2017). increasing the amount of glycerol in the hatching environment decreases the a. parthenogenetica hatching percentage (royan et al., 1987). bacteria are commonly found associated with the shell surface of most cysts. the degree of contamination varies considerably, but often bacteria are found completely coating the outer shell layer (wheeler et al., 1979). vibrio spp. has became dominant after 24 hours, probably because during hatching, artemia cysts are broken and a reserve organic substance, glycerol, is excreted to hatching water (sorgeloos et al., 1986). glycerol is an organic substrate that is utilized efficiently by vibrio spp. a very low inoculum of this population could became dominant, utilizing glycerol rather than the gram-positive population (lópeztorres and lizárraga-partida, 2001). bioflocs grown on glycerol as carbon source inhibit quorum sensing‐ regulated bioluminescence in v. harveyi and protect brine shrimp larvae from vibriosis (crab et al., 2010). the results of this experiment proved that under constant conditions for other factors influencing in hatching rate, increasing the amount of glycerol can increase the hatching and larval quality of these two species. the present study confirmed that the presence of glycerol in the incubation media of the p. spinosa and a. franciscana increase the hatching percentage in both species. however, in neither of these species, in the treatment of 10%, glycerol content had not any hatching and this issue can be indicative of several factors. by increasing the concentration of glycerol in the hatching media, the amount of exogenous pressure in the environment, outside and inside the cyst is equal and in cases of high levels of glycerol, by increasing the concentration of glycerol, the incubation medium increases the pressure in this environment and causes inhibition of water absorption by the cysts. on the other hand, it can be concluded that increasing the amount of glycerol to 10%, reduces the water quality factors for the species, which caused the hatching percentage in p. spinosa and a. franciscana to be zero. in another hand, the artemia was tested to study the combined effects of photoperiod, temperature, and salinity, each with three levels, on the hatching percentage of their cysts. photoperiod and temperature, and temperature-salinity interaction, were found to significantly affect the hatching percentage of artemia (arun et al, 2017). according to the outcomes of the current study, addition of glycerol in the incubation water enhances the hatching percentage and nauplii quality of p. spinosa and 240 gholamzadeh et al./ effect of glycerol on phallocryptus spinosa and artemia franciscana hatching a. franciscana cysts. in order to produce fine and qualified aquatic species, making a significant contribution to aquatic animal and living food production, it is suggested to benefit from the factors influencing the production and quality of p. spinosa and a. franciscana larvae. glycerol is a crucial material in hatching environment serving capabilities to obstruct or promote hatching rate. the results in this study presented either hatching stoppage and enhancement when hatching medium of p. spinosa and a. franciscana included high glycerol content and certain glycerol amount respectively. in conclusion, quality parameters of the p. spinosa and a. franciscana cysts were investigated in responses to variation of hatching medium glycerol level. p. spinosa responsed to the different levels of glycerol treatments and recorded the highest mortality percentage in the 1% glycerol treatment while in the a. franciscana irregular response recorded a significant mortality at 0.1% level of glycerol and no statistically different in mortality rate among the fifferent glycerol concentrations. in terms of hatching percentage, p. spinosa and a. franciscana cysts revealed positive trend of improvement as glycerol level increased, but reached the optimum amount at 0.1% glycerol level for p. spinosa and 1% for a. franciscana cysts. although, hatching rate and mortality percentage was affected by glycerol level variation, total length response of a. franciscana cysts remained unchanging as glycerol level varied. overall, according to the results, glycerol at 0.1% level for p. spinosa and 1% level for a. franciscana are suitable in the cysts hatching media to increase hatching rate and nauplii growth. references abatzopoulos t.j., el-bermawi n., vasdekis c., baxevanis a.d., sorgeloos p. 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(2019) 7(2): 95-99 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article effect of chlorella vulgaris as a biofertilizer on germination of tomato and cucumber seeds odgerel bumandalai*,1rentsenkhand tserennadmid microbial synthesis laboratory, institute of general and experimental biology, mongolian academy of sciences, mongolia. s article history: received 27 february 2019 accepted 16 april 2019 available online 2 5 april 2019 keywords: biofertilizer germination algae crop abstract: although inorganic fertilizers are known to raise environmental and health problems, the current agricultural practices are heavily dependent on the application of synthetic fertilizers and pesticides. in this study, we examined the effect of chlorella vulgaris strain on germination of tomato and cucumber seeds. seeds were germinated in culture medium containing algal strain and grown for 3, 6, 9 and 12 days to study its effect on growth parameters. as results, c. vulgaris suspension increased the seed growth compared to those of the control (sterilized culture medium) of seed germination. the best treatments were 0.17 and 0.25 g/l of algal suspension for the root and shoot lengths of tomato and cucumber seeds, respectively. introduction as the global food demand increases, agriculture sectors have been increasingly using chemical fertilizer. inorganic fertilizers are rich in chemical substances, such as nitrogen, phosphorus and potassium. the excess uses of chemical fertilizers in agriculture are costly and also have various harmful effects on both living organisms and environment (santos et al., 2012). for instance, residual chemicals reach to water bodies through rainwater and cause eutrophication in water bodies. it can also reduce water-holding capacity, soil fertility and disparity in soil nutrients. moreover, groundwater contamination could lead to gastric cancer goiter, metabolic disorder, birth malformations, hypertension and livestock poisoning (khandare, 2013; youssef and eissa, 2014). in this regard, organic fertilizers and biofertilizers have become alternative sources. biofertilizers are eco-friendly, cost effective and renewable source of plant nutrients to supplement and replace the chemical fertilizers for sustainable agriculture (raja, 2013). biofertilizers contain various microorganisms that provide all kinds of micro and macro-elements via nitrogen fixation, phosphate and *correspondence: odgerel bumandalai doi: https://doi.org/10.22034/ijab.v7i2.582 e-mail: odgerelb@mas.ac.mn potassium solubilization or mineralization, release of plant growth promoting substances, production of antibiotics and biodegradation of organic matter in the soil (goel et al., 1999; sinha et al. 2010). when biofertilizers are used continuously for many years, parental inoculums become sufficient for further multiplication (youssef and eissa 2014), hence they participate in nutrient cycling and benefit crop productivity (singh et al., 2011). main benefits of biofertilizers are (1) cheap source of nutrients, (2) suppliers of microelements, (3) suppliers of organic matter, (4) counteracting negative impact of chemical fertilizers, (5) secretion of growth hormone (gaur, 2010), (6) no adverse effects to ecosystem and (7) longer shelf life (sahoo et al., 2014). the main microorganisms used in biofertilizers are azotobacter, azospirillium, cyanobacteria, azolla, phosphate solubilizing microorganisms, mycorrhizae, sinorhizobium and plant growth promoting rhizobacteria (hegde et al., 1999; youssef and eissa 2014). algal biomass contains macronutrients as well as micronutrients, growth regulators, polyamines, natural enzymes, carbohydrates, proteins, amino acids, and vitamins implemented for improving 96 bumandalai and tserennadmid / effect of chlorella vulgaris as a biofertilizer on tomato and cucumber vegetative growth (abd el moniem and abd-allah, 2008; el-fouly et al., 1992; mahmoud and amara, 2000; shaaban and mobarak, 2000). in addition, algal biomass increases the yield due to the presence of vitamins, auxins and gibberellins. the algal varieties tested as biofertilizers primarily belong to the branch of blue-green algae (cyanophyta) and green algae (chlorophyta). it has been shown in many studies that green algae can (1) add organic matter, (2) synthesize and liberate amino acids, vitamins and auxins, (3) reduce oxidizable matter content of the soil, (4) provide oxygen to the submerged rhizosphere, (5) improve salinity and buffer the ph, (6) solubilize phosphate, and (7) increase the fertilizer use efficiency of crop plants (faheed and abd-el fattah, 2008; abd el moniem and abd-allah, 2008; bileva, 2013; dubey and dubey, 2010; grzesik and romanowskaduda, 2015; vig et al., 2012). the aim of this work was to study the effect of chlorella vulgaris strain on germination of tomato and cucumber seeds and to determine any potential application of c. vulgaris microalga as a biofertilizer to improve the yield quality and productivity. materials and methods plant material: the experimental plants were seeds of tomato and cucumber. these seeds were purchased from retailer store under ministry of food, agriculture, light industry, mongolia in 2017 and kept at -4°c under dark condition until the experiment. algal culture: microalga strain was obtained from the culture collection of microalgae at institute of general and experimental biology and cultivated using standard medium 04. the final ph of the medium was 6.8, after being autoclaved. the culture was grown with a light intensity of 8 klux provided by cool white fluorescent lamps and a temperature of 25±2°c under illumination regime of 8:16 light and dark cycle for a week. filtered air was let to bubble in the culture vessels to provide aeration and agitation. effect of culture media after growth of algal strain on seed germination. algal suspensions were collected at 3d, 6th, 9th and 12th days and examined for both cell count and dry biomass yield. determination of cell number and biomass content of alga. growth of c. vulgaris strain was measured in terms of cell number and dry weight biomass. cell concentrations were counted using a hemocytometer. data were given as cell per ml. the determination of dry biomass yield was performed using vladimirova’s method (sirenko, 1975). the culture suspensions were mixed well prior to the sampling. 5 ml of samples were collected in weighing bottles thrice weekly. the bottles were dried at 105°c oven until the weight of the bottles become constant. the dry biomass yield was determined using following formula: dw (g/l) = (a – b)/y × 200 where a is total weight of weighing bottle containing dried biomass (g), b=weight of the weighing bottle (g) and y=sample volume taken (ml). the data were given as mg/g algae mass. treatment of tomato and cucumber seeds. seeds were surface sterilized with 30% sodium hypochlorite for 8 min, then rinsed with distilled water several times before germination. the seeds of tomato and cucumber were placed in petri dishes containing 3 ml of sterilized culture medium as a control. 2 ml of algal suspension was collected after growing the algal strain for 3, 6, 9 and 12 days, and added to each petri dish containing tomato and cucumber seeds. petri dishes were maintained in thermostat at temperature of 18±2°c under the light regime of 8:16 light and dark for a week. at the end of the experiment, lengths of shoots and roots per plant were determined. statistical analysis. all experimental analyses were performed in triplicate and the mean values were calculated. the data were subjected to analysis of variance and student’s t-test and f-test were used to assess differences between means. results growth parameters (cell counts and dry biomass yield) of alga. the growth of algal strain was followed after 12 days. the cell counts and dry weight were recorded at 3, 6, 9, and 12 days. as shown in table 1, the dry biomass yields were 0.06, 0.12, 0.17 and 0.25 g/l at day 3, 6, 9 and 12, respectively. meanwhile, the 97 int. j. aquat. biol. (2019) 7(2): 95-99 cell counts were 26.6, 50.1, 77.2 and 109.8 million cell/ml in respective algal suspensions. seed germination and seedling growth of tomato and cucumber seeds: the results of growth parameters obtained for the germination of tomato and cucumber seeds subjected to culture media after growth of chlorella strain for 3, 6, 9, and 12 days treatments are given in figures 1 and 2. the lengths of shoot and root of tomato were highest at day 9, which were 17.4 and 44.6 mm, respectively (fig. 1). as compared to the control, the growths of roots gradually increased by 29.1, 52.8, 100% at days 3, 6 and 9, respectively. however, the length of root was 40% shorter than that of control at day 12. the lengths of shoot at days 6 and 12 were close to that of control. the lengths of shoots at day 3 and 9 were higher than that of control by 43.3 and 87.9%, respectively. the lengths of cucumber shoot and root were 2.1 and 2.4 times longer than that of control at day 12, table 1. concentrations of algal suspension used in this experiment. days 3 6 9 12 dry biomass (g/l) 0.06 0.12 0.17 0.25 cell counts (cells/ml) 26.6*106 50.1*106 77.2*106 109.8*106 figure 1. effect of culture medium containing chlorella grown for 3, 6, 9 and 12 days on growth parameters of seed germination of tomato. figure 2. effect of culture medium containing chlorella grown for 3, 6, 9 and 12 days on growth parameters of seed germination of cucumber. 98 bumandalai and tserennadmid / effect of chlorella vulgaris as a biofertilizer on tomato and cucumber which were 21.6 and 55 mm, respectively (fig. 2). the lengths of shoot were close to that of control at day 3, 6, and 9. the length of root was close to that of control at day 3. however, the lengths of roots were 41.5 and 80.8% higher than the control at days 6 and 9. discussions tomato and cucumber are considered as the most important and common vegetable plants in many countries. many studies have been conducted on tomato to develop bio-stimulants, which can improve lateral and longitudinal root formation, roots nutrient uptake, increasing total volume and vigor of the root system. among them, chlorella microalga was also tested for the tomato bio-stimulant. in our study, the result showed the significant growth of shoot and root in tomato plant. the similar results were also observed in other researchers’ studies. the application of c. vulgaris was tested on tomato plant, which showed strong stimulating effect on plant growth while inhibiting the development of meloidogyne arenaria nematode parasite (choleva et al., 2005). it was also observed in garcia-senin’s (2013) study that the use of irrigation water with c. pyrenoidosa and chlorella sp. cultures favored the production of tomato plants with special attention in poor soils. author also concluded that their results could lead to a lower environmental impact and a cost-effective tomato crop production. moreover, several ocean algae extracts were tested on tomato seedlings and their results also showed the enhanced seed germination, plant growth, and germination rate (hernández-herrera et al., 2014; shariatmadari et al., 2011). the similar phenomenon was also observed in cucumber plant. the results showed the growth of root in cucumber plant was increasing as the concentration of microalgal suspension increases. in the study of abd elhafiz et al. (2015), chlorella sp. cultures were shown to enhance the germination of cucumber seeds. conclusion it can be concluded that c. vulgaris suspension can enhance the germinations of tomato and cucumber seeds. algal suspensions of 0.17 and 0.25 g/l can improve the root and shoot lengths of tomato and cucumber seeds, respectively. acknowledgements we thank mr. ghorbanzade, ghane and darand for sampling. references faheed a.f., abd-el fattah z. (2008). effect of chlorella vulgaris as bio-fertilizer on growth parameters and metabolic aspects of lettuce plant. journal of agriculture and social sciences, 4(1965): 165-69. abd el moniem e.a., abd-allah a.s.e. (2008). effect of green alga cells extract as foliar spray on vegetative growth, yield and berries quality of superior grapevines. american-eurasian journal of agriculture and environmental science, 4(4): 427-33. choleva t., bileva y., tzvetkov barakov p. 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(2022) 10(4): 321-335 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article assessment of the embryotoxic potential of contaminated sediments using fish embryotoxicity tests for the river buriganga, dhaka, bangladesh md. baki billah1, shilpi sarkar2, meherun nesha3, md. inja-mamun haque4, lutfun nahar5, nowshaba hoque amrita5, muhammed alamgir zaman chowdhury3, md. kamrujjaman1, md. golam rabbane4 1department of zoology, jahangirnagar university, savar, dhaka-1342, bangladesh. 2bangabandhu sheikh mujibur rahman maritime university, dhaka-1216, bangladesh. 3institute of food and radiation biology, atomic energy research establishment, ganakbari, savar, dhaka-1349, bangladesh. 4department of fisheries, university of dhaka, dhaka-1000, bangladesh. 5srijon abash, ambagan, savar, dhaka-1342, dhaka, bangladesh. s article history: received 16 july 2021 accepted 25 april 2022 available online 2 5 august 2022 keywords: whole sediment sediment organic extract heavy metals zebrafish abstract: sediment samples from six different locations of buriganga river following exposure to zebrafish (danio rerio) eggs and larvae displayed prominent effects on both whole sediments and sediment organic extracts. the acute and sublethal effects during 96 h exposure period included (i) a significant (p<0.05) increase in morality and abnormalities in zebrafish eggs and embryos; (ii) a significant (p<0.05) reduction in hatching success and heart rate; (iii) increased frequency of helical tail and lordosis after 96 h exposure to sediment extracts; (iv) developmental delay and yolk sac edema after exposed to whole sediments at 96h exposure period. chemical analysis showed the increased concentrations of heavy metals (zn, cr, cu, pb, and cd) in downstream (s1, s2, and s3) compared to upstream (s4, s5, and s6), where some ions such as cd and cr exceeded the environmental protection agency’s threshold effect level (epa tel). the current study delineates the contamination of extremely toxic compounds in the sediment of buriganga river, which may initiate toxic effects on the early life stages of fish. therefore, integrating zebrafish embryo toxicity tests may be crucial for evaluating the sediment quality of polluted rivers. introduction greater dhaka, the capital city of bangladesh, has eighteen million people, of which only 25% of households absorb the sewage treatment facilities (islam et al., 2015a). dhaka city is surrounded by buriganga-turag-dhaleswari river systems (fig. 1) which are being polluted by the untreated waste of more than 30,000 factories (whitehead et al., 2018) situated on the bank of these rivers leading to extremely serious pollution scenario in bangladesh where industrial pollution contributes 60% pollution of dhaka watershed (islam et al., 2015a). to acclimatize with the economic development of other asian mega-cities, it is essential to improve the health of surrounding rivers, which support a number of communities, agriculture and industries of dhaka city. on a worldwide scale, the toxic effluents from correspondence: md. baki billah doi: https://doi.org/10.22034/ijab.v10i4.1290 e-mail: bakibillah29@gmail.com leather, textiles, bricks, steel, pharmaceuticals, paper, garments and battery factories cause severe metal and organic pollution leading to disruption of ecology and deterioration of water quality (whitehead et al., 2018). pollution takes away nine million lives (premature deaths) per year has been reported by the lancet commission on pollution (landrigan et al., 2018). the un sustainable development goal (sdg) 6.3 aims to achieve a good ecological and chemical status in surface waters (schiwy et al., 2015) before 2030 (whitehead et al., 2019). to achieve this goal, sediment ecotoxicology may play a crucial role because sediments can be a secondary source of pollution (schiwy et al., 2015). on a worldwide scale, chemicals of anthropogenic origin may be absorbed in particulate matter and finally aggregate in the sediment (ahlf, 1995l; zeng 322 billah et al./ embryotoxic potential of contaminated sediments using fish embryotoxicity tests et al., 2020). therefore, a comprehensive assessment of contamination of an aquatic ecosystem depends not only on the toxicity of the water phase (engwall et al., 1994) but also on the determination of the toxicity of sediment (ahlf, 1995; burton and macpherson, 1995). sediment is the major reservoir of pollutants (bartzke et al., 2010). the contaminants in the sediments can be remobilized by flooding or dredging and can affect organisms in the water column, thus representing a source of expansive pollution in the water compartment (hollert et al., 2003). the aggregation level of toxic compounds in sediment could be higher than in the free water column (hollert et al., 2002). as sediment pollutants are linked to aquatic organisms and human health via the food chain, it is imperative to give a comprehensive toxicity evaluation of river sediments and address strong environmental risks to support further management strategies for the restoration of the health of the whole river system. to see the effects of sediments on aquatic life, controlled toxicity and bioaccumulation tests are indispensable; however, we have succinct information on the concentration and bioavailability of water and sediment contaminants. in general, sediment contact assay using the whole sediment on zebrafish represents a real field-like exposure scenario. but it is necessary to extract sedimentbound chemicals to the dissolved phase to perform in vitro and in vivo tests using fish early life stage tests. to isolate more polar chemicals in the hydrous phase, it is necessary to elute sediments with artificial water, which mimics remobilization during flood events. in contrast, sediment could be extracted with an organic solvent to rearrange the non-polar substances (ho and quinn, 1993). as the extraction procedure of eluates and extracts results in a small volume of eluates and extracts, therefore precautions should be maintained in selecting bioassays, which require a small sample volume. under these preconditions, bioassays using primary or fish cells lines (lee et al., 2020; mennillo et al., 2020) and early life stages of fishes are appropriate (dar et al., 2019; cormier et al., 2021). as zebrafish, danio rerio is comparatively easy to breed and eggs could be available throughout the year; the use of zebrafish provides some advantages over other models. besides, the eggs and post-larvae of zebrafish are transparent to monitor continuously through a microscope, and embryonic development is relatively rapid and simultaneous (von nagel, 1986; kimmel et al., 1995), which intrigues the suitability of this model in toxicity study of sediment. the river buriganga, one of the most polluted rivers, runs past dhaka city, the capital of bangladesh. many tanneries in the hazaribagh area discharged the wastes to the buriganga until their shifting in 2017 (harris et al., 2016) in the savar area. heavy metal in the water and sediments induces harmful effects on biota (yi and zhang, 2012), including the chances of cancer and cancerrelated diseases (kim et al., 2015). heavy metal pollution in the buriganag river is reported but there is no research available for sediment-bound contaminants with embryotoxicity or teratogenic potential on the early stages of fish from this river. several studies have incorporated the early life stages of fish, especially zebrafish (hollert et al., 2003, strmac et al., 2002; wu et al., 2010) and the cell line model (mennillo et al., 2020) following exposure to sediments to extrapolate the chemical burden associated. with the benefit of hindsight, the purpose of the present study was (i) to estimate the concentration of some selected heavy metals from human-impacted regions of buriganga, (ii) to examine the toxicity of whole sediment and sediment organic extracts on zebrafish early developmental stages and iii) to evaluate the suitability of zebrafish for the assessment of the toxicity of sediment. materials and methods sampling locations: sediment samples (n=30) were drowned from six locations (within latitude 23°42'4.83"n and longitude 90°20'10.62"e of dhaka city) of buriganga river (fig. 1) during the winter season of 2019 using stainless steel shovels with five 323 int. j. aquat. biol. (2022) 10(4): 321-335 replicates at each station. among the sampling locations, uttar keraniganj (s1) has experienced massive industrial expansion from nearly 2000 smes in 2001 to 6766 in 2013 (bangladesh economic review, 2017). sadarghat biwta terminal (s2) is the largest terminal where millions of people aggregate daily to travel to different regions of bangladesh leaving the port polluted. one of the most pollution sources of the buriganga river is the hazaribag industrial area (s3) which released untreated waste from a number of tanneries to the buriganga river directly (asaduzzaman et al., 2016). from 1960 to 2017, 90% of the tanneries were located in the harzribagh area, downstream of the buriganga river. but to reduce pollution and to achieve the national standard and sdg 6.3, the government of bangladesh has decided to shift the tanneries from hazaribag to a new area in savar named harindhra on the bank of dhaleswari river, connected to buriganga (harris et al., 2016). therefore, to understand the effects of the relocation of tanneries on the ecosystem, it is urgent to study the toxicity of sediments associated with the hazaribag area. in contrast, rayer bazar (s4) is a densely populated area with a huge density of slums. the tannery effluent from the hazaribag area mobilizing to upstream of the buriganga river makes this area highly polluted. bosila (s5) is characterized by slums, brick fields and fertilizer industries. nearly 9000 square meters of effluents from 104 fertilizer industries, located in bosilla, demra, fatulla and faridabad, are discharged into the buriganga river per day (rahman and bakri, 2010). the station gabtoli (s6) is the largest bus terminal for transportation to the different districts of bangladesh. this area also contains dense population, numerous brick fields, landfills and many industries (ahmed et al., 2016). chemical analysis of heavy metals: the collected sediment samples were transported to the laboratory by maintaining a cold chain and kept at -20°c until further processing. heavy metals were determined by digesting the freeze-dried sediments with concentrated hno3 and hclo4 (yang et al., 2020; li et al., 2020) briefly, the homogenized sediment samples (~1g) were taken into a separate beaker with figure 1. location map of the river buriganga indicating sampling spots. 324 billah et al./ embryotoxic potential of contaminated sediments using fish embryotoxicity tests 8 ml of concentrated hno3 (65%) and 4 ml of hclo4 (70%) and digested at 70°c. when the brown fumes disappeared, the digested mixture was filtered by the whatman filter paper. then, deionized water was added to make a final volume of 50 ml and subjected to atomic absorption spectrophotometer (aas) (aa-7000, shmadzu, japan) analysis. organic matter extraction: acetone extraction method was imposed to extract the sediment organic matter from the freeze-dried sediment sample (hollert et al., 2002; strmac et al., 2002; wu et al., 2010). in short, dried sediment (20 g) was taken inside the thimble and extracted with acetone for 1216 h using a soxhlet apparatus. a vacuum rotary evaporator (rotavapor, 400 mbar, 38°c) was used to reduce the volume of the acetone extracts. the extracts were concentrated by nitrogen dry followed by re-dissolved in 1 ml dimethylsulfoxide (dmso) (i.e., 20,000 mg/ml as stock) and stored at 4°c for further sediment testing. dmso (0.3% v/v ratio) was maintained in the solution. rearing and egg production of zebrafish: 3month-old zebrafish with a ratio of 3:2 (males/females) were reared for egg production in fish breeding chambers according to the protocol of braunbeck and lammer (2006) with some modifications. fishes were fed commercially available dry flake food (tetra, melle, germany) and hard-boiled egg yolk two times daily. the surplus food particles and the faeces of zebrafish were removed twice a day while the aquaria screens were washed on each alternate day. the fertilized eggs were prudently transferred in petri dishes (18 cm diameter) and dipped in artificial water (iso 7346/3). the exposure tests were conducted using embryos within 2 h post-fertilization (hpf). testing of organic extracts: the methods described by viganò et al. (2020) were implied to organic extract exposure. artificial water (iso) was used to dilute the organic extract according to iso 7346/3. from the 20,000 mg/ml organic extract stock solution; 100 mg/ml working solutions were prepared. 2 ml working solution with 5 eggs was placed in every 20 wells while the other 4 wells of a 24-well plate were added 5 eggs with artificial water, used as the negative control. hatching rate, abnormalities in development, swimming activity and mortality of embryos and larvae were recorded after 24, 48, 72 and 96 h toxicological endpoints using a stereomicroscope. five parallel tests were performed for each sediment sample. a total of 350 eggs were used in this study: 5 rounds x 7 treatments (6 sites+ negative control) x 2 replicates x 5 eggs. additionally, the deceased embryos and larvae were instantly removed from the petri dishes. testing of whole sediments: the guidelines described by hollert et al. (2003) were followed to carry out the whole sediment exposure with slight modification. concisely, 3 g of freeze-dried whole sediment and 7.5 ml of 24 h ventilated artificial water (333 mg/ml h2o) were mixed in a glass petri dish. after 1 h, 5 fertilized eggs (2 h post fertilization) were transferred to each petri dish and incubated at 26°c with 14 h light/10 h dark photoperiod. five parallel tests were performed for each sediment sample. a total of 350 eggs were used in this study: 5 rounds x 7 treatments (6 sites+ negative control) x 2 replicates x 5 eggs. the developmental stages of fertilized eggs were recorded for lethal and sub-lethal endpoints during 24, 48, 72 and 96 h of post-fertilization. the observations were continued until the hatching of embryos and dead embryos were removed immediately. determination of cardiotoxicity: zebrafish embryos were exposed to sediments for 48 h from different stations of buriganga river to investigate the effects on heart rate (beat/min) in embryos (4850 hpf). a microscope (olympus, japan) coupled with olympus cellsens entry software was used to determine heart rate as previously described by babić et al. (2017). statistical inferences: differences among the sampling locations were tested using one-way analysis of variance (anova), with duncan’s multiple range test. spss 19.0 (spss, usa) was used to perform all statistical analysis and the significance was tested at p<0.05 level of 325 int. j. aquat. biol. (2022) 10(4): 321-335 significance. results chemical analysis of sediments: the heavy metal concentrations in the sediment of different sampling stations of buriganga river were found in the decreasing order of zn>cr>cu>pb>cd (table 1). of all the five metals investigated, the concentrations of zn in different stations are higher than that of other metals. the highest concentration of zn was found in s2 (252.4±31.4 µg/g) whereas the lowest was detected in s5 (118±4.62 µg/g). the concentrations of cr from the sediments of the present study of buriganga river were ranged from 35.72±2.61 (s4) to 72.91±5.47 µg/g (s2). except for s4, all stations exceeded the limit of epa tel. concentrations of cu were in decreasing order of s2>s1>s4>s3>s5. the highest (41.91±3.26 µg/g) and the lowest (14.51±1.5 µg/g) cu concentrations were found in station 2 and station 5, respectively. cu concentrations of all the stations were within the acceptable limit of epa tel except for the s2. the highest concentration of pb was found in s2 (27.6±1.18 µg/g) followed by s3 (24.2±2.94 µg/g), s4 (18.5±1.10 µg/g), s1 (13.2±2.14 µg/g) and s5 (11.3±1.02 µg/g), respectively. the concentration of pb from all the stations was below the acceptable limit of epa tel. similarly, cd was found maximum in s2 (1.64±0.03 µg/g) followed by s3 (1.12±0.43 µg/g), and s5 (0.59±0.03 µg/g). sediment samples from all the stations exceeded the epa tel limit except s5. embryo toxicity tests the survival rate in the whole sediment: in dmso control solution, zebrafish embryos developed normally and the survival rate was 99.5% in the whole sediment and sediment organic extract exposure. the whole sediments from six different stations (s1, s2, s3, s4, s5 and s6) revealed survival rates from 60±5.6 to 86±8.3 % after 96 h exposure period (fig. 2). among the six different stations, samples from both the downstream and upstream exhibited the significant (p<0.05) reduction in survival rate compared to the negative control, with the highest (86%) and the lowest (60%) survival rate in s5 and s3, respectively. the survival rates from six different stations were of decreasing order of s5>s6>s1>s2>s4>s3 (fig. 2). the survival rate in sediment organic extracts: the zebrafish embryos exposed to sediment organic extracts from six stations showed the highest survival rate ranging from 70 to 94%, which was 8% higher than the whole sediment exposure. the increased survival rate reflected the much better condition of zebrafish embryos exposed to sediment extracts. the highest survival rate (94%) was observed in s4 while the lowest (70%) in s3 (fig. 2). among the stations, s1, s2 and s6 exhibited moderate survival rates of 77, 78 and 88%, respectively (fig. 2). in comparison with the dmso control, organic extracts from both upward and downward stations displayed significant (p<0.05) reduction in survival rate (fig. 2). developmental abnormalities in the whole sediment: no abnormal embryo was observed in the control solution. but, some developmental abnormalities in the zebrafish embryos were observed when exposed to sediment samples from heavy metal concentration in downstream (µg/g) concentration in upstream (µg/g) epa tel s1 s2 s3 s4 s5 s6 zn 120.2±11.32 252.4±31.4 131.7±8.31 122.4±6.3 118±4.62 119±13.12 <90 cr 68.8±4.17 72.91±5.47 45.76±6.12 35.72±2.61 41.74±5.14 43.10±6.0 37.3 cu 23.9±2.74 41.91±3.26 19.70±1.13 21.±2.42 14.51±1.5 16.16±4.1 35.7 pb 13.2±2.14 27.6±1.18 24.2±2.94 18.5±1.10 11.3±1.02 13.0±3.2 35 cd 0.95±0.013 1.64±0.03 1.12±0.43 0.74±0.018 0.59±0.03 0.62±0.06 0.596 *sd, standard deviation, epa tel= environmental protection agency’s threshold effect level. table 1. heavy metal analysis (±sd) of sediments collected from six spots of buriganga river during winter. 326 billah et al./ embryotoxic potential of contaminated sediments using fish embryotoxicity tests different stations of the buriganga river. developmental abnormalities of zebrafish after exposure to whole sediments varied from 5(±0.91) to 12(±2.2)%. station 1 (s1) showed the highest (12%) while site 5 displayed the lowest rate (5%) of abnormalities (fig. 4). station 3 exhibited the abnormalities in between the highest and the lowest rates. significantly (p<0.05) higher abnormalities were recorded in zebrafish embryos after 96 h exposure to sediments from both upstream and downstream stations compared to the negative control. the sublethal effects in the whole sediments were lack of desorption of the yolk sac after 24 h exposure (fig. 4c), changes in the yolk sac severe pericardial edema after 48 h (fig. 4f), and enlargement of the yolk sac, pericardial edema and yolk sac edema after 72 h (fig. 4i), developmental delay, yolk sac edema, bent tail and haemorrahage in the gut region after 9 h expose (fig. 4l). developmental abnormalities in sediment organic extracts: embryos developed normally in the dmso control solution and no abnormalities were figure 2. survival rate of zebrafish embryos after the exposure to the (a) whole sediment and (b) organic sediment extracts for 96h exposure in the buriganga river. bar (mean±sd) with a different letter is significantly different (anova, p<0.05). a a c c d b bd cd bc d bd cd b bc a b figure 3. percentage of abnormalities observed among zebra fish embryos exposed to the (a) whole sediments and (b) organic extracts for 96h from six sites in buriganga river. bar (mean±sd) with a different letter is significantly different (anova, p<0.05). 327 int. j. aquat. biol. (2022) 10(4): 321-335 figure 4. changes in zebrafish embryos after exposure of sediment at specific time periods: (a) represented normal embryo after 24h; (b) exhibited developmental delay, lack of somite formation and failure of tail detachment after 24h exposure to sediment extracts; (c) showed lack of desorption of yolk sac after 24h exposure to whole sediments; (d) exhibited normal embryo after 48h; (e) demonstrated changes in yolk sac and larva with bent tail and lordosis after 48h exposure to organic extracts; (f) represented changes in yolk sac and severe pericardial edema after 48h exposure to whole sediments; (g) represented normal embryo after 72h; (h) highlighted the complete curvature of spinal cord, yolk sac edema and complete changes in the pigmentation after 72h exposure to sediment extracts; (i) demonstrated the enlargement of yolk sac, severe pericardial edema, and yolk sac edema after 72h exposure to whole sediment; (j) represented the normal embryo after 96h; (k) exhibited helical tail and lordosis after 96h exposure to sediment extracts; (l) showed developmental delay, yolk sac edema and helical tail after exposed to whole sediments. b, brain; s, somites; t, tail; ey, eyes; ye, yolk sac edema; tnd, tail not detached; wos, without somites; ldy, lack of desorption of yolk sac; er, ear; lo, lordosis; het, helical tail; spe, severe pericardial edema; ysc, yolk sac; cpcd, curvature of spinal cord; pced, pericardial edema; eysc, enlargement of yolk sac. 328 billah et al./ embryotoxic potential of contaminated sediments using fish embryotoxicity tests noted. the abnormalities of zebrafish embryos ranged from 5(±1.16) to 10(±2.31) % after exposure to the sediment organic extracts. the highest abnormalities were observed s1 while the lowest in s6. the abnormality rates of zebrafish embryos from six different stations were of decreasing level of s1>s3>s2>s4>s5>s6 (fig. 3). significant differences in abnormalities were also observed in sediments from the control and the sampling stations. sublethal effects in zebrafish embryos were also observed after exposure to the sediment organic extracts (fig. 4b, e, h and k). heart rate in the whole sediment: the heart rate of zebrafish embryos in response to the whole sediment was recorded after 48 h of the exposure period. the unexposed embryos exhibited heat rate of 155±17.2 beat/min. among the sampling stations, downstream sampling stations (s1, s2 and s3) exhibited a significant (p<0.05) reduction in heartbeats compared to the control, while the sediments from figure 5. percentage of heart rate observed among zebrafish embryos exposed to the (a) whole sediments and (b) organic extracts for 48h from six spots in buriganga river. bar (mean ± sd) with a different letter is significantly different (anova, p<0.05). figure 6. hatching rates of zebrafish embryos exposed to a) whole sediments and b) organic extracts for 96h from six sites of buriganga river. bar (mean ± sd) with a different letter is significantly different (anova, p<0.05). 329 int. j. aquat. biol. (2022) 10(4): 321-335 the upstream (s4, s5 and s6) showed similar indices to the negative control (fig. 5a). these results confirmed that the sediments from the downstream stations (s1, s2 and s3) were effective to induce the cardiotoxic effects. heart rate in sediment organic extracts: significant (p<0.05) differences in the average heartbeat of zebrafish embryos exposed to upstream and downstream (s1, s2, s3 and s4) sediment extracts were recorded compared to dmso control solution (fig. 5a). hatching rate in whole sediments: in the whole sediment and sediment extracts, in dmso control solution, the zebrafish embryos survived and hatched normally. the highest (89.5±14.4%) and the lowest (72±9.47%) hatching rates were found in sediments from s5 and s2 after 96h exposure, respectively (fig. 6). significant differences (p<0.05) in hatching rate for the exposure period of 96 h, were observed in sediments from all the sampling stations compare to the negative control. hatching rate in sediment organic extracts: in the control, the zebrafish embryos hatched normally. like the effects of whole sediments, s2 and s5 showed the highest (87±18.5%) and the lowest (81±15.52%) hatching rate, respectively after 96 h exposure period (fig. 6). significant differences (p<0.05) in the hatching rate for the exposure period of 96 h, were observed in sediments from all the sampling spots compare to the negative control. discussion in human-impacted regions, hundreds of thousands of chemicals from industry, domestic and household sources (viganò et al., 2015a) funnel into the aquatic environment leading to potential ecotoxicity on sediment inhabitant biota (dekker et al., 2006). the most sensitive stages in fish development are the early stages as demonstrated by 173 toxicity tests (suter et al., 1987). fry survival is usually critical for fish development (woltering, 1984). the early life stages are the most sensitive stages as exposure at these stages may result in serious alterations and permanent malformations (viganò et al., 2015b). thus, assessment of sediment toxicity using zebrafish embryos is of great importance because the fish community might be directly or indirectly exposed to sediment chemicals. this study showed that zebrafish embryos exposed to sediment extracts and whole sediment from buriganga river demonstrate different responses. the cytotoxic effects following exposure to the whole sediment were prominent and stronger than the effects obtained by the exposure to sediment organic extracts, which is similar to that observed by hollert et al. (2003) and wu et al. (2010). the effects of sediments from upstream (s4, s5 and s6) and downstream (s1, s2 and s3) sampling stations collaborate with the results of chemical analysis in this study. exposure of zebrafish embryos to whole sediment and sediment organic extracts for 96 h resulted in 40 and 30% mortality, respectively. these findings highlighted the early life stages test with brown trout exposed to sediment elutes and water samples of krahenbach and korsch for 60 days where mortality was reported 30% (luckenbach et al., 1999). in another study by wu et al. (2010), the whole sediment from the yangtze river induced 39% mortality similar to this study (40% mortality in s3). another study by hallare et al. (2005) on laguna lake demonstrated the highest mortality rate of embryos in sediment extracts compared to the whole sediments, which was opposite to the present finding. in the present study, the survival rate of whole sediment and sediment organic extracts was 60±5.6 to 86±8.3% and 70 to 94%, respectively, leading to high mortality in whole sediments. as whole sediment testing using zebrafish embryos represents a field-like exposure condition, the highest 40% mortality of zebrafish embryos exposed to whole sediment (s3) documents the severity of pollution of buriganga river, leading to the scenario of the complete decline of the whole fish population in the short-term exposure period. as sediment induces low oxygen concentration or oxygen depletion during whole sediment exposure (kuster and altenburger, 2008), the changes in the mortality 330 billah et al./ embryotoxic potential of contaminated sediments using fish embryotoxicity tests of embryos in this study, maybe not only to the effects of toxicants in sediments but also to the low oxygen as well (wu et al., 2010). the chorion may protect the embryos not by completely preventing but by slowing down the uptake of toxicants (van leeuwen et al., 1985). in contrast, lipothilic compounds (pahs and pcbs) in sediment extracts easily penetrate the embryos, but the heavy metals mostly remain on the surface of the chorion (mitchibata, 1981). moreover, due to the underdeveloped metabolic systems of embryos, the pollutants cannot interact with the enzymatic system leading to mortality (ensenbach and nagel, 1995). developmental abnormality rates of zebrafish embryos after exposure to whole sediments and sediment organic extracts varied from 5±0.91 to 12±2.2% and 5±1.16 to 10±2.3%, respectively. wu et al. (2010) found the abnormality rates in the six sites of the yangtze river varied from 1.33 to 9.33% in whole sediment and 4.71 to 11.81% in organic extracts that were nearly similar to the current study. besides acute cytotoxicity, the present study reported the sublethal effects on zebrafish embryos exposed to sediment extracts and whole sediments highlighting the changes in developmental delay, lack of somite formation, failure of tail detachment, lordosis, yolk sac and severe pericardial edema which were not only similar to crassostrea gigas embryos and larvae exposed to sediment-associated pahs and heavy metals (geffard et al., 2001) but also similar to zebrafish embryos exposed to contaminated sediments from nidda river, germany (schweizer et al., 2018). thus, the abnormal changes i.e. curvature of the spinal cord, enlargement of the yolk sac and helical tail may be due to unspecific reactions of zebrafish embryos to toxicants and can be better represented by the pronounced differences in the reaction of zebrafish embryos to sediment extracts (strmac et al., 2002). several studies have been carried out by different authors on different rivers for the assessment of sediment quality and contamination level using zebrafish embryos and they found induced developmental abnormalities in both whole sediments and sediment organic extracts (hollert et al., 2003; strmac et al., 2002; wu et al., 2010; li et al., 2016). during the preparation of the sediment organic extracts, the soxhlet extraction process desorbed the lipothilic substances, such as persistent organic pollutants (pahs, pcbs etc), which could easily penetrate the chorion and harm the embryos. on the other hand, whole sediment exposure contained hydrophilic substances such as heavy metals, which might be intercepted by the chorion on the surface (michibata, 1981), leading to the toxicity of the embryos (van leeuwen et al., 1995). regarding the hatching rate, distinct differences were observed in zebrafish embryos after exposure to sediment extracts and whole sediments from the buriganga river. in this study, zebrafish embryos exhibited significantly decreased hatching in response to whole sediments after 96 h exposure. in contrast, a significant decrease in the hatching rate was also found in sediment extracts after 96 h exposure. li et al. (2016) investigated the embryotoxicity of sediments from the yangtze river estuary using zebrafish embryos and showed reduced hatching rates varied from 87.5 to 95.83% after 96 h exposure; similarly, a decrease in hatching rate was also recorded in zebrafish embryos after 96 h exposure to surface water and wastewater (wu et al., 2010; thellmann et al., 2015; ribeiro et al., 2020). the increased hatching rate indicates exposure to low toxicants concentration, which can be compared with the symptoms of hysteresis (strmac et al., 2002). the decreased hatching rate can be explained by the remarkable differences in the reaction of embryos and larvae to sediment extracts and whole sediment, which might be due to the diverse composition of sediment extracts. zebrafish embryo chorion is responsible for the oxygen transport, nutrient in and excretory substances (rawson et al., 2001) out of embryos but the minute pores (500-700 nm) on chorion help to enter the toxic chemicals within embryos leading to changes in hatching rate (wang et al., 2017) by inhibition of hatching enzyme chorionase (strmac et al., 2002). it was reported that heavy metals in 331 int. j. aquat. biol. (2022) 10(4): 321-335 effluents can delay hatching by changing the expression of hatching-related genes (jezierska et al., 2008). zebrafish embryos were exposed to zn, ni and cr for 96 h (ansari and ansari, 2015) and to cu, cd and pb (jezierska et al., 2008) might delay hatching by inhibiting the expression of hatchingrelated genes. generally, zebrafish develop glands in their head region before hatching, which secrets chorionase, subsequently disrupting the chorion membrane to facilitate the hatching process. but heavy metal interferes with the development of this gland, thus involved in the decreased secretion of chorionase, resulting in delayed hatching (samson and shenker, 2000; williams and holdway, 2000). after 96h exposure, the sediments from downstream sampling sites (s1, s2 and s3) exhibited a significant reduction in heartbeats compared to the control while the sediments from the upstream (s4, s5 and s6) showed similar indices to the control. in contrast, the sediment extracts from all the sampling sites (s5 and s6) did not exhibit any cardiotoxic effects on zebrafish embryos. a similar result was postulated by strmc et al. (2002), where lower heartbeat frequency was observed in sediments exposed to zebrafish embryos for 96 h, similar to wu et al. (2010), where estuary sediments from the upstream have more cardiotoxic effects on zebrafish embryos than that from downstream. likewise, zebrafish embryo toxicity test showed that wastewater from upstream increased heartbeat and cardiotoxic effects after 96 h exposure (ribeiro et al., 2020). the present study confirms that heartbeat is a suitable biomarker (ribeiro et al., 2020) to access the toxicity of sediments on zebrafish embryos (babic et al., 2017). the changes in heartbeat frequencies may be due to toxicants associated with sediments that invoke lethal alterations (strmc et al., 2002) on embryos. chemical analysis data showed the increased concentrations of some heavy metal ions in downstream (s1, s2 and s3) stations compared to upstream (s4, s5 and s6) where some ions such as cd and cr exceeded the environmental protection agency’s threshold effect level (epa tel). high metal concentrations in the study area might be due to the obsessive industrial discharge from different industries and dismissal of domestic sewage combined with natural and anthropogenic processes (ahmad et al., 2010; islam et al., 2015b). the variation in metal concentration from diverse locations might be due to the river streaming and water disposal system of industries (alam et al., 2003). the high concentration of zn from this study may be due to the extensive discharge of industrial wastage as well as the mixing of domestic wastewater into the river (dong et al., 2012), while the concentration of pb and cu was much lower than the previous study (ahmed et al., 2010). gouva et al. (2020) found that exposure to various concentrations of heavy metals in zebrafish larvae decreased the hatching and survival rate. zebrafish embryos exposed 1 to 1000 μm cdcl2 or above result in reduced survival, delayed in hatching, lack of somite formation, detachment of tail, and pericardial edema (cheng et al., 2000) similar to that observed in zebrafish embryos exposed to sediment extracts from buriganga river. in contrast, exposures to cdcl2 in zebrafish early life stages includes delay in hatching and altered swimming ability (capriello et al., 2019) in zebrafish embryos. the lethal and sublethal effects of zebrafish embryos following exposure to sediments demonstrated a clear difference in downstream samples compared to upstream and collaborated conclusions drawn from chemical analysis. however, from the chemical analysis data, a clear gradient of contamination was not found among the different sediment sites in the buriganga river, suggesting the complex nature of the chemical mixture of sediments. therefore, the non-defined effects induced by sediments might be due to the presence of stressors, such as phytotoxins and bacterial toxins, responsible for the additive or synergistic toxic effects on the early life stages of zebrafish. conclusion the present study assessed the toxic effects of 332 billah et al./ embryotoxic potential of contaminated sediments using fish embryotoxicity tests polluted sediments on zebrafish early life stages. the reduced survival rate, hatching rate, and induced developmental abnormalities were observed after exposure to the whole sediment and sediment organic extracts. chemical analysis showed that cd and cr exceeded the environmental protection agency’s threshold effect level (epa tel). from the acute and sublethal toxicity, it was clear that the sediment effects on zebrafish embryos agreed with the chemical analysis results. therefore, the identification of pollution sources, especially heavy metals in the river buriganga is essential for the routine monitoring of river health. new policy measures on sediment toxicity should be adopted to protect the deteriorating sediment quality and public health especially in the river buriganga. fish early life stages especially with zebrafish embryos might be a sensitive indicator for the comprehensive evaluation of sediment quality of sediments from bangladesh rivers. acknowledgment this work was supported by the bangladesh bureau of educational information & statistics (banbeis) under grant of advanced research in education (ls 2018812). the author is highly grateful to lutfun nahar and nowshaba hoque amrita for their breeding and rearing of the zebrafish and for analyzing the data. references ahlf w. 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(2016) 4(1): 17-24 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article a comparative study of the histoarchitecture of endocrine pancreas in labeo bata (hamilton, 1822), sperata aor (hamilton, 1822) and chitala chitala (hamilton, 1822) saroj kumar ghosh1, 2, shrabani barun1, 3, padmanabha chakrabarti*1 1fisheries laboratory, department of zoology, the university of burdwan, burdwan-713104, west bengal, india. 2department of zoology, bejoy narayan mahavidyalaya, itachuna, hooghly-712147, west bengal, india. 3department of zoology, asansol girls’ college, asansol, burdwan-713304, west bengal, india. article history: received 12 november 2015 accepted 6 january 2016 available online 2 5 february 2016 keywords: histoarchitecture endocrine pancreas labeo bata sperata aor chitala chitala abstract: the disposition and cellular organization of the endocrine pancreas were studied in three species of freshwater teleosts viz., labeo bata (hamilton, 1822), sperata aor (hamilton, 1822) and chitala chitala (hamilton, 1822) using histological techniques. in l. bata, the endocrine pancreas tissues were mainly distributed in the adipose tissue among the intestinal coils and adjacent to extrahepatic bile duct, while in s. aor and c. chitala, the endocrine pancreas predominantly attached with wall of the stomach along with exocrine pancreatic part. histological analysis demonstrated that the endocrine components of all the three species were enclosed in a thin capsule provided with different cells, interspersed with blood sinuses. the cytoarchitectural analysis showed that in l. bata, β cells were usually arranged in groups while α cells were often interspersed with blood vessels. in s. aor and c. chitala, the rounded or oval α cells were usually arranged either in groups or scattered to the islets periphery and β cells which were densely granulated and typically stained with aldehyde fuchsin (af), romies azan (ra) and mallory’s triple (mt) were observed in the central areas of the islets and intercalated with blood vessels. the δ like cells were founded at a low frequency and intermingled with β cells and exhibited moderate cytoplasmic granules in l. bata, s. aor and c. chitala. despite being the subject of extreme controversy, the nature and function of different islet cells were discussed. introduction the endocrine pancreas present in different teleosts plays a momentous role in fish physiology. in fish, it exhibits various degrees of morphological, anatomical and cytological variations (lazarow, 1963; kudo and takahashi, 1973; falkmer and ostberg, 1977). morphocytologically, the patterns of endocrine pancreas in fishes exhibits numerous species variations and quite often consists of several prominent knots of tissue, more or less segregated from the acinar parenchyma known as principal islets (brinn and epple, 1972). a compact terapodlike organization of the pancreas with islets distributed throughout the exocrine parenchyma has been observed in clarias batrachus (khanna and mehrotra, 1968), mystus seenghala (khanna and * corresponding author: padmanabha chakrabarti doi: http://dx.doi.org/10.7508/ijab.2016.01.003 e-mail address: dr.pchakrabarti@yahoo.in gill, 1973) and conger japonicas (kobayashi and takahashi, 1974). tinctorial criteria, following granule staining and optical microscopic examination have usually been used for the identification of cells. location of b and d cells tend to occupy the central part of the islet whereas the a and f cells are more numerous at the periphery. in cottus scorpius and channa punctatus, the b and d cells generally occupy central part of the islet whereas α and granular cells are mainly located at the periphery (falkmer, 1961; khanna and singh, 1971). in the goldfish carassius carassius, b cells are usually arranged in groups or in cords which run irregularly or anastomose with each other to form a complicated network. the a and c cells are intermingled with these b cells cords of groups and 18 ghosh et al./ characterization of endocrine pancreas in l. bata, s. aor and c. chitala a cells often occur in small patches of few cells (kobayashi and takahashi, 1970). as regards the physiological role of the islet cells, ultimate proof have been obtained for the production of insulin by β cells in coho salmon, oncorhynchus kisutch (plisetskaya et al., 1985). the nature and function of a cell has been settled in favour of their being the source of glucagon in c. carassius (kudo and takahashi, 1973). sayrafi et al. (2011) differentiated α and β cells by haematoxylin and eosin staining in the islets of langerhans of pangasius sanitwongsi. the aim of the present work is to characterize the cellular details and functional aspects of the endocrine pancreas in labeo bata, sperata aor and chitala chitala having different feeding habits, by histological analysis. materials and methods healthy fishes of l. bata (16.02±1.89 cm in total length; n=16), s. aor (31.35±2.43 cm in total length; n=12) and c. chitala (46.38±3.17 cm in total length; n=14) were collected from local freshwater fish farm of burdwan (23.2333°n, 87.8667°e), west bengal, india during february-august, 2015. fishes were killed with an overdose of anesthetic (0.03% ms 222; tricaine methone-sulphonate, sigma chemical co.) following the guidelines given by the institutional ethical committee. for histological studies, the fishes were dissected and small pieces of the pancreas of s. aor and c. chitala and pancreatic tissues associated with adipose tissues among the intestinal coils of l. bata were removed and fixed in aqueous bouin’s fluid for 18 hrs. the tissues were then placed in 70% ethanol and subsequently dehydrated through the ascending series of ethanol, followed by acetone and cleared with xylene. tissues were embedded in paraffin wax (56-58°c). serial sections were cut at 4 µm thickness. deparaffinized sections were brought to distilled water through descending series of ethanol and were stained with delafield’s haematoxylineosin (he), mallory’s triple (mt) (mallory, 1936), aldehyde fuchsin (af) (halami, 1952), and romies azan (ra) stains. sections were dehydrated through ascending series of ethanol, cleared in xylene, mounted in dpx, observed and photographed under leica ec3 compound microscope. results the endocrine pancreas in l. bata (herbivore), s. aor (carnivore) and c. chitala (highly carnivore) exhibit a great deal of variations in relation to their occurrence as well as morpho-histological peculiarities. the endocrine pancreas in l. bata is associated with the adipose tissue located in between figure 1. endocrine pancreas in l. bata showing α cells at the periphery and β cells (arrow heads) in the central region. arrow indicates blood vessel (mt, 100x). figure 2. acinar cells (ac) separated from islets of langerhans by connective tissue septa (arrow heads) in l. bata. note homogenous cytoplasm and deep nucleus in β cells (solid arrows) and deep nucleus in α cells (broken arrows). bv indicated blood vessels (mt, 1000x). 19 int. j. aquat. biol. (2016) 4(1): 17-24 coils of the intestine around the blood vessels and the bile duct within the mesentry. however, in s. aor and c. chitala, the pancreas is found to occur as a welldeveloped compact organ situated in between the lobes of liver and seen to span from the border of the stomach to the anterior part of the intestine. the intrahepatic pancreatic tissue of hepatopancreas in l. bata is usually devoid of islets of langerhans. different cell types could be identified in the pancreatic islets, based mainly upon differences in the density of the cytoplasmic ground substance, in the shape and secretory granules. in l. bata, the pancreatic islet cells are separated from exocrine part by a thin capsule of connective tissue mainly of collagenous fibres. the distribution of α cells is restricted to the islet periphery facing towards acinar cells (figs. 1, 2). the islet of α cells contained sparse cytoplasm and densely stained nucleus (fig. 2). in the central portion of the islets the af positive β cells exhibit rounded moderately dense cytoplasmic mass and direct contact with blood cells (figs. 3, 4). distinct granules of β cells are not seen in mallory’s triple stain since the granules are soluble in ethanol. in s. aor, the pancreas is relatively more organized figure 3. showing α cells at the periphery (broken arrows) and inner β cells (solid arrows) adjacent to blood vessels (bv) and acinar cells (ac) in l. bata (ra, 400x). figure 4. endocrine pancreas in l. bata showing af positive β cells, α cells (solid arrows) and vacuolated δ cells (broken arrows) encircling blood vessels (bv) (af, 1000x). figure 5. comparatively light stained islet of langerhans (il) attached to the wall of the stomach (sw) and encircled by acinar cells (ac) in s. aor (he, 100x). figure 6. scattered elongated islet of langerhans (il) positive to af in between acinar cells (ac) and pancreatic duct (pd) in s. aor. arrow heads indicate septa in between ac and il (af, 400x). 20 ghosh et al./ characterization of endocrine pancreas in l. bata, s. aor and c. chitala in its structural architecture and patches of endocrine islets are surrounded by acinar cells (figs. 5, 6). the rounded α cells are positive to romies azan stain provided with conspicuous dense nucleus and homogenous cytoplasm which are situated at the surface of islets below the septa (fig. 8) adjacent to blood vessels. the β cells are comparatively larger in size and ovoid in shape and clustered in the central portion of islets and are stained with romies azan and mallory’s triple stain (figs. 7, 8). however, δ cells are solitary and few in number, provided with densely stained nuclei and scantly cytoplasm (figs. 7, 8). the small and large islet of langerhans in c. chitala scattered as irregular elongated or oval masses of comparatively pale staining cells adjacent to rich blood vessels and acinar cells (figs. 9, 10). α cells are at the periphery of the islet but occasional isolated cells may be found in its interior. β cells are numerous in the centre of the islets positive to af having prominent nucleus and homogenous figure 7. islet of langerhans (il) separated from acinar cells (ac) by septa (arrow heads) in s. aor. note peripheral α cells (solid arrows), intense red coloured β cells (broken arrows) and innermost δ cells adjacent to blood vessels (bv) (ra, 1000x). figure 8. islet of langerhans (il) in s. aor showing β cells with prominent nucleus and cytoplasm mass (broken arrows), α cells (solid arrows) and innermost δ cells in between acinar cells (ac). arrow heads indicate septa (mt, 1000). figure 9. light stained small elongated or large oval islet langerhans (il) in c. chitala in between acinar cells (ac) and blood vessels (bv). arrow heads indicate septa (ra, 150x). figure 10. higher magnification of islet langerhans (il) in c. chitala showing α cells at the periphery (solid arrows), β cells (broken arrows) and δ cells in central position. arrow heads indicate septa in between il and acinar cells (ac) (ra, 1000x). 21 int. j. aquat. biol. (2016) 4(1): 17-24 cytoplasm (figs. 11, 12). occasional isolated comparatively larger blue coloured agranular δ cells are scattered in between β cells (fig. 10). discussion in the present observation, the islet of langerhans are dispersed throughout the pancreas in l. bata, s. aor and c. chitala. in all the fish species, the islet of langerhans are enclosed in a thin capsule and consists of comparatively lightly stained fusiform cells with distinct nuclei and interspersed with blood sinuses. the endocrine portion of the l. bata is mainly distributed around intestinal bulb, intestine and bile duct. these descriptions resemble those given for islet aggregates in salmoniformes (wang et al., 1986). in s. aor and c. chitala, a number of lightly staining islet of langerhans of different sizes and shapes occur in between exocrine acinar cells and around the pancreatic duct. youson and almahrouki (1999) considered the islet of langerhans as principal islets of various sizes in fishes. they further opined that the intrahepatic pancreatic tissue is usually devoid of islets. in the present investigation in all the three fish species, the α cells are the most dominant cell types and most common on the periphery that appeared ovoid structure having oval nuclei and intense acidophilic cytoplasm. β cells are large in size, oval or rounded in shape with a granular or homogenous cytoplasm. on contrary, δ cells are solitary and few in number. shyamsundari et al. (2006) reported that ovoid islets of langerhans are usually with a central cluster of β cells and the α cells in the periphery in lizard fish saurida tumbill. mokhtar (2015) opined that the endocrine part of ctenopharyngodon idella, the ovoid α cells are dominate, β cells are polyhedral and they grouped in small clusters while δ cells are small, fusiform and argyrophilic cells. boquist and patent (1971) by ultrastructural analysis noticed that in the islet of the teleost scorpaena scropha the α cells are mostly abundant in the periphery of the islets and contained numerous secretory granules with electron dense core while β cells are central in position having medium sized rounded moderately dense secretory granules. in the present study, it has been observed that α, β and δ cells have contacts with the blood capillaries and sometimes the secretory contents of the cells are observed into the blood vessels. therefore, it may be conjectured that after being secreted from the endocrine cells the hormones are carried out into the capillaries and thereby promoting movement of glucose. iaglov (1978) emphasized that in c. carassius, cyprinus carpio, tinca tinca and silurus glanis α, β and δ cells figure 11. af positive β cells (solid arrows), α cells (broken arrows) and fairly stained δ cells (arrow head) in the islet of langerhans (il) of c. chitala interspersed with blood vessels (bv). ac indicates acinar cells (af, 600x). figure 12. higher magnification of af positive β cells (solid arrows) adjacent to blood vessels (bv) in c. chitala. note the presence of α cells (arrow heads) and solitary δ cell (broken arrow) (af, 1000x). 22 ghosh et al./ characterization of endocrine pancreas in l. bata, s. aor and c. chitala are shoot shaped and all have contacts with the capillaries and the hormones from the endocrine cells is carried out via emiocytosis into the blood vessels. in the present investigation, in all the three fish species the most important hormones secreted by the islet of langerhans are insulin from β cell and glucagon from α cells. both play a role in proper metabolism of sugars and starches in the fish body. insulin promotes the movement of glucose and other nutrients out of the blood and into cells. when blood glucose rises, insulin released from the β cells causes glucose to enter body cells to be used for energy. also, it sometimes stimulates conversion of glucose to glycogen in the liver. another hormone, glucagon from α cells promotes the movement of glucose into the blood when glucose levels are below normal. it causes the breakdown of stored liver glycogen to glucose, so that the sugar content of blood leaving the liver rises (sorokin et al., 1982; plisetskaya et al., 1985; 1986). however, immunological analysis to endocrine cells of the aforesaid three species under study is further needed for detailed cellular structure and functions. acknowledgements the authors are grateful to dr. sanjib ray, head of the department of zoology, the university of burdwan, burdwan for providing essential laboratory facilities. references boquist l., patent g. 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(2016) 4(1): 17-24 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی ی درون ریز پانکراس در ماهیانشناسی غدهای ساختار بافتمطالعه مقایسه labeo bata (hamilton, 1822) ،sperata aor (hamilton, 1822) وchitala chitala (hamilton, 1822) *1باراتیچ ، پادمانرا3 ،1شرابانی بارون ،2، 1کومار قوشساروج .هندوستان غربی، بنگال ،411327 بوردوان بوردوان، دانشگاه جانورشناسی، گروه شیالت، مایشگاهآز1 بنگال غربی، هندوستان. ، 232171 هوقلی ایتاچونا، ،بجوی نارایان ماهاویدیاالیا ،جانورشناسی گروه7 بنگال غربی، هندوستان. ،214413 بوردوان ،آسانسول کالج دختران آسانسول، ،جانورشناسی گروه4 چکیده: labeo bata (hamilton, 1822)، sperata aorشامل شیرینریز پانکراس در سه گونه ماهی استخوانی آبی درونوضعیت و سازمان سلولی غده (hamilton, 1822) و chitala chitala (hamilton, 1822) شناسی مورد مطالعه قرار گرفت. در گونه های بافتتکنیکاستفاده از ا بl. bata ه در دو ک، در حالیدپراکنده شده بودن واقع در باالی پانکراسوی اصفر مجرایای نزدیک بین توده رودهعمدتاً در بافت چربی های غده پانکراس بافت د کرد شناسی تائیریز پانکراس بود. تحلیل بافتبخش برونهمراه با به دیواره معده چسبیده غالب طوربه غده پانکراس، c. chitalaو s. aorگونه های خونی احاطه پراکنده شده در بین سینوسلف تهای مخلکه اجزای درون ریز هر سه گونه مورد مطالعه توسط یک کپسول نازک حاوی سلو اغلب αهای که سلولدر حالی داشتندصورت تجمعی آرایش به معموالً βهای سلول ،l. bataتحلیل ساختار سلولی نشان داد که در گونه شدند. می صورت تجمعی یا پراکنده در به معموالً αهای گرد و بیضی شکل سلول c. chitalaو s. aorهای. در گونهبودند های خونی واقع شدهبین رگ های آلدئید فوشین، رومیز آزان و تریپل مالوری طور معمول با رنگکه بهمتراکم مانند دانهدانه βهای ند و سلولشتاطراف جزایر النگرهانس آرایش دا تعداد اندک و آمیختهنیز به δهای شبیه های خونی مشاهده شدند. سلولو در اتصال با رگالنگرهانس ، در ناحیه میانی جزایر شوندمی یرنگ آمیز نشان دادند. در ضمن در این مقاله c. chitalaو s. aorهایگونه های سیتوپالسمی میانه را درسیتوپالسمی یافت شدند و گرانول βهای سلولبا مورد بحث قرار گرفت. سهانرهای مختلف جزایر النگها، طبیعت و عملکرد سلولبه جای تاکید بیشتر بر تفاوت .labeo bata ،sperata aor ،chitala chitalaی درون ریز پانکراس،ساختار بافتی، غده :کلمات کلیدی int. j. aquat. biol. (2015) 3(2): 68-71 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology short communication experimental study to evaluate the pathogenicity of streptococcus iniae in guppy (poecilia reticulata) milad adel*1, reza safari1, pedram moayeri2, ahmad nosrati movaffaq3, elham khalili4 1department of aquatic animal health and diseases, caspian sea ecology research center, sari, iran. 2department of clinical sciences, faculty of veterinary medicine, university shahrekord, shahrekord, iran. 3department of fisheries, sari university of agricultural sciences and natural resources, sari, iran. 4department of health and food quality control, faculty of veterinary medicine, university shahrekord, iran. article history: received 20 april 2014 accepted 9 january 2015 available online 2 5 april 2015 keywords: streptococcus iniae experimental stud guppy disease abstract: streptococcus iniae has emerged as an important fish pathogen over the last decade in farmed rainbow trout in iran. the main objective of this study was to evaluate the pathogenicity of s. iniae in poecilia reticulata. atotal of 60 apparently healthy p. reticulata were obtained from ornamental fish pet store and injected intraperitoneally with 1.5×106 cfu of bacteria. for 14 days after challenge, the rate of mortality and clinical signs were recorded. the first clinical signs was observed in challenged fish 48 hrs after injection of s. iniae and first mortality was observed 72 hrs after injection. no significant differences in mortality and clinical signs between both sexes were observed. streptococcus iniae was collected from internal organs of fishes challenged, and was confirmed using the conventional biochemical tests and pcr. it is concluded that, p. reticulata is susceptible to streptococcosis and can play an important role in transmission of the disease to other ornamental fish species and also cultured fish. introduction streptococcosis/lactococcosis is one of the most important bacterial diseases in farmed rainbow trout in iran. this disease was reported in wild and farmed fishes in fresh and salt water, and also in ornamental fishes (austin and austin, 2007). the main pathogenic species that have been associated with this disease include streptococcus iniae, s. agalactiae, s. parauberis, s. dysgalactiae, s. faecium, s. milleri, s. uberis, s. ictaluri, s. phocae, s. faecalis, lactococcus garvieae, l. piscium, carnobacterium piscicola and vagococcus salmoninarum (shoemaker et al., 2000; russo et al., 2006). streptococcus iniae and l. garvieae are the major pathogens of streptococcosis and lactococcosis in the cultured farms in iran (akhlaghi and keshavarzi, 2002; soltani et al., 2005). the occurrence of this disease * corresponding author: milad adel e-mail address: miladadel85@yahoo.com in the cultured rainbow trout in iran was reported by akhlaghi and keshavarzi (2002), soltani et al. (2005), soltani et al. (2008), sharifiyazdi et al. (2010), pourgholam et al. (2011), fadaeifard et al. (2012) and rahimi kia and mehrabi (2013). streptococcus iniae is the etiological agent of a hemorrhagic septicemia, characterized by anorexia, uni or bilateral exophthalmia, blackening of the skin, abdominal distension and hemorrhages in the internal and external organs (agnewa and barnes, 2007). also, s. iniae described as an important zoonotic bacterial disease causing cellulites and endocarditis in humans (agnewa and barnes, 2007). streptococcus iniae was isolated from different ornamental fishes; including red-tail black shark (epalzeorhynchos bicolor), rainbow shark (e. frenatum), erythrurus erythrurus, zebra danio (danio rerio), pearl danio (danio albolineatus), 69 adel et al./ pathogenesis of streptococcus iniae in guppy clown loaches (chromobotia macracanthus), botia macracanthus, barbus conchonius, hyphessobrycon sp., and african cichlids (russo et al., 2005; raissy et al., 2012; ferguson et al., 1994). in the last 10 years, the ornamental fish industry has developped in iran (meshgi et al., 2006). there are 150 species of aquarium fishes, and about 40 of them are bred and raised throughout the country (meshgi et al., 2006). the guppy (poecilia reticulata), is a small member of the live-bearing poeciliidae family (breden et al., 1987). despite the importance of s. iniae in streptococciosis outbreaks in iran, there are few researches about the agent of disease in ornamental fish. hence, the aim of this study is to evaluate the pathogenicity of s. iniae in p. reticulata. materials and methods fish: a total of sixty apparently healthy p. reticulata (each sex: 30 specimens) were obtained from an ornamental fish store (sari, mazandaran province, north of iran) in april 2013. the average weight of females and males were 0.8-1 (0.8 ± 0.2) g and 0.40.7 g (0.4 ± 0.3), respectively. also, the average length of female and males were 3-6 (4 ± 2) cm and 2.5-4 (2.5 ± 1.5) cm, respectively. fish were transferred to fish diseases laboratory at the sari university of agricultural sciences and natural resources, in aerated tank using a portable air pump and introduced to a 200 l glass aquarium. fish were divided into control and two treatments (including male and female groups) each includes 20 specimens. the water temperature, do and ph of aquariums were 28 ± 1°c, 4.9 ± 0.2 mg l-1 and 7.6 ± 0.2, respectively. poecilia reticulata were kept in these aquaria for one week for adaption period and fed by similar diet (taksiran company, iran). bacterial strains and challenging: streptococcus iniae was isolated from kidney of moribund p. reticulata showing the signs of streptococosis. the isolated bacteria were confirmed using the conventional biochemical tests and molecular methods (pcr). in preparation a stock culture, the bacteria were purified in tsa at 30°c for 20 hrs. a few of the resultant purified colonies were grown for 20 hrs at 30°c in three 150 ml brain heart infusion (bhi) broth. the isolate was adjusted to mcfarland turbidity standard no. 3, which was equivalent to 1.5×106 cfu/ml. all the fish in two groups were intraperitoneally challenged with 0.1 ml of s. iniae (1.5×106 cfu/ml). fourteen days after challenge, the mortality rate and clinical signs were recorded. data were analyzed using spss (version no. 18) and significant differences between to both sex were determined by a one-way analysis of variance (anova). isolation of bacterium and bacteriological examination: sampling was performed from kidney, brain and liver of challenged fish that showed clinical signs in the aseptic condition, then directly streaked by sterile swabs on blood agar supplemented with 1.5% nacl and tiosulfate citrate bile salt agar (tcbs). plates were incubated at 30°c for 48 hrs. then, the macroscopic and microscopic observation of the colonies, single colonies with pure culture growth were subcultured onto bhi and identified using the conventional biochemical tests. identification of the isolated bacteria by pcr: dna was extracted using a dna isolation kit (mbst, iran) according to the manufacturer's instructions. two pairs of primers, including f (5′-ctt acc tta gcc cca gtc taa cga c-3′) and r (5′ gtc gta aca agg taa gcc gta tcg –3) were used to identify s. iniae (soltani et al., 2005). the primers were synthesized by cinnagen company (tehran, iran). the expected 513 bp pcr amplification product confirmed the biochemical identification. the pcr was performed in a total reaction volume of 50 μl, containing 500 mm kcl, 100 mm tris-hcl (ph 9.0), 60 mm mgcl, 200 μm dntps, 1 μl of each primer and 0.2 u taq dna polymerase per 50 μl reacti, 1 μl of template dna and 40 μl of sterile distilled. distilled water was used as a negative control in each pcr reaction. the reaction was repeated for 37 cycles under the following conditions: 3 min at 94°c (1 cycle), 1 min at 94°c, 1 min at 45°c, 1.5 min at 72°c (35 cycle) 70 int. j. aquat. biol. (2015) 3(2): 68-71 and finally pcr were completed with the final extension step at 72°c for 10 min. pcr products were separated on 2% agarose gel in 0.5× tris– borate–ethylene diamine tetra acetic acid (edta) buffer and visualized using ethidium bromide and an uv illuminator. results no mortality and clinical signs were observed in the control group. the first signs of disease were observed in challenged specimens 48 hrs and first mortality 72 hrs after injection. the first clinical signs were the lethargy, anorexia, erratic swimming and bottom siting. also, blackening of the skin, abdominal distension, hemorrhoids and anal prolapse (fig. 1), uni and bilateral exophthalmia sometimes with hemorrhage (fig. 2), accumulation of blood fluids in the abdominal cavity, hemorrhages in the external surface (fig. 3) and in internal organs, including the liver, kidney and intestine were observed in some moribund fish. no significant differences in mortality and clinical signs between sexes were observed. the isolated bacteria were confirmed using the conventional biochemical tests and pcr (austin and austin, 2007). discussion streptococcus iniae is a major fish pathogen causing streptococcosis and zoonotic bacterial disease. streptococcosis in the cultured fish is considered as a major problem in iran causing a significant economic losses in the aquaculture industry, especially in the rainbow trout. the occurrence of streptococcosis in the rainbow trout farms was reported in different parts of iran (soltani et al., 2005). in addition, this disease was reported in different ornamental fish in some countries (russo et al., 2005; raissy et al., 2012; ferguson et al., 1994), whereas, there are few reports of this disease in the ornamental fish industry in iran (raissy et al., 2012). in this present study, the first signs of disease observed in the challenged fish two days after injection of s. inane and mortality observed in third day. raissy et al. (2012) evaluated the pathogenicity of s. iniae in silver shark and rainbow shark (e. frenatum). the clinical signs in these species were similar to the results of present study with differences in outbreak time of clinical signs and mortality (raissy et al., 2012) that could be a species specific or different strain of s. iniae. furthermore, the observed clinical signs of this study are similar to those of reported in rainbow trout and other ornamental fish (austin and austin, 2007; ferguson et al., 1994). in conclusion, p. reticulata is susceptible to streptococcosis and can play as carrier. this species is able to transmit of this bacteria to other ornamental fish species and also cultured fish. it is recommended an extensive studies to evaluate of pathogenicity of s. iniae in other ornamental fish. figure 1. blackening of skin, abdominal distension, hemoraghes and anal prolaps in the female p. reticulata challenged by s. iniae. figure 2. uni and bilateral exophthalmia in the male p. reticulata challenged by s. iniae. figure 3. hemorrhages in the external surface in the female p. reticulate challenged by s. iniae. 71 adel et al./ pathogenesis of streptococcus iniae in guppy acknowledgment this work was supported by the sari university of agricultural sciences and natural resources. references agnewa w., barnes a.c. (2007). streptococcus iniae: an aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination. veterinary microbiology, 122: 1-15. akhlaghi m., keshavarzi m. (2002). the occurrence of streptococcosis in the cultured rainbow trout of fars province. iranian journal of veterinary research, 2: 183-189. austin b., austin d.a. (2007). bacterial fish pathogens, diseases of farmed and wild fish. 3nd. chichester, uk, springer praxis publishing. 648 p. breden f., scott m., michel e. (1987). genetic differentiation for anti-predator behavior in the trinidad guppy, poecilia reticulata. animal behaviour, 35: 618-620. fadaeifard f., momtaz h., rahimi e., mirzakhani a. (2012). detection of streptococcus iniae and lactococcus garvieae by multiplex polymerase chain reaction (pcr) in some rainbow trout farms of iran. african journal of microbiology research, 11(2): 260-263. ferguson h.w., morales j.a., ostland v.e. (1994). streptococcosis in aquarium fish. diseases aquaculture organism, 19: 1-6. meshgi b., eslami a., yazdani h. (2006). study on the parasitic infections of aquarium fishes around tehran. journal of faculty of veterinary medicine, university of tehran, 61(1): 1-5. pourgholam r., laluei f., saeedi a.a., zahedi a., safari r., taghavi m.j., nasrollhzadeh saravi h., pourgholam h. (2011). distribution and molecular identification of some causative agents of streptococcosis isolated from farmed rainbow trout (oncorhynchus mykiss, walbaum) in iran. iranian journal of fisheries sciences, 10(1): 109-122. rahimi kia e., mehrabi y. (2013). detection and identification of different streptococcosis strains in farmed rainbow trout in boyerahmad and dena regions (north south of iran). world journal of fish and marine sciences, 5 (3): 315-321. raissy m., zandi s., ahmadi a., foroutan m.s., fadaeifard f. (2012). experimental evaluation of pathogenicity of streptococcus iniae in silver shark and rainbow shark. african journal of microbiology research, 6(14): 3560-3563. russo r., mitchell h., yanong r.p.e. (2006). characterization of streptococcus iniae isolated from ornamental cyprinid fishes and development of challenge models. aquaculture, 256: 105-110. sharifiyazdi h., akhlaghi m., tabatabaei m., mostafavi zadeh s.m. (2010). isolation and characterization of lactococcus garvieae from diseased rainbow trout (oncorhynchus mykiss, walbaum) cultured in iran. iranian journal of veterinary research, 11(4): 342350. shoemaker c.a., evans j.j., klesius p.h. (2000). density and dose: factors affecting mortality of streptococcus iniae infected tilapia, oreochromis niloticus. aquaculture, 188: 229-235. soltani m., jamshidi sh., sharifpour i. (2005). streptococcosis caused by streptococcus iniae in farmed rainbow trout (o. mykiss) in iran: biophysical characteristics and pathogensis. bulletin of the european association of fish pathologists, 25: 95107. soltani m., nikbakht g., ebrahimzadeh moussavi h.a., ahmadzadeh n. (2008). epizootic outbreaks of lactococcosis caused by lactoccoccus garviae in farmed rainbow trout (onchorhynchus mykiss) in iran. bulletin of the european association of fish pathologists, 28(5): 95-106. int. j. aquat. biol. (2017) 5(5): 328-335 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology short communication a first record of halodule pinifolia miki den hartog, and new locality of nationally endangered halophila beccarii asch, from the eastern coast of sri lanka susantha udagedara*,1daniel fernando, nishan perera, akshay tanna, rosalind bown blue resources trust, no. 86 barnes place, colombo 7, sri lanka. article history: received 26 august 2017 accepted 24 october 2017 available online 2 5 october 2017 keywords: seagrass sri lanka distribution conservation abstract: this study presents the first record of halodule pinifolia on the eastern coast of sri lanka, approximately 310 km along the coastline from the previously recorded location (kapparathota, weligama). further, this study describes the new locality of the nationally endangered seagrass species halophila beccarii to valaichchenai lagoon, 26.5 km north of batticaloa lagoon, where it was previously recorded, along with the presence of two other species, halophila ovalis and halodule uninervis. while the species composition the seagrass habitat of valaichchenai lagoon might change in the near future due to the impact of climate change, multiple threats already exist at these newly discovered seagrass habitats, including multiday fishing vessel movements, the collection of shells on the seagrass beds, and solid waste dumping into the lagoon. decline of seagrass abundance in valaichchenai lagoon would negatively impact the food security and income generation of fishers. therefore, a well-established legislative framework and systematic long-term monitoring of seagrass in valaichchenai lagoon are essential in order to develop seagrass conservation plans before populations decline significantly or become locally extinct. introduction seagrasses are an extensively distributed group of marine flowering plants having a relatively low number of species in the world (72) that are often overlooked due to their submerged environment (short et al., 2007; short et al., 2011). seagrass has been identified as one of the most productive ecosystems, second to mangroves and coral reefs (nadiarti et al., 2012). therefore, seagrasses are ecologically and economically important due to the ecosystem services and functions they perform, specifically in relation to atmospheric carbon absorption in mitigating climate change (fourqurean et al., 2012; kumara and udagedara, 2012). unfortunately, estimated annual loss of this valuable species may be as high as 7% of their total global area since 1990 (serrano et al., 2016). this is due to shifts in water quality, increased loading of sediment, contaminants, poor land management, nutrients, coastal development, impact from global climate *corresponding author: susantha udagedara doi: https://doi.org/10.22034/ijab.v5i5.358 e-mail address: susantha@blueresources.org change, and other changes, and fisheries overexploitation (orth et al., 2006; coles et al., 2011; nadiarti et al., 2012). the tropical indo-pacific represents one of the highest seagrass bioregions in the world, accounting for around 35% of total species (short et al., 2011). however, this region also has extensive gaps in knowledge related to seagrass distribution and species, accounting for around 24% data scarcity (data deficiency), particularly for population data (short et al., 2011). sri lanka is located in the indo-pacific bioregion. so far fourteen species belonging to six genera (60% of the indo-pacific bioregion seagrasses) have been recorded there: enhalus acoroides, halophila beccarii, h. decipiens, h. ovalis, h. ovata, h. minor, h. stipulacea, thalassia hemprichii, cymodocea rotundata, c. serrulata, halodule uninervis, h. pinifolia, ruppia maritime and syringodium isoetifolium (udagedara in preparation). the species h. stipulacea was recently added to the 329 int. j. aquat. biol. (2017) 5(5): 328-335 seagrass species list in sri lanka (lk6-2132oraka). in sri lanka, seagrasses are found primarily in shallow, sheltered marine and estuarine waters on sandy, salty, or clay substrate in the gulf of mannar, the lagoons around the jaffna peninsula and puttalam, the negombo estuary, weligama, mulathivu, trincomalee, chilaw lagoon, batticaloa lagoon, mawella lagoon, koggala lagoon, mullaitivu, valaichchenai lagoon (abeywickrama and arulgnanam, 1991; udagedara in preparation). since propagules are transferred by ocean currents and have a wide distribution pattern across coastal habitats (abeywickrama and arulgnanam, 1991), no species is endemic to sri lanka (abeywickrama and arulgnanam, 1991). distribution records of seagrasses for the eastern part of sri lanka are extremely limited, and mostly only available prior to 1990 due to the three-decade civil conflict that prevailed in this area until 2009. there is some information on seagrasses for the southern coast of sri lanka, but this data is also limited due to lack of research (silva et al., 2013; udagedara et al., 2017; udagedara et al., unpublished). information on population declines is also insufficient, however silva et al. (2013) highlighted a decline in the standing crop of seagrass ecosystems to be around 96% in the northern, eastern and western parts of negombo lagoon between 1997 and 2004. in addition, around 20% of the total seagrass cover in negombo lagoon has been lost owing to pollution and micro-algal proliferation on seagrass beds as a result of nutrient loading (joseph, 2011). therefore, it is highly likely that similar declines may be affecting other areas given similar anthropogenic impacts, suggesting that national declines may be very high and be likely to lead to extirpations before proper documentation is in place. halodule pinifolia has to date been recorded in a total of four localities from the northern, western, and southern parts of sri lanka: 1991 and 1996 in negombo estuary (abeywickrama and arulgnam, 1991; pinto and punchihewa, 1996); 2011 in kapparathota, weligama (bandara et al., 2011); 2014 in mannar (isea, 2014); and 2016 in mandaitheevu, jaffna lagoon (digamadulla et al., 2016). this species’ prominent characteristics include rhizome creeping with 2-3 roots and a short erect stem of 1-3 cm length at each inter node. its blades are 5-20 (-29) cm long and (0.03-) 0.06-0.125 (-0.15) cm wide, with apex rounded with minute serrations and two poorly developed to non-existing lateral teeth. however, h. pinifolia is often confused with narrow-leaved h. uninervis which can be distinguished by blade apex morphology (abeywickrama and arulgnanam, 1991; kuo and den hartog, 2001). the iucn global redlist has categorized this as a species of least concern with a declining population trend (short et al., 2010b). the seagrass h. beccarii is commonly known as ocean turf grass. it has a thin rhizome with two scales covering the base of the erect stem and a group of 412 leaves at the top. the blades are lanceolate, up to 13 mm long, 1-2 mm wide, with no cross veins, apex pointed. the lack of cross veins is a unique character among genus halophila (abeywickrama and arulgnanam, 1991; kuo and den hartog, 2001). halophila beccarii is categorized as endangered according to the 2012 national red list and vulnerable by 2011 global iucn red list as it is rare in nature, and when encountered only occurs in fragmented populations (short et al., 2010a; meo, 2012). due to its specific habitat requirements and narrow depth range, its global area of occupancy has been estimated to be less than 2,000 km² (short et al., 2010a). to date this species has only been recorded in four localities in sri lanka: in 1885 and 1991 at batticaloa lagoon (dassanayake et al., 1995); in 1932 at muthur, south of trincomalee (dassanayake et al., 1995); and in 1991 and 2003 at negombo estuary/lagoon (samarakoon and van zon, 1991; abeywickrama and arulgnanam, 1991). these biologically important seagrass ecosystems have not been studied over a long period of time, and there are considerable information gaps, particularly for the eastern coast of sri lanka. this paper presents some new data that can help fill in some of these information gaps, which it is hoped will provide a 330 udagedara et al. / a first record of h. pinifolia and new locality of h. beccarii in sri lanka baseline for future research. methods valaichchenai lagoon is situated between 81.5489527.941750 and 81.554221-7.942600 in the vicinity of passikudah bay in the batticaloa district of sri lanka, encompassing an area of about 13.21 ha (silva et al., 2013) (fig. 1). according to punyawardena. (2008), the lagoon area belongs to the dry low country (dl2b) agro ecological zone with less than 200 mm of mean annual rainfall. silva et al (2013) categorized the lagoon as having the highest index of annual fresh water influx per unit area (0.665 mm3ha-1yr-1) although it is located in the dry zone, largely due to large amounts of fresh water diverted from the maduru oya river together with the comparatively small size of the lagoon itself. preliminary observations, personal discussions with local communities (fishers) and personal communications with experts including fishery inspector were carried out to determine potential areas of distribution of seagrass meadows within the valaichchenai lagoon. in may 2017, rapid seagrass sampling surveys were conducted covering all habitat types in the lagoon. a random vegetation sampling method using a 50x50 cm2 quadrate (burdick and kendrick, 2001; mckenzie et al., 2001) was conducted on 50 m parallel transects with 5 m intervals between each transect. seagrass sampling and photography of each quadrat was done while skin diving. collected specimens were identified based on the key developed by abeywickrama and arulgnam, (1991) and kuo and den hartog, (2001). herbarium specimens of each species were collected, pressed and independently coded by members of the research team, with species identification verified in consultation with a globally recognized seagrass taxonomist. specimens were deposited in the national herbarium in peradeniya (pda), sri lanka, under voucher numbers brt/sg/sl/ne/vc/001 to 004. anthropogenic impacts were recorded through personal observations and discussions with local communities adjacent to the sampling area. results and discussion results of the rapid seagrass sampling indicated that seagrasses were distributed along both sides of valaichchenai lagoon, and extending inland approximately 2 km from the lagoon mouth, including a lagoon branch, nasivanthivu to puliyadora aru (~5 km). seagrass occurrence tended to be patchy, ranging from sparse growth to extensive meadows. a total of four species of seagrass were recorded during the surveys, namely: halophila beccarii, h. ovalis, 25m figure 1. map of valaichchenai lagoon, batticaloa district, sri lanka. 331 int. j. aquat. biol. (2017) 5(5): 328-335 halodule uninervis, and h. pinifolia (fig. 2). the species composition and distribution pattern showed similarities to that of the negombo estuary described by samarakoon and van zon (1991). the record of h. pinifolia from valachchenai lagoon is the first record of this species from the east coast of sri lanka, with previous records being confined to the west coast in weligama, the negombo estuary, mannar, mandaitheevu, and jaffna lagoon (fig. 3). halodule pinifolia is the most widely distributed species in the main lagoon of valachchenai, but does not extend in to the peripheral lagoon (nasivanthivu to puliyadora aru). closer to the lagoon mouth, h. pinifolia was found together with other recorded species. high numbers of epiphytes in h. pinifolia’s blades were observed in the area where it was located close to mangrove patches and shallow, muddy substrate. the abundance of h. pinifolia was similar to that in the negombo estuary, and may be due to species-induced high physicochemical tolerance limit and the high index of annual fresh water influx per unit area of both lagoon (de silva and amarasinghe, 2007; silva et al., 2013). halophila beccarii was recorded on both sides of the lagoon and towards the lagoon mouth up to approximately 1.5 km as a small, scattered, patchy population. halophila. beccarii was mainly found in sandy or muddy substrate, and in some locations mixed with h. ovalis, h. uninervis (similar to the observations of kanal and short (2009)) and h. pinifolia. different leaf morphology was observed during the study and narrow leaf blades were observed figure 2. clockwise from upper left: halodule uninervis, halodule pinifolia, halophila beccarii and halophila ovalis (photo copyright: susantha udagedara). 332 udagedara et al. / a first record of h. pinifolia and new locality of h. beccarii in sri lanka in shallow areas where tidal action was high (~15 to 30 cm), while wide leaf blades were identified in areas where water depth was comparatively high (~30 cm to >1 m). scattered h. ovalis populations were found in the main lagoon on predominantly sandy substrate, and in certain areas overlapping with other species. only h. ovalis was widely distributed along both sides of the peripheral lagoon (nasivanthivu to puliyadora aru ~5 km). however, it was observed that the majority of h. ovalis was present in shallower water, and in areas where it grew exclusively, the leaves had more red color pigmentation. sidik et al. (2010) highlighted that this might be due to uv-blocking pigments that protected these plants when exposed to direct sunlight during low tides. however, in mixed populations they had lower levels of red pigmentation as they were partially protected by species of halodule that attained greater vertical relief and provided some degree of shading. valachchenai lagoon is heavily impacted by anthropogenic activities that threaten seagrasses, other coastal ecosystems and associated species. the lagoon is an important anchorage for fishing boats with a fisheries harbor located ~3.1 km inside the lagoon. the wake caused by the constant movement of boats through the lagoon can have negative impacts on seagrasses. this newly discovered habitat is very vulnerable due to various anthropogenic impacts including wake impacts of multiday boats (120 to 300 boat movements per week depending on season fisheries inspector at valachchenai harbour) entering the lagoon to access the fishery harbor from the lagoon mouth. the center of the lagoon has been dredged to facilitate boat movement and may have resulted in the loss of seagrass habitat, and is the likely reason that no seagrasses were observed in the middle part of the lagoon. significant threats to the lagoon ecosystem include the collection of shells on the seagrass beds; indiscriminate disposal of solid waste into the lagoon; oil and chemical pollution from fishing boats; lagoon encroachments; anchoring of boats on seagrass beds; the use of outboard motors in shallow seagrass areas; the use of harmful fishing gears such as monofilament gill nets and drag nets. further, other recorded impacts were sedimentation and agricultural runoff from upriver sources. santharooban et al. (2012) recorded higher than accepted levels of nitrogen and phosphorous being discharged into valachchenai lagoon from the fisheries harbor, paper mill, rice mills, and shrimp farms. this may lead to eutrophication, which would result in the degradation of seagrass habitats within the lagoon (miththapala, 2008). conclusion this study documents the first record of h. pinifolia from the eastern coast of sri lanka, and provides a new locality of the globally vulnerable h. beccarii. the presence of h. beccarii is significant as this species has diminished in other areas of sri lanka. for example, de silva and amarasinghe (2007) reported the extirpation of this species at the same locations in figure 3. distribution map of halodule pinifolia and halophila beccarii (red circle and trundle indicates a new record) 333 int. j. aquat. biol. (2017) 5(5): 328-335 negombo estuary from where it was previously recorded by jayasuriya (1990). it is important that adequate management is carried out to prevent similar outcomes at valachchenai lagoon, where increasing anthropogenic impacts may result in severe population declines or extirpation in the near future. silva et al. (2013) estimated that the average monthly income for fishers in valaichchenai lagoon was lkr 17,000.00 (130.1 usd-conversion rate 130), which is lkr 1 4,441.00 of average monthly income of the sri lankan fishers. unsworth et al. (2014) highlighted the importance of seagrasses for high fisheries production due to their value as a critical nursery habitat. therefore, a decline of seagrass in the valaichchenai lagoon would negatively impact food security and income generation of artisanal and subsistence fishers. seagrass species composition and habitat of this lagoon may change due to anticipated climate change impacts in the future including increasing sea-surface temperature, rising sea levels, and other disturbance regimes highlighted by waycott et al. (2007). results from this study will help in the development of long-term monitoring programs and management strategies that take into account available information on the ecological biogeography of the population in addition to impacts from anticipated climate induced shifts. this data can also be fed into the mechanisms that enable the updating of the iucn red list status of these species for future conservation decision-making. it is also of note that although around 72 countries have some form of seagrass management or protection via various mechanisms, sri lanka is yet to develop dedicated management plans or legislation for the protection of seagrass. developing sound legal mechanisms is essential for the management of this critical habitat (unsworth and cullen-unsworth, 2014), and it is hoped that this and other ongoing research will contribute to the improved management of seagrass habitats in sri lanka. acknowledgments our thanks to ms. k. chartrand miller for the verification of h. uninervis, and prof. f.t. short and dr. l. mckenzie for the verification of h. ovalis. we also thank m. abeyratne and p. wijesinghe for their help with the field research. the fieldwork for this study was carried out with funding provided to blue resources trust by the tokyo cement group. references abeywickrama b.a., arulgnanam p. 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(2021) 9(3): 159-166 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article otolith shape analysis of lethrinus lentjan (lacepède, 1802) and l. microdon (valenciennes, 1830) from the red sea yassein a.a. osman* 1, snæbjörn pálsson2, ahmed f. makkey1 1national institute of oceanography and fisheries, niof, egypt. 2department of life and environmental sciences, university of iceland, reykjavík, iceland. s article history: received 14 february 2021 accepted 15 april 2021 available online 2 5 june 2021 keywords: otolith outline emperor fish morphometry abstract: otolith shape and morphology are used to identify fish species and population stocks. the aim of this study was to distinguish the lethrinus lentjan (lacepède, 1802) and l. microdon (valenciennes, 1830) (family: lethrinidae) using otolith shape. the analyses apply the shaper package in r which enables to extract the outline and otolith morphology from images and for statistical examining of individual variation. otoliths of 165 individuals from the two lethrinus species were collected during 2019 and 2020. the wavelet levels were examined by using 6 wavelets to collect 63 coefficients. the regression between width and fish length were b = -0.03 (t = 2.6, p = 0.01) for l. lentjan and was significantly different (t = 2.120, p = 0.036) for l. microdon (b = 0.018). introduction the family lethrinidae is one of the most important groups of fishes in coral reef fisheries in egypt, which includes 39 species with 29 emperor species of the genus lethrinus (carpenter and niem, 2001). the annual catch of this family is around 1,469 tonns representing 3.06% of the red sea fishery production (gafrd, 2020). generally, the emperors are longlived, reaching age greater than 20 years, with size less than 20.0 cm fl (l. variegatus, valenciennes, 1830) to 80.0 cm fl (l. nebulosus (forsskål, 1775) (randall, 1995; carpenter and niem, 2001). otolith comparison of lethrinids can be challenging due to lack of informative morphological characters to distinguish their species (carpenter and allen, 1989; carpenter, 2002; carpenter and randall, 2003). the identification of lethrinidae based on morphological characteristics could be solved by costly dna analyses, however, otolith shape analyses may offer a cheap and easily applicable method tin this regard (libungan and pálsson, 2015; libungan et al., 2016; mehanna et al., 2016; osman et al., 2020). otolith shape and dimensions are commonly used to identify fish species but may provide also important *correspondence: yassein abdel-maksoud osman doi: https://doi.org/10.22034/ijab.v9i3.1159 e-mail: yasseinahmed66@yahoo.com information such as stock, age and the growth of the fish during its lifespan (lecomte-finiger, 1999; tuset et al., 2003; jawad et al., 2017). elsherif et al. (2020) estimated the phylogenetic relationships and taxonomy of three species of family lethrinidae, including l. mahsena, l. nebulusus and l. grandiculis from northern red sea, showing that they lack discriminative morphological traits. therefore, the aim of the present study is to compare l. lentjan and l. microdon based on their otolith shape to distinguish them and the results could provide a tool to characterize other species of the family lethrinidae along the egyptian coast of the red sea. materials and methods sampling: a total of 165 specimens of l. lentjan (n=96) and l. microdon (n=69) were obtained at hurghada fishing port (27°13ꞌ43.32ꞌꞌn, 33°50ꞌ33.20ꞌꞌe), in northern red sea, egypt during 2019 and 2020. the fishes were sampled randomly from the commercial catch of the hook and line fishery. the fish total length (l) was measured to the nearest 0.1 mm; fish weight (w) to the nearest 0.01 g, and also the sex was recorded. sagittal otoliths were 160 osman et al./ otolith shape analysis of lethrinus lentjan and l. microdon extracted, cleaned and dried. all otolith images were estimated on the distal side using a stereomicroscopic with axiocam erc 5s camera (carl-zeiss-promenade 10; 07745 jena, germany) and the software of zeiss. the statistical analysis was performed with rstudio (r core team 2015) using the r packages of ade4 (dray and dufour, 2007), pixmap (bivand et al., 2011), ipred (peters and hothorn, 2013), vegan (oksanen et al. 2013), jpeg (urbanek, 2014) and shaper (libungan and palsson, 2015). otolith photos were read into r and the outlines were extracted using the conte function in r (fig. 1). feret length and width were measured to the nearest 0.1 mm. area and perimeters were obtained from the figures using shaper. analysis of species differences and otolith shape: the independence of the different otolith variables was evaluated with pearson correlation and summarized with descriptive statistics. the difference between the species was analysed for weight, transformed with square-root, and the different otolith characteristics was tested with a linear model taking length and sex into account. a regression line was fitted for both species and the success of these methods in distinguishing species was evaluated by looking at how many individuals of species l. lentjan where within the range of l. microdon and vice versa. the shape of each otolith was fitted with a function of independent wavelet shape coefficients, obtained with the wavethresh package in r (claude, 2008; nason, 2012; libungan and palsson, 2015a). differences in size among the otoliths were standardized to remove size differences. the number of wavelet coefficients increase by the power of 2 for each wavelet level; 63 coefficients were obtained for each outline using 6 wavelet levels. the quality of the reconstruction rises with the number of wavelet levels (fig. 6), and the shape of sprat otolith appears to be precisely described (with 98.5% accuracy with respect to the original otolith contour-line) by the sum of the first 5 wavelet levels. the difference in shape between the species was summarized by plotting the average otolith shape based on normalized wavelet coefficients (libungan and pálsson, 2015b). to investigate which areas and coefficients on the outline contribute most of the variations in shape, the mean shape coefficients and standard deviation were plotted against the angle of the outline from the coefficients using the plotci command in the gplots package (warnes et al., 2014). to determine which region contributed most to the differences between the species, the proportion variation between the species out of total variation (the intraclass correlation icc), was calculated along the outline of the otolith. the difference in otolith shape between the two species using length of the fish as a covariate was analysed using canonical analysis of principal coordinates (cap) (anderson and willis, 2003) using the vegan package (oksanen et al., 2013) on the standardized wavelet/fourier coefficients and tested with permanova. to classify individuals to their taxonomic classification based on the population variation within the two species, linear discriminant analysis (lda) was applied to the coefficients using the lda function in the mass package in r (ripley et al., 2014), and the misclassification error estimated using cross validation based on bootstrapped samples of the dataset as in libungan and palsson (2015). results morphological measurements: the length figure 1. original otolith shapes and the red outline marks the shape of the otolith which is extracted by shaper and forms the basis for the analysis of variation within and between the two species investigated. (a) lethrinus lentjan and (b) l. microdon, with scale bars (1 mm). anterior of the otolith is to the left. 161 int. j. aquat. biol. (2021) 9(3): 159-166 distribution of the l. lentjan varied from 164 to 507 mm tl, and showed three modes (220, 280 and 450 tl mm) which might present different cohorts, a single unimodal distribution was observed for l. microdon which range overlapped with l. lentjan (236-513 mm) (fig. 2, table 1). the average length for two species was estimated at 286.3±96.0, 368.01±65.70 mm and weight at 465.74±501.74, 643.54±346.71 g for l. lentjan and l. microdon, respectively. the correlation coefficient of otolith length, otolith width, otolith area and perimeter for the two lethrinus species were strongly correlated, with r varying between 0.88 and 0.97 (fig. 3). the square root of weight of the fishes could be explained by length and species but was independent of sex (r-squared = 0.987). the square root of the weight increased by b = 1.05 g per cm (t = 107.82, p<0.001) for both species, but l. lentjan weighted on average 2.97 g more than l. microdon (t = 16.49, p<0.001) for a given length (fig. 4). despite these differences, there is some overlap of the two distributions around the regression lines. nine l. microdon weighted less than the 97.5 percentile of l. lentjan and 18 of l. lentjan weighted more than 2.5% of the distribution of l. microdon. separate analyses of variation in length, width, perimeter and area of the otoliths resulted in similar patterns as expected due to their high correlation and table 1. descriptive statistics for lethrinus lentjan and l. microdon otoliths. n: sample size, f: females, m: males, tl: total length, bw: weight, ol: length, oh: height; oa: area op: perimeter. species l. lentjan l. microdon min.-max. average± sd min.-max. average± sd n 96 (66 f, 30 m) 69 (42 f, 27 m) tl range (mm) 164-507 286.3±96.9 236-513 368.01±65.70 bw (g) 70-1825 465.741±501.74 134.6-1582.8 643.54±346.71 ol (mm) 6.41-15.3 9.89±2.42 6.36-9.692 8.05±0.84 oh (mm) 4.68-11 6.99±1.57 3.781-5.773 4.78±0.49 oa (mm) 21.08-115.9 49.24±23.52 18.421-37.176 27.20±4.95 op (mm) 21.76-111 41.6±21.01 20.19-35.485 27.94±4.00 figure 2. length distribution of lethrinus lentjan and l. microdon; red line presents the mean values for the two species. 162 osman et al./ otolith shape analysis of lethrinus lentjan and l. microdon presented here just for the width of the otoliths which showed the largest difference between the species (t = 4.19, p<0.001), and gave the highest proportion of variation explained by the model (r2 = 0.45). differences were found between the species traits in all cases (p<0.01 or <0.001), but the difference was smaller with larger fishes as seen with significant differences in the regression slopes and was independent of sex. the regression slopes for width on fish length were b = 0.03 (t = 2.6, p = 0.01) for l. lentjan and was significantly different (t = 2.120, p = 0.036) for l. microdon (b = 0.018). however, the variance was much larger for l. lentjan, therefore, the significance should be taken with caution (fig. 5). an inspection of figure 5 shows the split of the two species but there are about 16 l. lentjan individuals with similar width or smaller than the width of l. microdon, the overlap was larger for the other traits. main shape features: the otolith shape of the two species differs (permanova f’ = 149.68, p<0.001, table 2) as reflected in the scatter of individual shapes in the ordination plot. the first discrimination axis of the cap analyses based on the wavelet coefficient showed 98.1% of the differences between the two table 2. variations in otolith shape between fish sex based on anova-like permutation test based on 1000 permutations. species df sum. sq f’ p-value l.lentjan v. l.micrododn fish length sex residual 1 1 1 162 19.896 0.238 0.172 31.373 102.73 1.23 0.890 0.001 0.250 0.409 figure 3. the correlation coefficients of otolith measurements for the two species; lethrinus lentjan and l. microdon. figure 4. relationship of weight and length of lethrinus lentjan and l. microdon. 163 int. j. aquat. biol. (2021) 9(3): 159-166 figure 5. relationship of otolith width and fish length of lethrinus lentjan (le) and l. microdon (mi). figure 6. quality of the wavelet, the red vertical lines show the level of wavelet and number of fourier harmonics needed for a 98.5% accuracy of the reconstruction. figure 7. differentiation of otolith shape of lethrinus lentjan (le) and l. microdon (mi), based on canonical analysis of principal coordinates with the wavelet coefficients. l. lentjan and l. mi-crodon are indicated by open and filled dots, respectively. figure 8. mean otolith shape based on wavelet reconstruction for two species lethrinus lentjan (le, n=96), l. microdon (mi, n=69). numbers represent angles in degrees (°) based on polar coordinates (see fig. 4). the centroid of the otolith (center of the cross) is the center point of the polar coordinates. 164 osman et al./ otolith shape analysis of lethrinus lentjan and l. microdon species and the second axis 1.3% (figs. 7 and 8), but the shape of few (~4) individuals of the two species overlapped. the misclassification error based on the lda-analyses was 2.4%. the differences in the mean shape (fig. 8) of the two species are mainly at certain regions along the edge of the otoliths, namely at 0-20, 80-140 and 170-190 angles (fig. 9), and interestingly there is a notable difference in the width. the average shape of otolith varied within species mainly at pararostrum at angle ca 30-40, 140 and 180° counted anti-clockwise from right to left. discussions the relationship between weight and length is very important to estimate the biomass from length and provides information on the condition factors of fish (moutopoulos et al., 2002; souza et al., 2019). the otolith morphology may provide better information to comparison between stocks or species as it is independent of conditions and can be used in diverse studies e.g. to characterize fish species in archaeological sites (aguilera et al., 2013; souza et al., 2019) or among prey where other information may be lacking. the length frequency may be used to study the age, growth, survival rate, mortality rate and stock differentiation and fisheries management (jones, 1984; pauly, 1984; pauly and morgan, 1987; athanassios et al., 2018; mehanna et al., 2018a, b; osman et al., 2020; liang et al., 2020; froese et al., 2020). the results may be explained by the fact which length of otolith is more sensitive to variation growth rate and relation to changes in fish metabolism (pawson, 1990; flecher 1991; osman et al., 2020). the otolith measurements of the two species were examined with fish length to get the relationship between the otolith width and fish size. the difference between the species indicate the otolith of l. lentjan may be large than l. microdon, and the differences in otolith measurement among the species may be due to variation in environmental condition and habitat, as well as water temperature and dissolved oxygen effect on fish growth (campana and casselman, 1993; cardinale et al., 2004; zischke et al., 2016). interspecific variation in otolith morphology can reflect live at different depth e.g. fishes live at large depth have generally large otoliths (tuset et al., 2003a; baniet al., 2013; zischke et al., 2016). the shape of otolith may be estimated with standard statistical methods. in the current study, we used two multivariate methods to distinguish l. lentjan and l. microdon i.e. canonical analyses of figure 9. mean and standard deviation (sd) of the wavelet coefficients for all combined otoliths and the proportion of variance among groups or the intraclass correlation (icc, black solid line). the horizontal axis shows angle in degrees (°) based on polar coordinate (see also fig. 1) where the centroid of the otolith is the center point of the polar coordinates. 165 int. j. aquat. biol. (2021) 9(3): 159-166 principal coordinates and linear discriminant analyses both revealed clear difference and the latter a high overall score of correct classification. the wavelet transform may be usefulness in otolith shape analysis for the morphological measurements that estimated by otolith outline (pararsotrum, postrostrum and exicura major) and the most variation between species and among the population. the correlation between species is high for the first canonical analysis. the multivariate method was used to the wavelet coefficient to get shape varietion between species. the anova a-like permutation analyses were significant between two species i.e. the two species differs. the mainly variation between species at pararostrum at angle 30-40, 140 and 180° counted anti-clockwise from right to left. the wavelet might prove to be better for explaining shape differences, while for others, the fourier method might be more powerful to distinguish populations. in addition, the evaluation of the applicability of the wavelet, in otolith shape analysis is warranted (libungan et al., 2015; libungan et al., 2016). the otolith comparison between two species was the first study to estimate the difference between two species and family in the egyptian coast of the red sea. therefore, this study encourages to more study to compare between the most commercial species of the red sea using otolith morphology and shape. finally, this study considers important by adding more details to food and feeding, stock assessment and paleontology studies. references ahmed m., ahmed m., madkour f., hanafy m. 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(2015) 3(2): 72-77 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article evaluation effect of dietary egg lecithin on digestive enzymes and body composition of juvenile binni (mesopotamichthys sharpeyi gunther, 1874) seiedeh maedeh seiedzadeh, vahid yavari, hamid mohammadiazarm*,1mohammad mosavi department of fisheries, khorramshahr university of marine science and technology, khorramshahr, khouzestan, iran. article history: received 5 november 2014 accepted 11 february 2015 available online 2 5 april 2015 keywords: growth egg lecithin binni enzyme. abstract: in this study, the effects of dietary egg lecithin on digestive enzymes and body biochemical composition of juveniles mesopotamichthys sharpeyi was evaluated. four experimental diets including control diet (with 0% egg lecithin) and three diets containing 2%, 4% and 6% egg lecithin were used. at the end of the experiment, digestive enzymes activity (lipase, amylase and alkaline phosphatase) and body biochemical compositions were assessed. the results showed no significant differences between experimental treatments in moisture and ash content. maximum content of the crude protein and crude lipid were recorded in 4% lecithin treatment and it had significant differences with control group. the digestive enzymes activity (lipase, amylase and alkaline phosphatase) showed significant differences between control and experimental groups. an increasing trend was observed in the digestive enzymes activity among treatments. based on the results, it was concluded that 4% to 6% dietary egg lecithin in the diet, can promote growth and survival rate of juvenile binni. introduction in the recent decade, the aquaculture industry had a fast growth (heydarnejad, 2012). the factors such as availability, stability, appropriate quantity, price and nutritional value of the diet are crucial in development of this industry (nasopoulou and zabetkis, 2012). lipids are the most important ingredient of fish diet. because, it acts as an energy source, contains essential fatty acids and fat-soluble vitamins (sargent et al., 1997). fish oil is a source of lipids in fish diets; that supply through catch (pike, 2005). estimates show that the amount of fish oil production will not be able to supply required fish oil through catch in next years (tocher et al., 2008). the productions of fish diets are based on soybean oils and fish oil, but researches have shown that use of animals and plant neutral oils in the diets of fish, especially during larvae stage leads the accumulation of fat in the intestinal enterocyte (rinchard et al., 2007). this topic also causes the reduction in uptake * corresponding authors: hamid mohammadiazarm and vahid yavari e-mail address: azarmhamid@gmail.com, yavarivahid@gmail.com of saturated and mono-unsaturated fatty acids as energy sources in the intestine and subsequently, decreases growth and survival of fish larvae (moraise et al., 2007). in addition, fish larvae have limited enzymatic ability for producing the lipoproteins; therefore, adding the phospholipids in the diet can provide larval requirements to phospholipids helping to produce lipoproteins (tocher, 2008). industrial source of phospholipids is lecithin, which produced during the purification process of crude oils such as soybean crude oil, sunflower, canola, turnip and chicken egg. mesopotamichthys sharpeyi is a cyprinid species (nikpey, 1996), known as binii or bunii. this species is native freshwater fishes of tigris basin found in iran, iraq, turkey and syria. binii has been recently considered as a proper candidate for aquaculture in iran due to its high resistance to a wide range of the environmental conditions, having proper features for rearing and marketability 73 seiedzadeh et al./ egg lecithin for binni fish (hamidian, 2003). it is also cultivated for stocking in its natural habitats. there is little information available about its physiological features, especially during larval development and growth performance. in addition, the appropriate diet needs to be formulated for this species based on its requirements and life stage. therefore, this study was carried out to investigate the effect of dietary egg lecithin on the digestive enzymes and body composition of juvenile binni. materials and methods diet preparation: the ingredients and composition of the experimental diets are given in table 1. four experimental diets containing chicken egg lecithin were prepared. all diets were formulated to isonitrogenic and isolipidic. to determine the effect of different levels of the lecithin on the growth performance; four levels of lecithin (0%, 2%, 4% and 6%) were used in reduction of soybean oil. dry ingredients were weighed and ground (100 µm particle sizes) and then mixed thoroughly. fish oil, soybean oil, chicken egg lecithin and water were added to the dry ingredients and mixed again, until a dough was formed, which was dried at room temperature for 24 hrs and grounded into desirable particle sizes. the diets were broken up and sieved into a proper pellet size, packed, and stored at -20°c. experiment fish and feeding conditions: the experiment was conducted from september until november 2013 in wet-lab of khorramshar university of marine science and thechnology. juveniles of m. sharpeyi were obtained from a local farm (maleki farm, khozestan, iran). the fish were treatment control egl2 egl4 egl6 ingredients diets(g 100g-1) fish meala 23.0 23.0 23.0 23.0 corn gluten meal 10.0 10.0 10.0 10.0 wheat meal 33.1 33.1 33.1 33.1 wheat bran 15.0 15.0 15.0 15.0 fish oilb 6.0 6.0 6.0 6.0 soybean oil 6.0 4.0 2.0 0 egg lecithinc 0 2.0 4.0 6.0 vitamin premixd 3.0 3.0 3.0 3.0 mineral premixe 2.0 2.0 2.0 2.0 binderf 2.00 2.00 2.00 2.00 antioxidantg 0.02 0.02 0.02 0.02 lysine 0.27 0.27 0.27 0.27 proximate composition (%dm) moisture 8 8.8 9.8 8.4 crude protein 32.25 32.28 32.75 32.36 crude fat 12 12.31 12.26 12.7 ash 8.42 8.67 8.77 10.40 lysine 2.00 2.00 2.00 2.00 aclopeonella meal, iran. bkilka oil, mazandaran co, iran. cchicken egg lecithin, merck, germany with purity 90% phosphatidylcholine. dvitamin premix (composition per 1kg): a=1600000 iu, d3=400000 iu, e=40000 mg, k3=2000 mg, b1=6000 mg, b2=8000 mg, b3=12000 mg, b5=40000 mg, b6=4000 mg, b9=2000 mg, b12=8 mg, h2=40 mg, c=60000 mg, inositol=20000 mg. emineral premix (composition per 1kg): iron:6000 mg, zinc:10000 mg, selenium:20 mg, cobalt:100 mg, copper:6000 mg, manganese:5000 mg, iodine:600 mg, cocl2:6000 mg. fbinder: amet binder (component: crude protein: 71.98٪, crude fiber: 0.9٪, ash: 17.8٪, moisture: 9.55٪). gantioxidant: butylated hydroxytoluene (bht). dm, dry matter. table 1. ingredient and proximate composition of experimental diets. 74 int. j. aquat. biol. (2015) 3(2): 72-77 acclimated to the laboratory condition for two weeks prior experiment. the fish (with initial mean weight of 3.1 ± 0.17 g) were introduced randomly into 300l circular plastic tanks with 40 fish per tank for the feeding trial after being collectively weighed. three replicate groups of fish were hand-fed to apparent satiation three times a day (9:00, 13:00 and 17:00) for 8 weeks. during the experimental period, water temperature, do and ph were 26 ± 1°c, 6.33 ± 0.073 mg l-1 and about 7, respectively. the photoperiod was natural conditions during experiment. at the end of experiment, the juvenile fish of each tank were collectively weighed after anesthetizing with a clove powder solution with a concentration of 30 mg l-1 after starvation for 24 hrs. in addition, at the end of experiment, fifteen specimens from each tank were transferred to tubes for assessment of digestive enzymes activity and body biochemical composition. chemical analyses: proximate analyses of the diets and fish were determined according to the aoac (1995). crude protein content was measured using the kjeldahl method by an auto kjeldahl system (kjeltec tm2300, foss, sweden). crude lipid was analyzed by soxtec system, moisture content by an oven (d-63450, heraeus, hanau, germany) drying at 105℃ for 24 hrs and ash by a furnace muffler (550℃ for 4 hrs). digestive enzymes activity: for assessment of enzyme activities, the intestine samples were homogenized in 100 mm tris-hcl, 0.1 mm edta and 0.1% triton x-100 (ph 7.8) with a homogenizer (ika®ti8 basic, germany), and centrifuged (12000 g for 30 min at 4℃). we used 100 mg tissue ml-1 buffer for homogenization and then the extracted supernatants were kept frozen in -80℃ to determine biochemical features. the activity of α-amylase, lipase and alkaline phosphatase were measured by the enzymatic photometric method using amylase, lipase and alkaline phosphatase pharmaceutical kits (pars azmoon, tehran, iran), respectively. protein was also determined by enzymatic colorimetric method using biuret kit (pars azmoon, tehran, iran). enzyme activity expressed as a specific activity of u mg-1 protein. statistical analysis: data were subjected to one-way anova to test the effect of sources and levels of lecithin on growth performance and lipoprotein fractions. when significant differences were found in one-way anova, duncan’s multiple range test was used to rank the groups. all statistical analyses were performed using spss version 16 (spss, chicago, il usa) with a significant level of p<0.05. the values presented are mean ± standard deviation (sd). results the results of digestive enzymes activity are shown in table 2. the activity of lipase indicated a significant difference between treatments and control groups. the activity of lipase increased with increasing dietary egg lecithin in diet (p<0.05). the activity of alkaline phosphates in brush border of intestinal epithelium was significantly increased with increasing the dietary egg lecithin compared to that of control group (p<0.05). activity of alkaline phosphates in juvenile fed the control diet was 231.24 ± 16.77 u mg-1 protein and 376.92 ± 10.17 umg-1 protein in juvenile fed 6% egg lecithin. also, the activity of amylase significantly increased as dietary egg lecithin was increased (878.97 ± 82.76 to 1085.09 ± 0.09 umg-1 protein) (p<0.05). treatment control 2% lecithin 4% lecithin 6% lecithin am(u/mgprotein) 878.97 ± 82.76a 1089.05 ± 0.05b 1094.69 ± 52.88b 1085.09 ± 0.09b alp(u/mgprotein) 231.24 ± 16.77a 312.28 ± 0.28b 300.25 ± 1.09b 376.92 ± 10.17c lip(u/mgprotein) 0.85 ± 0.07a 1.08 ± 0.05b 1.21 ± 0.01b 1.42± 0.03c am: amylase, alp: alkaline phosphatase, lip: lipase (mean ± se), n=3 with different letters in each row, indicate the presence of significant differences between the experimental groups (p<0.05). table 2. analysis of the digestive enzymes activity of juveniles mesopotamichthys sharpeyi fed different experimental diets for 56 days. 75 seiedzadeh et al./ egg lecithin for binni fish the result of the body biochemical composition is shown in table 3. the crude protein and crude lipid compositions of juvenile increased with increase amount of dietary egg lecithin (p<0.05). the highest crude protein content was observed in juvenile fed diets containing 4% egg lecithin. the crude lipid of juvenile fed 4% lecithin was the highest. others body biochemical composition such as moisture and ash showed no significantly different compared with control group (p>0.05). discussion the results showed that crude protein increase by increasing the levels of dietary egg lecithin. this result is in agreement with the results of zhao et al. (2013) on large yellow croaker (larmichthys crocea), hung et al. (1997) on juvenile of atlantic salmon (salmo salar), azarm et al. (2013) on rainbow trout (onchorynchus mykiss) and sink (2014) on channel catfish (ictalurus punctatus). generally, many factors are attributed to the body biochemical composition such as species, primary size and duration of experiment. but in the case of lecithin, it was reported that metabolism of many proteins is changed by phosphatidylcholine. therefore, high growth in treatments with high level of phospholipids is due to increasing demand for amino acids that induces increasing protein production (sotudeh et al., 2011). the higher level of egg lecithin in the diet showed an increase in the amount of lipid in the body of fish, that it was significantly higher in treatment with 4% egg lecithin. this result is similar to those of large yellow croaker (l. crocea) (zhao et al., 2013), caspian brown trout (salmo trutta caspius) (sotudeh et al., 2011) and atlantic salmon (s. salar) (hung, 1997). the lecithin induces the lipoprotein production and increases efficiency of nutrient absorption from the digestive tract via intestinal epithelium and then improves transferring of nutrients to the body tissues and consequently, increases consumption of fatty acids (morais et al., 2007). many studies showed that phospholipids has important role in lipid transfer inducing an increase in growth performance and access to energy and consequently, increase in the whole lipid of fish body (niu et al., 2008; sink, 2014). many works showed that phospholipid deficiency in larvae and juvenile diets is led to lipid accumulation in enterocyte. hence, the phospholipids are essential for transfer of lipids from enterocytes to blood and lymph of fish (teshima et al., 1986; tocher et al., 2008). different levels of egg lecithin had no significant effect on ash and moisture of fish. this result corresponds with those stated on juvenile flounder (paralichthys olivaceus) (kim et al., 2006), caspian brown trout (s. t. caspius) (sotudeh et al., 2011) and channel catfish (i. punctatus) (sink, 2014). higher levels of egg lecithin in diet showed significantly higher amylase, alkaline phosphates and lipase activity. the specific activity of amylase was significantly higher in egg lecithin treatments compared to that of control group that can be related to lysophospholipids which acts as an emulsifier in intestine (azarm et al., 2013). also, specific activity of amylase can be different due to feeding habits (hidalgo et al., 1999), sex (chakrabarti et al., 1995), amount of diet carbohydrate (kuzmina, 1996) and ion concentrations (munilla-moran and saboridorey, 1996). treatment control 2% lecithin 4% lecithin 6% lecithin ash 2.39 ±0.29a 2.33 ± 0.12a 2.19 ± 0.35a 2.50 ± 0.41a moisture 69.59 ± 1.31a 69.64 ± 0.83a 67.30 ± 0.18a 67.91 ± 0.4a lipid 9.51 ± 0.08a 11.67 ± 1.36ab 13.21 ± 0.84b 12.23 ± 0.47ab protein 14.92 ± 0.47a 15.94 ± 0.60ab 16.69 ± 0.07b 16.32 ± 0.15ab (mean ± se), n=3 with different letters in each row, indicate the presence of significant differences between the experimental groups (p<0.05). table 3. proximate composition (%) of the whole body of juveniles mesopotamichthys sharpeyi fed the experimental diets for 56 days (wet weight %). 76 int. j. aquat. biol. (2015) 3(2): 72-77 alkaline phosphates is a metal enzyme that mainly presents in the brush border epithelial cells of the intestine and its activity related significantly lipid, glucose, calcium and inorganic phosphorus absorption (zhao et al., 2013). thus, alkaline phosphates is used for absorption of nutrients by larvae intestinal of vertebrates (zhao et al., 2013). in the present study, the specific activity of alkaline phosphates increased significantly with increase of dietary egg lecithin. this result is similar to those of sander lucioperca (zambonino infante and cahu, 2001) and european sea bass (d. labrax) larvae (cahu et al., 2003) that application of the phospholipids in the diet had been led to higher alkaline phosphates activity, which reflects rapid maturation of the intestine. also, it was reported the ratio between alkaline phosphates and cytosole enzyme (leucine aminopeptidase) is a maturation index of enterocytes (wold et al., 2007; hamza et al., 2008). the activity of lipase increased significantly by higher level of the dietary egg lecithin which is consistent with the findings of cahu et al. (2003) on sea bass (d. labrax) larvae. cahu et al. (2003) pointed out that regulation of lipase and phospholipase enzymes activity is controlled by genetic (mrna amount) and hormonal factors. increased levels of phospholipids and triglycerides in the diet induce increasing the rate of mrna translation of relevant enzymes and the other hormonal factors such as cholecystokinin in the regulation of enzymes activity. this result is similar to that of azarm et al. 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(2021) 9(4): 226-233 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article comparative study of plasma biochemical parameters in mature male and female goldfish, carassius auratus marzieh abbasi1, bahram falahatkar* 1,2, ali bani2,3, behroz heidari2,3 1fisheries department, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. 2department of marine sciences, the caspian sea basin research center, university of guilan, rasht, iran. 3department of biology, faculty of science, university of guilan, rasht, guilan, iran. s article history: received 3 june 2021 accepted 23 august 2021 available online 2 5 august 2021 keywords: biochemistry blood plasma reproduction carassius auratus abstract: blood biochemical parameters are important factors that can show the changes in health offering critical feedback on physiological condition of fish. in the present study, we examined a comparative study on the blood biochemical parameters in mature male and female goldfish, carassius auratus. plasma samples of 72 male and female goldfish that have been kept at 24°c for 30 days were analyzed and their biochemical parameters levels were determined. there were significant differences in all measured parameters between genders so that, maximum concentrations of the glucose (73.45±0.68 mg/dl) and calcium (8.32±0.05 mg/dl) were found in female fish, while the highest levels of the total protein (3.14±0.01 g/dl), cholesterol (281.65±3.19 mg/dl) and triglyceride (428.31±1.17 mg/dl) were measured in males. based on the results, the plasma biochemical parameters changes vary considerably between male and female goldfish. introduction blood biochemical parameters are valuable, effective and sensitive indices to monitor physiological changes of animals (satheeshkumar et al., 2012) that offer critical feedback on state of body health and condition of aquatic organisms (edsall, 1999). recent attention has been given to the biochemical characterization of fish blood as an internal index because these parameters supplies valuable knowledge about internal organs and metabolic parameters of both wild and cultured fishes (edsall, 1999), thus they are used by fish physiologists. numerous studies have documented that several factors can influence blood biochemistry, such as diseases and toxic chemicals (silverira-coffigny et al., 2004), season (dawson, 1990), stress (morales et al., 2005), species (langston et al., 2002), temperature (magill and sayer, 2004), sexual maturity (hatami nasari et al., 2014) and sex (adel et al., 2016). fish sex and maturation influence blood parameters (gabriel et al., 2011) and sex differences is one of the most important factor that affects oscillation in blood *correspondence: bahram falahatkar doi: https://doi.org/10.22034/ijab.v9i4.1279 e-mail: falahatkar@guilan.ac.ir biochemical parameters (svoboda et al., 2001) and change the levels of various substances such as glucose, calcium, protein, cholesterol and triglyceride (rosety et al., 1992; pedro et al., 2005; asadi et al., 2006; zhang et al., 2014). on the other hand, plasma nutrient variations could be a result of gonad maturation and nutrient transportation to gonads by blood. for this reason, the levels of glucose, total protein, triglyceride and cholesterol are considered important indices of the internal milieu status of both sexes in teleosts. research on some species such as, caspian kutum, rutilus frisii (firouzbakhsh et al., 2013), pike, esox lucius (adel et al., 2016) and pikeperch, sander lucioperca (zakes et al., 2016) have revealed that biochemical parameters are affected by sex. determination of variation in biochemical analysis in male and female fish might provide some useful baseline information to enhance further studies on mechanisms of sex and effects of sexual processes. goldfish, carassius auratus, as a cyprinidae member, is widely distributed in the world and 227 int. j. aquat. biol. (2021) 9(4): 226-233 excellent laboratory model for study of the endocrine system and various experiments (popesku et al., 2008; munakata and kobayashi, 2010; blanco et al., 2018). in this study, we hypothesized that sex differences may exert changes on biochemical characteristics. in other words, the purpose of the present study was to characterize variations in blood biochemistry in both sexes of goldfish. materials and methods fish and sampling: this study was carried out on 72 mature goldfish (36 males and 36 females) supplied from agha-seyed sharif aquatic farm (guilan province, iran) in december 2018. the fish were transferred to the wet laboratory at the faculty of natural resources, university of guilan (sowmeh sara, guilan province, iran). they were maintained in a reservoir tank (150×50×50 cm) for a day before experiment, with a water circulation and filtration system. water temperature, dissolved oxygen and ph were 24±1°c, 7.5±0.4 mg/l and 7.5±0.3, respectively. the mean weight of mature male and female goldfish was 25.12±1.10 and 27.59±1.29 g and average length was 15.10±0.17 and 16.44±0.36 cm, respectively. experimental design: six 60 l aquaria were used for the experiment and 12 fish were introduced into each aquarium at ambient temperature of 24°c. the temperature was controlled by a thermostat (300 watt, aqua, tehran, iran). during the experiment, the fish were fed twice daily at a rate of 2% body weight (priestley et al., 2006; hafeez-ur-rehman et al., 2015) with a commercial carp pellet (faradaneh, shahrekord, iran; containing 35-38% crude protein; 48% crude fat; 7-11% ash; 5-11% moisture; 1-1.5% phosphorus; 4-7% crude fiber). fish were kept under 12l:12d light regime throughout the experiment. sampling: a total of 72 sexually mature goldfish (36 males and 36 females) were used in this experiment. two males and two females were sampled from each aquarium at days 0, 14 and 30. fish were anesthetized by 200 mg/l clove powder (abdulrahman et al., 2018), then the blood samples were quickly extracted from the caudal vein of each goldfish by a 2.5 ml heparinized syringe to measure the blood biochemical indices. blood samples were centrifuged for 10 min at 1500 g at 4°c (labofuge, heraeussepatch, germany) to plasma extraction, then they were stored at -80°c. the experiment was conducted in accordance by animal ethics handling (granstrom, 2003). to ensure the fish were mature, 6 males and 6 females at the initiation of the experiment were dissected and their gonads were removed and fixed in bouin's solution for histological analysis. biochemical analysis: blood glucose, calcium, total protein, cholesterol and triglyceride concentrations were measured using enzymatic colorimetric analysis based on morris and davey (2001). all parameters were measured using commercial available kits (pars azmun, karaj, iran) and spectrophotometer (uv2100 plus, unico, usa). optical absorption of samples was measured at 25°c for all parameters using spectrophotometry method. the absorbance of glucose, total protein, cholesterol and triglyceride were recorded at 546 nm and 570 nm for calcium. gonad histology: after 24 h of preserving of the samples in fixative solution, the gonads were dehydrated in alcohol, embedded in paraffin wax, sectioned at 5 μm thickness, stained with hematoxylin and eosin (h&e) (sanchez et al., 2011) and examined under light microscope. classification of gonad maturation stages was done according to criteria defined by brown-peterson et al. (2011). statistical analysis: data were examined for normality and homogeneity of variances using kolmogorov-smirnov and levene's tests, respectively. all data were analyzed by two-way and one-way anova test. sex and days of sampling were considered as independent variables and blood biochemical parameters were dependent factors. the accepted statistical significance level was p<0.05. the spss software (ver. 16.0, chicago, usa) was used for analysis and data are presented as the mean ± standard error (se). pearson correlation was used to detect any relationship between the analyzed parameters. results the blood biochemical variables in mature fish are 228 abbasi et al./ biochemical parameters in mature male and female goldfish shown in figure 1. variations between sex and days of sampling were found in the blood glucose, calcium, total protein, cholesterol and triglyceride values. there were significant differences in blood glucose, calcium, total protein, cholesterol and triglyceride between male and female sampled throughout the experiment (p<0.05; fig. 1; a-e). interaction between sex and day showed that males have higher concentrations of total protein, cholesterol and triglyceride, but the glucose and calcium levels in females were higher in all days. tables 1 and 2 show correlation coefficient of the blood plasma biochemical parameters of mature male and female, respectively. our results in mature male showed significant positive correlation of the triglyceride with cholesterol (table 1; r=0.946, p<0.05) and significant negative correlation of the triglyceride and cholesterol with glucose (r=-0.739, p<0.05; r=-0.821, p<0.05, respectively). pearson analysis revealed that there was significant positive correlation of calcium with cholesterol, total protein and triglyceride (rcholesterol = 0.957, rtotal protein = 0.904, rtriglyceride = 0.889, p<0.05) in female. furthermore, in mature female, there was a significant positive correlation of the triglyceride with cholesterol (r =0.889, p<0.05) and total protein (r= 0.886, p<0.05). figure 1. glucose (a), calcium (b), protein (c), cholesterol (d) and triglyceride (e) levels in mature male and female goldfish (carassius auratus) in day 0, 14 and 30. values are reported as mean±s.e.; n=12 fish for each group, m=male, f=female. numbers with different superscripts indicate significant differences among treatments (p<0.05). 229 int. j. aquat. biol. (2021) 9(4): 226-233 a significant negative correlation between the cholesterol, calcium, total protein and triglyceride with glucose (rcholesterol=-0.819, rcalcium= -0.797, rtotal protein=-0.796, rtriglyceride=-0.774, p<0.05) were recorded in female mature goldfish. histological examination of the female and male gonads showed the vitellogenesis (vitellogenic oocyte in the ovary) and spermatogenesis (spermatids in testis) as main characteristics corresponding to the maturation stage, respectively (fig. 2). discussion exogenous and endogenous factors such as diseases (chen et al., 2005), sex (adel et al., 2016) and reproduction (suljević et al., 2017) could induce major changes in the blood compositions. in the present study, we found female’s goldfish have higher concentrations of the glucose which is in consistent with previous studies on beluga sturgeon (huso huso) (asadi et al., 2006), atlantic bluefin tuna (thunnus thynnus) (percin and konyalioglu, 2008) and sterlet sturgeon, acipenser ruthenus (akhavan et al., 2016). glucose plays a key role during the reproductive period and is an important fuel for metabolism (gharaei et al., 2011). glucose levels vary considerably between species, sex, age, nutritional, size, sexual maturity and reproductive status (mcdonald and milligan, 1992). in our study, table 1. correlation of blood plasma biochemical parameters in mature male goldfish, carassius auratus. parameters glucose calcium total protein cholesterol triglyceride -0.739* 0.005 -0.278 0.946* cholesterol -0.821* -0.125 -0337* total protein 0.356* 0.202 calcium 0.119 table 2. correlation of blood plasma biochemical parameters in mature female goldfish, carassius auratus. parameters glucose calcium total protein cholesterol triglyceride -0.774* 0.889* 0.886* 0.889* cholesterol -0.819* 0.957* 0.926* total protein -0.796* 0.904* calcium -0.797* figure 2. histological sections of testis (a) and ovary (b) stained with hematoxylin & eosin in goldfish, (carassius auratus) in day 0, 14 and 30. for male and female: scale bar 30 µm, 20 × magnification; scale bar 200 µm, 40 × magnification, respectively. arrows in a and b represent spermatid (included possess dense nuclei) and vitellogenic oocyte (included lipid deposition), respectively. 230 abbasi et al./ biochemical parameters in mature male and female goldfish increased plasma glucose level in female goldfish might be a consequence of the gonad development and vitellogenesis processes because during vitellogenesis and ovary development glucose demand increase for energy supply and oocyte growth. in vitellogenesis, the hepatic glucose production increase due to glycogenolysis. glycogenolysis is the process of glycogen breakdown to glucose-1-phosphate and glycogen, an important strategy to increase the blood glucose levels (hemre et al., 2002). in the present study, the glucose in female was higher in day 0 compared with days 14 and 30. it seems that during ovary development, the glucose is utilized for energy supply of vitellogenesis process or stored in oocyte (akhavan et al., 2016). changes in the blood plasma calcium of fish species occur as a result of physiological parameters such as sex, development, sexual maturity, diseases and etc. (suljević et al., 2017). in the present study, interaction of sex and day showed that the highest level of calcium was found in female. moreover, the calcium levels have significantly increased in female in all days. baghizadeh and khara (2015) in common carp (cyprinus carpio) and adel et al. (2016) in pikeperch showed significant differences in the calcium concentrations between genders, and noted that females exhibited higher levels of calcium. the plasma calcium in female fish exhibits changes during the ovarian maturation (sumpter, 1985; tyler and sumpter, 1990) and during this phase, the plasma calcium displays a variation and increases (srivastava and srivastava, 1994), whereas in male, plasma calcium levels show no change at different stages of the gonadal maturation (balbontin et al., 1978). calcium is essential mineral for vitellogenesis; vitellogenin binds by calcium and deliver in oocyte. there is a positive correlation between the concentrations of calcium and ovarian stages in female fish but calcium levels have no changes in male during spermatogenesis (patino and sullivan, 2002). based on the results, interaction of sex and day showed that in all of days, and the blood plasma total protein increase in male compare to female goldfish. these results were in agreement with adel et al. (2016) in pikeperch. plasma total protein increased during the vitellogenesis as it is necessary for vitellogenin forming and increasing demand of protein is controlled by several sex hormonal mechanisms in this process (akhavan et al., 2016). in the present study, the accumulation of the protein in oocyte caused decreasing the total protein in female. however, due to the short time of the experiment, further studies are required to determine their reasons. the results also showed that the plasma cholesterol and triglycerides increase in male in day 30 compare with female in day 0. lund et al. (2000) in striped bass (morone saxatilis), yeganeh (2010) in common carp, zakes et al. (2014) in pikeperch and adel et al. (2016) in pike have revealed that cholesterol values significantly increase during maturation and reproductive cycle. in the current study, we observed cholesterol and triglyceride concentrations in females were lower than males. these finding may be caused by changing in physiological condition as a result of the oocyte maturation. the reason for the decrease of the plasma cholesterol in female is unknown, although it seems that the use of cholesterol for steroid synthesis, especially estrogenic hormones for vitellogenesis may be an explanation of the reduction in the lipid levels measured in female compared to male fish (kusakabe et al., 2009). because cholesterol is main precursor to the steroid hormones biosynthesis and its levels may change during the reproductive cycle between genders (mommsen et al., 1999; tokarz et al., 2015). in addition, cholesterol and triglycerides account important compounds during the gonadal development (firouzbakhsh et al., 2013). decrease in concentrations of the plasma cholesterol and triglycerides in the mature female may be due to the lipid mobilization towards oocytes for increased growth and prerequisite for gonadal development (young et al., 2004). in conclusion, the present study revealed that biochemical indices were different between male and female goldfish. glucose and calcium increased in females because females have more demand of the glucose and calcium than males during ovarian development. decrease of the blood total protein in https://www.scielo.br/scielo.php?script=sci_arttext&pid=s0102-09352016000501251#b11 231 int. j. aquat. biol. 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(2019) 7(2): 106-111 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article pectinase enzyme from streptomyces coelicoflavus gial86 isolated from meyghan salt lake, arak, iran ensieh salehghamari*†1, zohre nasrollahzadeh†1, mohammad tahmaseb1, mohammad ali amoozegar2 1department of cell and molecular sciences, faculty of biological sciences, kharazmi university, tehran, iran. 2department of microbiology, faculty of science, university of tehran, tehran, iran. s article history: received 1 august 2018 accepted 20 april 2019 available online 2 5 april 2019 keywords: actinomycete halotolerant pectin methyl esterase pectate lyase poly galactoronase abstract: aquatic saline ecosystems are suitable environments for isolation of microorganisms with high diversity and widely used biotechnology features. the pectinase enzyme is one of the most important commercial enzymes with high potential in food and pharmaceutical industries. therefore, the discovery of microorganisms with new characteristics has always been a focus of research. as such, a pectinase producing actinomycete streptomyces coelicoflavus gial86 was isolated from meyghan salt lake of arak located in markazi province of iran. this strain was screened among 35 isolates of halotolerant actinomycetes with the highest production of pectinase enzymes. it was also found that production of pectate lyase, pectin esterase and polygalacturonase increased simultaneously with the logarithmic growth of the strain and its maximum production is at the time of stationary phase beginning. also, some actinomycete strains with more pectinase activity were identified by molecular identification and their phylogenic relationships were investigated. introduction pectinase enzyme is commonly referred to a group of enzymes that play a role in pectin biodegradation (dai et al., 2018). pectins are high molecular weight polysaccharides composing of α-1→4 linked d galacturonic acid residues with a few rhamnose residues in the main chain and arabinose, galactose and xylose on its side chain (rangarajan et al., 2010). pectinase enzymes are included three enzymes viz. poly galacturonase, pectine sterase, and pectate lyase based on their role on the pectin degradation (khatri et al., 2015). pectinase enzymes are extensively used in food, cosmetic and pharmaceutical industries (jayani et al., 2005; mohandas et al., 2018). as extraction of medicinal plants seeds by chemical solvents are harmful to human health, enzymeassisted extraction is a green extraction technique. pectinase enables the release of cell components with cell wall degradation (kontiä, 2016). pectinases are produced by different organisms, including bacteria, fungi, plants and insects. microbial *correspondences: ensieh salehghamari doi: https://doi.org/10.22034/ijab.v7i2.481 e-mail: esaleh@khu.ac.ir these authors are equally contributed to this article. pectinases are superior to other organisms, due to genetic manipulation ability. although fungal pectinases have become more widely used in the industry, actinomycete pectinases have high industrial potential (kumar and suneetha, 2015). actinomycetes belongs to a high gc class of gram-positive bacteria of prokaryote and produce different industrially useful extracellular enzymes like cellulase, xylanase and pectinase. as they are efficient degraders of plant debris, these enzymes are valuable for food and pharmaceutical industries. also, actinomycetes have a high biotechnological significance in recent years due to the production of valuable metabolites produced by its various genera (ballav et al., 2015). however, many of the rare genera of actinomycetes have not been discovered for their biotechnological products. studies on the extreme aquatic environments can be useful to produce novel enzymes form their microorganisms. the present investigation focuses on production and analysis of pectinase enzymes of halotolerant actinomycetes isolated from 107 int. j. aquat. biol. (2019) 7(2): 106-111 meyghan saline lake and phylogenetic relationships of the strains. materials and methods bacterial strains and culture medium: the meyghan lake, locating 15 km north east of arak, markazi province of iran. a total of 35 actinomycete strains were received from iranian biological resource center (ibrc), including those of meyghan lake. in order to revive and maintenance of actinomycetes, the salt free isp2 medium was used (salehghamari and najafi, 2016). then, they were cultured and incubated at 28°c for 5 days. pure cultures of each strain was streaked on slants of isp2 media and stored at 4°c for further study. screening of pectinolytic activities: the actinomycetes strains were assayed for their pectinolytic activity in agar plates containing pectin as a sole carbon source. halos of hydrolysis were detected using i2/ki (0.3% i2 and 0.6% ki) (soares et al., 1999; aaisha and barate, 2016) in a solid medium containing (g/l): pectin 10, yeast extract 1, and bacteriological agar 15. the plates were incubated at 28°c for 5 days. clear zone formation due to pectin hydrolysis was evaluated around colonies. several pectinolytic strains were isolated, and one strain was selected for a larger clear zone of hydrolysis for further investigation. enzyme production and medium condition: in order to measure the selected pectinase enzyme, 1 ml of 0.5 mc farland suspension of the selected strain was inoculated into 100 ml of production medium containing (g/l): pectin 10, yeast extract 5 and peptone 5 (saoudi et al., 2015) and incubated at 28°c for 72 hours and 180 rpm. the culture medium was centrifuged at 4000 g for 10 min to precipitate mycelia and the suspension was used for further enzyme study. growth curve of the candidate was determined at the same production medium and condition by bradford protein assay (burgess and deutscher, 2009). enzyme assays, pectin methyl esterase, pectate lyase and poly galactoronase: pectate lyase activity was assayed by measuring the absorbance at 235 nm (collmer et al., 1988). 0.05 ml from supernatant containing enzyme was added to a reaction mixture consisting of 0.001 ml of 0.01 m tris–hcl buffer at ph 7, supplemented with 0.02 ml pectin 10% (w/v). one unit of enzyme activity was defined as the amount of enzyme required to produce unsaturated oligogalacturonides equivalent to 1 μmol/min under standard assay conditions. pectin methyl esterase activity (u) was determined as the enzyme amount which releases 1 μmol of methanol from pectin per min at ph 7 and room temperature. 1 ml from supernatant was added to a reaction mixture consisting 0.5 ml of 3 mm potassium phosphate buffer at ph 7, supplemented with 2 ml pectin 10% (w/v) and 0.15 ml bromothymol blue 0.01% (w/v). the enzyme activity was assayed by measuring the absorbance at 620 nm (hagerman and austin, 1986). poly galacturonase activity was determined by quantifying the amount of reducing groups expressed as galacturonic acid units, liberated during the incubation of a mixture of 0.5 ml of 10% pectin, 0.25 ml of 3 mm potassium phosphate buffer at ph 7, 1 ml dinitrosalicylic acid 3 mm and 0.5 ml of culture supernatant at 100°c for 15 min. the absorbance was measured at 540 nm (miller, 1959). one unit of poly galacturonase activity (u) was defined as the amount of enzyme that liberates 1 μm of galacturonic acid per min under standard assay conditions. dna extraction, 16s rdna amplification and phylogenetic analysis: some of the actinomycete strains with the best pectinase activity were grown for 4 days at 28°c and 200 rpm in isp-2 medium. strains biomass were precipitated at 4000 rpm for 10 min and washed twice with 10% sucrose. about 200 mg of pellets were used for dna extraction as described by kieser et al. (2000). all of the 16s rdna genes were amplified using primers 9f (5´ aagagtttgatca tggctcag 3´) and 1542r (5´ aggaggtgatcc aaccgca 3´). the reaction mix (25 μl) contained 1 μl of genomic dna, 25 μl of taq master mix (2x), 0.5 mm of each primer and 5% dmso. the reaction was started with an initial denaturation at 96°c for 300 second followed by 30 cycles of denaturation at 96°c for 30 second, annealing at 58°c for 30 second and extension at 72°c for 60 second, with a final extension 108 salehghamari et al./ pectinase enzyme from s. coelicoflavus isolated from meyghan salt lake, iran at 72°c for 300 second. the pcr products were purified and analyzed by macrogen inc. (seoul, korea). to identify phylogenetic neighbors and calculate pairwise 16s rdna sequence similarities, the eztaxon server (http://www.ezbiocloud.net/eztaxon) was used. to align the sequences, clustalx software (version 2.0, conway institute, usa) was used. phylogenetic tree was constructed by the neighborjoining method using mega software (version 6.0, biodesign institute, usa). the bootstrap was calculated from 1,000 replicates. results screening of pectinase producing strains: in the current study, 35 actinomycetes isolates were screened for their pectinase activities. fifteen isolates were shown the large clear zones of hydrolysis (fig. 1) and they were selected for further assay to produce pectinase in liquid culture. to select the candidate, the fifteen isolates were cultured into liquid medium to produce pectinase and after five days they were assayed for pectin methyl esterase activity. pectinase enzyme activity and growth curve: among pectinase producing strains, an actinomycete strain, called strain gial86 displayed the highest pectin methyl esterase activity (0.03 u/ml) after 5 days of incubation and therefore was selected for further assay of pectinase enzymes. to find the best time of methyl esterase production during the strain gial86 life time, the production of this enzyme was monitored along with its growth curve for 8 days. as shown in figure 2, the production of pectin methyl esterase enzyme increases simultaneously with the logarithmic phase, and its maximum activity is on the fifth day at the end of log phase. pectate lyase and polygalactoronase activity of strain gial86 were also assayed during 8 days. to determine production phase of these enzymes, enzyme activities were measured during growth curve of strain gial86 (figs. 3, 4). the production of pectate lyase and polygalactoronase were increased simultaneously figure 1. some of the strains exhibited the large clear zone of pectin hydrolysis. figure 2. pectin methyl esterase and growth curve of gial86 isolated strain. the empty circle is pectin methyl esterase and full circle is biomass absorbance. figure 3. pectate lyase activity and growth curve of gial86 isolated strain. the empty circle is pectate lyase and full circle is biomass absorbance. 109 int. j. aquat. biol. (2019) 7(2): 106-111 with the logarithmic phase. pectate lyase maximum activity was monitored on the seventh day and the maximum activity of polygalactoronase was measured on sixth day at the time of stationary phase beginning. bacterial identification and phylogenetic analysis: genomic dna of the 9 pectinase producing actinomycetes of meyghan lake were isolated and their 16s rdna genes were pcr amplified with specific forward and reverse primers. based on the 16s rdna gene sequence, meyghan pectinase producing actinomycetes shared high levels of sequence similarity with these genus, streptomyces and nocardia. they were more belong to genus streptomyces (80%) and lesser to nocardia (20%). isolate gial86 shared the high level of sequence similarity (100%) with the species streptomyces coelicoflavus ab(184650). dna sequence of gial86 strain was deposited in genbank under accession number mh685384. the dna sequence of other strains also were deposited in genbank under accession numbers mk918501, mk418651, mk418652, mk418653, mk418654, mk418655, mk418656 and mk418657, respectively. phylogenetic trees based on the 16s rdna sequences constructed with the nj method (fig. 5). discussions iran is among the countries where ecosystems, especially saline lakes, are found to be abundant, so that many arid and semi-arid regions of the country, including the central regions of iran, have salty soil and water. one of these lakes, which is the origin of halophilic or halotolerant microorganisms with high biodiversity is meyghan lake. these microorganisms, and in particular actinomycetes, are highly capable of producing enzymes resistant to extreme environmental conditions such as temperature, ph and high salt concentrations, which are of great value in the field of biotechnology (amoozegar et al., 2008). in this study, the bacterial strains were subjected into agar plate and submerged fermentation to identify potent isolate with highest enzyme activity. therefore, the production of pectinase in 35 actinomycetes of maygan lake was evaluated and 48% of them were pectinase producer. in 2009, rohban et al. (2009) were able to screen 28 pectinase producing bacteria (12%) out of 231 halophilic strains isolated from hoz_e soltan lake (rohban et al., 2009). in 2013, in an area of india, 10 strains of streptomyces isolated and all of them were a pectinase producer (arijit et al., 2013). oumer and abate (2018) determined 95 isolates from ethiopia with 35.5% of them having pectinase activity. at the present study, quantitative assay of s. coelicoflavus gial86 pectinase was shown that all three enzymes were produced simultaneously with the logarithmic growth of the bacterium and eventually stabilized during stationary phase which this determined that production of pectinase is affected by fermentation time. similar results were obtained with the other pectinase producing bacteria. some researchers also reported that pectinase in bacillus sp. is produced concomitantly with its growth curve (roosdiana et al., 2013; joshi et al., 2011). this coincidence also found between fungi producing pectinase. xia et al. (2009) indicated that the trend of the growth curve of aspergillus niger and the pectinase activity curve coincides in the exponential phase, which can be explained that the enzyme amount may change along with the growth of biomass. finally, due to the molecular identification of actinomycete producing pectinase, it was found that these bacteria are of the two genera, including streptomyces and nocardia. different industries figure 4. poly galactoronase activity and growth curve of gial86 isolated strain. the empty circle is poly galactoronase and full circle is biomass absorbance. 110 salehghamari et al./ pectinase enzyme from s. coelicoflavus isolated from meyghan salt lake, iran require high-temperature and a wide range of ph, since pectinases produced in streptomyces, have good resistance to high temperatures and extreme ph, so these enzymes are good for use in the industry (kumar and suneetha, 2015). for better enzymatic production, it is necessary to screen novel species of streptomyces from unique habitats. acknowledgements the authors gratefully acknowledge university of tehran and kharazmi for financial supports. references aaisha g.a., barate d.l. 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(2017) 5(5): 348-359 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article cholinesterase activity and histopatological changes in the mediterranean crab, carcinus maenas, exposed to environmental contaminants sana ben khedher*1, zohra houas2, hamadi boussetta1 1laboratory of biochemical and environmental toxicology, higher institute of agriculture, chott-mariem, 4042-sousse, tunisia. 2laboratory of histology cytology and genetics, faculty of medicine, monastir 5019, tunisia. article history: received 14 september 2017 accepted 24 october 2017 available online 2 5 october 2017 keywords: acetylcholinesterase chlorpyrifos-ethyl metals histopathological biomarkers abstract: marine environments are continuously being threatened by a large number of pollutants including heavy metals and organophosphorous pesticides from anthropogenic sources. these compounds can cause a serious environmental problem. the present study aimed: (1) to measure sensitivity of acetylcholinesterase (ache) activity to in vivo exposure to the organophosphorous chlorpyriphos-ethyl (cpf) and to the heavy metals cadmium (cd) and copper (cu) and (2) to use the histopathological lesions as tissue biomarkers for biomonitoring of different contaminations. the results clearly showed that the ache activity in different tissues (digestive gland, muscle and eyes) of carcinus maenas was relatively sensitive to the concentrations of cpf and tended to have different patterns in response to cd, cu and cu+cd mixture exposure. the transfer of treated crabs to the clean sea water allowed to recover totally or partially the lost activity depending on selected tissues and contaminant exposure (metals or organophosphorous compounds). histopathological biomarkers in c. maenas exposed to different contaminants showed the presence of different lesions which altered the digestive gland after 7 days of contamination. introduction the marine ecosystem is threatened by increasing levels of contaminants originating from anthropogenic activities (ali and sreekrishnan, 2001; chouksey et al., 2004). this situation endangers the health of organisms and human. among anthropogenic contaminants, pesticides are widely detected in freshwater and marine ecosystems. the organophosphates (op) and carbamates (cs) are modern synthetic insecticides and potent neurotoxic molecules (ghedira et al., 2009). these compounds reach the sea through rivers and lead to the contamination of different marine ecosystems (water and sediments) (mora et al., 1999). they can produce adverse effects on non-target aquatic organisms. the most widely used pesticide in agriculture to control insects and fungi is the op insecticide chlorpyrifos (cpf). because of its broad spectrum, cpf represents a threat to non-target species including aquatic organisms (botté et al., 2012). this toxicity has been demonstrated by numerous field and laboratory *corresponding author: sana ben khedher doi: https://doi.org/10.22034/ijab.v5i5.379 e-mail address: benkhedher.sana@yahoo.fr studies on temperate species (fulton and key, 2001). the mode of cpf action is cholinesterase (che) inhibition. chlorpyrifos induces irreversible che inhibition (fulton and key, 2001), triggering constant stimulation of the muscles which leads to paralysis and death (botté et al., 2012) and this at high doses of op exposure. an increasing number of studies provide evidence that che activities may be affected by a wide range of contaminants other than op and cs, including heavy metals (jebali et al., 2006; vioque-ferňandez et al., 2007; elumalai et al., 2007; bonacci et al., 2008). heavy metals such as cadmium and copper have the capability to induce harmful effects on living organisms at ecological relevant concentrations and have been considered as important environmental contaminants (cunha et al., 2007). copper and cadmium are of particular concern in marine ecosystem because several species are able to bioaccumulate and/or bioconcentrate them in the body tissues where they may reach toxic concentrations that 349 int. j. aquat. biol. (2017) 5(5): 348-359 cause deleterious effects (pereira et al., 2009). histopathological lesions are another environmental biomarker. however, whilst studies on finfish have shown that histopathology is a sensitive indicator of individual and population health status, and results from numerous controlled laboratory exposures of shellfish (crustaceans and molluscs) to toxicants have shown that histopathological changes also occur in the organ and tissue systems of these animals, relatively few field studies have included shellfish histopathology in the suite of employed monitoring tools (stentiford et al., 2005). in this study, we (1) measured the sensitivity of che versus ache activity to in vivo exposure to the ops (chlorpyrifos-ethyl) and heavy metals (cadmium and copper) and (2) to show the histopathological alterations after exposure to different contaminants. materials and methods chemicals: the following reagents were obtained from sigma-aldrich (villefranche, france): acetylthiocholine (asch) and 5,5’-dithio-2-nitrobenzoate (dtnb). crabs collection: crabs (carcinus maenas) were collected with hand at the kuriat island, which is an uncontaminated area (jebali et al., 2011) in september 2012. specimens (male) were immediately transported to the laboratory in aerated buckets filled with seawater. upon arrival at the laboratory, crabs were divided into 31 groups of 6 crabs and placed in plastic tanks with 10 l of natural sea water (36.89‰ salinity) and kept at 17.2°c. crabs were fed regularly with fish every three days during the acclimation and the exposure period. after 7 days of acclimation, crabs were used to study the kinetic effect of cd, cu and chlorpyrifos-ethyl pesticide (cpf) on crab ache activity and the histopathological biomarkers. in vivo effects of environmental contaminants: after 7 days of acclimation, groups of crabs were exposed to chlorpyrifos-ethyl (cpf) (4.8 μg/l) (gagnaire et al., 2008), cd (200 μg/l) (blasco et al., 1999), cu (200 μg/l) (roméo et al., 2006; vieira et al., 2009) and to their mixture cu+cd (200 μg/l cd+200 μg/l cu) for 0.5, 1, 2, 4 and 7 days (d); after each time, 6 crabs of treated and relative control groups were sacrificed and the main organs i.e. digestive gland, muscle and eyes, were carefully removed and frozen at -80°c until analysis. exposure to cpf, cd, cu and (cd+cu) was renewed every 3 days. chlorpyrifos-etyl is the active ingredient of pesticide dursban®, it was prepared by adding an organic solvent i.e. acetone. all treatments and controls received the same acetone concentration (0.01%) (tu et al., 2009). this acetone concentration is below the no-observed-effect concentration (noec) of 0.1% reported by mayer (1987). cadmium and copper were prepared by adding distilled water. in order to study the capacity of crabs to recover the ache activity; at the end of contamination period (7 day), six treated crabs of cpf, cu, cd and their mixture (cd+cu) groups were transferred into the clean water. after 7 days, crabs were sacrificed and the same organs were carefully removed and frozen at -80°c until analysis. biochemical determinations cell-free extract preparations: all steps for cell-free extract preparation were carried out at 4°c. organs (digestive gland, muscle and eyes) were homogenized in ice-cold phosphate buffer (100 mmol/l; ph 7.5; 1 mmol/l edta; 1 mmol/l reduced glutathione; gsh) at a rate of 3 ml/g (buffer volume/tissue weight). homogenates were then centrifuged at 9,000×g for 15 min. the supernatant of each sample was stored at – 20°c, for no longer than a week, until enzyme activity determination. total protein content in the supernatant (s9) was measured following the bradford method (bradford, 1976), at 595 nm, using bovine serum albumin as standard protein. ache activity to in vivo exposure to environmental contaminants: che activities were estimated by the method of ellman et al. (1961). acetylthiocholine (asch) was used as substrate for preliminary screening of che types in tissues. basal conditions in the reaction mixture (final volume 1150 µl) of the che assay were as follows: 100 mm phosphate buffer, ph 7.5; 50 µl of 8 mm dithiobisnitrobenzoate (dtnb) and 40 µl supernatant (enzyme solution). the reaction was started by adding 51.28 µl of 45 mm substrate (2 mm final concentration). 350 ben khedher et al./ che activity in carcinus maenas exposed to environmental contaminants the enzymatic reaction rate was quantified spectrophotometrically at 412 nm against a blank without substrate for each activity measurement. in order to subtract the spontaneous hydrolysis of substrate, a second blank was performed without sample. enzyme activity was recorded over 10 min after adding substrate. the ache activity was expressed as specific activity (nmol substrate hydrolysed/min/mg protein). histology: the digestive gland of the 6 crabs of treated and control groups sacrificed after 7 days of exposure to metals and cpf and after 7 days of recovery was removed and fixed in bouin’s fixative for 48 hrs. the preserved tissue was processed by a routine histological method; dehydrated in graded ethanol solution and embedded in paraffin. embedded tissues were cut into sections of 5 μm thickness by a rotary microtome (leitz wetzlar 1512). the thin sections of the digestive gland tissue were stained by trichrome masson and hematoxylin-eosin for observation by the light microscope. sections were photographed with a microscope (leica 132 dm 750) provided with a numerical camera (leica icc50 hd) and examined for lesions. statistical analysis: statistical analyses were performed using spss software. significant differences between means were determined using one-way anova followed by the duncan’s test. in order to determine kinetic parameters such as the apparent michaelis–menten constant (km) and the maximum substrate hydrolysis velocity (vmax), we used graphpad prism version 4 for windows (graphpad software). results in vivo effects of chlorpyrifos-ethyl on ache activity: during the seven days of experiment, no mortality was reported in controls. in vivo exposure to lower concentration of chlorpyrifos-ethyl (cpf) (4.8 μg/l) led to inhibition of ache activity in all tissues (fig. 1). a decrease of activity was not time dependent in any organ. in eyes, activity was decreased 29.46%. in muscle, the maximum decrease of activity was observed at 1 day (45.29%) and maintained lower than that of control. in digestive gland, the inhibition of ache activity reached its maximum at 7d (68.71%). figure 1. acetylcholinesterase activity measured in digestive gland (a), muscle (b) and eyes (c) of carcinus maenas exposed to chlorpyrifos-ethyl pesticide for 0.5, 1, 2 and 7 days then transferred to the clean sea water for one week (recovery period). for each condition n=6 and 3 repetitions for each extract. the results were expressed as the mean ± standard deviation. * = significant at p<0.05. 351 int. j. aquat. biol. (2017) 5(5): 348-359 after short time (7 days) of depuration, the digestive gland and the muscle were able to recuperate ache activity, while in the eyes, an increase of the ache activity observed as 42.77±1.06 nmol/min/mg protein. in vivo effects of cd, cu or their mixture on ache activity: ache activity in tissues (digestive gland, muscle and eyes) after in vivo exposure to cadmium, copper and their mixture for seven days is summarized in figure 2. che activity was affected by exposure to cd, cu and their mixture (cu+cd), but significant differences were observed compared to different metals. exposure to cd reduced ache activity in all tissues and the maximum reductions were observed at 7d (respectively 69.79% in digestive gland, 48.12% in muscle and 71.44% in eyes). in contrast to higher inhibition of ache activity in cd-treated crab, cu induced activity after 2 days (24.16±2.44 nmol/min/mg protein in muscle), then the activity figure 2. acetylcholinesterase activity measured in digestive gland (a), muscle (b) and eyes (c) of carcinus maenas exposed to cadmium, copper and their mixture for 0.5, 1, 2 and 7 days then transferred to the clean sea water for one week (recovery period). for each condition n=6 and 3 repetitions for each extract. the results were expressed as the mean ±standard deviation. different letters a, b, c and d indicated a significant difference between different treatments and relative controls at 0.05 confidence level. c cd cu cd + cu c op op b cells + +a +b + + + e cells + + + + + + f cells + + + + + + r cells + + + + + + abnormal lumen (alu) ++ +++ +++ +++ haemocytic infiltration in the interstitial sinus (hi) +++ +++ +++ +++ necrotic tubules of hepatopancreas (nt) ++ +++ ++ thickened basal laminae (tbl) ++ ++ ++ ++ coagulation in the thickened basal laminae (co) + walling off of the tubules by haemocytes around the thickened basal laminae + + ++ ++ reserve inclusion (ri) ++ ++ ++ ++ ++ ++ the following symbols were adopted to describe the lesions severity and structural changes: (-): no alteration, (+): low, (++): moderate and (+ + +): high alteration. a decrease of the number of b cells b cells with large vacuoles table 1. a semi-quantitative evaluation of the histopathological lesions in digestive gland of control carcinus maenas (c) and (c cpf) and crabs exposed to cadmium (cd), copper (cu), (cd+cu) mixture and chlorpyriphos-ethyl (cpf). 352 ben khedher et al./ che activity in carcinus maenas exposed to environmental contaminants 353 int. j. aquat. biol. (2017) 5(5): 348-359 decreased after 7days of exposure (16.2±2.62 nmol/min/mg protein). in digestive gland and eyes, different patterns in ache response were observed compared to muscle. the inhibition of ache activity was detected at 0.5d (16.77% and 48.53% in digestive gland and muscle, respectively), 1 day (22.7% and 51.89% in digestive gland and eyes respectively) and 2d (40.79% and 53.89% in digestive gland and eyes respectively) of cu-exposure, decreased to become critical at 7d and the activity was estimated at 45.5% and 30.96% of control in digestive gland and eyes, respectively. in addition to the observed ache activity increase in cu-treated crab, the exposure to mixture of cd and cu caused pronounced increases after 0.5d and 1d. compared to control, i observed a significant increase of ache activity in muscle at short exposure times (0.5d and 1 d) and then a high decrease at long exposure times (43.77% and 55.82% of ache activity were lost after 2 and 7 days, respectively). in digestive gland, an inhibition of ache activity was observed throughout the period of contamination which reaches its maximum after 7 days (75.03%). in eyes, a critical decrease of activity was observed at the early time of the exposure (0.5d; 70.39%). the mixture of cd and cu causes a rapid decline in ache activity at 0.5d which is maintained throughout the entire exposure. the transfer of contaminated crabs into the clean sea water for one week allowed to recuperate the totally lost activity in muscle of cd and cu-treated crab. in eyes, ache activity was partially recovered of cdand cu treated crabs. in mixture (cd+cu) treated crab, a partial ache activity was recuperated in selected organs. thus, 44.72%, 16.41% and 19.98% were recuperated in digestive gland, muscle and eyes organs, respectively. histopatogical lesions of crabs: we examined the digestive gland of crabs after 7d of exposure to metals and cpf and after 7 days of recovery. the digestive gland of control group (figs. 3a, b, c, d, q, r, s, t) showed the typical organization of glandular tubular structure as normally. it is composed of branched tubules and of different types of epithelial cells lining the tubules. the cells are embryonic (e) cells, fibrillar (f) or “dark” cells, restzellen (r) or “light” cells and blasenzellen (b) or extrusion cells (fig. 3b). the lumen (lu) of each tubule was found to have a ‘star’ like appearance (figs. 3b, d, h, q). the interstitial sinuses between tubules are normal (fig. 3a). the digestive gland exposed to metals (cd, cu and mixture) and cpf exhibited an abnormal lumen (alu) (figs. 3f, n, v) and haemocytic infiltration (hi) in the interstitial sinus (is) (figs. 3n, r, u). other pathological lesions were also observed in crabs exposed to contaminants in laboratory. in fact, we observed necrotic tubules (nt) (fig. 3e) of the digestive gland containing tissue debris (td) in the lumen (fig. 3j). the thickened basal laminae (tbl) (figs. 3i, u) and the walling off of the tubules by haemocytes (hc) around the thickened basal laminae (fig. 3n) were abundant in crabs exposed to mixture (cd+cu). the coagulation (co) in the thickened basal laminae was observed only in digestive gland of crabs exposed to mixture (cd+cu) (fig. 3m). we observed also the presence of four types of cells (b, e, f and r) in crabs exposed to metals and cpf. it was noted that the number of b cells in crabs exposed to cd were less than that those of digestive gland of control crabs and the b cells are vacuolated in crabs exposed to cu (table 1). however, we exhibited the presence of reserve inclusion (ri) in control group and those exposed to metals and cpf (figs. 3a, e, i, m, u). figure 3. digestive gland histopathology of carcinus maenas after 7 days of contamination and 7 days of recovery. a-d (control of metals) and q-t (control of cpf) typical organization of the digestive gland of control c. maenas. arrangement of digestive gland tubules and its cells. distal tubule tips with e cells (embryonic cells), b cells (blasenzellen cells), r cells (restzellen cells) and f cells (fibrillenzellen cells). the interstitial sinuses (is) between tubules were normal (a, c bar=x10; b, d bar=x40). e-p and u-x alterations in the histoarchitecture of the digestive gland of c. maenas exposed to cadmiuim (cd) e-h, copper (cu) i-l, cd+cu m-p and cpf (u-x). formation of abnormal lumen (alu) (f, n, v bar=x40) and haemocytic infiltration (hi) in the interstitial sinus (is) (n, r, u bar=x40). necrotic tubules (nt) (e bar=x10) of the digestive gland containing tissue debris (td) in the lumen (j bar=x40). the thickened basal laminae (tbl) (i, u bar=x10) and the walling off of the tubules by haemocytes (hc) around the thickened basal laminae (n bar=x40).the coagulation (co) in the thickened basal laminae (m bar=x10). the presence of reserve inclusion (ri) (a, e, i, m, u bar=x10) in control and exposed crabs. 354 ben khedher et al./ che activity in carcinus maenas exposed to environmental contaminants we noted the intensity of each histopathological alteration in digestive gland of crabs after 7 days of exposure to contamination in table 1. the results revealed that abnormal lumen (alu), haemocytic infiltration in the interstitial sinus (hi) and thickened basal laminae (tbl) were important histopathological lesions of the crabs exposed to metals and cpf. the study of the histology of crabs after 7 days of recovery showed that crabs return to their normal structure. it was noted the absence of alterations in digestive gland of this group (figs. 3g, h, k, l, o, p, w, x). discussion the measurement of the biological effects of toxicants have become of major importance for the assessment of the quality of the environment. the use of biochemical markers has been proposed as sensitive "early warning" tools for biological measurement effect (van der oost et al., 2003). the inhibition of acetylcholinesterase (ache) activity has been used widely as a biomarker of exposure to organophosphorous pesticides (ops). sensitivity to pollutants: in vivo exposure to priorities pollutants: crabs have often been proposed as possible bioindicators of marine pollution, but very few data regarding the effect of priorities pollutants such as heavy metals and organophosphorous on che activity in selected tissues are available. the present study is focused on enzyme sensitivity to exposure in vivo to pollutants, with the aim of validating che in tissues of c. maenas as a useful tool for monitoring environmental quality and exposure to pollutants in marine environments. the exposure to 4.8 μg/l of cpf caused a significant decrease of ache activity in main organs of crabs (muscle and eyes). similary, after 96 hrs, muscle che activity was significantly inhibited by 49% when fish (acanthochromis polyacanthus) were exposed to 10 μg/l of cpf (botté et al., 2012). varό et al. (2003) showed an important inhibition (76%) in nervous system in the fish (dicentrarchus labrax) exposed to 1 mg/l of dichlorvus after 96 hrs. very strong ache inhibition was observed in the digestive gland of blue mussel (mytilus trossulus) exposed for 2 days to 100 μg/l of dichlorvos (kopecka-pilarczyk, 2010). in addition, the transfer of contaminated crabs to the clean sea water for a short period (7 days) allowed to a rapid recovery of the ache activity. in fact, recovery is defined as a significant increase in the ache activity that occurs following cessation of exposure to anti-cholinesterase agents (kumar et al., 2010). several studies with shrimp, crab and lobster species have shown that ache inhibition in the animals still occurred days after exposure had ended (mchenery et al., 1991; abdullah et al., 1994). recovery from the effects of op compounds is both chemical and species specific (zinkl et al., 1991). de la torre et al. (2002) noted that the inhibition of fish brain ache can be detected soon after the beginning of "in field" exposure to ops but the time required for recovery of basal values after transfer fishes to unpolluted media can take several weeks. similarly, kumar et al. (2010) showed that recovery of ache activity following reduction in exposure to ops has been found to be a process that takes time, depending on factors such as the type of insecticide, tested species and the extent of depression of ache. indeed, organophosphorus compounds are generally irreversible inhibitors because the dephosphorylation rate of the bound enzyme proceeds at an insignificant rate. therefore, the inhibitory effects of op exposure may be long lasting, with recovery depending on new enzyme synthesis (habig and digiulio, 1991). when levels are depressed more, a greater amount of enzyme must be produced. some ops, including chlorpyrifos, can also be metabolically altered to more active ache inhibitors as an oxon analogue (kumar et al., 2010). regarding effects of the heavy metals such as cd, cu and their mixture on ache activity, it was dependent of investigated tissue and time of exposure. indeed, cd is a heavy metal commonly used in environmental studies because it is highly toxic (lane and morel, 2000), widely distributed in the environment and can adversely affect the organisms at relatively low exposure concentrations (waisberg et al., 2003; banni et al., 2009). the neurotoxicity effect of cd illustrated in many in-field and in-vivo studies used invertebrate organisms as bioindicators (roméo 355 int. j. aquat. biol. (2017) 5(5): 348-359 et al., 2006). the negative cd-effect with time of exposure on che activity in all tissues was clear. similary, jebali et al. (2006) showed inhibition of ache in fish seriola dumerilli after exposition of 100 and 250 μg/kg of cd. the copper is a trace element that plays a fundamental role in the biochemistry of organisms, including aquatic organisms that can take it up directly from water (grosell et al., 2003). however, it can become toxic at high concentrations (alquezar et al., 2008; vieira et al., 2009). in several aquatic animals, ache and other cholinesterases (che) have been found to be inhibited by copper in vivo conditions (roméo et al., 2006; elumalai et al., 2007). in fact, ache activity in hexaplex trunculus is decreased by exposure to copper in the digestive gland after 2d (roméo et al., 2006). however, no significant effect on ache was observed in the brain of seabream sparus aurata fingerlings exposed for 1 day to sublethal concentrations of copper sulphate (varó et al., 2007; frasco et al., 2008) and increases of ache activity were also reported in specimens of s. auratus exposed to sublethal copper concentrations for 20 days (romani et al., 2003). in the present study the results of in vivo exposure to cu were much more difficult to interpret, as significant enhancements of ache activity were detected in muscle tissues, at the early time exposure (2d) and dramatic repression at 7d. the study of romani et al. (2003) showed that the copper has been found to induce ache activity and catalytic efficiency in white muscle and brain tissues of s. aurata fishes exposed to cu without accumulation of metals. these results suggest an increase of free cu aliquot into the cells, likely due to mechanisms of metal homeostasis. similarly, cunha et al. (2007) showed an increase of ache activity in the gastropod nucella lapillus exposed to 44 mg/l cu, an increase which may be due to direct interaction of metal with the enzyme. the exposure to cu+cd led to a high increases in ache activity (23.88±1.75 nmol/min/mg protein in muscle) at 0.5d, then decrease time-dependent. this results support that cu is an essential metal and may allow a competitive effect with cd on ache activity and thus, the enhancement ache activity by cu exposure may cover or/ prevent the negative effect of cd. to sum up, our results clearly show that ache activity in different tissues of c. maenas tends to have different patterns in response to in vivo exposure to cd, cu and cu+cd. in eyes tissue, the ache activity was highly affected by cd, cu, and more critical affected by their mixture. this may be of particular concern for eyes, the primary organs of sensory and in contact with external environment, where the highest susceptibility was observed. as regard sensitivity of investigated tissues to heavy metals, eyes are the most sensitive tissue where the risk of neurotoxicity is therefore higher. during the depuration period, the ache activity was totally recuperated in muscle tissues of cu or cdtreated crab and partially in the mixture (cd+cu) treated crab. this increase of ache activity can be explained by the biochemical and physiological mechanisms such as increases of metallothionein synthesis, storage in lysosomes compartment and excretion of metals absorbed and distributed in their tissues and synthesized new molecules of ache (serafim and bebianno, 2009), so these animals are found in their normal physiological state. furthers studies focused on the kinetic uptake of metals and metallothionein responses in a longer depuration period of c. maenas are needed to confirm the advanced hypothesis. others studies on bivalve, showed that a short period of depuration no longer than 10 days was sufficient for the animal to return to its normal physiological state after exposure to cu and this depend in concentration exposure (gnassiabarelli et al., 1995; serafim and bebianno, 2009). histopatogical lesions of crabs: studies on crustacean health status have focused on the response of individual organ systems to laboratory exposure to a range of contaminants (victor, 1993, 1994; soegianto et al., 1999 a, b; bhavan and geraldine, 2000). the relative ease at which a holistic assessment of health can be made using histopathology and the suitability of these species as environmental sentinels provide support for the inclusion of crustaceans as indicators 356 ben khedher et al./ che activity in carcinus maenas exposed to environmental contaminants of aquatic environmental health. in fact, the exposure to a noxious chemical, such as pesticide, would be reflected in alterations in the structure of the tubules and epithelial cells. the essential metals, like copper, can also produce toxic effects when the metal intake is excessively elevated (wagner and boman, 2004; turkmen and turkmen, 2005). cadmium, nonessential metal, known by its purely toxic effect on the marine organism produces a general state of stress in contaminated organisms (olabarrieta et al., 2001; geffard, 2001). the crustacean digestive gland is a sensitive organ and liable to injury by pesticides and other water-born pollutants (baticados and tendencia, 1991; bhavan and geraldine, 2000). the digestive gland is essentially composed of branched tubules and of different types of epithelial cells (e-cells, r-cells, f-cells and b-cells) lining the tubules (ben khedher et al., 2014). several such structural alterations were noted in the digestive gland tubules of test crabs that had been exposed to cpf, cd, cu and cd+cu after 168 hrs in the present study. abnormal lumen, hemocytic infiltration in the interstitial sinuses and necrosis lesions were observed in crabs exposed to contaminants of this study. similar observations have been made in the freshwater prawn macrobrachium malcolmsonii exposed to endosulfan. the thickened basal laminae (tbl), another aspect of alterations, were also observed in crabs contaminated by cpf and metals in the present study. this thickening might have been due to production of collagenous fibers and melanin or due to the coagulation and walling off by hemocytes, as noted in penaeus monodon due to aflatoxin toxicosis (bautista et al., 1994); this thickening might also represent a defensive reaction against the toxicity of insecticides, a phenomenon noted in the tiger prawn p. monodon infected with vibrio harveyi (jiravanichpaisal and miyazaki, 1994). other distinct pathological alterations were also observed in crabs after 168 hrs of exposition to cpf and metals. we observed the walling off of the tubules by haemocytes around the thickened basal laminae and the coagulation in the thickened basal laminae (observed only in crabs exposed to cd+cu). these results were in accordance with those noted in digestive gland of c. maenas collected from bizerta lagoon (ben khedher et al., 2014). we showed also the presence of reserve inclusion (ri) in control and exposed c. maenas collected in september. in fact, ben khedher et al. (2014) observed the presence of ri cells both in control and polluted sites only in c. maenas collected in summer (july). the function of ri cells are likely associated with the synthesis and storage of haemocyanin and other products such as glycogen; these reserves are utilized during stressful periods such as during moulting, disease or hibernation and during normal reproduction (johnson, 1980; stentiford and feist, 2005; ben khedher et al., 2014). in the present study, we showed that after 7 days of recovery, crabs are not altered. in fact, we noted the absence of alterations in digestive gland of c. maenas. conclusions this work has clearly shown the ches activities in muscle and eyes tissues of the mediterranean crab c. maenas. the ache activity in different tissues (digestive gland, muscle and eyes) of c. maenas was relatively sensitive to the exposure of chlorpyrifosethyl and tends to have different patterns in response to exposure to cd, cu and cu+cd mixture. the present results showed that depuration experiments allow to recover totally or partially the lost activity in treated crabs depending in selected tissue and contaminant exposure (metals or organophosphorous compounds). further studies focused on the kinetic uptake of metals and ches activities responses in an increasing depuration period of c. maenas needs to be carried out to investigate the neurotoxicity effects of these compounds. to conclude, the exposure of c. maenas to metals and cpf allowed observing the histopathological alterations in digestive gland. it is imperative that contamination of the aquatic environment by metals and cpf (pesticide) should be prevented. acknowledgments the present study was financially supported by the ministry of scientific research and technology, 357 int. j. aquat. biol. 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(2014) 2(2): 58-68 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article alterations in biochemical parameters of the freshwater fish, alburnus mossulensis, exposed to sub-lethal concentrations of fenpropathrin mahdi banaee*1, antoni sureda2, fazel zohiery1, behzad nematdoust hagi1, daniela s. garanzini3 1aquaculture department, natural resource faculity, behbahan khatam alanbia university of technology, iran. 2laboratori de nutrició comunitària i estrès oxidatiu; department of biology science, balearic islands university, illesbalears, spain. 3laboratorio de ecotoxicología, instituto de investigaciones marinas y costeras (conicet/ universidad nacional de mar del plata), funes 3350 (b7602ayj) mar del plata – argentina. article history: received 11 december 2013 accepted 12 march 2014 available online 2 5 april 2014 keywords: fenpropathrin oxidative stress lipid peroxidation, biochemical parameters alburnus mossulensis abstract: fenpropathrin is a new pyrethroid insecticide used to control crop pests. the aim of this study was to evidence fenpropathrin-induced oxidative stress and alterations in biochemical parameters in the freshwater fish, alburnus mossulensis. total antioxidant capacity, malondialdehyde (mda), catalase activity (cat), aspartate aminotransferase (ast), alanine aminotransferase (alt), alkaline phosphatase (alp), lactate dehydrogenase (ldh), creatine phosphokinase (ck), acetylcholinesterase (ache) in the whole body extract were measured in a. mossulensis after exposure to sub-lethal concentrations of fenpropatrin (approximately equal to 1, 2, 3, 5 and 10% of 96 h lc50) for 15 days. the 24, 48, 72 and 96 h lc50 of the fenpropathrin for a. mossulensis was 562.28±45.19, 218.18±18.75, 136.18±11.90 and 121.38±11.84 µg/l at 24±2 °c. exposure to 2.75, 5.50 and 12.6 µg/l fenpropathrin significantly increased ast activity in fish. a significant increase in the alp and ldh activities was observed in fish after a 15 day exposure to 1.25, 5.50 and 12.60 µg/l fenpropathrin. fenpropatrin significantly induced lipid peroxidation and increased mda levels in fish. compared with the control group, total protein levels in fish decreased after exposure to 2.75, 5.50 and 12.60 µg/l fenpropathrin on day 15. total antioxidant capacity, ache and cpk activities in fish exposed to fenproparthin were significantly lower than control group. there was a significant increase in the cat and alt activities in fish after exposure to 5.50 and 12.60 µg/l fenpropathrin. in conclusion, fenpropathrin has the potential to disrupt biochemical parameters in a. mossulensis and to induce oxidative stress. introduction the use of pyrethroid insecticides has recently increased compared to organochlorine pesticides due to their high selectivity and lower persistence in the environment (goulding et al., 2013). fenpropathrin [2, 2, 3, 3tetramethylcyclopropanecarboxylic acid cyano (3-phenoxyphenyl) methyl ester] is a new pyrethroid insecticide used to control crop pests in tomatoes, tobacco, cotton farms and fruit orchards. pyrethroid insecticides enter surface water via air drift, leaching from agricultural land and surface runoff during or after the application of pesticides * corresponding author: mahdi banaee e-mail address: banaee@bkatu.ac.ir (gan et al., 2005; woudneh and oros, 2006; weston et al., 2009; goulding et al., 2013). although there is no report indicating the presence of fenproparthin in surface water and groundwater, the presence of other pyrethroid pesticides in sediment, surface water (amweg et al., 2006; holmes et al., 2008; feo et al., 2010), deltaic regions of big rivers (feo et al., 2010; weston and lydy, 2010) and estuaries (woudneh and oros, 2006) and agriculture wastewater (you et al., 2004) have been reported. pyrthroid pesticides are lipophilic compounds, which may be absorbed through gills, skin or 59 banaee et al./ int. j. aquat. biol. (2014) 2(2): 58-68 alimentary ducts. food poisoning with pyrethroids is another influential pathway of these pesticides on fish (macneale et al., 2010). after entering the body, pyrthroid pesticides can be accumulated in fat tissues (banaee, 2013). pyrthroid pesticides in animal's body are rapidly hydrolyzed in the body of the exposed animals and their metabolites excreted. carboxy esterases play an important role in the detoxification of these pesticides (sogorb and vilanova, 2002). reactive oxygen species (ros) produced during the detoxification process of pyrthroid pesticides are known as important agents that cause oxidative stress (ansari et al., 2011; eldemerdash, 2011). in addition, alterations in biochemical parameters (das and mukherjee, 2003; parvez and raisuddin, 2005), and hematologic factors (saxena and seth, 2002; muranli and güner, 2011), reproductive disorders (moore and waring, 2001), neurological disorders (soderlund et al., 2002), histopathological changes (velmurugan et al., 2007; korkmaz et al., 2009; kan et al., 2012), and mutations (ansari et al., 2011; beggel et al., 2011), and reduced survival rate of larvae and embryos (köprücü and aydin, 2004; ural and sağlam, 2005), are reported in several fish species exposed to various pyrethroid pesticides. werner and moran (2008) suggested that acute toxicity of pyrethroids to fish is usually higher than 200 ng/l. however, synthetic pyrethroid insecticides are highly toxic to fish and aquatic invertebrates (polat et al., 2002; datta, a. kaviraj, 2003; ural and sağlam, 2005; feo et al., 2010). there is little information on the toxicity of fenproparthin to aquatic organisms. alburnus species belong to the cyprinidae family that is found in most rivers in iran. the wide distribution of the freshwater fish alburnus sp. makes this species a potential and useful biomarker for monitoring aquatic ecosystems. therefore, the aim of the present study was to evaluate the changes in some biochemical parameters of the freshwater fish alburnus mossulensis after exposure to sublethal concentrations of fenpropathrin. materials and methods fish: freshwater fish, alburnus mossulensis, 7.36±1.60 g were netted from the maroon river, khuzestan province, iran. fishes were kept in glass aquaria with dechlorinated water to acclimatize them to laboratory conditions (24±2°c, ph: 7.4±0.2, hardness 355±25 mg/l of caco3 and 16 l: 8 d photoperiods) for two weeks prior to use. specimens were fed with live food and commercial diet for ornamental fish. acute toxicity test: the acute toxicity test was performed according to semi-static methods described in the oecd procedure. fishes were not fed 24 h before the experiments and during the acute toxicity test. the experiments consisted of a control group and five experimental groups. acute test was performed to determine the appropriate toxicity range for the sub-lethal assay. 10 fish per group were exposed to different fenpropathrin concentrations (0.0, 10, 50, 100, 250, 500 and 1000 µg/l fenpropathrin, purity 30%) in 85 l aquarium. during the 96 h acute toxicity experiment, water in each aquarium was aerated and had the same conditions as the acclimation period. test solutions were renewed every 24 h to maintain the chemical and the water quality. every 24 h the dead fish were removed and the number of survivals was recorded. the experiment was repeated in triplicate. lc50 values were calculated by the probit analysis test (banaee et al., 2011). sub-lethal toxicity test: for sub-lethal toxicity tests, the concentrations of fenpropathrin in water were maintained modestly below the 96 h lc50 value. based on this value, five sub-lethal concentrations 1, 2, 3, 5 and 10% of 96 h lc50 were chosen for the freshwater fish (alburnus mossulensis). the fish were divided into five treatments and a control group by triplicate (10 specimens per each aquarium). test solutions in each aquarium were renewed every 24 h. on the other hand, the twenty percent of water was changed daily to reduce the build-up of metabolic wastes and to keep concentrations of fenpropathrin near the nominal level. during the experiment, fishes 60 banaee et al./ int. j. aquat. biol. (2014) 2(2): 58-68 were fed twice a day with commercial food, and the fish mortality was recorded. at the end of the experimental period, on day 15, all fish per treatment were captured and anaesthetized with a clove powder extract. then, they were sacrificed and washed with buffered normal saline. fish were homogenized during two minutes in ice cold phosphate buffer (ph 7.4; 1:10, w/v) using a glass homogenizer and then centrifuged for 15 min at 15000 g at 4°c with a refrigerated centrifuge. supernatants were immediately used to measure biochemical parameters by using spectrophotometric assays. biochemical analysis: lactate dehydrogenase (ldh) activity determination is based on measuring the conversion of pyruvate to l-lactate by monitoring the nadh oxidation. aspartate aminotransferase (ast) was assayed in a coupled reaction with malate dehydrogenase in the presence of nadh. in the alanine aminotransferase (alt) assay, the enzyme reacts with alanine and αketoglutarate to form glutamate and pyruvate. ldh converts pyruvate to lactate and nad+. in the determination of creatine phosphokinase (ck) activity, the enzyme reacts with creatine phosphate and adp to form atp, which is coupled to the hexokinase/gdp reaction generating nadph. all these activities were monitored by measuring the change in absorbance at 340 nm. alkaline phosphatase (alp) assay is based on the enzymemediated conversion of p-nitrophenol phosphate to nitrophenol in an alkaline buffer at 405 nm (moss and henderson, 1999). acetylcholinesterase (ache) activity was determined by adding an adequate volume of the sample into a cuvette containing 0.1 m phosphate ph 8.0, and acetylcholine iodide (0.015 m) and dithiobisnitrobenzoic acid (0.01 m) as substrates. ache activity was recorded during 180 s at 405 nm (thomas, 1998). levels of protein in liver tissue were determined by standard procedures used in clinical biochemistry laboratories based on manual biochemical kits (parsazemon co, iran) (johnson et al., 1999). total antioxidant capacity was assayed according to the ferric reducing ability of plasma (frap) method. briefly, the frap reagent contained 5 ml of a (10 mmol/l) tptz (2,4,6tripyridylstriazine) solution in 40 mmol/l hcl plus 5 ml of fecl3 (20 mmol/l) and 50 ml of acetate buffer, (0.3 mol/l, ph=3.6) and was prepared freshly. aliquots of 100 µl supernatant were mixed with 3 ml frap reagent. the conversion rate of ferric tripyridyl-striazine (fe3+-tptz) complex to ferrous tripyridyls-triazine (fe2+-tptz) at ph 3.6 and temperature 25°c is directly proportional to the concentration of total antioxidant in the sample. fe2+-tptz has an intensive blue color that can be monitored up to 5 min at 593 nm by a uv/vis spectrophotometer. lc numerical value of lethal concentrations at different times 24 h 48 h 72 h 96 h lc10 149.28±45.74 58.045±20.15 56.04±11.31 49.34±10.15 lc20 291.05±39.32 113.01±17.25 83.55±10.19 74.07±9.12 lc30 393.28±38.67 152.65±16.66 103.39±10.26 91.90±9.49 lc40 480.63±40.99 186.52±17.30 120.34±10.90 107.14±10.48 lc50 562.28±45.19 218.18±18.75 136.18±11.90 121.38±11.84 lc60 643.92±50.88 249.84±20.86 152.02±13.21 135.62±13.48 lc70 731.27±58.11 283.71 ±23.65 168.97±14.86 150.85±15.45 lc80 833.50±67.56 323.35±27.38 188.80±17.02 168.69±17.94 lc90 975.27±81.79 378.32±33.09 216.31±20.26 193.42±21.59 lc99 1311.97±118.12 508.87 ±47.86 281.64 ±28.61 252.15±30.75 table 1. numerical value of lethal concentrations at different times 61 banaee et al./ int. j. aquat. biol. (2014) 2(2): 58-68 calculations were performed using a calibration curve of feso4•7h2o (100 to 1000 µm/l) (iris et al., 1996). malondialdehyde (mda) content was assessed by a modification of a thiobarbituric acid assay and was expressed as µmol/g tissue (placer et al., 1966). 500 µl of the supernatant was transferred to a pyrex tube and mixed with 2500 µl tricholoroacetic acid (20%) and 1000 µml triclorthiobarbituric acid (67%). the tubes were then placed in boiling water (100°c) for 15 min. after cooling, the mixtures were extracted with a solution containing 1000 µl of distilled water and 5000 µl n-butanol: pyridine (15: 1). the mixture was then centrifuged at 2000 g for 15 min at 4°c. the pink color produced by these reactions was measured spectrophotometrically at 532 nm to measure mda levels. mda concentration was calculated with an external standard of mda. tetraethoxypropane and absolute ethanol were used to prepare the mda standards. concentrations of mda in whole body samples are expressed in µm per g protein. catalase activity was measured by an assay with hydrogen peroxide based on formation of its stable complex with ammonia molybdate. 0.2 ml of supernatant was incubated in 1 ml reaction mixture containing 65 mm hydrogen peroxide in 60 mm sodium-potassium phosphate buffer, ph 7.4 at 25°c for 4 min. the enzymatic reaction was stopped with 1 ml of 32.4 mm ammonium molybdate and concentration of the yellow complex of molybdate and hydrogen peroxide was measured at 405 nm. all chemical materials were obtained from merck co, germany. all biochemical parameters were measured in duplicate by uv/vis unico spectrophotometer (model 2100). statistical analysis: statistical analyses were performed using spss (release 19 ibm) software. data are presented as mean ± sd. all the data were tested for normality (kolmogorov-smirnov test). data were analyzed by one-way of variance analysis (anova). the significant means were compared by duncan test and a p<0.05 was considered statistically significant. results numerical value of lc50 of fenpropathrin at 24, 48, 72 and 96 hours are presented in table 1. fish mortality was progressively elevated with increasing the concentration of fenpropathrin. sub-lethal concentrations of fenpropathrin were determined according to 96h lc50. no mortality was observed in fish exposed to sublethal concentrations of fenpropathrin and control group during experimental periods. loss of appetite, increased mucus secretion, increase in abnormal behavior, swimming in the surface of water and swimming vertically were the major changes observed in fish after exposure to the higher concentrations (5.50 and 12.60 µg/l) of fenpropathrin. changes in the enzymatic activities determined in the whole body of fish exposed to different concentrations of fenpropathrin are presented in table 2. ast activity in the whole body of fish exposed to 2.75, 5.50 and 12.60 µg/l fenpropathrin was significantly higher than in the control group. a concentration of fenpropathrin (µg/l) enzyme activity levels (unit per g protein tissue) ast (u/g) alt (u/g) alp (u/g) ldh (u/g) cpk (u/g) ache (u/g) 0.00 µg/l 2.28±1.04a 0.67±0.13a 2.28±0.36a 2.54±1.10a 13.47±5.70b 7.99±2.17b 1.25 µg/l 2.33±0.97a 0.51±0.14a 4.21±2.03ab 4.49±2.28ab 5.45±0.80a 5.66±2.14a 2.75 µg/l 4.11±1.25b 0.36±0.05a 2.52±0.54ab 4.64±2.26ab 7.81±2.75a 4.35±1.11a 3.15 µg/l 3.55±1.60ab 0.45±0.15a 3.72±0.69ab 4.05±1.34ab 5.30±1.18a 4.59±1.40a 5.50 µg/l 6.00±1.11c 2.40±0.50c 4.88±1.29b 5.94±3.07b 4.65±0.93a 4.29±0.48a 12.60 µg/l 4.06±1.38b 1.55±0.41b 4.71±2.14b 5.74±3.16b 6.94±2.33a 4.62±1.02a table 2. changes in the level of enzyme activities in the whole body of fish exposed to different concentrations of fenpropathrin 62 banaee et al./ int. j. aquat. biol. (2014) 2(2): 58-68 significant increase in alt activity was observed in the whole body of fish exposed to 5.50 and 12.60 µg/l fenpropathrin when compared with control group. ache and cpk activities significantly decreased in the whole body of fish after exposure to all concentrations of fenpropathrin. there was a significant increase in the alp and ldh activities in the whole body of fish exposed to 1.25, 5.50 and 12.60 µg/l fenpropathrin when compared with control group. alterations in the malondialdehyde, total protein, total antioxidant levels and catalase activity in the whole body of fish exposed to different concentrations of fenpropathrin are presented in figures 1 to 4. a significant increase in mda levels in the whole body of fish exposed to fenproparthin was observed in all treated groups when compared with control group (fig. 1). total antioxidant capacity in the whole body of fish exposed to fenproparthion progressively decreased with increased concentrations of the pesticide (fig. 2). there was a significant increase in the cat activity in the whole body of fish after exposure to 5.50 and 12.60 µg/l fenpropathrin respect to the control group (fig. 3). the results of this study showed that total protein levels in the whole body of fish exposed to 2.75, 5.50 figure 1. changes in the mda levels in the whole body of fish exposed to different concentrations of fenpropathrin. significant differences between values when compared with control groups were characterized by alphabet symbol (p<0.05). values represent mean ± s.d. figure 2. changes in the total antioxidant levels in the whole body of fish exposed to different concentrations of fenpropathrin. significant differences between values when compared with control groups were characterized by alphabet symbol (p<0.05). values represent mean ± s.d. figure 3. changes in the cat activity in the whole body of fish exposed to different concentrations of fenpropathrin. significant differences between values when compared with control groups were characterized by alphabet symbol (p<0.05). values represent mean ± s.d. figure 4. changes in the level of total protein in the whole body of fish exposed to different concentrations of fenpropathrin. significant differences between values when compared with control groups were characterized by alphabet symbol (p<0.05). values represent mean ± s.d. 63 banaee et al./ int. j. aquat. biol. (2014) 2(2): 58-68 and 12.60 µg/l fenpropathrin significantly decreased when compared with control group (fig. 4). discussion the present results show that fenpropathrin is highly toxic to freshwater fish, a. mossulensis. the toxicity of fenpropathrin on a. mossulensis increased with increasing the concentration and exposure time. when fishes were exposed to 10 µg/l fenpropathrin, only 3.34% died after 96 h, whereas all the fishes (100%) died after 48 h when were exposed to a concentration of 1000 µg/l fenpropathrin. in addition, the 24, 48, 72 and 96 h lc50 values of fenpropathrin of a. mossulensis were calculated as 562.3±45.2, 218.2±18.8, and 136.2±11.9 and 121.4±11.8 µg/l, respectively. imbalance, vertical swimming and swimming in the water, loss of appetite, bleeding at the base of the fins and eye balls were important clinical symptoms observed in fish exposed to high concentrations of fenpropathrin. behavior disorders in fish exposed to fenpropathrin maybe associated to the neurotoxicity potential of pyrethroid pesticides. neurological disorders in animals are often attributed to a dysfunction in ion channels of neurons, particularly sodium ions after exposure to pyrethroid pesticides (sogorb and vilanova, 2002; banaee, 2013). however, a decrease in ache activity may also play a role on the behavioral changes in fish after exposure to fenpropathrin. reduced ache activity in the whole body of a. mossulensis exposed to fenpropathrin may be due to changes in this enzyme function (tayebati et al., 2009; el-demerdash, 2011). decreases in the ache activity levels in different tissues of fish were observed after exposure tochlorpyrifos (halappa and david, 2009; sharbidre et al., 2011), diazinon (banaee et al., 2011), methyl parathion (sharbidre et al., 2011), monocrotophos (rao, 2006), and atrazine (santos and martinez, 2012), permethrin and deltamethrin (goulding et al., 2013). although, there is no information on the detoxification mechanism of fenpropathrin in fish, free radicals that are produced during the biotransformation of this pesticide in liver tissue may be the most important cause of oxidative stress in a. mossulensis. increases in mda levels in the whole body extract of fish may be an important bioindicator of lipid peroxidation in different tissues of freshwater fish, after exposure to fenpropathrin. an increase in mda levels were reported in different tissues of fish exposed to diazinon (oruç and usta, 2007; isik and celik, 2008), deltamethrin (yonar and sakin, 2011), methyl parathion (monteiro et al., 2006; isik and celik, 2008; sharbidre et al., 2011), chlorpyrifos (sharbidre et al., 2011), carbamazepine (li et al., 2010), and trazine (paulino et al., 2012). a decrease in total antioxidant capacity was an important detrimental response of the fish antioxidant defense system to increased free radicals after exposure to fenpaprothrin. similar results were observed in rainbow trout and carp after exposure to diazinon and cyfluthrin (sepici-dinçel et al., 2009; banaee et al., 2013). the overproduction of free radicals during pesticide detoxification may be associated with a decrease in the hepatic total antioxidant capacity (monteiro et al., 2006; banaee et al., 2013). impairment in the synthesis of enzymatic and non-enzymatic antioxidants may be the most important factor in reducing the levels of cellular total antioxidant. therefore, the decline in the cellular antioxidant capacity makes the cells more vulnerable to oxidative stress damage. a decrease in non-enzymatic antioxidant levels were observed in different tissue of fish exposed to chlorpyrifos (sharbidre et al., 2011) and andatrazine (santos and martinez, 2012). the elevated mda levels and total antioxidant capacity are indicative of increased oxidative stress in fish. the results in the present study indicated that the imbalance between oxidants and antioxidants in cells was the most important factor in causing oxidative stress in fish after exposure to fenpropathrin. similar results were observed in the sheepshead minnow (cyprinodon variegatus) (harper et al., 2008), channa punctata (sayeed et al., 2003; atif et al., 2005), medaka (oryzias latipes) (ingeborg et al., 2002) exposed to bifenthrin esfenvalerate, respectively. 64 banaee et al./ int. j. aquat. biol. (2014) 2(2): 58-68 catalase plays an important role in the elimination of hydrogen peroxide in cells (banaee et al., 2013). hydrogen peroxide may play a role in increasing mda levels in the fish exposed to fenpropathrin. moreover, an increase in catalase activity in the whole body of fish after exposure to high concentrations of fenpropatrin may be effective in the removal of hydrogen peroxide produced in cells during detoxification process. an increase in cat activity in different tissues of fish exposed to atrazine (paulino et al., 2012) was reported. ast and alt enzyme activities are important in cellular nitrogen metabolism, oxidation of amino acids, and liver gluconeogenesis (banaee, 2013). in stress situations, increased activity of liver enzymes such as ast and alt has stimulatory effects on gluconeogenic mechanism (banaee, 2012; 2013). thus, increased levels of these aminotransferasa activities may have played an important role in energy supply for fish exposed to fenpropatrin. similar changes were observed in labeorohita, after exposure to fenvalerate (prusty et al., 2011). increase in alt and ast activities were observed in plasma, liver and kidney of oreochromis mossambicus, cyprinus carpio and korean rockfish (sebates schlegeli) after exposure to monocrotophos (rao, 2006), bifenthrin (velisek et al., 2009) and cypermethrin (jee et al., 2005). the results showed that the protein of the whole body was reduced, which may be related with the increased transaminase activities. the increased activity of these enzymes may lead to protein breakdown to provide energy and regeneration of tissue damages. fish muscle breakdown under stress situation may be one of the main reasons for decreasing tissue protein. decreased total protein levels were reported in oreochromis mossambicus, o. niloticus, cyrinus carpio, and oncorhynchus mykiss exposed to endosulfan (kumar et al., 2011), malathion (patil and david, 2008), diazinon (banaee et al., 2008; banaee et al., 2011; banaee et al., 2013), lindane (saravanan et al., 2011), and cypermethrin (korkmaz et al., 2009), bifenthrin (velisek et al., 2009) and cypermethrin (jee et al., 2005). reduction of muscle mass of fish may decrease cpk activity in fish after exposure to fenpropathrin. rosalki (1998) believed that damage to connective tissues and a reduction of muscle mass are the main reasons for the decrease in the activity of the cpk enzyme. after fish exposure to pesticides, ldh may increase to supply energy to fish. ldh is an enzyme that participates in the anaerobic pathway of carbohydrate metabolism. the increase of ldh activity is a diagnostic index widely used to recognize increases of anaerobic metabolism resulting from depletion of energy under anaerobic and environmental stress conditions (banaee, 2012; 2013). the increase of ldh activity in the whole body extract of fish was a physiological mechanism to provide more energy to deal with the effects fenpropathrin on the freshwater fish, a. mossulensis. similar changes in ldh activity were observed in crayfish exposed to endosulfan (banaee and ahmadi, 2011). increased ldh activity in the gill and brain of tilapia, oreochromis mossambicus after exposure to monocrotophos was reported by rao (2006). in contrast, tripathi and shasmal (2011) showed an inhibitory effect of chlorpyriphos on ldh activities in different tissues of the fish. alp plays a significant role in phosphate hydrolysis and in membrane transport and it also acts as a good bio-indicator of stress in biological systems. increased alp activity in the whole body extract of fish may be due to the effects of fenpropathrin on transphosphorylation activity. increases in alp activity were reported in the whole body extract of crayfish after exposure to endosulfan (banaee and ahmadi, 2011). rao (2006) reported that the alp activities were increased in plasma, gills and kidneys of tilapia exposed to monocrotophos. it can be concluded that fenpropatrin was highly toxic to a. mossulensis. exposure to sub-lethal concentrations of fenpropatrin resulted in significant biochemical alterations and behavioral changes which may be potentially disruptive for the survivability of a. mossulensis. this fact should be taken into consideration when this pesticide is used 65 banaee et al./ int. j. aquat. biol. 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(2018) 6(1): 37-43 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article ectoparasitic infestation of the nile squeaker, synodontis schall (bloch and schneider, 1801) from the cross river estuary, nigeria victor oscar eyo*1, emmanuel offiong effanga2 1department of fisheries and aquaculture, faculty of environmental management and pollution, nigeria maritime university, okerenkoko, delta state, nigeria. 2department of zoology and environmental biology, faculty of biological science, university of calabar, p.m.b. 1115 calabar, cross river state, nigeria. article history: received 3 october 2017 accepted 24 february 2018 available online 2 1 april 2018 keywords: ectoparasitic infestation abundance intensity prevalence abstract: the objective of this study was to determine the abundance, intensity and prevalence of ectoparasites of synodontis schall from the cross river estuary, nigeria. a total of 150 fresh samples were collected between may and october, 2013 from the catches of the artisanal fisheries at nsidung beach. the skin, gill and fin scrapping were prepared from each specimen and examined following standard methods for microscopic analysis. the results showed that 21 specimens were infested with 77 ectoparasites belonging to five species, including trichodina sp., protozoan cyst, ergasilius lizae, dactylogyrus sp. and gyrodactylus sp. with an overall prevalence of 14.0%. infestation rate was highest in 10-14.9 cm class size (53.55%), followed by 15-19.9 cm (34.00%), ≥20.0 cm (8.00%) and 5-9.9 cm (4.67%) size classes. parasites were more prevalent in the gills (82%), followed by the skin (14%) and least (4%) in the dorsal fin. prevalence and abundance of ectoparasites was highest (28.57% and 0.71) in 5-9.9 cm size class, followed by 10-14.9 cm (16.25% and 0.60), 15-19.9 cm (9.80% and 0.41) and ≥20.0 cm (8.33% and 0.25) classes. prevalence and abundance of ectoparasites was higher in female (15.63% and 0.56) than male (11.11% and 0.43). dactylogyrus sp., trichodina sp., protozoan cyst and e. lizae exhibited organ specificity as they were specifically recovered from the gills whereas gyrodactylus sp. was found in the skin and fin. however, it was concluded that to prevent zoonosis, s. schall should be washed with clean water and cooked properly before consumption. introduction the nile squeaker, synodontis schall, belonging to the family mockokidae is one of the valuable food fish for the inhabitants of cross river estuary. this is attributed to its excellent taste and meat quality. according to steffens (2006), s. schall is rich in moisture, dry matter, protein, lipid, vitamins, minerals and caloric value, thereby increasing its acceptability and demand by fish consumers. this species is also popularly used in tropical aquaria due to its upsidedown swimming nature (otubisin, 1986; teugels, 1996). according to teugels (1996), s. schall is endemic to africa and generally very common all year round. they also contribute a significant percentage in the catches of artisanal fisheries of the cross river estuary. in fisheries science, abundance, intensity and prevalence of fish parasites is directly influenced by several factors such as size, age, sex, species, diet, *corresponding author: victor oscar eyo doi: https://doi.org/10.22034/ijab.v6i1.376 e-mail address: sirvick2003@yahoo.com season, climate change and environment of fishes. in nigeria, parasitic diseases of fish pose a serious economic and public health concern in the fishing industry due to the risk of transmission of parasite from fish to fish consumers (zoonosis). in most aquatic ecosystems, ectoparasites are common and free living organisms that are capable of causing diseases (madanire-moyo and barson, 2010). according to ekanem et al. (2014), ectoparasitic diseases affects the normal health and physiological conditions of fish leading to poor growth, abnormal metabolic activities and subsequently, death of affected fish. ectoparasites of some bonyfish species in the cross river estuary and other tropical waters have been documented (obiekezie and enyenihi, 1988; obiekezie et al., 1987; obiekezie, 1998; ekanem et al., 2011; olofintoye, 2006; ekanem et al., 2013; ekanem et al., 2014; eyo et al., 2015). presently, there is no specific report on ectoparasites 38 eyo and effanga/ ectoparasitic of synodontis schall from the cross river estuary, nigeria of s. schall in the cross river estuary. therefore, the objective of this study was to investigate the abundance, intensity and prevalence of ectoparasites of s. shall from the cross river estuary, nigeria and to recommend safety measures to curb the risk of zoonosis among fish consumers. materials and methods study area: the cross river estuary located in the southern part of nigeria, lies approximately between latitudes 4º and 8ºn and longitude 7º30 and 10ºe. it origins from the cameroon mountain and meanders west wards into nigeria and then south ward through high rainforest formation before discharging into atlantic ocean at the gulf of guinea (akpan and offem, 1993). the study area is rich in mangrove forest vegetation with climate characterized by long wet season from april to october and a dry season from november to march. it is also characterized by a cold, dry and dusty period between december and january which is known as harmattan season. temperature ranges from 22ºc in the wet to 35ºc in the dry season with a relative humidity ranging from 60% (dry season) to above 90% during the wet season (akpan and offem, 1993). collection and identification of s. schall and their sexes: a total of 150 freshly caught s. schall were collected between may and october, 2013 from the catches of the artisanal fisheries at nsidung beach, calabar (fig. 1) which is a major landing point of the artisanal fisheries of the cross river estuary. fish samples were transported immediately in ice-packed containers to fisheries and aquaculture laboratory, institute of oceanography, university of calabar, for further analysis. identification of s. schall was based on identification key given by fischer et al. (1981). differentiation of sexes were be based on external features (anal opening) and internal features such as gonad (eyo et al., 2013a). measurements of s. schall total length (tl-cm) and size class grouping: total length of s. schall was measured from snout to the base of the caudal fin rays using a measuring board to the nearest 0.1 cm. the fish were grouped into four size classes, including 59.9, 10-14.9, 15-19.9 and ≥20.0 cm. examination of s. schall for ectoparasites: each s. schall was carefully examined independently for ectoparasites. scrapings from the gills, skin and fin of s. schall were smeared on clean glass slides, covered with cover slip and examined under light microscopes for ectoparasites. ectoparasites recovered were fixed in 4% phosphate buffered formalin (pbf) for further processing and species identification (paperna, 1980, 1996). identification of ectoparasites was carried out according to roberts (2000), obiekezie and engenihi (1988) and obiekezie and ekanem (1995). evaluation of parasitological indices: parasitological indices evaluated include dominance (d), prevalence (p), mean intensity (i) and abundance (a). the dominance of endoparasites was calculated according to roohi et al. (2014) as given below: dominance = ( 𝑁 𝑁 𝑆𝑢𝑚 )*100 where n=abundance of endoparasite species and n sum=sum of the abundance of all endoparasite species found) and expressed as a percentage. the endoparasite were classified based on their dominance values according to niedbala and kasparzak (1993) as follows: eudominant (>10%), dominant (5.1-10%), subdominant (2.1-5%), recedent (1.1-2%) and subrecedent (<1.0%) of given species. prevalence (%), mean intensity and abundance were calculated figure 1. map of the cross river estuary showing the sampling area (nsidung beach). 39 int. j. aquat. biol. (2018) 6(1): 37-43 according to upadhyay et al. (2010) given below: prevalence (%) = ( 𝑁𝑜.𝑜𝑓 𝑖𝑛𝑓𝑒𝑐𝑡𝑒𝑑 𝑓𝑖𝑠ℎ 𝑇𝑜𝑡𝑎𝑙 𝑁𝑜.𝑜𝑓 𝑓𝑖𝑠ℎ 𝑒𝑥𝑎𝑚𝑖𝑛𝑒𝑑 )*100 mean intensity = 𝑁𝑜.𝑜𝑓 𝑐𝑜𝑙𝑙𝑒𝑐𝑡𝑒𝑑 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑠 𝑁𝑜.𝑜𝑓 𝑖𝑛𝑓𝑒𝑠𝑡𝑒𝑑 𝑓𝑖𝑠ℎ abundance = 𝑁𝑜. 𝑜𝑓 𝑝𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑠 𝑁𝑜.𝑜𝑓 𝑓𝑖𝑠ℎ 𝑒𝑥𝑎𝑚𝑖𝑛𝑒𝑑 results number of examined fish, infested and parasites recovered from s. schall: out of 150 examined samples, 21 samples were infested with 77 ectoparasites. in 5-9.9 cm size class, 2 out of 7 (4.67%) examined samples were infested with 5 ectoparasites. in 10-14.9 cm size class, 13 out of 80 (53.33%) were infested with 48 ectoparasites. in 1519.9 cm size class, 5 out of 51 (34.00%) were infested with 21 ectoparasites. in ≥20.0 cm size class, only 1 out of 12 (8.00%) was infested with 3 ectoparasites. table 1 shows the number of examined fish, number of fish infested and number of ectoparasites recovered. prevalence, intensity and abundance: the result obtained for prevalence (%), intensity and abundance of ectoparasites in relation to size class (cm) is shown in table 1. prevalence was highest (28.57%) in 5-9.9 cm size class, followed by 10-14.9 cm (16.25%), 1519.9 cm (9.80%) and ≥20.0 cm (8.33%) classes. intensity was highest (4.20) in 15-19.9 cm size class, followed by 10-14.9 cm (3.69), ≥20.0 cm (3.00) and 5-9.9 cm (2.50) classes. abundance was highest (0.71) in 5-9.9 cm size class, followed by 10-14.9 cm (0.60), 15-19.9 cm (0.41) and ≥20.0 cm (0.25) classes. prevalence, intensity and abundance in relation to sex: out of 150 examined specimens, 96 were females (64%) and 54 males (36%). out of 96 examined females, 15 specimens were infested with 54 ectoparasites with prevalence of 15.63%, intensity of 3.60 and abundance of 0.56. out of 54 examined males, 6 specimens were infested with 23 ectoparasites with prevalence of 11.11%, intensity of 3.83 and abundance of 0.43. table 2 shows the number of fish examined, prevalence, intensity and abundance of parasites recovered from s. schall in relation to sex. prevalence, intensity, abundance and dominance in relation to organ specificity: the prevalence in relation to organ specificity (table 3) showed that in 5-9.9 cm size class, ectoparasites were most prevalent in the gills and least in the skin. three trichodina sp. was recovered from the gills, whereas 2 gyrodactylus sp. was recovered from the skin. trichodina sp. recovered from the gills had a dominance value of 60.00 (eudominant parasite), prevalence (14.29%), intensity (3.00) and abundance (0.43). gyrodactylus sp. recovered from the skin and fin had a dominance value of 40.00 (eudominant parasite), prevalence (14.29%), intensity (2.00) and abundance (0.29). in 10-14.9 cm size class, ectoparasites were most prevalent in the gills and least in the skin. eighteen trichodina sp. was recovered from the gills with a dominance value of 37.50 (eudominant parasite), prevalence (6.25%), intensity (3.60) and abundance table 1. number and percentage of fish examined, prevalence, intensity and abundance of ectoparasites recovered from synodontis schall from the cross river estuary. size class (cm) no. and % of fish examined no. of fish infested no. of parasites collected prevalence intensity abundance 5 -9.9 7 (4.67) 2 5 28.57 2.50 0.71 10-14.9 80 (53.33) 13 48 16.25 3.69 0.60 15-19.9 51 (34.00) 5 21 9.80 4.20 0.41 ≥ 20.0 12 (8.00) 1 3 8.33 3.00 0.25 total 150 (100) 21 77 14.00 3.67 0.51 table 2. prevalence, intensity and abundance of parasites recovered from synodontis schall in relation to sex. sex no and % of fish examined no of fish infested no. of parasites collected prevalence intensity abundance female 96 (64) 15 54 15.63 3.60 0.56 male 54 (36) 6 23 11.11 3.83 0.43 total 150 (100) 21 77 1.0 3.67 0.51 40 eyo and effanga/ ectoparasitic of synodontis schall from the cross river estuary, nigeria (0.23). nine gyrodactylus sp. was recovered from the skin with a dominance value of 18.75 (eudominant parasite), prevalence (2.50%), intensity (4.50) and abundance (0.11). fifteen protozoan cyst was recovered from the gills with a dominance value of 31.25 (eudominant parasite), prevalence (3.75%), intensity (5.00) and abundance (0.19). six ergasilus lizae was recovered from the gills with a dominance value of 12.50 (eudominant parasite), prevalence (3.75%), intensity (2.00) and abundance (0.08). in 15-19.9 cm size class, ectoparasites were only prevalent in the gills. six e. lizae was recovered from the gills with a dominance value of 28.57 (eudominant parasite), prevalence (1.96%), intensity (6.00) and abundance (0.12). twelve trichodina sp. was recovered from the gills with a dominance value of 57.14 (eudominant parasite), prevalence (5.88%), intensity (4.00) and abundance (0.24). three dactylogyrus sp. was recovered from the gills with a dominance value of 14.29 (eudominant parasite), prevalence (1.96%), intensity (3.00) and abundance (0.06). in ≥20.0 cm size class, only 3 gyrodactylus sp. was recovered from the dorsal fin with a dominance value of 100.00 (eudominant parasite), prevalence (8.33%), intensity (3.00) and abundance (0.25). numerical abundance and percentage of ectoparasites in relation to organ: numerical abundance and percentage of ectoparasites in relation to organ (table 4, fig. 2) showed that 63 ectoparasites (82%) were recovered from the gills (33 trichodina sp., 15 table 3. dominance, prevalence intensity and abundance of ectoparasites in relation to organ specificity. size class (cm) no. of fish examined no. of fish infested parasite species no. of parasites collected organs dom pre int abn 5-9.9 cm 7 1 trichodina sp. 3 gills 60.00 14.29 3.00 0.43 1 grrodactylus sp. 2 skin 40.00 14.29 2.00 0.29 total 7 2 5 100.00 28.57 2.50 0.71 10-14.9 cm 80 5 trichodina sp 18 gills 37.50 6.25 3.60 0.23 2 grrodactylus sp 9 skin 18.75 2.50 4.50 0.11 3 protozoan cyst 15 gills 31.25 3.75 5.00 0.19 3 ergasilus lizae 6 gills 12.50 3.75 2.00 0.08 total 80 13 48 100.00 16.25 3.69 0.60 15-19.9 cm 51 1 ergasilus lizae 6 gills 28.57 1.96 6.00 0.12 3 trichodina sp. 12 gills 57.14 5.88 4.00 0.24 1 dactylogyrus sp. 3 gills 14.29 1.96 3.00 0.06 total 51 5 21 100.00 9.80 4.20 0.41 ≥20.0 cm 12 1 grrodactylus sp. 3 dorsal fin 100.00 8.33 3.00 0.25 total 12 1 3 100.00 8.33 3.00 0.25 *dom = dominance, pre = prevalence, int = mean intensity and abn = abundance table 4. numerical abundance and percentage of ectoparasites from synodontis schall in relation to organ. organs percentage of parasites in organs parasite species number of ectoparasite species gills 82 trichodina sp. 33 protozoan cyst 15 ergasilus lizae 12 dactylogyrus sp. 3 total 63 skin 14 grrodactylus sp 11 total 11 fin 4 grrodactylus sp 3 total 3 overall total 100 77 41 int. j. aquat. biol. (2018) 6(1): 37-43 protozoan cyst, 12 e. lizae and 3 dactylogyrus sp.), 11 ectoparasites (11 gyrodactylus sp.) from the skin (14%) and 3 gyrodactylus sp. (4%) from the dorsal fin. discussion the results showed that out of 150 examined specimens, 21 specimens were infested with 77 ectoparasites belonging to five parasitic species, including trichodina sp., protozoan cyst, e. lizae, dactylogyrus sp. and gyrodactylus sp. with an overall prevalence of 14.0%. this overall parasite prevalence is quite lower than prevalence of 72.6% reported by eyo et al. (2013b) for s. batensoda from rivers nigerbenue confluence, nigeria, 85.2% reported by auta et al. (1999) for synodontis sp. in zaria dam, nigeria. however, the overall prevalence reported in this study is higher than 3.33% reported by ekanem et al. (2011) for parasites of landed fishes from the great kwa river, nigeria and 13.6% reported by ugwuozor (1987) in imo river. this indicates that parasite prevalence in wild fish vary according to location. ekanem et al. (2011) attributed such variation to the river sanitary condition, the location of the river from residential areas, and the number of people visiting the river and their purposes. according to eyo et al. (2013b), occurrence of parasites are common in some localities although with variation in prevalence. in this study, the occurrence of trichodina sp., protozoan cyst, dactylogyrus sp. and gyrodactylus sp. in s. schall in the cross river estuary agrees with findings of other authors (ekanem et al., 2011; ekanem et al., 2014) and this indicates that these ectoparasites are common in the estuary. comparing the prevalence of ectoparasites according to class size, 10-14.9 cm had the highest infection rate of 53.55%, followed by 15-19.9 cm (34.00%), ≥20.0 cm (8.00%) and 5-9.9 cm (4.67%) size classes. this indicates that s. schall of 10-14.9 cm size class are more susceptible to ectoparasite infection in the cross river estuary. this observation corroborates with findings of anderson (1974) and ekanem et al. (2011) that fishes of lower class size are more susceptible to ectoparasites. generally, fish length is directly correlated to age and body size. according to poulin (2000), older fishes have longer time span to accumulate parasites than younger fishes and may provide more internal and external space for parasite to establish with a larger surface area for gill and skinattaching parasites. this could be the reason for a higher parasite intensity (4.20) observed in 15-19.9 cm. however, prevalence and abundance of ectoparasites were highest (28.57% and 0.71) in 5-9.9 cm size class, followed by 10-14.9 cm (16.25% and 0.60), 15-19.9 cm (9.80% and 0.41) and ≥20.0 cm (8.33% and 0.25). this findings agrees with ekanem et al. (2011) that fish of a lower class size are more susceptible to parasites because they have a weaker immune system compared to fishes of higher size class. abundance was highest (0.71) in 5-9.9 cm size class, followed by 10-14.9 cm (0.60), 15-19.9 cm (0.41) and ≥20.0 cm (0.25). according to eyo et al. (2013b), high infection of trichodonids in the skin and gills of fish hosts with mucus secretion in their gills could lead to irritation and breathing problems. similarly, klinger and floyd (2002) reported that these parasites cause serious skin and gill irritation, and infected fish are displayed some behavioral abnormalities such as flashing, rubbing, rapid breathing and excessive mucus secretion in gills. monogenean trematodes recovered in this study including gyrodactylus sp. and dactylogyrus sp. are also reported to cause behavioral abnormalities, figure 2. numerical abundance and percentage of ectoparasites of synodontis schall relation to organs. 42 eyo and effanga/ ectoparasitic of synodontis schall from the cross river estuary, nigeria including flashing, rubbing and rapid breathing (eyo et al., 2015). in this study, prevalence and abundance of ectoparasites in relation to sex revealed that female s. schall were more infected than male. female s. schall had prevalence of 15.63% and abundance (0.56) while males had prevalence of 11.11% and abundance (0.43). this findings agrees with eyo et al. (2015) for farmed clarias gariepinus and alam et al. (2010) for channa punctatus, that female fishes were more infected than the male fishes. this could be attributed to ecological habitat and sex hormones which are responsible for depressing the level of parasites infestation (alam et al., 2010). also, aloo et al. (2004) reported that differences in parasitic infestation in relation to sex is physiological. organ specificity of ectoparasites in s. schall showed that dactylogyrus sp., trichodina sp., protozoan cyst and e. lizae were specifically recovered from the gills whereas gyrodactylus sp. was found in the skin and fin. apart from trichodina sp. which are also found in the skin and fin of fish, paperna (1996) reports that dactylogyrus sp. and e. lizae are gill specific parasites. eyo et al. (2015) reported a similar findings on monogenean parasites in farmed c. gariepinus. the preference of dactylogyrus sp. in fish gills is related to feeding and attachment activities of the parasites while gyrodactylus sp. specificity on the skin and fins is linked to reproductive reasons (reed et al., 1996). generally, parasites were more prevalent in the gills (82%), followed by the skin (14%) and least (4%) in the dorsal fin indicating that the gills of s. schall is the most affected organ in the cross river estuary. in this study, single infestation of ectoparasites was dominant with few occurrence of multiple infestations. single infestation of parasites in fish will result in weakening the resistance of the host increasing its susceptibility to multiple parasitic infestation. according to amare et al. 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(2014) 2(2): 69-74 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article the relative eye size, visual cells, cone mosaic and retinal tapetum in the spotted barb puntius binotatus (valenciennes, 1842) leong-seng lim*1, audrey daning tuzan1, linus malitam2, julian ransangan1, gunzo kawamura1 1borneo marine research institute, universiti malaysia sabah, kota kinabalu, sabah, malaysia. 2innovasi sedia sdn. bhd. lot 37, block e, ground floor, new world commercial center, donggongon, 89500 penampang, sabah, malaysia. article history: received 1 january 2014 accepted 20 march 2014 available online 2 5 april 2014 keywords: spotted barb puntius binotatus eye retina visual capability abstract: the relative eye size, types of visual cell and mosaic, and the existence of retinal tapetum in the eyes of the spotted barb puntius binotatus were determined to gain baseline information on its visual capability. the p. binotatus acquired relatively larger eye size than the other similar sized freshwater fish species with its retina being contained both cone and rod visual cells (cone ellipsoid to outer nuclei ratio = 1: 5.7). three types of cone were identified (double-, central singleand corner singlecones), arranging in the square mosaic, and the retinal tapetum was determined to be existed. these results evidenced that the p. binotatus has good visual capability as it possessed both the photopic and scotopic visions. vision can be the primary sense for the p. binotatus. further study is needed to gain more information on the vision of this species. introduction vision is generally known as the primary sense for most fish species, as they rely on it to detect preys and predators for survival. fish visual capability is highly related to the eye structures. therefore, studying on the eye and its retinal morphology can provide insight to the visual capability of the fish (e.g. nag and bhattacharjee, 2002). the spotted barb puntius binotatus is a cyprinid (roberts, 1989; robins et al., 1991), found in the mountainous streams, rivers and lakes of the southeast asian countries, including cambodia, indonesia, laos, malaysia, philippines, thailand and vietnam and introduced species to singapore (talwar and jhingran, 1991; rainboth, 1996; jenkins et al., 2009). due to its common availability, p. binotatus has been recognized as an important bioindicator to the environmental bio-degradation status of these freshwater resources (baumgartner, 2005; zakeyudin et al., 2012). in addition, it is an * corresponding author: leong-seng lim e-mail address: leongseng999@hotmail.com economically important species in mindanao of philippines (dorado et al., 2012). despite of its common availability, little information is available about its biology. in sabah, p. binotatus is locally known as “turongou” and popular to the native people as a deep-fried delicacy. with aim to promote the delicacy at commercial level, the first hatchery and pond culture has been setup by a local company, innovasi sedia private limited, for the production of this fish (lim et al., 2013). such operation has provided a great opportunity for studying its biology, which is useful for understanding the ecology and to improve the culture techniques of this species. in the pond culture, the p. binotatus has been observed to swim to water surface and snap on the given floating commercial pelleted feed accurately. such feeding behaviour is similar to those observed in the visual feeder species, such as the trout and grouper (ahlbert, 1976; tan et al., 2013). therefore, the 70 lim et al./ int. j. aquat. biol. (2014) 2(2): 69-74 p. binotatus may rely much on its vision to detect objects. nonetheless, there has been no information on the vision of this species. for this reason, the present study aimed to examine the relative eye size and its retinal morphology to find baseline information on the visual capability of the cultured p. binotatus. materials and methods sampling and measurements: a total of 33 cultured p. binotatus were sampled from several ponds, managed by the local company innovasi sedia private limited. the specimens were then transferred to the wet laboratory in borneo marine research institute, universiti malaysia sabah. subsequently, each fish were measured for their standard length (sl, in mm), and eye diameter (ed, in mm). prior to measurement, the specimens were anesthetized with 200 ppm tricaine methanesulfonate (ms222) and measurements were carried out with a ruler. the relative eye size (res) of p. binotatus was calculated through dividing ed by sl. histological procedures: after the measurements, the specimens were decapitated, and the head were fixed in bouin’s solution for 24 hours. the samples were subsequently transferred into a series of ethanol with ascended concentrations (70 % to 100 %), cleared with xylene, and finally embedded into paraffin. the samples were sectioned at 6 µm and stained with haematoxylin and eosin. the sections were examined under a light microscope (eclipse 80i, nikon, japan) attached to a computer, and photos were taken using the imaging software niselements f 3.0 (nikon, japan). retinal observation: from the histological sections, the types of visual cell and cone mosaic were identified, and the existence of retinal tapetum was determined. for identifying the types of retinal cone and its mosaic, tangential sections of the retina were observed. retinal rods were difficult to resolve by light microscope hence its existence was determined using the calculation on the ratio of the cone ellipsoids (ce) number to the outer nuclei (on) number within 100 µm length of retina. the retinal rods were assumed existed if the ce to on ratio was more than 1: 1 (blaxter and staines, 1970). the presence of retinal tapetum was ascertained using a microscope with a guide light. results relative eye size: table 1 demonstrates the measurements on the standard length, eye diameter and res of the cultured p. binotatus. the size of fish examined in the present study was in the range of tl from 3.60 cm to 4.90 cm. the res value was 0.07 ± 0.006 (mean ± s.d.). cone and mosaic types: figure 1a shows the tangential section on the retinal temporal region of the p. binotatus. three types of cone: the double cone, the centraland the corner-single cones were identified, arranging in a square mosaic (fig. 1b). presence of retinal rods: table 2 shows the calculation on the number of cone ellipsoids (ce), outer nuclei (on) and the ratio of ce to on. the ce: on was determined to be 1: 5.7 from the mean minimum maximum mean standard deviation standard length, sl (cm) 3.60 4.90 4.22 0.320 eye diameter, ed (cm) 0.25 0.40 0.31 0.035 relative eye size, ed /sl 0.06 0.09 0.07 0.006 table 1. calculation of the relative eye size for p. binotatus (n = 33). cone ellipsoids (ce) outer nuclei (on) ce: on (from means) min max mean s.d. min max mean s.d. 22 33 25.6 4.3 133 158 145.2 11.3 1: 5.7 table 2. calculation of the cone ellipsoid-outer nuclei ratio within 100 µm for p. binotatus (n = 5). 71 lim et al./ int. j. aquat. biol. (2014) 2(2): 69-74 numbers of the ce and on, proving the presence of the retinal rods. existence of retina tapetum: the retina of p. binotatus was shining when it was torch-lighted under the microscope without a light source (figs. 2a-b). therefore, retinal tapetum existed in the eye of the p. binotatus. discussion this is the first report on the vision of the p. binotatus. in the present study, the res mean value was 0.07, indicating that the p. binotatus acquired quite large size of eyes, in comparison with some other similar or larger sizes of cyprinids, which their res ranged generally from 0.04 – 0.06 (table 3). the greater eye size commonly accommodates the larger eye lens than the smaller one. therefore, more light can be gathered and a higher resolution image can be generated in the brain. the greater eye size also provides the better visual sensitivity to the fish under dim light conditions with its better lightgather feature. the relative large eye size in p. binotatus therefore, suggested that this species may have good vision under bright condition, and also able to see in dim light. indeed, the presence of retinal rods and tapetum further supports the idea that the p. binotatus may possess scotopic vision. retinal rods is the visual cells, which is responsible for scotopic vision due to its wider area of outer segment for light absorption (evans, 2004) while the retinal tapetum is the light-reflecting system which reflects light back into the retinal visual cells layer hence more light can be absorbed by the retinal rods (bone et al., 2004), increasing the fish visual sensitivity under the dim light (somiya, 1980). the possession of several types of retinal cone suggested that the p. binotatus may have both the color and ultraviolet (uv) visions because each type of cone is sensitive to different light spectrum (evans, 2004), as demonstrated by jordan et al. (2006) on 15 species of the lake malawi cichlids through microspectrophotometry. the capability of uv vision in p. binotatus can be further assured as it possesses the corner cone, which was reported to be cyprinids sl (cm) ed (cm) ed /sl garra gotyla gotyla 12.0 0.52 0.04 garra lamta 9.0 0.38 0.04 barilius bendelisis bendelisis 8.0 0.43 0.05 barilius vagra vagra 8.0 0.45 0.06 schizothorax richardsonii 24.0 1.12 0.05 acrossocheilus hexagonolepis 7.0 0.42 0.06 danio aequipinnatus 7.0 0.41 0.06 table 3. reviewed information on the relative eye sizes of several cyprinids from nag and bhattacharjee (2002). figure 1. square mosaic in the retina (a) the square box with black line shows a basic square mosaic, scale 10 µm; (b) illustration on the basic square mosaic with labels on the types of cone visual cell. 72 lim et al./ int. j. aquat. biol. (2014) 2(2): 69-74 uv sensitive (bowmaker and kunz, 1987; beaudet et al., 1997). in the present study, well-arranged square cone mosaic with additional corner cones was identified in the retina of the p. binotatus. according to the reviews by evans and browman (2004), wellarranged square cone mosaics were found only in fish species rely typically on photopic vision. those fish species which live in habitats with reduced ambient light intensity and typically nocturnal possess usually less-arranged mosaics or absence of mosaic. with the possession of several types of retinal cone and the square cone mosaic, therefore, p. binotatus can be considered as a diurnal species. in fact, how the cone mosaic contributes to the fish visual capabilities is still not fully known, despite so many studies have been conducted to find out the answer (cook and chalupa, 2000). nevertheless, the square cone mosaic is believed to enhance many visual characteristics of the fish. several studies suggested that the square cone mosaic is predominant in acute vision (engström, 1963; kawamura and tamura, 1973; kawamura et al., 1981), although there are also studies suggested that it is important for the fish to detect motion (lyall, 1957; ahlbert, 1973). in wild, p. binotatus mainly feed on zooplanktons and insect larvae (rainboth, 1996) which are very tiny in size. therefore, good visual acuity appeared to be more essential than motion detection for the p. binotatus to detect its prey. the function of square cone mosaic in the p. binotatus should be mainly for acute vision. in conclusion, p. binotatus has good visual capability. they have big eyes, acquiring good visual acuity and both the color and uv visions under the photopic environments, allowing them to track on the tiny zooplanktons and insect larvae. in addition, they possess scotopic vision. it is confirmed that vision is an important sense to the p. binotatus. therefore, further studies should be carried out to gain more information on the visual capability of this species. acknowledgement the authors thank ms. henrieta lidya bt. lawrence and ms. veronica albert for their technical assistance. this study was supported by the innofund (c027) project, provided by the ministry of science, technology and innovative (mosti) of malaysia. references ahlbert i.b. 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(2023) 11(1): 59-68 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article a new distributional record of flying barb, esomus metallicus (actinopterygii: cyprinidae), from kapalo banda river, west sumatra, indonesia robin1, fitri sil valen1, destra ramadhanu1, aryok nomleni2, gilbert turnip2, liga insani3 1departement of aquaculture, faculty of agriculture fisheries and biology, university of bangka belitung, jl kampus terpadu ubb, balunijuk 33127, bangka belitung, indonesia. 2republic of indonesia defense university, ipsec area, sentul, sukahati, citeureup, bogor, west java, indonesia. 3marine and fisheries polytechnic of jembrana, fish aquaculture study program, jembrana 82218, bali indonesia. article history: received 19 november 2022 accepted 10 january 2023 available online 2 5 february 2023 keywords: coi, cyprinidae, distribution, dna barcoding, phylogenetic. abstract: a new locality was reported for esomus metallicus in kapalo banda river, 50 kota regency, west sumatra province, indonesia, about 74 km from maninjau lake. identification of species was made by morphological and molecular methods using the coi gene. we also applied the neighbor-joining method to construct the phylogenetic tree. based on morphological analysis, the specimens from kapalo banda river were e. metalicus with the unique feature of having long maxillary barbels. based on the dna sequence, specimens of west sumatra were identical to e. metallicus sequence of thailand specimens with a similarity of 99-100%, suggesting the probable origination of the kapalo banda river from thailand. introduction the striped flying barb, esomus metallicus, is a freshwater fish from southeast asia (kottelat, 2013), including thailand (siriwan et al., 2018; fu et al., 2021), laos (kottelat, 2015), vietnam (dao et al., 2017), and malaysia (ng et al., 2019; jamaluddin et al., 2022). it is an introduced species in indonesia that have recently been discovered (arbsuwan et al., 2012; hasan et al., 2020). in indonesia, e. metallicus was accidentally released during the shipping of fry by local fish farmers (hasan et al., 2020). in many cases, aquaculture is the cause of the exotic fish introduction (de silva et al., 2009). the exotic fishes change the pattern of food and niche competition in inland waters (albornoz-garzón and villa-navarro, 2017), though so far, there are no reports of the negative impact of e. metallicus on aquatic communities (arthur et al., 2010; pulungan et al., 2011; arbsuwan et al., 2012). however, more research is needed to ascertain the impact of e. metalicus on aquatic communities, especially correspondence: fitri sil valen doi: https://doi.org/10.22034/ijab.v11i1.1780 e-mail: fitrisilvalen@ubb.ac.id dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.8.8 native fish. striped flying barb is found in three localities, including the siak river, sumatra (pulungan et al., 2011), reteh river, riau province (arbsuwan et al., 2012), and maninjau lake, west sumatra province (hasan et al., 2020). in this study, we report a new locality for e. metallicus in kapalo banda river, 50 kota regency, west sumatra province, indonesia, about 74 km far from maninjau lake, 281 km from siak river, and 503 km from reteh river. kapalo banda river is a tourist spot and conservation area under customary law and societal rules. there is no punishment for fishing violations in this area; it is just a belief that has been passed down from generation to generation about the bad things that will happen if we damage the aquatic ecosystem, and catching fish is done only for personal use in that place. in this work, we also provide morphological and molecular data of the collected specimens to identify them. for molecular identification, we used dna barcoding based on cytochrome c oxidase 60 robin et al./ a new distributional record of flying barb subunit i (coi), a standard method for species identification with a high level of accuracy (hubert et al., 2008; luo et al., 2011; valen et al. 2019a; lutfiatunnisa et al., 2021). material and methods specimens were collected from an irrigation canal in kapalo banda taram (fig. 1), 50 kota regency, west sumatra province, indonesia. the fish are protected by local customary law and societal rules called “ikan larangan”. in this case, research specimens need permission from the local customary head. we also use friendly fishing gear to protect the aquatic environment and the natural habitat. the study was conducted from 26-28 january 2022, and 15 specimens were captured using a cast net and fish trap (serdiati et al., 2021). ten specimens were fixed in 10% formalin (serdiati et al., 2020) and deposited at the laboratory of universitas bangka belitung, indonesia. for subsequent dna analysis, five specimens were fixed in 95% ethanol (valen et al., 2019b). morphological identification of specimens was made based on rainboth (1996), arbsuwan et al. (2012), and hasan et al. (2020). dna extraction: dna extraction was done using a genomic dneasy blood and tissue kit (qiagen). a total of 25 mg of tissue samples were taken using sterile tweezers and put into a 1.5 ml tube, then 180 µl of atl buffer and 20 µl of proteinase k were added. afterward, the samples were vortexed and centrifuged for 20s and heated in a heating block at 56°c overnight. 200 µl buffer al was added, vortexed, and incubated at 56°c for 10 min. then 200 µl 96% ethanol was added and vortexed. the sample and reagent mixture was transferred to a dneasy mini spin column and placed in a 2 ml collection tube. samples were centrifuged at 8000 rpm for 1 min. then, the liquid was drained into the collection tube, and placed in the spin column in a new 2 ml collection tube, 500 µl of buffer aw1 was added and centrifuged at 8000 rpm for 1 min. the spin column was placed into a new 2 ml collection tube, and added 500 µl of buffer aw2, and centrifuged at 14,000 rpm for 3 min. the spin column was transferred to a new 1.5 ml tube. dna was eluted by adding 100 ul of ddh2o to the center of the membrane spin column, then was incubated at room temperature of 15-25°c for 1 min. then centrifuge was done at 8,000 rpm for 1 min. the final step was repeated by adding 100 µl of ddh2o to get a final volume of 200 µl. the extracted dna was amplified using the figure 1. kapalo banda river, the location where esomus metallicus found in 50 kota district, west sumatera, indonesia. 61 int. j. aquat. biol. (2023) 11(1): 59-67 hotstart method. the parameters used in this method are as follows: denaturation at 94°c for 3 min, denaturing at 94°c for 30s, annealing at 48°c for 30s, and extension at 72°c for 45s (valen et al., 2021; valen et al., 2022a), and the pcr process was repeated for 38 cycles. the two primers of fish f1 tca acc aac cac aaa gac att ggc ac and fish r1 tag act tct ggg tgg cca aag aat ca were used (ward et al., 2005). the pcr product was visualized in 1% agarose gel via electrophoresis by staining nucleic acid gel stain (gelred®) (hasan et al., 2021). the samples were sequenced using the sanger sequencing method at a sequencing service facility (sanger and nicklen, 1977). data analysis: molecular identification of species was done by comparing the sequences to the bold system (https://www.boldsystems.org) and blast at ncbi (https://blast.ncbi.nlm.nih.gov) to analyze the homology and similarity between sequences. to reconstruct the phylogenetic tree, we downloaded the sequences of esomus species from the genbank and sequences of castostomus commersonii as an outgroup (table 1). all sequences were initially aligned using the clustal w in mega6 software (tamura et al., 2013). the phylogenetic tree was reconstructed using the neighbor-joining method (saitou and nei, 1987) with a bootstrap test of 10000 replications (felsentein, 1985). the evolutionary distance was calculated using the uncorrected p-distance method (nei and kumar, 2000) in mega6 software (tamura et al., 2013). results and discussion a new record of e. metallicus was done from an irrigation canal of kapalo banda river, 50 kota district, west sumatera province, sumatera, indonesia, during 26-28 january 2021 by collecting 15 specimens (470-590 mm) (fig. 2). diagnosis: meristic characters of e. metallicus are shown in table 2. body compressed and elongated; eye moderately large; head small; mouth superior, upper jaw shorter than lower jaw; snout moderately long; maxillary barbels long reaching to ventral body, rostral barbels quite long; lateral line incomplete. the color of fresh specimen: yellow body; a black band running at dorsal midline of body from posterior end of opercle to caudal fin base; two light yellow bands present at lateral side of body, running parallel to black lateral band just above it and attaching to it at around caudal peduncle. all fin membranes colorless or transparent. the discovery of e. metallicus in the kapalo banda river is a new record in addition to its previous records (fig. 3). new records of fish contribute to understanding species biogeography (valen et al., 2022b, c) and the distribution range of species (ihwan et al., 2020; valen et al., 2020). species location accession number authors esomus metallicus thailand jf915604.1 collins et al. (2016) esomus metallicus thailand fj753495.1 britz, et al. (2016) esomus metallicus thailand kc456399.1 panprommin et al. (2013) esomus metallicus thailand mk049431.1 panprommin et al. (2019) esomus metallicus thailand kf805365.1 phuttawongk et al. (2015) esomus longimanus thailand hm224169.1 tang et al. (2015) esomus longimanus vietnam mk777274.1 thu et al. (2020) esomus thermoicos sri lanka mk214872.1 sudasinghe et al. (2018) esomus thermoicos sri lanka mk214871.1 sudasinghe et al. (2018) esomus cf ahli, ef452888.1 mayden et al. (2016) esomus danricus india kf511504.1 dhar et al. (2015) esomus danricus india kf511505.1 dhar et al. (2015) esomus danricus india kf511506.1 dhar et al. (2015) castostomus commersonii america hq557394.1 april et al. (2011) table 1. dna sequense of genus mystacoleuscus from genbank. 62 robin et al./ a new distributional record of flying barb kapalo banda river is not connected to maninjau lake, and their distance is about 74 km; therefore, e. metallicus are not naturally moved to this new record from that previously recorded site. therefore, e. metallicus was probably released into kapalo banda river accidentally by local farmers, although this area is not an aquaculture area. however, several streams connected to the kapalo banda river have some fish farming activities, so it is possible that fish have been introduced into kapalo banda river. esomus metallicus is an exotic species with 2 long beards in the mouth and used as an ornamental fish. esomus metallicus is exotic to indonesia and inhabits with natural and endemic fishes of sumatra e.g. rasbora spp. can be a competitor to those small fishes (hasan et al., 2020; albornoz-garzón and figure 2. specimen of esomus metallicus found in kapalo banda river in 50 kota district west sumatera province. meristics (counts) present study hasan et al. (2020) rainboth (1996) pored lateral line scales 13-14 10-14 dorsal fin rays 8 8 8 anal fin rays 8 8 8 pectoral fin rays 14 14 ventral fin rays 7 7 caudal fin forked forked forked forked table 2. meristic characteristics of esomus metallicus collected from the kapalo banda river in 50 kota district, west sumatera province, indonesia (n=15). figure 3. distribution of an invasive esomus mettalicus in sumatera. the triangle circles are the previous record. the square is the recent record from kapalo banda river, 50 kota district, west sumatera, indonesia. 63 int. j. aquat. biol. (2023) 11(1): 59-67 villa-navarro, 2017). alien or invasive fishes enter new areas either intentionally or not and can potentially disrupt endemic fishes by adapting to new environments and competing with natural fishes (radkhah et al., 2016; insani et al., 2020; bariyyah et al., 2021). hence, there is a need for an in-depth study of the impact of e. metallicus in waterbodies on endemic fish and their habitats for making policies to control invasive fishes. the establishment of non-native species has a severe impact on ecosystem functioning by affecting community levels of native species (sato et al., 2010; mousavisabet and eagderi, 2016). ecological risks of alien fishes are difficult to be estimated in the initial stages, but their harm is irreversible. moreover, the kapalo banda river is protected by customary law to maintain native and endemic fish, an ancestral heritage that must be preserved. dna-barcoding of striped flying barb from kapalo banda river successfully sequenced with a base-pair length of 680 bp (table 3). fragments with more than 658 base pairs of coi genes can be used as a standard for differentiating animals (hebert et al., 2003). the results revealed that the samples from the kapalo banda river are e. metallicus with a similarity of about 99-100% (table 4). to ensure species validity, we also performed species identification by bold system by specimen identification tools to check the species-level barcode records. the submitted sequence has been matched to e. metallicus with a similarity of 100% to the bin id: bold: aaf2628. according to hebert et al. (2003), species with 99-100% similarity levels are identical. in the phylogenetic tree, striped flying barb from west sumatra, indonesia (red circle) clustered within e. metallicus species (fig. 4) with 100% dna similarity with those of thailand. therefore, specimens of west sumatra probably originated from thailand, or they are from the same ancestor. the kapalo banda river and thailand were formerly connected to the sundaland river; however, this species was not collected before in sumatra (kottelat et al., 1993), and since there is no difference between their sequences, as is common for long-distant species, therefore striped flying barb from west sumatra has originated from thailand. according to hasan et al. (2020), e. metallicus in indonesia was introduced and accidentally released from fish farms. there are nine esomus species, including e. danrica (shangningam and kosygin, 2022; abujam et al., 2021), e. nimasowi (abujam et al., 2021), e. metallicus (jamaluddin et al., 2022), e. malayensis (kottelat et al., 2013), e.thermoicos (sudasinghe et al., 2019), e. longimanus (kottelat, 2001), e. caudiocellatus (kottelat, 2013), e. bengalensis (bhakat and sinha, 2020), e, altus table 3. sequence of striped flying barb, esomus metallicus from the kapalo banda river in 50 kota district, west sumatera province, indonesia. dna barcoding aagacattggcaccctttatttagtatttggtgcctgagctggaatagtgggaaccgccctgagccttcttatccgagctgaa ctaagccaacccggatcacttctaggcgatgaccaaatttacaatgttattgttactgcccacgcttttgtaataatctttttt atagttataccaattcttattggaggatttggaaactgacttgtaccattaatgattggagcaccagacatagcatttccacg aataaataatatgagtttctgactcctgcccccatcatttcttttactactagcctcatctggggttgaggctggagcaggaa cagggtgaacagtatatccacctcttgctggaaaccttgcccatgcgggagcatcagtagatctaactatcttttcgttacac ttagcaggtgtttcatcaattttaggagcaattaattttattactaccattattaatatgaagccccctgccatttctcaatat caaacaccactgtttgtttgagccgttttagtaacagccgttcttctcctactatcactaccagttttggcggctggaattaca atgcttcttacagaccgtaacctcaataccaccttctttgacccagcaggaggaggagacccaattctttatcaacacttatt ctgattctttggc specimen similarity species identified accession number family genus striped flying barb striped flying barb 100% esomus metallicus fj753495 cyprinidae esomus 100% esomus metallicus mk049431 cyprinidae esomus table 4. species identification and similarity. 64 robin et al./ a new distributional record of flying barb (kottelat, 2013) and e. ahli (kottelat, 2013). in the genbank, there are only five esomus species’ coi genes, including e. metallicus (fj753495) from thailand, e. danricus (kf511504) from india, esomus cf. alhi (ef452888), e. thermoicos (mk214871) from sri lanka and e. longimanus (hm224169) from vietnam. the genetic distance of e. metallicus from kapalo banda river was 0.00 to that of thailand. esomus metallicus is the closest species to e. longimanus, with a genetic distance of 0.028 (table 5). esomus metallicus is native to thailand (beamish et al., 2006) and southeast asia, and e. longimanus is native to asia (doi, 1997) and is also found in thailand, showing a close ancestor with e. metallicus. acknowledgments we thank the research and community service institute (lppm) of the university of bangka belitung for facilitating research and publications and indra mayendra dt. marajo nan hitam as our guide. references abujam s., gogoi b., das a.n., das d.n., biwas s.p., bleher h. 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(2021) 9(2): 115-123 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article the omani sandfish sea cucumber, holothuria scabra jaeger, 1833 (holothuroidea: holothuriidae): fishery, length-weight relationship and condition factor saud m. al jufaili*1, khalfan m. al rashidi2, iman s. al kharusi3 1department of marine science and fisheries, sultan qaboos university, po box 34, al-khod 123, sultanate of oman. 2directorate general of fisheries research, ministry of agriculture and fisheries wealth, po box 427, muscat 100, sultanate of oman. 3ministry of agriculture and fisheries wealth, po box 427, muscat 100, sultanate of oman. s article history: received 7 december 2020 accepted 22 february 2021 available online 2 5 april 2021 keywords: echinodermata holothuroidea fisheries management oman sea abstract: the fishery of omani sea cucumber, holothuria scabra, is limited so far to the mahout bay in al wusta governorate. this fishery supports an uncertain number of fishermen ranging from 100 to 150. this fishery is not well-studied and has no current management program. in order to prepare a management strategy, the government has suspended this fishery for 2 years from march 2018 to march 2021. apart from presenting a thorough review on the sea cucumber fishery in oman, the current work presents a detailed analysis of the length–weight relationship, sex ratio, and condition factor of the h. scabra collected from four different areas during the period february-may 2019. based on the results, the females were longer than males, that is 226±33.4 and 221±37.8 mm, respectively. the overall sex ratio of the entire/pooled sample was 0.49 which was not different from the anticipated theoretical sex ratio of 0.5. the mean kn and the empirical weight–length equations for the total sample were between 0.12±0.01 and 1.0±0.16, and w=0.03l1.6, respectively. introduction sea cucumbers are non-migratory space-specific species that are found in shallow protected seagrass and muddy sea beds around and within protected bays, coral reefs, and mangrove areas (hamel et al., 2001; agudo, 2006). they are reported to spawn throughout the year with enhanced peaks at certain period of the year (lokani, 1990; battaglene and bell, 2004). in addition, sea cucumbers reproduce asexually as well (conand, 1996; conand et al., 2002; dolmatov, 2014) and their life span ranges between 5 and 12 years depending on the species (conand, 1990). sea cucumber larvae spend 70 days in a planktonic stage before they settle into the ground where they develop into a juvenile. the growth rate of sea cucumbers is assumed to be slow and may take up to 5 years to mature (conand, 2008). it grows up to 0.5 cm per month (≈14 g) under favorable conditions and attains maturity between 23 and 26 cm (agudo, 2006; navarro et al., 2012; omar et al., 2013; yanti et al., 2020). sea cucumbers are scavengers or deposit *correspondence: saud m. al jufaili doi: https://doi.org/10.22034/ijab.v9i2.1168 e-mail: sjufaily88@gmail.com feeders (mercier et al., 1999). there are reports suggesting existence of sea cucumbers from long ago in many places, for example solomon islands, india, the philippines, and indonesia among others (conand, 1990; battaglene and bell, 1999) and are known to be harvested for decades from the indo-pacific region (james, 2001; bumrasarinpai, 2006). yet, there is not much information on sea cucumber fisheries around the world. with the increasing demand for sea cucumbers in the international market, the different types and sizes of sea cucumbers excavated in the absence of management resulted in more pressure for sea cucumber hunting and eventually to overfishing (conand, 2008; hair et al., 2018). overfishing has been documented in australia (skewes et al., 2006), fiji (preston, 1988), and new caledonia (conand, 1990). in several countries, it was heavily exploited, which led these countries to apply moratorium on the fishery and ban fishing like in the case of the egyptian sea cucumber (hasan, 2003), the papua new guinea 116 al jufaili et al./ fishery, length-weight relationship and condition factor of holothuria scabra (hair et al., 2018), and turkey (aydin, 2017). other countries have introduced zonation or sea cucumber marine-protected areas where fishing areas are closed for a period of up to 4 years before they are opened again. in addition, whenever open, closed season is always applied during reproductive season (purcell et al., 2016). the price and demand for sea cucumber increased 16.6% in the international markets from 2011 to 2016 (purcell et al., 2018). the increase in demand has an effect on the international fishing activities for sea cucumbers. the sea cucumber fishery and industry in oman is in the initial stage, and the fishery being so far limited to small areas of mahout bay and targeted by small number of traditional fishermen. omani sea cucumber, holothuria scabra is a valuable species and an excellent source of income for the local communities and the fishery so far is an open access and hardly studied. so far, in oman there are no fixed records of sea cucumber landings or about the actual number of fishermen involved. furthermore, there are no data available on the basic stock assessment or population distribution of the sea cucumbers. basically, there are plenty of missing information that needs to be understood and managed to develop the fishery. hence, the current study reviews the omani sea cucumber fishery and provides information on the length–weight relation and sex ratio from four main fishing areas in oman. materials and methods all the available publications and reports (published and unpublished) on sea cucumbers were reviewed to understand their fishery. fishing areas were visited and officials, fishermen, traditional processors, and traders were interviewed. the review present data on the sea cucumber landings, income, export, and any other information. as per the information obtained, the sandfish sea cucumber samples were collected from four major fishing areas viz. ashaghia, hofnat, al naqil, and ashaghia (fig. 1) in mahout bay during the period february-may 2019. the samples were measured to the nearest of 1 mm for total length (from mouth to anus) using a flexible plastic tape. an electronic balance was used to record the wet weight to the nearest 1 g for each sea cucumber. sea cucumbers were dissected for visual sex identification purposes. length-weight relationship was calculated using pauly’s (1984) equation of 𝑊𝑊 = 𝑎𝑎 × 𝐿𝐿𝑏𝑏, where w is the wet weight in g, l the total length in mm, a and b the regression constants, “a” the intercept, and “b” the slope. fulton’s condition factor (kn) was obtained by 𝐾𝐾n = 𝑊𝑊/(𝑎𝑎 × 𝐿𝐿𝑏𝑏) (le-cren, 1951). the length– weight relation, the condition factor, and sex ratio were obtained for the whole sample, for each area, males, females, and unsexed sea cucumbers. the sex ratios were also presented by different length intervals and per sampling area. results the fishery: the sandfish sea cucumber is the primary species among the other 21 different species that are identified in oman (claereboudt and alfigure 1. the mahout bay and the sea cucumber fishing areas. 117 int. j. aquat. biol. (2021) 9(2): 115-123 rashdi, 2011). according to the information available, it is distributed around the mahout bay in the gulf of masira along the al wusta governorate in sandy protected and seagrass bays. six main fishing areas are famous in this region, alnaqil, al-eigah, wadsumah, al-shaghia, al-hofnat, and ras-knasah (fig. 1). the fishery started in the 1960s at a low scale and the catch was exchanged for food. owing to the demand from the neighboring countries, the fishery expanded in 2003 and 2004 (al-rashdi et al., 2007). as it is not very difficult to catch sea cucumbers, this fishery encourages women and their kids also to help in catching practice, in many cases, they give a competition to the fishermen (fig. 2). to catch sea cucumbers easily, one requires to walk on shallow water, spot the sea cucumber, and catch it. it is reported that women make 50% of the total fishing community and men contribute up to 30%, while kids contribute 20% (maf, 2009). few of these people dive in traditional ways to about 3-m depth, especially during high-tide periods. of late, the number of divers were observed to increase due to higher market demands. the intensity of fishing increases during low and extreme low-tide periods. the actual number of total fishermen involved in the fishery is not known, however, it is expected to be around 100–150 (unpublished data). there is no information about the number of traders involved; however, a vast percentage of sea cucumbers that are eviscerated, boiled, dried, re-boiled, and re-dried are collected in woven bags and exported to neighboring countries in car trunks. collecting and selling sea cucumbers became popular among fishing people in mahout bay area and the landings increased from 3.6 t in 2013 to 39.4 t in 2015. targeting sea cucumber is open throughout the year and the intensity depends on the demand and request from the traders. every year, however, the fishermen target the sea cucumbers from november to march for 3-4 h per day with an average catch of 100 different sizes of sea cucumbers (maf, 2009). an increase in collecting and processing sea cucumber into beche-de-mer among the fishermen and even drying undersized sea cucumbers are also observed (fig. 3). the unlicensed number of fishermen doubled in 2004-2005, that is to 100-200, respectively, keeping in mind the increase in prices per kg to five times from 2000 to 2005 (al-rashdi et al., 2007). it was also noticed that the fishermen started to dry undersized sea cucumber widely within figure 2. women and kids wading for sea cucumbers. figure 3. samples of sea cucumber catches. sea cucumbers range from large to undersized. 118 al jufaili et al./ fishery, length-weight relationship and condition factor of holothuria scabra mahout bay area ultimately resulting in an increased number of fishermen. as a result, the government, as a precautionary approach, decided to suspend the fishery for 1 year starting from march 2018 to march 2019. the ministry later extended the suspension for another year until 27th march 2021. although the suspension still exists, few people were found with approximately 1.2 t of illegal sea cucumbers in 2018 (unpublished data). the price of each sea cucumber ranges from 0.5 to 2.00 riyal omani (or) (1 or = 2.6 $), while the price per kg for the dry sea cucumbers range between 35 and 55 or. it is worth mentioning that 100 sea cucumbers make up to almost 2 kg after drying (maf, 2009). length-weight relationship and sex ratio: during the present study, a total of 713 sea cucumbers were collected from four fishing areas, of which 121 were males, 116 females, and 494 unsexed. in all, 424 (59%) samples were collected from the hofnat area, with 374 unsexed samples, 26 males, and 24 females. a total of 142 (19%) samples were collected from al naqil area, with 70 unsexed samples, 39 males, and 33 females. a total of 84 (12%) samples were collected from ashaghia, with 41 unsexed samples, 23 females, and 20 males. finally, 81 (11%) samples were collected from aleigah with 36 males, 36 females, and 9 unsexed (table 1, fig. 4). the maximum length recorded was 320 mm for a male sample from al naqil, while the smallest sample was an unsexed sea cucumber of 70 mm length from hofnat area (table 1). overall, the females were found to be longer than males, that is 226±33.4 and 221±37.8 mm, respectively. on the other hand, the heaviest sea cucumber, wet weight of 600 g, was captured from al naqil area, while the lowest weight recorded was 280 g from ashaghia. overall, males were heavier than females, that is 271±81.49 and 264.48±64.61 g, respectively (table 2). the overall sex ratio of the entire/pooled sample was 0.49 which was not different from the anticipated theoretical sex ratio of 0.5 ( χ2=0.11; df=1; p>0.05). the sex ratio within the sampling areas al naqil, aleigah, ashaghia, and hofnat were 0.49, 0.53, 0.5, and 0.48, respectively, and all were not different from table 1. descriptive statistics for the sandfish sea cucumber, holothuria scabra from oman. sampling area mean median mode minimum maximum count al naqil male 237 240 210 170 320 39 female 252 260 260 190 300 33 unsexed 232 235 230 130 300 70 combined 238 240 250 130 320 142 aleigah male 221 220 220 160 310 36 female 224 220 230 170 290 36 unsexed 190 190 190 130 250 9 combined 219 220 230 130 310 81 ashaghia male 208 210 210 160 310 20 female 211 210 210 160 250 23 unsexed 173 180 180 100 250 41 combined 192 195 210 100 310 84 hofnat male 207 210 210 150 270 26 female 207 210 220 160 290 24 unsexed 142 140 140 70 270 374 combined 149 140 140 70 290 424 all data male 221 210 210 150 320 121 female 226 220 220 160 300 116 unsexed 158 150 140 70 300 494 combined 179 170 140 70 320 731 119 int. j. aquat. biol. (2021) 9(2): 115-123 the theoretical sex ratio of 0.5 (p>0.05). the monthly sex ratios ranged between 0.38 (al naqil/may sample) and 0.75 (ashaghia/february sample) (table 3). the sex ratio versus total sea cucumber length within the sampling areas showed no pattern and females could be more than male in any given length group or could be less than the males (fig. 5). however, the overall sample sex ratio did not show a pattern for sea cucumbers below 180 mm or above 240 mm. the overall sex ratio showed a pattern between lengths 180 and 230 mm and is explained by the power function (sex ratio=2.1*10-7 l 2.7, r=0.97) (fig. 6). in addition to the mean kn, the empirical weight-length equations for the total sample, female, male, unsexed, and combined male and female h. scabra per sampling area are presented in table 4. the length-weight relationship for the total samples collected was w=0.03l1.6 for a total of 680 male, female, and table 2. length (mm) and wet weight (g) data collected from the four different sampling areas during the period february–may 2019. sampling area mean median mode minimum maximum count l(mm) w(g) l(mm) w(g) l(mm) w(g) l(mm) w(g) l(mm) w(g) l(mm) w(g) al naqil male 237 295 240 270 210 240 170 170 320 600 39 39 female 252 309 260 300 260 300 190 180 300 500 33 33 unsexed 232 254 235 240 230 240 130 120 300 600 70 70 combined 238 278 240 255 250 300 130 120 320 600 142 142 aleigah male 221 256 220 250 220 250 160 140 310 550 36 36 female 224 256 220 250 230 230 170 170 290 430 36 36 unsexed 190 198 190 230 190 230 130 100 250 280 9 9 combined 219 219 220 220 230 230 130 130 310 310 81 81 ashaghia male 208 241 210 235 210 190 160 190 310 400 20 20 female 211 246 210 240 210 260 160 150 250 340 23 23 unsexed 173 185 180 200 180 210 100 50 250 280 41 41 combined 192 215 195 220 210 240 100 50 310 400 84 84 hofnat male 207 210 210 150 270 26 female 207 210 220 160 290 24 unsexed 142 125 140 120 140 130 70 30 270 450 374 374 combined 149 140 140 70 290 424 all data male 221 271 210 250 210 250 150 140 320 600 121 121 female 226 264 220 250 220 230 160 150 300 500 116 116 unsexed 158 150 150 130 140 130 70 30 300 600 494 493 combined 179 188 170 180 140 130 70 30 320 600 731 731 figure 4. the length–frequency distribution of male, female, and unsexed sandfish sea cucumber samples (top graph) and for the combined males and females (bottom graph). 120 al jufaili et al./ fishery, length-weight relationship and condition factor of holothuria scabra unsexed sea cucumbers. the linear regression resulted in a high goodness of fit (r2) which ranged from 0.61 to 0.9 (table 4). the mean condition factor (kn) for h. scabra, ranged between 0.12±0.01 and 1.0±0.16 (table 4). overall, the unsexed sea cucumbers seemed to be on the lower side of the wellbeing with kn of 0.89±0.16. per sampling area, the males in ashaghia had the lowest condition factor with a kn of 0.12±0.01. discussions the sandfish sea cucumber fishery in oman is advancing fast to its developing stage. the number of fishermen involved increased fast. there is a tendency among fishermen to explore more fishing grounds as the demand increases. enforcing regulations therefore table 3. monthly and overall sex ratios for sea cucumber samples collected from anaqil, ashaghia, aleigah, and hofnat sampling areas during the period february-may 2019. alnaqil ashaghia month sex ratio chi-square p-value sex ratio chi-square p-value feb-19 0.43 0.43 0.51 0.75 1 0.32 mar-19 0.5 0 1 0.55 0.14 0.71 apr-19 0.5 0 1 0.44 0.22 0.64 may-19 0.38 0.69 0.41 0.57 0.29 0.6 overall 0.49 0.5 0.48 0.53 0.21 0.65 aleigah hofnat feb-19 0.5 0 1 0.66 0.33 0.56 mar-19 0.47 0.07 0.8 0.56 0.53 0.47 apr-19 0.47 0.05 0.82 0.4 0.6 0.44 may-19 0.55 0.2 0.65 0.4 0.6 0.44 overall 0.5 0 1 0.48 0.08 0.78 feb-19 0.5 0 1 mar-19 0.52 0.15 0.7 apr-19 0.46 0.53 0.47 may-19 0.48 0.06 0.8 overall 0.49 0.1 0.74 figure 5. sex ratio distribution based on sandfish sea cucumber length (mm). 121 int. j. aquat. biol. (2021) 9(2): 115-123 could be an issue if stricter measures are applied. the overall sex ratio for samples collected for this study was not different from 1:1 (0.49), and this was different from the sex ratio within the sampling areas ranging 0.48-0.53. this result was supported by the h. scabra study from abu rhamada island in the red sea (hassan, 2005). the female-to-male ratio for the same species was 1.48:1 for samples collected from kudat, malaysia; however, the sex ratio was not significantly different (arsad et al., 2017). the sex ratio for samples collected from mahout bay in oman was reported to be 1:1 (al-rashdi et al., 2007). as far as the sex ratio among different length groups is considered, the sex ratio did not have a prevailing of one sex over the other. the sex ratio power function identified within this study can be used for reconstructing the sex structure from catch data and therefore can be used for sex-based management applications and assessment models. analysis of length-weight relationship indicated that the growth is negative allometric as all the bvalues obtained were lower than 3. the b-values ranged from 1.22 to 1.88 for the different sexes and for the unsexed in different sampling areas. in addition, the overall b-values were 1.4, 1.3, 1.65, and table 4. length-weight equations from different sampling areas with the mean condition factor provided. sampling area length-weight n r2 mean kn±sd gender relationship al naqil male w=0.07l1.53 39 0.82 1.01±0.13 female w=0.04l1.62 33 0.61 1.01±0.13 unsexed w=0.34l1.22 70 0.67 1±0.14 male+female w=0.07l1.52 72 0.73 1±0.12 combined w=0.11l1.43 142 0.7 1±0.14 aleigah male w=0.17l1.36 36 0.76 1±0.13 female w=0.3l1.47 36 0.78 0.4±0.04 unsexed 9 male+female w= 0.21l1.31 72 0.77 1.00±0.11 combined w=0.13l1.41 81 0.82 1±0.11 ashaghia male w=1.02l1.02 20 0.88 1.00±0.07 female w=0.43l1.18 23 0.63 1±0.12 unsexed w=0.08l1.5 41 0.84 1±0.16 male+female w=0.74l1.08 43 0.75 1±0.09 combined w=0.13l1.41 84 0.84 1±0.13 hofnat male female unsexed w=0.01l1.88 374 0.8 1±0.17 male+female combined w=0.01l1.82 424 0.8 1±0.21 total sample male w=0.17l1.4 95 0.8 1±0.21 female w=0.19l1.3 92 0.71 1±0.12 unsexed w=0.33l1.65 493 0.86 0.89±0.16 male+female w=0.18l1.34 187 0.746 1±0.12 combined w=0.03l1.64 680 0.9 1±0.16 the equations are given by sampling areas and by genders. figure 6. relationship between the lower total length interval (cm) and the total sample sex ratio for the omani sandfish sea cucumber, holothuria scabra. 122 al jufaili et al./ fishery, length-weight relationship and condition factor of holothuria scabra 1.64 for the male, female, unsexed, and for the combined sexes, respectively. these b-values were significantly lower than the regression coefficient of 3 (pauly, 1983) i.e. the length growth is greater than the weight growth, results that were supported by length– weight relationship of h. spinifera, bohadschia marmorata, stichopus naso, and h. atra samples collected from point pedro and mullaitivu in northeast region of sri lanka (veronika et al., 2018) and from gulf of mannar, india (venkataraman, 2007). the length-weight equations obtained by al-rashdi et al. (2007) was w=0.033l2.178 for h. scabra from the data collected from mahout bay, oman. this was also supported by h. scabra from fiji (w=0.1878l 2.5807) (lee et al., 2018). the results showed slightly higher b-values than the current study. different lengthweight results can be obtained by different studies depending on the sampling methodology, time of sampling, and area. the status of sea cucumbers cannot be concluded as underexploited or overexploited, since it is in its early stages of developing. it is recommended to form a liaison committee between the government and the sea cucumber fishermen society i.e. for a successful control over the fishery a strong co-management and cooperation between the government and the fishermen are highly recommended. acknowledgment we would like to thank the marine science centre for providing help to carry out this study. we are grateful to maryam saud al jufaili for her support with the maps. references agudo n. 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(2022) 10(5): 417-428 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article evaluation of pineapple waste crude extract in improving growth performance and resistance to aeromonas hydrophila in nile tilapia (oreochromis niloticus) priyaporn khumsrisuk, ratchaneegorn mapanao, nudtha nithikulworawong program of aquatic animal production technology, faculty of multidisciplinary studies, khon kaen university, nong khai campus, amphur muang, nong khai province, thailand. s article history: received 9 august 2022 accepted 24 october 2022 available online 2 5 october 2022 keywords: pineapple peel extract protein efficiency ratio mic relative percentage survival abstract: the study on the antibacterial activity of fruit, peel, stem, and core of the pineapple, ananas comosus crude extract against aeromonas hydrophila and the effect of supplementing the diet with pineapple waste crude extract on growth performance and resistance against a. hydrophila in nile tilapia, oreochromis niloticus were conducted. the best result of antibacterial screening was performed by disc method and the minimum inhibitory concentration was found in pineapple fruit extract with an inhibition zone of 7.96±0.20 mm and the minimum inhibitory concentration value was 125 mm per liter. the minimum bactericidal concentration value was not observed in all parts of the pineapple extract at any concentration. furthermore, nile tilapia (2.34±0.82 g) were stocked in 12 aquaria and fed a diet supplemented with 4 different doses of pineapple peel extract (0, 1, 2 and 3%) for 8 weeks. the results revealed that the best value of weight gain, average daily weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio and feed utilization efficiency was observed in fish fed 1% pineapple peel extract and additionally, the mortality of fish challenged by aeromonas hydrophila was recorded for 10 days after the challenge. the result showed that fish-fed diet containing 1% pineapple peel extract had significantly the lowest mortality and the highest relative survival percentage. introduction the total aquatic animal production is 82.1 million tons, of which 54.3 million tons of finfish (fao, 2020). forecasts of the total aquaculture production in thailand have been gradually growing from 0.6 to 0.9 million tonnes over the past 20 years (tiptiwa et al., 2020). in thailand, the most popular economic freshwater fish is nile tilapia, oreochromis niloticus. the highest tilapia production in 2019 was 228982 tons, or 53.58% of the total output, with a market value of 1143446 million baht (department of fisheries, 2021). nile tilapia is one of the species of global aquaculture because they tolerate the environment well and tolerance to low dissolved oxygen. due to the widespread adoption of intensive aquaculture systems in tilapia farming, which requires higher stock densities to achieve higher yields per unit area, the feed requirement in tilapia correspondence: nudtha nithikulworawong doi: https://doi.org/10.22034/ijab.v10i5.1744 e-mail: nudtha@kku.ac.th dor: farming has continued to increase. the quality and quantity of aquaculture feed are important in the aquaculture business. nutritious aquaculture feeds and appropriate daily feeding rates are important factors in increasing fish growth and survival rates (asma et al., 2016). over the past three decades, fishmeal production has been limited, and the price has continued to rise in line with demand, negatively affecting commercial aquaculture's profitability (naylor et al., 2000). therefore, several studies are being conducted to find alternative protein sources, including plant, single-cell, and animal proteins (hardy, 2010). the potential for replacing fishmeal with plant-based proteins is great, and it may eventually take precedence in aquaculture feed (olsen and hasan, 2012). it has anti-nutritional properties that decrease fish's nutrient utilization capacity and growth rate (francis et al., 2001). in 418 khumsrisuk et al./ evaluation of pineapple waste crude extract in improving growth performance addition, an exogenous enzymatic supplementary diet can improve nutrient utilization (kolkovski, 2011), and increase protein digestibility (yigit et al., 2018), and an appropriate nutritional value is an alternative solution that is necessary to be pursued for fish. additionally, health performance improvement and disease resistance of nile tilapia/ are significant challenges faced by aquarists. moreover, the factor affecting tilapia culture is a. hydrophila causes the death of freshwater fishes. management of aquatic animal health in aquaculture is essential for a successful production. the prevention and control of bacterial diseases in antibiotic-used aquaculture has increased antibiotic-resistant bacteria. it can also lead to human complications due to the consumption of antibiotic-treated fish. plant extracts are currently used in aquaculture for treating and controlling bacterial diseases instead of antibiotics, causing resistance to specific pathogenic bacteria (seyfried et al., 2010). the use of plant extracts is an alternative option for prophylaxis by increasing the immunity of fish, allowing it to control the spread of the infection. in addition, the plant extracts are environmentally friendly and do not cause any side effects. pineapple, ananas comosus, native to central and south america. it is grown in tropical and tropical countries such as thailand, the philippines, malaysia, indonesia, china, kenya, and india. in 2020, thailand was reported as the seventh pineapple producer worldwide, with 1.5 million tons. the export volume of canned pineapple and pineapple juice amounted to 290123 and 41914 tons, respectively (shahbandeh, 2022). during pineapple processing, approximately 60% of the waste is produced, including peel, core, stem, and crown (ketnawa et al., 2012). disposing of large amounts of waste from processing is an ongoing problem due to its tendency to spoilage by microorganisms and cause serious environmental problems. therefore, innovations leading to the utilization of this waste are useful in managing the large amount of waste generated by processing. according to the composition of physicochemical and nutritional values such as bromelain, organic acids, antioxidants and phenolic compounds, pineapple can be considered one of the most useful fruits in producing value-added substances (ali et al., 2020). pineapple by-products are an abundant source of bromelain (proteolytic sulfhydryl enzymes) belonging to the family of proteindigesting enzymes that can be extracted from pineapple in several native cultures (gregory and kelly, 2016). bromelain is a pricey combination of proteolytic enzymes with various biological properties that are utilized in the pharmaceutical, culinary, and cosmetic industries (ramli et al., 2017). moreover, pineapple has been used as a medicinal plant is attributed to bromelain. the pineapple extract also contains cytotoxic, antidiabetic, hyperlipidemia, antioxidant properties (das et al., 2019), anti-edematous, various closely related proteinases, fibrinolytic, anti-inflammatory activities, antithrombotic and anti-cancer activity and for a wide range of therapeutic benefits (pavan et al., 2012). pineapples are important in the medical, nutritional supplement to promote health, leather and textile industries, folk medicine and digestive assistance. it is appropriate to consider the pineapple as a feed additive for aquaculture. therefore, this research aimed to evaluate the utilization of pineapple properties to increase the growth efficiency and disease resistance of nile tilapia. material and methods pineapple crude extract preparation: the pineapples were bought from the market in si chiang mai district, nong khai province, thailand. the fruits were cleaned, air-dried, and its part was separated (fruit, peel, stem, and core) and then chopped into small pieces before being blended with cold distilled water in a 1: 1 ratio for 3 minutes. the resulting mixture was filtered through a cheesecloth and centrifuged at 10,000×g for 20 minutes and collected the obtained supernatant (crude enzyme extract). bacteria preparation: the bacteria isolated from 419 int. j. aquat. biol. (2022) 10(5): 417-428 the liver and spleen of the diseased fish and crossed steak on a nutrient agar plate (himedia) and incubated at 37°c for 18 hours. the predominant bacterial colony was selected and grown on na plates to obtain a pure culture. the species of bacteria were identified by morphology using gram’s stain and biochemical properties using api 20e strip (biomerieux). the isolated colonies were cultivated in nutrient broth (himedia) and incubated at 37°c for 18 hours. the bacteria’s turbidity was adjusted to 0.5 mcfarland turbidity standards no.0.5 (1.5x108 cfu/ml). this bacterial suspension was used for the antibacterial activity and challenge test. antibacterial activity of pineapple extracts by disc diffusion method: the antibacterial activity of different parts of pineapple extracts was conducted by the disc diffusion method (bauer et al., 1966). briefly, the prepared bacteria were inoculated onto mueller hinton agar (mha; himedia) surface and distributed evenly with a sterile l-shaped glass rod. the sensitivity test was performed after the plates were dried for 3 to 5 minutes. the different pineapple extracts (fruit, core, stem, and peel) in a volume of 20 µl were added to the filter paper discs (5 mm in diameter) and allowed to dry. then the paper discs were impregnated on the agar plate and incubated at 37°c for 24 hours. the diameter of the inhibitory zone surrounding the discs was measured to assess the antibacterial activity. all tests were carried out in triplicates and expressed in millimeters. determination of minimal inhibitory concentration (mic) and minimal bactericidal concentration (mbc): mic values were determined by the two-fold serial dilution method. briefly, one ml of mhb and one ml of pineapple extract were added to each test tube. after that, one ml of suspended a. hydrophila was inoculated into these test tubes, followed by incubation at 37oc for 24 h. the tube with the lowest concentration and no visible bacteria growth was considered as mic. two test tubes were used as negative and positive controls, including the extract with no bacteria and bacteria without the extract. all tubes with no visible growth were used to examine the presence or absence of growth by plating onto an agar plate and incubating at 37oc 24 h. the mbc was recorded as the lowest concentration with no detectable growth. experimental diets and experimental design: the experimental diet was prepared using a fish meal, ingredients (%) basal diet fishmeal (60%)1 33 soybean meal (45%)2 10 rice bran 36 corn meal 9 soybean oil 2.5 fish oil 2.5 α-starch 5 dicalcium phosphate 1 premix3 1 total 100 proximate composition by analysis (% dry weight on basis) protein 30.28 ash 11.39 fat 12.86 moisture 8.72 fiber 1.31 1fish meal and 2soybean meal obtained from faculty of agriculture, khon kaen university, thailand. 3vitamin and mineral mixture provided the following per 1 kg diet :vitamin a 4,00,000 iu, vitamin d3 450,000 iu, vitamin e 6,500 iu, vitamin k3 1,000 mg, vitamin b1 3,900 mg, vitamin b2 2,400 mg, vitamin b6 2,250 mg, vitamin c 15,000 mg, vitamin b12 5 mg, biotin 120 mg, and niacin 5,520 mg . table 1. ingredients and chemical composition of the experimental diet. 420 khumsrisuk et al./ evaluation of pineapple waste crude extract in improving growth performance soybean meal, rice bran, corn meal, soybean oil, fish oil, α-starch, dicalcium phosphate, and premix as ingredients which contain 30.28% protein, 12.86% lipid and 11.39% ash estimated by aoac (2010) (table 1). in the experimental diet, pineapple extract was sprayed on top of the experimental diet and varnished with fish oil by giving four different concentrations of 0, 1, 2 and 3% (v/w feed) pineapple peel extract (ppe). group i received 0% of ppe (control), group ii 1%, group iii 2% and group iiii 3% of ppe, respectively. the experimental design used a complete randomized design (crd), which consisted of 4 treatments. twelve aquaria containers (0.45×0.90×0.45 m, water volume 150 liters) were each stocked with 15 fish. each experimental diet was fed to fish in three aquaria. the fish were fed the experimental diet at 3% of their body weight twice daily at 09:00 and 15:00 for 8 weeks. the feed consumption was recorded weekly, and every two weeks was weighed to measure the growth of the fish in each tank. the amount of feeding was changed every two weeks at the end of the experiment. at the final weighing and growth performance was calculated. growth performance: fish from all groups were selected randomly every two weeks and the growth performance was assessed by calculating the following growth parameters (silva and anderson, 1995; tacon, 1987; bake et al., 2014). weight gain (wg) = final body weight initial body weight average daily gain (adg) = ((final body weight initial body weight))/ (total duration of experiment (days)) specific growth rate (sgr) = ((ln final weight-ln initial weight))/ (total duration of experiment (days)) x 100 relative growth rate (rgr) = [(final body weight initial body weight) / (initial body weight x total duration of experiment (day)] x 100 feed conversion ratio (fcr) = (total feed intake (g))/ (weight gain (g)) survival rate (sr) = (final fish number)/ (initial stocked number) x 100 protein efficiency ratio (per) = [(final body weight (g) initial body weight (g)) / protein consumed (g)] x 100 feed utilization efficiency (fue) = [(final body weight (g) initial body weight (g)) /total amount of feed consumed (g)] x 100 isi (%) = [wet weight of intestine (g)/wet weight to fish (g)] × 100 fish proximate composition: at the end of the 8week, fish were taken from each tank. proximate constituents, including protein, moisture, ash, and lipids, were evaluated based on aoac (2010). the moisture was determined by drying the sample in an oven at 105°c. crude lipid was determined by chloroform-methanol extraction (2:1 v/v). crude protein was determined (kjeldahl procedure: n x 6.25) using an automatic kjeldahl system. ash was measured by incineration in a muffle furnace at 500°c for 6 hours. challenge study with a. hydrophila: the challenge test with a. hydrophila was performed based on a modified method of dong et al. (2017). the fish from the four groups was selected randomly at 10 per group. they were challenged with 0.1 ml of intraperitoneal a. hydrophila containing 1.5x108 cfu/ml. the fish was then kept for another 10 days. mortality was recorded every day to day 10 of treatment. the data were calculated for mortality rate percentage and relative percentage of survival (rps) (ellis, 1988) as the following: rps = 1((% mortality in treatment / % mortality in control)) × 100 statistical analysis: all data were expressed as mean ± standard deviation (sd). the treatments were compared using one-way analysis of variance (anova), followed by duncan's new multiple range test (dmrt) in spss 27.0 software. the level of significant difference of p<0.05. results antibacterial activity: the results of the antimicrobial activity assay of all crude extracts against a. hydrophila are presented in table 2. the 421 int. j. aquat. biol. (2022) 10(5): 417-428 highest antibacterial activity was observed in pineapple fruit extract with an inhibition zone of 7.96±0.20 mm and significantly different from extracts from other parts (p<0.05). the highest mic value as 125 mg/ml was found in pineapple peel, fruit, and stem extract. the determination of mbc was not observed in all pineapple part extracts. growth performance: when nile tilapia were fed pineapple peel extract (ppe) for eight weeks, their growth response significantly improved (table 3). all fish groups fed the ppe-supplemented diet did not exhibit any growth response differences (p>0.05). the growth parameters and survival rate of all fish groups fed ppe were higher than those of the control group. all fish groups fed ppe showed significantly higher in weight gain (wg), average daily gain (adg), relative growth rate (rgr), protein efficiency ratio (per) and feed utilization efficiency (fue) than the control group (p<0.05). the best value of wg, adg, per, fue, specific growth rate (sgr) and feed conversion ratio (fcr) was observed in fish fed 1% ppe and had a significant difference with control (p<0.05). fish proximate composition: fish carcass composition are shown in table 4. there were significant differences in crude lipid, crude protein and ash between the experimental groups (p<0.05), but no differences were found in moisture between all experimental groups (p>0.05). challenge study: after the challenge test, the lowest mortality rate was found in fish fed pineapple pulp extract (ppe) in the diet at 1%, with an average of pineapple part inhibition zone (mm.) mic (mg/ml) mbc (mg/ml) peel 7.87±0.20a 125 na stem 7.10±0.10b 125 na fruit 7.97±0.20a 125 na core 6.97±0.15b 250 na p-value 0.00 na :no activity; the different superscript in column as significance difference (p<0 .05). table 3. growth parameters of nile tilapia fed diets containing different levels of pineapple pulp extract (ppe ( for 8 weeks . parameters different levels of ppe (%) p-value 0 1 2 3 initial wt (g) 2.77±0.05 2.76±0.05 2.85±0.11 2.84±0.09 0.430 final wt (g) 7.44±0.19a 8.26±0.33b 8.09±0.16b 7.87±0.18b 0.011 wg (g) 4.67±0.23a 5.49±0.39b 5.24±0.13b 5.03±0.25ab 0.029 adg (g) 0.083±0.004a 0.098±0.007b 0.094±0.002b 0.090±0.004ab 0.036 sgr (%) 1.76±0.074 1.95±0.109 1.86±0.053 1.81±0.091 0.122 rgr (%/day) 3.01±0.20a 3.76±0.48b 3.39±0.32ab 3.47±0.21b 0.019 fcr 1.66±0.11 1.41±0.13 1.53±0.07 1.59±0.13 0.127 sr (%) 84.44±7.70 91.11±10.18 86.67±6.67 93.33±6.67 0.535 per (%) 6.85±0.34a 8.09±0.28b 8.08±0.47b 7.91±0.47b 0.015 fue (%) 2.06±0.10a 2.43±0.08b 2.42±0.14b 2.37±0.14b 0.015 isi (%) 16.22±1.10 16.73±2.54 15.61±0.95 14.90±0.99 0.538 within a row, means with the difference letters are significantly different (p<0.05). table 4. carcass proximate composition of nile tilapia fed supplementary diets with different levels of peel pineapple extract (ppe) for 8 weeks . data are presented as mean±sd. proximate composition different levels of ppe (%) p-value 0 1 2 3 moisture (%) 4.96±2.18 6.13±1.08 4.67±0.36 4.88±0.13 0.663 crude lipid (%) 13.24±0.12a 13.62±0.32a 15.99±0.13b 13.57±0.34a >0.001 crude protein (%) 7.03±0.28c 6.07±0.67ab 5.75±0.33a 6.56±0.18bc 0.021 ash (%) 12.07±0.18c 12.38±0.31a 12.59±0.12c 12.38±0.21b 0.009 values in each row with different superscripts show a significant difference (p<0.05). table 2. antimicrobial activity of different part of pineapple crude extract against aeromonas hydrophila. 422 khumsrisuk et al./ evaluation of pineapple waste crude extract in improving growth performance 26.67±11.55%. although there were no significant differences across the treatments, all of them differed from the control significantly (p<0.05). fish fed diet containing ppe at 1% showed the highest relative percentage of survival (rps) at 50% compared to the other group supplemented with ppe (37.50% and 12.55%, in ppe 2% and ppe 3%, respectively) (table 5). while fish fed on ppe in the diet started to die after 48 hours, the control group developed clinical illness and died 24 hours after the challenge (fig. 1). discussion sustainable and ecologically friendly aquaculture is one of the most important aspects of any aquaculture business. one approach is to use agricultural byproducts as a component in aquaculture feed, encouraging high-quality aquatic feed and minimizing processing waste. therefore, the purposes of the current investigation were to determine how pineapple peel extract affected growth performance, feed utilization, and disease resistance against a. hydrophila in nile tilapia the growth performance showed that growth response was better among fish fed ppe mixes, especially with 1% ppe fed fish. fish-fed ppe supplemented increased wg, adg, rgr per and fur more than those of the control. these findings were in accordance with the finding of yuangsoi et al. (2018), who reported that nile tilapia fed 1% pineapple waste extract supplementation improved growth, protein digestibility and feed utilization were higher than those on the control diet. van doan et al. (2021) found that 10 g/kg pineapple peel powder was incorporated into the diets is clearly related to growth rate and feed efficiency in nile tilapia culture under biofloc system. sukri et al. (2022) reported that the addition of 30% pineapple waste powder in formulated diet can increase the growth rate of nile tilapia. furthermore, singh et al. different levels of ppe (%) mortality (%) relative percentage of survival (rps) 0 53.33±11.55 1 26.67±11.55 50.00 2 33.33±11.55 37.5 3 46.67±11.55 12.5 p-value 0.077 remark :within a column, means with the difference letters are significantly different (p<0.05). table 5. mortality rate and relative percent survival( rps (of nile tilapia after infected with aeromonas hydrophila for 10 days . figure 1. mortality rate of nile tilapia fed feeds incorporated with ppe and challenge with aeromonas hydrophila for 10 days. 423 int. j. aquat. biol. (2022) 10(5): 417-428 (2011) found that the common carp fingerlings that were supplemented with 1% pineapple waste extract had the best fish growth with the lowest fcr value. sharma et al. (2019) also revealed that common carp (cyprinus carpio) fingerlings fed pineapple peel extract-supplemented diet (ratio of 1:2) showed improved growth performances. subandiyono et al. (2018) showed that 1.5% pineapple extract in diet significantly affected the growth of java barb (puntius javanicus). deka et al. (2003) found that rohu (labeo rohita) fed a diet containing 25% pineapple waste powder showed significantly higher growth performance. tongsiri and pimpimol (2019) revealed that supplementation of pineapple crude extract in diet at 0.5 ml/kg was able to significantly increase growth rate and the average daily weight gain of the climbing perch (anabas testudineus). yusni et al. (2021) reported that the combination of 1.5% pineapple extract with commercial diet showed a significant increase in weight and length of catfish (clarias batrachus). the effect of exogenous protease supplementary in low fish meal (30 g/kg) pellets diet and extruded diets may improve growth, nutrient retention, and digestibility of the gibel carp (carassius gibelio) (shi et al., 2016), whereas supplementation of protease in diets with low fishmeal content can promote the growth of shrimp and tilapia like a diet with a high content of fishmeal (li et al., 2016). nilamsari et al. (2021) reported that a highly significant effect on feed utilization efficiency (epp), protein efficiency ratio (per) and relative growth rate (rgr) of milkfish (chanos chanos) was seen when fish were fed pineapple extract supplementation. the maximum dose of pineapple extract is 4.50, 4.26 and 4.39 ml can be produced at about 52.73%, per about 1.55% and rgr about 3.22%, respectively. pineapple peels contains a high concentration of bromelain followed by core, crown and stem (misran et al., 2019). the active ingredients in bromelain are the combination of cysteine proteases which would be hydrolyzing protein into amino acids thereby speeding up the feeding process. when protein in feed is easier to digest, fish will be able to use protein better that will make the growth better. so, fish protein utilization can be increased by supplementing the diet with the enzyme such as bromelain. this will result in increased fish growth and digestibility. improving growth efficiency is important by increasing protein digestibility and rapid absorption (wulandhari et al., 2017). as shown in the results, fish-fed a diet supplemented with ppe had a lower fcr than fish-fed a control diet, with the lowest fcr value occurring at 1% ppe. these findings could be attributed to the fish fed a diet supplemented with ppe having a quick metabolism, which enhances fcr. the protease enzymes can help in complete protein digestion. according to reports, 20-25% of proteins in animal feed are indigestible, however adding protease enzymes to the diet improves protein digestion (shi et al., 2016). protease enzymes are crucial for the digestive system's ability to break down proteins. it acts to hydrolyze proteins to release short-chain peptides in diet. this plays a key role in boosting growth factors, protein digestibility, and quick absorption. (rungruangsak-torrissen and sundby 2000). taqwdasbriliani et al. (2013) also mentioned that bromelain can be activated collagen conversion into gelatin by hydrolyzing the gelatin molecule. gelatin is a type of protein from the skin's collagen, which is the connective tissue of the bones or ligaments of fish. it acts as a component of bone. therefore, it may benefit bone growth. additionally, wiszniewski et al. (2018) reported that supplementation of bromelain enzyme improved the length of the villi and folds of the mucous membrane, activating lipase and pepsin and improving intestinal tissue and intestinal adsorbent cells, resulting in better intestinal absorption and digestion. on the contrary, if the amount of enzyme passes through the appropriate point, it may respond to negative effects and may inhibit the growth of fish (amalia et al., 2013). as shown in the present result, fish fed 3% ppe-supplemented diet decreased growth performance. this occurs because an excess 424 khumsrisuk et al./ evaluation of pineapple waste crude extract in improving growth performance of amino acids will affect how easily fish protein is digested, causing the amino acid-rich protein to be used for energy instead of growth (delima et al., 2017). wulandhari et al. (2017) also mentioned that an appropriate increase in the amount of pineapple affects the growth of the fish. based on the results of the antibacterial activity of crude pineapple extract in this study, all part of pineapple extracts showed antibacterial efficacy against a. hydrophila. pineapple peel extract (ppe) is a highly antibacterial waste product from pineapple processing and has no significance difference with pineapple pulp. from the previous results, pineapple peel extract can be further applied in aquatic feed for bacterial resistance. loon et al. (2018) studied different concentrations of pineapple extract as potential for an antibacterial agent against staphylococcus aureus, escherichia coli, corynebacterium spp., proteus spp., bacillus subtilis and s. pyogenes (ali et al., 2015), lubaina et al. (2019) reported that the pineapple peel with ethyl acetate extraction demonstrated significant antimicrobial activity against pathogens; staphylococcus aureus, e. coli, pseudomonas aeruginosa, vibrio cholera, and klebsiella pneumonia and the results were comparable with the synthetic antibiotic tetracycline as same in report of okoh et al. (2019). nahid hasan et al. (2021) also revealed that the ethanol and methanol extracts peel of pineapple showed antibacterial activity and the minimum inhibitory concentration (mic) values against p. aeruginosa and e. coli. additionally, the mic values of extracts of pineapple peel against s. aureus, p. aeruginosa, and salmonella typhi (okoh et al., 2019) and the mic of bromelain from pineapple against streptococcus mutans, enterococcus fecalis, aggregatibacter actinomycetemcomitans, and porphyromonas gingivalis were reported by praveen et al. (2014). phytochemicals, also known as phytonutrients, are naturally occurring secondary substances in plants that function as antioxidants, antiinflammatory and antimicrobial agents. they play an important role in the detoxification of damaging and deleterious chemicals from the body (jayaraman et al., 2008). the medicinal properties of a crude extract from pineapple are mainly derived from bromelain and phytochemical factors such as vitamin c and flavonoid and various combinations of thiolendopeptidases and other compounds such as carbohydrate, cellulase, phosphatase, glucosidase, glycoprotein, peroxidase, and many protease inhibitors (pavan et al., 2012). the antibacterial activity of the pineapple peel extracts may be due to the presence of polyphenols, saponins, flavonoids and other secondary metabolites in the extract. flavonoids play an important role in antimicrobial activity (namarata et al., 2017) and have antimicrobial potential by binding to adhesion or complexes to the cell wall and inhibiting enzyme activity (akinpelu et al., 2008). phenols can denature proteins and its lipophilic property allows them to attract lipid molecules present in cell membranes and destroy bacterial cell membranes (maurer, 2001). phenol alters membrane permeability that could result in oxidative phosphorylation uncoupling, active transport inhibition and metabolites losing due to membrane damage (lubaina et al., 2019). the ability to act as a non-ionic surface agent of phenolic compounds is the primary mechanism of antimicrobial activity, which can disrupt the lipidprotein interfaces or cause proteins deterioration and inhibition of the enzyme activity of pathogens. additionally, both bromelain and saponins act on bacterial cell walls and membranes. bromelain plays an important role in bacterial protein degradation. it is one of the key components in bacterial membranes that causes cell injury and death (praveen et al., 2014). bromelain may hydrolyze some of the peptide bonds present in the cell wall of bacteria. however, the mechanism by which bacterial growth is inhibited is still unknown (bhattacharyya, 2008). according to the results of the a. hydrophila challenge test, the mortality rates in all ppe diets decreased to 26.67% (1% ppe extract), 33.33% (2% ppe extract) and 46.67% (3% ppe extract) compared with the control (53.33%). furthermore, all fish groups fed a diet containing ppe had better 425 int. j. aquat. biol. (2022) 10(5): 417-428 relative percentages of survival (rps) than the control group, which is one of the most obvious criteria of immunological assessment in the challenge test. the rps of nile tilapia in diets containing 1-3% were 50.0, 37.5 and 12.5%, respectively. fish-fed diet supplemented with 1% ppe produced the highest rps and highest resistance towards a. hydrophila. from our findings, fish-fed diets supplemented with ppe showed an improvement in disease resistance, with the highest value observed in fishfeddiets supplemented with 1% ppe. dietary ppe supplementation could better support nile tilapia immune and pathogen resistance, increasing the fish's resistance to bacterial invasion. this was observed as an increase in rps values against a. hydrophila in the fish fed the ppe supplementation compared to the control group in this study. these results might be explained by bromelain's capabilities, which include a range of fibrinolytic, antiedematous, antithrombotic, antiinflammatory actions and a decrease in inflammatory response in the body. it may also relate to bromelain's interaction with the 3′ :5′ -cyclic monophosphatase adenosine, 3′ :5′ -cyclic monophosphatase guanosine and calcium-dependent signaling cascadesm, which are involved in gastrointestinal secretion signaling pathways. additionally, bromelain can prevent bacterial adherence. as a result, the bacteria cannot connect to different glycoprotein receptors in the gastrointestinal tract (praveen et al. 2014). similar findings were noticed by van doan et al. (2021) who reported that improvement disease resistance to s. agalactiae was found in fish-fed pineapple peel powder, with the highest value in fish-fed 10 g kg-1 pineapple peel powder. after a. hydrophila injection, the mortality of fish fed ppe decreased that was comparable with earlier investigations of other herbs for example, nile tilapia fed a diet that included morus alba linn (mapanao et al., 2019) and moringa oleifera (mapanao et al., 2021), l. rohita fed with a diet containing achyranthes aspera (kumar et al., 2019) and c. auratus fed with a diet containing phyllanthus niruri and aloe vera (ahilan et al., 2010), lates calcarifer fed citrus depressa hayata leaf meal (shiu et al., 2016). our findings suggested that using pineapple peel extract as a crucial dietary component for fish species to boost immunity against common fish diseases. conclusion the findings of the current research on the effectiveness of pineapple extract in inhibiting the bacterial a. hydrophila in nile tilapia demonstrated that the highest inhibition zones were pineapple extract from fruit at 7.96±0.20 mm. and the mic was 125 mg/ml. although it was not bactericidal, the pineapple extract was antibacterial. the result of growth performance showed that wg, adg, sgr, fcr, per, and fue were significantly higher in the fish group fed 1% pineapple peel extract (ppe) supplemented in diet. additionally, the significantly lowest mortality and the highest relative percentage survival (rps) were also observed in the fish group fed 1% ppe. based on nile tilapia performance, pineapple extract can be used as a natural ingredient in fish feed to promote growth and improve health. acknowledgments the authors sincerely thank for the partially supported by khon kaen university. references ahilan b., nithiyapriyatharshini a., ravaneshwaran k. 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(2021). effect of supplementation of pineapple extract in fish feed on the growth of catfish (clarias batrachus). international stem journal, 2: 43-52. international journal of aquatic biology (2014) 2(6): 330-336 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article morphological variation of shad fish alosa brashnicowi (teleostei, clupeidae) populations along the southern caspian sea coasts, using a truss system sara paknejad1, adeleh heidari2, hamed mousavi-sabet*21 1department of fisheries, islamic azad university, tonekabon branch, mazandaran, iran. 2department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, p.o. box: 1144, guilan, iran. article history: received 1 july 2014 accepted 5 october 2014 available online 2 5 december 2014 keywords: caspian sea alosa braschnicowi truss system shad fish morphometrics abstract: a 15-landmark morphometric truss network system was used to investigate the hypothesis of population fragmentation of shad fish alosa braschnicowi borodin, 1904 along the southern caspian sea. a total of 181 a. braschnicowi specimens were caught from six localities, respectively from the west to the east including; astara, rezvanshahr, anzali, tonekabon, sari and miankale. principal component analysis, canonical variates analysis and clustering analysis were used to examine morphological differences. univariate analysis of variance showed significant differences between the means of the six groups for 72 standardized morphometric measurements out of 105 characters studied. in canonical variates analysis, the overall assignment of individuals into their original groups was 71.46% and scatter plot of individual component scores between cv1 and cv2 showed fish specimens grouped into six areas. clustering analysis based on euclidean square distances among groups of centroids using upgma resulted into six main clusters indicating morphologically distinction populations of a. braschnicowi in the region. these populations of a. braschnicowi are distinguished especially by head shape, eye diameter, and pre-dorsal, pre-pelvic and pre-anal distances. therefore, it is suggested considering these morphologically different populations as distinct stock in the southern caspian sea coasts. introduction the study of morphological characters with the aim of defining or characterizing fish stock units, has a great interest in ichthyology (bektas and belduz, 2009). the morphometric characters is particularly important where the differences are mostly attributed to environmental influences rather than genetic differentiation (bektas and belduz, 2009). geographical isolation of populations and interbreeding can lead to morphometric variations between populations, and this morphometric variation can provide a basis for population differentiation (bookstein, 1991). the family clupeidae is found in warmer marine waters with some anadromous or permanent freshwater residents. this family has about 200 * corresponding author: hamed mousavi-sabet e-mail address: mousavi-sabet@guilan.ac.ir species in 56 genera worldwide (eschmeyer and fong, 2011; coad, 2014), with eight reported species in the caspian sea. alosa braschnicowi is an economically important clupeids of the caspian sea that widely distributed across this sea. this species distributes in the south in winter, moving north to spawn in spring (coad, 2014). the morphometric characters between male and female sexes in this species did not different (whitehead, 1985). the study of morphometrics using the truss network system is effective in capturing information about the shape of an organism (kocovsky et al., 2009). it covers the entire fish in a uniform network, and theoretically, increases the likelihood of extracting morphometric differences between specimens (kocovsky et al., 2009; cakmak and alp, 2010). 331 paknejad et al/ morphological variation of a. brashnicowi along the southern caspian sea coasts therefore, it has been used in the differentiation of various populations within a species and also various species (kocovsky et al., 2009). various effects of the geographical isolation on fish population in the southern caspian sea basin have been documented in the recent past (mousavi-sabet et al., 2011; mousavi-sabet et al., 2012; mousavi-sabet and anvarifar, 2013; kohestan-eskandari et al., 2014). however, the variability of the a. braschnicowi population parameters and its spatial distribution has not been studied in iranian waters of the caspian sea. therefore, propose of this study was to use a set of morphometric characters for examine whether specific ecological constraints, due to geographic variation, could affect the formation of stock separation for this species. materials and methods a total of 181 specimens of a. braschnicowi were randomly collected by beach seine from six fishing regions along the southern caspian sea coasts, including the miankale (36°54′10.89″ n, 53°48′48.33″ e; 31 individuals), sari (36°48′04.63″ n, 53°02′07.50″ e; 30 individuals), tonekabon (36°50′20.97″n, 50°50′17.25″e; 30 individuals), anzali (37°29′29.86″ n, 49°27′39.59″ e; 30 individuals), rezvanshahr (37°36′32.19″ n, 49°08′25.54″ e; 30 individuals) and astara (38°23′47.40″ n, 48°54′10.17″ e; 30 individuals) in november 2011 (fig. 1). the sampled fish were fixed in 10% formaldehyde at the sampling sites and transported to the laboratory. for extracting morphometric data, the left side of fishes were photographed by a 300-dpi, 32-bit color digital camera (cybershot dsc-f505; sony, japan) with dorsal and anal fins erected by pins. a total of 105 distance measurements between 15 landmarks were surveyed using the truss network system according to bookstein (1991) with minor modifications (fig. 2). images were saved in jpg format, and the defined landmark points were digitized using tpsdig2 (mustafic et al., 2008) on pictures. a box truss of 26 lines connecting the landmark points was generated for each fish to represent the basic shape of fish (bookstein, 1991; mustafic et al., 2008). all measurements transformed into linear distances for subsequent analysis (mustafic et al., 2008). an allometric method was used to remove size-dependent variation in morphometric characters using following formula: madj = m (ls / l0)b, where m is the original measurement, madj the size adjusted measurement, l0 the standard length of the fish, ls the overall mean of the standard length for all fish from all samples in each analysis, and b was estimated for each character from the observed data as the slope of the regression of log m on log l0 using all fish in any group. the results derived from the allometric method were confirmed by testing significance of the correlation between transformed variables and standard length (mustafic et al., 2008). the sex of specimens was determined macroscopically, and there were no significant differences in tested variables between the sexes within the same stock. therefore, the data for both figure 1. sampling site locations of a. braschnicowi along the southern caspian sea coast. figure 2. locations of the 15 landmarks for constructing the truss network on a. braschnicowi. 1tip of snout; 2anterior edge of eye; 3posterior edge of eye; 4posterior tip of maxillary; 5 forehead (end of frontal bone); 6end of operculum; 7dorsal origin of pectoral fin; 8origin of dorsal fin; 9origin of pelvic fin; 10termination of dorsal fin; 11origin of anal fin; 12 termination of anal fin; 13dorsal side of caudal peduncle, at the nadir; 14ventral side of caudal peduncle, at the nadir; 15end of body lateral line. 332 international journal of aquatic biology (2014) 2(6): 330-336 sexes were pooled for all subsequent analyses. univariate analysis of variance (anova) was performed for each morphometric character to evaluate the significant difference between the six populations (rodriguez et al., 2010). those morphometric characters which showed highly significant variations (p≤0.01) were used to achieve the recommended ratio of the number of organisms measured (n) to the parameters included (p) in the analysis of at least 3–3.5 (bookstein, 1991) to obtain a stable outcome from multivariate analysis. the principal component analysis (pca), canonical variates analysis (cva) and cluster analysis (ca) by adopting the euclidean square distance as a measure of dissimilarity and upgma (unweighted pair group method with arithmetical average) as the clustering algorithm (yakubu et al., 2011) were employed to discriminate the six populations. statistical analyses for morphometric data were performed using the spss version 16 software package. to determine the most effective morphometric characters f p characters f p characters f p characters f p 1-2 0.000 1.000 3-4 2.801 0.019 5-9 7.800 0.000 8-11 3.636 0.004 1-3 25.690 0.000 3-5 6.842 0.000 5-10 3.547 0.005 8-12 6.034 0.000 1-4 11.691 0.000 3-6 2.720 0.022 5-11 4.875 0.000 8-13 1.980 0.085 1-5 6.637 0.000 3-7 1.169 0.327 5-12 3.665 0.004 8-14 5.013 0.000 1-6 13.629 0.000 3-8 7.566 0.000 5-13 5.467 0.000 8-15 4.246 0.001 1-7 12.917 0.000 3-9 3.790 0.003 5-14 2.905 0.015 9-10 0.517 0.763 1-8 7.118 0.000 3-10 5.837 0.000 5-15 3.658 0.004 9-11 4.800 0.000 1-9 3.654 0.004 3-11 3.430 0.006 6-7 9.633 0.000 9-12 1.729 0.131 1-10 1.298 0.267 3-12 5.928 0.000 6-8 7.476 0.000 9-13 1.807 0.114 1-11 5.886 0.000 3-13 3.609 0.004 6-9 3.316 0.007 9-14 2.028 0.078 1-12 3.354 0.007 3-14 4.244 0.001 6-10 8.110 0.000 9-15 1.606 0.161 1-13 5.519 0.000 3-15 7.471 0.000 6-11 3.348 0.007 10-11 1.960 0.088 1-14 6.359 0.000 4-5 9.823 0.000 6-12 6.974 0.000 10-12 1.401 0.227 2-3 28.648 0.000 4-6 3.275 0.008 6-13 5.541 0.000 10-13 0.443 0.818 2-4 9.007 0.000 4-7 0.086 0.994 6-14 4.937 0.000 10-14 0.822 0.536 2-5 7.263 0.000 4-8 3.720 0.003 6-15 9.226 0.000 10-15 0.425 0.831 2-6 14.608 0.000 4-9 2.135 0.064 7-8 2.386 0.041 11-12 2.907 0.015 2-7 14.325 0.000 4-10 2.523 0.032 7-9 5.660 0.000 11-13 3.253 0.008 2-8 7.073 0.000 4-11 1.837 0.109 7-10 3.010 0.012 11-14 1.164 0.330 2-9 2.917 0.000 4-12 2.746 0.021 7-11 2.020 0.079 11-15 1.951 0.089 2-10 1.472 0.202 4-13 1.659 0.148 7-12 4.070 0.002 12-13 1.746 0.127 2-11 5.020 0.000 4-14 1.945 0.090 7-13 2.921 0.015 12-14 0.410 0.842 2-12 1.167 0.328 4-15 3.272 0.008 7-14 2.026 0.078 12-15 2.963 0.014 2-13 2.895 0.016 5-6 12.284 0.000 7-15 4.412 0.001 13-14 0.837 0.525 2-14 4.422 0.001 5-7 18.966 0.000 8-9 1.209 0.307 13-15 0.389 0.856 2-15 2.595 0.028 5-8 8.181 0.000 8-10 3.924 0.002 14-15 0.998 0.421 table 1. the results of anova for morphometric measurements of a. braschnicowi populations along the southern caspian sea. 333 paknejad et al/ morphological variation of a. brashnicowi along the southern caspian sea coasts measurement to differentiate studied populations, the contributions of variables to principal components (pc) were examined. to examine the suitability of the data for pca, bartlett’s test of sphericity and the kaiser–meyer–olkin (kmo) measures were performed. the bartlett’s test of sphericity, tests the hypothesis that the values of the correlation matrix equal zero and the kmo measure of sampling adequacy tests, whether the partial correlation among variables is sufficiently high (yakubu et al., 2011). the kmo statistics vary between 0 and 1 and the values greater than 0.5 are acceptable (nimalathasan, 2009; yakubu et al., 2011). results the correlation between transformed morphometric variables and standard length was not significant (p>0.05) which confirms that size or allometric signature on the basic morphological data was accounted. significant differences between six populations of a. braschnicowi were observed in terms of 72 morphometric characters out of 105 studied (table 1). of these 72 characters, 60 characters were found to be highly significant (p≤0.01) and were used for further multivariate analysis. in this study n:p ratio was 3.01 (181/60) that revealed samples size were adequate. the value of kmo for overall matrix is 0.695, and the bartlett’s test of sphericity is significant (p≤0.01). the results of kmo and bartlett’s suggest that the sampled data is appropriate to proceed with a factor analysis procedure. principal component analysis of 60 morphometric measurements extracted 12 factors with eigen values>1, explaining 94.52% of the variance (table 2). the first principal component (pc1) accounted for 25.94% of the variation and the second principal component (pc2) for 15.33% (table 2), and the most significant loadings on pc1 were 1-3, 1-4, 1-6, 1-7, 1-9, 1-11, 1-12, 1-13, 1-14, 2-3, 2-6, 2-7, 2-11, 3-13, 3-14, 3-15, 4-6, 5-6, 5-7, 5-9, 6-8, 6-10, 6-11, 6-12, 6-13 6-14, 6-15, 7-15 and on pc2 were 1-8, 2-8, 38, 4-8, 5-8, 6-8, 8-10, 8-11, 8-12, 8-14, 8-15. in this analysis, the characteristics with an eigen value exceeding 1 were included, and others discarded. the cv1 and cv2 were plotted to allow visual examination of the distribution of each locality sample along the cvs (fig. 3). in the scatter plot, the miankale, astara and anzali specimens were grouped together in the same quadrant with high value for cv1 and low value for cv2 (quadrate iv), tonekabon in a quadrant with low value for both cvs (quadrate iii), rezvanshahr in a quadrant with factor eigenvalues percentage of variance percentage of cumulative variance 1 15.567 25.945 25.945 2 9.203 15.338 41.283 3 7.332 12.220 53.503 4 6.235 10.392 63.895 5 3.961 6.601 70.496 6 3.198 5.330 75.826 7 2.662 4.437 80.263 8 2.609 4.348 84.611 9 1.907 3.179 87.789 10 1.771 2.952 90.741 11 1.178 1.963 92.704 12 1.094 1.823 94.527 table 2. eigen values, percentage of variance and percentage of cumulative variance of principal component analysis of morphometric measurements for a. braschnicowi populations along the southern caspian sea. 334 international journal of aquatic biology (2014) 2(6): 330-336 high value for both cvs (quadrate ii) and finally sari in a quadrant with low value for cv1 and high value for cv2 (quadrate i). the overall random assignment of individuals into their original groups was high (71.46%), indicating that these samples are probably divergent from each other (fig. 3). the dendrogram derived from ca of euclidean square distances among groups of centroids showed six main clusters based on morphometric characteristics. despite the geographical distance between the miankale and sari regions and rezvanshahr and anzali regions, morphometric clustering revealed that the individuals of these locations form the same clade with great homogeneity, while tonekabon and astara exhibited higher heterogeneity, confirming the results obtained from discriminant analysis for this species (fig. 4). discussion the multivariate analysis of morphometric characteristics classified the a. braschnicowi populations along the southern caspian sea coasts into six distinct groups. the results demonstrate that there are morphologically distinct populations of figure 3. scatterplot of group centroids of standardized canonical variates functions 1 (cv1) and 2 (cv2) for morphometric characteristics of six populations of a. braschnicowi along the southern caspian sea. figure 4. dendrogram derived from cluster analyses of morphometric measurements on the basis of euclidean distance for a. braschnicowi populations along the southern caspian sea. 335 paknejad et al/ morphological variation of a. brashnicowi along the southern caspian sea coasts a. braschnicowi particularly for tonekabon region. these morphological differences is solely related to body shape variation and not to size effects which were successfully accounted by allometric transformation. size-related traits play a predominant role in morphometric analysis and the results may be erroneous if not removed from data (bookstein, 1991; buj et al., 2008; torres et al., 2010). the analysis of variance also revealed significant phenotypic variation among the six populations. cva could be a useful method to distinguish different stocks of the same species (yakubu and okunsebor, 2011). the present study showed a high differentiation among the populations of a. braschnicowi in the studied areas. this segregation was partly confirmed by pca, where revealed that the populations were distinct from each other. the causes of morphological differences among populations are often quite difficult to explain (bookstein, 1991). it has been suggested that the morphological characteristics of fishes are determined by genetic, environment and the interaction between them (heidari et al., 2013; kohestan-eskandari et al., 2014). the environmental factors prevailing during the early development stages, when the individual’s phenotype is more amenable to environmental influence is of particular importance (eschmeyer and fong, 2011). the phenotypic variability may not necessarily reflect population differentiation at the molecular level (bookstein, 1991). apparently, different environmental conditions can lead to an enhancement of pre-existing genetic differences, providing a high interpopulation structuring (eschmeyer and fong, 2011; heidari et al., 2013; mousavi-sabet and anvarifar, 2013). the tonekabon (in the south-central part of the caspian sea), miankale (in the southeast part of the sea) and astara (in the southwest part of the sea) specimens are more distinct from the others. the distinctive environmental conditions of the tonekabon, miankale and astara relative to the other studied areas may underlie the morphological differentiation among these three populations. the studied populations are distinguished from each other by morphologic differences especially in head shape, eye diameter, and pre-dorsal, pre-pelvic and pre-anal distances. geographical isolation can also affect growth pattern and reproductive strategy of fish species. the importance of such factors on producing morphological differentiation in fish species is wellknown (yamamoto et al., 2006; pollar et al., 2007; heidari et al., 2013). as conclusion, the present study proposes high morphological differentiation among a. braschnicowi populations along the southern caspian sea coasts. the results also suggest these morphologically different populations should be considered as distinct stock in the southern caspian sea in fisheries management and commercial exploitation of this species and any stock enhancement program. nevertheless, future studies on determination of population structure will be elucidated using biochemical and molecular genetics methods. references bektas y., belduz a.o. 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(2019) 7(3): 132-139 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article modelling potential distribution of fluvial fish species for expanding conservation knowledge: case study of the genus barbus in iran hossein mostafavi*1, jafar kambouzia2 1department of biodiversity and ecosystem management, environmental sciences research institute, shahid beheshti university, gc, tehran, iran. 2department of agroecology, environmental sciences research institute, shahid beheshti university, gc, tehran, iran. s article history: received 9 february 2019 accepted 8 june 2019 available online 2 5 june 2019 keywords: barbel cyprinid conservation freshwater abstract: species inhabiting fresh waters are severely influenced by anthropogenic factors. effective management and conservation plans require high accurate and reliable species distribution forecasts. here, we modelled potential distribution of the genus barbus in iran, based on environmental variables using species distribution models (sdms). six environmental predictors (i.e. slope, bankfull width, elevation, mean air temperature, range of air temperature and annual precipitation) were applied for modelling. the models were selected among different technique (glm, gam, cta, sre, gbm, rf, mars, and fda) which their results were summarized through ensemble forecasting approaches. according to the tss (true skill statistic), the accuracy of the implemented models was greater than 0.8. the results showed that the projected distributions not only were observed in the same recorded basins but also in the new basins. presented results deepen the conservation knowledge in iran and act as a guidance for management decisions aimed at legal identification of critical habitats for species as well as informing them for translocation of threatened or captive-bred populations. introduction nowadays, there is a growing concern about the longterm viability of fish species due to human pressures especially in freshwater ecosystems. human activities such as excessive withdrawal, water pollution, alterations of river flow, habitat degradation, alien species, etc. can negatively affect the distribution of many species in their habitats (dudgeon et al., 2006; mostafavi et al., 2015). freshwater ecosystems, especially rivers are one of the most important habitats of the world. although they account for only 0.01 percent of global water resources and just cover small portion of the planet's surface, but they support 10 percent of all known wildlife species and one-third of all vertebrate species e.g. about 40 percent of the world's fish species (mcallister et al., 1997; pringle, 2003). therefore, conservation biologists and natural resource managers have been investigating for ways to protect these ecosystems and their species. for proper conservation and management of fish *correspondence: hossein mostafavi doi: https://doi.org/10.22034/ijab.v7i3.575 e-mail: hmostafaviw@gmail.com species in these ecosystems, we need to apply new methods for protecting them and provide practical and reliable information for managers and researchers. nowadays, with the advancement of science, especially computer science, the use of modeling methods along with other methods has provided many opportunities for them to accomplish better and more effective environmental decision making. in this regard, modelling the distribution of species has become an important component of conservation programs (thuiller et al., 2005; morid et al., 2016; amiri et al., 2017) and one of the most important environmental modelling methods is species distribution models (sdms). forecasting species’ distributions has become a significant part of conservation planning in the last decade, and there is a wide range of modeling techniques for this purpose (guisan and thuiller, 2005). the spatial distribution of environments that are suitable for the species can then be estimated across a study region. this approach 133 int. j. aquat. biol. (2019) 7(3): 132-139 is valuable for generating geospatial information that can be applied across a broad range of fields, including conservation biology, ecology, and evolutionary biology. sdms usually use the relationship between environmental variables and species records to identify the environmental conditions in which populations can be present. it means the spatial distribution of the suitable environment for species is modelled and then the distribution of species is estimated for the whole region (pearson et al., 2007). many countries in europe, the united states, australia and some asian countries have made extensive studies in this regard, however few studies have been done in iran in aquatic ecosystems (mostafavi et al., 2014). iran has high biological diversity, especially in freshwater fish species (esmaeili et al., 2018) but freshwater ecosystems in iran have been affected by a variety of human activities, such as damming, industrial development, urbanization, agriculture, etc., causing more destruction of these habitats that has resulted in serious threats to fish biodiversity (mostafavi et al., 2014). previous studies have been mostly carried out on identifying biodiversity of fishes in freshwater ecosystems (tabatabaei et al., 2015; zamani faradonbe and eagderi 2015; ghasemi et al., 2015; hoghoghi et al., 2016; morid et al., 2016; zamani faradonbe et al., 2017), but due to various reasons such as lack of facilities, budget and lack of access to all rivers, only the main rivers have been investigated and in many rivers the presence and absence of many species has not been thoroughly studied. in addition, in iran for the introduction of species into new environments, the potential of the regions has not been properly studied. according to guisan et al. (2013), sdms are a tool for differentiating habitat quality at a range-wide scale, therefore, based on our results, critical habitats were typically defined as habitats necessary for the persistence, or long-term recovery, of sensitive/threatened species. moreover, according to these results, sdms can potentially inform the translocation decision process. indeed, if translocation is deemed necessary, sdms can identify potential recipient sites, which may be climate refugia within the current range, or sites that are projected to become newly suitable. or sdms can be used to identify which local species may be at risk of impact from the introduction of a translocated species through predicted overlapping distributions, in the same way as they are used to identify conflict areas between native and invasive species. in addition, sdms are able to identify sites, where species may have been absent due to habitat degradation. consequently, based on more detailed future studies effective conservation and restoration measures can be undertaken to maintain and (re)establish species populations. this can be useful for conservation decision because the habitats of some species were already degraded due to human pressures (mostafavi et al., 2015). the widespread barbus populations from the southern tributaries of the caspian sea, lake namak and urmia lake basins, and the euphrates and tigris drainages previously identified as barbus lacerta (nikmehr et al., 2016; khaefi et al., 2017a; esmaeili et al., 2018). naseka and bogutskaya (2009) recognize b. cyri as a valid species from the caspian sea basin. khaefi et al. (2017b) revalidated b. miliaris, a nominal species found in the lake namak basin. furthermore, khaefi et al. (2017a) described b. karunensis from the karun river drainage in iran, increasing the number of barbus species in iran to four. the objective of this study are (1) identification of potential distribution member of the genus barbus over the extent of iran, (2) determining the importance of environmental variables on the distribution of members of this genus and (3) developing recommendations that can aid in the conservation of riverine species in iran. materials and methods study area, fish data and envronmenthal predictors: this study was conducted in iran which encompasses 19 basins (coad, 2019). member of the genus barbus widely distributed in iran and adjacent countries. they are found in the caspian sea, urmia lake, namak lake and tigris basins in iran (mostafavi et al., 2014; khaefi et al., 2017a). data used in this study covers 134 mostafavi and kambouzia / modelling potential distribution of fluvial fish species several time periods (1970-2000) obtained from databases originating from the previous field samplings, several museums and literature (berg, 1949; saadati, 1977). sites where fish were occurred/observed are called actual data. moreover, sites which had an unclear position to the river network or were outside the temporal period between 1970 and 2000, as well stocked with species population and located in any lakes and wetlands were excluded. the primary database contained approximately 1700 sites which were reduced to 1090 sites after a detailed quality check concerning the reliability of the biological and spatial information. after collecting the fish data, we considered three environmental variables at local scale (elevation (ele), stream slope (slo), and bank-full width (b_wid)), and five variables at the regional scale (maximum air temperature (max_tem), minimum air temperature (min_tem), mean air temperature (a_tem), the range of air temperature (r_tem) and annual precipitation (pre)). we extracted ele and b_wid from google earth (google inc. 2009, version 5). b_wid was maximum width the stream attains, typically marked by changes in vegetation, topography, or texture of sediment. slo was calculated for a 1 km stretch extending upstream of each site. climate variables were extracted from worldclim data (hijmans et al., 2005, 2007) to characterize annual climate trends based on records for 30 years of monthly means (1970 to 2000), and interpolated at 30 arc-seconds grid extent (approximately 1 km at the equator). climate variables were extracted in a circular buffer (5 km) around each sampling site (mostafavi et al., 2014) as a catchment layer similar to ccm2 (catchment characterization and modelling database) (vogt et al., 2003, 2007; de jager and vogt, 2010) is not available for iran. variable redundancy within environmental variables was tested by spearman's rank correlation (r). if two variables were highly correlated (r>|0.75|) (filipe et al., 2013; mostafavi et al., 2014); one of them was excluded to avoid co-linearity according to our expert judgement and literatures. modelling process (techniques, calibration, evaluation and ensemble forecasting): in this study, the biomod2 (biodiversity modelling) package (thuiller, 2003) was used within the r software (r development core team, 2011) for modelling. as above mentioned, our fish data is based on a heterogeneous data set containing information from several sources, therefore the “presence-background modelling” approach was used according to chefaoui and lobo (2008) and barbet‐massin et al. (2012). then, the following nine modelling techniques were applied: generalised linear models (glm), generalized additive model (gam), classification tree analysis (cta), artificial neural network (ann), surface range envelops (sre), generalized boosting method (gbm), random forest (rf), multivariate adaptive regression splines (mars), and flexible discriminant analysis (fda). we applied a cross-validation procedure by randomly splitting the data into calibration (80% of the data) and validating (20%) data sets with 10 repetition runs to assess model performance stability (mostafavi et al., 2014). model evaluation was based on the true skill statistic (tss) which corresponds to the sum of sensitivity and specificity minus 1, and is independent of prevalence (lobo et al., 2008; thuiller et al., 2009a, b). finally, all nine modelling techniques were combined in an ensemble-forecasting framework as recommended by araújo and new (2007). variable importance was calculated by a permutation procedure used in biomod2, which is independent of the modelling technique (thuiller et al., 2009a, b). we used the software arcgis desktop 10.3 (esri© 1999-2008) to map the spatial pattern of the predicted distributions of the studied fish species within the 19 drainage basins of iran. results after correlation test, seven environmental variables (b_wid, slo, ele, a_tem, r_tem, and pre) out of eight, remained as independent variables for the modelling. table 1 describe their characteristics. overall, tss values ranged from 0.52 to 0.99 for testing validity (fig. 1). in addition, the tss was good in average (>0.78). moreover, whilst sre showed the 135 int. j. aquat. biol. (2019) 7(3): 132-139 lowest accuracy and rf was highest (table 2). the relative importance of environmental variables for each model is showed in table 2. among all variables, the most important ones are mean air temperature, annual precipitation and elevation, respectively (≥15%), whereas other variables show the low values (<15%). moreover, the relative importance of predictors in each model was a little bit different to each other. according to figure 2, barbus species were predicted not only in the same recorded basins (i.e. caspian sea, urmia lake, namak lake and tigris basins) but also predicted in the new basins (i.e. hari river basin and upper tigris river drainage). moreover, in the same recored basins (i.e. caspian sea, urmia lake, namak lake and tigris basins) some new sites were identified which was not recorded in the literatures. discussions the results of this study have important implications for conservation activities and management. the modelling framework has the ability to highlight the potential areas of species occurrence, and as well to identify sites, where species may have been absent due to habitat degradation. model performance: the results showed that the performance of the models was acceptable. rf was performed the best while sre was performed the weakest. sre or bioclim (bioclimatic envelope table 1. mean and range (minimummaximum) of environmental variables. number of sites b_ wid (m) slo (‰) ele (m) a_tem (⁰c) r_tem (⁰c) pre (mm) 1090 mean 112.5 1.6 731 19.1 13.3 384.8 rang e 1.0-3539.8 0.0-28.0 (-)27-2708 5.5-29.5 6.9-18.5 53-1478 abbreviations: bank-full width (b_wid), wetted width (w_wid), stream slope (slo), elevation (ele), mean air temperature (a_tem), range of air temperature (r_tem), annual precipitation (pre). table 2. the order of relative importance of environmental predictors for genus barbus fish. abbreviations: bankfull width (b_wid), stream slope (slo), mean air temperature (a_tem), annual precipitation (pre), elevation (ele), range of air temperature (r_tem). figure 1. evaluation of model accuracy according to tss validation index 136 mostafavi and kambouzia / modelling potential distribution of fluvial fish species model) algorithm uses a rectangular envelope that enclose all (or a specified percentile) of the species records in the environmental space (guisan et al., 2017). it is known as one the simplest modelling methods that only uses the presence records to model species environmental niche. sre assumes no interaction between the environmental variables and has a simple response shape which is not capable of capturing all the complexity in species-environment relationship (merow et al., 2014). based on the number of selected environmental variables, sre might lead to over or under fitting (guisan et al., 2017). a comprehensive comparison study indicated the sre to have a low predictive power (elith et al., 2006). on the contrary, random forest (rf) has high transferability and predictive performance (yates et al., 2018). rf is a machine learning algorithm built based on an ensemble of classification tress. it uses bagging or bootstrap aggregation technique to reduce the variance in classification trees and by selecting only a subset of variables on each node avoids overfitting (hastie et al., 2009). having the classification trees as its base learner; rf is capable of including high order interaction in model fitting that makes it powerful to draw more realistic speciesenvironment relationship (merow et al., 2014). these features make rf a robust predictive model. moreover, it is essential to be indicated that all applied models in this study come from different families ranging from statistical to machine learning and tree based models. each of them have their own specific limitations such as ignoring interaction among the predictors (e.g, gam and glm) or assuming specific distributions, whereas the others can handle missing values, are insensitive to outliers and can handle large datasets. on the other hand, some models such as rf are quite flexible while a model like glm is less flexible. the results of gam and mars are interpretable, nevertheless, models such as ann belong to a black box category in which understanding the structure of the model is not straightforward (valavi et al., 2018). therefore, all nine modelling techniques were combined in an ensemble-forecasting framework as recommended by (araújo and new, 2007) to reduce the uncertainty. evaluation of selected variables: the most important part of species distribution modelling is to select and use appropriate environmental variables. these variables can be used to describe geographical and biological conditions of the species and predict correct distribution range of the species. it is not possible to identify all variables and factors affecting species niche characteristics in modelling. this might be due to the deficiencies of ecological knowledge in studied species, the lack of knowledge for recognizing all dimensions of the species niche, availability limitations and the use of all variables in the form of information layers in modelling (due to the lack of information layers or their inaccuracies). temperature appears to be one of the main determinant factors of spatial distribution for stream fishes (e.g. buisson et al., 2008; logez et al., 2012). freshwater fishes are ectothermic (cold-blooded) animals and particularly sensitive to temperature. fluctuation of temperature figure 2. prediction of genus barbus on the 19 basins of iran, a: name of the basins; b: actual data or sites where fish was observed; c: prediction/or sites where fish is expected according to modelling, (white circle: background data/sites, black circle: observed/predicted sites). 137 int. j. aquat. biol. (2019) 7(3): 132-139 effects the metabolism, breeding, development and growth of fishes (mann, 1996). in addition, mean air temperature has been widely reported as an important variable affecting fish distribution (e.g. pont et al., 2005; buisson et al., 2008; abdoli and naderi, 2009; mostafavi et al., 2014, 2015), which is in line with the results of this study. about the slope variable, it generally depends to the ecology of fishes which in this study it was not so much important for the species studied while in some studies (e.g. filipe et al., 2013; mostafavi et al., 2014) it was important for brown trout (salmo trutta). comparing the modelling results with available reports: comparing results of the studied species complex distribution modelling with existing reports (e.g. mostafavi et al., 2014; khaefi et al., 2017a) shows that the members of barbus species was predicted not only in caspian sea, urmia, namak and tigris basins, but also predicted in hari river basin and upper tigris river drainage. moreover, in the same recored basins, some new sites were identified. two reasons may/can be stated for this difference. first, sampling methods conducted by researchers might be incomprehensive because almost every year new species are described from remote and mountainous regions of iran (khaefi et al., 2017a; esmaeili et al., 2018). second, these differences can also be attributed to the limiting factors, which influence distribution of freshwater fish species. although we can consider the distribution of a species based on the relationship between the environmental conditions and the species inherent characteristics in terms of its potential distribution, but the various natural and artificial factors have constrained the realized distribution of species (sexton et al., 2009). since the stream network is the only corridor for distribution of fish species, the main limiting natural factor in freshwater ecosystems is the catchment border, by which movement of species between catchment would be restricted (schmutz et al., 2000; pont et al., 2005). whilst, the habitat suitability for a species may be beyond its main catchment area, distribution of species within the catchment area may be confronted with various obstacles, especially artificial constructions like dams, resulting in limited distribution of a species within the catchment area. species distribution can also be impacted by biological interactions in which the importance of some characteristics such as competition and predation is remarkable (araújo and new, 2007). in addition, the absence of species in the new predictions for the same basin might be related to different sampling and monitoring methods conducted in diverse habitats and rivers in iran as well as due to human pressures. conclusion sdms are important conservation and management tools because the models can reliably predict areas suitable for species occupation, and the species responses to particular environmental variables can suggest management alternatives. acknowledgment this study was funded by shahid beheshti university, g.c. special thanks and gh. amiri ghadi for their support during our fish sampling. references abdoli a., naderi m. 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(2022) 10(6): 537-542 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article cyanobacteria diversity in various waterbodies of mosul, iraq rawaa m. hmoshi1, mahmmoud ismail mohammed2 1department of environmental science, college of environmental science and technology, university of mosul, mosul, iraq. 2department of biology, college of science, university of mosul, mosul, iraq. s article history: received 21 october 2022 accepted 22 december 2022 available online 2 5 december 2022 keywords: algae bacteria asm-1 medium microcystis oscillatoria abstract: cyanobacteria are photoautotrophic bacteria that can adapt to various environments due to their extensive physiological adaptability. these bacteria are naturally distributed in diverse ecosystems, including freshwater, marshes, groundwater, lakes, brackish water (estuaries), salt water, moist soils, and dry land. this study was conducted to enlist cyanobacteria isolates in different waterbodies in mosul, iraq. for this purpose, 16 sites were selected and sampled. based on the results, the gloeocapsa nigrescens was the dominant species (10.34%), followed by microcystis robusta (6.69%), oscillatoria nigro-viridis (6.69%), and oscillatoria sp. (6.69%). mosul dam lake (station 12) was the most diverse one with six cyanobacteria species, including schizothrix sp., aphanocapsa koordesii, g. crepidium, o. trichoides, m. flos-aquae, and plectonema tomasinianum. introduction cyanobacteria are ancient negative gram-stain microorganisms that emerged during the volcanic age approximately 3.5 billion years ago (sergeev, 2018). they play a crucial role in transforming the earth’s atmosphere to an aerobic condition (demoulin et al., 2019). they are known as bluegreen bacteria or algae because of their blue-greenish hue (matheron and caumette, 2015; zahra et al., 2020). water and land are both suitable habitats for cyanobacteria (svirčev et al., 2019). they are found in various environments, including saltwater, freshwater, cold, hot, and terrestrial ecosystems (fitri et al., 2021; kostryukova et al., 2021). cyanobacteria have approximately 2,000 species in 150 genera in five orders (vincent, 2007). they are important primary producers playing a significant role in carbon and nitrogen cycles (vincent, 2007). cyanobacteria can be found in oil fields and oil pools around oil wells (radwan and al-hasan, 2000; chaillan et al., 2006; el-sheekh and hamouda, 2014) and in moist, shady atmospheres with heavy correspondence: rawaa m. hmoshi doi: https://doi.org/10.22034/ijab.v10i6.1783 e-mail: rawaahamoshi@uomosul.edu.iq dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.10.3 rainfall (wiśniewska et al., 2022). in addition, makhalanyane et al. (2015) reported these bacteria in the northern and southern polar, where environmental conditions are extremely harsh. in addition, they are a renewable energy source known as third generation biofuel (tgb) (teta et al., 2020; filatova et al., 2021; forchhammer and selim, 2022; sánchez-baracaldo et al., 2022). the cyanobacteria have distinct shapes, such as unicellular, colonies, and filamentous (tamulonis and kaandorp, 2014; herrero et al., 2016). they contain various cell types, e.g. the heterocysts are characterized by thick walls, particularly in nostocales and stigonematales, which bear the nitrogen enzyme for nitrogen fixation and converting nitrogen gas into ammonia and amino acids. the akinete type can reproduce new filaments under inconvenient conditions (kaushik and sharma, 2017). in recent years, interest in blue-green bacteria (cyanobacteria) has increased rapidly due to their ability to produce various materials such as vitamins, amino acids, fatty acids, proteins, various dyes, 538 hmoshi and mohammed / cyanobacteria diversity in various waterbodies of mosul enzymes, phenols, and alkaloids, which are applied in diverse fields including medicine, industry, and agriculture. for instance, in agriculture, they used to produce biological fertilizers and nitrogen fixation from the atmosphere by transforming the nitrogen to ammonium, which is necessary for plant growth and dissolving the phosphate and consequently enhancing and improving the production of the crop (kumar et al., 2019; kini et al., 2020). cyanobacteria can also remove crude oil, heavy minerals, and pesticides from wastewater (mona et al., 2020). furthermore, they possess secondary metabolic compounds used as anti-fungal, anti-bacterial, and anti-cancer agents (singh et al., 2016; kumar et al., 2019). despite the importance of cyanobacteria, there is little information available regarding cyanobacteria in iraqi natural ecosystems. hence, the current study aimed to survey and identify cyanobacteria isolates in different waterbodies in mosul, iraq. materials and methods from february to april 2020, water samples were collected from different water bodies (table 1). for transitional culture, liquid and solid assimilated medium-1 (asm-1) was used to isolate cyanobacteria. the solid asm-1 was prepared by adding agar at a concentration of 1% per 100 ml of liquid medium. the water samples were inoculated using the streaking method and incubated at 26±2°c under constant lighting of 2500 lux for four weeks. following the growth of the cyanobacteria colonies, the developing colonies were identified using a light microscope, according to waterbury (2006) and hossain et al. (2020). to obtain a pure culture, the identified colonies were transferred to new petri dishes containing the medium mentioned above and cultured in the incubator as mentioned above (waterbury, 2006; hossain et al., 2020). results and discussion based on the results, most samples were positive for the presence of cyanobacteria (table 1). previous studies have confirmed the presence and blue-green bacteria in the nineveh province (al-shakarchi and al-shahery, 2020), showing the diversity of cyanobacteria in these areas due to suitable conditions e for their growth. in addition, since cyanobacteria can accumulate pollutants, they are crucial indicators for environmental pollutants (paerl et al., 2011; mona et al., 2020; lu et al., 2021). the inventory of cyanobacteria species in the studied area is shown in table 2. based on the results, gloecapsa nigre-scens showed the highest percentage (10.34%), followed by osillatoria nigroviridis, microcystis robusta, and osillatoria sp. (6.89%), and the remaining taxa each consists 3.44% of the richness. six cyanobacteria were found in the no. site results 1 water from the waterfall that pours into lake al-qusour 2 soil from the house garden 3 al-qusour lake basins of the animal resources laboratory / college of agriculture + 4 subtrahends of the industrial area + 5 khoser al-kharazi near the college of environment + 6 al-qusour lake at a certain depth 7 lake of the presidential palaces at a depth + 8 the discard of the hammam al-alil cement plant 9 litter from the khosr estuary, which flows during the day in the tigris + 10 edge of the right side of the mosul dam lake + 11 the left edge of the mosul dam lake + 12 the middle of the mosul dam lake (from the surface) + 13 the middle of the mosul dam lake (from the depth) + 14 subtrahends of wadi akab estuary + 15 karasaray site + 16 khoser al-muthanna district (sewage water) + table 1. sampling stations and collection results cyanobacteria. 539 int. j. aquat. biol. (2022) 10(6): 537-542 surface water of mosul dam lake (station 12), the most diverse site. temperature and light conditions were optimum in this site along with fall turnover i.e. the circulation of nutrients from the lake’s bottom; then it had algae bloom during sampling (lürling et al., 2018; jiang et al., 2022). no cyanobacteria existed in sites 1, 2, 6, and 8, which could be due to unsuitable environmental conditions. in site 8, the cement factory’s drainage water was saturated with oil contamination. despite the report that cyanobacteria that break down hydrocarbons may live in such environments, they did not find in this study. the oils inhibit certain types of cyanobacteria, and it takes resistant species a relatively long time to adapt to these systems (zutshi and fatma, 2015; yadav et al., 2016; karmakar et al., 2018). in site nine, many microorganisms, including bacteria, fungi, algae, and diatoms, but no cyanobacteria. the absence of cyanobacteria could be attributed because of pollution and containing large waste, and the high nacl significantly reduces the growth rate of cyanobacteria by ionic (na+) stress (batterton and van baalen, 1971; zahra et al., 2020). some collected cyanobacteria had spherical colonies, such as microcystis robusta, m. flosaquae, and gloeocapsa nigrescens (fig. 1a-d); some branched filament shape, plectonema tomasinianum (fig. 1e), or unbranched filaments, lyngbya birgei (fig. 1f), and osillatoria trichoides (fig. 1g), and some filamentous, such as nostoc sp. and anabaena sp., containing heterocyst vesicles (fig. 1j, h). the heterocysts vesicles serve as communication elements; they bind together on the surface, and the no. site isolates % 1 water that flows from the waterfall and into the lake nd (not detected) nill 2 residential garden soil nd nill 3 basins from al-qusour lake of the animal resources laboratory/ college of agriculture gloeocapsa nigrescens nageli phytoeonis sp. lyngbya birgei calothrix parietina 10.34 3.44 3.44 3.44 4 subtrahends of the industrial zone spirulina subtilisima 3.44 5 khoser al-kharazi, near the college of the environment gloecapsa nigrescens green algae fragillaria sp. (ditoms) 10.34 6.89 3.44 6 al-qusour lake (depth) nd nill 7 al-qusour lake (surface) osillatoria nigro-viridis gloeocapsa nigrescens anabaena sp. anabaena spiroides 6.89 10.34 3.44 3.44 8 hammam al-alil cement factory water nd nill 9 litter from the khosr estuary, which flows into the tigris river nd nill 10 the right bank of mosul dam lake chlorogloea fritschii green algae phormidium sp. 3.44 6.89 3.44 11 the left bank of the mosul dam lake osillatoria nigrouviridis osillatoria lemmermann 6.89 3.44 12 the middle of the mosul dam lake (surface) schizothrix sp. aphanocapsa koordesii gloeocapsa crepidium osillatoria trichoides microcystis flos-aquae plectonema tomasinianum 3.44 3.44 3.44 3.44 3.44 3.44 13 the middle of the mosul dam lake (depth) microcystis robusta 6.89 14 subtrahends of the wadi akab mouth anabaena variabilis osillatoria sp. 3.44 6.89 15 karasaray area microcystis robusta 6.89 16 khosar hay al-muthanna nostoc sp. 3.44 table 2. sampling stations and collection results from various waterbodies of mosul. 540 hmoshi and mohammed / cyanobacteria diversity in various waterbodies of mosul heterocysts are sites for nitrogen fixation to survive in environments with low nitrogen (burnat et al., 2014; marino et al., 2020). according to the results, the identified cyanobacteria at various sites have different adaptations to environmental parameters and also vary in their growth rates (demay et al., 2019; filatova et al., 2021). studies showed that the possibility of isolating diverse cyanobacteria from various sites depends on the proper environmental conditions e.g. temperature, light, ph, and pollution (wijffels et al., 2013; ammar et al., 2014; kaushik and sharma, 2017). in conclusion, this study showed that g. nigrescens was the most common species in the study sites (10.34%) in waterbodies of mosul, followed by m. robusta, osillatorio sp., and o. nigroviridis (6.89%). mosul dam lake, with six species, was the most diverse site. references al-shakarchi h.k.s., al-shahery y.j. 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(2013) 1(3): 109-115 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article compensatory growth response of sailfin molly, poecilia latipinna (lesueur, 1821) to starvation and refeeding vahid morshedi1, 2, preeta kochanian*1, meysam ahmadi-niko1, maryam azodi3, hossein pasha-zanoosi1 1department of fisheries, faculty of marine natural resources, khoramshahr marine science and technology university, pb no:669, khoramshahr, iran. 2young researchers club of ilam azad university, ilam, iran. 3persian gulf research and study centre, persian gulf university, bushehr, iran. article history: received 25 april 2013 accepted 22 may 2013 available online 2 0 june 2013 keywords: catch-up growth body composition starvation hyperphagia ornamental fish abstract: compensatory growth response and body composition of male sailfin molly, poecilia latipinna subjected to short-term starvation and subsequent feeding were studied for 54 days. four feeding schedules were used in this study: c, control (were fed to apparent satiation throughout the experiment); t1, treatment 1 (3 days starvation and 6 days refeeding); t2, treatment 2 (6 days starvation and 12 days refeeding); t3, treatment 3 (9 days starvation and 18 days refeeding). at the end of the experiment, the starved fish gained a body weight comparable to that of the control fish. there were no differences in condition factor, specific growth rate and weight gain between the starved and control fish at the end of the experiment. daily feed intake was significantly higher in t3 than that in the control. short-term starvation did not influence protein, lipid and ash contents. moisture content of t2 and t3 fish were significantly higher than those of t1 and control one. the results indicated that complete compensation occurred in the starved fish and that this species can tolerate to short term starvation without any significant effects on growth and feeding performance. introduction culture of ornamental fish is an important industry in the world. the volume and value of ornamental fish export in the world are 47,548 tonnes and $703 million us dollars (fao, 2007). freshwater teleosts make up to 90-96% of the ornamental fish trade (livengood and chapman, 2007). mollies, the family poeciliidae, are very popular among the ornamental fish hobbyists worldwide and are cultured usually in outdoor earthen ponds or net cages (fernando and phang, 1994). sailfin molly, poecilia latipinna is a good candidate as an ornamental fish. a high reproductive potential, feeding from different types of feed and tolerance to changes in temperature and dissolved oxygen fluctuations makes sailfin molly a suitable species * corresponding author: preeta kochanian e-mail address: pkochanian@kmsu.ac.ir tel: +986324234725 for aquarium rearing (jacobs, 1971; snelson, 1982). as in other aquaculture operations, feed costs can affect the economics of an aquarium business. thus, a suitable feeding strategy that improves the growth performance may considerably reduce the cost of culture operations. compensatory (or catch-up) growth in fish is usually defined as a growth acceleration seen following the return of favorable conditions after a period of growth depression (dobson and holmes, 1984; jobling, 1994; ali et al., 2003). compensatory growth has a vital role in feed management and optimization in fish culture practices (lovell, 1980). there are several studies on the effect of starvation and refeeding in coldwater fishes (miglavs and jobling, 1989; quinton and blake, 1990; nicieza and 110 morshedi et al./ int. j. aquat. biol. (2013) 1(3): 109-115 metcalfe, 1997; nikki et al., 2004) and warm water fishes (russell and wootton, 1992; hayward et al., 1997; gaylord and gatlin, 2000, 2001; wang et al., 2000; zhu et al., 2001). sailfin molly, p. latipinna is a popular ornamental fish, but compensatory growth has not been examined in this species. thus, this study was conducted to investigate a compensatory growth response in sailfin molly subjected to shortterm starvation and refeeding. this study also aimed to evaluate the effects of feeding regimes on growth, feed utilization, and body composition of sailfin molly. materials and methods the experimental male fish, p. latipinna, were transported from a commercial farm (rahvand ltd, kashan, iran) to the laboratory. specimens were acclimated in 500 l tank for two weeks before the start of the experiment where they were fed with frozen bloodworms twice a day. during the experiment, data were collected every 9 days. fish were randomly selected and weighed to the nearest 0.01 g and measured to the nearest 0.1 mm. after adaptation, 400 fish (1.30±0.82 g) were randomly distributed into 20 rectangular glass aquaria (33.6×25×25 cm, 21 l). each aquarium was supplied with air stone and aeration. four treatment groups were established with five replicates. the control group (c) was fed ad libitum twice a day with a commercial formulated feed (manufactured by tetra, germany), containing 35% crude protein, 5% crude lipid, 4% crude fiber and 12% moisture, at 09:00 and 16:00 h throughout the experiment. fish in the other three treatments were starved for 3, 6, or 9 days followed by 6, 12 or 18 refeeding (referred to as t1, t2 and t3, respectively) in repeated cycles during 54 days the experiment. during the refeeding days, the specimens were fed ad libitum twice a day with the same commercial feed as described above. in each tank, the number of uneaten pellets was counted for calculation of daily food consumption. throughout the experiment, dissolved oxygen, temperature and ph were monitored weekly. water temperature was maintained at 28±1 °c, dissolved oxygen was > 6 mg l-1, water ph and ammonia were 7-7.6 and 1.01±0.12 mg l-1. a photoperiod of 14l: 10d using fluorescent lights was supplied throughout the experiment. at the end of the experiment following 16 h of starvation, fish were randomly sampled dried to constant weight at 105°c to measure the moisture content. the dried samples homogenized for determining the following parameters, which crude protein was determined by micro kjeldahl method (n×6.25) after acid digestion, lipid by etherextraction method using a soxtec system, fiber by acid and alkaline digestion then combustion in a muffle oven at 550°c for 5 h and moisture content by drying at an oven with a temperature of 120°c for 5 h (aoac, 1995). the following indices were calculated: specific growth rate (sgr % day-1) = 100[(lnwt-lnw0)/t]; percentage weight gain (%) = 100[(wt-w0)/ w0], where wt and w0 are final and initial weight (g) and t is the feeding duration (day); condition factor = 100[w/ l3], where l = length (cm); feed conversion ratio = intake (g, dry weight) / wet weight gain (g); protein efficiency ratio = wet weight gain / protein consumed (dry matter); daily feeding intake (g) = g feed day-1. statistical analyses were performed using spss, version 15.0 for windows. the normality of distribution of variables was tested using kolmogorov–smirnov test. the homogeneity of variances was tested using the levene’s f test. the possible differences in the variables among the treatments were tested using one-way anova. a significant difference between sample means was tested using the tukey test. data were expressed as mean±standard error (se) and differences were considered statistically significant at p<0.05. results survival of the experimental male fish ranged from 97 to 100% and did not differ among the treatments (p>0.05). at the end of the 54 days of experiment, there were no significant differences in mean final 111 morshedi et al./ int. j. aquat. biol. (2013) 1(3): 109-115 body weight between the treatments (p>0.05, fig. 1, table 1). there were no significant differences between the treatments in specific growth rate, weight gain or condition factor at the end of the experiment (p>0.05). however, these parameters increased with increase of starvation periods (table 1). at the end of the experiment, daily feed intake was significantly higher in t3 fish than that of the control fish (p<0.05, table 2). the highest daily feed intake levels were observed in t3, t2 t1and control fish, respectively. feed conversion ratio (fcr) varied between 3.6 and 5.3 and no significant difference was found between the control group and the starved group. however, fcr tended to decrease with parameters treatment c t1 t2 t3 initial weight(g) 1.32±0.78 1.41±0.86 1.35±0.90 1.29±0.72 final weight(g) 2.29±0.17 2.20±0.12 2.30±0.86 2.30±0.94 cf 2.06±0.33 1.93±0.24 1.98±0.24 1.99±0.02 sgr(% day-1) 1.00±0.13 0.81±0.09 0.99±0.28 1.07±0.12 wg (%) 73.33±6.28 55.41±1.50 74.49±11.27 79.17±4.12 table 1. growth performance of sailfin molly reared at four feeding regimes for 54 days (mean ±se). c, control (fed twice daily to apparent satiation); t1, treatment 1 (3 days starvation and 6 days refeeding); t2, treatment 2 (6 days starvation and 12 days refeeding); t3, treatment 3 (9 days starvation and 18 days refeeding). different superscript letters denote significant differences between the experimental groups. parameters treatment c t1 t2 t3 daily feed intake(g) 0.20±0.01a 0.23±0.01ab 0.21±0.02ab 0.24±0.01b fcr 4.03±0.98 5.37±0.04 4.04±1.35 3.69±0.19 per 0.78±0.15 0.53±0.00 0.78±0.24 0.77±0.03 table 2. feed utilization of sailfin molly reared in four different feeding regimes for 54 days (mean ±se). c, control (fed twice daily to apparent satiation); t1, treatment 1 (3 days starvation and 6 days refeeding); t2, treatment 2 (6 days starvation and 12 days refeeding); t3, treatment 3 (9 days starvation and 18 days refeeding). different superscript letters denote significant differences between the experimental groups. treatment parameters protein (%) lipid (%) ash (%) moisture (%) initial 13.87±0.01a 5.79±0.04a 3.08±0.03a 76.19±0.02a c 13.33±0/47a 5.71±0.08a 2.75±0.16a 76.75±0.74a t1 12.14±1.49a 5.12±2.78a 2.76±1.51a 78.76 ±0.28ab t2 11.77±0.49a 4.41±2.36a 2.95±1.16a 79.63 ±0.18bc t3 11.58±1.38a 4.28±1.56a 2.94±0.39a 79.97±0.34c table 3. body composition of sailfin molly subjected to four feeding regimes for 54 days (mean ±se, n=5, each n consist of measurements of five fish). c, control (fed twice daily to apparent satiation); t1, treatment 1 (3 days starvation and 6 days refeeding); t2, treatment 2 (6 days starvation and 12 days refeeding); t3, treatment 3 (9 days starvation and 18 days refeeding). different superscript letters denote significant differences between the experimental groups. 112 morshedi et al./ int. j. aquat. biol. (2013) 1(3): 109-115 increasing the duration of starvation (p>0.05, table 2). there were no significant differences in protein efficiency ratio (per) among different treatments (p>0.05, table 2). short-term starvation did not affect the whole-body protein, lipid and ash at the end of the experiment (p>0.05), and no significant differences was detected between the starved and the control fish (table 3). however, moisture content was significantly higher in t2 and t3 fish than that of the control fish (p<0.05), and the body’s water content tended to increase with longer starvation periods (table 3). discussion this experiment indicated that compensatory growth is occurred following short-term starvation periods in sailfin molly. at the end of the experiment, all starved fish fully compensated the lost weight, which was indicated by the similar final mean weights in the four treatments. the results of this study are in agreement with many other compensatory growth studies (e.g. gaylord and gatlin, 2000 (channel catfish, ictalurus punctatus), xie et al., 2001 (gible carp, carassius auratus), zhu et al., 2001 (threespined stickleback, gasterosteus aculeatus and minnow, phoxinus phoxinus), tian and qin, 2003 (barramundi, lates calcarifer), nikki et al., 2004 (rainbow trout, oncorhynchus mykiss fasted for 2 or 4 days), mattila et al., 2009 (pick perch, sander lucioperca fasted for 1 day)). in contrast, studies on gibel carp and chinese long snout, leiocassis longirostris (zhu et al., 2004), gilthead sea bream, sparus aurata (eroldoĝan et al., 2006), atlantic halibut, hippoglossus hippoglossus (heide et al., 2006) and white fish, coregonus lavaretus (kankanen and pirhonen, 2009) showed partial compensatory growth. the present differences among these experiment could be due to different experimental protocols or condition, temporal differences, physiological condition and severity of feed deprivation (jobling, 1987; jobling and koskela, 1996). in the present study, the specific growth rate, although not significantly, tended to increase with increase of the feed deprivation. this may be due to reduced metabolic rate during feed deprivation as a result of decreased activity (love, 1970; jobling, 1980; eroldoĝan et al., 2006) and increased daily food intake or both (heide et al., 2006). there were no difference in condition factor between the starved and control fish (table 1) indicating that compensatory mechanisms had occurred (kankanen and pirhonen, 2009). the fish in the t3 treatment had a significantly higher mean daily feed intake than other treatments, but there were no significant differences in feeding performance (as fcr and per) compared to the control fish during the period of refeeding (table 2). compensatory growth can be achieved by hyperphagia (wang et al., 2000; tian and qin, 2003, 2004; nikki et al., 2004; mattila et al., 2009) or combination of hyperphagia and improved feed efficiency (qian et al., 2000; gaylord and gatlin, 2001; li et al., 2005). as there were no differences in feed conversion ratio, it can be assumed that the mechanism to compensate for weight loss in the sailfin molly was probably hyperphagia during the period of refeeding. the present results consistent with the contention (heide et al., 2006) that for aquaculture purposes, an initial longer period of figure 1. mean weight of sailfin molly subjected to different cycles of starvation and refeeding for 54 days. c, control (fed twice daily to apparent satiation); t1, treatment 1 (3 days starvation and 6 days refeeding); t2, treatment 2 (6 days starvation and 12 days refeeding); t3, treatment 3 (9 days starvation and 18 days refeeding). no significant differences observed in four groups (error bars have been omitted for clarity). 113 morshedi et al./ int. j. aquat. biol. (2013) 1(3): 109-115 starvation is preferable to achieve a clear compensatory effect. the body composition of the fish subjected to a period of starvation was similar at the end of the experiment to that of the control fish. there was an exception in moisture content, which was significantly different between the deprived and control fish. moisture content was significantly increased in the deprived fish than in the control fish and there was a general tendency for lipid content, although not significantly, to decrease with increasing moisture content (table 3), indicating that the inverse relationship between lipid and moisture content that was probably caused by replacement of utilized lipid with an equal volume of water (grigorakis and alexis, 2005). this is in agreement with the results obtained in previous studies. however, different results have been reported for other fish species. for example, mattila et al. (2009) reported that moisture content in pikeperch subjected to longer starvation period was much higher than that of the control fish. the results on hybrid tilapia, oreochromis mossambicus × oreochromis niloticus (wang et al., 2000) and great sturgeon, huso huso (falahatkar et al., 2009) also indicated that starvation had a significant effect on moisture content. the effect of starvation on utilization of reserve protein and lipid seems to be species-specific (ince and thorpe, 1976; mehner and wiese, 1994), which may have caused the difference in the results. the present study indicated that sailfin molly adapted to shortterm period of starvation and can defend body composition (except moisture) in these periods. overall, this study showed that sailfin molly reared under different cycles of starvation and refeeding protocols for 54 days lead to complete compensation. the deprived fish were still undergoing compensatory growth at the end of the experiment. however, further research including physiological response is needed to confirm this finding. the tendency of decreased feed conversion ratio and increased specific growth rate in the deprived fish indicated that the use of starvationrefeeding cycles could decrease costs of labour, food and culture time in the commercial production of sailfin molly. in addition, these regimes could improve water quality in aquarium. acknowledgments the authors are grateful to the khoramshahr marine science and technology university for the financial support and providing the rearing facilities. references ali m., nicieza a., wootton r.j. 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(2015) 3(5): 323-330 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology review article review on the caspian shemaya, alburnus chalcoides (güldenstädt, 1772) bahram falahatkar*1,2, ali safarpour amlashi3, soheil eagderi4, hamed mousavi-sabet1,2 1fisheries department, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. 2the caspian sea basin research center, university of guilan, rasht, iran. 3departments of fisheries, faculty of marine science, tarbiat modares university, noor, iran. 4department of fisheries, faculty of natural resources, university of tehran, p.o. box 4314, karaj, iran. article history: received 18 july 2015 accepted 5 october 2015 available online 2 5 october 2015 keywords: alburnus chalcoides cyprinidae shemaya conservation abstract: alburnus chalcoides, caspian shemaya, is found in the river systems of the aral, black and caspian sea basins and is an economically valuable cyprinid from the southern caspian sea. this species has been reported as near threatened species in this basin due to damming, over and illegal fishing, destruction of its spawning grounds and water pollution. the caspian shemaya is an important species ecologically and economically, but information about its biology and ecology is widely scattered. hence, in this review, its taxonomy, general characteristics and morphology, distribution, habitat and ecology, age and growth, reproduction, conservation status and threats in the southern caspian sea is summarized, and a bibliography on this fish is provided. introduction alburnus rafinesque, 1820 is a widespread genus of the family cyprinidae with about 39 recognized species distributed in the europe and west asia (bogutskaya, 1997; bogutskaya et al., 2000; freyhof and kottelat, 2007a, b; kottelat and freyhof, 2007; özulug and freyhof, 2007; coad, 2009; bug et al., 2010; khataminejad et al., 2013a, 2013b; mousavisabet et al., 2013, 2015). this genus has eight confirmed species in iranian inland waters, including a. chalcoides (güldenstädt, 1772), a. filippii kessler, 1877 and a. hohenackeri kessler, 1877 in the south caspian sea basin, a. atropatenae berg, 1925 in the urmia lake basin, a. mossulensis heckel, 1843, a. zagrosensis coad, 2009 and a. caeruleus heckel, 1843 in the tigris river basin and a. amirkabiri, mousavi-sabet et al., 2015 in the namak lake basin (mousavi-sabet et al., 2014, 2015). in addition, a. doriae de filippi, 1865 and a. maculatus keyserling, 1861 have uncertain provenance and validity from iran (coad, 2009). * corresponding author: bahram falahatkar e-mail address: falahatkar@guilan.ac.ir alburnus chalcoides, caspian shemaya, is found in the river systems of the aral, black and caspian sea basins (bogutskaya, 1997). this species was described as chalcalburnus chalcoides, but bogutskaya (1997), bogutskaya and naseka (2004) and kottelat and freyhof (2007) suggested that the genera alburnus and chalcalburnus are the same. thus, these two genera were merged into one genus i.e. alburnus. therefore, this species is considered as phenotypic variant of a. chalcoides. the caspian shemaya inhabits southern coasts of the caspian sea from east to west (patimar et al., 2010), and is morphologically varied from those of the black and aral sea basins (berg, 1949; bogutskaya, 1997). alburnus chalcoides is a commercial or semicommercial species in iran (sattari et al., 2004). there are some studies on different biological and ecological aspects of a. chalcoides in the southern caspian sea basin (svetovidov, 1945; darabi, 1999; rajabi nezhad and azari takami, 2001; mohsen 324 int. j. aquat. biol. (2015) 3(5): 323-330 zadeh and bahadori, 2001; azari takami and rajabi nezhad, 2002; bagherian and rahmani, 2007; rahmani, 2006; 2008; bagherian and rahmani, 2009; shirvani and jamili, 2009; rahmani et al., 2009; patimar et al., 2010; khataminejad et al., 2013b; mousavi-sabet et al., 2014), but they are widely scattered. therefore, the aim of this study is to summarize the ecology and biology of this valuable species in the southern part of the caspian sea. morphology: the body is elongated, compressed, and moderately deep with moderate size. the mouth is terminal without barbels. its abdomen has an obvious scaleless sharp keel from vent to throat. there is a well-developed pelvic axillary scale. the branched rays of the dorsal fin 7-9, branched rays of the anal fin 12-19 after 3 spines, lateral line scales 54-74, gill rakers 18-25 and total vertebrae 42-45 (svetovidov, 1945; coad, 2015). unlike other populations of the caspian shemaya which the last unbranched rays in their dorsal fin are soft, those rays of iranian caspian shemaya transformed as a smooth sharp spine (svetovidov, 1945). in deepest part of the body, a narrow dark band runs in both sides of body from the head to the caudal fin (coad, 2015). inferior mandible is protruded. this species has two rows of the pharyngeal teeth with formula of 2.5-5.2 and rarely 2.4-5.2, 2.5-4.2 and 2.5-5.3 (svetovidov, 1945; coad, 2015). their teeth are protracted, thin, curved inward, and well-hooked at the tip. in addition, the teeth are strongly serrated on the anterior border of their length, and have a narrow and concave surface. its swim bladder is pointed posteriorly and its gut is elongated s-shape (svetovidov, 1945; coad, 2015). the caspian shemaya has a metallic silvery body and its dorsal part bears a contrasting olive-green colour (fig. 1). both dorsal and caudal fins are grayish, and the pectoral, ventral, and anal fins are colorless to whitish. the iris is bright silver and its peritoneum is light brown in colour (coad, 2015). in adults, its standard length (sl) is 14.20-19.99 cm (svetovidov, 1945). its average total length (tl) is 15.06 cm in males (1-4 years) and 17.57 cm in females (1-5 years) and the average weight is 24.7 g and 41.7 g in males (1-4 years) and females (1-5 years), respectively (rahmani et al., 2009). the reported maximum tl in siahroud river (central of the south caspian sea basin) is 24.20 cm and in the gorganroud river (the southeastern of the south caspian sea basin) is 24.30 cm, both being a fiveyear-old female (patimar et al., 2010). the reported maximum tl of males in siahroud river is 21.30 cm and in gorganroud river is 20.75 cm. all fish with tl ≥ 21.30 cm and ≥ 20.80 cm in siahroud and gorganroud rivers were female (patimar et al., 2010). this needs to be noted that the sex ratio reduces with increasing tl. bagherian and rahmani (2007, 2009) examined morphology of two populations, from haraz and shiroud rivers. their results showed that the males and females between the two populations were morphologically different. figure 1. alburnus chalcoides (güldenstädt, 1772) from shalmanroud. 325 falahatkar et al./ review of the caspian shemaya rahmani et al. (2007) pointed out that these two populations are almost separable based on meristic characters. rahmani et al. (2006) showed that the populations of gazafrud and haraz rivers are separated using morphometric characters but not by meristic ones. rahmani et al. (2009) used the 18s rrna gene and found populations from haraz, shiroud and gazafrud rivers are homogenous. alburnus chalcoides shows a remarkable morphological variation in the southern part of the caspian sea that maybe related to their adaptation to different habitats (mohadasi et al., 2013, 2014). distribution: alburnus chalcoides inhabits almost all iranian coasts of the caspian sea and its rivers, including the atrak, gorganroud, gharasu, tajan, babolroud, haraz, sardabroud, aras, tonekabon, polroud and sefidroud rivers, the anzali lagoon, and the gorgan bay (kozhin, 1957; svetovidov, 1945; holčík and oláh, 1992; shamsi et al., 1997; kiabi et al., 1999; abdoli, 2000; bagherian and rahmani, 2007; 2009; abdoli and naderi, 2009; patimar et al., 2010). habitat and ecology: the caspian shemaya lives in both brackish and freshwater, downstream, coastal lakes, estuaries, and adjoining areas of seas where salinity is lower than 14 ppt (kottelat and freyhof, 2007b). commonly, this fish lives near to surface, but knipovich (1921) reported this species from depths of 23.8-25.6 m in the iranian shore of the caspian sea. this semi-anadromous species migrates to the rivers and moves to upstream for spawning. in southern part of the caspian sea, its spawning grounds have been reported form the atrak river in southeast to the aras river in southwest of the caspian sea, but it also found predominantly in rivers of the central parts of the southern caspian sea basin (patimar et al., 2010). holčík and oláh (1992) reported a feeding migration in july to september in the anzali lagoon. alburnus chalcoides feeds on phytoplankton e.g. crysophyta, chlorophyta and cyanophyta and zooplankton e.g. copepoda and cladocera (abdurakhmanov, 1962). furthermore, it feeds on macrophytes (coad, 2015) and some larger organisms such as chironmidae larvae, gomphonema, crustaceans, terrestrial insects, and small fish (rajabi nezhad and azari takami, 2001). age and growth: it is reported that the caspian shemaya's longevity is over 5 years in the southern caspian sea basin (holčík and oláh, 1992; patimar et al., 2010). the growth of a. chalcoides in the southern caspian sea basin is related to the age and geographical location where it lives (patimar et al., 2010). it spawns in spring in the anzali lagoon at 1029.0 cm with a mean weight of 64.7 g (holčík and oláh, 1992; karimpour et al., 1993) in march and peaks in may and at the beginning of june (karimpour et al., 1993). all the spawning fish were 2-5 year-old but most of them (63%) were 3-yearold. the males will reach maturity one year earlier than females in 2-4 year-old i.e. the females mature in 3-5 year-old (holčík and oláh, 1992; karimpour et al., 1993). the fish growth rate at first three years of life is more than that of the second three years (holčík and oláh, 1992). in haraz and shiroud rivers, the age groups of 2+ years for males and 3+ years for females are the most abundant age groups (rahmani, 2008). rahmani et al. (2009) found that the growth rate is better in the shiroud river compared to the other populations in the southern caspian sea basin, because this river has proper biological parameters. patimar et al. (2010) compared the growth rate of a. chalcoides populations from the shiroud and gorganroud rivers and found a five-year life cycle, with a negative allometric growth pattern for males and a positive one for females in siahroud river. also, they found a positive allometric growth pattern for both sexes in the gorganroud river (patimar et al., 2010). mousavi-sabet et al. (2014) presented the length-weight relationship of the genus alburnus in iran and reported a negative allometric growth for a. chalcoides from the southern caspian sea basin. reproduction: alburnus chalcoides spawns intermittently while has three batches of eggs. it lays two of them only within a period of 18-19 days (svetovidov, 1945; coad, 2015). the caspian shemaya is a semi-anadromous species. female 326 int. j. aquat. biol. (2015) 3(5): 323-330 matures and spawns one year later than male, while they are larger than males (bagherian and rahmani, 2007). in addition, females form the majority (57%) of the migrated fish (coad, 2015). sometimes larger fish mature and spawn earlier (karimpour et al., 1993; rahmani et al., 2009). during spawning period, the caspian shemaya enters the rivers (for long distances upstream) in autumn and move upstream. then, it spawns in heavy current rivers on gravel bottom (svetovidov, 1945; sattari et al., 2004). males are marked with small tubercles scattered on top of the head and fine tubercles on the anterior flank scales during spawning periods (bagherian and rahmani, 2007). sexual proportion is unbalanced in females’ favor (1:1.54) in siahroud and gorganroud rivers (patimar et al., 2012) and shiroud river (1:2.36) (rahmani et al., 2009). males are somewhat territorial. they gather at spawning grounds together and stay for females, which arrive later (freyhof and kottelat, 2007a, b). alburnus chalcoides spawns from april to july in siahroud river, and from march to june in the gorganroud river, and its peak is in may in both rivers (patimar et al., 2010). nikoo et al. (2010) measured serum sex steroids of a. chalcoides during spawning in the valiabad river and concluded that this fish may be a multiple spawner. spawning occurs in 0.2-0.7 m depth, water flow rate about 1 m/s, and 18-26°c, often with a lot of splashing (kottelat and freyhof, 2007b). since adults were caught in july and february and young fish were found in the southern caspian sea throughout the year, it concluded that iranian populations spawn throughout the year (svetovidov, 1945). eggs stick and adhere on pebbles or stones. embryo developed for 2-3 days, larvae migrates to shallows and backwaters after the remaining among the gravels about 8-11 days (kottelat and freyhof, 2007b). the juveniles migrate to downstream in autumn of the same year or spring of the next year. parents go back to the sea, lakes, and/or estuaries soon after the spawning. rahmani et al. (2009) reported a peak gonadosomatic index for males in may and for females in early june in the shiroud river. alburnus chalcoides has 1.5 mm eggs in diameter as early as 13 march (with a standard length of 213.2 mm) and 1.7 mm on 4 june (with a total length of 154.6 mm) (karimpour et al., 1993). mean absolute fecundity of the caspian shemaya from the southern caspian sea basin has been reported 3568 eggs in haraz river (rahmani, 2006), 6630 eggs in the anzali lagoon with a mean relative fecundity is 140 eggs/g of body weight (karimpour et al., 1993), 3900 eggs with diameter reaching 1.17 mm in the shiroud river (rahmani et al., 2009), 8426 eggs (average 212 eggs/g) with mean diameters of 1.40 mm in the siahroud river (patimar et al., 2010) and 4215 eggs (average 112 eggs/g ) with mean diameters of 1.40 mm in gorganroud river (patimar et al., 2010) (table 1). conservation status: this species has been reported as near threatened species in the southern caspian sea basin due to damming, over and illegal fishing during spawning season and destruction of the spawning grounds (kiabi et al., 1999; naderi and abdoli, 2004). mostafavi (2007) reported that a. chalcoides is a near threatened species in the talar river in mazandaran province (north of iran). in addition, this species is listed as a vulnerable to endangered species in europe (lelek, 1987) and an study area mean (min.-max.) reference anzali lagoon 6630 (2951-11855) karimpour et al. (1993) sefidroud river 9960 (2929-18860) azari takami and rajabi nezhad (2003) shiroud river 3906 (1370-10387) rahmani (2006 and 2009) haraz river 3568 (1647-6932) rahmani (2006) siahroud river 8426 (1674-38340) patimar et al. (2010) gorganroud river 4215 (623-17263) patimar et al. (2010) table 1. absolute fecundity of alburnus chalcoides found by different authors in the southern of the caspian sea, iran. 327 falahatkar et al./ review of the caspian shemaya endangered species in turkey (fricke et al., 2007). however, a. chalcoides is ranked in red list category and criteria of iucn (gesner et al., 2010) as least concern. unfortunately, there is no information and ranking available about a. chalcoides in the caspian sea basin in iucn. threats: the main threat are water pollution especially in spawning grounds, damming the rivers which are the migration routes of this fish, over and illegal fishing during spawning and deterioration of the spawning grounds (kiabi et al., 1999; naderi and abdoli, 2004; abdoli and naderi, 2009). in addition, there are some reports about excessive levels of cadmium, lead and other heavy metals in this fish (shirvani and jamili, 2009). based on our study, further researches including habitat requirements, population genetics, migrations (number, duration, and reason), natural behavior during migration, ionic balance and osmoregulation, spawning and artificial breeding can help to better understanding of this species. references abdoli a. 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(2015) 3(5): 290-300 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی alburnus chalcoides (güldenstädt, 1772)کولی خزری، مروری بر شاه 1, 2 سیدحامد موسوی ثابت ،4ایگدری سهیل، 3علی صفرپور املشی، *1, 2بهرام فالحتکار .ایران، گیالن، 1111صندوق پستی ،صومعه سرا ،گیالن دانشگاهدانشکده منابع طبیعی، شیالت، گروه1 .ایران رشت، گیالن، دانشگاه خزر، دریای آبریز حوضه پژوهشکده2 .ایران نور، ،تربیت مدرس دانشگاه دانشکده علوم دریایی، شیالت، گروه3 .ایران ،1311صندوق پستی کرج، تهران، دانشگاه طبیعی منابع و کشاورزی پردیس دانشکده منابع طبیعی، شیالت، گروه1 چکیده: با یک گونه شود و دریاهای آرال، سیاه و خزر یافت میآبریز های ای حوضههای رودخانهدر سیستم، alburnus chalcoides ،کولی خزریشاه ویه و رواسطه سدسازی، صید بیعنوان یک گونه در معرض تهدید بهبه. این گونه باشدمیخزر جنوبی دریایحوضه دراز کپورماهیان ارزش اقتصادی اشد، اما بکولی خزری یک گونه مهم از نظر بوم شناختی و اقتصادی میهای تخمریزی و آلودگی آب گزارش شده است. شاهغیرقانونی، تخریب جایگاه ناسی، شهای عمومی و ریختشناسی، ویژگیرو در این مطالعه مروری، آرایه. از ایناستده شناسی آن بسیار پراکنشناسی و بوماطالعات در مورد زیست این مورد در منابع فهرستو یک بیان شدهخالصه به طور آن و تهدیدات یحفاظتوضعیت ، سن و رشد، تولیدمثل، شناسیبومپراکنش، زیستگاه و .گرددمی ارائه ماهی .فاظتح ،کولیشاهکپورماهیان، ، alburnus chalcoides :کلمات کلیدی int. j. aquat. biol. (2022) 10(4): 315-320 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology short communication effect of mint powder, mentha longifolia, and vitamin d administration on growth indices of juvenile rainbow trout, oncorhynchus mykiss albab fawwaz alfarras1, noora m. hameed2, huda sabah jabr3, salam ahjel4, mohamed kazem alaraji5, ahmed s. abed6 1medical lab. techniques department/ college of medical technology, al-farahidi university, iraq. 2anesthesia techniques, al-nisour university college, iraq. 3department of anesthesia techniques, al-mustaqbal university college, babylon. 4department of pharmacy, al-zahrawi university college, karbala, iraq. 5department of pharmacy, ashur university college/baghdad, iraq. 6department of prosthetic dental technology, hilla university college, babylon, iraq. . s article history: received 17 march 2022 accepted 20 june 2022 available online 2 5 august 2022 keywords: trout mint vitamin d fish diet antioxidants abstract: numerous studies have shown that herbs can be used as growth stimulants in aquaculture. this study aimed to investigate the effect of combined consumption of mint powder and vitamin d on the growth and health of juvenile rainbow trout, oncorhynchus mykiss. for this purpose, a total of 200 juvenile rainbow trout were divided into four groups and treated for 60 days as follows: the 1st group (control) used a standard diet; the 2nd group had 1.5% mint powder in their diet; the 3rd group having an additional amount of 120 mg/kg of vitamin d in their diet; the 4th group fed a diet having mint and vitamin d supplements as 2ed and 3rd groups. all groups' growth and health indices viz. condition factor, hepatic steatosis index, visceral sensitivity index, spleen somatic index, weight gain percentage, specific growth rate, feed conversion ratio, satiety index, food conversion efficiency, and protein efficiency ratio were measured and compared on days 20, 40, and 60. the results revealed that the use of mint powder in the juvenile rainbow trout diet not only had no remarkable effect on the growth and health of fish but also improved their growth. in addition, all the analyzed indications of the mint powder group outperformed than the vitamin d group. introduction the application of the plant essential oils and herbs in aquatic and livestock diets has attracted much attention in recent years (sońta et al., 2019; pouladi et al., 2020; molajou et al., 2021; paray et al., 2021; mucha and witkowska, 2021; nehme et al., 2021; angane et al., 2022). antioxidant properties are found in almost all essential oils and herbs, helping prevent microbial growth and toxin production in feed (ceylan et al., 2019; dorra et al., 2019). plant compounds also improve the performance of livestock and aquatic products, strengthen their immune system, and have antimicrobial properties (ahmadifar et al., 2021). by effectively releasing digestive enzymes, they increase nutrient digestion and absorption, and by enhancing nitrogen uptake, they help protein absorption into the cell (wainstein et al., 2012). correspondence: albab fawwaz alfarras doi: https://doi.org/10.22034/ijab.v10i4.1713 e-mail: a.ibrahim@uoalfarahidi.edu.iq several studies have shown adding herbs to food improves aquatic growth performance (manhas and gill, 2010; promya and chitmanat, 2011; hai, 2015; prabu et al., 2017). medicinal plants have emerged as an alternative and promising agent for controlling disease in fish (zam et al., 2019). medicinal herbs are used in aquaculture not only as chemotherapeutics but also as feed supplements. this is because they include a diverse range of nutrients in addition to chemical compounds (kalita and borthakur, 2010; dutta et al., 2020). therefore, they have been shown to promote growth, hunger stimulation, immunological stimulation, antibacterial activity, and anti-stress activity in aquaculture (shakya, 2017; caipang, 2020). the ease of access to many plants and their low cost encourages their application in aquaculture on a large scale (khosravi et 316 alfarras et al./ effect of mentha longifolia, and vitamin d on oncorhynchus mykiss al., 2018; bharathi et al., 2019; f ayoub et al., 2019). spices and condiments such as mint are used in foods and are natural sources of antioxidants and antibacterial chemicals in aquaculture (azizkhani and tooryan, 2015; abdul qadir et al., 2017; khan et al., 2018). mentha longifolia (mint), a member of the lamiaceae family, is found in the mediterranean region and north africa, australia, and europe (mahmoud et al., 2022). it has wide application in the food processing and pharmaceutical industries. mint's seeds, bark, flowers, stems, and leaves have all been used in traditional folk medicine as carminatives, stimulants, antispasmodics, antimicrobials, and antioxidants, and for the treatment of various ailments like digestive problems and headaches (asghari et al., 2018). the addition of mint in the diet of poultry improves growth performance and physiological conditions and raises antioxidant activity and feed conversion ratio (kumar and patra, 2017; vargassánchez et al., 2019). based on the above-mentioned background, this study aimed to investigate the effects of mint powder and dietary vitamin d on rainbow trout's (oncorhynchus mykiss) health and growth performance. methods and material in march 2021, 200 rainbow trout juveniles with an average weight of 36±2 g and a total length of 15.2±0.7 cm were purchased and transferred to a wet lab, at alfarahidi university. the fish were maintained in a 1500liter tank for two weeks to acclimatization. during the adaption period, they were fed a conventional diet of trout twice a day at a rate of 3% body weight, and 20% of the tank water was replaced daily. the dietary composition of the ratio used is presented in table 1. the fish were divided into four groups each with three replications and introduces randomly into twelve 300l tanks, each with a density of twelve fish. the 1st group (control) was fed a standard diet, the 2nd group fed 1.5% mint powder in their diet, the 3rd group fed a diet having 120 mg/kg of vitamin d and the 4th group fed a combination of mint and vitamin d supplements as mentioned in 2ed and 3rd treatments. the desired amount of mint powder (1.5%) and vitamin d (120 mg/kg dry weight) were mixed and converted as pellets with a diameter of approximately 3.5 mm by a meat grinder. the pellets were frozen at -18°c after being dry at 45°c for 30 hours (with 10% humidity). on days 20, 40, and 60, two fish from each tank (6 fish per treatment) were randomly sampled. after anesthesia using ms222, the fish were killed and following growth parameters viz. condition factor (cf) (osho and usman, 2019), the hepatic steatosis index (hsi) (ribeiro et al., 2013), visceral sensitivity index (vsi) (torstensen et al., 2011), spleen somatic index (ssi) (abdel-tawwab et al., 2021), weight gain percentage (wg%) (besson et al., 2016), specific growth rate (sgr) (korzen et al., 2016), feed conversion ratio (fcr), satiety index, food conversion efficiency (fce) (zhang et al., 2010), and protein efficiency ratio (per) (moogouei, 2014; taee et al., 2017; adeshina et al., 2018) were measured. after verifying the data for normality with the kolmogorov-smirnov test, a one-way analysis of variance (anova) was used to analyze the data. using duncan's multiple range test (mrt), mean differences between the groups were obtained. data analysis was performed using spss software version 25. data analysis was performed using spss software version 25. results and discussion no signs of disease or mortality were observed in the treatments during the experimental period. the findings revealed no differences between the experimental groups except for visceral and satiety indices on the 20th day. on this day, when comparing vitamin d treatment to others, vsi and satiety had increased significantly (p<0.05) (fig. 1). vsi shows gastrointestinal-specific anxiety, affective, and cognitive response to the fear of gastrointestinal symptoms, sensations, and the circumstances in which these visceral symptoms and sensations manifest themselves (torstensen et al., 2011). in addition, the satiety index describes the sensation of being full and losing one's appetite after eating (zhang et al., 2010) significant increases in the vsi, satiety coefficient, wg%, per, sgr, and fce were observed in the treatment of mint powder on the 40th day. on this day, other treatments revealed no significant differences from the control one (p<0.05) (fig. 2). there are reports on the positive growth effects of mint on the broiler and aquatic content amount (%) protein 39 fiber 3.7 raw fat 12 ash 11 moisture 10 phosphorus 1.3 table 1. contents of the fish feed ingredients. 317 int. j. aquat. biol. (2022) 10(4): 315-320 animals (hong et al., 2012). in line with our findings, emami et al. (2012) reported similar results. also, ssi in mint and vitamin d supplemented treatments was significantly lower (p<0.05). spleen somatic index is a critical metric for determining a fish's relative vulnerability to various stressors (serrat et al., 2019; abdel-tawwab et al., 2022). the vsi increased in the mint powder supplemented treatments on day 60 (p<0.05) (fig. 3). the satiety index for mint powder treatment was higher than others. compared to the control group, mint powder treatments significantly improved sgr, per, wg%, and fce (p<0.05). in these indices, there was no difference between the vitamin d group and the control one. regarding hsi and vsi, the supplement of the mint powder showed a nearly uniform trend over time. while little changes were found in the control and vitamin d groups regarding these indices, i.e. a significant decline was found in hsi and vsi in the vitamin d group on days 40 and 60. hsi is diagnosed when the amount of fat within the liver is greater than 5% of the total weight (eguchi et al., 2012; machado and cortez-pinto, 2013; abd el-kader and el-den ashmawy, 2015; liu et al., 2017), on day 60, vsi in the control group decreased significantly. on this day, there was a substantial decline in the satiety index in the control and vitamin d groups. in previous works, vitamin d has caused an increase in the immune system and other hematological factors (dehghanizadeh et al., 2014), despite a decrease in the studied growth parameters. no differences were found in the supplemented mint powder groups. except in the control group on day 40, no other changes in the ssi were seen. although there is no remarkable change in the weight gained in all study groups, the trajectory of change in this index in the mint powder group had a distinct tendency in the 40th and 60th days compared to the 20th day. until the end of the experiment, the condition factor increased significantly in all of the groups, including the control group. the condition factor of a fish reflects the fish's biological and physical circumstances, as well as oscillations in those circumstances caused by the interaction between feeding environments, parasite diseases, and physiological factors (osho and usman, 2019; jafari-patcan et al., 2018; mouludi-saleh and eagderi 2019; eagderi et al., 2020). although there were no significant differences in any of the studied groups regarding the feed conversion ratio, the reduction in this index was noticeable on days 40 and 60. but in the mint treated groups, the indices of specific growth rate, feed conversion efficiency, and protein figure 1. a comparison of growth indicators in juvenile trout fed with a variety of diets on day 20 of the experiment. figure 2. a comparison of growth indicators in juvenile trout fed with a variety of diets on day 40 of the experiment. figure 3. a comparison of growth indicators in juvenile trout fed with a variety of diets on day 60 of the experiment. 318 alfarras et al./ effect of mentha longifolia, and vitamin d on oncorhynchus mykiss efficiency ratio exhibit showed increasing trends, with a significant difference on the 40th day compared to the 20th day. conclusion according to the findings of this study, mint powderreceiving juveniles exhibit a considerable improvement in growth and health indices at the end of the trial period, i.e., day 60, compared to other groups. furthermore, the trend of changes in the group receiving mint powder during the experiment revealed that mint powder has a proper effect on the growth of juvenile rainbow trout. references abd el-kader s.m., el-den ashmawy e.m.s. 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(2017) 5(4): 282-285; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology short communication first report of the fireworm hermodice carunculata (annelida: amphinomidae) preying on a sea cucumber rômulo barroso1*, daniel filgueiras2, mariana contins3, jerry kudenov4 1departamento de biologia; pontifícia universidade católica do rio de janeiro, rio de janeiro-rj, brazil. 2rosamar mergulho; rua lucio bacelar, 40/802. praia da costa, vila velha-es, brazil. 3laboratório de echinodermata, museu nacional/ufrj, rio de janeiro, brazil. 4department of biological sciences, university of alaska anchorage, 3211 providence drive, anchorage, alaska 99508, usa. article history: received 6 august 2017 accepted 24 august 2017 available online 2 5 august 2017 keywords: amphinomidae hermodice isostichopus predation abstract: the annelid ‘fireworm’ hermodice carunculata is widely recorded in clear shallow waters of the tropical atlantic ocean and adjacent seas, where it inhabits hard substrata. it is an omnivore and opportunistic scavenger, feeding strategies considered central to the maintenance of its broad geographic distribution. hermodice carunculata also preys on various cnidarians, and starfish. this study represents the first report of active predation by h. carunculata on living specimen of the holothurian isostichopus badionotus, from the southwestern atlantic, brazilian coast. the fact that parts of living holothurians were consumed, excluded the possibility of scavenging behavior. such predatory behavior is described here for the first time, corroborates that h. carunculata feeds on echinoderms other than starfish. however, we cannot presently answer the question whether h. carunculata actively preys on healthy holothurians or opportunistically feeds on injured sea cucumbers. introduction the ‘fireworm’ hermodice carunculata (pallas, 1766) is a shallow-water species distributed in tropical atlantic waters including the gulf of mexico, caribbean, mediterranean and red seas (ahrens et al., 2013). these worms are often called ‘fireworms’ since they can deliver urticating neurotoxin(s) to defend themselves against some predators and the occasional hapless diver. nakamura et al. (2008) characterized the neurotoxin complanine in eurythoe complanata, but its method of delivery remains elusive. tilic et al. (2017) refuted the long-held notion that hollow syringe-like harpoon notochaetae inject the neurotoxin into tissues since such chaetae are solidly constructed. moreover, tilic et al. (2017) found no evidence of venom glands or pores associated with the liberation of a neurotoxin. in respect to feeding habits, h. carunculata is a generalist species that is both omnivorous and an *corresponding author: rômulo barroso doi: https://doi.org/10.22034/ijab.v5i4.359 e-mail address: barroso.romulo@gmail.com opportunistic scavenger (fauchald and jumars, 1979; wolff et al., 2014; jumars et al., 2015). marsden (1963) conducted the first study on the feeding behavior of h. carunculata by examining the contents of its digestive tract. she found cells and fragments of corals, eunicid jaws, radular ribbons and numerous annelid chaetae, and maintained her laboratory specimens by feeding h. carunculata pieces of fish flesh and eventually a dying crinoid (marsden, 1963). various reports describing cnidarian predation by h. carunculata include hermatypic corals (ott and lewis, 1972; miller and williams, 2007; wolf and nugues, 2013; miller et al., 2014); anemones (lizama and blanquet, 1975); gorgonians (vreeland and lasker, 1989); fire corals (whitman, 1988; lewis and crooks, 1996); zoanthids (sebens, 1982; francinifilho and moura, 2010); and upside-down jellyfish (stoner and layman, 2015). a relevant aspect of cnidarian predation by h. carunculata is that these 283 int. j. aquat. biol. (2017) 5(4): 282-285 worms function both as vectors and as reservoirs for coral pathogens (sussman et al., 2003). recently, barroso et al. (2016) reported the first occurrence of predation by h. carunculata on two species of starfish, although another fireworm, pherecardia striata, preys on acanthaster planci (linnaeus, 1758), the crown-of-thorn starfish (glynn, 1984; cortes, 1997). the purpose of this communique is to provide details about the first report of h. carunculata preying on a sea cucumber. materials and methods the present observation was made during scuba dives at escalvada island, guarapari, espírito santo state, (20°42'0.08"s, 40°24'27.60"w), a subtropical region on the brazilian south-eastern coast on march 18, 2017. the sandy habitat was 15 m deep, and visibility of water column was 10 m during the observations. feeding activity was observed and photographed between 9:45 and 10:00 am. results and discussion one specimen of h. carunculata was observed preying actively on a specimen of the sea cucumber, isostichopus badionotus (selekna, 1867) during a 15minute period (fig. 1a). the sea cucumber was ca. 40 cm long, while the worm was ca. 15 cm. the worm fed actively on an open gash in the anterior end of the sea cucumber which repeatedly swung its anterior end from side to side, apparently in response to the worm’s actions (fig. 1b). such predation by h. carunculata is noteworthy, and reinforces both its role as an omnivore and the fact that its predation on echinoderms is not restricted to starfishes, as reported by barroso et al. (2016). conversely, this observation also adds another taxon to known natural predators of sea cucumbers, which mainly includes fish, crustaceans and sea stars (francour, 1997). we began our observations after the worm’s predatory attack began, and thus cannot categorically state whether the sea cucumber’s initial injury was caused by the worm or whether the worm opportunistically exploited an open injury somehow sustained earlier. for instance, both glynn (1984) and cortes (1995) observed pherecardia striata accessed figure 1. hermodice carunculata feeding on the anterior end of the holothurian isostichopus badionotus. (a) entire specimens and (b) close up on the feeding action. 284 barroso et al./ annelid prey on sea cucumber the coelomic cavity to feed on internal organs of the starfish acanthaster planci through body wall incisions inflicted previously by harlequin and other shrimps. the fact that hermodice actively consumed a living isostichopus excludes the possibility that the worm displayed scavenging behavior, which of course, is a previously confirmed feeding strategy for h. carunculata (wolf et al., 2014; jumars et al., 2015). within this context, it is worth noting that h. carunculata may also function as a pathogen vector to starfishes and sea cucumbers as it does to cnidarians (sussman et al., 2003). finally, given limited field observations, we cannot answer the question whether h. carunculata is an active predator of healthy holothurians or an opportunistic feeder of previously injured sea cucumbers. clearly, additional observations and experimental data on the feeding biology and behavior of h. carunculata are needed to such questions. references ahrens j.b., borda e., barroso r., paiva p., campbell a.m., wolf a., nugues m.m., rouse g.w., schulze a. 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(2017). getting to the root of fireworms’ stinging chaetae—chaetal arrangement and ultrastructure of eurythoe complanata (pallas, 1766) (amphinomida) journal of morphology, 2017:1-12. vreeland h.v., lasker h.r. (1989). selective feeding of the polychaete hermodice carunculata pallas on caribbean gorgonians. journal of experimental marine biology and ecology, 129: 265-277. whitman j.d. (1988). effects of predation by the fireworm hermodice carunculata on milleporid hydrocorals. bulletin of marine science, 42: 446-458. wolff a.t., nugues m.m. (2013). predation on coral settlers by the corallivorous fireworm hermodice carunculata. coral reefs, 32: 227-231. wolff a.t., nugues m.m., wild c. (2014). distribution, food preference, and trophic position of the corallivorous fireworm hermodice carunculata in a caribbean coral reef. coral reefs, 33: 1153-1163. international journal of aquatic biology (2014) 2(6): 305-312 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2014 npajournals. all rights reserved original article fish assemblage structure and habitat use of the snow fed stream assiganga a major tributary of river bhagirathi in central himalaya (india) gurnam singh, naresh k agarwal*1 fish reproduction and conservation biology research lab., department of zoology, school of life science, hnb garhwal university, campus badshshithaul-249 199, tehri garhwal (uttarakhand) india. article history: received 3 september 2014 accepted 13 november 2014 available online 2 5 december 2014 keywords: stream habitat fish assemblage river bhagirathi himalayan stream abstract: assiganga stream is an important tributary of bhagirathi river in central himalaya (india). the stream is characterized by heterogeneity in habitat and substratum features harboring diverse fish fauna. at present this stream is facing threat of being fragmented by the construction of two hydroelectric projects. present study aimed to study fish diversity and their habitat use in assiganga stream. this study reports fifteen species (14 indigenous and 1 exotic) belonging to 8 genera, 4 families and 3 orders. snow trout, schizothorax richardsonii (cyprinidae family) and salmo trutta (salmonidae family) were the dominant species (> 65% of total fish catch) throughout the entire length of stream. the presence of rich benthic food, clear water, low turbidity (01-05 ntu), high do (8.75-10.75 mgl), and high water velocity (1.10-1.40 m-s) with characteristic rapids and cascades in upper reaches provides ideal habitat for the existence of native snow trout and exotic trout species. few cat fishes, loaches, tor spp. and lesser barils also have been reported during the study. introduction fishes are invariable living components of water bodies and important food resource and indicators of the ecological health of water body. india has heterogeneity in climatic conditions, therefore, has a large network of rivers, both in himalaya and plains harboring 2500 fish species (jayaram, 2010). these rivers always remain the site of most of our evolutionary history and human activities, and have wide range of diversity in terms of fish and other aquatic organisms. naturally functioning stable stream systems promote the availability in heterogeneity of habitats. the fresh water fishes show variations in relation to habitat and geographical condition. the study of the habitat parameters and diversity of fish population of a river lend support to fishermen and ichthyologists (kar, 2010). according to ‘convention on biological diversity’, information’s on aquatic biodiversity is * corresponding author: naresh k agarwal e-mail address: agarwalnareshk3@rediffmail.com lacking at global as well as at local level. in absence of this information, it is difficult to assess status of any species and to prepare its conservation and management plan. fish resources in the fluvial systems of garhwal (central himalaya) had not been completely explored because most of the streams are located in aloof mountainous steep terrain with dense forest cover. some important studies from view point of fish diversity have been conducted in central himalaya, garhwal (badola, 1975; sharma, 1984; singh et al., 1987; lakra et al., 1987; dobriyal and kumar, 1988; agarwal et al., 2005, 2011; bisht et al., 2009; agarwal and singh, 2012). in spite of these studies, there is still complete dearth of information on some of the important central himalayan streams. the stream assiganga, a major tributary of river bhagirathi (ganga) is one of the unexplored streams in central himalaya from view point of fish diversity and water quality. moreover stream habitat is facing 306 international journal of aquatic biology (2014) 2(6): 305-312 threat of being altered by the hydro-electric projects being constructed on it. hence an attempt is made to generate base line information on fish diversity and their habitat use in the assiganga stream. study area: assiganga stream is located between latitude 30°48'n and longitude 78°27'e in uttarkashi district of uttarakhand state (india). it is snow fed perennial stream with high water discharge during summer and monsoon seasons. the stream originates after joining of two small streams viz. the kaldi gad (elevation 4521 m asl) and gajoli gad (elevation 3836 m asl) at sangamchati (elevation 1505 m asl). thereafter stream traverse a distance of ~15 km before debouching with river bhagirthi at gangori (elevation 1160 m asl) upstream to the northern side of uttarkashi (latitude 31°27'34''n to 31'13'n and longitude 77°58'51''e to 78°53'e) (fig. 1). the water is pristine or near pristine with low depths, high transparency and dissolved oxygen. the morphometry of stream varies considerably from dodital to gangori. throughout the length, stream has torrential flow and passes through deep gorges at many places. it has low volume in the upper stretch which increases downward due to joining of several 1st and 2nd order tributaries. the uppermost reaches are gorge-like and rocky or full of huge boulders. in the middle stretch, streambed consists of partially or fully matured boulders (fig. 2), while pebbles, cobbles, and silt are observed in the lower stretch besides fully mature boulders of varying size. stream habitat is characterized by diverse microhabitats as pools, riffles, rapids, runs, and cascades. materials and methods regular monthly sampling of fishes and physicochemical parameters was carried out in the assiganga stream during the year 2010-12. fish collection was made with the help of skilled fisherman during daytime (6:00-18:00 hrs), while ‘baur’ (indigenous trap) and ‘gill net’ were also fixed during late evening hours (17:00 -18:00 hrs) and recovered in early morning hours (5:00-7:00 hrs). fishing methods employed were cast net (dia. 2.0 m, mesh size 1.8 x 1.8 cm), gill net (mesh size 1.2 x 1.2 cm, l x b = 12 m x 1.5 m), baur or phans (fine nylon loops knotted over a long nylon cord of 5-8 m length), scoop net and hook and lines. collected fish samples were preserved in 8-10% formaldehyde. small fish specimen (<150 mm in total length) were preserved directly while the large specimen (>150 mm in total length) were preserved with preservative injection or slitting the abdomen. fish identification was performed on the basis of morphometric and meristic characters (day, 1878; tilak, 1987; talwar and jhingran, 1991; shrestha, 2008; badola, 2009; jayaram, 2010). the physico-chemical variables (ambient and water temperature, velocity, ph, total figure 1. geographical location of assiganga stream. (a) india’s state map showing uttarakhand, (b) upper ganga river system in central himalaya and (c) assiganga stream from its origin to merging in river bhagirathi. figure 2. substratum and habitat features of assiganga stream. 307 singh and agarwal/ fish assemblage structure and habitat use of the snow fed stream assiganga dissolved solids, do, free co2, and turbidity) were analyzed using standard methods outlined in american public health association (apha, 1998). the temperature was measured using mercury thermometer, velocity by the float method and ph with the hanna made electronic digital ph meter. the total dissolved solids were calculated by digital tds meter, do with the winkler’s iodometric method while turbidity was measured by digital turbidity meter (elico model 331e). substratum material has been characterized as large boulder (>1024 mm size), small boulder (256-1024 mm), cobbles (64-128 mm), coarse gravels (16-64 mm), fine gravel (2-34 mm) and sand (0.062-2.0 mm) following (armantrout, 1999). stream habitat was classified as pools, riffles, rapid, run, and cascade habitat type description pools a segment of the stream with reduced current velocity, depth exceeding than surrounding habitats. riffles a relatively shallow area with gradient less than 4% with swift flowing water completely or nearly covering obstructions and substrate of smaller rock gravel or bedrock having surface or subsurface agitation. rapid a relatively deep stream section with swift currents and gradient exceeding 4% resulting in series of short drops, considerable surface agitation, pocket pools and rock and boulders exposed at all but high flows run an area of swiftly flowing water with gradient over 4% with minor surface agitation and in which slope of the water surface is roughly parallel to the overall gradient of the stream. cascade an area of continuous stepping with low water depth and swiftly flowing water. table 1. stream habitat types with their description. s. no. ichthyo species with order and family local name present status order cypriniformes family cyprinidae 1 schizothorax richardsonii maseen abundant 2 s. plagiostomus asela common 3 schizothoraichthys curvifrons chongu rare 4 s. progastus chongu rare 5 tor putitora khasra common 6 t. tor khasra rare 7 t. chilinoides mahseer rare 8 barilius bendelisis fulra rare 9 garra gotyla gotyla gunthala rare family cobitidae 10 noemacheilus rupicola gadiyal rare 11 n. montanus gadiyal rare order siluriformes family sisoridae 12 glyptothorax pectinopterus kathrua rare 13 glyptothorax madraspatanum kathrua rare 14 pseudecheneis sulcatus kathrua rare order salmoniformes family salmonidae 15 salmo trutta brown trout common table 2. status of ichthyofauna reported from assiganga with their local names. 308 international journal of aquatic biology (2014) 2(6): 305-312 (table 1). fishes have been categorized as abundant, common and rare based on their average abundance. the relative abundance (ra) of fish species across the study sites was worked out by the following formula. ra = (number of samples of particular species × 100)/ total number of samples. results fish composition: in the present study 15 fish taxa (14 indigenous and 1 exotic species) have been reported from entire stretch of assiganga stream. all the species reported belongs to 8 genera, 4 families and 3 orders (table 2). species richness pattern: the cyprinidae family was the dominant taxon in middle and lower stretches of the stream while in the upper stretch, salmonidae family predominated. the snow trout schizothorax richardsonii and salmo trutta were present throughout the stream and contributes > 65% of total fish catch. the s. plagiostomus and tor putitora contribute 15-20% of total fish catch and are reported only in lower and middle stretch. the relative abundance of these species was followed by tor, barilius and garra spp. (table 3). some cat fishes and loaches were recorded sporadically in few catches. physico-chemical parameters: the seasonal analysis of physico-chemical parameters of assiganga stream showed characteristic features (fig. 3). stream water showed high dissolved oxygen content (8.7 ± 0.36 to 10.80 ± 1.5 mg-l) throughout the year in all seasons. while free carbon dioxide was recorded low in all seasons (1.2 ± 0.133 to 1.45 ± 0.105 mg-l). the total dissolved solids were found in optimum range and little variation was recorded in different seasons. water was clear with low turbidity throughout the year with maximum value (05 ± 2.0 ntu) in monsoon months to minimum value (01 ± 0.0 ntu) in winter season. annual ph value ranged between 7.75 ± 0.49 to 8.1 ± 0.27. the water temperature was recorded within the highest limit of cold water fishes. it was recorded 20.0 ± 1.33°c in the monsoon while 10.0 ± 0.5°c in winter season. the water velocity was recorded high throughout the year. it ranged between 1.1 ± 0.132 to 1.4 ± 0.087 m-s in winter and monsoon season, respectively. discussion the fish assemblage and their relative abundance in assiganga stream varied in association with number of factors viz. flow rate, nature of substratum, watername of the species common name different seasons summer monsoon post monsoon winter schizothorax richardsonii maseen 11.56 5.78 9.10 5.05 s. plagiostomus asela 4.33 2.60 3.61 1.73 schizothoraichthys curvifrons chongu 0.00 0.28 0.72 0.00 s. progastus chongu 1.58 0.57 1.15 0.00 tor putitora khasra 3.17 2.16 2.89 2.16 t. tor khasra 1.73 0.00 1.44 0.00 t. chilinoides mahseer 1.44 0.00 1.73 0.57 garra gotyla gotyla gunthala 0.72 0.00 0.72 0.14 barilius bendelisis fulra 2.60 0.57 1.73 0.14 glyptothorax pectinopterus naou 0.86 0.14 0.57 0.00 g. madraspatanum naou 0.72 0.00 0.00 0.00 pseudecheneis sulcatus kathrua 2.02 0.72 0.57 0.28 noemacheilus rupicola gadiyal 1.73 0.00 0.72 0.00 n. montanus gadiyal 1.30 0.14 0.00 0.00 salmo trutta brown trout 6.50 4.33 3.61 4.33 species richness 14 10 13 8 table 3. relative abundance of fish fauna of assiganga stream. 309 singh and agarwal/ fish assemblage structure and habitat use of the snow fed stream assiganga depth, food availability, physico-chemical properties, stream length and seasons. it is reported that the abundance and composition of fish species is highly variable in space and time and closely related to environmental variables (vilar et al., 2011). high species richness along with high abundance (14 species) was recorded in summer season, whereas very low species richness as well as low abundance was recorded during winter season. contrary to this (bisht et al., 2009) has recorded high fish diversity in the monsoon season in a spring fed stream with low discharge, but the observation of low fish diversity in winter is similar in both the studies. comparatively high fish diversity in the summer season might be due to optimum temperature and moderate volume of water in asiganga. in monsoon season the stream is heavily flooded, while in winter season the water temperature is very low, which is not conducive for noemacheilus, barilius and tor spp. the fish distribution pattern also varied in the different stretches of stream. upper course of assiganga stream is most torrential and is frequented by salmo trutta, s. richardsonii, s. curvifrons, and s. plagiostomus. the rapid zone of the stream is inhabited by garra, glyptothorax and pseudecheneis spp. intermediate stretch of the stream is less torrential with comparatively high temperature in contrast to upper region and is found to be inhabited by schizothoraichthys progastus and tor putitora. the lower stretch of the stream is slow moving meandering zone and is frequently inhabited by the tor tor and barilius spp. while the noemacheilus spp. are found only in shallow area of stream and area of joining of other small stream in lower zone. present observation is in agreement of sehgal, (1999) that water temperature is always an important limiting factor affecting geographical distribution and local occurrence within one water system. sehgal, (1999) also reported that schizothorax sp. and salmo trutta having upper temperature tolerance limit of 20°c. the present study reveals that fish species with powerful muscular cylindrical bodies (snow trout and the exotic trout) inhabits most preferably the bottom water layers of deep fast moving segment of the stream. while the fishes (barilius and tor spp.) without any striking modifications to current are recorded mostly from the shallow and deep pools, respectively. the small loaches (noemacheilus spp.) with special attachment devices are found among the shallow water in pebbles and shingles. garra, glyptothorax and pseudecheneis spp. having adhesive organs on their ventral surface were found clinging to rocks and boulders in fast water currents. menon, (1954) also related the distribution pattern of himalayan fish to the morphological characteristics. hill stream fishes have special morphological modification which helps them to inhabit the torrential streams (singh and agarwal, 1991, 1993; singh et al., 1993). the reference stream is characterized with heterogeneity in habitat (cascade, falls, runs, rapids, riffles and pools) and substratum type (boulder, cobbles, gravels and sand). this habitat heterogeneity results into variation in the availability of fish fauna in different stretches of the stream. fish assemblage structure is strongly related to habitat structure (meffe and sheldon, 1988; schoener, 1974; galacatoes et al., 1996) where habitat have been identified as one of the primary criteria on which many biological communities are organized. the fish species richness often increases as habitat complexity increases, with depth, velocity and cover being the most important variables governing this relationship (schlosser, 1982; fellly and felly, 1987; figure 3. seasonal variation in physico-chemical variables (mean ± sd) of assiganga stream. 310 international journal of aquatic biology (2014) 2(6): 305-312 pusey et al., 1995). the shoals of tor and barilius spp. were found always in pools (shallow as well as deep pools). this pool habitat is favorable for tor putitora and t. tor and they prefers deep water in the adult stages and shallow water in the breeding seasons. schizothoracines spp. preferred mostly rapid and riffle habitat but occasionally reported from pools. true hill stream fishes, glyptothorax, pseudecheneis and garra spp. were recorded mostly from the rapids and cascades habitat. the noemacheilus spp. were found only from the shallow side pools of stream and its small tributaries having low velocity. the introduced exotic trout salmo trutta is thriving well in the assiganga especially in the upper region due to low temperature, fast current with high dissolved oxygen, and cascade and rapid type of habitat. it was frequently recorded from rapids and cascades habitat type with sporadic presence in riffles and pools. all these observations divulge that hill stream fishes are habitat specialists and the pool habitat is most preferable habitat. similar observation was found in streams of lower middle western himalaya by johal et al. (2002). various earlier studies (probst et al., 1984; mc clendon and rabeni, 1987; lakra et al., 2010) also observed that fish distribution is highly related to habitat composition. various anthropogenic activities have been taking place all along the stream. at present, 2 hydro power projects namely assiganga-i (5 mw) and assiganga-ii (3 mw) are under construction while one more project, assiganga-iii (3 mw) have been proposed on it. the construction of these hpp is obstructing the natural flow of assiganga stream. this obstruction is causing the dry up of fragmented stream segment, changes in substratum type, physico-chemical characteristics and the physiography of the stream. the substratum provides feeding and breeding ground to fishes and is major factor which influences the distribution and abundance of fish fauna. assiganga stream possess rocky substratum with boulder and cobbles and gravels favorable for some important hill stream fishes. the alteration in rocky and boulder substratum will be detrimental for many stream fishes. developing hatchlings hiding in the crevices of rocks, stone, cobbles and gravels react differently to the current and turbidity of the water (shreshtha, 1993). the forced flowing of stream through tunnel will also destruct the stream habitat which will directly affect the distribution and abundance of fish fauna. observations on the physico-chemical characteristics of assiganga stream very well corelate the occurrence and distribution of fish species. low temperature, high oxygen and fast flow of stream with riparian zone enriched with huge vegetation is highly supported by schizothorax sp. and s. trutta while the high velocity and oxygen with characteristic cascades, rapids and riffles favored the existence of cat fishes (glyptothorax and pseudecheneis spp.) and garra spp. comparatively high temperature in the lower stretch and side pools was preferential to the noemacheilus and barilius spp. bisht et al., (2009) has also reported that the seasonal distribution and relative abundance of fish fauna is directly related to change in physicochemical properties, channel course, water discharge and pattern and geometry of tributaries. vilar et al., (2011) also reported that abundance and composition of fish species is closely related to various environmental variables. all of these alterations may result into the extermination of some of the native species (agarwal et al., 2011). the alteration in physico-chemical properties controls the distribution of various sections of the biotic fauna and flora (bahuguna and badoni, 2002). acknowledgement the authors are grateful to university grant commission (ugc), new delhi for the financial support in the form of research project no. 37199/2009(sr). references agarwal n.k., singh g. 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(2019) 7(1): 27-34 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article effect of probiotic pediococcus acidilactici on growth, reproductive and bacterial count of marine rotifer brachionus plicatilis yosra abbasi1, nasrollah ahmadifard*1, amir tukmechi2 1department of fisheries, faculty of natural resources, urmia university, urmia, iran. 2department of microbiology, faculty of veterinary medicine, urmia university, urmia, iran. s article history: received 29 august 2018 accepted 22 february 2019 available online 2 5 february 2019 keywords: bacterial colony probiotic rotifer specific growth rate abstract: this study investigated the effects of different concentrations of probiotic pediococcus acidilactici on the total number, specific growth rate, attached egg number and microbial count (lactic acid and total aerobic bacteria) in culture water and body of marine rotifer brachionus plicatilis. the experiment was conducted as a completely randomized design in four groups: first group fed probiotic at 0.5×106 cfu ml-1 (p1), the second group fed probiotic at 1×106 cfu ml-1 (p2), the third group fed probiotic at 1.5×106 cfu ml-1 (p3), and the last group fed without probiotic (np, control). rotifers were cultured in standard conditions at an initial density of 30 ind.ml-1 using a 2.5×106 cell ml-1 of nanochloropsis oculta. based on the results, the maximum number (215±4.91 ind.ml-1) of rotifers were significantly obtained after ten days in the third group in comparison to other treatments (p<0.05). in addition, the highest growth rate (0.46±0.023 ind.ml-1) and shortest doubling time (1.5±0.14 days) were obtained in the treatment p3 but no significant difference was found between np and other groups (p>0.05). moreover, it was revealed that the total aerobic bacteria was significantly related to the 3rd group that found be 1.80×103 cfu rotifer-1 and 34.0×104 cfu ml-1 in rotifer body and culture water, respectively (p<0.05). nonetheless, lactic acid bacteria of rotifer body and culture water was not concentration-dependent and the highest number of lactic acid bacteria in rotifer body (50.58±6.08 cfu rotifer-1) and rotifer culture water (4.43±3.28×103 cfu ml-1) was obtained in the treatments of p1 and p2, respectively. in conclusion, the present study showed that the bacteria p. acidilactici with n. oculta algae provide a higher growth rate and total aerobic bacteria in rotifer b. plicatilis. introduction in the early stages of aquatic larvae, the initial establishment of microflora is associated with their foods that introduced into the system as live food and water of the culture system (makridis et al., 2000; planas et al., 2004). live foods, especially rotifer, are the main cause of transmission of the bacteria to the digestive tract of the marine larvae (bergh et al., 1994; makridis et al., 2000; ringø and birkbeck, 1999) and the transmission of pathogenic bacteria may result in high mortality rates (grisez et al., 1997). keys et al. (1935) suggested that bacteria may be a significant source of food for zooplankton. besides, it is expressed that in aquaculture system with high stocking densities, bacteria act as a direct food source for rotifers and may also contribute to the nutrition of *correspondence author: nasrollah ahmadifard doi: https://doi.org/10.22034/ijab.v7i1.489 e-mail: n.ahmadifard@urmia.ac.ir the target organisms (nevejan et al., 2018). in recent years, many efforts have been made to develop microbial control methods to reduce the use of chemicals and antibiotics and to promote the use of natural resources for sustainable aquaculture development (cabello, 2006). probiotics, defined as living organisms such as bacteria, yeast, and microalgae, when used at appropriate rates, have beneficial effects on host organisms and play a role in reducing mortality in aquatic organisms (kesarcodiwatson et al., 2008; verschuere et al., 2000). in aquaculture, probiotic bacteria may be introduced directly into the culture water or through live foods, including rotifers to aquatic animals. rotifers play an important role as live food for larvae, and their microbial community is important (støttrup and 28 ahmadifard et al./ effect of pediococcus acidilactici on marine rotifer brachionus plicatilis mcevoy, 2008). different kinds of food such as nanochloropsis, isochrysis and chlorella; and saccharomyces have been used in rotifers feeding. moreover in rotifer cultures, vibrio, pseudomonas and acinetobacter are common opportunistic bacteria which may be important additional food sources for rotifers (verdonck et al., 1994). however, gatesoupe et al. (1989) and gatesoupe (1989) indicated that inoculation of probiotic bacteria (bacillus toyoi) into disinfected rotifers increases the growth rate of japanese flounder, paralichthys olivaceus and turbot larvae, scophthalmus maximus, respectively. also, it is determined that adding lactobacillus plantarum to rotifer culture water inhibit the growth of aeromonas salmonicida and increases the population growth rate and the nutritional value of rotifers (gatesoupe, 1991). most applicable probiotics in aquaculture belong to the genera lactobacillus, carnobacterium, vibrio alginolyticus, bacillus, pseudomonas and pediococcus (verschuere et al., 2000). pediococcus acidilactici is a gram-positive lactic acid bacteria that can grow in a wide range of ph, temperature, and osmotic pressure and can colonize the digestive tract of aquatic animals (azimirad et al., 2016; bhunia et al., 1988; klaenhammer, 1993). this probiotic is a facultative anaerobic bacteria that has a lower sensitivity to oxygen and in the intestines has inhibitory effects on the growth of pathogens in fish due to the ability to produce bacterial and organic acids (acetic and lactic acid) (vázquez et al., 2005). moreover, p. acidilactici has the ability to overcome the normal microbiota of the digestive tract of endothermic animals and had shown promising results in animal and human experiments (barros et al., 2001). this group of bacteria is not usually found in fish, but an improvement of growth, reproduction, and immunity in angelfish, pterophyllum scalare were obtained by using of p. acidilactici-enriched artemia (azimirad et al., 2016). the growth and resistance to pathogenic bacteria in turbot (psetta maxima), by using p. acidilactici in culture water and through enriched rotifer were studied, and the results showed the increase of transmission of this bacterium to fish by enriched-rotifer (villamil et al., 2010). also, the positive effect on the weight of pollack, pollachius pollachius, was found using of p. acidilactici enriched-artemia nauplii (gatesoupe, 2002). based on resource overview, probiotic p. acidilactici may have the potential to convey a wide range of benefits but is less studied in rotifer culture, an important live food in the larval stages of fish. therefore, in this study, the effect of this probiotic on growth factors of rotifer b. plicatilis was investigated. in addition, the number of bacterial colonies in the rotifer body and in culturing medium was investigated. materials and methods preparation the organisms: the primary stock of algae nanochloropsis oculta, as a food source, and rotifer b. plicatilis were obtained from shrimp research institute of bushehr, iran. the algae were cultured in standard methods as suggested by nematzadeh et al. (2018) with valne culture medium, harvested 4-7 days after the culture period, counted with hemocytometer lam and stored in the refrigerator for the feeding of rotifer. commercial probiotic (pedi-guard®) was prepared from tak gene company (tehran, iran) contains 1×1013 cfu kg-1 of p. acidilactici. experimental design: the initial stock of rotifer b. plicatilis (210 µm average length) was up-scaled using algae n. oculta at a density of 1.5×106 cell ml-1 in a 4 l glass vessels. for rotifer cultivation, saltwater obtained from urmia lake, filtered using 0.2 μm mesh, autoclaved at 121°c for 15 minutes and finally diluted with sterilized distilled water. glass flasks (working volume: 200 ml) were filled with saline water of 30 ppt, and then rotifers were introduced with a density of 30 individual ml-1. the flasks were maintained at temperature 24±1°c with submersible heaters and provided with gentle continuous aeration using a central pump and without air stone. along with adding bacteria to the culture medium, rotifers were daily fed with 2.5×106 cell ml-1 of algae n. oculta. probiotic p. acidilactici with 24 h intervals was added to each flask with three replicates as follows: p1: 0.5×106 cfu ml-1, p2: 1×106 cfu ml-1 and p3: 1.5×106 cfu ml-1 and p4 (np): (none-probiotic, np, 29 int. j. aquat. biol. (2019) 7(1): 27-34 control). to evaluate the effect of probiotic on rotifer population, a total number of rotifers and attached eggs were daily counted via 3 replicates of each treatment for 10 days. lugols’ solution was used to fixation the rotifers (nematzadeh et al., 2018). finally, using the following equations, the specific growth rate (sgr) and the doubling time (dt) were calculated (krebs, 1995; planas and estevez, 1989). sgr = ln nt− lnn0 t dt = 𝐿𝑛 2 𝑆𝐺𝑅 = 0.693 𝑆𝐺𝑅 where n0 = initial population density and nt = density of population after time t (days). microbiological assays: 24 h after the feeding period, rotifer specimens were sieved and washed with autoclaved saltwater at a salinity of 30 ppt and then were homogenized under sterile conditions into falcon tube with sterile saline solution (%87 w/w, 1m). the homogenate at two serial dilutions of 10-1-10-2 was spread onto mrs agar (merck, germany) and tsa (merck, germany) media via 3 replicates for lactic acid bacteria and total viable heterotrophic aerobic bacteria cultivation, respectively. plates were placed at 28 °c for 24-48 h and colony forming units (cfu) were counted as cfu rotifer-1 (shiri harzevili et al., 1998). in addition, those mentioned bacteria were investigated in culture water of rotifer to obtain the viability of those in water. statistical analysis: after survey of the normalization of data by shapiro-wilk test, the homogeneity of variances was investigated by levene’s test. one-way anova was used to investigate the effects of different concentrations of probiotic and duncan's test was used for multiple comparisons of means. the minimum significance level of the test was considered as p<0.05. spss software version 21 was used to check the data and excel software was used to draw charts. results growth and reproductive factors: during the 10-day experiment, the addition of 3 concentrations of bacteria p. acidilactici along with algae had a significant effect on the population density, specific growth rate and doubling time of rotifers (fig. 1 and table 1; p<0.05). the highest population density (nmax) (215±4.91 ind.ml -1) was observed in the treatment p3 that was significantly more than the other groups; however, there was no significant difference in maximum density between np and p2 treatments. (p>0.05). among the examined treatments, the figure 1. the total number (mean±se) of rotifer fed with 3 concentrations (p1: 0.5×106 cfu ml-1, p2: 1×106 cfu ml-1 and p3: 1.5×106 cfu ml1) of pediococcus acidilactici compared to the np, control at 10 days. 30 ahmadifard et al./ effect of pediococcus acidilactici on marine rotifer brachionus plicatilis highest growth rate (0.46±0.023 ind.ml-1) and shortest doubling time (1.5±0.14 days) were related to rotifers fed with treatment p3. nevertheless, there was no significant difference between the growth rates of np and treatments p1 and p2 (p>0.05). based on table 2, the total number of attached eggs in rotifers fed with 3 concentrations of p. acidilactici fluctuated from initial day until 10th day; however on the second and fourth day the treatment p1 and on the 6th day of treatment p3 significantly showed the highest values of attached eggs (p<0.05). microbiological analysis: the total number of aerobic bacteria in culture water and rotifers fed with three concentrations of the probiotic p. acidilactici are shown in figure 2. as shown with increasing the concentrations of probiotic in treatments, the total aerobic bacteria of rotifer body increased and the maximum colony count of 1.80×103 cfu rotifer-1 was significantly observed in treatment p3 (p<0.05). also, the total aerobic bacteria in culture water was significantly concentration-dependent and the highest number (34×104 cfu ml-1 of culture water) of those was observed in treatment p3. in the group without probiotic was found the lowest total number of bacteria colony in rotifer culture water. the total lactic acid bacteria in different treatment of culture water and rotifers are shown in figure 3. contrariwise, the total number of lactic acid bacteria in both of culture water and rotifers was not significantly concentration-dependent. in rotifers, the higher amount of lactic acid bacteria was significantly observed in treatment p1 (p<0.05), and with increasing the concentration of probiotic, the colony count of bacteria was similar to np treatment. also, the maximum number (44.33±3.28×102 cfu ml-1 of culture water) of lactic acid bacteria in culture water table 1. specific growth rate (sgr), n max and doubling time (dt) (mean±se) in rotifer fed with 3 concentrations of pediococcus acidilactici compared to the np (control) at 10 days. treatment cfu ml-1 of probiotic n max (ind ml -1) dt (days) sgr (day-1) np 0 165±2.08c 2.04±0.55b 0.340 ± 0.044b p1 0.5×106 189±7.51b 1.78 ±0.16ab 0.389 ± 0.020ab p2 1×106 164±4.16c 2.13 ± 0.37b 0.326 ± 0.035b p3 1.5×106 215±4.91 a 1.50 ± 0.14a 0.461 ± 0.023a table 2. the total number (mean±se) of attached eggs in rotifer fed with 3 concentrations of pediococcus acidilactici at 10 days. experimental groups experimental days 1 2 3 4 5 6 7 8 9 10 np 19.0±1.00a 11.3±0.33b 19.0±3.46a 7.3±0.33b 13.0±0.58a 13.0±1.53b 9.3±1.20a 15.3±3.93a 11.3±2.33a 12.0±2.08a p1 18.7±1.76a 16.0±2.08a 16.0±1.00a 18.0±1.15a 18.7±7.42a 17.3±3.28ab 9.3±1.20a 12.7±2.40a 12.0±1.15a 10.3±0.88a p2 17.3±1.20a 11.7±0.88b 18.3±2.03a 8.0±2.08b 13.0±1.53a 13.7±0.88b 11.0±2.08a 10.3±0.88a 11.0±1.53a 10.3±1.67a p3 18.7±1.20a 17.0±1.00a 19.3±2.85a 9.3±0.88b 16.0±2.31a 21.7±2.19a 14.3±1.76a 11.0±4.58a 12.3±3.58a 15.0±2.65a figure 2. the cfu (mean±se) of total microflora in culture water (a) and rotifer (b) fed with 3 concentrations of pediococcus acidilactici at 10 days. np, control, p1: 0.5×106 cfu ml-1, p2: 1×106 cfu ml-1 and p3: 1.5×106 cfu ml-1 of p. acidilactici. 31 int. j. aquat. biol. (2019) 7(1): 27-34 was observed in treatment p2. there were no significant differences in the number of lactic acid bacteria between treatment np and treatments of p1 and p3 (p>0.05). discussions in the present study, lactic acid bacteria, pedicoccus acidilactici had a beneficial effect in term of population growth and microbial composition of rotifer b. plicatilis. the total population of rotifers as well as the specific growth rate at 1.5×106 cfu ml-1 of probiotic showed the maximum amount during 10 days of the culture period compared to other treatments. in finfish and shellfish larvae culture, adding the benefit bacteria not only improve the health of live foods but also can be transmitted to fish larvae (zambonino and cahu, 2010). these positive effects of probiotics on live foods (gatesoupe, 2002) and target organisms (gatesoupe, 2002; gatesoupe and lesel, 1998) have been indicated. shiri harzevili et al. (1998) has been reported that lactic acid bacteria had no effect on growth rate of rotifer b. plicatilis, but modified the micro-flora of them. also, survival of turbot larvae, scophthalmus maximus fed with lactic acid bacteria increased when exposed to pathogenic vibrio sp. (gatesoupe, 1994). nonetheless in the study of planas et al. (2004), by increasing the concentration of p. acidilactici in rotifer culture resulted in an increase in growth factors in comparison to the nonprobiotic treatment, and the optimum concentration of probiotic was reported at a rate of 1×109 cfu ml-1. similarly, in the current study, the sgr, doubling time, and attached egg of rotifer population increased but this was not concentration-dependent. first, at low concentration, positive effects of bacteria on population growth and sgr were observed, but with increasing the probiotic concentration, those factors were first reduced and then increased at 1.5×106 cfu ml-1 of p. acidilactici. these trends may be due to two major factors, including (1) bacteria as a food source and (2) as a microbial component of these animals is providing essential nutrients in the aquatic environment (nevejan et al., 2018). with using the high levels of bacteria, they can be used not only as a microbial component but also it acts as food sources. since rotifers can ingest bacteria forming an aggregate (abell and bowman, 2005), the increase of population growth rate and sgr in a high concentration of probiotic can be related to providing of carbon, protein, b-complex vitamins and polyunsaturated fatty acids from direct consumption of bacteria. planas and estevez (1989) indicated the correlation between reproductive factors and quality of food ingested, so with that, the former is shorter when the food quality is better. the selection of probiotic bacteria in long-term enrichment of live food is very important because probiotic bacteria can be unsuitable for aquatic nutrition with an antagonistic activity with algae (planas et al., 2004). in this study, lactic acid bacteria only increased at low concentration of p. acidilactici and the higher concentration of probiotic had no increased effect on lactic acid bacteria colony in comparison to np treatment. this no incremental figure 3. the cfu (mean±se) of lactic acid bacteria in culture water (a) and rotifer (b) fed with 3 concentrations of pediococcus acidilactici at 10 days. np, control, p1: 0.5×106 cfu ml-1, p2: 1×106 cfu ml-1 and p3: 1.5×106 cfu ml-1 of p. acidilactici. 32 ahmadifard et al./ effect of pediococcus acidilactici on marine rotifer brachionus plicatilis effect could be due to the antagonistic of algae with probiotic when fed to rotifer (shiri harzevili et al., 1998). however, based on the results, total number of aerobic bacteria colonies in the body of rotifers showed an increasing trend with increasing probiotic concentration, and the maximum number of total microflora (1.8×103 cfu per rotifer) was about 2.4 times higher than total microflora of np treatment. the methods and foods used for cultivation can effect on the microflora of rotifers (skjermo and vadstein, 1993). in this study, the positive trend of the probiotic concentration on the composition of the rotifers and the culture water, indicate a non-selective uptake of bacteria from the culture water by rotifer (skjermo and vadstein, 1999). on the other hand, low density of lactic acid bacteria in treatment with high amount of probiotic indicates that this bacteria in the environment by the production of compounds such as water-soluble vitamins (phillips, 1984) have been responsible for the growth of other bacteria in rotifer culture water, which was confirmed by high density of total microflora. the microbiological assay only measures the living cells and does not measure dead and digested cells. planas et al. (2004) used a combination of three probiotic pediococcus acidilactici, lactococcus casei and lactobacillus lactis in the culture water of rotifer b. plicatilis, and found that at the end of the trial the total aerobic bacteria of rotifers was approximately 5×103 cfu rotifer-1. the number of probiotic cells that incorporate to live food are depending on the probiotic type, exposure time, and the state (dead or live) of the living organism (gomes et al., 1998). if the level of bacterial load in live foods is too high, it can reach a level that negatively affects the health of the host. in the current study, the loading of the probiotic p. acidilactici at higher concentration without positive effect on colony count of lactic acid bacteria increased the population growth rate of rotifer as well as the total aerobic bacteria. in otherwise, olsen et al. (1999) found that overloading bacteria through artemia is caused poor growth of halibut larvae hippoglossus hippoglossus during the feeding period with live prey. on the other hand, gatesoupe (2008) stated that a microorganism, with long-term exposure in the environment, can multiply in the alimentary canal. for example, when bacillus sp. was added to water for 20 days, it increased the intestinal bacterial flora of treated microorganism in comparison to the non-probiotic group. also, ahmadifard et al. (2018) indicated that long-term enrichment of artemia with 1×105 cfu ml1 of b. subtillis increased the total microflora from 5.76 to 6.41 log cfu in bacillus-enriched artemia. in the present study, in culture water of rotifer, the maximum number of lactic acid bacteria was obtained in 1×106 cfu ml-1 of p. acidilactici. however, the total aerobic bacteria increased with positive correlation with increasing the probiotic concentration. bacteria p. acidilactici is a terrestrial bacterium, therefore its survival rate is important in saline water. this study showed that by increasing the concentration of this bacterium, the total aerobic bacteria concentration in the water environment increased, which indicates the positive of the survival rate of this bacterium within 10 days of the experiment. in conclusion, the present study showed that the bacteria p. acidilactici along with algae n. oculta through direct consumption or providing essential material could result in higher population growth rate and higher total aerobic bacteria in rotifer b. plicatilis. references abell g.c., bowman j.p. 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(2018) 7(1): 27-34 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology چکیده فارسی شور آب روتیفر هایباکتری تعداد و تولیدمثل رشد، بر pediococcus acidilactici پروبیوتیک تاثیر brachionus plicatilis 2چیکمهوامیر ت ،1*فردنصراهلل احمدی ،1یسرا عباسی .ایران ،ارومیه ،دانشگاه ارومیه ،دانشکده منابع طبیعی ،گروه شیالت1 .ایران ،ارومیه ،ارومیه دانشگاه ،دامپزشکی دانشکده ،میکروبیولوژی گروه2 چکیده: های متصل و تعداد بر تعداد کل، نرخ رشد ویژه، تخم pediococcus acidilacticiهای مختلف باکتری پروبیوتیکی در تحقیق حاضر تاثیر غلظت ( گروه تغذیه 1)مورد بررسی قرار گرفت. آزمایش حاضر در چهار گروه شامل brachionus plicatilisکلنی باکتریای در محیط کشت و بدن روتیفر cfu ml-1( گروه تغذیه شده با غلظت 3) ،از باکتری cfu ml 610×1-1( گروه تغذیه شده با 2، )از باکتری lcfu m 610×5/0-1شده با غلظت لیتری با میلی 500شد. روتیفرها در شرایط استاندارد و در ظروف پالستیکی ( گروه شاهد )گروه بدون پروبیوتیک( انجام 4)از باکتری و 5/1×610 لیتر بود. عدد در میلی 30کشت شدند. تراکم اولیه روتیفرها در تیمارهای مختلف ml cell 610×5/2-1با تراکم nanochloropsis ocultaجلبک داری بیشتر از سایر طور معنیهدست آمد که بهروز در تیمار سوم ب 10بعد از لیتر(عدد در میلی 215±5/4براساس نتایج حداکثر تراکم روتیفر ) ولی بین تیمار شاهد با ؛دست آمدهدر تیمار سوم ب و کمترین زمان دو برابر شدن جمعیت (. همچنین باالترین نرخ رشد ویژه>05/0pتیمارها بود ) ( و محیط کشت آن cfu rotifer 310 ×81/1-1های هوازی در روتیفر )تعداد کل باکتریداری یافت نشد. عالوه بر آن سایر تیمارها تفاوت معنی (1-cfu ml 410×0/34در تیمار سوم باکتری یافت شد. با این حال تعداد باکتری ) های اسید الکتیکی در محیط کشت و بدن روتیفر وابسته به ( و cfu rotifer 08/6±58/50-1های اسید الکتیکی در روتیفر )باالترین میزان باکتریغلظت باکتری پروبیوتیکی مورد استفاده در تیمارها نبود و همراه به .acidilactici p دست آمد. مطالعه حاضر نشان داد که باکتریهترتیب در تیمار اول و دوم ب( بهcfu ml 310×28/3±43/4-1محیط کشت ) نماید. ایجاد می b. plicatilisهای هوازی بیشتری را در روتیفر رشد و تعداد باکتری n. ocultaجلبک .ویژه رشد نرخ ،روتیفر پروبیوتیک، باکتری، کلونی :کلمات کلیدی int. j. aquat. biol. (2017) 5(6): 360-369 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article evaluation of oxidative stress induced by cadmium and comparative antioxidant effects of shirazi thyme (zataria multiflora boiss) and vitamin e in common carp (cyprinus carpio) mohammad mohiseni*1, dara bagheri2, mahdi banaee1, behzad nematdust haghi1 1department of fisheries, natural resources and environmental faculty, behbahan khatam alanbia university of technology, behbahan, iran. 2department of fisheries, persian gulf university, bushehr, iran. article history: received 11 september 2017 accepted 20 november 2017 available online 2 5 december 2017 keywords: fish antioxidant system heavy metal medicinal plant stress abstract: shirazi thyme is an active phytobiotc contains phenolic compounds and flavonoids which have strong antioxidant properties. this study was conducted to investigate the potential protective effects of shirazi thyme compared to that of vitamin e against cadmium toxicity. common carp juveniles (34±3 g) were divided into four groups and fed by three different diets, including commercial diet without any additive (for control and metal only group) and supplemented with either 1% ground shirazi thyme or 100 mg/kg vitamin e. all treatments except the control were exposed to sublethal concentration of waterborne cadmium (1.5 mg/l free ion) for 15 days and liver, kidney and gill were sampled 3, 7, 10 and 15 days after the exposure. the results showed that treatment of the fish with cadmium for 15 days resulted in a significant reduction in glutathione reductase (gr), glutathione-s-transferase (gst) and catalase (cat) and led to liver, kidney and gills dysfunction. on the other hand, the level of malondialdehyde (mda) significantly increased during metal exposure. supplementation of diets with shirazi thyme and vitamin e led to a significant protection against metal exposure in different tissues. moreover, shirazi thyme was found to be as effective as vitamin e. the current finding can provide a useful reference for stress protective effects of thyme and its beneficial role in aquaculture. introduction due to industrialization, the application of heavy metals in a variety of industrial and agricultural fields has been expanded (tan et al., 2010). heavy metals cannot be eliminated through biological degradation, therefore, potentially can accumulate in the tissues of aquatic animals making this toxicant deleterious to the aquatic environment. accumulation of heavy metals in the tissues may intensify the generating of reactive oxygen species (ros) which may lead to oxidative stress resulting in lipid peroxidation, dna damage and alteration in ion homeostasis (kalay et al., 1999; farombi et al., 2007). the toxicity generated by this contaminant generally involves neurotoxicity, hepatotoxicity and nephrotoxicity (stohs and bagchi, 1995). cells have equipped by enzymatic systems which have an ability to convert oxidants into nontoxic molecules, thus protecting the organism from the harmful effects of the xenobiotics. glutathione *corresponding author: mohammad mohiseni doi: https://doi.org/10.22034/ijab.v5i6.318 e-mail address: mohiseni@ut.ac.ir reductase (gr), glutathione-s-transferase (gst), superoxide dismutase and catalase (cat) are the main cell enzymatic defense systems (gate et al., 1999). cadmium is one of the most biologically toxic heavy metals with a high environmental concern (ruangsomboon and wongrat, 2006; mohiseni et al., 2016). cadmium can actively be accumulated and result in toxicity to liver, kidneys, brain and heart. several studies showed that using free radical scavengers and antioxidants can protect the organism against cadmium toxicity (ochi et al., 1987; fariss, 1991; mohiseni et al., 2017a). in this context, protective effects of some natural antioxidant including vitamins (especially e and c) are well documented (chaurasia and kar, 1997; gupta et al., 2004; cinar et al., 2010; mekkawy et al., 2011; harabawy and mosleh, 2014). phenolic compounds in phytochemicals are believed to promote optimal health relatively through 361 int. j. aquat. biol. (2017) 5(6): 360-369 their antioxidant and free radical scavenging effects, thereby, protecting cellular components against free radical-induced damages. but due to their diverse chemical structures, they are likely to possess different antioxidant capacities (hamzawy et al., 2012; soleimany et al., 2016; mohiseni et al., 2017b). thyme is a phytobiotic and used since ancient times as a medicinal herb. it has strong antimicrobial and antioxidant activity due to its very high contents of thymol, p-cymene, carvacrol, eugenol and 4allylphenol (alçiçek, 2011; yılmaz et al., 2012). shirazi thyme (zataria multiflora boiss) is a thymelike plant belonging to the lamiaceae family that geographically grows in central and southern parts of iran, pakistan and afghanistan (hosseinzadeh et al., 2000). the essential oil of shirazi thyme contains significant quantities of phenolic compounds such as thymol, carvacrol and monoterpenes, which have an antioxidant, antibacterial and antifungal activities (ehsani et al., 2014). there are some evidences about the positive effects of thyme on growth and feed utilization in broiler (hosseini et al., 2013; sadek et al., 2014) and fish (dorojan et al., 2014; sönmez et al., 2015), but despite the strong antioxidant capacity of these components, a little information are exist about using of thyme to improve stress resistance in fish (antache et al., 2014). due to the presence of active ingredients, medicinal herbs possess antioxidant properties and can be used as anti-stress in aquaculture. this will reduce the cost and side effects of synthetic or chemical products. they are also eco-friendly compounds and hence will not affect the environment. although various medicinal plants contain numerous antioxidant compounds, however, a few of them have been investigated so far. therefore, this study was conducted to evaluate the potential protective effects of shirazi thyme compared to vitamin e (a strong and well-known natural antioxidant) against cadmium exposure in juvenile common carp based on oxidative stress responses. materials and methods diet preparation: three experimental diets were prepared and used for feeding trial during the experiment. a commercial diet (table 1) for carp (naghshin kermanshah, iran) was milled by feed producer, feed additives were added, mixed thoroughly and finally, the diets re-pelletized with kitchen grinder using 3mm die (montero et al., 1999). for control and metal only (mo), milled commercial diet re-pelletized without any supplementation whereas in diets 2 and 3 the commercial diet was supplemented with either 10 g/kg dry feed ground shirazi thyme (t) (yılmaz et al., 2013) or 100 mg/kg of dry feed vitamin e (e) (kaushik, 1995; ortuño et al., 2001). they were placed through natural air flow and after drying were kept in the refrigerator (4°c). acclimation condition and experimental design: in autumn 2014, 180 healthy juveniles of common carp (average weight of 34±3 g) obtained from persian fish hatchery (ahvaz, iran) and transferred to the khatam alanbia, university of technology (behbahan, iran). two weeks before the experiment, juveniles were randomly divided into four groups (in triplicates) and transferred to the separate 300-l tanks, each containing 15 juveniles and individually equipped with air stone and heater (25±2°c, ph 7.3). the oxygen level was monitored daily (8±1.5 mg/l) and the fish were kept under photoperiod cycle of 12l:12d. initially, all fish were fed 3% of biomass per day with the commercial diet. water exchanging during both acclimation and the whole experiment periods was done daily at the rate of 30%. after the acclimation period, all groups were fed only by their own specially-prepared diets. except for control, all experimental groups were exposed to the sublethal waterborne concentration of cadmium (1.5 mg/l free ion) for 15 days (vinodhini and narayanan, 2009). table 1. proximate composition of experimental diet (naghshin, kermanshah, iran). chemical analysis proportion (%) crude protein 32 crude lipid 5.5 crude ash 10 carbohydrate 47-49 crude fiber 4 total phosphor 1.2 362 mohiseni et al./ shirazi thyme, a natural antioxidant in fish feed sampling was done on 3, 7, 10 and 15 days after cadmium exposure. 3 fish were caught from each tank (9 fish per treatment) at each sampling time. the fish were sacrificed by spinal cord dislocation, then they were dissected and liver, kidney and gills were removed and washed in an ice-cold kcl solution (1.15%). the samples were homogenized in 5 volumes of 100 mm phosphate buffer (ph=7.4). homogenate was centrifuged at 12000×g for 15 min at 4°c. the supernatant was separated and stored at 70°c until antioxidant analysis (oliveira et al., 2010). enzyme activity and mda assay: gr activity was determined using reduced nicotinamide adenine dinucleotide phosphate (nadph) and oxidized glutathione (gssg) as substrates by a technique described by cohen duvel (1988). gst activity was evaluated according to habig et al. (1974) using 1chloro 2,4 dinitrobenzene as substrate. cat activity was measured spectrophotometrically based on h2o2 decomposition as substrate (aebi, 1984). total protein content of aliquots was measured according to the bradford method (kruger, 1994). lipid peroxidation assay was carried out in terms of mda by measuring the thiobarbituric acid reacting substances (tbars) and expressed as nmol mda/g wet weight (ringwood et al., 2003). statistical analysis: all data were statistically analyzed by one-way analysis of variance (anova). turkey’s test was used to evaluate the mean difference among experimental groups (p<0.05). statistical analyses were performed using ibm spss statistics for windows (version 19). the data presented as mean±se. results the specific activity of liver gr, gst and cat, and mda levels are presented in figure 1. the results showed that cadmium exposure induced gr activity at the 7th day of exposure approximately in all treatments but after 15 days of exposure, the lowest and highest enzyme activity were recorded in the mo and t groups, respectively. although the gst in the figure 1. effect of experimental diets on enzyme activity and lipid peroxidation in liver of common carp during different times of metal exposure. different letters shows significant differences between groups (p<0.05). 363 int. j. aquat. biol. (2017) 5(6): 360-369 mo group showed the highest activity at the 3rd day, the enzyme level decreased by time and reached the lowest level at the end of the experiment. the t and e treatments had a significant higher gst activities compared to the control and mo groups. cat showed slight changes in all treatments during the experiment, however, the enzyme activity for the mo decreased significantly compared to the other treatments (except for the e) at the end of the experiment (the 15th day). a significant elevation in mda level was obtained for the mo group at all sampling times; however, there were not significant differences between the control and the other treatments. the increase in mda level in the mo group was more than 200% compared to the control in the most cases. despite the cadmium exposure, the levels of mda in the t and e groups did not exceed the normal range (p>0.05). except for 7th day that the mo group showed the highest activity, kidney gr significantly decreased compared to the control and t groups at the 10th and both 10th and 15th days after cadmium exposure, respectively (fig. 2). cadmium led to a significant elevation in kidney gst activity at the 3rd, 7th and 10th day in all the metal exposed groups. although the gst levels for the t and e groups tended to remain in normal range, the enzyme activity significantly decreased in the mo group at the final sampling time. the same pattern was observed in cat at the 15th day of cadmium exposure, where the lowest enzyme activity was observed in the mo group (p<0.05), although, the cat activity remained unchanged at the previous sampling times (3rd, 7th and 10th). based on lipid peroxidation analysis, thyme and vitamin e kept the kidney mda levels within the normal range in the cadmium-exposed fish. on the other hand, the mo group had the highest levels of kidney mda during the experiment. figure 3 shows the gills enzyme activity and lipid peroxidation at different times of the metal exposure. there was some slight fluctuations in gill gr activity in all cadmium exposed fish during the initial times post exposure (3rd, 7th and 10th), but at the final figure 2. effect of experimental diets on enzyme activity and lipid peroxidation in kidney of common carp during different times of metal exposure. different letters shows significant differences between groups (p<0.05). 364 mohiseni et al./ shirazi thyme, a natural antioxidant in fish feed sampling, the highest and lowest enzyme activity were recorded for the t and mo groups, respectively. similar trends were obtained about gst approximately. shirazi thyme seems to have more ameliorating effects than vitamin e on the gill cat activity and the highest enzyme activities were related to the t group at 10th and 15th days. similar to the mda levels in liver and kidney (figs. 1, 2), the gill mda levels of the mo group were significantly higher than that of the control at the most times (3rd, 10th and 15th), while the rate of gill lipid peroxidation in the t and e groups was not exceeded the control levels (p>0.05). discussion cadmium is a toxic metal that can promote an oxidative stress and because of its long retention in vital tissues (such as liver and kidney), subsequently contributes to the development of serious pathological effects (el-demerdash et al., 2004). the present study has clearly demonstrated the capability of cadmium to induce oxidative stress in different tissues of common carp. based on the results, although gr, gst and cat activities initially tend to increase after cadmium exposure, finally the most enzyme activity showed a clear reduction in the mo group in all experimental tissues. although the mechanism of the cadmiuminduced oxidative stress is still not completely clarified, some hypotheses have been proposed. cadmium potentially can inhibit the mitochondrial electron-transfer chain reaction and leading to the accumulation of semiubiquinones. this unstable component would transfer one electron to molecular oxygen to form superoxide anion, the high energy free radical. it has also suggested that cadmium could directly weaken some antioxidant enzymes including cat, gr and superoxide dismutase. this two phenomenon will intensify the cadmium toxicity and leading to oxidative stress (cinar et al., 2010). figure 3. effect of experimental diets on enzyme activity and lipid peroxidation in gill of common carp during different times of metal exposure. different letters shows significant differences between groups (p<0.05). 365 int. j. aquat. biol. (2017) 5(6): 360-369 although the cat activities showed significant decrease in the mo group at 10th and 15th days, the significant differences were only recorded at the latter day for liver and kidney. the iron deficiency may explain the dissimilar cat activities in different tissues. jurczuk et al. (2004) stated that cadmium has a potential effects to impair the intestinal absorption of iron which is required for the cat activity. as the fish were challenged by waterborne cadmium in the current study, the higher recorded fluctuation in gill cat activity for the mo group may be related to their direct exposure to the metal. several investigations have shown that the formation of oxygen free radicals or ros increased as a result of metal exposure (stohs and bagchi, 1995; almeida et al., 2002; tan et al., 2010). ros can elicit widespread damage to cell components mainly as lipid peroxidation of membrane lipids (almeida et al., 2002). the cell’s antioxidant system in response will increase the rate of antioxidant enzymes production to cope with the stressful condition. when the metal concentration or exposing duration increased and overcame the capacity of the natural detoxifying system, enzyme activities will eventually decrease and several detrimental effects may occur (stohs and bagchi, 1995; sevcikova et al., 2011). in agreement with our results, the potentially harmful effects of heavy metals in cell antioxidant depletion has been previously reported in several investigations (stohs and bagchi, 1995; romeo et al., 2000; el-demerdash et al., 2004; farombi et al., 2007; tan et al., 2010; sevcikova et al., 2011). our data on cadmium exposure in fish have shown that the rate of lipid peroxidation (mda) in the mo group was increased in different tissues which directly results in free radical-mediated toxicity. lipid peroxidation is one of the main indicators of oxidative damage incidence and has been found to have an important role in toxicity and carcinogenicity (elnekeety et al., 2011). the rate of lipid peroxidation is followed by the balance between the production of ros and the elimination potential of those radicals by an antioxidant (sayeed et al., 2003). the increased level of mda along with the decreased activity of gr, gst and cat in the mo group may be attributed to the free radical formation that initiated chain reaction of bond formation with vital macromolecules (such as nucleic acid, protein, lipids and carbohydrates), impairing crucial cellular process that may disrupt the normal metabolism of the cells and ultimately organs (el-nekeety et al., 2011). similar to the vitamin e, shirazi thyme was found to highly potentiate against cadmium toxicity. based on the results, shirazi thyme stimulated antioxidant enzyme activities after cadmium exposure. it can be implied that the antioxidant compounds in shirazi thyme may enhance or reserved the existing antioxidant system. the suggested role of thyme compounds in the prevention of cadmium toxicity can be explained by their ability in radical scavenging (miura et al., 2002) and also the promotion of antioxidant enzyme activities (sengül et al., 2008). flavonoids and phenolic compounds are the main components in many medicinal herbs (kandaswami and middleton, 1994; rice-evans et al., 1997). these biologically active chemicals are thought to promote optimal health via their antioxidant and radical scavenging properties, thereby keeping cellular organelles from induced damage by free radicals (ündeğer et al., 2009). shirazi thyme is mainly composed of monoterpenes and aromatic compounds that have antibacterial, antiviral and antifungal activities. the plants essential oil also contains considerable amounts of phenolic compounds, mainly including carvacrol, thymol and р-cymene (kavoosi et al., 2012). the antioxidant activity of these compounds has been previously reported in several investigations (miura et al., 2002; ündeğer et al., 2009; kavoosi et al., 2012; sajed et al., 2013). these antioxidants are able to protect or amend the liver health and function in unfavorable environmental conditions and therefore resulted in improved metabolism and better animal growth performance. our data on common carp support conclusions of researchers who attributed physiological protecting effects of thyme in animal diets. accordingly, hassan barakat (2008) was found that thyme pretreatment in mice leads protection against nickel toxicity more 366 mohiseni et al./ shirazi thyme, a natural antioxidant in fish feed effective than basil. el-nekeety et al. (2011) also reported that the essential oil of common thyme (thymus vulgaris) has potential antioxidant activities and may induce protective effects against aflatoxicosis in the male rat in dose-dependent manner. the same results on hepatorenoprotective effects of common thyme against aflatoxicosis in the rat have been previously reported (hamzawy et al., 2012). vitamin e has also shown inhibitory properties over harmful effects of cadmium. considering the lipid solubility of vitamin e, the biological function of the compound is interpreted by many reports as support for prevention of lipid peroxidation (mccay, 1985; chow, 1991; harabawy and mosleh, 2014). although this is considered as a primary role of vitamin e, however, there is some evidence that scavenging of lipid-based radicals may not be attributed the only form of its activity (chow, 1991). the oxidation of some amino acid-based radicals including radicals of tryptophan, tyrosine, methionine and histidine is also decreased in the presence of this antioxidant (mccay, 1985). supporting with this documentation, we also found the efficient role of vitamin e against lipid peroxidation as there was not a significant increase in liver, kidney and gill mda levels in vitamin e treated group. generally, the current results revealed that cadmium induced stressful effects on liver, kidney and gills function. by inducing antioxidant enzymes activities and reduction in lipid peroxidation of the tissues, shirazi thyme (10 mg/kg diet) showed high protective effects as those observed for vitamin e (100 mg/kg diet) in common carp juveniles. generally, the current finding can provide a useful reference for stress protective effects of thyme and its beneficial role in aquaculture. references aebi h. 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(2017) 5(6): 360-369 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی ازیشیر آویشن اکسیدانی آنتی اثر مقایسه و کادمیم سنگین فلز از ناشی اکسیداسیونی آسیب بروز ارزیابی (zataria multiflora boiss )ویتامین و e معمولی کپور در (cyprinus carpio) 1حقی دوست نعمت بهزاد ،1بنایی مهدی ،2باقری دارا ،*1محیسنی محمد 1گروه شیالت، دانشکده منابع طبیعی، دانشگاه صنعتی خاتم االنبیاء بهبهان، بهبهان، ایران. .ایران برازجان، فارس، خلیج دانشگاه طبیعی، منابع و کشاورزی دانشکده شیالت، گروه2 چکیده: با مطالعه نای. دهندمی نشان خود از باالیی اکسیدانیآنتی خصوصیات ترکیبات این که است فالوونوئیدی و فنلی ترکیبات حاوی شیرازی آویشن با هایماهی. است شده انجام شده شناخته طبیعی اکسیدانآنتی یک عنوانبه e ویتامین با مقایسه در شیرازی آویشن حفاظتی اثر بررسی هدف راکخو از استفاده با غذایی هایجیره. گرفتند قرار تغذیه مورد مشخص غذایی جیره سه با و شده بندی تقسیم گروه چهار به( 34±3) وزنی میانگین . شدند ساخته جیره کیلوگرم در گرممیلی 100 یا و شیرازی آویشن درصد 1 مکمل حاوی و( تنهایی به فلز و کنترل گروه برای) افزودنی بدون تجاری با مواجهه از پس 15 و 10 ،7 ،3 روزهای در و گرفته قرار کادمیم زیرکشنده غلظت معرض در روز 15 مدت به تیمارها کلیه کنترل، گروه از غیر به غلظت در داریمعنی کاهش کادمیم با مواجهه روز 15 طول در که داد نشان نتایج. گرفتند قرار بردارینمونه مورد آبشش و کلیه کبد، سنگین، فلز مواجهه اثر در آلدهیددیمالون سطح دیگر سوی از. شد دیده بررسی مورد هایبافت در کاتاالز و ترانسفرازاسگلوتاتیون ردوکتاز، گلوتاتیون هایآنزیم مواجهه در را داریمعنی حفاظتی اثر e ویتامین و شیرازی آویشن مکمل از استفاده. داد نشان داریمعنی افزایش شده یاد هایبافت در سنگین فلز با نقش بر بررسی این از حاصل هاییافته. بود بهتر e ویتامین از شیرازی آویشن حفاظتی اثر که داد نشان نتایج زمینه این در. داد نشان کادمیم با .کندمی تاکید پروریآبزی در آن کاربردی مزایای همچنین و استرس با مواجهه در شیرازی آویشن حفاظتی .استرس دارویی، گیاه سنگین، فلز ،اکسیدانیآنتی سیستم، ماهی :کلمات کلیدی int. j. aquat. biol. (2022) 10(6): 524-536 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article reproductive biology of greasy grouper, epinephelus tauvina and coral hind grouper cephalopholis miniata (family: serranidae) in the southern red sea, shalatien, egypt ezzat mohammed-abdallah1, azza el-ganainy2, mahmoud m. farrag1, mohsen a. moustafa1, alaa g.m. osman1 1department of zoology, faculty of science, al-azhar university, assiut branch, 71524 assiut, egypt. 2national institute of oceanography and fisheries, fisheries division, egypt. s article history: received 7 october 2022 accepted 21 december 2022 available online 2 5 december 2022 keywords: gonadosomatic index spawning season grouper abstract: groupers are the main component of commercial fisheries in the red sea. the reproductive biology of epinephelus tauvina and cephalopholis miniata collected at the shalatien landing site in the southern egyptian red sea were investigated from january to december 2017. a total of 212 specimens of e. tauvina with a total length range of 23.6 to 70.3 cm and 243 specimens of c. miniata with a total length range of 17.4 to 42.1 cm were examined. females of e. tauvina and c. miniata accounted for 61.7 and 61.9% of the total fish sampled, showing a 1:1.61 and 1:1.63 male-to-female sex ratio, respectively. the monthly distribution of maturity stages and gonadosomatic index values showed that the spawning season for both sexes was extended from april to october for e. tauvina and from april to september for c. miniata. the lengths at first sexual maturity of the males and females of e. tauvina were estimated at 49 and 48.1 cm, respectively, while those for c. miniata were estimated at 23.2 and 22.7 cm, respectively. all our results can help in the proper management of these valuable resources. introduction the shalatien fishing area is a productive area in the egyptian sector of the red sea. coral reef terraces characterize the coastal area of shalatien in many regions. these coral reefs help break the waves and form protected areas with abundant food sources for different fishes (mahmoud et al., 2009). in 2020, groupers (serranidae) were highly abundant in the shalatien fishery, representing approximately 34.5% of the fish production from the shalatien landing site (gafrd, 2020). because of their commercial importance, they suffer from strong fishing pressure, which extends to overexploitation (morris et al., 2000), resulting in a decrease in their population worldwide (sadovy de mitcheson et al., 2013; rajan, 2015; bulanin et al., 2017; ohta et al., 2017; de mitcheson et al., 2020). many groupers form spawning aggregations at specific locations and times that are often associated correspondence: ezzat mohammed-abdallah doi: https://doi.org/10.22034/ijab.v10i6.1774 e-mail: ezzatahmed1278.el@azhar.edu.eg dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.9.2 with the lunar cycle (sadovy de mitcheson and colin, 2012; osman et al., 2021). at these locations and times, they are especially vulnerable to fishing pressure from artisanal and commercial fisheries because of increased catchability (lethal effects) and decreased reproductive output at lowered animal densities (sadovy de mitcheson, 2016; ohta et al., 2017). many studies have reported that the total catch of groupers has decreased and that the stock has been overexploited (e.g., sadovy, 2005; tharwat, 2005; mohammad, 2007; grandcourt et al., 2009; mohammed-abdallah, 2015; el-ganainy, 2017; osman et al., 2021). the red list assessments for all 163 grouper species show that 25% of the species are considered to be at risk of extinction or categorized as being near threatened, but 30% of all species are considered to be data deficient (sadovy de mitcheson et al., 2013; iucn, 2021). an understanding of fish reproductive strategies 526 mohammed-abdallah et al./ reproductive biology of epinephelus tauvina and cephalopholis miniata is required to properly manage fisheries (komolafe and arawomo, 2007; dinh, 2018). knowledge regarding female maturity stages and spawning seasons is important to prevent catches during the spawning season (el-ganainy, 1997; osman et al., 2018). the sex ratio provides a useful tool for assessing fish's biological characteristics and estimating a population's reproductive potential (sossoukpe et al., 2013; sabrah et al., 2017). the gonadosomatic index (gsi) is widely used to measure the extent of reproductive investment, gonadal development and maturity of fish in relation to spawning (ogunola et al., 2018; khatun et al., 2021; sabbir et al., 2021). the length and age at first sexual maturity are important parameters in fisheries research for evaluating the optimum age of the first capture and determining the minimum legal size that may be needed to preserve a suitable spawning stock of a species and to ensure the occurrence of at least one spawning event among mature fish (lappalainen et al., 2016; osman et al., 2018; mawa et al., 2021). this work aimed to investigate the sex ratio, maturity, gsi and length at first sexual maturity of epinephelus tauvina and cephalopholis miniata in the shalatien fishery to provide proper data for the conservation of their stocks. materials and methods random samples of e. tauvina and c. miniata were collected monthly from the shalatien fishing port, which is one of the productive fishing grounds along the egyptian red sea coast and located in the southern red sea, at elba national park, 520 km south of hurghada, egypt (fig. 1). the samples were collected from january to december 2017. for each specimen, total length (tl) was measured to the nearest cm, and total weight (tw) was determined to the nearest 0.1 grams (g). specimens were sexed by macroscopic dissection, and the gonads were weighed to the nearest 0.01 grams. the sex ratio was calculated from the formula of sex ratio (m:f) = number of females/number of males. this ratio was determined during different months and in different length groups. the chisquare test at the 0.05 significance level was employed to determine the goodness of fit of the observed sex ratio to that of the theoretical sex ratio of 1:1 (m:f). the following formula of wootton )1998( was used for the chi-square test. χ2 = [(f-s)2/ s] + [(m-s)2/ s], where χ2 is the symbol for chisquare, f is the observed number of females, m is the observed number of males and s is the expected figure 1. map of the study area (shalatien). 527 int. j. aquat. biol. (2022) 10(6): 525-536 number of each sex (the hypothetical 1:1 ratio). the monthly variation in the maturity stage was determined. six maturity stages were identified according to the scale of gunderson (1993), with some modifications, as follows: (1) thread: sex cannot be determined at this maturity stage, and the gonads in individuals are filamentous, (2) stage i (immature or inactive): the gonads have a translucent appearance, and the testes are smaller and thinner than the ovaries, (3) stage ii (resting): the gonads are translucent and enlarged and the testes do not contain sperm, while the ovaries possess a few small eggs, (4) stage iii (active or developing): gonads are larger than stage ii, and the testes are opaque without sperm; ovaries are translucent with small eggs, (5) stage iv (activeripe or developed): testes are white, and some sperm are expelled from the core when cut, and ovaries are opaque and solid with fully formed eggs, (6) stage v (spawning or ripe-running): gonads are enlarged and occupy most of the body cavity; milt and eggs are expelled from the genital openings when we press slightly on the two sides of the body, and (7) stage vi (spent): ovaries are flaccid with few eggs, while the testes are almost empty. the gsi is an indirect method used to determine the spawning season of fish. it was calculated for each fish according to sokal (1995) using the formula of gsi = (gonad weight/total fish weight) x 100. the length at first sexual maturity (lm) represents the length at which 50% of individuals are mature during the spawning season. individuals at stages iii-vi were considered mature, and it was possible to determine their lm values. after plotting the percentage of mature individuals against their mid-lengths (king, 1995), lm was estimated as the point on the x-axis corresponding to the 50% point on the y-axis. the equation used to estimate lm from the logistic curve was as follows (erisman et al., 2010): p = 1/ (1 + exp −r (lt − lm)), where p is the proportion of mature individuals in a length class, lt is the total fish length, r is the intercept, and lm is the slope. results a total of 212 specimens of e. tauvina with a tl range of 23.6 to 70.3 cm, including 77 males, 124 female,s and 11 unsexed and 243 specimens of c. miniata with a tl range of 17.4 to 42.1 cm, including 91 males, 148 females and 4 unsexed were collected for reproductive biology studies (table 1). sex ratio: the overall sex ratio indicated the occurrence of more females than males for the two studied species. the ratio of males to females of e. tauvina was 1:1.61, and females accounted for 61.7% of the total sampled fish. the sex ratio of males to females of c. miniata was 1:1.63, and females accounted for 61.9% of the total sampled fish. the chi-squared analysis indicated a significant difference compared to the expected 1:1 ratio (χ2 = 10.53, df = 1, p<0.05 for e. tauvina and χ2 = 13.12, df = 1, p<0.05 for c. miniata). the monthly changes in the percentages of males and females are represented in figure 2 for e. tauvina and figure 3 for c. miniata. the variation in the male-to-female sex ratios of e. tauvina and c. miniata according to species sex n tl (cm) total weight (g) min max mean ± sd min max mean ± sd e. tauvina males 77 24.4 62.2 40.6±11.7 206.1 3350.3 1226.5±1050.0 females 124 27.5 70.3 44.3±12.5 259.9 5015.3 1615.0±1339.3 unsexed 11 23.6 26.6 25.3±1.0 189.5 265.1 231.8±25.2 pooled 212 23.6 70.3 42.0±12.6 189.5 5015.3 1402.1±1245.8 c. miniata males 91 20.2 42 29.5±5.9 135.4 1286.9 474.3±282.8 females 148 17.4 42.1 27.5±5.0 75.6 1277.9 398.4±283.7 unsexed 4 22.6 23.5 23.2±0.4 190 214.9 205.7±11.4 pooled 243 17.4 42.1 28.1±6.1 75.6 1286.9 420.6±284.1 table 1. descriptive statistics of means of epinephelus tauvina and cephalopholis miniata during (2017) from southern red sea, shalatien, egypt. 528 mohammed-abdallah et al./ reproductive biology of epinephelus tauvina and cephalopholis miniata length groups is presented in figures 4 and 5, respectively. monthly distribution of maturity stages: the seasonal variation in the maturity stage was determined for each individual. the e. tauvina spawning period began in april because ripe gonads could be observed in the samples at that time, and the percentages of spawning individuals were 40 and 0 10 20 30 40 50 60 70 80 jan feb mar apr may jun jul aug sep oct nov dec f re q u e n c y % month male female figure 2. monthly variation in the percentages of males and females of epinephelus tauvina from the southern red sea, shalatien, egypt. 0 10 20 30 40 50 60 70 80 jan feb mar apr may jun jul aug sep oct nov dec f re q u e n c y % month male female figure 3. monthly variation in the percentages of males and females of cephalopholis miniata from the southern red sea, shalatien, egypt. 0 10 20 30 40 50 60 70 80 90 100 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 f re q u e n c y ( % ) length group (cm) male female figure 4. percentages of males and females of epinephelus tauvina according to length group. 529 int. j. aquat. biol. (2022) 10(6): 525-536 45% of the males and females, respectively (figs. 6, 7). this stage extended until september, revealing a long spawning season, with a peak in may for males (66%) and in june for females (67%). the first appearance of ripe gonads of c. miniata occurred in april, indicating the beginning of the spawning 0 10 20 30 40 50 60 70 80 16 18 20 22 24 26 28 30 32 34 36 38 40 42 f re q u e n c y ( % ) length group (cm) male female figure 5. percentages of males and females of cephalopholis miniata according to length group. 0 10 20 30 40 50 60 70 80 90 100 jan feb mar apr may jun jul aug sep oct nov dec m a tu ri ty s ta g e % month i ii iii iv v vi figure 6. monthly distribution of maturity stages (i: inactive, ii: spent, iii: active, iv: active-ripe, v: ripe-running, vi: spent) in male epinephelus tauvina. 0 10 20 30 40 50 60 70 80 90 100 jan feb mar apr may jun jul aug sep oct nov dec m a tu ri ty s ta g e % month i ii iii iv v vi figure 7. monthly distribution of maturity stages (i: inactive, ii: spent, iii: active, iv: active-ripe, v: ripe-running, vi: spent) in female epinephelus tauvina. 530 mohammed-abdallah et al./ reproductive biology of epinephelus tauvina and cephalopholis miniata period. the percentages of individuals in this stage during this month were 15% and 17.3% of the males and females, respectively (figs. 8, 9). this stage extended until august, revealing a long spawning season, with a peak in june for males (57%) and in may for females (55%). gonadosomatic index (gsi): the overall gsi values were higher for females than for males in both 0 10 20 30 40 50 60 70 80 90 100 jan feb mar apr may jun jul aug sep oct nov dec m a tu ri ty s ta g e % month i ii iii iv v vi figure 8. monthly distribution of maturity stages (i: inactive, ii: spent, iii: active, iv: active-ripe, v: ripe-running, vi: spent) in male cephalopholis miniata. 0 10 20 30 40 50 60 70 80 90 100 jan feb mar apr may jun jul aug sep oct nov dec m a tu ri ty s ta g e % month i ii iii iv v vi figure 9. monthly distribution of maturity stages (i: inactive, ii: spent, iii: active, iv: active-ripe, v: ripe-running, vi: spent) in female cephalopholis miniata. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 jan feb mar apr may jun jul aug sep oct nov dec a v e ra g e g s i month males females figure 10. monthly variation in gsi values for epinephelus tauvina from the southern red sea, shalatien, egypt. 531 int. j. aquat. biol. (2022) 10(6): 525-536 species. the spawning season of e. tauvina was extended from april to october, while it spanned only from april to september for c. miniata. the highest value of this index was recorded in may for males and females of e. tauvina and c. miniata (figs. 10, 11), and the lowest values for both sexes were recorded in january for e. tauvina and in december for c. miniata. length at first sexual maturity (lm): the lm values for the males and females of e. tauvina were estimated at 49 and 48.1 cm (fig. 12), respectively, while those for the males and females c. miniata were estimated at 23.2 and 22.7 cm (fig. 13), respectively. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 jan feb mar apr may jun jul aug sep oct nov dec a v e ra g e g s i month males females figure 11. monthly variation in gsi values for cephalopholis miniata from the southern red sea, shalatien, egypt. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66p ro p o rt io n o f m a tu ri ty o f m a le total length (cm) p = 1/(1 + exp−0.2.8× (tl − 49.0)) (a) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 p ro p o rt io n o f m a tu ri ty o f fe m a le total length (cm) p = 1/(1 + exp−0.3× (tl − 48.1)) (b) figure 12. logistic curves for the proportions of mature males (a) and females (b) of epinephelus tauvina from the southern red sea, shalatien, egypt. 532 mohammed-abdallah et al./ reproductive biology of epinephelus tauvina and cephalopholis miniata discussion the application of the sex ratio is useful in studying spawning populations (faltas, 1983; sabrah et al., 2017). in the present study, the sex ratio of e. tauvina and c. miniata revealed that females dominated the catch during the whole period of study. our results agree with those recorded by ohta et al. (2017) for e. tauvina in the yaeyama islands and wahbeh (2005) for c. miniata in the aqaba gulf, jordan. according to wahbeh (1992), cherif et al. (2007) and sabrah et al. (2017), the predominance of females may be connected with the higher catchability of females than males or due to the high mortality of females, as they are more vulnerable to fishing pressure. chakroun-marzouk and ktari (2006) reported an unbalanced sex ratio favouring females in some species, revealing that natural mortality may be higher among males, while females may present high vulnerability to fishing. according to the length group’s evaluation, males of e. tauvina were dominant at smaller fish sizes and absent among larger fish sizes, while females of c. miniata were dominant at all sizes. the results are supported by nikolsky (1963), who stated that males predominate in fish populations at early life stages, while females predominate later due to probable adaptive mechanisms. the sex ratio in these species could vary greatly; however, no studies have explicitly addressed the potential mechanisms explaining those patterns. the monthly distribution of maturity stages showed that the ripe stage for males and females of e. tauvina began in april, ended in september, and increased to its highest peak in may for males and in june for females. on the other hand, the ripe stage for males and females of c. miniata began in april, ended in august, and increased to its highest peak in may for both males and females. these results 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 16 18 20 22 24 26 28 30 32p ro p o rt io n o f m a tu ri ty o f m a le total length (cm) p = 1/(1 + exp−0.66× (tl − 23.2)) (a) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 16 18 20 22 24 26 28 30 32 p ro p o rt io n o f m a tu ri ty o f fe m a le total length (cm) p = 1/(1 + exp−0.7× (tl − 22.7)) (b) figure 13. logistic curves of the proportions of mature males (a) and females (b) of cephalopholis miniata from the southern red sea, shalatien, egypt. 533 int. j. aquat. biol. (2022) 10(6): 525-536 indicate that both e. tauvina and c. miniata in the southern red sea, shalatien, have a prolonged spawning season that extends from april to october for e. tauvina and from april to september for c. miniata. the gsi refers to the relationship between the gonadal weight and the fish somatic weight (wootton, 1991), and the seasonal timing of reproduction is often identified based on changes in the gsi (biswas, 1993; king, 2013; ogunola et al., 2018). our results revealed that the gsi differed between males and females of e. tauvina and c. miniata; the gsi for males was lower than that for females. these results suggest that the energy invested in gamete production by females is likely more than that invested by males (patimar, 2008). the results showed that the gsi increased in both sexes of e. tauvina from april to october and in both sexes of c. miniata from april to september, indicating the timing of the spawning season in these species. these findings for e. tauvina agree to some extent with those found by ohta et al. (2017) in the yaeyama islands, japan, abu-hakima et al. (1982) in kuwait and el-ganainy (2017) in the red sea, egypt, whose investigations of the spawning season of e. tauvina indicated that it occurs from march to june. abu‐hakima (1987) stated that e. tauvina collected from kuwait spawned in april and may. el-sayed (1999) recorded that e. tauvina in the persian gulf spawns in may and june, while members of this species from kuwait spawn from march to may (mathews and samuel, 1987). the observed decrease in the gsi values during january for e. tauvina and december for c. miniata indicated the end of the spawning season and the beginning of the spent and resting periods. likewise, the spawning season of c. miniata in the aqaba gulf, jordan, was found to occur between may and october (wahbeh, 2005). el-ganainy (2017) stated that c. miniata collected from the red sea, egypt, spawned in may and june. the spawning season of c. miniata is relatively long, which is consistent with the general pattern of longer spawning seasons among smaller groupers (sadovy, 1996; wahbeh, 2005). differences between our results and the previous studies about the spawning seasons of investigated species may be attributed to differences in water temperature among locations (jansen and gislason, 2011; osman et al., 2018), sampling techniques, geographic disparity and the sampling year of the studies (soykan et al., 2020). knowing the lm of fish helps predict their harvestable size and hence has great value for fishery management (sabrah et al., 2017). in the present study, e. tauvina reached first sexual maturity at 48.55 cm tl. the lm values of e. tauvina found in the present study were longer than those reported by ohta et al. (2017) in the yaeyama islands, japan, species author region spawning season sex ratio (m:f) lm e . ta u v in a abu-hakima et al. (1982) kuwait water march to june 61.1 mathews and samuel (1987) kuwait water march to may el-sayed (1999) persian gulf off qatar may to june 53 shakeel and ahmed (1996) maldives 37.5 abu-hakima (1987) kuwait water april to may ohta et al. (2017) yaeyama islands, japan march to june 1:1.8 37.1 el-ganainy ( 2017) red sea, egypt may to june 36.6 present study red sea, shalatien, egypt april to october 1:1.61 48.55 c . m in ia ta shakeel and ahmed (1996) maldives 20 wahbeh (2005) aqaba, jordan may to october 1:1.28 el-ganainy (2017) red sea, egypt may to june 28.2 present study red sea, shalatien, egypt april to september 1:1.63 22.95 table 2. the spawning season, sex ratio and the length at first sexual maturity (lm) of epinephelus tauvina and cephalopholis miniata found in the present study and in various regions by different authors. 534 mohammed-abdallah et al./ reproductive biology of epinephelus tauvina and cephalopholis miniata shakeel and ahmed (1996) in the maldives (37.3 cm) and el-ganainy (2017) in the red sea, egypt (36.6 cm), but smaller than those reported by elsayed (1999) in the persian gulf (53 cm) and abuhakima et al. (1982) in kuwait waters (61.1 cm) (table 2). our results revealed that c. miniata reached first sexual maturity at 22.95 cm. these findings roughly correspond to those obtained by shakeel and ahmed (1996) for c. miniata collected in the maldives (20 cm) but are smaller than those estimated by el-ganainy (2017) in the red sea, egypt (28.2 cm). these differences in length and age at the first sexual maturity of investigated species in the present study and in the previous studies may be attributed to the length and age at first sexual maturity of fishes influenced by environmental factors like abundance and seasonal availability of food, predation, temperature, photoperiods and also the locality (osman et al., 2018; adekoya et al., 2019; hossen et al., 2019; soykan et al., 2020). it can be concluded that, both sexes of e. tauvina and c. miniata in the shalatien landing site in the southern red sea, egypt, showed a prolonged spawning season that extended from april to october in e. tauvina and from april to september in c. miniata. e. tauvina reached sexual maturity at 49 cm for males and 48.1 cm for females, while c. miniata reached sexual maturity at 23.2 cm for males and 22.7 cm for females. fishing of spawning aggregations leads to a reduction in the average size of the individuals caught and a remarkable decline in the sex ratio. establishing protected areas (or no-take zones) in the red sea is a valuable management option for protecting the size and the breeding populations of coral reef fish, especially groupers that aggregate to spawning. references abu-hakima r. 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(2022) 10(6): 504-514 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article total petroleum hydrocarbons in water, sediment, and redbelly tilapia, coptodon zillii in shatt al-basrah canal, iraq ali m. glou, rajaa n. al-yassein, amjed k. resin department of fisheries and marine resources, college of agriculture, university of basrah, iraq. s article history: received 7 june 2022 accepted 21 august 2022 available online 2 5 december 2022 keywords: tphs pollution muscles tissue sediments abstract: water pollution is one of the most common global problems resulting from increased industrial and agricultural activities. petroleum hydrocarbons have extremely dangerous to the aquatic environment. the total petroleum hydrocarbon (tphs) was investigated in water, sediment, and muscles of coptodon zillii at abu sakhir and alzubair bridge stations seasonally in the shatt al-basra canal. the results showed a variation in the tphs levels in the studied stations. in addition, a significant difference in the tphs was recorded during the seasons in the water, and sediments between stations. the results showed significant differences in the tphs in the muscles in the spring but no significant in other seasons between the two stations. the results of the lipid contents of fish revealed significant differences between the two studied stations in the fall, spring, and summer seasons but not significant in winter. introduction water contamination is a crucial global problem resulting from expanding industrial and agricultural activities (wang et al., 2019). many pollutants enter the aquatic environments directly or indirectly due to human activities, affecting aquatic organisms (li et al., 2019). one of the main environmental pollutants worldwide is crude oil-based hydrocarbons (ławniczak et al., 2020). petroleum hydrocarbon pollution is a significant environmental issue that threatens the aquatic environment, whether from benzene or other toxic organic materials (abha and singh, 2012). increased urbanization and intensive industrial activities have led to an increase in the consumption of oil and its products worldwide (liu et al., 2019). there are many ways to enter oil contaminants in the aquatic environment e.g. discharged through oil spill accidents or by-products for individual or commercial uses (cai, 2021). furthermore, navigation, transporting oil, washing loading docks, correspondence: rajaa n. al-yassein doi: https://doi.org/10.22034/ijab.v10i6.1782 e-mail: rajaa.alyassein@uobasrah.edu.iq dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.7.0 balance water, and export ports are the main sources of pollution of aquatic ecosystems that have been reported as approximately 6 million tons annually (chougule et al., 2009). in addition, there are other sources of hydrocarbon pollution, such as domestic, agricultural, and power plant releases (frena et al., 2017). therefore, when petroleum hydrocarbons are discharged into aquatic ecosystems, they cause harm to living organisms, with toxic effects ranging from acute to chronic depending on the metabolism and photo-oxidation process (kuppusamy et al., 2020). this damage may appear direct or indirect after a long period (rodrigues et al., 2010). releasing large quantities of petroleum hydrocarbons into rivers and coastal areas during oil production and transportation negatively impacts the environment and human health (ihunwo et al., 2021). it would be affected water in its dissolved particles, organisms, and sediments phase (raja et al., 2022). because of their hydrophobic nature, hydrocarbons tend to bind to organic particles in aquatic 505 int. j. aquat. biol. (2022) 10(6): 504-514 ecosystems where they are deposited in sediments (qiu et al., 2009). fish can rapidly absorb soluble petroleum hydrocarbons that are highly sensitive to lipids, which enhances living organisms’ ability to absorb petroleum hydrocarbons through the respiratory and gastrointestinal tracts (rodrigues et al., 2010). fish plays a significant role in the food chain and is an important indicator for many environmental pollutions in the aquatic system (wang et al., 2021). on the other hand, fish is an important part of the human diet due to having high-quality protein (holub and holub, 2004). in addition, it contains a wide variety of vitamins and minerals, containing vitamins a and d, magnesium, selenium, and phosphorus (fischer and glei, 2015). fishes can absorb petroleum hydrocarbons from contaminated water directly or indirectly through food and sediments (scheuhammer et al., 2016). the uptake of contaminants directly from the aquatic environment is known as bioconcentration (muijs and jonker, 2012). fish generally dissolve petroleum hydrocarbons due to their high lipid solubility (mcconville et al., 2018). tilapia can be found in freshwater or brackish waters such as rivers, lakes, estuaries, shallow streams, and ponds (iyabo, 2015). it is the second most important fish in the world aquaculture after common carp, with production reaching 6.3 million tons in 2018 (fao, 2019). redbelly tilapia, coptodon zillii has a widespread distribution within the iraqi inland waters. it might come from iraq's bordering countries, such as turkey, syria, and iran (mutlak and al-faisal, 2009). based on abovementioned background, this study aims to evaluate the total petroleum hydrocarbon levels in the water, sediment, and muscles of c. zillii in shatt al-basra canal, iraq. material and methods study area and sampling: the shatt al-basra canal is a drainage channel that starts north of basra and extends southeast. it covers about 37,157 km and is situated within the alluvial plain of basrah, located between latitudes 3027-3028 in the north and 47504749 in the east. it transfers flood waters from the al-hammar marsh to khor al-zubayr and then to the persian gulf. it is also used to withdraw saline water from irrigated agricultural lands, which later turned into marsh reclamation. moreover, this channel is a waterway connecting the southern marshes with the persian gulf from its northern part (hassan et al., 2018). the sampling was done in two stations, including (1) abu sakhir, in the northwest of basrah (47.702856n, 30.561467e), and (2) al-zubair bridge, in the southwestern part of the city of basrah in (47.760947n, 30.442322e). it is about 6,423 m from the center of basrah and about 15,272 m from the second station (fig.1). fish, water, and sediment samples were collected seasonally to measure total petroleum hydrocarbons from the selected stations from fall 2021 to summer 2022. fish were collected by gill net, and the samples were frozen and taken to the lab, where they were weighed, measured, and stored at -4oc. they were dissected using aseptic equipment to obtain the muscles, and the samples were labeled for analysis. water samples were collected from the surface in 2-liter amber glass bottles by adding 10 mm of ccl4, carbon tetrachloride. before water sampling, all bottles were washed with warm water and liquid detergent, then properly rinsed with tap water and dried for 24 hours, afterward the bottles were cleaned with acetone, dried for about 30 seconds, and finally rinsed with nhexane. sediments were collected during the low tide period from the slightly submerged riverbank at approximately 2-3 m using a small shovel and placed in plastic bags. total petroleum hydrocarbons extraction: fish and sediments extractions were performed according to goutx and saliot (1980). three gram of fish muscles was dried, weighted, and crushed in a ceramic mortar with a pestle. for sediment, 3 g of the dried sediment was passed through a sieve of 63 μm mesh and put into an extraction thimble. the fish and sediments' ground samples were placed separately in thimbles with 100 ml of methanol: benzene (1:1 v/v) 506 glou et al./ total petroleum hydrocarbons in water, sediment, and redbelly tilapia for 24 h, as the extraction solvent. the extract was saponified for two hours at 40°c by adding 15 ml of an aqueous 4n meoh (koh) solution. the contents were poured into a separating funnel, and then 50 ml of normal n-hexane was added; afterward, the sample was well-shaken and left to settle. the two layers were formed, and the layer containing hydrocarbons was taken and passed on a separation column from the bottom of the glass wool, topped with a layer of silica gel, alumina, then anhydrous sodium sulfate. the extraction of total hydrocarbons from the water was based on unep (1989), that in this procedure, 10 ml of ccl4 was added to 1 liter of sampled water. using an electric mixer, the sample was shaken for 30 min; then, the contents were poured into a separating funnel and left to settle. the organic layer (the lower layer containing carbon tetrachloride and hydrocarbons was collected and passed over a separation column containing glass wool at the bottom, topped with a layer of aqueous sodium sulfate na2so4 to remove water. the extracted muscles, water, and sediment samples were dried at laboratory temperature. finally, the total petroleum hydrocarbons were measured after dissolving them with pure hexane using a spectrofluorometer. for calibration, basrah crude oil was used by dissolving a known weight of oil with a specific volume of n-hexane to prepare standard solutions using a fluoridation device to determine the concentrations of total petroleum hydrocarbons in fish, water, and sediments. the emission intensity was measured at a wavelength of 310 nm and at an excitation of 360 nm. to determine the lipid concentrations in muscles, aliquots of the lipid extracts were dried and dissolved with a mixture of n-hexane (egan et al., 1981). statistical analysis: the statistical analysis was performed using spss 20 (statistical package for social sciences). lsd test was used to determine those differences under the significance level of p<0.05 through the anova test (snedecor and cochran, 1989). results the results showed differences in the total petroleum hydrocarbons in the water, sediment, and muscles for both stations in the shatt al-basra canal study areas. the highest tphs in the water were 21.58 and 30.53 µg.l-1 in winter in the first and second stations, respectively. the lowest values were 7.45 and 8.61 µg.l-1 in summer and fall in the first and second stations, respectively (fig. 2). the sediments results showed the highest level of tphs as 39.59 and 41.37 µg/g dry weight in spring in the first and second figure 1. map of sampling sites in the shatt al-basrah canal. 507 int. j. aquat. biol. (2022) 10(6): 504-514 figure 2. tphs in the water of shatt al-basrah canal. figure 3. tphs in the sediments of shatt al-basrah canal. figure 4. tphs in the coptodon zillii muscles of shatt al-basrah canal. figure 5. percent distribution of lipids in the muscles of coptodon zillii in shatt al-basrah canal. 508 glou et al./ total petroleum hydrocarbons in water, sediment, and redbelly tilapia stations, respectively. the lowest values were 8.17 and 6.98 µg/g dry weight in fall at the first and second stations, respectively (fig. 3). a highly significant difference (p≤0.05) at both stations in the tphs level in the muscles of c. zillii was recorded. the highest value was in spring at the first station as 36.56 µg/g dry weight, while the lowest value was 3.87 µg/g dry weight in fall at the second station (fig. 4). the highest lipids of c. zillii were 9.90 and 9.51% in spring at the first and second stations, but the lowest values were 3.41 and 4.61%, respectively in winter at the first and second stations (fig. 5). discussion petroleum hydrocarbons enter the aquatic environment through various sources, including the atmosphere, industrial sources, household waste, and untreated civil waste (eganhouse et al., 1981). the current study showed a seasonal variation in the tphs in water in all seasons in both stations. a highly significant difference in tphs was observed in the water of both stations. the least tphs in water was 7.45 µg.l-1 in summer in the first station (fig. 6). however, the highest tphs was 30.53 µg.l-1 in winter at the second station (fig. 7). however, a higher level of tphs recorded in the surface waters was observed in summer in the study of adeniji et al. (2017). depending on the regions and seasons, the amount of petroleum hydrocarbons in water varies e.g. the highest tphs were 4.92-46.4 µg.l-1 in khor abdullah water (nasir, 2005), but the lowest tphs at shatt albasrah was 0.05-26.44 µg.l-1 (atti, 2014), whereas in the current study, it was 7.45-30.53 µg.l1. conversely, the tph was 45.07 to 307 µg/l in the water of algoa bay in south africa, and the ranges of tph in a seasonal investigation were 45.07 to 273 g/l in surface water and 55.72 to 307 g/l in bottom water (adeniji et al., 2017). based on the results, the second station had the highest concentrations of tphs, which may be due to the accumulation of oil residues from the basrah oil refinery (aziz and sabbar, 2013). furthermore, the small fishing boats, and the presence of main drainage pipes that receive untreated domestic and sewage waste to the east of the canal, are the most important sources of petroleum hydrocarbon pollution (eganhouse et al., 1981). the ratio of tph was higher in the winter because of the low temperatures that prevented evaporating compounds (al-saad and al-timari, 1993). in addition, the effectiveness of microorganisms in breaking down hydrocarbons decreases with decreasing temperature (khillare et al., 2014; marić et al., 2020). due to the high temperatures, the oil loses 20-50% of its components through evaporation (harnstrom et al., 2009). light oxidation also leads to the breaking of oil compounds in the water column, which coincides with the high temperatures in the summer, as this factor depends on the length of the illumination period during the day and is one of the reasons for the low concentrations of tphs in the summer (talal, 2010). there were also highly significant differences in tphs in the sediments of both stations. the highest concentration of tphs in sediment was 41. 37 µg/g dry weight in spring in the second station. a consistent rise in tph in sediments was noted from summer to autumn (al-saad et al., 2017b). there was a seasonal variation, with the dry season having the most variability because of the higher level of increased oil activity during that time (clinton et al., 2009). massoud et al. (1996) reported tph in sediments between 10-15 mg/kg should be regarded as unpolluted and 15-50 mg/kg as slightly polluted. the levels of phcs in the sediments were high in our study, however 279 to 17 900 mg.kg-1 was reported by lindén and pålsson (2013), and the high level of phc in the surficial sediment of the inshore area of ratnagiri has been reported as 107.7 ppm, dry weight (chouksey et al., 2004). table 2 shows the petroleum hydrocarbon (phc) levels in the sediments of different water bodies in iraq. the values of phcs in iraqi waterbodies were 2.3-50.2, 4.76-45.24, and 19.43-49.09 µg/g dry weight (hantoush, 2006; al-hejuje, 2014; al-ali et al., 2016) and in our work, it was 6.98-57.5 µg/g dry weight. pollutants can enter the aquatic environment 509 int. j. aquat. biol. (2022) 10(6): 504-514 and settle in the sediment in various ways, including direct deposit because of their weight or longdistance water current transport because of their stability, which causes them to deposit when the speed of the currents decreases (liu et al., 2019). these compounds enter the living organisms and reach the sediments after death (al-saad, 1995). therefore, thp levels in the sediment were consistently higher than in surface water, showing that it accurately indicates pollution in the river systems (osuji et al., 2004). the reason for increasing phcs levels in sediment after many years is the establishment of shore refineries (aziz and sabbar, 2013)). similarly, variation in different regions could be related to the transfer of phcs loads from the inshore areas to the open-shore sediments and probably to an important role of benthic organisms in the biological transfer of phc into the sediment due to the remnants of an oil spill (chouksey et al., 2004). due to the particle size, the figure 6. tphs levels in different sample types at station 1. figure 7. tphs levels in different sample types at station 2. region tphs µg.l -1 references khor abdullah 4.6-22.6 al-timar et al. (2002) khor abdullah 0.9-23 al-timar et al. (2003) khor abdullah 4.92-46.4 nasir (2005) khor abdullah 11.23-26.57 nasir (2007) northwest persian gulf 6.38-13.36 al-imarah et al. (2007) shatt albasrah 0.05-26.44 atti (2014) shatt albasrah 7.45-30.53 current study table 1. phcs concentrations of water in different iraqi aquatic ecosystems. 510 glou et al./ total petroleum hydrocarbons in water, sediment, and redbelly tilapia sediments’ bottom structure with silt and clay has a greater potential to preserve tph. therefore, sediment analysis is a good indicator of the distribution of petroleum hydrocarbons (raja et al., 2022). an increase in the phc during the summer at both stations (31.82 and 37.21 µg/g dry weight, respectively) was recorded, maybe because of irregular oil leaks from industrial areas, the accumulation of oil residues from fishing boats that are active in the summer, municipal sewage water, and surface runoff from the land (al-saad et al., 2017a). water temperature also impacts the speed of organic matter sedimentation and the solubility of hydrocarbons (al-dossari, 2008). the lowest concentration of petroleum hydrocarbons in the sediments was in the fall (6.98 µg/g dry weight), and with the decrease in temperature. tphs gradually increased during the winter at station two due to the reduction in temperature. lower temperatures reduce the evaporation process, photo-oxidation, and biodegradation by microorganisms in the sediments, which can oxidize more than 70% of the crude oil in roughly 18 days (atlas and bartha, 1973). moreover, the higher phytoplankton mortality rates during the winter will result in higher organic matter levels in sediments, increasing the proportion of hydrocarbons that are adsorbed to the surface of these sediments (al-khafaji, 2007). petroleum hydrocarbons were found in all fish samples. there was a correlation between thc in the organisms and their surrounding water and sediments (clinton et al., 2009). the phcs in c. zillii muscles range from 3.87-57.5 µg/g dry weight; however, it was 2.545 to 48.16 µg/g dry weight in other iraqi waterbodies (table 3). differences in phc are found based on the seasons and fish species, e.g. in summer and winter seasons, tenualosa ilisha had the highest phc (6.85 and 7.65 g/g dry weight, respectively), whereas in summer and the winter, euryglossa orientalis had the lowest thc concentration (2.45 and 2.64 g/g dry weight, respectively) (al-ali et al., 2016). there is a significant fluctuation in the tphs of fish between the seasons (ansari et al., 2012). albaidani (2014) indicated that the abundance of phytoplankton, the primary food for fish, causes high tphs during the spring season. additionally, physical and chemical factors of water bodies affect the composition and spread of petroleum compounds (akaahan et al., 2014). due to their feeding habits, some fish have higher phcs in their muscle (veerasingam et al., 2011). nasir (2007) explained that the variations in thc concentrations in fish muscles in different seasons are caused by the influence of environmental factors, nutrition, type region tphs µg.l-1 references khor abdullah 34-192 nasir (2005) shat al-arab 2.3-50.2 hantoush (2006) north-west persian gulf 34.37-66.02 nasir (2005) iraqi coast regions 2.39-30.88 al-khion (2012) shat al-arab 4.76-45.24 al-hejuje (2014) north-west persian gulf 19.43-49.09 al-saad et al. (2017a) shatt al-basrah 6.98-57.5 current study table 2. phcs concentrations in sediments of different iraqi aquatic ecosystems. region tphs µg/g dry weight references shat al-arab 29.6-45.9 al-saad (1989) khor alzubair 8.3-40.6 al-saad (1990) shat al-arab estuary and northwest persian gulf 1.7-10.91 al-saad et al. (1997) shat al-arab estuary and northwest persian gulf 2.55-26 hantoush et al. (2001) iraqi marine waters 11.44-48.16 nasir (2007) north-west persian gulf 2.545-7.25 al-ali et al. (2016) shatt al-basrah 3.87-57.5 current study table 3. phcs concentrations in fishes of different iraqi aquatic ecosystems. 511 int. j. aquat. biol. (2022) 10(6): 504-514 and amount of available food, and the breeding season. chronic exposure to phcs causes physiological and histopathological alternations at all life stages of fish (rodrigues et al., 2010). the primary threat posed by these pollutants is accumulating in the tissues of aquatic species and then transferring to humans (wang et al., 2021). thus, phcs in fish tissues more than the normal level significantly negatively impact human health (lindén and pålsson, 2013). lipids had significant differences between the two studied stations at most seasons. the values in the first station were 3.41, 5.12, 5.61 and 9.90%, respectively (fig. 6) and4.61,7.80, 7.99 and 9.51% in the second station (fig. 7). the present results were higher than previous studies because the fish of the shatt al-basra canal are exposed to oil pollutants continuously. increasing the hydrocarbon levels in fish could be related to the high concentration of lipids in the fish muscle tissue (shriadah, 2001). the amount and nature of fat vary in different fish species (suzuki, 1981). there is a direct correlation between thcs ratio and fat percentage (jamoussi et al., 2022) i.e. fatty parts of fish possess higher concentrations of organic pollutants (shriadah, 2001). ansari et al. (2012) observed a decrease in the level of tph in the tissues of lean fish. feed type is also responsible for fish muscles’ high and low-fat content (tolosa et al., 1996). in addition, many factors, such as the exposure time, the lipid content of tissues, environmental conditions, age, and sex, might affect the accumulation of phs in fish (froehner epossess11). in conclusion, there were significant differences in the tphs levels of fish and their surrounding environment. tphs concentrations in fish samples are based on the expected levels of the tph; therefore, tilapia can be a good indicator to assess tph pollution in aquatic systems. acknowledgments the authors thank the fisheries and marine resources department and marine center laboratories. references abha s., singh c.s. 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(2017) 5(6): 413-424doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article composition and structure of phytoplankton community in ouémé river basin, republic of benin arsène mathieu houssou*1, 2, elie montchowui1, 2, clément agossou bonou3 1laboratoire de recherche en aquaculture et en biologie et ecologie aquatiques, ecole d’aquaculture de vallée, université nationale d’agriculture, bénin. 2laboratoire d’hydrobiologie et d’aquaculture, faculté des sciences agronomiques, université d’abomey-calavi, bénin. 3laboratoire de recherche en biologie appliquée, ecole polytechnique d’abomey-calavi, université d’abomey-calavi, bénin. article history: received 31 july 2017 accepted 2 december 2017 available online 2 5 december 2017 keywords: anthropogenic impact diversity potamoplankton specific composition upstream-downstream gradient abstract: this study aimed to assess the composition and structure of floating phytoplankton assemblage in ouémé basin. phytoplankton samples were collected monthly from october 2014 to september 2015. quantitative samples were taken with a horizontal van-dorn sampler and 20 μm mesh plankton net was used for additional qualitative sampling. microscopic observation of phytoplankton allowed identification of 208 species including 70 bacillariophyta species, 58 chlorophyta species, 24 charophyta species, 21 euglenophyta species, 18 cyanophytes species, 9 phyrrophyta species, 5 ochrophyta species and 3 cryptophyta species. the shannon diversity index varied from 2.4 bit.ind-1 and 3.1 bit.ind-1 showing a relatively good diversification of the community. the population appears largely dominated by 14 species which represent 83.8% of the total phytoplankton. aulacoseira granulata and euglena gracilis were the most predominant species with respectively 40.17% and 15.91% relative abundance. regarding the horizontal pattern of phytoplankton abundance, downstream stations have the greatest abundances. so, the results suggest that downstream stations are richer in phytoplankton which structure differs from that in upper stations. introduction phytoplankton in aquatic environments is an important resource due to maintaining of the food chain and consequently the maintenance of the ecosystem functioning. sterner and elser (2002) and twiss et al. (2010) reported that suspended phytoplankton is highly used in the food chain as a rich source of nitrogen and phosphorus relative to macroalgae, macrophytes and detritus. phytoplankton studies in africa, particularly in rivers, still very poor. african potamoplankton is therefore poorly known, whereas silva et al. (2001, 2005) reported that it is specifically very rich. potamophytoplankton is sensitive to physicochemicals factors, climatic factors and river current, and its study appear necessary if needing it as ecological indicator. the unidirectional current imposes a major constraint on the maintenance of its population. since water is continually transported in *corresponding author: arsène mathieu houssou doi: https://doi.org/10.22034/ijab.v5i6.327 e-mail address: arsnehous@yahoo.fr downstream, continuous supply of phytoplankton inoculum is necessary (reynolds, 2000). therefore, perennial population is dominated by species which can react rapidly, integrating the short water retention time in the river (kilham et al., 1986; reynolds, 2000). dominance by one or only a few numbers of species may therefore be observed (quiblier et al., 2008). these species, depending on population structure and control factors, may be used in ecosystem bio-monitoring (tavassi et al., 2008). to date, ouémé river’s potamophytoplankton remains little known; while this river is one of the biggest one in west africa. its ecosystem comprised of much diversified habitats allowing a rich biological community. in different areas, the river receives various substances (domestic, agricultural, industrial, etc.), which doubtless leads to its enrichment in nitrogenous and phosphorus elements. phytoplankton in this ecosystem would therefore be very rich and 414 houssou et al./ phytoplankton assessment in ouémé basin diversified. the present study is intended to be a first comprehensive assessment of phytoplankton throughout the ouémé basin. according to smayda (1980), the specific composition of the phytoplankton communities, the diversity and dominance of one population in relation to another are all evolving characters and phenomena characterizing succession in the community. the study therefore proposed to evaluate these aspects for the suspended phytoplankton community in ouémé river basin. materials and methods study area and sampling sites: the study was carried out in ouémé river basin, which is the longest and largest catchment area in benin. it is long about 510 km and its catchment (fig. 1) extends between 6°51' and 10°11' north latitude and 1°29' and 3°24' east longitude. it covers an area equivalent to half of benin territory (i.e. more than 50000 km²). a total of fifteen stations (fig. 1, table 1) were sampled. these are representative of both the river course and its main tributaries (okpara, zou, beffa and donga rivers). nine stations were retained on the river. the stations of affon, bétérou, atchakpabéthel, atchakpa-rejet (wastewater discharge point of the “sucrerie de complant du bénin (sucobe)” and atchakpa-pompage (water pumping point of sucobe) were selected to represent the upper course of the river. the three stations in atchakpa are also representative of the direct effects of sucobe on the ouémé river. the lower course was represented by stations such as bétékoukou (dassa), zagnanado, bonou and agonlon-lowé. the last two stations represented the deltaic zone of the basin (in downstream). six stations were chosen on the selected tributaries. the kpassa hydraulic dam and the kaboua station were representative of okpara river. toué and atchérigbé were retained on zou river while vossa (ouessè) and donga were chosen respectively on beffa river and donga river. phytoplankton sampling and processing: phytoplankton is sampled monthly at each of the fifteen stations between october 2014 and september 2015. the sampling protocol in great rivers applicable to the european water framework directive (laplace-treyture et al., 2010) has been used. quantitative samples were taken from the first meter of depth using a van dorn horizontal sampler (2 liters). at each station, three samples at three different points (horizontal plane) of 2 l each were taken. they were then mixed and lugol iodine (8 drops per 100 ml of sample) was added (druart and rimet, 2008). the mixture was packaged in polyethylene bottle and allowed to sediment for 24 hours (shadow). then, it was concentrated by removing water to have 100 ml of sample. additional fixation was done using 5% formalin (laplacetreyture et al., 2010). a complementary sample with a qualitative aim was also taken using 20 μm mesh plankton net. the samples were processed in a laboratory under light microscope. phytoplankton species were identified using guides and specific descriptive works such as prescott (1954), compère (1974 and 1975), vanlandingham (1982), nogueira and correia (2000), tsukii (2005), kinross (2007), bellinger and sigee figure 1. location of sampling sites. 415 int. j. aquat. biol. (2017) 5(6): 413-424 (2010), oyadomari (2011) and simic et al. (2014). a four-grid counting cell (burker turk) was used for cells enumeration for each identified species. the current name of each identified species was searched in algaebase, the global algae information database (guiry and guiry, 2016). the systematic classification of algaebase was thus used. minimum of 400 cells of each identified species were counted. in case of very abundant species (more than 400 cells in 1 ml of sample), they were counted in three consecutive 1 ml sub-samples. rare species were enumerated in the whole sample volume (houssou et al., 2016). during counting, only cells with an integral structure were taken into account (houssou et al., 2015). the phytoplankton density per liter of river water was then estimated using the equation below (eq1). eq1: 𝐷 = 1 6 ( 𝑁 𝑇𝑑 ∗ 100) where d is density of the species per liter of river water. n is the number of cells counted and td is the rate of sample volume corresponding to n. data analysis: the specific composition of phytoplankton in the study area was evaluated and explored with the occurrence frequency (f). the frequency was calculated according to equation (eq2). it allowed the assessment of species constancy in a given environment (dajoz 2000). depending on f value, three groups of species are distinguished: i-) constant species (f ≥ 50%); ii-) accessory species (25% ≤ f <50%) and iii-) incidental species (f <25%). the community structure was studied through the alpha and beta diversity indices. the shannon diversity index (eq3), the evenness (eq4), the margalef index (eq5) and the dominance index y (eq6) were calculated. also, spatial similarity of the zooplankton assemblage was studied with jaccard index (eq7). eq2 (dajoz, 2000): f=(µi x 100)/µt, eq3 (shannon and wiener, 1949): 𝐻′ = − ∑( 𝑛𝑖 𝑁 ) log 2 ( 𝑛𝑖 𝑁 ) 𝑆 𝑖=1 eq4 (buzas and gibson, 1969): 𝐸𝑣𝑒𝑛𝑛𝑒𝑠𝑠 = e𝐻′ s eq5 (margalef, 1958): 𝐷 = s − 1 ln 𝑁 eq6: 𝑌 = ( 𝑛 𝑁 ) 𝑓𝑖 eq7 (jaccard, 1901): nc/ (na+nb-nc) where μ is the number of samples in which species i is present, μt is the total number of samples. s is specific richness, ni is the abundance of species i and n is the total abundance of all species. fi is the frequency of species i in the samples. na and nb are respectively the number of species present in the sites a and b to be compared. nc is the number of common species to both sites. table 1. geographic coordinates of sampling sites. stations code river latitude longitude agonlin-lowé ag-l ouémé river 6°39'35.2"n 2°28'38.6"e bonou bon ouémé river 6°54'32.5"n 2°26'57.1"e zagnanado zag ouémé river 7°12'50.9"n 2°28'26.4"e dassa das ouémé river 7°37'17.0"n 2°27'59.1"e atchakpa-bethel atc-beth ouémé river 8°00'22.9"n 2°22'39.3"e atchakpa-rejet atc-r ouémé river 8°03'38.1"n 2°22'33.8"e atchakpa-pompage atc-p ouémé river 8°04'27.0"n 2°22'12.6"e bétérou bét ouémé river 9°11'55.2"n 2°16'04.6"e affon aff ouémé river 9°57'28.6"n 1°51'45.4"e kpassa kpa okpara river 9°16'59.7"n 2°44'13.4"e kaboua kab okpara river 8°10'49.8"n 2°45'05.5"e toué tou zou river 7°12'22.8"n 2°17'23.3"e atchérigbé atc zou river 7°33'44.8"n 2°07'57.7"e vossa vos beffa river 8°29'34.6"n 2°20'27.1"e donga don donga river 9°42'37.7"n 1°56'41.2"e 416 houssou et al./ phytoplankton assessment in ouémé basin results composition of phytoplankton: the identified phytoplankton community is composed of 208 species (table 2). they belong to 8 phyla such as bacillariophyta with 70 species in 39 genera, chlorophyta with 58 species belonging to 32 genera, charophyta with 24 species in 10 genera, euglenophyta with 21 species belong 6 genera, cyanophyta represented by 18 species in 15 genera, pyrrophyta with 9 species in 7 genera, ochrophyta with 5 species in 5 genera and cryptophytes represented by 3 species belonging to 2 genera. the species occurrence frequency showed that 137 species among the 208 identified are constant in the area (f≥50%). these include species such as microcystis aeruginosa, m. flosaquae, m. protocystis, anabaenopsis circularis (cyanobacteria), aulacoseira ambigua, a. granulata, gomphonema gracile, g. parvulum, cocconeis pellucida, amphora ovalis (bacillariophyta), euglena gracilis and lepocinclis oxyuris (euglenophyta). a set of 15 species are accessory to the area (25≤f<50). these include oscillatoria rubescens, g. vibrio, nitzschia rostellata and stephanodiscus sp.. fifty-five (55) species were accidental in the basin (f<25%). among these are marine species such as gyrodinium sp, prorocentrum denatatum, p. lima, and prorocentrum sp. (toxic dinoflagellates). these species have been found only in the ouémé delta. alpha and beta diversity of phytoplankton community: the phytoplankton specific richness and margalef index are presented in figure 2. higher richness was observed during low flow (from february to july). the temporal highest richness (190 species) was observed in march. the flood period (from october to december) was that of lower specific richness. the lowest richness (118 species) was observed in december. margalef's index had same pattern as the specific richness. it varied between 6.7 observed in december and 10.8 in july. the phytoplankton community was less diversified during the low flow (fig. 3). the smallest shannon index (2.4 bit.cell-1) was observed in march and may. in contrast, the community was more diversified in december (3.1 bit.cell-1). the evenness had same profile as shannon diversity with values ranged from 0.06 to 0.12. the jaccard index (table 3) showed an important similarity between the phytoplankton communities in all stations (j varying between 0.62 and 1). however, the value of the index was higher between the downstream stations on one hand and between upstream stations (lower limit: dassa) on the other hand. value decreases when the communities present in downstream stations are opposite to those present in upstream stations. dominant phytoplankton species: only 14 species including 2 bacillariophyta, 3 euglenophyta, 6 chlorophyta, 2 charophyta and 1 cyanophyta largely dominated the phytoplankton population (table 4). they account for 83.8% of the total phytoplankton abundance in the basin. the two bacillariophyta figure 2. temporal variation of the specific richness and margalef index of phytoplankton community in ouémé basin. figure 3. temporal variation of shannon diversity and evenness index of phytoplankton community in ouémé basin. 417 int. j. aquat. biol. (2017) 5(6): 413-424 table 2. occurrence frequency of identified phytoplankton species in ouémé river basin. phyla genera species occurrence frequency (%) cyanophyta anabaena anabaena oscillarioides 21.1 anabaenopsis anabaenopsis circularis 58.3 aphanocapsa aphanocapsa elegans 8.9 asterocapsa asterocapsa submersa 100 chroococcus chroococcus sp. 0.6 cyanosarcina cyanosarcina thalassia 7.8 dolichospermum dolichospermum spiroides 55 glaucospira glaucospira laxissima 75.6 lyngbya lyngbya sp. 1.1 merismopedia merismopedia glauca 18.3 microcystis microcystis aeruginosa 100 microcystis flosaquae 100 microcystis protocystis 100 oscillatoria oscillatoria rubescens 25 oscillatoria sp. 92.2 stigonema stigonema sp. 76.7 synechocystis synechocystis aquatilis 98.9 tychonema tychonema bornetii 10 bacillariophyta achnanthes achnanthes felinophila 86.7 achnanthidium achnanthidium minutissimum 66.1 amphora amphora ovalis 83.3 amphora sp. 32.8 amphipleura amphipleura sp. 2.2 aulacoseira aulacoseira ambigua 100 aulacoseira granulata 100 caloneis caloneis undulata 100 catenula catenula sp. 77.8 cocconeis cocconeis pellucida 96.7 cocconeis sp. 33.3 coscinodiscus coscinodiscus perforatus 1.1 coscinodiscus radiatus 2.2 coscinodiscus sp. 6.7 cyclotella cyclotella meneghiniana 97.2 cymbella cymbella lanceolata 94.4 cymbella prostrata 65 cymbella sp. 46.7 cymatopleura cymatopleura elliptica 70.6 diatoma diatoma sp. 100 entomoneis entomoneis alata 100 eunotia eunotia bilunaris 100 fragilaria fragilaria acus 23.3 fragilaria capucina 16.7 fragilaria sp. 100 gomphonema gomphonema gracile 100 gomphonema parvulum 100 gomphonema sp. 79.4 gomphonema vibrio 26.7 grammatophora grammatophora sp. 25 gyrosigma gyrosigma acuminatum 100 gyrosigma attenuatum 100 gyrosigma sp. 13.3 gyrosigma strigilis 8.9 hyalodiscus hyalodiscus radiatus 15 hyalodiscus sp. 7.8 418 houssou et al./ phytoplankton assessment in ouémé basin phyla genera species occurrence frequency (%) hyalosynedra hyalosynedra laevigata 1.1 melosira melosira moniliformis 2.2 navicula navicula gregaria 100 navicula peregrina 6.7 neidium neidium sp. 6.7 nitzschia nitzschia reversa 100 nitzschia rostellata 38.9 nitzschia sp. 68.3 nitzschia paradoxa 100 parlibellus parlibellus protractoides 2.2 placoneis placoneis constans 11.1 placoneis gastrum 100 pleurosigma pleurosigma obscurum 100 pinnularia pinnularia gibba 10.6 pinnularia cardinalis var. africana 100 pinnularia sp. 100 pinnularia tabellaria 13.3 pseudo-nitzschia pseudo-nitzschia seriata 2.8 rhizosolenia rhizosolenia setigera 2.2 sellaphora sellaphora pupula 19.4 stephanodiscus stephanodiscus alpinus 100 stephanodiscus hantzschii 100 stephanodiscus sp. 39.4 surirella surirella alata 100 surirella elegans 93.9 surirella capronii 100 surirella linearis 92.8 surirella robusta 100 synedra synedra superba 11.1 tabellaria tabellaria flocculosa 8.3 tabellaria sp. 10 thalassiosira thalassiosira sp. 57.8 ulnaria ulnaria ulna 95.6 urosolenia urosolenia eriensis 8.3 euglénophyta euglenaria euglenaria anabaena 8.3 euglena euglena gracilis 100 euglena sp. 6.7 lepocinclis lepocinclis acus var. longissima 100 lepocinclis oxyuris 96.7 lepocinclis sp. 27.2 phacus phacus helikoides 100 phacus longicauda 88.9 phacus longicauda var. torta 100 phacus orbicularis 93.3 phacus undulatus 75 strombomonas strombomonas acuminata 100 strombomonas confortii 100 strombomonas ferrazii 100 strombomonas fluviatilis 100 strombomonas rotunda 6.7 strombomonas scabra 9.4 strombomonas verrucosa 100 strombomonas verrucosa var. borystheniensis 86.7 trachelomonas trachelomonas acanthophora var. speciosa 13.3 trachelomonas sp. 25 table 2. continued 419 int. j. aquat. biol. (2017) 5(6): 413-424 phyla genera species occurrence frequency (%) charophyta chlorokybus chlorokybus sp. 7.8 closterium closterium acerosum 23.3 closterium acerosum var. tumidum 100 closterium braunii 72.2 closterium gracile 100 closterium parvulum 100 closterium setaceum 54.4 closterium tumidulum 23.3 cosmarium cosmarium botrytis 95 cosmarium contractum 92.8 cosmarium quinarium 2.2 cosmarium reniforme 85 cosmarium sp. 87.8 euastrum euastrum ansatum 65 gonatozygon gonatozygon brebissonii 2.2 klebsormidium klebsormidium sp. 68.3 micrasterias micrasterias fimbriata 4.4 pleurotaenium pleurotaenium ehrenbergii 2.2 staurastrum staurastrum anatinum 100 staurastrum leptocladum f. africanum 100 staurastrum longipes 100 staurastrum natator 100 staurastrum paradoxum var. parvum 100 staurodesmus staurodesmus glaber 100 chlorophyta actinastrum actinastrum hantzschii var. fluviatile 100 actinastrum hantzschii var. subtile 100 acutodesmus acutodesmus acuminatus 100 ankistrodesmus ankistrodesmus densus 100 ankistrodesmus falcatus 100 ankistrodesmus fusiformis 100 chlorogonium chlorogonium sp 10 chodatella chodatella quadriseta 4.4 characium characium oviforme 26.7 cladophora cladophora sp. 100 coelastrum coelastrum astroideum 100 crucigeniella crucigeniella apiculata 86.7 crucigenia crucigenia sp. 31.7 desmodesmus desmodesmus abundans 100 desmodesmus armatus var. bicaudatus 100 desmodesmus communis 100 desmodesmus intermedius 100 desmodesmus intermedius var. balatonicus 100 desmodesmus magnus 100 desmodesmus maximus 100 desmodesmus opoliensis 16.7 desmodesmus opoliensis var. mononensis 83.3 dicloster dicloster acuatus 83.3 eudorina eudorina carteri 100 eudorina sp. 78.3 eremosphaera eremosphaera sp. 1.1 eremosphaera viridis 2.2 lacunastrum lacunastrum gracillimum 23.3 lagerheimia lagerheimia sp. 4.4 micractinium micractinium bornhemiense 100 monactinus monactinus simplex var. echinulatum 92.8 monactinus simplex var. sturmii 100 table 2. continued 420 houssou et al./ phytoplankton assessment in ouémé basin species (a. granulate and a. ambigua) occupy respectively 40.17% and 6.28% of the total population (i.e. 46.45% for both species). euglena gracilis (euglenophyta) was the second most dominant species (15.91%). the dominance index y evolved according to species relative abundance. it varied between 0.40 for the most abundant species (a. granulata) and 0.01 for the least abundant species (acutodesmus acuminatus). the 14 species have a dominance of 0.83 out of a total of 1 for the identified 208 species. discussion the phytoplankton community recorded in ouémé basin is composed of 208 species. this specific phyla genera species occurrence frequency (%) chlorophyta neospongiococcum neospongiococcum sp. 56.7 pachycladella pachycladella zatoriensis 70 pectinodesmus pectinodesmus javanensis 100 pediastrum pediastrum angulosum 100 pediastrum boryanum 100 pediastrum duplex 100 pediastrum kawraiskyi 76.7 pediastrum simplex var. biwaense 100 pediastrum simplex var. duodenarium 100 quadrigula quadrigula lacustris 53.3 scenedesmus scenedesmus quadricaudata var. biornatus 85 scenedesmus obtusus 100 scenedesmus tropicus 100 selenastrum gracile 100 stauridium stauridium privum 86.1 stigeoclonium stigeoclonium aestivale 100 tetradesmus tetradesmus bernardii 93.3 tetradesmus obliquus 66.1 tetrastrum tetrastrum heteracanthum 16.7 tetraëdron tetraëdron incus 62.2 tetraëdron gracile 91.7 tetraëdron triangulare 53.9 tetraëdron trigonum 54.4 tetraspora tetraspora sp. 100 treubaria treubaria quadrispina 66.7 volvox volvox aureus 46.7 pyrrophyta ceratium ceratium carolinianum 70 gyrodinium gyrodinium sp. 5 peridiniopsis peridiniopsis quadridens 83.3 peridinium peridinium bipes 65 prorocentrum prorocentrum denatatum 7.8 prorocentrum lima 2.8 prorocentrum sp. 1.1 pyrocystis pyrocystis sp. 75 scrippsiella scrippsiella trochoidea 43.3 ochrophyta centritractus centritractus africanus 40 dinobryon dinobryon sertularia 92.8 gonyostomum gonyostomum sp. 66.7 tribonema tribonema sp. 75 vaucheria vaucheria sp. 100 cryptophyta campylomonas campylomonas sp. 94.4 cryptomonas cryptomonas ovata 88.9 cryptomonas sp. 86.7 table 2. continued 421 int. j. aquat. biol. (2017) 5(6): 413-424 richness is more or less stable, showing the ecological importance of this ecosystem. the numerous agroecological, industrial and residential areas crossed by the ouémé river and its tributaries justify this specific richness. good mineralization in water due to exogenous inputs, allows many species survival and multiplication in the environment. the observed specific richness is above that reported (89 species) on the kwa river in nigeria (victor et al., 2013) and 192 species on the coastal river in ivory coast (niamien-ebrottié et al., 2013). it is also superior to the specific phytoplankton richness (149 species) observed in the subtropical river of the lower iguaçu in brazil (perbiche-neves et al., 2011). it is below the 265 species of phytoplankton identified in the australian "daly" tropical river (townsend et al., 2012). geographical differences as well as the various levels of anthropization perfectly explain the deviations from these rivers. albert (2010) reported that a species distribution reflected in its geographical space (longitude, latitude), its ecological niche defined in environmental space (climate, soil, resource). so even in the absence of a significant difference in climate, soil and resources in the environment are important factors to the biodiversity composition. compared to african lakes, the phytoplankton richness observed in the ouémé basin is above the 39 species identified in hlan lake (houssou et al., 2016) ag-l bon zag tou atc das atcbéth atc-r atc-p kab vos kpa bét don aff ag-l bon 0.967 zag 0.846 0.867 tou 0.764 0.783 0.893 atc 0.764 0.783 0.893 1 das 0.726 0.744 0.858 0.845 0.845 atc-béth 0.692 0.709 0.818 0.825 0.825 0.954 atc-r 0.692 0.709 0.818 0.825 0.825 0.954 1 atc-p 0.692 0.709 0.808 0.814 0.814 0.941 0.973 0.986 kab 0.649 0.665 0.851 0.74 0.74 0.857 0.898 0.898 0.911 vos 0.668 0.685 0.79 0.784 0.784 0.921 0.965 0.965 0.965 0.916 kpa 0.62 0.635 0.733 0.756 0.756 0.879 0.909 0.909 0.909 0.985 0.914 bét 0.683 0.7 0.807 0.824 0.824 0.94 0.986 0.986 0.986 0.91 0.979 0.908 don 0.63 0.645 0.744 0.779 0.779 0.88 0.923 0.923 0.923 0.834 0.915 0.857 0.936 aff 0.683 0.7 0.807 0.813 0.813 0.94 0.986 0.986 0.972 0.91 0.979 0.908 1 0.936 sampling sites codes are same as in table 1 table 3. jaccard similarity between the phytoplankton communities of ouémé river basin species mean abundance (x104.cell.l-1) relative abundance (%) dominance y aulacoseira granulata bacil 52.82 40.17 0.40 euglena gracilis eug 20.92 15.91 0.16 aulacoseira ambigua bacil 8.25 6.28 0.06 lepocinclis oxyuris eug 5.21 3.96 0.04 pediastrum duplex chlo 3.16 2.40 0.02 pediastrum angulosum chlo 2.94 2.24 0.02 desmodesmus intermedius var. balatonicus chlo 2.85 2.17 0.02 staurastrum leptocladum cf. africanum charo 2.76 2.10 0.02 microcystis aeruginosa cyano 2.56 1.94 0.02 desmodesmus intermedius chlo 1.99 1.51 0.02 ankistrodesmus densus chlo 1.95 1.48 0.01 cosmarium botrytis charo 1.84 1.40 0.01 phacus longicauda eug 1.53 1.16 0.01 acutodesmus acuminatus chlo 1.39 1.06 0.01 total 83.77 0.83 table 4. list of dominant phytoplankton species in ouémé basin. 422 houssou et al./ phytoplankton assessment in ouémé basin and 51 species in azili lake in benin (houssou et al., 2015); these two lakes being strongly influenced by the overflows of ouémé river. the richness of 111 species of lake guiers in senegal (ngansoumana, 2006) is also smaller than that of ouémé. the phytoplankton community in ouémé basin was during low flow less diversified than during the flood period. the low flow period was that during which phytoplankton is greatly multiplied. this followed the reduction or even the cancellation of the river flow. weak nutrient diluted associated with high sun exposure have been major factors which increased phytoplankton development. all species have experienced significant population growth which has raised the specific richness. therefore, rarest species are sampled. margalef's specific index confirms the profile observed in the specific richness of the basin. the shannon index and evenness indicated a relatively good diversification of the phytoplankton community (chen et al., 1994; sun et al., 2004). however, few species number is dominant during low flow season. this confirms the high mineralization during this season. regarding the dominance, diatom a. granulata was more predominant (>40% of the population). this fact confirms observations in which diatoms are dominant in terms of abundance in tropical rivers with a predominance of a. granulata and other species of the same genera (hötzel and croome, 1996; decy et al., 2017). according to reynolds (2000) and decy et al. (2017), these diatoms are typically of the r strategy. they are able to withstand the nutritional variability associated with variations in water flows and able in achieving net growth within short time imposed by downstream transport. this justifies the dominance of the species even in upstream stations where the river current is more or less continuous throughout the year. kilham et al. (1986) qualified species of the genus aulacoseira as species adapted to low light conditions. chlorophycea species such as those of the genus ankistrodesmus, desmodesmus and pediastrum were also included in the dominant species. this group of species could become predominant in the case of good light penetration in the river (zalocar de domitrovic et al., 2014). euglena gracilis and lepocinclis oxyuris were also dominant. these two species are known for their selectivity of eutrophic environment, the anthropic impact in the basin then explains their abundance. as regards the similarity between phytoplankton communities in the different sampling stations, a horizontal stratification was observed. the community structure in the three stations in the delta area is similar and clearly differs from all other stations. this confirms the upstream-downstream gradient of mineralization in the basin. in addition to direct exogenous inputs, these stations also receive all substance or particle that is transported by the current making nutrients available for habitats variability in the area. it is also observed that community present in the stations from dassa to affon and then on beffa and okpara rivers are form equivalent. conclusion the floating phytoplankton assemblage in the ouémé basin is composed of 208 species grouped into 8 phyla: bacillariophyta, chlorophyta, charophyta, euglenophyta, cyanophyta, pyrrophyta, ochrophyta and cryptophyta. the population is relatively well diversified with lowest diversity during low flow. fourteen (14) species are dominant with more than 83% of the total phytoplankton population. a. granulata is the most predominant species. other species such as e. gracilis, a. ambigua and l. oxyuris are also strongly represented. it was also observed an ecological difference between ouémé delta and all other parts in the basin. acknowledgement we are grateful to the west africa agricultural productivity program (waapp) which funded this research. we thank all those who have contributed in one way or another to this study. we also thank the reviewers for their comments and contributions. references albert c. 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(2019) 7(1): 56-64 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article biochemical and physiological effect of dietary supplements of zno nanoparticles on common carp (cyprinus carpio) mahdi banaee*1, maryam vaziriyan1, azam derikvandy2, behzad nematdoost haghi1, mohammad mohiseni1 1department of aquaculture, faculty of natural resources and environment, behbahan khatam al-anbia university of technology, iran. 2department of environment, faculty of natural resources and environment, behbahan khatam alanbia university of technology, iran. s article history: received 16 december 2018 accepted 23 february 2019 available online 2 5 february 2019 keywords: common carp dietary exposure zno-nanoparticle plasma abstract: zno-nps, like other macromolecule sources, may provide the fish with sufficient amounts of zinc and be effective in regulating the biochemical function of cells in organisms. this study aimed to assess the possibility of using nanoparticles in common carp diet by evaluating alterations in blood biochemical parameters, as a clinical marker of fish health. in this study, fish were fed diets supplemented with 0 (control), 5, 10 and 15 mg kg-1 zno-nps for 21 days. the results showed that after 21 days admiration of zno-nps, 10 and 15 mg kg-1 concentrations significantly increased aspartate aminotransferase (ast) activity and glucose, cholesterol, triglyceride and creatinine levels in plasma of fish (p<0.05). also, the administration of 15 mg kg-1 zno-nps significantly (p<0.05) increased alanine aminotransferase (alt), lactate dehydrogenase (ldh) and alkaline phosphatase (alp) activities. no significant changes were observed in plasma total protein, albumin and globulin levels (p>0.05). in conclusion, the results showed that diets containing high concentrations of znonps supplement (10 and 15 mg) is caused severe cytotoxic effects, including changes in blood biochemical parameters. the primary toxic mechanism of zno-nps was possibly increasing the cellular oxidative stress and disrupting the biochemical function of cells. introduction zinc (zn) is the second trace element in organisms and cannot be stored in the body (kuma et al., 2018), therefore it needs to be provided regularly through the diet for physiological activities. the importance of zinc in fish and crustaceans health has been verified (olmedo et al., 2013; ma et al., 2014). also, it is a significant component of many enzymes, the cellular antioxidant system and hormones, and an essential element for many physiological functions (taheri et al., 2017; domínguez et al., 2019). zinc is involved in the natural growth, reproduction (uriu-adams and keen, 2010), regulating the reverse transcription and dna synthesis, cellular division and gene expression (swain et al., 2016; frassinetti et al., 2006), photochemical processes of the visual system (swain et al., 2016), recovering injuries (frassinetti et al., 2006), bone formation (swain et al., 2016), increasing the immune system (gharekhani et al., 2015; *correspondence author: mahdi banaee doi: https://doi.org/10.22034/ijab.v7i1.590 e-mail: mahdibanaee@yahoo.com beitsayah et al., 2019) through energy production, protein synthesis, protecting cell membranes against bacterial endotoxin and increasing the lymphocytes proliferation rate and producing antibodies (swain et al., 2016; velazquez-carriles et al., 2018). zinc absorption is very variable in different species of fish and differs depending on the organisms’ age, place of absorption in the digestive tract, and individual needs. zinc in diet can be found as mineral salts such as zinc oxide or zinc sulfate or as organic chelators, including zinc propionate and zinc acetate. although the bioavailability of inorganic zinc sources is more than inorganic zinc salts, the high cost of providing organic forms of zinc chelate has limited their use in the diet of the organism (frassinetti et al., 2006). in commercial diets of farmed fish species, the amount of zinc is proportional to the fish needs; however, the bioavailability of zinc in feedstuffs is usually low (davis and gatlin, 1996; bilandžić et al., 57 int. j. aquat. biol. (2019) 7(1): 56-64 2014). therefore, using the additional zinc in nutritional supplements (100 to 150 mg zn per kg feed) may overcome the inhibitory effect of compounds such as calcium phosphate in fish meal and phytate or phytic acid in soybean meal, and other oilseeds and grains (davis and gatlin, 1996; ma et al., 2014; bilandžić et al., 2014). the increased excretion of zinc in animals treated with zinc-supplemented diets has increased concerns about environmental pollutions (musharraf and khan, 2019). therefore, researchers try to find a source of zinc with a higher bioavailability to reduce zinc levels in food supplements for animals. among all possible strategies, using nanotechnology to produce nanoparticles could be a potential alternative for both organic and inorganic zinc sources. using zinc nanoparticles compared to conventional zn sources, as well as zn nanoparticles are more efficient and less toxic (taheri et al., 2017). due to their small size, zinc oxide nanoparticles (zno nps) can easily be absorbed by the digestive tract and are more effective in lower concentrations compared to the conventional zno (chupani et al., 2018). in the organisms’ body, mineral nanoparticles interact more effectively with organic and inorganic materials which are due to their larger surface area (chupani et al., 2018). the effect of zno nps on improving the growth, as well as enhancing the efficiency of the consumed feed and economic sources produced in farms for different species of farmed animals are reported (hongfu, 2008; lin et al., 2009; mishra et al., 2014). the results of studies show that administration of low concentration of nano-zinc oxide in enhancing the growth rate of different farmed animals can have similar results as administration of high concentrations of zno (macromolecules). therefore, this can be one of the benefits of zno nps (hongfu, 2008; taheri et al., 2017). since the physiological function of zn is influenced by its mode of transfer and storage in the aquaculture (bilandžić et al., 2014), using zinc supplement as nanoparticles can affect the physiological indicators and fish health. there is little information on the adverse effects of zinc oxide nanoparticles (nps) on farm animals such as fish (connolly et al., 2016). in most cases, the base of toxicological studies on metal nps is fish exposure with the soluble phase of nps (chupani et al., 2017, 2018; dekani et al., 2019) and there is still a lot to learn about the toxicology and potential hazards of different doses of zinc oxide nps in foodstuff of fish (swain et al., 2016). meanwhile, depending on the concentration, way, and duration of exposure, the cytotoxicity of zinc oxide nps can lead to oxidative stress (wang et al., 2014), lipid peroxidation, damage to cell membranes, and oxidative damage to dna (dekani et al., 2019). studies conducted so far are on the oral administration of zinc oxide nps 300 and 1000 mg kg-1 feed (connolly et al., 2016) and zinc oxide nps 30, 50, 100 and 500 mg kg-1 feed (chupani et al., 2018; dekani et al., 2019) in fish. therefore, investigating the probable toxicity of oral administration of these nps in lower doses seems necessary. therefore, this study aimed to evaluate oral administration of zinc nanoparticles on biochemical factors of blood, as the fish health assessment index, and investigating the possibility of using zinc nanoparticles in the feedstuff of common carp, cyprinus carpio. materials and methods fish: one hundred forty four immature common carp (mean weight: 20.5±2.5 g) were obtained from a local fish farm (ahvaz, khuzestan province, iran) and randomly distributed into 12 circular tanks of 80 l capacity (12 fish per each tank) at the department of aquaculture (khatam alanbia university of technology). the experiment was conducted following the national ethical framework for animal research in iran (mobasher et al., 2008). before the experiment, fish were acclimated in aerated freshwater (24±2°c; ph, 7.4±0.2; 50% water exchange rate/day) for two weeks. the fish were subjected to artificial light (16l/8d). during the acclimatization period, fish were fed with commercial pelleted feed (beyza feed mill, shiraz, iran) by the manufacturer’s recommendations. diet preparation: since oral administration of 50 and 1000 mg zinc oxide nps to common carp and rainbow trout proved to have adverse effects (connolly et al., 58 banaee and vaziriyan/ oral exposure of zno-nanoparticles on plasma biochemical parameters of carp 2016; chupani et al., 2017), this study used 5, 10 and 15 mg kg-1 zinc oxide nps which might have lower toxicity. the formulated fish feed was enriched with nanoparticles of zinc oxide (dekani et al., 2019). nanoparticles of zinc oxide were used at 5, 10 and 15 mg per kg feed for a total of three treatments. commercial zno nanoparticles, with an average primary particle size of 50 nm in the powder form, were purchased from iranian nano-materials pioneers company, iran (table 1). the tem, sem micrographs and the x-ray powder diffraction (xrd) curves of nano-crystalline zno are presented in figures 1-3. zno nanoparticles were prepared using distilled water and then ultrasonicated (10 min, 35 khz, 100/400w) using an ultrasound bath (elma, germany). then, solutions were added to powdered feed to obtain nominal concentrations of 5, 10 and 15 mg zno nps per kg. each supplemented diet was mixed in a mixer for 30 minutes and then homogenized into a paste by adding fish oil (20 ml kg-1) and distilled water into the food mixer. the amount of distilled water required for pelleting (20table 1. zinc oxide nanoparticles physicochemical proprieties, according to the iranian nano-materials pioneer’s manufacturer. zinc oxide zno purity +99.9 % average primary particle size (d50) 10-30 nm specific surface area (ssa) 60 m2 g-1 color white bulk density 5.606 g cm-3 figure 3. tem micrographs of the nano-zno powders (adapted from iranian nano-materials pioneers company’s catalog). figure 2. sem micrographs of the nano-zno powders (adapted from iranian nano-materials pioneers company’s catalog). figure 3. the x-ray powder diffraction (xrd) curves of nanocrystalline zno (adapted from iranian nano-materials pioneers company’s catalog). 59 int. j. aquat. biol. (2019) 7(1): 56-64 40% of feed weight) was then added to the mixture and further homogenized. this mixture was passed through a meat grinder, producing string shapes, which were dried in an oven at 55°c for 12 h and then broken to produce 10 mm long pellets. the pellets were packed and stored at -20°c in a freezer. the control diet was prepared by the same process, although no supplement was added. experimental design: during the experimental period, fish fed commercial pelleted feed enriched with 0 (control), 5, 10 and 15 mg kg-1 nanoparticles of zinc oxide supplement following the manufacturer’s recommendations for three weeks. at the end of the experiment, 12 fish per treatment were captured using a scoop net and anesthetized with clove powder solution (1:5,000). anesthetized fish were bled from the caudal artery/vein using 2 ml heparinized syringes. the collected blood was transferred into 2 ml microcentrifuge tubes. the blood sample was centrifuged for 15 min at 6000 g at 4°c. plasma samples were immediately stored at -25°c before biochemical analysis. sampling and analysis of blood biochemical parameters: all blood biochemical parameters were determined using a uv-visible spectrophotometer (unico 2100) and standard biochemical reagents (pars azmun company, tehran, iran). each biochemical blood parameter was measured by a particular method. total plasma protein concentration was measured at 540 nm by the biuret reaction. the albumin assay is based on the dye-binding properties of plasma albumin with a bromocresol green. an increase in the blue-green color was measured at 630 nm. the plasma globulin was calculated based on the ratio of albumin to total protein (johnson et al., 1999). plasma glucose was measured by the glucose-oxidase method at 500 nm (sacks, 1999). plasma cholesterol levels were measured by the chod-pap enzymatic method at 510 nm, triglyceride levels by gpo-pap enzymatic method at 546 nm (rifai et al., 1999) and creatinine by the jaffe method at 510 nm (fosterswanson et al., 1994). the activity of aspartate aminotransferase (ast) and alanine aminotransferase (alt) in plasma was determined by nadph consumption and its conversion to nad+ at 340 nm. lactate dehydrogenase (ldh) in plasma was determined based on the conversion of pyruvate to lactate at 340 nm, alkaline phosphatase (alp) based on converting nitrophenol phosphate into nitrophenol and phosphate at 405 nm, and based on optical density (od) absorption and the formula presented in the kits' manual (moss and henderson, 1999). data analysis: the significant difference in the biochemical parameters of fish fed enrich diet with different concentrations of zno nanoparticles was examined using one-way anova. all data were checked for normality (kolmogorov-smirnov test). means were compared by duncan’s test and a p<0.05 was considered statistically significant. statistical analyses were performed using spss (ibm, 19) software. data are presented as mean (sd). results during the experiment, mortality was not observed in the control group, and fish fed zno nps supplement. on day 21 of the experiment, the results indicated that ast activity at 10 mg kg-1 and 15 mg kg-1 zno nps supplement increased compared to the control (p<0.05). the findings demonstrated that alt, ldh, and alp activities statistically increased in the plasma of fish fed with 15 mg kg-1 zno nps supplement compared to the control group (p<0.05). however, alt, ldh and alp activities on day 21 of the study did not show any significant difference in fish fed with 5 mg kg-1 and 10 mg kg-1 zno nps supplement compared to the control group (p>0.05) (fig. 4). on day 21, in all the groups fed with zno nps supplement showed no significant differences in plasma total protein, albumin and globulin levels compared to the control group (p>0.05). on day 21, a statistically significant increase in plasma glucose, cholesterol, triglyceride, and creatinine levels was seen compared to the control group (p>0.05) in fish fed with 10 mg kg-1 and 15 mg kg-1 zno nps supplement (table 2). discussions the required zinc concentration in a feed of farmed 60 banaee and vaziriyan/ oral exposure of zno-nanoparticles on plasma biochemical parameters of carp common carp is between 15-30 mg per kg feed (davis and gatlin, 1996; wang and wang, 2015). apparently, zinc content (as mineral salts such as zinc oxide or zinc sulfate) in the formulated diet of common carp and produced in most factories of aquaculture feed in iran is 30 mg per kg feed; however, due to the use of oilseeds in the base diet, the bioavailability of zinc may reduce for fish (ma et al., 2014). that is why this study investigates the possibility of using zno nps in foodstuff of common carp to prevent zinc deficiency in the long term. this study aimed at evaluating the blood biochemical parameters, as a general health indicator, in common carp, treated with zno nps in 5, 10 and 15 mg kg-1 feed in a 21-day experiment. as far as we know, there are few studies on oral administration of zinc oxide nps in fish (connolly et al., 2016; chupani et al., 2018; dekani et al., 2019). evidence suggests that diets containing zinc oxide nps could provide a path to transfer this compound to upper levels of the food chains in aquatic organisms such as fish. therefore, adding zinc oxide nps may be useful to guarantee fish needs to zinc. however, it can have several potential complications, such as being intoxicated with zinc oxide nps. therefore, this study aimed at investigating the effects of oral table 2. alterations in the blood biochemical parameters of common carp, cprinus carpio oral exposure to zno nanoparticles. biochemical parameters concentrations of zno nano-particles (mg) per 1 kg feed 0.0 5.0 10.0 15.0 total protein 4.6±0.5b 4.2±0.5b 4.4±0.4b 3.8±0.4a albumin 2.8±0.5a 2.7±0.3a 2.7±0.4a 3.2±0.4b globulin 1.7±0.3b 1.5±0.4b 1.8±0.4b 0.7±0.2a cholesterol 193.9±22.0a 180.9±30.2a 260.6±25.9b 244.0±30.3b triglycerides 281.6±54.8a 354.9±51.7b 446.9±52.0c 375.6±44.1b glucose 41.1±3.5a 89.7±21.6b 89.8±5.9b 95.7±12.0b creatinine 0.2±0.1a 0.2±0.0a 0.4±0.1b 0.5±0.1c significant differences between values when compared with control groups were characterized by alphabet symbol (p<0.05). values represent mean ±s.d. figure 4. changes in the enzyme activities in plasma of fish with oral exposure to zno nanoparticles (significant differences between values when compared with control groups were characterized by alphabet symbol (p<0.05). values represent mean±s.d). 61 int. j. aquat. biol. (2019) 7(1): 56-64 administration of zinc oxide nps on specific blood biochemical parameters in common carp. in this study, oral administration of 5, 10 and 15 mg kg-1 zno nps did not cause any mortality during the experiment which indicates that zno nps (< 15 mg per kg feed) were not acutely toxic to the survival of common carps. ma et al. (2014) and connolly et al. (2016) also showed low toxicity of zno nps to scophthalmus maximus and oncorhynchus mykiss. in the present study, we analyzed changes in blood biochemical parameters of common carp. ast, alt, ldh, and alp are found in cells of different organs such as the heart, kidneys, liver, skeletal muscles, brain, intestine, and gills as well as erythrocytes. the release of intercellular enzymes into the blood and their increased activity in plasma are the most important clinical signs in diagnosing damage to cell membranes (rezaei shadegan and banaee, 2018; hatami et al., 2019; banaee et al., 2019). ast activity significantly increased in plasma of fish fed diets containing 10 mg kg-1 and 15 mg kg-1 zno nps, whereas its activity remained near the control level in fish fed with 5 mg kg-1 zno nps. thus, oral administration of more than 5 mg kg-1 zno nps increased the plasma ast activity which may reflect damage to liver tissue. zno nps may indirectly cause oxidative stress and damage hepatocytes (dekani et al., 2019). moreover, the results indicate the increasing probability of oxidative stress with increased concentration of zno nps in the diet. previous studies show that oral exposure to zno nps caused a significant increase in ast activity in plasma (fazilati, 2013; chupani et al., 2018). an increase in ast and alt in the liver, kidney, and blood of fish treated with zno nps is reported (taheri et al., 2017; chupani et al., 2018; dekani et al., 2019). the activities of increased alt, ldh, and alp in plasma of common carp significantly increases (p<0.05) indicating that liver damage might be induced by high levels of zno nps (15 mg kg-1) in the diet. the increased activities of ldh and alp in plasma of fish fed with 15 mg kg-1 zno nps may confirm the effect of zno nps supplements on the cellular metabolic functions. however, plasma alt, ldh and alp activities were not affected in fish fed with 5 and 10 mg kg-1 zno nps compared to the control group. therefore, due to antiradical and antioxidant properties of zno nps, its administration at doses lower than 15 mg kg-1 might prevent lipid peroxidation of cell membranes and inhibit the release of the enzymes described above into the plasma (swain et al., 2016). our findings support the previous results of sharma et al. (2012), fazilati, (2013), najafzadeh et al. (2013), and ansari et al. (2015). these authors observed a significant increase in the activities of liver enzymes in the blood of mice after oral exposure to zno nps. changes in biochemical parameters such as glucose, total protein, albumin, globulin, creatinine, cholesterol, and triglyceride are indices of the physiological functions in different organs, including the liver, kidneys, intestine, and gills of fish. therefore, any alterations in these clinical indices may indicate physiological disorders (ahmadi et al., 2014; nematdoost haghi and banaee, 2017). although more than 60% of zn binds albumin and is transported in the blood (suttle, 2010), the present study shows that diets containing zno nps have no obvious effects on total protein, albumin and globulin levels in plasma of common carp. the results also showed that administration of zno nps supplement had no significant effect on the plasma total protein, albumin and globulin levels. sobhanirad and naserian (2012) found that zinc supplement administration may have no increasing or decreasing effects on protein synthesis in the liver or total protein, albumin and globulin level (sobhanirada and naserian, 2012). our results also showed that 10 and 15 mg kg-1 zno nps in the diet caused a significant increase in plasma glucose levels in plasma of common carp. wijesekara et al. (2009), and chabosseau and rutter, (2016) found that zinc plays an important in insulin biosynthesis and secretion, and is concentrated in the pancreas. thus, adequate zn is essential for the regulation of blood glucose (wijesekara et al., 2009; chabosseau and rutter, 2016; olechnowicz et al., 2018). however, administration of high doses of zn in 62 banaee and vaziriyan/ oral exposure of zno-nanoparticles on plasma biochemical parameters of carp the diet can be toxic and increase blood glucose levels (wijesekara et al., 2009). moreover, an increase in blood glucose of fish may reflect an increased need for energy to counteract the effects of stress caused by zno nps toxicity. hyperglycemia or elevated blood glucose levels indicate impaired glucose uptake and lipid metabolism and degradation of glycogen in liver (banaee et al., 2019). the results of this study showed that the administration of 10 and 15 mg kg-1 zno nps had a significant effect on cholesterol and triglyceride levels in plasma of common carp. high doses of zinc in the foodstuff may increase cholesterol level by decreasing hdl-cholesterol and apolipoprotein (apo) a-1 (foster et al., 2010; olechnowicz et al., 2018). a decrease in plasma cu due to oral consumption of zinc may increase blood cholesterol level. cu deficiency increases the activity of hmg coa reductase and consequently increases cholesterol concentration (foster et al., 2010). zinc is effective in lipids catabolism and therefore in providing energy from stored fat (olechnowicz et al., 2018). a significant increase in plasma triglyceride in fish fed diets containing zno nps may be the result of a disturbance in lipoproteins biosynthesis, increased rate of lipids catabolism, severe liver and kidney damage, and an increased rate of cell membrane lipid peroxidation. degradation of fats stored in tissues to provide energy to deal with zno nps toxic effects may be another reason for blood triglyceride level in fish. any increase in plasma creatinine levels is a biomarker of kidney damage because creatinine is usually removed from the blood and then excreted from the body (soleimany et al., 2016; banaee et al., 2017). the administration of a high dose of zno nps (15 mg kg-1) for 21 days caused a significant increase in creatinine levels in plasma of fish, indicating damage in the function and structure of the kidney. other researchers reported similar results after zno nps oral exposure (ansari et al., 2015). conclusion following oral administration of zno nps for 21 days, our findings demonstrated that zno nps at 5 mg per kg feed did not have any adverse effects on the clinical characteristics of fish health. however, with an increase in zno nps (10 and 15 mg kg-1), significant changes were observed in specific blood biochemical parameters which may be due to zno nps cellular toxicity. therefore, by administering zno nps and the consequent increase in the bioavailability of zinc, disturbances may be found in the physiological function of cells. further research should be done on the effects of zno nps in nontoxic concentrations on other physiological indices such as growth, reproduction, the immune system in fish before using zno nps as a food supplement in fish foodstuff. acknowledgment this study was supported by grant from behbahan khatam alanbia university of technology. also, the authors are grateful to m. banaie for proofreading the manuscript. references ahmadi k., mirvaghefei a.r., banaee m., vosoghei, a.r. 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(2023) 11(1): 1-10 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology table original article use of ethanol (95%) extract of anacardium occidentale (linnaeus 1753) to control centrocestus formosanus (nishigori 1924) infection in xiphophorus hellerii (heckel 1848) udaya priyantha kankanamge epa, asha srimali premarathna department of zoology and environmental management, university of kelaniya, daulagama, kelaniya 11600, sri lanka. s article history: received 25 october 2022 accepted 2 december 2022 available online 2 5 february 2023 keywords: aquarium fish parasites trematode infection abstract: centrocestus formosanus (nishigori 1924) is a trematode parasite introduced into many parts of the world through the aquarium fish trade. its infection causes high fish mortalities and economic losses to fish farmers worldwide. in this study, the efficacy of ethanol (95%) extract of anacardium occidentale (linnaeus 1753) apple to control c. formosanus infection in xiphophorus hellerii (heckel 1848) was investigated. according to probit analysis, the estimated 96 h lc50 of plant extract for x. hellerii was 387.28 mgl 1. infected x. hellerii was treated with concentrations of 300, 320, 340, 360, and 380 mgl1 plant extract with exposure periods of 24 and 48 h. the behavior and mortality of treated and non-treated fish were observed for two weeks. mortality and parasitic intensity of treated fish were significantly lower than that of non-treated fish during the experiment and recovery period. the parasitic intensity in treated fish decreased significantly with increasing concentration of plant extraction in 24 h and 48 h exposure. the lowest dose of a. occidentale apple extract needed to reduce more than 70% of metacercariae infected to gills of x. hellerii within 24 h was 340 mgl-1. according to the findings, a. occidentale apple extract can effectively control c. formosanus infections in aquarium fish. introduction centrocestus formosanus (nishigori 1924) (trematoda, heterophyidae) is a small intestinal parasite of fish-eating birds and mammals. it uses fish as a second intermediate host in completing its complex life cycle (hernandez et al., 2003; ortega et al., 2009, pinto and melo, 2011; paula-andrade et al., 2012). infestations of c. formosanus in aquarium fish have been reported in asia (gjurcevic et al., 2007; rim et al., 2013), europe (gjurcevic et al., 2007; pinto and melo, 2010), and other parts of the world (mitchell et al., 2000; hernandez et al., 2003; salgado-maldonado et al., 2005; paula-andrade et al., 2012). its infection leads to pathogenic conditions and contributes to high mortalities (mitchell et al., 2000; mitchell et al., 2005) in aquarium fish, including, aplocheilus panchax (mardhavi, 1980), cyprinus carpio (hernandez et correspondence: udaya priyantha kankanamge epa doi: https://doi.org/10.22034/ijab.v11i1.1760 e-mail: epa@kln.ac.lk dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.1.1 al., 1998), danio rerio, hypostomus plecostomus, trichogaster trichopterus, poecilia sphenops (scholz and salgado-maldonado, 2000), carassius auratus (gjurcevic et al., 2007), xiphophorus maculatus, poecilia reticulata, cichlasoma nigrofasciatum and nimbochromis venustus (ortega et al., 2009). chemical substances, including acriflavine, chloramine, formalin, malachite green, methylene blue, etc., are widely used to control infectious parasitic fish diseases (madsen et al., 2000). however, resistant genes and residues of drugs remaining in the animal body lessen the wide usage of such chemicals in controlling fish diseases (swain and sahoo, 2003). further, using chemicals is expensive and discouraged by most ornamental fishimporting countries due to their adverse impacts on human health and the environment. recently, there 2 epa and premarathna / use of anacardium occidentale to control infection in xiphophorus hellerii has been increased research into utilizing traditional plant-based medicines to control bacterial and protozoan infections in cultured fish species (castro et al., 2008), mainly because phytomedicines are safe, widely available, and inexpensive. however, plant extracts to treat parasitic infections in fish have rarely been reported. anacardium occidentale (linnaeus 1753), cashew, is widely used in traditional medicine in asia. its leaves, bark, roots, and fruit are reported to have anti-microbial (goncalves and gobbo, 2012), anti-fungal (queiroz et al., 2011), and anti-filarial (suresh and ray, 1990) constituents. the antiparasitic activity of a. occidentale on gastrointestinal nematodes in sheep has also been previously reported (nery et al., 2010). apple of a. occidentale is considered a by-product of the cashew processing industry. the present study aimed to investigate the efficacy of ethanol (95%) extract of a. occidentale apple to control c. formosanus infected swordtail, xiphophorus hellerii (heckel 1848). xiphophorus hellerii is a small, popular ornamental freshwater fish of the family poeciliidae. materials and methods preparation of plant extraction: anacardium occidentale apples in the fully ripened stage were collected from home gardens in the puttalam district (8.0408°n, 79.8394°e), sri lanka, washed thoroughly, cut into small pieces and dried in the shade. the dried a. occidentale apples were ground using a mixer grinder (braun ag; type: mx 32). the resultant powder (370 g) was exhaustively extracted by a soxhlet apparatus using 100 ml of 95% ethanol. after the extraction, ethanol was evaporated using a rotary evaporator attached to a vacuum pump (ikar rv 10d s96). the extract’s stock solution (100 g.l-1) was prepared using deionized water and stored at 4°c. parasitic survey: a disease outbreak that caused mass mortalities of x. hellerii broodstock was observed in a fish breeding center at ginigathhena, rathnapura (6.9857°n, 80.4883°e) in january 2020. hundred x. hellerii with 3.88±0.12 cm length and 95.22±1.31 g weight were randomly caught from infected broodstock ponds and were anesthetized using ms 222 (tricaine methanesulphonate) and examined for external parasites (buchmann, 2007). wet mounts of gill tissues were observed under an optical microscope (olympus cx21fs1) to determine whether parasites were present. many metacercariae cysts, oval in shape, were randomly distributed along the entire length of the gill filaments of infected fish. the metacercariae were identified morphologically using histological slides and photographs (hernandez et al., 2003; gjurcevic et al., 2007; han et al., 2008). toxicity assay of plant extraction on fish: xiphophorus hellerii (length and weight of 3.55±0.15 cm and 88.42±0.36 g, respectively) were randomly collected from the infected broodstock tanks and used to estimate lc50 of a. occidentale apple extract. the acute toxicity tests were performed according to the procedure for static nonrenewal technique (usepa, 2002). according to the results of the range-finding test, the concentration of a. occidentale ethanol (95%) extract to be used for the static nonrenewal acute toxicity test ranged 200400 mgl-1. static non-renewal 96 h toxicity bioassay was carried out with varying extracts of 270, 300, 330, 360, and 390 mg.l-1. three replicates for each treatment and control were used in the study. glass tanks (40x20x20 cm) were filled with 10 l of aged tap water, and the stock solution of plant extract was added in required volumes to get a concentration series of 270, 300, 330, 360, 390 mg.l-1. water was pre-aerated for 15 min to full oxygen saturation before different volumes of plant extracts were added. ten infected broodstock fish were introduced into all the experimental tanks. tank water was wellaerated and fish were not fed during the experimental period. the experiment duration was 96 h and at each 24 h period, the total number of dead fish if any, was recorded and carcasses were discarded immediately. the concentration at which 50% of fish mortality occurred after 96 h was taken as the median lethal concentration (lc50) for 96 h. 3 int. j. aquat. biol. (2023) 11(1): 1-10 determination of the anti-parasitic activity of plant extraction: the experiment was conducted in glass tanks filled with 10 l of aged tap water. the stock solution of ethanol (95%) extract of a. occidentale was added in required volumes to get a concentration series of 300, 320, 340, 360, and 380 mgl-1. tanks were randomly arranged, with three replicates for each treatment and control. water was pre-aerated for 15 min to full oxygen saturation before the different volumes of plant extract were added. ten infected x. hellerii (length 3.65±0.15 cm and weight 83.62±0.26 g) were introduced into each aquarium. tanks were well-aerated, and fish were not fed during the experimental period. the treatment was conducted for 24 and 48 h of exposure times. after exposure, the treated and non-treated fish samples were anesthetized (ms 222), and their gills were removed. the gills were observed under a high-power microscope (olympus, cx21 fs1), and the number of metacercarial cysts in all the gill filaments was counted. the total number of metacercarial cysts present in a fish was taken as the parasitic intensity of that fish. the percentage reduction of parasitic intensity was calculated compared to the parasitic intensity of non-treated fish [(% reduction of parasitic intensity = average number of parasites in control fish – average number of parasites in a fish after the treatment / average number of parasites in control fish) x 100]. a concentration of plant extract was considered adequate if more than 70% of metacercariae were killed at a concentration not toxic to x. hellerii. after the experiment, the treated and non-treated fish were transferred into another set of glass tanks with aged tap water. water was continuously aerated, and fish were fed a commercially available diet daily (2% of body weight). excess feed and fecal matter were siphoned out every day. fish were observed for recovery for two weeks, and any dead fish were counted and discarded. histopathological studies: random samples of gills of fish treated with plant extract and non-treated fish were fixed in 10% phosphate-buffered formalin. samples were processed following standard procedure and 5 μm thick tissue sections (leica microtome rm2235) were stained with hematoxylin and eosin stains (eagderi et al., 2013). data analysis: the data were subjected to the anderson-darling test to confirm the normality. the interaction between ethanol (95%) extract of a. occidentale and exposure time in reducing parasitic intensity was determined by two-way anova. the mean parasitic intensity and mortality of treated and non-treated fish in 24 and 48 h exposure and water quality parameters in experimental tanks were compared using one-way anova followed by tuckey’s pairwise test. the parasitic intensity of fish in experimental tanks after 24 and 48 h exposure was compared using paired ttest. all the statistical tests were conducted using minitab 14 statistical software. results toxicity assay of plant extraction on fish: the 96 h lc50 value of ethanol (95%) extract of a. occidentale on x. hellerii at the 95% confidence level was 387.28 mg.l-1 (table 1). at exposure to high concentrations of plant extract, the symptoms of toxicity observed include initial inactivation, air gulping, slow opercula rate, and setting at the bottom motionless. then they exhibited body imbalance and surface floating, followed by death. mortality of exposure period lc50 (mg.l -1) standard deviation 95% confidence interval limits 24 540.18 229.65 430.94-8801.35 48 478.31 182.00 408.52-1137.37 72 395.54 113.98 367.08-475.95 96 387.28 126.92 358.79-470.04 table 1. median lethal concentration (lc50) of ethanol (95%) extract of anacardium occidentale on xiphophorus hellerii. 4 epa and premarathna / use of anacardium occidentale to control infection in xiphophorus hellerii x. hellerii increased with increasing concentration of ethanol extract of a. occidentale according to the curve obtained from probit analysis for 24, 48, 72 and 96 h exposure (fig. 1). symptoms of infection and identification of metacercariae: xiphophorus hellerii of the broodstock tanks showed symptoms of parasitic infection such as lethargy, respiratory difficulties such as air gulping, gathering near air stones, excess mucus on swollen gills, pale coloration in gills and hemorrhagic areas on the opercula (fig. 2a). the opercula were bulged out in some fish, even exposing gills. the prevalence of infection was 100%, and the mean intensity of encysted metacercariae was 496.8±127.9 (range 223-558) cysts per fish (fig. 2b). the same gill arch showed various developmental stages of metacercarial cysts, indicating continuous infection. the cyst contained larvae with two suckers, 32 circumpolar spines around the oral sucker arranged in two rows, and an x-shaped excretory bladder occupying a greater portion of the posterior body. a moderately small ventral sucker was positioned at about one-third of the anterior region (fig. 3a). histopathologically, the changes were characterized by moderate to severe hyperplasia of figure 1. probability plot for mortality obtained from probit analysis for 96 h exposure of xiphophorus hellerii to different concentrations of ethanol (95%) extract of anacardium occidentale. figure 2. (left: a) the swollen, reddish-colored opercula of an infected xiphophorus helleri, and (right: b) metacercarial cysts in a gill of an infected xiphophorus hellerii (100x h&e). 5 int. j. aquat. biol. (2023) 11(1): 1-10 the primary gill lamellae's cartilage that enveloped the metacercarial cysts (fig. 3b). the enclosed larvae in some cysts showed specific features of adult fluke, and they were surrounded by a thick capsule secreted by the host tissue. newly recruited cysts did not have such a thick capsule, and the larvae did not show features of an adult fluke. the antiparasitic activity of plant extraction: the mean parasitic intensity of treated fish was significantly lower than that of non-treated fish (table 2) after 24 h and 48 h exposure times (p<0.05). there was a significant interaction between the concentration of plant extract and exposure time in removing metacercariae from infected fish (p<0.05). the parasitic intensity of fish treated with 300 mg.l-1 plant extract after 24 h was significantly lower than that of control fish and higher than that of all the other treatments. the parasitic intensity of fish in all the treatments after 48 h was not significantly different (p>0.05). many inactive metacercariae were observed under a high-power microscope in the wet mounts of the gills in the treated fish. in contrast, the gills of control fish contained highly active metacercariae. there was a linear relationship between the parasitic intensity of fish and the concentration of plant extraction after 24 (fig. 4a) and 48 h (fig. 4b) exposure times (regression analysis, r2>0.5). the mean parasitic intensity of fish was significantly lower in 48 h exposure than 24 h exposure in all the treatments (p<0.05, paired t-test). there was a slight increase in the parasitic intensity of fish in control aquaria after 48 h compared to 24 h. this difference was not significant (p>0.05, paired t-test). a b c d a b figure 3. (a) centrocestus formosanus metacercaria in a primary gill filament of xiphophorus hellerii. (os) oral sucker, (eb) xshaped excretory bladder, (vs) ventral sucker, (a) wall of the cyst, (b) thick capsule secreted by host tissues (400x h&e), and (b) gills of x. hellerii show extensive hyperplasia due to the presence of metacercarial cysts of c. formosanus; a. hyperplasia due to cysts; b. the cartilage of the primary gill lamella; c. a newly recruited cyst; d. cysts at a stage of higher development (400x h&e). period the concentration of ethanol (95%) extract of a. occidentale control 300 mg.l-1 320 mg.l-1 340 mg.l-1 360 mg.l-1 380 mg.l-1 24 h 523.3±60.7a1 376.3±32.0b,1 (30%) 202.4±12.0c,1 (32%) 137.9±14.1c,1 (73%) 117.5±21.3c,1 (77%) 120.7±5.99c,1 (77%) 48 h 531.5±57.1a1 152.1±9.33b,2 (71%) 117.8±7.16b,2 (77%) 81.0±7.37b,2 (84%) 73.7±14.2b,2 (86%) 71.5±4.22b,2 (87%) different superscript letters in a row denote significant differences in parasitic intensity in treated and control fish (p<0.05). different superscript numbers in a column represent significant differences between different exposure times (p<0.05) indicated by paired t-test (p<0.05). table 2. mean ± sd and % reduction (within brackets) of metacercariae of c. formosanus infected gills of xiphophorus hellerii treated with ethanol (95%) extract of a. occidentale and control fish. 6 epa and premarathna / use of anacardium occidentale to control infection in xiphophorus hellerii according to histopathological observations, cavitation due to the removal of metacercariae was observed in the primary gill filaments of treated fish (fig. 5). the number of fish deaths in five treatment aquaria was significantly lower than that of control one (p<0.05). the mortalities of fish between 24 and 48 h were not significantly different in both treatment and control aquaria (p>0.05) (table 3). fish mortalities during the recovery period: during the recovery period, the skin pigmentation, swimming pattern, swimming position, activity and opercular rate of fish treated with plant extract were normal, while control fish showed symptoms of parasitic infection. fish mortalities in treated aquaria ranged from 1-2, while more than 50% of fish died in control aquaria. water quality in the experimental tanks: temperature, do, conductivity and tds did not vary significantly between the control and the treatment tanks (p>0.05). however, ph values were significantly lower in the treatment tanks than in the control tanks (p<0.05) during the experimental period (table 4). discussion all the ethanol extracts of a. occidentale apple used in the study effectively reduced metacercariae infection in x. hellerii. the observation of dead metacercariae and cavities of cysts produced from the gill tissues of the treated fish showed the impact of plant extract on the metacercariae of c. formosanus. the parasitic intensity of fish treated with 300-380 mgl-1 plant extract was significantly lower than in the control fish within the experimental period. the low parasitic intensity may have increased the chance of survival of treated fish, decreasing their mortality. the toxic constituents of a. occidentale apple extract may have inactivated and killed the metacercariae in treated fish. according to dao et al. (2021), fresh a. occidentale y = -2.9805x + 1204.3 r² = 0.75 0 50 100 150 200 250 300 350 400 300 320 340 360 380 400 a ve ra g e p a ra si ti c in te n ci ty concentration of plant extract (mgl-1) y = -1.0265x + 448.23 r² = 0.86 0 20 40 60 80 100 120 140 160 300 320 340 360 380 400 a ve ra g e p a ra si ti c in te n si ty concentration of plnat extract (mgl-1) a b figure 4. (a) the mean parasitic intensity of fish vs. the concentration of ethanol (95%) extract of anacardium occidentale (a) after 24 h exposure time, and (b) after 48 hour exposure time. period the concentration of ethanol (95%) extract of a. occidentale control 300 mgl-1 320 mgl-1 340 mg.l-1 360 mg.l-1 380 mg.l-1 24 h 5.33±0.88a (4-7) 0.67±0.33b (0-1) 0.67±0.67b (0-2) 0.67±0.33b (0-1) 1±0.58b (0-2) 1.33±0.33b (1-2) 48 h 5.67±1.45a (3-8) 0.67±0.67b (0-2) 1±0.58b (0-2) 0.67±0.67b (0-2) 0.33±0.33b (0-1) 1±1b (0-2) different superscript letters in a row denote significant differences (p<0.05) indicated by one-way anova followed by tukey’s pairwise comparison. table 3. mean±sd and range of mortality (within brackets) of xiphophorus hellerii treated with ethanol (95%) extract of anacardium occidentale and control fish. 7 int. j. aquat. biol. (2023) 11(1): 1-10 apple contains 2 mg.g-1 tannin and 76 mg.g-1 total phenolic substances. the anti-parasitic properties of tannins, especially on nematodes, have been previously shown in several in vitro and in vivo studies (hoste et al., 2006; molan et al., 2002; paolini et al., 2002). bath treatment of 340 mg.l-1 of plant extract reduced more than 70% of metacercarial cysts from infected gills of x. hellerii within 24 h. due to this high efficacy of plant extract in removing metacercariae, a 100% reduction of parasites may be achieved by extending immersion time or repeating the same treatment. the mean parasitic intensity of treated fish decreased with the increasing concentration of a. occidentale apple extract. a similar observation was made by buchmann et al. (2003) when garlic extract, allium sativum, was tested in controlling ichthyophthirius multifiliis infestation in fish. though the metacercariae infected to the gills of the control fish were active throughout the experimental period, the parasitic intensity in the same fish did not significantly vary during the experimental period. it indicates that the intensity of infection in experimental fish did not change within the experimental period. therefore, the rapid reduction of mean parasitic intensity with increasing exposure time (24 and 48 h) in the treated fish might be attributed to the toxic effects of different concentrations of ethanol (95%) extract of a. occidentale. the results of the present study are comparable with the findings of ekanem et al. (2004), who observed a 90% reduction in i. multifiliis infection in fish after treatment in baths of mucuna pruriens and carica papaya plant 1 a b figure 5. gills of xiphophorus hellerii after the treatment with the ethanol extract of anacardium occidentale (360 mgl-1) a. cavitation in the primary gill filaments due to removal of the metacercariae, and b. dead metacercariae (100x h&e). control 300 mg.l-1 320 mg.l-1 340 mg.l-1 360 mg.l-1 380 mg.l-1 ph 7.21±0.00a 7.17±0.3a 7.00±0.01b 6.97±0.04b 6.99±0.01b 6.96±0.02b temperature (c°) 24.54±0.00 24.47±0.02 24.45±0.02 24.52±0.05 24.56±0.01 24.73±0.01 dissolved oxygen (mgl-1) 7.04±0.00 7.05±0.06 6.90±0.06 6.84±0.10 6.99±0.05 7.00±0.09 conductivity (µcm-1) 846.27±0.03 835.87±2.26 840.20±3.24 843.13±5.02 834.53±6.11 827.47±3.41 tds (mgl-1) 410.63±0.03 409.40±1.15 411.53±1.45 413.07±2.77 408.87±3.11 406.87±1.27 different superscript letters in a row denote significance difference (p<0.05) indicated by one-way anova followed by tukey’s pairwise comparison. table 4. mean±se of physico-chemical parameters of water in aquaria treated with ethanol (95%) extract of anacardium occidentale and control aquaria during the experiment. 8 epa and premarathna / use of anacardium occidentale to control infection in xiphophorus hellerii extracts (200 mg.l−1) within 72 h and 96 h, respectively compared to untreated controls. centrocestus formosanus metacercariae infection damaged the primary gill lamellae and respiratory epithelium of infected x. hellerii. the gills of severely infected fish were swollen and paled in coloration, and hemorrhagic areas on the opercula were also observed. hypoxia due to these symptoms (mitchell et al., 2000; scholz and salgadomaldonado, 2000; mitchell et al., 2005) could be the reason for the significantly higher number of fish deaths observed in the control aquaria. gill arches of infected x. hellerii contained various developmental stages of larvae indicating continual infection. the reduction of water ph in the aquaria treated with plant extract could be attributed to ascorbic, gallic, and tannic acids present in the apple of a. occidentale (gordon et al., 2011; queiroz et al., 2011). ripe apples of a. occidentale were used in the experiment, and they contain more ascorbic acid (17.31 mg.g-1) than unripe apples (gordon et al., 2011). though organic acid can strongly influence the ph in water (munson and gherini, 1993), the narrow ph variation (6.9-7.1) observed in the experimental aquaria may not severely impact fish health or parasitic activity. the 96 h lc50 of ethanol extract of a. occidentale on x. hellerii (387.28 mg.l-1) was significantly higher than that of the 96 h lc50 of moringa oleifera seed extract (124.0 mgl-1) on cyprinus carpio (kavitha et al., 2012), nerium indicum leaf extract (96 mg.l-1) on channa punctatus (sudhanshu and singh, 2004), and tephrosia candida leaf extract (6.43 mg.l-1) on oreochromis niloticus (mohotti and epa, 2016). toxicity symptoms of fish treated with plant extract observed in the present study were similar to those of o. niloticus exposed to derris powder extracts (akinbulumo et al., 2005). during the recovery period, the skin pigmentation, swimming pattern, swimming position, activity and opercular rate of treated fish were normal. therefore, ethanol extraction of a. occidentale apple used in the study may not have any severe adverse effect on the behavior or health of x. hellerii due to its comparatively lower fish toxicity. it is necessary to conduct further experiments with the purified effective substances of a. occidentale in the vicinity of the lowest dose (340 mgl-1; 24 h) to establish the therapeutic amount needed for a good parasiticidal effect. references akinbulumo m.o. 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(2015) 3(6): 390-397 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article habitat use of alburnoides namaki in the jajroud river (namak lake basin, iran) melahat hoghoghi1, soheil eagderi*2,1bahman shams-esfandabad3 1department of biodiversity, environment faculty, science and research branch, islamic azad university, tehran, iran. 2fisheries department, natural resources faculty, university of tehran, karaj. iran. 3department of environment, arak branch, islamic azad university, arak, iran. article history: received 12 august 2015 accepted 25 november 2015 available online 2 5 december 2015 keywords: spirlin freshwater habitat parameters hsi abstract: a fish species prefer a particular habitat where provides its biological requirements, hence, understanding their habitat use and preferences are crucial for their effective management and protection. this study was conducted to assess the habitat use and selection patterns of alburnoides namaki, an endemic fish in jajroud river, namak lake basin, iran. the river was sampled at 18 equally spaced sites. a number of environmental variables, including elevation, water depth, river width, river slope, velocity, substrate type, average diameter of bed stone, riparian vegetation type and total dissolved solid (tds) and the relative abundance of a. namaki were recorded at each site. the results showed that a. namaki mostly selects upper parts of the river with higher slope, higher depth, lower width, lower velocity, bed rock substrate i.e. bed with boulder cover, tds of 100-150 ppm, and deciduous forest and residential area riparian type compared with the available ranges. this study provides the habitat use and environmental factors affecting on the distribution of a. namaki in the jajroud river. introduction increasing anthropological activities particularly in riverine ecosystems have changed their physicochemical features threatening the survival of their inhabitants. riverine ecosystems includes about 0.001% of water resources of our planet, nevertheless they include almost half of the fish species i.e. its species diversity is 7500 times greater than other aquatic systems in terms of fish biodiversity (helfman et al., 2003). a fish species prefer a particular habitat where provides its biological requirements (throw, 1997). many environmental parameters are considered to be important for influencing habitat preference of fish (yu and lee, 2002). hence, understanding their habitat use and preferences are crucial for their effective management and protection (torgersen and close, 2004; tejerina-garro et al., 2005; tabatabaei et al., 2015) due to providing the relationship between environmental factors and aquatic * corresponding author: soheil eagderi e-mail address: soheil.eagderi@ut.ac.ir communities (rashleigh et al., 1995). spirlins, the members of the genus alburnoides, a member of cyprinid family is found in europe, asia minor and central asia with 25 species (coad, 2015). until 2009, all populations of alburnoides species in iran were known as a. bipunctatus in iranian inland waters. based on the recent researches, seven species have been reported from iranian inland water (coad, 2015; mousavi-sabet et al., 2015; jouladeh-roudbar et al., 2015). among them, the endemic alburnoides namaki bogutskaya and coad, 2009 was found in the rivers of the namak lake basin. members of the genus alburnoides are lithophilic and rheophilic fishes, which inhabit in barbel and grayling zones (breitenstein and kirchhofer, 2000; tahami et al., 2015). they are very sensitive to human activities and levels of dissolved oxygen (čihăr, 1999). in european waters, spirlins are extremely threatened and nearly close to extinction 391 hoghoghi et al./ habitat use of alburnoides namaki in the jajroud river because of their sensitivity (kirchhofer, 1997; lusk et al., 1998). the freshwater fishes of iran are also faced to recent severe droughts, climate change, pollutions, introduction of exotic fishes and anthropogenic impacts, and as a consequence, many fish populations have been intensively affected especially sensitive fishes, like the members of the genus alburnoides (esmaeili et al., 2014a, b; tahami et al., 2015). little information is available regarding biology and ecology of the endemic a. namaki, and its habitat use and selection are unknown. therefore, this study was conducted to assess the habitat use and selection patterns with regard to habitat availability of this endemic species in the jajroud river. materials and methods study area: the jajroud river, in the namak lake basin, locates 30 km of the northeast of tehran (tehran province, iran) and originates from the central alborz mountains at klon-e bastak mountains at the north of the darbandsar village (fig. 1). this river drains to the latian dam and afterward flows toward the mamlou dam. the approximate length of the river is 40 km with a basin of about 710 km2 and the mean slope of 4%. the samplings were carried out between letian (n:35°46'10'', e: 51°41'25'') and mamlou dams (n: 35°43'42'', e: 51°41'49''), since the area before letian dam was not available because of its protection by environment department and the river after the mamlou dam was dried during sampling period. sampling was started after latian dam distributed in elevational profiles (to downstream) to cover all available habitats along the river in october and november 2012 through 18 equally spaced (about 100 m) sites, during daylight hours. the elevation (m) and geographic coordinates were recorded for each site to ±10 m using gps (global positioning system; garmin) (torgersen and close, 2004), and the river and sampling locations were mapped using arcgis 9.3 (fig. 1). fish were sampled in 10-15 m stretches of the river using a backpack electrofishing device (samus mp750, 45 cm diameter, aluminium ring anode) in the downstream–upstream direction using upstream and downstream stop-nets of 2 mm mesh. for sampling, one-removal method with similar catch-per-unit effort strategy was employed (klaar et al., 2004). fish specimens were collected from each site during 30 min, anesthetized in clove powder solution (1 g l-1), identified according to coad (2015), counted, photographed, and finally placed in slow moving water along the river bank to recover and return to the river. habitat data: since there is limited or no knowledge available on a. namaki studied here, the environmental variables were selected according to the results of other studies conducted on other fishes (chuang et al., 2006; rifflart et al., 2009; tabatabaei figure 3. location of tehran province, jajroud river, and sampling stations. substrate (mm) riparian vegetation type bedrock >4000 bv deciduous forest boulder 2564000 bm deciduous forest and residential area cobble 64-265 pebble 16-64 a grasslands or herbs gravel 2-16 ba largely unvegetated sand <2 table 1. explanation and abbreviation for each categorical habitat variable. 392 int. j. aquat. biol. (2015) 3(6): 390-397 et al., 2015). the habitat data were measured immediately after sampling (yu and lee, 2002). the measured variables include elevation (m), water depth (cm), river width (m), river slope (%), velocity (cm/s), substrate type (table 1), average diameter of bed stone (cm), riparian vegetation type and total dissolved solid (tds) (ppm). elevation of sampling sites were recorded by gps (garmin). the mean depth (cm) of each site was estimated by measuring depth at 20 random points across sampling site using a measuring bar, and their average was considered as river depth (lotfi, 2012). the mean width of river (m) was measured using a tapeline by measuring upper, middle and lower sections of each sampling site and their average was considered as river width. the mean slope (%) was measured by a clinometer (suunto pm-5/360 pc; ww.suunto.com) at the midline of the river in three areas (beginning, middle, and end of each site). the surface velocity (m/s) was estimated by a simple float based on hassanlie (1999), and repeated three times to minimize error. dominant substrate type was determined both visually and randomly via measuring the diameter of the riverbed stones in 20 selected quadrate (50×50 cm) based on lotfi (2012), and classified according to johnston et al. (1996) and tabatabaei et al. (2015). bed stone diameter average also were calculated by measuring diameter of bed stones in 20 selected quadrate (50×50 cm) based on lotfi (2012). riparian vegetation type (based on the type of vegetation growing at riparian parts of the river or absence thereof), were classified according to our observations, photographs, and standard procedures with some modifications (johnston et al., 1996). tds was measured using a portable water quality instrument (wtw gmbh). the first seven variables were continuous, and other variables were nominal. the abbreviation and description for each discrete variable are presented in table 1. habitat use and habitat selection: habitat use, availability, and selection were calculated over the range of each environmental variable. each environmental variable was divided into a series of intervals, and the mean relative abundance of each species in each interval was calculated using habitat selection (habsel) software 1.0 (jowett, 2014). the formula s= (%uc,i)/(%ac,i), where i is the interval of a given environmental variable c, %uc,i is the percentage of utilization of a specific interval of an environmental variable c utilized by fish, and %ac,i is the percentage of availability of this environmental variable (guay et al., 2000; waddle, 2001; tabatabaei et al., 2015), resulted in a selectivity value (s) at each interval. since no comparable study on microhabitat use of this species was available, therefore, only the specimens larger than 40 mm were selected and counted in each station for further analysis; because the habitat use of fishes in lotic systems can be strongly affected by ontogeny (copp and vilizzi, 2004; gillette et al., 2006). results all collected fish during sampling were belonging to four species viz. alburnoides namaki, capoeta busei, capoeta caculata and oxynoemacheilus bergianus that were returned to the river after identification and counting. a total of 66 specimens of a. namaki were collected. the studied habitats in jajroud river mostly occurred in an elevational range of 1422-1490 m above sea level, with 13-53 cm depth, 5-24 m width, 0.4-1.9 m/s water velocity, slope of 1.2-2%, stone diameter of 12-40 cm, and tds of 100-286 ppm and cobble and then boulder substrate type, deciduous riparian forest, and with most available cover type of boulder (fig. 2). the habitat-use pattern of a. namaki generally followed habitat availability (fig. 2). considering the availability of environmental variables and the selectivity, the habitat selection pattern of this species mainly had the following features: elevation 1480-1490 m, water depth 35-45 cm, channel width less than 10 m, channel slope 1.8-2%, water velocity less than 1 m/s, bed rock substrate, average diameter of bed stone larger than 40 cm, deciduous forest and residential area riparian type, and tds 100-150 ppm (fig. 2) the results revealed that a. namaki mostly selected upper parts of the river with higher slope and depth. 393 hoghoghi et al./ habitat use of alburnoides namaki in the jajroud river furthermore, this species selects lower width and velocity, bed rock substrate i.e. bed with larger elements, deciduous forest and residential area riparian type, and boulder cover compared with the available ranges. in some cases, the pattern of habitat use was different from the pattern of habitat selection i.e. in water depth, river width and velocity. discussion most of endemic fish species with limited distribution are threatened due to destruction of their habitats. hence, it is necessary to study their habitat use and selection patterns prior to endanger their survival in order to their effective management and protection (rashleigh et al., 2004). the current study provided the habitat use and selection patterns, and environmental factors affecting on the distribution of a. namaki, an endemic fish of iranian inland waters, in the jajroud river that supplies drinking and agricultural water of tehran province and khojir area. the members of the genus alburnoides are very sensitive to levels of dissolved oxygen. having low tolerance to water polluted by industrial, agriculture or urban wastes makes these cyprinid fishes a good biological indicator of the environment quality (čihăr, 1999). in recent years, the industrial effluents in jajroud region have been caused the disposal of industrial effluents and chemical figure 2. habitat availability (blue line), used (red line) and selected (black line) by a. namaki for environmental variables. 394 int. j. aquat. biol. (2015) 3(6): 390-397 pollution of surface and groundwater waters including jajroud river. therefore, the findings of the present study can show the importance factors for effective management and protection of this endemic species. the results revealed that a. namaki mostly selects deeper reaches with bed rock substrate and larger bed stones. instream habitat structures provide a variety of functions for stream fishes (quist et al., 2005; tabatabaei et al., 2015); cover features provide protection from predators or ameliorate adverse conditions of stream flow or seasonal changes in metabolic costs and thereby influence fish survival and movement (mackenzie and greenberg, 1998; tabatabaei et al., 2015). in addition, deep body shape of this specie can help to rapid turning and maneuvering in tight quarters as deeper reaches with substrate consisting large bed rocks that provides dead spaces to establish proper habitat. furthermore, substrate type can be important for fish spawning and feeding behavior (quist et al., 2005; tabatabaei et al., 2015). aburnoides namaki mostly uses area with lower river width i.e. less than 10 m. researches showed that habitat with higher river width have little suitability for fishes such as varicorhinus barbatulus (littlejohn et al., 1985; chuang et al., 2006). in addition, deeper reaches with lower current, less river width and lower tds along with larger bed stones can be provide transparent water to penetrate sunlight causing higher production of periphyton algae as main food items of the alburnoides species (treer et al., 2006). habitat use of the a. namaki is area with deciduous forest and residential area riparian type. this can be due to providing organic matters that considered as base of the primary production in the riverine ecosystems (wootton, 1999). in addition, high organic matter depends on the presence of proper condition and enough time to decompose of allochthons such as lower water velocity and appropriate substrate. hence, riparian type and bed cover are important factors in distribution of riverine fishes due to providing the major source of carbon and energy (smokorowski and pratt, 2007). furthermore, the deciduous forest riparian vegetation type can be stabilize fish habitats by providing source of carbon and detritus during low production season e.g. autumn. this detritus are a ground to develop small animals such as invertebrate as base of the riverine food chain (li et al., 2009). the limitations of using an electrofishing device (yu and lee, 2002; mercado-silva et al., 2008), considering the limited sampling period and the variability of the habitat features within each station, may affect the efficiency of the sampling procedure. fish habitat-use patterns may vary by changing the environmental conditions and be affected by seasonal patterns (copp and vilizzi, 2004; gillette et al., 2006). seasonal patterns were not assessed here, but the habitat use and selection patterns of a. namaki are indicative of autumn. therefore, we recommend investigation of the habitat use and preference patterns in other seasons as well. references breitenstein m.e., kirchhofer a. 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(2015) 3(6): 390-397 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی (ایران نمک، دریاچه حوضه) جاجرود رودخانه در alburnoides namaki ماهی زیستگاه ترجیح 3اسفندآباد شمس بهمن ،*2سهیل ایگدری ،1مالحت حقوقی 1گروه تنوع زیستی، دانشکده محیط زیست، دانشگاه آزاد اسالمی واحد علوم و تحقیقات تهران، ایران. 2گروه شیالت، دانشکده منابع طبیعی، دانشگاه تهران، کرج، ایران. .ایران اراک، اراک، واحد اسالمی آزاد دانشگاه طبیعی، منابع و کشاورزی دانشکده زیست، محیط گروه3 چکیده: ایهترجیح و استفادهی نحوه درک رو این از .دهدمی ترجیح را ،شودمی فراهم آن زیستی یهانیازمند که جایی ،ویژه زیستگاه یک ماهی، گونه یک مکی، نماهی خیاطه زیستگاه و الگوهای انتخاب استفاده نحوه ارزیابی منظوربه مطالعه این. است ضروری موثرشان حفاظت و مدیریت برای زیستگاه alburnoides namaki ، با فواصل تقریباً ایستگاه 11 از مجموع در. درآمد اجرا به ایران نمک دریاچه حوضه از جاجرود رودخانه دبومزا یگونه یک قطر ،بستر نوع سرعت، رودخانه، شیب رودخانه، عرض آب، عمق ارتفاع، شامل محیطی متغییرهای از تعداد یک .گردید بردارینمونه رودخانه دربرابر نتایج در هر ایستگاه ثبت گردید. a. namaki نسبی فراوان همراهبه محلول جامد مواد کل و رودخانهحاشیه گیاهی پوشش نوع بستر، سنگ متوسط به یسنگ بستر نوع کمتر، جریان سرعت کمتر، عرض بیشتر، عمق بیشتر، شیب با رودخانه باالدست هایبخش بیشتر a. namakiنشان داد که دسترس در هایمحدوده با مقایسه در را مسکونینواحی و برگریز جنگل ایحاشیه پوشش و tds 151-111 ،سنگتخته بسترهایی حاوی عبارت .کندمی فراهم را جاجرود رودخانه در a. namaki پراکنش بر موثر محیطی فاکتورهای و زیستگاه از استفاده نحوه مطالعه این .کندمی انتخاب .زیستگاه مطلوبیت شاخص زیستگاهی، پارامترهای شیرین،آب خیاطه،ماهی :کلمات کلیدی int. j. aquat. biol. (2021) 9(5): 333-343 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article long-term effect of zinc oxide nanoparticles on population growth, reproductive characteristics and zinc accumulation of marine rotifer, brachionus plicatilis shilan mohammadi1, nasrollah ahmadifard* 1, behrooz atashbar2, abbas nikoo3, ramin manaffar1 1department of fisheries, faculty of natural resources, urmia university, p.o. box: 57153-165, urmia, iran. 2department of artemia, artemia and aquaculture research institute, urmia university, p.o. box: 57153-165, urmia, iran. 3department of analytical and physics and applide chemistry, faculty of chemistry, urmia university, urmia, iran. s article history: received 7 july 2021 accepted 5 october 2021 available online 2 5 october 2021 keywords: biological model nanoparticles rotifer zinc bioaccumulation abstract: in the present study, the effects of zno nanoparticles (nps) on marine rotifer, brachionus plicatilis, was investigated in three separate experiments. firstly, the sensitivity and reproductive characteristics of b. plicatilis were studied at concentrations of 0, 0.1, 0.5, 1, 3, 5 and 10 mg l-1 of zno-nps for 10 days. based on the results, the total number of rotifers (tnr) significantly decreased at 5 and 10 mg l-1 of zno-nps. in addition, the specific growth rate (sgr) of animals was negative at two of the concentrations of zno-nps. in the second experiment, the tnr at 4 concentrations of zno-nps (0, 10, 13, 17, and 19 mg l-1) during 72 h were tested and the 24-72 h lc50 of zno-nps was calculated. after three days, the entire population of rotifers was generally lost at 19 mg l-1 of zno nps. the lc50 of zno-nps in animals at 24, 48, and 72 h intervals was registered as 18.2±1.34, 12.43±0.08, and 9.63±0.26 mg l-1, respectively. finally, the zinc accumulation in rotifers was measured at different concentrations (0, 0.1, 0.5, and 1.3 mg l-1) of zno-nps and maximum zinc (123 μg g-1 of rotifer dw) uptake by rotifers was observed in treatment 3 mg l-1 of zno-nps. in sum, it can be concluded that the b. plicatilis can be used as a biological model for studying marine water contaminants with nanoparticles, especially zno-nps. introduction in recent years, the release of nano-materials into the environment has been a source of concern. zno nanoparticles (zno-nps) are a kind of metal oxide used in various applications of catalysts, semiconductors, and paints (bhuvaneshwari et al., 2017). besides, zinc is required in low amounts as a nutrient for the activation of some enzymes and proteins in the body of organisms, for instance, phytoplankton; however, its high concentrations are toxic to many aquatic species (hogstrand, 2011). the zinc particle size is highly effective in its toxicity (bhuvaneshwari et al., 2017; sarkheil et al., 2018) and releasing toxic ions of zno-nps caused ecotoxicity risks (ates et al., 2013; ma et al., 2013). in addition, cross-sectional area and environmental factors, including ph, temperature, and organic matter affect *correspondence: nasrollah ahmadifard doi: https://doi.org/10.22034/ijab.v9i5.1292 e-mail: nasrollah@gmail.com the solubility of nanoparticles (ma et al., 2013; bhuvaneshwari et al., 2017). in general, the smaller particles have higher cross-sections; hence, the nanoparticles are more soluble than powdered (zno bulk). particle-solubility is one of the highest toxicity mechanisms of nanoparticles, compared to powdery states. nanoparticles produce toxicity by producing hydroxyl radicals and active oxygen. in addition, the photo-catalytic properties of the nanoparticles play an important role in their toxicity (ma et al., 2013). the ultimate destination of this kind of environmental pollutant is aquatic environment, especially the marine environment (sarkheil et al., 2018), which is necessary to examine their effects on marine organisms. rotifers are well-suited organisms for ecological relationships, biological properties, and practical 334 mohammadi et al./ effect of zinc oxide nanoparticles on brachionus plicatilis capability to check the toxicity of materials in aquatic organisms (gama-flores et al., 2004). in general, rotifers are one of the main components of freshwater and marine ecosystems frequently used in monitoring heavy metals (luna-andrade et al., 2002). brachionus genus of rotifers is particularly important as a laboratory organism for environmental studies due to its global distribution, rapid reproduction, short reproductive periods, easy cultivation, and easy access to their latent eggs. these creatures, due to their relatively short lifespan and high growth rates, are also favorable for chronic short-term toxicity studies (gama-flores et al., 2004; hagiwara and yoshinaga, 2017). this genus is used to determine the standard of contamination based on the standard biochemical standard (luna-andrade et al., 2002). the ecotoxicological studies of zno-nps on large aquatic organisms, including fish, common carp (hao and chen, 2012), zebra fish (ma et al., 2013), crustaceans (wong et al., 2010), and small creatures such as phytoplankton (aruoja et al., 2009) and zooplanktons, including daphnia (ma et al., 2013) and artemia franciscana nauplii (ates et al., 2013; bhuvaneshwari et al., 2017; sarkheil et al., 2018) have been conducted. moreover, the effects of different sizes of nanoparticles on the salt lake rotifer brachionus manjavacas were investigated by snell and hicks (2011). the effect of tio2 and cuo nanoparticles on b. plicatilis and b. calyciflorus was studied by clément et al. (2013) and manusadžianas et al. (2012), respectively. despite the fact that clément et al. (2013) used a maximum of 48 hours to investigate of nanoparticles effect on rotifers; in natural environments, rotifers are involved with entering pollutants on a daily basis. in aquatic water bodies, rotifers can also be fed when faced with any type of contaminant, and this may also be an antagonist to food. in this case, its impact will be different from that used by clément et al. (2013). therefore, in this study to simulate with the environment, we tried to use a long-term study along with the nutrition of rotifers with algae. in this regard, we studied some of the life-related and historical characteristics of b. plicatilis in response to different concentrations of zinc oxide nanoparticles (znonps). in addition, the toxicity threshold value and accumulation rate of zno nanoparticles in rotifer were investigated. materials and methods preparation of zno-nps solution: the zno nanoparticle powder with a purity of 99.98% and a particle size of 20-25 nm (2-13-314cas ≠) was obtained from the spanish company with the commercial name of tenan. to prepare the stock solution (1000 mg l-1), 1 g of zno-nps was dissolved in 10 ml of deionized water. then, it was placed in an ultrasonic bath (intersonic, is-2, 300w, 35 khz) for 10 min and eventually reached 1000 ml volume. the prepared solution was stored in a dark container and room temperature (sarkheil et al., 2018). the particle size of zno-nps suspension was measured using a transmission electron microscopy (tem) (philips biotwin, the netherlands). the electron micrographs were taken via electron microscopy section, urmia university, urmia, iran. the percentage of the zn-ions dissolved in the solution was determined by centrifuging at 3200 g for 20 min and the zinc content in the filtrate suspension was measured using atomic absorption (nov aa 400, analytic jena, germany). the soluble zinc was calculated by dividing the measured zinc (as zn) per the total used zno-nps (1 g) and multiplying by 100 (lowry and lopez, 1946). general experimental procedures: the marine water rotifer, b. plicatilis, müller cysts obtained from the shrimp research institute in bushehr, iran, and incubated in standard seawater at 20 ppt. the juveniles were scaled to mass density in a wet-lab using nannochloropsis oculta. the saltwater for rotifer culture was prepared with synthetic salt in accordance with oecd guideline 201 (oecd, 1984), passed through a 2 μm filter, and autoclaved at 121°c for 15 min to avoid any pathogens. the sterilized distilled water was also used to dilute saltwater. a glass vessel with a working volume of 100 ml was used to test the steps. to each test vessel, 50 rotifers per ml of water with 30 g l-1 were introduced. to control the temperature, all containers were performed in a 335 int. j. aquat. biol. (2021) 9(5): 333-343 temperature-controlled glass aquarium. light intensity (1000 lx) and temperature (27°c) were maintained during the test. test steps: this research was carried out in 3 separated tests, including (1) low concentration test (2) high concentration test, and (3) bioaccumulation of zn in the rotifer body. low concentration test: in this experiment, tests were set up in triplicate at nominal concentrations of 0, 0.1, 0.5, 1, 3, 5 and 10 zno nps mg l-1 for 10 days. during the experiment, the culture water daily renewed with the mentioned concentration of zno nps. rotifers were daily fed with 2.5×106 cell ml-1 of n. oculta algae. water quality parameters, including salinity (30±1 g l-1), ph (7.6-8.3) and temperature (24±1°c) were regularly checked. gentle and continuous aeration was performed to meet the needs of rotifers. the growth and reproductive characteristics of rotifers were evaluated at 24-h intervals. high concentration test: after determining the results of the first experiment, which showed chronic effects on the population of rotifers, at this stage, higher concentrations of zno-nps were used to investigate the possible acute effects on rotifers. for this purpose, the four concentrations of zno-nps including 10, 13, 17, and 19 mg l-1 along with a control treatment (with zero zno-nps) at 3 replicates were selected. the culture conditions were the same as in the first experiment. in this experiment, the number of rotifers for 72 hours was counted along with the control group (with a normal growth rate). zinc bioaccumulation: in this experiment, to determine zn uptake by rotifers, 50 ind ml-1 of rotifers were placed in saline water at 30 g l-1 and temperature of 27°c. then, these rotifers were exposed to 0.1, 0.5, 1, and 3 mg l-1 of zno-nps along with a control treatment (with zero zno-nps) at triplicate for 5 days. after 5 days, the rotifers were filtered using a 50 μm mesh, rinsed with filtered and sterilized saline water, and finally the rotifers as paste were kept in -20°c for zn content analysis. measuring factors total number and growth rate of rotifers: to monitor the growth rate of the rotifer, 1 to 2 ml of rotifer’s water samples were daily taken using a micro-pipette. then, 2 drops of lugol's solution were added to fix the specimens and counted by microscope using the hemocytometer (neubauer) chamber. through the following equation, specific growth rate (sgr) was calculated (krebs, 1995). sgr = (lnntlnn0) /t, where n0 and nt respectively shows the initial and final population of rotifer and t represents the experiment period (days). sgr value was calculated in the exponential phase of the population (kennari et al., 2008). biometric factors of rotifers: at the end of the first experiment, the effect of zno nps on possible changes in length and width of rotifers and eggs were also investigated. for this purpose, 15 adult amictic rotifers were randomly selected from each replicate and fixed by 6.5% hcl. then, the length and width of the lorica and the large and small diameter of the egg were measured under a microscope using a calibrated scale. from those measured factors, the lorica biovolume and egg volume were calculated as follows (kennari et al., 2008): lorica bio-volume (μm3) = 0.52 × a × b × c, where a= length, b= width and c= body height (is considered to be 0.4 a). egg volume (μm3) = 4/3 π (a2b + ab2) / 16, where a, and b are two dimensions of eggs diameter (μm). zno nps uptake by rotifers: freeze dried samples were weighed and wet-digested with nitric acid (65% hno3 suprapur®, merck, germany) using mls1200 mega microwave for 30 minutes under cold water and were then diluted with distilled water to reach the required volume. the zinc content was obtained using an atomic absorption device (nov aa 400, analytic jena, germainy) (lowry and lopez, 1946). statistical analysis: after ensuring the normalization of the data by shapiro-wilk test, the homogeneity of variances was investigated via levene’s test. one-way anova was used to investigate the effects of different concentrations of probiotic and duncan's test was used to find the differences among meanings. the probit analysis of spss software is used to calculate the lc50 of zno nps. the minimum significance level of the test was considered as 336 mohammadi et al./ effect of zinc oxide nanoparticles on brachionus plicatilis p<0.05. spss software version 21 was used to check the data and excel software to draw charts. results characterization of zno nanoparticles suspension: the tem image of zno nps from stock suspension is shown in figure 1. based on the electron micrograph, the particles of zno included electron dense oval and round shapes. the mean diameter of relative uniform zno nps was 24.28±12.3 nm. the percentage of the zn-ions dissolved in the stock solution was 56.84%. first experiment total number of rotifers: there is no report on the use of b. plicatilis as a biological model for zno nps toxicity testing. figure 2 shows the effect of zno nps on population growth of rotifer during 10 days. on the 10th day, the highest number of rotifers was significantly related to the control treatment, while, in the other groups, the rotifer number significantly decreased (p<0.05); thus, the total number of rotifers in the treatment with 10 mg l-1 of zno nps was less than other groups. in the treatment with 0.1 and 0.5 mg l-1 of zno nps, the number of rotifers showed an increasing trend; however, it was significantly lower than the control treatment. specific growth rate: the effect of zno nps on sgr of b. plicatilis is shown in figure 3. the changing trend of sgr was related to the amount of zno nps. the lowest sgr was observed at two concentrations of 5 and 10 mg l-1 of zno nps which was found to be negative (-0.823 and -0.1866, respectively). up to 3 mg l-1 of zno nps, the sgr of rotifers was not shown to be negative; however, it was significantly lower than the control treatment (p<0.05). biometric factors of rotifers: based on the results, the lorica biovolume was insignificantly affected by different concentrations of zno nps. however, the highest lorica biovolume of rotifer was observed at a concentration of 1 mg l-1. by increasing the zno nps concentration, the lorica biovolume of rotifer decreased; nevertheless, it was still more than the control group. however, zno nps significantly affected the egg volume of rotifer which was larger than those observed at 1 and 3 mg l-1 of zno nps figure 1. tem image of zno nps from stock suspension. table 1. lorica biovolume (lb) and egg volume (ew) of the rotifers exposed to different amount of zno nps in culture media (first experiment) (mean±sd, n=3). factors amount of zno nps in culture vessel (mg l-1) zero 0.1 0.5 1 3 5 10 lb (×103 μm3) 1994±115 2271±227 2476±202 2570±185 2378±169 2172±316 2269±140 ev (×103 μm3) 301±45c 480±117bc 793±117ab 854±155a 860±138a 617±96bc 805±78ab different letters in each row indicate a significant difference (p<0.05). table 2. lethal concentration (10, 30, 50 and 90) (mg l-1) of zno nps on brachionus plicatilis at different exposition times (hours), (mean±sd, n=3). lethal concentration (mg l-1) in different expose time (hours) 24 48 72 10lc 7.21±2.17 7.06±0.05 6.09±0.16 30lc 13.26±0.97 9.95±0.06 7.98±0.21 50lc 18.21±1.34 12.43±0.08 9.63±0.26 90lc 39.58±2.90 21.45±0.14 15.23±0.40 337 int. j. aquat. biol. (2021) 9(5): 333-343 compared to other groups (table 1). second experiment total number of rotifers: the effect of high concentrations of zno nps on the number of b. plicatilis is illustrated in figure 4. in the control treatment, an increase in density was observed, while, in the treated groups, the total number significantly decreased on the basis of nanoparticle content over three days (p<0.05). thus, three days after adding 19 mg l-1 of zno nps, the entire population of rotifers was generally lost. in the other treatments, the rotifer population reached its minimum level according to the figure 2. the total number of rotifers (ind ml-1) during 10 days of exposition to low concentrations of zno nanoparticles, bars with different letters are significantly different (mean±sd, n=3, anova, p<0.05). figure 3. specific growth rate (sgr) of rotifers (day-1) during 10 days of exposition to low concentrations of zno nanoparticles, bars with different letters are significantly different (mean±sd, n=3, anova, p<0.05). 338 mohammadi et al./ effect of zinc oxide nanoparticles on brachionus plicatilis concentration of zno nps. lethal concentration (lc) of zno nps on rotifer b. plicatilis: in table 2, the results of the lc10, lc30, lc50, and lc90 of zno nps are given at 24, 48, and 72 hours after being added to the culture media. the 24h and 72h lc50 of zno nps were 18.21 and 9.63 mg l-1 and the 24 and 72 h lc90 were 39.58 and 15.23 mg l-1, respectively. zno nps uptake by rotifers: figure 5 illustrates the uptake of zno nps by b. plicatilis after 5 days’ exposure to low concentrations of zno nps in seawater medium. in the control group, the rotifer samples showed 25.95±2.11 μg g−1 of zn which was accumulated from algae and seawater medium. exposure of zno nps to rotifer medium culture caused the accumulation of about 132.47±0.37 μg g-1 of zn at 3 mg l-1 of zno nps. the highest zinc uptake and the lowest zinc content were obtained in the group with 0.1 g of zno nps without any significant difference with the control treatment. figure 4. total number of rotifers (ind ml-1) within 72 h exposition to high concentrations of zno nanoparticles (second experiment), bars with different letters are significantly different (mean±sd, n=3, anova, p<0.05). figure 5. zinc content in the rotifer brachionus plicatilis exposed to 0 (control), 0.1, 0.5, 1, and 3 mg l-1 of zno nps for 5 days. bars with different letters are significantly different (mean±sd. anova, p<0.05) (third experiment). 339 int. j. aquat. biol. (2021) 9(5): 333-343 discussions brachionus plicatilis müller 1786 is a common inhabitant of the persian gulf or seasonal water in southern part of iran. growth and survival characteristics and chronic and acute mortality of these species were studied at different concentrations of zno nps (size < 30 nm) in the laboratory. in the case of the chronic effects, the total number of rotifers decreased with increasing the concentrations of zno nps. on the early days, zno nps had no negative effect on the rotifer population and positive effects were observed. the chronic toxicity effect of nanoparticle on the population growth of rotifers was observed after the 6th day. based on the results, the specific growth rate of the rotifers exposed to nanoparticles decreased with increasing zno nps concentration. at higher concentrations, the effect of nanoparticles caused a negative effect on specific growth rate. in previous studies on rotifer, the maximum population density and specific growth rate are two important bio-indicators (dahms et al., 2011). there is no study on the effects of zno nps on population growth rate of rotifer b. plicatilis, while chronic pb toxicity to the population growth rate of b. calyciflorus (grosell et al., 2006) and philodina rapida (esbaugh et al., 2012) was studied. in other invertebrates such as copepod amphiascus tenuiremis, the chorionic toxicity of carbon nanotubes was investigated by templeton et al. (2006) who obtained an increase in mortality and a decrease in molting success and fertilization rates. in another study, chronic toxicity of znso4 and zno nps in daphnia magna (bacchetta et al., 2017) was studied. after 21 days of exposure, the organisms exposed to low concentrations of zno nps showed complete improvement and full potential reproduction, while the organisms exposed to znso4 showed a dosedependent reduction and a reduced life span (bacchetta et al., 2017). planktons including phytoplankton and zooplankton are more sensitive to the surrounding environments, especially in a wide range of pollutants, than larger animals, including fish and crustaceans (suthers and rissik, 2009). they are often used as model organisms in the standard toxicity test to evaluate the environmental impact of chemical substances (lam and wang, 2008; snell and hicks, 2011; manusadžianas et al., 2012; clément et al., 2013; manfra et al., 2017; sarkheil et al., 2018). rotifers are one of the marine filter-feeding animals and constantly filter food particles, including nanoparticles. therefore, nanoparticles may become toxic in the ionic form or by bonding with the detritus. in this study, it seems that the zno nanoparticle had effects on life history of rotifer by bonding with algae. the study of esbaugh et al. (2012) showed that the pb bound to food items are more toxic than the case it was exposed only the rotifer p. rapida. in the study of zno nanoparticles toxicity on the acartia tunsa transmitted through a phytoplankton regimen, its dosedependence over time and in decreasing survival and reproduction in copepod was obtained (jarvis et al., 2013). it was shown that nanoparticle via absorption into tissues depresses the population growth rate of rotifers (snell and hicks, 2011). the mechanism of nanoparticle effect could be due to a decrease in food intake which consequently decreased reproduction in animals. most likely, the reduction of food intake is an adaptation mechanism to minimize the contact with toxic substances. such response behavior of the rotifer b. manjavacas is also reported by snell and hicks (2011) where it was exposed to a composition of 37 nm polystyrene nanoparticle. in their studies, it was stated that the nanoparticles with dimensions of 37 nm can be absorbed by rotifer b. manjavacas, while larger particles pass the intestine without absorption. they also indicated that nanoparticles are able to pass through the intestinal wall or up taken by epithelial phagocytosis. it was also found that sgr of b. manjavacas with increasing the nanoparticle concentration from 1 to 2 mg l-1 significantly decreased, while, in 0.5 mg l-1 of 37-nm nanoparticle or in 100and 200 nm particles, no significant difference was found in sgr of rotifer (snell and hicks, 2011). the body size of rotifers strongly correlated with physiological and ecological conditions (kirk, 1997; 340 mohammadi et al./ effect of zinc oxide nanoparticles on brachionus plicatilis hotos, 2002). the body size in rotifers and its plasticity in nature have been suggested to be an evolutionary response to predation (allan, 1976) as well as environmental factors (kennari et al., 2008). released nano-materials, as environmental factors, can lead to evolutionary responses. in the present study, the size-change of the rotifers in term of lorica biovolume and egg volume in response to chronic toxicity of zno nps was significantly indicated. this may suggest that the rotifers fed with zno nps physiologically responded to changing environmental conditions. as an emerging field, the effect of nanomaterials on body change need to be investigated in further research, since small organisms such as rotifers have high surface to volume ratio and are susceptible to contaminant exposure. in the present study, the 48 h lc50 value of zno nps on b. plicatilis was found to be 12.43 mg l-1. the 48 h lc50 of zinc metal on the freshwater rotifers p acutiformis species at 25 and 5°c was detected to be 500 and 1550 μg l-1, respectively (epa, 1987). the effect of tio2 nanoparticle on immobility of b. plicatilis showed that 48 h ec50 of 25 nm tio2 nanoparticle was 10.4 mg l-1; however, the toxicity was reduced using larger particles (clément et al., 2013). manusadžianas et al. (2012) investigated the acute toxicity of cuo nanoparticles to the freshwater rotifer b. calyciflorus. they showed that 24 h lc50 of cuo nanoparticles was 0.24 mg l-1 that is more sensitive than algae nitellopsis obtuse and shrimp thamnocephalus platyurus with 24h lc50 4.3 and 9.8 mg l-1, respectively. the ecotoxicity of polystyrene nps to b. plicatilis was demonstrated by manfra et al. (2017). in their study, the neonoate rotifers were exposed to 24-48 h polystyrene nps in the range of 0.5-50 mg l-1. two forms, namely anionic and cationic polystyrene nps, had different effects on the rotifer. they observed no mortality for anionic pscooh vs. a concentration-dependent increase in mortality for cationic ps-nh2. in natural seawater, the 48 h lc50 value of cationic ps-nh2 was 6.62 mg l-1. there is a difference in the toxicity of nanoparticles in fresh and salt waters as well as the marine and fresh water species. bhuvaneshwari et al. (2017) stated that the electrostatic repulsion between nanoparticles can reduce in higher salinity of seawater medium. however, in the b. plicatilis, the toxicity of zno nanoparticles is much higher than anionic polystyrene np and is lower than tio2 nanoparticle and cationic polystyrene nanoparticle. more comparative studies are still needed to determine the sensitivity of various species to the toxicity of nanoparticles including zno nps. in comparison, with d. magna, the lc50 nanoparticle was one third of that of b. plicatilis. in another study, for d. magan, the 48h lc50 zno nps was reported at 3.2 mg l-1 (heinlaan et al., 2008) and 2.6 mg l-1 (blinova et al., 2010). in a. salina, at 30 mg l-1, zno nps did not show any significant acute toxicity after 48 hours of exposure (sarkheil et al., 2018). khoshnood et al. (2017) reported that 24 h exposition of a. franciscana nauplii to 200 mg l-1 of zno nps had only 26.6 % mortality. in rotifers like other zooplanktons, nanoparticles with bioaccumulation can be transferred to higher trophic levels (holbrook et al., 2008). in the present study (bioaccumulation test/ third experiment), the zn uptake by the rotifers exposed to zno nps was assayed and indicated that, via increasing the nanoparticle in saltwater, the uptake by rotifers significantly increased and exposition to 3 mg l-1 of zno nps resulted in higher bioaccumulation compare to 0.1 mg l-1. marine rotifer is a non-selective filter feeder and can ingest particles up to 20 µm (sarma et al., 2011). in seawater, nanoparticle materials tend to aggregate (manfra et al., 2017), despite the fact that it can still be consumed by rotifers. nonetheless, the uptake of nanomaterial by rotifer have been reported by other researchers. snell and hicks (2011) stated that the nanoparticle of 37 nm is strongly absorbed by the exposed rotifer and subsequently transferred to amictic eggs. they also found that polystyrene nanoparticle with a size < 50 nm affect feeding rate and reproduction of rotifer through entering tissues and passing from mother to produced eggs. rubiofranchini and rico-martínez (2011) and alvaradoflores et al. (2012) indicated that diet is the main route of pb accumulation in rotifers such as b. calyciflorus and asplanchna brightwellii. the bioaccumulation of 341 int. j. aquat. biol. (2021) 9(5): 333-343 zn can be due to the passing of the zno nanoparticles through intestinal duct and being absorbed by epithelial phagocytosis. alvarado-flores et al. (2012) showed that accumulated metal had physiological effects on the freshwater rotifer brachionus calyciflorus. in this study, as shown in the first experiment of the present study, via increasing the concentrations of zno nps, its effects on the population growth of rotifers also increased. the bioaccumulation of zno nps has been reported for other marine species. della torre et al. (2014) showed a significant retention of anionic polystyrene np in sea urchin embryos. sarkheil et al. (2018) indicated that artemia nauplli can uptake the zinc oxide nanoparticle from saltwater in a concentration-dependent manner. additionally, ates et al. 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(2022) 11(1): 76-85 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article effect of selected retinoids on carbohydrate metabolism in the freshwater monsoon prawn, macrobrachium malcolmsonii kaduru venkaiah1, malapati hanuma reddy2, jangampalli pradeep kiran3, sri bhashaym sainath1 1department of biotechnology, vikrama simhapuri university, nellore-524 320, ap, india. 2department of marine biology, vikrama simhapuri university, nellore-524 320, ap, india. 3department of internal medicine, texas tech university of health science centre, lubbock, 79413, tx, usa. article history: received 22 october 2022 accepted 1 january 2023 available online 2 5 february 2023 keywords: eyestalks crustacean hyperglycemic hormone glucose glycogen phosphorylase abstract: retinoic acid isomers such as 9 cis retinoic acid (9cra) and all trans retinoic acid (atra) have been discovered in crustaceans. however, their physiological significance in the biological framework of crustaceans is unclear. the present study evaluates the effect of retinoic acid on the hemolymph glucose levels in the monsoon prawn, macrobrachium malcolmsonii. injection of 9cra into intact prawns significantly elevated the hemolymph glucose levels in a dose-dependent and time-dependent manner. however, such hyperglycemic response in 9cra-injected eyestalk ablated (esx) prawns was not observed. no changes in the hemolymph glucose levels were noticed in atra-injected intact or esx prawns. bilateral esx showed significant elevation in the total carbohydrates and glycogen levels with a significant reduction in the activity levels of phosphorylase activity in the crustacean hyperglycaemic hormone (chh) target tissues, hepatopancreas and muscle of prawns. injection of 9cra into intact prawns showed significant elevation in the activity levels of phosphorylase activity with a concomitant decrease in the total carbohydrates and glycogen levels in the chh target tissues compared to vehicle-injected prawns. no significant differences were observed in the selected biochemical variables in 9cra-injected prawns over 9cra-injected esx prawns. the expression levels of chh in the eyestalks of 9cra-injected prawns were significantly elevated over its respective control. it can be concluded that 9cra-induced hyperglycemia, at least in part, mediates chh in m. malcolmsonii. introduction among the top global exporters of edible crustaceans, such as crabs and prawns, india occupies the third position globally. because of their economic importance and high nutritional value, the momentum of culturing edible crustaceans on par with fisheries is happening at a pace in india. however, the limited seed availability is one of the major bottleneck problems associated with the crustacean aquaculture industry (sainath et al., 2013). eyestalk ablation (esx), a classical surgical operation technique, has been followed by aquafarmers to induce ovarian maturation in crustaceans. though esx is promising, it often leads to deterioration of seed quality and mortality in the broodstock. compared to fisheries, technologies to promote reproduction (such as injection of crude extract of the pituitary gland and gonadotropin correspondence: sri bhashaym sainath doi: ttps://doi.org/10.22034/ijab.v11i1.1724 e-mail: drsainath@simhapuriuniv.ac.in dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.10.0 releasing hormone) (hoga et al., 2018) are yet to be developed in the crustacean aquaculture industry. though clear-cut-picture is not available, many researchers investigated the signalling molecules that can promote ovarian maturation and, thereby seed in the hatchery industry (sainath et al., 2013). however, to convert successful experiments at the laboratory level to the land/field level, it is important to understand the interplay between endogenous hormones that control and coordinate crustacean reproduction. this provides a suitable platform to understand the crosstalk between various signalling molecules that regulate ovarian growth in crustaceans at multiple levels. glucose is one of the substrates for energy metabolism in crustaceans (chung et al., 2010). several studies indicated that circulating levels of glucose alter in 76 venkaiah et al./ effect of selected retinoids on carbohydrate metabolism in the freshwater monsoon prawn response to various biotic (parasitic infections) and abiotic (temperature, hypoxia, autotomy and salinity and environmental pollutants) factors (webster, 1996; chang et al., 1998; kuo and yang, 1999; stentiford et al., 2001; wilcockson et al., 2002; reddy et al., 2011). changes in circulatory levels of glucose indicate abnormal changes, including a reduction in survival rate, decreased immunity against bacterial infections, and altered behaviour (wanlem et al., 2011; sun et al., 2013; wang et al., 2017). the x-organ sinus gland complex in crustaceans is a major neuroendocrine system synthesizing peptide hormones, including crustacean hyperglycemic hormone (chh), a neuropeptide primarily involved in regulating glucose homeostasis (fanjal-moles, 2006). at the molecular level, the mature peptide of chh comprises 72-73 amino acid residues (soyez, 1997) and the amidation of chh is critical for its biological activity (katayama et al., 2002). though the synthetic sites, chemical nature, mode of action, and target sites for chh have been demonstrated in several crustaceans, the exact mechanism of chh release from the xo-sg complex into the circulation is not completely understood (fanjalmoles, 2006; ohira, 2016). many studies have shown that several signalling molecules such as biogenic amines (melatonin, and serotonin), opioids (methyl enkaphalin), and insulin-like factors can promote ovarian maturation in decapods crustaceans and at the same, they influence carbohydrate metabolism via triggered release of eyestalk peptide, chh. on the other hand, the involvement of eyestalk peptide chh in the regulation of crustacean reproduction is well recognized. not all molecules (for example, dopamine) that are involved in the stimulation of eyestalk chh, thereby hyperglycemia are involved in the promotion of ovarian growth in crustaceans. these studies indicate that the regulation of crustacean reproduction is complex as it is operated by several molecules and their crosstalk at multiple levels. thus, the identification of signalling molecules that influence physiological functions in crustaceans needs to be verified first. this information at a later stage might be helpful in designing experiments related to the crosstalk of signalling molecules in the regulation of crustacean reproduction (ohira, 2016). retinoic acid (ra) is the biologically active metabolite of vitamin a. the morphogentic effects of ra on organ differentiation during development are wellacknowledged (theodosiou et al., 2010; clagget-dame and knutson, 2011; andre et al., 2014; macejova et al., 2016). ra exerts in two isoforms, 9-cis retinoic acid (9cra) and all-trans retinoic acid (atra) and both exert their genomic actions via retinoid receptors [9cra binds retinoic acid x receptors (rxrs) and retinoic acid receptor (rars) and atra exerts its genomic action via rars] (mangelsdorf et al., 1995; theodosiou et al., 2010). rxrs and rars can form homodimers and/or heterodimers with other members of the nuclear receptor family, peroxisome proliferator activator receptor gamma (mangelsdorf et al., 1995; theodosiou et al., 2010). interestingly, the occurrence of ra across major phyla of metazoans, including invertebrates, at least in part suggests that ra is not a bonafide molecule associated with vertebrates (theodosiou et al., 2010; andre et al., 2014). after the ligand is bounded, the rar/rxr or rxr/rxr specifically binds the retinoid response elements on the dna and regulates the transcriptional expression of ra target genes (andre et al., 2014). in major invertebrate phyla (protostomes and non-vertebrate deuterostomes), the discovery of endogenous retinoids and identification of vertebrate-type counterparts of retinoid metabolism might suggest the existence of common signalling pathways (andre et al., 2014). in crustaceans, ra isomers, 9cra and atra and the counterparts of retinoid metabolism include a cellular retinoic acid binding protein that transports retinoids and retinoic acid signalling proteins retinoic acid x receptors in crustaceans (asazuma et al., 2007; hopkins et al., 2008; tang et al., 2014; nagaraju et al., 2011; cui et al., 2013; venkaiah et al., 2019). retinoid counterparts, though discovered in crustaceans, the physiological significance of retinoids in the biological framework is still in its infancy. in vertebrates, the role of ra in regulating carbohydrate metabolism is well-documented (rhee et al., 2013). ra, through the stimulation of insulin secretion and expression of the glucose transporter 2 gene regulates the glucose levels in vertebrates and these effects of ra are believed to be mediated by retinoid receptors (chertow et al., 1997; pan et al., 2014). interestingly, zou and bonvillian (2003) have shown that administration of 9cra caused a significant elevation in the hemolymph sugar levels, while administration of atra caused inhibitory effects on hemolymph glucose levels in the fiddler crab, uca pugilator. on the other hand, exogenous administration of 9cra induced hyperglycemic effect, 77 int. j. aquat. biol. (2022) 11(1): 75-85 whereas exogenous injection of atra did not result in hyperglycemia in intact freshwater crabs, oziotelphusa senex senex (reddy and sainath, 2008). further, studies of reddy and srilatha (2015) showed that 13cra induced hyperglycemia in the same experimental crab, o. senex senex, and ascribed to either metabolism of 9cra to 13cra or direct action of 9cra and/or 13cra. despite these limited studies, no information is available with respect to the studies linking retinoic acid and hyperglycemic response in crustaceans. macrobrachium malcolmsonii is a freshwater monsoon prawn known as poor man protein. the test organism is one of the most widely available prawns in the penna river, which drains to bay of bengal, nellore district, andhra pradesh, southern part of india. it is one of the important edible prawns with aquaculture importance. it is the second largest palaemonid prawn after the freshwater giant prawn, m. rosenbergii, and has a potential for aquaculture development in the inland waters of india (mishra et al., 2014). though it has a tremendous ability for culture in ponds and rivers (hossain et al., 2012), due to the limited availability of seed, the aquaculture of m. malcolmsonii is not expanded as expected (rao et al., 1986; mishra et al., 2014). therefore, understanding the physiological significance of signalling molecules that promote ovarian growth is of paramount importance. however, prior development of signalling molecules as tools to promote ovarian growth, it is important to understand their physiological significance in the biological framework of crustaceans. in accordance with this notion, the present study aims to investigate the effect of selected retinoids, 9cra, and atra on hemolymph glucose levels, tissue (hepatopancreas and muscle) carbohydrate metabolism and eyestalk peptide chh in the freshwater edible monsoon prawn, m. malcolmsonii. materials and methods collection and maintenance of animals: monsoon prawns, m. malcolmsonii was collected in and around penna river, nellore (14°27'52.9"n, 79°58'25.2"e), andhra pradesh, india and transferred from the collection site to our laboratory (distance: 14 km) in aerated plastic containers. without causing stress, they were transferred to aquaria or holding tanks and acclimatized to the controlled laboratory conditions (temperature: 25-32ºc; salinity: 0.5 ppt; dissolved oxygen: 5-7 ppm; total alkalinity: 50-100 ppm). filtered river water and continuous aeration facility were provided to the prawns, and during their sojourn, the prawns were fed ad libitum with a commercially pelleted diet (cp aquaculture ltd., chennai, india). after changing the ambient medium by at least 25 to 35%, prawns were fed pelleted feed once a day. further, the 1 g biomass to 1 l water ratio was constantly maintained throughout the experimental period. the male prawns at the intermolt stage (c4) with body size and weight of 10±1 cm and 11±2 g, respectively, were used. after acclimatization (10 days), feeding was stopped one day before the commencement of the experiment to avoid changes due to prandial activity. during the experiment period, no feed was provided to the prawns. eyestalk ablation: eyestalk ablation (esx) is a classical operation to deprive eyestalk peptides that control and coordinate metabolic events in crustaceans. in the present study, esx prawns were obtained by cutting at the base of the stalks, and post-ablation procedure, cauterization of the wound was performed to prevent fluid loss (sainzhernandez et al., 2008). after 24 hours, esx prawns were used for the present experiments. test chemicals and their preparations: the isomers of retinoic acid, 9-cis-retinoic acid and all trans retinoic acid (98% purity) were purchased from cayman chemicals, inc., ( ann arbor, mi). the chemical structures of retinoic acid isomers are shown in figure 1. different doses of retinoic acid isomers were prepared freshly. the required concentrations of retinoic acid isomers, 9cra, and atra were prepared by dissolving in ethanol and diluted accordingly in crustacean saline (van harreveld, 1936). the concentrations of retinoic acid isomers were prepared freshly on the day of experimentation. experimental design: retinoic acid isomers 9cra and atra or crustacean saline were injected into the prawns through the base of the coxa of the second pair of walking legs using a micro-syringe (hamilton syringe). dose-dependent effects of retinoic acid isomers: intact and esx prawns in groups 1 and 1a served as controls, whereas intact and esx prawns in groups 2 and 2a were treated as concurrent controls and received crustacean saline (10 µl volume). intact prawns in groups 3, 4, 5, 6, 7, 8, 9 and 10 received injections of 9cra in 10 μl volume at concentrations 10-12, 10-11, 10-10, 10-9, 10-8, 107, 10-6 and 10-5 mole/prawn, respectively. esx prawns in groups 3a, 4a, 5a, 6a, 7a, 8a, 9a and 10a received 78 venkaiah et al./ effect of selected retinoids on carbohydrate metabolism in the freshwater monsoon prawn injections of 9cra in 10 μl volume at concentrations 10 12, 10-11, 10-10, 10-9, 10-8, 10-7, 10-6 and 10-5 mole/prawn, respectively. similar experiments were designed to evaluate the dose-dependent effects of atra. for intact prawns in groups 11, 12, 13, 14, 15, 16, 17 and 18 received injections of atra in 10 μl volume at concentrations 10-12, 10-11, 10-10, 10-9, 10-8, 10-7, 10-6 and 10-5 mole/prawn, respectively. for esx prawns in groups 11a, 12a, 13a, 14a, 15a, 16a, 17a and 18a received injections of atra in 10 μl volume at concentrations 1012, 10-11, 10-10, 10-9, 10-8, 10-7, 10-6 and 10-5 mole/prawn, respectively. after 2 hrs, hemolymph was collected and analyzed for glucose levels (reddy and sainath, 2008), and to avoid circadian variations; all the experiments were carried out between 8 am and 11 am. time-course action of retinoic acid isomers: based on the dose-dependent effects of retinoic acid isomers, a certain concentration of retinoic acid (which induced hyperglycemia) was injected into intact prawns at different time points (i.e. 0, 60, 120, 180, 240 and 300 min). hemolymph was collected, and changes in glucose levels at respective time points were determined. six groups with n = 10 per group were maintained at corresponding time points to determine the time-course effects of selected retinoic acid isomer at selected concentration. analysis of selected biochemical variables: based on the results of time course action of selected retinoic acid isomer and at selected concentration, parameters such as hemolymph glucose levels, total carbohydrate levels, glycogen and glycogen phosphorylase in the chh target tissues, hepatopancreas and muscle of prawns were determined. determination of total hemolymph sugar levels: total carbohydrates in the hemolymph were quantified as per the method described by caroll et al. (1956). briefly, hemolymph was drawn (100 μl) from the arthrodial membrane of the third pair of walking legs using a hypodermal syringe from control and experimental prawns. hemolymph samples were mixed with trichloroacetic acid (tca; 10% w/v) and subjected to centrifugation at 4,000 rpm for 10 min. the protein-free samples (supernatant) were used for total carbohydrate analysis using anthrone reagent and the absorbance was measured spectrophotometrically at 620 nm. the concentration of total carbohydrates in the hemolymph was calculated using a standard graph prepared with known glucose concentration (analar grade). isolation of chh target tissues: chh target tissues like hepatopancreas and muscle were quickly isolated and after removal of adhering material, the tissues were stored at -80°c until further analysis of tissue glycogen levels, total carbohydrates, and glycogen phosphorylase activity. determination of total carbohydrates and glycogen levels: the total carbohydrates and glycogen content in hepatopancreas and muscle tissues were analyzed figure 1. dose-dependent effects of 9cra on hemolymph sugar levels in intact (open bars) and eyestalk-ablated (esx) prawns (solid bars). hemolymph was collected from animals for sugar quantification 2 hr after injection. each bar represents a mean sd of ten individual prawns. bars with same superscript do not differ significantly from each other p<0.001. 79 int. j. aquat. biol. (2022) 11(1): 75-85 according to the method described by caroll et al. (1956) and quantified in terms of anthrone-positive substances. to accomplish this task, the protein-free samples were prepared using tricholoroacetic acid (tca). briefly, 5% (w/v) tissue homogenates prepared in tca (10% w/v) followed by centrifugation (3000 rpm for 15 min at 4°c) were used for the analysis of total carbohydrates. on the other hand, after thoroughly mixing five volumes of 95% ethanol added to 1 ml of tca supernatant, followed by placing the mixture at 4°c overnight, facilitates precipitation of glycogen. after centrifugation (3000 rpm for 15 min), the resultant supernatant was removed and the obtained precipitate was dried by inverting the tubes at room temperature. after 10 min, the precipitate was dissolved in 0.5 ml of distilled water. finally, tissue glycogen content was determined from the ethanolic precipitate of tca supernatant. the methodology includes the following: to the 0.5 ml of supernatants, 5 ml of anthrone reagent was added, followed by boiling for 15 min. after cooling at room temperature, the absorbance of the colour developed was read at 620 nm spectrophotometrically (model: jasco v-750; mary’s court easton, md 21601) against a reagent blank. a standard with a known quantity of glucose using the same procedure was prepared in parallel to the experimental samples. assay of tissue phophorylase: the activity levels of phosphorylase in hepatopancreas and muscle tissues were estimated in accordance with the method described by cori et al. (1955). the selected tissues were homogenized (5% w/v) in ethylenediamine tetraacetic acid (0.037 m; ph 6.8) containing sodium fluoride (0.1 m) (guillory and mommaerts, 1962). after centrifugation (3000 rpm for 10 min) the resultant supernatant diluted four times (1:3) with a buffer 0.03 m cysteine hydrogen chloride and 0.07 m sodium glycerophosphate (ph 6.8) was used as enzyme source. initially 0.4 ml of enzyme source was incubated with 0.2 ml of 2% glycogen solution at 35ºc for 20 min. after incubation, the reaction was initiated by the addition of 0.2 ml of 0.016 m glucose-1-phosphate (g-1-p) to one tube (tube 1) and in another tube (tube 2), in addition to 0.2 ml of 2% glycogen solution, 0.2 ml of 1:1 ratio of 0.016 m g-1-p and 0.004 m adenosine-5-monophosphate was added. the contents in tubes of 1 and 2 were used to estimate the activity levels of phosphorylase ‘a’ and phosphorylase ‘ab’, respectively. the reactions were terminated by the addition of 5 ml of 5 n sulphuric acid. the liberated inorganic phosphate (pi) was determined based on subbarow and fiske (1925). briefly, the contents 1.0 ml of solution and 1.0 ml of 2.5% ammonium molybdate solution were mixed well, and 0.4 ml of 0.2% 1-2-4-amino naphthosulphonic acid reagent was added. the colour developed was diluted to 10 ml of distilled water. after 5 min, the blue colour developed was measured at 720 nm spectrometrically against the reagent blank. the phosphorylase activity was expressed as μmoles of pi liberated/mg protein/hr. the protein content in the tissue homogenates of hepatopancreas and muscle was estimated based on lowry et al. (1951) using bovine serum albumin as standard. expression of chh mrna levels: total rna was isolated from the eyestalks of control and experimental prawns, m. malcolmsonii. in this study, 50 eyestalks were pooled and used for the isolation of total rna from control and 9cra-injected prawns using trizol reagent (invitrogen). the purity of rna was analyzed using nanodrop 2000 and by formaldehyde gel electrophoresis. the first strand cdna synthesis was performed as per the manufacturer’s instructions of iscripttm cdna synthesis kit (biorad, india) using 1 µg of total rna. the first strand cdna was used to determine the expression levels of chh or internal standard, β-actin using semiquantitative pcr (applied biosystems, simpliamptm, thermal cycler). the reaction mixture contains 10 µl of phusion mixture (thermo scientifics), 1 µl of forward primer (fp), 1 µl of reverse primer (rp), 2 µl of cdna and 6 µl of nuclease free water. the pcr cycle conditions were as follows: step 1 includes 1 cycle of 95°c, for 30s followed by 30 cycles of 95°c for 5s, 55°c for 15sec (step 2) and final step includes 1 cycle of 72°c for 10 min. the primers for chh (forward primer, fp: 5-̍ ccttaagaaaagggccatcc-3̍; reverse primer, rp: 5̍-gttggcgtattcg tcgagtt-3̍ was designed based on the chh sequence from the experimental prawn (bankit gene accession no. mn372310). to avoid genomic contamination, the gene-specific primers for chh were designed from exon 2 and exon 3 (chen et al., 2004). the primers for β-tubulin, an internal standard/house-keeping gene (fp: 5′‐ cccttccctcgtctccac‐3′; rp: 5′‐ gccagtgtaccagtgaaggga‐3′) was designed from the m. rosenbergii (li and tsai, 2000). the amplified products were run on 1.8% agarose gels in tae buffer, and the chh amplicons' relative intensities were normalized against the corresponding β-tubulin band 80 venkaiah et al./ effect of selected retinoids on carbohydrate metabolism in the freshwater monsoon prawn intensities. statistical analysis: the results were expressed as mean±sd and statistically analyzed using an analysis of variance (one-way anova) followed by tukey’s posttest (spss version 16.0, spss inc, chertsey., uk). differences were considered to be significant at p<0.05. results effect of eyestalk ablation on carbohydrate metabolism: extirpation of both eyestalks resulted in a significant reduction in the total carbohydrates in the prawn, m. malcolmsonii (figs. 1, 2). total carbohydrates and glycogen content were significantly enhanced, while a significant reduction was observed in total phosphorylase activity in selected tissues of esx prawns (tables 1 and 2). effect of different concentrations of 9cra on hemolymph sugar levels: injection of 9cra induced hyperglycemia in intact prawns in a dose-dependent manner (fig. 1). a significant increase was noticed in hemolymph sugar levels as the concentration of 9cra was increased in intact prawns as compared to untreated, and saline-injected intact prawns. however, a significant elevation in hyperglycemic response was observed up to 10-7 mole 9cra/prawn compared to intact prawns, which received 9cra at doses 10-12, 10-10, 10-9 and 10-8 mole/prawn. on the other hand, intact prawns which received doses higher than 10-7 mole 9cra/prawn exhibited a saturated response in inducing hyperglycemia. hence, in the subsequent experiments, group tcho glycogen hepatopancreas muscle hepatopancreas muscle control 15.620.68 4.610.38 1.080.21 0.680.003 esx 20.411.09*a 7.28*a 0.18 2.59*a0.41 1.510.009*a 9cra-injected intact 11.291.27* 3.21*0.41 0.62*0.09 0.41*0.003 9cra-injected esx 19.61 1.38*a 7.39*a0.24 2.48*a0.96 1.61*a0.009 values are the mean (mg glucose/g tissue)  sd of ten individual prawns. for evaluation of ‘‘p’’ for esx, and 9cra injected prawns (normal and esx), prawns in control group served as controls; for 9cra injected esx prawns, esx prawns served as controls. *represents significant value at p<0.001. the same capital letters in a row indicate a non-significant difference between esx and 9cra injected esx prawns. table 1. effect of eyestalk ablation (esx) and injection of 9-cis retinoic acid (9cra) into normal and ablated prawns on hepatopancreas, muscle total carbohydrate (tcho), and glycogen levels in the prawn macrobrachium malcolmsonii. figure 2. dose-dependent effects of atra on hemolymph sugar levels in intact (open bars) and eyestalk-ablated (esx) prawns (solid bars). hemolymph was collected from animals for sugar quantification 2 hr after injection. each bar represents a mean sd of ten individual prawns. bars with same superscript do not differ significantly from each other p<0.001. 81 int. j. aquat. biol. (2022) 11(1): 75-85 10-7 mole/prawn was selected as the test dose. injection of 9cra at selected doses did not elicit hyperglycemic response in esx prawns. effect of different concentrations of atra on hemolymph sugar levels: injection of atra into intact or esx prawns did not induce hyperglycemic effect at selected doses (10-12 mole/prawn to 10-5 mole/prawn) (fig. 2). time course action of 9cra on hemolymph sugar levels: figure 3 represents time-course action of 9crainduced hyperglycemia in intact prawns. hemolymph sugar level increased significantly (p<0.001) after 9cra injection (10-7 mole/prawn) and reached the highest peak at 120 minutes, thereafter, a decline in hemolymph sugar levels was observed. effect of 9cra on tissue carbohydrates, glycogen content and activity of phosphorylase: injection of group hepatopancreas phosphorylase muscle phosphorylase “a” “ab” “a” “ab” control 2.690.42 4.380.31 3.890.22 6.620.38 esx 1.210.28*a 3.120.18*a 2.310.24*a 4.180.42*a 9cra-injected intact 3.640.62* 5.380.82* 4.910.28* 7.420.61* 9cra-injected esx 1.310.51*a 3.080.21*a 2.380.31*a 4.420.81*a values are mean (imoles of ip released/mg protein/hr)sd of ten individual prawns. for evaluation of ‘‘p’’ for esx, and normal+9cra injected prawns, prawns in the control group served as controls; for esx+9cra injected prawns, esx prawns served as controls. p<0.001; ns, not significant. the same capital letters in a row indicate a non-significant difference between esx and 9cra-injected esx prawns. table 2. effect of eyestalk ablation (esx) and injection of 9-cis retinoic acid (9cra) into normal and ablated prawns on hepatopancreas and muscle glycogen phosphorylase activity levels of macrobrachium malcolmsonii. figure 3. time course action of 9cra-induced hyperglycemia in intact prawns. hemolymph was collected from intact prawns after injection of 9cra (10-7 mol/prawn), at the time points indicated for sugar quantification. each point represents a mean sd of ten individuals. values in parentheses represent percent change from control (0 hr). figure 3. changes in the expression of chh mrna in the eyestalk of intact prawns injected with 9cra. each bar represents a mean±sem of ten individuals. asterisk (*) represent significant differences between the control and intact prawns injected with 9cra. 82 venkaiah et al./ effect of selected retinoids on carbohydrate metabolism in the freshwater monsoon prawn 9cra into intact prawns resulted in a significant decrease in the total carbohydrates (tcho) and glycogen levels in the hepatopancreas and muscle over controls (table 1). injection of 9cra into intact prawns significantly elevated the activity levels of phosphorylase ‘a’ (active) and ‘ab’ (total) in both hepatopancreas and muscle (table 2). bilateral eyestalk ablation resulted in a significant increase in tissue tcho and glycogen levels with a decrease in phosphorylase activity (tables 1, 2). however, injection of 9cra into ablated prawns did not cause significant changes in the levels of tcho and glycogen and the activity levels of phosphorylase in both tissues (tables 1 and 2). effect of 9cra on the expression of chh mrna in the eyestalks of prawns: injection of 9cra into intact prawns significantly enhanced the expression levels of chh mrna in the eyestalks of prawns compared to untreated or saline-injected prawns (fig. 4). discussion in the present study, we found that injection 9cra but not atra induced hyperglycemia in intact prawns as evidenced by elevated hemolymph sugar levels, reduction in the total carbohydrates, and glycogen content, enhanced phosphorylase activity in hepatopancreas and muscle. interestingly, 9cra did not elicit any hyperglycemic response in esx prawns, suggesting 9cra could mediate the eyestalk principle in inducing hyperglycemia. this was supported by enhanced expression levels of chh mrna (rt-pcr) in the eyestalks of 9cra-injected intact crabs. the xo-sg complex is a major neuro-endocrine system located in the eyestalks that synthesizes a range of peptides that can control and coordinate physiological functions, including carbohydrate metabolism in decapods (pillai et al., 2010; nithya et al., 2013). the findings of the present study demonstrated that bilateral esx caused a significant reduction in the hemolymph sugar levels in prawns, demonstrating the location and significance of eyestalk peptide, chh in prawns, m. malcolmsonii. the results are in agreement with previous studies (komali et al., 2005; reddy and sainath, 2008; sainath and reddy, 2010; yang et al., 2018). the primary function of chh is not directly to elevate hemolymph sugar levels but instead, the primary function of chh is to regulate the intracellular glucose levels through the breakdown of glycogen by stimulating the activity levels of phosphorylase in its target tissues like hepatopancreas and muscle (fanjal-moles, 2006). consequently, the leakage of glucose molecules into hemolymph leads to hyperglycemia, a view supported by telford (1975). therefore, reduced hemolymph sugar levels could be ascribed to enhanced levels of total carbohydrates and glycogen content accompanied by reduced activity levels of tissue phosphorylase system in chh target tissues of esx prawns (sainath and reddy, 2010; yang et al., 2018). the current findings indicated that injection of 9cra into intact prawns induced hyperglycemia in a dosedependent manner, while the such a response was not observed in 9cra-injected esx prawns. these findings support that the 9cra induced hyperglycemia occurs via eyestalk principle, chh in intact prawns. rt-pcr studies also revealed that the injection of 9cra elevates chh mrna expression levels in the eyestalks of intact prawns, suggesting 9cra-induced hyperglycemia could be indirect i.e. via triggering of eyestalk chh. the results are in agreement with previous studies where 9crainduced hyperglycemia in the intact fiddler crab, u. pugilator (zou and bonvillian, 2003) and also in freshwater rice field crab, o. senex senex (reddy and sainath, 2008). whereas, atra did not induce hyperglycemic response in intact or esx prawns, suggesting that 9cra could be a possible ra isomer associated with the eyestalks (reddy and sainath, 2008). further, reddy and srilatha (2015) demonstrated that the administration of 13cra, a metabolite of 9cra induces hyperglycemia in intact crabs, o. senex senex but not in esx crabs. significant reduction in the total carbohydrates and glycogen content associated with elevated activity levels of phosphorylase system in selected tissues of 9cra injected intact prawns could be ascribed to stimulatory effects of 9cra at the level of eyestalk peptide, chh. these events could indicate the mobilization of carbohydrate reserves from its target tissues, thereby elevating hyperglycemic response in 9cra-injected intact prawns. the pleiotropic effects of monsoon in vertebrates on various physiological functions vision formation, cell differentiation and proliferation, immune system response, embryonic development and metabolism (pan et al., 2014). with regard to glucose metabolism, the role ra and its cognate receptors in the regulation of glucose homeostasis is well-studied (chertow et al., 1997; rhee et al., 2013; pan et al., 2014; zhou et al., 2021). studies also indicated that ra isomers control and coordinate 83 int. j. aquat. biol. (2022) 11(1): 75-85 glucose homeostasis via insulin secretion from the beta cells of the pancreas and glucose transporters (clark et al., 1995; blumentrath et al., 2001; kane et al., 2010). in crustaceans, retinoic acid isomers (hopkins et al., 2008; venkaiah et al., 2019), insulin-related signalling system (rosen et al., 2013; li et al., 2015; huang et al., 2015; 2016; chandler et al., 2017), and glucose transporter 4 (li et al., 2017) have been discovered. indeed, a clear-cut picture of vitamin a metabolism provides a concrete basis for understanding the physiological significance of ra in vertebrates (chen and chen, 2014). however, current knowledge of retinoid signalling in crustaceans is far from beyond (hopkins et al., 2008; tang et al., 2014; nagaraju et al., 2011; cui et al., 2013). though ra has not been recognized as an endogenous hormone, retinoic acid and rxrs have been detected in crustaceans (andre et al., 2014). few studies demonstrated the possible link between the role of ra in the regulation of limb regeneration (hopkins et al., 2008), ovarian reproduction (cui et al., 2013; venkaiah et al., 2019), and carbohydrate metabolism (zou and bonvillian, 2003; reddy and sainath, 2008). the findings of this study also elaborate on the physiological role of ra in the regulation of carbohydrate metabolism in the monsoon prawn, m. malcolmsonii. further experiments, isolation and characterization of retinoid counterparts such as rxr, crabp receptors, identification of 9cra in the eyestalks and their crosstalk with other endogenous molecules provide insights into the regulation of carbohydrate metabolism by ra in the prawns. acknowledgements we thank, the head, department of biotechnology, vikrama simhapuri university, nellore, ap, india for providing laboratory space and instruments. the authors declare that the experiments conducted during these studies comply with the current laws of their country. one of the corresponding authors, sbs thank the dst-serb for providing financial support to complete part of the current research work. references andre a., ruivo r., gesto m., castro l.f.c., santos m.m. 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(2017) 5(6): 401-407 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article a comparative study of parasite communities of some endemic fish species in the alborz dam and the babol river in the southern caspian sea basin, mazandaran province ali taheri mirghaed*1, maryam barzegar1, hoseinali ebrahimzadeh mousavi1, hooman rahmati-holasoo1, abbas bozorgnia2 1department of aquatic animal health, faculty of veterinary medicine, university of tehran, iran. 2faculty of natural resources sciences, ghaemshahr branch, islamic azad university, ghaemshahr, iran. article history: received 10 september 2017 accepted 2 december 2017 available online 2 5 december 2017 keywords: caspian sea fish parasite babol river alborz dam abstract: the babol river is one of the main rivers in the southern caspian sea basin in mazandaran province. the alborz dam has been built along the river to provide annual irrigation and flood protection. this study aimed to identify and describe the prevalence and parasite intensity of some endemic fish species in babol river and alborz dam and to compare their parasite communities. the field investigations were carried out from june 2016 to march 2017 and approximately, 546 fish specimens, including alburnoides tabarestanensis, capoeta razii and squalius turcicus, were examined. a total of 13 parasite species, including ichthyophthirius multifiliis, trichodina gracilis, myxobulus minutus, dactylogyrus chalcalburni, d. vistulae, d. lenkorani, gyrodactylus gobioninum, g. prostate, paradiplozoon homoion, allocreadium isoproum, rhabdochona denudatai, ligula intestinalis and bothriocephalus acheilognathi, were isolated from the examined fishes. the results showed that host-parasite system in alborz dam is mainly influenced by the parasite’s fauna of babol river. the only exception in this regard is the plerocercoids of l. intestinalis, one of the most common parasites in alborz dam and found in the abdominal cavity of all fish species. the prevalence rate and the mean intensity of parasitic infection in this reservoir are far more than those in babol river. in addition this is the first report of trichodina gracilis from the gills and skin of c. razii in iran. introduction the babol river is one of main rivers in the southern caspian sea basin in mazandaran province. the river originates from the northern slopes of the alborz mountains and flows toward the caspian sea after passing through the cities of babol and babolsar. the river flows in the mountainous part as a valley river with a notably slope. but as the river enters the caspian coastal zone, the slope decreases rapidly and the river continues as a stream. the width of the babol river in the caspian coastal zone is about 40 kilometers (ramin, 2001). the alborz dam, with a height of 78 meters and volume of 150 million cubic meters, was built on the babol river in 2010 to provide annual irrigation; it also is beneficial for flood protection and fisheries activities (ghaffari et al., 2006). the babol river provides a suitable habitat for *corresponding author: ali taheri mirghaed e-mail address: mirghaed@ut.ac.ir numerous species of migratory and non-migratory fishes belonging to the families of petromyzonidae, cobitidae, nemacheilidae, esocidae, cyprinidae, mugilidae and gobiidae, but the largest number of fish species belongs to cyprinid family. alburnoides tabarestanensis mousavi-sabet, anvarifar & azizi 2015; capoeta razii jouladeh-roudbar, eagderi, ghanavi & doadrio 2017 and squalius turcicus de filippi, 1865 are the most abundant fish species in the babol river (ramin, 2001; esmaeili et al. 2017; jouladeh-roudbar et al., 2017) found in the alborz dam. several parasitological studies have been conducted on the fishes inhabiting in rivers of the southern caspian sea basin in mazandaran province. molnár and jalali (1992) have recorded the monogeneans dactylogyrus lenkorani in the tonekabon and tajan rivers of the caspian sea basin 402 barzegar et al./ comparative study of fish parasites from the babol river and alborz dam and d. pulcher in the tajan, tonekabon, and ghasemlu rivers of the caspian sea basin as parasites of c. capoeta. malek and mobedi (2001) have reported clinostomum complanatum from this species in the shiroud river. rohei aminjan and malek (2004) have found nine parasite species in fish from the shiroud, namely trematodes c. complanatum, diplostomum spathaceum, posthodiplostomum cuticola and allocreadium sp., the monogeneans d. pulcher, d. lenkorani and gyrodactylus mutabilitas, and nematodes rhabdochona fortunatowi and capillaria sp. miar et al. (2008) have reported myxobolus saidovi in the fish from the valasht lake and the chalus river, mazandaran. maleki and malek (2007) have reported infection of fish from the shirud river in the caspian sea basin with the digeneans p. cuticola, d. spathaceum, c. complanatum, and allocreadium sp. jalali et al. (2005) have summarized the occurrence of gyrodactylus species in iran and recorded these species from fish in sefid river. barzegar and jalali (2008) have reviewed crustacean parasites in iran and found ergasilus peregrinus, ergasilus sp., lamproglena compacta, lernaea sp., tracheliastes longicollis, and tracheliastes polycolpus. barzegar et al. (2008) have recorded the digenean eye parasite d. spathaceum from a. bipunctatus. the only research study on the fish parasites in the babol river has been carried out by hassanpour et al. (2004), who have recoded three metazoan parasite species, including hepaticola petruschewskii from the intestine of a. bipunctatus (tabarestanensis), s. cephalus (turcicus) and c. gracilis (razii), and capillaria sp., and corynosoma villosum from the intestine of rutilus kutum. so far, there is no report on fish parasites from the alborz dam. communities of fauna and flora and the related parasites differ among the rivers and other water body; thus, different parasites are found in single hosts from two different ecosystems (miar et al., 2008). these two water bodies, the babol river and the alborz dam, belong to a single region, biogeographically, but with different ecosystems: one is a lotic, but the other is a lentic ecosystem. this study aimed to identify protozoan and metazoan parasites in some endemic fish species in the babol river and the alborz dam in mazandaran province. it also aimed to compare parasite communities in the case of species diversity and the prevalence and intensity of parasite species among the infected members between the two different ecosystems. materials and methods parasitological study: field investigations were carried out in the babol river and the alborz dam from june 2016 to march 2017. the fishes were caught by hook or electrofishing and immediately transported alive in oxygen-filled plastic bags to laboratory to be kept in aquaria. identification of the fish was carried out according to berg (1965), coad (2017), and keivany et al. (2017). only fresh or immediately killed fish samples were examined for parasites. first, the fish were sedated using clove oil and then necropsied for parasitological analysis. the fish internal and external organs were examined. methods used for collecting, fixing, staining, and mounting of the parasite specimens were carried out in accordance with fernando et al. (1972), gussev (1983), and roberts (2001). the identification of parasites was carried following gussev (1985), lom and dykova (1992), yamaguti (1961), woo (2000), and jalali (1998). data collection and management: to describe the parasite populations, some indices of infection, such as parasite prevalence and mean intensity were determined. prevalence was defined as the number of individuals of a host species infected with one or more particular parasite species, divided by the total number of hosts examined for that parasite species (expressed as a percentage). the mean intensity was defined as the average intensity of a particular species of parasite among the infected members of a particular host species (bush et al., 1997). statistical analysis: to analyze of data, excel and spss version 19 software were used. in this regard, mann-whitney u was used to compare the mean intensity of parasitic infection between the babol river and the alborz dam. the values of p<0.05 403 int. j. aquat. biol. (2017) 5(6): 401-407 were considered as significant. results approximately 546 fish specimens belonging to the cyprinid family, include a. tabarestanensis, c. razii, and s. turcicus, were examined (table 1). a total of 13 protozoan and metazoan parasites were isolated from the examined fish of the babol river and alborz dam. the species identified in the host fish include ichthyophthirius multifiliis, trichodina gracilis, myxobulus minutus, dactylogyrus chalcalburni, d. vistulae, d. lenkorani, gyrodactylus gobioninum, g. prostate, paradiplozoon homoion, allocreadium isoproum, rhabdochona denudatai, ligula intestinalis and bothriocephalus acheilognathi (table 2). monogenean parasites were the most abundant parasitic group in both river fish and dam fish (fig. 1). the prevalence of the parasites among the examined fish in the babol river and alborz dam are compared in figure 2. different fish species showed different prevalence; also prevalence of the alborz dam and babol river is different. mean intensity of the parasite species collected from the examined fish from the babol river and alborz dam is also presented in figure 3. there were significant difference in mean intensity of different parasites table 1. examined fish species from the babol river and alborz dam in the southern part of the caspian sea basin, iran. examined fishes length (cm) weight (g) number of specimens number of infected fish a. tabarestanensis 6.9±0.2 7.20±0.6 114 94 c. razii 16.0±0.3 29.0±1.5 224 157.5 s. turcicus 12.3±0.4 14.8±0.6 208 187.5 table 2. the parasite communities of some endemic fish species in the babol river and alborz dam, in the southern caspian sea basin in mazandaran province. parasite host (s) infected organs ciliophora ichthyophthirius multifiliis fouquet, 1876 a. tabarestanensis gills alborz dam, babol river trichodina gracilis polyanski, 1995 c. razii gills babol river myxozoa myxobulus minutus nemeczek, 1911 s. turcicus gills alborz dam, babol river monogenea dactylogyrus chalcalburni dogiel & bychowsky, 1934 a. tabarestanensis gills babol river dactylogyrus vistulae prost, 1957 s. turcicus gills alborz dam, babol river dactylogyrus lenkorani mikhailov, 1967 c. razii gills alborz dam, babol river gyrodactylus gobioninum gussev, 1955 c. razii gills & skin babol river gyrodactylus prostae ergens, 1963 c. razii a. tabarestanensis gills & skin babol river paradiplozoon homoion byckowsky et nagibina, 1959 (diporpa & adult stage) a. tabarestanensis s. turcicus gills alborz dam, babol river digenea allocreadium isoproum looss, 1900 s. turcicus intestine babol river cestoda ligula intestinalis linnaeus, 1758 a. tabarestanensis s. turcicus c. razii abdominal cavity alborz dam bothriocephalus opsariichthydis yamaguti, 1934 a. tabarestanensis s. turcicus intestine alborz dam, babol river nematode rhabdochona denudata dujardin, 1845 s. turcicus intestine babol river figure 1. frequency of different parasitic groups in the fish from the babol river and alborz dam, in southern caspian sea basin in mazandaran province. http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatget.asp?genid=2867 http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatget.asp?genid=1720 http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatget.asp?spid=1989 http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatget.asp?genid=2867 http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatget.asp?genid=1720 404 barzegar et al./ comparative study of fish parasites from the babol river and alborz dam among the fish species and between the babol river and alborz dam. overall, mean intensity of i. multifiliis, m. minutus, d. vistulae, d. lenkorani and l. intestinalis in the tested fish from the alborz dam was significantly higher than those of babol river. however, t. gracilis, d. chalcalburni, g. gobioninum, g. prostate, a. isoproum and r. denudate in the tested fish from the alborz dam was significantly lower than those of babol river. discussion only eight parasite species were isolated from the fishes in the alborz dam, of which seven species, including i. multifiliis, m. minutus, d. chalcalburni, figure 2. prevalence of different fish parasites in different fish species from the babol river and the alborz dam in the southern caspian sea basin in mazandaran province. figure 3. mean intensity of fish parasite in different fish species from the babol river and alborz dam in the southern caspian sea basin in mazandaran province. asterisks show significant difference between the alborz dam and babol river. ** p<0.01; *** p<0.001; **** p<0.0001. 405 int. j. aquat. biol. (2017) 5(6): 401-407 d. vistulae, d, lenkorani, p. homoion and b. opsariichthydis, are similar to those of babol river. miar et al. (2008) found similar results during their investigation on parasitic fauna of fish in the valasht lake and the chaloos river. based on their result, of 12 parasite species only three were isolated from the fish of the valasht lake. the alborz dam is a newly constructed and therefore the fish-parasite system in this reservoir is not completely formed and mainly influenced by the parasitic fauna of the river (iziumova, 1987). the plerocercoids of l. intestinalis are of the most common parasites among examined fish which found only in the alborz dam. all the conditions for the incidence of ligulose, including the high density of cyclops as the first intermediate host, the extensive presence of host fish species that feed on the cyclops as their preferred diet, and the fish-eating birds as definitive host are available in standing water of lakes and reservoir of dams (jalali, 1998). in iran, l. intestinalis has been reported from almost all dam reservoirs and lakes (rouhani, 1998; yousefi et al., 2005; barzegar and jalali, 2005; jalali and barzegar, 2006; mohammadi hefzabad and ghare-daghi, 2012). in the present study the prevalence rate of infection in c. capoeta and s. cephalus were 33% and 10% respectively, while the highest infection was observed in a. tabarestanensis (81%). an important aspect of the damage of l. intestinalis infestation is its effect on fish reproduction. the size of gonads even slightly infested fish is always smaller and participation in reproduction always ceases, which ultimately leads to a decrease in fish host populations (parsa khanghah et al., 2011). besides direct losses caused by mortality, their presence may also reduce marketability of fish (sac et al., 2016). this issue is important in commercially valuable fish and may cause great economic losses. yousefi et al. (2005) recorded the occurrence of l. intestinalis in abdominal cavity of r. kutum with the prevalence rate of 100% in the aras dam. bothriocephalus acheilognathi was isolated from a. tabarestanensis and s. turcicus from both babol river and alborz dam. this parasite has spread throughout the world and grass carp and common carp are the hosts of this parasite (xi et al., 2016). this parasite commonly infects native and endemic fish species in iran. bothriocephalus acheilognathi can cause massive fish kills in cultivated fish. however, it is a pathogen in the fish from natural environments (salgado-maldonado and pineda-lopez, 2003). this is the first report of b. acheilognathi in the intestine of a. tabarestanensis in iran. the protozoan i. multifiliis was found on the skin of a. tabarestanensis in the babol river and the alborz dam. ichthyophthiriasis is recognized as one of the most pathogenic protozoan of fish resulting in significant economic losses in the affected fish (jalali, 1998) and has been reported from many iranian fish species (pazooki et al., 2006; jalali, 1998; raissy et al., 2010). high infection rate with this parasite will have negative effect on fish population, a phenomenon that may occur in the alborz dam. wurtsbaugh and tapia (1988) reported a mass mortality of fish in the lake titicaca associated with an epizootic of the protozoan parasite, i. multifiliis. during the present study, adult stage of a. isoporum was found in the intestine of s. turcicus. digeneans are heteroxenous, which means that they require more than one host to complete their life cycle and fish may also be infected by the metacercarial larval or adult stages (paperna, 1964). of course adult intestinal trematodes are normally considered unable to cause disease unless at high number (paperna, 1964). among the groups of parasites found in this study, monogeneans presented the highest number of species. six species of monogeneans were found in the fish with d. lenkorani as the most abundant one. it is likely that fish protozoan and metazoan parasites, which are not dependent on an intermediate host, will be presented in their hosts. their intensity may suddenly increase particularly when the hosts are under high stocking density (lom and hoffman, 1964). in the present study c. razii and a. tabarestanensis were introduced as a new host for p. homoion in iran. despite low number of the parasite species, the 406 barzegar et al./ comparative study of fish parasites from the babol river and alborz dam prevalence and mean intensity parasite infection in the alborz dam is far more than babol river. in most cases, the construction of a dam results in changes in fish biodiversity and stock abundance. usually, the number of migrating fish species and fast flowing water species decline while stocks of pelagic species and species that prefer slow moving water such as a. tabarestanensis c. razii and s. turcicus (i.e. preadapted to lacustrine conditions) increase. the impounded waters have often been managed by introducing species better adapted to lacustrine environments to develop fishery activities. the high fish density in lakes and reservoirs along with other conditions including fairly constant environmental conditions, increase the chance of meeting the free living stages of parasites with their hosts and develop the outbreak of protozoan and metazoan parasites. in conclusion, both rapid and gradual changes of the environment can modify host immune responses, parasite communities and the specificity of their interactions so continuing such studies are powerful tools to understanding of how biotic and abiotic factors affect fish species and their parasite communities. references barzegar m., jalali b. 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(1988). mass mortality in lake titicaca (peru-bolivia) associated with the protozoan parasite ichthyophthirius multifiliis. transaction of american fisheries society, 117(1): 213-217. xi b., li w., wang w., nie p., xie j. (2016). cryptic genetic diversity and host specificity of bothriocephalus acheilognathi yamaguti, 1934 (eucestoda: bothriocephalidea). zoological systematics, 41(2): 140-148. yamaguti s. (1961). systema helminthum, monogenea and aspidocotylea, interscience publishers. 699 p. yousefi m.r., sefid-gar a.a., maliji g., mousavi j., asnaashari m.y. (2005). report of several cases infection of rutilus rutilus by ligula intestinalis in aras dam. journal of medical sciences, 2(26): 80-83. int. j. aquat. biol. (2017) 5(6): 401-407 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی ریاید آبریز هضحو شرقی جنوب در البرز سد و بابلرود رودخانه بومی ماهیان برخی انگلی جمعیت ایمقایسه بررسی مازندران استان در خزر 2بزرگنیا عباس ،1هوالسو رحمتی هومن ،1موسوی زاده ابراهیم حسینعلی ،1برزگر مریم، 1*میرقائد طاهری علی ، ایران.تهران تهران، دانشگاه دامپزشکی دانشکده آبزیان هایبیماری و بهداشت گروه1 .ایران قائمشهر، قائمشهر، واحد اسالمی آزاد دانشگاه طبیعی، منابع دانشکده آبزیان و شیالت گروه2 چکیده: منظوربه رودخانه این امتداد در البرز سد و بوده مازندران استان در خزر دریای آبریز هضحو شرقیجنوب هایرودخانه ترینآب پر از یکی بابلرود میزان تشریح و شناسایی تحقیق این اصلی هدف. است شده احداث سیل خطر برابر در مناطق حفاظت و آبیاری جهت نیاز مورد آب آوردن فراهم تحقیقات. است مذکور ماهیان هایانگل مقایسه و البرز سد و بابلرود بومی ماهیان برخی پریاخته و یاخته تک هایانگل به آلودگی شدت و شیوع ورازی ماهیسیاه ،طبرستانی خیاطهماهی شامل کپورماهیان خانواده به متعلق ماهی نمونه 546 تقریباً و شده انجام 1396 سال طی در میدانی ،ichthyophthirius multifili، trichodina gracilis شامل انگل گونه 13 مجموع در. گرفتند قرار شناسیانگل بررسی مورد ایرودخانه سفید myxobulus minutus، dactylogyrus chalcalburni، d. vistulae، d. lenkorani، gyrodactylus gobioninum، g. prostate، paradiplozoon homoion، allocreadium isoproum، rhabdochona denudatai، ligula intestinalis و bothriocephalus opsariichthydis أثرمت اساساً البرز سد دریاچه در میزبان-انگل سیستم که دهدمی نشان نتایج. شدند جدا بررسی مورد ماهیان مختلف هایاندام از دریاچه در انگلی هایونهگ ترینفراوان از یکی که است اینتستینالیس لیگوال پلروسرکوئید زمینه این در استثناء تنها .است بابلرود رودخانه انگلی فون از مختلف هایگونه به آلودگی شدت و شیوع درصد که است حالی در این. شد یافت بررسی مورد ماهیان تمامی شکمی محوطه در و بوده البرز سد پوست و بششآ از گراسیلیس تریکودینا حضور گزارش اولین حاضر بررسی این، بر عالوه. است بابلرود از بیشتر مراتب به البرز سد دریاچه در انگلی .است ایران در ماهی سیاه .البرز سد بابلرود، ماهی، انگل ،دریای خزر :کلمات کلیدی int. j. aquat. biol. (2017) 5(6): 370-374 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article comparative ultrastructural study of general body epidermis of the hill-stream fishes; botia almorhae (teleosti: botiidae), homaloptera brucei (teleostei: balitoridae) and schizothorax richardsonii (teleostei: cyprinidae) hoshiyar singh*1, ila bisht2 1department of zoology, surajmal agarwal degree college pulbhatta, kichha, u.s. nagar, k.u. nainital, india. 2department of zoology s.s.j. campus almora, k.u. nainital, 262522, india. article history: received 9august 2017 accepted 23 october 2017 available online 2 5 december 2017 keywords: epidermis surface architecture hill-streams gbe abstract: the aim of the present study is to provide a basis for better knowledge of the surface architecture of the gbe of some hill-stream fishes. the skin of the hillstream fishes, on the dorsal surface of the body just behind the head, is densely set with scales and composed of an epidermis and a dermis supported by a hypodermis. noticeable differences exhibited in the patterns of microridges on epithelial cells, distribution of mucous cells and presence of tubercles on the general body epidermis of the hill-stream fishes may be considered as modifications relating to possible difference in the functional requirement at the different locations. the skin has long been recognized to protect the organisms from deleterious environmental impacts (physical, chemical, microbiological). it is also well-known to be crucial for the maintenance of temperature, electrolyte and fluid balance. introduction the hill-stream fishes are well-adapted to specialized conditions of their life in torrential environment, where velocity of water current is high. fishes living in hill-streams show several important modifications and may be conveniently divided into two groups. the members of one group are temporary inhabitants of the hill-streams and migrate upwards only at certain periods of their life for specific purposes such as spawning. these species move up by muscular effort and do not exhibit special modifications. whereas other live permanently in the rivers and streams of the hills such as members of the families cyprinidae (e.g. garra, schizothorax, schizothorax, barbus and crossocheilus), balitoridae (e.g. balitora), psilorhynchidae (e.g. psilorhynchus), nemacheilidae (e.g. nemacheilus), and sisoridae (e.g. glyptothorax pectinopterus and pseudocheneis sulcatus) that have specialized organs to live in such an environments. some fishes in the hill-streams of india are represented by the genera belonging to the families *corresponding author: hoshiyar singh doi: https://doi.org/10.22034/ijab.v5i6.330 e-mail address: singh.hoshiyar320@gmail.com cyprinidae, botiidae and balitoridae. these fishes show a remarkable uniformity in their body contours. dorsally their body is slightly arched, while ventrally it is usually flat from snout to anus. the aim of the present study is to provide a basis for better knowledge of the surface ultrastructural of the general body epidermis (gbe) of three hill-stream fishes, including botia almorhae, homaloptera brucei and schizothorax richardsonii. materials and methods the live b. almorhae (127-177 mm in total length) were collected from the kosi river at kakrighat of district nainital (1200 m. above sea level (asl)), h. brucei (76-101 mm in total length) from west ramganga at chaukhutia in district almora (1200 m asl) and s. richardsonii (152-203 mm in total length) from the kosi river at hawalbagh in district almora (1194 m asl) uttarakhand. the water current was very fast having the velocity between 0.5 to 2.0 m/sec (bhatt and pathak, 1991) and the river bed is rocky. 371 int. j. aquat. biol. (2017) 5(6): 370-374 the collected fishes were transferred to the laboratory in well-ventilated plastic containers and kept for 5-6 days in glass aquaria having an artificially prepared rocky bed with aquatic vegetation grown therein. the aquaria were cleaned and supplied with fresh spring water on alternate days. the fishes were fed on aqua feed (tropical fish food). the following procedure was adopted for the preparation of the specimen for sem. the maintained specimen in laboratory at 25±2°c were cold anesthetized based on mittal and whitear (1978), for sem preparation. skin fragments of about 10×10 mm were cut from their dorsal sides just behind their heads. tissue were excised and rinsed in 70% ethanol with one change of saline solution to remove debris and then fixed in 3% glutaraldehyde in 0.1m phosphate buffer at ph 7.4 overnight at 4°c in a refrigerator. the tissues were washed with 2-3 changes in phosphate buffer and dehydrated in ascending series of ice cold acetone (30%, 50%, 70%, 90% and 100% approximate 20-30 mins.) and dried at critical point using a critical point dryer (bio-rad england) with liquid carbon dioxide as the transitional fluid. tissues were glued to stubs, using conductive silver preparation (eltecks, corporation, india). the samples were coated with gold using a sputters coater (jfc 1600) and examined under (jeol, jsm6610 lv) scanning electron microscope and the images were observed on the screen. results the skin covering the general body surface of b. almorhae, h. brucei and s. richardsonii are rough and covered with a large number of scales. in b. almorhae, the entire external body surface is covered by minute scales; however those of in h. brucei and s. richardsonii have large number and small scales, respectively. each scale is covered externally by the epidermis which reaches the posterior free margins transversing a short distance on its inner surface and then continue to the outer surface of the underlying scale. the polygonal epithelial cells are shown in the gbe of b. almorhae, h. brucei and s. richardsonii (figs. 1, 2, 3); the free surface of the epithelial cells is differentiated into microridges, forming characteristic patterns. in b. almorhae, the epithelial cells bear numerous short, sinus and branched interwoven microridges (fig. 4). however the finger print-like patterns of microridges are often shown on the surface of the epithelial cells of h. brucei and s. richardsonii (figs. 5, 6). these type microridges are often interconnected with fine transverse connections, the microbridges (fig. 7), these microbridges shown only in gbe of h. brucei and s. richardsonii. in b. almorhae,the epithelial cells bear microridges and are commonly associated with mucus secreting cells, the mucous cells, which are scant in number figure 1. semph (scanning electron microphotograph) of the gbe of botia almorhae showing polygonal epithelial cells (marked by arrow) (scale bar=10 μm). figure 2. semph of the gbe of homaloptera brucei showing polygonal epithelial cells (marked by arrow) (scale bar=5 μm). figure 3. semph of the gbe of schizothorax richardsonii showing polygonal epithelial cells at high magnification (marked by arrow) (scale bar =5 μm). 372 singh and bisht / ultrastructural study of general body epidermis in three hill-stream fishes (fig. 8) while the mucous cell apertures are rare comparatively and occur at the border of three or four epithelial cells in h. brucei (fig. 9) but the mucous cells, though distributed throughout the epidermis are, in general, concentrated mainly on the outer layer of the epidermis, often releasing their secretory contents profusely at the surface through small pores in the s. richardsonii, (fig. 10). a large number of tubercles are found on the epidermal surface of h. brucei, these tubercles exist in a well-designed pattern. the unculi are equidistantly placed and supported by epithelial cells. polygonal outlining of the epidermal cells is seen at the base of the unculi, indicating unculi to be modified epithelial cells (figs. 11, 12), all these structures are not shown in the gbe of b. almorhae and s. richardsonii. discussion the epidermis is ectodermal in origin and consists of several layers of simple cells, of which the outer are being constantly worn away by wear and tear and replaced by newer ones which develop at their base. these layers of cells are composed of flattened cells, known as epithelium cells, of which the deepest layers are made up of columnar cells forming the stratum germinativum in which cells are always multiplying by mitotic division to replace the outer worn out cells. a superficial layer of dead horny cells, forming the stratum corneum is not present in fishes as an adaptation to life in water (khanna, 1993). the epidermis of the gbe of b. almorhae and the structures associated with them show considerable structural modifications. these may be considered as figure 4. semph of the gbe of botia almorhae epidermis showing microridges at the surface epithelium (scale bar=5 μm). figure 5. semph of the gbe of homaloptera brucei showing that the microridges are generally; finger printlike, and are often arranged in the form of small groups (marked by arrows) (scale bar =5 μm). figure 6. semph of the gbe of schizothorax richardsonii showing finger print-like patterns of microridges (marked by arrows) (scale bar=10 μm). figure 7. semph of the gbe of schizothorax richardsonii showing finger print-like patterns of microridges that have canaliculi and microbridges (marked by arrows) (scale bar =5 μm). figure 8. semph of the gbe of botia almorhae showing the opening of mucous cells (marked by arrows) (scale bar=50 μm). figure 9. semph of the gbe of homaloptera brucei showing the openings of mucous cells (marked by arrows) (scale bar=10 μm). 373 int. j. aquat. biol. (2017) 5(6): 370-374 adaptations in relation to its peculiar habit and habitat. homaloptera brucei is adapted to live in hill-streams characterized by fast flowing current under boulders. it is found in mountain streams (high gradient streams). the general body epidermis of h. brucei, exhibits compactly arranged microridges forming intricate mesh-like patterns, which are characteristic of the habitat under the boulders and stones. furthermore, these microridges may gain a firm base and support from a dense network of fine filaments. the free surface of each epithelial cell is characterized by the presence of a series of microridges. the microridges of the cells appear smooth and uniform in width. frictional force is less under boulder and stones; therefore, the requirement of lubrication is minimum in h. brucei. the epidermis of h. brucei possesses a large number of elevations distributed at irregular intervals. the epidermis with elevations alternates with that of the non-elevated surface. the average thickness of the epidermis varies in the two regions of h. brucei (non-elevated region: 61.7 μm, at elevated region: 85.9 μm) (bisht, 1999). breeding tubercles are keratin based epidermal nodules, which are found in at least fifteen families of fishes in four orders. breeding tubercles might offer a workable tool for examination of sexual selection among cyprinids. the large number of tubercles in males indicates increasing reproductive power of the fishes. the primary function of the epidermis is to provide protection against environmental hazards. in fish, this function is mainly attributed to the gland cells which secrete their contents on the surface (singh, 2014). in the general body epidermis of h. brucei and s. richardsonii finger print-like microridges, may in addition impart firm consistency or rigidity to the free surface of the epithelial cells. this could be considered as an adaptation to withstand mechanical stress and protect the surface of the fish, which has the characteristic habit of bottom dwelling. this specific pattern of microridges helps in the spreading of mucus from mucous cells over a wide area. the sudden spread of mucus is facilitated by numerous canaliculi formed by epidermal microridges. the abundance of mucus on the skin of s. richardsonii exhibits its habitat in open water or bottom dwellings, where frictional force is very high. this study indicates that the presence of mucus secretion is performing multifunctional activities, assisting the fish to adapt to their characteristic mode of life for their maintenance against adverse environmental conditions, to which these are exposed. on the other hand open water surfaces have more pathogenic agents, which affect the epidermis; therefore, s. richardsonii has a greater more requirement of mucus. it also renders the skin less permeable and prevents the entry of pollutant materials and micro-organisms, which would otherwise infect the fish. fish skin is a multipurpose tissue that serves numerous vital functions including figure 10. semph of the gbe of schizothorax richardsonii showing the mucous openings and their secretory contents profusely at the surface through a small pore. (marked by arrows) (scale bar=10 μm). figure 11. semph of the gbe of homaloptera brucei showing the well-developed tubercles at high magnification (marked by arrows) (scale bar =200 μm). figure 12. semph of the gbe of homaloptera brucei of showing polygonal epithelial cells and unculi on the tubercles (marked by arrow) (scale bar=10 μm). 374 singh and bisht / ultrastructural study of general body epidermis in three hill-stream fishes chemical and physical protection, sensory activity, behavioural purposes or hormone metabolism. further, it is an important first line defence system against pathogens, as fish are continuously exposed to multiple microbial challenges in their aquatic habitat (rakers et al., 2010). studies of fish skin indicated that epidermal cells follow separate pathways of differentiation in different fishes. in most of the fishes, the epidermis is related more to the deposition of slime over its surface and undergoes the process of mucogenesis and in some the epidermal cells undergo the process of keratinization forming a layer at the surface (singh and bisht, 2014). acknowledgments we are grateful to all the people who helped in different part of this work especially the department of college of veterinary and animal sciences, g.b. pant university of agriculture and technology, pant nagar (u.k.), for providing necessary instruments for this research work. thanks are also due to m.p. singh for his technical support. references bhatt s.d., pathak j.k. 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(2010). ‘fish matter’: the relevance of fish skin biology to investigative dermatology. experimental dermatology, 19: 313-324. singh h. (2014). characterization of snout in the hill stream fishes; botia almorhae (teleostei: cobitidae), homaloptera brucei (teleostei: balitoridae) and schizothorax richardsonii (teleostei: cyprinidae) of kumaunhimalaya: a scanning electron microscopic (sem) investigation. international journal of zoology and research, 4(1): 1-6. international journal of aquatic biology (2014) 2(6): 313-318 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2014 npajournals. all rights reserved original article seasonal and sexual variations of fatty acid composition in fillet of capoeta erhani yılmaz emre1, 3, kazım uysal2, faruk pak3, nesrin emre3, mustafa kavasoğlu*21 1the mediterranean fisheries research, production and training institute, kepez, antalya, turkey. 2dumlupınar university, faculty of arts and sciences, department of biology 43100, kütahya, turkey. 3akdeniz university, faculty of sciences, department of biology, 07058, campus, antalya, turkey. article history: received 25 march 2014 accepted 13 october 2014 available online 2 5 december 2014 keywords: fatty acid nutritional value season sex abstract: the lowest lipid levels of capoeta erhani observed in winter and vice versa in summer. the fatty acid composition of the fillets was significantly different among seasons (p<0.05), while the variations among sexes were not at the same degrees (p>0.05). the ratios of the unsaturated fatty acids (ufas) were higher than half of the total fatty acids among all seasons. the level of pufa was highest in autumn (25.91%), and lowest in summer (22.11%). among seasons and sexes, the levels of total n3 pufas in total fatty acids changed from 15.43% to 21.89% and n6 pufas from 3.8% to 7.97%, respectively. the level of n3 pufas was present in excess that of the n6 pufas. the ratios of the n3 pufas to n6 pufas in the fillets of c. erhani were highest in autumn for both sexes and remarkably influenced by seasons. introduction oil, especially fish oil is important food source because of providing physiological and energy needs of human. fats absorb hydrophobic vitamins and regulate cholesterol metabolism (connor, 2000; jabeen, 2010). the importance of fats is due to their polyunsaturated fatty acid content (aras, 2003; louly, 2011). fatty acids including eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) were found mostly in fish oil contributing to the reduction of cardiovascular diseases, regulating the synthesis of prostaglandins and helping cancer prevention (marichamy, 2012). it is known that neurons do not have regeneration capability and neuronal membranes have little generative capabilities. pufas comprise 1/3 of fatty acids content of nervous system (bourre, 1997). therefore, fatty acids of fish oils are not only essential for normal growth and development, but also contain anti-immune system properties. in addition, they are used in production of * corresponding author: mustafa kavasoğlu e-mail address: kavasoglu87@hotmail.com pharmacological drugs. fishes are the important sources of proteins, vitamins; essential minerals and rich n3 long chain polyunsaturated fatty acids (jabeen, 2010). the previous studies indicate that nutritional value, especially fatty acid composition of fish, changes with season, sex, species, salinity and temperature (gills, 1984; uysal, 2011). therefore, the determination of season that fish have the best nutritional quality in terms of fatty acid composition is important for human diet. capoeta erhani is a member of cyprinid family and found in the ceyhan river of eastern anatolia. it is a benthic freshwater species. since, there is no reports found concerning the fatty acid composition of this fish. therefore, we aimed to determine the seasonal and sexual nutritional values and the fatty acid compositions of c. erhani in this study. materials and methods fish sampling and experimental procedures: the 314 international journal of aquatic biology (2014) 2(6): 312-318 specimens of c. erhani were caught from the menzelet dam lake (37°41'12''n; 36°50'11''e). after biometric data analyses (i.e., weight and length), the specimens were transferred on ice from field to the laboratory. a total of 40 specimens including 10 specimens in each season (5 males and 5 females) were examined. the dorsal muscle of specimens from each sex were excised and homogenized in a blender for 3 min for subsequent lipid extraction. measurements: total lipid extraction was performed according to the procedure of bligh and dyer method (1959). methyl esters were prepared by transmethylation using 2m koh in methanol and hexane according to ichihara et al. (1996) with minor modifications. extracted lipids (10 mg) were dissolved in 2 ml hexane followed by 4 ml of 2 m methanolic koh. the tube was then vortexed for 2 min at room temperature. after centrifugation at 4000 rpm for 10 min, the hexane layer was taken for further analysis in gc analyses. gas chromatographic conditions: the fatty acid composition was analyzed by gc with auto sampler (thermo focus) equipped with a flame ionization detector and a fused silica capillary column (30 m x 0.32 mm, id x 0.25 μm film). the oven temperature was 140°c, held for 5 min, raised to 200°c at rate of 4°c/min and to 220°c at a rate of 1°c/min, while the injector and the detector temperature were set at 220°c and 280°c, respectively. the sample size was 5 μl and the carrier gas was controlled at 16 psi. the split used was 1:40. fatty acids were identified by comparing the retention times of fame mixture (supelco). the results were expressed in gc area fatty acids spring mean ± sd summer mean ± sd autumn mean ± sd winter mean ± sd c12:0 1.832 ± 0.17 0.762 ± 0.34 0.284 ± 0.16 1.234 ± 0.60 c13:0 0.102 ± 0.05 0.087 ± 0.04 0.07 ± 0.02 0.086 ± 0.04 c14:0 6.234 ± 0.60 5.105 ± 0.35 4.356 ± 0.72 5.938 ± 0.94 c14:1 0.148 ± 0.02 0.167 ± 0.04 0.13 ± 0.04 0.142 ± 0.02 c15:0 0.41 ± 0.11 0.882 ± 0.73 1.304 ± 0.51 0.47 ± 0.20 c15:1 0.062 ± 0.02 0.135 ± 0.08 0.182 ± 0.06 0.072 ± 0.04 c16:0 18.942 ± 0.89a 18.357 ± 1.80a 20.066 ± 1.43a 19.224 ± 0.63a c16:1 8.392 ± 2.28a 11.355 ± 3.01a 10.922 ± 3.60a 9.45 ± 2.37a c17:0 0.26 ± 0.09 0.37-0.14 0.492 ± 0.15 0.31 ± 0.20 c17:1 0.156 ± 0.03 0.255 ±0.12 0.304 ± 0.10 0.164 ± 0.06 c18:0 1.658 ± 0.39 1.852 ± 0.34 3.626 ± 1.00 1.72 ± 0.37 c18:1n9 21.464 ± 2.16b 14.867 ± 4.71a 10.734 ± 4.73a 20.124 ± 5.16b c18:1n7 2.836 ± 0.78 3.03 ± 0.38 2.798 ± 0.79 2.888 ± 0.88 c18:2n6 5.85 ± 1.19 3.415 ±1.31 1.328 ± 0.23 5.64 ± 1.97 c18:3n3 0.69 ± 0.22 1.137 ± 0.61 1,228 ± 0.42 0.602 ± 0.28 c20:0 0.126 ± 0.04 0.13 ± 0.01 0.134 ± 0.02 0,132 ± 0.02 c20:1 1.638 ± 0.13 1.422 ± 0.53 1.058 ± 0.41 1.716 ± 0.21 c20:3n6 0.196 ± 0.05 0.175 ± 0.0 0.116 ± 0.05 0.192 ± 0.06 c20:4n6 0.072 ± 0.02 0.07 ± 0.02 0.13 ± 0.02 0.06 ± 0.02 c20:2n6 0.834 ± 0.23 1.3 ± 0.39 2.226 ± 1.14 0.924 ± 0.54 c20:5n3 5.23 ± 0.73a 7.187 ± 1.50ab 9.172 ± 1.33b 5.626 ± 1.18a c22:1n9 0.066 ± 0.02 0.147 ± 0.09 0.15 ± 0.03 0.08 ± 0.01 c23:0 0.184 ± 0.03 0.24 ± 0.05 nd 0.196 ± 0.03 c22:6n3 9.518 ± 1.81a 10.515 ± 3.26a 10.568 ± 2.66a 10.122 ± 1.48a unidentified fatty acids 13.1 17.038 18.622 12.888 * averages followed by the same letter show no statistical differences (p>0:05). nd: not detected table 1. seasonal variation of fatty acid ratios in fillets of female c. erhani (% of total fatty acids). 315 emre et al/ seasonal and sexual variations of fatty acid composition in c. erhani as percentage value. statistical analyses: the results were presented as means ± sd (standard deviation). the data was analyzed by the spss package programme, version 12. the seasonal variations and fatty acid profiles of specimens were compered using one-way anova followed by tukey’s multiple comparison tests. the sexual comparison was performed by student t-test. the results were found significant at p<0.05. results and discussion the mean weights and lengths of the collected fishes were 168.76 g and 24.92 cm, respectively. the total lipid of the c. erhani ranged 64.62% to 68.45%. the lipid content of c. erhani was maximum in summer and minimum at winter. high lipid level in summer and low lipid level in winter have noted in most fishes (shirai et al., 2002; zlatanos, 2007). decreases fillet lipid content can be as result of feeding situation. similar and contradictory results have been reported for solea solea (gökçe, 2004), sander lucioperca (uysal, 2005), and cyprinus carpio (güler, 2008). the fatty acid composition of c. erhani in both sexes was presented in tables 1 and 2. mean of 24 fatty acids from c12:0 to c22:6n3 were identified. collectively, the 24 fatty acids were comprised about average 87% of the total fatty acids. there were significant differences (p<0.05) among seasons in the fatty acid concentrations. the fatty acids occurring in the highest proportions were palmitic (16:0), palmitoleic (16:1), oleic (18:1n9), eicosapentaenoic (epa) (20:5n3), docosahexaenoic (dha) (22:6n3) acids in all seasons. in particular, fatty acids spring mean±sd summer mean±sd autumn mean±sd winter mean±sd c12:0 1.172 ± 0.51 0.756 ± 0.51 0.366 ± 0.14 0.844 ± 0.07 c13:0 0.07 ± 0.01 0.122 ± 0.05 0.067 ± 0.01 0.05 ± 0.01 c14:0 5.48 ± 0.48 5.48 ± 0.64 4.254 ± 0.70 5.606 ± 0.70 c14:1 0.164 ± 0.02 0.168 ± 0.03 0.128 ± 0.01 0.142 ± 0.04 c15:0 0.444 ± 0.06 1.14 ± 0.58 1 ± 0.51 0.332 ± 0.08 c15:1 0.076 ± 0.02 0.162 ± 0.07 0.138 ± 0.09 0.046 ± 0,01 c16:0 18.478 ± 0.64a 18.906 ± 0.94a 19.388 ± 1.02a 20.134 ± 1.00a c16:1 9.4 ± 1.22ab 13.182 ± 1.61b 10.91 ± 2.29b 7.632 ± 1.08a c17:0 0.298 ± 0.06 0.488 ± 0.17 0.458 ± 0.11 0.202 ± 0.07 c17:1 0.18 ± 0.04 0.286 ± 0.10 0.264 ± 0.10 0.2 ± 0.15 c18:0 1.82 ± 0.25 2.026 ± 0.49 2.502 ± 0.66 1.742 ± 0.42 c18:1n9 20.242 ± 0.96b 12.314 ± 4.25a 11.878 ± 3.08a 23.128 ± 3.44b c18:1n7 2.988 ± 0.39 3.312 ± 0.30 2.832 ± 0.54 2.272 ± 0.54 c18:2n6 5.43 ± 0.97 2.718 ± 1.32 1.632 ± 0.36 6.822 ± 1.59 c18:3n3 0.726 ± 0.09 1.172 ± 0.47 1.126 ± 0.36 0.522 ± 0.13 c20:0 0.134 ± 0.01 0.194 ± 0.10 0.128 ± 0.01 0.114 ± 0.02 c20:1 1.916 ± 0.09 1.308 ± 0.32 1.174 ± 0.15 1.884 ± 0.15 c20:3n6 0.212 ± 0.05 0.154 ± 0.03 0.128 ± 0.02 0.202 ± 0.02 c20:4n6 0.068 ± 0.02 0.102 ± 0.04 0.108 ± 0.04 0.08 ± 0.01 c20:2n6 0.936 ± 0.22 1.79 ± 0.66 2.152 ± 0.76 0.872 ± 0.33 c20:5n3 5.452 ± 0.28a 7.182 ± 1.36b 9.186 ± 0.87c 5.056 ± 0.78a c22:1n9 0.076 ± 0.01 0.136 ± 0.05 0.13 ± 0.08 0.075 ± 0.01 c23:0 0.197 ± 0.07 0.207 ± 0.02 0.305 ± 0.03 0.16 ± 0.05 c22:6n3 10.372 ± 1.13a 9 ± 1.74a 11.58 ± 3.11a 11.602 ± 2.76a unidentified fatty acids 13.669 17.695 18.166 10.281 * averages followed by the same letter show no statistical differences (p>0:05). nd: not detected table 1. seasonal variation of fatty acid ratios in fillets of male c. erhani (% of total fatty acids). 316 international journal of aquatic biology (2014) 2(6): 312-318 c. erhani has more c16:0 and c18:1n9 than other fatty acids through study period. the highest concentration of saturated fatty acids (sfas) was found in summer and autumn, mainly in the form of palmitic acid (16:0). similar results were reported by aggelousis and lazos (1991) and rasoarahona et al. (2005) who noted that palmitic acid was in the highest level in autumn for three tilapia species. high values of sfas in summer and its low levels in winter could be due to adaptation of fish to cold temperature. logue (2000) reported that adaptation of fish species to cold water temperature is associated with increasing unsaturation. however, bulut et al. (2012) indicated that sfas are not affected by seasonal variations. in this study, the ratios of sfas of male reached to the highest level in summer and that of female in autumn, but in both sexes there were not significantly change (p>0.05). huynh (2007) noted that in both spawning and nonspawning periods, fatty acid contents decrease in the order of mufa>sfa>pufa. also, kozlova and klotimchenko (2000) indicated that this situation was characteristic for fatty fishes. similar result was found in present study for spring, summer and winter seasons that can be as a result of feeding period of c. erhani. the results also showed that the major mufa found in all samples is oleic acid (c18:1). many studies have reported similar finding in different species (huynh, 2007, prato, 2012; usydus, 2012). oleic acid (18:1n9) reached the highest level in female and male fillets in spring and winter, respectively (tables 1 and 2). changes of oleic acid among seasons were significant (p<0.05), but sexual variations did not show the same result (p>0.05). usydus (2012) noted that the percentages of mufa in baltic sprat (sprattus sprattus balticus) were higher during the spawning season than the feeding season. the results showed that total mufa contents and oleic acid were similar in spring and autumn. the oleic acid has drastic roles in human diet. lemaitre et al. (2006) reported that trans oleic acid reduces the risk of coronary heart disease. the palmitoleic acid was the second most abundant mufa ranged from 7.63% to 13.18% in male and from 8.39% to 11.35% in female with the highest values in summer and lowest in winter for male (tables 1 and 2). seasonal variations in the levels of palmitoleic acid were significant in male of c. erhani (p<0.05). the levels of pufas in total fatty acids were found in the range of 22.11% to 25.91% in males and 22.39% to 24.76% in females during all seasons (figs. 1 and 2). eicosapentaenoic (epa) and docosahexaenoic (dha) acids comprise a great part of pufas in fillets of c. erhani. the levels of epa in the fillets of c. erhani varied from 5.23% (spring) to 9.17% (autumn) for female; 5.05% (winter) to 9.18% (autumn) for male, and of the dha from 9.51% (spring) to 10.56% (autumn) for female; 9% (summer) to 11.60% (winter) for male. in the light of these values, the high levels of epa and dha is observed in autumn and winter seasons. therefore, it is concluded that c. erhani are more beneficial for human nutrition in those seasons. çelik et al. (2005) reported that epa contents of fishes in cold regions figure 1. seasonal variations of main fatty acid groups in fillets of male c. erhani. figure 2. seasonal variations of main fatty acid groups in fillets of female c. erhani. 317 emre et al/ seasonal and sexual variations of fatty acid composition in c. erhani are higher than those of warm regions. the results of present study also support this finding. the levels of epa and dha were influenced by seasons. but, only variations in the level of epa were significant (p<0.05). the levels of epa and dha between sexes were not different remarkably. usydus (2012) found similar results about epa and dha in feeding and wintering seasons. however the results on the seasonal variations of epa and dha were in a contrary with previous reports carried out on seasonal variations of fatty acid compositions in different species (gökçe, 2004; shirai et al., 2002). in addition, the percentage of dha always exceeds that of epa. the percentages of these n3 pufas in fillets depend on diet (sargent et al., 2002) and their variations might be related to changes in the nutritional habits of the fishes (norrobin et al., 1990). the results showed that the levels of n3 pufas ranged from 15.43% to 20.96% in female and 16.55% to 21.89% in male. the levels of n6 pufas ranged from 3.8% to 6.95% in female and 4.02% to 7.97% in male (fig. 3) indicating that the levels of n3 pufas reach the highest levels in autumn in both sexes. but the opposite situation was observed in n6 pufas levels especially in female. the levels of n3 pufas exceeded that of the n6 pufas. similar results were observed by huynh (2007), mnari (2006) and zlatanos (2007). the n3/n6 ratio is a proper indicator of the nutritional value of fishes (piggott and tucker, 1990). the high level of n3/n6 fatty acid ratio is essential in the diet of human beings to decrease plasma lipids and reducing heart disease and cancer risk (kinsella et al., 1990). the results of the present study showed that the n3/n6 ratios of c. erhani were relatively high. the n3/n6 ratios were highest (approximately 5.5) in autumn in both male and female. considering the n3/n6 ratios and high level of n3 pufas, this study revealed that c. erhani is a healthy food for human diet especially during autumn period. references aggelousis g., lazos e.s. 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(2014) 2(2): 99-104 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article dietary synbiotic improves the growth performance, survival and innate immune response of gibel carp (carassius auratus gibelio) juveniles fahimeh mahghani1, ahmad gharaei*1,2, mostafa ghaffari3, raza akrami4 1department of fisheries, faculty of natural resources, university of zabol, zabol, iran. 2department of fisheries, international hamoun wetland research institute, university of zabol, iran. 3department of fisheries, faculty of marine sciences, chabahar maritime university, chabahar, iran. 4department of fisheries, islamic azad university, azadshahr branch, iran. article history: received 17 october 2013 accepted 14 february 2014 available online 2 5 april 2014 keywords: synbiotic growth survival innate immunity gibel carp abstract: this study was conducted to evaluate the effect of dietary synbiotic (biomon imbo) on the growth performance, survival and innate immune response of gibel carp (carassius auratus gibelio) juveniles. fish with initially average weight 15.5±0.2 g were randomly distributed into tanks (20 fish/tank). basal diets (biomar) were supplemented with 0 (control), 0.5, 1.0, 1.5 and 2.0 g symbiotic per kg-1 in a totally randomized design trial in triplicate group. at the end of the experiment (60 days), innate immune parameters (serum ig levels and lysozyme activity) and growth factors (final weight, weight gain (wg %), specific growth rate (sgr) and food conversion ratio (fcr)) were assessed. according to our results, the growth performance and feed efficiency improve in fish fed on the diet containing 2.0 g kg-1 synbiotic (p<0.05). there were no significant differences in survival among all treatments (p>0.05). lysozym activity and serum ig levels in fish fed the 2.0 g kg-1 synbiotic diet was higher compared to other experimental groups (p<0.05). these results indicate that synbiotic can be considered as a beneficial dietary supplement for improving the immune response and growth performance gibel carp (carassius auratus gibelio) juveniles. introduction improvement and protection of fish health in commercial production practices is a major factor in the aquaculture industry. a novel approach to these goals is application of probiotics and prebiotics in fish farming industry (irianto and austin, 2002; wang and xu, 2006; wang et al., 2008). probiotic defined as organisms and substances which contribute to intestinal microbial balance and could enhance the growth and health of the host (gatesoupe, 1999; kesarcodi-watson et al., 2008). prebiotics are defined as non-digestible dietary ingredients that beneficially affect the host by selectively stimulating the growth and/or activating the metabolism of health-promoting bacteria in the gastrointestinal tract (manning and gibson, 2004). * corresponding author: ahmad gharaei e-mail address: agharaei551@gmail.com gibson and roberfroid (1995) have defined the mixture of preand probiotics as synbiotics that exert synergistic effects in promoting beneficial bacteria and the health of the gastrointestinal tract of the host, so their potential applications have spurred attention. although benefits associated with prebiotics and probiotics are desirable, researchers are concerned about a conclusive result, depending on type and amount of preand probiotic consumed. therefore more studies need to be conducted to provide a better understanding of their direct effects on health. the use of probiotics and prebiotics in aquaculture is now widely accepted but limited data is available regarding the application of synbiotics in aquaculture (li et al., 2009; rodriguez-estrada et al., 2009; daniels et al. 2010; zhang et al., 2010; ai et 100 mahghani et al./ int. j. aquat. biol. (2014) 2(2): 99-104 al., 2011; ye et al., 2011; mehrabi et al., 2012; nekoubin et al., 2012; nekoubin and sudagar, 2012; montajami et al., 2012). carassius auratus gibelio is an improved strain of carp and suitable fish model for carrying out in vivo aquaculture experiments. also, gibel carp known as goldfish and one of the most popular ornamental species in the world due to its varieties with attractive body shape and skin color (moreira et al., 2011). the aim of the present research was to study the effects of synbiotic (biomin imbo) on growth performance, survival, and immune response of gibel carp juveniles (carassius auratus gibelio). materials and methods gibel carp juveniles (carassius auratus gibelio) (average initial weight 15.5±0.2 g), obtained from a local ornamental fish farm (golestan province, iran), were randomly stocked into 12 tanks (70 l) at a density of 20 fish/tank (3 tanks/treatment). water temperature, dissolved oxygen and ph were monitored daily and maintained at 25.3±0.8°c, 5.2±0.3 mg/ l, and 7.24±0.15, respectively. continuous aeration was provided to each tank through an air stone connected to a central air compressor. the type of synbiotic was applied in this study was biomin imbo (biomin, herzogenburg, austria) in which was comprised of probiotic (entercoccus faecium 5×1011 cfu/g) and fructooligosaccharide (fos) as prebiotic. to prepare the diets, a commercial pellet diet (biomar) (containing 41% protein, 6% lipid, 11.1% ash and 23.2 mj/kg ge) was mixed with the supplementation with various levels of synbiotic (0.5, 1.0 ,1.5 and 2.0 g/kg) and water, and made again into pellets (cerezuela et al., 2008). the pelleted diets were air-dried, ground and sieved to produce a suitable crumble. then the feed stored at 4°c until feeding trial. the experimental fish were weighted every 15 days in order to adjust the daily feed rate which was 3% of the total biomass the fish were fed three times daily to apparent satiation for 60 days. in order to measure the growth parameters, weight and length of all fish were measured at every 15 days interval. after 8 week feeding period, weight gain (wg%), specific growth rate (sgr %/day), feed conversion ratio (fcr) and survival rate (%) were calculated according to following equations (bekcan et al., 2006): wg(%) = (wt−w0) × 100/w0 , sgr = (ln wt−ln w0) × 100/t , fcr = dry feed fed / wet weight gain, , survival rate = (nt/n0) × 100. here wt and w0 are final and initial body weights (g), respectively, t is duration of experimental days, n0 is the initial number of fish and nt is the final number of fish. at the end of trial, six fish/tank were sampled for blood. in order to provide sufficient blood for the subsequent assays fish were euthanized, the tail cut off and blood was removed. for serum isolation, blood samples were aliquoted into non-heparinized tubes and left to clot for 12 h (at 4°c), prior to centrifugation at 5000 g (3500 rpm) for 5 min in a clinical centrifuge. isolated serum was stored at 20°c until further analysis. serum total immunoglobulin (ig) levels were determined according to the method described by siwicki and anderson (1993). briefly, serum total protein content was measured using a microprotein determination method (c-690; sigma), prior and after precipitating down the immunoglobulin molecules, using a 12% solution of polyethylene glycol (sigma). the difference in protein content represents the ig content. serum lysozyme activity was determined by turbidometric assay according to the method of ellis with slight modifications (ellis, 1990). aliquots (1.75 m/l) of micrococcus lysodeikticus suspension (sigma) (0.375 mg/ml, 0.05 m pbs, ph 6.2) were mixed with 250 μ/l of each sample and the optical density was measured after 15 and 180 s by spectrophotometer (biophotometer eppendorf) at 670 nm. pbs was used as the blank and results were expressed in amounts of lysozyme μg/mg of sample calibrated using a standard curve determined with hen’s egg white lysozyme (sigma) in sterile sodium phosphate buffer. 101 mahghani et al./ int. j. aquat. biol. (2014) 2(2): 99-104 data were analyzed by one-way analysis of variance using the statistical software spss version 18.0. subsequent significance between groups was defined by duncan’s multiple range tests. data are presented as treatment means ± standard of deviation (sd). differences were considered significant when p value was less than 0.05. results the growth performance of gibel carp juveniles fed diets supplemented with various levels of dietary synbiotic is presented in table 1. compared to the control group, fish fed different dietary levels of synbiotic diet displayed improved (p>0.05) growth performance, including wg, sgr and fcr. there were no significant differences in survival among all treatments (p>0.05) however, fish fed different dietary levels of synbiotic had higher survival compared to the control. the effects of different dietary levels of synbiotic on the serum total immunoglobulin (ig) levels and lysozym activity of gibel carp juveniles are shown in figures 1 and 2, respectively. both innate immune responses measured (ig and lysozyme activity) were significantly higher (p<0.05) in synbiotic fed fish compared to the control group. fish fed 2.0 g kg-1 synbiotic displayed significantly elevated lysozyme activity (100.66 ± 2.51 μg ml-1) and total immunoglobulin (125 ± 10.53 μg ml-1) compared to the control. discussion in the present experiment, the growth performance (p>0.05), total immunoglobulin (ig) and lysozym activity of gibel carp were significantly (p<0.05) improved by supplementing the basal diet with synbiotic. this is in agreement with results some studies have revealed the positive effects of synbiotic on the growth performance in fish. similarly, synergistic effects application of synbiotic was found to enhance the growth performance and survival of white shrimp (litopenaeus vannamei) (li et al., 2009), rainbow trout (oncorhynchus mykiss) (rodriguez-estrada et al., 2009), european lobster (homarus gammarus) (daniels et al., 2010), sea cucumber (apostichopus japonicus) (zhang et al., 2010), caspian kutum (rutilus frisii kutum) fry (talibi haghighi et al., 2010), zebrafish (danio rerio) control 0.5 g kg-1 1.0 g kg-1 1.5 g kg-1 2.0 g kg-1 final weight (g) 31.5 ± 2.5 34.8 ± 1.7 34.4 ± 2.9 35.5 ± 2.6 35.9 ± 3.12 wg (%) 54.2± 10.9 70.03 ± 10.2 69.06 ± 16.9 72.5 ± 13.6 73.16 ± 14.4 sgr (%/day) 0.48 ± 0.07 0.58 ± 0.06 0.56 ± 0.1 0.6 ± 0.08 0.62 ± 0.1 fcr 4.23 ± 0.8 3.3± 0.16 3.3 ± 0.75 3.2 ± 0.51 3.1 ± 0.84 survival 90.2 ± 9.8 93.3± 6.6 96.6 ± 3.5 96.6 ± 3.5 97.6 ± 2.5 table 1. growth and feed utilization of gibel carp fed different dietary levels of synbiotic for 60 days figure 1. serum total immunoglobulin (ig) levels gibel carp juvenile fed with diets containing different levels of symbiotic. bars assigned with different superscripts are significantly different (p<0.05). figure 2. lysozyme activity of gibel carp juvenile fed with diets containing different levels of symbiotic. bars assigned with different superscripts are significantly different (p<0.05) 102 mahghani et al./ int. j. aquat. biol. (2014) 2(2): 99-104 larvae (nekoubin et al., 2012), rainbow trout (mehrabi et al., 2012), grass carp (ctenopharyngodon idella) (nekoubin and sudagar, 2012) and taxas cichlid (herichthys cyanoguttatus) (montajami et al., 2012). improved growth performance may be due to the elevation of digestive enzyme activities, possible improvements of intestine morphology or via synbiotic fermentation by endogenous gut microbes to produce short chain fatty acid (scfas). unlike this study, ai et al. (2011) reported that no significant interactions were observed between dietary bacillus subtilis and fructooligosaccharide (fos) on the growth performance, immune response and disease resistance of large yellow croaker (larimichthys crocea). the absence of interactions between fos and bacillus subtilis in their study was attributed mainly to the absence of the fos actions. moreover, the synbiotic effects may be also influenced potentially by the type of species and the environment. ye et al. (2011) demonstrated that feeding with fos, mos or bacillus clausii alone, or in various combinations, yielded best growth performance, feed efficiency and healthy status of the japanese flounder (paralichthys olivaceus) and, which was more pronounced in fish fed the synbiotics than those fed preand probiotics alone. synbiotics, the combined application of probiotics and prebiotics, is based on the principle of providing a probiont with a competitive advantage over competing endogenous populations; thus, effectively improving the survival and implantation of the live microbial dietary supplement in the gastrointestinal tract of the host (gibson and robefroid, 1995). the use of synbiotics it may be possible to produce greater benefits than the application of individual probionts (merrifield et al., 2010). stimulation of the immune response of fish through dietary supplements is of high interest for commercial aquaculture (staykov et al., 2007). in this respect, the innate immune system is very important since aquatic animals are continually vulnerable to numerous opportunistic pathogens and this part of immune response provides the first line of defense for the host (magnadóttir, 2006). the use of natural immunostimulants is a developing area in aquaculture because they are biodegradable, biocompatible and safe both for the environment and human health (ortuno et al., 2002). it is clear from the present study that dietary supplementation of synbiotic can modulate the innate immune responses of gibel carp. based on the results, fish fed with 2.0 g kg-1 synbiotic had significantly higher total immunoglobulin (ig) and plasma lysozyme compared to those fed 0.5, 1.0 and 1.5 g kg-1 synbiotic and control group. ye et al. (2011) also reported that lysozyme activity was significantly higher in japanese flounder (paralichthys olivaceus) fed a synbiotic diet (fos + bacillus clausii, mos+ bacillus clausii or fos + mos + bacillus clausii). another study on white shrimp (litopenaeus vannamei) also reported the synergistic effect between 108 cfu/g bacillus megeterium and 0.2% isomaltooligosaccharides on immune responses and disease resistance (li et al. 2009). the immunostimulatory nature of synbiotic may be attributed to stimulation of the growth of beneficial bacteria such as lactic acid bacteria (zhang et al., 2011). mourino et al. 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(2015) 3(1): 14-18 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article extended distribution of the invasive sucker catfish pterygoplichthys pardalis (pisces: loricariidae) to cauvery river system of peninsular india murugan muralidharan*1, kaliappan manikandan1, muthukaruppan gobi2 1sri paramakalyani centre for environmental sciences, manonmaniam sundaranar university, alwarkurichi – 627 412, tamil nadu, india. 2school of bio-engineering, srm university, kattankulathur – 603 203, tamil nadu, india. article history: received 7 november 2014 accepted 21 december 2014 available online 2 5 february 2015 abstract: this report provides the first record of the exotic sucker fish p. pardalis from the cauvery river system, tamilnadu state, india. information on the assumed presence of this invasive species based on sightings of a hitherto unknown species by local people and fishermen in stretches of cauvery river has been confirmed by our study. sailfin sucker fishes are popular among pet traders and aquarists in southeast asian countries and have been regarded as invaders worldwide. occurrence of p. pardalis in newer habitats due to its potential invasion in the south indian waters issues threat to native fauna. keywords: sailfin fish, pterygoplichthys, exotic species, cauvery river, india. introduction invasion of aquatic systems by exotic fishes, widely recognized as the consequence of human mediated environmental intervention, has become the prominent threat to biodiversity in the recent years. aquaculture and aquariums are responsible for introduction of ornamental and economically important fish species to newer environments (chavez et al., 2006). the family loricariidae includes catfish generally referred to as “plecs” that are widespread throughout south america. though the majority of the species available are from wild, these are also being bred through commercial breeding farms for the aquarium trade. as the reason they are often hybridized among stocks for better varieties, hence, the identity of individual species is always uncertain (wu et al., 2011). pterygoplichthys pardalis, suckermouth armored catfish of the family loricariidae is native to tropical america, occurring in the lower, middle and upper amazon river basin of brazil and peru * corresponding author: murugan muralidharan e-mail address: muralistream@gmail.com (weber, 2003; page and robins, 2006). this species is characterized by bony plates covering the body, a pair of subterminal barbels, sucking lips, usually a spine in front of the adipose fin, a flat-bottom body (page and burr, 1991) and uncoalesced dark spots on a light background (page and robins, 2006). being voracious algal feeders, the introduction of this species was solely for the aquaria. species of pterygoplichthys are widespread invasive fish known from many areas outside their native ranges, including hawaii, mexico, puerto rico and the continental united states (ludlow and walsh, 1991; page and burr, 1991; bunkley-williams et al., 1994; chavez et al., 2006). species of this genus have been recorded from several southeast asian countries, including singapore, malaysian peninsula, java, sumatra, vietnam and taiwan (liang et al., 2005; page and robins, 2006: levin et al., 2008), bangladesh (hossain et al., 2008) and india (daniels, 2006; krishnakumar et al., 2009; knight, 2010; sinha et al., 2010). in addition, p. pardalis has 15 muralidharan et al./ extended distribution of p. pardalis to south india been recorded from lake matano, sulawasi, indonesia (herder et al., 2012), malaysian peninsula (samat et al., 2008), phillipines (chavez et al., 2006) and sri lanka (sumanasinghe and amarasinghe, 2013). further, it has been regularly bred and exported from singapore. pterygoplichthys pardalis and p. multiradiatus are among the most popular and intensively marketed varieties of tropical aquarium fish species in south india (knight, 2010). the ecological impacts upon introduction of this species to the aquatic habitat are disruption of food chain by overgrazing of benthic algae (liang et al., 2005; chavez et al., 2006), competing with native species (nico and martin, 2001), modifying substrates and disrupting benthic communities (hoover et al.,2004) and damaging the banks by burrowing (bunkley-williams et al., 1994). information on the new ranges occupied and extension in distribution range would enable effective management. herein, we report the occurrence of p. pardalis in cauvery river of peninsular india. materials and methods fish collection was accomplished with cast nets. specimens were preserved in 10% buffered formalin and stored at sri paramakalyani centre for environmental sciences, manonmaniam sundaranar university, alwarkurichi. meristic characters and morphometric measurements were carried out following the methods of armbruster (2003). measurements were taken with digital slide calipers up to the nearest 0.1 mm and expressed as percentage of standard length. other external features and coloration were also examined. abbreviations of meristic counts: d dorsal, a anal, p pectoral, v ventral, c caudal, l.l lateral line scales. results eight specimens of p. pardalis (fig. 1) were collected from cauvery river (11°02'10.4''n, 78°08'45.2''e) at mohanur (fig. 2), namakkal district, tamilnadu on 24 october 2013. pterygoplichthys pardalis is diagnosed by discrete dark spots on the lateral and caudal peduncle with a pattern of uncoalesced dark spots on a light background, stout pectoral fins with rough surfaces and inferior disc-like protrusible mouth. fin ray counts for the fishes are d: i 12, a: i 4; p: i 6; v: i 5; c: 14; l.l: 26-32. morphometric details of the samples as provided in table 1. body behind head completely plated dorsally and laterally. belly naked, with the plates occurring on the ventral side of the body only at the caudal peduncle region. figure 1. dorsal and ventral view of p. pardalis collected from cauvery river, india. 16 int. j. aquat. biol. (2015) 3(1): 14-18 ventral surface of the pectoral girdle covered in skin mesial to the coracoid strut. caudal peduncle round in cross section. adipose fin present in the peduncle region. edge of snout covered with plates. postdorsal ridge inconspicuous, with the single, median, unpaired preadipose plate. body coloration, particularly on the abdomen, consists of dark spots on light background, however head exhibit linear patterns forming geometric shapes. most of the samples have a base color of light gray usually becoming lighter towards the ventral side. this is the first report of pterygoplichthys pardalis in a south indian river system. discussion in southern india, p. multiradiatus another species of the genus has been reported from vylathur and the chackai canal kerala (daniels 2006; krishnakumar et al., 2009) and wetlands of chennai, tamilnadu (knight, 2010). being a generalist with a wide spectrum of food, this opportunistic invader could be a threat to native species. it is hence regarded as potential threat to native fish diversity in western ghats (molur et al., 2011). occurrence of p. pardalis in open waters of peninsular india is concerning in terms of the mounting pressure on the already dwindling indigenous fishes. distribution of pterygoplichthys species along the stretches of the cauvery river basin may be expected as there are few news reports on the occurrence of this species in mettur reservoir and of being caught in the figure 2. distribution of the genus pterygoplichthys in south india. collection site of p. pardalis (red star). previous records of p. multiradiatus (black star). measurements range mean s.d. total length (mm) 142.5 189.1 163.91 12.8 standard length (mm) 107.3 138.4 125.38 10.0 expressed as %sl predorsal length 40.07 45.90 42.36 2.1 head length 22.46 27.03 24.50 1.6 snout length 10.55 14.42 12.23 1.3 mouth length 9.45 13.40 11.56 1.4 barbel length 4.03 6.50 5.10 0.8 pectoral spine length 28.13 31.48 29.01 1.0 pelvic spine length 17.61 23.62 20.25 1.8 anal fin spine length 11.62 15.46 13.49 1.8 dorsal spine length 25.79 32.14 28.33 2.0 dorsal fin base length 36.41 39.29 38.23 1.0 caudal peduncle depth 9.10 11.42 9.97 0.9 head depth 12.46 17.36 15.29 1.7 mouth width 10.63 13.22 12.01 0.8 orbit diameter 2.17 3.52 3.01 0.3 pre adipose length 83.72 90.34 86.87 2.5 table 1. morphometric measurements of p. pardalis collected from cauvery river, india. 17 muralidharan et al./ extended distribution of p. pardalis to south india downstream river. the reason for successful expansion and establishment could be due to the suitable habitat for feeding and nesting and the polluted segments with fewer disturbances from humans. acknowledgements first author (m.m) acknowledges the financial support from ugc available through major research project (f. no. 39-332/2010 sr). we thank m. sankar, mohanur for his assistance in the collection of specimens. we gratefully acknowledge l. m. page, university of florida museum of natural history for helping with the taxonomic identification. map of cauvery river was downloaded from open street map contributors. references armbruster j. (2003). the species of the hypostomus cochliodon group (siluriformes: loricariidae). zootaxa, 249: 1-60. bunkley-williams l., williams jr. e.h., lilystrom c.g., corujo-flores i., zerbi a. j., aliaume c. (1994). the south american sailfin armored catfish, liposarcus multiradiatus (hancock), a new exotic established in puerto rican fresh waters. caribbean journal of science, 30(1-2): 90-94. chavez m.j., de la paz r.m., manohar s.k., pagulayan r.c., vi c. (2006). new philippine record of south american sailfin catfishes (pisces: loricariidae). zootaxa, 1109: 57-68. daniels r.j.r. (2006). introduced fishes: a potential threat to the native freshwater fishes of peninsular india. journal of the bombay natural history society, 103(2 and 3): 346-348. herder f., schliewen u.k., geiger m.f., hadiaty r.k., gray s.m., mckinnon j.s., walter r.p., pfaender j. (2012). alien invasion in wallace’s dreamponds: records of the hybridogenic “flowerhorn” cichlid in lake matano, with an annotated checklist of fish species introduced to the malili lakes system in sulawesi. aquatic invasions, 7(4): 521-535. hoover j.j., killgore k.j., cofrancesco a.f. (2004). suckermouth catfishes: threats to aquatic ecosystems of the united states? aquatic nuisance species research program. engineers research and development center, vicksburg, ms. ansrp bulletin 4 (1): 1-13. hossain m.y., rahman m.m., ahmed z.f., ohtomi j., islam a.b.m.s. (2008). first record of the south american sailfin catfish pterygoplichthys multiradiatus in bangladesh. journal of applied ichthyology, 24: 718-720. knight j.d.m. (2010). invasive ornamental fish: a potential threat to aquatic biodiversity in peninsular india. journal of threatened taxa, 2(2): 700-704. krishnakumar k., raghavan r., prasad g., bijukumar a., sekharan m., pereira b., ali a. (2009). when pets become pests – exotic aquarium fishes and biological invasions in kerala, india. current science, 97: 4-25. liang s.h., wu h.p., shieh b.s. (2005). size structure, reproductive phenology, and sex ratio of an exotic armored catfish (liposarcus multiradiatus) in the kaoping river of southern taiwan. zoological studies, 44(2): 252-259. levin b.a., phuong p.h., pavlov d.s. (2008). discovery of the amazon sailfin catfish pterygoplichthys pardalis (castelnau, 1855) (teleostei: loricariidae) in vietnam. journal of applied ichthyology, 24: 715717. ludlow m.e., walsh s.j. (1991). occurrence of a south american armored catfish in the hillsborough river, florida. florida scientist, 54: 48-50. nico l., cannister m., neilson m. (2012). pterygoplichthys pardalis. usgs nonindigenous aquatic species database, gainesville, fl. nico l.g., martin r.l. (2001). the south american sucker mouth armored catfish, pterygoplichthys anisitsi (pisces: loricariidae), in texas, with comments on foreign fish introductions in the american southwest. the southwest naturalist, 46: 98-104. molur s., smith k.g., daniel b.a., darwall w.r.t. (2011). the status and distribution of freshwater biodiversity in the western ghats, india. cambridge, uk and gland, switzerland: iucn, and coimbatore, india, zoo outreach organisation, 110 p. page l.m., burr b.m. (1991). a field guide to freshwater fishes of north america north of mexico. peterson field guide series, houghton mifflin company, boston, ma, 432 p. page l.m., robins h.r. (2006). identification of sailfin catfishes (teleostei: loricaridae) in southeastern asia. raffles bulletin of zoology, 54: 455-457. 18 int. j. aquat. biol. (2015) 3(1): 14-18 samat a., shukor m.n., mazlan a.g., arshad a., fatimah m.y. (2008). length-weight relationship and condition factor of pterygoplichthys pardalis (pisces: loricariidae) in malaysia peninsula. research journal of fisheries and hydrobiology, 3: 48-53. sinha r.k., sinha r.k., sarkar u.k., lakra w.s. (2010). first record of the southern sailfin catfish, pterygoplichthys anisistsi eigenmann and kennedy, 1903 (teleostei: loricariidae), in india. journal of applied ichthyology, 26: 606-608. sumanasinghe h.p.w., amarasinghe u.s. (2013). population dynamics of accidentally introduced amazon sailfin catfish, pterygoplichthys pardalis (siluriformes, loricariidae) in pologolla reservoir, sri lanka. sri lanka journal of aquatic sciences, 18: 3745. yamamoto m.n., tagawa a.w. (2000). hawaii’s native and exotic fresh water animals. mutual publishing, honolulu, hawaii, 200 p. weber c. (2003). subfamily hypostominae (armored catfishes). in: r.e. reis, s.o. kullander, c.j. ferraris, jr. 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(2021) 9(3): 214-216 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article generalized modified levakovic growth function for aquatic species deniz ünal* 1 department of statistics, faculty of arts and sciences, çukurova university, 01330 adana, turkey. article history: received 24 april 2021 accepted 22 june 2021 available online 2 5 june 2021 keywords: aquatic species, curve fitting, growth function, fisheries, lag time. abstract: growth is a concept that includes social, economic and physical sub-processes and it also shows itself in the field of fisheries. it is extremely important to obtain the appropriate mathematical model for growth for aquatic species. the growth modelling for the aquatic species contains the phenomena of bio-diversity, formation and population dynamics of aquatic species or stock estimation for the creature. and this study aims to design and implement a new mathematical model for growth process for aquatic species as generalized modified levakovic growth (glm) model. introduction mathematical growth models are used to predict the growth of biological systems and intraspecific population dynamics. growth functions are simple, nonlinear mathematical equations that describe the change in the size of an individual or population over time. in some cases, simple exponential growth functions can model the growth. growth curves serve to express the development of growth parameters over time mathematically. it is unthinkable to show an ever-increasing trend for the concept of growth; because nothing can be expected to increase continuously (bertalanffy, 1938). in the simplest terms, even the population growth has a certain saturation level, so called "carrying capacity". this value assumes a numerical upper bound role for growth size. von bertalanffy (1938) proposed the first important research on the mathematical expressions of the size of the organism. in addition, the richards model (richards, 1959), obtained by the modification of the von bertalanffy model, has found a wide range of application. lifeng et al. (1998) was implemented richards model to various species such as amoeba proteus, rice, fish, cattle and deer. later, brich (1999) defined a new generalized logistic model and *correspondence: deniz ünal doi: https://doi.org/10.22034/ijab.v9i3.1271 e-mail: dunal@cu.edu.tr compared it to richards model. to compare and fit the models, brich (1999) applied the real data set which was also used in the richards study. the use of the growth models is not only limited to the field of biology. they can be applied to many other fields such as determining the market volume in economics (fisher and fry, 1971; fingleton and lópez‐bazo, 2006). levakovic (1935) model, one of the functions used in the modeling of growth, is actually the generalized form of the hossfeld (woollons et al, 1990) model (lee, 2000). since the levakovic model is not very useful in practice, this function is generally used unified with other growth models (bontemps and duplat, 2012). the model known as levakovic i is known as the best model among 4-parameter models (zeide, 1993). based on above-mentioned background, in the first part of this study, construction of a new growth model is defined by inspiring levakovic function called as generalized modified levakovic growth (glm) model to apply for aquatics. in the second section, the prominent features of glm model is discussed and finally conclusion section is given. (1) construction of generalized modified levakovic growth function 215 int. j. aquat. biol. (2021) 9(3): 214-216 statistical modeling with growth data creates a more efficient way to understand biological progress. in this section, a new growth model is constructed as generalized modified levakovic (glm) growth model. the relative growth rate: the relative growth rate is calculated as: 𝑧𝑧(𝑡𝑡) = 𝑑𝑑𝑑𝑑(𝑡𝑡) 𝑑𝑑𝑡𝑡 𝑦𝑦(𝑡𝑡) = d dt lnt, 𝑧𝑧(𝑡𝑡) > 0, and we define a new relative rate for growth as follows: 𝑧𝑧(𝑡𝑡) = c a b 𝑒𝑒−𝑎𝑎𝑡𝑡 (1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)−1 … … … … … (1) where 𝑦𝑦∞ = lim𝑡𝑡→∞ 𝑦𝑦(𝑡𝑡) and a, b, c are any real numbers with a, b, c ≥ 0, t is time variable, 𝑡𝑡 ≥ 0 and 𝑦𝑦(𝑡𝑡) is current size at time 𝑡𝑡. thus: 𝑑𝑑𝑦𝑦(𝑡𝑡) 𝑑𝑑𝑡𝑡 𝑦𝑦(𝑡𝑡) = c a b 𝑒𝑒−𝑎𝑎𝑡𝑡 (1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)−1 … … … … (2) from now on, it is easy to define the instant rate 𝑦𝑦(𝑡𝑡). to obtain the 𝑦𝑦(𝑡𝑡) function which gives the current size at time 𝑡𝑡, the integration of the equation 2 should be find. � 𝑑𝑑𝑦𝑦(𝑡𝑡) 𝑑𝑑𝑡𝑡 𝑦𝑦(𝑡𝑡) 𝑑𝑑𝑡𝑡 = � c a b 𝑒𝑒−𝑎𝑎𝑡𝑡 (1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)−1 𝑑𝑑𝑡𝑡 … (3) by using the change of variable technique, the integral in the equation 3 can be evaluated as: 𝑢𝑢 = 1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡 → 𝑑𝑑𝑢𝑢 = 𝑎𝑎 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡 𝑑𝑑𝑡𝑡 ln 𝑦𝑦(𝑡𝑡) = 𝑙𝑙𝑙𝑙(1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)𝑐𝑐 + 𝑙𝑙𝑙𝑙 𝑐𝑐1 where 𝑐𝑐1 is an integration constant. therefore, the formula for glm growth function is: 𝑦𝑦(𝑡𝑡) = 𝑐𝑐1 (1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)𝑐𝑐 … … … … … (4) to see the 𝑐𝑐1, let us analyze this equation as 𝑡𝑡 tends to infinity: 𝑌𝑌∞ = lim𝑡𝑡→∞ 𝑦𝑦(𝑡𝑡)= 𝑐𝑐1 is an asymptote, i.e. this number gives the carrying capacity. since 𝑐𝑐1 = 𝑌𝑌∞, the equation 4 should be arranged as in equation 5. then, the general formula for glm growth function would be: 𝑦𝑦(𝑡𝑡) = 𝑌𝑌∞ (1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)𝑐𝑐 … … … … … (5) growth rate: using equations 2 and 5, we define growth rate for the glm growth function as follows: 𝑑𝑑𝑦𝑦(𝑡𝑡) 𝑑𝑑𝑡𝑡 = 𝑦𝑦∞c a b 𝑒𝑒−𝑎𝑎𝑡𝑡(1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)𝑐𝑐−1 … (6) where 𝑦𝑦∞ = lim𝑡𝑡→∞ 𝑦𝑦(𝑡𝑡), a, b, c are any real numbers with a, b, c, t≥0 , where t is time variable and 𝑦𝑦(𝑡𝑡) is current size at time 𝑡𝑡. (2) properties of generalized modified levakovic growth function to obtain some identifier features of the glm model, the initial value of glm model is: 𝑦𝑦(0) = 𝑌𝑌∞ (1 − 𝑏𝑏 )𝑐𝑐 initial behavior of the growth rate can be reached by taking zero in the first derivative of glm model that is: 𝑦𝑦′(𝑡𝑡) = 𝑑𝑑𝑦𝑦(𝑡𝑡) 𝑑𝑑𝑡𝑡 � 𝑡𝑡=0 = 𝑦𝑦∞c a b (1 − 𝑏𝑏 )𝑐𝑐−1 as the initial behavior of the glm function. another important feature for a growth curve is the critical points that can be obtained by reaching the points where the first derivative is equal to zero. therefore, we should take dy(t)/dt = 0. 𝑑𝑑𝑦𝑦(𝑡𝑡) 𝑑𝑑𝑡𝑡 = 𝑦𝑦∞c a b 𝑒𝑒−𝑎𝑎𝑡𝑡(1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)𝑐𝑐−1 = 0 by exponential transformation, tcritical=ln b1/a can be calculated. then, the value of the glm growth model at critical point would be 𝑦𝑦(𝑡𝑡𝑐𝑐𝑐𝑐𝑐𝑐𝑡𝑡𝑐𝑐𝑐𝑐𝑎𝑎𝑐𝑐) = 0. inflection point: inflection points are the points where the graph of a function changes curvature i.e. the point where the curve turns to convex from concave or vice versa. therefore, it is one of the most important properties that gives information about the structure of a curve. finding these points is possible by setting the second derivative to zero and often requires a rather tedious calculation process. in this section, the inflection point of the glm function is calculated. 𝑑𝑑2𝑦𝑦(𝑡𝑡) 𝑑𝑑𝑡𝑡2 = −𝑦𝑦∞c 𝑎𝑎2 b 𝑒𝑒−𝑎𝑎𝑡𝑡(1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)𝑐𝑐−1[1 + 𝑏𝑏 (1 − 𝑐𝑐)𝑒𝑒−𝑎𝑎𝑡𝑡(1 − 𝑏𝑏 𝑒𝑒−𝑎𝑎𝑡𝑡)−1] … (7) for 𝑡𝑡1 = 𝑙𝑙𝑙𝑙(𝑏𝑏𝑐𝑐)1/𝑎𝑎 or 𝑡𝑡2 = ln𝑏𝑏1/𝑎𝑎 second derivative given in equation 7 is equal to 0, i.e. 𝑑𝑑2𝑦𝑦(𝑡𝑡) 𝑑𝑑𝑡𝑡2 =0 for 𝑡𝑡1 and 𝑡𝑡2. population size at inflection point: notice that 𝑡𝑡2 = ln𝑏𝑏1/𝑎𝑎 is the critical value for glm growth function, therefore by consideration of 𝑡𝑡1 = 𝑙𝑙𝑙𝑙(𝑏𝑏𝑐𝑐)1/𝑎𝑎, we can calculate y(t) at inflection point 𝑡𝑡1 = 𝑙𝑙𝑙𝑙(𝑏𝑏𝑐𝑐)1/𝑎𝑎as follows: y(𝑡𝑡1) = 𝑌𝑌∞ (1 − 1 𝑐𝑐 )𝑐𝑐 which gives the population size at point of inflection. and population at inflection point is: y(𝑡𝑡1) = 𝑌𝑌∞ (1 − 1 𝑐𝑐 )𝑐𝑐 … … … … … (8) 216 ünal / generalized modified levakovic growth function for aquatic species the maximum specific growth rate: using the value of 𝑡𝑡1 = 𝑙𝑙𝑙𝑙(𝑏𝑏𝑐𝑐)1/𝑎𝑎 in the first derivative of y(𝑡𝑡), that is calculating the following equation, y′(𝑡𝑡1) = 𝑌𝑌∞ 𝑎𝑎 �1 − 1 𝑐𝑐 � 𝑐𝑐−1 … … … … … (9) the slope of the curve at inflection point 𝑡𝑡1 = 𝑙𝑙𝑙𝑙(𝑏𝑏𝑐𝑐)1/𝑎𝑎 can be obtained. lag time: the points where the growth reaches its maximum and stops the lag time, denoted by λ. this point is the x-intercept of the tangent curve at the inflection point. again, by considering 𝑡𝑡1 = 𝑙𝑙𝑙𝑙(𝑏𝑏𝑐𝑐)1/𝑎𝑎 as an inflection point, the lag time λ can be reached by writing tangent equation about (t1, y(t1)) point. then using the equations 8 and 9, the tangent equation would be as: yy(𝑡𝑡𝑐𝑐𝑖𝑖𝑖𝑖)= 𝑦𝑦′(𝑡𝑡𝑐𝑐𝑖𝑖𝑖𝑖). (𝑡𝑡 -𝑡𝑡𝑐𝑐𝑖𝑖𝑖𝑖) at (λ, 0), this equation gives the lag time as: 0 − y(𝑡𝑡1)= 𝑦𝑦′(𝑡𝑡1). (λ -𝑡𝑡1) −𝑌𝑌∞ (1 − 1 𝑐𝑐 )𝑐𝑐 = 𝑌𝑌∞ 𝑎𝑎 (1 − 1 𝑐𝑐 )𝑐𝑐−1 (λ − 𝑙𝑙𝑙𝑙(𝑏𝑏𝑐𝑐)1/𝑎𝑎) and by performing some manipulations, λ − ln(𝑏𝑏𝑐𝑐) 1 𝑎𝑎 = 𝑐𝑐 − 1 𝑐𝑐 𝑎𝑎 equation can be reached. then the lag time is as follows: λ = 𝑐𝑐 − 1 + ln(𝑏𝑏𝑐𝑐)𝑐𝑐 𝑐𝑐 𝑎𝑎 conclusion the time-dependent evolution/variation seen in a population or living thing is expressed by growth functions. there is an enormous amount of empirical and theoretical study relating to the growth. because, as emphasized in this study, the importance of accurate modeling of growth is known by scientists. the model presented in the study will contribute to study of biological systems particularly aquatics. in addition, the application of this model to fisheries data sets in future studies will provide new expansions and will lead to progress in growth estimation. references birch c.p. 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(2018) 6(3): 162-169 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article hematology and plasma chemistry reference intervals for wild population of alburnus chalcoides (guldenstadt, 1772): influence of sex, habitat and seasonal variation azam moshfegh1, mahbubeh setorki*2,1vahide bahrpeyma1, akram tehranifard1, mohammadreza rahimibashar1 1department of biology, lahijan branch, islamic azad university, lahijan, iran. 2department of biology, izeh branch, islamic azad university, izeh, iran. article history: received 2 november 2017 accepted 7 june 2018 available online 2 5 june 2018 keywords: biochemical parameters, caspian sea blood indices seasonal habitat abstract: the study was aimed to establish reference interval for some blood biochemical (sodium, potassium, calcium, and magnesium) and hematological (total count of white blood cells, neutrophils, lymphocytes, monocytes, and eosinophils) indices in both sexes of alburnus chalcoides in response to its habitats with different physiochemical conditions and to seasonal variation. fish samples were collected from its seasonal habitats, including river, estuary, and caspian sea. the mean concentration of sodium, potassium, calcium, and magnesium in plasma of fish were 134.69±13.61 (mmol/l), 3.26±0.83 (mmol/l), 11.65±1.82 (mg/dl) and 2.13±1.12 (mmol/l), respectively. these electrolytes demonstrated significant differences between the male and female as well as between the fish collected from different habitats. total wbcs count was 6045.35±960.25 (per mm3) and the mean percentage of neutrophils, lymphocytes, monocytes and eosinophils were 26.55±3.37%, 67.74±3.72%, 3.04±0.70% and 0.98±0.04%, respectively. wbcs generally showed considerable differences between male and female. furthermore, a higher percentage of monocytes, neutrophils and eosinophils were observed in the fish collected from river and estuary during spring and summer. the study demonstrated that differences in sex, habitat and seasonal variation cause variation in hematology and plasma chemistry reference intervals for wild population of a. chalcoides when migrating between its natural habitats. introduction alburnus chalcoides is a commercially valuable anadromous and benthopelagic fish, inhabiting in rives, coastal lakes, estuaries, and brackish waters (up to 14 ppt). this species is distributed in the caspian sea basin, and its spawning occurs in small rivers or streams (kottelat and freyhof, 2007). the adult populations migrate upstream for spawning, and the embryonic development lasts 2-3 days, and then the larvae (with the age of 8-11 days) actively migrate downstream (kottelat, 1997). after spawning, the adults migrate back to lakes, estuaries, and seas (i.e., brackish water). recently, due to overfishing, expanding hydroelectric development, and strong ecological impacts, the population of a. chalcoides has considerably declined, especially in the southern and southwest coasts (kottelat and freyhof, 2007; nikoo *corresponding author: mahbubeh setorki doi: https://doi.org/10.22034/ijab.v6i3.377 e-mail address: doctor.setorgi@gmail.com et al., 2010; rajabi nezhad and azari takami, 2001), and hence this fish has been classified as a vulnerable in the iucn red list (kiabi et al., 1999; rahmani et al., 2007). knowledge of hematological or biochemical reference ranges is important in assessing, managing, and better understanding the health status animal populations (dickinson et al., 2002). diagnostic assessment of blood hematological and biochemical parameters have been used extensively for many terrestrial animal species (hrubec and smith, 2004). whereas this type of hematological utilization is limited for fishes. hence, it is necessary to utilize such an information to assess the health status of fish populations in their natural habitat as well as in the captive condition. owing to migration between three types of aquatic ecosystems (i.e., river, estuary, and caspian sea), 163 int. j. aquat. biol. (2018) 6(3): 162-169 a. chalcoides is exposed to different cyclical external conditions which in turn change its internal biochemical status (naderi and abdoli, 2004). the external cyclical conditions may influence blood biochemical and hematological parameters, and understanding normal health status of a. chalcoides during migration between these different aquatic ecosystems is ecologically of high importance. therefore, the present study was aimed to determine reference ranges of some hematological and plasma biochemical parameters of both sexes of a. chalcoides when migrating between its annual habitats (river, estuary, and the caspian sea). materials and methods sampling location: for this study, 84 healthy individuals of a. chalcoides (average total length: 16.5±4 cm; average weight: 45.69±0.27 gr; male=42 and female=42) were collected from three locations across its annual migration way (i.e., river, estuary, and caspian sea, with salinity of 0.4, 3.75, and 9.71 ppt, respectively). these locations are in the southern cost of the caspian sea and lale river in the chamkhaleh, guilan province (fig. 1). the sampling was done during 12 month in 2014; from the river, in may, june, july and august; from the estuary, in april and september; and from the caspian sea, in october, november, december, january, february, and march. some physicochemical parameters of water, including salinity, dissolved oxygen, ph, and temperature, at the sampling sites (table 1). sex and age determination: the collected fish were dissected and the sex determination was done according to gonadal characteristics. age was determined by scale reading from the fish. briefly, scale samples (8-15 scales) were removed from below table 1. the mean value of air temperature as well as water physiochemical parameters at the sampling location in river (lale river), estuary and the caspian sea. month location dissolved oxygen (mg/l) ph salinity (ppt) water temperature (°c) air temperature (°c) april lale river estuary 7.40 8.10 4 13.16 15.75 may lale river 7.50 8.30 1 19.08 20.13 june 9.60 8.40 0.00 24.48 25.08 july 9.20 8.30 0.00 25.50 26.02 august lale river estuary 9.00 8.20 0.00 27.39 28.04 september caspian sea 8.10 7.90 3.50 24.55 26.16 october 9.00 8.00 9.50 19.56 22.49 november 8.10 8.20 10.00 17.84 19.28 december 8.40 8.00 9.00 11.78 14.21 january 8.60 8.00 9.10 9.00 9.75 february 8.22 8.20 10.50 10.27 11.65 march 8.22 8.30 10.24 10.79 12.41 figure 1. sampling location in the southern coast of the caspian sea. 164 moshfegh et al./ hematological and biochemical reference intervals for a. chalcoides the lateral line and between the anal and caudal fins and cleaned in 5% sodium peroxide, and the scale radiuses were counted based on the von bertalanffy equation (biradar, 1989). fish with 3-4 years old were used for the study. blood collection and hematological and biochemical analysis: fish blood was drawn from the caudal vein using heparinized and unheparinized syringes. the heparinized samples were used for white blood cell counting (total count of white blood cells (wbc), neutrophils, lymphocytes, monocytes, and eosinophils) and the for plasma biochemical evaluation (sodium, potassium, calcium, and magnesium ions) was used unheparinized samples. after centrifugation at 2000 g, blood plasma was collected and stored at -20°c until further assay. total calcium and magnesium concentrations were determined with colorimetric assay kit and unico uv2100 spectrophotometer. sodium and potassium were analyzed via a flame photometer (model pfp7, jenway, essex, uk) (wong et al., 2001). to establish the percentage of leukocytes, wbcs were counted in each treatment with sysmex k-1000 analyzer (ballarin et al., 2004; clark and kruse, 1990). statistical analysis: data analysis was performed using spss software (spss, version. 22). all results are presented as the mean ± standard deviation (sd). significant differences were determined using oneway anova, followed by tukey test to compare the differences between the fish groups collected from the river (lale river), lale river estuary, and sea (p<0.05). t-test student was applied to elucidate the effect of sex and seasonal variation on plasma ions. in addition, pearson correlation coefficient was used to determine the relationship between the biometric indices (length and weight) and blood wbcs. results figure 2 shows the mean reference level of potassium, sodium, calcium, and magnesium in plasma of adult male and female a. chalcoides. the values of potassium and calcium in the female were significantly higher than those of males, whereas magnesium showed higher level in the males than females (p<0.05). no significant sex related difference was found in the mean plasma sodium level. the effect of seasonal variation and subsequent cyclical condition on the above mentioned plasma electrolytes in the male and female a. chalcoides is shown in table 2. the highest and lowest amount of calcium and sodium was measured in the fish collected from the caspian sea during autumn and spring, respectively. plasma potassium concentration was measured with the highest level in winter, but the lowest in spring. furthermore, these electrolytes (except magnesium) illustrated significant differences between the fish collected from the sampling habitats (i.e., river, estuary, and caspian sea). during sampling period, white blood cell count demonstrated no significant difference in the percentage of lymphocytes, monocytes and eosinophils between the male and female a. chalcoides (fig. 3), but the number of neutrophils in the female animals was higher than the males (p<0.05). however, significant differences were observed when compared to their respective ones collected from table 2. reference interval of four main blood plasma electrolytes (calcium, potassium, sodium, and magnesium) in both male and female alburnus chalcoides in different season. plasma ion season spring summer autumn winter sex m f m f m f m f calcium (mg/dl) 10.30 10.55 10.25 11.89 12.68 13.65 11.83 12.61 potassium (mmol/l) 1.63 2.69 3.18 3.31 3.06 3.73 3.48 4.53 sodium (mmol/l) 122.50 124.47 129.21 130.29 146.65 147.36 139.13 137.90 magnesium (mmol/l) 1.40 1.15 2.83 3.03 2.47 2.46 1.47 2.08 m= male, f= female 165 int. j. aquat. biol. (2018) 6(3): 162-169 other locations (fig. 4). the highest percentage of monocytes, neutrophils and eosinophils was found in the blood of the animals collected from the estuary (i.e., lale river estuary) (p<0.05), and the percentages in the fish sampled from different locations showed an order of estuary> river> caspian sea. the mean percentage of lymphocytes showed no significant difference between three habitats (p<0.05). the correlation between biometric indices (length and weight) and blood wbc is shown in table 3. pearson analysis showed a strong positive correlation between wbc, neutrophils, lymphocytes, and eosinophil with weight and length of the collected fish, but monocytes showed a negative correlation with weight and length (p<0.05 and p<0.01). discussion some environmental conditions such as temperature, table 3. the correlation between the biometric indices (length and weight) and blood wbc of alburnus chalcoides. parameter total weight gonadal weight total length wbc 0.251* 0.195 0.305** neutrophils 0.089 0.197 0.018 lymphocytes 0.028 -0.112* -0.099* monocytes -0.101* -0.142* -0.001** eosinophil 0.156 0.205 0.127 asterisk (*) and double asterisk (**) show significant difference at p<0.05 and p<0.01, respectively. figure 2. the mean reference level of potassium (a), sodium (b), magnesium (c), and calcium (d) in plasma of adult male and female alburnus chalcoides (data expressed as mean ± sd). 166 moshfegh et al./ hematological and biochemical reference intervals for a. chalcoides seasonality, oxygen, and salinity as well as internal factors (age, sex, reproductive state, genetic variation, and stage of development) have been documented to affect hematologic values (knowles et al., 2006). due to lack of information concerning some hematological and biochemical parameters of a. chalcoides, the present study aimed to determine a reference value for wbcs count and some plasma electrolytes predominantly involve in osmoregulation during presence in its environmentally different habitats. higher amount of potassium and calcium were observed in the female; the female fish collected during the autumn showed the highest concentrations of plasma calcium and the lowest level in the spring (breeding season). owing to a wide fluctuation in plasma calcium level in female fish during the breeding cycle, i.e., their gonadal maturation (webb et al., 2002), the observed difference in the plasma calcium concentration in the female fish can be an evidence for higher amount of plasma calcium in the non-breeding seasons (autumn and winter) of a. chalcoides. hence, plasma calcium level is a reliable indicator for oocyte maturation (involving in vitellogenesis) and its level gradually increases during the pre-spawning period and reaches to a peak value just before spawning, and then illustrates a sharp decline after the spawning (bromage et al., 1993). support for this result has come from previous reports in which the amount of plasma calcium in female atlantic flatfish hippoglossus hippoglossus were 4.5 and 2.5 mmol/l, respectively, before and after spawning period (björnsson et al., 1998; bromage et al., 1993). magnesium participates in many biological processes such as modulating a large number of enzymes as well as regulating energy metabolism and protein synthesis (davis and gatlin iii, 1996). magnesium deficiency is associated with deficiency figure 3. the mean percentage of monocytes (a), lymphocytes (b), neutrophils (c), and eosinophils (d) in the blood of male and female alburnus chalcoides (data expressed as mean ± sd). 167 int. j. aquat. biol. (2018) 6(3): 162-169 of other important minerals such as calcium, sodium and potassium which reflects the mg2+-dependence of ion exchange mechanisms such as sodium/potassium atpase pumps and positive canals (ellis et al., 2002). the present study demonstrated higher plasma magnesium concentration in the male a. chalcoides; however, no significant difference was observed associated with season and sampling location. hence, the acquired data regarding the difference in plasma magnesium level between male and female a. chalcoides can be considered as new physiological information about the fish. this data corroborate the findings of bagheri et al. (2007) who reported that both plasma calcium and magnesium are influenced by gender, growth phase and stage of gonadal maturity. plasma sodium and potassium increase in fish migrate from hyposmotic to hyperosmotic environments (mancera et al., 2017); therefore, the observed higher plasma sodium level in a. chalcoides collected from sea compared to those of river and estuary is an acceptable osmoregulational and physiological phenomenon. this value agrees with that observed in young green sturgeon (acipenser medirostris), an anadromous species, when migrating from freshwater to sea water (poletto et al., 2017). in addition, no sex dependent difference was observed in plasma sodium concentration, suggesting that plasma sodium concentration is independent to gonadal difference in males and females. similar results also have been reported by khodadadi (2009) who found no sex dependent difference in plasma sodium level of barbus sharpeyi. evaluating the number of is an important hematological index to evaluate immune system and also a biomarker for terrestrial and aquatic studies (modesto and martinez, 2010; sancho et al., 2000). in figure 4. the mean percentage of monocytes (a), lymphocytes (b), neutrophils (c), and eosinophils (d) in the blood of alburnus chalcoides collected from the caspian sea (sea), estuary (lale river estuary), and river (lale river) (data expressed as mean ± sd). 168 moshfegh et al./ hematological and biochemical reference intervals for a. chalcoides the present study, wbc count demonstrated the order of percentage as follows lymphocytes> neutrophils> monocytes> eosinophils. however, the fish collected from river and estuary during spring and summer illustrated significantly higher percentage of wbc (except lymphocytes) compared to those collected from sea in the autumn and winter. in addition, the percentage of neutrophils in the females was significantly higher. support for this results has come from previous studies in which significantly higher percentage of lymphocytes was recorded in the spawning season, and also the level of wbcs number in the females were higher than those in males; i.e., some sex-related differences in hematological indices was observed (adel et al., 2017; marijani et al., 2017; örün and erdemll, 2002). conclusion the study demonstrated that differences in sex, habitat and seasonal variation cause variation in hematology and plasma chemistry reference intervals for wild population of a. chalcoides when migrating between its natural habitats, and knowledge about health status of this fish species is better to be considered just according to it habitat environment and further investigation are need to determine precise other hematological and biochemical reference intervals for the fish in river, estuary, and caspian sea. acknowledgments the authors would like to acknowledge the islamic azad university of lahijan financial support. references adel m., dadar m., khajavi s.h., pourgholam r., karimí b., velisek j. 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(2018) 6(3): 162-169 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی (: alburnus chalcoides) کولی شاه ماهی وحشی جمعیت پالسمای شیمیایی و شناسیخون پارامترهای مرجع میزان فصلی تغییرات و زیستگاه جنسیت، تاثیر 1بشر رحیمی محمدرضا ،1فردتهرانی اکرم ،1بحرپیما وحیده ،2*سترکی محبوبه ،1مشفق اعظم .ایران الهیجان، الهیجان، واحد اسالمی، آزاد دانشگاه ،شناسیزیست گروه1 .ایران ایذه، ایذه، واحد اسالمی، دآزا دانشگاه شناسی،زیست گروه2 چکیده: کل تعداد) شناسیخون و( منیزیم و کلسیم پتاسیم، سدیم،) بیوشیمایی پارامترهای برخی از مرجعی و طبیعی میزان که دارد سعی حاضر مطالعه خصوصیات اب طبیعی هایزیستگاه در را کولی شاه ماهی ماده و نر جنس دودر ( ائوزینوفیل و مونوسیت فوسیت،نل نوتروفیل، خون، سفید هایگلبول صید زرخ یدریا و مصب رودخانه،) زیستگاه سه از ماهی هاینمونه. نماید گزارش فصلی تغییرات با ارتباط در همچنین و متفاوت فیزیکوشیمیایی 13/2±12/1 و 65/11±82/1 ،26/3±83/0 ،69/134±61/13 ترتیببه ماهیان پالسمای در منیزیم و کلسیم پتاسیم، سدیم، غلظت میانگین. شدند شده وریآجمع مختلف هایزیستگاه از که ماهیانی بین همچنین و ماده و نر هایجنس بین داریمعنی تفاوت هاالکترولیت این. بود لیتر بر مولمیلی هاتیمونوس ها،تیمفوسیل ها،لینوتروف درصد نیهمچن و مکعب متریلیم در عدد 35/6045±25/960 خون دیسف یهاگلبول کل. دادند نشان بودند، ماده و نر جنس بین داریمعنی تفاوت سفید هایگلبول. بودند 98/0±04/0 و 04/3±74/0 ،74/67±72/3 ،55/26±37/3 بیترتبه هالینوفیائوز و باالتری درصد بود، شده آوریجمع تابستان و بهار در رودخانه مصب و رودخانه از که ماهیانی هایائوزینوفیل و هانوتروفیل ها،مونوسیت. داشتند یعیبط زانیم در ییهاتفاوت و راتییتغ توانندیم یفصل راتییتغ نیهمچن و ستگاهیز ت،یجنس که است آن انگریب حاضر قیتحق. دادند نشان جادیا آن یعیطب یهاستگاهیز نیب مهاجرت هنگام در یکول شاه یماه یوحش یهاتیجمع نیب خون یپالسما یمیش و یشناسخون یپارامترها .ندینما . .فصلی زیستگاه خونی، هایشاخص خزر، دریای بیوشیمیایی، پارامترهای :کلمات کلیدی int. j. aquat. biol. (2021) 9(6): 370-382 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article effects of penconazole and copper nanoparticle fungicides on redbelly tilapia, coptodon zillii (gervais, 1848): reproductive outcomes mahmoud m.s. farrag*1, rashad e.m. said1, ibtisam m.h. elmileegy 2, nasser s. abou khalil2, eman s.a. abdel allah3, mohamed f. el-sawy1, alaa g.m. osman1 1department of zoology, faculty of science, al-azhar university, assiut, egypt. 2department of medical physiology, faculty of medicine, assiut university, assiut, egypt. 3department of physiology, faculty of veterinary medicine, assiut university, assiut, egypt. s article history: received 22 april 2021 accepted 4 august 2021 available online 2 5 august 2021 keywords: penconazole copper nanoparticles antioxidant reproductive outcomes histopathology abstract: several effective fungicides have recently been applied, but they can harm ecosystems and non-target organisms. the findings of this study should be helpful to evaluate the reproductive response of redbelly tilapia, coptodon zillii, of both sexes upon exposure to 0.8 and 1.6 μg/l penconazole and 7.5 and 15 mg/l copper nanoparticle as fungicides for three months. the gonadosomatic index of males increased in the penconazole groups in parallel with testosterone. a significant increase was observed in estradiol and progesterone of penconazole and copper nanoparticle groups. in testicular homogenate, there was a significant decrease in superoxide radical in penconazole (i) and penconazole (ii) groups and cat of penconazole (i) and copper nanoparticle (i) groups, along with a significant increase in nitric oxide (no) of cu-np (ii) group. in ovarian homogenate, a significant increase in no of penconazole (i) group and lipid peroxides of copper nanoparticle (i) group, along with decrease in cat of penconazole (ii) and copper nanoparticle (i) groups and sod of penconazole (ii) and copper nanoparticle (i) groups were found. the histopathological examination indicated gross deteriorations in the gonads of fish exposed to the fungicides except in the copper nanoparticle (ii) group. these findings suggested the reproductive burden in c. zillii following exposure to the investigated fungicides by disrupting gonadal sex hormones and inducing redox imbalance and cytopathological abnormalities. it is recommended to reduce the flow of these materials to aquatic areas particularly the natural fisheries and artificial hatcheries. introduction penconazole (pen) is a commonly used triazole fungicide for foliar pathogen control in the horticultural, agricultural, and forest industries, but it is poisonous to a variety of aquatic organisms, including fishes (kenyon et al., 1997; icoglu et al., 2018). its environmental persistence, bioaccumulation through the food chain, and resistance to degradation draw much attention to its health risk as an aquatic toxicant (güdücü et al., 2011; cabizza et al., 2012). triazole fungicide intoxication affects not only challenge fish's reproductive ability during their entire life cycle but also their progenies, implying that it has implications for the health of fish populations on a large scale (liao et al., 2014; liu et al., 2014; dong et *correspondence: mahmoud m.s. farrag doi: https://doi.org/10.22034/ijab.v9i6.1196 e-mail: mahmoudfarrag42@azhar.edu.eg, m_mahrousfarrag@yahoo.com al., 2018). penconazole, along with other members of the triazole family, has a negative effect on fish reproductive biology on several levels, including spermatogenesis, estrogen/androgen balance, reproductive performance, sexual differentiation, apoptosis, and testicular and ovarian histoarchitecture, in addition to their ill effects on human health (li et al., 2012; shen et al., 2013; liao et al., 2014; bhat et al., 2015; chu et al., 2016; dong et al., 2018). these outcomes are most likely caused by disruption of the hypothalamic-pituitary-gonadal (hpg) axis, transcript levels of steroidogenic enzymes and androgen and estrogen receptors, redox homeostatic status, and cytohistological features (skolness et al., 2013; dong et al., 2018). 371 int. j. aquat. biol. (2021) 9(6): 370-382 nanoparticles (nps), on the other hand, are used in the fishery and aquaculture industries for a variety of direct and indirect purposes. direct applications include the feeding industry and animal welfare, such as fish disease control, while indirect applications include water and wastewater treatment, fishpond sterilisation, and commercial fish packaging (khosravi-katuli et al., 2017). the use of nanoparticles (nps) in biomedical sciences and engineering has gradually expanded over the last decade (kaida, 2004; wang et al., 2007). despite its many applications, nps toxicity has drawn much attention from scientists due to its unique properties, such as a large surface-to-volume ratio and high reactivity toward chemical and biological molecules (wani et al., 2011). cu-nps and soluble cuso4 have reproductive effects in clarias batrachus in vivo and in vitro, even at low concentrations (murugananthkumar et al., 2016). on the other hand, female mice and rats showed oxidative stress and apoptosis-mediated ovarian dysfunction, as well as a disturbance in sexual hormonal balance (yang et al., 2017; zhang et al., 2018). however, it is still unknown how cu-np exposure affects fish female reproductive performance, especially when complex interrelationships of neuro-hormonal circuits are involved. the tilapia fish's ability to spread across african waters appears to be due in large part to their diet flexibility and ability to withstand harsh aquaculture conditions (lowe-mcconnell, 2000). in addition, their high fecundity, combined with their rapid growth rate, allows them to breed and establish in areas outside of their native range (martin et al., 2010). the purpose of this research was to find out more about the effects of pen and cu-np on c. zillii reproductive function in both sexes. materials and methods determination of 96 hrs lethal concentration (lc50) of pen: the lc50 of pen was determined according to turner (1965). the pre-acclimatized c. zillii specimens were held in six glass aquaria (25 l capacity), each containing six fish. dead fish were recorded every 12 h and removed. the acute toxicity test was conducted according to turner (1965). each group of fish was subjected to daily renewed single doses of pen at 10, 12, 14, 16, 18, and 20 µg/l. the lc50 for 96 hrs exposure was calculated according to: lc50 = largest dose − ∑ a × b n where a is the dose difference between two successive doses, b is the mean of dead fish between two successive doses, and n is the total number of fish per group. fish were exposed to 1/10 lc50 (1.6 μg/l) and 1/20 lc50 (0.8 μg/l) of pen for three months. the lc50 of cu-nps was 150 mg/l (1/10 lc50 = 15 mg/l and 1/20 lc50 = 7.5 mg/l) (abdel-khalek et al., 2015) (suplemntary 1). fish maintenance: in 160 l rectangular tanks containing dechlorinated tap water, 180 adults c. zillii of both sexes with average body weights of 30±5 g and average lengths of 14±4 cm were preserved. the water parameters were maintained as conductivity = 2000 µs/cm; ph ≈ 7.5; oxygen saturation = 90-95%; temperature = 25°c; and photoperiod = 12:12 light: dark. the fish were fed 3% of their body weight in commercial feed pellets (40% crude protein, 4.22% fat, 5.88% crude fiber, 10.30% ash, and 10.03% moisture) twice a day for 21 days, and the water was changed every two days. experimental groups and sampling: fish were randomly assigned to five groups with three replicates of 12 fish each at the end of the acclimatization period. the control group received no treatment, the pen (i) group received 0.8 µg/l pen, the pen (ii) group received 1.6 µg/l pen, the cu-np (i) group received 7.5 mg/l cu-np, and the cu-np (ii) group received 15 mg/l cu-np for three months. air stones connected to an air compressor were used to aerate the tanks. the fish were independently sampled after three months of exposure. the gonad weights, body weights (bw), and standard lengths (sl) were all measured. five males and five females from each group were gathered at the end of the experiment and anaesthetised with 0.02% benzocaine solution. blood 372 farrag et al./ reproductive outcomes of coptodon zillii influence by penconazole and copper samples were taken from the caudal vein based on blazer et al. (2000), then centrifuged at 300 rpm for 10 min, and the obtained serum was stored at -20°c until analysis of testosterone, estrogen (e2) and progesterone (p4). testes and ovaries were dissected out and preserved for the later histological examination. to prepare 10% w/v homogenates of testis and ovary tissues were homogenized in phosphate buffer (ph 7.4) using a homogenizer (ika yellow line di 18 disperser, germany). the homogenates were centrifuged at 8.000 rpm for 15 minutes, and the supernatants were kept frozen at 20°c for the subsequent biochemical assays. reproductive studies: the percentages of condition factor k % (cren, 1951), gonadosomatic index gsi% (solomon; ramnarine, 2007), fecundity (sturm, 1978), and hatchability (ali, 2001) were computed as follows: k % = (w/l3) ×100 gsi % = (gonad weight / total body weight) ×100 fecundity = (average number of ripe eggs × ovary weight)/ sample weight hatchability % = (number of hatched eggs / total number of eggs) ×100 estimating gonadal sex hormones: t enzyme-linked immunosorbent assay kits (catalogue number: 11teshu-e01, alpco, salem, nh, usa) were used to measure testosterone (t) according to the manufacturer's instructions. the concentrations were determined using a standard curve built from calibrator concentrations and optical densities. e2 and progesterone (p4) (catalogue numbers bc-1111 and bc-1113, respectively) were also measured using e2 enzyme immunoassay test kits from biocheck in foster city, california, following the manufacturer's instructions. estimation of oxidant/antioxidant parameters: catalase (cat) was evaluated according to aebi (1984), superoxide dismutase (sod) was determined according to (misra; fridovich, 1972) lipid peroxides (lpo) were estimated according to ohkawa et al. (1979). nitric oxide (no) was measured as nitrite concentration using the method of ding et al. (1988). superoxide radical (o2٠−) was measured according to podczasy and wei (1988). histopathological examination: after the experiment, testes and ovaries were dissected and fixed in 10% neutral formalin. the specimens were then washed in 70% ethyl alcohol three times (24 h each time). serial sections of 6-8 µm thickness were prepared according to normal histological techniques for staining with the hematoxylin and eosin method (levison, 1997). the sections were examined by an olympus bx51 microscope and the photographs were taken by an olympus dp72 camera adapted onto the microscope. statistical analysis: the statistics package for social sciences spss (v 21) program was used to analyze the results. to find the significant difference between means, the duncan post-test was chosen from the post hoc window. the significance of probability values between 0.05 and 0.01 (both included) was determined. results effects on the reproductive performance: there were no significant changes in k among all experimental groups (table 1). the condition factor was reduced in the treated groups compared to the control one. fish exposed to cu-np (ii) showed no obvious change compared to the control. regarding pen i, pen ii, and cu-np (i) treatments, they did not show any significant difference. in males, the gsi of pen (i) and pen (ii) groups were significantly higher than that of the control group. however, there was no significant change in the gsi of cu-np (i) and cu-np (ii) groups compared to the control group. the gsi of cu-np (i) and cu-np (ii) groups were significantly lower than that of pen (i) group, while the gsi of pen (ii) group was significantly higher than that of the cu-np (i) group. the comparisons between the gsi of pen (i) group and that of pen (ii) group, the gsi of pen (ii) group and that of cu-np (ii) group, and the gsi of cu-np (i) group and that of cu-np (ii) group did not reveal a significant difference. there were no significant differences in the gsi, fecundity, and hatchability percentage of female c. zillii among all the experimental groups. 373 int. j. aquat. biol. (2021) 9(6): 370-382 effects of the different treatments on the serum levels of the gonadal sex hormones: there were significant increases in the serum testosterone levels (table 2) of pen (i), pen (ii), and cu-np (i) groups compared with that of the control group. the serum testosterone levels of pen (i) and cu-np (i) groups were significantly lower than those of the pen (ii) group. on the other hand, the serum testosterone level of cu-np (ii) group did not significantly change compared to that of the control group. compared to the control group, e2 levels in pen (ii) and cu-np (ii) groups significantly increased, while those in pen (i) and cu-np (i) groups exhibited insignificant differences. e2 level in pen (ii) group was significantly higher than that in cu-np (ii) group. there was an insignificant difference in e2 level between pen (i) and cu-np (i) groups. e2 levels in pen (i) and cu-np (i) groups were significantly lower than those in pen (ii) and cu-np (ii) groups. regarding p4, pen (ii) and cu-np (ii) groups exhibited a significant increase in their levels compared to the control group. in contrast, a comparison between p4 level in pen (i) and cu-np (i) groups and that in the control group showed no significant difference. p4 level in pen (ii) group was significantly higher than that in cu-np (ii) group. p4 levels in pen (i), cu-np (i) groups were significantly lower than those in pen (ii) and cu-np (ii) groups. p4 level in pen (ii) and cu-np (ii) groups exhibited a significant elevation compared to the control one. effects on the gonadal oxidant/antioxidant balance: table 3 shows the changes in the redox homeostatic parameters in both testis and ovary of c. zillii exposed to either pen or cu-np. concerning the oxidant /antioxidant parameters in the testis, o2− levels of pen (i) and pen (ii) groups were significantly lower than those of the control group. however, no significant difference in o2− levels were found between the cu-np (i) and cu-np (ii) groups (and the control group). cu-np (i) and cu-np (ii) groups had slightly higher o2− levels than the pen (ii) group but were not substantially different from the pen (i) group. there was no significant difference in o2− levels between the pen (i), cu-np (i), and cunp (ii) groups. when compared to the control group, the no level of the cu-np (ii) group was significantly higher, whereas the other experimental groups showed no significant difference. there were no significant changes in lpo levels and sod activities among the experimental groups. compared with the control table 1. effects of penconazole and copper nanoparticles on the condition factor percentage (k %), gonadosomatic index percentage (gsi %), fecundity, and hatchability percentage of coptodon zillii. parameter group control 0.8 μg/l pen 1.6 μg/l pen 7.5 mg/l cu-np (i) 15 mg/l cu-np (ii) pvalue k % 1.88±0.11 1.48±0.01 1.66±0.046 1.55±0.008 1.89±0.23 0.068 gsi % male 0.325±0.10c 0.76±0.07ab 0.41±0.084c 0.53±0.04bc 0.53±0.047bc 0.000 female 1.52±0.45 3.634±0.39 2.73±0.121 3.17±0.92 3.17±0.92 0.431 fecundity 1171±498 817.6±92.9 991.3±238.5 520.1±61.9 944±182 0.533 hatchability % 98.23±0.64 72.30±6.86 71.76±5.84 85.11±10.45 81.94±4.06 0.161 a-c letters indicate significant differences at p<0.05 (one-way anova followed by duncan post-test). table 2. effects of pen and cu-nps on serum sex hormone levels. parameter group control 0.8μg/l pen (i) 1.6μg/l pen (ii) 7.5mg/l cu-np (i) 15mg/l cu-np (ii) pvalue t level (µg/l) 14.12±2.82c 30.32±3.77b 56.67±8.17a 29.90±4.43b 13.8±2.5c 0.000 e2 level (ng/l) 66.4±5.2c 44.5±6.9c 857.8±40.8a 94.2±3.8c 220.55±58.23b 0.000 p4 level (µg/l) 0.240±0.075c 0.03±0.009c 1.12±0.160a 0.180±0.037c 0.540±0.051b 0.000 a-c different letters indicate significant differences at p<0.05 (one-way anova followed by duncan post-test). 374 farrag et al./ reproductive outcomes of coptodon zillii influence by penconazole and copper group, the cat activity of pen (i) and pen (ii) groups significantly increased, and that of cu-np (i) and cu-np (ii) groups showed no significant difference. a significant reduction in cat activity was observed following exposure to pen at doses of 0.8 and 1.6 μg/l compared with exposure to copper nano formulated fungicide at 7.5 and 15 mg/l. no significant differences were found when comparing the cat activity of pen (i) group with that of pen (ii) group, and cu-np (i) group with that of the cunp (ii) group. concerning the oxidant/antioxidant parameters in the ovary, the comparison between the o2levels of all experimental groups revealed a lack of significant difference. no level in c. zillii exposed to pen at a dose of 0.8 µg/l was significantly higher than the other experimental groups. the comparison among no levels in cu-np (i), cu-np (ii) and pen (ii) groups, and also between these groups one side and the control one on the other side revealed the absence of significant difference. lpo levels of cu-np (i) and cu-np (ii) groups were significantly higher than that of the control group. lpo level of the pen (i) group was significantly lower than that of cu-np (i) group, while the lpo level of the pen (ii) group was significantly lower than that of cu-np (ii) group. there was no significant difference in lpo level between either pen (i) or pen (ii) groups and the control group. the comparison between lpo levels of pen (i) and pen (ii) and between cu-np (i) and cunp (ii) showed the absence of significant difference. compared to the control group, the cat activities in pen (ii) and cu-np (i) groups showed significant decreases, while those in pen (i) and cu-np (ii) exhibited insignificant differences. a significant decrease in cat activity was observed in cu-np (i) group when compared with cu-np (ii) group, while a comparison between cat activity of pen (i) group and that of pen (ii) group revealed the insignificant difference. cat activity of pen (i) group was significantly higher than that of cu-np (i) group while, cat activity of pen (ii) group was significantly lower than that of cu-np (ii) group. sod activity was significantly decreased in cu-np (i) and pen (ii) groups compared to the control group. there was no significant difference in sod activity of pen (i) and cu-np (ii) groups compared to the control group. sod activity of pen (i) group was significantly higher than that of pen (ii) group while, sod activity of cu-np (i) group was significantly lower than that of cu-np (ii) group. a significant increase in sod activity of pen (i) group was found when compared with that of cu-np (i) group while, a significant decrease in sod activity of pen (ii) group was found when compared with that of cu-np (ii) group. effects of different treatments on the histopathological feature of gonads: the histopathological alterations in the testes of the various experimental groups are shown in figure 1ae. the histoarchitecture of the control group's testes was normal, with capsules, seminiferous tubules, and table 3. effects of pen and cu-nps on the gonadal oxidant/antioxidant balance. treatment parameters testis control 0.8μg/l pen (i) 1.6μg/l pen (ii) 7.5mg/l cunp (i) 15 mg/l cu-np (ii) pvalue o2− level (nmol/mg protein) 0.07±0.01a 0.05±0.003bc 0.034±0.01c 0.061±0.003ab 0.069±0.001ab 0.007 no level (nmol/mg protein) 2.37±0.30bc 2.78±0.17ab 1.923±0.42c 2.59±0.063abc 3.17±0.234a 0.036 lpo level (nmol/mg protein) 0.11±0.030 0.074±0.019 0.139± 0.095 0.116±0.033 0.073±0.032 0.801 cat activity (u/mg protein) 0.06±0.01a 0.038±0.003b 0.02±0.005b 0.077±0.010a 0.084±0.010a 0.002 sod activity (u/mg protein) 3.58±0.69 4.301±1.160 2.380±0.013 5.028±0.58 5.574±0.889 0.134 ovary o2− level (nmol/mg protein) 0.36±0.05 0.24±0.074 0.31± 0.0322 0.411±0.0336 0.290±0.023 0.159 no level (nmol/mg protein) 2.58±0.08b 5.442±1.450a 2.24±0.22b 2.55±0.265b 2.943±0.824b 0.027 lpo level (nmol/mg protein) 0.47±0.06bc 0.418±0.072c 0.59±0.07bc 0.733±0.005a 0.657±0.097ab 0.044 cat activity (u/mg protein) 0.05±0.01a 0.04±0.021ab 0.02±0.003bc 0.021±0.001c 0.050±0.003a 0.028 sod activity (u/mg protein) 4.64±0.60a 5.287±0.678a 2.49±0.35b 2.245±0.001b 5.42±1.10a 0.015 375 int. j. aquat. biol. (2021) 9(6): 370-382 germ cells (fig. 1a). in the testes of the pen (i), however, gross degenerative changes in the seminiferous tubules, a decrease in the number of spermatozoa, and hemorrhage were discovered (fig. 1b). in the experiments of the pen (ii) group, signs of inflammatory responses appeared in the form of congestion in the seminiferous tubules, hemorrhage, and leukocyte infiltration (fig. 1c). the testes of the cu-np (i) group displayed normal spermatozoa, spermatogonia, and spermatocytes; at the same time, there was a marked decrease in the number of spermatozoa relative to spermatocytes and spermatogonia (fig. 1d). slight histopathological changes were observed in the cu-np (ii) testes group, including leukocyte infiltration at the margins while spermatozoa, spermatogonia, and spermatocytes maintained their normal appearance (fig. 1e). the histopathological changes in the ovary of the various experimental groups are shown in figure 2a. the structure in the control group was found to be normal. the vitellogenic oocyte/vitelline membrane appeared normal and intact to the outside, with easily distinguishable zona radiata and follicular epithelium. the size of the follicles increased proportionally at each stage of development. the follicular layers were not fully formed in the primary stage, but they were visible. the follicles were smaller in diameter at this point in their growth. the ooplasm was filled with figure 1. photomicrographs of histological changes in testes of coptodon zillii fish (h&e stain) exposed to different concentrations of penconazole and copper nanoparticles. a: testes of control group showing normal histological features of testis with normal seminiferous tubules, normal capsule (arrow), normal spermatozoa (st), and normal spermatogonia and spermatocytes (star): a: bar = 150 mµ, b: bar = 40 mµ. b: testes of pen (i) group showing gross degenerative changes in the seminiferous tubules (arrow), decrease in the number of spermatozoa (st), and hemorrhage (hg): bar = 40 mµ. c: testes of pen (ii) group showing congestion in the seminiferous tubules (arrow), hemorrhage (gh), and leucocyte infiltration (star): bar = 40 mµ. d: testes of cu-np (i) group showing normal spermatozoa (st), spermatogonia and spermatocytes (star), and decrease in the number of spermatozoa and increase in the number of spermatocytes and spermatogonia relative to the control. bar = 40 mµ. e: testes of cu-np (ii) group showing normal spermatozoa (st), spermatogonia and spermatocytes (arrowhead) with leucocytes infiltration at the margins (arrow): bar = 40 mµ. 376 farrag et al./ reproductive outcomes of coptodon zillii influence by penconazole and copper granular structures called cortical alveoli during the cortical alveolus stage. at this stage, the zona radiata began to form, and follicular epithelial cells appeared to form on the exterior. the next stage of development was the vitellogenic stage, and vitellogenesis was observed in the developing follicles. at the final stage of oocyte development, i.e., the mature stage, marked vitellogenesis was observed in the matured oocyte. figure 1. photomicrographs of histological changes in ovary of coptodon zillii fish (h.e stain) exposed to different concentrations of penconazole and copper nanoparticles. a: ovary of control group showing; normal primary stage (ps), cortical alveoli stage (cas), vitellogenic stage (vs) and mature oocyte (mo), follicular epithelium (fe), zona radiate (zr), nucleolus (no) and nucleus (n), bar = 150 µm. b: ovary of pen (i) group showing; decreased primary stage (ps) and cortical alveoli stage (cas), atretic vitellogenic and mature oocyte stages (ao), bar= 200 µm. c: ovary of pen (ii) group showing; degeneration (atretic) of all mature oocyte stage (ao) with membrane detachment of vitellogenic stage (arrow), bar = 40 µm. d: ovary of cu-np (i) group showing; normal primary stage (ps) and cortical alveoli stage (cas), atretic vitellogenic and mature oocyte stages (ao) with membrane detachment (arrow), bar= 150 µm. e: ovary of cu-np (ii) group showing normal primary stage (ps) and cortical alveoli stage (cas), atretic vitellogenic and mature oocyte stages (ao) with membrane detachment with mild degenerative changes and slight vitellogenic droplets (arrow), degenerated zona radiate (zr), bar= 200 µm. 377 int. j. aquat. biol. (2021) 9(6): 370-382 after being exposed to various concentrations of pen and cu-np, structural changes in the ovaries were observed. microscopic examination found mild ovarian changes after three months of cu-np exposure. the ovaries of the pen (i) group (fig. 2b) showed a decrease in the primary and cortical alveoli stages, as well as elongated ovarian follicles in the atretic vitellogenic and mature oocyte stages. degeneration in (atretic) of all mature oocyte stages was observed in the ovaries of the pen (ii) group (figure 2c), along with membrane detachment of the vitellogenic stage. the cu-np (i) group's ovaries (fig. 2d) revealed zona radiata detachment from the oocyte membrane in vitellogenic and mature oocytes. this condition, however, was not seen in the primary or cortical alveoli stages. similarly, oocytes in the primary and cortical alveolar stages appeared normal in the cu-np (ii) group's ovaries (fig. 2e). discussions according to the results, exposure to pen and cu-np caused an increase in testosterone production, indicating a possible disruption in the hpg axis. although testosterone levels in the pen and cu-np groups were higher, there were obvious negative changes in the spermatogenesis process, which could decrease fertility. adaptive changes in metabolic pathways to cope with contaminant stress were caused by the pen and cu-np challenges, as were organ and dose-specific changes in the oxidant/antioxidant profile to reduce oxidative stress. the biochemical measurements are supported by histopathological alterations in the testicular and ovarian histoarchitecture, which indicate a disruption in the cellular microenvironment and functionality. these findings should be useful in evaluating the potential ecological risk of fungicides, particularly in linking changes in endocrine function indicators to negative consequences in the entire organism. the gsi significantly increased following exposure of males to pen at doses of 0.8 and 1.6 μg/l in the current study, similar to pimephales promelas exposed to propiconazole (pcz) and danio rerio exposed to clt (baudiffier et al., 2013; skolness et al., 2013). histomorphometric analysis of the testes challenged with other members of triazole fungicide showed enlargement of the interstitial space, duplication of leydig cell mass, and distension of seminiferous tubules. the observed increase in gsi under the effect of pen might be due to the stimulation of the hpg axis leading to hypertrophy of the testicular tissues. the current findings of gsi disagreed with the observations of other dangerous materials such as ddt and chlorpyrifos (mlambo et al., 2009). gonadosomatic index is a sensitive marker, and its decrease indicates decreased hypothalamic, pituitary, or gonadal activity (kime, 1999; rahman et al., 2020). in this study, the significant increase in testosterone levels in the pen (i), pen (ii), and cu-np (i) groups corresponds to previous reports on the effects of other triazole family members and soluble and np forms of copper on other fish species (wu et al., 2014; murugananthkumar et al., 2016). the significant increase in e2 level in the pen (ii) group compared to the baseline level is similar to that seen in zebra fish (danio rerio) after exposure to difenoconazole (dong et al., 2018). cu-np stimulated basic hormonal functions in coptodon zillii exposed to 15 mg/l cu-np, as confirmed by an in vitro study on cultured porcine ovarian granulosa cells, suggesting the stimulatory effects of cu-np on their basic hormonal functions, taking into account differences in response in relation to cu-np shape, which play a role in modifying its activity and selectivity (sirotkin et al., 2020). compensatory responses include up-regulation of steroidogenic enzymes, transcription factors, sertoli cell genes androgen receptor, and testicular expression of the follicle-stimulating hormone gene, as well as the proliferation of gonadal tissues, may be responsible for the current results. these adaptive mechanisms may be important in overcoming pen and cu-np gonadotoxicity and also in preventing gonadal sex hormone overproduction. nonetheless, despite a significant increase in testosterone levels in the pen and cu-np groups. the apparent disruption in the spermatogenesis process based on the histopathological section of the current study should 378 farrag et al./ reproductive outcomes of coptodon zillii influence by penconazole and copper be kept in mind. the significant decreases in cat activity in the testis of pen (i) and pen (ii) groups in the present work are in harmony with that reported in rainbow trout (oncorhynchus mykiss) exposed to sublethal concentrations of pcz for 20 and 30 days (li et al., 2010b) and in goodeid fish (chapalichthys pardalis) exposed to ag-np for 21 days (valerio-garcia et al., 2017). the decrease in cat activity may be interpreted as a sign that the redox-sensitive condition improves. however, this conclusion will not be cutting-edge until other enzymatic and non-enzymatic antioxidants are estimated, reflecting the logic behind the recommendation that total antioxidant capacity analysis is the most relevant investigation for evaluating oxidant/ antioxidant capacity, rather than the simple sum of observable antioxidants, takes into account the cumulative synergistic action of all antioxidants present in the sample and provides an integrated parameter (ghiselli et al., 2000). the reduction in testicular o2− levels in the pen (i) and pen (ii) groups compared to the control group may represent an improvement in the antioxidant defensive mechanism; it is not necessary to look at the levels of studied antioxidants because the testis is armed with a wide range of enzymatic and nonenzymatic antioxidants. triazole fungicides cause low-intensity oxidative stress, which causes a compensatory increase in the antioxidant potential to counteract the negative effects of ros (husak et al., 2017). they also cause low-intensity oxidative stress, which causes a compensatory increase in the antioxidant potential to counteract the negative effects of ros. increased no levels in cu-np (ii) testicular homogenate compared to control and pen (ii) groups, as well as pen (i) ovarian homogenate compared to other groups, could be attributed to increased inducible no synthase (majewski et al., 2020). through its interaction with the superoxide radical to create highly reactive peroxynitrite, no is a potent mediator of cellular injury (pacher et al., 2007), highlighting its possible role in gonadotoxicity. the ability of cu-np to induce lipid peroxidation in the ovary is inconsistent with that observed in the liver and gills of juvenile epinephelus coioides (wang et al., 2014). this may be due to the catalytic production of ros on the np surface (srikanth et al., 2016). following endocytosis, the release of copper from cunp causes damage to macromolecules, especially polyunsaturated fatty acids, due to an excess of reactive oxygen species (ros) produced by fentontype reactions (heinlaan et al., 2008). the present study's findings of substantial inhibition of cat activity in the testis of pen (i) and pen (ii) groups, as well as sod in the ovary of pen (ii), corroborate previous findings in cyprinus carpio acutely exposed to various concentrations of tebuconazole (li et al., 2010a). according to li et al. (2010a), under triazole fungicides, excessive reactive oxidant production has been shown to suppress enzymatic antioxidants, shifting the oxidant/antioxidant balance to the oxidant side. as shown in this study, increased t levels may have a negative feedback effect on gnrh, fsh, and lh secretion, resulting in spermatogenesis inhibition. in pen (ii) group, the fungicide evoked inflammatory responses in testis as manifested in this study by congestion in the seminiferous tubules, haemorrhage, and leucocyte infiltration. the activation of nuclear transcription factors may promote the release and formation of various inflammatory chemokines due to increased ros production and decreased in antioxidant defence mechanisms. chemokines play a role in leukocyte migration during intoxication (bautista, 2002; reinke et al., 2000). increased leukocyte infiltration in the testes exposed to pen is likely due to upregulated expression of various chemokine system constituents. the separation of the zona radiata from the oocyte membrane in vitellogenic and mature oocytes after exposure to cu-np (i) is consistent with previous findings in zebrafish (danio rerio) challenged with gold np (dayal et al., 2016). one of the explanatory factors involved in this cytological modification following cu-np bioaccumulation in the ovarian tissue could be oxidative stress-induced cell cycle arrest and induction of the apoptotic cascade (yang et al., 2017; zhang et al., 2018; naeemi et al., 2020). the adverse 379 int. j. aquat. biol. 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(2018). copper nanoparticles show obvious in vitro and in vivo reproductive toxicity via erk mediated signaling pathway in female mice. international journal of biological science, 14(13): 1834-1844. 382 farrag et al./ reproductive outcomes of coptodon zillii influence by penconazole and copper suplemntart 1. determination of 96 hrs lethal concentration (lc50) of pen pen (µg /l) number of fish number of dead fish a b 0 6 0 0 0 0 12 6 0 12 0 0 14 5 1 2 0.5 1 16 3 3 2 2 4 18 1 5 2 4 8 20 0 6 2 5.5 11 sum 24 lc50 = 20 (24/6) = 16 µg/l int. j. aquat. biol. (2015) 3(1): 19-24 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article effect of actinobacteria as a single cell protein on growth performance of xiphophorus helleri selvakumar dharmaraj*1,2, kandasamy dhevendaran3 1department of aquatic biology and fisheries, university of kerala, kariavattom campus, thiruvananthapuram – 695 007, kerala, india. 2department of biotechnology, mohamed sathak college of art and science, sholinganallur600116, tamil nadu, india. 3school of chemical and biotechnology, sastra university, thanjavur 613401, tamil nadu, india. article history: received 25 january 2014 accepted 24 december 2014 available online 2 5 february 2015 keywords: single cell protein (scp) actinobacteria streptomyces ornamental fish xiphophorus helleri abstract: the potential of marine actinobacteria particularly streptomyces as a single cell protein (scp) feed for the growth of ornamental fish, xiphophorus helleri has been investigated. the streptomyces strains used as scp were isolated from the marine sponges, namely callyspongia diffusa, mycale mytilorum, tedania anhelans and dysidea fragilis. six scp feeds were prepared and their effects were compared with those of control diet. after 30 days of feeding trials, the growth parameters including absolute growth rate, specific growth rate and feed conversion efficiency were found to be significantly (p<0.001) higher in groups that received scp feed than those of control one, whereas feed conversion ratio was lower. thus it was found that in addition to being effective antibiotic agents against harmful pathogens, streptomyces could also promote the growth of fish effectively. marine actinobacteria, particularly streptomyces, could play an important role as a single cell protein (scp) in aquaculture nutrition and is a promising microbe for the development of marine biotechnology. introduction actinobacteria represents one of the largest taxonomic units among the 18 major lineages currently recognized within the domain bacteria, including five subclasses and 14 suborders (stackebrandt, 2000). among the five subclasses, actinobacteria belonging to the order actinomycetales (commonly called actinomycetes) account for approximately 7,000 of the metabolites reported in the dictionary of natural products and the vast majority of these compounds are derived from the single genus streptomyces. streptomyces species are distributed widely in marine and terrestrial habitats (pathomaree et al., 2006) and are of commercial interest due to their unique capacity to produce novel metabolites. marine streptomyces are widely distributed in biological sources such as fishes, molluscs, sponges, seaweeds, mangroves, besides seawater and * corresponding author: selvakumar dharmaraj e-mail address: biochem_selva@yahoo.com sediments (dhevendaran and anithakumari, 2002; dhevendaran and praseetha, 2004; qianqun et al., 2004; sivakumar et al., 2007; das et al., 2008; elshatoury et al., 2009; selvin et al., 2009). these organisms are gaining importance not only for their taxonomic and ecological perspectives, but also for their novel secondary metabolites production like antibiotics, enzymes, enzyme inhibitors, pigments and their biotechnological application such as probiotics and scp (single cell protein) (dharmaraj, 2010; das et al., 2010). algae, fungi and bacteria are the chief of microbial protein (single cell protein-scp) that has been utilized as a protein supplement (anupama, 2000). though marine actinobacteria particularly streptomyces are known to produce primary as well as secondary metabolites and also has advantages such as (1) the degradation of macromolecules, such as starch and protein in the culture pond water, (2) 20 int. j. aquat. biol. (2015) 3(1): 19-24 the production of antimicrobial agents and (3) the formation of heat and desiccation resistant spores. there are only few attempts on the usage of streptomyces spp., as scp in aquaculture. there has been report on streptomyces incorporated feed as a scp source, isolated from the fish gut of catla catla. these diets exhibited a remarkable increase in growth pattern and better conversion efficiency compared to control feed (suguna and rajendran, 2012). another report shows improved growth, feed conversion efficiency and higher protein content in juvenile prawns on 50 days of feeding trials of streptomyces as scp source (manju and dhevendaran, 1997). by considering the above facts, this study aimed to use streptomyces as scp feed for the ornamental fish xiphophorus helleri and to evaluate its effects on the growth performance based on absolute growth rate (agr), specific growth rate (sgr), feed conversion efficiency (fce) and feed conversion ratio (fcr). materials and methods experimental fish: red swordtails (x. helleri, cyprinidae) weighing about 1.5 g were stocked in seven 20-litre plastic troughs, each contains twenty fish. one trough fed with control feed, whereas the rest of the fish were supplemented with scp feed. the experiment was conducted for 30 days and repeated in triplicate. streptomyces as single cell protein (scp): single colonies of streptomyces strains were isolated from marine sponges namely callyspongia diffusa, mycale mytilorum, tedania anhelans and dysidea fragilis collected at a depth of 5 to 10 m from vizhinjam port, situated about 16 km to the south of trivandrum at 8°22'30" n latitude and 76°59'16" e longitude on the south-west coast of india. among 94 cultures isolated, only six streptomyces strains namely aqbcd03, aqbcd11, aqbmm35, aqbmm49, aqbta66 and aqbdf81 were used as scp source and the characterization of the isolates has been detailed in dharmaraj and sumantha (2009) and dharmaraj (2011). the strains were inoculated in 500 ml of starch casein broth in a 1 liter erlenmeyer flask and incubated at room temperature for seven days. the streptomyces grew as a mat on the surface of the broth (non-motile form). the mat was harvested and the cell mass was dried and mixed with the formulated feed ingredients (das et al., 2006). feed preparation: fish were fed with seven different feeds including the scp feed and control feed for 30 days. formulated diets were prepared according to the least square method (hardy, 1980). the protein content in the ingredients was estimated by kjeldahl method (belcher and godbert, 1954). the compositions of the ingredients used for preparing formulated diets and their protein mass fractions are shown in tables 1 and 2. control feed: the ingredients of control feed consist s. no. ingredients protein (%) 1 rice bran 10.522 2 chickpea flour 35.540 3 ground nut oil cake 40.010 4 tapioca flour 2.520 5 fish meal 55.004 6 streptomyces sp.aqbcd03 (f1) 55.110 7 streptomyces sp aqbcd11 (f2) 56.672 8 streptomyces sp aqbmm35 (f3) 55.342 9 streptomyces sp aqbmm49 (f4) 56.236 10 streptomyces sp aqbta66 (f5) 55.094 11 streptomyces sp aqbdf81 (f6) 55.872 table 1. concentration of protein in the ingredients added to the formulated feeds. 21 dharmaraj and dhevendaran/ effect of actinobacteria as scp on growth performance of x. helleri of fish meal, rice bran, groundnut oil cake and chickpea flour with tapioca flour as binder. the ingredients were ground to a fine powder and mixed thoroughly with sufficient water to obtain smooth dough. the dough thus prepared was steam cooked for 30 min and allowed to cool. this was extruded through a pelletiser. the pellets were dried and then stored in dry airtight containers at 28°c. single cell protein (scp) feed: the steam cooked dough was prepared and cooled as given above. to this dough, a known quantity dried cell mass of streptomyces, was added and mixed. this was extruded through a pelletiser, the pellets were dried and then stored in dry airtight containers at 28°c. determination of growth performance: the fish were fed with prepared feeds at the rate of 5% body mass once a day. the unconsumed feed was siphoned out 6 hours after feeding. the following morning, the faecal matter was collected from each trough. the unconsumed feed and faecal matter were dried in an oven at 60°c and their mass was recorded. about 75% of water of each trough was changed every day with minimum disturbances to the fish. the initial mass was measured before the experiment and the final mass on the 30th day after the feed supplementation. the growth parameter like absolute growth rate (agr), specific growth rate (sgr), feed conversion efficiency (fce) and feed conversion ratio (fcr) were calculated by the method described by halver (1972). the formulae for calculation are given by the eqs. 1-4, respectively: agr = (final mean mass–initial mean mass)/g [1] sgr = [(ln final mass–ln initial mass)/ rearing period] ×100 [2] fce = [(final mass–initial mass)/ (feed given– unconsumed feed)] ×100 [3] fcr = [(feed given–unconsumed feed)/ (final mass– initial mass)] [4] chemical analysis: the proximate composition of feed and tissue of the fish were analysed for moisture, crude protein, crude fat, crude fiber and ash and nitrogen-free extract according to the standard methods of aoac (2000). the proximate ingredients formulated feeds (g %) control feed scp feed rice bran 16.50 16.50 chickpea flour 18.50 18.50 groundnut oil cake 23.50 23.50 tapioca flour 16.00 16.00 fish meal 25.50 15.50 streptomyces dried cell mass 10.00 table 2. composition of ingredients in formulated feeds. formulated feeds proximate composition (%) moisture crude protein crude fat ash crude fibre nitrogen-free extract (nfe) control 10.87 35.04 12.02 13.80 2.45 25.82 f1 11.90 45. 20 14.20 13.34 2.85 12.51 f2 11.51 43.25 13.90 13.50 2.62 15.22 f3 11.91 44.22 13.21 13.66 2.78 14.22 f4 11.95 45.15 13.31 12.33 2.94 14.32 f5 11.48 44.08 13.28 13.58 2.70 14.88 f6 11.56 44.56 13.26 13.28 2.81 14.53 table 3. proximate composition of different feeds. 22 int. j. aquat. biol. (2015) 3(1): 19-24 compositions of the formulated feeds are displayed in table 3. statistical analysis: all experiments were performed in triplicate and the results are expressed as mean values with ± standard deviation (± sd). data were subjected to one way analysis of variance (anova; sigmastat v. 3.5, systat software inc, san jose, ca, usa) and means were separated by tukey test at p<0.001 significant level (hayter,1986). results thirty days of feeding trials with single cell protein (scp) result a marked increase in the growth performances of the fishes compared to control treatment. the proximate compositions of red swordtail fish fed on formulated diets are displayed in table 4. when comparing the fcr, the control feed given fishes showing the higher value of 3.724 ± 0.256 than those of others. the fce values of f3 (78.732 ± 0.946), f5 (67.326 ± 0.363) were higher than the control treatment (26.940 ± 1.875). there was significant increase in food conversion efficiency when fed with scp feed and decreased values of food conversion ratio. the scp feed was rich in protein and this may be the reason for the better growth rate. the scp feeds given fishes were found to be significant than the control fishes with respect to agr and sgr (p<0.001) (table 5). the formulated feeds proximate composition (%) moisture crude protein crude fat ash crude fibre nitrogen-free extract (nfe) control 77.00 15.00 3.00 3.20 1.00 0.80 f1 78.00 15.25 3.15 2.65 0.75 0.20 f2 77.65 15.14 3.09 2.90 0.85 0.37 f3 78.04 15.02 3.10 3.00 0.59 0.25 f4 77.97 15.31 3.02 2.67 0.75 0.28 f5 78.01 15.06 3.04 3.12 0.46 0.31 f6 78.05 15.00 3.02 3.10 0.48 0.35 table 4. proximate composition of red swordtail fish fed on different diets. formulated feeds initial weight (g) final weight (g) agr (g) fce (%) fcr(g) sgr (%) c 1.530 ± 0.020 1.840 ± 0.030 0.310 ± 0.010a 26.940 ± 1.875a 3.724 ± 0.256g 0.659 ± 0.012a f1 1.520 ± 0.010 2.050 ± 0.010 0.530 ± 0.010c 46.167 ± 0.871c 2.167 ± 0.041e 1.068 ± 0.030c f2 1.530 ± 0.010 1.993 ± 0.015 0.463 ± 0.006b 39.881 ± 0.947b 2.508 ± 0.059f 0.945 ± 0.006b f3 1.533 ± 0.025 2.540 ± 0.020 1.007 ± 0.006g 78.732 ± 0.946g 1.270 ± 0.015a 1.803 ± 0.031g f4 1.520 ± 0.020 2.243 ± 0.020 0.723 ± 0.003e 63.014 ± 0.812e 1.587 ± 0.020c 1.390 ± 0.016e f5 1.530 ± 0.010 2.35 ± 0.010 0.820 ± 0.005f 67.326 ± 0.363f 1.485 ± 0.008b 1.532 ± 0.016f f6 1.535 ± 0.005 2.23 ± 0.020 0.695 ± 0.018d 59.456 ± 0.685d 1.682 ± 0.019d 1.333 ± 0.028d values in the same column sharing a common superscript are not significant (p<0.001) table 5. agr, fce, fcr and sgr in xiphophorus helleri fed with control feed and scp feeds. source of variation degrees of freedom sum of squares mean square f between groups 6 0.985 0.164 1838.804*** within groups 14 0.00125 0.0000893 total 20 0.986 *** significant (p<0.001) table 6. one way anova for weight gain of xiphophorus helleri fed with control feed and scp feeds. 23 dharmaraj and dhevendaran/ effect of actinobacteria as scp on growth performance of x. helleri weight gain showed significant differences at the 1% level by tukey test, when comparing the control feed and scp feeds given fishes (tables 6). discussion there are reports in support of the streptomyces as scp in aquaculture. the juvenile prawns macrobrachium idella fed with streptomycesincorporated feed for 50 days, showed improved growth (140.54%) and feed conversion efficiency (45%), and higher protein content (54.72%) (manju and dhevendaran, 1997). furthermore, preliminary report on the growth increment of tiger shrimp, penaeus monodon on supplementation of streptomyces-incorporated feed showed high growth in terms of length (15.79%) and weight (57.97%) compared with those of control treatment (length (4.08%) and weight (32.77%)) (das et al., 2006). also, oreochromus mossombicus fed with single cell protein (scp) from azolla sp. showed better growth and conversion efficiencies (manju and dhevendaran, 2002). in the present work the scp feed replaced nearly 30-40% of the fish meal incorporated in the feed. in aquaculture, essential and expensive components of the feed are proteins, especially the fish meal. since the supply of fish meal has become uncertain and the prices have increased rapidly, the need for cheaper alternative protein sources has increased. among unconventional protein sources, microbial origin appears to be a promising substitute for fish meal, replacing up to 25-50%. this research in line with above-mentioned works, indicates the importance of marine actinobacteria, particularly the application of streptomyces in aquaculture. conclusions marine microorganisms synthesise macromolecules and vitamins which benefit animal nutrition. there are very limited reports on the application of actinobacteria, particularly streptomyces as scp in aquaculture. this is the first report on the application of streptomyces of marine origin act as scp feed for the growth of ornamental fish and this study proved that there is a significant increment in the growth of the ornamental fish x. helleri fed with scp feed. therefore, in the near future the applications of streptomyces as single cell protein (scp) will play an important role in aquaculture nutrition and is a promising microbe for the development of marine pharmaceutical biotechnology. acknowledgements the authors wish to thank the department of science and technology (dst), new delhi, india for financial support and the department of aquatic biology and fisheries, university of kerala, for providing necessary facilities. references anupama p. 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(2018) 6(5): 265-273 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article the al wathba wetland reserve lake in abu dhabi, united arab emirates and its ostracod (seed shrimp) fauna anitha saji *1, steffen mischke2, pritpal s. soorae1, shakeel ahmed1, shaikha al dhaheri1 1environment agency abu dhabi (ead), united arab emirates. 2faculty of earth sciences, university of iceland, sturlugata 7, askja, 101 reykjavík, iceland. s article history: received 26 july 2018 accepted 4 january 2019 available online 5 january 2019 keywords: heterocypris salina cyprinotus cingalensis salinity ramsar wetland site abstract: al wathba wetland reserve (awwr) lake, in the united arab emirates (uae), is an artificially created water body in a natural wetland region that experienced seasonal flooding before the establishment of the lake. the lake is mostly fed by treated waste water, and became a protected wetland reserve after the greater flamingo started to successfully breed in the area in 1998. detailed monitoring of several hydrochemical parameters and water depth at nine stations and two inlets of treated water in the lake was conducted over a period of seven years starting in january 2010. as a result, the seed-shrimps (ostracoda: podocopida) heterocypris salina, previously reported from a late miocene location in the uae, and cyprinotus cingalensis were recorded for the modern fauna of the uae for the first time. the presence of the ostracods only at the station with the lowest salinity in the awwr lake shows that their distribution is predominantly controlled by the salinity of the water which covered an extremely large range of more than two orders of magnitude (1.45-457‰) at the different sampling sites and inlets during the monitoring period. thus, the lake represents an interesting and important ecological research laboratory under semi-natural conditions. introduction terrestrial surface waters are rare in arid regions and they represent important biodiversity hotspots of the aquatic and terrestrial flora and fauna, including migratory birds, amphibians and mammals in lake, wetland and gallery forest regions. dryland lakes often support local fisheries and provide recreational services. natural surface waters in western and central asia are often endangered due to man-made hydrological changes such as water withdrawal for irrigation farming and due to increasingly frequent extreme weather conditions as a result of global climate change (lelieveld et al., 2012). the desiccation of the aral sea, the rapidly falling level of the dead sea and the disappearing lake urmia are the most prominent and serious examples (micklin, 1988; tourian et al., 2015; willner et al., 2015). knowledge of the state of local surface waters in arid regions is urgently required to understand their ecological and societal significance, and to assess their vulnerability to human impacts and global climate change. *correspondence: anitha saji doi: https://doi.org/10.22034/ijab.v6i5.483 e-mail: asaji@ead.ae an important component of aquatic ecosystems such as al wathba is the micro-crustacean fauna. among micro-crustaceans, the ostracods form a widely distributed and diverse group which occupies all types of aquatic or even semi-aquatic environments (the ocean, marginal marine settings such as lagoons and estuaries, saline and freshwater lakes, rivers, wetlands, springs, wet leaf litter, etc.). the ostracods have two calcitic valves that enclose the soft body and form a carapace typically 0.5-2.0 mm long. more than 65,000 living and fossil ostracod taxa at or below the species level have been described (kempf, 1997). we here present a study of a man-made lake in the uae with a focus on its spatial and temporal waterchemistry characteristics and a first record of its ostracod (micro-crustacean) fauna. materials and methods the al wathba wetland reserve (awwr) lake is located on the coastal sabkha plain ca. 40 km southeast of abu dhabi island (fig. 1). the partly 266 saji et al./ al wathba wetland in abu dhabi and its ostracod fauna dune-covered sabkha in the lake area lies 15-18 m above sea level. the region represented a seasonally flooded wetland with temporarily rising water levels probably resulting from increasing sub-surface water flow and hydrostatic pressure from stronger winds and higher tides in winter (al dhaheri, 2004). the construction of the mussafah al ain truck road between 1980 and 1984 dammed the southward flow of surface waters and increased the area and annual duration of emerging surface waters, and the abundance of waterfowl. the ecological potential of the wetland for local birdlife was recognised and awwr lake established as a perennial water body mostly fed by treated waste water from the mafraq wastewater treatment plant, which is the main sewage treatment plant of the city of abu dhabi (fig. 2). the permanent water body attracts a diverse birdlife and became protected as al wathba wetland reserve since 1998 due to the first successful breeding of the greater flamingo (phoenicopterus roseus) on the arabian peninsula since a last record in 1922 from kuwait (ticehurst, 1926). its high biodiversity including 260 bird species (soorae et al., 2014) led to the declaration as an internationally recognised and protected ramsar wetland site in 2013. the protected wetland has an area of ca. 5 km² and the lake surface covers ca. 1.6 km² (al dhaheri, 2004). the maximum water depth of the lake is 2.3 m. the awwr is managed by the environment agency abu dhabi (ead) and mainly used for research, education and eco-tourism including birdwatching today. the salinity of awwr lake is variable due to the fresh water input and the underlying sabkha substrate. however, most of the lake is hyperhaline with a mean salinity of ca. 200‰ between 2010 and 2014 (saji et al., 2016). during the same period, mean monthly values of water temperature, ph and dissolved oxygen concentration ranged from 22.8-35.9°c, 7.0-8.6, and 3.7-7.9 mg l-1, respectively (saji et al., 2016). mixed plant communities of zygophyllum qatarense, haloxylon salicornicum, dipterygium glaucum, anabasis setifera and tamarix cf. ramossisiima dominate the vegetation, with phragmites australis reed beds along the less brackish margins of the lake (al dhaheri, 2004) (fig. 3). the mean january and july temperatures are 19.8 and 35.8°c, respectively, and mean annual temperature is 28.1°c (ncms, 2017). mean annual precipitation in the region is ca. 100 mm, mostly occurring between december and february, and mean annual evaporation in the order of 2000 mm (abdelfattah and meharibi, 2005; scad, 2015). water samples were collected within the frame of an artemia research programme from nine stations figure 1. al wathba lake in the al wathba wetland reserve in the uae (green dot) and the location of a late miocene record of the ostracod heterocypris salina (triangle). figure 2. sampling stations st 1 to 10 and the inlet water valves i and ii of the wastewater treatment plant (wwtp). ostracods were exclusively recorded at station 8. 267 int. j. aquat. biol. (2018) 6(5): 265-273 (st 01 to st 10, st 09 excluded due to mostly dry conditions) in the lake and two inlets (inlets i and ii) on a quarterly basis between 2012 and 2015 and on an almost complete monthly basis in 2010, 2011 and 2016. this programme was established by the terrestrial environment research center of the environment agency abu dhabi in 2001 to monitor artemia as most important food source for birdlife and especially the greater flamingo in the awwr. the sampling sites for artemia were chosen based on a 100 m grid overlaid on awwr lake. the samples were collected with a van dorn water sampler (2.2 l) from the water column below the water surface. the two inlets were not examined for the presence of artemia or ostracods. the water was passed through a 20 µm plankton net and the retained organisms examined using a zeiss stemi 2000 low-power binocular microscope. the presence of ostracods was checked on a regular basis and the identification of taxa is based on two samples of 23 march (sample 1) and 22 june 2016 (sample 2) which were stored in 70% ethanol. identification was aided by the use of a zeiss supra 40vp scanning electron microscope at the institute of geological sciences of the free university of berlin (germany) and is based on descriptions of (malz, 1976; meisch, 2000). voucher specimens in ethanol are deposited in the wet collection of the invertebrate collection of the environment agency abu dhabi (icead). the water depth was recorded at each station with a measuring metre scale. a hand-held wtw 350i was used to measure ph and temperature. salinity was mostly measured in the lab using sub-samples of water samples diluted with distilled water. lab analyses of water samples focused on nitrogen (as nitrate, nitrite and ammonia), phosphate (as po4), total organic carbon (toc) and heavy metals copper (cu), cadmium (cd) and iron (fe), and followed standard procedures (u.s. environmental protection agency, 1983; saji et al., 2016). all the data were presented in graphs drawn using coraldraw v.12 and grapher (golden software v.3). figure 7 was prepared using corel photo-paint and coreldraw and the map of figure 9 was created in amcharts software. results the water depth at the sampling stations changed over the observation period by a minimum of 0.56 m at st 10 to a maximum of 2.10 m at st 04 (fig. 4). the salinity ranged from 1.45‰ at inlet i to 457‰ at st figure 3. typical scenery of the al wathba lake. figure 4. water depth data for the sampling stations (st) during the years 2010 to 2016. grey vertical bars indicate months during which water samples were unsuccessfully examined for the presence of ostracods and orange bars represent months during which ostracods were present at st 08. the two horizontal lines at the left show the water depth range at st 08 during the monitoring period and the vertical line in between these lines indicates the water depth range for the ostracod records at st 08. st 08 was dry in march 2014 and ostracods were not recorded. 268 saji et al./ al wathba wetland in abu dhabi and its ostracod fauna 07 (fig. 5). the ph value was lowest with 6.55 at inlet i and highest with 9.67 at st 10 (fig. 6). nitrate concentrations were lowest at st 08 with <0.03 mg l-1 and highest at st 10 with 771.66 mg l-1 (fig. 8a). nitrite concentrations ranged from <0.03 mg l-1 at st to 9.07 mg l-1 at inlet i (fig. 8b). ammonia concentrations were as low as <0.02 mg l-1 at sts 07 and 10 and as high as 18.7 mg l-1 at st 03 (fig. 8c). phosphate concentrations were in a range from <0.2 mg l-1 at sts 01 to 07 and st 10, to 20.88 mg l1 at inlet ii (fig. 8d). toc concentrations were between 2.4 mg l-1 at inlet i and 257.3 mg l-1 at st 10 (fig. 8e). concentrations of cd were often below detection limit (<0.0005 mg l-1) and reached a maximum of 1.62 mg l-1 at inlet i (fig. 8f). the cu concentrations were also often below detection limit (<0.005 mg l-1) and a maximum of 26.94 mg l-1 was determined for inlet i (fig. 8g). concentrations of fe remained also often below detection limit (0.05 mg l1). a highest value of 259.2 mg l-1 was recorded at inlet ii (fig. 8h). ostracods were exclusively recorded at st 08 during the course of the observations between the figure 5. salinity data and ostracod records in the years 2010 to 2016 shown in a similar way as for water depth in figure. 4. (refer to fig. 4 for symbols of the sampling stations). figure 6. ph data and ostracod records in the years 2010 to 2016. (refer to fig. 4 for symbols of the sampling stations). figure 7. the ostracods heterocypris salina (1-6) and cyprinotus cingalensis (7-10) from awwr lake. heterocypris salina: (1) right valve (rv) with anterior soft parts, 2 left valve (lv) with anterior soft parts, 3 rv, 4 carapace with rv fully visible, 5 juvenile rv, 6 lv; cyprinotus cingalensis: 7 rv, 8 lv, 9 rv with more pronounced “hump”, 10 juvenile rv. all images apart from 4 show internal views of valves. specimens housed at institute for geological sciences of the free university of berlin, germany. 269 int. j. aquat. biol. (2018) 6(5): 265-273 years 2010-2016. ostracods were present during two thirds (27) of the 41 surveys at st 08 (fig. 4). sample 1 collected on the 23 march 2016 contained juvenile and adult specimens of heterocypris salina (brady, 1886) (fig. 7). sample 2 from the 22 june 2016 included juvenile and adult specimens of h. salina and of cyprinotus cingalensis (sohn and morris, 1963) (fig. 7). the specimens of h. salina are more abundant than those of c. cingalensis in sample 2. discussion the occurrence of c. cingalensis in the awwr lake represents the first record of the species from the uae. valves or living specimens of c. cingalensis were recorded so far from saudi arabia, yemen including socotra island, sudan, palestine, sri lanka, india, indonesia, the philippines, japan, australia and hawaii given that some records as c. scholiosus and cheikella scholiosa represent junior synonyms of the figure 8. (a) nitrate, (b) nitrite, (c) ammonia, (d) phosphate, (e) toc, (f) cd, (g) fe and (h) cu concentrations and ostracod records in the years 2010 to 2016. 270 saji et al./ al wathba wetland in abu dhabi and its ostracod fauna species (brady, 1886; sars, 1889; vavra, 1906; klie, 1933; sohn and morris, 1963; hartmann, 1964; bhatia and khosla, 1967, 1975; malz, 1976; neale, 1979; bhatia, 1983; kempf, 1986; bhatia and singh, 1988; reeves, 2004; karanovic, 2008, 2012; mischke et al., 2012; mohammed et al., 2014) (fig. 9). cyprinotus cingalensis is apparently distributed in the african and australasian subtropical and tropical region. living specimens of c. cingalensis or eggs were probably introduced to the awwr lake by migrating birds. specimens or eggs may have travelled attached to the feathers or may have survived passage through the digestive tract (meisch, 2000; karanovic, 2012). however, it remains speculative whether the c. cingalensis population in the awwr lake possibly originates from specimens or eggs of southern origin (saudi arabia or yemen) or from other regions with known or even unknown c. cingalensis populations. the record of h. salina from the awwr lake is the first record of living ostracods from the uae. fossil valves of late miocene age (7 million years) were reported from the al gharbia region in the uae (mazzini et al., 2013) (table 1). the species is widely distributed in the holarctic region and occurs also in the southern hemisphere where it was probably introduced relatively recently (meisch, 2000). heterocypris salina is common in the region with records from iran, jordan, palestine, saudi arabia and sudan (schöning, 1996; griffiths et al., 2001; rosenberg et al., 2011; mischke et al., 2012). awwr lake experienced large fluctuations with figure 9. distribution of the records of fossil and living cyprinotus cingalensis (blue) and the record of living specimens from the uae (red). (map produced with amcharts.com). table 1. preliminary checklist of the non-marine ostracods from the uae. taxon age reference cyprideis torosa (jones, 1850) late miocene (7 ma) holocene mazzini et al., 2013; gebel et al., 1989; preston et al., 2015 cyprideis sp. holocene gebel et al., 1989 ; stewart et al., 2011 candona (lineocypris?) sp. holocene gebel et al., 1989 candona sp. late miocene (7 ma) mazzini et al., 2013 heterocypris salina (brady, 1868) late miocene (7 ma), living mazzini et al., 2013; this study vestalenula cylindrica (straub, 1952) late miocene (7 ma) mazzini et al., 2013 prolimnocythere sp. late miocene (7 ma) mazzini et al., 2013 cyprinotus cingalensis brady, 1886 living this study 271 int. j. aquat. biol. (2018) 6(5): 265-273 respect to water depth, salinity, ph and the other recorded parameters over the monitoring period. the large fluctuations probably result from the discharge of treated waste water at relatively irregular time intervals and the rise of the groundwater level as a results of stronger winds and higher tides in winter which is sometimes accompanied by precipitation. although generally a very shallow lake, water depth was changing by a maximum of 2.1 m at st 04. the smallest water depth variations were recorded at st 10 where depth varied between 0.17 and 0.73 m. the salinity gradient in the lake is extremely large, with a lowest value of 1.45‰ measured at inlet i and a maximum of 457‰ at st 07. this recorded maximum salinity is significantly higher than those of the dead sea brine (~340‰) and even higher than those of the most saline lakes in the dry valleys of antarctica (marion, 1997). fluctuations were also most extreme at st 07 where the salinity changed over a full order of magnitude (from 43‰ in october 2010 to 459‰ in june 2016). in contrast, lowest salinity variations occurred at st 08 (2.18-68.78‰). however, mostly low salinities of <5‰ were measured at the inlets i and ii, and between 5-10‰ at st 08. the ph values varied from neutral to highly alkaline conditions. lowest ph values were recorded at the inlets (mean ph 7.5) whilst predominantly high values occurred at st 08 (mean ph 8.2). the concentration of nitrate in the water of the awwr lake is mostly between 5 and 50 mg l-1. these values are typical for treated waters or generally for waters affected by industrial or agricultural activities. however, the nitrate concentrations are mostly not critical with respect to drinking water standards (who, 2011; saji et al., 2016). similarly to nitrate concentrations, nitrite levels are mostly under the recommended concentration of 3 mg l-1 in drinking water (who, 2011). mean ammonia concentrations are between 1.5 mg l-1 at inlet i and 2.8 mg l-1 at st 04. these values are relatively high and typical for anaerobic ground waters (who, 2011). the phosphate concentrations in awwr lake waters are almost exclusively above those of natural waters with geogenic phosphate concentrations of typically <0.05 mg l-1. however, mean concentrations between 2.7 mg l-1 at sampling station st 07 and 8.8 mg l-1 at inlet ii are not critically high. toc values are low for the inlets (inlet i: 6.1 mg l-1 on average, inlet ii: 7.0 mg l-1 on average) and at st 08 (13.0 mg l-1 on average), and between 25.954.4 mg l-1 on average at the other sampling stations. cadmium concentrations are mostly below detection limit (<0.0005 mg l-1) and thus, below the recommended threshold for drinking water of 0.003 mg l-1 (who, 2011). however, concentrations as high as an order of magnitude above this level were occasionally recorded during the first two years of the monitoring period. copper concentrations are mostly well below the recommended value of 2 mg l-1 (who 2011). iron concentrations in the water samples from awwr lake are mostly in the range of naturally occurring waters with the exception of water collected in may 2010. the results of our study demonstrate that salinity level at sampling station st08 was significantly lower as compared to other sampling stations at different locations in awwr. therefore, the occurrence of ostracods in awwr lake is apparently predominantly controlled by salinity level. on the other hand, the evaluation of other parameters at all sampling stations such as ph, toc and cadmium concentrations did not reveal significant relationships with the distribution of the recorded ostracods. the presence of the recorded ostracods is mainly controlled by the relatively low salinity at station st08 in awwr. based on our limnological analyses carried out in the years 2010 to 2016, the ostracod species c. cingalensis was recorded for the first time in uae. a second species h. salina was also recorded, which was reported from the late miocene of the uae before. cyprinotus cingalensis was not recorded for the country before. the ostracods were only recorded at one of nine regularly monitored sampling stations in the lake which is characterised by the lowest salinity of all stations. thus, salinity is probably the predominant parameter which controls the distribution of ostracods in awwr lake. ostracods were only 272 saji et al./ al wathba wetland in abu dhabi and its ostracod fauna recorded in a range of relatively low salinities between 2.18-13.74‰. future monitoring of the lake would benefit from additional analysis of the bottom-water oxygenation at the stations, from analysis of the ion chemistry of the lake water and of groundwater in the region, and from observations of the temporal occurrence of the individual ostracod species in the lake. acknowledgements we thank sirthaj khan for help with sampling and data accumulation and c. steuber from emirate natural history group (enhg) for continuous support of the project. we thank dr. richard perry (research committee, ead) for comments and editorial help that greatly improved the manuscript. the study was supported by environment agency abu dhabi (ead). references bhatia s.b. 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(2018) 6(2): 49-54 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article protective effects of prebiotic in zebrafish, danio rerio, under experimental exposure to chlorpyrifos samira yousefi*,1seyed hossein hoseinifar department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 22 july 2017 accepted 27 october 2018 available online 2 5 april 2018 keywords: chlorpyrfos prebiotic immunoglobulin immune response abstract: the current study estimated the immunotoxicological effects of the herbicide chlorpyrifos at sub-lethal concentration and the potential ameliorative effects of galactooligosaccharide (gos) in danio rerio. fish was sampled after a 56-days feeding with gos and then exposed to 15 mg/l chlorpyrifos for 7-days to assess the non-specific immune responses (total protein, immunoglobulin and alp activity). the results revealed that feeding zebrafish with 1% dietary gos increased total protein levels (p<0.05), but no significant effect was noticed between groups fed 0.5 and 2% gos and control (p>0.05). there were significant difference between total immunoglobulin levels 1% and control group (p<0.05). furthermore, in case of alp activity no significant alteration was noticed between gos fed fish and control (p>0.05). the present findings revealed that dietary supplementation with gos could be useful for modulation of the immunity in response to chlorpyrifos exposure, thereby presenting a promising feed additive in aquaculture. introduction aquatic ecosystems that run through agricultural areas have probability of contamination by several chemicals such as pesticides that are used extensively, which on entails multiple change to the aquatic ecosystem, resulting in multiple changes in organism, such as altering the growth rate, patterns of behavior, nutritional value and etc. aquaculture is a major part of the world’s food production, therefore it is essential to maintain fish health (tripathi et al., 2002). obviously, water contaminated with chemical pesticides and stressful conditions may increase the incidence of infectious diseases by opportunist bacteria (moraes et al., 2007; rudneva, 2006). chlorpyrifos (o,)-diethyl-o3,5,6-trichlor-2pyridyl phosphorothioate (cpf) is located in an expansive group of organophosphorus insecticides that is commercially used to control foliar insects and affects agricultural products (rusyniak and nanagas, 2004) and subterranean termites (venkateswara rao et al., 2005). it was first introduced in 1965 and used to control insects in agriculturally and in the home. organophosphorus compounds (op) are acetylcholin *corresponding author: samira yousefi doi: https://doi.org/10.22034/ijab.v6i2.364 e-mail address: yousefisamira72@gmail.com esterase (ache) inhibitors generally used to control snails. annually more than 3 million cases of acute poisoning whit organophosphorus compounds are reported, resulting deaths of 300,000 people (bertolote, 2006). exposure to op prevents ache activity leading to the accumulation of the neurotransmitter acetylcholine (ach), activation of ach receptors, and high stimulation of cholinergic neurons and seizures. it has been observed that vitamins and minerals could ameliorate pyrethroids effects on chicks (aslam et al., 2010). although, there is no report available on fish. lately, much attention has been paid to approaches for decreasing damaging effects of pesticides on fish. galactooligosaccharide (gos) is a prebiotic, produced through the enzymatic conversion of lactose and mainly consists of galactose and glucose molecules (sako et al., 1999). in recent years, zebrafish, danio rerio, raised a model for quick analysis of genes function and biological activities of organic molecules. in this way, possibility of compounds test will be performed (crawford et al., 2008). 50 yousefi and hoseinifar / protective effects of prebiotic in zebrafish using prebiotics can be a convenient and cost effective way to reduce the inevitable intake of pesticides in humans and wildlife. despite limited works on potential of prebiotics to reduce stress, several studies have reported beneficial effects on prebiotics and algae. those stress agents are transportation, temperature stress, environmental pollution and other stressful situations (carvalho et al., 2009; liu et al., 2010). dietary administration of probiotics improved fish immune parameters under stressful conditions (taoka et al., 2006; gomes et al, 2009). by inversely regulating the gut colonization of the probiotic bacterial strain and body cortisol level (carnevali et al., 2006). perhaps the mechanism of action of prebiotics is like probiotic. the use of probiotic alchem poseidon (containing a mix of bacillus subtilis, lactobacillus acidophilus, clostridium butyricum and saccharomyces cerevisiae) increased stress tolerance capacity in paralichthys olivaceus (taoka et al., 2006). in addition, spirulina platensis is used to combat organ toxicants by heavy meals, including lead and cadmium (taoka et al., 2006). despite the potential of prebiotics, there is very limited available information about possible ameliorative effects of prebiotics when fish exposed to pesticides. thus, the present study was performed to elucidate possible ameliorating effects of dietary prebiotic galactooligosaccharide immunelogical stress danio rerio as a result of chlorpyrifos exposure. materials and methods fish and experimental condition: the current study was conducted at aquaculture lab of gorgan university of agricultural science and natural resources. four hundred and twenty zebra fish fry were purchased from a private of ornamental fish center (golestan orovince, iran). fish (45±0.1 mg) were adapted for two weeks to experimental condition then randomly released into 12 aquarium (60 l), at a density of 35 fish per aquarium. treatments were under static aerated water conditions with 50% change every day during rearing. experimental diets and feeding trial: vivinal-gos® was kindly supplied by friesland foods domo company (zwolle, the netherlands). a commercial diet (biomar fish foods, protein 54%, fat 18%, moisture 10%, digestible energy 4 cal/gr) were supplemented by different levels of gos (0, 0.5, 1 and 2%). the prepared diets were kept 24 hours in room temperature, dried and stored in plastic bag at -4ᵒc. fish were hand-fed three times a day(09:00-11:0013:00) for 8 weeks to satiation. at the end of feeding trial, prebiotic fed fish were exposed to a predetermined lc50 of chlorpyrifos (15 mg/l) for one week. evaluation of immune parameters samples collection: six fish were randomly sampled under sterile conditions after anesthetized with clove solution with dose of 2000 ppm were cut head and fins, whole body were taken as sample according to holbech et al. (2001). then, they were homogenized and transferred to 1.5 ml sterile tubes, centrifuged (2000rpm for 10 min at 4°c) and the obtained supernatant were stored at -80°c until analyses. whole body total protein: whole body total protein levels were measured by biuret method using a commercial kit (pars azmun co, ltd., tehran, iran) according to company protocol and their absorption was at 560 nm. whole body total ig: the total protein was measured according to the method explained above and 12% polyethylene glycol solution was added to supernatant. after centrifugation immunoglobulin molecules were precipitated and total protein levels were measured again. whole body alkaline phosphatase activity (alp): alp activity of the whole body was determined using a commercial kit (pars azmun co, ltd., tehran, iran) (hoseinifar, 2015). samples were prepared following company protocol and their absorption was read at 405 nm. statistical analysis: after checking the normality of the data, statistical analysis was performed using one way anova followed by duncan's multiple-range test. the significant difference levels was considered when p<0.05. the statistical analysis was performed by spss software 19 (spss, usa). 51 int. j. aquat. biol. (2018) 6(2): 49-54 results the results of total immunoglobulin of gos fed zebrafish exposed to the chlorpyrifos are shown in figure 1. there were significant different between total immunoglobulin levels of 1% gos fed zebrafish and control group (p<0.05). however, no significant difference was noticed between 0.5 or 2% gos and control group (p>0.05). following pesticide exposure, in case of alp activity no significant was noticed between gos fed and control group (p>0.05) (fig. 2). although, alp levels were slightly decreased in gos fed fish. furthermore, feeding zebrafish on 1% dietary gos increased total protein levels (p<0.05), but no significant difference was noticed between groups feeding on 0.5 and 2% gos fed and control (p>0.05) (fig. 3). discussion fish are constantly exposed to chemicals released into aquatic system and therefore are organisms that are always exposed to disruption of innate and adaptive immune reasons (watzke et al., 2007). pesticides are considered as one of the main stressors in the environment which in living organisms cause induce oxidative, metabolic, immunological stress and growth retardation (slaninova et al., 2009; sahar et al., 2011). in fact, serum immune parameters are very good indicator of immune system (nayak, 2010). the immune response is a protective response against infective agents enter the body. hence it is host resistance challenge test determines the immune system function with highest level of communication biological relevance, because it measures an integrated immune reasons at the level of the whole organisms (köllner et al., 2002). immunoglobulins are natural antibodies that are produced in the absence of stimulant external antigens (magnadottir et al., 2011). in the present study, the total protein and immunoglobulin was found increased in group fed 1% gos and pesticide exposed. however, in case of alp activity, no significant was noticed between gos fed fish and control. however, being fed with prebiotic supplemented diets, the values increased considerably. this might suggest the positive role of the prebiotic in enhancing the immune status of the figure 1. the total immunoglobulin levels of gos fed zebrafish exposed to chlorpyrifos (values are presented as the mean±s.d.; the bars assigned with different letter denote significant difference between treatments (p<0.05)). figure 2. the alp activity of gos fed zebrafish exposed to chlorpyrifos (values are presented as the mean±s.d.; the bars assigned with different letter denote significant difference between treatments (p<0.05)). figure 3. the total protein levels of gos fed zebrafish exposed to chlorpyrifos. values are presented as the mean±s.d.; the bars assigned with different letter denote significant difference between treatments (p<0.05)). 52 yousefi and hoseinifar / protective effects of prebiotic in zebrafish pesticide exposed fish. although there are several reports of the effect of prebiotics on the immune system, so that reported increased immune response administration of gos in rainbow trout (hoseinifar et al., 2015) and goldfish (miandare et al., 2016) diet significantly increased level mucus total protein. however, there is a limited studies on protective effects of prebiotic in pesticide exposed fish. jayantha rao et al. (1984) reported that tilapia mossambica, treated with phosphamidon, an organophosphate insecticide, showed a higher serum bilirubin level and severe liver damage. also, mohapatra et al. (2012) reported that values of total protein and albumin were decreased in the fenvalerate exposed labeo rohita as to the probiotic supplemented fish. also, it has been reported that fenvalerate exposed fish in comparison to the non-exposed fish high level of serum alp and acp . the improved immune system caused using gos can be due to the production of short chain fatty acids (scfas) (guerreiro et al., 2015) that is result of gos fermentation by beneficial intestinal microbiota. scfas binding to receptors gpr43 which is modulated innate immune response and inflammatory cells (maslowski et al., 2015). furthermore, the effects of prebiotics on immune response attributed to modulation of intestinal microbiota and increase in lactic acid bacteria and bacillus which their cell walls (lipopolysaccharide) function as immunostimulant (song et al., 2014; hoseinifar et al., 2012). this study indicate the impacts of chlorpyrifos on fish. in addition, the role of gos in protecting immune cells against chlorpyrifos induced immunotoxicity by improving innate immune system to defend against infections through stimulation of innate immune responses, which underlines the important role of dietary gos supplements in aquaculture. further research using different concentrations of gos supplementation should be performed to provide a complete protection and help on understanding the role of gos in defending against chlorpyrifos induced immune-toxic impact. acknowledgments the authors would like to thank the staff at aquaculture lab of gorgan university of agricultural sciences and natural resources. references abdelkhalek n.k.m., ghazy e.m., abdel-daim m.m. 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(2018) 6(1): 49-54 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی کلرپیریفوس سم با مواجه در( danio rerio) زبرا ماهی بر پربیوتیک حفاظتی اثرات فرسیدحسین حسینی ،*ییوسفسمیرا .ایران گرگان، گلستان، گرگان، طبیعی منابع و شاورزیک علوم دانشگاه ،دانشکده شیالت و محیطزیست شیالت، گروه چکیده: زبرا ماهی رد ساکاریدالیگوگاالکتو پربیوتیک حفاظتی اثرات و کشنده تحت غلظت در کلرپیریفوس کشعلف ایمنی زیست اثرات حاضر مطالعه در (danio rerio) اختصاصی غیر ایمنی پاسخ بررسی جهت سم با مواجه هفته یک و پربیوتیک با تغذیه روز 56 از بعد. گرفت قرار بررسی وردم در کل پروتئین هک بود این دهنده نشان نتایج. شد انجام بردارینمونه ماهی بدن کل از( فسفاتاز آلکالین فعالیت و کل ایمونوگلوبولین کل، پروتئین) اختالف شاهد گروه با درصد 2 و 5/0 سطوح بین کهحالی در(. p<05/0) بود یافته افزایش ساکاریدالیگوگاالکتودرصد 1 سطح با شده تغذیه گروه دارییمعن اختالف شاهد گروه با مقایسه در درصد 1 سطح با شده تغذیه گروه در کل ایمونوگلوبولین میزان در(. p<05/0) نداشت وجود دارییمعن مشاهده شاهد گروه و پربیوتیک با شده تغذیه هایگروه بین معناداری اختالف نیز فسفاتاز آلکالین فعالیت میزان خصوص در(. p<05/0) داشت وجود سم اب مواجه در زبرا ماهی در ایمنی هایفاکتور بر مثبتی تأثیر جیره در ساکاریدالیگوگالکتو پربیوتیک از استفاده اینبنابر(. p>05/0) نشد .شود معرفی پروریآبزی صنعت در کننده امیدوار نتایج با افزودنی یک نوان واندتمی نتیجه در. داشت کلرپیریفوس .ایمنی پاسخ ،ایمونوگلوبولین پربیوتیک، کلرپیریفوس، :کلمات کلیدی international journal of aquatic biology (2014) 2(2): 85-90 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved review article feeding and breeding biology of amblypharyngodon mola – a review sandipan gupta*,1samir banerjee aquaculture research unit, department of zoology, university of calcutta, 35, ballygunge circular road, kolkata-700019, west bengal, india article history: received 11 march 2014 accepted 24 april 2014 available online 2 5 april 2014 keywords: food algae spawning fecundity sex-ratio abstract: amblypharyngodon mola is a popular food fish of indian sub-continent due to its high nutritional value. earlier many workers have carried out studies on feeding and breeding biology of this fish species but consolidated information on the same is not available. so, a survey of published literatures on the feeding and breeding biology of a. mola has been carried out to consolidate the available information. lacunae of information has been pointed out for further study mainly on age group wise variation in food preference and correlation of breeding periodicity with hydrological parameters and photoperiod. introduction amblypharyngodon mola, commonly known as mola carplet or pale carplet is a popular food fish mainly in indian sub-continent due to its high nutritional value (alam et al., 2004; saha et al., 2009) with high protein, vitamin and mineral content (zafri and ahmed, 1981; mazumder et al., 2008). it is also rich in fe, zn and ca (roos et al., 2007; kongsbak et al., 2008). earlier number of research works has been carried out on its feeding and breeding biology. the main purpose of this review work is to consolidate those scattered information and to highlight the gaps in the knowledge for further research work on this particular fish species. amblypharyngodon mola (cypriniformes: cyprinidae) is a freshwater fish species (fig. 1); a natural inhabitant of ponds, canals, beels, slowmoving streams, ditches, baors, reservoirs and inundated fields (rahman, 1989; talwar and jhingran, 1991; menon, 1999; saha et al., 2009). the species is distributed in india, bangladesh, pakistan * corresponding author: sandipan gupta e-mail address: sandipangupta2007@gmail.com tel: +919830082686 and myanmar (talwar and jhingran, 1991); also has been reported from afghanistan (coad, 1981). feeding biology mookherjee and basu (1946) have reported a. mola as a surface feeder; they have documented unicellular and filamentous algae, protozoans and rotifers as preferred food types for this fish species. das and moitra (1963) have reported a. mola as herbivorous fish. piska et al. (1991), mamun et al. (2004), gupta and banerjee (2013b) and mondal and kaviraj (2013) have also documented herbivorous feeding habit of this fish species. piska et al. (1991) also have reported it as a bottom feeder; algae have been documented as the main food for this fish species. mamun et al. (2004) have reported phytoplankton preference for this fish species; they also have documented chlorophyceae as the mostly preferred food class for a. mola; debris with mud which have been observed in the gut content of the studied specimens by mamun et al. (2004) have been 86 gupta and banerjee/ international journal of aquatic biology (2014) 2(2): 85-90 considered as the secondary food; while zooplankton and semi-digested food have been considered as incidental food for the fish species. gupta and banerjee (2013b) have reported phytoplankton as the basic food and chlorophyceae as the mostly preferred food class for this fish species. mondal and kaviraj (2013) have reported algae as the main food; they have documented high preference for chlorophyceae followed by myxophyceae, bacillariophyceae, cyanophyceae and euglenophyceae for this fish species. dewan (1973) and chowdhury (1999) have reported phytoplankton as the most dominant food group for this fish species. mustafa (1991) has documented blue green algae along with planktonic crustacean and rotifera to form the main food of a. mola. miah and siddiqui (1992) have reported mola as omnivore with higher feeding preferences for debris and plant foods. suresh et al. (2007) have reported that a. mola feeds predominantly on phytoplankton and myxophyceae has been reported as the mostly preferred food class for this fish species. mondol et al. (2013) have reported chlorophyceae as the mostly preferred food class for a. mola followed by bacillariophyceae, cyanophyceae and euglenophyceae in rice field ecosystem of bangladesh. piska et al. (1991) have reported size group wise variance of food preference in a. mola; small and medium size group fishes have been reported to prefer algae but in higher size groups algae consumption has been reported to be less than that of the higher aquatic plants. they have documented low consumption of diatoms in higher size groups. gupta and banerjee (2013b) have also studied the size group wise food preference, but have not found any significant change in food preference along different size groups. mondal and kaviraj (2013) have reported variation in food preference according to different weight class; lower weight class have been reported to prefer more algae than higher weight class; higher weight class though preferred algae have been reported to consume high amount of other plant materials, debris and mud than the lower weight class. mamun et al. (2004) have reported high feeding intensity in a. mola but have not correlated it with breeding periodicity. piska et al. (1991) have reported that feeding intensity is low during the spawning season and high during pre-spawning and post-spawning period of a. mola; this observation has later been supported by suresh et al. (2007), gupta and banerjee (2013b) and mondal and kaviraj (2013). piska and waghray (1986) also have reported high incidence of empty guts coincided with the spawning season of a. mola. breeding biology sexual dimorphism and sex ratio: except hoque and rahman (2008), none have documented any information on the sexual dimorphism of a. mola. figure 1. mola carplet, amblypharyngodon mola. 87 gupta and banerjee/ international journal of aquatic biology (2014) 2(2): 85-90 hoque and rahman (2008) have reported that males and females are different in color; males are comparatively brighter than females. the color of females is light and they are large in size. in case of mature female the abdomen is soft and swollen, pelvic fins are smooth and caudal fin is deeply forked. during the spawning season mature females are with distended abdomen by which they can be easily recognized. suresh et al. (2007) also have reported the comparative larger size of females than males. afroze and hossain (1983) have reported the average sex-ratio in population of a. mola as 1:1.67 for males and females of this fish species. piska and waghray (1986) also have observed predominance of females in the population of this fish species. afroze et al. (1991) have reported significant female dominance over male in a. mola. azadi and mamun (2004) also have reported the significant dominance of females over males in the population of a. mola. they have documented the monthly variation of sex ratio between males and females to be ranged from 1:1.15 to 1:3.64 with an average value of 1:2.078. suresh et al. (2007) have reported significant variation in sex ratio of a. mola in different months from the expected ratio of 1:1 and the ratio of male and female fish have been reported to vary from 1:1 to 1:12. number of males has been reported to be very low to nil during spawning and post-spawning season; and they have concluded that this may be due to the spawning and post spawning mortality of males. hoque and rahman (2008) have also reported the female dominance in population of a. mola; they have reported an average ratio of 1:1.71 for males and females of this fish species. saha et al. (2009) have reported the average sex-ratio in the population of a. mola as 1:2.03 for males and females. gupta and banerjee (2013a) have reported the average sex ratio of 1:3.04 for males and females of this fish species while mondal and kaviraj (2013) have documented the ratio of 1:1.9 for the same. length at first maturity: suresh et al. (2007) have reported the length at first maturity for male and female of a. mola as 5.1-5.6 cm and 3.9-4.4 cm, respectively; early maturation of female than male has been reported by them. on the other hand, hoque and rahman (2008) have reported early maturation of male than female; they have documented the length of smallest mature male and female mola as 4.8 cm and 5.5 cm, respectively. gupta and banerjee (2013a) later also have supported this view; they have reported 5-5.5 cm and 5.5-6 cm as length at first sexual maturity for male and female fish, respectively. fecundity: mookherjee and basu (1946) have reported the fecundity of amblypharyngodon mola as 500; same has been documented by bhuiyan (1964). dewan and doha (1979) have reported the fecundity to be ranged from 1,021-13,812. parveen (1984) has reported the average fecundity of a. mola as 3,601. misra and jain (1985) have documented the fecundity to be ranged from 1,210-16,072. afroze and hossain (1990) have reported the fecundity of a. mola to be ranged from 400-8,550. mustafa (1991) has documented the average fecundity of a. mola as 738. azadi and mamun (2004) have reported the fecundity to be ranged from 1,280-13,679 with an average of 5,182.67 ± 3,731.51. suresh et al. (2007) have reported the fecundity to be ranged from 21-16,867 while the relative fecundity range has been reported to be 7-2,122. hoque and rahman (2008) have reported the lowest and highest mean fecundity of mola as 1,023 ± 625 and 6,806 ± 125 in size groups ranging from 5.0-5.5 cm and 8.1-8.5 cm respectively in pond and 1,220 ± 550 and 6,923 ± 425 for the same size groups in beel of bangladesh. saha et al. (2009) have reported the fecundity to be ranged from 1,291-12,737 with mean value of 5,751.73 ± 3,321.73. gupta and banerjee (2013c) have reported the fecundity to be ranged from 1,0149,690 with an average of 4,592.64. mondal and kaviraj (2013) have documented fecundity range of 3,785-12,590 for the fish species. maturation and spawning: azadi and mamun (2004) have reported three gonadal maturity stages (immature, maturing and ripening) in female a. mola while hoque and rahman (2008) have documented five maturity stages (immature, maturing, mature, 88 gupta and banerjee/ international journal of aquatic biology (2014) 2(2): 85-90 ripe and spent) for both female and male mola. gupta and banerjee (2013a) have documented five maturity stages (immature, maturing, mature, ripe and spent) for female and four maturity stages (immature, mature, ripe and spent) for male mola. piska and waghray (1986) have reported breeding season of a. mola in andhra pradesh from february to july. in west bengal, april to october has been documented as the breeding season by suresh et al. (2007) while gupta and banerjee (2013a) have reported april to december as the breeding season with two spawning months in june and november. mondal and kaviraj (2013) have documented july as the spawning month for this fish species in west bengal. rahman (1989) has reported may to october as the spawning months for this species in bangladesh. according to afroze and hossain (1990), august is the peak breeding season of mola while parveen (1984) has reported the breeding season of mola from june to october/november in bangladesh. kohinoor et al. (2003) have reported may-july and september-october as spawning months for this fish species in bangladesh. azadi and mamun (2004) have reported mola as a multiple spawner and they have documented july, august, october and march as the spawning months in bangladesh. hoque and rahman (2008) have stated april to october as the breeding season in bangladesh; may and september have been reported as the spawning months by them. saha et al. (2009) have reported march to august as the breeding season in bangladesh for this fish species. conclusion from in-depth review of literatures, it is evident that number of works has been carried out on feeding and breeding biology of amblypharyngodon mola mainly in bangladesh and india but few lacunae are there which must be investigated to benefit fishery of this fish species. feeding habit and food preference have been properly documented only for the adult fishes along with size group wise and weight class wise food preference; but no such detail documentation is there regarding any such significant difference in food preference along with age groups. on the other hand, there is ample information existing on the breeding biology of this fish species; female dominance over male in the population has been reported by all the previous workers and most of them have reported mola as a high fecund fish. though differences in breeding periodicity and spawning frequency have been observed in the earlier documented information but that too is without any significant study on correlation of breeding periodicity with hydrological parameters and photoperiod. so, there is ample scope for study in this area for more meaningful contribution on feeding and breeding biology of amblypharyngodon mola. references afroze s., hossain m.a. (1983). the fecundity and sex ratio of amblypharyngodon mola (ham.) (cypriniformes: cyprinidae). university journal of zoology, rajshahi university, 2: 29-32. afroze s., hossain m.a. (1990). the reproductive cycle of the fresh water fish amblypharyngodon mola (ham.). (cypriniformes: cyprinidae). university journal of zoology, rajshahi university, 9: 17-21. afroze s., hossain m.a., parween s. (1991). notes on the size frequency distribution and length-weight relationship of freshwater fish amblypharyngodon mola (hamilton) (cypriniformes: cyprinidae). university journal of zoology, rajshahi university, 10 & 11: 103-104. alam m.j., dewan s., rahaman m.r., kunda m., khaleque m.a., kader m.a. (2004). study on the cultural suitability of amblypharyngodon mola with barbodes gonionotus and cyprinus carpio in a farmer’s rice fields. pakistan journal of biological sciences, 7: 1242-1248. azadi m.a., mamun a. (2004). reproductive biology of the cyprinid, amblypharyngodon mola (hamilton) from the kaptai reservoir, bangladesh. pakistan journal of biological sciences, 7 (10): 1727-1729. bhuiyan a.l. (1964). fishes of dacca. asiatic society of pakistan, dacca. 39 p. coad b.w. (1981). fishes of afghanistan, an annotated check-list. national museum of natural sciences publications in zoology, 14:1-26. 89 gupta and banerjee/ international journal of aquatic biology (2014) 2(2): 85-90 chowdhury f.a. (1999). effects of hypopthalmichthys molitrix and catla catla on the growth, survival and yields of amblypharyngodon mola in mixed culture. m.sc dissertation. bangladesh agricultural university, mymensingh. 131 p. das s.m., moitra s.k. (1963). studies on the food and feeding habits of some freshwater fishes of india. iv. a review on the food and feeding habits, with general conclusions. ichthyologica, 11 (1-2): 107-115. dewan s. (1973). investigations into the ecology of fishes of mymensingh lake. dissertation. bangladesh agricultural university, mymensingh bangladesh. dewan s., doha s. (1979). spawning and fecundity of certain pond fishes. bangladesh journal of agriculture, 4: 1-8. gupta s., banerjee s. (2013a). studies on some aspects of reproductive biology of amblypharyngodon mola (hamilton-buchanan, 1822). international research journal of biological sciences, 2 (2): 69-77. gupta s., banerjee s. (2013b). food and feeding habit of amblypharyngodon mola (hamilton-buchanan, 1822) in west bengal, india. international journal of scientific research, 2 (5): 293-297. gupta s., banerjee s. (2013c). fecundity study of amblypharyngodon mola (ham.-buch., 1822) from an undisturbed wetland of west bengal. fishing chimes, 33 (6): 67-69 hoque a.s.m., rahman m.r. (2008). reproductive ecology of mola (amblypharyngodon mola). journal of agriculture and rural development, 6 (1& 2): 165174. kohinoor a.h.m., islam m.s., thilsted s.h., wahab m.a. (2003). reproductive biology of three indigenous small fish, mola (amblypharyngodon mola), chela (chela cachius) and punti (puntius sophore). in: m.a. wahab, s.h. thilsted, m.e. hoq (ed). small indigenous species of fish in bangladesh: proceedings of bau-enreca/danida workshop on potentials of small indigenous species of fish (sis) in aquaculture and rice-field stocking of improved food and nutrition security in bangladesh. 30-31 october, 2002, bangladesh agricultural university, mymensingh, bangladesh & enrec/danida. 3-22 p. kongsbak k., thilsted s.h., wahed m.a. (2008). effect of consumption of the nutrient-dense, freshwater small fish amblypharyngodon mola on biochemical indicators of vitamin a status in bangladeshi children: a randomised, controlled study of efficacy. the british journal of nutrition, 99 (3): 581-597. mamun a., tareq k.m.a., azadi m.a. (2004). food and feeding habits of amblypharyngodon mola (hamilton) from kaptai reservoir, bangladesh. pakistan journal of biological sciences, 7 (4): 584588. mazumder m.s.a., rahman m.m., ahmed a.t.a., begum m., hossain m.a. (2008). proximate composition of some small indigenous fish species (sis) in bangladesh. international journal of sustainable crop production, 3 (4): 18-23. menon a.g.k. (1999). checklist freshwater fishes of india. records of zoological survey of india, occasional paper. no: 175: i-xxix, 1-366 p. miah m.j.u., siddique w.h. (1992). studies on the food and feeding habits of mola, amblypharyngodon mola. bangladesh journal of agricultural science, 19 (2): 165-170. misra s.d., jain k.k. (1985). a study on the fecundity of amblypharyngodon mola (ham.). indian journal of zoology, 13 (1): 1-8. mondal d.k., kaviraj a. (2013). feeding and reproductive biology of amblypharyngodon mola (cypriniformes: cyprinidae) from two floodplain lakes of india. international journal of aquatic biology, 1 (3): 125-131. mondol m.m.r., nahar d.a., dewan s., rahman m.m., jasmine s., hossain m.y. (2013). food and feeding habits of the mola carplet amblypharyngodon mola (hamilton, 1822) in rice field ecosystem with consideration of water quality parameters. our nature, 11 (1): 61-75. mookherjee h.k., basu s.p. (1946) life history of a. mola (ham.) a delicate food fish of bengal. science and culture, calcutta, 12: 54-56. mustafa g. (1991). composite culture and biology of some indigenous fishes of bangladesh. ph.d. dissertation. faculty of biological science, dhaka university, dhaka, bangladesh. 299 p. parveen s. (1984). studies on the culture methods and some aspects of culture biology of amblypharyngodon mola. m.sc. dissertation, dhaka university, bangladesh. 137 p. piska r.s., swamy b.r., devi p.j. (1991). food and feeding habits of freshwater cyprinid amblypharyngodon mola (ham.). indian journal of fisheries, 38: 126-128. 90 gupta and banerjee/ international journal of aquatic biology (2014) 2(2): 85-90 piska r.s., waghray s. (1986). some aspects of reproductive biology of amblypharyngodon mola (hamilton). geobios, 13 (5): 204-207. rahman a.k.a. (1989). freshwater fishes of bangladesh. the zoological society of bangladesh, department of zoology, university of dhaka, dhaka, bangladesh. 364 p. roos n., wahab m.a., chamnan c., thilsted h. (2007). the role of fish in food-based strategies to combat vitamin a and mineral deficiencies in developing countries. the journal of nutrition, 137 (4): 11061109. saha b.k., islam m.r., saha a., hossain m.a. (2009). reproductive biology of the mola carplet amblypharyngodon mola (hamilton) (cypriniformes: cyprinidae) from netrakona water. bangladesh journal of scientific and industrial research, 44 (3): 377-379. suresh v.r., biswas b.k., vinci g.k., mitra k., mukherjee a. (2007). biology of amblypharyngodon mola (hamilton) from a floodplain wetland, west bengal. indian journal of fisheries, 54 (2): 155-161. talwar p.k., jhingran a.g. (1991). inland fishes of india and adjacent countries. vol-1 and vol-2. oxford and ibh publishing co. pvt. ltd. new delhi, bombay and calcutta, 1063 p. zafri a., ahmed k. (1981). studies on the vitamin a content of fresh water fishes. content and distribution of vitamin a in mola (a. mola) and dhela (rohtee cotio). bangladesh journal of biological sciences, 10: 47-53. int. j. aquat. biol. (2018) 6(1): 44-48oi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology short communication effects of dietary cassava dough (fufu) kitchen waste as a replacement for maize on growth performance of clarias gariapinus fingerlings olusola adeniyi babalola*1, murisiku onigemo2, deborah olude1 1department of fisheries technology, lagos state polytechnic, ikorodu, lagos, nigeria. 2department of animal production technology, lagos state polytechnic, ikorodu, lagos, nigeria. article history: received 15 dec 2017 accepted 25 february 2018 available online 2 5 february 2018 keywords: feeding fingerlings cassava dough kitchen waste abstract: the study was carried out to determine the growth performance of clarias gariepinus fingerlings cultured in the circular plastic ponds and fed diets containing varying level of cassava dough (fufu) kitchen waste at 0, 2.5, 5, 7.5, and 10% levels of inclusion. two hundred and twentyfive fingerlings were randomly distributed into five treatments, which were replicated thrice-using completely randomize design. while the diet with 0% serves as a control. clarias gariepinus with average initial weight of 10 g were stocked at the rate of 15 fishes per replicates. the result shows that there were no significant different among the treatment as compared with mean weight gained, feed intake, specific growth rate, protein efficiency ratio, and feed conversion ratio. clarias gariepinus fingerlings fed with 2.5% level of inclusion of cassava dough kitchen waste had the highest weight gained with the value of 11.66 g, followed by treatments 1, 4, 3 and 5, respectively. the result shows that the feed with 4.96% of cassava dough kitchen waste has the highest feed intake, specific growth rate, protein efficiency ratio and feed conversion ratio. therefore, a diet with 4.96% level of inclusion of cassava dough kitchen waste is hereby recommended to be in the diet of c. gariepinus fingerlings for economic reasons over maize. introduction the growth of nigerian aquaculture sector is currently being undermined by the challenges of inadequate supply and prohibitive cost of quality fish feeds. feed accounts for over 60% of the cost of fish production and maize is the major source of carbohydrate or energy source in the fish diet (fagbenro et al., 2003). the cost of maize is relatively high compared to other sources because of its competitive use as food for human and feed for livestock and fish. in order to develop practical diets for farm fish at relatively low cost, research efforts must therefore be geared towards evaluating alternative feedstuff ingredient that can replace maize without compromising fish growth and health (olurin et al., 2006). cassava (manihot esculenta), the plant which is native to south america, especially brazil is widely cultivated in the tropics and appears to be the best possible alternative for overcoming the chronic high *corresponding author: olusola adeniyi babalola doi: https://doi.org/10.22034/ijab.v6i1.440 e-mail address: sola_ aug@yahoo.com feed cost in the livestock industry and its by-product such as fufu waste after processing is of little or no food value to man (ukachukwu, 2008). fufu is dough made from boiled cassava paste and a stable food in part of the west and central africa (obadina et al., 2006). fufu contained about 0.35-4.8% crude protein, 0.2-1.42% crude fiber, 1.19-1.32% fat, 77.97-87.24 soluble carbohydrate and 312.78-375.40 kcal/g of energy (lancaster et al., 1982; etudaiye et al., 2009; ojo et al., 2014; bamidele et al., 2015). the cooking of fufu in southwestern part of nigeria result in the generation of caked deposit at the base of the pot, which are often discarded and constitute nuisance in commercial fufu production centers. this caked deposit composed majorly of cooked and dried fermented soluble carbohydrates which is suggestive of high energy value. this research was therefore designed and conducted to evaluate the effect of replacing maize with dried fufu waste in the diets of clarias gariepinus 45 int. j. aquat. biol. (2018) 6(1): 44-48 so as to establish it as alternative source of energy in fish feed formultion. materials and methods the study was carried out in the aquaculture unit of the department of fisheries technology, lagos state polytechnic, ikorodu nigeria. the rearing chambers used for were 15 circular plastic tanks of 60 cm in diameter and 30 cm deep each, 225 mixed sex fingerlings of catfish, locally formulated feed, weighing scale, and water test kits. the tanks were placed in the aquaculture unit of fisheries technology department. clean water from an overhead tank that is connected to borehole was used throughout the experiment period and the essential physicochemical parameters of the water were observed before and during the experiment. the temperatures of the water were measured with thermometer, the dissolved oxygen was determined using digital oxygen meter and ph of the water was measured with ph meter. the tanks’ water was changed twice a week and fresh water is supplied to the tanks to improve water quality and avoid pollution from uneaten food and metabolites. experimental design: 225 fingerlings of catfish with average initial weight of 10 g were randomly allotted to five treatments with three replicates per treatment in a completely randomized design (crd) experiment. experimental diet and feeding regime: five diets were prepared. each diet contained: maize, soya meal, fishmeal, bone meal, vitamins minerals, premix and salt that were obtained from toll feed millers at sabo market ikorodu lagos nigeria, and fufu kitchen waste was obtained from reputable food processing unit. the brittle fufu kitchen waste was sun dried and fine crushed and mixed with other feedstuffs using the formulations as shown in table 1. the experimental fish were fed base on 5% body biomass and the quantity was adjusted as the fish increase in body weight. the feed was divided into two equal meals and fed at 07:00 and 16:00 hrs for 12 weeks. growth performance evaluation: weighing of fish was carried out early in the morning once in a week by transferring fish from the tanks into the weighing bowl. the weight of fish was taken and recorded using a digital weighing scale (ohaus scout pro balances models sp-601). these data were used to compute the growth parameters i.e. mean weight gain, specific growth rate, feed conversion ratio, protein efficiency ratio, and mortality rate according to amisah et al. (2009) and amoah (2012). data analysis: all data collected were subjected to analysis of variance (anova) using assistatstatistical assistance 7.6 beta software developed by silva and azevedo (2009). results and discussion the growth performances of fish fed with the experimental diets are shown in table 2. statistical analysis revealed that the levels of fufu meal did not significantly (p>0.05) influence the weight gain, feed table 1. composition of the experimental diets. ingredient diet 1 diet 2 diet 3 diet 4 diet 5 fish meal 36.72 36.72 36.72 36.72 36.72 soya bean meal 36.72 36.72 36.72 36.72 36.72 maize 19.83 14.87 9.915 4.96 0.00 fufu waste 0.00 4.96 9.915 14.87 19.83 methionine 0.56 0.56 0.56 0.56 0.56 lysine 0.56 0.56 0.56 0.56 0.56 vitamin c 0.56 0.56 0.56 0.56 0.56 bone meal 2.25 2.25 2.25 2.25 2.25 premix 1.40 1.40 1.40 1.40 1.40 salt 1.40 1.40 1.40 1.40 1.40 calculated analysis crude protein 44.38 44.17 43.95 43.74 43.52 metabolizeable energy 2627.62 2614.80 2601.99 2589.17 2576.36 46 babalola et al./ effects of dietary cassava on growth performance of clarias gariapinus intake, feed conversion ratio, protein efficiency ratio, and specific growth rate of the fish. the average physicochemical parameters of the rearing medium are presented in table 3 and all measured variables are within the values suitable for the culture of c. gariepinus. these results indicated that all the measured parameters were statistically similar for the fish fed with experimental diets. similar results were also obtained when cassava peel was used to replace maize in the diets of orechromis niloticus (fakunmoju et al., 2014). the highest weight gain was recorded in fish fed with diet 2 (11.66 g) while the lowest weight gain was recorded in fish fed diet 5 (8.64 g). the differences in the weight gain might be attributed to the feed intake of the fish, which follows a similar trend as the weight gain. feed consumption has been reported to influence weight gain and other growth parameter of fish (du et al., 2005; el-haroun, 2007). the similarity of the weight gain and feed intake of the fish could be because of the fact that the metabolizeable energy values of fufu waste and maize are within the same range (lancaster et al., 1982; honeyman and zimmerman, 1991; etudaiye et al., 2009; ojo et al., 2014; bamidele et al., 2015) and energy is the major determinant of feed intake in fish (boujard and médale, 1994; morales et al., 1994; yamamoto et al., 2000). the specific growth rate follow the same trend as the weight gain, hence, cassava dough waste could be used to replace maize in diet of c. gariepinus without compromising the growth of the fish. the data obtained from the study also show that the fish fed with the diet 5 had the highest protein efficiency ratio (1.31) while the lowest was observed among the fish fed with the diet 3 (1.23). the feed conversion ratio does not follow any particular trend, the best feed conversion ratio was recorded among the fish fed with the diets 1 and 2 (1.75); while, the numerical worst feed conversion ratio was recorded in fish fed with the diet 3 (1.84). the feed conversion ratio recorded in this study is within the range reported by many authors for pond-reared fish (bjorkli, 2002; robinson and li, 2015). this implied that the experimental diets are well utilized by the fish. the numerical differences in the performance indices may be due to the growth of any animal which is influenced by many factors such as the internal growth during their life cycle and external changes as influenced by environmental factors (babalola et al., 2016). table 3 showed the means of all essential water quality parameters recorded during the experiment. the water quality parameters were measured and found to be in the range of tolerance for c. gariepinus culture with a ph range of 6.5-6.8, dissolved oxygen range of 5.28-5.3 mg/l, a temperature range of 26.128.7°c, and a salinity range of 75.3-76.4 ppt (mustapha and omotosho, 2005; omotayo et al., 2011). this perhaps may be responsible for 100% survival rate of the c. gariepinus fingerlings during the experimental period. it is therefore expedient to conclude that fufu waste table 2. performance characteristics of fish fed different percentage of fufu kitchen waste. parameters t1 t2 t3 t4 t5 sem final weight (g) 20.43 21.66 19.21 20.28 18.64 weight gain (g) 10.43 11.66 9.21 10.28 8.64 1.17 feed intake (g) 18.29 20.35 16.97 18.15 15.18 1.90 feed conversion ratio 1.75 1.75 1.84 1.77 1.76 0.04 specific growth rate(%/day) 4.06 4.12 3.91 4.00 3.86 0.11 protein efficiency ratio 1.28 1.30 1.23 1.29 1.31 0.03 table 3. mean values of water quality parameters. parameters t1 t2 t3 t4 t5 sem ph 6.60 6.70 6.80 6.50 6.70 0.05 do (mg/l) 5.28 5.30 5.30 5.30 5.30 0.00 temperature (°c) 28.7 27.0 27.4 28.5 26.1 0.48 salinity(ppt) 76.3 75.3 76.4 76.3 75.5 0.23 47 int. j. aquat. biol. 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(2016) 4(1): 1-10; doi: e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article population dynamic parameters of the red mullet mullus barbatus (mullidae) in the arzew gulf, algeria lotfi bensahla talet*1, ahmed bensahla talet2, zitouni boutiba1 1laboratoire réseau de surveillance environnementale, department of biology, faculty of natural sciences and life, university of oran1, ahmed benbella, es-sénia, 31000 oran, algeria. 2laboratoire aquabior es-sénia, department of biotechnology, faculty of natural sciences and life, university of oran1, ahmed benbella, 31000 oran, algeria. article history: received 9 november 2015 accepted 4 january 2016 available online 2 5 february 2016 keywords: exploitation fishery mortality weight-length relationship von bertalanffy abstract: population dynamic parameters of red mullet, mullus barbatus (n=592) caught by trawlers operating in arzew gulf (algeria) in the western mediterranean were studied. samples were collected between february 2003 and january 2004. females ranged from 10.9-23.2 cm in total length and 12-149.2 g in weight whereas males length was comprised between 12.6 and 19.7 cm and weight between 21.3 and 99.9 g. the relationship between length and weight was w=0.00167l2.842 (r²=0.850). growth parameters of the von bertalanffy equation were: l∞=208.9 cm, k=0.66 year-1, and t0=-0.143 year for males, while for females: l∞=250.9 cm, k=0.6 year-1, and t0=-0.015 year. estimates of total (z), natural (m) and fishing (f) mortalities were: 1.58, 0.8 and 0.79, respectively and the estimated exploitation ratio 0.5 indicating that the resource is being exploited at its limit in this area. the virtual population analysis of m. barbatus by vit software showed that the number of recruits per year was estimated at 4447304.6 individuals, balanced total biomass was estimated at 168 486 tons, the gains are represented by recruitment 51.71 tons (30.69%) and growth 116.76 tons (69.31%), losses are represented by natural mortality 98.48 tons (58.45%) and fishing mortality 70 tons (41.55%). the current yield per recruit was estimated to y/r=15.74 g and maximum sustainable yield can be reached by increasing the current fishing effort to stabilize it at msy=17.91 g. introduction red mullet, mullus barbatus is a demersal fish living on muddy, sandy or gravel bottoms of the continental shelf between 10 to 500 m of depth. it is usually caught by trawlers (bauchot and hureau, 1986) and represents one of the economically most important species for trawl fishery at oranian coasts. several works have already been made on its biological features in the mediterranean basin (ardizzone, 1998; jukic-peladic, 1998; marano et al., 1998; morey et al. 2003; gharbi et al., 2004; ozbilgin et al., 2004; layachi, 2007; joksimović et al., 2008; maximov et al., 2008; mehanna, 2009; giacalone et al., 2010; sieli et al., 2011; aydın and karadurmuş, 2013; cherif, 2014; çiçek, 2015). arzew gulf is located between stidia and mostaganem towns in the algerian shoreline of * corresponding author: lotfi bensahla talet doi: http://dx.doi.org/10.7508/ijab.2016.01.001 e-mail address:btlotfi@yahoo.fr mediterranean with the geographical coordinates of 35°49'59"n and 00°10'00"w. it constitutes one of the biggest petroleum harbors and many industrial complexes are situated in this area making it an industrial and commercial hub for algerian economy. arzew fishery is the second one in the area after oran town fishery and supplies the surrounding towns in sea products. the aim of this work was to describe some population dynamical parameters of m. barbatus in the arzew gulf to ensure the renewal of the resource given that it is under over fishing pressure during these last decades (bensahla talet and boutiba, 2000) similar to benthic species of this gulf. also, this study will contribute enriching the available data on growth and fisheries of this species in the western mediterranean. 2 bensahla talet et al./ growth and fishery of mullus barbatus from arzew gulf materials and methods a total of 594 specimens of m. barbatus were caught by a commercial bottom trawlers, operating in the gulf of arzew (fig. 1) at depths of 150-300 m between february 2003 and january 2004. the total length (l) was measured with an ichtyometer to the nearest 1 mm; total weight (w) was measured with a precision balance to the nearest 0.1 g, and sex of individuals was determined after opening their abdominal cavity. the length-weight relationship (lwr) is generally expressed by the following equation: w = 𝛼*lb (ricker, 1973), where w is the total weight (g) and l= total length (cm). the parameters 𝛼 and b were estimated by the method of least squares obtained by logarithmic transformation to correct the nonlinearity of the original curve and the heterogeneity of variances: logw = b*logl + log𝛼 the null hypothesis for the isometric growth (h0: b=3) was analyzed by t-test, using the following statistical formula: ts = (b-3)/sb, where sb is the standard deviation of the parameter b, for α=0.05 (morey et al., 2003). the parameters of the equation of von bertalanffy growth were estimated using the elefan method (fisat ii gayanilo et al., 2005): lt = l∞ [1 –e -k(t –t0)], where lt is total length in cm at a given age l∞ is asymptotic length, k is growth curve parameter, and t0 is hypothetical age for a length equal to zero. the jones and van zalinge plot (jones and van zalinge, 1981) was used to estimate total mortality using fisat ii (gayanillo et al., 2005), the natural mortality was calculated using pauly’s empirical equation (pauly, 1980) as following: ln(m)=-0.0152-0.279 ln(l∞)+0.6543 ln(k)+0.463 ln(t) where t is the mean annual habitat temperature (in °c) considered here at 17.5°c, and f the fishing mortality was deducted from the subtraction of m from z (z=m+f). the selection length which corresponds to the probability of capture of each individual by the fishing gear was determined by plotting the cumulated catch curve (pauly, 1984). the exploitation ratio e was estimated from gulland formula (gulland, 1971): e=f/z. (e<0.5 underexploited stock, and e>0.5 overexploited stock). vit software (lleonart and salat, 1997) was developed initially for assessing mediterranean fisheries dealing with pseudo-cohorts when information about annual catch at length or age is limited to a short period of individual or few consecutive years (ratz et al., 2010). the program has as entry data: growth parameters of von bertalanffy (l, k, t0), lwr, natural mortality (m), fishing mortality (f), and length at first maturity. also it only requires knowledge of annual catches for a given exploited species (70 tons in the case of this study) instead of a long historical series of ten years (bouaziz et al., 2010). results from 594 collected specimens of m. barbatus, 258 were males (43.58%), 318 were females (53.37%) and 18 were unsexed (3.04%). the sex ratio was in favor of females 1:0.81 (fig. 2) but the χ2 test did not reveal any significant difference (χ2=2.93>χ2t 1,0.05=3.84). the total length of males ranged 12.619.7 cm and their weight 21.3-99.9 g. the total length of females ranged 10.9-23.2 cm and their weight 12-149.2 g. the length frequency distribution of the entire population of m. barbatus is presented in figure 3. the lwrs are shown in table 1 and figure 4a, b, c. males exhibits a positive allometric growth pattern, while females had a negative allometric figure 1. sampling area (arzew gulf). 3 int. j. aquat. biol. (2016) 4(1): 1-10 n 𝛂 b se of b r2 combined 594 0.0172 2.83* 0.015 0.83 males 258 0.0079 3.11* 0.041 0.84 females 318 0.0176 2.82* 0.057 0.86 n: number, se of b: standard error of b, *: significative test-t (t-test, p<0.05). table 1. parameters of the length-weight relationship of mullus barbatus caught in arzew gulf. figure 3. length frequency distribution of mullus barbatus caught in arzew gulf. figure 2. sex ratio distribution in relation to length of mullus barbatus caught in arzew gulf. l∞ (mm) k (yr-1) t0 (yr) z m f e combined 250.9 0.49 -0.185 1.580 0.794 0.786 0.490 males 208.9 0.66 -0.143 1.560 1.010 0.550 0.350 females 250.9 0.60 -0.150 1.700 0.906 0.794 0.467 table 2. parameters of the von bertalanffy growth equation, the rates of total (z), natural (m) and fishing (f) mortalities and exploitation ratio (e) of mullus barbatus caught in arzew gulf. class (cm) catch in weight (g) catch in number z f vpa parameters result 10.5 62226.66 4447304.6 0.808 0.008 total biomass balance, d 168.486 t 11.5 163381.13 3971900.97 0.817 0.017 12.5 1354669.35 3513191.49 0.917 0.117 spawning stock biomass (ssb) 79.48 t 13.5 2201490.45 3027695.76 0.965 0.165 14.5 6495585.79 2554243.99 1.246 0.446 stock mean age 1.537 year 15.5 8796137.83 2008513.55 1.392 0.592 16.5 13215384.38 1493064.51 1.752 0.952 stock mean length 13.872 cm 17.5 16378226.85 984290.21 2.265 1.465 18.5 8495328.58 534870.29 1.814 1.014 virgin stock mean age 1.674 year 19.5 4015651.3 304651.16 1.4 0.6 20.5 3785692.09 182491.4 1.526 0.726 virgin stock mean length 15 cm 21.5 3353111.66 92335.88 1.813 1.013 22.5 1083359.34 32727.46 1.401 0.601 biomass/rercuit 27.681 g 23.5 599754.59 10696.75 1.586 0.786 ssb/recruit 17.87 g total 70000000 23157978.02 table 3. virtual population analysis parameters of mullus barbatus caught in arzew gulf obtained by vit 1.3 software. 4 bensahla talet et al./ growth and fishery of mullus barbatus from arzew gulf growth pattern (t-test, p<0.05). the selection length at which 50% of individual can be captured by the fishing gear was determined graphically (fig. 5) and was evaluated at 16.08 cm of lt. the von bertalanffy growth parameters, mortalities (fig. 6a, b) and exploitation ratio are shown in table 2. the exploitation ratio (e=0.49) reflects a fishing situation near the equilibrium concerning the figure 4. parameters of the length-weight relationship of mullus barbatus caught in arzew gulf. (a) all individuals, (b) males, and (c) females. figure 5. gear selection ogive for mullus barbatus caught by trawl in arzew gulf (codend mesh size of 4 cm). 5 int. j. aquat. biol. (2016) 4(1): 1-10 exploitation of this mullidae. the virtual population analysis of m. barbatus by vit software showed that the mean number of m. barbatus specimen in arzew gulf is 23.15 106 while the number of recruits per year was estimated at 4447304.6 represented by size class 10.5 cm (table 3). individuals between 16.5 and 19.5 are the most affected by the fishing gear deployed (bottom trawl). the average age of the current stock is 1.53 years and the average size 13.87 cm. the total balanced biomass is equal to 168.48 tons with gains mainly represented by growth 116.76 tons (69.31%) and recruitment 51.71 tons (30.69%) and losses are represented by natural mortality 98.48 tons (58.45%) and fishing mortality 70 tons (41.55%). the current yield per recruit was estimated to y/r=15.74 g (for f=0.79) and maximum sustainable yield can be reached by increasing the current fishing figure 6. total mortality of mullus barbatus fished in arzew gulf. (a) males and (b) females. locality sex n 𝛂 b se of b r2 author turkey (black sea) c 1435 0.00880 3.03 aydın and karadurmuş, 2013 turkey (iskenderun bay, mediterranean) c 212 0.0072 3.1618 0.95 çiçek, 2015 romania (black sea) c 680 0.00842 3.12 0.99 maximov et al., 2008 montenegro (adriatic sea) c 0.00773 3.09 joksimović et al., 2008 m 0.00729 3.11 f 0.00767 3.10 italy (castellammare gulf) c 18932 0.00820 3.09 0.00 0.98 giacalone et al., 2010 m 9596 0.00660 3.17 0.01 0.94 f 4730 0.00630 3.19 0.01 0.98 egypt (mediterranean) 0.00770 3.10 mehenna, 2009 morocco (mediterranean) c 0,000009 3,03 0.98 layachi et al., 2007 algeria (arzew gulf) c 594 0.01720 2.83 0.150 0.83 present study m 258 0.00790 3.11* 0.041 0.84 f 318 0.01760 2.82* 0.057 0.86 n: number, m: males, f: females, c: combined sexes, se of b: standard error of b, and r2: coefficient of regression. table 4. length-weight relationship parameters for mullus barbatus in the previous studies. 6 bensahla talet et al./ growth and fishery of mullus barbatus from arzew gulf effort to stabilize it at msy=17.91 g (f=1.73). discussion the growth pattern of m. barbatus in the arzew gulf seems to be different between the two sexes. in fact, males of this species grow faster than females as reported by joksimović et al. (2008) from the adriatic. based on the results, females’ asymptotic length is important than that of males due to the fact that males do not attain lager sizes in their natural habitat given that the males (f=1.23) are under a more fishing pressure than females (f=0.79). furthermore, the females had a negative allometric growth pattern compared to the males (b>3) which confirms that males gain weight faster than length and this was in agreement with the results of papaconstantinou et al. (1981) for this species in termaikos gulf. growth differences between sexes can be explained by the fact that females undergo significant physiological changes compared to males during their sexual cycle. the positive allometric growth pattern have been reported for this species in some other areas of mediterranean (table 4) which is not the case of females caught in arzew gulf probably due to weight loss after the spawning period. the growth parameters of the von bertalanffy equation differ greatly from one region to another (table 5) probably due to the biotic and abiotic differences of their habitat. in fact, magnan (1988), walker (1997) and svanbäck (2004) noticed that inter-population variation in lwr parameters are correlated with differences in the availability of resources or by geographical differences in locality sex n lt∞ (mm) k (yr-1) t0 (yr) author tunisia (gabes gulf) c 267.3 0.51 -0.010 gharbi et al., 2004 montenegro (adriatic sea) c 671 301.2 0.11 -3.182 joksimović et al., 2008 m 178.1 0.28 -3.013 f 274.7 0.14 -2.688 romania (black sea) c 680 163.2 0.37 -1.390 maximov et al., 2008 turkey (izmir bay) c 110891 242.6 0.56 -0.305 ozbilgin et al., 2004 turkey (iskenderun bay) c 212 219.8 0.19 -1.168 çiçek, 2015 italy (castellammare gulf) f 595 221.2 0.38 -0.940 sieli et al., 2011 egypt (mediterranean) c 271.0 0.66 mehenna, 2009 morocco (mediterranean) c 270.0 0,43 -0.090 layachi et al., 2007 algeria (arzew gulf) c 594 250.9 0.49 -0.158 present study m 258 208.9 0.66 -0.143 f 318 250.9 0.60 -0.150 n: number, m: males, f: females, and c: combined sexes. table 5. growth parameters of mullus barbatus given by different authors. locality sex n z m f e author montenegro (adriatic sea) c 671 0.749 0.342 0.407 0.54 joksimović et al., 2009 italy (adriatic sea) 3.51 0.91 2.60 0.74 ardizzone, 1998 1.2-1.9 0.43-0.77 0.77-1.13 0.59-0.64 marano et al., 1998 croatia (adriatic sea) 1.48 0.58 0.90 0.6 vrgoč, 2000 turkey (izmir bay) c 110891 3.85 1.07 0.71-0.72 ozbilgin, 2004 turkey (iskenderun bay) c 212 1.39 0.45 0.93 0.67 çiçek, 2015 algeria (arzew gulf) c 594 2.86 0.82 2.03 0.71 present study m 258 f 318 table 6. total (z), natural (m), fishing (f) mortalities and exploitation ratio (e) of mullus barbatus in different areas. 7 int. j. aquat. biol. (2016) 4(1): 1-10 ecological conditions such as water temperature, salinity, and food supply. the maximum value of the growth constant (k) in the arzew gulf reflects fast growth of this species in agreement with findings of mehenna (2009). this shows the fertility of algerian waters probably due to entrance of the enriched water with plankton from the atlantic and coastal upwelling (taupier-letage and millot, 1988). mortier (1992), taupier-letage and millot (1988), and salas et al. (2001) found that the algerian current has a higher nutrient content than the other parts of the mediterranean being supplied directly from the entering atlantic current. furthermore, bosc et al. (2004) noted that nutrients concentrations are higher in the western mediterranean basin than the eastern basin allowing the development of phytoplankton and zooplankton. the values of exploitation ratio differ from one region to another (table 6) depending on the environmental factors affecting the natural mortality and the fishing effort deployed. conclusion based on the results, the stock of m. barbatus in arzew gulf is exploited at its maximum limit. in order to maintain a sustainable exploitation of this resource, it would be wise to increase reasonably the fishing effort for a better yield per recruit ensuring the renewal of this resource in arzew gulf. algerian legislation had already set a minimum landing size (15 cm of total length) to preserve the resource but to our knowledge, it is insufficient. acknowledgements the authors wish to thank the laboratory teams lrse and aquabior for their precious assistance in the collection and processing of samples in this study. references ardizzone g.d. 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(2016) 4(1): 1-10 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی در خلیج آرزو، الجزایر mullus barbatus (mullidae) جمعیت کفال قرمز، شناسیپارامترهای پویایی 1بوتیبة زیتونی، 2ثالث سهلة بن احمد، 1*ثالث سهلة بن لطفی .الجزایر ،0133اوران ،احمدبن بال -1دانشگاه اوران ،زیستیبیعی و طعلوم دانشکده ،شناسی زیست، گروه زیست آزمایشگاه تحقیق و کنترل محیط1 الجزایر. ،0133اوران ،احمدبن بال -1دانشگاه اوران ،زیستیطبیعی و علومدانشکده ،آزمایشگاه آکوابیور، گروه بیوتکنولوژی 2 چکیده: ( الجزایر) آرزو خلیج ( صید شده به وسیله ترال در292)به تعداد mullus barbatus قرمز، کفال جمعیت شناسیپویایی پارامترهایدر این تحقیق سانتیمتر و 9/13-2/20 ترتیبها بهانجام شد. طول کل و وزن ماده 2332و ژانویه 2330ها بین فوریه برداریشد. نمونه مطالعه در غرب مدیترانه هاوزن نمونه-رابطه طول. بودگرم 0/21-9/99سانتیمتر و 6/12-7/19ترتیب که طول کل و وزن نرها بهحالی گرم بودند، در 2/129-12 222/2l33167/3=w (22/3=2rبدست آمد. پارامترهای رشد معادله وان ) :برتالنفی عبارت بودند ازcm 9/232=∞l ،1-year 66/3 =k و سال 120/3=0t 9/223برای نرها و=∞l ،1-year 6/3 =k 0=312/3 و سالt هابرای ماده. ( مرگ و میر کلz( طبیعی ،)m و ) صیادی(f ) به ترتیب منطقه در این حد نهاییدر این منبع برداری تخمین زده شد که بیانگر بهره 2/3برداری نیز د و نسبت بهرهشدنتخمین زده 79/3و 2/3، 22/1 باشد، می در سالقطعه 6/2227032به میزان هاه تعداد بازسازینشان داد ک vitافزار توسط نرم m. barbatusباشد. تحلیل مجازی جمعیت گونه می 76/116 به میزان رشد هاسطوهب و درصد( 69/03)تن 71/21میزان ازی بهبازس واسطهبهتوده زیافزایش تن تخمین زده شد، 162226شده باالنستوده کل زی 22/21تن ) 73 میزانبه و میر صیادی مرگ هاسطوهبدرصد( و 22/22تن ) 22/92 میزانبه ه مرگ و میر طبیعیاسطوهب ، تلفاترا نشان داددرصد( 01/69) تن بدست وسیله افزایش تالش صیادیاند بهتومیکه و حداکثر محصول پایدارتخمین زده شد g 72/12=y/r صورتبه. بازده جاری به بازسازی دارا نشان د درصد( ثابت شود. g 91/17=msyید باید در حد آ برتالنفی.ناوزن، و-مرگ و میر، رابطه طول ،صیادی ،برداریبهره :کلمات کلیدی int. j. aquat. biol. (2022) 10(6): 474-477 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article green turtle (chelonia mydas) nesting area and hatchlings released at daran beach, jiwani district, gwadar, balochistan with some recommendations for its conservation asad ullah1, sohaib ahmed1, arz muhammad umrani*1, mukhtar ahmed2, shehr yar nasim3, nazeer ahmed4, abdul ghafar ghotia4, fayaz ahmad1, khair khatoon5, fehmeeda yousaf2, amber khalid4, ahmad zamir1, zile huma7 1pakistan forests institute peshawar, peshawar, pakistan. 2university of balochistan, quetta, pakistan. 3pir mehr ali shah airid agriculture university, rawalpindi, pakistan. 4lasbella university of agriculture, water and marine sciences, las bela, pakistan. 5balochistan university of information technology, engineering and management sciences quetta, quetta, pakistan. 6lahore college for women university, lahore, pakistan. 7sardar bahadur khan women university quetta, quetta, pakistan. s article history: received 11 september 2022 accepted 26 november 2022 available online 2 5 december 2022 keywords: conservation daran beach recommendation threats balochistan abstract: this study aimed to investigate the nesting area of green turtle, chelonia mydas, and hatchlings released in daraan beach, jiwani, pakistan, with providing recommendations for their conservation. survey trips were conducted along daran beach, jiwani, for the current study from september 2020 to august 2021. we examined the nests, nesting turtles, hatchlings, and turtle tracks during this period. in addition, interviews were conducted with representatives of the local community, fishing industry, and wildlife service to obtain further information. on daraan beach, the three green turtle nesting locations (rindani taak, dedalah taak and shaheed taak) were recorded. the results showed that september had the fewest hatchlings and january had the most hatchlings, whereas, in the months of may, june, july, and august, there was no hatchling record. the results also revealed increasing hatchlings during our study during 2020-2021 due to the wildlife department’s efforts to protect and conserve green turtles at daran beach. the green turtle population in jiwani is in danger due to human activities such as climate change, global warming, sea level rise, pollution, and poaching. introduction pakistan enjoys a strategic location in south asia, close to the arabian sea, iran, afghanistan, china, and india, with variable temperatures due to the arid and semi-arid environment (khan, 1999). it has a 1050 km long coastline, starting from sir creek and ending at jiwani, balochistan. balochistan has an 800 km coastline comprising two districts viz. lasbela and gwadar (waqas et al., 2011). there is diverse marine life found along the coast of pakistan, and to maintain the health of these coastal environments, marine fauna plays important role. the coastal waters of pakistan have recorded five species of marine turtle. the green turtle, chelonia mydas, is dominant among them and regularly breeds on balochistan and sindh's beaches. the correspondence: asad ullah; arz muhammad umrani doi: https://doi.org/10.22034/ijab.v10i6.1761 e-mail: asad.fst@gmail.com; arz.forest87@yahoo.com dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.4.7 green turtle is a species at risk of extinction. july till december is often the most productive period for the green turtle's egg-laying. the eggs are placed in confined chambers that have been excavated out, with each clutch containing between 75 to 120 eggs (sikandar et al., 2022). along the coast of karachi, major nesting grounds of the green turtle can be found at hawksbay and sandspit coasts. along the coast of balochistan, the green turtle may be seen in jiwani, astola island, and ormara beaches. approximately 600 km long gowadar coast stretches from ras jiwani to hingol river, comprised of jiwani, gowadar, pasni and ormara i.e. the makran coast (begam et al., 2016). sandy beaches of jiwani are best owed with suitable nesting habitats for marine turtles. chelonia mydas 475 int. j. aquat. biol. (2022) 10(6): 474-477 commonly, while lepidochelys olivacea occasionally visit this coast for nesting (waqas et al., 2011). astola island is also an important nesting site of c. mydas. according to the iucn red list, sea turtles are considered endangered (iucn, 2008). under the balochistan wildlife protection act of 1975, it is explicitly stated that all species of sea turtles are legally protected. the coastal wetland of jiwani is one of pakistan’s planned ramsar sites (waqas et al., 2011). this work aimed to study the nesting area of green turtles and hatchlings released in daraan beach, jiwani, pakistan, from september 2020 to august 2021, with providing recommendations for their conservation. materials and methods the study area is the valuable beach of the balochistan coastline, including the daraan village of jiwani. (fig. 1) the beach is the main nesting habitat for green turtles. the daraan beach is an eyecatching combination of golden sand and blue water located 15 km southeast of jiwani town. daraan village is the only residential area near the beach. it is considered one of the favorite beaches for marine turtle nests on the balochistan coast. green turtles move towards the beach to lay eggs, usually at night. nests of green turtles on daraan beach are protected by putting a wire cage in a circular pattern without disturbing the natural habitat and eggs of green turtles. daraan beach is looked after by wwfpakistan, balochistan wildlife department, and local communities. the current study was done from september 2020 to august 2021 by excursions along daran beach, jiwani. we examined the nests, nesting turtles, hatchlings, and turtle tracks during this period. furthermore, data were collected through interviews with local communities, fishermen, and members of the wildlife department. the nesting regions of green turtles were visited during the day. results on daraan beach, the three green turtle nesting locations (rindani taak, dedalah taak and shaheed taak) were recorded based on observations from september 2020 to august 2021, and the monthly numbers of hatchlings released at each site are presented in table 1. regarding the number of released hatchlings, rindani taak in january had the highest record. from september 2020 through august 2021, december had the greatest number of hatchlings released. at dedalah taak, no hatchlings were observed in the months of june, july, or august. based on observations, september had the fewest hatchlings, and december and january had the most hatchlings. in may, june, july, and august, there was no hatchlings record. discussion the current study concentrated to identify turtle habitat along daran beach, jiwani, outlining threats by suggesting recommendations for their figure 1. map of the study area. 476 ullah et al./ green turtle nesting area and hatchlings released at daran beach, pakistan conservation. sea turtle survival, nesting, and conservation are all suitable in pakistan's coastline. five of the seven species of marine turtles are present along pakistani coastlines. balochistan's nearly 800 km of coastline, which is blessed with sandy beaches, makes it an ideal location for turtles to live. an important coastline beach of balochistan is jiwani's daran beach, where rindani taak, dedalah taak and shaheed taak were found nesting locations. there are few reports about the times and locations where marine turtles nest on the coast of pakistan (murray, 1884; minton, 1966; hirth, 1971; ghalib and zaidi, 1976; frazier, 1980, kabraji and firdous, 1984; firdous, 1985; groombridge, 1985). nesting takes place on the coast of karachi from june to early november (minton, 1966). green turtles visit the beaches of west pakistan all year long (zwinenberg, 1975). ghalib and zaidi (1976) provided information regarding marine turtles from the karachi coast, but without species names. they recorded year-round breeding, with a surge between july and november. they observed a low nesting rate from november to march. according to groombridge (1985), 5000 females nest at the hawks bay and sands pit coasts yearly. based on the results, december had the greatest number of hatchlings released. in the nesting areas viz. rindani taak, dedalah taak and shaheed taak on the daran beach, hatchlings released were 12050, 8395, and 19177, respectively. according to waqas et al. (2011), 5445, 7119 and 8784 hatchlings were released at daran beach in 2006, 2007, and 2008, respectively. however, this number has increased in our study during 2020-2021 due to the wildlife department’s efforts for the protection and conservation of green turtles at daran beach. however, the international union for conservation of nature and natural resources considers sea turtles endangered due to their vulnerability to numerous anthropogenic and natural threats. jiwani's endangered green turtles face challenges, including climate change, global warming, sea level rise, pollution, and poaching, etc. recommendations for conservation: pakistan started its marine turtle conservation program in 1979. however, currently, all marine turtles are protected and placed in schedule-iii (protected animals) of the balochistan wildlife (protection, preservation, conservation and management) act2014. based on our field observations and collected data by interviews, the following recommendations are suggested for the conservation of endangered green turtles: (1) it requires public awareness and education regarding the role of green turtles in the ecosystem and biodiversity, and efforts have been made to spread the message through the media, (2) months hatchlings released during the year 2020 – 21 rindani taak dedalah taak shaheed taak september 2020 102 289 213 october 2020 635 523 2313 november 2020 2720 1386 2983 december 2020 2266 1703 4008 january 2021 3210 1509 4274 february 2021 1243 1285 2176 march 2021 1047 779 2280 april 2021 520 918 930 may 2021 307 0 0 june 2021 0 0 0 july 2021 0 0 0 august 2021 0 0 0 total 12050 8395 19177 table 1. hatchlings released during the year 2020-2021 in three studied sites of jiwani's daran beach. 477 int. j. aquat. biol. (2022) 10(6): 474-477 national and international ngos should be involved to increase awareness, education, and capacity building on marine turtle conservation among local communities and other stakeholders, (3) research is required for protecting marine turtle populations and, particularly, for setting management objectives for preserving vital ecosystems, (4) training to fishermen is suggested for the safe release of accidentally trapped turtles, (5) protection of eggs and hatchlings from predators, (6) pollution is one of the main threats, so pollution should be controlled by the public and the authorities concerned, (7) petrol and diesel trade on daraan beach is a factor for its pollution. therefore, illegal trades should be curbed for the protection of biodiversity, (8) balochistan wildlife act 2014 should be implemented with its true essence, (9) regular patrolling and monitoring is a need of the time, and (10) manpower of wildlife department should be enhanced for effective performance. references frazier j. (1980). exploitation of marine turtles in the indian oceans. human ecology, 8(4): 329-370. ghalib s.a., zaidi s.s.h. (1976). observations on the survey and breeding of marine turtles of karachi coast. agriculture pakistan, 27: 87-96. groombridge b. (1985). indian sea turtles in world’s perspective. symposium on endangered marine animals and marine parks, the marine biological association of india. pp: 1-16. hirth h.f. (1971). synopsis of biological data on the green turtle chelonia mydas (linn.). fao fisheries synopsis, 85(1): 1-8. kabraji a.m., firdous f. (1984). conservation of turtles, hawkesbay and sandspit, pakistan. world wildlife fund project, 1451: 52. khan m.s. (1999). herpetology of habitat types of pakistan. pakistan journal of zoology, 31: 275-289. khan, a. (2013). pakistan wetlands programme’s marine turtle conservation efforts on daran beach, jiwani, pakistan. indian ocean turtle newsletter. 26 p. minton s.a. (1966). a contribution to herpetology of west pakistan. bulletin of the american museum of natural history, 134: 27-184. murray j.a. (1984) the vertebrate zoology of sind. richardson & co, london. https://doi.org/10.5962/ bhl.title.11813 sikandar a., noreen m., nasir i. (2022). the status and nesting sites of the marine turtles of pakistan at karachi and makran coast: a literature review. international journal of biology and biotechnology, 19(2): 259-264. waqas u., hasnain s.a., ahmad e., abbasi m., pandrani a. (2011). conservation of green turtle (chelonia mydas) at daran beach, jiwani, balochistan. pakistan journal of zoology, 43(1): 85-90. zwinenberg a.j. (1975). the green turtle (chelonia mydas), one of the reptiles most consumed by man, needs immediate protection. bulletin of the maryland herpetological society, 11(2): 45-63. int. j. aquat. biol. (2017) 5(4): 263-267; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article biology of the west african fiddler crab, uca tangeri (eydoux, 1835) (decapoda: ocypodidae) from a mangrove wetland in lagos, nigeria rasheed olatunji moruf1*, teslim asafe ojetayo2 1department of marine sciences, university of lagos, akoka, nigeria. 2department of forestry, wildlife and fisheries, olabisi onabanjo university, ago-iwoye, nigeria. article history: received 3 june 2017 accepted 24 august 2017 available online 2 5 august 2017 keywords: growth pattern feeding habits sex ratio mangrove abstract: the size distribution, allometric growth pattern, stomach analysis and sex ratio of the west african fiddler crab, uca tangeri in a mangrove wetland were investigated. the carapace length ranged from 1.11-3.7 cm and the weight 7.7-25.5 g. the crab exhibited negative allometric growth (b<3). no linear relationship was found between length and weight of u. tangeri as correlation coefficient (r) was 0.2464 for combined sexes. the condition factor ranged between 4.7 and 12.1, varying in relation to size and sex. the crabs fed mainly on detritus, diatoms, and algae with higher preference for plant materials. the males were significantly more numerous than females with the ratio 1:0.6. uca tangeri is an ecologically important species in our marsh. therefore, mangrove ecosystem should be effectively monitored for the conservation of this species. introduction crabs of the family ocypodidae are characterized by their sexually dimorphic claws; the males’ major claw is much larger than the minor one while the females’ claws are both the same size (levinton et al., 1995). the subfamily ucinae known as fiddler crabs are composed of small crabs (callander et al., 2013), found along sea beaches and brackish inter-tidal mud flats, lagoons and swamps. the west african fiddler crab, uca tangeri, lives in the eastern atlantic ocean. its carapace is violet to black, or sometimes yellowish in females, while the appendages are yellowish brown. the abdomen is small and greatly reduced. it is carried permanently fixed below the cephalothorax into which it fits tightly within a shallow depression. the movement of the smaller claw from ground to mouth during feeding explains the crabs' common name; it looks as if the animal were playing the larger claw like a fiddle (levinton et al., 1995). although, the west african fiddler crab does not constitute a food item for the coastal communities but it has played ecological role in the mangrove *corresponding author: rasheed olatunji moruf doi: https://doi.org/10.22034/ijab.v5i4.328 e-mail address: tunjimoruf@gmail.com ecosystem where it helps to clean up the mangrove areas by its feeding habits on the fallen leaves (olafsson et al., 2002). therefore, the aim of this study is to provide baseline data on the biology of the west african fiddler crab in the mangrove swamps of abule-agege creek with particular emphasis on the size distribution, growth pattern, food and feeding habits and sex ratio. materials and methods study site and sampling: the study was carried out along the mangrove area lining the abule-agege creek, which is one of the several adjoining creeks of the lagos lagoon with latitude 6o26'-6o37'n and longitude 3o23'-4o20'e. the sampling area is a typical estuarine water zone with extensive mangrove but low transparency and alkaline (ph>7) in nature (moruf and lawal-are, 2015). one hundred and twenty two specimens of u. tangeri (fig. 1) were collected with hand and hand-net on weekly basis at the station between the hours of 6-8 am. specimens were immediately placed in an ice-chest and transported to 264 moruf and ojetayo/ biology of the west african fiddler crab the laboratory. they were then stored in the freezer (-20°c) after identification based on schneider (1990). the crabs were removed from the freezer, allowed to thaw and a towel was used to remove excess moisture from their body. after sex determination based on kwei (1978), the carapace length (cl) (from the edge of the frontal region to the tip of the carapace back wall) was measured using a 0.05 cm precision venier caliper. the total weight was taken to the nearest 0.1 g on a sartorius top loading balance (model: dt1001a). length-frequency distribution: the lengths of the crabs were plotted against their respective frequencies. carapace-length/weight relationship: the carapacelength/weight relationship was expressed using the equation (pauly, 1983): w=a lb where, w=weight of the crabs in grams, l=total length of the crabs in cm, a=regression constant and b=regression coefficient. the values of constant “a” and “b” were estimated from log transformation equation as follow (parsons, 1988): log w = log a + b log l the condition factor (k) of the crab was determined using the formula (bannister, 1976): k=100w/lb where, k=condition factor, w=weight of the periwinkle (g), l=length of the periwinkle (cm) and b = regression coefficient. food and feeding habits: the cardiac stomach of each specimen was dissected and the contents were extracted into a petri dish. the extracted contents were mixed with little water and examined under a binocular microscope for the food types using the numerical and occurrence methods based on hyslop (1980). sex ratio: the sex ratio was tested for any deviation from the expected 1:1 ratio using chi-square analysis. level of significance was tested at 5% level of significance (p<0.05). the chi square value (χ2) was calculated using the formula: χ2 = (observed – expected)² expected where, observed=number of females in the sample and expected=the total number of males in the sample. statistical analysis: data were analyzed using microsoft excel 2010 and spss software. results size composition: the ranges of carapace length, width and weight were 1.l1-3.70 cm, 1.30-3.80 cm and 7.7-25.50 g, respectively. the crab exhibited unimodal size distribution, revealed from size frequency distribution (fig. 2). the relative growth in u. tangeri showed no linear relationship between the length and weight. the length/weight relationship values for the male, female and combined sexes were given as follows: for male: log w=log 0.1002+1.1648 log l (r=0.1338) for female: log w=log -0.2241+1.2019 log l (r=0.3223) for combined sexes: log w=log 0.0063+1.1761 log l (r=0.2464) the values of “b” were 1.1648, 1.2019 and 1.1761 for the males, females and combined sexes which showed that the west african fiddler crabs exhibited a very low negative allometric growth pattern. the correlation coefficient (r) was 0.1338 for the males, 0.3223 for females and 0.2464 for combined sexes which showed a very low correlation (far from ‘1’) between the carapace length and weight in u. tangeri. condition factor: the variations in condition factor (k) by size and sex of u. tangeri from the abuleagege mangrove swamp are presented in table 1. figure 1. west african fiddler crab, uca tangeri. 265 int. j. aquat. biol. (2017) 5(4): 263-267 the k-values ranged from 4.7-10.8 (male), 5.5-12.1 (female) and 4.7-10.7 (combined sexes). the highest k-values were recorded for the medium size (2.0-2.4 cm) group. in term of sex, the female has the highest k-value (12.1). food analysis: a total of 33 (27%) of the 122 specimens of u. tangeri examined in this study had empty stomachs. the stomach contents consisted mainly of plant materials, detritus, algae, diatoms, pebbles and unidentified mass. the plant materials formed the most important food item occurring in 95.5% of the u. tangeri crabs examined. detritus and diatoms occurred in 78.7% and 48.3% of the stomachs, respectively (table 2). sex ratio: using the species conspicuous external morphological features, 78 were males and 44 were females giving a sex ratio of 1:0:6. a chi-square (χ2) test indicated that this ratio was significantly (p<0.05) figure 2. carapace length frequency distribution of uca tangeri from the mangrove area of abule-agege creek. table 1. condition factor by sex and size of uca tangeri from mangrove swamps of abule-agege creek. carapace length (cm) male female combined sex n cl(cm) wt(g) k n cl(cm) wt(g) k n cl(cm) wt(g) k 1.0-1.4 14 1.2 1.8 10.4 9 1.2 1.7 9.8 23 1.2 1.8 10.4 1.5-1.9 28 1.6 2.7 6.6 7 1.5 2.4 7.1 35 1.6 2.6 6.4 2.0-2.4 19 2.1 10 10.8 10 2 9.7 12.1 29 2.1 9.9 10.7 2.5-2.9 13 2.7 13 6.6 4 2.7 11.1 5.6 17 2.7 12.1 6.2 3.0-3.4 5 3.1 18 6 6 3.1 16.4 5.5 11 3.1 17.2 5.8 3.5-3.9 7 3.5 20 4.7 7 3.5 20 4.7 86 36 122 table 2. stomach contents of uca tangeri from the mangrove swamp of abule-agege creek food items numerical method occurrence method no % no % detritus 2619 48.6 70 78.7 algae 125 2.3 35 39.3 diatom 176 3.3 43 48.3 plant materials 2386 44.3 85 95.5 sand grain/pebble 36 44.4 unidentified mass 86 1.6 76 85.4 266 moruf and ojetayo/ biology of the west african fiddler crab different from the expected and theoretical ratio of 1male:1female. therefore, male u. tangeri are significantly more abundant than female. discussion the west african fiddler crab examined in this study showed a unimodal size distribution. unimodal size frequency was reported by lawal-are and nwankwo (2011) for sersema huzardii from the same mangrove habitat. this result suggest that there was only one predominant generation of crabs sampled and the specimens belonged to the same year class. the observed values of the regression coefficient (b) for the u. tangeri (1.1648-1.1761) which are less than 3 are indications of negative allometric growth. this result is in conformity with the findings of moruf and lawal-are (2015) for the mangrove prosobranch, tympanotonus fuscatus but contrary to the positive allometric growth recorded for brachyuran crab, portunus validus in moruf and lawal-are (2017a). the correlation coefficient (r), 0.1338 for the males, 0.3223 for females and 0.2464 for combined sex were not close to “1”, indicating that there was no linear relationship between length and weight of u. tangeri. this is contrary to the high correlation between length and weight reported by lawal-are and kusemiju (2000) for callinectes sp. the condition factor (k) for the west african fiddler crabs ranged from 4.7 to 12.1 and varied in relation to size and sex of the crabs. lawson and oloko (2013) reported values of 2.14 to 9.48 for the lagoon crab, callinectes amnicola from the yewa river. in studies of population dynamics, high “k” values of a crab show favorable environmental conditions such as habitat and prey availability (moruf and lawal-are, 2017a). it was observed from this study that the average sized group has the highest condition factors. this indicates successive growth as a result of molting activity. the older the crab, the more difficult it is for the crab to molt. on the average, the condition factors of the female of u. tangeri were higher than that of the male. stomach analysis revealed low percentage empty stomach which was due to the abundance food items during the season of collection. the crabs showed a carnivorous feeding habit as their stomach contents consisted of detritus, diatoms, algae and plant materials. they however showed preference for plant materials/ detritus. lawal-are and nwankwo (2011) had a similar opinion on the feeding habit of s. huzardii from the same mangrove wetland. the presence of sediment balls near the entrance to a burrow is a good indication of its occupation, as it was evidence in the stomach contents of u. tangeri as sand grains. the feeding habits of fiddler crabs play a vital role in the preservation of wetland environments by sifting through the sands; they aerate the substrate and prevent anaerobic conditions. the males were significantly more numerous than females. this observation however was in contrast with the work of moruf and lawal-are (2017b) who reported no significant difference in sexes (ratio of 1:0.98) of c. amnicola from lagos coast. warner (1977) was of the opinion that sex ratio is one of the major factors that determines the population of tropical brachyuran crabs that breeds continuously throughout the year. he noted the greater the difference in ratio the lesser the population. in conclusion, u. tangeri exhibits sexual dimorphism with males attaining larger sizes than females. frequency histograms with unimodal distribution are typical of many brachyuran crabs. the determination of age and growth rate had not attracted much attention due to effect of ecdysis resulting in the absence of annular structures in crabs. thus, no direct method of determination of ages in crabs. the mangrove ecosystem should be effectively monitored for the conservation of this species. acknowledgments we would like to thank dr. mobedi, m. ahoo and a.m. alavi for their kind assistances. references bannister j.v. (1976). the length-weight relationship, condition factor, gut contents in the dolphin fish, coryphaena hippurus (l) in the mediterranean. journal of fish biology, 9: 335-338. 267 int. j. aquat. biol. (2017) 5(4): 263-267 callander s., kahn a.t., maricic t., jennions m.d., backwell p.r.y. (2013). weapons or mating signals? claw shape and mate choice in a fiddler crab. behavioral ecology and sociobiology, 67: 1163-1167. hyslop e.j. (1980). stomach content analysis: a review methods and applications. journal of fish biology, 17: 411-414. kwei e.a. (1978). size composition, growth and sexual maturity of callinectes latimanus (rath) in two ghanaian lagoons. zoology journal of limnology society, 64(2): 151-175. lawal-are a.o., kusemiju k. (2000). size composition, growth pattern and feeding haibits of the blue crabs, callinectes amnicola (de rocherburne) in the badagry lagoon, nigeria. journal of scientific research and development, 5: 169-176. lawal-are a.o., nwankwo h. (2011). biology of the hairy mangrove crab, sersema huzardii (decapoda: graspidae) from a tropical estuarine lagoon. journal of american science, 7(7): 402-408. lawson e.o., oloko r.t. (2013). growth patterns, sex ratios and fecundity estimates in blue crab (callinectes amnicola) from yewa river, southwest nigeria. advances in life science and technology, 7: 24-33. levinton j.s., judge m.l., kurdziel j.p. (1995). functional differences between the major and minor claws of fiddler crabs (uca, family ocypodidae, order decapoda, subphylum crustacea): a result of selection or developmental constraint. journal of experimental marine biology and ecology, 193: 147-160. moruf r.o., lawal-are a.o. (2015). growth pattern, whorl and girth relationship of the periwinkle, tympanotonus fuscatus var radula (linnaeus, 1758) from a tropical estuarine lagoon, lagos, nigeria. international journal of fisheries and aquatic studies, 3(1): 111-115. moruf r.o., lawal-are a.o. (2017a). size composition, growth pattern and condition factor of two portunid crabs, callinectes amnicola (de rochebrune) and portunus validus (herklots) from lagos coast, nigeria. nigerian journal of fisheries and aquaculture, 5(1): 1521. moruf r.o., lawal-are a.o. (2017b). comparability in dietary elements, sex ratio and fecundity of portunidae crabs, callinectes amnicola (de rochebrune) and portunus validus (herklots) off lagos coast. journal of aquatic sciences, 32(1a): 23-71 olafsson e., buchmayer s., skov m.w. (2002). the east african decapod crab, neosarmatium meinerti (de man) sweeps mangrove floors clean of leaf litter. ambio, 31(7-8): 569-573. parson r. (1988). statistical analysisa decision making approach. second edition. harper and row publishers, new york.791 p. pauly d. (1983). some simple methods for the assessment of tropical stocks. fao fish. tech. pap., 234: 52. schneider w. (1990). field guide to commercial marine resources of the gulf of guinea. food and agricultural organization of the united, rome, italy. 186 p. warner g.f. (1977). the biology of crabs. the gresham press, old working, surrey birmingham, great britain. 179 p. int. j. aquat. biol. (2013) (1): 1-5 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology body shape changes during the early development of the beluga (huso huso) reza asgari1, soheil eagderi *1, gholamreza rafiee 1, hadi poorbagher1, naser agh2, hamid eshagh zadeh1 1 department of fisheries, faculty of natural resources, the university of tehran, p.o. box: 31585-4314, karaj, iran. 2 artemia and aquatic animals research institute, urmia university, urmia, iran. article history: received 2 march 2013 accepted 4 april 2013 available online 5 april 2013 keywords: acipenseridae body shape ontogeny morphometrics abstract: early body shape changes of beluga sturgeon were studied using landmark-based geometric morphometric approach to recognize its allometric growth pattern. sampling was done from hatching up to 50 days post hatching (dph). left side of specimens were photographed using digital camera and nine landmark points were digitized on two-dimensional images. total length (ti) was measured using the software imagej. to study of the body shape changes during early development, the mean procrustes distance between all specimens of same age, for all age groups, was calculated. the scores of relative warp analysis (rw) were used as descriptors for the variation in shape. rw analysis revealed a sharp body shape change during early ontogeny on 18 dph. growth trajectory was computed by plotting rw against tl. the inflection point of body shape corresponds to a tl of 23.3 mm (18 dph). results showed that ontogenetic shape changes encompassed a pre-inflection shape changes, which included the elongation of the head and tail regions i.e. positive allometric growth pattern and postinflection shape changes, with a nearly isometric growth pattern. introduction the early development of fish larvae is accompanied with very complex shape changes. different growth rates of various parts of the body or allometric growth is a common phenomenon during this period (osse and van den boogaart, 1995). during the early development, changes in body shape are related to the functions of different organs such as respiration, feeding and swimming (simonovic et al., 1999; russo et al., 2007). recognition of morphogenesis process and growth pattern of fishes may lead to better understanding of biological priorities during the early developmental stages and gives insights into biological, behavioral and ecological characteristics (gisbert, 1999). many studied have been carried out on change of the body shape during the early ontogeny of various fishes using traditional morphometric approaches but recently, geometric morphometric techniques * corresponding author: soheil eagderi e-mail address: soheil.eagderi@ut.ac.ir have been applied (bookstein, 1991; rohlf, 1998; zelditch et al., 2004). geometric morphometric methods are useful tools in developmental biology to extract shapes data and analyze using multivariate statistical tests, explaining how morphological structures are generated (zelditch et al., 2004). hence, this study was conducted to study the changes of the body shape in beluga sturgeon (huso huso) using landmark-based a geometric morphometric method covering a period from hatching up to 50 days post hatch (dph) that is synchronized with the transition of larvae from internal to external feeding. material and methods specimens were obtained from the dadman international sturgeon research institute (guilan, iran). newly hatched larvae were stored in 500 l fiberglass tanks with a water depth of 20 cm and 2 asgari et al./ int. j. aquat. biol. (2013) (1) 1-5 reared up to 50 dph. larvae were stocked in tanks with a density of 1800 tank 1. the water was supplied from a mixture of ground and river water with a discharge of 400 and 250 ml 1 min 1, respectively. the water temperature (°c), do (mg l-1) and ph of were 15.9, 7.7 and 7.8 during experiment, respectively. due to asynchrony in external feeding of pre-larvae and to avoid starving and cannibalism, feeding was started from 8 dph using artemia nauplii (500 nauplii larvae 1) which continued till 12 dph. the larvae were then fed using a mixture of artemia nauplii and daphnia till 25 dph. afterwards the larvae were fed using a mixture of commercial food pellet (biomar) and mashed chironomid larvae in a rate of 30% bw d 1 (4-6 times per day). ten specimens were sampled every day from each tank, anaesthetized with ms222, preserved in 10% buffered formalin and stored in 70% ethanol after 24 hours. before fixation, the total length (tl) was measured (in mm) as an independent data using the software imagej (version 1.240). the left sides of specimens were photographed using a stereomicroscope equipped with a cannon camera with a 5 mp resolution and nine landmarks points were digitized on two-dimensional images using the software tpsdig2 (fig. 1). the landmark data was tested using tpssmall which confirmed the suitability for further analysis and the figure 1. (1) anterior tip of the snout; (2) anterior margin of the eye ; (3) posterior margin of the eye; (4) posterior most point of the gill slit; (5) ventral point of the gill slit; (6) anterior of the yolk sac ; (7) posterior end of abdominal area ; (8) anus; (9) posterior end of vertebrate; (10) posterior most tail extremity; (11) pseudo-landmark on the dorsal edge of body in the front of the anus; (12) pseudo-landmark on the dorsal edge of body in the front of landmark 4; (13) insertion point on superior of yolk sac between landmark 7 and 12. figure 3. growth trajectory from 1 to 50 dph: rw1 and tl. the splines represent the shape at the extremes of the growth trajectory: the bottom one at the beginning and the upper at the end of development, respectively. figure 2. scatter plot of relative warp analysis, depicting rw1 and rw2. the youngest specimens labeled from 1, the older ones have higher numbers. 3 asgari et al./ int. j. aquat. biol. (2013) (1) 1-5 consensus configurations were computed for each age sample (rohlf, 2005). the data were analyzed using generalised procrustes analysis (gpa), in order to remove all non-shape related information and relative warp analysis using past soft (version 2.10). relative warp analysis is analogous to a principal component analysis (pca) for these sorts of data (rohlf and marcus, 1993). the relative warp scores (rw1 and rw2) were used as descriptors for the variation in shape (bookstein, 2005). the shape changes during growth were visualized as generating wireframe graph and deformation grids (splines) using the software morphoj. the correlation between shape descriptors and tl was tested with permutation tests with 1000 random permutations. growth trajectory was computed by plotting rw1 and rw2 against tl. allometric growth was calculated as a power function of total length using non-transformed data: y = axb where y was the independent variable, x the dependent variable, a the intercept and b the growth coefficient. isometric growth, positive and negative allometric growth are indicated by b=1, b>1 and b<1, respectively (zelditch et al., 2004). the inflexion points of growth curves were determined according to fuiman (1983) and van snik et al. (1997). inflexion point and regression analyses were extracted and performed using ms-excel 2007 (microsoft corporation). results the first two relative warps explained 91.06% of the body shape changes during ontogeny (rw1 63.86% and rw2 27.20%). figure 2 displays the morphospace defined by rw1 and rw2 spreading along rw1 according to age (youngest specimens on the right side of the graph; the older ones on the left side) (fig. 2). rw1 reflects (1) elongation of the head (including the snout and post-orbital region) and (2) elongation of caudal area i.e. allometric growth pattern of the trunk region, whereas rw2 indicated an increase in depth of the head and trunk (fig. 2). there was a weak correlation between rw1 scores and tl (r2=0.433) (fig. 3). however, the regression model showed that rw1 scores were strongly correlated to tl during early ontogeny upto 18 dph (r2=0.963) but no correlation onwards (r2=0.007; fig. 4). similar results were found before and after the absorption of the yolk sac (r2=0.345 and r2=0.355, respectively; fig. 5). the inflection point of body shape corresponds to a tl of 23.3 mm, which was coincided with the age of 18 dph. ontogenetic shape changes encompassed two phases: (1) pre-inflection shape changes, which figure 4. rw1 versus tl. the dotted line represents the inflexion point of growth. figure 5. (a) rw1 versus tl, (2) rw2 versus tl. = before yolk sac absorption and = after yolk sac absorption. the dotted line represents the inflexion point of growth. 4 asgari et al./ int. j. aquat. biol. (2013) (1) 1-5 included the elongation of the head and tail regions (positive allometric growth) and (2) post-inflection shape changes, with a nearly isometric growth pattern. in summary, main changes of the shape occurred during the absorption of yolk sac when a period started that the body shape became a miniature form of the adults. discussion the present study indicated that most important changes in shape of beluga larvae occur during early development up to 18 dph and involve the elongation of head and caudal area along with the increase of the body depth. during this period, the trunk had a negative allometric growth. as the first study of allometric growth in sturgeons using geometric morphometric approach, our study is in agreement with other researches that used traditional methods (gisbert, 1999; russo et al., 2007; fuiman, 1983; osse, 1990; osse and boogart, 1995; van snik et al., 1997) confirming that there is a correlation between changes of body shape and growth pattern, which is according to their functional importance. since predation and starvation are the main causes of mortality in fish larvae, development of feeding and swimming organs appears to be two important priorities during the early ontogeny (osse et al., 1997). the positive allometic growth of anterior and posterior regions of body in beluga larva is similar to other fishes indicating the importance of the organs that are related to respiration, feeding and movement (osse and van den boogart, 1995; gisbert et al., 2002). changes in body shape pattern of the beluga larvae at the length of 23 mm (18 dph), reflects full swimming ability that is necessary for external feeding. it has been suggested that ability to escape from predation and prey is synchronous with fully development of fins (hale, 1999, gibb et al, 2006). formation of fins in beluga is occurred at 15 dph being coincided with stiffening of fin rays (asgari, 2012). also the cephalic lateral line canals are completed at 18 dph (asgari, 2012). the neoromasts of these canals play a vital role in reception of environmental stimuli during feeding and swimming (omori et al., 1996). the present study show the importance of body shape changes during the early development of beluga larvae, which are associated with development of feeding apparatus, swimming, respiration and sense organs. based on the pattern of body shape changes, beluga larva can be considered fully developed or fry after 18 dph. since these body shape changes are associated with skeletal changes, the main structures of beluga skeleton are probably formed during this period. further studies on the ontogeny of musckloskeletal and digestive systems are recommended. references asgari r. (2012). early development of huso huso (acipenseridae): a case study for ontogeny of feeding apparatus and digestive enzyme (trypsin, amylase and lipase) activity up to 50 dph. phd. thesis, department of fisheries, university of tehran. 170 p. bookstein f.l. (1991). morphometric tools for landmark data. geometry and biology. cambridge: cambridge university press. fuiman l.a. (1983). growth gradients in fish larvae. journal of fish biology, 23: 117-123. gibb a.c., swanson b.o., wesp h., landels c., liu c. (2006). development of the escape response in teleost fishes: do ontogenetic changes enable improved performance? physiological and biochemicalal zoology, 79: 7-19. gisbert e. (1999). early development and allometric growth patterns in siberian sturgeon and their ecological significance. journal of fish biology, 54: 852-862. gisbert e., merino g., muguet j.b., bush d., piedrahita r.h., conklin d.e. (2002). morphological development and allometric growth patterns in hatchery-reared california halibut larvae. journal of fish biology, 61: 12171229. hale m.e. (1999). locomotor mechanics during early life history: effects of size and ontogeny on faststart performance of salmonid fishes. journal 5 asgari et al./ int. j. aquat. biol. (2013) (1) 1-5 of experimental biology, 202: 1465–1479. omori m., sugawara y., honda h. (1996). morphogenesis in hatchery-reared larvae of the black rockfish, sebastes schleeli, and its relationship to the development of swimming and feeding actions. ichthyological research, 43: 267-282. osse j.w.m. (1990). form changes in fish larvae in relation to changing demands of function. netherlands journal of zoology, 40: 362-385. osse j.w.m., van den boogaart j.g.m. (1995). fish larvae, development, allometric growth, and the aquatic environment. ices marine science symposium 201: 21-34. osse j.w.m., van den boogaart j.g.m., van snik g.m.j., van der sluys l. (1997). priorities during early growth of fish larvae. aquaculture, 155: 249-258. rohlf f.j. (1993). relative warp analysis and an example of its application to mosquito wings. in: marcus, l. f., e. bello and a. garcía-valdecasas (ed.). contributions to morphometrics. mardrid: c.s.i.c, pp.131-159. rohlf f.j. (1998). on applications of geometric morphometrics to studies of ontogeny and phylogeny. systematic biology, 47: 147-158. rohlf f.j. (2005). software by f. james rohlf. available from: http://life.bio.sunysb.edu/ee/rohlf/software.html. russo t., costa c., cataudella s. (2007). correspondence between shape and feeding habit changes throughout ontogeny of gilthead sea bream sparus aurata l., 1758. journal of fish biology, 71: 629–656. simonovic p.d., garner p., eastwood e.a., kovac v., copp g.h. (1999). correspondence between ontogenic shifts in morphology and habitat use in minnow phoxinus phoxinus. environmental biology of fishes, 56: 117–128. van snik g.m.j., van den boogaart j.g.m., osse j.w.m. (1997). larval growth patterns in cyprinus carpio and clarias gariepinus with attention to the finfold. journal of fish biology, 50: 1339-1352. zelditch m.l., swiderski d.l., sheets h.d., fink w.l. (2004). geometric morphometrics for biologists: a primer. elsevier (usa). http://life.bio.sunysb.edu/ee/rohlf/software.html int. j. aquat. biol. (2023) 11(1): 34-40 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023iranian society of ichthyology original article short-term assessment of heavy metals in surface waters of the shatt al-arab river ghassan a. al-najar, abdulkareem t. yesser, amir a. jabir, kadhim h. younis marine science center, university of basrah, basrah, iraq. s article history: received 24 october 2022 accepted 19 december 2022 available online 2 5 february 2023 keywords: heavy metals salinity seawater intrusion shatt al-arab surface water abstract: the shatt al-arab river's water quality deteriorates naturally due to salinity intrusion, and freshwater sources decrease. seven sampling locations along the shatt alarab river in southern iraq were used to examine the level of six heavy metals, including cu, cd, ni, co, mn, and fe, during januar-december 2021. salinity levels in the study area ranged from 1.55 upstream to 35.15 g/l downstream of the study area. the ph of surface water ranged 7.545-8.325, indicating alkaline conditions. the concentrations of six heavy metals, viz cu, cd, ni, mn, co, and fe in the study area were 3.741±4.219, 3.654±4.169, 7.700± 6.251, 2.551±3.898, 2.292±3.996, and 18.236±5.583 µg/l, respectively, which decreased in the order of fe > ni > cu > cd > mn > co. there was a considerable change in the quantity of heavy metals throughout the year, with the summer months having the highest concentration. there is a correlation between seawater intrusion and the concentration of heavy metals in the surface waters. the mean levels of the heavy metals were below the allowed values of who drinking water guidelines. introduction increasing heavy metals in river ecosystems is a significant global issue. over the last decades, there has been a significant rise in the quantity of heavy metals in river water (mishra et al., 2022). the shatt al-arab river starts at the confluence of the tigris and euphrates rivers in qurna, north of basrah, iraq, and flows approximately 200 km northwest of the iraqi marine waters, south of the al-fao city (aldoghachi, 2022). the river width ranges from 250 to 300 m, close to the euphrates and tigris confluence to about 700 m in basrah and over 800 m close to its estuary (abduljaleel et al., 2020). its salinity varies greatly depending on the season and quantity of freshwater discharge (abdallah, 2016). abduljaleel et al. (2020) reported that the salinity of the water in the euphrates, tigris, and shatt al-arab rivers dramatically increased over time. they emphasize how the lack of efficient river basin management programs causes the water quality of correspondence: ghassan a. al-najar doi: https://doi.org/10.22034/ijab.v10i6.1784 e-mail: ghassan.kamel@uobasrah.edu.iq dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.5.5 the euphrates, tigris, and shatt al-arab rivers to deteriorate. the shatt al-arab estuary and its upstream encountered increasing saltwater intrusion from the iraqi marine waters, which has a negative impact on ecosystem productivity. studies on the effects of urban contamination from the city and industry on the shatt al-arab surface water in the basrah region have been studied (douabul et al., 2013; al-aboodi, 2018). moyel (2014) used a water quality index to assess the water quality of the northern shatt al-arab region to determine its suitability for drinking, irrigation, and aquatic life. lafta (2014) and abdallah (2016) calculated the upstream flow to predict seawater intrusion into shatt al-arab, and it was only suitable for irrigation purposes. salt concentrations are higher than those permitted for a particular use of water should be categorized as pollutants (karamouz et al., 2003). under the influence of geochemical processes, heavy metal contamination will transfer 35 int. j. aquat. biol. (2023) 10(1): 34-40 with surface water and contaminate soil and water directly or indirectly (liu et al., 2018). however, most works focus primarily on the characteristics of heavy metal contamination in soil and pay little attention to heavy metal pollutants transfer in surface water. it is difficult to identify the distribution characteristics of heavy metals in agricultural ecosystems because of the integrated and cohesive substrate in the surrounding ecosystems (lermi et al., 2020). excessive pollutant discharge into surface rivers has caused major decreases in aquatic environmental indicators in recent years (liu et al., 2016). in this study, the shatt al-arab river is assessed regarding the influence of seawater intrusion from the iraqi marine water on the concentrations of heavy metals with the main objective of assessing the levels of heavy metals (cu, cd, ni, co, mn and fe) in the surface waters of the shatt al-arab based on temporal and spatial variability. materials and methods the main source of freshwater in basrah is shatt alarab, with about 200 km long and an estuary with a three km width. shatt al-arab extends 63 km till reaching al-fao. in the basrah region, it provides services for industry, agriculture, navigation, and ecosystem biodiversity. numerous small waterways branch out of both banks of the river in basrah city; most waterways are used for agriculture. table 1 shows the locations of the monitoring sites in this study. seven sampling sites in the shatt al-arab river were set up, and each site was positioned with a global position system (gps) to ensure the representativeness of the collected samples. the seven sampling sites were fao (sea area) (st1), fao city (st2), abu al-khaseeb (st3), basrah city (st4), sidebad island (st5), hartha (st6), and qurna (st7). surface water samples were taken at a depth of 30 cm in the middle of the river using a plastic bottle with a one-liter capacity that was fully filled. water samples were collected monthly (january-december 2021), divided into 6 periods: january-february (p1), march-april (p2) may-june (p3), july-august (p4), september-october (p5), and novemberdecember (p6). one-liter polypropylene sample containers were used to hold the water samples, which had been cleaned three times in distilled water. to prevent water evaporation, all the water samples were parafilm-sealed after being fixed with hno3 to a ph<2. (apha, 2005). the water temperature, salinity, and ph at the study sites were measured using a portable multi-meter water quality instrument (yasi, usa). the sample waters were transferred to the laboratory, kept in a lowtemperature incubator, and then examined for the presence of heavy metals. all samples were filtered using a 0.45 m cellulose acetate membrane filter to remove contaminants. the concentrations of heavy metals viz. co, ni, cu, cd, mn, and fe were measured using an atomic flame absorption spectrum technique at wavelengths of 248.3, 228.8, 240.7, 279.5, 264.88, and 324.8 nanometers, respectively. results the water quality of the shatt al-arab river deteriorates naturally due to salinity intrusion, and freshwater sources decrease. salinity levels in the study area ranged from 1.55 (st7) to 35.15 gm/l (st1). the ph of surface water samples ranged between 7.545 to 8.325, indicating alkaline conditions in all studied sites (table 2). while the temperature ranged between 14.85°c in the winter and 29.55°c in the summer months. the concentration of heavy metals (µg/l) in the study area is shown in table 3. heavy metals were found in all the surface water station name e n st1 fao (sea) 48°48'20.74" 29°47'36.81" st2 fao city 48°29'31.07" 29°58'43.92" st3 abu al-khaseeb 47°54'12.59" 30°28'41.65" st4 basrah city 47°48'49.03" 30°33'6.97" st5 sidebad island 47°46'43.38" 30°34'20.80" st6 hartha 47°44'47.87" 30°41'41.25" st7 qurna 47°27'8.93" 30°59'5.50" table 1. monitoring stations and their coordinates. 36 al-najar et al./ short-term assessment of heavy metals in surface waters of the shatt al-arab river sampling sites water quality periods p1 p2 p3 p4 p5 p6 st1 temperature(°c) 15.35 17.55 27.3 29.05 27.145 17.16 salinity (gm/l) 34.35 34.75 34.4 35.15 19.53 17.73 ph 7.65 7.85 8.05 7.81 7.795 7.76 st2 temperature(°c) 15.56 18.35 27.65 29.55 27.51 17.37 salinity (gm/l) 21.85 22.75 23.4 31.55 18.25 17.53 ph 7.80 8.06 8.05 7.80 7.59 7.88 st3 temperature(°c) 18.15 21.99 26.85 29.45 17.57 17.19 salinity (gm/l) 5.91 5.25 5.17 16.32 22.25 15.23 ph 7.96 8.14 7.66 7.82 7.595 7.88 st4 temperature(°c) 15.82 21.95 26.65 28.69 27.14 17.09 salinity (gm/l) 3.82 4.105 5.15 14.08 16.53 16.32 ph 7.54 8.27 7.89 7.83 7.795 7.88 st5 temperature(°c) 15.82 21.86 25.97 29.52 27.90 17.465 salinity (gm/l) 3.43 3.91 4.69 14.41 15.61 13.05 ph 7.54 8.325 7.68 7.84 7.75 7.955 st6 temperature(°c) 14.85 20.65 26.25 28.21 27.55 17.15 salinity (gm/l) 1.99 2.865 3.85 27.26 14.28 12.35 ph 7.62 8.26 7.65 7.86 7.78 7.97 st7 temperature(°c) 16.54 21.3 26.6 29.15 28 17.8 salinity (gm/l) 1.55 1.85 2.94 5.31 4.22 3.18 ph 7.65 8.14 7.65 7.95 7.85 8.03 table 2. water quality in surface water of the shatt al-arab river at different locations and times during study period. sampling time sampling site heavy metals concentration µg l-1 cu cd ni mn co fe january-february (p1) st1 12.5 12.45 12.5 12.31 9.33 25.36 st2 7.52 4.65 18.66 2.09 nd 17.968 st3 3.54 1.23 20.52 1.42 1 24.169 st4 1.77 0.615 10.26 0.71 0.5 18.282 st5 1.18 1.23 0.41 0.4733 0.3333 12.686 st6 0.885 0.3075 5.13 0.355 0.25 11.792 st7 0.708 0.246 4.104 0.284 0.2 11.23 march-april (p2) st1 13.15 12.815 13.26 11.305 12.605 24.185 st2 3.44 4.685 18.31 1.33 1.555 19.864 st3 2.515 2.145 16.265 1.54 1.525 26.719 st4 1.2575 1.0725 8.1325 0.77 0.7625 20.212 st5 0.8383 2.145 0.715 0.5133 0.5083 14.024 st6 0.6288 0.5363 4.0663 0.385 0.3813 13.036 st7 0.503 0.429 3.253 0.308 0.305 12.415 may-june (p3) st1 12.1 12.63 15.32 12.11 12.41 29.08 st2 3.24 9.21 21.12 2.32 1.52 19.728 st3 1.78 4.25 9.23 2.04 1.01 26.536 st4 0.89 2.125 4.615 1.02 0.505 20.073 st5 0.5933 4.25 1.4167 0.68 0.3367 13.928 st6 0.445 1.0625 2.3075 0.51 0.2525 12.947 st7 0.356 0.85 1.846 0.408 0.202 12.33 july-august (p4) st1 13.99 14.11 12.99 11.5 13.5 28.06 st2 5.88 6.55 14.36 1.52 3.01 20.656 st3 4.22 5.36 12.36 1.02 1.73 27.785 st4 0.89 2.125 4.615 1.02 0.505 20.073 st5 1.4067 5.36 1.7867 0.34 0.5767 14.583 st6 1.055 1.34 3.09 0.255 0.4325 13.556 st7 0.844 1.072 2.472 0.204 0.346 12.91 september-october (p5) st1 13.44 12.55 11.55 13.01 12.98 24.99 st2 3.69 2.34 11.25 3.66 0.55 18.448 st3 3.65 3.11 15.65 2.98 0.72 24.815 st4 1.825 1.555 7.825 1.49 0.36 18.771 st5 1.2167 3.11 1.0367 0.9933 0.24 13.025 st6 0.9125 0.7775 3.9125 0.745 0.18 12.107 st7 0.73 0.622 3.13 0.596 0.144 11.53 table 3. monitoring stations and their coordinates. 37 int. j. aquat. biol. (2023) 10(1): 34-40 samples with significant temporal and spatial variations. the average concentrations of six heavy metals, including cu, cd, ni, mn, co, and fe were 3.741±4.219, 3.654±4.169, 7.700±6.251, 2.551± 3.898, 2.292±3.996, and 18.236±5.583 µg/l, respectively. all the sampling sites showed an overall rising trend in the presence of heavy metals from upstream (st7) to downstream (st1) (fig. 1). the first station (st1) had the highest average concentrations of the six heavy metals of fe, co, mn, ni, cd, and cu, which were 25.70417, 11.73083, 11.90917, 13.15833, 12.67583, and 13.06667 µg/l, respectively. the concentrations decreased in the order of fe > cu > ni > cd > mn > co. there were significant differences in heavy metals abundance between sampling periods (fig. 2). the fe had the highest concentrations during study periods, and its concentration decreased in the sampling time sampling site heavy metals concentration µg l-1 cu cd ni mn co fe november-december (p6) st1 13.22 11.5 13.33 11.22 9.56 22.55 st2 8.11 1.16 6.22 1.23 1.33 17.856 st3 5.36 0.65 3.17 1.09 1.03 24.018 st4 2.68 0.325 1.585 0.545 0.515 18.168 st5 1.7867 0.65 0.2167 0.3633 0.3433 12.607 st6 1.34 0.1625 0.7925 0.2725 0.2575 11.718 st7 1.072 0.13 0.634 0.218 0.206 11.16 mean 3.741 3.654 7.700 2.551 2.292 18.236 ±sd 4.219 4.169 6.251 3.898 3.996 5.583 table 3. continued figure 1. the distribution of heavy metals in the surface waters of the shatt al-arab river at the study sites. figure 2. distribution of heavy metals in the surface water of the shatt al-arab river at different times. 38 al-najar et al./ short-term assessment of heavy metals in surface waters of the shatt al-arab river following order: p4 > p2 > p5 > p1 > p6 > p3. while nickel was in the following order in terms of concentration: p1 > p2 > p3 > p5 > p4 > p6, as the second rank after fe. for cu, the highest concentration was during periods 1 and 6. the highest concentration of cd was during periods 4 and 3 and the highest concentrations of mn and co were found during periods 4 and 3, respectively. discussion a spatial and temporal distribution of the surface water temperatures of the shatt al-arab river, showed typical seasonal variability, reflecting changes in air temperature and dynamics of the river flow. the ph value did not alter seasonally, but there was some spatial variability along the shatt al-arab river. these values were within the range of the usepa (1999) criterion for surface water (6.5-9). salinity in shatt al-arab is mostly caused by seawater entering the river from iraqi marine waters. salinity variation across seasons and sites can be correlated with flow regimes and seasonal influences (al-asadi et al., 2019). chemical ions entering the river from its major tributaries and sea salt entering from the sea both impact the quality of the shatt alarab river. the mean salinity concentration is due to the loss of most tributaries of the freshwater flow in the river combined with increases in seawater intrusion from iraqi marine waters (moyel and hussain, 2015; abdullah, 2016). thus, the mean salinity values at all studied sites along shatt alarab river were increased from qurna (st7) (5.31 g/l) to fao sea (st1) (34 g/l). these results indicate a tendency toward water stress due to rising water demand by population growth, the development of irrigated land, the building of dams within river basins, and other factors. reduced runoff and increased water evaporation losses have also been caused by climate change. heavy metals are highly persistent in the environment and can be toxic for living organisms. the current study showed that trace metal concentrations are below who recommendations (who, 2011). according to the salinity, the river can be divided into two regions. these regions could also be easily recognized due to their heavy metal concentrations. extremely low heavy metals were found in the first region, which comprised the qurna (st7), harth (st6), sidebaed island (st5), and basrah city (st4). the second region, which includes abu al-khaseeb (st3), fao city (st2), and fao sea (st1), had the highest concentration. therefore, there was clear evidence that seawater intrusion influenced the levels of heavy metals. salinity, ph, geological environment, and terrestrial runoff are a few variables that might have affected metal changes (raknuzzaman et al., 2016). cd, cu, and pb in lake water can be decreased by raising ph levels because proton binding decreases as ph rises, reducing the metal-binding capacity (tokalioglu et al., 2000). water volume and velocity are positively correlated with the level and distribution of heavy metals in surface water (alloway and steinnes, 1999). heavy metals in the surface water were diluted by the high-water volume and velocity, decreasing their concentration. one of the factors causing a decrease in the concentrations of heavy metals in the surface water is rainwater entering the surface of the water body in a short period (shamsuzzaman et al., 2012). the levels of heavy metals in the shatt al-arab river surface waters during the summer (p3, p4, and p5) showed a slight increase compared to the rest of the year. the shatt al-arab river experiences a major decrease in water volume and velocity during the dry seasons, and the physical and chemical characteristics of the water body differ noticeably from those during the rainy season. while land use patterns and levels of human activity on both banks of the shatt al-arab river have remained mostly unchanged throughout this period, the residual concentration of heavy metals in the surface water has increased dramatically. hence, to monitor and assess local ecological and environmental conditions, it is essential to know the spatial distribution of heavy metals in surface water (mohiuddin et al., 2011). in conclusions, the primary causes of the salinity 39 int. j. aquat. biol. (2023) 10(1): 34-40 of shatt al-arab waters are from marin water. this work provided baseline data on some toxic heavy metal concentrations in the surface water of this river at various times and locations following seawater intrusion. the results showed that heavy metal concentrations are below who recommendations. all the sampling sites showed an overall rising trend in the presence of heavy metals from upstream (st7) to downstream (st1). furthermore, there is a correlation between seawater intrusion and the concentration of heavy metals in the surface waters, with concentrations highest at sites with the highest salinity levels. references abdallah d.a. (2016), modelling approach to understanding salinity variation in a highly dynamic tidal river, the case of the shatt al arab river, phd dissertation, university of technology, delft, netherlands. 210 p. abduljaleel h.y., schüttrumpf h., azzam r. (2020). a gis-based water quality management for shatt alarab river system, south of iraq (no. rwth-202009237). lehrstuhl für ingenieurgeologie und hydrogeologie, rwth-2020-09237. al-asadi s.a., alhello a.a. (2019). general assessment of shatt al-arab river, iraq. international journal of water, 13(4): 360-375. al-aboodi a.h., abbas s.a., ibrahim h.t. (2018). effect of hartha and najibia power plants on water quality indices of shatt al-arab river, south of iraq. applied water science, 8(2): 1-10. aldoghachi a., jasim m. (2022). assessment of metals contents, petroleum hydrocarbons and physicochemical parameters in shat al-arab river. egyptian journal of aquatic biology and fisheries, 26(3): 7586. douabul a.a.z., maarofi s.s.a., al-saad h.t., alhassen s. (2013). gaseous pollutants in basra city, iraq. air, soil and water research, 6: aswr-s10835. alloway b.j., steinnes e. (1999). anthropogenic additions of cadmium to soils. cadmium in soils and plants. pp: 97-123. apha. (2005). standard methods for the examination of water and wastewater. american water works association and water environment federation, 21st ed.; american public health association: washington, dc, usa. karamouz m., szidarovszky f., zahraie b. (2003). water resources systems analysis. lewis publishers is an imprint of crc press llc. 608 p. lafta a.a. (2014). computer model and empirical models for prediction of salinity intrusion in estuaries, shatt al-arab estuary as a case study. basra science journal, 40(3): 161-147. lermi a., sunkari e.d. (2020). geochemistry, risk assessment, and pb isotopic evidence for sources of heavy metals instream sediments around the ulukışla basin, niğde, southern turkey. turkish journal of earth sciences, 29(7): 1167-1188. liu l., li w., song w., guo m. (2018). remediation techniques for heavy metal-contaminated soils: principles and applicability. science of the total environment, 633: 206-219. liu z., wang l., yi x., mao q., liu y. 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(2015) 3(2): 60-67 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article effect of dietary nanosilver on gut proteases and general performance in zebrafish (danio rerio) biplab sarkar1,2, manoj kumar1, suresh verma1, raja mansingh rathore*1,3 1school of biotechnology, kiit school of biotechnology, kiit university, bhubaneswar 751024, india. 2national institute of abiotic stress management, malegaon, baramati, pune, india. 3nutrimar as, kiit school of biotechnology, kiit university, bhubaneswar 751024, india. article history: received 15 december 2014 accepted 22 january 2015 available online 2 5 april 2015 keywords: silver nanoparticle fish feed zebrafish protease enzymes metalloprotease abstract: the present study was conducted to evaluate the effect of dietary inclusion of silver nanoparticle on general performance and digestive proteases in zebra fish. four experimental diets were designed with a concentration of 5, 20 and 40 parts per billion (ppb) nanoparticles in the diet (named s5, s20, and s40, respectively) and control group without any nanoparticles (s0). there was no significant difference was observed in fi and fcr between the dietary treatments. at the end of the experiment, s20 exhibited highest wg and sgr followed by s40 compared with other treatments while, there was no significant difference observed between s0 and s5. similar trends were also observed in total protease enzyme activity. to evaluate the protease enzyme patterns on gut extract, substrate sds-page was performed and the inhibition of zymogram was studied. the results showed that there was no difference in banding patterns between s0 and s5 with edta treated samples whereas two extra bands of molecular weight (mw) 67 and 37 appeared in s20 and s40, were inhibited by edta indicating the presence of metalloprotease in those dietary regimes. there were no differences in the banding patterns of pmsf treated samples suggesting that the total serine protease remains unaffected by the dietary regimes. to conclude, we found 20 ppb inclusion of silver nanoparticle in fish diet improves general performance and induces metalloprotease activity in fish. further detailed study is required before establishing dietary inclusion of silver nanoparticle for industrial purposes. introduction over the years, technological applications in aquaculture have been associated with intensification of the applied systems for increased production with economic profitability. besides high density culture systems, efforts are also being made to achieve high-growth performances and early weaning by shortening productive cycles. especially in a situation of juvenile stocking, the animals are often subjected to focus all their physiological resources for high performances thereby making them more sensitive to infection due to different pathological and environmental factors. the uses of antibiotics as a growth promoter in basal diets are in practice, particularly in case of monogastric animals * corresponding author: raja mansingh rathore e-mail address: r.m.rathore@gmail.com (cromwell, 1991). however, continuous use of antibiotics leads to its retention in animal tissues, which may provoke antibiotic resistance both in animals and consumers thereby raising questions for food security. in this scenario, dietary inclusion of trace elements and nanoparticles is an ideal option, where levels of inclusion must be enough to satisfy metabolic needs and most importantly must be within the permissible limits of tissue retention and environmental hazards. nanoparticles received considerable attention in the recent years because of their ability to deliver a wide range of molecules to the body and for a sustained period of time. so far, several nanoparticle-based therapeutic and diagnostic agents have been 61 sarkar et al./ dietary nanosilver and gut proteases developed (chakraborty et al., 2013). historically, silver compounds are in practice for controlling microbial proliferation. the modern-day applications of silver compounds are mostly for medical (antimicrobial agent in wound dressing, catheters, etc.) and as domestic appliances. silver nitrate is unstable and can be toxic to tissues (atiyeh et al., 2008) whereas nanosilver is a submicronic and colloidal form of metallic silver (1-100 nm size) which has presently got wide commercial attention (rai et al., 2009) due to its pronounced impact than bulk silver metal as antimicrobials (mohanty et al., 2012; sarkar, 2010). it is more stable to hydrochloric acid in the gut and absorbed at a much lower extent by cells. therefore, it is minimally toxic and exhibits a higher antimicrobial effect (choi et al., 2008). one of the studies shows silver nanoparticles is more effective against gram-positive and gram-negative pathogens as compared with acid-fast bacteria. furthermore, silver nanoparticles are not cytotoxic to macrophages at the bactericidal concentration and can augment intracellular killing potential of macrophages (mohanty et al., 2012). despite its potential effect on digestive microbial biodiversity and function, roles of nanosilver in relation with growth, immunological status, digestive enzyme activity, metalloproteinase regulation and intestinal structure of farmed animals are already published (wright et al., 2002). silver nanoparticle can cross the cell membranes and penetrate into the tissues to perform biological activities. however, this depends upon pathological and immunological status of the subject, size, structure and purity of the particle, dose and the method of delivery. some authors suggest that silver promotes an increase of zinc and copper concentration over epithelial tissue, thus indirectly stimulating its positive effects for metabolism (lansdown, 2002). there is very limited information on dietary application of silver nanoparticles in fish feed. therefore, this is a timely approach to evaluate the encouraging effects of dietary nanoparticles within nontoxic levels of administration. aim of this study was to verify the effects of the nanosilver in the feed for zebra fish juveniles. the main purpose was to evaluate the general digestive performances during the fast grow-out phase of the fish to provide basic information for use of nanosilver for nutritional research. in an ecotoxicological assessment study, 75% survival rate was observed for zebra fish even at dietary inclusion of 0.25 ppm (parts per million) nanosilver for 15 days (perello, 2013). even if there are no reports by now; however, there might be a risk of bio-magnification in such concentration and further research is required to verify. in this scenario, dietary concentrations in ppb (part per billion) can be considered as nontoxic level and may retain beneficial effects of nanoparticles. in one of the pathogenecity tests conducted at our laboratory, we observed that the zone of inhibition against gramnegative aeromonas hydrophila were evident from 5 ppb nanosilver concentration (unpublished data). for the present study, we have considered 5 ppb to 40 ppb of dietary inclusions of nanosilver in the diet. zebra fish (danio rerio) is selected for our current study as it not only mimics other aquaculture varieties, but also a preclinical model for many biological studies (hsu et al., 2007; chakaraborty et al., 2009, 2011; chakraborty and agoramoorthy 2010). in addition, genetics and molecular basis of zebra fish have been extensively mapped (hill et al., 2005). hence, any experimental trends, including nutritional exposition exhibited by this fish can be applied to other aquaculture species as well as the vertebrate regime. this research provides basic information about digestive performances of dietary nanosilver as a trace element in the diet. materials and methods the experiment was conducted at the fish rearing facility of kiit school of biotechnology, bhubaneswar, during autumn 2012. zebra fish juveniles (40 days after hatching, length 10.5 ± 1.6 mm) were obtained from local fish supplier, m/saquatech, bhubaneswar. fish were kept in 500 l frp (fiberglass reinforced plastic) tank for two 62 int. j. aquat. biol. (2015) 3(2): 60-67 weeks acclimatization before transferring to experimental tanks fitted with flow through fresh water exchange. after acclimatization, fish were randomly stocked at a density of 100 juveniles in 100 l polyfibre aquaria. three replicates were maintained for each feeding regime with 12-hour light: 12-hour dark photoperiod. the fish were reared in static water tanks with 100% water exchange on every third day. throughout the experiment, the rearing water was constantly supplied with oxygen by air stone diffusers to maintain the dissolved oxygen above 6 mg/l. major water quality parameters were monitored throughout the experiment, and all ethical protocol were followed during the entire experiment (table 1). organically coated silver nanoparticle was procured from sigma aldrich, kolkata, india. the size and shape of the silver nanoparticle (90 nm) were confirmed in scanning electron microscopy (jem 2100, jeol, japan) operating at 300 kv (fig. 1). the nanoparticle was suspended in ultrapure water (milli q) and its concentration was adjusted to 5, 20 and 40 µg/kg in three volumes of 5 ml. an artificial fishmeal based diet (40% protein) was formulated from wheat flour, mustard oil cake, vitamin mixes as per previous studies of rathore et al. (2005). the detailed composition of diets is presented in table 2. the pre-weighed dry ingredients were carefully blended using a laboratory food mixer. the mixtures were the primed with water to yield a suitable mash. moist diets were made into 500 µm pellet size and dried at 40°c in a fan assisted drying cabinet. this diet was treated as control (s0) as three experimental sets were formulated by adding 5 ml of silver nanoparticle in three different proportions of 5 ppb (s5), 20 ppb (s20) and 40 ppb (s40), respectively. the fish were feed 4% of body weight twice a day until visible apparent satiation. leftover feed in the tank were removed by siphoning, and their dry weight was recorded. the difference between initial water quality parameters 0 days 15 days 30 days s0 s5 s20 s40 s0 s5 s20 s40 ph 7.2  0.3 7.30.03 7.20.01 7.30.06 7.10.02 7.10.3 7.10.4 7.10.4 7.20.5 ammonia (ppm) 0.19  0.04 0.140.03 0.200.13 0.250.03 0.310.04 0.220.02 0.260.03 0.280.07 0.230.03 d.o. (ppm) 5.80.2 6.40.6 6.50.34 6.80.27 6.60.19 5.80.22 6.30.37 6.60.29 6.40.5 co2 (ppm) 0.150.02 0.140.04 0.120.03 0.090.02 0.220.03 0.130.03 0.100.04 0.120.01 0.080.04 nitrate (ppm) 0.060.01 0.070.01 0.140.04 0.200.02 0.160.03 0.090.01 0.130.04 0.110.03 0.160.03 alkalinity (ppm) 602.2 735 656 817 726 916.84 726.84 806.84 796.84 table 1. water quality parameters during experimental period. ingredients (g/kg) s0 s5 s20 s40 wheat flour 36 36 36 36 fishmeal 35 35 35 35 corn gluten 15 15 15 15 mustard oil 8 8 8 8 cod liver oila 2 2 2 2 vitamin premixb 2 2 2 2 mineral premixc 2 2 2 2 5 ml nanosilver solution adjusted to g/kg 0 5 20 40 nutritional analysis* crude protein 41.2 40.3 40.1 40.1 total lipid 8.4 7.8 8.2 8.6 ash 14.5 14.2 14.2 14.4 table 2. ingredients and crude nutrient composition of the experimental diets. 63 sarkar et al./ dietary nanosilver and gut proteases administered diet and the unfed diet were used to calculate the feed intake. ten fish were collected randomly from each tank at weekly intervals to estimate the food requirement of the growing juvenile. as general growth performances, the length and weight were measured every week and also for the final sampling at 30 days. fish were collected at 9 a.m., washed properly through a sieve and were immediately frozen at -20°c. the digestive systems of individual fish were removed using a glass plate maintained at 0°c under a dissecting microscope. dissected digestive tracts are pooled (100 mg) and washed in pbs (phosphate buffer saline ph 7.4). protein lysate was prepared in ripa buffer at 4°c. the lysate was centrifuged 15 min at 8000 rpm (4°c) and supernatant collected. total protein was estimated based on bradford et al. (1976). total protease activity was evaluated by using 1% azocasein in 50 mm tris–hcl, ph 7.5 (garcia carreno, 1992). ten microlitres of enzyme extract was mixed with 0.5 ml of buffer (50 mm tris–hcl, ph 7.5), 0.5 ml of substrate solution and incubated for 10 min at room temperature. the reaction was stopped by addition of 0.5 ml 20% trichloroacetic acid and then centrifuged at 14000 x g (11480 rpm) for 5 min. the absorbance of the supernatant was recorded at 366 nm. separation of proteins from extracts was done by 12% sds-page according to laemmli (1970). protein extract (15 µg protein/sample) was loaded into each well and electrophoresis was performed on a mini gel electrophoresis device (g biosciences, noida, india). the molecular weights of the proteins were determined by comparison of mobility with known marker proteins, plotting the log10 of rf values against the molecular weight. the protease composition was studied after separation of proteins by substrate sds-page (garcia-carreno and haard, 1993). enzyme preparations containing 5 mu activity were incubated with inhibitors to determine the class of enzymes in the gut extract of zebra fish juveniles. solutions of serine protease inhibitor pmsf (100 mm) and metalloprotease inhibitor edta (20 mm) were prepared and incubated with enzyme preparation 5 mu activity (1:1) at 25°c for 1 hrs prior to loading into the wells. then they were subjected to substrate sds-page with sample volume adjusted to 15 µl containing 5 mu activity loaded to each well. after electrophoresis, the gel was immersed in a solution of 3% casein in 50 mm tris–hcl, (ph 7.5) for 30 min at 5°c to allow the substrate to diffuse into the gel at low enzyme activity followed by incubation at 25°c for 60 min. the gel was then washed, stained and destained. clear bands (zymograms) were identified as protease activity bands that are compared bands of the enzyme preparation without inhibition. analysis of physico-chemical parameters of control and experimental sets of aquarium were assayed through standard protocol prescribed by apha (1992). feed intake (fi as % body weight) was the mean feed consumption per fish as a percentage of the fish body weight (calculated to intake per day) for the experimental period. specific growth rate (sgr) was estimated according to the formula: sgr = 100 x (ln(w2)-ln(w1))/t, where w2 = final fish weight, w1 = initial fish weight and t = duration of the experiment in days. feed conversion ratio (fcr) was estimated as: fcr = feed intake/ growth, figure 1. sem micrograph of silver nanoparticle used in the study. 64 int. j. aquat. biol. (2015) 3(2): 60-67 (where feed intake is the amount of feed consumed by the fish, and growth is the increase of fish biomass during the same period). biological and analytical data were subjected to oneway analysis of variance (anova) using microsoft excel programme for windows at a significance level of 0.05. results at the end of the experiment, as compared with that of control, s5 treatment did not show any difference in weight gain whereas, s20 and s40 treatments showed 28% and 14% increase in total weight gain (p<0.05), respectively. likewise, 24% and 14% increase in sgr were observed on s20 and s40 groups respectively (p<0.05). however, no significant differences observed on fi and fcr between treatments (table 3). among all the treatments, s20 exhibited better performance in terms of weight gain and sgr compared with the others. the specific protease activity was recorded as 0.598 ± 0.07 units-1protein-1 at the beginning of the experiment. by the end of the experiment, the activity increased 5.6 % in s0 (0.632 ± 0.04 units1protein-1) while s5, s20 and s40 showed 6.3% (0.636 ± 0.04 units1protein-1), 21.7% (0.726 ± 0.02 units1protein-1) and 18.8% (0.711 ± 0.03 units1protein-1) enhancement in activity at the end of experimental period (p<0.05) as compared to that of the beginning of the study. at 30 days of rearing, the protease activity showed no significant difference between s0 and s5 whereas that the fish treated with s20 and s40 showed 14.8 and 12.5% enhancement in activity (p<0.05)compared to that of control group (fig. 2). protease activity bands (zymograms) based on substrate sds-page analysis was compared among control and experimental samples to find out the differences in banding pattern of serine and metalloproteases in the current experiment is presented in table 4. pmsf treated intestinal extract treatments s0 s5 s20 s40 p< wg (mg) 43.0 ± 3.00 43.3 ± 3.32 55.0 ± 3.06 49.67± 4.98 0.05 fi 1.23 ± 0.01 1.24 ± 0.01 1.27 ± 0.01 1.26 ± 0.04 ns fcr 0.03 ± 0.002 0.03 ± 0.002 0.02 ± 0.001 0.03 ± 0.003 ns sgr 0.79 ± 0.04 0.84 ± 0.05 0.98 ± 0.04 0.9 ± 0.07 0.05 wg: average weight gain, fi: feed intake, fcr: feed conversion ratio, sgr: specific growth rate, ns: non significant, *: p< 0.05. table 3. general performance of zebrafish fed with different dietary levels of silver nanoparticle. predicted mw of protease bands control s0 band patterns in pmsf treated samples band patterns in edta treated samples s5 s20 s40 s5 s20 s40 86        73        67     45        40        37     34        24        21        18        molecular weights of bands are calculated by rf migration plot against medium range protein marker. presence of band marked () and inhibited bands are marked (). absences of bands are left blank. table 4. banding patterns of protease zymograms from gut extract of zebra fish treated with pmsf and edta by substrate sds-page. 65 sarkar et al./ dietary nanosilver and gut proteases did not show any significant differences in banding pattern among the treatments. this implies that the status of serine proteases remained unaffected by silver nanoparticles. interestingly, in the intestinal extract of s20 and s40, two extra bands of mw about 66 and 37 kda were observed which were inhibited by edta. this indicates the presence of metalloproteases in these dietary regimes. however, these two protease bands were found absent in s0 and s5 and neither any differences in banding pattern among them was observed. discussion growth and metabolic performances are related to homeostatic processes that may reflect the characteristics of nutrients administered in feed. the current study evaluated the effect of dietary nanosilver on general performance and gut proteases in zebrafish. it has been reported that silver nanoparticles applied into the egg (in ovo) can upregulate the expression of fibroblast growth factor (fgf2) and vascular endothelial growth factor (vegf) that may stimulate satellite cell proliferation and differentiation (hotowy et al., 2012). furthermore, chicken embryos injected with silver nanoparticles affected the expression of genes responsible for muscle development during embryogenesis (sawoz et al., 2012; pineda et al., 2012). growth stimulatory impacts as an additive which ameliorate sanitary profile of animal are inversely proportional to the surrounding environment (cromwell, 1991). as minute level of microbial load and biotic stresses is prevalent at commercial fish farms, the balancing role of silver nanoparticle is believed to be more apparent, which ultimately tends the fish towards homeostasis and growth. though there are no reports available on growth promoting role of colloidal silver in fish, it is noted from in vitro study that amount of coliforms in pig ileal lysate is reduced significantly after treatment with silver nanoparticles whereas no impact is seen on lactobacilli population when the application doses of silver nanoparticle are enhanced proportionately (fondevila et al., 2009). these results predicted the fact that colloidal silver in medium dose can inhibit harmful coliforms but do not influence beneficial lactobacilli which was corroborated also through in vivo study. fondevilla et al. (2009) suggested a numerical increase in daily growth of pig at nanosilver enriched diet. in the current study, silver nanoparticle at a dietary dose 20 and 40 ppb showed an indication of improved growth in zebra fish juveniles as compared to other dietary regimes. it may be possible that the nanoparticle exhibiting a probable antimicrobial activity in the digestive system keeps the fish less prone to pathogenic challenges at the gut which in turn provides better digestive activities. trace elements like zinc and copper have been shown to induce productive performances in farmed animals and higher vertebrates by reducing post weaning diarrhea, affecting pancreatic and intestinal digestive enzymes activities and maintaining morphology of the intestinal mucosa thereby increasing absorption potential of nutrients (zhou et al., 1994; li et al., 2001; hedemann at al., 2006; broom et al., 2006). potentially, silver nanoparticles are also expected to have a similar effect considering the chemical similarity of silver with other metals such as copper. in the present study, the increase in protease activity at 20 ppb and 40 ppb level can be attributed to the possible impact of silver nanoparticles towards digestive performances. figure 2. specific protease activity of zebra fish fed with the different dietary inclusion level of silver nanoparticle at 30 days of rearing. *: significance level p<0.05. 66 int. j. aquat. biol. (2015) 3(2): 60-67 studies related with the role of silver nanoparticles on metalloproteinases regulation have already been described (wright et al., 2002; warriner and burrell, 2005). the topic use of silver promotes an increase of zinc and copper concentration over epithelial tissue, thus indirectly stimulating its positive effects (lansdown, 2002). in the present study, the results from enzyme activity suggested that there is increase in number of metalloprotease related enzymes. the result supports the idea of silver nanoparticles inducing the metalloproteases in zebra fish. however, this study is subjected to further detailed characterization of proteases and the involvement of silver nanoparticles. in conclusion, the present study showed that nanosilver enriched formulated diet can influence the digestive performance in zebra fish. here, a 20 ppb dietary inclusion of silver trace metal based nanoparticle exhibited as suitable in terms of both protease and metallic enzymatic regulations. this also suggests that 20 ppb dose of silver nanoparticles may induce metalloprotease in zebra fish gut. however, its role as a growth promoter and nutrition is subjected to further studies. this information will contribute for further research on fish physiology and nanoparticles as a feed additive. acknowledgement the authors acknowledge department of science & technology, government of india, for financial support to this research. the authors are also thankful to the director, kiit school of biotechnology, kiit university, bhubaneswar, odisha for providing necessary facilities to carry out the experiments. references american public health association (apha), (1992). standard methods for the examination of water and waste water. 18th ed. washington dc, apha. atiyeh b.s., costagliola m., hayek s.n., dibo s.a. 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(2002). early healing events in a porcine model of contaminated wounds: effects of nanocristalline silver on matrix metalloproteinases, cell apoptosis and healing. wound repair and regeneration, 10: 141151. international journal of aquatic biology (2014) 2(6): 325-329 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2014 npajournals. all rights reserved original article some reproductive features of cobitis sp. from dough river in the southern caspian sea basin mahin sheikh*1, rahman patimar1, kiavash golzarianpour2 1department of fishery, faculty of natural resource, university of gonbad kavous, iran. 2department of biology, faculty of sciences, university of gonbad kavous, iran. article history: received 2 april 2014 accepted 30 october 2014 available online 2 5 december 2014 keywords: reproduction caspian sea basin egg diameter fecundity abstract: cobitis sp. is an endemic spined loach species from the dough river in the southern caspian sea basin, (golestan province, iran). to provide some reproductive features of this fish, sampling was performed at monthly intervals throughout the year and 417 individuals were collected. the specimens ranged in total length from 27.1 to 92.9 mm and total weight from 0.11 to 6.79 g. the spawning of spined loach of dough river occurs from march to june. the highest mean value of gonadosomatic index was observed in april as 1.96 for males and 6.61 for females. egg diameter ranged from 0.3 to 1.3 mm, with a mean value of 0.9 mm. absolute fecundity varied from 155.04 to 3212 eggs. fecundity relative to total weight fluctuated from 55.41 to 634.76 eggs g–1. this species is among those spawning early in spring compared to other species of this genus from southern caspian basin. introduction the genus cobitis have three valid species in iran including cobitis linea (heckel, 1849), c. faridpaki (mousavi-sabet, vasil'eva, vatandoust and vasil'ev, 2011) and c. keyvani (mousavi-sabet, yerli, vatandoust, ozeren and moradkhani, 2012). cobitis faridpaki and c. keyvani are found in the southern caspian sea basin. also, the presence of the spined loach c. tenia linnaeus, 1758 has been reported in this basin (abdoli and naderi, 2009). whereas others believe that c. taenia is rather a northern european species and its occurrence in the southern caspian sea basin is unlikely (kottelat and freyhof, 2007). cobitis linea heckel, 1849 is found in the kor river basin and the upper kul river drainage of the hormozgan basin (banarescu and nalbant, 1967; bianco and nalbant, 1980). the members of cobitidae are small benthic freshwater fishes with a wide distribution area covering large parts of eurasia and africa (perdices and doadrio, 1997). spined loach during day time remains buried in sand, mud or dense weed growths, * corresponding author: mahin sheikh e-mail address: m_sheikh66@yahoo.com being active at night, and is mostly solitary (coad, 2012). the loaches achieve sexual maturity in the first (males) or second (females) year of their life (boron and pimpicka, 2000; marconato and rasotto, 1989). since there is no information is available about reproductive biology of dough river population of genus cobitis, therefore this study was conducted to provide some reproductive features of the member of this population in southern caspian sea basin. the taxonomic position of the population of genus cobitis from dough river (gorganrood river basin) is unclear, therefore it is considered as cobitis sp. in the present study. material and methods a total of 417 specimens were collected monthly from march 2012 till february 2013 using electrofishing from dough river (55°44'n, 37°27'e) (fig. 1). in the field, all specimens were immediately preserved in 10% formaldehyde solution and transferred to the laboratory and then their total and standard lengths were measured to the nearest 0.01 326 international journal of aquatic biology (2014) 2(6): 325-329 mm using calipers. total weight and weight of gonads of both sexes were measured with an electronic analytical balance to the nearest 0.01 and 0.001 g, respectively. the gonadosomatic index (gsi) was calculated as gsi=wg/w×100 (nikolski, 1963), where wg is weight of gonad and w total weight, to estimate the spawning season. absolute fecundity (af) was estimated in 43 ovaries. the number of eggs was estimated by the gravimetric method, using three pieces of approximately 0.02 g each from the anterior, medial and posterior positions of both ovarian lobes. the relative fecundity index was calculated as rf = f/tw, where f is absolute fecundity and tw total weight (bagenal and tesch, 1978). to determine the oocyte diameter, the ovaries were preserved in 10% formalin solution. the diameters of 30 ova of each female were measured using a microscope outfitted with an ocular micrometer. all statistical analyses were performed with a significance level of p<0.05 using the spss 17 software package. results in total, 417 specimens of cobitis sp. were caught, ranging in total length from 27.1 to 92.9 mm and total weight from 0.11 to 6.79 g (table 1). the highest mean of gsi (±sd) was recorded 96 ± 1.16 for male and 6.61 ± 8.97 for female in april (fig. 2). the female’s gsi increased during march to april, peaking at the mid of spring and then decreased until august, then showed a slow increase in december. the mean value (± sd) of absolute fecundity was 1132.5 ± 674.66. the absolute fecundity was significantly related to total weight and also gonad weight (p<0.05), (fig. 3). the relative fecundity was 55.4 to 634.8 with a mean (±sd) of 279.5 ± 114.49 per gram body weight. there was no significant figure 1. map of southern caspian basin, dough river. figure 2. monthly change of gsi in male and female of cobitis sp. from the dough river. figure 3. relationship between absolute fecundity and (a) total length (mm) and (b) total weight (g) of female cobitis sp. from the dough river. 327 sheikh et al/ some reproductive features of cobitis sp. from dough river relationship between relative fecundity and length and weigh of specimens (p>0.05). egg diameter ranged from 0.3 to 1.3 mm, with a mean value (± sd) of 0.9 ± 0.88. size distribution of eggs indicated that the majority of oocytes ranged from 0.9 to 1 mm (fig. 4). discussion gonadosomatic index is an indirect method for estimation of spawning time in fishes (biswas, 1993). in spined loaches, this index increases in spring and summer, which are spawning seasons, and low in winter (wootton, 1979). in this study, gsi reached its peak in april in both sexes, which indicates concurrency of sexual maturity. as compared with other species of this genus from southern caspian sea basin, this species is among those spawning early in spring (early spring species). concurrency of the gonadosomatic index peak in males and females is among the characteristic of the population or species of studied southern caspian sea fishes. typically peaks of this index are observed in the fish with time difference (although little), while in this species, peak of the gonad growth occurs concurrently. single peak gonad growth indicates that this species spawns once a year. as borone et al. (2008) reported in c. taenia from klavoj lake, poland from may to july when water temperature is higher than 18.5ºc. a similar time was reported for c. bilineata (marconato and rasotto, 1989) in northern italy and for c. faridpaki and c. keyvani (mousavi sabet et al., 2012) for southern caspian sea basin. in the study conducted by patimar et al. (2011) in siahroud stream on c. cf. satunini species, spawning time were reported to be between april and june, which is similar to spawning time of european loaches i.e. c. elongatoides and c. trichonica. spawning time for eastern caspian sea species of the genus cobitis was from march to june (mousavi sabet et al., 2012) spawning time depends on various factors such as temperature and food supply. among various factors, temperature is the most important determinant of spawning time. in southern caspian sea basin, spawning was reported to occur during a period in which the largest food resources were supplied in the environment for the fish species living in the streams and rivers (abdoli and naderi, 2009). overall gonadosomatic index pattern is similar to that of other species and various populations of loaches, and there is no special difference in this regard. the only difference may be peak time of this index, which is in turn occurs in different months depending on environmental conditions, especially temperature. fecundity is among the important biological indices that indicate broad changes under diverse environmental conditions in different populations. in this study, absolute fecundity has a significant relationship with fish size (length and weight), but there was no significant relationship between sex n tl (mm) tw (g) mean ± sd range mean ± sd range male 289 55.5 ± 0.7 37.7-76.8 1.29 ± 0.49 0.34-3.69 female 128 50.4 ± 1.58 27.1-92.9 1.25 ± 1.39 0.11-6.79 table 1. length and weight (mean ± sd) of males and females of cobitis sp. from the dough river. figure 4. oocyte diameter size frequency distribution for cobitis sp. from the dough river. 328 international journal of aquatic biology (2014) 2(6): 325-329 relative fecundity and length and weigh, which is consistent with the studies conducted by oliva et al. (2002) on c. paludica and patimar et al. (2011) on c. cf. satunini. direct and significant relationship of absolute fecundity with fish sizes in the studied population indicate that the energy allocated to reproduction is directly related to the size (length and weight) of the fish; i.e. by increasing age and consequently length and weight, overall energy allocated to reproduction increases. but lack of a significant relationship between relative fecundity and fish size indicates that increased energetic investment in reproduction per weight or length unit does not follow a special rule. one of the most important cases in examination of reproduction activities and fecundity of the populations is examination of ovum diameter. it seems that increasing food availability in the living environment of fishes in pre-spawning periods has evident effect on increasing weight and ova sizes. in this study, observed ovum varied in size from 0.4 to 1.3 mm. in c. cf. satunini in southern caspian sea basin, ovum diameter was reported in the range of 0.44 to 1.02 mm (patimar et al., 2011), which is similar to the results of our study. the highest absolute fecundity rate in our study (3212) in 5 years old female fish was higher than 1400 ova (lobon et al., 1984), 1235 ova (soriguer et al., 2000) and 1984 ova (oliva et al., 2002) for c. paludica, and 1366 ova for c. faridpaki, and 2211 ova for c. keyvani (mousavi sabet et al., 2012), but lower than 4282 ova for c. taenia (bohlen, 1986) and 4666 ova for c. cf. satunini (patimar et al., 2011). if combined with increased ovum diameter, high fecundity in the populations indicates the increased energetic investment in reproduction. but if combined with decreased ovum diameter, the increased absolute fecundity indicates high energy allocation to reproduction per weight unit. since relative fecundity had no significant relationship, the latter cannot be easily concluded. but overall increase in reproduction energy in c. faridpaki and c. keyvani can be attributed to the studied population. however, high absolute fecundity in the population studied by patimar et al. (2011) and c. taenia (bohlen, 1986) also indicates that the studied population is in lower rank for allocation of reproduction energy as compared to some populations and/or species. references abdoli a., naderi m. (2009). biodiversity of fishes of the southern basin of the caspian sea. abzian scientific publication, tehran. 242 p. bagenal t.b., tesch f.w. (1978). age and growth. in bagenal t.b. methods for assessment of fish production in freshwater, 3nd. blackwell scientific publication, london. pp: 165-201. banarescu p., nalbant t. (1967). the 3rd danish expedition to central asia. zoological results 34. cobitidae (pisces) from afghanistan and iran. videnskabelige meddelelser fra dansk naturhistorisk forening. pp: 149-186. bianco p.g., nalbant t. (1980). re-description of cobitis linea, with some remarks on the subgenus bicanestrinia (cypriniformes: cobitidae). copeia, 4: 903-906. bohlen s.r., dollase w.a., wall v.j. (1986). calibration and applications of spinel equilibria in system feo2al2o3sio2. journal of petrology, 27: 43-56. boron a., pimpicka e. (2000). fecundity of spined loach, cobitis taenia from the zegrzynski reservoir, poland (osteichthyes, cobitidae). folia zoological, 49: 135140. coad b.w. (2012). freshwater fishes of iran. from: www.briancoad.com. retrieved 4/10/2012 kottelat m., freyhof j. (2007). handbook of european freshwater fishes. kottelat cornol, switzerland and freyhof, berlin, germany. 646 p. lobon-cervia j., zabala a. (1984). observation on the reproduction of cobitis paludicola de buen, 1930 in the jarma river. cybium, 8: 63-68. marconato a., rasotto m.b. (1989). the biology of a population of spined loach, cobitis taenia (l.). bollettino di zoologia, 56: 73-80. mousavi sabet h., vasil’eva e.d., vatandoust s., vasil’ev v.p. (2011). cobitis faridpaki sp. nova—a new spined loach species (cobitidae) from the southern caspian sea basin (iran). journal of ichthyology, 51(10): 925-931. mousavi sabet h., yerli s.v., vatandoust s., cevher 329 sheikh et al/ some reproductive features of cobitis sp. from dough river özeren s., moradkhani z. (2012). cobitis keyvani sp. nova—a new species of spined-loach from south of the caspian sea basin (teleostei: cobitidae). turkish journal of fisheries and aquatic sciences, 12: 7-13. mousavi-sabet h., kamali a., soltani m., bani a., esmaeili h.r., khoshbavar rostami h., vatandoust s., moradkhani z. (2012). reproductive biology of cobitis keyvani (cobitidae) from the talar river in south of the caspian sea basin. iranian journal of fisheries sciences, 11(2): 383-393. nikolsky g.v. (1963). the ecology of fishes. academic press, london. 352 p. oliva-paterna f.j., torralva m.m., fernández-delgado c. (2002). age, growth and reproduction of cobitis paludica in a seasonal stream. journal of fish biology, 60: 389-404. perdices, a., doadrio i. (1997). phylogenetic relationships and classification of the genera cobitis and sabanejewia (cobitidae) based on allozyme data. ninth international congress of european ichthyologists (cei9) "fish biodiversity", italy. book of abstracts, p: 71. soriguer m.c., vallespin c., gomez-cama c., hernando j.a. (2000). age, diet, growth and reproduction of a population of cobitis paludica (de buem, 1930) in the palarncar stream (southwest of europe, spain) (pisces: cobitidae). hydrobiology, 436: 51-58. int. j. aquat. biol. (2018) 6(5): 288-293 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article food risk of some heavy metals for adults and children via consumption of fish species: euryglossa orientalis, argyrops spinifer and sillago sihama samira mirmohammadvali, eisa solgi*1 department of environment, faculty of natural resources and environment, malayer university, malayer, iran. s article history: received 8 august 2018 accepted 25 october 2018 available online 2 5 october 2018 keywords: persian gulf shif island heavy metals thq abstract: this study aimed to investigate the concentration of some heavy metals in three fish species with high consumption in shif island, bushehr province, and calculation of the estimated daily intake (edi), estimated weekly intake (ewi), target hazard quotients (thq) and hazarded index (hi). three species viz. euryglossaorientalis, argyrops spinifer, and sillago sihama were collected from the bushehr coastal water using fishing boat. after the transfer of samples to the laboratory, the heavy metals were extracted and analyzed using atomic absorption spectrophotometer (aas). based on the results, the average concentrations of fe, zn, cu, mn and ni in e. orientalis were 10.02, 8.33, 1.18, 0.80, and 0.86 mg/kg, in a. spinifer 7.25, 5.75, 0.74, 0.43, and 0.37 mg/kg, and in s. sihama 6.20 , 8.27, 0.60, 0.47, and 1.28 mg/kg, respectively. daily and weekly intake values in all three studied species in the group of children were higher than the adult group. the highest and lowest daily and weekly intake rates were observed for fe in the e. orientalis and cu in a. spinifer. the target hazard quotient (thq) and hazard index (hi) for both adults and children showed less than 1. also, the comparison of metal concentrations showed that the concentration of mn in all three species and ni in e. orientalis and s. sihama were higher than the who standard. introduction heavy metals are dangerous pollutants in our natural environment due to their toxicity. when organisms are exposed to high metal levels in an aquatic environment, they can absorb the available metals directly from the environment, contaminated water and food, and thus, accumulate them in their tissues. further they can enter the food chain and extend the problem to humans (jovanović et al., 2017). fish is an important part of the human diet and therefore in many studies, the contamination of various tissues of fish by metals has been investigated (baramaki et al., 2012). in different regions of the persian gulf, oil pollution, along with other urban, agricultural and industrial pollution has degraded this valuable ecosystem and their resources such aquatic species are at risk of contamination (pourrang et al., 2005). bushehr area has particular economic importance due to having about 800 oil platforms and traffic about 25000 oil tankers each year (hosseini et al., 2014). humans are exposed to heavy metals through *correspondence: eisa solgi doi: https://doi.org/10.22034/ijab.v6i5.473 e-mail: e.solgi@malayeru.ac.ir various pathways, primarily via the food chain. metals such as iron, zinc, and copper are essential to the human body. these elements are cofactor of many enzymes and the human body needs a specific concentration of these elements (iron 8-18 mg/day, copper 0.9 mg/day, zinc 8-11 mg/day and nickel 0.5 mg/day). however, outside of this range, the effects of deficiency and toxicity may be observed (singh et al., 2006; fao, 1963). risk assessment is a process that probability and magnitude of damage, loss, or damage from a hazard and potential health hazard are estimated (yeganeh, 2012). the general objective of risk assessment is to pay attention to the status of soil, air, water or sediment contamination, investigation of all possible exposure ways of the studied organisms to contamination, estimating the amount of pollutants in body organisms and determination the adverse effects (mansouri et al., 2012). the risks of heavy metals are mainly divided into carcinogenic and noncarcinogenic effects. in an assessment of the non289 int. j. aquat. biol. (2018) 6(5): 288-293 carcinogenic effects of heavy metals, a function called risk ratio (thq, target hazard quotient) is used. thq is a ratio of a concentration of heavy metal content in the tissue to its rfd. the oral reference dose (rfd) is the daily exposure of persons to pollutants or toxins that can pose no appreciable risk during their lifetime. the unit of rfd is usually mg/kg body weight/day. the thq values below 1 show that there is no non-carcinogenic health risk to the consumer. albeit, if the frequency and extent of exposure to contamination are increased, the probability of adverse effects will be increased (yeganeh, 2012). with increasing population and subsequently increase in per capita fish consumption, human exposure to heavy metals has increased due to fish consumption. therefore, determination of the concentration of these metals and providing a suitable strategy for consuming food containing these pollutants, is important (zazouli et al., 2013; guang et al., 2015). many studies have been carried out on the risk assessment of heavy metals (zheng et al., 2007; kalani and riazi, 2014; who, 1985). sewage and oil spill from tankers are the sources of pollution in the coastal area and fish caught from these waters polluted with heavy metals. therefore, this research was conducted to determine the risks of heavy metals for humans associated with fish consumption in three fish species, including euryglossaorientalis, argyrops spinifer, and sillago sihama materials and methods study area: shif island is one of the small islands in the bushehr province. it locates 12 km northwest of the bushehr city and 6 km north of the bushehr port (29°4ʹ12"n, 50°53ʹ25"e). sampling: a total of 30 fishes (10 specimens of each species) of e. orientalis, a. spinifer and s. sihama were collected. fishes after collection were stored in ice and transported to the laboratory. then, they were washed with deionized water to remove external contamination. after the biometric characteristics (weight, length, and sex) were recorded, the muscle samples of fish species were separated. each sample was dried at 105°c until to reach a constant weight. chemical analysis: two grams of each muscle sample was digested with nitric acid and perchloric acid (4:1) at 40°c for 1 hour and 140°c for 3 hours (yap et al., 2006). the digested samples were filtered with whatman filter paper no. 42 and then diluted to 25 ml with deionized distilled water and analyzed for heavy metals using the atomic absorption spectrophotometer (aas). for the heavy metals standard solutions, the calibration curves fit was r2 = 0.9925-0.9976, that showed the extreme linearity. estimated daily intake and estimated weekly intake of heavy metals: estimated daily intake (edi) of studied heavy metals from the consumption of the three species was calculated according to following formula: edi= (cm× ir) / bw where estimated daily intake (edi) (mg/kg-day) is the edi of heavy metal, c (mg/kg) is the mean concentration of heavy metal, ir (g/day) is the amount of fish consumption which is taken as 20 g/day based on banagar et al. (2015), and bw (kg) is the body weight of the consumer (70 kg for adult). the estimated weekly intake was calculated by multiplying the daily intake of the heavy metal in the number of days of the week according to the following formula: ewi=edi× number of days of the week ptdi= provisional tolerable daily intake ptwi= provisional tolerable weekly intake target hazard quotient: thq (target hazard quotient) is the ratio between exposure and reference oral dose (rfding) and it is usually applied to show the risk of non-carcinogenic effects, which are calculated based on the following formula: thq = ef×ed×ir×c / rfd×bw×at where thq is target hazard quotient, ef = frequency of exposure (365 days per year), ed = total exposure time (60 years), rfd = reference dose (mg/kg/day) and at = average days (ef × ed). hazard index (hi): hazard index (hi) was calculated by summing all the calculated thq values of heavy metals in fish for each species. 290 mirmohammadvali and solgi / food risk of some heavy metals in fishes for adults and children hi= ∑ 𝑇𝐻𝑄 = 𝑇𝐻𝑄𝑐𝑢 + 𝑇𝐻𝑄𝑓𝑒 + 𝑇𝐻𝑄𝑧𝑛 + 𝑇𝐻𝑄𝑚𝑛 + 𝑇𝐻𝑄𝑛𝑖 statistical analyses: the data were statistically analysed with spss version 22 statistical package programs. the shapiro-wilk test was used to analyze the normality of data distribution. results the heavy metals concentration in fishes is shown in table 1. the highest and lowest concentrations of metals in e. orientalis, a. spinifer and s. sihama were fe-mn, fe-ni and znmn, respectively. the estimated daily intake values for both adults and children are presented in table 2. according to the results of daily intake values, in the adult group for cu and zn, the ascending line was a. spinifer < s. sihama < e. orientalis, for fe s. sihama < a. spinifer < e. orientalis, for mn s. sihama < e. orientalis < a. spinifer and for ni a. spinifer < e. orientalis < s. sihama, and for children group, the corresponding values for fe, cu, zn and ni were as follows a. spinifer < s. sihama < e. orientalis and for mn s. sihama < e. orientalis < a. spinifer. estimated weekly intake for both adults and children are shown in table 3. the highest amount of weekly intake in adults (24.99) and children (96.74) was found for fe in the e. orientalis. also, the lowest weekly intake values of cu in both adults and children were 1.05 and 1.4 in a. spinifer, respectively. the values of thq and hi (fe, zn, cu, mn and ni) for three studied fish species are given in table 4. according to the results, the highest and lowest risk indices are for s. sihama and a. spinifer. the target hazard quotient and hazard index for all metals were lower than 1. therefore there was no a potential health risk for adults and children via the consumption of table 1. concentration of fe, zn, cu, mn and ni (μg/g/ww) in muscle tissue of euryglossa orientalis, argyrops spinifer and sillago sihama in the shif island. muscle mn zn fe mn ni euryglossa orientalis 1.18 8.33 10.02 0.80 0.86 argyrops spinifer 0.74 5.75 7.25 0.43 0.37 sillago sihama 0.60 8.27 6.20 0.47 1.28 table 2. estimated daily intake (mg/ kg /day) of heavy metals (fe, zn, cu, mn and ni) for muscle tissue of euryglossa orientalis, argyrops spinifer and sillago sihama consumed by adults and children and the provisional tolerable daily intake (epa, 2005). edi fish species cu fe zn mn ni adults euryglossa orientalis 0.28 3.57 2.97 0.42 0.30 argyrops spinifer 0.15 2.58 0.26 2.05 0.16 sillago sihama 0.16 2.21 2.95 0.21 0.45 children euryglossa orientalis 1.10 13.82 11.48 1.62 1.18 argyrops spinifer 0.20 3.55 0.35 2.82 0.22 sillago sihama ptdi 0.64 40 8.55 500 11.40 300 0.82 140 1.76 table 3. estimated weekly intake of heavy metals (fe, zn, cu, mn and ni) in muscle tissue of euryglossa orientalis, argyrops spinifer and sillago sihama (adults and children) (mg/kg/week) and the provisional tolerable weekly intake (epa, 2005). ewi fish species cu fe zn mn ni adults euryglossa orientalis 1.96 24.99 20.79 2.94 2.10 argyrops spinifer 1.05 18.06 1.82 14.35 1.12 sillago sihama 1.12 15.47 20.65 1.47 3.15 children euryglossa orientalis 7.7 96.74 80.36 11.34 8.26 argyrops spinifer 1.4 24.85 2.45 19.74 1.54 sillago sihama ptdi 4.48 280 59.58 3500 79.80 2100 5.74 980 12.32 291 int. j. aquat. biol. (2018) 6(5): 288-293 these species. discussion as fish is an important part of the human diet, it is often deemed as the most suitable object among the bioindicators of an aquatic ecosystem. in recent years, fish consumption has increased significantly. similarly, the concentration of heavy metals has increased in fishes (ullah et al., 2017). in this study, the highest concentrations of zn, cu, fe and mn in fish were observed in e. orientalis. concerning the e. orientalis is benthic fish species that feed on sediments and benthic invertebrates tend to accumulate the highest concentrations of heavy metals in comparison to other species. in the case of ni, the highest concentrations were found in s. sihama and the lowest in a. spinifer. henry et al. (2004) investigated heavy metals in four fish species from the french coast, and concluded that the e. orientalis had the highest levels of cadmium, copper and lead in agreement with our findings. differences in the heavy metal concentrations are observed among the fish species depend on feeding habits, age, size, and length of fish, and habitat. considering the per capita fish consumption in the south of the country (20 gr/day) and the mean weight of adults and children (70 and 14.5 kg, respectively), the highest daily and weekly intake in adult group for fe, zn and cu were observed in the e. orientalis, while for mn in a. spinifer and ni in s. sihama. for the children group, the highest daily and weekly intake for fe, zn and cu were found in e. orientalis, for mn in a. spinifer fish and ni in s. sihama. generally, the daily and weekly intake rates of the studied metals in children were higher than adults, except for mn in a. spinifer and ni in all species, which adults were a higher than children. the results are in agreement with shahri et al. (2017) findings that studied ni, pb, cd and zn in the muscle of four fish species of in the chabahar region with highest daily intake of metals in children than adults. the risk of heavy metals due to the consumption of marine products is often calculated by thq. thq is based on the ratio of the metals in the food stuff to the reference amount (rfd) of those metals. if the amount is less than 1, it indicates a lack of food risk and vice versa, if more than 1, represents a risk of food intake (bajgiran et al., 2015). the thq of fe, cu, zn, and mn for the adults and children were less than 1 value. also, the thq for all of the metals, except mn in a. spinifer and ni in three other species, was higher for children compared with adults. in the study of table 4. target hazard quotient (thq) and hazard index (hi) values of heavy metals through fish consumption in the shif island. thq hi fish species cu fe zn mn ni adults euryglossa orientalis 0.039 0.005 0.009 0.003 0.015 0.039 argyrops spinifer 0.026 0.003 0.0008 0.014 0.006 0.026 sillago sihama 0.039 0.003 0.009 0.001 0.022 0.039 children euryglossa orientalis 0.151 0.019 0.036 0.011 0.058 0.151 argyrops spinifer 0.099 0.014 0.026 0.007 0.025 0.099 sillago sihama 0.159 0.012 0.038 0.005 0.088 0.159 table 5. comparison of fe, cu, zn, mn and ni concentrations in the muscle tissue of euryglossa orientalis, argyrops spinifer and sillago sihama with global standards (µg/g/ww). ni mn zn fe cu reference who 0.6 0.5 75 100 3 (who, 1985) maff 50 30 (maff, 1995) fao 40 100 30 (fao, 1963) nhmrc 150 10 (maher, 1986) euryglossa orientalis 0.86 1.18 8.33 10.02 0.80 present study argyrops spinifer 0.37 0.74 5.75 7.25 0.43 present study sillago sihama 1.28 0.60 8.27 6.20 0.47 present study 292 mirmohammadvali and solgi / food risk of some heavy metals in fishes for adults and children wang (2005) that health risk of heavy metals through the consumption of vegetables and fish in tianjin, china was assessed, the thq in children was 1.5 to 3.5 times higher than the adults. also, the hazard index (hi) of these metals was less than 1, indicating no health risk from the intake of these species for the consumer. karimi et al. (2014) examined the concentrations of chromium, nickel, zinc, and copper in the muscle and skin tissues of two edible fish species of alosa caspica and clupeonella cultiventris caspia in the southern caspian sea and indicating consumption of these fish species with a current rate of contamination is not a risk for consumers. ullah et al. (2017) studied heavy metals in 8 species of fish and the implications for human consumption in bangladesh. according to the results, the consumption of 140 gr/ week for the adult is not prohibited. in this study, to assess the risk of heavy metal accumulation, these values were compared with the standards in this field. the results showed that the concentrations of heavy metals of cu, fe and zn were lower than global values, but the concentration of mn in each of the 3 species, as well as the concentration of ni metal in e. orientalis and s. sihama, was higher than the who standard. considering that the fish species studied are in high-consumption in shif and bushehr, the concentration of metals such as mn and ni in these species is higher than who standard, it is necessary to manage the exposure of this metals around shif island and manage its input sewage. acknowledgments the authors gratefully acknowledge funding provided for this research by the malayer university of iran. also the authors are grateful to mr. mirshahvalad responsible of the central laboratory of malayer university. references alipour h., pourkhabbaz a., hassanpour m. 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(2018) 6(5): 288-293 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی کفشک، شانک و شورت فلزات سنگین در گروه کودکان و بزرگساالن مصرف کننده ماهی ریسک غذایی برخی *سلگی عیسی ،میرمحمدولی سمیرا ایران. مالیر، مالیر، دانشگاه زیست، محیط و طبیعی منابع دانشکده زیست، محیط گروه چکیده: ، (edi) گونه از ماهیان پرمصرف در جزیره شیف )استان بوشهر( و تخمین جذب روزانه 3سنگین در برخی عناصر غلظت این تحقیق با هدف بررسی های انجام شد. در این پژوهش سه گونه کفشک، شانک، شورت از آب (hi)( و شاخص خطر thq) ( و برآورد پتانسیل خطرewi) جذب هفتگی ه ها به آزمایشگاه عناصر سنگین مربوطه استخراج شده و با استفادبرداری شد. پس از انتقال نمونهساحلی استان بوشهر با استفاده از قایق صیادی نمونه با توجه به نتایج میانگین غلظت فلزات آهن، روی، مس، منگنز گیری شد.اندازه contraa700 مدل analytik jena)شعله(از دستگاه جذب اتمی ، 60/0، 27/8، 20/6( و شورت )37/0و 43/0، 74/0، 75/5، 25/7(، شانک )86/0و 80/0، 18/1، 33/8، 02/10) ی کفشکترتیب در گونهو نیکل به . بیشترین و کمترین بودگونه ماهی در گروه کودکان باالتر از گروه بزرگساالن 3مقادیر جذب روزانه و هفتگی در هر .دست آمدهب( 28/1و 47/0 ی کفشک و فلز مس در گونه ی شانک مشاهده شد. تخمین پتانسیل خطر و ترتیب مربوط به فلز آهن در گونهمقادیر جذب روزانه و هفتگی به بود. همچنین مقایسه 1گونه مقادیر پتانسیل خطر و شاخص خطر کمتر از 3الن و کودکان نشان داد که در هر شاخص خطر برای دو گروه بزرگسا های کفشک و شورت باالتر از استاندارد گونه و غلظت فلز نیکل در گونه 3غلظت فلزات با استانداردهای جهانی نشان داد که غلظت فلز منگنز در هر who دست آمد.همقادیر پایین تر از استانداردهای جهانی ب اما در سایر عناصر است .خطر ارزیابیجزیره شیف، فلزات سنگین، خلیج فارس، :کلمات کلیدی int. j. aquat. biol. (2023) 11(1): 11-19 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article some trace elements in the water, sediments and muscles of three fish species along the tigris river in misan, southern iraq majida sabah al-enazi1, israa ibrahim lazim2 1department of biology, college of education for pure science, university of basrah, basrah, iraq. 2department of biology, college of science, university of misan, misan, iraq. s article history: received 11 august 2022 accepted 14 october 2022 available online 2 5 february 2023 keywords: trace elements pollution accumulation bioconcentration factor abstract: this study aimed to study the concentrations of some trace elements viz. pb, ni, cd, co, and fe in the water, sediments and muscle of three fishes of liza abu, mesopotamichthys sharpeyi and carasobarbus luteus in the tigris river by selecting six stations distributed along the misan governorate, southern iraq. the study was conducted during two seasons, winter and summer. the concentrations of the studied trace elements in water followed the following order: fe > ni > cd > co > pb. fe had the highest concentration in the water (28.748), while the lowest onewas pb (0.0523). the results also recorded the trace elements in order of fe > ni > pb > cd > co. the highest concentration of fe in the sediments was 9690.39, and the lowest was cobalt (8.612). the bioconcentration factor (bcf) values in the studied fish species were much higher than the biosedimentation factor (bsf). we conclude that the concentrations of trace elements in the sediments were more than in the waters of the tigris river, as a storage place of pollutants in the aquatic environments. introduction several studies have been conducted on the pollution of rivers with trace elements, and the overcontamination of these elements has become a concern for the health of the aquatic ecosystem (abdullah et al., 2020; lazim et al., 2022). because the trace elements are characterized by their high stability and remain for a long time, and are not affected by other environmental factors. it depends on its ability to form multiple complex compounds. therefore, it is difficult to remove them (agbozu et al., 2007; al-enazi et al., 2020). thus, this pollution causes great environmental stress to aquatic organisms (kapahi and sachdeva, 2019). trace element pollution in aquatic environments has been increasing worldwide in recent years. this is because of human activities, which are the primary source of pollution in the environment, such as the dumping of industrial waste and chemical fertilizers, which play a significant role in the dissemination of correspondence: majida sabah al-enazi doi: https://doi.org/10.22034/ijab.v11i1.1718 e-mail: majida.abdalsaied@uobasrah.edu.iq dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.2.2 trace elements in the aquatic environment (huang et al., 2018; algül and beyhan, 2020). most of these elements reach the ecosystem by dumping directly or indirectly, such as industrial waste dumped into river waters directly without treatment (mulk et al., 2017; al-enazi et al., 2022). or by dumping agricultural residues due to pesticides, chemical fertilizers, and wastewater (al-zubaidi, 2012; salman, 2015; khamar et al., 2018). these minerals are considered among surface water's most critical environmental pollutants because of their accumulation within these ecosystems and their high reliability. they are characterized by the inability to decompose by microorganisms and other life processes. therefore, they constitute a significant threat to human health (alkam and jadan, 2010; al-naggar et al., 2018). high levels of elements in water often lead to elevated concentrations in sediments that are storage places for pollutants (juned et al., 2018). the elements are released from the sediments directly 12 al-enazi et al./ some trace elements in the water, sediments and fishes in tigris river through their release and dissolution through the water column or indirectly by feeding the various benthic organisms or pelagic species, accumulating in these organisms’ tissues (al-najjar, 2015). these elements may accumulate to very high toxic levels and cause multiple effects on aquatic organisms. thus, they pose a potential health risk to consumers (vanden broek et al., 2002; tabari et al., 2010). several studies have shown that trace elements are responsible for many severe and disastrous effects on fish and other aquatic animals (gundogdu et al., 2016). therefore, water and sediment quality monitoring must be performed periodically to assess the risks of these elements (ali et al., 2016; karikari et al., 2020). their effect on aquatic organisms varies depending on their intensity, between chronic effects that symptoms appear after some time and acute effects that cause a reduction in a number of aquatics and change in environmental conditions by impacting the initial stages of growth. the impact of pollution with these elements on living organisms varies according to age, development, and other physiological factors, and also, according to the type of tissue where the metabolically active tissues are the target tissues of these trace elements (olusola and festus, 2015; huseen and mohammed, 2019). many aquatic organisms, including fish, are useful bioindicators of trace element pollution in aquatic environments (al-khafaji and lazim, 2013; sobhanardakani et al., 2018; farhood and ali, 2020). tigris river receives many pollutants, especially trace elements, due to many human activities and factories that throw pollutants directly into the river without any treatment. therefore, the current study aimed to study bioaccumulation of some trace elements (pb, ni, cd, co, and fe) in water, sediments, and muscles of three species of fish found in the river, liza abu, mesopotamichthys sharpeyi and carasobarbus luteus as bioindicators of the pollution along the tigris river in the city of misan. materials and methods the study was conducted on the tigris river in misan governorate, southeast of iraq. this city is characterized by its agricultural wealth, which derives its source from the tigris river and its subsidiary rivers and contains many districts and sub-districts (fig. 1). six sampling sites were selected for the study characterized by different environmental characteristics (fig. 1, table 1). samples were collected from six stations as monthly in the winter and summer of 2021. water samples were collected from the middle of the river using plastic (polyethylene) 5 l bottles with three replicates. a few drops of hno3 were added as a stabilizing agent to stabilize the elements in the water. for the sediment, the samples were collected from the middle of the river using a grab sampler of van veen. they were kept in nylon bags and stored in boxes according to the imrp (2006). fish samples were collected from the study area using gill nets (25 mm mesh) and then placed in an insulated cork container containing ice until they reached the laboratory. the trace elements were extracted from the water after being digested using the method of apha (2005). the trace elements in the sediments were extracted using the method of yi et al. (2007). for fish samples, r.o.p.m.e. (1983) method was used to measure trace elements. preparation of blank solution: it was prepared by the same method for extracting trace elements but without adding any samples to understand the pollution problem resulting from using chemicals during working conditions in the laboratory as correction values. calculation of trace element: the trace elements were calculated using the calibration curve according to usco (1992). trace elements in water were calculated based on the equation of econ. = a×b/c×1000, where econ. = concentration of the element in water (µg/l), a = concentration of the element extracted from the calibration curve (μg/l), b = final volume of the filter sample (ml) and c = initial volume of the filter sample (ml). trace elements in sediments and muscles of fish: they were measured according to the equation of econ. = ab/ d, where econ. = concentration of the 13 int. j. aquat. biol. (2022) 11(1): 11-19 element in the sample (µg/g dry weight), a = concentration of the element extracted from the calibration curve (μg/l), b = final volume of the filter sample (ml), and d = dry weight of the sample (g). bioconcentration (bcf) and biosedimentation factor (bsf): since the purpose of this study is to understand transporting path of trace elements between water, sediments, and fish, the bcf and bsf were used to divide the total concentration rate of each element in fish (a) by its concentration in water (b) and sediment (c), respectively (kumar et al., 2009) using the equations of bcf = a/b, and bsf = a/c, where a = the total concentration of the element in the fish muscles, b = the concentration of the same element in water and c = the concentration of the element in the sediment. statistical analysis: the statistical program of spss was used to analyze the data. the significance of the differences between the means was tested using the least significant difference test (lsd) at the significance level of p≤0.05. results trace elements of water: the highest concentration of pb was 0.16 μg/l in the summer at the third site, and its lowest record was 0.02 μg/l at the first site in the winter (table 2). at the same time, the highest concentration of ni was 0.89 μg/l in the fourth site in summer and below 0.09 μg/l at the same site in winter. the highest cd was 0.69 μg/l in the third site during the summer and the lowest (0.003 μg/l) in the fourth site in winter. the highest co is 0.42 μg/l recorded in the third station in the summer, and the figure 1. the study sites in the tigris river, misan governorate. name y x s1 32.4501 46.7187 s2 31.8481 47.3876 s3 31.8269 47.1456 s4 31.7126 46.9593 s5 31.6077 47.1649 s6 31.5113 47.3423 table 1. coordinates of the geographical location of the study sites. 14 al-enazi et al./ some trace elements in the water, sediments and fishes in tigris river lowest (0.02 μg/l) at the fourth site in winter. the highest fe was 80.92 μg/l in the third site in summer and the lowest (9.43 μg/l) at the sixth site in winter. the highest cumulative rate of iron among the studied elements in water was 28.748, while lead recorded the lowest cumulative rate of 0.0523 (table 5). the concentrations of the studied trace elements appeared in the order: fe > ni > cd > co > pb. trace elements in the sediment: the highest value of lead was 80.22 μg/g at the third site in summer, and the lowest one was 10.04 μg/g at the first site in the winter, and the highest value of nickel was elements sites season t fe co cd ni pb 17.45 16.89 0.04 0.04 0.46 0.02 s1 winter 16.15 15.44 0.05 0.01 0.61 0.04 s2 65.03 63.95 0.09 0.31 0.63 0.05 s3 10.33 10.19 0.02 0.003 0.09 0.03 s4 18.024 17.66 0.076 0.018 0.22 0.05 s5 9.924 9.43 0.047 0.015 0.39 0.042 s6 133.56 0.323 0.396 2.4 0.232 t 22.26 0.053 0.066 0.4 0.0386 av 29.56 28.88 0.03 0.32 0.29 0.04 s1 summer 21.57 20.14 0.1 0.59 0.68 0.06 s2 82.9 80.92 0.42 0.69 0.71 0.16 s3 26.7 25.14 0.03 0.61 0.89 0.03 s4 24.12 23.18 0.4 0.22 0.24 0.08 s5 34.51 33.16 0.3 0.35 0.67 0.03 s6 211.42 1.28 2.78 3.48 0.4 t 35.236 0.213 0.46 0.58 0.066 av 28.748 0.133 0.263 0.49 0.0523 total average table 2. concentrations of the measured trace elements (μg/l) in water. elements sites season t fe co cd ni pb 1186.12 1120.43 1.11 12.55 41.99 10.04 s1 winter 1258.46 1091.93 5.44 12.66 115.31 33.12 s2 20650.54 20442.5 7.45 15.76 144.41 40.42 s3 2075.35 1962.21 4.96 2.33 76.41 29.44 s4 2229.98 2081.11 3.55 11.44 114.66 19.22 s5 11563.27 11431.13 6.12 13.81 95.22 16.99 s6 38129.31 28.63 68.55 588 149.23 t 6354.885 4.938 11.425 98 24.87 av 8916.94 8779.16 1.45 14.91 100.11 21.31 s1 summer 23215.76 23035.96 16.18 15.54 116.14 31.94 s2 9769.2 9435. 55 18.22 19.32 215.89 80.22 s3 12765.8 12562.11 11.25 12.12 133.13 47.19 s4 10319.9 10012.43 16.29 18.14 224.16 48.88 s5 14578.6 14330.16 10.33 9.31 184.39 44.41 s6 78155.37 73.72 89.34 973.82 273.95 t 13025.895 12.286 14.89 162.303 45.658 av 9690.39 8.612 13.157 130.151 35.264 total average table 3. concentrations of the measured trace elements (µg/g dry weight) in sediment. 15 int. j. aquat. biol. (2022) 11(1): 11-19 224.16 μg/g at the fifth site is in summer and the lowest (41.99 μg/g) at the first site in winter. cd had the highest concentration of 19.32 μg/g at the third site in summer and the lowest (2.33 μg/g) at the fourth site in winter. the highest co was 18.22 μg/g at the third site in summer and the lowest (1.11 μg/g) at the first site in winter. the highest fe in the sediments was 23035.96 μg/g in the second station in the summer and the lowest as 1091.93 μg/g in the winter (table 3). the current study recorded the values of trace elements in the sediments of the study sites according to the following pattern: fe > ni > pb > cd > co. the highest cumulative rate of fe in the sediments was 9690.39 and the lowest cumulative rate was for co (8.612) (table 5). trace elements in fish muscle: the highest lead concentration was recorded in c. luteus as 3.44 μg/g in winter and the lowest as 1.13 μg/g in l. abu in winter. the highest ni was 51.11 μg/g in c. luteus during summer and the lowest (29.66 μg/g) in m. sharpeyi during winter. at the same time, cd recorded the highest concentration of 16.87 μg/g in c. luteus during summer and the lowest (7.91 μg/g) in l. abu during winter. for co, the highest value was recorded at 6.76 μg/g in m. sharpeyi during summer, and the lowest value was 3.88 μg/g in l. abu during winter. for iron, the highest concentration was 150.12 μg/g in c. luteus during summer and the lowest (80.33 μg/g) in l. abu in winter (table 4). iron had the highest accumulative rate of 141.145 among the other studied elements in fish, and pb recorded the lowest accumulative rate of 1.5 among the other elements (table 5). bcf and bsf: table 5 shows the bcf values of the three fish species of l. abu, m. sharpeyi, and c. luteus that were higher than the bcf values. discussion the results showed that the highest concentrations of the studied trace elements in the water occurred during summer. the reason for this may be because elements season type t fe co cd ni pb 125.36 80.33 3.88 7.91 32.11 1.13 winter liza abu 151.82 101.55 4.19 9.44 34.77 1.87 summer 90.94 4.035 8.675 33.44 1.5 av 142.28 98.66 3.98 7.99 29.66 1.99 winter mesopotamichthys sharpeyi 171.29 114.26 6.76 10.52 36.93 2.82 summer 106.46 5.37 9.255 33.295 2.405 av 198.81 132.17 5.76 14.32 43.12 3.44 winter carasobarbus luteus 227.16 150.12 5.89 16.87 51.11 3.17 summer 141.145 5.825 15.595 47.115 3.305 av 677.09 30.46 67.05 227.7 14.42 t 112.85 5.077 11.18 37.95 2.40 total average table 4. concentrations of the measure trace elements (µg/g dry weight) in the studied fishes. b s f b c f elements conc. (μg/g) d.w in c. luteus b s f b c f elements conc. (μg/g) d.w in m. sharpeyi b s f b c f elements conc. (μg/g) d.w in l. abu elements conc. (μg/g) d.w in sediment elements conc. (μg/l) in water elements 0. 0 9 4 63 .1 9 3.305 0. 0 6 8 4 5. 9 8 2.405 0. 0 4 2 5 28 .6 8 1.5 35.264 0.0523 pb 0. 3 6 2 96 .1 5 47.115 0. 2 5 6 6 7. 9 5 33.295 0. 2 5 6 9 68 .2 44 9 33.44 130.151 0.49 ni 1. 1 8 5 59 .2 96 15.595 0. 7 0 3 3 5. 1 9 9.255 0. 6 5 9 3 32 .9 84 8 8.675 13.157 0.263 cd 0. 6 7 6 43 .7 96 1 5.825 0. 6 2 4 4 0. 3 8 5.37 0. 4 6 8 5 30 .3 38 3 4.035 8.612 0.133 co 0. 0 1 5 4. 99 141.145 0. 0 1 1 3. 7 0 106.46 0. 0 0 9 4 3. 16 33 90.94 9690.39 28.748 fe table 5. bcf and bsf for the trace elements studied in three types of fish during the study period. 16 al-enazi et al./ some trace elements in the water, sediments and fishes in tigris river of the decrease in the dilution factor of these elements in the water due to the low water levels during the summer and the high temperatures leading to an increase in evaporation rates and an increase in the decomposition processes of organic matter, which causes an increase in the concentration of trace elements in the water (obasohan, 2008; mahmood, 2015). in contrast, the winter season showed the lowest concentrations of all measured elements. this may be attributed to the increase in water levels due to rain or as a result of the change in the activities of some living organisms, such as feeding and reproduction (salman, 2015). most studied elements increased in the summer, especially in the third site, 82.9 μg/l. this is due to the entrance of diverse pollutants in this site that represents the center of the city of misan (saeed, 2014), and the industrial wastewater from some oil facilities whose wastes pour into the tigris river, and the high temperatures in the summer, which led to an increase in the evaporation of water (salman, 2006). the low concentrations of some trace elements in water in the current study may be due to their tendency to accumulate within aquatic organisms (vardanyan et al., 2008) or through their adsorption on the surfaces of sediments, or the formation of complexes when interacting with sulfates and silicates (dhir and kumar, 2010). considering that sediments are the final recipient of all the products of natural and human activities that are presented to the ecosystem and therefore represent the most important environmental indicators of pollution by trace elements that accumulate in them as a result of these activities (saeed and shaker, 2008). increasing concentrations of trace elements in sediments directly or indirectly negatively affect aquatic and benthic organisms because they are in direct contact with the sediments (vanden broek et al., 2002). the concentrations of the studied trace elements in the sediments were higher than their concentrations in the water. this result agrees with bazrafshan et al. (2015), as the concentrations of these elements on the surface are lower than in the deep layers (aradpour et al., 2020). according to the order of trace elements in the sediments, the cobalt had the lowest values (8.612 μg/g), while the highest values were for fe (9690.39 μg/g), and nini (130,151 μg/g). this indicates that these areas are polluted by sewage water from household, industrial and commercial wastes (abaychi and al-saad, 1988; al-enazi, 2014). the reason may be due to trace elements’ tendency to attach the suspended matter, including organic and clay particles, in sediments (kaiser et al., 2004). water's chemical and physical factors affect the sedimentation and adsorption of trace elements in sediments (remoudaki et al., 2003). the presence of higher trace elements in the sediments of aquatic systems is because they represent the main reservoir of those elements and can move from the phase of their presence in the sediment to the phase of their presence in the water column (al-saad et al., 1996). hence, the possibility of its transmission through the food chain by consumption through higher-level organisms such as humans is a crucial concern (abdullah et al., 2007). the accumulation of trace elements in fish negatively affects human health by consuming contaminated fish (fergusson, 1990). according to the results, the concentration of trace elements in the muscles of the three fish species was higher than the permissible limits. therefore, these species of fish can be used as bioindicators of pollution in the tigris river, which agrees with the findings of al-sanjari (2009). the accumulation of trace elements in fish is generally higher than in surrounding waters (olaifa et al., 2004). the high concentrations of trace elements in the muscles of three fish species in the summer are due to the high temperatures that increase their metabolic activities and feeding leading (barsytelovjoy, 1999; vanden broek et al., 2002). differences in the concentrations of trace elements in fishes may be due to differences in the species’ ecological needs, metabolism, age, size, length, and swimming behaviors (demark et al., 2006; al-ghanim et al., 2016). the results indicated that bcf in the fish were higher than bsf, which indicates that the trace 17 int. j. aquat. biol. 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(2017) 5(5): 295-309; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article phytoplankton diversity of floodplain lakes of the majuli river island of the brahmaputra river basin, assam, northeast india bhushan kumar sharma*,1mrinal kumar hatimuria freshwater biology laboratory, department of zoology, north-eastern hill university, permanent campus, shillong-793 022, meghalaya, india. article history: received 25 august 2017 accepted 14 october 2017 available online 2 5 october 2017 keywords: alluvial floodplain abundance composition density tropical wetlands abstract: phytoplankton of three floodplain lakes (beels) of the majuli river island of upper assam, northeast india (nei), sampled during september 2010–august 2012, revealed rich diversity (108 species) with ghotonga > holmari ≥ bhereki beels; richness of chlorophyta and of cosmarium > staurastrum > euastrum in particular. the monthly richness and community similarities affirmed heterogeneity in phytoplankton composition. phytoplankton comprised between 59.5±12.5, 57.1±12.3 and 48.6±13.5% of net plankton abundance of bhereki, holmari and ghotonga beels, respectively. bacillariophyta > chlorophyta showed quantitative importance in bhereki while chlorophyta > bacillariophyta recorded importance in holmari and ghotonga beels. cyanophyta showed sub-dominance and dinophyta > euglenophyta showed low densities in the three beels. phytoplankton richness and abundance followed oscillating monthly variations; anova registered insignificant richness variations amongst beels. the results are characterized certain monthly and annual variations but mean values depicted high species diversity, low dominance and high equitability. individual and cumulative influence (vide cca) yielded limited insight on the role of seventeen abiotic factors on phytoplankton in holmari and bhereki beels. introduction phytoplankton, an integral link of aquatic food-webs, is inadequately analyzed in various studies on the indian floodplain ecology due to incomplete inventories of taxa while the detailed studies on their diversity in these ecotones are yet limited (sharma, 2015). this generalization holds valid for the floodplain lakes which form an important component of inland aquatic resources of northeast india (nei) and the brahmaputra river basin of assam in particular. the fewer notable works on phytoplankton diversity from the former region are from selected floodplain lakes (pats) of manipur (sharma, 2009, 2010) and beels (sharma, 2004, 2012, 2015) of assam. majuli river island, the largest river island and a geographically interesting landform of fluvial geomorphology of the brahmaputra river system of assam state of nei, is dotted with the floodplain lakes (beels) which play an important role in the socioeconomic development of the region through *corresponding author: bhushan kumar sharma doi: https://doi.org/10.22034/ijab.v5i5.347 e-mail address: profbksharma@gmail.com significant fisheries potential. the wetlands of the majuli floodplains remain unexplored for their phytoplankton diversity and thus this study merits biodiversity and ecological importance. the observations are made on monthly variations of richness and abundance of phytoplankton and their constituent groups of three selected beels as well as their community similarities, species diversity, evenness and dominance. the individual and cumulative influence of abiotic factors vis-à-vis monthly variations of richness and abundance are analyzed to understand their ecological importance with reference to phytoplankton assemblages. materials and methods the present study is a part of a limnological survey undertaken during september, 2010–august, 2012 in bhereki (94o08′23.3″e, 26o55′40.4″n; 72 m asl), holmari (94o12′30.6″e, 26o59′17.3″n) and ghotonga (94o15′28.7″e, 27o01′52.7″n, 69m asl) beels of majuli river island located in the jorhat district (fig. 2 sharma and hatimuria/ phytoplankton diversity of floodplain lakes of majuli river island, india 1) of upper assam (nei). various macrophytes noticed in these wetlands included eichhornia crassipes, hydrilla verticellata, utricularia flexuosa, trapa natans, lemna major, l. minor, pistia striates, salvinia sp., nymphaea spp., nymphoides spp., potamageton spp., azolla pinnata, euryale ferox, and sagittaria sp. water samples collected at regular monthly intervals from the selected beels were analyzed for seventeen abiotic factors namely water temperature, rainfall, ph, specific conductivity, dissolved oxygen, free carbon dioxide, total alkalinity, total hardness, calcium, magnesium, chloride, dissolved organic matter, total dissolved solids, phosphate, nitrate, sulphate and silicate. water temperature, specific conductivity and ph were recorded by field probes, dissolved oxygen was estimated by winkler’s method and other parameters were analyzed following apha (1992). monthly qualitative and quantitative net plankton samples were collected by plankton net (# 30 µm) and were preserved in 5% formalin; the former collected by towing plankton net through the littoral and semi-limnetic regions of different beels and the latter by filtering 25 l water each at two sampling stations in each beel. qualitative samples were screened and phytoplankton was identified following islam and haroon (1980), adoni et al. (1985) and fitter and manuel (1986). quantitative samples were analyzed by using a sedegwick-rafter counting cell for enumeration of abundance (nl-1) of phytoplankton and its constituent groups. community similarities (sørensen’s index), species diversity (shannon’s index), evenness (pielou’s index) and dominance (berger-parker’s index) were calculated following ludwig and reynolds (1988) and magurran (1988). the hierarchical cluster analysis based on phytoplankton community similarities was done using spss (version 20). two-way anova was used to analyze the significance of temporal variations of biotic communities. ecological relationships between abiotic and biotic parameters of bhereki, holmari and ghotonga beels were determined by pearson correlation coefficients (r1, r2 and r3 respectively); pvalues were computed and their significance was ascertained after the use of bonferroni correction. the canonical correspondence analysis (xlstat, 2015) was done to analyze cumulative influence of 17 abiotic parameters (water temperature, rainfall, ph, specific conductivity, dissolved oxygen, free co2, total alkalinity, total hardness, calcium, magnesium, chloride, dissolved organic matter, total dissolved solids, phosphate, nitrate, sulphate and silicate) on phytoplankton assemblages. results the variations (ranges, mean±sd) in abiotic parameters of bhereki, holmari and ghotonga beels are indicated in table 1 and that of different aspects of phytoplankton diversity are included in table 2. we observed a total of 108 phytoplankton species, belonging to five groups, with 98, 99 and 103 species from bhereki, holmari and ghotonga beels, respectively (table 2). chlorophyta (79 species) included 69, 71 and 75 species in three beels, respectively and recorded richness of cosmarium > staurastrum > euastrum species. the monthly phytoplankton richness varied between 32-62, 34-71 and 29-60 species (figs. 2, 3); it recorded 42.5-78.3, 38.8-68.2%; 36.3-74.7, 53.3-79.0% and 38.0-76.7, 38.9-79.5% community similarities (vide sørensen’s index) in bhereki, holmari and ghotonga beels during the two years, respectively. the hierarchical cluster figure 1. district map of assam state indicating location of majuli river island (insert map of india showing assam state of northeast india). 3 int. j. aquat. biol. (2017) 5(5): 295-309 factors bhereki beel holmari beel ghotonga beel range mean±sd range mean±sd range mean±sd water temp. oc 21.5-27.5 23.7±1.7 21.0-27.5 23.6±1.7 21.5-27.5 23.9±1.7 rainfall (mm) 0.0-413.7 142.6±133.9 0.0-413.7 142.6±133.9 0.0-413.7 142.6±133.9 ph 6.29-7.41 6.67±0.23 6.56-7.13 6.87±0.13 6.17-6.85 6.51±0.16 conductivity (µscm-1) 102.0-189.0 140.7±24.4 111.0-220.0 173.6±32.5 73.0-182.0 121.4±26.8 dissolved oxygen (mgl-1) 4.8-8.0 6.3±0.9 5.6-8.0 7.1±0.8 4.0-8.0 6.2±1.0 free co2 (mgl-1) 6.0-24.0 13.6±4.0 6.0-16.0 10.2±2.8 6.0-20.0 13.8±3.4 alkalinity (mgl-1) 44.0-126.0 70.3±20.7 64.0-116.0 92.3±14.2 38.0-88.0 62.2±13.4 hardness (mgl-1) 42.0-128.0 69.8±20.3 56.0-122.0 89.3±16.9 38.0-84.0 60.8±13.6 calcium (mgl-1) 27.3 81.9 43.0±13.1 37.8-73.5 60.2±9.2 25.2-54.6 38.7±7.8 magnesium (mgl-1) 1.3-11.9 6.5±2.8 2.2-11.9 7.1±2.4 1.0-11.3 5.4±2.3 chloride (mgl-1) 6.0-33.0 11.0±5.2 4.0-22.0 8.9±3.5 7.0-40.0 13.1±6.5 dom (mgl-1) 0.041-0.319 0.162±0.062 0.026-0.278 0.113±0.047 0.038-0.353 0.166±0.063 tds (mgl-1) 0.088-0.172 0.137±0.023 0.080-0.160 0.115±0.022 0.104-0.180 0.147±0.020 phosphate (mgl-1) 0.145-3.619 0.963±0.697 0.093-1.582 0.761±0.393 0.165-1.499 0.845±0.414 nitrate (mgl-1) 0.501-4.522 1.855±1.047 0.544-4.411 1.800±1.030 0.499-3.566 1.758±0.838 sulphate (mgl-1) 1.387-17.78 8.789±4.161 0.793-14.075 6.473±3.741 0.925-13.282 7.219±3.600 silicate (mgl-1) 0.140-2.652 0.880±0.547 0.140-2.547 0.825±0.511 0.140-1.187 0.660±0.275 table 1. abiotic factors of bhereki, holmari and ghotonga beels (september 2010-august 2012). figure 2. monthly variations in species richness of phytoplankton (2010-2011). figure 3. monthly variations in species richness of phytoplankton (2011-2012). 4 sharma and hatimuria/ phytoplankton diversity of floodplain lakes of majuli river island, india analysis (figs. 4-9) indicated high phytoplankton affinities between december vs. january and again between june vs. july samples of bhereki beel during the first year; and between june vs. august and again between february vs. march collections during second year while peak divergence is noticed february > april and december > june during two years, respectively. in holmari beel, maximum affinity is recorded between june and july, 2011 and between february and april, 2012, during the two years respectively. in ghotonga beel, high affinities are indicated between june-july-august while maximum divergence is noted during january > april and during may > november in two years, respectively. phytoplankton abundance ranged between 88-936 (418±227), 207-1292 (407±249) and 93-1627 (335±302) nl-1 (figs. 10-11) and comprised between table 2. variations (ranges, mean±sd) of phytoplankton of bhereki, holmari and ghotonga beels (september 2010-august 2012). bhereki beel holmari beel ghotonga beel richness net plankton (total) 209 species 212 species 232 species net plankton (monthly) 77-113 95±11 81-137 102±12 83-134 104±11 phytoplankton total 98 species 99 species 103 species % similarity 38.8-78.3 36.3-79.0 38.0-76.7 phytoplankton (total) 32-62 41±8 14-71 46±9 29-60 42±7 chlorophyta 16-46 26±7 15-52 29±9 13-48 27±8 bacillariophyta 4-13 9±2 6-13 10±2 4-13 8±2 cyanophyta 2-6 4±1 2-6 5±1 1-6 4±2 quantitative net plankton 261-1253 663±261 449-1815 682±289 282-1923 628±320 phytoplankton nl-1 88-936 418±227 207-1292 407±249 93-1627 335±302 % composition 26.8-76.2 59.5±12.5 39.0-84.8 57.1±12.3 22.0-84.6 48.6±13.5 species diversity 2.256-3.567 2.941±0.311 2.547-3.743 3.042±0.243 1.555-3.541 2.894±0.469 dominance 0.086-0.464 0.201±0.086 0.091-0.317 0.194±0.049 0.069-0.676 0.254±0.169 evenness 0.611-0.932 0.793±0.072 0.716-0.907 0.799±0.046 0.462-0.947 0.779±0.123 chlorophyta nl-1 37-821 153±169 48-596 176±120 28-751 152 ±155 % composition 13.5-87.7 35.1±19.5 21.5-64.6 42.5±10.9 7.0-82.0 44.6±18.8 bacillariophyta nl-1 17-515 173±144 47-473 120±91 12-278 72±59 % composition 5.3-79.5 40.1±19.9 17.5-52.0 29.6±9.0 5.5-55.1 25.6±15.8 cyanophyta nl-1 6-209 68±58 14-350 100±77 4-293 59±75 % composition 2.4-34.7 17.1±10.9 3.9-47.5 24.9±12.6 1.4-73.6 20.4±22.4 dinophyta nl-1 0-47 7±11 0-82 7±17 0-680 40±138 % composition 0.0-17.2 2.3±4.1 0.0-22.9 1.6±4.6 0.0-41.8 4.9±9.0 euglenophyta nl-1 0-60 18±18 0-29 6±6 2-41 12±10 % composition 0.0-29.6 5.4±6.8 0.0-5.2 1.5±1.4 0.8-16.0 4.6±4.5 important taxa (nl-1) cosmarium spp. 8-277 52±61 4-102 24±22 2-104 24±30 closterium spp. 0-102 9±22 0-48 12±12 1-93 23±29 euastrum spp. 0-51 6±11 0-23 3±5 0-15 4±5 micrasterias spp. 0-51 6±10 0-42 7±9 0-27 8±7 staurastrum spp. 1-52 12±14 1-108 15±21 0-135 16±27 staurodesmus spp. 0-100 10±21 0-24 3±5 0-11 3±3 important species (nl-1) elakatothrix gelatinosa 0-67 22±15 5-125 39±30 0-88 22±22 tabellaria fenestrata 0-60 16±16 6-71 23±16 0-82 11±18 navicula cuspidata 0-61 16±17 0-61 12±15 0-109 8±22 selenastrum gracile 0-90 9±19 0-52 11±14 0-175 10±35 nostoc sp. 0-65 10±14 0-63 19±19 0-58 8±14 amphora normani 0-188 10±37 0-13 5±3 0-69 12±16 cymbella ventricosa 0-93 10±18 0-36 7±8 0-58 14±17 rhopalodia sp. 0-100 24±25 0-45 17±13 0-21 4±5 oscillatoria sp. 0-122 22±34 0-76 15±23 0-24 4±6 phormidium sp. 0-70 17±17 0-128 47±43 0-28 8±9 phacus pleuronectes 0-58 14±16 0-24 5±5 1-27 10±7 peridinium sp. 0-47 7±11 0-82 6±17 0-680 37±138 aphanocapsa sp. 0-25 3±6 0-51 8±15 0-269 35±75 5 int. j. aquat. biol. (2017) 5(5): 295-309 59.5±12.5, 57.1±12.3 and 48.6±13.5% of net plankton of bhereki, holmari and ghotonga beels, respectively. chlorophyta abundance ranged between 37-821 (153±169), 48-596 (176±120) and 28-751 (152±155) nl-1 and comprised 35.1±19.5, 42.5±10.9 and 44.6± 18.8% of phytoplankton of three beels, respectively (table 2). bacillariophyta formed 40.1±19.9% of phytoplankton in bhereki (173±144 nl-1) and 29.6± 9.0% in holmari (120±91 nl-1) and 25.6±15.8% in ghotonga (72±59 nl-1) beels (table 2). cyanophyta density varied between 68±58, 100±77 and 59±75 nl-1 while dinophyta density ranged between 7±11, 7±17 and 40±138 nl-1 and that of euglenophyta between 18±18, 6±6 and 12±10 nl-1 in bhereki, holmari and ghotonga beels, respectively. phytoplankton species diversity, dominance and evenness varied (table 2) between 2.256-3.567, 2.547-3.743 (figs. 12, 13) and 1.555-3.541; 0.086– 0.464, 0.091-0.317 and 0.069-0.676; and 0.611-0.932, 0.716-0.907 and 0.462-0.947 in the sampled beels, figure 4. hierarchical cluster analysis of phytoplankton of bhereki beel (2010-2011). figure 5. hierarchical cluster analysis of phytoplankton of bhereki beel (2011-2012). 6 sharma and hatimuria/ phytoplankton diversity of floodplain lakes of majuli river island, india respectively. this study registered insignificant influence of individual abiotic factors on phytoplankton richness and abundance. chlorophyta richness is positively correlated with sulphate (r2=0.549, p=0.0027) in holmari beel; chlorophyta abundance is positively correlated significantly with ph (r1=0.581, p=0.0029) in bhereki beel and it is inversely correlated with dissolved organic matter (r3=-0.586, p=0.0026) in ghotonga beel. no individual factor significantly influenced bacillariophyta density in any beel. cyanophyta density is significantly correlated directly with silicate (r1=0.550, p=0.0027) and inversely with total hardness (r1=-0.544, p=0.0030) and magnesium (r1=-0.614, p=0.0007) in bhereki beel; it is correlated indirectly with total hardness (r3=-0.610, p=0.0008) in ghotonga beel. this study registered insignificant influence of abiotic parameters on dinophyta in the sampled beels while euglenophyta recorded positive correlation in holmari beel (r2=0.547, p=0.0028). the cca ordination biplots of phytoplankton assemblages (figs. 14-16) recorded 51.79%, 51.67% and 74.81% figure 6. hierarchical cluster analysis of phytoplankton of holmari beel (2010-2011). figure 7. hierarchical cluster analysis of phytoplankton of holmari beel (2011-2012). 7 int. j. aquat. biol. (2017) 5(5): 295-309 cumulative influence of 17 abiotic factors along first two axes in bhereki, holmari and ghotonga beels, respectively. discussion water temperature concurred with the geographical location of the sampled beels. bhereki and holmari beels indicated slightly acidic to circum-neutral waters, whereas ghotonga beel showed slightly acidic waters. specific conductivity exhibited low ionic concentration; this interesting feature warranted their inclusion of all beels under ‘class i’ category of trophic classification vide talling and talling (1965). all three beels are characterized by moderately hard water character, moderate dissolved oxygen, low free co2, low chloride content, and low concentrations of dissolved organic matter, total dissolved solids and nutrients. a total of 108 species recorded in this study characterized species-rich nature of phytoplankton with ghotonga > holmari ≥ bhereki beels; the biodiverse character is hypothesized to habitat figure 8. hierarchical cluster analysis of phytoplankton of ghotonga beel (2010-2011). figure 9. hierarchical cluster analysis of phytoplankton of ghotonga beel (2011-2012). 8 sharma and hatimuria/ phytoplankton diversity of floodplain lakes of majuli river island, india diversity and environmental heterogeneity of the majuli beels. phytoplankton is more diverse than the reports from the floodplains of manipur (sharma, 2009, 2010) and assam (sharma, 2004, 2012, 2015), and is notably diverse than ‘ad-hoc’ ecology reports from certain beels of assam (barbaruah and dutta, 2014; gupta and devi, 2014) and bihar (baruah et al., 1993; sanjer and sharma, 1995). our results also indicated higher richness than the reports from nine lakes (zutshi et al., 1980), dal lake (zutshi and vass, 1982) and nilang lake (wanganeo et al., 1996) of the kashmir himalayas; and nainital lake, uttarakhand (negi and rajput, 2015). the comparisons affirmed biodiversity value of phytoplankton of the majuli beels. phytoplankton richness followed concurrent monthly variations in bhereki and ghotonga beels than marginally higher value in holmari beel. anova indicated insignificant variations of richness amongst three beels; it indicated significant annual variations (f1, 23=12.516, p=0.004) in holmari beel. this study showed indefinite periodicity of richness in the three beels and thus endorsed the reports from floodplain lakes of nei (sharma, 2004, 2009, 2010). chlorophyta, the speciose group, is characterized by richness of desmid genera cosmarium > staurastrum > euastrum in all beels. this feature is an indicator of waters with low ionic concentrations and low calcium content (woelkerling and gough, 1976; payne, 1986; sharma, 1995); this salient feature corresponded with figure 10. monthly variations in abundance of phytoplankton (2010-2011). figure 11. monthly variations in abundance of phytoplankton (2011-2012). 9 int. j. aquat. biol. (2017) 5(5): 295-309 the results from the floodplains of manipur (sharma, 2009, 2010, 2015) and assam (sharma 2012, 2015) and also with the results from meghalaya (sharma 1995; sharma and lyngskor, 2003; sharma and lyngdoh, 2003). the phytoplankton community similarities suggested heterogeneity in species composition in bhereki, holmari and ghotonga beels. the similarities recorded inter-annual differences in bhereki and holmari beels in particular. it recorded 51-70% and 60-70% similarities in ~94% and ~96% instances during two years, respectively in bhereki beel; 51-60% and 61-70% similarities in ~50% and ~54% instances during two years, respectively in holmari beel; and 41-60% and 51-60% similarities in ~70% and ~80% instances during two years, respectively in ghotonga beel. the cluster analysis affirmed more affinities in early monsoon particularly in bhereki and ghotonga beels but in general affirmed heterogeneity in their monthly composition in all beels. the results differed from phytoplankton homogeneity reported by sharma (2009, 2010, 2015). phytoplankton abundance followed the stated order: bhereki > holmari > ghotonga beel with wider range in the last beel; anova registered insignificant quantitative variations amongst beels and insignificant annual and monthly variations in individual beels. figure 12. monthly variations in species diversity of phytoplankton (2010-2011). figure 13. monthly variations in species diversity of phytoplankton (2011-2012). 10 sharma and hatimuria/ phytoplankton diversity of floodplain lakes of majuli river island, india this study recorded higher abundance than the results from wular lake of kashmir (ganai et al., 2010); bihar (baruah et al., 1993; sanjer and sharma, 1995); loktak lake (sharma, 2009) and two floodplain lakes (sharma, 2010) of manipur; and samuajan (sharma, 2004), ghorajan (sharma, 2012) and deepor (sharma, 2015) beels of assam. phytoplankton formed dominant component of net plankton in bhereki beel and holmari beels but registered sub-dominant role in ghotonga beel; the former concurred with the reports from floodplain of bihar (baruah et al., 1993; sinha et al., 1994; sanjer and sharma, 1995), assam (yadava et al., 1987) and maharashtra (patil, 2002). the sub-dominance in ghotonga concurred with the reports from nei floodplains (sharma, 2004, 2009, 2010, 2012, 2015) and suggested availability of other food resources such as organic matter absorbed in sediments, detritus, bacteria, etc. as hypothesized by sharma (2012). the phytoplankton followed oscillating monthly quantitative variations during two years with peak densities during winter, summer and post-monsoon in bhereki, holmari and ghotonga beels, respectively. the lack of periodicity concurred with the reports of sharma (2010, 2012) but differed from the trimodal patterns reported from loktak lake (sharma, 2009) and deepor beel (sharma, 2015). the winter peak in the first beel concurred with the reports of yadava et al. (1987), sanjer and sharma (1995), sharma and lyngdoh (2003), sharma and lyngskor (2003) and figure 14. cca ordination biplot of phytoplankton and abiotic factors (bhereki beel). abbreviations: abiotic: alk (alkalinity), ca (calcium), cl (chloride), con (conductivity), do (dissolved oxygen), dom (dissolved oxygen matter), fco (free carbon dioxide), hd (hardness), mg (magnesium), ph (hydrogen-ion concentration), no3 (nitrate), po4(phosphate), rain (rainfall), sio2 (silicate), so4 (sulphate), tds (total dissolved solids), wt (water temperature). biotic: an (amphora normani), bac (bacillariophyta), bcr (bacillariophyta richness), chr (chlorophyta richness), chl (chlorophyta), cv (cymbella ventricosa), clos (clostridium), cos (cosmarium), cyn (cyanophyta), din (dinophyta), eg (elakatothrix gelatinosa), euas (euastrum), eug (euglenophyta), gsp (gomphonema sp.), micr (micrasterias), np (net plankton), nsp (nostoc sp.), nv (navicula cuspidata), osp (oscillatoria sp.), phsp (phormidium sp.), phy (phytoplankton), pp (phacus pleuronectes), pr (phytoplankton richness), psp (pinnularia sp.), rsp (rhopalodia sp.), sg (selenastrum gracile), ssp (spirulina sp.), stau (staurastrum), staur (staurodesmus), tf (tabellaria fenestrata). 11 int. j. aquat. biol. (2017) 5(5): 295-309 sharma (2009, 2010), while summer peak observed in holmari beel is concurrent with the reports from certain beels of assam (sharma, 2004, 2012, 2015). chlorophyta recorded relatively high abundance than the reports from the floodplains of nei (sharma, 2004, 2009, 2010, 2015) while it broadly concurred with the results of sharma (2012). it formed the sole dominant fraction of phytoplankton of holmari and ghotonga beels and indicated sub-dominant role in bhereki beel. this group notably influenced phytoplankton abundance (r1=0.675, p=0.0001; r2=0.948, p<0.0001; r3=0.908, p<0.0001); peak densities of chlorophyta contributed to phytoplankton maxima in all beels. anova registered insignificant variations of chlorophyta abundance amongst three beels; it registered significant annual density variations in bhereki (f1, 23=5.002, p=0.046) and ghotonga (f1, 23=5.315, p=0.041) beels. chlorophyta is characterized by quantitative importance of cosmarium spp. in bhereki > ghotonga > holmari beels, respectively; closterium spp. and staurastrum spp. in ghotonga > holmari > bhereki beels, respectively while staurodesmus spp. deserved attention in bhereki beel. the significance of desmid taxa concurred with the results the floodplains of nei (sharma, 2009, 2010, 2015). amongst chlorophyta species, only elakatothrix gelatinosa indicated importance in holmari > ghotonga > bhereki while selenastrum gracile showed certain importance in holmari and ghotonga beels, and desmidium sp. is notable in holmari beel. bacillariophyta formed the dominant group in bhereki beel and recorded sub-dominance in holmari and ghotonga beels; the former concurred with the figure 15. cca ordination biplot of phytoplankton and abiotic factors (holmari beel). abbreviations: abiotic: alk (alkalinity), ca (calcium), cl (chloride), con (conductivity), do (dissolved oxygen), dom (dissolved oxygen matter), fco (free carbon dioxide), hd (hardness), mg (magnesium), ph (hydrogen-ion concentration), no3 (nitrate), po4(phosphate), rain (rainfall), sio2 (silicate), so4 (sulphate), tds (total dissolved solids), wt (water temperature). biotic: bac (bacillariophyta), bcr (bacillariophyta richness), chr (chlorophyta richness), csp (caloneis sp.), dsp (desmidium sp.), chl (chlorophyta), ct (cymbella tumida), clos (clostridium), cos (cosmarium), cyan (cyanophyta), din (dinophyta), eg (elakatothrix gelatinosa), euas (euastrum), eug (euglenophyta), micr (micrasterias), np (net plankton), nsp (nostoc sp.), nv (navicula cuspidata), osp (oscillatoria sp.), phsp (phormidium sp.), phy (phytoplankton), pp (phacus pleuronectes), pr (phytoplankton richness), rsp (rhopalodia sp.), sg (selenastrum gracile), stau (staurastrum), staur (staurodesmus), tf (tabellaria fenestrata). 12 sharma and hatimuria/ phytoplankton diversity of floodplain lakes of majuli river island, india report from deepor beel (sharma, 2015) while the latter concurred with the report from loktak lake (sharma, 2009). the diatoms recorded significant (f2, 71=7.143, p=0.0019) density variations amongst three beels but registered insignificant annual and monthly variations in individual beels. gomphonema sp. > rhopalodia sp. > navicula cuspidata ≥ tabellaria fenestrata > pinnularia sp. > amphora normani > cymbella ventricosa influenced the diatom abundance in bhereki beel; tabellaria fenestrata > caloneis sp. > rhopalodia sp. > navicula cuspidata > cymbella tumida deserved mention in holmari beel while cymbella ventricosa > amphora normani > tabellaria fenestrata > navicula cuspidata showed importance in ghotonga beel. cyanophyta played a sub-dominant role concurrent with the reports from deepor beel (sharma, 2010, 2015) and ghorajan beel (sharma, 2012). it registered insignificant variations amongst beels but registered significant annual (f1, 23=5.169, p=0.044) variations in ghotonga beel. dinophyta in bhereki and holmari beels, and euglenophyta in bhereki, holmari and ghotonga beels, respectively, are characterized by low densities. this generalization concurred with the results of singh et al. (1982), sharma and lyngdoh (2003), sharma and lyngskor (2003) and sharma (2010). the sub-dominance of dinophyta in ghotonga beel and its contribution to phytoplankton peak in october, 2010 with importance of peridinium sp. is, however, notable. phacus pleuronectes deserved attention in bhereki > holmari beels. phytoplankton is characterized high diversity with figure 16. cca ordination biplot of phytoplankton and abiotic factors (ghotonga beel). abbreviations: abiotic: alk (alkalinity), ca (calcium), cl (chloride), con (conductivity), do (dissolved oxygen), dom (dissolved oxygen matter), fco (free carbon dioxide), hd (hardness), mg (magnesium), ph (hydrogen-ion concentration), no3 (nitrate), po4(phosphate), rain (rainfall), sio2 (silicate), so4 (sulphate), tds (total dissolved solids), wt (water temperature). biotic: an (amphora normani), bac (bacillariophyta), asp (aphanocapsa sp.), chr (chlorophyta richness), chl (chlorophyta), cs (closterium setaceum), cv (cymbella ventricosa), clos (clostridium), cos (cosmarium), cyan (cyanophyta), din (dinophyta), eg (elakatothrix gelatinosa), euas (euastrum), eug (euglenophyta), micr (micrasterias), np (net plankton), phy (phytoplankton), pp (phacus pleuronectes), pr (phytoplankton richness), psp (peridinium sp.), sg (selenastrum gracile), stau (staurastrum), staur (staurodesmus), tf (tabellaria fenestrata). 13 int. j. aquat. biol. (2017) 5(5): 295-309 wider monthly variations; bhereki and ghotonga beels indicated high diversity (>3.0) during eleven months each and registered higher averages during first year while holmari beels indicated species diversity >3.0 during 15 months with higher mean during second year. these remarks reflected greater inter-annual variations in habitat diversity vis-a-vis phytoplankton diversity of the sampled beels in general and of holmari beel in particular. the features of high species diversity with relatively lower densities of large number of species is ascribed to fine niche portioning amongst inhabitant species in combination with high microand macro-scale habitat heterogeneity (sharma, 2012, 2015). our results registered low to moderate phytoplankton dominance without confirming to any definite monthly pattern; it recorded insignificant temporal variations amongst beels and indicated significant annual (f1, 23=9.143, p=0.011) as well as monthly (f11, 23=3.984, p=0.015) variations in bhereki beel. high dominance recorded for a specific period both during two years in ghotonga beel coincided with higher abundance of cyanophyta as well as with peaks of peridinium sp. during first year and of aphanocapsa sp. during second year, respectively. likewise, various taxa resulted in the periods of higher dominance in bhereki and holmari beels while low dominance with relatively lesser fluctuations during certain months indicated lack of quantitative importance of individual species (mcnaughton, 1967). following macarthur (1965), it is hypothesized that the majuli beels provided resources for utilization by fewer or majority of species and thus providing variable conditions from low to high amount of niche overlap. phytoplankton is characterized by moderate to high evenness in ghotonga > bhereki > holmari beels, with higher averages during first year and indefinite pattern of monthly variations in all beels. high evenness noticed during several months is attributed to equitable abundance of majority of taxa (washington, 1984) while dominance of certain species resulted in moderate values during february, 2012 in bhereki; october, 2010, january-march and may, 2012 in holmari; and during november, 2010 and august, 2012 in ghotonga beels. anova registered both insignificant variations of evenness amongst three beels; it exhibited significant annual variations (f1, 23=5.541, p=0.038). individual abiotic factors exerted insignificant influence on phytoplankton richness and abundance. chlorophyta abundance is positively correlated with ph in bhereki and inversely correlated with dissolved organic matter in ghotonga beel. cyanophyta density is significantly correlated directly with silicate and inversely with total hardness and magnesium in bhereki beel, and it is correlated indirectly with total hardness in ghotonga. the canonical correspondence analysis (cca) with 17 abiotic factors recorded low influence phytoplankton assemblages along first two axes in bhereki and holmari beels than in ghotonga beel. cca reflected the importance of water temperature, ph, hardness, dissolved organic matter, total dissolved solids, sulphate and silicate in bhereki beel; water temperature, rainfall, free carbon-dioxide, magnesium and total dissolved solids recorded importance in holmari beel; and reflected importance of water temperature, specific conductivity, hardness, calcium, magnesium, dissolved organic matter and total dissolved solids in ghotonga beel. in general, this study yielded limited insight regarding individual and cumulative influence of abiotic factors on phytoplankton diversity; the results thus suggested need to analyse factors associated with microhabitat variations of the sampled beels. to sum up, the speciose phytoplankton of bhereki, holmari and ghotonga beels, heterogeneity in their composition, richness of chlorophyta and of certain desmid genera merit biodiversity value. the quantitative importance chlorophyta in holmari and ghotonga beels and of bacillariophyta in bhereki beel; lack of any definite temporal variations of phytoplankton richness and abundance; and low to moderate dominance are notable. variations in composition, abundance, diversity, and dominance suggested habitat diversity during two years of this study. the limited individual and low cumulative influence on phytoplankton assemblages yielded 14 sharma and hatimuria/ phytoplankton diversity of floodplain lakes of majuli river island, india limited insight on overall role of abiotic factors. acknowledgments this study is partially undertaken under the “university for excellence program (upe)’ of northeastern hill university, shillong. thanks are due to the head, department of zoology, nehu, shillong, for providing the necessary facilities. the authors have no conflict of interests. references adoni a.d., ghosh g., chourasia s.k., vaishya a.k., yadav m., verma h.g. 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(2018) 6(4): 221-234 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article hemato-immunological, serum metabolite and enzymatic stress response alterations in exposed rainbow trout (oncorhynchus mykiss) to nanosilver ali taheri mirghaed*1, peyman yarahmadi1, mitra shabrang harehdasht2, paul m. craig3, hamed ghafari farsani4, nahid ghysvandi5, soheil eagdari2 1department of aquatic animal health, faculty of veterinary medicine, university of tehran, tehran, iran. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 3department of biology, faculty of science, university of waterloo, waterloo, ontario, canada. 4young reasearchers and elite club, shahrekord branch, islamic azad university, shahrekord, iran. 5department of fisheries, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 23 february 2018 accepted 2 june 2018 available online 2 5 august 2018 keywords: nanosilver hematology serum metabolites cortisol oxidative stress serum ions abstract: the aim of present study was to investigate the effects of sub-lethal concentrations of silver nanoparticles (agnp) on hematological parameters, differential tests of white blood cells, serum metabolite parameters, serum enzymes activity and serum ions in rainbow trout, oncorhynchus mykiss. healthy rainbow trout, were exposed to sub-lethal concentrations (0, 1.5 and 2.5 ppm) of nanosilver for 14 days. rbc, wbc and hct levels were significantly (p<0.05) increased in exposed groups. within the white blood cells, only neutrophils showed a significant increase at 7 and 14 days post exposure (p<0.05). serum triglyceride, total serum protein, albumin and globulin levels were decreased (p<0.05) in exposed fish, however, cholesterol levels increased in the 2.5 ppm group at 7 days after exposure (p<0.05). cortisol and glucose increased significantly at 7 and 14 days of exposure in both concentrations of agnps (p<0.05). decreases in serum ions level were observed, although reduction in chloride ions occurred earlier and more severe than other measured parameters (p<0.05). elevation in serum alp, ldh, alt and ast enzymes were observed during the experiment (p<0.05), although sod and cat activity were significantly decreased in exposed groups (p<0.05). the results revealed that agnp can affect the hematological, serum metabolite and enzymatic parameters of o. mykiss, as well as agnp exposure induce a general oxidative stress response in o. mykiss. introduction nanotechnology is rapidly expanded in applications ranging from electronics to biotechnology and involves materials and process that have at least one dimension in the range of 1-100 nanometers (moore, 2006). although the applications of nanotechnology is broad, there is increasing concern about nanomaterial impacts on the health of the environment, especially in aquatic ecosystems that act as a sink for many pollutants, including engineering nanomaterials (enms) (scown et al., 2010a). case studies examining the toxicology effects of enms, have shown that the interactions between nanomaterial and all levels of biological organization, from cells to ecosystems are remarkably complex (aschberger et *corresponding author: ali taheri mirghaed doi: https://doi.org/10.22034/ijab.v6i4.512 e-mail address: mirghaed@ut.ac.ir al., 2011). therefore, with worldwide increases in enm application, it is essential to investigate their impact on humans, animal health and the environment. nanotechnology has a broad potential to revolutionize all scientific fields (hood, 2004). among all of enms, silver nanoparticles (agnps) were acclaimed for their antibacterial properties (aschberger et al., 2011). it is well-documented that agnps are toxic to a variety of model and non-model aquatic organisms (asghari et al., 2012; asharani et al., 2008; behra et al., 2013; farkas et al., 2011; griffitt et al., 2008; lapresta-fernández et al., 2012). the toxic effects of nanosilver has been assessed and determined in different aquatic organisms with acute 222 taheri et al./ effect of nanosilver on hematological, enzymatic and stress of rainbow trout and chronic toxicity mechanisms of these compounds. these studies reported that silver nanoparticles result in decreased hatching rate, increased mortality, and hatching delay in zebrafish (danio rerio) embryos and larvae (massarsky et al., 2013; yeo and kang, 2008). furthermore, it has been demonstrated in zebrafish exposed to agnps that there is abnormal body axes, slow blood flow, pericardial edema and cardiac arrhythmia, and increased apoptosis in the gills of juveniles and adults (asharani et al., 2008; griffitt et al., 2009). furthermore, exposure of rainbow trout (oncorhynchus mykiss) to different sizes of agnps (10 nm, 35 nm and 600-1600 nm) induced several detoxifying and reactive oxygen scavenging transcripts, such as cyp1a2, cyp3a45, hsp70a, gpx, and g6pd (scown et al., 2010b), changes in plasma biochemical parameters (johari et al., 2013) and resulted in oxidative stress and cellular metabolic activity in primary gill cells of rainbow trout (farkas et al., 2011). although numerous studies have demonstrated the toxic effects of agnps, there is still a lack of understanding the impact agnps on aquatic ecosystems due to the difficulty in accurately quantifying agnps within a natural setting (malina et al., 2010). data, such as hematological, serum electrolytes, as well as serum enzymatic parameters are needed to assess the environmental risk posed by silver and the other nanoparticles. hence, the main objective of this study was to determine the effects of nanosilver exposure over time on serum metabolites, including triglyceride (tg), cholesterol (cho), total protein (tp), albumin (albu), globulin (glo), glucose (glu) and cortisol, and serum ions (ca++, k+, na+ and cl-), serum enzymes activity (alkaline phosphates (alp), lactate dehydrogenize (ldh), alnine aminitransferase (alt), aspartat (ast), sod (superoxide dismutase), and cat (catalase), and assess if these changes in hematogical parameters can be used as key biomarkers for agnp exposure in rainbow trout (oncorhynchus mykiss). materials and methods characterization of silver nanoparticles: the nanosilver colloidal product (commercial name: nanocid®) was purchased from nano nasb pars co., (u.s patent no. us/2009/0013825) which contained 4000 ppm nanosilver particles. prior to use, the shape and size distribution of the agnps was determined using a transmission electron microscopy (tem). two experimental concentrations of 1.5 and 2.5 ppm were obtained from this solution. fish: healthy, mixed sex rainbow trout (n=90; weight: 101.2±0.4 g; total length: 19.6±0.7 cm; mean±sd) were obtained from a local fish farm (mahisara fish culture; karaj, iran). the fish were acclimatized to the laboratory conditions in 100l tanks, which were equipped with a flow-through system with dechlorinated tap water for 14 days and fed (1% of bw) commercial rainbow trout diet (behparvar co. tehran, iran) twice a day. after acclimation period, fish were distributed into nine 100l fiberglass tanks, and assigned to three doses of agnps; 0 mg l-1 (control), 1.5 ppm and 2.5 ppm, based on the 96 hr lc50 values obtained in the acute toxicity test for this experiment (lc50=8.9 mg l -1) based on shaluei et al. (2012) and previous experiments in a number of fish species which is summarized in table 1. exposures were run in triplicate, at a constant water quality with a controlled photoperiod (12 hrs light; 12 hrs dark). fish were allowed to acclimate for 48 hrs prior to the start of the exposure regime. fish were sampled after 0, 1, 7, and 14 days exposure. experimental sampling: fish were fasted 24 hrs prior to sampling and three fish per replicate were anesthetized with clove powder at a concentration of 200 mg l-1. blood samples were collected through caudal vein puncture, and blood samples were allocated into two portions, one portion of was transferred to heparinized tubes for measurement of haematological parameters and another one to nonheparinized tubes that left to clot for 12 hrs (at 4°c), prior to centrifugation at 4ºc, 7000 rpm for 10 min. the serum was extracted from samples, placed in new tubes and stored at -80ºc until analysis. hematological parameters assay: heparinized blood samples were prepared for counting the number of red 223 int. j. aquat. biol. (2018) 6(4): 221-234 blood cells (rbcs) and white blood cells (wbcs) under a light microscope using neubauer hemocytometer slide based on sarder et al. (2001). the blood hematocrit (hct) was determined by microhematocrit capillary tubes, which were centrifuged at 4ºc, 3500 g for 10 min and hct was reported as percentage of packed cell volume (%pcv). furthermore, to determine the white blood cells, blood was smeared on slides and air-dried after staining. white blood cells were classified under light microscope with ×1000 magnification (härdig et al., 1988). serum parameters assay serum metabolite parameters: metabolite parameters, including glucose, triglycerides, cholesterol, total protein and albumin were determined with an auto analyzer hitachi 911 with reagents provided in standard analyses kits (pars azmon, tehran, iran). the serum glucose and cholesterol concentrations were measured based on glucose oxidase and cholesterol oxidase commercial kit (pars azmoon). the serum total protein were measured based on commercial available procedure at 27°c and 546 nm wave length, which employed bovine serum albumin as a standard (pars azmoon). serum triglycerides (tg) were determined using commercial assay based on enzymatic colorimetric method with glycerol phosphate oxidize at 546 nm and 37 °c (pars azmoon). the serum albumin concentrations were determined using a purchased assay (pars azmoon), and used acidic ph and bromocresol green as a reagent. the serum globulin concentration was calculated by subtracting the albumin values from the total serum protein. serum enzymes activity: serum enzyme activities, including lactate dehydrogenase (ldh), alkaline phosphatase (alp), alanine aminotransferase and aspartate aminotransferase (ast) activities were measured based on commercial kits protocol (pars azmoon, tehran, iran) and an autoanalyzer (eppendorf, epos, germany) based on shahsavani et al. (2010). serum cat and sod activity were measured based on methods previously described by goth (1991) and marklund and marklund (1974), respectively. serum ions: for determination the serum ion concentration, commercial available reagents were used (pars azmon co., tehran, iran). the chloride (cl-) were determined based on mercury thiocyanate colorimetric reaction according to available commercial kit protocol. serum calcium (ca2+) concentration were measured by using commercial pars azmoon based on o-cresolphthalein as regent. sodium (na+) and potassium (k+) were determined based on commercial pars azmoon kit procedure and a flame photometer (corning 410, united kingdom). statistical analysis: the results are presented as means±sd. differences between parameters were analyzed by one-way analysis of variance and significant means were subjected to a multiple comparison test (duncan) at the p=0.05 level. results silver nanoparticles characterization: transmission electron microscopy images of agnps showed uniform and spherical structure of particles (fig. 1a, b). the average size of particles was 23.06±1.91 nm (fig. 1c) and based on the data, 66% of the particles table 1. effective concentrations of nanocid® obtained from toxicity experiments on fish. reference tested organism effect measured shahbazzadeh et al. (2009) rainbow trout (o. mykiss) (average weight: 1.049 g) lc50 = 2.3 mg/l moaddab et al. (2011) osteoblast (g292) cell line ic50 = 3.42 μg/ml shahbazzadeh et al. (2011) fibroblast (hf2) mesenchymal stem cells ic50 = 6.68 μg/ml ic50 = 6.33 μg/ml shaluei et al. (2012) caspian roach (r. caspicus) (average weight: 3.5 g) lc50 = 0.028 mg/l johari et al., 2013) 3 stages of rainbow trout (o. mykiss): eleutheroembryos larvae (average weight: 154 mg), juvenile (average weight:15 g) lc50 = 0.25 mg/l lc50 = 0.71 mg/l lc50 = 2.16 mg/l shaluei et al. (2013) silver carp (h. molitrix) lc50 = 0.202 mg/l http://www.chemicalbook.com/chemicalproductproperty_en_cb6392671.htm 224 taheri et al./ effect of nanosilver on hematological, enzymatic and stress of rainbow trout had diameter less than 40 nm. the commercially purchased agnps were a water-soluble form of colloidal nanoparticles, and remained in suspension during exposure. figure 1. (a, b) transmission electron microscopy images of silver nanoparticles (c) size range of silver nanoparticles diameter. figure 2. effect of exposing the rainbow trout fish to 2 doses of silver nanoparticles on blood parameters. (a) rbc count, (b) wbc count, (c) hematocrit percentage. data are presented as mean±sd (n=9). plus signs (+) indicate a significant difference (p<0.05) between the same exposing dose from control time (day 0); multiplication signs (×) indicate a significant difference (p<0.05) from the same day’s control group. 225 int. j. aquat. biol. (2018) 6(4): 221-234 hematological parameters analysis: no mortality was observed during the sub-acute exposure for all treatments. there was a significant (p<0.05) increase in the red blood cells count in 1.5 ppm at 1, 7 and 14 days and in 2.5 ppm at 1 and 7 days exposure compared to control group (fig. 2a). the number of white blood cells did not show changes in the first sampling time point, although in days 7 and 14 of exposure, the wbc counts were significantly increased (p<0.05; fig. 2b). no significant changes (p>0.05) were observed in percentage of lymphocyte, monocyte and eosinophil cells (figs. 3 b, c, d), although the number of neutrophils increased (p<0.05) at 7 and 14 days in all exposure groups. serum metabolite parameters: after 1 and 7 days, triglyceride levels significantly decreased in the 2.5 ppm group. however, after 14 days of exposure, triglyceride level decreased in all doses of agnp. triglyceride levels in fish exposed to the 2.5 ppm dose showed a significant increase compared to previous sampling time (t2) of this dose (fig. 4a). cholesterol level of blood plasma increased in the 2.5 ppm group after 7 days of exposure (fig. 4b). a significant decrease of total protein was observed in all exposed fish during the experiment (fig. 4c). additionally, the plasma albumin level decreased significantly in the 2.5 ppm exposure group after 7 days and in both doses after 14 days (fig. 4d). furthermore, plasma glucose increased significantly in all exposure groups after 7 and 14 days (fig. 4e). serum ion analysis: a significant decrease (p<0.05) in calcium ions level was observed solely in the high dose after 14 days of exposure (fig. 5a). furthermore, the amount of k+ was significantly decreased (p<0.05) at 7 and 14 days of exposure (fig. 5b). there were no significant changes in na+ concnetrations at 1 or 7 days, although after 14 days of exposure, fish in both of exposure doses were showed significant decrease (p<0.05) in na+ compared to control group (fig. 5c). moreover, agnp exposure induced figure 3. white blood cell differential count in rainbow trout exposed to nanosilver for 14 days. (a) lymphocytes percentage, (b) neutrophils percentage, (c) monocytes percentage and (d) eosinophils percentage. data are presented as mean±sd (n=9). capital letters (a, b, c) indicate a significant difference (p<0.05) between the same exposing dose from control time (day 0); small letters (a, b, c) indicate a significant difference (p<0.05) from the same day’s control group. 226 taheri et al./ effect of nanosilver on hematological, enzymatic and stress of rainbow trout figure 4. serum biochemical parameters in rainbow trout exposed to silver nanoparticles for 14 days. (a) triglyceride level, (b) cholesterol level, (c) total protein level, (d) albumin level, (e) globulin level, (f) glucose level, and (g) cortisol level. data are presented as mean±sd (n=9). plus signs (+) indicate a significant difference (p<0.05) between the same exposing dose from control time (day 0); multiplication signs (×) indicate a significant difference (p<0.05) from the same day’s control group. 227 int. j. aquat. biol. (2018) 6(4): 221-234 significant decreases in cllevel at 1, 7 and 14 days (fig. 5d). among all investigated serum ions, clions did not change. serum enzymes activity: both concentrations of apnps induced significantly increased (p<0.05) alkaline phosphatase level of serum in all sampling times (fig. 6a). likewise, ldh levels increased (p<0.05) in all exposed fish except at 1.5 ppm after one day of exposure (fig. 6b). both aminotransferases i.e. alt and ast, showed significant increase (p<0.05) in all agnp exposed fish (fig. 6c, d). for serum antioxidant enzymes activities, cat demonstrated a significant decrease in activity showed at 2.5 ppm of agnp one day after exposure and there was a significant decrease in low and high dose of agnp on days 7 and 14 (p<0.05). likewise serum sod activity of exposure groups exhibited a significant (p<0.05) dose-dependent decreased compared to the control group (fig. 6e, f). discussion in ecotoxicological investigations, physiological responses like hemato-immunological, serum metabolites, enzymatic activity, ion homeostasis and endocrine disturbances provide valuable insight into the affects on aquatic species (kavitha et al., 2010; kim and kang, 2004; rao, 2006b). indeed, physiological assessments measure the responses of organisms exposed to pollutants and correlate these responses with pollutant effects (handy and depledge, 1999). the results presented here demonstrate that there were no mortalities in rainbow figure 5. serum ions concentration in rainbow trout exposed to nanosilver for 14 days. (a) calcium, (b) potassium, (c) sodium and (d) chloride. data are presented as mean±sd (n=9). capital letters (a, b, c) indicate a significant difference (p<0.05) between the same exposing dose from control time (day 0); small letters (a, b, c) indicate a significant difference (p<0.05) from the same day’s control group. 228 taheri et al./ effect of nanosilver on hematological, enzymatic and stress of rainbow trout trout after 14 days exposure with sub-lethal concentrations (1.5 and 2.5 ppm), however there were impacts on haematological parameters. we observed a significant increase in the number of red and white blood cells and hematocrit percentage. in agreement with our results, vinodhini and narayanan (2009) reported that combined (cd+pb+cr+ni) metal solutions increased the level of rbcs, htc and hb in cyprinus carpio after 32 days exposure. the increase level of rbcs could be attributed to stress in exposed fish and this is a response to supply oxygen demand in a high stress situations (yarahmadi et al., 2015). although in contrast, others have reported a significant decrease of rbc count, hct percentage and hemoglobin amount in nanoparticles exposed fish (karthikeyeni et al., 2013; shaluei et al., 2013; shaw et al., 2012). this could be explained by the dissimilarity in the differences in tested organism’s species or differences between the sizes of nanoparticles. figure 6. serum hepatic enzymes in rainbow trout exposed to silver nanoparticles for 14 days. (a) serum alkaline phosphatase (alp) level, (b) serum lactate dehydrogenase (ldh) level, (c) serum alanine aminotransferase (alt) level, (d) aspartate aminotransferase (ast) level, (e) serum catalase(cat) activity and (f) serum superoxide dismutase (sod) activity. data are presented as mean±sd (n=9). capital letters (a, b, c) indicate a significant difference (p<0.05) between the same exposing dose from control time (day 0); small letters (a, b, c) indicate a significant difference (p<0.05) from the same day’s control group. 229 int. j. aquat. biol. (2018) 6(4): 221-234 there were significant increases in both wbc count and percentage of neutrophils in the 7th and 14th days of exposure. the wbcs counts and especially neutrophil cells are one of the non-specific immune indicators in fishes (ainsworth, 1992; neumann et al., 2001). the increase in wbc counts is known as a normal reaction to pollutants which can alter the normal physiological processes in fish (handy and depledge, 1999) and it has been reported in some studies after exposing the fish to pollutants (weinreb, 1958). the increase of some hemato-immunological parameters such as leukocytes number and neutrophil level has been observed in hypophthalmichthys molitrix (shaluei et al., 2013) in tilapia nilotica (khalaf-allah, 1999) that were subjected to nanosilver and different pesticides. fish exposed to agnp showed remarkable change in the levels of serum metabolites; decreases in serum triglyceride, total protein and albumin level and conversely increases in serum cholesterol, glucose and cortisol were observed. there is limited information about effect of agnp on serum metabolite parameters in fish, although it is well-known that serum metabolite parameters are biomarkers to evaluate the relationship between pollution and fish physiological response in ecotoxicological study (adams et al., 1992; adams and ryon, 1994; folmar et al., 1993; öner et al., 2008). the reduction in serum triglyceride suggested that experimental animals are suffering from poor nutrition or starvation as a result of pollutant exposure-related stress (white and fletcher, 1986). in agreement with this study, öner et al. (2008) reported that serum triglyceride decreased in cuexposed oreochromis niloticus, although in contrast, serum triglyceride was increased in ag-exposed fish. however, ag, cd, cr, cu, zn exposures increased serum cholesterol concentration (handy and depledge, 1999), which is in agreement with the results of this study. the serum total protein is one of the humoral innate immune parameter in fish which mainly include albumin and globulin (zhang et al., 2013). the reduction of serum proteins in agnp exposed fish may be related to a decrease in small proteins found in the blood, lysozymes, and suppression of innate immune response (han et al., 2014). cortisol is a stress-related hormone and it is known that increased serum concentrations can be use as a first stress response bioindicator to environmental disruption (barton, 2002). the increased serum cortisol level in the present investigation is consistent with similar observations in h. molitrix exposed with agnp (nanocid) (shaluei et al., 2013). the results demonstrated revealed an increased glucose level after 7 and 14 days of exposure. blood glucose elevation during pollutant stress is well known as secondary stress response (handy and depledge, 1999). the serum glucose level is one of the secondary stress response associated with increased levels of circulating cortisol due to stress (barton, 2002), and is demonstrated here with the strong correlation between increased circulating cortisol and glucose levels. the results of present study indicated significant decreases in serum ca2+, k2+, na+ and cl-, ions levels during the experiment. among these four ions, only serum chloride ions level changed one day after exposure. pollutants are wel-known to disrupt the homeostatic balance of serum ions in fish (handy and depledge, 1999). there is limited information about effect of agnps on serum ion concentration. however, in agreement with our results, johari et al. (2013) reported one dose-dependent plasma reduction of chloride and potassium ions in rainbow trout juveniles after 3 h of exposure to agnps. compensating for this, apical sodium channels and the basolateral na+/k+-atpase in gill epithelial cells, facilitate active uptake of na+ from the dilute environment (evans et al., 2005). these specialized cells have an important role in ion transporting including nacl uptake. a previous study by katuli et al. (2014) demonstrated that agnps inhibit the activities of na+/k+ -atpase in gill of zebrafish (danio rerio). conversely, gill injuries which were observed in many experiments as an effect of agnp might lead to disruption in nacl uptake in exposed fish (farmen et al., 2012; scown et al., 2010b; wu and zhou, 2013). consequently, there would be a reduction in the concentration pf na+ and clions in 230 taheri et al./ effect of nanosilver on hematological, enzymatic and stress of rainbow trout the blood of agnps exposed fish. the induced stress response found in this study may be associated with the loss of ion homeostasis, as cortisol is known to alter ionoregulatory mechanism in the gill (barton, 2002). some serum enzymes such as alp, ldh, alt and ast were analyzed as useful biomarkers of liver injury and health status, and results revealed that the activity level of these enzymes increased in response to agnp exposure compared to the control (fig 6). previous studies have shown an increase in serum alp, ldh, alt and ast contents in response to toxicant exposure (das et al., 2004; gulumian et al., 2006). alp and ldh are cellular membrane enzyme and their activity is used as indicator of cell membrane damage (gulumian et al., 2006). rao (2006a) reported that the alp activities in plasma, gill and kidney of exposed o. mossambicus with insecticide increased the lysosomal mobilization, cell necrosis, localized hypoxic conditions and muscular harm. increases of ldh in serum may be a result of releasing the isozymes from the destroyed tissues and indicates cell lysis (agrahari et al., 2007; lemaire et al., 1991). two serum aminotransferases (alt and ast) which were measured in this study are well known as liver specific enzymes in hepatocellular necrosis condition (geeraerts and belpaire, 2010). our results showed a significant increase in both enzymes in agnps exposed fish. the increase of these enzymes has been observed in the gill, liver, and kidney as a response to pollutant exposure (oluah, 1999; rajyasree and neeraja, 1989; rao, 2006a). table 2. a summary of present experiment results. upward arrows () indicate significant increases (p<0.05) and downward arrows () indicate significant decreases (p<0.05) in experimental parameters. in dose-dependent group results compared with same sampling time’s control group; in time-dependent column results compared with same dose’s t0 sampling data. dose-dependent changes time-dependent changes t0 t1 t2 t3 1.5 ppm 2.5 ppm 1.5 2.5 1.5 2.5 1.5 2.5 1.5 2.5 t1 t2 t3 t1 t2 t3 b lo o d c e ll s r e d rbc           %hct           w h it e wbc          nut       mon lym eos b lo o d p la sm a io n s ca   k         na     cl             b io c h e m ic a l fa c to r s tri         chol   protot             alb       glo             glu         h e p a ti c e n z y m e s alp             ldh           alt             ast             sod 231 int. j. aquat. biol. (2018) 6(4): 221-234 the antioxidant enzymes assayed in the present study revealed that agnps affected oxidative defense system that impacted cat and sod activity. sod and cat are two key oxidative defense enzymes that provide primary antioxidant protection against oxidative stress through catalyze dismutation of superoxide anion radicals and hydrogen peroxide, respectively (kolayli and keha, 1999; wu and zhou, 2012, 2013). in the present study, we observed a significant decrease in serum sod and cat activity in exposure groups from day 1 to 14. in agreement with our results, wu and zhou (2013) reported the dose-dependent decrease in hepatic sod and cat activity in medaka (oryzias latipes) after 14 days of exposure. agnps induced ros generation and damage to various cellular components in human liver (piao et al., 2011). the reduction in the level of sod and cat activity lead to oxidative system damage affected by agnp (li et al., 2009). further to this, ros produced in tissue following pollutant exposure have a synergistic effect on the reduction of cat activity (stanic et al., 2006). conclusion a summary of all results drawn from this study is presented in table 2, in which hematological alterations of o. mykiss, sub-lethal exposed to agnp, suggest that exposed fish faced a catastrophic situation that results in increased levels of rbc, wbc and neutrophils as immune response. from analysis of serum metabolites and enzymatic parameters, it was concluded that sub-lethal agnp exposed fish had altered ion homeostasis and impacted serum enzymatic parameters. enzymatic findings indicate an increase in serum alp, alt, ast and ldh concentration which may be contributed to cell and tissue damages following agnp exposure. furthermore, sod and cat activity revealed the toxicant effect of agnps by suppressing oxidative stress system. as result, we suggest that blood analysis, serum metabolite and enzymatic activity are used as a good biomarker of agnp contamination. however we strongly encourage future studies in other aquatic species to confirm these findings. acknowledgments this study was support by university of tehran. references adams s.m., crumby w.d., greeley m.s., ryon m.g., schilling e.m. 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(2021) 9(4): 244-247 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology short communication two new localities for lobocheilos falcifer (valenciennes, 1842) (teleostei: cyprinidae), a rare and vulnerable freshwater fish species of java, indonesia veryl hasan1,2, lucas de oliveira vieira3,4, felipe polivanov ottoni*3,4,1endang dewi masithah5 1aquaculture department, fisheries and marine faculty, airlangga university, surabaya, indonesia. 2generasi biology indonesia foundation, zoology division, gresik, indonesia. 3laboratory of systematics and ecology of aquatic organisms, center of agrarian and environmental sciences (ccaa), federal university of maranhão, chapadinha-ma, brazil. 4federal university of maranhão, postgraduate program in environmental sciences, center of agrarian and environmental sciences, chapadinha-ma, brazil. 5marine department, fisheries and marine faculty, airlangga university, surabaya, indonesia. article history: received 12 june 2021 accepted 26 july 2021 available online 2 5 august 2021 keywords: ahan, endangered species, labeoninae, native species. abstract: lobocheilos falcifer is a rare and vulnerable freshwater fish species from java, indonesia and currently listed as vulnerable (vu) within the iucn red list. in 2019, three specimens of l. falcifer were collected and photographed on rawa pening lake, semarang regency, central java province and solo river, karanganyar regency, central java province as two new localities for this species, extending its distribution in about 80 km to southeast. we also provided the morphological data of this species as well. introduction lobocheilos bleeker, 1854 is a cyprinid endemic to southeast asia and the western indonesia archipelago (borneo, java and sumatra) (rainboth, 1991; ciccotto and page, 2016; ciccotto and tan, 2018). two species of lobocheilos viz. l. lehat bleeker, 1858 and l. falcifer (valenciennes, 1842) occur in java (fricke et al., 2021), but few information on these species are available, especially on their distribution. lobocheilos lehat is probably extinct (fricke et al., 2021) considering that it is not sampled since it was first discovered on parongkalong, west java province in 1853 (kottelat et al., 1993; kottelat and tan, 2008), whereas l. falcifer is currently listed as a vulnerable species (vu) based on the iucn red list status (lombantobing, 2019). lobocheilos falcifer was reported in the following freshwater localities in java: cisadane river, west java province (kottelat and tan, 2008; hadiaty, 2011), wadaslintang reservoir, central java province (hasan et al., 2019a), and tuntang river, semarang regency, central java province (hasan et al., 2019b). *correspondence: felipe polivanov ottoni doi: https://doi.org/10.22034/ijab.v9i4.1285 e-mail: fpottoni@gmail.com in this paper, we reported new records of l. falcifer in the rawa pening lake and solo river, central java province, a range extension of about 80 km southeast from the closest locality where the species was already known. materials and methods three specimens of l. falcifer (fig. 1) were collected using cast nets in 2019 in the outlet of the rawa pening lake, and in the upstream of solo river (figs. 2, 3). these two sites are located in semarang regency and karanganyar regency, respectively, at central java province, indonesia. the specimens were labeled and fixed in 96% ethanol (hasan et al., 2019). the material was deposited at the zoology laboratory, generasi biologi indonesia foundation (gbi). morphological identification followed diagnostic characters presented in kottelat and tan (2008). examined materials (all from indonesia: central java province): lobocheilos falcifer: gbi0002, 1 male, 161 mm tl, semarang regency, rawa pening 245 int. j. aquat. biol. (2021) 9(4): 244-247 lake, outlet of the lake, 7°18'23"s; 110°26'37"e, v. hasan, 10 nov. 2019. — gbi0003, 1 female, 176 mm tl, semarang regency, rawa pening lake, outlet of the lake, 7°18'23"s; 110°26'37"e, v. hasan, 10 nov. 2019. — gbi0004, 1 male, 168 mm tl, karanganyar regency, solo river, upstream of the river, 7°47'06.0"s; 110°57'07.0"e, v. hasan, 5 dec. 2019. results and discussions lobocheilos falcifer (valenciennes, 1842) (fig. 1) new records: see examined material section. identification: some morphometric and meristic data of the specimens examined here are presented in table 1. specimens collected on rawa pening lake and solo river (fig. 1) were identified as l. falcifer based on the following diagnostic features proposed by kottelat and tan (2008), listed below. lobocheilos falcifer is distinguished from the other species of lobocheilos occurring in borneo, sumatra, figure 1. specimen of lobocheilos falcifer gbi0002. a. lateral view of the body. b. lateral view of head. c. ventral view of head and position of the mouth (photograph by v. hasan). figure 2. the locations where lobocheilos. falcifer specimens were collected in this work. a. rawa pening lake. b. solo river; central java province, java, indonesia (photograph by v. hasan). 246 hasan et al./ two new localities for lobocheilos falcifer and java by the combination of the following features: no dark blotch on caudal peduncle; 30–31+2–3 lateral line scales; body uniformly silver and greenish on top of head to caudal peduncle in life and fresh specimens; faint mid–lateral strip in life and fresh specimens and all fins membranes colorless in life and fresh specimens. our identification was confirmed by tan heok hui (lee kong chian natural history museum, singapore). the discovery of l. falcifer from the rawa pening lake and solo river (figs. 2, 3), both in java, are new records for this rare and vulnerable species, extending its distribution in about 80 km to the southeast from the previous closest record (fig. 3). there are several studies on freshwater fishes in indonesia that are limited to a single or few rivers. in sumatra, kottelat and tan (2008) recorded l. ixocheilos kottelat & tan, 2008 on batang hari basin, and then iqbal et al. (2017) l. ixocheilos on musi basin, being sampled in two localities separated by more than 150 km. new table 1. morphometric and meristic data of lobocheilos falcifer. morphometric data (mm) present study kottelat and tan (2008) gbi0002 gbi0003 gbi0004 total length 161.0 176.0 168.0 136.8 standard length 134.0 143.0 137.0 115.1 head length 25.0 25.9 25.5 25.8 snout length 12.2 12.5 12.2 12.0 pre dorsal length 50.4 60.0 50.7 44.7 pre ventral length 60.2 60.5 60.3 51.3 pre anal length 95.0 100.0 95.0 75.0 meristic data dorsal-fin rays 11 11 11 12 pectoral-fin rays 16 17 16 17 ventral-fin rays 9 9 9 9 anal-fin rays 8 8 8 8 caudal-fin rays 16 16 17 17 figure 3. distribution of lobocheilos falcifer in java. black rhombus are the previous records of the species on cisadane river (west java province), wadaslintang reservoir (central java provnce), and tuntang river (central java province). black aquares are the new records of this study, on rawa pening lake and solo river (central java province). 247 int. j. aquat. biol. (2021) 9(4): 244-247 records of endemic species are an important contribution for the understanding of species diversity and biogeography, among other biological topics (hasan and islam, 2020; hasan et al., 2021). acknowledgments we thank pedro bragança (south african institute for aquatic biodiversity, south africa) for the english language review, generasi biologi indonesia foundation and andik setiawan as our guide; and the ministry of finance indonesian as the scholarship program sponsor and for funding our research (no. 20160221035555). fpo thanks coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes finance code 001), and fundação de amparo à pesquisa e ao desenvolvimento científico e tecnológico do maranhão (fapema) for providing the financial and infrastructure support to carry out his scientific research. finally, we thank tan heok hui from lee kong chian natural history museum, singapore for confirming the specimens identification. references ciccotto p.j., page l.m. (2016). from 12 to one species: variation in lobocheilos rhabdoura (fowler, 1934) (cyprinidae: labeonini). copeia, 104(4): 879-889. ciccotto p.j., tan h.h. (2018). a new species of lobocheilos (teleostei: cyprinidae) from east kalimantan, indonesian borneo. zootaxa 4399(4): 543552. fricke r., eschmeyer w.n., van der laan r. (2021). eschmeyer's catalog of fishes: genera, species, references. (http://researcharchive.calacademy.org/res earch/ichthyology/catalog/fishcatmain.asp). electronic electronic version accessed 07 jul 2021. hadiaty r.k. (2011). diversitas dan kehilangan jenis ikan di danau-danau aliran sungai cisadane. jurnal iktiologi indonesia, 11(2): 143-15. hasan v., tamam m.b. (2019). first record of the invasive nile tilapia, oreochromis niloticus (linnaeus, 1758) (perciformes, cichlidae), on bawean island, indonesia. check list, 15(1): 225-227. hasan v., gausmann p., nafisyah a.l., isroni w., widodo m.s., islam i., chaidir r.r.a. (2021). first record of longnose marbled whipray fluvitrygon oxyrhyncha (sauvage, 1878) (myliobatiformes: dasyatidae) in malaysian waters. ecologica montenegrina, 40: 75-79. hasan v., soemarno., widodo m.s., wiadnya d.g.r., akhmad t.m., bambang i. (2019a). distribution extension and first record of lobocheilos falcifer (cypriniformes, cyprinidae) in central java province, indonesia. ecology, environemnet and conservation 25 (july supplement issue): s119-s122. hasan v., soemarno., widodo m.s., wiadnya d.g.r. (2019b). lobocheilos falcifer (valenciennes, 1842) (cyrpiniformes, cyprinidae): distribution extension in java and first record from tuntang river, semarang regency, indonesia. ecology, environment and conservation, 25(4): 1713-1715. hasan v., islam i. (2020). first inland record of bull shark carcharhinus leucas (müller & henle, 1839) (carcharhiniformes: carcharhinidae) in celebes, indonesia. ecologica montenegrina, 38: 12-17. iqbal m., setiawan a., aprilia i., isa m., yustian i. (2017). first record of lobocheilos ixocheilos kottelat & tan, 2008 (cypriniformes, cyprinidae) in south sumatra province, indonesia. check list, 13(6): 931-933. kottelat m., tan h.h. (2008). a synopsis of the genus lobocheilos in java, sumatra and borneo, with descriptions of six new species (teleostei: cyprinidae). ichthyological exploration of freshwaters, 19: 27-58. kottelat m., whitten a.j., kartikasari s.n., wirjoatmodjo s. (1993). freshwater fishes of western indonesia and sulawesi. periplus editions, hong kong. 221 p. lumbantobing d. (2019). lobocheilos falcifer. the iucn red list of threatened species 2019: e.t91005744a91005782. available in: http://dx.doi. org/10.2305/iucn.uk.2019-2.rlts.t91005744a910 05782.en. access on: july 5, 2021. rainboth w.j. (1991). cyprinids of south east asia. in: i.j. winfield, j.s. nelson (ed.). cyprinid fishes: systematics, biology and exploitation. chapman and hall, london. pp: 156-210. int. j. aquat. biol. (2015) 3(1): 1-13 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article rotifer assemblages (rotifera: eurotatoria) of the floodplain lakes of majuli river island, the brahmaputra river basin, northeast india bhushan kumar sharma*,1sumita sharma, mrinal kumar hatimuria freshwater biology laboratory, department of zoology, north-eastern hill university, umshing, shillong 793 022, meghalaya, india. article history: received 24 october 2014 accepted 2 december 2014 available online 25 february 2015 keywords: beels composition interesting taxa new records richness similarities abstract: our plankton and semi-plankton collections from twelve floodplain lakes (beels) of majuli river island, upper assam reveal 124 rotifer species (32 genera and 17 families); these merit biodiversity value as ~52.0% and ~30.0% of species, ~68.0 and ~45.0% of genera and ~74.0 and ~65.0% of the families of the phylum known from northeast india (nei) and india, respectively. two species are new to india with trichocerca uncinata as new record to the oriental region. eleven species are new to the study area and we provide an updated list (144 species) for following meta-analyses of majuli rotifera. biogeographically important elements include one australasian, four oriental, four palaeotropical and one cosmo (sub) tropical species while several species are of regional distribution interest. the rotifer fauna is predominantly tropical and lecanidae > lepadellidae collectively include ~53.0% species but it records paucity of brachionus species. individual beels record total richness of 60-100 (77 ± 12) species, monthly richness between 24 ± 7-34 ± 7 species and maximum up to 54 species/sample. the results are characterized by high community similarities (59.7-90.4% vide sørensen’s index), more rotifer homogeneity amongst beels, lack of any pattern of temporal richness variations and much limited influence of abiotic parameters. introduction segers et al. (1993) hypothesized (sub) tropical floodplain lakes to be the globally rich habitats for rotifer diversity. this hypothesis is extended to the rotifer assemblages of the floodplain lakes (beels) of assam (sharma, 2005; sharma and sharma, 2005a, 2008, 2012a, 2014a) and pats of manipur (sharma, 2009a, 2009b) states of nei. various ad-hoc works and incomplete inventories from other indian floodplains, however, do not facilitate any generalization. the present study is an attempt to analyze this salient feature as well as to examine ecosystem diversity of rotifera in twelve beels of majuli (commonly called ‘majoli’ or land in the middle of two parallel rivers) a geographically interesting landform of fluvial geomorphology of the brahmaputra river system in assam state of nei. * corresponding author: b. k. sharma e-mail address: profbksharma@gmail.com this largest river island, the world-heritage site and hotspot for flora and fauna is alarmingly shrinking due to erosion and is threatened to cease existence on the world map. we present an inventory of the examined rotifer species with comments on richness, composition, interesting elements, community similarities and influence of abiotic parameters. this report is of biodiversity and ecology value besides providing an update for following meta-analyses on the rotifer diversity of majuli studied earlier vide sharma (2014). materials and methods our observations are based on water and plankton and semi-plankton samples collected, during september, 2010-august, 2012, from twelve floodplain lakes (beels) of majuli river island 2 int. j. aquat. biol. (2015) 3(1): 1-13 (long.: 93°95'e, lat.: 25°27'n), upper assam (fig. 1, table 1). the sampled beels possessed different aquatic macrophytes’ namely eichhornia crassipes, hydrilla verticellata, utricularia flexuosa, trapa natans, lemna major, l. minor, pistia striates, salvinia sp., nymphaea spp., nymphoides spp., potamageton spp., azolla pinnata, euryale ferox, and sagittaria sp. the collections were obtained monthly from six beels (marked with*) and seasonally from rest of the beels (table 1). water samples were examined for abiotic parameters. water temperature, specific conductivity and ph were recorded by the field probes; dissolved oxygen was estimated by winkler’s method while free co2, total alkalinity and total hardness were analyzed following apha (1992). the qualitative plankton samples were collected by towing a plankton net (# 50 µm) from the littoral, limnetic/semi-limnetic regions of different beels and were preserved in 5% formalin. all samples were screened with wild (m8) dissecting microscope, different rotifers were isolated and mounted in polyvinyl alcohollactophenol mixture, and micro-photographs were taken with leica (dm 1000) stereoscopic phase contrast microscope fitted with an image analyzer. the different species were identified following koste (1978), segers (1995) and sharma and sharma (1999, 2000, 2008, 2013). the percentage similarities between rotifer assemblages of the sampled beels were calculated vide sørensen’s index (sørensen, 1948) and hierarchical cluster analysis was performed using spss (version 20). ecological relationships between abiotic factors and richness were calculated by pearson’s correlation coefficients (r); their p values were determined and significance was ascertained after bonferroni corrections. the reference collections were deposited in the holdings of freshwater biology laboratory, department of zoology, north-eastern hill university, shillong. results the details of the sampled beels and their recorded abiotic parameters (mean ± sd) are indicated in table 1. water temperature ranged between 23.4 ± 1.9-24.2 ± 2.3°c; ph between 6.51 ± 0.16-7.04 ± 0.19; specific conductivity between 110.2 ± 20.8210.4 ± 41.3 µs/cm; dissolved oxygen between 5.1 ± 1.8-7.8 ± 0.7 mg/l; free co2 between 10.3 ± 2.414.8 ± 4.6 mg/l; total alkalinity between 62.2 ± 13.4113.5 ± 24.6 mg/l; and total hardness varied between 60.8 ± 13.6-113.0 ± 23.8 mg/l. we observed a total of 124 species including two new records from india namely testudinella amphora (fig. 2a) and trichocerca uncinata (fig. 2b) and globally interesting brachionus kostei (fig.3a); keratella edmondsoni (fig. 3b), lecane blachei (fig. figure 1. district map of assam state indicating location of majuli river island (insert map of india indicating assam state of northeast india). figure 2. (a) testudinella amphora hauer and (b) trichocerca uncinata (voigt). 3 sharma et al./ rotifera of the floodplain lakes of majuli river island 3c), l. niwati (fig. 3d) and filinia camasecla (fig. 3e). in addition, brachionus durgae, keratella tecta, colurella adriatica, conochilus unicornis, lecane bifurca, lepadella lindaui, l. minuta, l. triba, mytilina michelangellii, pleosoma lenticulare and trichocerca scipio are new records to majuli rotifera thus updating their total tally to 144 species (appendix i). total richness in individual beels (appendix ii) ranged between 60-100 species and recorded 59.7-90.4% community similarities vide figure 3. (a) brachionus kostei shiel (after sharma, 2014), (b) keratella edmondsoni ahlstrom, (c) lecane blachei berzins, (d) lecane niwati segers, kothetip and sanoamuang and (e) filinia camascela myers. figure 4. hierarchical cluster analysis of rotifer assemblages of different beels. 4 int. j. aquat. biol. (2015) 3(1): 1-13 sørensen’s index (table 2). the hierarchical cluster analysis between rotifer assemblages of different beels is shown in figure 4. discussion water temperature concurred with geographical location of the sampled beels. the slightly acidic to circum-neutral and well-oxygenated waters of different beels are notable for low ionic concentrations as reflected by specific conductivity values; the latter warrant inclusion of these ecosystems under ‘class i’ category of trophic classification vide talling and talling (1965). free co2 occurred in all beels during the study period which, in turn, are characterized by ‘bicarbonate alkalinity’ and ‘moderately hard’ to ‘hard-water’ character. a total of 124 species, belonging to 32 genera and 17 families, observed from beels of majuli river island reflect rich rotifera assemblage and are of biodiversity value as ~52.0% and ~30.0% species of the taxon known till date from northeast india (nei) parameters↓ beels→ bhereki* ghotonga* holmari* chela* chakuli* khorkhoria* latitude 26°57′09.1″n 27°01′52.7″n 26°59′17.3″n 27° 04′58.2″n 26°56′40.3″n 26° 56′47.4″n longitude 94°12′23.0″e 94°15′28.7″e 94°12′30.6″e 94° 17′51.9″e 94°09′01.9″e 94°12′28.8″e altitude 67 m asl 73 m asl 75 m asl 89 m asl 69 m asl 74 m asl water temperature o c 23.7 ± 1.7 23.9 ± 1.7 23.6 ± 1.7 23.4 ± 1.9 23.6 ± 1.9 23.9 ± 1.8 ph 6.67 ± 0.23 6.51 ± 0.16 6.87 ± 0.13 7.04 ± 0.19 6.82 ± 0.18 6.80 ± 0.24 sp. conductivity µs/cm 140.7 ± 24.4 121.4 ± 26.8 173.6 ± 32.5 210.4 ± 41.3 180.8 ± 37.8 172.1 ± 44.4 dissolved oxygen mg/l 6.3 ± 0.9 6.2 ± 1.0 7.1 ± 0.8 7.8 ± 0.7 6.2 ± 0.8 6.4 ± 1.2 free co2 mg/l 13.6 ± 4.0 13.8 ± 3.4 10.2 ± 2.8 10.3 ± 2.4 14.8 ± 4.6 13.9 ± 5.0 total alkalinity mg/l 70.3 ± 20.7 62.2 ± 13.4 92.3 ± 14.2 113.5 ± 24.6 105.8 ± 29.0 90.2 ± 29.9 total hardness mg/l 69.8 ± 20.3 60.8 ± 13.6 89.3 ± 16.9 113.0 ± 23.8 104.0 ± 26.2 88.8 ± 27.2 parameters↓ beels→ doriya dubori tuni baatomaari jur chereki latitude 26°57′27.7″n 26°57′01.9″n 26° 58′35.3″n 26°59′25.9″n 26°59′45.3″n 26°58′25.4″n longitude 94°10′02.4″e 94°16′13.8″e 94°15′57.8″e 94°13′08.0″e 94°14′34.4″e 94°10′38.7″e altitude 70 m asl 70 m asl 67 m asl 71 m asl 71 m asl 67 m asl water temperature o c 24.2 ± 2.3 24.1 ± 1.9 23.9 ± 2.1 24.0 ± 1.9 23.9 ± 2.5 24.2 ± 1.7 ph 6.70 ± 0.32 6.61 ± 0.19 6.69 ± 0.14 6.87 ± 0.13 6.71 ± 0.14 6.62 ± 0.21 sp. conductivity µs/cm 110.2 ± 20.8 132.4 ± 18.6 123.6 ± 23.0 114.2 ± 20.5 130.8 ± 24.6 128.2 ± 33.2 dissolved oxygen mg/l 5.8 ± 1.2 7.0 ± 0.9 5.1 ± 1.8 6.2 ± 0.9 5.9 ± 1.1 6.1. ± 1.0 free co2 mg/l 12.0 ± 5.2 11.8 ± 4.2 12.2 ± 1.9 11.4 ± 1.9 12.7 ±3.5 12.1 ± 3.6 total alkalinity mg/l 67.3 ± 12.2 72.2 ± 11.4 82.3 ± 12.3 91.5 ± 16.2 88.9 ± 12.9 90.8 ± 16.6 total hardness mg/l 62.8 ± 12.6 70.8 ± 10.6 79.1 ± 15.6 89.0 ± 12.8 81.4 ± 12.0 86.9 ± 17.0 * sampled monthly between september, 2010–august, 2012; others sampled during winter (december/january), pre-monsoon (march-may), monsoon (june-august) and post-monsoon (september-october) between september, 2010–august, 2012. table 1. the sampled beels of majuli and their abiotic parameters (mean ± sd). beels 1 2 3 4 5 6 7 8 9 10 11 12 1 89.9 83.8 90.4 87.7 86.7 73.4 66.2 66.2 71.7 71.1 71.8 2 84.1 84.7 80.6 88.2 74.2 70.0 72.7 75.3 69.6 76.0 3 87.0 81.0 83.5 67.9 65.3 63.8 66.7 74.5 73.5 4 80.0 84.1 72.0 65.8 62.9 65.7 75.0 71.5 5 85.7 67.9 61.0 62.2 66.7 69.1 63.8 6 73.3 64.9 64.9 73.0 68.4 70.5 7 73.3 66.7 73.6 80.9 82.4 8 79.5 65.4 70.0 77.3 9 72.0 59.7 75.4 10 67.5 75.4 11 76.0 12 1-bhereki, 2-ghotonga, 3-holmari, 4-chakuli, 5-chela, 6-khorkhoria, 7-doriya, 8-dubori, 9-tuni, 10baatomari, 11-jur, 12-chereki. table 2. percentage similarities between rotifera assemblages of majuli beels. 5 sharma et al./ rotifera of the floodplain lakes of majuli river island and india, respectively. besides, our collections from this limited geographical study area include rich higher diversity of the taxon comprising ~68.0 and ~45.0% of genera and ~74.0 and ~65.0% of the families known from nei and india, respectively. testudinella amphora and trichocerca uncinata are new additions to indian rotifera. the former is known from australian, neotropical and oriental regions (segers, 2007); it is examined from the last region from thailand (sa-ardrit et al., 2013) and vietnam (trinh dang et al., 2013), and the present report extends its distribution to the indian subregion. the holarctic trichocerca uncinata is a noteworthy new report from the oriental region; the eastern himalayan tropical-latitude populations of this cold-water species may represent glacial relicts as hypothesized by segers (1996). this study adds eleven species to the rotifer fauna of majuli studied vide sharma (2014), thereby, raising their total tally to 144 species and thus affirm our hypothesis (sharma, 2005, 2009a; sharma and sharma, 2008, 2014a) on habitat diversity and environmental heterogeneity of the brahmaputra river basin floodplains. the sampled beels are species-rich than 110 rotifer species (arora and mehra, 2003) known from the backwaters of yamuna river at delhi while these are distinctly diverse than the reports of 48 species from 37 beels (sarma, 2000) and 64 species from 12 beels of the pobitora wildlife sanctuary (sharma, 2006) of assam state of nei; 27 species from two floodplain lakes of kashmir (khan 1987); and 38 species from four ox-bow lakes and nine floodplain lakes of south-eastern west bengal (khan, 2003). we caution on over-emphasis on comparisons with some ad-hoc indian reports with incomplete species inventories and inadequate sampling. the occurrence of biogeographically interesting elements namely the australasian brachionus kostei; the oriental endemics keratella edmondsoni, lecane blachei, l. niwati and filinia camasecla; the palaeotropical lepadella discoidea, l. vandenbrandei, lecane lateralis and l. unguitata; and cosmo (sub) tropical brachionus durgae imparts biodiversity value to our report. of these, brachionus kostei, lecane niwati and lepadella vandenbrandei are observed till date from this country from nei with l. niwati known only from majuli and loktak basin (sharma and sharma, 2014b). amongst the species of regional distribution interest, lepadella benjamini, l. dactyliseta, l. elongata, l. quinquecostata, lecane doryssa, l. pusilla, macrochaetus longipes, mytilina michelangellii and testudinella tridentata are known to be restricted to nei. total richness in individual majuli beels (60-100, 77 ± 12 species) needs cautious analysis with two beels indicating 100 species each while below average richness noticed in seven beels; the latter is hypothesized to invasion of eichhornia crassipes in these wetlands. the results, however, broadly concur with 69-93 species listed from various beels of assam (sharma and sharma, 2008) and also with 71 and 75 species reported from utra and waithou pats, respectively of manipur (sharma, 2011) but differ from relatively lower richness (62-73 species) observed from 15 floodplain lakes (pats) of manipur (sharma, 2009b). on the other hand, majuli beels record lower rotifer diversity than the reports of 151 (koste 1974) and 148 species from rao tapajos and lago camaleao (koste and robertson, 1983), respectively; 130 species from lake guarana, brazil (bonecker et al., 1994); 136 species (iyi-efi lake) and 124 species (oguta lake) from niger delta (segers et al., 1993). further, majuli rotifers are less diverse than that of 106 taxa from thale-noi lake, thailand (segers and pholpunthin, 1997); 104 species from laguana bufeos, bolivia (segers et al., 1998); and 114 taxa from the rio pilcomayo national park, formosa, argentina (jose de paggi, 2001). high rotifer community similarities amongst the sampled beels (59.7-90.4% vide sørensen’s index) and ~ 61.0% instances in the matrix with similarity values between 71-90% reflect more homogeneity in their composition. this is attributed to common occurrence of several lecane and lepadella species in various beels in particular. peak similarity 6 int. j. aquat. biol. (2015) 3(1): 1-13 between bhereki and chakuli beels is corroborated by their cluster groupings while lowest similarity value is observed between tuni and jur beels. rotifera assemblages of holmari and khorkhoria beels with high similarity (88.4%) also exhibit closer affinity vide hierarchical cluster analysis. on the other hand, dubori beel indicates greatest divergence in its species composition and is followed by baatomari beel. the rotifer taxocoenosis (overall and in individual beels) is predominantly tropical with rich diversity of ‘tropic centered’ lecane (42 species; 21-37, 28 ± 5 species) and common occurrence of several cosmotropical and pantropical species while lecanidae > lepadellidae collectively contribute the bulk of species (66 species, ~53.0%) as well as in individual beels (31-57, 42 ± 8 species). the relative consistency of the importance of periphytic taxa of the two most diverse families (~51.0-59.0%) supports hypothesis of green (2003) on the possibility of assemblage rules for the periphytic rotifer community. on the contrary, paucity of brachionus spp. is noteworthy; it may partly be attributed to the lack of definite pelagic habitats (de manuel, 1994) in the floodplains, shallow nature and the growth of aquatic macrophytes. the specific observations are, however, desired to highlight factors limiting their occurrence in majuli beels particularly in light of the report of 18 brachionus species in assam state (sharma and sharma, 2014c) with 16 species occurring in the brahmaputra floodplains (sharma and sharma, 2014d). of the six monthly sampled beels, the monthly rotifer richness varied between 24 ± 7 species (chela beel) 34 ± 7 species (khorkhoria beel); it lacked any pattern of monthly or annual variations. amongst seasonally sampled six beels, the richness varied between 33 ± 5 species (doriya beel) 42 ± 7 species (chereki beel) with maximum up to 53 and 54 species/sample in doryia and chereki beels, respectively but indicated no pattern of seasonal variations. the individual abiotic factors indicate much limited significant on the richness with significant positive and inverse correlations with rainfall only in holmari (r = 0.551, p = 0.0026) and chela (r = -0.588, p = 0.0013) beels, respectively. this generalization concurred with the results of sharma (2005, 2009b) and sharma and sharma (2012b) but is in contrast to notable influence of abiotic factors reported by sharma and sharma (2005b) and sharma (2009a). in conclusion, this study indicates rich and diverse rotifera assemblage of majuli beels in the indian context with various interesting species and is a useful contribution to the rotifer diversity in the floodplain ecosystems of india. our collections are yet biased towards planktonic and semi-planktonic taxa while specific analyses of periphytic, benthic and sessile taxa are likely to up-date the species inventory. the rotifers-aquatic macrophytes associations in these fluvial wetlands, the flushing influence of the brahmaputra waters on the rotifer assemblages and their subsequent re-colonization merits attention due to lack of such works from india. acknowledgements the senior author (bks) acknowledges the support of the ministry of environment & forests (govt. of india) sponsored project no. 22018-09/2010-cs (tax) for collections of the samples for this study. the senior author also thanks the head, department of zoology, northeastern hill university, shillong for laboratory facilities. the authors have no conflict of interests. references a.p.h.a. 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(1996). the biogeography of littoral lecane rotifera. hydrobiologia, 323: 169-197. segers h. (2007). annotated checklist of the rotifers (phylum rotifera), with notes on nomenclature, taxonomy and distribution. zootaxa, 1564: 1-104. segers h., ferrufino n.l., de meester l. (1998). diversity and zoogeography of rotifera (monogononta) in a flood plain lake of the ichilo river, bolivia, with notes on little known species. internationale revue hydrobiologie, 83(5-6): 439448. segers h., nwadiaro c.s., dumont h.j. (1993). rotifera of some lakes in the floodplain of the river niger (imo state, nigeria). ii. faunal composition and diversity. hydrobiologia, 250: 63-71. segers h., pholpunthin p. (1997). new and rare rotifera from thale noi lake, pattalang province, thailand, with a note on the taxonomy of cephalodella (notommatidae). annals limnologie, 33(1): 13-21. sharma b.k. (2005). rotifer communities of floodplain lakes of the brahmaputra basin of lower assam (n. e. india): biodiversity, distribution and ecology. hydrobiologia, 533: 209-221. sharma b.k. (2009a). diversity of rotifers (rotifera: eurotatoria) of loktak lake, northeastern india. tropical ecology, 50(2): 277-285. sharma b.k. (2009b). rotifer communities of floodplain lakes of manipur (north-east india): biodiversity, distribution and ecology. journal of bombay natural history society, 106(1): 45-56. sharma b.k. (2011). zooplankton diversity of two floodplain lakes (pats) of manipur, northeast india. opuscula zoologica, budapest, 42(2): 185-197. sharma b.k. (2014). rotifers (rotifera: eurotatoria) from wetlands of majuli the largest river island, the brahmaputra river basin of upper assam, northeast india. check list, 10(2): 292-298. sharma b.k., sharma s. (1999). freshwater rotifers (rotifera: eurotatoria). in: fauna of meghalaya. state fauna series, 4(9): 11-161. zoological survey of india, calcutta. sharma b.k., sharma s. (2000). freshwater rotifers (rotifera: eurotatoria). in: fauna of tripura: state fauna series. zoological survey of india, calcutta, 7 (4): 163-224. sharma b.k., sharma s. (2005a). biodiversity of freshwater rotifers (rotifera: eurotatoria) from northeastern india. mitteilungen aus dem museum für naturkunde berlin, zoologische reihe, 81: 81-88. sharma b.k., sharma s. (2005b). faunal diversity of rotifers (rotifera: eurotatoria) of deepor beel, assam 8 int. j. aquat. biol. (2015) 3(1): 1-13 (n.e. india)-a ramsar site. journal of bombay natural history society, 102(2): 169-175. sharma b.k., sharma s. (2012a). deepor beel-a ramsar site of india: an interesting hot-spot with its rich rotifera biodiversity. acta zoologica academiae scientiarum hungaricae, 58(2): 105-120. sharma b.k., sharma s. (2012b). rotifera diversity of a floodplain lake of the brahmaputra river basin of lower assam northeast india. opuscula zoologica, budapest, 43 (1): 67-77. sharma b.k., sharma s. (2014a). northeast india an important region with a rich biodiversity of rotifera. in: b.k. sharma, h.j. dumont, r.l. wallace (ed.). rotifera xiii: rotifer biologya structural and functional approach. international review of hydrobiology, 99(1-2): 20-37. sharma b.k., sharma s. (2014b). indian lecanidae (rotifera: eurotatoria: monogononta) and its distribution. in: b.k. sharma, h.j. dumont, r.l. wallace (ed.). rotifera xiii: rotifer biologya structural and functional approach. international review of hydrobiology, 99 (1-2): 38-47. sharma b.k., sharma s. (2014c). the diversity of indian brachionidae (rotifera: eurotatoria: monogononta) and their distribution. opuscula zoologica, budapest, 45(2): 165-180. sharma b.k., sharma s. (2014d). floodplains of the brahmaputra river basin-globally interesting ecotones with rich rotifer (rotifera: eurotatoria) biodiversity. in: r.k. sinha, b. ahmed (eds.). rivers for life proceedings of the international symposium on river biodiversity: ganges–brahmaputra meghna river system, ecosystems for life, a bangladesh india initiative, iucn, international union for conservation of nature, 258-270. sharma s. (2006). rotifer diversity (rotifera: eurotatoria) of floodplain lakes of pobitora wild-life sanctuary, assam. records of the zoological survey of india, 106(3): 76-89. sharma s., sharma b.k. (2008). zooplankton diversity in floodplain lakes of assam. records of the zoological survey of india, occasional paper no. 290: 1-307. sharma s., sharma b.k. (2013). faunal diversity of aquatic invertebrates of deepor beel (a ramsar site), assam, northeast india. wetland ecosystem series, 17: 1-226. talling j.f., talling i.b. (1965). the chemical composition of african lake waters. internationale revue der gesamten hydrobiologie, 50: 421-463. trinh dang m., segers h., sanoamuang l. (2013). rotifers from thuy tien lake and nhu y river in central vietnam, with a description of ploesoma asiaticum new species (rotifera: monogononta). journal of limnology, 72 (2): 276-286. 9 sharma et al./ rotifera of the floodplain lakes of majuli river island family: brachionidae 1. anuraeopsis fissa gosse, 1851 2. brachionus angularis gosse, 1851 3. b. durgae dhanapathi, 1974** 4. b. calyciflorus pallas, 1766 5. b. dichotomus reductus koste & shiel, 1980 6. b. diversicornis (daday, 1883) 7. b. falcatus zacharias, 1898 8. b. kostei shiel, 1983 9. b. quadridentatus hermann, 1783 10. keratella cochlearis (gosse, 1851) 11. k. edmondsoni ahlstrom, 1943 12. k. lenzi hauer, 1953 13. k. tecta (gosse, 1851)** 14. k. tropica (apstein, 1907) 15. platyias quadricornis (ehrenberg, 1832) 16. plationus patulus (o.f. müller, 1786) family: euchlanidae 17. beauchampiella eudactylota (gosse, 1886) 18. euchlanis dilatata ehrenberg, 1832 19. e. incisa carlin, 1939 20. e. triquetra ehrenberg, 1838 21. dipleuchlanis ornata segers, 1993 22. d. propatula (gosse, 1886) 23. tripleuchlanis plicata (levander, 1894) family: mytilinidae 24. lophocharis oxysternon (gosse, 1851) 25. mytilina acanthophora hauer, 1938 26. m. bisulcata (lucks, 1912) 27. m. michelangellii reid & turner** 28. m. ventralis (ehrenberg, 1830) family: trichotriidae 29. macrochaetus collinsi (gosse, 1867) 30. m. longipes myers, 1934 31. m. sericus (thorpe, 1893) 32. trichotria tetractis (ehrenberg, 1830) family: lepadellidae 33. colurella adriatica ehrenberg, 1831** 34. colurella colurus (ehrenberg, 1830) 35. c. obtusa (gosse, 1886) 36. c. uncinata (o.f. müller, 1773) 37. lepadella acuminata (ehrenberg, 1834) 38. l. apsida harring, 1916 39. l. benjamini harring, 1916 40. l. biloba hauer, 1958 41. l. costatoides segers, 1992 42. l. dactyliseta (stenroos, 1898) 43. l. discoidea segers, 1993 44. l. elongata koste, 1992 45. l. eurysterna myers, 1942 46. l. latusinus (hilgendorf, 1899) 47. l. lindaui koste, 1981** 48. l. minuta (weber & montet, 1918)** 49. l. ovalis (o.f. müller, 1786) 50. l. patella (o.f. müller, 1773) 51. l. quinquecostata (lucks, 1912) 52. l. rhomboides (gosse, 1886) 53. l. triptera ehrenberg, 1832 54. l. triba myers, 1934** 55. l. vandenbrandei gillard, 1952 56. l. (heterolepadella) apsicora myers, 1934 57. l. (h.) ehrenbergi (perty, 1850) 58. l. (h.) heterostyled (murray, 1913) 59. squatinella lamellaris (o. f. müller, 1786) family: lecanidae 60. lecane aculeata (jakubski, 1912) 61. l. arcula harring, 1914 62. l. bifurca (bryce, 1892)** 63. l. blachei berzins, 1973 64. l. bulla (gosse, 1851) 65. l. closterocerca (schmarda, 1859) 66. l. crepida harring, 1914 67. l. curvicornis (murray, 1913) 68. l. decipiens (murray, 1913) 69. l. doryssa harring, 1914 70. l. elongata harring & myers, 1926 71. l. flexilis (gosse, 1886) 72. l. furcata (murray, 1913) 73. l. haliclysta harring & myers, 1926 74. l. hamata (stokes, 1896) 75. l. hornemanni (ehrenberg, 1834) 76. l. inermis (bryce, 1892) 77. l. inopinata harring & myers, 1926 78. l. lateralis sharma, 1978 79. l. leontina (turner, 1892) 80. l. ludwigii (eckstein, 1883) 81. l. luna (o.f. müller, 1776) 82. l. lunaris (ehrenberg, 1832) 83. l. monostyla (daday, 1897) appendix i: systematic list of rotifer taxa known from majuli river island phylum: rotifera class: eurotatoria subclass: monogononta order: ploima 10 int. j. aquat. biol. (2015) 3(1): 1-13 84. l. nitida (murray, 1913) 85. l. niwati segers, kotethip & sanoamuang, 2004 86. l. obtusa (murray, 1913) 87. l. ohioensis (herrick, 1885) 88. l. papuana (murray, 1913) 89. l. paxiana hauer, 1940 90. l. ploenensis (voigt, 1902) 91. l. pusilla harring, 1914 92. l. pyriformis (daday, 1905) 93. l. quadridentata (ehrenberg, 1830) 94. l. rhytida harring & myers, 1926 95. l. signifera (jennings, 1896) 96. l. simonneae segers, 1993 97. l. stenroosi (meissner, 1908) 98. l. styrax (harring & myers, 1926) 99. l. tenuiseta harring, 1914 100. l. thienemanni (hauer, 1938) 101. l. undulata hauer, 1938 102. l. unguitata (fadeev, 1925) 103. l. ungulata (gosse, 1887) family : notommatidae 104. cephalodella forficula (ehrenberg, 1830) 105. c. gibba (ehrenberg, 1830) 106. monommata longiseta (o. f. müller, 1786) 107. m. maculata harring & myers, 1930 108. notommata pachyura (gosse, 1886) family: scaridiidae 109. scaridium longicaudum (o.f. müller, 1786) family : trichocercidae13/1 110. trichocerca abilioi segers & sarma, 1993 111. t. bicristata (gosse, 1887) 112. t. cylindrica (imhof, 1891) 113. t. elongata (gosse, 1886) 114. t insignis (herrick, 1885) 115. t. insulana (hauer, 1937) 116. t. pusilla (jennings, 1903) 117. t. rattus (o.f. müller, 1776) 118. t. scipio (gosse, 1885)** 119. t. similis (wierzejski, 1893) 120. t. tenuior (gosse, 1886) 121. t. tigris (o.f. müller, 1786) 122. t. uncinata (voigt, 1902)* 123. t. voluta (murray, 1913) 124. t. weberi (jennings, 1903) family : asplanchnidae 125. asplanchna priodonta gosse, 1850 family : synchaetidae 126. pleosoma lenticulare herrick, 1885** 127. polyarthra vulgaris carlin, 1943 family : dicranophoridae 128. dicranophoroides caudatus (ehrenberg, 1834) 129. d. forcipatus (o. f. müller, 1786) order: flosculariaceae family: conochilidae 130. conochilus unicornis rousselet, 1892** family : floscularidae 131. sinantherina socialis (linne, 1758) 132. s. spinosa (thorpe, 1893) family : hexarthridae 133. hexarthra mira (hudson, 1871) family : testudinellidae 134. testudinella amphora hauer, 1938* 135. testudinella emarginula (stenroos, 1898) 136. t. patina (hermann, 1783) 137. t. tridentata smirnov, 1931 138. pompholyx sulcata hudson,1885 family : trochosphaeridae 139. filinia camasecla myers, 1938 140. f. longiseta (ehrenberg, 1834) 141. trochosphaera aequatorialis semper, 1872 sub-class : digononta order : bdelloidea family : philodinidae 142. philodina roseola ehrenberg, 1832 143. rotaria neptunia (ehrenberg, 1830) 144. r. rotatoria (pallas, 1766) ----------------------------------------------------------------------------------------------------------------------------------------------- * new record from india, ** new record from majuli river island 11 sharma et al./ rotifera of the floodplain lakes of majuli river island taxa↓ beels sr. no.→ 1 2 3 4 5 6 7 8 9 10 11 12 order: ploima family: asplanchnidae 1. asplanchna priodonta gosse + + + + + + + + + + family: brachionidae 2. anuraeopsis fissa gosse + + + + + + + + + 3. brachionus angularis gosse + + + + 4. b. durgae dhanapathi + + + 5. b. falcatus zacharias + + + + 6. b. kostei shiel + + 7. brachionus quadridentatus hermann + + + + + + + + + + + + 8. keratella cochlearis (gosse) + + + + + + + + + + + + 9. k. edmondsoni ahlstrom + 10. k. lenzi hauer + + + + + + + 11. k. tecta (gosse) + + + + + 12. k. tropica (apstein) + + + + + + + + + + 13. platyias quadricornis (ehrenberg) + + + + + + + + + + + + 14. plationus patulus (o.f. müller) + + + + + + + + + + + + family: euchlanidae 15. beauchampiella eudactylota (gosse) + + + + + + + + + 16. dipleuchlanis propatula (gosse) + + + + + + + + + + + 17. euchlanis dilatata ehrenberg + + + + + + + + + + + + 18. e. triquetra ehrenberg + + + + + + + 19. tripleuchlanis plicata (levander) + + + + + + + family: flosculariidae 20. sinantherina socialis (linne) + + + + + + + 21. s. spinosa (thorpe) + + + + + + + + + + + + family: lecanidae 22. lecane aculeata (jakubski) + + + + + + + + + + 23. l. arcula harring + + + + 24. l. bifurca (bryce) + + + + 25. l. blachei berzins + + + + + + + + + 26. l. bulla (gosse) + + + + + + + + + + + + 27. l. closterocerca (schmarda) + + + + + + + + + + + + 28. l. crepida harring + + + + + + 29. l. curvicornis (murray) + + + + + + + + + + + 30. l. decipiens (murray) + + + 31. l. doryssa harring + + + + + + + + 32. l. flexilis (gosse) + + + + 33. l. furcata (murray) + + + + + + + + + + + 34. l. haliclysta harring & myers + + + 35. l. hamata (stokes) + + + + + + + + + + + + 36. l. hornemanni (ehrenberg) + + + + + + + + + 37. l. inermis (bryce) + + + + + + + 38. l. inopinata harring & myers + + + + 39. l. lateralis sharma + + + + + + + + + + 40. l. leontina (turner) + + + + + + + + + + + + 41. l. ludwigii (eckstein) + + + + + + + + + + + 42. l. lunaris (ehrenberg) + + + + + + + + + + + + 43. l. luna (o.f. müller) + + + + + + + + + + + + appendix ii: species composition of rotifera of beels of majuli river island. 12 int. j. aquat. biol. (2015) 3(1): 1-13 appendix ii: species composition of rotifera of beels of majuli river island (contd.). taxa↓ beels sr. no.→ 1 2 3 4 5 6 7 8 9 10 11 12 44. l. monostyla (daday) + + + + + + + 45. l. nitida (murray) + + + + + 46. l. niwati segers, kotethip & sanoamuang + + + 47. l. obtusa (murray) + + + + + + + + + + 48. l. ohioensis (herrick) + + + + + + + + + 49. l. papuana (murray) + + + + + + + + + + + + 50. l. paxiana hauer + + + 51. l. ploenensis (voigt) + + + + + + + + + + + 52. l. pusilla harring + + + + + + 53. l. pyriformis (daday) + + + + + + + + + + 54. l. quadridentata (ehrenberg) + + + + + + + + + + + + 55. l. rhytida harring & myers + + 56. l. signifera (jennings) + + + + + + + + + + + + 57. l. stenroosi (meissner) + + + + + + + + + 58. l. styrax (harring & myers) + + + + + + 59. l. tenuiseta harring + + + 60. l. thienemanni (hauer) + + + 61. l. undulata hauer + + + + + + 62. l. unguitata (fadeev) + + + + + + + + + + + + 63. l. ungulata (gosse) + + + + + + + + + + + family: lepadellidae 64. colurella adriatica ehrenberg + + + 65. colurella obtusa (gosse) + + + + + + + + + + + + 66. c. uncinata (o.f. müller) + + + + + + + + + + + + 67. lepadella acuminata(ehrenberg) + + + + + + + + 68. l. apsida harring + + + + + + + 69. l. benjamini harring + + + + + + + + + + 70. l. biloba hauer + + + + + 71. l. costatoides segers + + + + + 72. l. dactyliseta (stenroos) + + + 73. l. discoidea segers + + + + + + + + + 74. l. elongata koste + + + + + + 75. l. lindaui koste + + + 76. l. minuta (weber & montet) + + 77. l. ovalis (o.f. müller) + + + + + + + + + + + + 78. l. patella (o.f. müller) + + + + + + + + + + + + 79. l. quinquecostata (lucks) + + + 80. l. rhomboides (gosse) + + + + + + + + + 81. l. triba myers + + + + 82. l. triptera ehrenberg + + + + 83. l. vandenbrandei gillard + + + + 84. l. (heterolepadella) apsicora myers + + + + 85. l. (h.) ehrenbergi perty + + + + + + + + + + + + 86. l. (h.) heterostyla (murray) + + + + + + + + + 87. squatinella lamellaris (o.f. müller) + + + + + + + family: mytilinidae 88. lophocharis oxysternon (gosse) + + + + + + 89. mytilina acanthophora hauer + + + + + + 90. m. bisulcata (lucks) + + + + + + + + 13 sharma et al./ rotifera of the floodplain lakes of majuli river island taxa↓ beels sr. no.→ 1 2 3 4 5 6 7 8 9 10 11 12 91. m. michelangellii reid & turner + + + + + 92. m. ventralis (ehrenberg) + + + + + + + + + + family: notommatidae + 93. cephalodella forficula + + + + + + 94. c. gibba(ehrenberg) + + + + + + + + + + 95. monommata longiseta(o.f. müller) + + + + + + + + + family: scaridiidae 96. scaridium longicaudum ( müller) + + + + + + + + + + + family: synchaetidae 97. pleosoma lenticulare herrick + + + + 98. polyarthra vulgaris carlin + + + + + + + + + + + + order: flosculariaceae family: conochilidae 99. conochilus unicornis rousselet + + + + + family: hexarthridae 100. hexarthra mira (hudson) + + + family: testudinellidae 101. testudinella amphora hauer + 102. testudinella emarginula stenroos + + + + + + + + + + 103. t. patina (hermann) + + + + + + + + + + + + 104. t. tridentata smirnov + + + + 105. pompholyx sulcata hudson + + + + + + + family: trichocercidae 106. trichocerca bicristata (gosse) + + + + + 107. t. cylindrica (imhof) + + + + + + + + + + 108. t. elongata (gosse) + + + + 109. t. insignis (herrick) + + + + + + + + + 110. t. rattus (o.f. müller) + + + + + + + + + + 111. t. scipio (gosse) + + + 112. t. similis (wierzejski) + + + + + + + + + 113. t. tigris (o.f. muller) + + + + + + + + 114. t. uncinata (voigt) + 115. t. weberi (jennings) + + + + family: trichotriidae 116. macrochaetus longipes myers + + + + + + 117. m. sericus (thorpe) + + + + + + + + + + + 118. trichotria tetractis (ehrenberg) + + + + + + + + + + + + family: trochosphaeridae 119. filinia camasecla myers + + 120. filinia longiseta (ehrenberg) + + + + + + + + + + 121. trochosphaera aequatorialis semper + + + sub-class: bdelloidea family: philodinidae 122. philodina citrina ehrenberg + + + + + + + + 123. rotaria neptunia (ehrenberg) + + + + + + 124. r. rotatoria (pallas) + + + + + total richness (species) 70 79 66 65 60 73 100 82 75 75 79 100 1-bhereki, 2-ghotonga, 3-holmari, 4-chakuli, 5-chela, 6-khorkhoria, 7-doriya, 8-dubori, 9-tuni, 10-baatomari, 11jur, 12-chereki. appendix ii: species composition of rotifera of beels of majuli river island (contd.). int. j. aquat. biol. (2021) 9(4): 258-263 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article body shape variation of garra rufa (teleostei, cyprinidae) populations in the tigris basin in iran using geometric morphometric analysis maryam saemi-komsari1, hamed mousavi-sabet*1, masoud sattari1, soheil eagderi2, saber vatandoust3, ignacio doadrio4 1department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 3department of fisheries, babol branch, islamic azad university, babol, iran. 4biodiversity and evolutionary group, museo nacional de ciencias naturales, csic, madrid, spain. s article history: received 2 june 2020 accepted 21 january 2021 available online 2 5 august 2021 keywords: shape variation phenotypic plasticity geometric morphometric iran abstract: geometric morphometric method was used to examine the body shape variations among the six populations of garra rufa, in iranian part of tigris basin. a total of 15 landmark-points was used for 170 specimens to hypothesize population differentiation of g. rufa in the six rivers and reservoir. in discriminant function analysis, 85.9% of original grouped cases correctly classified. principal component analysis (pca) and canonical variates analysis (cva) confirmed the significant difference between the populations. the results revealed that the studied populations are divided into three clades based on differences in body depth, caudal peduncle length, backward moving of anal fin. caudal peduncle showed shortening trend in five populations. narrower body shape was dominated among specimens of four regions. studies on body shape provide supporting data on fisheries, stock management, and conservative programs. introduction geographical isolation and interbreeding are resulted in morphometric variations among populations of a single species (bookstein, 1991; torres-dowdall et al., 2012; heidari et al., 2013, 2014; kohestaneskandari et al., 2014). body shape plays an important role in fish locomotion, feeding behavior, and predation reflecting evolutionary adaptation in response to environmental pressures (webb, 1982; guill et al., 2003; mousavi-sabet and anvarifar, 2013; mousavi-sabet et al., 2018). therefore, morphological variations of different populations as part of adaptation to their habitats can guarantee the survival of the population (nacua et al., 2010; paknejad et al., 2014; vatandoust et al., 2015). a main purpose of morphometric study is to test hypotheses about the factors affecting body shape. geometric morphometrics is an approach to study shape using landmark points (webster et al., 2010). the landmarkbased analysis uses various statistical techniques to exclude size, position, and orientation, therefore only *correspondence: hamed mousavi-sabet doi: https://doi.org/10.22034/ijab.v9i4.611 e-mail: mousavi-sabet@guilan.ac.ir shape variables can be extracted (webster et al., 2010; adams et al., 2004). the family of cyprinidae includes the most diverse taxa distributing in all basins of iran (esmaeili et al., 2018). one of the most phylogeographically interesting cyprinid genus is garra distributed in southern, southwestern and northwestern asia (esmaeili et al., 2016, 2018; mousavi-sabet et al., 2019). the genus garra hamilton, 1822 with 151 valid species, is one of the most diverse genera of the labeoninae, and has a widespread distribution ranging from east asia to africa (sayyadzadeh et al., 2015; mousavi-sabet and eagderi, 2016; froese and pauly, 2021). in iran, the genus is found in tigris, persis, hormuz, makran, mashkid, and sistan, jazmurian, kerman and lut basins (esmaeili et al., 2016). garra rufa inhabits harsh ecological conditions in different environment having high ability to tolerance a wide range of environmental factors that results its wide distribution and variation in body shape (coad, 2018). therefore, study of population of g. rufa could 259 int. j. aquat. biol. (2021) 9(4): 258-263 well describe its morphometric variation and phenotypic plasticity. hence, in the present study, we compare the body shape of different populations of g. rufa from the tigris basin using geometric morphometric method. materials and methods a total of 170 specimens of g. rufa were collected from six rivers of iranian part of the tigris basin, including godarkhosh (33°30′16″n, 45°54′3″e), sirvan (35°05'12.3"n 46°05'19.2"e), chardavol (33°45′n 46°34′e), leileh (35°03'30.9"n 45°57' 27.7"e), baneh rivers (36°00'31.1"n 45°54'15.6"e), and siah-gav twin lakes (32°52'03.1"n 47°42'03.7"e) using electrofishing device during september 2013 september 2015. the collected fish were preserved in 10% buffered formalin after anesthesia. sexual dimorphism does not appear in genus garra; therefore, sex determination has not been carried out. the left side of each individual (with dorsal and anal fins were fixed by pins) was photographed, then 15 homologous landmark-points were defined and digitized on 2d images using tpsdig2 software version 2.16 to extract body shape data (rohlf 2004) (fig. 1). generalized procrustes analysis (gpa) carried out on data to remove non-shape data. principal component analysis (pca) was used to explain the variance-covariance structure to summarize the variation among the specimens. the multivariate analysis of variance (manova) and canonical variates analysis (cva) were used to investigate power of distinction among groups. mahalonobis distance also calculate to reveal the distance between the studied populations in terms of morphology. all statistical analyzes were done using past software (hammer, 2012) at the 95% confidence limit. clustering analysis was performed as the euclidean square distance clustered algorithm (sneath and sokal, 1973). results pca showed 52.81% of shape variations of the first two components derived from the variance-covariance matrix. however, the screen plot in pca showed, four component situated above the jullife broken line (julliffe cut-off=5.996e-05) include 74.67% of variance with significant level (table 1). the cva/ manova identified significant differences in body shape among the studied populations of g. rufa (p=2.305e-60 wilkils lambda=2.67). the pairwise figure 1. defined landmark points to extract the body shape data. 1. anterior tip of the premaxilla; 2. center of the eye; 3. dorsal edge of the head vertical to the eye center; 4. end of operculum; 5. nape at the beginning of the scale; 6 and 7. origin and insertion of the dorsal fin; 7. upper margin of caudal peduncle; 9. center of caudal peduncle; 10 lower margin of caudal peduncle; 11 & 12. insertion and origin insertion of the anal fin; 13. origin of the pectoral fin; 14. the lower beginning of gill slit; 15. ventral edge of the head vertical to the eye center. figure 2. the results of canonical discrimination analysis (cva) of the six studied population of the garra rufa in tigris basin with respect to the first two canonical variables based on body shape extracted from landmarks. 260 saemi-komsari et al./ body shape variation of garra rufa populations in the tigris basin hotelling's test of the groups showed significant differences in all groups (p<0.0001). the cva plot depicted based on the first two cvs clustered g. rufa population into three distinct groups and all population, showed in some extends overlapping (fig. 2). mahalonobis distance showed the most distances of the chardavol population from others in terms of the body shape (table 2). the body shape changes were latero-dorsal shift of the pectoral fin, shorter caudal peduncle, and smaller head with longer snout in chardavol population. in sirvan population, lower body depth and shorter caudal peduncle became highlighted. population of baneh can be identified by having lower body depth and longer caudal peduncle, whereas that of leilerizan showed lower body depth, and shorter caudal peduncle. however, the godarkhosh population showed ventral position of the snout, shorter caudal peduncle, and deeper body depth. population of siahgav spring can be identified by the deepest body depth, ventral position of the snout and short caudal peduncle. discriminant analysis (da) revealed 85.9% of original grouped cases correctly classified. 67.6% of cross-validated grouped cases correctly classified (table 3). the dendrogram derived from cluster analysis of euclidean square distances showed that six populations of g. rufa segregated from each other into table 1. eigenvalue and variance of the first four principle component of six studied populations of the garra rufa in tigris basin of iran. pc eigenvalue % variance 1 0.000896986 35.16 2 0.000450501 17.659 3 0.000299303 11.732 4 0.000198041 7.7628 total 72.31% table 2. mahalanobis distance analysis for the six studied population of the garra rufa in tigris basin of iran. table 3. classification matrix showing the number and percentage of individuals that were correctly classified. sites godarkhosh sirvan siagav chardavol leilerizan bane total original (%) godarkhosh 89.3 3.6 3.6 0 3.6 0 100 sirvan 10.7 78.6 7.1 0 3.6 0 100 siagav 3.3 6.7 80 0 3.3 6.7 100 chardavol 0 0 4.8 90.5 4.8 0 100 leilerizan 0 3.1 6.2 0 90.6 0 100 baneh 0 3.2 6.5 3.2 0 87.1 100 cross-validate (%) godarkhosh 67.9 14.3 3.6 0 14.3 0 100 sirvan 25 53.6 14.3 3.6 3.6 0 100 siagav 3.3 16.7 50 0 16.7 13.3 100 chardavol 4.8 0 19 66.7 4.8 4.8 100 leilerizan 3.1 3.1 6.2 0 84.4 3.1 100 baneh 3.2 3.2 6.5 3.2 3.2 80.6 100 cross validation is done only for those cases in the analysis. in cross validation, each case is classified by the functions derived from all cases other than that case. 85.9% of original grouped cases correctly classified. 67.6% of cross-validated grouped cases correctly classified. 261 int. j. aquat. biol. (2021) 9(4): 258-263 three distinct clusters, g. rufa from the siah-gav and baneh appeared in one cluster, along with godarkhosh population, leileh and chardavol formed the second one, and the last one refers to the sirvan population (fig. 4). discussions the experimental phenotypic discrepancies among the g. rufa populations discovered morphologically separated stocks in the studied populations of tigris basin in iran. phenotypic plasticity can be implied by phenotypic variations among the population specimens. head and mouth shape variation can be considered as reflective of differences in selection of food items and direction of feeding (langerhans et al., 2003). a fish with a mouth oriented upward usually feeds in the water column vs from bentic feeding behavior (andersson et al., 2005). morphological adaptations in freshwater fishes according to wide variety of physiological and environmental conditions result in genetic divergence and/or phenotypic plasticity (gatz, 1979; wainwright et al., 1994; eklov et al., 2006). phenotypic plasticity responded to environmental variations refer to niche patterns of resource utilization, behavior, and/or habitat use (gatz, 1979; wainwright et al., 1994; eklov et al., 2006; langerhans, 2008). moreover, diet pattern could influence morphology, while particular dietary items induce morphological change within or among populations (wainwright et al., 1994). however, abiotic factors including food abundance, temperature (hossain et al. 2010), body shape (beacham, 1990), amount of food (currens et al., 1989), and type of food or feeding mode (pakkasmaa, 2001; peres-neto and magnan, 2004; proulx and magnan, 2004) as well as biotic factors affect phenotypic plasticity (he et al., 2013). as mentioned before, in our study siah-gav reservoir showed the deepest body depth after godarkhosh vs. narrower body in other riverine habit. similarly, haas et al. (2010) found that deeper-bodied cyprinella (cyprinidae) are indicative of reservoir compared to riverine habitats. our study on population of g. rufa in chardavol showed lately higher position of the pectoral fin. it is supposed that lateral positioning of the pectoral fins correlates with the locomotory characteristics of particular species to improve maneuvering (webb, 1982; bandyopadhyay et al., 1997). figure 2. dendrogram derived from cluster analyses of morphometric variables on the basis of euclidean distance of the six studied population of the garra rufa in tigris basin of iran. the mean shape of species in relation of consensus shape of the garra rufa are represented. 262 saemi-komsari et al./ body shape variation of garra rufa populations in the tigris basin overall, studies on some species confirmed that shape differences could be related to trophic ecology (costa and cataudella, 2007), regarding local adaptation and ecological radiations (schluter and mcphail, 1992; langerhans et al., 2003). overall, geographical isolation plays key role on producing morphological variation in fish species (yamamoto et al., 2006). importance of environmental conditions and biogeographical patterns on morphological differentiation within communities is linked to the zoogeographical history of a region (hoagstrom and berry, 2008). selective pressures influencing individual mechanisms revealed how the process of evolution moves within the population in response to adaptation in their habitat. references adams d.c., rohlf f.j., slice d.e. 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(2014) 2(2): 105-110 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article fluoxetine alters reproductive performance of female fighting fish, betta splendens mohammad navid forsatkar*1, tahmine latifi2, soheil eagderi1 1department of fisheries, faculty of natural resources, the university of tehran, karaj, iran. 2department of fisheries, faculty of natural resources, university of zabol, zabol, iran. article history: received 25 october 2013 accepted 17 january 2014 available online 2 5 april 2014 keywords: fighting fish fluoxetine egg quality reproduction. abstract: this study was aimed to investigate the effects of waterborne fluoxetine on the reproduction performance of female fighting fish (betta splendens). for this purpose, mature, ready for spawning females were exposed to concentrations of 0, 0.54 and 54.0 µg/l fluoxetine for 7 days. then they were introduced into the spawning tank containing pre-acclimated male and reproductive consequences including number of copulations per spawning, number of eggs per copulation, duration of spawning, fecundity and hatching rate were assessed. fluoxetine concentration of 54.0 µg/l, was significantly affected on the number of produced eggs per copulation, fecundity and hatching rate. in addition, the mean number of copulations per spawning was not different between treatments but significantly different for the spawning duration between control and 54.0 µg/l treatments. the results suggest that fluoxetine can impacts on reproductive performance of female fighting fish at concentrations greater than those found in the aquatic environments. introduction selective serotonin reuptake inhibitors (ssris) are common treatment for alleviating depressive disorders (stroud, 2012). ssris block the serotonin transporter from taking-up serotonin released by presynaptic serotonergic neurons from synaptic junctions within the brain. this action results increasing serotonin concentrations within the synapse (kwon and armbrust, 2006). fluoxetine is one of the most prescribed ssri antidepressant that metabolize in liver (mennigen et al., 2010). following human excretion, fluoxetine enter into the aquatic environments through sewage (kwon and armbrust, 2006). potentially, it has an ability to accumulate in body which brain and liver are the main sites for its accumulation in non-target organism including fish (brooks et al., 2005). fluoxetine has been identified in the list of chemicals of concern, suggested to have the potential to impact wildlife populations (weinberger ii and klaper, * corresponding author: mohammad navid forsatkar e-mail address: nforsatkar@ut.ac.ir 2014). several works have indicated the negative effect of fluoxetine on fish reproduction. mennigen et al. (2008) administered fluoxetine to female goldfish (carassius auratus) and found that plasma estradiol levels and expression of estrogen receptors are reduced significantly after its injections. fluoxetine significantly reduced cumulative egg production in zebrafish (danio rerio; lister et al., 2009). also, fluoxetine concentrations as low as 1 µg/l, that is found in many freshwater environments, was found effective on mating behaviour, specifically nest building and defending in male fathead minnow (pimephales promelas; weinberger ii and klaper, 2014). however, fluoxetine did not have a significant effect on reproductive properties of japanese medaka, oryzias latipes (foran et al., 2004). in addition, fluoxetine impairs the reproductive axis of male fish (mennigen et al., 2010), that were previously reported in human and 106 forsatkar et al./ int. j. aquat. biol. (2014) 2(2): 105-110 other mammals (e.g. bataineh and daradka, 2007; safarinejad, 2008). fighting fish (betta splendens), is a suitable model to study the impacts of pollutant on reproductive traits (e.g. clotfelter and rodriguez, 2006). female fighting fish spawns under bubblenest constructed by male that cares brood in the bubble and exhibits some levels of aggression in contrast to any intruder (jaroensutasinee and jaroensutasinee, 2003). fluoxetine in both administration forms of injection (clotfelter et al., 2007) and exposure (dzieweczynski and hebert, 2012), reduces the aggressive behaviour of male fighting fish; however, there is no information about the effect of fluoxetine on female reproductive traits. therefore in this study, female fighting fish exposed to sub-lethal waterborne fluoxetine and then its reproductive consequences including fecundity, hatching rate, the number of copulations with its mate, the number of released eggs per copulation and whole spawning duration were investigated. material and methods fish: forty mature female b. splendens were obtained from a local pet store. they kept into two 20 l tanks filled with decholorinated tap water for 14 days. temperature and photoperiod set as 27±1°c and 12l: 12d, respectively. fish were fed twice a day with commercial pellet (biomar, germany) and frozen blood worms. in addition, 44 males were purchased from a local pet store for future spawning activities. they also fed twice a day with same food as females. experimental protocol: thirty gravid females from the stock were selected and randomly distributed into three 20 l glass tank (ten fish per tank) exposing with concentrations of 0, 0.54 and 54.0 µg/l fluoxetine for 7 days before spawning. the concentration of 0.54 µg/l is considered as natural occurrence of fluoxetine (fent et al., 2006); also we applied 54.0 µg/l to examine the acute toxicity of fluoxetine. for spawning activity, thirty 36 l glass tanks equipped to a float substrate for building bubblenest, a half flower pot for female hiding and a heater to kept temperature at 27°c were used. after nest building by male, each female was introduced into the spawning tank. they were spawned after about one day; however, because of egg mustering behaviour in fighting fish, direct fecundity calculation was impossible. therefore, we calculated the number of copulations per each spawning activity and the number of laid eggs by female in copulations. finally, the fecundity was estimated in each spawning as: number of eggs per copulation × number of copulations. duration of spawning was evaluated as an indicator of female readiness for spawning activity. also, hatching rate was investigated to examine the quality of eggs (schreck et al., 2001). statistics: all data were tested for normality and homogeneity of variances using kolmogorovsmirnov and the levene test, respectively. then, they were examined using a one-way anova following duncan’s multiple range test (p<0.05). all statistical analyses were performed using the statistical software of spss, version 19. results there were no significant differences in the body length of females at the beginning of experiment (f= 0.280, p= 0.758). after 7 days exposure period, the fecundity of 54.0 µg/l treatment was significantly different in compared to another exposed treatment (f= 5.938, p= 0.007; fig. 1). however, the fecundity was lower in 0.54 µg/l treatment (346.3 ± 82.89) than figure 1. effect of 7 days fluoxetine exposure on the fecundity of female betta splendens. data are means ± sd (n = 10; **p< 0.01). 107 forsatkar et al./ int. j. aquat. biol. (2014) 2(2): 105-110 that of control one (377.2 ± 101.93), but not significantly different (p= 0.434). the duration of spawning were significantly different between control treatment and females exposed to 54.0 µg/l fluoxetine (f= 3.825, p= 0.034; fig. 2). this duration was recorded 98.50 ± 23.45, 118.5 ± 27.89, and 131.50 ± 28.96 min for control, 0.54 µg/l and 54.0 µg/l treatments, respectively. figures 3 and 4 show the number of copulations per spawning and average number of eggs per copulation, respectively. after 7 days exposure to fluoxetine, there was insignificant differences of the mean number of copulations per spawning between treatments (f= 1.478, p= 0.246), this index was higher (51.8 ± 13.17) in 54.0 µg/l treatment than two others (44.5 ± 13.46, and 43.2 ± 9.01 for control and 0.54 µg/l treatments, respectively). the mean number of eggs per copulation was significantly lower in 54.0 µg/l treatment than that of control and 0.54 µg/l treatments (f= 9.063, p= 0.001). this ranged from 5.26 ± 2.74 in fish exposed to 54.0 µg/l treatment to 8.56 ± 1.05 in control one. the hatching rate was significantly different among treatments (f= 8.404, p= 0.001; fig. 5). this was 70.05 ± 13.02, 82.9 ± 6.45 and 86.11 ± 6.80 for 54.0 µg/l, 0.54 µg/l and control treatments, respectively. discussion fluoxetine causes some disruption in different aspect of fish life (e.g. mennigen et al., 2010, 2008; clotfelter and rodriguez, 2006). based on the results, the fecundity of exposed females to 54.0 µg/l fluoxetine was significantly lower than that of control and 0.54 µg/l treatments. females are responsible for the production of good quality eggs that are sufficient to guarantee future generations. any disturbance to this task could alter offspring success and have population consequences. lister et al. (2009) founded the significant reduction in cumulative egg production of zebrafish after 7 days exposure to 32 µg/l fluoxetine. they showed that figure 2. spawning duration (± sd) of female betta splendens after 7 days exposure to fluoxetine (n= 10; *p<0.05). figure 3. means ± sd of the number of copulations per spawning in female betta splendens after 7 days exposure to fluoxetine (n= 10). figure 4. average total amount of eggs laid in copulations of female betta splendens after 7 days exposure to fluoxetine. data are means ± standard deviation (n= 10; **p<0.01). figure 5. means ± sd of the hatching rate in female betta splendens after 7 days exposure to fluoxetine (n = 10; **p<0.01). 108 forsatkar et al./ int. j. aquat. biol. (2014) 2(2): 105-110 fluoxetine decreases both ovarian estradiol concentration and expression of fshr and lhr receptors. in the present study, we did not measured the plasma levels of sexual hormones and expression of reproductive genes; however, it is unquestionable that fluoxetine decreases ejaculatory response in human (safarinejad, 2008), and milt release (mennigen et al., 2010), and egg production (lister et al., 2009) in fish. complex processes are involved to produce mature gametes (maruska and fernald, 2011); therefore, fluoxetine maybe reduce the fecundity of female fighting fish due to change in circulatory concentrations of the sexual hormones and disruption of reproductive axis. despite of the lower fecundity of females exposed to 54.0 µg/l fluoxetine, they had more number of copulations in compared to control and 0.54 µg/l treatments, but not significantly different. the average number of released eggs per copulation in 54.0 µg/l treatment was significantly lower than that of others. in addition, the spent time by females for spawning in 54.0 µg/l treatment was significantly higher than that of control one. these results indicates that higher levels of fluoxetine is caused females to produced lower eggs in longer spawning duration. based on results, the females of 54.0 µg/l treatment copulate with its mate discontinuously and many of their copulations are terminated without any egg production. occurrence of secondary sexual traits in mosquito fish, gambusia affinis delayed after exposure to fluoxetine (henry and black, 2008). also, there are reports showing indolence or less basal swimming (kohlert et al., 2012) and aggressive behaviour (dzieweczynski and hebert, 2012) in male fighting fish after fluoxetine treatment. therefore, many unsuccessful copulations resulting less eggs production even during longer spawning period could be due to demolition of reproductive axis that led to postponing secondary sexual traits and non-proper spawning behavior. these results suggest that acute concentration of fluoxetine causes reduction in libido and impotence of female fighting fish. the hatching rate was decreased significantly after exposure to 54.0 µg/l fluoxetine. schreck et al. (2001) reported that low quality gametes lead to reduction of hatching rate. also, maternal fluoxetine administration in mice showed side effect on her generation (santos gouvêa et al., 2008). however, fluoxetine exposure had insignificant effect on hatching of japanese medaka (oryzias latipes; foran et al., 2004). this controversial results may be explained by different concentration of fluoxetine. the maximum fluoxetine level used by foran et al. (2004) was 5 µg/l which approximately 11 folds lower than our maximum concentration. it is obvious that hatching success of fighting fish depends on environmental conditions such as temperature (forsatkar and nematollahi, 2013) and photoperiod (giannecchini et al., 2012). however, these factors were similar in treatments; therefore decreased hatching rate may be a secondary effect following reduces of female condition. in conclusion, this study demonstrated the exposure of sexually mature female fighting fish to acute concentration (54.0 µg/l) of fluoxetine is led to decrease of reproductive consequences in compared to 0 and 0.54 µg/l treatments. these findings suggest that fish can be affected by far below lethal doses of fluoxetine. also, the observed results of this study indicate a dose dependent effect of fluoxetine on reproductive traits of female fighting fish. in addition, these results invite more attention to fate of pharmaceuticals in the aquatic ecosystems. references bataineh h.n., daradka t. 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(2019) 7(5): 260-270 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article effects of three anesthetics of clove extract, sodium bicarbonate, and lidocaine on blood parameters and cortisol level on male and female broodstocks of caspian kutum (rutilus kutum) leila babaiinezhad, masoumeh bahrekazemi*,1 department of fisheries, qaemshahr branch, islamic azad university, qaemshahr, iran. s article history: received 21 january 2019 accepted 1 july 2019 available online 2 5 october 2019 keywords: anesthetic rutilus kutum stress aquaculture abstract: in this study, 32 male and female caspian kutum were anesthetized with clove extract (50 mg/l), lidocaine (150 mg/l), and sodium bicarbonate (300 mg/l). blood parameters and serum cortisol levels were investigated in the control (no anesthetic) and the treated groups containing both males and females in 10 min and 24 h after anesthesia. based on the result, difference in the red blood cells in the control group and treated groups males was not significant. in females, however, the red blood cells in all treated groups was different except lidocaine treatment after both sampling times (p>0.05). the white blood cells of males and females in control group was significantly different in the sodium bicarbonate group in 10 min and 24 hours after anesthesia (p<0.05). the hemoglobin and hematocrit did not change in male and female fish in lidocaine treatment after both times. cortisol changed in male and female in the sodium bicarbonate group after both times. the wbcs in females was significantly higher in clove extract and lidocaine treatments, whereas rbcs, hb, hct, and cortisol were significantly higher in males (p<0.05). therefore, due to irreversible stressful effects of the sodium bicarbonate, it is not a suitable anesthetic for the studied fish. although the stressful effects of lidocaine were lower than clove extract, especially in females, the clove extract has no irreversible effects and the stress-induced effects had been moderated 24 h after anesthesia. hence, lidocaine and then clove extract is recommended as suitable anesthetics, especially for female caspian kutum. introduction anesthetics have significant applications in aquaculture such as sampling, transportation, artificial reproduction, hormone injections, vaccination, surgery, and biopsy (ross and ross, 2008; lepic et al., 2014). in all above-mentioned applications, anesthetic agents decrease physiological activities, which reduce imposed stress and avoid illnesses and death (keene et al., 1998). nowadays, it seems necessary to explore anesthetic compounds with superior features such as rapid induction, easy and quick return, rapid disposal of tissues, and lack of toxicity at therapeutic levels, along with cheapness and availability. hence, both natural and chemical compounds have been tested for this purpose (hoseini et al., 2013; mazandarani et al., 2015; taheri mirghaed et al., 2016; mazandarani and hoseini, 2016; effati and bahrekazemi, 2017; yousefi *correspondence: masoumeh bahrekazemi doi: https://doi.org/10.22034/ijab.v7i5.319 e-mail: bahr.kazemi@gmail.com et al., 2018). the commonly used anesthetics in aquaculture are tricaine methane sulfonate (ms222), benzocaine, quinaldine, metomidate, clove extract and 2-phenoxy ethanol (lepic et al., 2014). an active ingredient in clove extract and eugenol is 4-allyl-2-methoxyphenol (ross and ross, 2008). the effect of clove extract and clove oil has been studied on different fish species, including the roach (rutilus rutilus), caspian kutum (r. kutum), common carp (cyprinus carpio), persian sturgeon (acipenser persicus), rainbow trout (oncorhynchus mykiss), and beluga (huso huso) (wagner et al., 2002; velisek et al., 2005; soudagar et al., 2009; imanpoor et al., 2010; imanpoor and farahi, 2011; farahi et al., 2011; hoseini, 2011; hoseini and ghelichpour, 2012; mazandarani and hoseini, 2017). sodium bicarbonate (nahco3) was first introduced in 1942 as an 261 int. j. aquat. biol. (2019) 7(5): 260-270 anesthetic in aquatic animals (fish, 1942). by producing co2 from carbonic acid, sodium bicarbonate causes numbness in aquatic animals (wagner et al., 2002). however, there are few studies on the anesthetic effect of this compound on fishes like rainbow trout, and common carp (booke et al., 1978; altun et al., 2009). lidocaine with 2-(diethylamino)n-(2, 6-dimethylphenyl) acetimide chemical compound, is insoluble in freebase water and it is generally used as a water-soluble hydrochloride salt form to anesthetize aquatic animals (ackerman et al., 2005). lidocaine has so far been used as an anesthetic in some species such as common carp, silver carp (hypophthalmichthys molitrix), tilapia (oreochromis mossambica), zebrafish (danio rerio) and catfish (ictalurus punctatus) (carrasco et al., 1984; ross and ross, 2008; collymore et al., 2014; effati et al., 2014). although anesthetics can reduce physiological and biochemical stress in fish, some of these compounds may have side effects on hematological and biochemical parameters of blood. accordingly, a side effect of such compounds on tensions in the aquatic species is expected (lepic et al., 2014). many studies have been conducted to describe physiological effects of various anesthetics in fishes; however, none of them compared physiological effects of the anesthetics and their differences in males and females. in addition, few researchers such as imanpoor and farahi (2011) have focused on the effects of three anesthetics in caspian kutum. therefore, this study aimed to investigate the effects of three anesthetics, namely clove extract, sodium bicarbonate, and lidocaine on the blood parameters and cortisol level in caspian kutum and determine the best anesthetic in male and female broodstocks. materials and methods experimental design: the experimental process was carried out in the shirood river (tonekabon, mazandaran) in the southern caspian sea. during the caspian kutum migration and their artificial reproduction in 2016, the broodstocks were collected. among fish which were ready for propagation, 20 males and 20 females with age of 3 years, and average weight and total length of 352.5±56.2 g and 34.88±5.2 cm, respectively, were randomly selected. preparation of anesthetic solution: lidocaine (2%) was obtained from the pasteur institute of iran, sodium bicarbonate from the soda kaveh chemical industry co. and clove extract from the giah essence pharmaceutical co. to prepare the anesthetic solutions, 50 mg/l clove extract, 150 mg/l lidocaine, and 300 mg/l sodium bicarbonate were added to a water tank and then, the resultant solution was completely stirred and homogenized (table 1). after preparing water containing anesthetic solution with the desired concentrations (ackerman et al., 2005; velisek et al., 2005, 2011; collymore et al., 2014), five male and five female broodstocks of each anesthetic group placed in it and the time of their introduction was registered. fish were anesthetized through bathing in small tanks for starting the anesthetic process and then, they were transferred to larger tanks to recover (velisek et al., 2006) (table 1). blood sampling and blood parameters analyses: the blood samples were taken from the caudal vein in two steps (10 min and 24 h after recovery). for cortisol measurements, 2 ml of the blood was transferred to non-heparinized tubes, while the remaining blood (2 ml) was poured into the heparinized tubes for blood cell counts. the blood samples of the control fish with no anesthetics were also taken. the numbers of white blood cells (wbc) and red blood cells (rbc) were counted using a neubauer hemocytometer (houston et al., 1996). hematocrit was also measured by microhematocrit method (rehulka, 2000), while hemoglobin was determined through the cyanmethaemoglobin method using spectrophotometer with 540 nm wavelength. (blaxhall and daisley, 1973). for serum preparation and cortisol assay, the blood was centrifuged at 3000 rpm for 10 min and the serum collected and kept frozen at -80°c. cortisol level was determined using radioimmunoassay (yaghobi et al., 2015). statistical analysis: the statistical model was designed as completely random which was performed by two way repeated anova. the differences between treatments were investigated with duncan’s 262 babaiinezhad and bahrekazemi / effects of clove extract, sodium bicarbonate, and lidocaine on caspian kutum test. the pairwise t-test was also employed to compare blood parameters and cortisol of the sampling times in 10 min and 24 h after anesthesia. pillai’s multivariable analyze was used to compare the number of rbcs and wbcs along with the values of cortisol, hemoglobin and hematocrit in male and female broodstocks. all analysis were done by spss (version 23) at a confidence level of 95%. all values were presented as mean±sd. results male broodstocks: the results showed that 10 min after anesthesia, number of rbcs in the control group was almost the same as other three treatments. 24 hours after anesthesia, a little decrease of rbcs was observed in the groups of clove extract and lidocaine with a little increase of rbcs in the sodium bicarbonate group. however, differences were not significant with that of the control group (p>0.05) (table 2). comparison of 10 min and 24 h after anesthesia for rbcs counts in males revealed significant differences. since the wbcs in the control male fish was 8833.33±152.7 in mm3, the only different group was those anesthetized with sodium bicarbonate (p<0.05), 10 min and 24 h after anesthesia (table 2). in addition, comparison of wbcs in male broodstocks showed significant differences only with lidocaine (p<0.05) (table 2). hemoglobin levels in male of the control group was estimated as 9.37±0.31 g/dl and it is increased in all anesthetics group, 10 min after anesthesia with a significant increase in clove extract (p<0.05). 24 h after anesthesia, the fish anesthetized with sodium bicarbonate were only group having significant increase (table 2). comparison of the hemoglobin in males revealed significant differences in 10 min and 24 h in lidocaine treatment (p<0.05) (table 2). moreover, the hematocrit in males of the control group was 27.67±0.58%, and it was significantly elevated in the group with clove extract in 10 min after anesthesia (p<0.05) and comparing to control group, the hematocrit levels were increased in male fish anesthetized with sodium bicarbonate and clove extract after 24 h (table 2). the results showed that the hematocrit in males anesthetized with clove extract at 10 min and 24 h after anesthesia was almost the same (p>0.05) (table 2). the cortisol levels in male fish of the control group was 313.00±9.89 ng/ml. although levels of the hormone elevated after anesthesia in all treatments, significant differences were observed only in sodium bicarbonate treatment in both times of the sampling (p<0.05) (table 2). comparisons of the cortisol level in male showed the same result (p<0.05) (table 2). female broodstocks: the number of rbcs in control female broodstocks was significantly different from table 1. the doses of the anesthetics and the used procedure of anesthesia in this study. anesthetic dose anesthesia time (min) recovery time (min) clove extract 50 mg/l 5 5 sodium bicarbonate 300 mg/l 5 10 lidocaine 150 mg/l 2 5 table 2. blood and hormonal parameters measured in the male broodstocks of caspian kutum under anesthesia treatments. rbc (n/mm3) wbc (n/mm3) hb (g/dl) hct (%) cortisol (ng/ml) control 1183333±76376.26abc 8833.33±152.75a 9.37 ±0.31ab 27.67±0.58ab 313.00±9.89a clove extract (10 min.) 1133333±28867.51ab 8266.67±1193.03a 12.87 ±0.21c 39 .00±1c 418.67±87.55a clove extract (24 h) 1140000±52915.03abc 7166.67±461.88a 11.93±0.38bc 37.67 ±0.58c 422.67±67.53ab lidocaine (10 min.) 1200000±111355.29abc 7566.67±503.32a 11.5±0.75bc 33.63±1.53bc 437.67±22.68ab lidocaine (24 h) 936666±49328.83a* 6066.67±404.14a* 9.63±0.15ab* 20.33±1.53a* 347.33±24.58a sodium bicarbonate (10 min) 1323333±256969.52bc 13533.33±3780.6b 11.33±2.71bc 34.33±7.37bc 700.00±50.91c sodium bicarbonate (24 h) 1463333±327159.49c 14800±3064.31b 12.5±3.24 c 40±11.27c* 530.79±55.69b* data are presented as means ± standard error. significant differences of the blood parameter between different anesthetics at two sampling times and the control group are indicated by unlike letters in each column (p<0.05; duncan,s test), and significant differences of blood parameter between 10 min and 24 h sampling for each anesthetic are shown by the asterisk (*) (p<0.05; t-test). 263 int. j. aquat. biol. (2019) 7(5): 260-270 those in treated groups with clove extract and sodium bicarbonate 10 min after anesthesia (p<0.05). comparing to the control group, the rbcs 24 h after anesthesia was markedly increased in female fish received sodium bicarbonate (table 3). comparisons of the rbcs count in females revealed a significant differences 10 min and 24 h after anesthesia only in clove extract treatment (p<0.05) (table 3). the wbcs in control female fish was 7333.33±207.26 in mm3; the females anesthetized with all three anesthetics exhibited incremental trends, which was significant in fish treated with sodium bicarbonate (p<0.05). comparing to the control group, the wbcs in all three anesthetic treatments was not significantly different 24 h after anesthesia (p>0.05) (table 3). comparisons of the wbcs in female broodstocks confirm significant differences only in lidocaine treatment in 10 min and 24 h (table 3). hemoglobin levels in the females of the control group was 7.3±0.46 g/dl, which significantly increased 10 min after anesthesia with clove extract and sodium bicarbonate (p<0.05). compared to the control group, a significant increases observed 24 h after anesthesia in sodium bicarbonate group (table 3). considerable differences after 10 min and 24 h after anesthesia are observed in all treatments (p<0.05) (table 3). also, the hematocrit in females of the control group was 23.33±0.58% and increased 10 min after anesthesia with clove extract and sodium bicarbonate (p<0.05). the hematocrit levels markedly table 3. blood and hormonal parameters in the female broodstocks of caspian kutum under anesthesia treatments. rbc (n/mm3) wbc (n/mm3) hb (g/dl) hct (%) cortisol(ng/ml) control 968333.3±75883.68a 7333.33±207.26a 7.3±0.46 a 23.33±0.58 a 325.50±31.82 a clove extract (10 min.) 1393333±187705.44b 12300±468.22ab 12.93±1.65 c 39.67±5.51 c 383.50±12.16 ab clove extract (24 h) 993333.3±66583.28a* 9166.67±445.09ab 8.47±0.49 ab* 25.67±1.53 a* 343.33±11.42 ab lidocaine (10 min) 966666.7±15275.25a 8066.67±378.59ab 6.5±0.2 a 22.67±2.52 a 361.00±39.59 ab lidocaine (24 h) 1026667±49328.28a 7450±353.55a* 8.57±0.3 ab* 26±1 a 302.33±41.64 a* sodium bicarbonate (10 min) 1413333±55075.7b 12900±100b 13.03±0.21 c 40.33±1.53 b 637.00±24.88 c sodium bicarbonate (24 h) 1403333±50332.23b 9433.33±602.77ab 10.47±2.76 b* 40±1 b 490.00±20.52 b* data are presented as means ± standard error. significant differences of the blood parameter between different anesthetics at two sampling times and the control group are indicated by unlike letters in each column (p<0.05; duncan,s test), and significant differences of blood parameter between 10 min and 24 h sampling for each anesthetic are shown by the asterisk (*) (p<0.05; t-test). table 4. multivariate pillai test results of the blood and hormonal parameters. effect formula red blood cell sampling time (10 min and 24 h) pillai=0.002, f (1,19)= 0.031, sig= 0.862 sampling time × anesthetic drug pillai=0.332, f (3,19)= 3.15, sig= 0.049 gender (male and female) pillai=0.028, f (1,19)= 0.555, sig= 0.465 hemoglobin sampling time (10 min and 24 h) pillai=0.013, f (1,19)= 0.248, sig= 0.624 sampling time × anesthetic drug pillai=0.328, f (3,19)= 3.088, sig= 0.052 gender (male and female) pillai=0.058, f (1,19)= 1.161, sig= 0.295 hematocrit sampling time (10 min and 24 h) pillai=0.006, f (1,19)= 0.106, sig= 0.748 sampling time × anesthetic drug pillai=0.360, f (3,19)= 3.563, sig= 0.034 gender (male and female) pillai=0.064, f (1,19)= 1.298, sig= 0.269 white blood cell sampling time (10 min and 24 h) pillai=0.473, f (1,19)= 17.03, sig= 0.001 sampling time × anesthetic drug pillai=0.422, f (3,19)= 4.62, sig= 0.014 gender (male and female) pillai=0.383, f (1,19)= 11.811, sig= 0.003 cortisol sampling time (10 min and 24 h) pillai=0.928, f (1,19)= 246.099, sig= 0.00 sampling time × anesthetic drug pillai=0.909, f (3,19)= 63.417, sig= 0.00 gender (male and female) pillai=0.037, f (1,19)= 0.735, sig= 0.402 264 babaiinezhad and bahrekazemi / effects of clove extract, sodium bicarbonate, and lidocaine on caspian kutum increased in females 24 h after anesthesia with sodium bicarbonate (table 3). comparisons of the hematocrit in female broodstocks anesthetized with clove extract resulted in significant differences between 10 min and 24 h post-anesthesia (p<0.05) (table 3). the serum cortisol level in female fish of the control group was 325.50±31.82 ng/ml. 10 min and 24 h after anesthesia by sodium bicarbonate, the levels of the hormone were elevated significantly (table 3). the results also showed a significant differences in the cortisol levels in the broodstocks at 10 min and 24 h after anesthesia with sodium bicarbonate (table 3). comparative results of pillai multivariable analyze: multivariate pillai test of the red blood cells showed no differences in 10 min and 24 h after anesthesia. however, the mean number of rbcs was different in two sampling times of 10 min and 24 h and various types of anesthetic agents. also, the mean number of the red blood cells was not significantly different between male and female in two sampling times (p>0.05) (table 4). applying multivariate pillai test on the hemoglobin and hematocrit showed similar results of the rbcs. despite significant differences between their mean values in two sampling times and three anesthetic drugs, no difference was observed between male and female (p>0.05) (table 4). however, the results on the wbcs were different from other parameters. the mean number of the white blood cells in terms of time after anesthesia, type of the anesthetic drugs, and gender was different significantly (p<0.05) (table 4). furthermore, a significant differences were observed between the mean value of the cortisol in terms of time after anesthesia and type of anesthetic drugs (p<0.05). however, the amount of cortisol based on the gender was almost unchanged in two sampling time (p>0.05) (table 4). the results between male and female broodstocks showed a significant difference between the rbcs in the control group and clove extract and lidocaine treatments only at 10 min, so that only in clove extract, the rbcs in females was higher than that of males (p<0.05) (fig. 1). furthermore, the differences in the wbcs was observed between control group and clove extract treatment at 10 min and 24 h after anesthesia, and the lidocaine and sodium bicarbonate treatments only in 24 h after anesthesia (p<0.05). the wbcs of females was lower in the control group and sodium bicarbonate, while the wbcs of males was lower in the clove extract and lidocaine (fig. 2). in the hemoglobin, the difference between male and female broodstocks was significant in the control group and clove extract at 24 h and lidocaine at 10 min and the hemoglobin in both anesthetics was lower in females figure 1. effect of anesthesia by clove extract, lidocaine, and sodium bicarbonate on rbcs count of male and female broodstocks in 10 min and 24 h after anesthesia. different lowercase letters show significant difference between rbcs count in the males in 10 min and 24 h sampling times with the control group and each other; whereas, different uppercase letters show significant difference between rbcs count in the females in 10 min and 24 h sampling times with the control group and each other. asterisks show significant difference between the males and females’ rbcs count in each treatment. 265 int. j. aquat. biol. (2019) 7(5): 260-270 (p<0.05) (fig. 3). in terms of the hematocrit, there was a significant difference between male and female in the control group, and clove extract in 24 h, bicarbonate sodium in 10 min, and lidocaine in both sampling times, so that the hct was higher in females in lidocaine in 24 h and sodium bicarbonate in 10 min (p<0.05) (fig. 4). however, the results of the cortisol were different since significant differences were observed between male and female broodstocks in clove extract in 24 h, sodium bicarbonate in 10 min, and lidocaine in both times. moreover, the hormone was lower in females in all three anesthetic groups figure 2. effect of anesthesia by clove extract, lidocaine, and sodium bicarbonate on wbcs count of male and female broodstocks in 10 min and 24 h after anesthesia. different lowercase letters show significant difference between wbcs count in the males in 10 min and 24 h sampling times with the control group and each other; whereas, different uppercase letters show significant difference between wbcs count in the females in 10 min and 24 h sampling times with the control group and each other. asterisks show significant difference between the males and females’ wbcs count in each treatment. figure 3. effect of the anesthesia by clove extract, lidocaine, and sodium bicarbonate on hb amount of male and female broodstocks in 10 min and 24 h after anesthesia. different lowercase letters show significant difference between hb amount in the males in 10 min and 24 h sampling times with the control group and each other; whereas, different uppercase letters show significant difference between hb amount in the females in 10 min and 24 h sampling times with the control group and each other. asterisks show significant difference between the males and females’ hb amount in each treatment. 266 babaiinezhad and bahrekazemi / effects of clove extract, sodium bicarbonate, and lidocaine on caspian kutum figure 4. effect of the anesthesia by clove extract, lidocaine, and sodium bicarbonate on hct amount of male and female broodstocks in 10 min and 24 h after anesthesia. different lowercase letters show significant difference between hct amount in the males in 10 min and 24 h sampling times with the control group and each other; whereas, different uppercase letters show significant difference between hct amount in the females in 10 min and 24 h sampling times with the control group and each other. asterisks show significant difference between the males and females’ hct amount in each treatment. figure 5. effect of the anesthesia by clove extract, lidocaine, and sodium bicarbonate on cortisol amount of male and female broodstocks in 10 min and 24 h after anesthesia. different lowercase letters show significant difference between cortisol amount in the males in 10 min and 24 h sampling times with the control group and each other; whereas, different uppercase letters show significant difference between cortisol amount in the females in 10 min and 24 h sampling times with the control group and each other. asterisks show significant difference between the males and females’ cortisol amount in each treatment. 267 int. j. aquat. biol. (2019) 7(5): 260-270 (p<0.05) (fig. 5). discussions since different species of fish show different responses to anesthetic substances, determination of the type and dosage for any species is necessary (lepic et al., 2014). analysis of the blood parameters is one of the most important and reliable methods that can provide important information about healthy conditions of an organisms (kristan et al., 2012). there is a close relationship between changes of rbcs, hemoglobin, and hematocrit and the stressful environmental factors. releasing of the cathecolamines are primary stress response causing erythrocytes to swell and spleen releases new erythrocyte to blood. this will consequently lead to increase of hct and rbcs as well as hemoglobin levels. all these changes lead to increase of oxygen carrying capacity of the blood to supply demanded oxygen under stressful conditions (wendelaar bonga, 1997). therefore, increasing the number of rbcs indicates high stress levels in fish (wedemeyer et al., 1990). in this study, no significant differences were observed between the experimental treatments and control group in the rbcs in male fish, 10 min and 24 h after anesthesia. for females, however, anesthetized fish with clove extract and sodium bicarbonate were significantly different in 10 min after anesthesia. 24 h after anesthesia, the rbcs of females anesthetized with sodium bicarbonate was the only case with significant increase. in addition, according to the anesthetic type, there was a considerable difference in the red blood cells in two sampling times as well as hb and hct. also, by comparing males and females, significant increase appears in the rbcs of females in the group of clove extract treatment 10 min after anesthesia. the dissimilar results between two genders may be due to differences in their physiology (farrell et al., 2011). imanpoor and farahi (2011) showed that using clove extract for anesthetizing male broodstocks of the caspian kutum had no significant effects on their rbc. velisek et al. (2006) worked on the catfish (silurus glanis) anesthetized with clove oil, showing that the rbcs were not significantly different in 10 min and 24 h after anesthesia. similar results were obtained by soudagar et al. (2009) on the roach (r. rutilus), farahi et al. (2011) on the adult males of caspian kutum, effati and bahrekazemi (2017) on grass carp (ctenopharyngodon idella), effati et al. (2014) on silver carp, and lepic et al. (2014) on vimba (vimba vimba), all of which are consistent with our results in male broodstocks but not with those found in female fish. in addition, like clove extract after 10 min, the use of clove oil in female persian sturgeon caused a significant increase in blood parameters (rbcs, hb, and hct) (mazandarani et al., 2015). the present study showed that 10 minutes after anesthesia, the hemoglobin was elevated in all treatments with a significant increase in the males and females in the clove extract treatment and female in the sodium bicarbonate treatment. 24 h after anesthesia, significant increase was observed in male and female of the sodium bicarbonate treatments. according to high levels of hemoglobin in the males and females anesthetized with sodium bicarbonate even 24 h after anesthesia, it can be concluded that stressful conditions remained after that period. velisek et al. (2005, 2006) used clove oil, not clove extract, to anesthetize common carp and catfish and concluded no significant effects on hemoglobin. the results showed a significant reduction of the hemoglobin in the female broodstocks in clove extract and lidocaine treatments. rbc, hb and hct for female broodstocks were lower than males even in the control group. in addition, the hematocrit significantly increased 10 min and 24 h after anesthesia with clove extract and sodium bicarbonate treatments. a study by chen (2012) showed that after anesthesia, the hct of goldfish (carassius auratus) was significantly increased by lidocaine treatment, which was in contrast to our results that could be due to difference in species type and dosage of lidocaine, as well as the physicochemical conditions of the experimental water. despite lower hct in clove extract treatment in females, in the two other anesthetics, the hct was higher in female. 268 babaiinezhad and bahrekazemi / effects of clove extract, sodium bicarbonate, and lidocaine on caspian kutum although roos and roos (2008) deduced that stress may increase the number of white blood cells, the fact is that wbcs may change during stress but the effect of stress on wbcs is inconsistent. the brain and body of fish have constant communication with each other. during acute or short-term stress, the first response of the brain is to help the body deal with the situation, which might increase number of wbcs. at the end of stressful event, the brain sends signals to reverse the initial stress reaction, allowing the wbcs to return to normal situation (roos and roos, 2008). in the present study, although the wbcs increased in 10 min sampling, it was significant only in sodium bicarbonate treatment in both genders. in addition, the number of wbcs was decreased 24 h after anesthesia in all treatments. our results are in agreement with above-mentioned arguments. therefore, by relieving anesthesia stress after 24 h, wbcs were decreased in all treatments. furthermore, the number of wbcs was significantly different between anesthetics in two sampling times and gender with lower amounts in females only in sodium bicarbonate. it is concluded that stress-inducing effect of sodium bicarbonate in females is lower. a study by farahi et al. (2011) on male c. gibelio anesthetized with clove extract yielded no significant changes in wbc counts, which is similar to the results of the present study in male fish anesthetized with clove extract. in addition, imanpoor and farahi (2011) showed that using clove extract for anesthetizing male caspian kutum fish no effect on wbcs. however, soudagar et al. (2009) in roach and imanpoor et al. (2010) in persian sturgeon reported that the number of wbcs has been elevated 10 min after anesthesia with clove extract, which was not significant 24 h after anesthesia. this result is in contrast to our finding. among possible reasons for these dissimilar results, species, age, gender, and condition of rearing condition can be mentioned (ross and ross, 2008). cortisol is the most important indicator of the stress (wagner et al., 2002). in the present study, the cortisol showed significant differences in the sodium bicarbonate treatment in male and female at both sampling times. there were also significant differences based on the sampling time and gender. the cortisol levels in females were lower, which was significant only in the lidocaine group at both times. the stress-induced effect of the sodium bicarbonate in both sexes is greater than those of the clove extract and lidocaine, in which stress levels in females were less than males. wagner et al. (2002) reported that serum cortisol levels decreased in rainbow trout treated with clove extract indicating diminished stress levels. clove extract may block the transmission of sensory information to the hypothalamus and prevent activation of the hypothalamus-pituitary-interrenal axis. in this regard, small (2003) studied the effects of clove extract on the channel catfish, showing high efficiency of the clove extract in suppressing the secretion of cortisol, which is in agreement with our results especially 24 h after anesthesia. effati and bahrekazemi (2017) reported that cortisol level was significantly higher in 10 min and 24 h times after lidocaine and sodium bicarbonate treatments comparing to the control group in grass carp which is similar to sodium bicarbonate results in the caspian kutum. in persian sturgeon, the non‐anesthetized female fish had significantly higher cortisol levels than those anesthetized with clove oil (mazandarani et al., 2015) which is in contrast to our finding. the reason might be large size of the persian sturgeon. conclusion according to the blood parameters, especially cortisol levels, sodium bicarbonate was not an appropriate anesthetic for male and female broodstocks since stressful effects were continued up to 24 h after induction of anesthesia. comparing other two anesthetics, i.e. clove extract and lidocaine, the effects of the lidocaine were lower than clove extract especially in females. however, the latter has no irreversible effects, and after 24 h of anesthesia, stressinduced effects had been greatly moderated. therefore, lidocaine and then clove extract can be recommended as suitable anesthetics, especially in female caspian kutum. 269 int. j. aquat. biol. 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(2022) 10(6): 438-450 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article effect of dietary lactococcus lactis and bacillus subtilis on the innate immunity, intestinal microbiota, histometrical indices, and resistance against aeromonas hydrophila in oscar, astronotus ocellatus agassiz, 1831 abbas hasaninia1, habib vahabzadeh roudsari1, hossein khara1, alireza shenavar masouleh2, mohaddeseh ahmadnezhad3 1department of fisheries, lahijan branch, islamic azad university, p.o. box: 1616, lahijan, iran. 2international sturgeon research institute, iranian fisheries research organization, agricultural research, education and extension organization (areeo), rasht, iran. 3inland waters aquaculture research center. iranian fisheries research organization, agricultural research, education and extension organization (areeo), bandar anzali, iran. s article history: received 11 september 2022 accepted 26 november 2022 available online 2 5 december 2022 keywords: bacillus immune system lactococcus lactis microbiota histology abstract: this work aimed to investigate the effect of dietary lactococcus lactis and bacillus subtilis on the immune responses, intestinal microbiota, and resistance to pathogens of oscar, astronotus ocellatus. during 70 days trial, 300 juveniles (8.96±0.033 g) were fed diets enriched with l. lactis and b. subtilis. the treatments included 150, 300, 450 mg kg-1 of dietary l. lactis (ll150, ll300, ll450); 150, 300, 450 mg kg-1 of dietary b. subtilis (bs150, bs300, bs450); 150, 300, 450 mg kg-1 of diet an equal mixture of l. lactis and b. subtilis (mix150, mix300, mix450); and a nonsupplemented control group. at the end of the rearing period, histological, immunological, and intestinal microbiota indices in treatments were investigated. to evaluate disease resistance, 15 fish in each treatment were infected in each treatment by aeromonas hydrophila. the results showed that adding b. subtilis and l. lactis, particularly in mix300, reduced the anaerobic heterotrophic bacterial microbiota and increased lactic acid bacteria (lab) in fish. the highest white blood cell (wbc) level was recorded in the ll150 group. the lymphocytes in fish fed ll150, ll300, mix150, and mix300 diets were changed and neutrophils of ll150, ll300, ll450, mix300, and mix450 were significantly increased. monocytes in fish fed mix300 and mix450 diets raised significantly. the igm, ach50, and lysozyme levels in fish-fed diets enriched by bacteria, especially in ll450, were significantly higher than the control treatment. the intestinal villi in ll450, bs150, and mix450 were significantly higher, showing lower damages than the other treatments. the survival rates of the infected fishes were higher in mix150 and mix300 groups. introduction oscars (astronotus ocellatus) are ornamental fish of distinct quality and value. they are known as everhungry fish interested in feeding even at satiation with a unique appearance, intelligence, and behavior, making them enjoyable to aquarium enthusiasts. these, combined with the fact that oscar can live just as long as a dog, make it more like a pet than most other fishes (yilmaz and arsalan, 2013). health and nutrition are two vital aspects of ornamental fish farming, as its annual international exports are around us$ 200 million, or less than 3% of the total world fish trade (ghosh et al., 2008; gobi correspondence: habib vahabzadeh roudsari doi: https://doi.org/10.22034/ijab.v10i6.1753 e-mail: habib.vahabzadeh@gmail.com dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.1.4 et al., 2018). the gastrointestinal tract of fish is a key area of interaction with pathogens in fish farms. it is important to enhance the cultured fish immune system by enriching the normal gut microbiota, as it affects a wide range of biological processes, including the development of gut-associated lymphoid tissue (galt) and the ability to combat infections (nayak et al., 2007). microbial population diversity will be altered by manipulating microbial populations and changing environmental conditions caused by the proliferation of selected bacteria (sayes et al., 2018). the competitive elimination of 439 int. j. aquat. biol. (2022) 10(6): 438-450 pathogenic bacteria by probiotics can effectively reduce or eliminate the prophylactic use of antibiotics in intensive systems (saputra et al., 2016). it has been well-established that the gi performance of aquatic organisms is modified via microbial modification (doan et al., 2021). providing probiotics via diet can improve the microbial balance of the host (nayak, 2010) and increase resistance against disease-causing bacteria in aquatics (al-dohail et al., 2011). lactic acid bacteria (lab) of lactococcus lactis are known to produce a wide range of antimicrobial compounds that can inhibit growth or kill a broad range of bacteria (loh et al., 2017). bacillus subtilis identified in the gi tract of several finfish species are spore-forming bacteria resistant to adverse environmental conditions, with various species showing unusual physiological features enabling them to survive in different environments (díazrosales et al., 2006). bacillus can affect nutrition, adherence, and colonization of pathogens, affecting fish’s immune system with probiotic potentials (soltani et al., 2019). the adhesion of gram-positive probiotics into fish feed can improve the immuno-physiological functions of hosts and enhance their disease resistance. probiotics multiply after settling in the intestine and use sugar to grow and produce unsaturated fatty acids (blottiere et al., 2003). the probiotic dosage is a missing factor in earlier studies (shenavar masouleh et al., 2016). in addition, insufficient data are available to demonstrate the behavioral growth of gram-positive probiotics, such as synergistic or antagonistic effects (doan et al., 2021). the effects of probiotics on the growth indices and nutrition of some fish species were reviewed in previous studies e.g. oscar and the nile tilapia (oreochromis niloticus) (safari and atash, 2013; won et al., 2020). the b. subtilis as a probiotic has recently been introduced to iranian fish farmers; however, its effect on oscar has not been studied. based on the above-mentioned background, the present study aimed to investigate the effects of dietary l. lactis and b. subtilis on immune responses, intestinal microbiota, and resistance to pathogens in oscar fish. materials and methods experimental conditions: during 70 days of the experimental period, 300 oscar juveniles were stocked in 50-liter aquaria, ten fish per aquarium (0.16 g l-1). there were 10 experimental treatments, each with three replicates. the mean weight and length of juveniles were 8.96±0.03 g and 8.23±0.02 cm, respectively. adaptation to the lab condition and feed was made for ten days. bacillus subtilis (persian type culture collection: ptcc no.:1204) and l. lactis (registered at ncbi under no. jf831150) were added to the basic diet (coppens, germany) in different concentrations, including 150 mg kg-1 (1.5×106 cfu g-1), 300 mg kg-1, (3×106 cfu g-1) and 450 mg kg-1 (4.5 ×106 cfu g-1), as an equal mixture of b. subtilis and l. lactis (table 1). the predicted powder-containing bacteria was dissolved in 50 ml of ringer solution and added per kg of diet. the control group received the same basal diets without added bacteria. the pelleted diet from coppens company (troco crumble he) with size of 0.8-1.2 mm was used as basic diet, containing 56% crude protein, 15% crude fat, 0.5% fibre, 8.4% ash, 2.3% calcium, 0.7% sodium, and 1.4% total phosphorus. the juveniles were fed at 3-5% of their body weight based on the water temperature and biomass of each aquarium (according to initial and mid-term biometric measurements) 3 times a day (8 a.m., 12 a.m., and 2 p.m.) for 70 days. the growing juveniles were transferred to 200 l aquaria to maintain their biomass. the physico-chemical parameters of water, including temperature, dissolved oxygen, ph, hardness (dh), and total ammonia before and after feeding, are shown in table 2. blood sampling and serum preparation: feeding was stopped for 24 hours, allowing gut evacuation, with three fish randomly selected from each aquarium. using a syringe, 2 ml of blood was taken from their caudal vein. then, 1.5 ml of blood was taken and 440 hasaninia et al./ effect of dietary lactococcus lactis and bacillus subtilis on the innate immunity of oscar saved in non-heparinized tubes for measuring immunological parameters (abarike et al., 2018). the blood serums of non-heparinized samples were separated by centrifuging at 1409 g force (3000 rpm) for 10 min (labofuge, manufactured by heraeus sepatech, germany), then stored at -80°c until analysis. the remaining blood samples (0.5ml) were used for blood cell counts. the blood sample was diluted with a natt-herrick solution to calculate white blood cells (wbc) by a neubauer hemocytometer slide. the blood smears on glass microscope slides were stained with giemsa for the differential leukocyte count, and the percentage of different leukocytes was determined (mohammadian et al., 2020). measurement of immunological parameters: the immunoglobulin m (igm) of the serum was measured by an immunoturbidimetric assay in a spectrophotometer (2100-vis, unico, the us) at a wavelength of 340 nm with distilled water as the control (teige et al., 2019). to determine lysozyme activity through the gradual lysis of a gram-positive bacterium (micrococcus lysodeikticus, sigma, usa), 50 μl of serum was added to 950 μl of the m. lysodeikticus solution (200 mg ml-1 of the bacterium in 5% sodium phosphate at a ph of 6.2). the solution turbidity observation was at 530 nm and 22°c after 0.5 to 4.5 minutes by a turbidimetric assay in an elisa reader (awareness, usa). to record the data (mg ml-1), egg lyophilized albumen lysozyme (sigma) was applied based on sharifuzzaman and austin (2009) and merrifield et al. (2010). the alternative complement pathway (ach50) was measured based on the photometric method at 414 nm using a spectrophotometer (awareness, usa) through hemolysis of rabbit red blood cells (rarbc; tcs biosciences botolph clydon, uk) (tukmechi et al., 2011). intestinal bacteria count: three samples from each treatment were randomly selected and euthanized with 1500 ml l-1 of clove oil (cpcsea, 2021). then intestine was removed under sterile conditions (nayak et al., 2007). the ringer solution was applied, making 101 to 108 dilutions of the intestinal fecal contents. after sampling, dilutions of 101 through 107 of intestinal extracts were prepared using a sterile ringer solution. then 0.1 ml of each dilution was inoculated on the tryptic soy agar (tsa) medium and de man, rogosa, and sharpe agar (mrs) media (lab specific medium) for the intestinal bacterial count. tsa incubation plates were in aerobic condition at 25°c and mrs plates treatment l. lactis added to basic feed (ml kg-1) b. subtilis added to basic feed (ml kg-1) control 0 0 ll150 150 ll300 300 ll 450 450 bs150 150 bs300 300 bs450 450 mix150 75 75 mix300 150 150 mix450 225 225 table 1. experimental treatments based on added bacteria to basic diet. nitrite (mg l-1) ammonia (mg l-1) hardness ph dissolved oxygen (ppm) temperature (ºc) <0.04 0.07-0.10 172.4±0.4 7.36 ±0.21 7.6±0.44 27.84±0.32 table 2. physico-chemical parameters of water during the 70-days of rearing 441 int. j. aquat. biol. (2022) 10(6): 438-450 were incubated at 30°c and under anaerobic conditions. the bacterial count (cfu) took place after the incubation. bacterial challenge test (bct): fish were exposed to pathogens based on al-dohail et al. (2011) and sharifuzzaman and austin (2009). after final sampling, aeromonas hydrophila (atcc:15309, obtained from pasture institute, iran) was injected into fishes to evaluate their resistance to pathogens. five specimens, 30-35 g., were randomly selected from each replicate in all treatments. upon anesthetizing, 0.1 ml of a. hydrophila (a dosage of 1×108 cells/fish) was injected intraperitoneal (mcfarland standard kit no. 1, dalynn biologicals). during the 14-day bct period, the water temperature of the tanks was 28°c. individual infections, mortalities, and disorders were monitored and recorded daily. at the end of this period, fins, skin, internal organs, liver, and swim bladder were sampled and examined to identify probable lesions. intestinal histology: after opening the abdomen, the anterior, middle, and posterior parts of the intestines were removed and fixed into buen's solution. following 48 hours, the fixed intestinal tissues were processed, and histological slides were prepared based on eagderi et al. (2013) and stained with hematoxylin-eosin. images were taken using a light microscope (bel, bio2, italy) equipped with a eurekam 10.0 camera. imagej 1.46r software was used for the histomorphometry of tissue expansions. mucous folds and other histometric parameters were measured in 6 random fields. measurements were made on the mucosal fold’s height, the thickness of the mucosal epithelium, the parine layer, submucosa, and the muscle layer. the tissue indices were compared between treatments based on the average size of the three parts of the intestine (suvarna et al., 2012). data analysis: the data normality of all measured parameters was determined using the shapiro-wilk test. the treatments were compared using the oneway analysis of variance (anova). after ensuring the homogeneity of variances, duncan multiple range test (dmrt) was used to compare treatments. statistical data analyses were done using spss-23, and the graph was plotted in excel-2016. results wbc and differential count: the lowest wbc was recorded in the control group, while the highest value was observed in the ll150 group (p<0.05; table 3). the lymphocyte of all treatments that were fortified with probiotics was lower than the control group (p<0.05) (table 3). neutrophils of all treatments receiving b. subitilis and l. lactis were higher than the control one (p<0.05; table 3). the highest monocyte percentage was recorded in mix300 and mix450 (p<0.05). there was no significant difference in eosinophile between treatments (p>0.05; table 3). serum immunological parameters: the serum lysozyme activity in the ll450 and mix450 was treatments wbc (mm3) lymphocytes (%) neutrophils (%) monocytes (%) eosinophils (%) control 3316.67±72.64c 82.67±0.88a 12.33±0.33b 4.67±0.33bc 0.33±0.33 ll150 5000.00±493.28 a 77.33±0.33c 16.00±0.57a 6.00±0.57abc 0.67±0.33 ll300 3633.87±0.007 bc 77.33±1.20c 16.00±1.52a 6.00±0.57abc 0.67±0.33 ll450 4033.87±284.80 bc 78.00± 0.53bc 15.67±0.06a 5.67±0.33abc 0.67±0.33 bs150 3816.87±120.19 bc 81.00±1.53ab 14.00±1.20ab 4.67±0.67bc 0.33±0.33 bs300 3500.00±208.17 c 79.00±1.00bc 14.67±1.15ab 5.33±0.33abc 1.00±0.58 bs450 3450.00±125.83 c 78.33±0.88bc 14.33±0.88ab 6.33±0.33ab 1.00±0.58 mix150 4033.33±272.84 bc 79.67±0.88bc 15.67±0.66ab 4.33±0.33c 1.00±0.58 mix300 4433.33±437.16 ab 77.00±1.15c 15.67±0.33a 6.67±0.88a 0.67±0.33 mix450 4133.33±176.38 bc 77.00±1.00c 14.93±0.30a 6.67±0.33a 0.67±0.33 values expressed as mean±sd. different letters in each column indicate the significance of the difference (p<0.05). table 3. total and differential wbc of lactococcus lactis and bacillus subtilis added diets for oscar after 70 days. 442 hasaninia et al./ effect of dietary lactococcus lactis and bacillus subtilis on the innate immunity of oscar significantly higher than the control treatment (p<0.05; table 4). the ach50 was higher in all treatments than in the control one (p<0.05; table 4). treatments of the ll450, bs300, and mix450 were the highest from others (p<0.05; table 4). based on the results, the igm, lysozyme, and ach50 were influenced by increasing bacterial dosage in all treatments (table 4). the igm level of ll450 and bs300 was significantly increased in treatments (p<0.05; table 4). intestinal microbiota counts: the results revealed that aerobic heterotrophic bacteria of intestinal mucus and lab were well-colonized, and their number in the intestine was significantly elevated by increasing bacterial dosage (p<0.05). considering the total intestinal microbiota on tsa and mrs media, there was a significant difference between all treatments and the control (p<0.05). the lowest and the highest bacterial counts were observed in the ll150 and the control, respectively. the total lab count in the intestine of all treatments significantly decreased compared with control and b. subitilis treatments. however, mix150 and mix300 exhibited a significant difference from the control (p<0.05). there was an increasing value in the number of intestinal mucus bacteria related to bacterial dosage of diets (table 5). intestinal histology: in the histometeric indices (table 6), based on the average size in the anterior, middle, and posterior portions of the intestine, the villius average height in mix450, villi epithelium average diameter, villi average diameter, the treatments igm (ml dl-1) lysozyme (µg ml-1) ach50 (u%) control 32.50±0.50c 28.00±1.00c 128.0±1.0d ll150 33.00±1.00 c 29.00±1.00c 129.0±1.0cd ll300 38.00±1.00 abc 30.00±1.00bc 135.0±2.0abc ll450 42.00±1.00 a 36.50±2.50a 137.0±1.0a bs150 34.00±1.00 c 28.00±2.00c 129.0±2.0cd bs300 40.50±3.50 a 34.50±2.50abc 136.5±4.5a bs450 38.50±0.50 ab 32.50±1.50abc 134.0±1.0abcd mix150 32.50±0.50 c 30.00±1.00bc 130.0±0.0bcd mix300 37.00±1.00 abc 31.00±1.00abc 135.0±1.0abc mix450 38.00±3.00 abc 37.50±3.50a 137.0±2.0a values expressed as mean±sd (n=81). different superscript lowercase letters within each column represent significant differences (p<0.05). table 4. the igm, lysozyme and ach50 value of a. ocellatus fed with diets containing lactococcus lactis and bacillus subtilis after 70 days. table 5. the number of aerobic heterotrophic bacteria of intestinal mucus on tsa and mrs (log cfu g-1±sd). treatments tsa (cfu g-1) mrs (cfu g-1) control 8.29±0.006a 3.54±0.06e ll150 5.71±0.05 g 4.50±0.01ab ll300 6.33±0.02 e 4.59±0.01b ll450 7.25±0.01 d 4.42±0.01c bs150 6.11±0.04 f 4.03±0.01d bs300 8.09±0.04 b 4.02±0.04d bs450 8.13±0.02 b 4.004±0.01d mix150 7.75±0.07 c 4.81±0.02a mix300 7.78±0.026 c 4.88±0.01a mix450 8.07±0.03 b 4.62±0.01b values were expressed as mean±sd (n=3): different superscript lowercase letters within each column represent significant differences (p<0.05). 443 int. j. aquat. biol. (2022) 10(6): 438-450 thickness of parine layer in ll300, the thickness of muscle layer in bs300, bs450, the thickness of the submucosal layer in ll450 and bs150 were significantly different compared to the control (p<0.05) (fig. 1). bacterial challenge test (bct): after two weeks of monitoring aeromonas-infected oscars, the highest mortality rate was detected in the control group (p<0.05), while the lowest mortality rate (p<0.05) was recognized in mix150 and mix300 (fig. 2). the first mortality was recorded after six days of injection in the control group. the most common lesions observed during bct, were bleeding in the anus, base of the anal, caudal fins, and around the mouth. limited necrospy hemorrhage was detected in the liver, swim bladder, peritoneal cavity, and hyperemia in the kidney (table 7). discussion the results showed a significant increase of wbc as an immunity index in the ll150, and mix300. increased levels of wbc were observed in rainbow trout and nile tilapia fed b. subtilis and l. lactis treated diets (balcázar et al., 2007; opiyo et al., 2019), indicating that an increase in probiotic supplementation may reflect improved immunity of fish. probiotics such as b. subtilis (lee et al., 2017) and l. lactis (xia et al., 2018) have been proven useful in aquaculture. the immune system response of fish can be greatly influenced by adding b.subtilis and l. lactis in different fish diets (nayak et al., 2010; dias et al., 2020). after colonizing bacillus in the mucosal epithelium of the intestine, protection signs show against pathogens by competitive exclusion or competition for available energy and the production of inhibitory compounds (soltani et al., 2019). the results showed a significant igm and lysozyme improvement in oscar, especially in treatments ll450, bs300 and bs450. these findings are similar to those reported for lactobasilus rhamnosus, carnobacterium maltaromaticum, and c. divergens in rainbow trout (panigrahi et al., 2004; kim and austin, 2006), pediococcus acidilactici in the nile tilapia (ferguson et al., 2010), and the oscar (safari and atash, 2013). according to nayak (2010), probiotics can stimulate the intestinal immune system by increasing the number of igm and acidophilic granulocytes. safari and atash (2013) reported that the lysozyme level significantly increased in the blood serum of oscar fed with diets enriched with p. acidilactici. the effects of b. subtilis and l. lactis on the immune responses of the nile tilapia showed that the immune system table 6. mean intestinal histomteric indices (µm) of oscar fed by lactococcus lactis and bacillus subtilis and mixed probiotic diets. treatments villus height (µm) epitelium diameter (µm) villus width (µm) muscular thickness (µm) lamina propria (µm) sub mucosa (µm) control 369.77±14.85 e 32.42±0.90f 68.57±1.99g 85.01±3.37c 13.42±0.38e 32.66±1.04d ll150 514.65±17.19 bc 47.65±1.16ab 98.77±2.74b 74.13±2.61d 46.87±1.88b 36.30±1.27d ll300 493.64±12.78 c 49.07±1.67a 105.58±3.76a 85.22±3.44c 58.55±1.89a 42.69±1.93c ll450 547.28±12.97 ab 44.73±1.26bc 92.01±2.32cd 86.91±2.49c 60.16±1.54a 50.37±1.32a bs150 546.48±17.72 ab 41.66±1.06cd 88.44±2.20cd 85.33±2.15c 47.83±1.41b 51.63±1.73a bs300 421.33±12.15 d 37.37±1.08e 78.38±2.14ef 117.20±5.35a 42.17±1.47c 49.46±1.60ab bs450 473.31±9.78 c 41.28±1.10cd 86.58±2.21cd 114.26±3.57a 42.67±0.96c 47.54±1.32ab mix150 495.22±18.27 c 34.13±0.95f 72.78±1.87fg 100.37±2.72b 38.16±1.15d 47.91±1.46ab mix300 503.54±16.63 bc 41.83±1.26cd 87.94±2.47cd 61.91±1.84e 47.06±1.63b 41.77±1.12c mix450 580.03±15.05 a 39.03±0.98de 82.23±1.91e 97.49±3.75b 46.31±1.33bc 45.44±1.36bc different letters in each column indicate significant differences between them (p<0.05). (mean±sd). 444 hasaninia et al./ effect of dietary lactococcus lactis and bacillus subtilis on the innate immunity of oscar performance, including lysozyme, became significantly efficient in the fishes fed-diets enriched with probiotic bacteria (won et al., 2020). the probiotic bacteria could improve the lysozyme level figure 1. histologic cross-section view of the posterior intestine of oscar in the control group (a&b) and the treatment 9 (mix450) (c& d) (abbreviations: gc: goblet cell, ec: enterocyte, lp: lamina propria, muscular thickness (mt), sm: submucosa. eosinhematoxylin staining. figure 2. the mortality rate of differently treated aeromonas ocellatus exposed to a. hydrophila after 14 days. 445 int. j. aquat. biol. (2022) 10(6): 438-450 in different species, including rainbow trout (balcázar et al., 2007), the malabar grouper (epinephelus coioides) (sun et al., 2012) and nile tilapia (han et al., 2015; abarike et al., 2018; opiyo et al., 2019). the use of lab in aquaculture can affect the igm level as an important humoral immunity against pathogens (panigrahi et al., 2005). it has been shown that b. subtilis in the diet of rainbow trout and rohu (labeo rohita) can improve the igm level (nikoskelainen et al., 2003; nayak, 2010), which is in line with the findings of the present study on oscar. our study results indicated that the igm level in ll450 and bs300 was significantly higher. the proof of higher igm levels in some treatments can be attributed to stimulating antibodies produced by lab (yu et al., 2020). panigrahi et al. (2004) reported that feeding rainbow trout with diets enriched with l. rhmanosus increased ach50 in the anterior part of the kidney. a similar increasing trend in ach50 has been reported in the brown trout (salmo trutta) (balcázar et al., 2006), rainbow trout (ramos et al., 2015) and the gilthead seabream (sparus aurata) (díaz-rosales et al., 2006) fed diets containing l honi, and l. lactis and also in rainbow trout fed diets enriched with l. rhamnosus (nikoskelainen et al., 2003). compared to the control, the increase of ach50 in all treatments indicated the positive effect of probiotics. the findings of the present study showed that the addition of l. lactis and b. subtilis to the basal diet of oscar could be due to the increasing colonization rate of microbiota or a significant reduction of the intestinal bacterial count. based on our results, diets with 1010 cfu b. subtilis, and l. lactis led the intestinal bacterial count to log 5.71 cfu ml-1 and the intestinal lab count ranged between log 3.33 to log 4.81 cfu ml-1. the highest bacterial count was found in mix300. feeding the persian sturgeon and beluga (huso huso) fry with diets enriched with two types of lab (l.mesenteroides and l. curvatus), at a dosage of 109, showed the intestinal bacterial count about log 3 cfu ml-1 and lab count was log 2.27 to log 3.02 cfu ml-1 (askarian et al., 2011). during the persian sturgeon feeding on diets enriched with l. lactis at a dosage of 108, the intestinal bacterial count reached log 4.05 cfu ml-1 and the intestinal lab count ranged between 3.35 and log 4.19 cfu ml-1 (shenavar masouleh et al., 2016). the inhibited growth of pathogenic bacteria by beneficial bacteria could be due to the individual or combined production of antibacterial metabolites (e.g., bacteriocins, siderophores, lysozymes, proteases), competition for essential nutrients, alteration of ph by organic acid production, and competitive exclusion (kim and austin, 2006; mukherjee and ghosh, 2016). in addition, the inhibitory effect is related to the organic acid excreted by l. lactis (loh et al., 2017). the differences in cfu are due to the feeding behavior of species, the initial dosage of added bacteria, its strain, and rearing environmental conditions, particularly water temperature (martínez cruz et al., 2012; sayes et al., 2018). the intestinal microbiota showed a significant difference between juveniles fed diets enriched with bacterial species and those in the control group. both added bacteria were effective in reducing the populations of common intestinal bacteria. moreover, the results showed that labs were wellcolonized in the intestine of oscar, and their population in the intestine significantly increased with the increase of enrichment. the results of probiotic bacteria in the mrs medium showed that the intestinal wall in the control group lacked these bacteria. adding probiotics to the diet caused a rise in the number of probiotic bacteria in the intestine, with the highest number observed in mixed diets i.e., a mixture of b. subtilis and l. lactis could easily survive in the gastrointestinal tract, attach to the mucosal surface of the intestine, proliferate, and act synergistically. histological examinations of the intestines show a significant extension of intestinal folds in the treatments containing l. lactis and b. subtilis. it was found that b. subtilis probiotic could improve the immunity, and digestive system, especially the fish intestinal morphology parameters (lee et al., 2017). probiotics increase food absorption and enzyme 446 hasaninia et al./ effect of dietary lactococcus lactis and bacillus subtilis on the innate immunity of oscar digestion process and improve the intestinal tissue of common carp (yanbo and zirong, 2006), sea bass using b. mojavensis (hamza et al., 2016), the barb fed by mixed probiotics (allameh et al., 2017) and nile tilapia fed by b. subtilis and l. lactis (liu et al., 2017; xia et al., 2018). also, using two probiotic strains (pdp11 and pdp13) from alteromonadaceae, similar results were reported i.e. including the increased size and number of microvilli in the intestine of solea senegalensis fry (saenz et al., 2009). probiotics have been reported to increase the thickness of the muscle layer due to the modulation of the physiological activities of intestinal mucosal cells (lazado and caipang, 2014). the combined use of lactobacillus, entrococous, pedicoccus and bacillus probiotics in feeding rainbow trout has increased the anterior surface of the intestine by increasing the length of the villi and the number of goblet cells in the diet containing probiotics (ramos et al., 2015). recent studies linked the improvement of intestinal histological indices in nile tilapia feddiet containing b. subtilis and l. lactis to better growth performance, feeding efficiency, and increased activity of intestinal tissues. probiotics could facilitate the absorption of effective nutrients by improving the length of the intestinal villi, the muscle layer's thickness, and the trypsin's activity (won et al., 2020). this study concerning the increased disease resistance induced by probiotic supplements confirmed the results of previous studies (raida et al., 2003; panigrahi et al., 2007; soltani et al., 2019). aeromonas hydrophila is a known pathogenic bacterium that causes diseases in the cichlids family (saputra et al., 2016). our findings indicated that adding probiotic bacteria to the basal diet improved oscar resistance against a. hydrophyla. the mortality rate after 14 days was lower in groups fed diets enriched with b. subtilis and l. lactis and started later than in control. in the nile tilapia, l. lactis improved immune responses and resistance against diseases (xia et al., 2018). the symptoms observed in intentionally a. hydrophila infected fishes were similar to those reported for persian sturgeon exposed to the same bacterium (soltani and kalbassi, 2001). the symptoms in both experiments were imbalance, lethargic swimming, bruising with bleeding spots on the external surfaces, and hemorrhage in internal organs. in the rainbow trouts receiving diets supplemented with 109 and 1012 cfu of l. rhamnosus, the survival rate increased by 33.7 and 6.3%, respectively, after exposing fish to a. salmonicida (nikoskelainen et al., 2001). kim and austin (2006) applied diets enriched with 107 cfu g-1 of clostridium maltaromatieum (b26) and c. divergens (b33) for two weeks. they reported that the immunological protection improved following rainbow trout exposure to a. salmonicida and yersinia ruckeri. brunt et al. (2007) supplemented diets with 2×108 cfu g-1 of bacillus (jb-1) and a. sobria (gc2) applied for 14 days on rainbow trout, showing lower mortalities than that in the control when the fishes were exposed to vibrio ordalii, v. anguillarum, streptococcus iniae, a. salmonicida, and y. ruckeri. won et al. (2020) listed many studies confirming improvements in e. malabaricus fed a diet containing l. plantarum, chinese drums (miichthys miiuy) with a diet added by c. butyricum (cb2), the nile tilapia diet enriched by b. subtilis and l. acidophilus (mixture of both) showed a reduced mortality rate among fish exposed to a. hydrophila, v. anguillarum, pseudomonas fluorescens, and s. iniae respectively. they also conducted 13 days of bct trial with a. hydrophila on the nile tilapia, revealing a significantly increased survival rate of the tilapia fed with diets enriched with l. lactis and b. subtilis than control one and the diets enriched with oxytetracycline (otc) (won et al., 2020). differences in the probiotic efficacy are related to bacteria strains and their concentration in basic diet, target organism criteria, including fish species, age, its diseasecausing intensity, challenge duration, and environmental condition, including water temperature, salinity, ph, and alkalinity (lee et al., 2017; elsabagh et al., 2018; won et al., 2020). 447 int. j. aquat. biol. (2022) 10(6): 438-450 conclusion based on the results of the immunological analysis, the two probiotics of l. lactis and b. subtilis improved the performance of oscar’s immune system. the oscar juveniles fed diets enriched with probiotic bacteria had more efficient intestines and showed higher resistance against stressors and diseases. the mixture of b. subtilis and l. lactis (150 mg+150 mg) per kg of feed could modulate the intestinal microbiota, immunity, and resistance in exposure to a. hydrophila. the better results of mixed treatments can be attributed to the symbiotic effect of using two different probiotics simultaneously. acknowledgments the authors would like to appreciate mehdi masoumzadeh and foruzan bagherzadeh for their valuable assistance in bacterial culture and jalil jalilpour, who advised us in statistical analysis. references abarike e.d., cai j., lu y., yu h., chen l., jian j., tang j., jun l., kuebutornye f.k.a. 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(2020). immunoglobulins, mucosal immunity and vaccination in teleost fish. frontiers in immunology, 11: 1-14. 1.1. 1.2. 1.3. 1.4. 1.5. international journal of aquatic biology (2013) 1(5): 215-220 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved technical note investigation of coastline change of the urmia lake using remote sensing and gis (1990 2012) shirkou jaafari*,1afshin alizadeh shabani, afshin danehkar department of environmental science, faculty of natural resources, university of tehran, karaj, iran. article history: received 2 june 2013 accepted 3 september 2013 available online 2 5 october 2013 keywords: urmia lake coastline satellite imagery remote sensing gis abstract: the study on water level fluctuation in the urmia lake has become of a great importance in recent years. this is due to the importance of the position of this lake as a natural heritage at an international level. the water level decrease at the urmia lake during the recent years has made it necessary to address the matter more than before. investigation and assessment of changes in the lake is necessary because it is the largest international wetland and one of the iranian national parks. the main purpose of the current study was to investigate changes in the lake water levels using satellite imagery and gis technique, preparing and processing multispectral landsat images in 1990, 1998, 2006 and 2011, classifying the images and extract the land use map for these four time periods. results of this study indicated that the urmia lake has faced significant decrease in water level during the past twenty years, especially in the past decade. also, the surface area of the lake decreased by 3052 km2 and the salt area increased from 1990 to 2011. the present study indicate the incidence and development of environmental crisis in the region. hence, it is essential to take into account the entire social, economic, and environmental considerations as well as all the macro-environmental issues at a regional scale to save the urmia lake introduction monitoring of coastal area is a necessary task in sustainable development and costal environment management and protection. for coastal area monitoring, the coastline mapping in different times is a fundamental work. the coastline is determined as the line of contact between the land and the water body (sugumaran et al., 2004). the coastline is one of the important linear features in the environment, which has a dynamic nature (rasoli et al., 2007; winarso et al., 2001). remote sensing performs an important task for spatial data acquisition from economical perspective (ozesmi and bauer, 2002). satellite images are simple to interpret and easily obtainable. furthermore, absorption of infrared wavelength region by water and its strong * corresponding author: shirkou jaafari e-mail address: sh.jaafari@ut.ac.ir tel: +989376923134 reflectance by vegetation and soil make such images an ideal combination for mapping the spatial distribution of land and water. these characteristics of water, vegetation and soil make the images containing visible and infrared bands being widely used for coastline mapping (dewitt et al., 2002). examples of such images are: tm (thematic mapper) and etm+ (enhanced thematic mapper) imagery (moore, 2000). lake urmia in the northwestern corner of iran is one of the largest permanent hyper saline lakes in the world and the largest lake in the middle east (zarghami, 2011; hassanzadeh et al., 2011; karbassi et al., 2010). it extends as much as 140 km from the north to the south and is as wide as 85 km from the east to the west during the high-water periods (jalili 216 jaafari et al/ international journal of aquatic biology (2013) 1(5): 215-220 et al., 2012). the lake was recognized as a wetland of international importance by the ramsar convention in 1971 and designated a unesco biosphere reserve in 1976 (birkett and mason, 1995). the lake itself is home of a unique brine shrimp species, artemia urmiana. it supports many species of reptiles, amphibians and mammals. lake urmia provides a very important seasonal habitat for many species of migratory birds. around 200 species of birds have been documented on and around the lake including pelicans, egrets, ducks, and flamingos (ebrahimi, 2010). the watershed of the lake is an important agricultural region with a population of around 6.4 million people. it is estimated that 76 million people is resident within a radius of 500 km. mapping changes of coastline for the urmia lake using tm imagery is the main aim of this paper. furthermore, a new semiautomatic approach for coastline extraction from tm imagery has been developed and presented. material and methods study area: the urmia lake, as the largest water body in iranian plateau is located between two major provinces of the eastern azerbaijan and west azerbaijan bounded between 37° 5´ 38° 16´ n and 45° 01´ 46° e at 1275 m above sea level (fig. 1). its surface area ranges from 4750 to 6100 km2 and the average and greatest depths account for 6 and 16 m, respectively (azari takami, 1993). more than 20 permanent and seasonal rivers as well as a few submarine streams and springs feed the lake. the average salinity of the lake ranges between 220-300 mg/l depending on temporal and spatial conditions, in recent years it has reached to more than 380 mg/l. due to the ecological heritage of the uremia lake, it has been recorded as a protected habitat in the world by the united nations (eimanifar and mohebbi, 2007). methodology: for this study, 12 predominantly cloud-free landsat tm images of the uremia lake (path 168, row 34; path 169, row 33; path 169, row 34) were selected. the landsat images covering the entire lake urmia coastline were acquired from the us geological survey (usgs) website. data collected within the same year and seasons were best for this kind of study. the satellite images were geometric corrected using 1:25000 topographic maps using maximum neighborhood correction and then radiometric (chander and markham, 2003) and atmospheric correction was operated by envi 4.5 (chavez, 1998). for each period of study (1990, 1998, 2006, 2012), three images were mosaicked and final images were prepared (fig. 2). then figure 1. the study area. figure 2. mosaicked satellite images of 1990 (a), 1998 (b), 2006 (c) and 2012 (d). 217 jaafari et al/ international journal of aquatic biology (2013) 1(5): 215-220 unsupervised classification and k-means were used for image classification. according to the study goals, two land use/cover classes were defined including water body and others (anderson et al., 1976) in envi 4.5. three bands were used (bands 3, 4 and 5) for landsat 4/5 tm images. then the water body of lake urmia was separated in arc gis 9.3 and finally the layout of maps was prepared. results image analysis showed significant changes during the study period (fig. 3). also the analysis of maps figure 3. prepared maps for 1990 (a), 1998 (b), 2006 (c) and 2012 (d). year 1990 1998 2006 2012 area (km2) 5418 5637 4125 2366 table 1. lake urmia area in study period. 217 218 jaafari et al/ international journal of aquatic biology (2013) 1(5): 215-220 showed that water surface area had increased by 219 km2 from 1990 to 1998, from 1998 to 2006, decreased by 1512 km2, and finally from 2006 to 2012 decreased by 1759 km2. the maximum decrease occurred during 1998 to 2012 (table 1). our results indicated that the urmia lake lost 56% of the total surface from 1990 to 2012 (fig. 4). discussion in the present study, coastline changes and water surface were determined using landsat-tm images and unsupervised classification method, respectively. whereas karimi and mobasheri (2011) in a similar study, used several remote sensing images containing landsat-mss, landsat-tm, landsat-etm+ and modis sensors. the authors have used supervised classification method to determine water surface. our results are very close to theirs. we also found a great decrease of coastline changes in 1998 to 2006 which has begun in 1998. in other research alesheikh et al. (2007) had similar results. great changes have happened as a result of 3 meters decrease in height of water of the lake. it seems that the role of human factors and impacts is more than natural factors in the destruction of the lake (reveshty and maruyama, 2010). to investigate the reasons of reduction decrease in depth of the lake, several theories have been brought up. for example, some workers believe that the shahid kalantary causeway, which divides the lake, prevents the water circulation and this has caused the salinization of the lake. however, recent study shows that the impact of this causeway on the salt content of the lake is not significant. construction of dams and diversion of surface water for agriculture, along with reduced precipitation and warmer temperatures over the basin, and to a lesser extent reduced inflow of groundwater are generally among the possible causes (hassanzadeh et al., 2011; hoseinpour et al., 2010; eimanifar and mohebbi, 2007). reduced water volume concentrates the salts in the lake making it very saline for the brine shrimp which is near the bottom of the simple food chain support the very diverse bird populations. water conservation within the basin might provide some relief. however, finding the volume of water needed to restore the lake, without going outside the watershed, would probably require water from important areas of irrigated agriculture. water transfer from the caspian sea would be very expensive and time consuming and may come too late to avert damage to the ecosystem by the historically low water levels and high salinity that are already occurring. diverting water from neighboring watersheds would be less costly and time consuming but also has some serious challenges. a comprehensive integrated water management plan would take all the elements of the basin's water budget into account, balancing demands for irrigation, ecosystem preservation, social and human impact and water quality as well as operating within the national and regional political events. figure 4. coastline change detection during 1990 to 2012. 219 jaafari et al/ international journal of aquatic biology (2013) 1(5): 215-220 references: alesheikh a.a., ghorbanali a., nouri n. (2007). coastline change detection using remote sensing. international journal of environmental science and technology, 4: 61-66. anderson j.r., hady e., roach e.j., wetter t., richard e. (1976). lands cover classification system for use with remote sensor data. united states government printing office, washington, pp: 80-85. azari takami, g. (1993). uraemia lake as a valuable source of artemia for feeding sturgeon fry. journal of veterinary faculty, university of tehran, 47: 2-14. birkett c. mason i. (1995). a new global lakes database for remote sensing programme studying climatically sensitive large lakes. journal of great lakes research, 21: 307-318. chander g., markham b. (2003). revised landsat-5 tm radiometric calibration procedures and postcalibration dynamic ranges. ieee transactions on geoscience and remote sensing, 41: 2674-2677. chavez p.s. (1998). an improved dark-object subtraction technique for atmospheric scattering correction of multi-spectral data. remote sensing of environment, 24: 459-479. dewitt h., weiwen feng j.r. (2002). semiautomated construction of the louisiana coastline digital land-water boundary using landsat tm imagery. louisiana’s oil spill research and development program, louisiana state university, baton rouge, la 70803. ebrahimi a. (2010). lake urmia and its challenges, jahad daneshgahi publishing. 169 p. eimanifar a., mohebbi f., (2007). urmia lake (northwest iran): a brief review. saline systems, 3: 5. hassanzadeh e., zarghami m., hassanzadeh y. (2011). determining the main factors in declining the urmia lake level by using system dynamics modeling. water resources management, 26: 129145. hoseinpour m., fakheri fard a., naghili r. (2010). death of urmia lake, a silent disaster investigating causes, results and solutions of urmia lake drying. paper presented at the 1st international applied geological congress, department of geology, islamic azad university-mashad branch, iran. pp: 147-154. jalili s., kirchner i., livingstone d., morid s. (2012). the influence of large-scale atmospheric circulation weather types on variations in the water level of lake urmia, iran. international journal of climatology, 32: 1990-1996. karbassi a., bidhendi g., pejman a., bidhendi m., (2010). environmental impacts of desalination on the ecology of lake urmia. journal of great lakes research, 36: 419-424. karimi n., mobasheri m.r. (2011). shoreline change analysis of urmia lake using multi-temporal satellite images. eighteenth national conference on geomatics, iran, tehran. pp: 88-96. moore l.j. (2000). shoreline mapping techniques. journal of coastal research, 16: 111-124. ozesmi s.l., bauer e.m. (2002). satellite remote sensing of wetlands. wetlands ecology and management, 10: 381-402. rasoli a., abasian sh., |jahanbakhsh s. (2007). monitoring of uremia lake water level fluctuations using multi-temporal satellite images processing. journal of modaress, 12: 53-71. reveshty m.a., maruyama y. (2010). study of uremia lake level fluctuations and predict probable changes using multi-temporal satellite images and ground truth data period (1976-2010) new challenge about climate change or human impact, map asia 2010, malaysia, kuala lumpur. pp: 179-194. sugumaran r., harken j., gerjevic j. (2004). using remote sensing data to study wetland dynamics in iowa, iowa space grant (seed) final technical report, university of northern iowa, cedar falls. pp: 1-17. winarso g., budhiman s. (2001). the potential application of remote sensing data for coastal study, proc. 22nd. asian conference on remote sensing, singapore, national university of singapore. pp: 8791. zarghami m. (2011). effective watershed management; case study of urmia lake, iran. lake and reservoir management, 27: 87–94. 219 220 jaafari et al/ international journal of aquatic biology (2013) 1(5): 215-220 zeinoddini m., tofighi m., vafaee f. (2009). evaluation of dike-type causeway impacts on the flow and salinity regimes in urmia lake, iran. journal of great lakes research, 35: 13–22. international journal of aquatic biology (2015) 3(3): 161-171 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article structure and seasonal dynamics of phytoplankton and zooplankton in lake azili, small lake of the pond of river ouémé, benin arsène mathieu houssou*1, hyppolite agadjihouédé1, 2, elie montchowui1, 2, clément agossou bonou3, philippe lalèyè1 1laboratory of hydrobiology and aquaculture, university of abomey-calavi, bp 526 cotonou, republic of bénin. 2national high school of agronomics and technics sciences of kétou, university of agriculture of kétou, bp 95 kétou, republic of bénin. 3polytechnic school of abomey-calavi, university of abomey-calavi, bp 2009 cotonou, republic of bénin. article history: received 2 november 2014 accepted 22 april 2015 available online 2 5 june 2015 keywords: cyanobacteria eutrophication ecological health plankton abstract: the early reaction of the plankton communities to environmental changes makes it a useful tool for monitoring the pollution of aquatic environments. lake azili is a small water body, the majority of which is strongly influenced by river ouémé during the floods time. its ecosystem is one of the most important for the country, due to its rich biodiversity, especially that of halieutics. by following the evolution of its health, due to the strong anthropological pressure, this study aims to estimate the structure and the dynamics of its planktonic biodiversity and to assess its current state. it was carried out for seven months between may to november, 2012, according to the hydrological seasons of benin. plankton samplings were monthly, taken in a vertical sense from the lake, from all depths, using plankton net. the diversity indices were calculated for the compartment of zooplankton, that of phytoplankton being a preliminary evaluation. a total of 51 species of phytoplankton and 36 zooplankton species were inventoried. instability is observed in the seasonal structure of both communities, especially for the period of transition between the floods and the floods recession. due to the specific composition and the diversity, the ecosystem of lake azili is perturbed. introduction the aquatic biodiversity has been used for several decades as an ecological health and environment pollution indicator. in this regard, several studies estimated the distribution of the aquatic flora and fauna to understand the environmental changes. nowadays, in all of africa’s waters, the monitoring of quality is much more based on the assessment of macroscopic organism (mainly halieutic fauna), although the microscopic organisms are more sensitive to the environmental variations. in continental waters, sensibility of microorganisms is more notable due to strong human influence (groga, 2012). the distribution of these organisms is dictated by the auto-ecological processes that result from the global changes and the anthropological factors (dolédec et al., 1999). several algorithms and other * corresponding author: arsène mathieu houssou e-mail address: arsenehous@yahoo.fr statistical designs currently exist and are developed based on the plankton structures at specific abundance levels to assess the quality of the aquatics environments. but these tools are developed in temperate conditions, and it is necessary to adapt them on the tropical environments. this adaptation requires a good knowledge of the structure of microorganisms (phytoplankton and zooplankton) as well as their ecology. thus, several african countries began the exploration of the planktonic populations in their waters. but in benin, little study regarding plankton was carried out, while the country has several water bodies, among which lake azili forms a particular ecosystem. some populations depend on this lake, in particular the village “agonvè”, surrounded with water and forming a small island within the country. over the past few years, a 162 international journal of aquatic biology (2015) 3(3): 161-171 continuous process of degradation of the ecosystem has been recorded (aklinon, 2005). therefore, it is important to involve different disciplines to evaluate its current state and to identify ways of rehabilitation. hence, this study aims to estimate the phytoplankton and zooplankton communities of the azili lake and to establish a base for future works of quality assessment. materials and methods study sites and sampling stations: this study was carried on lake azili, which is a small flood lake of ouémé river in the municipality of zangnanado, situated in the center of benin. the lake azili extends in period of flood recession from 7°15' to 7°20' n and from 2°20' to 2°30' e. its area is 200 ha of water and reach 300 ha of permanent deep swamp. it measures an average 1.7 km from north to south and 2 km from east to west. its volume is regularly influenced by the olougbé river and the channel houan. the latter is a tributary and outflow for azili lake, at the same time. along 5 km, the channel houan links the lake with the ouémé river which, during the overflow between july and october pours a part of its waters in the lake through the channel. the oulugbé river is 15 km long and 2-3 m wide, and it is a permanent tributary of the lake. the high water of lake azili is essentially caused by those of ouémé river. three sampling stations (fig. 1), with a different level of human impact and representing the various housing environments, were chosen so as to represent the lake area. sampling have been made from upstream to downstream, as follows: dohoundji (07°15'n, 002°27'e), whéliyamey (07°15'n, 02°27'e) and gbekpletin (07°15'n, 002°27'e). sampling methods: the sampling was carried out monthly, according to the hydrological seasons of benin. it covered the period from may to november, 2012. the period of recession was represented by three months (may, june and july) and the flood season by the period from august to november. to ensure the good representation of the various species, the composite sampling protocol was retained. a composite sampling unit was a mixture of three vertical lines (lake full vertical sampling) at the same station. the sampling effort was thus nine hauls a month. the samples are then immediately fixated in 5% formalin solution. both organisms groups were sampled by the same conical net with a mesh size of 30 µm. thus, the study allowed to estimate exactly the zooplankton diversity, while regarding the phytoplankton, the diversity is not precisely estimated due to the fact that several species are smaller than 30 µm. the hydrological and physico-chemical conditions during the sampling are presented in table 1. a hanna multi-parameter and a voltcraf oximeter have been used to survey the physico-chemical parameters of water, while the hydrology was evaluated using a secchi disc (diameter 30 cm) and a graded and weighted rope. laboratory analysis: in the laboratory, all the samples were concentrated in the same volume of 100 ml before observation on photonic microscopy. the fifth aliquots of every sample were analyzed by taking, after homogenization, 1 ml of water sample figure 1. localization of the sampling sites in lake azili. 163 houssou et al/ plankton assemblage assessment in lake azili (1 ml pipette fixed). the species identification, based on the morphological characters, was carried out for phytoplankton compartment based on nogueiracorreia and ferreira (2000), tsukii (2005), kinross (2007) and oyadomari (2011). the identification of zooplankton species implied according to beauchamp (1965), smith and fernando, (1978), pourriot and francez (1986) and lynne (2004). after identification, the individuals of every species were counted per compartment, on counting cell. for the algal group, three categories of species were considered in the enumeration, i) the species found in abundance in all the fields, which were counted in 20 compartments, ii) the frequent species, counted in 40 fields and iii) the rare species which were counted on all the cell. as zooplanktons, the individuals were counted in all the compartments. during the counting for both communities, only the individuals having a whole structure are considered. data analysis: after enumeration of the individuals of species identified in 20 ml (1/5 of sample), the abundances related to the filtered water volume were obtained with the following formulas: for phytoplankton species, the abundance by milliliter of sample was first calculated by: n = (n * 196 / x), where n is the total number by ml observed; n the number of individuals counted by ml, 196 total number of compartment on the counting cell and x number of observed compartment. then, the density of every species in the lake, as well for the phytoplankton as for zooplankton was calculated by: where d is the density (ind.m-3), ni the total number of individuals by ml for the species i, he the depth of the lake, sbf the basic area of the plankton net, 3 number of hauls by sample and 20 number of aliquot of 1 ml observed. after calculation of the abundances, the data were transformed before any treatment with log (x+1) to stabilize the variances of biological over-dispersal (frontier, 1973). these data were projected in the factorial design of the principal components analysis (pca) to study the seasonal dispersal. the univariate diversity was also studied only for zooplankton by calculating the indices below. it is not calculated for phytoplankton considering that all the species of the lake are not certainly represented. the diversity of shannon-wiener, to discriminate the various populations. it is obtained by: where h' is the index of diversity expressed in bit/individual, ni the number of the species, n the total number of individual constituting all the species, log2 the logarithm on base 2. the diversity index of simpson. it is obtained by: where sp is the diversity index, ni the specific effective and n the total number of individual in the sample merged all species. the diversity index of margalef, expressed by: where ns is the total species number and n the total abundance. the evenness index to estimate the regularity between the species. it is obtained by: low water time flood time may june july august september october november température (°c) 30.7 ± 0.1 28.4 ± 0.1 26.2 ± 0.1 27,5 ± 0.2 27.7 ± 0.1 28.2 ± 0.3 29.37 ± 0.3 dissolved oxygen (mg.l-1) 6.1 ± 0.1 5.6 ± 0.2 3.8 ± 0.1 3.4 ± 0.1 3.6 ± 0.1 4.0 ± 0.3 4.2 ± 0.3 ph 6.8 ± 0.1 6.8 ± 0.03 6.9 ± 0.2 6.6 ± 0.1 6.3 ± 0.2 6.8 ± 0.02 6.6 ± 0.04 tds (ppm) 19.0 ± 0.1 17.5 ± 0.0 26.2 ± 2.0 25.5 ± 1.3 28.7 ± 0.3 28.5 ± 2.2 26.3 ± 0.3 conductivity (µs.cm-1) 43.7 ± 4.2 34.5 ± 0.9 51.3 ± 3.5 51.8 ± 2.3 55.0 ± 0.9 56.3 ± 4.2 51.5 ± 0.5 secchi depth (m) 0.7 ± 0.01 0.5 ± 0.05 0.6 ± 0.04 0.37 ± 0.0 0.40 ± 0.02 0.34 ± 0.02 0.19 ± 0.0 table 1. physico-chemical parameters recorded during the study. 164 international journal of aquatic biology (2015) 3(3): 161-171 where h' is the shannon index, log2 the logarithm on base 2 and ns the specific richness. the anova one ways test is finally used to study the temporal variations in the zooplankton and phytoplankton abundance. results populating composition and occurrences: during the study, a total of 51 phytoplankton species, share out in five classes (table 2) are inventoried including, diatomophyceae represented by 24 taxa, cyanophyceae by eight taxa, chlorophyceae by 12 taxa, euglenophyceae by six taxa and pyrrophyceae by one species. as indicated in the methodology, this diversity represents probably not all the phytoplankton in lake due to the used net. nevertheless, it represents a preliminary result of this diversity study. as the presence/absence, of the diatoms species during the shallow depths season, all taxa were identified except aulacodiscus sp. the latter was identified only during the floods, contrary to navicula sp., which was absent in the samples only during recession period. about the cyanobacteria taxa, all were identified as well in flood as during the recession. in the chlorophytes group, it was noted the absence of cladophora sp. during the flood recession while it appeared in the floods time. an opposite distribution pattern was observed for cosmarium sp. as the euglenophytes, all the species were represented in both seasons with the exception of trachelomas sp. which had the same distribution as cosmarium sp. the only one pyrrophyceae species was identified during both seasons. according to the occurrence percentages of various taxa regarding all the algal population, it is obtained a net dominance of the diatoms melosira genera in the lake. as regard to zooplankton, 36 taxa made up 30 species of rotifers, three copepods and three cladocerans (table 2) were recorded. the presence/absence showed that three rotifers taxa, including brachionus patulus, lepadella sp. and proales daphnicola were appeared in the lake during the floods time. in addition, two other rotifers i.e. lecane leontina and lecane sp. were disappeared with the floods. as copepods, just acartia sp. was found only during the high depths. the cladoceran bosmina sp., contrary to moina dubia, had the same distribution similar to the previous copepod species. the occurrence percentage of each rotifers species regarding whole group and both seasons showed the dominance of species as anuraeopsis navicula, anuraeopsis sp., asplanchna brighwelmlii, brachionus falcatus, cephalodella giba, keratella tropica, polyarthra vulgaris and testudinella sp.. as the copepods, mesocyclops sp. was dominant, while daphnia sp. dominated the cladoceran population. plankton population’s abundance: the total phytoplankton harvested with the net, during both hydrologic seasons, knew globally a distribution in which the floods time was unfavorable (fig. 2a). the best densities were obtained between may and july with the peak in june, which was followed by a progressive decrease until october showing a slight rise in november. this temporal variation was significantly different between june and october (p<0.05). regarding the zooplankton (fig. 2b), the distribution of the abundance was similar with that of phytoplankton, except in september. the two lowest densities of total zooplankton observed in october and november was significantly different and from that of all other months (p<0.05). in addition, the total zooplankton in may (relatively low) was significantly different from that of the other months with the exception of september. seasonal distribution of phytoplankton: the abundances data of various phytoplankton taxa, according to the months, both hydrological seasons, reduced and centered by a factorial analysis of the main components are represented on figure 3. with eigenvalue of 5.61, the first factor explains 80.15% of the results. it is essentially formed by the lowwater period and the month of august, representing the floods inception. all the variables are negatively selected and well-represented on it. this period (may-august) is correlated to the strong representat 165 houssou et al/ plankton assemblage assessment in lake azili phytoplankton code water level zooplankton code water level low flood % low flood % diatomophyceae rotifers aulacodiscus sp. d1 + 0.03 anuraeopsis navicula r1 + + 4.21 caloneis sp. d2 + + 0.36 anuraeopsis sp. r2 + + 11.51 cocconeis sp. d3 + + 2.27 asplanchna brightwelmlii r3 + + 14.25 diatoma trenuis d4 + + 0.13 asplanchna girodi r4 + + 2.93 eunotia bilunaris d5 + + 2.03 asplanchna sp. r5 + + 2.57 eunotia sp. d6 + + 0.05 brachionus caudatus r6 + + 0.71 gomphonema amoenum d7 + + 0.42 brachionus falcatus r7 + + 4.73 gomphonema sp. d8 + + 1.63 brachionus patulus r8 + 0.15 gomphonema vibrio d9 + + 1.13 brahionus quadridentatus r9 + + 0.49 melosira ambigua d10 + + 29.86 brachionus sp. r10 + + 2.31 melosira granulata d11 + + 2.90 cephalodella giba r11 + + 20.27 melosira sp. d12 + + 27.39 colurella uncinata r12 + + 0.33 melosira varians d13 + + 5.16 filinia longiseta r13 + + 1.25 navicula sp. d14 + 0.002 filina opoliensis r14 + + 1.92 nitzchia paradoxa d15 + + 0.14 habrotrocha sp. r15 + + 1.84 nitzschia reversa d16 + + 0.27 hexarthra intermédia r16 + + 0.50 nitzschia sigma d17 + + 5.26 keratella tropica r17 + + 4.75 pinnularia cardinalis d18 + + 0.08 lecane leontina r18 + 0.21 suriella capronii d19 + + 0.02 lecane sp. r19 + 0.01 suriella linearis d20 + + 1.57 lepadella patella r20 + + 0.78 synedra acus d21 + + 2.68 lepadella sp. r21 + 0.01 synedra splendens d22 + + 1.40 lindia sp. r22 + + 2.17 thalassiosira rotula d23 + + 0.08 ploesoma sp. r23 + + 2.59 thalassiosira sp. d24 + + 0.74 proales daphnicola r24 + 0.46 cyanophyceae proales decipiens r25 + + 1.60 microcystis flos-aquae cy1 + + 0.62 proales sp. r26 + + 1.86 microcystis sp. cy2 + + 0.29 polyarthra vulgaris r27 + + 5.58 microcystis wesenbergii cy3 + + 0.27 pompholyx sulcata r28 + + 1.42 oscillatoria sp. cy4 + + 0.02 rotaria sp. r29 + + 1.13 raphidiopsis méditerranea cy5 + + 0.80 testudinella sp. r30 + + 7.46 spirulina sp. cy6 + + 0.02 copepods stigonema sp. cy7 + + 0.75 acartia sp co1 + 1.12 synechocystis aquatilis cy8 + + 0.89 afrocyclops sp. co2 + + 4.95 chlorophyceae mesocyclops sp. co3 + + 9.95 binuclearia eriensis ch1 + + 5.11 nauplui n + + 83.97 cladophora sp. ch2 + 0.003 cladocera closterium aciculare ch3 + + 0.61 bosmina sp. cl1 + 12.85 + presence, absence table 2. phytoplankton and zooplankton species occurrence according to the hydrological seasons. 166 international journal of aquatic biology (2015) 3(3): 161-171 -ion of diatoms (melosira granulata, melosira varians, melosira ambigua, melosira sp., nitzschia sigma, gomphonema sp., synedra acus,cocconeis sp. and suriella linearis) and of chlorophytes (binuclearia eriensis), in opposition to diatoms (nitzschia paradoxa, navicula sp., pinnularia cardinalis, suriella capronii and aulacodiscus sp.), cyanobacteria (oscillatoria sp.), chlorophytes (cladophora sp., cosmarium sp., scenedesmus sp., staurastrum sp.), and euglenophytes (trachelomas sp.). the high-water time (october and november) and table 2. continued. phytoplankton code water level zooplankton code water level low flood % low flood % closterium parvulum ch4 + + 0.62 daphnia sp. cl2 + + 87.00 cosmarium sp. ch5 + 0.01 moina dubia cl3 + 0.15 gonatozygon sp. ch6 + + 0.06 micrasterias sp. ch7 + + 0.06 scenedesmus quadricauda ch8 + + 0.13 scenedesmus sp. ch9 + + 0.03 staurastrum sp. ch10 + + 0.02 stigeoclonium aestivale ch11 + + 0.37 tetraedron sp. ch12 + + 0.07 euglenophyceae euglena sp. e1 + + 0.48 phacus caudatus e2 + + 0.15 phacus longicauda e3 + + 0.68 strombomona sp. e4 + + 0.33 trachelomas bituricensis e5 + + 0.03 trachelomas sp. e6 + 0.01 pirrophyceae peridinium bipes p1 + + 0.07 + presence, absence figure 2. temporal evolution of total phytoplankton (a) and total zooplankton (b) of lake azili (the mustache boxes with no common letters are significantly different (p<0.05)). 167 houssou et al/ plankton assemblage assessment in lake azili august compose the second axis, which explains 11.02% of the distribution. it opposes the floods period, which is positively selected with regard to the average depths. the species such as the diatoms aulacodiscus sp., the cyanobacteria microcystis flos-aquae, the chlorophyte scenedesmus sp. and the euglenophyte t. bituricensis are well-represented during the floods, contrary to the species as eunotia sp., gomphonema amoenum, thalassiosira rotula (diatoms), microcystis wesenbergii (cyanobacteria), closterium aciculare, stigeoclonium aestivale (chlorophytes) and peridinium bipes (pirrophyte). the phytoplankton distribution in the factorial design of pca, following the quality representation of various species, show that those of diatoms are represented well in both hydrological seasons, the flood recession period; however being more favorable. on the other hand, the period during which the lake receives the first influxes of river ouémé (august-september) knew the best may june july august september october november -1.0 -0.5 0.0 0.5 1.0 fact. 1 : 53.10% -1.0 -0.5 0.0 0.5 1.0 f a c t. 2 : 2 1 .7 1 % r13 r14 r30 r28 r16 r23 r7 r8 r6 r9 r10 r1 r2 r17 r3 r4 r5 r24 r25 r26 r11 r18 r19 r20 r21 r12 r29 r22 r27 r15 co1 co3 co2 n cl2 cl3 cl1 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 fact. 1 : 53.10% -4 -3 -2 -1 0 1 2 3 4 5 f a c t. 2 : 2 1 .7 1 % d11 d13 d10d12 d24 d23 d17 d15d16 d5 d6 d9 d7 d8 d18 d14 d2 d22 d21 d4 d20 d19 d3 d1 cy1 cy3 cy2 cy8 cy4 cy6cy5 cy7 ch8 ch9 ch4 ch3 ch7 ch10 ch5 ch6 ch1 ch12 ch2 ch11 e3 e2 e5 e6 e1e4 p1 -8 -6 -4 -2 0 2 4 6 8 fact. 1 : 80.15% -5 -4 -3 -2 -1 0 1 2 3 4 5 f a c t. 2 : 1 1 .0 2 % may june july august september october november -1.0 -0.5 0.0 0.5 1.0 fact. 1 : 80.15% -1.0 -0.5 0.0 0.5 1.0 f a c t. 2 : 1 1 .0 2 % figure 3. projection of phytoplankton species and the sampling time in the factorial design of principal component analysis. figure 4. projection of zooplankton species and the sampling time in the factorial design of principal component analysis. 168 international journal of aquatic biology (2015) 3(3): 161-171 representations of other groups (cyanobactera, chlorophytes, euglenophytes and the only pirrophyte species). the high-water months there were not so selective. seasonal distribution of the zooplankton: zooplankton distribution submitted to analysis in principal components is presented on figure 4. first axis which explains 53.10% of the results is characterized by the shallow depths season, all the variables being negatively correlated; it is composed by june, july and august. september is also well represented on it. therefore, it explains the microfauna distribution during the recession and a part of that of the transition period between both seasons. it includes the species such as rotifers testudinella sp., anuraeopsis sp., keratella tropica, polyarthra vulgaris and nauplii of copepods, which oppose their strong representation of shallow depths to that of: i) brachionus patulus, brahionus quadridentatus, lecane leontina, lecane sp. and lepadella sp. (rotifers), ii) acartia sp. (copepod) and iii) bosmina sp. and moina dubia (cladoceran). the second axis, which expresses 21.71% of the results, is formed by the floods period (september, october and november). it selects positively the last two months and negatively september, the latter being very well-represented in the factorial design. it includes essentially the rotifer species, among which filinia longiseta, filina opoliensis, pompholyx sulcata, brachionus falcatus, asplanchna brightwelmlii, proales decipiens and lindia sp. are strongly correlated to september. they oppose their spread to testudinella sp., ploesoma sp., brahionus quadridentatus and habrotrocha sp., which are selected by the floods period. according to the quality representation of various zooplankton species, selected and not selected by one or the other one, both axes of the pca design, it was observed that rotifers were distributed well on both seasons. nevertheless, august (at the floods beginning) was favorable to the abundance of several species. the same period was the one with the best representations of two other zooplankton groups (copepods and cladoceran), while the high depths were unfavorable. zooplankton specific diversity and dominance: the diversity indices of zooplankton populations are presented in table 3. several indices were calculated to assess the diversity. in none of the studied months, the specific richness was equal to the total value observed on the lake. thus, the ecological preference of the different species varied over the sampling period. regarding the diversity indices, no high variations have been observed between both hydrological seasons, nor between constituent months. in that, the specific richness varied between 18 and 29 species, while the simpson diversity is near of unity for all the study period. the shannon wiener index is varied between 2.74 bit.ind-1 and 3.29 bit.ind-1, the highest value coinciding with the biggest specific richness of august, the biggest simpson diversity and also that of margalef (29, 0.96 and 4.46, respectively). the samples uniformity measured by evenness, remained very high during both seasons. all the index consideration, shows a good diversity of zooplanktonic population in the table 3. monthly variation of the diversity index and the dominance degree of zooplankton species. rs sp h’ evenness marg dominants species %d may 25 0.95 3.09 0.88 3.95 brachionus falcatus, cephalodella giba 12.8 june 28 0.96 3.28 0.95 4.28 asplanchna girodi, cephalodella giba 10.3 july 27 0.96 3.28 0.98 4.12 polyarthra vulgaris, testudinella sp. 8.9 august 29 0.96 3.29 0.92 4.46 anuraeopsis navicula, asplanchna brightwelmlii 10.3 september 27 0.95 3.20 0.91 4.30 filina opoliensis, asplanchna brightwelmlii 12.4 october 19 0.93 2.74 0.82 3.46 habrotrocha sp., testudinella sp. 21.2 november 18 0.93 2.74 0.82 3.02 keratella tropica, testudinella sp. 18.9 rs: specific richness, sp: simpson diversity, h': shannon diversity, marg: margalef diversity 169 houssou et al/ plankton assemblage assessment in lake azili lake. the best diversifications were recorded during the transition period (august-september) while lower diversities were recorded during the high depths (october-november). this diversity has resulted in low levels of dominance by the next most represented species in the different months of the study (table 3). thus, the percentage of dominance varied between 8.9% and 21.2%. the lowest dominance does not correspond exactly to the most diversified, but altogether the tendency remains the same. discussion although the study of the phytoplankton compartment is a preliminary evaluation of the diversity of the microflora of lake azili, a significant number of species was identified. totaling 51 species, the found richness is widely below that of other watercourses of the sub-region (111 and 192 species, respectively on the lake guiers in senegal (ngansoumana, 2006) and in the coastal river of ivory coast (niamien-ebrottié et al., 2013)). the microflora sampled in the present study showed a structure dominated by diatoms, similar to onozeyi (2013) on ogun river in southwest of nigeria. azili lake is a particular ecosystem with its important vegetation cover and the strong anthropological pressure; it has a muddy bottom due to the organic decomposition. this, therefore, justifies the important presence of diatoms species, which, in continental environment, knows good diversifications due to the organic mineralization and re-suspension caused by human activities (groga, 2012). in the diatoms group the species selective of polluted environment were identified. the strong representation of the genera nitzschia, gomphonema, navicula, pinnularia, caloneis, eunotia, etc., shows that the ecosystem is perturbed and rich in organic elements. the cyanobacteria presence, generally characteristic of high nitrogenous and phosphated mineral concentration in thermal conditions from 25 to 30°c (erkaya et al., 2011), is not characteristic of the lake, even if the thermal conditions are filled all year (tropical environment). some identified species were more representative during the transition period between recession and floods. this dynamics gives two hypotheses. given that in this period, the lake receives the influxes of its tributary (the river ouémé), whether it had a direct contribution in specimens by the river or the mineral conditions were improved by its jet. the first hypothesis would give more a gradient upstream-downstream in the species distribution, not being the case in the results of this study. the second hypothesis thus remains the most convincing even if a more spatial study remains to be carried out. it would thus be a mineralized material re-suspension and a nutriment contribution while the physico-chemical conditions of the river are poor (hounsou, 2011). nevertheless, the presence and temporal distribution selective species for high eutrophication conditions (erkaya et al., 2011), shows the ecosystem disturbance even if a planktonic bloom is not observed at present. in addition, the chlorophytes have presented an ecological preference for the big floods period selecting particularly the transition period. thus, the arrival of new species and the disappearance of others previously inventoried species can be observed, which was the case in four phytoplankton phyla, the most representative of inventoried population. an instability in the algal community structure in the lake due to exchanges with its tributary is subsequently noted. regarding the zooplankton populations, the biodiversity of the lake is globally cosmopolitan. filinia longiseta, filina opoliensis, testudinella sp., brachionus caudatus, keratella tropica, etc. are generally well-represented in diverse environments (bozkurt and akin, 2012). its wealth is important, being slightly superior than that of cross river of nigeria (offem et al., 2009). it presents a characteristic structure of tropical fresh water by the wide dominance of rotifers. the seasonal structure of this community shows a tendency towards the transition period for several species. the diversity is also unstable due to the exchanges with the ouémé river, a classic structure being, nevertheless, 170 international journal of aquatic biology (2015) 3(3): 161-171 observed with the peak abundance shortly after that of phytoplankton. in the population of inventoried rotifers, many species are characteristic of polluted environment. selecting the strongly eutrophic ecosystems (bozkurt and akin, 2012), the distribution and temporal dominance of the species from the genera filina and lepadella, as of some phytoplankton species, show that the lake is in a degradation process. the health of an aquatic ecosystem can be estimated by several methods one of them being the calculation of the diversity index. it is recognized that species diversity increases with succession, while the characteristics of different species vary the spatial and temporal distribution according to the classification level of the food chain. a diversity index to explain the community characteristics will thus have to consider the functional structure of this one both at a spatial and temporal level. the work which we realized has a temporal consideration reduced and is valid only for the considered period. therefore, for all the calculated indices, the population presents an average diversity. the shannon index, dependent on the size of samples and on the type of habitat (grall and coïc, 2005), interpreted with evenness, classifies the lake in the category of the moderately polluted ecosystems during the floods recession and the transition period and strongly polluted during the floods, on the scale of the sandy/muddy ecosystems according to simboura and zenetos (2002), showing again the impact that the ouémé river has on the azili lake. the plankton biodiversity of the lake is a classic population of tropical environment with an unstable structure due to the rejection of the tributary ouémé river. considering the ecosystem established by the lake, pressures which are exercised and its planktonic biodiversity, a tendency to eutrophication is observed. the distribution of species along the two hydrological seasons, submitted with the classification of the lake on the simboura and zenetos (2002) pollution scale, highlighted in both group of phytoplankton and zooplankton, some species potentially bio-indicators of pollution. therefore, aulacodiscus sp., microcystis flos-aquae, scenedesmus sp. and trachelomas bituricensis are phytoplankton species candidates for bio-indication in the lake azili subject to a further study in this direction. regarding the zooplankton group, those are species such as: filinia longiseta, filina opoliensis, pompholyx sulcata, brachionus falcatus, asplanchna brightwelmlii, proales decipiens, lindia sp., testudinella sp., ploesoma sp., brahionus quadridentatus and habrotrocha sp. a complementary study assessing the actual state of the lake pollution and the direct effects of pollutants on these species so will confirm or refute species as bioindicator of pollution in the lake azili environment. acknowledgements we are grateful to all person who contributed to the good realization of this work. a particular thought to mr. yaovi rené for his contribution in the data collection and to the teams of the laboratories of hydrobiology and aquaculture of faculty of agronomic sciences and research in applied biology of polytechnic school of abomey-calavi. references aklinon s.y. 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(2015) 3(6): 379-389 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article a histo-morphological characteristics of gonads in mudskipper, periophthalmus waltoni koumans, 1941 from helleh estuary, southwestern iran somayeh ghasemian1, hamid reza esmaeili*1, mohsen nokhbatolfoghahai1, abdolrahim pazira2 1department of biology, college of sciences, shiraz university, shiraz, iran. 2department of fisheries, islamic azad university, bushehr branch, bushehr, iran. article history: received 14 june 2015 accepted 12 o c t o b e r 2015 available online 2 5 d e c e m b e r 2015 keywords: gobiidae reproduction indices gonad histology iran abstract: in this study, the morphological and histological studies of male and female gonads in mudskipper, periophthalmus waltoni koumans, 1941 from helleh estuary (bushehr province, southwestern iran) were performed to determine its gonadal development stages and histomorphological characteristics. sampling was done from april 2010 to march 2011 and a total of 81 individuals were collected by hand net. the gonads of specimens were removed, their sexes determined and then fixed in 10% formalin solution after checking their morphology and measuring their weights, lengths and widths. six stages of gonadal developments in females and four stages in males were determined based on macroscopic and microscopic observations and reproductive indices. in female, increasing of the ovary size is occurred because of the accumulation of yolk materials in oocytes, and in the last stages, little folding in ovary was observed. formation of zona radiata and yolk granules in the third stage, and increasing thickness of this layer and yolk granules were observed from stage three to stage six. in male, gradual developments of the sperm cells were observed from stage one onward. introduction there are sever debates on the taxonomic position of gobioid fishes at the order and family levels especially during the last few years. while most of the scientists consider these fishes in the order perciformes, however, thacker (2013) proposed gobiiformes and gobionellidae as distinct order and family. the family gobionellidae was identified by thacker (2003; there denoted the “expanded monophyletic gobionellinae”) and separated from gobiidae (thacker, 2009) as part of broad molecular phylogenetic analyses of the gobioid fishes (order gobiiformes; thacker, 2013). that analysis delineated the family gobionellidae composed of inshore, shallow-dwelling fishes, most of which are euryhaline; it includes stream gobies, mudskippers, and eel gobies, among others (thacker, 2013). however, mudskippers has been considered in the order perciformes (perch-likes), family gobiidae * corresponding author: hamid reza esmaeili e-mail address: hresmaeili22@gmail.com (gobies) and subfamily oxudercinae (see eschmeyer and fong, 2015). esmaeili et al. (2010) reported 40 species of the gobioid fishes from iran including mudskippers. mudskippers of iran are member of the subfamily oxudercinae (tribe periophthalmini, murdy, 1989), or periophthalmus lineage including periophthalmus, boleophthalmus and scartelaos (thacker, 2013). they are completely amphibious fish that can use their pectoral fins to "walk" on land (harris, 1960; swanson and gibb, 2004). these fishes spend extensive periods of time out of water and have numerous physiological, morphological and behavioral specializations for amphibious life style (gordon et al., 1969; clayton, 1993; graham, 1997; lee and graham, 2002). walton’s mudskipper, periophthalmus waltoni koumans, 1941 (sensu murdy, 1989) is a member of periophthalmus lineage known from a variety of intertidal habitats, including mudflats and mangrove 380 int. j. aquat. biol. (2015) 3(6): 379-389 ecosystems of the persian gulf and makran sea (ghanbarifardi and malek, 2007; ghanbarifard et al., 2014a, b). its distribution is from the western part of persian gulf to pakistan (murdy, 1989). walton’s mudskipper is not commercially valuable locally, but it is preyed by sea birds. it may play an important role as a bioindicator of anthropogenic impact on habitat in mangrove forests and intertidal areas (wong et al., 2000; polgar, 2008). study on the gonadal development of p. waltoni is scare. therefore, this study was conducted to provide a detailed description of the gonadal development and histo-morphological characteristics of p. waltoni from helleh estuary, southwestern iran. materials and methods a total of 81 individuals of walton’s mudskipper, p. waltoni (fig. 1) were collected by hand-net from helleh estuary in bushehr province (29°09'23.9'' n; 50°40´19'' e, 2 m a.s.l), southwestern iran between april 2010 to march 2011. after anesthesia, fish were fixed in 10% formalin, then labeled individually and deposited in the zoological museum, collection of biology department, shiraz university (zm-cbsu). some morphometric features were measured using digital caliper to the nearest 0.01 mm and body weights were determined using a digital balance to the nearest 0.1 g. the specimens were cut through the ventral position and gonads were taken out of the body, the sexes and stages of sexual maturation were determined as possible as by naked eye examination and under compound microscope. a chi-square test was used to assess deviation from 50:50 sex ratio based on robards et al. (1999). the gonads were then fixed into 10% formalin solution after checking their morphology and measuring their weights by a digital balance to the nearest 0.001 g, and the lengths and widths with a digital caliper at 0.01 mm. the histological sections of the gonads were prepared by a routine histology method based on bancroft and stevens (1991) as follow: the gonads were dehydrated in alcohol, cleared in xylene, and impregnated in paraffin wax at 56oc melting point, sectioned at 5-7 μm thickness and then stained using hemotoxylin and eosin (h&e). after preparing the histological slides, they were studied under a compound microscope and the photos were taken. sexual maturity of each specimen was classified according to macroscopic criterion based on ices (international council for exploration of the sea of 1963, 1999) and a microscopic criterion, based on morphological and histological analysis. results size range: a total of 81 individuals of p. waltoni ranging 49.81-125.88 mm (s.d: 15.38) in total length, 39.71-102.35 (s.d: 12.84) in standard length, and total weight of 0.85-17.35 g (s.d: 4.04) were collected. sex ratio: we determined sex in 50 female and 28 male specimens. the overall sex ratio was significantly female biased and deviates from the hypothetical distribution of 1:1 (1.8:1, f/m; chi square=6.205, df=1, p=0.01). the structure of ovaries: a pair of elongated ovaries were situated in the posterior of the gut and adhered to the vertebral column. anteriorly, the two lobes are free but adjoined together posteriorly and ended to the genital pore. during gonadal development, the weight, length and width of ovary increased and its color changed from cream to dark yellow. the oocytes were observed easily from 3rd stage by naked eye. macroscopic and microscopic characteristics of ovaries: macroscopically and microscopically the figure 1. walton's mudskipper, p. waltoni, from helleh estuary, southwestern iran. 381 ghasemian et al./ histo-morphological characteristics of gonads in periophthalmus waltoni maturity stages in the present study maturity stages used in ices female male i. immature i. immature i. virgin ii. immature and developing ii. immature and developing ii. virgin maturing vii. recovering-spent iii. maturing iii. maturing iii. maturing iv. maturing iv. ripe iv. maturing v. ripe v. maturing vi. spawning vi. spawning vii. spent vii. spent table 1. comparison of the used maturity criterion in the present study with criterion used in ices. stage measurements n mini max mean sd i gonad weight (g) 1 0.002 0.002 0.00200 . gonad length (mm) 1 8.38 8.38 8.3750 . gonad width (mm) 1 0.55 0.55 0.5483 . gl.acl (%) 1 47.16 47.16 47.1565 . ii (early) gonad weight (g) 23 0.001 0.027 0.01061 0.006236 gonad length (mm) 23 4.50 14.53 9.6293 2.19507 gonad width (mm) 23 0.83 1.51 1.1910 0.21419 gl.acl 23 36.20 75.46 55.9532 9.30253 ii (late) gonad weight (g) 7 0.016 0.050 0.02529 0.011940 gonad length (mm) 7 8.88 13.47 11.0557 1.68212 gonad width (mm) 7 1.25 1.84 1.5640 0.19575 gl.acl 7 49.58 71.33 59.6237 8.86275 iii gonad weight (g) 10 0.040 0.124 0.07210 0.032716 gonad length (mm) 10 10.32 14.19 11.7585 1.31446 gonad width (mm) 10 1.68 2.61 2.1268 0.33231 gl.acl 10 51.72 72.18 62.1627 7.40175 iv gonad weight (g) 4 0.185 0.330 0.23375 0.065749 gonad length (mm) 4 14.78 16.69 15.3238 0.91017 gonad width (mm) 4 3.20 3.64 3.3967 0.18149 gl.acl 4 65.76 95.84 77.1354 13.19831 v gonad weight (g) 4 0.620 0.680 0.64500 0.026458 gonad length (mm) 4 19.51 22.22 20.4800 1.26219 gonad width (mm) 4 4.74 5.24 4.9275 0.24001 gl.acl 4 81.29 93.53 89.0697 5.41776 vi gonad weight (g) 1 0.850 0.850 0.85000 . gonad length (mm) 1 24.09 24.09 24.0900 . gonad width (mm) 1 5.24 5.24 5.2383 . gl.acl 1 104.06 104.06 104.0605 . gl.acl: the ratio of gonad length to abdominal cavity length table 2. descriptive statistics of gonad measurements in p. waltoni (female) from helleh estuary, southwestern iran. 382 int. j. aquat. biol. (2015) 3(6): 379-389 ovaries were classified into seven developmental stages based on the size and weight, degree of occupation of the body cavity, presence or absence of ripe oocytes, diameter of the oocytes and histological observations. the stages were classified as immature (stage i), immature and developing (stage ii), maturing (stages iii and iv), ripe (stage v), spawning (stage vi) and spent (stage vii). no specimens of spent stage were observed. macroscopic classification was similar to those used in ices (table 1). the gonad measurements have shown in tables 2 and 3. ovary maturation stages: in the immature stage (i), the ovary was too small, thread-like and transparent so that sex determination was difficult. the mean diameter of oocytes was 0.05 mm. no histological section was prepared successfully for this stage. in the stage ii (immature and developing), the ovary was small, thin, long with smooth surface and soft texture. it was cream in color and had oocytes that could not be observed by naked eye. the mean diameter of oocytes was 0.07 mm (fig. 2a). in the histological observations (fig. 3a-b), two oocyte types (i and ii; mostly type ii) with many interstitial tissue were observed. there was an extensive, light violet nucleus in the center of oocyte with abundant nucleolus around the nucleoplasm. in larger oocytes, the lipid droplets were formed in the cytoplasm and stage measurements n min maxi mean sd 1 gonad weight (g) 1 0.001 0.001 0.00100 . gonad length (mm) 1 6.73 6.73 6.7300 . gonad width (mm) 1 0.41 0.41 0.4050 . gl.acl (%) 1 63.37 63.37 63.3710 . 2 gonad weight (g) 7 0.001 0.001 0.00100 0.00 gonad length (mm) 7 7.02 9.57 8.2707 1.01745 gonad width (mm) 7 0.37 0.52 0.4231 0.04715 gl.acl 7 46.75 69.35 61.5569 7.56434 3 gonad weight (g) 14 0.001 0.067 0.01157 0.018744 gonad length (mm) 14 4.05 12.96 8.5657 2.32659 gonad width (mm) 14 0.36 1.32 0.7448 0.28723 gl.acl 14 23.67 58.55 44.4367 9.96509 4 gonad weight (g) 6 0.002 0.093 0.01983 0.035891 gonad length (mm) 6 7.02 10.54 9.0350 1.36054 gonad width (mm) 6 0.68 1.30 1.0378 0.24428 gl.acl 6 35.45 62.16 48.4772 9.47384 gl.acl: the ratio of gonad length to the abdominal cavity length. table 3. descriptive statistics of gonad measurements in p. waltoni (male) from helleh estuary, southwestern iran. figure 2. morphological profiles of the female maturity stages of p. waltoni, helleh estuary, southwestern iran. (a) immature and developing stage, (b, c) maturing stages, (d) ripe stage, and (e) spawning stage. 383 ghasemian et al./ histo-morphological characteristics of gonads in periophthalmus waltoni follicular layer encloses oocyte. in the third stage (maturing), the size of ovary increased, it was lobulated and its color changed from cream to light yellow because of the accumulation of yolk materials. the oocytes were not visible by naked eye in this stage. the mean diameter of oocytes was 0.1 mm (fig. 2b). the histological sections showed three oocyte types (i, ii and iii; mostly iii) in the ovaries with developed interstitial tissues. oocyte size was increased in this stage, the ratio of nucleus to cytoplasm was decreased; the lipid droplets increased and formation of yolk granules was distinguished. furthermore, the follicular layer and zona radiata were appeared around the oolema (fig. 3c-d). in the maturing stage (iv), the ovary increased in size due to increasing of the yolk of the oocytes, its color changed to yellow, and wrinkled. the mean diameter of oocytes was 0.3 mm (fig. 2c). in this stage, the oocytes are known as follicles. their size were increased and the ratio of the nucleus to cytoplasm and interstitial tissue were decreased. in comparison to the lipid droplets, the yolk granule accumulations were significant. zona radiata was thickened and follicular layer developed compared to previous stages. a thickening structure was found between the zona radiata and follicular layer, which could be the site of the micropyle (fig. 4a-c). in the ripe stage (v), due to increasing the ovary size and abdominal cavity restriction a little folding was observed in gonad. following the figure 3. histological profiles of the female maturity stages of p. waltoni, helleh estury, southwestern iran. (a-b) immature and developing stage, (c-d) maturing stages n: nucleou, nu: nucleolus, c: cytoplasm, ld: lipid droplets, fl: follicular layer, yg: yolk granules, tl: theca layer, fl: follicular layer, zr: zona radiata. 384 int. j. aquat. biol. (2015) 3(6): 379-389 reduction of ovary interstitial tissue, cohesion between oocytes was decreased. the mean diameter of oocytes was 0.46 mm in this stage (fig. 2d). in microscopic observations, follicular sizes, density of yolk granules and thickness of zona radiata increased and the ratio of nucleus to cytoplasm decreased and some of the follicles were in absorbing stage. follicular cells changed their shapes from cuboid to pavement state (fig. 4d-f). in the spawning stage (vi), the ovary obtained its maximum size, having very thin epithelium with loosely oocytes connected to each other. the mean diameter of oocytes was 0.61 mm in this stage (fig. 2e). in microscopic sections, the follicle sizes increased and almost no nucleus was visible because of accumulation of yolk granules. in spawning stage, thickness of the zona radiata and theca layer was increased and decreased, respectively (fig. 4g-h). testes maturation stages: the position of the testes was the same as ovary. their weights, lengths and widths were increased during the gonadal developmental but not as large as ovary and their color was changing from grey to milky during gonadal development. the main histological characteristics which correspond to the maturation stages are the spermatogenesis process. in this study, five spermatogenic cells were diagnosed as follow: (i) spermatogonia stage: they were the largest figure 4. histological profiles of the female maturity stages of p. waltoni, helleh estuary, southwestern iran. (a-c) maturing stages, (d-f) ripe stage, (g-h) spawning stage ld: lipid droplets, fl: follicular layer, yg: yolk granules, tl: theca layer, fl: follicular layer, zr: zona radiata. 385 ghasemian et al./ histo-morphological characteristics of gonads in periophthalmus waltoni figure 5. morphological profiles of the male maturity stages of p. waltoni, helleh estuary, southwestern iran. (a) immature stage, (b) immature and developing stage, (c) maturing stage, and (d) ripe stage. figure 6. histological profiles of the male maturity stages of p. waltoni, helleh estuary, southwestern iran. (a) immature stage, (b) immature and developing stage, (c) maturing stage, (d) ripe stage s: spermatogonia, ps: primary spermatocyte, ss: secondary spermatocyte, st: sspermatid, sz: spermatozoa, sl: sertoli cell 386 int. j. aquat. biol. (2015) 3(6): 379-389 spermatogenic cell, with clear cytoplasm and large nucleus. the cysts or nests were absent. (ii) primary spermatocytes stage: these cells possess nuclei that were densely packed. (iii) secondary spermatocytes: they were similar to primary spermatocytes but smaller. (iv) spermatids: they were smaller in size than secondary spermatocytes and possess dense nuclei. (v) spermatozoa: the spermatozoa were the smallest and densest spermatogenic cells and product of modifications of spermatid cells. they were concentrated in the seminal lobules after breaking through the nest wall. the sperms, later acquired the ability to be mobile in the seminal fluid. this signifies the final product of the process of spermatogenesis. macroscopically and microscopically, testes were classified into four developmental stages, including immature (stage i), immature and developing (stage ii), maturing (stage iii) and ripe (stage iv). testes measurements are shown in table 3. in the immature stage (i), the testes were very small, thread like and grey (fig. 5a). spermatogonia and primary spermatocytes were differentiable in the histological sections (fig. 6a). in the second stage (immature and developing), the sizes of whitish cream gonads were increased (fig. 5b). in addition to pervious stage, the histological sections showed the presence of the secondary spermatocyctes and few accumulation of the spermatids (fig. 6b). in the maturing stage (iii), the testes were elongated, thickened and milky in colour (fig.5c). the number of the spermatogonia were reduced, a little spermatozoa were observed but spermatids were dominant (fig. 6c). in the stage iv (ripe), the testes were more elongated, thickened and milky (fig. 5d). in this stage, the gonads were ripe as milt released by cutting part of testis. in histological observations, nearly within all of the tubules were full of spermatozoa (fig. 6d). discussion based on the results, six stages of the ovary development, including immature, immature and developing, maturing, ripe, spawning and spent were observed in p. waltoni. lawson (2010, 2011) classified testis and ovary of periophthalmus papilio into seven stages. the general pattern of the histological development of the ovaries in p. waltoni conforms to that of the most teleosts (elgharabawy, 1996; assem, 2000, 2003). teleost ovaries can be divided into three types based on the pattern of oocyte development viz. synchronous, group synchronous and asynchronous (nagahama, 1983). in histological observations of p. waltoni ovaries, in each stage, the follicles were at different developmental stages, but at ripe and spawning stages, most of the follicles were developed. the simultaneous presence of three clearly distinct sizes of oocytes in ripe ovaries of gobius vittatus (gobiidae) indicated that this species is a multiple spawner (kovacic, 2007). testicular structure in teleosts can be classified into two types i.e. tubular and lobular, depending on the pattern of spermatogenesis (billard et al., 1982). the testis of p. waltoni is tubular that is the same as scartelaos gigas (kim et al., 2011). development of sperm or milt in fish passes through multiplication, growth and maturation stages. the testis of p. waltoni is similar to other species consisting seminiferous tubules, spermatogonium, spermatocyte, spermatids and spermatozoon. their presence was an indication that the gamete or sperm had gone through process of maturation (spermatogenesis) as were observed in figures 5-6. in the present study, there was variation in the monthly gsi values, the highest gsi values correspond to when the gonads were at ripe and ripe running stages (spawning phase), while the lowest gsi values indicate totally spent stage or starting developing stage (recovering stages). gsi of female was peaked in spring and autumn, and that of males were peaked in spring, but no peak was observed in autumn in male, maybe because of no enough collected male specimens in this season. the fish burrows in the mud flats that might be responsible for fewer collected specimens. the gsi for females was always higher than males. this has been reported in other fishes (pajuelo and lorenzo, 2000; 387 ghasemian et al./ histo-morphological characteristics of gonads in periophthalmus waltoni esmaeili and shiva, 2006). the mean of gsi was higher in female in reproduction season (mahomoud et al., 2011). the gsi varied with species, sex, seasons and availability of food (see pajuelo and lorenzo, 2000; esmaeili and shiva, 2006; mahomoud et al., 2011). this index has been widely used as indicator of the fish spawning season, but its use in reproductive biology studies is more suitable when it is associated with other reproduction indicators such as macroscopic and histological techniques. this is very important in males since differences in gonad size (length and weight) are less conspicuous than in females. our findings demonstrated that spawning season of p. waltoni is in spring and autumn interpreted from the results of reproductive index (e.g. gsi) and gonad histology. therefore, this study has provided information on the maturation process and reproductive cycle of mudskipper, p. waltoni in the coastal area of persian gulf which contribute baseline data towards management ecology, conservation and biological studies of this fish. acknowledgments we are thankful to s. babai, s. mirghiasi and r. zamaniannejad for their kind help in fish collection and shiraz university for financial support. references assem s.s. 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(2015) 3(6): 379-389 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی از ،periophthalmus waltoni koumans, 1941 خورکگل ماهی گنادهای شناسیبافت و ریختی هایویژگی ایران غربی جنوب هله، مصب 2پذیرا عبدالرحيم ،1الفقهایی نخبه محسن ،*1اسماعيلی حميدرضا ،1قاسميان سميه 1گروه زیستشناسی، دانشکده علوم، دانشگاه شیراز، شیراز، ایران. .ایران بوشهر، بوشهر، واحد اسالمی، آزاد دانشگاه شیالت،گروه 2 چکيده: یماه ماده و نر هایجنس گنادهای( میکروسکوپی و ماکروسکوپی) شناسیبافت و ریخت همطالع جنسی، بلوغ مراحل تعیین منظوربه تحقیق، این ماه ازبرداری نمونه. گرفت صورت ایران جنوب بوشهر، استان در هله مصب از ،periophthalmus waltoni koumans, 1941 خورک،گل از های ماهیی نمونهگنادها. گردید آوریجمع دستی، ساچوک وسیلههب نمونه 11 در مجموع تعداد و گرفت صورت 2211 مارس تا 2212 آوریل به. شدند تثبیت %12 فرمالین محلول در ها،آن عرض و طول وزن، گیریاندازه ،ریختی بررسی از بعد و گردید تعیین آنها جنسیت ،شدند خارج بدن در. گردید تعیین نرها برای مرحله 4 و هاماده برای جنسی بلوغ مرحله 6 ،تولیدمثلی شاخص بررسی و میکروسکوپی و ماکروسکوپی مشاهدات کمک شکمی حفره محدودیت دلیلهب آخر مراحل در و بود، یافته افزایش تخمک اندازه ،هااووسیت در ایزرده مواد تجمع افزایش دلیلهب ماده، گنادهای ضخامت افزایش و سوم مرحله در ایزرده هایگرانول و شفاف شعاعی الیه تشکیل. شد مشاهده تخمدان در خوردگیچین کمی گناد، وسعهت یط در ه قابل مشاهد اول مرحله از اسپرم هایسلول تدریجی وسعهت نر، گنادهای در. بودند مشاهده قابل ششم تا سوم همرحل از ایزرده هایگرانول و الیه این .بود .ایران گناد، شناسیبافت تولیدمثلی، هایشاخص ،gobiidae :کلمات کليدی int. j. aquat. biol. (2018) 6(6): 307-313 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article growth and condition factor of capoeta damascina in the azad dam lake and the komasi river in kurdistan province, iran hasan fazli*1, gholamreza daryanabard1, medi naderi jelodar1, rahman mirzaei2, hamid hosseinpour2 1caspian sea ecology research center (cserc), iranian fisheries science research institute (ifsri), agricultural research, education and extension organization (areeo), sari, mazandaran, iran. 2department of fisheries, jihad-agriculture of kurdistan organization, sanadaj, kurdestan, iran. article history: received 1 july 2018 accepted 8 december 2018 available online 2 5 december 2018 keywords: growth parameters length-weight relationship condition factor population abstract: the objective of the present study was to estimate the population parameters, including length-weight relationship (lwr), condition factor (kf), relative condition factor (kn), age, growth, and mortality of capoeta damascina in the azad dam and komasi river in the kurdistan province, iran. total length and weight were 51-350 (180.6±68.63) mm and w: 2.5-465 (93.3±110.89) g in the river and 120-260 (191.5±30.76) mm and 14.7-216 (75.9±39.89) g in the dam lake and river, respectively. the length-weight regression was w=0.0122×fl2.9338. the sex ratio (m:f) was 1:0.47, for adult c. damascina which differed significantly from the expected 1:1 (p<0.001). the von bertalanffy growth parameters were estimated as l∞ = 373.3 mm, k = 0.17 yr-1 and t0 = -0.58 yr. the instantaneous coefficient of natural mortality was estimated as 0.36 yr-1. the average kf values were 1.13±0.14 and 0.98±0.18, and kn were 1.11±0.12 and 0.98±0.18, in the river and the lake, respectively. in conclusion, c. damascina showed an isometric growth pattern, which the rapid growth is occurred during the two first years of life. the kn showed that the fish were at suitable welfare conditions in the azad dam. introduction the freshwater mesopotamian barb, capoeta damascina, is a cyprinid species native to asia, with a wide distribution. it is known as siahmahi and sardeh, which is mainly caught for consumption and sports fishing (froese and pauly, 2017; asadollah et al., 2017; esmaeili et al., 2016, 2018). knowledge of length-weight relationship (lwr), length-length relationship, condition factor (kf), growth and recruitment are important tools for the adequate management of fish species (king, 2007). the lwr parameters are important in fish biology providing information on the stock condition, condition indices and several aspects of fish population dynamics (bagenal and tesch, 1978; gonçalves et al., 1997; zamani faradonbeh et al., 2015). also, these relationships are used in the conversion of fish length and body weight to provide some measure of biomass (froese, 1998), to estimate the condition, *corresponding author: hasan fazli doi: https://doi.org/10.22034/ijab.v6i6.424 e-mail address: hn_fazli@yahoo.com reproduction, life cycle and general health of the fish species (pauly, 1983). the kf is used to compare the condition, fatness or well-being of the fish (bagenal and tesch, 1978). the relative condition factor (kn) is influenced by many environmental and biological factors (le cren, 1951). kf measures the deviation from a hypothetical ideal fish but kn measures the deviation from the average weight or length of fish (anderson and neumann, 1996). the determination of fish growth is fundamental for population modeling, stock assessments and managing exploited species (gulland, 1988). despite the wide distribution of c. damascina in iran, knowledge on the life history parameters of this species is limited. previous studies on the life history parameters of c. damascina in iranian inland waters were limited to distribution (keivany et al., 2016), age and growth (asadollah and soofiani, 2008; bahrami kamangar et al., 2015; asadollah et al., 2017). 308 fazli et al./ growth and condition factor of capoeta damacina therefore, the aim of the present study was to estimate the population parameters, including lwr, kf, kn, age, growth and mortality of c. damascina in the azad dam and the komasi river in the kurdistan province iran. materials and methods the study was performed at the azad dam and the komasi river located on 75 km west of the city sanandaj, at an altitude of about 1400 m above sea level (fig. 1). azad dam is an earthen dam with a clay core. the crest length and maximum height of the dam are 595 and 117 m, respectively. the total capacity of the dam is 300 million m3. samplings were carried out on august to november 2015 and february-may 2016. three sampling sites were selected along the dam using multi-mesh gill net (20 m length and 4 m height, with 14, 18, 22, 26, 30, 33 and 40 mm mesh sizes) and electrofishing in the komasi river. a total of 130 specimens of c. damascina were collected. the fork length (fl), standard length (sl) and total length (tl) were measured to the nearest 1 mm and total weight to the nearest 0.1 g (for overall individuals). sex was determined by visual inspection. scales were collected from the middle of the body behind the pectoral fins above the lateral line and preserved in the envelopes for future treatment in the laboratory. the scales were washed in petri dishes with tap water. the organic layers were removed by rubbing and washing the scales between the fingers in tap water. then, the age of fish was determined by counting the number of annuli on the scales. the length-weight relationship was derived by applying an exponential regression as the following equation: 𝑊 = 𝑎𝐹𝐿𝑏 where w is the total weight (g), fl = the fork length (mm), and a and b are parameters to be estimated (ricker, 1975). parameters estimation was conducted by least squares linear regression on loglog transformed data: ln(w) = ln(a) + b×ln(fl). t-test was used to investigate for the departure from isometry (b = 3) based on pauly (1984): 𝑡 = 𝑠.𝑑. ln⁡(𝐹𝐿) 𝑠.𝑑. ln⁡(𝑊) × |𝑏 − 3| √1 − 𝑟2 × √𝑛 − 2 where s.d.ln (fl) and s.d.ln (w) are standard deviations natural logarithm of the fork length (cm) and weight, respectively, and a and b are parameters and r2 is regression coefficient between length and weight and n is sample size. the condition factor (kf) was calculated as the following equation (froese, 2006): 𝐾𝐹 = 𝑊 𝑇𝐿3 × 100 where w (g) is weight and tl (cm) is the total length. the relative condition factor (kn) that compensates the changes in form or condition factor with an increase in length was calculated using the following equation (froese, 2006): 𝐾𝑛 = 𝑊 𝑎𝑇𝐿𝑏 where w is weight (g), tl is total length (mm), a and b are respectively the exponential form of the intercept and slope, of the logarithmic length–weight equation. the pearson correlation coefficient was calculated to investigate the relationship of kn and k figure 1. map of azad dam located at northwest of iran with sampling stations (st.). 309 int. j. aquat. biol. (2018) 6(4): 307-313 length. the von bertalanffy growth curve (von bertalanffy, 1938) was fitted to the observed lengths at age for the resulting age-length key using a nonlinear estimation method as following: where lt is the total length at age t, l∞ is the theoretical maximum length, k is a growth coefficient and t0 is the hypothetical age for lt=0. the parameter φ`, the growth performance index, was calculated as (pauly, 1983): φ`⁡ = 𝑙𝑜𝑔𝐾 + 2𝑙𝑜𝑔𝐿∞ where k is the growth coefficient and l∞ is the theoretical maximum length (cm). the instantaneous coefficient of natural mortality was estimated using the methods in pauly model (pauly, 1980) with von bertalanffy growth parameters. where m is the instantaneous coefficient of natural mortality, k is the growth coefficient and t is the mean annual habitat temperature, t=12.0°c. the comparison between the average values for sexes was carried out by t-test and for seasons by analysis of variance (anova). differences in sex ratios from the expected 1:1 were analyzed by chi-square tests (zar, 2010). results a total number of 130 specimens were caught in this study, with 90 specimens caught by gillnet in azad lake and 40 specimens caught by electrofishing in the komasi river. the tl and w of c. damascina ranged from 51 to 350 mm and 2.5 to 465.0 g and averaged (±sd) 188.2 (±45.85) mm and 81.3 (±69.86) g, respectively (table 1). total length ranges 51-350 and 120-260 mm in the river and lake, respectively. the length group of 181-190 mm was prevailing and formed 14.2%, followed by the length group of 171180, comprising 12.6% of the total catch (fig. 2). the mean tl and w were 180.6±68.63 mm, 93.3±110.89 g in the river and 191.5±30.76 mm and 75.9±39.89 g in the lake, respectively. no significant differences were found in the parameters between the river and lake samples (t-test; p>0.05; table 1). length-length relationships and the coefficient of determination r2 are given in table 2. the lengthlength relationships were found to be highly correlated (in all cases: r²=0.99, p<0.001). the tl and w regression of whole samples was: w=0.0122×tl2.9338 (r2= 0.955, n=130, standard error of b = 0.057 and a = 0.004). the value of b was 2.9338, significantly not different from 3.0 (t-test, p>0.05), indicating an isometric growth (fig. 3). the average of kf values were 1.13±0.14 and 0.98±0.18 in the river and lake, respectively. these averages were significantly different (t-test, p<0.001; table 1). kn were calculated in the river and lake using the lwr. there was a significant difference between )1( )( 0ttk t ell    )ln(463.0)ln(6543.0)ln(279.00152.0)ln( tklm   table 1. descriptive statistics of weight, total length, condition factor (kf) and relative condition factor (kn) of capoeta damascina in azad dam. parameter place n mean sd min max t-test weight, g river 40 93.3 110.89 2.5 465.0 t=0.96 lake 90 75.9 39.89 14.7 216.0 p>0.34 total 130 81.3 69.86 2.5 465.0 total length, mm river 40 180.6 68.63 51 350 t=0.97 lake 90 191.5 30.76 120 260 p>0.35 total 130 188.2 45.85 51 350 kf river 40 1.13 0.14 0.95 1.88 t=4.9 lake 90 0.98 0.18 0.59 1.61 p<0.001 total 130 1.03 0.18 0.59 1.88 kn river 40 1.11 0.12 1.0 1.7 t=5.0 lake 90 0.98 0.18 0.6 1.6 p<0.001 total 130 1.02 0.18 0.6 1.7 310 fazli et al./ growth and condition factor of capoeta damacina the average of kn values 1.11±0.12 and 0.98±0.18, respectively, with significant difference (t-test, p<0.001; table 1). the age of c. damascina ranged 1-7 years. ages 3 and 4 were the most dominant age groups, representing 55.9% of the samples. the von bertalanffy growth equation was estimated as shown in figure 4: 𝐿𝑡 = 373.3(1 − 𝑒 −0.17(𝑡−(−0.58))) the growth performance index (φ`) of c. damascina was computed as 2.37. estimates of the instantaneous coefficient of natural mortality for c. damascina obtained from the pauly method was 0.36/yr. the sex ratio (m:f) was 1:0.47, for adult c. damascina (n = 87), which differed significantly from the expected 1:1 (χ2=11.0, p<0.001). discussion study on the population characteristics of c. damascina in iranian inland waters are scarce and limited to the caspian basin, west and center of iran (asadollah and soofiani, 2008; bahrami kamangar et al., 2015; asadollah et al., 2017; table 3). the b value usually varies between 2 and 4 (tesch, 1971) or ranges figure 2. size distribution of capoeta damascina in azad dam, n = sample size. figure 3. length-weight relationship of capoeta damascina in azad dam. figure 4. theoretical growth curve calculated for total length of capoeta damascina in azad dam. 311 int. j. aquat. biol. (2018) 6(4): 307-313 from 2.50 to 3.50 (froese, 2006). according to the present study, the exponent b of lwr was 2.94, remained within the expected range and indicating an isometric growth. similar results were reported by asadollah et al. (2017). in contrast, bahrami kamangar et al. (2015) reported a lower b value (2.84 in gheshlagh reservoir and river, iran) and asadollah and soofiani (2008) reported a higher b value (3.23 for both males and females in hana dam, respectively). the sampling gear might influence the size range covered and cause deviations from existing values lwr parameters. in addition, geographical location and associated environmental conditions such as water temperature, which is the determining factor of feeding capacity, seasonality, stomach fullness, disease and parasite loads can affect the value of b (bagenal and tesh, 1978; froese, 2006). kf reflects information on physiological states of fish in relation to welfare (kumolu and ndimele 2010). higher kf value indicates favorable environmental conditions (blackwell et al., 2000). in the present study, the komasi river fish were in suitable condition. according to george et al. (1985), kn indicates the general well-being of the fish. if the values of kn>1 indicates that the well-being of the fish is good; whereas, value <1 reflects poor feeding activity and low well-being of the fish. in the present study, the kn of c. damascina were close to 1 (in the lake) and higher than 1 (in the river). these results suggested that the well-being of the fish was good in the azad dam region. similar results were reported for alburnus mossulensis in the azad dam (fazli et al., 2018a). in contrast, the average of kn carassius gibelio was lower than 1 (fazli et al., 2018b). according to helfman et al. (1997), knowledge of fish age and growth is necessary for stock assessment, develop management or conservation plans. the results showed that the rapid growth of c. damascina was found during the first two years of life, followed by a period of slow growth rate in the rest of life. the age of c. damascina varied from 1 to 7 yr. similar results were reported in the hana dam (asadollah and soofiani, 2008). in contrast, bahrami kamangar et al. (2015) found a lower (between 0 and 5) and asadollah et al. (2017) a higher age ranges (1+-9 and 1+-10+ years for males and females, respectively). holmgren and appelberg (2001) and bautista et al. (2012) reported that range of age distribution in a population is closely related to the nutritional status of the environment. the asymptotic length (l∞) of c. damascina was 37.3 cm is lower than that reported by asadollah and soofiani (2008), bahrami kamangar et al. (2015) and asadollah et al. (2017). also, the growth performance index (φ`) of c. damascina (2.37) indicates a lower growth rate in the azad dam. holmgren and appelberg (2001) and bautista et al. (2012) reported that the growth characteristics of the local populations of the same species change due to habitat variations, water quality and nutrients. the present study showed that the overall m:f ratio of c. damascina was 1:0.47, the overall sex ratio is unbalanced in favor of males. in contrast, the sex ratio table 2. length-length relationships of capoeta damascina in azad dam. equation n a b r2 fl=a+b×tl 113 -3.08 0.918 0.99 fl=a+b×sl 113 5.38 1.069 0.99 sl=a+b×tl 113 -7.16 0.854 0.99 table 3. the von bertalanffy growth parameters and length-weight relationships of capoeta damascina from different locations. study area sex age b l∞ k t0 φ` author (s) hana dam, iran m 1+-6+ 3.23 52.4 0.177 -0.30 2.69 asadollah and soofiani, 2008 f 1+-6+ 3.17 64.9 0.151 -0.45 2.80 gheshlagh reservoir and river, iran m+f 0-5 2.84 67.52 0.12 -0.79 2.74 bahrami kamangar et al., 2015 zayandehrud river, iran m 1+-9+ 2.95 56.2 0.098 -0.63 2.49 asadollah et al., 2017 f 1+-10+ 2.99 117.1 0.05 -0.43 2.84 azad dam, iran m+f 1-7 2.94 37.3 0.17 -0.58 2.37 present study 312 fazli et al./ growth and condition factor of capoeta damacina of this species in other iranian inland waters was in favor of females (asadollah and soofiani, 2008; bahrami kamangar et al., 2015). in general, the overall expected sex ratio in many species is 1:1, but it could be affected by some natural and environmental factors (bohlena and ritterbusch, 2000). in conclusion, c. damascina has an isometric growth pattern. the rapid growth occurred during the two first years of life. the kn of c. damascina were close to 1 (in the lake) and higher than to 1 (in the river) which suggested that the well-being of the fish was good in azad dam region. acknowledgments we are grateful to the jihad-agriculture of kurdistan organization which supported the study. we thank the staff of the department of stock assessment at the caspian sea ecology research center for providing samples used in this study. references anderson r.o., neumann r.m. 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(2018) 6(6): 307-313 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی در دریاچه سد آزاد و رودخانه کوماسی در استان capoeta damascinaرشد و ضریب چاقی سیاه ماهی کردستان، ایران 2پورحسین حمید ،2میرزائی رحمان ،1جلودار نادری مهدی ،1دریانبرد غالمرضا ،1*فضلی حسن .ایران ساری، کشاورزی، ترویج و آموزش تحقیقات، سازمان کشور، شیالتی علوم تحقیقات موسسه خزر، دریای اکولوژی پژوهشکده1 .ایران سنندج، کردستان، استان کشاورزی جهاد سازمان شیالت، مدیریت2 چکیده: (، سن، رشد و مرگ و nk(، ضریب چاقی نسبی )kf(، ضریب چاقی )lwrوزن )-طولهدف از این مطالعه برآورد پارامترهای جمعیت شامل رابطه مقادیر پارامترهای اندازه( در دریاچه سد آزاد و رودخانه کوماسی در استان کردستان، ایران بود. capoeta damascinaمیر طبیعی سیاه ماهی ) 120-260( گرم در رودخانه و طول کل: 3/93±89/110) 5/2-465متر، وزن: ( میلی6/180±63/68) 51-350گیری شده شامل طول کل: بود. نسبت جنسی fl 0122/0 =w×9338/2 وزن-( گرم در دریاچه بود. رابطه بین طول9/75±89/39) 7/14-216متر، وزن: ( میلی76/30±5/191) l∞ =3/373 . پارامترهای رشد برتالنفی شامل(p>001/0) داری داشتاختالف معنی 1:1بوده که با نسبت 1: 46/0)ماده:نر( برای ماهیان بالغ برآورد شد. در رودخانه و دریاچه میانگین ضریب yr 36/0-1محاسبه شد. ضریب مرگ و میر طبیعی yr 58/0 =0 t و yr 17/0 = k-1متر، میلی گیری، این ماهی عنوان نتیجههدست آمد. بهب 98/0±18/0و 11/1±12/0ترتیب و ضریب چاقی نسبی به 98/0±18/0و 13/1±14/0ترتیب چاقی به دارای رشد ایزومتریک می باشد. رشد در دو سال اول زندگی سریع است. ضریب چاقی نسبی نشانگر وضعیت مطلوب سالمت این ماهی در منطقه سد آزاد می باشد. .، جمعیتچاقی ضریب وزن،-طول رابطه رشد، پارامترهای :کلمات کلیدی international journal of aquatic biology (2013) 1(5): 202-208 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article evaluation of water quality using topsis method in the zaringol stream (golestan province, iran) zohreh mazaheri kohanestani*1, rasoul ghorbani 1, abdolazim fazel2 1department of fisheries, gorgan university of agricultural sciences and natural resources, p.o. box 45165-386, gorgan, iran. 2department of fisheries, university of guilan, guilan, iran. article history: received 8 may 2013 accepted 21 august 2013 available online 2 5 october 2013 keywords: pollution topsis method water quality zaringol stream abstract: in order to evaluate water quality condition, biochemical oxygen demand (bod), dissolved oxygen (do), temperature, ph, turbidity, total suspended solid (tss), phosphate (po4-), nitrate (no3-) and fecal coliform were measured seasonally from 9 sites from november 2009 to august 2010 in zaringol stream. water quality condition was estimated using topsis method. comparison of topsis values in different sampling stations showed the minimum (0.230) and maximum values (0.604) are in points 1 and 5, respectively. according this result, point 1 had the best water quality condition and point 5 had the lowest quality. also, seasonal results of topsis values showed that the maximum value was found in spring. discharge of effluents from land uses located along the stream specifically, trout farms and starting agricultural activity and production process in spring and summer are most important reasons for decreasing of water quality. topsis estimates values ≤0.5 for almost stations and seasons. it shows zaringol stream has an average water quality. introduction rivers play an important role in watersheds for carrying off wastewater and run off from farmland and are the most susceptible water body to pollutants (yu et al., 2003; singh et al., 2004; wang et al., 2007). the constant discharges of wastewater and seasonal surface run-off have a strong effect on the river, water quality, human health and aquatic organisms (kazi et al., 2009). water quality zoning is critical to identify preferred potential usages of water resources and source of pollution to optimize water usage and management, especially, in rivers and streams (simenove et al., 2003; karimian et al., 2007). making decision in the field of water resource management represents a task of high importance and responsibility (kirilov et al., 2009). choosing the best option is technically challenging as there is * corresponding author: zohreh mazaheri kohanestani e-mail address: zohremazaheri_65@yahoo.com no scientific tools to predict future impacts of alternative management actions (gao and hailu, 2011). since determining of water quality condition is done with numerous different parameters could effect on water quality with different ways as it seems antithetic sometimes, managers use valid index to combine different parameters and calculate a value for taking decision more easily such as nsfwqi, wqi, etc. (liou et al., 2003; heernandezromero et al., 2004; simoes et al., 2008). these indexes are comprehensive and common in water quality zoning (jonnalagadda and mhere, 2001), though they can throw us in making mistakes when their value are in border ranges of two water quality classes (for example the value belongs to class "bad" but is also so near to the next water quality class "average") . 203 mazaheri kohanestani et al/ international journal of aquatic biology (2013) 1(5): 202-208 multivariate criteria decision making (mcdm) is one of research fields of management sciences have expanded in different applied researches based on its need recently and it makes it possible to take decision wisely (saati et al., 2007). topsis is the most famous classical mcdm technique described by hwang and yoon (1981) for the first time. in topsis technique, the basic solution method is defining positive and negative ideal (non-ideal) solution (biorani and ghofran, 2009). positive ideal solution includes the best available value of parameters while the non-ideal one is made of the worst available value of parameters. finally, the best answer has both the shortest distance from the ideal solution and the longest from the non-ideal (saati et al., 2007). simplicity, rationality, comprehensibility, good computational efficiency and ability to measure the relative performance for each alternative in a simple mathematical form are some of the advantages of topsis methods (roszkowska, 2010). zaringol stream with 22 kilometer length is one of the gorgan-rud brunches and has important role in water supply of agricultural, aquaculture and domestic usages (abdoli and rahmani, 2002). in recent years, great changes have taken in marginal regions lead to severe effect of its water quality though, there is no report on water quality because of limited hydrometric station along the stream. so, the aim of this study is to evaluate water quality using topsis method in zaringol stream, the golestan province. materials and methods this study was carried out in zaringol streamgolestan province. water was sampled from 9 sites along the stream during december 2009 to september 2010, seasonally (fig. 1). according to figure 1, after station 1 as the nearest station to the spring and a less polluted station, there are two trout farms located at station 2 and 5 and their actual capacities are 15 and 7 tons, respectively. other stations are located along the stream where the agriculture is the dominated land use. nine water quality parameters were measured including biological oxygen demand (bod5), dissolved oxygen (do), nitrate (no-3), phosphate (po-4), temperature (δt), ph, turbidity, fecal coliform and total suspended solid (tss) by water checker u-10 and spectrophotometer. decision matrix (m×n) was made as: a1, a2, …, am are alternatives; c1, c2, …, cn are evaluation factors; gij means evaluation rate of ai related to factor cj and w is evaluation factor weight were calculated by ahp (eigenvector) method (gao and hailu, 2011) (table 1). 𝑊𝑖 = 𝑤1, 𝑤2, … , 𝑤𝑛 , ∑ 𝑊𝑖=1 next, data were normalized to remove variation of each factor (pimerol and romero, 2000). 𝑝𝑖𝑗 = 𝐺𝑖𝑗 √∑ (𝐺𝑖𝑗)2𝑚𝑖1 ∀𝑖 = 1, ,2, … , 𝑚 𝑗 = 1,2, … , 𝑛 figure 1. site of water sampling-zaringol stream. 203 204 mazaheri kohanestani et al/ international journal of aquatic biology (2013) 1(5): 202-208 then weight of evaluation factors was affected by multiplying its vector to decision matrix and provided a normalized weighted decision matrix. positive ideal (a+) and non-ideal (a-) solutions were determined and distance index calculated. "k" and "l" are associated with benefit and cost criteria, respectively (hwang and yoon, 1981). 𝐴+ = {(𝑀𝑎𝑥 𝑣𝑖𝑗 𝑗 ∈ 𝐽), (𝑀𝑖𝑛 𝑣𝑖𝑗 𝑗 ∈ 𝐽′); 𝑖 = 1,2, 𝐾, 𝑚} = {𝑣1 +, 𝑣2 +, . . , 𝑣𝑛 +} 𝐴− = {(𝑀𝑖𝑛 𝑣𝑖𝑗 𝑗 ∈ 𝐽), (𝑀𝑎𝑥 𝑣𝑖𝑗 𝑗 ∈ 𝐽′); 𝑖 = 1,2, 𝐾, 𝑚} = {𝑣1 −, 𝑣2 −, . . , 𝑣𝑛 −} 𝐽 = {𝐾|𝐾 = 1,2, 𝐾𝑛} 𝐽′ = {𝑙|𝑙 = 1,2, 𝐾𝑛} parameter unit weight do saturate (%) 0.17 fecal coli form colony/100ml 0.16 ph --0.11 bod5 ppm 0.11 δt ◦c 0.1 no3 ppm 0.1 po4 ppm 0.1 turbidity ntu 0.08 t.s.s ppm 0.07 table 1. weight of water quality parameters used in topsis method. parameter/station 1 2 3 4 5 6 7 8 9 log turbidity (ntu) 1.46a 2.36b 2.16ab 2.33b 2.49b 2.08ab 2.22b 2.05ab 2.03ab tss (ppm) 0.20a 1.06c 0.80b 0.87b 0.95b 0.82b 0.95b 0.84b 0.91b po4 (ppm) 0.36ab 0.88b 0.25ab 0.16a 0.2a 0.12a 0.08a 0.06a 0.11a no3 (ppm) 1.38a 2.18a 1.40a 0.63a 2a 2.08a 2.28a 1.25a 2.35a ph 8.75a 8.58a 8.75a 8.77a 8.36a 8.56a 8.66a 8.37a 8.30a t (◦c) 14.05a 16.65a 17.18a 20.82a 16.98a 18.68a 19.3a 17.95a 19.35a do (%) 66.50b 53.25a 66.25b 66b 54.75a 59.25ab 59.50ab 58.50ab 58.75ab bod5 (ppm) 1.78a 2.75bc 2.38ab 2.23ab 2.83bc 3.30c 3.45c 2.65bc 2.68bc fecal coli form×105 (counts/100 ml) 1a 2.2bc 2.13bc 1.63ab 2.75c 2.2bc 1.93bc 1.55ab 1.40ab data presented as mean. similar letter shows no difference between stations. table 2. water quality parameters of different stations zaringol stream. station 𝑑𝑖 + 𝑑𝑖 − 𝐶𝑖  rank 1 0.0538 0.0161 0.230 9 2 0.0286 0.0401 0.584 2 3 0.0336 0.0269 0.468 5 4 0.0383 0.306 0.444 6 5 0.0303 0.0461 0.603 1 6 0.0303 0.0335 0.525 4 7 0.0269 0.0371 0.580 3 8 0.0404 0.0221 0.353 8 9 0.414 0.0257 0.383 7 table 3. results of topsis method of different stations-zaringol stream. 205 mazaheri kohanestani et al/ international journal of aquatic biology (2013) 1(5): 202-208 finally after calculating the distance from positive ideal solution (𝑑𝑖+), non-ideal solution (𝑑𝑖−) and relative nearest vicinity from ideal answer (𝐶𝑖*), values were arranged as an ascending order (hwang and yoon, 1981). maximum value (nearest one to 1) has the highest preference. 𝑑𝑖 + = √∑(𝑉𝑗 − 𝑉𝑗 +) 2 𝑛 𝑗=1 ∀𝑖 = 1,2, … , 𝑚 𝑑𝑖 − = √∑(𝑉𝑗 − 𝑉𝑗 −) 2 𝑛 𝑗=1 ∀𝑖 = 1,2, … , 𝑚 𝐶𝑖 + = 𝑑𝑖 − (𝑑𝑖 − + 𝑑𝑖 +) ∀𝑖 = 1,2, … , 𝑚 0 ≤ 𝐶𝑖  ≤ 1 data checked for normality distribution with the kolmogorov-smirnov test. spatial and temporal variation of water quality parameter were analyzed using one-way anova and duncan's post-hoc test, assuming a significant level of α=0.05 by spss 17 software package. results data on water quality parameters of different stations are given table 2. considering that the point 1 does not expose to the pollution resources, so it was considered as the test station. according to table 2, maximum turbidity belongs to stations 2 and 5. also maximum values of total suspended solid, phosphate were measured in station 2. dissolved oxygen decreased in station 2 and 5 significantly. some of water quality parameters had no significant difference between different stations (i.e. ph, temperature and nitrate). as the aim of calculation was to detect polluted stations in zaringol stream, so it should be considered that the nearest value to 1 shows more water pollution (table 3). maximum and minimum relative nearest vicinity from ideal answer (identification polluted points) calculated for station 5 and 1 respectively. in the other hand the most polluted station is 5 and the least one is number 1. comparison water quality parameters between different seasons show significant increase in turbidity, temperature, bod5 and fecal coli form in spring and no3 in summer. ph decreased in summer, parameter/station spring summer autumn winter log turbidity (ntu) 2.24b 2.13ab 1.59a 2.40b tss (ppm) 0.6a 1.01b 1.11b 0.57a po4 (ppm) 0.39a 0.33a 0.15a 0.11a no3 (ppm) 1.73b 2.28b 2.51b 0.38a ph 8.56b 8.33a 8.67b 8.71b t (◦c) 25.1c 21.44b 10.76a 14.23a do (%) 56.78a 63a 60.33a 61.11a bod5 (ppm) 2.93b 2.71ab 2.91b 2.12a fecal coli form×105 (counts/100 ml) 2.43b 1.91b 1.60a 1.51a data present as mean. similar letter shows no difference between stations. table 4. water quality parameters at different seasons zaringol stream. table 5. results of topsis method of different seasons zaringol stream. seasons 𝑑𝑖 + 𝑑𝑖 − 𝐶𝑖  rank spring 0.0238 0.0685 0.742 1 summer 0.0443 0.0457 0.507 3 autumn 0.0459 0.0501 0.522 2 winter 0.0674 0.0210 0.237 4 205 206 mazaheri kohanestani et al/ international journal of aquatic biology (2013) 1(5): 202-208 too. po4 and cod and salinity did not show significant difference between seasons (table 4; p<0.05). relative nearest vicinity from ideal answer in different stations show that winter, summer, autumn and spring rank from high water quality to low, respectively. discussion results of water quality parameters at different stations of zaringol stream showed that the minimum values of total suspended solids, turbidity, biological oxygen demand, fecal coli forms and maximum value of dissolved oxygen were measured in station 1. according to these results, it was the least polluted station, as it is mentioned before. results of topsis ranking show the minimum value (or best water quality condition) in station 1, so it confirms our claim. after station 1, a decline could be observed in water quality and some of critical parameters such as biological oxygen demand and fecal coli forms which show water pollution clearly, increased significantly. similar to the results of water quality parameters, an increasing trend can be observed in the topsis values. based on the topsis ranking, station 2 had the second grade of water pollution and station 5 was the most polluted station and had the nearest topsis value to 1. since stations 2 and 5 are located after trout farms and the concentrations of some ions like phosphate (significantly) and nitrate (not significantly) increased in these stations, it can be infer that entrance of farms effluents had an important effect on water quality condition. the topsis values showed that station 5 was more polluted than station 2. it is reported that the severity of effect of fish farms effluents on water quality depends on volume and concentration of substances, flow rate of water and time of effluent discharge (pillay, 2004). although the actual capacity of first fish farm was more than second ones (located before station 5), station 5 was more polluted because the water loaded a lot of pollution from station 2. also adding a branch to the main stream flow and bringing a new volume of substances was another probable reason for the highest topsis value in station 5. comparison of increasing rate of topsis value between station 1 to 2 (0.354) and station 4 to 5 (0.159) confirms that first farm discharge more effluents into the stream than second one. the increasing trend in topsis values did not resume along the stream and water quality trend to better condition in other stations. self-purification power of stream improved water quality condition along the stream; though others land uses discharge along the stream prevented it to go back to the first condition again completely. there are some reports on environmental effect aquaculture (manoochehri et al., 2010; uzbilek kirkagac et al., 2009; pulatsu et al., 2004; mmochi et al., 2002) which confirm effect of fish farm effluents on water quality condition. our results agree with them. the topsis values show maximum value and the worst condition in spring. due to climate condition, rain fall decrease in warm season, so because of decreasing of stream flow, concentrations of contaminated components increase and generally stream is in acute condition. in addition to agricultural activity and reproduction period of trout farm start in spring and continue in summer. therefore water quality of warm seasons may affect more than cold seasons. based on relative nearest vicinity from ideal answer ranged from 0.23 to 0.6 and the theoretical highest value (ci* =1) we can conclude that zaringol water quality condition is almost average because most of the topsis values of stations and seasons are ≤0.5. in summary, the results of our research showed topsis method declared water quality variations clearly as they were expected based on analyzing water quality parameters and can determine polluted stations. therefore this is an applied simple method and can be used for managers to make decision easily while they are faced to several complicated parameters. 207 mazaheri kohanestani et al/ international journal of aquatic biology (2013) 1(5): 202-208 references abdoli a., rahmani h. 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(2018) 6(1): 31-36 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article the effects of dietary lysozyme on growth performance and haematological indices of rainbow trout (oncorhynchus mykiss) fingerling meysam shakoori*,1seyed hossein hoseinifar, hamed paknejad, valiollah jafari, roghieh safari department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 2 november 2017 accepted 17 february 2018 available online 2 5 february 2018 keywords: lysozyme rainbow trout growth performance haematology abstract: the present study investigates the effects of dietary lysozyme on growth performance and haematological indices of rainbow trout (oncorhynchus mykiss) fingerlings. one hundred and twenty juvenile rainbow trout fish (initial average weight 5.46±0.05 g) were fed on varying levels of dietary lysozyme (0, 0.5, 1 and 1.5 g kg-1) for 8 weeks. thereafter, growth performance as well as haematological indices including wbc, rbc and hct were measured. evaluation of growth performance showed no significant increase in fishes fed on different levels of dietary lysozyme (p>0.05). also, feeding on dietary lysozyme significantly increased wbc compared control (p<0.05); the highest level was detected in 1.5 g kg-1 treatment. furthermore, rbc of fish fed 1 or 1.5 g kg-1 lysozyme were significantly higher than other treatments. the same results were noticed in case of hct which was higher in fish fed treated diet. these results indicated that dietary lysozyme affect haematological parameters rather than growth performance. introduction fish and shellfish produced in aquaculture industry meet the market demands of increasing world population for healthy protein (fao, 2016). the main issues aquaculture industry facing with, are disease outbreak (azimirad et al., 2016), therefore, researches regarding fish health status and immunostimulants are of high importance (nawaz et al., 2018). considering the cross talk between nutrition and health status, modulation of fish immune response can be taken into account through dietary approaches (yarahmadi et al., 2016). besides, a major concern in aquaculture is the use of chemical therapeutics, such as antibiotics, because of their impact on the environment as well as on the fish product (hoseinifar et al., 2017). this concern can be resolved by using environment friendly feed additive. on the other hand, determination of the mode of action of immunestimulant will help in selection of the optimum kind and doses (wang et al., 2017). among various types of immunostimulant, one strategy for improving the fish feed safety and deprivation diseases is adding *corresponding author: meysam shakoori doi: https://doi.org/10.22034/ijab.v6i1.432 e-mail address: meysam.shakoori@gmail.com antimicrobial enzymes in diet (deng et al., 2012; chen et al., 2014; abdel-latif et al., 2017). in fish, lysozyme can be found in different organs and body fluids including mucus, serum, gills, alimentary tract etc. (chen et al., 2014). the main function of lysozyme is removing the pathogens through bacteriolytic activity (modanloo et al., 2017). this occurs by cleave of the glycosidic linkage in the peptidoglycan component of gram-positive bacterial cell walls (van doan et al., 2017a). it has been suggested that the abovementioned function will finally results in improved growth performance (abdel-latif et al., 2017). moreover, lysozyme along with other factors, including immunoglobulins, lactoferrin in antimicrobial activity and with lactoperoxidase, may limit the migration of neutrophils into a damaged tissue by behaving as an anti-inflammatory agent (chen et al., 2014). however, in spite of these benefits, there is limited information available about possible effect of dietary lysozyme on fish growth performance and health (deng et al., 2012, chen et al., 2014). therefore, the present work aimed 32 shakoori et al./ effects of lysozyme on growth performance and haematological indices of rainbow trout to study the effects of different levels of dietary lysozyme on growth performance and haematological parameters of rainbow trout. materials and methods experimental design: one hundred and twenty juvenile rainbow trout purchased from an aquaculture farms and transferred to the laboratory in gorgan university of agricultural and natural resources, then were acclimatized to the experimental condition for 2 week before the start of the main experiment. ten juvenile fish (with initial average weight of 5.46±0.05 g) were randomly released into 12 tanks. the capacity of each tank was 200l and the exchange of 50% water occurred every day. water temperature and dissolved oxygen were 16±3.1 and 7.5 mg l-1, respectively. preparation of experimental diets: a commercial trout feed was used in this study as control diet (coppens co., netherland). the chemical composition are shown in table 1. four experimental diets was prepared with different levels (0, 500, 1000, 1500 g kg-1) of dietary lysozyme (zhejiang aegis biotech co., ltd) in a dry basis. thereafter, the prepared diets were kept in plastic bag at -4°c. fish were hand-fed thrice (8:00 and 12:00 and 16:00) daily for 8 weeks with 3% of body weight. sampling: fish were not fed for 24 hrs prior to sampling at the end of the feeding trial. total number and mean body of fish in each tank were measured to calculate the growth, feed utilization and nutrient retention. for haematological parameters, six specimens were randomly sampled from each tank then blood was collected from the caudal vein with a heparinized syringe and transferred into a heparinized tube. grow performance: growth performance and survival rate of juvenile rainbow trout were enumerated using following equations: weight gain (wg) = final weight (g) initial weight (g) specific growth rate (sgr) = 100 (ln final weight-ln initial weight) / duration of experiment feed conversion ratio (fcr) = feed intake (g) / weight gain (g) protein efficiency ratio = weight gain (g)/protein fed (g). grow rate = final weight – initial weight / day survival rate = (final number / initial number) × 100 haematological analyses: red blood cells and white blood cells were counted using a neubauer hemocytometer according to martins et al. (2004). haematocrit was measured with microcentrifuge method, using standard heparinized microhaematocrit capillary tubes (75 mm at 7000 g for 10 min) (blaxhall and daisley, 1973). statistical analysis: after checking the normality of the data, statistical analysis was performed using one way anova followed by duncan's multiple-range test. the significant difference levels was considered when p<0.05. the statistical analysis was performed by spss software 19 (spss, usa). results grow performance: the growth indices of o. mykiss fed diets supplemented with different levels of dietary lysozyme shown in table 2. the diet with 1.5 g per kg dietary lysozyme has better growth performance but no significant differences were observed in the growth parameters of fish in the different treatments. after 60 days of feeding, the variation range of weight gain, sgr and gr also fcr and feed protein rate efficiency and survival rate summarized in table 2. according to the results, changes that was measured has not significant effect between different treatments. heamatological indices: the present results shown dietary lysozyme supplementation exerted a certain influence on the blood indices studied (table 3). respect to the values, number of white blood cells increased significantly in group with 1 and 1.5 g kg-1 dietary lysozyme compare to control diet group. number of total red blood cells showed a significant table 1. analysis of the commercial feed for rainbow trout (coppens, netherlands ultra). analyses composition protein 48% fat 22% crude fiber 0.9% ash 8.8% total p 1.45% 33 int. j. aquat. biol. (2018) 6(1): 31-36 increase in group with 1 g kg-1 dietary lysozyme compare to experimental treatment also control group diet. hematocrit value show a significant increase in experimental diet compare to control diet. discussion lysozyme is an antibiotic peptide and growth enhancer with positive bacteriolytic ability that can used in aquaculture and poultry industry (abdel-latif et al., 2017; gong et al., 2017). in this study, dietary lysozyme supplementation generally increased the growth performance parameters and survival rate of fish although no significant different was observed between treatment groups. in contrast with our study, previous researches shown antibacterial agents that used in growth promoting experiment has a significant effect in channel catfish (sanchez‐martínez et al., 2008) dietary apidaecin (apidaecintype peptides) on common carp (cyprinus carpio). in addition, dietary lysozyme (500 mg kg-1) significantly increased the growth performance of broiler chickens zhong-ke et al. (2008). likewise, administration of 100-300 mg kg-1 lysozyme broiler chickens diet resulted in improved growth parameters (yi-nan, 2010; lu et al., 2009; ding, 2010). similar results obtained in case of 70 mg kg-1 lysozyme in duck diets (gu and zhang, 2008), 100 mg kg-1 lysozyme in meat rabbit diets (guo et al., 2010) and 300-500 mg kg-1 lysozyme in weaned piglet diets (wang et al., 2008; lu et al., 2010; wu et al., 2010). based on the above results, it can be assumed that lysozyme is a growth promoter. however, the mode of action remained unclear. in chick, the antimicrobial activity of lysozyme could result in better intestinal health and improved digestion and it is a complex and integrated interaction between microbes and immune system, which is responsible for the development and maturation of the fishes immune system. chen et al. (2013) found that the absence of intestine microbiota could lead to depression of the host immune response status. in terrestrial animal research, it has long been established that dietary antibacterial material include lysozyme could result in morphology changes in intestinal (deng et al., 2012). when all this actions occurred in body, animals could save extra energy in intestine also can use instead for growth, or increasing the absorption of other nutrients (chen et al., 2014). however, some factors like stress, season, sex ,water temperature and quality, low ph and sedimentation can change lysozyme activities in fish (saurabh and sahoo, 2008). the relationship between nutrition with immunity has been recognized a long years ago for researchers but to explaining this mechanism, we need more experiment (hoseinifar et al., 2016). however, table 2. growth performance of rainbow trout fed with different dietary lysozyme levels (g kg-1) for 8 weeks experimental period (means±sd). index control 0.5 mg kg-1 1 mg kg-1 1.5 mg kg-1 initial weight (g) 5.44±01𝑎 5.42±03𝑎 5.50±04𝑎 5.48±07𝑎 final weight (g) 27.62±1.75𝑎 29.19±0.49𝑎 29.58±0.40𝑎 30.07±0.75𝑎 feed intake (g per fish) 1.22±76.6𝑎 1.32±57.5𝑎 1.66±0.00𝑎 1.66±0.00𝑎 weight gain (g) 25.81±3.56𝑎 28.10±0.46𝑎 28.83±0.38𝑎 29.43±0.79𝑎 fcr 0.65±0.09𝑎 0.59±0.01𝑎 0.57±0.007𝑎 0.56±01𝑎 sgr (% day) 2.70±0.10𝑎 2.80±0.0𝑎 2.80±0.02𝑎 2.83±0.03𝑎 per 0.74±0.10𝑎 0.81±0.01𝑎 0.83±0.01𝑎 0.84± 0.01𝑎 gr 0.43±0.05𝑎 0.46±0.02𝑎 0.48±0.02𝑎 0.49± 0.01𝑎 survival rate (%) 73.33±46.1𝑎 80±34.6𝑎 100±0.00𝑎 90.21± 21.2𝑎 table 3. effect of dietary lysozyme levels on hematological indices of rainbow trout (g kg-1). index control 0.5 1 1.5 wbc 13.53 ± 1.10𝑎 18.26 ± 0.70𝑏 20.53 ± 0.35𝑏𝑐 24.36 ± 3.17 𝑐 rbc 1.80±0.05𝑎 1.90±0.09𝑎 2.07±0.06𝑏 2.01±0.04𝑏 hct 26.83±3.21𝑎 39.63±6.80𝑏 44.5±2.17𝑏 47.5±1.32𝑏 means in the same row with different superscripts are significantly different (p<0.05) wbc white blood cell (×103 μl) rbc red blood cell (×106 μl) hct hematocrit (percent %) 34 shakoori et al./ effects of lysozyme on growth performance and haematological indices of rainbow trout possible reason for contradictory findings may be was effect of this agents on lysozyme activity that cause decreasing enzymes functions in intestine microbiota. generally, blood parameters are an important indicator of the physiological state of the internal organs. in other words, the haematology is an importance part of analysis the diagnosis in many diseases, metabolic studies as well as biological control of living organisms, such as aquatic organisms. also, haematological parameters of fish are useful tool to assess the health status and as well providing information on nutrient status, digestive function and routine metabolic level of fish (yarahmadi et al., 2016). blood composition changes can be result of changes occurred in diet quality and nutrient compounds (hoseinifar et al., 2014). hematological parameters are important health indicators whose study reveals the health conditions of fish regarding diseases and immune system conditions before and after an experiment. according to this study, dietary lysozyme supplementation shown a significantly effect on hematological indices between treatment group and control group. number of white blood cells was counted has increased significantly in group with 1 and 1.5 g kg-1 dietary lysozyme compare to control diet group. blood leucocyte phagocytic activity and serum lysozyme deceased with the reduction in intestine microbiota. it indicated that the absence of the micro-organisms reduce the conjunction between pathogen associated molecular patterns (pamp) and pattern recognition receptors (prr) in the gut-associated lymphoid tissue (galt) (magnadottir, 2010). also rymuszka et al. (2005) found that lysozyme may enhance the fish immune functions by the non-specific modulation of leucocyte activities. possibly lysozyme activity in blood and fishes’ organs such as gut, liver and kidney is a reason for increasing white blood cell due to increasing phagocytosis performance with presence lysozyme on treatment diet and finally stimulating the production of white blood cells increases the ability of the body to remove pathogens such as bacteria, fungi and viruses as well as gastrointestinal tract function. red blood cells, hematocrit and hemoglobin are responsible to carried oxygen in body. lysosomal enzymes such as alp plays an important role in the mineralization of skeleton of aquatic animals and in membrane transport activities (van doan et al., 2017b). it is often employed to assess the integrity of plasma membrane, and the decrease in activity may be taken as an index of disturbance of membrane transport system (öner et al., 2008). in this study red blood cells count increase in diet with 1 and 1.5 g kg-1 dietary lysozyme also hematocrit levels has a significant difference between treatment groups and control diet. however dietary lysozyme in rainbow trout diet is associated with an increase in rbc number and hematocrit level in plasma. suggestion reason for this influence may be attributed to the increased alp enzyme activity in liver, that cause improve immune system function as well as transport activity. in conclusion, the present study revealed beneficial effects of dietary lysozyme on growth performance and haematological parameters of rainbow trout. acknowledgments the author would like to thank the staff at aquaculture lab of guasnr. the study was funded by guasnr as master thesis. references abdel-latif m.a., el-far a.h., elbestawy a.r., ghanem r., mousa s.a., abd el-hamid h.s. 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(2018) 6(1): 31-36 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی ماهی بچه خونی هایشاخص و رشد عملکرد بر خوراکی لیزوزیم اثرات ( oncorhynchus mykiss) کمان رنگین آالیقزل ولی اهلل جعفری، رقیه صفری ،نژادحامد پاک ،فرسیدحسین حسینی ،*میثم شکوری .ایران گرگان، گلستان، گرگان، طبیعی منابع و شاورزیک علوم دانشگاه ،دانشکده شیالت و محیطزیست شیالت، گروه چکیده: تعداد. پردازدمی کمان رنگین آالی قزل ماهی بچه در خونی هایشاخص و رشد عملکرد بر جیره در خوراکی لیزوزیم اثرات بررسی به حاضر عهلمطا صفر) مختلف سطوح حاوی آزمایشی هایجیره با هفته هشت مدت به گرم 46/5±05/0 وزنی میانگین با کمان رنگین آالیقزل ماهی بچه قطعه120 گلبول تعداد شامل) خونی هایشاخص و رشد هایشاخص دوره پایان در. شدند تغذیه خوراکی لیزوزیم (وگرملکی بر گرم 5/1 و 1 ،5/0 ،(شاهد) زیملیزو مختلف سطوح با شده تغذیه های ماهی در داریمعنی افزایش رشد هایشاخص بررسی. شد گیریاندازه( هماتوکریت و قرمز گلبول سفید، رد سفید گلبول تعداد افزایش سبب داریمعنی طورهب لیزوزیم حاوی جیره با تغذیه اگرچه،(. p>05/0) نداد نشان شاهد تیمار با مقایسه در خوراکی ماهی قرمز هایگلبول تعداد عالوه،هب. گردید مشاهده لیزوزیم کیلوگرم در گرم 5/1 تیمار در میزان بیشترین ،(p<05/0) شد شاهد گروه با مقایسه سطوح خصوص در مشابهی نتایج(. p<05/0) بود تیمارها سایر از بیشتر دارییمعن طورهب لیزوزیم کیلوگرم ازای به گرم 5/1 یا 1 با شده تغذیه بر اگرچه است خونی هایشاخص بر جیره لیزوزیم اثرگذاری از حاکی مطالعه این نتایج. گردید مشاهده تیمار تحت هایماهی در هماتوکریت .نداشت اثری رشد هایشاخص .شناسیخون رشد، کارایی کمان، رنگین آالیقزل خوراکی، لیزوزیم :کلمات کلیدی int. j. aquat. biol. (2021) 9(1): 15-22 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article ultramicroscopy of structures involved in the posterior region of scales in two flathead fishes (teleostei: perciformes) azad teimori*, 1mina motamedi*, vahid amiri, majid askari hesni department of biology, faculty of sciences, shahid bahonar university of kerman, 76169-14111 kerman, iran. s article history: received 21 july 2020 accepted 19 december 2020 available online 2 5 february 2021 keywords: sem development scale ctenii persian gulf abstract: morphology of structures involved in the posterior region of scales in two flathead fish species viz. platycephalus indicus and grammoplites suppositus was studied using ultramicroscopy. the fish individuals were divided into three size groups based on their standard lengths and their scales were removed from four body regions. the microscopic observations indicated that the scales of both species were ctenoid. the posterior margin of all scales was formed by two rows of ctenii. typically, the shape of the posterior region of scales in p. indicus was crescent, while it was triangular in g. suppositus. the number of ctenii in the scales of p. indicus was minimum 12 and maximum 60, while in g. suppositus it was minimum 6 and maximum 38. moreover, the results indicated that the number of ctenii was increased during fish development because the smaller fishes have fewer ctenii in their scales than the adults, while, their general morphology has not been changed properly. this developmental change was significantly higher in p. indicus than g. suppositus. the increase of ctenii during fish development allows greater flexibility in movement. in conclusion, modification in the ornamentations of the posterior region has a hydrodynamic function and they are subject to modification during the fish development. the ctenii varying considerably in the number and could be easily counted, therefore, could be used as an appropriate taxonomic character at least in flathead fishes or even other fish groups. introduction the flatheads of perciformes are one of the members of small to medium fish species with a notably flat head distributing in the marine waters of tropical indowest pacific regions (mastrototaro et al., 2007). from this order, the family platycephalidae includes about 20 genera (imamura, 1996) and 85 valid species (froese and pauly, 2015; fricke et al., 2020). the members of this family are characterized by having an elongated body, dorso-ventrally depressed head, and a large-mouth. usually, the lower jaw is longer than the upper one (imamura and mcgrouther, 2008). these fishes are benthic in nature, frequently found on the sandy and muddy bottoms at depths of 10 to 300 m, more often in shallower than 100 m (mastrototaro et al., 2007). the flathead fishes of the family platycephalidae are particularly very interesting owing to the dramatic ornamentation that exists in the posterior region of their scales from the microscopic view (hughes, 1981; *correspondence: azad teimori & mina motamedi doi: https://doi.org/10.22034/ijab.v9i1.950 e-mail: a.teimori@uk.a.ir & m.motamedi@uk.ac.ir motamedi et al., 2020). nevertheless, little information is available on the microscopic characterization of the posterior region in the scales of these fishes (hughes, 1981; roberts, 1993; wonsettler and webb, 1997). the only comprehensive study on the scales of the family platycephalidae has been done by hughes (1981). he described the formation, development, and resorption of ctenii in the scales of flathead fishes of family platycephalidae. in the present study, the posterior region of the scales in two species of the family platycephalidae was investigated from the ontogenetic view using the scanning electron microscopy (sem). to do this objective, the scales from four body regions of each species were removed and the microscopic ornamentations in their posterior regions were described. materials and methods studied fishes and sampling: the studied materials 16 teimori et al./ scale ultramicroscopy of flathead fishes are two flathead fishes, including bartail flathead platycephalus indicus (linnaeus, 1758) and spotfin flathead grammoplites suppositus (troschel, 1840) were collected from the iranian coastal waters of the persian gulf (fig. 1). a total of 42 specimens (21 per each species) were collected by the trawl net and transferred to the laboratory for further examinations. after anesthesia by 1% clove solution, fishes were fixed in 5% formaldehyde and later stored in 70% (for the study of scale morphology). all fish materials and the examined scales were deposited at the zoological museum of shahid bahonar university of kerman (zm-sbuk). scale preparation: the fish specimens were divided into three range sizes based on their standard lengths (seven specimens per size class) and their scales were extracted from four body regions for microscopic examination. to do this objective, the left-hand side of the fish was divided into four regions along the longitudinal axes of the body as follow: region h (head), region d1 (from the 3rd to 4th rows below the dorsal fin or above the lateral line), region d2 (the region below the second dorsal fin) and region c (caudal peduncle region) (fig. 1). scales were immediately rinsed in distilled water, cleaned mechanically to remove irrelevant matter using a fine brush, and transferred into a 1% koh solution for 40 min to remove soft tissues from the surface. ultramicroscopy and scale terminology: the cleaned scales were dehydrated through an ascending ethanol series (30, 50, 70, and 90%) at 30 min intervals (lippitsch, 1990), dried on whatman filter paper, kept for several hours between two glass slides to avoid curling of the margins of the scales. scanning electron microscopy images from the posterior region of scales were prepared for each size class. scales were mounted dorsal-up on sem stubs. sem images were captured with a camscan mv2300 sem at shahid bahonar university of kerman (sbuk). for defining the posterior region and its structure types and distribution in the posterior region, we follow hughes (1981). to determine the appearance of cetnii in the most posterior region of scales we follow hughes (1981) and bräger and moritz (2016). concerning the number of ctenii, three types were considered as follows: the number of 0-9 ctenii (as few), the number of 10-30 ctenii (as moderate), and the number of ctenii>30 (as many). results typically, the posterior region of body scale in the studied flathead fishes is covered by comb-like (or tooth-like) structures so-called ctenii (or the spine-like ornamentations in the posterior margin of scale). generally, ctenii are defined as structures consisting of a base and a spine. these tooth-like structures that ossify separately are more or less detached from the main body of the scale. the ctenii appear in one or more rows marginally or sub-marginally at the posterior field, and in the studied fishes, they were figure 1. photographs of (a) grammoplites suppositus and (b) platycephalus indicus showing four studied body regions where scales were removed. h, head region; d1, the region below the first dorsal fin; d2, the region below the second dorsal fin; c, caudal peduncle region. 17 in t. j. a qu at . b io l. (2 02 1) 9 (1 ): 1 522 c ha ra ct er s f is h bo dy r eg io n (s ee f ig . 1 ) h ea d (h ) d or sa l 1 (d 1) d or sa l 2 (d 2) c au da l p ed un cl e (c ) c la ss i c la ss i i c la ss i ii c la ss i c la ss i i c la ss i ii c la ss i c la ss i i c la ss i ii c la ss i c la ss i i c la ss i ii p la ty ce ph al us in di cu s c te ni in po st er io r re gi on pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i sh ap e of ct en i c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d n o. o f ct en i m od er at e (1 720 ) m od er at e (1 725 ) m an y (4 751 ) m od er at e (1 216 ) m an y (3 646 ) m an y (5 458 ) m od er at e (2 627 ) m an y (3 738 ) m an y (3 845 ) m od er at e (1 719 ) m an y (3 436 ) m an y (4 560 ) t he r eg io n co ve re d by ct en i c ov er ed th e w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed al m os t t he w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed h al f of th e po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on g ra m m op lit es s up po si tu s c te ni in po st er io r re gi on pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i pr es en t/ t ra ns fo rm in g ct en i n ot o bs er ve d pr es en t/ t ra ns fo rm in g ct en i sh ap e of ct en i c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ tr un ca te c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d c om blik e/ po in te d __ __ c om blik e/ po in te d n o. o f ct en i fe w (6 -7 ) m od er at e (1 013 ) m an y (3 335 ) m od er at e (1 315 ) m od er at e 16 -1 9 m od er at e 19 -2 3 fe w (8 -9 ) m od er at e 25 -2 8 m an y (3 638 ) m od er at e (1 012 ) __ __ m an y (3 537 ) t he r eg io n co ve re d by ct en i r es tr ic te d to th e m id dl e re gi on r es tr ic te d to th e m id dl e re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on r es tr ic te d to m id dl e pa rt c ov er ed th e m os t p ar t o f po st er io r re gi on c ov er ed al m os t t he w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed th e w ho le po st er io r re gi on c ov er ed al m os t t he w ho le po st er io r re gi on __ __ c ov er ed th e w ho le po st er io r re gi on t ab le 1 . m or ph ol og ic al d es cr ip tio n of th e m ic ro st ru ct ur es in vo lv ed in th e po st er io r r eg io n of tw o st ud ie d fl at he ad s pe ci es in th re e si ze c la ss es . t he d es cr ip tio n is p re se nt ed fo r t he s ca le s fr om fo ur bo dy re gi on s. 18 teimori et al./ scale ultramicroscopy of flathead fishes found to be in transforming ctenii type i.e., separate ossifications that arise as whole spines in two or more alternating rows marginally and transform into truncated spines sub-marginally. characteristics of the posterior region of scales: the sem images of the posterior region for the scales of two studied species are shown in figures 2 and 3, and the details for the posterior region of the scales are summarized in table 1. the most common characteristic for the scales of the studied flathead fishes is the presence of transforming ctenii in their posterior part (figs. 2, 3). also, the sem imaging indicated that the posterior region of the scales in the fishes from all three size classes in both species was covered by ctenii. the only exception, however, was the scales from the caudal peduncle region (region c) in young individuals (class ii) of g. suppositus that had not ctenii in the posterior region (fig. 3h). generally, the shape of the posterior region of the scales in p. indicus was crescent (fig. 2), while it is typically triangular in g. suppositus (fig. 3). the number of ctenii in the posterior region of the scales in p. indicus was minimum 12 (in class i of region d1) and maximum 60 (in class iii of region c), while in g. suppositus it was minimum 6 (in class i region h) and maximum 38 (in class iii region d2) (table 1). the posterior margin of all ctenoid scales examined in this study was formed by two rows of ctenii, while the three complete rows of ctenii were not observed in any of the examined scales. the most frequent state, however, was the two incomplete rows with one complete row between them. the ctenii of both rows were similar in morphology (figs. 2, 3). typically, a ctenus in the posterior part of the figure 2. sem images of the posterior region in the scales of platycephalus indicus and pattern of changes in ctenii during fish growth. 19 int. j. aquat. biol. (2021) 9(1): 15-22 studied scales consists of a long, narrowing spine with generally a pointed or a truncate end, bifurcating anteriorly into two arms (fig. 4). the arms and lobes of the base of a ctenus were joined and formed the sides of an arch. the lobes were sculptured to form articulation grooves which provide a suitable tool of interlocking with nearby ctenii (fig. 4). also, several pores were observed on the surface of the base of a ctenus (fig. 4). the ctenii in the posterior region of scales in both species and three classes were generally similar, with a bit variation regarding space between two lobes at the base of each ctenus. furthermore, four types of distinct, bony structures, each with a specific distribution in the posterior field of the scales of studied flathead fishes were recognized (fig. 4). the first structure was recognized as ctenii. the ctenii from the more posterior row were figure 3. sem images of the posterior region in the scales of grammoplites suppositus and pattern of changes in ctenii during fish growth. figure 4. microstrutures involved in the posterior region of scale in grammoplites suppostius. structures in the posterior field of scale in g. suppositus. ctenii (c), subctenii (sc), cteniial base (cb), subcteniial base (scb). (a) degenerating ctenus, (b) older row ctenus and, (c) younger row ctenus. 20 teimori et al./ scale ultramicroscopy of flathead fishes different from those of the more anterior row. the second type of structure exists at the ends of each row of ctenii was distinguished as a subctenus. the third type of structure was termed as ctenial base and makes up the bulk of the posterior field. the fourth type of structure is similar to, but usually distinct from, a ctenial base. it formed the anterior border of the posterior field and represented the base of a subctenus. this structure was termed as a subctenial base (fig. 4). discussions like other members of the flathead fishes, the two studied species have rough and ctenoid scales. it should also be noted that we found some scales from the caudal peduncle region in young individuals of g. suppositus, which had not ctenii in the posterior region. this exception is more likely because of the deformed and regenerated scales, which have been examined from this region. the ctenii in the scales of studied flathead fishes were of transforming type, which arises as whole spines in two or three alternating rows marginally and transforms into truncated spines submarginally (johnson, 1984). the two other types of ctenii in fish scales are “peripheral ctenii”, which occur as whole spines in one row at the scale margin (e.g. in the gobioid gobiomorus) (figure 3b in roberts, 1993, p. 67), and the “whole ctenii”, which has separate whole spines marginally and submarginally (e.g. in the scale of the black cardinal fish epigonus telescopus, see also figure 5. comparison of the posterior region of the scales from region ii, among the three size classes of platycephalus indicus. 21 int. j. aquat. biol. (2021) 9(1): 15-22 figure 20a in roberts, 1993, p. 86). in further examination, we compared the scales from region ii, among the three size classes because usually, scales from this region are the most grown scales (see also fig. 5). our observations indicated that the smaller fishes had fewer ctenii in their scales than the adults. therefore, it is likely that the number of ctenii is increased during fish development of these fishes (table 1, fig. 5). however, the general morphology of ctenii had not been changed properly during development (fig. 5). the developmental change in the number of ctenii was found to be significantly higher in p. indicus than g. suppositus. in p. indicus, the number of ctenii in the fish scales of class i was moderate (12-27), while they were increased to many ctenii (38-60) in the fishes of class iii. in g. suppositus, the number of ctenii in the fish scales of class i were few (6-9) or moderate (12-27), while they were increased to moderate (19-23) or many ctenii (33-38) in the fish scales of class iii. it has been documented that the growth of the posterior region of scale is happened by adding new ctenii to the posterior margin (hughes, 1981). however, our microscopic observations revealed that the developing ctenii were morphologically similar to mature ctenii but were relatively smaller in size. by growing a new ctenus, the spine of the ctenus behind which it is growing is degenerating. therefore, the spine of the older ctenus is replaced by the new growing ctenus. hughes (1981) concluded that the spinal degeneration of older ctenii when new ctenii develop behind them is a mechanism for conserving and recycling scale material. functionally, ctenii in the posterior region of scales may improve hydrodynamic efficiency of swimming by affecting the profile of the overlying epidermis, and thus assisting in breaking vortices caused by the swimming fish and thereby reducing drug (burdak, 1969; sire, 1986). as a result, increasing the number of ctenii in the posterior margin of scales in the studied fishes during their development may allow greater flexibility in fish movement. however, the number of ctenii in the scales of small and large fishes in g. suppositus was found to be obviously lower than p. indicus. this means that g. suppositus is not probably very active in swimming as p. indicus. therefore, it can be assumed that modification in the ornamentations of the posterior region has a hydrodynamic function and they are subject to modification during the growth of the fish (burdak, 1986; sire, 1986; sire and arnulf, 2000). besides, variation in the number of ctenii in the posterior region of the scales in the adult flathead fishes was found to be suitable for discrimination of the flathead species. takagi (1953) has also been accentuated that ctenii are the most conspicuous structures of the posterior field of fish scales. these structures varying considerably in the number and could be easily counted, therefore, could be used as an appropriate taxonomic character at least in flathead fishes or even other fish groups. acknowledgment shahid bahonar university of kerman has supported financially this work (grant id: 1395-96 to the first author and grant id: 1396 to the second author). references bräger z., moritz t. (2016). a scale atlas for common mediterranean teleost fishes. vertebrate zoology, 66: 275-386. burdak v.d. (1969). ontogenesis of squamation in the mullet (mugil saliens risso.). zoologicheskii zhurnal, 48: 241-247. burdak v.d. (1986). morphologie fonctionnelle du tegument ecailleux des poissons. cybium, 10: 147. fricke r., eschmeyer w.n., van der laan r. (2020). eschmeyers catalog of fishes: genera, species, references. last access in 08/08/2020. http:// researcharchive.alacademy.org/research/ichthyol ogy/catalog/fishcatmain.as. froese r., pauly e. (2015). fishbase. world wide web electronic publication. www.fishbase.org. hughes d.r. (1981). development and organization of the posterior field of ctenoid scales in the platycephalidae. copeia, 3: 596-606. johnson g.d. (1984). percoidei: development and relationships. in: h.g. moser., richards w.j., cohen d.m., fahay m.p., kendall a.w., jr. richardson s.l. (ed.), ontogeny and systematics of fishes. american society of ichthyologists and herpetologists no. i. 22 teimori et al./ scale ultramicroscopy of flathead fishes imamura h. (1996). phylogeny of the family platycephalidae and related taxa (pisces: scorpaeniformes). species diversity, 1: 123-233. imamura h., mcgrouther m. (2008). new records of a flathead fish, onigocia grandisquama (regan, 1908) (teleostei: platycephalidae) from australia. memoirs of the queensland museum, 2: 239-243. lippitsch e. (1990). scale morphology and squamation pattern in cichlids (teleostei, perciformes): a comparative study. journal of fish biology, 37: 265291. mastrototaro f., roberto c., francesca c., letizia s. (2007). first record of dwarf flathead elates ransonnetii (platycephalidae) in the mediterranean sea (northwestern ionian sea). cybium, 31: 393-394. motamedi m., teimori a., amiri v., askari hesni m. (2020). characterization of age-dependent variability in the flank scales of two scorpaeniformes fishes by applying light and scanning electron microscopy imaging. micron. 128 p. roberts c.d. (1993). comparative morphology of spined scales and their phylogenetic significance in the teleostei. bulletin of marine science, 1: 60-113. sire j.y. (1986). ontogenetic development of surface ornamentation in the scale of hemichromis bimaculatus (cichlidae). journal of fish biology, 28: 713-724. sire j.y., arnulf i. (2000). structure and development of the cteniial spines on the scales of a teleost fish, the cichlid cichlasoma nigrofasciatum. acta zoologica, 81: 139-158. takagi k. (1953). a study of the scale of the gobiid fishes of japan. journal of the tokyo university of fisheries, 39: 231-257. wonsettler a.l., webb j.f. (1997). morphology and development of the multiple lateral line canals on the trunk in two species of hexagrammos (scorpaeniformes: hexagrammidae) journal of morphology, 233: 195-214. international journal of aquatic biology (2015) 3(3): 191-198 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article study of fungicidal properties of colloidal silver nanoparticles (agnps) on trout egg pathogen, saprolegnia sp. seyed ali johari1, 2, mohammad reza kalbassi*2,1mehdi soltani3, il je yu4 1fisheries department, natural resources faculty, university of kurdistan, sanandaj, iran. 2department of aquaculture, marine sciences faculty, tarbiat modares university, mazandaran, noor, iran. 3department of aquatic animal health, faculty of veterinary medicine, university of tehran, tehran, iran. 4institute of nanoproduct safety research, hoseo university, asan, korea. article history: received 2 march 2014 accepted 14 april 2014 available online 2 5 june 2015 keywords: antifungal in vitro saprolegnia silver nanoparticles rainbow trout abstract: silver nanoparticles (agnps) are known to have bactericidal and fungicidal effects. since, there is few information available on the interaction of colloidal nanosilver with fish pathogens. hence, the current study investigated the effects of colloidal agnps on the in vitro growth of the fish pathogen saprolegnia sp.. before the experiments, various important properties of agnps were wellcharacterized. the antifungal activity of agnps was then evaluated by determining the minimum inhibitory concentrations (mics) using two-fold serial dilutions of colloidal nanosilver in a glucose yeast extract agar at 22ºc. the growth of saprolegnia sp. on the agnps agar treatments was compared to that of nanosilver-free agar as controls. the results showed that agnps have an inhibitory effect on the in vitro growth of the tested fungi. the mic of agnps for saprolegnia sp. was calculated at 1800 mg/l, which is equal to 0.18 percent. it seems that agnps could be a proper replacement for teratogenic and toxic agents, such as malachite green. in addition, the indirect use of agnps could be a useful method for providing new antifungal activity in aquaculture systems. introduction the reduction in fish diseases is undoubtedly very important for the future success of the aquaculture industry. indeed, the largest cause of economic losses in aquaculture comes from diseased fish, and oomycete (water mould) infections are second only to bacterial diseases in their impact (meyer, 1991). oomycetes such as saprolegniales, including the saprolegnia, achlya, and aphanomyces species, have been found responsible for fish infections in aquaculture, fish farms, and hobby fish tanks (baldauf et al., 2000; bruno and wood, 1999; daugherty et al., 1998; hussein and hatai, 2002; neish and hughes, 1980; okumuş, 2002; willoughby and pickering, 1977). saprolegnia is one of the most destructive oomycete pathogens for fishes, being endemic to all freshwater habitats * corresponding author: mohammad reza kalbassi e-mail address: kalbassi_m@modares.ac.ir around the world and partly responsible for the decline of cultured and natural populations of salmonids, cyprinids, acipensers, and other freshwater fish (van west, 2006). although malachite green is very effective for controlling fungal infections on the surface of fish and fish eggs (okumuş, 2002), its suspected teratogenicity (meyer and jorgenson, 1984) has limited its use to the treatment of nonfood fish under an investigational new animal drug application held by the u.s. fish and wildlife service, and its re-registration for the treatment of fungal infections in food fish is highly unlikely (bruno and wood, 1999). currently, there are few registered aquatic fungicides other than malachite green. formalin is not completely effective for controlling fungal infections in fish or fish eggs (bruno and wood, 192 international journal of aquatic biology (2015) 3(3): 191-198 1999; okumuş, 2002), plus there are also concerns about its effect on both the environment and the personnel who handle it (fitzpatrick et al., 1995). furthermore, the use of other fungicides, such as ozone, hydrogen peroxide, sodium chloride, iodophores, and copper is not widespread (bruno and wood, 1999; forneris et al., 2003; rach et al., 1998; schreier et al., 1996). therefore, more research is needed to identify new secure and effective aquatic fungicides. in this regard, the efficacy of many potential fungicides has been tested using in vitro screening methods (bailey, 1984; bailey and jeffrey, 1989; bruno and wood, 1999), and it was found that in vitro tests correlated well with the in vivo conditions of surface infections of fish (bailey, 1983a, b; fereidouni et al., 2013). recently, various inorganic antibacterial and antifungal materials containing silver have been developed and some are already in commercial use (hansel et al., 1998; kawahara et al., 2000; palenik & setcos, 1996; wang et al., 2007; yamamoto et al., 1996; johari et al., 2014a). among various antibacterial metals, silver is known to have a wide antibacterial spectrum and be relatively safe (cho et al., 2005; mohan et al., 2007; oya, 1996; tic, 1998). one of the antimicrobial silver species that has been known for a long time, yet received little attention in aquaculture, is nanometer-sized silver particles (agnps) which exhibit both bactericidal and mycocidal effects (cho et al., 2005; mohan et al., 2007). also recently antimicrobial efficacy of agnps have been studied against some fish pathogens (vaseeharan et al., 2010; antony et al., 2013; mahanty et al., 2013; dananjaya et al., 2014; swain et al., 2014). since our previous study confirmed the prevention effect of silver zeolite (sz) against saprolegnia sp. (johari et al., 2014a), in the present study we examined the inhibitory effect of agnps on the growth of the water mould saprolegnia and found that nanosilver is effective against saprolegnia in vitro. suggestions are also given on techniques for the indirect application of agnps in fish production systems. materials and methods silver nanoparticles and characterizations: colloidal agnps, type l (commercial name: nanocid), were donated by nano nasb pars co. (tehran, iran). the colloid product was synthesized using a process involving the photo-assisted reduction of ag+ to metallic nanoparticles, registered under united states patent application no: 20090013825 (rahman nia, 2009). according to information provided by the manufacturer, the product was a water-based colloid containing 4000 mg/l spherical agnps (average size 16.6 nm). the detailed specifications of this colloid have been analyzed and reported previously (asghari et al., 2012; johari et al., 2013). also, prior to using the colloid product in the present study, tem analyses of the undiluted agnp suspension (4000 mg/l) were performed using an h-7100fa transmission electron microscope (hitachi, japan) with an acceleration voltage of 125kv. the diameters of 700 randomly selected particles were measured at a magnification of 100,000 using axio vision digital image processing software (release 4.8.2.0, carl zeiss micro imaging gmbh, germany). antifungal activity tests: a pure stock of fish saprolegnia sp. previously purified from rainbow trout eggs and characterized by the department of aquatic animal health, veterinary medicine faculty, university of tehran (shahbazian et al., 2010) was cultured on a glucose yeast extract agar (gya) and stored at 4°c until use. the composition of the gya included: 20 g/l agar, 10 g/l glucose, 2 g/l yeast extract, 2.04 g/l kh2po4, and 0.596 g/l na2hpo4.12h2o. the antifungal effects of the agnps were evaluated by determining the minimum inhibitory concentrations (mics) using the agar concentration method (bailey, 1983a, b). briefly, agar plugs containing fungal hyphae of saprolegnia (5 mm in diameter) were removed from the edge of the pure stock and placed in the middle of depression spots on plates containing various concentrations of agnps and incubated at 22ºc. the maximal growth of saprolegnia (colony diameter) was determined after 193 johari et al/ effect of agnps on growth of the saprolegnia 24, 48, and 72 hours. to determine the inhibitory concentration range, twelve test concentrations of agnps, including 4000, 2000, 1000, 500, 250, 125, 62, 31, 15, 8, 4, and 2 mg/l plus a control were prepared on gya plates in triplicate. the growth of saprolegnia in the presence of the agnps was compared to that of the control. according to the antifungal activity observed in the range-finding tests (inhibitory effects were observed between 1000–2000 mg/l), more six concentrations of agnps, including 1000, 1200, 1400, 1600, 1800, and 2000 mg/l, were selected and their inhibitory effects against saprolegnia checked after 24, 48, and 72 hours in the same way as described above. as a criterion for evaluating the saprolegnia growth with the treatments, the area over which the saprolegnia hyphae grew in the petri dishes was calculated and compared to that in the positive control as follows. in all cases, the mean and standard deviations were calculated using microsoft office excel 2007.  (%)index growth asaprolegni (growth area of saprolegnia on the plates in the agnps treatments/growth area of saprolegnia on the plates in the control) x 100 results silver nanoparticles and characterizations: the agnps observed by tem were spherical in shape, with a maximum diameter of 129 nm (fig. 1): 65.14% of the particles had diameters between 1 and 13 nm (just 2.28% of the particles had diameters more than 100 nm) and the cmd (count median diameter) for the particles was 6.47 nm (fig. 2). also, the geometric mean diameter (gmd) and geometric standard deviation (gsd) of the agnps were 12.65 nm and 1.46, respectively. antifungal effects of silver nanoparticles: the silver nanoparticles exhibited dose-dependent effects on the colony size of the saprolegnia. the colonies grew well in the controls, where after 72 hours the whole surface of the culture media (on 90 mm petri dishes) was covered by saprolegnia sp. hyphae. the figure 1. tem micrograph of silver nanoparticles. figure 2. size distribution of silver nanoparticles in undiluted suspension (4000 mg/l) based on transmission electron microscope data. a: number frequency and b: cumulative frequency (cmd: cumulative median diameter). 194 international journal of aquatic biology (2015) 3(3): 191-198 results of the range-finding tests showed no difference in the saprolegnia growth with concentrations of 2 and 4 mg/l agnps compared to the positive control (without agnps). yet with agnp concentrations of 8 to 1000 mg/l, the saprolegnia growth gradually began to decrease, and no growth was observed with an agnp concentration of 2000 mg/l. therefore, it was concluded that growth of saprolegnia sp. was limited within an agnp concentration range of 1000 to 2000 mg/l. the results of the mic determination tests showed figure 3. growth of saprolegnia in different concentrations of agnps, compared to control after 24, 48, and 72 hours (mic determination test). figure 4. growth of saprolegnia on agar plates containing different concentrations of agnps (mic determination test, after 72 hours). 195 johari et al/ effect of agnps on growth of the saprolegnia that with 1200 and 1600 mg/l agnps, the saprolegnia growth was less than 50 and 20%, respectively, compared to the control; and finally with 1800 mg/l, the saprolegnia growth was zero (figs. 3 and 4). thus, the mic of the agnps (at 22ºc) was determined to be 1800 mg/l for saprolegnia sp., which was approximately equal to 0.18% silver nanoparticles. discussion silver particulates, and especially nanosilver in colloidal form, are known worldwide as a cure or inhibitor of bacterial, fungal, and viral diseases (murr, 2009). thus, silver is already commercially used to take advantage of its antibacterial properties (kawashita et al., 2000). for example, silver nanoparticles are widely used in the production of antifungal and antibacterial ceramics, textiles, and paints. the mechanisms involved in the antimicrobial activity of agnps have previously been reported (kim et al., 2007; rai et al., 2009; morones et al., 2005; sanpui et al., 2008) and include: (1) changing and damaging the membrane structure of a microorganism, which increases its permeability and disrupts the transportation functions, resulting in cell death, (2) penetration of a microorganism and interaction with phosphorus and sulfur-containing compounds, such as dna and proteins, (3) loss of the replication ability of the dna, (4) inactivation of certain enzymes, (5) attacking the respiratory chain, (6) generating hydrogen peroxide and free radicals, and (7) the release of the silver ions from the nanoparticles, the antimicrobial activity of which is well known (feng et al., 2000; song et al., 2006; yamanaka et al., 2005; yoshihiro, 2002). in the present study, agnps were found to inhibit the in vitro growth of the water mould saprolegnia, making agnps a good candidates for indirect use in the aquaculture industry. it is important to note that the direct use of agnps in any form can pose several toxic effects on aquatic biota (asghari et al., 2012; johari et al., 2013; johari, 2014; johari et al., 2014b; kalbassi et al., 2011; salari joo et al., 2012, 2013; sharifian et al., 2013; hosseini et al., 2014; johari et al., 2015a; tavana et al., 2014) and therefore the indirect use is the only way for peaceful use of this material in aquaculture industry. the mic value for the agnps was high compared to that for other antifungal materials used for the direct treatment of eggs or larva (for example, malachite green), yet since agnps can be easily mixed or coated on other substances for indirect treatment, they may be a useful disinfectant against saprolegnia. for instance, agnps can be included in the polymeric structures of aquaculture equipment, such as fiberglass or polyethylene troughs, trays, culture tanks, and other propagation and rearing instruments, as an antimicrobial and even antifouling agent that can reduce the use of anti-pathogen ingredients in the water environment. therefore, in terms of potential use, the incorporation of agnps into the surfaces and other objects in the aquaculture industry is conceivable. based on the mic results, mixing approximately 0.2% agnps into the structure of aquaculture equipment may completely inhibit saprolegnia growth. now it is necessary to find the best applicable methods to use agnps in aquaculture systems, such as fish ponds, hatcheries, and aquariums. silver nanoparticles can be coated on the surface of different media (such as activated carbon, zeolite, and foams) and then used in the water filtration systems of recirculation systems and hatcheries to reduce bacterial and fungal diseases transmitted and spread through water. in this regard, johari et al. (2015b) recently used a silver nanoparticles layer on the clinoptilolite and used this material in the water filtration system of salmonid's egg to prevent saprolegniasis. in conclusion, further investigation of the antibacterial, antiviral, and antifungal activities of agnps against other fish pathogens is needed. furthermore, since the current experiment was carried out at 22ºc, the results correlate with the culture conditions of shrimp and warm water fish (such as cyprinids and acipensers). meanwhile, for cold water fish, such as salmonids, the current study needs to be repeated at lower temperatures (i.e. 10196 international journal of aquatic biology (2015) 3(3): 191-198 12ºc) to understand the inhibitory effects of agnps for such species. acknowledgments the authors gratefully acknowledge the support of the tarbiat modares university of i. r. iran, who funded this research through a ph.d. thesis project. also, this research was partially supported by the green nanotechnology program through the national research foundation of korea funded by the korean ministry of education, science and technology. we thank dr. ji hyun lee for his technical assistance in the analysis of the tem images and mrs. saba asghari for her assistance in the experiments. the authors are also grateful to the mr. mansour fotovat (director of nano nasb pars co. ltd, tehran, i. r. iran), for providing the silver nanoparticles for the current research. references antony j.j., nivedheetha m., siva d., pradeepha g., kokilavani p., kalaiselvi s., sankarganesh a., balasundaram a., masilamani v., achiraman s. 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(2020) 8(3): 216-223 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology review article the life story of tgfβs superfamily: from the beginning to the end shaghayegh hasanpour, soheil eagderi*,1hadi poorbagher department of fisheries, faculty of natural resources, university of tehran, karaj, iran. s article history: received 7 january 2020 accepted 24 may 2020 available online 2 5 june 2020 keywords: tgfβ smad canonical pathway non-canonical cascades abstract: tgfβ-superfamily consists a plethora of extracellular growth factors, modulating developmental procedures and homeostasis in vertebrates and invertebrates. tgfβ-superfamily ligands, synthesized as the large inactive precursors, transform into active ligands following by their interaction with extracellular proteolytic enzymes. principally, tgfβs ligation to their responsive receptors can trigger two distinct transduction cascades, including 1smad dependent or canonical pathway and 2smad independent or non-canonical ones. r-smads are substrates for the type i receptors, as their gs domains act as a docking site for r-smads. in the canocical pathway, upon phosphorylation of ssxs of mh2, two phosphorylated-smads (p-smads) in accordance with receptor tetra-dimerization, homo or heterodimerize and then form a trimer complex by smad4. the trimers translocate to the nucleus, where in association with other transcription factors (activators and repressors) modulate their target genes expression. the purpose of this review is to provide a comprehensive information about these cascades and their downstream effectors with an emphasis on the canonical one. introduction transforming growth factor β (tgfβ) superfamily consists a broad spectrum of extracellular growth factors, regulating development and homeostasis processes in vertebrates and invertebrates. its highly conserved members have been identified in sea urchins, nematodes, flies and vertebrates (raftery and sutherland, 1999; ten dijke and hill, 2004; cavaleri and schöler, 2009; wu and hill, 2009; pauklin and vallier, 2015). the common feature of tgfβsuperfamily members is a conserved cysteine knot structure (liang and rubinstein, 2003; gordon and blobe, 2008). this superfamily, containing over 40 ligands, is divided into a number of families, including tgfβs (1-3), activin (a & b), bmps (1-20), gdfs, lefty (1-2), nodal and inhibin (reddi, 1997; sutherland, 1999; kramer, 2002; liang and rubinstein, 2003; ober et al., 2003; field et al., 2003a, b; ten dijke and hill, 2004; james et al., 2005; kondo, 2007; itoh and ten dijke, 2007; gordon and blobe, 2008; perrett, 2008; cavaleri and schöler, 2009; *correspondence: soheil eagderi e-mail: soheil.eagderi@ut.ac.ir raftery and schier, 2009; seuntjens et al., 2009; chng et al., 2011; lonardo et al., 2011; oshimori and fuchs, 2012; quail et al., 2013; itoh et al., 2014; vallier, 2016; fathi et al., 2017) . some of these factors are synthesized by a wide variety of cells, whereas others are produced by a specific cell type. furthermore, in contrast with some tgfβs, which are active for a short period, others are transcribed throughout the life (kramer, 2002). tgfβ-superfamily members participate in a plethora of pleiotropic functions divided into the cellular and physiological works. the cellular functions managed by tgfβs are as follows: (1) pluripotency maintenance of stem cells, (2) proliferation (growth), (3) changes in cell shape (migration and adhesion), (4) apoptosis and (5) differentiation. the physiological processes controlled by tgfβs encompass: (1) early axial patterning, (2) inductive interactions during organogenesis, (3) wound healing and (4) tissue homeostasis. according to their wide roles, abnormal expression of tgfβs provokes cancer stem cells 217 int. j. aquat. biol. (2020) 8(3): 216-223 (csc), neoplasia, developmental disorders and vascular disease (raftery and sutherland, 1999; ten dijke and hill, 2004; james et al., 2005; itoh and ten dijke, 2007; gordon and blobe, 2008; schier, 2009; lonardo et al., 2011; donahue and dawson, 2011; oshimori and fuchs, 2012; beyer et al., 2013; quail et al., 2013; itoh et al., 2014; pauklin and vallier, 2015; vallier, 2016). synthesis and primary process of tgfβs: tgfβsuperfamily ligands are synthesized as the large inactive precursors, consisting four main domains (in an amino to carboxyl terminal direction), including (1) the signal or leader peptide, (2) the latency associated peptide (lap) or pro-domain, (3) a furin convertases cleavage sequence and (4) the biologically active mature domain. the lap involves in folding, stability and dimerization of tgfβs in the intracellular space (raftery and sutherland, 1999; kramer, 2002; ten dijke and hill, 2004; jing et al., 2006; itoh and ten dijke, 2007; gordon and blobe, 2008; schier, 2009; seuntjens et al., 2009; chng et al., 2011; oshimori and fuchs, 2012; beyer et al., 2013; pauklin and vallier, 2015; vallier, 2016). after their entrance in extracellular space, the tgfβ precursors are transformed into active ligands following the lap separation by (1) extracellular proteolytic enzymes, including plasmin, matrix metallo-proteinases (mmp2/9), cathepsin-d and thrombospondin or (2) integrin αvβ6 and αvβ8 and or 3furin convertases (furin (spc1) and pace4 (spc4)) (raftery and sutherland, 1999; kramer, 2002; tam et al., 2003; jing et al., 2006; gordon and blobe, 2008; seuntjens et al., 2009; chng et al., 2011; oshimori and fuchs, 2012; beyer et al., 2013; sun et al., 2014;). however, the bmps do not lose their lap conferring them stability in extracellular space as well. therefore, their modulation merely depends on the antagonists compared to other tgfβs. bmps antagonists regulate bmps activity by preventing their interaction with receptors. based on the size of their cysteine knot, bmps antagonists are clustered into (1) can, (2) twisted gastrulation (tsg) and (3) chordin/noggin families (kramer, 2002; minchiotti et al., 2002; field et al., 2003a, b; gordon and blobe, 2008; perrett, 2008; wu and hill, 2009; chng et al., 2011; beyer et al., 2013; pauklin and vallier, 2015; vallier, 2016) . canonical and non-canonical tgfβ pathways: tgfβs can trigger two distinct and independent pathways including (kodjabachian et al., 1999; munoz-sanjuan and a, 2001; stemple, 2001; whitman, 2001; kramer, 2002; liang and rubinstein, 2003; ten dijke and hill, 2004; james et al., 2005; sun et al., 2006; itoh and ten dijke, 2007; kondo, 2007; blobe, 2008; jia et al., 2008; gordon and xu et al., 2008; cavaleri and schöler, 2009; chan et al., 2009; de robertis, 2009; harvey and smith, 2009; schier, 2009; seuntjens et al., 2009; wu and hill, 2009; zhendong, 2009; chng et al., 2011; oshimori and fuchs, 2012; beyer et al., 2013; ramel and hill, 2013; stewart et al., 2014; itoh et al., 2014; pauklin and vallier, 2015; fathi et al., 2017): (1) smad dependent or canonical transduction cascade and (2) smad independent or non-canonical signaling pathways. the second one, itself, encompasses (1) mapk pathway (e.g. mapk/erk, p38 and jnk), (2) pi3k/akt pathway and (3) nf-kb pathway (fig. 1). non-canonical tgfβs pathways: in mapk pathway, ligand binding phosphorylates traf6 (e3 ligase tnf receptor-associated factor 6) that in turn induces the activation of tak1. the activated tak1 then transmits the signal by activating mek that phosphorylates p38/jnk/erk1/2. these transcription factors translocate to the nucleus to transcribe genes modulating proliferation (carmanyrampey and moens, 2006; gordon and blobe, 2008; perrett, 2008; seuntjens et al., 2009; wu and hill, 2009; oshimori and fuchs, 2012; quail et al., 2013; vallier, 2016). tgfβ type i receptors can also enable the scaffold protein (shca) to bind with grb2-sos to activate ras-erk1/2 signaling pathway. furthermore, the activated type ii receptors can also triggered par6 that consequently induces tight junction destruction and epithelial mesenchymal transition (emt) (carmany-rampey and moens, 2006; gordon and blobe, 2008; perrett, 2008; seuntjens et al., 2009; wu and hill, 2009; oshimori and fuchs, 2012; quail et 218 hasanpour et al./ the life story of tgfβs al., 2013; vallier, 2016). canonical tgfβs pathways 1. introduction of the components, involved in the canonical tgfβs signaling cascades tgfβs superfamily receptors: tgfβs recruit serine/threonine protein kinase transmembrane receptors to relay the signal into their downstream effectors. based on the structural and functional characteristics, the receptors are subdivided into two subfamilies including the type i and type ii receptors. there are seven type i (activin-receptor like kinase; alk1-7) and five type ii receptors in the human genome. these glycoprotein receptors have a single transmembrane span and an intrinsic serine/threonine kinase domain in their c-terminal segment. however their main difference refers to the gs domain of type i receptor which is associated by immunophiline fkbp1a (raftery and sutherland, 1999; lele et al., 2001; whitman, 2001; kramer, 2002; ober et al., 2003; field et al., 2003a; gordon and blobe, 2008; seuntjens et al., 2009; wu and hill, 2009; chng et al., 2011; oshimori and fuchs, 2012; beyer et al., 2013; pauklin and vallier, 2015). following ligand binding, tgfβ type ii receptor, as a constitutively active kinase, hetero-dimerize with an appropriate type i receptor. in fact, the active receptor complex includes two type i and two type ii receptors (tetra-dimerization) due to its recruitment by a dimeric ligand. upon ablation of the immunophilin fkbp1a/fkbp12, the gs domain is phosphorylated figure 1. canonical and non-canonical tgfβ transduction cascades. 219 int. j. aquat. biol. (2020) 8(3): 216-223 by the type ii receptor. the phosphorylated gs domain acts as a docking site for receptor-regulated smads (r-smads), the unique substrates of type i receptors (raftery and sutherland, 1999; kramer, 2002; gordon and blobe, 2008; perrett, 2008; xu et al., 2008; schier, 2009; seuntjens et al., 2009; wu and hill, 2009; chng et al., 2011; oshimori and fuchs, 2012; beyer et al., 2013; pauklin and vallier, 2015; vallier, 2016). in addition to the main receptors, there are three accessory or co-receptors, including; (1) tgfβr3 (known as β-glican), (2) endoglin (co-receptor in bmps signaling) and (3) tdgf1 (namely crypto). crypto belongs to the egf-cfc family (epidermal growth factorcrypto, frl1, and cryptic). egfcfc family includes crypto and cryptic in mice, hcripto and h-criptic in human, crypto in chick, one-eyed pinhead (oep) in zebrafish and frl-1 in frog (xenopus laveis). members of this family are anchored in the lipid membrane by peptidoglycans. they can be detached from the membrane as a soluble molecule, in turn; competent cells (even oep mutant one) will be able to respond properly to nodal signaling. the co-receptors merely reinforce the signal transduction because their intracellular domain are devoid of any sequence motif involving in signal transduction (mullins, 1998; kodjabachian et al., 1999; raftery and sutherland, 1999; shen and schier, 2000; chen and schier, 2001; schier, 2001; whitman, 2001; minchiotti et al., 2002; kramer, 2002; liang and rubinstein, 2003; tam et al., 2003; ten dijke and hill, 2004; bartscherer and boutros, 2008; gordon and blobe, 2008; xu et al., 2008; schier, 2009; wu and hill, 2009; chng et al., 2011; lonardo et al., 2011; beyer et al., 2013; nozawa et al., 2013; pauklin and vallier, 2015; tuazon and mullins, 2015; vallier, 2016). smads: mad (mothers against dpp (bmp orthologue)) is known as the smad orthologue in drosophila melanogaster. it transduces the dpp signal from the receptor to the nucleus (raftery and sutherland, 1999; neave et al., 1997; reddi, 1997; kramer, 2002; kondo, 2007; wu and hill, 2009; quail et al., 2013). smads, a family of conserved transcription factors, are clustered into third class according to their phylogenetic relationships and functional evaluations: (1) r-smads (receptorregulated smad: smad1/2/3/5/8), (2) co-smad (common-mediator smad: smad4) and (3) ismads (inhibitory smads: smad6/7). in general, r-smads have a central role in the canonical transduction cascade and smad4 enhances the signaling triggered by tgfβs. however, smad6/7 down regulate the signal transduction. r-smads and smad4 show sequence homology in two unrelated regions, including the n-terminal mad homology (mh1) domain to bind with dna and the c-terminal mh2 domain to bind with other transcription factors. these domains are separated by a poorly conserved prolin-rich linker region. furthermore, r-smads contain a ssxs motif in their mh2 domain to bind with the type i receptors. in this regard, r-smads accession to type i receptors is facilitated by auxiliary proteins such as smad anchors for receptor activation (sara). in addition, r-smads encompass a ppxy motif in their linker region that can be phosphorylated by erk, gsk3 as well as cdk8/9; thereby creating a docking site for ww domain containing proteins. therefore hecte3 ligases as the ww-domain containing proteins (including smurf1/2, ectodermin (e3 ubiquitin ligase), wwp2 and nedd4l) marked r-smads for proteasome degradation (raftery and sutherland, 1999; kramer, 2002; liang and rubinstein, 2003; field et al., 2003a, b; ober et al., 2003; ten dijke and hill, 2004; kondo, 2007; gordon and blobe, 2008; xu et al., 2008; cavaleri and schöler, 2009; schier, 2009; seuntjens et al., 2009; wu and hill, 2009; chng et al., 2011; oshimori and fuchs, 2012; quail et al., 2013; beyer et al., 2013; itoh et al., 2014; pauklin and vallier, 2015). r-smads linker region provide a platform to converge tgfβ signaling pathway and other biological transduction cascades like wnt, fgf and igf. other ww domain containing proteins such as yap1 transcription factor and sin1 coactivator help to smads maintenance in the nucleus. latent conformation of r-smads and smad4 is due to a reciprocal intramolecular interaction between 220 hasanpour et al./ the life story of tgfβs their mh1 and mh2 domains during the absence of signaling factors. smads phosphorylation precludes this auto-inhibition and makes their mh1 and mh2 domains available to interact with dna and other transcription factors respectively (raftery and sutherland, 1999; kramer, 2002; field et al., 2003a, b; liang and rubinstein, 2003; ober et al., 2003; ten dijke and hill, 2004; kondo, 2007; gordon and blobe, 2008; xu et al., 2008; cavaleri and schöler, 2009; schier, 2009; seuntjens et al., 2009; wu and hill, 2009; chng et al., 2011; oshimori and fuchs, 2012; beyer et al., 2013; quail et al., 2013; itoh et al., 2014; pauklin and vallier, 2015). i-smads exclusively retain the mh2 domain without its ssxs motif. they alleviate phosphorylation of r-smads by type i receptors. principally i-smads terminate the tgfβs signaling pathway in several different ways. first, i-smads mark the type i receptors or r-smads by recruiting e3-ubiquitinligases known as smurf1/2 (smad ubiquitination regulatory factors). in this regard, smad7 and smad6 suppress all r-smads and smad1/5/8 respectively. second, smad7 inactivates type i receptors by recruiting gadd34 complex and catalytic subdomain of protein phosphatase i (raftery and sutherland, 1999; kramer, 2002; ten dijke and hill, 2004; james et al., 2005; mizoguchi et al., 2006; bennett et al. 2007; gordon and blobe, 2008; schier, 2009; seuntjens et al., 2009; wu and hill, 2009; beyer et al., 2013; itoh et al., 2014; vallier, 2016; fathi et al., 2017). the canonical tgfβs signaling pathway: r-smads are substrates for the type i receptors, as their gs domains act as a docking site for r-smads. upon phosphorylation of ssxs sequence of mh2, two phosphorylated-smads (p-smads) in accordance with receptor tetra-dimerization, homo or heterodimerize and then form a trimer complex by smad4. the trimers translocate to the nucleus, where in association with other transcription factors (activators and repressors) modulate their target genes expression (raftery and sutherland, 1999; munoz sanjuan and a, 2001; kramer, 2002; ten dijke and hill, 2004; itoh and ten dijke, 2007; gordon and blobe, 2008; xu et al., 2008; cavaleri and schöler, 2009; schier, 2009; wu and hill, 2009; oshimori and fuchs, 2012; beyer et al., 2013; itoh et al., 2014; pauklin and vallier, 2015). functional analysis of smad proteins suggests that, the canonical or smad dependent tgfβ signaling is clustered into two distinct branches with respect to the r-smads. in this regard, bmps and gdfs exclusively transmit signal through alk1/2/3/6/8 receptors and smad1/5/8 whilst tgfβs, activin and nodal (activin-like factors) transmit signal through alk4/5/7 and smad2/3. however, tgfβs itself robustly activate both smad1/5/8 and smad2/3 in many cell types and gdf8/9/11 can also transduce signal through alk4/5/7. therefore, the above-mentioned category is almost over simplification. additionally, due to high affinity between the type ii receptors and all tgfβ members except bmps, the ligand-receptor interaction is highly cooperative and upon their binding, both type of receptors in direct association transduce the signal into smads. while in the case of bmps, the type ii receptors indirectly binds to the type i by recruiting the bmp ligands (kodjabachian et al., 1999; munoz-sanjuan and a, 2001; stemple, 2001; whitman, 2001; kramer, 2002; liang and rubinstein, 2003; ten dijke and hill, 2004; james et al., 2005; sun et al., 2006; itoh and ten dijke, 2007; kondo, 2007; gordon and blobe, 2008; jia et al., 2008; xu et al., 2008; de robertis, 2009; cavaleri and schöler, 2009; chan et al., 2009; harvey and smith, 2009; schier, 2009; seuntjens et al., 2009; wu and hill, 2009; zhendong, 2009; chng et al., 2011; oshimori and fuchs, 2012; beyer et al., 2013; ramel and hill, 2013; itoh et al., 2014; stewart et al., 2014; pauklin and vallier, 2015; fathi et al., 2017). smad2/3 binds to 5'-agac-3' or its complement gtct known as smad binding elements (sbe), whereas, smad1/5/8 preferentially binds to ggcgcc or ggagcc namely bmp response elements (bre). except the smad2, all r-smads and smad4 can directly bind to dna with low affinity and specificity. hence, to achieve high affinity and selectivity, smad proteins must thus interact 221 int. j. aquat. biol. (2020) 8(3): 216-223 with other transcription factors (raftery and sutherland, 1999; munoz-sanjuan and a, 2001; kramer, 2002; ten dijke and hill, 2004; itoh and ten dijke, 2007; gordon and blobe, 2008; xu et al., 2008; cavaleri and schöler, 2009; schier, 2009; wu and hill, 2009; oshimori and fuchs, 2012; beyer et al., 2013; itoh et al., 2014; pauklin and vallier, 2015). the regulator factors of tgfβ signaling: the modulator factors of tgfβ signaling divide into two main parts. the first one is extracellular factors, including (a) antagonist agents, (b) agonist agents like co-receptors and diffusible ligand binding proteins, which promote the ligand accessibility, (c) processing enzymes like furin and (d) secreted protein acidic rich in cysteine (sparc). in fact, tgfβs increase the sparc transcription to intensify tgfβs signaling, because acidic condition promotes tgfβs transformation into their mature ligands (gordon and blobe, 2008; schier, 2009; seuntjens et al., 2009; wu and hill, 2009; beyer et al., 2013; pauklin and vallier, 2015; vallier, 2016). the second one encompasses intracellular factors such as (a) smad6/7, (b) the shuttling system of smads (ran/gtpase export/import system), (c) proteins involved in receptor trafficking, (d) sara, (e) erb2/her2 that sequester smad2/3 away from smad4, (f) transcription factors, coactivators and corepressors and (g) mirnas (gordon and blobe, 2008; schier, 2009; seuntjens et al., 2009; wu and hill, 2009; oshimori and fuchs, 2012; beyer et al., 2013; pauklin and vallier, 2015; vallier, 2016). additionally, trim33/ectodermin alleviates tgfβs signaling through mono-ubiquitination of smad4. whereas smad4 de-ubiquitination by fam/usp9x attenuates the effect of trim33/ectodermin (schier, 2009; seuntjens et al., 2009; oshimori and fuchs, 2012; beyer et al., 2013; pauklin and vallier, 2015; vallier, 2016). during unstimulated condition, smads interaction with ran gtpase export/import system creates a highly dynamic equilibrium in which unphosphorylated smads continuously shuttle between the cytoplasm and nucleus. upon smads phosphorylation and trimer complex formation by means of smad4, they are accumulated in the nucleus. this accumulation is due to their higher import rate in comparison with monomeric unphosphorylated smads. in addition, downstream effectors of the hippo pathway e.g. yap and taz transcription factors maintain the trimer complex in the nucleus (schier, 2009; seuntjens et al., 2009; wu and hill, 2009; pan, 2010; beyer et al., 2013; pauklin and vallier, 2015; vallier, 2016). to turn off the smad signaling, protein phosphatases (e.g. ppm1a, pyruvate dehydrogenase phosphatases (pdp) and small c-terminal phosphatase (scps1, 2 & 3)) dephosphorylate rsmads, thereby trimer complexes disruption. dephosphorylated smads is then recognized by ranbp3 and exported from the nucleus (raftery and sutherland, 1999; ten dijke and hill, 2004; gordon and blobe, 2008; schier, 2009; seuntjens et al., 2009; wu and hill, 2009; pan, 2010; beyer et al., 2013; pauklin and vallier, 2015; vallier, 2016). references bartscherer k., boutros m. 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(2009). nanog in the twin fish models medaka and zebrafish: functional divergence or pleiotropy of vertebrate pluripotency gene. 265 p. international journal of aquatic biology (2013) 1(5): 233-239 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article lc50 and bioaccumulation of lead nitrate (pb(no3)2) in goldfish (carassius auratus) mahdi banaee*,1behzad nematdoust haghi, fazel zoheiri aquaculture department, natural resource faculty, behbahan khatam alanbia university of technology, behbahan, iran. article history: received 12 june 2013 accepted 3 september 2013 available online 2 5 october 2013 keywords: lead nitrate gills viscera muscle goldfish bioaccumulation abstract: lead is a metal with no known biological benefit to organisms. the present study focused on bioaccumulation of lead in various organs of gold fish (caracius auratus). fishes were exposed to lead nitrate [pb(no3)2] at a series of concentrations 0.0 mg/l (control group), 0.09, 0.15, 0.24, 0.3, 0.36 and 0.45 mg/l, which were equivalent to approximately 2, 3, 5, 6, 7 and 9% of 96 h lc 50 for 28 days. after 28 days of exposure, 10 fish per treatment were captured and anesthetized under aquatic solution of clove powder (200 mg/l). fishes were euthanized and the gill, viscera and muscle tissue were sampled and weighed. then, lead concentrations were measured in different tissues of goldfish using icp. viscera had the highest lead bioaccumulation potential, followed by the gill. the muscles were least preferred site for detecting the bioaccumulation of pb. in conclusion, although lead was found in all tissues tested, pb bioaccumulation potential is variable depending on the tissue structure. introduction major adverse effects on environmental quality, ecosystem integrity and human health have often been associated with mismanagement of chemical materials and the eliminations of hazardous substances. contamination of aquatic ecosystem with heavy metal is one of the biggest environmental concerns (agrahari, 2009). heavy metals reach to the aquatic environment from natural and anthropogenic sources and distributed in the water bodies, suspended solids and sediments during the course of their transportation (olajire and imeokparia, 2001; adeniyi et al., 2005; aderinola et al., 2009). fishes are major sources of protein. they constitute major components of most aquatic habitats acting as bio-indicator of heavy metal levels in the aquatic environment. they have been recognized as good bio-accumulators of organic and inorganic pollutants (king and jonathan, 2003). although several * corresponding author: mahdi banaee e-mail address: banaee@bkatu.ac.ir tel: +986712221230 adverse health effects of heavy metals have already been known, exposure to heavy metals is increasing in many parts of the world, particularly in developing countries, though emissions have declined in most developed countries over the last century (järup, 2010). an increase of heavy metals toxicity and its bio-accumulation in various tissues of aquatic organisms threatens the biodiversity of ecosystems and health of consumers (adeniyi et al., 2008; vinodhini and narayanan, 2008a; george et al., 2011). however, accumulation of metals in fish tissues depends on many factors including environmental factors, type of heavy metals, metal concentration, time of exposure and biological characteristics of fish (jezierska and witeska, 2006). lead is one of the non-essential and toxic heavy metals which can be found as galena (pbs), cerussite (pbco3) and anglesite (pbso4) in earth’s crusts, rocks, soil and water (jalili et al., 2002). lead may reach to the environment due to anthropogenic 234 banaee et al/ international journal of aquatic biology (2013) 1(5): 233-239 activities such as mining, coal burning, cement industry, daily usage of gasoline and gas oil, batteries and paints. since lead may enter aquatic ecosystems through discharge of industrial waste waters, such as painting, dying, battery manufaction units and oil refineries (ramesh et al., 2009). lead, like most heavy metals, find ultimately its way into surface waters influencing the health of aquatic animals such as fishes. environmental pollution with heavy metals can cause serious risk such as genetic abnormality, physiological and biochemical problem and behavioral disorder in aquatic organisms (scott et al., 2003). among the aquatics inhabiting the polluted waters, fishes are sensitive to heavy metal pollution compared to other aquatics (alinnor, 2005). lead damages various body organs including the nervous, reproductive systems and excretory systems (kidneys) causing dysfunction of blood cells (frumkin and geberding, 2007; vinodhini and narayanan, 2008b). unfortunately, lead has been recently detected in groundwater and surface water supplies in different areas of iran, arising concern about its effects (charkhabi et al., 2005; kazemi et al., 2013). as most fishes are the food source for human, bioaccumulation of lead in different tissues of these organisms may threaten the human’s health. therefore, the purpose of the present study were to investigate the pb bioaccumulation potential in the viscera, gills and muscles of goldfish exposed to sublethal concentrations of lead nitrate. materials and methods fish: goldfish (mean weight 16.66 ± 3.09 g) were purchased from local sellers and transported to the laboratory. fish were acclimated to the laboratory conditions for 2 weeks before the initiation of experiments. fishes were randomly allocated to 12 closed 200 l recirculating tanks supplied with oxygenated water with constant dissolved oxygen (6.5 ± 0.5 mg/l), temperature (22 ± 2 ºc), ph (7.4 ± 0.2), water hardness (150 ± 5 mg/l caco3) and photoperiod (16l:8d). during acclimation and at all experiments, fishes were fed with commercial aquarium fish diet at the manufacturer’s recommended rate (2% of their body weight twice a day). fishes were starved for 1 day before the start of the experiments, and 24 h before sacrifice. acute toxicity experiments: one hundred and eighty goldfish, carassius auratus, were used in acute toxicity tests. the acute toxicity test was conducted following the organization for economic cooperation and development (oecd) guideline no. 203 under static-renewal test conditions. the lead solutions used in the experiment were prepared using dissolving lead nitrate [pb(no3)2] (merck co., germany) with 99.5% purity in distilled water at nominal concentrations of 0 (control), 2.5, 5, 10, 15, 20 mg/l. ten acclimatized specimens were randomly selected and allocated to each glass aquarium (100 l) with three replicates with 30 individuals being tested at each lead concentration. during the 96 h acute toxicity experiment, water in each glass aquarium was aerated and had the same conditions of the acclimation period (dissolved oxygen 6.0 ± 0.5 mg/l, temperature 22 ± 2 ºc, ph 7.4 ± 0.2, water hardness 225 ± 5 mg/l caco3). for the static-renewal tests, the specimens were exposed to the metal 96 h with replacement of the test solution every 24 h (all stock solutions were made immediately prior to use). the water was changed daily to reduce the build-up of metabolic wastes to keep concentrations of pb(no3)2 close to the nominal levels. fish mortality was recorded at 0, 24, 48, 72, and 96 h after exposure to pb(no3)2. fishes were considered dead when the gill opercula and body movement ceased. dead fish were immediately removed using a dip net. lc50 values were calculated using the probit analysis (banaee et al., 2011). sub-lethal toxicity experiments: 315 gold fish were randomly allocated to 21 aquaria (100 l), with 7 treatments and three replicates and sub-lethal toxicity tests were performed over 28 days. every tank containing 15 fishes were exposed to test solutions with the following concentrations of lead nitrate [pb(no3)2]: 0.0 (control), 0.09, 0.15, 0.24, 235 banaee et al/ international journal of aquatic biology (2013) 1(5): 233-239 0.3, 0.36 and 0.45 mg/l, respectively. sub-lethal concentrations were selected according the previous acute toxicity test. the water was changed daily to reduce the build-up of metabolic wastes and keep concentrations of lead nitrate near the nominal levels. ten fishes per each exposure concentration were captured and anesthetized with the extract of clove powder (1:5000) 28 days after the exposure. lead levels analysis in different tissues: after dissecting the specimens, gill, viscera and muscle were separated. the samples were weighed and were placed in an electrical furnace to obtain the ashes for 8 hours at 550 °c. then 1 g of ash was mixed with hno3 and hcl (1:1). for isolation of ash particle the solution was filtered, mixed with deionized water and diluted to 25 ml. lead concentration was measured using icp-oesperkin elmer (7300-bd). after calibration with standard soluble at 0.5, 1, 2, 5, 8 and 10 mg/lit, calibration diagram of lead was drawn and metal level in these prepared soluble measured. samples were filtered using whatman filter (0.22 µm) and at last pb concentration in each sample detected with icp instrument. pb levels were measured in triplicate and measurements were repeated three times. statistical analysis: data are presented as mean ± sd. all the data were tested for normality (kolmogorov-smirnov test) and analyzed using a one way analysis of variance (anova). the linear regression was used to examine the relationship between the concentration of lead in water and its accumulation in various tissues. results acute toxicity of pb(no3)2 was determined in goldfish after 24, 48, 72 and 96 hours of toxicity. lc50 values significantly decreased over exposure time from 16.93 ± 1.29 mg/l at 24 h to 5.02 ± 0.54 mg/l at 96 h. results of this study have been shown in table 1. according to figure 1, the toxicity of lead exposure time to pb(no3)2 lc50 (mg/l) range 24 h 16.93±1.29 14.74-20.20 48 h 12.47±0.94 10.72-14.57 72 h 7.92±0.68 6.57-9.32 96 h 5.02±0.54 3.93-6.14 table 1. median lethal concentrations of pb(no3)2 to goldfish. concentration of pb(no3)2 in water (mg/l) bio-concentration of pb(no3)2 in different organs (mg/g) muscle visceral gills 0 0.000±0.000 0.000±0.000 0.000±0.000 0.09 0.067±0.005 0.437±0.022 0.240±0.012 0.15 0.060±0.002 1.130±0.057 0.276±0.014 0.24 0.075±0.004 1.732±0.087 0.318±0.016 0.3 0.108±0.005 2.040±0.102 0.378±0.019 0.36 0.226±0.011 3.645±0.182 0.546±0.027 0.45 0.222±0.021 3.636±0.182 0.706±0.035 table 2. lead bio-concentrations of different organs at exposure times of 28 days. figure 1. changes of mortality of goldfish exposed to various concentrations of lead at different exposure times. 235 236 banaee et al/ international journal of aquatic biology (2013) 1(5): 233-239 nitrate on goldfish and mortality of fishes increased with increase of concentration and exposure time. sub-lethal concentrations of pb(no3)2 were equivalent to approximately 2, 3, 5, 6, 7 and 9% of 96 h lc50 value (0.09, 0.15, 0.24, 0.30, 0.36, and 0.45 mg/l of aqueous solution of lead nitrate, respectively) for 28 day toxicity testing. therefore, no mortality and no significant changes was found in clinical indicators and feeding behavior in treated fishes during the experimental periods. the bioaccumulation of lead in different organs of goldfish after exposure to contaminated water with different concentrations of pb(no3)2 are presented in table 2. bio-concentration of pb in the viscera and the gill were higher than that of the muscle in all of treatments. linear regression between concentrations of lead in water and its bio-accumulation in various tissues was graphically illustrated in figure 2. discussion pollutants enter the fish body through a number of routes: directly through the digestive tract due to consumption of contaminated water and food or nondietary routes across permeable membranes such as the gill or skin (burger et al., 2002). on absorption, pollutants are transported in the blood stream to either a storage place (i.e bone) or to the liver for transformation and/or storage (nussey et al., 2000) or excreted by the kidney, the gill or stored in an extra hepatic tissues such as fat (dimari et al., 2008). lead, as one of heavy metals, is toxic for wildlife, experimental animals and humans (wirth and mijal, 2010). results of our research showed the direct effect of pollution levels on bio-accumulation of lead in goldfish tissues. the bioaccumulation of pb in visceral tissue of exposed fish obviously was higher than the other tissues. it seems reasonable because the viscera organs such as the liver and digestive organs are important organs for the uptake of heavy metals from diets. ganbi (2010) reported that pb bioaccumulation potential in the visceral tissues is higher than the muscle. vinodhini and narayanan (2008a, b) found that the kidney, the liver and the spleen accumulate more metals compared with muscle tissues of common carp in metal polluted media. this result agrees with those of other authors (adeniyi et al., 2008; sharma et al., 2011). there are numerous proteins (e.g. metallothionein) in visceral tissue which may be bound to heavy metals (frumkin and geberding, 2007). the high amount adipose tissue surrounding visceral tissues may be effective in the bioaccumulation of pb. in fish, gills are considered to be the dominant site for contaminant uptake because of their anatomical and/or physiological properties that maximize absorption efficiency (takarina et al., 2012). in our research gill accumulation was lower than the viscera and higher than the muscle. there are records of similar bio-concentration of lead was noted by łuszczek-trojnar et al. (2013). muscles are the edible part of fish; therefore bioaccumulation of heavy metals in muscle could be a threat to consumers. although ozturk et al. (2009) believed that muscle is not an active tissue in accumulation of heavy metals, our results confirmed accumulation of lead in muscles. studies conducted by other researchers on lead bioaccumulation in the other species (falusi and olanipekun, 2007; korai et al., 2008; ozturk et al., 2009; ganbi, 2010; victor et al., 2012) confirmed our results. figure 2. linear regression between of water concentrations and tissue levels of pb after 28 days of exposure. 237 banaee et al/ international journal of aquatic biology (2013) 1(5): 233-239 although different bio-accumulation abilities observed in different tissues, the levels of pb in of goldfish tissues indicated its high bioavailability. the correlation coefficients (r2) for three types of tissues, including gills, viscera and muscle were 0.9330, 0.9588 and 0.8716 for 28 days of exposure. the linear regression between the concentration of lead in water and its accumulation in goldfish's tissue may indicate that various tissues could serve as a useful tool for the evaluation of heavy metal exposure. generally, positive correlation exists between pb concentrations of the tissues and exposure levels. physiological differences in the function and structure of various tissues may be influenced on the bioaccumulation of metals. yap et al. 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(2004). assessment of different soft tissues of the green lipped mussel perna viridis (linnaeus) as biomonitoring agents of pb: filed and laboratory studies. water, air, and soil pollution, 153: 253268. 239 © 2022 iranian society of ichthyology short communication effect of parity and calf gender on milk yield and composition of buffalo, bubalus bubalis inhabiting southern iraqi wetlands muayad abdulwahid jaber al-fayad*,1 hussein majeed shareef department of animal production, faculty of agriculture and marshlands, university of thi-qar, nasiriyah, iraq. s * 75 : 76 references . density % ash % lactose % protein % solid nonfat % fat % total solid % daily milk production/k birth seq. 31.86±0.27a 0.65±0.006a 5.14±0.045a 3.38±0.029a 9.2±0.076a 5.96±0.18a 15.15±0.198a 3.74±0.128d 1 32±0.36a 0.64±0.01a 4.9±0.21a 3.26±0.065a 8.8±0.17a 5.21±0.082b 14.02±0.237b 5±0.26c 2 30.6±0.19a 0.61±0.039a 4.75±0.034a 3.14±0.023a 8.49±0.061a 5.09±0.095b 13.61±0.138bc 4.7±0.73d 3 30.73±0.32a 0.64±0.0086a 4.69±0.047a 4.69±0.042a 8.37±0.083a 4.84±0.053b 13.23±0.122c 11.14±0.165a 4 and more . density % ash % lactose % protein % solid nonfat % fat % total solid % daily milk production/k gender 30.83±0.18b 0.63±0.0046a a4.83±0.032 a18±0.021. a61±0.005. a17±0.073. b13.79±0.16 a8.74±0.32 male 31.51±0.2a 0.64±0.0054 a4.9±0.043 a3.24±0.028 a8.79±0.077 a5.42±0.077 a14.22±0.1 b6.4±0.26 female . 77 : the buffalo is one of the ancient and indigenous animals in iraq. it was domesticated in the middle of the third millennium bc in 2500 bc, approximately in the sumerian city of ur in mesopotamia (borghes, 2008). the iraqi buffalo spreads throughout ir... int. j. aquat. biol. (2018) 6(2): 88-94 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article the effects of zinc-enriched saccharomyces cerevisiae on the growth and mineral composition of marine rotifer, brachionus plicatilis kaveh nematzadeh1, nasrollah ahmadifard*1, naser samadi2, naser agh3, sirwe ghaderpour1 1department of fisheries, faculty of natural resources, urmia university, p.o. box: 46414-356, urmia, iran. 2department of analytical and physics and applide chemistry, faculty of chemistry, urmia university, urmia, iran. 3department of artemia, artemia and aquaculture institute, urmia university, urmia, iran. article history: received 18 january 2018 accepted 25 april 2018 available online 2 5 april 2018 keywords: saccharomyces cerevisiae zinc enrichment mineral composition abstract: rotifers are important zooplankton in commercial finfish hatcheries. however, due to the limited variety of food available, zinc content of cultured rotifers in artificial environments may not meet the requirements of fish larval. it has been reported that direct addition of soluble zinc to culture media was not effective on the zinc content of rotifer. thus, in this study, the effect of zinc-enriched saccharomyces cerevisiae was investigated on the growth and mineral composition of rotifer, brachionus plicatilis. four different food treatments, including (1) yeast without enrichment (control), (2) yeast containing 21.23 mg g-1 of zinc, (3) yeast containing 56.25 mg g-1 of zinc, and (4) yeast containing 132.93 mg g-1 of zinc, were used to produce rotifer for a period of 10 days. afterwards, specific growth rate (sgr), the total number of rotifers, total eggs attached to rotifers, and the total number of eggs were measured. finally, the mineral composition of rotifer in different treatments was analyzed. the findings revealed that yeast enriched with 56.25 mg g-1 of zinc significantly improved the growth of rotifers. the maximum number of rotifers (274 ind ml-1), total eggs attached to rotifers (29.3 number ml-1), and the total number of eggs (36 number ml-1) were found in the third treatment. the highest zinc content was observed in the fourth treatment (about 822.5 μg g-1 of rotifers). the maximum values of fe (13.84 μg g-1 of rotifers) and mn (15.22 μg g-1 of rotifer) were related to the treatment 4 and control, respectively. however, the amount of cu did not significantly differ among the treatments. in conclusion, this study found that zinc-enriched yeast improved the growth, reproduction, and body composition of b. plicatilis. introduction zooplankton such as rotifers are important in the first feeding of fresh and marine fish larvae (isik et al., 1999). euryhaline rotifer, brachionus plicatilis is a suitable feed item for the larvae of marine fish and shellfish in terms of their sizes (150-350 µm) and high reproductive rate (hamre et al., 2008; lubzens, 1987; kennari et al., 2008). in marine fish hatcheries, rotifers should be considered not only regarding their density but also their nutritional value, especially mineral composition (støttrup and mcevoy, 2008). the nutritional value of live feeds affects the growth and survival of fish larvae (watanabe et al., 1983). the minerals are responsible for the development of nervous system, growth and survival of aquatic *corresponding author: nasrollah ahmadifard doi: https://doi.org/10.22034/ijab.v6i2.443 e-mail address: n.ahmadifard@urmia.ac.ir larvae, bone formation, maintenance and adjustment of the colloidal system and acid-base balance of aquatic organisms (matsumoto et al., 2009). moreover, minerals play an important role in hormones and enzymes system (watanabe et al., 1978b). apines-amar et al. (2004) indicated that zinc is an important mineral in fish nutrition and plays a vital role in bone health. although a small amount of zinc in the body of aquatic organisms is present, it is known as an essential element in the fish feed and as a cofactor for over 300 enzymes (watanabe et al., 1978b). it has been demonstrated that the levels of zinc in rotifers were lower than levels found in copepods by five folds (hamre et al., 2008). the cultured rotifers in 89 int. j. aquat. biol. (2018) 6(2): 88-94 artificial environments have about 80 μg g-1 of zinc (matsumoto et al., 2009), while its value in a planktonic copepods have about 700 μg g-1. the zinc requirement of marine fish is about 15 to 40 mg kg-1 (dry mater), and the amount of zinc in cultured rotifers does not meet the needs of marine fish larvae (matsumoto et al., 2009). the nutrients required by larvae of fish can be increased in rotifers through direct (short-term enriching) and indirect (through rotifer diets) methods. in the direct one, enrichment materials (including minerals) can be added to culture media of rotifers, but it has been reported that the direct addition of soluble materials such as zinc to the culture media was not effective on zinc content of rotifer (matsumoto et al., 2009). in the indirect method, the minerals are stabilized in rotifer body through the diet such as algae or yeast (dhert et al., 2001). since many years ago, different kinds of yeasts have been used in rotifers nutrition along with algae. yeast has been used as an intermediary to deliver nutrients to rotifers. the use of omega-3-rich oil along with baker's yeast makes rotifers rich in omega-3 fatty acids (penglase et al., 2011). rotifers fed on yeast are able to synthesize unsaturated fatty acids and to elongate the chain (lubzens et al., 1985). moreover, yeast enrichment with zinc can be a practical method for increasing the zinc content of rotifer. therefore, given the need for sufficient levels of minerals in live foods such as rotifers, in the present study, the effects of zincenriched baker's yeast saccharomyces cerevisiae were investigated on the growth and the mineral content of rotifers. materials and methods zinc-enriched yeast preparation: zinc sulfate (znso4.7h2o) was purchased from sigma-aldrich, usa. baker's yeast was provided by artemia and aquaculture research institute at urmia university, urmia, iran. the growth media (yepd) contained yeast extract, peptone, and glucose, which was obtained from the jahan kimia company in urmia. in this study, the method described by wang et al. (2012) was adopted to enrich yeast in non-growth phase with zinc sulfate. briefly, 1 g of s. cerevisiae was initially cultured in 200 ml of yepd medium at 27°c, ph=5.8 and 160 rpm for 24 hrs on a shaker incubator (n-biotech, nb-205v, south korea). then, zinc sulfate was added into the medium at the concentrations of 0.5, 1 and 1.5 g per 200 ml of medium. the incubation of yeast cell was performed for 24 hrs under the same conditions. next, the yeast cells were centrifuged (3000 rpm for 1 min) and washed with normal saline to remove the additional zinc sulfate. following enrichment, the amount of zinc in the yeast was found to be 21.23, 56.25 and 132.93 mg g-1 of the yeast. finally, the zinc-enriched yeast cells were used for rotifer feeding. treatments and test methods: brachionus plicatilis was obtained from the iran shrimp research center (bushehr, iran) and up-scaled to mass density in a wetlab using nannochloropsis oculta. the rotifers receiving yeast in four groups, including (1) yeast without enrichment (control group), (2) yeast containing 21.23 mg g-1 of zinc, (3) yeast containing 56.25 mg g-1 of zinc, and (4) yeast containing 132.93 mg g-1 of zinc. each group had three replicates, and all groups were cultured in 2l flask for a period of 10 days. after disinfection of the flasks, the rotifers with a density of 50 ind ml-1 were added. they were fed with 1×106 cell ml-1 of algae and a 0.5 g of yeast per million rotifers on a daily basis. water quality parameters, including salinity (30±1 ppt), ph (7.6-8.3) and temperature (24±1°c) were regularly checked. gentle and continuous aeration was performed to meet the needs of rotifers. the maximum number of rotifers, total eggs attached to rotifers, and total egg numbers were measured on a daily basis. through the following equation, specific growth rate (sgr) was calculated based on krebs (1995). sgr=(lnnt-lnn0)/t, where n0 and nt are respectively for the initial and final population of rotifer, and t stands for experiment period (days). sgr value was calculated in the exponential phase of population. at the end of the feeding period, the biometric factors in each treatment were measured using a microscope equipped with 90 nematzadeh et al./ the effect of zinc-enriched yeast on rotifer brachionus plicatilis micrometer lenses. analysis of minerals: after centrifuging the yeast cells and rotifers (2000 rpm for 20 min) and discarding the supernatant, the precipitant was used for the analysis of minerals. through the use of mls-1200 mega microwave, the samples were digested with nitric acid under cold water for 30 minutes. using an atomic absorption (nov aa 400, analytic jena, germany), the mineral concentrations (zn, mn, cu and fe) of digested sample were measured (lowry and lopez, 1946). statistical analysis: spss statistical software, version 21 was used to run the analyses. using levene's and shapiro-wilk test, the data were analyzed for homogeneity of variances and normality, respectively (p<0.05). one way anova was used for the analysis of groups and was followed by duncan honest significant difference test. differences among the means were considered significant at p<0.05. the data are displayed with mean±sd. results after the eighth day till the end of the culture period, the total numbers of rotifers in the treatment groups were higher than that in the control group (table 1). however, no significant differences were observed among the groups receiving zinc-enriched yeast. at the 10th day, the maximum total number of rotifers was related to those fed with the yeast containing 56.25 mg g-1 zinc. the findings revealed that the enriched-yeast improved sgr of the rotifer compared to the control group (p<0.05). no significant differences were found in sgr of the groups fed with zinc-enriched yeast. however, the rotifers fed with enriched-yeast containing 56.25 mg g-1 zinc showed higher sgr than other treatments (table 2). the numbers of total eggs in rotifers fed with the enriched-yeast for 10 days are given in table 3. the maximum number was found in the group containing 56.25 mg zinc. in addition, the numbers of these factors in the rotifers fed with the enriched yeast were higher than the control group. based on the findings, the maximum total egg numbers attached to the rotifers were found in the treatment of 56.25 mg zinc which was significantly different compared to the other groups at the 8th day (p<0.05) (table 4). the maximum zinc content (about 822.5 μg g-1 table 1. mean numbers of rotifers fed with zinc-enriched yeast with different contents (21.23, 56.25 and 132.93 mg g-1 of zinc in yeast) for 10 days (mean±sd, n=3). days* yeast zinc content (mg g-1) 21.23 56.25 132.93 un-enriched yeast 1 72.6±3.51 a 72.6±3.79 a 75.0±3.61 a 75.0±1.00a 2 87.0±4.36 a 78.6±8.33 b 89.3±4.04 a 78.6±4.04 b 3 89.6±3.06 ab 76.0±8.89 b 92.0±4.58 a 77.0±4.58 b 4 99.3±5.51 a 83.3±6.81 a 97.3±7.02 a 90.3±11.24 a 5 110±3.79 a 107 ±6.51 a 99±10.5 a 104±4.51 a 6 127±7.0 a 122±10.8 a 91.6±12.1 a 121±11.3 a 7 165±5.03 a 173±4.00 a 153±6.81 a 153±6.56 a 8 189±3.06 ab 197±11.2 a 181±8.08ab 172±3.61 b 9 214±9.87 ab 223±16.0 a 201±2.65 ab 190±2.52 b 10 257±10.9 ab 274±33.2 a 271±10.5 a 216±9.53 b *data was analyzed in each day separately. different letters in each row have a significant difference (p<0.05). table 2. the sgr of rotifers fed with zinc-enriched yeast in different contents (21.23, 56.25 and 132.93 mg g-1 of zinc in yeast) after 10 days (mean±sd, n=3). treatments sgr of zinc 1-mg gyeast containing 21.23 a±0.0080.1265 of zinc 1-mg gyeast containing 56.25 a0.1317±0.003 of zinc 1-mg gyeast containing 132.93 a±0.0120.1295 un-enriched yeast b0.1071±0.013 different letters in each row have a significant difference (p<0.05). 91 int. j. aquat. biol. (2018) 6(2): 88-94 rotifer) was obtained at the highest zinc supplementation level. the maximum fe content (13.84 μg g-1 of rotifer) and mn (15.22 μg g-1 of rotifer) were obtained in treatment 4 and control, respectively, but cu content did not differ among the treatments (fig. 1). discussion minerals such as zinc are present in aquatic environments and in addition to providing the needs of living organisms, they can be accumulated in aquatic organisms. in artificial environments (namely marine fish hatcheries), the growth of rotifers, due to the limited variety of food available, may result in inadequate concentrations of minerals, especially zinc. access to trace minerals for fish larvae is vital since they have important roles in immunity enhancer, stress releaser, cellular metabolism, formation of skeletal structures, disease resistance and other physiological functions, as well as, serve as cofactors and/or activators of a variety of enzymes (failla, 2003; satoh, 2003; antony jesu prabhu et al., 2016). due to the solubility of zinc, its enrichment is not the same as fat-soluble materials. therefore, in this study, initially, the baker's yeast was exposed to various concentrations of zinc and in this way, the amount of zinc increased in yeast after 24 hours. the results of the first step demonstrated that it is possible to enrich baker's yeast. the enriched yeast was used as a feed to increase the zinc content of rotifers. this method of enrichment has already been used to increase zinc levels in chlorella by matsumoto et al. (2009) and selenium enriched yeast in rotifer culture by penglase et al. (2011). these two studies found that indirect enrichment of rotifers with minerals had positive effects on the growth rate of rotifers than table 3. total number of eggs in rotifers fed with zinc-enriched yeast in different contents (21.23, 56.25 and 132.93 mg g-1 of zinc in yeast) for 10 days (mean±sd, n=3). days* yeast zinc contents (mg g-1) 21.23 56.25 132.93 un-enriched yeast 1 12.6±2.89a 13.0±6.08 a 12.3±3.21 a 13.3±6.11 a 2 11.6±4.73 a 12.3±3.06 a 17±6.08 a 7.67±2.52 b 3 17.0±7.00 a 14.0±6.25 a 11.3±3.51 a 17.6±4.62 a 4 10.6±2.08 a 12.6±3.21 a 16.6±4.04 a 13.0±3.46 a 5 20.6±2.08 a 19.33±10.41 a 23.0±6.08 a 13.0±7.21 ab 6 19.0±5.20 a 24.3±4.16 a 22.0±4.36 a 14.6±3.21 a 7 27.3±1.53 a 30.6±11.72 a 20.6±5.69 a 18.6±11.06ab 8 34.6±3.21 ab 36.6±3.21 a 18.3±8.08 cb 16.3±5.69 c 9 31.0±8.19 a 31.0±11.14 a 34.6±4.16 a 23.6±7.51 b 10 31.0±6.56 a 35.0±5.29 a 30.3±1.15 a 28±12.70 a *data was analyzed in each day separately. different letters in each row have a significant difference (p<0.05). table 4. total number of attached eggs in rotifers fed with zinc-enriched yeast in different contents (21.23, 56.25 and 132.93 mg g-1 of zinc in yeast) (mean±sd, n=3). days* yeast zinc contents (mg g-1) 21.23 56.25 132.93 un-enriched yeast 1 7.0±1.00 a 10.0±6.56 a 6.3±1.53 a 10.6±3.79 a 2 5.3±3.06 b 13.0±6.25 a 10.6±2.52 a 14.6±4.93 a 3 15.3±7.09 a 12.0±6.00 a 7.3±5.03 b 13.6±4.51 a 4 9.3±1.53 a 10.0±5.29 a 11.6±5.51 a 9.0±2.65 a 5 16.0±4.35 a 14.3±9.50 a 14.6±6.66 a 8.6±6.51 b 6 12.6±3.21 a 17.3±3.06 a 15.3±6.66 a 9.3±0.58 a 7 18.6±1.53 a 25.0±10.82 a 14.6±7.02 a 13.6±7.02 a 8 21.0±2.65 ab 24.6±4.73 a 9.6±7.64b 10.6±5.69 b 9 20.3±8.50 a 22±1.45 a 21.6±5.51 a 16.3±7.64 a 10 21.6±9.07 a 29.3±5.69 a 23.3±1.53 a 22.3±11.8 a *data was analyzed in each day separately. different letters in each row have a significant difference (p<0.05). 92 nematzadeh et al./ the effect of zinc-enriched yeast on rotifer brachionus plicatilis direct methods. thus, minerals bound to an ingestible particle can be absorbed very efficiently (penglase et al., 2011). the findings of the present study corroborate this conclusion. also, nguyen et al. (2008) reported that the direct addition of zinc had no effect on the zinc content of rotifers, but when enriched chlorella was used, significant differences were found among the experimental groups. the effectiveness of using insoluble zinc as zinc-yeast in aquatic organisms is very important because the soluble zinc is rapidly released into the environment (li and robinson, 1996). in this study, zinc-yeast had a positive effect on the population growth of rotifer, b. plicatilis, in the first three groups, whereas it had an adverse effect on the fourth group. although positive effects of zincenriched chlorella on the growth rate of the rotifer b. plicatilis have already been reported in the literature (matsumoto et al., 2009), they found no change in the lorica length of zinc-enriched rotifers. zinc raises enzyme activities and immune system function and affects the composition of rotifers (watanabe et al., 1978a). minerals in natural environments such as aquatic ones have a significant effect on the growth and composition of all microorganisms (nguyen et al., 2008 ; fujita, 1972). in our research, the maximum density of rotifers was obtained in the day 10th in the 56.25 mg g-1 of zinc group and sgr and reproductive factors were improved in the rotifers fed with this amount of zincyeast. this finding is consistent with those reported by penglase et al. (2011). they also found that the growth of rotifers is affected by minerals such as zinc, selenium, and iodine. sarma and tamborenea (1991) reported that sgr of b. plicatilis is within the range of 0.1-2, but most species show sgr of less than 0.5 of the days. our findings revealed that rotifers fed with enriched-yeast possessed greater sgr compared to that of the control group. improvements in growth could be attributed to better metabolism, enzyme activities, and immune system resistance (nordgreen et al., 2013). in particular, the sgr of rotifers depends on the amount of food mineral (watanabe et al., 1978a; hamre-srivastava et al., 2008). figure 1. the amounts of zinc, fe, cu and mn of the rotifers fed with zinc-enriched yeast. different letters above the bars mean significant difference. data are displayed in mean±sd, n=3. 93 int. j. aquat. biol. (2018) 6(2): 88-94 in the present study, the highest amount of zinc content (822.5 μg g-1 of zinc in rotifer) was observed in the group fed with enriched yeast containing 132.93 mg g-1 zinc. in line with the present results, the zinc content of daphnia has been investigated in different concentrations of zinc in media culture (muyssen et al., 2002). in that study, a high level of zinc increased the daphnia zinc content, while its low amount had no effect on daphnia. the zinc content of enriched rotifers fulfils the larvae requirements of marine fish [15-40 mg kg-1 of the food (nrc, 1993)]. watanabe et al. (1978a) reported that proximate composition of rotifers varied in different food sources such as yeast and algae, but their mineral composition remained constant. moreover, they reported that zinc content of rotifers without enrichment was approximately 80 μg g−1 dry weight. on the contrary, others have reported positive effects of minerals through the enrichment of yeast or algae (matsumoto et al., 2009; fujita, 1972; takahashi et al., 2005). the amounts of fe in treatments corresponded to the rises in zinc level, but the trend of mn content was in reverse to the fe and zinc contents. this finding is in line with that reported by matsumoto et al. (2009). they found that mn and cu levels decreased with an increase in zinc levels of rotifers. these effects of minerals in rotifers may indicate an antagonistic interaction to keep mineral homeostasis. in addition, presumably, the mn content of rotifers is affected to a great extent by a host of nutrients or materials. additional studies are warranted to elucidate on this finding. likewise, nguyen et al. (2008) found an antagonistic effect of zinc and mn on artemia. they reported that, in enriched artemia nauplii with both zinc and mn, the level of mn reduced, but in the enrichment solely with mn, the mn content of artemia nauplii was more than previous one. conclusion this study found that yeast with 56.25 mg g-1 of zinc had an improved effect on the growth and reproductive factors of rotifers. on the other hand, the highest amount of zinc content was obtained in rotifers fed with yeast containing 132.93 mg zinc. thus, the employment of this indirect method for the enrichment of rotifers was found to have no negative effect on the fe and cu contents of rotifers. on the other hand, the amount of mn decreased compared to the one found in the control group. references abasali h., mohamad s. 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(2018) 6(2): 88-94 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی brachionus plicatilis شور آب روتیفر معدنی مواد ترکیب و رشد بر روی با شده غنی نانوایی مخمر تاثیر 1روه قادرپور، س3، ناصر آق2ناصر صمدی ،1دفر، نصراله احمدی1کاوه نعمت زاده .ایران ،ارومیه ،دانشگاه ارومیه طبیعی، منابعدانشکده شیالت، گروه1 .ایران ،ارومیه ،دانشگاه ارومیه ،دانشکده شیمی ،شیمی تجزیه، فیزیک و کاربردی گروه2 .ایران ،ارومیه ،دانشگاه ارومیه ،پروریپژوهشکده آرتمیا وآبزی ،آرتمیا گروه3 چکیده: دلیل باشند. مقادیر روی موجود در روتیفرهای پرروشی بههای تجاری ماهیان میمهم در تغذیه الرو ماهیان در تفریخگاه روتیفرها زئوپالنکتون ا هکند. گزارش شده که افزودن مستقیم روی به محیط کشت روتیفر تاثیری بر میزان روی آننیاز الرو ماهیان را تامین نمیمحدویت مواد غذایی مورد بررسی brachionus plicatilisر تحقیق حاضر تاثیر مخمر غنی شده با روی بر رشد و ترکیب مواد معدنی روتیفر آب شور ندارد، بنابراین د ، میلی گرم روی در گرم مخمر 23/21( مخمر حاوی 2، )( مخمر بدون غنی سازی )گروه شاهد(1)تیمار غذایی شامل 4قرار گرفت. برای این منظور های ر و هر کدام با سه تکرار در کشتگرم روی در گرم مخممیلی 93/123( مخمر حاوی 4و )میلی گرم روی در گرم مخمر 25/56( مخمر حاوی 3) های چسبیده به ها، تعداد تخماستفاده شدند. در پایان آزمایش در هر کدام از تیمارها نرخ رشد ویژه، تعداد کل روتیفر، تعداد تخم 10مدت روتیفر به ه دمورد ارزیابی قرار گرفت. همچنین مقادیر مواد معدنی روتیفرها با استفاده از جذب اتمی مورد سنجش قرار گرفت. نتایج نشان داد که استفاروتیفر دسبب بهبود شاخص های رشد و تولید مثل گردید. حداکثر تعداد روتیفر، تعدا یدارطور معنیگرم روی در گرم مخمر بهمیلی 25/56از مخمر حاوی لیتر( )تعداد در میلی 36لیتر( و )تعداد در میلی 3/29لیتر(، )روتیفر در میلی 274ها به ترتیب های چبسیده به روتیفر و تعداد کل تخمکل تخم ر مقدار آهن میکروگرم بر گرم روتیفر بود. حداکث 822 به مقدار 4دست آمد. براساس آنالیز ترکیب مواد معدنی بیشترین مقدار روی در تیمار هب و شاهد حاصل شد اما مقدار مس در بین 4ترتیب در تیمارهای میکروگرم بر گرم روتیفر( به 22/15میکروگرم بر گرم روتیفر( و منگنز ) 84/13) ع روی در بدن توان گفت که مخمر غنی شده با روی باعث بهبود فاکتورهای رشد و تجمداری را نشان نداد. در جمع بندی میتیمارها تفاوت معنی روتیفر می گردد. .معدنی مواد ترکیب شده، غنی مخمر سولفات، روی نانوایی، مخمر :کلمات کلیدی int. j. aquat. biol. (2021) 10(4): 344-348 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article biochemical and molecular identification of the isolated bacteria from the beach soil and saline water of sawa lake, iraq alyaa adnan makki1, sofia jabar jasim2, ahmed ayad al nuaimy2, ansam saad al-khafaji*21 1agricultural extension and training office, al-muthana, iraq. 2department of desert studies center and sawa lake, al-muthanna university, al-muthana, iraq. article history: received 7 june 2022 accepted 15 july 2022 available online 2 5 august 2022 keywords: bacteria, soil, water, sawa lake. abstract: this work was conducted to study the biochemical characteristics of the bacterial species isolated from the beach soil, and water of sawa lake using molecular and morphological markers. samples were collected from the water, and surrounding beach area. the results showed the presence of isolates, including proteus mirabilis, providencia vermicola, alcaligenes aquatilis, raoultella planticola, enterobacter cloacae, klebsiella pneumonia, and p. rettgeri. the molecular diagnosis confirmed those of biochemical traditional markers using 16s gene primers. introduction natural lakes have different origins; however, those artificial lakes are created by dams on rivers or other water systems (abu samour and al-khatib, 1999). sawa lake is located southwest of samawah city, with a surface area of about 3-5 km and a length of 10 km, a depth of 3-5.5 m surrounded by lime, and a salinity of up to 35 g/l as tds (jamil, 1977). it has no inlet or outlet system, but it gets water from the euphrates through joint cracks and fissures transporting water to aquifers beneath it. its water level fluctuates during dry and wet seasons but does not dry up. the presence of their special microorganisms characterizes most lakes. some bacteria grow optimally on media containing 3-15% salt and are widely distributed in salt lakes. these bacteria receive great attention because of their potential for producing of solutes and degrading enzymes (ventosa and oren, 1998). the salt-tolerant bacteria form a heterogeneous physiological group that includes gram-positive or gram-negative bacteria (arahal and ventosa, 2002). they also can be classified based on their need for sodium chloride. bacteria possess several characteristics that helped *correspondence: ansam saad al-khafaji doi: https://doi.org/10.22034/ijab.v10i4.1719 e-mail: ansam.saad@mu.edu.iq them to exist in saline media, such as enzymes that work in saturated salts, the purple membrane that allows light growth, and gas vesicles that help their buoyancy, and high osmosis in saline conditions can be harmful. in cells, the water goes out to the external medium to achieve an osmotic balance to prevent cellular water loss (galinski, 1993). some molecular characteristics of the different bacteria present in saline environments have an effective role in helping bacteria to overcome such a problem. therefore, this work aimed to study the biochemical characteristics of such bacterial species isolated from the soil, and water of sawa lake using molecular and morphological markers. materials and methods a total of 50 samples were collected from the water and soil of sawa lake from randomly selected sites. the samples were diluted using chrom agar medium. the petri dishes were incubated at 28℃ for 48 hours (saravanan et al., 2004). the colonies were purified and the streaking method was used for culturing nutrient agar under sterilization conditions. the petri dishes were incubated for 24-48 hours until 345 int. j. aquat. biol. (2022) 10(4): 344-348 the appearance of single colonies. biochemical diagnosis of bacterial isolates: the isolates were diagnosed based on morphological characteristics (baron and finegold, 1990; collee et al., 1996), and biochemical tests (cruickshank et al., 1975). the tests include gram stain, motility, voges proskauer, starch hydrolysis, nitrate reduction, indol, oxidase, catalase, urease, methyl red, gelatin hydrolysis, citrate vitalization, and h2s. molecular diagnosis of bacterial isolates: bacterial isolates were diagnosed using pcr technique. the dna of the seven isolates was extracted using an extraction kit for gram-negative and gram-positive bacteria (cat. no. d6005, usa; zymo research company) according to its protocol. dna concentration was measured using a spectrophotometer with a wavelength of 260 nm, and calculated using the equation of dna concentration (μg/ml) = light absorption at a wavelength of 260 nm × 50 × dilution factor. to determine the purity of dna, the following equation was applied (william et al., 1997): dna purity = dna purity of absorption at a wavelength of 260 nm / absorption at a wavelength of 280 nm. polymerase chain reaction to double 16s rrna gene was carried out under aseptic conditions for isolates using the maxime pcr premix (i-taq, cat. no. 25026) kit (intron, south korea) using the nonspecialized primers of 16s gene including. rrna. 27f: 5'-aga gtt tga tcc tgg ctc ag-3' and 1492r: 5'ggt tac ctt gtt acg act t-3' (1492) (islam and sar, 2011). the pcr steps and conditions are shown in table 1. the pcr products were examined using 1% agarose gel for one hour until the blue-colored loading solution reached approximately 2 cm before the end of the gel. then the gel containing the pcr product was examined using uv light to determine the product and match it with the measurement scale used kb. the multiplexed bacterial isolates' dna products (pcr amplicons) were sequenced (macrogen company, south korea) in the forward and reverse directions. all sequences of nitrogenous bases were analyzed by blast basic local alignment search tool) to compare with the available data in genbank (national center for biotechnology information, ncbi). the neighbor-joining phylogenetic tree was drawn using mega7 software (kumar et al., 2018). time temperature repeat cycle pcr step 3 min. 94°c 1 initial denaturation 45 sec. 94°c 35 denaturation 45 sec. 56°c annealing 60 sec. 72°c extension 7 min. 72°c 1 final extension table 1. initiator program of 27 f, and 1492 r (green and sambrook, 2012). p. rettgeri k. pneumoniae e. cloacae r. planticola a. aquatilis p. vermicola p. mirabilis isolations tests gram stain + + + + + + motility + + voges proskauer + + + + starch hydrolysis + + + nitrate reduction indol + + + + + + + catalase + + oxidase + + + + + + urease test + + + + methyl red gelatin hydrolysis + + + + citrate vitalization k/a/-/ h2s a/a/+/a/a/-/k/a/-/k/a/-/ h2s k/a/-/k/a/-/h2s t.s.r table 2. the results of biochemical tests for the isolated bacteria from the water and sediment of sawa lake. 346 makki et al./ bacteria of the beach soil and saline water of sawa lake results biochemical tests: the results of biochemical tests are presented in table 2, showing bacteria isolates from the water and sediment of sawa lake that describes their colony’s morphology. proteus mirabilis: a gram-negative bacterium, its medium-sized colonies form transparent white concentric circles on moving nutrient agar. providencia vermicola: a gram-negative bacterium, its colonies form circular, opaque from the inside and transparent from the edges, mucous viscous on nutrient agar. alcaligenes aquatilis: gram-negative bacteria with spherical colonies appear in smooth mucous yellow color with flat, regular, motile edges on nutrient agar. raoultella planticola: gram-negative bacteria whose colonies are small in size, flat, yellow, transparent, and with irregular edges on the nutrient agar. enterobacter cloacae: its colonies are large, circular, convex, translucent, smooth, mucous, and motile, as gram-negative bacilli on the nutrient agar. klebsiella pneumonia: non-motile gram-negative bacillus, as clear, round, convex, and mucous colonies on the nutrient agar. providencia rettgeri: it was shown as a small, opaque, convex, mucous, and gram-negative bacilli on the nutrient agar. molecular diagnostics: the results of the molecular analysis showed the presence of seven isolates viz. p. mirabilis, p. vermicola, a. aquatilis, r. planticola, e. cloacae, k. pneumonia, and p. rettgeri in the sediment and soil of lake sawa confirm the results of figure 1. the sequence alignments of the seven isolated bacteria from sawa lake: 1. proteus mirabilis, 2. providencia vermicola, 3. alcaligenes aquatilis, 4. raoultella planticola, 5. enterobacter cloacae, 6. klebsiella pneumonia and 7. p. rettgeri. 347 int. j. aquat. biol. (2022) 10(4): 344-348 the traditional biochemical methods (figs. 1, 2). discussions the gene of rrna 16s sequences is globally used to identify bacteria isolates (sambrook and maniatis, 1989). anzai et al. (2000) indicated the presence of similarities between the bacterial isolates in many regions of the world. our results showed some differences in the bases of the isolated bacteria. the reconstructed neighbor-joining tree using registered genes in the national center for biotechnology information ncbi (wisnieweski, 2008; zakaria et al., 2005) revealed their differences. however, the differences in the sequence of proteus mirabilis were figure 2. the neighbor-joining tree shows the phylogenetic relationships between the isolates of 1. proteus mirabilis, 2: providencia vermicola, 3: alcaligenes aquatilis, 4: raoultella planticola, 5: enterobacter cloacae, 6: klebsiella pneumonia and 7: p. rettgeri isolated in this study and other isolates of the same bacteria registered at the national center for biotechnology information (ncbi) 348 makki et al./ bacteria of the beach soil and saline water of sawa lake significant compared to others (fig. 1). this may be due to their evolution to adapt to environmental features of their habitats, such as contamination with heavy metals, which prompted the genetic change to tolerate pollution to ensure avoiding their toxicity, or the adapting to the high level of salinity of the water and sediments of lake sawa (naik and dubey, 2011). references abu samour h., khatib h. (1999). geography of water resources, dar al-safa publishing and distribution, amman, hashemite kingdom of jordan. anzai y., kim h., park j.y., wakabayashi h., oyaizu h. (2000). phylogenetic affiliation of the pseudomonads based on 16s rrna sequence. international journal of systematic and evolutionary microbiology, 50(2): 1563-1589. arahal d.r., ventosa, a. (2002). moderately halophilic and halotolerant species of bacillus and related genera. in appbacteriaions and systematics of bacillus and relatives, pp. 83–99. baron e.j., finegold s.m. (1990). diagnostic microbiology. 8th ed. the c.v.mosby company. collee j.g., miles r.s., watt b. (1996). tests for the identification of bacteria. in: mackie and mccartney practical medical microbiology by colle, j.g.; fraser,a.g.; marmion,b.p. and simmons,a. 14th ed, churchill livingstone, singapore. p:131-149. galinski e.a. (1993). compatible solutes of halophilic eubacteria: molecular principles, water-solute interactions, stress protection. experientia, 49: 487-496. jamil a.k. (1977). geological and hydrogeochemical aspects of sawa lake, s. iraq. ull.coll. kumar s. (2018). mega x: molecular evolutionary genetics analysis across computing platforms. oxford journal, 35(6): 1547-1549. naik m.m., dubey s.k. (2011). lead-enhanced siderophore production and alteration in cell morphology in a pb-resistant pseudomonas aeruginosa strain 4ea. current microbiology, 62(2): 409-414. sambrook j., fritgah e., maniatis t. (1989). molecular cloning, a laboratory manual, cold spring harbour. laboratory, 145(3): 1365-1373. saravanan t., bhaskaran r., muthusam m. (2004). pseudomonas fluorescens induced enzymological changes in banana roots (cv. rasthali) against fusarium with disease. the plant pathology journal, 3: 72-80. ventosa a., nieto j.j., oren a. (1998). biology of moderately halophilic aerobic bacteria. microbiology and molecular biology reviews, 62: 504-544. wisniewski-dyé f., vial l. (2008). phase and antigenic variation mediated by genome modifications. antonie van leeuwenhoek, 94(4): 493-515. zakaria l., kulaveraasingham h., guan t.s., abdullah f., wan h.y. (2005). random amplified polymorphic dna (rapd) and random amplified microsatellite (rams) of g node infected oil palm and coconut stump in malaysia. asia pacific journal of molecular biology and biotechnology, 13(1): 236 34. islam e., sar p. (2011). culture-dependent and independent molecular analysis of bacterial community within uranium ore. journal of basic microbiology, 4: 1-13. kumar s. (2018). mega x: molecular evolutionary genetics analysis across computing platforms. oxford journal, 35(6): 1547-1549. int. j. aquat. biol. (2017) 5(5): 336-341doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article determination of helminth parasites in abdominal cavity of alosa caspia (eichwald, 1838) from the southeast part of the caspian sea ali taheri mirghaed*1 department of aquatic animal health and disease, faculty of veterinary medicine, university of tehran, iran. article history: received july 2017 accepted 22 october 2017 available online 2 5 october 2017 keywords: helminths parasites shad alosa caspia caspian sea abstract: alosa caspia (eichwald, 1838) belongs to clupeidae family, is considered as one of the main fish in the southern coast of the caspian sea. the aim of the present study was to evaluate the helminthic parasite infections in abdominal cavity of a. caspia from southeastern part of the caspian sea. in this regard, 30 fish were caught from bandar-torkaman and transferred alive to the parasitological laboratory. then parasites specimens were fixed and transferred to the national museum of parasitology, faculty of veterinary medicine at university of tehran for identification. a total of two parasite species including anisakis simplex and pronoprymna ventricosa were isolated from the fish. 100% of the fish were infected with at least one helminthic parasite species. pronoprymna ventricosa has the highest infection prevalence rate and was isolated from pyloric caeca, intestine and stomach of 93.33% of the fish specimens. anisakis simplex is found in abdominal cavity of 33.33 % of the studied fish. intensity of a. simplex and p. ventricosa was calculated as 8.4±5.31 and 91.4±21.46, respectively. based on the statistical analysis, there were no significant differences in total parasites burden, parasite prevalence and parasite intensity between male and female of the studied fish (p>0.05). introduction alosa caspia (eichwald, 1838) belongs to clupeid, is considered as one of the main fish in the southern coast of the caspian sea. due to lack of a. caspia farming (aquaculture) as well as difficulties with conducting research on marine environments, the study on diseases and parasitic infections of the fishes in these environments has not been taken into consideration. on the other hand, infected fish can act as a source of infection and easily be hunted by sea creatures and fish-eating birds or marine mammals. this fact helps the evolution of life cycle of some fish parasites. this type of study also is important from “public health” aspect as always there is the danger of transferring the disease and the infection to human. depending on the host species, parasites in the abdominal cavity contribute to damage, pathogenicity and disruption of the natural activities of the body. the parasites found in the abdominal cavity of the fish during clinical studies are mainly helminthes *corresponding author: ali taheri mirghaed doi: https://doi.org/10.22034/ijab.v5i5.393 e-mail address: mirghaed@ut.ac.ir parasites, such as cestodes, nematodes, trematodes and acanthocephalans (nezafat rahimabadi et al., 2008). although many of the parasites, particularly nematodes and trematodes, have also zoonotic importance and eating raw or improperly cooked or processed fish is the main source of these infections for humans, and this has been reported from various geographical regions. for example, anisakis species are important in terms of public health and can cause gastroenteritis in humans (pahlavan et al., 2014). alosa caspia is a type of valuable edible fish which is caught from the sea, so studying and evaluating its diseases and parasitic infections have always been faced with problems and limitations and a few studies have been conducted in this field. during a study on parasitic infections of a. caspia, jalali and vatandoust (1989) for the first time separated monogenean parasite, mazocras alosae from gills of the examined fish in khezerabad, mazandaran province. later, kornijchuk and barzegar (2006) reported the isolation 337 int. j. aquat. biol. (2017) 5(5): 336-341 of digenean pronoprymna ventricosa from the intestine of a. caspia from shahid madani center. during the evaluation of the parasitic infection of iranian herrings, bozorgnia et al. (2012) found m. alosae on the gills and metacerceria of diplostomum spathaceum from the eyes of the fish as well as an unknown nematode species of hysterothylacium sp. from the intestine of the examined fish in the khazarabad. undoubtedly, in order to manage the rebuilding and preservation of natural resources, as well as to manage health and quarantine of the fish farms, having precise information about the status of fish health in each region is a key and valuable factor. the main objective of the present study was to investigate the prevalence and intensity of parasitic infestation of a. caspia from the southeast coast of the caspian sea. materials and methods a total number of 30 specimens of a. caspia were caught from the southeast coast of the caspian sea, bandar-torkaman and immediately transported alive in oxygen-filled plastic bags to laboratory. identifying of the fish species was carried out in accordance with naderi jolodar and abdoli (2001). the sex of the fish was determined by examination of the gonads after opening of the abdominal cavity. then, abdominal cavity and gastrointestinal tract of the fish were monitored to find the helminthes parasites using a stereomicroscope. in the case of parasitic infections, the parasites were carefully separated and counted and the results were recorded. after separation and washing with normal saline (0.6% nacl), trematodes were fixed in 70% alcohol and finally were stained by acetocarmen (georgiev et al., 1986). while the nematodes were fixed in 10% formalin, then they were transferred to 70% alcohol and sent to the museum of parasitology in faculty of veterinary medicine, university of tehran for accurate identification. parasites were stained with lactofenol after clearing with glycerin (moravec, 1994) and they were mounted with entellan on the slide and examined microscopically. identification of the genus and species of parasites was carried out using identifying keys (hanek and fermando, 1972; roberts, 2001). in order to do a parasitic evaluation, the following equations were used: percentage of parasitic infections: prevalence rate = (the number of parasitic infected fish/the total number of examined fish) × 100 the average intensity of parasitic infection: the average intensity of parasitic infection = the number of counting parasites/ the total number of parasites of infected fish average frequency of parasite: average frequency of parasite = the total number of parasites/ the total number of examined fish average parasitic burden: average parasitic burden = the total number of counting parasites from all examined fish statistical calculations: data of evaluating of the parasites were expressed as mean±standard deviation. in order to analyze the data, software spss version 16 was used. in this regard t-test was used to compare the parasitic burden of male and female fish. furthermore chi-square test and fisher and s exact test was used to compare the prevalence and the intensity of parasitic infection in both male and female and the values of p<0.05 were considered as significant. results specimens of a. caspia with an average length of 28.4±3.92 cm and average weight of 363±36.2 g were examined (table 1) and a total of two parasite species including a. simplex and p. ventricosa were isolated from the examined fish (table 2). 100% of the fish samples were infected at least by one of the parasites species. the highest prevalence rate of parasite infection was belong to p. ventricosa, which was isolated from the pyloric area, intestine and stomach of 93.33% of the examined fish while a. simplex were found on the intestine, mesenteric and liver (abdominal area) of 33.33% of the infected fish (table 2). according to the results, 33.33% of the intestine, 30% of the liver and 3.33% of the mesenteric were infected with a. simplex and 26.66% of the stomach, 93.33% of the pyloric ceca and 36.67% of the intestine 338 taheri mirghaed / helminthes parasites in abdominal cavity of alosa caspia of the fish were infected with p. ventricosa (table 3). the mean intensity of a. simplex and p. ventricosa in a. caspia is presented in tables 2 and 3. accordingly, the highest rate of intensity of infection was related to infection of the pyloric ceca with p. ventricosa. the prevalence, infection intensity, range of parasite number and average frequency of parasites of the examined fish are presented in table 3. at the same time, no significant difference was found in total parasitic burden and isolated parasites between males and females (p<0.05) (table 4). discussion parasites are a large group of pathogens infecting various fish species and seafood (woo, 2006). many parasites may infect other vertebrates, including humans, in addition to aquatic animals. in fact, the fishes act as carriers of several parasites transferring them to other vertebrates. several studies have been carried out on parasitic infections of the caspian sea fish species in iran, which is mainly related to the iranian coasts. information about fish infestation is scarce in the southeast coast of the caspian sea. in this study, two parasitic species including a. simplex and p. ventricosa were isolated from a. caspia in the southeast part of the caspian sea. the migration of fish between the northern and southern parts of the caspian sea, and reproduction and feeding patterns increase the risk of exposure to various parasites (naderi jolodar and abdoli, 2004). in this survey, all samples were infected by at least one parasite species. in the other words, 100% of samples were infected with parasites. at the same time, no differences were found between the male and female individuals which is probably due to the type of feeding and the same immigration behavior of the male and female. despite the numerous reports of parasitic infections in the caspian sea, the relationship between trematode standard deviation±averaging minimum maximum weight (g) 363±36.2 245 423 total length (cm) 28.4±3.92 24 31 standard length (cm) 24.3±13.2 21.5 26 table 1. biometric characteristics of alosa caspia from the southeast part of the caspian sea. prevalence (%) infection intensity range frequency parasite infection 100 114±2.2 4-121 114.22±2.2 a. simplex 33.33 8.5±4.31 6-18 2.8 1±0.17 p. ventricosa 93.33 94.21±4.46 4-121 89.22±11.34 table 2. prevalence, infection intensity, range and frequency of parasites of alosa caspia from the southeast part of the caspian sea. table 3. prevalence, infection intensity, range and frequency of parasites in different organs of alosa caspia from the southeast part of the caspian sea. parasite species prevalence (%) infection intensity range frequency intestine a. simplex 33.33 8.43±4.61 7-18 2.1±31.1 intestine p. ventricosa 36.67 57.8±13.4 73-4 17.5±56.2 pyloric p. ventricosa 93.33 102±16.5 121-25 98.2±16.3 stomach p. ventricosa 26.66 15.1±6.24 5-36 4.2±42.0 liver a. simplex 30 8.28±3.64 7-15 2.1±48.3 mesenteric a. simplex 3.33 6±0 6 0.2±0 table 4. relationship between sex and parasite load in alosa caspia from the southeast part of the caspian sea. sex number of samples number of infected fish parasitic load a. simplex female 13 5 43 male 17 6 54 p. ventricosa female 13 12 1310 male 17 16 1742 339 int. j. aquat. biol. (2017) 5(5): 336-341 infections is very limited. pronoprymna ventricosa is commonly reported from the pyloric and intestine of various fish species, and particularly from clupeidae family in the world. for example, this parasite has been reported from various species of the alosa spp. in the north atlantic ocean, the mediterranean sea (bray and gibson, 2000) and the black sea (gaevskaya and kornijchuk, 2003; popjuk, 2009; ozer et al., 2013). in iran, although reports are limited in this regard, this parasite has been reported only from the clupeidae in the caspian sea. for example, shamsi and dalimi (1996) isolated pseudopentagramma symmetrica which is synonymous for p. ventricos from c. engrauliformis, c. cultriventris and c. grimmi. in another report, varshoie et al. (2010) again found this trematode from 53 to 58% of various species of kilka (sprat) in the caspian sea. the p. ventricosa parasite was first isolated in 2005 from a. caspia, (kornijchuk and barzegar, 2005). however, in two separate studies, yousefi et al. (2011) and barzegar et al. (2012) isolated and reported the parasite from a. caspia in the caspian sea. in the recent review of barzegar et al. (2012), 100% of the samples studied in the caspian sea were infested with p. ventricosa. in the present study, 96.66% of the fish were infected with this parasite. in general, due to the similar nutritional and migration habits among clupeids, it is not surprising to expect the infestation of this parasite in other members of the family in the caspian sea. the trematode p. ventricosa is one of the parasites that can be detected by the naked eye. this parasite is not important in terms of the public health. meanwhile, in the case of mild to moderate infection in the fish host, it is also not very problematic, and serious damage occurs only when the intensity of the infection and the number of parasites are increased (jalali, 1998). another parasite isolated from a. caspia is nematode a. simplex from anisakidae family. crustaceans are the first intermediate host of the nematode, and the fish mainly act as the second intermediate and sometimes the final hosts for the parasite. when fish are infected by infectious larval stages of the parasite, the larvae can be found as cyst or free organism in the gastrointestinal tract, abdominal cavity, muscles and other organs. serious damages will occur if the intensity of the infection is high and due to the migration of the parasite to other organs (jalali, 1998). so far, this parasite has been reported from various fish species on the northern and southern coast of the caspian sea. the larvae stages of a. simplex are isolated from sprats (shamsi et al., 1998), acipenser stellatus (mokhayer, 1974) and rutilus kutum (eslami and kohneshahri, 1978). it seems that this is the first official report on infections of young a. caspia with this parasite in iran. the most important thing about a. simplex, along with the possible damage to the host fish is its pathogenicity to human. anisakis species are able to infect humans who eat raw or undercooked fish and may causes serious allergies and digestive problems (robert, 2001). jeddy et al. (2012) surveyed the parasitic infestation of the kilka fish in the caspian sea during whole year period. they examined 64 fish per season and a total of 4 parasites including pseudopentagrama symmetrica, bunocotyle cingulate, larval stages of anisakis sp. and contracaecum sp. were found from the examined fish. their results shows that p. symmetrica (p. ventricosa) had the highest percentage rate of infection and the most intensity of infection among other parasites. they also found that the parasites induced no serious damages in the fish, but only reduced their growth; also no significant effects were found on the fish reproduction. the researchers reported that the highest levels of parasite infection was in the gastrointestinal tract, followed by abdominal cavity and gonads while in the present study, the highest prevalence rate of parasites was observed in pyloric area. according to the present results, it is concluded that the infection of helminthes parasites in abdominal cavity of a. caspia from the southeast part of the caspian sea is significantly high, but the parasitic diversity is low. at the same time, due to the isolation of the zoonotic parasite a. simplex from the examined fish, consideration should be given to the consumption of the fish. therefore, consuming of the raw or semicooked fillet of this fish is strictly forbidden. 340 taheri mirghaed / helminthes parasites in abdominal cavity of alosa caspia references azizi h., tahmasebi kohiani a., nematolahi a., adel m., borjian a., jafari m. 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(in persian) varshoie h., mobedi i., aghazadeh meshgi m., jalali b. (2010). survey on the digenean and monogenean helminthes of clupeidae (teleostes) from southern part of caspian sea. research journal of parasitology, 5(3): 148-155. woo p.t.k. (2006). fish diseases and disorders. volume 1: protozoan and metazoan infections. 2nd ed, cabi, uk. 800 p. yousefi m., sefidgar s., maliji g., mousavi s., asna ashari m. (2005). infection of river whitefishes (rutilus rutilus) by ligula intestinalis parasite in aras dam. journal of babol univarsity of medical sciences, 7(2): 80-83. (in persian) yousefi m.r., hosseinifard s.m., halajian a., nasiri amiri m., shokrolahi s. (2011). pronoprymna ventricosa (digenea: faustulidae) in alosa caspia fish in north of iran. world journal of fish and marine sciences, 3(2): 104-106. int. j. aquat. biol. (2017) 5(5): 336-341 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی جنوب سواحل در alosa caspia (eichwald, 1838) زالون ماهی شگ شکمی محوطه کرمی هایانگل شناسایی خزر دریای شرقی *میرقائد طاهری علی .ایران ،. ایران تهران، تهران، دانشگاه دامپزشکی دانشکده آبزیان هایبیماری و بهداشت گروه چکیده: از هدف. آیدمی شمارهب خزر دریای جنوبی سواحل در اصلی ماهیان از یکی و ماهیانشگ خانواده اعضاء از( alosa caspia) خزر دریای ماهیشگ . باشدمی خزر دریای شرقی جنوب سواحل در( a. caspia) زالون ماهیشگ شکمی محوطه کرمی هایانگل آلودگی وضعیت بررسی حاضر تحقیق هب نهایی شناسایی جهت هاانگل جداسازی از پس و شدند منتقل آزمایشگاه به و صید ترکمن بندر منطقه از ماهیشگ نمونه 30 تعداد منظور بدین شامل انگلی گونه 2 نهایت در و بودند؛ مبتال انگل نوع یک به حداقل هانمونه % 100 حاضر بررسی در. شدند منتقل تهران دانشگاه شناسیانگل موزه anisakis simplex و pronoprymna ventricosa آلودگی شیوع باالترین نتایج اساس بر. گردید جداسازی مطالعه مورد ماهیان شکمی محوطه از شکمی محوطه از نیز a. simplex انگل و شد جدا هانمونه درصد 33/93 یمعده و روده پیلوریک، زوائد از که بود p. ventricosa به متعلق انگلی برای 4/94±46/21 و 4/8±31/5 ترتیببه p. ventricosa و a. simplex هایانگل آلودگی شدت. شد جدا بررسی مورد ماهیان درصد 33/33 جداسازی هایانگل شدت و شیوع کل، انگلی بار برای داریمعنی اختالف هیچ آماری هایبررسی اساس بر حال عین در. گردید محاسبه آلوده ماهیان (.p>05/0) نگردید مشاهده مطالعه این در ماده و نر جنس دو بین شده .خزر دریایزالون، ،alosa caspia کرمی، هایانگل :کلمات کلیدی int. j. aquat. biol. (2021) 9(3): 200-206 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article modelling the time series of capture fishery and aquaculture production in iran hadi poorbagher*1, soheil eagderi1, reza nahavandi2 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2animal science research institute of iran, agricultural research, education and extension organization (areeo), karaj, iran. s article history: received 18 february 2021 accepted 27 may 2021 available online 2 5 june 2021 keywords: times series capture fishery aquaculture forecasting abstract: the trend of capture fishery and aquaculture production in iran shows an ascending trend. an estimate of future production may be useful for management purposes and to provide some clues about the effectiveness of the current plans to reach the goals. we used the data provided by the food and agricultural organization of the united nation (fao) to model the time series of the production of aquatics in both sectors. the data covered the years 1980-2018. we predicted the production of aquatics until 2025 using autoregressive integrated moving average models. several techniques were used to estimate the parameters of the model. however, searching the all possible values of the parameters provided the model with the best predictability. according to the selected model, the production of capture fishery will have an ascending trend and increase to 1,513,533 tons in 2025. aquaculture production will also have an increasing trend, however, the rate of change will be lower than that of the capture fishery. aquaculture production will reach to 552944 tons in 2025. the forecast is based on the assumption that the rate of changes in the development of capture fishery and aquaculture will remain in the present status. sudden changes in management practice or environmental conditions may have a remarkable influence on future production. introduction the caspian sea in the north, and the persian gulf and oman sea in the south have provided a unique status for the iran in terms of fisheries, transport and trade. the caspian sea is the habitat of many precious sturgeon fishes though their present status is not promising. the indigenous fish fauna of the caspian sea basin comprises 159 species with 62% are considered endemic to the basin (naseka and bogutskaya, 2009; esmaeili et al., 2018) indicating the importance of this sea in terms of species diversity. the capture fishery is one of the main economic activities of the southern caspian sea that many people in the region is relied on. nowadays, cage culture, along with capture fishery, is being practised in this water body providing additional job opportunity for the locals and is gradually taking an important place in the production of fishery products. the catch in the caspian sea has decreased from 98000 in 2000 to 44279 tons in 2009 indicating a 54% *correspondence: hadi poorbagher doi: https://doi.org/10.22034/ijab.v9i3.1220 e-mail: poorbagher@ut.ac.ir decrease in the total catch (iranian fishery organization, 2010). despite the invaluable importance of the caspian sea, it is being suffered from pollution, overfishing, and ballast water problems (eagderi et al., 2013). the ecological problems of the sea will threaten the long term sustainable fisheries and subsequently the living status of the locals residing on its coastal areas. the close cooperation of five countries around the sea to improve the present status of the sea is indispensable. the persian gulf is a shallow semi-enclosed water and is connected to the oman sea through the strait of hormuz with the fishery being an important activity of the region, second only after the oil industry (carpenter et al., 1997). among the neighboring countries, iran has the greatest exclusive economic zone and the longest coastline. this body of water is the living and spawning ground of many marine fishes harbouring reef ecosystems and also pearl oysters. the total fish species of persian gulf comprise 743 201 int. j. aquat. biol. (2021) 9(3): 200-206 species, of them, many are important in terms of fisheries (eagderi et al., 2019). several important species live in the persian gulf and oman sea including tunas and mackerels (karimpour et al., 2013). the brittle ecosystem of the persian gulf has experienced several major damages by the oil wars and tanker sinking (alavian petroody et al., 2017). also, overfishing, illegal and unmonitored catch by foreign fleets are among its current problems. despite the damages incurred to the ecosystem of the persian gulf, the catch had an ascending trend increasing from 260500 in 2000 to 348122 tons in 2009 indicating a 33% increase in the catch. aquaculture is a growing industry in iran. the major part of aquaculture production belongs to warm water fishes especially chinese carp, and in lower stands, rainbow trout, freshwater and marine shrimp (karimpour et al., 2013). the aquaculture production has increased from 66000 in 2000 to 207353 tons in 2009 indicating over 214% growth in this sector (iranian fishery organization, 2010). a time series a sequence of data listed in time order. time series and the related analyses are used in various fields, including statistics, economics, earth science and engineering (amiri et al., 2018). modelling the time series provides a tool to forecast future values. times series analyses have been extensively used in fishery science for forecasting the catch and landings (stergiou et al., 1997; koutroumanidis et al., 2006; amiri et al., 2017, 2018). the autoregressive integrated moving average model (arima) is one of the widely-used time series models in fisheries forecasting studies (stergiou, 1989; tsitsika et al., 2007; bako et al., 2013). the knowledge about the probable future trend of data is of high importance in management practice. the present study thus aimed to predict the future trend of capture fishery and aquaculture productions in iran using an arima model. we used several methods to estimate the parameters of the model. materials and methods all calculations were made using r 4.0.3. the data of capture fishery and aquaculture production were downloaded from the fao website (www.fao.org) for 1980-2018. the data were transformed into time series using the function ts() with the frequency = 1, and examined for outlier using the function tsclean(). since no outlying data were found, all data were used for modelling. the data of the first 28 years (i.e. 19802007), covered 70% of all data and were used as training data, the rest were used as testing data. model specification: the autocorrelation (acf) and partial autocorrelation function (pacf) plots indicated some significant autocorrelation in some lags. therefore, autoregressive integrated moving average (arima) model was used for modelling and forecasting of the time series (jebb et al., 2015). the arima needs the time series to be stationary (coghlan, 2015), that is, the mean, variance and autocorrelation of the time series should be constant. to examine the stationary of the time series the augmented dickey-fuller test (adf) was used. the adf test indicated that time series were not stationary. they had no constant variance over time, natural logarithm was thus used. differencing was used to detrend the time series. after transformation with logarithm and differencing, the adf test was used again to examine the stationarity of the transformed data. non-stationary data were transformed using the box-cox method (nelson and granger, 1979) with adf being used again. parameters estimation: the arima model has three components: autoregressive (ar), integrated (i) and moving average (ma). the components ar and ma are the predictors explaining the autocorrelation in the time series. the integrated component indicates the differencing used to detrend the time series making it stationary. the orders of these components are shown by p, d and q. there are some methods to determine p, d and q: (1) transforming the data using natural logarithm, differencing or both, then performing adf test to examine the stationarity of data and finding d. the acf and pacf plots are then used to find p and q (coghlan, 2015); (2) there is a function (auto.arima()) in the package forecast that find automatically p, d and q (hyndman et al., 2007). it is worthy to note that the default values in the 202 poorbagher et al./ time series of capture fishery and aquaculture production in iran function for p, d and q are 5, 2 and 5. therefore, these values can change if required. the method to estimate the parameters can change, too. the adequacy of the model is examined after parameter estimation using the acf and pacf plots; (3) estimating p and q using the extended autocorrelation function (eacf) using the function eacf() from the package tsa (cryer and chan, 2008); (4) estimating p and q by the best arima subset model using the function armasubsets() in the package tsa (cryer and chan, 2008). the latter method may suggest several values for p and, therefore, further investigation may be required to find the best values for p and q. the above methods were used for the estimation of an arima model. to examine the adequacy of the selected model, root mean square error (rmse) was used with the data predicted by the model and testing data. finally, we examined all possible values of p, d and q (in the range of 0-10 and 1331 times totally) to find the model with the smallest rmse as the best model. evaluating the model: the residuals of the selected model were evaluated using acf and pacf graphs to find if all variance of the data has been explained by the model. normality, independence and constant variance of the residuals were examined using the shapiro-wilk, ljung-box and mcleod-li tests, respectively. results temporal changes of catch and aquaculture products: the products of capture fishery and aquaculture are shown in figure 1. the capture from the fishing had stable ascending trend from 1980 to 2010, while the slope of the trend increased after 2010. figure 1. temporal changes of capture fishery (a) and aquaculture production (b) in iran during 1980-2018. t im e se ri es a s ig ni fi ca nt a ut oc or re la ti on in ti m e se ri es p v al ue o f t he a d f te st b ef or e da ta tr an sf or m at io n p ar am et er e st im at io n m et ho d transformation parameter p v al ue o f t he a d f te st a ft er d at a tr an sf or m at io n residual analysis rmse n at ur al lo ga ri th m d if fe re nc in g b ox -c ox p d q l ac k of s ig ni fi ca nt au to co rr el at io n in a c f pl ot l ac k of s ig ni fi ca nt au to co rr el at io n in p a c f pl ot p va lu e of th e sh ap ir ow ilk te st p va lu es o f t he l ju ng -b ox te st s at d if fe re nt la ge s > 0. 05 p va lu es o f t he m cl eo dl i t es ts at d if fe re nt la gs > 0 .0 5 catch yes 0.933 acf and pacf plots yes yes no 0 1 0 0.010 yes yes < 0.001 yes yes 225170.00 auto.arima yes yes no 0 2 1 0.010 yes yes 0.001 yes yes 159732.00 eacf yes yes no 0 1 0 0.010 yes yes < 0.001 yes yes 225170.00 armasubsets yes yes no 0 1 0 0.010 yes yes < 0.001 yes yes 225170.00 armasubsets yes yes no 4 1 0 0.010 yes yes < 0.001 yes yes 185287.50 searching all possible values yes yes no 4 1 9 0.010 yes yes < 0.001 yes yes 32230.17 table 1. the analyses on time series of catch to estimate parameters of the arima model. 203 int. j. aquat. biol. (2021) 9(3): 200-206 the product from aquaculture had gentle positive slope from 1980 to 2000 but the slope increased with greater slope until 2018 (source: www.fao.org). the acf plot indicated significant autocorrelation in time series of capture fishery and aquaculture (fig. 2). hence, an arima model was used. estimation of arima model parameters: the data were analysed using various methods (table 1). among the various methods, searching the all possible values of p, d and q resulted in a model with the best predictability. however, the shapiro-wilk test rejected the null hypothesis assuming normality in the residual. among the various methods, searching the all possible values of p, d and q resulted in a model with the best predictability for aquaculture data (table 2). the performance of the final model in the prediction of testing data is shown in figure 3. the model based on searching all possible values of the parameters had the best predictability for both catch and aquaculture time series. prediction: the predicted values of catch and aquaculture products are depicted in figure 4. both catch and aquaculture are predicted to have an ascending trend. the catch will reach to over 1.5 million tons and the production of aquaculture sectors will reach to over 550000 tons in 2025. discussions estimating the future trend of production is largely helpful for fishery managers. one of the widely-used tools in forecasting the catch is the time series analysis and in particular, the arima models. the present study used an arima model to forecast fishery catch and aquaculture production in iran and estimated its parameters using several methods. of the various table 2. the analyses on time series of aquaculture data to estimate parameters of arima model. t im e se ri es a s ig ni fi ca nt a ut oc or re la ti on in ti m e se ri es p v al ue o f t he a d f te st b ef or e da ta tr an sf or m at io n p ar am et er e st im at io n m et ho d transformation parameter p v al ue o f t he a d f te st a ft er da ta tr an sf or m at io n residual analysis rmse n at ur al lo ga ri th m d if fe re nc in g b ox -c ox p d q l ac k of s ig ni fi ca nt au to co rr el at io n in a c f pl ot l ac k of s ig ni fi ca nt au to co rr el at io n in p a c f pl ot p va lu e of th e sh ap ir ow ilk te st p va lu es o f t he l ju ng -b ox te st s at d if fe re nt la ge s > 0. 05 p va lu es o f t he m cl eo dl i te st s at d if fe re nt la gs > 0 .0 5 aquaculture yes 0.990 acf and pacf plots yes yes no 7 3 7 0.010 yes yes 0.433 yes yes 343929.60 auto.arima yes yes no 0 1 0 0.329 yes yes 0.148 yes yes 61523.82 auto.arima yes yes yes 0 1 0 0.815 yes yes 0.170 yes yes 74211.00 eacf yes yes no 5 3 6 0.010 yes yes 0.333 yes yes 273927.70 armasubsets yes yes no 0 3 7 0.010 yes yes 0.336 yes yes 273927.70 searching all possible values yes yes no 1 3 8 0.010 yes yes 0.142 yes yes 27856.05 figure 2. the acf plot for the time series of the catch from fishing (a) and aquacultural production (b). 204 poorbagher et al./ time series of capture fishery and aquaculture production in iran types of estimating the parameters of the arima models, searching for all possible values of the parameters resulted in the best model in terms of predictability. such a method is time-consuming, particularly, with a high number of data. lack of this method in the literature may be related to the problem of over-fitting. further examination of the model with a second unused series of data can further evaluate the quality of the model and investigate if the model made in this method may be over-fitted. in the present study, the number of the data was low and thus we had no chance to examine the quality of the model with a set of data in addition to those we used as testing data. the fishery catches of iran increased from 43659 in 1980 to 828872 tons in 2018 indicating 180% growth. however, this increase is mainly related to the latest years, i.e. after 2016. according to the model used in the present study, it is predicted a 82% increase in capture fishery of iran in 2025 compared with 2018. while the current trend is promising, our prediction is based on the data presented to fao. according to an overview by fao, the iranian marine resources seem to have reached to their biological limits (fao, 2020). therefore, the trend of the forecasted figure about the future capture fishery production may shift downward or remain stable. the precision of the method to gather the data is a crucial step that should always be scrutinized by the related organizations and our prediction is hence surely influenced by the quality of the data (watson and pauly, 2001). the predicted increasing trend of capture fishery of iran hints an increasing by-catch the iranian fishing fleets may encounter in upcoming years. the by-catch has always been one of the main environmental problems of the capture fisheries. the rate of fishery by-catch may be dramatically high. for instance, 3.7 million tonnes of fish were landed in the united states while 1.06 million tonnes (~30%) of the caught fishes figure 3. the performance of the final model in prediction of the testing data for time series of catch and aquaculture products. the black, red and green points indicate the training, testing and predicted data, respectively. figure 4. the prediction of aquatics production through capture fishery and aquaculture until 2025. the hollow points indicate the predicted values from 2019 to 2025. the dotted lines indicate 0.95 confidence intervals for the predicted values. 205 int. j. aquat. biol. (2021) 9(3): 200-206 were discarded (harrington et al., 2005). the by-catch in iranian fishery depends on the target species, for example, in case shrimps, it may reach to > 86% (farrokhi et al., 2014). therefore, despite the promising figures for the capture fisheries, the forthcoming pressure on the iranian marine ecosystems should be taken into account in planning for the future. the aquaculture has had a steady growth in iran. based on our model, it will reach to over half a million tonnes in 2025, i.e. ~ 26% growth compared with that of 2018. aquaculture is becoming a substitute or replacement for the capture fishery production. aquaculture has a special place in five-year plans of the country and various encouragements have been granted for private sectors. with regards to the pressure on the marine sources, it seems that the forecasted growth of aquaculture is quite realistic. in conclusion, our study indicates a growth both for capture fishery and aquaculture production in iran. further study is recommended to forecast the production of aquatics at the species level. references balon e.k. 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(2013). predictive modeling of pelagic fish catch in malaysia using seasonal arima models. agriculture, forestry and fisheries, 2: 136-140. carpenter k.e., krupp f., jones d.a., zajonz u. (1997). living marine resources of kuwait, eastern saudi arabia, bahrain, qatar and uae. fao species identification field guide for fishery purposes. fao, rome. 293 p. coghlan a. (2015). a little book of r for time series, 0.2. ed. published under creative commons attribution 3.0 license. cryer j.d., chan k.-s. (2008). time series analysis: with applications in r. springer science & business media. eagderi s., fricke r., esmaeili h.r., jalili p. (2019). annotated checklist of the fishes of the persian gulf: diversity and conservation status. iranian journal of ichthyology, 6: 1-171. eagderi s., mojazi amiri b., adriaens d. (2013). description of the ovarian follicle maturation of the migratory adult female bulatmai barbel (luciobarbus capito, guldenstadt 1772) in captivity. iranian journal of fisheries sciences, 12(3): 550-560. esmaeili h.r., sayyadzadeh g., eagderi s., keyvan abbasi (2018). checklist of freshwater fishes of iran. fishtaxa, 3(3): 1-95. fao. (2020). fishery and aquaculture country profiles, the islamic republic of iran. available: http://www. fao.org/fishery/facp/irn/en (accessed 11.20.20). farrokhi e., kamrani e., akbarzade a., raeisi h., solaimani a. (2014). species composition of bycatch trawl commercial trawler from fishing grounds in hormozgan province. journal of fisheries, 67: 375-392. (in farsi) harrington j.m., myers r.a., rosenberg a.a. (2005). wasted fishery resources: discarded by-catch in the usa. fish and fisheries, 6(4): 350-361. hyndman r.j., khandakar y. (2007). automatic time series for forecasting: the forecast package for r. monash university, department of econometrics and business statistics. journal of statisical software, 27(3): 10.18637/jss.v027.i03 iranian fishery organization (2010). the statistical yearbook 2000-2009. available at https://www.amar. org.ir (accessed 11.20.20).. jebb a.t., tay l., wang w., huang q. (2015). time series analysis for psychological research: examining and forecasting change. frontiers in psychology, 6: 727. karimpour m., harlioglu m.m., khanipour a.a., abdolmalaki s., aksu ö. (2013). present status of fisheries in iran. journal of fisheries, 7: 161. 206 poorbagher et al./ time series of capture fishery and aquaculture production in iran koutroumanidis t., iliadis l., sylaios g.k. (2006). timeseries modeling of fishery landings using arima models and fuzzy expected intervals software. environmental modelling and softwar, 21: 1711-1721. naseka n.m., bogutskaya n.g. 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(2013) (1): 14-21 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology serum biochemical characteristics of carassius auratus (l) following short-term formalin or nacl treatment seyyed morteza hoseini*1, reza tarkhani1 1 department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 2 march 2013 accepted 12 april 2013 available online 14 april 2013 keywords: therapeutic goldfish stress biochemistry abstract: goldfish (carassius auratus) were subjected to either formalin (250 ppm) or nacl (10 ppt), over a 0.5-h period followed by 24-h freshwater exposure. serum biochemistry was monitored before exposure (0), immediately after (0.5) plus 3 and 24 h after exposure. results showed that both formalin and nacl treatments caused rapid increase in cortisol with a peak at 3 h, which did not recover until 24 h. likewise, glucose showed similar patterns, however, returned to initial levels at 24 h after exposure. formalin caused significant decrease in sodium and chloride levels which returned to initial levels at 24 h after exposure. both formalin and nacl caused calcium and total protein elevation at 3 and 24 h after exposure. albumin and globulin levels were significantly affected by formalin and nacl at 3 and 24 h after exposure. it is suggested that formalin and nacl at the therapeutic concentrations cause rapid stress in goldfish which is eliminated after 24 h in freshwater. in addition, formalin causes slightly osmotic disturbance which is eliminated after 24 h recovery in freshwater. both formalin and nacl cause serum calcium and protein alteration after a while, which lasts until, at least, 24 h. more studies are needed to explain underlying mechanisms. formalin and nacl treatment, although advantageous in ectoparasite removal, are stressful in goldfish, which should be considered if they are going to be used. since formalin causes osmotic disturbance and more stress response, nacl treatment is suggested as an alternative. introduction goldfish is an ornamental fish and the most common one in iran. breading and rearing of this species is performed by locals in some regions of iran (hoseini and tarkhani, 2012). ecto-parasites are common in fish culture activities, including goldfish culture, causing huge economic loss. certain chemical compounds are used as therapeutic agents to control ecto-parasits infection. formalin and nacl are two effective therapeutics to control ecto-parasites. 250 ppm formalin and 10 ppt nacl over a 30-min is effective to remove ectoparasites in fish (klinger and francis-floyd, 1998). beside the anti-parasitic effects of these therapeutics, they may be used as a prophylactic agents in healthy fish. although therapeutics have the health benefit for fish, they might cause adverse effects on fish. for * corresponding author: e-mail address: seyyedmorteza.hoseini@gmail.com instance, formalin is a reducing agent forming methylene cross-link in proteins (chang and gershwin, 1992). also, formalin treatment causes pathological symptoms in fish gills (wedemeyer, 1971). similarly, nacl exposure causes osmotic disturbance and stress in freshwater fish. there are several studies on the effect of formalin treatment (wedemeyer 1971; nieminen et al., 1983; powell et al., 1996; sykes et al., 2011) and nacl exposure on freshwater fish (bœuf and payan, 2001; gupta and hanke, 1982; hoseini and hosseini, 2010). however, no study has investigated the effect of therapeutic dose of formalin and nacl on stress response and serum characteristics in goldfish. thus, in the present study, adult goldfish were subjected to 250 ppm formalin or 10 ppt nacl over a 30-min period (klinger and francis-floyd 1998) and 15 hoseini and reza tarkhani/ int. j. aquat. biol. (2013) (1) 14-21 changes in their serum characteristics were investigated. materials and methods fish and maintenance conditions: a total number of 90 fish [90.3 ± 3.1 g (mean ± sd)] were randomly distributed into 9 glass aquaria (1 × 0.4 × 0.5 m) filled with 200 l well water. fish were fed twice a day at a rate of 2% of body weight. all aquaria were aerated continuously and 90% of the water was exchanged daily. water temperature, ph, total hardness and dissolved oxygen were 26 ± 1°c, 7.05 ± 0.1, 190 ± 7.1 ppm and 6 ± 0.71 ppm (mean ± sd). fish were maintained under these conditions for 1 mouth to acclimate experimental condition. experimental design and sample collection: feeding was ceased 24 h before the first blood sampling. one fish was removed from each aquarium (three samples per treatment) for blood sampling. after the initial sampling, the aquaria were assigned as: control, formalin-treated (ft) and nacl-treated (st) with three replicates. ft and st groups were exposed to 250 ppm formalin (merck, frankfurter, germany) or 10 g l-1 nacl (purchased from local supplier; 95% purity) over a 0.5 h period (klinger and francis-floyd, 1998), respectively. after 0.5 h, second blood samples were collected from all groups. 3 and 24 h after exposure, further blood samples were collected from all groups. for blood sampling, fish were anesthetized using 3000 ppm clove solution over less than 1 min (hoseini et al., 2011). thereafter, blood samples (0.7 ml) were collected by caudal severance. blood samples were poured in non-heparinized plastic tubes and remained at 4°c for 2 h prior to centrifugation (2000 × g, 6 min). all serum samples were stored at -20°c until further analyses. serum analyses: serums were analyzed for cortisol using elisa method (ruane et al., 2002) using commercial kite (ibl, gesellschaft für immunchemieund immunbiologie, germany). inter assay coefficient of variation was found to be 9.2% calculated by measuring three known concentrations of cortisol for 5 times. glucose (glucose oxidase method), calcium (cresolphethalein complexone method), total protein (biuret method) and albumin (bromocerol green method) were determined according to thomas (1998) using commercial available kits (pars azmun co. ltd, tehran, iran). chloride levels were measured spectrophotometerically via thiocyanate method (thomas, 1998) using available kit (zist chem co. tehran, iran). sodium was measured using flame photometer (seac, florence, italy; hoseini and hosseini, 2011). globulin levels were calculated by subtraction of albumin from total protein. statistical analyses: data were examined for normality and homogeneity of variances using the shapiro-wilk's and levene's test, respectively. accordingly, cortisol and glucose values were logtransformed. all data were analyzed using 2-way anova and lsmeans' test with treatment (control, ft and st) and time (0, 0.5, 3 and 24 h) as factors. data are presented as the mean ± standard deviation. results treatment and sampling point had a significant effect (p<0.0001) on cortisol level (fig. 1). in the control group, cortisol had no significant difference among 0, 0.5 and 3 h, however, at 24 h, it was significantly lower than 0.5 and 3 h (fig. 1). in both ft and st groups, cortisol elevated significantly (~ 20 folds) at 0.5 h compared to 0 h and stabled at 3 and 24 h, despite the significant decrease compared to 0.5 h (fig. 1). both ft and st groups, showed significantly higher cortisol level compared to that of control at 0.5, 3 and 24 h (fig. 1). there was no significant difference in glucose level of control group at any sampling point (fig. 2). both ft and st groups showed significant elevation in glucose levels at 0.5 and 3 h which returned to 0 h levels at 24 h. there was no significant difference between 0 and 24 h values among the groups. ft group showed significantly higher glucose levels at 0.5 and 3 h compared to control, while, st group showed significantly higher levels only at 3 h. sodium and chloride levels did not show any changes over the time in the control and st groups 16 hoseini and reza tarkhani/ int. j. aquat. biol. (2013) (1) 14-21 (fig. 3 and 4). conversely, in the ft group, sodium levels was significantly lower at 0.5 h compared to 0 h, however, increased at 3 and 24 h and reached the value of 0 h (fig. 3). chloride levels were significantly low at 0.5 and 3 h compared to 0 and 24 h, in ft group (fig. 4). there was no significant change in sodium and chloride levels at 0 h among the treatments (fig. 3 and 4). the ft group had the lowest level of sodium, then, the control and st groups, at 0.5 h. the chloride levels of ft group were lower than those of the control and st groups, at 0.5 h. ft group had lower sodium and chloride levels than the control and st group, at 3 h. the sodium levels of st group were significantly higher a ab a c a bc a a a a b c b b b b a a b c b b b b 0 50 100 150 200 250 300 before exposure immediately after exposure 3 h after exposure 24 h after exposure c o rt is o l (n g / m l) time (h) pvalue time < 0.0001 treatment < 0.0001 time × treatment < 0.0001 control formalin nacl a a a a a a a a a a b b c b a a a a a b b b a a 0 50 100 150 before exposure immediately after exposure 3 h after exposure 24 h after exposure g lu co se ( m g / d l) time (h) pvalue time < 0.0001 treatment < 0.0001 time × treatment < 0.0001 control formalin nacl figure 1. effect of formalin and nacl treatments on serum cortisol levels of goldfish c. auratus over the time. different lowercase letters over the bars show significant difference in cortisol levels over the time in each treatment, separately. different uppercase letters over the bars show significant difference in cortisol levels between the treatments at each time point, separately. figure 2. effect of formalin and nacl treatments on serum glucose levels of goldfish c. auratus over the time. see figure 1 for elucidation. a a b a a a ab a a a a b b ab a a a a c a a a b a 0 50 100 150 200 250 300 before exposure immediately after exposure 3 h after exposure 24 h after exposure s o d iu m ( m e q / l) time (h) pvalue time < 0.0001 treatment = 0.37 time × treatment = 0.005 control formalin nacl figure 3. effect of formalin and nacl treatments on serum sodium levels of goldfish c. auratus over the time. see figure 1 for elucidation. a a a a a a a a a a b b b b a a a a a a a a b a 0 50 100 150 200 before exposure immediately after exposure 3 h after exposure 24 h after exposure c h lo ri d e ( m e q / l) time (h) pvalue time < 0.0001 treatment = 0.19 time × treatment = 0.006 control formalin nacl figure 4. effect of formalin and nacl treatments on serum chloride levels of goldfish c. auratus over the time. see figure 1 for elucidation. a a a a a a a a a b a a b c b d a a a a c b b b 0 10 20 30 before exposure immediately after exposure 3 h after exposure 24 h after exposure c a lc iu m ( m g / d l) time (h) pvalue time = 0.0004 treatment < 0.0001 time × treatment < 0.001 control formalin nacl figure 5. effect of formalin and nacl treatments on serum calcium levels of goldfish c. auratus over the time. see figure 1 for elucidation. a a a a a a a aa a b a ab b b b a a a ab b bc b c 0 1 2 3 4 5 6 before exposure immediately after exposure 3 h after exposure 24 h after exposure t o ta l p ro te in ( g / l) time (h) pvalue time < 0.0001 treatment = 0.19 time × treatment = 0.006 control formalin nacl figure 6. effect of formalin and nacl treatments on serum total protein levels of goldfish c. auratus over the time. see figure 1 for elucidation. 17 hoseini and reza tarkhani/ int. j. aquat. biol. (2013) (1) 14-21 than that of ft group, at 24 h, however, chloride levels were significantly higher in the st group than both control and ft groups, at this point. calcium levels did not change significantly over time, in the control group (fig. 5). ft group showed a significant decrease at 0.5 h followed by an increase at 3 and 24 h, compared to 0 h values. st group showed significant increase at 3 and 24 h compared to 0 and 0.5 h. there were no significant differences among the groups at 0 and 0.5 h. however, at 3 h, the levels of calcium significantly increased in ft group followed by an increase in st group, compared to control. at 24 h, both ft and st groups had significantly higher levels compared to control. serum levels of total protein are shown in the fig. 6. control group showed no significant change over time. ft group showed significant increase at 3 h which remained elevated until 24 h. st group showed steady increase in total protein over time. there was no significant difference among the groups at 0 h, however, ft group showed significantly lower values at 0.5 h compared to both control and st groups. at 3 h, st group had higher levels compared to control and at 24 h both ft and st groups showed higher levels compared to control. albumin levels had no change over time, in control group (fig. 7). ft group showed significantly lower values at 0.5 h compared to the other sampling points. st group showed ~ 2 folds increase at 3 h compared to the other sampling points. there was no significant difference among groups at 0 h. at 0.5 h, ft showed significantly lower levels compared to control. at 3 h, both ft and st groups showed significantly higher levels compared to control, furthermore, the values of st group was significantly higher than ft. control and st groups showed similar albumin levels which were significantly lower than ft group, at 24 h. there was no significant difference in globulin levels over the time in the control group (fig. 8). globulin levels at 3 and 24 h were significantly higher than 0 and 0.5 h in the ft group. st group showed significantly higher globulin levels at 24 h compared to the other sampling points. there were no significant differences among the groups at 0, 0.5 and 3 h, however, st group showed significantly higher levels compared to the control, at 24 h. discussion chemicals may be stressful for fish being reflected in changes of serum biochemical's levels (hoseini and tarkhani, 2012). serum cortisol elevation has been known as a primary stress response which increases rapidly after stress and cortisol is routinely used as an indicator of the stress in fish (wendelaar bonga, 1997; barton, 2002). hyperglycemia is secondary stress response, which is stimulated by primary stress responses (release of catecholamines and corticosteroids in to the circulation) to supply energy needed to cope with stress (wendelaar bonga, 1997; barton, 2002). formalin was found to a a a a a a a a a a b b b a b a a a ab a c b a a 0 0.5 1 1.5 2 before exposure immediately after exposure 3 h after exposure 24 h after exposure a lb u m in ( g / l) time (h) pvalue time = 0.0004 treatment < 0.0001 time × treatment < 0.001 control formalin nacl figure 7. effect of formalin and nacl treatments on serum albumin levels of goldfish c. auratus over the time. see figure 1 for elucidation. a a a a a a a aa a a a a b ab b a a a a a a b b 0 1 2 3 4 5 6 before exposure immediately after exposure 3 h after exposure 24 h after exposure g lo b u li n ( g / l) time (h) pvalue time = 0.35 treatment < 0.0001 time × treatment = 0.04 control formalin nacl figure 8. effect of formalin and nacl treatments on serum globulin levels of goldfish c. auratus over the time. see figure 1 for elucidation. 18 hoseini and reza tarkhani/ int. j. aquat. biol. (2013) (1) 14-21 damage gill tissue (wedemeyer, 1971; smith and piper, 1972) which could cause respiratory distress. the present results demonstrated that formalin and nacl exposure over 0.5 h were stressful for goldfish, since near 20 folds increase in circulating levels of cortisol was observed after treatment (0.5 h). it seems that ft group experienced severe stress compared with the st group because of higher glucose levels than st group at 0.5 and 3 h. also, results showed that ft and st groups did not recover from stress at 3 h, cortisol levels did not reach the pre-treatment levels at this point, despite a marked decrease. on the other hand, glucose levels remained high, particularly ft group which showed peak at this point. the higher cortisol level at 24 h in both ft and st groups seems not to be attributed to stress, since glucose levels reached pre-treatment levels at this point. glucose levels remain higher during stress (wendelaar bonga, 1997; barton, 2002) to ensure energy supply to cope stress. on the other hand, it was found that glucose levels remained elevated for a while even after stress termination (ruane et al., 2002; hoseini, 2010). higher cortisol levels in ft and st groups at 24 h might suggest that the fish were stressed as a result of formalin and nacl treatment. present results are in agreement with the previous studies (nieminen et al., 1983; kakuta et al., 1991; sanchez et al., 1997; hoseini and hosseini, 2010) which showed stress elevation as a result of formalin or nacl treatment in rainbow trout o. mykiss (walbaum) and common carp cyprinus carpio l. sodium and chloride are the most abundant blood ions. maintenance of hydromineral balance is crucial in fish. there are many studies reporting increase in sodium and chloride concentrations as a result of nacl exposure (abohegab and hanke, 1984; van der linden et al., 1999; hoseini and hosseini, 2010; hosseini and hoseini, 2010). passive ion transfer into and from the body lead to hemoconcentration during nacl exposure. no significant change in sodium and chloride levels in st group might be due to low salinity and short-term exposure. the results suggest osmotic disturbance occurrence due to formalin treatment since decrement in both ions were observed at 0.5 and 3 h. previous studies showed formalin treatment led to gill damage (wedemeyer, 1971; smith and piper, 1972) and gill is the most important organ in ion transport. the sodium and chloride levels returned to initial levels at 24 h, could suggest that gill damage was not irreversible over this period. on the other hand, stress might cause ion loss in freshwater fish. under stressful condition, fish need more oxygen, increase the respiration rate, which increases the gill permeability and passive ion loss (wendelaar bonga, 1997). thus, stress caused by formalin exposure could be, at least in part, responsible for lower sodium and chloride levels in ft group. calcium levels showed different patterns compared to sodium and chloride, after formalin and nacl treatment. it was expected that ions show similar pattern as a result of gill potential damage or stress. thus, the reason of the change in calcium levels is different of that of sodium and chloride. based on the measured parameters in this study, it is not clear why calcium showed such patterns. however, prolactin and somatolactine as well as corpuscles of stannius are involved in calcium regulation in fish (kaneko and hirano, 1993). likewise, flik and perry (1989) found hypercalcemic effect of cortisol via act on calcium pump in fish gill. measurement of prolactin, somatolactin and calcium pump activity might explain the observed changes in calcium levels. serum levels of protein change as a result of hemoconcentration or hemodilution (wood et al., 1983) as well as stress (almeida et al., 2005). however, present results are hard to interpret precisely. in ft group, decrease in protein at 0.5 h might be due to hemodilution, as the levels of sodium and chloride were significantly low at this point. however, increase in protein levels of both ft and st group, might be as a result of stress, as cortisol and glucose levels were high at this point. amino acids are an important source for gluconeogenesis in fish and the levels might increase during stress, although previous work showed inconstancy in total protein change during stress 19 hoseini and reza tarkhani/ int. j. aquat. biol. (2013) (1) 14-21 (wells et al., 1986; laidley and leatherland, 1988; pickering and pottinger, 1995; di marco et al., 2008). surprisingly, protein levels in both ft and st group were high at 24 h, when serum glucose, sodium and chloride levels had returned to initial levels. so that this elevation in serum protein might be due to another factor rather than stress or osmotic disturbance, which needs further research. on the other hand, patterns of albumin and globulin did not follow the pattern of total protein, suggesting change serum protein profile and alteration in liver protein synthesis. further work on serum protein profile and liver protein synthesis might illustrate the change observed in this study. in conclusion, formalin and nacl treatment at therapeutic concentrations caused rapid stress response in goldfish which was eliminated after 24 h recovery in freshwater, however, treated fish might be stressed at this point. formalin treatment, in addition, causes slightly osmotic disturbance which was eliminated after 24 h recovery in freshwater, too. however, formalin and nacl treatments cause serum calcium and protein profile alteration after a while, which lasted until, at least, 24 h and more detailed studies are needed to explain underlying mechanisms. formalin and nacl treatment, although advantageous in ecto-parasite removal, are stressful in goldfish which should be considered when are used. likewise, since formalin causes osmotic disturbance and more stress response (glucose levels), nacl treatment is suggested as alternative. refrerences abo hegab s., hanke w. 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(2021) 9(4) 264-267 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology short communication evaluation of length-weight relationship for a native goby, awaous jayakari (teleostei: gobiidae) in the middle east amir hassan masoumi1, saud m. al jufaili *2,1hamid reza esmaeili*1,3 1ichthyology and molecular systematics research laboratory, zoology section, department of biology, school of science, shiraz university, shiraz, iran. 2department of marine science and fisheries, sultan qaboos university, muscat, oman. 3center for hydrobiology and aquatic biotechnology, shiraz university, shiraz, iran. s article history: received 10 june 2021 accepted 22 august 2021 available online 2 5 august 2021 keywords: native fish, middle east, inland waters, oman, lwr. abstract: parameters of length-weight relationship of freshwater and marine fishes have long been of interest as a longstanding theme fisheries sciences and have been estimated for many fishes around the world. in this study, for the first time, length weight relationship (lwrs) was estimated for a native fish, the long nose goby, awaous jayakari (gobiidae) collected from the inland waters of oman in 2020. the parameter of b for male and female specimens based on standard length (sl) was within the range of 2.666-4.377 and 2.852-3.374, respectively. as proposed for different fishes there was high and significant correlation coefficients with r2≥0.97 between length and weight (0.9700.983). bailey’s ‘t’ test revealed that b value significantly deviated from 3 for both males and females showing their positive allometric growth pattern. introduction parameters of length-weight relationship of freshwater and marine fishes have long been of interest as a longstanding theme fisheries sciences and have been estimated for many fishes around the world. length-weight relationships (lwrs) have been implemented to assess status of fish populations in fisheries since the beginning of the 20th century (froese, 2006; jellyman et al., 2013; purrafee dizaj et al., 2020). lwrs of fishes act as a significant tool in fisheries science (esmaeili and ebrahimi, 2006; esmaeili et al., 2014, 2015; hossain and sultana, 2014; sadeghi and esmaeili, 2018; mouludi-saleh and eagderi, 2019; al-jufaili et al., 2021) and be able to convert length into weight, since during field studies the weight cannot always be determined accurately. also, in conjunction with several other parameters (e.g. sex ratio, age at first maturity, longevity, fecundity), population dynamics can be investigated. length and weight are biometric data easily taken and available in most datasets from *correspondence: saud m. al jufaili and hamid reza esmaeili doi: https://doi.org/10.22034/ijab.v9i14.1315 e-mail: hresmaeili@shirazu.ac.ir, sjufaily88@gmail.com monitoring studies (zuchi et al., 2020). awaous jayakari commonly referred to as the arabian or long-snout freshwater goby, is known so far from one locality in the desert area (wadi) of oman, and information on its detailed morphology and molecular systematics using fresh specimens is scare (eagderi et al., 2019; esmaeili et al., 2020). review of literatures revealed that no information is available on lwrs of a native goby fish of the middle east, awaous jayakari (boulenger, 1888) (see aljufaili et al., 2021). therefore, this study was carried out to describe and discuss its lwrs. materials and methods sampling site and procedure: the awaous jayakari specimens were collected from darsait al toyan, muscat, oman (fig. 1) using mostly foldable shrimp and crab fishing traps (mesh size of 3*3 mm, 3 nets for overnight). the sampling was conducted twice in winter 2020. total length (tl) and standard length (sl) of the specimens were measured to the nearest 265 int. j. aquat. biol. (2021) 9(4): 264-267 0.1 mm using digital calipers attached to computer. the specimens were weighed to the nearest 0.01 g (total weight, tw) using a digital electronic balance. fish individuals were classified according to sex based on the shape of urogenital papilla. to do this, urogenital papilla was examined under a stereo microscope (zeiss stemi sv8). the parameters of the length–weight relationships were calculated using formula of w = al b and expressed by linear regression of the log-transformed weight and length which gives the linear equation (koutrakis and tsikliras, 2003). logw = loga + b logl where w = total weight in grams, l= length in cm, a = a constant being the initial growth index, and b = growth coefficient. prior to regression analysis, log–log plots of length and weight values were performed for visual inspection of outliers (froese, 2006). the significance of the regression between lwrs of fishes was tested by anova. the regression coefficients for male and female specimens were compared using students ‘t’ test to study the variations in the b values between sexes (zar, 1974). bailey's ttest was used to find out whether 'b' value significantly deviated from the expected cube value of 3. data were analyzed statistically by using ibm spss (version 22) statistical software package. results and discussion the length–weight parameters for a. jayakari are given in table 1 including the length range and weight ranges, as well as the equation parameters a and b together with their 95% confidence intervals and the correlation coefficients. lwr was significant (p<0.001) with a high correlation coefficient with r2≥0.97. bailey’s ‘t’ test showed that b value significantly deviated from 3 in both sexes showing positive allometric growth pattern (p<0.05). in the present study, the data on length-weight relationship for the fish a. jayakari is presented to clarify some characteristics of its population dynamics. the mean b value of length-weight relationships based on tl was 3.723 for males and 3.282 for females, being within the expected range of 2 to 4 (tesch, 1971). the mean b value based on sl was 3.521 for males and 3.113 for females being within the expected range. higher b value for males might be due to low number of available specimens (see froese, 2006). however, different in the b value in sexes in high number of specimens, indicating that one sex might be heavier than another one with the same length which might be due to difference in fatness, gonadal development and less metabolic activity as stated by hossain and sultana (2014). it has been already reported that variation in b value in fishes might be due to several factors, including season, figure 1. collection site of awaous jayakari. 266 masoumi et al./ length-weight relationship for a native goby, awaous jayakari species, habitat, sex, gonad maturity, diet, stomach fullness, health, preservation techniques and locality (esmaeili, 2001; le cren, 1951; sadeghi and esmaeili, 2018; purrafee dizaj et al., 2020; eagderi et al., 2020; al jufaili et al., 2021). differences in the lwrs could be due to the combination of one or more of the above factors. in conclusion, this study provides the first basic information on lwrs of a. jayakari from the middle east which will be useful in their fisheries and conservation management. acknowledgment we are thankful to sultan qaboos university (squ) and shiraz university for financial supports. references al jufaili s.m., sayyadzadeh g., jawad l., esmaeili h.r. (2021). length-weight relationships of five fish species from the inland waters of oman. iranian journal of ichthyology, 8(1): 63-67. eagderi s., mouludi-saleh a., cicek e. (2020). lengthweight relationship of ten species of leuciscinae subfamily (cyprinidae) from iranian inland waters. international aquatic research, 12(2): 133-136. eagderi s., fricke r., esmaeili h.r., jalili p. (2019). annotated checklist of the fishes of the persian gulf: diversity and conservation status. iranian journal of ichthyology, 6: 1-171. esmaeili h.r. ( 2001). biology of an exotic fish, silver carp, hypophthalmichthys molitrix (val., 1844) from gobindsagar reservoir, himachal pradesh, india. phd thesis. panjab university, chandigarh, india. esmaeili h.r., ebrahimi m. (2006). length-weight relationships of some freshwater fishes of iran. journal of applied ichthyology, 22(4): 328-329. esmaeili h.r., gholamifard a., vatandoust s., sayyadzadeh g., zare r., babaei s. (2014). length– weight relationships for 37 freshwater fish species of iran. journal of applied ichthyology, 30(5): 1073-1076. esmaeili h.r., masoudi m., sayyadzadeh g., mehraban h. r., gholami z., teimori a. (2015). length–weight relationships for four aphanius species of iran (teleostei: cyprinodontidae). journal of applied ichthyology, 31(3): 578-579. esmaeili h.r., sadeghi r., larson h.k. (2020). the longsnout freshwater goby awaous jayakari (boulenger, 1888)(teleostei: gobiidae), an additional fish element for the iranian waters. zoology in the middle east, 66(1): 29-36. froese r. (2006). cube law, condition factor and weight– length relationships: history, meta‐analysis and recommendations. journal of applied ichthyology, 22(4): 241-253. hossain m., sultana n. (2014). morphometric characters and length-weight relationship of bele, (glossogobius giuris) from mithamoin haor, kissorgonj, bangladesh. journal of the bangladesh agricultural university 12(2): 389-395. jellyman p.g., booker d.j., crow s.k., bonnett m.l., jellyman d.j. (2013). does one size fit all? an evaluation of length–weight relationships for new zealand's freshwater fish species. new zealand journal of marine and freshwater research, 47(4): 450-468. koutrakis e., tsikliras a. (2003). length–weight relationships of fishes from three northern aegean estuarine systems (greece). journal of applied ichthyology, 19(4): 258-260. le cren e. (1951). the length-weight relationship and seasonal cycle in gonad weight and condition in the perch (perca fluviatilis). the journal of animal ecology, 201-219. mouludi-saleh a., eagderi s. (2019). length-weight relationship and condition factor of ten fish species (cyprinidae, sisoridae, mugilidae, cichlidae, gobiidae and channidae) from iranian inland waters. journal of table 1. descriptive statistics and parameters of lwrs for awaous jayakari from oman. sex n tl/sl range (cm) w range (g) a 95% ci of a b 95% ci of b r 2 tl.f 19 5.73-13.03 1.40-19.74 0.0052 0.0027-0.0099 3.282 2.987-3.577 0.970 sl.f 19 4.55-10.90 1.40-19.74 0.014388 0.0086-0.0242 3.113 2.852-3.374 0.974 tl.m 5 6.08-12.50 1.79-21.92 0.002183 0.0003-0.0174 3.723 2.778-4.668 0.981 sl.m 5 4.84-10.27 1.79-21.92 0.007244 0.0013-0.0396 3.521 2.666-4.377 0.983 m, male; f, female; n, number of specimens; tl, total length; sl, standard length; w, weight; a, intercept; b, regression slope; r2, correlation coefficient. 267 int. j. aquat. biol. (2021) 9(4): 264-267 wilidlife and biodiversity 3(4): 12-15. purrafee dizaj l.p., esmaeili h.r., abbasi k., valinassab t., salarpouri a. (2020). does length-weight equation fit clupeid fishes? an evaluation of lwrs for six clupeids from iran (teleostei: clupeiformes). international journal of aquatic biology, 8(2): 126131. sadeghi r., esmaeili h.r. (2018). length‐weight relationships of three gobiid species (perciformes: gobiidae) along the iranian intertidal coast of the persian gulf and makran sea. journal of applied ichthyology, 5: 1233-1234. tesch f.w. (1971). age and growth. in: w. e. ricker (ed.), methods for assessment of fish production in fresh waters. blackwell scientific publications, oxford. pp: 99-130. zar h.j. (1974). biostatistical analysis. prentice hall, new jersey. 718 p. zuchi n., röpke c., shibuya a., farago t., carmona m., zuanon j., amadio s. (2020). length‐weight relationship of fish species from central amazon floodplain. journal of applied ichthyology, 36: 837841. int. j. aquat. biol. (2022) 10(5): 429-437 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article fish diversity of the euphrates river between medina and qurna cities, southern iraq sajad a. abdullah1, abdul hussein j. abdullah2, yasser w. ouda3 1department of biology, college of education-qurna, university of basrah, iraq. 2department of marine vertebrate, marine science center, university of basrah, iraq. 3department of biology, college of education-qurna, university of basrah, iraq. s article history: received 5 june 2022 accepted 15 august 2022 available online 2 5 october 2022 keywords: ecology biodiversity euphrates inland waters abstract: the present study was conducted to evaluate the fish community and diversity status in the euphrates river between medina and qurna cities, northwest of basrah province, iraq. in addition, the relationship between some environmental parameters, including water temperature, dissolved oxygen, salinity, ph, and turbidity, and fish abundance were investigated by sampling from november 2020 to october 2021. based on the results, there were no significant differences (p>0.05) in the environmental factors between the two studied sites except for salinity and turbidity. during the study, a total of 7387 fishes were collected belonging to 12 families, 21 genera, and 22 species. the cyprinidae was dominated by seven species, followed by the luciscidac with three species, the cichlidae and mugilidae families with two each, and the rest with one species each. there were ten natives, eight exotics, and four marine fish species. planiliza abuc was dominat species with 26.11 % of the total catch. significant differences (p<0.05) were found in species richness and number of the collected individuals between two sites. based on the results, fish species can be classified into three groups based on their distribution; common species with nine species, accounting for 98,66%; seasonal species with four species accounting for 0.798% and rare species, with nine species accounting 0.541% of total richness. three species of p. abu, coptodon zillii and carassius auratus were dominant species with 63.110% of the total catch. introduction a study of fish communities gives a clear picture of their stocks (dwivedi et al., 2016) that are used in managing aquatic systems (simonovic et al., 2017; pyron et al., 2019). the fish community is affected by environmental factors that can be observed using diversity indices (abdullah et al., 2019b; al-helli et al., 2019; zarei and krakhmalnyi, 2019) such as richness, diversity, and evenness (abbas et al., 2017). several studies have been performed in the inland waters of iraq to investigate the environmental and biological effects the fish composition e.g. in the euphrates river, al-noor et al. (2009) analyzed fish population structure and recorded 21 species, hussein et al. (2015) worked the fish structure in the euphrates river between chibyaish and mudyna and recorded 24 species belonging to seven families, and 12 species, khaddara (2014) studied the physiochemical of fish correspondence: sajad a. abdullah doi: https://doi.org/10.22034/ijab.v10i5.1762 e-mail: sajad.aballah@uobasrah.edu.iq dor: 20.1001.1.23830956.2022.10.5.10.1 structure in the euphrates river, abdullah (2017) documented the fishes of the tigris river at alqurna city and abood (2018) reported the structure of fish assemblages in the shatt al-arab river. in this regard, the current study aimed to assess the fish structure of the euphrates river's medina and qurna cities, southern iraq using diversity indices by providing their relationships by some environmental parameters. material and methods the euphrates river has a length of 2775 km as the 27th longest river in the world (vander, 1975). it feeds large agricultural land in the northern basrah province of iraq. for this study, two sampling sites were selected between the cities of medina and qurna, including (1) the northwest site in the almedina bridge (718852n, 3425256e) and (2) al qurna city at pipe bridge (729605n, 3431444e). https://ij-aquaticbiology.com/index.php/ijab/article/view/1762 430 sajad et al./ fish diversity of the euphrates river between medina and qurna cities water samples were collected from the study area monthly from the middle of the river at a depth of 20 cm. water physiochemical parameters were measured during fish sampling between november 2020 and october 2021. water temperature (°c) was measured using a thermometer, dissolved oxygen (do) using a ysi55 device, salinity as psu using extech 31156 device, ph using lovibond-senso direct 150 device, turbidity as nephelometric turbidity unit (ntu) by hanna hi93703 device. several fishing methods, such as bottom net (160 m in length with 6.4 mm mesh size), gill nets (1x60 m2 with 15 mm mesh size), cast net (with a diameter of 4 m with 1.5 mesh size) and a hand net for small fishes were used to collect fishes. in addition, some data are based on the catches of local fishermen. the fish were identified based on carpenter (1997), coad (2015), and eagderi et al. (2019, 2022). the relative abundance of fish species was calculated according to odum (1979). biodiversity indices, including diversity (shanon, 1949), evenness (j) (pielou, 1977) and richness index (d) (margalef, 1968) were also calculated. fish were also divided based on their occurrence in the monthly samples (tyler, 1971), and dominance (d3) (kwak and peterson, 2007). spss (ver. 19) was used for statistical analysis by applying anova to assess differences in ecological indices, the number of species and individuals (p≤0.05) between the two sites and principal component analysis (pca) to analyze the relationship between physicochemical characteristics and the study period's months. results water temperatures, salinity and do during the study period are presented in figure 1. water temperatures were between 11°c in january and 36.5°c in july, with a mean of 21.3±8.5°c. do was recorded between 7.4 mg/l in july and 8.9 mg/l in january, with a mean of 8.07±0.43 mg/l. salinity levels ranged from 1 g/l in february to 2.6 g/l in july, with a mean of 1.7±0.5 g/l. ph was within the alkaline range ranging from 6.9 in july to 8.5 in march, with a mean of 7.85±0.4 (fig. 2). the turbidity ranged from 7.8 ntu in august to 23.6 ntu in december, with a mean of 7.85±0.49. there were no significant differences between the environmental parameters of the two sites (p>0.05) except salinity and turbidity. in the pca, the first two pcs accounted 82.79 and 11.11% of the variances, respectively (in total 93.90%). the pc1 mostly includes factors of temperature and salinity. there was a correlation between july and september. some parameters, such as do and ph were correlated with january, february, march, april, and november. based on the results, there is a positive correlation between turbidity and december and may (fig. 3.) figure 4 shows the correlations between the abundance and fish richness and water temperature, dissolved oxygen, salinity, ph, and turbidity. the water temperature positively correlated with the figure 1. monthly variations in water temperature, dissolved oxygen and salinity in the study area of the euphrates river during 2020-2021. 431 int. j. aquat. biol. (2022) 10(5): 429-437 richness and species abundance (r = 0.430, r = 0.07, respectively). the richness and fish’s abundances had weak positive correlation (r = 0.45, r = 0.08, respectively) with salinity and negative correlation (r = -0.31, r = -0.01, respectively) with do and turbidity (r = -0.69, r = -0.12, respectively). ph showed a negative correlation (r = -0.38) with richness and a positive correlation (r = 0.25) with the fish’s abundances. a total of 7387 fish individuals were collected during the study period belonging to 12 families, 21 genera, and 22 species, of which 10 were native, eight exotic fish species, and four marines. the cyprinidae was dominated group by seven species, followed by the luciscidac with three, the cichlidae and mugilidae with two species each, and the rest with one species each (table 1). the monthly variation in the abundance and richness in the two study sites are shown in table 2. at the northwestern sites, the total number of individuals was 3515, ranging from 154 in june to 423 in march, including 17 species ranging from 8 in february to 13 in may. the total number of individuals in southeast sites was 3872, ranging from 223 in january to 426 in may. there were 19 collected species in this site, ranging from ni9ne in may to 15 in august. the results showed significant differences (p<0.05) in species and individuals between the two studied sites. the relative abundance of fish species ranges from 376 in june to 724 in may, with 22 species and 21 genera. the species ranged from ten in december 2020 to 15 in july, with the lowest numerical abundance as 5.1% in june and 9.8% in may. panaliza abu was dominated species, with 1929 specimens and 26.11% of the catch, ranging from 9.9% in june to 29.8% in february. coptodon zilli had the second position with 1807 individuals and 24.46 %, ranging from 8.5% in june to 24.9% in july. the collected c. auratus was 926, accounting for 12.54% of the total catch, fluctuating from 5.9% in november to 18.4% in december. alburnus sellal constitutes 12.33% of the total composition ranging from 3.5% in july to 14.7% in november, with 911 specimens. oreochromis auratus had 11.09% of the total catch, then carasobarbus luteus with 9.46%, cyprinus carpio with 1.11%, and siluris triostegus with 0.80%. the rest species ranged from 0.76%, representing a. marmiad species, to c. idella and g. rufa, which accounted for 0.01% of the total catch (table 3). the monthly variation in the number of species is depicted in figure 5. the number of native species fluctuated from four in november to eight in january 2021. the alien species number differs between figure 2. monthly variations in hydrogen ion and turbidity in two sites of the euphrates river during 2020-2021. nove. 2020 dece. janu. 2021 febr. march april may june july augu. septe. octo. temp. salin. do turb. ph -2 -1 0 1 2 3 4 5 6 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 f 2 ( 1 1 .1 1 % ) f1 (82.79 %) biplot (axes f1 and f2: 93.90 %) figure 3. principal component analysis between physicochemical factors and months in the euphrates river during 2020-2021. 432 sajad et al./ fish diversity of the euphrates river between medina and qurna cities three in december and seven in july. the abundance of marine species at the second station was notable, with one species recorded in december, january, march, may, july, and october and four in november. the results of biodiversity indices, including diversity (h), evenness (j) and richness (d), are presented in figure 6. the lowest rate was recorded as 1.432 in december and the highest as 3.204 in july. the range of the evenness index was 0.365 in november to 0.754 in september. the values of the richness index were between 1.48 in august and 3.57 in october. the fish assemblage in the study area was divided into three groups (fig. 7). the common species comprised nine, accounting for 98.695% of the total catch, including p. abu, c. zillii, c. auratus, a. sellal, o. aureus, c. luteus, c. carpio, s. triostegusa and a. marmid. relative abundance% family habitat species no 23.40 cyprinidae f f f f f f f carassius auratus + 1 carasobarbus luteus* garra rufa * 2 3 ctenopharyngodon idella+ cyprinus carpio + hemiculter leucisculus + 4 5 6 mesopotamichthys sharpeyi* 7 35.55 cichlidae f coptodon zillii + 8 oreochromis aureus+ 9 26.22 mugilidae f m planiliza abu* 10 planiliza subviridis 11 13.60 leuciscidae f f f acanthobrama marmid* alburnus sellal * 12 13 leuciscus vorax* 14 0.798 siluridae f silurus triostegus* 15 0.09 clupeidae m tenualosa ilisha* 16 0.08 poeciliidae f gambusia holbrooki + 17 0.07 mastacembelidae f mastacembelus mastacembelus* 18 0.05 heteropneustidae f heteropneustes foosilis + 19 0.05 sparidae m acanthopagrus arabicus 20 0.04 engraulidae m thryssa whiteheadi 21 0.03 cyprinodontidae f aphanius dispar* 22 * = native species, + = alien species, f=fresh water species, and m= marine water species. table 2. monthly variations in the number and richness of fish species in the euphrates river from november 2020 to october 2021. richness number of individuals months site 1 site 2 site 1 site 2 november 2020 10 14 321 393 december 9 12 221 279 january 2021 12 10 275 223 february 8 13 298 358 march 11 14 423 272 april 10 12 350 332 may 13 9 298 426 june 10 13 154 231 july 12 14 265 323 august 11 15 297 371 september 11 12 271 401 october 9 13 342 263 total 3515 3872 table 1. fish species were collected from november 2020 to october 2021. 4 3 3 in t. j . a q u at . b io l. ( 2 0 2 2 ) 1 0 (5 ): 4 2 9 -4 3 7 t ab le 3 . m o n th ly v ar ia ti o n s in t h e re la ti v e ab u n d an ce o f sp ec ie s in t h e e u p h ra te s r iv er f ro m n o v em b er 2 0 2 0 t o o ct o b er 2 0 2 1 ( e u p h ra te s r iv er ). o c t. s e p . a u g . ju ly ju n e m a y a p r. m a r. f e b . ja n . 2 0 2 1 d e c . 2 0 2 0 n o v . 2 0 2 0 t . n u m % n u m . % n u m . % n u m . % n u m . % n u m . % n u m . % n u m . % n u m . % n u m . % n u m . % n u m . % n u m . 1 9 2 9 2 5 .1 1 7 5 1 9 .5 1 7 8 1 8 .9 1 5 6 2 6 .7 1 7 8 9 .9 5 5 1 8 .2 1 7 5 1 1 .4 1 2 6 2 8 .1 2 5 9 2 9 .8 2 2 0 1 7 .8 1 0 2 1 9 .7 1 2 0 2 2 .4 1 9 0 p e n a li za a b u 1 8 0 7 2 0 .6 1 4 4 1 8 .5 1 6 7 1 9 .5 1 6 1 2 4 .9 1 6 6 8 .5 4 7 1 7 .2 1 6 5 2 0 .0 2 2 1 2 1 .9 1 9 9 2 2 .4 1 6 5 2 0 .9 1 2 0 1 2 .3 7 5 2 0 .8 1 7 7 c o p to d o n z il i 9 2 6 1 4 .0 9 8 8 .6 7 8 1 2 .7 1 0 5 1 2 .0 8 0 1 3 .7 7 6 8 .2 7 9 5 .0 5 5 6 .. 6 6 0 8 .9 6 6 1 1 .7 6 7 1 8 .4 1 1 2 5 .9 5 0 c a ra ss iu s a u ra tu s 9 1 1 9 .6 6 7 9 .4 8 5 8 .8 7 3 3 .5 2 3 9 .7 5 4 7 .9 7 6 1 0 .4 1 1 5 8 .6 7 8 1 0 .2 7 6 1 3 .1 7 5 1 0 .7 6 5 4 .7 1 2 5 a lb u rn u s se ll a l 8 1 9 8 .0 5 6 5 .5 5 0 1 2 .2 1 0 1 8 .9 5 9 1 2 .1 6 7 8 .8 8 5 7 .1 7 9 6 .2 5 6 8 .9 6 5 3 .8 2 5 5 .7 3 5 1 6 .8 1 4 3 o re o c h ro m is a u re u s 6 9 9 6 .2 4 3 6 .8 6 1 5 .0 4 1 8 .1 5 4 1 0 .8 6 0 8 .9 8 6 5 .2 5 7 3 .8 3 5 6 .9 5 1 1 8 .3 1 2 0 1 4 .3 8 7 2 .2 1 9 c a ra so b a rb u s lu te u s 8 2 0 .3 2 3 .9 3 5 1 .2 1 0 1 .2 8 1 .3 7 0 .6 6 0 .6 7 0 .1 1 0 .5 3 0 .4 3 c y p ri n u s c a rp io 5 9 0 .3 2 0 .2 2 1 .0 8 0 .9 6 2 .8 2 7 0 .5 6 0 .5 5 0 .2 1 0 .2 2 s il u ru s tr io st e g u s 5 6 0 .3 2 1 .1 1 0 1 .1 9 0 .9 5 0 .7 7 0 .5 6 1 .1 8 1 .0 6 0 .5 3 a c a n th o b ra m a m a rm id 3 8 1 .7 1 2 0 .4 4 0 .6 4 1 .2 1 2 0 .3 3 0 .3 2 0 .2 1 l e u c is c u s v o ra x 1 6 0 .8 5 0 .4 4 0 .4 4 0 .2 2 0 .2 1 h e m ic u lt e r le u c is c u lu s 8 0 .1 1 0 .1 1 0 .1 1 0 .2 1 0 .1 1 0 .1 1 0 .2 1 0 .1 1 p e n a li za s u b v ir id is 7 0 .1 1 0 .1 1 0 .2 1 0 .2 1 0 .1 1 0 .1 1 0 .1 1 t e n u a lo sa i li sh a 6 0 .1 1 0 .1 1 0 .2 1 0 .2 1 0 .1 1 0 .1 1 g a m b u si a h o lb ro o k i 5 0 .2 1 0 .2 1 0 .1 1 0 .1 1 0 .2 1 m a st a c e m b e lu s m a st a c e m b e lu s 4 0 .1 1 0 .1 1 0 .1 1 0 .1 1 a c a n th o p a g ru s a ra b ic u s 4 0 .1 1 0 .2 1 0 .1 1 0 .1 1 h e te ro p n e u st e s fo ss il is 4 0 .1 1 0 .1 1 0 .2 1 0 .2 1 m e so p o ta m ic h th y s sh a rp e y i 3 0 .1 1 0 .2 1 0 .1 1 t h ry ss a w h it e h e a d i 2 0 .2 1 0 .1 1 a p h a n iu s d is p a r 1 0 .1 1 c te n o p h a ry n g o d o n id e ll a 1 0 .2 1 g a rr a r u fa 6 0 5 6 7 2 6 6 8 5 8 8 3 7 6 7 2 4 6 8 2 6 9 5 6 5 6 5 0 7 5 0 0 7 1 4 n u m b e r o f in d iv id u a ls 8 .2 9 .1 9 .0 8 .0 5 .1 9 .8 9 .2 9 .4 8 .9 6 .9 6 .8 9 .7 r e la ti v e a b u n d a n c e 1 4 1 4 1 3 1 5 1 3 1 3 1 4 1 3 1 2 1 4 1 0 1 3 n u m b e r o f s p e c ie s 434 sajad et al./ fish diversity of the euphrates river between medina and qurna cities seasonal species included four species accounting 0.798% of the total catch: l. vorax, p. subviridis, t. ilisha, and g. holbrooki. the occasional species consist of night species and by 0.541% formed h.leucisculus, m. mastacembelus, a. arabicus, h. foosilis, m. sharpeyi, t. whiteheadi, a. dispa, c. idella and g. rufa. three species of p. abu, c. zillii and c. auratus were 63.1% of the total catch and p. abu with 26.11 % had the highest dominance index (d3) (fig. 8) -1 -0.5 0 0.5 1 water temp. do salinity ph turbidity no. of species no.of individuals figure 4. some ecological factors that influenced the richness and individuals of fishes in the euphrates river during 2020-2021. figure 5. monthly variation in the number of exotic, marine, and native species in study sites during november 2020 to october 2021 in the euphrates river. figure 6. monthly variations in the values of biodiversity indices in the study area in the euphrates river during 2020-2021. 435 int. j. aquat. biol. (2022) 10(5): 429-437 discussion ecological factors directly impact fish distribution in aquatic ecosystems (roa-fuentes and casatti 2017). based on the results, do was negatively correlated with water temperature and salinity, but do and ph had little fluctuation despite the temperature. salinity is changed based on various factors, including evapotranspiration, agriculture fertilization, soil erosion, runoff water and other anthropogenic activities, and its value was within the permissible limits for the collected fishes (al-helli et al., 2019). turbidity can be caused by a variety of factors, including dissolved organic compounds, bacteria, microscopic algae, suspended clay particles and colloidal solids, and it was in two sites were to be above 5 ntu, indicating a higher rate (who, 2010). the fish fauna of the studied area was 22 species belonging to 21 genera and 12 families, including 10 natives, 8 exotics, and 4 marines; the family of cyprinidae was dominated taxa. lazem and atte (2016) in their work to assess the fish structure in the euphrates river at al-samawa city, recorded 24 fish species that some of them not found in our work, including a. grypus, c. sublimus, c. kais, and l. xanthopterus. mohamed and abood (2017) on the assemblage of shatt al-arab structure in the supremacy of cyprinidae and the difference of species number caught and ecological factors during his study. the rest of the families, such as the luciscidac family, have three species each, the cichlidae and mugilidae families have two species, while the remainder of the families has only one species; this study coincided with many researchers in recording several families in iraqi inland waters such as hossain et al. (2012), coad (2015). at both sites, p. abu, c. zillii, c. auratus and a. sellal were the most abundant species forming 69.3% of the total catch. the rest of the species had little abundance (30.7%) of the total number of individuals in the study area. the native fish species constituted 50.142% of the total catch, but eight exotic species consist 49.56% of the total catch. alamari et al. (2012) reported some of these fishes in the euphrates river near al-hinia town, showing a high occurrence of exotic fishes that can threaten the native fishes as they grow faster by declining the water quality of the euphrates river. the second site had four marine species, accounting for 0.297% of the total catch. abdullah et al. (2021) reported increasing marine species in the lower euphrates river. the biodiversity indices are used as a single number to describe the diversity of a system (magurran, 1988). the biodiversity index values (h) in this study range from poor to medium, according to shannon-weaver index values, revealing differences between sites due to the appearance of marine species (younis et al., 2010) in the second site. the result of the evenness index (j) indicates figure 7. fish species occurrence in fishing samples from the two study sites from november 2020 to october 2021 in the euphrates river. figure 8. dominated three fish species in the study area from november 2020 to october 2021 in the euphrates river. 436 sajad et al./ fish diversity of the euphrates river between medina and qurna cities that two sites are within the half balanced to the unbalanced range, with a minor increase in the second site, due to the presence of some marine species in the lower of the euphrates river (mohamed et al., 2015). the richness (d) of the fish structure in the euphrates river, according to jorgensen et al. (2005), varied between disturbed and semi-complete, as found in the current work. according to tyler (1971), the fish assemblage in the studied area was divided into three groups; common, seasonal, and occasional fish species. panaliza abu, c. zillii, c. auratus, a. sellal, o. aureus, and c. luteus were common species, i.e they are collected during the 12 months sampling period. mohamed and al-jubouri (2017) have also reported the presence of these species in a study of the fish community in the al-diwaniya river. other species found in the monthly fishing samples were c. carpio, s. triostegusa, and a. marmid in this river (al-zaidy et al., 2019). four seasonal fish species (l. vorax, p. subviridis, t. ilisha, and g. holbrooki) were recorded i.e. their presence varied monthly. the rest of the fishes were rare species. the three most abundant fish species of p. abu, c. zillii and c. auratus were dominant (d3) in the euphrates river as reported by other researchers in iraqi inland waters (salman, 2012; jawad et al., 2021). acknowledgment the authors thank o. saleh in the qurna agriculture directorate for mapping the study area, and the department of life sciences at the college of education qurna, university of basra, for their continuous support of this work. references abbas l.m., abu-elhine a.j., radhy a.g., hassan a.h. 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(2010). ecological assessment of fish assemblage in the shatt al-arab river-karmat ali, basrah using integrated biological index (ibi). journal of the university of karbala, 2010 special issue 1: 22-31. zarei darki b., krakhmalnyi a.f. (2019). biotic and abiotic factors affect the population dynamics of ceratium hirundinella, peridinium cinctum, and peridiniopsis elpatiewskyi. diversity, 11(8): 137. international journal of aquatic biology (2014) 2(6): 292-298 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2014 npajournals. all rights reserved original article the effects of different levels of beta plus on growth performance, microbial flora and blood parameters of caspian trout, salmo caspius (kessler, 1877) sadegh karimzadeh1, abdolsamad keramat amirkolaie*2,1soodabeh parhizkar miandehy3 1department of animal sciences, roudaki higher education institute, tonekabon, mazandaran, iran. 2department of fisheries, faculty of animal sciences and fisheries, sari agricultural sciences and natural resources university, km 9 darya boulevard, p.o.box: 578, sari, iran. 3biochem tehran co., postal code: 14197-987, tehran, iran. article history: received 25 march 2014 accepted 29 october 2014 available online 2 5 december 2014 keywords: microbial flora caspian trout growth performance survival rate abstract: this study was conducted to evaluate the effects of beta plus probiotic, a mixture of bacillius leicheniformic and bacillius subtilis, on the growth performance and intestinal microbial flora of caspian trout (salmo caspius). a basal diet was formulated and supplemented with beta plus at 0, 0.5 and 1 g kg-1, leading to three experimental diets. each experimental diet was randomly assigned to quadruple 1500 l tanks. the caspian trout with an initial weight of 108.7 ± 1.8 g were randomly distributed in the experimental tanks. the results showed that inclusion of dietary beta plus significantly increased the final weight and specific growth rate (sgr) of caspian trout compared to those the control treatment. the feed conversion ratio (fcr) was also improved significantly after probiotic administration to the experimental fish. however, the body composition and blood parameters were not influenced by the probiotic inclusion. total count of gram positive and negative bacteria in the intestine of the fish increased by feeding on diet contained 1 g kg-1 beta plus (p<0.05). in conclusion, administration of the probiotic beta plus can improve the nutrient efficiency and growth performance of caspian trout confirming the positive effect of a mixture of bacillus spp. introduction the caspian trout, salmo caspius, is a valuable commercial species and known as one of the nine brown trout species (quillet et al., 1992). caspian trout is distributed mainly at the western and southern cost of caspian sea (kiabi et al., 1999) and showed a better growth and higher weight gain in compare to other brown trout populations (sedgwick, 1995). the population of this species shows a steady decline over the past two decades and considered to be an endangered species in the southern of caspian sea (kiabi et al., 1999). recent development in aquaculture industry may lead to introduction of the caspian trout as a new candidate. this species can be cultured in cage, concrete pond and flow-through system (kalbassi et * corresponding author: abdolsamad keramat amirkolaie e-mail address: amirkola@yahoo.com al., 2006; sarvi et al., 2006). intensive production system may deteriorate water quality, increasing stress level and incident of disease (losordo et al., 1999). these conditions can threat fish immune system leading to more vulnerability to bacterial infection (liltved and cripps, 1999; irianto and austin, 2002). in such a condition, antibiotics have been introduced as a solution to prevent disease and enhance growth performance in fish (burr et al., 2005). however, an increase in antibiotic use may have negative side effects such as the development of antibioticresistant bacteria which makes the use of antibiotic growth promoters undesirable. in addition, some antibiotics make fish more susceptible to bacteria, viral, and parasitical disease (gatlin, 2006). the 293 karimzadeh et al/ beta plus and caspian trout public concern of antibiotic concentration in food has been resulted in the regulation of strict measures on antibiotic usage for farm animal treatment (patterson and bourkholder, 2003). probiotics are usually live microorganisms that may confer a health benefits on host (fuller, 1992). in aquaculture, probiotics have been used to control diseases, enhance specific and non-specific immunity (mccracken and gaskins 1999; verschuere et al., 2000) providing nutrients and enzymatic functions (wache et al., 2006), and improve water quality (balcazar et al., 2006). the lactic acid bacteria, including bacillus spp. are widely used as probiotics in humans and their application have led to health benefits against gastrointestinal disorders including diarrhea, inflammatory bowel disease, and lactose (madsen, 2001). in addition, bacillus and lactobacillus have been supplemented as an immune-stimulator to enhanced the growth, innate immune responses, and disease resistance of a number of fish species (sun et al., 2010; geng et al., 2011; geng et al., 2012). the lactic acid bacteria are also known to be present in the intestine of healthy fish (balcazar et al., 2007). bioplusis a commercial probiotic composing naturally occurring bacteria strains including, bacillus subtilis and bacillus licheniformis, which improved growth parameters of rainbow trout larvae (alizadeh et al., 2011). a mixture of bacillus subtilis, bacillus licheniformis, lactobacillus spp. and arthrobacter spp. improved immune system, growth and lower mortality against the pathogens of cobia, rachycentronc anadum, (geng et al., 2012). bacillus sp. also changes gut microbial composition in rainbow trout, oncorhynchus mykiss (ramos et al., 2013) since some work is available on the effects of different probiotics on fish performance and health parameters, but a little information is available on the effect of a mixture of bacillus sp. on those parameters in caspian trout. therefore, this study was conducted to assess the effects of beta plus as a probiotic on the fish performance, survival rate and microbial flora in the caspian trout materials and methods experimental system and animal: this study was carried out at an experimental facility of rainbow trout farm located in the northern part of iran (savadkoh, mazandaran province, iran). the caspian trout juveniles were propagated at a nearby reproduction facility and adapted to a commercial diet (chineh. company, iran) and new environment for a week prior the experiment. after adaptation period, the fish with initial weight of 108.7 ± 1.8 g were divided randomly into twelve 1500 l tanks, with a stocking density of 25 individuals per tank. the experiment was lasted 8 weeks. the used probiotic i.e., beta plus (bp), composes bacillius leicheniformic (dsm 5749), bacillius subtilis (dsm 5750) and betaien with a commercial name of beta plus (biochem co., germany). a basal diet was formulated with estimated gross energy and protein levels of 12.56 mj kg-1 and 433 g kg-1, respectively (table 1). three levels of bp (0, experimental diet amount soybean meal 15 fish meal 42 meat meal 15 corn 10 wheat 7 fish oil 5 molasses 2 dl-methionin 0.84 nacl 0.4 lysine 0.76 premix 2 nutrient composition of the experimental diet percentage (dry weight) moisture 6.5 crudeprotein 43 crude fat 12 crude ash 10 energy (kcal/kg) 3650 table 1. the percentage of ingredient used in the experimental diet on percentage dry matter weight basis. 294 international journal of aquatic biology (2014) 2(6): 292-298 0.5 and 1.5 g kg-1 prebiotic diet) were added to the basal diet to prepare three experimental diets. all ingredients were finely ground, mixed and pelletized. a pellet maker with 4 mm diameter die was used for producing the diets. all three experimental diets were air-dried at 50°c at the same time and stored at -20°c. experimental procedure: fish were weighed on the 1st and the last day of the experiment. they were hand fed with the experimental diets at the rate of 1.2-1.6% of biomass, three times a day (8.00, 13.00, and 18.00 h). the experimental diets were randomly assigned to three treatments including 12 tanks i.e. four replicates per diet. water quality parameters were monitored daily to ensure being in appropriate range. the water temperature and ph were 9-11°c and 7.3-7.9, respectively, during the experiment. the oxygen concentration was always above 8 mg l-1. in addition, water flow was 12 l min-1 for each tank. on day 56, all fish were weighed and five specimens were randomly sampled from every experimental tank. they were sacrificed using an overdose of clove essence solution and analyzed their body composition. three specimen were randomly selected from each tank for bacterial count. for analyzing blood parameters, three fish were randomly sampled from each tank before weighing and immediately anaesthetized using clove essence solution and one ml of blood was collected by caudal vein puncture using syringes containing 3 mg of na2edta. then, they were stored at 4°c after gently shaken for further examination. red blood cell and white blood cell counts were estimated following the method of schalm et al. (1975). haemoglobin (hb) and haematocrit (ht) concentrations were determined based on barros et al. (2002). chemical analysis: the food samples were collected and pooled at regular intervals during the experimental period and grounded using a 1 mm screen grinder before analyses. dry weight of food and fish body were measured for dried samples for 24 hrs at 103°c (iso 6496 1983). ash content was determined by incineration in a furnace for 4 hrs at 550°c (iso 5984 1978). crude protein (n×6.25) was measured by the kjeldahl method after acid digestion according to iso 5983 (1979). lipid was extracted with petroleum ether in a soxhlet apparatus. energy content was measured by direct combustion in an adiabatic bomb calorimeter (ikac-7000, fa. ikaanalysentechnik, weitersheim, germany). fish performance: weight gain was determined by the difference between initial and final body weights. feed conversion ratio (fcr) was calculated as follow: fcr = feed consumed (g) / wet body weight gain (g). specific growth rate (sgr) was calculated as follows and expressed as a percentage: sgr = 100 (ln wfinalln winitial) × days -1. the calculations were based on the dry weight of the diets. statistical analysis: data are presented as means of each treatment ± standard deviation (sd). normality of data after transformation (asin) and homogeneity of variances were tested using diets parameters control bp 0.5 bp 1 initial weight (g) 110.11 ± 7 107.14 ± 4 108.78 ± 5 fcr 1.55 ± 0.17b 1.54 ± 0.88b 1.37 ± 0.05a final weight (g) 297.3 ± 0.83b 298.5 ± 3.6b 323.9 ± 2.3a sgr 1.16 ± 0.96b 1.17 ± 0.13b 1.57 ± 0.06a mortality 0 0 0 different superscript letters show significant differences. table 2. growth performance in caspian trout, salmo caspius, feeding on different levels of beta plus (bp; g/kg) over eight weeks experimental period. all values are means of four replicates (tanks)/ treatment ± sd (standard deviation). 295 karimzadeh et al/ beta plus and caspian trout levene’s f test. one-way anova was used to determine the effects of bp levels on fish performance, blood parameters and bacterial count using the sas software. the means were compared by a tukey’s post hoc test. for all statistical analyses, each tank was considered as the experimental unit. results there were no mortality observed throughout the study. the results revealed a positive impact of bp on the growth performance of caspian trout. inclusion of 1 g kg-1 probiotic improved final weight compared to control one (p<0.05). however, lower level of bp supplementation (0.5 g kg-1) did not have a significant effect on final weight (table 2). also fcr and sgr were improved due to feeding on 1 g kg-1 probiotic diet (p<0.05). however the lower levels of the probiotic did not affect those parameters (table 2). dietary administration of bp affected bacterial count in the intestine of caspain trout (table 3). both gram positive and gram negative counts were increased significantly by addition of 1 g kg-1 of bp to the diet (p<0.05). however, lower level of bp supplementation (0.5 g kg-1) did not induce any change in bacterial count (table 3). dietary bp did not influence body composition of caspian trout over eight weeks experimental period (table 4). likewise, blood parameters of caspian trout were not affected by supplementation of bp (table 5). nevertheless, there was a trend toward a greater red and white blood cells concentrations and bacterial community diet control bp 0.5 bp 0.1 bp 1 bp 0.1 gram positive* gram negative** 2.3×104 ± 45b 2.4×104 ± 62b 2.6×104 ± 50b 2.5×104 ± 23b 6.8×105 ± 40a 3.6×105 ± 25a different superscript letters show significant differences. table 3. bacterial counts in the caspian trout, salmo caspius, feeding on different levels of beta plus over eight weeks experimental period. all values are means of four replicates (tanks)/ treatment ± sd. parameters diets control bp 0.5 bp 1 dry mater 26.54 ± 2.33 26.51 ± 2.54 26.56 ± 2.77 protein 15.82 ± 1.32 15.79 ± 1.40 15.80 ± 0.88 fat 7.18 ± 0.89 7.19 ± 0.78 7.21 ± 0.96 ash 3.11 ± 0.11 3.09 ± 0.17 3.07 ± 0.14 table 4. body composition in caspian trout, salmo caspius, feeding on different levels of beta plus over eight weeks experimental period. all values are means of four replicates (tanks)/treatment ± sd. parameters diets control bp 0.5 bp 1 hematocrit (%) 39.77 ± 1.00 41.33 ± 2.08 41.89 ± 1.52 hemoglobin (gr/dl) 13.60 ± 0.36 13.10 ± 0.72 13.87 ± 0.40 white blood cells (×103/lµ) 11.52 ± 1.37 11.99 ± 2.65 12.55 ± 2.58 red blood cells (×106/lµ) 0.68 ± 0.26 0.71 ± 0.37 0.78 ± 0.65 table 5. body composition in caspian trout, salmo caspius, feeding on different levels of beta plus over eight weeks experimental period. all values are means of four replicates (tanks)/treatment ± sd. 296 international journal of aquatic biology (2014) 2(6): 292-298 hematocrit in treatment fed 1 g kg-1 bp diet. discussion the present study showed that supplementation of two bacillius species improve growth performance including final weight, biomass gain, sgr and fcr in caspian trout. a large number of previous works also showed positive effects of different dietary probiotics on fish growth performance and feed efficiency of rainbow trout (bagheri et al., 2008; alizadeh et al., 2011; ramos et al., 2014), african catfish (clarias gariepinus) (al-dohail et al., 2009), gilthead sea bream (sparus aurata) (suzer et al., 2008) and zebra fish (danio rerio) (sedaghat et al., 2012). the beneficial effect of the probiotic on growth may be related to its effect on the microbial flora of intestine. the mechanisms by which probiotic bacteria stimulate growth rate are not yet clearly elucidated. administration of bacillus group may change the proportion of bacillus count in the intestine thereby leading to a larger count of germ positive bacteria. lactic acid bacteria inhibit specific fish pathogens by production of bacteriocins (thompson et al., 1999; ebrahimi et al., 2012). the positive effects of b. subtilis and b. licheniformis on the immune system and resistance to some pathogens were also reported in salmo salar (austin et al., 1992). a better growth performance of caspian trout in the current study may be also attributed to a lower proportion of gram negative bacteria to gram positive ones. the promotion of lactobacillus population can limit pathogenic bacteria colonization (ringo and gatesoupe, 1998; thompson et al., 1999; verschuere et al., 2000). low level bp administration (0.5 g kg-1 ), in this study, did not change growth parameters in caspian trout that was in agreement with those of rainbow trout (ramos et al., 2014). enzyme secretion ability of bacillus spp. such as protease may increase with proliferation of bacteria population (fuller and perdigon, 2003). this condition can elevate digestive enzyme activity (askarian et al., 2011), thus leading to a better nutrient digestion achieving a better growth. the results also showed that presence of probiotic in the diet affect the gut microbiota of fish. the similar observation was also made by balcazar et al. (2006) and merrifield et al. (2010) in salmonids. the present experiment suggests that supplementation of bp modulates gut microbiota and through that significantly improves growth performance of juvenile caspian trout. in addition, multi-species probiotics seems to be more effective than singlestrain probiotics, because different strains are more likely to survive and find their specific niche in the gut as suggested by bezkorovainy (2001). blood analysis revealed that the measured blood parameters were not affected by the dietary probiotic supplementation. this finding is similar to that of dotta et al. (2011) who observed that probiotic supplementation did not influence the red blood cell and hematocrit in nile tilapia, oreochromis niloticus. in contrast, other results show that probiotic diet increases hematological parameters in african catfish, clarias gariepinus, (ayoola et al., 2013). the different results may indicate that some other parameters such as experimental condition, fish species and diet composition can influence the impact of probiotic on the hematological response. in conclusion, the results demonstrated that the administration of bp as a dietary probiotic can improve growth performance and feed efficiency in the caspian trout by promotion of gram positive bacterial growth that may indicate growth improvement. based on the findings, supplementation of bp at a level of 1.5 g kg-1 diet is suitable for feeding of caspian trout to support fish growth and health. references al-dohail m.a., hashim r., aliyu-paiko m. 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(2018) 6(2): 95-103 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article contrasting fish size distributions between neotropical run-of-river and storage reservoirs louise cristina gomes*1, pitágoras augusto piana2, diesse aparecida de oliveira sereia3 1pós graduação em biologia comparada, universidade estadual de maringá, brazil. 2centro de engenharias e ciências exatas, universidade estadual do oeste do paraná, toledo, paraná, brazil. 3centro de ciências biológicas, universidade tecnológica federal do paraná, dois vizinhos, paraná, brazil. article history: received 7 march 2018 accepted 24 april 2018 available online 2 5 april 2018 keywords: aquatic environments dam fish assemblage size spectrum abstract: the spectrum size has been used to assess environmental disturbances and to understanding the energy flow in ecosystems. the objective of this work was to investigate if the operation regimes of reservoirs, including run-of-river and storage systems, interfere with the biomass spectra of fish fauna. we tested the hypothesis that the run of river reservoirs present higher proportions of large individuals than storage system. samplings of fish fauna were carried out between january 2005 and december 2007 at six neotropical reservoirs belonging to the sub-basin iguaçu river and coastal basin in são jorge river, brazil. the spectrum calculation was performed using the pareto type i continuous distribution model. reservoirs operated under run-of-river regime had significantly higher values than those operated under storage regimes. this study has elucidated some impacts of reservoir operating regime on biomass spectra and indicated differences in size spectra of fish assemblages among the sampled reservoirs. therefore, it is important to incorporate management plans that take dam operating mode into account so that conservation of aquatic fauna, especially fish, is more effective. introduction body size is a fundamental trait of living organisms, being linked with many important physiological and ecological processes (giacomini et al., 2016; wheeland and rose, 2016; brown, et al., 2004; elton, 1927). the relationship between body size and abundance denominated size spectrum, is a concept initially developed by sheldon et al. (1972), in which the authors assessed the distribution from bacteria biomass to whales and hypothesized that aquatic biomass is uniformly distributed among logarithmic class sizes. this approach provide important information about trophic ecology (petchey and belgrano, 2010; gaedke, 1993; gaedke, 1992) and environmental disturbances (gamble et al., 2006; shin et al., 2005), besides that, it has contributed for a better understanding of aquatic communities function and the regulatory processes of energy flow in these environments (zhou et al., 2010; layman et al., 2005; thiebaux and dickie, 1992; rodríguez and mullin, 1986). *corresponding author: louise cristina gomes doi: https://doi.org/10.22034/ijab.v6i2.464 e-mail address: louise-cristina@hotmail.com as approaches based on body size are independent of the taxon, individuals with the same body size are considered energetically equivalent, and used as a reference for simplification of food webs. size-based indicators has been used for studies of fish assemblages to assess impacts caused by fishing activity on stock size, marine food web dynamics (gamble et al., 2006; shin et al., 2005; rice and gislason, 1996) and fish communities in reservoirs (kantoussan et al., 2009). thus, the use of spectrum size models are relevant to fisheries science in the context of the ecosystem approach to fisheries management. the theory behind of spectrum size includes several mathematical derivations and estimation methods that are described in a number of detailed publications and their appendices (andersen et al., 2015; sprules and barth, 2015; reuman et al., 2008, white et al., 2007; andersen and beyer, 2006; brown and gillooly, 2003). freshwater ecosystem are vulnerable to anthropogenic disturbance due to landscape 96 gomes et al. / contrasting fish size distributions between neotropical run-of-river and storage reservoirs influences on habitat and fishes (van der lee and koops, 2015). reservoirs are historically recent environments and considered as complex ecosystems, with characteristics distinct from those of natural rivers and lakes (fernando and holcik, 1991). the main neotropical water basins are altered by dams, which were built for various purposes such as power generation, irrigation and urban water supply (stenberg, 2006; agostinho et al., 1999). these systems are exposed to natural and artificial forces that determine dynamic characteristics; in addition to climatological and hydrological events, reservoir operational regime, manner of construction, and use also interfere with the system dynamics and spatial and temporal organization (straskraba et al., 1993). further, reservoir operation significantly impacts river hydrology, changing the magnitude, frequency, duration, and timing of the flow regime (suen, 2011). in this context, the reservoir operation mode can be classified according to the control mechanism for everted and turbined water. run-of-river systems whose characteristics are more similar to natural lakes and storage systems in which water stock is kept high mainly in high rainfall periods, enables the maintenance of activity in dry periods. therefore, the storage system tend to cause more disturb than run-ofriver system due to higher water level fluctuations (poff and hart, 2002). these characteristics promote fragmentation and transformation of the river, as well as changes in species diversity, trophic structure and community composition (poff et al., 1997; ward and stanford, 1995). the conceptual model about differences in water level fluctuations in reservoir are provided in figure 1. in this context, the aim of this study was to assess the size spectrum distributions in fish in neotropical reservoirs, specifically to answer the following question: is there difference in fish size spectrum in reservoirs with different operating regimes? for this, we tested the hypothesis that the run of river reservoirs present higher proportions of large individuals than storage system. materials and methods study area: this study was conducted in six reservoirs located in the state of paraná, southern brazil. four reservoirs were analyzed in the paraná river basin, belonging to the following sub-basin: iguaçu river and two reservoirs belonging to the coastal basin in são jorge river (fig. 2). the iguaçu river present an area of approximately 72.000 km2 and the high unevenness of the basin constituted a great attraction for the hydroelectric exploitation, resulting in a cascade of five reservoirs that alters their physical, chemical and biological attributes (baumgartner et al., 2012). the fish fauna of iguaçu river present a high degree of endemism due to geographic isolation caused by iguazu falls. the coastal basin is formed by rivers draining directly into the atlantic ocean and present a total area of 5.630,8 km2. they are rivers with great slope, rapids and greater speed of current, as a result of this, several reservoirs were designed for the production of figure 2. location of the six neotropical reservoirs sampled from january 2005 to december 2007. so=salto osório, ss=salto santiago, se=segredo, fa=foz do areia, vo=vossoroca and sm=salto do meio. figure 1. conceptual model about differences in water level fluctuations. 97 int. j. aquat. biol. (2018) 6(2): 95-103 energy (rodrigues et al., 2005). these reservoirs present wide heterogeneity of morphometric and limnological characteristics. for analysis criteria, the reservoirs were grouped in pairs with one run-of river and one storage system each, according to the subbasin in which they belong, area and proximity to the operating system (table 1). sampling: samplings were carried out between january 2005 and december 2007 (table 1). data collection took place in non-adjacent bank regions at points located upstream the dam in six reservoirs using mesh gill nets between 2.4 cm and 16 cm. nets were exposed for 24 hours and collections took place at 8, 16 and 22 hours. fish were anesthetized with benzocaine hydrochloride (250 mg/l), as required by resolution no. 714/cfmv as of july 20, 2002, which regulates procedures and methods of euthanasia in animals (cfmv, 2002). after anesthesia fish were fixed in 10% formalin and 70% alcohol (shibatta and cheida, 2003) and assigned id based on location, shift, and sampling month. specimens were subsequently taken to the laboratory for analysis. analysis were performed at the laboratory of ichthyology at the state university of west paraná, toledo campus. specimens were classified according to graça and pavanelli (2007), britski et al. (1999) and eschmeyer (1990). weights and length were obtained for all specimens. the list of species and their respectively abundances are provided in table 2. data analysis: the richness of species was assessed using the number of individual captured (see supplementary material). to represent the size spectrum, we used the biomass of all individuals captured in each reservoir. the spectrum calculation was performed using the pareto type i continuous distribution model for each reservoir. according to this model, the probability of finding individuals that are older or of a specific size decays on a logarithmic scale as size increases. this has been used for the analysis of biomass frequency distributions (white et al., 2008; vidondo et al., 1997). the derivation of the pareto type i model is described by the following equation: log2(prob(s ≥ s)) = c* (log2(k) – log2(s)) where prob(s≥s) is the fraction of individuals over a given size (s) taken at random (s). the constant c is the slope of the size spectrum, representing the frequency distribution of fish body size. thus, when c=-1, the frequency distribution is uniform between individual fish of different sizes; if c<-1, the frequency of smaller individuals is higher than large individuals; if c>-1, the frequency of large individuals is greater that smaller individuals. the pareto model was fitted by non-linear regression using the least squares method. the possible effects of reservoir operation regime on the biomass spectra of fish assemblages were assessed using paired t test, both using the software statistica 7.1® (stat soft, 2005). results were considered significant at p<0.05. results the slopes of the biomass spectra of fishes observed in each reservoir indicated that reservoirs foz do areia (storage), salto santiago (storage), salto osório (run of river) and vossoroca (storage) tended to have higher concentrations of small individuals (c<-1), while segredo (run of river) show equal distribution between large and small (c~-1). salto do meio (run of table 1. physical factors of the analyzed reservoirs. locations were paired as follows: pair 1: fa and se; pair 2: ss and so; pair 3: vo and sm (fa=foz do areia, se=segredo, ss=salto santiago, so=salto osório, vo=vossoroca, sm=salto do meio and *=data not found). physical factors fa se ss so vo sm operation regime storage run-of-river storage run-of-river storage run-of-river sub-basin iguaçu iguaçu iguaçu iguaçu coastal coastal fill year 1980 1992 1980 1975 1931 1931 area (km2) 139 80.6 208 55 5.1 0.1 sampling quarterly quarterly bimonthly bimonthly quarterly quarterly average depth (m) 40.0 36.6 35 25.5 4.0 * altitude 742 607 506 397 814 722 residence time (days) 105 47 50.8 16 110 * 98 gomes et al. / contrasting fish size distributions between neotropical run-of-river and storage reservoirs table 2. species collected from january 2005 to december 2007 at six neotropical reservoirs and their respective abundances (fa=foz do areia, se=segredo, ss=salto santiago, so=salto osório, vo=vossoroca and sm=salto do meio). taxon common name fa se ss so vo sm atheriniformes odontesthes bonariensis (valenciennes, 1835) “peixe-rei” 174 282 30 characiformes apareiodon vittatus (garavello, 1977) “canivete” 67 716 88 294 astyanax altiparanae garutti & britski, 2000 “tambiú” 33 28 331 38 61 61 astyanax bifasciatus garavello & sampaio, 2010 “lambari-do-rabo-vermelho” 13902 2100 5270 1812 astyanax dissimilis garavello & sampaio, 2010 “lambari” 647 24 12 7 astyanax gymnodontus (eigenmann, 1911) “lambarizão” 184 256 1047 265 astyanax janeiroensis (eigenmann, 1908) “lambari-do-rio” 4 astyanax longirhinus garavello & sampaio, 2010 “lambari” 4 2 astyanax minor garavello & sampaio, 2010 “lambari-do-rabo-amarelo” 1651 1363 4100 292 astyanax serratus garavello & sampaio, 2010 “lambari” 7 bryconamericus ikaa casciota, almirón & azpelicueta, 2004 “lambarizinho” 206 529 18 10 bryconamericus pyahu azpelicueta, casciotta & almirón, 2003 “lambarizinho” 2 characidium sp. “charutinho” 4 cyphocarax santacatarinae (fernández-yépez, 1948) “saguaru” 4 deuterodon iguape eigenmann, 1907 “lambari” 2265 1475 deuterodon sp. a “lambari” 1262 384 deuterodon sp. d “lambari” 12413 460 hoplias malabaricus (bloch, 1794) “traíra” 180 24 213 165 hoplias aff. malabaricus (bloch, 1794) “traíra” 38 18 leporinus elongatus (valenciennes, 1850) “piapara” 3 leporinus friderici (bloch, 1794) “piau” 2 leporinus macrocephalus (garavello & britski, 1988) “piauçu” leporinus obtusidens (valenciennes, 1837) “piau” 4 leporinus octofasciatus (steindachner, 1915) “piau-listrado” 2 leporinus sp. “piau” 1 oligosarcus longirostris menezes & géry, 1983 “saicanga” 1372 262 256 306 prochilodus lineatus (valenciennes, 1837) “corimba” 116 salminus brasiliensis (cuvier, 1816) “dourado” 1 serrapinnus sp. “piabinha” 1 cypriniformes cyprinus carpio (linnaeus, 1758) “carpa-comum” 5 2 2 1 hypophthalmichthys nobilis (richardson, 1845) “carpa-cabeça-grande” 1 gymnotiformes gymnotus inaequilabiatus (valenciennes, 1839) “morenita” 6 2 1 gymnotus sylvius (albert & fernandes-matioli, 1999) “morenita” 2 perciformes australoheros kaaygua (casciotta, almirón & gómez, 2006) 3 australoheros sp. “acará” 2 crenicichla iguassuensis (haseman, 1911) “joaninha” 69 122 27 30 crenicichla sp. “joaninha” 17 15 6 14 geophagus brasiliensis (quoy & gaimard, 1824) “cará” 227 168 50 15 842 260 micropterus salmoides (lacepède, 1802) “boca-grande” 77 8 oreochromis niloticus (linnaeus, 1758) “tilápia-do-nilo” 1 3 1 tilapia rendalli (boulenger, 1897) “tilápia” 17 24 4 3 136 1 siluriformes ancistrus sp. “cascudo-roseta” 4 3 corydoras ehrhardti (steindachner, 1910) “limpa-fundo” 53 26 corydoras paleatus (jenyns, 1842) “limpa-fundo” 889 1072 540 133 1 glanidium ribeiroi (haseman, 1911) “bocudo” 26 93 4 18 hypostomus ancistroides (ihering, 1911) “cascudo-pintado” 2 2 hypostomus aspilogaster (cope, 1894) “cascudo” 1 hypostomus boulengeri (eingenmann & kennedy, 1903) “cascudo” 1 hypostomus commersoni (valenciennes, 1836) “cascudo” 80 223 13 2 88 30 hypostomus derbyi (haseman, 1911) “cascudo” 36 73 4 9 hypostomus myersi (gosline, 1947) “cascudo” 7 6 1 2 hypostomus sp. 1 “cascudo” 3 hypostomus sp. 2 “cascudo” 1 isbrueckerichthys sp. “cascudinho” 1 pimelodus britskii garavello & shibatta, 2007 “mandi-pintado” 1024 1397 pimelodus ortmanni haseman, 1911 “mandi-pintado” 44 181 459 685 pseudoplatystoma corruscans (spix & agassiz, 1829) “pintado” 1 rhamdia branneri haseman, 1911 “bagre, jundiá” 3 5 1 7 rhamdia quelen (quoy & gaimard, 1824) “bagre-amarelo” 30 42 99 int. j. aquat. biol. (2018) 6(2): 95-103 river), showed a higher proportion of large individuals (c>-1) (fig. 3). the reservoirs operated under run-ofriver regime had significantly higher values than did those operated under storage regimes (paired t test: t=3.777, p=0.032). discussion the results indicated differences in size spectra of fish assemblages among the sampled reservoirs. here, abiotic events that could to determine some relationships between body size distributions and the environment were not evaluate. however, the physical characteristics differences were considered in each reservoir and how these aspects can change the communities and to provide some information about ecological processes which regulate these environments. fluctuations in reservoir water level due to table 2. continued. taxon common name fa se ss so vo sm rhamdia voulezi haseman, 1911 “bagre, jundiá” 34 18 4 2 rineloricaria sp. “cascudo-espada” 237 259 steindachneridion melanodermatum (garavello, 2005) “surubim” 1 tatia jaracatia pavanelli & bifi, 2009 “bagre-sapo” 1 total richness 40 29 26 25 16 21 figure 3. size spectra of fish assemblages in the studied reservoirs, adjusted using the pareto type i model. 100 gomes et al. / contrasting fish size distributions between neotropical run-of-river and storage reservoirs accumulation operating regime cause flooding of bank areas considered critical for fish feeding (hahn et al., 1998) and may act as a disturbance factor, altering the structure and stability of fish assemblages (tundisi et al., 2003). the increase in nutrients from the decomposition of flooded vegetation provides an increase in primary productivity, and a consequent increase in smaller sized individuals. according to agostinho et al. (2001), flooding may favor juveniles and may thus influence the size distribution patterns. the success of higher proportions of small individuals in accumulation regime reservoirs possibly is likely due to fluctuations in water level that provide better conditions for some species. for example, the genus astyanax have adhesive eggs (sato et al., 2006) and fractional spawning (agostinho, 1999), facilitating the use of margin areas as sites for spawning and juvenile shelter when the reservoir contains abundant water (i.e., high water level). when the opposite occurs, that is, lower water level, the edges and vegetation are exposed and there is a tendency toward increased turbidity, which reduces the visibility for piscivorous fish and consequently, decreases predation rate (robertis et al., 2003). important ecological interactions and physiological processes in aquatic ecosystems are dependent on body size (e.g., mortality, transfer efficiencies, metabolism and growth) (kerr and dick, 2001). consequently, researchers have been incorporating biomass spectrum theory into models designed to estimate size-dependent ecological processes. for example, benejam et al. (2015) examined the effect of land use on the size structure of fish communities in subtropical streams. van der lee and koops (2015) investigated correlations between body size of fish with habitat loss. jennings et al. (2002) quantified trophic transfer efficiencies using a combination of biomass spectrum data and production-body size and trophic level-body size relationships. furthermore, the current understanding of regulatory processes and trophic dynamics of aquatic ecosystems has been focused on ecological studies (jansson et al., 2007; shin and cury, 2004) and approaches using body size have been used to primarily to provide information concerning conservation of aquatic environments (petchey and belgrano, 2010), trophic structure, and biotic interactions in fish assemblages (emmrich et al., 2011; jennings et al., 2001). in this sense, body size is crucial for predicting responses to energy flow, species diversity and population densities (layman et al., 2005). from an ecological point of view, trophic dynamics are highly sensitive to environmental disturbances resulting from fluctuations in water level (tundisi et al., 2003), and these changes are key components for the reorganization of fish assemblages for which feeding grounds and reproduction are affected (agostinho et al., 2007). although reservoirs are dominant components of hydrological landscapes, studies of the effects of operating regime on biomass spectra are scarce, making it difficult to make inferences regarding size distribution and energy flow in communities. besides that, others factors can be associate to the differences in sizes distributions. the size of individual in ecological community is affected by many kinds of processes, for example, environmental changes, species invasion and management (petchey and belgrano, 2010). according to metabolic theory of ecology, a greater part of variation among organisms, including their ecological roles and life history, is limited by their body sizes, chemical composition and operating temperatures (brown et al., 2004). this study has elucidated some impacts of reservoir operating regime on the size distributions and this suggests that energy transfer occurs more efficiently through fish fauna in reservoirs with lower fluctuations in water level, such as run-of-river regimes. however, more researches is needed to unreavel relationships between body size distributions and the ecological processes regulating the energy flow in reservoirs. in addition, researches which consider abiotic factors and others relevant aspects are need considering the acceleration of transformations at the landscape caused by impoundments. finally, it is important to incorporate management plans that take dam operating mode into account so that conservation of aquatic fauna, especially fish, is more 101 int. j. aquat. biol. 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(2020) 8(5): 300-310 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article feeding ecology of the endemic freshwater puffer fish carinotetradon travancoricus (hora & nair, 1941) in western ghats hotspot, india chelapurath radhakrishnan renjithkumar*,1 kuttanelloor roshni, kutty ranjeet department of aquatic environment management, kerala university of fisheries and ocean studies (kufos), panangad, kochi, kerala, india. s article history: received 9 june 2020 accepted 5 september 2020 available online 2 5 october 2020 keywords: puffer fish chalakudy river diet preference omnivorous feeding intensity abstract: the study aimed to investigate the feeding ecology of a vulnerable freshwater puffer fish, carinotetradon travancoricus from the chalakudy river of kerala, western ghats, a biodiversity hotspot of india. fishes were sampled from october 2018 to september 2019. stomach condition of the fishes showed the percentage of empty gut to be significantly higher during all seasons (p< 0.01). feeding intensity depicted the fish to follow an ‘active’ feeding strategy (31.59±10.32%) during premonsoon season. diet composition and relative length of gut analysis indicated that the fish during its early stages relied on an omnivorous diet however preferring autochthonous food materials such as insects (27.91%) and crustaceans (25.30%) during its adult stages. a perceptible variation in the feeding strategy associated with the spawning season of c. travancoricus was also noticed. during their spawning season (may-august), a greater preponderance towards animal matter (52.18%) was noticed in their diet. the results of gastrosomatic index indicated that feeding activity of c. travancoricus is considerably reduced (2.99) during the spawning period. the present study provides the baseline information on the feeding ecology of c. travancoricus which could be helpful to aquarists for breeding and rearing of this species in captivity and thereby reducing their fishing pressure in wild. introduction puffer fishes of the family tetradontidae are one of the most diverse groups in tropical seas and freshwater areas of the world. among them, only four genera (27 species) are known to adapt to freshwater environments, occurring in three tropical regions of the world (southeast asia, central africa and south america) (ebert, 2001; nelson, 2006). in india, three species of freshwater puffer fishes are reported viz. carinotetradon travancoricus, c. imitator and tetradon cutcutia (yamanoue et al., 2011). carinotetradon travancoricus (hora & nair, 1941) is commonly known as malabar puffer fish, an endemic fish inhabiting the rivers and lakes of western ghats, a biodiversity hotspot in southern india (remadevi et al., 2000; dahanukar, 2011). freshwater puffer fish trade from india substantially increased since 1990’s, ever since they were marked as aquarium fish due to its yellowish body colour, ovoid body shape and *correspondence: chelapurath radhakrishnan renjithkumar e-mail: renjith.kumar347@gmail.com puppy dog eye (prasad et al., 2012; liya and ramachandran, 2013). the species in their native range is presently being impacted by the severe modifications in their habitats due to damming, deforestation, conversion of forest area in to agriculture and rubber plantation and also due to over harvesting for aquarium pet trade (raghavan et al., 2008). the population of puffer fishes have since then declined and is now listed as ‘vulnerable’ in the iucn red list of threatened species due to the over exploitation and habitat loss (dahanukar, 2011). the global aquarium fish trade industry is large, diverse and involving nearly 5300 freshwater and 1802 marine fishes (hensen et al., 2010; rhyne et al., 2012). nearly 90% of the export market involve tropical freshwater fishes and among them only 10% are captive bred and the remaining are wild caught (olivier, 2001). in india, the hub for the collection of wild caught ornamental fishes is the western ghats 301 int. j. aquat. biol. (2020) 8(5): 300-310 and eastern himalayan biodiversity hotspots known for their exceptional freshwater biodiversity and endemism (allen et al., 2010; raghavan et al., 2013). the export market of freshwater ornamental fishes from india accounts to over 1.5 million of fishes belonging to 30 threatened species mostly contributed by botia striata, c. travancoricus, sahyadria denisonii and s. chalakkudiensis (raghavan et al., 2013). being mostly gregarious, bright coloured and small, the malabar puffer fish, c. travancoricus are easily caught and hence they outnumber other indigenous species exported from india (raghavan et al., 2013). indiscriminate collection of this threatened species from natural waters for export trade resulted in the severe population decline (dahanukar, 2011). in order to reduce their fishing pressure, knowledge regarding the nutritional requirements of c. travancoricus in wild is essential to develop technologies for their captive breeding and rearing. information on the feeding habits of fishes in a given ecosystem have considerable importance in fisheries conservation and is a key factor to determine their growth rate, condition and population level (begum et al., 2008; saikia, 2016). the analysis of diet of fishes is also important to better understand the behavior of the organisms and permits a comprehensive understanding of ecosystem functioning that is required for its in-situ conservation (braga et al., 2012; tonella et al., 2018). till date any comprehensive study on the feeding ecology of c. travancoricus especially from the wild is lacking. on this background, the present study was undertaken to examine the feeding intensity, gastrosomatic index (gasi), relative length of gut (rlg), index of relative importance (iri) and gut content analysis of c. travancoricus in chalakudy river, western ghats of india. materials and methods sample collection: sampling was done monthly from the chalakudy river (10°18'40''n, 76°38'10''e), a perennial river system in kerala state of india from october 2018 to september 2019 using seine nets (mesh size 5-8 mm) and scoop nets (mesh size 2-4 mm). the region receives an annual rainfall of 3000 mm during the monsoon season extending from juneseptember followed by post-monsoon (octoberjanuary) and pre-monsoon season (february-may). after collection, the fishes were preserved in ice and brought to the laboratory where total length (tl) was measured to the nearest 0.01 cm and total weight (tw) was taken to an accuracy of 0.01 g. a total of 278 specimen (10.5 to 30.0 mm tl and 0.12 to 0.62 g tw) were analyzed for the study. healthy fishes without any sign of injury or infection were dissected out for stomach content analysis. the weight and volume of the gut contents of each stomach were measured and preserved in 4% neutral formaldehyde solution for further analysis. prey items were removed from the dissected stomach and the contents were examined under a stereomicroscope (motic smz 168, china) (x10) and individual food items were identified to lowest possible taxonomic level. feeding intensity, gastrosomatic index (gasi) and relative length of gut (rlg): the intensity of feeding was determined based on the degree of fullness and points were allotted to each gut. depending on the fullness of stomachs, points were assigned as 20, 15, 10, 5 and 0 for full, 3/4 full, 1/2 full, 1/4 full and empty stomachs (pillay, 1952). fishes with stomach full and 3/4 full were considered as ‘active’ feeders, 1/2 full as ‘moderate’ feeders and 1/4 and empty stomach are ‘poor’ feeder. the sum of allotted points based on different degrees of fullness of stomach was divided by the number of stomach samples for that month. monthly gastrosomatic index (gasi) was calculated following desai (1970) using the formula: gasi = weight of the gut/total weight of the fish × 100. fishes were classified based on their body length as juveniles (<20 mm) and adults (>20 mm) and their relative length of gut (rlg) was calculated following al-hussaini (1949): rlg = length of the gut/ total length of the fish gut content analysis: gut content analysis was calculated using frequency of occurrence following the formula after hynes (1950): 302 renjithkumar et al./ feeding ecology of carinotetradon travancoricus fi= 100 * ni/ n where, fi is the frequency of occurrence of the ith food item in the sample; ni = number of stomachs in which the ith item was found and n= total number of stomachs (with food) examined. in order to ascertain the seasonal variations in the gut contents, monthly data pertaining to a particular season were grouped together and analysed. index of relative importance (iri): the contribution of each prey items to the diet of fishes was determined following three relative metrics of prey quantity: percentage frequency of occurrence (%oi= number of guts containing prey i/total number of guts containing prey*100), percentage composition by number (%ni =number of prey i/total number of prey*100) and percentage composition by volume (%vi=volume of prey i/total volume of prey*100) (hynes, 1950; pillay, 1952; bowen, 1996). the index of relative importance (iri) of each prey taxon (pinkas et al., 1971) was calculated using the following formula: irii= (% ni +% vi) % oi statistical analysis: seasonal variations in the gut contents were analysed statistically through one-way anova followed by duncan’s multiple range test (dmrt) using spss 20.0 software of ibm to determine the significant difference of a food item between seasons (gomez and gomez, 1984). significance was measured at p<0.05 level. results feeding intensity: an assessment of the feeding intensity of c. travancoricus indicated that the percentage of empty and quarter full guts were considerably high (48.96±6.98) throughout the year, while only very few percentages of guts (3.99±3.80) were found to be full. further assessment on the stomach fullness condition, that relates to the feeding intensity showed that although the proportion of ‘poor feeders’ were significantly higher (f=48.55, p<0.01) during most part of the year, the percentage of ‘active feeders’ relatively improved (31.59±10.32) during the pre-monsoon season (fig. 1). an in-depth analysis on the monthly variation of the feeding intensity depicted that a greater percentage of fishes with full stomach were observed during february to april (fig. 2). likewise, a very high percentage of the empty stomach was observed during june to august (monsoon season). gastrosomatic index and relative length of gut: mean values of gasi for c. travancoricus ranged from 2.89 (july) to 4.66 (april). monthly variation in gastrosomatic index (gasi) of c. travancoricus is depicted in figure 3. the variations in gasi values coincided with the patterns of feeding intensity with highest mean values observed during pre-monsoon season (4.38). while corroborating these observations with the spawning season of the species, it is figure 1. feeding intensity of carinotetradon travancoricus in chalakudy river. 303 int. j. aquat. biol. (2020) 8(5): 300-310 understood that the gastro-somatic index was high during post-spawning season (december-april) and the reduction in the gasi during may to november were in tune with the broader spawning season, which indicates that the fish follows voracious feeding during post spawning season amassing required energy reserves for the next spawning period. the average rlg of c. travancoricus were found to range from 0.67-1.57 in juvenile fishes and 0.70-1.32 in adult fishes. it was seen that 55.35% of the juvenile fishes and 25.64% of the adult fishes had an rlg value greater than 1, indicating with age c. travancoricus shifts its food preference from a predominantly omnivorous diet to a preferably carnivorous diet. gut content analysis: the percentage composition of food items in the gut of c. travancoricus were categorized in to seven groups (fig. 4). insects (18.52%) and crustaceans (18.07%) were the most dominant food items in the gut. insects were represented by the larvae of odonata (dragonflies and damselflies), ephemeroptera (mayflies), hemiptera figure 2. monthly variations in the feeding intensity of carinotetradon travancoricus in chalakudy river. figure 3. monthly variations in gastrosomatic index of carinotetradon travancoricus in chalakudy river. 304 renjithkumar et al./ feeding ecology of carinotetradon travancoricus (aquatic bugs) and diptera (flies), while copepods, cladocerans (moina sp. and daphnia sp.) and ostracods formed the major group of crustaceans in the diet. this was followed by annelids (14.30%) represented mainly by chironomid larvae. the fish also devoured a wide array of phytoplankton’s namely the diatoms (14.02%) and green algae (12.55%) such as navicula sp., cymbella sp., syndera sp., cocconeis sp., pinnularia sp., fragillaria sp., nitzschia sp., spirogyra sp., ulothrix sp., shizogonium sp., pleurodiscus sp., uronema sp. and oedogonium sp.. the remaining fraction in the diet included sand (10.78%) and miscellaneous items (11.77%), that were mostly composed of detritus. seasonal variations in diet composition: the diet of c. travancoricus composed predominantly of invertebrates, followed by algal matter in all the periods, but with significant seasonal difference in the diet (fig. 5). the results indicate that c. travancoricus is a specialistic feeder favouring insect larvae, however, during monsoon season when the availability of such prey decreases, they rely upon crustaceans and annelids as alternate feed. this was further supported by the results of dmrt analysis indicating that the preference for insects significantly varied (f=21.945, p<0.001) between seasons (table 1). the fish consumed more insects during premonsoon than any other season. in contrast, the occurrence of annelids in the diet was more during monsoon especially in june (18.39%). the preference for crustaceans however, did not vary significantly (f=2.086; p>0.05), but was found to be higher during june to september. an interesting observation noticed in the diet preference was the greater availability of phytoplankton in the diet of c. travancoricus during the post monsoon months. hence our study suggests table 1. seasonal variation of different food items in the gut of carinotetradon travancoricus from chalakudy river. season food items crustaceans insects chlorophyceae diatom annelids sand others pre-monsoon 18.733a 20.010c 9.790a 13.158a 13.185a 12.240a 12.888b monsoon 18.305a 16.708a 13.848b 13.293a 16.043b 10.115a 11.688a post monsoon 17.170a 18.833b 14.003b 15.600b 13.668a 9.988a 10.735a f value 2.086 21.945 52.389 16.137 4.360 2.755 10.607 significance 0.18 0.001 0.001 0.001 0.047 0.117 0.004 a, b and c means with different superscript alphabets in a column indicate significant difference them are homogeneous (dmrt; p<0.05). figure 4. percentage composition of food items in the diet of carinotetradon travancoricus. 305 int. j. aquat. biol. (2020) 8(5): 300-310 that the fish has a preference for specific food items during certain period of the year. index of relative importance: monthly variation in the percentage of iri for different food items is indicated in table 2. month-wise percentage of iri for different food items indicated that insects were the dominant food item in every month with maximum values observed during march (31.93%). the occurrence of crustaceans was observed round the year with maximum value during june (29.87%) and minimum in november (19.64%). the composition of diatoms in the gut varied from 10.35 to 16.89% in may and december, respectively while for green algae it was 7.27% in may to 14.15% in july. peak index value for annelids was recorded in july (15.28%) and lowest value in april (6.42%). discussions chalakudy river, the fifth largest river in kerala state, india originates in the western ghats, one of the 34 global biodiversity hotspots (myers et al., 2000). the river system harbors a rich and diverse fish fauna of 98 species and many of them are endemic and threatened (ajithkumar et al., 1999). the river also faces serious threats in the form of habitat degradation, overexploitation, and invasion of alien fish species (raghavan et al., 2008). knowledge on the feeding biology of endemic and threatened fish species in an ecosystem is important to link scientific knowledge with biodiversity conservation issues to table 2. monthly changes in the index of relative importance (iri) of different food items in carinotetradon travancoricus from chalakudy river. months oct nov dec jan feb mar apr may jun jul aug sep crustaceans 23.38 19.64 22.09 24.53 23.02 26.19 26.07 27.68 29.87 26.40 27.80 26.96 insects 28.31 28.99 29.92 29.92 30.27 31.93 30.88 29.31 24.32 22.69 24.38 24.05 chlorophyceae 12.63 12.50 12.18 10.79 9.34 7.69 7.95 7.27 11.46 14.15 14.10 12.43 diatoms 13.19 15.21 16.89 15.80 14.83 13.23 11.43 10.35 11.94 12.23 11.06 14.55 annelids 13.56 9.37 8.88 6.68 7.70 6.45 6.42 9.02 15.28 14.58 14.18 11.36 sand grains 2.62 5.71 3.55 6.17 6.42 6.38 7.22 8.02 2.78 4.63 3.47 4.16 miscellaneous items 6.31 8.57 6.49 6.10 8.43 8.13 10.03 8.35 4.34 5.32 5.00 6.48 figure 5. seasonal variation in the frequency of occurrence of different food items in carinotetradon travancoricus. 306 renjithkumar et al./ feeding ecology of carinotetradon travancoricus prepare suitable management measures (huo et al., 2014). freshwater fishes have a broad range of feeding characteristics and adaptations, consuming a variety of food items and improving their diet by using the more energetic or easily available food resources (de oliveira et al., 2019). limited knowledge is available on the feeding profile and ecology of the malabar puffer. our study shows the feeding intensity of c. travancoricus to follow a distinct seasonal pattern whereby the fish was relatively a ‘poor feeder’ during the monsoon season, which on a closer observation coincided with the gonadal development of c. travancoricus. in the present study, most the fishes during may–august were either mature or ripe, indicating the period to be the peak spawning season for this species. feeding intensity of fish can be influenced by maturity, spawning and the availability of food items in its environment (ikusemiju and olaniyan, 1977). the feeding habits of fishes are greatly decreased during the reproductive season and a high feeding activity reported during pre-spawning period should therefore be to store food as energy reserves during the spawning period. the ripe gonads occupying more space in the peritoneal cavity compresses the gut during the spawning season leading the fish to follow a low feeding regime (serrajuddin et al., 1998). feeding pattern of fishes is determined by a number of other factors such as intensity of light, temperature, ph, salinity, time of day, season, and any internal rhythm that may exist (lagler, 1956). the gastrosomatic index (gasi) encountered in our study were relatively lower during the months of juneoctober indicating the poor feeding activity of c. travancoricus which in turn corresponds to their spawning period (anupama et al., 2019). the low gasi value during the spawning season has been observed in many tropical freshwater species (sarkar and deepak, 2009; mondal and kaviraj, 2010; gupta and banerjee, 2014; alam et al., 2016; roshni et al., 2016; manorama and ramanujam, 2017). the gasi thereafter increased gradually along with their feeding activity. availability of preferred food item is also a factor that may influence the gasi in omnivorous fishes. our observations showed that for meeting its pre-spawning nutritional requirements, c. travancoricus preferentially feeds on larvae and juveniles of insects and crustaceans. since these developmental stages of insects are not available throughout the year, they shift to a more phytoplankton rich diet during their post spawning phase. following the classification of kramer and bryant (1995) reporting the rlg values for carnivorous, omnivorous and herbivorous fish species to be 0.6-0.8, 0.8-1.0 and 2.5-16.4, respectively, our results suggest c. travancoricus to be an omnivore. the average rlg of c. travancoricus in this study was close to 1.00 indicating that this species is an omnivore preferring more of animal matter in their diet. the results are comparable to the results of karmaker and biswas (2015) in t. cutcutia (0.670.86). our study also suggests that the juvenile individuals prefer phytoplankton or zooplanktonbased diet while the adults favour insects and annelids. hence there is a discernable shift in the feeding strategy with development in this fish. the present study revealed that c. travancoricus in chalakudy river feeds only a few types of food items and therefore can be categorized as a stenophagic feeder. the main food constituents of the gut contents were aquatic invertebrates and algal matter. seasonal changes in the availability of prey items was found to significantly affect the diet composition in this species. hence the changes in the feeding habits of fishes are closely related to the changes in the food availability due to the changes in the environmental parameters and physiological variation (wootton, 1990). littoral zones in the river systems are occupied by extensive macrophytes that provide suitable habitat for small sized fishes which have distinct behavior pattern, such as low swimming activity (priyadarshana et al., 2001; thomaz et al., 2008; dibble and thomaz, 2009; grzybkowska et al., 2018). the macrophytes habitat is a favorable environment for these fishes because of the high availability of food resources like invertebrates and algae (quirino et al., 2015; grzybkowska et al., 2018). the favorable 307 int. j. aquat. biol. (2020) 8(5): 300-310 macrophyte habitat of c. travancoricus is cabomba in riverine areas of western ghats region (anupama and harikrishnan, 2015). medium densities of aquatic macrophytes, including c. caroliniana enhance fish diversity, feeding, growth and spawning (dibble et al., 1996; grzybkowska et al., 2018). this plant is abundantly found in the downstream floodplains of chalakudy river which also provided good protection to this fish. the higher consumption of microcrustaceans during the monsoon period has been reported in various riverine environments (nandy and mandal, 2020). the increase in the content of copepods and cladocerans in the gut of c. travancoricus is a strong indication of the increased abundance of these microcrustaceans in the river during the monsoon season. cladocerans (daphnia and moina) may be preferred over copepods and rotifers due to their lower swimming speed in aquatic ecosystem and slow prey avoidance response (lazzaro, 1987). the thick overhanging vegetation of the forest in western ghats brings in a wide array of allochthonous organisms, including insects in their larval forms and other invertebrates into the aquatic environment, most of them are microcrustaceans and aquatic insects that utilize this wet spell for their development (bonato et al., 2012; prasad et al., 2012). chiromonid larvae, the main insect items found in the gut of puffer fish, are frequently associated with the submerged macrophytes which use as support, shelter and food source (pinder, 1995). in dry season, with a decrease in the volume of water inflow and increase of water temperature favours a higher productivity in the ecosystem and growth of microalgae mainly diatoms (johnson and arunachalam, 2012; lopes et al., 2016). the presence of sand grains and small amount of detritus indicate the bottom feeding habit of the fishes. sand grains and detritus could be accidently ingested along with other food items such as insects and crustaceans. our observations are in tune with the earlier studies of joshy (2004) from puzhakkal river and prasad et al. (2012) from kallar river, india, indicating the most preferred food item of c. travancoricus as crustaceans (cladocera, rotifers, copepods) and insects. a comparison on the feeding habits of c. travancoricus with that of other freshwater puffer species revealed similarities as well as differences in the diet. krumme et al. (2007) observed that freshwater banded puffer colomesu spsittacus to be carnivorous, preferring molluscs and crustaceans. the emerald puffer, t. cutcutia from assam, india mainly feeds on insects, and mollusks (karmaker and biswas, 2015). the present finding therefore provides a salient understanding on the feeding biology of c. travancoricus in a lesser studied chalakudy river with impetus on their preferential feeding strategy. our study also suggests that this species has established well to the lotic ecology of chalakudy river by modifying its feeding habits, which includes diversifying its prey preference, adjusting to seasonal fluctuations in food availability and synchronizing its spawning period in such a manner that their young ones has ample phytoplankton or zooplankton based diet during their early developmental stages. later in the reproductive phase they selectively feed on insects especially their larvae which have 10-60% fat in their dry matter (kouřimská and adámková, 2016), required for the gonadal development and spawning. conclusion study of feeding ecology is vital to understand the ecological adaptation of the species to the environment. it also provides knowledge on the feed preference, seasonal variabilities in feed availability associated with the environment and also relates them to other biological factors impacting the fishes. our result provides the first account on the feeding ecology of the malabar puffer fish from a tropical river in a biodiversity hotspot. carinotetradon travancoricus is an omnivore, feeding mainly on insects, crustaceans and algal matter. the variations in the feeding intensity, diet composition and other food indices indicate that environment plays an important role in altering the feeding biology of this fish. being a widely traded ornamental aquarium fish, we hope the inputs on its feeding strategy will help develop an artificial diet for its breeding and rearing in captivity thereby reducing their capture pressure from the wild. 308 renjithkumar et al./ feeding ecology of carinotetradon travancoricus acknowledgments the first author acknowledges funding from the postdoctoral fellowship of the kerala state council for science, technology & environment (kscste), government of kerala, india. the authors are also thankful to local fishers at the chalakudy river for providing fish samples. references ajithkumar c.r., remadevi k., thomas k.r., biju c.r. 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(2018) 6(2): 66-74 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article length-weight, condition factor and gonadosomatic index of blackspot snapper lutjanus fulviflamma (forsskal, 1775) (perciformes: lutjanidae) in the northern persian gulf alireza razi, ahmad noori*1 department of fisheries science, faculty of marine science and technology, university of hormozgan, bandar abbas, iran. article history: received 10 january 2018 accepted 25 may 2018 available online 2 5 april 2018 keywords: allometric growth gonadosomatic index lutjanidae reproductive season abstract: the present study aimed to evaluate the length-weight and length-length relationships as well as the condition factor and gonadosomatic index of the blackspot snapper, lutjanus fulviflamma in the northern persian gulf, hormozgan province, iran. the specimens were collected monthly from april 2016 to march 2017. the size (tl, total length; fl, fork length; sl, standard length) were measured and weighted (bw, total body wet weight). a total of 446 individuals were analyzed. the tl-bw relationship indicated isometric growth pattern in both sexes. in females, the means for condition factor was higher than males. in both sexes, the lowest value of both condition factor and gonadosomatic index were detected in autumn with ascending trend in the next seasons reaching the peak in spring. the oscillation in condition factor, as well as gonadosomatic throughout the sampling period, was most prominent in females which may be related to the reproductive cycle. the information reinforces data to define fishing closed seasons in this important fish that is used in many places in the world. introduction the blackspot snapper, lutjanus fulviflamma (lutjanidae), is a widespread species throughout the indo-pacific regions. this fish occurs from the red sea and the persian gulf to south africa to the east to australia, and the ryukyu islands in the west pacific as far as samoa (carpenter and niem, 2001). this species mainly inhabits coral reefs or rocky substrata at depth of 3-35 m. their juveniles sometimes found in brackish water or mangrove estuaries or in the lower reaches of fresh-water streams. the diet of this species mainly consists of fishes, shrimps, crabs, and other crustaceans. at new caledonia and east africa spawning occurs mainly from august to march (spring and summer) (carpenter and niem, 2001). lutjanus fulviflamma commonly utilized in subsistence fisheries in iran with an uninterrupted fishery through the entire year, seen frequently fresh in local markets. catching is mainly performed with handlines, traps, and gill nets. despite its importance *corresponding author: ahmad noori doi: https://doi.org/10.22034/ijab.v6i2.454 e-mail address: nooryahmad@gmail.com in different regions, there are few published studies on its biology and life history. some studies on this species have done including investigations about age, growth, and reproduction performed around okinawa island, japan (shimose and tachihara, 2005), yaeyama island, japan (shimose and nanami, 2015), kenyan inshore waters (kaunda-arara and ntiba, 1997), and mafia island, tanzania (kamukuru and mgaya, 2004) and some aspects of its life cycle in the persian gulf (grandcourt et al., 2006). the length-weight relationship (lwr) assumes an important prerequisite in studies of biology, physiology, and ecology, especially in species with commercial value (froese, 2006). this relationship allows converting one variable to another, estimating the expected weight for a certain size, or detecting ontogenetic morphological changes related to maturation of fishes (lima-junior et al., 2002; zamani-faradonbeh et al., 2015a, b). such knowledge can be useful for further studies on the life history of 67 int. j. aquat. biol. (2018) 6(2): 66-74 the species and in the development of its fishery, resource management, and culture (radkhah and eagderi, 2015). the most frequently regression model used to evaluate this relationship is the power function y=axb (huxley, 1950) which is also known as allometric growth equation. the exponent b is considered equal to 3 as a benchmark for a fish with isometric growth. those with values above or below 3 are considered as positive or negative allometric growth, respectively (hartnoll, 1982). condition factor is commonly used as a quantitative indicator of the general status or ’well-being’ of the individual (lloret et al., 2013). it is based on the principle that individuals of a given length, exhibiting higher weight, are in a better condition. the condition factor is deeply affected by a series of factors, including individual, exogenous parameters like environmental factors and those endogenous such as feeding condition and growth rate, the degree of parasitism, reproductive cycle, etc. therefore, this quantitative value may vary according to seasons, geographical location and populations (lima-junior et al., 2002). the age, growth and reproduction traits of some lutjanids, including l. synagris (luckhurst et al., 2000), l. analis (burton, 2002; teixeira et al., 2010), l. argentimaculatus (russell and mcdougall, 2008a), l. griseus (fischer et al., 2005), l. bohar (marriott et al., 2007), l. carponotatus (kritzer, 2004), l. vitta (ramachandran et al., 2013), l. guttatus (amezcua et al., 2006), l. campechanus (patterson iii et al., 2001; white and palmer, 2004; wilson and nieland, 2001), l. sebae (newman et al., 2010), l. fulvus (shimose and nanami, 2014), l. erythropterus (fry and milton, 2009), l. malabaricus (fry and milton, 2009) and l. fulviflamma (grandcourt et al., 2006; kaundaarara and ntiba, 1997; shimose and nanami, 2015; shimose and tachihara, 2005) have been previously studied. in almost all above-mentioned studies, the relationship between age and otolith weight or length have been provided. although lwr was only considered in a few studies, however, it is not compared seasonally in detail. the evaluation of this relationship in recent studies is restricted to compare between sexes. hence, the purpose of the present study was to describe the length-weight (lwr) and length-length relationships (llr) and to clarify some of the life history parameters i.e. condition factor and gonadosomatic index of the blackspot snapper l. fulviflamma inhabiting the northern persian gulf. this study will provide useful information during interpretation of these relationships among growthrelated traits, management plans and to monitor populations of this species. in addition, the results will help the understanding the reproductive cycle of this fishery resource, and aid to complete information about the biology of snappers in the persian gulf through comparison of the results with previously published studies. materials and methods study area and specimens sampling: specimens of l. fulviflamma were sampled monthly from april 2016 to march 2017. a total of 446 individuals captured by hook and line from the persian gulf (qeshm island) (26°53'58"n, 56°10'03"e; 26°44'10"n, 56°00'27"e), in hormozgan province, iran. the specimens were identified according to allen (1985) and sexed macroscopically by visual observation of the gonads. each specimen was weighed (bw, total body weight) to the nearest 0.1 g using a digital balance. the length parameters, including total length (tl), fork length (fl) and standard length (sl) were measured with a biometric ruler to the nearest 1.0 mm. databases and calculations: to estimate the relationship between bw and tl, the empirical points were submitted to regression analysis using the allometric growth power function (y=axb), according to ricker (1973), where ‘y’ is the total expected body weight (g), ‘x’ is the total length (mm), ‘a’ is the yintercept, and ‘b’ is the slope. these parameters are easily estimated by linear regression analysis based on logarithmic transformation (ln) of variables with a fit equation (lny=lna+blnx) that was evaluated by the coefficient of determination (r2). fulton’s condition factor (k) was estimated for 68 razi and noori / some biological characteristics of blackspot snapper each sex using the formula of k=(100bw)/tlb, where: k is condition factor, bw is mean total body weight (g), tl is mean total length (mm), and ‘b’ is the weight growth coefficient from the length-weight relationship. gonadal development was assessed in terms of gonadosomatic index through the formula of gsi=(100gw)/bw (busacker et al., 1990) where: gsi is gonadosomatic index, gw is mean total gonad weight (g), and bw is mean total body weight (g). statistical analysis: all data were checked for normality by shapiro-wilk test. the growth pattern was defined by the coefficient ‘b’, which had its equality to 3 tested by the student’s t-test. student’s ttest was used for analyzing the differences between the sexes. the mann-whitney-u test was applied to compare the variables between sexes since data were heteroscedastic. all data in percentage were first transformed and arcsin of data was used in the analytical comparison (zar, 2010). all analysis was carried out by spss (version 15.0) packet program. type i error was accepted as 0.05. average values are given as mean ±sem. results from a total of 446 sampled specimens, 262 (58.74%) were females and 184 (41.26%) males and the sex ratio significantly biased in favor of females table 1. monthly descriptive statistics and estimated parameters of length-weight relationships for both sexes of lutjanus fulviflamma in the persian gulf (qeshm island) from april 2016 to march 2017. total length (cm) total body weight (g) regression parameters season sex n min max min max a 95% cl of a b 95% cl of b r january f m 22 13 14.00 12.70 25.80 26.00 45.9 31.1 270.1 252.1 0.0145 0.0142 0.0139 0.0151 0.0135 0.0149 3.0741 3.0769 3.0655 3.0828 3.0678 3.0859 0.995 0.993 february f m 20 11 18.50 20.90 26.50 25.50 95.3 123.6 272.8 238.2 0.0142 0.0128 0.0136 0.0147 0.0119 0.0138 3.0680 3.0571 3.0604 3.0756 3.0432 3.0710 0.958 0.844 march f m 15 20 14.50 17.00 25.70 24.90 46.3 58.0 243.5 222.7 0.0140 0.0131 0.0132 0.0147 0.0127 0.0136 3.0656 3.0623 3.0551 3.0760 3.0558 3.0688 0.970 0.972 april f m 23 29 17.50 18.40 28.70 25.10 95.5 107.2 342.1 238.0 0.0149 0.0145 0.0143 0.0155 0.0141 0.0148 3.0766 3.0803 3.0688 3.0844 3.0753 3.0853 0.957 0.964 may f m 22 10 15.00 15.00 26.90 24.00 57.0 58.1 348.6 219.5 0.0157 0.0155 0.0148 0.0166 0.0148 0.0163 3.0880 3.0962 3.0765 3.0995 3.0862 3.1063 0.969 0.994 june f m 32 14 13.20 14.90 26.70 23.00 35.7 46.6 324.8 163.2 0.0145 0.0137 0.0140 0.0150 0.0130 0.0144 3.0752 3.0727 3.0684 3.0820 3.0629 3.0824 0.981 0.985 july f m 31 14 14.00 13.70 27.30 22.00 37.3 33.7 309.4 122.1 0.0138 0.0128 0.0133 0.0143 0.0121 0.0134 3.0659 3.0587 3.0590 3.0728 3.0489 3.0685 0.987 0.981 august f m 17 16 14.10 14.50 22.60 18.90 43.8 47.6 146.7 89.9 0.0133 0.0131 0.0125 0.0142 0.0124 0.0137 3.0593 3.0636 3.0463 3.0722 3.0534 3.0737 0.942 0.933 september f m 24 14 13.90 13.70 23.30 19.30 42.5 41.8 167.1 93.6 0.0137 0.0135 0.0131 0.0142 0.0127 0.0144 3.0644 3.0705 3.0558 3.0730 3.0576 3.0833 0.968 0.908 october f m 19 17 15.00 14.30 21.00 23.10 48.0 40.3 123.5 159.7 0.0130 0.0122 0.0125 0.0134 0.0117 0.0126 3.0539 3.0494 3.0467 3.0611 3.0425 3.0564 0.968 0.985 november f m 18 16 12.40 14.60 23.70 24.40 29.9 44.1 183.5 196.4 0.0130 0.0129 0.0125 0.0135 0.0123 0.0135 3.0541 3.0605 3.0460 3.0621 3.0506 3.0705 0.993 0.980 december f m 19 11 13.50 14.80 24.50 25.50 37.5 47.1 209.3 262.7 0.0136 0.0133 0.0131 0.0141 0.0128 0.0138 3.0629 3.0661 3.0554 3.0704 3.0595 3.0727 0.990 0.997 all f m b 262 185 447 12.40 12.70 12.40 28.70 26.00 28.70 29.9 31.1 29.9 348.6 262.7 348.6 0.0141 0.0135 0.0138 0.0139 0.0143 0.0133 0.0137 0.0137 0.0140 3.0682 3.0656 3.0680 3.0656 3.0708 3.0650 3.0706 3.0661 3.0699 0.983 0.983 0.983 figure 1. the relationship between total length (mm) and body weight (g) for both sexes of lutjanus fulviflamma in the persian gulf (qeshm island) from april 2016 to march 2017. 69 int. j. aquat. biol. (2018) 6(2): 66-74 (χ2=13.641, p<0.001). length-weight relationships indicated isometric growth pattern in both females (bw=0.00001tl3.01, r2=0.97) and males (bw= 0.00001tl3.02, r2=0.97) (fig. 1). the results revealed no significant differences between sexes for b value (females: b=3.01, t262 = 0.23, p>0.05; males: b=3.02, t184=0.52, p>0.05). monthly lwr is presented in table 1 showing range of b 3.0539-3.088 in females (cv%=0.32), and 3.05-3.10 in males (cv%=0.41). in both sexes, the minimum allometric coefficient (b) was recorded in october and maximum one in may. lwr was highly significant correlated (p<0.001), with all coefficient values being more than 0.989 (table 2). the comparison between the parameters a and b in the l. fulviflamma with other lutjanus species is also shown in figure 2. the mean fulton’s condition factor in relation to tl size class (kmean) is depicted in figure 3. the kmean displayed a descending trend in females up to 16-18 cm tl and increased onwards. in males, the value of the kmean is decreased until 18-20 cm tl and after that, the trend showed an increasing manner. the kmean in females was significantly lower than that of the males in two size classes; 16-18 cm tl with the value of 1.41±0.03 in the females and 1.49±0.02 in the males (t70=-1.995, p=0.049) and 20-22 cm tl with the value of 1.48±0.02 and 1.57±0.03 in the females and the males, respectively (t62=-2.308, p=0.024). the fulton's condition factor (k) ranged 0.0103 to 0.0184 in females and 0.0104 to 0.0179 in males. the average k in the females (0.0141) was significantly higher than that of males (0.0135) (t444=4.293, p=0.000). there was a significant difference in k between months in both sexes (females: f11,261=7.154, p=0.000; males: f11,183=9.350, p=0.000). in the females, the lowest mean k was found in october and table 2. length–length relationships between total length (tl), fork length (fl) and standard length (sl) of lutjanus fulviflamma in the persian gulf (qeshm island) from april 2016 to march 2017. sex equation n a b r2 female tl = a + bsl sl = a + bfl fl = a + btl 262 10.016 -6.201 -0.215 1.142 0.907 0.952 0.988 0.988 0.996 male tl = a + bsl sl = a + bfl fl = a + btl 184 8.383 -6.305 1.196 1.154 0.908 0.942 0.991 0.991 0.994 both tl = a + bsl sl = a + bfl fl = a + btl 446 9.440 -6.230 0.294 1.147 0.907 0.948 0.989 0.989 0.995 figure 2. test plot of log (a) against b for some lwrs of fishes belong to lutjanidae. =present study parameters of lutjanus fulviflamma; = parameters of lutjanus argentimaculatus; = parameters of lutjanus quinquelineatus; = parameters of lutjanus russellii; = parameters of lutjanus fulviflamma; = parameters of lutjanus fulvus; = parameters of lutjanus lutjanus; = parameters of lutjanus monostigma; = parameters of lutjanus vitta; = parameters of lutjanus bohar; = parameters of lutjanus gibbus; = parameters of lutjanus sebae; = parameters of lutjanus kasmira; = parameters of lutjanus adentii. dotted line = regression line, r2 = 0.95. figure 3. mean fulton’s condition factor (kmean) per length (total length) class for both the sexes of lutjanus fulviflamma in the persian gulf (qeshm island) from april 2016 to march 2017. asterisk (*) indicated significant difference (p<0.05) between females and males kmean values. 70 razi and noori / some biological characteristics of blackspot snapper november (0.0130) and highest in may (0.0157); in the males, the lowest in october (0.0122) and highest in may (0.0155) (fig. 4). the monthly variations of the gonadosomatic index of females and males are shown in figure 5. the mean value of gsi was significantly higher in the females compared to that of the males (t114=3.134, p<0.05). in the females, the gsi fluctuated during the sampling months with the minimum level of 0.289 in september. the value onwards increased gradually with an abrupt increase in may reaching the maximum levels of 2.498. in males, the value of gsi revealed some minor variations throughout the sampling time with the maximum level of gsi (1.6498) in may (fig. 5). discussion the results revealed no significant difference between female and male regarding total length and body weight. both sexes have the same growth pattern i.e. isometric one as 3.008, 3.02 and 3.01 for females, males and combined, respectively. the measured bvalues were within the expected value for most of fishes (froese, 2006) and in accordance with other studied populations of l. fulviflamma (grandcourt et al., 2006; shimose and nanami, 2015; shimose and tachihara, 2005) as well as other members of the family lutjanidae (grandcourt et al., 2011; kritzer, 2002; newman, 2002; newman and dunk, 2002; ramachandran et al., 2013). plotting the log (a) against regression coefficient 'b' provides a comparison of the estimates found in this study on l. fulviflamma with the other lutjanus species. considering the coefficient of determination (r2=0.95) extracted from the assumed plot clearly revealed a highly significant correlation which approved the assumption of the same b-value in the species of the family lutjanidae. the results from comparing the growth parameter estimation between sexes in this study is also consistent with the findings on l. synagris (luckhurst et al., 2000), l. argentiventris (piñón et al., 2009), l. erythropterus (mcpherson, 1992), l. bohar (marriott et al., 2007), l. campechanus (patterson iii et al., 2001), l. analis (burton, 2002), l. guttatus (amezcua et al., 2006) and l. alexandrei (fernandes et al., 2012). although some studies demonstrated similar b-value but show a significantly different sexrelated body size between sexes (grandcourt et al., 2011; newman et al., 2000; shimose and nanami, 2015). in some species e.g. l. griseus (fischer et al., 2005), l. gibbus (nanami et al., 2010), l. carponotatus (kritzer, 2004; newman et al., 2000) l. adetii (newman et al., 1996), l. quinquelineatus (newman et al., 1996), l. malabaricus (mcpherson, 1992; newman, 2002), l. sebae (mcpherson, 1992; newman and dunk, 2002) and l. vitta (davis, 1992; newman et al., 2000) male grows larger than female figure 4. fulton’s condition factor (k) for both the sexes of lutjanus fulviflamma in the persian gulf (qeshm island) from april 2016 to march 2017. bars indicate standard error of mean values. figure 5. monthly mean gonadosomatic index (gsi) for females and males of lutjanus fulviflamma in the persian gulf (qeshm island) from april 2016 to march 2017. bars indicate standard error of mean values. 71 int. j. aquat. biol. (2018) 6(2): 66-74 while in l. fulvus (shimose and nanami, 2014), l. argentimaculatus (russell and mcdougall, 2008b), l. fulviflamma (grandcourt et al., 2006; kamukuru et al., 2005; shimose and nanami, 2015; shimose and tachihara, 2005) and l. ehrenbergii (grandcourt et al., 2011) the growth pattern is biased towards the females. different sex-related body size is a common phenomenon reported among lutjanids. those from different geographical regions demonstrated various growth patterns. it is assumed that in species found in atlantic, caribbean, and hawaiian regions, females usually show larger size while those from the indopacific possesses a general growth pattern with a larger size in favor to males (grimes, 1987; nanami et al., 2010). although this presumption looks like true, is not credited for all studies (fischer et al., 2005; russell and mcdougall, 2008b; shimose and nanami, 2014) and the same growth pattern was also shown in both regions (amezcua et al., 2006; fernandes et al., 2012; fry and milton, 2009; piñón et al., 2009). in the majority of these studies, the correlation between the fish age with length or body weight is analyzed and higher growth rate in females or males has been concluded. almost in all of these studies, the growth pattern is not distinctive between sexes during the first years of life but differences will prominent with age increment. in the present study, the same growth pattern between males and females is probably due to restricted analysis to length-weight relationship without considering the fish age classes. further studies on the age-length or age-weight of this species in this region will provide complementary data to have a better picture of this fish. based on the results, the fulton’s condition factor of l. fulviflamma fluctuates throughout the year. although the females demonstrated higher value than males, but almost the same. this species in the present studied area showed the lowest condition factor in october with gradual increasing trend peaked in may. the maximum value of gonadosomatic index measured in may in both sexes and decreased to reach the lowest level in september and october. then, the value fluctuated with ascending trend toward april and may. these findings are in agreement with the results of other studies on this species (grandcourt et al., 2006; shimose and nanami, 2015). grandcourt et al. (2006) demonstrated an augmentation of the gonadosomatic index value for both males and females of l. fulviflamma from january to may and a descending pattern from may to september. shimose and tachihara (2005) also showed the higher value in this species from april to july, with a peak in may and june for both sexes and low values from october to march with no exception of both sexes. also, the finding of the present study in agreement with the results of shimose and nanami (2015) showing the mean gonadosomatic values for both sexes increase in april, reaching a peak in may and then decreased from june to august and remained in low levels from september to march. in the present study, the spawning periods was from april to august with a peaked in may and june based on the gonadosomatic index and the fulton's condition factor. the higher value of the fulton's condition factor could be credited to the deposition of lipids and fats as an energetic source for the coming spawning periods. the highest value in the fulton's condition factor during the spawning periodicity are shown in other species (mir et al., 2012; rahman, 2017; ramachandran et al., 2013). in conclusion, both sexes of blackspot snapper have isometric growth pattern in the northern persian gulf with no difference in mean fulton's condition factor between various length classes in both sexes but different monthly with the same pattern in males and females. this value was significantly high in april to august with a peak in may. as the gonadosomatic index was revealed the same oscillation as that of the fulton's condition factor, it can be suggested that april to june can be considered as reproductive months in this species in the northern persian gulf. acknowledgments we are grateful to the editor and anonymous reviewers for their insightful comments, which greatly improved the manuscript. the special thanks to e. rahimi for his helps for sampling. the authors are thankful to the 72 razi and noori / some biological characteristics of blackspot snapper university of hormozgan, especially the department of fisheries science, for providing all the laboratory facilities for this study. this work was performed according to the iranian society for the animal welfare. references allen g.r. 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(2018) 6(2): 66-74 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی سیاه خال زرد سرخو ماهي در گناد رشد ضریب و چاقي ضریب وزن،-طول رابطه بررسي lutjanus fulviflamma (forsskal, 1775) فارس خلیج شمالي بخش در *نوری احمد راضي، علیرضا ،نایرا بندرعباس، هرمزگان، دانشگاه دریایی، فنون و علوم دانشکده شیالت، گروه چکیده: و ضریب رشد گناد در ماهی سرخو زرد خال سیاه ضریب چاقیطول و همچنین -وزن و طول-هدف از انجام این مطالعه بررسی رابطه طول (lutjanus fulviflamma) در بخش 95هر دو جنس نر و ماده بر اساس نمونه برداری ماهانه از فروردین تا اسفند باشد. این خصوصیات برایمی های طولی شامل طول کل، طول چنگالی و طول شمالی خلیج فارس در استان هرمزگان مورد تجزیه و تحلیل قرار گرفت. برای هر نمونه شاخص مورد ارزیابی قرار گرفت. بررسی رابطه بین طول و وزن عدد ماهی 446ر مجموع استاندارد و همچنین وزن کل اندازه گیری شد. در این تحقیق د و ضریب چاقیها بیش از نرها بود. کمترین میزان در ماده ضریب چاقیباشد. می ایزومتریکنشان داد که رشد این ماهی در هر دو جنس نر و ماده و ماده در فصل پاییز مشاهده گردید که در فصل زمستان روند صعودی به خود گرفته و در بهار به نیز کمترین ضریب رشد گناد در هر دو جنس نر و ضریب رشد گناد نشان ضریب چاقیها در مقایسه با نرها به مراتب میزان نوسان بیشتری را در اوج خود رسید. در طی مدت زمان مطالعه، ماده د در های ممنوعیت صیتواند در زمینه تعیین زمانیانه در آنها بستگی داشته باشد. این اطالعات میی تولید مثل سالدادند که ممکن است به چرخه مورد این گونه مهم مورد استفاده قرار گیرد. .مثل تولید فصل ،سرخوماهیان گناد، رشد ضریب ناهمگون، رشد :کلمات کلیدی international journal of aquatic biology (2015) 3(4): 225-235 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article food and feeding habits of silver carp, hypophthalmichthys molitrix (val., 1844) in gobindsagar reservoir, india. hamid reza esmaeili*1, mohinder singh johal2 1department of biology, college of sciences, shiraz university, shiraz 71454, iran. 2department of zoology, panjab university, chandigarh, 160014, india. article history: received 25 april 2015 accepted 12 june 2015 available online 2 5 august 2015 keywords: phytoplankton diet overlap index costello method silver carp abstract: investigation on the food and feeding habits of silver carp in gobindsgar reservoir indicated that the diet of silver carp was dominated by cyclotella spp. (diatom) followed by chlorophyceae, cyanophyceae, crustacea, dianophyceae and rotifera. the size range of cyclotella spp. varied between 5-25 µm indicating that silver carp is capable to collect the food particles smaller than distance between its gill rakers. probably, excretion of mucus plays an important role in collecting such small particles. by applying costello method, it was concluded that cyclotella spp. is very important food item while the others are general food items in the diet of silver carp in this water body. study of diet overlap index of different size groups of silver carp revealed that the value of “d” varied between 0.461 (moderate) to 0.972 (high), indicating that the diet of small size groups was significantly different from those of large size groups. the present observations indicated that the diet overlap index between female and male specimens of silver carp was very high (0.915), clearly indicating that the diet of male and females specimens did not differ significantly. analysis of gut contents of silver carp indicated that zooplankton comprised only 7.7% by number and 19.3% by volume in the foregut contents of this fish, hence, silver carp can be considered as microphytoplankton feeder. introduction food types as well as feeding habits of a specific species are significance in relation to their growth and propagation under specific biological conditions. identification of principal types of food items and their occurrence form basis relating to the studies concerning predation, competition, trophy dynamic, feed webs and the trophic relationships in aquatic ecosystems; therefore play an important role both in culture as well as in wild condition (murphy and willis, 1996; wakjira, 2013; omondi et al., 2013). in the past numerous studies have described the details of food and feeding habits and digestion processes of different fish species as well as silver carp (dos santos and jobling, 1991; boujard and leatherland, 1992; firs and horn, 1993; marnane and bellwood, 1997; beyst et al., 1999; pederson, * corresponding author: hamid reza esmaeili e-mail address: hresmaeili22@gmail.com 1999; de pirro et al., 1999; morita and suzuki, 1999; boyce et al., 2000; horppila et al., 2000; vamosi et al., 2000; domaizon et al., 2000; wakjira, 2013; sarkhanizadeh et al., 2014). silver carp, hypophthalmichthys molitrix (val., 1844) is known for its culture in confine waters along with native fishes, hence most of the information is available on the culture fishery of this fish. although the food and feeding habits of silver carp by inspection of its gut contents and gill rakers have been documented in the past (wilamovski, 1972; dunseth, 1977; cremer and smitherman, 1980; smith, 1985, 1989; shei and liu, 1990; feng and zhou, 1995; gu et al., 1996; xie, 1999; domaizon et al., 2000) but still there is ongoing debate on the feeding habits of this important cultivable fish species, especially concerning the digestibility of algae in this 226 international journal of aquatic biology (2015) 3(4): 225-235 stomachless filter-feeding carp (gu et al., 1996; xie, 1999). in the present study, the gut contents of silver carp were analyzed both qualitatively and quantitatively in a natural water body to ascertain the food habits and food selectivity in feeding behavior of this exotic carp. study area: gobindsagar reservoir is one of the biggest and the deepest reservoirs of india situated in the foot hills of shivaliks (western himalaya) located at latitude 31°25' north and longitude 76°25' east. it lies in bilaspur and una districts of himachal pradesh, india. water spread area of gobindsagar is 16,867 hectares at full storage level, 5063 ha at dead storage level with an average level of 10,965 ha. mean depth of reservoir is 188.98 meters at full storage level, 117.35 m at dead storage level with an average level of 153. 17 m (katiha et al., 2000). the reservoir receives a number of rivulets like lunkhar, sir, gambhar, gamrola and ali (fig. 1). lunkhar and sir arms of the reservoir are important from the commercial fishing operation point of view. materials and methods in this study, gut content of 457 specimens of silver carp (between the size range 235-1000 mm total fish length) were analyzed monthly from the commercial catches of gobindsagar reservoir. for gut analysis, the intestinal tract of individual fish was removed. contents from anterior end of the intestine to its first loop (in some cases, slightly beyond the curvature) were collected as foregut contents (shei and liu, 1990). foregut contents were immediately preserved in 5% formaldehyde solution in separate plastic vials. the gut contents were analyzed qualitatively and quantitatively by drop method as described by murphy and willis (1996). identification of food items was facilitated by the analysis of water samples taken from gobindsagar reservoir. prey was recognized at the genus level, when possible, but for the frequency-abundance analysis, they were grouped in to families or higher other taxa (de pirro et al. 1999). percent number, percent biovolume, and frequency of occurrence were used to estimate the dietary importance of each food category. percent number is the number of individuals of a prey type divided by the total number of individuals have been expressed as a percentage, after pooling the gut contents of all fishes. percent biovolume is the equivalent measure for biovolume data. frequency of occurrence is the percentage of guts where a food item was present. in mathematical terms, the percentage abundance (% ai) and the percentage occurrence (% fi) of prey i has been described by the figure 1. gobindsagar reservoir, himachal pradesh, india. 227 esmaeili and johal/food and feeding habits of silver carp following equations: % ai = 100        st si % fi =   100 n ni where, si is the gut content (volume, weight or number) composed by prey i, st the total gut content of all intestines in the entire sample, ni is the number of predators with prey i in their intestine, and n is the total number of predators with intestine contents (amundsen et al., 1996). costello’s (1990) graphical method was used to describe prey importance and feeding strategy of this fish. overlap in the diets of males and females of silver carp was assessed using the schoener’s (1970) index as described by krebs (1989): schoener’s index, d = 1-0.5  n 1 |px,i – py,i|, where, “d” is overlap index, px,i is the proportion of food category i in the diet of male, py,i is the proportion of food category i in the diet of female and n is the number of category. this index gives values from 0 (no overlap) to 1 (complete overlap). according to zaret and rand (1971), wallace (1981) and vamosi et al. (2000), a “d” value>0.60 is thought to be biologically significant. however pederson et al. (1999) defined “d” above 0.74 as arbitrarily to mean high overlap, 0.25-0.74 as moderate and “d” less than 0.25 as low overlap. results based on the qualitative analysis, the following phytoplankton and zooplankton have been observed to be present in the intestine of silver carp during the period of investigations. (a) phytoplankton: (1) chlorophyceae: actinastrum spp., ankistrodesmus spp., botryococous spp., chlamydomonas spp., chlorella spp., chlorococcum spp., coelastrum spp., cosmarium spp., eudorina spp., oocystis spp., pandorina spp., pediastrum spp., rhizoclonium spp., scenedesmus bijugatus, scenedesmus spp., staurastrum spp. and tetraedron spp. (2) bacillariophyceae: cocconeis spp., cyclotella spp., cymbella spp., diatoma spp., fragillaria spp., gomphonema spp., gyrosigma spp., melosira spp., meridion spp., navicula spp., nitzschia spp., pinnularia spp., pleurosigma spp., stauroneis spp., stephanodiscus spp., synedra spp. and tabellaria spp. 3) dinophyceae: ceratium spp., glenodinium spp. and peridinium spp. (4) cyanophyceae: aphanocapsa spp., aphanotheca spp., chroococcus spp., gloeocapsa spp., merismopedia spp., microcystis spp. and oscillatoria spp. 5) euglenophyceae: euglena spp. (b) zooplankton: (1) rotifera: brachionus spp., keratella spp. and trichocerca spp. (2) cladocera: bosmina spp., bosminopsis spp. and daphnia spp. (3) copepoda: cyclops spp. during the period of investigations the following plankton have been collected from the water samples, but all of them did not occur in the gut contents of fishes., clearly indicating that the silver carp is selective in the selection of food items which are available in the surrounding water. (1) chlorophyceae: chlorococcum spp., staurastrum spp., cosmirium sp., oocyscis spp., ankistrodesmus spp., tetraedron spp., pediastrum spp., closteriopsis spp., zygnema spp., botryococuus spp., pandorina spp., edorina spp., scenedesmus spp., chlorella spp., chlamidomonas spp., uronema spp., rhizoclonium spp. and coelastrum spp. (2) bacillariophyceae: cyclotella spp., navicula spp., synedra spp., diatoma spp., cocconius spp. and fragillaria spp. (3) dinophyceae: ceratium spp., gymnodinium spp. and peridinium spp. (4) cyanophyceae: microcystis spp., gloeocapsa spp., anabena spp., gloeotheca spp. and merismopedia spp. (5) rotifera: keratella spp., asplanchna spp., splanchnopus spp., polyarthra spp. and brachionus 228 international journal of aquatic biology (2015) 3(4): 225-235 spp. (6) cladocera: daphnia spp., diaptomus spp. and bosmina spp. (7) copepoda: cyclops spp. (8) euglenophyceae: euglena spp. the results showed that the food components of silver carp can be divided into categories, viz., digested and undigested. the undigested phytoplankton include green algae, blue-green algae, rotifera, crustacea (cladocera and copepoda), diatoms (except cyclotella spp.), dinophyceae and cyclotella. the digested material were mainly consisted of the unidentified matters in decayed and semi-digested state. it formed 58% by volume in the foregut of silver carp (pooled data). it ranked first in term of volume (table 1, fig. 2). of the identifiable planktonic items, cyclotella spp. was the most abundant organism. this diatom formed 62.25% by number (fig. 3), 12.97% by volume (fig. 2) and 93% by frequency occurrence (fig. 4) in the foregut. thus cyclotella spp. was the most dominant plankton in terms of number and abundance and the second item in terms of volume in foregut contents of h. molitrix (table 1). next important food component in the gut contents of silver carp was green algae represented by different genera. this category was represented by 17.97% by number, 3.31% by volume and 93% by frequency occurrence in the forgut of this fish (table 1). blue green algae constituted amongst the food items as 7.89% by number, 4.06% by volume and 83% by abundance in the foregut. it ranked third in term of number in the gut contents. microcystis spp. was the most dominant cyanophyceae found in the gut contents of h. molitrix. crustaceans which included cladocera and copepoda constituted 5.18% by number, 14.39% by volume and 82% by frequency occurrence of the foregut contents (figs. 2-4). daphnia spp. and cyclops spp. were most dominant crustaceans food items female male pooled female male pooled female male pooled %v %v %v %n %n %n % o % o % o green algae 2.98 3.07 3.31 14.01 18.59 17.97 77 84 93 blue green algae 5.12 3.02 4.06 11.10 4.50 7.89 57 77 83 rotifera 3.25 4.05 4.9 0.96 2.10 1.89 36 60 76 crustacea 14.41 14.01 14.39 5.13 7.10 5.81 56 77 82 diatoms 0.17 0.31 0.23 1.23 1.71 1.48 39 63 80 dianophyceae 2.23 2.18 2.15 2.59 2.91 2.72 57 78 83 cyclotella 14.79 11.84 12.97 64.98 63.09 62.25 87 94 93 digested material 57.06 61.51 58 table 1. percentage biovolume (v), number (n) and occurrence (o) of the major food items in the foregut of silver carp. g re e n a lg a e b lu e g re e n a lg a e d ig e s te d m a te ri a l r o ti fe ra c ru s ta c e a d ia to m s d ia n o p h y c e a e c y c lo te lla female male pooled0 10 20 30 40 50 60 70 p e rc e n ta g e b io v o lu m e food item figure 2. percentage biovolume of different food items in the forgut of silver carp. g re e n a lg a e b lu e g re e n a lg a e r o ti fe ra c ru s ta c e a d ia to m s d ia n o p h y c e a e c y c lo te lla female male pooled0 10 20 30 40 50 60 70 p e rc e n ta g e n u m b e r food item figure 3. percentage number of different food items in the foregut of silver carp. 229 esmaeili and johal/food and feeding habits of silver carp consumed by the fish. the members of rotifera group were another group of zooplankton found in the gut and they represented themselves as 1.89% by number, 4.9% by volume and 76% by frequency occurrence in the foregut. amongst rotiferan zooplankton keratella spp. was the most abundant genus in the gut contents (table 1, figs. 2-4). another important food item in the gut contents of this fish was the members of bacillariophyceae (diatoms). this group of plankton included all the diatoms mentioned earlier except cyclotella spp. these diatoms were present in the forgut content as 1.48% by number, 0.23% by volume and 80% by frequency occurrence silver carp. amongst this group of diatoms, navicula spp. appeared as the major contributor to the diet of this carp. another group of phytoplankton, dianophyceae represented 2.72% by number, 2.15% by volume and 83% by frequency occurrence in the foregut contents. amongst dianophyceae, ceratium spp. was most dominant in the gut contents followed by peridinium spp. percentage number and biovolume of the major food items of different size groups are given in figure 5 and table 2, respectively. study of food items of various size groups (at 100 mm interval) showed that the percentage number of green algae decreased with increase in the size of the fish (fig. 5), while the percentage number of cyclotella spp. almost showed increasing trend with size of the fish except size group 200-300 mm and size group 600-700 mm (fig. 5). the present investigations on food overlap between size groups showed that significantly high overlap values were observed between large size groups or between small size groups. the moderate overlap values were observed between small and large size groups. the observations showed that the size group (mm)/ food items 200-300 300-400 400-500 500-600 600-700 700-800 800-900 900-1000 green algae 10.52 7.34 11.56 2.80 2.90 2.11 1.94 0.96 blue green algae 0.00 6.38 0.53 4.69 7.13 2.21 2.44 4.97 digested material 43.55 37.13 58.30 59.92 59.54 38.29 64.40 71.26 rotifera 38.81 11.08 9.06 4.80 1.74 4.89 2.95 1.64 crustacea 0.00 34.36 8.55 11.05 18.26 23.20 12.81 12.30 diatoms 0.00 0.42 0.93 0.13 0.14 0.28 0.10 0.45 dianoflagelata 0.91 1.33 1.10 2.34 1.77 2.10 2.26 3.83 cyclotella 6.21 1.95 9.97 14.26 8.51 26.91 13.09 4.61 table 2. percentage biovolume of the major food items in the foregut of different size groups of silver carp in gobindsagar reservoir. g re e n a lg a e b lu e g re e n a lg a e r o ti fe ra c ru s ta c e a d ia to m s d ia n o p h y c e a e c y c lo te ll a fem ale male pooled0 10 20 30 40 50 60 70 80 90 100 p e rc e n ta g r o c c u rr e n c e food item 2 0 0 -3 0 0 3 0 0 -4 0 0 4 0 0 -5 0 0 5 0 0 -6 0 0 6 0 0 -7 0 0 7 0 0 -8 0 0 8 0 0 -9 0 0 9 0 0 -1 0 0 0 green algae b lue green algae ro tifera crustacea diato ms diano phyceae cyclo tella 0 10 20 30 40 50 60 70 80 p e rc e n ta g e n u m b e r size group (mm) figure 4. percentage occurrence of different food items in the foregut of silver carp. figure 5. percentage number of the major food items in the foregut of different size groups of silver carp. 230 international journal of aquatic biology (2015) 3(4): 225-235 overlap index varied between 0.461-0.972. the minimum value (0.461) was observed between size groups 200-300 mm and 900-1000 mm. the highest value was observed between size groups 500-600 and 700-800 mm. the results revealed that the diet overlap index between females and males was very high (0.915 and 0.951 for food items found in foregut and hind gut, respectively), showed that the diet of female and male specimens did not differ significantly (table 3). relationship between percentage number and frequency occurrence of the food items in the foregut of silver carp (pooled data) based on the costello's (1990) method is given in figure 6. data showed that cyclotella spp. with high percentage number and frequency of occurrence represented the dominant prey taxon and thus was very important item in the diet of h. molitrix. green algae with high frequency of occurrence and less percentage number could be considered to be indicative of generalized diet. as the frequency of occurrences of the other food categories were high and their percentage number were low, hence, these items could be considered as general diet of this fish. however, the results showed that there was no preference for certain specialized taxa by silver carp. estimation of relative gut length of 45 specimens of silver carp (size range 399-985 mm) showed that gut length of this fish was 3.41-8.61 times greater than total length, with a mean of 5.59 and there was no significant difference between relative gut length of different size groups (anova, n = 45, df. = 6, f = 1.057, p = 0.405). discussion the results of the present study are in conformity with the earlier findings on the diet composition of silver carp. plankton identified in the gut contents of h. molitirx have been found to be similar in the report of cremer and smitherman (1980). plankton genera in water samples, with the exception of a few genera, have been observed in samples from intestine of silver carp, indicating no selectivity of specific types of plankton by this species from gobindsagar. lin (1969) and cremer and smitherman (1980) also reported no selectivity for specific types of plankton by silver carp, although chiang (1971) reported that silver carp selectively fed on cyanophyceae and chlorophyceae. the presence of few zooplankton genera e.g. polyarthra spp. and splanchnopus spp. in the water sample collected from gobindsagar and their absence in the gut contents may be attributed to the size group (mm) 200-300 300-400 400-500 500-600 600-700 700-800 800-900 900-1000 200-300 1.000 0.781 0.904 0.616 0.612 0.615 0.540 0.461 300-400 0.781 1.000 0.869 0.589 0.645 0.589 0.539 0.479 400-500 0.904 0.869 1.000 0.664 0.673 0.667 0.584 0.515 500-600 0.616 0.589 0.664 1.000 0.852 0.972 0.912 0.833 600-700 0.612 0.645 0.673 0.852 1.000 0.858 0.817 0.813 700-800 0.615 0.589 0.667 0.972 0.858 1.000 0.909 0.839 800-900 0.540 0.539 0.584 0.912 0.817 0.909 1.000 0.848 900-1000 0.461 0.479 0.515 0.833 0.813 0.839 0.848 1.000 table 3. diet overlap index (foregut) between different size groups of h. molitrix. figure 6. relationship between percentage number and frequency occurrence of the food categories in the foregut of silver carp based on the costello’s (1990) method. 231 esmaeili and johal/food and feeding habits of silver carp fact that these zooplankton appeared only for a limited time period of the year (april-may) or may there may be other possibility that these might have been digested in the intestine. the possibility of the later is very remote. the results of the present study showed that the diet of silver carp was dominated by cyclotella spp. (diatom) followed by chlorophyceae, cyanophyceae, crustacea, dianophyceae and rotifera. dominance of diatoms in the gut of silver carp has also been reported by ruzicka and ruzickova (1987) and xie (1999). according to xie (1999), the share of cyclotella spp. to the total phytoplankton biomass in the foregut contents of silver carp cultured in the net cage in the donghu lake (yangtze river) was 58-92% (mean 73%), which confirm the present investigations on the food of silver carp. however, ruzicka and ruzickova (1987) reported the abundance of different genera of diatoms (melosira spp, asterionella spp. and fragillaria spp.) in the gut of silver carp from podhora water reservoir. analysis of gut contents of silver carp from gobindsagar (pooled data) indicated that zooplankton comprised only 7.7% by number and 19.3% by volume of the foregut contents of this fish, hence, silver carp could be considered almost as phytoplankton feeder. tang (1970) also reported the similar results and classified silver carp as phyto–and microzooplankton feeder, with zooplankton at less than 7% of the total plankton organism. according to ruzicka and ruzickova (1987) zooplankton comprised less than 10% of food biomass of this fish which is in agreement with the present study. according to cremer and smitherman (1980), phytoplankton was the major contributor of intestine as food of silver carp and zooplankton were rarely present. the present studies showed that the majority of the plankton found in the intestine of silver carp was cyclotella spp. (62.25%) having the size range of 525 µm. almost similar results have been reported by xie (1999). they reported that cyclotella spp. and cryptomonas spp. (dinoflagellate) were dominant phytoplankton in the gut of silver carp, and the size of cyclotella spp. was usually smaller than 20 µm (mostly varied between 6-15 µm), while that of cryptomonas spp. was usually smaller than 30 µm (mostly ranged between 10-20 µm). the present results, together with those of smith (1989), li and dong (1996) and xie (1999) clearly pointed out that silver carp is able to collect food particles smaller than the distance between its gill rakers. the distance between gill rakers (dgr) of silver carp has been recorded by earlier workers. hampl et al. (1983) reported that dgr of silver carp was 12 µm at the narrowest part and 26 µm at the widest part, which do not change with age. liu (1981) reported dgr of silver carp to be 15-25 µm when the total fish length was 1.5-10 cm and 34-41 µm when the total fish length was 34-47 cm. spataru et al. (1983) found that dgr of silver carp was 36 µm (ranging between 33.9-37.2 µm). these observations indicate that the distances between gill rakers of silver carp are larger than 15 µm, hence, it can be concluded that h. molitrix is able to collect food particles smaller than its filtering net meshes and these observations confirm the present observations. the exact mechanism is still not understood. probably, excretion of mucus plays an important role in collecting such small food particles. according to li and dong (1996) silver carp can feed on cyclotella spp. (3.2 µm in diameter) efficiently through the dogging effect in conjunction with mucus. as described earlier cyclotella spp. was found to be the major food item in the gut of silver carp from gobindsagar reservoir and by applying costello method, it can be concluded that cyclotella spp.is important food item while the others are general food items in the diet of silver carp from gobindsagar. costello’s (1990) analysis is based on a two dimensional representation, where each point represents the frequency of occurrence and the abundance of a prey taxon. study of diet overlap index of different size groups showed that the value of “d” varied between 0.461 (moderate) to 0.972 (high). moderate overlap values were found between small size groups and large size groups but high overlap values were observed 232 international journal of aquatic biology (2015) 3(4): 225-235 between large size groups and between small size groups, indicating that the diet of small size groups was significantly different from those of large size groups. in general, a high value of overlap index means that the groups under comparison shared a number of abundant prey, therefore, competition for food was possible. however, according to pedersen (1999) diet overlap indices do not show the actual competition, as these values depend on how the food categories are defined and therefore must be used with caution (hurlbert, 1971; horppila et al., 2000). the present observations indicated that the diet overlap index between female and male silver carp was very high (0.915), indicating that the diet of male and females specimens did not differ significantly. the question of the digestibility of plankton consumed by silver carp is also an important aspect of feeding ecology of this species. presence of some undigested plankton e.g. scenedesmus spp., staurastrum spp. and microcysis spp. in the hindgut of silver carp showed that these food items could not be digested completely, while most parts of the body of cladoceran and copepods are digested. the present observations confirm the observations of earlier workers such as bitterlich and gaigner (1984) who reported that zooplankton and cladocerans are digested in 20 min after their intake as foods, but the same is not true in the case for the members of certain phytoplankton genera like cyanobacteria and certain chlorophyceae. these authors reported that the digestive efficiency of microcystis aeruginosa in the silver carp was 32.6% and the conversion efficiency was low at 4.6%. the findings of beveridge et al. (1993) indicated that the same species of cyanobacteria which constituted the food of silver carp in the present studies were digested poorly by the silver carp. similarly some genera e.g., scenedesmus, tetraedron, pediastrum and coelastrum belonging to chlorophyceae seem to be digested poorly (voros et al., 1997). on the contrary, the same authors reported an excellent digestibility for cryptophyceae, diatoms and the cyanobacterian aphanizomenon flosaquae, which accounted for more than 70% of the phytoplankton biomass ingested by silver carp, was assimilated poorly. according to them, from a qualitative point of view, cryptophycea, diatoms, and especially zooplankton were much more valuable food for the silver carp than cyanobacteria and desmid chlorophycea which were poor in long-chain polyunsaturated fatty acids. as far as the relative gut length is concerned, it has been observed that the value of relative gut length varied 3.41-8.61 with a mean of 5.59 and there was no significant difference between relative gut lengths of different size groups. however, due to lack of small specimens in the commercial netting, it is difficult to arrive to an exact conclusion about the change in diet of different size groups of silver carp in gobindsagar reservoir. according to cremer and smitherman (1980) gut length of silver carp was 3.57.3 times greater than total length, with a mean of 5.0, which is in agreement with the results of the present study, and this confirms phytoplanktivorus habits of silver carp. generally, change in gut length is believed to be related to change in the quality of food consumed (al-shamma, 1986; hofer, 1991) but, according to fuentes and cancino (1990) and xie et al. 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(1971). competition in tropical stream fishes: support for competitive exclusion principle. ecology, 52: 336-342. international journal of aquatic biology (2015) 3(4): 225-235 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved چکیده فارسی در hypophthalmichthys molitrix (val., 1844) ايکپورنقره ماهي غذايي عادات و غذا هند گوبيندساغر سد درياچه حميد رضا اسماعيلي1*، موهيندر جوهال سينگ2 1گروه زيست شناسي، دانشکده علوم، دانشگاه شيراز، ايران. .هند پنجاب، دانشگاه ،جانورشناسي گروه 2 چکيده: ماهی اين غالب غذاي رژيم .cyclotella spp دياتومه که داد نشان هند گوبیندساغر سد درياچه در اي کپورنقره ماهی غذايی عادات و غذا بررسی دامنه. گیرندمی قرار بعدي هايرتبه در ترتیب به( گردانتنان) روتیفرها و ها دينوفیسه پوستان،سخت ها، سیانوفیسه ها، کلروفیسه و داده تشکیل را از ترکوچک غذايی ذرات است قادر ايکپورنقره ماهی که است موضوع اين بیانگر که بوده متغیر میکرون 55 تا 5 بین .cyclotella spp اندازه با. باشد داشته مهمی نقش ريز ذرات اين آوريجمع در موکوس ترشح که رسدمی نظر به. نمايد آوريجمع را خود آبششی هايپاالينده بین فاصله غذايی ارقام غذايی، مواد ساير که حالی در بوده مهم بسیار غذايی رقم يک .cyclotella spp که شد گیرينتیجه چنین کاستلو روش بکارگیري که داد نشان مختلف هاياندازه در ايکپورنقره ماهی غذايی رژيم همپوشانی شاخص مطالعه. شوندمی محسوب درياچه در ماهی اين براي عمومی جنتاي. است بزرگ و کوچک ايکپورنقره ماهیان غذايی رژيم دارمعنی اختالف بیانگر که بوده متغیر( زياد) 275/0 تا( متوسط) 164/0 بین d مقدار آنالیز. ندارد وجود جنس دو بین ريدامعنی اختالف و( 245/0) بوده باال ماده و نر ايکپورنقره غذايی رژيم همپوشانی شاخص که داد نشانهمچنین جلويی گوارش لوله محتواي حجم از درصد 3/42 و تعداد نظر از درصد 7/7 تنها جانوري هايپالنکتون که داد نشان ايکپورنقره گوارش لوله محتواي .گرفت نظر در گیاهی خوارريزپالنکتون يک را اي کپورنقره توانمی ينابنابر ،دهندمی تشکیل را ماهی اين .ايکپورنقره کاستلو، روش غذايی، رژيم همپوشانی شاخص گیاهی، پالنکتون: کلمات کليدي int. j. aquat. biol. (2017) 5(3): 128-192 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology review article review of the herrings of iran (family clupeidae) brian w. coad1 canadian museum of nature, ottawa, ontario, k1p 6p4 canada. article history: received 4 march 2017 accepted 5 may 2017 available online 2 5 june 2017 keywords: morphology, biology, alosa, clupeonella, tenualosa, kilka, shad. abstract: the systematics, morphology, distribution, biology, economic importance and conservation of the herrings (kilkas and shads) of iran are described, the species are illustrated, and a bibliography on these fishes in iran is provided. there are 9 native species in the genera clupeonella, alosa and tenualosa in the caspian sea and rivers of southern iran. introduction the freshwater ichthyofauna of iran comprises a diverse set of families and species. these form important elements of the aquatic ecosystem and a number of species are of commercial or other significance. the literature on these fishes is widely scattered, both in time and place. summaries of the morphology and biology of these species were given in a website (www.briancoad.com) which is updated here for one family, while the relevant section of that website is now closed down. other families will also being addressed in a similar fashion. family clupeidae herrings, shads, sardines, sprats, pilchards and menhadens are moderate-sized fishes, usually less than 25 cm long, found in warmer marine waters with some species anadromous or permanent freshwater residents. there are about 64 genera and about 218 species world-wide (nelson et al., 2016), with 8 species in the caspian sea and one commonly found in persian gulf drainages. some other, primarily marine, species are known to enter rivers in southern iran (e.g. nematalosa nasus (bloch, 1795) and sardinella sindensis (day, 1878)) but are not dealt with here (hashemi and ansary, 2012; jouladeh-roudbar et al., 2015). the diversity of this * corresponding author: brian w. coad doi: https://doi.org/10.22034/ijab.v5i3.282 e-mail address: bcoad@mus-nature.ca family in the caspian sea is seen in the number of subspecies which have been described, rather than in genera. at the species level these are caspian sea endemics. a study by pourrafei et al. (2016) based on the nuclear gene rag1 did not support the monophyly of clupeidae but, as an abstract, details are lacking. these fishes are dealt with as a single family here. curiously, the species and subspecies in the caspian sea are generally of larger size than their relatives in the black sea basin. these observations are attributed to the variable environment in the caspian sea over time, with repeated changes in salinity and temperature which the fish could not avoid. black, mediterranean and atlantic species lived under more stable conditions and could, in any case, retreat from lowered temperatures for example. in addition, the caspian sea clupeids lacked the competitors which entered the black sea from the mediterranean and atlantic and some (clupeonella spp., alosa caspia) could occupy the pelagic, planktivore niche taken up by other species in the black sea. there are no other pelagic fish but these herrings in the stable salinity areas of the caspian sea. these fishes usually have modified scales on the belly forming abdominal scutes with a saw-like 129 int. j. aquat. biol. (2017) 5(3): 128-192 edge. most species have two, long, rod-like postcleithra. the lateral line is usually absent or on only a few scales. silvery cycloid scales are easily detached and are found only on the body. the mouth is usually terminal with jaws about equal in length. teeth are small or absent but gill rakers are long and numerous for sieving plankton. fins lack spines and there are no barbels. there is no adipose fin. the pectoral and pelvic fins have a large axillary scale. the caudal fin is deeply forked. the eye is partly covered by an adipose eyelid. the flesh is particularly oily and is highly nutritional. members of this family often form immense schools in surface waters of the ocean and the caspian sea where they feed on plankton. schooling is an anti-predator device making it difficult for a predator to pick out an individual from a tight mass of fish. there is also a "sentry effect" where awareness is increased by the presence of many fish. the school is maintained by a balance between visual attraction and lateral line stimulus repulsion. herring can feed on the smaller plankton, less than 300-400 µm, at night by filter-feeding but during the day can also use particulate feeding. in the latter, they select larger plankton using the area temporalis, a specialised ventro-posterior region of the retina which improves vision as herring approach food items from slightly below. herring are easily caught and are extremely valuable to commercial fisheries. they are the most important fishes economically, both as food for man and also for many other commercial fish species. wars have been fought over fisheries for herrings. in one year, members of the herring family made up 37.3% of all fish caught in the world. some are used for fish meal, as fertiliser and as an oil source. the 1994-1995 catch of clupeids in the iranian caspian was 98.3 tonnes by beach seine and 671.5 tonnes by gill nets, a decrease of 200 tonnes in total over the previous year's catch (iranian fisheries research and training organization newsletter 10: 4-5, 1995) (but see below where the catch is much higher – sources tend to vary). the caspian sea shads accounted for about 35% of total inland production in iran which was 117,300 tonnes in 1995 (bartley and rana, 1998). these fish are used in a high value form as frozen whole consumer packs, as fish meal for poultry and in aquaculture, and in canning (food and agriculture organization, fisheries department 1996). catches in the caspian sea for 1991 and 1992 were respectively 13,817 and 21,527 tonnes of kilka (clupeonella spp.) compared with 3,036 tonnes and 2,692 tonnes of sturgeons and 18,571 and 16,873 tonnes of bony fishes. the catch reached 51,000 tonnes in 1994 from none 10 years previously (food and agriculture organization, fisheries department, 1996). the kilka catch in gilan province in 1997 was 36,077 tonnes (total catch 39,154 tonnes for the province) and in mazandaran in 1998 was 31,583 tonnes (total catch 48,027 tonnes). the caspian environment programme (1998) gives the following catches in tonnes for the iranian caspian sea for kilka (clupeonella spp.) and for alosa pontica (= kessleri), respectively as 1,013 and 2 (1973), 1,170 and 2 (1974), 1,286 and 4.5 (1975), 900 and 5.5 (1976), 1,261 and 2 (1977), 771 and 0 (1978), 836 and 0 (1979), 619 and 0.1 (1980), 1,341 and 0.4 (1981), 798 and 10.4 (1982), 621 and 1.6 (1983), 1,517 and 20.3 (1984), 1,828 and 34.8 (1985), 2,450 and 71.9 (1986), 4,389 and 13 (1987), 4,700 and 16 (1988), 7,902 and 30 (1989), 8,814 and 30 (1990), 13,817 and 35 (1991), 21,527 and 35 (1992), 28,730 and 893 (1993), 51,000 and 720 (1994), 41,000 and 490 (1995) and 57,000 and 330 (1996). abdolmalaki and psuty (2007) give figures over a wide range of years for iranian coastal catches in the southern caspian sea with selected comparative species as presented in table 1. overfishing of clupeids in the caspian sea and stock collapses caused by the invasive ctenophore mnemiopsis leidyi have also had deleterious effects on the caspian seal (pusa caspica) which fed on these fishes (harkonen et al., 2012). a major source for the biology and systematics of caspian clupeids remains svetovidov (1952), now inevitably dated but not yet updated. whitehead (1985), hoestlandt (1991), coad (1997) and coad et 130 coad/ herrings of iran al. (2003) gave brief overviews of iranian herrings. there has been no recent, careful systematic and taxonomic study of these species in the caspian sea basin as a whole and extensive new material was not available for examination here. key to clupeidae: caspian sea species have numerous nominal subspecies and keys to these may be found in berg (1948-1949) and svetovidov (1952). 1a. upper jaw without a median notch, rounded when viewed from in front; last two anal fin rays enlarged; lower jaw articulation with skull below or anterior to posterior eye margin; caspian sea species; clupeonella spp. …………………………2 1b. upper jaw with a median notch; last two anal fin rays not enlarged; lower jaw articulation with skull behind posterior eye margin; caspian sea and persian gulf; alosa and tenualosa spp. ………….4 2a. pectoral fins rounded at tips; head large and narrow (interorbital width 15.5% or less of head length)…………………………clupeonella grimmi 2b. pectoral fins pointed at tips; head short and wide (interorbital width 16% or more of head length)…..3 3a. body and belly compressed (body depth about 21-27% of standard length); keeled belly scales evident …………………………clupeonella caspia 3b. body cylindrical and belly rounded (body depth 16-19% of standard length); keeled belly scales weakly developed……...clupeonella engrauliformis 4a. branched pelvic fin rays 7; upper gill rakers not overlapping lower gill rakers at angle of first arch; persian gulf basin………………… tenualosa ilisha 4b. branched pelvic fin rays 8; upper gill rakers overlap lower gill rakers at angle of first arch; caspian sea species; alosa spp. ………………… 5 5a. body deep and compressed; head large and deep, wedge-shaped in anterior view; caudal peduncle short; pectoral fins long……………………………6 5b. body not deep and not compressed; head not large and deep, not wedge-shaped in anterior view; caudal peduncle not short; pectoral fins short…….8 6a. gill rakers on first arch 50 or more, thin and long, much longer than gill filaments; teeth weakly developed…...…………………………alosa caspia 6b. gill rakers on first arch 48 or less, shorter, equal to or somewhat longer than gill filaments; teeth well developed …………………………………………7 7a. upper and lower profiles of head straight; lower jaw protruding and its upper edge straight ………………………………alosa saposchnikowii 7b. upper and lower profiles of head rounded; jaws equal in length and lower jaw has a crescentic upper edge………………………… alosa sphaerocephala 8a. gill rakers 49 or less, thick and coarse………………………… alosa braschnikowii 8b. gill rakers 57 or more, may be thin and long but can be coarse and short……………… alosa kessleri genus alosa linck, 1790 the caspian sea species of alosa were formerly placed in the genus caspialosa berg, 1915. svetovidov (1952) synonymised caspialosa with alosa. there are 5 species in iranian waters and the caspian sea as a whole but numerous subspecies have been described. alosa species are also found in the black sea, mediterranean sea and atlantic ocean. often distinguished by gill raker counts which in any case overlap, the various subspecies are difficult to identify. morphometric characters are of little help and zamakhaev (1944) points out that some named taxa are merely different age groups. alosa species are distinguished from sympatric clupeonella species by larger size (up to 75 cm total table 1. iranian coastal catches in the southern caspian sea with selected comparative species (abdolmalaki and psuty, 2007) catch and frequency 19271936 19371946 19471956 19571966 19671976 19771986 19871996 19972003 total recorded catch (t) 8,959 7,224 4,986 3,262 5,547 5,384 16,903 16,201 sturgeon meat + caviar (%) 13.4 8.8 16.3 50.9 40.9 34.2 9.4 5.0 rutilus kutum (%) 12.2 43.0 24.9 25.8 17.8 19.8 53.2 45.4 rutilus rutilus (%) 20.7 25.5 18.8 0.7 0.8 2.3 5.8 6.1 alosa spp. (%) 1.9 6.2 14.7 2.9 0.3 0.2 3.2 3.9 131 int. j. aquat. biol. (2017) 5(3): 128-192 length compared to 20 cm), a large mouth, a black spot on the flank behind the operculum and sometimes a row of such spots, an elongate scale or ala at the upper and lower base of the caudal fin, a notch at the mid-line of the upper jaw and by the last two anal fin rays not being elongated. caspian sea species have a laterally compressed belly with 29-36 spiny scutes running from the throat to the anal fin, the dorsal fin origin is closer to the snout tip than the caudal fin base, the dorsal fin lies in a groove formed by enlarged scales, scales are easily detached, the pelvic fin origin lies below or slightly posterior to the dorsal fin origin, teeth are usually present on the jaws, roof of the mouth (on the palatine bone and always on the vomer bone), and on the tongue, the opercular bone is distinctly striated, eggs are demersal, semi-pelagic, and lack an oil globule, gill rakers highly variable in shape and number (18-180), dorsal fin branched rays 11-16, anal fin branched rays 10-21, scales in lateral series 49-60, and vertebrae 43-55. afraei bandpyi et al. (2004) examined 336 specimens alosa species from mazandaran and golestan provinces and found the distinguishing characters presented in table 2. in addition, hosseini et al. (2011) examined 132 specimens of alosa species from gilan province and found the distinguishing characters presented in table 3. the two characters used in combination distinguish the species. the general farsi name for these fishes is shag mahi or zalun (both in gilaki). alosa volgensis (berg, 1913) was recorded from iranian waters by kottelat and freyhof (2007) but presence needs confirmation by specimens. alosa curensis (suvorov, 1907) is poorly known and described from kyzylagach bay of azerbaijan. it may eventually be recorded from iranian waters. these herrings migrate from the north caspian sea to overwinter in the central and southern parts, returning north in the spring. alosa braschnikowii (borodin, 1904) (fig. 1) common names: shagmahi, shagmahi-ye khazari. [dolkii siyanayn, agraxan siyanayi, sara siyanayi, irikoz siyanak, hasangulu siyanayi, agbas siyanak, all in azerbaijan; caspian marine shad, kurinskaya sel'd or kura herring, poloschataya sel'd or striped herring, agrakhanskaya sel'd or agrakhan herring, bol'sheglazaya sel'd or bigeye herring, dolginskaya sel'd or dolginka herring, belogolovaya sel'd or whitehead herring, astrabadskaya sel'd or astrabad herring, sel'd-gonets or driver, zheltospinka or yellow-back, gasankulinskaya sel'd or gasan-kuli herring, kiselevichevskaya sel'd or kiselevitch herring, krasnovodskaya sel'd or krasnovodsk herring, vostochnaya sel'd or eastern herring, obzhorka or glutton, sarinskaya sel'd or sara herring, maiskaya sel'd or may herring, brashnikovskaya sel'd or brashhnikov's shad, all in russian]. systematics: originally described as clupea caspiopontica var. braschnikowii. reshetnikov et al. (1997) revert to the original double "i" ending to the table 2. distinguishing characters of alosa species from mazandaran and golestan provinces, southern caspian sea (afraei bandpyi et al., 2004). species gill rakers ratio of eye diameter to total length (%) a. braschnikowii 20-40, mean 30.93 2.9-5.82, mean 4.72 a. caspia 110-125, mean 118.3 5.73-7.46, mean 6.21 a. pontica (= kessleri) 60-73, mean 66.82 4.27-6.48, mean 5.46 a. saposchnikowii 20-48, mean 32.83 6.0-9.33, mean 7.3 table 3. distinguishing characters of alosa species from gilan province, southern caspian sea (hosseini et al., 20114). species gill rakers ratio of eye diameter to head length (%) a. braschnikowii 22-49, mean 36.6 15.91-21.88, mean 18.53 a. caspia 74-128, mean 106.3 19.36-24.3, mean 22.48 a. kessleri 49-73, mean 58.5 17.75-22.4, mean 20.88 132 coad/ herrings of iran specific name although the catalog of fishes (downloaded 13 january 2017) has a single “i” ending. a lectotype from fort shevchenko (aleksandrovsk) is in the zoological institute, st. petersburg (zisp 13051) and paralectotypes were designated by svetovidov (1952) (zisp 13051). clupea caspio-pontica is an unneeded new name according to eschmeyer et al. (1996). alosa braschnikowii was regarded as a subspecies of alosa caspia by some authors. clupeonella leucocephala berg, 1913 from sumgait and gyurgenchai, azerbaijan is a synonym (as caspialosa brashnikovi leucocephalia (sic). it is listed as a synonym of c. b. grimmi in mikhailovsky (1941)), as is caspialosa caspia nigra kisselevitsh, 1923 from the caspian sea opposite dzambai (the material also included specimens of alosa saposchnikowii) (whitehead 1985, eschmeyer et al. 1996). alosa braschnikowii has several subspecies in the caspian sea, namely agrachanica (mikhailovsky, 1941) (author also spelt mikhaylovsky or mikhailovskaya; dated 1940 in eschmeyer et al. (1996) but 1941 in the online catalog of fishes and 1941 on the paper itself and in svetovidov (1952) and berg (1948-1949); species also spelt agrakhanika in berg (1948-1949); caspialosa brashnikovi morpha elata is a synonym according to mikhailovsky (1941)), the agrakhan herring; autumnalis (berg, 1915), the bigeye herring; braschnikowii (borodin, 1904) (also spelt brashnikovi in svetovidov (1952) and berg (19481949)), the dolginka herring; grimmi (borodin, 1904), the whitehead or astrabad herring, driver or yellow-back; kisselevitschi (bulgakov, 1926) (spelt kisselevitschi on the plate in bulgakov (1926), kisselevitschi in mikhailovsky (1941), kisselevitshi in svetovidov (1952) and whitehead (1985) and kisselewitschi in berg (1948-1949)), the gasan-kuli or kiselevitch herring; nirchi (morosov, 1928) (author also spelt morosow in mikhailovsky (1941) and morozov in eschmeyer et al. (1996)) (with caspialosa brashnikovi kenderlensis budamshin, 1938 from kendyrli bay as a synonym in svetovidov (1952) and berg (1948-1949)), the krasnovodsk herring; orientalis (mikhailovsky, 1941), the eastern herring or glutton; and sarensis (mikhailovsky, 1941), the sara or may herring. caspialosa brashnikovi derzhavini tarasevich, 1946 described from the caspian sea near the apsheron peninsula, azerbaijan may be another subspecies. caspialosa kiselevitschi morpha elata morozov, 1928 from the caspian sea, krasnovodsk bay, turkmenistan is an infrasubspecific taxon and its availability and validity as a taxon have not been examined (eschmeyer et al., 1996). this high number of subspecies is an indication of the populational variation of this shad and not all subspecies may be valid. a modern revision is required to assess this problem. in light of this uncertainty and the lack of adequate sample sizes to determine which of the subspecies occurs in iranian waters or which taxa are valid, reference is made here mostly to the species level. additionally, it figure 1. line drawing of alosa braschnikowii. 133 int. j. aquat. biol. (2017) 5(3): 128-192 should be noted that hybrids between the various subspecies, and between this species and other species, do occur to complicate matters even further. jaafari et al. (2013) compared fish from anzali and sari morphologically and found standard length, postorbital length and anal fin base length were significantly different. jafari et al. (2014) used microsatellite data to study fish from golestan province between gomishan and miankaleh. low genetic differentiation between the two populations could be due to natural migration, genetic diversity within populations was only 1% and among populations was 99%, and it was considered possible that a genetic bottleneck could arise in the future. paknejad et al. (2014) examined morphological variation between 6 localities along the whole iranian coast and found populations to be distinct, particularly the tonekabon region. head shape, eye diameter and predorsal, prepelvic and preanal lengths were the main characters. heidari et al. (2015a) examined fish from the miankaleh gulf for morphometric and meristic characters and suggest, in this brief abstract, that morphological variations should be taken into account in fisheries management and stock enhancement. the neotype of caspialosa brashnikovi agrachanica was designated by svetovidov (1952) as the specimen described by berg as caspialosa brashnikovi m. elata taken in front of the sulak river mouth, agrakhan bay and housed in the zoological institute, st. petersburg under zisp 7334. however, this neotype was not validly designated because qualifying conditions 75.3.1, 75.3.4 and 75.3.5 of the international code on zoological nomenclature were not met (n.g. bogutskaya, pers. comm. 23 january 2013). this also applies to the following 6 taxa. the neotype of caspialosa braschnikovi autumnalis was designated by svetovidov (1952) as a specimen 26.9 cm long from the eastern shore of the caspian sea at gasan-kuli (just north of the iranian border in turkmenistan) caught on 8 april 1948 and housed under zisp 31749. the neotype of caspialosa kisselevitschi is also from gasan-kuli caught on 30 june-1 july 1926 and was housed in the faculty of zoology, central-asian state university (sredne-aziatskogo gosudarstvennogo universiteta), tashkent. the neotype of clupea caspio-pontica var. grimmi was designated by svetovidov (1952) as a specimen 34.0 cm long found at ashur-ade (= ashuradeh) near astrabad bay (= gorgan bay or khalij-e gorgan) on 23 april 1903 is under zisp 13045. the neotype of caspialosa nirchi as designated by svetovidov (1952) is from the southern part of the caspian sea opposite north cheleken spit and is under zisp 31780. the neotype of caspialosa brashnikovi orientalis as designated by svetovidov (1952) is from the southern part of the caspian sea opposite karaashly and is under zisp 32187. the neotype of caspialosa brashnikovi sarensis from sara island is under zisp 32184 as established by svetovidov (1952). key characters: characterised by a relatively elongate and rounded body likened to a "herring" shape, not as deep as in some related species which are likened to a "shad" shape. total gill rakers 18-49 and short (about equal to gill filaments in length, sometimes shorter). teeth are well-developed in both jaws. morphology: dorsal fin with 3-5 unbranched and 12-15, mostly 14, branched rays, anal fin with 2-4, usually 3, unbranched rays and 10-20, mostly 18, branched rays. scales in lateral series 51-54. teeth are well-developed on the jaws, tongue and roof of the mouth. ghotbi jokandan et al. (2014) briefly distinguish this species from a. saposchnikowii on morphometric characters in gilan. the distance between the posterior end of the dorsal fin to the anterior caudal peduncle, distance between origins of pelvic and anal fins, head length, pectoral fin height and ratio of anal fin height to standard length were greater than in a. saposchnikowii. alavi-yeganeh and razavi (2016a, 2016b) found 26-47 gill rakers in 50 specimens 15-45 cm long from iran and number increased with total length, although only fish 15-20 134 coad/ herrings of iran cm and 40-45 cm long had significantly different counts. gill raker length, length/width ratio and ratio of raker to filament length decreased with increase of total length. equal lengths of rakers and filaments were found in fish greater than 25 cm. evidently, some alosa species may overlap in counts depending on size. the accompanying table (table 4) summarises characters of the subspecies and is taken from svetovidov (1952) and mikhailovsky (1941) but identification to subspecies should be done with the keys from these works. some of the characters used in the keys are not in the table as they do show individual variation and are difficult to summarise. an example is the nature of the gill raker (thin, thick, blunt, pointed, bent, straight, curved, branched, broken off, forked, swollen at the tip, etc.); another is the degree of protrusion of the lower jaw. the subspecies grimmi is quite specialised in association with its benthic mode of life, feeding mostly on gobies (gobiidae). it has a unique character in the well-developed callus on the tip of the lower jaw which adults acquire from rubbing the jaw on the sea bed while feeding, gill rakers are low in number as fine food is not taken, and the tips are broken off, broadened, and split owing to abrasion, and the rakers on the lower arch are reduced in number so the first raker is far from the tongue base. the subspecies nirchi is similar. in contrast, the subspecies kisselevitschi has a high gill raker count, rakers are pointed and not split at the tips, and the first raker is close to the tongue base. this species lives in surface waters feeding on clupeonella, atherina, shrimps, gammarids, and gobies (gobiidae). sexual dimorphism: none reported. colour: the back and top of the head are dark with a green or blue tint and may be grey-green. some subspecies are paler in colour with a grey or grassgreen back and pale flanks, nirchi has a whitish bluegreen head, light grey back with a slight greenish tint, and lower jaw and pectoral fins light, while grimmi is also quite pale with a grey-blue back and top of the head and whitish anterior head and pectoral fins. there is a dark spot behind the operculum but no series of spots along the flank in most subspecies, except in rare cases when there may be up to 7, occasionally 12-13. the subspecies grimmi regularly has a row of diffused, grey spots almost merging into a stripe, and nirchi occasionally. pectoral fins are dark on some subspecies (braschnikowii, sarensis, and kisselevitschi), pale or whitish on the others, although there is confusion in the literature over this, perhaps indicative of individual variation (cf. sarensis in mikhailovsky (1941)). the back and upper part of the head may become a deep black at spawning. the flanks and belly are silvery. size: attains 50.0 cm standard length but average lengths are about 27-34 cm. distribution: all the caspian subspecies are found widely distributed in the sea but chiefly in the south in winter, moving north to spawn in spring. the subspecies sarensis is reported from the lenkoran coast and from southwest of gasan-kuli (in turkmenistan just north of the iranian border), the subspecies orientalis from gorgan or astrabad bay, autumnalis from coastal waters at gasan-kuli, character / subspecies gill rakers (mostly) pectoral fins as % body length head as % of body length vertebrae agrachanica 20-46 (28-33) 13.1-15.6 22.6-25.2 47-54 autumnalis 21-37 (28-30) 16.4-19.9 26.0-29.2 45-53 braschnikowii 24-47 (30-33) 14.3-16.7 23.5-26.6 48-55 grimmi 18-28 (20-22) 12.9-15.2 22.9-26.4 45-52 kisselevitschi 29-49 (36-40) 13.9-16.8 24.2-26.9 43-53 nirchi 20-31 (23-26) 10.9-14.7 23.4-26.3 48-52 orientalis 20-35 (27-32) 13.5-18.0 25.0-27.8 45-53 sarensis 20-33 (24-27) 14.1-16.2 23.8-26.6 45-53 table 4. characters of the subspecies in alosa braschnikowii based on svetovidov (1952) and mikhailovsky (1941). 135 int. j. aquat. biol. (2017) 5(3): 128-192 kisselevitschi from astara and gasan-kuli, and grimmi from astara and gorgan bay. esmaeili et al. (2014) record it from the anzali wetland or talab. zoogeography: this species is endemic to the caspian sea. habitat: in winter, this species moves into deeper water towards the iranian coast. in march, it approaches coastal waters (vetchanin, 1984) including brackish waters but does not enter fresh water. it never enters rivers in the south of the caspian sea (jolodar and abdoli, 2004). salinities up to 47.6‰ are survived by this species. spawning and feeding grounds are in the north caspian for some populations but others live permanently in the south caspian sea and are of smaller size. the subspecies kisselevitschi, for example, lives off gasan-kuli in winter at depths below 25 m, not migrating or feeding. in march, they move north to feed and then return south to spawn but live almost entirely as a pelagic species in the southern caspian sea. knipovich (1921) reports this species from depths of 80-98 m in iranian waters. the density of this species increased from east to west in a 19992001 study in iranian waters (afraei et al., 2006). abdoli and naderi (2009) list it as from the southwest, southeast and south-central caspian sea in iranian waters. age and growth: maturity is attained at age 2-5 and life span is up to 10 years, although this varies with the subspecies. most south caspian forms apparently mature at age 2 according to svetovidov (1952). growth rates also vary between subspecies, orientalis being one of the slowest growing herrings in the caspian sea and reaching 10 years of age. the catch near astara of sarensis, for example, is mainly 4-5 year olds but this too varies with the subspecies and also with the year-class strength. vetchanin (1992) reported on grimmi catches from the southeastern caspian where the average length was 27.8-28.6 cm and the average weight 294-313 g. there is a tendency for length and weight to fall in catches as the summer progresses, from april to july. length and weight are less in southern, compared to northern waters. afraei et al. (2006) found this species to be the largest alosa in iranian waters on average at 395 mm and 760.3 g. males predominate at 55.8% in mazandaran and 69.4% in golestan catches. six age classes were present (1+ to 6+) with the 2+ class being the most common at 28.9% and 6+ the rarest at 8.9%. alavi-yeganeh et al. (2013) found mazandaran fish were 1-6 years old and had a b value of 3.693 showing positive allometric growth. ghotbi-jokandan et al. (2015) examined 147 fish from anzali, noor and torkeman and found a b value of 3.1. mousavi-sabet et al. (2016) gave a b value of 3.24 for iranian fish from 6 fishing areas from astara to miankaleh. food: diet in the southeastern caspian sea in winter comprises 85% clupeonella engrauliformis with some gobies (neogobius) and shrimps (vetchanin, 1984). from march to november the diet is dominated by clupeonella caspia, atherina boyeri (= caspia) and shrimps. juvenile liza saliens, syngnathus caspius, molluscs, crabs and higher aquatic plants are also recorded along with foreign objects such as rice husks, pieces of wood, foil, polyethylene, etc. this species is a cannibal. the more southerly populations examined favour atherina boyeri (= caspia) and neogobius species and some of these populations favour benthic invertebrates. the subspecies grimmi is the most benthic one and takes primarily gobies with some molluscs as well as clupeonella. feeding intensity rises sharply after spawning. while some herrings, like alosa pontica (= kessleri), feed poorly on their migration, this species feeds intensively on its spring migration. azizov et al. (2015) examined fish from the western caspian sea north of iranian astara in azerbaijan waters and found the main foods were kilkas, gobies and shrimps with some zoobenthos. intensive feeding took place in spring (march-april) before spawning and in july after spawning. the feeding regime altered after the invasion of the ctenophore, mnemiopsis leidyi. a shift was observed from 85% clupeonella engrauliformis to 65% atherina boyeri (= caspia). other fishes were also eaten including clupeonella grimmi, c. caspia, cyprinus carpio, liza saliens, as well as palaemon 136 coad/ herrings of iran spp. (iranian fisheries research organization newsletter 49: 2, 2006). afraei bandpei et al. (2012) examined 357 fish from beach seines in the southcentral caspian sea and found the diet was dominated by atherina boyeri (= caspia) at 58% with gobiidae (26%), clupeidae (11%), cyprinidae (3%) and mugilidae (1%). atherina boyeri (= caspia) was the most frequent prey at 78.6% index of relative importance, followed by neogobius fluviatilis (= pallasii) at 18.2%. a wider variety of items was fed on in november, december and february with lowest feeding in october and january. there were also differences in diet related to fish size. increases in the gastrosomatic index in february and march coincide with the spawning season and energy preservation for gonadal development. reproduction: vetchanin (1984) reports spawning of this species in the southeastern caspian sea north of iranian waters to begin in early may, continuing to july as it is intermittent. the subspecies sarensis spawns along the lenkoran coast from mid-april to the end of june. the subspecies orientalis spawns in gorgan bay from the end of march to the beginning of april, spawning schools forming at 17-18°c or higher. the subspecies autumnalis spawns at the same time off gasan-kuli near the iranian border with turkmenistan. the subspecies grimmi spawns in may-june in gorgan bay. the subspecies kisselevitschi has the latest spawning date, june to july and even in august off gasan-kuli when temperatures exceed 25°c. spawning takes place in shallow water (1.8-5.8 m) in the sea over sand or silt bottoms at 15-18°c (some subspecies and populations at 20-22°c, others beginning as low as 11°c), and a salinity of 8-13‰. fecundity is up to 178,400 eggs, average 66,000 per fish. there is no feeding while spawning. early maturers, like the south caspian populations, can reproduce up to 7 times in their life. parasites and predators: the caspian seal, pusa caspica, is a predator on this species (krylov, 1984). economic importance: the catch for all species of "caspialosa" in iran varied between 5,337 kg and 419,518 kg for the years 1956/1957 to 1961/1962 (vladykov, 1964). in the anzali region, the catch for the years 1933/1934 to 1961/1962 varied from 1,553 kg to 539,710 kg (vladykov, 1964). the catch has been as high as 126,900 centners or 12,690 t in the sea as a whole for the type subspecies alone (1 centner=100 kg (svetovidov, 1952)), taken chiefly in spring. other subspecies were not fished for as extensively although kisselevitschi was the most numerous of the south caspian forms of alosa braschnikowii, forming 70% of the drift net catch. conservation: reputedly depleted in iranian waters. kiabi et al. (1999) consider this species to be data deficient in the south caspian sea basin according to iucn criteria. criteria include medium numbers, medium range (25-75% of water bodies), absent in other water bodies in iran, and present outside the caspian sea basin. afraei bandpei et al. (2012) noted that fishing is forbidden in the southern caspian sea from may to september. sources: iranian material: cmnfi 1970-0581, 5, 226.0-245.0 mm standard length, gilan, caspian sea near hasan kiadeh (37º24'n, 49º58'e); cmnfi 1979-0431, 1, 297.2 mm standard length, mazandaran, bazaar at now shahr (no other locality data); cmnfi 1980-0126, 1, 245.8 mm standard length gilan, caspian sea near bandar-e anzali (37º28'n, 49º27'e); cmnfi 1980-0150, 1, 222.4 mm standard length, gilan, safid river estuary (37º24'n, 49º58'e). comparative material: bm(nh) 1938.8.2:1, 1, 245.9 mm standard length, kazakhstan, caspian sea, kaidak bay (no other locality data); bm(nh) 1938.8.2:2, 1, ca. 337.5 mm standard length, kazakhstan, caspian sea, kaidak bay (no other locality data); bm(nh) 1939.2.21:17-18, 2, 285.0305.2 mm standard length, caspian sea (no other locality data); bm(nh) 1939.2.21:19-20, 2, 222.9273.4 mm standard length, caspian sea (no other locality data). alosa caspia (eichwald, 1838) (figs. 2, 3) common names: shagmahi-ye shekambozorg (= big belly herring fish), shagmahi-ye chekameh dar, 137 int. j. aquat. biol. (2017) 5(3): 128-192 shagmahi-ye darya-ye khazar (= caspian sea herring fish), shah mahi (= king fish), zalun (in gilaki), puzanok. [xazar sisgarini, sara sisgarini in azerbaijan; kaspiiskii puzanok or caspian shad, severokaspiiskii puzanok or north caspian shad, srednekaspiiskii puzanok or central caspian shad, il'mennyi puzanok or il'men shad, enzeliiskii puzanok or enzeli (= anzali) shad, sarinskii puzanok or sara shad, bakinskii puzanok or baku shad, astrabadskii puzanok or astrabad shad, all in russian]. systematics: clupea caspia was originally described in latin from "hab. in caspio mari, meridiem versus" (caspian sea, towards the south). alosa caspia has 3 subspecies in the caspian sea basin, namely caspia (eichwald, 1838) (= north caspian, central caspian, caspian or il'men shad); knipowitschi (iljin, 1927) with natio knipowitschi (iljin, 1927) (= enzeli or anzali shad) and natio saraica (berg, 1948) (= sara or baku shad); and persica (iljin, 1927) (= astrabad shad). the differences between natio knipowitschi and natio saraica are small (e.g. gill rakers 122-166 versus 140-150, both averaging 145; vertebrae 43-49 versus 45-51, both with mostly 47 or 48; growth differences are known, the former grows faster in the first 2 years of life but the latter reaches a greater size) and they probably have no taxonomic significance being simply separate breeding populations. the differences between alosa caspia caspia natio caspia (the north or central caspian shad) and natio aestuarina berg, 1932 (the il'men shad) were found to be based on geography and growth rate (svetovidov, 1952). alosa rossica kessler, 1870 described from the volga river is a nomen nudum and is this species. other taxa now considered as synonyms of alosa caspia are caspialosa caspia salina svetovidov, 1936 from mertvyi kultuk and kaidak bays in the northeast caspian sea and caspialosa caspia kaidakensis kazancheev, 1936 (spelt kajdakensis in svetovidov (1952)) from kaidak, the latter being in any case a synonym of the former subspecies. c. caspia m. elongata berg, 1913 is also a synonym. alosa caspica yakovlev, 1871 is presumably a misspelling. figure 2. line drawing of alosa caspia. figure 3. alosa caspia courtesy of a. abdoli. 138 coad/ herrings of iran knipovich (1921) records a species, caspialosa enzeliensis iljin, from the southern caspian sea which he places as a subspecies of caspia. i have been unable to locate the original description of this taxon, which presumably is found in the anzali mordab (= talab) of iran. it is probably an unused manuscript name for what iljin later described as knipowitschi. as of 15 july 2007, this scientific name returned no hits on google and as of 12 december 2016 the only hit was for www.briancoad.com. the lectotype of caspialosa knipowitschi is a specimen 21.2 cm long from anzali in iran caught on 15 april 1915 and housed in the zoological institute, st. petersburg (zisp 31892). the lectotype of caspialosa caspia var. persica is a specimen 147.5 mm long from the caspian sea bay of asterabad (= gorgan bay or khalij-e gorgan) north of ashur-ade (= ashuradeh) at 36°53'n, 53°55'e caught on 25 april 1904 on the caspian expedition of 1904 and housed in the zoological institute, st. petersburg (zisp 16413). the lectotype of caspialosa caspia knipowitschi n. saraica is from near sara island and is under zisp 32183. the lectotype of caspialosa caspia salina is from mertvyi kultuk bay, 10 km west of cape kizil-kair and is under zisp 25813. these taxa were designated by svetovidov (1952) as none were before or material was not preserved. key characters: characterised by a relatively deep and compressed body likened to a "shad" shape, not as elongate and rounded as in some related species which are likened to a "herring" shape. total gill rakers 50-180, variously reported as thin or thick, long (obviously longer than the gill filaments), and forming a convex outline on the lower arch. teeth are poorly developed in both jaws. morphology: dorsal fin with 3-4 unbranched and 12-15 branched rays, anal fin with 3-4, usually 3, unbranched and 15-20 branched rays. scales in lateral series 49-54. the characters distinguishing subspecies all overlap widely and are given in table 5 after svetovidov (1952) and hoestlandt (1991). sexual dimorphism: females are longer and weigh more than males of the same age. colour: the back is blue-green to dark and the flanks silvery. there is a black spot on the flank behind the upper operculum margin and sometimes up to 7 spots extending along the upper flank to a level of the rear of the dorsal fin. size: reaches 28.0 cm standard length for caspia, to 29.6 cm for knipowitschi, and to 33.8 cm for persica. distribution: found in the caspian sea. the subspecies caspia is found mostly in the western half of the caspian sea basin but is the most widely distributed subspecies, found throughout almost the whole sea. the subspecies knipowitschi is found in the south near anzali, astara and the baku archipelago, near the northern shore of the apsheron peninsula in autumn with a few reaching the gorgan bay in fall and winter; natio saraica is found north of astara and spawns near sara island, natio knipowitschi spawns in the anzali wetland. the subspecies persica is found in the southeast, near gorgan or astrabad bay. holčík and oláh (1992) report persica from the western basin of the anzali wetland and this species is reported from the safid river and anzali wetland as subspecies persica and from the anzali wetland as knipowitschi (abbasi et al., 1999). abdoli and naderi (2009) list it as from the southwest, southeast and south-central caspian sea, the anzali wetland and gorgan bay in iranian waters for both knipowitschi and persica. reported characters / subspecies head length as % of body length pectoral length as % of body length vertebrae (mostly) gill rakers (mostly) caspia 25.5-28.1 15.5-18.1 45-52 (49-51) 68-150 (100-140) knipowitschi 18.3-24.1* 16.0-19.1 43-51 (47-48) 120-180 (130-160) persica 25.6-27.1 16.5-17.7 45-51 (47-49) 50-120 (60-90) * the numbers cited in svetovidov (1952: 256 in the english version) and hoestlandt (1991: 128) in the keys to subspecies do not agree with the numbers on p. 148 and p. 265 respectively in the species descriptions. table 5. characters of the subspecies in alosa caspia based on svetovidov (1952) and hoestlandt (1991). 139 int. j. aquat. biol. (2017) 5(3): 128-192 from the siahdarvishan river as a. caspia caspia (abbasi et al., 2007). esmaeili et al. (2014) record a. knipowitschi and a. persica as species from the anzali wetland but these natio are listed as a. caspia in the catalog of fishes (downloaded 15 december 2016). zoogeography: this species is a caspian sea endemic. habitat: the type subspecies prefers open waters. caspian shad winter at depths of 30-40 m or more and prefer temperatures not less than 8-11°c. they rise to surface waters in spring, moving north along the western shore of the caspian sea in waters of about 9-11°c according to kushnarenko (1986) while heckman in hoestlandt (1991) states that this shad begins to migrate at the end of march at 5-6°c water temperature with a peak at 9-14°c in mid to late-april, ending in early may. males migrate in large numbers at the beginning and end of the migration, females in the middle (pushbarnek, 1987) while heckman in hoestlandt (1991) states that two waves of migration occur, one usually in late april at 7.6-10.2°c comprised of over 80% males and the second in the first half of may at 10.8-14.0°c comprised of over 70% females. the young, which hatch in the spring, leave the summer feeding grounds before the adults and migrate south before october-november. adults follow as temperatures fall. some populations do not migrate north and spend their whole life in the southern caspian sea. this subspecies will enter fresh waters to spawn in addition to spawning in the open caspian sea. the subspecies knipowitschi prefers water warmer than that of all other caspian sea clupeids except for alosa caspia persica. its sea movements are not well known but spawning fish favour waters with freshwater input and some fish enter rivers so it is classified as semi-anadromous. this subspecies was common in the anzali wetland but is now replaced by persica (holčík and oláh, 1992). the rise in caspian sea level since 1977 is gradually returning the wetland to its supposed, natural brackish state and may improve the fisheries situation which had declined over the last 50 years. emergent and submergent aquatic macrophytes were decreasing and alosa caspia were increasing in numbers since 1989 (mandych, 1995). however, the fishery will require extensive engineering and management innovations to recover. it is also reported westwards to astara and eastwards to gorgan bay. the winter habitat of persica is unknown. it is semi-anadromous and remains in the southern caspian sea near the shore. from spring to fall this subspecies moves northward along the eastern caspian shore towards krasnovodsk bay and westwards to the anzali talab. age and growth: pushbarnek (1987) found shad of the type subspecies up to 7 years of age on the western coast of the middle caspian sea. in the spawning population, the predominant sizes and weights for males were 16-21 cm and 60-130 g and for females 18-23 cm and 70-140 g. males and females usually mature at 2-3 years although most spawn for the first time at 3 years. females grow faster than males. shad may spawn up to four times as the period of sexual maturation may continue for 2-5 years. the age composition of the spawning population is dependent on year-class strength. first spawners constitute 75.9% of 3-year-olds, 41.7% of 4-year-olds and 23.5% of 5-year-olds. the caspian shad is a slow-growing species compared to a. braschnikowii and a. saposchnikowii, its mean length being 21.2 cm compared to 32.2 cm and 25.6 cm for the two other species respectively (shubina, 1981). dmitriev (1947) briefly examined the anzali, iran population and found 6 age groups but life span is noted by heckman in hoestlandt (1991) to be up to 9 years. maturity is attained as early as 2 years although most fish appear to mature later as most spawners are 4-5 years old. the subspecies persica is the slowest growing of the shad species in the caspian sea, sexually mature fish being 13-21 cm long. some fish become mature at 2 years of age. life span is up to 8 years. the populations of both knipowitschi and persica are small compared to caspia. abbasi and sabkara (2004) studied 180 fish 140 coad/ herrings of iran from the southeast caspian sea coast of iran and found fork length to be 103-232 mm, mean 158.8 mm, weight 16-130 g, mean 52.2 g and age 2-5 years, mean 2.64 years. afraei et al. (2006) found this species to be the smallest alosa in iranian waters on average at 110 mm and 109 g. patimar et al. (2011) found iranian fish to reach age 5+ years, with positive allometric growth in the southeast caspian, and negative allometric growth in the central and southwest caspian. females had a b value larger than males. von bertalanffy growth functions were variable, different between males and females of each area and between sexes from different areas. the largest l∞ was in the southeast for males and in the central area for females. the highest k value was for males and the lowest for females in the southeast area. t0 was negative in all areas with lowest value for females (-0.531) in the southeast and the highest for males (-0.145) in the central area. alavi-yeganeh et al. (2013) found mazandaran fish were 2-5 years old and had a b value of 3.022. ghotbi-jokandan et al. (2015) examined 113 fish from anzali, noor and torkeman and found a b value of 2.98. mousavisabet et al. (2016) gave a b value of 3.12 for iranian fish from 6 fishing areas from astara to miankaleh. food: the most intensive feeding period occurs after reproduction, beginning in june and the highest condition factor is found at the end of this summer feeding period. little food is eaten in winter. temperature (affecting metabolic rate) and zooplankton biomass (decreases engender competition with c. engrauliformis and other planktivores) are important factors governing catches of this species (shubina 1981). food is chiefly copepods, more than 70%, with mysids at 20%, but some phytoplankton and small fishes are taken. food in rivers after spawning is mostly cladocerans and other crustaceans. the above refers to the type subspecies; food of the other two subspecies is assumed to be similar. the southeast caspian sea fish studied by abbasi and sabkara (2004) fed on phytoplankton (rhizosolenia and sprirogyra) at 4.5%, zooplankton (foraminifera, copepoda, cirripedia, bivalvia larvae) at 95.0%, and bony fish larvae and eggs at 0.5%. the presence of the ctenophore, mnemiopsis leidyi, a food competitor reduced the index of fullness and fish growth was reduced. abdollapour bereya et al. (2007) studied diet in fish from beach seines and gill nets in gilan. 98.0% of the stomach contents were zooplankton (ostracods, rhizopods, cladocerans, rotatarians, copepods, cirripedes, mysids, bivalve larvae and bony fish larvae and eggs), 1.8% was phytoplankton (notably rhizosolenia and spirogyra), and 0.2% was benthic items (foraminiferans, sponges, cumaceans, amphipods, insect larvae and palaemonids). acartia spp. (copepods) at 83.1%, and balanus (cypris larvae of the cirripede) at 12.9% were the most abundant. the zooplankton have declined drastically from predation by m. leidyi, the invasive ctenophore, and the fish have shown a great reduction in the index of fullness and in growth recently. reproduction: most spawning of the type subspecies occurs in the north caspian sea near the outflow of the major rivers, particularly the volga, and the fish overwinter in the south caspian, migrating between the two areas (shubina, 1981). this subspecies spawns successively, 3 times within a week. some fish enter fresh water to spawn. spawning takes place at the favoured water temperature of 13.824.1°c, with mass spawning at 18-22°c, beginning as early as late april or as late as mid-may and continuing to midor late june. most eggs are released in the upper 3m of the water column. fecundity reaches 41,000 eggs. the eggs are 1.111.38mm when ripe but unfertilised and 1.92-2.91 mm in diameter when fertilised, and are semi-pelagic to demersal. the subspecies knipowitschi spawns in the anzali talab (and probably the chemkhaleh river to the east of the safid river) in may and june after a spring migration from the sea, leaving in the fall. spawning of the subspecies persica takes place in gorgan bay and holčík and oláh (1992) suspect from catches of mature and spent fish that it also occurs in the anzali talab. parasites and predators: naem et al. (2002) found the monogenean trematode mazocraes alosae on the 141 int. j. aquat. biol. (2017) 5(3): 128-192 gills of this species in the western branch of the safid river. barzegar et al. (2008) record the digenean eye parasite diplostomum spathaceum from this fish. youssefi et al. (2011) report the digenean pronoprymna ventricosa from fish in the babol river. barzegar et al. (2012) found mazocraes alosae, d. spathaceum, p. ventricosa (digenean), and hysterothylacium sp. (anisakid). the caspian seal, pusa caspica, is a predator on this species (krylov, 1984) and it forms a substantial part of the diet of silurus glanis in the anzali talab (holčík and oláh, 1992). ashoori et al. (2012) found that grey herons (ardea cinerea) in the siahkeshim protected area of the anzali wetland or talab ate this species. economic importance: the type subspecies was the most important subspecies in the herring family in the caspian sea. it was caught off the coasts of dagestan and azerbaijan for research purposes and comprises 85% of the clupeid catch (pushbarnek, 1987), and 80-90% of the caspian commercial catch (kushnarenko, 1986). during the 1970s, it was only 2% of the total caspian fishery production. these herrings dominated the commercial catch in the caspian sea until the 1960s when commercial fishing was banned except on the western coast of the central caspian. many young of other commercial species were being killed in the herring fishery, entangled in the gill nets used. soviet catches have weighed as much as 75,000 t. this fish is fattier than other caspian clupeidae, except for a. kessleri, up to 18.1% of the body weight. the fat content decreases on the spring migration. the catch of the subspecies knipowitschi is of minor economic importance and had been little exploited when svetovidov (1952) summarised biology, as the age of captured fish indicated. about 420 tons (sic, possibly tonnes) were caught in the anzali talab in 1933 and 1934, but this may be an error in the report by vladykov (1964) according to holčík and oláh (1992) although berg (1948-1949) reports 4,200 centners for the same period. the fishing season in the talab began in mid-april and ended in mid-june when spent fish appeared. there appears to be no fishery data on the subspecies persica in the sea. holčík and oláh (1992) report catches of persica, which replaced knipowitschi, in the anzali talab from the end of april to the beginning of june but in 1990 this comprised only 5 kg. it is regarded as of inferior quality in iran. the caspian shad is the dominant fish catch in the iranian caspian, comprising 51,000t in 1994 rising from nothing a decade earlier (food and agriculture organization, fisheries department, 1996). robins et al. (1991) list this species as important to north americans. importance is based on its use as food. ershad langroudi (2004) investigated the durability of polycyclic aromatic hydrocarbons (pah), a carcinogen, in cold smoked fish and found relatively large amounts in the skin and flesh of this fatty shad compared to cyprinids. the pah density declined with storage time. nemati et al. (2012) determined that the protein hydrolysate produced from by-products (head, skin, viscera) using the enzyme alcalase was the best, with high nutritional value and was useful as a protein source in fish food. soleimani et al. (2012) found the amounts of fat, protein, ash and dry matter were higher in this species than in c. engrauliformis and it was a rich source of omega-3 fatty acid. salting and drying methods were compared. najm et al. (2014) determined the concentration of the heavy metals cadmium, chromium and lead in fish from the babol sar coast and found cadmium levels exceeded international standards for human consumption although a risk assessment concluded the fish were safe for consumers. sadeghi bajgiran et al. (2016) assessed this species for muscle content of nickel, vanadium and zinc and found levels not to be dangerous to consumers. conservation: the stocks of this species in the anzali talab are likely to increase if the lagoon becomes more saline (holčík and oláh 1992). kiabi et al. (1999) consider this species to be of least concern in the south caspian sea basin according to iucn criteria. criteria include abundant in numbers, widespread range (75% of water bodies), absent in other water bodies in iran, and absent outside the 142 coad/ herrings of iran caspian sea basin. sources: iranian material: cmnfi 1970-0524, 11, 58.7-88.9 mm standard length, gilan, caspian sea at bandar-e anzali (37º28'n, 49º27'e); cmnfi 19700532, 1, 113.0 mm standard length, gilan, caspian sea near bandar-e anzali (37º28'n, 49º27'e); cmnfi 1970-0543a, 1, 85.9 mm standard length, gilan, caspian sea at hasan kiadeh (37º24'n, 49º58'e); cmnfi 1970-0586, 1, 77.5 mm standard length, mazandaran, gorgan talab at ashuradeh-ye kuchak (36º50'n, 53º56'e); cmnfi 1970-0587, 2, 107.4-108.6 mm standard length, mazandaran, babol sar (36º43'n, 52º39'e); cmnfi 1971-0343, 1, 95.5mm standard length, gilan, langarud at chamkhaleh (37º13'n, 50º16'e); cmnfi 19790430, 1, 118.0 mm standard length, mazandaran, river east of now shahr (36º39'n, 51º31'e); cmnfi 1979-0431, 7, 120.8-155.1 mm standard length, mazandaran, now shahr bazaar (no other locality data); cmnfi 1979-0686, 2, 119.7-126.9 mm standard length, gilan, safid river (37º24'n, 49º58'e); cmnfi 1980-0146, 2, 106.9-171.8 mm standard length, mazandaran, gorgan talab at ashuradeh-ye kuchak (36º50'n, 53º56'e). comparative material: bm(nh) 1938.8.2:3, 1, 203.8 mm standard length, caspian sea (no other locality data); bm(nh) 1939.2.21:22-23, 2, 175.6-179.2 mm standard length, caspian sea (no other locality data); bm(nh) 1954.6.24:5-7, 3, 164.1-189.1 mm standard length, caspian sea (no other locality data). alosa kessleri (grimm, 1887) (figs. 4, 5) common names: shagmahi-ye poshtsiah, shagmahi darya-ye siah, shagmahi-ye moohajer or shagmahie-mohajer, zalun (in gilaki), puzanok. [volga siyanayi, garabel siyanak in azerbaijan; arkasy gara takgas in turkmenistan; blackback, caspian anadromous shad; chernospinka or black-spined herring, chernonosik or blacknose, beshenka, zalom, poluzalom, zheleznitsa, veselka, volzhskaya mnogotychinkovaya sel'd or volga many-rakered herring, volzhskaya sel'd or volga herring, astrakhanskaya sel'd or astrakhan herring, all in russian]. systematics: clupea kessleri was originally described from the volga river delta, astrakhan. clupea pontica (eichwald, 1838) was originally described in latin from "hab. in ponte euxino prope odessam" (= black sea near odessa). alosa kessleri was formerly considered as a subspecies of a. pontica. alosa pontica then had two subspecies in the caspian sea, namely kessleri (grimm, 1887) (chernospinka or black-spined herring, chernonosik or blacknose, beshenka, zalom, poluzalom, zheleznitsa, veselka, blackback), and volgensis (berg, 1913) (volzhskaya mnogotychinkovaya sel'd or volga many-rakered herring, volzhskaya sel'd or volga herring, astrakhanskaya sel'd or astrakhan herring, zheleznitsa, beshenka, veselka). kottelat and freyhof (2007), abdoli and naderi (2009) and figure 4. line drawing of alosa kessleri. 143 int. j. aquat. biol. (2017) 5(3): 128-192 naseka and bogutskaya (2009) consider alosa kessleri and a. volgensis to be valid species. a lectotype of kessleri, 40.1 cm long, was designated from the volga delta by l. s. berg under zisp 15925 (in the zoological institute, st. petersburg). a lectotype of volgensis, 34.8 cm long, is under zisp 15926 and is from the volga river at chernyi yar (svetovidov, 1952). a paralectotype of kessleri is under zisp 15922. caspialosa volgensis bergi tanasiichuk, 1938 described from the volga delta is a synonym of alosa kessleri (heckman in hoestlandt, 1991). eschmeyer et al. (1996) give author and date for alosa volgensis bergi as tanassiychuk, 1940, the variation probably being due to transliteration of a russian name and to year of actual publication rather than year on the journal. caspialosa kessleri infraspecies volgensis imitans berg, 1948 from the caspian sea (see berg (19481949) for further details) is not available because of its infrasubspecific rank (eschmeyer et al., 1996). clupea caspio-pontica borodin, 1904 is an unneeded new name (eschmeyer et al., 1996). key characters: characterised by a relatively elongate and rounded body likened to a "herring" shape, not as deep and compressed as in some related species which are likened to a "shad" shape. total gill rakers 49-158 in the caspian sea, with ranges about 57-95 in kessleri and 87-158 in volgensis. rakers are usually longer than the gill filaments in volgensis, shorter in adult kessleri. teeth are well developed in both jaws in kessleri and can be felt with a finger, poorly developed in volgensis such that they sometimes cannot be felt. morphology: dorsal fin with 3-5 unbranched and 12-16 branched rays, anal fin with 2-4, usually 3, unbranched and 15-21 branched rays. vertebrae 4750 in kessleri (also a report of 50-54, both in svetovidov (1952)), 48-54 in volgensis. pyloric caeca 21-62. scales in lateral series 53-56. gill rakers in adults are thick and often broken off at the tip or near the base in kessleri, unbroken in volgensis. the tips of the gill rakers may be swollen and they are arranged in a straight line. young fish have long and thin gill rakers with strong lateral spines. spines are lost with age. sexual dimorphism: none reported. colour: the overall coloration is dark with a black back which has a violet tinge in spring in kessleri, light olive-green in volgensis. there is dark, sometimes vague, spot on the flank behind the operculum and sometimes a series of spots in kessleri, but these are absent in volgensis. the pectoral fin is black on top. spawning kessleri become grey or grey-green on the back and flanks with bronze spots on the operculum and flanks. a greenish-yellow circle forms around the eye after spawning. size: reaches 52.0 cm total length and 2.0 kg for kessleri, 40.0 cm for volgensis. distribution: found in the caspian sea, entering northern rivers to spawn. abdoli and naderi (2009) list it as from the southwest, southeast and southcentral caspian sea in iranian waters. esmaeili et al. (2014) list this species from the anzali wetland or talab. zoogeography: this species is a caspian sea endemic. habitat: both subspecies are found in the open sea but kessleri ascends rivers much higher than figure 5. alosa kessleri courtesy of a. abdoli. 144 coad/ herrings of iran volgensis which spawns in the delta region. both subspecies overwinter in the southern caspian sea off the iranian coast and then migrate north to enter the volga and other northern rivers to spawn. the subspecies volgensis is absent from the southern caspian in summer. the subspecies kessleri shows a greater affinity than volgensis for cold water. the subspecies kessleri begins to migrate northward in march and april mostly along the western shore of the caspian sea, beginning to arrive in northern waters when temperatures are still below 5°c, most arriving when temperatures are 6-8°c compared to 10-13°c for volgensis. a mass migration into the lower volga takes place in late april or early may for both subspecies when water temperature reaches 9°c and the peak run begins at 12-15°c, ending at 22°c. the run of volgensis is about 10 days later than that of kessleri and spawning takes place earlier as they do not travel as far upriver. speed is up to 70 km/day for kessleri and depends on temperature. this fish used to run 2,000 km up the volga river. sexually immature fish remain in the south and do not migrate. knipovich (1921) reports kessleri as deep as 235-300 m in iranian waters. temperatures up to 25ºc are tolerated. age and growth: males are sexually mature at 3 years and females at 4 years, other reports give 4-5 years for both sexes in kessleri. many fish die after spawning but some survive to spawn two or three times. four and five-year-olds dominate on kessleri spawning runs with some older fish also present. females predominate in older fish making the spawning run. life span is between 7 and 8 years. growth of the volgensis subspecies is slower than in kessleri, which apparently grows faster than any other caspian clupeid. life span in volgensis is 7-8 years with females living longer than males. most spawners are 3-4 years old although in some years, 5 year old fish are abundant. males may mature at 2 years, females later. most fish spawn again the next year after their first time but some may miss a year. an individual may spawn up to four times during its life. heidari et al. (2015b) examined fish from the tonekabon fishing area and found b values of 3.687 and 3.571 for total and standard length, indicating positive allometric growth. alavi-yeganeh et al. (2013) found mazandaran fish were 2-5 years old and had a b value of 2.993. ghotbi-jokandan et al. (2015) examined 103 fish from anzali, noor and torkeman and found a b value of 2.88. mousavisabet et al. (2016) gave a b value of 3.13 for iranian fish from 6 fishing areas from astara to miankaleh. food: cladocerans are the main food item of young kessleri which have a feeding peak at 1800-2200 hours and another at about 0800 hours. adults in the sea take fishes such as clupeonella and atherina with some crustaceans and insect larvae. clupeonella caspia makes up 92% of the diet of kessleri in the northern caspian in may, with sander lucioperca at 6.6% and gammarids at 1.0%. there is said to be little feeding on the spawning run although some fish sampled contained cladocerans, copepods, insects, bryozoans and fish fry. the food of volgensis is similar to the other subspecies, taking copepods when young and larger items with growth. the main items are copepods, mysids, cumaceans, amphipods and small fishes. this subspecies feeds on the spawning migration. reproduction: spawning in kessleri occurs in rivers from mid-may to mid-august, either the delta or lower reaches when entering in a ripe condition, or as much as 500 km upriver when entering in an unripe condition. larger fish have spawning grounds further upriver than smaller fish and predominate earlier in the run. the spawning grounds in the volga river cover a considerable stretch. spawning usually occurs at 18-20°c between 0300 and 0600 hours or from 1600 hours to sunset. spawning occurs in the main channel, over shallow sand banks, or in backwaters. batches of eggs are laid at intervals of several days. eggs are pelagic as in other caspian alosa and develop as they drift downriver near the bottom. at 22.7°c incubation takes about 40 hours. the young fish descend in late summer and early fall. fecundity in kessleri reaches 344,000 eggs and egg diameter 1.51 mm. shed eggs are up to 4.1 m in diameter. some fish may return to spawn in total 145 int. j. aquat. biol. (2017) 5(3): 128-192 three times. spawning of the first batch of eggs in volgensis may occur in the sea with the subsequent 2 batches at 7-10 day intervals in the delta and river. this takes place from mid-may to the beginning of august. up to 281,000 eggs are shed. peak spawning occurs at 15-19°c and ends at 25-27°c. most spawning takes place in the evening between the 1600 and 2200 hours. the young appear in the pre-estuarine area of the volga river in july and towards october begin to migrate south. parasites and predators: the caspian seal, pusa caspica, is a predator on this species (krylov, 1984), larval shad are fed on by other fishes and by various invertebrates, and adults by various fishes and birds. economic importance: the subspecies kessleri and volgensis were caught on the spawning run with as much as 5,750 t being taken annually pre-world war ii. it is the biggest shad in the caspian sea. the subspecies kessleri was the most important and valuable herring in the caspian sea. early spring catches were mostly kessleri but as the run of volgensis built up it formed an increasingly significant part of the catch, forming as much as 92% of the total. the catch of volgensis has declined from this period until the 1970s when the fish taken were mostly kessleri. the catch of alosa pontica (= kessleri) on the north caspian fishing grounds in 1965-1972 has declined to 2-4% of the 1938-1943 catch. the subspecies volgensis was one of the most important caspian herrings, 23-29% of the total catch from 1936-1939, as high as 69,100t in 1939. the subspecies kessleri is said to be the tastiest caspian clupeid because of its high fat content, averaging 18.9% of weight along the coast of azerbaijan, while in volgensis it was 9.6%. postspawners of kessleri may have a fat content as low as 0.5%. catches are processed as canned, salted and pickled fish. beach seines are used to catch this fish. akhondzadeh basti et al. (2006) found the bacterial pathogen vibrio haemolyticus in fresh and smoked alosa kessleri. tavakoli et al. (2012) found a frequent contamination rate with staphylococcus aureus and vibrio parahaemolyticus, human pathogens, in fresh and smoked shad and they may cause health problems. robins et al. (1991) list this species as important to north americans. importance is based on its use as food. conservation: stocks in iranian waters are said to be depleted. the subspecies volgensis was in category i on the "red list" of the russian republic (pavlov et al., 1985). kiabi et al. (1999) consider this species to be data deficient in the south caspian sea basin according to iucn criteria. criteria include commercial fishing, numbers unknown, range unknown absent in other water bodies in iran, absent outside the caspian sea basin. sources: iranian material: none available. comparative material: bm(nh) 1879.11.14:22-23, 2, 255.9-259.1 mm standard length, caspian sea (no other locality data); bm(nh) 1939.2.21:21, 1, 388.6 mm standard length, caspian sea (no other locality data). alosa saposchnikowii (grimm, 1885) (figs. 6, 7) common names: shagmahi-ye cheshmdorosht, shagmahi, kilka (incorrectly), herring. [irikoz sisgarin in azerbaijan; bol'sheglazyi puzanok or bigeye shad, sapozhnikovskii puzanok or saposhnikovi shad, all in russian]. systematics: the lectotype of clupea saposchnikowii from the volga delta is in the zoological institute, st. petersburg under zisp 15921 (berg 1948-1949; eschmeyer et al. 1996). the name is often spelt saposchnikovi, in error, or with a single terminal "i"; reshetnikov et al. (1997) revert to the original spelling of the specific name. caspialosa caspia nigra kisselevitsh, 1923, in part, from the caspian sea opposite dzambai is a synonym with a lectotype in the zoological institute, st. petersburg (zisp 15938) (kisselevitsh is also transliterated kiselevich and kisselevitz). the material also included specimens of alosa braschnikowii (whitehead, 1985; eschmeyer et al., 1996). key characters: characterised by a relatively deep and compressed body likened to a "shad" shape, not 146 coad/ herrings of iran as elongate and rounded as in some related species which are likened to a "herring" shape. the upper and lower head profiles are straight. the upper edge of the lower jaw is straight. total gill rakers 20-48, short (obviously shorter than the gill filaments), and thick. teeth are well developed in both jaws. morphology: dorsal fin with 3-5, usually 4, unbranched rays and 12-15, mostly 13, branched rays, anal fin with 2-4, usually 3, unbranched rays and 15-21, mostly 18, branched rays. lateral series scales 52-55. vertebrae 47-53. pyloric caeca 36-59. sexual dimorphism: none reported. colour: fish from the southern caspian sea are more intensively coloured than those from the north. the back is violet with a green sheen and the flank has 4 dark stripes which merge with the dark on the back. there is a spot posterior to the operculum, which may be absent. size: reaches 36.0 cm total length and 650 g. distribution: found mainly in the north caspian sea and the coast of dagestan but entering iranian waters. abdoli and naderi (2009) list it as from the southwest, southeast and south-central caspian sea in iranian waters and see below under sources for some specific localities. zoogeography: this species is endemic to the caspian sea. habitat: this species spends its whole life in the caspian sea and never enters rivers. it favours colder water and is one of the first clupeid species to migrate north in spring, principally along the western coast. large fish migrate first. fish first approach the shore of azerbaijan in mid-march with a mass approach from late march to mid-april. it is less frequently encountered in the southern part of the caspian sea, overwintering in the central caspian and only moving south if winters are cold. a caspian sea biodiversity database (from www.caspian environment.org) has it at 400-600 m in the southern caspian in cold winters but later states it keeps at 15figure 6. line drawing of alosa saposchnikowii. figure 7. alosa saposchnikowii courtesy of a. abdoli. 147 int. j. aquat. biol. (2017) 5(3): 128-192 32 m. winter temperatures at which this species is found are 6-7°c. depths are 25-32 m in winter, more shallow in summer but below 9 m. knipovich (1921) reports this species in a depth range of 52-77 m in iranian waters. it tolerates a range of 3-25°c and spawns at salinities of 0.7-11.0‰, although preferring 4.0-7.5‰. the caspian sea biodiversity database (from www.caspian environment.org) estimates a population of 1.1125 billion fish. age and growth: life span is about 9 years and female lengths and weights exceed those of males throughout life. on average, males weigh less than half the weight of females since females carry a heavy egg load. growth is most intensive in the first two years of life and slows thereafter (chang, 1972). males mature at age 2 and females at age 3. ghotbijokandan et al. (2015) examined 40 fish from anzali, noor and torkeman and found a b value of 3.14. mousavi-sabet et al. (2016) gave a b value of 2.99 for iranian fish from 6 fishing areas from astara to miankaleh. food: a rapacious fish which takes young herrings and kilka, atherina and even benthophilus (lönnberg, 1900) as well as large crustaceans such as mysids and gammarids. it is a cannibal. this shad overwinters and feeds in the south caspian sea (chang, 1972). reproduction: the spring spawning migration (end of april to end of may) enters the north caspian sea and fish are mostly 15-25 cm in body length. males mature at a younger age than females as evidenced by fish 3-4 years old predominating among females and fish 2-4 years old among males in the north caspian catch. spawning takes place in may (peaking in the first 10 days) and most fish are returning for the second time. spawning temperatures are lower than in alosa caspia, being only 13-14°c although the peak is at 19-20°c. spawning occurs in il'mens, the sea where there is a freshwater discharge such as near the volga river mouth, and in the northeastern sea. females may spawn up to 6 times and males up to 5 times (chang 1972). spawning takes place in shallow water at 16m depths. fecundity is up to 318,852 eggs. the young migrate southwards. parasites and predators: mazandarani et al. (2016) found a trematode (pronoprymna ventricosa) and two nematodes (anisakis simplex and eustrongylides sp.) in the abdominal cavity of fish from behshahr, mazandaran province. the latter two are pathogenic to humans. the caspian seal, pusa caspica, is a predator on this species (krylov 1984). economic importance: an important commercial species in the central and northern caspian, taken on their way to, and on, the spawning grounds. the fishery in azerbaijan during 1937 caught fish on average 17cm long and 62g in weight, most fish being 2-3 years old. the caspian catch in the period 1936-1939 reached a peak of 8,800t annually. fish are caught with beach seines, stationary nets and drift nets. conservation: stocks in iranian waters are reputed to be depleted. kiabi et al. (1999) consider this species to be data deficient in the south caspian sea basin according to iucn criteria. criteria include numbers unknown, range unknown, absent in other water bodies in iran, absent outside the caspian sea basin. sources: iranian material: cmnfi 1970-0531, 15, 49.9-108.7mm standard length, mazandaran, larim river (36º46'n, 52º58'e); cmnfi 1970-0532, 1, 137.4mm standard length, gilan, caspian sea near bandar-e anzali (37º28'n, 49º27'e); cmnfi 19700543a, 2, 78.8-80.2mm standard length, gilan, caspian sea at hasan kiadeh (37º24'n, 49º58'e); cmnfi 1970-0581, 1, 102.1mm standard length, gilan, caspian sea near hasan kiadeh (37º24'n, 49º58'e); cmnfi 1979-0788, 3, 96.0-114.9mm standard length, mazandaran at khadje nafas (37º00'n, 54º07'e); cmnfi 1980-0136, 3, 107.3127.6mm standard length, mazandaran, fereydun kenar river (36º41'n, 52º29'e); cmnfi 1980-0157, 2, 96.6-101.1mm standard length, mazandaran, gorgan river estuary (36º59'n, 53º59'30"e); cmnfi 1980-0908, 1, 77.9mm standard length, gilan, safid river estuary (ca. 37º28'n, ca. 49º54'e). comparative material: bm(nh) 1954.6.24:8-10, 3, 150.5-177.0mm standard length, caspian sea (no 148 coad/ herrings of iran other locality data). alosa sphaerocephala (berg, 1913) (fig. 8) common names: shagmahi-ye agrakhan. [kruglogolovyi puzanok or roundheaded shad, agrakhanskii puzanok or agrakhan shad, both in russian]. systematics: the holotype of clupeonella sphaerocephala from agrakhan bay, at tyulenii island, turali in the northern part of the caspian sea is in the zoological institute, st. petersburg under zisp 15928 with more than 30 paratypes (eschmeyer et al., 1996). key characters: characterised by a relatively deep and compressed body likened to a "shad" shape, not as elongate and rounded as in some related species which are likened to a "herring" shape. the upper and lower head profiles are obviously rounded. the upper edge of the lower jaw is crescent-shaped. total gill rakers 25-45, long (equal to or longer than the gill filaments), and thin. teeth are well developed in both jaws. morphology: dorsal fin with 3-4, usually 4, unbranched rays and 13-15 branched rays, anal fin with 3-4, usually 3, unbranched rays and 17-20 branched rays. vertebrae 47-51. sexual dimorphism: none reported. colour: the back is dark with an olive tint, the tip of the snout is occasionally black and the pectoral fins are dark. there is a black spot behind the operculum and occasionally a row of such spots. size: reaches 25.0 cm standard length. distribution: found in the caspian sea including iranian waters. zoogeography: this species is endemic to the caspian sea. habitat: this species does not enter fresh waters. it is most common along the eastern shore of the northern part of the sea in spring where spawning occurs and along the northern shore of the northern part of the sea in summer. knipovich (1921) reports this species from iranian waters in a depth range of 52-77 m. age and growth: unknown. food: unknown, although presumably similar to other shads. reproduction: spawning takes place in the northeastern caspian from mid-may to the end of june peaking at 18-20°c, most frequently in a salinity of 8-11‰ and in depths around 3-8 m. the young move south in late autumn, as late as november, the last clupeids to leave this area. fecundity is about 20,000 eggs. parasites and predators: the caspian seal, pusa caspica, is a predator on this species (krylov, 1984). economic importance: this species is caught only in small numbers. conservation: the status of this species is unknown. sources: iranian material: none available. comparative material: bm(nh) 1954.6.24:11-13, 3, 145.6-162.1 mm standard length, caspian sea (no figure 8. line drawing of alosa sphaerocephala. 149 int. j. aquat. biol. (2017) 5(3): 128-192 other locality data). genus clupeonella kessler, 1877 this genus is found in the black and caspian seas basins with 5 species, 3 of which are in the caspian sea and in iranian waters. clupeonella species are distinguished from sympatric alosa species by smaller size, a small and toothless mouth, adipose eyelids are small or rudimentary, no spots on the flank, no elongate scales (ala) at the base of the caudal fin, no vomerine teeth, the lack of a notch at the mid-line of the upper jaw, and by the last two anal fin rays being elongated. species in this genus live entirely in the sea, or in fresh water, or migrate between the two. eggs are pelagic and have a large oil globule. the general farsi name for these fishes is kilka or kelka, i.e. "sprat", although sprat is erroneous according to berg (1948-1949) who uses tyulka for these fishes). the three clupeonella species have been fished in modern iran since december 1971 but the commercial catch did not exceed 15,000 tonnes. earlier catches date back only to 1939, with an annual catch of about 100t in 1943-1949, exported in a marinated form to the soviet union (alam, no date). curiously, the abundance of kilka has long been known as kinneir (1813) records "and herrings are in such abundance, that after a storm, the shores of ghilan and mazanderaun are nearly covered with them". caddy (1984) refers to the kilka fisheries of the iranian caspian by the scientific name sprattus sprattus but this is an error. caddy (1984) indicated that there were problems in marketing and utilizing these fishes in iran even though up to 50,000 t could be caught annually (200,000 t elsewhere in the same article). their best use was probably as food for predators such as sander lucioperca, esox lucius and salmo caspius. a study by razavi sayad (1993) suggested a ceiling of 100,000 t was possible. the caspian sea resources of kilka are estimated at 800,000 t from which 340,000 t can be exploited (abzeeyan, tehran, 6(8): iv, 1995). the catch reached 51,000 t in 1994 from none 10 years previously (food and agriculture organization, fisheries department, 1996) and was 36,000 t in 1997-1998 (irna, 31 march 1998) and 85,000 t in 1998-1999 (fazli and roohi, 2002a). the catch for the first 6 months of the iranian year was 17,000 t, taken by 70 trawlers and a 10% increase over the previous year (irna, 20 october 1998). fishermen in gilan caught 50,000t annually in the late 1990s (tehran times, 5 september 1999). a reported catch of 56,000 t was made in 1999-2000, a 13% increase over the previous year (irna, 27 march 2000). a later estimate expects the kilka catch to reach 66,000 t by the year 2000 (abzeeyan, tehran, 5(9): iv, 1995). fazli (2006) records that kilka fishing ships discharge their catches at three ports, babol-sar and amirabad in mazandaran and anzali in gilan. the catch decreased from 28,000 t to 19,600 t in mazandaran and from 57,000 to 42,600 t in gilan from 1999 to 2000. the catch per unit effort also decreased from 3,900 kg to 2,500 kg over the two years. anchovy kilka dominated the catch but the frequency fell from 85-90% to 76% of the catch and common kilka sharply increased. common kilka had been caught in spring and summer but in 2000 they were taken in all months. the average length of anchovy kilka declined from 96.3 mm in 1997 to 87.3 mm in 2000 and this was also reflected in the age structure, 5+ and 6+ fish being rare. the presence of the ctenophore, m. leidyi, was thought to be damaging stocks (fazli and roohi 2002a; ahmadpour et al., 2012). darvishi et al. (2004) studied dietary overlap between the ctenophore and the anchovy kilka (see below). barati (2009) found that the diet of chicks of the great cormorant, phalacrocorax carbo, at ramsar comprised only 2% clupeidae by frequency, perhaps a result of the decline in clupeonella stocks. fazli (2002) studied kilka catches off mazandaran in 1996-2000. fishing occurred at night and lasted 7.78-8.22 hours. the maximum catch at 42.8% was taken in october, november and december with a minimum catch in june. the least annual catch per vessel occurred in 1999-2000 (499,401 kg). 150 coad/ herrings of iran a study utilizing an echo-sounder and a pelagic trawler concludes that the maximum biomasses for the three clupeonella species in the southern caspian sea were in winter (422,300 t) and autumn (326,900 t) while in summer and spring values were lower at 275,100 t and 260,800 t, respectively. the population consisted of 66.1% anchovy kilka (c. engrauliformis), 18.9% bigeye kilka (c. grimmi) and 15% common kilka (c. caspia) (iranian fisheries research and training organization newsletter, 14: 6, 1996). note that later, the iranian fisheries research and training organization newsletter (17: 3, 1997) gives kilka biomass in the southern caspian sea as winter 22,300 t, autumn 26,900 t, summer 75,100 t and spring 60,800 t, presumably lacking the initial digit, and the percentages of kilka species in the biomass are also wrong. this is corrected in a subsequent newsletter (iranian fisheries research and training organization newsletter, (18: 43, 1997) but the corrected percentage biomasses are given as 66% for c. engrauliformis, 19% for c. caspia (as c. delicatula) and 15% for c. grimmi. it is unclear whether grimmi or caspia is the second most important kilka species. poorgholam et al. (1996) gave a stock assessment for kilkas in 1995-1995 using the hydro-acoustic method. clupeonella engrauliformis dominated the catch in iran at 91.8%, followed by c. grimmi at 6.84% and by c. caspia at only 1.35% (coad 1997). the 2+ and 3+ year classes accounted for 69.95% of c. engrauliformis, 81.06% of c. grimmi and 80.88% of c. caspia catches. catch rates of kilka on the top ranking 17 fishing grounds of 56 studied range from 800 to 1,200 kg per unit effort per hour while traditional grounds have rates of 400-800 kg per unit effort per hour. the kilka are caught by attraction to lights and netting or pumping the catch into specially constructed ships (fig. 9). the kilka fishing fleet of iran expanded in the 1980s and 1990s. there were 30 active vessels in mazandaran in 1994, each with a capacity up to 30tons (sic, probably tonnes here and elsewhere for modern catches) (abzeeyan, tehran, 4(10): iv, 1994). the mazandaran kilka cooperative companies union had 75 boats in 2000 (tehran times, 31 december 2000). gilan planned to construct 12 fish meal factories each with an annual capacity of 1,000 t and 10 kilka canneries also with 1,000t capacities (abzeeyan, tehran, 4(4): iii, 1993). catches off gilan alone from april 1994 to january 1995 increased 59% compared to the same period in 1993-1994, exceeding 20,000 t (abzeeyan, tehran, 6(1): ii, 1995). the catch off mazandaran from march 1994 to march 1995 was 15,400t, an increase of 10% over the previous year. about 1,000 t were processed for human consumption and the rest for fishmeal production (abzeeyan, tehran, 6(2): v, 1995). the total kilka catch for iran has increased to 45,000 t annually and efforts were being made to increase it to 110,000 t (abzeeyan, tehran 4(5): iv, 1993). the catch in 1995 was 32,000 t with 64.7% from mazandaran and 35.3% from gilan, with the maximum catch occurring in april (abzeeyan, tehran, 7(6): ii, 1996). catches declined from 95,000 t in 1999 to 15,497 t in 2003 (sayyad bourani et al., 2008). annual soviet catches reached 37,000 t in 1956 but this declined to 300-1,500 t by the end of the 1970s or 0.2-0.8% of all kinds of tyulka or kilka in the caspian sea. turkmenistan harvested 7,660 and 8,500 t in 1995 and 1996 although previously almost 45,000t valued at $22.5 million had been taken before equipment deteriorated (http://bisnis. doc.gov/isnis/isa/9805fish.htm, downloaded 14 march 2000). stocks remained large even though kilka were heavily fished. kohani et al. (2012) figure 9. conical lift net or kilka net after www.farsnews.com. 151 int. j. aquat. biol. (2017) 5(3): 128-192 reviewed fishing in gilan and noted the number of vessels used increased from 9 in 1989 to 110 in 1999, resulting in the highest catch at 55,900 t. mismanagement, the invasive ctenophore, pollution and temperature changes caused a decline in catches to 4,603 t in 2009. the kilka fisheries were threatened by the comb jelly (a ctenophore, m. leidyi), which arrived in the caspian sea in 1995 in the ballast water of ships and spread through the entire sea by the year 2000, feeding voraciously on zooplankton. it is now known as the "caspian monster" despite its small size of 5cm (j. muir 2001, http://news.bbc.co.uk/2/ hi/middle_east/1453117.stm). it doubles in size in one day, reaches maturity in two weeks and then produces 8,000 young each day. the fisheries collapsed by 50% in a few months, catches by one fisherman falling from being 3-6 t a night to half a tonne. bilio and niermann (2004) review the interplay of overfishing, pollution and the ctenophore invasion on the kilka fisheries of the black and caspian seas, kideys et al. (2005) gave an overview of ctenophore impacts on fisheries, and roohi et al. (2010) compared the effects on phytoplankton, zooplankton, zoobenthos and kilka. ghadirnejad (2003) reported that c. engrauliformis originally dominated the kilka catch at 85-90% but has dropped to 55% through the impact of the comb jelly which has up to 2,285 individuals per cubic metre in the southwest caspian sea. roohi et al. (2010) stated that iranian landings of kilka dropped about 70% from 69,070 t in 1995-2000 to 23,430 t in 2001-2006, a loss of at least u.s. $125 million. parafkandeh (2006) gave similar figures for declines in catches. fazli et al. (2009a) describe a multi-species approach for stock management, allowing for the decline of c. engrauliformis and increase in c. caspia in iranian waters through competition with the ctenophore. the fisheries may recover somewhat after the comb jelly population collapses (tidwell, 2001) or if a predator, beroe ovata, is introduced and can survive in the less saline waters of the caspian sea (j. muir 2001, http://news.bbc.co.uk/2/hi/ middle_east/1453117.stm). studies indicate it can survive the brackish caspian sea water, feed on the comb jelly and not feed on other plankton (iranian fisheries research organization newsletter, 36: 35, 2003). the catch records for the total kilka catch in mazandaran in tonnes is courtesy of f. darvishi (pers. comm. 2003) (table 6) and shows the drastic decline caused by the ctenophore, as well as monthly variations in catches. the species composition of kilkas changed after the introduction of the comb jelly comparing the year 2000 and before with the year 2002 the common kilka changed from about 1-5% to about 30%, the bigeye from about 10-15% to 0.2% and the anchovy kilka from about 85-90% to about 70% (iranian fisheries research organization months/years 1998 (1377) 1999 (1378) 2000 (1379) 2001 (1380) 2002 (1381) mean march-april (farvardin) 2,848 2,703 4,644 1,217 876 2,458 april-may (ordibehesht) 1,116 607 972 1,422 195 862 may-june (khordad) 370 763 1,819 125 158 647 june-july (tir) 1,392 919 194 425 444 675 july-august (mordad) 2,152 2,306 433 614 249 1,151 august-september (shahrivar) 3,117 2,010 581 528 336 1,314 september-october (mehr) 3,103 6,184 1,785 432 575 2,416 october-november (aban) 4,120 3,468 2,305 3,051 1196 2,828 november-december azar) 3,835 3,410 2,655 993 2,723* (2,179) december-january (dey) 2,754 1,735 620 1,082 1,548* (1,238) january-february (bahman) 3,968 1,262 2,146 1,586 2,241* (1,792) february-march (esfand) 2,815 1,667 1,192 1,903 1,894* (1,515) total 31,590 28,034 19,648 13,378 4,029 * = averaged over 4 and (5) years. table 6. the catch records for the total kilka catch in mazandaran in tonnes is courtesy of f. darvishi (pers. comm. 2003). 152 coad/ herrings of iran newsletter, 36: 2, 2003). the catch per unit effort (catch per vessel per fishing night) fell from 4 t to 1 t. the catch during 1997-1999 of anchovy kilka fell from 51,300 t to 491 t and bigeye kilka from 7,600 t to 309 t while common kilka rose from 1,500 t to 24,600 t (iranian fisheries research organization newsletter, 65: 4, 2011). parafkandeh haghighi and kaymaram (2012) found, for the years 2006-2007, that the common kilka was the dominant species (89.7%) while the anchovy kilka was at only 8.7% after previously being the dominant species. this was attributed to the comb jelly which occupied the anchovy kilka habitat at depths greater than 50 m. the total catch of kilkas fell from 95,000 t in 1999 to less than 20,000 t in 2007. the fishery moved to areas with depths less than 50m, the main reason for the change in species composition. in 2004, more than 200 fishing boats had been forced to stop operations. the kilka stock has been reduced from 400,000 t to 80,000t over the past 4 years and the catch fell by 34,000 t (www. iranmania.com, downloaded 4 october 2004). mamedov (2006) gives details of the biology and decline of kilkas in azerbaijan waters. the caspian seal was once a major predator on kilkas but the number of seals has declined on the kazakhstan and iranian coasts from 300,000 to 5,000 in recent years through ddt pollution, viral infections and food shortages (hashemi, 2001). fazli (2011) evaluated changes in species composition, catch and catch-per-unit-effort from 1961 to 2009. he found a negative correlation between long-term sea level changes and total catch which significantly declined in 1995 when sea level increased to its highest level. the catch-per-uniteffort before the ctenophore invasion (1996-1999) and after (2000-2009) showed significant declines in c. caspia and c. engrauliformis while c. grimmi increased significantly. overfishing and environmental factors after the introduction of the ctenophore were the major factors in species composition changes. catches are presented in table 7. fazli et al. (2013) also examined the stock status of iranian kilkas. the anchovy kilka and all three species together showed no overfishing from 1991 to 2000 but in 2000 the catch-per-unit-effort neared an “in danger” region and from 2001 to 2007 stocks were overfished. during 2008-2010 the biomass increased and the stocks became non-overfished. the anchovy kilka fishery generally reduced since 1998 and collapsed to the lowest level in 2010. for the anchovy stock to recover, the catch of all three species would have to be restricted during the spawning period and an international effort made to manage stocks. the food and agriculture organization (www.fao.org/fishery/facp/irn/en, downloaded 16 january 2017) summarised catches in the south caspian sea (table 8), showing the relative year c. caspia catch (t, %) c. caspia cpue* c. engrauliformis catch (t, %) c. engrauliformis cpue* c. grimmi catch (t, %) c. grimmi cpue* 1997 52,246, 86.5 3.35 6,704, 11.1 0.5 1,450, 2.4 0.09 1998 61,880, 72.8 2.92 18,445, 21.7 0.87 4,675, 5.5 0.22 1999 67,450, 71.0 3.12 14,535, 15.3 0.67 13,015, 13.7 0.6 2000 57,486, 73.7 2.26 9,828, 12.6 0.39 10,686, 13.7 0.42 2001 37,590, 83.2 1.31 2,801, 6.2 0.1 4,789, 10.6 0.17 2002 17,358, 69.5 0.75 25, 0.1 0 7,592, 30.4 0.33 2003 7,530, 50.5 0.57 89, 0.6 0.01 7,291, 48.9 0.55 2004 5,089, 26.9 0.4 227, 1.2 0.02 13,602, 71.9 1.06 2005 4,249, 18.8 0.31 542, 2.4 0.04 17,808, 78.8 1.3 2006 1,896, 8.5 0.15 1,048, 4.7 0.08 19,356, 86.8 1.51 2007 910, 6.0 0.1 379, 2.5 0.04 13,881, 91.5 1.49 2008 440, 2.7 0.06 277, 1.7 0.04 15,573, 95.6 2.13 2009 454, 1.8 0.06 302, 1.2 0.04 24,444, 97.0 3.35 *cpue = catch-per-unit-effort (tonnes per vessel per night) table 7. the catch after after the introduction of the ctenophore (after fazli, 2011). 153 int. j. aquat. biol. (2017) 5(3): 128-192 importance of kilka. the by-catch from the kilka fishery on the grounds at anzali comprised 0.2% of the catch (moradinasab et al., 2015). this low by-catch was attributed to the use of lantern nets and the positive phototropism of kilkas. by-catch species were a. caspia, the mullet mugil cephalus, the cyprinids alburnus chalcoides, cyprinus carpio and rutilus kutum, and the catfish silurus glanis. kilka are smoked, salted, canned in sauce and oil and marinated according to a traditional recipe and seasoned with fruits, herbs and vegetables in iran (keivany and nasrollahzadeh, 1990; www.netiran. com/business.html, downloaded 31 october 2003). moini and koochekian (2003) gave details of fish sauce production from kilkas using traditional, microbial and enzymatic methods, along with taste tests. vacuum packaging of fresh, smoked and salted kilka has been investigated in iran (annual report, 1995-1996, iranian fisheries research and training organization, tehran, p. 45-46, 1997) and studies on processing kilkas as fish balls have also been carried out (annual report, 1994-1995, iranian fisheries research and training organization, tehran, p. 40, 1996). koochekian sabour and moini (2009) described investigations on using iranian kilkas to produce a fermented fish sauce for marketing in southeast asian countries. one company markets kilka in a clear package which gives the product a bright and colourful appearance. kilka have even been made into crackers (iranian fisheries research and training organization newsletter, 18: 6, 1997, shojaei 1998). kilka have also been made into oil as a by-product of the fish meal industry (iranian fisheries research and training organization newsletter, 27: 3, 2001). omega-3 fatty acids have been extracted from kilka oil under laboratory conditions (salmani joloudar et al. 2009). m. shivazad, h. john mohammady, a.a. yousef hakimi and h. fazaely (http://iman.ut.ac.ir /news/agr.htm, downloaded 12 december 2004) discuss the use of c. engrauliformis as fish meal in animal nutrition and analyse the protein quality and faeed et al. (2006) studied spoilage in kilka meal from bacteria and fungi. nassiri moghaddam et al. (2007) studied commercial samples of kilka fish meal and found differences in protein efficiency ratio and net protein ratio, attributed to the species used, conditions and length of storage and processing methods. agh et al. (2012) showed that replacing kilka fish oil with vegetable oil in the diet of rainbow trout (oncorhynchus mykiss) broodstock significantly reduced total fecundity and hatching rate, and increased larval mortality and deformed larvae rate. jalili et al. (2012) compared diets with clupeonella fish meal and plant protein in various proportions on growth of rainbow trout fry and found, for example, replacing 50% of the fish meal with plant protein did not affect growth nor did a complete replacement with poultry by-product and plant proteins. the iranian fisheries research and training organization newsletter (20: 4, 1998) and rezaei et al. (2003) report on methods of transporting kilka in cold water and crushed ice to processing factories which were better than traditional methods. salmani et al. (2001) recommended chilled sea water for preservation for human consumption. motamedzadegan et al. (2009) found that partial hydrolysis of fish myofibrillar proteins using papain improves its functionality. motalebi et al. (2010) investigated the use of whey protein coating on quality and shelf life of kilkas; it can enhance quality and increase frozen shelf life in fish stored for up to 4 months. khanedan 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 bony fish 16,573 15,665 21,845 23,802 23,538 20,045.5 18,665 16,601 17,034 16,160 17,146 16,733 sturgeon 463 500 416 330 225 178.5 131 94 80 68 56 41 kilka 15,497 19,610 22,626 22,303 15,411 16,743 25,483 27,110 20,717 24,086 23,221 22,873 total 32,533 35,775 44,887 46,435 39,174 36,967 44,279 43,805 37,831 40,314 40,423 39,647 table 8. the summarised catches in the south caspian sea (food and agriculture organization, 2017) 154 coad/ herrings of iran et al. (2011) found that an edible film of sodium alginate on dressed kilka increased shelf life. seyfzadeh et al. (2013) used an edible film of whey protein and sodium alginate to coat gutted kilka, enhancing quality and shelf life for up to 6 months in frozen storage. valipour meri et al. (2011) showed that bacillus licheniformis can decrease significantly the aflotoxins in kilka fishmeal. khoshkoo (2008) and khoshkoo et al. (2010, 2012) studied deterioration of fish protein concentrate made from kilkas caught near anzali in different packaging and temperatures, stored in light and darkness. vacuum packaging and storage in darkness at lower temperatures was the best method to avoid deterioration. ebadi and shokrzadeh (2006) examined clupeonella species in mazandaran for the organochlorine pesticide lindane and found levels in muscle tissues to be less than fao and who recommended permissible intake and so were not a public concern. an account on the biology and identification of caspian kilka in farsi is given by emadi (1991) and fazli (1990). fazli and besharat (1998) and poorgholam et al. (1996) give accounts of biology and catches in iran in farsi. clupeonella caspia svetovidov, 1941 (figs. 10, 11) common names: rizeh keraye, rizeh kuli, kilka-ye ma'muli or kilka-e-maamooli (= common shad). [xazar kilkasi in azerbaijan; adaty kulke balyk in turkmenistan; kaspiiskaya tyul'ka or kil'ka (i.e. caspian tyulka or kilka), tyulka, obyknovennaya tyul'ka (i.e. common tyulka), all in russian; common kilka, common caspian kilka, sardelle, caspian sprat]. systematics: formerly identified as clupeonella cultriventris (nordmann, 1840), originally described from the northern shore of the black sea. clupeonella delicatula (nordmann, 1840), described from odessa market on the black sea, is a synonym of c. cultriventris and a lectotype is in the zoological museum st. petersburg under zisp 2254 with paralectotypes also under zisp 2254, as designated by svetovidov (1952). clupeonella delicatula caspia svetovidov, 1941 was considered to be a synonym and was described as from the "caspian sea, where it is met with almost everywhere, from very saline parts (kaydak bay) to quite fresh. enters the mouths of the volga and ural rivers, ascending sometimes very far upstream". the holotype is from the volga delta and is under zisp 15883 (svetovidov, 1952). kottelat and freyhof (2007) considered this subspecies to be a distinct species found in the caspian sea with cultriventris restricted to the black sea. reshetnikov et al. (1997) considered recognition of this subspecies as questionable. the caspian sea taxon, clupeonella caspia, has a lectotype, 152 mm long, designated by svetovidov (1952) in the zoological institute, st. petersburg (zisp 15883). clupea cultriventris is spelled cultiventris in some parts of eschmeyer et al. (1996), apparently in error. three syntypes of clupea cultriventris may be in the muséum national d'histoire naturelle, paris under mnhn 3681 (svetovidov, 1952; eschmeyer et al., figure 10. line drawing of clupeonella caspia. 155 int. j. aquat. biol. (2017) 5(3): 128-192 1996). clupea cultriventris var. tscharchalensis borodin, 1896 from lake charkhal in the ural river basin is variously listed as a variety, morpha or a distinct species (see svetovidov (1952); kottelat and freyhof (2007)). mtdna studies of fish from mazandaran and from gilan showed statistically significant differences in haplotype frequencies, indicating genetically different populations (lalouei et al., 2006). norouzi et al. (2012, 2013) examined microsatellite markers in fish from anzali collected in spring and summer and found evidence for two differentiated populations in the southwest caspian sea. norouzi et al. (2014) also sampled spring and summer populations at anzali and also at babol-sar and found significant genetic differentiation among seasons and regions. key characters: this species has a moderately deep body (21-27% of standard length), a short and wide head (interorbital width 16% or more of head length), a sharply keeled belly, and pointed pectoral fin tips. morphology: the dorsal fin has 3-4 unbranched rays, usually 3, followed by 11-14 branched rays and the anal fin has 1-3 unbranched rays, usually 3, and 14-19 branched rays. scales in lateral series 42-55. there are 24-30 belly scutes and 41-62 (rarely to 64), usually 51 or more, gill rakers. vertebrae 40-44 (rarely to 45) compared to 44-47 in the anchovy kilka and usually 46-48 in the bigeye kilka, probably as a result of higher water temperatures during development compared to other kilka species (prikhod'ko, 1979b). sexual dimorphism: sexual dimorphism is only evident during egg development when the belly of females is swollen. colour: the back is blue-green or light-green, the flanks silvery and the belly silvery-white or goldenyellow. fins are hyaline except the dorsal fin which has a central dark but faint stripe and the caudal fin which is darkish at the base. the iris is black. size: reaches 14.5 cm standard length and 19.0 g. distribution: found in the caspian sea, tributary rivers and some adjacent lakes. in iran, it is reported from sea and also the confluence of the pasikhan and pir bazar rivers of the anzali talab and the anzali talab and its outlets by holčík and oláh (1992), and from the safid river and anzali talab by abbasi et al. (1999). abdoli and naderi (2009) list it as from the southwest, southeast and south-central caspian sea in iranian waters. esmaeili et al. (2014) recently list it from the anzali wetland or talab. also in gorgan bay. zoogeography: this species is a caspian sea endemic. habitat: the habitat of this species in the caspian sea is the coastal zone of the sea at depths less than 100m, more usually less than 50-70 m, over a wide range of temperatures (2.6-27.6°c for adults, higher for larvae, and possibly lower temperatures since they are found under ice and probably over 28°c according to some reports), and in fresh and figure 11. clupeonella caspia courtesy of a. abdoli. 156 coad/ herrings of iran hypersaline waters (to 36‰). the young can develop in water at 16‰. southern populations live in a more saline habitat than northern and central caspian populations which are mostly in fresh water. this tyulka may not migrate far but does move between summer-winter feeding and spring-early summer spawning grounds. large schools are found 0.5-2.0 km from shore at depths of 20-25 m on the eastern coast of the caspian sea, descending deeper if water temperatures rise and coming up to about 8 m in autumn as temperatures fall. in winter this species is found at about 30-40 m deep where the temperature range is 7-10°c, warmer than surface waters. larvae and young remain in shallow coastal areas. knipovich (1921) reports a fish from a depth range of 235-300 m in iranian waters but populations at these depths are small (iranian fisheries research and training organization newsletter, 14: 6, 1996). the caspian sea biodiversity database (from www.caspianenvironment.org) states that the largest concentrations are found at 3-7‰ with most intensive spawning at 2-4‰. it is the most widely distributed kilka and with the other kilka species the most abundant fish in the caspian sea (prikhod'ko, 1979b). large schools can be found by day but these disperse at night. it overwinters in the southern caspian sea and some individuals move north to spawn and feed in april. the caspian sea biodiversity database (from www.caspianenvironment.org) estimates the population to number 224 billion fish, with 96 billion fish in the south caspian. the south and north caspian sea stocks are about equal in number after a decline in copepod biomass in the north. the relative frequency of this species compared to other kilkas increased after the invasion of m. leidyi, by more than 10% (fazli et al., 2006). the rise in caspian sea level since 1977 is gradually returning the anzali talab to its supposed, natural brackish state and may improve the fisheries situation which had declined over the last 50 years. emergent and submergent aquatic macrophytes were decreasing and such fish as c. cultriventris (= caspia) were increasing in numbers since 1989 (mandych, 1995). however the fishery will require extensive engineering and management innovations to recover. age and growth: osipov and kiyashko (2008) found that using otoliths gave more reliable estimates than using scales for ageing. this caspian species grows faster than its black sea relative. together with the sturgeons, this species comprises 82.1% of the fish biomass in the caspian sea. condition in this species is better in winter because of the summer-autumn feeding period after spring spawning compared to c. engrauliformis in the big kizil-agach (= bol'shoy kyzylagach or imeni kirova) bay of azerbaijan (badalov, 1972). local populations have differing growth regimes depending on the productivity of these areas (prikhod'ko, 1979b) and there are great variations on a yearly basis too. southern populations grow faster than northern ones in their first year. females grow somewhat faster than males (9.0 g versus 7.3 g average weight along the dagestan coast for example), and life span is about 6 years. this species is mature there at 1 year and average life span is about 3 years. females dominated the population in iran and sexual maturity was attained usually at age 2 and 24 year olds dominated catches but life span is up to 8 years (iranian fisheries research and training organization newsletter, 14: 6, 1996; abtahi et al., 2002). fazli et al. (2006) found age classes 0+ to 5+ in iranian waters with 0+ to 3+ making up 95% of the fish in 1997-1999. in 2000, age classes 0+ and 1+ were reduced in numbers and 2+ to 4+ fish comprised 93.8%. parafkandeh haghighi (2010) examined iranian populations in 2006-2007 and found a mean length of 102.4 mm, negative allometric growth, a mean age of 3.6 years, a male:female sex ratio of 0.6:1, the growth coefficient (k) was the highest of the three species at 0.321, total mortality (z) was 1.28/yr, natural mortality (m) was 0.622/yr, fishing mortality (f) was 0.658, exploitation rate (e) was 0.514, and the maximum constant yield for the kilka fishery was 14,100 t. fazli et al. (2013) studied the status of stocks in iranian waters from 1996 to 2011. the length range with optimum yield was 80-100 mm, mega-spawners were larger than 105 mm, the 157 int. j. aquat. biol. (2017) 5(3): 128-192 percentage of mature fish increased, optimum size declined since 2005, the percentage of mature fish decreased from 100% to 58% in 1999 and increased to 90% in 2001, and this kilka stock was in better condition than the other two species. fazli et al. (2017) found that the biomass of this species increased from 22,000 t in 1997 to 112,000 t in 2009 and declined to 83,300 t in 2013. over this time span the instantaneous coefficient of fishing mortality varied between 0.246/yr and 1.64/yr and exploitation rates were 0.327 and 0.764. the reference points at f0.1 and f40% were estimated at 0.92 and 0.8/yr, respectively. the maximum sustainable yield (msy) and fmsy were estimated at 22,670t and 8,690 vessel x nights (a unit of effort). the acceptable biological catch should be restricted to 17,500 t. abtahi et al. (2004) examined fish from the conical net and light catch at babol-sar, mazandaran and found average fork lengths were 69.82 mm, 83.56 mm, 88.38 mm and 88.43 mm while weights were 2.2 g, 4.18 g, 4.77 g and 5.06 g for fishes at maturity stages i, ii, ii and iv. fazli (2007) and fazli et al. (2007) studied this species from 1995 to 2004 in iranian waters, sampled at landing sites at amirabad and babol-sar in mazandaran and anzali in gilan. growth parameters were l∞=132 mm, k=0.259/yr. t0=-1.285/yr. the instantaneous coefficient of natural mortality was 0.506/yr, the instantaneous coefficient of total mortality (z) was 1.62/yr and the instantaneous coefficient of fishing mortality varied over 10 years from 0.125/yr to 1.487/yr. annual survival rate (s) was 0.2/yr. the exploitation rate was 0.092-0.64 and the acceptable biological catch was 7,450 t. age at first capture was 2.8 years. ages ranged from 1 to 7 years with age groups 2, 3 and 4 dominating at different periods. mean fork lengths were 59.3, 77.5, 87.4, 97.2, 104.5, 111.9 and 116.8 mm. females dominated in each month except april, averaging 0.47:1, possibly due to differing attraction to lights used in the fishery. biomass increased from 16,000 t in 1995 to more than 41,000 t in 2002, declining to less than 28,000 t in 2004. the increase was simultaneous with a sharp decline in anchovy kilka, changes in zooplankton composition and abundance, and especially an increase in zooplankton species favoured by this kilka. this kilka was overfished. janbaz and abdolmalaki (2009) found this species comprised over 86% of the total catch at babol-sar and amirabad in mazandaran, and age groups were 1+ to 6+ with age class 4 predominating at 35.3%. von bertalanffy growth parameters were l∞=114.92mm, k=0.339/yr, t0=-1.878/yr, lt=114.92(1-e 0.339 (t+1.878)), total mortality (z)=0.84/yr, natural mortality (m)=0.41/yr, fishing mortality (f)=0.44/yr and exploitation rate (e)=0.51. karimzadeh et al. (2010) and karimzadeh (2011a) examined fish from the babol-sar region and calculated growth parameters as l∞=143.5mm, k=0.30/yr -1 and t0=1.02/yr, instantaneous coefficient of natural mortality was 0.671 yr-1, fishing mortality was 0.849 yr-1, and the current exploitation rate was estimated as 0.55 and this species is now overfished. akbari miarkolae et al. (2014) examined 175 fish from babol-sar and found a b value of 2.71 and an average condition factor of 2.33 with maximum and minimum values in january and june respectively. condition factors were significantly different in all months studied and greater in winter than spring. an increased mean length and decreased mean weight in recent years was attributed to the ctenophore invasion limiting access to zooplankton. aliasghari et al. (2012) examined 3,774 fish from mazandaran and found a b value of 2.92, growth parameters were l∞=128.7 mm, k=0.41/yr, t0=0.59/yr, age groups were 1-6 years, average age was 3.27 years, 3-year-old fish dominated (45.24%) of the catch), male:female sex ratio was 1:0.779, survival rate was 0.239/yr, natural mortality was 0.448/yr, fishing mortality 0.983/yr, and exploitation rate was 0.687 indicating overfishing. the mean length and weight of fish had increased but mean age had decreased in recent years. moradinasab et al. (2012) studied fish caught in lift nets in gilan and found b values of 2.37 in males and 2.57 in females (both negatively allometric growth) with variations between sexes and seasons. 158 coad/ herrings of iran khedmati bazkiaei et al. (2013) studied 547 fish from the gilan coast and found most fish were 4-5 years old, older fish dominated and fewer younger fish were being caught, the growth equation was lt=142.1(1-exp -0.248(t+1.63)), natural mortality (m) was 0.506/yr, fishing mortality (f) was 0.994/yr and total mortality (z) was 1.5/yr. food: plankton is the main food and copepods predominate but diet also includes cladocera, balanus larvae and clam larvae. the dominant food item is the copepod eurytemora grimmi, particularly in winter when plankton biomass is lowered in the bol'shoy kyzylagach bay of azerbaijan. the food of the common kilka is more varied than the other kilka species simply because of its habitat in shallow coastal areas (badalov, 1972; prikhod'ko, 1979b). older fish take larger and faster crustaceans and consume less food in proportion to body size as they grow. the most intensive feeding is in summer and autumn, decreasing in winter and during reproduction. food is taken during the day. roushan tabari et al. (2009) examined fish from fishing vessels of mazandaran and found highest feeding activity in april with 280±153 prey items per fish weighing 2.9±1.6 mg. balanus nauplii and cypris larvae comprised 93% and acartia 7% at this time with increasing spring temperatures and reproduction, but the copepod acartia biomass dominated from october to february. khedmati bazkiaei et al. (2014) found fish from the anzali shore had copepods, balanus cypris and larvae, cladocerans, and azolla in their gut, dominated by copepods at 94.5% in number. reproduction: spawning occurs in january-february in the southern caspian, later in the north, mainly in depths less than 10m and where salinity is low to average for the caspian sea (badalov, 1972; prikhod'ko, 1979b). the largest southern caspian population spawns near the mouths of the volga and ural rivers (kozlovsky in hoestlandt, 1991). spawning is most intensive at 11°c, but occurs at 1020°c. spawning is intermittent and lasts from midapril to july. peak spawning in iranian waters of mazandaran province is april-may with an average fecundity of 28,240 eggs (abtahi et al., 2002). fazli et al. (2006) recorded mass spawning in iranian waters in april, continuing on until august. eggs are released in water 0.5-9.0 m deep at a salinity range of 0.02-15‰, perhaps as high as 29.15‰. fecundity reaches 60,000 eggs and egg diameter 1.0 mm, 0.48-1.46 mm for fertilised eggs. relative fertility is 4-13 times greater than in alosa species. holčík and oláh (1992) consider that it may spawn in rivers entering the anzali talab. the studies of fazli et al. (2006, 2007) showed that reproduction started in march, peaked in may and finished at the end of august. half the females were mature at 84.3 mm fork length. aliasghari et al. (2012) found from gonadosomatic indices and sexual maturity stages that spawning in iran began in february and peaked in may and june. karimi et al. (2013) examined 800 fish from the anzali coast and found batch spawning with group-synchronous oocyte development. maximum gonadosomatic indices were found in march-april and may in different years suggesting temporal variation in spawning. estimated relative batch fecundity was 6,718 eggs/g, with a range from 3,646 to 10,198 eggs. a low level of atresia (4.7%) during spawning showed that this species was in optimal condition. female common kilka were in better condition in 2008 than in 1994-1996. the invasive ctenophore m. leidyi did not appear to affect competitively the somatic condition of this kilka and overfishing is the probable cause of decline in numbers caught. khedmati bazkiaei et al. (2013) studied fish from the gilan coast and found the highest gonadosomatic indices in april-may and the lowest in novemberdecember. aliasghari and parafkandeh haghighi (2013) examined 3,774 fish caught in 2010 from mazandaran and found a male:female sex ratio of 1:0.779, the sex ratio varied between months (females appear to be less attracted to fishing lights with development of gonads as they cease feeding), and spawning began in march and ended in july with a peak in may. 159 int. j. aquat. biol. (2017) 5(3): 128-192 parasites and predators: samples of this species from babol sar and bandar anzali contained the digenean parasites p. symmetrica (probably p. ventricosa after youssefi et al. (2011)) and b. cingulata, the acanthocephalan c. strumosum, metacercariae of a bucephalus species, and larvae of a contracaecum and an anisakis species (iranian fisheries research and training organization newsletter, 11: 4-5, 1996, annual report, 1995-1996; iranian fisheries research and training organization, p. 28, 1997; shamsi and dalimi, 1996; shamsi et al., 1998). ghayoumi (2009) used the helminth parasites c. strumosum and p. ventricosa as bioindicators of the heavy metals lead and cadmium. ghayoumi et al. (2009) found that fish from babol-sar harbour contained the intestinal helminths c. strumosum, p. ventricosa, contracaecum sp. larvae and raphidascaris sp. larvae, diet being the main factor affecting diversity of the parasites. varshoie et al. (2010) record the helminths pseudopentagramma symmetrica (see above), b. cingulata, mazocreas alosae, c. strumosum, contracaecum sp. and anisakis sp. in this species from iranian waters. clupeonella species are an important food fish for sturgeons (59.4% by weight of acipenser stellatus diet in the middle caspian), sander, herrings (clupeidae) and the caspian seal (badalov, 1972; krylov, 1984) as well as salmo caspius and stenodus leucichthys (kosarev and yablonskaya, 1994). economic importance: it is caught by attraction to underwater electrical lights (prikhod'ko, 1979b) and in school seines in spring and purse seines in summer. in gilan, fish are caught at 12-43 m depths by lift nets equipped with underwater lights (moradinasab et al., 2012). in iranian waters, this species formed only a small proportion (1.35%) of the total kilka catch in a study by razavi sayad (1993) and fazli et al. (2006) gives values of 1.34%, 2.5% and 5.5% for the years 1990-91, 1997-98 and 1998-99, respectively. however, as the anchovy kilka catch declined, this species increased from 13.7% of the total catch in 1999 to 48.9% in 2003 (sayyad bourani et al., 2008). jorjani et al. (2014) detailed the chemical composition and fatty acid profile of this species from specimens landed at anzali. this species was rich in essential unsaturated fatty acids of the omega3 family especially docosahexanoid acid which is nutritionally attractive for humans. catches should be used for human consumption rather than as fish meal. gasan-kuli or hasan kuli is a town in turkmenistan near the iranian border referred to in fishery reports from this area. the catch of rutilus caspicus, c. carpio and sander marinum was nearly 1.44 x 104 tonnes with only 1.9% being accounted for by c. cultriventris (= caspia). however, by 1972 the catch of the commercially important species had declined to 1.5% and the less desirable clupeonella had increased to 5.73 x 104 t or 98.3% of the catch. the causes were reduction in the atrak runoff through irrigation withdrawals, pollution from agriculture, overfishing in the sea and the drop in sea level. flows of the atrak did not reach the sea in 1984, 1986, 1990 and 1991and spawning of species using the lower reaches did not occur (caspian environmental programme, 2000). ebadi and shokrzadeh (2006) examined c. delicatula (= caspia) for lindane at chalus, babol sar, khazarabad and miankaleh but levels detected were less than the fao/who recommended permissible intake and were no cause for public concern. similar studies on d.d.t. and d.d.e. and on chlorobenzilate from the same sites found levels were also less than the permissible intake (shokrzadeh and ebadi, 2005, 2006). shokrazadeh et al. (2009) also found that levels of lindane in dorsal muscle of kilka species were less than fao/who recommended intake. shokrzadeh lamuki et al. (2012) evaluated residues of d.d.t. and d.d.a. in fish from localities in mazandaran and found values (0.017-0.025 mg/kg d.d.t. and 0.0190.026 mg/kg d.d.a.) in the middle of the range of other species, cyprinids and a mullet, and varying between localities. iraj et al. (2014) examined concentrations of chromium, copper, nickel and zinc in fish from the southern caspian sea and found levels of chromium and zinc exceeded international 160 coad/ herrings of iran standards. najm et al. (2014) determined the concentration of the heavy metals cadmium, chromium and lead in fish from the babol-sar coast and found cadmium levels exceeded international standards for human consumption although a risk assessment concluded the fish were safe for consumers. ojagh et al. (2004) showed that natural antioxidants beta-carotene with ascorbic acid and green tea polyphenols were useful in preservation during storage with ice powder. naseri et al. (2005) found that a fluorescence detection method was a good technique for assessing degradation in steamcooked kilka. naseri et al. (2006) also used fluorescence detection to compare the quality of oil and brine filling material in canned kilka. naseri et al. (2009) studied chilling times on quality of the canned product but deterioration was not well shown. fluorescence detection of interaction compounds provided a good technique to assess product quality. naseri et al. (2010) studied lipid changes in canned kilka after long-term storage. motalebi and seyfzadeh (2011) found that a 20% whey protein coating on kilka frozen at -18ºc maintained quality for 6 months while control samples lost their quality after 3 months. zamani et al. (2012) found that belly bursting (enzymatic decomposition leading to unmarketable fish) can be prevented by trypsin inactivation through application of low temperature (4ºc), acidic ph, certain metals (aluminium, copper and zinc ions) and trypsin inhibitors. dorvaj et al. (2013) showed that protein hydrolysates from this kilka can be used as an alternative substrate for the culture of microorganisms. khanipour et al. (2014) found that the shelf life of breaded kilka with tempura batter was about 3 months at -18ºc. navidshad (2014) used an oil supplement from this fish in combination with soybean oil in chicken food to enrich the product. sahari et al. (2014) showed loss of certain vitamins in fish stored at -24ºc including a, d and k and significant losses in c and e vitamins. dehbandi et al. (2015) found that encapsulated nisin z (a polycyclic antibacterial and antioxidant peptide) in liposome improved shelf life of kilka surimi. robins et al. (1991) list this species as important to north americans. importance is based on its use as food and as bait. conservation: stocks on the iranian coast are said to have been depleted but its ecological specialisation on zooplankton means there is comparatively little competition with other fishes. it is probably not in any immediate danger. kiabi et al. (1999) consider this species to be of least concern in the south caspian sea basin according to iucn criteria. criteria include commercial fishing, abundant in numbers, widespread range (75% of water bodies), absent in other water bodies in iran, and present outside the caspian sea basin. sources: iranian material: cmnfi 1970-0531, 14, 78.0-88.6 mm standard length, mazandaran, larim river (36º46'n, 52º58'e); cmnfi 1980-0146, 7, 79.9-96.2 mm standard length, mazandaran, gorgan bay at ashuradeh-ye kuchak (36º50'n, 53º56'e); cmnfi 1993-0146, 3, 80.2-98.2 mm standard length, mazandaran, gorgan bay (no other locality data); cmnfi 1993-0167, 1, 96.6 mm standard length, mazandaran, caspian sea, 10 km offshore (ca. 36º49'n, ca. 52º39'e); cmnfi 1993-0168, 3, 84.9-88.0 mm standard length, mazandaran, caspian sea, 10 km offshore (ca. 36º49'n, ca. 52º39'e). clupeonella engrauliformis (borodin, 1904) (fig. 12) common names: rizeh keraye, kilka-ye anchovy or kilka-e-anchovi. [ancousabanzar kilka in azerbaijan; ancous sekilli kulke balyk in turkmenistan; anchousovidnaya tyul'ka or anchovylike tyulka, sardelle or sardel'ka, "sardinka" but incorrectly, all in russian; anchovy kilka, anchovy sprat]. systematics: no major synonyms. originally described from buinak, central part of the caspian sea. the lectotype is in the zoological institute, st. petersburg (zisp 13860) with paralectotypes as established by svetovidov (1952) (eschmeyer et al., 1996). eschmeyer et al. (1996) gave the date as 1906 but reshetnikov et al. (1997) and the online catalog 161 int. j. aquat. biol. (2017) 5(3): 128-192 of fishes give 1904. key characters: this species has a slender body (body depth 16-19% of standard length), a short and wide head (interorbital width 16.0-18.5% of head length), a rounded belly with keel scales weakly developed, and pointed pectoral fin tips. morphology: dorsal fin with 3 unbranched and 1214 branched rays, anal fin with 3 unbranched and 1519 branched rays. scales in lateral series 45-49. vertebrae 44-47, rarely to 48 compared to 40-45 in the common kilka (c. caspia). gill rakers number 56-67. belly scutes 23-31. sexual dimorphism: none reported. colour: the back and head are dark blue with violet, green or olive tints. these colours become brighter or turn black in dead fish. the fins are hyaline except the caudal fin which has a black base and the dorsal fin which has a central dark stripe. size: attains 15.5 cm standard length. distribution: found in the central and southern caspian sea, and in iranian waters the southeast caspian sea, southwest caspian sea and the southcentral caspian sea (kiabi et al., 1999) as well as the anzali talab, babol-sar beach and gorgan bay (armantrout, 1980; abdoli and naderi, 2009). zoogeography: this species is endemic to the caspian sea. habitat: the anchovy kilka, along with other kilkas, is the most abundant fish in the caspian sea forming large concentrations in the central and southern caspian wherever water depth exceeds 30m. the anchovy kilka is estimated to be the most numerous kilka at about 77% (ivanov and katunin, 2001; daskalov and mamedov, 2007). it is generally found in the upper water layers but may descend to 120 m. nearshore areas, inlets and water of salinity below 8‰ are avoided. they can tolerate a salinity range of 8-14‰ but the main part of the population is found at 10-12‰ (fazli et al., 2007). overwintering takes place in the southern caspian and the southern part of the central caspian sea at 8.5-9.0°c and up to 13.5°c. schools extend their range into the central and northern caspian in spring to feed (prikhod'ko, 1979b). this species has a hibernation period in the south caspian sea, a spring migration of part of the population to the central caspian, a feeding period in the central and south caspian and an autumn prespawning migration to the south caspian (sedov and rychagova, 1983). in iran, larvae are found mostly in surface layers at 5-20 m while adults are found in deeper zones. males dominate in winter while females dominate in other seasons. the maximum juvenile density (fish <75 mm), comprising 36% of the population, is seen in the summer (iranian fisheries research and training organization newsletter, 20: 7, 1998). jolodar and abdoli (2004) state it is most abundant at 100-150 m. zare et al. (2017) investigated acoustic target strength in iranian waters for calculating biomass, stock assessment and management strategies. mean target strength was correlated with indices of female maturity status and depth of fish occupancy, but not size. there was a high variability in mean target strength estimates despite a small range in fish size (3.2 cm). biological characteristics other than size were important drivers of this figure 12. line drawing of clupeonella engrauliformis. 162 coad/ herrings of iran variation. age and growth: abundance of young anchovy kilka, and hence future year-class strengths, depends on water temperature in autumn (octobernovember). falling water temperatures, in the eastern caspian for example, are caused by upwelling which brings nutrients to surface waters and promotes growth of plankton on which the kilka larvae feed (prikhod'ko, 1979a). females are somewhat larger than males in the spawning areas. sexual maturity is attained usually at age 2 and 2-4 year olds dominate catches but life span is up to 8 years (iranian fisheries research and training organization newsletter, 14: 6, 1996). this species shows the fastest rate of growth in the genus. of the 8 age classes, 0+, 1+, 2+ and 3+ form 99.91% of the whole population (iranian fisheries research and training organization newsletter, 20: 7, 1998). the same study showed that 18.6% of the population matures in the first year of life while 81% matures in the second. the mean age in coastal areas is 2.9 years, slightly higher than that in deep zones below 200 m where 0+ fish are more abundant. the caspian sea biodiversity database (from www.caspian environment.org) gives a population of up to 293 billion fish in the caspian sea. fazli et al. (2007) and sayyad bourani et al. (2008) studied these kilkas from catches with conical liftnets carrying underwater lights in the fisheries of gilan and mazandaran in the 1995-2004 period. fish were aged using the sagittal otoliths. length and weight ranges were 40-140 mm and 0.4-18.4 g with averages of 94.0 mm and 5.7 g (89.2-100.4 mm from 1999 to 2003 in sayyad bourani et al. 2008). the age range was 1-7 years (rarely to 8+ years in parafkandeh haghighi and kaymaram (2012)). the dominant age group varied from age 2 to age 4, making up 40.6% to 57.7% of the catch (fazli et al., 2007) or 5+ years with 4+-5+ making up 84.6% for 1999-2003 (sayyad bourani et al., 2008). growth was high for the first year of life and then gradually decreased. the von bertalanffy growth equation was lt=148(1-e -0.238(t+1.340)) (fazli et al., 2007, and following data). the sex ratio varied with season and was significantly different from equal at male:female=0.78:1 for adults. females were more abundant from january to june and males predominated from september to november. condition factors differed significantly between years, increasing from 1995 to 1996, being lowest in 1998 and then increasing to 2004, and between months, being lowest in january and february and then increasing in march. 50% of fish were mature at 84.5 mm fork length. annual survival rate was estimated at 0.32, the instantaneous coefficient of total mortality (z) was 1.14/year, and natural mortality was 0.473/year. age at first capture was estimated as 2.92 years. the total biomass declined from 186,000 t in 1996 to less than 12,000 t in 2004 and the exploitation rate for 1995-2004 varied between 0.34 and 0.815. fazli (2007) examining fish for the 10-year period 1995-2004 gave slightly different figures: estimated survival rate (s) was 0.174, fishing mortality was 0.65-2.93/yr, biomass declined from 185,000 t in 1996 to 8,320 t in 2004, exploitation rate was 0.390.83, maximum sustainable yield (msy) and fmsy were 44,652 t and 18,609 vessel x nights (a unit of effort), and the acceptable biological catch was 2,190 t. sayyad bourani et al. (2008) give a k value of 0.598/year and a l∞ of 110.13 mm. natural, fishing and total mortality coefficients were 0.69, 0.31 and 1 per year respectively and the sex ratio was female:male=68.2:31.8. these latter results for the 1999-2003 period show how values can change when subsets of data are used. karimzadeh et al. (2010) examined fish from the babol-sar region off mazandaran and calculated growth parameters as l∞=151.9 mm, k=0.28/yr -1 and t0=-1.12/yr, instantaneous coefficient of natural mortality was 0.633/yr-1 and the current exploitation rate was estimated as 0.41. parafkandeh haghighi (2010) and parafkandeh haghighi and kaymaram (2010) studied fish from anzali and babol-sar in 2006-2007 and found the mean length was 117.8 mm, mean age was 4.5 (or 4.6) years, a male:female sex ratio of 0.52:1, and 80% of fish were in the 4 and 5 years old age classes. growth parameters were 163 int. j. aquat. biol. (2017) 5(3): 128-192 l∞=147.45 mm and t0=-1.73/yr, b=2.56 (negative allometric growth), k=0.245/yr, z=1.067/yr, m=0.503/yr and f=0.564/yr and e=0.528/yr. aliasghari et al. (2011) estimated growth parameters for fish from babol-sar and found an increase in mean length and weight over previous years attributed to weak stock construction consequent on the ctenophore invasion, l∞=160.5 mm, k=0.25/yr 1 and t0=-0.92/yr for males and l∞=138.7 mm, k=0.55/yr-1 and t0=-0.42/yr for females, b values for both sexes were 2.5, males had age groups 1-7 years and females 2-7 years, mean age of males was 3.86 years and 4.58 for females, and four-year-old fish predominated at 26.55%. fatemi et al. (2009) examined fish taken from commercial vessels in 2007 using lift nets and lights. age structure ranged from 2 to 7 years and was dominated by the third year class (38.6%). backcalculation methods were validated using otoliths to determine lengths. janbaz et al. (2012) gave values for iran from 2005 to 2007 of k=0.375/year, l∞=131.7 mm, instantaneous coefficient of natural mortality was 0.49/year, fishing mortality was 0.51/year and total mortality 1.0/year, and exploitation rate was 0.51. abundance declined over the three years from 18.8% to 8.5% and to 6%, catch per unit effort declined from 0.3 to 0.1 t, and the condition factor declined. overfishing and the competitive ctenophore were the main causes of the decline. fazli et al. (2013) studied the status of stocks in iranian waters from 1996 to 2011. the length range with optimum yield was 85-105 mm, megaspawners were larger than 100mm, the percentage of mature fish increased, optimum size declined since 2004, the percentage of mature fish were more than 78% (often more than 95%), and the length structure of this kilka stock is a matter for concern. food: plankton is the main food and copepods predominate but diet also includes cladocera, balanus larvae and clam larvae. the dominant food item is the copepod eurytemora grimmi, particularly in winter when plankton biomass is lowered (badalov, 1972). it can make up over 70% of its food. this copepod is more characteristic of the diet of this kilka compared to the other two species and the daily vertical migrations and seasonal movements of the copepod are mirrored by the kilka. the most abundant fish species in the caspian depends on the most abundant member of the crustacean zooplankton (prikhod'ko, 1979b). this species feeds in winter, unlike c. caspia. intensive feeding begins in spring as a preparation for spawning (sedov and rychagova, 1983). spawning males show a positive response to light and so feed during the spawning season, while females do not. f. darvishi (pers. comm. 2003) has demonstrated that the this species has a similar feeding niche as the exotic ctenophore m. leidyi in iran and esmaili sari et al. (2002) also determined that there is a similar diet suggesting that a decline in stocks of the fish is the result of competition. darvishi (2003) and darvishi et al. (2004) studied catches of the anchovy kilka and the ctenophore in the southern caspian sea from august 2001 to october 2002. dietary overlap was >89 in babol-sar samples and >84 in nowshahr samples using the schoener index (presumably 0.89 and 0.84 where 0 is no dietary overlap and 1 is an identical diet). the ctenophore was also feeding on fish eggs but the effect of this was less than competition for food. rahimi bashar and alipour (2009) listed a wide range of phytoplankton and zooplankton species in the diet of fish from the southwest caspian sea. reproduction: spawning ends in late autumn and winter food requirements are higher than in springspawning c. caspia (badalov, 1972). areas for spawning in this species are extensive. spawning is most intensive in july when temperatures are 1324°c and salinity 8-13‰ although the caspian sea biodiversity database (from www.caspian environment.org) gives peak spawning (70%) as in october-november. fazli (2006) gives spawning in iran as spring and autumn but mass spawning takes place in in autumn. spawning takes place in the central and southern caspian along both eastern and western shores both in coastal regions and the open sea from late april to november. mass spawning takes place at depths of 50-200 m and as a result eggs 164 coad/ herrings of iran and larvae are carried over a wide area by the caspian gyral current at these depths (prikhod'ko, 1979b). young hatch mainly in autumn and reach 4.5-8.0 cm at an age of 8-10 months (prikhod'ko, 1979a). eggs are up to 1.82 mm in diameter and fecundity reaches 39,900 eggs. in iran, 80% of the population spawns in autumn and the remainder in spring. accordingly the fishery should be closed in october and november (iranian fisheries research and training organization newsletter, 19: 5, 1998). the subsequent iranian fisheries research and training organization newsletter (20: 7, 1998) states that 89% of the population spawns in autumn with september, at 68.3%, the major month. fazli et al. (2007) found reproduction to start in june, peaking in october and then declining. janbaz et al. (2012) found an absolute fecundity of 12,625 eggs in iranian waters, more than 50% of fish were mature when length exceeded 92.5 mm, and spawning was in autumn but overfishing and the ctenophore invasion pushed the kilka to spawn in winter which caused stocks to collapse. year-round spawning occurred as a strategy to mitigate these problems. aliasghari and parafkandeh haghighi (2013) examined 1,659 fish caught in 2010 from mazandaran and found a male:female sex ratio of 0.569:1, the sex ratio varied between months (females appear to be less attracted to fishing lights with development of gonads as they cease feeding), and spawning was from april to november with a peak in november. parasites and predators: samples of this species from babol sar and bandar anzali contain the digenean trematode parasites p. symmetrica (probably p. ventricosa after youssefi et al. (2011)) and b. cingulata, the acanthocephalan c. strumosum and larvae of the nematode contracaecum sp. (iranian fisheries research and training organization newsletter, 11: 4-5, 1996; shamsi et al., 1996b; annual report, 1995-1996; iranian fisheries research and training organization, p. 28, 1997; shamsi and dalimi, 1996; shamsi et al., 1998a, 1998b). ghayoumi (2009) used the helminth parasites c. strumosum and p. ventricosa as bioindicators of the heavy metals lead and cadmium. ghayoumi et al. (2009) found that fish from babolsar harbour contained the intestinal helminths c. strumosum, p. ventricosa, contracaecum sp. larvae and raphidascaris sp. larvae, diet being the main factor affecting diversity of the parasites. varshoie et al. (2010) record the helminths p. symmetrica (see above), b. cingulata, mazocreas alosae, c. strumosum and contracaecum sp. in this species from iranian waters. clupeonella species are an important food fish for sturgeons (59.4% by weight of sevryuga diet in the middle caspian), sander (percidae) and herrings and the caspian seal (badalov, 1972; krylov, 1984) as well as other fishes. economic importance: this species forms 80-90% of the catches of kilkas in former soviet waters (sedov and rychagova, 1983) and, as noted above, 91.8% of catches in an iranian study (razavi sayad, 1993; rezaei et al., 2003). high catches are related to the larger spawning and foraging range of this species compared to other kilkas and to its habitat in the caspian gyre, an area of increased biological productivity (prikhod'ko, 1979b). it is caught in former soviet waters by attraction to underwater electrical lights attached to the middle of the mouth of a fine-mesh conical net or the sides of a fish pump (ben-yami, 1976). fishing is suspended at full moons as the fish are dispersed. both large and small individuals are taken by these non-selective methods (prikhod'ko, 1981). incidental catches include mugilidae (common), and alosa spp., atherinidae and the cyprinid pelecus cultratus (all occasional) (ben-yami, 1976). the catch per unit effort for funnel nets and midwater trawls is 2,321 and 1,014 respectively (iranian fisheries research and training organization newsletter, 20: 7, 1998). fazli et al. (2009c) estimated the maximum sustainable yield at 44,652 t and 18,609 vessel x nights (as a unit of effort). the acceptable biological catch was estimated at 2,190 t in 2004 but in 2005 it was 4,300 t. in 2008, the catch declined to 220 t through overfishing combined with sea level changes, invasive species and pollution. in 1999, the 165 int. j. aquat. biol. (2017) 5(3): 128-192 catch was 67,450 t in iranian waters. janbaz et al. (2012) recorded a decline in catches from 4,250 t in 2005 to 924 t in 2007. it is regarded as a valuable and cheap food resource in iran where it is canned, made into sausages and surimi, and processed as fish meal (shamsi et al., 1996; moeini, 2002; shabanpour et al., 2002, 2006). ovissipour et al. (2103) showed that protein hydrolysates from this fish had high antioxidant activity and amino acid content and low heavy metal concentrations. this species has a high potential for use in animal and human diets. various studies on its preparation and storage as food have been carried out, e.g. rezaei et al. (2002, 2003), rezaeian et al. (2002), rezaie et al. (2007) and moeini et al. (2009). shelf life in cold storage at -18ºc was 60 days for packed and 30 days for unpacked fish and at -30ºc lipid quality was higher. moeeni et al. (2006) determined the proper formulation of soup powder and found that shelf life of the product was about 60 days. sarhadi et al. (2012) and sarhaddi et al. (2015) studied the proximate composition, amino acid profile, fatty acid and mineral composition of this species and found it to have good nutritional quality such that powdered bone may be used as an additive in food industries. robins et al. (1991) list this species as important to north americans. importance is based on its use as food and as bait. conservation: prikhod'ko (1981) recommends fishing in deeper waters where larger fish are concentrated to avoid an excessive take of young fish which favour the upper water layers. stocks in the southern caspian sea are said to be depleted. kiabi et al. (1999) consider this species to be of least concern in the south caspian sea basin according to iucn criteria. criteria include commercial fishing, abundant in numbers, widespread range (75% of water bodies), absent in other water bodies in iran, absent outside the caspian sea basin. daskalov and mamedov (2007) studied commercial catch data in the caspian sea generally and found a period of high catches from 1991 to 2000 with high spawning-stock biomass and relatively good recruitment. catches peaked at 271,400 t, fishing mortality reached 1.8y-1 in 1999 and overfishing occurred. from 2001 to 2004, the stock collapsed, recruitment failed in 2001 and catches fell to 54,300 t in 2005, of which the iranian fishery was about 8%. this was attributed to the spread of the ctenophore m. leidyi, with contributions from overfishing. fazli et al. (2007) also concur that both overfishing and the invasive ctenophore caused the collapse of stocks. the catch in iran declined from 71% of the total kilka catch in 1999 to 52.5% in 2003 (sayyad bourani et al., 2008). janbaz et al. (2012) give figures for the collapse in iran from 4,250 t in 2005 to 924 t in 2007. sources: iranian material: cmnfi 1993-0167, 1, 99.5 mm standard length, mazandaran, caspian sea (ca. 36º49'n, ca. 52º39'e); cmnfi 1993-0168, 4, 89.3-107.6 mm standard length, mazandaran, caspian sea (ca. 36º49'n, ca. 52º39'e). clupeonella grimmi kessler, 1877 (fig. 13) common names: kilka-ye cheshmdorosht (= bigeye kilka). [irikoz kilka in azerbaijan; sardelle or sardel'ka, bol'sheglazaya tyul'ka or bigeye tyulka, bol'sheglazaya kil'ka or bigeye kilka, all in russian; bigeye kilka, southern caspian sprat]. systematics: clupeonella grimmi was originally described from the central part of the caspian sea. the lectotype is in the zoological institute, st. petersburg under zisp 10934 as designated by svetovidov (1952). harengula macrophthalma knipovich, 1921 is a synonym. four syntypes are in the natural history museum, london under bm(nh) 1897.7.5:41-44 (when examined were numbered 42-44, 3 fish, 29.9-33.5 mm standard length in poor condition, september 2007), with many others apparently in the zoological institute, st. petersburg (eschmeyer et al., 1996). key characters: this species has a moderately slender body (17-22% of standard length), a long and narrow head (interorbital width 13.0-15.5% of head length), a sharply keeled belly, and rounded pectoral fin tips. morphology: dorsal fin unbranched rays 3-4, 166 coad/ herrings of iran usually 3, branched rays 13-15, and anal fin unbranched rays 3, branched rays 14-21. there are 44-49, usually 46-48, vertebrae, more than in the other two kilka species and probably a consequence of the low water temperature larvae develop in. belly with 26-32 scutes. gill rakers 42-51. the bigeye kilka is adapted to life in deeper water having, as its name indicates, big eyes with more rod cells and a weaker retina but also more transparent body tissues than other kilkas. sexual dimorphism: none reported except size. colour: the back and top of the head are dark. size: reaches 14.5 cm standard length and 15.2 cm fork length (karimzadeh, 2011b). distribution: found in the caspian sea and concentrated in the south including iranian waters. abdoli and naderi (2009) list it as from the southwest, southeast and south-central caspian sea in iranian waters. zoogeography: this species is endemic to the caspian sea. habitat: the bigeye kilka is found further away from the coast than the anchovy kilka at depths over 5070 m, down to 450 m, with large schools down to 130m. it does not enter fresh water or low salinity areas, staying well away from the shore. there is a daily vertical migration, avoiding sunlight, and following food items. larvae live in water temperatures of 5°c. overwintering occurs in the southern caspian at temperatures of 9-11°c, a migration to the central caspian takes place in spring, with a return south in autumn (prikhod'ko, 1979b). age and growth: sexual maturity is attained usually at age 2, and 2-4 year olds dominate catches, but life span is up to 8 years (iranian fisheries research and training organization newsletter, 14: 6, 1996). the female is larger than the male at the same age. growth is slower than in c. engrauliformis. males dominate the population (iranian fisheries research and training organization newsletter, 14: 6, 1996; fazli et al., 2005) but this study may have sampled spawning fish (see below). fazli et al. (2005) examined fish from the main landing ports (babol-sar, amirabad and anzali) and found the mean fork length of fish increased from 95.87 mm in 1997 to 105.0 mm in 2000 but then decreased to 102.3 mm afterwards. over this time period, fork length range became wider with specimens in the upper length classes representing most of the catch. six age classes were present, 1+ to 6+ years. during 1998-1999, age classes 1+ to 3+ comprised more than 90% of the catch. in 2000, there was a decrease in age classes 1+ and 2+ and an increase in 3+ to 5+ classes. in 2001, age classes 3+ and 4+ decreased and classes 5+ and 6+ increased. the relative frequency of the bigeye kilka has decreased in recent years as a result of the introduction of the ctenophore, m. leidyi, a food competitor and predator on kilka eggs and young. janbaz et al. (2016) examined 262 fish from babol-sar, amirabad and anzali stations caught between may 2006 and april 2007. the largest length-groups were found from december to figure 13. line drawing of clupeonella grimmi. 167 int. j. aquat. biol. (2017) 5(3): 128-192 february and the lowest in april, coinciding with spawning and feeding during wintering respectively. the male:female sex ratio was 0.33:1 which differed significantly from the expected ratio of 1:1. the length-weight regression was w= 0.0000744 fl3.14 for females and w=0.0000341fl3.16 for males, indicating isometric growth. age based on otolith readings showed that the population was composed of age groups from 2 to 7 years old with rapid growth during the second year. four-year-olds with a mean fork length and weight 115.5 mm and 11.9 g were the most abundant at 40.1%. the condition factor was 0.7-0.8 which varied in different months and coincided to the gonad development. the k value and l∞ of females was more than that of males. this was a rapidly-growing species in the southern parts of the caspian sea. khorashadizadeh et al. (2006) found fish in the babol-sar area off mazandaran to have 5 age classes, dominated by the 4+ class. karimzadeh et al. (2010a) examined fish from babol-sar and calculated growth parameters as l∞=148.6 mm, k=0.46/yr -1 and t0=0.18/yr, instantaneous coefficient of natural mortality was 0.881/yr-1 and the current exploitation rate was estimated as 0.26. karimzadeh (2011b) determined mortalities for these babol-sar fish as natural and fishing mortality 0.881 and 0.309/yr respectively, annual survival rate 0.304/yr, instantaneous coefficient of total mortality 1.19/yr and exploitation rate 0.26/yr. age groups were 2-6 years with ages 3 and 4 comprising 72% of the catch. average age of females was 4.194 years and males 3.347 years. catches decreased from overfishing, predators and ecological disturbances. aliasghari et al. (2011) also examined fish from babol-sar numbering 1,043 specimens. growth parameters were l∞=149.7 mm, k=0.32/yr -1 and t0=-1.2/yr for females and l∞=136.5mm, k=0.65/yr -1 and t0=0.24/yr for males. b values were 3.043 in females and 3.079 in males, both positively allometric. the fish were aged at 2-7 years with mean age 4.09 for females and 3.61 for males. four-year-old females dominated at 33.34% and three-year-olds in males at 47.45%. the male:female sex ratio was 0.656:1. this kilka had not been affected by pelagic changes in the caspian sea since it inhabits deeper waters. in recent years the population became older and length and weight increased as karimzadeh (2011b) noted also. this was attributed to use of nets with a standard mesh, overfishing and change in living depth. fazli (2007) examining fish for the 10-year period1995-2004 gave von bertalanffy parameters l∞=144 mm, k=0.265/yr, t0=-1.422/yr, estimated survival rate (s) was 0.298, natural mortality (m) was 0.46/yr, fishing mortality was 0.46-0.98/yr, biomass increased from 36,000 t in 1995 to more than 52,000 t in 1998 and declined to less than 5,900t in 2001, exploitation rate was 0.3-0.52, and the acceptable biological catch was 2,210 t. fazli et al. (2009) examined changes in the population biology of this kilka over the period 1995 to 2001, attributed to the invasive ctenophore. the overall sex ratio was 1.65:1 in favour of males, length-weight regressions were w=0.00922l2.851 for females and w=0.008021 l2.907 for males, indicating a negative growth for both sexes, growth parameters were l∞=142 mm, k=0.28 year-1, and t0=-1.39 years, the instantaneous coefficient of natural mortality was 0.460 year-1, and the instantaneous coefficient of fishing mortality varied between 0.469 and 0.980 year-1. biomass increased from 36,900 t in 1995 to more than 53,500 t in 1998 but declined to less than 5,900t in 2001. this was attributed to overfishing and the appearance of the ctenophore, a competitor for zooplankton food. parafkandeh haghighi (2010) studied fish in 2006-2007 and found the mean length was 119.5 mm, mean age was 4.6 years, a male:female sex ratio of 1.6:1, growth was negative allometric, k=0.267/yr, z=1.015/yr, m=0.537/yr and f=0.478/yr and e=0.471/yr. fazli et al. (2013) studied the status of stocks in iranian waters from 1996 to 2011. the length range with optimum yield was 90-110 mm, mega-spawners were larger than 110 mm, the percentage of mature fish increased, optimum size declined since 2002, the percentage of mature fish were more than 78% (often more than 95%), and the length structure of this kilka 168 coad/ herrings of iran stock is a matter for concern. food: migratory mysids often predominate in the planktonic diet of this species. fish fry are also eaten. its foods are less diverse than that of other kilkas because the variety is less in the deeper waters this fish inhabits during the day. the three kilkas share the available habitat and its foods, the common kilka in shallow, coastal waters, the anchovy kilka in the upper layers of the open sea and the bigeye kilka in deeper water of the open sea (badalov, 1972; prikhod'ko, 1979b). reproduction: spawning is extended, from january through to september but is most intense in spring and autumn (prikhod'ko, 1979b). males predominate in the spawning areas, remaining there while females leave immediately after spawning. males are mainly at 10-20 m and females at 20-25 m during the spawning season. water temperatures are at 6-13°c and salinity 12.6-13.0‰. fecundity is 28,300 eggs. in iranian waters, mature fish ready to spawn are always present in catches in winter and early spring (fazli et al., 2005). khorashadizadeh et al. (2006) found fish in the babol-sar area of the iranian coast to have peak spawning in early january. aliasghari and parafkandeh haghighi (2013) examined 1,034 fish caught in 2010 from mazandaran and found a male:female sex ratio of 0.656:1, the sex ratio varied between months (females appear to be less attracted to fishing lights with development of gonads as they cease feeding), and spawning occurred year round with a peak in october. parasites and predators: samples of this species from babol sar and bandar anzali contain the digenean parasites p. symmetrica (probably p. ventricosa after youssefi et al. (2011)), b. cingulata, the acanthocephalan c. strumosum, eustrongylides excisus, and larvae of a contracaecum and an anisakis species (iranian fisheries research and training organization newsletter, 11: 4-5, 1996, annual report, 1995-1996; iranian fisheries research and training organization, p. 28, 1997; shamsi and dalimi, 1996; shamsi et al., 1998, 1998a). ghayoumi (2009) used the helminth parasites c. strumosum and p. ventricosa as bioindicators of the heavy metals lead and cadmium. ghayoumi et al. (2009) found that fish from babolsar harbour contained the intestinal helminths c. strumosum, p. ventricosa, contracaecum sp. larvae and anisakis sp. larvae, diet being the main factor affecting diversity of the parasites. varshoie et al. (2010) record the helminths p. symmetrica (see above), b. cingulata, mazocreas alosae, c. strumosum, contracaecum sp. and anisakis sp.in this species from iranian waters. clupeonella species are an important food fish for sturgeons (59.4% by weight of sevryuga (acipenser stellatus) diet in the middle caspian), sander (percidae) and herrings and the caspian seal. predators consume 590 million kg of the three kilka species which themselves are the main consumers of zooplankton. kilkas are a very important element in the life of the caspian sea (badalov, 1972; prikhod'ko, 1979b; krylov, 1984). this species is taken to a lesser extent than other clupeonella species because it is relatively sparse. economic importance: the bigeye kilka catch amounts to about 70 million kg a year in former soviet waters of the caspian by means of electric light. all three kilka species are caught by using underwater electric lights and fish pumps (nikonorov, 1964) but in the case of the bigeye the effect is avoidance used to drive it to the bottom where it can be caught. other kilkas are attracted to the light but the bigeye is a vertical migrator, avoiding sunlight (prikhod'ko, 1979b). lightassisted catches of kilkas damages young shad (alosa) stocks which are an incidental catch (zakharyan and teruni, 1979). catches in iranian waters are only 6.84% of the total kilka take (razavi sayad, 1993). the relative frequency of the bigeye kilka in iranian catches was ranked second after anchovy kilka in 1990-1991 at 6.84%, increasing to 12.6% and 21.7% in 1997 and 1998 and then decreasing. the catch in 2008 was 490.8 t with maximum catch-per-unit-effort 0.19 t per vessel per night in february and minimum 0.03 t in january. omega-3 fatty acids from fish oil of this species have been tested as a dietary supplement and were 169 int. j. aquat. biol. (2017) 5(3): 128-192 found to relieve symptoms of dysmenorrhoea (moghadamnia et al., 2010). conservation: stocks in iranian waters are said to be depleted. kiabi et al. (1999) consider this species to be of least concern in the south caspian sea basin according to iucn criteria. criteria include commercial fishing, abundant in numbers, widespread range (75% of water bodies), absent in other water bodies in iran, and absent outside the caspian sea basin. sources: iranian material: cmnfi 1993-0167, 1, 93.0 mm standard length, mazandaran, caspian sea (ca. 36º49'n, ca. 52º39'e); cmnfi 1993-0168, 2, 91.8-94.0 mm standard length, mazandaran, caspian sea (ca. 36º49'n, ca. 52º39'e). genus tenualosa fowler, 1934 this genus comprises 5 species found from the indian ocean to indonesia and china. a single species enters rivers of southern iran. the genus is defined by a series of characters listed below under key characters: these fishes form part of local, artisanal fisheries throughout their range. tenualosa ilisha (hamilton, 1822) (figs. 14, 15) common names: sobour, soboor, sobur, sabur, zobur, zabur, zamur or zomur, all variants of the same word), bari, barak; mahi-ye khor kuchiku (= small bone fish, at abadan from www.abadan. com/abadanhistory.html, downloaded 15 march 1998). [zoboor, soboor, sbour in arabic; hilsa, indian shad or river shad; palo, palla or pulla and tikki-palwar in pakistan]. systematics: clupanodon ilisha was originally described from the ganges estuaries in india. formerly placed in the genus hilsa regan, 1917. alhassan (1982), citing a personal communication from a mr. al-abaychi in 1973, suggests that shatt al arab fish are distinct from those in pakistan on morphometric and meristic grounds but no data have been published. milton and chenery (2001) used genetic and otolith chemistry data that provided strong evidence for a distinct stock in kuwait, compared with stocks from india to sumatra. alhassan (1999) mentions that people in basrah can distinguish two kinds of sobur, based on taste. one is the tastier and pricier shatt al-arab form and the other is the less desirable estuarine/sea form. this has not been confirmed by systematic studies. jorfi et al. (2008, 2009) found differences between populations in iran and iraq using molecular techniques. key characters: this species is distinguished from other indian ocean clupeids by the upper jaw with a median notch, the anal fin ray count being less than figure 14. line drawing of tenualosa ilisha. 170 coad/ herrings of iran 30 rays, a terminal mouth (lower jaw not prominent nor flared at the corners), scales in lateral series are not perforated posteriorly, last dorsal fin ray not filamentous, weakly developed lines (the frontoparietal striae) on top of the head (usually covered by skin and not visible), gill rakers on inner arches straight not curled, a long head 28-32% of standard length, and 30-33 ventral scutes forming a keel along the belly, 15-18 being prepelvic and 11-15 postpelvic (al-nasiri and al-mukhtar, 1988a, 1988b; marammazi et al., 1995). morphology: dorsal fin with 4-5 unbranched rays followed by 14-16 branched rays, anal fin with 2-3 unbranched rays followed by 16-20 branched rays, pectoral fin branched rays 12-15 and pelvic fin branched rays 7. lateral series scales 44-51. gill rakers are fine and numerous, up to about 275 on the lower arch. iranian fish examined by marammazi et al. (1995) from the bahmanshir river in khuzestan have 30-32 total scutes along the belly, 16-18 prepelvic scutes, 13-15 postpelvic scutes, 19-21 dorsal fin rays, 19-24 anal fin rays, 13-15 pectoral fin rays, 8 pelvic fin rays and 44-51 scales. sexual dimorphism: none reported. colour: the back is grey-blue, bluish to green and the sides are silvery with golden, purplish or pink highlights. the dorsal fin is grey, the caudal fin greyblue with a silvery tinge and darkened margin, and the anal fin is light blue with some silvery tinges. paired fins are hyaline. the area behind the gill cover in young fish and many adults have a dark blotch followed by a series of spots or blotches running along the upper flank, for a total of 6-7. the blotches may take the form of bars. the eye is yellow to red. young have a bronze back, silvery flanks and a caudal fin margined in black. size: attains 60.6 cm total length and 2.49 kg for females and 43.0 cm and 0.68 kg for males. a sample of 233 moribund fish from the ashar canal, a branch of the shatt al arab, iraq examined by al-nasiri and al-mukhtar (1988a, 1988b) had a total length range of 70-152 mm. hussain et al. (1994) record fish migrating to the shatt al arab for breeding at 21-38 cm for males and 33-43 cm for females. mature females in the shatt al arab weighed about 0.5-1.1 kg (jabir and faris, 1989). fishes from kuwait attained 57.0 cm (al-baz and grove, 1995). fishes from the arvand, bahmanshir, karun and dez rivers of iran were 12-50 cm long (marammazi et al., 1998; ghafleh marammazi et al., 2004). nasri tajan (2009) gave a maximum length and weight for 344 fish from the bushehr coast as 45.1 cm and 953 g. distribution: reportedly found from the red sea and persian gulf through the indian subcontinent to the malayan archipelago in some general works, or more narrowly from the persian gulf to myanmar. it enters the shatt al arab and tigris river, once as far north as baghdad (kanazawa, 1955), but the northernmost distribution today in iraq is the hawr figure 15. tenualosa ilisha from kuwait by j. e. randall after fishbase. 171 int. j. aquat. biol. (2017) 5(3): 128-192 al hammar. before the construction of dams on the euphrates the migration was up to yaou and meshkhab and up to qal`at salih (31°31'n, 47°16'e) in the tigris of iraq (van den eelaart, 1954). the lower reaches of the tigris and euphrates rivers were connected by a channel to the khor alzubair in iraq during 1983. as a consequence the khor became oligohaline (at less than 10‰) rather than hypersaline (at more than 40‰), becoming an estuary with heavy reed growth. the catch of sobour in the khor by 1997 exceeded that in the shatt al arab and may involve diversion of stocks from the original habitat of the shatt (hussain, 1997). in iran, it is recorded as far north as the gargar shoteit on the dez river (marammazi, 1994). hussain, jabir and yousif (in litt. 1995) record this species from the shatt al arab in iraq and the bahmanshir, hilleh, jarrahi and zohreh rivers in iran. marammazi (1994), najafpour (1997), marammazi et al. (1998) and ghafleh marammazi et al. (2004) report this species from the arvand, bahmanshir, dez, karun and zohreh rivers. it may be found in the hormuz basin but this has not been verified with specimens. esmaeili et al. (2014) record it from the shadegan wetland and esmaeili et al. (2015) from the persis basin. hashemi et al. (2014) caught a single specimen from the shadegan wetland in a year-long survey. hashemi et al. (2015) listed this species as an exotic in the shadegan wetland but in the sense of a migrant from the sea. in the sea, they are found from bushehr around to kuwait in coastal waters (blegvad and løppenthin, 1944; hussain et al., in litt. 1995). zoogeography: al-hassan (1982) mentions a study comparing a population of this species from basrah, iraq with one from pakistan and finding significant meristic and morphometric differences, perhaps indicative of distinct stocks. habitat: sobour enter the shatt al arab in february and march during high tides and feed there until the fall according to a study by al-nasiri and almukhtar (1988a, 1988b) working on fish taken from the ashar canal, basrah, iraq. van den eelaart (1954) reported that most fish entered the shatt al arab in april during the last and first phase of the moon and anecdotal reports indicate the end of march to be the peak period of entry. they ascended into the hawr al hammar and from there into the euphrates as well as into the tigris (van den eelaart 1954). significant numbers were recording as entering the recovering hawr al hammar in 20052006 (hussain et al., 2006). small specimens (50100 mm) were observed in the east hawr al hammar in june 2005 and july 2006 (www.iraqmarshes.org, downloaded 29 august 2005, n.a. hussain in litt. 2006). in mid-april sobour were found below the yaou and meshkhab regulators which formed the limit of their migration on the euphrates in the early 1950s. the limit in the tigris was beyond amara. the main spawning grounds in the euphrates were probably somewhere between shinafiya and samawa and in the tigris between amara and qalat saleh. the last ones leave the shatt in july and fry are found in the rivers of iraq at the end of the june. hussain et al. (1994) recorded sobour ascending the shatt al arab during march with a continuing migration upstream through april to july for spawning and a return migration to the sea during august to october. al-hassan (1993) notes that local people believe that sobour ascend the shatt al arab during spring to marshes north of basrah for spawning, suggesting that they are the fluvial anadromous type. al-hassan (1999) considers they migrate to the sea in september-november, when they are landed in kuwait, and they then migrate to the iranian coast during december-january. males and females move upriver in separate groups according to iraqi fishermen (al-hassan, 1999). jorfi et al. (2008) suggest, based on molecular studies, that a population in the persian gulf chooses the karun river for spawning and migrates via the bahmanshir river, while others migrate up the tigris and euphrates rivers in iraq via both the bahmanshir and the arvand rivers. blegvad and løppenthin (1944) mention this species on sale at khorramshahr on 28-29 april. the spawning migration in iran occurs in spring 172 coad/ herrings of iran (sharifpour, in litt. 1991). it is only found in the zohreh river in spring and summer (marammazi, 1994). ghobeishavi et al. (2016) found a depression is some haematological and immunological parameters during the upstream migration in the karun river, possibly a result of hormonal and environmental effects. dastan et al. (2017) described gill and kidney tissue changes on the migration into the bahmanshir and karun rivers, chloride cells decreasing in number and area for example. they may be found in deep water, over 18 m, or in shallows, on their spawning migration. large concentrations of sobour occur below dams blocking their migration. young occur in side branches of the shatt al arab near food, shelter and the spawning grounds (hussain et al., in litt. 1995). this species occurs in river estuaries and coastal waters and appears to be restricted to the northern end of the persian gulf because this is the only part with large spawning rivers (hussain et al., in litt. 1995). these authors also suggest that an anadromous stock from the shatt al arab migrates to warmer waters off bushehr during january, february and march. at the same time there is a winter decline of kuwaiti stocks. there may also be a marine stock inhabiting coastal waters of kuwait since larvae have been found in kuwait bay during june and november and catches are made in the bay year round. hussain (1997) notes the changing conditions in the khawr az zubayr, which became oligohaline from hypersaline after it was connected to the tigriseuphrates basin by the shatt al basrah canal. in 1994, fishermen began catching sobour in the khawr az zubayr and by 1997 the numbers caught exceeded the catch in the shatt al arab. migrations in the indus river of pakistan (islam and talbot, 1968) may last over 7 months and the migration up the ganges river extends over 1,287 km. fish may move as much as 70.8 km in one day and may jump out of the water on the migration. age and growth: in the bahmanshir river, iran most fish are 4-5 years old. the minimum total length and age at maturity are 26.2 cm, 200 g and 2 years for males and 32.18 cm, 450 g and 3 years for females. von bertalanffy growth parameters in iranian females are l∞=57.78 cm and k=0.282 and in males 46.37 cm and 0.252 (marammazi, 1995; iranian fisheries research and training organization newsletter, 12: 5, 1996, annual report, 1995-1996; iranian fisheries research and training organization, p. 53-54, 1997). nasri tajan (2009) found bushehr coast fish were 2-4 years old with most 2+ years. hashemi et al. (2009) studied fish landed at hendijan and abadan and recorded l∞ as 42.81 cm, k was 0.9, m was 1.37, f was 2.41, z was 3.78 and e was 0.64. y'/r was 0.048 and b'/r was 0.19, exploitation rate (u) was 0.61, annual stock at the beginning of the year (p) was 7,615 t, annual standing stock (b) was 1,927 t and msy was 3,642 t. the stock was overfished. hashemi et al. (2009), also studying fish at hendijan and abadan, recorded l∞ as 54.6 cm, k was 0.96, m was 1.34, f was 2.8, z was 4.2, e was 0.68 and t0 was -0.14, and the stock was overfished. hashemi et al. (2010) studied 9,317 fish from the landings at abadan and hendijan. size range was 20-39 cm. the von bertalanffy growth parameters were l∞=43.32 cm, k=0.78yr -1, φ' was 3.16 and t0 was -0.18. mortality rates were m=1.29 and z=4.53, and fishing mortality (f) was 3.24yr-1. the exploitation rate (e) was 0.72 and the stock was overfished. values of the sizes where the probability of capture was 50% (l50) and 100% (l100) were 22.3 and 28.5 cm total length respectively. fish were recruited to the fishery at a mean size of l100=22.3 cm. the relative yield per recruit (y'/r) was 0.062, relative biomass per recruit (b'/r) was 0.12 and exploitation rate (u) was 0.76. the values for annual catch, total annual stock, standing stock and maximum sustainable yield were 4,645t, 6,635.71 t, 1,433.64 t and 3,274.19 t, respectively. the fishing pressure must be reduced from 3.24yr-1 to about 0.97yr-1for this population to be adequately managed. another study apparently based on the same or similar samples (hashemi seyed et al., 2010) found slightly different parameters: l∞=42.81 cm, k=0.0yr-1, t0=-0.25, z=3.78, m=1.37, f=2.41, 173 int. j. aquat. biol. (2017) 5(3): 128-192 e=0.64 and the values for annual catch, annual average standing stock and maximum sustainable yield were 7,615 t, 1,927 t, and 3,624 t, respectively. hashemi et al. (2010) assessed production in the shadegan wetland, khuzestan as 0.73 kg/ha/year. roomiani and jamili (2011) examined fish landed in iran from a northern persian gulf fishery. growth was isometric. maximum total length was 43 cm and weight 949 g. von bertalanffy growth parameters were l∞=42.74 cm total length, k=0.77 and t0=-0.21 years-1. total mortality (z) was 2.55 years-1, natural mortality (m) was 0.75 years-1, fishing mortality (f) was 1.8 years-1, and exploitation rate (e) was 0.7 years-1, and parameters indicate overfishing. maximum sustainable yield was calculated to be 2,653t. roomiani et al. (2012), presumably working with a partially similar dataset, found l∞=42.8 cm, k=0.7, z=3.45, m=1.24, f=2.21 and e=0.64. al-nasiri and al-mukhtar (1988a, 1988b) give a length-weight relationship of w=3.9 x 10-6 l3.16 or logw=3.16 logl-5.4 for fish aged at 0+ from the ashar canal at basrah. the mean condition factor was 0.87. fishes in the shatt al arab are in age groups 5 to 6 for the period may to august (hussain et al., 1991). in contrast, a later study on the shatt al arab fish showed there are 5 age groups and the second and third age groups dominate in catches (hussain et al., 1994). in this latter study, shatt al arab fish mature at 25 cm for males and 33 cm for females, similar to an iranian study (see below). the length-weight relationship was logw=-4.7074+ 3.0479 logl for females and logw=-4.5802+3.0193 logl. condition factor gradually increased with length groups in males, peaking at 32-33 cm followed by a sharp decline while females had a nearly stable condition factor from 34 to 43cm. mohammed et al. (2001) gave a von bertalanffy growth equation as l∞=60.47 cm and a condition factor of 0.32, slower growth than in indian and bangladesh populations and probably maturing later. amodeo (1956) gives lengths of 25 to 35 cm for fish caught in the shatt al arab on their spawning migration. young grow rapidly, 4.3 cm in octobernovember. most fish on the migration in the indus river were in age groups 3 and 4. life span is up to an estimated 7 years with maturity as early as 1 year. jawad et al. (2004) found haematocrit level to increase with body length up to 40 cm after which it decreased, males showed higher levels than females, and levels were higher pre-spawning than during spawning and increased slightly post-spawning, a general correlation with fish activity in iraqi waters. al-baz and grove (1995) studied fish taken from kuwait fish markets. females dominated the catch, male:female ratio being 1:2.4, perhaps because the sexes moved in different schools. the smallest mature female was 34.4 cm and 50% of the females are mature at 41.5 cm. they estimated natural mortality (m) based on von bertalanffy growth parameters (l∞ and k) and mean annual water temperature as logm=-0.0066 -0.279 logl∞+0.6543 logk + 0.4634 logt. the length-weight relationship was w=0.011l2.983 for males and w=0.007l3.104 for females. growth in the sexes follows different patterns. five age groups were detected using otoliths and fish were fully recruited to the fishery at 3 years of age. von bertalanffy growth parameters were l∞=52.70 cm and condition factor (k)=0.28 per year while using allen's method they were l∞=52.50 cm and condition factor (k)=0.36 per year. annual total mortality was estimated to be 1.2 using the k value of 0.36. a fishing mortality was calculated to be 0.8 per year. mohamed et al. (2001) studied 7,535 fish from landings at fao, iraq in 1998-1999 and found growth and mortality parameters were l∞=60.47 cm, k=0.32, z=1.28, m=0.62 and f=0.66. the exploitation rate (e) was 0.52, a bimodal recruitment pattern of unequal strength was observed (peaks in march at 54.65% and october at 45.35%), maximum yield per recruitment was achieved at emax=0.63 and lc=25.0 cm. they recommended fishing nets should be adjusted to capture fish over 25 cm to avoid overfishing. mohamed and qasim (2014a) assessed the stock in 2012-2013 in iraqi marine waters close to iran. they found fish were 12.2-48.0 cm total length with >93% of fish 26-44 cm. age span was almost 6 years. the b value was 3.2683 (allometric 174 coad/ herrings of iran growth), asymptotic growth (l∞)=61.47 cm, growth rate (k)=0.275, annual instantaneous rate of total mortality (z)=1.66, natural mortality (m)=0.55, fishing mortality (f)=1.11 and exploitation rate (e)=0.67. a bimodal pattern of unequal recruitment was observed with april fish being migrant breeders and july fish juveniles from the previous year recruiting to the adult stock. the maximum yield per recruitment was achieved at emax=0.72 and lc=27.8 cm. the stock was overexploited. food: the ashar canal study found them to feed on phytoplankton such as dinoflagellates and diatoms and on zooplankton, mainly copepods, as well as their own young. the sieve-like gill rakers are used to strain out planktonic organisms without selection. presence of some sand grains indicates that feeding can occur on the river bed. feeding intensity may decrease or cease on the spawning migration and is very high after spawning. the bahmanshir fish feed principally on copepods and diatoms. shatt al arab juveniles feed mostly on filamentous algae and diatoms with some organic matter, fish eggs and zooplankton while adults have empty stomachs on the spawning migration (hussain et al., in litt. 1995). in the indus river, the newly hatched larvae and juveniles graze for five to six months in fresh waters before they migrate to the sea (www.janggroup.com/thenews/feb2003-daily/18-02-2003/busi ness/b2.htm, downloaded 18 february 2003). the prime food in the sea off the iranian coast at deylam, bushehr is phytoplankton, principally bacilliariophyta followed by pyrrophyta. zooplankton was also taken (nasri tajan et al., 2008; nasri tajan, 2009). reproduction: the spawning migration depends on the flood regime of the rivers. turbid water and fast current are probably stimulants to egg deposition. the sobour depends on river-edge vegetation for egg deposition. spawning grounds in iraq are probably located near the beginning of the side branches of the northern sector of the shatt al arab, 120 km from the sea (hussain et al., 1994). this species is gonochoristic (blaber et al., 1997). males may ascend the river before females but females become dominant in indian populations. males dominate in march in the shatt al arab and the sex ratio reaches equilibrium in the spawning months of may-july (elsewhere in the same communication spawning is given as june to august) (hussain et al., 1994; jawad et al., 2004). spawning may occur more than once in a season in india. this has not been demonstrated for iran but could occur. the gonadosomatic index for fishes in the iraqi shatt al arab indicates peaks in march-may and july-august, suggesting two spawnings (hussain et al., 1991) although a later report (hussain et al., 1994) gives spawning as june to july and july to august as evidenced by two modes of juveniles found in september. sex ratio is equal during this period. all females entering the shatt al arab were mature with smallest female being 33.0cm long. males less than 25.0 cm were immature, the population reaching 100% maturity at 31-32cm (hussain et al., 1994). the kuwait fish studied by al-baz and grove (1995) indicated spawning between may and july with a peak in june. fecundity in the indus river population was estimated to be up to 2,917,000 eggs per female, egg diameters reached 0.89mm, and the hatching takes place in about 23 to 26 hours (www.janggroup.com/thenews/feb2003-daily/18-02-2003/busi ness/b2.htm, downloaded 18 february 2003). estimates for the hooghly river of india reach 13,230,500 eggs per female (al-hassan, 1993). fecundity in the shatt al arab ranges between 444,960 and 1,616,560 eggs for fish 33.0-41.5 cm total length although 2 fish 37.3 and 2 fish 39.0 cm total length had a range in egg numbers of 109,000233,840, showing that great variations in fecundity occur between individuals; possibly some fish had partially spawned before capture (jabir and faris, 1989). this latter study gave a relationship between absolute fecundity and total length as f=1.3699 l3.6681 and logf=0.1367 + 3.6681 logl and between fecundity and weight f=302.8214 w1.2087 and logf=2.4812 + 1.2087 logw. fecundity increased significantly with body weight, ovary weight and total length. relative fecundity (ova/gramme body weight) varied from 737 to 1,721, mean 1,216. hatching can occur within one day at an average 175 int. j. aquat. biol. (2017) 5(3): 128-192 temperature of 23°c. eggs, larvae and young are found on the spawning grounds but with growth the young move into estuarine and foreshore areas during winter months. hussain et al. (1994) record the appearance of juveniles from the northern shatt al arab from june to november. adults return to their original habitat in the sea after spawning. there is some evidence for freshwater resident populations in india which migrate upriver to spawn but do not descend to the sea. the bahmanshir fish are thought to spawn from april to july. only adults enter the bahmanshir (iranian fisheries research and training organization newsletter, 12: 5, 1996). absolute fecundity of fish from the arvand, bahmanshir, dez and karun rivers ranges from 374,892 to 1,954,144 eggs for total lengths of 380 to 500 mm, respectively and is related to age. ova with diameters 0.64-0.795 mm were released spontaneously in a study of this fish in khuzestan province, in several batches along its migration route (ghafleh marammazi et al., 2004). spawning begins on entry to the bahmanshir and arvand rivers in khuzestan in april, continuing to september and the end of their migration at the cities of shushtar and dezful higher upriver. males enter these rivers first in march, followed by females in april (ghafleh marammazi et al., 2004). nasri tajan (2009) found bushehr coast fish were batch spawners in april-may. parasites and predators: none reported from iran other than nematode larvae by ebrahimzadeh and nabawi (1975) for fish from the karun river. bannai and muhammad (2016) studied the parasite fauna of this shad as it immigrated to iraqi waters from fao in the estuary of the shatt al arab to the al hammar marshes. they concluded that these fish do not come from the indian ocean and are endemic to the persian gulf (unless parasites are lost on the migration from the indian ocean). economic importance: the ashar canal study cites 996,308 kg reaching the ashar fish market from october 1975 to june 1977 (see also sharma (1980)). the catch landed at fao on the shatt al arab estuary of iraq was 6,576 t in 1990-1991 (l.a.j. al-hassan in litt. 1995; however this seems much too high although the estimate is from the food and agriculture organization). this species forms the most important commercial fishery in the basrah region of southern iraq, average catches being 491.086, 319.661 and 267.988 t in 1977, 1978 and 1979 respectively (sic, jabir and faris, 1989). there is a drift-net and stake-net ("hadra") fishery in the sea by kuwait in kuwait bay and around falaikah island (al-baz and grove, 1995). the fishing season on the tigris-euphrates is march to august with a peak in april, or late april to early june (jabir and faris, 1989) or to november (ali et al., 1998). van den eelaart (1954) gave the fishing season for this species as march-august (peaking in april) in rivers, and march-may (peaking in april) in hawr al hammar, iraq. fish are caught at the mouth of the shatt al arab as they enter the river with stationary gill nets, drifting gill nets, and in "mailan" and "odda" traps from march to august. the catch averaged 150-180 kg per ten odda and in march 1953 the total catch at the mouth of the shatt al arab was about 25,000 kg (amodeo, 1956). large fish are only caught in the summer (alhassan, 1999). the catch at abadan in iran from february to november in 1943 was about 401.42 t and from january to june about 336.67 t (pillay and rosa, 1963). this species is seen on markets at ahvaz, khuzestan in november but these are sea-caught fish. marjan iran company was selling 600-800 g fish for u.s.$1.40/kg, 800-1,000 g fish for u.s.$1.60/kg, 1,000-1,200 g fish for u.s.$1.70/kg, and 1,200 g and larger fish for u.s.$1.80/kg in august 2003 (http://groups.yahoo.com/groups /hilsa/message/25). the catch in khuzestan province in 2000 was 2,688 t (ghafleh marammazi et al., 2004) and in 2006 was 4,989.83 t (about 15% of khuzestan's total commercial fish landing) (roomiani and jamili, 2011). the catch in khuzestan province in 2008 was 4,645 t (hashemi et al., 2010). these fish are caught with traps, weirs, gill nets and other devices in rivers on the spawning 176 coad/ herrings of iran migration. they are excellent eating until spawning occurs after which they lose their flavour. however this species has been implicated in clupeotoxic poisoning. hindi et al. (1996a) give the chemical composition of flesh of this species as 66.41% moisture, 12.12% fat, 18.72% protein and 1.98% ash, indicating a valuable food fish characterised as fatty. hindi et al. (1996b) give chemical indices for assessing fish freshness according to the month of capture and marketing (ph 6.06, total volatile nitrogen bases 15.32 mgn/100g fish, thiobarbituric acid 1.35 mg, and free fatty acids 1.33%). biogenic and anthropogenic sources were noted for the hydrocarbons in this species from the shatt al arab; n-alkanes attained 31.11 µg/g and hydrocarbons 10.91 µg/g, the highest for the fish species studied (al-saad et al., 1997). the fat content of this shad is a factor in these high levels (al-saad, 1990). salari and sadough (2009) compared heavy metal (cd, pb, cu, co, ni) content in muscle, liver and gill tissues of fish from the karun river and found levels less than those considered dangerous in iran. sadough niri et al. (2010) examined fish from the arvand river and found some of the above listed heavy metals exceeded international limits however. in pakistan, the indus river fishermen number between 8,000 and 9,000. jafri (1994) reviews the indus fishery which had yields up to 2,694mt. it is the most important indo-pacific shad species. the failure of the indus river fishery in 2003 through drought resulted in iranian fish being flown to pakistan for marketing there at rupees150-400 per piece (www.jang-group.com/thenews/feb2003-daily /18-02-2003/business/b2.htm, downloaded 18 february 2003). robins et al. (1991) list this species as important to north americans. importance is based on its use as food, in aquaculture and in textbooks. conservation: hussain et al. (in litt. 1995) report a decline in catches over the previous two decades in the shatt al arab. al-nasiri and al-mukhtar (1988a, 1988b) mention that fish enter the polluted ashar canal, a side tributary of the iraqi shatt al arab, during high tide when waters are diluted. a low tide in october resulted in severe oxygen depletion and fish suffocated. das et al. (1977) found samples from the ashar fish market in basrah to be contaminated with hydrocarbons, emitting a kerosene smell and being unfit for human consumption. al-saad (1990) found petroleum hydrocarbon residues to be high in khawr az zubayr fish at 40.6 μg/g as this species is one that accumulates fat. mohamed and qasim (2014b) found iraqi landings decreased from 90.2% of total landings in 1965-1973, to 52.9% in 19911994, to 41.8% in 1995-1999 and to 30.7% in 20002006. they recommended establishment of closed areas to protect spawning such as the shatt al arab for 30 days in may, catch of small fish up to 23 cm in spawning areas banned, reduction of pollution, and cooperation within iraq and with iran on fisheries management. evidently, overfishing and pollution are major factors in the conservation of this species, to which must be added variations in freshwater flow and quality from the marshes and tigris-euphrates through human processes. sources: some aspects of the biology of this species were based on pillay and rosa (1963) and al-hassan (1993) writing mostly on indian and pakistani populations. bhaumik (2015) also reviews studies on the biology of this species. specimens on markets in ahvaz, khuzestan examined. iranian material: cmnfi 1991-0153, 1, 243.3 mm standard length, khuzestan, zohreh river (no other locality data). comparative material: bm(nh) 1875.1.14:11-13, 3, 118.8-135.8 mm standard length, iraq, tigris river (no other locality data); bm(nh) 1920.3.3:178-182, 6, 103.3-132.4 mm standard length, iraq, basra (30º30'n, 47º47'e); bm(nh) 1989.1.13:1-3, 3, 53.959.9 mm standard length, iraq, khawr az zubayr (no other locality data); bm(nh) 1989.1.13:4-5, 2, 66.669.8 mm standard length, iraq, khawr az zubayr (no other locality data). references abbasi k. 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(2016). population structure of alosa linck, 1970 (sic) in the southern coast of caspian sea (guilan province). journal of animal researches, 28(4): 457465. (in farsi) zakharyan g.b., teruni v.n. (1979). the influence of light-assisted fishing of the "kilka" of the genus clupeonella on stocks of the caspian shad of the genus alosa (family clupeidae). journal of ichthyology, 19(5): 129-132. zamakhaev d.f. (1944). k voprosu o sistematicheskom polozhenii prokhodnykh sel'dei kaspiya [on questions concerning the systematic position of the caspian anadromous herrings]. zoologicheskii zhurnal, 23(2-3): 65-81. zamani a., rezaee m., madani r. (2012). in-vitro effects of biochemical factors on trypsin activity from intestine and pyloric caeca of common kilka (clupeonella cultriventris caspia) for inhibition of belly bursting. iranian scientific fisheries journal, 20(4): 53-62. (in farsi) zare p., kasatkina s.m., shibaev s.v., fazli h. (2017). in situ acoustic target strength of anchovy kilka (clupeonella engrauliformis) in the caspian sea (iran). fisheries research, 186(1): 311-318. int. j. aquat. biol. (2017) 5(3): 128-192 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی ماهیان(مروری بر شگ ماهیان ایران )خانواده شگ برایان دبلیو کد .کانادا ، k1p 6p4،انتاریو ،اتاوا، موزه تاریخ طبیعی کانادا چکیده: هاشود، گونهتوصیف می ماهیان ایرانشناسی، اهمیت اقتصادی و وضعیت حفاظتی شگزیست شناسی، پراکنش،در این مقاله سیستماتیک، ریخت ، clupeonellaهای سز جنبومی اگونه 9گردد. در مجموع تعداد مورد بررسی قرار گرفته و یک مرور منابع در مورد این ماهیان در ایران فراهم می alosa وtenualosa شود.یافت میهای جنوب ایران در دریای خزر و رودخانه .زالون ،کیلکا ،alosa، clupeonella، tenualosa شناسی، شناسی، زیستریخت :کلمات کلیدی int. j. aquat. biol. (2016) 4(3): 143-170: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology review article review of the perches of iran (family percidae) brian w. coad1 canadian museum of nature, ottawa, ontario, k1p 6p4 canada. article history: received 5 april 2016 accepted 7 june 2016 available online 2 5 june 2016 keywords: iran, biology, morphology, perca, sander. abstract: the systematics, morphology, distribution, biology, economic importance and conservation of the perches of iran are described, the species are illustrated, and a bibliography on these fishes in iran is provided. there are three species, perca fluviatilis, sander lucioperca and s. marinus, found naturally in the caspian sea basin, with s. lucioperca translocated. introduction the freshwater ichthyofauna of iran comprises a diverse set of families and species. these form important elements of the aquatic ecosystem and a number of species are of commercial or other significance. the literature on these fishes is widely scattered, both in time and place. summaries of the morphology and biology of these species were given in a website (www.briancoad.com) which is updated here for one family, while the relevant section of that website is now closed down. other families will also being addressed in a similar fashion. the perches, darters, pike-perches and their relatives comprise a family, the percidae, of mostly freshwater species found across the northern hemisphere. there are about 10-11 genera and about 226 species (nelson, 2006; eschmeyer and fong, 2011) with estimations up to 275 species (kestemont et al., 2015). there are three species in two genera in iran. maximum size approaches 1 m but many species (the darters particularly) are small. they are characterised by ctenoid scales; a dorsal fin with an anterior spiny portion and a soft rayed posterior portion; an anal fin with 1-2 spines (rather than 3 as in related families) and a few soft rays; pelvic fins thoracic in position, with 1 spine and 5 soft rays; branchiostegal membranes not attached to * corresponding author: brian w. coad e-mail address: bcoad@mus-nature.ca the isthmus; branchiostegal rays 5-8; teeth on the jaws, vomer and palatines in patches, sometimes with canine teeth; and the operculum has a sharp spine. perches are found in warm southern waters to subarctic ones, in both flowing and still water. some larger species are commercially important while smaller species make attractive aquarium fishes. the small darters of north america rival coral reef fishes for colour in their breeding condition. perches have a variety of reproductive strategies which include broadcasting, stranding, burying, attaching, clumping and clustering. during the breeding season tubercles develop, particularly on the male. these may be on the body, fins or head and are used to maintain contact and enhance grip between males and females during the spawning act. genus perca linnaeus, 1758 this genus comprises two species, one found in north american and one in eurasian, fresh waters. the body is compressed, scales are small and ctenoid, cheeks and gill covers are scaled, the opercular bone carries a single flat spine, the preopercle is serrated posteriorly and has spikes ventrally, there are no canine teeth, branchiostegal rays 7, the lateral line does not continue onto the 144 coad/ perches of iran caudal fin, and the body usually has strong bars. perca fluviatilis linnaeus, 1758 (figs. 1-2) common names: mahi-ye khardar, bacheh suf (=baby suf), mahi suf rudkhanehi astrakhan (=astrakhan river suf fish, presumably an old name at this russian locality, suf-e haji tarkhan (=astrakhan suf, an old name no longer in use), sufe rudkhaneh'i (=river suf), hashtarkhan suf. [xanibaligi in azerbaijan; okun' in russian; perch, european perch, eurasian perch, river perch]. systematics: perca fluviatilis was originally described from europe. a syntype (bm(nh) 1853.11.12.3) is a right half-skin. collette and bănărescu (1977) refute earlier workers who maintain that this species and the north american yellow perch (perca flavescens (mitchill, 1814)) were the same or at best subspecies, e.g., see svetovidov and dorofeeva (1963) and čihar (1975) for opposing views. .collette and bănărescu (1977) base their conclusion on the observation that the predorsal bone is anterior to the first neural spine in fluviatilis rather than extending between the first and second neural spines as in flavescens. other characters also exist to separate the two species. data on the north american perch cannot therefore be used uncritically as a summary of biology for the figure 1. perca fluviatilis, line drawing by s. laurie-bourque. figure 2. perca fluviatilis, anzali wetland june 2012, courtesy of k. abbasi 145 int. j. aquat. biol. (2016) 4(3): 143-170 iranian perch. systematics: no major synonyms. key characters: characters of the genus serve to identify the single, distinctive species in iran. morphology: lateral line scales 40-78; scale rows above lateral line 7-10; rows below lateral line 1222; and predorsal scales 10-21. scales have very fine circuli, few anterior radii, a posterior focus and a markedly incised anterior margin where about 5-7 radii terminate. the exposed part of the scale is coarse and is the base for ctenii, best developed on the margin. dorsal fin spines 12-18; dorsal soft rays 8-17, usually 12-15, after 0-5, usually 2-3 spines; anal fin soft rays 6-11 after 1-3 spines; pectoral rays 9-17; and pelvic rays 4-6, usually 5 after 1 spine. gill rakers 23-25, reaching between the third and fourth rakers below when appressed usually, but variable in length with diet, shortest when feeding on fish, longer when food is zooplankton. the gut is s-shaped with a large anterior loop and there are 3 pyloric caeca. vertebrae 38-44, and gill rakers 14-29. the chromosome number is 2n=48 (klinkhardt et al. 1995). meristic values for iranian specimens are: lateral line scales 59(4), 60(2), 61(2); scale rows above lateral line 9(7); rows below lateral line 17(4), 18(2), 19(1); scales between lateral line and pelvic fin 6(2), 7(5); predorsal scales 10(1), 11(2), 12(1), 13(2), 14(1); and caudal peduncle scales 24(1), 25(1), 26(1), 27(3), 28(1); dorsal fin spines 13(1), 14(2), 15(1), 16(4); dorsal soft rays 13(3), 14(5) after 2 spines; anal fin soft rays 8(3), 9(3), 10(1) after 2 spines; pectoral rays 11(2), 12(2), 13(3), 14(1); pelvic rays 5(8) after 1 spine; vertebrae 40(2), 41(3), 42(1). sexual dimorphism: males have longer paired fins than females and are brighter in colour. females are larger than males of the same age. colour: colour can be affected by diet, especially in the fins which are reddest when feeding on certain crustaceans, and by habitat depth but generally the colour is stable. fish from along the shore in weedy habitats are greenest, those in open water a pale yellow, and at depth are darker. the body is an overall greenish-yellow with 5-9 black bars on the flanks although in some fish bars are very faint. the first dorsal fin is grey with black markings on the membranes. the first spine is often black and deep black membranes are evident between spines 1 and 2 and the last 4 to 5 spines. the second dorsal fin is greenish-yellow with melanophores on the rays and membranes, the pectoral fin yellowish and other fins pinkish to yellow to silvery-white. paired and caudal fins have much sparser melanophores than the second dorsal fin. the lower part of the caudal fin is orange to red. the peritoneum is silvery and speckled with melanophores. size: 68.0 cm and 7.0 kg, possibly 10.4 kg but most are much smaller than this (machacek, (1983-2012), accessed 27 july 2012). distribution: found from the british isles across northern eurasia to eastern siberia. their presence in the caspian sea basin of iran is their most southerly natural distribution. also introduced to south africa, australia and new zealand. in iran, it has been reported from the aras dam, the hendeh khaleh swamp in gilan, anzali wetland (= mordab or talab) and its outlets, ab kenar and siah keshim protected region in the anzali talab, at bandar anzali, bandar anzali beach, the lower safid river, the amirkelayeh wetland, and the golshan, pesikan, shahrud and sheikan rivers (derzhavin, 1934; holčík and oláh, 1992; riazi, 1996; karimpour, 1998; abbasi et al., 1999; abdoli, 2000; abdoli and naderi, 2009; hamzei et al., 2013; esmaeili et al., 2014). jolodar and abdoli (2004) restrict its presence to the anzali wetland and rivers draining into it. anderson (1880) reports perch to be abundant in the lar river near tehran, but this is probably a misunderstanding at this early date. zoogeography: its closest relative is found in north america and they were once thought to be the same species on both continents. habitat: distribution is limited by an inability to survive a temperature of 31°c for more than a few hours, by an inability to tolerate salinities above about 10-12‰ and by avoidance of waters with an oxygen level of less than 3ml l-1. the upper lethal 146 coad/ perches of iran temperature is 33.5°c (collette et al., 1977). fresh water is required for spawning. riazi (1996) reports that this species is native (resident) to the siah keshim protected region of the anzali wetland and it is also reported from swamps near hendeh khaleh in gilan. optimal conditions are large, weed-free, moderately deep, mesotrophic waters with food fish such as rutilus rutilus readily available. turbidity is a limiting factor for this species which depends on sight to feed. it is found only in the lower reaches of rivers along the iranian shore and does not penetrate upstream (berg, 1948-1949). nevertheless it can be found in both running and still water and in both small and large water bodies. perch are a schooling fish, arranged by size and age. schools form in the morning and disperse as dusk falls. schools usually number about 50-200 fish but schools in the thousands are reported. there is a nocturnal resting area and perch move from it to a diurnal active area. the perch may move short distances within a lake and in large water bodies over 90 km but show strong homing tendencies. seasonal movements are between feeding, spawning and overwintering areas. different morphs are found in some areas, depending on habitats: one small, slow-growing, dark and gregarious, feeding on small crustaceans, and found in reed beds, the other large, fast-growing, light and solitary, feeding on fish, and found in open waters. populations in the safid river were supposedly increased after construction of the dam which reduced water flow and raised temperatures. age and growth: maturity in males is usually attained in the second year of life (at only 5-12 cm long) with females maturing 1-2 years later (at 12-18 cm or larger). however some males may mature during their first year or as late as their third. females grow slightly faster than males after the first 1-2 years. eutrophication may reduce the age of first maturity because of increased growth rates. perch in different habitats within the same water body, e.g., weeds beds as opposed to open water, will show different growth rates and body forms. growth over the whole range of the species varies markedly. generally fish at age 2 which are greater than 20 cm total length are characterised as having very good growth, moderate growth would be fish at age 3 greater than 16 cm total length while a very poor growth would be evidenced by all fish in the population being less than 16 cm total length. life span is up to at least 21 years and under artificial conditions up to 27 years, perhaps even 50 years. a maximum age of 11 years is given for a volga delta population examined by makarova (1986). nezami et al. (2004) found fish in the amirkelayeh lagoon or wetland on the caspian coast of iran were in age groups 1+ to 6+, had a total length of 9.5-33.5 cm and a weight of 10.5-350.0 g. heydarnejad (2009) gave the length-weight relationship for an iranian sample as w=0.0145tl3.011. saemi et al. (2012) determined growth rate and maximum length for anzali wetland fish were 0.31 and 21.17 cm in males and 0.33 and 21.0 cm in females, there was no sex ratio variation throughout a year although some months fluctuated, and length-weight relationships were w=0.011 fl3.055 for males and w = 0.024fl2.82 for females. differences with perch from other geographical regions were attributed to lack of appropriate food and the degraded environment in the anzali wetland. vafajooy dianati et al. (2013) examined 265 fish from the amirkelayeh wetland and found fish were age 2+ to 6+ years, age groups 2+ to 3+ with average instantaneous growth rate of 0.461 grew fastest while age 5+ to 6+ with 0.231 grew slowest, males outnumbered females, females had the highest condition factor (2.49) and males the lowest (1.06), and growth rates were almost isometric with a tendency to positive allometric for males, females and all fish. in the anzali wetland, ashja et al. (2010) found females (63%) outnumbered males (37%). vafajo dyanti et al. (2013) found fish in the amirkelayeh and anzali wetlands matured at age 23 years and fish up to 5 years were caught, males having an average total length of 22.5 cm, 158 g and gonad weight 7.15 g. there was no significant 147 int. j. aquat. biol. (2016) 4(3): 143-170 difference in gonadosomatic index between male broods. komsari et al. (2015) determined the von bertalanffy growth functions for 286 anzali wetland fish were lt=21.0(1-e -0.33(t-0.9)) for females and lt= 21.17(1-e -0.31(t-0.8)) for males, the lengthweight relationship was w=0.011tl3.1 for males and w=0.024tl2.8 for females, positive and negative allometry, respectively, there were no differences in sex ratio throughout the year although there was in some months attributed to gathering of spawning males and post-spawning feeding behavior of females, age was 2 to 7 years with a median of 4.6 for females and 4.8 years for males. food: food for small perch is zooplankton such as rotifers, switching to insect larvae, crustaceans, molluscs and leeches with growth (larger than about 20 mm). growth is enhanced if fish and crayfish are available. fish predominate in the diet at a range of sizes between 10 and 25 cm. in the caspian basin, rutilus rutilus, blicca bjoerkna, pungitius platygaster and gobies (gobiidae) are eaten (makarova, 1986). some slow-growing perch may feed on plankton until 2-3 years old. cannibalism is common. maximum feeding levels occur in summer and, by autumn, has fallen to a maintenance level (collette et al., 1977; popova and sytina, 1977). feeding is a daylight and highly visual activity. feeding is more effective in shoals as the perch attempts to seize other fish by the head and, if an individual perch misses, then other members of the shoal have an opportunity to seize the prey. large perch lie in wait for passing prey items and then dart out to seize them. unlike northern pike, perch will pursue a prey item if it tries to escape. in the amirkelayeh lagoon or wetland diet varied according to age, season and sex, and comprised a wide variety of organisms such as water bugs, odonates, gammarids, plant materials, chironomids, tinca tinca, hemipterans, perca fluviatilis, snails, syngnathus caspius, gambusia holbrooki, pungitius platygaster, dipterans, branchiopods, trichopterans, tubifex, frogs and shrimps. the species is an omnivore here and a cannibal (nezami et al., 2004). reproduction: spawning occurs in the spring in shallow water, 0.5-3.0 m deep, end of march to early june in the volga delta (makarova, 1986). in dagestan, spawning is from the end of march to the beginning of april and lasts 10-15 days (shikhshabekov, 1978), elsewhere only 2-3 days. water temperatures are around 11°c on the volga (lönnberg, 1900) and above 8°c in dagestan (shikhshabekov, 1978) but can occur under ice at 4°c. up to 80% of the spawning population in the volga delta is female (makarova, 1986). there is a spawning migration from deepwater resting areas to shallow spawning areas. males precede females onto the spawning ground by days or weeks and remain behind after spawning. brackish-water populations migrate into fresh water. spawning itself can occur by day or by night. absolute fecundity in the anzali wetland reached 35,942.93 eggs and egg diameter reached 42.25 μ (sic) based on 254 fish (ashja et al., 2010) or 18,312.6 for 5-year-old fish from a 60 fish sample (hayatbakhsh et al., 2010). the latter study found maximum average relative fecundity at 122.9 and gonadosomatic index at 16.11 was for three-year-old fish. komsari et al. (2014) looked at the reproduction of 324 anzali wetland fish and found a long vitellogenic process (october to february) and a short spawning season (january and february, about two weeks long), maximum gonadosomatic index was in january for females (15.57) and minimum was in august (0.2) and for males maximum was in january (6.84) and minimum in june (0.04), groupsynchronous ovarian development occurred, ovarian development occurred in only one clutch of oocytes (700-900 µm) with no maturation of any subsequent clutch, the average realized fecundity was 16,177 eggs in the late vitellogenic stage, lower than potential fecundity at 17,188 eggs, and mean relative fecundity was 141.12 eggs/g. although temperature is the major factor affecting the timing of spawning, the occurrence of spring floods is significant in some populations as it gives access to inundation zones of large rivers. eggs are twisted around plants, roots and logs in an egg148 coad/ perches of iran strand, a cylindrical, hollow, twisted structure up to 3.75 m long, 3.8 cm thick and 8 cm wide. this structure offers protection from predators, fungal infections, desiccation, mechanical damage and smothering in the mud. egg diameters reach 2.5 mm and fecundity 300,000 eggs. fecundity increases with age and depends on food supply as in most fish species. as many as 15-25 males queue up to fertilize the egg-strand, following the female as she twists around the logs and plants, rubbing against the plants to void the eggs. the female drives the males away from the egg-strand after fertilisation and may guard the egg-strand for some hours. parasites and predators: mokhayer (1976) records the protozoan trypanosoma percae from this species in the caspian sea basin, the nematode larva eustrongylides excisus and the annelid piscicola geometra. khara et al. (2006) record the eye fluke diplostomum spathaceum for this fish in the amirkelayeh wetland in gilan. sattari et al. (2002) and sattari (2004) records the presence of the nematode, eustrongylides excisus. this parasite can damage muscles in commercial species and render them unsuitable for sale. sattari et al. (2006; 2007) surveyed this species in the anzali and amirkelayeh wetlands, recording raphidascaris acus, eustrongyloides excisus and camallanus lacustris. khara et al. (2005) examined this species in the amirkelayeh wetland and found its diversity of parasites to be less than other predatory species such as esox lucius. parasites recorded were camallanus lacustris, diplostomum spathaceum, lernaea sp., argulus sp., and dactylogyrus sp.. sattari et al. (2007) record the nematode eustrongylides excisus and the digenean diplostomum spathaceum in this species in the anzali wetland of the caspian shore. barzegar et al. (2008) record the digenean eye parasite diplostomum spathaceum from this fish. barzegar and jalali (2009) reviewed crustacean parasites in iran and found achtheres percarum on this species. anvarian et al. (2012) and hamzei et al. (2013) isolated the bacteria lactobacillus fermentum and l. plantarum from aras dam fish intestines using molecular techniques. these bacteria play an important role in producing antimicrobial substances, immune response enhancement and increasing nutrient availability. anvarian et al. (2014) characterised the human pathogen aeromonas hydrophila, which causes gastroenteritis, on the skin and in the intestine of 35% of fish sampled from the aras dam, presumably from sewage contamination. sander lucioperca, esox lucius and lota lota are predators on perch in the caspian basin and doubtless other large fishes and birds take this species. ashoori et al. (2012) found that grey herons (ardea cinerea) in the siah keshim protected area of the anzali wetland ate this species. cannibalism begins as early as the fry stage when fish only 2.1 cm long eat smaller fry. economic importance: holčík and oláh (1992) report a catch of only 15 kg in the anzali wetland in 1990. this species has been studied for aquaculture in iran. rahbar et al. (2011) and hayatbakhsh et al. (2014) surveyed the haematology of anzali wetland perch, useful in assessing, for example, stress levels when handled. rustaiyan et al. (2012) found that muscle tissue of male and female perch is rich in the omega-3 fatty acid docosohexanaenoic acid, important in the human diet, as well as other significant fatty acids. salimi and esmaeili (2012) fed larval perch experimental diets with various protein levels and found growth performance and feed conversion increased with protein level from 246 to 550 g/kg, protein efficiency ratio declined linearly with increased protein, survival was not affected by dietary composition and the protein requirement ranged between 489 and 530 g/kg. ashja ardalan et al. (2009) found differences between fish from the abkenar and sheyjan areas of the anzali wetland in heavy metal content (copper, lead, mercury and zinc) in muscle and liver tissues, indicative of environmental pollution loads at the two localities. baramaki yazdi et al. (2012) found metal concentrations in this species from the anzali wetland in descending order were zn, cu, pb, cr 149 int. j. aquat. biol. (2016) 4(3): 143-170 and cd and all except cr are below levels of concern for humans. robins et al. (1991) list this species as important to north americans. importance is based on its use in aquaria and aquaculture, as food, in sport, in textbooks, in experiments and because it has been widely introduced outside its natural range. it has been implicated in ichthyootoxism (coad, 1979). conservation: illegal fishing and non-standard nets threaten stocks of this species (annual report, 19951996, iranian fisheries research and training organization, tehran, p. 55, 1997). kiabi et al. (1999) consider this species to be vulnerable in the south caspian sea basin according to iucn criteria. criteria include commercial fishing, sport fishing, medium numbers, habitat destruction, limited range (less than 25% of water bodies), absent in other water bodies in iran, and present outside the caspian sea basin. shapoori and gholami (2005) found the fishery not to have changed despite millions of fry being stocked. the iucn red list of threatened species (2015) lists this species as of least concern. sources: general biology is based on thorpe (1977) and craig (1987), and there is extensive european angling literature on this species. further details on collections examined can be found in the museum catalogues. iranian material: cmnfi 1970-0510, 1, 156.8 mm standard length, gilan, golshan river (37º26'n, 49º40'e); cmnfi 1979-0685, 1, 71.5 mm standard length, gilan, safid river around mohsenabad below dehcha (no other locality data); cmnfi 1980-0123, 1, 68.9 mm standard length, gilan, safid river around dehcha above mohsenabad (no other locality data); cmnfi 1980-0127, 1, 152.8 mm standard length, gilan, caspian sea near hasan kiadeh (37º24'n, 49º58'e); cmnfi 1980-0148, 2, 118.2-142.1 mm standard length, gilan, pir bazar roga (37º21'n, 49º33'e); cmnfi 2008-0110, 1, 89.9 mm standard length, gilan, swamp near hendeh khaleh (37º23'n, 49º28'e). genus sander oken, 1817 this genus is found in both north america and eurasia and contains five species. there are three species in the caspian sea basin, two of which are reported from iran. the shahid beheshti hatchery on the safid river breeds the third species, s. volgense (gmelin, 1789), a northern caspian sea species, according to raymakers (2002) but it is unclear if these have been released and have established populations in the southern caspian sea. the genera stizostedion rafinesque, 1820 and lucioperca schinz in cuvier, 1822 are junior synonyms of sander (see kottelat 1997). the pikeperches are elongate and compressed, have large jaws reaching back beyond mid-eye level, canine teeth on the jaws and palatines, the preopercle is figure 3. line drawing of sander lucioperca by s. laurie-bourque. 150 coad/ perches of iran serrated posteriorly and has spines ventrally, the opercle has a weak, flat spine at its postero-dorsal corner, cheeks are naked or scaled only dorsally, there are 7-8 branchiostegal rays, adult gill rakers are densely denticulated tubercles, in young denticulated rods, and the lateral line continues onto the caudal fin with accessory lateral lines on the upper and lower caudal lobes. divergence between north american and eurasian members of this genus may have occurred in the middle to late miocene or in the pliocene (billington et al., 1990, 1991; faber and stepien, 1998). sander lucioperca (linnaeus, 1758) (figs. 3-4.) common names: suf, suf-e ma'muli or soof-emaamooli, meaning common suf, sibey(ak) in gilaki, ? sevideh. this may be the fish referred to as sevideh from the langarud mordab (=wetland) by holmes (1845). however, the word may be sepideh which means "the white one" and may in fact refer to rutilus kutum, the safid mahi. [sif, caysifi or quaysifi in azerbaijanian; adaty silebalyk in turkmenian; sudak in russian; pike-perch, european pikeperch, zander]. systematics: perca lucioperca was originally described from european lakes, no types are known. akbarzadeh et al. (2007, 2009, 2009) identified three distinct populations in the southern caspian sea and aras dam using a truss analysis with morphometric characters (body heights and caudal peduncle measures) showing better discrimination than meristic characters. gharibkhani et al. (2009, 2014) examined the population genetic structure of fish from the talesh coast, anzali wetland, aras dam and chaboksar coast and found them to be genetically differentiated in spite of low genetic variation, with consequences for conservation. the greatest genetic distance was between anzali and aras populations. gharibkhani et al. (2011) used microsatellite markers to study the genetic diversity of fish from the anzali wetland and found it to be low, attributing this to artificial propagation of the species. mirza ahamdi et al. (2015) used microsatellite markers to examine genetic structure and variation in fish from aras dam lake and the anzali wetland, finding significant genetic differentiation. key characters: this species is separated from s. marinum by the dorsal fins being close together, obviously much less than the eye diameter apart, the anal fin spines are not closely joined to the soft rays, the interorbital width is less than, or equal to, the vertical eye diameter in adults, the upper jaw extends rearwards on a level behind the posterior eye margin in adults (under the rear of the eye in young), there are usually more than 18 soft rays in the dorsal fin, and the spiny dorsal fin bears large spots. morphology: lateral line scales 75-150 (this wide range from various literature sources presumably includes counts of scales in the lateral line, and other counts which are of smaller scales to one side of the lateral line; berg (1948-1949) gives a count of 80-97 figure 4. sander lucioperca, aras reservoir, march 2012, courtesy of k. abbasi. 151 int. j. aquat. biol. (2016) 4(3): 143-170 for example, which is inherently more reasonable). scales above the lateral line 12-17 and scales below the lateral line 16-24. scales are incised on the anterior margin where about 6-9 radii terminate, although not as incised as in perca fluviatilis. the focus is posterior. ctenii are well-developed. first dorsal fin spines 11-17; second dorsal spines 1-4 and soft rays 16-27, usually 19-24. anal fin spines 1-3, soft rays 9-14. pectoral fin branched rays 11-18 and pelvic fin branched rays 5. stubby, spinulose gill rakers number 10-17 and vertebrae 40-48, usually 45-48, mode 46. there are 4-9 pyloric caeca. the gut is relatively short but has a long anterior loop. the chromosome number is 2n=48 (klinkhardt et al., 1995; miri nargesi et al., 2007) although the latter authors found a different karyotype formula in iranian fish compared to other populations (2n=1m+13sm+4st+6a). meristic values for iranian specimens are:lateral line scales 82(1), 86(1), 89(1), 90(1), 91(3); scales above the lateral line 13(1), 14(4), 15(1); scales below the lateral line 21(2), 22(1), 24(2), 25(1); caudal peduncle scales 33(3), 34(3); first dorsal fin spines 13(3), 14(4); second dorsal soft rays 20(2), 21(2), 22(1), 23(2); anal fin soft rays 11(4), 12(1) 13(2); pectoral fin branched rays 13(2), 14(2), 15(3); pelvic fin branched rays 5(7); and vertebrae 45(3) or 46(3). sexual dimorphism: females have a much lighter, whitish belly than that of males which is marbled blueish in the spawning season. the genital papilla of females protrudes. colour: the back and flanks are green to blue-grey to brown-black, the belly is white to bluish and fins are yellow-grey. the dorsal and caudal fins have rows of black spots on the membranes, largest and most distinctive on the spiny dorsal fin. other fins are pale yellow. the eye is silvery because of reflection from the tapetum lucidum. young have 813 brown to blackish-brown bars but these usually fade with maturity. the peritoneum is silvery to brownish in preserved fish. size: up to 1.5 m and 20.0 kg. the iranian commercial catch in the 1950s was 24-60 cm long and weighed 1.6-2.7 kg, declining to 0.6 kg (faridpak, no date). distribution: found in the basins of the baltic, aegean, black, caspian and aral seas. in iran, reported in the aras river (and aras dam lake) and along the whole caspian coastal plain from astara to the atrak river including the talesh and chaboksar coasts, amirkelayeh wetland near lahijan, gomishan marshes or wetland, gorgan bay, anzali wetland and siah keshim protected region, the babol, gorgan, haraz, harisak, langarud, marbureh, nek, qareh su, rasteh, safid and shahrud rivers, as well as in the southeast caspian sea, southwest caspian sea and south-central caspian sea (nedoshivin and iljin, 1929; derzhavin, 1934; kozhin, 1957; griffiths et al., 1972; holčík and oláh, 1992; nejatsanatee, 1994; riazi, 1996; karimpour, 1998; abbasi et al., 1999; kiabi et al., 1999; abdoli, 2000; ghasempouri and esmaili sari, 2002; jolodar and abdoli, 2004; abdoli and naderi, 2009; gharibkhani et al., 2009, 2014; tabatabaie et al., 2011; esmaeili et al., 2014). this species has also been stocked in the zarreinerud, 70 km upstream of miandoab in 1971 (griffiths et al., 1972) and in the talkheh river, both in the lake urmia basin, in the valasht lake near marzanabad, evan lake northeast of qazvin, ghorigol lake near tabriz, marivan lake in kordestan, the chamzarivar river in the tigris river basin, and the haft barm lakes west of shiraz (anonymous, 1977). teimori et al. (2010) record it as introduced in the kor river basin of fars. although this species has been introduced to the manjil reservoir on the safid river (nümann, 1966; griffiths et al., 1972), this reservoir is drained to remove excess silt and no fishery exists (j. holčík, pers. comm. 1992). nümann (1966) recommended introduction of s. lucioperca to the dez dam and the karaj reservoir. however, introductions to reservoirs in khuzestan did not survive (m. almukhtar, pers. comm. 1995). cirspe (2006) lists s. lucioperca as being present in sistan but this may be an error. it is also found in the karakum canal and 152 coad/ perches of iran kopetdag reservoir of turkmenistan (sal'nikov, 1995) and may eventually reach the iranian tedzhen (=hari) river basin. zoogeography: the relationships of this species are discussed under the genus. habitat: generally this species is found in small schools near sandy and stony bottoms in deeper water of rivers. ideally there should be some concealment. it can also live in reservoirs. this species has both freshwater and semi-anadromous forms in the caspian sea basin. it has a limited migratory behaviour such that morphologically distinct stocks may exist in larger water bodies. the population of the anzali wetland represents a separate stock (hydrorybproject, 1965). riazi (1996) and karimpour (1998) report that this species migrates into the siah keshim protected region of the anzali wetland and is also resident there. most movements of this species are within 10-20 km although distances up to 300 km have been recorded in the volga river. suf show strong homing tendencies. the upper lethal temperature is 35°c. high turbidity levels are preferred. during the day, suf shelter from strong light by descending in the water column (collette et al., 1977; marshall, 1977). knipovich (1921) reports this species from depths of 11.0-11.9 m, possibly deeper, in the iranian caspian sea. in dagestan, this species prefers areas where there is flowing water well-supplied with oxygen (shikhshabekov, 1978) and avoids vegetation and therefore competition with esox lucius. suf will tolerate low salinities and can be found around river mouths in the caspian sea basin but the sea itself is too saline. ahmadnezhad et al. (2012) found that 1 g fingerlings can tolerate up to 12‰ salinity after a 10 day acclimation period. ahmadnezhad et al. (2014) showed development of an osmoregulatory function in the larval gill is not complete until the eleventh day and skin ionocytes are responsible for this function until then, useful data for optimising release of larvae in stocking. ahmadnezhad et al. (2014) found 2 g fingerlings tolerated salinity better than 1 g fingerlings but even the latter survived 12‰ caspian sea water and could be used for restocking. abstraction of water for irrigation (60% of water use) has severely reduced water levels and runoff rates necessary for reproduction of fishes along the caspian shore. estuarine habitats have been degraded inhibiting the survival of eggs, larvae and juveniles of anadromous and semi-anadromous fishes (the latter are species which spawn in the lower stretches and deltas of rivers where salinity is optimal at 8 g l-1 for many commercial species such as the suf). over 90% of coastal streams along the caspian shore are dry in july in iran because of irrigation demands. as a result larvae of spring spawners are flushed into fields where they die, and nursery and reproductive areas are confined because of their low tolerance to salinities above 7-8‰. without an adequate runoff, the sea encroaches on the estuary. age and growth: males mature at 2-6 years (32 cm) and females at 3-6 years (42-44 cm). life span may exceed 19 years, although in lake eğirdir, turkey only 7 age groups were recorded (becer and i̇kiz 1999a). optimum growth occurs at 28-30°c (marshall, 1977). during 1932-1933 in the anzali wetland, 5-7 year old fish dominated in catches and weighed 2.67.4 kg but by the 1960s this had declined to 2-5 years and 1.6-2.7 kg (hydrorybproject, 1965). catches in 1971/72 in the commercial fishery of iran were 3-7 years old, 33.0-55.0 cm long and weighed 370-2,100 g (razivi et al., 1972). abdolmalaki (2005) found age groups in the iranian caspian to be 2-5 years with 2-3 year olds forming 78.5% of the catch. the von bertalanffy growth equation was lt=52.5*[1exp-0.158*(t+1.852)]. the instantaneous rate of total, natural and fishing mortality was 0.95 year-1, 0.31 year-1 and 0.64 year-1, respectively. the calculated exploitation ratio was 0.67, the estimated biomass was 31.56 tons, the minimum sustainable yield was 13.89 tons (lower than the total catch), and the fishery return coefficient was 2.87%. abdolmalaki and psuty (2007) report 6 age groups for coastal waters of the southern caspian sea, the length-weight relationship was w=-0.020606l2.85, and the von bertalanffy parameters were l∞=55.05 153 int. j. aquat. biol. (2016) 4(3): 143-170 cm fork length (substantially less than in the volga river delta at 79.0 cm and aras lake in iran at 73.3 cm), k=0.15, t0=-2.59 and m=0.31. more than 90% of the beach-seine caught fish were smaller than the minimum legal length. these authors also provide details of recruitment and fishing mortality for this population which is enhanced by introduction of fingerlings. rahimibashar et al. (2008) found that the fish in the aras dam lake had allometric growth and age classes caught with cast nets and gill nets were 2+ to 5+ years. food: the suf is an ambush-pursuit predator. feeding on fish begins at a length of 5-10 cm (2-3 months of age) depending on the relative abundance of zooplankton, invertebrates and forage fish. in the volga delta, spawning rutilus rutilus (presumably r. caspicus) in april-may is the most important food, up to 80% of the annual ration. in the 1960s and 1970s when the population of suf was 7 million fish, they ate 53,000 tonnes of rutilus rutilus (presumably r. caspicus) (caspian sea biodiversity database, www.caspianenvironment.org). adults are solitary but young fish feed in schools on nauplii, copepods and some rotifers. some adults are cannibals (collette et al., 1977; marshall, 1977; popova and sytina, 1977) and balik (1999) reports that in lake beyşehir, turkey, a suf can eat its own species with a mean size of 35.9% of its length. apparently many prey fish are seized and swallowed tail first. one iranian specimen contained a neogobius melanostomus and gobies are an important food item generally in the caspian sea, 17.8% of the diet compared to 59.9% for rutilus rutilus (presumably r. caspicus). rahimibashar et al. (2008) found that the fish in the aras dam lake were carnivorous, almost gluttonous, feeding principally on bony fishes (92% food preference) with some crab larvae and aquatic insects. reproduction: the spawning migration begins in late march-early april in dagestan with spawning in early to mid-april (shikhshabekov, 1978). in eğirdir lake, turkey spawning took place from april to june and becer and i̇kiz (1999b) give details of fecundity, egg diameters, and the relationships between length, weight and gonad weight and fecundity for fish that mature as young as ages 1-2. the spawning season over the range of this species is late february to late july, usually april-may at 12°c (range 6-22°c) as deep as 17 m. there are distinct spawning stocks. in the anzali wetland, the main spawning area in the southern caspian, the spawning run usually starts in the first 10 days of march at water temperatures of 8.0-9.5°c, ending at 12-14°c (hydrorybproject, 1965; razivi et al., 1972). apparently, natural spawning has stopped completely in the wetland and this lagoon is stocked with fingerlings from spawners held at aras, a border reservoir lying between iran and azerbaijan (abdolmalaki and psuty, 2007). males build nests in depths of 30 cm to several metres on hard bottoms usually in turbid water. each nest is a flat pit edged by gravel or shell. plant roots are often exposed as a spawning substrate on which eggs are laid individually. the nest is guarded by the male and eggs are fanned. the female is driven away after spawning. male suf are so devoted to protecting the nest that they will remain on site even if water levels fall and their backs stick out of the water. in addition they will try to bite humans if they approach the nest. spawning is intermittent over several days and usually takes place at dawn. maximum fecundity is 2.5 million eggs and egg diameters are up to 1.5 mm. hatching occurs from 4 to 26.5 days, depending on temperature (collette et al., 1977; marshall, 1977). females descend to the sea first from the anzali wetland after spawning and fry there are 19-33 mm long by the end of may (hydrorybproject, 1965). parasites and predators: eslami and mokhayer (1977) examined 100 specimens of suf and found 20% to be infested with larvae of the nematode anisakis. ataee and eslami (1999, www.mondialvet99.com accessed 31 may 2000) report the helminth anisakis from the gastro-intestinal tract of fish from the anzali wetland. this parasite can infest man if fish is eaten smoked, salted or fried at temperatures below 50°c. mokhayer (1976) records the 154 coad/ perches of iran acanthocephalan corynosoma caspicum. jalali and molnár (1990) record the monogenean ancyrocephalus paradoxus from this species in the safid river. masoumian et al. (2005) recorded the protozoan parasite trichodina perforata from this species in the aras dam in west azarbayjan. pazooki et al. (2007) and rasouli (2013) recorded various parasites from localities in west azarbayjan province, including diplostomum spathaceum and argulus foliaceus from this species. barzegar et al. (2008) record the digenean eye parasite diplostomum spathaceum from this fish. barzegar and jalali (2009) reviewed crustacean parasites in iran and found achtheres percarum on this species. movahed et al. (2009, 2012, 2014, 2015) examined fish from the anzali wetland and found eustrongylides excisus (nematode), achtheres percarum, dactylogyrus sp. and diplostomum spathaceum (platyhelminths) and trichodina sp. (ciliophore). white blood cell and lymphocyte counts increased in parasitised fish, a defense mechanism. the bacterium clostridium botulinum is present in fish from coastal areas of northern iran and is a potential food hazard (botulism) if preservation is inadequate. contamination rate was 10% in (tavakoli and razavilar, 2003; tavakoli and tabatabei, 2005). the bacterium listeria monocytogenes and other listeria species were isolated from 9.7% of suf obtained in urmia fish markets, the lowest prevalence of seven species examined. nonetheless, listeriosis is a foodborne disease with a high fatality rate in humans. the caspian seal, pusa caspica, is a significant predator on this species (krylov, 1984) as are predatory fishes such as esox lucius, perca fluviatilis and silurus glanis. adult suf have few predators. economic importance: there is some opportunity for sport fishing for this species in the anzali wetland and potentially in various lakes around the country where it has been introduced (anonymous, 1977). it is a very popular food fish in iran (razivi et al. 1972) and one of the most important commercial bony fishes (karimpour et al., 2013) although this has declined over the 1927-2013 period (fazli, 2013; fazli et al., 2013). it has also been studied in iran as a control species for undesirable fishes (annual report, 1995-1996, iranian fisheries research and training organization, tehran, p. 80, 1997). nevraev (1929) reports on catches in various regions of iran in the early years of the twentieth century. there were no evident trends of increase or decrease. in the astara region from 1901-1902 to 1913-1914, the catch varied irregularly from 154 to 31,931 fish, in the anzali region from 1901-1902 to 1918-1919, the catch varied from 608,300 to 3,367,000 fish, in the safid river region from 18991900 to 1917-1918, the catch varied from 9,983 to 125,182 fish, and in the astrabad region from 19001901 to 1912-1913, the catch varied from 1,400 to 22,900 fish. stocks of this species are known to fluctuate in iran, as obviously do the catch statistics. most fish are caught in beach seines, although some are caught in gillnets, both legally and illegally (see below). the main fishing ground is coastal waters in the anzali region. catches in the 1920s were at 3,000-4,000 tonnes for the coastal zone of the southern caspian sea but declined drastically afterward (razavi, 1999). the commercial catch in iran from 1956/1957 to 1961/1962 varied from 206 kg to 20,945 kg (vladykov, 1964; ralonde and walczak, 1972), from 1965/66 to 1968/69 it varied from 7 to 77 tonnes (andersskog, 1970) and from 1963 to 1967 ranged from 0 to 14.6 t (ralonde and walczak, 1970). in the 6 years from 1980 to 1985 catches were recorded by the food and agriculture organization, rome as respectively 0, 0, 0, 12, 13 and 10 t. catches in 1990 were about 5-10 t and in 1996 about 35-40 t (bartley and rana, 1998b). in 2000-2001, the catch was 18 t or 11% of the total commercial catch in the iranian caspian sea basin. twelve tonnes were caught by beach seine along the coast, 3 t were taken in the anzali wetland and the rest was an estimated amount of unlicensed captures (abdolmalaki, 2005). in 2003-2004, the catch was 38 t, a decrease in comparison to the previous year, with 15 t of this from beach seine cooperatives. most fish were 155 int. j. aquat. biol. (2016) 4(3): 143-170 immature and undersized and the catch was based on release of fingerlings (abdolmalaki, 2006). there are minimum sizes for fish retention, e.g., 34 cm fork length for sander lucioperca, but fisheries do retain smaller ones for home consumption or even marketing (abdolmalaki and psuty, 2007). the caspian sea off the atrak river was an important fishery economic zone and included sander marinum. however, by 1972 the catch of commercially important species had declined to 1.5% and the less desirable kilkas (clupeonella spp.) had increased to 98.3% of the catch. the causes were reduction in the atrak runoff through irrigation withdrawals, pollution from agriculture, overfishing in the sea and the drop in sea level. flows of the atrak did not reach the sea in 1984, 1986, 1990 and 1991 and spawning of species using the lower reaches did not occur (caspian environmental programme, 2000). summaries of catches of this species in the coastal southern caspian sea over 8 decadal periods is given in abdolmalaki and psuty (2007) (table 1). in addition, recent catches of this species is shown in table 2 (abdolmalaki and psuty, 2007). in the period from 1933/34 to 1961/62 in the bandar-e anzali region catches varied from about 3,483 t at the earlier date to 33 kg at the later one, with large variations between years. holčík and oláh (1992) report a catch of only 22 kg in the anzali wetland in 1990, and from 1932-1964 reported catches varied from 1 to 2,581 t annually. hedayatifard and jamali (2008) showed that this fish is a good source of polyunsaturated fatty acids and one of the best sources for omega-3 fatty acids, useful in preventing cardiovascular disease. khaval (2007) investigated polyculture of this species with silver, bighead, grass and common carp at the safidrud fisheries research station. carp density was 3,000 fish per hectare with 60% silver carp, 20% grass carp and 10% each for common and bighead carp. suf weighed 2.1 g and were stocked at 250 fish per hectare. survival rates were 93.33% for the suf and 83.77% for the chinese carps. over the period april to november, suf fingerlings reached 54.4 g on average and production was 4,446.66 kg per hectare compared to 3,212.8 kg without polyculture. robins et al. (1991) list this species as important to north americans. importance is based on its use in aquaria and aquaculture, as food, in sport, in textbooks, and because it has been widely introduced outside its natural range. the suf or pike-perch is catch and frequency 1927-1936 1937-1946 1947-1956 1957-1966 1967-1976 1977-1986 1987-1996 1997-2003 total recorded catch (t) 8,959 7,224 4,986 3,262 5,547 5,384 16,903 16,201 sander lucioperca (%) 29.7 1.7 1.0 0.2 0.4 0.1 0.1 0.2 table 1. summaries of catches of sander lucioperca in the coastal southern caspian sea (abdolmalaki and psuty, 2007). year total catch (t) catch by beach seine (t) number of beach seine cooperatives beach seine efforts (hauls) number of illegal gillnets confiscated 1990 4.0 4.0 68 20,975 no data 1991 12.3 12.3 81 27,200 104,828 1992 10.0 8.7 88 30,239 109,446 1993 12.3 7.3 93 33,986 138,026 1994 40.2 22.6 91 27,868 215,381 1995 10.1 4.0 101 34,055 204,831 1996 8.0 2.8 109 42,847 270,727 1997 8.1 2.9 111 45,263 205,999 1998 95.0 54.8 125 52,574 222,897 1999 17.5 11.5 139 50,953 130,849 2000 18.0 12.0 147 56,913 82,678 2001 26.0 21.5 150 60,006 113,729 2002 30.0 20.3 150 57,310 141,506 2003 23.8 15.0 151 53,846 179,656 2004 22.5 14.4 151 49,809 261,875 table 2. recent catches of sander lucioperca in the coastal southern caspian sea (abdolmalaki and psuty, 2007). 156 coad/ perches of iran also fish-farmed in europe for stocking purposes as eggs or young. conservation: the decline in catches from about 3,000-4,000 tonnes to about 30 t noted above has never been adequately explained. overfishing, degradation of spawning grounds, fluctuating water levels and salinity variations in coastal waters, the volume of freshwater input, oxygen concentrations and input of organic material to estuaries and the sea, are all possible factors (abdolmalaki and psuty, 2007). hydrocarbon pollution occurs in the gomishan marsh (ghasempouri and esmaili sari, 2002). the stocks of this species in iranian waters also declined in the 1960s (walczak and ralonde, 1970; ralonde and walczak, 1972). causes were reduction in estuarine habitat needed for spawning, man-made habitat changes and overfishing of the younger age classes and first year spawners. the catch declined from 125 tonnes annually in the 1940s to 14.6 t in 1967. vladykov (1964) considered stocks all along the iranian shore to be at dangerously low levels. griffiths et al. (1972) suggested that stocks in iran were on the verge of extinction and recommended a three-year ban on catching this species. artificial raising of this species is difficult but more than 6 million fingerlings were raised and released into the anzali wetland in the iranian year 1991-1992, a 100% increase over the previous year (abzeeyan 4(2): vi, 1993). fingerling production rose from 0.12 million in 1990, to 1.50 million in 1991 and 2.50 million in 1992 (emadi, 1993) (note that matinfar and nikouyan (1995) give 1.63 and 2.44 million fingerlings for 1991 and 1992). fingerling production in 1995 was 2.269 million, in 1996 2.4 million and for 1996-1997 8 million (bartley and rana, 1998a; 1998b). the sturgeon international research institute, which opened in 1994 near rasht, released 5-8 million fry in 19961997 (bartley and rana, 1998b). the caspian environment programme (1998) gives annual production (in thousands) of cultured suf in government and private hatcheries as follows: 118 (1990), 1,630 (1991), 2,443 (1992), 1,160 (1993), 2,888 (1994), 2,270 (1995) and 2,414 (1996). karimpour et al. (2013) give production of fish fry and fingerlings in millions as 2.4 (in 1997), 3.8 (1998), 3.6 (1999), 4.3 (2000), 3.9 (2001), 7.4 (2002), 5.6 (2003), 11.0 (2004), 7.5 (2005), 13.5 (2006), 12.9 (2007) and 16.0 (2008). there is some variation in statistics from different sources. the release in 1999 numbered 5 million "juveniles" (i.f.r.o. newsletter, 23:4, 2000). billard and cosson (2002) cite an annual production of 5-10 million, mostly released in the anzali wetland and moghaddam (2006) gives a figure of 5.13 million fingerlings for 2002. the highest number of fingerlings released in the anzali wetland was 6,604,000 in 2003 with the lowest being 1,160,000 in 1993 for the period 1991-2003 according to abdolmalaki and psuty (2007). the latter authors also note that the beach seines used in iran do not protect young fish. there is a heavy mortality of discarded fish even when legal landing size is enforced and resources are inadequate to manage the fishery effectively. the minimum mesh size of the cod-end of the seines should be increased and its use monitored. ramin (1996) has studied semi-artificial propagation and rearing of fry of this species in iran. broodstock spawning occurs in march-april at 1214°c on artificial nests of green wool bunches on wooden frames placed in ponds at 5 m intervals. nests close to the bottom are preferred and eggs are dropped on them with an average fertilization of 3090%. the nests with eggs on them are kept in a mist chamber and the eggs collected and placed in jars. eyed eggs appear on day 3 or 4 of incubation. yolksac absorption lasts 9-13 days and exogenous feeding fry measure 4-6 mm. golmoradizadeh et al. (2011) and ershad langeroudi et al. (2012) injected fish with the hormones cpe (carp pituitary extract), hcg (human chorionic gonadotropin), lhrha2 (luteinizng hormone-releasing hormone analogue) and saline as a control, recommending the first two in induction of artificial reproduction in this species, with different dosages for males and females. ghafouri salah et al. (2008) studied physiological 157 int. j. aquat. biol. (2016) 4(3): 143-170 stress in this species and its effects on cortisol and muscle compounds. falahatkar et al. (2012) and sarameh et al. (2013) showed this species to be sensitive to stress during transportation as indicated by blood parameters. long-term exposure to stressors led to the fish becoming adapted to stressful conditions. sarameh et al. (2012) found that broodstock spawning performance could be improved considerably by using an effective photoperiod (24h light:0h darkness spawned earlier for example), and handling stress can lead to poor reproductive performance and inhibition of spawning. falahatkar et al. (2013) tracked changes in biochemistry, sex steroids and haematology pre and post-spawning, relating them to stress and reproduction. movahed et al. (2011) described haematological parameters of fish caught on the bandar anzali coast, noting no differences between age groups. haematology parameters are useful in assessing stress when the fish are handled and the effects of pollutants and parasites. azimirad et al. (2012) investigated the best levels of the amino acid betaine supplementation in the diet of larvae and found no effect on growth or survival rate. zakipour rahimabadi et al. (2012) examined the effects of different levels of dietary betaine and found that it can increase palatability and acceptability of food and thus could be used to wean fingerlings to an artificial diet. a treatment of 2% betaine with biomar (a fish feed) showed the highest increment in body weight, specific growth rate and food efficiency but a decrease in food conversion ratio and greatest cannibalism compared to other treatments, and the highest survival rate was with biomar and no betaine. mansouri taee et al. (2012, 2012) studied the optimal transition from live to artificial food of larvae. the best growth and highest survival rate were in larvae fed live food (zooplankton and artemia larvae) only for 34 days post-hatching, although fish weaned at 28 days posthatching had noticeable growth performance. rasooli karegar et al. (2014) examined density and food regime in larval culture, finding fish fed with daphnia at a density of 25 l-1 had the highest survival rate, for example, and a larger food size (daphnia rather than artemia) and a mixture of the two with a density of 50 l-1 from the third week of culture led to better growth. sharifpour et al. (2011) detailed damage to various organs in fish from the caspian sea of iran, suspected to be due to long-term exposure to toxic materials and considered the fish unsuitable for human consumption. tabatabaie et al. (2011) found mercury levels in muscle tissue of 0.06 µg/g in anzali wetland fish and 0.15 µg/g in gomishan wetland fish, less than in cyprinus carpio. nabavi et al. (2012) showed that caspian sea samples had levels of cadmium, lead and nickel above acceptable limits while essential metals (copper, iron, manganese and zinc) were below those limits. ehsani et al. (2012) examined biogenic amines (which cause food intoxication) and bacteria in ungutted fish during 2 days prestorage icing and 90 days frozen storage at -24ºc, finding all values were within acceptable limits. sahari et al. (2014) found losses in vitamins a, c, d, e and k during cold storage (-24ºc). lelek (1987) classifies this species as intermediate to vulnerable in europe. kiabi et al. (1999) consider this species to be vulnerable in the south caspian sea basin according to iucn criteria. criteria include commercial fishing, sport fishing, few in numbers, limited range (less than 25% of water bodies), absent in other water bodies in iran, and present outside the caspian sea basin. nezami et al. (2000) consider this species to be endangered because of overfishing, habitat destruction and spawning ground degradation. the iucn red list of threatened species (2015) lists this species as of least concern. sources: deelder and willemsen (1964) reviewed the biology of this species as did craig (1987). robins (1970) gave a bibliography of the genus stizostedion (=sander). further details on collections examined can be found in the museum catalogues. iranian material: cmnfi 1970-0532, 1, 209.3 mm 158 coad/ perches of iran standard length, gilan, caspian sea near bandar-e anzali (37º28'n, 49º27'e); cmnfi 1979-0431, 2, 211.5-241.0 mm standard length, mazandaran, now shahr fish bazaar (no other locality data); cmnfi 1979-0455, 1, 68.5 mm standard length, markazi, manjil dam (36º45'n, 49º17'e); cmnfi 1980-0127, 1, 266.1 mm standard length, gilan, caspian sea near hasan kiadeh (37º24'n, 49º58'e); cmnfi 1980-0130, 1, 197.4 mm standard length, mazandaran, river near iz deh (36º36'n, 52º07'e); cmnfi 1980-0150, 1, 280.8 mm standard length, gilan, caspian sea at safid river estuary (37º24'n, 49º58'e). sander marinus (cuvier, 1828) (figs. 5-6) common names: suf-e daryai (=sea suf), souf-edaryaee. [daniz sifi in azerbaijan; morskoi sudak or sea pike-perch, erroneously bërsh by fishermen in the caspian sea, both in russian; sea zander, estuarine perch]. systematics: lucioperca marina was originally described from the black sea at feodosiya. no types are known (eschmeyer et al., 1996). key characters: this species is distinguished from s. lucioperca by the dorsal fins being well-separated, but by a distance less than eye diameter, anal fin spines are weak and closely joined to the soft rays, interorbital width in adults is much greater than eye diameter, the upper jaw extends back level with the posterior pupil edge or almost to the posterior eye edge in adults, the dorsal fin soft rays are 18 or less, and the spiny dorsal fin lacks large spots. morphology: first dorsal fin spines 12-14, second dorsal fin spines 1-4 followed by 12-18 soft rays, anal fin spines 2-4 followed by 9-12 soft rays, pectoral fin with 1 spine and usually 15 soft rays, and pelvic fin with 1 spine and 5 soft rays. lateral line figure 5. line drawing of sander marinus by s. laurie-bourque. figure 6. sander marinus, anzali shore, may 2006, courtesy of k. abbasi. 159 int. j. aquat. biol. (2016) 4(3): 143-170 scales 75-88, scales above the lateral line 9-13 and caudal peduncle scales 34-36. scales have few anterior radii, a crenulate anterior margin and a posterior focus. gill rakers on the upper arch number 12-20, on the lower arch 11-17, reaching the second raker below when appressed, elongate and spinulose. vertebrae number 38-44. there are 5-7 pyloric caeca. cheeks are scaleless or almost scaleless. there are canines on the jaws and palatines. these counts are combined for several literature sources and include eastern and western populations of the southern caspian sea which show evident differences indicating distinct stocks. the gut is short and s-shaped. meristic values for iranian specimens are:first dorsal fin spines 13(2) or 14(1), second dorsal fin spines 2 (3), soft rays 16 (3); anal fin spines 2(3), soft rays 12(3); soft pectoral rays 15(3), pelvic fin with 1(3) spine and 5(3) soft rays, lateral line scales 79(2) or 82(1), scales above lateral line 10(1), 12(1) or 13(1), caudal peduncle scales 34(2) or 36(1); and total gill rakers 12(1), 13(1) or 15(1). sexual dimorphism: unknown. colour: the back is light grey and the flanks have 12-15 dark bars, which are sometimes indistinct and may be absent, e.g., in females from the eastern caspian which had small, irregular, dark-brownish speckles. some fish are almost black and lack bars. the first dorsal fin lacks the strong spots seen in sander lucioperca and is dark grey to black with patches of concentrated melanophores and clearer areas forming irregular and incomplete stripes, or darkly fringed and with a dark spot at the posterior base. the second dorsal fin and the caudal are finely spotted. other fins are grey with some melanophores on rays. eyes are silvery due to the tapetum lucidum. the peritoneum is brownish. size: attains 62.0 cm (65.0 cm total length in jolodar and abdoli (2004)) and 2.2 kg. distribution: found only in the northwestern black sea and the caspian sea. reported from near gasankuli in turkmenistan (berg, 1948-1949) and from the southeast caspian sea in iran (kiabi et al., 1999). jolodar and abdoli (2004) and abdoli and naderi (2009) record it from the central, southwestern and southeastern regions of the caspian sea including in astara. k. abbasi records it from the anzali shore (see photograph above). an old record from the anzali wetland is cited below under habitat. zoogeography: the relationships of this species are discussed under the genus. habitat: this species lives in the caspian sea proper and rarely enters rivers. de filippi (1865), however, did record "un molto bello e grosso individuo....in un canale di murdab, ove l'aqua era del pari sensibilmente dolce". it favours areas with rocky bottoms and does not migrate. in winter, part of the population moves into deeper water at depths of 30 m, rarely 100 m, while another part remains near the shore. the major concentration of this pike-perch is found near the shores of turkmenistan, and secondly of azerbaijan. the caspian sea off the atrak river is an important fishery economic zone. gasan-kuli or hasan kuli is a town in turkmenistan near the iranian border referred to in fishery reports from this area. the catch of rutilus caspicus, cyprinus carpio and sander marinum was nearly 1.44x104 tonnes with only 1.9% being accounted for by clupeonella cultriventris (= caspia). however by 1972 the catch of the commercially important species had declined to 1.5% and the less desirable clupeonella had increased to 5.73x104 t or 98.3% of the catch. the causes were reduction in the atrak runoff through irrigation withdrawals, pollution from agriculture, overfishing in the sea and the drop in sea level. flows of the atrak did not reach the sea in 1984, 1986, 1990 and 1991 and spawning of species using the lower reaches did not occur (caspian environmental programme, 2000). age and growth: sexual maturity is attained at 3-4 years for most fish with a few fish maturing at 2 years (guseva, 1975). life span is at least 12 years. growth is slightly faster in females up to age 5, evens out later and males become larger (kuliyev, 1981). males on the spawning grounds average 41.2 cm and females 42.9 cm, with an average weight of 0.94 kg (berg, 1948-1949). 160 coad/ perches of iran food: the principal foods are gobies (gobiidae), young herring and tyulkas (clupeidae), silversides (atherinidae), and crayfish. reproduction: the male prepares a nest site and protects the eggs. spawning takes place at 3-12 m on open, stony bottoms or in "nest-caves" and eggs are laid in a continuous layer. the male constructs and guards the nest. spawning usually begins in the second half of april and ends in mid-may at temperatures of 10-17°c, and is most intense at 1315°c (guseva, 1974a). up to 126,000 adhesive eggs are laid (guseva, 1974a, 1975; kuliyev, 1981) and are larger than in s. lucioperca. fertilised eggs are 2.6-3.8 mm and at water temperatures of 14.7°c, incubation takes 12-17 days (gusev, 1974a). parasites and predators: none reported from iran. economic importance: the sea pike-perch was commercially fished off the turkmenistan coast in the 1930s and 1940s with catches of 19 thousand centners (1 centner=100 kg) or 2,271,000 fish. in 1927-1929 the annual average on the shores of azerbaijan was 7,000 centners and in 1930-1932 10,300 centners. in 1930, the catch for the whole caspian sea was 909 thousand centners (zenkevitch, 1963). the development of offshore oil deposits has drastically reduced stocks throughout the caspian sea (guseva, 1974b, 1975; kuliyev, 1981). robins et al. (1991) list this species as important to north americans. importance is based on its use in textbooks. conservation: this species has been proposed for inclusion in the "red book of the u.s.s.r." which forms the basis for measures to protect species (pavlov et al., 1985). kiabi et al. (1999) consider this species to be data deficient in the south caspian sea basin according to iucn criteria. criteria include possibly few in numbers, limited range (less than 25% of water bodies), absent in other water bodies in iran, and present outside the caspian sea basin. the iucn red list of threatened species (2015) lists this species as data deficient. sources: morphology based in part on svetovidov and dorofeeva (1963) and kuliyev (1981). further details on collections examined can be found in the museum catalogues. iranian material: cmnfi 1970-0532, 3, 156.3-165.5 mm standard length, gilan, caspian sea near bandar anzali (37º28'n, 49º27'e). acknowledgements i am indebted to the department of biology, shiraz university and the canadian museum of nature, ottawa for funding of research. numerous colleagues and co-authors assisted in developing the website on iranian fishes, providing specimens, data and photographs and are listed at www.briancoad. com. references abbasi k. 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(2018) 6(1): 21-24 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology short communication investigations on the age, growth and mortality parameters of kawakawa, euthynnus affinis (cantor, 1849) from the north west coast of india vinod kumar mudumala1, mahesh kumar farejiya1, kiran s. mali1, karri rama rao*2,1pradnya sawant ankush1, siva anandhan1 1fishery survey of india, facility research centre, plot 2a, unit no. 12, sassoon dock, colaba, mumbai – 400005, india. 2department of zoology, govt. degree and pg college, jammikunta, karimnagar dt. telangana state, india. article history: received 9 october 2017 accepted 23 january 2018 available online 2 5 february 2018 keywords: asymptotic length mortality exploitation ratio length at age abstract: kawakawa, euthynnus affinis contributes to the tune of 35,466 tonnes forming 1% to the total marine fish landings of india. investigations on growth and mortality were of this species carried out based on the length frequency data during 2008-2012. the length-weight relationship for the pooled samples were derived for intercept a=0.0286, slope b=2.857 and coefficient of determination r2=0.917. the values obtained for growth parameters of l∞ was 67.86 cm, for growth coefficient (k) 0.70 and for to -0.26y-1. the natural, total and fishing mortalities recorded as 0.86, 1.48 and 0.62 y-1, respectively. the exploitation ratio (f/z) was observed as 0.42. the m/k ratio was estimated at 1.23. the average fork length of 46 cm and weight of 2024 g were estimated in the samples collected. the length at the end of 1st year to 5th year was observed to be 33, 52, 64, 70 and 75 cm, respectively. the fishing mortality derived in this study is more than the optimum and limit value indicated the sign of overexploitation. introduction among the neritic tunas occurring along the indian coastal waters, the kawakawa, euthynnus affinis is one of the dominant species. though, this species is reported to be distributed in the indo-pacific region (froese and pauly, 2007), india’s contribution was estimated to the tune of 35,466 tonnes forming 1% to the total marine fish landings of india (cmfri, 2017). these estimates are almost closer to the revalidated potential estimates of this species which is 38646 tonnes (moa, 2011). as per fao (2017), the kawakawa contribution in the world oceans was 3,68,978 tonnes during 2015. the parameters of age, growth, and mortality are essential for deriving the strategies for sustainable exploitation. as per the literature, considerable investigations have been carried out by pauline and janaka (1991) on the assessment of this species along the west coast of sri lanka, ahmed et al. (2015) on fishery, bionomics, seasonal elemental variations, health risk assessment and conservational *corresponding author: karri rama rao e-mail address: drkarriramarao@gmail.com management from karachi waters, ahmed et al. (2016) on the growth parameters along the pakistan coast of arabian sea, johnson and tamatamah (2013) from the coastal waters of tanzania, al-zibdah and odat (2007) from the gulf of aqaba, red sea, valeiras et al. (2008) from the western mediterranean sea. in indian waters, these studies are limited rather scanty in recent years except the works of kasim and abdussamad (2003) and khan and zafar (2004) from the maharashtra coast and rohit et al. (2012) from indian waters. further, the studies on population characteristics in the north west coast of india have been found to be inadequate and hence, these investigations are carried out so as to understand and monitor the changes in the population characteristics. materials and methods the specimens of e. affinis were collected from commercial landings at porbandar in gujarat, sassoon dock and ferry wharf, mumbai in maharashtra, during 2008-12. the area chosen for collection of 22 mudumala et al./ age, growth and mortality parameters of kawakawa from the north west coast of india samples in different landings centres occurring on the northwest coast of india is shown in figure 1. a total of 958 specimens with the size ranging 32-64 cm and weight ranging from 0.642-3.70 kg were considered for this study. the formula w=alb was used to calculate the statistical relationship between length and weight. the mathematical model of von bertalanffy equation l(t)=l∞[1-e –k(t–to)] was applied to derive the growth parameters, where, l(t) is the length of fish at time ‘t’, l∞ is the asymptotic length that the fish will achieve, k is the growth coefficient, to is the time at which the fish had zero length. a popular technique of computer analysis of monthly length frequency distributions. fisat-ii software programme developed by gayanilo et al. (2005) was used for estimation of growth parameters. the data was grouped on monthly intervals and used for the estimation of age and growth parameters by adopting model progression analysis (mpa). the total mortality (z) was estimated using beverton and holt model (1956). the growth performance index was drawn through the phi prime test (⏀) by applying the equation lnk+2*lnl∞ to evaluate the reliability the equation. results length weight relationships: the mean fork length of 46 cm and weight of 2024 g were observed in the samples collected for the pooled data during 20082012. the length weight relationship was estimated as w=2.860x10-2fl2.8577 (r2: 0.92). the value of ‘b’ is 2.86 which is close to the value of 3 indicating isometric growth. slight difference in value of ‘b’ in the present case could be more related to the absence of younger juvenile specimens. the coefficient of determination‘r2’ value is 0.92 which is indicative of high degree of correlation and better fit of length weight relationship. a graphical representation of length-weight relationship of e. affinis is given in figure 2. growth parameters and mortality: the growth parameters l∞, k value and to were estimated as 67.86 cm, 0.70 and -0.26, respectively (fig. 3). the growth performance index was derived based on growth parameters in the present study, the phi prime test (⏀) developed by munro and pauly (1983) and pauly and munro (1984) was applied to understand the reliability, evaluate the growth parameters and compare with earlier studies. the length at age revealed that, this species attains length at the end of 1st year to 5th year observed to be 33, 52, 64, 70 and 75 cm, respectively. sustenance of this fishery was observed for 1 to 3 years at the size range of 33-64 cm. the natural mortality (m) was recorded as 0.86 y-1, total mortality (z) was 1.48 y-1 and fishing mortality (f=z-m) was 0.62 y-1. the exploitation ratio (f/z) was observed as 0.42. m/k ratio was estimated at 1.23. the maximum yield per figure 1. the north-west coast of indian eez. figure 2. graphical presentation of length-weight relationship for euthynnus affinis. 23 int. j. aquat. biol. (2018) 6(1): 21-24 recruit obtained at the exploitation rate of 0.42 y-1 at mid class length of 46 cm. the assessment of resource status revealed that the fishing mortality at 0.62 y-1 is more than the targeted optimum fishing mortality (fopt) which is 0.42 y -1 and limit of fishing mortality (flimit=0.58 y -1) as per the biological reference point of view. these results indicating that this species is showing the sign of overexploitation. discussion the investigations on the length-weight relationship would help in understanding the species habitat condition, history of reproductive behaviour, life cycle and health of the fish species (froes, 2006; froese et al., 2011). in the present study, the value of ‘b’ is 2.86 which is very similar to the value obtained by kaymaram and darvishi (2012) wherein the value of ‘b’ derived was 2.87 for the pooled samples from the northern part of persian gulf and oman and defined that this species is showing isometric growth which is in confirmative with the present study. the growth parameters l∞ (asymptotic length) derived was 67.86 cm; k (growth coefficient) was 0.70 y-1and to was -0.26 y -1 in the present investigation. the results derived by ahmedet al. (2016) from the offshore waters of pakistan coast in the arabian sea for l∞ was 67.10 cm which is similar to present study. whereas k (1.049 y-1) and to (-0.84 y-1) values are showing significant difference. phi prime value (⏀) derived by ahmed et al. (2016) was 3.67 from the offshore waters of pakistan coast of arabian sea, whereas in the present study, the value obtained was 3.52. it is evident that the value does not show any significant difference. rohit et al. (2012) from indian waters derived total mortality (z) value of 1.68 y-1, fishing mortality (f) 0.75 y-1, natural mortality (m) 0.93 y-1 and exploitation ratio 0.36 y-1. the values obtained in the present study are 1.48 y-1, 0.62 y-1, 0.86 y-1 and 0.42 y-1, respectively. these values are closer to the values of rohit et al. (2012). the exploitation rate obtained this study (0.42 y-1) at 46 cm mid class length. these results are almost similar to the results of johnson and tamatamah (2013) from coastal waters of tanzania. the fishing mortality derived in this study is more than the optimum and limit value indicated the sign of overexploitation. periodic assessments, monitoring of characteristics of each fishery is essentia to evolve strategies for sustainable harvest, conservation and management of the fishery. this study may help planers and policy makers to take appropriate measures required to be addressed to minimise the exploitation levels. therefore, it is necessary to bring out proper management strategies to sustain this resource in north-west coast of india so as to meet the food requirement for growing human population of the country. acknowledgements authors are very much thankful to the fishery survey of india and the dept. of animal husbandry, dairying and fisheries, min. of agriculture and farmers’ welfare for extending constant support for accomplishing this work. references ahmed q., yousuf f., sarfraz m., ali q.m., balkhour m., safi s.z., ashraf m.a. (2015). euthynnus affinis (little tuna): fishery, bionomics, seasonal elemental variations, health risk assessment and conservational management, frontiers in life science, 8(1): 71-96. ahmed q., bilgin s., bat l. (2016). length based growth figure 3. graphical presentation of length-at-age for euthynnus affinis. 24 mudumala et al./ age, growth and mortality parameters of kawakawa from the north west coast of india estimation of most commercially important scombridae from offshore water of pakistan coast in the arabian sea. turkish journal of fisheries and aquatic sciences, 16: 155-167. al-zibdah m., odat n. (2007). fishery status, growth, reproduction biology and feeding habit of two scombrid fish from the gulf of aqaba, red sea. lebanese science journal, 8(2): 3-20. beverton r.h.j., holt s.j. (1956). a review of methods for estimating mortality rates in exploited rates in exploited fish populations with special reference to source of bias in catch sampling. rapports et procès-verbaux des reunions, ciem, 140: 67-83. cmfri (2017). central marine fisheries research institute, cochin, annual report, 2016-17. 289 p. fao (2017). fao year book. fishery and aquaculture statistics. 2015. rome/ italy. 107 p. froese r. (2006). cube law, condition factor and weight length relationships: history, meta-analysis and recommendations. journal of applied ichthyology, 22: 241-253. froese r., pauly d. (2007). fishbase. www.fishbase.org, version (07/2007). froese r., tsikliras a.c., stergious k.i. (2011). editorial note on weight-length relations of fishes. acta ichthyologica et piscatoria, 41: 261-263. gayanilo f.c., sparre p., pauly d. (2005). fao iclarmstock assessment tools ii (fisat ii), user’s guide, computerized information series (fisheries) no. 8, (revised version), food and agriculture organization of the united nations, rome, italy. johnson m.g., tamatamah a.r. (2013). length frequency distribution, mortality rate and reproductive biology of kawakawa (euthynnus affinis-cantor, 1849) in the coastal waters of tanzania. pakistan journal of biological sciences, 16: 1270-1278. kasim h.m., abdussamad e.m. (2003). stock assessment of coastal tunas along the east coast of india. in: v.s. somvanshi, s. varghese, a.k. bhargava (eds.). proc. tuna meet-2003. pp: 42-53. kaymaram f., darvishi m. (2012). growth and mortality parameters of euthynnus affinis in the northern part of the persian gulf and oman sea. indian ocean tuna commission (iotc), working party on neritic tunas (wpnt), iotc-2012-wpnt02-14. 14 p. khan m.z. (2004). age and growth, mortality and stock assessment of euthynnus affinis (cantor) from maharashtra waters. indian journal of fisheries, 51(2): 209-213. moa (2011). report of the working group for revalidating the potential of fishery resources in the indian eez, ministry of agriculture, new delhi. 69 p. munro j.l., pauly d. (1983). a simple method for comparing growth of fishes and invertebrates. iclarm fishbyte, 1: 5-6. pauline d., janaka d.s. (1991). an assessment of kawakawa (euthynnus affinis) stock on the west coast of sri lanka. asian fisheries science journal, 4: 219226. pauly d., munro j.l. (1984). once more on the comparison of growth in fish and invertebrates. iclarm fishbyte, 2: 21. rohit p., chellappan a., abdusssamad e.m., joshi k.k., said koya k.p., sivadas m., ghosh s., margaret muthu rathinam a., kemparaju s., dhokia h.k., prakasan d., beni n.(2012). fishery and bionomics of the little tuna, euthynnus affinis (cantor, 1849) exploited from indian waters. indian journal of fisheries, 59(3): 33-42. valeiras x., macias d., gomez m.j., lema l., godoy d., ortiz de urbina j.m., de la sernaj. m. (2008). age and growth of atlantic little tuna (euthynnus alletteratus) in the western mediterranean sea. iccat, 62(5): 16381648. int. j. aquat. biol. (2021) 9(6): 388-392 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology short communication alkanes in the sediments of al-gharraf river, southern iraq shaimaa talib abedali* 1, snaa talib jawed2, wesan fadhel khalef3 1department of biology, college of education for women, university of thi qar, thi qar, iraq. 2department of biology, college of education for pure sciences, university of thi qar, thi qar, iraq. 3department of biology, college of sciences, university of thi qar, thi qar, iraq. s article history: received 20 september 2021 accepted 217 december 2021 available online 2 5 december 2021 keywords: petroleum anthropogenic biogenic gas chromatography abstract: the study aimed to determine the concentrations of alkanes compounds using a gas chromatograph with high-precision separation techniques. samples were collected from four stations in the al-gharraf river in southern iraq during 2018-2019. a seasonal variation was observed in the concentrations of alkanes, which is the highest in the winter. the total alkanes concentrations were the lowest at 0.079 μg/g at station 1 in summer. the highest total concentration of alkanes was in station 3 in winter reaching 2.215 μg/g. there was a dominance of individual carbon compounds of c17, c19, and c21 indicating the source of hydrocarbon from phytoplankton, plant, and bacteria. the presence of carbon compounds higher than c25 reveals that organic matter is derived from land and aquatic plants. the results also indicate that the source of petroleum hydrocarbons in the sediments of the al-garraf river is a common biogenic and anthropogenic based cpi index and the pri/phy guide that was less than 1 in the second and third stations for all seasons, and greater than 1 in the first and fourth stations. introduction one of the important pollutants in the aquatic environment is petroleum hydrocarbons which affect their living organisms (nrc, 1985). hydrocarbons enter the aquatic environments through several sources and aquatic organisms absorb them during their feeding; however, the quantity of oil compounds is scare in the aquatic systems (cajeravelli et al., 1995). the regular alkanes with carbon numbers of c15, c17, and c19 are formed from marine biological sources e.g. phytoplankton; while alkanes with carbon numbers of c33 and c25 are composed of terrestrial vascular plants (sakari et al., 2008). the presence of high-chain carbon compounds (c25-c30) indicates the presence of flowering plants. the most individual alkane compound found in the sediments of the marshes is c17, which is produced by the sulfur bacterium of desulfovibrio desulforicans .(al-saad and ali-timari, 1993) chromatography is used for detecting the type of pollution and its source, whether it is biogenic or anthropogenic by measuring the carbon preference *correspondence: shaimaa talib abedali doi: https://doi.org/10.22034/ijab.v9i6.1497 e-mail: shaimaa.talib@utq.edu.iq coefficient (cpi). cpi is the sum of the odd-numbered carbon compounds to the sum of the concentrations of the even-numbered carbon compounds; if it is greater than one, indicating that the origin of the pollution is biogenic and if less than one, indicates the anthropogenic origin of the pollution, and if its values are close to one shows the common origin (canton and grimalt, 1992). the other source of hydrocarbons is oil and its derivatives are made up of a mixture of organic compounds constituting 50-98% of the total oil composition, in addition to other elements such as sulfur, nitrogen, and oxygen (leighton, 2000). oil also contains some trace elements such as cadmium (cd), vanadium (v), nickel (ni), and cobalt (co) (albagies, 1989). therefore, this study aimed to investigate the pollution of petroleum hydrocarbons in the al-garraf river, a tributary of the tigris river. materials and methods the al-gharraf river, a tributary of the tigris river, is located in the southeastern part of iraq, within the alluvial plain area before the kut dam (ghazi, 2004). 389 int. j. aquat. biol. (2021) 9(6): 388-392 it flows towards the southwest between the tigris and euphrates, passing through the al-hayy, qalaat sukar, al-figer, al-nasr, and al-shatra towns. then it splits into two branches, one of them i.e. shatt albid’ah ends in the marshes leading to marsh alhammar (directorate of water resources, 2008). four stations were selected for sampling: the first station (32°08'29.4"n 46°02'38.3"e) in an area characterized by the presence of villages and agricultural lands on both sides that were wide with the high water level, the second station (31°41'21.6"n 46°06'53.9"e) was about 50 km far from the first station, after the river exiting al-figer district in about 4 km. the river in this area is characterized by the abundance of water plants and with a narrow width, as well as the presence of villages and agricultural lands, the third station (31°22'50.8"n 46°10'51.0"e) is located after the river exits the city of shatrah, at a distance of 40 km from the second station. on both sides of the river in this station, there are villages and agricultural lands, with the narrow course less than in the first and second stations, with a decrease in the water level. the fourth station (31°09'55.0"n 46°36'36.6"e) was located at the islah area near the entrance of the river into the marshes (72 km away from the third station) and characterized by the presence of agricultural land and high water level. sediment samples were collected from the four stations in four seasons using a van veen grab and then kept in aluminum foil. drying the sediment was done by freeze dryer and then they grind with a mechanical mortar and afterward sieved with a metal sieve with 63 µm diameter (goutx and saliot, 1982). a gas chromatography device (hp-hewlett packard5890, usa) was adopted to determine the concentrations of aliphatic hydrocarbons (alkanes) of the petroleum from sediments in the laboratories of the marine science center, university of basra. it is table 1. total sum and type of n-alkanes (mg/l) in the sediment of the al-gharraf river in summer. n-alkane s1 s 2 s 3 s 4 c13 0.003 0.02 0.004 0.003 c14 0.002 0.01 0.005 0.002 c15 0.03 0.002 0.007 0.006 c16 0.002 0.003 0.006 0.004 c17 0.006 0.002 0.013 0.021 c18 0.004 0.083 0.032 0.011 c19 0.005 0.024 0.012 0.042 c20 0.006 0.038 0.035 0.046 c21 0.005 0.003 0.062 0.053 c22 0.003 0.049 0.025 0.032 c23 0.002 0.002 0.024 0.028 c24 0.004 0.032 0.093 0.002 c25 0.002 0.015 0.071 0.043 c26 0.008 0.061 0.102 0.025 c27 0.007 0.052 0.025 0.136 c28 0.004 0.064 0.064 0.102 c29 0.002 0.02 0.092 0.006 c30 0.005 0.025 0.21 0.063 c31 0.007 0.003 0.043 0.003 c32 0.002 0.002 0.009 0.005 c33 0.001 0.002 0.016 0.003 c34 0.002 0.001 0.006 0.002 sum 0.079 0.513 0.956 0.638 pristine 0.005 0.072 0.008 0.01 phytine 0.004 0.095 0.04 0.01 prstine/phytine 1.25 0.757 0.2 1 even 0.042 0.368 0.587 0.304 odd 0.043 0.145 0. 369 0.494 odd/even 1.023 0.394 0.628 1.625 table 2. total sum and type of n-alkanes (mg/l) in the sediment of the al-gharraf river in autumn. n-alkane s1 s 2 s 3 s 4 c13 0.002 0.034 0.005 n.d c14 0.002 0.025 0.004 n.d c15 0.001 0.043 0.007 0.021 c16 0.005 0.062 0.003 0.034 c17 0.001 0.093 0.082 0.042 c18 0.071 0.154 0.081 0.032 c19 0.028 0.054 0.095 0.068 c20 0.001 0.012 0.092 0.041 c21 0.011 0.047 0.12 0.052 c22 0.002 0.068 0.098 0.021 c23 0.032 0.084 0.11 0.092 c24 0.003 0.054 0.125 0.074 c25 0.001 0.082 0.095 0.062 c26 0.016 0.074 0.182 0.052 c27 0.085 0.042 0.121 0.063 c28 0.012 0.098 0.21 0.082 c29 0.052 0.024 0.071 0.054 c30 0.001 0.032 0.092 0.023 c31 0.062 0.011 0.021 0.021 c32 0.002 0.021 0.043 0.02 c33 0.041 0.003 0.002 n.d c34 0.003 0.009 0.003 n.d sum 0.434 1.126 1.662 0.854 pristan 0.084 0.07 0.03 0.062 phytan 0.06 0.12 0.051 0.054 pristine/phytan 1.4 0.583 0.588 1.1481 even 0.118 0.609 0.933 0.379 odd 0.316 0.517 0.729 0.475 odd/even 2.6 0.848 0.83 1.253 390 abedali et al./ alkanes in the sediments of al-gharraf river equipped with a type hp (3396a) integrator with injection type of splitless. the type of separation shaft was capillary column j+w db5, with a diameter of 0.25 mm and a length of 30 meters. results and discussions the total concentrations of total alkanes in the study stations ranged between 0.079 μg/g in the first station in summer and 2.215 μg/g in the third station in winter (tables 1-4; fig. 1). there were two types of regular alkanes, first with low molecular weights and others with high molecular weights, and their sources are multiple. compounds with double carbon atoms such as c16, c18, and c20 indicates the presence of plant and animal plankton and bacteria (volkmon, 1980) and the presence of carbon compounds more than c25 e.g. c31, c25, c27, and c29 evidence the organic matter is derived from land and aquatic plants (simoneit, 1993). the results showed that the concentrations of total alkanes in the winter are higher than in summer in all stations since the high evaporation reduces alkanes as well as microorganisms break them into normal alkanes (alsaad, 1996). microorganisms are less active and effective in winter (douabul et al., 2012). al-imarah et al. (2006) studied the seasonal changes in the content of total petroleum hydrocarbons in the water and sediments of the southern iraqi marshes showing the concentrations ranging 29.12-48.14 µg/g dry weight in its sediment samples and those of ranged 0.6-17.41 µg/l. the concentrations of hydrocarbons also were 0.012 µg/g dry weight in the sediments of al-baraka to 0.96 µg/g dry weight in the albaghdadiya sediments (nasir 2007) and it was also found that the concentrations of hydrocarbons in the water were lower than sediments. the results indicate that most of the cpi values are table 3. total sum and type of n-alkanes (mg/l) in the sediment of the al-gharraf river in winter. n-alkane s1 s 2 s 3 s 4 c13 0.002 0.004 0.004 0.008 c14 0.003 0.005 0.002 0.009 c15 0.023 0.007 0.074 0.019 c16 0.018 0.006 0.002 0.063 c17 0.082 0.002 0.102 0.232 c18 0.096 0.003 0.126 0.181 c19 0.092 0.012 0.075 0.173 c20 0.082 0.027 0.097 0.123 c21 0.065 0.042 0.041 0.176 c22 0.043 0.052 0.095 0.092 c23 0.086 0.026 0.086 0.163 c24 0.094 0.092 0.178 0.102 c25 0.092 0.071 0.093 0.103 c26 0.043 0.182 0.162 0.098 c27 0.096 0.178 0.086 0.146 c28 0.085 0.043 0.198 0.088 c29 0.0 89 0.064 0.093 0.096 c30 0.083 0.143 0.173 0.073 c31 0.032 0.013 0.063 0.022 c32 0.021 0.001 0.172 0.01 c33 0.032 0.112 0.021 0.003 c34 0.021 0.002 0.031 0.006 sum 1.28 1.66 2.215 1.988 pristane 0.046 0.012 0.062 0.062 phytane 0.015 0.082 0.076 0.193 pristane/phytane 3.06 0.14 0.815 3.112 even 0.589 0.55 1.236 0.839 odd 1.091 0.531 0.738 1.138 odd/even 1.85 0.96 0.597 1.356 table 4. total sum and type of n-alkanes (mg/l) in the sediment of the al-gharraf river in spring. n-alkane s1 s 2 s 3 s 4 c13 0.002 0.004 0.004 0.008 c14 0.003 0.005 0.002 0.009 c15 0.023 0.007 0.074 0.019 c16 0.018 0.006 0.002 0.063 c17 0.082 0.002 0.102 0.232 c18 0.096 0.003 0.126 0.181 c19 0.092 0.012 0.075 0.173 c20 0.082 0.027 0.097 0.123 c21 0.065 0.042 0.041 0.176 c22 0.043 0.052 0.095 0.092 c23 0.086 0.026 0.086 0.163 c24 0.094 0.092 0.178 0.102 c25 0.092 0.071 0.093 0.103 c26 0.043 0.182 0.162 0.098 c27 0.096 0.178 0.086 0.146 c28 0.085 0.043 0.198 0.088 c29 0.0 89 0.064 0.093 0.096 c30 0.083 0.143 0.173 0.073 c31 0.032 0.013 0.063 0.022 c32 0.021 0.001 0.172 0.01 c33 0.032 0.112 0.021 0.003 c34 0.021 0.002 0.031 0.006 sum 1.28 1.66 2.215 1.988 pristane 0.046 0.012 0.062 0.062 phytane 0.015 0.082 0.076 0.193 pristane/phytane 3.06 0.14 0.815 3.112 even 0.589 0.55 1.236 0.839 odd 1.091 0.531 0.738 1.138 odd/even 1.85 0.96 0.597 1.356 391 int. j. aquat. biol. (2021) 9(6): 388-392 greater than 1 showing that the main source of the hydrocarbons in the sediments of the al-gharrrf river is biological, and there is little contribution from human sources with the values of cpi less than 1 in the second and third stations. these pollutants can originate from different sources, including household waste, untreated heavy waters, atmospheric deposits carrying hydrocarbon atoms, and vehicle exhaust. the results also indicated that the values of pri/phy are less than 1 in the second and third stations for all seasons and greater than one in the first and fourth stations indicating the source of hydrocarbons in the sediments of the al-gharraf river from anthropogenic origin. references al-marah f.g., hantoush a.m., nasir a.m., al-yaseri s.t. (2006). seasonal variations of the total petroleum hydrocarbons in water and sediments of southern iraqi marshland after rehabilitation 2003. marsh bulletin, 1(1): 1-8. al-saad h.t., al-timari a.k. 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(2018) 6(2): 61-65 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology short communication shell choice and occupation by the hermit crab (crustacea: diogenidae) in laboratory environment cícero diogo lins de oliveira*1, ana maria siqueira quirino1, erasmo andrade da silva1, elvira florentino costa1, dejinalva da silva santos1, anastácia novais carvalho menezes1, mauro de melo junior2, francisco marcante santa da silva1 1universidade federal rural de pernambuco, unidade acadêmica de serra talhada, avenida gregório ferraz nogueira, s/n, bairro: josé tomé de souza ramos, cep: 56909-535, serra talhada-pe, brasil. 2 departamento de biologia, universidade federal rural de pernambuco, dois irmão – recife, pernambuco, brasil. article history: received 6 february 2018 accepted 24 april 2018 available online 2 5 april 2018 keywords: behaviour shells occupation behavioral ecology ethogram abstract: the neotropical hermit crabs’ behaviour is rarely studied, although it is an important tool for the conservation of these invertebrates. in this context, the present study aimed to describe the hermit crab ethogram on its behaviour in the choice and dispute by shells. 60 specimens of hermit crabs were collected that were occupying mollusc shells cerithium atratum, in sandstone reefs. in the laboratory, 30 hermit crabs were removed from their host shells by heating, the rest remained in their shells. the specimens were submitted to five experimental tests: (i) behaviour, (ii) shell dispute, (iii) shell preference, (iv) specimen behaviour with and without shell, and (v) shell contention between individuals with and without shell. the ethogram was built and evaluated by the ad libitum type of observational sampling methods and by the scanning type, for 30 uninterrupted minutes, records every two minutes, in total 15 observations per each observer. there were eight behavioural acts divided into 4 categories: immobility, interaction between specimen, environment exploration, and shell occupation. the most frequent behaviour was "walking", for both hermit crabs (54.2%) and those without shell (59.3%), followed by the rest of the individuals with and without shell, 16.9% and 12.4%, respectively. all hermits preferred mollusc shell bulla striata, when offered in conjunction with the shell of astrea tecta. however, all specimens returned to c. atratum shells when it was experimentally offered with all shells. studies with hermits have shown that the species has preferences of gastropods shell c. atratum. introduction diogenidae is a hermitages family of the decapoda that includes approximately 425 species, making it the second most diverse of the families of the anomura infraorder. it has a cosmopolitan distribution and is considered fragile due to non-calcification of its abdomen. its life strategy is closely related to the use of shells, being essential to its survival (vance, 1972; hazlett, 1981; matos and couto, 2010). the association between hermit crabs and gastropod shells is quite specific (sant’anna et al., 2006). hermits can move around with their shells due to the twisting of their abdomen, which is associated with the presence of modified uropods, allowing them to attach to the shell columella (narchi, 1973). shells *corresponding author: cícero diogo lins de oliveira doi: https://doi.org/10.22034/ijab.v6i2.439 e-mail address: linsdiogoc@gmail.com are of great importance to hermit crabs, for protection against predators, resistance to desiccation, protect against the force exerted by the waves tidal, protection of eggs in female, in some cases even against the other hermit crabs and a moderating function of environmental factors (bach et al., 1976; burggren, 1979; pinheiro et al., 2005). most hermit crabs use shells of different gastropods. the diversity of shells used was related to the number gastropod species present. the pattern of shell utilization varies among populations, which demonstrate preference for some shell types that can bring advantages and maximize their abilities (markham, 1968). the process of shell occupation was influenced by several factors such as availability 62 oliveira et al. / shell choice and occupation by the hermit crab in nature, weight, shape, architecture, internal volume and epibionts occurrence (rotjan and blum, 2004). in this perspective, the present work aims to identify the preferred shells to be occupied by hermit crabs, as well as characterization of their behaviour aspects in the choice and dispute by shells, collected on the jaguaribe beach, in the ilha de itamaracá – pe, brazil, observed in microcosm with the elaboration of an ethogram for the behavioural registers. materials and methods material collection was performed in july 2016 during low tide. a total of 60 individuals of hermit crabs were randomly collected from sandstone reefs. the captured individuals were conditioned in a tray containing substrate and water from the collection environment to simulate their natural environment (microcosm). at the same time, the empty shells of the gastropods, including bulla striata (bruguière, 1792), astraea tecta (phillipi, 1846) and cerithium atratum (born, 1778) of different sizes were collected (fig. 1). then, the hermit crabs were removed from their host shells by heating i.e. keeping the apex of the shells on fire flame. in the first four experiments, the ethological method of observational sampling type ad libitum (altmann, 1974) was used based on the continuous record of what the animal does, where the rule is the absence of rules (del claro, 2004). in the fifth experiment, the instantaneous scanning method was used (altmann, 1974) based on the discontinuous register, where at each predefined time interval, two minutes, all hermit crabs were observed to verify the interaction between hermit crabs with and without shell. this interval must be as small as possible, in order to act as if a photographic record was taken at each moment of observation (del claro, 2004). in order to compare the frequencies of the behaviours was used t-student test with significance level p=0.05 (zar, 1996). experiment i: consisted in observing how the hermit behaved when being offered a shell with aperture inferior to its size. the hermit without a shell was inserted into the microcosm before an empty shell of b. striata, with an opening smaller than its size. five replicates were used, whose hermit crab behaviours were observed for five minutes in each replicate. experiment ii: the preference of the hermit was verified by shells of different shape. ten replicates were used with two moments and duration of ten minutes each. at the first moment, the hermit without shell was inserted with an empty shell of b. striata and another of a. tecta and observed to their choice between the offered shells. in the second moment, after the individual had stayed in one of the shells, was inserted the shell of the c. atratum, type of shell that normally the hermit occupies in natural environment. experiment iii: competition verification by shells among hermit crabs. five specimens of hermit crabs without shell were inserted and only one shell of c. atratum, in the microcosm and observed the reactions. in this experiment, five replicas were used, with duration of 20 min, with the purpose of observing the competition between the individuals of hermit crabs by the shell. experiment iv: the behaviour among crabs with and without shells within the same microcosm was observed and thus the etogram made. four specimens were inserted, two with and two without shell, with the use of five replicas, whose hermit crab behaviours were observed for ten minutes. experiment v: was observed if there were differences in the behavioural patterns among the hermit crabs in the dispute for the shell. five replicas were used, where each replica simulated a microcosm where it figure 1. gastropods' shells used in the experiment, collected in jaguaribe beach, ilha de itamaracá – pe, brazil. (a) bulla striata (bruguière, 1792), (b) astraea tecta (phillipi, 1846), and (c) astraea tecta (phillipi, 1846). 63 int. j. aquat. biol. (2018) 6(2): 61-65 contained three hermit crabs with their respective shells and three without shells. the observation time was 30 minutes, with behaviour recorded at twominute intervals, to the total of 15 observations for each replica. t-student tests were still performed with significance level of 5%, to identify if there was significant difference in the frequency of behaviour between the individuals with and without shell. results and discussion in the first experiment, it was observed that 100% of the hermit crab turned the shells in the first contact. according pinheiro et al. (2005), this type of behaviour is classified with specific exploratory, by which these animals analyse their conservation, size, shape, weight, size of its aperture, colouration and its internal volume. in relation to the attempt to occupy the shells, 80% of the hermits insisted two or more times, even though they did not succeed in hosting it, because they are shells smaller than their size, and 20% tried to enter only once, after examining it. bertini and fransozo (2000) claim that the choice of shells is not random, thus exhibiting specific exploratory behaviours such as rotating the shells with the chelipods, exploring the opening and the inner space of the shell. for the second experiment, in the first stage it was recorded that all hermit crabs preferred the shell b. striata, analysing it before occupying it, clearly showing its preference rather than a. tecta. in the second moment, when inserted the shell that he occupied previously (c. atratum), they migrated to the introduced shell, thus being noticeable the preference for the latter. according to other studies (pereira et al., 2009), the species of hermit crabs tend to prefer elongated shells narrow spirals, such as of the genus cerithium. in the five replicates of the third experiment, there were complications being that in 80% of these, three or more disputes took place by the shell and only in a replica were verified two disputes by the shell, in this have the expulsion of the first host by another greater individual, who remained until the end of the treatment. in natural environments, which offer shelter are larger, it is usually not prospective this type of competition, however, in low availability of shells may occur (scully, 1983), being able to conclude that larger individuals stand out better than smaller specimens do. in the fourth experiment were registered 14 behavioural acts, divided into 14 categories and built the ethogram. the categories registered were: walk, enter the shell, enter in the shell empty, drive out the intruder, run away with the shell, run away without a shell, fight with shell; fight without shell, persecution, resting with shell, resting without shell, turn gravel and turn shell (table 1). it was found that the most common categories were those that were most frequently repeated, walking, turning shell, enter in the empty shell and resting with shell, actions classified as exploratory, corroborating with pinheiro et al. (2005), where he mentions that the main initial table 1. description of the behavioural acts of the hermit crabs in microcosm, collected in ilha de itamaracá, pernambuco, brazil. category behavioural act walk individuals with and without shells walking in the environment. enter the shell hermit without shell trying to get into the shell. enter in the shell empty shell-less specimen when occupying shell. drive out the intruder the specimen with shell expelling another without shell. run away with the shell the specimen with shell escaping from another without a shell. run away without a shell the specimen running away from individuals with shell. fight with shell confrontation between specimens with shell. fight without shell confrontation between specimens without shell. persecution individual chasing another. resting with shell specimen with shell still and isolated. resting without shell specimen without shell still and isolated. turn gravel individual stopped revolving the substrate. turn shell the specimens looked at the shells, exploring the opening and the inner space of the shell. 64 oliveira et al. / shell choice and occupation by the hermit crab activities are to recognize the environment (walking) and beyond the search for more adequate shelter, in the case of the experiment were turn shell and enter the shell. for the fifth experiment, a total of 11 categories was registered (table 2). among the behaviours registered the most frequent was the walk, with 59.3% of the without shell and 54.2% with shells, where the specimens walked on the substrate of the microcosm. then, the most frequent was the resting of the individuals with and without shell, 16.9% and 12.4%, respectively. it is noted that individuals without shells were in greater movement, because they are in search of shells, in which it is indispensable for their survival, thus being more in movements than those that already have shell. when analyzing the level of significance between the behavioural categories shared between the specimens with and without a shell, it was observed that none of the categories analyzed showed a significant difference between the behaviours of individuals with and without a shell (table 3), even though they presented different frequencies. according bertness (1981), this is because even though the specimens have shells they continue to look for new ones that best suit their size and other characteristics. based on the results, it was observed that there is a very strong relationship between the crabs in the choice of their shells, showing that this choice is related to the availability of the resource in the environment, and the search for a suitable shell is constant. note that there is a preference for shells of the species c. atratum, however, where it is not available in the environment, it may be replaced by a. tecta. and that prior to occupying them, there are exploratory behaviours such as turning the shell and analyzing the opening of it. on the other hand, the shared behaviours between individuals with and without shells have little distinction, since the shell search, which is ideal for the size of the individual, is consistent for both cases. acknowledgments the graduate program in biodiversity and conservation and all the team that make up the base in ilha de itamaracá of universidade federal rural de pernambuco, by the support during the study table 2. frequency of specimen categories, without and with shells, collected in ilha de itamaracá, pernambuco, brazil. category frequency with shell (%) frequency without shell (%) walk 54.2 59.3 enter the shell 12.0 0.0 run away with the shell 4.9 0.0 run away without the shell 0.0 5.6 fight with shell 5.8 1.4 fight without shell 0.0 5.6 persecution 0.9 4.5 resting with shell 16.9 0.0 resting without shell 0.0 12.6 turn shell 5.3 11.0 table 3. level of significance between the behavioural categories of the hermit crabs, with and without shells, collected ilha de itamaracá, pernambuco, brazil. category p-value walk 0.70 run away with the shell x run away without the shell 0.92 fight with shell x fight without shell 0.95 persecution 0.16 resting with shell x resting without she 0.51 turn shell 0.22 65 int. j. aquat. biol. 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(1996). biostatistical analysis. 3d ed., prentice hall, new jersey. 662 p. international journal of aquatic biology (2015) 3(3): 177-182 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article paraschistura ilamensis, a new species of loach from the tigris river drainage (teleostei: nemacheilidae) saber vatandoust*1, soheil eagderi2 1department of fisheries, babol branch, islamic azad university, babol, iran. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 7 november 2014 accepted 27 january 2015 available online 2 5 june 2015 keywords: paraschistura, morphology, new species, tigris, iran. abstract: paraschistura ilamensis, new species, is described from the tigris river drainage, iran. it is distinguished from other species of paraschistura in iran by a combination of the following characters: emarginate caudal fin, stout, deep and scaled body, deep caudal peduncle, 7-10 irregular and interrupted vertical pale brown bars on flanks, two obvious dark spots on the upper and lower caudal fin unbranched rays, and moveable protuberance at the antero-ventral corner of the eye in males. introduction nemacheilid loaches with about 72 genera and about 793 species are found across eurasia with one species in northeast africa (eschmeyer and fong, 2011). this family has a great diversity in iranian interior waters (coad, 2014). among the members of nemacheilids, paraschistura prokofiev, 2009 is a newly described genus, therefore, not all of its species fully examined and ascribed to it or related genera (coad, 2014). these small nemacheilid loaches, with a dark black spot or strip at the base of the anterior dorsal fin rays, are distributed from the upper tigris river basin, interior water bodies of turkmenistan and from iranian baluchistan east to the upper reaches of the indus river in afghanistan and pakistan (prokofiev, 2009; coad, 2014). many species of this genus were formerly included in the genus schistura mcclelland, 1838 (coad, 2014). hence, it can be concluded that the genus paraschistura remains poorly studied, and a comprehensive taxonomic revision is still not available. according to kottelat (2012), fourteen valid species * corresponding author: saber vatandoust e-mail address: s.vatandoust @gmail.com are belong to the genus, including p. alepidota, p. bampurensis, p. chrysicristinae, p. kessleri, p. lepidocaulis, p. lindbergi, p. microlabra, p. naseeri, p. nielseni, p. pakistanica, p. prashari, p. punjabensis, p. sargadensis, and p. turcomanus. in addition, p. turcmenicus described from turkmenistan suggested a synonym of p. sargadensis (berg 1948-1949) but bănărescu and nalbant (1966) consider it to be a valid subspecies, and p. baluchiorum described from pakistan is treated as synonym of p. bampurensis according to nalbant and bianco (1998). during the year 2014, we collected and examined materials of all known iranian species throughout iran. comparing the collected loach from the middle part of the tigris river drainage with nominal species by morphological characters, as well as colour patterns, it became clear that it represents an unnamed species which is described here. material and methods sampling and measurements: specimens were collected by electrofishing in 2014. they were fixed 178 international journal of aquatic biology (2015) 3(3): 177-182 in 5% buffered formaldehyde after anaesthesia, and stored in 72% ethanol. morphometric characters were measured by a dial caliper to the nearest 0.1 mm. all measurements are made point to point, never by projections. methods for counts and morphometric measurements were performed based on kottelat and freyhof (2007). standard length (sl) is measured from the tip of the snout to the end of the hypural complex. the length of the caudal peduncle is measured from behind the base of the last anal-fin ray to the end of the hypural complex, at mid-height of the caudal-fin base. the last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are noted as "1½". abbreviations used: sl, standard length. hl, lateral head length. vmfc, vatandoust and mousavi-sabet fish collection, tehran. zmmu, zoological museum, moscow state university, moscow. results paraschistura ilamensis, new species (figs. 1-5) holotype: vmfc psi3-h, 43 mm sl. iran, ilam prov.: spring siahgav, the tigris river drainage, 32°51'54"n 47°42'2"e. s. vatandoust, h. mousavisabet, h. bagherpour, m. cheraghpour, m. nourmohammadi and a. jouladeh. paratypes: vmfc psi3-p, 30 specimens, 26 42 mm sl, same data as holotype. diagnosis: paraschistura ilamensis is distinguished from the congeners in iran by a combination of none unique characters including emarginate caudal fin, stout and deep body, scaled body, deep caudal peduncle, 7-10 irregular and interrupted vertical pale brown bars on flanks, two obvious small dark spots on the upper and lower caudal fin unbranched rays, and moveable protuberance at the antero-ventral corner of the eye in males. description: for general appearance see figs. 1-5. measurements of holotype and paratypes are given in table 1. body stout (body depth at dorsal fin origin 14.1-18.5% sl, 16.4 ± 1.6). lateral line incomplete, extending to the mid of the dorsal fin base. mouth arched, upper jaw with developed processus dentiformis. maxillary barbel long, reaching to the end of the eye and passes it. outer mandibular barbel extending mid of the eye. inner mandibular barbel reaching to the maxillary barbel origin. nostrils proximate to the eyes, with a relatively long tube on the anterior nares opening. anterior dorsal fin origin located mid dorsum, or slightly posterior. ventral fin insertion below a vertical of the dorsal fin spines. developed axillary lobe at the base of pelvic fin, attached to the body. figure 1. paraschistura ilamensis, vmfc psi3-h, holotype, 43 mm sl; iran: spring siahgav. 179 vatandoust and eagderi/ paraschistura ilamensis, a new species of loach caudal fin emarginate with round lobes. very-small scales scattered on all over body. moveable protuberance at the antero-ventral corner of the eye in males. dorsal fin with 7½ branched rays (simple rays cannot be counted). anal fin with 5½ branched rays (simple rays cannot be counted). pectoral fin with 9 branched rays. pelvic fin with 7 branched rays. caudal fin with 8+8 branched rays. colouration: body and head pale cream to yellow. 710 vertical dark cross bars on flanks, sometimes irregular and interrupted. a pale dark bar at the caudal fin base. two obvious small dark spots on the upper and lower caudal fin unbranched rays. 2-3 rows of small dark speckle on caudal fin. an obvious dark blotch present at anterior dorsal fin origin, not appear in life, only a short dark bar is appear on the first spine of the dorsal fin in live specimens. some specimens have de-pigmented body in life, with no any obvious dark pigmentation (fig. 5). remarks: paraschistura ilamensis is distributed in the middle part of the tigris river drainage, which is geographically separated and morphologically distinct from the congeners. paraschistura ilamensis is the second confirmed species of this genus in the tigris river drainage, where is the westernmost figure 2. paraschistura ilamensis, vmfc psi3-p, paratype; iran: spring siahgav; a, 42 mm sl; b, 42 mm sl; c, 41 mm sl; and d, 34 mm sl. 180 international journal of aquatic biology (2015) 3(3): 177-182 figure 3. paraschistura ilamensis, vmfc psi3-p, paratype; iran: spring siahgav; a, 42 mm sl; b, 42 mm sl; c, 41 mm sl; and d, 34 mm sl. figure 4. paraschistura ilamensis, vmfc psi3-p, paratype; 41 mm sl; iran: spring siahgav. 181 vatandoust and eagderi/ paraschistura ilamensis, a new species of loach distribution of this genus. paraschistura chrysicristinae (nalbant, 1998) is the only nominal species from this basin, which is described from eastern anatolia, turkey (the upper tigris basin). both of these species are characterized by incomplete lateral line, but lateral line in p. ilamensis not reaching to the posterior origin of the dorsal fin vs. lateral line in p. chrysicristinae passes the dorsal fin and reaching till above the anal fin. paraschistura ilamensis is further distinguished from p. chrysicristinae by stout body (vs. elongate), 7-10 vertical dark cross bars on flanks (vs. 13-14), caudal fin with 8+8 branched rays (vs. 8+7), eyes placed in the mid of the head (vs. in the first half of head), and suborbital flap in front of the eye in males (vs. apparently no sexual dimorphism) (nalbant, 1998). the second closest geographical species with p. ilamensis is p. nielseni from the persian gulf basin. paraschistura ilamensis is distinguished from p. nielseni by having a deeper body (body depth figure 5. paraschistura ilamensis, vmfc psi3-p, paratype; 39 mm sl; iran: spring siahgav. holotype paratypes min max mean sd standard length (mm) 43.3 25.6 49.5 in percent of standard length head length 23.1 23.5 26.8 25.1 1.5 body depth at dorsal-fin origin 16.9 14.1 18.5 16.4 1.6 prepectoral length 27.3 27.1 30.1 28.6 1.2 predorsal length 52.4 52.6 58.2 54.9 2.1 postdorsal length 35.6 35.5 39.5 37.9 1.6 preanal length 79.0 76.7 81.0 78.6 1.7 prepelvic length 53.3 53.5 57.4 55.5 1.7 distance between pectoral and pelvic-fin origins 30.0 30.2 32.7 31.5 1.0 distance between pelvic and anal-fin origins 24.9 25.3 29.7 26.9 2.1 distance between vent and anal-fin origin 3.0 2.6 3.3 3.1 0.3 depth of caudal peduncle 13.2 10.5 13.3 12.6 1.2 length of caudal peduncle 18.0 14.6 18.8 16.9 1.6 dorsal-fin depth 18.9 19.0 22.6 20.7 1.5 anal-fin base length 6.9 6.8 8.6 7.7 0.6 pectoral-fin length 21.9 19.0 21.7 20.6 1.2 pelvic-fin length 17.8 14.8 17.5 16.4 1.1 in percent of head length head depth at eye 49.0 37.3 48.8 43.7 4.3 snout length 39.0 37.4 40.0 38.9 1.0 eye diameter 19.0 19.3 25.8 21.4 2.8 postorbital distance 50.0 45.9 56.5 46.0 4.1 maximum head width 61.0 48.1 58.0 51.2 4.2 interorbital width 33.0 23.3 33.0 28.1 4.4 table 1. morphometric data of paraschistura ilamensis (n = 17). 182 international journal of aquatic biology (2015) 3(3): 177-182 14.1-18.5% sl, 16.4 ± 1.6 vs. 13.8-15.5% sl, 14.6 ± 1.3), 7-10 vertical dark cross bars on flanks (vs. 717), dorsal fin located in the posterior half of the body (vs. dorsal fin placed in the mid dorsal), relatively longer barbels, emarginate caudal fin (vs. deeply emarginate), bigger head (head length 23.526.8% sl, 25.1 ± 1.5 vs. 21.0-24.2% sl, 22.8 ± 1.1), and caudal fin with 2 rows of dark spots (vs. 3 rows). paraschistura ilamensis is further distinguished from p. sargadensis by having a scaled body (vs. scaleless), and a dorsal-fin origin in front a vertical of the pelvic fin origin (vs. slightly behind the pelvic fin origin). paraschistura ilamensis is further distinguished from the other species of paraschistura in iran by a combination of characters, none of them unique. in p. ilamensis the body has small scales (vs. scales absent in p. kessleri), and obvious vertical dark bars are presented on flanks (vs. not regular cross bars in p. bampurensis). distribution: paraschistura ilamensis is found in siahgav spring near abdanan, in ilam province, in the tigris river basin, west of iran. etymology: the species name ilamensis comes from the ilam province, the region where it was found. comparative material: metaschistura cristata: vmfc msc, 10 specimens, 59-68 mm sl, iran, khorasan prov.: a stream near mashhad, hari river basin, h. mousavi-sabet a. jouladeh & b. ganjbakhsh. paraschistura bampurensis: vmfc psb-b, 12 specimens, 39-43 mm sl, iran, sistanand-baluchistan prov.: a qanat near bampour, s. eagderi and m. nasri. paraschistura kessleri: vmfc psk, 9 specimens, 33-42 mm sl, iran, sistan-and-baluchistan prov.: mashkid river, near sarbaz town, s. eagderi. paraschistura nielseni: vmfc psn, 21 specimens, 31-49 mm sl, iran, bushehr prov.: shapur river, s. eagderi and h. mousavi-sabet. paraschistura sargadensis: vmfc pss, 18 specimens, 29-41 mm sl, iran, sistan-&baluchistan prov.: a stream, near zaboli town (not zabol), in mashkid basin, s. eagderi and m. nasri. paraschistura turcomanus: zmmu p-57353, 1 specimen; zmmu p.5734, 3 specimens; syntypes. acknowledgments we are pleased to thank hamed mousavi-sabet (vmfc) and ekaterina vasileva (zmmu) for providing pictures and materials, arash jouladeh, manouchehr nasri, hasan jahani, gholamreza abdali, hamd hemmati, behrouz miran, hamidreza bagherpour, mehrdad cheraghpour, hashem nowferesti and mahdi nourmohammadi for helping with fish collection. references bânârescu p., nalbant t.t. (1966). the 3rd danish expedition to central asia. zoological results 34. cobitidae (pisces) from afghanistan and iran. videnskabelige meddelelser fra dansk naturhistorisk forening i københavn, 129: 149-186. berg l.s. (1948-1949). freshwater fishes of the u.s.s.r. and adjacent countries. part. 2. fourth edition. izdatelstvo akademii nauk sssr, moskva and leningrad, pp. 470-925. coad b.w. (2014). freshwater fishes of iran. available from: www.briancoad.com. retrieved 9/10/2014. eschmeyer w.n., fong j.d. (2011). animal biodiversity, an outline of higher-level classification and survey of taxonomic richness. zootaxa, 23:26-38 kottelat m., freyhof j. (2007). handbook of european freshwater fishes. kottelat, cornol and freyhof, berlin, 646 pp. kottelat m. (2012). conspectus cobitidum: an inventory of the loaches of the world (teleostei: cypriniformes: cobitoidei). the raffles bulletin of zoology, 26: 1199. nalbant t.t., bianco p.g. (1998). the loaches of iran and adjacent regions with description of six new species (cobitoidea). italian journal of zoology, 65: 109-123. prokofiev a.m. (2009). problems of the classification and phylogeny of nemacheiline loaches of the group lacking the preethmoid i (cypriniformes: balitoridae: nemacheilinae). journal of ichthyology, 49: 874-898. zoobank: urn:lsid:zoobank.org:pub:65237333-99fe-4866828b-bd47217f6f49 int. j. aquat. biol. (2018) 6(3): 114-121 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article circulatory system of red tail catfish (phractocephalus hemioliopterus bloch & schneider, 1801): a corrosion cast study ali taheri mirghaed*1, hooman rahmati-holasoo1, mehrdad ardeshiri1, hoseinali ebrahimzadeh mousavi1, javad sadeghinezhad2 1department of aquatic animal health, faculty of veterinary medicine, university of tehran, tehran, iran. 2department of basic sciences, faculty of veterinary medicine, university of tehran, tehran, iran. article history: received 21 february 2018 accepted 24 june 2018 available online 2 5 june 2018 keywords: circulatory system red tail catfish corrosion cast physiology abstract: red tail catfish, phractocephalus hemioliopterus, in one of the popular ornamental fish. the present study is aimed to describe and visualizes the cardiovascular system of this species with corrosion cast study method. for this purpose, 10 red tail catfish with 580 gr average weight were obtained and were filled their blood vessels and heart with fluid artificial resin made on the basis of methylmetacrylate after anaesthetizing and euthanizing. for complete polymerization and hardening of the methylmetacrylate, the fish were further submersed for 12-24 hrs in water bath following by 24-48 hrs submersion in a 25% solution of koh to full maceration. based on the results we describe the cardiovascular system i.e. the afferent and efferent vessels of gill, different parts of the heart, ventral aorta, dorsal aorta, intestinal and gastric vessels, liver, anterior and posterior parts of the kidneys, spleen, portal and hepatic vein. introduction the circulatory system plays an important role in many modeling approaches, therefore understanding its anatomy can be useful for many basic and diagnostic studies (nematollahi et al., 2003, 2005; noestelthaller et al., 2005; ghadam et al., 2016). vascular corrosion casting consists of injecting a liquid polymer into the circulatory system of a whole animal or an organ, allowing it to harden and then corroding away the overlying tissue (typically with potassium hydroxide solution) to produce a 3d cast of the internal structure of the vessels (hossler et al., 1986, 1991; zeindler et al., 1989). it will be possible to study the vascular structures in 3d by corrosion cast studies. this would give more information about the complexity of the vascular system in various species (schmidt et al., 1980). this method has been used in many studies on human and animals. few studies have been performed on the cardiovascular system of fishes (seyed ali et al., 1987; passantino et al., 2000; basten et al., 2009) such as light and scanning electron microscopic observations on methyl-methacrylate corrosion casts *corresponding author: ali taheri mirghaed doi: https://doi.org/10.22034/ijab.v6i3.445 e-mail address: mirghaed@ut.ac.ir of the blood vessels in the gills of channel catfish (ictalurus punctatus) (boland and olson, 1979), corrosion cast of the entire blood vascular system by a single injection of resin from the heart of a japanese catfish (silurus asotus) (iwamizu and itazawa, 1986), gill vasculature of yellow stingray (urolophus jamaicensis) using resin casts under sem (sherman and spieler, 1998), spatial organization of the microcirculation in gills of striped mullet (mugil cephalus) by scanning electron microscopic analysis of corrosion cast prepared by intravascular injection of methylmethacrylate (passantino et al., 2000), structural variation in gill vasculature among some elasmobranchs using corrosion casting and sem (sherman et al., 2005), vascular pattern of the recirculating system of rainbow trout (oncorhynchus mykiss) by corrosion cast methodology (nematollahi et al., 2011), the efferent branchial arteries and splanchnic arteries in the yellow stingray (urobatis jamaicensis) using sem to study the vascular corrosion casts (basten et al., 2009), and comparative study of the circulatory system in common carp (cyprinus carpio) and beluga (huso huso) with 115 int. j. aquat. biol. (2018) 6(3): 114-121 emphasis on the heart and main blood vessels, employing the corrosion cast methodology (ghadam et al., 2016). red tail catfish, phractocephalus hemioliopterus is a large neotropical catfishes of the family pimelodidae, inhabiting the amazon and araguaiatocantins river basins. this fish is characterized by having a strong skull ossification, a huge and bony predorsal plate, and red-orange tail and dorsal fins (goulding, 1980). it has a great economic importance as an ornamental fish, sport fishing or fish production, providing food and a valuable source of income for coastal populations, and also in some regions of the amazon, its fat and leather extracted are used as folk medicine for treating respiratory problems (souza et al., 2012). the present study describes and visualizes a 3d view of the circulation system of this fish using the corrosion cast methodology as an important figure 1. injection of artificial resin fluid into the heart (a), branchial vessels (b), liver (c), spleen (d), anterior kidney (e) and posterior kidney (f). (l) liver, (sb) swim bladder, (st) stomach. 116 taheri mirghaed et al/ circulatory system of phractocephalus hemioliopterus using corrosion cast study ornamental fish. materials and methods ten red tail catfish with average weight of 580 g were obtained from ornamental fish supply stores in tehran, iran. fish were kept for 2 weeks prior to the experiment in 1000-l tanks supplied with filtered, recirculated tap water (23±1°c). dissolved oxygen and ph were 6.5±0.5 mg/l and 7±0.3, respectively. wet smears from skin, fins and gills was prepared for parasitological examination under light microscope (nikon, e600). no ectoparasite were observed. the fish were anesthetized using pi222 (pars imen daru, iran) in dosage of 100 ppm and transferred to the operating table. to prevent the formation of blood clots in the vessels, the fish were injected intraperitoneally with heparin (5000 iu/kg) and euthanized with an overdose of the aforementioned anesthetic solution. then the heart and blood vessels were filled with artificial resin fluid made on the basis of methylmetacrylate. this solution was injected in the heart, branchial vessels, liver, spleen, anterior, and posterior part of the kidney (fig. 1a-f). fish were left for 12-24 hrs in a 20-24°c tap water bath to complete polymerization of the injected resin. then dipped in 25% potassium hydroxide (koh) for 24-48 hrs to maceration of the tissues (ghadam et al., 2016). the resin did not dissolve in potassium hydroxide, thus the heart and vascular casts remained in their natural positions. these structures were studied by gross examination and stereomicroscope. results the results are illustrated in figures 1-12 to provide information on the cardiovascular system. different parts of the heart and related vessels, including atrium (a), ventricle (v), bulbus arteriosus (ba) and ventral aorta (va) were shown in figure 2a, b. the va continues from the heart to the afferent branchial arteries (abas) (fig. 2c). the length of va is short. in a short distance from the heart, a common trunk (ct) is separated from va. the common trunk is divided into two branches (aba 1 and 2) and the rest of va separately made aba 3 and 4 (fig. 2c). the da continues along the vertebral column and supplied different parts of the body. in a short distance from the beginning of da, a branch viz. subclavian artery (sca) was separated to supply the cranial parts of body such as pectoral fins (fig. 5). in the middle of the body, it passed through a longitudinal groove (lg) in wb (fig. 6a). it supplies this part of the body through some pores (p) in the bones (fig. 6a, b, c). in caudal area, the da passed through haemal arches (ha). da and caudal vein (cv) which carried the figure 2. a, b and c: the main parts of the heart and related vessels. a (atrium), ba (bulbus arteriosus), ct (common trunk), v (ventricle), va (ventral aorta) and abas (1-4). each abas itself constituted afferent filament arteries (afas) (secondary lamella), that provide more surface for oxygen exchange (fig. 3a and b). 117 int. j. aquat. biol. (2018) 6(3): 114-121 deoxygenate blood, are passed together in ha (fig. 7). the venous blood from the caudal area such as caudal fin, ribs, trunk muscles and bones is collected by cv. many small vessels collect the venous blood, forming cv that goes to the anterior parts of the body. cv in its path is unified with the venous vessels from the spleen, stomach, intestine, gall bladder, and mesenteric vein and then carries as portal vein (pv) to the liver (fig. 8). the collected blood in the liver is entered to the heart via hepatic vein (hv) (fig. 8). the blood from the posterior kidneys is collected by the renal portal vein (rpv) and then is carried by the right post cardinal vein (rpcv) and left post cardinal vein (lpcv) to the common cardinal vein (ccv) and then to the heart. rpcv and lpcv are passed inside the bone plates on both right and left sides of the body. in total, rpcv is thicker than lpcv (fig. 8).on the figure 3. aba (afferent branchial arteries) and afa (afferent filament arteries) are seen. after oxygen exchange, abas joined together and made efferent branchial arteries (ebas). ebas joined together and formed dorsal aorta (da) at the beginning of weber bone (wb) (fig. 4). some branches such as orbital artery (oa) and encephalic artery (ea) separated from ebas and supplied blood to different anterior parts of the body. (fig. 4). figure 4. formation of dorsal aorta by joining of ebas. da (dorsal aorta), eba (efferent branchial arteries), ea (encephalic artery) and wb (weber bone). abas and afas were removed. figure 5. da (dorsal aorta), ebas (efferent branchial arteries), sca (subclavian artery) and wb (weber bone) are seen. 118 taheri mirghaed et al/ circulatory system of phractocephalus hemioliopterus using corrosion cast study other hand, the blood from anterior part of the kidneys (ak) and subclavian vein (scv) are joined to the rpcv and lpcv entering ccv (fig. 9). the pecardinal vein (prcv) transports the major parts of the blood from the head and brain to the heart through figure 6. lg (longitudinal groove) and wb (weber bone) and p (pores of the weber bone and other vertebrae). figure 7. vessels passing through the haemal arches. cv (caudal vein), da (dorsal aorta), ha (haemal arch) and s (spleen). figure 8. the path of the blood vessels from caudal area to the heart. a (atrium), abas (afferent branchial arteries), ba (bulbus arteriosus), ccv (common cardinal vein), cv (caudal vein), da (dorsal aorta), gv (gastric vein), hv (hepatic vein), iv (intestinal vein), lpcv (left post cardinal vein), pv (portal vein), rpcv (right post cardinal vein), rpv (renal portal vein), scv (subclavian vein) and v (ventricle). figure 9. ak (anterior kidney), ccv (common cardinal vein, pv (portal vein), prcv (precardinal vein), rpcv (right post cardinal vein), rpv (renal portal vein) and scv (subclavian vein). figure 10. ak: anterior kidney, ccv: common cardinal vein, hv: hepatic vein, jv: jugular vein, prcv: precardinal vein, rpcv: right post cardinal vein and pv: portal vein. 119 int. j. aquat. biol. (2018) 6(3): 114-121 ccv. the right prcv and rpcv are joined as a common part constituting ccv (figs. 9, 10). jugular vein (jv) transports the rest part of the blood from the head to ccv separately (fig. 10). finally, the collected blood in ccv is transported to the heart on right and left sides. the casts from liver showed wide vessels plexus (fig. 11a, b, c). bone plates had a dorso-ventral appearance extending from the head to the anterior part of the tail (fig. 12). discussion the corrosion cast study method can be used in pathological malformations and comparative anatomy (zeindler et al., 1989; hossler and douglas, 2001; nematollahi et al., 2011). in present study, the circulatory system of p. hemioliopterus including the different parts of heart and its vessels such as branchial arteries, dorsal aorta, common cardinal vein and hepatic vein were studied by corrosion cast methodology. we obtained a full casts of blood vessels perfusing various organs such as liver, stomach, spleen, posterior kidney and anterior kidney. according to observations, the length of the ventral aorta was short and after a short distance from the heart, a common trunk is separated from the ventral aorta. this branch is divided into two branches forming the branchial artery 1 and 2. in the following, the branchial artery 3 and 4 divided separately. these observations is correspond with those vascular pattern of common carp, but different with those of beluga and yellow stingray, showing similar pattern in bony fishes. however, there are some structural differences with common carp. the results also showed that the liver has two lobes of the same size locating behind of the heart. the caudal vein joins to the portal vein at the left side. as regards one of the most important problem in this fish is swallowing foreign bodies and subsequently it needs to surgical or non-surgical procedures (chansue and tangtrongpiros, 2005; ebrahimzadeh et al., 2006; wildgoose, 1998), therefore the results suggests that during surgical procedures it would be better that the incision must be done at right side of midline of this catfish. in most studies, the remained casts from liver show a wide vessel plexus because of high metabolic activity of this organ. according to the result, the right post cardinal vein (rpcv) was thicker than left post cardinal vein (lpcv) in p. hemioliopterus showing that major part of the blood of the renal portal vein is carried by (rpcv) that has not been reported in figure 11. vessels plexus in the heart and the liver. (a and b): liver and (c) heart. figure 12. dorsal view of the predorsal plate of red tail catfish. 120 taheri mirghaed et al/ circulatory system of phractocephalus hemioliopterus using corrosion cast study previous studies. however, presentation of a complete blood vessel system using a single corrosion cast can be led artifacts due to the extremely fine capillaries. furthermore researches are suggested to be done on other catfishes for comparison purpose. acknowledgments authors are thank s. ezhari for assisting with the figures. references basten b.l., sherman r.l., lametschwandtner a., spieler r.e. (2009). a unique vascular configuration among the efferent branchial arteries and splanchnic arteries in the yellow stingray (urobatis jamaicensis). microscopy and microanalysis, 15: 194-196. boland e.j., olson k.r. (1979). vascular organization of the catfish gill filament. cell and tissue research, 198: 487-500. chansue n., tangtrongpiros j. 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(2018) 6(3): 114-121 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی مطالعه(: phractocephalus hemioliopterus bloch & schneider, 1801) ردتیل ماهی گربه خون گردش سیستم عروق تحلیلی گیریقالب 2نژادصادقی جواد ،1موسوی زادهابراهیم حسینعلی ،1اردشیری مهرداد ،1هوالسو رحمتی هومن، 1*میرقائد طاهری علی .ایران تهران، تهران، دانشگاه ،دامپزشکی دانشکده ،آبزیان هایبیماری و بهداشت گروه1 .ایران تهران، تهران، دانشگاه دامپزشکی، دانشکده پایه، علوم گروه2 چکیده: هدف از این تحقیق مشاهده و توصیف سیستم . استعروف ( یکی از ماهیان زینتی مphractocephalus hemioliopterus) گربه ماهی ردتیل گرم استفاده 580عدد ماهی ردتیل با متوسط وزن 10. برای این منظور ازباشدمی گیری تحلیلی عروققالب گردش خون این گونه با استفاده از روش برای تکمیل پلیمریزه و سخت شدن متیل متاکریالت پر شد. عروق خونی ماهیان با رزین مصنوعی ساخته شده از متیلشد. پس از بیهوشی، قلب و غوطه ور شدند. در نهایت koh %25ساعت در محلول 48-24ساعت در حمام آب و سپس به مدت 12-24مدت متاکریالت، ماهیان در ادامه به ورت پشتی، عروق روده، معده و کبد، عروق ئسیستم گردش خون به شکل عروق آوران و وابران آبشش، بخش های مختلف قلب، آئورت شکمی، آ ه، طحال و نیز سیاهرگ پورتال و کبدی مشخص و نشان داده شد.خلفی و قدامی کلی .فیزیولوژی عروق، تحلیلی گیریقالب ردتیل، ماهی گربه خون، گردش متسیس :کلمات کلیدی international journal of aquatic biology (2013) 1(5): 240-244 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article body shape comparison of cyprinion macrostomum (heckel, 1843) and cyprinion watsoni (day, 1872) using geometric morphometrics method manoochehr nasri1, soheil eagderi*1, hamid farahmand1, iraj hashemzadeh segherloo2 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2department of fisheries and environmental sciences, faculty of natural resources and earth sciences, university of shahre kord, shahre kord, 115, iran. article history: received 16 september 2013 accepted 6 october 2013 available online 2 5 october 2013 keywords: cyprinidae cyprinion geometric morphometrics morphology abstract: this study investigated shape differences in two species of cyprinion macrostomum from tigris basin and cyprinion watsoni from hormuz basin using discriminate function analysis. coordinates of 17 external landmark points on 2d pictures were used for the analysis. there were significant differences of the two species. c. macrostomum have longer head length, snout length, preventral distance, head height, body height and length of pectoral fin bases than those of c. watsoni. the caudal peduncle length, caudal peduncle depth and anal fin base length in c. watsoni are longer than those of c. macrostomum, the pectoral fin in c. macrostomum was originated more posteriorly than that c. watsoni. based on the geometric morphometrics differences, the two species can be well recognized and differentiated. introduction species identification is the basic component of biodiversity conservation and fisheries management (ibañez et al., 2007). many biological aspects of an organism such as feeding efficiency, locomotion performance, vulnerability to predators, and reproductive success can be studied using body shape analysis (guill et al., 2003). fishes can adopt to the environment conditions in various ways to enhance their viability (nacua et al., 2010). hence, quantifying phenotypic differences among species may help to understand its natural history across a species’ geographic range, which have implications for both theoretical and applied works in ecological and fishery science. the traditional morphometrics is time consuming having lots of errors and low accuracy. genetic methods are costly and not readily available in the field (e.g., hutchinson et al., 2001; keyvanshokooh and kalbassi, 2006; ghasemi et al., 2007). in * corresponding author: soheil eagderi e-mail address: soheil.eagderi@ut.ac.ir geometric morphometrics, data is obtained from the coordinates of landmark-points (rohlf and marcus, 1993; adams et al., 2004), which are morphological meaningful points of specimens (richtsmeier et al., 2002). geometric morphometrics techniques have been used in many aspects of ichthyology including identification of fishery stocks (cadrin, 2000; mohadasi et al., 2013), studying the body shape variation within and between fish populations (nacua et al., 2010; heidari et al., 2013), analysis of head shape variation (cavalcanti, 2004), scale shape analysis to identifying species, genera, and local populations (ibañez et al., 2007), and body shape variation due to rearing temperature (sfakianakis et al., 2011). five species of the genus cyprinion have been reported from iran and there are some complexity in their taxonomy (bianco and banarescu, 1982; howes, 1982; banarescu and herzig-straschil, 1995; abdoli, 2000; coad, 2013). some works were 241 nasri et al/ international journal of aquatic biology (2013) 1(5): 240-244 performed on the morphological and biological aspects of this genus in iran, turkey, iraq and pakistan (kafuku, 1969; bianco and banarescu, 1982; banarescu and herzig-straschil, 1995; yilmaz et al., 2005; patimar and nasri, 2007; nasri, 2008; nasri et al., 2008; yüksel and gaffaroğlu, 2008) but all of them are based on traditional or descriptive methods. meanwhile, all members of the genus cyprinion in iran have relatively similar appearance (i.e. body shape) requiring subtle meristic data to distinguish them. even within these characters, there is not a considerable degree of variation. geometric morphometrics methods has not been used to assess the body shape variation among the genus cyprinion, therefore, this study was conducted to compare the body shape of two confirmed cyprinion species i.e. c. watsoni and c. macrostomum with visualization techniques afforded by the geometric morphometrics. the results of the present study can help to find morphological distinctions that may also be used to differentiate these two closely related species and better understanding of body shape pattern among the members of the genus cyprinion. materials and methods in total 64 specimens including, 24 c. macrostomum from the kashkan river (tigris basin) and 40 c. watsoni from the goodar river (hormuz basin) were collected using electrofishing (table 1). the specimens were fixed into 4% buffered formaldehid after anesthetizing in 1gl-1 clove solution and were transported to the laboratory for further examinations. in the laboratory, the specimens were identified using the mouth form, dorsal fin ray characters and the number of gill rakers according to abdoli (2000) and coad (2013). then, the left side of each specimen were photographed using digital kodak (6 mega pixels) camera. seventeen landmark-points were defined and digitized on 2d images using the tpsdig2 software version 2.16 (rohlf, 2010) (fig. 1). the adequacy of tangent shape for statistical analysis where investigated using the tpssmall (rohlf, 2003). the non-shape information were removed from landmark configurations using general procrustes analysis (gpa), the covariance matrices were generated and the shape differences between the two species analyzed using discriminant function analysis (dfa) in morphoj 1.02j (klingenberg, 2011). the patterns of body shape differences were illustrated in the wireframe in relation to each other for the quantification and visualization purposes. results the sum of digitization and orientation errors was 15% and the correlation between procrustes and tangent distances was 1, therefor tangent space approximation could be used for statistical analysis. the two species where separated based on discriminate function analysis with mahalanobis distance 8.3239 and p-value <0.0001 (fig. 2). depicting the differences in body shape between species number river basin province/town latitude longitude cyprinion macrostomum 24 kashkan tigris lorestan, pole-dokhtar 33°09'28"n 47°42'50"e cyprinion watsoni 40 goodar hormuz hormuzgan, bastak 27°19'27"n 54°27'46"e table 1. the geographical information of sampling sites. figure 1. seventeen defined landmarks on the left side of specimens. 1: the anterior-most point on the head; 2:the margin of head at the vertical nearest distance to the upper margin of the orbital; 3:the junction of the head and trunk; 4: the front edge of dorsal fin base; 5:the posterior edge of dorsal fin base; 6: the upper edge of caudal fin base; 7: the most distant point of lateral line at the base of caudal fin; 8: the lower edge of caudal fin base; 9: the posterior edge of anal fin base; 10: the front edge of anal fin base; 11: the lateral edge of pelvic fin base; 12: the outer edge of pectoral fin base; 13: the lower corner of opercular opening; 14: the lower margin of orbital; 15: the center of orbital; 16: the upper margin of orbital; 17: the end of the head. 241 242 nasri et al/ international journal of aquatic biology (2013) 1(5): 240-244 consensus body shapes of two species is presented in figure 3. discriminate function analysis indicated that c. macrostomum have longer head length, snout length, preventral distance, head height, body height and length of pectoral fin base to those of c. watsoni. the caudal peduncle length, depth of caudal peduncle and anal fin base length in c. watsoni are longer than those of c. macrostomum. the pectoral fin in c. macrostomum was originated more posteriorly than that of c. watsoni (figs. 3 and 4). discussion the results of this study showed that the body shape of cyprinion macrostomum and cyprinion watsoni are significantly different. this result was in agreement with previous works that used classical morphology of the genus cyprinion (e.g, banarescu and herzig-straschil, 1995; abdoli, 2000; nasri, 2008; nasri et al., 2008a; nasri et al., 2008b; coad, 2013). the observed differences can be divided in two categories. first a higher head height of c. macrostomum and, second, a deeper body and longer dorsal fin base in c. macrostomum, plus a longer caudal length in c. watsoni. whereas, based on previous findings, these two species could identified using the shape of mouth and dorsal fin ray that are more plastic characters. some authors considered the shape of species as a result of the environments and genetics (costa and cataudella, 2007; costa et al., 2010). environmental factors influence the shape of organisms via natural selection (chan, 2001). the kashkan river is a relatively large river with high productivity providing a lot of food resource, but the goodar river is a small stream with low productivity that is sometimes dried in summer. hence, some morphological differences between these two species may be related to phenotypic plasticity and responses to environmental conditions and depict different habitats. however, further studies are required to explain differences with different localities and all five species being included. the key characters to distinguish these two species are the strength and serration of the last unbranched dorsal fin ray and branched dorsal fin rays in c. macrostomum (coad, 2013). fishes like c. macrostomum living in high current rivers need more efforts for survival. there is no previous comparative study on body shape among cyprinion but coad (2013) noted that the dorsal fin in figure 2. the histogram of discriminate analysis separating the two species based on geometrics. figure 3. visualization of the relative shape differences among species based on wireframes. figure 4. visual differences between the two species (left: c. macrostomum, right: c. watsoni). 243 nasri et al/ international journal of aquatic biology (2013) 1(5): 240-244 c. macrostomum has been originated from the front of the pelvic fins. the dorsal fin as a rudder has more fin rays in c. macrostomum than c. watsoni. the origination of dorsal fin than pelvic fins in c. macrostomum may be due to the high number of dorsal fin rays and its importance for adaptation to the environment. in summary, this study was in agreement with the coad (2013) but also in c. watsoni the dorsal fin origin is in front of the pelvic fins. also our results have provided some morphological information to differentiate these two species more precisely. acknowledgments this study was supported by university of tehran (grant no: 25782/1/1). references abdoli a. 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(2018) 6(1): 15-20 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article the indian species of testudinella (rotifera: flosculariacea: testudinellidae) and their distribution bhushan kumar sharma*,1sumita sharma department of zoology, north-eastern hill university, shillong-793022, meghalaya, india. article history: received 13 november 2017 accepted 23 february 2018 available online 2 5 february 2018 keywords: biodiversity interesting elements littoral-periphytic rotifer richness abstract: our plankton and semi-plankton collections from india revealed 14 species of testudinella including two undetermined species yet awaiting descriptions. the oriental endemic t. insinuata is a new record from india while the australasian t. walkeri and the palaeotropical t. brevicaudata and t. greeni are other globally interesting species. testudinella amphora, t. brevicaudata, t. dendradena, t. greeni, t. parva semiparva, t. tridentata and t. walkeri are characterized by distribution restricted till date to northeast india (nei); t. insinuata is restricted to the floodplains of the kashmir valley of jammu and kashmir state of western himalayas; and t. incisa and t. mucronata are known for valid reports from tamil nadu and jammu and kashmir, respectively. t. emarginula, t. patina and t. tridentata are believed to be cryptic species-complexes and thus desired ecological and genetic analysis of local populations. this study merits interest vis-à-vis biodiversity and distribution of the indian rotifera. introduction rotifera had been documented and described from different states of india since pioneering taxonomic survey of anderson (1889) yet testudinella species (family: testudinellidae) were characterized by their little known nature in the indian waters (sharma and michael, 1980). this generalization holds valid even till date although sharma (1990) firstly gave an account of species from nei while sharma and sharma (2014a) maintained status-quo on the diversity of the taxon from this region. our more extensive plankton and semi-plankton collections from nei as well as elsewhere from india resulted in some interesting additions. we present an account of testudinella spp, including two undetermined species yet pending descriptions, known till date from this country. various taxa are illustrated and briefly commented with remarks on their distribution. the report is of interest for biodiversity and distribution of rotifera of the indian sub-region. materials and methods this present study is based on analysis of plankton and *corresponding author: bhushan kumar sharma doi: https://doi.org/10.22034/ijab.v6i1.401 e-mail address: profbksharma@gmail.com semi-plankton collections examined from wide range of habitats from various states of india including our extensive collections from seven states of northeast india (nei). the samples were collected by towing a nylobolt plankton net (50 µm) and were preserved in 5% formalin. individual specimens were screened with wild-stereoscopic binocular and mounted in polyvinyl alcohol-lactophenol mixture. illustrations are made with a leica dm 1000 phase contrast microscope using an image analyzer. testudinella spp. was identified following koste (1978), sharma (1990) and sharma and sharma (2008, 2013, 2015). results presented below is the systematic list of testudinella spp. observed from india (# new record from india; * examined in our collections from northeast india): phylum: rotifera class: eurotatoria subclass: monogononta order: flosculariacea family: testudinellidae 1. testudinella amphora hauer, 1938* 16 sharma and sharma / the indian species of testudinella 2. t. brevicaudata yamamoto, 1951* 3. t. dendradena de beauchamp, 1955* 4. t. emarginula (stenroos, 1898) s. lato* 5. t. greeni koste, 1981* 6. t. incisa (ternetz, 1892) 7. t. insinuata hauer, 1938 #* 8. t. mucronata (gosse, 1886) 9. t. parva parva (ternetz, 1892)* t. parva bidentata (ternetz, 1892)* t. parva semiparva hauer, 1938* 10. t. patina (hermann, 1783) s. lato* 11. t. tridentata smirnov, 1931 s lato* 12. t. walkeri koste & shiel, 1980* 13. t. sp. figure 1. testudinella spp. from india: (a) t. insinuata hauer (ventral view), (b) t. walkeri koste & shiel (ventral view), (c) t. brevicaudata yamamoto (ventral view), (d) t. greeni koste (ventral view), (e) t. amphora hauer (ventral view), (f) t. dendradena de beauchamp (ventral view), (g) t. parva parva (ternetz) (ventral view), (h) t. parva bidentata (ternetz) (ventral view) and (i) t. parva semiparva hauer (ventral view) (after sharma, 1990). 17 int. j. aquat. biol. (2018) 6(1): 15-20 14. t. sp. 1 testudinella insinuata hauer (fig. 1a) is a new record from india. testudinella walkeri (fig. 1b) and, the palaeotropical t. brevicaudata (fig. 1c) and t. greeni (fig. 1d) are species of global distribution interst. testudinella amphora (fig. 1e), t. dendradena (fig. 1f), t. incisa, t. parva parva (fig. 1g), t. parva bidentata (fig. 1h), t. parva semiparva (fig. 1i), t. mucronata (fig. 1j) and t. tridentata (fig. 1k) are examples of the regional distribution interest in the indian subcontinent while t. patina (fig. 1l) is widely known from this country. in addition, testudinella sp. (fig. 1m) and t. sp. 1 (fig. 1n) are un-determined species awaiting descriptions pending examination of more specimens. the former is characterized by its elongated oblong lorica morphologically distinct from other known species of testudinella while t. sp. 1 is notable for its pear-shaped lorica with closer affinity with t. patina species-complex. discussion a total of 14 species of testudinella (including two undetermined species awaiting descriptions) documented from india assert the most speciose nature of the genus known till date from south and southeast asia and from the indian sub-region in particular. this report assumes additional interest in view of the occurrence of 15 species of the taxon from the oriental region (segers, 2008) amongst nearly four dozen valid species known globally (segers, 2007) while jersabek and leitner (2013) raised totally tally to 60+ species. the richness of the indian testudinella spp. is higher than 10 species reported (sa-ardrit et al., 2013) from otherwise well studied thai rotifera. testudinella insinuata is a new record from india; originally described from botanical garden in buitenzorg, java, indonesia, this species was considered a synonym of t. parva (ternetz) by sanoamuang and savatenalinton (2001), segers et al. figure 1. continued: (j) t. mucronata (gosse) (ventral view), (k) t. tridentata smirnov (ventral view), (l) t. patina (hermann) (ventral view), (m) t. sp. (ventral view) and (n) t. sp. 1 (ventral view). 18 sharma and sharma / the indian species of testudinella (2004) and segers (2007). we designate it as a valid species following jersabek and leitner (2013) and in view of distinct morphological differences between two species. this oriental endemic is observed in our collections from the floodplains of the kashmir valley of jammu and kashmir state (bks, unpublished) of western himalayas. the australasian t. walkeri is an interesting recent addition to the indian fauna from mizoram state (sharma and sharma, 2015a). the report of this species endorsed affinity of rotifera assemblage of nei (sharma and sharma, 2014a, 2014b, 2017) with the faunas of southeast asia and australia. the palaeotropical t. brevicaudata and t. greeni are other two species of global distribution interest; the former was described from japan and is now known from the afrotropical, oriental and palaearctic regions (segers, 2007). testudinella greeni, described from australia, is reported from the afrotropical, australian, neotropical and oriental regions (segers, 2007). interestingly, this species was noticed to be restricted to the floodplain lakes of the brahmaputra river basin of assam (sharma and sharma, 2014a, 2014b) while it is now observed from certain small wetlands of mizoram (sharma and sharma 2015a) and nagaland (sharma et al., 2017) states thus indicating distinct distribution in nei. testudinella amphora, t. dendradena, t. incisa, t. parva parva, t. parva bidentata, t. parva semiparva, t. mucronata and t. tridentata are examples of the regional distribution interest in the indian subcontinent. of these, t. amphora, t. brevicaudata, t. dendradena, t. greeni, t. parva bidentata, t. parva semiparva, t. tridentata and t. walkeri are characterized by distribution exclusively restricted to northeast india (nei). further, t. parva semiparva recorded rare occurrence with the sole report from assam (sharma, 1990) and t. parva bidentata, is known till date from assam (sharma, 1990; sharma and sharma, 2001, 2008, 2014a) while t. dendradena is observed from assam (sharma and khan 2016; sharma et al., 2017) and nagaland (sharma et al., 2017). testudinella mucronata is validly known only from jammu and kashmir (edmondson and hutchinson, 1934); this species is also observed in our collections from the kashmir valley. on the other hand, we categorize questionable its ‘routine’ invalidated listings from andhra pradesh, karnataka, maharashtra and tamil nadu and west bengal. in addition, t. incisa is reported only from tamil nadu (edmondson and hutchinson, 1934) but was not noticed in our collections from this state (sharma and sharma, 2009). our collections from nei revealed two undescribed species of testudinella awaiting descriptions pending examination of more specimens. in addition, we consider t. emarginula, t. patina and t. tridentata to be variable morphospecies that probably represent cryptic species-complexes and thus recommend ecological and genetic analysis of local indian populations vis-à-vis cryptic diversity following jersabek and leitner (2013) and fontaneto (2014). we report species-rich testudinella (11 species) from northeast india (nei); this salient feature reiterates importance of this region as a part of the himalayan and the indo-myanmar biodiversity ‘hotspots’. high richness of the taxon is also endorsed by individual reports from seven states of nei with high records from arunachal pradesh (6 spp.: bks unpublished), assam (8 spp.: sharma and sharma, 2014a; bks unpublished); manipur (7 spp.: sharma et al., 2016; bks unpublished); nagaland (7 spp.: sharma and kensibo, 2017; sharma et al., 2017); meghalaya (7 spp.: sharma and sharma, 1999; sharma et al., 2016); tripura (7 spp.: sharma and sharma, 2000; bks unpublished) and mizoram (7 spp.: sharma and sharma, 2015a; bks unpublished). interestingly, all testudinella spp. known from nei are observed from the floodplain lakes of assam (commonly called as beels) and manipur (commonly called as pats) and thus highlight ecosystem diversity interest of these wetlands. this salient feature is well supported by the report of high richness (7 spp.) from three beels of the dibru-saikhowa biosphere reserve, upper assam as well as the reports of 6 spp. each from two ramsar sites located in nei namely deepor beel (sharma and sharma, 2015b; bks unpublished) and loktak lake (sharma et al., 2016; bks unpublished). on the other hand, our collections from the floodplains of the kashmir valley, jammu and 19 int. j. aquat. biol. (2018) 6(1): 15-20 kashmir state of western himalayas revealed 4 spp. with t. insinuata as a new record from india while t. emarginula is a new record from north india. testudinella is represented by lower richness in the collections from eastern india with only two species each known from west bengal (sharma, 1998) and bihar (sharma et al., 1992), and 3 species recorded from orissa or odisha state (sharma and sharma, 2005). further, we report (bks, unpublished) 2 species from madhya pradesh and 3 species from maharashtra from central india while we observed (bks, unpublished) 3 species from tamil nadu and two species each in our collections from andhra pradesh, telangana, karnataka and kerala of southern india. we caution on over-emphasis of the stated comparisons because of the differences in the sampling intensity. nevertheless testudinella is more species-rich in nei and in even our limited collections from the kashmir valley of the western himalayas than elsewhere from india. to sum up, this study provides interesting information on biodiversity, distribution and ecosystem diversity of the indian species of testudinella. we estimate richness of the taxon to be still higher pending analysis of more collections from the indian himalayas (western, central and eastern) with emphasis on samples from the states himachal pradesh, uttrakhand, jammu and kashmir, sikkim and higher latitudes of arunachal pradesh as well as western ghats. ecological and genetic analysis of cryptic diversity of t. emarginula, t. patina and t. tridentata species-complexes shall merit interest for analysis of genetic diversity of the indian rotifera. acknowledgements thanks are due to the head, department of zoology, nehu, shillong, for laboratory facilities. we are pleased to dedicate this study collectively to research students and to all those who helped us in our field collections during our long work experience of more than three decades of research interest on the biodiversity of indian rotifera. the authors have no conflict of interests. references anderson h.h. 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(2015). rotifer assemblages (rotifera: eurotatoria) of the floodplain lakes of majuli river island, the brahmaputra river basin, northeast india. international journal of aquatic biology, 3(1): 1-13. sharma s., sharma b.k. (2008). zooplankton diversity in floodplain lakes of assam. records of the zoological survey of india, occasional paper no., 290: 1-307. sharma s., sharma b.k. (2013). faunal diversity of aquatic invertebrates of deepor beel (a ramsar site), assam, northeast india. wetland ecosystem series, 17: 1-226. zoological survey of india, kolkata. international journal of aquatic biology (2014) 2(5): 223-228 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article effect of different cooking processes on the fatty acid profile of grass carp (ctenopharyngodon idella) fillets during chill storage ensieh hayati-jafar beigi1, ebrahim alizadeh1*,1eshagh zakipour rahimabadi1, mostafa yousef elahi2 1department of fisheries, faculty of natural resources, university of zabol, zabol 98615-538, iran. 2department of animal science, faculty of agriculture, university of zabol, zabol 98615-538, iran. article history: received 29 may 2014 accepted 23 september 2014 available online 2 5 october 2014 keywords: grass carp fatty acid cooking chill storage abstract: the effects of different cooking methods (deep fat frying, boiling and steaming) on lipid content and fatty acid composition of grass carp (ctenopharyngodon idella) fillets during chill storage were investigated. fillet samples were cooked and then stored at + 4°c for 4 days. the control and the cooked fillet samples were analyzed for their chemical characteristics. twelve fatty acids were identified with 39.11, 15.37 and 45.52 g/ 100 g of saturated (sfa), monounsaturated (mufa) and polyunsaturated fatty acids (pufa), respectively. the n-3/n-6 ratios of raw, deep fried, boiled and steamed samples were 1.12, 0.10, 1.45 and 0.72, respectively. the pattern of changes in fatty acid groups was different in fried, boiled and steamed samples after 1 and 4 days of chill storage. the epa+dha/c:16 ratio was higher in boiled and boiled-chill stored samples than steamed and fried samples. epa+dha/c:16 ratio for cooked, stored for 1 day and then 4 days were as 0.051, 0.003 and 0.017 for fried, 0.492, 0.583 and 0.489 for boiled and 0.247, 0.037 and 0.149 for steamed samples, respectively. these results showed that the boiled process is better than other cooking processes on the fa pattern of grass cap. introduction different aspects of beneficial effects of fish lipid on human health have been widely established (arts et al., 2001; connor, 2000) because of its long chain polyunsaturated fatty acids of n-3. various environmental and biological factors and different processing method can effect on the content of lipid and composition of fatty acids of fish species (sigurgisladóttir and pálmadóttir, 1993; love, 1997). it is well-known that changes in lipid content and fatty acid composition in frying process are higher than other cooking processes (gladyshev et al., 2006; gladyshev et al., 2007; bakar et al., 2008; larsen et al., 2010). therefore, the pattern changes of long chain polyunsaturated omega-3 fatty acids are different based on fish species and cooking procedure (garcía-arias et al., 2003; gladyshev et * corresponding author: ebrahim alizadeh e-mail address: ebi_alizadeh2003@yahoo.com al., 2007; bakar et al., 2008; larsen et al., 2010; nikoo et al., 2010a). the cooking process, chill storage and reheating is a common practice in large catering operations, restaurants, and homes (bakar et al., 2008). the changes in lipid content and fatty acid composition have studied in cook-chill-reheat or cook-freezereheat by garcía-arias et al. (2003), bakar et al. (2008) and nikoo et al. (2010a, b). although there are many references regarding the effects of cooking on fish fatty acid composition, data related to the effect of cooking and chill storage on fatty acid composition and lipid content of grass carp (ctenopharyngodon idella) is limited. therefore, this research was aimed to study the effects of different cooking processes on lipid content and fatty acid profile of grass carp fillets during chill storage. 224 international journal of aquatic biology (2014) 2(5): 223-228 materials and methods sample preparation: twenty fresh grass carp with weight of about 1.5 kg were purchased from a local market (zabol, iran). they were transported in isothermal iceboxes to the laboratory 5 hrs after catching. then, they were cleaned and filleted and same weight of fillets (about 100 g) were used for cooking process (deep fat frying, boiling and steaming). the cooked samples were placed in moisture-impermeable plastic bags, stored in chill room (+4°c) and analyzed on 0, 1 and 4 days. the experiences were performed with three replicates. cooking process: fish fillets were fried in frying oil (sunflower oil, bahar, iran) using a deep fryer (aaura, tefal, iran) for 6-7 min which was preheated to 180ºc. the fillets were boiled in a small water under 85-90ºc for 10-15 min (gladyshev et al., 2006). for steaming, samples were placed in a steamer (panasonic, japan) and steamed for 5–6 min. after cooking all samples were drained gently on a stainless steel grills and air cooled. chemical analysis: soxhlet apparatus with diethyl ether as solvent was used to measuring the total lipid content (aoac, 2000) and lipid extraction was performed based on bakar et al. (2008). lipid samples were converted to their constituent fatty acid methyl esters according to metcalf et al. (1966). analysis of fatty acid methyl esters was performed by a unicam 4600 with a bpx 70 capillary column (30.0 m x 0.25 mm i.d.) and quantified by fid detector. the split ratio was 10:1. the gc condition was as follows: injection port temperature was 300ºc and fid temperature was 350ºc. oven temperature program was set at an initial temperature of 160ºc for 6 min, then raised to 180ºc at 20ºc min-1 and held for 9 min and again was raised to 190ºc at 20ºc min1 and held for 14 min (metcalf et al., 1966). the carrier gas was helium. the column flow rate was 1.9 ml min-1. in the detector, helium gas flow rate was 30 ml min-1. the sample size was 1 μl. statistical analysis: the data were analyzed using one-way analysis of variance (anova) followed by tukey’s test. the significance of results was at 5%. all data are expressed as mean ± s.d. results the total lipid content of raw grass carp fillet was 1.44% (based on wet weight). deep frying significantly (p<0.05) increased the fillet lipid content to 26.7%. there was slight increase in lipid content of boiled and steamed samples (table 1). the fatty acid composition of raw samples is shown in table 2. twelve fatty acids were identified with 39.11, 15.37 and 45.52 g/100 g of saturated (sfa), monounsaturated (mufa) and polyunsaturated fatty acids (pufa), respectively. in raw samples, the abundance of fatty acids (in decreasing order) were palmitic acid (c16:0, with 29.27 %), oleic acid (c18:1 n-9, with 14.65 %), linoleic acid (c18:2 n-6, with 12.44 %), docosahexaenoic acid (c22:6 n-3, with 9.20 %) and arachidonic acid (c20:4 n-6, with 9.02 %). n-3 and n-6 fatty acids constitute about 52.06 and 47.14 % of pufas, respectively, exhibiting an n-3/ n-6 ratio of 1.12. the abundance of fatty acid groups (in decreasing order) in fried, boiled and steamed samples were mufas > pufas > sfas, pufas > sfas > mufas similar to raw samples and mufas > sfas > pufas, respectively (table 3). in boiled samples, content of higher polyunsaturated fatty acids was decreased, while c18:3 (n-3) significantly increased (p<0.05). the patterns of changes in fatty acid groups was different in fried, boiled and steamed samples after 1 and 4 days of chill storage (table 3). after 1-day storage of fried samples, the sfas levels were increased significantly (p<0.05), samples raw cooked deep fried boiled steamed lipid content (%) 1.44 26.7 1.94 1.94 table 1. lipid content of raw and cooked of grass carp fillets (% basis on wet weight) 225 hayati-jafar beigi et al/ effect of different cooking processes on the fatty acid profile of grass carp whereas slight decrease were observed in mufas and pufas. a significant increase were found in mufas levels of boiled samples after 1 days of storage (p<0.05), while the content of pufas were significantly decreased (p<0.05) (table 3). longer storage of steamed samples in chill room was led to little increase in sfas, significant increase in mufas and also significant decrease in pufas, respectively (p<0.05) (table 3). discussion the average lipid content of raw samples in this study was slightly less than the values reported by wu and mao (2008) and ojagh et al. (2009) for grass carp. among the biological and environmental factors, diet has a great effect on the lipid content of fish flesh (sigurgisladóttir and pálmadóttir, 1993). based on the classification by suriah et al. (1995), grass carp may be classified as lean fish with lipid content below 5%. after frying, fat content of fillet samples significantly (p<0.05) increased. this result is in agreement with those of garcia-arias et al. (2003), gokoglu et al. (2004), weber et al. (2008) and hakimeh et al. (2010), although the value obtained in this study was higher. the increase of total lipid table 2. fatty acid composition of grass carp fillets changes after cooking and chill storage. values are mean ± standard deviation of two determinations. capital letters (a, b, c) in the same line indicate significant differences (p<0.05) of storage. small letters (a, b, c, d, e) in the same line indicate significant differences (p<0.05) of treatment. fatty acid (g/100 g fatty acids) raw fillet fried samples boiled samples steamed samples day 0 day 1 day 4 day 0 day 1 day 4 day 0 day 1 day 4 ∑ sfa 39.112 30.300 33.774 31.233 32.443 32.940 34.887 33.662 32.939 33.992 ∑ mufa 15.370 35.576 33.267 32.919 17.767 39.613 39.263 41.729 32.373 35.441 ∑ pufa 45.517 34.125 32.959 35.848 49.790 27.447 25.850 24.610 34.689 30.567 ∑ n-3 24.061 3.211 1.855 3.151 29.450 19.335 19.498 10.288 4.515 11.749 ∑ n-6 21.456 30.914 31.104 32.697 20.340 8.112 6.352 14.322 30.174 18.818 n-3/ n-6 1.121 0.104 0.060 0.096 1.448 2.380 3.069 0.718 0.150 0.624 table 3. changes in fatty acid groups of grass carp fillets after cooking and chill storage. 226 international journal of aquatic biology (2014) 2(5): 223-228 content was not significant in boiled and steamed samples. similar findings have reported by weber et al. (2008) and gokoglu et al. (2004) for boiling and by hakimeh et al. (2010) for steaming. cooking induces water loss in the food, which in turn increases its lipid content (hoffman et al., 1994; garcía-arias et al., 2003). fat increase in fried sample could be due to oil penetration into the food after water is partially lost by evaporation. ågren and hänninen (1993) have concluded that additional oil in frying, mainly determines the small and lean fish lipid content. the fatty acid profile in this study was similar to that found by ojagh et al. (2009) and wu and mao (2008) for grass carp, although in present study, the content of pufas and mufas were higher and lower, respectively. based on the results, the content of c18:1 n-9c and c18:2 n-6c increased significantly after frying, while the content of other fatty acids decreased, especially docosahexaenoic acid and arachidonic acid. garcíaarias et al. (2003) and bakar et al. (2008) reported similar observations for shallow fat frying where the cooking process had significantly affected the fatty acid composition of fish, increasing oleic and linoleic acids and decreasing eicosapentaenoic and docosahexaenoic acids. it has reported that fatty acid profiles of fish in frying processes became similar to those of the culinary fat used (arias et al., 2003; bakar et al., 2008). the saturated fatty acid content decreased significantly in boiled samples. this could be explained by the fact that sfas are largely represented in neutral lipids and are more prone to migration (enser et al., 1996; badiani et al., 2002). in steamed samples, higher polyunsaturated fatty acid content decreased significantly, whereas the content of c18:1 (n-9) was increased. this finding is not in agreement with finding of bakar et al. (2008) and larsen et al. (2010) who reported no differences between steamed and raw samples. the n-3/n-6 ratio of deep fried, boiled and steamed samples were 0.10, 1.45 and 0.72, respectively. the longer storage of fried samples in chill room was caused a slight decrease in sfas and mufas and slight increase in pufas. the changes in fatty acid profile during storage in chill room could be due to changes in sfa and mufa content which are neutral lipids and more prone to migration (enser et al., 1996; badiani et al., 2002) and also oxidation progress during storage (bakar et al., 2008; nikoo et al., 2010a). the sfas levels increased after 4 days of storage of boiled samples, while the levels of pufas decreased. nikoo et al. (2010b) observed that the content of polyunsaturated fatty acids of rutilus frisii kutum decrease, whereas the content of saturated fatty acids increased after 2 days of refrigerated storage. a significant decrease and increase of mufas and pufas were observed for the steamed king mackerel after 1-day of storage, respectively, while the decrease of sfas was not significant (bakar et al., 2008). nutritional value of lipids is determined not only by the composition of fatty acids, but also by their ratio (simopoulos, 1999; schmitz and ecker, 2008). imbalance in the ratio of n-6 and n-3 acids (which should be about 3-5:1) can contribute to the development of cancer and the various kinds of inflammation (el-badry et al., 2007). this research demonstrates that, the n-3/n-6 ratio decreased to 0.060 and then increased to 0.096 after 1 and 4 days of storage in fried samples. this ratio increased to 2.380 and 3.069 after 1 and 4 days of storage in boiled samples. in steamed samples, n-3/n-6 ratio decreased to 0.150 and then increased to 0.624, respectively. these results indicate that the fatty acid composition of fried sample was affected by the frying oil agreeing with previous studies (amira et al., 2010; kitson et al., 2009). the epa+dha/c:16 ratio was higher in boiled and boiledchill stored samples than steamed and fried samples. epa+dha/c:16 ratio of cooked, stored for 1 day and then 4 days were 0.051, 0.003 and 0.017 for fried samples, 0.492, 0.583 and 0.489 for boiled samples and 0.247, 0.037 and 0.149 for steamed 227 hayati-jafar beigi et al/ effect of different cooking processes on the fatty acid profile of grass carp sample, respectively. some researchers reported that there was a significant effect of frying on the epa and dha levels in fried fish (özogul et al., 2009; gladyshev et al., 2006). the decrease in the epa and dha levels after frying may have been resulted from susceptibility of highly unsaturated fatty acids (hufa) to oxidation during heating. also, this reduction may have been caused by the absorption of oil during frying. the results show that cooking and chill storage affect the total lipid and fa profile of samples. this study concluded that boiling method retain the fatty acid composition such as n-3 better than other cooking methods. therefore it is recommended for cooking of grass carp. references ågren j.j., hänninen o. (1993). effects of cooking on the fatty acids of three freshwater fish species. food chemistry, 46: 377-382. amira m.b., hanene j.h., madiha d., imen b., mohamed h., abdelhamid c. (2010). effects of frying on the fatty acid composition in farmed and wild gilthead sea bream (spaurus aurata). international journal of food science and technology, 45(1): 113–123. aoac. (2000). official methods of analysis (17th ed.). association of official analytical chemists, procedure. washigton. dc, usa. arts m.t., ackman r.g., holub b.j. (2001). essential fatty acids in aquatic ecosystem: a crucial link between diet and human health and evolution. canadian journal of fisheries and aquatic science, 58: 122-137. badiani a., stipa s., bitossi f., gatla p., vignola g., chizzolini r. (2002). lipid composition, retention and oxidation in fresh and completely trimmed beef muscles are affected by common culinary practices. meat science, 96: 169-186. bakar j., zakipour rahimabadi e., cheman y.b. (2008). lipid characteristics in cooked-chill-reheated fillets of indo-pacific king mackerel (scomberomorous guttatus). food science and technology, 41: 21442150. connor w.e. (2000). importance of n-3 fatty acids in health and disease. american journal of clinical nutrition, 71: 171s -175s. el-badry a.m., graf r., clavien p.a. (2007). omega 3 – omega 6: what is right for the liver? journal of hepatology, 47(5): 718-725. enser m., hallett k., hewitt b., fursey g.a.j., wood j.d. (1996). fatty acid content and composition of english beef, lamb and pork at retail. meat science, 42(4): 345-358. garcía-arias m.t., álvarez pontes e., garcıá-linares m.c., garcıá-fernández m.c., sánchez-muniz f.j. (2003). cooking-freezing-reheating (cfr) of sardine (sardina pilchardus) fillets. effect of different cooking and reheating procedures on the proximate and fatty acid compositions. food chemistry, 83(3): 349-356. gladyshev m.l., sushchik n.n., gubanenko g.a., demirchieva s.m., kalachova g.s. (2006). effect of way of cooking on content of essential polyunsaturated fatty acids in muscle tissue of humpback salmon (oncorhynchus gorbuscha). food chemistry, 96(3): 446-451. gladyshev m.l., sushchic n.n., gubanenko g.a., demirchieva s.m., kalachova g.s. (2007). effect of boiling and frying on the content of essential polyunsaturated fatty acids in muscle tissue of four fish species. food chemistry, 101: 1694-1700. gokoglu n., yerlikaya p., cengiz e. (2004). effects of cooking methods on the proximate composition and mineral contents of rainbow trout (oncorhynchus mykiss). food chemistry, 84: 19-22. hakimeh j.a., akram a.a., bahareh s., alireza s.m. (2010). physicochemical and sensory properties of silver carp (hypophthalmichthys molitrix) fillets as affected by cooking methods. international food research journal, 17: 921-926. hoffman l.c., prinsloo j.f., casey n.h., theron j. (1994). effects of five cooking methods on the proximate, fatty acid and mineral composition of fillets of the african sharptooth catfish, clarias gariepinus. die sa tydskrif vir voedselwetenskap en voeding, 6(4): 146-152. kitson a.p., patterson a.c., izadi h., stark k.d. (2009). pan-frying salmon in an eicosapentaenoic acid (epa) and decosahexaenoic acid (dha) enriched margarine prevents epa and dha loss. food chemistry, 114(3): 927-932. larsen d., quek s.y., eyres l. (2010). effect of cooking method on the fatty acid profile of new zealand king 228 international journal of aquatic biology (2014) 2(5): 223-228 salmon (oncorhynchus tshawytscha). food chemistry, 119: 785-790. love r.m. (1997). biochemical dynamics and the quality of fresh and frozen fish. in: g.m. hall (ed.). fish processing technology, london, blackie academic and professional. pp 1-31. metcalfe i.d., schmitz a.a., pelka j.r. 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(20212 10(3): 229-233 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article effect of mixed and artificial feeding on the growth performance of gattan, luciobarbus xanthopterus heckel, 1843 larvae nawras a. al-faiz1, ghassan a. al-najar1, faleh m. al-zaidy2, kadhim h. younis2, amir a. jabir2 1college of marine sciences, university of basrah, basrah, iraq. 2marine science center, university of basrah, basrah, iraq. s article history: received 11 march 2022 accepted 3 may 2022 available online 2 5 june 2022 keywords: feeding native fish algae barbus gattan larvae abstract: this work aimed to study the rearing feasibility of luciobarbus xanthopterus larvae using artificial and mixed (live (chlorella sp.) + artificial) food and their effects on their growth performance during early development. larvae (1.65 cm in length and 0.02 g weight) were obtained from a marine science hatchery and cultured in indoor tanks for 35 days. the larvae fed mixed feed t1 and artificial feed t2 (fish meal + soybean meal). the results showed that the larvae of t1 treatment outperformed significantly in final length, final weight, final weight gain, daily and specific growth rate, which amounted to 3.44 cm, 0.3568 g, 0.3368 g, 0.0096 g/day, and 8.2185 % weight/day, respectively. also, the results showed that larvae fed on t1 grew faster. the present study showed that applying a mixture of artificial and live food after four weeks’ age i.e. after absorption of the yolk sac for feeding larvae can reduce the costs of producing and providing better growth and survival rates. introduction food security is a serious issue because of the rapid population increase. aquaculture is the fastestgrowing sector in animal protein production (garcia et al., 2005; daniel, 2018). it is one of the world's most important high-quality protein sources (fao, 2020; stankus, 2021). the larvae rearing stage is the most challenging practice in the aquaculture industry. fish larvae are cultured under highly controlled circumstances, particularly regarding feeding strategies (lavens and sorgeloos, 1996). cyprinids are of great economic importance taxa since it includes the most important farmed fishes globally (fao, 2014). gattan, luciobarbus xanthopterus is an important species in the inland waters of iraq. it has high economic value and is a favorite fish among iraqi consumers. in the iraqi part of the tigris and euphrates river system, it is prevalent, especially in the central and southern regions (al-rudainy, 2002). introducing alien fish species, and overfishing have declined the native fishes, such as gattan, and even correspondence: nawras a. al-faiz doi: http//doi.org/10.22034/ijab.v10i3.1591 e-mail: nawras.abdulzahra@uobasrah.edu.iq this species has disappeared in some regions (alnajare, 2020). the production of freshwater fishes in iraq consists of the common carp, grass, and silver carp, and there are no sufficient attempts to develop those native fish candidates to rear due to the limitation of supplying the quality larvae (fao, 2011). according to bawazeer (1981) in lake habbaniyah, and polservice consulting engineers (1983) in lake tharthar, gattan did not attract the interest of fish farmers, because of its slow-growing in natural waters (al-rudainy et al., 1997). later studies showed that gattan is suitable for the culture as commercial production in ponds (almukhtar et al., 2009) based on the results of its growth performance indicators, nutrition, and environmental factors tolerance (al-mahdawi et al., 1996; al-rudainy et al., 1997; al-azawi et al., 1999; al-rudainy et al., 2002; al-rudainy et al., 2004). larvae rearing is one of the most crucial phases in fish farming. this stage is the most challenging stage in the life history of fishes since the mortality is high 230 al-faiz et al./ effect of mixed and artificial feeding on the growth performance of gattan during this stage. this study aimed to investigate the rearing feasibility of gattan larvae in indoor systems using artificial and mixed (live + artificial) food and their effects on their growth performance during early development. materials and methods gattan larvae were obtained from the fish hatchery of the marine sciences center, university of basrah, and transferred to the marine sciences center's aquaculture laboratory using three-liter plastic containers equipped with aerators. the larvae were acclimated to the laboratory conditions using live food for 14 days. the experiments were performed in 200 l fiberglass tanks equipped to aerators. after the acclimatization period, the larvae were distributed into the rearing tanks (200 l) at 50 larvae/tank (larvae had an average weight of 0.020 g, and an average total length of 1.65 cm) i.e. a density of 1 larva/4 liter. throughout the 35-day experiment period, the larvae were fed to satiation using two food treatments, including t1 (a mixture of fish meal, soybean meal, and algae (chlorella sp.)) and t2 (artificial food consisting of fish meal and soybean meal) as twice a day. larvae’ total length and weight were measured weekly using a ruler and sensitive balance (type mettler model ae163), respectively. some of the physical and chemical environmental factors, including temperature, dissolved oxygen, ph, and salinity, were monitored during the rearing period using a german-made livebond water quality meter. the larvae's growth performance was evaluated using weight gain, daily growth rate (dgr), and specific growth rate (sgr) based on the following formulas: weight gain (g) = final weight initial weight dgr (g / day) = final weight initial weight / time (days) sgr (% weight / day) = 100 × (ln final weight – ln initial weight) / time (days) statistical analysis: the data was analyzed using spss version 22.0. one-way analysis of variance (one-way anova) was used to compare the treatments. the significant difference between means was examined by the least significant difference test (lsd test). all the differences were considered significant at p<0.05. results during the rearing period, the temperature was 25.6227.32ºc, dissolved oxygen 6.84-6.96 mg /l, salinity 1.70-2.55‰ and ph 7.46-7.55 (table 1). the total length, final weight, and weight gain rates, and the daily and specific growth rates of larvae fed t1 and t2 treatment are shown in table 2. the results showed a significant increase in the mean total length of t1 (3.440 cm) compared to t2 (3.18 cm) (p<0.05). the highest final weight and weight gain were found in larvae of t1 (0.3568 and 0.3368 g, respectively), with a significant difference (p<0.05) compared to t2 (0.2796 and 0.2596 g, respectively). the highest daily growth rate (dgr) was 0.0096 g/day, and the highest specific growth rate (sgr) was 8.2185% weight/day in the larvae of t1. in t2, they were 0.0074 g/day and 7.5281% weight/day, respectively, having significant differences (p<0.05) between the treatments. figure 1 shows weekly changes in mean length, mean weight, (mean weight gain, dgr, and sgr of l. xanthopterus larvae during five weeks rearing period. the larvae's mean total length, weight, and weight gain were increased during five weeks of the rearing period. the highest values of daily and specific growth rates during the experiment period were recorded in larvae of t1. environmental variables time 7 days 14 days 21 days 28 days 35 days temperature ◦c 25.62±0.16 25.77±0.23 26.48±0.15 26.88±0.12 27.32 ±0.35 dissolved oxygen (mg/l) 6.85±0.11 6.84±0.13 6.96±0.17 6.87±0.11 6.90±0.08 salinity ‰ 1.70±0.22 2.09±0.08 2.55±0.38 2.50±0.35 2.47±0.40 ph 7.51±0.08 7.54±0.17 7.46±0.11 7.47±0.12 7.55±0.19 table 1. measurements of some environmental factors of the rearing tanks (data expressed as mean±sd). 231 int. j. aquat. biol. (2022) 10(3): 229-233 discussion the environmental factors in the present study showed that temperature, dissolved oxygen, salinity, and ph values were within the suitable ranges for rearing gattan larvae (ghazi, 2009; yesser et al., 2016). it is crucial to determine the nutritional requirements of fish larvae to enhance their growth performance and survival rates (radhakrishnan et al., 2020). live food is the best food for fish cultivation since it has essential fish nutrition (bengtson, 2003). studies have indicated the importance of using protein sources having essential amino acids, phospholipids, and fatty acids in feeding aquatic organisms (daniel, 2018). when live food suffers from a lack of essential nutrient components, it cannot be adopted as a single diet for larvae to enhance their growth performance, therefore, it needs to use mixing food (radhakrishnan et al., 2020). prolonged use of live food for rearing fish larvae is often impractical and expensive, and it is necessary a transformation to an artificial diet (akbari diets parameters t2 t1 1.65±0.01a 1.65±0.01a initial length (cm) 0.020±0.002a 0.020±0.002a initial weight (g) 3.1800±0.1483b 3.4400±0.1140a final length (cm) 0.2796±0.0232b 0.3568±0.0407a final weight (g) 0.2596±0.0232b 0.3368±0.0407a weight gain (g) 0.0074±0.0007b 0.0096±0.0012a dgr (g / day) 7.5281±0.2389b 8.2185±0.321a sgr (% weight / day) the means in the same row with different letters mean show significant differences (p<0.05). table 2. the growth performance parameters of luciobarbus xanthopterus larvae fed experimental diets after 5 weeks (data expressed as mean± sd). figure 1. change in (a) mean length, (b) mean weight, (c) mean weight gain, (d) dgr, and (e) sgr of luciobarbus xanthopterus larvae during five weeks rearing period in two feeding treatments. 232 al-faiz et al./ effect of mixed and artificial feeding on the growth performance of gattan et al., 2010). to ensure the sustainability of aquaculture operations, it is crucial to search for feed sources containing similar levels of all nutrients that live food provides to meet the nutritional needs of the larvae and replace the live food and rely on it to improve growth and survival rates (salama, 2000; daniel, 2018). some studies have indicated the importance of algae as a source of protein in the feeding of fish as chlorella algae are one of the rich protein sources. the content of some dried chlorella algae had a crude protein of 50% compared to a fish meal whose protein level was 60% and soybean meal containing 44% protein (lubitz, 1963; campanella et al., 1999). the results showed that t1 feed improved all growth performance indicators compared to t2. a mixture of live and artificial feed for fish may provide the required nutritional components for larvae. this indicates the possibility of using a mixture of live and artificial feed for improving the growth of larvae after the fourth week i.e. after yolk sac absorption. it is necessary to provide protein-rich foods for fish larvae to enhance their growth and survival (mandal et al., 2009). phang (1992) pointed out the possibility of using algae as alternatives to animal protein sources due to its high production rates and nutritional value. venkataraman and becker (1985) showed the algae’s nutritional value is estimated by its quantitative and qualitative content of amino acids. the results of this study agree with the findings of ghazi (2009), which indicated a decrease in growth rates and weight gain of larvae of gattan fed live foods after four weeks of hatching and recommended the necessity of providing artificial foods along with live diets for larvae to improve their growth rates. akbari et al. (2010) also showed a decrease in growth rates and weight gain of rainbow trout larvae fed on live foods compared to those given artificial diets or mixed diets (artificial and live foods) after four weeks. this study showed an increase in growth rates and weight gain compared to the results of previous studies (ghazi, 2009; yesser et al., 2016). in conclusion, the present study showed that applying a mixture of artificial and live food after four weeks’ age from absorption of the yolk sac for feeding by gattan larvae can reduce the costs of producing and providing better growth and survival rates. references akbari p., imanpoor m., sudagar m., makhdomi n.m. (2010). comparison between live food and artificial diet on survival rate, growth and body chemical composition of oncorhynchus mykiss larvae. iranian journal of fisheries sciences, 9(1): 19-32. al-azawi a.h., al-rudainy a.j., rassoki r.h., abbas l.m. 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(2020) 8(5): 337-343 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article feed-nutrients uptake rates of different weight groups of red tilapia (oreochromis sp.) in a recirculating aquaculture system gholamreza rafiee*1, che ros saad2, mohd saleh kamarudin2, mohd razi ismail2, kamaruzaman sijam2 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2department of agrotechnology, faculty of agriculture, university of putra, selangor, malaysia. s article history: received 8 january 2019 accepted 25 march 2020 available online 2 5 october 2020 keywords: recirculating aquaculture red tilapia nutrient retention feed water quality abstract: the role of different weight groups of the red tilapia on nutrient uptake of the feed supply was investigated in a recirculating aquaculture system (ras). five weight groups of red tilapia viz. 20 (200.0), 40 (39.70.44), 80 (80.380.41), 120 (113.621.92), and 180 (177.671.81) g in triplicate treatments were designed. the studied nutrients uptake rates by red tilapia were significantly different (p<0.05) between the treatments. it was found that the red tilapia could assimilate 11.46% fe, 13.43% zn, 6.81% mn, 3.55% cu, 26.81 ca %, 20.29% mg, 32.53% n, 7.16% k and 15.98% p content of the feed supply during three weeks culture period. the specific growth rate (sgr) and food conversion ratio (fcr) indices showed significant differences (p<0.05) between the treatments. the highest growth rate was recorded in 120 g treatment. it was concluded that nutrient requirements of the red tilapia are changed in different growth stages. introduction fishes show different characteristics on nutrient uptake of the feed supply and metabolic excretions during their life cycle. the feeding habitat, stocking density, feed input rate and water quality effect the assimilation of nutrient by fish (tacon, 1995; rafiee et al., 2019). for desirable growth of fish, minerals in the feed supply must be nutritionally balanced, and elements and mineral concentrations in the water and fish body are major biological factor affecting dynamics of nutrient inputs in an aquaculture ecosystem e.g. recirculating aquaculture system (ras) (rafiee and saad, 2005; rafiee et al., 2019). a factorial deduction is known as the simplest method to determine nutrient requirements of fish; however, it does not comprise the changes in the absorption of the nutrient by fish during different life stages. hence, the main objective of this study was to evaluate the role of different weight groups of the red tilapia (oreochromis sp.) on nutrient uptake of the feed supply in a recirculating aquaculture system. *correspondence: gholamreza rafiee doi: https://doi.org/10.22034/ijab.v8i5.410 e-mail: ghrafiee@ut.ac.ir materials and methods five weight groups of red tilapia viz. 200.00 (20), 39.700.44 (40), 80.380.41 (80), 113.621.92 (120), and 177.671.81 (180) g in triplicate treatments were designed and fish randomly introduced into the experimental units. these weight classes were chosen based on our previous study using same culture system (rafiee et al., 2002, 2019). it was estimated that the mean individual fish weight in each group would attain to next mean individual weight group after three weeks, for example 20 g fish would grow to 40 g in 3 weeks, thereby, duration of the study was 3 weeks. each experimental system was a fiberglass tank (110x84x100 cm) equipped with three hydroponic troughs (110x30x5 cm), and a submersible pump (model aqua, 1500). a pump was used to circulate the water from the fish tank through the hydroponic troughs, then to fish tank again (fig. 1). the hydroponic troughs were used to control the penetration of light to the fish tank and reduce algal growth and as a bed for water recycling (providing the 338 rafiee et al./ feed-nutrients retention rates during different weight groups of red tilapia similar condition to fish in an aquaponic system without plant). at the beginning, each rearing tank was stocked with 75 red tilapias. water supply: each tank was filled with 640 l of water and aerated continuously with two circular air stones (3 l min-1). the characteristics of water supply were followed: aged tap water: ph = 7.17, ec = 0.16 mmhos, ca = 5 mg l-1, mg = 2.5 mg l-1, k = 0.00041 mg l-1, p = 0.00031 mg l-1, fe = 0.578 mg l-1, mn = 0.033 mg l-1, zn = 0.00015 mg l-1 and cu = 0.00013 mg l-1. feed and feeding: the feed was a commercial diet floating pellet (car-gill company), with 24% protein, 6% fat, 6% fiber and 11% moisture. the fish were fed twice a day ad libitum at 09.00 and at 13.30 h. the minerals content of feed was measured before initiation of the study. the characteristics of feed are given in table 1. sampling and water quality measurements: dissolved oxygen (do) and water temperature (t) in rearing tanks were measured twice a week (ysi model 57). electro conductivity (ec) was determined twice a week using ec meter (hana instrument conductivity meter hi 8033). two 100 ml of water were sampled from each tank to determine ph using orion model 410a ph meter. total ammonia (nh4nh3) was measured (taking two samples of water from tanks with 10 times dilution using distilled water) weekly (parsons, 1984). nitrite (no2) was weekly measured (ahph, 1980). fish weight measurement: the individual and biomass of fish were measured at the beginning and end of experiment using gravimetry and correction for water values. daily growth rate (dgr) feed consumption (fc), and feed conversion ratio (fcr) were calculated based on sirimannaa and dissanayaka (2019). sampling and dry fish weight measurements: before the experiment, each fish group acclimated with experimental feed for 1 month, 5 fish were sampled and weighted, then cut into the small pieces and were put in a dry dish, then it was put inside an oven at 70c and dry weight was recorded. the samples were reweighed until getting the fixed weight for each fish sample; this process was also done for fish sample at the end of the experiment (ahph, 1980). nutrient (minerals) content of the dry fish and feed measurements: the dry fish and feed were pulverized and homogenized prior to mineral composition analysis process. a 0.25 g of dry feed and fish flesh (four replicates) were taken and digested by kejeldahl method (hatch company, cat. no.23130-18, instruction manual). then, the volume of digested samples was brought up to 100 ml using distilled water. the concentration of total nitrogen (modified bertholet method) and phosphorous in each sample was measured using an auto analyzer (chem lab system 4). the percentage of elements in dry samples was measured using the following the equation of a%= n × 0.04; where a is percentage of element and table 1. the (mean±sd) percentage (%) of minerals* (nutrients) content of the supplementary fish feed. growth indices concentrations of apple cider vinegar control 1% 2% 4% bwi (%) 98.89 ±8.83 101.68±21.45 99.02±59.42 90.47±42.17 fcr 2.53±0.24 2.84±0.44 3.12±0.09 2.4±0.18 sgr (%) 1.64±0.81 1.75±0.35 1.65±0.99 1.50±0.70 cf (%) 1.24±0.38 1.23±0.08 1.30±0.09 1.23±0.95 *fe (ferrous), mn (manganese), zn (zinc), cu (cupper), ca (calcium), mg (magnesium), n (nitrogen), p (phosphorous) and k (potassium). figure 1. schematic feature of the used recirculating aquaculture system: 1. the fish tank 2. the hydroponic troughs 3. the water pump (rafiee and saad, 2005). 339 int. j. aquat. biol. (2020) 8(5): 337-343 n= auto analyzer reading. the mean feed nutrients accumulated in the body of the fish calculated using the fish growth rate (final mean fish weight initiation mean fish weight) × a% and extended to the fish biomass considering density of 75 fish per unit of system. concentration of minerals in the water: the concentrations of dissolved nutrients in the fish tank was measured weekly by auto analyzers and atomic absorption devices. the unit of measurement was reported in mg l-1. data analysis: percent values were transferred to arc sin values. data were subjected to one-way anova, significant differences between the means were compared based on duncan’s new multiple range test, and statistical significance were tested at 0.05 level using spss (version 10.0). results fish growth: growth parameters showed significant differences (p<0.05) between the treatments. the highest growth rate was recorded in 120 g treatment where the fish attained a mean individual weight of 172.8 g with the daily growth rate (dgr) of 2.82 g/day. the feed consumption (fc) in this treatment (3579 g) was higher with a mean feed conversion ratio (fcr) of 0.9, lower than others. the highest fcr and lowest growth rate were recorded in 180 g the treatment with average of 3.38 and 0.65 g/day, respectively (table 2). nutrient content of feed uptaken by red tilapia: table table 2. the fish weight (mean±sd) at the harvest time (fwt), feed conversion ratio (fcr), total feed consumption (tfc), daily growth rate (dgr) and feed consumption (g) per experimental unit per day (fcd). fish groups (g) fwt (g) fcr tfc (g) dgr (g) fcd (g) 20 44.730.53 1.10.02a 2025000a 1.180.03b 96.40.00a 40 63.020.00 1.180.05a 2167670a 1.160.04b 103.20.30a 80 112.256.31 1.180.16a 2702197a 1.520.29b 128.639.37a 120 172.761.11 0.90.01a 3579386c 2.820.12c 170.418.35c 180 191.264.40 3.380.92b 2868282b 0.650.18a 136.613.40b values with the same superscript letters in a column are not significantly different at the 0.05 level. table 3. percentage of mineral (nutrients) composition (mean±sd) of dry weight of red tilapia sampled at the beginning and end of the experimental period. fish groups (g) zn (%) fe (%) cu (%) mn (%) ca (%) mg (%) n (%) p (%) k (%) beginning 20 0.0047 0.00023d 0.0052 0.0.001a 0.0004 0.000018b 0.00045 0.00130b 2.32 0.24a 0.100 0.007bc 5.60 0.220a 1.09 0.625a 0.19 0.005a 40 0.0031 0.00023abc 0.0300 0.0084a 0.0004 0.000036abc 0.00053 0.00023a 2.61 0.41 a 0.102 0.005c 5.21 0.222a 1.09 0.110a 0.19 0.027a 80 0.0043 0.00230bcd 0.0320 0.0055a 0.00040 0.000100c 0.0004 0.00020a 2.81 0.02 a 0.091 0.002a 5.21 0.170a 1.05 0.611a 0.18 0.060a 115 0.0029 0.00220ab 0.0320 0.0010a 0.00058 0.000120ab 0.00013 0.00023a 2.87 0.09ab 0.093 0.004ab 5.17 1.110a 1.12 0.141a 0.16 0.009a 177 0.0032 0.0000abc 0.0370 0.0078a 0.00055 0.000230c 0.0004 0.00040a 2.60 0.11a 0.092 0.004ab 5.21 0.470a 1.08 0.130a 0.16 0.027a end 44 0.0033 0.00023abcd 0.0320 0.0150a 0.00037 0.000037abc 0.00065 0.00024a 2.75 0.53a 0.980 0.070c 5.27 0.860a 1.09 0.102a 0.19 0.034a 63 0.0029 0.00033ab 0.0330 0.0110 a 0.00040 0.000062ab 0.0007 0.00023a 2.83 0.08a 0.101 0.005bc 5.37 0.025a 1.26 0.095a 0.19 0.036a 112 0.0037 0.00061abcd 0.0310 0.0031 a 0.00053 0.000160ab 0.0003 0.00027a 2.45 0.27a 0.093 0.088ab 5.23 1.260a 1.12 0.150a 0.18 0.009a 172 0.0044 0.00040cd 0.0360 0.0084 a 0.00043 0.000230c 0.0004 0.00040a 2.61 0.32a 0.092 0.004ab 5.21 0.470a 1.08 0.130 a 0.16 0.027a 191 0.0031 0.00061abc 0.0340 0.0230a 0.00053 0.000260a 0.0004 0.00040a 2.48 0.31a 0.093 0.004ab 5.10 0.250a 1.03 0.140a 0.16 0.024a values with the same superscript letters in a column are not significantly different at the 0.05 level. 340 rafiee et al./ feed-nutrients retention rates during different weight groups of red tilapia 3 shows the percentage of nutrient content of red tilapia in different weight groups. the mineral composition in different weight groups indicated that the body weight significantly (p<0.05) has affected the assimilation rate of zn, cu, mn and mg. mean total feed nutrients uptaken by different weight classes reported in table 4. the percentage of feed nutrients uptake by fish (20 to 191 g) varied in different treatments. water quality parameters: total ammonia nitrogen ranged 0.73 to 9.66 mg l-1, nitrate-n 0.03 to 9.77 mgl-1, total inorganic nitrogen 6.50 to 19.34 mg l-1, ec 0.16 to 0.50 mmhos/cm and ph 7.26 to 5.89 (with a decrease in all treatments) during the experimental period. macro-elements concentration: the phosphorus (p) increased in all the treatments during the experiments. after one week, it showed significant differences (p<0.05) between treatments. the highest concentration was recorded in the treatment with 20 g fish i.e. p was lower in the treatments with higher weight and it reached to 19.33 mg l-1 in 80 g treatment after 2 weeks (table 5). mg showed significant differences (p<0.05) in all treatments during the experiment. the mg increased in 80 and 120 g treatments with 10.83 and 11.9 mg l-1, respectively (table 5). calcium was not different (p>0.05) between treatments and its lowest concentration was measured in 120 g group, and the highest values in 20 and 180 g groups, (24.67 mg l-1). the concentration of k in 80, 120 and 180 g groups increased during the experiment and it was higher (39.33 mg l-1) in 180 g table 4. average percentage of feed nutrients captured by different weight groups of red tilapia at the end of the experimental period. fish groups (g) zn (%) fe (%) cu (%) mn (%) ca (%) mg (%) n (%) p (%) k (%) 20 0.75 10.01 27.21 5.34 34.66 80.85 27.87 14.08 6.85 40 8.33 6.30 30.41 6.04 33.65 4.23 31.22 19.14 7.00 80 9.83 6.18 8.10 0.57 22.10 5.37 36.56 20.36 8.00 120 38.38 11.55 1.37 5.79 33.77 5.98 34.48 19.16 8.59 180 0.015 0.019 0.065 0.02 9.85 5.03 32.53 7.18 5.38 table 5. changes in the concentration (mean±sd) of total phosphorous (p) and magnesium (mg) in the fish tanks during a 3 -week experimental period. treatments (g) phosphorus (mg l-1) magnesium (mg l-1) week 1 week 2 week 3 week 1 week 2 week 3 20 10.551.20abc 11.370.86 a 13.131.80a 1.200.32bcd 2.330.79bc 3.431.81abc 40 13.33.00cd 14.500.70 a 16.601.10a 1.300.40abc 2.800.20bc 3.131.89a 80 9.716.80abc 11.293.72 a 19.334.8 a 1.47097ab 2.670.72ab 3.830.85c 120 5.006.25ab 8.901.46 a 15.631.70a 1.500.51a 2.870.58abc 3.981.96bc 180 2.590.70a 5.901.00 a 18.494.01a 1.90.98d 12.573.03c 4.700.17c values with the same superscript letters in a column are not significantly different at the 0.05 level. table 6. changes in the concentration (mean sd) of total calcium (ca) and potassium (k) in fish rearing tanks during the experiment. treatments (g) ca (mg l-1) k (mg l-1) week 1 week 2 week 3 week 1 week 2 week 3 20 13.002.00 a 17.331.53 a 24.671.53 a 5.001.70a 6.001.31 6.401.30a 40 14.332.10 a 18.330.58 a 27.002.00 a 4.501.00 a 6.701.70a 6.831.80a 80 12.71.85 a 17.00 2.19a 22.003.54a 5.001.13 a 6.701.51 a 5.941.60a 120 12.672.10 a 19.003.00a 27.002.15a 4.000.58 a 5.201.30 a 7.002.00a 180 15.001.17 a 16.003.93a 24.671.86a 4.780.21 a 6.702.90 a 8.331.20 0a values with the same superscript letters in a column are not significantly different at the 0.05 level. 341 int. j. aquat. biol. (2020) 8(5): 337-343 treatment (table 6). discussions based on the results, the nutrients uptake by fish per unit (kg) with the same biomass was different in different weight classes, showing that the nutrient requirements of red tilapia changed during its life cycle. it was estimated that the red tilapia on average can assimilate 11.46% fe, 13.43% zn, 6.81% mn, 3.55% cu, 26.81 ca %, 20.29% mg, 32.53% n, 7.16% k and 15.98% p of feed supply during rearing period. the assimilation rate of 32% (quillere et al., 1993), and 40-43.2% (siddiqui et al., 1988; el sayed, 1990) for feed nitrogen reported for the fingerling and breed of tilapia in the conventional system while this was 20% for a male population in ras without using plant (suresh and lin, 1992), 31% with a mixed population (rakocy et al., 1993) and 37.4% for a male population (zweig, 1986) and 29% by fish and shrimp (avnimelech and ritvo, 2002). it was calculated that averagely 88.54% fe, 93.19% mn, 86.57% zn, 96.44% cu, 73.19% ca, 79.71% mg, 67.47% n, 92.84 % k and 84.02% p of input feed were released to the culture system in the form of faecal material, urine and ammonia gas excretion forms. other works have reported the rates of 20-40% of feed nitrogen as nitrogen excretion in the form of ammonia-n by fish. if 39.29% of feed nitrogen excreted in the form of ammonia by the red tilapia (rafiee et al., 2019), a simple calculation shows that 25% of the feed nitrogen remained in the fecal materials as organic nitrogen. the composition i.e. the chemical form in which the minerals are present and the quantity of minerals in feed affect the feed availability to the fish. in this experiment, different weight groups of red tilapia were exposed to constant dietary nutrient input. the results showed that body size (biomass changes or density) affects the assimilation rates of some minerals supplied by the feed. the levels of mg in the feed supply have more effect on fish growth when combined with the adequate protein supply and its uptake reaches a higher level when it combined with a low protein supply (dabrowska et al., 1991). the concentration of mg in a whole body of carp fed with a diet with protein levels of between 24-34% ranged from 590 to 1000 mg kg-1. an increase in the mg concentration in the diet can causes more uptake of mg (dabrowska et al., 1991). the mg concentration in the whole body of rainbow trout linearly increases with increasing the mg content in fish diet and the optimum uptake between 299 to 385 mg kg-1 for wet weight of rainbow trout has been reported (shearer, 1989). the mean concentration of mg in the diet used in this experiment was 4.240 mg kg-1 and its uptake in the whole body as significantly different between treatments ranging 920 to 1010 mg kg-1 (dry weight), with the highest level in the 63 g group. increasing mg concentration in water led to a significant increase in mg uptake by rainbow trout, and it depends on dietary mg supply as well (shearer and asgard, 1992). in contrast, the lowest concentration of mg recorded in the 40 g group (63 g at the harvest time). based on these results, fish weight affects mg retention in red tilapia and the mg content of the diet was at the optimum level for red tilapia growth. the requirement of fish for phosphorous depends on the fish species, feed composition and form of phosphorous supply. phosphorous from vegetable feed components has an availability of 20-50% in carp and trout (nrc, 1983). the p concentration in the whole body of rainbow trout increase with an increase in dietary p content, and its concentration in a diet containing 3.4-8.6 g kg-1 (dry matter) causing p uptake between 3 to 4.3 g kg-1 of body weight (wet weight). the concentration of p in the present work was 11.8 g kg-1, and uptake was measured 9.4 to 12.6 g kg-1 (dry weight). the highest concentration was recorded in the 63 g group. the concentration of p in water was significantly different between the treatments, but not significant in weight classes. comparing these results to the previous work on rainbow trout (nakamura, 1982), indicates that p requirement of red tilapia is greater. there is a negative linear correlation between calcium content of the diet and the amount of p absorbed by carp (nakamura, 1982). this relationship 342 rafiee et al./ feed-nutrients retention rates during different weight groups of red tilapia in 63 g group can be related to the high concentration of ca in the water compared to others. zn deficiency affects the fish growth rate (hidalgo et al., 2002). a considerable excretion of zn via the gills has been reported with faecal zn excretion, which consists of both unabsorbed zn and endogenous zn. an amount of 5 to 20 mg kg-1 zn in the diet is considered for trout and carp (ogino and yang, 1978) and that in our experiment was higher (56 mg kg-1). the uptake of zn in the body of different weight groups ranged 24 to 51 mg kg-1 showing significant differences between groups. the zn in the whole body of rainbow trout has been reported 36 to 120 mg kg -1, when the zn in the diet and water supply were 1 to 590 mg kg-1 and 0.007 to 0.148 mg l-1, respectively (spry et al., 1988). zn concentrations in the water linearly increase with an increase in the zn content of diet and has a high turnover (wekell et al., 1992). high concentration of ca-phosphate is considered the reason for the low availability of zn in diets containing higher fishmeal. the direct addition of different levels of ca-phosphate resulted in a significant decrease in zn concentration in the trout (spry et al., 1988; scarpa and gatlin, 1992). severe dietary deficiency of the elements such as cu, mn, and fe in the fish is generally resulted a decrease in the growth rates (ogino and yang, 1978). experiments with semisynthetic ratios suggest a diet containing 12-20 mg mn kg-1 to cover the requirements of carp and rainbow trout (ogino and yang, 1978). the concentration of mn in the supplied feed of the current experiment was 30 mg kg-1, higher than the levels suggested for carp and rainbow trout (ogino and yang, 1978). the mn uptake in red tilapia ranged 3 to 7 mg kg-1, higher in 63 g group. the availability of mn in inorganic compounds differ according to the form of the bond within a compound (satoh et al., 1987), with use of mnso4 and mncl2 showing higher availability than mnco2 or mno2. the fe storage in the liver is significantly reduced in the cases of fe deficiency (walker and fromm, 1976). the fe concentration in the diet was 1090 mg kg-1. the recommended level of fe in the diet of carp and rainbow trout varies 60 to 50 mg kg-1 (cho and cowey, 1991). fe in different treatments was not significantly different, indicating that the high levels of fe in the diet met the requirements of red tilapia and fish weight was not significantly affected by fe retention. reductions in the fe, mn, cu, and zn may be attributed to chemical interactions leading to precipitation or low 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(2021) 9(6): 423-431 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article ecotoxicological responses of two planorbarius corneus s. lato (mollusca, gastropoda) allospecies to exposure of heavy metals oleksandr harbar* 1, diana harbar2, agnessa stadnychenko2, yuliia babych2 1department of ecology and geography, zhytomyr ivan franko state university, zhytomyr 10002, ukraine. 2department of zoology, biological monitoring and nature conservation, zhytomyr ivan franko state university, zhytomyr 10002, ukraine. s article history: received 7 june 2021 accepted 24 december 2021 available online 2 5 december 2021 keywords: allospecies gastropods heavy metals ecotoxicological indicators abstract: planorbarius corneus (linnaeus, 1758) is the most widespread and numerous gastropods in central-european waters, which range covers from the atlantic to the ural and outside the latter to the ob river basin. before the beginning of 21 century, malacologists had no doubts about its species status. this situation changed when genetic labeling showed that p. corneus is not a species, but a superspecies complex, planorbarius (superspecies) corneus s. lato, according to the centromere indices of the 12th pair chromosome. this complex consists of two vicarious genetic allospecies, western and eastern one, which ranges are separated by a narrow (up to 100 km) zone of the introgressive hybridization lying just in ukraine. ecotoxicological features of p. corneus s. lato allospecies under the influence of any pollutants have not been studied yet. our research focused on how different concentrations (0,001–1000 mg l-1) of some heavy metal ions (cu2+, zn2+, ni2+, mn2+) in the water environment affected the main ecotoxicological parameters of each vicarious allospecies, and on the limits of toxic effects for studied pollutants. we found the duration of the latency period, time-to-death, and mean time-to-death, as well as the coefficients of persistence and adaptation for each allospecies. according to our results, eastern allospecies are more sensitive to the heavy metals in the aquatic environment than western one. that creates a significant threat to the eastern allospecies populations, because the concentrations of these pollutants in the ukrainian waters remain rather high, despite some positive downward trends. introduction heavy metals are ecologically toxic substances that are found in ukrainian water bodies. this type of pollution is one of the limiting factors for the functioning and bioproductivity of aquatic ecosystems. heavy metals participate in hydroecosystem biogeochemical cycles and persist for a long time playing a key role in energy metabolism processes that provide the viability of aquatic organisms (afanasyev and grodzinsky, 2004; grubinko et al., 2012). their concentrations in the ukrainian waters are reported to be rather high, despite some positive downward trends (giriy et al., 2011). as microelements, heavy metals (cu, zn, ni, mn, etc.) are included in many organic compounds that are biologically important affecting basic biochemical processes of aquatic organisms *correspondence: oleksandr harbar doi: https://doi.org/10.22034/ijab.v9i6.1356 e-mail: o.v.harbar@gmail.com (hochachka and somero, 1988). zn2+ is an essential component of the carbonic anhydrase enzyme that catalyzes the reaction of carbonic acid decomposition and the reverse reaction of its formation. it provides normal growth and development of the mollusks by regulating the reactions that mitigate the effects of haemolymph stability disruption (kyrychuk, 2010). сu2+ is also an essential component of their respiratory pigments, mn2+ affects their growth and development, and ni2+ influences the processes of haematopoiesis in mollusks (romanenko, 2001). however, these pollutants may accumulate in aquatic animals and are highly toxic at relatively low concentrations. there are several factors that determine the accumulation of heavy metal ions: hydrochemical conditions of the environment, bioavailability for animals, type of 424 harbar et al./ ecotoxicological responses of two planorbarius corneus s. lato nutrition and nutrient digestibility level, genetic differences, and physiological status of affected species (oros and gomoiu, 2010). when heavy metal concentrations in the water bodies exceed the threshold limits, the boundary between their physiological and toxic effects blurs (gorovaya and stolyarova, 1987) with the detrimental consequences for aquatic organisms due to pathogenic (toxic or cancerogenic) effects. planorbarius corneus (linnaeus, 1758) is a widespread and often numerous mollusk (didukh, 2012) as only representative of bulinidae family in european waters. genetic labeling has recently proved (mezhzherin et al., 2005) that p. corneus is not a species but a superspecies complex that consists of two spatially separated vicarious allospecies, "western" and "eastern" one. they significantly differ genetically and by some conchological, anatomical and ecological characteristics (mezhzherin et al., 2006; garbar and garbar, 2007; garbar, 2009; stadnychenko et al., 2020). these taxa are welldifferentiated by gene frequency distribution peculiarities, namely by the centromere index of the 12th chromosome pair (mezhzherin et al., 2006; garbar and garbar, 2007; garbar, 2009; stadnychenko et al., 2020). western allospecies are marked by slightly larger shell size and its dark brown or almost black color. eastern allospecies have a smaller and lighter shells. these allospecies also significantly differ by having six numerical indices that characterize the whorl expansion rate of their shells and the relative size of apertures. the conchological parameters of both allospecies have significant geographical variability (garbar, 2009): their shell sizes decrease in the west-east direction and increase in the north-south one. the allospecies of p. corneus s. lato also significantly differ by several linear parameters of their reproductive system (8 out of 10 usually measured ones) and by some dimensional anatomical parameters, namely the size of the vagina, sperm reservoir, and its ducts (mezhzherin et al., 2005). the ranges of both allospecies are spatially separated. western allospecies are common in the western and central part of the right bank ukraine, and eastern allospecies inhabit the northeast and east of left bank and the extreme south of ukraine from the severskiy donets to the danube inclusive. between their habitats lies a zone of introgressive hybridization (about 100 km wide). the chorological separation of these allospecies is due to the different environmental conditions in their habitats, first of all, the different rates and duration of summer droughts in these areas. the purpose of this research is to find out the differences in the ecotoxicological characteristics of "western" and "eastern" p. corneus s. lato allospecies affected by various cu2+, zn2+, ni2+, and mn2+ concentrations. materials and methods a total of 1480 specimens of p. corneus s. lato (748 specimens of western allospecies and 732 specimens of eastern allospecies) were collected in summer 2020 (fig. 1). the first allospecies was collected from miropol vicinity (49°40'17"n, 33°45'39"e) in sapohivka river, the latter one from bilotserkivka figure 1. the shells of planorbarius corneus s. l. allospecies: а – «western»; в – «eastern»; 1 – top view; 2 – bottom view; 3 – side view. 425 int. j. aquat. biol. (2021) 9(6): 423-431 (50°6'27"n, 27°41'45"e) in psel river (fig. 2). identification of allospecies was performed by their conchological peculiarities (fig. 3) (garbar, 2009). in each river, the material was collected in the biotopes of four types: (1) coastal shallow waters, covered with clusters of filamentous algae (cladophora sp.), with the almost imperceptible flow and depth up to 5-7 cm, (2) coastal thickets of hard semi-submerged aquatic vegetation (scirpus sp. and typha sp.), overgrown with periphyton, that was concentrated in the river littoral 15-20 cm deep with the almost imperceptible flow, (3) deeper littoral zone (up to 1-1.2 m) with a well-developed phytal, mostly soft macrophytes (myriophyllum sp., nymphaea sp., lemna sp., etc.), and with a flow velocity of 0.1-1 m/sec, and (4) benthic zone, without vegetating macrophytes, covered with a layer of plant detritus. mollusks were acclimated for 14 days (khlebovich, 1981) in 10 l the aquarium, with a planting density of 5 specimens l-1, water temperature 21-23°с, рн 7.2-7.8, do 7.8-8.6 mg о2 l-1. the water was replaced every day. the mollusks were fed by soft vegetation from their sampling places (cladophora sp. and myriophyllum spicatum). the toxicological experiment was done according to alekseev (1981). the volume of the aquarium was 100 l, water temperature 20-23°с, рн 7.2-8.2, do 7.6-8.9 mg о2 l-1. toxic substances were cucl2·2h2o, zncl2·2h2o, nicl2·6h2o and mncl2·4h2o (puriss. p. a.) in concentrations of 0.001– 1000 mg l-1 (calculated by the cation). exposition time was 48 hours, with daily water replacement. the results were evaluated in 10 and 30 minutes, 1, 2, 4, 6, 24 and 48 hours after the beginning of the experiment. toxic effects on experimental animals were indicated by visual observation of their behavior and general table 1. the main toxicological characteristics of planorbarius corneus s. lato allospecies under the exposure of heavy metal ions (48 hours). indicator, mg l-1 "western" allospecies "eastern" allospecies cu2+ zn2+ ni2+ mn2+ cu2+ zn2+ ni2+ mn2+ threshold concentration 0.0001 0.001 0.01 0.1 0.00001 0.0001 0.001 0.01 lc0 0.001 0.05 0.1 50 0.0001 0.01 0.05 40 lc50* 0.05 0.4 20 250 0.005 0.2 2 150 lc100 0.01 1 50 1000 0.001 1 10 1000 toxicity rate 0.05 0.4 20 250 0.005 0.2 2 150 *graphically defined. figure 2. map showing the type localities of planorbarius corneus s.l. allospecies: black circle – «weastern»; black diamond – «eastern». 426 harbar et al./ ecotoxicological responses of two planorbarius corneus s. lato condition. as indicators, we evaluated the degree of the body covering epithelium damage, the intensity of mucus secretion by the outer epithelium cells, motion behavior, and nutrition intensity. persistence coefficient (pc) was calculated according to vyskushenko (2002) by the formula of pc=еk/еn, where еk is a time to the death of the last test animal; en is the time to the death of the first test animal. adaptation coefficient was calculated according to malacea (1968) by the formula of ac=еe/еc, where еe is an average time to the death of all the test animals, еc is an average time to the death of all the control group animals. results western allospecies (sapohivka river, pripyat basin) were collected on 11.08.2020. the average shell size (mm) were 25.99±0.32, the aperture height 13.28±0.11. eastern allospecies (psel river, dnieper basin) were collected 05.09.2020, the shell diameter (mm) was 24.96±0.15, and aperture height 12.29±0.08. the threshold concentrations of all toxicants for eastern allospecies were an order of magnitude lower than for western allospecies (table 1). the same order was for the main toxicological indicators, lc0, lc50 and lc100. we also found the difference in mortality between the allospecies under heavy metal ion poisoning (table 2). thus, in the water with 0.001 mg l-1 cu2+, western allospecies had 100% survival, while 10% of eastern allospecies specimens died at the end of the experiment. the duration of the latent table 2. mortality (%) of allospecies under the exposure of heavy metal ions. concentration, mg l-1 "western" allospecies "eastern" allospecies cu2+ zn2+ ni2+ mn2+ cu2+ zn2+ ni2+ mn2+ 0.0001 0 0 0 0 0 0 0 0 0.001 0 0 0 0 10 0 0 0 0.01 40 0 0 0 100 10 0 0 0.1 100 20 0 0 100 40 20 0 1.0 100 100 20 0 100 100 40 0 10 100 100 70 0 100 100 100 0 100 100 100 100 30 100 100 100 40 1000 100 100 100 100 100 100 100 100 table 3. latent period (h) of planorbarius corneus s. lato allospecies under the exposure of heavy metal ions. concentration, mg l-1 "western" allospecies "eastern" allospecies cu2+ zn2+ ni2+ mn2+ cu2+ zn2+ ni2+ mn2+ 0.001 14 24.1 25.5 12 21.0 23.1 0.01 4.2 10.3 24.2 25 1.3 8.3 21.0 24 0.1 1.4 2.1 20.5 4.1 0.5 1.5 16.5 4.0 1.0 0.5 1.5 2.1 2 0.3 1.1 1.4 1.5 10 0.3 1.0 1.1 1.1 0.15 0.4 0.5 1.0 100 0.1 0.1 0.1 0.35 0.1 0.1 0.1 0.3 1000 0.3 0.25 figure 3. measurements of the shells (scheme): hs – shell height; ws – shell width; hm – mouth hight; wm – mouth width; wt 1t – width of the last whorl (top view); wt 1b – width of the last whorl (bottom view); wt2 – width of the penultimate whorl; wt3 – width of the third whorl; ditt – diameter of the internal whorl (top view); ditb – diameter of the internal whorl (bottom view); rs – shell radius (garbar, 2009). 427 int. j. aquat. biol. (2021) 9(6): 423-431 poisoning periods also differs (table 3). under the toxicant concentrations of 0.001-10 mg l-1, the latent periods for eastern allospecies were lower than for western one. adaptation coefficients in the environment polluted by cu2+, zn2+, and ni2+ (table 4) were also lower for eastern allospecies. as for mn2+, there was no difference between the allospecies. the absolute values of the persistence coefficient (table 5) were higher for eastern allospecies. for both allospecies, the values of the persistence coefficient increased with the increase of toxicant concentration in the water. the data presented in table 6 show the toxic resistance of each allospecies under the exposure of heavy metals in concentrations from subthreshold to acutely lethal. for all the studied toxicants, in eastern allospecies the symptoms of poisoning appeared earlier and were caused by lower concentrations. time-to-death decreased when the toxicant concentration in the environment increased (table 7). table 4. adaptation coefficient (h) of planorbarius corneus s. lato allospecies under the exposure of heavy metal ions. concentration mg l-1 "western" allospecies "eastern" allospecies cu2+ 4 3.1 zn2+ 1.30 1.20 ni2+ 1.15 1.10 mn2+ 1 1 table 5. persistence coefficient (h) of planorbarius corneus s. lato allospecies under the exposure of heavy metal ions. concentration, mg l-1 "western" allospecies "eastern" allospecies cu2+ zn2+ ni2+ mn2+ cu2+ zn2+ ni2+ mn2+ 0.1 1.14 1.25 1.20 1.24 1 1.32 1.34 2.05 1.40 1.42 2.25 10 2.47 1.56 3.12 3.15 2.30 3.34 100 3.12 2.87 2.21 3.41 3.05 5.53 1000 3.57 3.73 table 6. rating of heavy metal ion concentrations (mg l-1) according to the effect on planorbarius corneus s. lato allospecies ion subthreshold sublethal chronic lethal acutely lethal "western" allospecies cu2+ 10-5 and lower 0.001-0.0001 0.05-0.01 1-0.1 zn2+ 0.01 and lower 1-0.01 2-1 5-3 ni2+ 0.01 and lower 2-0.1 6-13 40-10 mn2+ 0.3-0.003 10-1 40-20 80-50 "eastern" allospecies cu2+ 10-6 and lower 10-4–10-5 0.01-0.001 1-0.05 zn2+ 10-5 and lower 0.001-0.0001 0.1-0.01 3-1 ni2+ 10-4 and lower 0.01-0.001 0.4-0.1 10-1 mn2+ 0.01 and lower 5-0.1 30-5 70-35 table 7. time-to-death (h) of planorbarius corneus s. lato allospecies under the exposure of heavy metal ions. concentration, mg l-1 "western" allospecies "eastern" allospecies cu2+ zn2+ ni2+ mn2+ cu2+ zn2+ ni2+ mn2+ 0.001 37 35 0.01 30 41.0 43.3 27 39.2 39.3 0.1 12 23.2 29.1 10 21.3 25.3 1.0 0.5 18.4 19.3 0.3 17.1 17.3 1 10 0.3 6.3 8.1 29.1 0.15 6.0 7.2 27 100 0.1 0.4 0.5 16 0.1 0.1 0.1 15 1000 1.4 1.1 428 harbar et al./ ecotoxicological responses of two planorbarius corneus s. lato at the same concentrations, the time-to-death of eastern allospecies was higher. for the average timeto-death, a similar pattern was observed (table 8). discussions heavy metals enter the body of aquatic organisms mostly percutaneously in an osmotic way (golovko et al., 2018) and much less with their food objects (romanenko, 2001). they are able to accumulate in mollusks with subsequent redistribution between organs and tissues with haemolymph. the primary accumulation of heavy metals occurs in hepatopancreas. the heavy metals used in our experiments (cu, zn, ni, and mn) are biophilic, i.e. extremely necessary for viability processes of these aquatic organisms. however, they are extremely dangerous and often lethal at high concentrations, because their accumulation in the tissues and organs disrupts a number of important biochemical processes and physiological functions (frіas-espericueta et al., 2003; wong and pak, 2004; kyrychuk, 2010, grubinko, 2011). for each studied allospecies, we clarified the action limits of the above-mentioned toxicants depending on concentrations (from subthreshold to acutely lethal), which allowed to determine the zones of their toxic effects on test animals according to dudnik and yevtushenko (2011). the symptoms for each phase of the poisoning pathological process on aquatic mollusks are well-known (vyskushenko, 2002; pinkina, 2010; stadnychenko et al., 2021). however, the expressiveness and overtime changes of these symptoms for p. corneus s. lato has not been studied yet. we found that cu2+, zn2+, ni2+, and mn2+ below the threshold concentrations did not affect the ethological or physiological parameters of the studied mollusks. such a reaction to the polluted environment is usually regarded as a latent phase that is an initial stage of the poisoning process (veselov, 1968, 1984). this is the longest phase of this process, which is typical for all endogenous toxicants, including heavy metals. the first ethological and physiological changes were observed when experimental specimens were kept in an environment with sublethal toxicant concentrations. the response to the toxic effect includes reversible pathological changes both in the mollusks’ behavior and their physiological systems' functioning. the first ones were manifested by a significant increase of motion activity in attempts to leave the environment, and the second ones in the suppression of their food and sexual behavior. these indicators are the ground for the determination of toxicants’ threshold limit values for aquatic organisms. in ukraine, for fishery reservoirs, they are equal to 0.005 mg l-1 for cu2+, 0.01 mg l-1 for zn2+ and ni2+, 1 mg l-1 for mn2+ (resolution of the cabinet of ministers of ukraine, 2013). all the abovementioned pathological changes in mollusks were registered in the next phase of the poisoning, stimulation. in eastern allospecies, such changes occur 1-1.5 hours earlier and at much lower toxicant concentrations than in western allospecies. however, they developed cumulative toxicosis after prolonged chronic exposure to the same sublethal concentrations. during this phase, both allospecies demonstrated the stimulation of feeding and sexual behavior. the protective physiological response included increased secretory function of outer epithelial glandular cells that decreased the rate and the volume of toxicants' percutaneous penetration into the body. over time, the table 8. mean time-to-death (h) of planorbarius corneus s. lato allospecies under the exposure of heavy metal ions. concentration, mg l-1 "western" allospecies "eastern" allospecies cu2+ zn2+ ni2+ mn2+ cu2+ zn2+ ni2+ mn2+ 0.01 43 40 0.1 23.1 40 42 21 37 40 1.0 9.2 20 29 8.2 18 28 10 3.2 11 15 2.5 10 13 100 1.1 3 5 46 1 2.5 4 43 1000 3 429 int. j. aquat. biol. (2021) 9(6): 423-431 layer of skin mucus did not thicken, but thinned due to its partial coagulation, exfoliation, and split-off of different shape and size coagulants, laying bare affected areas of the mollusk body surface. under zn2+ exposure, only 55% of western allospecies specimens and 63% of eastern allospecies specimens remained covered by intact mucus at the end of the experiment. under the exposure of chronic lethal concentrations, the mortality at the end of the experiment was 36% for western allospecies, and 45% for eastern allospecies. the surviving mollusks were clearly depressed having a mild to moderate swelling of the body, which indicated the disruption of their water balance. this is one of the animals’ rapid protective physiological reactions (pinkina, 2010; pinkina and pinkin, 2018), aimed to decrease the toxicant damaging effects by diluting the toxic substances. in experiments with acute lethal concentrations, mollusks tried to avoid the toxic environment crawling on the aquarium walls, and, being above the surface of the water, immobilized, clinging to the walls. some individuals, exhausted by unsuccessful attempts to get out of the poisoned environment, sank to the bottom and lay there motionless. in this case, 45% of western allospecies specimens and 55% of eastern allospecies specimens had a dropout reaction. their body volume increased (1.5-2 times above normal) due to its watering, the columellar muscle tone decreased, and they could not retract the body into the shell, so their head and leg hung out through the aperture. this reaction is usually associated with impaired renal function due to decreased osmotic concentration of their excreta (kolupaev, 1989). in such cases, the continuity of the tissue structures, especially cell membranes, is usually injured, which in turn leads to the destruction and death of cells and tissues. such damages indicated the depressive and sublethal phases of the poisoning process. for the eastern allospecies, these symptoms were much clearer and started earlier than for the western allospecies. at the end of this experiment, during the lethal phase, the mortality reached 100%. thus, we established the time-to-death values for each allospecies. its duration decreased proportionally for both allospecies with increasing heavy metals concentrations. most early the death had come under the cu2+ poisoning and latest in mn2+ treatment. eastern allospecies were more sensitive to the effects of all the toxicants used in the experiments: with increasing concentrations, its time-to-death was much less (1-2 hours) compared to western allospecies. under the same concentrations of all heavy metal ions, eastern allospecies mortality was always higher than the western one. we assume that one of the reasons for this is the different ecological conditions in their usual natural habitats. after all, they are usually much more difficult for eastern allospecies due to the greater aridity of left bank ukraine climate, especially in the extreme south of the steppe zone. therefore, the toxic action limits from threshold to maximum lethality of p. corneus s. lato allospecies differ significantly for each heavy metal ion. eastern allospecies are affected by lower concentrations and earlier than western allospecies indicating their lower toxic resistance. eastern allospecies are more sensitive and less resistant to all heavy metal ions used in the experiment. this fact is confirmed by such ecotoxicological indicators as the latent period duration, time-to-death, and mean time-to-death, as well as adaptation and persistence coefficients. according to the latter, the first symptoms of poisoning and the symptoms of irreversible poisoning appear much earlier in eastern allospecies, and western allospecies are more viable than the eastern one. with increasing toxicant concentrations, the latent period begins earlier for both allospecies, and the time-to-death decreases. the toxicological reactions’ comparison between western and eastern genetic allospecies of p. corneus s. lato was the first study to estimate the physiological differences between them. therefore, this study encourages further investigations of the ecology and especially ecotoxicology of these mollusks. references afanasyev s.a., grodzinsky m.d. 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(2004). acute and subchronic toxicity of the heavy metals copper, chromium, nickel and zinc, individually and in mixture, to the freshwater copepod mesocyclops pehpeiensis. bulletin of environmental contamination and toxicology, 73(1): 190-196. international journal of aquatic biology (2013) 1(5): 245-253 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article benthic community structure in the gorgan bay (southeast of the caspian sea, iran): correlation to water physiochemical factors and heavy metal concentration of sediment mahmood saghali*1,2, rauf baqraf3, seyed abbas hosseini4, rahman patimar5 1department of zoology, university of national academy of sciences. baku, azerbaijan. 2iranian fisheries organization, bandar-e-turkman, iran. 3ecology department of azerbaijan oil company, baku, azerbaijan. 4department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. 5department of fisheries, gonbad-e-kavoos university, gonbad-e-kavoos, iran. article history: received 2 september 2013 accepted 14 october 2013 available online 2 5 october 2013 keywords: macrobenthos biomass frequency gorgan bay caspian sea heavy metal abstract: macrobentos frequency and biomass was investigated in the gorgan bay in 2011. five sampling sites were chosen to collect benthos and sediment from the bay using a van veen grab sampler. samples were collected seasonally. macrobenthos were indentified and their biomass was recorded. sediment heavy metals concentration were measured using atomic absorption spectroscopy. a total of 11 families belonging to three phyla of invertebrates were found. the phyla were annelids (nereidae, naididae, ampharetidae, lumbriculidae, tubificidae and amphiporidae), arthropods (pontogammaridae, balanidae and chironomidae) and mollusks (cardiidae and scrobicularidae). lumbriculidae (413 individuals m-2, corresponding to 18.7%) and cardiidae (55.2 g m-2, corresponding to 82.4%) had the highest frequency and biomass, respectively. annelids with an average of 1557 individuals m-2 was the most frequent groups, while, mollusks with the average of 141 g per m2 had the highest biomass. results showed that macrobenthos frequency in summer was significantly higher than those of the other seasons, however, in the case of biomass, there was a significantly higher biomass in the spring than the other seasons. the maximum metal concentration was related to zn and pb, whereas, cr and cd had the lowest values. there was no significant difference in zn and cr concentrations among the sampling seasons. pb concentration in winter was significantly lower than the other seasons, whereas, cd concentrations in the spring and summer were significantly lower than the autumn and winter. there were some correlations between benthos frequency and water physiochemical characteristics and sediment heavy metal levels. this study indicated that benthic fauna of the gorgan bay and the caspian sea are not similar. also, results showed that benthic fauna communities are affected by sediment heavy metal concentrations and water physiochemical characteristics, however, different benthos groups show unsimilar relationship with heavy metal concentration. introduction studying the biological and ecological aspects of water resources is very important in the ecosystem studies. the initial step of such surveys is the identification of organisms living in the ecosystem (ahmadi and mousavi, 2002). macrobenthic communities are a part of food chain and supply the required energy for the majority of aquatic species, * corresponding author: mahmood saghali e-mail address: m_saghali18@yahoo.com particularly fishes (soleimanroodi, 1994). thus, as the secondary producers, they are one of the energy suppliers and distributor in the ecosystem. the aquatic organisms are exposed to anthropogenic disturbances and natural changes in their habitats, which make them react in different ways. therefore, aquatic organisms have an important role in bioassessment (mooraki et al., 2009; girgin, 2010). 246 saghali et al/ international journal of aquatic biology (2013) 1(5): 245-253 benthic fauna's structure is affected by environmental factors such as temperature, ph, dissolved oxygen and pollution. the relationship between benthic fauna's structure and heavy metal contamination has been studied previously (rygg, 1985; hall et al., 1996; warwick, 2001; guerragarcía and garcía-góme, 2004; harriague et al., 2007; dauvin, 2008). rygg (1985) studied the relationship between benthic fauna diversity and concentrations of cu, pb and zn in the sediments from norwegian fjords and found a strong negative correlation between species diversity and heavy metals concentration. hall et al. (1996) studied the effects of metal contamination on macrobenthos in two north sea estuaries and found a decrease in the community at the polluted sites. warwick (2001) studied the effects of metal contamination on the intertidal macrobenthic communities at the fal estuary and reported that sediments' heavy metal concentrations were correlated most strongly with the composition of the communities. the gorgan bay is located at the southeast of the caspian sea with an area about 400 km2. due to the clayey bed, shallow depth and lack of heavy waves, the gorgan bay is considered as a suitable habitat for benthic communities. the bay is the major site of fish stock rehabilitation in the southern caspian sea, receiving huge number of fish fingerlings originated from the artificial propagation and rearing, yearly. the fish species are sturgeons (acipenseridae), caspian kutum (rutilus frisii kutum), common carp (cyprinus carpio) and caspian roach (rutilus rutilus caspicus), all feed on macrobenthic fauna. thus, condition of macrobenthic fauna in the gorgan bay is important for stock rehabilitation program that is performed by iranian fisheries organization. in addition, some species use the grorgan bay as the spawning and nursery ground with their off springs being associated with benthic fauna as food. despite the aforementioned importance, there are limited studies on the bay benthic fauna communities, necessitating the need for such studies. therefore, the aim of this study was to investigate frequency, biomass and seasonal variation in the macrobenthic fauna of the gorgan bay and its correlation with water physiochemical factors and sediment heavy metals (zn, cd, cr and pb) levels. materials and methods samples were collected during 2011, from 5 sampling sites (fig. 1). samples were collected monthly from each site. monthly data were averaged to represent seasonal data of the site, thus, there were 5 samples per season for the whole study area. benthos and sediment sampling was performed using a van veen grab (area = 225 cm2). benthos samples were sieved using a 63-micron sieve and macrobenthic organisms were fixed with 4% formalin. macrobenthos were identified taxonomically and their number and biomass were determined per m2. figure 1. the sampling sites in the gorgan bay. 247 saghali et al/ international journal of aquatic biology (2013) 1(5): 245-253 sediment sample preparation and heavy metals concentration (atomic absorption device) was measured according to saghali et al. (2013). water physicochemical characteristics including temperature, ph, dissolved oxygen, turbidity and salinity were recorded at each sampling site. water dissolved oxygen, salinity and ph were determined using a portable multiparameter meter (sension 156, usa). turbidity was measured using a secchi disk. data were subjected to shapiro-wilk's test to assess normality. normal data were analyzed by one way anova and duncan's tests, whereas non-normal data were analyzed by kruskal-wallis and mann– whitney u. to examine correlation between benthos frequency and water and sediment factors, data were analyzed using the spearman's test. data are presented as mean ± se. p<0.05 considered as the significant difference. results heavy metal concentrations of sediment are presented in table 1. the maximum metal concentration was related to zn and pb, whereas, cr and cd had the lowest values. there was no significant difference in zn and cr concentrations among the sampling seasons. pb concentration in winter was significantly lower than that of the other seasons, whereas, cd concentrations in the spring and summer were significantly lower than the autumn and winter. a total of 11 families were found belonging to 3 phyla of invertebrates (table 2). the phyla were annelids (nereidae, naididae, ampharetidae, lumbriculidae, tubificidae and amphiporidae), arthropods (pontogammaridae, balanidae and chironomidae) and mullusks (cardiidae and scrobicularidae). the dominant groups, in the case of frequency, were lumbriculidae (413 individuals m-2, corresponding to 18.7%), followed by ampharetidae (338 individuals m-2, corresponding metal season concentration zn spring 731.6 ± 78.6 a summer 787.8 ± 55.8 a autumn 816.6 ± 89.5 a winter 766.2 ± 91.4 a cr spring 165.6 ± 18.2 c summer 159.6 ± 22.5 c autumn 230.8 ± 34.7 c winter 192.4 ± 41.3 c cd spring 103.8 ± 5.89 d summer 107.2 ± 10.7 d autumn 178.8 ± 21.9 c winter 184.2 ± 30.8 c pb spring 771.8 ± 89.6 a summer 693.4 ± 120.7 a autumn 683.4 ± 79.9 a winter 520.4 ± 71.2 b table 1. sediment heavy metal concentrations in different seasons. different latin letters show significant difference. (anova and duncan's test; p< 0.05). a b b 0 200 400 600 800 1000 1200 1400 1600 1800 2000 annelids arthropods mollusks f re q u e n cy ( in d iv id u a ls / m 2 ) benthos figure 2. mean yearly frequency of different phyla of macrobenthos collected from the gorgan bay. n = 20. different letters above the bars show significant difference (kruskal-wallis and mann whitney u tests, α<0.05). figure 3. mean yearly biomass of different phyla of macrobenthos collected from the gorgan bay. n = 20. different letters above the bars show significant difference (kruskal-wallis and mann whitney u tests, α=0.05) 247 248 saghali et al/ international journal of aquatic biology (2013) 1(5): 245-253 to 15.3%), amphiporidae (311 individuals m-2, corresponding to 14.1%), tubificidae (258 individuals m-2, corresponding to 11.7%) and balanidae (230 individuals m-2, corresponding to 10.4%) (table 2). the other families contributed less than 10% of the total number of collected macrobenthos. annelids with an average of 1557 individuals per m2 was the most frequent groups, spring summer autumn winter year annelids nereidae 186 ± 55.1 173 ± 47.2 122 ± 26.1 280 ± 36.2 190 ± 23.5 naididae 44.4 ± 34.4 17.6 ± 5.54 61.2 ± 22.6 53.2 ± 8.95 44.3 ± 10.5 ampharetidae 274 ± 80.40 656 ± 102 241 ± 31.1 181 ± 47.6 338 ± 52.9 lumbriculidae 391 ± 134 710 ± 96.9 264 ± 36.1 289 ± 40.6 413 ± 57.1 tubificidae 289 ± 107 457 ± 56.6 122 ± 13.5 166 ± 28.1 258 ± 41.3 amphiporidae 279 ± 92.3 623 ± 128 146 ± 11.90 198 ± 28.6 311 ± 56.5 arthropods pontogammaridae 177 ± 29.2 124 ± 24.9 146 ± 33.5 111 ± 18.9 balanidae 417 ± 135 228 ± 33.4 285 ± 55.8 17.8 ± 17.8 230 ± 47.6 chironomidae 8.80 ± 8.80 2.20 ± 8.80 mollusks cardiidae 293 ± 113 196 ± 50.6 188 ± 38.9 135 ± 33.1 203 ± 33.5 serobiculariidae 17.8± 17.8 246 ± 47.6 115 ± 30.2 44.8 ± 11.8 110 ± 25.9 table 2. seasonal and yearly frequencies of different macrobenthos groups collected from the gorgan bay. spring summer autumn winter year annelids nereidae 0.86 ± 0.25 0.80 ± 0.22 0.56 ± 0.12 1.29 ± 0.17 0.87 ± 0.11 naididae 0.01 ± 0.007 0.003 ± 0.001 0.01 ± 0.005 0.01 ± 0.001 0.01 ± 0.002 ampharetidae 0.10 ± 0.03 0.24 ± 0.04 0.09 ± 0.01 0.06 ± 0.01 0.12 ± 0.02 lumbriculidae 0.22 ± 0.07 0.39 ± 0.05 0.15 ± 0.02 0.16 ± 0.02 0.23 ± 0.03 tubificidae 0.24 ± 0.09 0.37 ± 0.05 0.10 ± 0.01 0.14 ± 0.02 0.21 ± 0.03 amphiporidae 0.15 ± 0.05 0.35 ± 0.07 0.08 ± 0.01 0.10 ± 0.02 0.17 ± 0.03 arthropods pontogammaridae 0.09 ± 0.01 0.06 ± 0.01 0.07 ± 0.02 0.05 ± 0.01 balanidae 17.1 ± 5.54 9.38 ± 1.37 10.6 ± 2.29 0.73 ± 0.73 9.45 ± 1.95 chironomidae 0.02 ± 0.02 0.005 ± 0.005 mollusks cardiidae 115 ± 30.2 15.0 ± 5.69 49.5 ± 17.8 40.6 ± 19.8 55.2 ± 16.1 serobiculariidae 0.13± 0.13 1.98 ± 0.36 0.86 ± 0.23 0.34 ± 0.08 0.83 ± 0.19 table 3. seasonal and yearly biomass of different macrobenthos groups collected from the gorgan bay (g m-2). figure 4. mean (± sd) seasonal frequency (left figure) and biomass (right figure) of total macrobenthos collected from the gorgan bay. n = 5. different letters above the bars show significant difference (duncant's test for benthos frequency and kruskal-wallis and mann whitney u tests for benthos biomass, α=0.05). 249 saghali et al/ international journal of aquatic biology (2013) 1(5): 245-253 followed by arthropods (344 individuals m-2) and mollusks (314 individuals m-2) (fig. 2). table 3 shows the biomass of different macrobenthos groups. the dominant groups, in the case of biomass, were cardiidae (55.2 g m-2, corresponding to 82.4%), followed by balanidae (9.45 individuals m-2, corresponding to 14.1%). the other families had less than 2% of the total biomass (table 3). mollusks with the average of 141 g per m2 had the highest biomass, followed by arthropods (9.5 g m-2) and annelids (1.6 g m-2) (fig. 3). results showed that macrobenthos frequency in summer was significantly higher than that of the spring, which was significantly higher than those of the autumn and winter (fig. 3). however, there was significantly a higher biomass in the spring than those of the autumn and winter, which were significantly higher than that of the summer (fig. 3). the most frequent macrobenthos were arthropods during the spring and annelids during the summer, followed by annelids during the spring and winter (fig. 4). the highest biomass was observed in mollusks and arthropods during spring, followed by mollusks in autumn and winter (fig. 4). water physicochemical characteristics are presented in figure 5. range of temperature, salinity, turbidity, ph and dissolved oxygen were 11.7-29 ºc, 12.7-14.8 (ppt), 47.5-74.2 (cm), 7.6-8.3 and 7-9.9 (ppm), respectively. there were significant correlations between benthos frequency and water physiochemical characteristics and sediment heavy metal levels (table 4). the following correlations were found: between figure 4. mean (± sd) seasonal frequency (left figure) and biomass (right figure) of different macrobenthos collected from the gorgan bay. n = 5. different letters above the bars show significant difference (kruskal-wallis and mann whitney u tests, α=0.05). figure 5. (left figure): mean (± sd) seasonal temperature (ºc), salinity (ppt) and turbidity (cm) of the gorgan bay. (right figure): mean (± sd) seasonal ph and dissolved oxygen (ppm) of the gorgan bay. n = 5. 249 250 saghali et al/ international journal of aquatic biology (2013) 1(5): 245-253 ampharetidae and water dissolved oxygen, between lumbriculidae and sediment cr, between tubificidae and sediment cr, between amphiporidae and sediment cr, between cardiidae and sediment cd, and between serobiculariidae and water ph. discussion the gorgan bay is an important in terms of fisheries. it is the habitat of commercial fishes, which are consumed local people. in addition, it is an important habitat for fish stock rehabilitation program conducted by the iranian fisheries organization. macrobenthos of the bay play an important role in feeding of the fish inhabiting this region. however, there are limited studies on macrobenthos communities of the gorgan bay, which makes it difficult to compare the results. in this study, annelids were the most diverse group (6 families) and mollusks were the less diverse one (2 families). reverse trend was observed in the case of biomass. akrami et al. (2007) studied on the benthic fauna of northern coasts of the gorgan bay at 2003. they reported that there were 10 families in the bay and that the most and less frequent groups were ostracoda and amphipoda, respectively. zn pb cr cd t do ph tur sal nereidae correlation coefficient -0.8 -0.2 -0.2 0.2 -0.8 0.4 0.6 0.5 -0.8 sig. 0.2 0.8 0.8 0.8 0.2 0.6 0.4 0.5 0.2 naididae correlation coefficient 0.4 -0.6 0.3 0.6 -0.4 0.8 0.2 -0.2 -0.4 sig. 0.6 0.4 0.7 0.4 0.6 0.2 0.8 0.8 0.6 ampharetidae correlation coefficient 0 0.8 -0.8 -0.8 0.8 -1* -0.4 -0.4 0.8 sig. 1 0.2 0.2 0.2 0.2 0.000 0.6 0.6 0.2 lumbriculidae correlation coefficient -0.4 0.6 -1* -0.6 0.4 -0.8 -0.2 0.2 0.4 sig. 0.6 0.4 0.000 0.4 0.6 0.2 0.8 0.8 0.6 tubificidae correlation coefficient -0.4 0.6 -1* -0.6 0.4 -0.8 -0.2 0.2 0.4 sig. 0.6 0.4 0.000 0.4 0.6 0.2 0.8 0.8 0.6 amphiporidae correlation coefficient -0.4 0.6 -1* -0.6 0.4 -0.8 -0.2 0.2 0.4 sig. 0.6 0.4 0.000 0.4 0.6 0.2 0.8 0.8 0.6 pontogammaridae correlation coefficient 0.4 -0.4 -0.4 0.4 0.4 -0.2 -0.8 0 0.4 sig. 0.6 0.6 0.6 0.6 0.6 0.8 0.2 1 0.6 balanidae correlation coefficient -0.2 0.8 0 -0.8 0.2 -0.4 0.4 -0.4 0.2 sig. 0.8 0.2 1 0.2 0.8 0.6 0.6 0.6 0.8 chironomidae correlation coefficient -0.93 0.32 -0.10 -0.32 -0.74 0.21 0.94 0.73 -0.73 sig. 0.07 0.68 0.90 0.68 0.26 0.78 0.06 0.26 0.26 cardiidae correlation coefficient -0.4 -0.3 -0.6 -1* 0.4 -0.8 0.2 -0.2 0.4 sig. 0.6 0.7 0.4 0.000 0.6 0.2 0.8 0.8 0.6 serobiculariidae correlation coefficient 0.8 -0.2 -0.2 0.2 0.8 -0.4 -1* -0.6 0.8 sig. 0.2 0.8 0.8 0.8 0.2 0.6 0.000 0.4 0.2 table 4. correlation of benthos frequency with water physiochemical characteristics and sediment heavy metal concentrations. asterisks show significant correlations (t = temperature, do = dissolved oxygen, tur = turbidity, sal = salinity). 251 saghali et al/ international journal of aquatic biology (2013) 1(5): 245-253 hashemian (1998) reported that the highest and lowest frequencies of benthos was observed in spring (7356 individuals m-2) and winter (4309 individuals m-2), respectively, in the southern coast of the caspian sea. these differences between the present study and others may be due to changes in the benthic fauna structure over time as well as differences between the gorgan bay and the caspian sea environment, plus ecological and hydrological features. kousari (2009) reported that the most frequent macrobenthos group on the caspian sea (mazandaran province) was annelids followed by mollusks and arthropods. in addition, chironimids formed a small part of the macrobenthic fauna of mazandaran province coasts (kousari, 2009). such higher frequency of mollusks, in kousari (2009) study compared to the present study, shows difference in the environmental condition of the gorgan bay and the caspian sea. factors such as food availability, substrate structure, physicochemical conditions of the habitat, organic materials, competition and hunting affect macrobenthos structure (rosenberg et al., 1992; nyhakken, 1993; dobson, 1998; akrami et al., 2007) the increased frequency of macrobenthos in the summer can be associated with the rise in temperature in the late spring and summer, when the number of phytoplanktonic products increases. therefore, greater amounts of food will be available to organisms due to the fall of planktons. thus, these organisms will intensify their activities such as nutrition and reproduction during this period of time. hence their frequency and variance will certainly rise (burstein et al., 1968). however, the lowest biomass observed in summer could be related to increased benthivorous fish feeding activity due to rise in temperature and metabolism. the higher biomass of mollusks compared to the other groups seems to be related to their heavy shells, increasing their biomass. in contrast, annelids are small benthos making their biomass less than other groups, despite their great frequency. there were large statistical variations in some data including mollusks' biomass in the spring, autumn and winter, annelids frequencies in the spring and summer, and arthropods frequency in the summer. this shows that there was a large variation among the sampling sites, which needs to be considered in the future studies. ampharetidae showed a significant negative correlation with water dissolved oxygen, indicating the high tolerance of this family to low oxygen. serobiculariidae had a significant negative correlation with water ph suggesting the suitability of lower ph for this species. lumbriculidae, tubificidae and amphiporidae were significantly and negatively correlated to the sediment cr, while, cardiidae were significantly and negatively correlated to the sediment cd. it is well established that heavy metal contamination seriously affects benthos richness (dauvin 2008). similarly, chen et al. (2010) reported significant and negative correlation between heavy metals (copper, cd and pb) and benthic fauna richness. the present study indicated that annelids had the highest frequency and lowest biomass in the gorgan bay. mollusks had the highest biomass and lowest frequency in the bay. the highest macrobenthos frequency was observed in summer and the lowest was observed in autumn and winter. the highest biomass of macrobenthos was observed in spring and the lowest was observed in summer. the highest heavy metal concentration was related to pb and zn. zn and cr showed no significant variability among the sampling seasons, whereas, cd and pb did. among the studied heavy metals and water characteristics, sediment cr showed a greater role in benthic community structure. references ahmadi m., mousavi netekaran i. 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(2022) 10(3): 201-208 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article evaluation of pseudomonas stutzeri am1 and pseudomonas oleovorans st1.1 isolated from shrimp pond sediments as probiotics for whiteleg shrimp, litopenaeus vannamei culture thi cam tu phan, ngoc ut vu, thi tuyet ngan pham, hung hai vu, truong giang huynh college of aquaculture and fisheries, can tho university, campus ii, 3/2 street, ninh kieu district, can tho city, vietnam. s article history: received 26 march 2022 accepted 27 may 2022 available online 2 5 june 2022 keywords: probiotics pseudomonas litopenaeus vannamei water quality growth performance abstract: this study aimed to isolate the probiotic potential of nitrifying bacterial strains and to evaluate their effects on water quality and growth performance of the whiteleg shrimp, litopenaeus vannamei. based on an initial screening of 100 isolates identified from sediment samples, 12 strains could remove nitrogen compounds and two strains (pseudomonas stutzeri am1 and p. oleovorans st1.1) showed highly efficient nitrogen removal ability. within 96 h, total ammonia nitrogen (tan) removal efficiency in the two strains was 28.0-31.6% and 21.5-24.9%, respectively. the water addition of 103 cfuml-1 of p. stutzeri am1 (t1) and p. oleovorans st1.1 (t2) effectively reduced tan, nitrite, nitrate, and total sulfide and increased the survival rate and biomass of shrimp. however, no significant differences were found between the control (t0) and treatment groups (t1 and t2) in the final weight, weight gain and specific growth rate of shrimp. overall, p. stutzeri am1 (t1) and p. oleovorans st1.1 used as water supplements improved water quality and the survival rate of whiteleg shrimp. introduction shrimp farming is one of the fastest-growing aquaculture sectors in many countries worldwide. in vietnam, the whiteleg shrimp, litopenaeus vannamei is a hugely popular farmed shrimp; in 2020, this species's farming area and production reached 113,418 ha and 900,000 tons, respectively, increasing by 12% over the same period in 2019 (mard, 2021). in recent years, environmental problems caused by poor water quality are one of the threats to shrimp production (wang et al., 2018). through several studies, probiotics have demonstrated significant potential as therapeutic options for improving water quality (cai et al., 2019; nimrat et al., 2019; kim et al., 2021). probiotics in aquaculture can be administered through water additives or feed supplements during the rearing of aquatic species (zhou et al., 2009; leyva-madrigal *correspondence: truong giang huynh doi: http//doi.org/10.22034/ijab.v10i3.1580 e-mail: htgiang@ctu.edu.vn et al., 2011). a variety of bacteria (bacillus, lactobacillus, lactococcus, entercoccus, clsotridium, aeromonas, and pseudomonas), yeasts, and unicellular algae have been studied for use as probiotics in aquaculture (irianto and austin, 2002; vijayan et al., 2005). members of the genus pseudomonas are commonly found in aquatic environments, including shrimp culture ponds (preetha et al., 2015). the members of this genus are gram-negative rod with polar flagella providing motility, oxidase-positive, and catalase-positive, and it is obligate respiratory (jurat-fuentes and jackson, 2012). based on the results of pseudomonas application in several studies, these species have been proven to control pathogens, enhance growth performance, immune system function, and improve environmental conditions in the culture systems favorable to the 202 phan et al./ pseudomonas stutzeri and p. oleovorans as probiotics for whiteleg shrimp species being cultured (preetha et al., 2015; hai et al., 2009). in addition, dou et al. (2021) suggested that pseudomonas species possess great potential to remove nitrogen from wastewater based on aerobic autotrophic nitrification and anaerobic heterotrophic denitrification processes. for example, jin et al. (2015) reported that pseudomonas sp. adn-42, isolated from soil, has good nitrogen removal performance. similar results were found in p. stutzeri (wen et al., 2010), p. putida y-9 (xu et al., 2017), and p. putida zn1 (zhang et al., 2018). the present study aimed to isolate and identify efficacious probiotic pseudomonas spp. from shrimp pond sediments and examine their efficacy to remove nitrogen and enhance growth performance, survival, and water quality in whiteleg shrimp in vitro and in vivo conditions. two strains, pseudomonas stutzeri am1 and p. oleovorans st1.1, obtained in this study, exhibits huge potential as a probiotic candidate, as they effectively improved shrimp survival and water quality. the present study's findings can be applied to enhance health status and water quality in the shrimp culture industry. materials and methods isolation of candidate probiotic bacteria: sediment samples were collected from extensive shrimp ponds in soc trang, tra vinh, and kien giang provinces, and pseudomonas spp. were isolated from the collected samples using aob and nob media (chankaew et al., 2017). pseudomonas isolates were screened by applying the nitrogen removal tests (yang et al., 2011). they were identified using 16s rrna sequencing according to the manufacturer’s instructions after pcr amplification using a 27f-cm primer (5′ agagt ttgatcmtgg ctcag 3′) and a 1492r primer (5′ tacggytaccttgtt acgactt 3′) (frank et al., 2008). the purified products were sequenced by the nam khoa biotek company, ho chi minh city, vietnam. sequences were analyzed through http://www.ncbi.nlm.nih .gov/blast by comparing them with bacterial 16s rrna sequences in genbank to identify species. experimental design experiment 1: in vitro evaluation of nitrogen removal efficiency of p. stutzeri am1 and p. oleovorans st1.1: wastewater was collected from shrimp pond and analyzed for initial concentrations of tan, no2 --n, and no3 --n. a single colony of strains am1 and st1.1 were cultivated in a luria bertani broth medium and incubated with shaking until the stationary phase. the bacterial cells were harvested by centrifuging at 3000 rpm for 10 min at 4℃ and washed twice with 0.9% sterile nacl solution. then, the cells were resuspended in sterile saline (0.9% nacl) and adjusted to achieve a bacterial cell density of 1×108 cfu ml1 used for experiments (lami et al., 2020). the two-candidate probiotic bacteria were tested on the duran bottle scale for nitrogen removal efficiency. pseudomonas stutzeri am1 (t1, t2, and t3) and p. oleovorans st1.1 (t4, t5, and t6) at different concentrations (103, 104, and 105 cfuml1) were inoculated into 200 ml of wastewater in 250 ml duran bottle shaken at 150 rpm, 30ºc for four days. each treatment was run in triplicates. water parameters, including tan, no2 --n, no3 --n, ph, and temperature, were measured daily to calculate the nitrogen removal efficiency using the formula of nitrogen removal (%) = ((ci -ct) / ci) × 100, where ci is the initial concentration of nitrogen and ct is the concentration of nitrogen after sampling time. experiment 2: evaluation of p. stutzeri am1 and p. oleovorans st11 on water quality and growth performance of whiteleg shrimp: based on experiment 1, p. stutzeri am1 and p. oleovorans st1.1 at a concentration of 103 cfuml-1 were used for experiment 2. this experiment was carried out in the wet lab of the college of aquaculture and fisheries, can tho university. shrimp were fed a commercial diet (40% crude protein, 6% crude lipids, and 4% ash) for 7 days. after acclimatization, 100 shrimp (0.66±0.01 g) were randomly stocked into each composite tank, with a capacity of 500 l. the groups were given three treatments: the control group (t0) was not supplemented with probiotics, 203 int. j. aquat. biol. (2022) 10(3): 201-208 and the two other experimental groups were cultured in water supplemented every three days with probiotics (103 cfuml-1) containing p. stutzeri am1 (t1) and p. oleovorans st1.1 (t2), respectively. each group consisted of three replicates. the commercial feed was supplied to the shrimp three times daily at 3-5% body weight. experiments were performed with a 50% water exchange twice a week and lasted for 30 days. each tank was supplied with constant aeration during the experimental period to maintain the dissolved oxygen concentration (>4 mgl-1), and the salinity was maintained at 20 ppt. the water temperature and ph were 28.8-30.0℃ and 7.8-8.3, respectively. total alkalinity, tan, no2 --n, no3 —n, and total sulfide (s2⁻) were analyzed two times a week following the procedures of the standard methods (apha, 2017). at the end of the experiment, the final weight (fw), weight gain (wg), daily weight gain (dwg), specific growth rate (sgr), survival rate (sr), and shrimp biomass (b) for all groups were measured and calculated following the method described by niu et al. (2015): wg (g) = final weight initial weight dwg (g day-1) = [final weight initial weight]/days of culture sgr (% day-1) = 100 × (ln final mean weight ln initial mean weight)/days of culture sr (%) = 100 × [number of final shrimp / number of initial shrimp] b (kg.m-3) = [final weight × final population]/volume of water statistical analysis: statistical analysis was carried out using spss ver. 22 (spss inc. chicago, il, usa), and the results were presented as mean ± standard error (se). all data were subjected to oneway analysis of variance (anova), and tukey’s multiple comparisons test was then used to identify significant differences between treatments. all statistical significance tests were at the p<0.05. results isolation of probiotic bacteria: hundreds of microbial strains were isolated from the samples collected from the sediment of shrimp ponds. six strains, cn2.1, cn6.1, cn8.1, st1.1, tb7.2, and tv3.1 showed a high ammonium removal ability (aob), whereas strains am1, cn6.2, cn7.1, tb3.2, tb7.1, and tv4.1 demonstrated high nitrite removal efficiencies (nob) (table 1). the st1.1 and am1 strains had the highest nitrogen removal ability among the twelve bacterial strains and were selected for further study. after blast analysis, partial 16s rrna gene sequences analysis revealed 99% homology of isolates st1.1 and am1 with p. oleovorans and p. stutzeri, respectively. the nitrogen removal performance of strains am1 and st1.1: temperature and ph ranged 28.829.5ºc and 7.5-7.8, respectively. the aerobic inorganic nitrogen removal capacity of the strains st11 and am1 was shown in figure 1. in both strains st1.1 and am1, the removal efficiencies of tan, no2 --n, and no3 -n during 96 h were 21.531.6, 3.5-14.4, and 8.1-19.2%, respectively. although tan, no2 --n, and no3 --n were reduced in all treatments after 24 and 48h, no significant differences were found between treatments (p>0.05). after 96 h of incubation, tan in the treatments supplemented with strains st1.1 and am1 at different concentrations (t1, t2, t3, t4, t5, and t6) was significantly reduced than the control treatment. there were no significant differences aob strains ammonia removal (%) nob strains nitrite removal (%) cn2.1 69 am1 76.6 cn6.1 66.3 cn6.2 74.7 cn8.1 69.8 cn7.1 74.7 st1.1 73.2 tb3.2 72.1 tb7.2 64.6 tb7.1 70.5 tv3.1 62.1 tv4.2 71.3 table 1. nitrogen removal performance of the isolated nitrifying bacteria. 204 phan et al./ pseudomonas stutzeri and p. oleovorans as probiotics for whiteleg shrimp between t1, t2, t3, t4, t5, and t6 (p>0.05). the higher removal efficiency and faster removal rate of nh4 +-n indicated that strains st1.1 and am1 possessed a higher potential for heterotrophic nitrification. effects of p. stutzeri am1 and p. oleovorans st1.1 on water quality parameters: water temperature, ph, do, and alkalinity in each group were in a figure 1. nitrogen removal performances of strains am1 and st11 during the experimental period. a a ab b b0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 1 3 6 9 12 15 18 21 24 27 30 t a n ( m g l -1 ) days of culture t0 t1 t2 a a a a b b b 0 1 2 3 4 5 6 7 8 1 3 6 9 12 15 18 21 24 27 30 n o 2 n ( m g l -1 ) days of culture t0 t1 t2 b a a ab b b 0 1 2 3 4 5 6 7 1 3 6 9 12 15 18 21 24 27 30 n o 3 n ( m g l -1 ) days of culture t0 t1 t2 c 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 1 3 6 9 12 15 18 21 24 27 30 s 2 (m g l -1 ) days of culture t0 t1 t2 d figure 2. concentrations of tan, no2--n, and no3-n in different treatments during the experimental period. 205 int. j. aquat. biol. (2022) 10(3): 201-208 proper range for shrimp growth and survival from the beginning until the end of the experiment, and there were no significant differences between groups (table 2). tan in all groups reached the highest concentrations on day 9 of the experiment, and significant differences were found among treatments. tan in the control group was significantly higher than the t1 and t2 (p<0.05). the tan concentrations in all groups decreased later on day 12 and remained quite stable until the end of the experiment (fig. 2a). the concentrations of no2 --n in all groups gradually increased during the experimental period, and reached a peak at the end of the experiment. no2 —n concentrations in two experimental groups inoculated with probiotics (t1 and t2) were significantly lower than those of the control group on days 24, 27, and 30, but no significant difference was found between the two probiotic treated groups (p>0.05) (fig. 2b). the concentrations of no3 --n in all treatments varied from 10 to 8 mg l-1. no3 --n in the t1 and t2 were significantly lower than those in the control group on days 21 and 24 (p<0.05). however, there were no significant differences among all treatments on the other sampling days (p>0.05) (fig. 2c). total sulfide (s2-) in all treatments gradually increased from the beginning of the experiment until day 15 and decreased sharply later. no significant differences in total sulfide were found between treatments (fig. 2d). effects of p. stutzeri am1 and p. oleovorans st1.1 on growth performance of the whiteleg shrimp: table 3 shows the effects of inoculating p. stutzeri am1 and p. oleovorans st1.1 into the experimental tanks’ water on the survival and growth performance of whiteleg shrimp. the results showed that the experimental group inoculated p. stutzeri am1 (t1) showed a significantly higher survival rate and biomass compared with the control group (p<0.05). however, no significant differences were found in these parameters between t2 and the control groups. similarly, there were no significant differences in the fw, wg, and sgr between all groups (p>0.05). discussion the role of probiotics in controlling the colonization table 2. water temperature, ph, do, and alkalinity during the experimental period. parameters treatments t0 t1 t2 ph 7.87±0.05a 7.79±0.07a 7.8±0.08a temperature (℃) 28.1±0.3a 28.1±0.3a 28.1±0.3a do (mg l-1) 4.2±0.07a 4.21±0.08a 4.19±0.06a alkalinity (mgcaco3 l -1) 137.8±4.0a 137.5±4.1a 137.4±4.0a values shown are mean ± se. mean values within a row followed by the same letters show that there is no significant difference among the groups (p>0.05). table 3. growth performance and survival rate of the whiteleg shrimp. parameters treatments t0 t1 t2 initial weight (g) 0.66±0.01a 0.67±0.01a 0.65±0.01a final weight (g) 5.28±0.09a 5.54±0.09a 5.45±0.14a wg (g) 4.62±0.08a 4.87±0.08a 4.8±0.13a dwg (g day-1) 0.154±0.003a 0.162±0.003a 0.16±0.004a sgr (% day-1) 6.92±0.04a 7.06±0.06a 7.07±0.05a survival rate (%) 74.3±1.8b 88.7±3.5a 78.3±2.2ab biomass (kg m-3) 0.98±0.01b 1.23±0.03a 1.07±0.04b values shown are mean ± se. mean values within a row followed by the same letters show that there is no significant difference among the groups (p>0.05). 206 phan et al./ pseudomonas stutzeri and p. oleovorans as probiotics for whiteleg shrimp and overgrowth of pathogens has been documented in many studies (moriarty, 1999). in addition, the application of probiotics in aquaculture also enhances innate immunity (chauhan and singh, 2019), prevents microbial diseases (meidong et al., 2018), promotes growth and improves water quality (ma et al., 2009; kolndadacha et al., 2011). zhang et al. (2011) and tran et al. (2019) reported that several pseudomonas species could remove nitrogen. in the present study, two bacterial isolates, am1 and st1.1 showed the highest efficiency in removing nitrogen. according to 16s rrna sequencing, these candidates were identified as p. stutzeri and p. oleovorans. based on in vitro results, the concentration of nh4 +-n, no2 --n, and no3 --n decreased significantly after 96 h incubation, suggesting that strains am1 and st11 have a strong ability to oxidize and assimilate nitrogen in the water. similar results were also reported with strains of p. putida y-9 (xu et al., 2017), pseudomonas sp. (tran et al., 2019), and p. fluorescens nb14 (duo et al., 2021). yang et al. (2017) reported that the removal efficiency of total nitrogen was directly related to bacterial growth. in addition, the results of this study indicated that strain am1 has a strong ability to remove nitrogen compounds than st11. in water-supplemented conditions, the effects of p. stutzeri and p. oleovorans application had no impact on shrimp growth parameters (final weigh, wg, dwg, and sgr), but the survival rate and biomass of shrimp in the treatment supplemented with p. stutzeri am1 (t1) were significantly greater than those in control one. these results suggest that the application of p. stutzeri am1 via water addition improved the survival of whiteleg shrimp. this observation is similar to balcázar et al. (2007) report, where the application of p. aestumarina slv22 at 105 cfu g-1 diet for 28 days enhanced the survival rate of whitelegs shrimp. ngo and ravi (2009) also reported that penaeus latisulcatus shrimp fed p. synxantha and p. aeruginosa (2106 cfu kg-1 diet) diets for 84 days had a significantly higher survival rate. some previous studies demonstrated that the increased survival of shrimp supplemented with probiotics (lactobacillus plantarum, bacillus sp., pediococcus acidilactici, p. fluorescens) might be related to the decreased abundance of vibrio spp. in the digestive tract (lone et al., 1999; wang et al., 2007; castex et al., 2008; vanmaele et al., 2015). in addition, borges et al. (2008) reported that probiotics promoted the health of animals through improved water quality. in our study, water quality parameters, including ph, temperature, do and alkalinity were within suitable ranges for the culture of whiteleg shrimp (law 1988; wybanet al., 1995; nonwachaiet al., 2011; boyd, 2007) and were not significantly different between the treatments. however, the tan, no2 --n, no3 --n, and total s2concentrations were lower when the probiotic (p. stutzeri or p. oleovorans) was applied compared with no probiotic application. this could be suggested that p. stutzeri and p. oleovorans played an important role in improving water quality. in conclusion, based on the in vivo and in vitro experiments, the two candidate probiotics p. stutzeri am1 and p. oleovorans st11 isolated from the shrimp pond sediment, had the clear ability to remove nitrogen compounds in wastewater from shrimp culture. additionally, these isolates also effectively enhanced the survival rate of shrimp and water quality, which is very important for application in shrimp farms. therefore, these strains can be used as a potential probiotic in aquaculture. acknowledgments this research was partly funded by the can tho university improvement project vn14-p6, supported by a japanese oda loan. references apha (2017). standard methods for the examination of water and wastewater, 23rd edition. american public health association, american water works association, water environment federation, denver. 1504 p. balcázar j.l., tyrone r., david p.c. 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(2018) 6(6): 314-320 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article acute and chronic toxicity of ammonia in persian sturgeon, acipenser persicus, fingerlings shahram naghizadeh raiesi*1, mohammad mazandarani2,1seyed-ahmad shahidi1, azade ghorbani-hasansaraei1, , fatemeh khani2, sjad fatahi2 1department of food science and technology, college of agriculture and food science, ayatollah amoli branch, islamic azad university, amol, iran. 2department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 29 april 2018 accepted 10 december 2018 available online 2 5 december 2018 keywords: sturgeon ammonia lc50 growth performance toxicity abstract: in the present study, susceptibility of persian sturgeon, acipenser persicus, to un-ionized ammonia (uia) was studied. fish with an average weight of 5.52±0.45 g were exposed to different concentrations of un-ionized ammonia (uia) to determine median lethal concentration (96-h-lc50). the fish were exposed to six different concentrations of uia (control, 0.16, 0.31, 0.62, 0.94 and 1.25 mg/l uia). the control group was not challenged with ammonia (0.0008 mg/l uia). the results showed that the 96-h-lc50 of uia in persian sturgeon was 0.46 mg/l. according to 96-h-lc50, midterm exposure to sub-lethal uia was examined. in the chronic examination, the fish were exposed to different levels of sub-lethal ammonia; control (0.0008 mg/l uia), 10% of lc50 (0.05 mg/l uia), 5% of lc50 (0.025 mg/l uia) and 1% of lc50 (0.005 mg/l uia) for 30 days. final length, final weight, weight gain, specific growth rate, daily weight gain and body weigh index decreased significantly in the fish exposed to 0.05 and 0.025 mg/l uia. according to the results, conversion ratio and condition factor indices were higher in the fish exposed to 0.005 mg/l uia and control group compared with the fish exposed to 0.05 and 0.025 mg/l uia. introduction sturgeons’ rearing centers have been developing rapidly because catching the sturgeons has dramatically declined in the recent years. these species are reared in intensive and semi-intensive ponds, thus, water ammonia elevation is one of negative factors which can affect growth performance parameters of fish (el-shafai et al., 2004). agricultural and industrial run off, and decomposition of organic and biological compounds are the external sources of ammonia (barbieri and bondioli, 2015; benli et al., 2008). however, if the fish rearing water is un-polluted, ammonia elevation in intensive fish culture is related to protein catabolism of fish (walton and cowey, 1977; hasanalipour et al., 2013). in aquatic environments, there are two forms of ammonia, ionized and un-ionized (uia), which the un-ionized ammonia is more toxic to fish. transformation of the ionized and un-ionized ammonia forms depend on water temperature and ph (emerson et al., 1975). catabolism of protein in fish *corresponding author: shahram naghizadeh raiesi doi: https://doi.org/10.22034/ijab.v6i6.425 e-mail address: shahram9112006@outlook.com leads mostly to un-ionized ammonia production, which is mainly excreted by gills (wilkie, 1997). although ammonia has a great affinity to be ionized, there is an equilibrium between ionized and unionized ammonia transformation in aqueous environment. for instance, one unit ph elevation lead to 10 folds increase in coefficient of ionized to unionized ammonia transformation (emerson et al., 1975). the ammonia accumulation in intensive aquaculture is particularly unavoidable because of high protein catabolism (person-le ruyet et al., 1995; handy and poxton, 1993). ammonia elevation may affect the fish growth and reproduction performance parameters (guan et al., 2010; ip and chew, 2010). in aquaculture, it is important to know the concentrations of toxic ammonia under certain water temperature and the other physic-chemical conditions. also, fish vulnerability to ammonia is depend on their species, age and size. there are several studies on the ammonia toxicity in fishes (barbieri and bondioli, 2015; benli et al., 2008; dong et al., 2013; el-shafai 315 int. j. aquat. biol. (2018) 6(6): 314-320 et al., 2004; lemarie et al., 2004; person-le ruyet et al., 1997; rodrigues et al., 2014; wajsbrot et al., 1991). reporting different range of ammonia safe concentrations in several studies confirm that it is depend on different fish species and physic-chemical conditions. therefore, having information on uia susceptibility of each species is essential. although there are some reports on susceptibility of some sturgeon species to ammonia such as siberian sturgeon, acipenser baeri (salin and williot, 1991) and shortnose sturgeon, a. brevirostrum (isely and tomasso, 1998), there is a lack of information on susceptibility of persian sturgeon, a. persicus to water ammonia. considering that persian sturgeon is cultured in iran, determining the susceptibility of this species to water ammonia seems crucial. materials and methods ammonia preparation: to determine the water uia concentration, water ph and temperature should be controlled. water ph was fixed at 8.2 with koh (0.1 n) and water temperature was fixed at 25°c with heater (aquent co, usa). nh4cl (merck, darmstadt, germany) was used to prepare different concentrations of the ammonia. stock ammonia solution was prepared for each treatment. water total ammonia was measured based on le and boyd (2012) and the proportion of ionized and uia was determined according to water temperature and ph (emerson et al., 1975). determination of un-ionized ammonia median lethal concentration (lc50): a total of 300 persian sturgeon fingerlings (5.52±0.45 g) were provided from the shahid marjani sturgeon hatchery (golestan province, iran). the fish were stocked in three 900l tanks (1.5×1.5×0.4 m). after one week acclimation, susceptibility of these fish to un-ionized ammonia was studied. 144 fish were distributed into 18 aquaria with 60 l volume (0.5×0.3×0.4 m) and acclimatized to the experimental conditions for another one week. all aquaria were aerated continuously during experimental period (do: 6.2±1.11 mg/l). during adaptation, the fish were fed with commercial feed (energy, mahiran co, iran). feeding was performed 3% of the fish biomass twice a day. to determine ammonia median lethal concentration (lc50), the fish were exposed to six concentrations of ammonia (0.16, 0.31, 0.62, 0.94 and 1.25 mg/l uia and a control group) for 96 hours, which assigned in three replication for each treatment. the fish were exposed to the aforementioned ammonia concentrations in semi-static condition, during which, water was daily renewed and required amounts of ammonia were added to each aquarium to keep the ammonia concentration constant. feeding was ceased 24 h before ammonia exposure and thereafter. fish mortality, clinical signs and behaviors were recorded every 12 h (at 8 am and 8 pm). during the trial, the water hardness was 267±5.48 mg/l. temperature, dissolved oxygen and ph were controlled at 25±1°c, 6.2±1.11 mg/l and 8.2±0.11, respectively. mid-term exposure to sub-lethal concentrations of ammonia: chronic experiment was conducted according to the lc50, and fish were exposed to 1, 5 and 10% of 96-h-lc50. in this regard, 120 fish were randomly divided into 12 aquaria with 45 l water. after one week acclimation period, the aquaria were classified into four groups (triplicate per treatment). no ammonia was added to control group (un-ionized ammonia determined 0.0008 mg/l). the ammonia treated groups were exposed to 0.05 mg/l (10% of 96h lc50), 0.025 mg/l (5% of 96h lc50) and 0.005 mg/l (1% of 96h lc50) un-ionized ammonia for 30 days. during experiment, fish were fed at 3% of biomass twice a day by commercial food (energy, mahiran co, iran). the maintenance conditions are the same as which is mentioned in acute toxicity of the experiment. fish growth characteristics were recorded every 10 days as follows (el-husseiny et al., 2008): survival rate (sr) = final number of fish ×100 / initial number of fish specific growth rate (sgr) = [(ln final weight ln initial weight×100) / days] food conversion ratio (fcr) = the total food consumption (g) / weight gain (g) weight gain (wg) = final weight initial weight condition factor (cf) = final weight ×100 / (final length)3 body weight index (bwi) = (final weight initial 316 naghizadeh raiesi et al./ acute and chronic toxicity of ammonia in persian sturgeon weight) ×100 / initial weight daily weight gain (dwg) = [final body weight (g) initial body weight (g)] / days water physic-chemical characteristics: water total ammonia was measured by salicylate method (le and boyd, 2012). uia was calculated according to emerson et al. (1975). other water physic-chemical characteristics were measured by portable water checker and photometer with commercial kits provided by the manufacturer (wagtecch portable photometer 7100, berkshire, uk). statistical analyses: lc50 was determined using spss v. 22 software and probit test. the lowest observed effect concentration (loec) and no observed effect concentration (noec) were determined according to rand (1995). data of growth parameters were analyzed by using one-way anova and duncan test to find significant differences. p<0.05 was considered as significance. data are presented as mean ± standard deviation (sd). results the mortality of the fish exposed to different uia concentrations (0.16, 0.31, 0.62, 0.94 and 1.25 mg/l) during 96 h is presented in figure 1. there was no mortality in the control group and fish that exposed to 0.16 mg/l uia. in the 0.31 mg/l uia treatment, mortality was started at 48 h and reached 25% after 96 h. in the 0.62 mg/l uia treatment, mortality was started after18 h and it reached 75% up to 72 h post challenge. in the 0.94 mg/l uia and the highest concentration (1.25 mg/l) treatments, 100% of fish died after 6 hours exposure (fig. 1). according to the results, 0.46 mg/l (037-0.50 mg/l confidence limits) was calculated as 96-h-lc50 of un-ionized ammonia in persian sturgeon. loec and noec were 0.31 and 0.16 mg/l, respectively (table 1). behavioral symptoms during acute exposure: the control fish showed normal behavior including normal swimming with no abnormal reactions. the fish exposed to uia were generally trying to jump out of the aquaria and highly-responsive in the elementary hours of the test. the fish exposed to 1.25 mg/l uia showed abnormal behaviors such as fast and irregular swimming just after 1 h exposure. then, they became immobile at the aquaria bottom, followed by loss of equilibrium and imbalance lateral swimming. fast and irregular swimming was observed in the other table 1. lc50, loec and noec of un-ionized ammonia in persian sturgeon fingerlings. 95% confidence limits concentration (mg/l) upper lower slope±s.e intercept±s.e loec noec lc50 0.46 0.37 0.50 5.82±0.92 2.17±0.36 0.31 0.16 figure 1. mortality rate of persian sturgeon fingerlings during 96 h of exposure to different un-ionized ammonia concentrations (0.16, 0.31, 0.62, 0.94 and 1.25 mg/l). 317 int. j. aquat. biol. (2018) 6(6): 314-320 treatments, after 5 h exposure. also, the fish exposed to 0.31-0.96 mg/l uia showed fast swimming. in the 0.16 mg/l uia treatment, the fish showed slow swimming and bottom sitting in the aquaria. clinical signs during acute exposure: opened-mouth and skin petechia was the most frequent clinical signs of the freshly-dead fish. all moribund fish showed moderate hemorrhage at base of pectoral fins and barbels. great amount of mucus was observed on the skin of fish exposed to ammonia. after 12 h exposure, congestion in intestine and some internal organs was recorded. accumulation of bloody fluid in the abdominal cavity was also observed in dead fish after 30 h ammonia exposure. hemorrhage around anus and at the terminal part of the gut was observed in the some moribund fish within 48-96 h post challenge (table 2). growth performance: the growth performance parameters in the persian sturgeon exposed to different concentrations of unionized ammonia during 30 days are presented in table 3. no difference was observed in growth performance parameters between control group and the group exposed to 1% 96-h-lc50 (fish exposed to 0.005 ppm uia). decrease in growth performance parameters was reported by increasing the uia concentration to 0.025 and 0.05 mg/l. according to the result, most growth parameters (final length, final weight, weight gain, specific growth rate, daily weight gain and body weigh index) decreased significantly (p<0.05) in the fish exposed to 0.05 and 0.025 mg/l uia. feed conversion ratio and condition factor were higher in control and 1% 96-h-lc50 treatment in comparison with 5% and 10% 96-h-lc50 treatments. on the other hand there was significant difference in growth performance parameters between 5% and 10% 96-h-lc50 treatments (table 3). discussion in aquatic environments, dissolved ammonia is table 2. behavioral symptoms of persian sturgeon fingerlings exposed to different water un-ionized ammonia concentrations. un-ionized ammonia concentration (mg/l) behavioral responses control 0.16 0.31 0.62 0.96 1.25 nervousness ─ + 5 + 5 + 5 + 5 1+ irregular swimming ─ + 5 + 5 + 5 + 5 1+ loss of equilibrium ─ ─ ─ ─ ─ + imbalance swimming ─ ─ ─ ─ ─ + fast swimming ─ ─ + + + ─ responsive to external stimuli ─ ─ + + + ─ bottom sitting ─ + ─ ─ ─ 5+ +: observed; ─: not observed; + 5: observed after 5 h; + 1: observed after 1 h. table 3. growth performance of juvenile persian sturgeon, asipenser persicus, exposed with different level of un-ionized ammonia (uia) for 30 days. parameters control 0.05 mg/l uia 0.025 mg/l uia 0.005 mg/l uia initial weight (g) 6.53±0.81 a 6.74±0.70a 6.71±0.64a 6.49±0.61a final weight (g) 15.50±0.92a 10.19±1.46d 10.65±1.91 c 14.12±1.85b initial length (mm) 12.38±0.82a 12.54±0.74a 11.98±0.64a 12.11±0.72a final length (mm) 17.21±0.79a 13.6±1.08c 14.83±1.32b 16.74±1.05a weight gain (g) 8.64±1.22a 3.45±1.52d 4.60±1.20c 7.24±2.91a dwg 1 0.29±0.04a 0.11±0.051d 0.15±0.04c 0.24±0.16a bwi2 (%) 125.36±28.57a 52.44±2 5.60c 66.92±27.41c 105.24±43.21a sgr3 (% day− 1) 1.18±0.18a 0.33±0.28 c 0.72±0.29b 1.04±0.23a cf 4 0.31±0.03c 0.41±0.0a 0.35±0.0b 0.30±0.02c fcr 5 0.94±0.14c 2.35±0.34a 1.76±0.24b 1.11±0.32c survival rate 100a 100a 100a 100a a, b, c, d mean values in the same row with different superscript are significantly different (p<0.05). 1: daily weight gain, 2: body weight index, 3: specific growth rate, 4: condition factor, 5: feed conversion ratio. 318 naghizadeh raiesi et al./ acute and chronic toxicity of ammonia in persian sturgeon presented in two forms, ionized and un-ionized. unionized ammonia has higher permeability in the organism’s epitelium, thus, it is more toxic. if it accumulates in high concentrations, it causes severe toxicity in fish (shingles, 2001; ip and chew, 2010). ammonia concentration can increase to toxic levels in intensive rearing ponds and recirculating water systems. therefore, exposure to high uia concentrations in persian sturgeon is not unlikely. one of the methods to investigate the susceptibility of different fish species to toxicants is the lc50 determination. several studies were conducted on this topic (ruffier et al., 1981; wajsbrot et al., 1991; persole royet et al., 1995). different fish species showed different susceptibility to acute un-ionized ammonia exposure. person-le royet et al. (1995) studied on some marine species and reported that 96h-lc50 was 1.79, 2.73 and 3.32 mg/l for sea bass (dicentrarchus labrax), sea bream (sparus aurata) and turbot (scophthalmus maximus), respectively and sea bass was more susceptible than the other species exposed to un-ionized ammonia. 96-h-lc50 of ammonia was recorded 0.32 mg/l in fry rainbow trout (oncorhynchus mykiss) (wajsbrot et al., 1991). ruffier et al. (1981) found 96-h-lc50 of uia 3.1 mg/l for catfish (ichtalarus punctatus). as mentioned, the sensitivity to uia is variable depending on the fish species and conditions of fish culture. in the present study, 96-h-lc50 of uia in persian sturgeon was 0.46 mg/l, and compare to the above studies, it seems that persian sturgeon fingerlings are high susceptible in exposure to the acute toxicity with uia. in the present experiment, after 30 days, 5% and 10% of lc50 had no effect on survival rate but they caused the lowest growth performance parameters. although persian sturgeon fingerlings can tolerate high concentration of uia (10% of lc50), their weight gain and growth performance parameters significantly decreased even in lower concentration of uia (5% of lc50). several studies show that sub-lethal concentrations of ammonia have negative effects on growth parameters in bony fish. foss et al. (2003) showed that 96 days exposure to 0.17 mg/l uia reduced the growth of juvenile atlantic cod (gadus orhua) significantly. they explained that the growth reduction is attributed to a decrease in food intake. remen et al. (2007) studied on the interactive effects of oxygen and ammonia on growth and physiological status of juvenile atlantic cod (gadus orhua). the fish were exposed to nine different combinations of ammonia and oxygen concentrations for 64 days. according to the results, high ammonia concentrations caused a significant decrease in growth throughout the experiment. although, foss et al. (2003) showed that 69 days exposure to 0.11 mg/l uia has no significant effect on growth parameters in juvenile spotted wolffish, anarhichas minor. because the fish were able to be adapted to the new condition. the effects of chronic and periodic exposure to unionised ammonia on growth and food conversion efficiency in juvenile atlantic halibut, hippoglossus hippoglossus were examined by paust et al. (2011). they reported that chronic medium and high levels of ambient ammonia had a deleterious effect on growth performance but periodic exposure had no significant effect. alderson (1979) reported the threshold concentration of uia had no significant effect on juvenile dover sole (solea solea) and turbot growth. but decreasing ph have a negative effect on the growth of the fish. during last decade sturgeon catch from the caspian sea critically has decreased so rearing center for these valuable fish are developing. in this regard, ammonia accumulation in rearing pond is unavoidable and every knowledge abaut its deleterious effect can lead to better culture. conclusion persian sturgeon fingerlings are sensitive in exposure to uia moderately and its 96-h-lc50 was determined 0.46 mg/l. this fish can tolerate chronic toxicity with sub-lethal concentrations of uia without any mortality but these concentrations caused decrease in growth performance parameters. acknowledgment the authors gratefully acknowledge ayatollah amoli branch, islamic azad university, amol, iran, for their financial and commercial support in this project. references alderson r. 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(1999). update of ambient water quality criteria for ammoniatechnical version-1999. epa-823-f-99024. usepa, washington dc, usa. wajsbrot n., gasith a., krom m.d., popper d.m. (1991). acute toxicity of ammonia to juvenile gilthead seabream, sparus uuratu under reduced oxygen levels. aquaculture, 92: 277-288. walton m.j., cowey c.b. (1977). aspects of ammoniogenesis in rainbow trout, sulmo gairdneri. comparative biochemistry and physiology, 57: 143149. wilkie m.p. (1997). mechanisms of ammonia excretion across fish gills. comparative biochemistry and physiology, 118: 39-50. international journal of aquatic biology (2014) 2(6): 299-304 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2014 npajournals. all rights reserved original article spawning season and larval occurrence of blood cockle (anadara granosa) off the selangor coast, peninsular malaysia tatsuya yurimoto*1, faizul mohd kassim2, alias man3, reiko fuseya4 1japan international research center for agricultural sciences, tsukuba, ibaraki, japan. 2penang office of japan international research center for agricultural sciences, penang, malaysia. 3fisheries research institute, department of fisheries, penang, malaysia. 4national research institute of fisheries engineering, fisheries research agency, kamisu, ibaraki, japan. article history: received 20 april 2014 accepted 25 october 2014 available online 2 5 december 2014 keywords: blood cockle, spawning season, larva, anadara granosa, malaysia. abstract: we performed a plankton survey of blood cockle larvae, anadara spp., and histological observations of the gonads of adult blood cockle, anadara granosa, in the coastal waters off sungai buloh, peninsular malaysia from october 2010 to april 2011. the histological observations indicated the main spawning period in both sexes was from november to march. in addition, the occurrence of blood cockle larvae peaked in november and march. furthermore, a comparison of the distribution between umbo-stage and full-grown larvae suggested the umbo-stage larvae diffused offshore and return to coastal sites where they settle and mature into full-grown larvae. introduction blood cockle, anadara granosa, is one of the most popular bivalves for sowing aquaculture in southeast asia and the selangor coast off peninsular malaysia (pathansali and song, 1958; broom, 1985). regional fishermen collect natural spats of blood cockle in tidal flats of the selangor coast and cultivate them by sowing culture. therefore, elucidating the current status of blood cockle reproduction in the selangor coast is important for ensuring the sustainable production of this species in this region. however, information about the relationship between larval occurrence and the spawning period of blood cockle is insufficient, preventing fisheries management from achieving sustainable production. therefore, this study evaluated the main spawning season of blood cockle, performed histological observations of the gonads, and monitored the density of bivalve larvae including blood cockle larvae at 4 stations of the selangor coast. the results will help establish a * corresponding author: tatsuya yurimoto e-mail address: yurimoto@outlook.com monitoring system for blood cockle larvae. materials and methods field survey: a total of 4 sampling surveys at sungai buloh off the west coast of peninsular malaysia were carried from october 2010 to april 2011 (fig. 1). four survey stations (stations 1-4) were set along the coast and the adult blood cockles were collected with a dredge of 1.5 cm mesh. at these 4 stations, bivalve larvae samples were collected with a zooplankton net (diameter: 30 cm (0.07 m2), side length: 100 cm, netting: nxx13 (0.1 mm mesh opening)) by vertical hauling from the seabed to surface. the average±sd depth at stations 1-4 during the survey period was 2.3 ± 1.1, 4.0 ± 1.5, 7.4 ± 1.4, and 5.2 ± 1.4 m, respectively. water temperature, salinity, the dissolved oxygen concentration in bottom-layer water (~20 cm above the seabed), and the average concentration of chlorophyll a in the water column of station 1 were measured with a water quality 300 international journal of aquatic biology (2014) 2(6): 299-304 sensor (aaq-rinko, jfe advantech co., ltd., japan). in addition, during the surveys in november and march, when blood cockle larvae peaked, the vertical gradients of water temperature and salinity in 4 stations (except station 2 in november) were measured to determine the relationship between water mass and the distribution of blood cockle larvae. gonad observation: adult blood cockles (a. granosa) collected from the survey field were transferred to a temporary laboratory. then the shell length measurement and gonadal tissue was removed. tissue blocks fixed in 10% seawater formalin solution were substituted in 100% lemosol after alcohol dehydration and embedded in paraffin. sections 7-10 μm thick were observed under a light microscope after hematoxylin–eosin staining. histological observations of the gonads were performed to determine sex and maturity according to 5 stages: the immature, developing, mature, spawning, and spent stages—to determine the main spawning period. these stages were determined on the basis of gonad observation in blood cockle, a. granosa (yurimoto et al., 2014). larval observation: the morphology of fresh larvae samples collected from the field was identified based on yoshida (1964) and muthiah et al. (1992), and the numbers of individuals were counted under a light microscope. the items counted were the number of total bivalve larvae, bivalve larvae excluding blood cockle larvae, blood cockle larvae (anadara spp.; individuals between the umbo and full-grown stages at the genus level), and umbo-stage and full-grown blood cockle larvae (anadara spp.). in addition, the density of individuals in the water column, sampled using a zooplankton net, was determined. results environment: water temperature at station 1 ranged from 29.8-30.9°c (fig. 2a). the temperature increased gradually after a nadir at the early of february. on the other hand, salinity decreased from 30.3 to 29.1 psu during october and november (fig. 2b) and subsequently increased to 29.7 psu in april. the concentration of dissolved oxygen remained at 2.7 mg/l throughout the survey period (fig. 2b). meanwhile, the concentration of chlorophyll a increased to 10 μg/l over on average in november and march. gonad condition: the average shell length of the blood cockles collected during the survey period was 29.1 ± 3.5 mm (n = 92), and the male:female ratio was 0.47 : 0.53. regarding sexual maturation, males were mainly classified into the developing and mature stages in october, the mature and spawning stages from november to march, and the spawning and spent stages in april (table 1). on the other hand, females were mainly classified into the mature stage from october to november; the spawning stage from february to march; and the developing, spawning, and spent stages in april. larval occurrence: the greatest density of bivalve larvae occurred at station 2, ranging from 425431/m3 during november and february; the density peaked at 1,351/m3 in march and decreased rapidly to 381/m3 in april (fig. 3a). station 3 had the next highest density, ranging from 245-624/m3. in addition, the densities at stations 1 and 4 fluctuated but remained low; the peak densities of stations 1 and figure 1. sampling location of adult blood cockle and zooplankton at (c) sungai buloh (b) off the selangor coast, (a) peninsular malaysia. station 1, adult cockle sampling site; stations 1–4, zooplankton sampling sites. 301 yurimoto et al/ spawning season and larval occurrence of blood cockle 4 were 340/m3 in november and 461/m3 in april, respectively. the greatest density of bivalve larvae excluding blood cockle larvae occurred at station 2, peaking at 637/m3 in march. this was followed by 480/m3 at station 3 in march (fig. 3b). similar to block cockle larvae, the densities at stations 1 and 4 fluctuated but remained low, showing small peaks in november and april, respectively. in addition, high densities of blood cockle larvae occurred at station 2, increasing from 6/m3 in october to 264/m3 in november; the low density in february increased again to 714/m3 in march and decreased to 54/m3 in april (fig. 3c). the densities at stations 1, 3, and 4 fluctuated but remained low: 0-163, 48-154, and 2– 230/m3, respectively. in addition, blood cockle larvae were divided into 2 stages umbo-stage and full-grown larvae to determine the differences in the distributions of each stage (fig. 4). the highest density of umbo-stage larvae occurred at station 2, peaking at 225 and 669/m3 in november and march, respectively. the densities at the other stations fluctuated below 216/m3 (fig. 4a). in addition, the density of full-grown larvae increased at stations 1, 2, and 3 in november and station 2 in march; the densities at those sites ranged from 1447/m3 (fig. 4b). next, we calculated the mean ± se densities of umbo-stage and full-grown larvae at each station. the mean densities of umbo-stage larvae at stations 1, 2, 3, and 4 were 49 ± 28 (n = 5), 206 ± 108 (n = 5), 90 ± 17 (n = 5), and 61 ± 45/m3 (n = 4), respectively (fig. 5). in contrast, densities of full-grown larvae at stations 1, 2, 3, and 4 were 12 ± figure 2. environmental changes at station 1, sungai buloh. (a, ●) water temperature, (a, ○) salinity, (b, ▲) dissolved oxygen in the bottom layer (~20 cm from the bottom), and (c, ◆) average concentration of chlorophyll a in the water column; error bars indicate standard deviation. figure 3. changes in the density of (a) bivalve larvae, (b) bivalve larvae except blood cockle larvae, and (c) blood cockle larvae (anadara spp.) from the umbo stage to the full-grown stage. 302 international journal of aquatic biology (2014) 2(6): 299-304 8 (n = 5), 17 ± 9 (n = 5), 12 ± 5 (n = 5), and 6 ± 3/m3 (n = 4), respectively; the highest and lowest densities were at stations 2 and 4, which were coastal and offshore sites, respectively. finally, we analyzed vertical gradients of water temperature and salinity in november and march, when the density of blood cockle larvae was high. however, no thermocline or halocline was observed in either month. water temperature and salinity decreased gradually with increasing depth at the coastal and offshore sites (fig. 6a-d). discussion environment and sexual maturation: in culture experiments, a. granosa grew well in kakinada bay, india, in which water temperature and salinity ranged from 27.8-33.5°c and 13.69-34.40 psu, respectively, and dissolved oxygen levels were kept at 4.45 mg/l (narashimham, 1983). in addition, salinity affects the survival of blood cockle at <20 psu in the laboratory (davenport and wong, 1986). however, little is known about how low dissolved oxygen concentrations affect blood cockle. the lc50 of many bivalves is <2 mg/l (raquel and carlos, 2008). in the present survey, water temperature and salinity ranged from 29.8-30.9°c and 29.0-30.3 month n unknown male female i ii iii iv v ii iii iv v october 18 2 7 9 november 20 8 2 10 february 18 3 4 11 march 18 3 5 10 april 18 1 6 2 2 2 5 n, number of samples per month; i, immature stage; ii, developing stage; iii, mature stage; iv, spawning stage; v, spent stage. table 1. sexual maturation of blood cockle (anadara granosa) collected at station 1 off the selangor coast, peninsular malaysia. figure 4. density changes of (a) umbo-stage and (b) full-grown larvae of blood cockle (anadara spp.). figure 5. comparison of average densities of (a) umbo-stage and (b) full-grown blood cockle larvae at each station in sungai buloh. error bars indicate standard error; numbers in parentheses indicate numbers of samples. 303 yurimoto et al/ spawning season and larval occurrence of blood cockle psu, respectively, and the dissolved oxygen concentration exceeded 2.7 mg/l. therefore, the environment of the present survey area is considered suitable for blood cockle culture. mature blood cockle mainly occurs along the selangor coast from september to april (broom, 1983). in the present study, mature cockles of both sexes were observed in october and spawning-stage individuals occurred from november to march; thus, the spawning period of blood cockle is long, similar to that reported by broom (1983). in addition, the chlorophyll a concentration in the water column at station 1 increased in november and march. the densities of blood cockle larvae and the number of adults in the mature and spawning stages increased simultaneously. these results suggest food availability is closely related to the larval survival and sexual maturation of adult blood cockle. planktonic larva: the occurrence of bivalve larvae was similar to that of blood cockle larvae. the density of bivalve larvae increased at stations 2 and 3 in march, and that of blood cockle larvae also increased at station 2 from february to march. these results suggest the spawning of other kinds of bivalves as well as blood cockle peak in march off the selangor coast. in addition, a high density of blood cockle larvae was observed in november. these results show blood cockle larvae occur for a long period in this region. histological observations of adults showed the gonads of many individuals had reached the mature and spawning stages from november to march corresponding well with the occurrence of blood cockle larvae. furthermore, when blood cockle larvae were divided into the umbo and full-grown stages, umbo-stage larvae composed more half of all larvae. however, as the numbers of full-grown larvae peaked in november and march, blood cockle may have multiple settlement peaks in a year. in an experiment, the planktonic period of a blood cockle larva from hatching to settlement was approximately 18 days; furthermore, it took 14-16 days for them to become full-grown larvae (muthiah et al., 1992). in the present study, umbo-stage larvae were distributed mainly at stations 2 and 3, and were distributed more in offshore areas than station 1, where more adult cockles were found. on the other hand, low density full-grown larvae were collected from station 4, an offshore site, because the center of distribution was at stations 1 and 2. these results indicate that after spawning at the coastal sites, blood cockle larvae are distributed to offshore sites before returning to coastal sites where they become fullgrown larvae. then, the blood cockle larvae settle around muddy tidal flats where they live as adults. the present results support this notion, as the total average density at each station during the survey period showed there were high densities of umbostage larvae at stations 2 and 3, and full-grown larvae at stations 1 and 2. regarding water mass structure in november and march, when blood cockle larvae peaked, larvae were mainly distributed in mixing zones between fresh water and offshore water. thus, figure 6. vertical gradients of (a, c) water temperature and (b, d) salinity in the survey stations in (a, b) november and (c, d) march. station 1, ×; station 2, ●; station 3, △; station 4, ◇. 304 international journal of aquatic biology (2014) 2(6): 299-304 these results suggest blood cockle larvae are mainly distributed in water masses influenced by fresh waters (i.e., low-temperature and/or low-salinity water) to prevent improper dispersion due to influence of offshore water. the relationship between water mass and the distribution of bivalve larvae, such those of clams and oysters, is wellknown (matsumura et al., 2001; hofmann et al., 2004; sakurai and nakao, 1996). in the case of blood cockle larvae living in brackish waters, the present results also suggest an association between water mass structure and larval distribution. references broom m.j. (1983). gonad development and spawning in anadara granosa (l.)(bivalvia: arcidae). aquaculture, 30: 211-219. broom m.j. (1985). the biology and culture of marine bivalve molluscs of the genus anadara. worldfish, iclarm studies and reviews, 12: pp. 37. davenport j., wong t.m. (1986). response of the blood cockle anadara granosa (l.) (bivalvia: arcidae) to salinity, hypoxia and aerial exposure. aquaculture, 56: 151-162. hofmann e.e., powell e. n., bochenek e. a., klinck j.m. (2004). a modelling study of the influence of environment and food supply on survival of crassostrea gigas larvae. ices journal of marine science, 61: 596-616. matsumura t., okamoto s., kuroda n., hamaguchi m. (2001). temporal and spatial distributions of planktonic larvae of the clam ruditapes philippinarum in mikawa bay; application of an immunofluorescence identification method. japanese journal of benthology, 56: 1-8. muthiah p., narashimhan k.a., gopinathan c.p., sundarajan d. (1992). larval rearing spat production and juvenile growth of the blood clam anadara granosa. journal of the marine biological association of india, 34: 138-143. narasimham k.a. (1983). experimental culture of the blood clam anadara granosa (linnaeuas) in kakinada bay. proceedings of the symposium on coastal aquaculture, part 2: pp. 551-556. pathansali d., song m.k. (1958). some aspects of cockle (anadara grunosa l.) culture in malaya. proceedings of the indo-pacific fisheries, pp. 26-31. raquel v.s., carlos m.d. (2008). thresholds of hypoxia for marine biodiversity. proceedings of the national academy of sciences, 105: 15452-15457. sakurai i., nakao s. (1996). distribution of planktonic larvae of the japanese surf clam, pseudocardium sachalinensis at tomakomai, hokkaido. suisanzoshoku, 44: 17-23． yoshida h. (1964). seedling of shellfish, hokuryu-kan co., ltd. tokyo, pp.221. yurimoto t., mohd kassim f., man a. (2014). sexual maturation of the blood cockle, anadara granosa, in matang mangrove estuary, peninsular malaysia. international journal of aquatic biology, 2: 115-123. int. j. aquat. biol. (2022) 10(5): 398-405 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article effect of broccoli, brassica oleracea extract on growth performance and some blood parameters of common carp, cyprinus carpio asaa h. kadhim1, abdulkareem t. yesser2, khalidah s. al-niaeem3 1basrah agriculture directory, basrah, iraq. 2department of marine vertebrates, marine science center, university of basrah, basrah, iraq. 3department of fish and marine resources, agriculture college, university of basrah, basrah, iraq. s article history: received 1 may 2022 accepted 15 august 2022 available online 2 5 october 2022 keywords: brassica oleracea growth hematological parameters medical herbs abstract: the herb extract of broccoli, brassica oleracea was used to feed common carp (19.75±0.057 g) at four incremental levels of 0, 1, 2, and 3% of the diet for 56 days. fish were fed twice a day at a rate of 3% diets containing 32.7-33.7% crude protein and digestible energy (16.037-16.595 mj/kg). at the end of the study, the groups fed 2% broccoli extract were significantly better in weight gain (3.36±0.16 g), relative growth rate (17.02±0.77%), specific growth rate (0.30±0.01% day), food conversion ratio (7.74±0.39) and food conversion efficiency (12.93±0.65%). in terms of hematological parameters, albumin showed significant enhancements in 1, 2, and 3% broccoli extract (p≤0.05). however, globulin levels were not affected by broccoli extract inclusion (p≥0.05). total protein and a/g ratio showed significant enhancements in the 2% broccoli extract group (p≤0.05). according to the findings, using 2% broccoli extract was the best option for growth and blood profile in common carp rearing. therefore, the present study recommends adding 2% of broccoli extract to improve nutrient efficiency, growth performance, and hematological parameters in c. carpio fingerlings. introduction aquaculture has grown substantially to meet human demand (srivastava and panadey, 2015; ringø and song, 2016). aquaculture is one of the fastestgrowing industries, accounting for nearly half of all aquatic products (fao, 2020). diseases cause severe losses leading to economic losses in aquaculture. pathogens such as bacteria, viruses, fungi, and protozoa are major contributors to these losses (srivastava and panadey, 2015). antibiotics and chemotherapy have been widely used to reduce disease risk in fish farms (abdel-tawwab et al., 2009; van hai, 2015; tiengtam et al., 2015). in contrast, excessive antibiotic use in aquaculture can harm public health and the water environment (cabello, 2006; done et al., 2015). hence, aquaculture is under pressure to reduce the use of synthetic antibiotics and chemotherapeutics, both of which endanger food safety and hygiene, and disease correspondence: asaa h. kadhim doi: https://doi.org/10.22034/ijab.v10i5.1754 e-mail: asaahanoon84@gmail.com dor: 20.1001.1.23830956.2022.10.5.7.8 control in humans, due to the risks posed by chemical residues in foods and antibiotic resistance passed on to human pathogens (serrano, 2005; chen et al., 2020). however, many countries have prohibited the use of antibiotics in aquaculture (vignesh et al., 2011). efforts are being made to use plants, plant extracts, and natural plant compounds as potential alternatives to synthetic chemicals to stimulate immune responses and disease resistance (chakraborty and hancz, 2011). fish, unlike vertebrates, lack adaptive immunity and rely entirely on innate immunity (arala-chaves et al., 2000; esteban et al., 2005; little et al., 2005). the products that can increase host immunity and disease resistance, such as immunostimulants, are being used in disease prevention and have received much attention recently (srivastava and panadey, 2015). plants are the oldest form of healthcare known to https://ij-aquaticbiology.com/index.php/ijab/article/view/1754 399 int. j. aquat. biol. (2022) 10(5): 398-405 man due to the widespread presence of various compounds such as alkaloids, flavonoids, phenols, terpenes, steroids, and essential oils. plants have anti-stress, growth-promoting, appetite-stimulating, immune-stimulating, and anti-microbial properties (hassan et al., 2018). because of the properties of their derivatives, they are widely used in fish diets as a growth enhancer and immune stimulator (citarasu, 2010), in addition to being low-cost, widely available, and easy to prepare (harikrishnan et al., 2005). broccoli, brassica oleracea, is a member of the brassicaceae family, known as "cruciferae" due to its distinctive flower confirmation of four petals arranged in a cross-shape. brassica varieties are among the top 10 economic crops in the world (i.e., broccoli, kale, cauliflower, and chinese cabbage) (francisco et al., 2017). since of their high levels of nutrients and health-promoting phytochemicals (such as phenolics, glucosinolates, vitamins, and minerals), these vegetables have been identified as important components of a healthy diet (francisco et al., 2017). broccoli (brassica oleracea l. var. italica) has recently gained popularity among wellness consumers. it is a nutritious vegetable with high vitamins and minerals, bioactive phytochemicals (glucosinolates, phenolic compounds, and flavonoids), and antioxidants (dominguez-perles et al., 2010). plant-based foods containing significant amounts of bioactive phytochemicals, such as b. oleracea extract, may provide desirable health benefits to common carp, cyprinus carpio, growth performance, and some immune-hematological indicators that are tested as the aim of this study. materials and methods preparation of aqueous extracts of brassica oleracea: broccoli was obtained from a local market in basrah, iraq, and thoroughly washed under running water to remove dust particles. the soft parts were cut into small pieces and thawed using a home mincer. to make an aqueous extract, 50 g of the minced broccoli was weighed and placed in one liter of ellen meyer flasks, 250 ml of deionized water was added, and the mixture was placed in a vibrating incubator at 35 °c for 24 hours. the mixture was filtered through filter paper (whatman no1) to remove the residue, and the filtrate was stored at 20°c until used. diet preparation: four isonitrogenous and isocaloric experimental rations (diets 1, 2, 3, and 4) were made with varying amounts of b. oleracea extract (0, 1, 2, and 3%, respectively). the feed ingredients were ground and milled into pellets. the b. oleracea extract was added at the end of the pelleting process. pellets were packed in polyethylene bags, sealed airtight, labeled after drying and frozen until ready to use. the ingredients and chemical analysis of experimental diets containing varying levels of extracted b.oleracea are shown in table 1. rearing system: inside the laboratory, four recycling aquaculture systems (ras) units were used. each unit had three plastic tanks (30×30×60 cm) arranged in two rows on an iron holder, each with a glass tank (30×40×90 cm) for water filtration. each tank was provided with air pumps for aeration, and heating equipment was used to maintain the temperature of the water. the study lasted for eight weeks (56 days), at the fish labs of the vertebrates department (marine science center, university of basrah). fingerlings of common carp were obtained from the fish culture unit of agriculture college, the university of basrah. fish were placed in plastic tanks and acclimatized for two weeks before the experiment. fish had a mean body weight of 19.75±0.057 g. the fish were introduced randomly into four treatments, including diet 1 (0% broccoli extract) as control and diets with broccoli extract at three levels 1, 2, and 3% broccoli extract (diet 2, diet 3, and diet 4, respectively). each treatment was replicated using three tanks containing 10 fish. the fish were fed twice daily at 09.00 am and 02.00 pm, at 3% of their body weight. cleaning of the tanks and water changes were done weekly. water quality measurement: a water quality multimeter (taiwan) was used to measure the 400 kadhim et al./ effect of broccoli, brassica oleracea extract on growth performance and some blood parameters temperature, dissolved oxygen (mg/l), ph, and salinity (psu) of the water in the recycled aquaculture system (ras). the experimental tanks' water was replaced 50% weekly to maintain a healthy environment. growth parameters: at the end of the feeding period (56 days), fish were weighed and counted to calculate: weight gain (wg), relative growth rate (rgr), specific growth rate (sgr), feed conversion ratio (fcr), food conversion efficiency (fce), and survival rate as they served as indicators for growth performance, using the formula described by jobling and koskela (1996). weight gain (g) =final average weight (g) – initial average weight (g). relative growth rate (%) = weight gain (g) / initial weight (g) × 100 specific growth rate (% per day) = 100 × (ln final weight / ln initial weight) / days of the experiment feed conversion ratio = feed consumed (g) dry weight / weight gain (g) feed conversion efficiency (%) = weight gain (g) / feed consumed (g) dry weight × 100 survival rate (%) = number of fish survived / number of fish stocked × 100 blood collection: after 56 days of the experiment, four fish per treatment were used for blood analysis, and 2.5 ml blood samples were collected by cardiac puncture using 3 ml disposable syringes pre-rinsed with 0.5 m edta as an anticoagulant. the blood was kept at -20°c before analysis. total protein (g/dl), albumin (g/dl), globulin (g/dl), and albumin/globulin ratio (a/g ratio) are all measured (velisek et al., 2009). statistical analysis: statistical differences between treatments were evaluated by analysis of variance followed by least significant differences (lsd). the significance level was set as p≤0.05. all statistical analyses were performed using spss program version 26. results table 2 shows the mean water quality parameters of the treatments during the experiment. water quality ingredients control diet % b.oleracea water extract % in the diets control (diet 1) diet 2 diet 3 diet 4 fish meal 32 32 32 32 soya meal 22 22 22 22 wheat brain 30 29 28 27 yellow corn 15 15 15 15 vitamin premix 0.5 0.5 0.5 0.5 minerals premix 0.5 0.5 0.5 0.5 b.oleracea extract 0 1 2 3 chemical analysis % moisture 3.2±0.18a 3.5±0.21b 3.6±0.24b 3.7±0.26b crude protein 33.98±0.054a 33.92±0.057a 33.86±0.59a 33.80±0.06a crude fat 13.8±2.52a 13.6±2.87a 13.3±3.12a 13.1±3.43a ash 1.7±0.12a 1.6±0.15a 1.2±0.18a 1.5±0.16a nfe 41.2±0.63a 40.91±0.71a 40.88±0.87a 39.1±0.92b fiber 5±0.32a 5.17±0.30ab 5.16±0.29ab 5.8±0.24b ge (mj/ kg) * 16.595±0.032a 16.476±0.039a 16.361±0.041a 16.037±0.042a *according to smith's equation (1971): protein × 18.5 + fat × 33.5 + nfe × 13.8. table 1. ingredients and chemical analysis of experimental diets containing varying levels of extracted (on a dry matter basis). treatments temperature (ºc) dissolved xygen (mg/l) ph salinity (psu) diet 1 (0 %) 25.14±0.16 5.22±0.26 7.36±0.04 2.94±0.05 diet 2 (1%) 25.01±0.10 5.38±0.29 7.38±0.02 2.91±0.04 diet 3 (2%) 24.81±0.38 5.54±0.15 7.31±0.04 2.93±0.04 diet 4 (3%) 25.13±0.18 5.03±0.74 7.93±0.04 2.94±0.06 table 2. the physical and chemical properties of the water used in experimental tanks of common carp fish fed different levels of b.oleracea. 401 int. j. aquat. biol. (2022) 10(5): 398-405 parameters were not different within the recommended ranges for the culture of c. carpio (fao, 2020), positively reflecting the increased survival rate, which reached 100% for all treatments. figure 1 illustrates the trend of body weight in fishfed various levels of broccoli extract during the experiment. the fish feddiets 1 and 3 have grown faster than those fed diets 2 and 4. the best growth performance was recorded in the fish-fed diet 3, and the least growth in the fish-fed figure 1. average body weight of common carp as affected by diets containing different levels of brassica oleracea extract. items control diet % b. oleracea water extract % in the diets diet 1 (0%) diet 2 (1%) diet 3 (2%) diet 4 (3%) initial weight (g) 19.75±0.07a 19.74±0.06a 19.75±0.07a 19.74±0.04a final weight (g) 22.85±0.12ab 22.58±0.12bc 23.11±0.23a 22.20±0.38c weight gain (g) 3.10±0.17ab 2.84±0.19bc 3.36+ 0.16a 2.46±0.34c rgr (%) 15.73±0.88ab 14.38±1.01b 17.02±0.77a 12.47±1.71c sgr (% day) 0.28±0.01ab 0.26±0.01b 0.30±0.01a 0.23±0.03c fcr 8.03±0.45a 8.76±0.52a 7.74±0.39a 10.49±1.34b fce % 12.46±0.70 a 11.43±0.70 a 12.93±0.65 a 9.63±1.32 b survival rates % 100a 100a 100a 100a the same letter in the same row means it is not significantly different at p≥0.05. table 3. growth performance (means ± sd) of common carp fish fed diets containing different levels of brassica oleracea extract. figure 2. the effect of brassica oleracea extracts on serum total protein (g/dl), albumin (g/dl), globulin (g/dl), and a/g ratio (%) in comparison with the control diet of common carp. 402 kadhim et al./ effect of broccoli, brassica oleracea extract on growth performance and some blood parameters diet 4 (table 3). dietary supplementation with 2% broccoli extract resulted in significantly higher final weight and weight gain, rgr and sgr. fcr and fce% in the fish-fed diets with 3% broccoli extract differ (p≤0.05) from the control one; while a similar value was observed in the fish fed 1 and 2% which did not differ significantly (p≥0.05) with the control treatment. total protein and albumin levels increased significantly (p≤0.5) with broccoli extract, reaching the highest value at 2% of the diet (5.70±0.13 g/dl and 4.38±0.11, respectively). there were no significant differences between dietary supplementation with 3% broccoli extract and the control one globulin levels at 1, 2, and 3% of broccoli extracts did not differ significantly from the control. the albumin/globulin (a/g) ratio was highest in diet 2, which differs significantly (p≤0.05) from other treatments (fig. 2, table 4). discussion a variety of feed additives are used in aquaculture to promote growth, stimulate immunity, prevent disease, and improve fish antioxidant status (bilen et al., 2020). infectious diseases have recently been identified as a major issue in the aquaculture industry, resulting in significant losses for farmers (erguig et al., 2015; syahidah et al., 2015; karga et al., 2020). the use of antibiotics and chemicals in aquaculture is frequently expensive and unacceptable because it leads to antibiotic and chemical resistance (erguig et al., 2015; syahidah et al., 2015; bulfon et al., 2017). hence, immunostimulants such as medical plant extracts or products have been used to control fish and shellfish diseases and increase phagocytic activity in various fish (gopalakannan and arul, 2006; kim et al., 1999). in aquaculture, many immunostimulants are used to improve fish immune profiles that have the potential to replace antimicrobial agents. this study showed the effect of dietary plant supplementation of broccoli extract on growth performance and some blood parameters of common carp reared in a recycling aquaculture system. environmental factors of the systems were within recommended ranges (fao, 2020), which was contributed by the increased survival rate of fish in all treatments. fish fed a 2% broccoli extract diet increased their weight gain (wg), relative growth rate (rgr), and specific growth rate (sgr). positive growth results could be attributed to nutrient protection in the intestine as a result of the b.oleracea extract's effectiveness against bacterial pathogens, and increased levels of digestive enzymes, improved cellular respiration, and absorbed nutrients (citarasu, 2010; awad and awaad, 2017; begnami et al., 2018; rudiansyah et al., 2022). food conversion ratio (fcr) and food conversion efficiency (fce) in fish-fed diets containing 1% and 2% broccoli extract did not differ significantly (p≥ 0.05) from the control treatment (diet 1). while the fish-fed diet 4 showed the least growth compared to the control treatment. oke et al. (2017) proposed that including b.oleracea powder in the diet of nile tilapia at 0.5g/100g would improve feed utilization, survival rate, and non-specific immune infections. herb active principles have the growth-promoting ability and act as appetizers to trigger the immune system, act as a broad-spectrum antimicrobial, and include anti-stress properties, all of which will play a significant role in fish cultivation (mahdavi et al., 2013). many authors treatments (% extract) total proteing/dl albuming/dl globuling/dl a/g ratio% diet 1 (0%) 3.45±0.32b 2.42±0.31c 1.06±0.17a 2.28±0.24b diet 2 (1%) 3.58±0.44b 3.12±0.55b 1.15±0.23a 2.71±0.39b diet 3 (2%) 5.70±0.13a 4.38±0.11a 1.16±0.26a 3.77±0.18a diet 4 (3%) 3.46+ 0.38b 2.45+ 0.35bc 1.07+ 0.16a 2.29+ 0.25b the same letter in the same column means it is not significantly different at p≥ 0.05. table 4. the effect of brassica oleracea extracts on serum total protein (g/dl), albumin (g/dl), globulin (g/dl), and a/g ratio (%) in comparison with the control diet of common carp. 403 int. j. aquat. biol. (2022) 10(5): 398-405 have demonstrated that immunostimulants can also promote growth. influences of dietary medical plants supplementation on growth have been evaluated with several species with different results. gynostemma pentaphyllum is a customary chinese herbal medicine blended into fish feed, causing a raised weight gain, feed conversion efficiency, and specific growth rate in grass carp, ctenopharyngodon idella (wu et al., 1998). dietary supplementation of quillaja saponin increased the growth rate of c. carpio (francis et al., 2002). the results could indicate that broccoli extract enhances fish growth, feed utilization, and immune function. dietary broccoli appeared to significantly induce non-specific immune parameters in common carp fish, albumin values showed significant enhancements by 1, 2, and 3% broccoli extract (p≤0.05). however, broccoli extract inclusion did not affect globulin levels (p≥0.05). total protein and a/g ratio showed significant enhancements by 2% broccoli extract compared to other treatments (p≤0.05). according to the findings of this study, a 2% broccoli extract was the best option for growth and blood profile. therefore, the present study’s data recommends adding 2% of broccoli extract to improve nutrient efficiency, growth performance, and hematological parameters in c. carpio fingerlings. hematological parameter analysis can be used to monitor health status, detect illnesses, and track disease progression and response to therapy (clauss et al., 2008; alexander et al., 2010). all the levels of included broccoli in the diet of common carp gave protection in terms of reduced percent mortality which is reflected in the increased relative survival rate values. the results indicate the immunostimulatory and disease resistance properties of broccoli aqueous extract and so its potential to be used as an immunoprophylactic in finfish aquaculture. as a conclusion, this study found that b.oleracea extract has a positive effect on the growth performance of common carp. brassica oleracea extract in the diet can act as a growth promoter, appetite stimulator, and immunostimulant, as well as reduce stress, reduce food losses, and protect fish, resulting in better fish growth. references ayshwarya m., sudha rameshwari k. 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(1998). studies on gynostemma pentaphyllum used as fish feed additives. journal of shanghai fisheries university, 7: 367-370. international journal of aquatic biology (2015) 3(3): 155-160 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article antibacterial activities of bioactive compounds extracted from marine algae gracilaria salicornia against aeromonas hydrophila somayeh rasooli1, masoud sattari*1, zohreh ramezanpour2, javid imanpour namin1 1department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, iran. 2department of ecology, international sturgeon research institute, rasht, iran. article history: received 13 february 2015 accepted 22 april 2015 available online 2 5 june 2015 keywords: red algae extract fish pathogen antibacterial gracilaria salicornia abstract: herbal medicinal products have attracted significant research interest in recent years. considering the efficiency of algae products in controlling pathogenic bacteria and also easy access to large resources of algae, this study was conducted to evaluate the effects of methanolic, chloroformic and aqueous extracts of gracilaria salicornia against aeromonas hydrophila, a heterotrophic, gramnegative, rod-shaped bacterium found mainly in warm climate. algae samples were collected from qeshm island coastlines and transferred to the laboratory. standard methods were used to obtain the algae extract. antibacterial activities of various extracts were tested against the bacterium using well diffusion assay method. significant differences were observed in antibacterial activities of different extracts (p<0.05). the diameter of zone of growth inhibition varied in correlation with concentration of the extracts (50, 100, 200 and 300 mg.ml-1). the best inhibition zone was observed at 100, 200 and 300 mg.ml-1 methanolic and 300 mg.ml-1 aqueous extracts. introduction application of herbal medicinal products with minimal environmental impacts, without causing resistance in bacteria and easy availability have attracted considerable research interests (aoki, 1992; marino and spiewak, 1999; maunchid et al., 2005; perrucci, 1994). in addition, high costs of using antibiotics in aquaculture, environmental concerns and difficulties in prescribing antibiotics have resulted in herbal medicinal products to be considered as a replacement for commercial antibiotics (raa, 1996; iwama, 1996). algae are low calorie food sources rich in vitamin, minerals, protein, steroids, polysaccharides and fiber (ito and hori, 1989; lahaye, 1993) and used in traditional medicine since 3000 bc (smit, 2004). they also contain substances for developing new antibiotics (faulkner, 2002; newman et al., 2003). although, the effects of algae extracts on gram * corresponding author: masoud sattari e-mail address: msattari@guilan.ac.ir positive and negative bacteria are influenced largely by species, organs used for extraction, target microorganisms, season and growth condition (paniagua et al., 2009; la barre et al., 2010; kellmann et al., 2010; guven et al., 2010). the genus gracilaria is a member of red algae with a life cycle in three phases inhabiting tropical and sub-tropical regions (guiry, 2011; skriptsova et al., 2001). some biological functions have been reported for bioactive compounds extracted from this genus of algae, including anti-oxidative, antiinflammatory, anti-fungal, anti-viral, anti-spasm and cytotoxic activities. these bioactive compounds may be used to treat disorders in nervous system, cardio vascular and blood circulatory system. they may also be used as anti-hyperglycemic and contraceptive agents (de almeida et al., 2011). these algae have also been subject of few studies in iran (gharanjik et al., 2000; saeidnia et al., 2009; 156 international journal of aquatic biology (2015) 3(3): 155-160 nasir et al., 2011; plubrukarn eet al., 2012; zandi et al., 2007; jassbi et al., 2013; saeidnia et al., 2011). aeromonas hydrophila is a gram negative, motile, facultative aerobic and anaerobic bacteria found in water bodies and also in digestive systems of healthy fishes. under stressful conditions, these bacteria are caused mortality in freshwater fishes through dermal and visceral hemorrhagic septicemia and enteritis (tavakoli and akhlagi, 2009). biological and antibacterial activities of bioactive compounds obtained from marine macro-algae have been subjects of several studies worldwide (sastry and rao, 1994) including studies against a. hydrophila (rebecca et al., 2012; al-laham and al-fadel, 2014; aruna et al., 2010; vijayabaskar and shiyamala, 2011). in addition, antibacterial activities of some vascular plants against a. hydrophila have been studied in iran (ali shahi et al., 2009; ali shahi, 2010). considering the efficiency of bioactive compounds obtained from algae to control pathogenic bacteria and availability of a large amount of algae in northern and southern parts of iran, the present study was conducted to evaluate the effect of methanolic, chloroformic, aqueous extracts of g. salicornia against a. hydrophila. materials and methods samplings were carried out during high tide at kaveh harbour in the qeshm island (south iran at 55'57ºe and 26'56ºn) from late october 2013 till december 2014. the algae samples were washed in sea water and cleaned of epiphytic algae, sand, debris and transported to the laboratory in the ice box. methanolic extract: ten grams of the algae powder (powdered by liquid nitrogen) were covered in filter paper and placed in soxhlet apparatus containing 100 ml methanol (80%) in a mantle at 65°c (boiling point of the solvent). the extractions were repeated four successive times (approx. 4 hrs). the obtained extract was placed in 50 ml falcon tubes and kept at 4°c for further experiment (souza, 2011). chloroformic extract: to obtain chloroformic extract, 100 ml of raw methanolic extract and 100 ml chloroform were mixed in a container. within few minutes, two separate phases were formed. the chloroform phase was removed after 3 days and stored at 4°c for further tests. the obtained extracts were centrifuged, passed through whatman filter paper and freeze dried. the used solvent for extraction was evaporated and the remaining part was kept at -17°c for antibacterial tests. aqueous extract (polysaccharide): algae powder with proportion of 1:10 was boiled in distilled water (100°c) under reflux for 2 hrs. the hot extract was passed through filters with 24 micrometer mesh size. the filtered aliquot was condensed using a rotary evaporation condenser to the lowest volume, called row polysaccharide extract. concentrations of methanolic and chloroformic extracts: methanolic extracts of 50, 100, 200 and 300 mg.l-1 were prepared by dissolving algae samples in 0.5 ml dimethyl sulfoxide (dmso) solvent. the desired concentrations of the extracts were stored at -17˚c for further tests. concentrations of polysaccharide extract: polysaccharide extracts of 50, 100, 200 and 300 mg.l-1 were prepared. algae aqueous extract were dissolved in 0.5 ml distilled water and the desired concentrations were kept at -17˚c in labeled sterile micro tubes for further tests. antibacterial tests: aeromonas hydrophila isolates was obtained from fish health laboratory of international sturgeon research institute. antibacterial activities of algae extracts were assayed using well diffusion assay method. bacterial cultures (nutrient agar and nutrient broth) were incubated at 37°c for 24 hrs. then, 30 µl of bacterial strain cultured in nutrient broth was uniformly added on nutrient agar followed by punching 3 holes (4mm in diameter) aseptically in the medium using the tip of pipette. these holes were loaded with different concentrations of the extracts and incubated at 37˚c for 24 hrs. the zones of inhibition were measured after 24 hrs using a digital caliper (indu, 2006; sharma and sharma, 2011). the whole process was carried out aseptically. commercial tetracycline and dmso were used as positive and 157 rasooli et al/ antibacterial activities of g. salicornia extracts against a. hydrophila negative controls, respectively. data analyses: data are presented as mean ± sd of triplicate samples. one-way anova was used to test the significance of differences between four algae extract concentrations and also between algae extracts and commercial antibiotics. duncan post hoc test was used to assess differences among sample means. statistical package of spss ver. 16 was used for data analysis. diameter of the inhibition zone was measured according to thomson et al. (1985). zones of inhibition around the wells were measured and scored as follow: triply positive (+++) if a zone of inhibition was greater than 7 mm, doubly positive (++) if greater than 2 mm, positive (+) if less than 2 mm, and negative (-) if no inhibition of bacterial growth was occurred (thompson et al., 1985). results the results of antibacterial tests of the algae extracts against a. hydrophila are presented in table 1. different responses were observed and the bacterium was sensitive to commercial antibiotic (positive control). negative control (dsmo) did not have any effect on the bacterium. the results revealed significant differences among activities of various concentrations of the methanolic, chloroformic and aqueous extracts (p<0.05). a significance differences between antibacterial activities of algal extracts and the positive control were also found except 100 and 200 mg.ml-1 methanolic (fig. 1). antibacterial effect of all studied concentrations of the methanolic extracts as well as 300 mg.ml-1 aqueous extract were significantly higher than those of other extracts (fig. 1). discussion in the present study, all concentrations of the methanolic extracts and 300 mg.ml-1 aqueous extract concentration mg.ml-1 methanolic extract chloroformic extract aqueous extract 50 +++ ++ ++ 100 +++ ++ ++ 200 +++ ++ ++ 300 +++ ++ +++ positive control +++ +++ +++ negative control table 1. antibacterial activities of g. salicornia extract against a. hydrophila, triply positive (+++), doubly positive (++), positive (+) and negative (-). figure 1. comparison of antibacterial activities of algae extract with various concentrations and commercial antibiotic against a. hydrophila. 158 international journal of aquatic biology (2015) 3(3): 155-160 showed considerable antibacterial activities. other concentrations of aqueous and chloroformic extracts showed poor antibacterial activities compared to the methanolic extracts. antibacterial activities of 100 and 200 mg.ml-1 methanolic and aqueous extracts did not have significant differences with commercial antibiotics, hence they can be categorized as similar as tetracycline i.e. commercial antibiotics. hornsey and hide (1974) studied antibacterial activities of 151 species of marine algae and reported no such activity for gracilaria (hornsey and hide, 1974). in another work, polar and non-polar extracts of gracilaria species exhibited some effects on certain pathogenic bacteria (de almeida et al., 2011). adaikalaraj et al. (2012) studied the potentials of antibacterial activities in red algae gracilariacaea and reported higher activity for methanolic extracts (adaikalaraj et al., 2012). rebecca et al. (2012) also reported antibacterial activities of methanolic extract of centroceiod sp. against a. hydrophila (rebecca et al., 2012). the findings of this study confirm, the results of those above mentioned works regarding antibacterial activities of gracilaria extract. the present study is the first report on antibacterial activities of bioactive compounds extracted from g. salicornia against a. hydrophila. according to the results, g. salicornia extracts can be used to control pathogenic microorganisms in aquaculture. from ecological point of view some metabolites produced by marine plants could be useful in growth and resistance of fish against certain diseases. marine algae uphold an advantage to develop antibacterial agents for application in aquaculture. from economic viewpoint, it is possible to establish standard methods for extracting various bioactive compounds from algae with higher antibacterial activities. acknowledgments authors extend their sincerest gratitude to mr. rasa a senior lecturer at the department of biology and mrs. fatemeh ghanbari, phd students of organic chemistry of the department of chemistry at university of guilan for their supports. references adaikalaraj g., patric r.d., johnson m., janakiraman n., babu a. 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(2011). antioxidant potential of two red seaweeds from the 160 international journal of aquatic biology (2015) 3(3): 155-160 brazilian coasts. journal of agricultural and food chemistry, 59(10): 5589-5594 tavakoli e., akhlagi m. (2009). the amount of lysozyme, immunoglobulins, blood cell count and hematocrit levels in rainbow trout following experimental infection with a pathogenic aeromonas hydrophila. journal of veterinary research, 64 (2): 157-162. thompson j.e., walker r.p., faulkner d.j. (1985). screening and bioassays for biologically-active substances from forty marine sponge species from san diego, california, usa. marine biology, 88(1): 1121. vijayabaskar p., shiyamala v. (2011). antibacterial activities of brown marine algae (sargassum wightii and turbinaria ornata) from the gulf of mannar biosphere reserve. advances in biological research, 5(2): 99-102. zandi k., salimi m., sartavi k. (2007). in vitro antiviral activity of the red marine algae from persian gulf against herpes simplex virus type 2. journal of biological sciences, 7(7): 1274-1277. international journal of aquatic biology (2015) 3(4): 199-207 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article acute toxicity and effects of malathion exposure on behavior and hematological indices in indian carp, cirrhinus mrigala (hamilton) abdul rauf*1 department of zoology, govt. superior degree science college, shah faisal colony, karachi, pakistan. article history: received 1 april 2015 accepted 4 june 2015 available online 2 5 august 2015 keywords: malathion acute toxicity behavior hematology indian carp abstract: malathion is one of the most commonly used pesticides in agriculture. this study was aimed to investigate the acute toxicity of malathion as an aquatic pollutant on the behavior and hematological indices in indian carp (cirrhinus mrigala). a static experiment was conducted and 1, 24, 48, 72 and 96 hrs lc50 values of malathion for the test fish were estimated as 14.55 mg/l, 12.48 mg/l, 11.56 mg/l, 10.85 mg/l and 9.32 mg/l, respectively. during 96 hrs exposure to 9.32 mg/l of malathion, behavioral abnormalities such as hyperactivity, cough, convulsions, erratic swimming, loss of balance, rapid opercular movements, gill mucous secretion, surfacing and gulping of air were observed in the test fish. the hematological changes in exposed fish after 96 hrs exposure to malathion included a significant decrease in erythrocyte count, hemoglobin content, hematocrit, leukocyte count and a significant increase in neutrophils count as compared to the control fish. in conclusion, acute exposure to 9.32 mg/l of malathion provoked behavioral and hematological abnormalities in indian carp which offers a valuable tool to monitor malathion induced toxicity in fish. introduction among different fresh water pollutants, pesticides are one of the most potentially harmful chemicals introduced into the environment. organophosphate pesticides are the most commonly used pesticides in the world and require more awareness because of their possible toxic effects on non-target animals (aktar et al., 2009). malathion (c10h19o6ps2) is an organophosphate pesticide widely used in both agriculture and households to control insect pests affecting a number of crops, stored grains and livestock feed via ground and aerial sprays and aerosols. after its application, malathion can contaminate surface water through accidental spillage, spray drift and runoff following rain. the aquatic distribution of malathion can cause harmful effects on aquatic environment and its organisms. malathion is less persistent in an environment under alkaline conditions and has a higher degree of * corresponding author: abdul rauf e-mail address: abdulrauf75@hotmail.com persistence under acidic conditions. the half-life of malathion is up to 11 days and it degrades into malaxon which is more toxic than the parent (martinez and leyhe, 2004). aquatic distribution of malathion causes significant adverse health effects on fish and other non-target animals. the toxicity potential of malathion varies in different fish and depends upon the differences in absorption, detoxification and inhibition of enzyme acetylcholinesterase (ache) that breaks down the neurotransmitter acetylcholine so that subsequent impulses can transmit across the synapse. inhibition of ache drastically affects growth, feeding and reproductive behaviors and can lead to death of fish usually by asphyxiation (sparling and fellers, 2007). behavioral changes are one of the most important indicators of environmental stress; while hematological parameters are important indicators of disease and are frequently used tools in toxicological 200 international journal of aquatic biology (2015) 3(4): 199-206 research, environmental monitoring and in the evaluation of the pathophysiological changes in fish under different stressful conditions (pimpao et al., 2007). in the last few years, there have been several studies to investigate the malathion toxicity in different fish species including glossogobius giuris (venkataramana et al., 2006), opheocephalus punctatus (pugazhvendan et al., 2009), oreochromis niloticus (al-ghanim 2012), clarias gariepinus (ahmed, 2012) and heteropneusteus fossillis (deka and mahanta, 2012), but most of these studies were confined to reporting histological changes in fish exposed to sub-lethal concentrations of malathion and very little attention has been paid to the acute effects of malathion exposure on fish hematology. malathion is a commonly used pesticide and its presence in fresh water reservoirs of pakistan has previously been reported (mastoi et al., 2008). however, there is a paucity of scientific documentation regarding the effects of malathion on local fish species. indian carp (cirrhinus mrigala) is an important edible fresh water fish with substantial economic importance and wide distribution in fresh water reservoirs of pakistan. considering the growing use of malathion in pakistan and lack of knowledge about its potential toxicity in local fresh water fish fauna, present study was carried out to determine acute toxicity of malathion and its effects on behavior and hematological indices of c. mrigala. materials and methods juveniles of c. mrigala weighing 20.4 ± 1.8 g and total length of 5.5 ± 1.8 cm were obtained from jokhio fish farm (24°16′5″n, 67°35′55″e) thatta, located northeast of karachi, pakistan. fish were carried to the laboratory in plastic containers and were acclimated to the laboratory conditions for two weeks in a fiberglass tank containing 500 l of continuously aerated fresh water. during this period, temperature, ph, dissolved oxygen and photoperiod were 22.5 ± 1.9°c, 7.5 ± 0.3, 6.8-7.5 mg/l and 12:12 hrs dark-light cycles, respectively. fish were fed with a commercial fish food twice a day during the period of acclimation but were starved for 24 hrs prior to the experiment and throughout the experiment. technical grade malathion with 95% active ingredient was purchased from edgro (pvt.) ltd. karachi and a stock solution at a concentration of 200 mg/l was prepared by dissolving it in the acetone. the stock solution was stored in dark bottles at 4°c and its different dilutions were used to determine acute toxicity, behavioral and hematological effects on experimental fish. after two weeks of acclimation, a static acute toxicity test was performed. the juvenile specimens were exposed to each of the seven different concentrations (0.5, 1, 2, 4, 8, 16 and 32 mg/l) of malathion to determine 96 hrs lc50 values. the experiment was performed in triplicates in 100 l glass aquaria each containing 20 juvenile c. mrigala. the physicochemical indices of diluting water used were: temperature 22.5 ± 1.9°c, ph 7.5 ± 0.3, salinity 107 ± 3.2 mg/l, total hardness 112 ± 2.1 mg/l and dissolved oxygen 6.8-7.5 mg/l. these water quality parameters were determined according to the procedures described in standard methods (apha, 1992). prior to the introduction of fish, the required volume of malathion was added and water in the aquaria was aerated for one hour to obtain a homogenous mixture of the toxicant. two control sets were also run containing the same number of fish in the same volume of water but without malathion. water was renewed daily and fish mortalities were recorded after 1, 24, 48, 72 and 96 hrs of exposure. fish were considered dead if their gill opercular movement ceased and they could not respond to the stimulus provided by a glass rod. dead fish were immediately removed from the aquaria and lc50 values were calculated using probit analysis test (finney, 1971). at the end of the acute toxicity test, twenty juvenile c. mrigala were exposed to 9.32 mg/l (96 hrs lc50) for 96 hrs in 100 l glass aquaria to determine behavioral and hematological alterations. the experiment was run in triplicate and two control sets were also run concurrently. fresh test media were provided daily to maintain the concentrations of malathion near to the 80% of nominal concentration. 201 rauf/ malathion toxicity in cirrhinus mrigala the physicochemical characteristics of water were maintained the same as mentioned above. the behavioral changes of fish were recorded at every 12 hrs during the study and dead fish were removed from the aquaria. at the end of 96 hrs exposure period, a total of twelve surviving fish from experimental aquaria and same number of fish from control group were randomly collected using a small dip net with minimum disturbance in the water. blood sample of each fish was collected by cardiac puncture using a 18 g needle attached to a plastic syringe. blood was transferred into heparanized glass vials (50 iu sodium heparin/ ml of blood) and was immediately used for hematological examinations. the hematological indices examined included erythrocyte count (rbc), hemoglobin concentration (hb), hematocrit (hct), mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch), mean corpuscular hemoglobin concentrations (mchc), leukocyte count (wbc), leukocyte differential counts and were determined according to the unified methods for hematological examination of fish (svobodova et al., 1991). all statistical analyses were carried out using spss 17.0 computer software for windows (spss inc., chicago, il, usa). the difference in fish mortalities was analyzed by χ2 test. the values of hematological indices were presented as mean ± sd. the data for these parameters was tested for normality (kolmogorov-smirnov test) and analyzed by one way analysis of variance (anova) to test significant differences between the hematological parameters of controlled and exposed groups and p<0.05 were considered statistically significant. results cumulative mortality of c. mrigala after exposure to different concentrations of malathion at different durations of exposure is summarized in table 1. the data clearly indicates a time and concentrationdependent significant increase in fish mortality rate (p<0.05 in each case). the highest and fastest fish mortalities were recorded after 1 hrs exposure to the highest concentration of malathion (32 mg/l), while lowest fish mortalities were recorded after 96 hrs exposure to 1 mg of malathion. the lc50 values with 95% confidence limits of malathion for c. mrigala were estimated as 14.55 mg/l (13.98-15.32) for 1 hrs, 12.48 mg/l (11.91-13.39) for 24 hrs, 11.56 mg/l (10.89-12.89) for 48 hrs, 10.85 mg/l (no data as p>0.05) for 72 hrs and 9.32 mg/l (no data as p>0.05) for 96 hrs; and significant differences were observed in the lc10, lc50 and lc90 values recorded for different times of exposure (p<0.05; table 2). the behavioral response observed every 12 hrs of malathion exposure in test fish included hyperactivity, cough, convulsions, erratic swimming, loss of balance, rapid opercular movements, gill mucous secretion, surfacing and gulping of air. subsequently, exhausted fish sank to the bottom of aquaria and died. compared to the control group, fish exposed to 9.32 concentrations (mg/l) number of dead fish (%) 1 hrs 24 hrs 48 hrs 72 hrs 96 hrs control 0.5 1 4 2 8 11 18 24 32 4 20 24 30 32 40 8 28 32 38 41 47 16 78 84 90 96 100 32 100 nd χ2 1.78 2.01 2.29 2.78 3.17 p <0.05 <0.05 <0.05 <0.05 <0.05 nd= no data because of 100% mortality; (–) no dead fish. table 1. cumulative mortality of juvenile cirrhinus mrigala exposed to different concentrations of malathion. 202 international journal of aquatic biology (2015) 3(4): 199-206 mg/l of malathion for 96 hrs showed significantly lower values for rbc count, hb, hct and wbc count (p<0.05); but the values recorded for mcv, mch and mchc were not significantly different between the two groups (table 3). neutrophils of exposed fish group increased significantly (p<0.05) while no significant difference was observed in the lymphocytes, monocytes, eosinophils and basophils counts of both groups (table 4). discussion in the present study, the 1 and 96 hrs lc50 values of malathion for juvenile c. mrigala were found to be 14.55 mg/l and 9.32 mg/l, respectively. in view of this, malathion can be included in a group of moderately toxic substances for this fish. an increase in the toxicity of malathion for the test fish was observed with increase in duration of exposure and concentration of pesticide. for instance 4% fish died when they were exposed to 1 mg/l of malathion for 96 hrs, whereas 100% fish died when exposed to 32 mg/l of malathion for 1 hrs. the acute toxicity of malathion varies in different fish species and ranges from few ppb to several mg/l. office of the pesticide programme (usepa, 2006) has reported 96 hrs lc50 values of malathion for western mosquito fish (gambusia affinis) as 0.7 ppb, for rainbow trout (onchorhynchus mykiss) as 4.1 ppb, for bluegill sunfish (lepomis macrochirus) as 30 ppb, for cutthroat trout (o. clarki) as 174 ppb, for yellow time lethal concentration values with 95% confidence limits (mg/l)* lc10 lc50 lc90 1 hrs 2.41a (2.98-2.32) 14.55a (13.98-15.32) 17.52a (17.11-18.92) 24 hrs 2.18b (2.34-1.95) 12.48b (11.91-13.39) 15.27b (14.85-15.92) 48 hrs 1.75c (1.92-1.59) 11.56c (10.89-12.89) 13.39c (12.64-14.02) 72 hrs 1.56d (−) 10.85d (−) 11.21d (−) 96 hrs 1.32e (−) 9.32e (−) 10.52e (−) (−) no data because of p< 0.05, *lethal concentration values in columns with different letters significantly differ (p< 0.05). table 2. exposure time dependent lethal concentrations of malathion (mg/l) for juvenile cirrhinus mrigala. variables control group (n = 12) exposed group (n = 12) rbc (106/mm3) 1.71 ± 0.11a 1.10 ± 0.20b hb (g/dl) 4.94 ± 0.22a 2.48 ± 0.37b hct (%) 13.42 ± 2.07a 8.94 ± 1.32b mcv (fl) 214.75 ± 8.39a 211.34 ± 4.74a mch (pg) 29.42 ± 2.18a 30.76 ± 1.87a mchc (%) 24.33 ± 2.48a 26.56 ± 1.96a wbc (103/mm3) 36.41 ± 2.68a 24.44 ± 1.65b rbc = erythrocyte count; hb = hemoglobin concentration; hct = hematocrit; mcv = mean corpuscular volume; mch = mean corpuscular hemoglobin; mchc = mean corpuscular hemoglobin concentration; wbc = leukocyte count. different superscript letters indicate significant (p<0.05) difference between the groups. table 3. hematological indices of cirrhinus mrigala after 96 hrs exposure to 9.32 mg/l of malathion. variables control group (n = 12) exposed group (n = 12) neutrophils (%) 13.74 ± 3.85a 18.62 ± 2.15b lymphocytes (%) 73.12 ± 4.42a 72.42 ± 3.25a monocytes (%) 11.56 ± 1.23a 11.74 ± 1.65a eosinophils (%) basophils (%) 7.12 ± 0.95a 3.41 ± 0.55a 7.68 ± 1.05a 3.52 ± 0.84a different superscript letters indicate significant (p<0.05) difference between the groups table 4. leukocytes differential count of cirrhinus mrigala after 96 hrs exposure to 9.32 mg/l of malathion. 203 rauf/ malathion toxicity in cirrhinus mrigala perch (perca flavescens) as 263 ppb, for tilapia (tilapia mosambica) as 2000 ppb, for common carp (cyprinus carpio) as 6590 ppb and for medaka (oryzia latipes) as 40,000 ppb. on the other hand, very high 96 hrs lc50 values of malathion for black bullhead (ameiurus melas) as 11.8 mg/l and for indian catfish (hetereopneustes fossilis) as 15.3 mg/l have also been documented (martinez and leyhe, 2004). the toxicity potential of pesticides changes with respect to age, size and exposure time in different fish species. the difference in the toxicity of malathion among different fish species can be attributed to the difference in susceptibility and tolerance regarding absorption, biotransformation and excretion of pesticide (dutta et al., 1992). behavioral changes and hematological profile are sensitive indicators that may provide information about potential toxic effects of pesticides and other pollutants in aquatic organisms. the investigation of behavioral and hematological changes has become an important means of understanding toxicological impacts and pathological processes in fish (singh et al., 2009). the results of present study showed that malathion exposure at a concentration of 9.32 mg/l for 96 hrs exerted a certain influence on fish behavior that include hyperactivity, seizures, erratic swimming, loss of buoyancy, increased cough rate and gill mucous secretion. these behavioral changes correspond to the observations by other authors dealing with the toxicity of organophosphate pesticides. it regards abnormal fish behavior in danio rerio exposed to chlorpyrifos (levin et al., 2004) and c. mrigala exposed to diazinon (rauf and arain, 2013). behavioral changes in the exposed fish appear to be the manifestation of malathion toxicity. increased surfacing and gulping of surface water appear to be an attempt by fish to overcome hypoxia and to avoid breathing in the toxic water. erratic swimming, convulsions, loss of equilibrium and abnormal swimming in exposed fish are caused by deficiency in nervous and muscular coordination which can be attributed to the neurotoxic effects of malathion. similar observations have been reported in c. carpio and fingerling silurus glanis after acute exposure to dimethoate and diazinon, respectively (singh et al., 2009; koprucu et al., 2006). increased mucous secretion in malathion exposed fish during present study is probably an adaptive response of fish to reduce the irritating effect of the toxicant to eliminate it through epidermal mucous secretion. similar fish response has also been reported in c. carpio exposed to dimethoate (singh et al., 2009), c. mrigala exposed to diazinon (rauf and arain, 2013) and labeo rohita exposed to malathion (patil and david, 2008). in fact, malathion as other organophosphate pesticides exerts its effects by inhibiting the activity of enzyme acetylcholinesterase that leads to the accumulation of acetylcholine in cholinergic synapses and ends up with hyperstimulation; this decreased activity of acetylcholinersterase affects optomotor responses of fish that can disrupt the overall survivability of the animal in their natural environment (dutta et al., 1994). in the present study, the main hematological response of c. mrigala after acute exposure to malathion include significantly lower rbc count, hb concentration, hematocrit and wbc count as compared to the control. a similar response in these hematological indices to give evidence for suppressed hemotopoiesis, followed by anemia induction has previously been reported in fish exposed to other organaophosphate pesticides. it regards, changes in erythrocyte indices after exposure to chloropyrifos in clarias gariepinus (nwani et al., 2013), monocrotophos in catla catla (jeyapriya et al., 2013) and diazinon in fingerlings of s. glanis (koprucu et al., 2006). similarly, a significant decrease in erythrocyte count, hemoglobin content, hematocrit and wbc count after 96 hrs exposure to 8.15 mg/l of diazinon and 30 days exposure to 0.815 mg/l of diazinon has also been reported in immature c. mrigala (rauf and arain, 2013; haider and rauf, 2014). ahirwar et al. (2012) reported decreased rbc count, hemoglobin content and wbc count in notopterus notopterus following 3 hrs exposure to lethal concentrations of 204 international journal of aquatic biology (2015) 3(4): 199-206 malathion, while decreased rbc count, hematocrit and hemoglobin content have also been reported in c. gariepinus following sub-lethal exposure to malathion (ahmed, 2012). in consistence with the present study, a decreased rbc count, hemoglobin content and hematocrit levels in channa punctatus following three days exposure to malathion and a similar response in h. fossilis after four days exposure to malathion has previously been reported (parveen and shadab, 2011; singh et al., 2009). malathion induced decreased rbc count, hb content and hematocrit values in fish are indicator of anemia that can be a result of disruptive ironsynthesizing mechanism, destruction of mature erythrocytes and malfunctioning of the hemopoeitic system (adhikari et al., 2004). since hemopoeitic system of fish is mainly located in the interstitium of the kidney, the hematological response of test fish can be a result of malathion induced morphological alterations in renal interstitium of fish (dutta, 1992). changes in leukocyte differential counts are recognized as sensitive indicators of environmental stress as monocytes and neutrophils generally increase while lymphocytes decrease in response to a stressor (shah and altindg, 2005). in the present study, the decrease in total leukocytes count and alterations in the leukocyte differential count of fish after acute exposure to malathion are in consistence with the previous reports. it regards, decreased wbc count after acute exposure to diazinon in c. mrigala (rauf and arain, 2013) and fingerlings of s. glanis (koprucu et al., 2006), oreochromis mosambicus exposed to phosalaone (ali and rani, 2009) and o . mykiss treated with deltamethrin (velisek et al., 2007). similar to the present study, a decrease in total leukocyte count and increase in neutrophil count after five days exposure to diazinon has been reported in c. gariepinus (nwani et al., 2012). svoboda et al. (2001) reported a similar decreased non-specific immunity in c. carpio, following acute exposure to diazinon. in contrast to the present study, three and four days exposure to malathion induced increase in total leukocyte count in n. notopterus and c. punctatus, respectively (ahirwar et al., 2012; parveen and shadab, 2011) and authors attributed this increase to the acute response of immune system due to tissue injury caused by malathion exposure in fish. however, the decrease in total wbc count of test fish after acute exposure to malathion during present study may be attributed to the rapid destruction or failure in the delivery to the circulation due to reduced production of these cells; while, increase in neutrophil count may be due to the defensive response of fish to overcome malathion induced stress in fish. in conclusion, malathion can be classified as a moderately toxic substance for c. mrigala. exposure to malathion in a concentration of 9.32 mg/l for 96 hrs was caused severe stress and resulted in significant behavioral and hematological alterations in the test fish. malathion exposure, even in small concentrations has the potential to impair physiological activities leading to observed behavioral and hematological pattern and can ultimately lead to the death of fish. this fact should be taken into consideration when this pesticide is used in agricultural fields surrounding fresh water reservoirs to avoid malathion related toxicity in aquatic organisms. acknowledgement author is thankful to miss. qurat-ul-ain aslam of bahria university islamabad, for her valuable comments to improve manuscript and prof. m. ayub qureshi, head department of zoology and principal govt. superior science college, shah faisal colony, karachi, pakistan, for providing all the facilities for the successful completion of this study. references adhikari s., sarkar b., chatterjee a., mahapatra c.t., ayyappan. s. 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(2007). comparative toxicity of chlorpyrifos, diazinon, malathion and their oxon derivatives to larval rana boylii. environmental pollution, 147: 535-539. svoboda m., luskova v., drastichova j., zlabek v. (2001). the effect of diazinon on haematological indices of common carp (cyprinus carpio). acta veterinaria brno, 70: 457-465. svobodova z., pravda d., palackova j. (1991). unified methods of haematological examination of fish, 1st ed. research institute of fish culture and hydrobiology, vonday. 31 p. u.s.e.p.a. (united states environmental protection agency). (2006). reregistration eligibility decision for malathion: case no. 0248. office of prevention, pesticides, and toxic substances. office of pesticide programs, united states environmental protection agency. 196 p. velisek j., jucikova j., dobsikova r., svobodova z. (2007). effects of deltamethrin on rainbow trout (oncorhynchus mykiss). environmental toxicology and pharmacology, 23: 297-301. venakataramana g.v., sandhya rani p.n., murthy p.s. (2006). impact of malathion on the biochemical parameters of gobiid fish, glossogobius giuris (ham). journal of environmental biology, 27: 19-122. international journal of aquatic biology (2015) 3(4): 199-207 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved چکیده فارسی شناسی های خونسمیت حاد و اثرات در معرض قرارگیری ماالتونین بر رفتار و شاخص (cirrhinus mrigalaکپور هندی مریگال ) عبدول رئوف ، کلنی شاه فیصل، کراچی، پاکستان.عالی علومگروه جانورشناسی، کالج دولتی چکیده: ی از عنوان یکون بهیباشد. این مطالعه با هدف بررسی سمیت حاد ماالتمورد استفاده در کشاورزی می یهاکشون یکی از مهمترین آفتیماالت ( اجرا شد. یک آزمایش cirrhinus mrigalaشناسی کپور هندی مریگال )های خونآبی بر روی رفتار و شاخص هایهای اکوسیستمآالینده ، 24/14، 55/12های ترتیب در غلظتساعت ماالتونین برروی ماهی مورد آزمایش به 96و 50lc 1 ،42 ،24استاتیک به اجرا درآمد و مقادیر گرم در لیتر ماالتونین، میلی 24/6ساعت در معرض قرارگیری در غلظت 69تخمین زده شد. در طول گرم در لیتر میلی 24/6و 45/11، 59/11 آبششی، ترشح موکوس های رفتاری شامل تحرک باال، سرفه، تشنج، شنای نامنظم، از دست دادن تعادل، حرکات سریع سرپوشناهنجاری 69 از بعد ماالتیون سم معرض در ماهیان در خونی تغییراتیش مشاهده شد. سطح آب آمدن و بلعیدن هوا در ماهیان مورد آزماآبششی، به قایسهم در نوتروفیل تعداد در دارمعنی افزایش و لوکوسیت و هماتوکریت هموگلوبین، اریتروسیت، تعداد در دارمعنی کاهش یک شامل ساعت هایناهنجاری ماالتیون لیتر در گرممیلی 69 معرض در ماهیان قرارگرفتن که داشت بیان توانمی گیرینتیجه عنوان به. بود تیمار ماهیان با ماهیان در ماالتیون از ناشی سمیت پایش برای ارزش با ابزار یک عنوانبه که کندمی تحریک مریگال هندی کپور در را شناسیخون و رفتاری تواند مدنظر قرارگیرد.می شناسی، کپورهندی.، سمیت حاد، رفتار، خونماالتونین: کلمات کلیدی international journal of aquatic biology (2014) 2(5): 275-285 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2014 npajournals. all rights reserved original article effects of dietary administration of yarrow extract on growth performance and blood biochemical parameters of rainbow trout (oncorhynchus mykiss) mahmoud nafisi bahabadi1, mahdi banaee2*,1marzieh taghiyan1, behzad nematdoust haghi2 1aquaculture department, faculty of agriculture and natural resources, persian gulf university, bushehr, iran. 2aquaculture department, natural resource faculity, behbahan khatam alanbia university of technology, behbahan, iran. article history: received 4 june 2014 accepted 24 august 2014 available online 2 5 october 2014 keywords: medicinal plants yarrow extract rainbow trout biochemical factors growth performance abstract: the present study was conducted to investigate the clinical effects and possible side effects of yarrow extract (achillea millefolium l.) as feed additive on biochemical blood parameters and growth performance of rainbow trout (oncorhynchus mykiss). fishes were treated with 0 (control), 0.1, 0.5 and 1% of yarrow extract for 30 days. plasma alanine aminotransferase (alt), aspartate aminotransferase (ast), lactate dehydrogenase (ldh), alkaline phosphatase (alp), creatine kinase (ck), peroxidase activity, total complement and lysozyme activity, glucose, total protein, triglyceride and cholesterol were measured after 15 and 30 days of yarrow treatment. there were no significant changes in the lysozyme activity and glucose levels. total protein and globulin levels were significantly higher in the fish fed with diets enriched with 1% yarrow extract on day 30. triglyceride and cholesterol levels was significantly decreased in the fish fed with diets containing 0.5% and 1% yarrow extract on day 30 (p<0.05). ldh, ck and peroxidase activities in the fish fed with diets having 1% yarrow extract were significantly decreased at the end of the experiment (p<0.05). in contrast, a significant increase in ast, alp and total complement activity was observed in the fish fed with 1% yarrow extract diet, on day 15 (p<0.05). the weight gain and specific growth rate increased and food conversion ratio decreased in in the fish fed 1% yarrow extract on day 30. condition factor in the fish fed with yarrow extract was significantly higher than control group on 30 day. in conclusion, on the basis of these results, oral administration of yarrow extract up to 0.5% have not side effect on blood biochemical and clinical parameters of fishes. however, oral administration of 1% of yarrow extract caused cytotoxicity and modifications in blood biochemical parameters of fish. introduction compared with chemical drugs, the medicinal plants have effective agents and other compounds which accelerate gastrointestinal absorption process, improve the therapeutic effects and reduce side effects and drug toxicity (platel et al., 2002). although the results reported by laboratory animals evidence beneficial effects in the application of medicinal plants in treatment and prevention of disease in fish (sivaram et al., 2004; rao et al., 2006; divyagnaneswari et al., 2007), the contradictory effects of some medicinal plants has decelerated using them for fish in a commercial scale. the lack * corresponding author: mahdi banaee e-mail address: mahdibanaee@yahoo.com of knowledge about the biological effects of compounds in medicinal plants' extracts on the individual characteristics of different fish species are the main problems in application of these compounds (banaee, 2010). therefore, study of the effects of herbal derivatives as a drug on the health of fish may increase our knowledge which can favour the use of certain compound for disease treatment (banaee et al., 2011). yarrow (achillea sp.) has an important role in medicinal plants derived from its antioxidant (yakhkeshi et al., 2012), analgesic, anti-bacterial (mazandarani et al., 2007), anti-parasitic (khalili et 276 international journal of aquatic biology (2014) 2(5): 275-285 al., 2011), anti-fungal (ayatollahi mousavi et al., 1996), anti-inflammatory (benedek et al., 2007), anti-hypertensive and lipid-lowering (asgary et al., 2000), anticonvulsant and antispasmodic (lemmens-gruber et al., 2006), antitumor (csuporlöffler et al., 2009), anti-ulcer skin (cavalcanti et al., 2006), and antiarrhythmic (asgary et al., 2000) properties. the antibacterial properties of yarrow extract are attributed to flavonoids, saponins, and terpens (table 1) (mothana et al., 2009). moreover, the administration of medicinal plants with antibiotic properties can change intestinal bacterial flora, increase growth and improve carcass quality (tekeli et al., 2008). therefore, the present study was carried out to determine whether yarrow (achillea millefolium l.) would influence on the blood biochemical parameters, growth performance and immune parameters of rainbow trout. materials and methods dried yarrow extract preparation: yarrow powder was extracted with hydro-alcoholic mixture (ethanol 70% and water in 1:1 proportion) at room temperature by cold maceration method (asgary et al., 2003). then, this extract was filtered through whatman filter paper. finally, the extract was concentrated in an incubator at 50°c obtaining the dry extract. animal treatments: one hundred and twenty rainbow trout (with average weight and length of flavonoid essential oil cyaroside tricyclene n-heptanol cosmosiin α-pinene 3-octanone luteolin β-ocimene δ-3-carene apigenin γ-terpinene α-terpinene centaureidin terpinolene p-cymene quercetin linalool pinocarvone 3´-methoxy luteolin trans-thujone borneol luteolin 7-o-glucoside allo-ocimene α-copaene 5-hydroxy 3´,4´,6´,7´-tetra methoxy flavone terpin-1-ol geranyl acetate camphor longifolene salvigenin (z)-tagetone α-gurjunene galangin isoborneol α-guaiene eupatilin α-terpineol n-pentadecane α-himachalene α-cadinene γ-gurjunene γ-eudesmol β-chamigrene α-eudesmol γ-muurolene bornyl acetate β-selinene neryl acetate viridiflorene α-cyclogeraniol cis-pinocarveol β-calacorene isobornylformate (z)-cubenol α-terpinyl acetate β-bisabolol caryophyllene alcohol phytol (e)-cinnamaldehyde nootkatin patchouli alcohol occidol acetate α-bisabolene oxide a abietadiene (e,e)-farnesol n-heneicosane chamazulene methyl octadecanoate table 1. phytochemical compounds isolated from the dried yarrow extract adopted from saeidnia et al. (2011) and ebrahimi et al. (2012). 277 nafisi bahabadi et al/ effects of yarrow extract on rainbow trout 146.9 ± 18.1 g; 25.9 ± 2.6 cm, respectively) were randomly introduced into 4 treatments, 3 replicates with 10 fish in the 12 fibreglass tanks (200 l) with semi-closed water recirculating systems for two weeks to acclimate to the laboratory conditions (15 ± 2°c; ph: 7.4 ± 0.2; photoperiod: 16l: 8d; 20% water exchange rate/day) prior to experiments. during acclimation and experimental periods, fish were fed with prepared pellets according to commercial formulations at 2% of their body weight twice a day (hinshaw, 1999). the enriched diet was prepared with formulated fish feed obtained from chineh company, karaj, iran (table 2) and yarrow extract according to the method developed by banaee et al. (2011). each supplemented diet was mixed with distilled water until obtaining a homogenous mixture. this mixture was passed through a meat grinder, producing extruded string shapes, which were dried in an oven at 55°c for 12 hrs and then broken to produce pellets with approximate 10 mm length. the prepared pellets were packed and stored at -20°c in a freezer until be used. the control diet was prepared using the same process, although no supplement was added. in day of 15 and 30 of the experiment, 12 fish from each treatment were randomly sampled and anesthetized with clove powder solution (200 mg/l). then, the blood of fish from each experimental and control groups were taken by puncturing of the caudal vessels with a syringe and the blood was stored in sterilized glass vials containing the anticoagulant 1% edta at 4°c. the blood was centrifuged for 15 min at 4000 rpm. the contents of digestive tract, nutritional status (gastro-intestinesomatic index) and clinical symptoms such as changes in the size of gall-bladder and liver (hepatosomatic index) as well as feeding behavior of treated fish were evaluated. biochemical analysis: biochemical parameters of plasma including glucose, total protein, albumin, globulin, cholesterol and triglyceride; aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ldh), alkaline phosphatase (alp) and creatine kinase (ck) were analyzed using the biochemical diagnostic kit (parsazemon company, karaj, iran). alternative complement activity (ach50) was evaluated based on yano (1992) using rabbit red blood cells (rarbc). in this method, the absorbance of the haemolysate was measured at 414 nm against distilled water to acquire the 100% lysis value. the volume of plasma producing 50% haemolysis (ach50) was determined. lysozyme activity was determined by the lysis of bacterial cells of micrococcus lysodeikticus (lange et al., 2001). the decrease in turbidity at 570 nm after 0.5 min and 4.5 min was recorded by spectrophotometer. lysozyme of sample was calibrated using a standard curve determined with hen’s egg white lysozyme (sigma) in pbs. the total peroxidase activity was determined according to cuesta et al. (2007) with modification by measuring the optical density of 3,3′,5,5′– tetramethylbenzidine hydrochloride oxidation products during particular intervals of time at 450 nm. all biochemical parameters were measured in duplicates by uv/vis spectrophotometer (model unico 2100). growth performance: the growth parameters including weight gain percentage, specific growth rate, and feed conversion ratio and condition factor were calculated using the following formulas, on day15 and 30 day. weight gain (%) = final weight − initial weight initial weight × 100 specific growth rate (sgr%) = [(ln (final body weight)-ln (initial body weight))/(experimental periods)]×100 nutrient composition reference diet dry material 92 metabolize energy (kcal/g) 350 crud protein 40 ether extract (lipid) 10.5 ash 7.9 crude fiber 5.8 carbohydrate 27 table 2. nutrient composition of the commercial diets (chineh company, iran). 278 international journal of aquatic biology (2014) 2(5): 275-285 feed conversion ratio (fcr) = feed intake (g) wet weight gain (g) condition factor (cf) = weight (g) (length (cm))3 × 100 statistical analysis: a significant difference in the biochemical parameters of specimens treated with the different concentrations of a. millefolium extracts was examined using one-way anova. all the data were examined for normality (kolmogorovsmirnov test). the means were compared by duncan’s test and a p<0.05 was considered statistically significant. statistical analyses were performed using spss (ibm, release 19) software. data are presented as mean ± sd. results the hepato-somatic index (ih) was significantly decreased in fish fed with a diet containing 1% yarrow extract. no significant changes were observed during the test in spleen-somatic index (is) and gastro-intestine-somatic index (ig) in the fish fed with diets containing yarrow extract when compared with control group. the contents of the digestive system of fish as satiety index indicated that there were no significant difference between amounts of feed was fed by experimental fish and control group (fig.1a, b, c). significant changes in the wg and sgr was observed in the fish fed with a diet containing 1% yarrow extract during the experiment. the fcr was significantly decreased in fish with diets having 1% yarrow extract. the cf in the fish fed with yarrow extract was significantly higher than the control group at the end of experiment (table 3). there was no significant difference in glucose levels between fish fed yarrow extract and control group during experimental period. although, oral administration of 1% yarrow extract resulted in a significant increase in total protein levels, a significant decrease in the albumin levels was observed in plasma of fish fed with 0.1% yarrow extract on day 30. supplementing feed with 0.1 and 1% yarrow extract resulted in significantly increased in globulin levels on day 30 (p<0.05). triglyceride levels in the plasma of the fish fed with diets having 0.5% and 1% yarrow extract showed a significant decrease during the test. cholesterol levels figure 1. effects of different concentrations of yarrow extract as feed additive on (a) percentage ratio of spleen to total weight as spleen-somatic index (is), (b) liver weight to total weight as hepato-somatic index (ih) and (c) gastrointestinal tract weight to total weight (ig) as a satiety index or gastro-intestine-somatic index. significant differences between treatment and control groups were represented by alphabets (one-way anova, p<0.05). values represent mean ± s.d. 279 nafisi bahabadi et al/ effects of yarrow extract on rainbow trout significantly decreased in plasma of fish fed with diets having 0.5% and 1% yarrow extract on day 30 of the test (p<0.05), (table 4). there was a significant increase in the total complement activity in plasma of fish fed with food enriched with 1% of yarrow extract on day 30 of trial (p<0.05). no significant changes were observed in the ach50 activity on day 15 (table 4). the activity of ast significantly decreased in plasma of fish fed with 0.1% and 0.5% yarrow extract on day 30 of experiment (p<0.05). however, ast activity in plasma of the fish fed with diets having 1% yarrow extract significantly increased on day 15 of test (p<0.05). the activity of alt significantly increased in plasma of fish fed with 0.5 and 0.1% yarrow extract on 15 and 30 days when compared with control group, respectively. ldh activity in plasma of the fish fed with diets enriched with 0.1% and 1% yarrow extract was significantly lower than control group on day 30 of the experiment. the activity of alp significantly increased in plasma of fish fed with 1% yarrow extract on day 15 of experiment. the activity of cpk significantly decreased in plasma of fish fed with 1% yarrow extract during experimental period (p< 0.05), (table 5). a significant decrease in the peroxidase activity was observed in plasma of fish fed with 0.5% and 1% yarrow extract on day 15 (p< 0.05). also, there was no significant change in peroxidase activity on day 30 of test. no significant changes in lysozyme activity in plasma of fish fed with yarrow extract supplement were observed when compared with control group during experimental periods (p> 0.05), (table 5). discussion in this study, rainbow trout were fed with diets enriched with 0.1%, 0.5%, and 1% yarrow extract for 30 days. during the experimental periods, no mortality or changes in the appetite of the fish were observed. platel et al. (2002) evidenced the favorable effect of medicinal plants on digestion and a stimulating effect on bile secretion and the activity of pancreatic enzymes. moreover, adding plants extracts to the diet can affect the fish ability to find growth performance ye% of g/kg experimental periods 15th day 30th day weight gain (%) 0.0 27.45±7.45a 56.63±8.09a 0.1% 35.80±11.85ab 70.81±18.77b 0.5% 28.33±6.53a 67.23±11.43ab 1% 39.57±8.74b 69.79±9.36b specific growth rate (%/day) 0.0 1.61±0.38a 1.49±0.17a 0.1% 2.02±0.59ab 1.77±0.37b 0.5% 1.66±0.33a 1.71±0.23ab 1% 2.21±0.42b 1.76±0.18b food conversion rate 0.0 1.73±0.54b 1.59±0.24b 0.1% 1.40±0.62ab 1.33±0.37ab 0.5% 1.63±0.34ab 1.35±0.27ab 1% 1.18±0.33a 1.28±0.17a condition factor 0.0 0.85±0.06a 0.89±0.06a 0.1% 0.92±0.07a 1.00±0.09b 0.5% 0.93±0.13a 0.98±0.06b 1% 0.93±0.0.9a 0.98±0.08b effects of different concentrations of yarrow extract on growth index were analyzed using a one-way anova. significant differences between treatment and control groups were represented by alphabets (p<0.05). values represent mean ± sd. table 3. growth performance of rainbow trout fed with yarrow extract as a supplement. 280 international journal of aquatic biology (2014) 2(5): 275-285 food by stimulating their sense of smell and encourage them to eat more (adams, 2005). our results indicate that feeding of fish with 1% yarrow extract significantly improved growth performance. some compounds in medicinal plants extracts including bioflavonoids can positively induce effects on growth performance and general health of fish (yilmaz et al., 2006). similar results are found in cichlid, cryptoheros nigrofasciatus, (cek et al. 2007); red seabream, pagrus major (ji et al. 2007); and rainbow trout (bohlouli oskoii et al., 2012) which were fed with diets supplemented with medicinal plants extracts. although, the results indicate that administration period of yarrow extract had a significant effect on glucose levels in plasma, hypoglycemic effect of yarrow on rats with diabetes mellitus indicates this plant's effects on against in regulating blood sugar in these animals (sadeghi et al., 2009). these results may be attributed to the hypoglycemic activity of biochemical parameters ye% of g/kg experimental periods 30th day 15th day glucose (mg/dl) 0.0 % 74.64±26.41a 89.58±4.42a 0.1% 64.53±11.46a 83.46±7.41a 0.5% 73.32±22.68a 87.20±8.30a 1% 62.59±13.17a 83.08±5.56a total protein (g/dl) 0.0 % 4.54±0.55a 4.77±0.87a 0.1% 4.31±0.42a 4.52±0.59a 0.5% 4.32±0.48a 4.45±0.41a 1% 4.84±0.47b 4.37±0.91a albumin (g/dl) 0.0 % 3.00±0.40b 2.84±0.18a 0.1% 2.36±0.56a 2.89±0.09a 0.5% 3.00±0.51b 2.88±0.48a 1% 2.77±0.62ab 2.88±0.11a globulin (g/dl) 0.0 % 1.53±0.36a 1.93±0.82a 0.1% 1.95±0.67b 1.63±0.61a 0.5% 1.32±0.35a 1.56±0.39a 1% 2.07±0.43b 1.49±0.90a cholesterol (mg/dl) 0.0 % 222.11±41.41b 220.77±29.07ab 0.1% 226.75±34.86b 258.18±52.80b 0.5% 182.22±36.49a 178.16±42.49a 1% 168.43±38.39a 191.25±49.81a triglyceride (mg/dl) 0.0 % 167.38±39.68b 166.97±33.09b 0.1% 125.01±38.83a 169.77±12.43b 0.5% 104.04±40.61a 129.60±17.07a 1% 114.29±28.02a 126.02±26.34a ach50 (u/ml) 0.0 % 311.89±9.05a 321.44±7.86a 0.1% 321.89±43.96a 315.67±11.05a 0.5% 344.45±19.18a 314.34±10.06a 1% 344.45±46.22a 366.00±30.56b effects of different concentrations of yarrow extract on biochemical parameters. significant differences between treatment and control groups were represented by alphabets (one-way anova, p<0.05). values represent mean ± sd. table 4. plasma biochemical parameters in rainbow trout fed with yarrow extract as a supplement. 281 nafisi bahabadi et al/ effects of yarrow extract on rainbow trout herbal extracts to increase the level of plasma insulin and improvement of peripheral metabolism of glucose (awad, 2010) or may be attributed to the activation of glycogen synthesis and healthy hepatic function (ji et al., 2007). decreased glucose levels in the blood of rainbow trout fed with silymarin extract (banaee et al., 2011) and african catfish fed with onion and garlic extracts are reported (al-salahy, 2002). albumin and globulin make up most of the protein within the body and are measured in the total protein of the plasma. total protein, albumin and globulin tests are used to monitor the course of diseases in immune disorders, liver dysfunction and impaired kidney activity. at the end of the test, the levels of albumin in plasma of the fish fed with diets containing 0.1% yarrow significantly decreased. the 0.1% of yarrow extract was able to significantly increase the total protein levels on day 30. oral administration of 0.1 and 1 % yarrow extract significantly increased the globulin levels in plasma of fish on day 30. although an increased level of total protein, albumin and globulin in plasma of the fish fed with diets enriched with echinacea purpurea and silybum marianum was reported by bohlouli oskoii et al. (2012) and banaee et al. (2011), no significant changes are reported in the levels of albumin and total protein in plasma of fish fed with diets enriched with onion and garlic extract (alsalahy, 2002; naeiji et al., 2013).. quercetin is the most important flavonoid in yarrow (saednia et al., 2009) that prevents the biosynthesis of cholesterol by inhibiting the activity of fatty acid synthesis (yamamoto and oue, 2006). therefore, the biochemical parameters ye% of g/kg experimental periods 15th day 30th day ast (u/l) 0.0 % 87.27±12.65a 87.49±32.49b 0.1% 97.49±13.94a 63.23±16.61a 0.5% 96.75±16.18a 63.15±10.65a 1% 121.89±36.22b 69.91±13.59ab alt (u/l) 0.0 % 12.86±3.67a 17.27±2.65a 0.1% 12.57±4.39a 23.60±8.39b 0.5% 19.11±5.52b 17.64±4.44a 1% 12.79±3.87a 18.08±4.45a ldh (u/l) 0.0 % 808.89±103.17a 735.60±237.85b 0.1% 884.44±169.41a 523.05±203.21a 0.5% 882.22±116.76a 700.57±251.85ab 1% 890.37±205.86a 493.96±94.42a cpk (u/l) 0.0 % 177.31±23.81b 207.27±27.47b 0.1% 167.11±12.29ab 199.15±27.53b 0.5% 160.34±31.61ab 199.99±37.76b 1% 150.85±28.84a 151.19±31.27a alp (u/l) 0.0 % 18.89±5.59a 25.93±4.45a 0.1% 25.12±9.63a 23.69±12.64a 0.5% 34.41±20.40ab 21.95±4.12a 1% 45.44±25.92b 20.22±11.65a peroxidase (u/ml) 0.0 % 138.11±10.78b 139.44±10.49a 0.1% 129.67±11.68ab 126.11±11.21a 0.5% 120.00±10.10a 119.56±9.41a 1% 120.44±9.38a 163.44±86.12a lysozyme (u/ml) 0.0 % 123.68±8.38a 120.67±10.83a 0.1% 124.78±9.13a 125.11±11.95a 0.5% 128.22±11.10a 126.00±14.01a 1% 129.44±10.10a 124.22±18.46a effects of different concentrations of yarrow extract on biochemical parameters (enzyme activity). significant differences between treatment and control groups were represented by alphabets (oneway anova, p<0.05). values represent mean ± sd. table 5. plasma enzymatic activities in plasma of rainbow trout fed with yarrow extract as supplement. 282 international journal of aquatic biology (2014) 2(5): 275-285 decreased cholesterol levels in the blood of fish fed with 0.5 and 1% yarrow extract at the end of the test, and decreased levels of triglycerides in plasma of the fish fed with diets having 0.5% and 1% yarrow extract during the test may be attributed to the influence of quercetine on the synthesis of cholesterol. the influence of long term use of yarrow in decreasing triglyceride, total cholesterol and lowdensity lipoprotein (ldl) and increasing the high density lipoprotein (hdl) in the blood of human volunteers verifies this issue (asgary et al., 2000). the cholesterol synthesized in liver transfers to other tissues of the body through ldl, but hdl transports the cholesterol of peripheral tissues to liver. so, the increased excretion of cholesterol through bile (asgary et al., 2000) decreases the cholesterol level in blood of the fish fed with yarrow. decreases in cholesterol and triglyceride levels are also reported in blood of rainbow trout and catfish respectively fed with silymarin extract (banaee et al., 2011) and onion and garlic extracts (al-salahy, 2002). the complement system is an essential and effective part of the innate immune system. it can rapidly distinguish and opsonize bacteria for phagocytosis by specialized phagocytes or destroy them directly by membrane disorder (ahmadi et al., 2014). the increase in the complement activity (ach50) observed in plasma of fish fed with diets enriched with 1% of yarrow extract on day 15 may help to identify and eliminate bacterial agents by phagocytosis. increases in the total complement activity were reported in fish fed with a diet enriched with punica granatum, chrysanthemum cinerariaefolium and zanthoxylum schinifolium extracts (harikrishnan et al., 2010), s. marianum (ahmadi et al., 2012) and nasturtium nasturtium extracts (asadi et al., 2012). ast, alt, ldh, alp and ck are found in various tissues. when diseases or injuries affect these tissues, the cells are destroyed and these enzymes are released into plasma (banaee et al., 2011). although, oral administration of 0.1 and 0.5% yarrow extract resulted in a significant decrease in the activity of ast on day 30, a significant increase in ast activity in plasma of fish fed with enriched with 1% yarrow extract on day 15. treatment with 0.5 % yarrow extract resulted in a significant increase in ast activity on day 15. our results show that alp activity significantly increased in the plasma of fish fed with enrich diet with 1% yarrow extract when compared with the control group at day 15, no significant changes were observed in alp activity at day 30. ldh measurement is used to detect tissue disorders and as an aid in the diagnosis of tissue damage. the ldh activity also significantly decreased after the oral administration of 0.1 and 1% yarrow extract on day 30. a significant decrease were observed in the ck activity in plasma of common carp fed with enrich diet with 1% marshmallow flower extract when compared with the control group during experimental period. the most important of the biological mechanisms of flavonoids have been attributed to their antioxidant properties (oteiza et al., 2005). due to anti-radical and anti-oxidant properties of the yarrow extract, its administration might prevent lipid peroxidation of cell membranes and inhibit the release of foresaid enzymes into the plasma. therefore, yarrow may be useful in treatment and prevention of diseases caused by oxidative stress derived from its rich content in flavonoids. a low concentration of silymarin (banaee et al., 2011) and garlic (al-salahy, 2002; naeiji et al. 2013) in diet of fish has been evidenced to regulate the plasma activities of ast, alt, alp, ck and ldh. more hydrophilic flavonoids can interact at the membrane surface through hydrogen bonding; can act to reduce the access of oxidants and per-oxidants and protect the structure and function of cellular membranes (oteiza et al., 2005). lysozymes are a family of enzymes with antibacterial activity characterized by the ability to damage the cell wall of bacteria (ahmadi et al., 2014). our results showed that fish fed with diets containing yarrow extract had no effects on lysozyme activity. studies of sivaram et al. (2004) showed that no significant changes were observed in serum lysozyme activity of cultured grouper, epinephelus tauvina, fed with diets enriched with 283 nafisi bahabadi et al/ effects of yarrow extract on rainbow trout ocimum sanctum, withania somnifera and myristica fragrans extracts. peroxidases play an important role in defense system against extracellular bacterial and parasitic pathogens. myeloperoxidase and eosinophil peroxidase are important active peroxidases in immune system of fish and found in the cytoplasmic granules of neutrophils and eosinophil, respectively. if the leukocytes are stimulated, peroxidase activity will increase in plasma (ahmadi et al., 2014). the results revealed that the administration of yarrow extract to fish significantly decrease the peroxidase activity. antibacterial compounds that exist in the yarrow extract may be prevented stimulation of leukocytes. ahmadi et al. (2012) and asadi et al. (2012) found that the administration of s. marianum and n. nasturtium extract had no effects on peroxidase activity of fish. since the change in blood biochemical parameters is a useful clinical tool to predict and monitor the health in organisms, these factors can also be used for determining drug safety. preclinical research on the biochemical factors and growth performance as pharmacology indices of yarrow revealed that this herbal medicine may be useful by providing benefit in the treatment of some disease of aquatic animals. therefore, in clinical studies should be focused on the medical effects of yarrow. acknowledgements special thanks are extended to research assistant of faculty of natural resources, persian gulf university for their financial support. the authors are also grateful to the dr. antoni sureda for their cooperation and guidance throughout the writing manuscript. reference adams c. 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(2018) 6(5): 258-264 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article brachionus falcatus and platyias patulus indicating organic pollution in ouémé river’s basin, republic of benin arsène mathieu houssou*1,2, clément agossou bonou3, elie montchowui1,2 1laboratoire de recherche en aquaculture et en biologie et ecologie aquatiques, ecole d’aquaculture de vallée, université nationale d’agriculture, porto novo, bénin. 2laboratoire d’hydrobiologie et d’aquaculture, faculté des sciences agronomiques, université d’abomey-calavi, bénin. 3laboratoire de recherche en biologie appliquée, ecole polytechnique d’abomey-calavi, université d’abomey-calavi, bénin. s article history: received 9 june 2018 accepted 4 october 2018 available online 2 5 october 2018 keywords: bio-indication brachionus falcatus environmental pollution platyias patulus abstract: zooplankton is a biological compartment widely used in the bio-monitoring of aquatic ecosystems. it allows early detection of environmental disturbances even before reaching the upper compartments of interest to humans. the present study evaluated the ability of two rotifer species brachionus falcatus and platyias patulus to indicate organic pollution in the ouémé river basin. sampling was done between october 2014 and september 2015. plankton net of 20 μm mesh size was used. parameters such as nh4+, no2and po43were measured in water and used for the calculation of the organic pollution index (ipo). the results showed that the occurrence of b. falcatus and p. patulus was 96.66% and 81.11%, respectively. both species were particularly abundant in sites such as kaboua and vossa as well as downstream of the basin (agonlin-lowé and bonou). these strong abundances are linked to a very strong organic pollution in kaboua and vossa then to a high organic pollution at downstream. the least organically polluted station (kpassa) had the lowest abundance of both species. b. falcatus and p. patulus are therefore two indicator species of organic pollution in the ouémé river’s basin in benin. introduction aquatic ecosystems are invaluable, contributing substantially to a country's economy. but human activity generally affects their integrity. thus, there is change in the general functioning of these ecosystems and consequently a significant drop in productivity. ouémé river in benin is the first aquatic ecosystem in terms of occupied area. throughout its basin, there are sources of disturbance e.g. agriculture, industry, urbanization. simeonov et al. (2003) reported that anthropogenic influences such as urban, industrial and agricultural activities degrade surface waters that become hazardous to drinking water, industrial use, and agricultural use as well as for other types of use. these sources of disturbance at upstream of a stream are drained along the basin (calandre and jacono, 2006) and lead to a greater alteration at downstream and in the receiving water body (arimoro, 2009). ouémé river basin is thus impacted from upstream to downstream in evolving dynamics other time. *correspondence: arsène mathieu houssou doi: https://doi.org/10.22034/ijab.v6i5.465 e-mail: arsnehous@yahoo.fr to preserve the richness of the ouémé river ecosystem, it is necessary to be able to follow the evolution of its ecological status so that management decisions will be made at the right time. biological monitoring is a comprehensive integrating method to assess both the quality of the ecosystem and its impact on biocenosis. zooplankton is one of the most important biological compartments for ecological processes. they are good environmental indicators that can express different responses to different disrupters (houssou et al., 2017a). these responses are also very variable between the component species of the zooplankton community. some species are known to be common to eutrophic environments while others are very sensitive (el-enany, 2009). eutrophication affects the zooplankton composition shifting the dominance of large species (copepods) to the smaller species (rotifers) (el-shabrawy, 2000). it is therefore a very appropriate community for the monitoring of environmental changes. 259 int. j. aquat. biol. (2018) 6(5): 258-264 brachionus falcatus and platyias patulus are both rotifers species of the family of brachionidae. they are among the most abundant species in the ouémé basin (houssou et al., 2017b). mola (2011) showed that rotifers of brachionidae family are indicators of eutrophication; a phenomenon resulting from organic pollution and the mineral charge of water. the present study therefore assessed the ability of b. falcatus and p. patulus to indicate organic pollution in the ouémé river basin in benin. materials and methods the study was conducted in the ouémé river basin in benin. the catchment area (fig. 1) extends between 6°51' and 10°11' north latitude and 1°29' and 3°24' east longitude. it covers an area equivalent to half of the territory of benin republic (i.e. more than 50000 km²). fifteen sampling sites were selected from upstream to downstream and taking into account the main tributaries of the river (okpara, zou, beffa and donga rivers) (fig. 1). the variability of habitats and anthropization in the basin were key factors in the choice of sites. sampling of zooplankton was done monthly over a 12-month period (october 2014 to september 2015). a 20 μm mesh plankton net was used with a combination of vertical and horizontal sampling techniques. samples were fixed in-situ with 5% formaldehyde. in laboratory, the samples were treated under a photonic microscope. both rotifer species (b. falcatus and p. patulus) were identified and counted. the ahlstrom (1940) guide was used for identifications. the count was made milliliter per milliliter over the entire volume of the samples by using a four-grid enumeration cell of burker turk. a photograph of the species (fig. 2) was also made. a sample of water was collected biweekly at each station for determined parameters such as nh4 +, no2 and po4 3(houssou et al., 2017c). standards methods (apha, 2005) were used for determination of these parameters. the presence/absence of the two species was studied in the different stations and over time. the distribution profile of the abundances in the different sites was studied with the attribute symbol plot in canoco for windows v.4.5 software. then, the organic pollution in the different sites was evaluated with the organic pollution index (ipo) of leclercq and vandevenne (1987) modified by leclercq (2001). this index is based on the classification of parameters such as ammonium, nitrite and orthophosphate into pollution classes which mean gives the ipo. the classification grid is presented in table 1. the distribution spectrum of the mean abundance of species in the various status of organic pollution was established and the tukey test was used to test the difference between the statuses. the spearman correlation was also calculated between the species and the organic pollution index. results and discussion spatial and temporal distribution of species: the two figure 1. geographical map of sampling sites in ouémé river’s basin. figure 2. (a) brachionus falcatus and (b) platyias patulus. 260 houssou et al./ bioindication in oueme river basin species had a perfect distribution between december and then may and in june and july for b. falcatus (table 2). platyias patulus was also found in all stations in november. in september, it was absent in the basin while it was present only at agonlin-lowé and bonou in october. platyias patulus was absent in zagnanado in august, in kpassa in june, july and august and then in donga in june and august. as for b. falcatus, it was not found in vossa in october and november and then in kpassa in august, september, october and november. this distribution confers an occurrence of 96.66% to b. falcatus and 81.11% to p. patulus. they are therefore constant species in the ouémé basin (dajoz, 2000) and are characteristic of its ecosystem. the two species being cosmopolitan with a preference for warm waters, their strong presence in the ouémé basin is justified. they have already been found in beninese waters houssou et al. (2015 and 2016). the rarity of p. paltulus during the months of september and october (high water period) shows that it is strongly affected by the volume of water. it was only observed in october at the two stations in downstream (bonou and agonlin-lowé). the topography of the basin giving a relatively high stability to the downstream water compared to the upstream zone, favored the maintenance of the species in downstream even if water volume is high. the water flow is therefore a strongly limiting factor in the distribution of p. patulus in the ouémé basin. species abundance distribution: the abundance of b. falcatus and p. patulus in the study sites is shown on figure 3a and b, respectively. the two species presented a perfectly equivalent profile of their abundances. the growth/decline of one is systematically followed by that of the other. brachionus falcatus appeared most abundant with densities between 209 ind.m-3 and 927 ind.m-3. for p. patulus, the densities ranged from 39 ind.m-3 to 185 ind.m-3. the two extremes of density of each species were observed on the okpara river (kaboua and table 1. classes limits of parameters including in ipo calculation (leclercq, 2001). classes nh4 + (mg.l-1) no2 (µg.l-1) po4 3(µg.l-1) 5 < 0.1 < 5 <15 4 0.1-0.9 6-a0 16-75 3 1-2.4 11-50 76-250 2 2.5-6 51-150 251-900 1 >6 >150 >900 ipo = mean of classes numbers of the three parameters; ipo = 5.0-4.6: no organic pollution; ipo = 4.5-4.0: less organic pollution; ipo = 3.9-3.0: moderate organic pollution; ipo = 2.9-2.0: strong organic pollution; ipo = 1.9-1.0: very strong organic pollution. table 2. spatial and temporal distribution of brachionus falcatus and platyias patulus in the oueme river basin. sites site code oct14 nov14 dec14 jan15 feb15 mar15 apr15 may15 jun15 jul15 aug15 sep15 b p b p b p b p b p b p b p b p b p b p b p b p agonlin-lowé ag-l bonou bon zagnanado zag toué tou atchérigbé atch dassa das atchakpa-béthel atch-b atchakpa-rejet atch-r atchakpa-pompage atch-p kaboua kab vossa vos kpassa kpa bétérou bét donga don affon aff bbrachionus falcatus, pplatyias patulus. the colored boxes show the present of the species. 261 int. j. aquat. biol. (2018) 6(5): 258-264 kpassa station respectively for the highest and lowest abundance). the stations of agonlin-lowé, atchakparejet, vossa and bonou also had high densities of both species. the okpara river, which is the main tributary of the ouémé river, gave in this study the lowest and highest density of the two studied species. both species being primary consumers, their development is strongly linked to that of phytoplankton. phytoplankton in tropical condition is limited by the mineral charge; the insolation being mostly sufficient. thus the okpara river respectively in the kpassa and kaboua sites had the lowest and highest mineralization. at the kpassa sites (low mineralization), a hydrological dam is set up for the production of drinking water for parakou city. the pollutant discharges into this site are therefore very limited. as for the kaboua sites, located further downstream and in a forest area, it is a border between benin and the federal republic of nigeria. it is therefore highly used for trade between the two countries. at the mineral level, this station reflects not only the directly induced pollution but also the load of the whole river from the upstream under the influence of the transfers (calandre and jacono, 2006). during the low-water periods, there was a strong phytoplankton production coinciding with the bloom of the two studied species of rotifer. the removal of water during the dry season therefore created the conditions for multiplication of the two species (zébzé-togouet et al., 2005). organic pollution: in terms of organic pollution in the basin, three statuses were observed (table 3). moderate pollution has been observed in sites such as zagnanado, toué, atchérigbé, dassa, atchakpabéthel, atchakpa-pompage, kpassa, bétérou, affon and donga. a status of strong pollution was then observed at agonlin-lowé, bonou and atchakparejet. the stations of kaboua and vossa were very strongly polluted. ammonium, nitrites and phosphates which have been used for the determination of organic loading are products of mineralization of organic matter in water (leclercq, 2001; wang et al., 2006; abai et al., 2014). their concentration in water therefore indicates the degree of organic pollution. the organic inputs in the ouémé basin are from various sources. agriculture is currently the most important source of inputs, crop residues and processing waste. increasing urbanization in the ouémé basin places domestic wastewater as the second most important source of organic matter in the surface waters of the basin. the industry also figure 3. plot of brachionus falcatus (a) and platyias patulus (b) abundances throughout sampling sites. sites codes are same as in table 2.values represents species abundances (ind.m-3). 262 houssou et al./ bioindication in oueme river basin contributes in some places to the organic pollution of water. at stations with moderate organic pollution (such as affon, donga, etc.), urbanization is less and domestic waste is not an important source of pollution. the organic matter in the water of these sites comes mainly from agriculture. particularly in atchakpabetehel, the organic load could have been more important than moderate. self-purification between atchakpa-rejet (the discharge point of the “sucrerie du complant du benin (sucob)” plant) and this station by macrophytes, mainly eichhornia crassipes, contributed to the reduction of pollution (kone, 2002). in the discharge station of sucob where pollution is high, the main source of organic pollution is therefore the sewage from the sugar production plant. as for the other two sites (bonou and agonlin-lowé) with strong organic pollution, they are in downstream of the basin, thus presenting an accumulation of the pollutant load from upstream. these are also areas with significant urbanization. the direct anthropogenic impact is therefore important. the station of kaboua for the reasons mentioned above and the station of vossa have a very high organic pollution. anthropization and agriculture are the two sources of organic matter in the station of vossa. ecological spectrum of species: the distribution spectrum of the abundance of the two species in the three statuses of organic pollution observed shows an increase in both species with the increase in pollution (fig. 3). for each of the two species, the density in the moderate pollution status was significantly smaller than in the other two status (p<0.05). a significant correlation (p<0.05) is observed between the ipo and the abundance of the two species (table 4). the spearman correlation was -0.676 for b. falcatus and 0.707 for p. patulus. brachionidae rotifers are known to be strongly linked to water eutrophication (serafimjunior et al., 2010; mola, 2011). the phenomenon of eutrophication is a strong biological multiplication thanks to an enrichment of water in organic and mineral matter. thus the profile of the two species of brachionidae (b. falcatus and p. patulus) in the present study shows that they are strongly related to eutrophication. since organic pollution is the main source of eutrophication parameters, the two species perfectly indicate the organic load in the basin of the ouémé basin. table 3. organic pollution level of studied sites. stations parameter’s classes ipo pollution level nh4 + no2 po4 3 agonlin-lowé 2 3 2 2,33 strong bonou 3 3 2 2,67 strong zagnanado 4 3 3 3,33 moderate toué 3 3 3 3,00 moderate atchérigbé 4 3 3 3,33 moderate dassa 4 3 3 3,33 moderate atchapka-bethel 3 3 3 3,00 moderate atchakpa-rejet 2 2 2 2,00 strong atchakpa-pompage 4 2 3 3,00 moderate kaboua 2 1 1 1,33 very strong vossa 3 1 1 1,67 very strong kpassa 4 3 3 3,33 moderate bétérou 4 3 3 3,33 moderate affon 3 3 3 3,00 moderate donga 3 3 3 3,00 moderate table 4. spearman’s ranks correlation between species and ipo value. species ipo brachionus falcatus -0,676* platyias patulus -0,707* 263 int. j. aquat. biol. (2018) 6(5): 258-264 conclusion rotifers b. falcatus and p. patulus both from the family of brachionidae have a profile strongly related to organic pollution in the ouémé river basin. their maximum density is associated with the very strong organic pollution in the sites of kaboua and vossa. the two species are therefore interesting bioindicators of organic pollution in the ouémé river basin. acknowledgements we are grateful to the west africa agricultural productivity program (waapp) which founded this research. we thank all people who have contributed on any way to the effectiveness of this study. we also thank any reviewer who contributed to the scientific level of the paper. references abai e.a., ombolo a., ngassoum m.b., mbawala a. (2014). suivi de la qualité physico-chimique et bactériologique des eaux des cours d’eau de ngaoundéré, au cameroun. afrique science: revue internationale des sciences et technologie, 10(4): 135145. ahlstrom e.h. 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(2022) 10(3): 209-217 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology short communication structure and health status of the sand crab, emerita taiwanesis hsueh, 2015 from sangchan beach, thailand: the histopathological approach sinlapachai senarat*1, natthakitt to-orn2, chanyut sudtongkong1, gen kaneko3, natthawut charoenphon4, somrudee meprasert jitpraphai5, jes kettratad5 1department of marine science and environment, faculty of science and fisheries technology, rajamangala university of technology srivijaya, trang campus, trang, 92150, thailand. 2program of fisheries science, faculty of agricultural technology and agro-industry, rajamangala university of technology suvarnabhumi, ayutthaya 13000, thailand. 3college of natural and applied science, university of houston-victoria, victoria, texas 77901, usa. 4department of anatomy, faculty of medical science, naresuan university, 65000 thailand. 5department of marine science, faculty of science, chulalongkorn university, bangkok 10330, thailand. s article history: received 11 august 2021 accepted 4 april 2022 available online 2 5 june 2022 keywords: bioindicator sand crab gill coastal health histopathology abstract: although the impacts of environmental problems on aquatic organisms have been broadly reported in thailand, the literature has not covered the sand crab, emerita taiwanesis hsueh, 2015. in this study, we focused on the structure and health status of e. taiwanesis, an economically important crab species, living close to human activity areas in sangchan beach, rayong province, thailand. a total of 60 individuals were collected from the conservation and restoration of coastal resource project in ban rue leg kao yod-based participatory during december 2016 – january 2017. we identified histopathological changes in the gill structure, but not in other vital organs, including ganglion, stomach, intestine, hepatopancreas and muscular bundles. the histological alterations in the gill include hematocyte infiltration, pyknotic nuclei and degeneration of pillar cells in the gill (50% prevalence), suggesting that the gill is a sensitive organ to environmental changes. our observation provided a better understanding of e. taiwanesis morphology and its overall healthy state on sangchan beach. additionally, we suggest that the sand crab would be a suitable sentinel species for monitoring the environment of coastal areas in thailand. introduction benthic invertebrates such as shrimps, crabs, mussels and sea stars have been documented as useful sentinel marker species in biomonitoring programs, which evaluate the health status of animal populations under a serious threat of environmental illness (viarengo, 1993; fowler et al., 2004; hodkinson and jackson, 2005; carew, pettigrove et al., 2013; el-gammal, al-madan and fita, 2016; munroe et al., 2018). these species have suitable characteristics, including small size, common prevalence, relatively sessile behavior, and a strong tendency to bioaccumulate pollutants from environments (lazorchak et al., 2002; chiarelli and roccheri, 2014). the histopathological alterations in these sentinel species can be quantitatively analyzed, enabling the fast and efficient assessment of the *correspondence: sinlapachai senarat doi: http//doi.org/10.22034/ijab.v10i3.1316 e-mail: sinlapachai.s@rmutsv.ac.th environment (moore et al., 1987; wedderburn et al., 2000). multiple fields and laboratory studies have associated the histopathological alteration with the presence of heavy metals and toxic contaminants, demonstrating increased health risks for aquatic organisms in the environment (sarojini et al., 1993; victor, 1994; soegianto et al., 1999a, b; bhavan and geraldine, 2000). the sand crab emerita taiwanensis (hippoidea) is a newly found species described by hsueh (2015). it is reported to live on sandy beaches with a grain size of 0.25-1.0 mm or brackish water environment in taiwan (hsueh, 2015). biaklai (2016) also reported the presence of e. taiwanensis in thailand as a dominant species in the y-shaped breakwater area on sangchan beach, located close to human communities and industrial areas. the anthropogenic 210 senarat et al./ structure and health status of the sand crab, emerita taiwanesis using histopathological approach activities negatively affect aquatic life in general (silarat et al., 2014; thai-tourism thailand, 2015). indeed, the male to the female sex ratio of e. taiwanensis in sangchan beach is 1: 1.24, and fecundity ranges from 144-1,293 eggs, indicating the presence of reproductive problems (biaklai, 2016). the gametogenic maturation and embryonic development of e. taiwanensis have been explored (senarat et al., 2018), but the overall health status of the sand crab is still poorly known. in the present study, we investigated the structure and assessed the health status of e. taiwanensis as a sentinel species in sangchan beach, thailand. the histopathological methodology was used for the assessment of health status. materials and methods sand crab collection: the dead e. taiwanensis samples with a mean carapace width of 4-5 cm were collected from december 2016 january 2017 from the conservation and restoration of coastal resource project in ban rue leg kao yod-based participatory, sangchan beach, rayong province, thailand (12°39'46''n, 101°14'50''e). these samples have been deposited as voucher specimens from the work of biaklai (2016), and we used a total of 60 fixed samples of them. species identification was performed according to the decapod taxonomic studies of boyko and harvey (1999) and hsueh et al. (2015). morphology and histological techniques: the morphology was observed externally and internally under a stereomicroscope (sz760b2l). the samples were then processed with standard guidelines of histological techniques (presnell and schreibman, 1997). four-micron-thick sections of each block were stained with harris’s hematoxylin and eosin (h&e) and cresyl violet (cv) (presnell and schreibman, 1997; suvarna et al., 2013). structural and histopathological features were microscopically evaluated and photographed using a leica digital 750 light microscope. each histopathological alteration on each organ was assessed as percent prevalence (%). results and discussion the examinations of histological sections showed that the central nervous system (cns), digestive system (ds), and muscular system (ms) did not have any apparent histopathological lesions (figs. 1-4). on the contrary, the respiratory system (rs) had several histopathological alterations (fig. 5). the cns of the sand crab was comprised of the brain (supraesophageal ganglion) located in the head region (fig. 1a-b) and the ventral nerve cord (vnc) (fig. 1a-f) as seen in other crabs (sandeman et al., 1992; saetan et al., 2013). this system is involved in the endocrine regulation of the growth and reproduction of the crustaceans (diwan, 2005; ramachadra, 2018). stalked compound eyes were present in the anterior part of the head (fig. 1a, g). the optic nerve connecting the eyes to the brain was observed (fig. 1g). longitudinal sections showed that the eye’s surface is organized by a layer of facet lenses. a wide crystalline cone layer was visible beneath the lens, and the layer of retinal cells was arranged throughout the rhabdom layer. the pigment cells were widely extended between the crystalline cones and retinal cells (fig. 1h). each part of the brain (fig. 1c-d) and ganglion (fig. 1e-f) contained the neuropils (or supporting cells) associated with neuronal cell clusters. a compact arrangement of numerous neurons was observed in the brain, and each cell had a prominent nucleus surrounded by the basophilic nucleoplasm (fig. 1e-f). the secretory granules were scattered throughout the cytoplasm, which is referred to nissal bodies (also called nissl substances or nissl materials) (fig. 1f). a previous observation showed that the neurons produce the gnrh-like peptide to control reproductive activity (saetan et al., 2013). the neuropil contained a single nucleus, which was covered with homogeneously deep basophilic cytoplasm (fig. 1d-f). the ds of e. taiwanesis consisted of the foregut, midgut and hindgut. the major function of this 211 int. j. aquat. biol. (2022) 10(3): 1-217 system includes the ingestion, transit of nutriments and mechanical digestion (ceccaldi, 1989). in the cardiac stomach, various folds projecting into the lumen were observed (fig. 2a-b). the epithelium of figure 1. morphology and light microscope of the central nervous system (cns) and eye structure (ey) of emerita taiwanesis a: head (he) connected to compound eyes. b-d: the brain (br) contained neuropils (ne) and clusters of neurons (nu). neuronal fibers (nf) were also observed. e-f: a representative ganglion in the brain. ganglions are contained in both neuropils (ne) and the cluster of neurons (nu). the nissal bodies (asterisk) were seen in the neucleoplasm. each ganglion was connected to the nerve tract with neuronal fibers (nf). g: longitudinal section showing the eye (ey) connected to the optic stalk (os). h: the detailed structure of the eye is composed of muti-cellular layers including a layer of facet lenses (fc), crystalline cone layer (cc), retinula cell (rc), and rhabdom layer (rh). the pigment cells (pc) were broadly scatted among retinula cells and the rhabdom layer. b, c, e = harris’s hematoxylin and eosin (h&e), d, f, g-h = cresyl violet (cv). 212 senarat et al./ structure and health status of the sand crab, emerita taiwanesis using histopathological approach figure 2. light microscope showing the structure of stomach and intestine of emerita taiwanesis. a: the representative histology of cardiac stomach (cs) and the lateral teeth (lt). b: high magnification showing longitudinal folds projecting into the lumen that was lined by cuticle (cu) and a simple culumnar epithelium (em). the gastric mill teeth (gm) were also observed. c: high magnification of lateral teeth (lt) containing the thick epithelium (ep). d-e: deep longitudinal fold (lf) of intestine (in) lined with the simple columnar epithelium (ep) and tegumental glands (tg). the thin muscular layer (ml) surrounded of the intestinal wall. 213 int. j. aquat. biol. (2022) 10(3): 1-217 the mucosal layer in the cardiac stomach was lined with a simple columnar epithelium and covered by a thin cuticle (fig. 2b). the ossicle system was composed of gastric mill teeth (fig. 2b) that comprised two lateral teeth (lt) (fig. 2a), the thickened cuticle and epithelium of the teeth (fig. 2c) in line with the observation of other crustaceans (jantrarotai et al., 2005; lumasag et al., 2007; melo et al., 2006). the gastric mill teeth are considered to help the digestion of the hard-shelled prey (anger, 2001). meanwhile, the intestine was a tube with deep longitudinal folds (fig. 2d-e) lined by a simple columnar epithelium (fig. 2f). numerous tegumental glands were scattered throughout the intestinal epithelium (fig. 2d-e). it was also covered by a thin muscular layer (fig. 2d-e). the hepatopancreas (or the digestive gland) of e. taiwanesis was morphologically visible as yellowish-brown tissue within the cephalothoracic cavity (fig. 3a). this organ has a key role in metabolism and xenobiotic detoxification in crustaceans (johnston et al., 1998; sousa and petriella, 2001). it is constituted by a great number of oval or circular acini (tubules) (fig. 3b-d). each acinus was covered with simple epithelial cells, which were prominently separated from the neighboring ones by a thin sheet of connective tissue (fig. 2b). the cross-sectional observation found that the acinus lumen has a star-like shape (fig. 2b). the acinus was classified into four main cell types (e, r, b, and f cells) (fig. 3b-d) based on the detailed features reported in other crustaceans (ceccaldi, 1989; maharajan et al., 2015). the embryonic or embryonalzellen cell (e-cell) was the first cell close to the basement membrane (fig. 3b). a round to oval nucleus was found in the middle region of the cytoplasm of this cell. restzellen cell (r-cell) was a tall columnar cell with an apical microvillar border figure 3. morphology and light microscope showing the hepatopancreas of emerita taiwanesis. a: the yellowish-brown hepatopancreas (hp) was observed in the cephalothoracic cavity. b-c: circular acini (ac) were separated from neighboring structures by a thin sheet of connective tissue (cnt). the lumen (lm) of the acinus had a star-like shape. the epithelium of this organ was composed of four main types of cells, including embryonalzellen cell (e-cell), restzellen cell (r-cell), blasenzellen cell (b-cell), and fibrillenyellen (f-cell). abbreviations: arterisk = an apical microvillar border, vc = vacuoles. 214 senarat et al./ structure and health status of the sand crab, emerita taiwanesis using histopathological approach (fig. 3c) with a basal nucleus (fig. 3c). the numerous small lipid vacuoles were easily found in this cell (fig. 3c). blasenzellen cell (b-cell) was the large cell containing a giant, single secretory granule (fig. 3c). in addition, a spindle-shaped fibrillenyellen cell (f-cell) was identified between b-cells and f-cells (fig. 3c). the nucleus was centrally located in this cell. the cytoplasm of this cell had a non-vacuolated structure (fig. 3c-d). the muscular bundles were widely scattered along the body (fig. 4a) and tightly packed as the muscle segments known as “myomeres” (fig. 4b). it was formed by striated muscle and mainly contained skeletal muscle fibers. each myofibril comprises several myofilaments. the presence of multinucleate cells was observed in the skeletal muscle fibers, where the flattened nuclei were seen in the periphery in parallel to each other for the whole length of the fiber (fig. 4b). the gills of e. taiwanesis were organized along the body (fig. 4a) with several normal lamellae structures (fig. 5a). it could be classified into primary and secondary lamellae (fig. 5a). the surface of the secondary lamella was covered with a thin layer of the cuticle (fig. 5b). irregular intervals of pillar cells were observed in the primary lamellae parallel to the surface (fig. 5b). the secondary lamella had uniform interlamellar and normal haemocoelic spaces with an optimum number of haemocytes (fig. 5b). however, a few types of histopathological alterations were present: the hemocytes infiltration with 10 percent prevalence (fig. 5c) and degeneration of hemocytes and both pyknotic nuclei and degeneration of pillar cells (50% prevalence) (fig. 5c-d). the gills are the primary respiratory organ in crabs, which are also responsible for many physiological functions such as excretion of nitrogenous wastes, regulation of acid-base balance and ion regulation (wilkens, 1981; redmond, 1995). the alteration of gill structure is related to the functional impairment of homeostasis often caused by environmental pollutants (alazemi et al., 1996; kumar and tembhre, 2010). the observed histopathologies in gills are minor and can normally be found in healthy e. taiwanesis. however, these lesions might be the defense responses to some pollutants such as nickel (abraham and radhakrushnanm 2002), a combination of chlorpyrifos and cypermethrin (maharajan et al., 2015) and industrial effluent environment (jerome and chukuka, 2016). conclusions although sangchan beach, rayong province, thailand, is a major human activity area, many figure 4. light microscope images showing the muscular buddles of emerita taiwanesis. a: the muscle (m) was parallel to the gill structure (gi). b: high magnification showing the tightly packed myomeres (my) that formed the striated muscle. the presence of multi-nucleate cells (cycles) of the skeletal muscle fibers (asterisks) was observed. 215 int. j. aquat. biol. 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(2022) 10(2): 131-144 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology review article qanat system, an ancient water management system in iran: history, architectural design and fish diversity ghazal esmaeili1, amin habibi1, hamid reza esmaeili2 1department of architecture, faculty of art and architecture, shiraz university, shiraz, iran. 2ichthyology and molecular systematics research laboratory, zoology section, department of biology, school of science, shiraz university, shiraz, iran. s article history: received 19 january 2022 accepted 5 march 2022 available online 2 5 april 2022 keywords: ancient iran kariz, water-related technology habitat suitability and biodiversity al-azim marsh abstract: ancient iran is one of the leading civilizations that actively appear to water resources management, especially by the invention of “qanat”, an artificial underground system/ subterranean tunnel-wells system where the water flows through gravity on a slight slope in arid and semi-arid regions at least 5000 years ago. qanats were innovated in ancient iran, spread throughout much of the middle east, and extended into north africa, spain, italy, and south asia. tools preparation, size selection, digging the first and deepest vertical shaft known as “mother-well”, digging several other vertical shafts along a line between the mother-well and qanat outlet, and constructing a horizontal connection between vertical shafts (known as the main tunnel), which guides the water out through an outlet, are the main steps in qanat construction. by this innovation, iranian solved their water-related problems using the basic concepts of hydraulics. in the same way, water-related infrastructures were built using locally available materials to make a better life for humans and other wonderful well-designed and well-adapted organisms in dry and semi-dry regions, yielding great civilization with a simple, but a fantastic architecture that provides cold water in hot summer and warm water in cold winter. by means of these ancient underground structures, water was funneled from mountainous areas and aquifers to lower lands and thus alluvial fans could be opened up to settlement, and an agrarian civilization developed and evolved. in addition, qanat provides a continual flow suitable for many aquatic organisms, including crabs, amphipods (gamarids), freshwater shrimps, and fishes. qantas are home and refugia to about 42 fish species (36 native and 6 exotic species) belonging to 20 genera, 7 families, and 2 orders. the qanat ichthyofauna is dominated by cyprinidae with 19 species (45.2 %) followed by leuciscidae and nemacheilidae (6 species, 14.28% each), poeciliidae and aphaniidae (4 species, 9.52%), and cobitidae (1 species 2.38%). it is about 14% of the total ichthyofauna of iran. the qanat ichthyofauna comprises 36 natives (including 20 endemics) and 6 exotic species. qanat fauna dominates by species that are generally of small size, are broadcast spawners, nonmigratory, and have a wide tolerance of environmental conditions. introduction water is one of the most important components in nature and daily life. the study of this very common substance is a subject of many different disciplines (berking et al., 2019), including engineering, mathematics, art, agriculture, biology, and health. water is crucial for the life support of all organisms and health promotion, food security, social development, economic improvement, political stability, and environmental function and sustainability. in terms of function and purpose, water  correspondence: hamid reza esmaeili e-mail: hresmaeili@shirazu.ac.ir is used as freshwater (domestic, drinking, tap, and portable water); for food production (water for irrigation and animal husbandry); fishery; navigation (transport); cult practices; energy (hydropower); status (political power); hygiene; and for entertainment, protection, cooling, and recreation (berking et al., 2019). especially in arid and waterscarce regions, water management is a fundamental need for humans and societies. in arid areas like iran, water resources management for sustainable development is far more noteworthy. 132 esmaeili et al./ qanat system, an ancient water management system in iran to warrant proper water resources development, establishing a rational connection between the available resources (surface water and groundwater), spatiotemporal requirements, geo-climatic conditions, cultural values, legal rights, technological tools, political power, religious beliefs, and socioeconomic privileges is a critical issue (berking et al., 2019; mahan et al., 2019; saatsaz and rezaie, 2021). in this regard, ancient iran is one of the leading civilizations that actively appear to water resources management, administration, and investment in infrastructure and technology, not merely water availability. although there has been a long history of water governance in iran, it has difficulty solving current water problems. in this context, understanding how water-related technology, rules, society, economics, and political systems worked in the past provides us with more profound knowledge of how our history shaped the present, allowing us to form our future (mahan et al., 2019; saatsaz and rezaie, 2021). several historical studies have investigated ancient water-related topics of the world and iran (e.g., esmaeili and teimori, 2006; kuros and labbaf khaneiki, 2007; mays, 2010; mahmoudian and mahmoudian, 2012; gholikandi et al., 2013; moteahed et al., 2014; esmaeili et al., 2015; hashemzadeh segherloo, 2015; alemohammad and gharari, 2017; sparacio et al., 2017; manual et al., 2018; berking et al., 2019; labbaf khaneiki, 2019; mahan et al., 2019; saatsaz and rezaie, 2019; teimori and esmaeili, 2020). most of these investigations focus on (i) water infrastructure such as qanats, dams, weirs, water-mills, (ii) water resources management, (iii) socioeconomic aspects of water, (iv) architectural design of water-related systems, and (v) habitat suitability and biodiversity. one of the ancient water management systems is qanat. qanat is one of the important inland aquatic ecosystems commonly containing freshwater in arid and semi-arid regions. it is an underground tunnel, almost horizontal, which bores into an aquifer and guides the water out through an outlet. qanat provides a continual flow suitable for many aquatic organisms, including crabs, amphipods (gamarids), freshwater shrimps and fishes (esmaeili and temori, 2006; esmaeili et al., 2012, 2015, 2018; hashemzadeh segherloo, 2015; sparacio et al., 2017; teimori and esmaeili, 2020). based on the above-mentioned background, this study was conducted to (i) review the history of qanat formation in iran, (ii) characterize the architectural design of qanat, and (iii) update qanat fish biodiversity. materials and methods for the history and architectural design of qanat several studies have been consulted (e.g., kuros and labbaf khaneiki, 2007; mays, 2010; mahmoudian and mahmoudian, 2012; gholikandi et al., 2013; salehi moteahed et al., 2014; alemohammad and gharari, 2017; sparacio et al., 2017; manual et al., 2018; berking et al., 2019; labbaf khaneiki, 2019; mahan et al., 2019; saatsaz and rezaie, 2019). the data collected for the fish diversity are based on the previous works done by saadati (1977), armantrout (1980), coad (1995, 1996, 2006), esmaeili and temori (2006), rezaei tavabe and azarnivand (2013), esmaeili et al. (2010a, b; 2015, 2017, 2018), teimori and esmaeili (2020) and several field trips. results and discussion qanat definition: qanat (also known as karez in afghanistan and pakistan, kanerjing in china, falaj in the arabian peninsula, qanat romani in jordan and syria, fogarra (fughara) in north africa, khettara in morocco, and galeria in spain), is an artificial underground system/ subterranean tunnel-wells where the water flows through gravity on a slight slope in the arid lands of the old world/eurasia (fig. 1). in fact, qanats are gently sloping tunnels dug far enough into alluvium or water-bearing sedimentary rock for extracting groundwater (english, 1968; waterhistory, 2022). once constructed, groundwater filters into the channel, run down its gentle slope, and emerges at the surface as a stream (english, 1968). history of qanat formation: ancient water mechanisms changed and evolved in nature as “one species turns into another”, due to geographical advantages or disadvantages, analogous to “natural 133 int. j. aquat. biol. (2022) 10(2): 131-144 selection”, as described by charles darwin (1809– 1882), but driven deliberately by the people of that time. especially in arid and water-scarce regions such as the iranian plateau, water management has been a fundamental need for humans. such evolution and changes in water management might have been coupled with the dispersal of modern humans to the iranian plateau. probably, living on the iranian plateau started with the dispersal of early modern humans from africa, dated between at least ∼90,000 and ∼50,000 years ago in the middle-paleolithic of the stone age (delson, 2019; saatsaz and rezaie, 2019). later, agricultural communities started to appear in the southwestern, western, and northwestern of the iranian plateau where perennial rivers, rainfall, and fertile alluvial soils allowed agrarian societies to develop (riehl et al., 2013; saatsaz and rezaie, 2019). this was followed by animal domestication, construction, stone cutting, woodworking, mining, metalworking, trading, and other services (saatsaz and figure 1. a simple schematic showing a typical qanat system; (a) cross-section, (b) aerial view. 1, mother well; 2, vertical shaft; 3, tunnel (gallery); 4, qanat outlet; 5, storage pond; 6, field canal (saatsaz and rezaie, 2021). figure 2. one possibility for the diffusion of qanat technology. modified from waterhistory (2022). 134 esmaeili et al./ qanat system, an ancient water management system in iran rezaie, 2019). almost 6,000 years ago, the southwestern and western parts of the iranian plateau were a part of the "fertile crescent” under the control of the mesopotamian civilization (pollock and susan 1999). in this region, the low gradient meandering karun, karkheh, jarrahi, and dez rivers flow in vast floodplains, underlain by thick alluvial deposits. meander cut-offs (oxbow lakes), marshes, and abandoned streams are developed alongside these rivers. this area, however, is susceptible to low, erratic rainfall and drought; only irrigated agriculture was feasible. hence, to meet full irrigation, the mesopotamians developed a complex of water systems, including canals with different sizes (one central canal and a network of secondary, tertiary, quaternary, and field canals), head-gate, distributors, regulators, inlets and outlets, weirs, levees, and storage reservoirs (tamburrino, 2010). birth of qanat: the exact dating of qanats is difficult unless their construction was accompanied by documentation or, occasionally, by inscriptions (waterhistory, 2022). most of the evidence we have for the age of qanats is circumstantial; a result of their association with the ceramics or ruins of ancient sites whose chronologies have been established through archeological investigation or the qanat technology was introduced long ago by people whose temporal pattern of diffusion is known (waterhistory, 2022). based on different written records, the qanat system has a persian origin (biancone and tusa, 1997; todaro, 2002; de feo et al., 2010; sparacio et al., 2017; waterhistory, 2022). in fact, written records leave little doubt that ancient iran (persia) was the birthplace of the qanat. as early as the 7th century bc, the assyrian king sargon ii reported that he had found an underground system for tapping water during a campaign in persia. his son, king sennacherib, applied the "secret" of using underground conduits in building an irrigation system around nineveh (waterhistory, 2022). diffusion of qanat technology: during 550-331 bc, when persian rule extended from the indus to the nile, qanat knowledge spread throughout the empire (fig. 2). the achaemenid rulers provided a major incentive for qanat builders and their heirs by allowing them to retain profits from newly-constructed qanats for five generations. as a result, thousands of new settlements were established and others expanded (english, 1968; waterhistory, 2022). to the west, qanats were constructed from mesopotamia to the shores of the mediterranean, and southward into parts of egypt. to the east of persia, qanats were constructed in afghanistan, the silk route oases settlements of central asia, and chinese turkistan i.e. turpan (waterhistory, 2022). during roman-byzantine era (64 bc to 660 ad), many qanats were constructed in syria and jordan. from here, the technology appears to have diffused north and west into europe. there is evidence of roman qanats as far away as the luxembourg area (see sparacio et al., 2017; waterhistory, 2022). architectural design of qanat: in the early part of the first millennium b.c., persians started constructing this elaborate tunnel system to extrac groundwater in the dry mountain basins of present-day iran. many researchers have discussed the architectural design of qanat (e.g., english, 1968; karki et al., 2017; saatsaz and rezaie, 2019; waterhistory, 2022). a review is provided here under several headings: specialist qanat diggers: most qanats in iran are constructed by a group of professional/ specialist diggers known as muqannis. they inherited this task from the slaves and captives of the achaemenid and sassanian kings (english, 1968; waterhistory, 2022). these diggers form a community of traveling artisans, migrating from place to place as floods destroy qanats in one area or a lowered water table demands that qanat tunnels be extended in another (english, 1968; waterhistory, 2022). the techniques of qanat construction have changed little from birth to the present day (fig. 3). the muqannis must work with water flowing around them, ventilation is poor, and the chances of cave-ins are great. tools for qanat construction: the tools for qanat construction are primitive and include a broad-bladed pick, a shovel, a windlass, a long rope, a small lamp and bucket (fig. 3). 135 int. j. aquat. biol. (2022) 10(2): 131-144 site selection: site selection is the first and critical step in the construction of a qanat. local slope conditions, landscape, anomalies in soil color and moisture, ground-water supplies, vegetation cover and the proposed location of the new settlement are conventional indicators to locate a qanat construction point. ancients likely knew groundwater could be available in foothills, wadies, dry riverbeds, and alluvial fans (english, 1968; saatsaz and rezaie, 2019; waterhistory, 2022). these factors are weighed by an expert, usually one of the older, more wellknown muqannis, who chooses where a trial well figure 3. constructing a qanat using reinforcing rings. modified from waterhistory (2022). figure 4. 3d cross-section of a typical qanat (estaji and raith, 2016). 136 esmaeili et al./ qanat system, an ancient water management system in iran should be dug. a favorable site often lies near the mouth of a wadi, but where the water table is deep and the qanat long, the general topographic setting and variations in vegetation are used as indices of the likely location of underground water supplies (english, 1968). figure 5. number and percentage of different fish families in qanats of iran. figure 6. map of iran showing major drainage basins of iran. cyprinidae 19 (45%) gobionidae 2 (5%) leuciscidae 6 (14%) nemacheilidae 6 (14%) aphaniidae 4 (10%) poeciliidae 4 (10%) cobitidae 1 (2%) 137 int. j. aquat. biol. (2022) 10(2): 131-144 after viewing evidence, the first and deepest vertical shaft known as “mother-well” is dug by a team of skilled muqannis using only simple hand tools. the mother well is sunk into the saturated zone for locating the water table and checking the quality, quantity, and regularity of the groundwater flow (kheirabadi, 2000). in this stage, the aesthetic parameters of water (i.e., temperature, turbidity, color, taste, and odor) are detected by qanat diggers through the senses. the sense of hearing is occasionally used to detect water movement. the length of the qanat is measured from the mother well to the point where water surfaces. the qanat length could also be short or long, varying from a few hundred meters to ∼100 km. the depth of the mother well may vary from ten to several hundred meters usually 30-100 meters based on the water table depth, qanat length, earth slope, and the owner’s capital for excavation (english, 1968; karki et al., 2017; saatsaz and rezaie, 2019; waterhistory, 2022). the qanat length and the mother-well depth are often high in dry regions. in qanat design, the slope is one of the most critical parameters that control a qanat length. in the next step (figs. 1, 3, 4), along a line between the mother-well and qanat outlet, the muqannis dig vertical shafts with a diameter ranging from 80 to 100 cm (semsar yazdi and labbaf khaneiki, 2016). at intervals of ∼20 to 200 m, the shafts are used to remove the excavated materials from the main tunnel (a connection between vertical shafts), air circulation and provide access for repair and maintenance. the gently sloping main tunnel transports water from groundwater wells to the surface some distance away. during the digging process, excavated soils are dumped all-over the shaft opening to prevent entering surface runoff into the shaft and the main tunnel. if the soil is loose and unstable, the tunnel and shaft lining is necessary to improve the qanat durability. if the soil is firm, no lining is required for the main tunnel. in loose soil, reinforcing rings are installed at intervals in the tunnel to prevent cave-ins. these rings are usually made of burnt clay. mineral, salt, and other deposits which accumulate in the tunnel bed need periodic cleaning and maintenance work. architecture and social aspects of qanats: the qanat gadget changed into concerned with the social systems of nearby groups and cities (english 1966; bonine 1982). for instance, wealthier households often had non-public and direct get entry to water networks, or they lived toward the opening or upstream of it. at the identical time, the relaxation of a metropolis`s population used the collective and public offerings to be had in every mahalleh which may encompass that neighborhood`s water storage. sharing and keeping the not unusual place primary sources helped shape near social relationships inside a neighborhood. furthermore, the principal public establishments together with the bazaar, mosques, baths, and faculties commonly had direct get entry to freshwater. this method that the financial shape of the metropolis had a near courting with the qanats and the water community in general. in short, a multiplicity of interwoven elements has formed how human beings assemble and keep qanats throughout the records in their use, growing precise methods of residing and inhabiting the territory. the use of qanat water became observed via way of means of a collective agency of manufacturing control, in the location around tehran, the typical qanat primarily based totally collective device is known as boneh. the volume of improvement of the social and technical agency relies upon manufacturing boneh advanced out of the want for cooperation in building and keeping qanat and its water control prolonged to cowl agriculture manufacturing and associated socioeconomic device. a typical quantity of irrigation is cultivated on all bonehs, and normal collective water divisions primarily based totally on crop desires are made among the bonehs. within the collective device, a social hierarchy defines roles and duties. number of qanats: it seems that about 120,000 qanats have been constructed in iran, of which about 37,000 are still in use in arid and semi-arid regions of the country, producing some 7 billion cubic metres of groundwater and forming around 11% of the aquifer discharge which is annually mined across the country (estaji and raith, 2016). 138 esmaeili et al./ qanat system, an ancient water management system in iran persian qanats in the unesco world heritage list: at its 40th meeting in istanbul, turkey on july 10, 2016, the world heritage committee (whc) listed eleven iranian qanats as the 20th in the list of iran’s unesco world heritage site due to the historical importance of iran’s aqueducts, their sophisticated and civilized systems, and their environmental benefits. these registered qanats are qassabah (in gonabad), baladeh (in ferdows), zarch (in hassan abad), mirza nasrollah water mill (in mehriz), juppar (in kerman), akbarabad and qasemabad (in bam), moon (in ardestan), wezwan and mazdabad (in isfahan) and ibrahim abad (in arak). qanat fish diversity: the data collected from different sources, including several field works, figure 7. live specimen of capoeta aculeata, iran: lake namak basin. figure 8. live specimen of capoeta fusca, iran: sharifabad qanat, birjand. figure 9. live specimen of capoeta macrolepis, iran: kor river basin. 139 int. j. aquat. biol. (2022) 10(2): 131-144 showed that there are 42 fish species (fig. 5) in qanats of iran in several basins (fig. 6) belonging to 20 genera, 7 families, and 2 orders. the qanat fish species are listed in table 1 and a selection of species, showing variation in body form, is given in figures 714, and a representative fish habitat is given in figures 15-16. the qanat ichthyofauna is dominated by cyprinidae with 19 species (45.2%) followed by figure 11. paraschistura cristata, iran: zanglanlou river. figure 12. male (upper) and female (lower) aphanius pluristriatus, iran: jahrom, qanat, mond river drainage. figure 10. paraschistura turcmenica, zm-cbsu j2929, 36 mm sl; iran: khiyaban qanat. 140 esmaeili et al./ qanat system, an ancient water management system in iran leuciscidae and nemacheilidae (6 species, 14.28% each), poeciliidae and aphaniidae (4 species, 9.52%), and cobitidae (1 species 2.38%). it is about 14% of the total ichthyofauna of iran. the qanat ichthyofauna comprises 36 natives (including 20 endemics) and 6 exotic species. qanat fauna dominates by species that figure 13. live specimen of garra nudiventris, zm-cbsu h1500, 54.5mm sl; iran: kalat e baba qanat. figure 14. alburnoides qanati, iran: moshkan qanat, kor river basin. figure 15. kalat e baba qanat. habitat of garra nudiventris. 141 int. j. aquat. biol. (2022) 10(2): 131-144 are generally of small size, are broadcast spawners, non-migratory, and have a wide tolerance of environmental conditions (coad, 1996). conclusion qanat is one of the ancient water management systems and is one of the important inland aquatic ecosystems order family species status qanat/basin cypriniformes cyprinidae barbus miliaris de filippi, 1863 e namak capoeta aculeata (valenciennes, 1844)* n kavir, namak capoeta birunii zareian & esmaeili, 2017* e zayandehrud capoeta buhsei kessler, 1877 e namak, kavir capoeta capoeta (güldenstädt, 1773) n urmia capoeta ferdowsii jouladeh-roudbar, eagderi, murillo-ramos, ghanavi, doadrio, 2017 e zohreh capoeta fusca nikolskii, 1897 n sistan,tedzhen, bedjestan, lut capoeta macrolepis (heckel, 1847)* n kor capoeta saadii (heckel, 1847) e kor, maharlu, makran, persis, kerman–na’in, sirjan, lut, and hamun-e jaz murian carasobarbus luteus (heckel, 1843) n hormuz, persis carassius auratus (linnaeus, 1758) ex kor, maharlu cyprinion microphthalmum (day, 1880) n lut, mashkid, hormuz, jazmurian, kavir cyprinion tenuiradius heckel, 1847 e persis garra nudiventris (berg, 1905) e lut garra persica berg, 1913 e hamun-e jaz murian garra rossica (nikol’skii, 1900) n hari, bejestan, sistan, lut, hamun-e jaz murian garra rufa (heckel, 1843) n persis schizothorax pelzami kessler, 1870 n hari, kavir tariqilabeo adiscus (annandale, 1919) n sistan gobionidae gobio nigrescens (keyserling, 1861) n hari pseudorasbora parva (temminck & schlegel, 1846) ex kavir leuciscidae acanthobrama persidis (coad, 1981) e kor, persis alburnoides idignensis bogutskaya & coad, 2009 e tigris alburnoides namaki bogutskaya & coad, 2009 e namak, kavir alburnoides qanati coad & bogutskaya, 2009 e kor, sirjan alburnus sellal heckel, 1843 n hormuz, persis, kor, maharlu alburnus ulanus (günther, 1899) e urmia nemacheilidae oxynoemacheilus persa (heckel, 1847) n persis paraschistura abdolii freyhof, sayyadzadeh, esmaeili & geiger, 2015 e sirjan paraschistura bampurensis (nikolskii, 1900) n hamun-e jaz murian, mashkid paraschistura cristata (berg, 1898) n hari paraschistura naumanni freyhof, sayyadzadeh, esmaeili & geiger, 2015 e persis, maharlu paraschistura turcmenica (berg, 1932) n hari, kavir cyprinodontiformes aphaniidae aphanius farsicus teimori, esmaeili & reichenbacher, 2011 e maharlu aphanius pluristriatus (jenkins, 1910) e persis aphanius isfahanensis hrbek, keivany & coad, 2006 e zayandehrud aphanius sophiae (heckel, 1847) e kor, sirjan poeciliidae gambusia holbrooki girard, 1859 ex widely distributed poecilia latipinna (lesueur, 1821) ex namak poecilia reticulata peters, 1859 ex namak xiphophorus hellerii heckel,1848 ex persis, namak table 1. fish biodiversity in qanats of iran. e, endemic; n, native; ex, ecotic. 142 esmaeili et al./ qanat system, an ancient water management system in iran commonly containing freshwater in arid and semi-arid regions and must have been started at least 5000 years ago in iran, working mainly based on the cooperation (in construction, maintenance, operation and technology transfer) which is, in fact, inherent in the technical traits and mechanism of qanat (fig. 17). this unique ecosystem with specific architectural design provides habitat to about 40 fish species, about 13% of the total natural fish fauna of the country, and many other aquatic organisms (e.g., invertebrates, amphibians, reptiles). at present, many factors threaten the qanat systems in iran. climate changes and increasing risks of desertification, overconsumption of freshwater resources due to population growth and introduction of new technologies, pollution, as well as inadequate policies have all contributed towards the degradation of the ingenious system of qanat construction and figure 16. abdolrahamati qanat, birjand, habitat of capoeta fusca. figure 17. aspects of cooperation in qanat, arrows do not denote causal relationships (modified from semsar yazdi and labbaf khaneiki, 2016). 143 int. j. aquat. biol. (2022) 10(2): 131-144 maintenance. traditional qanats systems are being replaced by pump-wells which are faster and easier to excavate but do not offer a fish habitat. pump wells often dry up qanats and natural springs by lowering the phreatic level. in ancient iran, water-related problems were solved by basic concepts of hydraulics. in the same way, water-related infrastructures were built using locally available materials to make a better life for humans and other wonderful well-designed and well-adapted organisms in dry and semi-dry regions, yielding great civilization with a simple, but a fantastic architecture that provides cold water in hot summer and warm water in cold winter. by means of these ancient underground structures, water was funneled from mountainous areas and aquifers to lower lands and thus alluvial fans could be opened up to settlement, and an agrarian civilization developed and evolved. acknowledgments we are thankful to shiraz university for its financial support. references alemohammad s.h., gharari s. 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(2015) 3(1): 25-27 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology short communication length-weight relationships of garra rufa, in the tigris and persian gulf basins of iran iraj hashemzadeh segherloo*1, seyedeh narjes tabatabaei2, aghil mansouri1, asghar abdoli3, mahdi ghalenoei3, kiavash golzarianpour4 1department of fisheries and environmental sciences, faculty of natural resources and earth sciences, shahr-e-kord university, shahr-e-kord, iran. 2fisheries department, natural resources faculty, university of tehran, karaj, iran. 3department of biodiversity and ecosystem management, environmental research institute, university of shahid beheshty, tehran, iran. 4faculty of sciences, gonbad kavous university, gonbad, iran. article history: received 3 november 2014 accepted 8 january 2015 available online 2 5 february 2015 keywords: tigris basin, persian gulf basin, length-weight relationship, morphometrics. abstract: garra rufa, a bottom dwelling freshwater fish and native to the middle east, is distributed in the southwestern iran and the tigris basin. considering the importance of length-weight relationships data of a species in different habitats, the length-weight relationship of g. rufa from 13 rivers in the persian gulf and the tigris basins was explored. the value of exponent b ranged from 2.74 to 3.19 with average of 2.99 in the tigris basin and 2.96 in the persian gulf basin which was in normal range (2.5-3.5). as the length-weight parameters were concluded for each location separately, this information would be useful for further population dynamics researches. introduction garra rufa, a bottom dwelling freshwater fish, belongs to the family cyprinidae and native to the middle east (jarvis, 2011). this species is distributed in the southwestern regions of iran in the tigris, persian gulf, lake maharlu, kor river and the hormuz basins (coad, 2013). a scale-less head, two pairs of barbels, an adhesive mental disc and cycloid scales are some characteristics of this species (jarvis, 2011). garra rufa feeds mostly on algae and diatoms (coad, 2013) and its maximum total length has been recorded at 13 cm in iran (esmaeili and ebrahimi, 2006). to estimate weight corresponding to a given length, growth rates, length and age structures, and other components of fish population dynamics, length and weight data are needed (cherif et al., 2008; froese, 2006). also, the length-weight data of a species in different habitats will be useful to compare life history and morphological aspects of populations * corresponding author: iraj hashemzadeh segherloo e-mail address: ihashem@nres.sku.ac.ir inhabiting different habitats (cherif et al., 2008). esmaeili and ebrahimi (2006) studied length-weight relationship of 291 specimens of g. rufa in iran but they did not mentioned anything about the localities and it remained unclear whether the specimens were from a single locality or different localities. therefore, due to the lack of length-weight relationship of different populations of g. rufa complex in iran, this study was conducted to explore the length-weight relationships in 13 populations of g. rufa collected from tigris and persian gulf basins. materials and methods the specimens were collected from 13 rivers in the persian gulf and tigris basins by electrofishing. the collected specimens were fixed in 4% formalin, after over-anaesthetization. the locality and sampling data are provided in table 1. in the laboratory each specimen measured to the nearest 1 mm (total 26 int. j. aquat. biol. (2015) 3(1): 25-27 length, tl) and weighted to the nearest 0.1 g (weight, w). the length-weight relationship of g. rufa was determined by the method of least squares using the equation w = αlb (keys, 1928) and logarithmically transformed into log w = log α + b log l (keys, 1928), where w is the weight of the fish in grams and l is the length of the fish in centimeters. results and discussion the number of specimens analyzed in each locality and the mean body length and weight are presented in table 1. the length-weight relationship parameters α, b, and r2 are denoted in table 2. the results revealed that there was a significant relationship (p<0.001) between length and weight for fishes in each river. the normal distribution of river coordinate s n tl min tl max tl m w min w max w m tange haft 32° 55' n 48° 45' e 27 50.70 95.96 68.26 ± 13.58 2.45 13.32 5.95 ± 3.34 beshar 30° 40' n 51° 32' e 21 54.82 101.62 75.21 1.97 13.98 5.78 ± 2.99 mazoo 32° 48' n 48° 24' e 19 58.92 123.36 88.96 ± 18.51 2.66 28.31 10.51 ± 6.70 palangan 35° 5' n 46° 37' e 51 63 178 137.1 ± 2.65 11.5 239 129.92 ± 60.15 sirvan 35° 14' n 46° 18' e 27 60 148 96.2 ± 1.9 3.6 57.8 10.85 kheirabad 30° 31' n 50° 27' e 70 50.74 124.84 82.32 ± 16.81 1.96 28.1 8.26 ± 5.07 gamasiab 34° 22' n 47° 55' e 10 52.51 105.74 72.11 ± 16.85 2.10 18.06 6.34 ± 5.55 ghalate 34° 30' n 47° 36' e 31 69.4 153.86 109.68 ± 25.97 4.46 55.85 21.93 ± 14.62 cheshme gerdab 33° 29' n 47° 57' e 12 75.96 169.58 122.37 ± 29.80 5.87 73.71 28.52 ± 20.90 maroon 30° 40' n 50° 18' e 12 53.04 100.16 73.50 ± 12.08 1.84 13.45 5.40 ± 3.01 dashte chenir 28° 43' n 51° 47' e 17 48.18 120.62 82.44 ± 18.20 1.53 23.74 8.22 ± 5.40 kheirak-shekarak 29° 14' n 51° 37' e 20 54.82 111.12 79.39 ± 18.18 1.91 15.78 6.10 ± 4.06 tange faryab 29° 13' n 51° 27' e 47 49.38 105.06 78.95 ± 11.32 1.64 14.15 6.11 ± 2.54 n: sample size; tl min: minimum total length; tl max: maximum total length; tl m: average total length; w min: minimum weight; w max: maximum weight; w m: average body weight. table 1. sampling sites, number of examined specimens, total length and weight data (min, max, mean ± standard deviation (sd)) of the studied populations of g. rufa. basin river b α r2 tigris tange haft 2.74 0.00005 0.95 beshar 2.86 0.00002 0.93 mazoo 2.99 0.00001 0.99 palangan 2.95 0.05 0.98 sirvan 3.00 0.01 0.97 kheirabad 2.96 0.00002 0.98 gamasiab 3.19 0.00001 0.98 ghalate 3.16 0.00001 0.99 cheshme gerdab 3.08 0.00001 0.99 persian gulf maroon 3.14 0.00001 0.97 dashte chenir 3.02 0.00001 0.99 kheirak-shekarak 2.82 0.00002 0.98 tange faryab 2.86 0.00002 0.98 table 1. the length-weight relationship parameters (a, b, and r2) for g. rufa in different rivers. 27 hashemzadeh segherloo et al./ length-weight relationships of garra rufa exponent b usually is 2.5-3.5 (froese, 2006), and for g. rufa in all rivers it was similar to the noted range. the average of parameter b was 2.99 in tigris basin and 2.96 in the persian gulf basin, and for all specimens, the overall value was 2.98 (2.74-3.19), this value is concordant with the b calculated by gerami et al., (2013) and esmaeili and ebrahimi (2006) for g. rufa in cholvar branch of the karun river, since there are similar values in some populations. this results can be useful for further research on g. rufa. refrences cherif m., zarrad r., gharbi h., missaout h., jarbout o. (2008). length-weight relationships for 11 fish species from the gulf of tunis (sw mediterranean sea, tunisia). pan-american journal of aquatic sciences, 3(1): 1-5. coad b. (2013). fresh water fishes of iran. available from: www.briancoad.com. retrieved 6/1/ 2013. esmaeili h.r., ebrahimi m. (2006). length-weight relationships of some freshwater fishes of iran. journal of applied ichthyology, 22: 328-329. froese r. (2006). cube law, condition factor and weightlength relationships: history, meta-analysis and recommendations. journal of applied ichthyology, 22: 241-253. jarvis p.l. (2011). biological synopsis of garra rufa. canadian manuscript report of fisheries and aquatic sciences 2946, 20 p. keys a.b. (1928). the weight-length relationship in fishes. proceeding of the national academy of science. washington, dc, pp.922-925. patiyal r.s., sharma r.c., punia p., goswami m., lakra w.s. (2010). length-weight relationship of tor putitora (hamilton, 1822) from the ladhiya river, uttarakhand, india. journal of applied ichthyology, 26: 472-473. gerami m.h., abdollahi d., patimar r. (2013). lengthweight, length-length relationship and condition factor of garra rufa in cholvar river of iran. world journal of fish and marine sciences, 5 (4): 358-361. int. j. aquat. biol. (2021) 9(2): 79-87 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article rearing catfish heteropneutes fossilis on feed supplemented by fermented leaf meal of ipomoea aquatica saheli ali, anilava kaviraj*1 department of zoology, university of kalyani, india. s article history: received 12 september 2020 accepted 25 february 2021 available online 2 5 april 2021 keywords: fish meal replacement nutrients growth digestibility enzymes abstract: replacement of fish meal by cost effective and sustainable plant resources in the formulation of feed for fish is a viable option to alleviate the current crisis in fish feed industries arising due to high cost and non-availability of fish meal. the present research was aimed to evaluate if fermented leaf meal of the aquatic plant ipomoea aquatica could be used as a fish meal alternative in the formulation of feed for the catfish heteropneustes fossilis. fresh green leaves of i. aquatica were sun dried and finely ground to make ipomea leaf meal (ilm), which was then fermented for 12 days by the phytase producing bacteria stenotrophomonas maltophilia strain kuaksp1 (genbank accession no. ky790423) isolated from rumen of goat. four iso-proteinous, iso-lipidous and isoenergetic feeds were formulated by replacing 0, 25, 50 and 75% of fish meal by the fermented ipomoea leaf meal (film). protein digestibility of the feeds was evaluated within 12 days in an indoor experiment in glass aquaria and growth performance of the fish was evaluated after 8 weeks rearing in outdoor cement tanks. h. fossilis grew better on film supplemented feed as compared to fish meal based control feed. maximum apparent protein digestibility (apd) of the feed, maximum weight gain (wg) and specific growth rate (sgr) and minimum fcr of the fish were found in 50% replacement group. however, crude protein (cp) and crude lipid (cl) deposition in the muscle of the fish and activity of protease in the gut was higher in 25% replacement group. it is concluded that h. fossilis accepts and grows well on the plant based film supplemented feed. for better growth management of the fish, incorporation of film in the feed should be restricted to 25 to 50% of fish meal. introduction freshwater aquaculture has been unprecedentedly growing in indian subcontinent during the last few decades due to application of modern tools and techniques. fish farmers are coming forward with options of culture of various species of freshwater fish and contributing towards augmentation of aquaculture production. however, the main hurdle behind maximizing aquaculture production still remains the use of nutritionally balanced formulated feed, which constitutes about 70% of the total cost in most successful aquaculture practices. cost of the commercial fish feed is gradually escalating and becoming out of reach of the poor and marginal fish farmers due to increasing cost and unavailability of fish meal, the main source of protein in the formulated feed. as a result, replacement of fish meal by cost *correspondence: anilava kaviraj doi: https://doi.org/10.22034/ijab.v9i2.999 e-mail: akaviraj@gmail.com effective, eco-friendly and environmentally sustainable plant products have become priority research since last two decades (kaushik et al., 2004; hardy, 2010; perez-velazquez et al., 2018; daniel, 2018). although initial researches found soybean and other legumes as nutritionally sound feedstuff to replace fish meal in the formulation of feed for fish, they were proved non-viable due to high cost of the legume by-products and relative non-availability due to large scale human consumption (gatlin et al., 2007; dan et al., 2017). in india, several attempts have been made to utilize easily available and low cost terrestrial (mondal et al., 2012, 2015; saha and ghosh, 2013; roy et al., 2016) and aquatic plants (hasan and chakrabarti, 2009; saha and ray, 2011; debnath et al., 2018) to replace fish meal as a source of protein to develop nutritionally 80 ali and kaviraj / rearing heteropneutes fossilis with fermented leaf meal of ipomoea aquatica sound feed for carps and catfishes. about 50 species of aquatic plants have been found as potential feedstuffs for fish culture in india (mandal et al., 2010). ipomoea aquatica, a semi-aquatic tropical plant, that grows profusely in canals, rivers, lakes, ponds and low lands in india and other asian countries (austin, 2007), offers a promising and alternative feedstuff in the formulation of fish feed. the plant has hardly been explored as feedstuff for fish, despite being rich in crude protein and crude lipid (mandal et al., 2010). recently, we used fermented leaf meal of this plant as fish meal alternative in the formulation of feed for an herbivorous carp, labeo rohita (ali and kaviraj, 2018). in this study, the fermented leaf meal of this plant was used as fish meal alternative in the formulation of feed for the catfish h. fossilis. mandal et al. (2011) successfully utilized fermented mulberry leaf meal in the diet of h. fossilis, but they used the mulberry leaf meal along with fish offal, an animalbased feedstuff. the main problem in using plant resources as feedstuff is the presence of anti-nutritional factors (anfs) which create several adverse effects on the physiology of fish. phytic acid is a most important anf, which binds with necessary cations rendering them unavailable to fish, binds to proteins, affects digestive enzyme activities, and reduce digestibility of the feed stuff (reddy et al., 1989; afinah et al., 2010; krogdahlet al., 2010; khan and ghosh, 2013). therefore, several researchers have used fermentation as an effective tool to remove phytic acid from the plant ingredients before using the plant resources as feed stuff (roy et al., 2009; mondal et al. 2015; dey et al., 2016; ali et al., 2017). selective use of phytase producing bacteria in the fermentation process serves dual purpose of removing phytic acid and restoring nutrient quality of the plant feedstuff (roy et al., 2009; dey et al., 2016; ali et al., 2017). in this study, we used phytase-producing bacteria isolated from goat rumen to ferment leaf meal of i. aquatica and the fermented products were used to partially replace fishmeal in formulation of h. fossilis feed and monitored growth performance, digestibility and digestive enzymes’ activities of the fish. materials and methods experimental feed: we used stenotrophomonas maltophilia strain kuaksp1 (genbank accession no. ky790423), a phytase-producing bacteria isolated from goat rumen (ali et al., 2017) to ferment leaf meal of i. aquatica. fresh green leaves of the plant were collected from local ponds, sun dried and finely grounded to make the meal before fermentation. the detailed fermentation process has been described by ali and kaviraj (2018). after 12 days of fermentation, the wet fermented products were dried in a hot air oven. the proximate composition, phytic acid, tannic acid and some essential mineral contents of the fermented i. aquatic leaf meal (film) have been reported by ali and kaviraj (2018). four isoproteinous (30% crude protein), iso-lipidous (8% crude lipid) and iso-energetic (15 kj/g energy) experimental feed were formulated by replacing 0% (t1), 25% (t2), 50% (t3) and 75% (t4) of fish meal by the film (table 1). to balance the level of nutrients, non-proteinous dietary supplements like fish oil, starch, dextrin and α-cellulose were added to each formulated feed at required amounts. carboxymethyle table 1. formulation of experimental feed by replacing fish meal (fm) with fermented ipomea leaf meal (film). fmrl (%) t ingredients (g/kg) proximate composition fm film st dx αc fo vm mm cmc cr2o3 cp (% dw) cl (% dw) energy (kj/g) 0 t1 750 0 100 120 15 11 1 1 1 1 29.90 7.92 10.38 25 t2 630 210 70 74 5 6 1 1 1 1 30.18 7.90 9.83 50 t3 478 478 20 14 2 3 1 1 1 1 29.93 7.98 9.92 75 t4 228 682 37 85 8 1 1 1 1 1 30.12 7.88 10.36 fmrl = fishmeal replacement level: t = treatment; st=starch; dx= dextrin; αc= α-cellulose; fo= fish oil; vm= vitamin mixture; mm= mineral mixture; cmc= carboxymethyl cellulose (binder); cr2o3 (marker); cp = crude protein; clcrude lipid. 81 int. j. aquat. biol. (2021) 9(1): 79-87 cellulose was used as a binder and cr2o3 was used as a non-absorbent marker. after feed formulation, four types of feed were ground and pelleted separately using a hand pelletizer fitted with a 2 mm diameter to prepare the final experimental feeds. then, the pelleted feeds were dried before using in the experiments. experiments: fingerlings of h. fossilis (average l=6.5 cm; w=2.8 g) were procured from naihati fish farm and were acclimatized for one week in cement tanks, each containing 250 liters of water. during the acclimatization period, the fish were fed ad libitum a feed containing 30% crude protein. two separate experiments were conducted using h. fossils fingerlings: (a) an indoor feeding and digestibility experiment and (b) an outdoor growth experiment. the feeding and digestibility experiments were conducted in the laboratory in glass aquarium of 20-l capacity. each aquarium contained 10-l of clean deep tube-well water supplied from an overhead tank and five numbers of acclimatized fingerlings of h. fossilis. for each experimental feed type three replicates were maintained. temperature (22-25°c), ph (7.2±0.2), dissolve oxygen (6.3–6.5 mg l−1), free co2 (1.99±0.2 mg l−1), total alkalinity (72±0.2 mg l−1) and total hardness (82±0.2 mg l−1) of the aquarium water were maintained regularly. every day at 8 am, fish of each aquarium were hand fed using the specific experimental feeds based on 5% of the aqurium biomass. then, the fish were allowed to feed for 6 hours. left over feed and fecal matter were collected in a particular time interval and processed according to the method of samaddar et al. (2015) and roy et al. (2016). the experiment was continued for 12 days. the pooled samples of feed and feces from each aquarium were digested, using a mixture of sulfuric acid and perchloric acid, as described by bolin et al. (1952), and chromium contents in the samples were determined by flame atomic absorption spectrophotometer (varian spectra aa240) according to guhathakurta and kaviraj (2000). two parameters were determined from the above experiment: (a) feed intake rate (fir) and (b) apparent protein digestibility (apd). fir was calculated from the amount of feed given and amount of uneaten feed siphoned out after 6 h and was expressed as gram per 100 g body weight per day (g 100 g−1 bw d−1). apparent protein digestibility (apd) of the feed was calculated from the proportion of cr and protein in diet and feces based on ellestad et al. (2002). the growth experiments were conducted in 400-l outdoor cement circular tanks (diameter 85 cm and average depth 50 cm) under 12 h day-light photoperiod. each tank contained 4.0 cm thick soil at the bottom, 350-l of the same deep tube-well water, which was used in the feeding and digestibility experiment, and 20 acclimatized fingerlings of h. fossilis. the tanks were arranged as complete randomized block design (gomez and gomez, 1984); so that, there were three replicates for each of the four fish meal replacement levels (treatments). the experimental fish were hand fed twice daily by the formulated feeds up to satiation. 50% of water of each experimental tank was weekly renewed by fresh tubewell water. water quality parameters of each experimental tank were determined every week by standard methods (apha, 1995). the range of values observed during the experiment was: dissolved oxygen (5.55-6.32 mg l-1), free carbon dioxide (0.052.08 mg l-1), ammonia-n (0.05-2.05 mg l-1), alkalinity (20.12-82.00 mg l-1 as caco3), hardness (88-130 mg l-1 as caco3) and ph (6.76-7.02). the experiment continued for eight weeks, after which, all fish were sampled from the experimental tanks. length and weight of the sampled fish were recorded. then, the sampled fish of each replicate of a treatment were divided into two different sets. one set was used for proximate composition analyses (crude protein, crude lipid and ash). for this purpose, five fish were randomly selected and a small portion of the muscle below the first ray of the dorsal fin from the dorsal side of the fish was first removed, washed with distilled water and preserved at −20°c until proximate composition analyses according to aoac methods (helrich, 1990). daily protein retention (dpr) in the body was determined by [(fbp-ibp) /d], where fbp and ibp are final body protein and initial body protein, respectively, and d is the days of the experiment. the 82 ali and kaviraj / rearing heteropneutes fossilis with fermented leaf meal of ipomoea aquatica data of length and weight, feed consumption rate and crude protein levels were used to determine specific growth rate (sgr), feed conversion ratio (fcr), protein efficiency ratio (per) and apparent net protein utilization (anpu) by the experimental fish following the methods of castell and tiews (1980). another set of five fish sampled from each tank was used for determination of digestive enzyme activities. for this purpose, the sampled fish were kept in clean water containing glass aquaria (20 l) for 24 h to starve the fish. after 24 h of starving, the fish were fed the experimental feed up to satiation. following 4 h of feeding, they were taken out from each treatment. then, they were dissected out to collect the digestive tract. activities for α-amylase, protease and lipase were measured in the homogenized and sonicated samples of the digestive tract by the method of bernfeld (1955), marks and lajtha (1963) and josep and kurup (1999), respectively. protein content of the samples was determined by the method of lowry et al. (1951). the nature of distribution of the observations for each parameter was evaluated by kolmogorov– smirnov (k–s) and shapiro–wilks (s–w) tests (johnson and wichern, 2001). since all observations were found normally distributed, all data were subjected to single factor anova, without any transformation, followed by least significant difference (lsd) test to compare mean among the treatments (gomez and gomez, 1984). results fir, apd, wg, sgr, fcr, per and anpu of the fish have been presented in table 2. the results of one-way anova among the treatment means exhibited that fir and apd decreased significantly in the replacement groups except in 50% replacement level (t3), which showed similar feed intake rate and significantly higher apd as compared to control (t1). wg, sgr and anpu of the fish significantly (p<0.05) increased and fcr significantly decreased in all film supplemented feed (t2-t4), compared to t1. per was similar between t1 and t2 (25% replacement group), but increased significantly in t3 and t4 (75% replacement group) as compared to t1. best growth was exhibited by 50% replacement of fish meal by film (t3). crude protein (cp), crude lipid (cl) and ash in fish muscle also increased significantly (p<0.05) in the fish fed filmsupplemented feeds (t2-t4). there was no significant difference in cp and ash among the treatments of film-supplemented feeds (t2-t4). however, cl deposition was significantly higher in t2 as compared to t3 and t4. dpr by h. fossilis has been presented in figure 1. dpr significantly (p<0.05) increased in all supplemented feed (t2-t4) in comparison to control (t1). maximum retention was found in 25% table 2. digestibility of the feed, growth and nutrient deposition in heteropneutes fossilis fed film supplemented feeds for 60 days. data are mean±standard deviation (n = 9); means with dissimilar superscripts in the same row indicates least significant difference (lsd) between the means at p<0.05. t1 t2 t3 t4 feed intake and digestibility feed intake rate (g. 100g -1bw.d -1) 3.22±0.14a 2.39±0.04c 3.09±0.09a 2.83±0.01b apparent protein digestibility(apd, %) 78.39±0.43b 66.90±0.86c 82.35±0.20a 65.05±0.10d growth total weight gain (%) 237.15±9.73c 284.17±24.32b 345.41±18.81a 312.15±17.19ab specific growth rate (sgr, % d-1) 2.02±0.04c 2.24±0.10b 2.48±0.07a 2.35±0.06ab feed conversion ratio (fcr) 2.32±0.09a 1.94±0.16b 1.59±0.08c 1.76±0.09bc protein efficiency ratio (per) 1.44±0.05 d 1.71±0.14cd 2.09±0.11a 1.88±0.10ab apparent net protein utilization (anpu, %) 103.35±15.80b 246.68±24.6a 247.91±9.84 a 211.49±8.42a nutrient deposition1 moisture (%) 75.11±0.77a 67.99±0.73b 68.58±0.24b 69.75±0.05b crude protein (%) 20.1±0.56b 25.33±0.89a 25.30±0.35a 24.03±0.30a crude lipid (%) 3.06±0.07c 3.95±0.02a 3.62±0.04b 3.64±0.17b ash (%) 1.72±0.28 b 2.72±0.13a 2.49±0.06a 2.57±0.07a 1, initial values: moisture 79.12±1.28; crude protein 16.4±0.98 %; crude lipid 2.77±0.17%; ash 1.30±0.11 83 int. j. aquat. biol. (2021) 9(1): 79-87 replacement level (t2). activities of the digestive enzyme, α-amylase and protease, also significantly increased (p<0.05) over control (t1) in t2 (fig. 2). the α-amylase activity significantly decreased in t3 and t4 (50% and 75% replacement level), but there was no significant difference (p>0.05) in protease activity between control (t1) and the higher replacement levels (t3 and t4). the lipase activity did not vary significantly (p>0.05) between control (t1) and filmsupplemented feed treatments (t2-t4). discussions the results of the present study indicate that fir and apd of h. fossilis fed the experimental feeds supplemented by film decreased as compared to control feed (t1) except in t3 (50% replacement level). heteropneutes fossilis, being an omnivorous fish, can digest high level of protein in feed containing animal-based feedstuffs or mixture of both plant and animal feedstuffs, but digestibility of protein decreases when feed predominantly contains plant based materials (mondal et al., 2008, 2011). this was also evident from the digestibility experiment of mystus vittatus, a predominantly carnivorous fish, showing high range of apd (91-96%) when fed fishmeal or animal-based feed, but showed a decline in apd (86-88%) when fed by plant-based feed (kaviraj et al., 2013). however, h. fossilis fed silk worm pupae, an animal protein source, showed only 78.8 to 86.72% apd (hossein and jauncy, 1993), indicating that digestibility depended not only on the source of protein, whether animal or plant, but also on other factors like amino acid composition, proportion of lipid etc. usmani et al. (2003) observed apd of h. fossilis to vary widely between practical feed ingredients with rice bran showing the least value (61.1%) and soybean meal the highest value (95.4%). interestingly, despite low digestibility and feed figure 1. daily protein retention (dpr) in the body of heteropneutes fossilis fed fiml supplemented feed. data are mean of three replicates (n=9). the error bars indicate ± standard deviation. different letters above the error bar indicate least significant difference (lsd) between two treatments at p<0.05 figure 2. digestive enzyme activities of heteropneutes fossilis fed film supplemented feed. data are mean of three replicates (n=9). the error bars indicate ± standard deviation. different letters above the error bar indicate least significant difference (lsd) between two treatments at p<0.05 84 ali and kaviraj / rearing heteropneutes fossilis with fermented leaf meal of ipomoea aquatica intake rate h. fossilis grew well with film supplemented feed and deposited higher level of crude protein and crude lipid in the muscle of the fish as compared to fishmeal-based control feed (t1). fm based feed is always balanced in amino acids and superior to feed containing fermented products as alternative to fm (samaddar et al., 2015, 2020). but fermentation results in hydrolyzed nature of the amino acids and most of the catfish can quickly accumulate a high level of amino acids from the feed containing fermented products without any symptom of hyperaminoacedemia due to presence of a proper stomach (dabrowski et al., 2010; samaddar et al., 2020). in the present study, higher deposition of cp and increased dpr in fish fed film supplemented feed probably resulted from higher assimilation of amino acids in these treatment groups. heteropneustes fossilis fed fermented fish offal supplemented feed also showed a tendency of accumulating lipid at a high rate, which provided a protein sparing effect (mondal et al., 2008). moreover, h. fossilis is known to consume and digest wide variety of foods (chondar, 1999). it has been found that the fish grows well on detritus-based food (kohli, 1996) or feed comprising of composted weed with crude protein level as low as 15-20% (biswas and kaviraj, 2003). the experimental tanks in the present study contained 4.0 cm thick bottom sediment, which was the store house of the uneaten feed and its subsequent transformation into nutrient rich detritus. heteropneutes fossilis could well utilize this detritus, in addition to direct consumption of the experimental feed. the results of the present study also indicate that based on wg, sgr and fcr, h. fossilis exhibits maximum growth in t3 (50% replacement level), although there is no significant difference of these parameters between t3 and t4. the fish exhibits maximum deposition of cp, cl and ash in t2 (25% replacement level), although cp and ash did not vary between t2-t4 treatments. there is also no significant difference in dpr between any two film supplemented feed (t2-t4). however, protease activity significantly increased in t2 and remaining similar in t3 and t4 as compared to control (t1). on the other hand, amylase activity was similar in t2, but decreased in t3 and t4 as compared to t1. it indicates that h. fossilis can utilize maximum amount of cp and carbohydrate of the feed when the feed is supplemented by film at low level (25%). carbohydrate is easily available in feed, but utilization of carbohydrate by fish depends on level, source and complexity of carbohydrates as well as carbohydrate metabolizing enzymes (nrc, 1983; mollah and alam, 1990; tung and shiau, 1991; wilson, 1994; stone et al., 2003; orire and sadiku, 2014; zhou et al., 2015). the results indicate that h. fossilis can utilize limited amount of carbohydrate of film (t2). rahman et al. (2017) observed that h. fossilis could utilize dextrin better than glucose. many herbivorous and omnivorous fishes have been found to use oligosaccharides and polysaccharides easier than disaccharides or monosaccharides (tung and shiau, 1991; shiau and peng, 1993). the implication is that fish meal can be supplemented up to 75% by the film in the formulation of feed for h. fossilis. but based on apd, growth parameters, nutrient deposition and digestive enzyme activities the best growth of h. fossilis can be obtained when fish meal replacement level is restricted to 25 to 50%. conclusions: the results of the present study reveal that the film is a potential fishmeal alternative in the formulation of feed for catfish h. fossilis. despite low digestibility of this feed and low feed intake rate by h. fossilis, the fish grew well with film supplemented feed and deposited higher level of crude protein and crude lipid in the muscle of the fish as compared to fishmeal-based control feed, because the fish could better utilize the nutrients contained in the film supplemented feed through direct consumption as well as through detritus formed from the uneaten feed at the bottom of the experimental tanks. heteropneutes fossilis could utilize maximum amount of cp and carbohydrate when replacement of fishmeal by the film was low. based on apd, growth parameters and digestive enzyme activities it is concluded that 25 to 50% of fishmeal can be replaced by film in the formulation of feed for h. fossilis. 85 int. j. aquat. biol. 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(2016) 4(1): 31-42 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article morpho-histological characteristics of gonads and reproductive index in an endemic fish species, oxynoemacheilus persa (heckel, 1847) (teleostei: nemacheilidae) from kor river basin, iran soroor mirghiyasi, hamid reza esmaeili*,1mohsen nokhbatolfoghahai ichthyology and developmental systematics lab., department of biology, college of sciences, shiraz university, shiraz, iran. article history: received 8 november 2015 accepted 19 january 2016 available online 2 5 february 2016 keywords: reproduction gonad morphology gonad histology sexual maturation gonado-somatic index abstract: this study presents the first details on morphological and histological characteristics of gonads, gonadal development stages and reproductive index of the persian loach, oxynoemacheilus persa (heckel, 1847), an iranian endemic species. sampling was done from april 2010 to april 2011 using electrofishing device and a total of 196 individuals were collected. the gonads of specimens were removed, their sexes determined and then were fixed in 10% formalin solution after checking their morphology and measuring their weights, lengths and widths. based on the size, shape and weight of the gonads, degree of occupation of the body cavity, presence or absence of ripe oocytes or milt, diameter of the oocytes in the ovary, and histological observations, five stages of sexual maturation in females and four stages in males were determined by macroscopic and microscopic criteria. the results of the gonadal stages showed that the o. persa spawns in the middle of spring and these stages were correlated to the gonado-somatic index (gsi). introduction the order cypriniformes with 11 family and 4298 species is one of the large order of fishes (eschmeyer and fong, 2016). the family nemacheilidae comprises about 659 species which is the second largest family in the order cypriniformes (eschmeyer and fong, 2016). the nemacheilid fishes are found throughout eurasia and northeast africa (berra, 2001; nelson, 2006). they are small fishes, live in fast flow hill streams with quite secretive life hiding under stones or in mud. the genus oxynoemacheilus with about 47 species, is a species-rich genus of nemacheilids known from albania eastwards to central iran (golzarianpour et al., 2011; erk'akan, 2012; kottelat, 2012; kamangar et al., 2014; mafakheri et al., 2015). the persian loach, oxynoemacheilus persa (heckel, 1847) which is generally known as persian sag mahi (= dog fish), louch (= louch) or mar mahi parsi (= snake fish) in farsi, is an endemic poorly known loach distributed in endorheic kor and * corresponding author: hamid reza esmaeili doi: http://dx.doi.org/10.7508/ijab.2016.01.005 e-mail address: hresmaeili@shirazu.ac.ir exorheic persis basins in southern iran (esmaeili et al., 2010, 2015) and information about different aspects of its biology and ecology including its reproduction is scare. reproductive studies of fishes, such as assessment of size at maturity, duration of the spawning season and fecundity, require knowledge of the state of gonad development and a large number of macroscopic maturity scales in individual fish (carrasson and bau, 2003). although macroscopic staging can enable detailed recording of the seasonal occurrence of different reproductive stages, histological analysis of the gonads can provide a more precise determination. cellular substructures can be recognized in the growing follicles and ovarian tissue and allow for unambiguous grading and interpretation of reproductive status. description of the general pattern of histology and development of teleost are given by wallace and selman (1981), tylerand sumpter (1996), and tomkiewicz et al. (2003). since 32 mirghiyasi et al./ morpho-histological characteristics of gonads in oxynoemacheilus persa there is no data available regarding the gonadal morphology and histology of oxynoemacheilus persa, therefore, this study was conducted to presents a detailed description of gonad morpho-histology of this species. materials and methods a total of 196 specimens of o. persa (fig. 1) (81 males and 111 females) were collected monthly from kor river basin, fars province (30°36'16.9'' n; 52°56'40.1'' e) from april 2010 to april 2011 using electrofishing device. after anesthesia, fish were fixed into 10% formalin in the field, then labeled individually and deposited in the zoological museum, collection of biology department, shiraz university (zm-cbsu). some morphological measurements were made using a digital caliper to the nearest 0.01 mm and body weights were determined by a digital balance to the nearest 0.001 g. for histological studies, the specimens were dissected and their ovary or testis were removed. the sexes and stages of sexual maturation were determined as possible as by naked eye examination and under a compound microscope (olympus). the chi-square test was used to assess deviation from 50:50 sex ratio based on robards et al. (1999). weight, length, width, color, and shape of each gonad were recorded and the maturity stage of them was recognized macroscopically based on nikolsky (1963). the histological sections of ovary or testis of each maturing stages were prepared by routine histology method (bancroft and stevens, 1991; eagderi et al., 2013) as follow: they dehydrated in alcohol, cleared in xylene, imbedded in paraffin wax at 56°c melting point, sectioned at 5-7 μm thickness, and then the sections were stained by hemotoxylin and eosin (h&e) staining method. the histological slides were studied under a light compound microscopy and their pictures were taken by a compound microscope equipped to a digital camera. the gonado-somatic index (gsi) was calculated by dividing the gonads weight by the whole body weight and multiply by 100 (nikolsky, 1963) to examine the seasonal changes in the gonads for estimating spawning season. results size range: a total of 196 individuals of o. persa were collected ranging 25.19-78.24 mm (s.d: 12.48) in total length, 24.29-74.22 mm (s.d: 12.02) in fork length, 21.34-66.01 (s.d: 10.55) in standard length and 0.085 to 4.50 g (s.d: 0.90) in total weight. sex ratio: the collected 81 male and 111 female specimens giving an overall sex ratio of 1m: 1.37f. it was significantly female biased (chi square =12.629, p<0.001). gonado-somatic index: the results showed that female invest more in gonads than males (anova, p<0.001). a significant differences were observed between female and male’s gonado-somatic index (anova, p<0.05). both female and male gonadosomatic indices peaked in april showing that figure 1. oxynoemacheilus persa, from kor river basin, fars province. 33 int. j. aquat. biol. (2016) 4(1): 31-42 figure 2. monthly variation of mean gsi in female and male specimens of oxynoemacheilus persa. 34 mirghiyasi et al./ morpho-histological characteristics of gonads in oxynoemacheilus persa o. persa spawns in the middle of spring (fig. 2). macroscopic and microscopic characteristics of ovaries: the left and right ovaries of o. persa were stuck to each other and made an integrated ovary extending along the body cavity in dorsal position above the gut. based on the size and weight of the ovary, degree of occupation of the body cavity, presence or absence of ripe oocytes, diameter of the oocytes in the ovary, and histological observations, we described 5 maturation stages in the ovary. the stages were classified as immature (stage i), immature and developing (stage ii, early and late), maturing (stage iii), ripe (stage iv), spawning (stage v). no specimens of immature stage were observed and the stage ii was divided into two stages: early and late. gonad measurements of o. persa have shown in tables 1 and 2. ovary maturation stages: no specimens of immature stage (i) were observed. in the stage ii, early immature, the ovary was small and cream in color. oocytes were not visible to the naked eye. the mean diameter of oocytes was 0.073 mm (fig. 3a). in histological sections of the ovary, small and round oocytes with a large nucleus and abundant nucleoli around the nucleoplasm were observed. no lipid droplets were found in this stage. the oocytes had basophilic cytoplasm and an acidophilic nucleus. the ratio of nucleus to cytoplasm volume was high (fig. 4a, b). in the developing stage (ii, late), the ovary was larger and the oocytes were not visible by naked eye. the mean diameter of oocytes was 0.131 mm (fig. 3b). in histological observations, the oocyte size increased and the ratio of nucleus to cytoplasm decreased. in larger oocytes, the lipid droplets were formed in the cytoplasm and follicular and theca layers covered each oocyte. in addition, a very thin zona radiate appeared between oolema and follicular layer. the zona radiata was distinguished in two parts: one part as finger-like projections which is placed under the follicular cells and another stage measurements n minimum maximum mean std. deviation 2 early gonad weight (g) 19 0.002 0.013 0.00532 0.002868 gonad length (mm) 19 5.010 9.600 6.87211 1.150447 gonad width (mm) 19 1.273 2.543 1.74737 0.297476 gl.acl (%) 19 44.97 76.49 57.8247 7.54236 2 late gonad weight (g) 39 0.030 0.188 0.06713 0.025999 gonad length (mm) 39 10.050 19.380 14.04615 1.932407 gonad width (mm) 39 2.257 5.237 3.18667 .472447 gl.acl (%) 39 51.65 78.38 68.9231 5.69150 3 gonad weight (g) 14 0.017 0.202 0.10521 0.054044 gonad length (mm) 14 10.050 20.170 15.40643 2.931255 gonad width (mm) 14 2.033 4.597 3.67643 .847645 gl.acl (%) 14 48.41 96.72 74.6653 13.82134 4 gonad weight (g) 18 0.144 0.423 0.24367 0.073964 gonad length (mm) 18 13.690 20.880 18.14833 1.783800 gonad width (mm) 18 4.493 6.093 5.14926 .426514 gl.acl (%) 18 63.26 99.06 89.9383 9.06568 5 gonad weight (g) 21 0.234 0.795 0.42562 0.131349 gonad length (mm) 21 18.140 29.190 20.60238 2.261541 gonad width (mm) 21 4.907 10.367 6.70921 1.133205 gl.acl (%) 21 79.94 99.43 91.7612 5.21365 gl.acl: the ratio of gonad length to abdominal cavity length. table 1. descriptive statistics of gonad measurements in oxynoemacheilus persa (female) from kor river basin, fars province. 35 int. j. aquat. biol. (2016) 4(1): 31-42 figure 3. morphology of ovary of oxynoemacheilus persa in different stages. (a) immature stage (ii, early), (b) developing stage (ii, late), (c) maturation stage (iii), (d) ripe stage (iv), and (e) spawning stage (v). stage measurements n minimum maximum mean std. deviation 1 gonad weight (g) 24 0.001 0.002 0.00113 0.000338 gonad length (mm) 24 4.28 11.20 6.2642 1.61155 gonad width (mm) 24 0.060 0.302 0.16389 0.055755 gl.acl (%) 24 0.92 4.29 2.1816 0.77465 2 gonad weight (g) 19 0.001 0.021 0.01011 0.004665 gonad length (mm) 19 3.62 11.19 7.3453 2.02354 gonad width (mm) 19 0.413 1.175 0.61404 0.176038 gl.acl (%) 18 3.07 9.25 4.4624 1.51573 3 gonad weight (g) 18 0.010 0.028 0.01689 0.005075 gonad length (mm) 18 6.16 11.85 8.2925 1.32283 gonad width (mm) 18 0.492 0.912 0.65546 0.123627 gl.acl (%) 18 3.22 6.45 4.5645 0.89245 4 gonad weight (g) 19 0.016 0.070 0.03642 0.014261 gonad length (mm) 19 7.73 16.48 11.1745 2.25921 gonad width (mm) 19 0.613 1.158 0.90167 0.168485 gl.acl (%) 19 4.08 7.25 5.8294 .96958 gl.acl: the ratio of gonad length to abdominal cavity length. table 2. descriptive statistics of gonad measurements in oxynoemacheilus persa (male) from kor river basin, fars province. 36 mirghiyasi et al./ morpho-histological characteristics of gonads in oxynoemacheilus persa uniform thin part on the oocyte membrane. this stage is called returning stage, because after spawning, the ovaries, return to this stage to start oogenesis (fig. 4c, d). in the third stage i.e. maturing stage, the ovary size was clearly larger than the previous stage and its color changed from cream to light yellow due to accumulation of the yolk materials in the oocytes. yellow oocytes with an average diameter of 0.237 mm were visible to the naked eye in this stage (fig. 3c). the growing oocytes were characterized by small acidophilic yolk granules and many clear lipid droplets that had entirely filled the cytoplasm and also thickened two parts of zona radiata. the finger-like projections were significantly elongated (fig. 4e, f). in the ripe stage (iv), the size and weight of figure 4. microphotographs of ovaries of oxynoemacheilus persa in different stages. (a-b) immature stage (ii, early), (c-d) developing stage (ii, late), (e-f) maturation stage (iii), (g-h) ripe stage (iv), and (i-j) spawning stage (v). n: nucleus, nu: nucleoli, ld: lipid droplets, tl: theca layer, fcl: follicular cell layer, flp: finger–like projections, and zr: zona radiata. 37 int. j. aquat. biol. (2016) 4(1): 31-42 gonads increased but had not achieved their maximum size. their color was yellow, and also they wrinkled a little. the mean of ova diameter was 0.442 mm (fig. 3d). in this stage, the oocytes are known as follicles. their size increased and the ratio of nucleus to cytoplasm decreased. the large follicles were full of lipid droplets more than pervious stages. furthermore in this stage, a coalescence of lipids and yolk granules were occurred. the nuclear envelope broke down and the thickness of the zona radiata was higher than previous stages (fig. 4g, h). in the spawning stage (v), the ovary was yellow and occupied most of the body cavity. it achieved its maximum weight. large and yellow oocytes, full of yolk with an average diameter of 0.518 mm were distinguishable in this stage (fig. 3e). cohesion between oocytes decreased and some of them were observed separately in the body cavity. oocytes were characterized by large mass of yolk and numerous large lipid droplets. the zona radiata was completely thick in this stage (fig. 4i, j). testes maturation stages: based on macroscopic and microscopic observations, 4 stages of maturation were distinguished for males of o. persa, as follow: immature stage (i): the testes were very thin, threadlike and grey in color (fig. 5a). spermatogonia were the dominant cells. these cells were the largest spermatogenic cells, with clear cytoplasm and large nucleus. some primary spermatocytes were also observed (fig. 6a). developing stage (ii): the size of testes increased (fig. 5b). spermatogonia, primary and secondary spermatocytes were more remarkable in the histological sections. secondary spermatocytes were similar to the primary spermatocytes but smaller (fig. 6b). maturing stage (iii): the testes were more elongated, flat and milky (fig. 5c). the number of spermatogonia were significantly reduced. primary and secondary spermatocytes, and spermatids were observed in the tubules. these cells were located in clusters. spermatids were smaller than the secondary spermatocytes (fig. 6c). ripe stage (iv): testes were quite milky and massive (figure 5d). tubules were characterized by figure 4. continued. 38 mirghiyasi et al./ morpho-histological characteristics of gonads in oxynoemacheilus persa having secondary spermatocytes, large numbers of spermatids and spermatozoa. the predominant cells were spermatozoa with a dark blue stain related to their nucleus. they were the smallest spermatogenic figure 5. morphology of testes of oxynoemacheilus persa in different stages. (a) immature stage (i), (b) developing stage (ii), (c) maturation stage (iii), and (d) ripe stage (iv). figure 6. microphotographs of testes of oxynoemacheilus persa in different stages. (a) immature stage (i), (b) developing stage (ii), (c) maturation stage (iii), and (d) ripe stage (iv). s: spermatogonia, ps: primary spermatocytes, ss: secondary spermatocytes, st: spermatids, and sz: spermatozoa. 39 int. j. aquat. biol. (2016) 4(1): 31-42 cells (fig. 6d). discussion this study provided the details on gonad morphology and histology of o. persa, an endemic loach species of iran. oxynoemacheilus persa does not exhibit a clear external sexual dimorphism as found in the cyprinodontid and poecilid fishes. however, banarescu and nalbant (1964) report that pectoral fin rays 2-5 are widened and thickened in males of persian loach. also according to coad (2016), the males have numerous fine tubercles on the dorsal surface of their pectoral fin rays in bands similar to our observations. an unequal sex ratio was observed, which may reflect different survival rates for males and females. it appears that the strategy of this species, in terms of the sex ratio, is the "investment" in females. this is brought about through the selective predation of males or the higher survival rate and greater longevity of females, or the greater endurance of females to environmental stress which is reported in other fish species (see esmaeili and shiva, 2006). the results of the present study showed that o. persa spawns in the middle of spring. the mean values of gonado-somatic index, percentage of late gonad maturation stages (iv, v) and high frequency of large oocytes confirmed the spawning season. the season of spring reported as the spawning season for some other loaches. for example, the gonadosomatic index of paracobitis malapterura indicated that reproduction of this species occurred in april– may (pattimar et al., 2009). mousavi-sabet et al. (2011) also reported the spawning of cobitis keyvani from talar river occures from may to late july. the reproductive index of females was higher than males. this matter is justified according to more volume of female’s gonad (mahomoud et al., 2011). this index has been widely used as indicator of the fish spawning season, but its use in reproductive biology studies is more suitable when it is associated with other reproduction indicators such as macroscopic and histological techniques (ghasemian et al., 2015). in the present study, based on the size, shape and weight of the gonads, degree of occupation of the body cavity, presence or absence of ripe oocytes or milt, diameter of the oocytes in the ovary, oocyte shape, vitellogenesis, size of oil droplets and yolk vesicles, five stages of ovary development and four stages of testis development were observed. many studies have been performed on histological and morphological changes of ovary in fishes (biswas, 1993). generally, the process of teleost oogenesis may be divided into 5-8 stages (west, 1990; fishelson et al., 1996; ünver and ünver-saraydin, 2004). the results of the histological study of gonad development in o. persa is basically similar to other teleost. the process of oogenesis has been divided into 5 stages which resemble many species such as aphanius farsicus (monsefi et al., 2007). the testes development process of o. persa revealed 4 sexual stages corresponding to a. farsicus and cyprinus carpio. the oocyte reaches maximum size of 1.11 mm in o. persa, this is directly found that the nutritional state of the fish affects the vitellogenic and maturation of oocyte because there is a close relationship between the vitellogenesis and oocyte size (wallace and selman, 1978). the ecological factors, such as temperature, photoperiod and nutrition are important environmental factors for regulations of reproductive pattern in most teleost (de vlaming, 1972). as conclusion, the persian loach, o. persa demonstrates some reproductive strategies, including significantly female biased sex ratio, little sexual dimorphism and spring spawning in response to its habitat. the small body size is another significant factor in the life history of o. persa which allows this loach to colonise and exploit microenvironments. however, the limited distribution makes the species highly vulnerable and could result in significant loss if habitats are disturbed or destroyed. the provided information in this study on the sex ratio, reproductive index and maturation process of persian loach contribute baseline data towards management ecology, 40 mirghiyasi et al./ morpho-histological characteristics of gonads in oxynoemacheilus persa conservation and biological studies of this fish. acknowledgments we are thankful to s. ghasemian, s. babai and r. zamaniannejad for their kind help in fish collection and shiraz university for financial support. references banarescu p., nalbant t. 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(2016) 4(1): 31-42 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی ,oxynoemacheilus persa (heckel پارسی ماهیلوچ در مثلیتوليد نمایه و اگناده بافتی -ریختی هایویژگی ایران کر، رودخانه حوضه از( خار بدون جویباری ماهيانلوچ: عالی استخوانی ماهيان) ،(1847 الفقهایی نخبه محسن ،*اسماعيلی حميدرضا ميرغياثی، سرور .ايران شیراز، شیراز، دانشگاه علوم، دانشکده شناسی،زيست بخش تکوينی، سیستماتیک و شناسیماهی آزمايشگاه چکيده: oxynoemacheilus persa پارسی ماهی لوچ ماده و نر گنادهاي جنسی بلوغ مراحل و تولیدمثلی نمايه بافتی،-ريختی هايويژگی مطالعه، اين در (heckel, 1847)، در و انجام الکتريکی صید وسیلههب ،0200 آوريل تا 0202 آوريل ماه از بردارينمونه. است گرديده ارائه ،ايران بومزاد گونه يک عرض و طول وزن، گیرياندازه ريختی، بررسی از پس و گرديده جنسیت تعیین خارج، بدن از گنادها. گرديد آوري جمع ماهیقطعه 091 تعداد کل رب. گرديد آماده شناسیبافت مقاطع ائوزين، و هماتوکسیلین آمیزيرنگ معمول روش از استفاده با و تثبیتدرصد 02 فرمالین محلول در ها،آن رد میلت رنگ شیري مايع يا و ماده گناد در تخمک وجود عدم يا و وجود شکمی، حفره طولبه نسبت گناد اندازه گنادها، وزن و شکل اندازه، اساس مراحل بررسی نتايج. گرديد منظور نرها براي مرحله 4 و هاماده براي جنسی بلوغ مرحله 5 شناسی،بافت مشاهدات و تخمدان در تخمک قطر نر، گناد -يگناد میانه میزان با جنسی بلوغ مراحل و نموده ريزيتخم بهار میانه در ماهی اين که داد نشان پارسی ماهی لوچ ماده و نر گنادهاي جنسی بلوغ .است مرتبط بدنی .بدنی-گنادي نمايه جنسی، بلوغ گناد، شناسیبافت گناد، شناسیريخت تولیدمثل، :کلمات کليدی int. j. aquat. biol. (2020) 8(6): 447-454 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article removal of coliform bacteria from dairy wastewater using graphene-silver nanocomposite mahmood bahri1, amir hossein hamidian* 1,2, yu zhang2,3, min yang1,2,3 1department of environmental science and engineering, faculty of natural resources, university of tehran, karaj, iran. 2state key laboratory of environmental aquatic chemistry, research center for eco-environmental sciences, chinese academy of sciences, beijing, china. 3university of chinese academy of sciences, beijing, china. s article history: received 9 august 2020 accepted 27 november 2020 available online 2 5 december 2020 keywords: nanocomposite coliform bacterium removal aqueous solution abstract: nanoparticles are widely used in removing bacteria from water and sewage. this study evaluated the effect of graphene/silver nanocomposite on the removal of coliform from a dairy effluent. the composition was synthesized and its properties were determined using different techniques such as sem, xrd and ftir. the effects of various factors, including ph, adsorbent dose and contact time on coliform removal from solution were studied. antimicrobial activity of the nanocomposite was examined by pour plate method in a vrbl medium. after preparing the vrbl medium from the sewage effluent, 1 ml of nanocomposite solution and 1 ml of bacterial suspension containing 9×104 cfu/ml were added into each of the plates. according to the results, the rate of silver ion release is faster as ph decreases, which naturally results in the increase of disinfection. moreover, a higher percentage of removal occurred with an increase in contact time and nanocomposite dose due to higher exposure to nanoparticles and their higher penetration into bacterial cell membrane. therefore, nanoparticles produced by this method exhibited good antibacterial activity, so that 100% of bacteria were eliminated at a nanocomposite concentration of 3.4 g/l and ph=5, after 90 min. introduction about 80% of diseases in developing countries are caused by poor water quality (rajaei et al., 2012). eleven percent of the world’s populations still do not have access to clean water resources. hygiene hazards caused by water pollution in developing countries are much more serious than those in developed countries (khazaee et al., 2016; wang and yang, 2016;alavian et al., 2017). some researchers predicted that nearly 135 million people will die by drinking contaminated water by 2020 (singh et al., 2016). discharging untreated sewage into environment causes adverse effects to human (thamilselvi and radha, 2017) and direct (mansouri et al., 2013; mirzajani et al., 2016b; hamidian et al., 2016) and indirect (mirzajani et al., 2016a, b) effects on ecosystems. conventional disinfection methods, including chlorination, ultraviolet, and ozonation are used to prevent or decrease risk of waterborne diseases. the use of *correspondence: amir hossein hamidian doi: https://doi.org/10.22034/ijab.v8i6.1025 e-mail: a.hamidian @ut.ac.ir chemical disinfectants such as chlorine produces harmful byproducts, when chlorine reacts with other pollutants in water (mthombeni et al., 2012). formation of disinfection by-products, such as trihalomethanes is suspected to have a negative effect on human health, especially in terms of carcinogenicity and fetal abnormalities (hong et al., 2007; kanan and karanfil, 2011). studies over last few decades have indicated a relationship between disinfection of water and formation of disinfection byproducts. thus, it is necessary to identify and use new technologies for water disinfection (li et al., 2008; mojoudi et al., 2018, 2019). pollutants found in water include organic pollutants, heavy metals, nitrates and radionuclides. in addition, water may contain many problematic organisms, such as bacteria, viruses and parasites, which should be removed or deactivated for the safety of those who are in contact with water (zarpelon et al., 448 bahri et al./ removal of coliform bacteria using graphene-silver nanocomposite 2016; mccullagh et al., 2007). despite developments in water management practices, the presence of bacteria and parasite in water is a major threat to human and animal health. escherichia coli is the most important factor in detecting fecal contamination in water resources. coliform bacteria are considered as indicators of drinking water pollution and known as appropriate indicators of water quality. therefore, the index of coliform bacteria is now monitored to control microbiological quality in water treatment plants (wiley and sonse, 2005). production of nanoparticles with antimicrobial properties provides a suitable ground for water decontamination. nanoparticles with different mechanisms of chlorine remove bacteria, which is of high importance in water treatment (chitra and annadurai, 2013). antimicrobial properties of metal nanoparticles are related to their small size and high ratio of surface to volume, which allows them to react closely with microbial membrane (ruparelia et al., 2008). due to their proper surface area and excellent adsorption capacity, silver nanoparticles are widely used for disinfecting water. several researchers approved antibacterial effects of silver nanoparticles and their potential against a wide range of microbes, including antibiotic-resistant bacteria (nanda and saravanan, 2009; tang et al., 2017). conventional techniques for producing nanoparticles include physical, chemical and biological methods (mojoudi et al., 2018). advantages of biological methods such as lower cost, faster speed, high production and safe environment led to more attention to these methods compared to two other types (parikh et al., 2008). based on above-mentioned background, this study investigated the effect of graphene-silver nanocomposite on coliform bacteria in dairy effluents. moreover, it studied the effects of parameters such as ph, adsorbent dose and duration on the rate of bacterial removal. materials and methods this study used 5-10 nm dark gray graphene oxide nanoparticles with 99% purity and silver nitrate (merck, germany). universal 320 bench-top centrifuge (hettich, germany) were used in the experiment and a up50h ultrasonic homogenizer (hielscher, germany) to homogenize the solid phase of nanocomposite in the solvent. nitric acid and 0.1 m sodium hydroxside were also used to adjust acidity. synthesis of ag-rgo nanocomposite: to prepare graphene-silver nanocomposite, 1.7 g of silver nitrate was initially dissolved in 100 ml of deionized water and then 0.62 g of naoh was added into the solution. the resulting brown ag2o deposition was filtered and dissolved in 100 ml of ammonia. [ag (nh3)2+] complex was formed in this stage; then, 7.2 g of oleic acid added into the stirring solution. after stirring for 90 min, 0.1 g of graphene oxide was added (lan et al., 2014). in addition, 90 ml of mango extract was poured into the solution, and it was stirred for half an hour. finally, it was placed under uv lamp for 8 hours. then, the solution was centrifuged at 6000 rpm for 15 min and dried at 50°c in an oven for 12 hours. several techniques such as sem, xrd and ft-ir were used to verify the synthesis of ag-rgo nanocomposite synthesis of ag-rgo nanocomposite: to prepare graphene-silver nanocomposite, 1.7 g of silver nitrate was initially dissolved in 100 ml of deionized water and then 0.62 g of naoh was added into the solution. the resulting brown ag2o deposition was filtered and dissolved in 100 ml of ammonia. [ag (nh3)2+] complex was formed in this stage; then, 7.2 g of oleic acid added into the stirring solution. after stirring for 90 min, 0.1 g of graphene oxide was added (lan et al., 2014). in addition, 90 ml of mango extract was poured into the solution, and it was stirred for half an hour. finally, it was placed under uv lamp for 8 hours. then, the solution was centrifuged at 6000 rpm for 15 min and dried at 50°c in an oven for 12 hours. several techniques such as sem, xrd and ft-ir were used to verify the synthesis of ag-rgo nanocomposite. disinfection test: samples of sewage effluents were collected from pegah tehran dairy co. and used to evaluate the efficiency of the synthesized disinfectant. the samples were collected before entering the chlorination unit (iso 19458:2006 water quality 449 int. j. aquat. biol. (2020) 8(6): 447-454 sampling for microbiological analysis, 2006). after transferring to the laboratory, pour plate procedure (mixed culture) was used for microbial cultivation using violet red bile lactose (vrbl). the effects of different parameters such as ph, adsorbent dose and contact time on the removal efficiency of the nanocomposite were investigated (ph = 5, 6, 8 and 9 with a volume of 50 ml solution, concentrations of 0.8, 1.7 and 3.4 g/l of composite; contact time = 0, 60 and 90 min). nitric acid and 0.1 m sodium hydroxide were used to adjust the ph of aqueous solution. after preparing vrbl medium from wastewater, 1 ml of composite solution and 1 ml of bacterial suspension containing 9×104 cfu/ml were added into each of the plates. then it was incubated at 37°c for 24 hours. after cultivating bacteria, brilliant-green bile lactose (bgbl) medium was used to confirm coliform of the grown colonies. formation of turbidity and gas in durham tube confirmed the existence of coliform of grown colonies, and finally, colonies were counted (iso 4832:2006 microbiology of food and animal feeding stuffs horizontal method for the enumeration of coliforms colony-count technique, 2006). the experiment was conducted with three replicates. results determination of ag-rgo nanocomposite properties using sem images: scanning electron microscope (sem) images of ag-rgo nanocomposite in different sizes are shown in figure 1. the presence of the reduced graphene oxide-silver nanocomposite is clearly visible. in addition to graphene plates, silver nanoparticles were accumulated in aggregates as seen as a mass due to their small size (fig. 1). analysis of ag-rgo nanocomposite using ft-ir spectroscopy: figure 2 shows the peaks of ag-rgo nanocomposite using ftir spectroscopy. peaks of oh and c=o symmetric stretching vibration and c-oc asymmetric stretching vibration, as well as c=c binary bond appeared at 3637, 1759, 1051, and 1655 cm-1, respectively. since this study aimed to reduce graphene oxide to graphene using mango extract, width of oh spectrum was reduced by decreasing graphene. analysis of ag-rgo nanocomposites using xrd pattern: xrd patterns of graphene/silver nanocomposite is portrayed in figure 3. the results of infrared spectroscopy confirmed the presence of c-c bonds on the surface of graphene/silver nanoparticles. characteristic peaks of the silver nanoparticles were observed in 400-800 nm. moreover, true peak in a nanometer wave number could be a reason for the existence of the nanoparticles in nanocomposite. effect of ph on the efficiency of silver/graphene figure 1. different magnifications of sem image of ag-rgo nanocomposite. figure 2. ft-ir spectrum of ag-rgo nanocomposite: the peaks of oh, c=o, c-o-c and c=c bonds appeared at 3637, 1759, 1051, and 1655 cm-1, respectively. 450 bahri et al./ removal of coliform bacteria using graphene-silver nanocomposite nanocomposite in coliform removal: the efficiency of ag-rgo nanocomposite for the removal of coliform bacteria increased by a decrease in ph (fig. 4). maximum adsorption capacity was observed at ph=5. figure 5 depicts adsorption capacity of graphenesilver nanocomposite for coliform bacteria. adsorption capacity of ag-rgo nanocomposite increased by decreasing ph, and maximum adsorption capacity was observed at ph=5. effect of time on the removal of coliform bacteria by graphene-silver nanocomposite: the effect of time on the percentage of coliform removal from solution using graphene-silver nanocomposite is shown in figure 6. as time increased, adsorption capacity increased and the highest removal percentage was observed after 90 min. figure 7 indicates adsorption capacity of graphene-silver nanocomposite in removing coliform bacteria from solution. maximum adsorption capacity was found at 90 min. effect of adsorbent dose on the removal of coliform bacteria using graphene-silver nanocomposite: this study was conducted using concentrations of 0.8, 1.7 and 3.4 g/l of nanocomposites and constant concentrations of coliform colonies (9×104cfu/ml) in a fixed volume of solution (50 ml) and after 0, 60 and 90 minutes. percentage of the removal of coliform bacteria was increased by increasing the dose of nanocomposite in solution. the highest removal percentage was observed in the adsorbent dose of 3.4 g/l (fig. 8). figure 9 depicts absorption capacity of graphene-silver nanocomposite in different doses for removing coliform bacteria. as nanocomposite dose increased, the absorption capacity increased. discussions based on the results, bacterial removal efficiency figure 3. xrd pattern of ag-rgo nanocomposite: the peaks shown in 400-800 nm confirm the proper formation of nanoparticles. figure 4. the effect of ph on the removal of coliform bacteria from aqueous solution by ag-rgo nanocomposite at ph = 5, 6, 8, and 9. the minimum and maximum removals were observed at ph 5 and 9, respectively. figure 5. adsorption capacity of ag-rgo nanocomposite against the coliform bacteria at ph = 5, 6, 8, and 9. the minimum and maximum removals were observed at ph 5 and 9, respectively. figure 6. the effect of exposure time on the removal of coliform bacteria from aqueous solution by ag-rgo nanocomposite at 0, 60 and 90 minutes. the minimum and maximum removals were observed at 0 and 90 minutes, respectively. 451 int. j. aquat. biol. (2020) 8(6): 447-454 increased by decreasing ph, so that the maximum removal rate of coliform bacteria was observed at ph=5. song et al. (2016) conducted a study on the removal of e. coli and staphylococcus aureus using graphene-silver nanocomposite at ph=5.5 to 5.8. they found that the highest percentage of bacterial removal is observed at ph=5.5. shao et al. (2015) also studied antibacterial activity of silver/cellulose nanocomposites for removing e. coli, s. aureus, bacillus subtilis, and candida albicans at ph=5.5 to 5.8. their study demonstrated that the highest removal efficiency is occurred at ph=5.5. li et al. (2018) found that graphene-based nanocomposites at ph=5 have the highest disinfection efficiency of e. coli and s. aureus. in general, ph has a significant effect on the use of nanocomposites during the removal process of coliform bacteria. silver nanoparticles (agnps) are converted into silver ions in aqueous solutions. the amount of free silver ion has a direct relationship with the amount of agnp oxidation. it is notable that oxidation of these particles is higher in aqueous solutions with lower ph. silver ion causes the death of these microorganisms by contacting bacteria and damaging cell membrane. therefore, lower ph leads to the greater and faster release of silver ions and naturally results in the increase of disinfection property (shao et al., 2015; li et al., 2018). another important factor in the removal process of coliform bacteria is the time to expose organic waste with disinfectant. in our results, the bacterial removal rate increased as time increased, so that the highest percentage of bacterial removal was observed at 90 min. chang et al. (2016) compared antimicrobial behavior of silver-coated carbon nanocomposites against e. coli and s. aureus at a time duration of 0 to 25 min indicating that bacterial removal increased as the time increased and maximum (99.99%) bacterial removal was obtained after 25 min. biswas and bandyopadhyaya (2016) investigated disinfection of water using activated carbon impregnated with silver nanoparticles. they sought to remove e. coli bacteria with a constant number of bacterial colonies (104cfu/ml) over a period of 0 to 30 min. their figure 7. adsorption capacity of ag-rgo against coliform bacteria during time contact of 0, 60 and 90 minutes. the minimum and maximum removals were observed at 0 and 90 minutes, respectively. figure 8. the effect of nanocomposite dose on the removal of coliform bacteria from aqueous solution by ag-rgonanocomposite at concentrations of 0.8, 1.7 and 3.4 g/l of nanocomposite and 96 × 104 cfu/ml of bacterial suspension. minimum and maximum removals were observed at concentrations of 0.8 and 3.4 g/l, respectively. figure 9. adsorption capacity of ag-rgonanocomposite against coliform bacteria at concentrations of 0.8, 1.7 and 3.4 g/l of nanocomposite and 96 × 104 cfu/ml of bacterial suspension. minimum and maximum removals were observed at concentrations of 0.8 and 3.4 g/l, respectively. 452 bahri et al./ removal of coliform bacteria using graphene-silver nanocomposite results showed that maximum amount of bacteria removal is occurred within 30 min with a adsorption does rate of 29.8 μg/l. moteriya et al. (2017) examined the composition of silver nanoparticles synthesized with leaf of cassia roxburghii against fungi, gram-positive, and gram-negative bacteria. they conducted their experiment at contact time of 0 to 120 min. their results showed that the removal process of bacteria experienced an increasing trend as time increased, however it did not significantly change from 30 to 120 min. zazouli et al. (2016) studied deactivating e. coli bacteria in water via silver nanoparticles with ultraviolet radiation over a period of 10 to 60 min. it was found that bacterial removal process increased as the contact time increased, so that maximum efficiency (100% removal of e. coli bacteria) was achieved at 60 min. in general, when the contact time increases, the amount of nanoparticles binding to the bacteria and their penetration into the cell increase. moreover, by increasing the contact time more silver ions are produced and thus the bacterial removal process increases. the results of antimicrobial property determination of ag-rgo nanocomposite showed that there is a direct relationship between concentration of nanocomposite and percentage of bacterial removal. the higher concentration of nanocomposite in solution results in the higher removal rate of coliform. in the present work, the highest removal efficiency was observed at a concentration of 4.3 g/l. zhang et al. (2016) used 25.6 to 50 μg/ml of separated silver nanoparticle-decorated magnetic graphene oxide to remove e. coli and s. aureus from a solution. their results suggested that the removal percentages of these bacteria increase by increasing adsorbent dose. the highest removal percentage applying an adsorbent dose of 50 μg/ml was 99.99 and 99.96% for e. coli and s. aureus, respectively. parandhaman et al. (2015) investigated antimicrobial behavior of silica-silver nanocomposite in disinfecting gram-negative bacteria of e. coli and pseudomonas aeruginosa. antibacterial activity of this nanocomposite was examined at a dose range of 0.1 to 2 μg/ml. thepr results indicated that increasing the adsorbent dose increases the bacterial removal efficiency, so that the highest removal percentage of bacteria with an adsorbent dose of 2 μg/ml was 99.98 and 99.73% for e. coli and s. aureus, respectively. chandraker et al. (2017) studied antibacterial properties of amino acid functionalized silver nanoparticles decorated on graphene oxide sheets. antibacterial activity of this nanocomposite was performed for gram-negative bacteria of e. coli and gram-positive bacteria of s. aureus with an adsorbent dose of 20 to 100 μg/ml. based on their results increasing the selected adsorbent dose increases antibacterial activity, so that the highest bacterial removal (100%) was achieved in the adsorbent dose of 100 μg/ml for both e. coli and s. aureus. conclusion this study investigated the effect of graphene-silver nanocomposite on the removal of coliform bacteria from dairy effluents. analysis of different variables on the antibacterial behavior of ag-rgo nanocomposite indicated that there is an increasing trend in the removal of coliform bacteria by increasing ph, so that the highest removal efficiency of coliform bacteria was observed at ph=5. moreover, as time increased, an increasing trend was observed in the removal of coliform bacteria, so that the highest bacterial removal process occurred at 90 min. as the selected dose increased, an increasing trend occurred in the removal of bacteria, and the highest removal rate of coliform bacteria was observed in the adsorbent dose of 3.4 g/l. in general, graphene-silver nanocomposite has high ability to remove coliform bacteria, and its disinfectant activity has a direct relationship with ph of solution, exposure time and disinfectant dose. abbreviations: trihalometanes (thm); ultrasonic homogenizer (uh), crystal violet neutral red bile lactose agar (vrbl), brilliant green lactose bile broth (bgbl), scanning electron microscope(sem), fourier transform infrared spectroscopy (ftir), and. x-ray diffraction (xrd). 453 int. j. aquat. biol. (2020) 8(6): 447-454 acknowledgement this manuscript was supported by iran national science foundation (insf) under the contract no. 97002416 and chinese academy of science president’s international fellowship initiative. grant no. 2021vea0004. references alavian s.s., hamidian a.h., ashrafi s., eagderi s., khazaei m. (2018). study on age-related bioaccumulation of some heavy metals in the soft tissue of rock oyster (saccostrea cucullata) from laft port – qeshm island, iran. iranian journal of fisheries sciences, 16: 897-906. bitton g. (2005). wastewater microbiology. 3rd. new york: john wiley and sons. 746 p. changy.n., gong j.l., zeng g.m., ou x.m., song b., guo m., zhang j., liu h.y. (2016). antimicrobial behavior comparison and antimicrobial mechanism of silver coated carbon nanocomposites. process safety and environmental protection, 102: 596-605. chitra k., annadurai g. 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(2021) 9(1): 55-65 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology review article an overview of the pesticides’ impacts on fishes and humans eric amenyogbe1,2, jian sheng huang*1,2, gang chen*1,2, zhongliang wang1,2 1college of fisheries, guangdong ocean university, zhanjiang 524025, china. 2guangdong provincial key laboratory of aquaculture in the south china sea for aquatic economic animal of guangdong higher education institutes, laboratory of fish aquaculture, zhanjiang 524025, china. s article history: received 16 august 2020 accepted 12 november 2020 available online 2 5 february 2021 keywords: pesticides immune system genotoxicity bioaccumulation abstract: agrochemicals, also known as pesticides include nematicides, molluscicides, rodenticides, herbicides, fungicides and insecticides, can control pests, weeds, fungi, rodents, etc. the accumulation of pesticides in the food chain and water has harmful effects on humans and animals. despite the advantages provided by pesticides, aquatic organisms and human health are affected as the results of continuous usage of pesticides and issues of building up of chemical substances in aquatic organisms, such as fish. pesticides must be lethal to the targeted species without any effect on non-targeted ones. pesticides have harmful effects on the nervous system. other pesticides are known to be carcinogenic substances. this review discussed the effects of pesticides on the immune system, protein, chromosomes, behavior, enzymes, growth, bioaccumulation, genotoxicity and changes in blood biochemical parameters of fish and humans and suggested some possible ways of mitigating such effects. introduction the human population has rapidly increased in the last few decades leading to suburbanization, mechanization, or industrialization (al-mamun, 2017). the nutrient fortification of water environments, climate change, acid rain and contamination by insecticides, metals and manufactured toxic constituents cause such anthropogenic troubles (kamble, 2014). the death of wildlife, including marine and freshwater organisms, a growing hazard to human wellbeing, includes chronic respiratory disease, damage to organs (e.g., brain, lung, heart, liver and kidneys) and algal bloom in many water bodies, are some of the pesticidal effects (al-mamun, 2017). increasingly, water bodies are getting exposed to contaminants or pollutants owing to human actions, such as farming, industrialization and domestic activities (lu et al., 2015; al-mamun, 2017). the use of agrochemicals is essential for controlling pests and increasing food production for the universal population (khan et al., 2013; al-mamun, 2017). globally, serious concerns have been raised about the existence of these toxic *correspondence: jian sheng huang and gang chen doi: https://doi.org/10.22034/ijab.v9i1.972 e-mail: gdoucg@126.com, huangjs@gdou.edu.cn chemicals in both aquatic and terrestrial environments or ecosystems (alrumman et al., 2016). according to recent studies, industrial chemicals, pesticides, and other metals affect the endocrine system of several organisms, including humans (haseena and malik, 2017). the presence of these chemicals may negatively affect the normal function of the endocrine system and cause growth disorders in many species, including invertebrates and complex animals, also in humans; it can cause cancer, genetic disorders, respiratory diseases and neurological effects (bibi et al., 2016; haseena and malik, 2017). this review discussed the effects of pesticides on fish and human focusing on the immune system, protein, chromosomes, behavior, enzymes, growth, bioaccumulation, genotoxicity and changes in blood biochemical parameters of fish and humans and suggested some possible ways for mitigating such effects. pesticides: well-thought-out as a distinctive class of chemical compounds, according to who, pesticides are utilized to kill a wide array of pests that comprises 56 amenyogbe et al./ impacts of pesticides on fishes and humans rodents, weeds and insects. they are utilized with the objectives of improving yield and qualities of farm products (sharma et al., 2020). pesticides are wellthought-out as hypothetically toxic elements released into the environment to kill fungi, weeds, insects, rodents, etc. the physical and chemical properties of pesticides are different. they are categorized based on their properties (us environmental protection agency, 2018). currently, there are three most common categories of pesticides that are extensively used, and the categories are based on the type of the entrance, pesticide utility and the entity (yadav et al., 2017). based on the harmfulness of pesticides, the world health organization (who) grouped them into four categories, including exceedingly hazardous, greatly hazardous, abstemiously dangerous and considerably harmful (al-mamun, 2017). the most popular pesticides among these are herbicides (grace communications, 2018). the majority of the pesticides is anticipated to aid as crop protection, which generally protects crops from weed growth, fungal infestation and insect attacks. pesticides are categorized based on the target animal (organism) and chemical structure (organic, inorganic, synthetic and biological substances) (yadav et al., 2017; li and jennings, 2017). generally, pesticides are biological agents, such as a virus, bacterium, or fungus that discourages, debilitates, eradicates, or depresses pests (us environmental protection agency, 2018). despite the advantages, pesticides have harmful effects on humans and other organisms (hong et al., 2019). benefits of pesticides: the beneficial effects of pesticides are important. controlling of pests, weeds, rodents, etc. results in the high productivity and better quality of crops, which also results in additional economic revenue. the above benefits might help in the medical and educational needs of children of farmers, leading to better education and good health of the general public of a nation. general acknowledged commercial incentives are dependent on the polluter pays standard or principle, comprising taxes, user fees and the licensing fees. nevertheless, the benefits are mainly social benefits, including risk reduction and capacity to increase labour, hence by creating opportunities for employment (popp et al., 2013). the increases in outputs and production have been related to some factors comprising the use of pesticides (fertilizers). the reduction of losses caused by the diseases, insect pests and weeds can reduce the quality, market value and the number of harvestable crops (aktar et al., 2009). following warren (1998), there was a remarkable increase in yield of crops in the usa during the 20th century, and considerable economic losses were observed without pesticidal use (aktar et al., 2009). usa spent 9.2 billion usd yearly on pesticides for crop protection and saved approximately 60 billion usd on crops leading to a net profit of 6.5 usd for every donated dollar on pesticides (gianessi, 2009). the pesticides can generate substantial environmental and socio-economic remunerations in the form of safe, healthy and affordable food, which contributes to secure farm incomes and enable sustainable farm management by improving the efficiency of natural resources, such as soil, and water (popp et al., 2013; al-mamun, 2017). it can be opposite when pesticides are inappropriately used. the pesticides have many advantages and play a significant role in the management of weeds, rodents, fungi, etc. and improving crop quality, yield protection and food affordability, thereby providing economic benefits to farmers and the consumers. certain ingredients of pesticides such as azadirachtin and/or glufosinate are compounds that occur naturally and are created using processes of synthetic chemistry (schwartz et al. 2004; ressel 2015; fernandes et al., 2019; mesnage et al., 2019). additionally, several plants, of which several are utilized as eatable crops, have advanced resistance means against predators like microorganisms as well as insects through the production of compounds performing like pesticides (ujváry, 2010). these naturally occurring pesticides are commonly established to have favorable wellbeing influences in humans due to the fact that they stimulate cellular stress adaption response pathways (martel et al., 2019; mesnage et al., 2019). 57 int. j. aquat. biol. (2021) 9(1): 55-65 since 1945, the utilization of pesticides has prevented approximately 7 million people from death as a result of killing pests, which spread diseases, including yellow fever, typhoid fever, bubonic plague and encephalitis (http://study.com/academy/lesson/ use-of-pesticides-benefits-and-problems-associatedwith-pesticides.html). the use of pesticides prevents the outbreaks of diseases, which contributes to improving human health. for example, malaria is reported to be responsible for the death of over 5000 a day worldwide (ross, 2005). toxic nature of pesticides: world health organization (who) reported that approximately 300,000 people died per annum as a result of pesticide poisoning (gunnell and eddleston, 2003). this happened as a result of inhalations or consumption of pesticides by a person above the threshold via accident or occupation (sharma et al., 2020). pesticide poisoning occurs as a result of relatively ignorance or unawareness of detrimental effects via inhalation of these chemicals by people. the literature available has indicated that during the gestation period, when women are exposed to ddt, there were traces of the ddt in their breast milk and umbilical cord (wolff et al., 2007; debost-legrand et al., 2016; sharma et al., 2020). it has also been observed that there was some relationship between exposure to pesticides and changes in body weight at the birth time (kezios et al., 2013). on the other hand, work-related poisoning known as occupational poisoning occurs as a result of the usage of these chemicals in the field of agriculture, manufacturing industries by the people in their daily activities (gangemi et al., 2016; darçın et al., 2017). work-related poisoning of pesticides can happen in several ways, such as ingestion, skin contacts, and inhalation (calvert et al., 2008; sharma et al., 2020). the majority of people also consumed pesticides for suicide (gunnell et al., 2007). the existence of several agrochemicals popularly known as pesticides in water bodies such as dams, lakes, streams, and rivers generates a multifarious exposure of these chemicals hypothetically poisonous to aquatic organisms (covert et al., 2020; norman et al., 2020). when fish species are exposed to poisonous material such as pesticides, unexpected and extreme death may occur as the results of acute toxicity. the most ostensible signs of serious poisoning in fish as the result of pesticide usage consist of severe reaction to external stimuli, muscle spasms, and sudden fast swimming in circles, lethargy, pallor, and forward extension fins (sabra and mehana, 2015). signs such as respiratory dysfunction and suffocation, disruption of nerve functions, and neurological disorder are some of the major clinical signs that can result in the mortality of fish (banaee et al., 2011, 2012). who reported the median lethal dose (ld50) of different pesticides in rats and other laboratory animals (table 1). pesticidal effects on humans: pesticides and their residues can cause harmful effects on human health. the pesticides can enter into the human body and the food chain through direct contact, foods, and polluted air and water are not in dispute. as a result, human health is currently under threat of continuous use of pesticides. pesticides from the agricultural field result in dire ailment in humans (lee et al., 2011b). many signs or symptoms, such as skin rashes, dizziness, poor concentration, nausea, body aches, cramps, impaired vision and headache occurred because of pesticide poisoning (pan-germany, 2012; al-mamun, 2017). these signs and symptoms are related to the amount and toxicity of the specific pesticide, mode of activity, application mode, frequency and duration of direct contact with person and chemicals. the direct contact or exposure to sub-lethal amount of these chemicals for a long period, such as several months, years and decades, could lead to chronic diseases (pan-germany, 2012). with regard to the chronic poisoning of pesticides, the signs cannot be observed. table 1. the hazard ratings of a lethal concentration of the relative toxicity of the agricultural pesticides. toxicity lc50(ml) minimal >100 slight 10-100 moderate 1-10 high 0.1-1.0 extreme 0.01-0.1 super < 0.01 source: https://www.beyondpesticides.org/programs/wildlife/fish 58 amenyogbe et al./ impacts of pesticides on fishes and humans personnel working in the field of agriculture can be affected in great jeopardy, but others are also at risk through the food chain and polluted air and water (almamun, 2017). the prevalence of chronic illnesses has begun to increase as pesticide usage has grown in the last few decades. several studies reported that pesticide exposure has harmful effects on the nervous system, reproduction, respiratory systems and cardiovascular system in humans (mostafalou et al., 2012). chronic illnesses, such as adult brain cancer, lymphocytic leukemia (cll), prostate cancer, childhood cancer, alzheimer's disease (ad), parkinson's disease (pd), reproductive disorders and hormonal imbalance, respiratory diseases, breast pain and infertility were also reported in humans due to pesticide exposure (lee et al., 2011a; tanner et al., 2011; abdullah et al., 2011; andersen et al., 2012; cocco et al., 2013; zaganas et al., 2013; schinasi and leon, 2014; al-mamun, 2017; polanco rodriguez et al., 2017; bonner et al., 2017; sabarwal et al., 2018). some adverse effects of pesticides on aquatic ecosystems and pesticidal effects on fish: currently, the use of herbicides has been increased tremendously and studies reported that it influences the environment and non-targeted organisms (pérez et al., 2011; pérezparada et al., 2018). although it was first intended to control only unwanted plants (stenersen, 2004), it has an adverse effect on non-targeted aquatic organisms and other animals according to the recent studies (tulgar and arınç, 2018; pérez-parada et al., 2018). water sources, such as rivers, ponds, etc., hold pesticides at the bottom, which affects the reproduction and behaviors of living organisms in the aquatic ecosystem leading to the extinction of species (tulgar, 2018). several living organisms are dependent on planktons as food sources. these planktons mostly collect the pesticidal residues, which are transferred to fish and other invertebrates (pereira et al., 2013; tulgar, 2018). the entrance of pesticides into aquatic environments has many implications. fish are affected directly or indirectly by the use of pesticides. the vulnerable parts of fish to chemical exposure are liver, kidney, brain and gills (mahmood et al., 2016). the widespread usage of chlorpyrifos increase the lethal load in aquatic ecosystems, causing antagonistic effects on non-targeted species, including fish (palanikumar et al., 2014). severe and long-lasting toxic effects of chlorpyrifos in diverse species of fish were widely observed (anita et al., 2016). sublethal toxic effect of chlorpyrifos in aquatic ecosystems can prompt developmental, hematological, histopathological, biochemical, neurobehavioral, oxidative and morphological changes, whereas the lethal intensities are responsible for mass mortalities of nontargeted species, weight loss, low disease resistance and sterility in fish (sunanda et al., 2016; banaee et al., 2019a, b, 2020; hatami et al., 2019). according to jadhav and pawar (2018), the concentration of endosulfan is more toxic to fish than any other aquatic organisms. one of the extensively used groups of insecticides in agriculture is organophosphates (ops). sublethal doses of organophosphates resulted in different physiological injuries, such as reproductive failure, predator avoidance and feeding problem (little et al., 1990). the continuous utilization of fungicides, herbicides and insecticides have been associated with the reduction of animal and fish population. due to overuse of pesticides, the population of different species (peregrine, falcon, bald eagle and osprey) are declining (scholz et al., 2012). the extensive usage of pesticides has several implications, such as bioaccumulation, genotoxicity, the immune system effects, protein effects, chromosomal effects, behavioral effects, enzymes effects, growth and changes in blood biochemical parameters on fish and humans. bioaccumulation of pesticides: pesticides could accrue in aquatic animals via several means; directly from water through skin or gills (bioconcentration), the ingestion of polluted/contaminated food (biomagnification) and suspended particles uptakes (ingestion) (van der oost et al., 2003; banaee et al., 2015). bioaccumulation of several kinds of pesticides directly affects fish (rao and pillala 2001; clasen et al., 2018). the effect of pesticides in fish results in fish behavioral changes. sluggish movement of fish and alteration of their swimming ability makes them more 59 int. j. aquat. biol. (2021) 9(1): 55-65 susceptible to the predators, reduce their feeding ability, maintain their position and defend their territories (ullah et al., 2014; srivastava et al., 2016). the behavior of an organism offers an exceptional viewpoint to connect physiology and natural balance between animals and their surroundings (srivastava and singh, 2013; srivastava et al., 2016). the behavior of the organism permits them to adapt internal and external stimuli, which enables them to adjust to ecological variables (srivastava et al., 2016). according to srivastava and singh (2014) and ullah et al. (2014), the sub-lethal concentration of pesticides in water ecosystems resulted in structural and functional variations in organisms. the bioaccumulation effects of several kinds of pesticides on aquatic organisms, such as fish, are immeasurable. immune system: the low concentration of pesticides disturbs the fish immune system (farid and el-sayed, 2015) and also work as an impersonator of sex hormones, and trigger uncharacteristic sex growth, the feminization of males, uncharacteristic sex ratios and infrequent coupling behavior. indirectly, pesticides have impacts on fish through interfering with the diet source and sporadic habits (satyavardhan, 2013). the function of the immune system is modified by the pesticides leading to immune depression, unrestrained cell growth and modification of host resistance comprising innate immunity and assimilated immunity. the most vital feature or possession of fish is its immune system against pathogens. fish become vulnerable to infectious diseases and pathogens when sub-lethal concentrations of pesticides are introduced (zelikoff et al., 2000). protein: several studies have been reported the decrease of protein content under stress in fish and other organisms (tiwari and singh, 2009). as reported by tiwari and singh (2004), there was a significant reduction in the levels of protein in gills, blood, intestine, muscles and liver of channa punctatus when exposed to oleandrin and the introduction of c. carpio to endosulfan. also, there was a significant reduction in protein content of colisa fasciatus and tor putitora when exposed to cypermethrin (singh et al., 2010; ullah et al., 2014). there was a substantial reduction of protein content in c. carpio when exposed to endosulfan (bibi et al., 2014). the introduction of clarias batrachus and labeo rohita to cypermethrin and malathion also revealed the reduction in the protein content (thenmozhi et al., 2011). a study by bose et al. (2011), revealed the decrease in protein content of the liver of oreochromis niloticus when exposed to thiamethoxan. several studies showed the reduction of protein contents in fish when exposed to pesticides. enzymes: all metabolic processes in the cell need enzyme catalysis to sustain. metabolic pathways are dependent on enzymes. enzymes help body to break down large complex molecules into small molecules, such as glucose. pesticides in animals provoke some enzymatic pathways. as reported by srivastava et al. (2016) and das and mukherjee (2003), brain acetylcholinesterase action was observed to be reduced after 45 days of exposure to cypermethrin. lactate dehydrogenase action in the brain and liver were elevated but inhibited in the kidney, and succinate dehydrogenase (sdh) and atpase activities were depleted in brain, kidney and liver because of the cypermethrin toxicity (das et al., 2003). ogueji and auta (2007) reported the variations in biochemical parameters, such as alkaline phosphatase, glutamic oxaloacetic acid transaminase, glutamic pyruvic acid transaminase, triglycerides, cholesterol, protein and serum glucose in c. gariepinus under lamda-cyhalothrin exposure. genotoxicity: the genotoxic effect has become the key biomarkers for assessing contamination related damages. biomonitoring programs have revealed the genotoxicity and wellbeing effects (hughes and hebert, 1991). genotoxicity is defined as an asset possessed by specific elements, which is dangerous to the genetic evidence confined in animals. several dynamisms can damage dna, rna and other genetic resources (hong et al., 2020). the assets of genotoxicity can be applied only to the constituents that damage the genetic statistics. an element that causes genotoxicity is identified as genotoxin (srivastava et al., 2016). many studies revealed that genotoxins are mainly birth-defect factors, mutation60 amenyogbe et al./ impacts of pesticides on fishes and humans causing agents, mutagens and carcinogens. however, there is evidence linked to pesticides (srivastava et al., 2016). cancer is the unrestrained development of cells inside the body (kushwaha et al., 2012). the perfect model for checking wastewater quality and genotoxins is fish, since fish can metabolize toxic substances. fish have small size micronuclei because their chromosomes are considerably small (nagpure et al., 2007). the micronuclei are shaped in the fish cells. the development of micronuclei is based on the degree of propagation of cells that is also based on the fish species, target tissues, conditions of environments and the type of harmful waste. the rapd profile and comet assay in c. punctatus was changed when the fish was introduced or exposed to sub-lethal concentration pff, a sign of its genotoxic possibility to aquatic animals (pandey et al., 2018). however, processes for the collaborative influence of contaminants in the cells of fish are still not completely understood. chromosomes: chromosomal abnormalities are triggered by dichlorvos at 0.01 ppm concentration in the form of stubbed arms, extra fragments, attenuation, chromatid breaks, sub-chromatid breaks, chromatid gaps, centromeric gaps and pycnosis in kidney cells of c. punctata after 24, 48, 72 and 96 hours of exposure (farid and el-sayed, 2015). similarly, dichlorvos toxicity was associated with the changes in dna duplication that triggered the mutation and cellular hyper propagation (sabra and mehana, 2015). changes in blood biochemical parameters: the biochemical parameters of blood can be the indicators of pesticidal toxicity. when severe damages are caused to some tissues with special reference to the liver, a combination of various biochemical parameters could shrink considerably in cells, which can reduce specific biochemical dynamics in fish blood when introduced to pesticides (banaee et al., 2008). distractions and injuries of tissue in organs could lead to the reduction in survival, development, fitness and high probability of vulnerability to pathological alteration. the rate of recurrence and tissue lesion intensity is based on the pesticidal concentrations and the duration of the toxins introduced to fish (sabra and mehana, 2015). as reported by fanta et al. (2003), histopathological lesions were noted in the liver of c. carpio and cirrhinus mrigala, (velmurugan et al., 2009). according to banaee et al. (2012), the histopathological alterations were observed because of the exposure of dichlorvos and diazinon treated fish for 10 to 30 days. behavioral variations: fish behavior is mostly affected by the uptake of contaminants. fish eat and accumulate various pollutants, including pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons and heavy metals (srivastava et al., 2016). according to srivastava and singh (2013), the acetylcholinesterase action of pesticides disturbed the existence and caused the death or mortality of c. batrachus when exposed to propiconazole and mancozeb. srivastava and singh (2013) also observed that compounds that hinder cholinesterase action disturbed the regular movements of fish. as a result, fish were further predisposed and vulnerable to infections and pathogens (satyavardhan 2013; gill and raine, 2014; srivastava et al., 2016). imbalanced swimming, darting and erratic movements and hypoexcitability can be observed in l. rohita, c. carpio, o. mossambicus, c. catla and c. mrigala due to the introduction of sodium cyanide (ullah et al., 2014). growth disorders: deficiency in standard growth and development can diminish the fish survival rate. larvae of fish can be directly exposed to pesticides through parenteral in viviparous fish (viant et al., 2006). according to todd and leeuwen (2002), there was an influence of carbaryl pesticides on the fish embryo. decreased development of fish comprises of anomalies in nourishing or eating behaviors, such as dysfunction in the metabolism process and waste of energy caused by insecticide exposure (sabra and mehana, 2015). conclusion and future directions: in the water cycle, chemicals contaminate groundwater, and most of these are transferred to lakes, rivers and ponds. they also hinder the existence of aquatic life by 61 int. j. aquat. biol. (2021) 9(1): 55-65 polluting the sources of food of the aquatic organisms. aquatic organisms and humans are affected directly or indirectly due to the ingestion of contaminated fruits, vegetables, fish and water (simone et al., 2018; grace communications, 2018), which are also the main sources of food and energy for organisms. several studies have reported the activities of agriculture as the primary sources that release toxic components into the aquatic environments. the intake of pesticides by fish accumulates in human body systems and bio-intensification through the food chain causes environmental and health concerns. the nontarget organisms have been continually affected by persistent organic pollutants in the ecosystem and increase probabilities of the disruption of the endocrine system, immune system, protein, chromosomes, behavior, enzymes, growth of fish and humans, bioaccumulation, genotoxicity and changes in blood biochemical parameters as long-term chronic effects. biological sustainability of water environments is under serious threat due to heavy pesticidal usage (mahmood et al., 2016). eliminating the risks from the use of pesticides is impossible. however, the risks can be reduced e.g. there is a need for environmentally friendly pesticide formulation, which can minimize the destructive effects of pesticide utilization. the appropriate dosage of pesticides is essential and required to reduce the risks. crops that are grown without chemicals can be useful for human and aquatic organisms. furthermore, the harmful pesticides, which are prohibited, must be strictly enforced by imposing jail terms and massive fines. it is believed that these approaches will help to save aquatic lives as well as humans. acknowledgement we acknowledge the china agriculture research system (cars-47) and southern marine science and engineering guangdong laboratory (zhanjiang) (zjw-2019-06) authors’ contributions: all authors contributed equally to this study. references abdullah n.z., ishaka a., samsuddin n., mohd rus r., mohamed a.h. 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(2020) 8(3): 224-227 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology short communication checklist of gastropod molluscs in west coast of algeria khadidja meziane1, ahmed kerfouf1, affef baaloudj*21 1laboratory of eco-development of spaces, university of djillali liabes, sidi bel abbes 22000, algeria. 2laboratory of biology, water and environment (lbee), university of 8 may 1945, guelma 24000, algeria. article history: received 29 may 2020 accepted 24 june 2020 available online 2 5 june 2020 keywords: gastropod, intertidal zone, molluscs, rocky, algeria. abstract: samplings were performed during 2015-2019 from the rocky intertidal zone of the west coast of algeria to determine its gastropods’ inventory. the regular survey from 18 stations revealed the presence of 28 species belonging to 12 families. the patellids was most diverse family with 17.85% of the total sampled gastropods, followed by buccinidae (14.28%), trochidae (14.28%), muricidae (10.71%), mitidae (7.14%), certhiidae (7.14%), vermetidae (7.14%), aplysiidae (7.14%), cymatiidae (3.57%), haliotidae (3.57%), calliostomatidae (3.57%) and turbinids (3.57%). in addition, the crustaceans living under these mollusk shells were reported. introduction aquatic malacological fauna is not well-known in the west coast of algeria. most of the pervious inventories of the benthic fauna of continental shelf in the west coast of algeria have not been updated (dautzemberg, 1895; pallary, 1900; llabador, 1935; kerfouf, 2007). the absence of synthesis work led us to update the list of gastropod mollusks in the substrate of the west coast of algeria by sampling these regions. materials and methods regular explorations were conducted at 18 stations in a natural or artificial hard substrate of the west coast of algeria (table 1). visible species were searched with the naked eye in all the preferred environments and the specimens were collected by hand (lewis and magnuson, 2000; micharlik-kucharz et al., 2000; falkner et al., 2001; bonham et al., 2002; darby et al., 2002; labaune and magnin, 2002; cameron and pokryszko, 2004; kisset et al., 2004; pokryszko et al., 2006; boschi and baur, 2007a, b). the sampling performed at the infra littoral and midlittoral levels, in a depth not exceeding 3 m. in each station, the surveyed surface was between 1 and 6 m². an additional list of the mollusk shell hosts was provided. *correspondence: affef baaloudj e-mail: bafef@yahoo.fr the 18 surveyed stations were chosen randomly and depending on the site accessibility. each station has its own characteristics, by the nature of the substrate, hydrogen supplies and terrigenous and its biodiversity, and subject to anthropogenic pressures, that impacted the malacological diversity of each station. results and discussions a total of 28 species of gastropod mollusks belonging to 12 families were collected. checklist: family patellids: 17.85% with 5 species (17.85%) • patella nigra (da costa, 1791) • patella ferruginea (gmelin, 1791) • patella rustica (linneaus, 1758) • patella ulyssiponensis (gmelin, 1791) • patella caerulea (linneaus, 1758) family buccinidae: 4 species (14.28%) • buccinulum corneum (linnaeus, 1758) • pisania striata (gmelin, 1791) • pisania maculosa (lamarck, 1819) • cantharus dorbignyi (payraudeau 1826) 225 int. j. aquat. biol. (2020) 8(3): 224-227 family trochids: 4 species (14.28%) • monodonta articulata (lamarck, 1822) • monodonta turbinata (born, 1780) • gibbularari lineata (michaud, 1829) • gibbula richardi (payraudeau 1826) family muricidae: 3 species (10.71%) • stramonita haemastoma (linneaus, 1767) • muricopsis cristata (brocchi, 1814) • phyllonotus trunculus (linneaus, 1758) family mitidae: 2 species (7.14%) • pusia ebenus (lamarck, 1811) • mitra cornicula (linneaus, 1758) family certhiids: 2 species (7.14%) • certhium vulgatum (bruguière, 1792) • cave certhium (risso, 1826) family vermetidae: 2 species (7.14%) • vermetus triquetus (bivona-bernardi, 1832) • dendropoma petraeum (monterosato, 1884) family aplysiidae: 2 species (7.14%) • aplysia punctata (cuvier, 1803) • aplysia fasciata (poiret, 1789) family cymatiidae: 1 species (3.57%) • cymatium cutaceum (linneaus, 1767) family haliotids: 1 species (3.57%) • haliotis tuberculata lamellosa (lamarck, 1822) family calliostomatidae: 1 species (3.57%) • calliostoma zizyphinum (linneaus, 1758) family turbinidae: 1 species (3.57%) • astraea rugosa (linneaus, 1758) the hosted decapod of collected shells of the gastropod were as following: • mitra zonata (marryatt, 1817) • corraliophylameyondorffi (calcara, 1845) • cerithium alucastrum (brocchi, 1814) • semcassis saburon (bruguière, 1791) • phalium granulatum (von born, 1778) • cassidaria echinophora (linneaus, 1758). the presence of a large number of gastropods in certain stations coincides with the absence of socioeconomic structures and low urbanization and a minimum of trampling. we noticed that the strong diversity of gastropods is accompanied by a remarkable floristic diversity, while the diversity of table 1. sampling stations in the west coast of algeria. station position town 1 35° 18’04.58’’ n1°24’09.29’’o béni saf plage du puit 2 35° 18’17.01’’ n 1°23’36.69’’o béni saf plage du puit 3 35° 18’24.73’’ n 1°22’46.96’’o béni saf plage de sidi boucif. 4 35° 21’21.44’’ n 1°17’13.59’’o plage de sidi djelloul 5 35° 17’58.00’’ n 1°28’08.16’’o plage de rechgoun 6 35° 17’50.49’’ n 1°28’26.15’’o plage de rechgoun 7 35° 19’06.43’’ n 1°28’36.78’’o ile de rechgoune 8 35° 26’10.45’’ n 1°14’20.98’’o terga plage 9 35° 33’21.56’’ n 1°11’40.96’’o sbiaat plage 10 35° 33’12.25’’ n 1°11’51.40’’o presque-ile de sbiaat 11 35° 44’31.88’’ n 0°50’12.54’’o bousfer plage. oran 12 35° 44’24.15’’ n 0°50’17.07’’o bousfer plage. oran 13 35° 44’29.95’’ n 0°50’13.68’’o bousfer plage. oran 14 35° 44’34.88’’ n0°45’16.39’’o ain el turk (digue artificielle). oran 15 35° 42’29.20’’ n 0°54’21.13’’o les andalouses plage 16 35° 42’39.44’’ n 0°54’28.34’’o les andalouses plage 17 35° 34’23.15’’ n1°09’32.35’’ o bouzedjar plage. digue artificielle. 18 35° 34’25.52’’ n1°09’24.87’’ o bouzedjar plage. 226 meziane et al./ checklist of gastropod molluscs in west coast of algeria gastropods was minimum in anthropized areas, despite the important density of certain species which, by their number of individuals, created a sort of competition for other species that find some difficulties to settle in these disturbed environments. climate change has an influence on the spatial distribution of temprature-sensitive species, and thus is likely to impact the marine environment, particularly the intertidal habitats (helmuth et al., 2010). the induced thermal stresses result frequently in detrimental physiological and behavioral responses, and in extreme cases, lead to mortality (verdelhos et al., 2015). as conclusion, a total of 28 species of gastropod molluscs were identified. since the substrate of the harvested areas was rocky, certain gastropods lead a sedentary life or with very little movement. the identified mollusc shells, which shelter crustaceans, gives us an overview of the gastropods existing in the deeper areas; these shells were rejected by the waves during winter storms, to end up in the surveyed foreshore. the richest stations in gastropod molluscs are those where the algal carpet is more remarkable. these algae serve as food for grazers and refuge for others i.e. in sites undergoing strong anthropogenic activities (pollution, discharge of waste water, etc.), the diversity of gastropods is clearly low. acknowledgments we thank reviewers for their comments and suggestions. we are grateful to dr. khelifa rassim (university of british columbia, vancouver, canada), for helpful comments. we thank the mesrs and the dgrsdt for supporting and funding this work. references bonham k.j., mesibov r., bashford r. 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(2018) 6(3): 147-156 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article effects of stocking density on the growth, survival and production of endangered bata, labeo bata (hamilton, 1822) in primary nursing mohammad rafiqur rahman*1, sirajum monira shanta1, md. abul kalam azad2, mohammad kamruzzaman hossain2, shabnam mostary2, azhar ali3, mns mamun siddiky3, md. anamul haque4 1department of fisheries biology and genetics, bangladesh agricultural university, mymensingh-2202, bangladesh. 2department of fisheries, bangladesh. 3bangladesh fisheries research institute, mymensing-2201, bangladesh. 4spectra hexa feeds limited, bangladesh. article history: received 18 april 2018 accepted 12 june 2018 available online 2 5 june 2018 keywords: labeo bata density growth survival production abstract: effect of stocking densities on the growth, survival and production of bata, labeo bata fry and fingerlings were tested in a primary nursery rearing system. the experiment was conducted for a period of 4 weeks in six earthen nursery ponds having an area of 0.032 ha each. four-day-old fry stocked at 1.0 million/ha was designated as treatment-1 (t1), 1.5 million/ha as treatment-2 (t2) and 2.0 million/ ha as treatment-3 (t3). at stocking, all fry were of same age with a mean length and weight of 1.03±0.03 cm and 0.12±0.01 g, respectively. fry in all the treatments were fed with mega commercial fish feed. physico-chemical parameters such as water temperature, transparency, dissolve oxygen, ph and total alkalinity in all the treatments were suitable ranges for fry and fingerling rearing. plankton population (both phytoplankton and zooplankton) were found to be at optimum level for fish culture. highest weight gain was observed in t1 (3.46±0.08) and lowest in t3 (1.98±0.03). final length, final weight and survival of fingerlings also followed the same trends as weight gain. fingerlings in t1 produced significantly higher specific growth rate (12.15±0.08) than t2 (11.31±0.03) and t2 (10.22±0.05). feed conversion ratio was significantly lower in t1 (0.26±0.01) than t2 (0.42±0.02) and t3 (0.65±0.01). significantly higher number of fingerlings was produced in t3 (1177700±4700) than t2 (963300±9900) and t1 (717850±7350), respectively. despite of this, consistently higher net benefits were found from t1 than t2 and t3. overall, highest growth (3.60±0.16 g), survival (71.79 ± 1.04%) and net benefits (tk. 127,087.00) of fingerlings were obtained at a density of 1.0 million hatchlings/ha. therefore, out of three stocking densities, 1.0 million fry/ha appears to be most suitable stocking density for nursing and rearing of l. bata fry and fingerlings in primary nursing. introduction the bata, labeo bata (hamilton, 1822) is one of the most important species of minor carps under cyprinidae family. it has great demand as table fish due to its deliciousness, flavor and less spiny structure (mahfuj et al., 2012). it is a freshwater subtropical species which is commonly known as ‘bangon bata’. it is well-known as different name in different countries, bata in bangladesh (rahman, 2005), bhagan in india (nath and dey, 1989), and bata labeo in nepal (shrestha, 2008). this fish contains about 15.42% of protein and 3.73% of lipid (ahmed et al., 2012). it feeds on phytoplankton, algae and soft leaves of aquatic grasses. at the first sexual maturity attained *corresponding author: mohammad rafiqur rahman doi: https://doi.org/10.22034/ijab.v6i3.458 e-mail address: mrafiqurrahamn@yahoo.com 14.12 and 14.60 cm in total length for male and female l. bata, respectively (hossen et al., 2014). like tropical cyprinids, it normally breeds in streams, rivers and flood plains during monsoon months of april to july and fecundity of females is estimated around 23,000 to 81,000 per kg body weight (hussain and mazid, 2001). labeo bata is one of the major aquaculture species in bangladesh. earlier, the fish was widely available throughout the rivers, haors, baors, beels, jheels, canals and ponds of bangladesh, but it has been seriously declining in the main streams (rahman, 2005; dahanukar et al., 2004). loss of habitat and overexploitation, indiscriminate killing of fry and 148 rahman et al./ effects of stocking density on the growth, survival and production of labeo bata fingerlings, pollution, siltation and other ecological changes are local threats to wild populations of l. bata (hossain et al., 2009; devi and ali, 2013; hossain, 2014). international union of conservation of nature (iucn), bangladesh (2000) listed l. bata as one of the endangered minor carps. many scientists have suggested that special attention is needed to protect this fish from extinction (hussain and hossain., 1999; hussain and mazid, 2001). therefore, it is very much essential to protect this fish from extinction through development of appropriate nursing and rearing technique of fry and fingerlings. the fry and fingerlings of l. bata rearing have not yet been well-developed due to its improper care and lack of appropriate rearing technology. so, a suitable culture technology for nursing and rearing of l. bata fry are very important to ensure reliable and regular supply of fingerlings. stocking density is an important aspect to take into account when ranking families or progeny groups for growth performance. fish density is a key factor affecting growth and maturation of wild and cultured fish besides food supply and its quality, genetics and materials and methods study area and experimental design: the experiment was carried out for a period of 28 days from 01 june 2017 to 28 june 2017 in six earthen ponds at the private nursery ponds of fish seed farm, trishal, mymensingh, bangladesh. the area of each pond was 0.032 ha with an average water depth of 1 meter. all the ponds were of similar shape, size, basin conformation and bottom type. three treatments differing in stocking densities of fry were employed with two replicates each. the designed treatments were 1.0 million fry/ha (treatment-1, t1), 1.5 million fry/ha (treatment-2, t2) and 2.0 million fry/ha (treatment-3, t3). proximate composition of feeds: there are two types of commercial mega fish feeds (pre-nursery and nursery) were purchased from local dealer of mega feed seller. proximate composition of the feeds (moisture, protein, lipid, ash and fiber) was analyzed according to aoac (1984) method and nitrogen free extract (nfe) by subtraction (castell and tiews, 1980). proximate composition of experimental feeds is shown in table 1. pond preparation, stocking and management: at first, the ponds were dewatered, freed from aquatic vegetation, exposed to full sunlight and had a welldesigned system of inlet and outlet. after drying, quicklime (caco3, 247 kg/ha) was spread over the pond bottom. all the ponds were filled with ground water to a depth of about 1 m. five days subsequent to liming, the ponds were fertilized with organic manure (cowdung) at the rate of 2470 kg/ha, urea 25.0 kg/ha, and tsp 12.5 kg/ha to stimulate the primary productivity of the ponds. seven days after manuring, the pond water was sprayed with dipterex at the rate of 1 ppm to eradicate harmful insects and predatory zooplankton (rahman et al., 2005). all the experimental ponds were stocked with four-day-old hatchlings of l. bata having an initial average length of 1.03±0.04 cm and weight of 0.12±0.01 g, respectively. the stocked hatchlings were fed four times in a day with commercially available mega prenursery and nursery feed commencing from the first day of stocking. the feeding rate was 24 kg/million fry/day for the first 1 week, 28 kg for the second 1 week, 32 kg for the third 1 week and 36 kg for the fourth 1 week. fingerlings were collected weekly by a fine-meshed (hapa) net for growth studies. for proper feeding, the feed was broadcasted homogenously in each time. water quality parameters: the water quality parameters such as water temperature, transparency, dissolved oxygen (do), ph and total alkalinity were monitored weekly throughout the experimental period table 1. proximate composition (% dry matter) of the supplementary feeds supplied in experimental ponds. brand name of feed type of feeds crude protein crude lipid crude fiber ash nfe* mega fish feed pre-nursery 40.57 6.97 4.89 15.58 30.05 nursery 35.52 7.03 4.95 17.56 34.94 *nitrogen free extract (nfe) was calculated as 100% (moisture + crude protein + crude lipid + crude fiber + ash) 149 int. j. aquat. biol. (2018) 6(3): 147-156 between 09.00 and 10.00 hr. water temperature was recorded using a celsius thermometer and transparency (cm) was measured by using a secchi disc of 20 cm diameter. dissolved oxygen were determined (mg/l) directly by a digital water quality analyzer hanna do meter (model-hi 9146, romania), ph by a digital ph-meter (milwaukee ph meter, model-ph55/ph56, usa), and total alkalinity was estimated following the standard method (stirling, 1985; apha, 1992). plankton monitoring: quantitative and qualitative estimates of plankton in the nursery ponds were taken weekly. ten liters of pond water were collected from different locations and depth of the pond and filtered through fine mesh (0.04 mm) plankton net. filtered water was taken into a measuring cylinder and carefully made up to standard volume with distilled water. using a plastic tubing, water was siphoned off from the measuring cylinder and water sample were concentrated into 50 ml and preserved using 5% buffered formalin in small plastic vials for subsequent studies. from each 10 ml preserved sample, 1 ml subsample was examined using a sedgwick–rafter (s–r) cell was used under a calibrated compound microscope for plankton counting. plankton count (number of cells/l of water sample) was made using the formula proposed by stirling (1985) and rahman (1992). estimation of growth, survival, production and feed utilization study of fishes: twenty individuals from each pond were sampled weekly during the experimental period. growth in terms of length and weight, specific growth rate (sgr) and food conversion rate (fcr) was estimated. sgr and fcr were calculated according to brown (1957), castell and tiews (1980) and gangadhara et al. (1997), respectively. after 4 weeks, the fingerlings were harvested by repeated netting, followed by draining the ponds. the fingerlings were counted and weighed. survival (%) and production (number/ha) of fingerlings were then calculated and compared among the treatments. analysis of experimental data: the collected data of growth, survival, production, water quality patrameters and plankton abundance under different treatments were analyzed through one way analysis of variance (anova) followed by duncan's new multiple range test to find out whether any significant difference existed among treatment means (duncan, 1955; zar, 1984). the statistical analysis was performed by spss software 19 (spss, usa). the level for significance was set at 0.05%. a simple costbenefit analysis was done to estimate the net benefits from different treatments. results and discussion water quality parameters: the ranges and mean values of various physic-chemical parameters, such as water temperature, transparency, dissolved oxygen (do), ph and total alkalinity recorded during the experimental ponds are shown in table 2. during the study period, the water temperature ranged from 26.00 to 32.50°c with a mean value of 30.19±0.73, 29.48±0.64 and 29.28±0.76°c in t1, t2 and t3, table 2. physico-chemical characters of water in the earthen nursery ponds during the experimental period. parameter treatments t1 t2 t3 water temperature (°c) 30.19±0.73a (27.00-32.50) 29.48±0.64a (27.00-32.00) 29.28±0.76a (26.00-32.00) transparency (cm) 28.75±1.35c (23.00-35.00) 35.50±1.77b (28.00-42.00) 45.38±2.69a (33.00-55.00) dissolved oxygen (mg/l) 5.74±0.13a (5.23-6.30) 4.77±0.17b (4.10-5.42) 3.86±0.0.10c (3.50-4.38) ph 7.56±0.22a (6.80-8.30) 7.94±0.23a (7.20-8.60) 7.88±0.21a (7.10-8.60) total alkalinity (mg/l) 129.25±3.90a (115.00-145.00) 124.75±2.18b (112.00-133.00) 110.00±2.42c (102.00-121.00) values in the same row having the same superscript are not significantly different (p>0.05). values in the parenthesis indicate the range. 150 rahman et al./ effects of stocking density on the growth, survival and production of labeo bata respectively, which were not differed significantly (p>0.05). similar results were observed by haque et al. (1991, 1993), kohinoor et al. (1994) and samad et al. (2016). mean transparency were 28.75±1.35, 35.50±1.77 and 45.38±2.69 cm in t1, t2 and t3, respectively which were differed significantly (p<0.05). transparency was significantly higher (p<0.05) in t3, where stocking density was high, may be due to reduction of plankton population by the higher density of fish (haque et al., 1993). the dissolved oxygen (do) in the morning hours varied from 3.50 to 6.30 mg/l, with mean values of 5.74±0.13, 4.77±0.17 and 3.86±0.0.10 mg/l in t1, t2 and t3, respectively, the value of t1 was differed significantly (p>0.05) from t2 and t3. comparatively low do was recorded in the morning in ponds stocked with higher density of fish as compared with the ponds stocked with lower stocking density (haque et al., 1993; rahman and rahman 2003; ahmed, 2015). it seemed that the reason behind this was the high consumption of oxygen by the fishes as pointed out by saha et al. (1988). the level of dissolved oxygen (do) is within the acceptable range in all the experimental ponds. during the study, the ph values varied from 6.80 to 8.60 with the mean values of 7.56±0.22, 7.94±0.23 and 7.88±0.21 in t1, t2 and t3, respectively, which were the characteristics of good water quality and suitable for fish culture (rahman and rahman, 2003; hossain et al., 2007; ahmed, 2015; samad et al., 2016). the mean values of total alkalinity were 129.25±3.90, 124.75±2.18 and 110.00±2.42 mg/l in t1, t2 and t3, respectively and the ph value of t1 was differed significantly (p>0.05) from t2 and t3. significantly lower (p>0.05) alkalinity was observed in t3 than t1 and t2. higher total alkalinity values might be due to higher amount of lime doses during pond preparation and frequent liming during the experimental period (jhingran, 1991). similar results were also recorded in the studies of kohinoor et al. (1997), chakraborty and mirza (2007), ahmed (2015) and samad et al. (2016). water quality was widely acknowledge to be one of the most important rearing conditions that could be managed to reduce diseases exposure and stress in intensive fish culture (wedemeyer, 1996). in the present study, all of the water quality parameters were suitable ranges for rearing of fry and fingerlings (dof, 2010). plankton enumeration: the mean number of phytoplankton and zooplankton under three stocking densities were 311.57±1.85 and 89.30±1.16, 273.84±1.76 and 75.85±0.90, 226.79±1.62 and 63.27±1.01 in t1, t2 and t3, respectively (table 3). table 3. mean values (±se) and ranges of plankton abundance (cell/ml ×103) of pond water under different treatments during 4-week nursing period. plankton group treatments t1 t2 t3 plankton (cell/ml×103) bacillariophyceae 135.24±3.34a (125.48-150.08) 118.33±1.62b (112.01-123.89) 100.27±1.94c (92.87-107.24) chlorophyceae 102.34±1.22a (98.12-107.54) 93.65±1.56b (87.09-98.92) 77.57±1.20c (74.02-82.98) cyanophyceae 67.35±1.30a (62.05-71.55) 57.84±1.54b (53.02-63.58) 45.02±1.33c (38.59-49.01) euglenophyceae 6.64±0.86a (4.95-8.99) 4.02±0.41b (2.98-6.59) 3.93±0.32b (2.74-5.23) total phytoplankton 311.57±1.85a 273.84±1.76b 226.79±1.62c zooplankton (organism/ml×103) crustacea 48.47±1.27a (44.12-53.42) 40.21±1.41b (35.22-46.54) 34.52±0.84c (31.59-37.84) rotifera 40.83±1.15a (36.23-45.21) 35.64±0.86b (32.06-39.01) 28.75±0.99c (25.47-34.88) total zooplankton 89.30±1.16a 75.85±0.90b 63.27±1.01c values in the same row having the same superscript are not significantly different (p>0.05). values in the parenthesis indicate the range. 151 int. j. aquat. biol. (2018) 6(3): 147-156 twenty seven genera of phytoplankton were recorded in four groups viz. chlorophyceae, bacillariophyceae, cyanophyceae and euglenophyceae. the mean abundance of total phytoplankton in t1 was significantly higher (p>0.05) than those of t2 and t3. the zooplankton population consisted of 12 genera, including nauplii in two groups viz. crustacean and rotifera. crustacea were dominant over the entire experimental periods in all treatments. the abundance of zooplankton differed significantly (p<0.05), decreasing from t1 to t3. in the present study, the quantity of both phytoplankton and zooplankton was inversely related with the stocking density of fry. the quantity of phytoplankton and zooplankton was higher in t1 where stocking density of fry was low. it seems that in the ponds where the stocking density of high, consumption of plankton by the fishes also high. it was also found that in all the ponds that phytoplankton production was higher in comparison with zooplankton. higher phytoplankton concentrations in pond water normally indicate higher productivity. the higher abundance of phytoplankton compared to zooplankton might be due to fertilization of ponds with urea and tsp and excess uneaten feed (keshavanath et al., 2002). more or less similar results have also been recorded in various food fish and fry or fingerling rearing ponds (wahab et al., 1994; haque et al., 1998; kohinoor et al., 1999; chakraborty et al., 2003, rahman et al., 2005; chakraborty and mirza, 2007; ahmed, 2015). growth, feed utilization and production of fish: weekly growth of l. bata fry under different stocking densities at the end of the experimental period are summarized in table 4. the growth in length and weight of fry are depicted in figures 1 and 2, respectively. the initial length and weight of fry stocked in all the ponds were the same i.e. 1.03±0.03 cm and 0.12±0.01 g, respectively. it is evident the data that fry after 4 weeks of rearing attained a size of 3.25 to 3.61 cm (mean 3.43±0.15 cm) in length and 3.52 to 3.67 g (mean 3.60±0.16 g) in weight in ponds with lowest stocking density of 1.0 million/ha, while the fry attained a size of 2.89 to 2.93 cm (mean 2.91±0.08 cm) in length and 2.82 to 2.87 g (mean 2.85±0.09 g) in weight in ponds with 1.5 million/ha, and 2.40 to 2.44 cm (mean 2.42±0.08 cm) in length and 2.07 to 2.12 g (mean 2.09±0.09 g) in weight in ponds stocked with table 4. growth performance, survival and production of labeo bata fry or fingerlings after 4 weeks of rearing; mean ± se with ranges in parentheses. parameters treatments t1 t2 t3 initial length (cm) 1.03±0.03a (0.90-1.10) 1.03±0.03a (0.90-1.10) 1.03±0.03a (0.90-1.10) final length (cm) 3.43±0.15a (3.61-3.25) 2.91±0.08a (2.93-2.89) 2.42±0.08b (2.40-2.44) initial weight (g) 0.12±0.01 a (0.09-0.13) 0.12±0.01a (0.09-0.13) 0.12±0.01a (0.09-0.13) final weight (g) 3.60±0.16a (3.52-3.67) 2.85±0.09b (2.82-2.87) 2.09±0.09c (2.07-2.12) net weight gain 3.46±0.08a (3.40-3.55) 2.73±0.03b (2.70-2.75) 1.98±0.03c (1.95-2.00) specific growth rate (sgr) (%/day) 12.15±0.08a (12.07-12.22) 11.31±0.03b (11.28-11.34) 10.22±0.05c (10.17-10.26) food conversion ratio (fcr) 0.26±0.01c (0.25-0.26) 0.42±0.02b (0.40-0.43) 0.65±0.01a (0.64-0.64) survival rate (%) 71.79±1.04a (71.05-72.52) 64.22±0.66b (63.56-64.88) 58.89±0.24c (58.65-59.12) production (number/ha) 717850±7350c (710500-725200) 963300±9900b (953400-973200) 1177700±4700a (1173000-1182400) values in the same row having the same superscript are not significantly different (p>0.05). values in the parenthesis indicate the range. 152 rahman et al./ effects of stocking density on the growth, survival and production of labeo bata 2.0 million/ha (table 4). from the present study, it is clearly indicated that the maximum growth in length and weight was attained at the lowest stocking density with 1.0 million/ha with the growth gradually decreasing with increase in density. final average size in length and weight of fry attained in ponds with different stocking densities was observed to be significantly different (p<0.05). from table 4, it clearly indicated that the t1 (3.46±0.08 g) showed the highest average gain in both length and weight followed by t2 (2.73±0.03 g) and t3 (1.98±0.03 g). stocking density had previously been observed to have a direct effect on the growth of fish (haque et al., 1993; kohinoor et al., 1994; rahman and rahman, 2003: ahmed, 2015; samad et al., 2016). high density of fingerlings in combination with increased concentration of food in the rearing system might have produced a stressful situation and toxic substance which could be the probable cause for poor growth in t2 and t3 (haque et al., 1994; rahman and rahman, 2003). the range of specific growth rate (% per day) was found 10.22 to 12.22 with a mean value of 12.15±0.08, 11.31±0.03 and 10.22±0.05 in t1, t2, and t3, respectively. specific growth rate (sgr) in t1 was significantly higher (p>0.05) than in t2 and t3 due to lower stocking density. similar result was found by chakraborty and mirza (2007), ahmed (2015) and samad et al. (2016). feed conversion ratio was significantly lower in t1 (0.26±0.01) than t2 (0.42±0.02) and t3 (0.65±01). therefore, sgr and fcr were best for fish in t1 where lowest number of hatchlings (1.00 million/ha) was reared. however the lower fcr value in the present study indicates better food utilization efficiency, despite the values increased with increasing stocking densities. fingerlings of l. bata had significantly higher survival in t1, where, the stocking density was lower than t2 and t3. the reason for reduced survival rate in these treatments was probably due to higher stocking density of fry as well as competition for food and space in the experimental ponds. the fcr values of present findings are lower than the values reported by islam (2002) and islam et al. (2002), chakraborty and mirza (2007), ahmed (2015) and samad et al. (2016). the highest survival rate was also observed in t1 (71.79±1.04) and the lowest in t3 (58.89±0.24). there was a significant variation (p>0.05) in the survival rate in l. bata fry and fingerlings among different treatments. the present findings compared with the values reported by chakraborty and mirza (2007), ahmed (2015) and samad et al. (2016) where they found similar survival rate of endangered l. bata at lower stocking density. in this experiment, crude protein levels (40.57% and 35.52% dry weight) in supplementary feeds are higher than the dietary protein of 31% for the optimal growth of labeo rohita (de silva and gunasekera, 1991). however, growth in terms of length, weight, net weight gain, sgr and survival rate of fingerlings of l. bata was significantly higher in t1 where the stocking density was low compared to those of t2 and t3 although the same food was supplied in all the treatments at an equal ratio. the productions of fingerlings (number/ha) were figure 1. weekly mean length (cm) gain of labeo bata under different stocking densities. figure 2. weekly mean weight (g) gain of labeo bata under different stocking densities. 153 int. j. aquat. biol. (2018) 6(3): 147-156 710500 to 725200 (mean 717850±7350), 953400 to 973200 (mean 963300±9900) and 1173000 to 1182400 (mean 1177700±4700) in t1, t2 and t3, respectively (table 5). production of fingerlings differed significantly (p<0.05) among the three treatments. significantly higher numbers of fingerlings were produced in ponds stocked with 2.0 million fry/ha than those from the ponds stocked with 1.0 and 1.5 million fry/ha, respectively. on the other hand, cost of production in t1 was consistently lower than those t2 and t3 (table 5). highest net benefit (in term of bangladeshi tk. /ha and one us$ = bangladeshi tk. 80.00) was obtained in t1 (127,087.00) followed by t2 (71,272.00) and t3 (12,744.00), respectively. despite this, consistently higher net benefits were obtained from ponds stocked with 1.0 million fry/ha than those from 1.5 and 2.0 million fry/ha. the higher market price of the larger fingerlings produced in ponds with 1.0 million fry/ha, substantially increased the net benefit compared to smaller fingerlings that produced in other ponds with higher stocking densities. overall, highest growth, survival and benefits of fingerlings were obtained at a density of 1.0 million fry/ha. water quality parameters of pond water during the study period were within the acceptable limits. growth of fingerlings to be greater extent depended on the quality and quantity of food available. in the present investigation, the amount of supplementary feeds given in different treatments was based on the number of hatchlings stocked and amount of feed provided per fry was kept at the same level. hence, the observed low growth at higher stocking densities could be due to less availability of natural food and some variations in environmental parameters (kohinoor et al., 1997). the results in the present experiment are very similar to those of saha et al. (1988), kohinoor et al. (1994), hossain (2001), rahman and rahman (2003), chakraborty et al. (2003, 2006), chakraborty and mirza (2007). from the present study, it can be concluded that the table 5. cost and benefits from the nursing of bata, labeo bata fingerlings in 1-ha earthen ponds for a nursing period of 4 weeks. item amount (tk)/ha/ 2 months remarks treatments t1 (tk)a t2 (tk) t3 (tk) a. variable cost: 1. price of hatchlings (@ tk. 0.15/piece) 150,000 225,000 300,000 2. feeds a. pre-nursery (@tk. 80.00/kg) 23,360 34,960 46,640 b. nursery (@tk. 55.00/kg) 20,955 31,405 41,855 3. human labour cost (@tk. 200.00/day) 12,000 12,000 12,000 02 labour/day 4. fertilizer (urea, tsp and cowdung) 5,950 5,950 5,950 5. lime (247 kg/ha, @tk. 20.00/kg 4,950 4,950 4,950 6. miscellaneous 10,000 10,000 10,000 dipterex, oxy flow, carrying, netting etc total variable cost (tvc) 227,215 324,265 421,395 b. fixed cost: 1.pond lease value 49,400 49,400 49,400 tk. 200.00/decimal/year (local rate, mymensingh region) 2.interest of operating capital 27,008 36,713 46,426 10% interest according to bangladesh krishi bank, bangladesh total fixed cost (tfc) 76,408 86,213 95,826 total cost (tc= tvc+tfc) 303,623 410,378 517,221 total return (tr)b 430,710 481,650 529,965 price is related with size and weight gross margin (gm= tr -tvc) 203,495 157,385 108,570 net return (tr-tc) 127,087 71,272 12,744 *atk 80.00 =1 us$ bselling price of fingerlings tk. 0.60/ piece (size 3.60 g, t1), tk. 0.50/piece (size 2.85 g, t2) and tk. 0.45/piece (size 2.09 g, t3). 154 rahman et al./ effects of stocking density on the growth, survival and production of labeo bata survival, growth and production of l. bata fingerlings were inversely related to the stocking densities of fry. stocking density of 1.0 million fry/ha may be advisable for rearing of l. bata fingerlings for 4 weeks in primary nursing. production of adequate quality seeds through application of our present findings might be extremely helpful towards the protection of l. bata from extinction as well as for its conservation and rehabilitation. references ahmed s., rahman a.f., hossain m.g., nahar n. 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(1984). biostatistics. prentice–hall, inc., englewood cliffs. 718 p. international journal of aquatic biology (2013) 1(5): 209-214 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article effects of different levels of live food replacement with microdiet on growth factors, survival and resistance to salinity stress of indian white shrimp post-larvae (fenneropenaeus indicus) seyed hossein hoseinifar*,1parviz zare department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 11 may 2013 accepted 21 september 2013 available online 2 5 october 2013 keywords: fenneropenaeus indicus post-larvae artemia replacement microdiet growth abstract: in this study, partial and complete replacement of live food (artemia nauplii) with a microdiet was investigated in post-larval indian white shrimp, fenneropenaeus indicus. post-larvae (pl1) were stocked into nine 20-l vessels (5-l water volume) at a density of 50 l-1. shrimp were fed six times a day over 10 days. at the end of the feeding period, there was no significant difference in quality index (stress-test survival) among treatments. statistical analysis of results showed that partial or complete replacement of live food with microdiet significantly decreased survival, total length, carapace length, weight, growth rate, performance index, and the number of spines in upper rostrum of the post-larvae. however, substitution of live food with microdiet had no negative effects on resistance to salinity stress. these results showed that the commercial microdiet used in this study is not a good replacement of live food; further studies are required to determine the nutritional requirements of indian white shrimp larvae and post-larvae before microdiets can be utilized effectively. introduction crustacean larval stage is considered as a critical stage in their life history. success of larval rearing depends mainly on the availability of suitable diets that are readily consumed, efficiently digested and that provide the required nutrients to support good growth and health (giri et al., 2002). larval diets are typically composed of different species of microalgae and newly hatched artemia nauplii (robinson, 2005). while supplying live food is difficult and requires considerable space and expense, microdiets are easier to maintain and usually have lower production costs (jones et al., 1993; person-le ruyet et al., 1993; wang et al., 2005). sorgeloos and leger (1992) described finfish and crustacean larviculture diets for the first feeding stages as being the major bottleneck for complete * corresponding author: seyed hossein hoseinifar e-mail address: hoseinifar@gau.ac.ir tel: +98 1716622251 replacement of live feeds. the development of costeffective and nutritionally complete formulated larval diets would be of tremendous benefit to commercial hatchery operations (robinson et al., 2005). the possibility of replacing live food with formulated diets has been investigated in several studies (kanazawa, 1982; kanazawa, 1985; jones et al., 1989; jones et al., 1997; samocha et al., 1999; gallardo et al., 2002; wouters et al., 2003; robinson et al., 2005; tang et al., 2010). microparticulate foods are in various forms (e.g. microencapsulated, microbound and microcoated) have yielded some degree of success in commercial hatcheries as partial replacements for live foods (langdon et al., 1985) but have deleterious effects deterioration on water quality (muir et al., 1991). nutrients leaching from microparticulated foods can 210 hosseinifar and zare / international journal of aquatic biology (2013) 1(5): 209-214 affect growth and survival of shrimp post-larvae and depredate the water quality via proliferation of bacteria (jones et al., 1987; muir et al., 1991). growth and survival data are powerful tools for understanding the effects of both live and formulated diets on first-feeding larvae (cannavate and fernandez-diaz, 1999). therefore, in the present study, growth, survival and resistance to salinity stress were used to evaluate the effects of partial and complete replacement of artemia nauplii with commercial microdiet on indian white shrimp (fenneropenaeus indicus) post-larvae. materials and methods source of shrimp and stocking: this study was conducted at the abziparavar chabahar hatchery, chabahar. larvae were obtained from three eyestalkablated spawners (47.6, 37.16, 45.2 g). shrimp nauplii were kept in spawning tanks (1000 l) containing natural sea water (37 g l-1 salinity, 30.5 oc, 8.1-8.2 ph) supplemented with a mixture of the microalgae chaetoceros and tetraselmis, which was added daily at a rate of 2×106 cells ml-1 (ziaei nejad et al., 2006). beginning at post larval stage 1 (pl1), shrimp post-larvae were harvested and stocked into nine 20-l vessels (5-l water volume) at a density of 50 individual l-1. feeding trial: three treatments, each with varying levels of live food substitution [0% (treatment a), 50% (treatment b) and 100% (treatment c)] with commercial microdiet (royal™ (bernaqua bvba, belgium; with particle size of 100-200 µ; table 1) were used with three replicates. indian white shrimp post-larvae were fed with experimental treatment from pl1 to pl11 stage. post-larvae in treatment a were fed with newly hatched artemia franciscana nauplii at a rate of 15 nauplii for each post larvae, 6 times a day. along with post-larvae growth the number of nauplii was increased to 25 nauplii per post-larvae (ziaei nejad et al., 2006). to evaluate the effects of complete replacement of live food postlarvae fed with microdiet according to feeding schedule suggested by shakoori (2005) (table 2). in treatment c this amount divided by half and live food was used in combination with microdiet. indices: at the beginning and the end of trial 20 specimens were sampled randomly from each vessel to determine the following growth factors: carapace length, total length, and number of spines over the rostrum. survival was calculated on the basis of the final number of post-larvae (pl11) in relation to the initial number of post-larvae (pl1). the final number of post-larvae was obtained by counting all individuals in each treatment. specific growth rate (sgr) was determined using the following:   100 lnln 0    t ww sgr t where t is the culture period in days, lnw0 is the natural logarithm of the weight (g) of the shrimp at beginning of the experiment, and lnwt is the natural logarithm of the weight (g) of the shrimp at day t. quality index (qi) was calculated from surviving post-larvae (%) after an acute salinity challenge (from 37 to 15 g l-1) (gallardo et al., 2002): qi =     100 plb pla where pla is the number of surviving post-larvae (%) after the sudden change of salinity and plb is the total number of post-larvae before the sudden change of salinity. ingredient approximate amount crude protein 50% crude fat 15% crude fiber 2% ash 20% calcium 2% phosphorous 1.5% vitamin a 20000 iu/kg vitamin d3 4000 iu/kg vitamin e 400 iu/kg vitamin c 1100 ppm hufa 25 mg/gr dha 12 mg/gr epa 8 mg/gr table 1. nutritional profile of the commercial microdiet royal™ (bernaqua bvba, belgium) according to manufactures. 211 hosseinifar and zare / international journal of aquatic biology (2013) 1(5): 209-214 the performance index (pi) was calculated according to gallardo et al. (1995): pi = [growth ×survival ×ql] statistical analysis: all statistical analyses were conducted using spss statistical package version 10.0 (spss inc., chicago, il, usa). data were examined for normality and homogeneity of variance, and were subjected to a one-way analysis of variance (anova). when significant differences were observed, duncan's multiple range tests were performed (zar, 1994). mean values were considered significantly different at p<0.05. data are expressed as mean values ± sd. results growth and development: measurement of growth factors showed that post-larvae fed live food had significantly higher weight, carapace length, sgr and total length than both other treatments (p<0.05) (table 3). there was no significant difference between growth factor of post-larvae in treatment b or c (p>0.05) survival, quality index and performance index: survival data analysis showed that feeding with microdiet significantly decreased survival but the survival of shrimp fed 50% or 100% microdiet were not significantly different compared to the other (fig. 1). quality index calculation demonstrated that postlarvae fed with different dietary treatments had same resistance to salinity stress (fig. 2). shrimp fed on live food had significantly higher performance index but no differences were observed between the 50% and 100% groups (table 1). discussion brine shrimp nauplii are widely used in hatcheries throughout the world. the annual available brine shrimp cysts were 800 metric tons, or 40%, of the total demand from aquaculture (li, 2003). stage pl 1 pl 2 pl 3 pl 4 pl 5 pl 6 pl 7 pl 8 pl 9 pl 10 pl 11 feeding amount (mg md l-1 day-1) 3 6 9 12 15 18 21 24 27 30 33 md= microdiet table 2. feeding schedule of indian white shrimp during the experiment (shakoori, 2005). total length (mm) carapace length (mm) weight (mgr) w/cl ratio sgr (mm day-1) live feed 12.76 ± 0.82a 2.58 ± 0.18a 11.7 ± 1.3a 0.40 ± 0.09a 0.88 ± 0.09a 100% replacement 10.35 ± 1.00c 2.29 ± 0.20c 6.1 ± 1.0b 0.26 ± 0.04b 0.54 ± 0.06b 50% replacement 11.04 ± 0.73b 2.46 ± 0.14b 7.8 ± 0.2b 0.31 ± 0.02b 0.61 ± 0.03b values (mean ± se) in the same column not sharing a common superscript are significantly different (p<0.05). table 3. growth factors of fenneropenaeus indicus post-larvae fed different level of live food replacement with microdiet. figure 1. the number of spines in upper rostrum of the post-larvae of fenneropenaeus indicus fed with different treatment (p<0.05). 211 212 hosseinifar and zare / international journal of aquatic biology (2013) 1(5): 209-214 fluctuations in the supply of cysts have resulted in unstable prices and higher production costs for the hatchery (sorgeloos et al., 2001). in response to the limitations of supplying live food for early stages of shrimp culture several attempts have been made to reduce dependency of hatchery to live food and varying levels of success achieved in these trials (kanazawa, 1982; kanazawa, 1985; jones et al., 1989; jones et al., 1997; samocha et al., 1999; gallardo et al., 2002; wouters et al., 2003; robinson et al., 2005; tang et al., 2010). the result of our study showed that complete (100%) or partial (50%) replacement of artemia nauplii with a commercial microdiet decreased growth and performance index of indian white shrimp postlarvae. similar result was reported by robinson (2005) who replaced live food with zeigiertm microbound diets in farfantepenaeus aztecus postlarvae culture. moreover replacement of live food with crumbled experimental microbound diets in zoea-mysis stages of litopenaeus setiferus (gallardo et al., 2002) or with microencapsulated diet frippak® in p. monodon zoea-pl culture (wouters et al., 2003) was not successful and resulted in reduction weight gain. one possible reason of growth reduction is due to nitrogenous products including ammonium, nitrite and nitrate which are toxic to several marine organisms at extremely low concentrations (spotte, 1979). robinson et al. (2005) found that sub-lethal ammonia–nitrogen concentrations were detected within a short time after feeding (24 h). in contrast, feeding of l. vannamei post-larvae with low level crumbled microbound diet frippak® had no negative effect on growth (wouters et al., 2003). they assessed varying replacement levels of live food with crumbled microbound diet for litopenaeus vannamei mysis and post-larvae and observed that replacement levels below 25% did not impact growth performance. the result of the present study showed that 50% or 100% replacement of artemia nauplii with microdiet resulted in lower survival. similar result was reported in several studies (gallardo et al., 2002; wouters et al., 2003; robinson, 2005). however, samocha (1999) indicated that only complete replacement resulted in a reduction of survival and that 25% and 50% replacement had no negative effects on litopenaeus vannamei post-larvae survival. in contrast, substitution of artemia with microencapsulated diet frippak® had no negative figure 2. survival (a, s, %), quality index (b, qi, %) and performance index (c, pi, mmday-1) of the post-larvae of fenneropenaeus indicus fed with live food and different level of live food substitution. different letters indicate significant differences (p<0.05). 213 hosseinifar and zare / international journal of aquatic biology (2013) 1(5): 209-214 effects on p. monodon post-larvae survival (wouters et al., 2003). the contradictory nature of the obtained results is possibly attributed to the type of microdiet, route of administration or species. resistance to salinity stress test shows the quality of shrimp post-larvae (palacious et al., 2007) and our results revealed that substitution of live food with a microdiet in post-larval rearing did not affect their quality in this respect. in addition there was no significant difference in resistance of post-larvae fed with different level of live food replacement (p>0.05). however, previous studies on f. azetcus and l. setiferus showed that post-larvae feeding with microdiet diet significantly decreased quality index (p<0.05) (gallardo et al., 2002; robinson et al., 2005). it is concluded that replacement of live food (50 or 100%) the present microdiet negatively affects growth performance and survival. future studies are necessary to evaluate the potential of lower level replacements. acknowledgments the author's kindly thank mr. madani, owner of abziparvar chabahar hatchery and the staff at abziparvar chabahar shrimp hatchery for supplying diet and post-larvae. special thanks also to dr. bagher mojazi amiri, jahangir ejdaha kosh and ashkan ejdaha kosh for their kind help during the experiment. references: cannavate j.p., fernandez-diaz c. 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(2023) 11(2): 131-140 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article length-based fishery status and population dynamics of spiral babylon, babylonia spirata, linnaeus, 1758, stock in the northern waters of the oman sea, sistan and baluchestan province of iran seyed ahmad reza hashemi1, mastooreh doustdar2, elnaz erfanifar1 1offshore fisheries research center, iranian fisheries science and research institute, agricultural research education and extension organization, chabahar, iran. 2iranian fisheries science and research institute, agricultural research education and extension organization, tehran, iran. s article history: received 19 june 2022 accepted 26 march 2023 available online 2 5 april 2023 keywords: spiral babylon exploitation coefficient mortality fishing oman sea abstract: in the present study, the population characteristics of spiral babylon, babylonia spirata, were evaluated by sampling at four sites in the northern oman sea, iran, including pozm, konark, beries, pasabandar from march 2021 to march 2022. a total of 2779 babylonian snail specimens (1489 males, and 1290 females) were measured and about ten percent of the specimens were described. the mean length and weight of males and females were 36±5, and 32±5 mm and 14±6, 10±4 g, respectively. growth and mortality indices for females and males including infinite length (l∞ = 68 and 76 mm), growth coefficient (k = 0.54 and 0.3 (yr-1)), natural mortality (m= 0.7 and 0.4 (yr-1)), fishing mortality (f = 2.30 and 1.79 (yr-1)), total mortality (z = 3 and 2.19 (yr-1)) and exploitation coefficient (e = 0.77 and 0.82 (yr-1)) were estimated. based on the lbspr assessment model, estimated to be about 0.3, and a ratio of pmega<0.1, lmean/lopt <1 and lmean /lf=m<1 show considered undesirable. the present study showed that the spiral babylon stock has reached 'overfished' status. introduction population dynamics are driven by changes in the abundance or biomass of a population through time by a series of life-history traits such as fecundity, successful recruitment, growth rates, and mortality rates. estimates of population dynamics can provide insights into a harvested marine population species. it can indicate how a population arrived at its current state and how it might change in the future (brown and guy, 2007). recruitment, growth, and mortality rates are the primary population dynamics parameters that explained the harvestable part of a fish population (brown and guy, 2007). mollusca is the second phylum in the number of living species, after arthropods (pandian, 2017). of the seven molluscan classes, the 78,000 known gastropod species contribute 85% of all mollusks and the 60,000 species of prosobranchs contribute a significant part of this group (pandian, 2017). the correspondence: seyed ahmad reza hashemi doi: http//doi.org/10.22034/ijab.v11i2.1627 e-mail: seyedahmad91@gmail.com dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.2.6.9 genus babylonia (mollusca: gastropoda: buccinidae) possesses 11 extant species, two of which polytypic with two subspecies each, and 12 fossil and extinct species (gittenberger, and goud, 2014). the three most common species, viz. babylonia areolata, b. japonica and b. spirata, have continuous ranges (gittenberger and goud, 2014). the oman sea is a region of the northern indian ocean that connects the arabian sea with the strait of hormuz, which then runs to the persian gulf. it borders iran and pakistan on the north, oman on the south, and the united arab emirates on the west (taghavimotlagh and shojaei, 2017). the oman sea, with its unique ecological conditions, hosts a wide variety of marine species that provide livelihood, employment, and vast economic activities for the settlers. iran has more than 120,000 fishermen. therefore, fishing has played a major role in creating employment in coastal areas, as well as in 132 hashemi et al./ length-based fishery status and population dynamics of spiral babylon economic activities for post-harvest operations (taghavimotlagh and shojaei, 2017). some studies on the biological characteristics of shell species in different parts of the world include mohan (2007), cob et al. (2009), arrighetti et al. (2012), daza-guerra et al. (2018), and matos et al. (2020). recently, the illegal fishing of babylonia spirata in the northern waters of the oman sea has increased significantly. it is not used for food in the region and is caught only for export. there is currently no specific management or restriction for this species in this area. despite the economic importance of this species, little is known about the stock of b. spirata population. therefore, this study investigates several life-history traits and fish assessment measures of b. spirata from the oman sea to provide fundamental information for proper identification and management. hence, first, the structure of the population and growth rate of b. spirata from the oman sea will be studied; then, a series of fisheries assessment data will be calculated such as mortality and exploitation coefficient to evaluate the sustainability of its population in the oman sea. materials and methods sample sites and specimen collection: based on the location in the northern oman sea, iran, four sites were selected for sampling b. spirata in the ports of pozem (60˚15´e, 25˚35´n), konark (60˚28´e, 25˚60´n), beris (61˚10´e, 25˚82´n) and pasabandar (61˚25´e, 25˚70´n) (fig. 1). samplings were carried out monthly from march 2017 to march 2022. samples were collected with the help of fishermen using special baskets from subtidal areas (sandy and pebble shore) at depths of less than 50 meters. a total of 2779 specimens of b. spirata specimens were collected (fig. 2). biometric data: the shell weight and length of all specimens were measured. the total length was determined by a biometric ruler with 1 mm precision (fig. 2) and wet weight to the nearest 1 gr. the equation of wi = a × li b (1) was used to calculate the relationship between the total length and wet weight, where, wi is the shell weight (g), li is the shell length (mm), a is a constant coefficient, and b, figure 1. map of babylonia spirata sampling stations in the northern oman sea, iran. 133 int. j. aquat. biol. (2023) 11(2): 131-140 an equation power. equation (2) was used to decipher significant differences between the calculated b from equation (1) and b = 3 for a shell of similar growth with s.dx, the standard deviation of total length natural log, s. dy standard deviation of weight natural log,b slope, r2 coefficient of determination, and n is sample sizes (zar, 2010). t = [(s.dx)/ (s.dy)] × [(lb-3l)/ (√ (l-r2)] × [√ (n-2)] (2) population dynamic parameters of b. spirata: the data were pooled monthly from different stations, and subsequently grouped into classes of three centimeters interval. the elefan method was used to analyze the data for growth rates. the estimation of l∞, the infinite length was obtained using equation (3) and the maximum length of samples (lmax) based on froese and binohlan (2000) log l∞ = 0.044 + 0.9841 * log (lmax) (3) the growth rate was obtained by applying the elefan method (optimization model), using the rstudio software with the tropfishr package (mildenberger et al., 2017). the optimum value of t0 (time that length is zero) was calculated by the experimental formula of pauly equation (equation (4)) with k, the growth factor (froese and binohlan, 2000). log (-t0) = 0.3922 0.2752 log l∞ 1.038 log k (4) the maximum lifespan was estimated based on froese and pauly (2017 and the equation of tmax = t0 + 3 / k (5). mortality: natural mortality (m) was calculated based on the brey equation (6) with, m as the annual natural mortality coefficient, amax as the life expectancy based on the year, bmmax as the maximum body weight (30*3.81) based on k j (for this species the coefficient is 3.81 kj /g) and t (kelvin) is the average ambient temperature (brey, 1999; mohan, 2007). log (m) = 1.672 +( 0.993* log (1/amax))) – (0.035* log (bmmax)) – (300.447/ t+273) (6) the mean annual temperature of the sea surface of the northern oman sea was estimated at 26°c or 299 (k) (hashemi, 2020). total mortality (z) was calculated based on the length-converted catch curves data. the fishing mortality was estimated using equation (7), with z, the total mortality, f, the fishing mortality, and m the natural mortality. f = z – m (7) the exploitation rate (e), which is the ratio of fishing mortality to total mortality, was calculated using equation (8) based on sparre and venema (1998) e = f / z (8) the relative yield per recruitment (y/r) and relative biomass per recruitment (b'/r): the relative yield per recruitment (y/r) was estimated against the fishing mortality coefficient or exploitation rate. in this equation (9), e is the exploitation coefficient, u is the exploitation rate, m is the natural mortality coefficient, f is the fishing mortality coefficient and lc (length at first capture) is the same as lc50 (gayanilo et al., 2003). figure 2. the babylonia spirata species in the northern oman sea, iran. 134 hashemi et al./ length-based fishery status and population dynamics of spiral babylon y' / r = eu m/k ( -3 u / (1 + m) + 3 u2 / (1 + 2m) + u3 / (1 + 3m ) with u = 1 (lc / l∞) ; m = (1 e) / ( m / k) = ( k / z) ; e = f / z (9) in addition, the relative biomass per recruitment (b'/r) was calculated using the equation of b' / r= y' / r / f (10). length-based indicators (lbi): in the length-based indicators (lbi), popt is the percentage of fish caught at the optimum length for harvest, pmega, is the percentage of mega-spawners (length between 1.1 lopt and lmax on the catch length composition) in the catch, and lf=m is values for optimal fishing length that were calculated using the formula of 11, 12, and 13, respectively (froese and binohlan, 2000; cousido-rocha et al., 2022): lopt=linf (3/(3+m⁄k)) (11) lf=m = (0.75lc+0.25linf) (12) + %10 (13) optl= megap length-based spawning potential ratio (lbspr): lbspr is one of the biological reference points for determining the stock status of a species in the population. the reference points of lbspr used are: lbspr 20% is as the limit reference point and lbspr 40% is a target reference point (hordyk et al., 2015) that is calculated as: lbspr = ∑(1−𝐿𝑋)(𝑀 𝐾⁄ )[(𝐹 𝑀⁄ )+1])𝐿𝑥 𝑏 ∑(1−𝐿𝑋) 𝐿𝑥 𝑏 𝑀/𝐾 (14) where lx is fork length, m is natural mortality; k is the growth rate, f is fishing mortality, and b is exponent usually close to 3. estimating spr with those functions needs the simple assumptions of asymptotic or logistic selectivity and no variation in length at age. the f/m ratio can be estimated from the length composition of the catch (hordyk et al., 2015). the relationship between f/m and spr is asymptotic and determined by the selectivity parameters (carruthers and hordyk, 2018). statistical analyses: comparison of population dynamic values between male and female lengths, and weights were tested by student’s test (t-test) with paired t-test and independent t-test, respectively. the normality of this data was assessed using the kolmogorov–smirnov test. data analyses were performed using fisat ii and r studio softwares (1.1.46) with the tropfishr package. results length frequency distribution: the mean ± standard deviation of total length (lm) and total weight (wm) for male (1489 specimens) and female (1290 specimens) were 36±6 (18-58 mm), and 32±5 (19-50mm), and 14±6 (12-63 g), 10±5 (21-50 g), respectively. the differences between total length and total weight in both sexes were not significant (t = 1.45, p= 0.05; t = 1.44, p>0.05, respectively). the length (tl) data were categorized into 3-mm groups which the highest frequency belonging to individuals with 27 to 30 mm (fig. 3a). length-weight relationship: the relationship between length and weight give a=0.0008 and b= 2.66 (r2 = 0.89) for female, and for male, a=0.008 and b=2.71 (r2 = 0.88) and for both sexes a=0.0008 and b=2.70 (r2 = 0.88). the results showed significant differences between estimated b and b=3 at the level of 0.05 (fig. 3b), which means a negative allometric growth pattern for both sexes of b. spirata in the northern waters of the oman sea. growth studies: the population dynamic parameters of male, female, and both sex of b. spirata are detailed in table 1. growth parameters for both sexes together were l∞ = 71 mm (w∞ = 79 gr), k = 0.35 (yr-1), and t0= -0.2. the growth curve (fig. 4) highlighted 6 cohorts and age groups and the growth performance index as ф = 3.11. there is recruitment throughout the year and the highest rate of recruitment is observed in the winter and summer season’s time. based on the results, the maximum lifespan of this species was nearly 8 years. mortality estimate (m): the natural mortality (m), fishing mortality (f), and total mortality (z) were estimated as 0.5 (yr-1), 1.75 (yr-1), and 2.25 (yr-1), respectively (table 1). the exploitation coefficient was estimated as 0.78 (yr-1) (fig. 5). based on the calculated parameters, the von bertalanffy equations for this species (length and weight) in the northern oman sea, iran, are as follows. 135 int. j. aquat. biol. (2023) 11(2): 131-140 figure 3. length and frequency (a) as well as length weight relationship (b) of total babylonia spirata in the northern oman sea, iran. species (sex) method l∞ (mm) k ) 1-yr ) ot m f z e (male) spirata b. elefan 76 0.3 -0.42 0.4 1.79 2.19 0.82 b. spirata (female) elefan 68 0.54 -0.24 0.7 2.3 3 0.77 b. spirata (total) elefan 71 0.35 -0.37 0.5 1.75 2.25 0.78 table 1. comparison of population dynamics values of babylonia spirata with two methods (shepherd method and elefan method) (l∞= infinite length, k= growth rate, to= time that length is zero, m= natural mortality, f= fishing mortality, z= total mortality, e= exploitation rate). figure 4. growth curve derived from the structure of population of babylonia spirata from the northern oman sea (iran). the growth curve plot shows reconstructed frequencies, with negative and positive values as white and black colored histograms, respectively. the background shading shows runs of peaks, with positive peaks in blue, negative peaks in red, and values of zero in white. the different color backgrounds were added in order to help visualizing sign and magnitude of the bin values. the sum of all positive peaks is called the “available sum of peaks” (asp), which represents a maximum possible score. the “estimated sum of peaks” (esp) is the sum of peak values crossed by the growth curves. 136 hashemi et al./ length-based fishery status and population dynamics of spiral babylon lt = 71 (1 exp (-0.35 (t + 0.2))) wt = 79 (1 exp (-0.35 (t + 0.2))) ^ 2.70 the relative yield per recruitment (y/r) and relative biomass per recruitment (b'/r): based on length at first capture (lc = 29 mm), which is 50% the probability of catching shell, relative production per recruitment and relative biomass per recruitment were estimated as y'/r = 0.009 and b'/r = 0.2, respectively. the results demonstrate an exploitation rate (u) of 0.70 and the fishing mortality at maximum sustainable yield (fmsy) equaled to 1 for this stock (fig. 5). length-based indicators (lbi) and length-based spawning potential ratio (lbspr): length-based reference point is the percentage of shell caught at the optimum length for harvest (lopt=48 mm, popt=0.10), (lf=m=40 mm, popt=0.13) and percentage of mega-spawners in the catch (lmega=53 mm, pmega=0.04), respectively. based on the lbspr assessment model (fig. 6), lopt was estimated at about 0.3 (0.27-0.33). the ratio of pmega<0.1, lmean/ lopt <1 and lmean /lf=m <1 were estimated. a b figure 5. exploitation coefficient curve (a) and relative yield per recruit (y'/r) and relative biomass per recruit (b'/r), fmsy (b) of babylonia spirata (total) in the northern oman sea, iran (fmsy = fishing mortality rate of maximum sustainable yield, f 0.5= fishing mortality rate at which the slope of the yield-per-recruit curve is only half the slope of the curve at its origin, f0.1= fishing mortality rate at which the slope of the yieldper-recruit curve is only one percent the slope of the curve at its origin). figure 6. the lbspr of babylonia spirata (total) in the northern oman sea, iran. 137 int. j. aquat. biol. (2023) 11(2): 131-140 discussion we have managed to study a series of population dynamics values of the species b. spirata and this is the first time in the study area despite economic and ecological importance. babylonia spirata is one of the economically valuable species in the southern and northern oman sea, iran. life-history traits of b. spirata: the l-w relationship showed negative allometric growth and the female was heavier than males in the same length group. it seems the growth curve (length and weight) of b. spirata (total) slows down after two years and allometric growth is common in gasterpoda (carare and surugiu, 2020). the relationship between the length and weight of strombus canarium was calculated as a = 0.0000018 and b = 3.2 (r2 = 0.85) for males, and for females, a = 0.0000015 and b = 3.3 (r2 = 0.81) (cob et al., 2009). the observed size pattern was influenced by latitude and local spatial responses to factors such as the substrate changing the morphometric variables of the snail. the morphological characteristics such as length and weight relationship, are greatly affected by latitude, and it seems that as the latitude increases, the size of the snail becomes larger (matos et al., 2020). in addition, the morphological characteristics of snails are influenced by various factors such as temperature, available food, intra-group competition, substrate, climate, tidal conditions, and specific characteristics of habitat (matos et al., 2020). the l-w relationship is of great importance in fishery assessments (haimovic and velasco, 2000). according to marthin (1994), the range of "b" could be from 2.5 to 4 and it is believed b = 3 with isometric growth. also, the functional regression bvalue shows the body form, and it is directly related to the weight affected by ecological factors such as temperature, food supply, spawning conditions, and also other factors, such as sex, age, fishing time, and area and fishing vessels. population dynamic of b. spirata: comparisons of the population parameters of b. spirata with other studies in different parts of the world (table 2) show that the infinite length and growth coefficient of males from different species are smaller than females. also, the infinite length and growth coefficient of different species changes in various regions (table 2). differences in the range of the infinite length and growth rate are influenced by the ecological differences of each region (king, 2007). the growth rate is expected to be higher in the tropical zones. higher k-values for species are common in tropical waters due to their poikilothermic nature and result in a higher metabolic rate in relation to high temperate (hashemi, 2020). in general, the difference in the infinite length and growth rate in various regions might be due to the quantity and quality of food and climatic conditions (bartulovic et al., 2004). various factors can also affect holothurian growth including age, sex, season, year, type of feeding, physiological conditions, differences in food availability, and reproductive period (lalèyè, 2006). references species/region l∞ (mm) k (yr-1) m f z e mohan (2007) b. spirata, india 68 1.08 1.61 4.44 6.05 0.73 mohan (2007) b. zeylanica, india 76 1.15 1.65 3.37 5.02 0.67 cob et al. (2009) strombus canarium, malaysia 70 (f) 69 (m) 1.5 (f) 1.2 (m) 0.95 (f) 0.86 (m 1.61 (f) 1.86 (m) 2.56 (f) 2.86 (m) 0.62 (f) 0.65 (m arrighetti et al. (2012) olivancillaria deshayesiana, argentina 38 0.14 0.36 0.29 0.65 0.44 daza-guerra et al. (2018) cittarium pica, colombia 94 0.32 0.60 1.11 1.71 0.65 present study (2022) b. spirata, oman sea (iran) 71 0.35 0.5 1.75 2.25 0.78 table 2. comparison of population dynamic values of babylonia spirata with other studies around the world. for details of the abbreviation name of the values see table 1. 138 hashemi et al./ length-based fishery status and population dynamics of spiral babylon the natural mortality rate of b. spirata in this study was less than that of fishing mortality. the ratio of fishing mortality to maximum sustainable yield (f/fmsy) was over than one. to recall that if the value of f/fmsy is more than 1, it indicates that there is overfishing (arrizabalaga et al., 2012). the exploitation coefficient was over 0.5, indicating that the amount of capture fisheries was more than the optimum level. the exploitation coefficient should not be greater than 0.5 or, the fishing mortality should not exceed natural mortality otherwise they indicate overfishing (king, 2007; hashemi et al., 2021; hashemi and doustdar, 2022). the most important factors affecting the pressure on stocks are, first, the amount of catch and harvest of the stocks, and second, the environmental factors that affect survival and access to the fishery resources (mateus and estupinan, 2002). length-based indicators (lbi) and length-based spawning potential ratio (lbspr): the lmean/lopt index has values less than one (about 0.7), which means the presence of overfishing, and also lmean/lf=m index has values less than one (about 0.8) and pmega less than 0.1 is calculated which the show considered undesirable and index range optimal of lmean/lopt and lmean/lf=m is close to one and pmega is about 0.3-0.4 (cousido-rocha et al., 2022). the lbspr rate of this species was calculated at 0.3 (30%). lbspr gives estimates of spawning potential ratio (spr), where values below 0.2 (≈ 0.5 b/bmsy) indicate depletion and values above 0.4 (≈ 1.0 b/bmsy) indicate good stock status. the 95% confidence limits provided by lbspr are unrealistically narrow, sometimes close to deterministic, which partly explains their very low matching score. in conclusion, the lbspr model is a promising tool for the length-based assessment of data-limited fish stocks (hordyk et al., 2015). with regards to cheap and simple to collect measurements of the length composition of an exploited stock and also life-history information, models, and methods have been developed in recent years, for instance, the length-based spawning potential ratio method (lbspr) (hordyk et al., 2015). management and conservation of b. spirata in the oman sea: it is recommended that appropriate instructions be established for the harvesting and management of this species in the area. this parameter is needed for fisheries management and conservation of exploited this species. the present study showed that these stocks have reached 'overfished' status. the results of the present study could help in the management and sustainable exploitation of this species’ stocks. acknowledgments we would like to thank prof. bahmani, the manager of the iranian fisheries science research institute (ifsri), and very grateful to the experts of the offshore fisheries research center (ofrc, chabahar), for helping with the project work. references arrizabalaga h., murua m., majkowski j. 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(1996). biostatistical analysis. 3rd edition. prentice-hall inc., new jersey, usa. 662 p. u = 1 (lc / l∞) ; m = (1 e) / ( m / k) = ( k / z) ; e = f / z (9) int. j. aquat. biol. (2022) 10(5): 349-365 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article diatom community structure in relation to physico-chemical factors in a tropical soda lake shala and inflowing hot-springs, ethiopia solomon wagaw1,2 seyoum mengistou2, abebe getahun2, assefa wosnie3, yirga enawgaw1 1department of biology, wolkite university, wolkite, ethiopia. 2department of zoological sciences, addis ababa university, addis ababa, ethiopia. 3department of biology, dilla university, dilla, ethiopia. s article history: received 27 may 2021 accepted 22 october 2022 available online 2 5 october 2022 keywords: diatom community lake shala hot spring rda analysis abstract: diatoms are highly diverse and versatile, with members growing under different environmental conditions including extreme environments. although diatom communities in some extreme environments have been investigated recently, little is known about their community structure within the hot springs of soda lakes in ethiopia. the study aimed to assess the diversity and distribution of diatoms from lake shala and inflowing hot springs in relation to physico-chemical variables. water and diatom samples were collected from lake shala and three inflowing hotsprings. the mean ph, temperature, ec, salinity, tds, do, no3-+no2-, nh3+nh4+, srp, tp and sio2 were significantly different among the stations. the significant variations in these factors could be attributed to their heterogeneous geological characteristic and the hydrology of the study area. a total of 45 diatom taxa were identified, with the highest species observed in shala hora mid hotspring sites (37) and the lowest in shala gike hotspring (29). diatom community structure was also examined and it was found that the diatom community of lake shala and inflowing hotsprings are highly influenced by environmental water conditions. characteristic taxa including anomoeoneis sphaerophora, nitzschia spp., rhomboids gibberula, r. gibba, r. acuminata, r. operculata, navicula spp. and frustulia rhomboids, showed a wide tolerance to ph, salinity, ec, tds, temperature, nitrogen and phosphate. rda analysis found a number of discriminating taxa and salinity, conductivity, ph, do srp and temperature were key factors that accounted for a significant variation in the diatom community structure. introduction soda lakes of the east african rift valley are among the world’s most productive ecosystems (ogato et al., 2016). the lesser flamingo (phoeniconaias minor geoffroy saint-hilaire, 1798) population inhabits these lakes and can gather in flocks of over one million birds when the phytoplankton community of the lake is dominated by arthrospira fusiformis, their primary food sources (matagi, 2004; kumssa and bekele, 2014). in ethiopia, the main flamingo lakes are abijata, shala, metehara, chitu, and aranguade, which provide a preferred feeding and breeding habitat for large populations of avifauna (kumssa and bekele, 2014). despite the ecological, economic and scientific research values of these lakes, only a few are correspondence: solomon wagaw doi: https://doi.org/10.22034/ijab.v10i55.1229 e-mail: wagawsolomon2@gmail.com dor: 20.1001.1.23830956.2022.10.5.1.2 subjected to active conservation (matagi, 2004). these ecosystems are highly sensitive to environmental change mainly because of seasonal alterations of hydrological conditions and human‐ induced disturbances, which have been demonstrated to affect biological community structure in saline lakes (oduor and schagerl, 2007a; krienitz and kotut, 2010). saline lakes, especially those in the tropics, remain poorly studied, particularly in the processes controlling species distribution (mengistou, 2016; ogato and kifle, 2017). diatoms are considered the main component of aquatic ecosystems because they are usually the dominating primary producers (krienitz and kotut, 2010) and functionally important in sequestering and transforming many inorganic nutrients into organic https://ij-aquaticbiology.com/index.php/ijab/article/view/1229 350 wagaw et al./ diatom community structure in lake shala, ethipoia forms (krienitz and kotut, 2010). the benthic diatoms constitute a significant energy source for higher trophic levels (bate et al., 2002; carvalho et al., 2002). despite their ecological importance, the various factors that regulate community distribution and structure are poorly understood compared to pelagic phytoplankton communities (carvalho et al., 2002). many studies have reported a wide distribution of benthic diatoms and their tolerance to gradients of diverse environmental variables (gasse, 1986; wood and talling, 1988; kebede et al., 1994; oduor and schagerl, 2007a; krienitz and kotut, 2010). saline lakes and the nearby hot springs are complex ecosystems in which many environmental factors vary on spatial and/or temporal scales. these factors include geomorphic characteristics, nutrient and ionic concentrations and other physico-chemical properties, manifesting great ecological variability in species composition (gasse, 1986; wood and talling, 1988; gebre‐mariam, 2002; oduor and schagerl, 2007b; krienitz and kotut, 2010). there have been few studies on the ecology of benthic diatom in ethiopian rift valley lakes (gasse, 1986; woldesenbet, 2019; wondmagegn, 2019). however, compared with other east african rift valley lakes, only a few data are available on diatoms of saline lakes in ethiopia (e.g. gasse, 1986). previous studies on lake shala have focused on the primary productivity, algal biomass and water chemistry (talling et al., 1973; baumann et al., 1975; wood and talling, 1988; kebede et al., 1994; gebre-mariam, 2002) and on the taxonomic identification and classification of the phytoplankton species, with limited studies, i.e., ogato (2015), focusing on the relationships between physicochemical variables and phytoplankton. however, the physicochemical variable-dependent diatom distributions have not been well-studied in lake shala and associated hot-springs. accordingly, the present study focuses on patterns of benthic diatom community structure in relation to environmental (chemical and physical) and spatial factors in this alkaline saline environment. lake shala is one of the east african soda ecosystems and is fed by heated groundwater rising from deep aquifers along established fault lines (grant and jones, 2016). these hot springs and their drainage channels provide remarkable ecosystems and exhibit spatio-temporal variation in biotics with fascinating gradients of ph, salinity, dissolved oxygen (do), electrical conductivity (ec) and temperature. the purpose of this work was to answer whether the diatom of lake shala and inflowing hot spring habitats differ or not; whereas the lakes are extreme in terms of increased salinity and ph, the hot springs show extreme temperatures. material and methods description of the study area: lake shala lies between 7°24’-7°33’n and 38°23’-38°39’e at altitudes of approximately 1558 m within the abijata-shala lakes national park, some 287 km south of addis ababa in the main ethiopian rift valley. the lake is volcano-tectonic (woldegabriel et al., 2016) and found in the hydrologically closed system of the ziway-shala basin. lake shala is the deepest among the ethiopian rift valley lakes; it has approximately length of 28 km, 12 km width, and an average depth of 87 m (maximum 266 m), with a surface area of around 329 km2 and a vast catchment area (3920 km2) (von damm and edmond, 1984; baxter, 2002). lake shala receives its water from river adabat and gidu (baumann et al., 1975; baxter, 2002). the lake is also surrounded by numerous hot springs of varying salinity, temperature, size and discharge rate, which feed the lake (baxter, 2002). lake shala is characterized by a high ph, saline-alkaline conditions and high phosphate content, but with very low nitrogen levels (ogato et al., 2014). its water is rich in sodium (na+), carbonate (co3 2-), bicarbonate (hco3 -) and chloride (cl-), attributable to their presence in large concentrations in the trachytic and rhyolitic rocks of the ethiopian rift (klemperer and cash 2007), although they are poor in divalent cations (calcium [ca2+] and magnesium [mg2+]) (ogato et al., 2014). the surface water temperatures 351 int. j. aquat. biol. (2022) 10(5): 349-365 of lake shala range from 22 to 26oc (gebremariyam, 2002). despite its hostile nature, lake shala supports phytoplankton and is dominated by diatoms (kebede et al., 1994) and cryptophytes (ogato and kifle, 2017). lake shala also supports sparse zooplankton communities and is dominated by rotifers such as brachionus dimidiatus, b. pliciatilis and hexarthra (mengistou, 2016). the benthic macroinvertebrate community of the lake comprises tubificidae, ostracoda and chironomidae (tudorancea and harrison, 1988). the lake supports a rich diversity of avifauna, mainly pelicans and lesser flamingos inhabiting the lake and its volcanic island. oreochromis niloticus and other small-sized fish such as aplocheilichthys sp., were reported for the lake in 2002 (golubtsov et al. 2002). water physico-chemical analysis: sampling for physicochemical parameters was carried out at the same time during diatom sampling. dissolved oxygen, electrical conductivity, ph, and temperature were measured in situ with an hq40d hach lange multi-meter. for nutrient analyses, water samples were filtered through whatman gf/c) paper. ammonium (nh4-n) was analyzed by the indolphenol blue method, nitrate (no3-n) was determined with the sodium-salicylate method and nitrite (no2-n) was analyzed based on the reaction between sulphanilamide and n-naphthyl-(1)ethylendiamin-dihydrochloride (apha, 1999). soluble reactive phosphorus (srp) was determined by the ascorbic acid method, while total phosphorus (tp) was determined by the ascorbic acid method after the persulfate digestion of unfiltered samples. diatoms data collection: benthic diatoms were collected from the onshore habitat of lake shala and inflowing hot springs during april, may, and july 2018. a total of four sampling sites were selected based on the nature of the lake and inflowing hot springs (fig. 1). at each sampling station, diatoms were sampled by brushing stones with a toothbrush, following kelly (2000). at least cobles (5-15 cm) sized stones were brushed and the resulting diatom suspensions were put in a small plastic bottle. the samples were preserved in ethanol (70%) and transported to addis ababa university, limnology laboratory. at the laboratory, each diatom sample was acid cleaned with potassium permanganate figure 1. map of lake shala showing the study sites/sampling points. (abbreviation: (sl: shalla lake; shh1: shalla hora hot spring 1; shmh: shalla hora mid hot spring; sgh: shalla gike hot spring). 352 wagaw et al./ diatom community structure in lake shala, ethipoia (kmno4) and hydrochloric acid (hcl) to oxidize organics and remove the carbonates (cvetkoska et al., 2018). diatom were identified to the species or to genus levels, when possible, sub species level using appropriate keys under an inverted nikon microscope equipped with digital camera at a magnification of 200x and 400cx (gasse, 1986; komárek and kling, 1991; kelly, 2000; komárek et al., 2003; bellinger and sigee, 2010). statistical analysis: a nonparametric test, kruskalwallis, analogous to analysis of variance (ibm spss statistics 20) was used to compare means of physicochemical parameters among the four sampling sites. the association between diatom species distribution and physicochemical variables was evaluated by canonical multivariate analysis using canoco for windows 4.5 version software (ter braak and smilauer, 2002). detrended correspondence analysis (dca) was employed to check the response of the data, and it was found that the longest gradient (lg) length was 0.859. therefore, redundancy analyses (rda) were used to elucidate the relationships between species assemblages and environmental variables. diatom species with a total percent abundance <1% were not included in assessing the association between diatom taxa distribution and physicochemical variables. results physico-chemical parameters: a summary of spatial variations in physicochemical parameters of lake shala and its inflowing hot springs are shown in figures 2 and 3. the highest mean values of ph (10.17) were recorded at lake shala (sl), while the lowest value was recorded at shala hora hot-spring 1 (shh1) and differences are significant between the stations (p<0.05). dissolved oxygen (do) values were within the range of 0.75-8.11 mg l-1 and showed some variability among the studying sites (p<0.05). electrical conductivity (ec), salinity and total dissolved solids (tds) across all studying stations were significantly different (p<0.05). ec varied from 26.74±3.8 ms cm-1 in sl to 5.67±7.8 ms cm-1 in shh1. the highest conductivity mean value was recorded at sl (26.74 ms cm-1) and followed by shmh (15.85 ms cm-1). salinity and tds showed a trend similar to that of conductivity and were significantly different (p<0.05) between the studied sites. nutrient concentrations along the sampling stations are reported in figure 3 and their distribution showed spatial heterogeneities with studying stations. no3 -+no2 and nh3+nh4 + concentrations varied from 0 to 2.8 μg l-1 and from 0 to 4.15 μg l1, respectively and were insignificantly different among the stations (p>0.05). the mean value of soluble reactive phosphorus (srp) ranged from 37 μg l-1 to 211.5 μg l-1. in sl, the concentration of srp was greater and showed a significant difference (p<0.05). the distribution of total phosphate (tp) indicates that the levels of phosphate variations among the studied sites (p<0.05). highest tp was recorded at sl (8165 μg l-1) and the lowest mean values were documented in shmh (835 μg l-1). there was a significant difference in dissolved sio2 between sampling stations (p>0.05). the lowest mean value of dissolved sio2 (58.75 mg l -1) was recorded at sl and the highest at sgh (124.5 mg l1). diatom composition and their relative abundance: forty-five (45) identified diatom species from lake shala, and its associated hot springs are presented in table 1. the number of taxa (species richness) among the samples ranges between 29 and 37. highest taxa were recorded from shmh (37) (table 1). the taxa showed a clear gradient along the physicochemical variables and the number decreased to 36, 30 and 29 from sl, shh1 and sgh, respectively. dominant species in most of the samples were anomoeoneis sphaerophora, frustulia rhomboides, nitzschia spp., navicula spp., epithemia frickei, e. operculata, rhopalodia gibberula, r. rupestris and achnanthes spp. taking into account all samples, 20 (about 44.4%) diatom species such as amphora spp., a. sphaerophora, campylodiscus clpeus var bicostata, cyclotella meneghiniana, epithemia adnata, e. frickei, e. gibba, e. hyndmanii, 353 int. j. aquat. biol. (2022) 10(5): 349-365 e. operculata, frustulia rhomboides, navicula spp., nitzschia spp., rhopalodia acuminata, r. acuminate var. protracta, r. brebissonii, r. gibberula, r. rupestris and stephanodiscus spp. were common to the studied water bodies (table 1, fig. 4). species that were exclusive to each sampling site were represented by a few individuals. species found only in lake shala were campylodiscus hibernicus, cymatoppleura solea, encynonema spp., gomphonema spp. and pleurosigma spp. while species found only in shala hora mid hot spring (shmh) were as follows: epithemia turgida var. westermannii, rhopalodia constrica and r. vermicularis. distribution of diatom species in relation to physicochemical variables: the rda showed that the first two axes sufficiently (96.5%) explained the cumulative percentage variance in the diatom species-environmental variables relation in studied sites (fig. 5, table 2). figure 2. spatial variation of physical environmental variables along the studied stations. (sl: shalla lake; shh1: shalla hora hot spring 1; shmh: shalla hora mid hot spring; sgh: shalla gike hot spring; tep: temperature; do: dissolved oxygen; tds: total dissolved solids) (values with different letters (a, b, c, d) within a column are significantly different at p< 0.05 level (tukey test is applied)). 354 wagaw et al./ diatom community structure in lake shala, ethipoia figure 4. venn diagram representing the number of diatoms that are unique and shared between the samples from 4 different sampling sites. (sl: shalla lake; shh1: shalla hora hot spring1; shmh: shalla hora mid hot spring; sgh: shalla gike hot spring. figure 3. spatial variation of inorganic nutrients along the studied stations. (sl: shalla lake; shh1: shalla hora hot spring 1; shmh: shalla hora mid hot spring; sgh: shalla gike hot spring; srp: soluble reactive phosphorus; tp: total phosphorus) (values with different letters (a, b, c, d) within a column are significantly different at p< 0.05 level (tukey test is applied)). 355 int. j. aquat. biol. (2022) 10(5): 349-365 the results indicated that ph was the most important environmental variable accounting for species distribution in the first axis. the distribution of diatom species was also positively correlated with ec, salinity, tds and do in axis 1 and contributed 71.8% of the variance. relative abundance of anomoeoneis sphaerophora, rhomboids operculata, stenopterobia spp., epithemia argus, stephanodiscus spp., e. frickei, e. hyndmanii, e. adnata, amphora spp., campylodiscus clpeus var bicostata, navicula spp., cyclotella. meneghiniana and achnanthes spp. had a strong positive association diatom species relative abundances (%) all stations sl shh1 shhm sgh achnanthes spp. 4.2 6 5.8 4.3 amphora spp. 2.0 1.5 3.5 1.8 1.9 anomoeoneis sphaerophora 15.7 9.2 15.3 19.9 15.4 a. styriaca (grunow) hustedt 0.6 0.6 0.7 0.5 0.7 campylodiscus clpeus var bicostata w. sm. 1.7 1.2 1.2 1.2 2.6 c. hibernicus (ehrenberg) 0.1 0.2 cyclotella iris brun & héribaud 0.7 1.6 2 c. meneghiniana kützing 3.0 3.4 3.2 2 4.8 cyclotella spp. 2.3 4.3 3.6 cymatoppleura solea (brébisson) w. smith 0.1 0.2 cymbella spp. 0.3 0.1 0.3 0.3 0.6 diatoma spp. 0.2 0.2 0.3 0.2 epithemia adnata (kützing) brébisson 2.5 1.7 3.3 2.6 2.6 e. argus (ehrenberg) kützing 1.7 1.3 2.3 2.4 e. argus var. alpestris (w. smith) grunow 0.2 0.4 e. frickei krammer 3.8 2.5 3.3 5 3.3 e. hyndmanii w. smith 1.7 0.9 0.7 2.7 1.9 e. smithii carruthers 0.6 0.6 1.1 e. sorex var. gracilis hustedt 0.2 0.4 0.6 e. turgida (ehrenberg) grunow 0.3 0.3 1.3 e. turgida var. westermannii (ehrenberg) grunow 0.1 0.3 encynonema spp. 0.1 0.2 encyonopsis microcephala (grunow) krammer 0.4 0.9 0.4 0.4 eunotia spp. 0.3 0.4 0.3 0.3 frustulia rhomboids (ehrenberg) de toni 5.6 3.6 10.4 3.8 8.2 gomphonema spp. 0.1 0.6 hantzschia spp. 0.4 0.5 0.5 0.4 melosira ambigua (grun.) müller 0.5 0.4 1 1.7 navicula spp. 5.4 2 7.3 6.8 4.6 nitzschia spp. 9.1 18.3 8.5 5.6 4.6 pinulariaa spp. 0.3 0.4 0.7 0.4 pleurosigma spp 0.1 0.5 rhopalodia acuminata krammer 7 9.2 7.1 5.2 8.4 r. acuminate var. protracta (grunow) 2.3 3.1 1.8 2 2.6 r. brebissonii krammer 1.7 2.8 2.7 0.7 1.5 r. constrica (w. smith) krammer 0.1 0.2 r. gibba (ehrenberg) o. müller var. gibba 2.1 2.9 0.7 2.3 1.9 r. gibberula 7.8 13.1 6.8 5.9 5.6 r. operculata (agardh) håkansson 7.3 7.4 3.2 7.9 10.4 r. rupestris (w. smith krammer) 3.3 5.4 2.3 2.4 3.7 r. vermicularis (o. müller) 0.1 0.4 surirella ovalis brébisson 0.3 0.5 0.3 0.7 surirella turgida w. smith 0.7 0.4 1 0.9 stenopterobia spp. 1.4 1.2 0.7 2.3 stephanodiscus spp. 1.7 1.5 1.7 1.7 2 total no. species 36 30 37 29 table 1. list of the 45 diatom taxa identified in the lake shalla and inflowing hot springs. 356 wagaw et al./ diatom community structure in lake shala, ethipoia figure 5. redundancy analyses (rdas) triplots showing the relationship between diatoms communities, environmental parameters and sites (show with different color). diatom species relative abundances (%) all stations sl shh1 shhm sgh achnanthes spp. 4.2 6 5.8 4.3 amphora spp. 2.0 1.5 3.5 1.8 1.9 anomoeoneis sphaerophora 15.7 9.2 15.3 19.9 15.4 a. styriaca (grunow) hustedt 0.6 0.6 0.7 0.5 0.7 campylodiscus clpeus var bicostata w. sm. 1.7 1.2 1.2 1.2 2.6 c. hibernicus (ehrenberg) 0.1 0.2 cyclotella iris brun & héribaud 0.7 1.6 2 c. meneghiniana kützing 3.0 3.4 3.2 2 4.8 cyclotella spp. 2.3 4.3 3.6 cymatoppleura solea (brébisson) w. smith 0.1 0.2 cymbella spp. 0.3 0.1 0.3 0.3 0.6 diatoma spp. 0.2 0.2 0.3 0.2 epithemia adnata (kützing) brébisson 2.5 1.7 3.3 2.6 2.6 e. argus (ehrenberg) kützing 1.7 1.3 2.3 2.4 e. argus var. alpestris (w. smith) grunow 0.2 0.4 e. frickei krammer 3.8 2.5 3.3 5 3.3 e. hyndmanii w. smith 1.7 0.9 0.7 2.7 1.9 e. smithii carruthers 0.6 0.6 1.1 e. sorex var. gracilis hustedt 0.2 0.4 0.6 e. turgida (ehrenberg) grunow 0.3 0.3 1.3 e. turgida var. westermannii (ehrenberg) grunow 0.1 0.3 table 2. list of the 45 diatom taxa identified in the lake shalla and inflowing hot springs. 357 int. j. aquat. biol. (2022) 10(5): 349-365 with ph and positively correlated with ec, salinity, tds and do while no3 -+no2 -, nh3+nh4 +, sio2 and tp showed a negative association in axis 1. nitzschia spp., rhopalodia gibberula and r. rupestris were strong and positively correlated with ph (0.86), ec (0.99), tds (0.99), sal (0.99) do (1) and tp (0.99) and showed negative association with tep (-0.98), no3 -+no2 (-0.27), nh3+nh4 + (-0.23) and sio2 (-0.30) in axis 2 (table 2). nitzschia spp., rhopalodia gibberula, r. rupestris r. acuminata r. acuminata var protracta, cyclotella meneghiniana and r. gibba are representative taxa in lake shala abundantly (sl) (fig. 5). these taxa typically occurred in habitats with high specific ec, salinity, tds, do, ph and tp. the diatom assemblage in shala hora mid hotspring (shmh) had intermediate ec, salinity, tds, do, ph and tp mean values and was represented by anomoeoneis sphaerophora, epithemia frickei, e. hyndmanii, e. argus, e. adnata, stenopterobia spp, cyclotella spp, rhopalodia operculata, campylodiscus clypeus, achnanthes spp., amphora spp., navicula spp. and frustulia rhomboids. discussion spatial physico-chemical dynamics in lake shala and its associated hot springs: there has been significant interest in finding life in extreme environments with high temperatures, salinity and ph like hot springs and soda lakes. hence, this is the first report on the investigation of diatom diversity and community structure at different physicochemical factors of ethiopian saline-alkaline lake shala and inflowing hot springs. this ecological study aimed to examine the influence of the spatial variations in the environmental variables on the diatom distribution within lake shala and its inflowing hot springs. the value of high ph, salinity, ec and tds diatom species relative abundances (%) all stations sl shh1 shhm sgh encynonema spp. 0.1 0.2 encyonopsis microcephala (grunow) krammer 0.4 0.9 0.4 0.4 eunotia spp. 0.3 0.4 0.3 0.3 frustulia rhomboids (ehrenberg) de toni 5.6 3.6 10.4 3.8 8.2 gomphonema spp. 0.1 0.6 hantzschia spp. 0.4 0.5 0.5 0.4 melosira ambigua (grun.) müller 0.5 0.4 1 1.7 navicula spp. 5.4 2 7.3 6.8 4.6 nitzschia spp. 9.1 18.3 8.5 5.6 4.6 pinulariaa spp. 0.3 0.4 0.7 0.4 pleurosigma spp 0.1 0.5 rhopalodia acuminata krammer 7 9.2 7.1 5.2 8.4 r. acuminate var. protracta (grunow) 2.3 3.1 1.8 2 2.6 r. brebissonii krammer 1.7 2.8 2.7 0.7 1.5 r. constrica (w. smith) krammer 0.1 0.2 r. gibba (ehrenberg) o. müller var. gibba 2.1 2.9 0.7 2.3 1.9 r. gibberula 7.8 13.1 6.8 5.9 5.6 r. operculata (agardh) håkansson 7.3 7.4 3.2 7.9 10.4 r. rupestris (w. smith krammer) 3.3 5.4 2.3 2.4 3.7 r. vermicularis (o. müller) 0.1 0.4 surirella ovalis brébisson 0.3 0.5 0.3 0.7 surirella turgida w. smith 0.7 0.4 1 0.9 stenopterobia spp. 1.4 1.2 0.7 2.3 stephanodiscus spp. 1.7 1.5 1.7 1.7 2 total no. species 36 30 37 29 table 2. continued. 358 wagaw et al./ diatom community structure in lake shala, ethipoia recorded in the studied lakes shala and inflowing hot-springs did reach previously reported in saline alkaline lakes of east africa (talling et al., 1973; melack et al., 1982; wood and talling, 1988; kebede et al., 1994; talling and lemoalle, 1998) and inflows hot springs (mpawenayo and mathooko, 2004; owen et al., 2004). saline alkaline nature of these soda lakes is further influenced by the heterogeneous surficial geology characteristic, climate and hydrology of the region (legesse et al., 2004). east african soda lakes are characterized by high concentrations of carbonate salts, especially sodium carbonate and related salt complexes (wood and talling, 1988; kebede et al., 1994). they also contain high concentrations of sodium chloride and other dissolved salts, making them saline-alkaline lakes (gebre-mariam, 2002; klemper and cash, 2007). during the present investigation, the mean ph values were significantly different over the studied sites. the highest mean values of ph (10.17) were recorded in lake shala surface waters, with these high values indicating the influence of potential photosynthetic activity and increased salinity concentrations, both possible causes of alkaline conditions (talling, 2011). while lower mean ph value recorded in hot springs might be due to less concentration of sodium and carbonate species in the cation and anion-dissolved solutes, respectively. this was supported by the results, which found that the majority of lakes and hot springs of soda lakes in the kenyan rift valley (grant and jones, 2016; salano et al., 2017). however, the relationship between ph and other factors is complex, being influenced by chemical and biological processes (hammer, 1986; williams, 1998). dissolved oxygen levels were significantly lower in hot spring sites with a mean value ranging between 0.75-3.73 mg l-1. the observed higher amount of do in the lake shala site is probably related to the activities of photosynthetic organisms. lower oxygen concentrations in hot springs may arise from the high rate of respiration by decomposers such as bacteria and fungi that anchor the gravel and small rocks of the hot springs (renaut et al., 2008; grant and jones, 2016; salano et al., 2017). extremely high microbial load within the great east africa rift valley soda lakes is described by lanzen et al. (2013) and grant and jones (2016). wood et al. (1984) also suggested the depletion of do in tropical soda lakes by algal biomass decomposition and high microbial activity. although salinity, ec and tds were significantly different across the studied sites, they show considerable strong gradients from hot springs with lower salinity, tds and ec or diffuse groundwater inflows along the shores towards the lake with higher mean values. high mean values of salinity (14.21 g l-1), ec (26.74 ms cm-1) and tds (11.7 g l-1) in lake shala could be associated with the accumulation of solutes that originate from rain falling directly on the surface of the lake and drainage basin, weathering reactions between runoff and groundwater, hydrothermal fluids and their interactions with subsurface rocks (baumann et al., 1975; von damm and edmond, 1984; ayenew and legesse, 2007). similarly, east african soda lakes inflowing hot spring water also show considerably lower salinity, ec and tds than soda lakes; for example, the salinity of elmentaita and bogoria hot springs have been recorded between 1.6-2.3 g l-1 and 3.5-3 g l-1, respectively. while in lake elmentaita (3.8-4.6 g l-1) and lake bogoria (10-20 g l-1), the surface water salinity was higher (krienitz and schagerl, 2016). ogato (2015) also reported the low concentration of salinity (4.5 g l-1) and conductivity (8.2 ms cm-1) of inflowing hot springs around lake shala. concentrations of soluble and largely inorganic forms of the element’s nitrogen, phosphorus and silicon were surveyed and significantly different in the four sampling sites. nitrogen limitation has been suggested for some other tropical african soda lakes (melack et al., 1982; wood and talling, 1988; kebede et al., 1994; talling and lemoalle, 1998). in the present study, nitrate and ammonia-nitrogen were less and not detected in lake shala and inflowing hot springs, which is an indication of 359 int. j. aquat. biol. (2022) 10(5): 349-365 nitrifying bacteria occurrence. high diversity and abundance of ammonia and nitrite-oxidizing organisms are widely distributed and responsible for low nitrogen levels in african soda lakes (sorokin, 1998; grant and jones, 2000; grant and jones, 2000; sorokin et al., 2001; grant, 2006). however, in shh1 higher concentration of the no3 -+no2 (2.8 μg l-1) and nh3+nh4 + (4.15 μg l-1) was recorded. the possible reason could be the visitors of livestock around the hot springs which cause considerable inputs of nitrogenous wastes. the concentration of phosphorus was quite high in the lake and inflowing hot springs and was significantly different between sites. high mean soluble reactive phosphate (srp) and total phosphate (tp) concentrations were recorded in lake shala. there is a general trend of increasing phosphate content concentration with increasing salinity and conductivity. this result is nearly comparable with the work done in ethiopian inland waters by wood and telling (1998). the significant variations in these factors could be attributed to the specific abiogenic and biogenic transformations (wood and telling, 1998), the predominance of phosphate mineral-rich rocks (talling and talling, 1965), and the release from the anoxic water column (oduor and schagerl, 2007b). high total phosphate and soluble reactive phosphate concentrations are also reported in east african saline-alkaline lakes (melack et al., 1982; kalff, 1983; oduor and schagerl, 2007b). in the present study, silicate concentration significantly varied between the sites. previously the concentration of sio2 in lake shala (56 mg l -1) (kebede et al., 1994) and the main hot springs located on the shore of lake shala (64.8 mg l-1) (ogato, 2015) were reported. however, the mean concentration sio2 observed in the present study is remarkably high in hot springs. the contributor could be silicate soils, porous volcanic lavas and the enhanced dissolution of solid silicates in saline waters of high alkalinity and ph (wood and telling, 1998). the silicate concentration of lake shala in this study (58.75 mg l-1) was comparable with the value (56) reported by kebede et al. (1994) and (49.55) reported by gebre-mariam (2002). the silicate concentration is notably low in lake shala, indicating the dominance of diatoms in the lake. several studies on tropical african lakes (hecky, 1993; gebre-mariam, 2002) reported the association of depletion with the abundance of diatoms. wood and talling (1988) and kebede et al. (1994) suggested that sio2 could be significantly removed from the solution in lake shala, which was dominated by diatoms. diatom community structure in lake shala and hot springs: the extremely inhospitable conditions in alkaline, saline lakes mean that the biodiversity in these systems is limited to organisms with special adaptations to survive such extreme conditions (matagi, 2004). in the present study, forty-five diatom taxa were identified from lake shala and its inflowing hot spring with rhopalodiaceae (9 taxa) and epithemiaceae (9) being best represented. the most abundant species were a. sphaerophora, nitzschia spp., r. acuminata, r. gibberula, r. operculata, navicula spp. and f. rhomboids. this is in agreement with earlier studies done in alkaline, saline habitat which reported anomoeoneis, rhopalodia, nitzschia and epithemia to be the most dominant (hecky and kilham 1973; gasse et al., 1983; mpawenayo and mathooko, 2004; owen et al., 2004) in contrast with the reported dominance of algal biomass by spirulina platensis (blue-green algae, cyanobacteria) in alkaline-saline lakes ethiopia and kenya (talling et al., 1973; harper et al., 2003; ballot et al., 2004; oduor and schagerl, 2007a). the dominance of anomoeoneis, rhopalodia, nitzschia and epithemia species in these harsh environments can be attributed to their ability to withstand extreme water conditions like very high temperatures, ph and salinity. there was a difference in the taxa number and individual abundance of diatoms among the sampling sites. low taxon number and individual abundance were recorded in sgh (29) and shh1 (30). this might be due to the special physical features of the habitat, such as high temperature and 360 wagaw et al./ diatom community structure in lake shala, ethipoia low dissolved oxygen; living in such ecosystems has tolerated and adapted to this hostile environment. owen et al. (2004) also argue that the hot spring’s low diversity values are due to extreme environmental conditions. contrary, the highest number of diatom species (37) were recorded at shmh and this might be due to the exchange and contact of the lake organisms with the surrounding hot spring because depending on the lake shala water level and wave, this hot spring area can be covered by lake water, which enables direct exchange of organisms. even minor changes in the lake water level can impact the ecology of the hot springs and adjacent habitats (renaut et al. 2008, 2013), impacting species diversity (krienitz et al., 2005). diatoms have complex spatial dynamics within aquatic ecosystems (cvetkoska, 2018). numerous researchers (e.g., gasse et al., 1983; hecky and kilham, 1973; mpawenayo and mathooko, 2004; owen et al., 2004) have demonstrated different diatom assemblages across a range of different alkaline habitats. differences in diatom community structure among the studding site were investigated and there were dissimilarities in the diatom communities in lake shala and its inflowing hot springs. similarly, owen et al. (2004) and mpawenayo and mathooko (2004) reported variations in the diatom populations in lake bogoria, lake elmentaita and their inflowing hot springs. during the current investigation, several diatom species, e.g., a. sphaerophora, nitzschia spp., r. gibberula, r. operculata, r. acuminata, navicula spp., f. rhomboids, c. meneghiniana, e. adnata and e. frickei, occurred across a range of physico-chemical variables, suggesting that most species have high tolerance ranges of environmental conditions. distribution of diatom in relation to physicochemical variables: numerous studies conducted the influence of physico-chemical and nutrient variables on diatom communities and have shown the importance of water ph and related variables (e.g., salinity, alkalinity, conductivity) as main drivers structuring diatom communities (gasse et al., 1983; hecky and kilham, 1973; smol and stoermer, 2010). in the present study, rda analysis demonstrated that the diatom community structure of lake shala and inflowing hot springs was highly influenced by ph and also to some extent by ec, do, salinity, tds and srp in axis 1, which explained 96.5% of the variance in diatom community composition. moreover, the analyses indicated strong spatial variability, highlighting the importance of the different environmental factors in structuring the benthic diatom community, which could largely be attributed to variations in physicochemical features and used as an indicator of lake development, erosion, alkalinization, acidification, salinization, climate change, and especially eutrophication. according to hecky and kilham (1973) and gasse et al. (1983), the distribution of diatom species is essentially influenced by salinity and conductivity. diatoms in lake shala and inflowing hot springs were affected by salinity and nutrients as indicated by the rda (fig. 6), with owen et al. (2004) and mpawenayo and mathooko (2004) reporting similar findings. owen et al. (2004) highlighted the effect of variations in physico-chemical variables of soda lakes and their inflowing hot springs on diatom communities. this result was consistent with a study in east african soda (e.g. gasse et al., 1983; hecky and kilham, 1973; mpawenayo and mathooko 2004; owen et al., 2004). conclusion marked variability in physico-chemical features was observed among the studying sites in lake shala and its inflowing hot springs. this variation was related to inter-site differences in geomorphic and hydrological characteristics, resulting in uneven diatom species distribution. a total of 45 diatom taxa were identified, of which 20 (about 44.4%) diatom species were common across the study area. this indicates that the studied hot springs of soda lakes in ethiopia are important niches that harbor an unexpectedly high richness of diatom species. the 361 int. j. aquat. biol. 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(2019). water quality assessment of lake hawassa, ethiopia, using macroinvertebrate and diatom based multimetric index. ph.d. thesis. addis ababa university, addis ababa, ethiopia. 214 p. material and methods despite its hostile nature, lake shala supports phytoplankton and is dominated by diatoms (kebede et al., 1994) and cryptophytes (ogato and kifle, 2017). lake shala also supports sparse zooplankton communities and is dominated by rotifers such as brac... water physico-chemical analysis: sampling for physicochemical parameters was carried out at the same time during diatom sampling. dissolved oxygen, electrical conductivity, ph, and temperature were measured in situ with an hq40d hach lange multi-meter... diatoms data collection: benthic diatoms were collected from the onshore habitat of lake shala and inflowing hot springs during april, may, and july 2018. a total of four sampling sites were selected based on the nature of the lake and inflowing hot s... statistical analysis: a nonparametric test, kruskal-wallis, analogous to analysis of variance (ibm spss statistics 20) was used to compare means of physico-chemical parameters among the four sampling sites. the association between diatom species distr... results physico-chemical parameters: a summary of spatial variations in physicochemical parameters of lake shala and its inflowing hot springs are shown in figures 2 and 3. the highest mean values of ph (10.17) were recorded at lake shala (sl), while the lowe... nutrient concentrations along the sampling stations are reported in figure 3 and their distribution showed spatial heterogeneities with studying stations. no3-+no2and nh3+nh4+ concentrations varied from 0 to 2.8 μg l-1 and from 0 to 4.15 μg l-1, res... diatom composition and their relative abundance: forty-five (45) identified diatom species from lake shala, and its associated hot springs are presented in table 1. the number of taxa (species richness) among the samples ranges between 29 and 37. high... distribution of diatom species in relation to physicochemical variables: the rda showed that the first two axes sufficiently (96.5%) explained the cumulative percentage variance in the diatom species-environmental variables relation in studied sites (... the results indicated that ph was the most important environmental variable accounting for species distribution in the first axis. the distribution of diatom species was also positively correlated with ec, salinity, tds and do in axis 1 and contribute... discussion diatom community structure in lake shala and hot springs: the extremely inhospitable conditions in alkaline, saline lakes mean that the biodiversity in these systems is limited to organisms with special adaptations to survive such extreme conditions (... distribution of diatom in relation to physico-chemical variables: numerous studies conducted the influence of physico-chemical and nutrient variables on diatom communities and have shown the importance of water ph and related variables (e.g., salinity... conclusion references int. j. aquat. biol. (2023) 11(1): 20-29 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article population dynamics of two sympatric native and exotic cichlids in a tropical microtidal estuary, india kuttanelloor roshni1, chelapurath radhakrishnan renjithkumar1,2 1department of aquatic environment management, kerala university of fisheries and ocean studies, kochi 682506, india. 2st albert’s college (autonomous), ernakulam, kerala, india. s article history: received 14 june 2022 accepted 24 december 2022 available online 2 5 february 2023 keywords: alien species growth mortality overfishing vembanad lake abstract: population dynamics of two sympatric cichlids, the native etroplus suratensis and the alien oreochromis mossambicus, were determined from the vembanad lake, the largest estuarine system of southern peninsular india. based on annual length-frequency data, the results revealed that e. suratensis has a higher asymptotic length (l∞) (302 mm vs. 262mm), but a lower growth constant (k) (0.68 year-1 vs. 1.1 year-1) than o. mossambicus, suggesting the faster growth rate of the alien species. natural mortality (m) rates recorded for e. suratensis (0.69 year-1) indicate lower levels of predation and other natural stressors in the lake. the exploitation ratio (e) was estimated to be 0.83 for e. suratensis and 0.78 for o. mossambicus, both values greater than the predicted optimum and suggestive of overexploitation. our study provides the first information on the demography of wild e. suratensis, which would help inform future management strategies in the vembanad lake. introduction biological invasion is the second-most important threat to global biodiversity after habitat loss (welcomme, 1988; courtenay and robins, 1989). the invasion and establishment of non-native species can impair ecosystem functioning through adverse impacts at the individual, population and community levels of native species, eventually leading to their population decline or extinction (nyman, 1991; sato et al., 2010). ecological risks caused by alien fish species are difficult to be estimated in the initial stages, though the impacts are far-reaching and irreversible (stapp and hayward, 2002). despite the high rate of global biological invasion, the ecological, economic and social impacts of alien species are poorly known to effectively address and manage the issue (gu et al., 2015; xia et al., 2019). mozambique tilapia, oreochromis mossambicus (peters, 1852) is a benthopelagic, eurytopic and salt correspondence: kuttanelloor roshni doi: https://doi.org/10.22034/ijab.v11i1.1621 e-mail: roshni.phd@gmail.com dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.3.3 tolerant mouth-brooding cichlid inhabiting slowflowing rivers and streams, as well as the lakes and lagoons of eastern africa (skelton, 1993). due to its perceived utility as a candidate aquaculture species, it is one of the world's most translocated and widely distributed alien fish species (canonico et al., 2005). the species survives in many unsuitable environments due to its huge ecological plasticity characterized by adaptable life-history traits, trophic flexibility, tolerance to extreme and unfavorable environmental conditions, immense breeding potential, sound parental care, high growth rate, and aggressive and territorial behaviour (pérez et al., 2006; maddern et al., 2007). considering its ecological impacts, o. mossambicus has been listed as one of the 100 most invasive species in the world (global invasive species database, 2019) and is believed to create the most adverse ecological effects (lowe et al., 2000). the pearlspot, etroplus suratensis (bloch, 1790), 21 int. j. aquat. biol. (2023) 11(1): 20-29 is a medium-sized euryhaline substrate-spawning fish inhabiting estuaries, coastal lagoons, and natural and man-made freshwater habitats of peninsular india and sri lanka (munro, 1955; baensch and riehl, 1985). this species is a major contributor to inland fisheries in peninsular india (padmakumar et al., 2012) and sri lanka (jones et al., 2018), supporting the livelihoods of thousands of subsistence fishers. etroplus suratensis has high acceptance in domestic and international markets and has recently been considered an ideal aquaculture species (padmakumar et al., 2009). though assessed as ‘least concern’ on the iucn red list of threatened species™, localized population declines of this species have been recorded (abraham et al., 2019). vembanad lake is the largest tropical estuarine system located on the southwest coast of india, harboring a rich ichthyodiversity (maitra et al., 2018). etroplus suratensis, one of the most abundant species in the lake ecosystem (samuel, 1969), has shown drastic population reductions due to a synergy of anthropogenic stress in the habitat (kurup et al., 1995; padmakumar et al., 2012). the landing of the species decreased from 1252 tonnes in the 1960s (samuel, 1969) to 458 t in the middle 1980s (kurup et al., 1995) and 135.28 t in 2012-2013 (roshni et al., 2017). the reported reduction in e. suratensis has been concomitant with an increase in catches of the exotic o. mossambicus (kurup et al., 1993; padmakumar et al., 2012). this co-existence of e. suratensis and o. mossambicus results in competition for food and nesting habitats since both species belong to the same family (cichlidae) and have similar habitat preferences (benthopelagic), feeding behaviour (omnivores) and reproductive requirements (mouth and substrate brooders requiring sufficient space for spawning) (ward and samarakoon, 1981; bindu and padmakumar, 2008; russel et al., 2012; froese and pauly, 2019) leading to the potential competitive displacement of e. suratensis (odum, 1971). in this context, this work aims to estimate the population characteristics of native e. suratensis and alien o. mossambicus in vembanad lake to inform appropriate management plans. materials and methods the vembanad lake (9°28′, 10°10′n, 76°13′, 76°31′e) is a shallow, complex bar-built micro-tidal estuary covering an area of 241 km2 with a catchment area of 14500 km2, depth range of 1-12 m and total length of 80 km, stretching from munambam in the north to alappuzha in the south in the state of kerala having open permanent connections with arabian sea (martin et al., 2011). it is listed as a ramsar site (no. 1214) by the convention of wetlands of unesco in 1981, considering its ecological significance and global conservation importance. it is one of the fastdegrading estuaries in india, and its fish fauna is under severe depletion pressure due to lake reclamation, bottom dredging, pollution, overexploitation of fishery resources and alien species introductions (maitra et al., 2018). samples of e. suratensis and o. mossambicus were collected from commercial catches at major fish landing centers along the vembanad lake monthly from june 2017 to may 2018. fish were caught using gillnets of different mesh sizes ranging from 2-9 cm and cast nets with a mesh size of 1.5 cm. data were collected randomly from well-mixed catches to avoid size-based bias in the population estimation. the total length of each individual fish was measured (lt, to the nearest 0.01 mm), and data were grouped into 10 mm intervals for further analysis. the growth and mortality parameters were estimated for the pooled population. asymptotic length (l∞) and growth coefficient (k) were estimated using von bertalanffy growth formula (vbgf) (von bertalanffy, 1938; gayanilo et al., 2005) using elefan-1 (electronic length frequency analysis) incorporated in fisat-ii (fao-iclarm stock assessment tools) (see http://www.fao.org/fishery/topic/16072/en#4) software (gayanilo et al., 2005). based on the l∞ and k values, growth performance index () and potential longevity (3/k) were estimated (munro and 22 roshni and renjithkumar / population dynamics of two sympatric native and exotic cichlids pauly, 1983) using the formula:  = log 10 (k) + 2 + log 10 (l∞). total mortality (z) was estimated using the lengthconverted catch curve method (pauly, 1984). natural mortality (m) was calculated using the empirical formula of pauly (1980): in m = 0.0152-0.279 in (l∞) + 0.6543 in (k)+0.4634 in (t), where l∞ is the asymptotic length in mm, k is the growth constant in year–1 and t is the annual mean temperature (29◦c). the instantaneous fishing mortality rate (f) was computed as f = z-m. the exploitation rate (e) was estimated as e = f/z (gulland, 1970). emax (maximum yield per recruit) and e50 (exploitation that retains 50% of the biomass) were calculated from relative yield per recruit (y/r) and relative biomass per recruit (b/r) analysis using the knife-edge selection method (pauly and soriano, 1986). the recruitment pattern was determined by reconstructing the recruitment pulses from a time series of lengthfrequency data (gayanilo et al., 2005). results a total of 745 specimens of e. suratensis (67-300 mm) and 865 specimens of o. mossambicus (62-260 mm) were analysed during the study period. using elefan-ii, the growth parameters, i.e. asymptotic length (l∞), growth constant (k) and age at which length equals 0 (t0) were estimated as 302 mm, 0.68 year-1 and -0.01527 year for e. suratensis, and 262 mm, 1.1 year-1 and -0.0041 year for o. mossambicus, respectively (fig. 1a, b). the growth performance index () was estimated as 4.87 and 4.79 for e. suratensis and o. mossambicus, respectively. based on the growth equation, e. suratensis attained a length of 151 mm and 225 mm at the end of the first and second years of growth, respectively, while o. mossambicus reached 175 mm and 233 mm during the same period. total (z), natural (m) and fishing (f) mortality coefficients estimated for e. suratensis were 4.04 year-1, 0.69 year-1 and 3.35 year-1, and z, m and f for o. mossambicus were estimated as 4.42 year-1, 0.99 year-1 and 3.44 year-1 (fig. 2a, b). the exploitation rate (e) of e. suratensis was estimated as 0.83 and for o. mossambicus as 0.78. the exploitation rate at which marginal increase in relative yield per recruit figure 1. (a and b). von bertalanffy growth curve of etroplus suratensis and oreochromis mossambicus from vembanad lake, india. 23 int. j. aquat. biol. (2023) 11(1): 20-29 becomes 1/10th of its value (e10), the exploitation rate at which the stock would be reduced to 50% of its unexploited biomass (e50), and exploitation rate at which yield per recruit reaches maximum (emax) were estimated as 0.550, 0.368 and 0.675 for e. suratensis, and 0.600, 0.386 and 0.722 for o. mossambicus (fig. 3a, b). the size at first capture for e. suratensis was estimated as l25 = 136.38 mm, l50 = 153.25 mm and l75 = 170.12 mm, and for o. mossambicu as l25 = 136.30 mm, l50 = 149.48 figure 2. (a and b). length-converted catch curve for etroplus suratensis and oreochromis mossambicus from vembanad lake, india. figure 3. (a & b). relative yield-perrecruit (y/r) and relative biomassper-recruit (b/r) analysis using knife-edge method for etroplus suratensis and oreochromis mossambicus from vembanad lake, india. figure 4. (a & b). recruitment pattern for etroplus suratensis and oreochromis mossambicus from vembanad lake, india. 24 roshni and renjithkumar / population dynamics of two sympatric native and exotic cichlids mm and l75 = 162.12 mm. recruitment of e. suratensis and o. mossambicus to the fishery was continuous and took place throughout the year, with around 64% of the recruitment occurring between july and september for both species (figs. 4a, b). discussion though o. mossambicus attains a maximum length of 390 mm (wohlfarth and hulata, 1983), no fish in this size range were obtained in the present study. it has been suggested that o. mossambicus can alter the life history traits in favour of the surrounding environment; hence, a standard growth pattern with respect to a particular ecosystem or geographical area is difficult to fix (pérez et al., 2006; maddern et al., 2007). menon (1999) estimated a maximum attainable length of 400 mm for e. suratensis; however, individuals encountered in the present study showed a significantly lower length range which may be due to the high levels of anthropogenic stress and their subsequent impacts limiting the growth of the species (padmakumar et al., 2012). bindu and padmakumar (2014) reported a maximum length of 340 mm for e. suratensis from vembanad lake, comparable with the maximum estimated length observed in the present study. there is no published information on the growth (k, l∞ and ) and mortality parameters (z, m and f) of e. suratensis which makes comparing different geographic populations impossible. the estimated asymptotic length of e. suratensis is higher and the growth coefficient lower than that of o. mossambicus indicating that o. mossambicus grows much faster than e. suratensis facilitating its rapid colonization in the lake (table 1). slow-growing species are more vulnerable to collapse, especially when climatic variability and harvest dynamics contribute unfavorably to fish survival rates (pinsky and byler, 2015). the slow growth of e. suratensis could probably increase the depletion pressure of the species if extreme climatic variations and severe overexploitation continue in their natural habitat. the l∞ estimated in the present study is almost similar to that of o. mossambicus populations in african waters (weyl and hecht, 1998), while the species showed their highest growth rate in vembanad lake than any other region, which suggests that the lake provides the best habitat requirement for o. mossambicus, facilitating its range expansion and population explosion. the growth performance index value () values observed for e. suratensis (4.87) and o. mossambicus (4.79) is high, reflecting excellent environmental conditions and high food availability in the habitat, favouring rapid growth of the species. the growth performance () values recorded for o. mossambicus was higher than those observed in previous studies (moreau et al., 1986; de silva et al., 1988; de silva and amarasinghe, 1989), which may be attributable to the ability of the species to utilize a wide range of food items (maitipe and de silva, 1984) in the vembanad lake. the higher natural mortality of o. mossambicus in the present study could be due to active fish predators in the lake (gosavi et al., 2019), and the higher fishing mortality suggests high exploitation (glamuzina et al., 2007; panda et al., 2018). overfishing is a major threat to inland waters (allan et al., 2005). in the present study, a higher fishing mortality was observed for o. mossambicus compared to populations from sri lanka (amarasinghe and de silva, 1992; amarasinghe, 2002; amarasinghe et al., 2017). for an exploited fish stock of optimal utilization, the rate of fishing mortality (f) should be equal to the rate of natural mortality (m), giving an exploitation rate (e) of 0.5 (gulland, 1970). the relative yield per recruit (y/r) and biomass per recruit (b/r) determined based on knife-edge selection estimated the exploitation rate (e) of e. suratensis and o. mossambicus to be greater than 0.5. also, the computed value of exploitation was greater than the predicted emax, indicating that both the fishes are under excessive fishing pressure. etroplus suratensis is exploited more intensively, particularly due to a targeted fishery due to their high economic value and market demand. bindu and padmakumar (2014) reported that 25 int. j. aquat. biol. (2023) 11(1): 20-29 e. suratensis attains maturity at a length of 195 mm in males and 200 mm in females in vembanad lake. the length at first capture (lc) of e. suratensis was estimated as 153.25 mm in the present study, indicating that the exploited stock mainly comprises immature individuals. the capture of smaller-sized individuals before reaching maturity can affect population stability and recruitment (gwinn et al., 2015; van overzee and rijnsdorp, 2015). however, allen et al. (2002) reported o. mossambicus reaches sexual maturity at a length of 150 mm, and the length at first capture (lc) estimated for the species in the present study was 149.48 mm, almost reaching reproductive maturity. for e. suratensis, the overexploitation of immature fishes could be a major threat in the lake, affecting recruitment and leading to population decline. meanwhile, the overexploitation of near-mature individuals of o. mossambicus is unlikely to threaten its population size due to the species continuous spawning, stable recruitment, and rapid growth. recruitment of both e. suratensis and o. mossambicus was continuous throughout the year, with a major pulse during julyseptember producing 64.30 and 64.24% recruitment, respectively. spawning and recruitment of the two fish species occurring in the same month could lead to severe inter-specific competition for available food resources and space due to overcrowding of fingerlings. our study indicates the presence of a wellestablished population of o. mossambicus in vembanad lake, contributing significantly to the total annual fishery. this will lead to the rapid occupation of a vacant spatial and trophic niche by the species resulting in the competitive displacement of many native fishes, especially those sharing similar ecological resources like e. suratensis and pseudetroplus maculatus (raghavan et al., 2008). conclusion currently, e. suratensis in vembanad lake is experiencing severe fishing pressure, as evident from the high rate of exploitation, including a greater proportion of juveniles in the catch. this is in addition to threats from alien species such as atractosteus spatula, clarias gariepinus, pangasianodon hypophthalmus, piaractus brachypomus and pterygoplichthys multiradiatus (krishnakumar et al., 2011; roshni et al., 2014; bijukumar et al., 2019). there is an urgent need to reduce the fishing effort and the mesh sizes of fishing gears to avoid the future collapse of this fish species. also, a more detailed investigation is recommended to understand the life history traits and threats to species l∞ k  z m f e e50 lc location source o. mossambicus 448 0.52 2.8 1.39 1 0.394 0.282 18.8 kadulla reservoir, sri lanka amarasinghe and de silva (1992) o. mossambicus 460 0.45 2.77 3.03 0.955 2.076 0.685 19.6 minneriya reservoir,sri lanka amarasinghe and de silva (1992) o. mossambicus 266. 0.23 chicamba lake, mozambique weyl and hecht (1998) o. mossambicus 378 0.51 2.69 2.83 1.1 1.73 0.61 15.9 tabbowa reservoir, sri lanka amarasinghe (2002) o. mossambicus 314 0.84 salton sea, california, usa caskey et al. (2007) o. mossambicus 402 0.42 2.64 2.15 0.93 1.22 0.57 0.75 22.4 minneriya reservoir,sri lanka amarasinghe et al. (2017) o. mossambicus 424 0.31 2.55 2.34 0.75 1.59 0.68 0.75 22.5 udawalawe reservoir, sri lanka amarasinghe et al. (2017) o. mossambicus 432 0.29 2.54 1.96 0.69 1.27 0.68 0.75 23.7 victoria reservoir, sri lanka amarasinghe et al. (2017) o. mossambicus 262 1.1 4.79 4.42 0.99 3.44 0.78 0.368 149.5 vembanad lake, india present study e. suratensis 302 0.68 4.87 4.04 0.69 3.35 0.83 0.386 153.3 vembanad lake, india present study l∞, asymptotic length (mm); k, growth coefficient (year-1); , growth performance index; z, total mortality (year-1); m, natural mortality (year1); f, fishing mortality (year-1); e, current exploitation rate, e50, exploitation rate where stock is reduced to half its virgin biomass; lc, length at first capture (mm). table 3. growth, mortality and exploitation parameters of oreochromis mossambicus and etroplus suratensis from various geographical regions. 26 roshni and renjithkumar / population dynamics of two sympatric native and exotic cichlids e. suratensis for implementing proper conservation plans. reward-based encouragement to the local fishers for collecting large quantities of o. mossambicus, detailed scientific research on the impacts on native species, and the development of novel eradication measures are also required for managing the fast-growing population of o. mossambicus. acknowledgments the authors are thankful to the head, department of aquatic environment management, kufos, kochi, india, for providing the necessary facilities for the research. we express our sincere gratitude to rajeev raghavan, assistant professor, kufos, kochi, india, for his constructive comments on the draft manuscript. the first author thanks the kerala state council for science technology and environment (kscste), india, for the financial support to carry out the research. references abraham r., de alwis goonatilake s., fernado m, kotagama o. 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(2021) 9(4): 254-257 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology quality analysis and effect of reused water on some biological characteristics of phaseolus vulgaris and pisum sativum batool m.h. al-adily *,1noor m. naji, shaymaa o.h. al-mamoori collage of sciences, babylon university, hillah, iraq. article history: received 7 april 2021 accepted 21 august 2021 available online 2 5 august 2021 keywords: reused water, p. vulgaris, p. sativum, stem anatomy. abstract: seeds of phaseolus vulgaris l. and pisum sativum were planted in separated pots, then these pots divided into two groups, each group divided into four subgroups. four different wastewater were used to irrigate the treatments. the results showed the effects induced by irrigated wastewater on p. vulgaris l. and p. sativum. the degree of water salinity induced increasing sod, protein content and the area of xylem elements, but reduced chlorophyll-α in p. vulgaris. the cat, protein and chlorophyll-α were increased in in p. sativum as a result to salinity which also reduces the area of xylem elements. introduction phaseolus vulgaris l. is a legume species with a wide distribution, therefore it can be grown in a high range of environmental conditions except those of extremes (singh, 1999). also, pisum sativum is an edible leguminous seed that need a particular soil feature i.e. clay with slight acidity (vural et al., 2000; mani, 2015). in this species, salinity is more effective on germination of the bean (ghezal et al., 2016). in iraq, especially in past years, the regression of fresh water and increased the area with saline soil (al-adily, 2014) has been led to use other unusual water resources for irrigation (al-maamori and al-adily, 2018). many works done regarding the effects of using wastewater for irrigation (khurana and singh, 2012; nwaokobia et al., 2018). since both p. vulgaris and p. sativum are sensitive to salinity (maas and hoffman, 1977; de pascales et al., 1997; munns and tester, 2008), therefore, this work aimed to study the effects of different reused water for the irrigation of p. vulgaris and p. sativum as alternative for fresh water. materials and methods the seeds of p. vulgaris and p. sativum were planted in pots each with 1 kg soil, then seed pots of each *correspondence: batool m.h. al-adily doi: https://doi.org/10.22034/ijab.v9i4.1347 e-mail: batool_aladily@yahoo.com species were divided into four subgroups each with three replicates. they were irrigated with four different wastewaters (table 1). these wastewaters were collected from four different puncture water within babylon governorate, iraq. standard methods were used to measure of the water characteristics (apha, 2005). in addition, the standers methods of soil labs were used to measure the important features of the used soil in the palnted pots (icarda, 2001). the measure characteristics are shown in table 1. the biochemical measurement of the plants with ages of 45 day were chlorophyll-α (yash, 1998), proline content (bates, 1973), sod (marklund and marklund, 1974), total protein (bradford, 1976) and catalase content (aeibi, 1984). the method to study the anatomical variations was based on jahanson (1940). results and discussions the biochemical results of p. vulgaris l. and p. sativum showed their different strategies to adapt with water quality (tables 1, 2). the high salinity of type 3 water (ec=3320 μs/cm) reduce chlorophyll-α and cat in p. vulgaris, which may be due to concentration of chloride because bean is sensitive to salinity ions (bouzid and rahmoune, 2012; anna et 255 int. j. aquat. biol. (2021) 9(4): 254-257 al., 2015) causing defect in amount of gas exchange and the producig chlorophyll-α (kauymakanova and stoeva, 2008). whereas, p. sativum do not showed an increase in chlorophyll-α content with increase salinity of irrigated water and high concentration recorded in group 4 containing high concentration of organic matter (0.28%). pisum was able to tolerant salinity by increasing proline formation and protein content which was in a positive relationship with cat. this results are in agreement with other studies done on pisum on other type of soil and under different weather conditions (mani, 2015; artega et al., 2020). the results also give a good indicator to prove ability of pisum agriculture in iraq. histological sections of the stem in the studied species (figs. 1 and 2) revealed that thickness of epidermal cells was higher in group 2 which irrigated with wastewater containing lower concentrations of total ions and salinity, while the high levels of salinity induced increasing of the epidermal layer numbers and no increase found in group 3, so in this group showed an increasing in area of xylem elements and the higher increase was in group 4. bean is adapted to salinity in many different adaptation, some were table 1. some characters of four types of wastewater used in irrigated of phaseolus vulgaris and pisum sativum. wastewater parameters 4 3 2 1 8.3 8.5 8.1 8.6 ph 3210 3320 950 1438 e.c (μs/cm) 1450 2340 698 1030 tds (mg/l) 1740 2930 786 1240 salinity (mg/l) 920 1480 840 700 total hardness (mg/l) 368.74 593.18 336.67 280.56 calcium (mg/l) 134.51 216.38 122.81 102.34 magnesium (mg/l) 499.85 779.76 409.76 349.89 chloride (mg/l) 320 400 240 140 alkalinity (mg/l) 241.18 153.92 122.55 67.65 sulphate (mg/l) 0.28 0.12 0.16 0.07 organic matter (%) figure 1. anatomical variation of stem of phaseolus vulgaris induced by different wastewater sources (20x). 256 al-adily et al./ effect of reused water on some biological characteristics of p. vulgaris and p. sativum biochemically or phonologically (al-hassan et al., 2016) and in this study anatomical adaptations, including decrease the layers of collenchyma tissue was observed. in contrast, in pisum, there was no significant variations in the area of epidermis, while organic matter may be induced the producing of the parenchyma tissue and the width of vascular bands (as seen in group 4) and the number of the xylem elements but it reduced the diameter of xylem vessel. plants can tolerant elevated concentrations of salinity ions by increasing the fibers of xylem (al-adily et al., 2018). salinity causes some plants to change the anatomy of vascular system according to degree of ec of water or soil (tietjen et al., 2017; qaderi et al., 2019). references aeibi h. 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(2016) 4(3): 189-201; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article effect of aquatic plants upon planktonic and periphytic organisms: a microcosm-based approach svetlana kurbatova*,1nina lapteva, svetlana bykova, igor yershov, yelena borisovskaya laboratory of experimental ecology, i.d. papanin institute for biology of inland waters, russian academy of sciences, 152742 borok, yaroslavskaya region, russian federation. article history: received 11 january 2016 accepted 27 may 2016 available online 2 5 june 2016 keywords: aquatic plants stratiotes aloides bacteria periphyton plankton abstract: aquatic plants have a major influence upon other aquatic organisms, by altering both water chemistry and spatial structure of the habitat in shallow water bodies. some of them, such as stratiotes aloides l., may suppress algal growth. but how aquatic plants would ultimately influence the heterotrophic community and the aquatic ecosystem as a whole is far from clear. our microcosmbased study demonstrated that even a modest density of s. aloides caused a decline in phytoplankton chlorophyll concentration and periphytic algae abundance, including cyanobacteria, whereas diatoms appeared to be immune to the plant influence. photosynthetic rate remained unaltered despite decreased chlorophyll concentration. while bacterial counts remained largely unchanged, more bacteria were observed forming microcolonies as well as associating with particulate organic matter. numbers of periphytic heterotrophic organisms did not differ significantly between the planted and plant-free control microcosms. zooplankton diversity increased and cladocerans assumed a more prominent position within the microcosms with macrophytes. we assume that the presence of plant’s leads to increased importance of bacteria and protists in the functioning of the food webs. therefore, decreasing of algal abundance does not involve reducing the number of heterotrophic planktonic and periphytic organisms. introduction aquatic plants affect water flow, cycles of organic and inorganic nutrients, water quality and aquatic community structure in the shallow waters. macrophytes change food interaction and production of different trophic levels. plants often reduce the abundance and generic richness of algae (muylaert et al., 2010) and their role as primary producers. rooney and kalff (2003) revealed an increase in the bacterial production and violation of the classical interaction between the bacteria and phytoplankton due to the changes in the turnover of phosphorus and dissolved organic carbon caused by submerged macrophytes. as other food resources (bacteria and protozoa) are more important in the diet of zooplankton, their production may significantly exceed that of phytoplankton within stands of * corresponding author: svetlana kurbatova e-mail address: kurb@ibiw.yaroslavl.ru macrophytes (burks et al., 2006). aquatic plants significantly increase the amount of detritus in the aquatic ecosystems. as a result, the importance of the detritus-based food chains rises. the organic matter and energy from detritus decomposers flows to detritivores and bacteriophages, and to predators (moore et al., 2004). heterotrophic organisms use aquatic plants as shelter or feeding sites. features of the structure of heterotrophic organisms’ communities within stands of macrophytes often link with the complexity of spatial structure of the habitat (mcabendroth et al., 2005). in addition, some aquatic plants can significantly alter water chemistry (brammer and wetzel, 1984; pokorný et al., 1984). this aspect of the effect of macrophytes on the aquatic communities’ structure has received little attention. 190 kurbatova et al./ effect of aquatic plants on plankton and periphyton we hypothesized that changes in chemical parameters of the environment caused by the vital activity of the macrophytes may influence the abundance of some species. for our research, we have chosen water soldier (stratiotes aloides l.) because this plant has a marked effect on the cationic composition of the water (brammer and wetzel, 1984) and has a high allelopathic potential (burks et al., 2006). stratiotes aloides is a vascular aquatic plant commonly found in the shallow mesotrophic and eutrophic water bodies. under the right conditions, water soldier can occupy a major portion of the water body. the influence of water soldier upon planktonic and periphytic algae is well established (jasser, 1995; mulderij et al., 2005; hilt, 2006; mohamed and alshehri, 2010) and is likely to alter the structure of the heterotrophic consumers’ community. in the recent decades, a small number of studies looking at zooplankton community of water soldier mats (bittel, 1980; irvine et al., 1990; strzałek and koperski, 2009) and heterotrophic organisms associated with water soldier periphyton (mieczan, 2010) have been published. the objective of the present study was to investigate the effect of the changes in the environment (such as cationic composition and excretion of allelochemicals) caused by aquatic plants on the development of plankton and periphyton. materials and methods microcosm design: open-air microcosms were set up in square (1x1 m) shallow containers. to limit daily water temperature fluctuations, those containers were placed in larger water-filled concrete tanks. containers were filled with filtered water (64 µm mesh to remove of zooplankton) probably contained algae and bacteria, to the depth of 0.3 m (total volume 300 l). microcosms were covered by netting to prevent insects, leaf litter and mollusks entrance. after zooplankton collecting, the equal amounts of its concentrations were placed into individual microcosms. stratiotes aloides was collected from the local habitats and acclimated for two weeks under the experimental conditions. in accordance with moderate density observed in the field, ten plants per microcosm were introduced a week after the addition of zooplankton (10 ind. m-2). average wet weight of an individual plant was 173 g; at the beginning of the experiment, the plants were in the flowering or early fruiting phase. two days after plant introduction, periphyton development was monitored using microscope slides. each treatment was done in triplicate, with plant-free microcosms as controls. prior to introduction of the plants, n-no3 and nnh4, as well as soluble reactive phosphorus (srp) levels were measured. to avoid n limitation of plants and algae n-no3 was added to adjust n: p ratio to 30: 1, typical for mesotrophic lakes of the region (datsenko, 2007). those measurements were repeated after 30 days, and 0.11 mg l-1 n-nh4 (as ammonia chloride) was added in each microcosm. during the experiment, the levels of nitrogen and phosphorus were maintained within their typical range of water soldier habitats (tarkowskakukuryk, 2006; michalska-hejduk et al., 2009). the experiment was monitored for 60 days. sample collection and analysis: water temperature and ph values were measured daily between 09:0010:00. every 10 days, the concentration of oxygen, major cations (na+, k+, ca2+, mg2+), biochemical oxygen demand (bod5), chemical oxygen demand (cod), chlorophyll concentration, the number of bacteria, the plankton photosynthesis rate and rate of organic matter decomposition were assessed. every five days, the microcosms were sampled for srp and zooplankton. once a week, one suspended slide per microcosm was sacrificed for periphyton community analysis. oxygen, bod5, srp (as orthophosphate) and cod (using permanganate procedure) were measured according to alekin et al. (1973). major cation concentrations were determined by the use of flapho-4 flame photometer and aas-1 atomic absorption spectrophotometer. the chlorophyll was measured by the spectrophotometer lambda-25 191 int. j. aquat. biol. (2016) 4(3): 189-201 (perkin-elmer) following acetone extraction (sirenko and kureishevich, 1982). bacterial cells were collected on the membrane filters (pore size 0.17 µm), stained with dapi (porter and feig, 1980) and counted under the epifluorescent microscope. bacterial biomass was calculated based on cell counts and average cell size; cell density was assumed 1 g cm-3. n-nh4 and n-no3 was measured by the photometric procedure using nessler's reagent and salicylic acid, respectively, at the begining and mid-experiment (alekin et al., 1973; cataldo et al., 1975). the rate of plankton photosynthesis and organic matter degradation was assessed by measuring oxygen level changes in the light and dark bottles (kuznetsov and dubinina, 1989). zooplankton was collected with a 0.5 l plankton sampler at 6 locations per microcosm (3 l total), fixed with formaldehyde and counted by the standard hydrobiologycal methods. periphyton algal, protist and rotifer community makeup and the organisms’ numbers were determined by direct microscopic counts on unstained unfixed suspended slides at 280x magnification. organisms were identified to the rank of the species, but some of them only to the genus. periphytic organisms’ biomasses were calculated using dimensions and approximated geometric shape, assuming a specific gravity of 1. in order to assess the biomasses changes of zooplankton, algae and bacteria, the approximate phytoplankton biomass was calculated, based on the chlorophyll a values and applying coefficient 0.4 (mineeva and shchure, 2012). data analysis: for all figures, the mean±one standard deviation is shown (mean±sd). taking into account the small sample size, nonparametric methods were used in data processing. the effects of s. aloides on the chlorophyll a, cations concentrations, zooplankton and periphyton abundance were analyzed by friedman anova (repeated measures) test. pairwise comparisons were made using wilcoxon test and bonferroni corrected. calculations were made using past (hammer et al., 2001). correlations between parameters were evaluated by spearman's rank correlation coefficient using statistica 6.0 software package. results chemical parameters: during the first 47 days, the microcosm’s water temperature averaged 24ºс in the morning and it was rising to 30-31ºс by midday. the temperature declined towards the end of the experiment, averaging 15ºс in the morning over the final 10 days. the water ph values in the control microcosms steadily increased from 8.0 to 9.3 over the experiment; in the planted microcosms, water ph remained close to neutral (7.5-7.7), increasing in the final week to 8-8.2. the experimental microcosms have shown lower oxygen concentration (average 6.7 mg l-1) compared to that of the controls (9.2 mg l-1). by midday, the oxygen concentration decrease to 4.24.6 mg l-1 both in the planted and control microcosms. in the planted microcosms, waterborne oxygen concentration correlated with the water temperature (r=-0.83, p=0.042, n=7); in the controls, oxygen correlated with the ph values (r=0.93, p=0.008, n=7). the potassium and magnesium cation concentrations in the planted microcosms were significantly lower (friedman anova: p<0.001) compared to the controls (fig. 1). early in the experiment, the sodium and calcium cation concentrations in the controls exceeded those of the treatments. then the concentrations of these ions were above the control’s level in the tanks with s. aloides. there was a negative correlation between the calcium concentration and ph in the controls (r=-0.79, p=0.033, n=7). in the planted microcosms, a positive correlation was observed between ph and sodium (r=0.86, p=0.012, n=7), and a negative between ph and magnesium (r=-0.83, p=0.022, n=7). srp levels over the first 40 days of the experiment were comparable between the controls and planted microcosms. average srp concentration in the control and treatments were 16.3 and 17.6 µgp l-1, respectively. on day 44, in the 192 kurbatova et al./ effect of aquatic plants on plankton and periphyton controls, srp peaked at 50.4 µgp l-1, declining to 8.3 µgp l-1 by day 60. in the treatments, srp concentration steadily declined to 6.5 µgp l-1 from day 44 towards the end of experiment. srp concentration correlated with the sodium (r=0.93, p=0.008, n=6), and calcium (r=0.83, p=0.042, n=6) concentrations in the controls and treatments, respectively. prior to adjustment of the n:p ratio, the initial levels of waterborne nitrogen in the microcosms were 0.009 mg l-1 n-no3 and 0.14 l -1 n-nh4. after 30 days, the controls contained 0.023 mg l-1 n-no3 and 0.13 mg l-1 n-nh4; while the planted microcosms contained 0.013 mg l-1 n-no3 and 0.19 mg l-1 n-nh4, at which point n:p ratio was adjusted again. bod5 and cod of the planted microcosms were two fold of those of the controls in the beginning and at the end of the experiment. at other times, bod5 and cod values in the former were below or insignificantly different from the controls. average bod5 values over the entire experiment were similar in the controls and treatments (2.2 mg l-1), ranging from 1.3 to 3.5 and 1.7 to 2.8 mg l-1, respectively. cod values averaged 10.1 mg l-1 (ranging from 4.5 to 15.9 mg l-1) and 11.1 mg l-1 (4.0-15.5 mg l-1) in the controls and treatments, respectively. bod5 to cod ratio throughout the experiment was below 0.5, suggesting that much of the organic matter present in the system is made up of recalcitrant compounds. bod5 correlated with the phytoplankton photosynthesis rate (r=0.93, p=0.008, n=6) in the figure 1. cation concentrations in the experimental systems (mean±sd). 193 int. j. aquat. biol. (2016) 4(3): 189-201 controls; no such correlation was observed in the planted microcosms. phytoplankton: over the first 40 days of the experiment, lower concentrations of chlorophylls α, b and c were observed in the water of planted microcosms compared to controls (fig. 2). after 50 days, the chlorophyll α and c concentrations increased in the treatments and declined in the controls. the differences in the chlorophyll α concentration between the planted microcosms and plant-free controls were statistically significant (friedman anova: p<0.05). despite two to threefold lower chlorophyll concentrations in the treatments, the planktonic photosynthetic rates were about 0.5 mg o2•l-1•d-1 in both the treatments and controls. photosynthesis correlated with srp levels in the controls (r=0.84, p=0.036, n=6). no significant correlations between the photosynthesis rate and other measured parameters were detected in the planted microcosms. e480/e664 ratio stayed below 1.4, averaging 0.75 and 0.67 in the controls and planted microcosms, suggesting that the phytoplankton was not nitrogen limited (watson and osborne, 1979). bacteria: bacterial counts were comparable in the planted microcosms and controls. bacterial numbers and biomass averaged 5×106 cells ml-1 (ranging 3.47.7×106 cells ml-1), and 0.7 mg l-1 (ranging 0.4-1.0 mg l-1), respectively. in the planted microcosms, most bacteria were associated with detritus particles or formed micro aggregates (fig. 3a, b). the bacterial community was dominated by small rods until 30th days and with other forms appearing by the end of the experimental period (fig. 3c-f). in the planted microcosms, the bacterial numbers and figure 2. chlorophyll a, b and c concentrations (mean±sd). figure 3. bacterial aggregates (a and b) and various bacterial morphologies (c–f) from the planted microcosms. 194 kurbatova et al./ effect of aquatic plants on plankton and periphyton biomass correlated with the biomass of copepods (r=0.79, p=0.036, n=7 and r=0.86, p=0.014, n=7, respectively) and with the decomposition rates of organic matter (r=-0.79, p=0.036, n=7). unlike bacterial counts, peaks of the organic matter decomposition coincided in the planted microcosms and controls. the rate of organic matter decomposition over the whole period of the experiment was 0.31±0.07 and 0.35±0.05 mg o2•l -1•d-1 in the controls and planted microcosms, respectively. the rate of planktonic production exceeded the rate of organic matter decomposition by a factor of 1.7 and 2 in the controls and planted microcosms, respectively. microperiphyton: submerged slides revealed the presence of algae from bacillariophyta, chlorophyta and chrysophyta divisions, cyanobacteria, protists from sarcomastigophora and ciliophora phyla, as well as rotifers. algal cell numbers and biomass in the controls were ranged 18-585×103 cells cm-2 (fig. 4a), and 0.12-1.36 mg cm-2, respectively (fig. 4c). the total number of species was 4-11. at the beginning of the experiment, in the control, the periphytic community was dominated by the green algae (coleochaete scutata brebisson, hormidium sp.) and cyano figure 4. periphytic algal number (a, b) and biomass (c, d) in controls and planted microcosms (mean±sd). 195 int. j. aquat. biol. (2016) 4(3): 189-201 figure 5. periphytic heterotrophic organisms abundance (a, b), biomass of rotifers and ciliates (c, d) and biomass of sarcomastigophora (e, f) in controls and planted microcosms (mean±sd). 196 kurbatova et al./ effect of aquatic plants on plankton and periphyton bacteria (aphanizomenon flos-aquae (l.) ralfs ex born. et flah). navicula pupula kütz. var. pupula (fig. 4a) was the dominant species in number at 19th day, but c. scutata and synedra ulna (nitzsch.) ehr. var. ulna contributed significantly to the biomass as well. in the second half of the experiment, the algal community was dominated by rivularia sp. cyanobacteria. in the planted microcosms, algal cell number and biomass were from 49-265×103 cells cm-2 (fig. 4b) and 0.3-0.6 mg cm-2, respectively during 47 days and the algal biomass peaked to 1.92 mg cm-2 on 60th day (fig. 4d). normally, 8 to 11 species of the algae were found on the submerged slides of the treatments, except during 33-47 days, when only 3 to 4 species were detected. at the early stages of the experiment, c. scutata, stigeoclonium sp. and a. flos-aquae were detected in the periphytic community in the planted microcosms. coinciding with 26th day, diatoms, especially n. pupula, along with s. ulna and cocconeis placentula her. var. placentula dominated the algal community, with increase of c. scutata by the end of experiment. the number and biomass of cyanobacteria differed significantly (friedman anova: p<0.01) between the planted microcosms and plant-free controls. the differences in diatoms were not statistically significant (friedman anova: p=0.09). heterotrophic organism numbers were somewhat lower in the planted microcosms compared to controls (fig. 5a, b); the differences were not statistically significant (friedman anova: p=0.06). they were 0.7-2.5×103 ind. cm-2 in the treatments and 0.9-6.7×103 ind. cm-2 in controls. the biomass of heterotrophic periphytic organisms ranged 0.014-0.37 mg cm-2 and 0.006-0.97 mg cm-2 in the planted microcosms and controls, respectively (fig. 5c-f). the biomass of the most abundant organism represented by heterotrophic nanoflagellates (hnf), was very low. the most numerous organisms were desmarella irregularis stokes, codonosiga botrytis (ehrenberg) kent, bodo saltans ehrenberg and anthophisa vegetans (o.f.m.) stein. ciliates were present in small numbers, but their biomass in the controls was noticeable at the end of the experiment. in the controls, the ciliate community was dominated by sedentary genera of vorticella ehrenberg, vaginicola lamarck et ehrenberg and stentor polymorphus müller. in the planted microcosms, peak ciliate numbers were reached on 47th day, with vorticella sp. and chilodonella uncinata ehrenberg present. rotifers were philodina acuticornis murray and ph. сitrina ehrenberg both in the planted and controls microcosms. zooplankton: a total of 48 species of zooplankton were found in this experiment; 34 of them were common in both the planted microcosms and controls. diversity shannon' index values calculated on the base of both numbers of individuals and biomass, were more than 50% higher in the plantcontaining microcosms during 35 to 50 days. on average, the zooplankton numbers and biomass were 86 ind. l-1 and 2.7 mg l-1 in the planted microcosms vs. 70 ind. l-1 and 2 mg l-1 in the controls. the portion of the cladocerans was greater in the planted microcosms (91% of total biomass), compared to that of the control one (79%). daphnia longispina o.f. müller was the dominant species in both control and planted microcosms (fig. 6a). at the beginning 10th day, alona rectangula sars, (chydoridae) found in greater numbers in the planted microcosms (fig. 6b). in addition, alonella exigua (lillijeborg), graptoleberis testudinaria (fischer) and chydorus sphaericus (o.f. müller) were observed in appreciable numbers. numbers of chydoridae correlated with the numbers of periphytic hnf (r=-0.94, p=0.005, n=6). since 40th day, substantial (up to 47 ind. l-1) numbers of ceriodaphnia quadrangula (o.f. müller) was observed in the planted microcosms (fig. 6c); those numbers were significantly higher compared to the controls (friedman anova: p<0.001). ceriodaphnia quadrangula counts correlated with water ph values (r=0.87, p=0.001, n=7), sodium ion (r=0.9, p=0.00002, n=7) and chlorophyll a (r=0.77, p=0.04, n=7) concentrations. 197 int. j. aquat. biol. (2016) 4(3): 189-201 copepods averaged 30 ind. l-1 in both controls and planted microcosms; however, the biomass was 0.33 mg l-1 in the former and 0.23 mg l-1 in the latter. in both the controls and treatments, the vast majority of copepod numbers was represented by the juvenile stages of cyclopoidae, whereas much of the biomass was made up by the adult and copepodite stages of calanoidae (eudiaptomus gracilis (sars), e. graciloides (lillijeborg), acanthodiaptomus denticornis wierzejski). overall pattern of calanoidae copepodite dynamics was similar in the controls and planted microcosms, with lower numbers in the latter (friedman anova: p<0.01). copepod numbers reversely correlated with potassium ion concentration (r=-0.79, p=0.036, n=7) in the treatments. this relationship was not found in the controls. biomass of rotifers were low in the controls (approx. 0.0006 mg l-1) and planted microcosms (approx. 0.0011 mg l-1). the higher species richness was observed in the planted microcosms (22 species vs. 13 in the control). lecane luna (müller), polyarthra vulgaris carlin, keratella cochlearis (gosse) and k. quadrata (müller) were found in both the controls and planted microcosms; lecane (m.) bulla (gosse), l. (m.) arcuata (bryce), l. (m.) lunaris (ehrenberg), colurella obtuse (gosse) and testudinella patina (hermann) were only present in the planted microcosms. predatory zooplankters (adult cyclopoida and polyphemus pediculus (l.)) represented about 1% and 2% of the total zooplankton numbers in the controls and planted microcosms respectively. discussion algal community exhibited the most prominent response to inclusion of water soldier plants in the microcosms. the presence of macrophytes caused declining the planktonic chlorophyll levels, reduction in periphyton abundance and changes in the algal community. macrophytes may directly influence the algae by shading, altering water chemistry, competing for nutrients, and allelopathic action or indirectly, by influencing consumers. none of the above mechanisms was identified as the dominant one. water soldier is known to absorb and accumulate potassium ion (brammer and wetzel, 1984). during the vegetation season, potassium is used to build up the plant's tissues (renman, 1989); on the other figure 6. cladoceran biomass in controls and planted microcosms: (a) daphnia longispina, (b) chydorus sphaericus and (c) ceriodaphnia quadrangula (mean±sd). 198 kurbatova et al./ effect of aquatic plants on plankton and periphyton hand, potassium is released from the decaying tissues slower, than other cations, such as calcium or sodium (brammer and wetzel, 1984). as a result, s. aloides clumps depress the potassium levels compared to open water (mulderij et al., 2009). this effect was also observed in our experiments. brammer (1979) suggested that the planktonic chlorophyll reduction is brought about by alteration of the water ionic composition, as well as competition for nutrients. jaworski et al. (2003) demonstrated that low potassium concentrations failed to limit the growth of two species of diatoms and one species of photosynthetic flagellates, suggesting that potassium absorption by water soldier plants is unlikely to directly influence phytoplankton growth. however, potassium concentrations may correlate with the trophic status. potassium requirements vary among different groups of algae (gerloff and fishbeck, 1969). we found appreciable numbers of the diatoms in the periphyton in the planted microcosms. at the same time, significant concentrations of chl c typical of diatoms were found in plankton samples, suggesting importance of those algae in phytoplankton as well. in the presence of water soldier, cyanobacterial numbers in periphyton were significantly lower compared to the controls. toporowska et al. (2008) indicated seasonal changes in relative abundance of cyanobacteria in s. aloides periphyton: cyanobacteria were more abundant in spring, compared to summer and fall. diatoms dominated water soldier periphyton in all seasons, showing greater species richness (77 species total) compared to periphytom of other plants investigated. several studies (mulderij et al., 2006; strzałek and koperski, 2009) have shown that algal biomass is reduced in the presence of water soldier even the nutrients are not limited and the light is sufficient. it is recognized that, s. aloides produces strong allelopathic effects upon planktonic and periphytic algae (jasser, 1995; mulderij et al., 2005; hilt, 2006; mohamed and al-shehri, 2010); however, published reports on how different algae respond to the plant are contradictory. different strains of the same species of algae may differently respond to the chemicals produced by water soldier (mulderij et al., 2005; al-shehri, 2010). the majority of works studied only the influence of plant extracts; however such approach reflects only potential effects. less researchers studied effects of plant exudates. the latter approach is more correct since the metabolites of live plants particularly may really change the dynamics of some species and structure of aquatic communities. although there is little doubt about allelopathic influence of plant upon the algal community, the exact mechanisms and targets of such an influence remain to be identified. similarly, aquatic plants can repel or suppress zooplankton (pennak, 1973; burks et al., 2000). our experiment detected no such influence, as zooplankton numbers, biomass, and species richness were somewhat higher in the planted microcosms compared to the controls. similar results were obtained in s. aloides mats in lake bużysko (strzałek and koperski, 2009). growth of chydoridae, as well as c. quadrangula is responsible for the observed increase of cladoceran fraction among the zooplankton. euplanktonic d. longispina, a dominant species in the controls, remained numerous in the planted microcosms as well. those organisms are known to be present sporadically among s. aloides (irvine et al., 1990; strzałek and koperski, 2009). whereas irvine et al. (1990) concluded that daphnia abundance is reversely correlated with the density of s. aloides, other authors failed to find that relationship (strzałek and koperski, 2009). although daphnias prefer open water, they may develop among water soldier plants if the conditions (including food availability) are right. it is also unlikely that lower numbers of calanoids in the planted microcosms is due to allelopathic influence of plants. calanoids are well-known to avoid floating and submerged plant areas (dorgelo and koning, 1980) which may be probably related to their locomotion manner. we attribute the changes in the zooplankton community primarily to restructure of the 199 int. j. aquat. biol. (2016) 4(3): 189-201 environment (bittel, 1980) and changes in the trophic resources. macrophytes, suppressing phytoplankton, reorganize the classical food web and facilitate the transfer of organic material from bacteria and protozoa to the higher trophic level (to metazooplankton). the share of zooplankton in the sum plankton biomass was twice as higher in the microcosms with plants. in control, the ratios of biomasses of bacteria, phytoplankton and zooplankton were 1: 1.2: 3, respectively; in the planted microcosms, 1.5: 1: 6, respectively. this suggests the importance of dissolved organic matter recovery and reutilization at the higher trophic levels via microbial loop. the overall bacterial numbers remained comparable between the controls and planted microcosms. this was due to a balance between the organic nutrient supply enhancement on one hand and the grazing pressure increase on the other. bacterial growth can be stimulated by organic plant metabolites, nutrients excreted by zooplankton (ejsmont-karabin, 1984) and organic matter leaching from senescent plant parts (efremov et al., 2012) and phytoplankton. at the same time, formation of the bacterial aggregates and microcolonies and association of bacteria with detrital particles indicates that bacteria are likely to have experienced substantial grazing pressure from hnf. according to langenheder and jürgens (2001) up to 50% of the microbial biomass may be found in the form of such aggregates under those conditions. simultaneously, zooplankton can directly feed on larger bacterial aggregates. thus, we hypothesize that bacteria supports a fair portion of zooplankton growth, either directly, by zooplankton feeding on bacterial aggregates, or in a less direct way, via heterotrophic flagellates (carrik et al., 1991). the latter pathway seems to be more plausible, as protists are an important food source for zooplankton, especially when algal numbers are low (sanders and wickham, 1993). this link is well-established for daphnia (carrik et al., 1991, sanders et al., 1996); it is likely that hnf serves as an important food source to c. quadrangula as well, since šimek et al. (1997) noted a significant decline in hnf in the presence of c. quadrangula and diaphanosoma brachyurum (liévin). likewise, numbers of chydoridae increased among s. aloides and correlated with numbers of periphytic hnf, allowing us to suggest a trophic link between hnf and chydoridae. though water soldier clearly influenced the algal abundance and community composition, its effects upon the heterotrophic organisms of plankton and periphyton were limited. species forming the zooplankton or periphyton were eurybionts, and environmental changes not were beyond limits of their tolerance. however, influence of plants on abundance of individual species and groups of heterotrophic organisms is possible, since a significant correlations with some hydrochemical parameters were detected. the present experiment proves that the maintenance of zooplankton biomass in the macrophytes may occur due to the additional support from the bacteria and protozoa. perhaps, plant effects would be more significant at higher plant densities. acknowledgments authors thank d. pavlov (institute for biology of inland waters russian academy of sciences, ibiw ras) and d. sobolev (university of houstonvictoria, usa) for their assistance in preparation of the english translation of this manuscript. we are grateful to i. stepanova (ibiw ras) for the chemical analysis of nitrogen forms. we wish to thank s. bolotov (ibiw ras) for his consultation on data analysis. references alekin o.a., semenov a.d., skopintsev b.a. 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(2020) 8(4): 246-252 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article an investigation on the helminth parasites of caspian turtle (mauremys caspica) with a taxonomic note on recovered falcaustra lane, 1915 (nematoda: kathlaniidae) and spiroxys schneider, 1866 species (nematoda: gnathostomatidae) ehsan rakhshandehroo*1, amin gholamhosseini2, amin ahmadi1, mostafa rakhshaninejad2, amir ali heidari2 1department of pathobiology, school of veterinary medicine; shiraz university, shiraz, iran. 2department of aquatic animal health and diseases, school of veterinary medicine, shiraz university, shiraz, iran s article history: received 15 april 2020 accepted 21 august 2020 available online 2 5 august 2020 keywords: caspian turtle falcaustra spiroxys phylogeny abstract: in this study, the digestive tracts of the caspian turtles (mauremys caspica) were investigated for the presence of helminth infections. specimens of roundworms were recovered from the large intestine and stomach of the caspian turtles. the morphologic measures revealed the infection with nematodes of the genus falcaustra and spiroxys. however, some differences were found in the collected falcaustra specimens compared to the previous descriptions. in parallel, samples were analyzed by sequencing of the ribosomal gene targets. the phylogenetic analysis showed that the falcaustra species as members of superfamily cosmocercoidea were grouped with some members of ascaridoidea and spiruroidea. despite the significant morphologic differences, spiroxys species formed a sister group with ascaroid and cosmocercoid roundworms. therefore, our molecular findings revealed that the taxonomic position of both nematodes need be revised. introduction nematodes of the genus falcaustra lane, 1915 and spiroxys schneider, 1866 have been reported from different species of turtles around the world. bursey et al. (2000) listed a total of 68 falcaustra species in reptilian hosts from which 29 species infect turtles. in recent two decades, a number of new species of falcaustra were introduced from the digestive tract of turtles (bursey and kinsella, 2003; bursey and rivera, 2009; bursey and brooks, 2011) and other reptiles (bursey et al., 2007; bursey et al., 2009). spirurid nematodes of the genus spiroxys are also parasitic in the alimentary canal (mainly stomach) of this type of hosts (baker, 1987) with more than 17 recorded species (roca and garcía, 2008). most of the taxonomic knowledge on both abovementioned genera are based on morphological features. this has been led to describing many new taxa that are often difficult to identify (baker et al., 1986). in addition, the taxonomy of the genus falcaustra is somewhat confused because of several provided descriptions of some species and the fact that *correspondence: ehsan rakhshandehroo e-mail: rakhshandehroo@shirazu.ac.ir the information on some species is inadequate. although molecular-based methods have developed for the identification and taxonomy of helminths, data for molecular diagnosis of falcaustra and spiroxys is rare (li et al., 2014; rajabloo et al., 2017). therefore, the integration of genetic markers with morphology could be important for accurate identification. in the present study, the helminths recovered from the stomach and large intestine of the caspian turtle (mauremys caspica) (testudines: geoemydidae) were examined by conducting a morphological study and molecular analyses of their targeted regions in ribosomal rna gene. materials and methods the parasite specimens and diagnosis: a total of 5 infected turtles of m. caspica were collected in the sepidan (29°54'27"n 52°32'48"e) and beyza (29°58'19"n 52°24'4"e) regions, fars province, southern iran. turtles were subjected to standard euthanasia and post mortem examination using protocols described by mader and garner (2006) and 247 int. j. aquat. biol. (2020) 8(4): 246-252 underwood and reymond (2020). the alimentary canal including the esophagus, stomach, small and large intestines of turtles were examined separately for the presence of helminthes. some of the recovered specimens were preserved and fixed in 70% ethanol, cleared in lactophenol, mounted and examined for morphological examinations according to bursey et al. (2009) and some were fixed into 96% ethanol for molecular study. molecular procedures: genomic dna was extracted from the adult nematodes using a commercial kit (mbst, iran) according to the manufacturer’s protocol. our investigation showed infection of the examined turtle specimens with the species of falcaustra and spiroxys in the large intestine and stomach, respectively. the search for the recorded falcaustra in the genbank® revealed few available items mainly ribosomal and also mitochondrial (cytochrome oxidase) genes. on the basis of sequences for the partial 18s rdna, a pair of primers: f (5'-agaaacggctaccacatc-3') and r (5'ttacggtcagaactaggg-3') were designed and used for recovered falcaustra sp.. the pcr condition was 94°c for 4 min (initial denaturation), followed by 35 cycles of 94°c for 1 min (denaturation), 54.6°c for 1 min and 72°c for 1 min (extension) and a final elongation at 72°c for 2 min. for molecular diagnosis of the stomach worms i.e. spiroxys sp., the partial 18s rdna and internal transcribed spacer (its) regions were considered and amplified using primers: f (5'-agaggt gaaa tt cgtggacc-3') and r (5'-atatgcttaagttca gcgggt-3') (hasegawa et al., 2009). pcr condition was 94°c for 4 min (initial denaturation), followed by 35 cycles of 94°c for 1 min, 55.7°c for 1 min and 72°c for 1 min (extension) and a final elongation at 72°c for 25 min. the pcr reactions were adjusted in a final volume of 25 μl, including 12.5 μl of pcr premix (ampliqon, denmark, cat. no. a180301) (containing tris-hcl ph: 8.5, (nh4)2s04, 3 mm mgcl2, 0.2% tween 20, 0.4 mm of each dntp, 0.2 units/µl taq dna polymerase and inert red dye and stabilizer), 10 pmol of each primer, 6.5 μl h2o and 4 μl of dna extracted as template. the presence of the expected amplicons and their size were assessed by electrophoresis on each reaction product in 1.2% (w/v) tris-acetate/edta agarose gel and visualized under ultraviolet illumination. products from each assay were sequenced (abi 3730 dna analyzer; bioneer, korea). because the specimens were found for the first time in the region, no positive control was available to conduct in molecular assay. to determine the phylogenic positions of recovered parasites, the sequences were compared with previously reported other nematodes in the genbank. creating multiple-sequence alignment was performed using the clustalw program embedded in the mega 6.0 software. data was also used for construction of the phylogenetic trees using maximum likelihood method (tamura et al., 2007). results morphologic measurements: the large intestines of all turtles were highly infected with round worms (falcaustra sp.); however only three nematodes (one intact female and two broken) (spiroxys sp.) were recovered from a gastric section. according to the morphologic characters, nematodes from the large intestine were belonged to the family kathlaniidae lane, 1914, the genus falcaustra (bursey et al., 2009). those worms have a cylindrical body tapered gently towards the tail end. at the anterior margin, the mouth opening bounded by lips, each with conspicuous pairs of large fork-like papillae (fig. 1b). the mouth opens into the pharynx with relatively thick walls. the esophagus has subspherical isthmus and a spherical bulb at the end (figs. 1a, c). in males (n=18), length was 15.6±1.4 (13.2-17.2 mm) and the width (at level of esophageal-intestinal junction) was 420.7±33 (372.2-459.9 µm). the esophagus consisting of vestibule (pharynx) 74.3±6.2 (65-80 µm); corpus 1939.7±236 (1608.5-2256.3 µm); isthmus 154.1±12.8 (133.2-165 µm) and a valved bulb 222.9±27.4 (173.4-244.3 µm) in length. nerve ring was 369.4±6.5 (363.9-376.6 µm) from the anterior end. one pseudosucker was supported by 18-20 pairs of the muscle bands terminating on the border of the structure was seen in all males (fig. 1d). pairs of the 248 rakhshandehroo et al. / helminth parasites of caspian turtle obliquely arranged muscle bands were observed beginning near posterior lip of the pseudosucker and terminating anterior to the cloaca. five pairs of the caudal papillae, including one pair precloacal, one pair adcloacal and three pairs postcloacal (3-1+1) were observed. spicules were similar in shape, curved, alate (which extended almost to the blunt end of the spicule), distal end pointed, 1.68±0.16 (1.48-1.9 mm) in length. gubernaculum 178.9±17.9 (154.1-202.7 µm) in length, blunt distal tip. tail (from the anus to the posterior end) was conical, terminated with a relatively sharp end, 574.5±68.7 (488.9-678.2 µm) in length (fig. 1e). females’ (n=15) length was 15.1±1.3 (13.2-17.6 mm) with width (at level of esophageal-intestinal junction) of 374.1±38.1 (319.9-455.1 µm). the esophagus consisting of vestibule (pharynx) 86.4±8.9 (70-100 µm); corpus 1689.5±163.4 (1510.1-2074.7 µm); isthmus 159.5±22.9 (138.2-212.3 µm) and the valved bulb 216.8±32.2 (174.5-288.9 µm) in length. nerve ring was 416.8±67.8 (367.5-478.1 µm) from the anterior end. vagina directed anterodorsally giving rise to two divergent amphidelphic uteri. vulva, slightly prominent slit, 5.3±0.3 (5-5.4 mm) from the posterior end; eggs were oval to elliptical, thick shelled, unembryonated and 114.8±9.8 × 69.7±6 in size. tail was conical, 1098.9±106.2 (976.5-1164.9 µm) (figs. 2a, b). major morphologic measurements of falcaustra species reported having one pseudosucker structure are listed in table 1. according to described features, figure 1. falcaustra sp. (the iranian isolates); (a) the anterior end (dorsal view); (b) the head region, showing large forked papillae on the head (arrows); (ph) pharynx (vestibule) and proximal part of the esophagus (eso); (c) esophagus (distal part). isthmus (is) and bulbus (b) parts; (d) male, the pseudosucker structure showing radiating muscles (arrow); (e) caudal end of male. oblique muscles (om); caudal papillae (cp) spicules and gubernaculum (gub). scale bar~300 µm. figure 2. falcaustra sp., female. (a) distal part of reproductive system; (b) female, conical tail (arrow points to the anus); (c) spiroxys sp., anterior part include the esophagus, muscular (m) and glandular (g) parts; (d) trilobed pseudolabia forming a tooth on median part (arrow); (e) the female reproductive system; (f) caudal end of female, arrow points to the anus. scale bar~300 µm. 249 int. j. aquat. biol. (2020) 8(4): 246-252 our specimens were identified as f. ararath massino, 1924, described from a species of fresh water turtle, emys orbicularis, in armenia however, differences are seen in the papillae pattern and the average spicule size in males. in this study, only one intact female nematode of table 1. main (selected) characterizations of some species of falcaustra which are relatively closed to the specimens recovered in the present study. species host body length (mm) spicule length (mm) pseudo sucker papillae pattern country falcaustra ararath (current study) caspian turtle (mauremys caspica) 15.3 1.68 (1.48-1.9) 1 3-1 +1 iran f. ararath (massino, 1924) european pond turtle (emys orbicularis) 15.0 1.54 1 8–6–10 +1 armenia f. kaveri (karve and naik, 1951) cyprinid fish (barbus carnaticus) 13.7–15.5 2.00–2.23 1 6–0–16 +1 india f. armenica (massino, 1924) european pond turtle (emys orbicularis) 7.2–8.9 1.10–1.30 1 6–2–10 armenia f. dubia (yuen, 1963) fanged river frog (limnonectes macrodon) 13.5–14.0 1.54–1.69 1 6–2–12 +1 malaysia f. kempi (baylis & daubney, 1922) elongated tortoise (indotestudo elongate) 10.9–12.8 2.90 1 10–0–8 +1 india f. simpsoni (johnston and mawson, 1944) bell frog (litoria aurea) 13.0–13.5 2.00 1 8–0–12 +1 australia f. chelydrae (harwood, 1932) common snapping turtle (chelydra serpentina) 10.0–12.5 3.4–3.9 1 6–0–14 usa figure 3. phylogenetic position of the falcaustra (a) and spiroxys (b) isolates from caspian turtles among nematode species in different hosts inferred from partial ribosomal gene targets using the maximum-likelihood method. bootstrap values (2000 psuedoreplicates) represented at the nodes. the horizontal distance is proportional to evolutionary change as indicated (scale bar). 250 rakhshandehroo et al. / helminth parasites of caspian turtle the genus spiroxys was found in the stomach content and investigated. the morphology was indicative of the genus spiroxys schneider, 1866 (figs. 2c-f). its body length is 27.4 mm, the maximum width 676.5 µm, the oesophagus (2543.2 µm), thickened from the first third to the posterior and divided into anterior muscular, and a long posterior glandular portion (fig. 2c). the cephalic end surrounded by the trilobed pseudolabia projectinh anteriorly in each median lobe to form a tooth (fig. 2d). thin-shelled sub-elliptical eggs were seen with a mean length of 56.96±6.4 × 41.04±6.6 µm (fig. 2e). tail was short, stout and conical, 333.18 µm in size. according to these features, our specimen was identified as spiroxys ankarafantsika, described by roca and garcía (2008) from madagascan pleurodiran turtles (pelusios castanoides and pelomedusa subrufa). molecular analysis: the sequences of ribosomal gene targets were detected in samples and successfully amplified. for falcaustra samples, dna fragments of about 600bp was obtained for partial 18s rrna gene (recorded in the genbanks as mt160412). the comparison between our data and the three available sequences for falcaustra in the genbank revealed about 98.4 percent identity. our samples had also a high identity with two other nematodes, paraquimperia africana (the intestinal parasite of a eel species) (98.4%) and ichtyobronema hamulatum from burbot, lota lota (97.9%). the phylogenetic analysis confirmed the close relationship between our samples and f. araxiana and f. catesbeianae separated from turtle and frog hosts, respectively (fig. 3a). it was unexpected that the falcaustra species as members of superfamily cosmocercoidea were grouped with members of superfamilies ascaridoidea (paraquimperia) and spiruroidea (ichtyobronema); however, the other cosmocercoid nematodes were located in a separate clade. furthermore, our specimens formed a sister group with some members of seuratoidea (cucullanus, dichelyne and truttaedacnitis). the targeted ribosomal region was amplified in spiroxys genetic material leading to fragments of about 1140 bp. our specimens had nearly complete identity with s. japonica (ab818381 and ab818382) and s. hanzaki (ab818383) found in frog (lithobates catesbeianus) and salamander (andrias japonicus) hosts, respectively. in addition, a great identity was observed between our specimens, as members of gnathostomidea, with anguillicola crassus from superfamily dracunculoidea. according to the phylogenetic tree, both nematodes are grouped with each other (fig. 3b). based on the ribosomal region, spiroxys sp. formed a sister group with ascaroid and cosmocercoid nematodes, whereas there are significant differences between those taxa according to the morphologic characteristics. discussions different species of nematodes have been described occurring in the digestive tract of turtles (bursey et al., 2009; bursey and brooks, 2011). of the prevalent roundworms, the infection with the species of falcaustra and spiroxys were found in caspian turtle. previously, f. armenica from caspian turtles (youssefi et al., 2015) and f. araxiana from european pond turtles (emys orbicularis) (rajabloo et al., 2017) were reported in north and southwest regions of iran, respectively. unexpectedly, despite the similarity of the host, our samples had different morphology compared to those species. this indicates the diversity of the parasite in different localities. having similar turtle hosts, f. ararath, f. araxiana and f. armenica were reported from emys orbicularis in armenia (the northwest neighbor of iran), f. donanaensis from mauremys leprosa in spain (hidalgo-vila et al., 2006) and f. manouriacola from impressed tortoise (manouria impressa) (bursey et al., 2009). all the species had differences in main morphologic features, including the body size, spicule length and the number of pseudosuckers. in this study, the structure of the esophagus, head and presence of one pseudosucker agreed with those previously described f. ararath massino, 1924. in contrast, the caudal papillae pattern was not exactly unique to the previous description. in addition, regarding the average spicule length, our specimens had relatively higher measurements (1.68 comparing 251 int. j. aquat. biol. (2020) 8(4): 246-252 to 1.54 µm). nonetheless, these differences were not significant enough to assign our isolates into a new taxon. although numbers of species have explained in pervious literature for the genus falcaustra, many of reports have re-described them or introduced new species during the past decades. in turtles, in addition to 29 species listed by bursey et al. (2000), several new species have also assigned as f. manouriacola from m. impressa (bursey and rivera, 2009), f. kutcheri from geoemyda yuwonoi (bursey et al., 2000), f. greineri from orlitia borneensis (bursey and kinsella 2003) and f. donanaensis from m. leprosa (hidalgo-vila et al., 2006). regarding the controversial and/or overlapping morphological measurements in the past, the taxonomic investigation of those species is difficult. this was also the case for the spiroxys samples. although we did not recover male worms, the only recovered female had characteristics close to s. ankarafantsika separated from madagascar freshwater turtles (roca and garcía, 2008). many of the spiroxys species have found in fresh water turtles within the suborder cryptodira (berry, 1985). the caspian turtles belongs to cryptodirans, however, s. ankarafantsika separated from pleurodiran turtles (belong to suborder pleurodira) (roca and garcía, 2008). according to reports on the presence of spiroxys spp. in pleurodiran turtles in australian (berry, 1985), madagascan (roca et al., 2007) and ethiopia (berry, 1985), it was hypothesized that this nematode has been acquired from non-marine cryptodirans (roca et al., 2007). terefore, if we consider our samples as a type of s. ankarafantsika, it can be concluded that the transfer of spiroxys sp. between two suborders of fresh water turtles have previously occurred. despite the presence of accurate morphologic characterization, molecular data on falcaustra and spiroxys is rare. in the genbank, few records exist for f. araxiana and f. catesbeianae and for s. japonica and s. hanzaki. nevertheless, on the basis of partial ribosomal gene, the cosmocercoid nematodes formed a separate clade; however, the falcaustra species of turtles, as representatives of cosmocercoidea, were grouped with paraquimperia (of ascaridoidea) and ichtyobronema (from spiruroidea), both separated from kinds of fish. these groups possess significantly different morphology at least on their head region. in addition, a great identity was observed between the spiroxys, as member of gnathostomidea, with anguillicolaoïdes crassus from dracunculoidea and with ascaroid and cosmocercoid nematodes, both with a distinct structure. we found also the close relationship between f. araxiana and paraquimperia africana showing the ambiguity on the present classification and phylogenetic relationship (rajabloo et al., 2017). this controversial maybe because partial sequences have been considered and the number of available records is too limited to have an accurate molecular comparison. in this study, the sequence data for the spiroxys specimen was so close to s. japonica, but the morphologic characters were not agreed with this species well. on the other side, it was stated that the 18s rdna and its regions revealed significant nucleotide differences comparing the two prevalent species, s. japonica and s. hanzaki (li et al., 2014). hence, it seems that the selected regions could be used for the identification at the level of species instead of the genus or higher taxa. as a conclusion, these results criticize the morphologic data. thus, it is believed that more molecular studies should be done to introduce a firm taxonomic framework for the prevalent nematodes of turtles. in addition, the phylogeny of both nematodes could be reconstructed according to the genetic investigations. references baker m.r. (1986). falcaustra species (nematoda: kathlaniidae) parasitic in turtles and frogs in ontario. canadian journal of zoology, 64: 228-237. baker m.r. (1987). synopsis of the nematoda parasitic in amphibians and reptiles. memorial university of newfoundland occasional papers in biology, 11: 1325. berry g.n. (1985). a new species of the genus spiroxys (nematoda; spiruroidea) from australian chelonians of 252 rakhshandehroo et al. / helminth parasites of caspian turtle the genus chelodina (chelidae). systematic parasitology, 7: 59-68. bursey c.r., platt s.g., rainwater t.r. (2000). falcaustra kutcheri n. sp. (nematoda: kathlaniidae) from geoemyda yuwonoi (testudines: emydidae) from sulawesi, indonesia. journal of parasitology, 86: 344349. bursey c.r., kinsella j.m. (2003). falcaustra greineri n. sp. (nematoda: kathlaniidae) from orlitia borneensis (testudines: emydidae). journal of parasitology, 89: 961-964. bursey c.r., goldberg s., kraus f. (2007) a new species of falcaustra (nematoda, kathlaniidae) and other nematodes from sphenomorphus simus (squamata, scincidae) from papua new guinea. acta parasitologica, 52: 142-145. bursey c.r., goldberg s.r., kraus f. (2009). new species of falcaustra (nematoda: kathlaniidae) in nyctimystes cheesmani (anura: hylidae) from papua new guinea. journal of parasitology, 95: 146-151. bursey c.r., rivera s. (2009). new species of falcaustra (nematoda; ascaridida: kathlaniidae) in the impressed tortoise, manouria impressa (testudines: testudinidaae). comparative parasitology, 76: 141148. bursey c.r., brooks d.r. (2011). nematode parasites of five species of turtles from the area de conservación guanacaste, costa rica, with description of a new species of falcaustra. comparative parasitology, 78: 107-120. hasegawa h., hayashida s., ikeda y., sato h. (2009). hyper-variable regions in 18s rdna of strongyloides spp. as markers for species specific diagnosis. parasitology research, 104: 869-874. hidalgo-vila j., ribas a., florencio m., pérez-santigosa n., casanova j.c. (2006). falcaustra donanaensis sp. nov. (nematoda: kathlaniidae) a parasite of mauremys leprosa (testudines, bataguridae) in spain. parasitology research, 99: 410-413. li l., hasegawa h., roca v., xu z., guo y.n., sato a., zhang l.p. (2014). morphology, ultrastructure and molecular characterisation of spiroxys japonica morishita, 1926 (spirurida: gnathostomatidae) from pelophylax nigromaculatus (hallowell) (amphibia: ranidae). parasitology research, 113: 893-901. mader d.r., garner m.m. (2006). euthanasia and overview of biopsy and necropsy techniques. in: d.r. mader (ed.). reptile medicine and surgery. elsevier, st. louis, missouri. pp: 564-578. rajabloo m., sharifiyazdi h., namazi f., shayegh h., rakhshandehroo e., farjanikish g. (2017). morphological and molecular analyses of the spiruroid nematode, falcaustra araxiana massino, 1924 (= spironoura araxiana) from the european pond turtle (emys orbicularis). journal of helminthology, 91: 356359. roca v., garcía g., montesinos a. (2007). gastrointestinal helminths found in the three freshwater turtles (erymnochelys madagascariensis, pelomedusa subrufa and pelusios castanoides) from ankarafantsika national park, madagascar. helminthologia, 44: 1-6. roca v., garcía g. (2008). a new species of the genus spiroxys (nematoda: gnathostomatidae) from madagascan pleurodiran turtles (pelomedusidae). journal of helminthology, 82: 301-303. tamura k., dudley j., nei m., kumar s. (2007). mega4: molecular evolutionary genetics analysis (mega) software version 4.0. molecular biology and evolution, 24: 1596-1599. underwood w., raymond a. (2020). avma guidelines for the euthanasia of animals. version 2020.0.1. pp: 92-94. youssefi m.r., mousapour a., nikzad r., gonzalez-solis d., halajian a., rahimi m.t. (2016). gastrointestinal helminths of the caspian turtle, mauremys caspica (testudines), from northern iran. journal of parasitic diseases, 40: 65-68. int. j. aquat. biol. (2022) 9(2): 145-xx issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology technical note minimally invasive blood collection techniques as a source of gdna for genetic studies on turtles and tortoises mohd hairul mohd salleh1,2, yuzine esa*1,3 1department of aquaculture, faculty of agriculture, universiti putra malaysia, 43400 serdang, selangor, malaysia. 2royal malaysian customs department, persiaran perdana, presint 2, 62596 putrajaya, malaysia. 3international institute of aquaculture and aquatic sciences, universiti putra malaysia, lot 960 jalan kemang 6, 71050 port dickson, negeri sembilan, malaysia. article history: received 15 february 2022 accepted 22 april 2022 available online 2 5 april 2022 keywords: venipuncture procedure, southern river terrapin, subcarapacial venous plexus, jugular vein, genomic. abstract: this technical note aims to describe the venipuncture procedure used to collect blood from southern river terrapins via the subcarapacial venous plexus (svp) and jugular vein. on uncooperative terrapins, svp was applied while the jugular vein was reversed. 1.5 ml blood was preserved in 0.5 ml edta and stored at -20°c. reliapreptm blood genomic dna miniprep was used to extract dna. thermo scientifictm nanodrop 2000c was used to determine the concentrations of extracted dnas. the greatest concentration of dna is 136.3 g/l, and the highest purity is 1.90. the treatment is safe, minimally invasive, and effective. introduction oral scrapings and cloacal swabs have been used to recover dna from living reptiles (turtles, terrapins, and tortoises) (wendland et al., 2009; garcía–feria, 2015). although whole blood is the ideal supply, numerous blood extraction procedures have been developed (gottdenker and jacobson, 1995). however, chelonian members vary considerably in their vascular anatomy, degree of cooperation, and the possibility to cause injuries (mans, 2008). blood collection techniques formerly employed on freshwater turtles included highly invasive and fatal operations such as heart puncture and decapitation (mcdonald, 1976). non-lethal and less invasive blood collection methods include venipuncture of prominent veins (e.g., jugular, brachial, femoral, and iliac) (mader, 2005), the subcarapacial venous plexus (hernández–divers et al., 2002), the occipital sinuses (hernández–divers et al., 2002), and cardiac puncture (fowler, 1995). the jugular vein is the blood collection site for chelonians and should be used whenever possible. in most mammals, the jugular vein is precise and has a lower chance of lymph infection than other locations. this sample site is inappropriate for uncooperative or aggressive species without prior chemical restriction *correspondence: yuzine esa e-mail: yuzine@upm.edu.my to enable adequate head extension (kuchling, 2012). the (external) jugular vein is a superficial vein that runs from the dorsal part of the tympanum to the coelomic cavity on both sides of the neckline in a caudodorsal path. some animals have a dorsal and ventral external jugular vein (barrows et al., 2004; mcarthur et al., 2008). the venous contact of the cranial arteries from the azygous veins and the cervical anastomosis of the left and right jugular veins forms the subcarapacial venous plexus (svp). this blood sampling site is frequently used in uncooperative species where jugular venipuncture without sedation is impossible, in neonates and other small chelonians, and in individuals too ill to withstand the burden of restriction or sedation (barrows et al., 2004). the svp anatomy is found near the midline of the cranial side of the carapace, where the cervical vertebrae enter the shell. several vessels, including the normal intercostal vessels and the caudal cervical branch of the external jugular veins, form a venous sinus at this site. as a result, there is a higher risk of lymph contamination (barrows et al., 2004; mans, 2008). this blood collection study in batagur affinis aimed to explain and demonstrate the efficacy of two minimally invasive venipuncture approaches until the 146 mohd salleh and esa / blood collection techniques in turtles and tortoises quality of genomic dna (gdna) was assessed. as a result, we are the first to provide a complete methods and protocols note on batagur sp. blood collection. until the gdna quality was confirmed, it was the most comprehensive technical discovery from blood draws. the capacity to manage a sample volume without considerable sample contamination solved one of the primary concerns in the field after the invasive approach issue (avataneo et al., 2019). several research studies have described the use of gdna sequences to examine genetic variation in b. affinis (çilingir et al., 2019). materials and methods this study comprised 120 b. affinis individuals from four populations across the east and west malaysia peninsula: pasir gajah, kemaman (ke), terengganu (4.2524°n, 103.2957°e); bukit pinang (bp), kedah (4.2221°n, 100.4370°e); bota kanan (bk), perak (4.3489°n, 100.8802°e); and bukit paloh, kuala berang (kb), terengganu (5.0939°n, 102.7821°e). a total of 30 individuals of b. affinis were sampled at each location. the arena permit approval number is b00335-16-20, which was rewarded by the department of wildlife and national parks, peninsular malaysia. the venipuncture blood collection technique and procedures consist of the following steps (table 1). blood storing method: the vial should be shaken vigorously before being stored away from direct sunlight and preferably in cool place to ensure proper mixing. although drawn-out preservation is best accomplished by keeping samples at +4 to –8°c, samples in the buffer can be kept up at encompassing temperature for quite a long time if necessary (dutton, 1996) (fig. 1). dna extraction protocol: nucleic acids were extracted from 200 µl of each edta whole blood sample. after cell lysis and protein denaturation, extractions were performed with automated systems using reliaprep™ blood gdna miniprep system (promega, madison, usa) with binding column technology. a final extraction volume of 200 µl was obtained from the 200 µl input volume of the edta whole blood sample. the stages were as follows: the blood sample was shaken for at least 10 min at room temperature in a rotisserie shaker. 20 µl of proteinase k (pk) solution, 200 µl of blood, and 200 µl of cell lysis buffer (cld) were added to a 1.5 ml microcentrifuge tube, vortexed for at least 10 seconds. the solution was incubated at 56°c for 10 minutes. 250 µl of binding buffer (bba) was added and mixed by vortexing for 10 seconds. the contents were added to the binding column and located in a microcentrifuge at maximum speed for one minute. the flow-through in the collection tube was removed and discarded. 500 µl of column wash solution (cwd) was added to the new binding column and centrifuged at maximum speed for 3 minutes. this washing procedure was repeated for a total of three washes. the column was placed in table 1. the venepuncture technique and procedures. step svp jugular vein 1 the target area is swabbed with an alcohol swab to minimise the risk of infection the alcohol swab we use contains 70% isopropyl alcohol and 30% water. the head is slowly pulled out, and the neck is swabbed with an alcohol swab to minimise the risk of infection the alcohol swab we use contains 70% isopropyl alcohol and 30% water. 2 the venipuncture person either extends the head and bends the neck ventrally or pushes the head back into the shell. the head is placed in the appropriate position by slowly pulling it out from its retracted position underneath the carapace until the sampling site is easy to see. 3 the needle is directed dorsocaudally and inserted on the midline at the point where the skin attaches to the ventral aspect of the cranial carapace. the needle is introduced in a craniocaudal direction, maintaining mild negative pressure. 4 the needle is bent upwards at a right angle of about 60 degrees (fig. 2a) and advanced in a caudodorsal direction, creating intense negative pressure. the needle is directed dorsocaudally and inserted on the midline at the point where the skin attaches to the ventral aspect of the cranial carapace. 5 if no blood is aspirated, the needle is slightly withdrawn and redirected until blood appears in the syringe hub. immediately after piercing the skin, the syringe is lightly aspirated. then the needle is slowly inserted further into the neck until blood is seen to enter the syringe hub (fig. 2b). 6 more blood is obtained by gentle suction throughout the operation than by strong suction, probably because the latter causes the vein to collapse. more blood is obtained by gentle suction throughout the operation than by strong suction, probably because the latter causes the vein to collapse. 7 swab the area of the needle puncture wound again with the alcohol swab. swab the area of the needle puncture wound again with the alcohol swab. 147 int. j. aquat. biol. (2022) 10(2): 145-150 a clean 1.5 ml microcentrifuge tube. 200 µl of nuclease-free water was added to the column and centrifuged at maximum speed for one minute. the binding column was discarded. this study obtained a 200 µl volume of extracted dna from the 200 µl input volume of whole blood. dna quality assessment: the concentration of the isolated dnas was determined using the thermo scientifictm nanodrop 2000c spectrophotometer model nd-2000 (thermo fisher scientific, waltham, usa). the automated systems ability to extract nucleic acids of high purity was proven by direct gel loading. following nanodrop quantification of the isolated nucleic acids, the results were put directly into the 1% agarose gel with molecular markers. loading the extracted dnas from the same sample was used to test the repeatability of each system. results and discussions our approach is comparable to that outlined for sea turtles (owens and ruiz, 1980). blood sampling methods such as ours have been defined as drawing blood from the cervical sinus, dorsal cervical sinus, occipital sinus, and subcarapacial vein. but true blood sinuses do not exist in jawed vertebrates (randall et al., 1997), and these techniques draw blood from the external jugular vein or one of its branches. because of the varying proximity of the external jugular vein to the dorsal surface and other morphological aspects, some variant in blood collection practice is required. therefore, we choose two blood collection techniques (collection from the jugular vein and svp) and recommend them because they are more successful than other procedures (mans, 2008). at the same time, we used two different-sized needles. first, we successfully collected more blood via the lateral jugular vein using a 23g x 1¼ inch needle. then, we also tried a 3 ml syringe with a 23g x 1¼ inch needle and a 21g x 1¼ inch needle. mans (2008) recommends using the smallest needle size appropriate for the animal's size and sample volume. utmost chelonian species require a 22to 27-gauge hypodermic needle of sufficient length. the amount of blood to be obtained will determine the size of the syringe used. to avoid excessive negative pressure, which may cause the sampled vein to collapse, it should be used the smallest needle possible. this minimally invasive blood sample procedure, successfully applied to batagur species, has proven effective. even for people who have only received a single training session and have little or no previous expertise with animals, the entire technique takes no more than two minutes to complete. the tension experienced by the animal is maintained to a bare figure 1. (a) svp blood sampling angle is approximately 60 degrees, and (b) blood collection from the right jugular vein of a malaysian southern river terrapin (batagur affinis). 148 mohd salleh and esa / blood collection techniques in turtles and tortoises minimum, and there is virtually little risk of hurting the river terrapin as a result of this procedure. furthermore, the current strategy has reduced the risk of sample contamination, which has been identified as one of the most pressing challenges in the field (avataneo et al., 2019). after sampling from the external jugular vein, there is generally a little superficial damage or scarring of terrapins actual bleeding of the needle puncture site, but this stops 5-10s after the region is swabbed with 70% alcohol. as a result, no physical harm or scaring of terrapins has been recorded. some extracellular fluid contamination is acceptable when assessing isotope dilution and osmolality of bodily fluids. when evaluating the haematological qualities of blood, however, it is unacceptable. if blood contamination is intolerable, another sample can be collected from the opposite side of the head. as an alternative, we suggested a tissue collection procedure (about 2-4 mm of clipped scute of the shell), which is also a successful way of obtaining gdna for genetic studies on turtles and tortoises (praschag et al., 2009; ismail et al., 2016). the most commonly used solution for terrapin blood preservation consists of 100mm tris-hci, ph 8; 100mm edta, ph 8; 10mm nacl and 1-2% sodium dodecyl sulphate (sds). bowen et al. (1996), encalada et al. (1996), and seutin et al. (1991) recommend a 1:10 dilution of blood to buffer, and white and densmore, (1992) suggest a 1:5 ratios. however, higher blood concentrations up to a 1:1 ratio may be employed, mainly if a higher concentration (2%) of sds is employed to achieve sufficient cell lysis (dutton, 1996). we collected 1.5 ml of blood and added 0.5 ml of edta for preservation in a 2 ml microcentrifuge tube at a ratio of 1:3 and stored at 20°c. the dna extraction protocol was efficient in extracting gdna from whole blood based on an analysis of total gdna in the agarose gel (fig. 2). spectrophotometer measurements showed dna concentration and purity differences depending on the population (table 2). higher concentrations and purity were obtained from samples number 6 and 3, and vice versa for samples 4 and 2. the concentration and 260/280 ratio of the extracted dna samples corresponded to the recommended amount of 1 µg of suitable quality dna table 2. results of gdna extraction from whole blood samples. sample number dna concentration (ηg/µl) purity (od 260/280) 1 120.8 1.82 2 39.3 1.80 3 42.7 1.90 4 35.2 1.83 5 71.4 1.88 6 136.3 1.87 figure 2. genomic dna was loaded into a 1% agarose gel and separated by electrophoresis for 40 minutes at 75 volts, lane m: 1 kb ladder size standard marker, lanes 1-3: batagur affinis affinis gdna, lanes 4-6: batagur affinis edwardmolli gdna. 149 int. j. aquat. biol. (2022) 10(2): 145-150 (concentration ≥ 20 ng/µl according to the protocol 'illumina library construction services sample requirements' (brajković et al., 2018; uc davis genome center, 2018). furthermore, the values of purity ratio optical density (od) 260/280 followed the values proposed by biase et al. (2002), ≥ 1.8. conclusions this protocol proved that the blood collection method is advantageous due to its simplicity, rapidity and affordable reagents, apart from the high molecular weight dna and purity achieved in all samples. acknowledgments i want to acknowledge the universiti putra malaysia and the royal malaysian customs department for funding my studies through a graduate research fellowship and my study leave. in addition, i'd like to express my gratitude to the genetic and breeding laboratory, family, and friends for their assistance and support in this research project. furthermore, the collaboration between the turtle conservation society of malaysia and the department of wildlife and national parks, peninsular malaysia, was awarded a research permit (b-00335-16-20). last but not least, we thank the anonymous reviewers and editor for their insights and suggestions to improve this paper. references avataneo v., d’avolio a., cusato j., cantù m., de nicolò a. 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(2018) 6(5): 254-257 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology short communication effects of dimethoate and bacilar fertilizer on biochemical and immunological parameters in common carp, cyprinus carpio fatemh mohseni1, mahdi banaee*1, parvaneh shokat1, mohammad mohiseni2 1department of aquaculture, faculty of natural resources and the environment, behbahan khatam alanbia university of technology, behbahan, iran. 2department of fisheries science and engineering, faculty of agriculture and natural resources, lorestan university, khorramabad, iran. article history: received 19 may 2018 accepted 23 october 2018 available online 2 5 october 2018 keywords: agrochemicals carp immunotoxicity abstract: fish exposure to agrochemicals can suppress their immune system and survival. therefore, this study investigates adverse effects of sublethal concentrations of dimethoate alone or combined with bacilar (an organophosphorus pesticide and bio-fertilizer) on the innate immune parameters of common carp, cyprinus carpio, within 14 days. no significant changes were found in levels of total protein, immunoglobulin, the activity of lysozyme and complement c3 in fish exposed to bacilar alone; however, globulin and complement c4 level indicated a significant reduction. fish exposure to dimethoate alone or combined with bacilar resulted in a decrease in the activity of ach50, lysozyme, complement c3, c4 and levels of total protein, globulin, and immunoglobulin in compared with the control group. in conclusion, the results of this study showed that innate immune parameters decreased in fish exposed to dimethoate and/or bacilar. as consequences: dimethoate or/and bacilar have the immunosuppressive effect on fish. introduction the presence of different pollutants, such as pesticides and chemical or organic fertilizers in aquatic ecosystems near agricultural farms can be a serious threat to aquatic organisms (fazilat et al., 2017). dimethoate, with the chemical formula of o, odimethyl s-methyl carbamoyl methyl phosphorodithioate and the organophosphorus pesticide – bacilar – which is the biological product of bacillus subtilis, along with some micronutrients are used in agriculture. therefore, the introduction of these compounds into surface waters can bring about alterations in biochemical parameters of blood (fazilat et al., 2017), cause oxidative stress (dogan et al., 2011) and behavioral disorders in fish. changes in the activity of innate immune parameters (ahmadi et al., 2014), alterations in the gene expression of cytokines il-6, il-8, il-10 and tnf-α (chen et al., 2014), and an increase in expression of gene atrophies lc3-ii, dynein (chen et al., 2015) and hsp60, hsp70, and hsp90 genes (xing et al., 2015) in the head kidney and spleen, alterations *correspondence: mahdi banaee doi: https://doi.org/10.22034/ijab.v6i5.404 e-mail: mahdibanaee@yahoo.com in expression of immunoglobulin m (igm), complement c3, and lysozyme (lyz) (ma and li, 2015) are a number of damages incurred to the immune system of fish exposed to different pesticides and agrochemical compounds. these changes can increase fish sensitivity to biological and environmental infections. thus, this study evaluates changes in the immune parameters of common carp treated with varied concentrations of either dimethoate or dimethoate and bacilar. materials and methods this study is conducted according to the ethics of animal experimentation in iran. common carp, cyprinus carpio, were divided into 9 groups and tested for 14 days under different conditions: fish under standard environmental conditions, or the control group (group i); fish treated with 16 and 32 g l-1 of dimethoate (respectively groups ii and iii); fish treated with 0.1 and 0.2 ml l-1 of bacilar bio-fertilizer (respectively groups iv and v); fish treated with 16 g l-1 of dimethoate + 0.1 ml l-1 of bacilar (group 255 int. j. aquat. biol. (2018) 6(5): 254-257 vi); fish exposed to 32 g l-1 of dimethoate + 0.1 ml l-1 of bacilar (group vii); and fish exposed to 32 g l-1 of dimethoate + 0.2 ml l-1 of bacilar (group ix). blood samples were collected from all groups. the following methods measured immune parameters of plasma: total immunoglobulin by amar and colleagues’ method (amar et al., 2000), alternative complement pathway activity (ach50) by yano’s method (yano, 1992) and based on hemolysis of sheep red blood cells (shrbc) (baharafshan research company), lysozyme activity in blood plasma via micrococcus luteus (actinobacteria: micrococcaceae) (lange et al., 2001), the activity of complements c3 and c4 via immunoturbidimetry (abdollahi et aal., 2016), and total protein, albumin and globulin (johnson et al., 1999) by pars azmun kits. after checking the normality of data via shapiro-wilk, data analysis was done by one-way anova, using spss 22. the means were compared by duncan test at a 99% confidence level (𝛼 = 0.01%). results no mortality was observed in fish during the experiment; however, an increase in mucus secretions, changes in color and behavior, such as unbalanced swimming, swimming on the surface, and neurological reactions are among the significant behavioral changes in fish exposed to dimethoate alone or combined with bacilar on the final days of the experiment. biochemical parameters of common carp exposed to dimethoate alone or combined with bacilar for 14 days are presented in table 1. the results of this study indicated that bacilar alone had no significant effects on levels of total protein, immunoglobulin, lysozyme activity, and complement c3 in fish. however, levels of these parameters in fish treated with either dimethoate or dimethoate and bacilar were significantly reduced. fish exposure to bacilar and dimethoate decreased globulin and complement c4 levels. there was a significant increase in albumin levels in fish treated with bacilar and dimethoate compared to that of the control. no significant changes were found in ach50 activity in fish treated with 0.2 ml l-1 of bacilar; however, ach50 activity was significantly reduced in other experimental groups. discussion the immunotoxicity of agrochemicals on fish has been widely investigated (marchand et al., 2017; chen et al., 2015; ma and li, 2015). in the present study, immunological parameters in plasma of fish exposed table 1. biochemical parameters of cyprinus carpio exposed to dimethoate alone or combined with bacilar for a period of 14 days. treatments total protein (g/dl) albumin (g/dl) globulins (g/dl) immunoglobulins (mg/ml) lysozyme (u/ml) ach50 (u/ml) complement c3 (mg/dl) complement c4 (mg/dl) control 4.8±0.2c 2.1±0.6a 2.7±0.5b 15.6±3.3c 23.3±2.4b 92.0±8.5e 15.5±2.9c 16.3±0.4c 16 µg/l dimethoate 4.0±0.1ab 3.0±0.5bc 0.9±0.5a 2.7±1.1a 13.5±3.0a 81.2±3.6cd 10.9±1.8a 10.2±0.6a 32 µg/l dimethoate 3.9±0.4ab 2.7±0.3b 1.13±0.4a 8.0±4.0b 14.0±3.6a 70.6±2.0b 8.4±1.2a 8.9±1.9a 0.1 ml/l bacilar 4.6±0.7c 3.0±0.3bc 1.6±0.8a 15.8±5.6c 22.3±4.5b 83.0±3.7cd 15.7±4.7c 14.1±0.9b 0.2 ml/l bacilar 4.4±0.3bc 3.5±0.4c 0.9±0.5a 17.8±4.7c 19.9±0.5b 90.6±5.9e 14.1±2.8bc 13.6±0.6b 16 µg/l dimethoate & 0.1 ml/l bacilar 4.1±0.7ab 3.1±0.7bc 1.0±0.8a 6.3±3.6ab 12.9±2.2a 84.8±5.0d 10.8±1.9a 13.8±0.4b 16 µg/l dimethoate & 0.2 ml/l bacilar 4.0±0.3ab 3.1±0.6bc 0.9±0.6a 7.3±3.7ab 15.7±3.2a 78.0±1.8c 11.4±1.1ab 14.0±1.5b 32 µg/l dimethoate & 0.1 ml/l bacilar 4.1±0.4ab 3.2±0.4bc 0.9±0.4a 6.5±3.7ab 12.2±0.9a 61.4±2.0a 10.4±1.4a 10.5±2.0a 32 µg/l dimethoate & 0.2 ml/l bacilar 3.7±0.2a 2.8±0.3b 0.9±0.4a 4.0±1.9ab 12.2±0.8a 62.6±2.1a 9.9±1.8a 10.4±2.3a data are presented as mean±s.d. significant differences between values when compared with control group were characterized by alphabetical symbol (p<0.01). 256 mohseni et al./ effects of dimethoate and bacilar fertilizer on common carp to dimethoate and/or bacilar fertilizer are evaluated. a significant decrease in levels of total protein may indicate liver necrosis or disturbance in the physiological performance of fish treated with dimethoate or dimethoate and bacilar. dimethoate can reduce levels of total protein in plasma, especially globulins, by reducing appetite, causing a disturbance in the absorption of amino acids in intestines, increased activity of proteolytic enzymes, the increased rate of proteins break down in the liver and preventing protein synthesis in the liver (narra, 2017). liver failure, malnutrition as well as a biochemical reaction between agrochemicals and the amino acid sequences of proteins found in blood may account for lower plasma total protein. similar results are reported in fish exposed to different pesticides (ahmadi et al., 2014). an increase in albumin can be attributed to its role in distributing the pesticide in the blood (tarhoni et al., 2008). total immunoglobulin is a main element of blood adaptive immunity in bony fish and a well-known biomarker in evaluating the immunotoxicity of fish exposed to environmental pollutants (li et al., 2013). in this study, a decrease in total immunoglobulin levels is due to disturbance in the adaptive immune system in fish exposed to dimethoate alone or both dimethoate and bacilar. a reduction of total immunoglobulin levels in the blood may be due to an insufficient synthesis of immunoglobulins, or changes in the gene expression involved in the biosynthesis of ig’s subunits (ghazy et al., 2017). similar effects were observed in other species exposed to different agrochemical compounds (narra, 2017). lysozyme, a vital element of the innate immune system of bony fish, is very sensitive to pollutioninduced stress (ahmadi et al., 2014). lysozyme is expressed to a great extent in hematopoietic cells, granulocytes, monocytes, and macrophages (merlini and bellotti, 2005). therefore, a reduction in the activity of lysozyme in fish exposed to dimethoate alone or dimethoate and bacilar indicates the influence of this pollutant on lysozyme biosynthesis and thus disturbance in the innate immune system of common carp. furthermore, the significant decrease in lysozyme activity in plasma of fish exposed to dimethoate and bacilar may indicate debility of defense mechanisms against bacterial agents. the activity of lysozyme is reduced in fish treated with different pesticides (li et al., 2013). the complement system, as the first element of the innate immune system, plays a vital role in the immune system and affects the adaptive immune system by stimulating the reproduction of b cells (li et al., 2013; pushpa et al., 2014). both c3 and c4 are glycoproteins that are produced by liver cells, macrophages, and monocytes. new molecular and cellular findings indicate that complement proteins are synthesized in different parts (løvoll et al., 2007). therefore, the reduced activity of ach50, c3, and c4 in fish exposed to dimethoate or dimethoate and bacilar may disturb the biosynthesis of these elements in hepatocytes, macrophages, and monocytes. a reduction in total complement and its elements in fish exposed to different pollutants demonstrate a deficiency in the innate immune system (ahmadi et al., 2014). in general, our results indicate that exposure to agrochemicals, such as dimethoate and bacilar can suppress the innate immune system in common carp. a decrease in ach50, total protein, globulin, immunoglobulin, lysozyme, and complement c3 and c4 activity may increase fish sensitivity to infectious pathogens. acknowledgments this study was supported by grant from behbahan khatam alanbia university of technology. also, the authors are grateful to maryam banaie for proofreading the manuscript. references abdollahi r., heidari b., aghamaali m. 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(2018) 6(5): 254-257 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی cyprinusهای بیوشیمیایی و ایمنی در ماهی کپور معمولی )اثرات دایمیتوات و کود باسیالر بر فراسنجه carpio) 2، محمد محیسنی1، پروانه شوکت1*، مهدی بنایی1فاطمه محسنی نبیا ص( ببببا،، ایرا، گروه شیالت، دانشکده منابع طبیعی و محیط زیست، دانشگاه صنعتی خاتم اال1 ، خرم آباد، ایرا، روه علوم و مبندسی شیالت، دانشکده کشاورزی و منابع طبیعی، دانشگاه لرستا،گ2 چکیده: تواند موجب سرکوب سیستم ایمنی و کاهش بقای آنبا گردد بنابراین در قرار گرفتن ماهیا، در معرض مواد شیمیایی مورد استفاده در کشاورزی می یستی کش فسفره آلی و یک کود زهای زیرکشنده دایمیتوات به تنبایی و توام با کود باسیالر صیک آفتامطلوب غلظتاین مطالعه به بررسی اثرات ن داری در سطح پروتئین تام، ایمنوگلبولین و فعالیت روز، پرداخته شده است تغییر معنی 14های ایمنی ذاتی ماهی کپور معمولی در طی بر فراسنجه داری کاهش طور معنیبه c4با این حال، سطح گلبولین و کمپلما، در ماهیا، در معرض کود باسیالر به تنبایی یافت نشد c3لیزوزیم و کمپلما، ، لیزوزیم، کمپلما، ach50نشا، داد قرار گرفتن ماهیا، در معرض دایمیتوات به تنبایی و یا توام با کود باسیالر موجب کاهش فعالیت کمپلما، تام c3 ،c4 پروتئین تام، گلبولین و ایمنوگلبولین تام در مقایسه با ماهیا، گروه کنترل گردید نتایج این مطالعه نشا، داد که پارامترهای ایمنی و سطح هیا، اذاتی در ماهیا، در معرض دایمیتوات و یا باسیالر کاهش یافت در نتیجه، دایمیتوات و باسیالر دارای اثر مبار کنندگی بر سیستم ایمنی م هستند مواد شیمیایی کشاورزی، ماهی کپور، مسمومیت ایمنی :کلمات کلیدی int. j. aquat. biol. (2018) 6(5): 248-253 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article assessment of the primary production statues of the international gavkhooni wetland, iran mohammad hadi abolhasani*1, niloofar pirestani2, saeed ghasemi2 1environmental science, waste and wastewater research center, department of environmental science, islamic azad university, isfahan (khorasgan) branch, isfahan, iran. 2department of environmental science, islamic azad university, isfahan (khorasgan) branch, isfahan, iran. s article history: received 27 march 2018 accepted 4 july 2018 available online 2 5 october 2018 keywords: chlorophyll-a assessment trophic state physicochemical factors abstract: wetlands are of the unique and productive ecosystems in the world. the present study was conducted to determine primary production statues of the international gavkhooni wetland, based on chlorophyll-a. sampling was performed seasonally from march 2017 to february 2018. four sampling stations were assigned based on latitude and longitude. water salinity, temperature, ph, nitrate, phosphate, bod5, tds, ec, tss and dissolved oxygen were determined in triplicate in each station. trophy level was determined using index tsi. the results showed that there was no significant difference in mean chlorophyll-a content between the seasons. the highest and lowest chlorophyll-a contents were observed in spring and winter, respectively; and the significantly highest content was measured in the station a (shakh kenar). there was no significant difference in the water physicochemical parameters between the seasons (p>0.05). the mean water nitrate level of the sampling stations was 4.255 mg/l; the highest (5.07 mg/l) and lowest (3.35 mg/l) levels were recorded in the summer and autumn, respectively. the mean water phosphate level of the sampling stations was 1.082 mg/l; the highest (1.75 mg/l) and lowest (0.57 mg/l) levels were recorded to the winter and summer, respectively. the mean dissolved oxygen level during the study was 5.64 mg/l. according to the results, nitrate is the limiting factor for production in the gavkhooni wetland. based on index tsi, the wetland is oligotrophic in the spring, autumn and winter, but mesotrophic in the summer. introduction wetlands are of the unique and productive ecosystems in the world; as the annual ecological value of wetlands have been estimated 10 and 200 folds higher than those of the forests and agricultural fields, respectively (kamandari, 2014). wetland are marshes, pools and natural and artificial ponds, which have static or running water permanently or temporary, both saltwater and freshwater, and marine waters that have a depth of lower that 6 m in the lowest tide (majnoonian, 2015). gavkhooni wetland is located in 140 km southeast of isfahan and 30 km of the varzaneh city (najjari, 2003). the area climate is categorized as hyper-arid zone with annual average rainfall of 50-100 mm and average temperature of 15°c and average evaporation of more than 2800 mm (najjari, 2003). *correspondence: mohammad hadi abolhasani doi: https://doi.org/10.22034/ijab.v6i5.446 e-mail: hadi.mha2001@yahoo.com phytoplanktons include many cyanobacteria and single-cell algae that have chlorophyll-a (polladi et al., 2013). diatoms and dinoflagellates are well-known marine phytoplanktons; whereas, diatoms, dinoflagellates, desmidials and blue-green algae are dominated in freshwaters. despite that brown, green and blue-green single-cell flagellates are the main producers in a water body, it is hard to identify them, particularly when are presented in low number (zaki et al., 2004). phytoplanktons’ density is seasondepended. silica phytoplanktons (diatoms) are dominated often in the spring and autumn; however, green algae are dominated in the late spring and early summer. phytoplanktons can be used as water quality indicators, especially estimation of nutrient load in water or eutrophication. in addition, they are biological indicators of water quality change due to 249 int. j. aquat. biol. (2018) 6(5): 248-253 regional or global impacts such as invasion or toxic pollutants, temperature and increase in ultraviolet radiation (rolland et al., 2009). phytoplanktons are important for ecosystems in the case of organic materials production and placing at the base of the energy pyramid (davis, 1955; newell and newell, 1977). primary production is the biological base of an aquatic ecosystem; thus, determination of regional and global distribution of primary production is important (martin, 2004; leymarie et al., 2010). chlorophyll-a is the major photosynthetic pigment in plants and its value can be used for determination of the plants biomass and an ecosystem trophic state (brown et al., 1998; kalytyte, 2007). hence, this study was conducted to determine primary production statues of the international gavkhooni wetland based on chlorophyll-a content. materials and methods the gavkhooni wetland is a shallow and saline lake located in dry and arid climate of the central plateau of iran. the wetland area and depth at the time of birds’ migration is 63000 ha and 5 m, respectively (abadi et al., 2008) and is considered as the only permanent saline lake in the central plateau of iran. water basin of the wetland is located at 33 and 11 to 42 and 33 northern latitudes and 50 and 2 to 53 and 24 eastern longitudes (montazeri and karimpoor, 2011). sampling was performed seasonally from march 2017 to february 2018. four sampling stations were assigned based on the distance (12 km): station a (shakh kenar; 48 and 52 e to 20 and 32 n), station b (49 and 52 e to 19 and 32 n), station c (50 and 52 e to 19 and 32 n) and station d (the wetland estuary; 54 and 52 e to 16 and 32 n). triplicate samples were taken from each station, from 10 am to 2 pm. for measurement of some water physicochemical parameters, 1 l water was taken from each station and transferred to laboratory, avoiding light and heat. water salinity, temperature and ph were measured at the stations using a digital water checker apparatus (hach, wtw 2000). water nitrate and phosphate were measures according to standard methods using spectrophotometer (clesceri et al., 1989). bod5, tds, ec and tss were measures based on standard methods (standard method, 2012). the index tsi was used to determine the wetland trophic state (kerbs, 1989) as following formula: tsi(chla) = 9.81 ln(chla) + 30.6, where chla is chlorophyll-a concentration(mg/m3). this index categorize a water body into 3 classes (oligotrophic, mesotrophic and eutrophic) using digit scale (0-100). tsi near zero is ultra-oligotrophic water and the value near 100 is hyper-eutrophic water. the most common method to determine primary production in aquatic ecosystems is measurement of chlorophyll-a based on the following formula (kerbs, 1989): 7.3 c k r p where p = phytoplanktons’ photosynthesis (g carbon/m2/day), r = relative amount of light radiate to the water, k = reflection coefficient (m) and c = chlorophyll-a concentration (mg/m3). the value 3.7 is based on gram on fixed carbon per gram of chlorophyll during 1 hour photosynthesis. the coefficient k is 0.3 for shallow wetlands (kerbs, 1989). statistical analysis: comparison of the primary production among the seasons and stations was conducted using completely randomized block (sampling stations). data were analyzed by two way anova followed by duncan test at significance levels of 0.05. principal components analysis (pca) was used to analyze the main factors of the physicochemical factors. multivariate stepwise regression was used to find relationship between chlorophyll-a content and environmental factors. all analyses were performed in software sas. results table 1 shows water physicochemical parameters, trophic index and carlson index (tsi) in different seasons in the gavkhooni wetland. there was no significant difference in the water physicochemical parameters between the seasons (p>0.05). the mean water nitrate level of the sampling stations was 4.255 mg/l; the highest (5.07 mg/l) and lowest (3.35 mg/l) levels were recorded in the summer and autumn, 250 abolhasani et al./ primary production statues of gavkhooni wetland respectively. the mean water phosphate level of the sampling stations was 1.082 mg/l; the highest (1.75 mg/l) and lowest (0.57 mg/l) levels were measured in the winter and summer, respectively. the mean dissolved oxygen level during the study was 5.64 mg/l with the highest value in the spring (6.95 mg/l) and the lowest level in the winter (4.87 mg/l). the mean of water salinity was 12.18 ppt and the maximum and minimum levels were observed in the autumn (19.78 ppt) and winter (8.5 ppt), respectively. the highest and lowest temperatures were recorded in the summer (21.75) and winter (9), respectively; the mean value was 15.11. based on index tsi, the wetland is oligotrophic in the spring, autumn and winter, but mesotrophic in the summer. pca analysis was used to determine the significant water physicochemical parameters in chlorophyll-a content. three factors presented in table 2 encompass 60.43 percent of total variance; thus were considered as the main factors. these factors were used in stepwise regression to find a reliable equation among the main factors. dissolved oxygen, nitrate, ec and tds had higher cumulative variance compared to the other factors (table 3). the stepwise test revealed that among these parameters, nitrate was the only significant factor in chlorophyll-a content: chlorophyll-a = 26.24 – 3.45 no3 (r 2=0.77) based on the results, water nitrate is the limiting table 1. water physicochemical parameters, trophic index and carlson index (tsi) in different seasons in the gavkhooni wetland (2017-2018). parameter winter fall summer spring dissolved oxygen (mg/l) 0.81±4.87 1.1±5.32 1.14±5.45 0.67±6.95 bod(mg/l) 0.3±2.62 0.61±2.32 0.8±1.92 0.73±2.82 primary production (g/m2day c) 1.34±11.75 1.43±13.25 1.7±12.5 1.25±14.5 ph 1.2±7.87 1.2±7.79 1.23±7.92 1.54±7.22 temperature (°c) 0.89±9 1.78±12.46 2.45±21.75 2.45±17.25 salinity (ppt) 0.76±8.5 2.1±19.78 1.1±10.07 1.6±10.37 nitrate (mg/l) 0.65±3.6 0.56±3.35 1.1±5.07 1.13±5 phosphate (mg/l) 0.23±1.75 0.1±0.64 0.1±0.57 0.46±1.37 ec (ms/cm) 4.3±62 4.76±51.18 4.32±49 3.98±48 tds (mg/l) 2.45±24.5 2.2±16.42 2.3±30.25 2.45±21.57 depth (m) 0.23±1.72 0.43±1.72 0.63±2.27 0.53±2.2 trophy index (tsi) 38 (oligotrophic) 39.2 (oligotrophic) 43.2 (mesotrophic) 35.3 (oligotrophic) table 2. results of pca analysis in decrease in water physicochemical characteristics in the gavkhooni wetland (2017-2018). cumulative variance percentage of variance total component 21.3 21.76 2.97 1 43.31 20.1 1.76 2 60.43 15.97 1.32 3 table 3. output of cumulative percentage of water physicochemical characteristics in the gavkhooni wetland. factor cumulative percentage dissolved oxygen 0.7 bod 0.65 ph 0.69 temperature 0.41 salinity 0.52 no3 0.72 po4 0.43 ec 0.96 tds 0.93 depth 0.62 tss 0.49 figure 1. mean natural log of chlorophyll-a (mg/m3) in different seasons in the gavkhooni wetland (2017-2018) 251 int. j. aquat. biol. (2018) 6(5): 248-253 factors for production in the gavkhooni wetland. there was no significant difference in chlorophyll-a concentrations between the stations (p>0.05) (fig. 1). the highest and lowest chlorophyll-a contents were observed in spring and winter, respectively. the significantly highest content was related to the station a, but the lowest one in the station c. discussion wetlands are of the most important ecosystems in the world, which have unique biodiversity and high biomass, and controlling role in hydraulic systems, temperature balance, flood and storm, and role in biocontrol diseases, relation, transportation, tourism, creation, science and biosphere stock (bennett, 2002). lack of a significant difference in water physiochemical parameters between the seasons could be due to the similarity of the environmental conditions. nitrate is the limiting factors for plankton growth in the oceans; whereas, phosphate is the limiting factors in freshwaters (camacho, 2003). according to the results, nitrate was the only limiting factor in the gavkhooni wetland, which is in accordance with findings of abolhasani et al. (2013) in the shadegan wetland. varela and penas (1985) also found a strong relationship between nutrient content and phytoplankton growth. khalife nilsaz (2009) found no relationship between nutrient content and chlorophyll-a content in the wetland shadegan. the highest chlorophyll-a and dissolved oxygen concentrations were observed in the spring; the lowest found in winter. studying the vertical distribution of dissolved oxygen in the strait of hormoz, bahrami and ebrahimi (2008) found that the highest oxygen content was at the depth 10-15 m, where the highest chlorophyll-a content was observed. the results of the present study showed that station a had higher chlorophyll-a content than the others due to the presence of floating plants. the plants absorb water nitrate and phosphate (li et al., 2009) and these parameters were low in the growth season in the station a. on the other hand, phosphate absorption by plant biomass and formation of insoluble and precipitated phosphorous decrease water phosphate content (southwood and henderson, 2000). the lowest chlorophyll-a content was observed in the station c, which may be because trees shading the water surface. primary production is not often variable during a year, especially in the regions with narrow climate fluctuations. in the wetlands with narrow climate change, there is often a minimum production level in the middle of winter to the early spring and a maximum level in the late spring or the autumn (naz and turkman, 2005). the highest chlorophyll-a content in the wetland gavkhooni was observed in the spring, which might be due to the deposition of nutrient in the winter, increasing water temperature and photoperiod. it is suggested that water nutrient levels increases in the winter because phytoplanktons’ populations decrease markedly. this nutrient load promotes significantly the planktonic bloom in the spring (king et al., 2002). study in the southern part of the caspian sea showed that the highest phytoplankton density and peak is observed in the spring (ganjian and makglogh, 2004). in addition, the lowest production was observed in the winter that might be due to decline in phytoplankton population. in the baikal lake, it was found that temperature and thermocline are effective factors on regional distribution patterns and seasonal succession (fietz and welch, 2005). in the summer, rotifer of the genus brachionus were dominated because of water salinity elevation and other zooplanktons are low in number (kotani et figure 2. mean content of chlorophyll-a (mg/m3) in different stations in the gavkhooni wetland (2017-2018). 252 abolhasani et al./ primary production statues of gavkhooni wetland al., 2005). as a result, high production was observed in the summer and autumn. according to the wetlands trophic classification based on chlorophyll-a (camacho, 2003), the gavkhooni wetland, with chlorophyll content of 13.18 mg/m3, is categorized as mesotrophic. as the wetland receives agricultural wastewaters, this may increase the water nutrients; this might be positive now, but in the future, this may lead to ecological revolution in the wetland (galbraith, 2005) and it is potentiated to reach eutrophic state. references abadi m., rostani n., bagherzadeh karimi m. 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(2018) 6(5): 248-253 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی ایران گاوخونی المللی بین تاالب اولیه تولید ارزیابی وضعیت 2قاسمی سعید ،2پیرستانی نیلوفر ،1*ابوالحسنی محمدهادی .، ایرانخوراسگان(، اصفهان) دانشگاه آزاد اسالمی اصفهان، کشاورزی و منابع طبیعی مهندسی دانشکدهگروه محیط زیست، ،پژوهشکده پسماند و پساب 1 .اصفهان، ایرانخوراسگان(، )گاه آزاد اسالمی واحد اصفهاندانشکده کشاورزی و منابع طبیعی، دانش گروه مهندسی محیط زیست،2 چکیده: ای تولید اولیه بر مبن وضعیت تعیین. این تحقیق با هدف باشندهای جهان میاز پر تولیدترین محیط و فردهب منحصر هایاکوسیستم جمله از تاالب ها ایستگاه با 4در مجموع انجام گرفت. 96الی اسفند 96طور فصلی ازفروردین هگیری بنمونه تاالب بین المللی گاوخونی انجام گرفت. در aکلروفیل 5bod ،tds ،ce ،tss، نیترات، فسفات، phر هر ایستگاه پارامترهای شوری، دمای آب، در نظر گرفته شد. د طول و عرض جغرافیاییتوجه به میانگین در داریمعنی استفاده شد. نتایج نشان داد تفاوت tsiاز شاخص برای تعیین سطح تروفی گیری شد.و اکسیژن محلول با سه تکرار اندازه ن در فصل زمستان آدر فصل بهار وکمترین a که بیشترین میزان کلروفیل دادتغییرات فصلی کلروفیل نشان . نداشت ودوج هافصل بین a کلروفیل آب شیمیاییداری در پارامترهای فیزیکوتفاوت معنی. ها بودشاخ کنار( بیشتر از دیگر ایستگاه) aدر ایستگاه a میانگین کلروفیلین چنباشد، هممی یز پای فصل تابستان و در و به ترتیب بود گرم در لیترمیلی 255/4 مختلف هایایستگاه در نیترات میانگین .(p<05/0) در فصول مختلف مشاهده نشد رد فسفات مقادیر کمترین و بیشترین. مشاهده گردید آن مقادیر (در لیترمیلی گرم 25/3و گرم در لیتر میلی 07/5 به میزان) کمترین و بیشترین بدستمیلی گرم در لیتر 082/1 آن میانگین و (میلی گرم در لیتر 57/0و میلی گرم در لیتر 75/1 به میزان) شد تابستان مشاهده فصل زمستان و هایی است کهنتایج نشان داد که تاالب گاوخونی از دسته تاالب بود.گرم در لیتر میلی 64/5 برداری نمونه دوره کل در محلول اکسیژن میانگین .آمد تاالب در فصل بهار، پاییز و زمستان در ، tsiوسیله شاخص رود. در بررسی سطح تروفی تاالب بهشمار میهدر آن، نیترات عامل محدودکننده تولید ب ضعیت مزوتروف بود. ر ووضعیت الیگوتروف و در فصل تابستان د .شیمیاییفیزیکو فاکتورهای اولیه، تولیدوضعیت ،ارزیابی آ، کلروفیل :کلمات کلیدی © 2020 iranian society of ichthyology original article first record of thuridilla indopacifica gosliner, 1995 (sacoglossa: plakobranchidae) from the gulf of oman, iran yaser fatemi, mohammad reza taherizadeh*,1adnan shahdadi 1department of marine biology, faculty of marine sciences and technology, university of hormozgan, bandar abbas, iran. 422 references 423 : int. j. aquat. biol. (2016) 4(3): 179-188; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article rich rotifer assemblage (rotifera: eurotatoria) of a sub-tropical wetland of meghalaya, northeast india: ecosystem diversity and interesting features bhushan kumar sharma*,1kyapaomai role sounii pou, sumita sharma freshwater biology laboratory, department of zoology, north-eastern hill university shillong 793 022, meghalaya, india. article history: received 10 november 2015 accepted 29 may 2016 available online 2 5 june 2016 keywords: composition interesting taxa richness similarities abstract: a total of 90 species, belonging to 29 genera and 15 families, observed from an urban wetland of meghalaya is the species-rich rotifera assemblage known till date from any sub-tropical ecosystem of the indian sub-region. total richness (s) merits biodiversity value as ~56.0, ~38.0 and ~23.0% of the species recorded from meghalaya, northeast india (nei) and india, respectively while several rotifers of global and regional importance impart biogeographic interest. one species is new to nei and 19 species (including two unidentified) are new to meghalaya; this study resulted in an earlier report of five and two new records from india and nei, respectively. the diverse lecanidae > lepadellidae together comprising ~57.0% of s; largely littoral periphytonic nature of taxa; and distinct paucity of the brachionidae and lack of brachionus are notable features. the speciose lecane, high richness of cosmopolitans and occurrence of several tropicopolitan and pantropical species impart broadly ‘tropical’ character to the fauna. with low monthly richness and low community similarities, our results affirm heterogeneity of rotifer species composition; the richness is positively influenced only by water temperature. introduction rotifera, an integral link of aquatic food-webs and valuable contributors to freshwater productivity, have been documented from india since the work of anderson (1889) but the related literature yet indicated lacunae on biodiversity and ecosystem diversity of the taxon from different states of india (sharma, 1996, 1998; sharma and sharma, 2008) in general and in sub-tropical waters of nei (sharma and sharma, 2014a) in particular. referring to the latter region, sharma and sharma (1999) examined the rotifer fauna from the hill state of meghalaya with certain additions by sharma (2008, 2010) and sharma and sharma (2011) while sharma (2006) only dealt with their diversity of the paddy-field ecosystems. the present study is an attempt to analyse ecosystem diversity of the taxon of a subtropical wetland vis-a-vis update biodiversity of meghalaya rotifera. a list of 90 species documented * corresponding author: bhushan kumar sharma e-mail address: profbksharma@gmail.com from our collections is presented and interesting taxa are illustrated for validation. the diversity of rotifera assemblage is discussed with reference to the richness, composition, community similarities, interesting species and distribution of various taxa, and the influence of abiotic factors. materials and methods this study is a part of limnological reconnaissance of a sub-tropical wetland located at the campus of north-eastern hill university, shillong (25°36' 32.8''-25°36'36.3''n and 91°53'46.9''-91°54'01.5''e; alt. 1400 m asl), meghalaya state of nei undertaken during august 2014-july 2015. water samples were collected monthly for various basic abiotic parameters. water temperature, specific conductivity and ph were recorded by the field probes; dissolved oxygen was estimated by winkler’s method while free carbon dioxide, 180 sharma et al./ rich rotifer assemblage of a sub-tropical wetland of meghalaya alkalinity, hardness, calcium and chloride were analyzed following apha (1992). the qualitative plankton and semi-plankton samples were collected monthly by towing a nylobolt plankton net (#50 µm) and preserved in 5% formalin. all collections were screened with a wild stereoscopic binocular microscope; the rotifers were isolated and mounted in polyvinyl alcohol-lactophenol, and were observed with leica (dm 1000) stereoscopic phase contrast microscope fitted with an image analyzer. interesting taxa were illustrated and the measurements were given in micrometers (µm). various taxa were identified following koste (1978), koste and shiel (1989), segers (1995), sharma (1998) and sharma and sharma (1999, 2000, 2008, 2011, 2013, 2014b, 2015a). the percentage similarities between monthly rotifer assemblages were calculated vide sørensen’s index (sørensen, 1948) and spss (version 20) was used for the hierarchical cluster analysis. results the variations (ranges, mean ± sd) of certain abiotic parameters of the wetland are indicated in table 1. our collections revealed 90 species of phylum rotifera spread over 29 genera and 15 families (appendix 1). notommata copeus (fig. 1a) is a new record to nei. notommata spinata (fig. 1b) is an australasian species; lecane blachei (fig. 1c) is an oriental endemic; keratella javana (fig. 1d), lepadella bicornis (fig. 1e), l. discoidea (fig. 1f), l. vandenbrandei (fig. 1g), lecane simonneae (fig. 1h), l. unguitata and trichocerca siamensis (fig. 1i) are paleotropical species. mytilina michelangellii (fig. 1j), testudinella amphora (fig. 1k), trichocerca edmondsoni (fig. 1l), lecane aspasia (fig. 1m) and l. elegans (fig. 1n) are other interesting species. keratella tecta, lecane aspasia, l. elegans, l. obtusa, l. simonneae, l. stenroosi, l. undulata, lepadella bicornis, l. vandenbrandei, macrochaetus subquadratus, notommata spinata, mytilina michelangellii, testudinella amphora, trichocerca bidens, t. edmondsoni, t. scipio and t. siamensis, and two unidentified species are new records to meghalaya. colurella tesselata, lecane stichaea, parameters ↓ range mean±sd water temperature (oc) 12.0-22.5 17.4±3.2 ph 6.02-6.99 6.59±0.19 specific conductivity (µs cm -1) 31.0-51.0 37.4±5.4 dissolved oxygen (mg l-1) 5.6-7.6 6.7±0.5 free carbon dioxide (mg l-1) 6.0-22.0 11.3±4.3 total alkalinity (mg l-1) 18.0-30.0 24.0±3.5 total hardness (mg l-1) 20.0-32.0 26.3±3.4 calcium (mg l-1) 8.4-23.1 14.4±4.6 chloride (mg l-1) 25.0-38.0 32.5±4.1 table 1. variations in basic abiotic parameters. aug sep oct nov dec jan feb mar apr may jun jul aug 43.4 26.0 27.1 31.5 28.5 46.1 27.4 32.1 24.5 44.0 56.7 sep 61.7 34.4 28.5 29.0 50.9 44.0 47.2 28.5 48.2 57.5 oct 44.8 32.1 32.7 27.4 28.0 36.3 28.5 48.2 42.4 nov 47.8 26.6 39.0 40.0 44.4 60.8 41.6 28.5 dec 32.5 25.4 26.3 32.5 36.3 34.7 33.3 jan 36.8 27.0 19.0 27.9 40.0 33.9 feb 42.4 36.8 41.0 34.1 36.7 mar 48.6 31.5 30.0 33.3 apr 23.2 26.6 33.9 may 39.1 22.2 jun 28.5 jul table 2. percentage similarities (sørensen’s index) of rotifera. 181 int. j. aquat. biol. (2016) 4(3): 179-188 figure 1. (a) notommata copeus ehrenberg (dorsal view), (b) notommata spinata koste & shiel (lateral view), (c) lecane blachei bērziņš (ventral view), (d) keratella javana hauer (ventral view), (e) lepadella bicornis vasisht & battish (ventral view), (f) lepadella discoidea segers (ventral view), (g) lepadella vandenbrandei gillard (ventral view), (h) lecane simonneae segers (dorsal view), (i) trichocerca siamensis segers & pholpunthin (lateral view), (j) mytilina michelangellii reid & turner (lateral view), (k) testudinella amphora hauer (dorsal view), (l) trichocerca edmondsoni (myers) (lateral view), (m) lecane aspasia myers (dorsal view) and (n) lecane elegans harring (ventral view). 182 sharma et al./ rich rotifer assemblage of a sub-tropical wetland of meghalaya gastropus minor, stephanoceros fimbriatus and dissotrocha aculeata are recent new additions (sharma, 2016) to the indian rotifera while lecane dorysimilis and cupelopagis vorax are new additions to the rotifer fauna of nei (sharma, loc cit.). the rotifer richness ranged between 16-35 (25±6) species (fig. 2) with lecanidae > lepadellidae as main components, the community similarities (table 2) ranged between 19.0-61.7% (sørensen, 1948) and the hierarchical cluster analysis is shown in figure 3. discussion water temperature affirmed sub-tropical nature of the wetland concurrent with its geographical figure 2. monthly variations in species richness of rotifera and important families. figure 3. hierarchical cluster analysis of rotifera assemblage. 183 int. j. aquat. biol. (2016) 4(3): 179-188 location. the ‘slightly acidic circum neutral’, ‘soft’ and ‘calcium poor’ waters are characterized by low ionic concentrations; the latter warranted inclusion of this wetland under ‘class i’ category of trophic classification vide talling and talling (1965). further, it showed well-oxygenated water and low free carbon dioxide while chloride indicated certain influence of human impact in this seepage and rainwater fed biotope. the abiotic parameters broadly concurred with the reports from sub-tropical waters of meghalaya (sharma, 1995, 2001) and mizoram (sharma and pachuau, 2013) hill states of nei. a total of 90 species (s), belonging to 29 genera and 15 families, documented from this small urban wetland is the richest rotifera assemblage known from any sub-tropical ecosystem of the indian subregion and one of the richest from any subtropical wetland of south asia. this interesting feature is hypothesized to habitat diversity and environmental heterogeneity of the sampled wetland. our report merits biodiversity interest as ~56.0, ~38.0 and ~23.0 % of the species known from meghalaya, nei and india, respectively. notommata copeus is a new record to nei; it is known elsewhere from india (bks, unpublished) from andhra pradesh, jammu and kashsmir, kerala, punjab and tamil nadu while its unverifiable reports from karnataka and maharashtra lacked any validation. keratella tecta, lecane aspasia, l. elegans, l. obtusa, l. simonneae, l. stenroosi, l. undulata, lepadella bicornis, l. vandenbrandei, macrochaetus subquadratus, n. spinata, mytilina michelangellii, testudinella amphora, trichocerca bidens, t. edmondsoni, t. scipio and t. siamensis, and two unidentified species are new records to meghalaya fauna. besides, our collections resulted in recent additions (sharma, 2016) of colurella tesselata, lecane stichaea, gastropus minor, stephanoceros fimbriatus and dissotrocha aculeata to the indian rotifera while lecane dorysimilis and cupelopagis vorax are additions to the rotifer fauna of nei. this study thus raised overall rotifer richness of meghalaya to 161 species spread over 40 genera and 20 families; the former represented ~40.0 % of the diversity of indian rotifera and corresponded with 162 species reported recently from mizoram (sharma and sharma, 2015b) another hill state of nei. biogeographically interesting elements formed a notable fraction (~14.0% of s) and included the australasian notommata spinata; the oriental endemic lecane blachei; the indo-chinese lecane dorysimilis; the palaeotropical keratella javana, lepadella bicornis, l. discoidea, l. vandenbrandei, lecane simonneae, l. unguitata and trichocerca siamensis. besides, mytilina michelangellii, testudinella amphora and trichocerca edmondsoni are other interesting species. of these, lecane simonneae, l. unguitata, lepadella discoidea and l. vandenbrandei are categorized as eastern hemisphere elements following segers (2001) and sharma and sharma (2005, 2015b). the member of the first category affirmed affinity of the rotifer fauna of meghalaya with southeast asia and australia and thus affirmed earlier remarks on nei rotifera (sharma, 2005; sharma and sharma, 2005, 2008, 2013, 2014a, 2014c, 2015b). further, n. spinata is known from india only from nei with reports from assam, manipur and presently from meghalaya. the oriental l. blachei deserved attention for its distribution in nei (assam, meghalaya and manipur) and from west bengal in eastern india. the indo-chinese lecane dorysimilis is yet restricted to nei (assam and meghalaya) and delhi with likely wider distribution in the former region (sharma, 2016). of the palaeotropical species, keratella javana, lepadella vandenbrandei and trichocerca siamensis are known till date from india from nei; lepadella bicornis from nei (assam and manipur) and chandigarh (north india) while its unverifiable report as l. bicornis müller (non vasisht and battish) from kashmir (ticku and zutshi, 1993) is invalid (sharma and sharma, 2015a). lepadella discoidea (nei, delhi and kerala) and lecane simonneae (nei, kerala and tamil nadu) exhibited disjunct distribution in india while l. unguitata indicated relatively wider occurrence in this country. 184 sharma et al./ rich rotifer assemblage of a sub-tropical wetland of meghalaya amongst other interesting elements, lecane aspasia is known from assam (nei) and delhi; lecane elegans, mytilina michelangellii and testudinella amphora are restricted to nei while trichocerca edmondsoni is so far known kashmir himalayas and mizoram (nei). in addition, our collections included various species of regional distribution value namely lecane bifurca, l. doryssa, l. elegans, l. pusilla, l. stichaea, l. tenuiseta, lepadella costatoides, l. dactyliseta, l. quinquecostata and t. tridentata with lecane elegans, l. stichaea and t. tridentata known from india only from nei. the most diverse lecanidae (30 species) > lepadellidae (21 species) together comprised ~57.0% of s; trichocercidae ≈ notommatidae = testudinellidae collectively formed (~21.0% of s) while rest of the fourteen eurotatorien families recorded low richness. the species-rich genera lecane > lepadella formed main component (~54.0 % of s); trichocerca > testudinella together (13.3% of s) deserved certain attention and the rest of 25 genera recorded poor richness. the speciose nature of the `tropic-centered’ lecane affirmed the role of this thermophile in our collections concurrent with the rotifer fauna nei (sharma and sharma, 2014a, 2014b). the said feature along with occurrence of a large component of cosmopolitans (~73.0% of s) and of various tropicopolitan and pantropical species (~16.0%) impart a general ‘tropical character’ to the rotifer fauna. the notable feature of the littoral-periphytic composition of rotifer fauna, with lack of planktonic taxa, is hypothesized to the lack of definite pelagic habitats (de manuel, 1994) in spite of certain semilimnetic conditions in a part of the sampled wetland. our collections are interestingly characterized by poor brachionidae richness (4 species) in general and absence of brachionus spp. in particular; these interesting aspects deserved further analysis for causative factors in light of the record of 22 and 12 species of the two taxa, respectively from aquatic biotopes of meghalaya (sharma and sharma, 2014c). the low monthly richness (16-35, 25±6 species) affirmed heterogeneity of rotifer species composition; only three species occurred throughout the study period while other three indicated 50-83% frequency of occurrence and the rest indicated low frequencies of occurrence. this generalization is endorsed by low community similarities (19.061.7%) with similarity values below 50% in ~92 % cases in the similarity matrix. the heterogeneity in species composition is affirmed by hierarchical cluster which reflected affinity between november vs. may rotifer communities while january > march collections reflected greater divergence. our results indicated lowest richness during winter-spring and high richness of rotifers as well as important constituent families during monsoon in particular. of the various abiotic factors, the richness of rotifera (r=0.874, p=0.0009), lecanidae (r=0.651, p= 0.415) and lepadellidae (r=0.794, p=0.0061) is positively influenced by water temperature. conclusions the rich and diverse rotifera assemblage affirmed environmental heterogeneity of the ‘slightly acidic circum neutral’, ‘soft’ and ‘calcium poor’ waters of the sampled wetland characterized by low ionic concentrations and thus merits biodiversity and ecological interests. various new records and the report of species of global and regional interest, and eastern hemisphere elements impart biogeographic value to this study. the specific sampling of periphytic, sessile and benthic taxa are desired for further update and we estimate occurrence of 125+ from this wetland. the interesting features of rotifera assemblage provide scope for studies on the biodiversity and ecosystem diversity of the taxon in sub-tropical waters of nei in general and wetlands of the region in particular. acknowledgments thanks are due to the head, department of zoology, north-eastern hill university, shillong for laboratory facilities. the authors have no conflict of interests. 185 int. j. aquat. biol. 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(2016) 4(3): 179-188 order: ploima family: brachionidae 1. keratella cochlearis (gosse, 1851) 2. k. javana hauer, 1937 3. k. tecta (gosse, 1851)* 4. plationus patulus (o.f. muller, 1786) family: euchlanidae 5. dipleuchlanis propatula (gosse, 1886) 6. euchlanis dilatata ehrenberg, 1832 family: mytilinidae 7. mytilina michelangellii reid & turner, 1988* 8. m. ventralis (ehrenberg, 1830) family: trichotriidae 9. macrochaetus sericus (thorpe, 1893) 10. m. subquadratus perty, 1850* 11. trichotria tetractis (ehrenberg, 1830) family: lepadellidae 12. colurella sulcata (stenroos, 1898) 13. c. tesselata (glascott, 1893)** 14. c. uncinata (o.f. muller, 1773) 15. colurella sp. 16. lepadella acuminata (ehrenberg, 1834) 17. lepadella bicornis vasisht & battish, 1971* 18. l. biloba hauer, 1958 19. l. costatoides segers, 1992 20. l. cristata (rousselet, 1893) 21. l. dactyliseta (stenroos, 1898) 22. l. discoidea segers, 1993 23. l. ovalis (o.f. muller, 1786) 24. l. patella (o.f. muller, 1773) 25. l. quinquecostata (lucks, 1912) 26. l. rhomboides (gosse, 1886) 27. l. triptera ehrenberg, 1832 28. l. vandenbrandei gillard, 1952* 29. l. (h.) apsicora myers, 1934 30. l. (h.) ehrenbergi (perty, 1850) 31. l. (h.) heterostyla (murray, 1913) 32. squatinella lamellaris (o.f. müller, 1786) family: lecanidae 33. lecane aculeata (jakubski, 1912) 34. l. arcula harring, 1914 35. l. aspasia myers, 1917 * 36. l. bifurca (bryce, 1892) 37. l. blachei bērziņš, 1973 38. l. bulla (gosse, 1851) 39. l. closterocerca (schmarda, 1859) 40. l. decipiens (murray, 1913) 41. l. doryssa harring, 1914 42. l. dorysimilis trinh dang, segers & sanoamuang, 2015** 43. l. elegans harring, 1914* 44. l. flexilis (gosse, 1886) 45. l. furcata (murray, 1913) 46. l. hamata (stokes, 1896) 47. l. hornemanni (ehrenberg, 1834) 48. l. inermis (bryce, 1892) 49. l. ludwigii (eckstein, 1883) 50. l. lunaris (ehrenberg, 1832) 51. l. obtusa (murray, 1913)* 52. l. papuana (murray, 1913) 53. l. pusilla harring, 1914 54. l. pyriformis (daday, 1905) 55. l. quadridentata (ehrenberg, 1830) 56. l. signifera (jennings, 1896) 57. l. simonneae segers, 1993* 58. l. stenroosi (meissner, 1908)* 59. l. stichaea harring, 1913*** 60. l. tenuiseta harring, 1914 61. l. undulata hauer, 1938* 62. l. unguitata (fadeev, 1925) family: notommatidae 63. cephalodella gibba (ehrenberg, 1830) 64. c. mucronata myers, 1924 65. c. ventripes (dixon-nuttall, 1901) 66. monommata grandis tessin, 1890 67. notommata copeus ehrenberg, 1834# 68. n. spinata koste & shiel, 1991* family: gastropodidae 69. gastropus minor (rousselet, 1892)*** family: trichocercidae 70. trichocerca bidens (lucks, 1912)* 71. t. edmondsoni (myers, 1936)* 72. t. pusilla (jennings, 1903) 73. t. scipio (gosse, 1886)* 74. t. siamensis segers & pholpunthin, 1997* 75. t. similis (wierzejski, 1893) appendix 1: systematic list of the examined rotifer taxa phylum: rotifera super-class: eurotatoria subclass: monogononta 188 sharma et al./ rich rotifer assemblage of a sub-tropical wetland of meghalaya family: synchaetidae 76. ploesoma lenticulare herrick, 1885 77. polyarthra vulgaris carlin, 1943 family: dicranophoridae 78. dicranophorus forcipatus (o.f. müller, 1786) 79. dicranophorus sp. order: flosculariaceae family: floscularidae 2 80. sinantherina spinosa (thorpe, 1893) 81. stephanoceros fimbriatus (goldfusz, 1820)*** family: testudinellidae 82. pompholyx sulcata hudson, 1885 83. testudinella amphora hauer, 1938* 84. t. emarginula (stenroos, 1898) 85. t. parva (ternetz, 1892) 86. t. patina (hermann, 1783) 87. t. tridentata smirnov, 1931 order: collothecaceae family: atrochidae 88. cupelopagis vorax (leidy, 1857)** subclass: bdelloidea family: philodinidae 89. dissotrocha aculeata (ehrenberg, 1832) 90. rotaria neptunia (ehrenberg, 1830) ---------------------------------------------------------------------------------------------------------------------------------------------------- # new record from northeast india (nei); * new record meghalaya state; ** new record from nei (sharma, 2016); *** new record from india (sharma, 2016) int. j. aquat. biol. (2017) 5(6): 387-392 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology original article dna damage and hematological changes in common carp (cyprinus carpio) exposed to oxadiazon seyede asal zanjani1, hossein emadi*1, shahla jamili2, ali mashinchian1 1department of marine biology, faculty of marine science and technology, science and research branch, islamic azad university, tehran, iran. 2iranian fisheries research organization, agriculture research education and extension organization (areeo), tehran, iran. article history: received 18 august 2017 accepted 7 december 2017 available online 2 5 december 2017 keywords: oxadiazon carp comet assay erythrocytes biochemical parameters abstract: this study was carried out to investigate the genotoxic, and hematological and serum biochemical effects of a widely used herbicide, oxadiazon in common carp (cyprinus carpio) fingerling. fish were exposed to different concentrations (0, 1, 1.5 and 2 ppm) of the herbicide for 30 days. blood samples were collected, then comet assay in circulating erythrocyte cells was applied. erythrocytes cells of fish exposed to 1, 1.5 and 2 ppm of oxadiazon showed dna damage (21.3%, 22.9%, and 28.4%, respectively) significantly higher than the control group. moreover, exposure to oxadiazon significantly decreased wbc, rbc, hb, hct as well as serum albumin, glucose, and total protein levels, while serum alp was significantly increased in the exposed fish groups. no significant differences were found in mcv, mchc and mch levels between oxadiazon treatments and control groups. in conclusion, this study shows that oxadiazon is highly toxic to c. carpio and causes significant changes in hematological and biochemical parameters as well as indicates the mutagenic potential of oxadiazon in the erythrocyte cells of this fish. introduction herbicides can be introduced into aquatic environment due to runoff and leaching, cussing adverse effects on non-target organisms, particularly fishes (wany et al., 1992). oxadiazon (ronstar) is a widely used herbicide in rice fields against both mono and dicotyledonous weeds, as well as in fruit trees, vines, grasses, cotton, soybeans, onions, and sunflowers (ahmed et al., 2008). different oxadiazon residue concentrations ranged 0.4 to 7.24 μg/l in different water bodies (mamun et al., 2009; kim et al., 2014), as well as up to 0.442 ppm in different tissues of fishes and shellfishes have also been reported (imanaka et al., 1981). exposure to oxadiazon have been revealed a retarding growth in african catfish (clarias gariepinus (ajani et al., 2015), adverse effects on serum biochemical profile in common carp (cyprinus carpio) (saravanan et al., 2017) and platy fish (xiphophorus maculatus) (sadeghi and imanpoor, 2015), and induced peroxisome proliferation in the rodents *corresponding author: hossein emadi https://doi.org/10.22034/ijab.v5i6.417 e-mail address: emadihossein@yahoo.com (richert et al., 1996). exposure of aquatic organisms even to low environmentally-relevant concentrations of pesticides can result in severe effects on genetic and physiological parameters that can be considered as biomarkers for evaluation of fish health as well as monitoring of environment pollutants (wendelaarbonga, 1997; blahova et al., 2014; ahmadivand et al., 2016; mitkovska et al., 2017). comet assay is a sensitive technique for detection of dna damage, practically applied in all nuclear eukaryotic cells, especially for biomonitoring and confirming dna damage in aquatic organisms (jin et al., 2004; kim and hyum, 2006; klobucar et al., 2010; mitkovska et al., 2017). the method allows to detect a wide variety of dna damage, including dna single-strand breaks, double-strand breaks, alkalilabile sites and reparation, as well as oxidatively induced base damages, even when exposed to low concentrations of toxicants (lee and steinert, 2003; frenzilli et al., 2009). common carp is one of important and valuable 388 zanjani et al./ genotoxic effect of oxadiazon in common carp commercial fish species in iran that farmed in the caspian sea basin of iran, area that high amount of herbicides are used (salehi, 1999). moreover, this species is widely used in the evaluation of physiological and genotoxic effects of pesticides in both laboratory and field conditions (poleksic and karan, 1999; jin et al., 2004; kim and hyum, 2006; klobucar et al., 2010; blahova et al., 2014; mitkovska et al., 2017). in this study, we examined the dna damage in erythrocyte cells of common carp fingerling using the comet assay, and its hematological and serum biochemical changes after 30 days exposure to different concentrations (1, 1.5 and 2 ppm) of the herbicide, oxadiazon. materials and methods chemicals: for this study, oxadiazon (12% ec) manufactured by behkesht company (tehran, iran) was used. the stock solution was prepared in acetone and tap water based on the manufacturer’s protocol. fish: common carp with the mean weight of 18.27±2.3g and body length of 11.4±0.7 cm were obtained from a local fish farm (guilan, iran), and acclimated to laboratory conditions in 1000 l tanks filled with dechlorinated tap water for two weeks. the fish were fed commercial ffc-extruded fish food (faradaneh company, iran) twice a day and starved for 24 h before sampling. experimental design: a number of 120 fish were selected and divided into four duplicate groups (15 fish per replicate) in 100 l tanks and were exposed to concentrations 0, 1, 1.5 and 2 mg/l of oxadiazon. during the experiment the physicochemical characteristics of the water including, water temperature (°c), dissolved oxygen (mg/l), ph, and total hardness (mg/l as caco3) were 25.4±0.9, 6.5±4, 7.8±0.1 and 110±5, respectively. the water was renewed daily and the dead fish were counted and removed. hematological and serum biochemical analysis: after 30 days exposure to different concentrations (0, 1, 1.5 and 2 mg/l) of the oxadiazon, five fish from each replication were anesthetized with clove powder (200 mg/l), and blood was collected from caudal vein puncture. hematological parameters including red blood cells (rbcs), white blood cells (wbcs), hematocrit (ht) and hemoglobin (hb), mean erythrocyte volumes (mcv), mean color concentration (mchc) and mean erythrocyte hemoglobin (mch) were determined based on svobodova et al. (1991). serum alkaline phosphatase (alp), glucose, total protein and albumin were determined by commercial kits (parsazmon co. iran) according to the manufacturer protocol. comet assay: the dna damage in the collected blood samples of exposed carp was determined by comet assay (alkaline single cell gel electrophoresis) method as previously described by singh et al. (1988). briefly, a mixture of 5 μl of blood sample with 95 μl of 0.5% (w/v) low-melting agarose was added into degreased microscope slides previously covered with 1% normal melting agarose and covered with a coverslip. after agarose solidification (4°c for 20 min), the embedded cells were lysed in lysing buffer (2.5m nacl, 100mm na2edta, 10mm tris-hcl, 1% triton x-100 and 10% dmso, ph=10) at 4°c overnight. after a 30 min incubation in electrophoresis buffer (300 mm naoh, 1 mm edta, ph≥13) electrophoresis was carried out at 20 v and 300 ma for 30 min. subsequently, neutralization was performed in three washing steps in 0.4 m tris-hcl (ph=7.5). to visualize dna strand breaks, slides were stained with ethidium-bromide and observed using a fluorescence microscope (e600; nikon). the dna damage was quantified as the percent of tail dna. statistical analysis: the data was analyzed statistically at p<0.05 by one-way analysis of variance (anova) using the ssps 20 software (chicago, il, usa). results hematological and serum biochemical parameters: the results of hematological and serum biochemical parameters are shown in table 1. the wbc levels of fish exposed to 1.5 and 2 ppm were significantly lower than the control group (p<0.05), however, no significant changes were observed in the fish exposed to 0.1 mg/l of oxadiazon. similarly, significant 389 int. j. aquat. biol. (2017) 5(5): 387-392 decreases in hematocrit and hemoglobin levels were only found in fish exposed to 1.5 and 2 ppm. also, exposure to the highest concentration (2 ppm) of oxadiazon significantly decreased rbc levels (p<0.05). no significant differences were found in mcv, mchc and mch levels between oxadiazon treatments and control groups (p>0.05). moreover, exposure to oxadiazon significantly decreased serum albumin, glucose and total protein levels, while serum alp was significantly increased in the exposed fish groups (𝑃<0.05). dna damage: the results of the dna damage (% tail dna) in the erythrocyte cells of the control and treated groups are shown in figures 1 and 2. cell viability was found to be more than >80% in all treatments, allowing the comet assay to be performed. almost (92.5%) of the erythrocyte cells in the control group presented no dna damage, however, that the fish specimens exposed to different test concentrations exhibited significantly higher dna damage (p<0.05). among the tested concentrations, the highest damage (28.4%) was observed in erythrocyte cells of fish exposed to 2 ppm of oxadiazon followed by 1.5 ppm (22.9%) and 1 ppm (21.3%) trial groups. discussion this study describes genotoxic effects, and hematological and serum biochemical changes in common carp exposed to different concentrations of the herbicide oxadiazon. our findings confirmed the potential of oxadiazon herbicide to induce dna damage in erythrocytes cells of this fish. for assessment of genotoxic contamination of the aquatic environment, klobucar et al. (2010) and mitkovska et table 1. hematological and serum biochemical parameters of cyprinus carpio after 30 days exposure to different concentrations (0, 1, 1.5 and 2 ppm) of oxadiazon. dose (ppm) 0 1 1.5 2 wbc (103 /µl) 33±1.99a 31.03±0.4a 28.26±0.49b 27.16±0.56b rbc (106 /µl) 1.24±0.2a 1.20±0.4a 1.17±0.21a 1.03±0.5b hb (g/dl) 6.9±0.36a 6.56±0.24a 5.7±0.26b 4.07±0.58c ht (%) 33±1a 31.1±0.68ab 30.2±1b 25.83±1.4c mcv (fl) 227.39±1.61a 224.44±6.4a 228.76±2.28a 231.76±16.73a mch (pg) 66.22±1.53a 65.69±0.98a 65.08±1.51a 64.63±4.43a mchc (%) 20.93±1.57a 21.11±0.47a 18.90±1.49a 18.73±1.92a alp (u l) 64.33±2.51b 75±4.35a 70.66±6.65a 80.33±7.09a albumin (g/ dl) 0.96±0.02a 0.63±0.02d 0.78±0.02b 0.72±0.01c tp (g/ dl) 2.74±0.02a 1.97±0.05d 1.83±0.02b 1.45±0.03c glucose (mg/ dl) 89.33±7.02a 58±1b 59.33±1.15b 55.33±4.73b figure 1. dna damage in the blood samples of common carp on day 30 of exposure to different concentrations (0, 1, 1.5 and ppm) of oxadiazon. different letters indicate significant differences between the groups at p<0.05. 390 zanjani et al./ genotoxic effect of oxadiazon in common carp al. (2017) found in vivo genotoxicity by comet assay in carp, confirming this method as a reliable biomarker and common carp as a suitable bioindicator for water quality. many studies, assessing the genotoxic effects of pesticides found significant increase in the dna damage of erythrocytes by enhancing concentrations and exposure time (cavas and konen, 2007; guilherme et al., 2012; moreno et al., 2014). in contrast, cavalcante et al. (2008) observed that dna damage in gill cells of prochilodus lineatus exposed to roundup was not persisted over time. in the current study, the incidence of dna damage in erythrocytes cells after 30 days of exposure to oxadiazon could be attributed to intrinsic differences in the repair enzyme system and/or turnover cell in erythrocytes. however, further studies are still necessary to confirm this hypothesis. in response to a stressor such as pesticide exposure, the fish undergo a series of biochemical and hematological changes in an attempt to compensate the challenge imposed on them and thus cope with stress (wendelaar-bonga, 1997). similarly, significant changes in hematological and serum biochemical parameters of oxadiazon exposed fish were observed in this study. saravanan et al. (2017) assessed the acute toxicity effects of 0.5, 5 and 50 μg/l concentrations of oxadiazon on carp for 96 h, found that this herbicide causes a significant decrease in rbc, hb, and hct whereas the mcv, mch, wbc and serum alp were higher in treated group. however, the value of total protein, albumin, globulin, and mchc did not show any change in treatments. the effects of oxadiazon on some hematological and figure 2. comet assay of blood samples from common carp showing grades of dna damage in erythrocytes after 30 days exposure to oxadiazon. a: control; b: 1 ppm; c: 1.5 ppm, and d: 2 ppm. 391 int. j. aquat. biol. (2017) 5(5): 387-392 serum biochemical parameters in the recent report (saravanan et al., 2017), are in contrast with our finding which may be due to exposure time, herbicide concentration as well as fish health condition. the decrease rbc, hct and hb content in this study could be explained as a compensatory response that reduces the oxygen carrying capacity to maintain gas transfers and indicates a change in the water blood barrier for gas exchange in the gill lamellae (jee et al., 2005). also, change in wbc levels observed in current study indicates the immunotoxic potential of the herbicide as well as its xenoandrogens activity since oxadiazon has targeted the leukocytes profile (milla et al., 2011; ahmadivand et al., 2015). moreover, change in serum alp, albumin, glucose and total protein levels might have resulted from the hepatic dysfunction and immunosuppressive effect of the herbicide (nayak et al., 2004). in summary, this study shows that oxadiazon is highly toxic to the fish organism and causes significant changes in hematological and biochemical parameters as well as indicate the mutagenic potential of oxadiazon in the erythrocyte cells of c. carpio, suggesting that its use should be carefully monitored considering its potential impact on aquatic fauna. the current study also indicates that the comet assay is very sensitive tools for evaluating the genotoxic effect of pesticides on fish as well as further recommends its combined use with other biomarkers to monitoring aquatic environmental pollutions. further studies are necessary to elucidate its toxic effects on different biological parameters of fish, especially reproduction and immune system. references ahmadivand s., farahmand h., mirvaghefi a., eagderi s., zargar a. 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(2017) 5(6): 387-392 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2017 iranian society of ichthyology چکیده فارسی معموليشناسي در ماهي کپور و پارامترهای خون dnaبررسي اثرات سم اگزاديازون بر میزان آسیب ((cyprinus carpio 1مرادی ماشینچیان علي ،2جمیلي شهال ،*1عمادی حسین ،1زنجاني عسل سیده .ایران تهران، اسالمی، آزاد دانشگاه تحقیقات، علوم واحد دریا، بیولوژی گروه1 .ایران تهران، کشاورزی، ترویج و آموزش تحقیقات، سازمان کشور، شیالتی علوم تحقیقات موسسه2 چکیده: شناسی و بیوشیمیایی در ماهی کپور و همچنین پارامترهای خون dnaاگزادیازیون بر میزان آسیب کش این مطالعه با هدف بررسی اثرات علف متر( سانتی 4/11 ±7/0گرم و طول 27/18 ±3/2)میانگین وزنی عدد ماهی کپور 120انجام شد. بدین منظور تعداد (cyprinus carpio) معمولی گرم در میلی 2و 5/1، 1، 0روز در معرض غلظت های 30مدت لیتری به 100ماهی در تانک های 15در چهار گروه هر کدام با دو تکرار و تراکم ا هشناسی و بیوشیمیایی سرم بین گروهقرار گرفتند. در انتهای دوره آزمایش از ماهیان نمونه خون تهیه و پارامترهای خون اگزادیازیونلیتر سم دار بررسی گردید. دنباله dnaو تعیین میزان درصد کامت آزمونبا استفاده از گلبول قرمز های سلولدر dnaمقایسه شد. همچنین میزان آسیب 2 غلظت در بیتخر زانیم نیشتریب. (p< 05/0)داری بیشتر از گروه کنترل بود در تیمارهای سم بطور معنی dnaبر اساس نتایج میزان آسیب های عالوه در گروههب .شد مشاهده( %3/21و % 9/22بترتیب ) تریل بر گرمیلیم1 و 5/1 یهاغلظت در بیترتهب سپس و( % 4/28) تریبرل گرمیلیم ، پروتئین کل و آلبومین سفید و همچنین میزان گلوکز هایتعداد گلبول قرمز، هایهماتوکریت، هموگلوبین، تعداد گلبولاگزادیازیون میزان تیمار .(p< 05/0)داری در میزان آنزیم کبدی آلکالین فسفاتاز نشان دادند ها افزایش معنیدر حالی که این گروه داری مشاهده شد،سرم کاهش معنی (. بر اساس نتایج این مطالعه اگزادیازیون <05/0p)های آزمایش مشاهده نگردید در بین گروه mcv و mch ،mchcداری در میزان تغییر معنی ذارد.گشناسی و بیوشیمیایی این ماهی میبوده و اثرات شدیدی بر پارامترهای خون برای ماهی کپور بسیار سمی و دارای قابلیت ژنوتوکسیکی .بیوشیمیایی فاکتورهای کامت، آزمون کپور، اگزادیازیون، :کلمات کلیدی international journal of aquatic biology (2014) 2(5): 238-245 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article effect of microbial load on the condition index of the edible oyster, saccostrea cucullata in the sundarbans, india harekrishna jana1, keshab chandra mondal*1, chiranjit maity1, arpan das1, suman kumar halder1, abhijit mitra2, bikas ranjan pati1 1department of microbiology, vidyasagar university, midnapur (w) 721102, west bengal, india. 2department of marine science, university of calcutta, 35, b.c. road, kolkata –700019, west bengal, india. article history: received 1 may 2014 accepted 17 september 2014 available online 2 5 october 2014 keywords: livelihood total bacterial count total coliform fecal coliform. abstract: the effect of microbial load on the condition index of the edible oyster, saccostrea cucullata were analysed on monthly basis during 2010 and 2011 from the three different stations (namkhana, frasergaunge and sajnekhali) of indian sundarbans. the results showed significant variation with respect to microbial load between stations and seasons, which is reflected in the tissue of edible oyster. significant positive correlations were observed between microbial load of the ambient environment and the tissue system of oyster. the condition index of the oyster species also exhibited negative correlation with the microbial load of oyster tissue, which confirms the negative stress induced by microbes on the growth and survival of the species. introduction the condition index (ci) is used to estimate the effect that different environmental factors on oyster meat quality. the growth or health status of oyster is generally represented by its condition index value. the indices (ci) compare the dry meat weight of the animal to the interior of the carbohydrate and protein fractions, lipid and mineral contents and has been related to pollution effect (austin et al., 1993). the ci is an inexpensive, quick, representative and responsive tool for monitoring pollution (scott and lawrence, 1982) and has also been used to estimate growth differences among oysters living in different environmental conditions (austin et al., 1993; schumacker et al., 1998). the better condition index of the oyster is a reflection of the good environmental quality of the water (bhattacharyya et al., 2010). a number of studies revealed that the sources of fecal pollution mainly include municipal sewage system, storm water runoff, marine and boaters, * corresponding author: keshab chandra mondal e-mail address: mondalkc@gmail.com recreationalists, farm animals, pets and wild life (glasoe and christy, 2004). a primary concern in shellfish growing areas, which are generally located in the intertidal and shallow sub-tidal coastal zone, is contaminated from human sewage and animal wastes and the related health risk. krishnakumari et al. (1990) observed that condition index and percentage edibility values were higher at less polluted stations along maharastra coast. on this background, no study in indian sundarbans has been conducted to evaluate the microbial population in relation to condition index of oyster. the indian sundarbans is presently under stress due to discharge of untreated sewage and industrial wastes from the highly urbanized and industrialized city of kolkata, howrah and the newly emerging haldia portcumindustrial complex. according to unep report, 1125 million lit. of waste water is discharged per day through hooghly estuary. the lower stretches receive waste and wastewater load of 396 ×108 km3 per hour along with the annual run off 239 jana et al/ effect of microbial load on saccostrea cucullata 493 km3. the total volume of sewage discharge from the city of kolkata has been estimated 350 million tons (mitra et al., 2009). in addition, a large number of shrimp culture farms are situated at the out skirt of the kolkata city area (like malancha, minakhan block etc.) discharging huge organic load and trigger the microbial growth. in this context, it is extremely important to evaluate the impact of microbial load on the growth (ci value) of the oyster. in the present investigation for the first time, the impact of microbial load on the ci index of oyster (saccostrea cucullata) was evaluated. for this study, three potent pollution indicating bacterial group viz. total bacterial count (tbc), total coliform (tc) and fecal coliform (fc) in the oyster flesh, water and sediment were measured from three different station of sundarbans to evaluate the degree of contamination and its subsequent impact on the condition index of the oyster. the studies were conducted for two consecutive years in the three different sites of indian sundarbans namely namkhana, frasergaunge and sajnekhali, which are exposed to different level of environmental pollution. this analytical study will have great impact to the farmer and consumer who are dependent on this important sea food. materials and methods study area: the present investigation was carried for consecutive two years (2010-2011) in the three different stations like namkhana, frasergaunge and sajnekhali of indian sundarbans, which are exposed to varying degree of anthropogenic pollution (fig. 1). station-i (namkhana: 21°45'48.54''n, 88°13'52.55''e) is situated in the western sector of sundarbans, where high concentration of heavy metals was observed in water and the oyster. stationii (frasergaunge: 21°36'55.72''n, 88°12'33.15''e) is located further from pollution sources than station i. station-iii (sajnekhali: 22°07'36.21''n, 88°49' 50.60'' e) is situated in the eastern sector of indian sundarbans, which is considered to be relatively low in sources of pollution (barua et al., 2011). sample collection: water, sediments and six healthy oysters (4 to 5 cm) samples were collected in each month from the intertidal zone of each sampling station. each oyster was scrubbed, rinsed with distilled water (several times) and stored in a container, preserved in crushed ice and brought to the laboratory. the meat was aseptically extracted using a sterile knife and immediately used for the microbial analysis of oyster flesh (cleseri et al., 1998). water samples were collected using sterile water sampler. the sediments were collected using peterson grab, then put into sterile polythene bags and transported to the laboratory under ice bag for bacteriological examinations (cleseri et al., 1998). condition index measurement: condition index (ci) of oyster is recognized as the degree of fatness or the extent to which the meat fills in the shell cavity. it was measured as the ratio of dry oyster meat (oven dried at 60ºc for 48 hrs) to the volume of the shell cavity (abbe and albright, 2003). condition index (ci) = dry tissue weight (g) shell cavity volume (ml) × 100 bacteriological analysis: total coliform (tc), fecal coliform (fc) and total bacterial load (tbc) in the flesh of oyster, sediment and water of three different stations were enumerated. for bacterial analysis, 10 g oyster sample (meat) was blended with 90 ml of sterile 0.5% (w/v) peptone buffer (ph = 7) and different dilutions (10-1 to 10-2) were made. for enumeration of coliform, fecal coliform, the standard figure 1. map of indian sundarbans showing the location of sampling stations. three stations viz., namkhana, frasergaunge and sajnekhali. 240 international journal of aquatic biology (2014) 2(5): 238-245 mpn (most probable number) procedure (cleseri et al., 1998) was adopted using ltb (lowryl tryptose broth) and ec (escherichia coli) culture broth, respectively. briefly, 10 ml of 10-1 dilution was added in test tube containing 10 ml volume of double strength and 1 ml of each dilution (10-1 and 10-2 dilution) was added separately in test tube containing 10 ml volume of single strength ltb broth. the total sets were incubated at 35 ± 0.5°c for 24 hrs and examined for the presence of growth accompanied by gas production. the mpn was calculated and the results were expressed as “presumptive coliform mpn/100 g.” then the positive culture was inoculated in to brilliant green lactose bile broth and the tubes were incubated at 35 ± 0.5ºc for 24 hrs and examined for growth with gas production. the mpn of total coliform (tc) was calculated and the results were expressed as “confirmed coliform mpn/100 g”. to enumerate fecal coliform (fc), inocula from 24 hrs positive presumptive tubes were aseptically transferred to tubes of ec medium. these tubes were incubated at 44.0 ± 0.5°c for 24 hrs and examined for the presence of growth with gas production. the results were expressed as “fecal coliform mpn/100 g”. similarly, for total coliform and fecal coliform in the water and sediment samples were also enumerated. the total bacterial count (tbc) in all samples was done by standard plate counting method using tryptose glucose beef extract agar (tgbea) media. statistical analysis: the relationship among the parameters was analyzed by spss-10.0. each sample was analyzed and data were represented as mean ±sd. results the result indicated that a unique seasonal trend in all the three stations with respect to total bacterial figure 2. variation of total bacterial count (tbc) (a) in water, (b) sediment and (c) oyster at all the sampling stations. 241 jana et al/ effect of microbial load on saccostrea cucullata count (tbc), total coliform (tc) and fecal coliform (fc) in oyster flesh, water and sediment. relatively higher levels of total bacterial count (tbc), total coliform (tc) and fecal coliform (fc) in water, sediment and in oyster tissue were observed during the monsoon (july-october) season and lowest level during the pre-monsoon (march-june) season. the highest and lowest quantity of tbc in water samples during monsoon and pre monsoon season recorded in namkhana as 4.6x108 cfu/ml and 1.5 x107 cfu/ml, in frasergaunge 7x108 cfu/ml and 8x106 cfu/ml and in sajnekhali 4.8x108 cfu/ml and 7.8x106 cfu/ml, respectively (fig. 2a). the highest and lowest population density of total bacteria in sediment samples during monsoon and pre-monsoon season were recorded in namkhana as 9x1010 cfu/g and 3.5x107 cfu/g, in frasergaunge 8.8x1010 cfu/g and 2.2x107 cfu/g and in sajnekhali 1.8x1010 cfu/g and 1.5x107 cfu/g, respectively (fig. 2b). in relation to the environmental sample, the quantity of tbc in oyster during monsoon and premonsoon was recorded in namkhana as 8.9x108 cfu/g and 1.1x108 cfu/g, in fresergaunge 10x108 cfu/g and 8x107 cfu/g and in sajnekhali 6.6x108 cfu/g and 10x106 cfu/g, respectively (fig. 2c). the maximum density of total coliform (tc) in the water, sediment and oyster tissue was recorded in monsoon period as in namkhana 1600 mpn/100 ml, 110 mpn/g and 180000 mpn/100 g, in frasergaunge 1600 mpn/100 ml, 110 mpn/g and 160000 mpn/100 g and in sajnekhali 1600 mpn/100 ml, 110mpn/g and 91000 mpn/100 g, respectively (fig. 3). during pre-monsoon season, a comparatively low amount of tc was enumerated in namkhana 430 mpn/100 ml, 2.4 mpn/g and 2400 mpn/100 g, in frasergaunge 110 mpn/100 ml, 0.9 mpn/g, and 41400 mpn/100 g and in sajnekhali 110 mpn/100 ml, 0.9 mpn/g and 2000 mpn/100 g figure 3. variation of total coliform (tc) (a) in water, (b) sediment and (c) oyster at all the sampling stations. 242 international journal of aquatic biology (2014) 2(5): 238-245 (fig. 3) in the water, sediment and oyster tissue, respectively. the fecal coliform load (fc) was highest in water sample during monsoon and lowest during premonsoon season recorded as in namkhana 1600 mpn/100 ml and 220 mpn/100 ml, in frasergaunge 1600 mpn/100 ml and 80 mpn/100 ml and in sajnekhali 920 mpn/100 ml and 33 mpn/100 ml (fig. 4a). the highest fecal coliform (fc) in sediment samples during monsoon and pre-monsoon season were recorded as in namkhana 110 mpn/g and 2.4 mpn/g, in frasergaunge 110 mpn/g and 10 mpn/g and in sajnekhali 46 mpn/g and 0.7 mpn/g (fig. 4b). in oyster tissue, the fecal coliform (fc) recorded during monsoon and pre monsoon season recorded as in namkhana 180000 mpn/100g and 2400 mpn/100 g, in frasergaunge 91000 mpn/100 g and 2140 mpn/100g and in sajnekhali 35000 mpn/100 g and 1400 mpn/100g (fig. 4c). the microbial load in the ambient water and sediment exhibited significant positive correlation (p<0.01) with that in the oyster tissue except tc and fc of sediment at namkhana and frasergaunge (table 1). the seasonal variation of condition index has been found to be relatively higher in oyster during the premonsoon and lowest value during the monsoon in all the three stations. the station wise order of highest ci of oyster was as sajnekhali > frasergaunge > namkhana (fig. 5). highest ci of the oysters were 36.0 at sajnekhali, 32.73 at frasergaunge and 19.25 at namkhana found during the pre-monsoon season and 10.72, 6.88 and 5.0 during the monsoon at sajnekhali, frasergaunge and namkhana, respectively. the condition index of edible oyster showed figure 4. variation of fecal coliform (fc) (a) in water, (b) sediment and (c) oyster at all the sampling stations. 243 jana et al/ effect of microbial load on saccostrea cucullata significant negative correlations with total bacterial load (r = -0.664, p<0.01 at station-i; r = -0.454, p<0.05 at station-ii and r = -0.685, p<0.01 at station-iii), coliform load in the oyster flesh (r = 0.401, p<0.05 at station-i; r = -0.430, p<0.05 at station-ii and r = -0.555, p<0.01 at station-iii) and the condition index (ci) value showed insignificant negative correlation (r = -0.386) with fecal coliform in the oyster flesh from station-i, while it has significant negative correlation with fecal coliform at station-ii (r = -0.405, p<0.05) and at station-iii (r = -0.711, p<0.01), respectively (table 1). discussion relatively higher values of microbial load in all samples were observed in monsoon months (july to october), which may be related to storm water runoff and increased runoff from adjacent landmasses. the estuarine water budget in the sundarban estuary is regulated by dilution caused by fresh water discharge (mitra et al., 2009) that bring huge microbial load in the system. as a result, significant fresh water volume during monsoon in the present study area increases the microbial load in monsoon season. the station-wise order of microbial contamination in the study area was namkhana > frasergaunge > sajnekhali. this spatial variation may be attributed to the varying level of anthropogenic stress. the level of microbial load is a reflection of environmental pollution that is highest in station-i owing to fish landing and marketing activities. the lowest microbial load at station-iii is reflective of the minimul pollution sources. namkhana station receives the wastewater from kolkata and nearby character namkhana frazergaunge sajnekhali ‘r’-value ‘p’-value ‘r’-value ‘p’-value ‘r’-value ‘p’-value tbc of water × tbc of oyster. 0.436 <0.05 0.532 <0.01 0.473 <0.05 tbc of sediment × tbc of oyster. 0.813 <0.01 0.450 <0.05 0.727 <0.01 tc of water × tc of oyster. -0.033 is 0.531 <0.01 0.524 <0.01 fc of water × fc of oyster. -0.066 is 0.686 <0.01 0.607 <0.01 tc of sediment ×tc of oyster. 0.387 is 0.376 is 0.656 <0.01 fc of sediment × fc of oyster. 0.318 is 0.385 is 0.710 <0.01 tbc of oyster× ci of oyster -0.664 <0.01 -0.454 <0.05 -0.685 <0.01 tc of oyster × ci of oyster. -0.401 <0.05 -0.430 <0.05 -0.555 <0.01 fc of oyster × ci of oyster. -0.386 is -0.405 <0.05 -0.711 <0.01 table 1. inter-relation between different variables at three different stations viz., namkhana, frasergaunge and sajnekhali. figure 5. variation in condition index of oyster at all the sampling stations. 244 international journal of aquatic biology (2014) 2(5): 238-245 haldia port-cum-industrial complex but frasergaunge station receives the discharge of several hotel and tourism units located at bakkhali. on the other hand, sajnekhali station is situated in eastern sector of indian sundarbans, which is noted for its wilderness, anthropogenic stress is minimum in this sampling station owing to reserve mangrove forest (jana et al., 2013). similar results were also obtained in coastal south carolina, where scientists have employed a variety of technique to monitor and compare land uses and ecosystem responses in highly urbanized murrells inlet and relatively undeveloped north inlet (scott et al., 1996; white et al., 2004). murrells inlet also had higher occurrences of e. coli bacteria, fewer coliform free stations, and fewer bacterial species comprising the coliform group-findings that the researchers attributed to urban influences and higher densities of onsite sewage systems in the murrells inlet water shed (booth and jackson, 1997). subsequent analysis of murrells inlet by kelsey (kelsey et al., 2004, 2003) identified concentrations of on-site sewage systems, a rain fall events, and runoff from urban areas as key predictors of fecal coliform levels. the microbial load in the ambient water and sediment exhibited significant positive correlation with that in the oyster tissue except tc and fc of sediment at namkhana and frasergaunge. as the microbes from the ambient media often get accumulated in shellfish through filter feeder activity, hence the microbial load in ambient environment is directly affected this important sea food (mitra et al., 2007). the lowest ci value at station-iii because of the nonexistence of industries and anthropogenic activities in the reserved forest area. less pollution and anthropogenic stress may favour the maximum deposition of glycogen in oyster tissue and hence increase the ci value of oyster at station-iii than other stations. krishnakumari et al. (1990) also observed that condition index and percentage edibility values were higher at less polluted stations along maharastra coast. oyster condition and gonadal indices declined in areas receiving high levels of bacterial pollution, adjacent to known pollution sources such as marinas and highways (scott, 1976). condition index of oyster decreases with increase the microbial load in tissue of oyster because of markedly decline the assimilation efficiency of oyster and also oyster would expand considerable energy at high coliform concentration. similar results were also obtained by scott (1976) while working in little river, sc and north inlet, sc. he observed significant declines in oyster condition index when total coliform bacterial densities were > 70/100 ml. at these densities, the assimilation efficiency of the oyster markedly declined resulting in overproduction of gill mucous to deal effectively with the increased particulate load that generally accompanies elevated coliform bacterial densities. in the present case study, the lowest condition index values of oysters at namkhana and also in the monsoon months coincided with high microbial load. the present result indicates an alarming situation with respect to microbial load in the oyster tissue and its inter-relationship with condition index values. therefore proper water classification on the basis of microbial load is of extreme importance. oyster, being an edible product needs continuous monitoring with respect to coliform load to overcome the barrier of consumer acceptability, which may otherwise poses an adverse effect on the human health. the ability to produce, transport and market, healthy disease-free shellfish is crucial to the success of the indian oyster industry. under such circumstances, results of the present work may serve as baseline information for initiating oyster industry in the maritime state of west bengal. acknowledgments we are thankful to the ugc, government of india for providing financial assistance through minor research project (ugc project no: psw-144/09-10). references abb g.r., albright b.w. (2003). an improvement to the 245 jana et al/ effect of microbial load on saccostrea cucullata determination of meat condition index for the eastern oyster crassostrea virginica. journal of shellfish research, 22: 747-752. austin h., haven d.s., moustafa m.s. (1993). the relationship between trends in a condition index of american oysters crassotrea virginica, and environmental parameters in three virginia estuaries. estuaries, 16: 362-374. barua p., mitra a., banerjee k., chowdhury m. (2011). seasonal variation of heavy metals accumulation in water and oyster (saccostrea cucullata) inhabiting central and western sector of indian sundarbans. journal of environmental research, 5: 121-130. bhattacharyya s., panigrahi a., mitra a., mukherjee j. (2010). effect of physico-chemical variable on the growth and condition index of the rock oyster, s. cucullata (born) in the sundarbans. india. indian journal of fisheries, 57: 13-17. booth d.b., jackson c.r. (1997). urbanization of aquatic systems: degradation thresholds, storm water detention and the limits of mitigation. journal of the american water resources association, 33: 1077-1090. cleseri l.s., greenberg a.e., eaton a.d. (1998). standard methods for the examination of water, 20th ed. american public health association/ american water work association/ water environment federation, washington, dc. pp 9000-9221. glasoe s., christy a. (2004). coastal urbanization and microbial contamination of shellfish growing areas. washington, pp 1-28. jana h.k., mondal k.c., maity c., ghosh k., mitra a., banerjee k., dey s., pati b.r. (2013). variation of antioxidant biomarkers in the edible oyster saccostrea cucullata collected from three different water bodies of sundarbans. chemistry and ecology. doi: 10.1080/o2757540.2013.827669. kelsey h., porter d.e., scott g., neet m., white d. 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(2007). quality of edible oyster with respect to coliform load: a report from indian sundarbans. seshaiyana, 15: 1-2. schumcekr e.j., dambauld b.r., kauffmen b.e. (1988). investigation using oyster condition index to monitor the aquatic environment of wilapa bay of wasington. journal of shellfish research, 17: 338-339. scott g.i. (1976). oyster condition index as a monitor of biological pollution in south carolina coastal waters: a pilot study, university of south carolina, marine science program, p 101. scott g.i., lawrence d.r. (1982). the american oyster as a coastal zone pollution monitor: a pilot study. estuaries, 5: 40-46. scott g.i., fulton m.h., strozier e.d., key p.b., daugomah j.w., porter d., strozier s. (1996). the effects of urbanization on the american oyster, crassostrea virginica (gmelin). journal of shellfish research, 15: 523-524. white d.l., porter d.e., lweitus a.j. 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(2020) 8(4): 228-245 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology review article reviews on the biology and culture of silver pomfret, pampus argenteus (euphrasen, 1788) sandipan gupta*1 department of industrial fish and fisheries, brahmananda keshab chandra college, kolkata, west bengal, india. s article history: received 17 june 2020 accepted 24 august 2020 available online 2 5 august 2020 keywords: silver pomfret feeding habit breeding biology culture abstract: silver pomfret, pampus argenteus (euphrasen, 1788) is one of the most valuable and desired table fishes of the world. it has high economic value and has been reported to contribute significantly in the commercial fishery in its native ranges. considering its high economic value, it is really necessary to have scientific knowledge on its biology and culture so as to continue its fishery in long run. substantial research has so far been conducted to gather information on its biology and culture, but till date no such consolidated report is available on these aspects. the present work has been focused to gather the already documented information on its biology and culture, and to point out the scope of further research to support its fishery and trade. considering the information summed up in the present report, it is evident that ample information is available on its feeding and reproductive biology, but information on its culture methodologies is scanty. hence, further study is needed to gather more information on its culture and rearing methodologies to support its fishery and trade in coming days. introduction silver pomfret (pampus argenteus) belongs to the family stromateidae under the order scombriformes. it is one of the most valuable and desired food fishes found worldwide due to its soft and tender flesh, lesser number of bones and good taste, high protein and fat content (masuda et al., 1975; solanki et al., 1976; fei et al., 2011; hossain et al., 2011a, b, c, 2012a, b, 2016; peng et al., 2012a, b). it is an important fisheries resource due to its high economic value and export market; and for a long time, has been reported to significantly contribute to the commercial fishery of its native countries (pati, 1980; khan, 1982; dwiponggo, 1984; white, 1984; kim and lee, 1992; mohamed and ali, 1992; luo et al., 1993; al-qattan, 1998; ali et al., 2000; al-husaini, 2003; almatar and james, 2007; mohamed et al., 2008; shi et al., 2009a, b; liu et al., 2014a, b; din et al., 2015). its utilization in preparation of chinese medicine has also been reported (tang, 1987). considering the high economic value of silver pomfret, it is really necessary to gather scientific knowledge on its biology and culture, so as *correspondence: sandipan gupta e-mail: sandipangupta2007@gmail.com to continue its fishery and trade in a sustainable manner. earlier, considerable researches have been conducted on its biology and culture; however consolidated report on these aspects is unavailable till date. this report has been focused to summarize the already available information on its biology and culture and to explore the field of further research by specifying the information lacunae to support its fishery and trade in the coming days. morphological characters: the body is strongly compressed and oval in shape. the dorsal and ventral profile, both are almost equally convex. caudal peduncle is short, deep and strongly compressed; scute-like scales or fleshy keels are absent. the mouth is small, sub-terminal, slit-like and curved downward posteriorly. upper jaw is covered with skin and unmovable. single row of villiform teeth are present on both the pre-maxillary and dentary bones. the teeth on the pre-maxillary bone are mostly uni-cuspid in nature, conical in shape and blunt, and are interspersed with a few tri-cuspids, while those on the dentary are mostly tri-cuspid in nature, narrow, sharp, and are 229 int. j. aquat. biol. (2020) 8(4): 228-245 interspersed with a few uni-cuspid teeth. the snout is blunt, projected over the mouth; the maxilla is reaching below the first third of the orbit. eyes small, central and are with feeble adipose lids. gill membranes are joined to belly; gill slit short; total gill rakers on first gill arch ranges from 10-13. fin: the dorsal and anal spines are in a truncated form above the skin. anterior portion of the soft dorsal fin is elevated, but not that much extent as that for the anterior part of the anal fin which in the immature forms reaches to below the middle of the caudal fin; but as age advances it gradually becomes shortened. dorsal fin with 37-43 fin-rays while anal fin with 3443 fin-rays. the pelvic fins are absent. the pectoral fins are elongated and pointed with 24-27 fin-rays. the caudal fin is deeply forked; the lower lobe is much longer in the immature, sometimes being twice as long as the upper lobe. scales are small, cycloid and deciduous in shape; extend to the base of all the fins; slightly enlarged near the caudal peduncle. vertebrae 34-37 in number. colour: upper surface of the head and back as low as the lateral line is neutral gray with purplish shine; sides of the head and body is silvery gray that is fading to white on the abdomen. a dark spot is present on the upper portion of the opercle. dorsal and anal fins are minutely dotted with black; the outer half being the darker. caudal and pectoral fins are yellowish-white, also are minutely dotted with black, the outer half being the darker. the iris is silvery. the young are much darker; the vertical fins are being nearly black (day, 1878; talwar and jhingran, 1991; al-qattan et al., 2000). distribution: pampus argenteus is widely distributed throughout the indo-western pacific region, the eastern part of china, the western and south-western korean peninsula and the western asia including the persian gulf (day, 1878; pati, 1982; haedrich, 1984; davis and wheeler, 1985; kagwade, 1988; cho et al., 1989; kim and lee, 1992; alhussaini, 2003; azad et al., 2007; almatar and james, 2007; shi et al., 2009a,b; zhao et al., 2011; siyal et al., 2013; egderi et al., 2019). habitat: pampus argenteus is an inshore species; used to inhabit mainly brackish waters and estuaries and occurs in shoals over the muddy bottoms (abdurahiman et al., 2006). it is used to live at a depth range up to 110 m (haedrich, 1984; khan, 2000; shafi and quddus, 2003; mohamed et al., 2008). feeding biology food and feeding habit: to analyze the food and feeding habit of p. argenteus, so far plenty of research has been done in countries of its native range (suyehiro, 1942; rege, 1958; kulkarni, 1958; chopra, 1960; kuthalingam, 1963; nath, 1966; rao, 1967; pati, 1980; mohamed and ali, 1994; dadzie et al., 2000a; khan, 2000; sivakami et al., 2003; abdurahiman, 2006; peng et al., 2011; thangavelu et al., 2012). suyehiro (1942) has documented the presence of some gelatinous substances and medusae in the gut content of silver pomfret in his study in japan. similar kind of observations has been made further by kulkarni (1958) and chopra (1960). high percentage of tunicates, copepods, isopods, medusae and fishes has been noted by kulkarni (1958) while chopra (1960) has reported the presence of tunicates, jelly fish and medusae in the gut content of p. argenteus. rege (1958) has recorded gelatinous material in the diet of silver pomfret from bombay waters in addition to the occurrence of salps, hydromedusae, amphipods, copepods, shrimps and other small fish groups. he further has reported zooplankton and phytoplankton as the most favourite diet for this fish species. kuthalingam (1963) has reported crustaceans to form the main bulk of silver pomfret diet. larger crustaceans like penaeus spp., acetes spp., squilla spp. and anomurans have been reported to form the main bulk of adults’ diet. other components of the diet include copepods (oithona spp., euterpina spp. and eucalanus spp.), copepod nauplii, ostracods, amphipods, lucifer, zoea larvae, larval crustaceans, polychaetes, sagitta sp., larval bivalves, fish scales, vertebrae, flat fish, foraminifera, algal filaments and diatoms with good quantity of mucous. high percentage of copepods and cypris along with amphipods, ostracods, cladoceran, decapod larvae, 230 gupta / reviews on silver pomfret, pampus argenteus crustacean remains, gastropod larvae, calcareous remains, fish eggs, fish scales, polychaete remains has been documented by rao (1967) in the gut content of p. argenteus in his study from andhra-orissa coast. pati (1980) has documented copepod as the main food item for p. argenteus. acrocalanus spp., centropages spp., euterpina spp., nannocalanus spp., oithona spp., temora spp. and acartia spp. have been documented as the mostly preferred copepods while paracalanus spp., eucalanus spp., corycius spp. and pseudodiaptomus spp. are the less preferred copepods. apart from copepod, presence of ctenophores and medusa, diatoms, polychaete larvae, polychaete remains, copepod eggs, copepod nauplii and fish scale have been documented in the stomach content almost throughout the season and formed a fair percentage in the diet. decapod remains, ostracod, amphipod, decapod larvae, cladoceran, bivalve larvae, gastropod larvae, fish eggs, fish larva and algal filaments have been reported as occasional food items while rare occurrence of acetes spp., lucifer spp., marine insect and sagitta spp. in the diet has been documented. unidentified content in the form of semi-digested pulpy mass has been reported to form the maximum part of the diet. nath (1966) in his study at kerala coast has considered silver pomfret as a macro-plankton feeder; feeds mainly on crustaceans and polychaetes while mohamed and ali (1994) have documented silver pomfret to feed mainly on crustaceans with copepods as the dominant group. other components which have been reported in its diet are tunicates, medusa, jellyfish, fish larvae, eggs and scales. dadzie et al. (2000a) in their study from kuwait waters have reported total 19 types of crustacean items, three genera of bacillariophyceae (coscinodiscus sp., rhizosolenia sp. and hemidiscus sp.), one species of sagitta and one species of noctiluca in the gut content of silver pomfret along with lamellibranch veliger larvae, polychaete larvae, fish larvae, eggs of fish and invertebrates, fish scales and filamentous algae. white pulpy masses have also been documented in the gut contents. copepods and their eggs have been reported as the major food component followed by bacillariophyceae, other crustaceans, mollusc and fish scales. fish eggs and larvae have been recorded as the least common items. khan (2000) has reported the presence of mostly digested material and gelatinous substance in the stomach content of p. argenteus. copepods and amphipods have been documented in few occasions while young ones of some fish species like apogon spp.; myctophum spp.; nematopalaemon tenuipes and fish scales have been rarely documented. sivakami et al. (2003) have reported crustaceans such as copepods (oithona spp., euterpina spp., and eucalanus spp.), copepod nauplii, ostracods, amphipods, lucifer sp. and zoea larvae along with larger crustaceans (penaeus spp., acetes spp., squilla sp. and anomurans) in the diet of adult p. argenteus. other food items encountered are polychaetes, larval decapods, foraminiferans, sagitta spp. and sand particles. abdurahiman (2006) have reported crustaceans and detritus as the major diet components of p. argenteus. among crustaceans, copepods have been documented as the most important food item followed by amphipods and nauplii larvae. presence of fish items (cycloid and ctenoid scales, fish eggs and fish remains), diatoms (nitzschia spp. and coscinodiscus spp.) and worms has also been reported with decreasing order of importance in the diet. peng et al. (2011) have documented sagitta sp. as the most important food item along with shrimps, jellyfish, cephalopods, fish larva and zooplanktons for silver pomfret. thangavelu et al. (2012) have reported detritus and copepods to share the maximum part of the diet in silver pomfret. penaeid prawns, acetes sp. and fish larvae have also been recorded as other food items in the gut content. seasonal and depth wise variation in feeding: investigating the seasonal variation in the diet of silver pomfret in indian water, kuthalingam (1963) has reported that crustaceans used to form the bulk of the diet throughout the year, with highest percentage contribution in the month of february. the reduction in the occurrence of the crustaceans in the diet during march has been reported to become compensated with 231 int. j. aquat. biol. (2020) 8(4): 228-245 an increase in uptake of teleosts, polychaetes, molluscs and miscellaneous items. availability of various food items in the particular habitat has been concluded as the main factor behind this kind of fluctuation in diet. depth wise variation in diet of silver pomfret has also been reported by kuthalingam (1963); fishes captured from the range of 5-15 fathoms had maximum percentage of crustacean items while those captured from the range of 15-40 fathoms had polychaetes and foraminifera as the dominant items of the diet. dadzie et al. (2000a) in their study observed seasonal variation in percentage frequency of occurrence of different food items in the stomachs of p. argenteus. more variety (22 items with frequent occurrence) of food items have been documented in summer than in winter (11 items with frequent occurrence) and this variation has been reported to be due to the variation in availability of the prey organisms in the environment. abdurahiman (2006) has reported seasonal variation in feeding mainly in respect to pre-monsoon and post-monsoon season. detritus followed by copepods, diatoms and fish items have been reported as the mostly preferred food items in pre-monsoon while in post-monsoon season, detritus, copepods and fish items have been documented as the mostly preferred food. ontogenetic shift in food preference: pati (1980) has reported a striking change in the diet from post-larval stage to the adult. he has reported that the post-larvae are dominantly phytoplankton feeders, feeding on diatoms like coscinodiscus centralis, thallasiothrix frauenfeldii and pleurosigma normanii. with gradual growth, the juveniles have been reported to change their preference over copepods, copepod eggs and nauplii, and smaller crustaceans. polychaete remains have been documented first in the size group of 8-9 cm and has been reported from rest of the successive size groups. with further growth, increasing number of items in the diet has been documented with proportional decrease in the amount of copepod and diatom elements, but this has been reported to become compensated by other crustacean elements, jelly fishes, polychaete larvae and chaetognaths. kuthalingam (1963) has reported high percentage of large crustaceans like penaeus spp., acetes spp., squilla spp. and anomurans in the diet of adult silver pomfret while juveniles have been reported to feed mainly on copepods and other small crustaceans. dadzie et al. (2000a) have reported that in the size groups 145-164 to 205-224 mm, copepods and other crustaceans have high preference over other food items while size group 225-244 mm has maximum preference for bacillariophyceae. in size group 245264 mm, again high preference for copepods and other crustaceans has been reported while size group 265284 mm prefers only copepods and other crustaceans. sivakami et al. (2003) have reported size specific difference in food preference; small sized fishes within size range of 18-26 mm has been reported to prefer copepods (paracalanus parvus, oithona spp., euterpina spp., macrosetella spp., temora spp., acartia spp., harpacticoid copepods), ostracods, amphipods, larval stages of squilla sp. and lucifer sp. while adults prefer small crustaceans along with larger crustaceans. ontogenetic shift in food preference has also been reported by abdurahiman (2006). detritus and copepods have been reported to be highly preferred in almost all the size groups. the size group of 91-120 mm length has been reported to prefer detritus and copepods followed by nitzschia sp. and mysids. in size group of 121-150 mm length, detritus, oratosquillanepa, copepods and fish eggs have been documented as preferred food items. fishes in size group 151-180 mm length have maximum preference for detritus followed by copepods and nitzschia sp. in the size group of 181-210 mm length, detritus, copepods, cycloid fish scales and coscinodiscus spp. have been documented as preferred food items. the size group of 211-240 mm length has been reported to prefer detritus and copepods followed by mysids and nitzschia sp. in the size group of 241-270 mm length, detritus, copepods, coscinodiscus spp. and amphipods have been documented as preferred food items while for size group of 271-300 mm length, copepods, detritus, fish remains and amphipods have been 232 gupta / reviews on silver pomfret, pampus argenteus reported for the same. in size groups above 180 mm, preference for copepods has been reported to increase simultaneously with relative decrease in preference for detritus in the diet. thangavelu et al. (2012) have also reported an ontogenetic shift in diet with increased consumption of copepods in large length groups with decreasing proportion of detritus matter. change in feeding intensity: reduced feeding intensity during the breeding season has been reported for mature specimens of p. argenteus by kuthalingam (1963), pati (1980), dadzie et al. (1998, 2000a) and hossain et al. (2011a). pati (1980) further has documented that increasing surface water temperature has inhibitory effect on feeding intensity of immature silver pomfrets. reproductive biology sex ratio: most of the earlier researchers (mohamed and ali, 1993; al-abdul-elah et al., 2002; almatar et al., 2004; narges et al., 2007; nasir, 2016) have reported male dominance in the natural populations of silver pomfret except oda and namba (1982), dadzie et al., (2000b) and ghosh et al. (2009) who have reported female dominance in their studied populations. sivakami et al. (2003) and shi et al. (2006) have reported equal proportion of male and female in their studied populations. length at first maturity: all the earlier researchers have documented early maturation of male than the female in silver pomfret. gopalan (1967) has reported 22 and 26 cm as lengths at first maturity for male and female of silver pomfret from arabian sea while pati (1982) in his study at bay of bengal, reported 15 (male) and 17 cm (female). from their study at eastern part of china, lee and jin (1989) have documented 16.7 and 18.6 cm as lengths at first maturity for male and female, respectively. dadzie et al. (2000b) also have observed early maturation of males (minimum size at maturity 12.5-14.4 cm) than females (minimum size at maturity 20.5-22.4 cm) in populations of silver pomfret from kuwait waters. narges et al. (2007) also have documented early maturation of males (18 cm) than females (22.2 cm), but that is based on fork length (fl). mean lengths of 11.2 and 15.5 cm have been documented as length at first maturity for male and female, respectively in iraqi water by mohamed et al. (2008). sivakami et al. (2003) have reported 15 and 17 cm in standard length as length at maturity for male and female, respectively. almatar et al. (2004) have documented smallest mature male and female at the standard lengths of 12.7 and 16.5 cm, respectively in kuwait waters. gonad maturity stages: dadzie et al. (1998) have reported six maturity stages based on some macroscopic and microscopic characters to identify different maturity stages of testes which have been summarized in table 1. only two maturity stages namely immature and mature has been identified by lone et al. (2008a); they have reported that the immature fishes have very thin, streak like organ which become steadily thicker and bigger with maturity. the mature testes are creamy in colour and with increased blood supply. almatar et al., (2004) and lone et al. (2008b) have documented eight gonad maturity stages namely (i) developing virgin, (ii) developing, (iii) developed, (iv) gravid, (v) runningripe, (vi) partially spent, (vii) recovery and (viii) resting in female. the macroscopic and microscopic characters which have been documented for these different maturity stages have been summarized in table 2. dadzie et al. (1998) have reported seven maturity stages namely (i) immature, (ii) maturing virgin/ recovering spent, (iii) developing, (iv) maturing, (v) mature, (vi) running and (vii) spent depending on macroscopic and microscopic characters; these have been summarized in table 3. fecundity: pati (1981) has reported fecundity range of 40,610-90,460 for this species. mohamed and ali (1993) have reported fecundity range of 51,3162,45,356 in their study while fecundity range of 7,65,675-25,37,744 and 28,995-4,55,661 have been documented by abu-hakima (1984) and dadzie et al. (2000b), respectively. almatar et al. (2004) have assumed total fecundity of 3,58,542 for an average sized (~500 gm in weight). absolute fecundity of 90,071.1±29,750.9 and relative fecundity of 349.34±119.11 eggs/gm of body weight has been reported by qinman et al. (2009) in their study at 233 int. j. aquat. biol. (2020) 8(4): 228-245 bohai bay, china. sivakami et al. (2003) have reported 40,610-90,640 as the fecundity range for silver pomfret in their study. pati (1981), abu-hakima (1984) and dadzie et al. (2000b) have reported pampus argenteus as having determinate fecundity (i.e. the potential annual fecundity is fixed prior to the onset of spawning) though almatar et al. (2004) have documented it as an indeterminate fecund fish. spawning seasonality: most of the earlier researchers (gopalan, 1967; hussain and abdullah, 1977; bapat et al., 1982; pati, 1982; abu-hakima, 1984; mohamed and ali, 1993; safikhani, 1998; dadzie et al., 1998, 2000b; sivakami et al., 2003; almatar et al., 2004; mohammad and ehsan, 2007; narges et al., 2007; lone et al., 2008a,b; ghosh et al., 2009; nekuru et al., 2013) have reported a prolonged spawning season for p. argenteus except kuthalingam (1963) and shi et al. (2006). hussain and abdullah (1977) have observed april to september as the breeding season with two spawning peaks, one in april-may and another in september in kuwait waters while abu-hakima (1984) have documented march to august as the breeding season with the first peak in april and the second one in october. dadzie et al. (1998, 2000b) have reported may to august as the breeding period with two spawning peaks; one in may and another in august in kuwait waters. almatar et al. (2004) have documented late may to october as the breeding season with two spawning peaks in july and october in kuwait waters. later, lone et al. (2008a, b) have documented june to october as the breeding season with two spawning peaks, one in june and another in october in kuwait coastal waters. prolonged spawning season from may to october with two spawning peaks, one during may-july and another during october has been reported for this fish species in northwest of persian gulf by narges et al. (2007) while mohammad and ehsan (2007) have reported march to september as the breeding season in persian gulf. nekuru et al. (2013) have reported april to table 1. distinguishing characters of different maturity stages of male pampus argenteus as has been documented by dadzie et al. (1998). maturity stages macroscopic characters microscopic characters immature testis is thin, tiny, translucent thread like. numerous primary germ cells and spermatognia, which are mostly occupying the periphery of the testis, can be observed. groups of primary and secondary spermatocytes confined to cysts within the lobule can be observed at the centre of the organ. maturing virgin/recovering spent testis is still translucent, greyish in colour and used to occupy about 8% the peritoneal cavity. primary germ cells and spermatognia can be seen with increasing number of the primary and secondary spermatocytes. appearance of few cysts containing spermatids can also be observed. maturing testis is enlarging, opaque and cream-grey in colour with few blood capillaries around it. it used to occupy about 12% of the peritoneal cavity. germ cells at all stages of spermatogenesis in varying quantities are present. cysts of spermatozoa, mainly confined within lobules, appear late in this stage. mature testis is enlarged, turgid and whitish in colour with undulating margin. it used to occupy about 14.4% of the peritoneal cavity. large numbers of lobules are present packed with spermatozoa. lobules bounded by seminiferous tubules contain germ cells at different stages of spermatogenesis. running testis is pinkish white in colour, used to occupy about 17% of the peritoneal cavity. with slight pressure on the peritoneum, oozing of milt can be observed. lobules containing spermatozoa are still dominate though empty ones can also be encountered. spent testis is shrunken, flaccid and greyish in colour with visible blood capillaries. testis used to occupy about 7% of the peritoneal cavity. no evidence of undulating margin of the testis is visible and with slight pressure on peritoneum, oozing of milt cannot be observed. numerous convoluted lobules are present in the testis; some of them are revealing the presence of residual and relict spermatozoa. 234 gupta / reviews on silver pomfret, pampus argenteus table 2. distinguishing characters of different ovarian maturity stages of pampus argenteus as has been documented by almatar et al. (2004) and lone et al. (2008b). maturity stages macroscopic characters microscopic characters developing virgin ovary is very thin and thread like. the tunica albugenia is thick and the stroma is very well developed. oogonia are very common. the cytoplasm of the oocytes is very basophilic and the nucleus to cytoplasm ratio is lower than one. all oocytes are in the chromatin nucleolus stage or in the early peri-nucleolus stage. the sizes of the oocytes never surpass the 100 μm mark and majority of the oocytes is in the range of 50 to 70 μm. developing ovary is pale yellow in color with quite visible blood vessels on the surface. the texture of the ovary is compact and hard. opaque oocytes are present. the primary oocytes dominate, but some early stages of secondary oocytes in the yolk vesicle stage can also be seen. oocytes are with increasing size due to yolk vesicle deposition. the nucleus-to cytoplasm ratio is increased. the cytoplasm is less basophilic and looks pinkish-blue rather than dark blue, as is seen in very early oocytes. in the secondary oocytes, zona radiata is established. the size distribution of the oocytes at this stage ranged up to a maximum of 600 μm. however, this size distribution is season dependent. early in the season, oocytes in the smaller range can be found but during the peak breeding month comparatively large sized oocytes dominate over small oocytes. these are arranged generally in two groups; the first peak can be seen around 200 μm, while the second peak is in between 450 and 550 μm. some atresia can be observed. developed/maturing ovaries are large, yellowish in colour. opaque oocytes can be visible. the ovarian texture is still solid with increased blood circulation. the entire surface of the ovary is covered with big and small arteries. three groups of oocytes are present. a very small group of primary oocytes is present between the crevices made by the growing oocytes. some are in the secondary stage, but by far, the largest group is of tertiary-stage oocytes. these oocytes are characterized by a well-developed zona radiata. this is further divided into zona externa and zona interna. the theca and granulosa cells are seen around the zona externa. the tertiarystage oocytes have well developed, true yolk granules and some fat droplets. the nucleus or germinal vesicle has nucleoli in it. the stroma is thinner and all stages of atresia (α, β, γ and δ) of the tertiary yolked oocytes can be commonly seen. the tunica is thinner than in the previous stages, and the yolked oocytes can be seen through the tunica. the maximum size of the oocytes never surpasses 800 μm. some old post ovulatory follicles (pof) may also be seen. this is most true of those fish that have spawned earlier. gravid/ripening ovary at this stage exhibits a softer, speckled appearance when seen through the surface because the tunica is thin, completely stretched and transparent. this speckled nature is due to the appearance of hydrated oocytes in the ovaries. the overall color is light yellow with a spotted appearance of yellow (yolked oocytes) and white (hydrated oocytes). the blood supply is at its peak, and the surface is completely covered with the thick arteries and their capillaries. no eggs can be released with light pressure on the abdomen. only two types of oocytes can be seen; the primary oocytes present in the crevasses, and the tertiary stage oocytes and oocytes entering the final oocyte maturation (fom) stage. this stage is characterized by the movement of the (nucleus) germinal vesicle. along with this, there is sequestration of the oil droplets and the yolk granules. in the later stages, the nuclear membrane of the germinal vesicle breaks down and the chromosomes are free in the cytoplasm. this stage is a clearcut indication of the final maturity of the oocyte. by this time, the yolk is completely liquefied and nuclear elements are completely masked by this. these stages are very much interrelated and occur in quick succession, and may take 8 to 12 h before ovulation and actual spawning. the oocytes of this type are tightly packed and are at the same stage of development. the hydrated oocytes are filled with liquefied yolk, and the tunica is very thin. size wise, three groups of oocytes can be seen in such ovaries. one group, or peak, is around 200 μm for primary oocytes, the second peak is around 500 to 600 μm for tertiary oocytes ready to enter the fom stage, and the third peak is between 900 and 1200 μm for hydrated oocytes and those oocytes that are at different stages of fom. 235 int. j. aquat. biol. (2020) 8(4): 228-245 september as the breeding season with two spawning peaks in may and july in persian gulf. mohamed and ali (1993) have reported may-september as the breeding season with a single spawning peak in junejuly in iraq waters while march to september has been documented as the breeding season with two spawning peaks in june and september in mahshahr estuary, persian gulf, iran by safikhani (1998). gopalan (1967) has reported february to august as its breeding season with a single spawning peak during april-june in arabian sea while pati (1982) has documented february to august as the breeding season with two spawning peaks in april and august in the bay of bengal. in northwest coast of india, bapat et al. (1982) have reported two spawning periods for this fish species, one in february and another in august. sivakami et al. (2003) have reported february to august as the breeding period at orissa with two peaks, during february-april and june-august. ghosh et al. (2009) have reported silver pomfret as a perennial breeder with a spawning peak in june-november at arabian sea. kuthalingam (1963) has documented winter breeding (januaryfebruary) for silver pomfret in bay of bengal. shi et al. (2006) have documented early april to early june as the breeding season in china. lee and jin (1989) observed one peak from june to july in the eastern china sea. in silver pomfret, spawning occurs in the east china sea from early april to late may, but occurs later (from may to june) in the yellow sea (shi et al., 2005; zhao et al., 2010). an interesting phenomenon of spawning in silver pomfret has been reported by almatar et al. (2004) from kuwait waters. they have documented semi-lunar spawning table 2. continued. maturity stages macroscopic characters microscopic characters running/spawning ovary is whitish and jelly-like because of the overwhelming presence of the hydrated oocytes. the tunica is very thin, and oocytes can be clearly seen through it. the oviduct is well developed and full of mature, ovulated oocytes or eggs. blood supply to the ovaries is similar to that described for ripening stage. some ovaries are bloodshot because of the bleeding that ensued at the time of ovulation. oocytes are freely oozing out naturally or with light pressure. only two types of oocytes i.e. primary and tertiary oocytes are present. occasionally, a secondary oocyte can also be seen. the major difference between ripening and spawning ovaries is the absence or presence of pofs. the pofs are absent in ripening fish but are an integral part of the spawning ovary. the oocytes in the spawning ovary generally form two groups, but in some fish, a third peak of mature eggs is also present. the first peak of primary cells is around 200 μm, the second peak of tertiary oocytes is around 600 μm, and third one is around 900 to 1300 μm. this third peak is due to residual oocytes or eggs that have not been spawned and are present in the ovarian lumen. these eggs are to be involuted and recycled by the process of atresia. partially spent grossly, the ovarian outline is saggy and flaccid, and sometimes bloodshot. the tunica is wavy, and oocytes can be seen through them. however, in bloodshot ovaries, this is somewhat difficult. three groups, primary, secondary and tertiary oocytes are present. pofs of different types nearly always present. nearly all stages of atresia of tertiary and hydrated oocytes can be seen. atresia is more common in ovulated eggs that have not spawned during previous spawning. recovery ovaries are flaccid and reddish in colour. the ovarian outline is loose and baggy, and the tunica is thick. nothing can be seen through it. this may be because of involution of the ovarian parts and the diminishing blood supply. only the primary oocytes predominate in the ovarian cavity; these are in the peri-nucleolar or chromatin-nucleolar stage. in fishes where some development used to occur because of favourable temperatures, secondary and tertiary oocytes can also be seen; though majority of these are undergoing atresia. a lot of empty spaces can be seen in the ovary and in between the ovigerous folds. stroma can be seen in developing status again, and occasionally, old pofs can also be seen. size wise, only one peak of oocytes between 150 and 300 μm can be observed. resting ovary creamy to pale yellow in colour. black spots may be seen scattered on the ovary of older individuals. no opaque oocytes are visible. majorities of the oocytes are small and is comprised of oogonia and primary oocytes. some oocytes are in the chromatin nucleolar stage. the biggest oocytes present are in the early peri-nucleolus stage. the sizes of the oocytes never surpass 200 μm; however, the majority can be observed in the range of 75 to 150 μm. the oogonia are basophilic in nature. the nucleus is quite big, while the cytoplasm is in the form of a narrow rim around the nucleus. the stroma of the ovary is well developed, while the tunica albugenia is thick. 236 gupta / reviews on silver pomfret, pampus argenteus frequency (spawning occurs in first and third quarters of the moon phase) rather than a continuous daily spawning in this fish species. spawning habitat preference: inshore waters have been reported to be preferred by silver pomfret for spawning (rege, 1958; kuthalingam, 1963; gopalan, 1967; pati, 1982; khan, 2000; almatar et al., 2004). gopalan (1967) has reported 25.2-28.6°c as preferable temperature and salinity of 28.3‰ for spawning while almatar et al. (2004) have documented 26-32.5°c and salinity of 39‰ for the same in silver pomfret. al-abdul-elah et al. (2002) in their experiment have also reported 28-30°c as preferable temperature range for spawning and salinity range of 35-40‰ as the best to get maximum hatching rate. dadzie et al. (1998) have speculated that the spawning of p. argenteus is triggered by the low salinity discharge of the river mouth in the northern persian gulf (north of kuwait). aquaculture of silver pomfret the wild stock of silver pomfret is under threat mainly due to over-fishing that is putting high pressure on mature stocks and the young fishes smaller than their first maturity length (wen et al., 2006; narges et al., 2007, 2011; ghosh et al., 2009; siyal et al., 2013). apart from over-fishing, ecological alterations like changes in salinity and nutrients status (saad, 1982), reduction in freshwater inflow to the river due to drought and other influential factors (archangi et al., table 3. distinguishing characters of different ovarian maturity stages of pampus argenteus as has been documented by dadzie et al. (1998). maturity stages macroscopic characters microscopic characters immature ovary is thin, tiny and translucent thread like. ovigerous folds with spaces are oriented towards the centre of the ovary containing both oogonia (8.6±1.9 µm) and primary oocytes at the early perinucleolar (43.3±13.6 µm) and few late perinucleolar stages. maturing virgin/recoverin g spent ovary is creamy to pale yellow in colour; used to occupy 15% of the peritoneal cavity. oocytes are not discernible. oogonia are still present as also the spaces between the ovigerous folds. primary oocytes are present at all stages, especially late perinucleolar oocytes (74.0±8.8 µm). a few ovaries can also be seen with small lipid vesicles in cytoplasm, characteristic of early secondary phase. developing ovary is light yellow in colour with a reddish hue due to the presence of distinct network of blood capillaries. ovary is used to occupy 19% of the peritoneal cavity. oocytes are discernible. majority of the oocytes are at the late perinucleolus stage; a number of the primary oocytes can be seen to initiate the secondary growth phase (124.6±79.4 µm). maturing ovary is large in size and bright yellow in colour with conspicuous blood capillaries. ovary is used to occupy 24% of the peritoneal cavity. oocytes are clearly discernible. oocytes are at the vitellogenic stage (304.0±74.8 µm) with yolk granules and lipid vesicles present. mature ovary is very much enlarged and yellowish in colour; used to occupy 48% of the peritoneal cavity. active vitellogenesis can be observed with many oocytes attaining their maximum size (404.54±71.5 µm). large lipid vesicles coalesce to form lipid globules. the process ends with nuclear polarization. running ovary is greatly distended, yellow in colour and jelly like in appearance; used to occupy 64% of the peritoneal cavity. ripe eggs can be seen through the thin tunica. ova with oil globules can be seen to extrude out with slight pressure on the abdomen. oocytes are at nuclear polarization phase undergoing preovulatory nuclear changes, followed by ovulation. spent ovary is flaccid, shrunken, purplish in colour and bloodshot; used to occupy 46% of the peritoneal cavity. ovigerous folds are convoluted. residual, ruptured and atretic follicles are visible in the ovaries as well as primary oocytes. many oocytes are with small lipid vesicles in the cytoplasm, characteristic of the early secondary growth phase. 237 int. j. aquat. biol. (2020) 8(4): 228-245 2013) have been documented as important factors behind reduction in stock of silver pomfret. the decreasing trend of the market size of p. argenteus with deteriorating resources has been reported from different countries (liu and zhan, 1999; al-hussaini, 2003). so, in this regard, the most desirable solution can be “aquaculture” which will reduce the pressure of over-fishing and will allow the wild stocks some time to recover (peng et al., 2012b). considering its potential as an aquaculture species (shi et al., 2005; james and almatar, 2008; peng et al., 2012b); so far researches have been conducted on various aspects, such as artificial breeding (shi et al., 2009b), larval rearing in hatchery condition (al-abdul-elah et al., 2002, 2008; shi et al., 2009a,b), feeding behaviour and growth under tank culture conditions (cruz et al., 2000, 2003; almatar and james, 2007) and health management (azad et al., 2007) to standardize the techniques to get success in its culture. seed transportation is an important issue related to aquaculture, and research (peng et al., 2012a) has also been made to quantify the optimum stocking density to get maximum survivability during the seed transportation of silver pomfret. captive culture and rearing: experiments to formulate the proper methodologies for rearing of silver pomfret larvae and juvenile stages have been initiated long back in the end part of 1960’s. mito and senta (1967) after obtaining fertilized eggs through stripping wild broodstock studied the egg and embryonic development but the hatched out larvae did not survive beyond five days. in further study by oda and namba (1982), larvae survived only for 28 days. the first complete larval developmental stages of laboratory reared silver pomfret were reported by almatar et al. (2000). possibility of producing silver pomfret in hatchery condition first has been reported by al-abdul elah et al. (2002) with hatching rate of 36.5-51.8% in temperature range of 29-30°c and salinity range of 3540‰. they also have reported no significant difference in larval survivability between stocking densities of 20, 30 and 40 larvae/l; though with further increase in stocking density, increased rate of larval mortality has been reported. in 45 days culture period, they have reported achievement of 1.5% larval survivability. their further study (al-abdul elah et al., 2008) has reported an increased larval survivability (3.6%) with supply of nutritionally enriched live feed [rotifers cultured with mixed algae (chlorella, isochrysis and nannochloropsis) plus commercial enrichment media `super selco' and `dha protein selco'] after 38 days of rearing. the high larval survivability has been reported to be due to the presence of more ω-3 highly unsaturated fatty acid (ω-3 hufa) in the rotifers. the presence of microalgae in the larval rearing media has been suggested to enrich the nutritional quality of the rotifers. shi et al. (2009b) have documented 1618.5°c water temperature and 28.0±0.5‰ salinity as conducive for hatching of fertilized eggs following the results of their experiment to breed silver pomfret in captivity. fertilization rate of 18.50-33.50% and hatching rate of 43.83-51.00% have been documented by them in their experiment. shi et al. (2009a) have further tried to standardize the techniques for rearing larvae in captivity. the hatched-out larvae were reared in concrete tanks containing saline water (salinity 25-28 ppt; temperature 19-24°c) and were fed with rotifers (brachionus plicatilis), artemia's nauplii and formulated feed. larval survivability of 5-14% was achieved after the completion of 50 days’ rearing. cruz et al. (2000) first tried to analyze the acceptability of commercial feed for the culture of silver pomfret fingerlings. they used commercially available turbot feed (ecostart 15 by biomar of france; with 10.08% moisture, 51.0% crude protein, 14.46% crude lipids and 8.13% ash content) for the experiment and observed better food conversion ratio (fcr) and growth rate with the use of dry pellets than the moist pellets. they did not achieve optimum growth for silver pomfret fingerlings in the study and recommended for further analysis using other commercially available foods. based on their observation, they suggested that better performance can be obtained if silver pomfret fingerlings are fed with dry pellets and fed based on the natural feeding 238 gupta / reviews on silver pomfret, pampus argenteus rhythm, i.e., providing more feeds towards the end of the day and fed at least five times a day. as the smaller fishes tend to feed on the upper column while bigger fishes prefer to feed below the smaller fish and only the big fishes used to feed at the bottom of the tank; a need to install the feed catching tray has also been suggested by them when the fishes are small in size. further study (cruz et al., 2003) using semi moist salmon feed and re-pelletized salmon feed mixed with shrimp meat, encapsulated larval diets, fish oil, vitamin, and mineral mix provided comparative better result; though no conclusive statement was made by them on the efficiency of these feed components on the growth of silver pomfret. almatar and james (2007) studied the efficiency of using salmon feed mixed with cyclopeeze (commercially available copepods; marketed by argent chemical laboratory, redmond, wa, usa), salmon feed mixed with shrimp meat, salmon feed alone, and shrimp meat alone in the diet and concluded that the inclusion of shrimp meat in the diet either with salmon feed or alone can provide significantly high fcr and weight gain compared to that of feeds without shrimp meat in silver pomfret juveniles. further they compared the efficiency of three commercially available marine fish feed namely gemma (marketed by skretting, fontaine les vervins, france), salmon feed (marketed by dana feed a/s), and pompano feed (marketed by tsai seafoods inc., kaohsiung, taiwan) and showed that feeding with gemma feed (54.0% crude protein and 19.0% crude fat) or salmon feed (41.4% crude protein and 23.9% crude fat) can provide significantly high growth rates compared to that of pompano feed (with 43.0% crude protein and 6.0% crude fat). the results of these investigations although showed high growth rate; they concluded that there is yet further need to formulate a suitable feed with optimum protein and fat content for commercial ventures of this fish species. in this context, the finding of hossain et al. (2011a) can be put up; they have reported dietary lipid level of 16% in a 49% protein diet corresponding to a protein to energy ratio of 22.6 is optimum for better growth, feed utilization and whole-body fatty acid profiles in silver pomfret. they further have reported the higher requirement of dha (hossain et al., 2012a) and increasing dietary supplementation of vitamin e in high lipid diets (hossain et al., 2016) for optimum growth and survival of silver pomfret juveniles. utilization of lipid content in the diet as an energy source in silver pomfret has been reported by peng et al. (2012b). they additionally have reported that significant higher growth can be obtained by supplying a diet mixture of fish meat, artificial feed, agamaki clam and copepods. liu et al. (2014a) have reported for the first time that jellyfish (aurelia aurita and rhopilema esculentum) can be used as prey for artificial rearing of silver pomfret juveniles. later, in their experiment (liu et al., 2014b), they have reported that artificial diet mixed with jelly fish is more appropriate than the pure artificial diet in the rearing of silver pomfret juveniles to achieve good growth as jellyfishes have high amino acid contents. successful reproduction and offspring survival depend on lipid content and fatty acid composition of the brood-stock diets (izquierdo et al., 2001). lund et al. (2008) have reported the importance of poly unsaturated fatty acids (pufas) with 20 or more carbon atoms on fish maturation and steroidogenesis. thus, proper formulation of feed for the brood stock is also essential to get success in captive culture. hossain et al. (2011c) have reported the requirement of higher proportions of pufa, epa and dha in the diet of silver pomfret brood-stock. hossain et al. (2011b) have further reported the importance of higher levels of arginine and leucine, two essential amino acids in the silver pomfret brood stock diet. seed transportation: peng et al. (2012a) have reported that depletion of dissolved oxygen along with increasing ph and ammonia concentration may be the possible reasons behind juvenile mortality at the higher loading densities and temperatures. keeping the temperature of the transport bags’ water at low level may decrease the ammonia excretion rate; thus, may reduce lower ammonia concentration and reduce mortality. they have reported that to achieve 100% survivability of juvenile silver pomfret to be transported in plastic bags for 8-hour transport period, 239 int. j. aquat. biol. (2020) 8(4): 228-245 a loading density of 5 gm/l and temperature range of 15-25°c should be used while for 4-hour transport duration, a loading density of 20 gm/l and temperature of 15-20°c should be used. concluding remarks the present review represents that ample information is available on the food, feeding habit and reproductive biology of silver pomfret; though research on the aquaculture sector of this species has just started its journey. feeding biology: earlier researchers have conducted extensive studies on the food and feeding habit of this fish species and most of them have reported it mainly as a copepod and detritus feeder. the exceptional observations in some cases may be due to difference in availability of food components in the respective habitats or difference in food preference. gut content analysis, study of the types of extra-cellular enzymeproducing bacteria present in the digestive tract, study of the digestive tract histology are some of the modern techniques to conclude firmly on the food and feeding habit of any fish species (gupta, 2016, 2018; gupta and banerjee, 2015, 2016a, b). the earlier researchers have concluded on the feeding habit and food preference of silver pomfret mainly based on gut content analysis. thus, further study should be conducted focusing on the rest two techniques to strengthen the already concluded observations. this will also help to remove the so far existing little controversy regarding the mostly preferred food items of the species. interesting observations on the seasonal and depth wise variation in feeding, change in feeding intensity with increasing temperature and breeding season and ontogenetic shift in food preference have been documented by many researchers which will definitely help in designing appropriate rearing technologies for captive culture of silver pomfret. seasonal and depth wise variation in the diet has been reported to be due to seasonal variation in availability of food items in the particular habitat. ontogenetic shift in food preference has been studied by number of researchers; but there is no such synchronized report available so far on this particular issue. high preference for copepods in juveniles and crustaceans in adults has been reported by pati (1980), kuthalingam (1963) and sivakami et al., (2003); dadzie et al., (2000a) have documented maximum preference for copepods and crustaceans in most of the size groups studied with little shift in preference in the middle size group towards bacillariophyceae; abdurahiman (2006) and thangavelu et al. (2012) have reported high preference of copepod and detritus in all size groups with comparative high preference for copepods and low preference for detritus with increasing size groups. this kind of variation in observation may be due to difference in availability of the food items in the different habitats. age-wise, stage-wise and sex-wise study on feeding habit using enzymatic analysis of the digestive tract can be effective technique to delineate the underlying reasons for this opposing trend. in a nut shell, it can be concluded that overall satisfactory information on the feeding habit and food preference of this fish species is available but further study can be conducted to fill up the information gap which has been pointed out here in this report. reproductive biology: plenty of information is available on the different aspects of reproductive biology of silver pomfret such as sex ratio, length at first maturity, gonadal maturity stages, fecundity, spawning seasonality and spawning habitat preference. female dominance over male has been reported by maximum of the earlier researchers except few contradictory information from oda and namba (1982), dadzie et al. (2000b), sivakami et al. (2003), shi et al. (2006) and ghosh et al. (2009). high metabolic strain of spawning, specific strategy to regulate the population etc. (fagade et al., 1984; lambert and dutil, 2000) may be the reasons behind this contradiction. thus, further studies in this aspect are needed to clarify the confusion. all the earlier documentations have reported early maturation of male than female in this fish species; though the information on length at first maturity in different sexes documented by different researchers is varying. change in food availability, health status, 240 gupta / reviews on silver pomfret, pampus argenteus physico-chemical parameters etc. may be the reasons behind this kind of variations. high fecundity has been reported by all the earlier researchers for this fish species; though the difference in the range of fecundity documented may be due to variation in age, condition, health status, food availability etc. prolonged breeding season for silver pomfret has been reported by all the earlier researchers; though variation has been observed in respect to the number of spawning peaks. most of the researchers have documented two spawning peaks for this fish species except gopalan (1967), lee and jin (1989) and mohamed and ali (1993). the presence of variation in respect to the duration of the breeding season in different countries and number of spawning peaks may be due spatial and temporal fluctuations in climatic and physico-chemical parameters. information availability on the preferred habitat and environmental cues that stimulate breeding initiation of any fish species are also important. silver pomfret has been reported to prefer inshore water for spawning. most of the researchers have documented moderate temperature range and high salinity requirement for the breeding initiation in silver pomfret except dadzie et al. (1998) who have reported low salinity as the stimulant for the initiation of breeding. further study in this aspect can be done to resolve the contradiction. captive culture and rearing: till date, not much research has been performed to assess the suitability of silver pomfret for captive culture. al-abdul elah et al. (2002) first tried the captive culture of silver pomfret followed by shi et al. (2009b). though there was not much difference in results regarding the achieved hatching percentage, shi et al. (2009b) have reported the suitability of comparative low temperature and low salinity to achieve good hatching rate. the reasons behind this difference between the tested parameters are not clear; and thus, further study is required to determine the ambient temperature, salinity and other physico-chemical parameters conducive for hatching in silver pomfret. studies to determine the specific food to enhance the larval survivability in silver pomfret were performed by al-abdul elah et al. (2002) and shi et al. (2009a); the former researchers reported the suitability of nutritionally enriched live feed i.e. rotifer cultured with mixed algae while shi et al. (2009a) documented further addition of artemia nauplii and formulated feed with rotifer to enhance the larval survivability. further cruz et al. (2000) and almatar and james (2007) studied the acceptability and suitability of commercial feed in respect to better food conversion ratio (fcr) and growth rate for the culture of silver pomfret fingerlings. while cruz et al., (2000) suggested the suitability of turbot feed, almatar and james (2007) reported the suitability of shrimp meat alone or with salmon feed to achieve good growth rate. almatar and james (2007) further reported that formulated feed should be with optimum protein and fat content and that observation was further supported by hossain et al. (2011a). hossain et al., (2012a, 2016) further documented that growth rate in silver pomfret juveniles can be enhanced with increasing supplementation of vitamin e in high lipid diet and dha. liu et al. (2014a, b) have reported that jelly fish along with artificial feed can produce good result than pure artificial food in silver pomfret juveniles. considering all the earlier experiments on captive culture and rearing, it can be concluded that though researchers achieved success in enhancing the hatching rate nevertheless they failed to get the ultimate success due to the low survivability of the larvae. even after supplying rotifers, artemia nauplii and formulated feed, they did not manage to achieve more than 15% of larval survivability. the reasons behind low survivability may be due to high stocking density, change in physico-chemical parameters etc. thus, further study is needed to assess the reasons behind low survivability of silver pomfret larvae in captivity. the good acceptability of the commercial fish feed along with their suitability in enhancing growth rate has been reported. high percentage of lipid with increasing amount of vitamin e and protein in diets has been reported to promote growth in silver pomfret juveniles. thus, feed formulated with optimum ratio of protein and lipid with vitamin e 241 int. j. aquat. biol. (2020) 8(4): 228-245 supplement can be supplied in captive culture of silver pomfret to enhance the growth rate so as to achieve the marketable size quickly. on the other hand, brood stock feed should be formulated keeping higher proportions of pufa, epa and dha. finally, it can be concluded that further studies are needed to gather more information to fill up the information gap pointed out in this report; more specifically in the areas like improvement of larval survival and rearing techniques, brood stock management for successful captive spawning, suitable feed developments for different life history stages etc. pilot scale study can be supported to aid in mass production of the fry and commercial culture of the species using tanks, cages and costal ponds etc to support its fishery in long run. references abdurahiman k.p. 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(2022) 10(5): 370-377 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology short communication effect of parasitism on the relative condition factor of astyanax bimaculatus (characiformes: characidae) a freshwater fish from the caatinga domain, brazil dhenes ferreira antunes, bruno anderson fernandes da silva, fabio hideki yamada laboratory of parasitic ecology, department of biological science, regional university of cariri, crato, ceará state, brazil. s article history: received 3 april 2022 accepted 7 august 2022 available online 2 5 october 2022 keywords: freshwater fish neotropical region metazoan parasite upper jaguaribe river welfare abstract: the present study aimed to evaluate the effect of parasitism on the condition factor of astyanax bimaculatus (linnaeus 1758) (characiformes, characidae), in batateiras river, salgado river basin, northeastern brazil. a total of 242 host specimens were collected between august 2018 and february 2020. the host presented a community of metazoan parasites of 14 taxa, totaling 1,750 specimens collected, with a mean total abundance of 7.23 specimens per fish, being the class monogenea, the most predominant taxonomic group. the relative condition factor (kn) differed significantly between parasitized and non-parasitized individuals, in which the parasitized hosts presented higher values of kn. the abundance of the monogeneans characithecium costaricensis and diaphorocleidus sp. showed positive and significant correlations with the kn. considering the sex of the host, males had a higher parasite burden than females, although females present higher values of kn. the parasitic burden of hosts did not show significant differences between seasonal periods. introduction parasites are key organisms of biodiversity and play an important ecological role, whether in population dynamics, species coexistence, or trophic interactions (poulin, 1999; hugot et al., 2001; lefèvre et al., 2009). the parasite-host relationship can affect the entire community through its effects on species distribution and abundance (horwitz and wilcox, 2005). according to bauer (1961), gibbs (1985), and le cren (1951), parasites may have a negative effect on their hosts, which is reflected in a decrease in health conditions, reproductive fitness, and food conversion for use in cyclic activities. astyanax bimaculatus (linnaeus, 1758) (characiformes: characidae), popularly known as “lambari do rabo amarelo” (mirande, 2010; frick et al., 2018). its distribution extends from northeastern brazil and eastern south america to the prata river basin (sterba, 1973; lima et al., 2003). according to cordeiro et al. (2019), this species possesses adaptive plasticity associated with the reproductive mechanisms and strategies developed during its correspondence: fabio hideki yamada doi: https://doi.org/10.22034/ijab.v10i5.1560 e-mail: fhyamada@hotmail.com dor: 20.1001.1.23830956.2022.10.5.3.4 lifetime, allowing survival in the most varied habitats. to date, it has been recorded several parasitic associations to a. bimaculatus in several aquatic ecosystems in brazil: clinostomum complanatum (rudolphi, 1814), procamallanus (spirocamallanus) hillari (pinto & deli, 1976) and polymorphus sp. luhe, 1911 in the guandu river, rio de janeiro state (abdallah et al., 2004); magnivitellinum simplex (kloss, 1966) in the paraná river, paraná state (kohn et al., 2011); prostosthenhystera obesa (diesing, 1850) in the paraná river, são paulo state (kohn et al., 1997); p. (spirocamallanus) inopinatus (artigas & pereira, 1928), p. (s.) hillari, halipegus sp. looss, 1899 in the mogi-guaçu river, são paulo state (kohn and fernandes, 1987); rhabdochona acuminata (molin, 1860) in the lajes reservoir, rio de janeiro state (paraguassú and luque, 2007); lernaea cyprinacea (linnaeus, 1758), weir in antonio prado municipality, rio grande do sul state (gallio et al., 2007); myxobolus sp. in the dantas river, maranhão https://ij-aquaticbiology.com/index.php/ijab/article/view/1560 371 int. j. aquat. biol. (2022) 10(5): 370-377 state (silva et al., 2019); l. cyprinacea, urocleidoides sp. mizelle & price, 1964, rabdochona sp. railliet, 1916, p. (s.) hilarii, p. obesa, dolops sp. audouin, 1837 in the são francisco river, sergipe state (vasconcelos et al., 2013); and quadrigyrus torquatus van cleave 1920 and quadrigyrus nickoli schmidt & hugghins, 1973 in the chumucuí river, pará state (fujimoto et al., 2013). for fish, the relative condition factor (kn) may explain the health and welfare aspects of these organisms (mozsár et al., 2015), food resources (bolger and connoly, 1989), nutritional status and response to environmental factors (brown and murphy, 2004) as well as seasonal changes in environmental conditions (gomiero and braga, 2005). in this context, the present study aimed (1) to characterize the metazoan parasite community of a. bimaculatus from batateiras river, caatinga domain; and (2) to evaluate the effects of the parasitism on its relative condition factor (kn). materials and methods the specimens of a. bimaculatus were collected from august 2018 to february 2020 in batateiras river, salgado river basin, municipality of crato, ceará state (7º13'57.52''s; 39º26'25.46''w) (fig. 1), in two seasonal periods (dry – from august to october and rainy – from november to february). the individuals were measured (standard length (sl) to the nearest 0.1 mm) and weighed (to the nearest 0.1 g) at the laboratory. all the specimens were examined for ectoparasites (eyes, nostrils and gills) and endoparasites (stomach, intestine, liver, gonads, swim bladder and muscles) according to the parasitological methods of collection, fixation, preservation and preparation described by eiras et al. (2006). the parasite identification was performed according to moravec (1998), thatcher (2006) and cohen et al. (2013). the prevalence, mean abundance and mean intensity of the component communities were calculated according to bush et al. (1997). the standard length (ls) and total weight (wt) of each host specimen were fitted in the wt/ls ratio. the values of the regression coefficients a and b were used in the estimates of expected weight values (we), using the equation we = a.lsb. the relative figure 1. sampling area of astyanax bimaculatus collected from august 2018 to february 2020, batateiras river, salgado river basin, municipality of crato, ceará state, brazil. 372 antunes et al./ effect of parasitism on the relative condition factor of astyanax bimaculatus condition factor (kn) was then calculated, which is the quotient between the observed weight and the expected weight for a given length (kn= wt/we) (le cren, 1951). spearman’s rank correlation coefficient (rs) was employed to verify correlations between kn and parasite abundance (zar, 2010). the mann-whitney test (u) was employed to verify the differences between the kn of parasitized and non-parasitized hosts, hosts collected in dry and rainy seasonal periods, and males and females hosts. also, the mann-whitney test (u) was used to verify differences in parasitic burden between males and females (zar, 2010). the chi-square test (x2) was performed to verify differences in the prevalence of parasites between the two seasonal periods (dry and rainy) and the sex of the host. the statistical analyzes were performed using the statistica software package version 7.1 (statsoft inc., 2005) and the statistical significance level adopted was p≤0.05. results and discussion the parasitic community of a. bimaculatus was composed of 14 taxa, totaling 1,750 specimens of the parasite, with a mean total abundance of 7.23 specimens per fish, being the class monogenea, the taxonomic group more representative. of the 242 host specimens examined, 132 (average size of 5.06±0.109) and 110 (average size of 5.98±0.117) were collected in the dry and rainy seasons, respectively. the parasites anachantocotyle anachantocotyle, characithecium costaricensis, characithecium sp.1, characithecium sp.2, diaphorocleidus sp., urocleidoides trinidadensis, p. (spirocamallanus) hilarii and wallinia caririensis were present in both seasonal periods. while the ascocotyle sp., henneguya sp., quadrigyrus sp., dactylogyridae gen. sp., diplostomidae gen. sp. and spiroxys sp. were present in only one season. characithecium costaricensis, characithecium sp. 1, diaphorocleidus sp., u. trinidadensis, wallinia caririensisand p. (s.) hilarii presented a prevalence higher than 10% (table 1). parasitized individuals of a. bimaculatus showed kn higher (0.89±0.03) than non-parasitized (0.65±0.02) (z(u) = 4.7972; p<0.0001). the parasitic prevalence of characithecium sp.1, diaphorocleidus sp., a. anachantocotyle and w. caririensis were more prevalent in the rainy season. the host specimens presented an average kn of 0.81±0.03 and 0.85±0.04 in the dry and rainy seasons, respectively; however, not significant (z(u) = 1.5593; p<0.1189). the monogeneans c. costaricensis and diaphorocleidus sp. showed positive and significant correlations between its abundance and the kn of the analyzed hosts in both seasonal periods (table 2). of the 242 fish examined, 80 were females (average size of 6.26±0.195), 157 were males (average size of 5.90±0.06), and five undefined sex. the female specimens presented an average kn of 1.00±0.05, significantly higher than the male (0.75±0.03) (z(u) =4.9088; p<0.0001). the male hosts showed a significantly higher parasitic burden (978 specimens) than females (772 specimens) (z(u) 1.9485; p<0.0514). the parasitic prevalence of c. costaricensis, characithecium sp.1, and u. trinidadensis were more prevalent in the female host. the parasitic fauna of a. bimaculatus showed new occurrences and was predominantly by gill ectoparasite of class monogenea. the second representative group was the digenetic trematodes recovered from the intestine, gills and eyes. the metacercariae of ascocotyle sp. recovered from the gills of a. bimaculatus in batateiras river, have already been described to satanoperca pappaterra (heckel, 1840) (cichlidae) and crenicichla niederleinii (holmberg, 1891) (cichlidae) in the paraná river basin (yamada et al., 2008). furthermore, the parasites procamallanus (s.) hilarii and diplostomidae gen. sp. have already been registered for this host in other brazilian ecosystems (kohn and fernandes, 1987; abdallah et al., 2004, vasconcelos et al., 2013). in the present study, the relative condition factor (kn) of the parasitized hosts has been shown greater 373 int. j. aquat. biol. (2022) 10(5): 370-377 than non-parasitized. this finding corroborates with lizama et al. (2006) that found kn significantly higher in parasitized fish than in non-parasitized. although parasitism negatively affects the condition of the hosts (bauer, 1970), the fish parasitized by the monogeneans c. costaricensis and diaphorocleidus sp. showed positive and significant correlations between host kn and their abundance in both seasonal periods. in this context, moore (1987) pointed out that larger fish with high kn can be able to harbor larger numbers of parasites and can tolerate greater intensities of infestations. considering the seasonal periods, characithecium sp.1, diaphorocleidus sp., w. caririensis and a. anachantocotyle were more dominant in the rainy period. several studies indicate that limnological factors could influence the dynamic of host-parasite interactions (barker and coneb, 2000; lizama et al., 2006). the study area exhibits an intermittent regime (rosa et al., 2004). therefore, the kn of a. bimaculatus showed no significant differences during the rainy and drought seasons. this plasticity indicates that a. bimaculatus is probably a resilient species and well-adapted to this aquatic ecosystem. considering the sex of the host, males presented a higher parasitic burden than females. however, female specimens presented an average kn significantly higher than males. the prevalence of three monogeneans species (c. costaricensis, characithecium sp.1 and u. trinidadensis) was more prevalent in females, probably due to higher body mass and size, consequently, higher kn than males. the distinction of parasitism about the sex of the parasite species dry rainy p(%) ma±se mi±se p(%) ma±se mi±se phylum myxozoa class myxosporea henneguya sp. – – – 1.81 0.1727±0.1408 9.5±5.500 phylum platyhelminthes class monogenea anachantocotyle anachantocotyle 4.54 0.09±0.046 2.0±0.683 13.63 0.29±1.007 2.1±0.496 characithecium costaricensis 34.84 1.189 ±0.238 3.4±0.553 26.36 0.627±0.163 2.3±0.494 characithecium sp.1 34.84 1.795±0.326 5.1±0.712 15.45 0.436±0.157 2.8±0.819 characithecium sp.2 6.06 0.09±0.033 1.5±0.189 10.90 0.136±0.417 1.2±0.13 dactylogyridae gen. sp. – – – 0.90 0.009±0.009 1.0±0.000 diaphorocleidus sp. 21.21 0.931±2.526 4.3±0.737 54.54 2.918±0.477 5.3±0.743 urocleidoides trinidadensis 23.48 0.651±0.140 2.7±0.411 33.63 0.636±1.254 1.8±0.25 class trematoda diplostomidae gen. sp. 1.51 0.045±0.033 3.0±1.00 – – – ascocotyle sp. (metacercariae) 4.54 0.09±0.042 2.0±0.516 – – – wallinia caririensis 13.63 0.924±0.370 6.7±2.324 28.18 3.209±0.803 11.3±2.285 phylum nematoda class chromadorea procamallanus (spirocamallanus) hilarii 10.60 0.143±0.04 1.3±0.169 13.63 0.272±0.078 2.0±0.323 class secernentea spiroxys sp. (larvae) 2.27 0.03±0.018 1.3±0.333 – – – phylum acanthocephala class eoacanthocephala quadrigyrus sp. – – – 1.81 0.018±0.012 1.0±0.000 table 1. ecological descriptors of the parasitic community of astyanax bimaculatus, batateiras river, salgado river basin, municipality of crato, ceará state, brazil, in the dry and rainy seasonal periods. ma = mean abundance; mi = mean intensity; p(%) = prevalence and se = standard error. 374 antunes et al./ effect of parasitism on the relative condition factor of astyanax bimaculatus hosts may be linked to energy needs, different feeding habits or physiological differences of the individual (gonzález and acuña, 2000). for instance, males of cichla monoculus agassiz, 1831 (cichlidae) from the upper paraná river floodplain presented intensities of infestation of cestodes significantly higher than the females (machado et al., 2000). males of astyanax altiparanae garutti & britski, 2000 (characidae) from the chavantes reservoir showed a higher parasitic burden than females (zica, 2008). on the other hand, females of salminus brasiliensis (cuvier, 1816) (bryconidae) from the upper paraná river floodplain presented high levels of infestation by the acanthocephalans and nematodes (karling et al., 2013). in summary, the parasite communities of a. bimaculatus were characterized by high ectoparasite species richness, with monogeneans being numerically dominant; parasitized hosts showed the kn higher than non-parasitized. females could withstand a higher parasitic burden than males, and differences in kn between seasonal periods. in general, it is expected that parasites cause deleterious effects on their host; therefore, it is difficult to define and measure those effects. this corroborates the finding of kennedy (2009) and price (1980) in which that there is no pattern or order in space or time in fish parasitological studies and that most of the populations of fish parasites live in conditions of imbalance, dominated by stochastic events, respectively. acknowledgments this work was supported by the fundação cearense de apoio ao desenvolvimento científico e tecnológico (funcap) under the grant # bp3parasite species dry rainy rs p rs p phylum myxozoa classe myxosporea henneguya sp. – – 0.033 0.7280 phylum platyhelminthes class monogenea anachantocotyle anachantocotyle -0.026 0.7674 -0.140 0.1429 characithecium costaricensis 0.466 0.0001 0.266 0.0049 characithecium sp.1 0.131 0.1344 0.148 0.1227 characithecium sp.2 0.241 0.0053 0.008 0.9282 dactylogyridae gen. sp. 0.164 0.086 diaphorocleidus sp. 0.396 0.0001 0.305 0.0011 urocleidoides trinidadensis 0.266 0.002 0.163 0.0886 class trematoda diplostomidae gen. sp. 0.124 0.1559 – – ascocotyle sp. (metacercariae) -0.109 0.1095 – – wallinia caririensis 0.473 0.0001 0.133 0.1630 phylum nematoda class chromadorea procamallanus (spirocamallanus) hilarii -0.025 0.7727 0.338 0.0003 class secernentea spiroxys sp. (larvae) 0.106 0.2257 – – phylum acanthocephala class eoacanthocephala quadrigyrus sp. – – 0.220 0.0204 table 2. spearman’s rank correlation coefficient (rs) between the relative condition factor (kn) and the parasitic abundance of astyanax bimaculatus, batateiras river, salgado river basin, municipality of crato, ceará state, brazil, in the dry and rainy seasonal periods. 375 int. j. aquat. biol. (2022) 10(5): 370-377 0139-00039.01.06/19 (d.fa.) and bp3-0139 00039.01.00/18 (f.h.y.); and conselho nacional de desenvolvimento científico e tecnológico (cnpq) under the grant # 143996/2019-3 (b.a.f.s). references abdallah v.d., azevedo r.k., luque j.l. 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(2023) 11(1): 50-58 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2023 iranian society of ichthyology original article daily vertical migrations of lake baikal amphipods: major players, seasonal dynamics and potential causes dmitry yurevich karnaukhov1, ekaterina mikhailovna dolinskaya1, sofya aleksandrovna biritskaya1, maria aleksandrovna teplykh1, yana konstantinovna ermolaeva1, viktoria aleksandrovna pushnica1, lidia borisovna bukhaeva1, ilya andreevich makhov2, arina vitalievna lavnikova1, eugene anatolievich silow1 1irkutsk state university, irkutsk, lenin str., 3, 664025, russian federation. 2zoological institute, russian academy of sciences, st. petersburg, universitetskaya emb., 1, 199034, russian federation. article history: received 3 may 2022 accepted 11 january 2023 available online 2 5 february 2023 keywords: amphipods nocturnal vertical migrations lake baikal light pollution stratification abstract: daily vertical migrations of hydrobionts are widespread in all water bodies, such as small wells and vast oceans. this phenomenon is most common among crustaceans, especially in amphipods. lake baikal is one of the most biodiverse freshwater centers containing 354 species and subspecies of amphipods, and almost all of them are endemics. the study’s goal was to follow the year-round dynamics of daily vertical migrations of amphipods and pinpoint various environmental factors that affect this process. observations of amphipod migration were obtained using a remote video system. based on the results, the vertical migratory activity of amphipods can be tracked during the year. the key factor that reduces migratory activity was found to be the start of the reverse stratification period. it is assumed that lake baikal amphipods have no single general reason for vertical migrations, and a few of the driving forces that make different amphipods groups migrate are accumulations of regions positive temperatures that facilitate sex development, food foraging, passive dispersal, search for a partner, and pairing. introduction unique characteristics of large water ecosystems include the wide range of hydrobionts and the mutual living patterns of organisms, and interaction with various environmental factors. one such interaction is the daily vertical migration (dvm) of hydrobionts. dvm is the periodical migration of bottom and pelagic organisms toward the upper layers during the night. observations of dvms have been conducted for many years, and the content and structure of the migratory community in small water bodies (blinn et al., 1988; henri, 2018) to large lake ecosystems, seas, and oceans have been studied (greze, 1965; nishihama and hirakawa, 1998; hays et al., 2001; iguchi and ikeda, 2004; krapp et al., 2008; pacheco et al., 2014; last et al., 2016; vereshchaka and anokhina, 2017). the main group that performs dvm is crustaceans, consisting of amphipods (drolet and barbeau, 2009), isopods correspondence: dmitry yurevich karnaukhov doi: https://doi.org/10.22034/ijab.v11i1.1587 e-mail: karnauhovdmitrii@gmail.com dor: https://dorl.net/dor/20.1001.1.23830956.2023.11.1.7.7 (macquart-moulin, 1992), antarctic krill (okkonen et al., 2020), mysis (vereshchaka and anokhina, 2017), water fleas (griffin et al., 2020), ostracods (pacheco et al., 2014) and copepods (takahashi et al., 2009). the usual reason for such behavior in pelagic organisms is the protection–food factor because it is thought that migration towards upper water layers during the night for forage helps avoid predators that are less active during this time (kozhova, 1987). dvms are widely studied in the pacific ocean region, such as the reaction of the pelagic amphipod, phronima sedentaria (forskal, 1775) to temperature and the possible effect of temperature stress on the vertical migrations process (elder and seibel, 2015). another example is the study of the pacific ocean’s role in the global carbon cycle, which is based on the carbon transfer by plankton crustaceans, metridia pacifica brodsky, 1950 and m. okhotensis brodsky, 51 int. j. aquat. biol. (2023) 11(1): 50-58 1950 via vertical migrations (takahashi et al., 2009). a study of differences between dayand night-time zooplankton content was done in chilean patagonia that showed that plankton biomass and its migration depend on water body stratification (diaz-astudillo et al., 2017). also, hydrobiont-based dependence of dvm on ice coverage, moonlight, wind intensity, and eutrophication levels were studied in various water bodies (falk-petersen et al., 2008; krapp et al., 2008; pacheco et al., 2014; last et al., 2016; okkonen et al., 2020). lake baikal is an ancient freshwater body with a large number of endemic species. one of the most species-rich hydrobionts group is amphipods. 354 species and subspecies of amphipods are found in this lake (takhteev et al., 2015). lake baikal amphipods occupy many ecological niches, including a pelagic zone inhabited by the only described freshwater pelagic amphipod, macrohectopus branickii (dybowsky, 1874) (rusinek et al., 2012). both pelagic and benthic amphipods are active participants in dvms. studies of dvms in lake baikal have been conducted for more than 20 years; however, most observations were done once in various parts of the lake. despite this fact, this study revealed several important ecological aspects of the group, for example, the gathering of semi-pelagic amphipods near underwater slopes during the winter months (karnaukhov et al., 2016b) and different reactions of the pelagic species to artificial lighting (karnaukhov et al., 2019). in addition, the absence of amphipods in the littoral zone of the lake in the daytime has been repeatedly confirmed (karnaukhov et al., 2016a; takhteev et al., 2019). considering the importance of m. branickii in the pelagic community and the function and role of benthic amphipods in maintaining the equilibrium of the littoral community, it is crucial to conduct more rigorous studies of the dvms by lake baikal amphipods (karnaukhov et al., 2019). therefore, the goal of this study was to track the year-round dynamics of dvms and reveal various environmental factors affecting the migratory activities of amphipods. material and methods observation sites: the first video recording site of the lake baikal amphipod migratory complex was located in bolshie koty bay (table 1). the site is the gentle littoral platform in the deep part of the bay. video recordings were conducted every two hours during one full night in the migratory season. also, additional video recordings were obtained in different months. the second video recording site was located in listvenichny bay near the angara river’s source, which is why the site is not covered by ice during the winter. therefore, video recordings were done once every month during the year. sampling and processing of the material: the video recording system contained a metal frame, a gopro hero 4 action camera, and one light source, and a thermo-logger ibutton was put on the substrate. the video was then recorded for 15 min. during the video processing, done manually every 5 sec, the video was stopped, and the number of migratory organisms was counted. thus, the measurement unit was the number of specimens per freeze-frame (karnaukhov et al., 2016a; takhteev et al., 2019). also, catching migratory organisms with a juday plankton net (0.25 m diameter inlet port) was conducted along with video recordings. sample processing was done in the laboratory according to standard hydrobiological methods (salazkin, 1984). organism counting was done in bogorov counting tray with a stereomicroscope (мбс-10). for each migratory organism, the results were logged in a № site coordinates substrate depth ice coverage 1 bolshie koty bay 51°54'09.9"n 105°04'11.6"e small or and medium size pebbles and boulders 3 (3,5) m january – april 2 listvenichniy bay 51°52'05.0"n 104°49'47.0"e small or and medium size pebbles and boulders 2 (2,5) m not present year-round table 1. video recording sites. 52 karnaukhov et al./ daily vertical migrations of lake baikal amphipods separate file. later, the calculations of hydrobionts per m2 and m3 were conducted. statistical analyses: statistical analyses of the video recording results and samples were performed using past.3x software. the kruskal-wallis test and mannwhitney tests with bonferroni correction were used. results analysis of the video recording results in bolshie koty and listvenichny bays showed the presence of cottoid fish and amphipods, including pelagic amphipod m. branickii, in the migratory community (tables 2, 3). in most cases, the mean number of migrating benthic amphipods was more dominant than the mean number of fish and pelagic m. branickii. the maximum mean number of amphipods detected in video recording of the bolshie koty bay was 21.16±0.657 specimens per freeze-frame (night-time on august 16-17, 2018 at 00:00). however, earlier, we had witnessed 80 and sometimes more during the summer and fall (karnaukhov et al., 2016b). the maximum mean number of amphipods in listvenichny bay during the year was 6.967±0.286 specimens per freezeframe (december 20, 2017). the dynamics of amphipod abundance during different seasons in the video recordings of bolshie koty bay are shown in figure 1. high intensity of migratory activity of amphipods was detected in november 2017 and august 2018 (60 specimens per freeze-frame). at the same time, the main peak time of migratory activity cannot be established and is most likely absent in our case. the maximum numbers were detected during 22:00 and 4:00. migratory activity may be uniform during the night and fluctuations in abundance are caused by biotic (approaching young fish from coregoninae and cottoidae families), abiotic (temperature fluctuations, underwater streams, wave activity), and anthropogenic factors (shipping and, as a consequence, artificial lighting). based on the additional video recordings, an increase in the intensity of migratory activity of amphipods in bolshie koty bay was detected from june to december 2018 and reached 30-46 specimens per freeze frame (irrespective of the month). data for listvenichny bay were organized similarly to bolshie koty, for example, according to the seasons (fig. 2). increased migratory activity was also detected during the winter. date / time amphipoda fish macrohectopus branickii 20-21.11.2017 22:00 11.53±0.505 0.392±0.46 0.05±0.016 00:00 5.139±0.201 0.060±0.018 0.077±0.02 02:00 3.381±0.165 0.436±0.048 0.022±0.01 04:00 4.122±0.174 0.348±0.046 0.039±0.014 06:00 0.116±0.026 0.403±0.039 0.028±0.012 23-24.02.2018 22:00 1.502±0.14 0 0 00:00 0.116±0.025 0.696±0.034 0 02:00 0.547±0.054 0 0.00415±0.00413 04:00 0.564±0.072 0 0.025±0.01 06:00 0.122±0.026 0.166±0.009 0 27.05.2018 02:00 0.768±0.062 0.06±0.017 0.033±0.014 04:00 0.039±0.015 0.193±0.03 0 16-17.08.2018 00:00 21.16±0.657 5.74±0.355 0 02:00 14.409±0.489 7.475±0.482 0 04:00 5.182±0.207 15.365±0.953 0.028±0.012 06:00 24-25.11.2018 22:00 0.287±0.04 0.011±0.008 0 00:00 0.541±0.056 0 0 02:00 04:00 06:00 table 2. mean number of migratory community specimens, bolshie koty bay (2017-2018). 53 int. j. aquat. biol. (2023) 11(1): 50-58 the results also showed significant differences between months and seasons, and the mann-whitney tests with bonferroni correction identified strongly expressed differences between months and seasons (fig. 3). samples showed the presence of several groups of hydrobionts, including calanoida, cyclopoida, harpacticoida, and amphipoda (tables 4, 5). the dominant group of copepoda collected in bolshie koty bay was harpacticoida. its abundance was 2870 specimens per m2 of the bottom. the subdominant group was calanoida (251 specimens per m2 of the bottom). the mean abundance of cyclopoida was 125 specimens per m2. the dominant group in listvenichny bay was calanoida, with mean abundance during the whole period of sampling with 1758 specimens per m2. the subdominant group was harpacticoida (947 specimens per m2) and specimens of cyclopoida had the lowest abundance (71 specimens per m2). among the collected amphipods, several taxa were identified, viz. echiuropus levis bazikalova, 1945, e. macronychus sempercarinatus date / time amphipoda fish macrohectopus branickii 13.11.17 21:00 2.883±0.127 0.15±0.028 0.906±0.028 20.12.17 21:00 6.967±0.286 0 0 19.01.18 21:00 0.672±0.06 0 0 14.02.18 21:00 18.03.18 21:00 0.033±0.013 0 0 20.04.18 21:00 0 0.044±0.015 0 20.05.18 22:00 0.111±0.027 0.094±0.022 0 19.06.18 23:00 0.917±0.077 0.011±0.008 0 18.07.18 23:00 0.834±0.086 0 0 21.08.18 23:00 1.37±0.086 0.486±0.04 0.011±0.008 20.09.18 23:00 0.105±0.023 0.796±0.03 0.006±0.005 21.10.18 22:00 0.718±0.69 0 0.017±0.009 22.11.18 21:00 0.144±0.026 0.022±0.011 0.094±0.022 table 3. mean number of migratory community specimens, listvenichny bay (2017–2018). figure 1. abundance of amphipods at the observation site in bolshie koty bay during the night across seasons. 54 karnaukhov et al./ daily vertical migrations of lake baikal amphipods (bazikalova, 1975), gmelinoides fasciatus (stebbing, 1899), micruropus wohlii wohlii (dybowsky, 1874), micruopus sp., eulimnogammarus cyaneus (dybowsky, 1874), e.verrucosus (gerstfeldt, 1858), eulimnogammarus sp., pallasea sp., and brandtia sp. these species were found in samples at both sites during the entire observation period. however, only g. fasciatus, date / time amphipoda calanoida cyclopoida harpacticoida м2 м3 м2 м3 м2 м3 м2 м3 23-24.02.2018 22:00 73.3 24.4 0 0 200 66.6 3166.6 1055.5 00:00 66.6 22.2 373.3 124.4 66.6 22.2 293.3 97.7 02:00 120 40 0 0 100 33.3 513.3 171.1 04:00 180 60 0 0 0 0 806.6 268.8 06:00 40 13.3 6.6 2.2 0 0 133.3 44.4 27.05.2018 02:00 13.3 4.4 360 120 200 66.6 520 173.3 04:00 0 0 240 80 180 60 160 53.3 16-17.08.2018 00:00 60 20 20 6.6 93.3 21.1 7506.6 2502.2 02:00 80 26.6 33.3 11.1 80 26.6 10593.3 3531.1 04:00 86.6 28.8 1720 573.3 286.6 95.5 4520 1506.6 06:00 26.6 8.8 460 153.3 153.3 51.1 4826.6 1608.8 12.08.2018 00:00 320 106.6 326.6 108.8 506.6 168.8 12406.6 4135.5 24-25.11.2018 22:00 0 0 186.6 62.2 33.3 11.1 126.6 42.2 00:00 13.3 3.8 166.6 47.62 40 11.43 193.3 55.24 02:00 0 0 33.3 11.1 13.3 4.4 60 20 04:00 0 0 93.3 31.1 46.6 15.5 93.3 31.1 06:00 0 0 0 0 0 0 0 0 table 4. abundance of species from migratory communities obtained via the plankton net in bolshie koty bay (per m2 and m3). figure 3. statistical significance (p) in pair comparisons calculated using mann–whitney tests with the bonferroni correction. figure 2. abundance of amphipods at the observation site in listvenichny bay grouped according seasons: a – winter; b – spring; c – summer; d – autumn. 55 int. j. aquat. biol. (2023) 11(1): 50-58 m. wohlii wohlii, and micruropus sp. were found in bolshie koty bay during the winter (ice-covered period), while during the same time frame, e. cyaneus and eulimnogammarus sp. were found at listvenichny bay. discussion daily vertical migrations of lake baikal hydrobionts are complex phenomena, and the dominant part consists of amphipods’ migrations. the signal factor in starting migration is most likely the level of illumination (ringelberg, 1995). it is worth noting that the lighting sources used during experiment affect the migratory community in proportion to its increase compared to shooting without lighting. similar video surveillance without lighting has been carried out repeatedly (takhteev and didorenko, 2015). for example, the level of sensitivity to the light for the lake baikal amphipods is 0.0001 lux (bessolitsyna, 2002; rudstam et al., 1992; volkova, 1984). based on our results, the migration does not stop during the year, but their intensity varies. the observations during the different seasons in listvenichny bay vary significantly. however, since there is no ice coverage on this site year-round, it made sense to compare migratory activity during the ice-covered period and period without ice in bolshie koty bay during the same months as in listvenichny bay. such a comparison showed that migratory activity in listvenichny bay did not differ during the ice-free and -cover periods in the rest of the lake. meanwhile, during the ice-free period in listvenichny bay, migratory activity did not differ from during the ice-covered period in bolshie koty bay. at the same time, a comparison of migratory activity during the seasons in bolshie koty bay revealed no significant differences for such activity between february and may (lake baikal completely becomes ice-free in may and starts spring mixing the water temperature is relatively low). our observations showed that migratory activity was low during the february to march period and high during june to december. based on these facts, we concluded that the main limiting factor for migratory activity is the start of the reverse stratification period. this corresponded to the previous temperature hypothesis of lake baikal amphipods dvms, which states that crustaceans accumulate in the upper water layers in higher temperature regions to speed up their sexual maturation. for example, migrating specimens of the genus eulimnogammarus were only observed in immature individuals. they feed mainly on the food located at the bottom (morino et al., 2000). however, the dvms of the lake baikal amphipods cannot be explained by only the temperature hypothesis because we can witness migratory activity even during the ice-covered period, even if this event is not intense. based on the results, part of the amphipods in the date / time amphipoda calanoida cyclopoida harpacticoida м2 м3 м2 м3 м2 м3 м2 м3 13.11.17 21:00 1100 550 440 220 0 0 4120 2060 20.12.17 21:00 160 80 400 200 0 0 3760 1880 19.01.18 21:00 20 10 560 280 0 0 240 120 14.02.18 21:00 0 0 106.6 53.3 140 70 93.3 46.6 18.03.18 21:00 6.6 3.3 13.3 6.6 6.6 3.3 106.6 53.3 20.04.18 21:00 0 0 0 0 6.6 3.3 353.3 176.6 20.05.18 22:00 6.6 3.3 66.6 33.3 26.6 13.3 26.6 13.3 19.06.18 23:00 40 20 1466.6 733.3 53.3 26.6 46.6 23.3 18.07.18 23:00 20 10 13.3 6.6 0 0 40 20 21.08.18 23:00 240 120 0 0 240 120 720 360 20.09.18 23:00 0 0 12340 4936 0 0 1880 752 21.10.18 22:00 180 72 4400 1760 0 0 500 200 22.11.18 21:00 30 12 3050 1220 450 180 420 168 table 5. abundance of species from migratory communities obtained with plankton net in listvenichny bay (per m2 and m3). 56 karnaukhov et al./ daily vertical migrations of lake baikal amphipods plankton net samples were g. fasciatus and specimens of the genus micruropus. mating in the water column was already mentioned for some nonlake baikal amphipods, for example, pontoporeia affinis lindström, 1855 and p. rosttrata (marzolf, 1965; yu et al., 1998). besides, g. fasciatus can hunt plankton crustaceans (bekman, 1962). at both sites, besides amphipods, calanoida (represented in our study by one species, e. baikalensis sars, 1900), cyclopoida (cyclops kolensis lilljeborg, 1901), and harpacticoida (a number of species) were collected. the dominance of e. baikalensis over c. kolensis is due to the natural fluctuations of these species in lake baikal, while the dominance of harpacticoida in bolshie koty bay was due to the local distribution. in contrast to the first two taxa, harpacticoida in lake baikal are benthic organisms but actively engaged in daily vertical migrations (evstigneeva et al., 1991). the subspecies of m. wohlii wohlii and m. wohlii platycercus (dybowsky, 1874) are semi-pelagic amphipods (takhteev and didorenko, 2015), often gatherings at night far away from the shore. they were passively transferred by streams (also, the descent to the bottom can be passive without energy costs), including underwater ones and thus can move great distances. similar migration into pelagic zones can be found among other crustaceans, for example, copepods, ostracods and the amphipod hornellia (metaceradocus) occidentalis jl barnard, 1959 (aldredge and king, 1985). in addition, according to some information, hatching m. wohlii wohlii and m. wohlii platycercus occur in pelagic zones, which excludes the mass mortality due to predators (nikolaeva, 1967) and helps juvenile dispersal. the pelagic amphipod m. branickii regularly migrates from a depth of 200-700 m to follow its food source, e. baikalensis. however, often individuals of this species can be seen near shore (karnaukhov et al., 2016a). one reason for this is thought to be upwelling (didorenko et al., 2020). however, it does not completely explain the migration of m. branickii to the shore. macrohectopus branickii migrating to the shore becomes the part of the complex that we can observe in the lake littoral in which both exclusively benthic organisms (benthic amphipods, harpacticoida, and cottoidei fish) and exclusively pelagic ones (pelagic amphipod m. branickii, e. baikalensis, c. kolensis, and coregonidae fish) can be concurrently found. such conditions facilitate matter and energy exchange between two separate, disconnected lake systems with benthic and pelagic zones. in addition, one more factor that must be considered affecting the dvm of lake baikal amphipods is light pollution. benthic amphipods are actively attracted to the artificial light sources on the lake shore. these light sources might be visible to potential predators, leading to the depletion of the quality and quantity at some sites (gliwicz, 1986; karnaukhov et al., 2021). besides, artificial light with different wavelengths might also affect pelagic organisms, thus attracting them to the specific lake baikal shore locations (karnaukhov et al., 2019). conclusion vertical migratory activity of hydrobionts, in particular lake baikal amphipods, creates specific conditions for matter and energy exchange between normally disconnected zones of the lake: benthic and pelagic. the exchange did not stop during the year; however, establishing the reverse stratification period caused a significant decrease in the intensity of the vertical migrations. an explanation of migratory activity of all lake baikal amphipod diversity with one general cause is not reasonable. different groups of amphipods have different reasons, such as the search for a mating partner and mating itself, accumulation of positive temperature regions that speed up sexual maturation, search for food, and passive distribution with the help of underwater streams. the general migrants are specimens of the echiuropus, gmelinoides (one species g. fasciatus), micruropus, eulimnogammarus, pallasea, and brandtia genera. however, migratory activity is an inherent property of specimens from other lake baikal amphipod genera. it should be noted that migration to the 57 int. j. aquat. biol. 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(2018) 6(2): 104-113 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article the effects of different grain sources on gut evacuation rate and nutrient digestibility in common carp, cyprinus carpio zahra mazahery tehrany, abdolsamad keramat amirkolaie*,1hossein ouraji department of fisheries, faculty of animal sciences and fisheries, sari agricultural and natural resources university, sari, iran. article history: received 7 january 2018 accepted 25 april 2018 available online 2 5 april 2018 keywords: intestine carbohydrate degradation nutrient emptying time abstract: the main objective of this study was to investigate whether dietary cereal grains of different carbohydrate sources can change nutrient digestibility, evacuation rate and the number of bacterial colony in gut. common carp with an average weight of 244.7±6.3 g were divided randomly into sixteen 500-l tanks with a stocking density of 18 fish per tank. four experimental diets were formulated by inclusion of four cereal grains (wheat meal, barley meal, corn meal and rice meal) in a basal diet in a ratio of 40%. the four experimental treatments with four replicates were assigned in 16 tanks. inclusion of different types of cereal grain affected growth related parameters in c. carpio. corn and wheat diets led to larger weight gains and better feed conversion ratios compared to barley diet (324 and 321 versus 305 g for final weight; 1.93 and 1.90 versus 2.25 for fed conversion ratio). protein and dry matter digestibility in the common carp fed rice diet were lower in comparison to other cereal grains (73 and 58 versus 79-82 and 67-70%). the maximum and minimum bacterial colony numbers (133 and 63 cfu.gr-1×10-7) were observed in fish fed wheat and corn diets, respectively. evacuation time showed a delay by feeding on barley diet and almost all dry matter left in part i of the intestine after 30 min (first sampling), but this rate was recorded 70% for corn diet .in conclusion, although dietary grains change evacuation time and bacterial colonial number in common carp, this condition does not have a great impact on nutrient digestibility. introduction common carp, cyprinus carpio, as an omnivorous species generally feeds on pond bottom. however, in intensive systems, most fish nutrients requirement are met by component feed and fast growth in aquaculture production systems are mostly dependent on pellet diet (olsen and hasan, 2012). a review on the global use of commercial diets revealed that the ratio of commercial feed used for carps reached 50% in 2010, while it was 20% in 1995 (tacon et al., 2011). grains and their byproducts are the most important carbohydrate sources in fish feed and these ingredients can reach 50% of feed content in omnivorous fish. an increase in carbohydrate content is always associated with an integrated part of cell wall mainly composed of hexoses and pentoses, which is called non-starch polysaccharide (nsp) (van barneveld, 1999). these component generally cannot be digested because of *corresponding author: abdolsamad keramat amirkolaie doi: https://doi.org/10.22034/ijab.v6i2.429 e-mail address: amirkola@yahoo.com lack and/or shortage of enzyme secretion by monogastric animals, including fish (kuzmina et al., 1996; ravindra et al., 1999). increasing nsp has a negative effect on body weight and feed conversion ratio (smeets et al., 2018). in addition, nsp in diet can influence fish gastro-intestinal anatomy and its development (leenhouwers et al., 2006), gastric evacuation rate (lee et al., 1992; storebakken et al., 1999), microbial activity (amirkolaie et al., 2006; leenhouwers et al., 2007a, b), nutrient digestibility (refstie et al., 1999; amirkolaie et al., 2006; leenhouwers et al., 2007b), digesta viscosity (storebakken, 1985; refstie et al., 1999; leenhouwers et al., 2006, 2007a) and also viscosity and moisture content of the faeces (tran-tu et al., 2017). these impacts, however, can vary depending on the physicochemical properties of nsp (sinha et al., 2011). 105 int. j. aquat. biol. (2018) 6(2): 104-113 increasing nsp content in fish feed can depress digestive function in the gastro-intestinal tract (storebakken, 1985; refstie et al., 1999). because of enzyme limitation, a big part of nsps remains indigested in the gut and cannot be used as a source of energy for fish (sinha et al., 2011), and so many nutrients are wasted as feces (tran-tu et al., 2017). thereby a lower growth performance is expected with high-nsp diets. the binding of nsps to intestinal brush border membrane (de lange, 2000) increases intestinal secretion of water, nutrient and electrolyte (choct, 1997). feed ingredients with low digestibility may potentially influence the gastrointestinal emptying by increasing the volume of digesta (storebakken et al., 1999). gut evacuation rate can be also affected by type of nsp. an increased viscosity induced by soluble dietary nsp reduced digesta passage rate (johansen et al., 1996). the delay of digesta evacuation rate induced by viscose ingredient may also stimulate microbial degradation leading to volatile fatty acid production as end product (amirkolaie et al., 2006; leenhouwers et al., 2007a, b; sinha et al., 2011). these products may lower the ph of intestine and through that changes normal microbial community in the intestine (sinha et al., 2011). an increase in bacterial activity was also observed in mono-gastric animals fed diet contained soluble nsp (salanitro et al., 2001). due to limited availability of fishmeal and fish oil, plant ingredients of aqua-feed have been increasing. nsps constitute an important part of cereal grains (sinha et al., 2011) which are commonly used as feed ingredients in omnivorous fish (leenhouwers et al., 2007b). while some literature is available on the effects of carbohydrate sources, e.g. cereal grains on nutrient digestibility and digesta viscosity in african catfish (leenhouwers et al., 2006, 2007a), nile tilapia (leenhouwers et al., 2007b) and atlantic salmon (refstie et al., 1999), there is little information related to the impacts of cereal grains on common carp. therefore, the main goal of the current study was to investigate whether or not dietary cereal grains with different nsp sources can change nutrient digestibility, evacuation rate and the number of bacterial colony in common carp. materials and methods experimental diets, animal, and system: common carp fingerlings were bred at the reproduction facility of a nearby farm and adopted to the experimental conditions for seven days before the start of the experiment. afterward, the juveniles with an average weight of 224.7±8.3 g were randomly divided into sixteen 500-l tanks with a stocking density of 18 fish per tank. a basal diet was formulated using locally grown feed ingredients (behdaneh, kaboli factory, mazandaran) (table 1). four cereal grains, including wheat, barley, corn and rice were used on the basis of their nsp contents to have different types of nsp in the experimental diets. corn contains very low soluble nsps while barley has a large amount of soluble nsps (sinha et al., 2011). the other two grains contain moderate levels of soluble nsps (englyst, 1989; sinha et al., 2011). the experimental diets were formulated by inclusion of the four cereal grains to the basal diet in a ratio of 40%. before pelleting, the diet components were finely grounded and mixed. to prepare the diet, first water was added to the mash and mixed to form dough. afterward, the dough was pressed by screwing in a pasta maker through a die (3 mm). the wet pellets were then dried in an air dryer (50°c) and kept refrigerated until use. experimental procedure: the four experimental treatments were randomly distributed in 16 tanks with 4 replicate treatment. the fish were allowed to acclimatize with the experimental tanks and diets one week before the experiment initiation. to begin the experiment, the fish were weighted and randomly distributed in the experimental tanks. the fish were fed by hand twice a day (8.00 and 16.00) at a ratio of 2.5% biomass. the water supply was maintained from an underground source and aerated before use. the photoperiod regime during the experiment was 12 h light and 12 h dark. water quality parameters were monitored daily to 106 mazahery tehrany et al./ effects of grain sources on gut evacuation and nutrient digestibility in common carp ensure that they were in an appropriate range for the fish. water temperature was maintained at 26±1°c and water flow rate was set at 6 l min-1 and ph was 7.3-7.9, during the experiment. oxygen concentrations were measured in the tanks using a digital oxygen detector being > 6.1 mg l-1. at the day 56th, all fish were weighed. there was no mortality thorough the experimental period. in the course of the final week of the study, faeces were amassed from the tank bottom by pipette nearly 3 hrs post feeding. the daily faeces samples were pooled to obtain the required volume of faeces. to calculate apparent digestibility coefficients, the indicator method was employed with chromic oxide (cr2o3) as a marker (6 g kg−1). apparent digestibility (%) of nutrients is stated as a ratio of absorption of dietary nutrients according to below: adc=(1-[markerdiet/markerfaecesx nutrfaeces/nutrdiet ])x100 where adc= parent digestibility coefficient, aiadiet =dietary marker concentration, markerfaeces=faecal marker concentration, nutrdiet=nutrients of the diet, and nutrfaeces=nutrients of the faeces. gut evacuation rate measurement started following 72 hrs of fasting. after this period, the fish were fed once at 1% of body weight. the sampling was conducted at 0.5, 4, 8, 16, 24, 36 and 48 hrs after feeding. at every sampling time, one fish from each tank was killed by overdosed clove essence solution. the whole gut was removed from abdominal cavity of the fish and separated to three equal segments: the proximal, middle, and distal part of the intestine (part i, ii, and iii, respectively). the division of the intestine was conducted based on having equal lengths per intestine section. the contents of these three parts were collected separately for dry matter measurement. the evacuation rate calculation was based on dry weight of the samples (adamidou et al., 2009) by the use of formula introduced by elliot (1972): logwt = loga − rt. where wt is the geometric average weight of intestine dry matter content (digesta) at time t, a is a factor measured from the regression formula and r is the rate of intestine evacuation. the dry weights of intestine content (intestine i, ii, and iii) were table 1. feed ingredients and nutrients composition (% dry weight) of the four experimental diets. each value is the mean of three sub-samples. diets ingredients 40% corn 40% wheat 40% rice 40% barley corn meal 40 wheat meal 40 rice meal 40 barley meal 40 fish meal 15 15 15 15 soya bean meal 18 18 18 18 wheat gluten 15 15 15 15 wheat bran 5 5 5 5 canola oil 4 4 4 4 mineral premixa 1.5 1.5 1.5 1.5 vitamin premixb 1.5 1.5 1.5 1.5 proximate composition (%) dry matter 93.6 94.2 94.3 94.1 crude ash 16.1 14.2 14.3 18.2 crude protein 32.8 33.2 31.7 32.8 crude lipid 8.0 8.2 8.3 8.1 total nsp 9.1 10.9 4.5 12.9 soluble nsp 3.3 4.8 1.3 6.8 insoluble nsp 5.8 4.1 3.2 4.2 a mineral premix consisted of (mg kg-1 premix): 2600 mg mn, 600 mg cu, 6000 mg fe, 4600 mg zn, 50 mg se, 100 mg iu, 50 mg co, 100000 mg cholin chloride, up to 1 kg carrier. b vitamin premix consisted of (mg kg-1 premix): 1200000 iu vitamin a, 400000 iu vitamin d3, 3000 iu vitamin e, 1200 mg k3, 5400 mg c, 200 mg h2, 200 mg b1, 3360 mg b2, 7200 mg b3, 9000 mg b5, 2400 mg b6, 600 mg b9, 4 mg b12. 107 int. j. aquat. biol. (2018) 6(2): 104-113 regressed against time in order to examine a possible fit to a model for calculating time and rate of intestinal evacuation. at the end of the experimental period, common carps were also subjected to bacterial colony analysis after 72 hrs fasting. prior to dissection and homogenization, the fish were rinsed in sterilized distilled water, cleaned with ethanol (70.0%) and then washed again with sterilized distilled water to eliminate all superficial bacteria. the intestinal samples were dissected out under sterile conditions. subsequently, three samples from the mid segment of each intestine were collected and diluted with sterilized normal saline solution (0.85% nacl w/v). the diluted samples were transferred to nutrient agar plates to count the number of colony by a colony counter device (rogosa et al., 1951). chemical analysis: the dry matter analyses of feed and faeces samples were conducted by drying samples at 103c for 24 hrs until constant weight (iso 6496, 1983). a muffle furnace determined the ash content through incineration at 550c for 4 hrs (iso 5984, 1978). by applying the kjeldahl method (iso 5983 1979), crude protein (n×6.25) was measured after acid digestion. extracted in a soxhlet apparatus, lipid was derived by petroleum ether. chromic oxide was measured spectrophotometrically using method introduced by furukawa and tsukahara (1966). total and insoluble dietary nsp were analyzed according to englyst and cummings (1984). soluble nsp were calculated as the difference between total and insoluble nsp. fish performance: weight gain was determined by the difference between initial and final body weights. feed conversion ratio (fcr) was calculated per tank from feed intake data and weight gains: fcr = feed consumed (g) / wet body weight gain (g). specific growth rate (sgr) was calculated as follows and expressed as a percentage: sgr=100 (ln wfinal-ln winitial)×days -1(%/da) the calculations were based on the dry weight of the diets. data analysis: the data are presented here as means± sd for each treatment. proportional data (as %) were arcsine transformed and tested for normality (kolmogorov-smirnov test). one-way anova was used to detect significant effects of the grain treatments on fish performance parameters and digestibility. tukey's test was used for multiple pairwise comparisons between means. significance effects were identified when p<0.05 in each of the statistical tests applied. individual tanks were treated as experimental units in all analyses. non-linear regression model (exponential) was applied to relate the values of dry matter of intestine and evacuation time at three parts of intestine using microsoft excel 2010. results data on the growth performance of common carp are presented in table 2. inclusion of different types of cereal grain affect weight gain, sgr and fcr in c. carpio (p<0.05). both corn and wheat diets increased weight gain and sgr and improved fcr compared to diet with barley (p>0.05). however, final weight of common carp was not influenced by inclusion of the grain sources (p>0.05). the results also demonstrated that the inclusion of table 2. growth performance in common carp fed with different types of cereal grain over 8 weeks experimental period. different letters show significant difference among the treatments. growth parameters diets corn wheat rice barley initial weight (g) 229.8±9.1 220.0±3.4 220.3±3.4 228.5±6.38 final weight (g) 324.7±12.9 321.8±10.8 306.9±10.4 305.7±9.4 weight gain (g) 94.9±9.1a 101.8±9.6a 86.5±6.3ab 77.2±4.5b sgr (%/day) 0.62±0.03a 0.68±0.04a 0.59±0.03ab 0.52±0.03b fcr 1.93±0.12a 1.90±0.13a 2.20±0.09b 2.25±0.08b values are means of triplicate groups±sd. 108 mazahery tehrany et al./ effects of grain sources on gut evacuation and nutrient digestibility in common carp different types of cereal grain affected dry matter and protein digestibility of c. carpio (p<0.05; table 3). protein and dry matter digestibility were observed lower in common carp fed rice diet in comparison to the other cereal grains examined (p<0.05). however, fat digestibility was not affected by the type of the cereal grains (p>0.05). the evacuation rate of carp intestine was influenced by the cereal grain types (table 4). although most intestine content was evacuated 18-30 hrs after feeding, corn diet needed 18 hrs to evacuate 90% of the intestine content followed by rice, 20 hrs, wheat, 23 hrs with the longest evacuation time of 30 hrs observed in barley treatment. data collected from three segments of the intestine revealed that grain types changed evacuation rate in common carp (fig. 1). evacuation time showed chorological delays by feeding on barley diet and almost all dry matter left in part і after 30 min (first sampling), but this rate was recorded 70% for corn diet. the other two treatments table 3. nutrient digestibility in common carp feeding on different types of cereal grains over 8 weeks experimental period. different letters show significant difference among the treatments. parameters diets barley wheat rice corn dry matter 70.2±2.02a 70.9±0.89a 58.0±1.15b 67.27±1.25a protein 82.4±0.91a 82.8±1.31a 73.5±1.45b 79.9±1.55a fat 93.3±1.10 93.6±1.04 92.6±1.6 95.7±2.50 values are means of triplicate groups±sd. means with the different letters are significantly different (p<0.05). table 4. evacuation time in common carp intestine fed with different types of cereal grain and correlation coefficient (r) of the regression lines based on exponential model of gastric evacuation. treatments evacuation time (hrs) r 50% 75% 90% barley (meal) 9.24 15.35 30.70 -0.9313 wheat (meal) 6.93 11.51 23.02 -0.9548 rice (meal) 6.13 10.18 20.37 -0.9515 corn (meal) 5.47 9.10 18.20 -0.9668 figure 1. the evacuation rate (dry matter to the body weight) of different intestine segments in common carp over 48 h sampling period in the fish fed the four types of cereal grains. diet symbols: ■ = barley; ♦ = wheat; ▲= rice; ● = corn 109 int. j. aquat. biol. (2018) 6(2): 104-113 were somewhere in between corn and barley records. the slops of equations related to four cereal grains also showed a longer evacuation time for barley in comparison to other diets being similar for three parts of the common carp intestine. intestinal bacterial colony was affected by the cereal sources (fig. 2). the highest bacterial colony number (133 cfu.gr-1×10-7) was observed in fish fed wheat diet followed by rice and barley diets (p<0.05). the lowest number of bacterial colony (63 cfu.gr-1 ×10-7) was counted in common carp fed corn diet (p<0.05). discussion in the current study, type of cereal grain affected evacuation rate of intestine in common carp. this finding is similar to the results of adamidou et al. (2009) who observed that replacement of fishmeal by faba bean and chickpea affected gastrointestinal evacuation time in european sea bass. similarly, different gastric evacuation rates were also observed in gilthead sea bream when fed raw corn and wheat compared with extruded ones (venou et al., 2003). the results also revealed that feeding on barley diet led to the highest residence time of digesta in common carp. it appears that high content of beta glucan in barley (bach knudsen, 1997) as a soluble nsp can increase viscosity reflected in the reduction of intestinal evacuation time (spiller, 1984; storebakken, 1985). similar results were also reported by bach knudsen (2001) and adamidou et al. (2009) in european sea bass. in contrast, low soluble nsp content of corn diet, especially beta glucan (bach knudsen, 1997) reduces digeta viscosity (leenhouwers et al., 2007b), thereby intestinal emptying time is delayed. similarly, singh et al. (2011) observed that fast evacuation rate of soya and /or corn based diet to be caused by low digesta viscosity. the evacuation rates were almost similar for both rice and wheat diets in the current study, although there is a trend toward a lower evacuation rate in wheat diet. high solubility of arabinose and xylose in wheat diet (bach knudsen, 1997) may reduce evacuation rate by inducing a higher digesta viscosity (spiller, 1991). the type of cereal grains affected bacterial colony number. in general, dietary nsp is expected to increase bacterial colony through a negative impact on digestibility thereby a larger remaining material for bacterial degradation in the intestine (metzler-zebeli et al., 2010). the previous results showed that bacterial colony is highly related to soluble nsps content of grains (choct, 1996; choct et al., 2004). besides the substrate, the bacterial degradation is dependent on fish species and its gastrointestinal tract structure. while bacterial community can be easily figure 2. bacteria colony number in common carp intestine feeding on different types of cereal grains over 8 weeks experimental period. different letters above the bars show significant difference among the treatments. 110 mazahery tehrany et al./ effects of grain sources on gut evacuation and nutrient digestibility in common carp colonized by wheat diet in carp intestine, microbial degradation of wheat diet was reported to be low for african catfish (leenhouwers et al., 2007a). the lowest bacterial colony in corn diet can be related to low nsps content of this grain (sinha et al., 2011). furthermore, a high evacuation rate of corn diet does not allow the microbial community to establish in the fish intestine. bacterial colony numbers in rice diet were almost similar to those in barley treatment. lower bacterial activity in rice diet may be related to lower fiber content of rice in comparison to the other grain sources (pluske et al., 1998). the digestibility data revealed that protein and dry matter digestibility of rice diet were lower than those of other diets were. because of low fiber and high starch content of rice (jantrarotai et al., 1994; guimarães et al., 2008), we expected a larger digestibility values for this ingredient. however, the results did not meet our expectation. in addition, few studies on the evaluation of rice as a feed ingredient suggested a larger dry matter digestibility in comparison to other cereal grains (e.g., in nile tilapia, guimaraes et al. 2008; in mirror carp, ufodike and matty, 1983). nevertheless, it seems that rice protein is not digestible enough to fish (palmegiano et al., 2008). this may be related to a lower amino acid digestibility in rice protein (oujifard and seyfabadi, 2012). the type of cereal grains did not affect fat digestibility in common carp. this is similar to the results of leenhouwers et al. (2007b) who observed that cereal grains did not change protein and fat digestibility in african catfish. it seems that the negative impacts of nsp contents of grains on fat digestibility are not very large on fish (amirkolaie et al., 2005; leenhouwers et al., 2007a), although extensive impacts were previously proved for chicken (pasquier et al., 1996; singh et al., 2012). the present result also revealed that nutrients digestibility was not influenced by both gastrointestinal evacuation time and intestinal bacterial colonization. it appears that a delay in evacuation time, which may be associated with a longer exposure of feed to enzymes, did not change significantly nutrient absorption in common carp. similar observations were made in atlantic salmon (sveier et al., 1999) and european sea bass (adamidou et al., 2009) when dietary factors changed gastric evacuation rate. similar to delaying time, bacterial degradation does not seem to improve digestibility in common carp. a larger number of bacterial colony caused by wheat diet did not lead to an enhanced digestibility coefficient. it seems that intestinal microflora, which are largely responsible for degradation of digesta (amirkolaie et al., 2006), do not have a significant impact on nutrient digestion. therefore, it can be suggested that the effect of intestinal microbial degradation on nutrient digestibility is negligible to change nutrient digestibility. moreover, lower level of degradation in fish intestine might have caused such a little impact of bacterial colony number on nutrient digestibility. the concentration of short chain fatty acids, which are indicators of degradation, is much lower in fish intestine (18 mmol; amirkolaie., 2006) in comparison with warm blood animals like rat and cow (160 and 150 mmol respectively; rechkemmer et al., 1998). in conclusion, addition of dietary grains with different nsp sources changes evacuation time and bacterial colonial number in common carp. however, these changes were not reflected in nutrient digestibility. fat digestibility is not affected by cereal grain sources containing different levels of nsps in common carp. the current result may confirm the idea that due to structure of fish gastrointestinal tract, the impacts of microbial degradation and/or digesta movement rate in the gut is not considerable to have a great effect on nutrient digestibility. references adamidou s., nengas l., alexis m., foundoulaki e., nikolopoulou d., campbell p., karacostas l., rigos g., bell g.j., jauncey k. 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(2018) 6(2): 104-113 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی ماهی در پذیری هضم قابلیت وغذا عبور سرعت بر مختلف کربوهیدراتی منابع اثر بررسی (cyprinus carpio) معمولی کپور اورجی حسین کرامت، عبدالصمد تهرانی، مظاهری زهرا .ایران ساری، ساری، طبیعی منابع و کشاورزی علوم دانشگاهدانشکده علوم دامی و شیالت، شیالت، گروه چکیده: انماهی .ددستگاه گوارش بونی باکتریایی در تخلیه و تعداد کلو نرخ، مواد مغذی پذیریهضم بر مختلفکربوهیدراتی هدف اصلی این تحقیق بررسی اثرات منابع یشی با افزودن چهار . چهار غذای آزماندقطعه در هر تانک پخش شد 18لیتری با تراکم 500در شانزده تانک گرم 67/244±32/8کپور معمولی با وزن اولیه چهار تکرار در شانزده تیمار آزمایشی هر کدام با تهیه شد. چهار %40به یک غذای پایه در یک نسبت رد گندم، آردجو، آرد برنج و آرد ذرت( محصول غالت )آ وافزودن منابع مختلف کربوهیدراتی فاکتورهای رشد را در کپور معمولی تحت تاثیر قرار داد. ماهیان تغذیه شده با ذرت و گندم رشد باالتر . تانک قرار گرفتند در 90/1و 93/1 :گرم در وزن نهایی305مقایسه با گرم در 321و324جو نشان دادنند )غذای حاوی ن تغذیه شده با یب تبدیل بهتری در مقایسه با ماهیاضر در %58و 73) پروتئین و ماده خشک در غذای برنج در کپور معمولی کمتر از بقیه غالت بودپذیری قابلیت هضم(. غذایی ضریب تبدیل برای 25/2 مقایسه با های تغذیه شده از گندم و ذرت دیده به ترتیب در ماهی ( 63و u.grcf 133-101×-7) های باکتریاییتعداد کلونی . بیشینه و کمینه(%70-67و 82-79مقایسه با دقیقه )اولین نمونه 30همه ماده خشک در قسمت ابتدایی روده بعد از تاخیر در نرخ تخلیه دستگاه گوارش شده و تقریباً استفاده از غذای حاوی جو موجب شد. اگرچه غالت موجود در غذا توانست سرعت تخلیه ،توان گفتمیدر جمع بندی بود. %70ای برای غذای حاوی ذرت نرخ تخلیه که باقی مانده بود، در حالی( گیری معمولی را تحت تاثیر قرار دهد، ایجاد چنین شرایطی اثر بزرگی بر هضم پذیری مواد مغذی ایجاد نکرد. تگاه گوارش ماهی کپورهای باکتری دسکلونی دو تعدا .تخلیه سرعت ،مواد غذایی، تجزیه، کربوهیدرات، روده :کلمات کلیدی international journal of aquatic biology (2014) 2(6): 337-345 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2014 npajournals. all rights reserved original article description of skeletal structure and cranial myology of cobitis keyvani (cypriniformes: cobitidae) parya jalili1, soheil eagderi*1, hamed mousavi-sabet 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, p.o. box 1144, guilan, iran. article history: received 7 october 2014 accepted 11 december 2014 available online 2 5 december 2014 keywords: osteology muscle keyvan’s spined loach caspian sea basin abstract: cobitis keyvani is recently described from the sourhern caspian sea basin. limited information is available about morphological features of c. keyvani, therefore this study was conducted to provide osteological characteristics and cranial myology of this species. for this purpose, nine specimens of c. keyvani were collected from the talar river. the specimens were cleared and stained with alizarin red s and alcian blue for osteological examinations. the detailed skeletal structure and cranial muscles of c. keyvani were provided. based on the results, c. keyvani can be distinguished from other members of the genus cobitis by a contact between sphenotic and supraoccipital and a contact between pterosphenoid, parasphenoid, prootic and sphenotic in terms of osteological features. introduction the genus cobitis, a member of family cobitidae, is widespread from northern africa, throughout europe to eastern siberia, southeast to central vietnam (bohlen and ráb, 2001; vasil'eva and vasil'ev, 2006; kottelat and freyhof, 2007). this genus has three valid species in iran, including cobitis linea (heckel, 1849), c. faridpaki (mousavi-sabet, vasil'eva, vatandoust and vasil'ev, 2011) and c. keyvani (mousavi-sabet, yerli, vatandoust, ozeren and moradkhani, 2012) (mousavi-sabet et al., 2012). however, c. tenia (linnaeus, 1758) have been reported from iran including southern caspian sea basin (abdoli and naderi, 2009); there are believes that c. taenia is a northern european species (bohlen and ráb, 2001) and its occurrence in the southern caspian sea basin is unlikely (see in mousavi-sabet et al., 2012). this genus characterized by an elongated and compressed body, erectile spine below the eye, three pairs of short barbels, minute scales cover the body, * corresponding author: soheil eagderi e-mail address: soheil.eagderi@ut.ac.ir small dorsal and anal fins, caudal fin rounded or truncated (coad, 2014). males also have bony extensions of their pectoral fin rays, known as lamina circularis. cobitis keyvani was recently described from talar river (sourhern caspian sea basin) (mousavi-sabet et al., 2012). this species is distinguished from c. linea by lack of second lamina canestrini at the base of the first pectoral fin ray, and from c. faridpaki by large, dark and obvious spots along the mid-flank (coad, 2014). since, limited information is available about morphological features of c. keyvani, therefore, this study was conducted to provide a detailed osteological characteristics and cranial myology of this species. the results will provide a basis for further phylogenetic study of iranian member of the genus cobitis using osteological and morphological data. material and methods for osteological examination, nine specimens of 338 international journal of aquatic biology (2014) 2(6): 337-345 c. keyvani (fig. 1) with mean standard length of 58.7 ± 0.6 mm (mean ± sd) were collected from the talar river (in the sourhern caspian sea basin, iran). the caught specimens were anaesthetized using 1% clove solution and then fixed in 4% buffered formalin. for osteological examination, the specimens were cleared and stained with alizarin red s and alcian blue according to the protocol of taylor and van dyke (1972). muscle fibers of the dissected specimens were stained according to bock and shear (1972). the stained specimens were studied using a stereomicroscope (leica mc5), and different skeletal elements were dissected and scanned by a scanner equipped with a glycerol bath (epson v700). the obtained images were drawn using coreldrawx6 software. the terminology of skeletal elements follows rojo (2010) and sawada (1982) and muscle terminology follows winterbottom (1974). the scale bar of all drawings shows 1 mm. result and discussion osteology of cobitis keyvani neurocranium: in dorsal view, the posterior part of the neurocranium is oval-shaped and its anterior part is longer and narrower (fig. 2). the ethmoid region consists of the lateral ethmoid, preethmoid ii, supraethmoid-ethmoid, kinethmoid and pre-vomer (figs. 2a, b, c). the supraethmoid-ethmoid is bladeshaped positioning on the middle of the pre-vomer and has a relatively deep depression on its anterior margin and a rounded and downward projection under this depression similar to c. taenia (vasil’eva, 1984). the anterior part of the pre-vomer is wide; pointed posteriorly and overlaps with the anterior part of the parasphenoid. the two narrow preethmoid ii are positioned at the anterior part of the prevomer. the preethmoid ii bears two processes posteriorly viz. the dorsal and ventral processes; the dorsal process is articulated with the anterior part of the autopalatine and the ventral one with the anterior part of the pre-vomer. the preethmoid also bears a latero-external pointed process and an anterior facet which articulates with the maxilla. the lateral ethmoid is a long bone that has two posterior processes and one vertical process anteriorly which is connected to the orbitosphenoid (fig. 2b). the lateral ethmoid is moveable and projected from skin. a free broad kineethmoid is located between two maxillae (fig. 4a). the orbital region is composed of the frontal, orbitosphenoid, pterosphenoid, parasphenoid, lacrimal and sclerotic bones. the frontals have long and narrow anterior part. the frontals are separated posteriorly by the fontanle. the orbitosphenoid is dorsally connected to the anterior part of the frontal and ventrally to the parasphenoid. the pterosphenoid is ventrally connected to the parasphenoid and dorsally to the posterior part of the frontal. sawada (1982) mentions the pterosphenoid is attached only to the parasphenoid in cobitis, niwaella, sabajenewia, lepidocephalus and acanthopsoides, but in c. keyvani this bone meets the sphenotic, prootic and parasphenoid. the parasphenoid extends from the ethmoid region to the occipital region. the middle part of this bone is wide and connected to the pterosphenoid. two lateral processes present in the figure 1. lateral view of cobitis keyvani from talar river. 339 jalili et al/ osteology and cranial myology of cobitis keyvani middle part of the parasphenoid directing posteriorly (fig. 2b). the parasphenoid is bifurcate posteriorly (fig. 2b). a long lacrimal bone is situated at the anterior part of the lateral ethmoid. the otic region comprises of the parietal, epiotic, sphenotic, pterotic, and prootic (fig. 2a, b). the parietal is almost square-shaped bone and its posterior edge sutured with supraoccipital. the sphenotic is trapezoid in shape and ventrally connected to the prootic and posteriorly to the pterotic. according to sawada (1982) in the genus cobitis, the contact between the sphenotic and supraoccipital is absent; while in c. keyvani, the posterodarsal margin of the sphenotic is attached to the anterolateral part of supraoccipital (fig. 2a). there is no connection between the sphenotic and figure 2. the neurocranium of cobitis keyvani. (a) dorsal, (b) ventral and (c) lateral views. eo: exoccipital; bo: basioccipital; ep: epiotic; fr: frontal; le: lateral ethmoid; os: orbitosphenoid; pa: parietal; pv: prevomer; pro: prootic; ps: parasphenoid; pt: pterotic; so: supraoccipital; sp: sphenotic. eo: exoccipital; bo: basioccipital; ep: epioti; fr: frontal; fon: fontanel; pa: parietal; pts: pterosphenoid; sec: supraethmoid-ethmoid; so: supraoccipital; sp: sphenotic. 340 international journal of aquatic biology (2014) 2(6): 337-345 epiotic (fig. 2a). the ventral edge of the sphenotic forms the anterior facet of the neurocranium for articulating to the hyomandibular. the anterior part of the prootic is bifurcated which its medial branch is covered with the parasphenoid and the lateral branch attached to the pterosphenoid and sphenotic. this bone also has a large pore in its middle part. the pterotic is triangular in shape and enclosed by the epiotic, sphenotic and parietal bones. the epiotic is oval-shaped and attached to the supraoccipital posteriorly. the occipital region consists of the supraoccipital, exoccipitals and basioccipital. the supraoccipital is pentagon in shape, and its anterior part is concaved (fig. 2a, b). this bone is posteriorly connected to the exoccipital. the exoccipital froramen is located on its dorsal face and two other pores are located on its ventral part. a pointed process presents in its lateroventral part of the exoccipital. the basioccipital possesses two posterior processes, which are not fused with each other (fig. 2b). on the lateral part of the neurocranium, two facets present for articulating with the hyomandibular. the anterior facet is formed by the prootic and sphenotic, the posterior one forms by the sphenotic, prootic and pterotic bones. suspensorium: the suspensorium consists of the hyomandibular, ectopterygoid, endopterygoid, metapterygoid, symplectic, quadrate and palatine (fig. 3). the dorsal part of the hyomandibular is wider than its ventral part. the anterior rim of the hyomandibular is almost triangular in shape, and its dorsal margin has a pointed process. also, a large pore presents in its middle part of the hyomandibular. the opercular condyle is situated on the dorsolateral margin of the hyomandibular. a fossa is present on the dorsal part of the quadrate (fig. 3). the symplectic is elongated and narrow and located at the posterior part of the quadrate. the metapterygoid is a flat bone overlapping the endopterygoid anterodorsally and concaved ventrally. the endopterygoid is thin and bears an anterior facet to articulate with the autopalatine. the autopalatine is slightly wide and has an abdominal facet which is articulated with the pre-vomer. this bone also has two anterior and posterior protuberances on its dorsal part which letter one is larger (fig. 3). opercular series: the opercular series includes the opercle, preopercle, interopercle and subopercle (fig. 3). the anterodorsal part of the opercle bears a process; and the hyomandibular condyle is positioned under this process. the anteroventral part of the opercle is pointed and overlapped by the subopercle. the subopercle has two anterior processes; its dorsal process is longer than ventral figure 3. lateral view of the neurocranium in cobitis keyvani (left side). ect: ectopterygoid; end: endopterygoid; hy: hyomandibulare; io: interopercle; mp: metapterygoid; op: opercle; p: palatine; po: praeopercle; pre: preethmoid; q: quadrate; so: subopercle; sym: symplecic. 341 jalili et al/ osteology and cranial myology of cobitis keyvani one. the anterior part of the preopercle is pointed and covers the anterior part of the interopercle. the medial margin of interopercle is connected to the interhyal. jaws: the upper jaw consists of the maxilla and premaxilla. the premaxilla is l-shaped and thin and its horizontal part is wider than the vertical part. the horizontal part of this bone is directed dorsally (fig. 4a). the maxilla bears a well-developed mediolateral downward process whereas in c. taenia, this process is small (vasil’eva, 1984). this bone has an anterodarsal articular, which is connected to the preethmoid ii. a long process presents in the anteroventral part of the maxillae (fig. 4a). the lower jaw is composed of the dentary, anguloarticular and retroarticular. the dentary possesses an anteroventral process that is wide and has the coronoid process in the middle part of its dorsal portion. in c. keyvani, the coronomeckelian is absent. a short triangular retroarticular is positioned at the dorsoventral side of anguloarticular. the anguloarticular has a facet posteriorly which is articulated with the quadrate. branchial apparatus: the branchial apparatus includes five pairs of the ceratobranchial, four pairs of the epibranchial, three pairs of the hypobranchial, two pairs of the inphrapharyngobranchial and four unpaired basibranchial bones (fig. 5a). the 4th basibranchial is small. there is a tooth-like process on the ventral surface of fifth ceratobranchial, and on its dorsal surface; there is a row of 7 to 9 pharyngeal teeth. hyoid arch: the hyoid arch comprises of the paired epihyals, hypohyals and ceratohyals, unpaired urohyal and basihyal, and three pair of the branchiostegal rays (fig. 5b). the horizontal part of the urohyal has two lateral triangular processes and its vertical part is blade-shape situating on the dorsal part of horizontal portion (fig. 5b). a small pore presents in the ventral margin of the vertical part of the urohyal. the basihyal is long with narrow middle part positioned between the hypohyals. the hypohyal consists of two parts viz. ventral and dorsal hypohyals that are connected medially. the anterior part of the ceratohyal is connected to the dorsal and ventral part of the hypohyals. the first branchiostegal ray is connected to the medial part of the ceratohyal. the second branchiostegal ray is connected to the joint of the ceratohyal and epihyal. the 3rd branchiostegal ray is attached to the epihyal. the dorsal part of the epihyal is pointed. the interhyal is a cylindrical bone positioning on the dorsal edge of the epihyal (fig. 5b). the branchiostegal rays extend to the dorsal end of the subopercle (fig. 7). pectoral girdle: the pectoral girdle consists of the cleithrum, supracleithrum, coracoid, mesocoracoid, scapula, posttemporal and radials (fig. 5c). the cliethrum is l-shaped and has a blade-shape process on its posteroventral part. the dorsolateral part of the cleithrum is articulated with the ventral part of the supracleithrum. the supracleirthrum is long and dorsally connected to the posttemporal which attaches to the pterotic. the scapula has a large foramen in middle part and ventrally connected to the coracoid. the mesocoracoid is ventrally figure 4. (a) internal view of upper jaw and (b) lower jaw in cobitis keyvani. aa: anguloarticular; d: dentary; ke: kinethmoid; mx: maxillae; pmx: premaxillae; ra: retroarticulare. 342 international journal of aquatic biology (2014) 2(6): 337-345 connected to the coracoid and dorsally to the scapula and cliethrum (fig. 5c). the pectoral fin bears three radials that first one is broader and second one has the ventral and dorsal processes. the third radial is widest one. the pectoral girdle has one unbranched and seven branched rays. in males, the second ray of pectoral girdle is broader and forms the lamina circularis (fig. 5c). pelvic girdle: the pelvic girdle includes the paired pelvic bones, pelvic splint and radials. the posterior part of the pelvic bone is broader and its anterior part is bifurcate. three radials present posterior to the pelvic bone. the second radial is the smallest one. the two pelvic splint bones are positioned in the lateral side of the pelvic bones. the pelvic fin has one unbranched and six branched rays (fig. 5d). axial skeleton: the number of vertebrae is 42. the weberian apparatus and swim bladder capsule is formed by the four anterior centra (fig. 6a). also, four pair ossicles, including the tripus, intercalarium, scaphium and claustrum present in the weberian apparatus. the swim bladder capsule is formed from fourth centrum. in the lateral margin of the swim bladder capsule, there are two large pores that the posterior one is rounded and anterior one ovalshaped. the two pointed processes present in the latero-external portion of the swim bladder capsule. there are plentiful small pores on the wall of the swim bladder capsule. the cranial and caudal parts of vertebral column have 21 centra. in c. keyvani, similar to other members of cobitinae including cobitis taenia taenia, c. taenia striata, c. koreensis, acanthopsoides graciroides and lepidocephalus guntea (sawada, 1982), the number of cranial figure 5. (a) dorsal view of branchial apparatus, (b) hyoid arches, (c) medial view of pectoral girdle and (d) pelvic girdle of cobitis keyvani. bb: basibranchial bh: basihyal; ch: ceratohyale; cb: ceratobranchial; co: coracoid; eh: epihyal; hh: dorsal and ventral hypohyal; eb: epibranchial; hb: hypobranchial; ipb: inphrapharyngobranchial; ih: interhyal; uh: urohyal; cl: cleitherum; lc: lamina circularis; mco: mesocoracoid; r: radial; sc: scapula; pb: pelvic bone; ps: pelvic splint. 343 jalili et al/ osteology and cranial myology of cobitis keyvani vertebrae is about equal to those of caudal ones. unpaired fins: the dorsal fin has seven pterygiophores, one stay, three unbranched and seven branched rays (fig. 6b). the first pterygiophore is the largest one and supports the unbranched rays. four free supraneural bones present in the front of the dorsal fin. the anal fin possesses three unbranched and five branched rays, six pterygiophores and a small stay (fig. 6b). the first and largest pterygiophore supports the unbranched rays. the skeleton of caudal fin consists of the epural, parhypural, pleurostyle, and six hypurals bones (fig. 7). in c. keivany, the hyporal-1 is fused with the hypural-2 (sometimes the first three hypurals are fused). the caudal fin bears 17 branched rays and various numbers of the procurrent rays. finally, based on the osteological results, c. keyvani can be distinguished from other members of the genus cobitis by a contact between sphenotic and supraoccipital and a contact between pterosphenoid, parasphenoid, prootic and sphenotic. other features of c. keyvani are similar to other members of this genus reported by vasil’eva (1984) and sawada (1982). myology of cobitis keyvani muscles of the cheek: the adductor mandibulae of c. keyvani comprises of a1, a2 and aω sections, while in the studied cyprinids by matthes (1963), aω was absent, and the adductor mandibulae comprised a1, a2 and a3. in the studied species, the section a1 is the largest section of the adductor mandibulae and originates musculously from the dorsolateral margin of the hyomandibular. this section inserts as a wide tendon on the anterior portion of the maxillae. but in the studied cyprinids by matthes (1963), a1 originates from the preoperculum and quadrate and runs forward to insert on the antero-external face of the maxillary. the section a2 originates from the ventral part of both hyomandibular and anterior rim of the preopercle and inserts tendinously onto the medial part of the coronoid process of the dentary. the posterior fibers of a2 are covered by a1. the sections aω and a1 are attached anteriorly to a single tendon. the section aω inserts on the posterodorsal edge of the anguiloarticular (fig. 8). figure 6. (a) lateral view of the weberian apparatus and (b) dorsal and anal fins in cobitis keyvani. dr, distal radial; mr, medial radial; ns, neural spine; pr, proximal radial; na: neural arch; nc: neural complex. figure 7. (a) lateral view of the caudal fin in cobitis keyvani. e: epural; h: hypurals; ph: parhypurale; pls: pleurostile. 344 international journal of aquatic biology (2014) 2(6): 337-345 the adductor arcus palatinii occupies the lateral face of the hyomandibular. this muscle originates musculously from the ptersphenotic and inserts on the medial face of the hyomadibular. in other cypriniid fishes, it observes that the adductor arcus palatine consists of three parts and arises from the prootic and parasphenoid and insert on the inner dorsal surfaces of the palatine, pterygoid and hyomandibular bones (matthes, 1963). the levator arcus palatini muscle originates musculously from the lateromedial margin of the parasphenoid and the ventral portion of the ptersphenotic and inserts musculously on the anterodorsal rim of the metapterygoid and the posterodorsal rim of the endopterygoid. the anterior fibers of levator arcus palatini is attached to the dorsal part of a2 muscle. the levator opeculi muscle is conical in shape and originates with muscle fibers from the lateral face of the sphenotic. this muscle inserts as a tendon on the dorsal part of the opercular prominent process. the adductor opeculi originates musculously from the sphenotic and posterodorsal edge of the hyomandibuar and its fibers insert to the dorsal rim of opercle (fig. 8). ventral muscles of the head: the intermandibularis muscle lies between the lower jaws. the protractor figure 8. lateral view of the head muscles in cobitis keyvani. a1: subsection of the mandibulae aw: subsection the adductor mandibulae; a2: subsection the adductor mandibulae; ao: adductor operculi; d: dentary; hy: hyomandibular; lo: levator operculi; mx: maxillae; op: opercle; pt: pterotic; so: subopecle; sp: sphenotic. figure 9. ventral view of the head muscles in cobitis keyvani. ch: ceratohyal; d: dentary; eh: epihyal; had.: hyohyoidei adductors; hin: hyohyoides inferioris; hab.: hyohyoidei; imd: intermandibularis; pr.hy.: protractor hyoides; sth: sternohyoideus; uhy: urohyal. 345 jalili et al/ osteology and cranial myology of cobitis keyvani hyoides muscle is anteriorly attached to the medial face of the dentary and posteriorly to the ventral face of the ceratohyal and epihyal. in the family cyprinidae, the intermandibularis is sometimes absent or modified into a protractor hyoidei (matthes, 1963). the hyohyoides inferioris connects two side of hyoid arch and its fibers are attached to the dorsal face of the hyoid arch similar to other cyprinid fishes (matthes, 1963). the hyohyoideus abductor connects the left and right branchiostegal rays. the hyohyoidei addauctor has developed between the branchiostegal rays. the sternohyoideus is a large muscle and bears two bundles which are connected together. they originate tendinously from the anterior part of the urohyal and connect the hyoid arch to the cleithrum. references abdoli a., naderi m. (2009). the biodiversity of fishes of the southern basin of the caspian sea. abzian scientific publication. tehran. 238 pp. bock w.j., shear r.c. (1972). a staining method for gross dissection of vertebrate muscles. anatomischer anzeigerbiodiversity, 130: 222-227. bohlen j., rab p. (2001). species and hybrid richness in spined loaches of the genus cobitis l. (teleostei: cobitidae), with a checklist of european forms and suggestions for their conservation. journal of fish biology, 59(a): 79-85. coad b. (2014). fresh water fishes of iran. available from: http://www.briancoad.com. retrieved 3/9/2014. kottelat m., freyhof j. (2007). handbook of european freshwater fishes. kottelat, cornol, switzerland and freyhof, berlin, germany, 646 pp. mousavi-sabet h., vasil’eva e.d., vatandoust s., vasil’ev v.p. (2011): cobitis faridpaki sp. nova—a new spined loach species (cobitidae) from the southern caspian sea basin (iran). journal of ichthyology, 51 (10): 925-931. mousavi-sabet h., yerli s. v., vatandoust s., ozeren s. c., moradkhani z. (2012). cobitis keyvani sp. nova— a new species of spined-loach from south of the caspian sea basin (teleostei: cobitidae). turkish journal of fisheries and aquatic sciences, 12: 7-13. rojo a.l. (1991). dictionary of evolutionary fish osteology, crc press. boca raton, florida, 273 pp. matthes h. (1963). a comparative study of the feeding mechanisms of some african cyprinidae (pisces, cypriniformes). bijdragen tot de dierkunde, 33:3-35. sawada y. (1982). phylogeny and zoogeography of the superfamily cobitoidea (cyprinoidei, cypriniformes). memoirs of the faculty of fisheries of hokkaido university, 28(2): 65-223. taylor w.r., van dyke g.c. (1985). revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study. cybium, 9: 107-119. vasil'eva e.d., vasil'ev v.p. (2006). cobitis pontica sp. nova-a new spined loach species (cobitidae) from the bulgarian waters. journal of ichthyology, 46(1): 1520. vasileva e.d. (1984). comparative morphological analysis of two spiny loach populations (genus cobitis, cobitidae) differing from each other in the number of patches at the base of caudal fm. journal of ichthyology, 24: 43-53. winterbottom r. (1974). a descriptive synonymy of the striated muscles of the teleostei. proceedings of the academy natural science philadelphia, 125: 225-317. © 2022 iranian society of ichthyology short communication the effect of non-prepared ecological data on the decision of multivariate analysis: a case study of water quality data of the shatt al-arab river, iraq mujtaba a.t. ankush*1 college of agriculture, university of basrah, basrah, iraq. s . . 26 table 1. parameter unit site1 site2 site3 mean min-max mean min-max mean min-max wt °c 24.47 13.4-31.8 14.00 14-32.8 25.14 16.9-33 ph 8.14 7.3-8.94 7.20 7.2-8.9 8.38 7.1-9.2 ec μs/cm 4173.67 3260-5350 2958.00 2958-4920 3357.79 2530-4135 do mg/l 5.37 4.4-5.96 4.20 4.2-5.7 5.66 4.1-6.5 bod mg/l 2.16 1.82-2.9 1.26 1.26-3 2.04 1.24-2.72 turbidity ntu 14.49 6.9-28.5 6.00 6-36 27.33 12-100 th mg/l 1460.83 920-2000 880.00 880-2400 1370.56 780-2800 no3 mg/l 1.60 0.74-2.5 0.66 0.66-2.74 1.57 0.54-2.72 po4 mg/l 2.55 1.02-3.88 1.00 1-4.02 2.62 1.4-3.84 27 : 28 table 2. variable comp.1 comp.2 comp.3 comp.4 comp.5 comp.6 comp.7 comp.8 comp.9 wt -0.89612 0.15160 0.21736 -0.18784 0.11226 0.00968 0.08946 -0.02665 0.26466 ph -0.37803 0.31667 0.76967 0.20686 0.28523 0.02449 0.10016 0.11054 -0.13205 ec 0.73212 0.14407 -0.27285 0.07326 0.43610 -0.17989 0.35619 0.10097 0.06172 do 0.31449 0.68948 -0.12701 0.30545 0.00818 0.55726 0.00119 -0.05929 0.04644 bod 0.01971 -0.78733 0.05734 0.53074 -0.09034 0.14547 -0.03142 0.23660 0.09199 turbidity 0.31010 0.60198 0.19826 0.23904 -0.58604 -0.29291 0.08749 0.06762 0.05950 th 0.76349 0.01652 0.34981 0.18994 0.28069 -0.18166 -0.34398 -0.13384 0.10165 no3 0.53381 -0.55734 0.44429 -0.10603 -0.19022 0.15596 0.29089 -0.22472 0.01234 po4 0.64625 0.07272 0.24539 -0.63882 -0.06604 0.18226 -0.09638 0.24624 0.03558 29 : 30 references table 3. variable before transformation after transformation wt 0.0267 0.0374 ph 0.1838 0.2488 ec 0.7338 0.8816 do 0.187 0.187 bod 0.3572 0.3512 turbidity 0.0001 0.7793 th 0.0462 0.6852 no3 0.444 0.4429 po4 0.0294 0.03 table 4. variable comp.1 comp.2 comp.3 comp.4 comp.5 comp.6 comp.7 comp.8 comp.9 std wt -0.90701 0.01783 0.10440 0.23376 0.16852 0.08562 0.11574 0.03572 0.24712 std ph -0.45433 0.26018 0.77007 0.08724 0.27229 0.14204 0.01865 0.08511 -0.15285 std ec 0.71776 0.26478 -0.22065 -0.13878 0.47312 0.00789 0.31990 0.14291 0.01036 std do 0.20290 0.74736 -0.05139 -0.28826 -0.15952 0.52782 -0.07760 -0.04322 0.05117 std bod 0.13363 -0.73155 0.27187 -0.50247 -0.18669 0.10477 -0.01298 0.26686 0.05815 std turbidity -0.06640 0.73301 0.26422 -0.15574 -0.45397 -0.33950 0.19517 0.06325 0.02803 std th 0.71862 0.15041 0.47719 -0.16954 0.27098 -0.21327 -0.22966 -0.12086 0.13508 std no3 0.60579 -0.46154 0.38399 0.26347 -0.22158 0.18744 0.27578 -0.20591 0.02120 std po4 0.63137 0.17522 0.03015 0.67915 -0.15262 0.05164 -0.13897 0.24928 0.03335 31 : int. j. aquat. biol. (2018) 6(2): 55-60 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article evaluation of toxicity and biochemical effects of the oxadiargyl in common carp (cyprinus carpio l.) hossein emadi*1, somayeh shariatzadeh1, shahla jamili2, ali mashinchian1 1department of marine biology, faculty of marine science and technology, science and research branch, islamic azad university, tehran, iran. 2iranian fisheries research organization, agriculture research education and extension organization (areeo), tehran, iran. article history: received 10 november 2017 accepted 29 march 2018 available online 2 5 april 2018 keywords: carp growth performance biochemical parameters histopathology liver abstract: this study aimed to investigate the effects of sublethal concentrations of oxadiargyl herbicide on growth performance, serum biochemical parameters and liver histology of the common carp, cyprinus carpio, during 30 days exposure period. carp fingerlings were randomly divided into four duplicate groups. experimental groups were exposed to the concentration of 0.1, 0.3 and 0.5 ppm of oxadiargyl, while the control group was kept in toxicant free. during the experiment, none of the control fish died. weight gain and condition factor decreased, while food conversion ratio increased in fish exposed to 0.5 ppm of oxadiargyl. exposure to oxadiargyl significantly increased serum alt, ast and alp, as well as glucose levels, while cholesterol, total protein and albumin were significantly decreased in the exposed fish groups depend on oxadiargyl concentrations and exposure time. diffuse and focal necrosis mainly as perivascular necrosis fibrosis, increase in size and number of melanomacrophage centers, bile duct hyperplasia, hyperemia and hemorrhage, fatty changes in the hepatocytes, fibrocyte aggregation and focal inflammatory cells were also the histological lesions observed in the liver of exposed fish. these results showed that oxadiargyl is highly toxic to common carp and had negative effects on the growth performance, serum biochemical parameters and the liver tissue of common carp. introduction contamination of aquatic environments by runoff and leaching of agricultural pesticides and herbicides poses serious toxicological risks to non-target organisms such as fish and shellfish species (steinberg et al., 1995; grung et al., 2015). the exposure of fishes even to low concentrations of herbicides may result in various physiological disorders and cause severe effects on behavior (steinberg et al., 1995), growth performance (sweilum, 2006), immune system and susceptibility to infectious diseases (ahmadivand et al., 2015; xing et al., 2015), reproduction and endocrine system (ahmadivand et al., 2016), hematological and biochemical parameters, and histological changes of vital organs (poleksic and karan, 1999; blahova et al., 2014), as well as show genotoxic effects and devastating fish deaths (bálint et al., 1997; zanjani et al., 2017). oxadiargyl (c15h14cl2n2o3) belonging to the *corresponding author: hossein emadi doi: https://doi.org/10.22034/ijab.v6i2.466 e-mail address: emadihossein@yahoo.com oxadiazole group, is one of the herbicides extensively applied to control weeds in rice fields in asia and north europa as well as in the north of iran. oxadiargyl is a broad-leaf herbicide and acts by blocking porphyrin biosynthesis by inhibiting protoporphyrinogen oxidase (hwang et al., 2004). its half-life in soil is 20-30 days, however, its low aqueous solubility led to rapid accumulation and contamination in soil and water bodies (mahmoudi et al., 2013). many experiments investigated the phytotoxic effects of oxadiargyl (nethra and jagannath, 2011; ahmed and singh-chauhan, 2015; monjezi et al., 2015). however, in spite of the genotoxic and hematological effects (zanjani et al., 2017), as well as the retarding growth (ajani et al., 2015) induced by the other member of oxadiazole chemical family, oxadiazon in fish, there are a limited data concerning the lethal and sublethal effects of this herbicide on 56 emadi et al. / toxicity and biochemical effects of oxadiargyl in common carp aquatic organism, particularly freshwater fishes. the common carp, cyprinus carpio, is one of the most important and valuable commercial fish species in iran that mainly farmed in the north of iran i.e. guilan, mazandaran and golestan provinces, the area that the oxadiargyl is widely used to control weeds in paddy fields (mahmoudi et al., 2013). hence, the present study was aimed to investigate the effect of oxadiargyl on growth performance and serum biochemical parameters in common carp fingerling. histopathological changes in the liver, the main organ for detoxification was also investigated. materials and methods chemicals: technical grade, oxadiargyl herbicide (3% ec) manufactured by saveh herbicide company (arak, iran) was used in this study. fish and experimental design: common carp with the mean weight of 19.21±2.3 g and total length of 10.27± 0.47 cm were obtained from a local fish farm (guilan province, iran), and acclimated to laboratory conditions in 1000l tanks for two weeks. the fish were randomly selected and introduced into four duplicate 100l tanks (n=30) and exposed to concentrations of 0.1, 0.3 and 0.5 ppm of oxadiargyl. a control with toxicant free water was also maintained. the dechlorinated tap water was used and renewed daily. physiochemical characteristics were monitored before and after water exchange. the fish were fed (3% of body weight) commercial ffcextruded fish food (faradaneh company, iran) twice a day and starved for 24 hrs before sampling. growth performance: at the end of the experiment, the body weight and total length of individual fish of control and exposed groups were measured, and the feed conversion ratio (fcr), weight gain (wg) and condition factor were calculated based on kane et al. (2016). biochemical analysis: at day 30 after sublethal exposure, five fish from each replication were anesthetized with clove powder (200 mg.l-1), and blood was collected from caudal vein puncture. the blood was allowed to clot and serum was obtained by centrifugation at 4000×g for 5 min. then, serum aspartate aminotransferase (ast), alkaline phosphatase (alp), alanine aminotransferase (alt), albumin, cholesterol, total protein, and glucose were determined by commercial kits (parsazmon co. iran) according to the manufacturer protocols. histopathology: at the end of experiment, three fish from each replication were sampled and the liver tissue was removed for histological examinations. then the tissues were fixed in 10% nbf, dehydrated and embedded in paraffin, 5 µm sections were prepared and stained with hematoxylin and eosin (h&e) according to hewitson et al. (2010) and eagderi et al. (2013), and then examined under a light microscope (nikon e600). statistical analysis: the data were analyzed using the statistical package spss20 software (chicago, il, usa) by one-way analysis of variance (anova) at p<0.05. results growth performance: the results of growth performance parameters after 30 days sublethal exposure are shown in table 1. body weight and total length in the initial control were 19.26 ±1.26 g and 10.3±0.25 cm, and reached to 23.8±1.83 g and 10.8±0.23 cm, respectively, on day 30. however, the table 1. growth performance in common carp at day 30 after exposure to different concentrations of oxadiargyl (mean±se, n=30) dose (ppm) 0 0.1 0.3 0.5 initial weight (g) 19.26±1.26 a 18.91±1.39 a 19.29±1.47 a 19.4±0.92 a final weight (g) 23.8±1.83a 24.18±1.73a 22.5±2.11a 19.73±0.87b weight gain (g) 4.54 ±1.07a 5.27± 0.38a 3.21±0.67 ab 0.33±0.19b fcr 1.06±0.12a 1.04±0.1a 1.1±0.13a 1.25±0.08b initial size (cm) 10.3±0.25 a 9.96±0.05 a 10.48±0.43a 10.35±0.43a final size (cm) 10.8±0.23a 10.57±0.45 a 10.66±0.65 a 10.5±0.65 a condition factor 1.9±0.11 a 2.04±0.08 a 1.86±0.2 ab 1.7±0.1b different letters indicate significant differences at (p<0.05) between the experiment groups. 57 int. j. aquat. biol. (2018) 6(2): 55-60 increase of the weight and length were occurred in the control fish, they were inhibited in the treatments. a significant decrease in weight gain and condition factor, and increased food conversion ratio (fcr) were observed in fish exposed to 0.5 ppm of oxadiargyl (p<0.05). biochemical parameters: the results of serum biochemical parameters are presented in table 2. exposure to oxadiargyl significantly increased serum alp, ast and alt, as well as glucose levels, while cholesterol, total protein and albumin were significantly decreased in the exposed fish groups depend on oxadiargyl concentrations and exposure time (𝑃<0.05). however, no significant changes in the examined serum biochemical parameters of the fish exposed to 0.1 mg/l of oxadiargyl were observed. liver histopathology: the histological details of the carp liver (hepatopancreas) tissue is shown in the figure 1a. diffuse and focal necrosis mainly as perivascular necrosisfibrosis, hyperemia and hemorrhage, fatty changes in the hepatocytes, focal inflammatory cells and fibrocyte aggregation were the histological lesions in the liver of exposed fish. sever bile duct hyperplasia and increase in size and number of melanomacrophage centers depend on oxadiargyl concentration were also observed (fig. 1b, 1d). discussion oxadiargyl is the main herbicide, extensively applied to control weeds of the rice fields in the southern caspian sea basin (mahmoudi et al., 2013). the present work studied the toxicity effects of oxadiargyl on different physiological parameters of common carp. based on the results, histopathologically, oxadiargyl induced mild to severe alternations in liver of common carp depend on herbicide concentrations which may be a result of increasing cell activities, and is in agreement with previous studies reporting different histopathological changes in fish following exposure to herbicides (ahmadivand et al., 2014; poleksic and karan, 1999; blahova et al., 2014). moreover, the observed changes in size and number of melanomacrophage centers (mmcs) in liver tissue depend on the herbicide concentration further table 2. biochemical parameters in common carp after exposure to different concentrations of oxadiargyl. dose (ppm) 0 0.1 0.3 0.5 10 days alt (u l) 26.3±2.1a 25.9±1.3a 28.32±1.56b 32±1c ast (u l) 143±12.12a 144±11a 178.3±8.7b 236.67±7.6c alp (u l) 40.3±3.6a 39.14±2.2a 45.6±1.23b 47±1.2c albumin (g dl) 0.96±0.02a 1.01±0.05a 0.98±0.02b 0.78±0.04c cholesterol (mg dl) 256±2a 249.67±3.06a 216.67±5.86b 166.67±2.08c tp (g dl) 2.74±0.1a 2.78±0.21a 2.2±0.14b 2.06±0.12b glucose (mg dl) 59.33±7.02a 62.67±2.08a 74±1b 81.67±0.58c 20 days alt (u l) 25.8±1.2a 26.09±1.4a 30.1±1. 3b 33.8±1.3c ast (u l) 141±2.2a 141±9.7a 181.43±5.01b 251.61±4.9c alp (u l) 39.8±2.12a 38.9±3.2a 44.67±1.53b 46±3c albumin (g dl) 0.99±0.02a 0.95±0.03a 0.86±0.02b 0.69±0.02c cholesterol (mg dl) 259±5a 241.61±3.06a 191.67±4.31b 129.67±5.86c tp (g dl) 2.71±0.01a 2.68±0.24a 2.15±0.1b 1.98±0.03b glucose (mg dl) 60.12±4.01a 67.33±2.08ab 74±1b 106.34±0.51c 30 days alt (u l) 25.33±2.52a 24.19±1.53a 30.33±1.53b 36±0.8c ast (u l) 140±12.12a 141±14.8a 187.33±10.07b 267.67±8.96c alp (u l) 39.33±3.06a 38.17±2.52a 45.07±2.57b 49±2.8c albumin (g dl) 1.03±0.02a 0.94±0.05a 0.75±0.01c 0.67±0.02d cholesterol (mg dl) 257±6a 239.67±3.05a 198.67±5.86b 157.66±2.1c tp (g dl) 2.72±0.02a 2.63±0.24a 2.06±0.12b 1.95±0.02b glucose (mg dl) 59.29±5.32a 66.35±3.04 a 78±1b 96±1c different letters indicate significant differences between the groups at (p<0.05). 58 emadi et al. / toxicity and biochemical effects of oxadiargyl in common carp confirmed that mmcs can be considered as a biomarker of environmental stress such as pesticides (ribeiro et al., 2011). the toxic effects of pesticides on hepatic cells may result in the body metabolic alteration as well (wolf and wheeler, 2018). a significant change in serum biochemical parameters, including total protein, albumin, glucose, cholesterol as well as in serum alp, ast and alt levels were observed over a 30-day period and the higher concentration of herbicide caused more effects, which is in agreement with the findings of saravanan et al. (2017), evaluating the acute toxicity effects of oxadiazon on carp. decrease of total protein and albumin might have been resulted from the impaired protein synthesis due to hepatic dysfunction and immunosuppressive effect of the herbicide (nayak et al., 2004; gokcimen et al., 2007). moreover, change in glucose and cholesterol levels indicate the metabolic alteration in fish energy sources, especially carbohydrate metabolism due to liver dysfunction (barton, 2002). also, the observed increase in serum alp, alt and ast activity may be related to tissue damage resulting in the amino acid and protein metabolism disruption. detrimental histopathological and biochemical effects can have a negative effect on the growth performance of reared fish and can lead to a decrease in growth and economic losses. here, the growth performance of exposed fish was inhibited, which is in agreement with previous reports studying growth parameters of fish (ajani et al., 2015), as well as the rodents and dog (richert et al., 1996) following exposure to oxadiazole group. similarly, ajani et al. (2015) found a significant decrease in the growth and feed intake and utilization of african catfish (clarias gariepinus) exposed to 0.008 ml.l-1 and 0.02 ml.l-1 of figure 1. histopathological changes of the liver tissue of the common carp after 30 days sublethal exposure to oxadiargyl. (a) liver tissue of control fish, (b) fish exposed to 0.1 ppm, (c) 0.3 ppm, and (d) 0.5 ppm of oxadiargyl; p: pancreas, ht: hepatocytes, ss: sinusoidal spaces, fc: fatty changes, f: fibrosis, mc: melano-macrophage centers, n: necrosis, h: hyperemia, hp: hyperplasia. 59 int. j. aquat. biol. (2018) 6(2): 55-60 oxadiazon, the other member of oxadiazole group. also, saravanan et al. (2017) assessed the acute toxicity effects of 0.5, 5 and 50 μg.l-1 oxadiazon on carp for 96 hrs, and reported that this herbicide negatively affected the hematological and biochemical parameters of the fish. however, regarding the effects of oxadiargyl on fish growth no data was found. therefore, the growth inhibition by exposure to oxadiargyl in the current study may be due to the reduced fish appetite, dysfunction in metabolism process and waste of energy to overcome the stress caused by the herbicide (abdul-farah et al., 2004). in conclusion, the results showed that oxadiargyl has a significant negative effect on growth performance and induces significant biochemical and histopathological alterations in the liver of common carp, and it is suggested that this herbicide is highly toxic for c. carpio. however, further studies need to find out its toxic effects on the other physiological parameters of the fish. acknowledgments the authors would like to thank m. ahmadpoor for assisting with the histological sections. references abdul-farah m., ateeq b., ali m.n., ahmad w. 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(2018) 6(2): 55-60 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی (cyprinus carpio) معمولي کپور ماهي در بیوشیمیایي و رشد پارامترهای بر اکسادیارژیل کشعلف اثرات بررسي 1، علي ماشینچیان2، شهال جمیلي 1زاده، سمیه شریعت*1حسین عمادی .گروه بیولوژی دریا، واحد علوم تحقیقات، دانشگاه آزاد اسالمی، تهران، ایران1 .موسسه تحقیقات علوم شیالتی کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، تهران، ایران2 چکیده: عمولی، مبر پارامترهای رشد و بیوشیمیایی و همچنین میزان آسیب بافتی کبد در ماهی کپور اکسادیارژیلکش این مطالعه با هدف بررسی اثرات علف cyprinus carpio، گرم در چهار گروه هر کدام با دو تکرار و 27/19±3/2)میانگین وزنی عدد ماهی کپور 120ین منظور تعداد رای اانجام شد. ب قرار گرفتند. اکسادیارژیلمیلی گرم در لیتر سم 5/0و 3/0 1/0، 0روز در معرض غلظت های 30لیتری به مدت 100ماهی در تانک های 15تراکم غییر ها مقایسه شد. ترهای رشد بیوشیمیایی سرم بین گروهدر انتهای دوره آزمایش بیومتری انجام و از ماهیان نمونه بافت کبد و خون تهیه و پارامت (.>05/0p)در مقایسه با گروه کنترل مشاهده گردید های تیمارمیانگین افزایش وزن، ضریب تبدیل غذایی و فاکتور وضعیت گروهداری در میزان معنی در حالی داری مشاهده شد،سرم افزایش معنیalp و alt ، astآنزیم کبدی گلوکز و همچنین میزاناکسادیارژیل های تیمار عالوه در گروههب فیبروز بافت تشکیل و صفراوی مجاری هایپرپالزی (.>05/0p)داری نشان دادند ها کاهش معنی، پروتئین کل و آلبومین این گروهکلسترولکه ای و همچنین افزایش هستههای التهابی تکو نفوذ سلولها هپاتوسیتتغییر چربی در تعداد زیادی از ها، فیبروسیت و کالژن هایرشته حاوی بر اساس نتایج این مطالعه اکسادیارژیلروزه با سم مذکور مشاهده شد. 30و تعداد مراکز مالنوماکروفاژ در بافت کبد در پایان دوره اندازهمتوسطی در گذارد.برای ماهی کپور بسیار سمی بوده و اثرات شدیدی بر اندام کبد، میزان رشد و پارامترهای بیوشیمیایی این ماهی می .کبد ،شناسی آسیب بیوشیمیایی، پارامترهای رشد، پارامترهای کپور، :کلمات کلیدی int. j. aquat. biol. (2020) 8(4): 281-287 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article stock enhancement and density-dependence of caspian kutum (rutilus kutum kamensky, 1901) in iranian waters of the caspian sea hasan fazli*,1gholam reza daryanabard caspian sea ecology research center (cserc), iranian fisheries science research institute (ifsri), agricultural research, education and extension organization (areeo), iran. s article history: received 27 may 2020 accepted 9 july 2020 available online 2 5 august 2020 keywords: fingerlings benthic production ricker model caspian kutum abstract: the caspian kutum (rutilus kutum kamensky, 1901) is an important economic species in the southern, uniquely iranian waters of the caspian sea. the present study aimed to assess the desirable levels of the caspian kutum for stock enhancement with two scenarios using fingerlings released (fr) and recruitments (r) density-dependence and macrobenthic production (p). based on the results, in the years 1989-2018, the fr increased from 72 million in 1989 to 400 million in 2009 and then declined to 176 million in 2018. in contrast, the r with a lag of 2 years declined from 44.53 million in 1991 to 25.77 million in 1998, increased to 65.07 million in 2005, and then collapsed to 25.01 million in 2018. based on fr-r relationships of ricker and segment regression models, the lowest level of fr, which resulted in the highest r (39 million), was about 200 and 150 million fingerlings, respectively. based on the p/biomass ratio of macrobenthic species, the annual production was 241.6 thousand mt. it concluded that the desired number of the caspian kutum fingerling concerning stock enhancement could be lower than 150 million to prevent overcompensation in the iranian waters of the caspian sea. introduction the carrying capacity (cc) is a useful method to consider sustainable development in water resources management. the cc is the result of all the environmental components of the ecosystem, densitydependent (dd) factors, and sets the long-term maximum level of population density. according to einum and nislow (2005), if the population size of an area approaches the cc, emigration, and mortality will increase. after adjusting to a specific condition, mortality is influenced mainly by density-dependent factors (milner et al., 2003; su et al., 2004). stock enhancement is releasing of the cultured juveniles to increase the natural stock of juveniles and optimize harvest. stock enhancement of caspian kutum, rutilus kutum, in iranian waters of the caspian sea (iwcs) is conducting on a massive scale since 1989 (abdolhay et al., 2011; fazli et al., 2012). the caspian kutum is a bottom feeder and migratory anadromous species. this fish contains *correspondence: hasan fazli e-mail: h.fazli@areeo.ac.ir >70% of the annual iranian bony fish catches in the caspian sea (yousefian and mosavi, 2008; fazli et al., 2013). it has mostly recorded in the southern waters of this sea, especially iranian waters (valipour and khanipour, 2006). with regards to the recent collapse of two species of kilka and sturgeon stocks (khodorevskaya and krasikov, 1999; pourang et al., 2016; tavakoli et al., 2019) in the caspian sea, catch of caspian kutum (as an endemic species) is also declined in iran in recent years. from the 1950s to the 1980s, the annual catch of caspian kutum ranged 100-2100 mt. in this period, the seawater levels declined, which caused a drastic decline in the stock of caspian kutum (valipour and khanipour, 2006). since 1981, after a decrease in the stock of caspian kutum, the iranian fisheries organization (ifo) has been started artificial propagation for the restocking of this species in the iwcs. the fingerlings releasing increased from 2 million in 1981 (yousefian and mosavi, 2008) to more 282 fazli and daryanabard / stock enhancement and density-dependence of caspian kutum than 100 million in 1993 (abdolhay et al., 2011). afterward, the catch of iran reached >10000 mt and reached to the highest level in 2007-2008 (17,200 mt) (fazli et al., 2012). ifo has successfully done the restocking of caspian kutum in the iwcs, and abdolhay et al. (2011) concluded that due to the restocking program, the landings from capture fisheries as well as the number of fishers have increased. in contrast, fazli et al. (2012) reported that there is a significantly negative correlation between fingerlings released and condition factor. they concluded that more research needs to determine the desired level of artificial propagation. also, perceiving how the density-dependence of caspian kutum has impacted by anthropogenic activities (e.g., fishing and stock enhancement) is required for reasonable stock management. therefore, the main objective of the present study was to assess the desirable levels of the caspian kutum in the stock enhancement with two scenarios using (1) fingerlings released (fr) and recruitments (r) densitydependence and (2) macrobenthic production (p) in the iwcs. materials and methods study area and input data: the caspian sea, with an area of about 436×103 km2, can be divided into three parts, including the northern, middle, and southern. this study focuses on the deeper southern part i.e. iranian waters (fig. 1). two different scenarios are applied, the “recruitment/fingerlings” and the “macrobenthic production” scenarios. for scenario (1) two datasets used: r numbers and fr over the period of 1989-2018. the data series achieved from the final reports of the iranian fisheries science research institute (ifsri). the r numbers calculated by dividing the biomass at age 2 (the youngest fish in the catch; table 1) by the average weight of the age (estimated from growth parameters and length-weight relationship equations). one of the primary goals of the iranian fisheries organization (ifo) is to produce fingerling and releasing them in the rivers of the caspian sea (abdolhay et al., 2011). the data on released fingerlings used as input data (table 1; http://www. shilat.com). nowadays, due to degradation of the spawning ground, most of the stocks of caspian kutum (more than 90%) have been recovered by artificial propagation. therefore, in the present study, the fr set as spawning stock biomass (ssb) in the relationship between r and ssb. the data on released fingerlings used as input data (table 1; http://www.shilat.com). the fr-r models: the fr-r relationships of caspian kutum were estimated from time series of fingerlings released and recruitment. among several functions to fit fr-r, three models are used in this study as follows: (1) ricker fr-r model fit: 𝑅 𝐹𝑅 = 𝑎exp(−𝑏𝐹𝑅) table 1. the number of caspian kutum fingerlings released and recruit numbers in iranian waters of the caspian sea during the years 1989-2018. year fingerlings 106 biomass (mt) at age 2 year recruit num-bers (106) at age = 2 1989 72.0 1990 84.3 1991 109.8 5526.6 44.53 1992 96.6 5315.3 42.83 1993 100.0 4400.7 35.46 1994 142.7 4193.1 33.78 1995 125.1 4075.6 32.84 1996 142.1 3905.0 31.46 1997 154.4 3698.0 29.80 1998 143.3 3197.9 25.77 1999 147.8 3461.6 27.89 2000 147.4 4508.0 36.32 2001 233.0 5610.1 45.20 2002 225.2 5915.5 47.66 2003 155.5 7311.0 58.91 2004 179.4 7769.4 62.60 2005 229.1 8076.4 65.07 2006 174.5 7080.7 57.05 2007 262.5 6797.5 54.77 2008 187.1 6447.6 51.95 2009 400.5 6261.0 50.45 2010 279.5 6167.2 49.69 2011 272.7 5269.6 42.46 2012 249.6 4145.1 33.40 2013 200.9 3819.3 30.77 2014 176.3 3893.6 31.37 2015 185.6 3980.7 32.07 2016 192.6 3590.1 28.93 2017 3323.9 26.78 2018 3104.5 25.01 283 int. j. aquat. biol. (2020) 8(4): 281-287 (2) beverton-holt fr-r model fit: 𝑅 𝐹𝑅 = 𝑎 1 + 𝑏𝐹𝑅 (3) segmented regression fr-r model fit: 𝑅 = 𝑖𝑓 𝐹𝑅 ≤ 𝑏 𝑡ℎ𝑒𝑛 𝑎 × 𝐹𝑅 𝑒𝑙𝑠𝑒 𝑎 × 𝑏 where a is the intercept and b is the negative initial slope. all models with assuming lognormal error structures carried out using the fisheries library in r (flr) framework (kell et al., 2007). the relationship with the best overall fit based on the akaike information criterion selected the best fitting. in this study, the recruits are the fish of age two years; hence, the lag between fr and recruits is two years. the starting years for fl is 1989, whereas for recruits it is 1991. caspian kutum is a bento-pelagic species and carnivorous. this fish feeds on a variety of prey items, however, bivalves and cirripeds as the main prey groups consisting of 80% of the diet (bandpei et al., 2009). therefore, for scenario 2, the archival data were used on macrobenthic biomass collected in the years 2008-2010 (soleimani roudi, 2013; hashemian kafshgari et al., 2015) to estimate productivity. the data were collected seasonally using veen grab sampler (0.1 m2) at eight transects (astara, anzali, sefidrood, tonekabon, noshahr, babolsar, amirabad and torkman) with five stations at each transect located depths 5, 10, 20, 50 and 100 m (fig. 1). a rapid method suggested by nilsen et al. (2006) and gray and elliott (2009) used to estimate annual production. the production/biomass, p/b ratio set to 0.75, 0.35, and 0.35 for polychaeta, mollusca, and total macrobenthic, respectively. to estimate the biomass of caspian kutum based on macrobenthic annual production, the biomass transfer efficiency between macrobenthic and caspian kutum set between 10 and 20%. results for scenario one, under recruitment (r) and fingerlings released (fr), the fr was 72 million in 1989, and the r with a lag of 2 years (1991) was 44.53 million. the fr had two trends, first, increased from 72 million (in 1989) to the highest level (400 million) in 2009, then declined to 176 million in 2018. in contrast, r had different trends. during the years 1991-2018, it was declined to 25.77 million in 1998 and then increased to the highest level i.e. 65.07 million in 2005. in 2011, it was 42.46 million, and then it declined to the lowest level (25.01 million) in table 2. the annual mean wet weight of different macrobenthic groups in of iranian coastal waters of the caspian sea during 2008-2010. area surface km2 mean wet weight (g/m2) bivalvia polychaeta total macrobenthos astara 3705.5 40.22 3.40 52.56 anzali 682.9 15.28 1.52 22.43 sefidrood 814.7 13.48 1.49 17.65 tonekabon 1269.7 11.00 1.83 17.35 nohahar 1023.9 13.75 2.50 17.64 babolsar 1400.2 37.07 1.17 42.97 amirabad 1691.8 45.96 2.86 53.32 torkman 3876.5 26.88 3.37 59.41 figure 1. map of macrobenthic sampling stations in the iranian waters of the caspian sea. 284 fazli and daryanabard / stock enhancement and density-dependence of caspian kutum 2018 (fig. 2). three different fr-r relationships, ricker, beverton–holt, and segment regression models, were very similar (fig. 2). this study showed that the lowest level of fr, which resulted in the highest r (about 39 million), was about 200 and 150 million fingerlings for ricker and segment regression models, respectively. however, the ricker model resulted in giving the overall best fit for fingerlings and r of caspian kutum, based on the aic (table 3). for scenario two, under the macrobenthic p/b ratio of polychaeta, mollusca, and other microbenthic species, the annual production in eight strata was estimated (fig. 3). the results showed that the minimum of production occurred in the anzali strata, and the maximum one in torkman and astara strata in east and west regions, respectively. in general, the annual production of polychaeta, mollusca, other groups, and total macrobenthos was 29.5, 151.3, 60.8, and 241.6 thousand mt, respectively. if the biomass transfer efficiency is 10-20 and 80% of the caspian kutum diet belonged to macrobenthic, the annual biomass of caspian kutum could be about 20-40 thousand mt. based on table 1, the average r/total biomass ratio of caspian kutum is 12.1%, and the r estimated numbers ranged between 20 and 38 million. discussions this study indicated that during the last three decades, over restocking of caspian kutum have been occurred in the iwcs. several marine fish populations have strong density-dependent (myers and cadigan, 1993; andersen et al., 2017; mirzaei et al., 2019). densitydependent regulation is related to the size of the habitat. in small habitats (e.g., lakes), the regulation happens late in life, when exploiting the juveniles fish maximizes yield. in contrast, in marine habitats, the regulation is occurred early in life, when the yield maximized by the exploitation of matures (andersen et al., 2017). overcompensation is a competition between juveniles and adults when juveniles are the superior competitors (de roos et al., 2008), usually occurred is small habitat, results in stunted growth (andersen et al., 2017). therefore, caspian kutum exists mostly in the iwcs (haghi vayghan et al., 2013, 2015; valipour and khanipour, 2006) has a small habitat with late in life regulation. persson et al. (2007) reported that the presence of predators (e.g., sturgeon fishes, caspian seal (phoca capsica), pike-perch (sander lucioperca) in the case of the caspian sea) can decrease the population from the density-dependent competition and remove the stunting. however, several works (raisa khodorevskaya et al., 2014; pourang et al., 2016; cites, 2017; fazli et al., 2017; tavakoli et al., 2019) indicated that during the last decades the top predator's stocks collapsed in the caspian sea. therefore, at least during the two last decades, the predators could not affect the density of caspian kutum in the caspian sea. according to fazli et al. (2012), during 1991-2011, the average condition factor of both sexes of caspian kutum had a decreasing trend, and they concluded that table 3. the estimates of fingerlings released-recruitment, fr-r relationships using ricker, beverton-holt and segmented regression models for rutilus kutum in iranian water of the caspian sea. models performance ranked by ascending order of aic. model log-likelihood aic ricker r = 5.1×10-4 × fr × exp(-4.8×10-9 × fr) 23.508 -43.015 beverton-holt r = 46198 × fr/(31651333 + fr) 23.102 -42.204 segment r = if fr≤150×106 then 2.6×10-4 ×fr else 2.6×10-4 ×150×106 23.066 -42.132 figure 2. fingerlings released–recruitment (fr–r) relationships for rutilus kutum in the caspian sea. ricker fit (dash line), beverton–holt fit (dotted line) and segment fit (solid line). 285 int. j. aquat. biol. (2020) 8(4): 281-287 condition factor is adversely influenced by the number of fingerlings released (fazli et al., 2012) which confirm the results of the present study. the average depth of the northern part is about 3.5 m, comprised of fresh-water benthic species. the average depth of the middle and southern parts is 190 and 300 m, respectively, and occupied by brackishwater benthic species (khodorevskaya et al., 2009; karpinsky, 2010). the most concentration of benthos biomass (more than 95%) was reported at depths <100 m. the highest biomasses are observed at the eastern and western coast of the middle caspian sea (more than 1.0 kg/m2). in contrast, the lowest biomass (less than 30 g/m2) remarked in the southern caspian sea (khodorevskaya et al., 2009) at depths <100 m, whereas the most stocks of caspian kutum settle here. furthermore, since the 1990s, the new invader (mnemiopsis leydei) affected all components of the caspian sea (pourang et al., 2016) and its environment shifted to new conditions (kashkooli et al., 2017). during this period, the most important anthropogenic pressures are such as invasive species and pollution, which have affected in abundance and then extinction of some benthic species (lattuada et al., 2019). the primary benthos consumers are sturgeon fishes and cyprinids in the caspian sea. the fry of all sturgeons feed on crustaceans and worms and adult sturgeon of russian sturgeon, acipenser gueldenstaedtii, and persian sturgeon, a. persicus prefer to feed on mollusks. it revealed that during very long periods at depths of 100 m, the benthos biomass and species diversity are sharply decreased due to the influence of severe grazing pressure by sturgeons (karpinsky, 2010). however, during a few decades, the sturgeon stocks collapsed to the lowest level and even extinction (cites, 2017). on the other hand, the catch of other benthos feeders such as common carp (cyprinus carpio) and roach (rutilus lacustris) had a decreasing trend. the portion of these species from the total catch of bony fishes declined from 17.5% in 1997 to 3.5% in 2013 in the iwcs. these decreases could be the influence of severe grazing pressure by the over fingerlings released of caspian kutum during the last years. in conclusion, due to changing the ecosystem of the caspian sea, anthropogenic pressures, and extinction of benthic species, to protect this unique habitat, it actively encourages to decline the fingerlings released of caspian kutum. based on two scenarios in the present study, fr-r relationship, and macrobenthic production, the desirable number of the caspian kutum fingerling concerning stock enhancement is lower than 150 million. acknowledgments this research was funded by the iranian fisheries science research institute (ifsri). we thank the staff figure 3. annual macrobenthic production in different strata in the iranian waters of the caspian sea. 286 fazli and daryanabard / stock enhancement and density-dependence of caspian kutum of the department of stock assessment at the caspian sea ecology research center, inland water aquaculture research center and inland water stocks research center of ifro for providing samples used in this study. references abdolhay h., daud s., ghilkolahi s.r., pourkazemi m., siraj s., satar m.a. 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(2020) 8(4): 253-261 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article diversity in attachment devices of aquatic insects in a torrential hill stream of mid himalaya rajinder jindal1, devender singh*1, 2 1department of zoology, panjab university, chandigarh-160 014, india. 2department of zoology, govt. college, bhoranj (tarkwari), hamirpur, himachal, india. s article history: received 10 january 2020 accepted 8 june 2020 available online 2 5 a u g u s t 2 0 2 0 2020 keywords: adaptations adhesive apparatus sem slope abstract: present study was carried out to explore the morphological attachment devices od seven aquatic insect larvae or naiads (representing four orders of ephemeroptera, plecoptera, trichoptera, and hemiptera) along the swift water current of binwa, a mid-himalayan hill stream using scanning electron microscopy (sem) images. water current showed direct relation with slope/gradient and volume of water in stream. samples were collected at four sites located at different attitude along the stream. the collected larvae or naiads have many morphological features, including adhesive pad, friction pad, serrated tarsal claw, bifid tarsal claw, tarsal claw with clamp, sucker, and serrated spines which serve as their adaptations for the fast-flowing waters. principal component analysis revealed the importance of water current for assemblage and abundance of these aquatic insects, as it directly related to tds, electric conductivity, turbidity and nutrients level in stream water, and cumulatively affect these aquatic macroinvertebrates. similarities in structures of different species suggest the convergent evolution, while slight differences inferred as specializations for species specific niche of these organisms in stream habitat. introduction streams play an important role in the ecology of rivers and reservoirs (sharma, 2019). hill streams of mid himalayan region are swift, beside high-water velocity; support a vast community of the macroinvertebrates, in which aquatic insects dominate (goswami and singh, 2018). aquatic insects represent just a fraction of class insect but taxonomically they are very diverse. they have remarkable ability to attach with the substratum and works of some workers in the line of attachment devices are worth mentioning (nielsen, 1950; merrit and cummins, 1996; nelson, 2009; ditsche-kuru et al., 2010). little information is available on attachment devices used by insect larvae or naiads of western himalayan streams, so the present study on aquatic insect fauna of binwa stream has been undertaken. in this study, scanning electron microscopy (sem) was made on baetis sp., ecdyonurus sp., baetis himalayana, b. bifurcatus, *correspondence: devender singh e-mail: devendergndu@gmail.com cryptoperla sp., rhyacophila sp. and naucoris sp. on tarsal claws and ventral side of these organisms, which were supposed to help in attachment on substratum. adhesive apparatus, friction pads, tarsal claws with comb like serration and setae were reported, which might be useful for their survival by helping in attachment, clinging and resistance generation (frictional) in the swift waters. materials and methods study area: for the present study, collections have been done from the binwa, a hill stream of the dhauladhar range in north-west part of india. from its head to its mouth, the binwa stream covers a distance of about 48 km. four observation/collection sites have been selected on the basis of altitudinal differences i.e. kharli as s1 (2822 m above msl), baijnath as s2 (945 m above msl), near chobin as s3 (746 m above msl) and triveni as s 4 (572 m above msl) (fig. 1, table 1). the main habitat 254 jindal and singh / attachment devices in aquatic insects is comprised of runs and riffles with some pools and a substrate of boulders and cobbles. in some regions, the riparian vegetation is dense, which adds allocathonous organic matter to the stream, and serves as feeding material for the larval forms. drainage network in the study area, all the streams/nullah have been ordered as per strahler’s scheme (1957) and rosgen (1996). as longitudinal profile is constructed along the flow line of a stream, thus it effectively reflects the change in downward slope and knick points. slope of the river bed governed the changes in characteristic of water current such as formation of run, riffles and pool. similarly, the longitudinal profile of binwa (fig. 2) has been constructed from head (upstream kharli) to its mouth (at triveni) covering all four observation sites. this profile has been created by using 30 m digital elevation model (dem) of the study area and qgis 3.4 software. field collection: aquatic insects and water samples have been collected and observed on four sites for a period of two years i.e. march-2011 to feb-2013. water samples were analyzed for different parameters such as electric conductivity, tds, turbidity, dissolved oxygen, ph, nitrates and phosphates (apha, 1998). as the water current is the most important parameter for determining the diversity and abundance of aquatic insects in fast waters, therefore only the monthly average values of it given (table 2). during the present study, water current measured in the field on monthly basis using a float, and the time of the float was noted between the known distance, and then water current was measured using formula v = d/t, where v = velocity, d = distance traveled by table 1. stations and associated geographic characteristics. observation sites elevation distance from previous station (in m) slope in degree entrenchment ratio width/depth ratio head of the river 3560 nil nil 1.8 12.7 s1 (kharli) 2822 2200 18.55 1.4 8.8 s2 (baijnath) 945 18200 5.87 2.2 14.6 s3 near chobin 746 15700 0.689 1.6 12.5 s4 (triveni) 572 13200 0.94 2.3 15 figure 1. map showing study area and catchment region of binwa stream. 255 int. j. aquat. biol. (2020) 8(4): 253-261 the float and t is the time taken by the float to travel the distance. principal component analysis have been performed using spss.16 software to observe the influence of water current and associated parameters on abundance of aquatic insect naiads. naiads were collected from cobbles, large wood debris and other hard surfaces into surber sampler net by making disturbance in the substratum of the stream, unsettling them, and then collection in bucket and at last preserved in 4% formaldehyde solution. laboratory sorting and identification of the insect larvae were accomplished based on leckhmkuhl (1979), mccafferty (1981), merrit and cummins (1996) and subramanian and sivaramakrishnan (2007) with a binocular stereomicroscope. samples were then prepared for sem studies with the following procedure: preserved specimens were washed with double distilled water (3-4 times) and dehydrated in different grades of acetone i.e. 50% (15 min), 70% (10 hrs), 90% (15 min), 100% (2 shifts each after 20 min), amyl acetate: acetone sol. (1:1) for 30 min. and shifted to amyl acetate sol. (30 min). samples were subjected to critical point drying (cpd) and stubs of the samples were prepared. a fixed sample was mounted on metal stub, either made of aluminium or brass with the help of double stick tape. care was taken to avoid trapping of air bubbles under the tape. then the sample was coated with a thin layer (100 å) of gold in a gold sputter coating unit. finally, the sample was viewed under vacuum in the jeol jsm-6100 scanning electron microscope at an accelerating voltage of 15-20 kv at low probe current. when not being examined, the specimens were stored in a desiccator. on the basis of field observations for attachment of organisms with the substratum, and the results inferred from table 2. monthly average value of water current (cm/sec.) on four sites for study period 2011-13. observation sites study period mar. april may june july aug. sept. oct. nov. dec. jan. feb. s1 (kharli)* 2011-12* 93 102 110 109 105 83 73 65 2012-13* 90 105 110 109 105 83 73 65 s2 (baijnath) 2011-12 86.45 90.5 95.7 98.5 124.8 120.5 115.6 105.4 82.5 80.12 80 85.7 2012-13 88.5 91.5 95.8 98.3 129.8 124.5 114.6 103.4 83.5 79.23 81.2 84.3 s3 near chobin 2011-12 75.2 80.5 85.2 89.1 113 110.5 105.4 94 72.4 70 67.3 71.2 2012-13 74.5 78.5 83.2 87.1 114.5 108.5 103.4 96.5 71.5 68.7 67.4 70.4 s4 (triveni) 2011-12 70 72.1 72.5 78.6 90.7 96.4 81 72.1 70.2 60.1 65.3 72.4 2012-13 71 72.3 72.7 79.1 91.2 96.5 81.3 72.5 70.3 61.2 64.5 73.1 *water samples were collected only for eight months (april-november) due to non-accessibility in winter months. figure 2. generalized longitudinal profile of the binwa stream. 256 jindal and singh / attachment devices in aquatic insects sem studies, imaginative drawings have been drawn to show the suction generation, clinging and attachment mechanisms of these organisms with substratum. results longitudinal profile of the binwa stream throws light on characteristics of terrain along the river bed (fig. 2). upstream of s1 the slope of the stream bed was highest i.e. ~ 19° while between s1 to s2, s2 to s3, and s3 to s4 it was ~ 6°, 0.7° and 0.9°, respectively (table 1). beside longitudinal profile of channel, sinuosity (1.4), average width/depth ratio (12.7) and average entrenchment ratio (1.8) categorized binwa stream as b3-type (rosgen, 1996). as stream channel is narrower, high slope and have low sinuosity upstream, this implies high water discharge as well as high velocity (table 1). upstream boulders were numerous, and relatively more angular and rougher. roughness of boulder surface and angularity relatively decreased downstream. additionally, the progressive decrease was observed in the average size of river bed stone especially downstream. at s3 and s4 cobbles and pebbles were dominating the river bed with gradual increase in the proportion of sand. gradient of the table 3. seasonal rainfall record (in mm) of himachal pradesh and kangra for study period (2012). study period jan. feb. mar. april may june july aug. sept. oct. nov. dec. 2012 in hp* 112.1 66.4 43.2 64.1 11.5 25.5 207.1 311.6 152.1 2.9 6.7 31.8 2012 in kangra* (season wise) 216.5 100.5 1889.9 44.7 *data taken from india meteorological department, ministry of earth science, govt. of india (kaur and purohit, 2012). figure 3. principal component analysis (pca) of different physico-chemical parameters and aquatic insects of binwa stream: a. (s1), b. (s2), c. (s3), and d. (s4). (abbreviations: do dissolved oxygen, ta total alkalinity, th total hardness, wt water temperature, wc water current, cond. electric conductivity, turb. turbidity, tds total dissolved solid and aq.ins. aquatic insects) 257 int. j. aquat. biol. (2020) 8(4): 253-261 stream bed is in direct relation with water velocity. it is evident from above observations that water velocity is very high in the stream, to prevent their wash away in swift waters of stream, insect naiads have slight modifications in their structures which might be used as adaptations. water current in the stream was greatest during monsoon period (julyaug) as more rain recorded during this time period figure 4. different attachment devices in the studied aquatic insect naiads (e) baetis bifurcatus, (f) baetis himalayana, (g and h) cryptoperla sp., (i) rhyacophila sp., (i) naucoris sp. and (j) baetis himalayana. (abbreviations: cl-claw, s-spine, ug-unguitractor, sst-serrated setae) 258 jindal and singh / attachment devices in aquatic insects of year (tables 2, 3). pca shows the clustering of factors influenced by water current, as their maxima in same season when water current observed highest. surface runoff brings with it so many nutrients (no3 and po4), ions and solid waste into the stream during monsoon. therefore, during monsoon high water current is responsible for increased values of electric conductivity, tds and turbidity. aquatic insects inversely affected by these factors. abundance of aquatic insects observed in winter when water velocity was moderate, which pose less danger of their wash away and at high do due to low water temperature (fig. 3). scanning electron microscopy of tarsal claws with serration on inner side, and a sensilla near the upper surface of claw of baetis bifurcatus and b. himalayana are represented in figure 4e, f. in plecopteran, cryptoperla sp. has strong, paired (bifid) tarsal claw (fig. 4g) and proleg claw (fig. 4h). in trichopteran, rhyacophila sp. bears tarsal claw with clamp like structure (fig. 4i); hemipteran, naucoris sp. has long, thin, paired mesoleg and metaleg tarsus (fig. 4j). friction pads in ecdyonurus sp. on lateral sides, constituted of numerous serrated setae (fig. 5b, d). in b. himalayana, suckers and serrated setae are reported on ventral side near the external gills (fig. 5c). adhesive apparatus in baetis sp. that is used to make stronger contact with rough substratum (fig. 5a), is reported first time. discussions the diversity and abundance of aquatic insects’ naiads in torrential waters is determined by two main factors (i) water current and (ii) substratum. the biological significance of rate of water current has been emphasized by many workers (thorp and covich, 2001; jindal et al., 2012). water current is also influenced by two factors viz. (1) rate of precipitation that determines volume, and (2) slope of the substratum. high water current poses physical and mechanical danger to larvae and it also wash away the nutrients (nelson, 2009; jindal et al., 2012). in the upper parts of the studied catchment, terrain is very rugged and steep. steepness of the slope decreases dramatically downstream so as the water velocity. from its head to its mouth, the binwa covers a distance of about 48 km. however, in this relatively short distance it experiences an impressive fall of about 3100 m (3560 m elevation at its head and 539 m figure 5. different attachment devices in the studied aquatic insect naiads (a) baetis sp., (b and d) ecdyonurus sp. and (c) baetis himalayana. (abbreviations: ap-adhesive apparatus, su-sucker, sst-serrated setae, fp-friction pads, s-spine) 259 int. j. aquat. biol. (2020) 8(4): 253-261 at its mouth). therefore, it experiences a vertical fall of about seven meters for a horizontal distance of every 100 m, as one moves downstream from the head of the river. in terms of slope, the average slope of the river bed is about 4°. however, this fall in elevation is not uniform along the entire stretch of the river as is evident. fall in the elevation of river bed is the highest in the upper most segment of the stream (upto a distance of ~4 km from the head). it is moderate in middle part (from a distance of ~4 to 15 km) and the least in lower part of the profile (downstream from a distance of ~15 km from head to the mouth of the river). in other words, gradient of stream bed is highest in the upstream and it decreases exponentially downstream, however, there is slight increase in the gradient of river bed at the terminal part of catchment which may be attributed to deep vertical erosion by relatively much larger beas river near the mouth of binwa stream. evidence in this regard is provided by presence of ~60 m high river terrace at the mouth of the stream. as a result, to keep pace with level of trunk stream i.e. beas river, binwa has excavated the earlier mentioned river terrace by down cutting it and hence created relatively steeper slope. the stream slope/gradient decreases from upstream to downstream, this is in consonance with singh et al. (1993). on the basis of our findings, three types of approaches have been reported, and inferred from field observations and literature consulted as: (i) clinging devices (tarsal claw), (ii) frictional devices (different type of setae and friction pads), and (iii) fixing devices (sucker). hora (1930) while studying the torrential fauna of the indian subcontinent, reported particular forms and shapes (dorso-ventraly flat and streamlined body) and curved claws in aquatic insects. in b. bifurcatus and b. himalayana tarsal claws have comb-like serration on inner side, which might increase the surface area for attachment on rough substratum, and a sensilla was noticed near the claw, help to judge the environment. similar serrated claws have been reported in epeorus assimillis as an attachment device with rough surface of substratum (ditsche-kuru et al., 2010). in plecopteran, cryptoperla sp. have strong, paired tarsal claw and proleg claw that increase the area and reach for anchoring on substratum and applying in unsettling the substratum during detritivore feeding, respectively. rhyacophila sp. possesses a tarsal claw with clamp like structure for helping in clinging and holding the rough substratum. hemipteran, naucoris sp. has long, thin, paired mesoleg claw that can help in clinging and climbing the macrophytes, and metaleg tarsus act as oar during swimming. an imaginative diagram showed how the tarsal claws of ephemeropterans and trichopteran employed for clinging and holding the rough substratum in figures 6 and 7). similar claws and hooks have been reported in caddisfly (waringer, 1993), mayfly (kellog and kellog, 1994), and in stonefly (gorb, 1996; merrit and cummins, 1996; downes et al., 2000; beutel and gorb, 2001; nelson, 2009) as attachment devices on substratum. friction pads were observed in ecdyonurus sp. on figure 6. interactions between tarsal claw and different type of substrate ranging from smooth to rough. figure 7. interactions of tarsal claw of ephemeropterans and trichopteran naiads with rough substratum. 260 jindal and singh / attachment devices in aquatic insects lateral sides, constituted of numerous serrated setae, that can increase the frictional force and prevent the wash away by withstanding the water drag. in b. himalayana, suckers and serrated setae are found on ventral side near the external gills. the sucker help in attachment by generating suction, while serrated setae form fine contact with minute irregularities/unevenness of substratum and prevent weakening of suction by water rinsing (avoid water to reach upto sucker rim, as water weak the contact of rim with substratum) the sucker (see the imaginative diagram in fig. 8). in himalayan hill stream, similar sucker was reported in isoperla sp. (johal et al., 2011). the adhesive apparatus is reported first time in baetis sp., and is used to make firm contact with exposed rough substratum during sitting or resting position. as these observations were made on immature stages of aquatic insects, so these claws are not sexual dimorphism. it is clear that tarsal claw can easily cling to rough surface than smooth surface and roughness increases their chances of survival. most of these structures such as serrated setae, spines, sucker, adhesive apparatus and tarsal claws exert a cumulative effect to fix/attach these naiads with the substratum, and with stand the water force (jindal et al., 2012). while during winter, when water current is low their abundance observed in the stream (sharma and dhanze, 2012; jindal and singh, 2013). presence of similarity in the structures, such as tarsal claws, sucker, spines and serrated setae, represents convergence for same habitat. while slight variations such as tarsal claw of ephemeropteran have comb-like teeth on inner side, a clamp like structure for holding on inner side of trichopteran claw, bifid or paired tarsal claw in plecopteran help these organisms to explore different niches in the stream such as riffles, pools and swift velocity region. ephemeropterans with comb-like serration on inner side of tarsal claw and trichopteran with clamp like structure enable them to explore the riffle section of the stream where water velocity is high and they can roam on the exposed surface of rock to water current. while elongated and slender shaped meso-leg tarsal claw help hemipteran to climbing, and meta-leg modified for swimming. adhesive pad and friction pads help in fixing the organisms on substratum and resisting the water drag. most of these organisms inhabit the upstream (high slope, more water current) and the centre of the channel (high velocity with more volume) region. as this section (centre of channel) has least chance of predation, maximum chances to get food (as wash away material with high velocity), might be the reason for their adaptation to high water velocity. these structures are modified and arranged in such a way, so that naiads can cling with minute crevices, increase their area of attachment, increase the frictional forces and help to fix these organisms on substratum. conclusion entire course of binwa stream from its head to middle part is riffled and water flow is turbulent and very rapid. studied insects have developed many morphological characteristics namely adhesive pad, friction pad, serrated tarsal claw, bifid tarsal claw, tarsal claw with clamp, sucker, and serrated spines that act as adaptation mechanisms against the swift waters and serve as resistance increasing, hooking and vacuum generating devices. while adhesive apparatus reported for the first time in baetis sp. of western himalayan stream and it cumulatively increases the survival rate of these insect naiads in swift waters of western mid himalaya. acknowledgements the authors are thankful to the chairperson, department of zoology, panjab university, figure 8. image of sucker operating on the smooth surface as in baetis himalayana. 261 int. j. aquat. biol. (2020) 8(4): 253-261 chandigarh, for providing necessary research facilities; a. kumar and g.c bhoranj for helping determination of slope and the director central instrumentation lab, panjab university, chandigarh for providing sem facilities. references apha. (1998). standard methods for the examination of water and waste water, 20 ed. american public health association, washington, dc, 874p. beutel r.g., gorb s.n. (2001). evolution of locomotory attachment pads of hexapods. naturwissenschaften, 88: 530-534. ditsche-kuru p., koop j.h.e., gorb s.n. (2010). underwater attachment in current: the role of setose attachment structures on the gills of the mayfly larvae epeorus assimillis (ephemeroptera, heptageniidae). the journal of experimental biology, 213: 1950-1959. downes b.j., lake p.s., schreiber e.s.g., glaister a. (2000). habitat structure, resources and diversity: the separate effects of surface roughness and macroalgae on stream invertebrates. oecologia, 123(4): 569-581. gorb s.n. (1996). design of insect unguitractor apparatus. journal of morphology, 230: 219-230. goswami g., singh d. (2018). water quality and function of mandakini river ecosystem of central himalaya. international journal of biosciences, 12(6): 102-116. hora s.l. (1930). ecology, bionomics and evolution of the torrential fauna, with special reference to the organ of attachment. philosophical transactions of the royal society of london, series b, 218: 171-282. jindal r., singh d. (2013). biodiversity of seer stream (district bilaspur, h.p.) in relation to hydrobiological factors. ecology enviroment and conservation, 19(2): 245-249. jindal r., singh d., sharma c. (2012). sem studies on morphological adaptations of trichoptera and ephemeroptera of a western himalayan hill stream. international journal of environmental sciences, 2(4): 2454-2461. johal m.s., kumar m., rawal y.k. (2011). ultrastructure of adhesive organs of two aquatic insects inhabiting highland hillstreams. journal of environmental and bio-sciences, 25(1):15-19. kaur k., purohit m.k. (2012). rainfall statistics of india2012. hydromet division, india meteorological department (ministry of earth science), lodi road, new delhi. kellogg l., kellogg l. (1994). monitor’s guide to aquatic macro invertebrates, izaak walton league of america gaithersburg, md. 60 p. leckhmkuhl d.m. (1979). how to know the aquatic insects. i.a. dubuque (ed.), wm. c. brown company publishers. 168 p. mccafferty w.p. (1981). aquatic entmology: the fisherman’s and ecologist’s illustrated guide to insects and their relatives. science books international, boston, ma. 448 p. merritt r.w., cummins k.w. (1996). an introduction to the aquatic insects of north america, 3rd ed. kendal / hunt, duduque, ia. 862 p. nelson c.h. (2009). surface ultrastructure and evolution of tarsal attachment structures in plecoptera (arthropoda: hexapoda). aquatic insects, 31(1): 523-545. nielsen a. (1950). the torrential invertebrate fauna. benjamin / cumming, mento park, california. pp: 127-131. rosgen d.l. (1996). applied river morphology. pagosa springs, colorado. 385 p. sharma i., dhanze r. (2012). evaluation of macrobenthic fauna in hill stream environment of western himalaya, india. journal of threatened taxa, 4(9): 2875-2882. sharma i. (2019). ichthyodiversity of beas river system north western himalaya (h.p.), india. journal of environmental and bio-sciences, 33(1): 11-17. singh n., bahuguna s.n., bhatt k.c. (1993). the profile of river ecosystem, food and feeding habits of hill stream fishes and consequences of recent environmental degradation in garhwal himalaya. acta ichthyologica et piscatoria, 23(1): 3-30. strahler a.n. (1957). quantitative analysis of watershed geomorphology. transactions of american geophysical union, 38: 913-920 subramanian k.a., sivaramakrishnan k.g. (2007). aquatic insects of india-a field guide, ashoka trust for ecology and environment (atree), bangalore, india. 60 p. thorp j.h., covich a.p. (2001). ecology and classification of north american freshwater invertebrates, 2nd ed. acadmic press, san diego, ca. 1056 p. waringer j.a. (1993). the drag coefficient of cased caddies larvae from running waters: experimental determination and ecological application. freshwater biology, 21: 411-420. int. j. aquat. biol. (2013) (1): 33-35 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology effects of season and altitude on copper and zinc concentrations in benthos (chironomids and gammarids) and sediment arash javanshir *1 department of fisheries, faculty of natural resources, the university of tehran, p.o. box: 31585-4314, karaj, iran. article history: received 6 march 2013 accepted 12 april 2013 available online 14 april 2013 keywords: heavy metals, gammarus pluex chironomidae namrood river abstract: concentration of heavy metals in aquatic ecosystems is considered as an important environmental issue. in this study, the namrood river located in firoozkooh (tehran province, iran) was assessed for the existence of heavy metals. the namrood river is situated by the main road being by pollutants from tourism and recreational centers, gas stations, sewage of villages, agricultural wastewater and fish culture effluent. the water is extremely contaminated in some parts and possibly contains heavy metals. in the present study, two stations upand downstream were determined to sample the sediments and chironomidae and gammarus pluex in both cold and warm seasons of the year (middle of march and middle of august). the copper and zinc were measured in sediment and benthos. the results showed that copper and zinc concentrations ranged 0.170-0.966 and 0.187-3.846 ppm, respectively. sediments of the upstream station had the highest copper concentration among the samples in both cold and warm seasons of the year. introduction heavy metals are one of the components of the earth crust. they are a group of elements with atomic weights of 63.546 – 200.59 whose special weight is more than 6 g/cm3. they exist in the biospher following to natural source or anthropogenic ones (javanshir and shapoori, 2011). selecting biological indices is considered as one of the most important biological assessments which is used to determine the qualities of the water bodies. biological indices may be live organisms, which are able to live and survive according to their adaptations in various environments (pennak, 1979). otherwise, they cannot survive and are eliminated from the ecosystem (wright, 1995). macrobenthic animals may be used in indices that are used to monitor the sewage entering aquatic ecosystems. their biomass and species diversity is lower than those of planktons due to their longer lifetime. therefore, benthic macrofauna can reflect effects of all kinds of * corresponding author: arash javanshir e-mail address: arashjavanshir@hotmail.com wastewater and sewage (hilsenhoff, 1981). even frequent sampling (once or twice per months) shows significant changes of water status. the namrood river is located in southern part of alborz mountain in northern iran. it is a permanent river thus benthic animals may have a complete life history. the river covers a catchment area of about 51 km2, whilst melting of snow, originating from mountains, constitutes the main source of water. the present study aimed to investigate heavy metal contamination of two namrood river habitants, chironomids and gammarids. materials and methods sampling was carried out in summer (mid-june to mid-september) and winter (mid-november to midfebruary) in upstream (2101m) and downstream (1819m from sea level). in each station three points on a transect across the river were determined (two on the shallow and one on the central deep part of 34 javanshir / int. j. aquat. biol. (2013) (1) 33-35 river). samples were collected once per month. benthos and sediment were sampled using surber and ekman grab, respectively. all samples were fixed with 4% formaldehyde and kept in glass bottles of 200 ml and kept <16°c. tissue and sediment samples were prepared in laboratory following a protocol from moopan (1999). the benthos were identified using the keys from pennak (1978). heavy metals concentrations were measured using atomic absorption method. the samples were dried in an oven of 60 °c for 24 h. then, the particles smaller than 63 µm were separated using a sieve and the extraction was separated in a 200 ml glass bottle using watmann 0.45 µm filter paper. data were analyzed one-way anova followed by the duncan multiple range test. all statistical analyses were performed using spss 15 (spss inc.). results copper concentration of sediment, chironomids and gammarids suggest different concentrations from up to downstream which may differ among seasons (fig. 1). winter concentrations of copper in sediments were significantly different (p<0.05) between upstream (0.927 µg-1) to downstream (0.827 µg-1). also, there was a significant difference between upand downstream in summer (0.966 µg-1 and 0.701 µg-1, p<0.05). however, there was no significant difference in zinc of sediment among sampling stations and seasons (fig. 2). the lowest concentration of copper in chironomids bodies was measured in upstream and during the winter (i.e. 0.17 µg-1) but in other cases no significant differences were observed (fig. 1). gammarids were totally absent in upstream station contrarily to chironomid which were present in all seasons and stations. there was no difference in concentration of copper among seasons (0.469 µg-1 compared to 0.492 µg-1). concentration of zinc in gammarids, which were present in downstream show significant differences when winter is compared to summer (0.18 vs 1.61 µg-1). copper concentrations accumulated in macrobenthos were less than those of the sediments in stations and differences were significant (p<0.05). the concentrations of copper at up and down stream stations in summer and winter do not show any differences. concentration of copper in chironomids of upstream was more than those of downstream ones (p<0.05). the amount of copper accumulated in gammarids does not show a significant difference in winter and summer. zinc concentration shows fewer concentrations at downstream stations than the upstream ones. among benthic animals, downstream chironimids contained more zinc concentrations compared to upstream ones (p<0.05), but no significant differences was detected between seasons. figure 2 suggests that, zinc concentration of the upstream chironomids in the summer was more than that of the winter. such trend also can be seen in downstream station (p<0.05). 0.927 0.17 0.827 0.242 0.469 0.966 0.221 0.701 0.231 0.492 sed sed sedsedsedchir chirchirchir gam gam upstream winter downstream winter upstream summer downstream summer c o p p e r c o n c e n tr a ti o n p p m . g r1 0 1 3.84 0.76 3.15 1.45 0.18 3.58 2.63 3.22 3.61 1.61 sed sedsedsedchir chirchirchir gam gam upstream winter downstream winter upstream summer downstream summer z in c c o n c e n tr a ti o n p p m . g r1 0 4 2 1 3 figure 1. the average concentration of copper in upand downstream, sediment and benthos. sed: sediment; chiro; chironomus; gamm: gammarus. figure 2. the average concentration of zinc in upand downstream, sediment and benthos. sed: sediment; chiro; chironomus; gamm: gammarus. 35 javanshir / int. j. aquat. biol. (2013) (1) 33-35 the amount of zinc accumulation in gammarus had a significant difference in summer compared to winter (p<0.05). discussion the comparison of two main results about copper and zinc observed concentrations in up and down stream in all sampling seasons suggest that the amount of copper in sediments were higher than that of benthos, chironomid and gammarid. the exception may be observed in chironomids of downstream and in summer. at the end of summer and beginning of the fall an important amount of leaves were transferred to the river from the catchment basin. as the river originates from mountains one may suggest that the population of shredders is limited by humic and dead leaf. that is why in upstream gammarids were totally absent. in the case of copper concentration in body, gammarids showed higher concentrations than chironomids. chironomid may feed on plankton and fine particulate organic materials (fpom) (strayer, 2006; faria et al., 2007). in our study the low concentrations of copper in chironomid bodies may be due to low quantities of this metal in their food which is benthic algae (such as chantransia sp.). the bioaccumulation of copper in these algae may be less than autumnal leaves due to their short life span (tachet, 2002). this is not the case for other metal bioaccumulation such as zinc in this study. measurements suggest that the concentration of zinc in chironomids was higher than gammarids. it is possible that natural sources of zinc were transferred from upstream to downstream. chiromimids are filter feeders (feed on plankton and fpom) and thus may assimilate more zinc content than gammarids (shredders of dead leaves). zinc had a constant concentration in sediments (upstream vs downstream, p>0.5). other studies have shown that heavy metals are absorbed in different concentrations due to their concentration in ambient water (volk et al., 1997; townsend, 1989; vanni et al., 2001; javanshir et al., 2011). in conclusion, the present study showed differences between zinc concentrations in summer and winter both of them in downstream station. this can be in addition to food, stemmed from differences of solubility of this metal in cold and warm waters (javanshir et al., 2011). references faria m.s., lopes r.j., malcato j., nogueira a.j.a., soares, a.m.v.m. (2007). in situ bioassay with chironomus riparius larvae to biomonitor metal pollution in rivers and to evaluate the efficiency of restoration measures in mine areas. environmental pollution journal, 151: 213221. javanshir a., shapoori m., (2011). influence of water hardness (calcium concentration) on the absorption of cadmium by the mangrove oyster crassostrea gaster (ostreidae; bivalvia). journal of food, agriculture and environment, 9(2): 724-727. javanshir a., shapoori m., moezzi f. (2011). impact of water hardness on cadmium absorption by four freshwater mollusks physa fontinalis, anodonta cygnea, corbicula fluminea and dreissena polymorpha from south caspian sea region. journal of food, agriculture and environment 9(2): 763-767. hilsenhoff, w.l. (1981). aquatic insects of wisconsin: keys to wisconsin genera and notes on biology, distribution and species. natural history council, university of wisconsin-madison. 60 p. pennak r.w. (1978). freshwater invertebrates of united states, 2nd ed. john wiley & sons. 803 p. strayer d.l. (2006). challenges for freshwater invertebrate conservation. journal of the north american benthological society, 25: 271-287. tachet h. richoux p, bournaud m., usseglio-polatera p. (2002). invertebrés d’eau doucem systematique, biologie, ecologie. cnrs édition. paris. 588 p. townsend c.r. (1989). the patch dynamics concept of stream community ecology. journal of north american benthological society, 8: 36-50. vanni m.j., renwick w.h., headworth j.l., auch j.d., schaus m.h. (2001). dissolved and particulate nutrient flux from three adjacent agricultural watersheds: a five year study. biogeochemistry, 54: 85-114. volk c.j., volk c.b., kaplan l.a. (1997). chemical composition of biodegradable dissolved organic matter in streamwater. limnology and oceanography, 42: 39-44. wright d.a. (1995). trace metal and major ion interactions in aquatic animals. marine pollution bulletin, 31: 8-18. international journal of aquatic biology (2013) 1(6): 306-315 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article use of minimum legal size in managing black clam (villorita cyprinoides) fishery in india nagampoozhi suja*1, kolliyil sunil mohamed 1 molluscan fisheries division central marine fisheries research institute, p. b. no. 1603, tatapuram p. o, kochi 682018, india. article history: received 12 july 2013 accepted 4 december 2013 available online 2 5 december 2013 keywords: economic loss juvenile exploitation minimum legal size size at first maturity abstract: using size at first maturity (sfm) as a biological reference point, the minimum legal size (mls) for the black clam, villorita cyprinoides was fixed at 20 mm. corresponding minimum legal weight was calculated as 3.4 g. the reproductive load and mean generation time were determined as 0.35 and 0.72, respectively. of the total quantity of clams exploited during 1996-97, 50% were juveniles. the percentage was reduced to 7.9% during 2009-10. the loss in harvest weights due to exploitation of juvenile clam was 50%, 23% and 37% and the economic loss caused was us$ 4.6 million, us$ 1.5 million and us$ 1.1 million for the periods 1996-97, 2000-01 and 2002-03, respectively. if the undersized clams were permitted to grow up to mls, the harvest weights could have improved by up to 24% and hence the economic gain is to the tune of us$ 1.40 million during 2009-10. the difference between the present lmean in the fishery and the lopt is 4 mm. introduction the state of kerala leads india in the production of clams with estimated annual landings of about 66,000 tonnes in 2008-09. the black clam, villorita cyprinoides (family corbiculidae) contributes 45,000 tonnes, or about two-thirds of this total (cmfri, 2009; narasimham et al., 1993). during 2009-10, india exported 560 tonnes of clam meat in frozen, dried, freeze-dried and cooked forms, earning a foreign exchange of us$ 1 million (mpeda, 2010). most of the annual production of black clams, about 25,000 tonnes, comes from vembanad lake where almost 4,000 fishermen harvest them. the lake holds large sub-fossil deposits of black clam shells that are mined for commercial use (kripa et al., 2004). the other clams harvested in the lake are the grey clam, meretrix casta, and to a much lesser extent, the yellow clam, paphia malabarica, and another, the sunetta scripta. the lake also has commercially-important finfish. the fisheries for the * corresponding author: nagampoozhi suja e-mail address: nsuja_r@yahoo.co.uk tel: +919947186457 clams and the finfish provide the major livelihood for coastal communities around the lake (sathiadhas et al., 2004). being a rich and cheap protein source, clams are regularly fished from vembanad lake and the meat is sold in local as well as export markets for consumption. besides, the shell also holds commercial importance being the raw material for the manufacture of cement, calcium carbide and sand lime bricks. they are also used for lime burning, for construction, in paddy field / fish farms for neutralizing acid soil and as slaked lime. the shell is used as a raw material for the manufacture of distemper, glass, rayon, paper and sugar. harvesting of the black clams continues throughout the year (kripa et al., 2004). each fisherman harvests black clams about 20 days a month. harvesting method is using a hand rake with a pole attached (“kolli” or “varandi”) and dragging it on the bottom while the fisherman standing in his canoe. the pole is one inch in diameter and made of iron. the clam fishermen are organized into professional societies. 307 suja and mohamed/ international journal of aquatic biology (2013) 1(6): 306-315 there are eight black clam societies distributed around the lake in the kottayam and alappuzha districts (fig. 1). fishing rights and licenses for harvesting in the lake are issued by the state department of mining and geology. the total harvesting area leased out to the societies comprises about 18.65 km² (laxmilatha and appukuttan, 2002). the societies handle the sales of the clam shells by purchasing from the fishermen at a rate of us$ 13-15/ton. each fisherman and his family produce about 14 kg of meat and 130-140 kg of shells on his best harvesting days. fishermen’s wives usually sell the meat. meat is sold in the local village door-to-door, where customers prepare them to include as an important component in their evening meal for its high nutritional value. agents sometimes collect the meats directly from the fishing villages and sell them to retail food markets and large restaurants. the larger the meat is the higher the price. a kilogram of large meat brings about us$ 0.88. fishermen receive us$ 0.11-0.22/kg for the juvenile clam meat. an increasing price in the meat and shell of hard clams drove substantial jumps in fishing effort. in order to harvest more and more clams from the lake, modifications to the fishing gear, especially in the mesh size of the net was applied. considerable amounts of juvenile black clam are exploited every year from vembanad lake, which results in economic loss, in terms of harvest weights. earlier, the mesh size of the hand rake was 30 mm or above and it has been noticed that now fishing is done with hand rakes of mesh size 20 mm or below. the situation recalls a report on the juvenile fish capture in india by using trawls of mesh size less than 35 mm. najmudeen and sathiadas (2008) estimated the economic loss due to juvenile fishing in india by trawls as us$ 15.84 billion. a similar study on the cephalopods such as loligo duvauceli, sepia pharaonis and octopus membranaceous reported that the quantity of juvenile exploited from india by small meshed trawl fishing was 1817 tonnes, 2281 tonnes and 527 tonnes, respectively (mohamed et al., 2009). good fisheries management generally requires that fishing gears retain large fishes while allowing juveniles to escape (armstrong et al., 1990). this also avoids recruitment over fishing by allowing animals to spawn at least once before becoming vulnerable to capture. growth over fishing occurs when the fishery targets fishes of a size below the optimal harvestable size. when the fishes are removed before the cohort has had the opportunity to achieve its maximum biomass level, the fishery will lose much of the potential yield that could be achieved by catching them in the near future. disproportionately large scale removal and destruction of young and juveniles of fishes are detrimental to the fishery because when juvenile mortality is increased the future catches and subsequent recruitment will be affected (crowder and murawski, 1998; diamond et al., 1999). exploitation of juveniles of commercially important species results in considerable economic loss, in terms of what could have been obtained had the fishermen waited for a few months and allowed the animal to grow in size and weight. although, this phenomenon called as growth over-fishing has been figure 1. map of vembanad lake with the major harvesting locations of v. cyprinoides. 307 308 suja and mohamed/ international journal of aquatic biology (2013) 1(6): 306-315 reported in many works (pauly, 1988), assessments of economic loss due to such fishing are few (najmudeen and sathiadhas, 2008). concerns about over fishing rarely enter into management plans for invertebrate fisheries, especially those for bivalve mollusks (hancock, 1973). the rationale used to support exclusion of stock-recruit relationships from management plans for bivalve and other invertebrates is based on their high fecundity, long larval period and widely dispersed adult distribution. yet, management of most bivalve fisheries worldwide assumes that high fecundity will sustain historic levels of recruitment without need to protect (jamieson, 1993). a minimum legal size (mls) is seen as a fisheries management tool with the ability to protect juvenile fish, maintain spawning stocks and control the sizes of fish caught. the mls sets the smallest size at which a particular species can be legally retained if caught (hill, 1992) and in doing so helps to regulate the average age at first capture (walker, 1992). winstanley (1992) suggested that a mls could be used to protect immature fish ensuring that enough fish survive to spawn, control the numbers and sizes of fish landed, maximize marketing and economic benefits, and promote the aesthetic value of fish. the ices advisory committee on fisheries management recommended that fishing gears should retain only 25% of the fish at the minimum legal size (reeves et al., 1992). altering the selectivity of fishing gears (i.e. changing minimum mesh sizes) can play a crucial role in reducing the numbers of fish caught below the mls, and thus the selectivity of fishing gears must be considered alongside any potential mls. besides juvenile exploitation, the black clam fishery of vembanad lake is further affected by externalities like excessive fishing pressure, inflow of pollutants, shallow water industrial activities etc. all of which challenge the very sustainability of black clam resources and livelihood securities of black clam fishermen. hence, this study aims to quantify the amount of juvenile black clams exploited by hand rake from vembanad lake and arrive at minimum legal sizes for the black clam. in addition, an assessment on the economic loss to the fishermen by exploitation of these juvenile black clams is attempted. here we exploit long term data sets for black clam harvest to test whether the fishery changed over a two decade period characterized by greatly increased harvest pressure and declining clam stocks. materials and methods study area: the vembanad lake (9o44.34’n and 76o24.87’e) is the largest brackish water lake on the west coast of india. narrow and sinuous in the north and much broader in the south, the lake parallels the coast of the arabian sea. it is 96 km long and 14 km wide at its widest point and has a surface area of 24,000 km2. it consists of estuaries, lagoons, some man-made canals, marshes, and mangroves (ravindran et al., 2006). the salinity ranges from 0.3 at the lower end of the southern part to 18 ppt near the inlets. the water temperature ranges from 26 to 33.5 oc. aside from some shipping channels that are maintained to a 10-13 m depth, the major portion of the lake has a depth range of 2-7 m (menon et al., 2000). two major rivers, the pamba and the periyar, and four smaller rivers that all originate in the sahya mountains to the east, flow into the lake. the lake opens to the arabian sea through in two locations, one at azheekode, that is at least 100 m wide and fairly deep, and the other at cochin gut that is 450 m wide (menon et al., 2000). at the two openings, the rise and fall of tide is from 0.6 to 0.9 m. the bottom sediments where the black clams occur are a mixture of fine sand, clay, and silt and they extend over wide areas. broad wetlands surround the lake. they are included in the wetlands of international importance, as defined by the ramsar convention for the conservation and sustainable utilization of wetlands in 2002, in part because they support more than 20,000 waterfowl in the winter. kerala has a tropical climate with two rainy seasons, the heavy southwest monsoon from june to september and the lighter northeast monsoon from october to november. the total annual rainfall is 309 suja and mohamed/ international journal of aquatic biology (2013) 1(6): 306-315 about 300 cm. the maximum water temperature, at least 30 oc, occurs during pre-monsoon and the minimum is 24 oc which occurs in august (ravindran et al., 2006). during the monsoon, the flood discharge from the rivers can reach 2,500 m3/s (ravindran et al., 2006). outside of the monsoons, the weather is dry and winds are light. known for its scenic beauty, the vembanad lake and surroundings attracts many tourists. the lake has both brackish and nearly freshwater environments. they are separated from each other by a man-made bund or barrier, the thanneermukkom, which runs across the middle of the lake. constructed in 1974 and functional since 1976, it is about 2 km long. the government keeps it open to allow brackish water to flow to the southern part of the lake for six months, but then closes it for six months, december to may each year. its purpose is to prevent the entry of substantial amounts of salt water to the southern area because it used to reduce the production of rice in paddy fields off the southeast side of the lake. the paddy fields are extensive, totaling about 20200 ha in area. when the water remains fresh or nearly fresh (less than 0.5 ppt), two crops of rice can be produced each year. in some of the lowest land areas in this part of the lake, rice is grown for half a year for one crop, and then shrimp are grown in the same location for half a year. the shrimp larvae swim into the areas during the highest tides and remain to feed and grow to harvestable sizes. however, the black clams cannot reproduce well in low salinity and large areas in this southern region, otherwise suitable for the clams, cannot support them now. also, the average size of the black clams has diminished. in the northern region, salinities from 8 to 18 ppt are usual in march to may but the water salinity can be as low as 0 ppt during the monsoon. the floodwaters during the monsoon rains carry silt and clay into the lake. especially in the southern section of the lake, live black clams in some habitats have been buried by the sediments. over the centuries, this annual process has led to the accumulation of large deposits of black clam shells. the shells, black when the clams are alive, become white after being buried. the deposits are found varying in thickness from 22 to 50 mm and are under a sediment burden of 20 to 60 mm. the shells are also found under some lands that surround parts of the lake including the rice paddy fields. this shows that the lands were once part of the lake, but were covered by sediments (rasalam and sebastian, 1976). data: clam production data sets were collected from central marine fisheries research institute (cmfri). these data sets were provided by the eight different black clam co-operative societies for the periods 1996-97, 2001-02, 2002-03 and 2009-10. the societies keep accounts of the daily harvest by the respective licensed clam fishermen of the different localities surrounding vembanad lake. this accounted the annual production data of black clam from vembanad lake. cost details for the respective years were also obtained from the societies. the length frequency data sets for these years were obtained by sampling the clams from the landings at eight stations. monthly sampling of 200 clams from each landing locality of eight societies was done and they were measured for their length along the anterior – posterior axis and was recorded to the nearest of 0.1 mm using digital vernier calipers. analysis: to determine the proportion of juveniles exploited, the size at transition from juveniles to adult has to be determined. the method used to decide this was by plotting size-wise percentage of mature clams and determining the size at first maturity (sfmsize at which 50% of the animals are mature). sfm was considered as the size of transition from juvenile to adult and was taken as the cut-off length for fixing the mls. in doing so, it is assumed that most animals in the population would have an opportunity to become mature and spawn without prejudice to the selection factor of the gear. the weight corresponding to mls is the minimum legal weights (mlw). this was determined by converting mls into mlw using the standard l-w relationships. the asymptotic length (l∞) and 309 310 suja and mohamed/ international journal of aquatic biology (2013) 1(6): 306-315 growth rate (k) were estimated using length frequency analysis (elefan). these were taken as input parameters to determine the optimum length of capture (lopt) and mean generation time (tg). the reproductive load, which is the ratio between sfm and l∞, was determined to gain insight into the relationship between growth and reproduction of these animals (freose and pauly, 2000). the economic loss due to the capture of juveniles was estimated for each year by considering the price of adult and juvenile for that particular year and loss in harvested weights of juveniles if they were permitted to grow up to mls. results the annual production data (in tonnes) with corresponding mid length (minimum mid length: 13.5 mm and maximum mid length: 46.5 mm) for different years is given in table 1. the smallest size at which both males and females of black clam mature (sfm) is 20 mm (fig. 2).using sfm as a biological reference point, the mls for the black clam was fixed at 20 mm. corresponding weight, mlw was calculated as 3.4 g (fig. 3). the growth parameters l∞ and k were estimated as 56.2 mm and 0.935, respectively. lopt was determined as 28.05 mm (fig. 4). the lopt value is 50% of l∞. the reproductive load and mean generation time were determined as 0.35 and 0.72 respectively. lmean for the four year classes are given in table 2. the difference between the present lmean in the fishery and the lopt is 4 mm. of the total quantity of clams exploited during 1996-97, 50% were below mls. the percentage was reduced to 7.9% during 2009-10 (table 3). the loss in harvest weights due to exploitation of juvenile clam was 50%, 23% and 37% and the economic loss caused was us$ 4.6 million, us$ 1.5 million and us$ 1.1 million for the periods 1996-97, 2000-01 and 2002-03, respectively. if the undersized clams were permitted to grow up to mls, the harvest weights could have improved by up to 24% and hence the economic gain is to the tune of us$ 1.40 million during 2009-10. however, it is evident that the economic loss has come down over a period of 14 years. discussion in many of the developed countries, much of the nontargeted catches and juveniles are discarded in the water, whereas, in the developing countries, the non-targeted catches and under sized fishes are also brought to the shore. marine fishing in india has been contributing significantly to the country’s macro-economic standards and the coastal livelihood security. the analysis of costs and figure 3. length-weight relationship of v. cyprinoides. figure 2. size at first maturity of v. cyprinoides. figure 4. age – length weight key of v. cyprinoides. 311 suja and mohamed/ international journal of aquatic biology (2013) 1(6): 306-315 earnings of the numerous crafts and gears along the indian coast reveals the economic importance and strength of the fishery sector. though literature is available on the economic implications of by-catch, juvenile proportion of economically important fishes has not been quantified in most of the studies done in other countries which have addressed the destructive fishing behavior of different crafts and gears (najmudeen and sathiadhas, 2008). there is an interesting debate on the utilization of by-catch or mid length (mm) 1996-97 2000-01 2002-03 2009-10 13.5 153.358 486.4 14.5 439.89 190.57 15.5 3239.02 345.38 451 212 16.5 3694.99 1338.64 195 359 17.5 4988.51 1411.87 1022 414 18.5 4309.57 1226 1005 1806 19.5 2033.33 1087 1287 1009 20.5 1637.38 1192.22 2419 1006 21.5 1517.84 522.26 1127 1610 22.5 1780.44 1029.71 1710 2524 23.5 1354.95 1362.59 935 2411 24.5 2137.75 2406.86 136 2408 25.5 630.78 2922.12 442 2302 26.5 535.44 3238.01 682 2341 27.5 259.41 2552.56 817 2265 28.5 1546.34 761.98 951 2217 29.5 900.4 877.79 559 1169 30.5 1696.75 914.39 855 2258 31.5 831.7 1772.24 1279 2624 32.5 554.44 2190.55 421 2516 33.5 816.74 718 3623 34.5 1560.7 377.78 824 2478 35.5 314.08 2025 36.5 872.6 505 2156 37.5 448.63 203 2036 38.5 496.74 448 1652 39.5 40.5 672 41.5 42.5 743.19 500 414 43.5 626.78 745 44.5 251 45.5 141 46.5 total 37173 31156 19490 47644 juvenile clam meat 18858 6085 3960 3800 adult clam meat 18315 25071 15530 43844 table 1. length class wise average harvest weights (in tonnes) of v. cyprinoides from vembanad lake during 1996-1997, 2000-2001, 20022003 and 09-2010. horizontal line indicates the set mls. table 2. lmean values for the four year classes of v. cyprinoides. year lmean (mm apm) in fishery 1996 27 2000 29 2002 31 2009 32 lopt was calculated as 28.05 mm apm 311 312 suja and mohamed/ international journal of aquatic biology (2013) 1(6): 306-315 juvenile fishes landed as this would affect the longterm conservational measures of the fisheries (clucas, 1997). peterson (2002) opined that changes in hard clam management in north carolina and elsewhere throughout the species’ range and for bivalve fisheries generally are important to achieve sustainability. most studies use sfm as a reference point to differentiate between juveniles and adults. however, this is fraught with the error of including a substantial portion of adults as juveniles. indeed, when the objective is protection of juveniles and prevention of growth over-fishing, sfm is an apt matrix to fix mls. mls has the advantage of being relatively simple to understand (hill, 1992). it also has the capacity to reduce such problems as recruitment over-fishing by allowing fish to spawn once before being vulnerable to capture. in this study, the proportion of juveniles was observed to decrease from 1996-97 to 2009-10. this augers well for the fishery, although the exact reasons for it are unclear, particularly when effort expanded has increased over the years. a decrease in the juvenile exploitation over the years show that clam fishermen of vembanad lake are increasingly aware of the negative impact of growth over-fishing. they are organized under eight different black clam societies and are vocal and proactive in issues related to clam fisheries and sustainability. in some areas, the clam fishermen restock the juvenile clams back into the lake for further growth and future harvest. minimum legal sizes have been used as an effective fisheries management tool for more than 100 years with the chosen sizes being reviewed, but not always changed, from time to time. the purpose for setting legal sizes in australia was reviewed by hancock (1990) and he found protection of immature animals or juveniles and allowing individuals to spawn at least once as the chief reasons. control of fishing until optimum market size was cited next in importance, followed closely by the objective of controlling harvesting. in australia, the total usage of minimum legal sizes involves 125 species (hancock, 1990). in india, minimum legal weight (mlw) has been officially notified for export only for the very valuable rock lobsters, based on sfm (radhakrishnan et al., 2005). in most cases the fixation of mls appears to be based on empirical information and in some cases on specific scientific studies on the sfm. setting a mls and implementing the same would increase the economic efficiency of the fishery, besides affording protection table 3. year wise quantity (tones) and price of juvenile and adult v. cyprinoides meat (us$/kg) and the calculated economic loss (in us$) if the juveniles were allowed to grow. year quantity of juvenile clam meat exploited (in kg) value obtained for juvenile clam meat exploited* (in us$) quantity of adult clam meat exploited (in kg) value obtained for adult clam meat exploited** (in us$) value that might have obtained if the juveniles were allowed to grow (in us$) economic loss due to juvenile clam harvest (in us$) percentage of juvenile clam exploited 1996-97 18858000 1532174 18315000 6128697 6128696.78 4596523 50.73 2000-01 6085000 791030.2 25071000 2274212 2274211.89 1483182 19.53 2002-03 3960000 514787.1 15530000 1608710 1608709.78 1093923 18.92 2009-10 3800000 617484.6 43844000 2274943 2274943 1234969 7.98 *unit price of juvenile clam meat remained static as us$ 0.0812/kg during 1996-2010. **unit price of adult clam meat 1996-97: us$ 0.325/kg 2000-01: us$ 0.374 /kg 2002-03: us$ 0.406/kg 2009-10: us$ 0.487/kg 313 suja and mohamed/ international journal of aquatic biology (2013) 1(6): 306-315 to juveniles, allowing them to grow in weight and length. the present study shows that if the undersized clams were allowed to grow up to mls, the harvest weights could have improved by up to 24% in 2009-10 and would result in higher income. the economic advantage of preventing growth overfishing in black clam is very clear from this study. such a conclusion was also brought out by najmudeen and sathiadhas (2008). however these authors estimated the losses using sfm as the criteria to differentiate between juveniles and adults. in tropical waters, where growth and maturity in fishes is relatively faster, fishes mature at very small sizes, as evident from the very low reproductive loads. in fishes, generally the reproductive load values range between 0.4 and 0.7 with higher values being characteristic of smaller fishes (froese and pauly, 2000). it is very apparent that the reproductive loads in the present study are comparatively very low indicating their resilience to high fishing pressure and their relative success in the ecosystem. while bivalve mollusk fisheries and those for marine invertebrates more generally, are seldom managed to conserve spawning stock biomass, there is accumulating evidence to suggest that marine invertebrates can suffer recruitment limitation despite their high fecundity and complex life cycle. since fishing is an economic activity, the species that fishers target, the level of exploitation and the gear that they use are all influenced by the benefits they receive (i.e., the revenue) and the costs they incur (pascoe, 2006). as the immediate benefits from the juvenile fishing by destructive gears in india are lucrative, the fishermen will be more adherent to use such gears unless strict regulations are imposed on them with proper monitoring of their implementation. if the fishermen realize that there is no chance of getting economic incentives for undersized clams, he would not waste his effort by catching juveniles. clucas (1997), while discussing some of the critical points relevant to this issue, highlighted the importance of conserving marine resources than just utilizing everything that is caught. while fishing, when the fishermen realize that the catch contains higher percentage of juveniles, they would try to change the fishing ground and migrate to other places where more percentage of bigger clams is available. in the seas, adjacent to the british isles, demersal fish are exploited mainly by various forms of trawls and seines. these gears are selective in capturing fish which enter them. fish below some small size escape from the gear while fish above some larger size are all retained. between the lengths of zero and total retention, the proportion retained increases along an s-shaped curve, often referred to as the selection ogive. if the length at first capture is correctly determined, the yield from the stock is optimized. (armstrong et al., 1990). traditional methods of fisheries management, regulating effort through gear restrictions, harvest amount, size restrictions, and seasonal openings so as to sustain spawning stock biomass of wild stocks, have proven difficult to enforce and ineffective in most fisheries (boltsford et al., 1997). even though the fishermen in india have succeeded in improving the fishing efficiency with the help of technological advancements, they failed to achieve economic efficiency since huge amount of future income in the form of large quantity of juveniles are being destroyed every year (najmudeen and sathiadhas, 2008). lack of linkage between institutional policies and fishery management administrators for the long-term sustainability of marine fisheries in india is another concern. for example, the fishery research institutions in the country are concentrating in developing novel value added products for the utilization of trash fishes and fishery by-catch. utilization of by-catch provides some immediate monetary benefit to the fishermen and the processing industry. however, complete utilization of the by-catch, particularly when it is dominated by juveniles, may lead to increased pressure on some stocks of economically important species. this will eventually encourage the fishermen to target the juveniles of commercially important food fishes while fishing in shallow inshore waters. 313 314 suja and mohamed/ international journal of aquatic biology (2013) 1(6): 306-315 in some fashion, managers of exploited bivalves and other marine invertebrates must reflect with their management actions the real potential for recruitment and growth overfishing. in order to conserve the black clam wealth of vembanad lake from depletion and for regeneration and rejuvenation, the government has to implement measures similar to trawl ban (seasonal closure of mechanized trawling), which is effective enough up to a certain extend to protect the marine fishery resources of india. the mls fixed for black clam shall become a regulatory practice, so that juvenile exploitation and economic loss due to this can be controlled. an effective gear monitoring mechanism should be implemented. fisheries departments, research institutions and ngo’s should take initiatives to conduct awareness programmes among fishermen and make them understand the threats of juvenile exploitation. acknowledgements authors wish to acknowledge the director, cmfri, cochin for the facilities provided to carry out the study. the first author wishes to acknowledge the financial assistance provided by the department of science and technology, govt. of india. references armstrong d.w., ferro r.s.t., maclennan d.n., reeves s.a. 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(1992). a fisheries biologist’s application of minimum legal lengths. in: d.a. hancock (ed.), workshop on legal sizes and their use in fisheries management. bureau of rural resources proceedings no. 13. australia government public service, canberra, australia, 4750. winstanley r.h. (1992). a fisheries manager’s application of minimum legal lengths. in: d. a hancock (ed.), workshop on legal sizes and their use in fisheries management. bureau of rural resources proceedings no. 13. australia government public service, canberra, australia, 918. 315 int. j. aquat. biol. (2021) 9(1): 11-14 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology short communication misidentification of gymnotus interruptus (gymnotiformes: gymnotidae) leads to erroneous karyotype addressing filipe s. rangel-pereira1, felipe p. ottoni*21 1laboratory of systematics and evolution of teleostei fishes, department of zoology, federal university of rio de janeiro, po box 68049, cep 21944-970, rio de janeiro, rj, brazil. 2laboratory of systematics and ecology of aquatic organisms, center of agrarian and environmental sciences (ccaa), federal university of maranhão, br-222, km 04, s/n, boa vista, cep 65500-000, chapadinha, ma, brazil. s article history: received 4 october 2020 accepted 21 december 2020 available online 2 5 february 2021 keywords: cytogenetic, gymnotidae, gymnotus bahianus, knifefish, sex chromosome. abstract: recently an article entitled “a unique sex chromosome system in the knifefish gymnotus bahianus with inferences about chromosomal evolution of gymnotidae” has presented the chromosomal structure of a gymnotus population inhabiting the contas river basin, eastern brazil. we believed that the examined specimens were misidentified as g. bahianus. hence in this work we examined the used material of the above-mentioned work and found that they belong to the species g. interruptus. this conclusion was based on a careful examination of materials from both species (type, recently collected, and additional materials). gymnotus interruptus can be distinguished from g. bahianus by possessing a colouration pattern in which conspicuous dark obliquely oriented bands occur under lateral line at entire extension of trunk. introduction karyotype data are relevant informative characters in fish taxonomy and systematics (bertollo et al., 1986). although rarely used, it has yielded satisfactory results in some neotropical freshwater fishes, especially to better understanding of the taxonomic status of species complexes and cryptic species (e.g. milhomem et al., 2008). gymnotus linnaeus, 1758 is a genus of weakly electric fishes occurring from southern mexico (15ºn) to northern argentina (36ºs) (albert et al., 2005). current studies unveiled a high diversity of the karyotype arrangements in gymnotus species (2n = 34-54), as well as multiple sex chromosome systems (see almeida et al., 2015). however, karyotype information of only 10 out of 46 described gymnotus species is available (almeida et al., 2015). researchers are working to provide such data for more gymnotus species, although this process requires accurate species identification. the species g. bahianus campos-da-paz and costa, 1996 and g. interruptus rangel-pereira, 2012 *correspondence: felipe p. ottoni doi: https://doi.org/10.22034/ijab.v9i1.1019 e-mail: fpottoni@gmail.com occur as allopatric in the coastal atlantic basins of north-eastern brazil (see campos-da-paz and costa, 1996; rangel-pereira, 2012). recently an article entitled “a unique sex chromosome system in the knifefish gymnotus bahianus with inferences about chromosomal evolution of gymnotidae” has presented the chromosomal structure of a gymnotus population inhabiting the contas river basin, eastern brazil. we believed that the examined specimens were misidentified as g. bahianus. therefore, the present work aimed to clarify this issue by careful examinations of the materials from both species (type, recently collected, and additional materials). materials and methods morphological examination follows albert et al. (2005). material of g. bahianus and g. interruptus, including the type and recently collected materials, those examined by almeida et al. (2005), and other additional specimens were examined (see examined material). collection's acronyms follow fricke and 12 rangel-pereira and ottoni / misidentification of gymnotus interruptus eschmeyer (2020), except for those of ufrj. almeida et al. (2005) examined 34 specimens that were deposited in the mzusp) and ufba collections. abbreviations: bnd: dark pigment bands; mnrj: national museum of rio de janeiro; mzusp: museum of zoology at universidade de são paulo; pll: pored lateral-line scales; plr: pored lateral-line scales to first lateral-line ventral ramus; sal: scales above lateral line; ufba, natural history museum of bahia, at universidade federal da bahia; ufrj: biology institute of universidade federal do rio de janeiro. results and discussions we identified the materials of almeida et al. (2015) as g. interruptus. gymnotus interruptus is distinguished from g. bahianus, even in life, by possessing a colour pattern obliquely oriented wavy margin dark band pairs, presented below lateral line along trunk (vs. dark bands broken into rounded or vermiculate irregularly distributed dark spots in adults (>120 mm) and absent in anterior 60% of body length of juveniles (<110 mm) in g. bahianus) (fig. 1). in addition, counts of the examined material of ufba 6325 match those of g. interruptus, i.e. bnd: 23; pll: 95; plr: 35 and sal: 9 (fig. 1). more attention should be given to the accuracy of specimens’ identification to avoid taxonomic confusion. this step is essential for the establishment of a reliable karyotype database as well as for securing the taxonomic advances (wheeler, 2008). since cytogenetic data is applied in the gymnotus taxonomy (albert et al., 1999; milhomem et al., 2008, 2012), such misidentifications are potential sources for taxonomical confusion and errors in taxonomic studies. addressing alien characters to a given species can cause, for example, splitting or lumping bias in which populations are wrongly distinguished from it or grouped with it. such confusions, if perpetuated, can result in the description of synonyms or in the synonymization of valid species. therefore, since the specimens examined by almeida et al. (2015) are actually g. interruptus, as showed here, the karyotype pattern published by almeida et al. (2015) should be addressed to g. interruptus. examined materials (all from brazil. bahia state): gymnotus. bahianus: mnrj 12316, holotype. — mnrj 4346, paratypes, 31, fazenda almada, ilhéus municipality, g. pereira, 16 february 1945. — mnrj 4188, 2, same locality as holotype; j.g. dos santos, november 1944. — mnrj 4381, 3, urucutuca, ilhéus municipality, g. pereira, 1945. — mnrj 4382, 10, pirataquicé, ilhéus municipality, g. pereira, february 1945. — ufrj 10105, 5, rio água preta do mocambo, road ba–655, uruçuca municipality, f.s. rangel–pereira, j.l.o. mattos, r.c. rizzieri, w.j.e.m. costa and o.c. simões, 20 february 2014. — ufba 4452, 2, córrego do luxo between ibicaraí and floresta azul, ibicaraí municipality, a.m. zanata, p. camelier, a.b. a. goés, r. burger, 12 february 2008. — ufba 5498, 2, wetland near lagoa encantada, ilhéus municipality, p.h. carvalho, s.m.q. lima, d.p. almeida, 13 february 2009. gymnotus interruptus: ufrj 8218, holotype. — ufrj 8219, 1. — ufrj 8243, 1 (c&s), riacho cambiriba, rio de contas basin, iguaí municipality, p. bragança et al., 18 june 2011. — ufrj 10193, 3 (sequenced), rio mariana, acima da cahoeira da pancada grande, ituberá municipality, rangel-pereira et al., february 2014. — ufba 6325, 1, rio das pedras, rio de contas basin, jequié municipality, p.r.a.m. afonso, august 2008. — ufba 6493, 1, riacho serra grande, riacho do canto drainage, rio das almas basin, povoado nova esperança, wenceslau guimarães municipality, a.m. zanata et al., 10 october 2010. — ufba 6494, 2, riacho patioba, rio petro drainage, rio das almas basin, bridge at the border of wenceslau guimarães ecological station, wenceslau guimarães municipality, a.m. zanata et al., 09 october 2010. — mnrj 32273, 2, rio das velhas, close to serraria 2, ubaira municipality, m. cetra and m. barbosa-filho, 29 january 2007. — mnrj 32186, 2, rio preto tributary, close to the rebio on tres braços village, wenceslaus guimarães municipallity, m. cetra and m. barbosa-filho, 30 january 2007. — mnrj 32258, 3, tributary of rio uruba, close to valentim village, boa nova municipality, m. cetra et al., 01 february 13 int. j. aquat. biol. (2021) 9(1): 11-14 2007. — mnrj 32184, 1, tributary of rio jequiriça, under road to jequiriça, jequiriça municipality, m. cetra, 30 january 2007. — mnrj 32260, 6, tributary of rio preto, close to wenceslau guimarães state reserve, m. cetra et al., 31 january 2007. — mnrj 32270, 7, tributary of rio jequiriça, under road to jequiriça, jequiriça municipality, m. cetra et al., 30 january 2007. acknowledgements we are grateful to a.m. zanata, m.r. britto and p.r.a.m. affonso for material loan, and to a.m. katz and j.l.o. mattos for help with photographs. this study was financed in part by the coordenação de aperfeiçoamento de pessoal de nível superior brasil (capes) finance code 001, and fundação de amparo à pesquisa e ao desenvolvimento científico e tecnológico do maranhão (fapema). this paper is part of the ph.d. requirements of filipe da silva rangel pereira at the biodiversity and evolutionary biology graduate program of the federal university of rio de janeiro. figure 1. colour patterns of g. interruptus (a and b) and g. bahianus (c and d). note that dark brown bands are conspicuous in g. interruptus, despite being faded, while impossible to discriminate in, at least, the anterior two thirds of g. bahianus. (a) juvenile specimen 91.0 mm tl (ufrj 8218), (b) adult specimen 148.6 mm tl (ufba 6494), (c) juvenile specimen 82.0mm tl (ufrj 10105) and (d) adult specimen 192.0 mm tl (ufrj 10105). (scale bar = 10 mm) 14 rangel-pereira and ottoni / misidentification of gymnotus interruptus references balon e.k. (1984). reflections on some decisive events in the early life of fishes. transactions of the american fisheries society, 113: 178-185. albert j.s., fernandes-matioli f.m.c., de almeida-toledo l.f. (1999). new species of gymnotus (gymnotiformes, teleostei) from southeastern brazil: toward the deconstruction of gymnotus carapo. copeia, 1999: 410-421. albert j.s., crampton w.g.r., thorsen d.h., lovejoy n.r. (2005). phylogenetic systematics and historical biogeography of the neotropical electric fish gymnotus (teleostei: gymnotiformes). systematics and biodiversity, 2: 375-417. almeida j.s., migues v.h., diniz d., affonso p.r.a.m. (2015). a unique sex chromosome system in the knifefish gymnotus bahianus with inferences about chromosomal evolution of gymnotidae. journal of heredity, 106: 177-183. bertollo l.a.c., moreira–filho o., galetti j.r.p.m. (1986). cytogenetics and taxonomy: considerations based on chromosome studies of freshwater fish. journal of fish biology, 28: 153-159. campos-da-paz r., costa w.j.e.m. (1996). gymnotus bahianus sp. nov., a new gymnotid fish from eastern brazil (teleostei: ostariophysi: gymnotiformes), with evidence for the monophyly of the genus. copeia: 937944. fricke r., eschmeyer, w.n. (2020) guide to fish collections. available from: http://researcharchive. calacademy.org/research/ichthyology/catalog/collectio ns.asp. retrieved 2020. milhomem s.s.r., pieczaka j.c., crampton w.g.r., silva d.s., souza a.c.p., carvalho j.r., nagamachi c.y. (2008). chromosomal evidence for a cryptic species in the gymnotus carapo species-complex (gymnotiformes, gymnotidae). bmc genetics, 9: 75. milhomem s.s.r., crampton w.g.r., pieczarka j.c., shetka g.h. silva d.s., nagamachi c.y., (2012). gymnotus capanema, a new species of electric knife fish (gymnotiformes, gymnotidae) from eastern amazonia, with comments on an unusual karyotype. journal of fish biology, 80: 802-815. rangel-pereira f.s. (2012). gymnotus interruptus, a new species of electric fish from the rio de contas basin, bahia, brazil (teleostei: gymnotiformes: gymnotidae). vertebrate zoology, 62: 363-370. wheeler q.d. (2009). the new taxonomy. crc press. boca raton, 237 p. int. j. aquat. biol. (2019) 7(4): 218-223 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article some biological properties of kura goby, ponticola cyrius (kessler, 1874) (gobiiformes, gobiidae) from kura river, turkey erdoğan çiçek*1, selda öztürk1, sevil sungur2 1department of biology, faculty of art and sciences, nevşehir hacı bektaş veli university, nevşehir, turkey. 2health services vocational school, nevşehir hacı bektaş veli university, 50300, nevşehir, turkey. s article history: received 7 june 2019 accepted 21 august 2019 available online 2 5 august 2019 keywords: growth parameters ardahan çot creek caspian sea abstract: this work presents some biological properties, including length-frequency, length-weight relationships, fulton’s condition factor, growth parameter, and growth performance and mortality indices of kura goby, ponticola cyrius. a total of 73 specimens were collected from the çot creek a tributary of kura river, from may to september 2015. age of p. cyrius varied from 0 to iii age with age group i as the most frequent (56.16%). the total length and weight ranged 4.3-16.4 cm, and 1.1050.10 g, respectively. the length-weight relationship was w=0.0145l2.9795 with b-value indicating isometric growth pattern. the estimated von bertalanffy growth parameters are l = 26.36 cm, k = 0.187 year-1 and to = -1.33 year. growth performance index (φ') and mean fulton’s condition factor were estimated as 2.115 and 1.43, respectively. instantaneous rate of total, natural and fishing mortalities were 0.588, 0.371 and 0.217 year-1, respectively and the exploitation rate was calculated as 0.369. introduction the genus ponticola iljin, 1927 consists 14 valid species inhabiting the black and caspian sea basins (van der laan, 2019) that 8 of them viz. p. constructor, p. cyrius, p. eurycephalus, p. kessleri, p. rattan, p. rizensis, p. syrman and p. turani are found in turkey (çiçek et al., 2015). among them, kura goby, p. cyrius (kessler, 1874) is endemic to the kura river drainage (kuru, 1975; çiçek et al., 2016) and its absence in the lower kura river could have been prevented by prior penetration of p. gorlap (vasil'eva and vasilev, 1995). the kura goby prefers slow current and muddy bottoms. this species reaches a maximum total length of about 13.0 cm (coad, 2019). few population dynamic parameters of the kura goby have been provided by zamani faradonbeh et al. (2015) and asadi et al. (2017) from the southern caspian sea basin. recently vasileva et al. (2015) described p. iranicus from of sefid river drainage including shahre bijar, totkabon and gisum rivers, guilan province which probably is those previously reported *correspondence: erdoğan çiçek doi: https://doi.org/10.22034/ijab.v7i4.706 e-mail: erdogancicek@nevsehir.edu.tr p. cyrius in iranian part of the caspian sea basin, that need to be confirmed (nikmehr et al., 2018, 2019). the growth is the determination of the body size as a function of age and the stock assessment methods work essentially with age composition data (sparre and venema, 1998; hawk and allen, 2014). hence, age and growth information are crucial for management of the exploited fish stocks, and in this regard, the determination of the biological characteristics of a species is of great importance. therefore, this study aimed to provide some population parameters of p. cyrius inhabiting kura river part of turkey. materials and methods a total of 73 specimens were collected from the çot creek (41.17566121n, 42.94917989e), a tributary of the kura river, eastern anatolia region, turkey using an electrofishing device in may-september 2015 (fig. 1). the specimens were collected in slow flowing creek over rock-stony and muddy substrates. during sampling, stream width was 16 m, mean water depth 219 int. j. aquat. biol. (2019) 7(4): 218-223 24 cm, water temperature 21°c and flow rate of 0.67 m/s. the specimens were fixed into 10% formalin, after anesthesia, transferred to the laboratory and then stored in 70% ethanol. measurements were taken using a digital calipers and data recorded to the nearest 0.1 mm. meristic characteristics were counted using a stereomicroscope. the taxonomic key given by berg (1949), kuru (1975) and coad (2018) were used to identify the samples. the total length and total weight were measured and weighed to the nearest 1 mm and 0.01 g, respectively. the scale samples were removed from the nape area (cycloid) and the left side of the body ventral to the dorsal fin (cteniod) for the age determination. scales were soaked in water and examined independently twice with no reference to the previous readings and without any knowledge of the length or weight of the fish under the stereomicroscope. the precision was measured by the percentage of agreement between the two readings (chang, 1982). the assessment of age was based on the determination of the number of annuli on each scale. the length-frequency data were plotted with 1 cm length intervals. the length-weight relationships were determined according to the equation of w = a*lb, (sparre and venema, 1998), where w is total weight, a and b are regression constants and l is total length. growth in length and weight were expressed in terms of the von bertalanffy equation of lt = l1-e-k(t-to). the growth parameters l, k and to were estimated using the least squares method (sparre and venema, 1998). correspondence between empirical data and an expected distribution was tested by khi2 test. the b value was tested by t-test to verify that it was significantly different from the isometric growth (b = 3). the growth performance index (φ') was calculated figure 1. general body shape of ponticola cyrius. (a) pelvic disc fraenum with angular lobes, and (b) ctenoid and (c) cycloid scales. 220 çiçek et al./ biological properties of kura goby using the formula (pauly and munro 1984) of φ' = logk+2logl∞ and fulton’s condition factor (k) by equations of 𝐾 = 100 𝑊 𝐿𝑏 where; w = total weight, l = total length and b = regression constant (sparre and venema, 1998). the instantaneous rate of total mortality coefficient z was estimated using beverton and holt (1956)’s z equation using the formula of 𝑍 = 𝑘 (𝐿∞−�̅�) (�̅�−𝐿′) , where �̅� is the mean length of the entire catch, and l' = the lower limit of corresponding length intervals (sparre and venema, 1998). the natural mortality coefficient (m) was estimated following pauly’s empirical formula (pauly, 1980), linking the natural mortality with the von bertalanffy parameters, l∞ (cm), k and mean annual temperature (t, °c) of water in habitat (in this case 9.3°c. log10m = -0.0152 0.279log10l∞ + 0.6543log10k + 0.463log10t. fishing mortality rate (f) was calculated as the difference between z and m (z = f+m). the value of the average annual exploitation rate (e) was obtained by e=f/z (sparre and venema, 1998). results and discussions age and growth: age was determined by comparing the growth increment readings on scales by two readers. it was observed that the number of annuli counted for each specimen was similar for the two readers and there was a high harmony (89.05%) between the age estimations. age of p. cyrius varied from 0 to iii age groups and group i was the most frequent groups containing 56.16%, whereas the age group iii was the least i.e. 2.74%. indeed, life span of this species reported up to 3 years (coad, 2018). the total length ranged 4.3-16.4 cm with a mean (±sd) of 9.63±2.08 cm. in the previous studies, range of total length reported as 4.9-11.0 cm (8.1±2.1) by table 1. age, length and weight-frequency distribution of ponticola cyrius from kura river drainage. age n %n mean length (cm) range growth rate % mean weight (g) range 0 9 12.33 5.54±0.91 4.3-7.3 2.50±1.28 1.10-5.34 i 41 56.16 9.45±0.82 8.2-11.6 70.56 12.12±3.26 7.48-19.41 ii 21 28.77 11.25±0.81 9.7-12.7 19.05 20.05±4.73 11.48-30.91 iii 2 2.73 14.75±2.33 13.1-16.4 31.11 39.31±15.27 28.51-50.10 overall 73 9.63±2.08 4.3-16.4 13.96±7.91 1.10-50.10 figure 2. length distribution of ponticola cyrius from kura river drainage. 221 int. j. aquat. biol. (2019) 7(4): 218-223 zamani faradonbeh et al. (2015) and 2.3-11.9 cm (7.5±2.4) by asadi et al. (2017). the longest observed specimen in this study was 16.4 cm. total weight varied between 1.10-50.10 g with a mean of 13.96±7.91 g. based on the results, p. cyrius grows rapidly in their first year, and then this rate declines (table 1). according to vasil'eva and vasil'ev (2003) maturity of p. cyrius is attained in the second or third year. based on our examination, ii year’s old specimens had eggs. the total length-frequency distribution of kura goby is given in figure 2. the distribution was unimodal and the highest frequency (27.4%) was observed in the 10 cm length class followed by the 9 cm length class (26.0%). length-weight relationship: the length-weight relationship for p. cyrius is presented in figure 3. the correlation (r2) for regression analysis indicated strong relationship between the variables. the total lengthweight relationship was as w=0.0145l2.9795 with the b-value was not significantly different from 3.0 (p<0.001) indicating isometric growth of p. gorlap. in the previous studies, length-weight relationships of ponticola c.f. cyrius were reported as w=0.00001*l2.938 and w=0.0006*l3.214 by asadi et al. (2017) and zamani faradonbeh et al. (2015), recpectively. however, the confidance intervals of the b-value were not estimated in both of these studies. the length-weight relationship may be influenced by sex, maturity, geographical location and environmental conditions given year (balon, 1984). the von bertalanffy growth parameters estimated as follows l = 26.36 cm, k= 0.187 year-1 and to = 1.33 year (table 2). growth performance index (φ') and mean fulton’s condition factor were estimated 2.115 and 1.43, respectively. the back-calculated lengths were determined using von bertalanffy growth parameters and the observed and calculated growth in total length is presented in figure 4. the growth curves were not significantly different between the observed and calculated length (p<0.05). mortality: instantaneous total (z), natural (m) and table 1. length-weight relationship and von bertalanffy estimated growth parameters for ponticola cyrius from kura river drainage. b 95% ci of b a r2 l∞ (cm) k (year-1) l∞ (g) t0 (year) φ' k type of growth author 2.9795 2.919-3.041 0.0145 0.993 26.36 0.187 248.20 -1.33 2.115 1.43 i this study 2.938 0.00001 0.983 1.11 -a asadi et al. (2017) 3.214 0.0006 0.861 +a zamani faradonbeh et al. (2015) figure 3. length-weight relationship for ponticola cyrius from kura river drainage. 222 çiçek et al./ biological properties of kura goby fishing (f) mortalities were estimated as 0.588, 0.371 and 0.217 year-1, respectively. the exploitation rate (e) was calculated as 0.369 using estimated mortality rates. there is no published estimation of the natural and total mortality for p. cyrius for comparison. instantaneous natural mortality was estimated higher than fishing mortality. the species is fished only as by-catch, since has no commercial value. therefore, it is not fishing pressure on the species, however, the exploitation rate was calculated as 0.369. in addition, freyhof (2014) reported that p. cyrius is widespread occurring in more than 10 independent populations. hence, its iucn category assessed as lc (freyhof, 2014). indeed, no treat observed don the population during the sampling period. references balon e.k. (1984). reflections on some decisive events in the early life of fishes. transactions of the american fisheries society, 113: 178-185. berg l.s. (1965). freshwater fishes of the u.s.s.r. and adjacent countries. volume 3, 4th edition. israel program for scientific translations ltd, jerusalem. (russian version published 1949). chang w.y.b. (1982). a statistical method for evaluating the reproducibility of age determination. canadian journal of fisheries and aquatic sciences, 39: 12081210. çiçek e., birecikligil s. (2016). ichthyofauna of the turkish parts of kura-aras river basin. fishtaxa, 1: 14-26. çiçek e., birecikligil s., fricke r. (2015). freshwater fishes of turkey; a revised and updated annotated checklist. biharean biologist, 9: 141-157. coad b.w. (2018). freshwater fishes of iran. http://www.briancoad.com. revised: accessed 10.10.2018. freyhof j. (2014). ponticola cyrius. the iucn red list of threatened species 2014: e.t19513722a19849649. http://dx.doi.org/10.2305/iucn.uk.2014-1.rlts.t19 513722a19849649.en. downloaded on 10 october 2019. kuru m. (1975). dicle-fırat, kura-aras, van gölü ve karadeniz havzası tatlısularında yaşayan balıkların (pisces) sistematik ve zoocoğrafik yönden i̇ncelenmesi. doçentlik tezi, atatürk üniversitesi, erzurum, 181 p. (in turkish) nikmehr n., eagderi s., poorbagher h. (2018). taxonomic statue of p. iranicus based on coi cytochrome gene. figure 4. von bertalanffy length-at-age growth curve for ponticola cyrius from kura river drainage. 223 int. j. aquat. biol. (2019) 7(4): 218-223 journal of applied biological sciences, 12(2): 49-51. nikmehr n., eagderi s., poorbagher h., abbasi k. (2019). morphological variation of ponticola iranicus in the anzali wetland drainage. journal of wildlife and biodiversity, doi: 10.22120/jwb.2019.114328.1089 pauly d. (1980). on the interrelationships between natural mortality, growth parameters, and mean environmental temperature in 175 fish stocks. conseil international pour l'exploration de la mer, 39: 175-192. pauly d., munro j.l. (1984). once more on the comparison of growth in fish and invertebrates. fishbyte, 2: 21. sparre p., venema s.c. (1998). introduction to tropical fish stock assessment. part 1. manual. fao fisheries technical paper. no. 306.1 rev.2, rome, fao. 407 p. van der laan r. (2019). freshwater fish list. 27th. edition. almere, the netherlands, 20.8.2019. vasilieva e.d., vasiliev v.p. (2003). mugilidae, atherinidae, atherinopsidae, blenniidae, odontobutidae, gobiidae 1. in: miller p.j. (ed.). the freshwater fishes of europe. 8/i. aula, wiebelsheim. vasil'yeva ye.d., vasil'yev v.p. (1995). systematics of caucasian freshwater gobies (gobiidae) in the light of contemporary data, with a description of a new species, neogobius rhodioni, sp. nov. journal of ichthyology, 35(2): 139-157. vasil’eva e.d., mousavi-sabet h., vasil’eva v.p. (2015). ponticola iranicus sp. nov. (actinopterygii: perciformes: gobiidae) from the caspian sea basin. acta ichthyologica et piscatoria, 45(2): 189-194. asadi h., sattari m., motalebi y., zamani-faradonbeh m., gheytasi a. 2017. length-weight relationship and condition factor of seven fish species from shahrbijar river, southern caspian sea basin, iran. iranian journal of fisheries sciences, 16(2): 733-741. zamani faradonbeh m., eagderi s., ghojoghi f. 2015. length-weight relationship and condition factor of seven fish species of totkabon river (southern caspian sea basin), guilan, iran. international journal of aquatic biology, 3(3): 172-176. int. j. aquat. biol. (2022) 10(3): 224-228 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology short communication length-weight relationships of nine goatfish species (teleostei: mullidae) from the persian gulf and oman sea sorour echreshavi1, hamid reza esmaeili1, saud m. al jufaili2, mohsen safaie3 1ichthyology and molecular systematics laboratory, zoology section, biology department, school of science, shiraz university, shiraz, iran. 2department of marine science and fisheries, sultan qaboos university, muscat, oman. 3department of fisheries, faculty of natural resources, university of hormozgan, bandar abbas, iran. s article history: received 20 april 2022 accepted 22 june 2022 available online 2 5 june 2022 keywords: mathematical models growth parameters allometric growth persian gulf and oman sea ecoregions abstract: goatfishes (teleostei, mullidae) are one group of mainly coastal fishes that form an important part of food chains, and also have commercial value. mathematical models of fish growth provide an objective and practical method for description of growth parameters and estimation of fish weight at different time series. this study presents and describes length-weight relationships for nine goatfish species belonging to three genera of mulloidichthys, parupeneus and upeneus collected from the persian gulf and oman sea. the slope (b) of lwrs for all mullid species fell within the expected range of 2-4 varying from 2.370 for parupeneus heptacanthus to 3.179 for upeneus vittatus based on total length and from 2.391 for u. sundaicus to 3.804 for p. rubescens based on standard length, and r2 values ranged from 0.927 for u. tragula to 0.992 for u. pori. all the lwrs were highly significant, with p<0.005. introduction mathematical models of fish growth provide an objective and practical method for describing growth parameters and estimating fish weight at different time series (silva et al., 2015). a precise and accurate length-weight relationship (lwrs) equation allows for the conversion of growth-in-length to growth-inweight in fish stock assessment models and also estimation of biomass from the length frequency distribution, condition factor, and morphological characteristics of different fish populations (silva et al., 2015). the relationship equation also is an important aquaculture management tool. lwrs are commonly used as a fundamental tool for estimation of weight and biomass of the species understudies, where weighing fish in the field is often not possible to provide sufficient precision for lwr estimates (esmaeili and ebrahimi, 2006; esmaeili et al., 2014; hossain and sultana, 2014; sadeghi and esmaeili, 2018). also, in conjunction with several other parameters (e.g. sex ratio, age at first maturity, correspondence: hamid reza esmaeili doi: https://doi.org/10.22034/ijab.v10i3.1631 e-mail: hresmaeili@shirazu.ac.ir longevity, and fecundity), lwr can be used in population dynamics studies. length and weight are biometric data easily taken and available in most datasets from monitoring studies (zuchi et al., 2020). this relationship is generally expressed by the equation w = alb. in this formula, coefficient a describes body shape, and coefficient b gives information about the balance of the dimensions. values of b can be smaller than 3 (negative allometry = the fish grows faster in weight than in length), bigger than 3 (positive allometry = the fish grows faster in length than in weight), or equal to 3 (isometry) (koutrakis and tsikliras, 2003; froese, 2006). despite the usefulness and significance of the length-weight relationship in fisheries management programs and the economic importance of mullid fishes, little comparative information on these parameters is available for these fishes in the persian gulf and oman sea. goatfishes are predominantly benthivorous fishes that inhabit marine and brackish waters above sandy 225 int. j. aquat. biol. (2022) 10(3): 224-228 to muddy bottoms and coral reefs. they are distributed worldwide in tropical, subtropical, and temperate habitats between the upper littoral and the upper slope (uiblein, 2007; echreshavi et al., 2021). mullids comprise an important part of food chains in coastal ecosystems and have commercial value in many regions worldwide (pavlov, 2012). they are valuable fishes in ecosystem monitoring and management programs because they are sensitive to human-induced activities such as fisheries and habitat modification (uiblein, 2007; echreshavi et al., 2021). the present study was conducted to determine the length-weight relationship parameters for nine mullids, mulloidichthys vanicolensis (valenciennes, 1831), parupeneus heptacanthus (lacepède, 1802), p. margaritatus randall & guézé, 1984, p. rubescens (lacepède, 1801), upeneus doriae (günther, 1869), u. pori ben-tuvia & golani, 1989, u. sundaicus (bleeker, 1855), u. tragula richardson, 1846, and u. vittatus (forsskål, 1775) collected from the persian gulf and the oman sea. materials and methods the fishes were collected from seven localities in the persian gulf and oman sea, including hendijan (29°94'n, 49°62'e), dayyer (27°49'n, 51°56'e), bandarabbas (27°10'n, 56°16'e), minab (26°54′n, 56°41′e), jask (25°41'n, 57°53'e) chabahar (25°21'n, 60°36'e) and seeb (23°43′n, 58°11′e), using artisanal fishing gear (gill net and trammel net) (fig. 1). the sampling was done between august 2017 and february 2022. fish species were identified (fig. 2) according to ben-tuvin and kissil (1998), randall and kulbicki (2006), barman et al. (2007), and uiblein and heemstra (2010). the identifications were confirmed by dna barcoding. the collected specimens were fixed in 70% alcohol. the total length (tl), and standard length (sl) of the specimens were measured to the nearest 0.1 mm using digital calipers attached to the computer. using a digital electronic balance, the specimens were weighed to the nearest 0.01 g (total weight, tw). the relationship between length and weight was estimated using the formula of w = alb, where w is the total weight (g) and tl is the total length (cm). the parameters a and b were calculated by functional regression. the b-value for each species was tested by t-test at the 0.005 significance level to verify that it was significantly different from isometric growth (beverton and holt, 1996; froese, 2006). the lwr helps to determine whether it is an isometric (b=3) or an allometric growth pattern (positive if b>3 or negative if b<3) (morey et al., 2003). the extent of association between the variables were computed by figure 1. map of study area showing the location of the persian gulf and the oman sea. http://en.wikipedia.org/wiki/bernard_germain_de_lac%c3%a9p%c3%a8de http://researcharchive.calacademy.org/research/ichthyology/catalog/getref.asp?id=2710 226 echreshavi et al./ length-weight relationships of nine goatfish species determining the regression coefficient (r2) and its significance level and confidence limit of 95% of parameters a and b were calculated. results parameters of length-weight relationships for the nine studied species of mullids, including the length and weight ranges, and the equation parameters a and b, together with their 95% confidence intervals and the coefficient of determination, are given in table 1. lwrs were significant for all species (p<0.005) with high correlation coefficients with r2≥0.927. the slope (b) of lwrs for all mullid species fell within the expected range of 2-4, varying from 2.370 for p. heptacanthus to 3.179 for u. vittatus based on tl (table 1) and from 2.391 for u. sundaicus to 3.804 for p. rubescens based on sl (table 1). discussion the b-value of length-weight relationships based on tl from 2.370 for p. heptacanthus to 3.179 for u. vittatus based on sl and from 2.391 for u. sundaicus to 3.804 for p. rubescens is within the expected range of 2-4 (bagenal and tesch, 1978). this parameter is usually encountered in fin fishes, which lie between 2 and 4 according to bagenal and tesch (1978) or 2.5 and 3.5 based on froese (2006). according to carlander (1977), the values of b<2.5 or >3.5 are frequently caused by samples with narrow size ranges. for length-weight relationships, the value of parameter b depends primarily on the shape and fatness of the fish species (gubiani and agostinho, 2009). parupeneus heptacanthus presented a b value of 2.370 (based on tl), which shows an overproportional length increase relative to weight growth. according to froese (2006), it is reflected when b<2.5 (froese, 2006). however, p. rubescens presented bvalue of 3.804 (based on sl) that reveals an overproportional increase in weight relative to growth in length. in addition, u. sundaicus with b-value of 2.391 (sl) is characterized by having a moderately elongated body compared to other studied goatfishes (fig. 2), which shows the effect of its body form on the b value. in contrast, p. rubescens has a deep body with a high b-value of 3.804 (based on sl). it has been already reported that variation in bvalue in fishes might be due to several factors, including species, sample size, fish length (tl, sl, fl), season, habitat, sex, gonad maturity, diet, stomach fullness, health, preservation techniques and locality (le-cren, 1951; esmaeili, 2001; froese, 2006; lakshmikanth et al., 2021). differences in the lwrs could be due to the combination of one or more of the above factors. goatfishes of the family mullidae are characterized by having a moderately elongated and somewhat compressed body (size to 50 cm). table 1. descriptive statistics and parameters of lwrs for nine fish species from the persian gulf and the oman sea. r2 95% ci of b b 95% ci of a a w range (g) tl/sl range (cm) n tl/sl species 0.965 2.613-3.031 2.822 0.0112-0.0444 0.0223 126.20-330.45 21.79-31.03 30 tl m. vanicolensis 0.974 2.830-3.213 3.021 0.0119-0.0390 0.0216 17.91-24.67 30 sl 0.951 2.161-2.578 2.370 0.00543-0.02281 0.0111 68.18-229.40 17.44-25.51 30 tl p. heptacanthus 0.951 3.269-3.899 3.584 0.00177-0.01298 0.00479 13.56-20.92 30 sl 0.945 2.574-3.055 2.815 0.0113-0.0524 0.0224 147.90-600.21 19.46-37.46 35 tl p. margaritatus 0.971 2.564-3.311 2.938 0.02168-0.05171 0.0647 17.26-26.84 35 sl 0.976 2.627-2.965 2.796 0.0163-0.0537 0.0296 218.30-998.65 24.85-42.60 30 tl p. rubescens 0.976 3.571-4.037 3.804 0.00133-0.00592 0.002809 19.59-29.65 30 sl 0.941 2.596-2.979 2.788 0.0124-0.0339 0.0205 12.42-82.40 10.55-22.60 55 tl u. doriae 0.946 3.103-3.539 3.321 0.006784-0.01915 0.01139 8.71-15.18 55 sl 0.993 2.312-2.464 2.388 0.00709-0.01141 0.09001 90.50-247.21 18.21-27.62 30 tl u. pori 0.992 2.837-3.045 2.941 0.02666-0.04827 0.03588 14.41-20.21 30 sl 0.927 2.612-3.006 2.809 0.002611-0.09571 0.04999 91.40-255.13 17.26-29.84 30 tl u. sundaicus 0.956 2.276-2.507 2.391 0.05411-0.02652 0.03793 12.64-25.75 30 sl 0.942 2.400-2.699 2.550 0.005688-0.09571 0.03643 90.60-398.49 11.53-25.17 30 tl u. tragula 0.927 2.253-2.559 2.406 0.02704-0.01158 0.01770 9.03-21.65 30 sl 0.969 2.961-3.398 3.179 0.00401-0.0144 0.00761 37.20-140.25 13.66-21.70 30 tl u. vittatus 0.958 2.907-3.596 3.252 0.000419-0.00259 0.00104 11.73-16.00 30 sl 227 int. j. aquat. biol. (2022) 10(3): 224-228 in conclusion, this study provides basic information on lwrs of nine goatfishes species from the persian gulf and the oman sea, with a b value within the expected range of 2-4, which will be useful in their fisheries and conservation management. acknowledgments we would like to thank f. owfi for helping with fish collection and h. hashemi for providing fish survey facilities. the research work was funded by shiraz university and was approved by the ethics committee of biology department (su-9531462). references bagenal t.b., tesch f.w. (1978). age and growth. in: t.b. bagenal (ed.), methods for assessment of fish production in freshwater, 3rd ed. blackwell scientific publication: oxford, uk. pp: 101-136. barman r.p., mishra s.s., kar s., mukherjee p., saren s.c. (2007). marine and estuarine fish fauna of orissa. zoological survey of india journal, 260: 1-186. ben-tuvia a., kissil g.w. 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(2016) 4(1): 51-56 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article threatened fishes of the world: paracobitis vignai nalbant and bianco, 1998 (nemacheilidae) hamed mousavi-sabet*1, keyvan abbasi2, saber vatandoust3, soheil eagderi4 1department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, p.o. box 1144, guilan, iran. 2iranian fisheries sciences research, inland waters aquaculture research center, agricultural research education and extension organization (areeo), bandar anzali, iran. 3department of fisheries, babol branch, islamic azad university, babol, iran. 4department of fisheries, faculty of natural resources, university of tehran, karaj, alborz province, iran. article history: received 1 december 2015 accepted 30 january 2016 available online 2 5 february 2016 abstract: paracobitis vignai is a nemacheilid loach endemic to the sistan basin. it occurs in the helmand river and its related reservoirs in sistan-va-baluchistan province in southeastern iran and probably in afghanistan. this species is currently endangered due to habitat loss or degradation, damming and droughts. therefore, this paper reviews the available data on taxonomy and distribution of p. vignai, provides its morphometric features, and recommends actions for its conservation. keywords: endemic species, sistan basin, conservation, iran. introduction the loaches with a high dorsal dermal-crest have been placed in the genus paracobitis bleeker, 1863 for many years, specifically those from central asia (banarescu and nalbant, 1964), vietnam (nguyen, 2005), the middle east (prokofiev, 2009) and china (min et al., 2010). the genus paracobitis was appointed by bleeker (1863) for cobitis malapterura. this genus is restricted to near east and middle asia, and the species of paracobitis from china should be assigned to the genera homatula and schistura (nalbant and bianco, 1998). the species of the genus paracobitis are comparatively largesized loaches inhabiting freshwaters of western asia (bănărescu and nalbant, 1995; nalbant and bianco, 1998). there are thirteen valid species of the genus paracobitis in the world, which eleven of them are reported from iran (kottelat, 2012; coad, 2016; mousavi-sabet et al., 2014; freyhof et al., 2014; jouladeh-roudbar et al., 2015). according to freyhof et al. (2014), mousavi-sabet et al. (2015), and jouladeh-roudbar et al. (2015b), the valid * corresponding author: hamed mousavi-sabet doi: http://dx.doi.org/10.7508/ijab.2016.01.007 e-mail address: mousavi-sabet@guilan.ac.ir paracobitis species in iran comprise p. atrakensis esmaeili, mousavi-sabet, sayyadzadeh, vatandoust & freyhof, 2014, p. basharensis freyhof, esmaeili, sayyadzadeh & geiger, 2014, p. hircanica mousavisabet, sayyadzadeh, esmaeili, eagderi, patimar & freyhof, 2015, p. iranica nalbant and bianco, 1998, p. longicauda kessler, 1872, p. malapterura (valenciennes, 1846), p. molavii freyhof, esmaeili, sayyadzadeh & geiger, 2014, p. persa freyhof, esmaeili, sayyadzadeh & geiger, 2014, p. rhadinaea (regan, 1906), p. smithi (greenwood, 1976), and p. vignai nalbant and bianco, 1998. however, freyhof et al. (2014) believe that p. iranica is a synonym of p. malapterura. since there is little information available about various biological and taxonomical aspects of p. vignai, a nemacheilid loach endemic to the sistan basin, this paper reviews the available data on its taxonomy and distribution, provides its morphometric features (along with those of its sympatric species i.e. p. rhadinaea that is superficially similar to p. vignai), and recommends 52 mousavi-sabet et al./ threatened fishes of the world: paracobitis vignai actions for its conservation. materials and methods a total of 15 specimens of p. vignai and 14 specimens of p. rhadinaea were collected from chahnimeh reservoirs, near zahak town, sistanva-baluchestan province, in september 2014 by gillnet (fig. 1). after anesthesia with 1% clove solution, the specimens were fixed in 10% buffered formaldehyde and transferred to the laboratory for further processing. morphometric characters were measured by a caliper to the nearest 0.1 mm (table 1). all measurements are made point to point according to kottelat and freyhof (2007). the percentage ratios of morphometric characters in relations to standard length (sl) and head length (hl) were calculated. meristic characteristics of the specimens were counted using a stereomicroscope. the last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are noted as “1½”. results and discussion paracobitis vignai nalbant and bianco, 1998 (fig. 2) common names: sagmahi-ye sistan (persian); sistan’s loach (english). the species is endemic to sistan basin and named for professor augusto vigna taglianti, la sapienza university, rome. the holotype collected from "nahr taheri, zabol, seistan" by a. vigna (nalbant and bianco, 1998). morphology: according to original digenesis by nalbant and bianco (1998), paracobitis vignai is characterized by a completely scaleless body and large brown blotches in the middle of flanks. the lateral line extends to the base of the caudal fin. additionally, caudal fin is deeply emarginate or slightly furcate and posterior nare opening is slitshaped (freyhof et al., 2014). paracobitis vignai is superficially similar to p. rhadinaea from which it is distinguished by a completely naked body (vs. presence of scales in p. rhadinaea) and deeply emarginate or slightly forked caudal fin (vs. moderately or slightly emarginate in p. rhadinaea). morphometric characters of p. vignai and p. rhadinaea are provided in table 1. for general appearance of p. vignai see figure 2. meristic values for the studied specimens caught from iranian parts of the sistan basin are: dorsal fin unbranched rays 3, dorsal fin branched rays 7½, anal fin unbranched ray 2, anal fin branched ray 5½, pectoral fin branched rays 9, pelvic fin branched rays 7, and caudal branched rays 9+8. in addition, coad (2016) reported 6-8 branched rays of dorsal-fin, 5-6 anal-fin, 8-10 pectoral-fin rays and 6-7 pelvic-fin for p. vignai. the body is slender and compressed, particularly posteriorly. the head is long and the eyes are small. the dorsal adipose crest or keel on the caudal peduncle is well-developed. a slightly developed axillary lobe at the ventral-fin base. mouth well arched, surrounded by three pairs of short barbels. maxillary barbels not reaching to the eye origin. outer mandibular barbels reaching to the nostrils origin. inner mandibular barbels reaching to maxillary barbels origin. dark pigmentations at the base and on the mandibular barbels (inner and outher). lips are well-furrowed and interrupted in their middle. mental lobes are enlarged. mental lobes has dark grey pigmentations. the processus dentiformis is slightly developed. lateral line is figure 1. map of sampling station (δ, chahnimeh reservoirs) in the sistan basin. 53 int. j. aquat. biol. (2016) 4(1): 51-56 complete, reaching to base of caudal-fin, with 118121 pores. cephalic lateral line system bears 9 supraorbital, 4+15 infraorbital, 13 preoperculomandibular and 3 supratemporal pores. nostril is slit-shape and flap does not covers the nostril. coloration: in preserved specimens, the body is pale brown to cream with a row of dark-grey blotchs on middle of the back and on the mid-sides of the body may be occasionally fused (fig. 2). in live specimens the flanks are pale olivaceous with 9-14 dark grey blotches on dorsal and lateral, which are connected together by a row of pale grey small blotches. the blotches extend onto the adipose crest. blotches are line up in flank row anteriorly and posteriorly. all over the head, opercula and snout covered by dark grey blotches. the caudal fin base has a distinct dark bar. the belly is whitish. no distinct dark stripe from eye to snout. all fins are orange (in live specimens) or hyaline (in preserved specimens); dorsal, pectoral and caudal fins with dark spots on rays; pelvic and character paracobitis rhadinaea paracobitis vignai range mean ± sd range mean ± sd total length mm 147.15 193.85 108.69 117.99 standard length mm 126.97 168.38 92.48 101.11 in % standard length head length 22.49 25.64 24.36 ± 1.05 18.07 21.11 19.89 ± 0.87 head depth 9.23 11.60 10.79 ± 0.75 10.48 11.23 10.66 ± 0.45 maximum body depth 13.87 16.92 14.97 ± 1.03 13.82-15.40 14.61±1.11 predorsal length 47.73 50.88 48.16 ± 1.35 44.48 49.83 46.66 ± 1.29 postdorsal length 39.91 43.53 41.11 ± 1.20 41.82-45.40 43.61±1.11 pre anal length 73.30 77.51 75.05 ± 1.33 72.54 75.80 74.17 ± 1.18 caudal peduncle depth 7.53 9.24 8.64 ± 0.45 8.56 9.85 9.21 ± 0.49 caudal peduncle length 17.30 – 23.59 19.57 ± 2.23 17.00 – 20.11 18.50 ± 2.15 caudal fin length 12.59 15.63 13.89 ± 1.15 13.35 14.72 13.54 ± 0.75 dorsal fin length 13.73 16.05 14.80 ± 0.62 13.01 15.95 14.13 ± 0.79 dorsal fin base 9.20 11.98 10.62 ± 0.81 9.72 10.94 9.91 ± 0.77 anal fin length 7.90 10.08 9.09 ± 0.65 8.76 10.07 9.47 ± 0.39 anal fin base 8.07 9.82 9.12 ± 0.62 7.59 9.68 8.21 ± 1.01 pectoral fin length 12.79 15.51 14.86 ± 1.15 12.88 15.42 14.55 ± 0.79 ventral fin length 11.72 13.41 12.43 ± 0.63 11.64 14.24 12.31 ± 1.23 pecto-ventral distance 26.86 32.07 28.79 ± 2.56 27.73 32.13 31.93 ± 1.27 ventral-anal distance 20.87 23.92 21.97 ± 1.03 21.82 24.40 23.61 ± 1.11 pecto-anal distance 49.49 52.94 51.48 ± 1.28 48.09 53.44 52.77 ± 1.45 crest length 25.26 32.06 28.11 ± 3.85 30.69 32.45 31.07 ± 0.83 head length % snout length 38.15 41.19 39.87 ± 1.04 34.95 37.17 35.56 ± 0.86 eye diameter 7.67 10.58 9.57 ± 0.88 9.15 12.42 11.21 ± 0.89 head depth 44.07 49.51 47.28 ± 1.91 47.41 54.36 52.89 ± 2.08 post orbital distance 44.29 54.67 49.10 ± 3.43 46.29 54.92 51.11 ± 3.98 inter orbital length 20.99 25.38 22.56 ± 1.35 22.38 25.72 23.55 ± 1.23 inter nasal length 17.68 22.81 18.87 ± 1.90 17.61 ± 22.41 19.01 ± 1.56 table 1. morphometric characters of paracobitis rhadinaea (n = 10) and paracobitis vignai (n = 10). figure 2. paracobitis vignai, about 25 cm in tl, sistan basin, iran. 54 mousavi-sabet et al./ threatened fishes of the world: paracobitis vignai anal fins without dark pigmentation. when touching the dorsal margin of the crest, the reticulations make the crest there dark, otherwise the crest is light cream along the margin. the lateral line is light, sometimes in marked contrast to the rest of the flank. the rostral barbels and their bases are strongly dark pigmented, but the maxillary pair is almost immaculate (slightly pigmented in some specimens). size: the maximum size of the recent caught materials is about 25 cm in tl. distribution: this endemic species is restricted to the sistan basin of iran and presumably afghanistan. the species was distributed in the hirmand (helmand) river, the hamoun wetland and its related reserviours (in southeast of iran and probabely southwest of afghanistan). conservation status: iucn red list: not evaluated; iran: unknown. abundance: low. habitat and ecology: the species typically prefers sandy or gravel bottoms in the chahnimeh reservoirs and related streams. we caught some of specimens from the hirmand (helmand) river, at its entrance to the chahnimeh reservoirs, a shallow and narrow (up to 10 m wide; and 30-70 cm depth at the sampling time) river with diverse structures (pools, muddy shore, etc.), at 493 m altitude, and the water was unclear and muddy with slow current at the time of sampling. details of its ecology, reproduction and feeding are unknown. co-existing species: fish species of the iranian part of the sistan basin comprised 24 species in 19 genera, 4 families, and 3 orders. they comprises 4 endemic species, including paraschistura alta (nalbant & bianco 1998), schizothorax zarudnyi (nikol'skii, 1897), paracobitis rhadinaea (regan, 1906) along with p. vignai, 11 exotic species, including alburnus hohenackeri kessler, 1877, carassius auratus (linnaeus, 1758), c. gibelio (bloch, 1782), ctenopharyngodon idella (valenciennes, 1844), cyprinus carpio linnaeus, 1758, hemiculter leucisculus (basilewsky, 1855), hypophthalmichthys molitrix (valenciennes, 1844), h. nobilis (richardson, 1844), pseudorasbora parva (temminck and schlegel, 1846), oncorhynchus mykiss (walbaum, 1792) and gambusia holbrooki girard, 1859, and 9 native species, including cyprinion watsoni (day, 1872), garra rossica (nikol'skii, 1900), gonorhynchus adiscus (annandale, 1919), schizocypris alitidorsalis bianco and bănărescu, 1982, schizothorax intermedius mcclelland, 1842, s. zarudnyi (nikol'skii, 1897), schizopygopsis stoliczkai steindachner, 1866, paraschistura kessleri (günther, 1889), and triplophysa stoliczkai (steindachner, 1866) that coexists with p. vignai. threats: above mentioned 11 co-existing exotics species with p. vignai could may have major effects on the population of this endemic species through competition, habitat changes and introduction of parasites. furthermore, water pollution due to agricultural and industrial activities and hydrological alterations such as dam construction (about 10 main earth and concrete dams on the helmand river in afghanistan) and pumping water from this river which feeds the hamoun wetland have effected p. vignai populations. since the construction of dams on the helmand river in afghanistan, the hamoun wetland was dried during the last decade and the fishes has only survived in related reservoirs (three main water reservoirs naming as chahnimeh 1 to 3). natural drought in recent decades is the most important natural disturbance of freshwater fishes of iran (esmaeil and valavi, 2016; jouladeh-roudbar et al., 2015a). due to severe drought, many rivers of the iranian part of the sistan basin dried out causing the habitat loss of p. vignai populations. unintended poaching is another main treat for this endemic species. local people do not eat the fish, therefore it is not economically or nutritionally important but its population is damaged as incidental catch or bycatch in fishing from the chahnimeh reservoirs. conservation action: not protected in iran and afghanistan. conservation recommendations: urgent habitat protection is suggested. the ecological right of water of the hamun wetland and the related reservoirs should be respected (which is ignored by the 55 int. j. aquat. biol. (2016) 4(1): 51-56 damming) to prevent drought. education of local fishermen should be initiated. fishing programs should be forbidden or at least should be limited in the habitat of p. vignai, especially during its reproduction season. also fishing equipment and methods should be edited according to minimum bycatch (mousavi-sabet et al., 2013). finally, developing a conservation strategy for the species, habitat monitoring, conducting ecological and biological studies, and captive breeding could conserve this endemic crested loach having their future generations. acknowledgements we are pleased to thank j. freyhof and h.r. esmaeili for their help and helpful comments on the paracobitis research project, and the university of guilan for financial support. many thanks to a. juladeh-roudbar for helping with fish collection. also thanks to environment departments of sistanva-baluchistan province for supporting field surveys. references bănărescu p., nalbant t. (1964). süßwasserfische der türkei. 2. teil cobitidae. mitteilungen aus dem hamburgischen zoologischen museum und institut, 61: 159-201. bănărescu p., nalbant t. (1995). a generical classification of nemacheilinae with description of two new genera (teleostei: cypriniformes: cobitidae). travaux du muséum d'histoire naturelle grigore antipa, bucurešti, 35: 429-496. bleeker p. (1863). sur les genres de la famille des cobitioïdes. nederlands tijdschrift van dierkunde, 1: 361-368. coad b.w. (2016). fresh water fishes of iran. retrieved from http://www.briancoad.com. 2 january 2016. esmaeili h.r., valavi h. (2016). threatened fishes of the world: paracobitis persa freyhof, esmaeili, sayyadzadeh & geiger, 2014 (teleostei: nemacheilidae). fishtaxa 1: 29-34. freyhof j., esmaeili h.r., sayyadzadeh g., geiger m. 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(2010). paracobitis nanpanjiangensis, a new loach (balitoridae: nemacheilinae) from yunnan, china. environmental biology of fishes, 87: 199-204. mousavi-sabet h., gharaei a., ghaffari m. (2013). threatened fishes of the world: paracobitis rhadinaeus (regan, 1906) (nemacheilidae). croatian journal of fisheries 71(2): 51-55. mousavi-sabet h., gharaei a., nasrollahzade a., habibi a., eagderi s. 2014. redescription of paracobitis rhadinaea (regan, 1906) from sistan basin, iran (teleostei: nemacheiliidae). international journal of aquatic biology, 2(5): 286-291. mousavi-sabet h., sayyadzadeh g., esmaeili h.r., eagderi s., patimar r., freyhof j. (2015). paracobitis hircanica, a new crested loach from the southern caspian sea basin (teleostei: nemacheilidae). ichthyological exploration of freshwaters, 25: 339346. nalbant t.t., bianco p.g. (1998). the loaches of iran and adjacent regions with description of six new species (cobitoidea). italian journal of zoology 65 (supplement): 109-123. nguyen v.h. (2005). freshwater fishes of vietnam. v. 2. 760 p. prokofiev a.m. (2009). problems of the classification and phylogeny of nemacheiline loaches of the group lacking the preethmoid i (cypriniformes: balitoridae: nemacheilinae). journal of ichthyology, 49: 874-898. int. j. aquat. biol. (2016) 4(1): 51-56 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی paracobitis vignai nalbant and bianco, 1998 (nemacheilidae) ماهیان در معرض تهدید دنیا: 4ایگدری سهیل، 3دوست، صابر وطن2کیوان عباسی ،1*ثابت موسوی حامد سید سرا، ایران.صومعه گیالن، طبیعی، دانشگاه منابع دانشکده، شیالت گروه1 .ایران، انزلی بندر ،کشاورزی آموزش و تحقیقات سازمان، داخلی هایآب پروریآبزی تحقیقات مرکز، کشور شیالتی علوم تحقیقات موسسه2 ایران ،بابل ،اسالمی آزاد دانشگاه ،بابل واحد ،شیالت گروه3 دانشگاه تهران، کرج، ایران. طبیعی، منابع دانشکده، شیالت گروه4 چکیده: paracobitis vignai آن در استان سیستانبا در رودخانه هلمند و منابع آبی مرتبط که آبریز سیستان است حوضه بومی یجویبار ماهیسگ کی سالی سدسازی و خشکواسطه تخریب و ازدست دادن زیستگاه، شود. این گونه اخیراً بهو بلوچستان در جنوب شرق ایران و احتماالً افغانستان یافت می های ویژگیو کند، را مرور می p. vignaiشناسی و پراکنش در مورد آرایه های در دسترسدر معرض تهدید قرار گرفته است. بنابراین این مقاله داده نماید.ارائه می ای برای حفاظت آن را هسنجی آن و اقدامات توصیریخت .ایران ،حفاظت ،سیستان آبریزگونه بومی، حوضه :کلمات کلیدی international journal of aquatic biology (2013) 1(5): 221-227 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article cadmium induced histopathology in the olfactory epithelium of a snakehead fish, channa punctatus (bloch) debraj roy, debasree ghosh, dipak kumar mandal*1 department of zoology, visva-bharati university, santiniketan-731 235, west bengal, india. article history: received 5 august 2013 accepted 21 september 2013 available online 2 5 october 2013 keywords: cadmium toxicity histopathology olfactory organ channa punctatus abstract: histopathology on the olfactory organ of a snakehead fish, channa punctatus (bloch, 1793) were assessed after exposing the fish to 2.5 mg/l and 5mg/l of cdcl2 for 15 days, 30 days and 45 days. cellular organization of the epithelium was affected severely with degeneration of sensory and supporting cells and hyperplasia of basal cells and mucous cells. mucous cell proliferation indicates the upregulation of mucous secretion to protect the epithelium from toxic effect of cadmium. the olfactory epithelium was endowed with the multipotent basal cells which differentiate into sensory cells, supporting cells and other cell types of the epithelium during normal cells turn over and in the event of cell death. however, due to cadmium exposure proliferating basal cells failed to differentiate into normal cells and the undifferentiated proliferated cell formed lump and intraepithelial lesion altering the composition of the entire epithelium. present study indicates that in prolonged exposure to cadmium chloride olfactory functions of the fish might be impaired due to loss of all sensory cells. introduction fish are dependent on the olfactory sense for discriminating a wide array of odorous molecules that are involved in survival-linked behaviors such as food searching, predator avoidance, migration, reproduction and parental care (hara, 1992). structural organization of the olfactory organ has been described in channa punctatus (mandal et al., 2005) and in other fishes (chakrabarti and ghosh, 2010). olfactory organs of fish are particularly vulnerable to aquatic toxicants since their receptor cells are directly exposed to the environment and any damage in the olfactory system may adversely affect to the survival of fish species. in an aquatic environment, chemical wastes including heavy metals are common pollutants. mercury causes severe histological damage in the kidney (ghosh and mandal, 2012) and in the olfactory system of fish * corresponding author: dipak kumar mandal e-mail address: dkmandal.vb@gmail.com tel: +919474166574 (ghosh and mandal, 2013). contaminants altered normal olfactory mediated behaviors by modifying odorant perception (tierney et al., 2010). copper exposure causes ciliary loss and cell death in the olfactory epithelium of fish (moran et al., 1992; julliard et al., 1996). aluminum also causes damage to the olfactory epithelium of fish (klaprat et al., 1998). cadmium is known toxic heavy metal and common contaminants in aquatic system. cadmium and other divalent metal ions were taken up by olfactory sensory neurons of both rodent and fish and then transported through axon towards the olfactory bulb (tjälve and henrickson, 1999). cadmium inhibited ca++ influx by interfering ca-atpase activity (verbost et al., 1989). cadmium interferes with cellular signaling or suppresses the apoptotic response, which doubtlessly explains the widely recognized carcinogenic properties of this heavy 222 roy et al/ international journal of aquatic biology (2013) 1(5): 221-227 metal (shimada et al., 1998). cadmium causes histopathology in the liver, kidney and other organ system of fish. the present work has been designed to study the effect of cadmium on the histopathology of the olfactory epithelium of channa punctatus. material and methods maintenance of test animal: freshwater snakehead fish, channa punctatus (bloch, 1793) (22 to 27 cm in length and 100 to120g in weight) were procured from the freshwater ponds at santiniketan (23.68o n 87.68o e), west bengal, india and 120 adult, healthy fish were acclimatized under laboratory conditions for 15 days. fish were fed ad libitum with commercially available feed pellet and water was changed daily. physico-chemical parameters of water was analysed following the methods of apha (2010). the water parameters in the experimental aquarium were temperature 20-22 oc, ph 7.2; dissolved oxygen 4 to 4.2 mg/l; total alkalinity 160 mg/l and total hardness 166 mg/l. no mortality was observed during this period. fish were maintained and experiments were conducted following the guideline of animal ethics committee, department of zoology, visva-bharati university, india. experimental design: after acclimatization fish were selected randomly and transferred to experimental aquarium. total nine aquarium with 10 fish and 30 liters of water in each were taken for experiments. experimental aquarium were grouped into 3 sets containing 3 aquarium in each set. in each set of 3 aquarium, one aquarium was kept as control and other two were contaminated with 2.5 mg/l and 5 mg/l of cdcl2, respectively. in the first set, fish were exposed for 15 days and in the second and third sets fish were exposed for 30 days and 45 days respectively. the two sublethal dose of cdcl2 were selected for the experiments as 5% and 10% of the 72 hour lc50 value of cdcl2 for the fish (72h lc50 value 50.05 mg/l) which was determined following probit analysis method (finney, 1977). fish were kept under continuous aeration. water was changed and doses were renewed at 2 days interval. after completion of experiment fish were anaesthetized with 100 mg/l ms 222 and sacrificed. intact olfactory tissues were collected from both control and treated fish and processed for histology. tissue preparation: olfactory tissues were fixed in aqueous bouin’s fluid for 20 hours at room temperature. fixed tissues were washed in 70% ethanol repeatedly and dehydrated through graded series of ethanol and cleared in xylene. tissues were then infiltrated in paraffin wax (56-58 oc, merck, germany) for 45 minutes under a thermostat vacuum paraffin embedding bath for 1 hour. serial thin (4 µm) sections of the paraffin block of the tissue were obtained using a rotary microtome. six sections in two rows were taken in albumenized glass slide and stretched using a thermostat hot plate. the sections were deparaffinized with xylene, hydrated through downgraded ethanol to distilled water. the tissue were stained with delafield haematoxylin and counterstained with eosin (1%). stained slide dehydrated with graded ethanol, cleared in xylene and mounted with dpx. histological study: stained slides were examined, compared to control group and photographs were taken under bx51 olympus research microscope. for each treatment group 10 slides were prepared from the tissue of five fish. measurement of cell diameter and thickness of epithelium were taken by ocular micrometer. histopathological alterations in the olfactory epithelium were assessed semiquantitatively by using score ranging from – to +++ depending on the degree of alteration: (-) no alteration (+) mild alteration, (++) moderate alteration and (+++) severe alteration. results olfactory epithelium of control fish: olfactory epithelium (oe) of c. punctatus was a thick (30-40 µm) sheet of pseudostratified epithelium consisting of columnar sensory and non-sensory cells, round basal cells and mucous cells. olfactory epithelium infolded into 18-20 lamellae and formed olfactory rosette. in lamellae, the epithelium enclosed a stromal sheet containing blood vessels, connective tissues and nerve fibers. basal lamina (bl) separated 223 roy et al/ international journal of aquatic biology (2013) 1(5): 221-227 stromal sheet from the epithelial cells. sensory cells were found in the mid lateral side of the lamellae whereas non-sensory epithelium occupied the tip and basal part of the lamellae. sensory epithelium was composed of ciliated (corc) and microvillous (morc) olfactory receptor cells, supporting cells (sc) and basal cells (bcs). orcs and scs were arranged in alternate rows. basal cells were of two figure 1. sensory olfactory epithelium (oe) of control fish showing the normal arrangement of olfactory receptor cells (orc), supporting cells (sc), globular basal cells (gbc) and horizontal basal cells (hbc). basal lamina (bl) in between the epithelium and central core (cc) (channa punctatus, h&e). figure 2. oe of the fish exposed to 2.5 mg/l cdcl2 for 15 days, showing the proliferation of mucous cells (mcs), normal arrangement of ciliated orc (corc) and scs and a few vacuoles (arrow). bl remained distinct and intact (c. punctatus, h&e). figure 3. oe of the fish exposed to 5 mg/l cdcl2 for 15 days showing hyperplasia of bcs (arrows). bl distinct and intact (c. punctatus, h&e). figure 4. oe of the fish exposed to 5 mg/l cdcl2 for 30 days showing altered cellular organization and proliferative lesion (pl). proliferating cells with large nucleus (arrows). vacuoles indicated the degeneration of cells (c. punctatus, h&e). 223 224 roy et al/ international journal of aquatic biology (2013) 1(5): 221-227 morphological types including globular basal cells (gbc) and horizontal basal cells (hbc) (fig. 1). non-sensory epithelium possessed ciliated columnar cells (cnsc), mucous cells (mc) and bc. olfactory epithelium after 15 days exposure to cdcl2: mucous cells hyperplasia was found all over the epithelium. in 2.5 mg/l cdcl2 exposed fish, orcs, scs, bcs and bl were as normal as control figure 5. olfactory epithelium (oe) of the fish exposed to 5 mg/l cdcl2 for 30 days showing the proliferative lesion with large, round undifferentiated cells (arrow heads) and small cells with pyknotic nucleus around the lesion (c. punctatus, h&e). figure 6. oe of the fish exposed to 5 mg/l cdcl2 for 45 days showing thickening and outgrowth of epithelium, vacuoles (arrowheads), complete degeneration of columnar sensory and non-sensory cells and blood vessels (bv) formation within epithelial cells (c. punctatus, h&e). figure 7. oe of the fish exposed to 5 mg/l cdcl2 for 45 days showing altered cellular organization. bv within epithelium and above bl (c. punctatus, h&e). figure 8. oe of the fish exposed to 5 mg/l cdcl2 for 45 days showing extensive ramification of bv (arrowheads) and invasion of bv inside the lesion, disruption of bl, mixing of proliferating cells with the blood cells (arrow) (c. punctatus, h&e). 225 roy et al/ international journal of aquatic biology (2013) 1(5): 221-227 except a few vacuoles in the basal cell region (fig. 2). cell degeneration in sensory and nonsensory epithelium as well as basal cells hyperplasia were found when the fish exposed to 5 mg/l cdcl2. proliferating basal cells formed cluster along the basal lamina. these cells were oval and larger than the normal basal cells. cell degeneration were evident with the vacuoles formation abnormal proliferation of basal cells altered the organization of the epithelium (fig. 3). basal lamina and stromal sheet remained unchanged. olfactory epithelium after 30 days exposure to cdcl2: degeneration of sensory and non-sensory cells and hyperplasia of basal cells and mucous cells were observed when fish exposed for 30 days even at low concentration (2.5 mg/l) of cdcl2. neoplasia of basal cells formed intraepithelial lesions. the proliferative cells were larger (10±1.24 µm) than the normal basal cells (6.66±0.66 µm). nucleus to cytoplasmic ratio of these cells was higher than the normal basal cells. columnar sensory and nonsensory cells were completely degenerated and the epithelium was occupied by the proliferating undifferentiated cells. intraepithelial lesions grew further and formed lumps at an exposure to 5 mg/l of cdcl2 (fig. 4). a few of these lumps were protruded out of the epithelial surface. intercellular vacuoles within the lesion indicated the degeneration of proliferating cells. however, no blood vessel formation within the epithelium was found. basal lamina and stromal sheet were remained unaltered (fig. 5). olfactory epithelium after 45 days exposure to cdcl2: olfactory epithelium encounter with severe histopathology. even at 2.5 mg/l of cdcl2 exposure, proliferating cell mass increased the thickness of the epithelium and protruded out of the surface (fig. 6). sensory cells and columnar non-sensory cells were completely degenerated. enlarged intraepithelial lesions were occupied the epithelium at 5.0 mg/l cdcl2 exposure. angiogenesis within the epithelium and lesion and basal lamina disruption occurred (figs. 7, 8). discussion this study provided a clear evidence of the toxic effects of cdcl2 on the olfactory epithelium of c. punctatus. the structural organization of the epithelium altered due to degeneration of sensory and non-sensory cells and proliferation of basal cells. the change or damage to the olfactory system might strongly influence its functions as well as the fish behavior (hernadi, 1993; bettini et al., 2006). the immediate response of olfactory epithelium to cadmium toxicity was the hyperplasia of mucous cells. julliard et al. (1993) found that goblet cells increased in number following metal treatment. increase of mucous cell population was also reported by dang et al. (1999) in the gill epithelium following exposure to copper. immunohistochemical study revealed that mucous bound with metallothionein cadmium conjugate took an active role in eliminating the metal from the system (roy et al., 2012). therefore, hyperplasia of mucous cells and its enhanced secretion as observed in the present study was a protective mechanism. however, mucous secretion did not succeed to protect the olfactory table 1. semi quantitative scoring of the histopathology in the olfactory epithelium of cdcl2 exposed channa punctatus. histopathology cdcl2 exposure 15 days 30 days 45 days 2.5 mg/l 5.0 mg/l 2.5 mg/l 5.0 mg/l 2.5 mg/l 5.0 mg/l hyperplasia of mucous cells ++ +++ +++ +++ +++ ++ degeneration of sensory cells and supporting cells ++ ++ ++ +++ +++ hyperplasia of basal cells ++ ++ +++ +++ +++ proliferative lesion + + ++ +++ +++ alteration in the cellular organization of epithelium + + ++ +++ +++ lump formation + + ++ angiogenesis ++ score: (-) no alteration, (+) mild alteration, (++) moderate alteration, (+++) severe alteration. 225 226 roy et al/ international journal of aquatic biology (2013) 1(5): 221-227 epithelium when concentration of the metal and duration of exposure were enhanced. cadmium chloride exposure for a short period caused less histological damage to the olfactory epithelium due to its excellent regenerating power. basal cells differentiate into sensory and nonsensory cells of the epithelium during normal cell turn over and in the event of cell death. however cdcl2 exposure for a long period caused severe histopathology in the olfactory epithelium of the fish including degeneration of all sensory cells. julliard et al. (1993) observed that sublethal exposure to copper caused specific degeneration of all mature receptor cells as well as numerous immature neurons in the olfactory epithelium of oncorhynchus mykiss. hansen et al. (1999) showed that the number of receptor cells in the olfactory epithelium significantly reduced following exposure with 15 µg cu/l. cell death stimulate the proliferation of basal cells to repair the damage. basal cell hyperplasia and numerous clusters of undifferentiated basal cells were found in the olfactory epithelium of cadmium exposed fish groups. similar result was found by julliard et al. (1993) who observed numerous clusters of cells in the basal region following the exposure to copper. bettini et al. (2006) proved that low level of cu2+ was responsible for the death of fish olfactory receptor neurons. cu2+ increased the mitotic activity of the basal region of the epithelium. this study revealed that at prolonged exposure to cdcl2, uncontrolled proliferating basal cells and their failure to differentiate into normal sensory and non-sensory cells caused alteration in the normal composition of the olfactory epithelium. columnar sensory and non-sensory cells were replaced by stratified non-sensory cells. these results indicated that cadmium disrupted the normal cell cycle regulation. in this work, hyperplasia of basal cells, inflammation, proliferative lesion formation and angiogenesis in the olfactory epithelium of cadmium exposed fish indicated that cadmium chloride disrupted normal cell cycle regulation and carcinogenic at prolonged exposure. several reports supported that cadmium induced cell transformation. cadmium induced tumour in pituitary, testes and lungs (waalkes et al., 1999) and induced malignant transformation of human prostate epithelial cells (achanzar et al., 2001). dally and hartwig (1997) observed that cadmium inhibited the dna damage repairing. méplan et al. (1999) showed that cadmium suppressed cell cycle regulatory protein, p53 response to dna damage repairing. this inhibition of p53 function could play a role in cadmium induced cell transformation. this study revealed that cadmium chloride was toxic to the olfactory epithelium of channa punctatus and at prolonged exposure it caused irreversible damage to the olfactory sensory epithelium that might impair the olfactory function of the fish. acknowledgement the authors are grateful to the university grants commission, new delhi for financial support and are grateful to the former and present head of the department, department of zoology, visva-bharati university prof. s.k. maitra and prof. s.p. sinhababu for providing all laboratory and infrastructural facilities. references achanzar w.e., bhalchandra a.d., liu j., quader s.t., webber m.m., waalkes m.p. (2001). cadmium-induced malignant transformation of human prostate epithelial cells. cancer research, 61: 455-458. apha (2010). standard methods for the examination of water and wastewater, 20th edition (ed.). lenore s.c., arnold e.g., andrew d.e. (washington: apha). bettini s., ciani f., franceschini v. (2006). recovery of olfactory neurons in the african tilapia, tilapia mariae following exposure to low copper level. aquatic toxicology, 76: 321-328. chakrabarti p., ghosh s. (2010). histological and scanning electron microscopical study of the olfactory epithelium of the indian major carp, catla catla (hamilton). folia morphologica, 69: 24-29 227 roy et al/ international journal of aquatic biology (2013) 1(5): 221-227 dally h., hartwig a. (1997). induction and repair inhibition of oxidative dna damage by nickel (ii) and cadmium (ii) in mammalian cells. carcinogenesis, 8: 1021-1026. dang z., robert a.c., flik g., sjored e., wendelaar bonga s.e. (1999). metallothionein response in gills of oreochromis mossambicus exposed to copper in freshwater. american journal of physiology, 277: 320-331. finney d.j. (1971). probit analysis 3rd edition. cambridge university press, pp: 25-26. ghosh d., mandal d.k. (2012). histopathological effects and bioaccumulation of mercury in the kidney of an indian major carp, labeo rohita (hamilton). bulletin of environmental contamination and toxicology, 89: 479-483. ghosh d., mandal d.k. (2013). mercury induced toxicity response in the olfactory epithelium of labeo rohita (hamilton): a light and electron microscopic study. fish physiology and biochemistry. doi 10.1007/s10695-013-9826-2 hansen a., zippel h.p., sorensen p.w., caprio j. (1999). ultrastructure of the olfactory epithelium in intact, axotomized and bulbectomized goldfish, carassius auratus. journal of microscopic research and techniques, 45: 325-338. hara t.j. (1992). mechanism of olfaction, in: t.j. hara (ed.). fish chemoreception. chapman and hall, london. pp: 150-170. hernadi l. (1993). fine structural characterization of olfactory epithelium and its responses to divalent cataion cd2+ in the fish alburnus alburnus (teleostei, cyprinidae): a scanning and transmission electron microscopic study. neurobiology, 1: 11-31. julliard a.k., saucier d., astic l. (1993). effects of chronic low level copper exposure on the ultrastructure of the olfactory system in rainbow trout (oncorhynchus mykiss). histology and histopathology, 8: 665-672. mandal d.k., roy d., ghosh l. (2005). structural organization of the olfactory epithelium of a spotted snakehead fish, channa punctatus (bloch). acta ichthyologica et piscatoria, 35: 45-50. méplan c., mann k., hainaut p. (1999). cadmium induces conformational modifications of wild-type p53 response to dna damage in cultured cells. journal of biological chemistry, 274: 31663-31670. roy d., ghosh d., mandal d.k. (2012). induction of metallothionein in the olfactory epithelium of channa punctatus (bloch) in response to cadmium exposure: an immunohistochemical study. proceedings of zoological society, 65: 40-44. shimada h., shiao y.h., shobata m.a. (1998). cadmium suppresses apoptosis induced by chromium. journal of toxicology and environmental health, 54: 159-168. tierney k.b., baldwin d.h., hara, t.j., ross p.s., scholz n.l., kenedy c.j. (2010). olfactory toxicity in fishes. aquatic toxicology, 96: 2-26. tjälve h., henricksson j. (1999). uptake of metals in the brain via olfactory pathway. neurotoxicology, 20: 181-195. verbost p.m., van rooij j., flik g., lock r.a.c., wendelaar bonga s.e. (1989). the movement of cadmium through freshwater trout branchial epithelium and its interference with calcium transport. journal of experimental biology, 145: 185-197. waalkes m.p., miriam a., bhalchandran a.d. 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(2016) 4(1): 25-30 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article acute toxicity bioassay of the mercury chloride and copper sulphate in rutilus caspicus and rutilus kutum alireza pourkhabbaz1, borhan mansouri2, 3, mohammad hosein sinkakarimi4, ghasem rajaei*1, robabeh vajdi5 1department of environmental sciences, faculty of natural resource and environment, birjand university, birjand, iran. 2student research committee, kurdistan university of medical sciences, sanandaj, iran 3environmental health research center, kurdistan university of medical sciences, sanandaj, iran 4department of environmental science, faculty of natural resource and environment, malayer university, malayer, iran. 5department of environmental science, faculty of environmental, university of tehran, tehran, iran. article history: received 12 july 2015 accepted 22 january 2016 available online 2 5 february 2016 keywords: toxicity lc50 static bioassay fish abstract: the purpose of the present study was to determine the acute toxicity (lc50) of hgcl2 and cuso4 in caspian roach (rutilus caspicus) and cuso4 in caspian kutum (rutilus kutum). the caspian roach lc50 values for hgcl2 at 24, 48, 72, and 96-hrs of exposure, were 0.64, 0.61, 0.42, and 0.28 mg l-1, respectively, and for cuso4 were 11.55, 5.08, 2.49, and 1.47 mg l-1, respectively. the caspian roach lc50 values for cuso4 at 24, 48, 72, and 96-hrs of exposure, were 5.31, 4.17, 3.20, and 2.25 mg l-1, respectively. the results of this study showed that the toxicity of hgcl2 is higher than that of cuso4 for the studied species. the mortality decreased with time, and most of the deaths were occurred during the first 24 hrs. introduction pollution pressures are associated with urbanization endangering the coastal ecosystems. the biota may be stressed by discharged specific point sources (e.g. sewage effluents and industrial wastes) and general non-point pollution (e.g. harbor activities, storm drainage and agricultural drainage). the aquatic ecosystems that receive pollutants are often important fishery and recreational areas (pirbeigi et al., 2016; nasrolah pourmoghadam et al., 2015). it is therefore essential that techniques are established to monitor the pollutants that pose a danger to the aquatic biota and humans (gopalakrishnan et al., 2008). metals are an important group of the water pollutants that disturb the integrity of biochemical and physiological mechanisms in aquatic organisms, such as fishes. among metals, copper and mercury are of special concern since they are considerably toxic to aquatic animals at ecologically relevant concentrations (zhang et al., 2005; allen et al., * corresponding author: ghasem rajaei doi: http://dx.doi.org/10.7508/ijab.2016.01.004 e-mail address: ghasem.rajaei19@yahoo.com 1994). copper is a trace element that plays a fundamental role in the biochemistry of organisms, including aquatic organisms that can take it up directly from water (grosell et al., 2003; erickson et al., 1996). however, it can become toxic at high concentrations (alquezar et al., 2008). among metals, mercury is of special concern since it is considerably toxic to aquatic animals at ecologically relevant concentrations (vieira et al., 2009; oliveira et al., 2002). mercury is considered one of the most dangerous metals in the aquatic environment (goyer et al., 1995; ribeiro et al., 1996), mainly its organic forms that can be biomagnified in trophic chains representing an increased risk for top predators (macdougal et al., 1996), including humans consuming contaminated fishes. chronic exposure and accumulation of these chemicals by aquatic biota can result in tissues that produce adverse effects not only in the exposed organisms, but also in human beings (iarc., 1990; karthikeyan et al., 2007). therefore, it is essential to study the detrimental 26 pourkhabbaz et al./ acute toxicity of the mercury and copper in r. caspicus and r. kutum effects of such hazardous pollutants so as to formulate the strategies for safeguarding aquatic organisms. caspian roach (rutilus caspicus) and caspian kutum (rutilus kutum) are found in the southern part of the caspian sea, particularly in the coastal waters of iran, turkmenistan and azerbaijan. these species are economically important fishes (coad, 2015). hence, this study aimed to investigate the toxic effects of the mercury chloride and copper sulphate on the caspian roach and caspian kutum by determination of 96-hour lc50 values materials and methods the specimens of caspian roach (n=120) and caspian kutum (n=120), with mean weight of 3±0.6 g were obtained from sijval restocking center (bandar turkaman, north of iran) during august to september 2009. the fishes were transported in polythene bags to the fisheries laboratory of the gorgan university of agricultural sciences and natural resources. then, they were maintained in 20 l pre-cleaned glass aquaria filled with dechlorinated tap water with water temperature of 26±1°c in order to adaptation to the laboratory conditions. thereafter, sets of 10 specimens (in triplicate) were randomly introduced to the aquarium systems. the exposure time to hg (as hgcl2) and cu (as cuso4) were 96 hours, without any feed. control group was designed with three replicates. no mortality was observed during the experimental period in control groups as well. in addition, four different concentrations of the mercury (as hgcl2) in geometric decreasing amounts of 0.02, 0.03, 0.04, and 0.05 mgl-1 were used. also, four different concentrations of copper (as cuso4) in geometric decreasing amounts of 1, 4, 7, and 10 mg l-1 in threereplicates were designed. for determination of the mortality limits of the mercury and copper as well as survival rate, the treatments and replications were considered based on oecd (oecd, 1988). stock solutions (1000 mg l-1) were prepared by dissolving analytical-grade of the mercury (as hgcl2, merck) and copper (as cuso4; merck) in distilled water. preliminary tests were carried out to estimate the minimum lethal and maximum nonlethal concentrations of the mercury (as hgcl2) and copper (as cuso4). dissolved oxygen (do) (mg l 1), temperature (°c), total hardness (mg l-1) and ph were recorded in each aquarium during experiment. the recorded water parameters were as: temperature=26±1°c, ph=7.7±0.2; total hardness=205 mg l-1 as caco3, and do=7 mg l -1. water quality of the experimental tank was determined according to standard procedures. lethal concentration for 50% (lc50) values were calculated using the epa computer probit analysis program (version 1.5) (vieira, 2009; das et al., 2005; pyle et al., 2002). statistical analyses were performed using spss software (ver. 16.0, spss co., chicago, il, usa). all the data were tested for normality using kolmogorov-smirnov test. results the lc50 value for hgcl2 and cuso4 in the caspian roach and caspian kutum at 24, 48, 72, and 96-hrs of exposure period were presented in tables 1 and 2. the lc50 value in caspian roach for hgcl2 at 24, 48, 72 and 96-hrs of exposure period were 0.64, 0.61, metal lc50 values (mg l -1) 24 hours 48 hours 72 hours 96 hours copper lc10 0.69 0.56 0.48 0.43 lc50 5.31 4.17 3.20 2.25 lc90 40.07 30.11 21.04 11.36 table 1. lethal concentration (lc50) of mercury and copper estimated by epa method on the caspian roach (rutilus caspicus). 27 int. j. aquat. biol. (2016) 4(1): 25-30 0.42, and 0.28 mg l-1, respectively; while for cuso4, the lc50 values at 24, 48, 72 and 96-hrs were 11.55, 5.08, 2.49, and 1.47 mg l-1, respectively (table 1). discussion the 96-hrs lc50 values of fish is species and metal dependent. in this study, the 96-hrs lc50 for hgcl2 was determined to be 0.28 mg l-1. in other studies the acute toxicity thresholds for inorganic mercury (typically as hgcl2) in freshwater organisms vary from approximately 0.005-0.230 mg l-1 in crustaceans, to 0.06-0.8 mg/l in fish (ramamoorthy and baddaloo, 1995). the 96-hrs lc50 values for freshwater fish ranges 0.033-0.4 mg l-1, while the lc50 is generally higher for marine fish (boening, 2000). the 96-hrs lc50 values of hgcl2 on capoeta fusca was 0.154 mg l-1 (mansouri and baramaki, 2011), on gambusia holbrooki was 0.36 mg l-1 (ebrahimpour et al., 2010a, b; pourkhabbaz et al., 2011), and on acanthoparus latus was 0.648 mg l-1 (hedayati and safahieh., 2010). in addition, the 96hrs lc50 values of hgcl2 on heteropneustes fossilis, oncorhynchus mykiss, roccus saxatilis, and salvelinus fontinalis were found to be 0.35, 0.22, 0.09, and 0.075 mg l-1, respectively (pandey et al., 2005). the acute toxicity of the copper sulphate decreased with increasing exposure time in both species. copper sulphate was significantly (96-hrs lc50 values) more toxic on r. caspicus than r. kutum. the observed differences in the acting copper might be species dependent and their susceptibility rates to the test chemical, which resulted in their subsequent toxicity values. copper salts combine with proteins present in the mucus of the fish's mouth, gills, and skin, preventing respiration causing death (richey and roseboom 1987; peres et al., 1991). as conclusion, the mercury chloride was more toxic than copper sulphate on the caspian roach. in general, the toxicity of hgcl2 was higher than that of cuso4 in both studied species. acknowledgements the authors wish to express their gratitude to many people who have devoted their time and expertise to this project. we are also grateful to r. tahergoorabi and s.v. hosseini for their assistance in the preparation of the manuscript. references abdullah s., javed m. 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(2016) 4(1): 25-30 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی rutilus caspicus مس برروی کلمه ترکمنی سنجی سمیت حاد کلرید جیوه و سولفاتزیست rutilus kutumماهی سفید، و 5ربابه واجدی، *1رجایی، قاسم 4، محمدحسین سینکاکریمی3،2برهان منصوری ،1علیرضا پورخباز .ایران ،بیرجند ،بیرجند دانشگاه طبیعی، منابع دانشکده ،علوم محیط زیستی گروه1 .ایران ،سنندج ،علوم پزشکی کردستان دانشگاه ،کمیته تحقیقات دانشجویی2 .ایران سنندج، ،علوم پزشکی کردستان دانشگاهمرکز تحقیقات بهداشت محیط، 3 .ایران ،مالیر ،مالیر دانشگاه ،و محیط زیست طبیعی منابع دانشکده ،علوم محیط زیستی گروه4 .ایران ،تهران ،تهران دانشگاه ،محیط زیست دانشکده ،علوم محیط زیستی گروه5 چکیده: سفید ماهی و( rutilus caspicus) کلمه ترکمنی ماهی در مس سولفات و جیوه کلرید( 50lc) حاد سمیت تعیین حاضر مطالعه از هدف (rutilus kutum )50 میزان. بودlc و ،42/0 ،91/0 ،94/0 ترتیببه مواجهه ساعت 69 و ،22 ،44 ،24 در جیوه کلرید برای ترکمنی کلمه ماهی ماهی 50lc مقادیر همچنین. آمد بدست لیتر در گرممیلی 42/1 و 46/2 ،04/5 ،55/11 ترتیب به نیز مس سولفات برای و بودند گرممیلی 24/0 مطالعه این از حاصل نتایج. بود لیتر درم گرمیلی 25/2 و 20/3 ،12/4 ،31/5 ترتیببه مواجهه ساعت 69 و ،22 ،44 ،24 در مس سولفات برای سفید ترینباال و یافت، کاهش زمان روند با میر ومرگ میزان. است بوده مطالعه مورد هایگونه برای مس سولفات از باالتر جیوه کلرید سمیت که داد نشان .داد رخ اول ساعت 24 طول در میر و مرگ میزان .ماهی استاتیک، سنجی زیست ،50lc سمیت، :کلمات کلیدی int. j. aquat. biol. (2013) (1): 36-41 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology effects of different photoperiods on the survival and growth of beluga sturgeon (huso huso) larvae hamid eshagh zadeh*1, gholamreza rafiee1, soheil eagderi1, rezvanollah kazemi2, hadi poorbagher1 1department of fisheries, faculty of natural resources, the university of tehran, p.o. box: 31585-4314, karaj, iran. 2 international sturgeon research institute, p.o. box 41635-3464, rasht, iran. article history: received 16 march 2013 accepted 4 april 2013 available online 15 april 2013 keywords: acipenseridae beluga photoperiod morphometrics abstract: effect of different photoperiodic regimes was evaluated on growth performance and survival rate of the beluga (huso huso) prelarvae and larvae. newly hatched prelarvae were stored in 5 round fiberglass 500 l tank with different photoperiod (24l:00d, 18l:06d, 12l:12d, 06l:18 d, 00l:24d) till 50 days post hatch with three replicates. light intensity was 200 lux during the experiment. feeding was started from 8 days post hatch using live artemia nauplii. higher total length, survival rates and lower body area, yolk area for beluga prelarvae obtained in long light photoperiods (24l:00d, 18l:06d). also, higher growth parameters of the beluga larvae observed in long light photoperiods while different photoperiods had no effect on survival rate. the present study indicated that growth performance and survival rates of larvae are significantly influenced by photoperiod. the photoperiod 18l:06d resulted in the best growth performance and survival rate during early development of the beluga. introduction decline of sturgeon stocks in the caspian sea has urged the development of artificial propagation programs in hatcheries for both aquacultural and restocking proposes (bronzi et al., 1999). during artificial propagation of sturgeon fishes, the production of larvae is the most critical and important stage (yasemi et al., 2011). among the sturgeon fishes of the caspian sea, production of beluga larvae (huso huso) has drawn a great attention due to its high economic value and suitability for aquacultural propose. since the main focus of aquaculture is to use the techniques that increase fertilization success, survival rate and growth performance (verhaegen et al., 2007). the environmental factors such as light intensity and photoperiods, temperature and salinity are considered as important parameters effecting growth and survival of fish larvae (puvanendran and brown, * corresponding author: hamid eshagh zadeh e-mail address: hamidshil@yahoo.com 2002; martıńez-palacios et al., 2004; van eenennaam et al., 2005). light was found an important stimulant during the early development of sturgeon fishes in chinese sturgeon (acipenser sinensis), siberian sturgeon (a. baerii) and shortnose sturgeon (a. brevirostrum) (zhuang et al., 2002; richmond and kynard, 1995). also, a swim up behavior toward light zone and ontogenetic behavior has been reported in genus acipenser, scaphirhynchus and huso at the time of downward migration indicating important role of light and vision in their ontogenetic behavior (boglione et al., 1997; zhuanga et al., 2003). the role of vision and light preference in prelarvae and larvae of sturgeon fishes have been demonstrated in their behaviors such as orientation, schooling, rheotaxis, phototaxis, avoiding predators and habitat selection. however, there are differences in those behaviors, depending on the ecological 37 eshagh zadeh et al./ int. j. aquat. biol. (2013) (1) 36-41 requirements of species, species-specific, their life stage, feeding habitats (olla et al., 1996; kynard and horgan, 2002; rodrý´guez and gisbert, 2002; arnold, 1974). during the absorption of yolk-sac, sturgeon larvae have a low density of rod cells in their retina but density of the cells increase later to strengthen the vision under dark conditions (blaxter and staines, 1970; chai et al., 2006). whereas in most marine fish larvae, light and vision play vital role in early feeding stage because they do not feed at night (fielder et al., 2002; villamizar et al., 2009). hence, this study was conducted to survey the effect of different photoperiodic regimes as an important environmental factor on survival rate and growth performance of farmed-beluga sturgeon from 0 dph (day post hatch) until 50 dph. material and methods this experiment was accomplished at the dadman international sturgeon research institute (guilan, iran). the specimens were obtained from the artificial propagation of four (three males and one female), nine years old farmed-beluga sturgeon. newly hatched larvae i.e. eleutheroembryo were immediately introduced into 15 round fiberglass 500-l tanks (105×102×52 cm). the larvae were exposed to the photoperiods d24l:00d, 18l:06d, 12l:12d, 06l:18d and 00l:24d until 50 dph. the experiment was carried out in three replicates with a stocking density of 33 g larvae (with an average weight of 18.2 ± 0.001 mg and total length of 11.20 ± 1.1 mm, [mean ± sd]) per 500 l tank with a water depth of 20 cm till 12 dph and, after that, the water depth increased to 30 cm until the end of experiment. a 60 watt light fluorescent bulb (nama noor, iran) was used as a light source in all treatment. the bulb was suspended over each tank at same height to produce 200 lux light intensity during the experiment. a timer was used to set the dark and light periods. light intensity was measured using a light meter (tes, model 1336a, taipei, taiwan). the water was supplied from ground and river water with a discharge of 400 and 250 ml min-1, respectively. the water temperature, do and ph of treatments were recorded 15.9, 7.7 and 7.8 during the experiment, respectively. due to asynchrony in external feeding of prelarvae and to avoid starving and cannibalism, the larvae were fed a diet of artemia nauplii, daphnia and formulated diets (biomar 0.5 mm diameter contained 58% protein, 15% fat, 9.8% carbohydrate, 11.9% ash, 1.7% total phosphorus). feeding was started from 8 dph using live artemia franciscana nauplii (500 nauplii/larvae six times a day). later, a mixture of daphnia and artemia were used from 12 dph to 25 dph. from 20 dph, the percentage of live food decreased gradually and the percentage of formulated diets increased so that from 25 dph to 50 dph, the larvae were fed 100% formulated diets at a rate of 30% total biomass four times a day. dead larvae were removed and counted daily at each tank to measure the survival rate. effects of photoperiod on the growth performance and survival rate was investigated in two different stages, i.e. at the end of yolk sac absorption stage (12 dph) and at the end of the experiment (50 dph). at the end of each stage, 30 larvae from each tank were sampled and fixed in 5% buffered formalin. wet weight of larvae (ww) was measured to the nearest 0.001 g. left side of specimens were photographed using a stereomicroscope equipped with a 8mp digital camera (cannon) and their total length (tl), body area (ba) and area of yolk sac (ya), were measured using image j software. then, specific growth rate (sgr) and condition factor (cf) were calculated. prior to the analysis, normality and homogeneity of variance were checked and percentage data were transformed using arcsine. data were analysed using one-way anova to examine the effect of photoperiod on growth parameters, survival rate of beluga larvae, followed by duncan’s multiple range test (zar, 1994). data are presented as mean ± sd. results at the hatching time (0 dph), ya had a mean of 6.58 mm2 and decreased to 1.252 mm2 at 12 dph and there 38 eshagh zadeh et al./ int. j. aquat. biol. (2013) (1) 36-41 was no significant difference among treatments (p < 0.05). the treatment with longer light period (24l:00d, 18l:06d) had a greater effect on the absorption of the yolk sac than those with shorter light period (12l:12d, 06l:18d, 00l:24d). maximum and minimum of ya in the prelarvae were recorded in 00l:24d with 1.49 mm2 and 18l:06d with 1.03 mm2, respectively. the mean ba was measured 14 .7160 mm2 at 0 dph that reached to 42.486 mm2 in 12 dph and there was a significant difference between photoperiods (p<0.05). a higher ba was recorded in 00l:24d, which may be due their larger yolk sacs compared to others. there was a significant difference between photoperiods in tl and cf (p<0.05). the maximum and minimum tl were recorded in 24l:00d (24.82 ± 0.56 mm) and 00l:24d (22.83 ± 0.47), respectively. while maximum and minimum cf were recorded in 00l:24d (0.416 ± 0.30) and 24l:00d (0.324 ± 0.36), respectively. there was no significant difference between photoperiods in wet weight of larvae and sgr (p>0.05). mortality of prelarvae started at 0 dph and significant difference was detected between photoperiods in survival rate (p<0.05). as shown in table 1, the long light photoperiods (24l:00d, 18l:06d) increased the survival rate. the maximum and minimum survival rates were found in 18l:006d (95.86%) and 00l:24d (60.83%), respectively. as shown in table 2, there were significant differences between photoperiods in growth parameters, whereas no significant difference was detected between photoperiods in survival rate after the absorption of yolk sac. a higher tl and ww was recorded in the photoperiods with longer duration of light (24l:00d, 18l:06d), i.e. in 24l:00d and 18l:06d higher tl (9.82 ± 1.02) and ww (3.47± 1.45) were recorded, respectively. in 12l:12d and 00l:24d lower tl (9.08 ± 0.9) and ww (2.74 ± 1.03) were measured, respectively. 12l:12d and 00l:24d resulted in significantly higher (0.435 ± 0.03) and lower (0.319 ± 0.05) cf (p < 0.05). photoperiod had no significant effect on sgr. discussion the present study indicated that different photoperiods have significant effects on growth parameters and survival rate during and after yolk sac absorption. at the first stage, the earlier yolk sac absorption, lower body surface area, highest total length and survival rates of eleutheroembryo were observed in long light photoperiods, but these photoperiods had no significant effect on the wet weight and sgr. therefore, according to the results, long light photoperiodic regime result in faster absorption of yolk sacs, i.e. minimum ya and maximum ba in prelarvae. since the absorption of yolk sac is related to morphogenesis and metabolic processes, long light time photoperiodic regime can increase metabolism and growth performance (blaxter and hempel, 1966), which was observed in table 1. average (± sd) of growth parameters between photoperiods during yolk sac absorption. photoperiod 00l:24d 06l:18d 12l:12d 18l:06d 24l:00d final weight (mg) 49.46 ± 2.25a 48.80 ± 4.7a 49.73 ± 3.2a 50.83 ± 4.8a 49.56 ± 5.7a initial weight (mg) 18.2 ± 0.001 18.2 ± 0. 001 18.2 ± 0.001 18.2 ± 0.001 18.2 ± 0.001 final total length (mm) 22.83 ± 0.47a 23.54 ± 0.88ab 23.61 ± 0.62ab 23.82 ± 0.47ab 24.82 ± 0.56c initial total length 11.20 ± 1.1 11.20 ± 1.1 11.20 ± 1.1 11.20 ± 1.1 11.20 ± 1.1 yolk sac area (mm2) 1.89 ± 0.54 c 1.68 ± 0.46bc 1.68 ± 0.38bc 1.43 ± 0.384a 1.58 ± 0.37ab body area (mm2) 45.36 ± 1.6 b 42.44 ± 2.4a 41.75 ± 1.4a 41.43 ± 1.8a 41.44 ± 2.5a specific growth rate (%) 8.33 ± 0.97 8.21 ± 1.7 8.37 ± 0.94 8.55 ± 1.2 8.34 ± 1.2 condition factor (%) 0.416 ± 0.30c 0.377 ± 0.55b 0.379 ± 0.38b 0.374 ± 0.40b 0.324 ± 0.36a survival (%) 60.83 ± 22.27a 76.86 ± 10.49a 83.71 ± 15.82ab 95.86 ± 0.60b 91.15 ± 5.23b 39 eshagh zadeh et al./ int. j. aquat. biol. (2013) (1) 36-41 beluga prelarvae. our results are consistent with previous studies on the eleutheroembryo of hadduck (melanogrammus aeglefinus) as those exposed to the photoperiod 24l:00d had smaller ba compared to those exposed to 18l:06d and 12l:12d. in this study, increasing the light intensity decreased ya, but had no effect on ba (downing and litvak, 2002). in cod larvae (gadus morha) the different photoperiods had no effect on absorption the yolk sac (puvanendran and brown, 2002). maximum tl i.e. best growth performance and survival rate were occurred in 24l:00d and 18l:06d during prelarval development, respectively. whereas the best growth performance observed was not coincided with higher survival rate. this finding is in agree with light preference (positive phototaxis) and selection of white substrates in siberian sturgeon (a. baerii) and chinese sturgeon (a. sinensis) (chai et al., 2006; gisbert et al., 1999) whereas, the low survival of surubim (pseudoplatystoma corruscans) in the 24l:00d photoperiodic regime has been assigned to increasing swimming activity and energy consumption (campagnolo and nuñer, 2008). also, puvanendran and brown (2002) found an increase of survival rate of cod (gadus morha) larvae in the d24l:00d photoperiods during 0 dph to 28 dph compared with 18l:06d and 12l:12d. however, from 28 dph to 42 dph, there no difference between photoperiods in this species (puvanendran and brown, 2002). in addition, our result showed that photoperiod had positive influence on growth and survival rate in prelarval stage, but only on growth performance in larval stage. similar to previous studies, our study indicated that optimum photoperiods for growth and survival are different (fielder et al., 2002; el-sayed and kawanna, 2004). however in different species continuous light (campagnolo and nuñer, 2008), dark (piaia et al., 1999) and intermediate (solbakken and pittman, 2004) photoperiod may act better in comparison to other photoperiods. other studies on 6-month and one-year old beluga showed that 12l:12d had the best influence on growth performance and survival rate (bani et al., 2009; ghomi et al., 2010). finally, it is need to be mentioned that dark-light cycles in fish farming changes depend on species based on its habitat and life history. marine fish larvae dependent on light and vision (fielder et al., 2002) but sturgeon feeding is not dependent on vision. nevertheless, presence of light is essential in addition to other factors such as chemical and electrical for optimum growth and feeding. references arnold g. 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(2003). comparative ontogenetic behavior and migration of kaluga, huso dauricus, and amur sturgeon, acipenser schrenckii, from the amur river. environmental biology of fishes, 66: 37-48. international journal of aquatic biology (2015) 3(4): 236-244 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article histoarchitectural and surface ultrastructural analysis of the olfactory epithelium of puntius ticto (hamilton, 1822) saroj kumar ghosh*1,2, bappaditya pan3, padmanabha chakrabarti2 1department of zoology, bejoy narayan mahavidyalaya, itachuna, hooghly-712147, west bengal, india. 2fisheries laboratory, department of zoology, the university of burdwan, burdwan-713104, west bengal, india. 3university science instrumentation centre, the university of burdwan, burdwan-713104, west bengal, india. article history: received 10 april 2015 accepted 7 july 2015 available online 2 5 august 2015 keywords: olfactory organ functional anatomy histology scanning electron microscopy abstract: organization of various cells lining the olfactory mucosa of puntius ticto (hamilton, 1822) were described by light as well as scanning electron microscopy. the paired olfactory chambers located antero-dorsal to the eyes and communicated outside through anterior and posterior nasal openings. the oval shaped olfactory rosette lied at the bottom of chamber and composed of 18-20 lamellae arranged on either side of median raphe. sensory and non-sensory regions were distributed separately on each lamella. the sensory epithelium consisted mainly of two distinct morphological forms: ciliated and microvillous receptor cells. the non-sensory epithelium contained ciliated nonsensory cells, stratified epithelial cells with concentric microridges and mucous cells. basal cells were situated at the deeper part of the epithelium, adjacent to the central core. the functional significance of cellular components of the olfactory epithelium was discussed with the habit and habitat of fish. introduction olfaction is one of the most important senses driving basic patterns of behavior in fishes and involved in food searching, nest finding, predator-prey relationship, mother-infant recognition, kin identification, reproductive behavior, homing behavior and in many other ways (farbman, 1994). functional neuroanatomy of olfactory apparatus is attracting to study as it is the only organ in teleost where receptor neurons are directly exposed to aquatic environment. the receptor cells lining the olfactory epithelium are stimulated when they come into contact with water containing chemicals pass from nostrils to the olfactory mucosa and transmit signals to the nervous system (lara, 2008). the olfactory organs of fish exhibits extensive array of diversification relying upon systematic groups, life styles and ecological adaptations (zeiske et al., 2009). extensive investigations and information on * corresponding author: saroj kumar ghosh e-mail address: saroj.fisherylab@gmail.com the microarchitecture of the olfactory epithelium of different teleosts are well-documented using light and electron microscope (zeiske et al., 1994; byrd and brunjes, 1995; diaz et al., 2002; hansen and zielinski, 2005; lazzari et al., 2007; chakrabarti and ghosh, 2009; 2011; atta, 2013; ghosh and chakrabarti, 2014). still lacunae exist in some aspects of the studies relating to the cellular basis of olfaction among cyprinids. the present study attempts at finding out the detailed functional organization of the olfactory system in freshwater ticto barb, puntius ticto (cypriniformes, cyprinidae), found mostly at surface layer of waters and feeds on crustaceans, insects, planktons etc. (banik and saha, 2012). materials and methods tissue collection: a total of 24 adult live specimens of p. ticto (ranged 5.84 ± 0.06 cm in total length) 237 ghosh et al/ organization of the olfactory epithelium in puntius ticto were procured from regional freshwater pond of burdwan (23.2333°n, 87.8667°e), west bengal, india during september-december, 2014. fish were anesthetized with an aqueous solution of benzocaine (4 mg l-1) and sacrificed following the guidelines given by the institutional ethical committee. intact olfactory organs were attentively dissected out from the olfactory chamber under a zeiss stemi 2000-c stereoscopic binocular microscope and further processed for respective studies. histological preparation: the olfactory tissues were immediately fixed in aqueous bouin’s fixative for 16-18 hrs. after that, the fixed tissues were washed thoroughly in 70% ethanol, dehydrated with graded series of ethanol and cleared in xylene. then, the tissues were infiltrated in paraffin wax of 56-58˚c under a thermostat vacuum paraffin-embedding bath for one hrs. serial paraffin sections were cut at 4 µm thickness using leica rm 2125 rt microtome. after routine histological process deparaffinized sections were stained with delafield’s haematoxylin-eosin (he) and mallory’s triple (mt) stain (mallory, 1936) and observed under an olympus-tokyo pm-6 compound microscope. scanning electron microscopical (sem) preparation: after dissecting the olfactory chamber the olfactory rosettes were immersed in vivo with 2.5% glutaraldehyde solution in 0.1 m phosphate buffer (ph 7.4) for 20 mins. then, the olfactory rosettes were carefully dissected out and rinsed in 0.1 m phosphate buffer (ph 7.4). the samples were fixed with 2.5% glutaraldehyde for 24 hrs at 4°c and post fixed with 1% osmium tetroxide in 0.1 m phosphate buffer (ph 7.4) for two hrs. fixed tissues were washed repeatedly in same buffer and dehydrated in ascending series of acetone followed by isoamyl acetate. the tissues were dried with critical point drier (hitachi 8cp2), mounted on metal stubs, coated with gold palladium (20 nm thickness) and observed under a scanning electron microscope, hitachi s530. results puntius ticto bears a pair of the nasal pits located on the antero-dorsal surface of the head and slightly above the level of the eyes (fig. 1). each olfactory figure 1. dorsal view of head of p. ticto showing the position of olfactory pits (arrows). figure 2. dissection of head from the dorsal side showing the anatomical relationship of the brain and the olfactory rosettes (or) along with olfactory nerves (arrows) and olfactory bulbs (ob). ch represents cerebral hemisphere; opl: optic lobe; c: cerebellum; mo: medulla oblongata. 238 international journal of aquatic biology (2015) 3(4): 236-244 chamber is occupied by an olfactory rosette and communicates outside by two openings which lie close to each other. the olfactory chamber along with olfactory rosette is resided in a fossa framed in the ethmoid region of the skull and is affixed to the neighboring bones by connective tissues. olfactory rosettes along with olfactory bulbs are connected to the telencephalon by narrow olfactory nerves (fig. 2). the olfactory rosette is an oval texture with a convex ventral and a concave dorsal surface, occupying the entire space in the olfactory chamber (fig. 3). the rosette holds a central median raphe which is framed with 18-20 bilaterally arranged olfactory lamellae. the lamellae are closely set and conjoined to the wall of the olfactory chamber by their convex inner edges where as to the raphe by their proximal ends. the magnitude of lamellae differs in relation to their position in rosette. the lamellae are large in the middle and reduce in size towards anterior and posteriors ends of the olfactory rosette (fig. 3). the anterior and posterior lamellae are adhered obliquely to the raphe while the middle portions are nearly perpendicular. olfactory lamellae are distinguished into sensory and non-sensory epithelium. the outer concave margins of the lamellae bear welldeveloped linguiform processes (fig. 3) covered with sensory epithelium (fig. 4), whereas resting part of the lamellae are characterized with nonsensory epithelium. histologically each olfactory lamella consists of the olfactory epithelium and a central lamellar space, figure 3. oval shaped olfactory rosette supported with olfactory lamellae (ol) radiating from median raphe (r). arrows mark linguiform processes of ol (sem 60x). figure 4. linguiform process of olfactory lamellae (ol) showing sensory epithelium covered with dense aggregation of receptor cells (rc) (sem 600x). figure 5. olfactory lamellae (ol) based on raphe (r) showing organized olfactory epithelium (oep) separated by the central core (cc) (he 150x). 239 ghosh et al/ organization of the olfactory epithelium in puntius ticto the central core (fig. 5). the central core is composed of the loosely connective tissue containing nerve fibers (figs. 6, 7) and blood vessels. the olfactory epithelium is made up of the compactly arranged receptor cells having long dendrite reaching up to the free epithelial surface. in between the receptor cells, the mucous cells and stratified epithelial cells are present (fig. 6). the sensory epithelium is composed of a primary, secondary and few microvillous receptor cells. the primary receptor cells have long dendrite processes reaching up to the free epithelial surface and cell bodies with rounded, deeply stained nuclei located deep in the epithelium (fig. 7). the secondary receptor cells are differentiated by their elongated oval nuclei, present below the primary receptor cells and extend up to the central core below. microvillous receptor cells are small in number, more superficial in the olfactory epithelium having lightly stained nuclei but without cilia (fig.7). in some areas of the olfactory epithelium, the receptor cells are characterized with bipolar dendrites (fig. 6). scanning electron microscopic investigations have shown that the sensory epithelium consists of the morphologically distinct two types of receptor cells: ciliated and microvillous (fig. 8). ciliated receptor cells are provided with cluster of long cilia, rise above the epithelial surface. on contrary, microvillous receptor cells contain short dendrons and exhibits carving texture. ciliated receptor cells are the dominant over microvillous receptor cells. non-sensory epithelium is comprised of patches of ciliated non-sensory cells and polygonal stratified epithelial cells with irregularly arranged microridges showing finger print like architecture (fig. 9). secreted mucins from mucous cells are present over the microridges of the stratified epithelial cells. figure 6. olfactory epithelium (oep) showing the arrangement of prominent receptor cells (rc), stratified epithelial cells (arrow heads), mucous cells (mc) and basal cells (broken arrows) adjacent to central core (cc). solid arrows indicate bipolar dendrites of rc. note prominent nerve fibers (n) in cc (mt 600x). figure 7. higher magnification of sensory olfactory epithelium (oep) showing primary (solid arrows), secondary (broken arrows) and microvillous (arrow heads) receptor cells. note the presence of nerve fibers (n) in central core (cc) (he 1000x). 240 international journal of aquatic biology (2015) 3(4): 236-244 under optical microscope, the non-sensory epithelium is mainly lined with stratified epithelial cells having prominent nuclei and scattered mucous cells (fig. 10). basal cells are almost round in shape having centrally placed prominent nuclei and chromophobic cytoplasm, located in the deeper part of the epithelium (figs. 6, 10). the surface epithelium of raphe is marked by compactly arranged stratified epithelial cells with concentric microridges interspersed with mucous cells (fig. 11). discussion olfactory organ of fish is essentially a chemoreceptor and plays a meaningful role not only locating food but also detecting the presence of odoriferous substances in the aquatic ecosystem (datta and das, 1980). fish possesses a good sense of smell and are able to detect odour with the help of a pair of olfactory rosettes connected to the olfactory lobes of the brain by means of olfactory tracts (singh, 1977). in p. ticto, the olfactory rosettes are paired, located at the bottom of olfactory chamber which contain two openings through which water enters and leaves. during forward progression of fish the unidirectional circulation of water containing figure 8. surface view of sensory epithelium showing microvillous receptor cells (arrow heads) circumscribed by cluster of ciliated receptor cells (rc). solid arrow indicates blood cell (sem 5000x). figure 9. surface architecture of non-sensory epithelium showing patchy distribution of ciliated non-sensory cells (broken arrows) and polygonal stratified epithelial cells (sec) with fingerprint like microridges. note the presence of scattered mucous cells (solid arrows) and retention of mucin droplets (arrow heads) over sec (sem 4500x). figure 10. non-sensory epithelium (oep) lined with stratified epithelial cells (arrow heads) and mucous cells (solid arrows). note the presence of basal cells (bc) near the central core (cc) provided with blood vessels (bv) (mt 600x). 241 ghosh et al/ organization of the olfactory epithelium in puntius ticto odorants through the olfactory chamber is brought about by the activity of cilia present in the anterior nasal opening and the olfactory epithelium (goel, 1978). the oval shaped rosette of p. ticto, with 18-20 lamellae arranged on either side of the central median raphe, can be classified with bateson’s (1889) rosette type 3 or under burne’s (1909) rosette column i. the feeding habits of fish are reflected on the structural organization and size of sense organs particularly eye and olfactory apparatus (atta, 2013). puntius ticto possess equally developed vision and olfaction is thus a mediosmatic species and belongs to teichmann’s (1954) 1st group “eye-nose fishes”. olfactory rosette in p. ticto is multi lamellar, which increase the surface area of epithelium as well as the sensitivities and efficacy of the olfactory organ (zeiske et al., 1976). the dorsal edges of the lamellae bear particularly well-developed linguiform processes which slow down the water flow over the olfactory mucosa and facilitate better interaction of odorants particles with receptor cells. sensory and non-sensory olfactory epithelium exhibits variation in distribution among teleosts (yamamoto, 1982). in p. ticto, sensory regions are confined mainly in the linguiform processes while non-sensory regions are scattered rest of the lamellae. sensory epithelium consists of different type receptor cells whereas the non-sensory epithelium is made up of stratified epithelial cells and mucous cells. ciliated receptor cells are known as generalists; respond to varying species of odorants including amino acids, bile salts and other chemical cues while microvillous cells are called as specialist, which respond specifically to amino acids and nucleotides (hansen et al., 2003). the dendrite process of receptor cell which terminates at the free surface of the epithelium is the locus where olfactory transduction is initiated by contact with odoriferous substances (reese, 1965). in the present study, bipolar neurons with cylindrical process have a special interest because they probably form a different olfactory transduction mechanism stimulated by odour bearing substances. the surface of non-sensory epithelium is covered with a dense mat of non-sensory cilia which propel streams of incoming water and/or dissolved chemicals between the lamellae and the cilia of receptor cells (singh and singh, 1989). in p. ticto, the non-sensory epithelium is lined with principally of stratified epithelial cells characterized with finger print like microridges are thought to perform a role in supporting tissues exposed to abrasive forces (uehara et al., 1991). the stratified epithelial cells have been suggested to perform several functions: secretory, absorbing and glial (theisen, 1972; yamamoto and ueda, 1978; hernadi, 1993; hansen and zeiske, 1998). mucous cells secrete mucin which holds by the microridges of stratified epithelial cells, protects the olfactory epithelium from external hazards and lubricates the epithelial surface to smooth flow of water during ventilation. the mucus layer may also serve as an ion trap, which delays the access of heavy metals and salts to underlying organs (waryani et al., 2013). the basal cells are situated adjacent to the central core in both sensory and non-sensory olfactory epithelium of p. ticto, assumed to be the progenitor figure 11. surface feature of raphe characterized with stratified epithelial cells (sec) having concentric microridges. note the presence of mucous cells (solid arrows) and mucin mass (arrow heads) over sec (sem 5500x). 242 international journal of aquatic biology (2015) 3(4): 236-244 cells of the receptor (graziadei and metcalf, 1971) and supporting cells (ojha and kapoor, 1973). zeiske et al. 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(1982). comparative morphology of the peripheral olfactory organ in teleosts. in: t.j. hara (ed.). chemoreception in fishes. elsevier, amsterdam. pp: 35-59. zeiske e., mellinkat r., breucker h., kux j. (1976). ultrastructural studies on the epithelia of the olfactory organ of cyprinodonts (teleostei, cyprinodontoidae). cell and tissue research, 172: 245-267. zeiske e., theisen b., breucker h. (1992). structure, development and evolutionary aspects of the peripheral olfactory system. in: t.j. hara (ed.). fish chemoreception. chapmann and hall, london. pp: 1339. zeiske e., theisen b., breucker h. (1994). the olfactory organ of the hardhead sea catfish, arius felis (l.): gross morphology and fine structure. acta zoologica, 75: 115-123. zeiske e., bartsch p., hansen a. (2009). early ontogeny of the olfactory organ in a basal actinopterygian fish: polypterus. brain, behavior and evolution, 73: 259272. international journal of aquatic biology (2015) 3(4): 236-244 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved چکیده فارسی puntius ticto (hamilton, 1822) ماهی بویایی اپیتلیوم ارتفراساخ و بافتی ساختار تحلیل و تجزیه 1، پادمانابها چاکرابارتی3اپادیتیا پانب، 2،1*ومارگوشکساروج ، بنگال غربی، هندوستان.bejoy narayan mahavidyalaya ،itachuna ،hooghly 721217 جانورشناسی، گروه ، بنگال غربی، هندوستان.721211جانورشناسی، دانشگاه بوردوان، بوردوان گروهآزمایشگاه شیالت، ، بنگال غربی، هندوستان.721211، دانشگاه بوردوان، بوردوان مرکز ابزار دقیق علوم دانشگاه چکیده: نوری و الکترونی های ( براساس مشاهدات میکروسکوپhamilton, 1822) puntius tictoبویایی گونه مخاط های پوششساختار سلول منافذ قدامی و خلفی با بیرون در ارتباطو از طریق نداپشتی چشم واقع شده-قدامیدر ناحیه های بویاییتوصیف گردید. جفت محفظهرویشی حی اباشد. نوتیغه آرایش یافته در طرفین ستیغ میانی می 21-11غنچه بویایی بیضی شکل در کف این محفظه قرار دارد و شامل باشد.می ل از لحاظ ریختی شام کلی بر دو نوع متمایزطور و اپیتلیوم حسی به اندها پراکنش یافتهحسی و غیرحسی به صورت مجزا در هر طرف تیغه با اپیتلیالی های مطبق دار غیرحسی، سلولهای مژه. اپیتلیوم غیرحسی شامل سلولباشدمیمیکروویلی دار وهای گیرنده مژهسلول ند. اتر اپیتلیوم، نزدیک هسته مرکزی واقع شدههای عمیقهای پایه در قسمتباشد. سلولهای مخاطی میهای متحدالمرکز و سلولریزبرجستگی اجزای سلولی اپیتلیوم بویایی در ارتباط با رفتار و زیستگاه گونه مورد مطالعه بحث شد.در ادامه نیز اهمیت عملکردی آناتومی عملکردی، بافت شناسی، میکروسکوپ الکترونی رویشی.، اندام بویایی: کلمات کلیدی int. j. aquat. biol. (2018) 6(1): 25-30 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article cadmium and arsenic bioaccumulation and bio-concentration in the endemic toothed carp aphanius arakensis in salt water masoumeh ariyaee1, amir hossein hamidian*1, soheil eagderi2, sohrab ashrafi1, hadi poorbagher2 1department of environment, faculty of natural resources, university of tehran, karaj, iran. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 28 march 2017 accepted 7 december 2017 available online 2 5 february 2018 keywords: toxicity heavy metals organs bcf abstract: heavy metals are released to aquatic ecosystems from natural and anthropogenic recourses and accumulate to the body of organisms. this study aimed to assess the accumulation of as and cd in the gill, liver, and muscle of the toothed carp aphanius arakensis in salt water exposed to three concentrations of cd and as (5, 10 and 20 mg l-1) for 18 days. the specimens were collected from the shoor river with an average weight of 1.5±0.3 g (mean±sd) and length of 3.4±0.4 cm. the findings showed that the bio-concentration factor (bcf) of cd and as were in the following order: liver > gill > muscle, however, for 5 ppm of as the order was gill > liver > muscle. bcf in as concentrations were more than cd concentrations. also, the highest bcf was found at 5 ppm. the present study showed that the liver is the organ that accumulates the highest concentrations of as and cd. introduction fishes are suitable indicators for monitoring the landbased pollutions, particularly heavy metals which accumulate in fish tissues, being absorbed through the skin or indirectly through food (kamrine, 2000). heavy metals are important threat to the aquatic environments because of chemical persistent, low degradation, bioaccumulation and bio-magnification (anbiaee et al., 2009). among heavy metals, cadmium is very toxic, 2-20 times more than those of others (perez-lopez et al., 2008). arsenic is another hazardous heavy metal originating from fertilizers and effluents that enter to the surface and groundwater (philip, 1978). the heavy metals accumulate mostly in the muscle, liver and gill tissues of fishes due to their high metabolism rates (khodabande, 2000). the liver plays an important role in accumulation of heavy metals and detoxification of wastes and its histopathological changes may be used as indicators of heavy metals (wong et al., 2001). the gill is in direct contact with water and thus accumulates heavy metals (oliveirafilho et al., 2010). bioconcentration is a process that *corresponding author: amir hossein hamidian doi: https://doi.org/10.22034/ijab.v6i1.447 e-mail address: a.hamidian@ut.ac.ir chemicals affect the organisms and bioconcentration factor (bf) is determined by dividing concentration of heavy metals in the organism tissues to concentration of heavy metals in water (otitoloju et al., 2009). the genus aphanius is the only representative of the cyprinodontidae (teleostei, cyprinodontiformes) in eurasia. iran and central anatolia show the highest diversity of aphanius, and 14 extant and one fossil species are known to occur in iran based on data derived from fish morphometry and meristics, otoliths, scales and mtdna sequences (coad, 2016; teimori et al., 2014 ). twelve out of 14 iranian aphanius species are endemic to this country. this genus occurs in coastal (brackish) and landlocked (freshwater to saline) water bodies in the mediterranean and persian gulf basins from iberian peninsula as far eastwards as iran and pakistan (teimori et al., 2014). aphanius arakensis is a newly described species of this genus from the namak lake basin in iran (teimori et al., 2012, 2014). this species tolerates a wide range of physicochemical parameters (coad, 2016), then it can be an appropriate model to study the effect of heavy metal pollution since it is 26 hamidian et al./ cadmium and arsenic bioaccumulation and bio-concentration in aphanius arakensis found in both salt and fresh water in the namak lake basin. hence, this study was conducted to examine the accumulation of cd and as in the muscle, gill and liver of a. arakensis and their bioconcentration factor in different concentrations of as and cd in the laboratory condition. materials and methods in this study, a total of 375 female specimens of a. arakensis with an average weight of 1.5±0.3 g (mean±sd) and length of 3.4±0.4 cm were collected from the eshtehard shoor river (35º36'31'' n, 50º48'23'' e), (alborz province, iran) in june 2011 (fig. 1). dissolved oxygen (do), salinity, water temperature and ph were measured during the sampling, which were 11.68 mg l-1, 11-12 g l-1, 12.85±6.22°c and 7-8.5, respectively. the fish samples were transported to the fisheries laboratory of university of tehran in polythene bags. prior to the experiment, the fish were acclimatized to the laboratory conditions for five days in pre-cleaned 30 l glass aquaria (30×30×40 cm) filled with dechlorinated tap water. during the experiment, the fish were fed with a commercial fish food (biomar) at a rate of 3-5% body weight twice a day. the physicochemical conditions of the aquaria were as follows: temperature, 27.5±1.2°c, ph, 7.7±0.5, caco3 hardness, 295±18 mg l -1 and do, 7.9±0.1 mg l-1. cd and as were used as arsenic oxide (as2o3) and cadmium chloride (cdcl2) salts (merck, germany). twenty-one aquaria were filled with salt water of the sampling area and aerated throughout the experiment. the animals were randomly allocated to 21 aquaria as seven treatments each with three replicates containing 10 fish. three aquaria received no chemical and was figure 1. the sampling area () in the shoor river. 27 int. j. aquat. biol. (2018) 6(1): 25-30 the control. the other 18 aquaria received only a concentration of cd (5, 10 and 20 mg l-1) or as (5, 10 and 20 mg l-1) for 18 days. at the end of the experiment, the animals were anaesthetized and euthanized. a piece of their gill, liver and muscle were removed. five specimens from each aquarium were pooled to make an appropriate weight of sample (the gill and muscle≈1 g, the liver≈0.5 g). the organ samples were digested in a mixture containing hno3 and hclo4 (mansouri et al., 2012c). the tissues were then weighed accurately and put into 150 ml erlenmeyer flasks. then, 10 ml nitric acid (65%) were added to each sample. the samples were left overnight to be digested slowly (baramaki et al., 2012; mansouri et al., 2012). finally, 5 ml perchloric acid (70%) were added to each sample. digestion was performed on a hot plate (sand bath) at 90ºc. the digested samples were diluted with 25 ml deionized water. the concentration of cd and as were measured using icp-oes (gbc integra xl, australia) and presented as mg g-1 wet weight (ww). all the measurements were repeated three times and the average of the values was reported along with their standard deviations. the following equation was employed to determine the bioconcentration factor in different tissues of a. arakensis in different concentrations of metals. bcf=c tissues/ c water where c tissues are average concentration of metals in different tissues and c water: average concentration of metals in water. the analyses of data were performed using spss (version 16). results only 5% of the specimens died during the experiment. the detection limits, blanks and recoveries of the measurements of metals in the samples are presented in table 1. average accumulation of as and cd in different tissues of a. arakensis in salt water are shown in figures 2. the metals were accumulated in different tissues in the following order of magnitude: liver < gill < muscle. the figures 2 and 3 show that the highest accumulated cd and as was found in the liver. on the other hand, the lowest accumulated cd and as found in the muscle. the average accumulation of as and cd in different tissues of a. arakensis in salt water are shown in the figures 3 and 4. the lowest accumulation of as and cd was observed in the control group and in the muscle, while the highest accumulation of as and cd were found in the liver, and 10 and 20 ppm, respectively. bio-concentration factor (bcf) was calculated in different tissues of the specimens. the highest bcf of as was found in the gill and in the concentration of 5ppm. the highest bcf of cd was found observed in the liver. the lowest bcf of cd and as were detected in the muscle and in the concentration of 20 ppm (fig. table 1. detection limits, blanks and recoveries of the measurements. element detection limit (µg/g) recovery mean (%)±sd blank mean (µg/g)±sd) cd 0.29 96.10±3.77 0.054±0.006 as 0.19 97.50±1.46 0.0±0.0 figure 2. mean (±sd) as and cd average accumulation in different organs of female aphanius arakensis. 28 hamidian et al./ cadmium and arsenic bioaccumulation and bio-concentration in aphanius arakensis 4). bcf of as was more than that of cd. the maximum bcf of as and cd were found at the concentration of 5 ppm (5 ppm>10 ppm>20 ppm). discussion accumulation of metals in the aquatic environment suggests that fishes can serve as a suitable bioindicator for contaminating metals in aquatic environment, because they respond to variations with great sensitivity in aquatic ecosystems (mansouri et al., 2011). our findings confirm differences in accumulation of heavy metals in the different tissues. based on the results of this study, the liver identified as target organ in salt water aquaria (liver > gill > muscle) which is in agreement with kojadinovic et al. (2007), monsef zadeh (2008), naseri et al. (2005), norouzi et al. (2012) and mansouri et al. (2012). similarly, many studies showed that the liver can accumulate high concentrations of heavy metals due to its key role in the metabolism of the body. therefore, the liver is suitable indicator for the assessment of impact of environmental pollutants (yılmaz, 2009). after the liver, the gill showed a high accumulation of cd and as concentrations in salt water tanks. the gills are places that accumulate water ions and one of the first tissues that have direct contact with environmental pollutants. the gill is also exposed to the pollutants to a greater extent compared with other organs, causing a higher rate adsorption and accumulation of the pollutants than those of other organs. as in the liver, the gill can be considered as an indicator of pollutants in both marine and freshwater habitats (oliveira-filho et al., 2010). the present study showed that accumulation of cd and as was lower in the muscle than those of the liver and gill. this finding is similar to those of mansouri et al. (2012) and majnoni et al. (2013), where the rate of bioaccumulation in the muscle was lower than those of the liver and gill. generally, tissues which have low metabolic activities accumulate high concentrations of heavy metals (amini ranjabr and sotoodenia, 2005). however, the muscle is not an active tissue to accumulate heavy metals which has been confirmed in this study and by others (tekin-ozan and kir, 2007; figure 3. mean (±sd) as and cd accumulation in different organs of female aphanius arakensis in concentrations of 5, 10, 20 ppm and control group. figure 4. bcf of as and cd in different tissues (ng g-1 wet weight). 29 int. j. aquat. biol. (2018) 6(1): 25-30 karadede, 2000; wong et al., 2001). terra et al. (2008) indicated that low concentrations of heavy metals in the muscle may be a result of a low level of binding proteins. according to the finding of this study and other studies (subathra and karuppasamy, 2008; senthil murugan et al., 2008), the order of the magnitude of bcf for cd and as in tissues were liver > gill > muscle but this order in 5 ppm of as was gill > liver > muscle which is similar to findings of jayakumar and issac paul (2006) and asagba et al. (2008). the liver is a place for storage, detoxification and biological transmission of heavy metals in fishes. therefore, the highest accumulation of heavy metals is normally found in the liver (kalay and canli, 2000). it is shown that a large amount of metallothionein induction happens in the liver of fishes. transmission of a large volume of water through the gills to obtain oxygen in stressful condition may increase rapidly toxic heavy metals in the gills (karuppasamy, 2004). hamidian et al. (2013) ignored results of this study that bcf of as was more than that of cd. however, metabolic processes such as biological transmission, active uptake and fat metabolism affect bioaccumulation plus other factors including contamination gradients of water, temperature, salinity and interacting agents (van geest, 2010). acknowledgments we would like to thank m. khazaee for his help. this study was financially supported by the university of tehran. references amini ranjbar g., sotudenia f. (2005). accumulation of heavy metals in muscle of caspian sea golden mullet (mugil auratus) in relation with biometric characteristic (standard length, weight, age and genus). iranian scientific fisheries journal, 13-18. anbiaee a., keykhosro a., vatandoost j. 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(2011). experimental studies on concentration and depuration of cobalt in the selected organs of fresh water fish capoeta fusca. world journal of fish and marine sciences, 3: 387-392 monesef zadeh f., 2008. determination of pb, cd, hg, zn and cu in liver and muscle of kutum fish (rutilus frisiikutum) in the coast of guilan province. master of science thesis in environmental engineering, university of guilan. naseri m., rezaee m., abedi a., afsharnaderi a. (2005). investigation of some heavy metal concentrations (fe, cu, zn, mg, mn, hg, pb and cd) in the edible and nonedible of lizadussumieri tissues in the coast of bushehr. journal of marine science and technology, 4: 59-67. nowrouzi m., mansouri b., hamidian a.h., zarei i., mansouri a. (2012). metal contents in tissues of two fish species from qeshm island, iran. bulletin of environmental contamination and toxicology, 89: 1004-1008. oliveira-filho e.c., muniz d.h.f., ferreira m.f.n. 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(2008). seasonal variations of heavy metals in some organs of carp (cyprinus carpio l., 1758) from beyşehir lake (turkey). environmental monitoring and assessment, 138: 201-206. terra b.f., araujo f.g., calza c.f., lopes r.t., teixeira t.p. (2008). heavy metal in tissues of three fish species from different trophic levels in a tropical brazilian river. water, air and soil pollution, 187(1): 275-284. van geast j. (2010). bioaccumulation of sedimentassociated contaminants in freshwater organism: development and standardization of a laboratory method, phd thesis, university of guelph, canada. 232 p. wong c.k., wong p.p.k., chu l.m. (2001). heavy metal concentrations in marine fishes collected from fish culture sites in hong kong. environmental contamination and toxicology, 40: 60-69. yilmaz f. 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(2018) 6(1): 25-30 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی شور آب در aphanius arakensis بومی گورماهی در آرسنیک و کادمیوم زیستی تجمع و تغلیظ 2پورباقر هادی ،1اشرفی سهراب ،2ایگدری سهیل ،1*حمیدیان حسین امیر ،1آریایی معصومه .ایران کرج، تهران، دانشگاه طبیعی، منابع دانشکده زیست، محیط گروه1 .ایران کرج، تهران، دانشگاه طبیعی، منابع دانشکده شیالت، گروه2 چکیده: شوند. هدف از این مطالعه ارزیابی انباشت انباشته می زنده بدن موجودات درشوند و منتقل می یهای آباکوسیستمفلزات از منابع طبیعی و انسانی به as وcd کبد و عضله آبششدر ،aphanius arakensis در معرض سه غلظت شور ودر آبcd وas (5 ،10 برای میلی 20و )18گرم در لیتر ها نشان داد که یافته. شد آوریجمع مترسانتی 4/3±4/0 ( و طولsd±میانگین) گرم 5/1±3/0 با میانگین وزنی ها از رودخانه شورنمونه بود. روز عضله است. < کبد <آبششترتیب به asاز ppm5 اگرچه در غلظت عضله، <آبشش <: کبدبه این ترتیب است asو cdضریب تغلیظ زیستی bcf بود. همچنین بیشترین میزان کادمیومبیشتر از آرسنیکbcf غلظت در ppm 5 است اندامی. مطالعه حاضر نشان داد که کبد مشاهده شد دهد.را تجمع می cdو asغلظت بیشترینکه .تغلیظ زیستی ،هااندام ین،گسنسمیت، فلزات :کلمات کلیدی int. j. aquat. biol. (2021) 9(4): 217-225 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article lecane (rotifera: lecanidae) community in psammon habitat in central coast vietnam: diversity and relation to environmental condition hung quang duong1, nhat-truong phan1, quynh anh tran-nguyen1,2, minh van vo 1,2, mau trinh-dang* 1,2 1danang environmental and biology resources teaching research team (dn-ebr), the university of da nang, da nang, vietnam. 2the university of da nang—university of science and education, da nang, vietnam. s article history: received 6 may 2021 accepted 5 july 2021 available online 2 5 august 2021 keywords: lecane ph psammon sandy coast abstract: characteristics of the lecane (rotifera) community in psammon in central coast vietnam were investigated. a total of 50 taxa were identified in samples collected at hygropsammon zones of temporary pools, contributing 4 new species to rotifers’ record of vietnam. psammonxenic species accounted for the largest percentage of lecane community with 82%, followed by psammophiles (12%) and psammonbionts (6%). influences of some environmental factors on the distribution of psammic lecanids were also observed. this group of organisms showed a slight tendency towards sand with grain sizes larger than 125 µm. besides, other abiotic factors including ph, total phosphorus (tp) and total dissolved solids (tds) were also found to significantly related to the distribution of some common lecane species. introduction psammon is a highly unstable environment due to aperiodic influences of water (schmid-araya, 1998). nevertheless, this dynamic habitat has been proven to host a wide range of biota, including rotifers (pejler, 1995; schmid-araya, 1998; fontaneto and ricci, 2006; covazzi harriague et al., 2013; lokko et al., 2014; lokko and virro, 2014). appearing with a high abundance in psammic zone of lakes and beaches’ shore, rotifer is believed to have a significant contribution to ensure the flows of energy and matter between terrestrial and aquatic ecosystems (wallace, 2002). in addition to some common species similar to those in other environments, psammon was found to have the presence of some specific rotifer species, which have been regarded as “pronounced specialists on a psammic life” (pejler, 1995). however, knowledge on the structure and functioning of rotifer communities in this type of habitat is still quite limited. the dominance of the lecane in psammic rotifers community was reported in many studies worldwide (radwan and bielańska-grajner, 2001; segers and *correspondence: mau trinh-dang doi: https://doi.org/10.22034/ijab.v9i4.1213 e-mail: tdmau@ued.udn.vn chittapun, 2001; karabin and ejsmont-karabin, 2005; muirhead et al., 2006; trinh-dang et al., 2015), indicating a possibly important role of this genus in biochemical processes in psammon. therefore, this study was conducted with the aims: (1) to examine the diversity of lecane in psammic habitat and (2) to explore correlations between the lecane community and environmental conditions. these results are expected to contribute not only to taxonomic data but also to knowledge about the ecology of rotifers in general and of the lecane in specific. materials and methods sample collection and analysis: samples were collected at 12 temporary pools on the sandy coast of quang nam province, vietnam in dry and rainy seasons 2019 (fig. 1). at each pool, two samples of psammon rotifers were taken for qualitative and quantitative analysis. the sand was collected from the top 2 cm of the hygropsammon zone using a pvc bailer and then mixed with filtered water (through a 30 µm mesh-size net) to re-suspend the rotifers. rotifer suspension was retrieved by filtering the mixture 218 duong et al./ lecane community in psammon habitat through a 50 µm mesh-size net. samples were preserved in formaldehyde 5% for further analysis in the laboratory. for quantitative analysis, samples were retrieved from sand collected in a 0.5x0.5 m sampling frame using the above-described protocol. rotifer specimens were sorted from samples and examined using a hund wetzlar h600 microscope. identification was based on taxonomy and nomenclature of the rotifers as in segers (2007). the rotifers were classified into three categories: psammobiont, psammophile, and psammoxene following bielańska-grajner (2005), myers (1936), and wiszniewski (1937, 1934a, b). additionally, the species not on those lists were assigned to subjectively categories based on our observations of their habitats and literature data, as appropriate. water quality parameters, including temperature, ph, total dissolved solids (tds), conductivity were directly measured in-situ by a multiparameter water quality sonde ysi 6920. concentrations of total nitrogen (tn) and total phosphorus (tp) in water were analyzed in the laboratory by standard methods (apha, 2017). sand samples were also collected to determine grain size, which categorized into 3 groups based on the average grain size of sand: very fine sand (<125 µm), fine sand (125-250 µm) and medium sand (>250 µm) (wentworth, 1922). ejsmont-karabin (2004) and lokko et al. (2017) suggested that psammon rotifer community are likely to be dependent on grain size. data analysis: the species accumulator and species richness estimators were calculated using the vegan package (oksanen et al., 2013) in r program (r core team, 2014). relationship between abundances of taxa and environmental data was investigated through canonical correspondence analysis (cca). environmental variables tested were tn, tp, tds, conductivity, and ph of pool water and grain sizes of sand. explanatory environmental variables were selected by the forward selection in the cca, selecting only those variables that were significantly related to taxonomic abundances (p≤0.05) according to monte carlo permutation tests. lecane taxa that were observed in less than 10% of all samples were excluded from cca to prevent these rare taxa from having an inordinate influence on the statistical results. results lecane community in study area: in total, 50 species of lecane were identified in which 4 species were new to vietnam, specifically l. blachei bērziņš figure 1. study area sandy coast in central vietnam. 219 int. j. aquat. biol. (2021) 9(4): 217-225 1973, l. elsa hauer 1931, l. lateralis sharma 1978 and l. monostyla daday 1897 (fig. 2). among these, only 3 species were classified as psammonbionts, including l. minuta, l. phapi and l. spiniventris; 6 species were psammophiles; and 41 species belonged to the psammoxenic group, accounting for 6, 12 and 82% of the total species number, respectively (table 1). species diversity in the rainy season was slightly higher than that in the dry season, with the number of species recorded was 39 taxa and 33 taxa, respectively. this result was also reflected by the species accumulation curve, where the fitted accumulation curve slope in the rainy season (12.47) was higher than that in the dry season (10.23) (fig. 3). moreover, the estimators, which estimated the maximum number of taxa could be present in the lake, again confirmed the higher species diversity in the rainy season. in detail, according to the chao 2 estimator index, the number of taxa that could appear in the rainy season was 49±7 taxa, and in the dry season was 43±7 taxa. according to the jackknife 2 index, these numbers were 59 taxa in the rainy season and 50 taxa in the dry season. for the bootstraps index, the numbers of taxa were 46±4 and 38±3 taxa in the rainy and dry season, respectively (fig. 3). figure 2. species habitus: (a) lecane elsa hauer 1931, (b) lecane lateralis sharma 1978, (c) lecane blachei bērziņš 1973 and (d) lecane monostyla (daday, 1897). 220 duong et al./ lecane community in psammon habitat table 1. composition, occurrence, abundance of psammic lecane species (abundance of taxa: ind.m–3, +: present, : absent, *: new to vietnam). species very fine sand fine sand medium sand ecological group l. abanica segers, 1994 + 15.291 pph l. aculeata (jakubski, 1912) + 7.645 + px l. aeganea harring, 1914 + px l. batillifer (murray, 1913) 3.581 px l. bifurca (bryce, 1892) + pph l. blachei bērziņš, 1973* + px l. bulla (gosse, 1851) 11.470 138.252 64.914 px l. closterocerca (schmarda, 1859) + 3.581 13.339 pph l. crepida harring, 1914 + 91.743 px l. curvicornis (murray, 1913) + + 5.495 px l. dorysimilis trinh-dang, segers & la-orsri, 2015 + pph l. doryssa harring, 1914 1.194 + px l. elsa hauer, 1931* + 7.645 px l. eswari dhanapathi, 1976 + px l. furcata (murray, 1913) + + 7.645 px l. haliclysta harring & myers, 1926 + 7.645 + px l. hamata (stokes, 1896) 8.043 13.187 34.149 px l. hornemanni (ehrenberg, 1834) + 1.194 px l. inermis (bryce, 1892) + pph l. inopinata harring & myers, 1926 15.291 95.566 19.114 px l. kunthuleensis chittapun, pholpunthin & segers, 2003 + px l. lateralis sharma, 1978* 15.291 px l. leontina (turner, 1892) 137.615 7.645 px l. ludwigii (eckstein, 1883) 15.291 10.033 px l. luna (müller, 1776) 7.645 23.701 px l. lunaris (ehrenberg, 1832) 7.645 163.099 79.002 px l. minuta segers, 1994 + pb l. mitis harring & myers, 1926 7.645 px l. monostyla (daday, 1897)* + px l. namatai segers & mertens 1997 + px l. obtusa (murray, 1913) + 2.387 22.936 px l. papuana (murray, 1913) 7.645 96.840 20.387 px l. pertica harring & myers, 1926 45.872 + px l. phapi dang, segers & sanoamuang, 2015 + pb l. pyriformis (daday, 1905) + 22.936 px l. rhenana hauer, 1929 3.581 754.332 7.645 px l. signifera (jennings, 1896) 7.645 32.826 8.541 px l. simonneae segers, 1993 + + px l. sola hauer, 1936 42.049 px l. sp. + px l. spiniventris segers 1994 7.645 7.645 pb l. stichoclysta segers, 1993 + + px l. subtilis harring & myers, 1926 + px l. superaculeata sanoamuang & segers 1997 15.291 px l. tenuiseta harring, 1914 + pph l. thailandensis segers & sanoamuang, 1994 + px l. thienemanni (hauer 1938) 7.645 px l. undulata hauer, 1938 + 7.944 13.538 px l. unguitata (fadeev, 1925) 15.291 px l. ungulata (gosse, 1887) 2.387 px total 21 34 36 221 int. j. aquat. biol. (2021) 9(4): 217-225 the result also showed that the number of species was higher in samples characterized by larger grain size, specifically 21 taxa in the very fine sand group, 34 taxa in the fine sand group, and 36 taxa in the medium sand (table 1). nevertheless, in the consideration of unique species, the fine sand size group comprised the largest number of unique species (10 species), followed by the medium sand group with 7 unique species. the very fine sand group had the least unique species with only 2 species (table 1). moreover, the total average density of lecane species in the fine sand group was recorded as the highest (1.63x106 ind.m–3), followed by the medium sand group with the density of 0.57x106 ind.m–3, and especially lowest in the very fine sand group (0.07x106 ind.m–3). it was noticeable that most lecane species appeared with a very low density in the very fine sand group (less than 1x104 ind.m–3), excepting for 2 species of l. bulla and l. inopinata whose densities were high in all three sand size groups. species with high densities (over 1x105 ind.m–3) such as l. bulla (1.38x105 ind.m–3), l. leontina (1.37x105 ind.m–3), l. lunaris (1.63x105 ind.m–3), and l. rhenana (7.54x105 ind.m–3) only appeared in the fine sand group (table 1). in order to compare species diversity among sand types, species accumulation curves were used. fine and medium sand groups showed a similar pattern which increased rapidly in the first 6 samples with a slope of 11.87 and 11.73, respectively, indicating a comparable diversity level. however, the average number of lecane species found per sample was higher in samples belonging to medium type compared to the fine type (8.8>5.47). besides, richness estimators suggested that the estimated species richness of lecane in fine and medium sand could be up to approximately 40-60 species (fig. 4). in addition, the number of very fine sand samples were not enough to form a clear pattern. nonetheless, it was quite obvious that the diversity of lecane inhabiting this sand type was relatively lower than two other groups, expressed by a slope of 10.51. however, the average number of species recorded per sample of this sand group in our study was fairly high (about 9 species/sample). total species richness was expected to be 20-35 species in 3 samples according to the models. relation to water parameters: the value of water quality parameters surveyed tends to be higher in the dry season than those in the rainy season, most notably the ph value. cca plot demonstrated that sand samples collected in two seasons were discriminated by two main environmental gradients (fig. 5). interestingly, the abundance of lecane species in figure 3. species accumulator of species richness at study sites during rainy (left) and dry season (right), with the fitted curve and estimator curves. 222 duong et al./ lecane community in psammon habitat psammon habitat was significantly related to water ph, tds, tn, tp and conductivity (p = 0.031), with 44.53% of total variance being attributed to these environmental variables. a total of 79.64% of the cumulative variance in species was explained by the first two cca axes, yet only the first one was statistically significant at a level of 10% (p = 0.077). this gradient was likely to be defined mainly by tp and tds (scores were 0.88 and 0.66, respectively (p<0.1)) even though tn and conductivity also established strong correlations. meanwhile, the second ordination axis was strongly related to the water ph (p = 0.01). regarding the distribution of lecane, l. papuana and l. closterocerca were likely to be more abundant in higher tp and tds conditions while the density of l. luna was considerably enhanced by high ph value. discussions other than taxonomic studies, research on lecane group is relatively scarce with two typical publications investigating the biogeography and ecology of lecane (pejler and bērziņš, 1994; segers, 1996) even though lecane was often mentioned as an important part of rotifer communities in many studies. our work was one of the first studies focusing on the lecane community in psammon habitat and its relationship to some abiotic factors. in vietnam, trinh-dang et al. (2015) reported 89 taxa of rotifers in psammon habitat in thua thien hue province in which lecane contributed 42 taxa. our investigation focused solely on this genus reported a number of 50 species in the central coast of vietnam, higher than the previous record by 8 species. in our list, 35 taxa were similar to those in thua thien hue and 4 taxa were new records for vietnam. sand grain structure is considered as one of important factors structuring psammon communities (arov, 1990; ejsmont-karabin, 2004). according to giere (2009), sand grain size characterizes psammon habitat as it directly determines spatial and structural conditions, and thus indirectly determines the physical and chemical characteristics. arov (1990) suggested that larger rotifer species tend to inhabit sand with bigger grain size while smaller species occur more frequently in sand with smaller grain sizes. such relationship was not observed in the study on psammon rotifers in polish natural and artificial lakes (bielańska-grajner, 2005), yet it was partly supported by ejsmont-karabin (2004) who reported a positive significant correlation between the rotifer numbers and the share of grain size fraction 0.25-1.00 mm. in our study, the occurrence of lecane species was different among 3 grain-size groups despite an indifference in the biodiversity level. very fine sand figure 4. species accumulator with fitted curve and estimator curve of species richness at study sites in 3 types of sand: a) very fine sand (<125 µm); b) fine sand (125 250 µm); c) medium sand (>250 µm). 223 int. j. aquat. biol. (2021) 9(4): 217-225 seemed to provide a less favorable condition for lecane as the number of species found in this type of sand was much lower compared to the two other groups. lecane doryssa was identified as one of the most restricted species by fine sand. meanwhile, l. bulla and l. hamata seem to be well-adapted to a wide range of sand-size as their high densities were found in all samples. nevertheless, ejsmont-karabin (2004) suggested that the body-size of rotifers was not a significant factor defining the preference to different size classes of sand grains. her results showed that large sand grains (250-1000 µm) were favorable to all three size groups of rotifers whereas larger or smaller classes were not preferred. rotifers inhabiting psammic habitats have to experience an extremely unstable environment which is strongly impacted by various physical and chemical factors outside the sand (schmid-araya, 1998) such as wave exposure, the quantity of organic matter, sand grain morphometry, amount of pore water, water ph, temperature, and oxygen (pennak, 1940; neel, 1948; ruttner-kolisko, 1953; evans, 1982). our result indicated a clear difference in water characteristics of temporary pools in the studied area between two seasons. a general decrease in values of all parameters in the rainy season could be explained by the dilution of rainwater. as a result, these changes were very likely to alternate the psammic lecane communities’ structure and composition. correlation between the trophic state of the water body and community structure of planktonic rotifers has been investigated in many studies (as overviewed in ejsmont-karabin, 2012). however, the relationship between the water trophy and psammic rotifers, especially the lecanids, has not been examined in detail yet. myers (1936) suggested that beaches of lakes with the higher trophy are likely to have higher numbers of psammon rotifers. the results of a study conducted by ejsmont-karabin (2003) in 18 polish lakes showed that psammobionts were more abundant in lakes with the lower trophy while psammoxenes dominated the beaches of nutrient-rich lakes. figure 5. a triplot of species scores, site scores, and environmental variables. 224 duong et al./ lecane community in psammon habitat moreover, the density of psammon rotifers was reported to significantly correlate with the concentration of chlorophyll a, pheophytin, total and mineral phosphorus (ejsmont-karabin, 2006). our results were generally in agreement with these findings. tp and tds were significantly influential to the distribution of the most common lecane species found in our study. besides, tn and conductivity also showed a relative correlation though was not significant. moreover, our study found that l. signifera, l. closterocerca and l. papuana tended to present in hygropsammic zones at higher levels of nutrients concentration, tds and salinity in water. ph is not only an important factor influencing the distribution of planktonic rotifers (edmondson, 1944) but also may play a critical role in structuring psammic rotifers communities (wiszniewski, 1936). our cca analysis results were well in agreement with this statement as ph established a high correlation with the second ordination axis, which could explain 36.23% variation in lecanids distribution. for example, l. luna showed a high preference for an alkaline environment whereas lower ph conditions seem to favour the growth of l. lunaris, l. ludwigii, and l. bulla. this is comparable to the study result conducted by bērziņš and pejler (1987), which showed that l. luna prefers ph from 6.5-9 while l. lunaris is able to tolerate a wider ph range from 49.5. however, it is noticeable that aside from morphological traits, functional adaptations, such as moving and feeding behaviors of rotifers should be taken into account in understanding rotifers’ habitat selections (arov, 1990). besides, competition and predation might also play a role in the distribution of rotifers (green, 1987). conclusion a total of 50 species of lecane in psammic habitats of the central coast vietnam was recorded, in which 4 species are new to vietnam. psammonxenes group composed the largest part of the lecane community with a proportion of 82%. regarding relations to environmental conditions, lecane species showed a minor preference for fine and medium grain-size sand over a very fine type though the diversity of these groups is comparable. canonical correspondence analysis revealed the statistically significant influences of some environmental factors, specifically ph, tp, and tds, on the distribution of some commonly-found lecanids in psammon. more intense studies are required to deepen the knowledge about the diversity of lecane communities, and also of rotifers in general, and to fully understand their ecological importance in psammic habitat. acknowledgements we would like to thank the faculty of biology and environmental science, university of science and education udn for providing research facilities. this research is funded by ministry of education and training under project number b2020-dna-08. references apha (american public health association) (2017). standard methods for the examination of water and wastewater, 23rd edition, washington d.c. arov i. 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(2014) 2(4): 215-222 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article the relationship among spawning period, length at first maturity and depth distribution of mullus barbatus and upeneus moluccensis inhabiting the northeastern mediterranean coast of turkey caner enver özyurt1, erdoğan çiçek*2,1dursun avşar1, hacer yeldan1, meltem manaşırlı1 1çukurova university, faculty of fisheries, 01330 adana, turkey. 2nevşehir haci bektas veli university, faculty of art and sciences, department of biology, 50300 nevşehir, turkey. article history: received 10 may 2014 accepted 22 july 2014 available online 2 5 august 2014 keywords: mullus barbatus upeneus moluccensis fisheries management northeastern mediterranean abstract: this study examined some spawning characteristics and distribution of mullus barbatus and upeneus moluccensis in babadillimani bight, in the northeastern mediterranean (near mersin, turkey) between of may 1999 and april 2000. sampling was carried out monthly at depths of 0-50 m, 50-100 m and >100 m using commercial trawl net mesh size 22 mm knot to knot. the results showed no difference between the length at first maturity of males and females (p>0.001) in either m. barbatus or u. moluccensis. this length for the combined sexes was calculated to be 11.7 cm and 10.9 cm in m. barbatus and u. moluccensis, respectively. when monthly changes in the gonadosomatic index (gsi) values were evaluated, the spawning period was determined as july-november for m. barbatus and may-august for u. moluccensis. the mean total lengths from the individuals belonging to m. barbatus from depth layers of 0-50 m, 50-100 m and >100 m were calculated as 8.65 cm, 8.70 cm and 12.70 cm, respectively. total lengths for u. moluccensis were calculated as 8.40 cm, 11.66 cm and 13.32 cm, respectively. the mean total length of m. barbatus and u. moluccensis increased from coastal areas to deeper waters. therefore bottom trawl fishing must be conducted in waters deeper than 100 m for both m. barbatus and u. moluccensis. introduction for fisheries management, each individual fish should be given the chance to reproduce at least once (beverton and holt, 1957). therefore, it is necessary that catching of target fish species occur only after reaching to its length at first maturity. it is known that the size of fish changes as their distributed area varies (sparre and venema, 1992). in particular, smaller sized groups prefer to stay in coastal area (bone et al., 1995; pitcher and hart, 1982; bingel, 2002). thus, in fisheries management, prohibition is the preferred means through which commercial fisheries protect the immature individuals inhabiting in coastal area. in turkey, deep trawl fisheries are prohibited in the waters within three miles of the coastal strip (anonymous, 2006). these issues * corresponding author: erdoğan çiçek e-mail address: erdogancicek@yahoo.com become more important because mediterranean-type deep trawl nets have low selectivity and are commonly used along the mediterranean coasts of turkey (tokaç et al., 1998; özbilgin and tosunoğlu, 2003). between 1990 and 2001, mullid species comprised 16% of the demersal fish catches from the mediterranean coasts of turkey (anonymous, 19902005). both the native species mullus barbatus and the lessepsian immigrant upeneus moluccensis constitute the majority of this production. hence, this study was aimed to determine the length at first maturity and the spawning season of m. barbatus and u. moluccensis. in addition, this study aimed to determine the size groups of these species, the differences between their size distributions in 216 int. j. aquat. biol. (2014) 2(4): 215-222 relation to depth and the depth ranges in which the mature individuals are distributed. in light of these data, our aim in this study was to determine the most appropriate depth layers for sustainable fisheries. materials and methods this study was carried out at three depth layers including 0-50 m, 50-100 m and >100 m in babadillimani bight of the northeastern mediterranean, near silifke-mersin, between may 1999 and april 2000 (fig. 1). sampling was performed monthly during daytime in three above mentioned depth layers, using a traditional mediterranean-type deep trawl net with one-hour haul duration. after identifying of fish species in each catch, the quantity of the catch was assessed. if the quantity of fish in the catch was low, the whole catch was used for analysis. alternatively, if the quantity was high, then the catch was sub-sampled based on holden and raitt (1974). the samples were transported to the laboratory after fixation into 4% formaldehyde solution buffered with borax. the total length of specimens was measured to the nearest 0.1 cm. the gonads were removed and measured to the nearest 0.01 g to determine spawning season and length at first maturity. the age of specimens was determined using sagittal otoliths and the criteria recommended by chilton and beamish (1982). the monthly change of gsi values were used to determine the spawning season (gibso and ezzi, 1978). the gonadal development of both sexes was analyzed using the five-stage sexual maturity scale recommended by holden and raitt (1974). in order to determine the length at first maturity, which is the size where 50% of the individuals have reached sexual maturity, the individuals in the 1st stage were considered immature and specimens in the 4th and 5th stages were considered mature. specimens in 2nd and 3rd stages were thought to have reached this stage for the first time, therefore they were considered immature, and alternatively, if they laid eggs and returned to the 2nd and 3rd stages, they were denoted as mature. thus, for both sexes, a two-stage scale of maturity and immaturity was created. using this scale, the size group in which 50% of the individuals reached sexual maturity was determined. in fishes, the sexual maturity follows a normal distribution, which can be modeled with a logistic function (king, 1995; welcomme, 2001). the length at first maturity of male and female individuals of m. barbatus and u. moluccensis was modeled with the equation given by sparre and venema (1992): lba e lm )*( 1 1 )(    in this equation, m(l): percentage of matured individuals, which is in “l” length group, l: length group (cm), a and b: regression constants. the size group in which 50% of the individuals in the stocks has reached sexual maturity was determined using the equation below: b a lm )( 50 one-way analysis of variance (anova) was applied to determine the differences between depth layers with regard to size groups and duncan test was performed to determine if there were differences between the depth layers. results the monthly changes in the gsi values of m. barbatus and u. moluccensis are given in figures 2 and 3. figure 2 indicates that the gsi value of m. barbatus increases from november, reaching the highest level between may and august and then decreased. the alterations in the gsi value shows that the gonad of m. barbatus matures between figure 1. sampling stations in babadıllimanı bight ((1): 0-50 m, (2): 50-100 m and (3): >100 m depth ranges). 217 özyurt et al./ spawning characteristics and distribution in m. barbatus and u. moluccensis november and may and spawning is occurred between may and august. figure 3 shows the monthly changes in gsi of u. moluccensis that is started to increase in january and after reaching to the highest value in july, decreases again. these changes in the gsi values suggest that the gonads started maturing in january and began to empty from july through november. therefore, we conclude that u. moluccensis spawned between july and november. the monthly changes in the sexual maturity stages of m. barbatus are shown in table 1. the months in which the most of individuals in the 4th maturity stage were april and june and the period in which the individuals had just laid eggs was from may through august. therefore, the result supports the findings obtained from the monthly changes of the gsi values. monthly changes in the sexual maturity stages of u. moluccensis are shown in table 2. although the individuals in 4th maturity stage were commonly observed between may and october, we did not encounter specimen at 5th maturity stage in may or june, therefore we concluded that this species starts laying eggs in july. the 5th maturity stage individuals are those that have just laid eggs and these began to appear in july till october. thus, the results obtained both from table 2 and figure 3 confirm the conclusion that u. moluccensis reproduces between july and october. the length at first maturity for males and females of m. barbatus and u. moluccensis were determined as 11.9 cm and 11.1 cm and 11.0 cm and 11.4 cm, respectively. no significant difference was observed for the length at first maturity between male and female of m. barbatus and u. moluccensis (t-test, p<0.01). therefore, we combined both sexes to calculate the length at first maturity. the lengths at first maturity for combined sexes of m. barbatus and u. moluccensis were calculated as 11.7 cm and 10.9 cm, respectively (fig. 4). the distribution of age groups for m. barbatus and figure 2. monthly changes in gsi values for mullus barbatus. figure 3. monthly changes in gsi values for upeneus moluccensis. months n sexual maturity stages 1st 2nd 3rd 4th 5th may 41 1 2 2 18 18 june 42 18 24 july 26 2 24 august 43 12 2 29 september 68 45 23 october 40 34 6 november 67 36 31 december 41 14 27 january 122 14 105 3 february 55 1 37 16 1 march 60 51 9 april 81 20 16 45 table 1. monthly changes of maturity stage for mullus barbatus. 218 int. j. aquat. biol. (2014) 2(4): 215-222 u. moluccensis in different depth layers is shown in table 3. the percentages of the individuals of m. barbatus in the first age group and below in the 0-50 m, 50-100 m and >100 m depth layers were calculated as 90.24%, 85.87% and 44.35%, respectively. therefore, in the 0-50 m and 50-100 m depth layers, the 0 and first age groups were dominant, while the first and second age groups were dominant in waters deeper than 100 m. at depths greater than 100 m, 56.65% of individuals were more than 2 years old. the proportion of u. moluccensis individuals belonging to 0 and the first age group in 0-50 m depth layer was determined as 92.31 %, this value decreased with increasing water depth and was calculated as 44.91% at 50-100 m and 21.92% at depths greater than 100 m. the proportions of individuals that were in the second age group and had reached sexual maturity in the three depth ranges were 6.95%, 42.68% and 63.01%, respectively. therefore, the age of individuals of m. barbatus and u. moluccensis increase with increasing depths i.e. months n sexual maturity stages 1st 2nd 3rd 4th 5th may 34 1 21 9 3 june 32 1 2 10 19 july 26 1 7 14 4 august 64 5 15 25 19 september 65 4 16 29 10 6 october 59 16 22 1 20 november 1 1 december 44 16 28 january 67 37 30 february 53 14 39 march 50 50 april 8 8 table 2. monthly changes of maturity stage for upeneus moluccensis. figure 4. length at first maturity of mullus barbatus and upeneus moluccensis for combined sexes. 219 özyurt et al./ spawning characteristics and distribution in m. barbatus and u. moluccensis the younger individuals are found in the coastal area, while the older ones distributed in the deeper waters. there was no significant difference between the mean total length of m. barbatus for 0-50 m and 50100 m depth layers. however, the mean total length at >100 m depth was significantly different than those of other depth layers (anova, p<0.01). in u. moluccensis, significant differences were found between the mean total lengths in the different depth layers (anova, p<0.01). the changes in the mean total length for m. barbatus and u. moluccensis by depth layer are given in table 4. the mean total lengths from the individuals belonging m. barbatus for these three depth layers were calculated as 8.65 cm, 8.70 cm and 12.70 cm and for u. moluccensis, they were calculated as 8.40 cm, 11.66 cm and 13.32 cm, respectively. similar to the age distribution, the mean total length of m. barbatus and u. moluccensis increased from the coastal area to the deeper waters (table 4). there were no significant differences between the mean total length of m. barbatus for 0-50 m and 50100 m depth layers. however, the mean total length at >100 m depth was quite different than that of other depth layers (anova, p<0.01). in u. moluccensis, significant differences were found between the mean total lengths in the different depth layers (anova, p<0.01). discussion the results indicate that m. barbatus spawns between may and august. in previous studies, the spawning period of m. barbatus in iskenderun bay was reported to occur between april and august (mümann and denizci, 1955), may and august (akyüz, 1957) and may and august in antalya bay (mert et al., 1983). therefore, the data on reproductive period determined in this study is in agreement with previous studies. the length at first maturity for u. moluccensis was calculated as 10.9 cm and spawning is occurred between july and october. moreover, the average size group of m. barbatus in depths greater than 100 m depth was higher than that of other two depth ranges and the mean length of u. moluccensis increased in the deeper areas. species age groups depth ranges 0-50m 50-100m 100m> n % n % n % m. barbatus 0 248 25.46 225 35.32 15 4.13 i 631 64.78 322 50.55 146 40.22 ii 94 9.65 78 12.24 166 45.73 iii 1 0.10 8 1.26 25 6.89 iv 2 0.31 9 2.48 v 2 0.31 2 0.55 u. moluccensis 0 120 32.97 8 2.55 i 216 59.34 133 42.36 32 21.92 ii 24 6.59 134 42.68 92 63.01 iii 3 0.82 13 4.14 16 10.96 iv 1 0.27 13 4.14 6 4.11 table 3. age groups of mullus barbatus and upeneus moluccensis for each depth range. species depth ranges (m) n mean length (cm) standard deviation m. barbatus 0-50 974 8.65 2.5456 50-100 637 8.70 3.1686 >100 390 12.70 2.5626 u. moluccensis 0-50 486 8.40 2.4398 50-100 325 11.66 2.7892 >100 146 13.32 2.2093 table 4. mean total length of mullus barbatus and upeneus moluccensis for each depth range. 220 int. j. aquat. biol. (2014) 2(4): 215-222 the spawning period of u. moluccensis was reported occurring between june and september in the eastern mediterranean (golani, 1990), in antalya bay (mert et al., 1983) and in the aegean and mediterranean coasts of turkey (kaya et al., 1999). in this study, we determined the spawning period to occur from july till october. whereas, we observed that in babadillimani bight the spawning starts and finishes one month later than in previously described areas. however, spawning may show delays of several months with changing environmental factors (woynarovich and horváth, 1980). the reproductive periods of m. barbatus and u. moluccensis suggest that deep trawl fisheries should be banned from may till october to create a sustainable stock. our data indicate that delaying the start of fishing season one month and expanding the restrictions (anonymous, 2006) on deep trawl fishing along the mediterranean coasts of turkey to occur between 1st april and 15th september would be beneficial for fisheries management. mert et al. (1983) and çelik and torcu (2000) reported that m. barbatus reached its size at first sexual maturity in their first age group. the length at first maturity is not reported in these studies, however, the size distribution of the individuals belonging to the first age group was reported as 10.213.5 cm by mert et al. (1983) and 10-13 cm by çelik and torcu (2000). in addition, i̇şmen and i̇şmen (2001) and özyurt (2003) reported that the length at first maturity for m. barbatus is 11 cm. the previous findings are supported by our results, which indicate the length at first maturity is 11.7 cm. the age at first maturity for u. moluccensis was reported to fall within the first age group by mert et al. (1983) and kaya et al. (1999). the length range for individuals from the first age group was reported as 10-13 cm by mert et al. (1983) and the mean length for individuals from the first age group was reported 11.3 cm for females and 10.0 cm for males (kaya et al., 1999). these results are in agreement with finding of this study, which determined the length at first maturity to be 10.9 cm for u. moluccensis. the mean lengths of m. barbatus in 0-50 m and 50100 m depth layers were 8.65 cm and 8.7 cm, respectively, which are significantly smaller than the 11.7 cm length at first maturity we recorded for this species. however, the mean total length for water layers deeper than 100 m (12.7 cm) was greater than the length at first maturity. although the 8.39 cm mean length for u. moluccensis in the 0-50 m depth layer was smaller than the length at first maturity (10.9 cm), it should be noted that in the 50-100 m and <100 m depth layers, the mean lengths (11.66 and 13.32 cm, respectively) were higher than the length at first maturity. in addition, the percentage of individuals belonging the 0 age group for m. barbatus was 25.46%, 35.32% and 4.3% in the 0-50 m, 50-100 m and >100 m depth layers, respectively. because the 1st age group in this species was thought to have reached sexual maturity (mert et al., 1983; çelik and torcu, 2000). it is obvious that 60.78% of the individuals in 0-100 m depth layer have not reached sexual maturity. when the 1st age group of m. barbatus is taken to be representative of 64.78%, 50.55% and 40.22% of individuals in the 0-50 m, 50-100 m and >100 m depth layers, respectively, it is apparent that 59.16% of the individuals in the 0-100 m depth layer have just reached sexual maturity. yet, in the waters deeper than 100 m, the 0 age group of m. barbatus is represented by 4.13 % of total individuals and the 1st age group represents 40.22 % of the total sample. while the individuals belonging to the 0 age group for u. moluccensis represented 32.97% and 2.55% of the total sample in the 0-50 m and 50-100 m depth layers, respectively, this age group was not found at depths greater than 100 m. the 1st age group of this species was found to represent 59.35 %, 42.36 % and 21.92% of the total individuals in the 0-50 m, 50-100 m and >100 m depth layers, respectively. these data shows that the 0 age group of u. moluccensis at 0100 m was represented by 19.25% of the individuals at this depth and that the first age group represented 52.48% of the total. therefore, while the 0th and first age groups of u. moluccensis represented 71.73% of 221 özyurt et al./ spawning characteristics and distribution in m. barbatus and u. moluccensis the individuals in the 0-100 m depth layer, this proportion decreased to 21.92% at depths below 100 m. it is known that the distribution of fish stocks in a fishing area is more concentrated along the coast, especially for small-sized groups (bone et al., 1995; pitcher and hart, 1982; bingel, 2002). the results of this research clearly showed that smaller individuals are common in the coastal areas. indeed, valioni et al. (1998) reported that the m. barbatus individuals dispersed within areas shallower than 100 m in depth were smaller than 14 cm and that individuals distributed between 100 and 200 m in depth were generally 19-21 cm in length. similarly, somarakis and machias (2002) reported that mature individuals of this species were common in 60-80 m depth layers, while their juveniles were generally found in the shallower waters. in light of these data, it is obvious that fishing should be conducted in deeper waters. this is because the fish in deeper waters are more likely to have reproduced. therefore, the length and age at first maturity determined for m. barbatus and u. moluccensis indicate that the fishing conducted in the 0-100 m depth layer using a traditional mediterranean-type deep trawl net with poor selectivity excessively exploits newly matured individuals on the mediterranean coast of turkey. this type of fishing causes extensive over-fishing of these fish stocks. in this study, the average distances of 50 m and 100 m depth waters from the coastal strip were 0.8 miles and 2.5 miles, respectively. the average distances of the deep trawl stations from the coastal strip (0-50 m, 50-100 m, >100 m depth layers) were determined as 0.5, 1.6 and 2.9 miles, respectively. therefore, as individuals longer than the length at first maturity are common the waters deeper than 100 m, deep trawl fishing in the waters shallower than 2.9 miles can impose over-fishing pressure on m. barbatus and u. moluccensis stocks. in this area, deep trawl fishing within the two-mile distance from the coast is forbidden (anonymous, 2006). therefore, expanding deep trawling prohibition to three miles offshore in babadıllimanı bight (as in some other parts of mediterranean coast) is an appropriate and necessary action for the protection and sustainability of m. barbatus and u. moluccensis stocks. references akyüz e. (1957). observation on the iskenderun red mullet (mullus barbatus) and its environment. general fisheries commission for the mediterranean, proceedings and technical papers, 4: 305-326. anonymous. (1990-2005). fisheries statistics. republic of turkey, prime ministry state institute of statistics, ankara, 893 p anonymous. (2006). regulation of commercial fishery in marine and freshwater fishery 2006-2008 37/1. republic of turkey, ministry of agriculture and rural affairs, general directorate of protection and control, ankara, 31 p. beverton r.j.h., holt s.j. (1957). on the dynamics of exploited fish populations. fishery investigations, ministry of agriculture, fisheries and food, great britain, series 2, 19: 533 p. bingel f. (2002). investigations of fish populations. baki press, adana, 404 p. bone q., marshall n.b., blaxter j.h.s. (1995). biology of fishes. chapman and hall, london, 332 p. celik o., torcu h. 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(2015) 3(4): 208-217 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article development and application of wetland zooplankton index to assess the degree of eutrophication in sri lankan reservoirs mudiyanselage jayantha sisirakumara wijeyaratne*,1sangakkara mudiyanselage ayanthi indrachapa sangakkara department of zoology and environmental management, university of kelaniya, kelaniya, sri lanka. article history: received 8 february 2015 accepted 4 june 2015 available online 2 5 august 2015 keywords: wetland zooplankton index eutrophication nitrate phosphorus abstract: wetland zooplankton index (wzi) was developed for the low country intermediate zone of sri lanka using 20 reservoirs located between latitudes 7°20'22.081"n 7°48'33.558"n and longitudes 80°1'44.55"e 80°9'51.509"e. wzi ranged from 1.56 in anukkane reservoir which is located in a low flat terrain in the midst of agricultural lands to 3.69 in tampana reservoir which is located in a hilly area with a watershed mainly covered with forests. wzi showed a significant negative correlation with the nitrate-n content (r = -0.797) and cumulative content of nitrate-n and total phosphorus (r = -0.795) indicating that it can be used as an indicator of the degree of eutrophication of inland reservoirs in the low country intermediate zone of sri lanka. introduction sri lanka is a country that has a culture entangled with ancient irrigation systems with many cascading reservoirs and canals. due to these man-made reservoirs, sri lanka has a very high density of inland wetlands, which is about 4 ha for every km2 of land. about 12000 of these man-made wetlands are located in the dry zone and the intermediate zone of sri lanka (jayasena et al., 2011). these play a significant role in economy by providing water for irrigation, livestock, fisheries, aquaculture and generation of hydropower. in addition, these water bodies supply drinking water as well as for domestic purposes (imbulana et al., 2006). eutrophic conditions have been reported in many reservoirs in sri lanka mainly due to uncontrolled and excessive use of agrochemicals (imbulana et al., 2006). major types of fertilizer used for agricultural activities in sri lanka are sulphate of ammonia, urea, rock phosphate, muriate of potash, triple super phosphate and mixed fertilizers (mubarak, 2000). due to high costs of the chemical tests and the * corresponding author: m.j.s. wijeyaratne e-mail address: zoomjs@kln.ac.lk chemical waste generated by these methods, several alternatives are being used today to assess the water quality (barra et al., 2012). these include the use of indicator organisms (gannon and stemberger, 1978; el-shabrawy and khalifa, 2002; füreder and reynold, 2004; yantsis, 2009) and water quality indices (chow-fraser, 2006; seilheimer et al., 2009). wetland zooplankton index (wzi) is one of such indices (lougheed and chow-fraser, 2002). although some studies on the relationship between the abundance of some zooplankton species and water quality have been carried out (kamaladasa and jayatunga, 2007; gammanpila, 2010), development and use of a wzi to indicate water quality of inland water bodies is a novel approach in sri lanka. developing a wzi to find out whether it can be used to assess the eutrophic conditions of the reservoirs is highly appropriate in the present-day context as sri lanka is reported to have a high fertilizer consumption of 101.5 kg ha-1 (mubarak, 2000). therefore, the objective of the present study was to develop a wzi and to find out whether it can be used 209 wijeyaratne and sangakkara/ wetland zooplankton index for sri lankan reservoirs to assess the degree of eutrophication in the reservoirs in the low country (elevation: <300 m msl) intermediate zone (annual rainfall: 1,750-2,500 mm) of sri lanka. materials and methods this study was carried out in 20 reservoirs located in the low country intermediate zone of sri lanka between latitudes 7°20'22.081"n-7°48'33.558"n and longitudes 80°1'44.55"e-80°9'51.509"e (table 1). the surface area of the reservoirs varied from 1 ha to 756.3 ha (table 1). the low country intermediate zone was selected for this study as this area is a heavy agricultural area with paddy fields and coconut plantations. in addition, this area has a very high density of inland reservoirs. the reservoirs used in the present study except for two, namely tampana and kurunegala reservoirs provide water for irrigation purposes. tampana and kurunegala reservoirs are used to provide water for the households in the urban and suburban areas of kurunegala town. zooplankton of these reservoirs were sampled from june to october 2013 using "patalas-schindler" plankton sampler of 10-l capacity. sampling was carried out at least 5 m away from the aquatic vegetation and at least 3 m away from the shore as recommended by lougheed and chow-fraser (2002). samples were immediately preserved using 5% formalin based on dhargalkar and verlecar (2004). zooplankton were identified under the optical microscope using the taxonomic keys provided by fernando (1990). the abundance of each species was determined by sub-sampling using a sedgewick rafter. at the time of sampling, water samples were also collected in dark plastic-bottles for the determination of nitrate-n and total phosphorus levels. the water samples were immediately preserved at the site by adding 0.5 ml of concentrated sulphuric acid and the nitrate and total phosphorus contents were determined using apha (1998). on each sampling occasion, three samples were taken from each site. when the reservoirs were <20 ha in surface area, samples were taken from three sites, and when the reservoirs were 20-50 ha in surface area, samples were taken from five sites. the number of sample sites in each of the kimbulwana (278 ha) and bathalagoda (255 ha) reservoirs were eight. from wendaru (92 ha) and dewahuwa 756 ha) reservoir number name of the reservoir location surface area (ha) 1 adukkane 80°08'14.233"e 7°31'28.100"n 2.9 2 anukkane 80°07'14.380"e 7°30'43.170"n 9.1 3 bathalagoda 80°26'50.246"e 7°31'59.222"n 255.2 4 bodhimulla 80°05'51.912"e 7°31'35.360"n 3.9 5 dewahuwa 80°32'26.782"e 7°48'33.558"n 756.3 6 galewela 80°34'34.283"e 7°45'41.707"n 5.9 7 galpihilla 80°09'30.015"e 7°20'52.894"n 1.0 8 gaiyawa 80° 01'44.55"e 7°28'46.210"n 16.2 9 kanogama 80°09'51.509"e 7°32'35.510"n 22.4 10 karangamuwa 80°10'21.512"e 7°32'50.654"n 8.9 11 kimbulwana 80°28'19.837"e 7°39'50.338"n 237.9 12 kurunegala 80°21'43.271"e 7°29'36.949"n 48.0 13 makandura 79°59'38.603"e 7°20'22.081"n 36.0 14 metiyagane 80°10'59.642"e 7°23'37.450"n 5.2 15 munamaldeniya 80°03'51.940"e 7°32'57.392"n 17.4 16 polpitiya 80°11'90.675"e 7°31'40.407"n 11.2 17 saragama 80°20'10.126"e 7°30'37.575"n 20.1 18 tampana 80°24'25.140"e 7°26'55.036"n 1.5 19 umangawa 80°10'50.130"e 7°32'01.440"n 3.0 20 wendaru 80°22'30.099"e 7°27'55.695"n 92.0 table 1. location and the surface area of the reservoirs studied. 210 int. j. aquat. biol. (2015) 3(4): 208-217 reservoirs, samples were taken from six and ten sites, respectively. sampling sites were selected to cover the entire water body. wzi for each reservoir was calculated using the following equation of lougheed and chow-fraser (2002). where, yi =abundance of species i, ti = tolerance value (1-3) of species i, ui = optimum value (1-5) of species i and n = number of zooplankton species in the reservoir. the values for wzi ranged from 1.0, indicative of low water quality (high eutrophication) to 5.0, indicative of high quality (low eutrophication) (lougheed and chow-fraser, 2002). tolerance values (t) indicate tolerance or the niche breadth of a species. species that have a narrow range of distribution were given a low tolerance score as recommended by lougheed and chow-fraser (2002). in the present study, if a species was present in less than 33% of the studied reservoirs, i.e., ≤ 7 reservoirs, a t value of 1 was assigned. for the species that were found in 8-13 reservoirs, i.e., 3367% of the studied reservoirs, t value of 2 was assigned and for the species that were found in more than 13 reservoirs, i.e., in more than 67% of the studied reservoirs, the assigned t value was 3. to assign u values, three clusters of reservoirs were identified based on nitrate-n and total phosphorus contents. clustering was done based on bray curtis similarity using primer 5.0 software package. reservoirs with comparatively low nitrate-n and total phosphorus contents were grouped in cluster 1 and reservoirs with comparatively high nitrate-n and total phosphorus contents were grouped in cluster 3. the reservoirs with comparatively intermediate nitrate-n and total phosphorus levels were grouped in cluster 2. the assigning of u values for different zooplankton species is summarized in table 2. the product-moment correlation coefficients of wzi with the nitrate-n level, total phosphorus level and cumulative values of nitrate-n and total phosphorus were calculated. when the correlation coefficients were significantly different from zero, linear regression algorithms were developed taking wzi as the independent variable. results table 3 gives the mean ± standard error of the mean (sem) and the range of the nitrate-n and total phosphorus values of the reservoirs studied. the highest nitrate-n content was recorded at anukkane reservoir (5.624 ± 0.072 mg l-1) while the lowest nitrate-n content was recorded at adukkane reservoir (1.186 ± 0.013 mg l-1). total phosphorous levels varied from 0.003 ± 0.002 mg l-1 recorded in bodhimulla reservoir to 0.529 ± 0.036 mg l-1 recorded in karangamuwa reservoir the separation of reservoirs into clusters based on cluster/clusters of reservoirs where a particular zooplankton species is present u value assigned 1 5 1 and 2 4 1 and 3 3 2 3 1, 2 and 3 3 2 and 3 2 3 1 (cluster 1comparatively low nitrate-n and total pphosphorus level; cluster 2 intermidiate nitrate-n and total phosphorus level; cluster 3 – comparatively high nitrate-n and total phosphorus level). table 2. summary of the allocation of u values for different zooplankton species. 211 wijeyaratne and sangakkara/ wetland zooplankton index for sri lankan reservoirs the nitrate-n and total phosphorus contents is shown in figure 1. three clusters of reservoirs were identified at 61% similarity level. the reservoirs with relatively low nitrate-n and total phosphorus levels positioned in cluster 1. only three reservoirs were grouped in this cluster. reservoirs with relatively moderate levels of nitrate-n and total phosphorus were grouped in cluster 2. there were 7 reservoirs in this cluster. in cluster 3, reservoirs with relatively high levels of nitrate and total phosphorus were included. there were 10 reservoirs in this cluster (fig. 1). a total of 31 species of rotifers, 3 species of copepods and 9 species of cladocerans were identified in the samples (table 4). the optimum values (u) assigned to different zooplankton species are also given in table 4. two species of rotifers namely brachionus donneri and testudinella elliptica were found only in the reservoirs of cluster 1. therefore, a u value of 5 was assigned to them. one species of copepods, 3 species of cladocerans and 7 species of rotifers were found only in the reservoirs of cluster 3 and a u value of 1 was assigned to them. two species of cladocerans and 12 species of rotifers were found only in the reservoirs of cluster 2 and 3 and were assigned a u value of 2. no zooplankton species was found in the reservoirs of both clusters of 1 and 2. hence, a u value of 4 was not assigned to any species. balance 16 species were in the reservoirs categorized either under cluster 2 or both in cluster 1 and 3 or in all three clusters and therefore, they were assigned a u value of 3 (table 4). the tolerance value (t) assigned to each zooplankton species are also given in table 4. only 1 species namely diacyclops nanus was present in more than 66.6% of the reservoirs studied. therefore, a t value of 3 was assigned to this species. t value of 2 was assigned only to 4 species namely diaptomus nadus, filinia terminalis, hexarthra mira and trichocerca cylindrica. all other species were found in less than 33.33% of reservoirs studied and therefore those species were assigned a t value of 1. reservoir nitrate n (mg l-1) total phosphorus (mg l-1) 1 adukkane 1.186 ± 0.013 (1.199-1.173) 0.046 ± 0.004 (0.05-0.042) 2 anukkane 5.624 ± 0.072 (5.696-5.552) 0.086 ± 0.008 (0.094-0.078) 3 bathalagoda 2.403 ± 0.370 (2.773-2.033) 0.075 ± 0.007 (0.082-0.068) 4 bodhimulla 3.886 ± 0.215 (4.101-3.671) 0.003 ± 0.002 (0.005-0.001) 5 dewahuwa 3.416 ±0.227 (3.643-3.189) 0.060 ± 0.012 (0.072-0.048) 6 galewela 4.936 ± 0.038 (4.974-4.898) 0.190 ± 0.022 (0.212-0.168) 7 galpihilla 2.548 ± 0.076 (2.472-2.624) 0.042 ± 0.008 (0.05-0.034) 8 gaiyawa 3.923 ± 0.133 (4.056-3.79) 0.104 ± 0.01 (0.114-0.094) 9 kanogama 2.626 ± 0.100 (2.726-2.526) 0.071 ± 0.009 (0.080-0.062) 10 karangamuwa 3.452 ± 0.442 (3.894-3.01) 0.529 ± 0.036 (0.565-0.493) 11 kimbulwana 4.213 ± 0.256 (4.469-3.957) 0.147 ± 0.006 (0.153-0.141) 12 kurunegala 2.620 ± 0.344 (2.964-2.276) 0.164 ± 0.018 (0.182-0.146) 13 makandura 1.570 ± 0.390 (1.96-1.180) 0.065 ± 0.011 (0.076-0.054) 14 metiyagane 4.574 ± 0.345 (4.919-4.229) 0.065 ± 0.009 (0.056-0.074) 15 munamaldeniya 4.213 ± 0.094 (4.307-4.119) 0.068 ± 0.007 (0.075-0.061) 16 polpitiya 2.294 ± 0.687 (2.981-1.607) 0.309 ± 0.019 (0.328-0.290) 17 saragama 3.344 ± 0.108 (3.452-3.236) 0.038 ± 0.008 (0.046-0.030 18 tampana 1.932 ± 0.074 (2.006-1.858) 0.046 ± 0.006 (0.052-0.040) 19 umangawa 2.765 ± 0.193 (2.958-2.572) 0.057 ± 0.004 (0.061-0.053) 20 wendaru 2.294 ± 0.241(2.535-2.053) 0.030 ± 0.005 (0.035-0.025) table 3. mean ± sem values of nitrate-n levels and total phosphorus levels of the reservoirs studied. ranges are given within brackets. 212 int. j. aquat. biol. (2015) 3(4): 208-217 the values for wzi calculated for the 20 study sites are given in table 5. the highest wzi was obtained for tampana (3.69) while the lowest wzi was obtained for anukkane (1.56). wzi showed a significant negative correlation with nitrate-n content (r = -0.797; p = 0.00) while they were not significantly correlated with the total phosphorus content (r = 0.11; p = 0.64). nevertheless, wzi species cluster present u value no. of reservoirs present t value copepoda acanthocyclops vernalis 3 1 2 1 diacyclops nanus 1, 2 and 3 3 15 3 diaptomus nadus 1, 2 and 3 3 10 2 cladocera alona monocantha 3 1 1 1 chrydorus eurynotus 2 and 3 2 2 1 chydorus parvus 1, 2 and 3 3 4 1 diaphanosoma brachyurum 2, 3 2 3 1 diaphanosoma singhalense 2 3 1 1 karualona karua 3 1 1 1 leptodora kindti 2 3 1 1 moinodaphnia macleayi 3 1 1 1 pseudosida szalayi 2 3 1 1 rotifera asplanchna priodonta 3 1 1 1 brachionus angularis 2 3 1 1 brachionus budapestinensis 2 and 3 2 2 1 brachionus caudatus 1, 2 and 3 3 6 1 brachionus donneri 1 5 1 1 brachionus falcatus 2 and 3 2 3 1 brachionus forficula 2 and 3 2 5 1 brachionus patulus 2 and 3 2 3 1 brachionus rubens 2 and 3 2 2 1 brachionus urceus 3 1 2 1 coelopus tenuior 1 and 3 3 2 1 eothinia elongata 2 and 3 2 3 1 euchlanis dilatata 2 and 3 2 5 1 filinia terminalis 2 and 3 2 8 2 hexarthra mira 2 and 3 2 8 2 kellicottia longispina 3 1 1 1 keratella earlinae 1 and 3 3 2 1 keratella lenzi 2 3 1 1 keratella quadrata 3 1 1 1 lecane curvicornis 2 and 3 2 4 1 monostyla bulla 1, 2 and 3 3 6 1 polyarthra dolichoptera 3 1 1 1 polyarthra vulgaris 2 and 3 2 6 1 rattulus tigris 3 1 1 1 rotaria citrine 2 3 1 1 testudinella elliptica 1 5 1 1 testudinella patina 1 and 3 3 2 1 trichocerca cylindrica 1, 2 and 3 3 10 2 trichocerca dixon-nuttalli 2 3 1 1 trichocerca similis 3 1 1 1 trichotria pocillum 2 and 3 2 2 1 table 4. optimum values (u) and tolerance values (t) assigned for the zooplankton species recorded in the present study. 213 wijeyaratne and sangakkara/ wetland zooplankton index for sri lankan reservoirs showed a significant negative correlation with the cumulative content of nitrate-n and total phosphorus figure 1. clusters of reservoirs based on nitrate-n and total phosphorus levels [cluster1comparatively low nitrate-n and total phosphorus levels; cluster 2moderate nitrate-n and total phosphorus levels; cluster 3comparatively high nitrate-n and total phosphorus levels]. figure 2. simple linear regression algorithm between wzi and nitrate-n levels of the reservoirs studied with 95% confidence band. figure 3. simple linear regression algorithm between wzi and cumulative levels of nitrate-n and total phosphorus of the reservoirs studied with 95% confidence band. 214 int. j. aquat. biol. (2015) 3(4): 208-217 contents (r = -0.795; p = 0.00). because of the significant correlation of wzi with nitrate-n and cumulative value of nitrate-n and total phosphorus, the linear regression algorithms were developed using wzi as the independent variable and these are as follows (p = 0.00). nitrate-n (in mg l-1) = 9.35 – 2.31 wzi (r2 = 66.4) nitrate-n + total phosphorus (in mg l-1) = 9.55 – 2.34 wzi (r2 = 66.1) the 95% confidence band for these two algorithms are given in figures 2 and 3, respectively. discussion zooplankton respond rapidly to the changes in environmental conditions (schindler, 1987). however, they have been hardly used in sri lanka to study the environmental conditions of aquatic habitats (kamaladasa and jayatunge, 2007; gammanpila, 2010). although some individual species of zooplankton are used as indicator organisms of water quality (slȧdeĉek, 1983; perbiche-neves et al., 2013), studies on the use of zooplankton communities as a whole to indicate water quality are sparse (lougheed and chow-fraser 2002, yantsis 2009). all the reservoirs used in this study are located in a cascading system (jayasena et al., 2011) and except for tampana and kurunegala reservoirs, the land-use in their watersheds contain many paddy field and coconut lands. hence, storm water from these agricultural fields and excess water for wet paddy cultivation lands ultimately flow into these reservoirs. the watershed of tampana and kurunegala reservoirs are mostly covered with forests and urban residential areas, respectively. sri lanka is among the highest fertilizer users in south asian region (mubarak, 2000). in addition, the kurunegala district where most of these reservoirs are located is accounting for the highest extent of agricultural production (dcs, 2013). according to world bank statistics, fertilizer consumption in sri lanka was measured as 257.93 kg ha-1 of arable land in 2009. the most used fertilizers are nitrogenous, potash and phosphate fertilizers, including ground rock phosphate (trading economics, 2013). farmers use fertilizer excessively in fields due to lack of knowledge. large amounts of nutrients especially nitrate and phosphates are added to these inland water bodies due to agricultural runoff resulting in high nitrate-n and total phophorus levels. according to the nitrate-n and total phosphorous levels, all the reservoirs used in the present study are eutrophic (nürnberg, 1996). many negative environmental and social impacts have been reported in some sri lankan reservoirs due to eutrophication (wijesundara et al., 2012; ariyawansa et al., 2012; azmy et al., 2012). hence, it is important to assess the degree of eutrophication in inland fresh water bodies in order to take suitable mitigation measures to reduce further eutrophication and improve the water quality. the wzi recorded in the present study are within the range recorded by lougheed and chow-freser (2002) for laurentian great lakes basin although the water bodies are under contrasting ecological extremes. the u values for different zooplankton species were assigned in the present study using a simple method of clustering the reservoirs based on the nitrate-n and total phosphorus levels where as name of the reservoir wzi adukkane wewa 3.07 anukkane wewa 1.94 bathalegoda reservoir 3.30 bodhimulla wewa 3.13 dewahuwa reservoir 3.00 galewela wewa 2.99 galphilla wewa 3.11 gaiyala wewa 2.97 kanogama wewa 3.38 karangamuwa wewa 3.25 kimbulwana wewa 3.13 kurunagala wewa 3.77 makandura wewa 3.45 metiyagane wewa 3.39 munamaldeniya wewa 3.02 polpitiya wewa 3.00 saragama wewa 3.52 thampana reservoir 4.33 umangawa wewa 3.12 wedaru wewa 3.06 table 5. the values for wzi of the reservoirs studied.zooplankton species. 215 wijeyaratne and sangakkara/ wetland zooplankton index for sri lankan reservoirs lougheed and chow-fraser (2002) used partial canonical correspondence analysis. wzi closer to 1 indicates high degree of eutrophication while the values closer to 5 indicate least degree of eutrophication. the highest wzi value was recorded for tampana reservoir (3.69) which indicates least eutrophication. this reservoir has a comparatively low nitrate-n level of 1.932 mg l-1 and total phosphorus level of 0.046 mg l-1. the watershed of this water body, which is located in high elevation contains many forest areas and therefore does not receive agricultural runoff. the lowest wzi (1.56) was recorded for anukkane which is located in a flat terrain surrounded by agricultural lands. the highest nitrate value was recorded in this reservoir. this low wzi indicates high degree of eutrophication probably due to addition of large amount of nutrients from the agricultural fields in the watershed. the algorithms developed in the present study may be used to estimate the nitrate-n level and cumulative level of nitrate-n and total phosphorus in the inland reservoirs in this region of the country using the wzi. the u values and t values developed for each zooplankton species in the present study can be used in the estimation of wzi. the algorithms developed in the present study may not be applicable in the other regions due to differences in zooplankton diversity. hence area specific algorithms have to be developed for other regions of the country. references apha (1998). standard methods for examination water and wastewater. american public health association, washington, dc, 20th edition. 1325 p. ariyawansa j.k., mawjood m.i.m., silva e.i.l. 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(2015) 3(4): 208-217 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی توسعه و کاربرد شاخص زئوپالنکتون تاالب برای ارزیابی درجه یوتریفیکاسیون در منابع آبی سریالنکا و س. م. آ. سانگاککارا *م. ج. س. ویجیارانته کالنیا، سریالنکا.، دانشگاه کالنیا، و مدیریت محیط زیست گروه جانورشناسی چکیده: منبع آبی که در 02میانی سریالنکا با استفاده از سرزمینی برای ناحیه( wetland zooplankton index=wziتاالب ) زئوپالنکتون شاخص ، توسعه داده ندواقع شده بود e 80°9'51.509"e"44.55'1°80 جغرافیایی و عرض n 7°48'33.558"n"22.081'20°7طول جغرافیایی واقع شده های کشاورزی که در یک منطقه کم ارتفاع در میانه زمین، anukkane برای منبع آبی 65/1از wzi ، مقادیرشد. براساس نتایج یک wzi، متغیر بود. نتایج واقع شده استای با پوشش جنگلی ای با حوضهکه در ناحیه تپه tampanaبرای منبع آبی 56/3تا است ( نشان داد که بیانگر این موضوع r= -766/2) کل ( و محتوای تجمعی نیترات و فسفرr= -767/2همبستگی منفی را با محتوای نیترات ) ورد ممیانی سریالنکا سرزمینی ناحیهمنابع آبهای داخلی در برای ارزیابی عنوان شاخص درجه یوتریفیکاسیون تواند بهمی wziباشد که می استفاده قرار بگیرد. .فسفر، نیترات، یوتریفیکاسیون ، تاالب زئوپالنکتون شاخص :کلمات کلیدی int. j. aquat. biol. (2021) 9(2): 71-78 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article some ecophysiological information on geloina expansa (bivalvia: cyrenidae) population in santiago river, northern philippines: prelude to aquaculture technique development francis albert t. argente*, 1sotero m. aban, emmanuel federico c. capinpin jr., armando c. garcia, lemark m. bautista pangasinan state university – center for hydrology, aquaculture, natural and ocean sciences (psu – chanos), psu – binmaley campus, binmaley, pangasinan 2417, philippines. s article history: received 19 april 2020 accepted 13 november 2020 available online 2 5 april 2021 keywords: bivalve pangasinan estuarine growth reproduction abstract: knowledge on the optimum environmental conditions for a potential aquaculture species is necessary to simulate an artificial setting. the current study investigated the influence of several environmental parameters on the physiology of the mud clam geloina expansa under natural condition. highest cis were recorded during the months of may to august, averaging to 179.79 (±6.47 se) while the rest of the months averaged only 158.53 (±2.68). geloina expansa population monthly gsi in santiago river ranged from 9.64 to 26.51, low values were observed from november to february (12.00±0.36) and from june to july (12.24±2.59). chlorophyll a concentration and sediment tom have significant influence in ci variations. do significantly affected the changes in gsi. these ecophysiological information are vital for the development of aquaculture techniques for g. expansa in northern philippines. introduction the aquaculture industry is an important fisheries sector in the philippines, providing cheap but nutritious food for many filipinos. in 2018, the industry produced a total of 2.30 m metric tons of various commodities (psa, 2019). however, the philippine aquaculture industry may now be in a critical state due to climate change (santos et al., 2011; macusi et al., 2015). several high-valued cultivable species in southeast asia have shown signs of vulnerability to the changing environmental conditions (piamsomboon et al., 2016; guerrero, 2019). one of the proposed measures to mitigate the undesirable effects of climate change in the aquaculture industry is the search for new and better adapted species (d’abramo and slater, 2019). facts on the biology of a potential aquaculture species should be generated to prelude the development of aquaculture techniques. ideal environmental conditions for optimal physiological responses are vital biological information to establish a sound cultivation technology. knowledge on the optimum environmental conditions for a potential aquaculture *correspondence: francis albert t. argente doi: https://doi.org/10.22034/ijab.v9i2.858 e-mail: francisargente@psu.edu.ph species is necessary to simulate such situation in an artificial setting. geloina expansa (fig. 1) is one of the usual mud clam species found in santiago river, dagupan city, northern philippines. it is a suspension filter-feeder which thrives in muddy bottoms of shallow estuarine waters. economically, g. expansa supports an artisanal fishery in santiago river as well as in other parts of the philippines (argente, 2013). this bivalve also plays a major role in its environment as a bioremediation agent, having the capacity to accumulate various heavy metals and persistent organic pollutants in its natural habitat (dsikowitzky et al., 2011; elvira et al., 2016; ali and yep, 2016). it has also been reported to be resilient to adverse environmental conditions (morton, 1976; argente et al., 2014). these features make this clam species as a potential candidate for aquaculture. therefore, gaps on the biology of this bivalve should be addressed. biological indices such as the condition index (ci) and gonadosomatic index (gsi) were used to characterize various physiological activities of cyrenid clams such as growth and reproduction 72 argente et al./ ecophysiological information on g. expansa under given environmental conditions (gimin et al., 2005; rahim et al., 2012; castañeda et al., 2018). these ecophysiological parameters can provide significant information for the development of aquaculture techniques for g. expansa in northern philippines. as of the moment, limited physiological information is known on this bivalve species. hence, the current study investigated the influence of several environmental parameters on the physiology of g. expansa under natural conditions. materials and methods clam collection: mud clams utilized in this study were harvested from santiago river, dagupan city (16.018665°n, 120.317598°e). the area is an identified gleaning site for g. expansa which supports artisanal fishery. moreover, the river is a site for various aquaculture activities. it has brackish hydrographic features with muddy substrate, which is an ideal environment for g. expansa since this species is a known tropical estuarine inhabitant (morton, 1976; argente, 2016). clams were gathered on a monthly basis (november, 2014 to december, 2015) with the aid of a commissioned bivalve gleaner. no discrimination of sizes was done during the monthly collection. condition (ci) and gonadosomatic (gsi) indices: thirty clams of various sizes were collected every month to estimate the ci of g. expansa population in the river. internal shell capacity (isc) and dry weight of the soft tissues were obtained. the calculation of ci was based on ilano et al. (2007): 𝐶𝐶𝐶𝐶 = 𝑑𝑑𝑑𝑑 𝐶𝐶𝐼𝐼𝐶𝐶 × 1000 where dw is the dry weight of the soft tissue and isc is the internal shell capacity derived from the difference between the total weight and shell weight. another 30 clams of different sizes were used to determine the gsi of g. expansa in santiago river. individual soft tissue weights were taken using digital weighing balance. the soft tissue of each clam was further dissected to determine gonad weight. the gsi of each individual was computed according to the equation used by del norte-campos (2004): 𝐺𝐺𝐼𝐼𝐶𝐶 = 𝑠𝑠𝑠𝑠𝑑𝑑 𝑔𝑔𝑑𝑑 × 100 where stw is the soft tissue weight and gw is the gonad weight of the clam. environmental parameters: water temperature, salinity, dissolved oxygen (do), chlorophyll a (chl a) concentration and total organic matter (tom) of the sediment were monitored on weekly basis during the period of the study. observations of all parameters, except for tom, were done three times (5 am, 12 pm and 6 pm) on the day of monitoring. monthly water temperatures (°c) were recorded based from the readings of a field thermometer. monthly salinity (ppt) measurements were done with the use of a field refractometer. monthly dos (mg l-1) were monitored using a do meter. monthly estimation of chl a was based on the trichromatic method described by aminot and rey (2000) and argente and estacion (2014). three replicates of 1 l water samples were collected at different times during the day of monitoring and immediately filtered using whatmantm gfc (47 mm diameter) filters. pigment extraction was done by macerating the filters in 90% acetone producing 10 ml extracts. extracts were mixed thoroughly and centrifuged for 10 minutes at 500 rpm. extracts were transferred to glass cuvette and absorbance at 750, 664, 647 and 630 nm against a figure 1. geloina expansa in santiago river, pangasinan, northern philippines. 73 int. j. aquat. biol. (2021) 9(2): 71-78 90% acetone blank were measured with the use of a spectrophotometer. chl a concentration (mg m-3) was calculated according to the equation of jeffrey and humphrey (1975) as cited by aminot and rey (2000): 𝐶𝐶ℎ𝑙𝑙 𝑎𝑎 = ([11.85 × {𝐸𝐸664 − 𝐸𝐸750}] − [1.54 × {𝐸𝐸647 − 𝐸𝐸750}] − [0.08 × {𝐸𝐸630 − 𝐸𝐸750}]) × 𝑉𝑉𝑒𝑒 𝐿𝐿 × 𝑉𝑉𝑓𝑓 where l is the cuvette light-path in cm; ve is the extraction volume in ml; and vf is the filtered volume in liters. three weekly (12 in one month) replicates of sediment samples from the clam bed were used to measure the tom using the loss-on-ignition technique (argente et al., 2013). two-gram samples from each replicate were placed in pre-weighed crucibles, burned inside a muffle furnace at 650°c for 5 hrs, cooled down, weighed and recorded, accordingly. tom (% loss) was determined by the equation: 𝑇𝑇𝑇𝑇𝑇𝑇 = 𝐼𝐼𝑖𝑖 − 𝐼𝐼𝑓𝑓 𝐼𝐼𝑖𝑖 × 100 where si is the initial weight of the sediment before ashing and sf is the final weight of the sediment after ashing. environmental influence to ci and gsi: standard multiple regression analyses were done to determine which among these environmental parameters had the most influence on the monthly variations of ci and gsi in g. expansa population in santiago river. the significance level was set at p≤0.05. results condition (ci) and gonadosomatic (gsi) indices: the observed monthly ci values (fig. 2) for g. expansa in santiago river varied from 146.51 to 199.12 with an average of 164.61 (±3.53 se). highest cis were recorded during the months of may to august, averaging to 179.79 (±6.47) while the rest of the months averaged only 158.53 (±2.68). geloina expansa population monthly gsi (fig. 3) in santiago river ranged from 9.64 to 26.51, averaging to 16.04 (±1.54). it appeared that the trend of monthly gsi have two pulses with different strengths and duration. high gsi values were recorded during march to may (17.04±2.15 average) and august to october (25.64± 0.52). consequently, reduced gsi values were observed from the months of november to february (12.00±0.36) and june to july (12.24±2.59). environmental parameters: generally, water temperature increased from november 2014 to succeeding months with mean value (30.57°c±0.28) figure 2. monthly mean ci of geloina expansa population in santiago river. 74 argente et al./ ecophysiological information on g. expansa peaking in july (32.57°c), then decreased rapidly from august to december (28.23°c±0.29) (fig. 4a). mean monthly water temperature was 29.73°c (±0.41). salinity in the clam bed oscillated from 5.50 to 23.50 ppt (14.32 ppt±2.08) (fig. 4b). maximum salinity was detected from january to june (22.34 ppt ±0.27) while minimum salinity values were monitored during july to december (8.31 ppt ±1.41). gradual increase in do (4.38 to 8.62 mg l-1) was observed from november 2014 to may 2015 then values fluctuated thereafter (fig. 4c). the monthly mean do in the clam bed was 6.72 mg l-1 (±0.44). the water chl a concentration (17.54±2.43 mg m3) in the g. expansa clam bed exhibited high variability throughout the duration of the study (fig. 4d). elevated chl a concentration was recorded from may to august (29.00±1.94 mg m-3) while only a mean of 12.96 mg m-3 (±1.80) were observed for other months. the monthly monitoring of sediment tom in the clam bed recorded an average of 6.44% (±0.49) with highest record in august (8.34%) and least in may (2.01%) (fig. 4e). environmental influence to ci and gsi: all environmental parameters monitored in the clam bed contributed to the monthly variations of g. expansa ci (r2=0.93) in santiago river (table 1). among the parameters observed, only chl a concentration and sediment tom have significant influence in ci variations. between the measured parameters, chl a concentration has better association with ci. all parameters, except for salinity, have positive correlation with ci. figure 3. monthly mean gsi of geloina expansa population in santiago river. table 1. standard multiple regression analysis of geloina expansa condition index (ci) with the environmental parameters in santiago river. parameter standard estimate (β) standard error parameter estimate (b) standard error p-level intercept 39.07 45.79 0.418 water temp. 0.33 0.16 2.97 1.42 0.070 salinity -0.24 0.11 -0.42 0.20 0.063 do 0.21 0.12 1.77 0.99 0.113 chl a 0.46 0.19 0.69 0.28 0.039 tom 0.40 0.16 3.01 1.19 0.035 n = 14; r2 = 0.93; p<0.00024 75 int. j. aquat. biol. (2021) 9(2): 71-78 the results of the multiple regression analysis for gsi and environmental parameters (table 2) showed that all environmental parameters contributed in the monthly variations of gsi (r2=0.83). however, only do significantly affected the changes in the g. expansa population’s gsi. variations in do were directly associated with the changes gsi. discussions the ranges of the monitored environmental parameters in the g. expansa clam bed in santiago river suggest the threshold of the clam. the mean values of environmental characters may indicate the ideal conditions for g. expansa. such information should be validated under laboratory conditions. in figure 4. mean monthly variations in environmental parameters in geloina expansa clam beds in santiago river, including (a) water temperature, (b) salinity, (c) do, (d) chl a concentration and (e) sediment tom. 76 argente et al./ ecophysiological information on g. expansa this study, ci was used to characterize the quality of the soft tissue of g. expansa population at given environmental conditions. the current study showed relatively high ci values during wet season and moderate values for the rest of the year which indicated the good health status of the clams. similar trends were reported with other geloina species and cultured bivalves (mohite et al., 2009; mcfarland et al., 2016; ransangan et al., 2019). an ideal bivalve species for aquaculture should have high quality meat as indicated by their condition in their natural habitat (yildiz et al., 2011; filguiera et al., 2013). moreover, there have been reports that high ci in bivalve populations were related with the abundance of available food in their natural environment (galvao et al., 2015; pantea et al., 2018) which may be the case of santiago river. the results of this study revealed that the increase in chl a concentration and tom were associated with the increase in ci values of g. expansa population in santiago river. the significant influence of chl a concentration in water and tom in sediments on ci variations postulates the importance of the plankton and detritus in the diet of g. expansa. similar observations were reported by bachok et al. (2003) on the corbiculid mud clam, geloina coaxans, a synonym of g. expansa (poutiers, 1998). likewise, clemente and ingole (2011) suggested that the availability of phytoplankton and microalgae influenced the recruitment of polymesoda erosa (also synonym of g. expansa) in a mangrove forest. the gsi of g. expansa in santiago river was utilized to describe the reproductive activities of the clams, revealing the gonadal development and spawning season. reports on p. erosa (g. expansa) and other bivalves have claimed that increasing gsi values indicated gonadal development while reduced values implied spawning events (clemente and ingole, 2009; cabacaba et al., 2018; asaduzzaman et al., 2019). it appeared in this study that there were two peaks of annual reproductive events for g. expansa population in santiago river. similar observations were reported on p. erosa (g. expansa) from various regions which exhibited two pulses of annual recruitment pattern and spawning period (gimin et al., 2005; dolorosa and dangan-galon, 2014). population spawning of g. expansa as depicted by reduced gsi values was observed during the northeast monsoon months and on the onset months of the rainy season (southwest monsoon). similar trends in gsi observations were reported in other philippine bivalves (del norte-campos, 2004; morillo-manalo et al., 2016). in the philippines, northeast monsoon or “amihan” is characterized by cool and dry weather while the southwest monsoon or “habagat” is typified by rainy weather condition (oliveros et al., 2019). the monsoon regimes may have influenced the environmental conditions within the river which affected the variations in the monthly gsi values of g. expansa population. although all the environmental parameters measured in this study contributed in the fluctuation of gsi values, only the do observations significantly influence the changes in gsi. it appeared that the decrease in do concentration in the water triggered the spawning activities of the clam. this information is vital in the development of hatchery techniques for g. expansa as well as in the management of the clam beds. table 2. standard multiple regression analysis of geloina expansa gonadosomatic index (gsi) with the environmental parameters in santiago river. parameter standard estimate (β) standard error parameter estimate (b) standard error p-level intercept 62.01 29.64 0.070 water temp. -0.53 0.24 -2.00 0.92 0.061 salinity -0.07 0.17 -0.05 0.13 0.682 do 0.53 0.18 1.88 0.64 0.019 chl a 0.45 0.28 0.29 0.18 0.150 tom -0.17 0.24 -0.55 0.77 0.495 n = 14; r2 = 0.83; p<0.00652 77 int. j. aquat. biol. (2021) 9(2): 71-78 this study provided environmental and biological information that may be used to prelude the development of culture techniques for g. expansa in northern philippines. the environment influenced the reproductive and feeding activities of the mud clam. the observed environmental parameters in the clam beds may be utilized to determine the ideal conditions for g. expansa in an artificial setting. the soft tissue of the mud clam appeared to be of good quality which is preferred in aquaculture. the plankton and detritus assumed to be important in the diet of g. expansa. it appeared the decrease in do concentrations in the water may trigger the spawning of g. expansa. these results should be validated under laboratory conditions. acknowledgement gratitude is expressed to psu president, d.r. buted and the psu board of regents for granting financial support on this study. this is an aquatic science contribution of psu-chanos. references ali s.a.m., yep y.m. 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(2019). population dynamics of marsh clam, polymesoda spp. (bivalvia: corbiculidae) in marudu bay, malaysia. aacl bioflux, 12(2): 395-403. santos m.d., dickson j.o., velasco p.e.l. (2011). mitigating the impacts of climate change: philippine fisheries in focus. fish for the people, 9(2): 101-110. yildiz h., berber s., acarli s., vural p. (2011). seasonal variation in the condition index, meat yield and biochemical composition of the flat oyster ostrea edulis (linnaeus, 1758) from the dardanelles, turkey. italian journal of animal science, 10: 22-26. international journal of aquatic biology (2014) 2(5): 229-237 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article catch composition of the bottom trawl fishery along the coasts of karataş-adana (northeastern mediterranean sea) erdoğan çiçek1, mehmet karataş2, dursun avşar3, mohammad moradı*41 1nevşehir haci bektas veli university, faculty of art and science, department of biology, 50300 nevşehir, turkey. 2karamanoglu mehmetbey university, faculty of art and science, department of biology, karaman, turkey. 3cukurova university, faculty of fisheries, 01330 adana, turkey. 4department of biology, faculty of science, university of zanjan, zanjan, iran. article history: received 8 july 2014 accepted 18 september 2014 available online 2 5 october 2014 keywords: trawl fishery species composition karataş iskenderun bay mediterranean abstract: the catch composition of the bottom trawl fishery along the coasts of karataş was evaluated in the 2002-2003 fishing season. a total of 110 species were registered, the fishes showed the highest diversity (90 species) followed by 15 crustaceans species and 5 species of cephalopods. the highest catch per unit effort (cpue) value (66.8 kg h-1) was recorded in september when the fishing season was opened and decreased to the lowest value in march (12.5 kg h-1). the average cpue was 26.3 ± 18.9 kg h-1. the result showed that catch of fish decrease with increasing depth. the highest fish catch (47.42%) was found in 0-20 m depth range. 35.58 percent of the catch was between 20-50 m, and 17.00% between 50-100 m depth. lessepsian fish comprise 18.90% of all fish in terms of the number of species and 26.66% of the total fish catch. introduction the northeastern mediterranean is highly suitable fishing area for bottom trawling because of wide continental shelf and sandy and muddy bottom substrate. the bottom trawl hauls in the mediterranean coast of turkey are generally performed at depths above 70 m, although the hauls are occasionally performed at depths reaching 150 m depth (bingel, 1987). with bigger boats being added to the fishing fleet after the 1980s, regional stocks have been under ever-intensifying fishing pressure (gücü, 1994). therefore, the fishing effort has exceeded the sustainable production level in the region because of ineffective policies. the effects of this worsening situation were observed through decreases in catch, catch per unit effort (cpue), average size of landed species, average size of widely caught species in the past, substitution of species with a high commercial value by those with * corresponding author: mohammad moradı e-mail address: moradi_g@yahoo.com lower value and most importantly, a decrease in the number of species caught (gücü, 2000). one of most important peculiarities of the ne mediterranean coast of turkey is its higher diversity similar to those of tropical waters but with a quite low biomass. another point to be considered in the mediterranean, especially for northeastern mediterranean fisheries, is the lessepsian fish migration, which began with the opening of the suez canal in 1869. from the first record of lessepsian fish in 1902 (ben-tuvia, 1985; tillier, 1902), new records were reported, and the number of lessepsian species were increased to 65 (golaniet al., 2002). some of the species adapted to the mediterranean ecosystem (gücü, 2000), and those that reached trading densities include saurida undosquamis, leiognathus klunzingeri, upeneus moluccensis and upeneus pori. these are often the most abundant species in the main catch (bingel, 1987; avşaret al., 2000; çiçeket al., 2002). for this 230 international journal of aquatic biology (2014) 2(5): 229-237 reason, lessepsian migration should be monitored year by year (gücü, 2000). although some researches were carried out in turkey during the 1980s (bingel, 1981, 1987) and 1990s (anonymous, 1993; bingelet al., 1993), but no research on this topic has been performed for the last 15 years. therefore this study, even though it is limited in scope, aimed to report the numerical data on bottom trawl fishing along the coasts of karataş in the northeastern mediterranean. materials and methods the karataş coasts are situated between the mersin and iskenderun bays, where bottom trawl fishing is carried out intensively. for this reason, it can be claimed that the coasts of karataş can be a representative of the mentioned two bays. this study was carried out in one station in three depth levels from 0-20 m, 20-50 m and 50-100 m along the karataş coast on a monthly interval during fishing season between september 2002 and april 2003 with a commercial trawling vessel named coşkun reis (270 hp, 19.2 m) (fig. 1). the effective duration of the tows was one hour. throughout the sampling studies, the mediterranean-type commercial bottom trawl net consists of 700 round mouth meshes with a 22 mm (knot-to-knot) diamond-shaped cod-end (bingel, 1987). the towing speed ranged from 2.7 to 3.2 knots. the large species (rhinobatos rhinobatos, raja spp., gymnura altavela, dasyatis pastinaca etc.) were separated, and weighed to the nearest 0.1 g on the trawling vessel and others were transformed to the laboratory in iceboxes (packed in plastic bags) and then were maintained in deep freeze at -18°c. the samples were weighed and then identified based on fischer (1987), whitehead et al. (1984, 1986a, 1986b) and froese and pauly (2004). then numerical trawl fishery data was tabulated. similarity in proportions of species composition by month and trawl was analyzed using the “weighted pair–group method with arithmetic averages” based on davis (1973) by spss statistical software. the area (a, km2) covered in one hour of trawling was calculated according to avşar (2005): α=d*h*x2, where d is the length of swept area (m), h is the length of the buoy line head rope of trawl (m), and x2 is the constant opening of buoy line head rope (0.5) (pauly, 1980). in one hour of trawling, 0.0263 km2 was covered. the biomass per square kilometers (b) was estimated following the equation b = wc /(α*q) (avşar, 2005), where wc is the catch value by hour (g), α is the area covered by the trawl net (km2), and q is the catchability coefficient of the trawl net (=1) (bingel, 2002). the first 10 species with the highest biomass in the main catch were evaluated (bingel, 1987). results and discussion throughout the study, 15 crustacean (13.6%), 5 cephalopod (4.6%), and 90 fishes (81.8%, 8 cartilaginous, 82 teleost) species were identified. figure 1. sampling area and sampling stations along the karataş coasts (1: 0-20 m; 2: 20-50 m; 3: 50-100 m depth range). 231 çiçek et al/ catch composition of the bottom trawl fishery along the coasts of karataş the maximum number of species (43) was caught in october at a 20-50 m depth range, while the lowest caught (19 species) was in november at 50-100 m depth range. 87 species occurred in the depth zone 020 m, 92 species in the zone 20-50 m and 41 species in 50-100 m. similarity between months and depths: considering the similarities between months in terms of fish species, it was obvious that 5 different groups could be identified (fig. 2). the species in the first group consist of fish that live in the 50-100 m depth range; those in the second group live in the 20-50 m depth range, and third, fourth and fifth groups live at 0-20 m depth. therefore, monthly species and density differences between months were highest at the depths of 0-20 m. on the contrary, the differences figure 2. catch composition similarity dendogram according to depth ranges and months (i: 0-20 m; ii: 20-50 m; iii: 50-100 m depth range). figure 3. catch composition similarity dendogram according to monthly catch. 232 international journal of aquatic biology (2014) 2(5): 229-237 between months at other depth ranges were low. this may have occurred because the depth range of 0-20 m is near the coast and is used by some species for biological needs such as reproduction, feeding, or overwintering. for monthly grouping of species composition, five different groups were observed at a similarity level of 50% (fig. 3). september was a group on its own, followed by october and november in the next group. the third group, december, which is a transition month between autumn and winter, was observed to have similarities with upcoming months instead of the previous months. the months after december comprised two additional groups (january and february, and march and april). time-series of catch per unit effort (cpue): in total, a catch of 631.4 kg was obtained for the entire study period. the highest cpue value (66.8 kg h-1) was observed in september (fig. 4). in the following months, cpue decreased and reached its lowest value (12.5 kg h-1) in march, but it showed a tendency to increase in april. the average cpue for the entire fishing season was 26.3 ± 18.9 kg h-1, and the average yield per km2 was 1,000.3 ± 720.7 kg. the results showed that fish comprised about 79.10% of the total catch while the amount of crustaceans were 13.73%, and that of cephalopods was 7.17%. the monthly cpue changes were occurred based on depth i.e., the highest cpue value was recorded in september at the 20-50 m depth range and the lowest at the 50-100 m depth range for the entire fishing season (fig. 5). the data also shows that 42.33% of the total catch was obtained at a depth of 20-50 m, 39.46% at 0-20 m, and the rest (18.21%) at 50-100 m depth. charybdis longicollis had the highest proportion (31% of total catch at the depth range of 20-50 m). monthly changes in cpue values for fish: 79.10% of the total catch consisted of fish and a total of 499,421.13 g of fish were caught. the highest value in cpue (52.2 kg h-1) was in september when the fishing season opened, and it decreased in the following months, reaching the lowest value of 12.5 kg h-1 in march (fig. 6). an increase was observed in cpue in the last month of the fishing season (april). the average cpue for the entire fishing season was as 20.8 ± 14.4 kg h-1, and the average catch per km-2 was 791.2 ± 606.8 kg. for seasonal changes in monthly cpue, a rapid decrease in biomass was observed following the opening of the fishing season. this situation was caused due to the cohort from the previous year i.e. fishing in an area happens because of increases in the previous year’s cohort. this was observed in greece, where the trawl fishing regime is very similar to that of the fishery along the coast of turkish ne mediterranean (stergiou et al., 1997). somarakis and machias (2002) reported that prohibiting fishing during summer was an effective way to protect immature fish because these regulations make it possible to prevent the overfishing of mature fish. monthly changes in cpue for fish at various depths: when monthly cpue changes in fish at different depth ranges are taken into consideration (fig. 7), except for february, march, and april, the highest figure 4. time series of cpue and standard deviations. figure 5. monthly cpue fishes in depth ranges. 233 çiçek et al/ catch composition of the bottom trawl fishery along the coasts of karataş and lowest cpue were observed at 0-20 m and 50100 m depth range, respectively i.e. cpue decreases from inshore to offshore. thus, the fish biomass caught at 0-20 m and 20-50 m were 47.42%, and 35.58%, respectively, while it was 17.00% at 50-100 depth range. the average cpue values according to those depth ranges were 29.6 ± 27.1 kg h-1, 22.2 ± 13.7 kg h-1, and 10.6 ± 6.0 kg h-1, respectively. in addition for three depth ranges, the catch per km2 was 1,125.5 ± 1,029.4 kg, 844.7 ± 520.9 kg, and 403.5 ± 227.6 kg, respectively. based on the results, the highest number of fish species caught for the entire fishing season was at a depth range of 20-50 m (table 1). in october, 43 fish species were observed at the depth range of 20-50 m. the lowest number of species was 19 at the depth range of 50-100 m. an average of 32 fish species were caught for the whole study period in a trawl operation. a previous study found that the average number of species caught in a trawl for this area was 44 (bingel, 1987), and the results obtained from this study are parallel to those of bingel (1987), which suggests that this area has a rich diversity of species. monthly changes in percent occurrence of fish in the total catch: the proportion of fish in the total catch was lowest in april (67.45%), and the highest was in january (90.08%) (fig. 8). therefore, fish species were more than 2/3 of the total catch. lessepsian fish: a total of 17 lessepsian fish species, belonging to 14 families were spotted in this study (table 2). lessepsian fish consists 18.90% of the total number of species. except for january, the highest number of lessepsian fish species was at a depth of 0-20 m while the lowest number was recorded at the depth of 50-100 m. the highest number of species was in october with 13 species, and then with 0 species in september, december, and march. there was no significant difference in the number of lessepsian fish species between months (p<0.05). monthly changes in cpue for lessepsian fish: the highest average cpue for lessepsian fish was 11.73 kg h-1 in september, and the lowest in november with 3.39 kg h-1. the average cpue for lessepsian fish for the whole fishing season was 5.28 ± 3.32 kg h-1.also, the highest average cpue was 10.84 ± 9.87 kg h-1 at 0-20 m, followed by 4.40 ± 1.95 and 0.84 ± 0.49 kg h-1 at 20-50 m and 50-100 m depths, respectively. when the whole study period was considered, 68.18% of the total lessepsian fish biomass was caught at 0-20 m, 25.60% at 20-50 m, and the rest (6.22%) at 50-100 m depths. this shows that lessepsian fish disperse along the coast and that figure 6. mean monthly changes in the cpue value of fish. figure 7. monthly cpue fishes in depth ranges. figure 8. monthly changes of percentage occurrence of fish in total catch. 234 international journal of aquatic biology (2014) 2(5): 229-237 their biomass decreases commensurate with depth. both ben-tuvia (1985) and gücü and gücü (2002) reported that lessepsian fish use the coast as their preferred habitat. similarly, ben-yami and glaser (1974) pointed out that lessepsian species biomass decreases with depth. the 68.18% of the total lessepsian fish biomass at 0-20 m depth range of the study area is in agreement with the results of the above mentioned works. during 1983-1984, the proportion of lessepsian fish in the total catch in iskenderun bay was 62% (gücü and bingel, 1994). in this study, we caught 20.37% showing rapid decline of the proportion of lessepsian fish since then. gücü (2000) reported that the cpue for saurida undosquamis decreased about ten-fold from 1984 to 1996. this difference is assumed to have been caused by overfishing in the area. the lowest (14.01%) proportion of lessepsian fish in the total fish biomass was in november, and the highest (33.47%) in march with the average proportion of months depth ranges mean 0-20 m 20-50 m 50-100 m september 29 37 33 33 october 35 43 31 36 november 38 42 19 33 december 37 32 23 31 january 33 33 24 30 february 31 37 25 31 march 29 30 26 28 april 34 34 28 32 mean 33 36 26 32 table 1. number of this species obtained from each trawl operation. species months and percentage occurrence (%) average (%) 2002 2003 sep oct nov dec jan feb mar apr apogon nigripinnis 0.39 0.01 0.22 0.01 0.11 callionymus flamentosus 1.94 3.31 0.16 0.55 0.92 0.22 0.18 0.89 1.46 cinoglossus sinusarabici 0.66 2.22 1.76 1.23 1.61 0.30 0.62 1.80 1.23 dussumeria elopsoides 0.14 1.46 0.85 0.22 etrumeus teres 0.18 1.72 0.29 0.19 fistolaria commersonii 0.38 0.17 0.26 2.07 1.56 0.30 0.48 leiognathus klunzingeri 18.82 11.13 3.73 3.36 14.82 11.89 19.22 4.05 12.79 lagocephalus suezensis 1.87 2.63 2.76 0.30 0.83 pelates quadrilineatus 0.14 0.03 sphyraena chrysotaenia 0.37 1.43 1.38 0.98 0.79 0.48 stephanolepis diaspros 0.87 0.41 2.86 3.16 0.69 0.49 1.19 0.95 saurida undosquamis 28.38 43.64 71.49 64.03 45.31 47.79 54.61 50.79 45.56 sargocentron rubrum 0.29 0.02 siganus luridus 0.06 0.19 0.17 0.04 siganus rivulatus 0.11 0.49 0.17 -0 0.17 0.21 0.42 0.15 upeneus moluccensis 1.38 4.00 3.27 7.82 4.49 2.76 1.48 3.04 upeneus pori 47.11 32.48 11.62 20.73 25.70 34.10 19.47 37.57 33.11 table 2. monthly changes of each lessepsian fish species in their total catch. 235 çiçek et al/ catch composition of the bottom trawl fishery along the coasts of karataş 26.66% for the whole study period. lessepsian fish were consisted18.90% of the total species number. actually, in this research and in the above mentioned studies, species such as s. undosquamis, upeneus moluccencis, upeneus pori, and leiognathus klunzingeri have been included in the main catch and become important component of the regional trawl fishery (bingel, 1987; gücüet al., 1994). whereas, in other researches in the studied area (bingel, 1987; anonymous, 1993; gücü et al., 1994), s. undosquamis, u. moluccensis, and l. klunzingeri were the main catch. in recent years, u. pori (çiçek et al., 2002) has also been increasing in the main catch. in this study, we found that above mentioned species were included in the main catch except for u. moluccensis. although it was caught previously and not come across in our work. ben-yami and glaser (1974) reported that there were a significant annual fluctuations in cpue for this species (during1956 to 1970). therefore, it can be said that one can face a similar situation in the coasts of karataş even though no record of u. pori was reported in the main catch before 2000, however, it was found in both the study by çiçek et al. (2001, 2002) and in the present study. catch composition: teleost fish consist 76.98% of the main catch, followed by crustaceans with 15.98% and cephalopods with 7.04% (table 3) in the main catch. the prevalent species in the main catch were m. barbatus (19.48%), c. longicollis (15.98%) and s. undosquamis (15.56%). lessepsian fish consist 29.80% of the main catch. if c. longicollis is considered to be a lessepsian crustacean species, this proportion increases to 44.88%. number species cpue (kg h-1) biomass (kg km-2) percentage in total catch (%) percentage in main catch (%) 1 mullus barbatus 3.11 74.74 11.83 19.48 2 charybdis longicollis 2.56 61.31 9.70 15.98 3 saurida undosquamis 2.49 59.70 9.46 15.56 4 pagellus erythrinus 1.71 41.03 6.50 10.70 5 upeneus pori 1.58 37.88 6.00 9.87 6 bothus podas 1.22 29.27 4.64 7.63 7 sepia officinalis 1.16 27.02 4.28 7.04 8 spicara smaris 8.08 19.40 3.07 5.06 9 merluccius merluccius 7.64 18.35 2.91 4.78 10 leiognathus klunzingeri 6.90 16.77 2.66 4.37 total 61.05 100 table 3. cpue values, biomass, percentage occurrence of species in main catch (l: lessepsian). number 1983 1984 species cpue (kg) % species cpue (kg) % 1 saurida undosquamis 10.97 26.79 saurida undosquamis 16.08 41.85 2 stephonalepis diaspros 5.96 14.57 citharus linguatula 3.51 9.14 3 leiognathus klunzingeri 4.48 10.94 mullus barbatus 3.31 8.61 4 siganus rivulatus 2.51 6.14 carybdis longicollis 1.90 4.95 5 dasyatis pastinaca 2.35 5.74 merluccius merluccius 1.55 4.02 6 pagellus erythrinus 2.35 5.74 parapenaeus longirostris 1.45 3.78 7 mullus barbatus 1.48 3.61 squatina squatina 1.24 3.22 8 mullus surmuletus 1.34 3.28 arnoglossus laterna 1.22 3.18 9 diplodus annularis 1.05 2.57 myliobatos aquila 0.925 2.41 10 callionymus flamentosus 0.87 2.11 sephia officinalis 0.800 2.08 table 4. cpue values, percentage occurrence of species in main catch in between 1983 and 1984 from iskenderun bay. 236 international journal of aquatic biology (2014) 2(5): 229-237 when the proportion of total catch and cpue of species that made up the main catch (gücü and bingel, 1994) were compared with the results gathered from iskenderun bay between 1983 and 1984 in our study area, a great difference was observed between them (tables 3 and 4). while 6 species included in the main catch in 1983 and 5 species in 1984 were observed, none of these species were included in the main catch in the 2002-2003 fishing season. moreover, there were remarkable differences in the order of species that were included in the main catch, in terms of cpue and proportions. for instance, m. barbatus, which constituted 19.48% of the catch and weighed 3.1 kg h-1 for cpue in 2002-2003, was seventh in 1983, with 3.61% of the catch with1.5 kg h-1 for cpue. in 1984, it was the third most important component with 8.61% of the catch with 3.3 kg h-1 for cpue. this situation is an indicator of the lack of a remarkable difference in cpue of this species. saurida undosquamis, which was third in this study with 15.56% of the total catch and 2.5 kg h-1 cpue, was the main component of the catch in 1983 and 1984 with 26.79% and 41.85% of the total catch, respectively. when its cpue was considered in our study, it was 4 times lower than that of 1983 and 6 times lower than 1984. charybdis longicollis, which was not in the main catch in 1983, became fourth in abundance with 4.95% with 1.9 kg h-1 cpue in 1984. this species was second in abundance at 15.98% of the total catch and 2.6 kg h-1 cpue in 2002-2003 showing a substitution of species with little or no commercial value as result of decreasing commercially important fish due to fishing pressure. references anonymous. 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(1986b). fishes of the north-eastern atlantic and the mediterranean. unesco, paris, iii: 1008-1473. international journal of aquatic biology (2015) 3(3): 183-190 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article genetic variation and population structure of spottybelly greenling (hexagrammos agrammus) in korean coasts analyzed by dna markers emphasizing on microsatellites kazi ahsan habib*1, mohammad nazrul islam2, shirin sultana3, youn-ho lee4 1department of fisheries, sher-e-bangla agricultural university, sher-e-bangla nagar, dhaka-1207, bangladesh. 2department of biotechnology, sher-e-bangla agricultural university, sher-e-bangla nagar, dhaka-1207, bangladesh. 3fisheries biotechnology division, national institute of biotechnology, savar, dhaka-1349, bangladesh. 4korea institute of ocean science and technology (kiost), sadong, ansan, 426-744, south korea. article history: received 16 january 2015 accepted 27 february 2015 available online 2 5 june 2015 keywords: genetic diversity population structure mitochondrial dna microsatellite dna spottybelly greenling abstract: two nuclear microsatellite dna loci along with partial sequences of mitochondrial dna trnapro-d-loop region were analyzed to assess the genetic diversity and population genetic structure of spottybelly greenling (hexagrammos agrammus). a total of 85 individuals were investigated from two different locations at the east and the south coasts of korea which are imwonjin (im) and tongyeong (tn), respectively. in the analysis of genetic diversity, nucleotide diversities were low showing 0.01 whereas haplotype diversities were as high as 0.92 and 0.97 in im and tn populations, respectively indicating population bottleneck after rapid growth of these populations. no significant genealogical branches or clusters were recognized on the neighbor-joining phylogenetic tree. pairwise population statistics fst and the exact test of population differentiation from the analysis of microsatellite dna loci demonstrated no significant genetic difference between two populations investigated in the korean coasts. in addition, significant deviations from hardy-weinberg equilibrium and heterozygote deficiencies were found from the microsatellite dna loci. the results of the study will help to make a plan for fisheries management of the species. introduction spottybelly greenling, hexagrammos agrammus (temminck and schlegel, 1843), a member of the family hexagrammidae, is a commercially important fish species in korea. this fish is distributed along coastal areas of japan and korean peninsula, and somewhat found in the yellow sea. the species lives in shallow seaweed beds and reproduce from september to december on seaweeds as spawning substrates (chung and kim, 1994). during the breeding season, multiple females visit in the breeding territory of a male and release eggs. afterward, male fertilizes the eggs and look after these until hatching (munehara et al., 2000). marine organisms generally show low levels of genetic differentiation over large geographic distances. lack of physical barriers, high fecundity, * corresponding author: kazi ahsan habib e-mail address: ahsan_sau@yahoo.com dispersal of eggs and larvae by ocean current, and migration of adults in the open ocean contribute to the high gene flow, eventually lead to panmictic populations in many marine fishes (palumbi, 1994; walpes, 1998; grant and bowen, 1998). on the other hand, physical barriers to dispersal, such as depth, temperature, or salinity boundaries; or hydrodynamic eddies favoring larval retention; or strong philopatry are the factors that can promote population differentiation and genetic structure (grant and bowen, 1998). thus, the reason of forming population genetic structure of many marine organisms is sometimes difficult to understand (machado-schiaffino et al., 2009). assessment of population genetic structure is essential to make strategies for conservation and management of the fisheries’ resources. failure to detect population 184 international journal of aquatic biology (2015) 3(3): 183-190 units can lead to local overfishing and ultimately to severe declines (hutchings, 2000; knutsen et al., 2003). analysis of genetic diversity and population genetic structure also provide insights into historical demography and evolutionary process of the populations in the species. in this study, we investigated the genetic diversity and population genetic structure of spottybelly greenling in the east and the south coast of korea by analyzing microsatellite dna (msdna) and partial concatenated trnapro-d-loop region of mitochondrial dna (mtdna) markers. microsatellite markers are very useful for assessing genetic structure of fish populations due to their selective neutrality and high mutation rates providing greater allelic diversity and heterozygosity (o’connell and wright, 1997). mitochondrial dna d-loop region is particularly sensitive to assess genetic diversity and demographic history of the population in marine fish (buonnacorsi et al., 2001). materials and methods samples collection and dna extraction: a total of 85 spottybelly greenling individuals were collected from two locations of which 35 fish from imwonjin (im) and 50 fish from tongyeong (tn) of the south and the east coasts of korea, respectively (fig. 1) in 2007 and 2008. fishes were collected with the help of local fishermen and preserved in 95% ethanol until dna extraction. genomic dna was extracted from the tissue of preserved samples using a dneasy blood and tissue kit (qiagen, germany) according to the manufacturer’s protocol provided in the kit box. pcr amplification and sequencing: from the genomic dna, concatenated 3’ region of trnapro gene and 5’ region d-loop region (hyper-variable portion) was amplified using the primers hagf2: 5′– ccctaactcccaaagctaggattc–3′ and hagr2: 5′–atgctgggtctctcggagatgta– 3′, respectively through polymerase chain reaction (pcr) (habib et al., 2014). all the pcr reactions contained 50 μℓ reaction mixture having 2.5 unit of taq dna polymerase, 5 μℓ of 10× pcr buffer, 10 mm each of the dntps (2.5 μℓ each), 25 pmoles of each primer, and 0.5–1.0 μg template dna. the temperature profile of pcr was set at 95°c for 5 min as preheating, followed by 35 cycles of denaturation at 95°c for 1 min, annealing at 58°c, extension at 72°c for 1 min, with a terminal elongation at 72°c for 7 min. pcr products were examined by 1% agarose-gel electrophoresis with a standard-size marker and purified by a pcr purification kit (takara, japan). the purified dna was sequenced bidirectionally using automated dna sequencer 377 or 3100 (applied biosystems, usa). microsatellite loci amplification and scoring: two microsatellite loci (heot06 and heot17) developed for the most closely related species h. otakii (chen et. al., 2009) were amplified from the 85 samples of the korean coasts. pcr amplifications consisted of 3 min pre-heating at 94°c followed by 35 cycles of denaturation at 95°c for 45 sec, annealing at 55°c for 45 sec, and extension at 72°c for 1 min. pcr ended with a final extension at 72°c for 7 min. the pcr reaction mixture contained 1.5 μℓ of 10× pcr reaction buffer, 1.5 μℓ of 10 mm dntp, 1 μℓ of 0.2 mm of each primer, 3 units of super taq polymerase (super bio, osong, korea), and 0.5 μg of template dna with the total volume of 15 μl brought up with sterilized water. forward primers were labeled with figure 1. map showing sampling locations of h. agrammus samples and the major warm currents around the sampling area. abbreviations as follows: im– imwonjin (sea of japan) and tn– tongyeong (south sea of korea). 185 habib et al/ genetic variation and population structure of spottybelly greenling a fluorescent dye (genotech, korea) and the amplified products were run for size detection on the abi prism_310 automated sequencer. data collection and sizing of alleles were carried out using peak scanner v31.0 software (applied biosystem). data analysis: the trnapro-d-loop sequence data were edited and aligned with clustal w (thompson et al., 1994) and dnassist 2.5 (patterton and graves, 2000). the level of polymorphism was estimated as the haplotype diversity (h), nucleotide diversity (π), number of haplotypes, transitions (ti) and transversions (tv) for both populations separately and also for pooled samples using the program arlequin (version 3.5, schneider et al., 2000). in the microsatellite analysis, microsatellite polymorphism was evaluated by the number of alleles (n), allelic richness (ar), observed heterozygosity (ho), and expected heterozygosity (he). population genetic structure was evaluated by pairwise estimates of the fst values and by the exact test of population differentiation. exact test gives more weight to rare alleles and can therefore be more sensitive to the detection of weak population differentiation. all of these parameters were computed using the arlequin software except ar which was calculated using the fstat software (ver. 2.9.3.2, goudet, 2001). deviation from the hardy–weinberg equilibrium (hwe) was tested using popgene (ver. 1.31; yeh et al., 1999). phylogenetic relationship among haplotypes of trnapro-d-loop region was reconstructed with mega 3.1 (kumar et al., 2004) using the neighbor joining (nj) method (saitou and nei, 1987) of phylogenetic inference with 1000 replications. genetic distances were corrected following the model of tamura and nei, which was specifically conceived to reproduce the evolution of the mitochondrial d-loop region (zardoya et al., 2008). results genetic diversity: sequences of all of the 85 individuals of ij and tn populations were obtained after sequencing. a total of 500 nucleotide long sequences were acquired after removing the confusing sequences near the primer ends. these 500 bp long sequences included a portion of the trnapro (31 bp) sequence in the 3′ end and the rest 469 bp of the partial d-loop region at the 5′ end. most of the nucleotide substitutions were transitional in the studied sequences (table 1). about 30% of the nucleotide substitutions were transversion. no indels were detected in all the sequences analyzed. variations in the d-loop region defined 43 haplotypes with 39 polymorphic sites of which ten haplotypes were shared by two populations. the nucleotide diversities (π) were as low as 0.01 nucleotide differences per site. on the other hand, the haplotype diversities (h) were relatively high providing the values of 0.92-0.97 (table 1). in the msdna analysis, two microsatellite loci revealed a moderate level of polymorphism in the korean populations. in the locus heot06, 10 and 12 alleles were found from the im and tn populations, respectively, and 17 and 22 alleles in the locus heot17. the observed heterozygosities (ho) were found significantly lower than expected heterozygosities (he) for all loci in both populations. the average ho was around 0.5 and 0.6 for im and tn populations, respectively whereas he was estimated over 0.85 for both populations (table 2). in all cases, the 1– ho/he values were positive, meaning that im and tn populations were deficient population no. of haplotypes substitutions h π ti tv im 18 15 5 0.92 0.01 tn 35 28 11 0.97 0.01 pooled 43 30 12 0.95 0.01 * h: haplotype diversity, ti: transitions, tv: transversions, π: nucleotide diversity table 1. genetic diversities of im and tn populations. 186 international journal of aquatic biology (2015) 3(3): 183-190 in heterozygosities at both loci. the locus heot17 in both populations and heot06 in the tn population showed significant deviations from hwe. only nonsignificant deviation (p>0.05) from hwe was detected for the locus heot06 in im population. a summary of the statistical data, including n, ar, ho, he, and agreement to the hwe, for im and tn populations are presented in table 2. population differentiation: estimates of fst between im and tn populations for msdna was nearly zero (0.013) with no significant difference (p= 0.07) from random mixing, indicating that no population genetic structure (i.e. a single genetic stock) was established over the range of investigation. exact test of population differentiation also did not give any significant differentiation between two populations with the p-values for microsatellite loci 0.932 between im and tn. phylogenetic relationships of haplotypes: the topology of the neighbor joining tree of 43 haplotyp sequences of h. agrammus was shallow with short branches (sum of branch length, sbl = 0.121), and there were no genealogical clades or internal branches corresponding to any particular sampling locality (fig. 2). haplotypes from individuals of a population were scattered throughout the tree being well mixed with those from another population. discussion spottybelly greenling is a demersal fish which inhabits in coastal reef area. the fish shows restricted movement and releases sticky eggs on the seaweed beds. such limited dispersal characteristic is responsible for genetic differentiation among populations in several marine fish species (han et al., 2008a; mukai et al., 2009). however, the present study clearly indicates genetic homogeneity among two regional population samples, i.e. im and tn population of spottybelly greenling. the fst statistics for msdna markers revealed no significant genetic structure between two sampling sites suggesting substantial genetic exchange along the coast of korea. moreover, non-significant exact pvalue in the exact test of population differentiation reveals population panmixia for the fish in the korean coastal water. lack of clade with a specific geographic location (east coast or south coast) in the phylogenetic tree further supports high rate of gene flow between two populations (han et al., 2008a). many marine fishes show lack of genetic differentiation among the geographic regions due to higher dispersal potential during planktonic egg and larvae (palumbi, 1994). the adult spottybelly greenling lives in the bottom however, the larvae and fry spend planktonic stage for around two months in the shallow or subsurface waters (cho et al., 2001). therefore, transportation of planktonic larvae and fry by the ocean currents possibly caused this genetic homogeneity of spottybelly greenling along the korea coasts. the tsushima current would carry the microsatellite locus parameters population im tn heot06 n 10 12 ar 10.95 11.031 ho 0.6 0.764 he 0.804 0.837 deviation from hwe ns * heot17 n 17 22 ar 18.959 19.492 ho 0.4 0.418 he 0.911 0.915 deviation from hwe * * average value over two loci ho 0.5 0.59 he 0.858 0.876 n 13.5 17 *significant; ns = not significant table 2. summary statistics for two microsatellite loci in the im and tn populations of h. agrammus. (n = no. of alleles, ar = allelic richness, ho = observed heterozygosity and he = expected heterozygosity) 187 habib et al/ genetic variation and population structure of spottybelly greenling fries and larvae from the south to the northern parts. conversely, the coastal and the subsurface counter currents can transport these young fish from the northern to the southern areas and cause extensive genetic exchange between the regions. several marine fishes around korea as well as in northwest pacific exhibited similar pattern of genetic characteristics. as for example, fat greenling (h. otakii), a sister species of the spottybelly greenling did not show any genetic separation among the populations of the west coast (yellow sea), the south coast and the east coast (east sea) (habib et al., 2011). japanese sea bass lateolabrax japonicus and blackfin flounder glyptocephalus stelleri formed a single genetic population around the korea and the japan coastal waters (liu et al., 2006; xiao et al., 2010). in an another study, red tilefish (branchiostegus uitions), a subtropical demersal fish showed no population genetic structure among the samples collected from the east china sea and the japan coast (nohara et al., 2010). in all of these fishes, dispersal of pelagic eggs and larvae by the currents were considered as a probable cause for such genetic homogeneity. in the microsatellite study, no significant genetic structure was found between any of the populations but expected heterozygosity was significantly high across both locations. strong heterozygote deficiencies also have been reported for a number of fish species (waldman and mckinnon, 1993) such as common sole solea vulgaris (kotoulas et al., 1995) and plaice pleuronectes platessa (hoarau et. al., 2002), and the reasons were mentioned as the result of null alleles, wahlund effect or biological processes such as inbreeding and selection. inbreeding would be unlikely for the observed heterozygote deficiency in spottybelly greenling fish with large populations since the fish is widely distributed in the coasts of korea as well as in northwestern pacific ocean. wahlund effects or selection might be the reason for observed heterozygote deficiencies for this fish. wahlund effects occur when two or more subpopulations are unintentionally sampled as a single population. in case of selection, it might have occurred as a very recent event since microsatellites reflect very recent demographic history below last ten thousand years. null alleles are a common problem with microsatellite loci that can lead to high heterozygote deficiencies (hare et al., 1996; o’connell and wright, 1997). they occur when one allele is not amplified due to mutations in one of the primers, and/or when technical problems associated with amplification and scoring arise. the primers that we used in the present study were originally developed for the sister species, fat greenling (h. otakii) which might be one of the causes for heterozygote deficiency. significant deviations from hardyweinberg equilibrium in the present study were possibly caused due to the loss of heterozygosity in the population. very low nucleotide diversity with high haplotype diversity in both im and tn population of spottytbelly greenling (π = 0.01; h>0.9) indicates that the fish has experienced rapid population growth after bottleneck and accumulation of mutation (grant and bowen, 1998). habib et al. (2014) showed that this population expansion of spottybelly greenling has started during the pleistocene climatic figure 2. a neighbor-joining tree of trnapro-d-loop region haplotypes of h. agrammus. bootstrap supports of less than 50% in 1000 replicates are not shown. im and tn represent geographical distribution of each haplotype. 188 international journal of aquatic biology (2015) 3(3): 183-190 changes. in case of sudden population growth, the rate of stochastic loss of haplotypes decelerates and maintains more haplotypes than the loss by genetic drift (avise et al., 1984). high haplotypic diversity further suggests large, stable and effective population size over time in the spottybelly greenling since it is widely distributed in coastal waters of korea and the japanese archipelago (stepien, 1999). this kind of genetic make-up has been detected in a number of fish species, including fat greenling (h = 0.69 – 96, π = 0.002 – 0.004) (habib et al., 2011), redtile fish (h = 0.93, π = 0.008) (nohara et al., 2010), blackfin flounder (h = 0.99, π = 0.014) (xiao et al., 2010) and north pacific light fish, maurolicus japonicas (h = 0.58, π = 0.003) (habib et al., 2012). on the other hand, the shallow phylogenetic tree reveals very recent origin of the haplotypes and rapid population growth. moreover, the short branch length in the nj tree suggests a recent (young) population genetic divergence for the populations of h. agrammus (zhang et al., 2006). another greenling species, h. otakii, a shallow coastal water demersal fish distributed along the coast of korea, japan, and china in the northwestern pacific went through recent expansion (habib et al., 2011). since these two sister species, i.e. h. agrammus and h. otakii are distributed almost in the same geographic region and their habitats are also same, similar demographic pattern i.e. rapid population growth after a bottleneck was found. population expansion has been also reported for a number of other benthic fishes such as the yellow drum (han et al., 2008b), willowy flounder (xiao et al., 2008), blackfin flounder (xiao et al., 2010). assessment of genetic diversity and population genetic structure are essential for maintaining a productive fishery through effective management (seeb et al., 1990). different populations with unique genetic structure and/or geographically isolated stock should be managed as distinct units, and such units require separate monitoring and management (salgueiro et al., 2003). our study of msdna variations in spottybelly greenling did not show any evidence for genetic subdivision and revealed a single population between the east and south coast of korea. therefore, the study suggests rather simple management strategy for this fish, i.e. ‘one stock’ management policy in the study areas. however, caution must be taken for one stock management policy until the population genetic structure and the current migration pattern between the populations were thoroughly understood. for this, more microsatellite loci can be developed and analyzed for the samples incorporating more sampling sites. references avise j.c., neigel j.e., arnold j. 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(2008). genetic variation and population structure of willowy flounder tanakius kitaharai collected from aomori, ibaraki and niigata in northern japan. african journal of biotechnology, 7(21): 3836-3844. yeh f.c., yang r.c., boyle t. (1999). popgene, version 1.31: microsoft window-based free ware for population genetic. analysis. http://www.ualberta.ca/~fyeh zardoya r., castilho r., grande c., favre-krey l., caetano s., marcato s., krey g., patarnello t. (2004). differential population structuring of two closely related fish species, the mackerel (scomber scombrus) and the chub mackerel (scomber japonicus) in the mediterranean sea. molecular ecology, 13: 17851798. zhang j., cai z., huang l. (2006). population genetic structure of crimson snapper lutjanus erythropterus in east asia, revealed by analysis of the mitochondrial control region. ices journal of marine science, 63: 693-704. int. j. aquat. biol. (2018) 6(6): 296-302 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article cloning and characterization of promoters of vascular specific genes in zebrafish harinder vishwakarma1, jayanand manjhi*2,1anvesha sinha1 1school of biological sciences, shobhit university, nh-58, modipuram, meerut-250110, india. 2research and innovation centre, noida international university, gautam buddha nagar, greater noida, india. s article history: received 18 january 2018 accepted 14 december 2018 available online 2 5 december 2018 keywords: gene expression promoters red fluorescent protein zebrafish abstract: gene promoters located at the 5′ end of genes are instrumental in regulating the gene expression in a ubiquitous or tissue specific manner. the objective of the study was to identify, clone and characterize promoters involved in gene expression in specific tissues such as the blood and blood vessel of zebrafish. three genes, known to express in the blood and blood vessel lineage in zebrafish, were selected viz. pak2a, rac1 and cdc42. approximately 800 bp of putative promoter region of pak2a and 826 bp putative promoter region of cdc42 were cloned into plasmid vectors. this putative promoter did not show any expression in zebrafish embryos. however, approximately 716 bp of putative promoter region of rac1 showed red fluorescent protein expression. while study cmlc2 was used as a positive control. introduction the zebrafish, danio rerio, is a powerful model organism to study the vertebrate biology, well suited for the genetic basis of embryonic developments and genetic analysis (dooley and zon, 2000) in both vertebrate and invertebrate species (fire, 1986; flytzanis et al., 1985; jaenisch, 1988; rubin, 1988). zebrafish exhibits a rapid developmental pattern with the major organs, including a functional heart, vasculature, circulating blood cells, liver, brain etc. being developed within 30 hours after fertilization. the rapid external development and transparency of zebrafish embryos allow direct observation of developmental processes and the deployment of fluorescent reporter genes to facilitate examination of spatial and temporal gene expression patterns in living embryos (arnaout et al., 2007; bai et al., 2007). attributes such as external fertilization, optically transparent embryos, high fecundity, ease of maintenance, half of the genome size as compared to humans, knowledge of whole genome sequence along with the ease of production, maintenance, analysis of mutants and transgenic lines are in a relatively short period of 3 to 4 months generation time make the *correspondence: jayanand manjhi doi: https://doi.org/10.22034/ijab.v6i6.419 e-mail: jayanand2005@gmail.com zebrafish well-suited animal model of transgenic studies. introduction of foreign genes into zebrafish genome has been found comparatively easy with respect to other vertebrate model organisms to produce transgenic zebrafish. the transgenic zebrafish is widely used to study spatio-temporal gene expression using reporter genes like rfp (red fluorescent protein), and gfp (green fluorescent protein). transgenic animals are also found valuable model to study tissue/organ development in different types of mutagenesis screens (chávz et al., 2016). the availability of efficient techniques for manipulation of gene expression enables studies of gene function. there are different methods like: micro injection, electroporation, retroviral vectors delivery etc. which are widely used to generate transgenic zebrafish. out of these microinjection is found to be one of the most efficient techniques for gene delivery. gene transfer, utilizing microinjection technique, in fish began in the mid 1980’s (chourrout, 1986; dunham et al., 1987). microinjection of dna constructs into single celled fertilized zebrafish embryos has proven successful in the generation of transgenic zebrafish. efficiency of transgenesis in 297 int. j. aquat. biol. (2018) 6(6): 295-302 zebrafish has been achieved nearly 70-100% by retroviral vectors (e.g. pseudoretroviral vector) and transposase (e.g. tol2, sleeping beauty) (kawakami et al., 2007; stuart et al., 1990). insertions made in the genome using tol2 can be selected in f1 generation by outcrossing the injected fish. on an average, transgenic f1 offsprings obtained from 50-70% of the injected fish, at frequencies of 3100%. although the frequencies are lower than those with retroviral vectors (nearly 100%), the tol2 transposon system has advantages over the pseudotyped retrovirus system as the handling and modification of retroviral vectors is laborious, which makes the application of psudotyped retrovirus for transgenesis method difficult. tol2 is a transposable element obtained from medaka fish. it is an autonomous transposon that encodes a fully functional transposase enzyme that catalyzes transposition of a transposon construct that has 200 and 150 bp of dna from the left and right ends of the tol2 sequence, respectively. the tol2 transposon system has been shown to be active in all vertebrate cells, including zebrafish. tol2 integrates as a single copy through cut and paste mechanism and it does not cause any rearrangement or modification at the target site (kawakami et al., 2007) and transgenes created by tol2 mediated transgenesis may not suffer from gene silencing effects. to develop a tissue specific transgenic line, gene specific promoters have been used. promoters contain specific dna sequences and response elements which provide a binding site for rna polymerase and for proteins called transcription factors involved in the recruitment of rna polymerase. many genes promoter have been used to create transgenic fish line, e.g. zhu et al. (2005) cloned different fragments ranging from 2.5 kb to 69 bp upstream of translation start site of lmo2 gene in gfp vector construct and finally concluded that 174 bp proximal promoter was sufficient to recapitulate lmo2 expression. by using lmo2 promoter they created two transgenic lines, tg (lmo2:egfp) and tg(lmo2:dsred) both of which exhibited embryonic blood and endothelial expression. similarly, park et al. (2000) determined that 2.8 kb of the 5’-flanking sequence of huc was sufficient to restrict gfp gene expression to neurons, the core promoter was only 251 bp. meng et al. (1997) revealed that a 1.1 kb dna sequence was critical for expression of gata-2 in neurons. likewise promoters of many genes viz: kdr, gata1, gata2, eno2, spi1 etc. have been characterized for their expression using reporter genes like gfp, rfp etc. some other examples are summarized in the table 1. several promoter prediction programs like: bdgp (berkeley drosophila genome project), prosom, promoter prediction server etc. are available which help in the promoter analysis. these bio-informatics tools provide us an idea of the existence of putative promoter region of a gene of interest, based on which required primers for the amplification of dna region of interest can be designed by ligation to a fluorescent protein spatio-temporal expression of a particular gene can be checked. the present study is aimed at identification and characterization of promoter vascular specific genes to be employed for the generation of transgenic zebrafish species. materials and methods sample collection: experiments were carried out in accordance with institutional animal use and care committee regulations and approvals. the zebrafish embryos were collected at 24 hpf (hours post fertilization), immediately frozen in liquid nitrogen and stored at -80°c for future use. the study was carried out at zebrafish lab facility (institute of genomics and integrative biology, delhi, india). isolation of genomic dna from zebrafish embryos: wild type zebrafish were allowed to breed in breeding tanks and embryos were collected and frozen at 36 hours post fertilization (hpf). extraction of dna was performed from 50 embryos using the protocol given below and stored at -20c. the embryos were homogenized in 0.75 ml lysis buffer using a 2 ml dounce homogenizer. the volume was brought upto 20 ml (on ice). to this, 100 μl of proteinase k was added and incubated for 30 min at 65c (20 mg/ml). potassium acetate (8m) was added immediately after taking out of 65c. the reaction mixture was 298 vishwakarma et al./ cloning and characterization of promoters of vascular specific genes in zebrafish immediately incubated on ice for 30 min. it was then centrifuged at 12,000 rpm for 10 min. the supernatant obtained after centrifugation was transferred into a new tube, to which 0.6x isopropanol (13 ml) was added and was incubated for 5 minutes at room temperature. this was then centrifuged at 15000 rpm for 10 min. the supernatant was removed and pellet was washed with 100% ethanol. the air dried pellet was resuspended in 100 μl elution buffer and incubated overnight at 4°c. the optical density of dna was quantified by spectrophotometer. the extracted genomic dna was verified by electrophoresis on 0.8% agarose 1x tae gel. isolation of putative promoters, cloning and sequencing: putative promoter regions of selected genes involved in rhogtpase pathway like pak2a, cdc42 and rac1 were amplified using the primer pairs as given in table 1. to amplify 800 bp putative promoter region of pak2a gene forward primer i.d.ssb_p1248 with bglii site and reverse primer i.d.ssb_p1249 with xbai site was used. to amplify 826 bp putative promoter region of cdc42 forward primer i.d.-ssb_p1244 with xbai site and reverse primer i.d.-ssb_p1246 with xbai site. 716 bp putative promoter region of rac1 gene was amplified using following primer pairs: forward primer i.d.ssb_p1275 with bglii site and reverse primer i.d.ssb_p1276 with xbai site (table 1). these putative promoter fragments were cloned in vector pss536 by ligating at the appropriate site. the ligated product was transformed in e. coli dh5α, clones were checked by pcr using vector back bone specific primers and insert specific primers. the positive clones were sent for sequencing to tcga (the centre for genomic application, delhi, india). expression analysis: 3 nl of the dna (vector construct + insert) sample were microinjected into the 1-2 celled fertilized eggs, which were incubated at 29°c in dishes containing low concentration of methylene blue solution. dechorionated embryos were euthanized using tricaine. the red fluorescent protein (rfp) in transgenic embryos were observed in vivo using fluorescence microscope (carl zeiss, germany). images were captured at 2-3 days post fertilization with a zeiss axiocam camera using the zeiss axiovision software (carl zeiss, germany). promoter of cmcl2 gene was used as positive control (huang et al., 2003). results putaive promoters isolation from zebrafish genomic dna: the isolated genomic dna from zebrafish embryos were checked on 0.8% tae gel, showed the presence of heavy band just near to wells, no rna contamination was found and 260/280 ratio was nearly 1.9 (fig. 1a). the zebrafish genomic dna was used as template to amplify putative promoters related to blood vasculature. the pak2a putative promoter amplification was observed just below 1 kb as expected (promoters of vascular specific genes). the rac1 and cdc42 gene putative promoter amplified fragments were also observed at the expected size (fig. 2). for validating a promoter fragment in zebrafish, the strategy shown in figure 3 was followed. cloning and sequencing of putative promoters: the amplified putative promoter region of three genes i.e. pak2a, rac1 and cdc42 were cloned in topo-ta table 1. list of primers. s. no primer name 5′to 3′ sequence 1 i.d.-ssb_p1210 aaaagatctatttagcggccggaattcgc 2 i.d.ssb_p951 aaaagatctgcaggtttaaacgaattcgc 3 i.d.-ssb_p1195 tccttctccttctccctcaatctc 4 i.d.-ssb_p1248 aaaagatctagtacctcgaggctgtgagatt 5 i.d.-ssb_p1249 aaatctagaaatgatgcaatccagcgccca 6 i.d.-ssb_p1244 aaatctagacttgaatatgcgcatcttct 7 i.d.-ssb_p1246 aaatctagacgttggtacatattcggagg 8 i.d.-ssb_p1275 aaaaagatctgagcaaacagttgaagcggt 9 i.d.-ssb_p1276 aaatctagaccgtagctgtatctgtgaa 299 int. j. aquat. biol. (2018) 6(6): 295-302 vector (invitrogen, usa) as per manufacturer’s instructions. after cloning, these constructs were sent for sequencing to tcga by using primers t7 and ssb_p1195. the sequencing results were analyzed using blast-n (www.blast.ncbi.nlm.nih.gov.in) and assembled using cap3 assembler software tool. the sequencing results, alignment and blast results are shown in supplementary file (figs. s1-s8). the alignment showed 97, 97 and 96% similarity for pak2a, rac1 and cdc42, respectively. the putative figure 1. (a) gel shows genomic dna isolated from zebrafish embryos, m is the ƛ hind iii marker, lane 1 and 2 is the genomic dna and (b) gel picture showing pcr amplification of pak2a. genomic dna was used as template dna. figure 2. gel picture showing pcr amplification of rac1 and cdc42 genomic dna was used as template dna. figure 3. basic strategy to clone the putative promoter region in tol2 plasmid. the topo-ta step was skipped for pak2a, rac1 and cdc42 putative promoter region. 300 vishwakarma et al./ cloning and characterization of promoters of vascular specific genes in zebrafish promoter regions were cloned in a promoter less vector pss536 for expression analysis. the constructs were named as tg(rac1:dsred), tg(pak2a:dsred) and tg(cdc42:dsred). promoter expression analysis in zebrafish through fluorescence microscopy: three constructs tg(rac1:dsred), tg(pak2a:dsred) and tg(cdc42: dsred) were injected in zebrafish embryos (2-4 celled stage). tg(cmlc2:dsred) was used as positive control (for heart specific expression analysis), rac1 putative promoter region was able to drive the expression of rfp in zebrafish embryos and it was observed throughout the body while pak2a, cdc42 putative promoters were not able drive any expression (fig. 4). based on the literature, the expected expression and observed pattern of rfp in zebrafish embryos has been shown in table 2. discussion rho gtpases are guanine nucleotide binding proteins that cycle from the active gtp bound state and inactive gdp-bound state (buchner et al., 2007). they are known to play key roles in the modulation of a wide range of cellular processes like proliferation, apoptosis, cell migration, membrane trafficking, cytoskeletal rearrangements etc. (vidal et al., 2003). there are 32 zebrafish rho genes representing one or more homologs of 17 of the 23 predicted rho genes in human. in zebrafish, most of the rho genes are expressed in wild type healthy adults, but only a subset is expressed early in zebrafish development and rac1 is one of them, this might represent that it plays most critical roles before and around epiboly gastrulation. it has been shown that rho genes are conserved from lower to higher vertebrates. in general, central coding exons are the most conserved region and the rac family members are amongst the most conserved genes between zebrafish and humans (other genes are rho and cdc42) (dumont et al., 2009). the overall protein similarity between human and zebrafish rac family members is 98.4% (liu et al., 2007; vidal et al., 2003). this degree of conservation indicates that their functions are important in all vertebrates. different rhogtpases are known to participate in normal animal development. however, it is also known that the deregulation of their activities contributes to the generation of different human pathologies, including cancer progression, neurodegenerative disorders and infectious diseases. the transgenic zebrafish tg(rac1:dsred) may help table 2. expected and observed expression of different constructs. construct expected expression observed tg(cmlc2:dsred) heart specific heart specific tg(pak2a:dsred) ubiquitous no expression tg(rac1:dsred) ubiquitous ubiquitous tg(cdc42:dsred) ubiquitous no observation figure 4. the heart specific expression for cmlc-2 and ubiquitous expression for the rac1 gene. 301 int. j. aquat. biol. (2018) 6(6): 295-302 us to study the importance of molecular rhogtpase signaling pathway and can also form the basis of research to treat some of the lethal human diseases like: cancer progression, neurodegenerative disorders etc. tg(cmlc2:dsred) construct was used as positive control, the cardiac myosin light chain (cmlc) has been identified as a major contractile component of cardiac and other striated muscles; cmlc-2 is a regulatory light chain and potential modulator of contractile activity in heart and skeletal muscle cells (rottbeaur et al., 2006). chen et al. (2008) used zebrafish to identify the cmlc2 as the main rlc (regulatory myosin light chain) orthologue. they further showed that in cmlc2 morphants the sarcomere length is shorter and mutation in the genes encoding for rlcs have been casually linked to ~1% human cardiomyopathies (hsu et al., 2004; huang et al., 2003). the zebrafish is a powerful vertebrate model used to dissect molecular pathways of cardiovascular development and disease. because fundamental electrical properties of the zebrafish heart are remarkably similar to those of the human heart, the zebrafish may be an appropriate model for studying human inherited arrhythmias. heart disease is currently one of the most common causes of human death. a large number of researchers are currently searching for a simple animal model to assist in finding either a cure for heart disease or a novel gene that regulates heart development (mingjun et al., 2008). zebrafish is viewed as having advantages for heart related research. heart specific transgenic zebrafish will be valuable as research model for tracing the development the for the fate of heart cells, finding new heart specific genes and functions, establishing biological indices of environmental pollutants, and study the efficacy of therapeutic drugs (leskow et al., 2006). references arnaout r., ferrer t., huisken j., spitzer k., stainier d.y.r., tristani-firouzi m., chi n.c. 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(1986). integrative transformation of c. elegans. the embo journal, 5: 2673-2680. flytzanis c.n., mcmahon a.p., hough-evans b.r., katula k.s., britten r.j., davidson e.h. (1985). persistence and integration of cloned dna in post-embryonic sea urchins. developmental biology, 108: 431-442. hsu k., traver d., kutok j.l., hagen a., liu t., paw b. h., rhodes j., berman j.n., zon l.i., kanki j.p., look a.t. (2004). the pu.1 promoter drives myeloid gene expression in zebrafish. blood, 104: 1291-1297. huang c.j., tu c., hsiao c., hsieh f., tsai h. (2003). germline transmission of a myocardium-specific gfp transgene reveals critical myosin light chain 2 promoter 302 vishwakarma et al./ cloning and characterization of promoters of vascular specific genes in zebrafish of zebrafish. developmental dynamics, 228: 30-40. jaenisch r. (1988). transgenic animals. science, 240: 1468-1474. kawakami k. 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(2008). isolation and characterization of the zebrafish danio rerio insulin-like growth factor binding protein-3 promoter region. fisheries science, 74: 153-166. park h., kim c., bae y., yeo s., kim s., hong s., shin j., yoo k., hibi m., hirano t., miki n., chitnis a.b., huh t. (2000). analysis of upstream elements in the huc promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. developmental biology, 227: 279-293. rottbauer w., wessels g., dahme t., just s., trano n., hassel d., burns c.g., katus h.a., fishman m.c. (2006). cardiac myosin light chain-2 a novel essential component of thick-myofilament assembly and contractility of the heart. circulation research, 99: 32333. rubin g.m. (1988). drosophila melanogaster as an experimental organism. science, 240: 1453-1459. stuart g.w., vielkind j.r., mcmurray j.v., westerfield m. 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(2020) 8(4): 272-280 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article prey identification of invasive peacock bass from telabak lake malaysia using dna barcoding technique aliyu garba khaleel1,2, najlaa nawwarah rusli1, nurul izzati mohd radzif1, aiman syafiq muhd nasir1, mohamad zulkarnain mohd dali1, norshida ismail1, hou chew ha1, ahmad-syazni kamarudin*1 1school of animal science, faculty of bioresources and food industry, universiti sultan zainal abidin, besut campus, 22200 besut, terengganu, malaysia. 2department of animal science, faculty of agriculture and agricultural technology, kano university of science and technology, wudil, p.m.b. 3244 kano state, nigeria. s article history: received 7 june 2020 accepted 21 august 2020 available online 2 5 august 2020 keywords: conservation feeding habit invasive species abstract: invasive peacock bass cichla spp. have recently invaded freshwater habitats across malaysia. stomach contents of 135 peacock bass captured from the telabak lake of east coast of peninsular malaysia were analysed. the preys were examined using visual identification method and mitochondrial dna barcoding technique to identify the partial digested and decaying preys in the stomach. the current study identified 7 prey species (6 fishes 43.0% and 1 shrimp 5.1%) belongs to 5 families in fishes’ stomach. the results revealed that peacock bass is highly predator and generalist feeder with an opportunistic feeding behaviour. it is highly important to reduce and monitor the abundance of this species for future survival of native species in the lake. introduction the existence of an invasive fish species especially in inland waters is regarded as a crucial challenge in the conservation of tropical fish biodiversity (clavero and garcia-berthou, 2005; agostinho et al., 2005; cucherousset and olden, 2011; matsuzaki et al., 2016). non-native fish species are intentionally or accidentally introduced to a new habitat by human activities (radkhah et al., 2016; mousavi-sabet and eagderi, 2016; eagderi et al., 2018). peacock bass cichla spp. are highly predatory fishes originated from the amazon and introduced to many countries (fugi et al., 2008; kovalenko et al., 2009; marques et al., 2016). these fishes were intentionally introduced into malaysian freshwater by anglers in early 1990s (rahim et al., 2013). since then, it spreads to many freshwater bodies such as temengor reservoir and lake, raban lake, kapal tujuh lake, kampar river (hamid and mansor, 2013; desa and aidi, 2013; saat et al., 2014; tan and sze, 2017; yap et al., 2016; ng et al., 2018). peacock bass exert high predation on prey fish population which may lead to the decreasing of the prey fish abundances and diversity in a *correspondence: ahmad-syazni kamarudin e-mail: ahmadsyazni@unisza.edu.my particular area (zaret and paine, 1973; santos et al., 2001; pelicice and agostinho, 2008; franco et al., 2017). these fishes are daytime active piscivorous that consume a wide range of prey and tend to ingest the whole prey (zhao et al., 2014). to date, there is no documentation regarding their prey species across malaysian freshwater bodies. thus, diet composition study of this invasive fishes is necessary for better understanding of their ecological impacts on native biodiversity (garvey and chipps, 2012). study on piscivorous fish diet composition is traditionally based on stomach contents analysis. visual identification methods have been widely used in taxonomic identification of fish diet content (morris and akins, 2009; layman and allgeier, 2012; côté et al., 2013). however, this method has failed to identify 70% prey content to the lowest taxonomic species level in the stomach content due to high digestion effect and prey degradation (morris and akins, 2009; côté et al., 2013). this weakness, especially at low sample sizes may bias the ecological impact predictions since the detected prey might not represent the unknown percentage (côté et al., 2013). 273 int. j. aquat. biol. (2020) 8(4): 272-280 therefore, dna barcoding technique is used for high taxonomic resolution of fish diet as a supplement to traditional method (leray et al., 2011). analysis of mitochondrial dna is proven to be a useful tool for the study of genetic diversity (ahmadsyazni et al., 2017; ha et al., 2017; khaleel et al., 2019) and species identification (li et al., 2019; golani et al., 2019). matching of a short dna sequences from unknown samples to known sequences in global databases such as national centre for biotechnology information (ncbi) and barcode of life database (bold) is known as barcoding (ratnasingham and hebert, 2007; moran et al., 2015). this approach has been used effectively to classify dietary components in fishes (côté et al., 2013; moran et al., 2015), small body sized larval fish (riemann et al., 2010), rare deep-water sharks (dunn et al., 2010), and coral reef fish with rich generalist diet (leray et al., 2011). telabak lake is a man-made freshwater lake which play a pivotal socio-economic and ecosystem role for the people living in the surrounding area (khaleel et al., 2020). freshwater lakes in malaysia are known for the vast diversity of the aquatic live and fishes (shahabudin and musa, 2018). however, the introduction of invasive species such as peacock bass which preying on native fishes might give a threatening effect on the fish diversity. in this regard, current study aimed to provide first information concerning the prey identification and feeding habit of peacock bass in telabak lake, malaysia using dna barcoding technique. materials and methods sampling: a total of 135 peacock bass samples with average total body length of 24±2.1 cm and body weight of 244±2.3 g were collected from the telabak lake (5°37'56.9"n, 102°28'24.5"e), east coast of peninsular malaysia from october 2018 to january 2019. the samples were immediately transferred to the aquatic laboratory, faculty of bioresources and food industry, university sultan zainal abidin malaysia for further analyses. taxonomic classification and feeding habit: fish were dissected to remove the stomach content based on barbato et al. (2019). following the protocol of côté et al. (2013) with some modification, all prey items in the stomach were identified to the minimum taxonomic level. the highly digested preys with difficulty to identify were classified as fish and invertebrates, labelled separately and frozen. the feeding regime of cichla spp. was measured in qualitative and quantitative methods based on hynes (1950) and sahtout et al. (2018). the following indices were used to evaluate the importance of different prey items in the diets of cichla spp. 𝑉𝐶 (%) = 𝑁𝑜. 𝑜𝑓 𝑒𝑚𝑝𝑡𝑦 𝑠𝑡𝑜𝑚𝑎𝑐ℎ × 100 𝑁𝑜. 𝑜𝑓 𝑓𝑢𝑙𝑙 𝑠𝑡𝑜𝑚𝑎𝑐ℎ𝑠 (peyami et al., 2018) 𝐹𝑂 (%) = 𝑁𝑜. 𝑜𝑓 𝑠𝑡𝑜𝑚𝑎𝑐ℎ 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑖𝑛𝑔 𝑝𝑟𝑒𝑦 × 100 𝑁𝑜. 𝑜𝑓 𝑓𝑢𝑙𝑙 𝑠𝑡𝑜𝑚𝑎𝑐ℎ𝑠 (ashelby et al., 2016) 𝑁𝐼 (%) = 𝑁𝑜. 𝑜𝑓 𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙 𝑝𝑟𝑒𝑦 𝑖𝑡𝑒𝑚𝑠 × 100 𝑇𝑜𝑡𝑎𝑙 𝑁𝑜. 𝑜𝑓 𝑝𝑟𝑒𝑦𝑠 (karimi et al., 2019) 𝑉𝐼 (%) = 𝑊𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑝𝑟𝑒𝑦 𝑖𝑡𝑒𝑚𝑠 × 100 𝑇𝑜𝑡𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑡𝑜𝑚𝑎𝑐ℎ 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (karimi et al., 2019) 𝐼𝑅𝐼 = (%𝑁 + %𝑉) × %𝐹 (barbato et al., 2019) where vc is vacuity coefficient, fo = frequency of occurrence, ni = number of individuals, vi = volume of individuals and iri = index of relative importance. barcoding sample preparations: a small piece of the muscle tissue (2-3 mm3) was used from every frozen prey item identified as fish and invertebrates, respectively. then, all samples were carefully taken from each prey (preferably from dorsal muscle). to minimize the sample contamination by peacock bass cells, approximately 1 mm top layer of the tissue muscle of the prey that has direct contact to stomach fluids were removed prior to sampling for barcoding. all tools were sterilized using 95% ethanol and bunsen burner flame between each sample removing to avoid any possible contamination. prey dna extraction, amplification and sequencing: the total genomic dna of each prey item was isolated using favorgen dna extraction kit (favorgen biotech corp., ping-tung 908, taiwan) by following manufacturer’s protocol. the partial coi gene of mitochondrial dna was amplified by pcr using the universal primers coi-fish2 f (5’tcgactaatca 274 khaleel et al./ invasive peacock bass prey identification in malaysia taaagatatcggcac3’) and coi-fish2 r (5’acttcagggtgaccgaagaatcagaa3’) (ward et al., 2005) for unidentified fish samples and lco1490: 5'-ggtcaacaaatcataaagatatt gg-3' and hco2198: 5'-taaacttcagggtgac caaaaaatca-3' (folmer et al., 1994) for unidentified invertebrates. for both fish and invertebrates preys, the pcr was carried out in a 25 μl reaction volume containing 18.2 μl sterile distilled water, 2.5 μl taq buffer, 2.0 μl dntp mix (2.5mm), 0.5 μl of each primer (10 μm), 0.3 μl of 5 unit/μl taq polymerase (takara) and 1 μl template dna (1-50 ng/μl) on a thermal cycler pcr machine veriti 96 well thermal cycler (applied biosystem, california, usa), under the following thermal cycling conditions. initial denaturation at 95°c for 5 min, 35 cycles including denaturation at 95°c for 30s, annealing at 50°c for 30s and elongation at 72°c for 10 min, followed by final extension for 10 min at 72°c and the pcr product was maintained at 4°c. sequencing was succeeded using bigdye terminator v3.1 cycle sequencing kit (applied biosystems) following manufacturer's instructions, performed on an abi prism 3730xl genetic analyser (applied biosystems). data analysis: unknown sequences from fish and invertebrates were aligned and edited using clustalw multiple sequence alignment program in mega 7 (kumar et al., 2016). dnasp software was used to determine the variable sites among the sequence (librado and rozas, 2009). to discover the taxonomy of each prey species, the obtained haplotypes were queried using basic local alignment search tool (blast) against national center for biotechnology (ncbi) nucleotide database. a top species match was identified with a sequence similarity of at least >94% to avoid false positives. number of observed and detected prey species in the stomach of peacock bass was computed in percentages using minitab 16 software. results feeding intensity of peacock bass: among 135 examined stomach contents monthly from october 2018 till january 2019, 70 were empty (average 51.8%) with high value in december (87%) and sudden decline in january (26%) (fig. 1). using visual identification method, the remaining prey samples were successfully identified as fish and invertebrates with their percentages (fig. 2b, c, respectively). table 1. monthly variations of peacock bass dietary items with respect to their percentage frequency of occurrence (%fo), percentage number of individual (%ni), percentage volume of individuals (%vi) and percentage index of relative importance (%iri). variables prey oct. nov. dec. jan. male female fo (%) fish 68.4 85.7 66.7 78.6 42.2 04.7 invertebrates 42.1 25.0 33.3 42.9 26.6 32.8 ni (%) fish 64.5 80.5 20.0 65.2 38.0 31.0 invertebrates 35.5 19.5 80.0 34.8 05.0 26.0 vi (%) fish 67.1 42.1 62.7 57.5 23.5 31.1 invertebrates 04.1 01.1 04.4 02.5 01.9 00.7 iri (%) fish 84.4 87.2 32.8 85.8 92.6 96.3 invertebrates 15.6 12.8 67.2 14.2 07.4 04.7 figure 1. monthly variations of vacuity index of peacock bass stomachs examined between october 2018 and january 2019. 275 int. j. aquat. biol. (2020) 8(4): 272-280 however, it failed to classify prey to their lowest taxonomy due to high ingestion effect and degradation. table 1 provides the overall results of the classification and diet composition on prey of the examined peacock bass between october 2018 and january 2019. the fish preys dominated the entire diet with the exception of december at which high number values of invertebrates were recorded. mitochondrial dna barcode: a total of 656 base pair (bp) of coi for fish species and 679 bp of coi for invertebrates were obtained after deletion of lowquality nucleotides at the 5’ and 3’ ends. six different sequences were obtained from coi of fishes (psc01, psc02, psc03, psc04, psc05, and psc06) and one sequence for invertebrate (psc07). after blasting, in ncbi, following our criterion of >94% sequence similarities, 7 prey species belong to five families were cichla ocellaris, pristolepis fasciata, parambassis ranga, rasbora trilineata, cyprinus carpio and cyclocheilichthys enoplos (table 2). the percentage of each prey species identified (% n0) using dna barcode were also recorded with high value of 15.2% in p. fasciata. discussions the introduction of peacock bass into telabak lake was accompanied by a sharp and gradual decline of small-sized fishes (personal communication). this study showed that the decrease in fish diversity might be associated with feeding habit of peacock bass in the lake, since the observed prey items in the peacock bass stomach confirm its piscivorous feeding habit on targeted native prey species. we used traditional visual identification method and further dna barcoding technique to identify its preys. the highest vacuity coefficient was observed in december indicating their breeding and spawning season (gomiero et al., 2009) which limits their hunting time. high vacuity coefficient during breeding season was also reported from other fish species such as diplodus vulgaris (pallaoro et al., 2006), caranx rhonchus (sley et al., 2008), pagellus erythrinus (šantić et al., 2011). table 2. blast sequence match showing percentage identity of prey in peacock bass using barcode. sequence family species accession no. % identity % n0 psc01 cichlidae cichla ocellaris ku878410 99.20 5.1 psc02 pristolepididae pristolepis fasciata mk049486 99.07 15.2 psc03 ambassidae parambassis ranga mk448145 94.87 10.1 psc04 cyprinidae rasbora trilineata ku569018 99.99 2.5 psc05 cyprinus carpio ln591958 94.03 2.5 psc06 cyclocheilichthys enoplos ku692459 99.68 7.6 psc07 palaemonidae macrobrachium lanchesteri kp759429 98.18 5.1 % n0 = species percentage number identified after barcoding figure 2. visual identification of 135 peacock bass stomach content from october 2018 to january 2019 captured in telabak lake, (a) 51.8% empty stomach, (b) 43.1% unidentified fish species and (c) 5.1% unidentified invertebrate species. 276 khaleel et al./ invasive peacock bass prey identification in malaysia the lowest vacuity coefficient observed in january (26.3%) revealed its feeding initiation after breeding period. full recovery to feeding activity helps fishes to compensate the energy used during breeding (derbal et al., 2007). fish dominates the entire diet of the peacock bass in all months, except in december where invertebrates (prawn) were dominated. macrobrachium lanchesteri (prawn) spawns throughout the year with a peak at november (phone et al., 2005). therefore, it could be more available in december for peacock bass. another explanation for high predation of peacock bass on fish prey species might be related to naturally clear transparency of telabak lake. it is reported that peacock bass thrive well in clear freshwater for excellent predation (kovalenko et al., 2010). visual identification has failed to identify the prey items to the lowest species level due to the degradation of essential features such as fin ray shape and body coloration. however, only 48.2% of the ingested prey into fish and invertebrates were visually distinguished. this percentage is closer to the results of other similar studies (côté et al., 2013; dahl et al., 2017). only few species were successfully identified to the lowest species taxonomic level using visual method similar to other works (morris and akins, 2009; côté et al., 2013; moran et al., 2015; mzaki et al., 2017; sahtout et al., 2018). all identified prey species were native to malaysia freshwater except for c. carpio and cichla spp. opportunistic feeding habit of cichla spp. is one of the serious aspects that helped them adapt to a new environmental condition. the presence of cichla spp. in the stomach as a prey item might be due to cannibalism, and as proof of its opportunistic feeding habit in nature. it was previously examined that cannibalism is more pronounced during the spawning periods with scarcity of alternative foods, like small indigenous species of fish (junior and gomiero, 2010). in addition, low rates of cannibalism observed in this study might be due to native prey abundance (carvalho et al., 2014). once cichla spp. is introduced into a lake, they prey on variety of available fish species, shrimps and cichlids (pereira et al., 2015; mendonça et al., 2018). all native species found in the stomachs are of least concern (iucn red list, 2012) but they contribute largely in aquaculture, and as a source of income for the local community e.g. m. lanchesteri is used as food by locals (phone et al., 2005; aznan et al., 2017). the studies on introduction peacock bass have indicated a negative effect on local fish species (zaret and paine, 1973; molina et al., 1996; pinto-coelho et al., 2008; pelicice and agostinho, 2008; rahim et al., 2013). previous works of the fish population in lake redonda of cuba from 1989 to 1990, documented that many local fish species have been extinct after the introduction of peacock bass (molina et al., 1996). recently, menezes et al. (2012) reported that the introduction of peacock bass in the coastal lakes of rio grande do norte brazil had reduced native fish abundantly with a negative impact on their diversity. the invasion and adaptation of peacock bass in telabak lake might likely lead to the reduction of native fish species. the existence of these highly adaptive and fast-growing piscivorous fish may cause severe damages to the local aquatic populations through competition, predation and cascade effects across the trophic chain. although peacock bass attracts recreational anglers (mendonça et al., 2018), but local people depends on native aquatic species in the telabak lake. the lake plays a significant role for their daily needs and incomes. as our finding, 48.2% of the prey items submitted for barcoding were 100% identified to species level. other studies identified less than 70% when submitted for barcoding (morris and akins, 2009; côté et al., 2013), which is due to species differences. without using dna barcoding technique, most of the prey items could have been labelled as partially digested unidentified prey, leading to missing information, misidentification and less understanding of invasive peacock bass impact in the lake. conclusion this study provided useful information about feeding habits of cichla spp. for better understanding of the relationship between fish species and other living organisms in telabak lake. the presence of this invasive species may affect the government effort on 277 int. j. aquat. biol. 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(2019) 7(4): 239-244 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article barbus urmianus a new species from urmia lake basin, iran (teleostei: cyprinidae) soheil eagderi*1, nasrin nikmehr1, erdogan çiçek2, hamid reza esmaeili3, saber vatandoust4, hamed mousavi-sabet5 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2department of biology, faculty of art and science, nevsehir hacı bektas veli university, nevsehir, turkey. 3ichthyology and molecular systematics research laboratory, zoology section, department of biology, college of sciences, shiraz university, shiraz, iran. 4department of fisheries, babol branch, islamic azad university, babol, iran. 5department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. article history: received 7 july 2019 accepted 21 august 2019 available online 2 5 august 2019 keywords: taxonomy, freshwater, middle east, urmia barb, orumiyeh, cypriniformes. zoobank: urn:lsid:zoobank.org:pub:e28a975c-2cac-4ab5-90b9-3cb0501d8a2f urn:lsid:zoobank.org:act:5e7fc771-15ee-4b4b-9d3a-05ea1bd26d47 abstract: barbus urmianus from the mahabad-chai river in urmia lake basin, is distinguished from its congeners in the b. lacerta group by a well-developed middle pad of the lower lip, a shorter postdorsal length (25.2–42.0 vs. 46.4–60.7% sl), long anal fin (11.0–23.0 vs. 6.0–10.4% sl), short dorsal-fin base (9.2–15.6 vs. 16.1–22.6% sl), less scales in the caudal peduncle (14–23 vs. 25–35), and 64–85 scales on the lateral line (vs. 52–70). introduction the barbus populations of the southern caspian sea, lake namak, and urmia (orumiyeh) lake basins, euphrates and tigris drainages, and the qweik endorheic basin in syria have been previously considered as b. lacerta heckel, 1843 (berg, 1949; coad, 1995; khaefi et al., 2017a; çiçek et al., 2018). naseka and bogutskaya (2009) recognized b. cyri de filippi, 1865 as a valid species in the caspian sea basin. two other species, viz. b. miliaris de filippi, 1863, a nominal species found in the lake namak basin and b. karunensis, khaefi, esmaeili, geiger & eagderi, 2017 from the karun river drainage are found in the iranian inland waters (radkhah and eagderi, 2015; jalili et al., 2015; ghasemi et al., 2015; khaefi et al., 2017a, b; esmaeili et al., 2018). barbus cyri is also known from the urmia lake basin (khaefi et al., 2017b). a recent study on the ichthyofauna of the urmia lake basin revealed that the barbus population from the mahabad-chai river represents an undescribed species. hence herein, we describe it as barbus urmianus sp. nov. *correspondence: soheil.eagderi@ut.ac.ir doi: https://doi.org/10.22034/ijab.v7i4.725 e-mail: soheil.eagderi@ut.ac.ir materials and methods after anesthesia, the collected fishes were fixed in 10% buffered formaldehyde. measurements were made with a dial caliper and recorded to the nearest 0.1 mm. all measurements were made point to point never by projections. methods for counts and measurements follow kottelat and freyhof (2007). standard length (sl) is measured from the tip of the snout to the end of the hypural complex. the length of the caudal peduncle is measured from behind the base of the last anal-fin ray to the end of the hypural complex, at mid-height of the caudal-fin base. the width of the upper lip is measured ventrally at the anterior tip of the lip. the scales in the lateral line are counted as total scales from the first scale on flank to the last scale on the caudal-fin base. the last two branched rays articulating on a single pterygiophore in the dorsal and anal fins are counted as "1½". abbreviations used: sl, standard length; hl, lateral head length; imnrf-ut, ichthyological museum of natural resources faculty, university of tehran; zmcbsu, zoological museum of shiraz university, 240 eagderi et al./ barbus urmianus (teleostei: cyprinidae) a new species from iran collection of biology department, shiraz; vmfc, vatandoust and mousavi-sabet fish collection, tehran. results barbus urmianus, new species (figs. 1-4, table 1) holotype: imnrf-ut-1079-8, 117.7 mm sl; iran: western azerbaijan prov.: mahabad-chai river at miriseh village, beytas city, 36°29'55.14"n 45°33'54.26"e. paratypes: imnrf-ut-1079-1-15, 14, 101.1–187.6 mm sl; same data as holotype. vmfc b1388, 30, 95.0-184.2 mm sl; same data as holotype. diagnosis: barbus urmianus sp. nov. is distinguished from the other species of the b. lacerta group in iran by having short postdorsal length (25.2–42.0% sl vs. 50.5–53.2% sl in b. karunensis; 52.3–58.7% sl in b. miliaris; 46.4–59.1% sl in b. lacerta; and 47.8– 60.7% sl in b. cyri), short dorsal-fin base (9.2–15.6% sl vs. 16.8–21.8% sl in b. karunensis; 18.4–22.6% sl in b. miliaris; 16.2-21.8% sl in b. lacerta; and 16.1–21.2% sl in b. cyri), long anal fin (11.0–23.0% sl vs. 7.1-8.6% sl in b. karunensis; 6.8–9.1% sl figure 1. live specimen of barbus urmianus sp. nov., vmfc b1388, paratype, 130.1 mm sl, iran: western azerbaijan prov.: mahabad-chai river at miriseh village, beytas town, urmia lake basin. figure 2. barbus urmianus sp. nov., imnrf-ut-1079-8, holotype, 117.7 mm sl, iran: western azerbaijan prov.: mahabad-chai river at miriseh village, beytas town, urmia lake basin. 241 int. j. aquat. biol. (2019) 7(4): 239-244 in b. miliaris; 6.0–9.1% sl in b. lacerta; and 6.1– 10.4% sl in b. cyri), and less scale in caudal peduncle (14–23 vs. 26–29 in b. karunensis; 28–35 in b. miliaris; 25–32 in b. lacerta; and 28–33 in b. cyri). it is also discriminated from the other species of the b. lacerta group except b. karunensis by having a well-developed middle pad of lower lip (vs. poorly developed or absent). furthermore, b. urmianus is distinguished from b. karunensis by having 64–85 (mean 79.6) scales in lateral line (vs. 60–70 (63.1)), more scales below lateral line (12–15 vs. 9–11), less scales of predorsal (25–31 vs. 33–42), maxillary table 1. morphometric and meristic data of barbus urmianus sp. nov. (imnrf-ut-1079-8, holotype; imnrf-ut-1079-1-15, 15 paratypes, iran: western azerbaijan prov.: miriseh village at beytas town, mahabad-chai river, urmia lake basin). barbus urmianus sp. nov. holotype paratypes min max mean sd total length (mm) 121.7 101.3 187.6 135.5 23.9 in percent of standard length head length 22.0 16.9 27.6 24.5 2.4 pre orbital distance 8.8 6.7 12.8 10.6 1.4 post orbital distance 9.7 7.5 12.7 11.3 1.2 inter orbital distance 3.5 2.7 5.8 3.9 0.7 predorsal length 46.0 35.4 57.4 51.0 4.9 postdorsal length 32.8 25.2 42.0 35.6 4.7 base of dorsal-fin length 11.9 9.2 15.6 13.3 1.6 dorsal-fin length 18.2 14.0 21.8 18.0 2.2 base of anal-fin length 6.7 5.2 12.1 8.3 1.6 anal-fin length 14.4 11.0 23.0 17.7 3.2 preanal length 64.1 49.3 76.1 69.5 8.6 pectoral-fin length 15.2 11.7 19.7 17.1 2.0 pelvic-fin length 14.5 11.2 17.7 14.9 1.4 minimum body depth 8.8 6.8 20.5 10.7 3.0 maximum body depth 16.9 13.0 22.2 18.9 2.1 distance between pectoral and anal-fin 42.3 32.5 54.6 49.3 5.1 distance between pectoral and pelvic-fin 23.9 18.4 30.3 26.9 2.9 distance between pelvic and anal-fin 22.3 17.1 26.9 23.4 2.4 rostral barbel 3.1 2.4 5.8 4.1 0.8 maxillary barbel 5.3 4.1 7.6 6.0 0.9 in percent of head length head depth 58.3 51.5 63.7 57.0 3.4 pre orbital distance 43.6 38.5 49.4 43.9 2.8 post orbital distance 48.6 41.8 49.6 45.2 2.2 inter orbital width 40.5 34.2 65.9 42.1 7.2 eye diameter 13.5 11.4 17.1 13.8 1.5 maximum body depth 76.8 59.4 87.3 77.3 6.1 length of caudal fin 74.1 71.1 100.9 80.8 8.6 mouth width 51.7 27.2 51.7 32.1 7.2 meristic data dorsal fin unbranched rays 3 2 4 2.9 0.6 dorsal fin branched rays 7 7½ 10½ 7.8 0.9 pectoral fin rays 15 12 16 14.9 1.0 pelvic fin rays 9 6 10 8.3 1.2 anal fin unbranched rays 2 2 3 2.1 0.3 anal fin branched rays 7 5½ 7½ 5.5 0.8 lateral line scales 79 64 85 79.6 6.3 caudal peduncle scales 19 14 23 19.5 2.4 scales above lateral line 15 15 18 16.1 1.0 scales below lateral line 12 12 15 13.2 1.2 predorsal scales 26 25 31 27.5 1.9 gill rakers 7 6 9 7.2 0.9 242 eagderi et al./ barbus urmianus (teleostei: cyprinidae) a new species from iran barbels reaching beyond middle of eye (vs. not reaching), tip of anal fin reaching beyond middle of distance between base of last anal-fin ray and lower caudal-fin origin when pressed to body (vs. not reaching). barbus urmianus sp. nov. is also distinguished from b. cyri by having 64-85 (mean 79.6) scales in lateral line (vs. 52–69 (59.4)) and more scales below lateral line (12–15 vs. 9–13). in addition, b. urmianus sp. nov. is distinguished from b. lacerta by having 64–85 (mean 79.6) scales on lateral line (vs. 56–7 (62.9)) and less scales of predorsal (25–31 vs. 35–48). barbus urmianus sp. nov. is also distinguished from b. miliaris by having less scales of predorsal (25–31 vs. 37–45) and having wider mouth (17.2–51.7 vs. 12.9– 27.2% hl). description: for general appearance see figures 1–3; mouth structure figure 4 and morphometric and meristic data are provided in table 1. body elongate and cylindrical, greatest body depth somewhat before dorsal-fin origin decreasing towards middle of caudal figure 3. barbus urmianus sp. nov., imnrf-ut-1079-3, 5, 11, paratypes, a. 124.0 mm sl, b. 138.2 mm sl, c. 113.4 mm sl. iran: western azerbaijan prov.: mahabad-chai at miriseh village, beytas town, urmia lake basin. figure 4. ventral side of head of barbus urmianus sp. nov. 243 int. j. aquat. biol. (2019) 7(4): 239-244 peduncle, predorsal body profile convex, ventral profile slightly convex. head deep tapering towards rounded, blunt snout. dorsal profile of head slightly convex, with no marked hump between head and body. caudal peduncle 1.7–2.9 times longer than deep. triangular and pointed axillary scale at pelvicfin base. pelvic-fin origin below vertical of last unbranched dorsal-fin ray. caudal fin forked. posterior dorsaland anal-fin margins straight or slightly concave. tip of anal fin, when pressed to body, passing middle of caudal peduncle. pectoral fin reaching approximately 55–75% distance from pectoral-fin origin to pelvic-fin origin. pelvic fin not reaching anus. snout 48–72% of body depth at dorsalfin origin. width of upper lip 4.5–7.5% hl. lower lip thicker than upper lip, with a well-developed median pad (fig. 4). rostral barbel short, not reaching nostril; maxillary barbel 27–45% hl, reaching beyond the middle of the eye. largest known individual 187 mm sl. dorsal fin with 2–4 (mode 3) unbranched rays and 7½–10½ branched rays, 50–65% of posterior margin of last unbranched dorsal-fin ray covered with denticles. anal fin with 2–3 (mode 2) unbranched and 5½–7½ branched rays. pectoral fin with 12–16 (mode 15) rays. pelvic fin with 6–10 (mode 9) rays. lateral line with 64–85 (mean 79.6) scales. scale rows between dorsal-fin origin and lateral line 15–18 (mode 16). scale rows between pelvic-fin origin and lateral line 12–15 (mode 12). caudal peduncle scale 15–23 (mode 21). predorsal scales 25–31 (mode 28) and 6–9 (mode 7) gill rakers on first gill arch. pharyngeal teeth 1,4-4,1. coloration: in live specimens: body and head yellow to brown, flanks brown, lighter below lateral line; numerous small, irregular dark-brown spots and small blotches on dorsum and flanks, lower number of irregular dark-brown spots in fins; dorsal, pectoral and ventral fins brownish with fade orange colour anteriorly, caudal and anal fins brownish; belly yellowish white; barbell yellowish. in preservation: body and head brown with flanks darker above lateral line; belly light brown without spots; dark brown irregular spots and small botches scattered on entire body; fins cream to yellowish; barbels light brown. distribution: barbus urmianus is known from the mahabad-chai river, urmia lake basin, iran (fig. 5). etymology: the species is named for the urmia lake basin where the type materials were collected. figure 5. mahabad-chai river at miriseh village, urmia lake basin, type locality of barbus urmianus sp. nov.. 244 eagderi et al./ barbus urmianus (teleostei: cyprinidae) a new species from iran material examined: all from iran. barbus miliaris: imnrf-ut-1082, 2, 110.8–119.7 mm sl; iran: tehran prov.: nem river at harandeh village, dashtee kavir basin, 35°43´20.9˝n 52°39´18.4˝e. — zmcbsu g1101, 24, 70–97 mm sl; markazi prov.: qara chai (gharehchai) river, at jalayer, 34°53'13.9"n 50°02'10.9"e. barbus karunensis: zm-cbsu g1038, 12, 47.0– 121.0 mm sl; iran: kohgiluyeh and boyer-ahmad prov.: bashar river at talegah village 10 km north of yasuj city, 30°47'27.5"n 51°25'13.3"e. barbus lacerta: imnrf-ut-1080, 10, 67.7–97.8 mm sl; iran: kurdistan prov.: sefidbarg (leyleh) river sefid-barg village, tigris river drainage, 34°51´75.7˝n 46°20´17.6˝e. — zm-cbsu d111, 8, 57–139 mm sl; iran: lorestan prov.: karkheh river at kashkanrud 25 km west of khoramabad, 33°35'14"n 47°52'55"e. — zm-cbsu g964, 4, 104– 136 mm sl. iran: kermanshah prov.: leyleh river at shervineh west of javanrud, 34°52'29"n 46°21'06"e. barbus cyri: mnrf-ut-1081, 15, 44.0–94.5 mm sl; iran: guilan prov.: sefid river at, rostam-abad, caspian sea basin. — zm-cbsu g1125, 24, 69–127 mm sl. iran: mazandaran prov.: tajan river at sari, 36°12'13.8"n 53°05'10.7"e. acknowledgments we are pleased to thank university of tehran for financial support. references berg l.s. 1949. freshwater fishes of iran and adjacent countries. trudy zoologicheskogo instituta akademii nauk sssr, 8: 783-858. çiçek e., fricke r., sungur s., eagderi s. 2018. endemic freshwater fishes of turkey. fishtaxa, 3(4): 1-39. coad b.w. 1995. freshwater fishes of iran. acta scientiarum naturalium academiae scientiarum bohemicae, brno, 29(1): 1-64. esmaeili h.r., sayyadzadeh g., eagderi s., abbasi k. 2018. checklist of freshwater fishes of iran. fishtaxa, 3(3): 1-95. ghasemi h., jouladeh roudbar a., eagderi s., abbasi k., vatandoust s., esmaeili h.r. 2015. ichthyofauna of urmia basin: taxonomic diversity, distribution and conservation. iranian journal of ichthyology, 2(3): 177193. jalili p., eagderi s., nikmehr n., keivany y. 2015. descriptive osteology of barbus cyri (teleostei: cyprinidae) from southern caspian sea basin. iranian journal of ichthyology, 2(2): 105-112. khaefi r., esmaeili h.r., geiger m.f., eagderi s. 2017a. taxonomic review of the cryptic barbus lacerta species group with description of a new species (teleostei: cyprinidae). fishtaxa, 2(2): 90-115. khaefi r., vatandoust s., esmaeili h.r. 2017b. redescription of barbus miliaris de filippi, 1863 (teleostei: cyprinidae) from the endorheic namak lake basin of iran. fishtaxa, 2(1): 33-42. kottelat m., freyhof j. 2007. handbook of european freshwater fishes. kottelat, cornol and freyhof, berlin. 646 p. naseka a.m., bogutskaya n.g. 2009. fishes of the caspian sea: zoogeography and updated check-list. zoosystematica rossica, 18(2): 295-317. radkhah a., eagderi s. 2015. length-weight and lengthlength relationships and condition factor of six cyprinid fish species of zarrineh river (urmia lake basin, iran). iranian journal of ichthyology, 2(1): 61-64. int. j. aquat. biol. (2022) 10(3): 218-223 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article toxicity of fipronil and atrazine on metapenaeus affinis (milne-edwards, 1837) and their effects on oxygen consumption rajaa n. al-yassein department of fisheries and marine resources, college of agriculture, university of basrah, iraq. s article history: received 7 april 2022 accepted 2 may 2022 available online 2 5 june 2022 keywords: pesticides toxicity sublethal concentrations oxygen consumption biodiversity al-azim marsh abstract: increasing the use of pesticides has led to declines in crustaceans in aquatic systems. in this study, the effects of acute doses of fipronil insecticides and atrazine herbicides on the toxicity in sublethal concentrations and their effect on oxygen consumption of adult metapenaeus affinis at 20±1°c and 4 psu salinity were investigated. series of fipronil (0.05-2 µg.l-1) and atrazine (625-2000 µg.l-1) lethal concentrations were used. fipronil showed high toxicity to adult shrimp m. affinis compared to the atrazine. the median lethal concentration (lc50) for 96h of fipronil and atrazine were 0.47 and 8280.02 µg.l-1, respectively. a decrease in the rate of oxygen consumption with increasing sublethal concentrations after 24h was observed in fipronil and atrazine exposed shrimps. a significant difference in oxygen consumption was found between the control and the experimental treatments. the oxygen consumption of high concentration of fipronil 0.2 µg.l-1and high concentration of atrazine 6000 µg.l-1were 0.190 and 0.373 µg.l-1/o2/gm/h, respectively, compared to control one (0.540 µg.l-1/o2/gm/h). introduction human activities such as agricultural operations, industrial effluents, and increased urbanization posed a serious threat to the freshwater ecosystem (meijide et al., 2018). pesticides are extensively used to control and decrease unwanted plants and animals (choung et al., 2013). they can enter aquatic environments and cause negative effects on non-target species (bradley et al., 2017). fipronil is a phenylpyrazole insecticide used to control insect pests in agricultural and residential environments (gunasekara et al., 2007). the mode of action in fipronil is different from other insecticides like organophosphates, carbamates, and several pyrethroids, all traditional insecticides, to which many insects have evolved resistance. (cole et al., 1993). fipronil has adverse effects on aquatic organisms with high acute and chronic toxicity and the ability to accumulate (ngim and crosby, 2001). its environmental fate is unusual due to desulfinyl derivatives, which are two times more toxic to aquatic invertebrates (schlenk et al., 2001). fipronil has a low correspondence: rajaa n. al-yassein doi: http//doi.org/10.22034/ijab.v10i3.1589 e-mail: rajaa.alyassein@uobasrah.edu.iq to moderate water solubility, favors lipophilic (organic) matrices, and is stable at ambient temperatures (aajoud et al., 2003). atrazine is a common triazine herbicide used worldwide, mostly in developing countries (roustan et al., 2014). although atrazine has been banned for many years, it remains one of the most frequent herbicides with extreme persistence in water bodies (jablonowski et al., 2011). therefore, it has become one of the main concerns of aquatic life (solomon et al., 2008). due to high levels in soil and widespread application, atrazine has been found in various environmental samples, like ground and surface water, at levels substantially over the legal limits (tappe et al., 2002). therefore, it became one of the main concerns for aquatic life (solomon al., 2008). in most environments, atrazine concentrations have been recorded as one µg.l-1 or lower (de albuquerque et al., 2020). crustaceans serve an important role in the coastal ecosystem, having high economic importance 218 al-yassein / toxicity of fipronil and atrazine on metapenaeus affinis (mehanna et al., 2012). metapenaeus affinis inhabits the northern part of the persian gulf and migrates up the estuary of shatt al-arab to the iraqi inland waters (salman et al., 1990). it is the most abundant commercial shrimp on the iraqi coast (saoud et al., 1993). shrimp are commonly used in toxicity experiments to assess the potential hazards of poisonous chemicals on different aquatic organisms (key et al., 2003). therefore, this work aimed to study the toxicity of two pesticides of, fipronil and atrazine, as sublethal concentrations and their effect on oxygen consumption in m. affinis. material and methods collection and maintenance: adult m. affinis was collected from aquariums in the marine science station, university of basrah, iraq. under laboratory settings of 20±1°c and 4 psu salinity, the shrimps were acclimated in 10-gallon aquaria using a 14h light: 12h dark photoperiod. the weight of the specimens was 7.8-10.5 g, with a mean length of 5.48.2 cm. the shrimps were acclimated for ten days before experiments. throughout the acclimation period, water quality parameters such as dissolved oxygen (oxygen meter do-5509), salinity (american optics refractometer), temperature, and ph) were monitored daily. shrimp were fed daily during the acclimation period, and their feeding was stopped during acute toxicity tests (buikema et al., 1980). stock solution preparation: one gram of highly purified fipronil (98% purity) was dissolved in 1000 ml of pesticide grade acetone 98.0%. a similar procedure was used for atrazine. a diluted daily stock in deionized water was prepared for toxicity tests. acute toxicity tests: the dilute fipronil daily stock solution was used to prepare fipronil exposure concentrations of 2, 1, 0.40, 0.20, 0.10 and 0.05 µg.l1and atrazine exposure concentrations of 20000, 10000, 5000, 2500, 1250 and 625 µg.l-1. each beaker (1l) was wrapped in acetone-rinsed tin foil to eliminate any contamination and reduce evaporation before use (konwick et al., 2005). each beaker was stocked with five shrimps and each treatment with three replicates of fipronil and atrazine concentration and controls without any pesticide. water changes were done every 24 hours. toxicity studies were performed at regular conditions of 20±1°c, 4 psu salinity, ph=7.5-7.8, and do >7.2 mg.l-1. cumulative mortality of the animals was recorded for each dose/replicate. oxygen consumption measurement: adult m. affinis were exposed to fipronil or atrazine and their oxygen consumption in treatments and control groups was measured after 24h. shrimps were placed in an oxygen consumption detection flask filled with the test concentrations of pesticide. at intervals of 30, 60, 90, 120, and 180 minutes, the oxygen consumption rate was determined as mgo2/g/h based on chinni et al. (2000). statistical analysis: the probit method was used to determine the lc50 values with 95% confidence limits (ci) and the used concentrations to estimate the 2496h lc50 concentrations (razzaghi, 2013). also, data were analyzed using spss (22.0 version) for mean and standard deviation and evaluating the regression coefficient at a 5% level of significance. results in acute toxicity tests, increasing concentrations of fipronil and atrazine showed rising mortality in adult m. affinis. at 96h of exposure to fipronil, the mortality of 0.05, 0.10, 0.20, 0.40, 1 and 2 µg.l-1 treatments were 10, 20, 40, 70, 100, and 100%, respectively (fig. 1). in the atrazine treatments of 625, 1250, 2500, 500, 10000 and 20000 µg.l-1, the mortality were 5, 10, 20, 45, 70, and 100%, respectively (fig. 2). based on the results, fipronil was more toxic than atrazine. the lc50 values of fipronil at 24, 48, 72, and 96 h were calculated as 0.96, 0.68, 0.47 and 0.47 µg.l-1, respectively (table 1). atrazine lc50 were 20432.31, 12913.22, 8280.02 and 8280.02 µg.l-1, in the 0.05, 0.10, 0.20, 0.40, 1 and 2 µg.l-1 treatments, respectively (table 2). a significant reduction in oxygen ratio consumption was observed in exposure shrimps to sublethal concentrations of fipronil and atrazine. there was a significant difference in oxygen consumption (p<0.01) in m. affinis at all treatments. 219 int. j. aquat. biol. (2022) 10(3): 218-223 oxygen consumption rates in the shrimps with sublethal treatments of fipronil were 0.330, 0.250 and 0.190 µg.l-1 /o2/gm/h, respectively, and in the sublethal atrazine treatments were 0.445, 0.441 and 0.373 µg.l-1/o2/gm/h (figs. 3, 4). that of the control group was 0.540 µg.l-1/o2/gm/h. discussion the mortality ratio of m. affinis exposed to various concentrations of fipronil and atrazine raised as concentration increased. based on the results, fipronil was more toxic to the m. affinis than atrazine in 96h lc50. each toxic chemical has a different effect related to specific mechanisms of its action (barbieri et al., 2013). the toxicity of fipronil to the adult grass shrimp, palaemonetes pugio was 96h lc50 (key et al., 2003). after 72 hours of exposure, fipronil was found highly toxic to daphnia magna at concentrations of 0.1, 1, 10, and 100 µg.l-1 (bownik and szabelak, 2021). chandler et al. (2004) found that fipronil is highly toxic to the adult copepod amphiascus tenuiremis with lc50 of 6.8 mg.l -1 after 96 h. a decrease in survival of shrimp, penaeus monodon by 24h mortality exposed to fipronil has also been reported (hook et al., 2018). montagna and collins (2008) found that mortality increased with increasing figure 2. adult metapenaeus affinis mortality in each atrazine concentration (µg.l-1) after 2496h of exposure. figure 1. adult metapenaeus affinis mortality in each fipronil concentration (µg.l-1) after 2496h of exposure. 220 al-yassein / toxicity of fipronil and atrazine on metapenaeus affinis concentration of organophosphate chlorpyrifos and endosulfan insecticides in freshwater crab, trichodactylus borellianus in 24h of exposure. atrazine was less toxic to the m. affinis as reported by key et al. (2007) when exposed to p. pugio, with an lc50 96h of 9000 µg.l -1. atrazine has been recorded to be moderate to severely toxic to aquatic species e.g. its toxicity values for atrazine in freshwater shrimp, paratya australiensis with 96h lc50 value was 6500 to 9900 µg.l-1 (phyu et al., 2005). however, values of lc50 have been reported at 6100 and 4900 µg.l -1, respectively for mussels perna viridis and paphia malabarica (iqbal and navalgund, 2021). atrazine was reported to be less toxic compared to three other insecticides (carbofuran, dichlorvos, and malathion) in copepodids, tigriopus brevicornis with lc50 value of 153.2 μg/ l−1 (forget et al., 1998). there was no mortality in the marine copepod t. japonicus in response to 20000 µg. l-1 of atrazine (yoon et al., 2019). measurement of oxygen consumption is an important indicator of sublethal stress levels for predicting chemical substance risks (ansari et al., 2010). according to barbieri et al. (2013), the presence of pollutants in the ecosystem is the main cause of a decline in the regular amount of oxygen and impairment of the crustacean respiratory system. in many crustaceans, oxygen consumption is reduced because of a greater number of pesticides being figure 3. oxygen consumption rate in metapenaeus affinis at different concentrations of fipronil. time (h) lc50 (µg.l -1) std error 95% confidence limits 24 0.96 1.09 6.042-10.315 48 0.68 0.272 3.016-4.038 72 0.47 1.76 2.199-2.88 96 0.47 1.76 2.199-2.88 table 1. logarithm of fipronil lc50 (µg.l-1) values, standard error and 95%confidence limits (cl) to the adult metapenaeus affinis at 24, 48, 72 and 96h of exposure. time (h) lc50 (µg.l -1) std error 95% confidence limits 24 20432.31 0.581 6.085-5.361 48 12913.22 0.425 3.772-5.437 72 8280.02 0.157 2.008-2.662 96 8280.02 0.157 2.008-2.162 table 2. logarithm of atrazine lc50 (µg.l-1) values, standard error and 95%confidence limits (cl) to the adult metapenaeus affinis at 24, 48, 72 and 96h of exposure. 221 int. j. aquat. biol. (2022) 10(3): 218-223 absorbed through the gills, which are vulnerable and more susceptible to damage by pollutants (montagna and collins, 2008). the current study revealed that increasing the concentration and exposure time of fipronil and atrazine decreases the amount of oxygen consumption. oxygen consumption in the highest concentrations of fipronil and atrazine (2 µg.l-1; 95% ci, 0.1875-0.1925 µg.l-1 and 6000 µg.l-1; 95% ci, 0.3705-0.3755) were 0.190 and 0.373 µg.l-1/o2/gm/h, respectively. dillon (1983) recorded a decline in the rate of oxygen consumption in p. pugio exposed to dimethyl-naphthalene concentrations of 0.24 µg.l-1 at 18-22°c. a decrease in oxygen consumption has also been reported in t. borellianus exposed to chlorpyrifos and endosulfan (montagna and collins, 2008). in contrast, there was an increase in the respiratory rate of m. affinis, up to 44.4% when exposed to different concentrations of naphthalene (ansari et al., 2010). the reduction in the oxygen consumption rate of post-larvae penaeus indicus after 24h at 29±1°c during exposure to different concentrations of lead has been reported by chinni et al. (2000). in another study, inhibition in the oxygen consumption rate of 52.63% was proved in penaeid shrimps, xiphopenaeus kroyeri at 20°c when exposed to cadmium and zinc (barbieri et al., 2013). conclusion the results indicated that mortality increases with increasing fipronil and atrazine concentrations. fipronil was more toxic to adult shrimp m. affinis pesticides cons (µg.l-1) mean±std 95% confidence limits (cl) f-value fipronil control 0.5400±0.001 (0.5375-0.5425) 70075.000 0.6 0.3300±0.001 (0.3275-0.3325) 0.12 0.2500±0.001 (0.2475-0.2525) 0.25 0.1900±0.001 (0.1875-0.1925) atrazine control 0.5400±0.001 (0.5375-0.5425) 15446.750 1500 0.4500 ±0.001 (0.4475-0.4525) 3000 0.4100±0.001 (0.4075-0.4125) 6000 0.3730±0.001 (0.3705-0.3755) table 3. measured concentrations (mean±standard error), confidence limits (cl) with fvalue of fipronil and atrazine in sublethal test concentrations solutions of m. affinis. figure 4. oxygen consumption rate in metapenaeus affinis at different concentrations of atrazine. 222 al-yassein / toxicity of fipronil and atrazine on metapenaeus affinis compared to atrazine, with lc50 96h of fipronil and atrazine were 0.47 and 8280.02 µg. l-1, respectively. oxygen consumption rate decrease with increasing sublethal concentrations in shrimp exposed to fipronil or atrazine. acknowledgments the author is grateful to a. aldubakel for his help with the data analysis. references aajoud a., ravanel p., tissut m. 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(2022) 10(2): 169-180 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article impact succession of drought and flood on diversity indices, abundance, and size range of fish assemblage in al-shafi marsh, southern iraq abdul hussein j. abdullah1, sajad a. abdullah2, yasser w. ouda2 1department of marine vertebrate, marine science center, university of basrah, iraq. 2department of biology, college of education-qurna, university of basrah, iraq. s article history: received 16 january 2022 accepted 21 april 2022 available online 2 5 april 2022 keywords: drought flood abundance fish assemblage exotic abstract: this study was conducted in the al-shafi marsh, north of basrah city to investigate the effects of succession drought and flooding on the abundance and size spectrum of fish assemblage. three stations were selected and samples were collected on a monthly basis using various fishing gears such as electrofishing, gill nets, and cast nets. the temperature of the water was 12.50-34.53⁰c, and the salinity fluctuated from 1.64 psu in may to 4.03 psu in october. the ph ranged from 7.38 in september to 8.17 in march. the water depth in the low flat areas ranged from 33 cm in october to 71 cm in may, while the average depth in the seasonal flat areas ranged from zero in june, july, august, september, and october to 34cm in april. a total of 19 fish species were collected, representing 17 genera and 11 families. eight fish species were native, eight exotics, and two marines. cyprinidae was the most abundant family, with four species. the most abundant species were the abu mullet, planiliza abu, prussian carp, carassius gibelio, and blue tilapia, orechromis aureus (28.42, 20.97, and 14.90%, respectively). the d3 dominance index was 64.29%. the length groups of the most important sixth commercial fish species ranged from 5 cm in p. abu to 31 cm in leuciscus vorax. the diversity index ranged between 1.84 and 2.25, the evenness index 0.74 and 0.88, and the richness index between 1.63 and 2.37. the resident species accounted for 94.82% of the total catch. seasonal species account 2.76% of all samples. the occasional fish species account for 2.42% of total collected fishes. based on the results, there was insufficient flood pulsation to stimulate native species to reproduce, combined with overfishing, which resulted in a depletion of the species' biomass, and an increase in the abundance of small medium-sized fish species and small invasive species. introduction the marshes are unique environments characterized by high diversity and productivity; they contain various living organisms, whether plants or animals (al-zaidy et al., 2019; elsey-quirk et al., 2019; zou et al., 2021). they serve important functions for humans, including social and economic value, biodiversity resources, suspended matter filtering and sedimentation, pollution capture, and tourist and recreational opportunities (xu et al., 2019; zhang et al., 2020; vasquez et al., 2022). in marsh habitats, biogeochemical processes benefit the ecological system and species’ life cycles by sustaining and organizing correspondence: abdul hussein j. abdullah e-mail: abdulhassain.abdulah@uobasrah.edu.iq them (hammer, 2020). they also play a vital role in maintaining the balance of hydrological processes by serving as storage areas during the rainy season, flood control, erosion protection, water supply (moor et al., 2017), and economical source for food production (abdullah, 2019). marshes are stable habitats providing enough food, shelter to protect from predators, and a proper environment for breeding wildlife and conserving the ecosystem's health (balwan and kour, 2021). anthropogenic activities that increase environmental stress can affect the biological productivity of marsh, resulting in changes in 170 abdullah et al./ impact succession of drought and flood on diversity indices community composition, i.e. a decline in richness, a decrease in the occurrence of sensitive and native species, and an increase in exotic species (aziz et al., 2021). most primary production in aquatic ecosystems is produced by phytoplankton (andersen et al., 2016). previous works have shown a rise in the salinity in southern iraqi marshes due to a reduction in water discharge from the tigris and euphrates rivers. these changes have resulted in decreased fish species such as arabibarbus grypus, luciobarbus xanthopterus, mesopotamichthys sharpeyi and carasobarbus luteus (hintz and relyea, 2019). there is no study on the al-shafi marsh, but there are several studies on other marshes in southern iraq. mohamed et al. (2012) assessed the ecological and biological aspects of the fish assemblage in the chybayish marsh. abdullah (2019) investigated the fish assemblage and the impact of drowning and drought oscillations on the fish size spectrum in the al-chibyaish marsh in southern iraq. al-thahaibawi et al. (2019) studied the fish assemblage structure in al-huwaizah marsh in southern iraq. the ecological impacts of exotic and marine migratory species on the fish composition assemblage in east hammer marsh were discussed by abdalhsan et al. (2020). fish body size is frequently used as the primary criterion for determining the physiological characteristics of a fish species and its role in the ecosystem (heneghan et al., 2019). therefore, this study aimed to evaluate the fish assemblage in the al-shafi marsh and the impact of succession drought and flooding on fish abundance and size distribution. materials and methods al-shafi marsh is located north of al-basrah city and stretches from southern al-qurna to about 20 km to the al-deer region and nearly 15 km from east to west. the main water source for the marsh is the al-shafi river, which receives its water from the shatt al-arab river. it is approximately 7 km long and 30 km wide. to collect the samples, three stations were selected, including station 1 (30°51'n, 47°25'e), station 2 (30°51'n, 47°29'e), and station 3 (30°47'n, 47°27'e). the collected fishes' total length (cm) and weight (g) were measured after their catch. from january to december 2020, fish samples were collected monthly. gill-nets (with a length of 30-40 m and mesh size of 19-45 mm), cast nets, and electrofishing (500v, 10a) were used to catch the fish. fricke et al. (2020) and froese and pauly (2022) were used to identify fish species. the environmental factors were measured at the time of sampling, including water temperature (ºc) measured with a mercurial thermometer, salinity (psu) and ph were measured with a lovibond sensor (direct 150 germany). the following formulae were used to study biodiversity indices of the fish assemblage. relative abundance % = (ni / n) * 100 (walag et al., 2016), where ni = number of individuals of the species and n = total number of individuals of all species. fish diversity h = -∑ pi ln pi (huang et al., 2019), where h = diversity index, and pi = a proportion of species individuals in the sample. richness index d = s-1/ln n (nyitrai et al., 2012), where d = richness index, s = number of species and n = total number of species. evenness index j = h/lns (nyitrai et al., 2012), where j = evenness index and h = diversity index s: number of species. the species were split into length groups to determine the length frequency of each length group versus the number of fish. the occurrence of species was calculated based on tyler (1949): common species are found in monthly catch samples in 9 to 12 months; seasonal species appeared in the monthly catch samples in 6 to 8 months, and occasional fishes catch in 1 to 5 months. response to succession drought and flooding on the size spectrum of fish assemblage, composition patterns, and species lifehistory strategies are divided into equilibrium, periodic, and opportunistic groups (winemiller, 2005; escalera-vazquez et al., 2017). the dominance index for the abundant three fish species was calculated using the formula of d3= [∑ 𝑝𝑖3𝑖=1 .] 100, where pi = percentage of the abundant to a total number of individuals * 100. all statistical analysis was performed in spss (version 20). 171 int. j. aquat. biol. (2022) 10(2): 169-180 results based on the results, the mean water temperature showed high seasonal variations (25.76±7.56°c) (fig. 2). in january, the lowest water temperature was 12.50°c and rose gradually to the highest (34.53⁰c). it declined slowly to 14.0°c in december. in january, the water salinity was 2.78 psu and reached to the lowest, i.e. 1.64 psu, in may. it recorded as 4.03 psu in october and declined to 2.90 psu in december. its mean was 2.86±0.79 psu during the study period. the ph had slight changes ranging from 7.38 in september to 8.17 in march with a mean value of 7.78±0.30. no significant differences (p>0.05) were found between the three stations in water temperature, ph, and salinity. the relationship between water temperature and salinity was a weak positive (r = 0.243). a significant negative correlation (r = 0.638) was found between water temperature and ph, whereas a significantly negative correlation (r = 0.752**) was found between salinity and ph. figure 1. map of al-shafi marsh showed the three selected stations. 0 1 2 3 4 5 6 7 8 9 0 5 10 15 20 25 30 35 40 s li n it y ( p s u ) a n d p h t e m p e ra tu re ( ºc ) water temperature salinity ph figure 2. monthly variations in some ecological factors in al-shafi marsh. 172 abdullah et al./ impact succession of drought and flood on diversity indices marsh hydrology: the hydrology of al-shafi marsh is divided into two categories: permanent inundation areas, including low flat areas, rivers, canals, and small pits, and seasonal inundation areas. the water depth in the low flat areas ranged from 33 cm in october to 71 cm in may, with a mean of 46.83±14.11 cm. the depth in small rivers, canals, and small pits fluctuated from 77 cm in october to 124 cm in april, with a mean of 96.75±16.08 cm. in the seasonal areas, the depth varied from zero in june, july, august, september, and october to 34 cm in april, with a mean of 12.75±11.84 cm. the effect of the tidal events on the marsh was weak (fig. 3). the water depth had a weak correlation (r = 0.026) (p<0.05) with temperature. a significant negative correlation (r = 0.94**) was found between salinity and water depth of permanent inundation area. a significant positive correlation (r = 0.584*) was detected between ph and water depth of the permanent inundation area. species composition: a total of 19 species were collected from al-shafi marsh, belonging to 17 genera and 11 families. eight fish species were native, nine exotics, and two marines. cyprinidae was the most abundant family with four species, followed by cichlidae and leuciscidae, each with three species, poeciliidae with two species, and clupeidae, engraulidae, heteropneustidae, mastacembelidae, mugilidae, siluridae, and xenocyprididae with one species each (table 1). the total number of species collected from the al-shafi marsh ranged from nine in december to 14 in june. there were no significant (p>0.05) differences in the number of fish species 0 20 40 60 80 100 120 140 w a te r d e p th ( cm ) low flat area river, canals and pits seasonal areas figure 3. monthly changes in the water depth in low flat area, river, canals and pits and seasonal area of al-shafi marsh. 0 2 4 6 8 10 12 14 16 0 50 100 150 200 250 300 350 400 450 n u m b e r o f sp e ci e s n u m b e r o f in d iv id u a ls number of individuals number of species figure 4. monthly changes in the number of species and individuals in al-shafi marsh. 173 int. j. aquat. biol. (2022) 10(2): 169-180 between the stations (fig. 4). a total of 2685 fish specimens were collected, with 107 specimens in december and 385 specimens in may (fig. 3). relative abundance: three dominant species were formed 64.29% of the total number of species (table 2), including p. abu with 28.42% (19.50% in february to 37.65% in january), c. gibelio constituting 20.97% (15.88% in january to 28.11% in april) and o. aureus formed 14.90% (6.80% in june to 21. 50% in december). seven species were recorded as less than 1% of the total catch, including c. sublimus (0.97%), a. marmid (0.78%), h. leucisculus (0.78%), m. mastacembelus (0.26%), t. ilisha (0.19%), h. fossilis (0.11%), and t. whiteheadi (0.07%). three families had higher relative abundance at the family level, forming 85.14% of the total catch viz. cichlidae, mugilidae, and cyprinidae (30.76, 28.42, and 25.96%, respectively). the family leuciscidae recorded 28.42% 20.97% 14.90% 35.71% planiliza abu carassius gibelio oreochromis aureus other species figure 5. dominance index (d3) of the most abundance three species in the al-shafi marsh. family species native exotic marine cyprinidae carassius gibelio + carasobarbus luteus + carasobarbus sublimus + cyprinus carpio + xenocyprididae hemiculter leucisculus + leuciscidae acanthobrama marmid + alburnus sellal + leuciscus vorax + siluridae silurus triostegus + heteropneustidae heteropneustes fossilis + mastacembelidae mastacembelus mastacembelus + cichlidae coptodon zillii + oreochromis aureus + oreochromis niloticus + poeciliidae gambusia holbrooki + poecilia latipinna mugilidae planiliza abu engraulidae ttrayssa whiteheadi + clupeidae tenualosa ilisha + table 1. families and fish species with pointed native, exotic and marine species. 174 abdullah et al./ impact succession of drought and flood on diversity indices 8.49%, siluridae 2.94%, poecilidae 2.02%, xenocyprididae 0.78%, mastacembelidae 0.26%, clupeidae 0.19%, heteropneustidae 0.11%, and engraulidae 0.07% of the total catch. the dominance index (d3) for the three dominant species (p. abu, c. gibelio, and o. aureus) was 64.29%, while the remaining species accounted for 35.71% of the total caught (fig. 5). fish size-spectrum: figure 1 depicts the fish size range versus the number of fish for important commercial fishes (6 species). the total length of p. abu ranges 5-18 cm, with 9 cm being the most common size group (n = 181). the size range of c. carpio was 10-28 cm, with the dominant length group of 13 cm for nine individuals. the size range of c. luteus ranges was 7-25 cm, with the longest length group of 11 cm in 13 fish. the size range of c. gibelio was 8-25 cm, with a dominance group of 12 cm in 89 individuals. the monthly samples of 57 fish of l. vorax species varied 11-31 cm, with the dominance length group of 17 cm (n = 8). the size range of o. niloticus was 8-27 cm, with a dominant length group of 12 cm (n = 33). ecological indices: monthly changes in the fish assemblage's diversity, evenness, and richness indices in al-shafi marsh are shown in figure 7. with a mean of 1.99, the diversity index (h) ranged from 1.84 in august to 2.25 in february. the evenness index (j) ranged from 0.74 in june to 0.88 in february, with an average value of 0.81. the richness index (d) ranged from 1.63 in april to 2.37 in february, with an average of 2.05. occurrence of species: fish species are divided into three categories based on their frequency in monthly figure 6. distribution of fish size-spectrum length groups of the important commercial fish species in al-shafi marsh from january to december 2021. 175 int. j. aquat. biol. (2022) 10(2): 169-180 fishing samples. the resident fish group formed 94.82% of the total catch, including ten fish species, two of them collected in 12 months (p. abu and o. aureus); four species appeared in 11 months (a. sellal, c. gibelio, c. zillii, and s. triostegus); four species were caught in nine months (c. carpio, c. luteus, l. vorax, and o. niloticus). the seasonal species formed 2.76% of overall fish, with three species that two of them recorded in seven months (a. marmid, and g. holbrooki), and one species appeared in six months (c. sublimus). the occasional fish species comprise 2.42%, with six species that three of them caught in four months (h. leucisculus, m. mastacembelus, and p. latipinna), whereas one species was collected in three months (t. ilisha). the h. fossilis species in this group was observed in two months, but the lowest appearance was t. whiteheadi which was caught in one month (table 3). the cluster's temporal variations revealed two main groups; the first group was divided into two secondary groups. the first secondary group consisted of two groups: september, november, january, and august, but the other groups included october only. the second secondary group was parted into may, july, figure 7. monthly variations in the ecological indices in the al-shafi marsh from january to december 2021. figure 8. analysis of cluster for temporal variations of fish species groups in the al-shafi marsh north basrah city. 176 abdullah et al./ impact succession of drought and flood on diversity indices february, and march, while the second was june. the second mean group consisted of april and december (fig. 8). discussion the shatt al-arab river is the only source that supplies the marsh; therefore, the salinity concentration of its water depends on this river. the present work documented 19 fish species in the marsh, however, abdullah (2019) recorded 15 fish in al-chbyish marsh species, and 19 species were recorded by al-thahaibawi et al. (2019) in alhuwaizah marsh. hydrology is the main driving factor in the wetland’s ecosystem dynamics, and the seasonal rainfall raises the water level in the marsh (grubh and winemiller, 2018). temperature is the main controlling factor for water level and conductivity. the rise of water depth increases fish biodiversity, positively affecting food element availability, e.g. allochthonous items associated with plus flooding in the wetlands (rabuffetti et al., 2017; yang et al., 2020). in the al-shafi marsh, water depth was the most influential factor limiting fish assemblage. during the flood season, seasonal inundation habitats are critical for spawning, feeding, and nursery grounds for most species, and re-submersion determines fish species' access to breeding, nursery, and foraging habitats (chea et al., 2020). species composition in al-shafi marsh was correlated by hydrological alteration and other factors, e.g. the current seasonal flood pulsation covered the seasonal inundation areas that are important for spawning, and feeding of native cyprinids (wang et al., 2015), that were the core of the fish community before the introduction of exotic species (abdullah et al., 2021). a reduction in seasonal flood pulsing and decreasing available spawning grounds are led to insufficient flood periods to stimulate reproduction and a suitable condition for egg incubation and hatching (yang et al., 2020). in addition, the long-term overfishing caused a decrease in the abundance of fish species jan. feb. mar. april may jun. jul. aug. sep. octo. nov. dec. total planiliza abu 37.65 19.50 31.86 27.31 29.09 27.60 27.48 33.93 26.20 21.31 30.40 26.17 28.42 carassius gibelio 15.88 20.75 23.04 28.11 19.48 25.60 20.86 20.00 23.25 24.04 19.63 20.97 orechromis aureus 15.88 15.09 14.22 12.05 19.22 6.80 14.24 14.64 12.55 20.22 16.80 21.50 14.90 coptodon zillii 6.47 10.06 9.31 12.85 14.03 3.60 10.93 12.14 9.59 15.20 11.21 9.87 orechromis niloticus 5.29 7.55 6.43 5.97 4.80 6.29 8.86 14.75 15.20 6.00 alburnus sellal 4.71 5.66 3.92 1.61 2.08 19.20 5.63 7.86 6.56 4.80 7.48 5.59 silurus triostegus 2.94 3.77 2.94 4.02 3.12 4.80 2.98 3.32 2.19 3.20 1.87 2.94 leuciscus vorax 3.53 5.03 2.94 2.41 0.78 1.66 4.29 2.21 4.67 2.12 cyprinus carpio 2.35 3.77 2.94 0.00 1.30 1.32 2.86 4.06 3.28 3.20 2.01 carasobarbus luteus 2.94 3.77 4.42 1.20 2.65 2.58 3.83 2.40 3.74 2.01 gambosia holibrooki 1.47 0.80 1.56 0.80 2.14 1.48 3.74 1.01 poecilia latipinna 1.89 1.96 3.64 3.32 1.01 carasobarbus sublimus 1.89 2.45 1.04 1.20 1.85 1.64 0.97 acanthobrama marmid 1.18 1.26 1.96 1.04 1.43 0.74 2.40 0.78 hemiculter leucisculus 1.30 2.80 1.66 3.20 0.78 mastacembelus mastacembelus 1.18 0.71 1.09 0.80 0.26 tenualosa ilisha 0.98 0.40 0.66 0.19 heteropneustes fossilis 0.40 1.09 0.11 ttrayssa whiteheadi 0.80 0.07 table 2. monthly variations in the relative abundance of specie in the al-shafi marsh. 177 int. j. aquat. biol. (2022) 10(2): 169-180 species with the absence of some sensitive species (mesopotamichthys sharpeyi) due to changes in flood pulsing and destruction of spawning grounds (mao et al., 2016; ding et al., 2016). the flood pulsing during the rainy season is insufficient to stimulate some native species to spawn. therefore, the native species decrease and competition with exotic species such as tilapia with high abundance results in the decline of important sensitive native species (abdullah et al., 2021). mugilidae was the most abundant native family, represented by abu mullet, p. abu, in al-shafi marsh. this species has adapted to live under extreme temperatures, low oxygen, and feeding on organic detritus and diatoms available at the bottom of the marsh (mohamed, 2014; abdullah, 2019). the exotic fish species of cyprinidae and cichlidae families (c. gibelio, o. aurea, c. zillii, and o. niloticus) were the most abundant and are common in southern iraq marshes (al-thahaibawi et al., 2019; abdullah et al., 2021). the exotic species are omnivores foraging on low-quality items available in the marsh’s habitats, and they have a long active reproductive season reproducing at a young age (yu et al., 2019). the fish size range refers to the distribution of biomass in fish populations based on body size classes, and it is a common feature of the fish community (abdullah, 2019; vila-martinez et al., 2019). the results show that the size spectrum of fish appears to be stable due to the fishing, consistent with other findings (andersen et al., 2016; rabuffetti et al., 2017; benoit et al., 2021). the construction of dams at the head of the tigris and euphrates river system have reduced the flood pulsation in the southern marshes of iraq that is crucial to stimulate the reproduction of native species, and this also has been led to an increase in marsh’s’ salinity, resulting in increasing exotic species and reducing native species (al-zaidy and parisi, 2018; chea et al., 2020). the absence of native fish species representing the historical core of fish assemblage and the introduction of invasive species with high abundance can minimize diversity, richness, and evenness indices in the aquatic systems. these indices were within the range of 0 0.5 1 1.5 2 2.5 0 10 20 30 40 50 60 70 80 d iv e rs it y a n d r ic h n e ss i n d ic e s w a te r d e p th ( cm ) water depth of low flat areas richness diversity figure 9. monthly fluctuations of diversity and richness indices values with water depth in the low flat area in al-shafi marsh. group species occurrence months no. of species resident fish species p. abu, o. aureus 12 2 a. sellal, c. gibelio, c. zillii, s. triostegus 11 4 c. carpio, c. luteus, l. vorax, o. niloticus 9 4 seasonal fish species a. marmid, g. holbrooki 7 2 c. sublimus 6 1 occasional fish species h. leucisculus, m. mastacembelus, p. latipinna 4 3 t. ilisha 3 1 h. fossilis 2 1 t. whiteheadi 1 1 table 3. fish species occurrence in al-shafi marsh from january to december 2020. 178 abdullah et al./ impact succession of drought and flood on diversity indices previous studies conducted in inland waters of iraq e.g. (ab dalhsan et al., 2020; abdullah et al., 2021). several studies showed an increased diversity and richness with rising water depth. however, in the current study, diversity and richness values changed with the water depth in the areas of permanent inundation and relatively became high. this may be attributed to the ease of fishing at low water depth and the efficiency of fishing means (paller, 2018; ngor et al., 2018). most sensitive species have disappeared and been replaced by native tolerant and invasive species, such as the mullet fish (p. abu) and the prussian carp (c. gibelio). three species of the cichlidae (c. zillii, o. aureus, and o. niloticus) are occurred because of their tropical origins that are similar to the conditions of the current habitats (zhang et al., 2020), and these species are dominant in most months of the year (mohamed and abood, 2017; al-thahaibawi et al., 2019; abdullah et al., 2021). conclusions the current study revealed that the succession of droughts and flooding and lack of adequate flood pulses during the wet season decline the biomass. this event along with overfishing reduces the fish size spectrum, and increases the abundance of small and medium-sized fish species, resulting in the extinction of commercially important native species and the raising the exotic fishes. references abdalhsan h.t., hussain n.a., abduijaleel s.a. 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(2020) 8(4): 288-295 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article effects of different levels of chamomile (matricaria recutita) extract on growth and antioxidant parameters of zebra fish (danio rerio) zohreh fazelan1, reza akrami*1, seyyed morteza hoseini2 1department of fisheries, azadshahr branch, islamic azad university, azadshahr, iran. 2inland waters aquatics resources research center, iranian fisheries sciences research institute, agricultural research, education and extension organization, gorgan, iran. s article history: received 18 june 2020 accepted 24 august 2020 available online 2 5 august 2020 keywords: chamomile extract growth antioxidants zebrafish abstract: an eight-week feeding trial was conducted to investigate the effects of different levels of dietary chamomile (matricaria recutita) aqueous extract (cae) on growth performance and antioxidant status of zebra fish, danio rerio. fish (initial weight of 0.20±0.02 g) were stocked at 12 tanks (50 fish per tank) and fed with a basal diet (32% protein and 8% lipid) supplemented with 0 (control), 0.5, 1, and 2% cae in triplicate groups for 8 weeks. the results showed that fish fed 2% cae had a significantly higher growth and biomass gain compared to the control group (p<0.05). condition factor had a significant increase in 2% cae treatment compared with other treatments (p<0.05). there were no significant differences in catalase, glutathione peroxidase, and superoxide dismutase activity amongst treatments (p<0.05). finally, it seems the use of a 0.5-2.0% cae can be effective in improving the growth and antioxidant system of zebra fish, d. rerio. introduction under intensive and super-intensive fish culture systems, fish are exposed to a variety of stresses such as poor water quality, high density, and bad handling, which play a main role in fish disease. hence, preventing disease by chemical drugs is crucial to deal with the high cost of treatments (goda et al. 2008; volpatti et al., 2013). indeed, in intensive systems, improved nutritional condition plays an important role in promoting growth and maintaining fish health and welfare (lee et al., 2015; abdel-latif et al., 2020a, b; abdel-tawwab et al., 2020a, b). therefore, the most important challenge of the aquaculture industry to date is improving the diets formulated for optimal growth and promotion of aquatics health (akrami et al., 2010). nutritional manipulations and the addition of various supplements can increase fish health and safety (lee et al., 2015; abdel-latif et al., 2020a, b; abdel-tawwab et al., 2020a, b). so far, many studies have been done on food additives such as plant extracts, immune stimulants, enzymes, prebiotics, probiotics, and organic acids as alternatives to *correspondence: reza akrami e-mail: akrami.aqua@gmail.com antibiotics (lee et al., 2015). valuable herbal medicines have a special place in the treatment because of some features as economic value, low-cost production, non-destructive effects on the environment, few side effects compared to chemical drugs, lack of relative resistance to pathogens, uniqueness in the treatment of some diseases, and the existence of various clinical experiences in this field (ghasemi pirbaluti et al., 2011). herbal chemicals, including alkaloids, flavonoids, terpenes, phenolic compounds, pigments, and steroids have positive effects comprising increased growth, reduced stress, stimulating the immune system, and antimicrobial and antioxidant properties in fish (abdel-razek et al., 2019). these medicinal resources not only can be used as new drugs and bioactive compounds in increasing fish production, product quality, and fish health, but also they are eco-friendly due to their plant origin (chakraborty et al., 2014). chamomile recutita is known as german chamomile (mckee and bloomberg, 2006; harbren et 289 int. j. aquat. biol. (2020) 8(4): 288-295 al., 2009). medicinal and health-promoting properties of chamomile are mainly attributed to two groups of bioactive compounds present in chamomile flowers, which include essential oil (essence) and polyphenols. flavonoids are the major polyphenols in chamomile that have antioxidant properties (srivastava and gupta, 2009). the use of antioxidants reduces the rate of oxidation. the detection of toxicity and carcinogenicity of synthetic antioxidants has drawn the attention of scientists to the exploration of natural antioxidants (ghanavati et al., 2008). in addition to flavonoids, coumarins, terpenoids, and bisabolol are the most important compounds of chamomile flowers that are medically considerable. chamomile extract has antioxidant, disinfectant, antibacterial, sedative, antispasmodic, anti-diabetic, analgesic and anticancer activities, and protective effects on the gastrointestinal tract, nervous system and immunogenic modification (izadi et al., 2013). over the past decades, the global trade in ornamental fish has significantly grown (ranjan et al., 2017). one of the farmers' favorite ornamental fish is zebra fish (danio rerio hamilton, 1822) (hill et al., 2011). because of simplicity of reproduction method and omnivorous diet, zebra fish has known as an experimental model in laboratories to answer important research questions in various fields like biomedical, genetics, growth biology, pharmacology, toxicology, and evolutionary physiology (vascotto et al., 1997; sundin et al., 2019). regarding the importance of zebra fish in aquaculture and its value as a research model on the other hand, this species was selected as a target fish in the present study. therefore, the present study was aimed to evaluate the effect of chamomile aqueous extract on growth and antioxidant parameters of zebra fish, d. rerio. materials and methods experimental conditions: the experiment was conducted in the gorgan-mahi company (gorgan, golestan province, iran). fish were stocked in experimental aquariums (40x25x25 cm) and to adapt, they were fed with a control diet (32% protein and 8% lipid) for two weeks. after that, 600 fish (with a mean weight of 0.20±0.02 g) were randomly assigned into 12 aquaria, 50 fish per aquarium with three replicates per treatment. fish were fed with the experimental diets, until satiety, three times a day (08:00, 12:00, and 16:00 h) for 8 weeks. during the experiment, the water temperature, dissolved oxygen, and ph were recorded at 23°ϲ, 6.5 mg. l-1, and 7-8.5, respectively, by portable apparatus (hach hq40d, loveland, colorado, usa). preparation of chamomile extract and experimental diets: as the dietary additive, a commercially available chamomile powder (giahine industrial co., ltd. isfahan, iran) was used. first, 10 g of chamomile powder was boiled in 200 ml of the boiled water for 2 hours (wu et al., 2010), then, it was filtered by filter paper. the required concentrations, including 0 (control), 0.5, 1.0, and 2.0% was added to the control diet to prepare treatments. the control diet was formulated according to table 1 containing 32% protein and 8% fat (o'brine et al., 2015). after that, the ingredients were powdered and passed through a mesh (0.5 mm), and mixed thoroughly well and then, water, soya oil, and chamomile aqueous extract (in exchange the water) were added to the mixture. the obtained dough was passed through a mesh (1 mm) to form sticks which were allowed to dry for 24 h (at room temperature) and finally stored at 4°c until use. growth performance: for growth assessment, individuals (n15/tank) were randomly selected to measure the weight and length, at least 12 h after the last feeding (akrami et al., 2013, 2015). weight gain (wg; g) and (wg; %), specific growth rate (sgr; % day -1), condition factor (cf), survival rate (%), and biomass gain (bg; g) were calculated on the basis of the following formulas (htun‐han, 1978; tacon, 1990; watanabe et al., 1993; kamali-sanzighi et al., 2019): wg (g) = fw – iw wg (%) = 100 (fw – iw) / iw sgr = 100 (ln fw – ln iw) / t cf = 100 fw / l3 sr = 100 (number of fish at the end of the experiment/ number of fish at the beginning of the experiment) 290 fazelan et al./ effects of chamomile on zebra fish bg = fb – ib. sampling and measurement of antioxidant status: at the end of trial, due to the small size of fish, the activity of antioxidant enzymes was measured using the whole body. for this purpose, 3 fish were taken from each tank and anesthetized with clove powder (500 ppm). after that, the head and fins were separated and homogenized (holbech et al., 2001) and then centrifuged (10,000 rpm) in 3 stages at 4°c. the high phase was finally used to measure antioxidant activities. the superoxide dismutase (sod), glutathione peroxidase (gpx), and catalase (cat) enzymes were evaluated using commercial kits (navand, tehran, iran). sod level was estimated by measuring the rate of cytochrome c reduction (mccord and fridovich, 1969), catalase level was measured based on góth (1991) method by measuring the rate of hydrogen peroxide decomposition, and glutathione peroxidase level was measured by estimating the glutathione oxidation rate (yousefi et al., 2018). statistical analysis: growth performance and antioxidant status were analyzed using one-way analysis of variance (one-way anova) with a probability of 5% error in a completely randomized experimental design. the significance of the difference between the mean of different factors, with duncan's follow-up and multi-domain tests was done at p<0.05 using spss 13 software. results the effect of the dietary chamomile aqueous extract levels on weight gain (wg g), percentage of weight gain (wg%), specific growth rate (sgr), biomass gain (bg), condition factor (cf), and survival rate (sr) are shown in table 2. fish fed with the 2% chamomile diet demonstrated a significantly higher bg and wg (g) compared to fish fed with the 1% chamomile and control group (p<0.05). wg (%) and sgr significantly improved as dietary chamomile levels increased from 0.5 to 2% (p<0.05). although there was no significant difference of cf among the control, 0.5, and 2% treatments, cf recorded a significant increase in 2% treatment compared to 1% table 1. foodstuffs and chemical composition of the control diet (% dry matter basis). ingredients amounts (%) fishmeala 21 soybean mealb 25 wheat flour 43.6 meat mealc 5 soybean oil 4 mineral mixd 0.5 vitamin mixe 0.5 lysinef 0.2 methionineg 0.2 total 100 proximate composition (%) crude protein 32.40 crude lipid 8.63 crude fiber 3.13 crude ash nfeh ge (mj/kg)i 5.66 50.18 1958.585 a 66.21% protein; 13.59% lipid; b gorgan soya co., gorgan, iran (45% protein; 1.5% lipid); c 40% protein; 22.61% lipid; d the premix provided following amounts per kg of diet: mg: 350 mg; fe: 13 mg; co: 2.5 mg; cu: 3 mg; zn: 60 mg; nacl: 3 g; dicalcium phosphate: 10 g; e the premix provided following amounts per kg of feed: a: 1000 iu; d3: 5000 iu; e: 20 mg; b5: 100 mg; b2: 20 mg; b6: 20 mg; b1: 20 mg; h: 1 mg; b9: 6 mg; b12: 1 mg; b4: 600 mg; c: 50 mg; f sigma, st. luise, mo, usa; g mad tiour co., sanandaj, iran. 291 int. j. aquat. biol. (2020) 8(4): 288-295 one (p<0.05). survival rate also was 100% in all groups. after the 8-week trial, the glutathione peroxidase (gpx) and catalase (cat) activities improved in those fish fed with chamomile 2% diet over the control group (p>0.05). however, there were no significant differences between the treatments compared with the control group (table 3). also, there was no significant difference in activity of superoxide dismutase in all treatments compared to the control group (p<0.05) (table 3). discussions the present study confirmed that short feeding on a diet rich in chamomile (2%) led to a significant increase in growth indices of zebra fish. this result is consistent with the previous studies in neil tilapia (oreochromis niloticus) (abdel-wahhab et al., 2001; abdel-maksoud et al., 2002; khalafalla et al., 2009; zaki et al., 2012) and african catfish (clarias gariepinus) (abdelhadi et al., 2010). in addition, administration of different levels of chamomile powder (matricaria chamomilla l.) showed a significant increase of biomass and improved growth performance in hybrid red tilapia (oreochromis sp.) (nordin, et al., 2017). medicinal plants contain phytochemicals, including alkaloids, flavonoids, pigments, phenols, terpenes, steroids, polysaccharides, and essential oils which involved in different activities such as growth, nutrition, immune and anti-stress stimulation, and antimicrobial function (citarasu et al., 2010; chakraborty and hancz, 2011). additionally, bioactive compounds such as polysaccharides and terpenes regulate the immune system and act as prebiotics, which increase growth in fish (wang et al., 2009; ringo et al., 2010). also, monoterpene phenols, including thymol and carvacrol act as growth promoters in channel catfish (ictalurus punctatus) (zheng et al., 2009). as potential bioactive substances of chamomile i.e. alkanoids, coumarins, and triterpenoids can affect the digestion process and increase the activity of digestive enzymes to improve digestion, food absorption, and growth in fish (ell, 1965; samy et al., 2008; immanuel et al., 2009; citarasu et al., 2010; kaleeswaran et al., 2010; abdeltawab et al., 2010; hashemi and davoodi, 2011). table 2. ingredients and chemical composition of the experimental diets (% dry matter basis) containing different levels of chamomile aqueous extract. parameter dietary chamomile extract (%) control 0.5 1 2 iw (g) fw (g) 0.21±0.003 a 0.30±0.01b 0.20±0.012 a 0.34±0.01ab 0.18±0.006 a 0.29±0.01b 0.21±0.016 a 0.36±0.01a wg (g) 0.09±0.015c 0.13±0.006ab 0.11±0.005bc 0.15±0.005a wg (%) 42.13±7.07b 65.66±1.01a 59.96±1.62a 71.26±6.53a sgr (% day -1) 0.58±0.08b 0.84±0.01a 0.79±0.01a 0.89±0.05a bg (g) 4.5±0.76c 6.67±0.33 ab 5.5±0.28bc 7.5±0.28a cf 1.02±0.05ab 1.08±0.02ab 0.99±0.04b 1.14±0.03a sr (100) 100 100 100 100 data are represented as mean±se. the values of means in columns were significantly different (p<0.05). table 3. changes in activities of glutathione peroxidase (gpx), sodium dismutase (sod), and catalase (cato of zebra fish fed diets containing dietary chamomile extract for 8 weeks. parameter dietary chamomile extract (%) control 0.5 1 2 gpx (u/mg) 4.89±0.07a 4.95±0.15a 4.92±0.02a 5.05±0.21a sod (u/mg) 4661.22±375.51a 3416.32±48.98a 3181.62±459.18a 3010.20±606.11a cat (u/mg) 0.36±0.024a 0.35±0.008a 0.35±0.041a 0.37±0.059a data are represented as mean ± se. the values of means in columns were significantly different (p<0.05). 292 fazelan et al./ effects of chamomile on zebra fish moreover, essential fatty acids, including linoleic, linolenic and arachidonic acids in the composition of plant additives, were detected to be essential to growth (murray et al., 1991; abdel-latif et al., 2004). in the present work, chamomile treatment's failure to significantly affect antioxidant activities may be explained by the method of extract preparation, i.e. boiling chamomile in boiled water. it is likely that heating chamomile is accountable for not affecting antioxidant activities, since abaee et al. (2018) documented that although the polyphenols of chamomile aqueous extract were resistant to heat at temperatures of 70-120°c for 5 and 15 min, the antioxidant activities significantly decreased, even at a temperature of 70°c for 5 min. similarly, davidovpardo et al. (2011) found that the antioxidant activity of grape seed extract decreased by heating. svehlikova et al. (2004) also reported that while increasing temperature, thermal degradation of flavonoids increased in chamomile extract. chamomile's antioxidant activity is mainly attributed to flavonoids and especially to 7-glycoside apigenin and its derivatives. therefore, the destruction of flavonoids decreases antioxidant activity. at higher heat, more damage is introduced to the structure of flavonoid which decreases the antioxidant activities (srivastava et al., 2009). comparing the findings related to the total content of polyphenol compounds and antioxidant activity revealed that although heating chamomile extract does not affect the total content of phenolic compounds, it reduces the antioxidant activity. the likely reason for this phenomenon was attributed to the fact that not all phenolic compounds of the extract behave similarly under heating conditions (abaee, 2018). in previous works, the content of phenolic compounds in galangal a spice in the ginger family – increased by heating at 100°c for 10-30 min, while their values decreased in red pepper. heating degraded and hydrolyzed some flavonoids and converted their glycoside forms to aglycone ones (ayusuk et al., 2009). in addition to the aforementioned points, other factors such as method of fish sampling, using the whole body instead of blood, doses used, short duration of administration, and environmentally suitable conditions may be reasons for not observing differences in antioxidant parameters in the present study. in conclusion, as a considerable medicinal herb, chamomile requires further investigation to be determined the effective doses of chamomile to improve the antioxidant defense. the results indicated that chamomile aqueous extract had a significant, potential efficacy on growth parameters in zebra fish. so, with respect to the absence of adverse effects of chamomile on the activity of antioxidant enzymes and its positive effect on growth performance, the use of this supplement in the diet of zebra fish is recommended. references abaee a., mohammadian m., jaberipour s. 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(2013) 1(4): 185-187 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology short communication length-weight relationship and condition factor in giant harpiosquillid mantis shrimp, harpiosquilla raphidea (crustacea: stomatopoda) in banten bay waters, indonesia mugi mulyono *1, mufti petala patria1, abi abinawanto1, ridwan affandi2 1department of biology, faculty of mathematics and science, university of indonesia, depok, indonesia. 2departement of aquatic resources management, faculty of fisheries and marine sciences, bogor agricultural university, bogor, indonesia. article history: received 3 june 2013 accepted 30 july 2013 available online 2 0 august 2013 keywords: mantis shrimps harpiosquilla raphidea stomatopoda length-weight relationship abstract: length-weight relationship of harpiosquilla raphidea from banten bay waters was studied from 146 males and 186 females ranging from 38 to 354 and from 37 to 348 mm, respectively. the following equations were obtained ln w = 5.164 + 2.478 ln x (for males), ln w = 5.333 + 2.596 ln x (for females) and ln w = 5.264 + 2.549 ln x, ln x (pooled). the ‘b’ values (2.5497) found not near to 3 indicating allometric growth being matched up with the cube law. the ancova indicated not much variation between the ‘b’ values for males and females. in general, the growth rate of the mantis shrimp h. raphidea almost the same or not much different from other mantis shrimp species of the order stomatopoda. introduction stomatopods are a group of crustaceans coming under the class malacostraca and commonly known as ‘mantis shrimp’. the spearer mantis shrimp, harpiosquilla raphidea lives on muddy bottoms in coastal waters around indonesia. in a mudflat develop in the banten bay of province banten, this species is exploited commercially, mainly by small bottom-trawlers and gill net due to its economic value. little is known about this group and distribution of the adults. weight of the fish is a function of length. information on length-weight relationship is essential for studies on growth and sexual maturity of animals. our literature review shows that except few works by antony et al. (2004) in harpiosquilla harpax; sukumaran (1987); james and thirumilu (1993); tanuja (1996) in oratosquilla nepa and lyla et al. (1993) in harpiosquilla melanoura, the works concerned with the assessment of weight-length relationship in mantis shrimps from the indian * corresponding author: mugi mulyono e-mail address: mulyonomugi@gmail.com waters are scanty. moreover no work has so far been carried out on the length weight relationship of harpiosquilla raphidea. thus, the present study aimed to explain the mathematical relationship between two variables namely the length and the weight. the present study provides detailed information on the biology of h. raphidea along banten bay waters. materials and methods around 146 males ranging from 38 to 354 mm and 186 females ranging from 37 to 348 mm were collected from the banten bay waters from december 2011 to january 2012. the length was measured from the tip of the rostrum to the end of telson using vernier calipers. the weight was measured to the nearest 0.01 g using an electronic balance. the length-weight relationship was calculated separately for each sex as well as the pooled. the logarithmic equation for this relationship is ln (w) = ln a + b ln (l) i.e., y = a + 186 mulyono et al./ int. j. aquat. biol. (2013) 1(4): 185-187 bx, according to wardiatno and mashar (2010), where ‘w’ is weight (in gram), ‘a’ and ‘b’value are the constant and the regression coefficient, which were estimated using the least-squares regression analysis. the condition factor (k) determines the effects of seasonal and habitat difference in the robustness and general well-being of the species being calculated using the equation k = 100w/l3 (gayanilo and pauly, 1997), where: l = length (cm); w = weight (g); k = condition factor (g/cm3). the linear equation was fitted separately as well as pooled for males and females of h. harpax. analysis of covariance (ancova) was employed to test the significance of difference between regression coefficients (b) of both sexes (welcome, 2001). results the log values corresponding to the length and weight of males and females are plotted in the figures 1 and 2. there was linear relationship between the weight and length. the following relationships were found: males: ln w = 5.164 + 2.478 ln l females: ln w = 5.333 + 2.596 ln l there were differences in the condition factors for males and females, i.e. the k values for females and males were 1.21 and 1.19, respectively. the k value for the combined sexes was 1.20. the results of covariance analysis on the length-weight equation revealed significant difference between the regressions of males and females at 5% level (f = 2.8134; p>0.05). also, the length-weight relationship of the pooled was found: pooled: ln w = 5.265 + 2.549 ln l. discussion the ‘b’ values of both males and females were less than 3, which is in agreement with antony et al. (2004) in harpiosquilla harpax of parangipettai waters of india and sukumaran (1987) who also described an allometric pattern of growth in o. nepa of south kanara coast. rocket et al. (1984) investigated the length-weight relationship in squilla empusa in the northwestern gulf of mexico, where they concluded that the species shows an isometric growth pattern as the ‘b’ values (2.9574 for males and 2.9362 for females) did not deviated much from the cube value 3. giovanardi and manfrin (1984), tanuja (1996) and lyla et al. (1998) also presented separate equations for males and females in different species of stomatopods. james and thirumilu (1993) also recorded a similar kind of observation in o. nepa off the madras coast and found a similar equation for males and females (b value, 2.9661). the mean condition factor obtained from this study showed significant difference between sexes, thus, indicating sex dependent between female and male. figure 1. logarithmic relationship between the length and weight in male harpiosquilla raphidea. figure 2. logarithmic relationship between the length and weight in female harpiosquilla raphidea. figure 1. logarithmic relationship between the length and weight in male harpiosquilla raphidea. 186 mulyono et al./ int. j. aquat. biol. (2013) 1(4): 185-187 acknowledgement authors are thankful to ai setiadi, nurlaela, sigit and mbak riri for their assistance in the laboratorial works. we are indebted to prof. m. kasim moosa due to his help for the identification of the shrimps. references antony p.j., mani e.p., khan s.a. (2004). length-weight relationships in mantis shrimp harpiosquilla harpax (de haan) (crustacea: stomatopoda). journal of aquatic biology, 19: 39-42. gayanilo f.c., pauly d. (1997). fao-iclarm stock assessment tools (fisat). fao computerized information series (fisheries) no. 8, rome, 262 pp. giovanardi o., piccinctti-manfrin p. (1984). summary of biological parameters of squilla mantis l. in the adriatic sea. fao fisheries and aquaculture report, 290: 131134. james u.b., thirumilu p. (1993). population dynamics of oratosquilla nepa in the trawling grounds off madras. journal of the marine biological association of india, 35: 135-110. lyla p.s., panchatcharam k., khan s.a. (1998). age, growth and length-weight relationship in the stomatopod harpiosquilla melanoura (manning). in: proc. symp. advances and priorities in fish. tech., k.k. balachandran, t.s.g. iyer, p. madhavan, j. joseph p.a. perigreen m.r. raghunath and m.d. varghese (ed.). society of fisheries technologists (india). cochin, 44-47. rocket m.d., standard g.w., chittenden m.e. (1984). bathymetric distribution, spawning periodicity, sex ratios, and size compositions of mantis shrimp squilla empusa in the northwestern gulf of mexico. fishery bulletin, 82: 418-426. sukumaran k.k. (1987). study on the fishery and biology of the mantis shrimp oratosquilla nepa (latreille) of south kanara coast during 1979-1983. indian journal of fisheries, 34: 292-305. tanuja r. (1996). some aspects of biology and utilization of the mantis shrimp oratosquilla nepa from cochin waters. ph.d. thesis. cochin university of science and technology, india. wardiatno y., mashar a. (2010). biological information on the mantis shrimp, harpiosquilla raphidea (fabricius 1798) (stomatopoda, crustacea) in indonesia with a highlight of its reproductive aspects. journal of tropical and conservation, 7: 63-73. welcome r.l. (2001). inland fisheries, ecology and management. london: fishing news book, blackwell science. 358 pp. 187 int. j. aquat. biol. (2021) 9(6): 360-369 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article growth and exploitation parameters of the west african ladyfish elops lacerta valenciennes, 1847 in the ivorian’s exclusive economic zone, côte d’ivoire angelina gbohono loukou* 1,2, soumaïla sylla1, olivier assoi etchian1, ida akissi konan1, célestin boua atse2 1university nangui abrogoua, formation unit and research of nature sciences, laboratory of biology and animal cytology, 02 bp 801 abidjan 02, côte d’ivoire 2centre of oceanological research, b.p.v 18 abidjan, côte d’ivoire. s article history: received 25 september 2021 accepted 28 november 2021 available online 2 5 december 2021 keywords: growth parameters von bertalanffy model population dynamics exploitation rate west africa abstract: west african ladyfish, elops lacerta is a pelagic fish present in the coastal waters of west africa from mauritania to angola. this study is conducted to provide data on the growth and exploitation parameters of e. lacerta important for the management and conservation of this species in the ivorian’s exclusive economic zone (eez). these parameters were estimated by the indirect method using length frequency data collected in the ivorian’s eez during january 2019 to december 2020. monthly length frequency data were analyzed by fisat ii software. a total of 865 specimens were examined. the growth parameters from von bertalanffy growth function (vbgf) estimations were l∞ = 60.38 cm fork length, k = 0.39 year−1, and t0 = 0.46 year. the estimated potential longevity (tmax) was 7.69 years. the exploitation parameters showed that the total mortality rate (z), natural mortality rate (m) at 28.35°c and fishing mortality rate (f) were 1.57 year−1, 0.80 year−1 and 0.77 year−1, respectively. the exploitation rate (e = f/z) was 0.49. this value is lower than the optimal exploitation value (e50), thus expressing a case of under-exploitation of the species in the ivorian’s eez. the size of first capture (lc) below the size of first sexual maturity (fl50) and the optimal size of capture (lopt) requires the implementation of an adequate mesh size management policy to allow fish to reproduce several times before being captured. introduction studies on the growth and exploitation parameters of fishes are received a major impetus in recent years owing to the need to provide an adequate scientific basis for conservation of the resources. several methods are used to determine the growth of fish, methods direct by the mark-recapture technique using chemical marks or external or internal implants (dortel et al., 2014; hamel et al., 2014; eveson et al., 2015) and direct reading of hard structures (spines, otoliths, scales and vertebrae (campana, 2014; kumbar and lad, 2016; heimbrand et al., 2020; izzo et al., 2021). the method indirect estimate based of length distribution data over time (gayanilo et al., 2002; panfili et al., 2002). growth is an important aspect of the biology and life history of fish. information on biology and population dynamics are essential for monitoring the rational exploitation and management of the stock. *correspondence: angelina gbohono loukou doi: https://doi.org/10.22034/ijab.v9i6.1362 e-mail: loukouangelinna@gmail.com elops lacerta is a teleost fish of the family elopidae. it is pelagic species found in the east atlantic, precisely in the west african coastal waters, from mauritania to angola (iucn, 2019). it lives at depths between 1 and 50 m in marine environment and it is a migratory species whose biological cycle is divided into three phases. the larval and juvenile phases take place in brackish water or in estuaries (marine, intermediate and freshwater estuary). then, individuals in early sexual maturity migrate to the marine environment to continue growing and reproducing (hie-daré, 1982; n’dour, 2007; lawson and aguda, 2010; abdul et al., 2015). most of the works on e. lacerta have been done in lagoon environments (hie-daré, 1982; ikomi, 1994; ecoutin and albaret, 2003; konan et al., 2007; niyonkuru et al., 2007; lawson and aguda, 2010; adams et al., 2013; abdul et al., 2015; abdul et al., 2016; eyi et al., 2016; n’dour et al., 2017; coulibaly et al., 2018; 361 int. j. aquat. biol. (2021) 9(6): 360-369 dienye et al., 2021). in côte d’ivoire, few studies have been carried out in the lagoon environment i.e. there are none in the marine environment. therefore, the objective of this study is to provide data on the growth and exploitation parameters of e. lacerta for its management and conservation in the ivorian’s exclusive economic zone (eez) using length frequency data. materials and methods study area: côte d’ivoire is located in west africa in the intertropical zone belonging to the vast gulf of guinea ecosystem (bassou, 2016). the ivorian fishing area is located between latitudes 4°n and 5°n and longitudes 2.30°w and 8°w (fig. 1). fishing zone concerns the exclusive economic zone (eez) with a limit of 200 nautical miles (370.4 km). the study area has four marine seasons (assan et al., 2018): a short cold season (scs) in january to february (minor upwelling), a long warm season (lws) between march to june, a long cold season (lcs) from july to october (major upwelling) and a short warm season (sws) in november to december. the variation of salinity at the surface of the ivorian gulf is influenced by the intensity of freshwater inflow and rainfall. two periods of high desalination are recorded, the first from february to march and the second from may to june (gougnon et al., 2018). data collection: the specimens were obtained during january 2019 to december 2020 from the commercial catches at abidjan fishing harbour from the industrial trawlers operating in the eez. once a month, at least 30 individuals were collected and rapidly transported to center of oceanological research laboratory (cro). the fork lengths were measured on the 865 specimens were grouped by month. after measurements, dissection of each specimen was performed in the laboratory. the sex was determined and the different stages of sexual maturity were identified according to the macroscopic scale of fontana (1969), based on color, consistency, volume, shape, vascularity, oocyte size and the presence or absence of milt in the gonads. the class interval was determined according to sturge’s rule (scherrer, 1984). mature individuals were grouped by size class and the percentage of the mature individuals for each size class calculated. the size of first sexual maturity (fl50) was determined by sex and for the whole population (sex combined) according to ghorbel et al. (1996). data analysis: the size frequencies were used to create a dynamic cross-tabulation table using excel software. the data were analysed using fisat ii software (fao-iclarm stock assessment tools) (version 1.2.2) (gayanilo, 1996). growth parameters: the parameters of the von bertalanffy growth function (vbgf), asymptotic length (l∞) and growth coefficient (k) were estimated by means of elefan-i (electronic length frequency analysis). growth in length was studied using the von bertalanffy model (1938). this model describes the growth in length by the function: 𝐿𝐿𝐿𝐿 = 𝐿𝐿∞(1 − 𝑒𝑒−𝐾𝐾(𝑡𝑡−𝑡𝑡0)) where l∞ = asymptotic length (cm), k = growth coefficient (year-1), t; t0 = age at zero length (year) and t = age (year). pauly (1979) empirical equation for the theoretical age at length zero (t0) was used to obtain this parameter as: 𝐿𝐿𝐿𝐿𝐿𝐿(−𝐿𝐿0) = 0.392 − 0.275 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿∞ − 1.038 𝐿𝐿𝐿𝐿𝐿𝐿𝐿𝐿 where t0 = age at zero length (year), l∞ = asymptotic length (cm) and k = growth coefficient (year-1). the maximum age was calculated by the formula of pauly (1980a): 𝐿𝐿𝑚𝑚𝑚𝑚𝑚𝑚 = 3 𝐿𝐿 where tmax = maximum age (year) and k = growth coefficient (year-1). the asymptotic weight (w∞) was determined using the values of the intercept, the figure 1. fishing zone of côte d’ivoire. 362 loukou et al./ population parameters of elops lacerta in the ivorian’s exclusive economic zone asymptotic length and the allometry coefficient in the following formula: 𝑊𝑊∞ = 𝑎𝑎𝐿𝐿∞𝑏𝑏 where w∞ = asymptotic weight (g), a = intercept, l∞ = asymptotic length (cm) and b = allometry coefficient. the growth performance index (ø’) population in terms of length was determined using the index of pauly and munro (1984) and compared the index of baijot and moreau (1997): ø’ = log (k) + 2 log (l∞) where ø’ = growth performance index, k = growth coefficient (year-1) and l∞ = asymptotic length (cm). exploitation parameters: the natural mortality (m) was calculated as a function of asymptotic length (l∞), growth constant (k) and mean environmental temperature (t°c) (pauly, 1980b): 𝐿𝐿𝐿𝐿𝐿𝐿 𝑀𝑀 = −0.0066 − 0.279𝐿𝐿𝐿𝐿𝐿𝐿 𝐿𝐿∞ + 0.6543 log k + 0.4634 log t where m is natural mortality, l∞ = asymptotic length (cm), k = growth coefficient (year-1) and t = the mean habitat water temperature (°c). total mortality rate (z) and the fishing mortality (f) was computed using the relationship: z = f − m according to barry and tegner (1989), the ratio of total mortality to the growth coefficient is an indicator of the state of the population. if, z/k < 1: there is a predominance of growth over mortality in the population. if, z/k > 1: mortality predominates over growth. if, z/k = 1: the population is in a state of equilibrium. therefore, the m/k ratio should be between 1.5 and 2.5 (beverton and holt, 1959). the catch probability provides a direct estimate of the length at which 25, 50 and 75% of fish would be vulnerable to the fishing gear (pauly, 1984) through analysis of the catch curve converted to length. the optimal catch size is estimated for a given cohort according to the following equation beverton (1992): 𝐿𝐿𝑜𝑜𝑜𝑜𝑡𝑡 = 𝐿𝐿∞ 3 3 + 𝑀𝑀/𝐿𝐿 where lopt = optimal catch size (cm), m = natural mortality (year-1) and k = growth coefficient (year-1). the annual recruitment pattern was produced from routine of moreau and cuende (1991). this routine reconstructs the recruitment pulses from time series of length-frequency data to determine the number of pulses per year and the relative strength of each pulse (gayanilo et al., 2005). the exploitation rate (e) was quotient between fishing mortality and total mortality (beverton and holt, 1966): 𝐸𝐸 = 𝐹𝐹/𝑍𝑍 where f = fishing mortality (year-1) and z = total mortality (year-1). the ratio between natural mortality (m) and fishing mortality (f) corresponds to a level of exploitation of the species stock. three types of categories defined the level of exploitation of a species. according to gulland (1971), when the exploitation rate (e) is equal to 0.5, the exploitation of the stock is optimal (f = m). an exploitation rate below 0.5 describes a level of under-exploitation of the species (f<m). on the other hand, when the exploitation rate is less than 0.5, it indicates overexploitation (f>m). from the analysis, emax, e0.1 and e0.5 were also estimated from the modified form of beverton and holt (1964) relative yield per recruit (y’/r) analysis by pauly and soriano (1986). relative yield per recruit (y’/r) and relative biomass per recruit (b’/r) values were carried out using the fisat ii software. results growth parameters of e. lacerta: a total of 865 specimens (494 males and 371 females) were analysed. the size of first sexual maturity size was 26.56 cm (fl50) for males, 33.11 cm (fl.) for females and 27.75 cm for population in the ivorian’s exclusive economic zone (fig. 2). the smallest mature individual is 23.4 cm. the fish sampled were classified into three groups, juveniles (sizes < 23.4 cm), sub-adults (23.4 cm < sizes < fl50) and finally adult (sizes ≥ fl50). the restructured von bertalanffy growth curve and length frequency plot (fig. 3) and the estimation growth coefficient have made it possible to determine the values of the asymptotic length and the growth coefficient. the von bertalanffy growth model for e. lacerta in the ivorian’s exclusive economic zone is described as lt = 60.38 (1 e (0.39 (t to))). the values 363 int. j. aquat. biol. (2021) 9(6): 360-369 of the growth parameters of e. lacerta are presented in table 1. the number of oblique curves representing the number of generations of e. lacerta. the start of the growth curve approximates the breeding period of the species. this allows the time or month of birth for any individual length surveyed to be located at a wellknown date. the breeding of e. lacerta takes place from january. table 1. growth parameters of elops lacerta. growth parameters fl min-max l∞ k rn t0 tmax ø’ w∞ population 20-57 60.38 0.39 0.176 0.458 7.69 3.153 2269.86 fl: fork length (cm); l∞: asymptotic length (cm); k: growth coefficient (year-1); rn: response surface; t0: age at zero length (year); tmax: maximum age (year); ø’: growth performance index; w∞: asymptotic weight (g). table 2. exploitation parameters of elops lacerta exploitation parameters m f e (f/z) lc lopt e10 e50 emax population 0.80 0.77 0.49 25.28 35.86 0.57 0.34 0.673 m: natural mortality; f: fishing mortality; e: exploitation rate; lc: size at length at first capture; lopt: optimal catch size; e10: exploitation level at 10%; e50: exploitation level at 50%; emax: exploitation level maximal. figure 2. size of the first sexual maturity (fl50) of elops lacerta population in the ivorian’s exclusive economic zone. figure 2. restructured von bertalanffy growth curve and length frequency plot of elops lacerta. 364 loukou et al./ population parameters of elops lacerta in the ivorian’s exclusive economic zone exploitation parameters of e. lacerta: the total mortality (z), the natural mortality (m) at 28.35°c and the fishing mortality (f) were respectively 1.57, 0.80 and 0.77 year-1. exploitation level is estimated as 0.49 (fig. 4). the size at first capture (lc), obtained from the probability of capture was 25.28 cm. the lengths at which 25 and 75% of fish are captured were respectively 24.01 and 26.56 cm (fig. 5). the optimal catch size is 35.86 cm. the recruitment pattern’s continuously throughout the year. the percent recruitment varied from 1.15 at 22.75% (fig. 6). the size distribution of the catches suggests two peaks of recruitment, the first in may (22.75%) and the second in november (5.21%) belonging to the long and short marine warm seasons, respectively. the histogram of the virtual population analysis (fig. 7) indicates that natural mortality is highest in juveniles and sub-adults. it decreases as the size of the individual’s increases. smaller individuals survive the most in this environment. fishing mortality is more pronounced in adult individuals (sizes ≥ 27.75 cm). figure 8 shows the evaluation of yield and biomass per recruit as a function of fishing mortality. the curves for yield and biomass per recruit are on the x axis and fishing mortality on the y axis. the exploitation rate (fig. 9) at different levels as e10, e50 and emax have been determined using the reports of lc/l∞ and m/k. the exploitation parameters of table 3. the relative yield per recruit and relative biomass per recruit predicted at different rate of exploitation in elops lacerta. e 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 y’/r 0.008 0.015 0.02 0.023 0.024 0.024 0.023 0.02 0.016 0.013 b’/r 0.816 0.65 0.502 0.373 0.263 0.174 0.104 0.053 0.020 00 e: exploitation rate; y’/r: yield per recruit; b’/r: biomass per recruit. figure 4. length-converted catch curve of elops lacerta. figure 5. probability of capture of elops lacerta. figure 6. recruitment pattern of elops lacerta. 365 int. j. aquat. biol. (2021) 9(6): 360-369 e. lacerta are presented in table 2. the yield per recruit reached a maximum at an exploitation rate of 0.6 and decrease as it goes along the exploitation rate increased (table 3). discussions the ivorian fishing area is located in the tropical region characterized by warm waters and abundant light that favors the growth of phytoplankton, which figure 7. virtual population analysis of elops lacerta. figure 8. yield per recruit (y’/r) and biomass per recruit (b’/r) of elops lacerta in function of the fishing mortality rate (f). (fmax: value of f which gives the maximum possible yield). 366 loukou et al./ population parameters of elops lacerta in the ivorian’s exclusive economic zone provides most of the primary production on which marine food webs depend. the asymptotic length obtained in this pelagic fish would be linked on the one hand to an important availability of food in our studied environment necessary for the essential functions of the organism. the protein and energy requirements of a fish depend on the size, age, physiological condition and environment of the animal. thus, as an individual grows, its needs increase and needs to feed more. on the other hand, the high temperature of the environment would lead to an increase in the length of the fish. indeed, when the temperature of the environment is high, the organism undergoes a rapid digestion accelerating the process of anabolic which induces a rapid growth to the increase of the voluntary ingestion and the conversion index. high temperatures would have a positive influence on the asymptotic length. climate change influences living conditions in the oceans. however, many fish species developed adaptive strategies to varying environmental temperatures. the warming of the water would cause an increase in metabolic rates which would lead to a regression of growth rates. moreover, the growth rate of e. lacerta is relatively low compared to those reported by niyonkuru et al. (2007), abdul et al. (2015) and dienye et al. (2021). the growth coefficient obtained would be related to the sexual maturation of the gonads. indeed, the migration of these individuals to the marine environment to continue their growth and reproduction leads to a predominance of adult individuals in the environment. in addition, wagué and papaconstantinou (1997), explain that sexual maturation is probably one of the most important factors in the fall of the growth rate in fish. thus, the growth rate would be high if the population is composed of more juveniles and low when there are more old individuals. hie-daré (1982), also indicated that immature e. lacerta in lagoon has a rapid growth in this environment. the calculated size growth performance index (ø') is higher than that reported by niyonkuru et al. (2007) and dienye et al. (2021). the results of abdul et al. (2015) are slightly higher than our results. this difference can be explained by the methods for estimating the l∞ and k parameters. similar observations have been made by pauly (1991). baijot and moreau (1997), also reported that the mean values figure 9. relative yield per recruit (y’/r) and biomass per recruit (b’/r) of elops lacerta. 367 int. j. aquat. biol. (2021) 9(6): 360-369 of the size growth performance index of several african fish species are between 2.65 and 3.32 are considered low. elops lacerta can be considered as having a low growth performance. the maximum life span in our study differs from that those of niyonkuru et al. (2007) and abdul et al. (2015). it is important to mention that better living environment conditions significantly impact the life expectancy of any organism; this could justify the longer lifespan found in this species in the ivorian zone. the recorded mortality parameters indicate that natural mortality is higher than fishing mortality. this predominance of natural mortality over fishing mortality would be related to environmental conditions, diseases, interspecific responses such as predation and pollution. the size at first capture is lowest than the size of first sexual maturity and the optimum size of capture. this species living at depths between 1 and 50 m in marine environment, would not escape the different fishing gears. the individuals of e. lacerta caught have not had the opportunity to reproduce several times. the sustainability of the e. lacerta population is threatened in the exclusive economic zone. the recruitment of e. lacerta marked by important peaks observed during the long and short marine warm season would be explained by a preference of warm waters of the species. the massive arrival of individuals in the exploitable stock at these times of the year would be explained by the fact that the environmental conditions are more favorable for these individuals, which would also be strategy to provide more opportunities of individual to survive in at environment. in addition to temperature, salinity is an abiotic factor that can influence the biological cycle of the recruits. our work revealed that fishing mortality is more pronounced in adult individuals. indeed, the majority of juveniles and sub-adults would escape the various fishing gears, unlike the larger and more robust adult individuals. thus, the continuous fishing pressure on adult individuals could lead to a low rate of spawning stock in the environment, hence the inability to ensure the sustainability of the species. conclusion this study provides the first information on the growth and exploitation parameters of e. lacerta in the marine environment. it appears that the stock of e. lacerta exploited in the exclusive economic zone is dominated by large individuals. however, the implementation of a management policy for this resource prohibiting the capture of specimens of e. lacerta at sizes below the optimum catch size. acknowledgements we would like to thank all the staff of the aquaculture department of the center of oceanological research for their contribution to this work and in particular sylla soumaïla. references abdul w.o., omoniyi i.t., adekoya e o., adeosun f.i., odulate o.o., idowu a.a., olajide a.e; olowe o.s. 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(1984). biostatistique. gäetan morin. 850 p. uicn. (2019). elops lacerta. the iucn red list of threatened species. available from: https://www. iucnredlist.org/fr/species/182549/135034555. retrieved 26/05/2021. von bertalanffy l. (1938). a quantitative theory of organic growth (inquiries on growth laws ii). human biology, 10(2): 181-213. wagué a., papaconstantinou c. (1997). age, croissance et mortalité du serran hepate, serranus hepatus (linné, 1758) (poisson, serranidae) dans le golfe de thermaikos (mer egée, grèce). marine life, 7 (1-2): 39-46. international journal of aquatic biology (2014) 2(6): 346-350 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2014 npajournals. all rights reserved original article assessment of essential elements in the wild beluga sturgeon (huso huso) caviar from caspian sea seyed vali hosseini1, seyedeh fatemeh monsef rad *2,1hamed kolangi miandare2, mohammad harsij3, saeed sharbaty2 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2department of fisheries, gorgan university of agricultural sciences and natural resources, gorgan, iran. 3department fisheries, gonbad-e-kavoos university, gonbad-e-kavoos, iran. article history: received 21 october 2014 accepted 4 december 2014 available online 2 5 december 2014 keywords: beluga sturgeon caviar caspian sea risk assessment essential metals abstract: in this study, the concentration of calcium (ca), cobalt (co), chromium (cr), copper (cu), iron (fe), potassium (k), magnesium (mg), manganese (mn), selenium (se) and zinc (zn) as essential metals, were determined in caviar of wild beluga sturgeon caught from the caspian sea in march 2012. potassium (4885.51± 17.81 µg g-1) and magnesium (346.61± 6.6 µg g-1) had the highest concentration and cobalt and manganese levels were less than 0.01 mg kg-1 wet weight. the mean concentration of copper and zinc in the caviar samples were under the permissible limits proposed by the uk’s ministry of agriculture, fisheries and foods. the caviar maximum allowable daily consumption rate was calculated. however, the health risks from caviar consumption are uncertain because the amount of caviar consumed by heavy users is not known. introduction trace metals (e.g., arsenic, copper, iron, molybdenum, tin, etc.) are essential components of the hydrosphere and are necessary for normal metabolism of organisms in low concentration; however, elevated concentrations in tissues might be toxic for biological activities (joyeux et al., 2004). the rapid development of industry and agriculture has increased the heavy-metal pollution, which can be intensely accumulated and biomagnified in the water, sediment, and aquatic food chains (yi et al., 2011). sturgeons are the principal source of the most valuable food, i.e. caviar (speer et al., 2000) and the caspian sea have supplied more than 95% of world’s caviar output (mashroofeh et al., 2012). on the other hand, this lake receives the huge amount of pollutants including petroleum products, organic substances, metals and nitrogen compounds (unep, 2008). six sturgeon species also exist in the caspian * corresponding author: seyedeh fatemeh monsef rad e-mail address: monsefrad@gmail.com sea that four of them, including persian sturgeon, acipenser persicus; russian sturgeon, a. guildensteidti; stellate sturgeon, a. stellatus; and beluga sturgeon, huso huso are commercially valuable (agusa et al., 2004; pourang et al., 2005). overfishing of sturgeon for caviar and meat, loss of habitat due to the construction of dams on the caspian sea tributaries, and elevated levels of pollutions have been led to great decline in their population (iucn, 2012a). caviar of beluga is the most coveted of all caviar (speer et al., 2000). on the other hand, beluga as a predator, is at the top trophic level feeding on many fish species such as caspian roach (rutilus caspicus l.), common carp (cyprinus carpio l.) and kilka (clupeonella), and have the longest life time (mashroofe et al., 2012; iucn, 2012b). because of bioaccumulation through the food web, it is expected high levels of contaminants, such as metals, in beluga sturgeons (wang et al., 2008). some methods 347 hosseini et al/ essential metals in wild beluga sturgeon caviar have been proposed for potential risks estimation to human health due to heavy metals in food. these risks may be divided into two groups, including carcinogenic and non-carcinogenic impacts (yi et al., 2011). risk assessment is one of the fastest methods, which is required to investigate the effect of the hazards on human health and to determine the level of treatment, which tends to solve the environmental issues that occur in daily life (amirah et al., 2013). therefore, the objectives of the present study were to determine the concentration of ca, co, cr, cu, fe, k, mg, mn, se and zn in the caviar samples of wild beluga sturgeon and also compare the results obtained in other studies regarding metal levels; moreover, the potential human health risk due to the caviar consumption is evaluated. material and methods sample collection: a total of nine wild beluga sturgeons (with mean weight of 73.2 ± 8.2 kg) were caught by the shahid marjani hatchery center (gorgan, north of iran) from the southeastern coast of the caspian sea in march 2012. the specimens were washed and gutted on the site under hygienic conditions. then, 65 g of roe (“caviar”) free of the egg sack membrane were removed from each female, placed into separate 100 ml plastic containers with lids and immediately transported to the laboratory in an insulated box with a suitable quantity of flaked ice to completely cover the containers and kept frozen at -40°c in a freezer until analyzed. determination of trace elements: the samples were analyzed three times for ca, co, cr, cu, fe, k, mg, mn, se and zn by inductively coupled plasmaoptical emission spectrophotometer (icp-oes) (optima 2100dv, perkin elmer inc., waltham, ma, usa) as described by türkmen et al. (2009). briefly, about 5 g of homogenized caviar (using a blender panasonic, mj-w176p, osaka, japan) was mixed with 50 ml of ultrapure concentrated hno3. the mixture was heated on a lab digital heater (ika, staufen, germany) to 100-150ºc for about 2 hrs until the tissue dissolved and the solution evaporated to near dryness. all organic materials in each sample were completely digested by repeating the digestion twice more. after cooling, 5 ml of 1 n hno3 was added to the digested residue. afterward, it was transferred to a 25 ml volumetric flask and brought to level with by purified water with conductivity under 0.05 μs (millipore system, eschborn, germany). the recoveries of the metals were assessed by adding increasing amounts of each element to samples (spiking method), which were then taken through the digestion procedure. the resulting solutions were analyzed for their metal concentrations. recoveries of the metals ranged from 96.8-102%. in this study, metal concentrations were determined as mg kg-1 wet weight of roe. risk assessment: since, there is not information about caviar consumption, the maximum allowable daily consumption rate (madcr) for each metal was used for chronic non-cancer health risk assessment. the madcr (g/day) for caviar consumption was calculated using the following equation (wang et al., 2008): madcr= 106 × hq × rfd × (bw/c), where hq (hazard quotient) = 1; rfd is the reference dose (mg/kg/day) set by us epa(2011); bw is the body weight of 70 and 14.5 kg for an adult and child, respectively, and c is the metal concentration in the caviar samples (mg kg-1) and the constant adjusts for concentration conversions. also, the metal levels in caviar samples were compared with the guidelines proposed by maff (2000). results and discussion metal concentration: mean levels of essential metals in caviar of wild beluga are shown in table 1. among the measured metals, k (4885.51 ± 17.81 µg g-1) and mg (346.61± 6.6 µg g-1) had the highest levels, and se (0.9 ± 0.042 µg g-1) had the lowest level. also, the averages of co and mn were below detection limits in the samples. in another experiment conducted by wang et al. (2008), k (1.4 ± 0.2 µg g-1), mg (0.25 ± 0.02 µg g-1), ca (0.10 ± 0.03 µg g-1), and fe (0.06 ± 0.05 µg g-1) had the 348 international journal of aquatic biology (2014) 2(6): 346-350 highest levels in the caviar samples. some nutrients such as calcium, iron, magnesium, and potassium are essential elements required to maintain cellular function in the roe (hui, 2006; wang, 2008). gessner et al. (2002) detected cu and zn levels of 1.45 and 11.60 µg g-1 wet weight in beluga fresh caviar samples from the caspian sea. sadeghi rad et al. (2005) also found cu and zn levels of 1.74-6.8 and 33.8-131.2 µg g-1 wet weight, respectively, in caviar samples of wild persian sturgeon from southern caspian sea that were higher than the results of the present study. in the current finding, zn level was higher than the results were reported by wang et al. (2008) in caviar of beluga sturgeon from southern caspian sea; however, our finding showed that cu and zn concentration were lower than the dietary guidelines for fish, as represented by maff (2000) (standard: cu: 20 µg g-1ww ; zn: 50 µg g-1 ww). according to literature, cu and zn levels in fish gonads may increase during the pre-spawning period, and eggs contain large amounts of both metals (atukorala and waidyanatha, 1987; zubcov et al., 2012). it seems that contents of some metals such as copper and zinc in fish and other aquatic vertebrates are modified by diet, age of the organism, reproductive state, and other variables (elsier, 1993). in comparison to our results, levels of cu (0.7-1.6 µg g-1 ww), k (1017-1603 µg g-1ww), mg (230.1-266.0 µg g-1ww) and zn (16.8-24.0 µg g-1ww) were detected by wang et al. (2008) in eurasian caviar that were lower than our results, and concentrations of ca (56.8-173.9 µg g-1ww), co (5.2-23.9 ng g-1 ww), mn (0.8-1.4 µg g-1ww) and se (1.0-2.1µg g-1 ww) that were higher, and fe levels (21.6-155.1 µg g-1ww) that was comparable. these differences may be interpreted by many factors such as habitat, seasonal variations, individual affinity for metal uptake and dietary habits that influence on variations observed in metal concentrations among sturgeon species (papagiannis et al., 2004). on the other hand, the difference of physiological features of different fish tissue can affect on the accumulation of a particular metal (pourang et al., 2005). in other words, biomonitoring using caviar may not be effective for some contaminants, including some essential metals such as ca, co, cu, k, mg and zn that may accumulate in other organs like liver (wang et al., 2008), and the increased levels may reflect the ca co cr cu fe 51.7(±0.62) <0.01 0.28(±0.026) 1.27(±0.15) 65.61(±0.45) k mg mn se* zn 4885.51(±17.81) 346.61(±6.6) <0.01 0.90(±0.042) 24.32(±0.23) *ng g-1 wet weight table 1. average (±sd) metal levels (µg g-1 wet weight) in caviar of wild beluga sturgeon (huso huso; n=9). metals rfd2 (mg/ kg/ day) madcr (g/day) children adult cr(iii) 1.50e+00 77700 375000 cu 4.0e‐02 460 2210 fe 7.0e‐01 155 747 mg 4.90e+01 205 990 se* 5.00e-03 80600 389000 zn 3.00e-01 180 863 1data arranged by three significant figure 2reference doses of metals set by epa (2011) *ng g-1 wet weight table 2. maximum allowable daily consumption rate (madcr) estimates for children and adults using median concentration (mg kg-1 wet weight)1. 349 hosseini et al/ essential metals in wild beluga sturgeon caviar elevated requirement of sturgeons for these components (gessner et al., 2002). concentrations of essential metals are regulated by physiological mechanisms in fish (pourang et al., 2005); however, they are regarded as potential risks that can endanger both fish and human health (yilmaz et al., 2007). health risks: table 2 shows the toxicity information and the estimated madcr. analysis of risk is important due to the health benefits of consuming caviar, which is considered to be a nutritious food. the rate of caviar consumption is unknown; therefore, the health risks calculation of caviar consumption are impossible. however, the madcr provides an approach to evaluate its potential importance (wang et al., 2008). the madcr shows an average daily consumption rate that would not be expected to cause adverse non-carcinogenic health effects (epa, 2000). in this study, for adult chronic non-cancer health effects using a hq of 1, the madcr is relatively low for fe and zn especially for children. in comparison to our result, wang et al. (2008) estimated the madcrs for cr (358×104 g/day for children; 1727×104 g/day for adult) that was higher, zn (200 g/day for children; 980 g/day for adult) that was comparable, and se (53 g/day for children; 980 g/day for adult) that was more restrictive. because of the difference in body weight, the madcrs for children are about five times lower than for adults; although the same per kg values are used as insufficient work has been done to generate separate values for children. in conclusion, this study analyzed the concentration of some essential metals (co, cr, cu, fe, k, mg, mn, se and zn) in caviar samples of wild beluga sturgeon. the caviar consumption rate is expected to be very low, and regarding to health benefits of consuming caviar, it seems that consumption of this food does not pose any danger for human health. references agusa t., kunito t., tanabe s., pourkazemi m., aubrey d.g. 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(2021) 9(1): 41-54 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article food habits, ecomorphological patterns and niche breadth of the squeaker, synodontis schall (pisces: siluriformes: mochokidae) from niger river in northern benin hamidou arame1, alphonse adite* 1, kayode nambil adjibade1, rachad sidi imorou1, edmond sossoukpe2, sonon p. stanislas1 1laboratory of ecology and aquatic ecosystem management, department of zoology, faculty of science and technology, university of abomey-calavi, cotonou, benin. 2laboratory of wetland researches, department of zoology, faculty of science and technology, university of abomey-calavi, cotonou, benin. s article history: received 17 august 2020 accepted 19 january 2021 available online 2 5 february 2021 keywords: conservation diet sustainable exploitation omnivore mochokidae abstract: the squeaker, synodontis schall dominates the mochokid fish sub-community in niger river in northern benin and shows a great economic and commercial importance. the diet of s. schall has been analysed to evaluate the food habit and resource utilization in this regional river. fish samplings were made monthly from february 2015 to july 2016 using unbaited longlines and traps, seines and experimental gillnets. the results indicated that s. schall is an omnivore foraging in benthic and pelagic habitats with diet dominated by aquatic insects (34.32%), sand particles (18.768%), macrophytes (13.415%), seeds (8.549%), roots (8.319%), detritus (5.344%), mollusks (1.204%) and phytoplankton (0.6255%). the omnivore food habit depicted was also shown by the ecomorphological analysis mainly the relative gut length (gl/sl) varying between 0.8 and 5. the species showed high diet flexibility with high niche breadth ranging between 1.86 and 5.74. synodontis schall exhibited an ontogenetic diet shift that was also confirmed by pianka’s diet overlap indexes ranging between øjk=0.54-0.93. the conservation and the sustainable fisheries exploitation of s. schall require the reinforcement of fishing regulation, habitat protection and ecosystem followup. introduction knowledge on diet composition and food habits of fishes is important for habitat protection, species conservation, fisheries management and fish culture (rosecchi and nouaze, 1987; adite et al., 2007; kone et al., 2007). furthermore, dietary analysis gives a better understanding of how food resources are shared and constitutes a basic tool for assessing trophic structure and fish’s capability to adapt to environmental changes (rosecchi and nouaze, 1985; hajisamae et al., 2003; adité et al., 2006; berté et al., 2008). in west africa, catfishes are represented by 124 species, 24 genera and 8 families among which the family of mochokidae made about 48 species (paugy et al., 2004). among mochokids, synodontis is the most diverse genus comprising about 36 species (paugy and roberts, 2004) with s. schall, the most widespread and dominant species that occurs in most african rivers such as senegal, gambia, volta, tchad, and niger (paugy et al., 2003). in niger river, paugy *correspondence: alphonse adite doi: https://doi.org/10.22034/ijab.v9i1.973 e-mail: alphonseadite@gmail.com and levêque (2004) reported 28 species for the genus synodontis, with s. schall, the dominant and the most tolerant species to adverse environmental conditions (lowe-mcconnell, 1987). in some african water bodies, s. schall is described as an omnivore feeding on macro-invertebrates, macrophytes and detritus (willoughby 1974; hickley and bailey, 1987; ofori-danson, 1992). the species reproduces all seasons with peaks in wet and flood periods. in niger river in benin, to date, there is a gap of information on the feeding patterns of s. schall and no published work is available on its trophic ecology. in benin, s. schall dominated the mochokid subcommunity in many rivers and streams such as oueme, okpara, zou, sô, hlan, tove, mono, and niger (lalèyè et al., 2004; montchowui et al., 2007; djidohokpin et al., 2017; hazoume et al., 2017; sidi imorou et al., 2019). in niger river in northern benin, arame et al. (2019) reported only the genus synodontis comprising fourteen valid species, with 42 arame et al./ food habits of synodontis schall from niger river in northern benin s. schall, the most abundant species making numerically 74.50% of the mochokid fish assemblages (arame et al., 2020). despite the abundance and fisheries importance of s. schall that is intensively exploited in niger river in benin, nothing is known about the feeding ecology and resource utilizations. successful fisheries management and fish culture require knowledge on trophic ecology of the target species. the current research aims to study the diet composition and feeding habits of s. schall in niger river in bénin. materials and methods study area: the research area is the niger river in northern benin around malanville township. this region is located between 11°52'05″n and 3°22′59″e at an altitude of 200 m, and extended on about 3,016 km². the niger river is a regional running water that stands as a frontier between the two neighbor countries, benin and niger republics. in benin, the three tributaries, mékrou, sota and alibori of niger river caused severe inundations with a peak flood reaching 275 km2 that boosted a high fish productivity (welcomme, 1985; moritz et al., 2006; adjovi, 2006; adite et al., 2017). the niger river valley shows sandy-clayish and ferruginous soils showing plant communities comprising rooted, floating and submerged vegetation. a multi species artisanal fisheries occurred on floodplains, pools, river channels and involved many ethnic groups (hauber, 2011; arame et al., 2019; adjibade et al., 2019). collection sites: four stations were selected on niger river for the sampling of the fish species (arame et al., 2020): (1) tounga village located at 11°52'216"n, 3°23'907"e constitutes a highly degraded site, (2) behind dry port, situated at 11°52'216"n, figure 1. (above) internal organs and (below) structure of the digestive tract of synodontis schall from niger river in northern benin (1, esophagus; 2, stomach; 3, cardiac stomach; 4 (above), intestine; 4 (below), anterior intestine; 5 (above), rectum; 5 (below), medium intestine; 6 (above), anal orifice; 6 (below), rectum; 7, posterior intestine; 8, anal orifice). 43 int. j. aquat. biol. (2021) 9(1): 41-54 3°23'907"e, is also degraded, (3) money village, located at 11°52'987"n, 3°20'819"e, is a less degraded site, and (4) gaya village, less degraded, is located at 11°52'675"n, 3°25'329"e in the river at niger republic side. mochokid fish collections: fish samplings were performed monthly from february 2015 to july 2016 in aquatic vegetation and in open water habitats at the four stations. unbaited longlines and traps, seines and experimental gillnets were used for the samplings. collection procedures follow adite et al. (2013). mochokid samplings were also performed from fishermen artisanal catches on the basis of 1/3 of species abundance when the abundance exceeded 50 individuals. all individuals were retained for the sample when the abundance is less than 50 for a given species (kakpo, 2011; okpeicha, 2011). the fishes were then identified in situ based on van thielen et al. (1987) and levêque and paugy (2006). identified fishes were preserved in 10% formalin and transported to laboratoire d’ecologie et de management des ecosystèmes aquatiques (lemea), at the faculty of sciences and technics, in the university of abomeycalavi. in the laboratory, fish individuals were transferred into 70% ethanol to facilitate biological observations. http://www.fishbase.org (froese and pauly, 2018) were used to confirm fish species. dietary analysis: after sampling, each individual was measured for total length (tl) and standard length (sl) to the nearest 0.1 mm with an ichtyometer, and weighed to the nearest 0.01 g with an electronic balance (camry 0.1 g / 500 g; aws). each individual was then dissected and the gut was removed and measured (adite et al., 2007; gbaguidi et al., 2016). the stomach was then opened and food resources were removed and spread on a glass slide for examination first under a binocular to identify macroscopic foods items. then, a photonic microscope was used to identify fine food resources and algae (adite et al., 2017). references such as needham and needham (1962), bourrelly (1985, 1990) and tachet et al. (2010) were used to identify prey items at the lowest possible taxonomic level. the volume (v) of each food resource identified from the 1505 stomach dissected was estimated by water displacement following adite and winemiller (1997). data analysis: the estimated volume (v) and counts of each identified food item were recorded on excel software spreadsheet and the proportional volumetric consumption (%v) of each food item was computed using the formula (adite and winemiller, 1997): %v =vi vt × 100 where, %v is the proportional volumetric consumption of food item i in the diet, vi is the total volume of the food item i in n stomachs, vt is the total volume of food resource ingested by n stomachs, and n is the total number of stomachs dissected. the volumetric proportions of each food item consumed were calculated for different size classes to examine ontogenetic diet shifts. the analysis of variances (anova) was run with spss software (morgan et al., 2001) to assess spatial and seasonal variations in diet. empty stomach indexes (ce) were computed as the ratio of number of empty stomachs to total number of stomachs examined: ce = ni nt ×100 where, ce is the coefficient of emptiness, ni is the number of empty stomachs and nt is the total number of stomachs examined. the following formula of bahou et al. (2007) was used to estimate the occurrence frequency (of) of each food resource in the diet: of= ji nt ×100 where, ji is the number of stomachs containing prey item i and nt is the total number of non-empty stomachs. according to sorbe (1972), the prey is classified “accidental”, “secondary” and “preferential” when of<10%, of between 10 and 50% and of>50%, respectively. diet breadth (db) was computed following simpson (1949): diet breadth (db) = ∑ = n i ip 1 21 where, pi is the proportion of food resource i in the diet and n is the total number of food items in the diet. in general, the db ranges from 1 (when only one food resource is ingested), to n in case all food resources 44 arame et al./ food habits of synodontis schall from niger river in northern benin are ingested in equal proportions. the values of db were submitted to anova using spss software version 21 (morgan et al., 2001) to show the variation among life stage. pianka's diet overlap index (øjk) (1976) was computed between fish size classes to examine diet similarities and ontogenetic diet shift: ∅jk = ∑ ∑ ∑ = = = × n i n i ikij n i ikij pp pp 1 1 22 1 where, øjk is the pianka’s dietary overlap between species j and species k, pij is the proportion of resource i used by species j, pik is the proportion of food item i used by species k and n is the number of food resource ingested. the eco-morphological analysis of the diet was evaluated using the linear regressions between gut length (gl) and body weight (w) and between gut length (gl) and standard length (sl). likewise, the ratio (gl/sl) was computed as a measure of relative gut length and compared to published reference ratios 0.8 to 5 for omnivores (al-hussaini, 1947; kapoor et al., 1975). also, the eco-morphological patterns were examined to document the food habit of s. schall. results the digestive tract of s. schall: the morphology of digestive tract showed a thick-walled esophagus followed by a well-developed fork-like shape stomach, an intestine (anterior, medium and posterior), a rectum and an anal orifice (fig. 1). the pyloric caecum is absent. diet composition: in niger river, s. schall foraged mainly in pelagic and benthic habitats where the species consumed about 221 food resources dominated by aquatic insects (34.32%), sand particles (18.768%), macrophytes (13.415%), seeds (8.549%), roots (8.319%), detritus (5.344%), worms (4.735%) and rice hulls (3.681%), aggregating 97.131% of the stomach content (table 1). minor preys were mollusks (1.204%), fish scals (0.796%), crustacean (0.161%), zooplankton (0.083%) and phytoplankton (0.6255%) (table 1). seasonal variations of diet: figure 2 shows volumetric percentage (%) of preys consumed by s. schall according to seasons. the results of the food preys ingested showed significant (p<0.05) seasonal variations for insects (f2,1502=5.855, p=0.003), seeds (f2,1502=19.865, p=0.001), roots (f2,1502=18.029, p=0.001), detritus (f2,1502=16.24, p=0.001), phytoplankton (f2,1502=11.875, p=0.001), zooplankton (f2,1502=15.142, p=0.001), macrophytes (f2,1502=5.466, p=0.004). indeed, the highest volumetric percentage of insects (44.67%) ingested was recorded during flood while those of seeds (19.34%), roots (11.63%) and detritus (8.57%) were figure 2. seasonal variations of food resources consumed by synodontis schall in niger river in northern benin. 45 int. j. aquat. biol. (2021) 9(1): 41-54 table 1. volumetric, occurrence and numeric percentages of prey items ingested by synodontis schall from niger river in northern benin. prey category prey / family/genus / species volumetric percentage (%v) number (n) numeric percentage (%n) phytoplankton blue algae cyanophyceae 0.096 435 5.226 green algae chlorophyceae 0.181 942 11.318 trebouxiophyce 0.004 22 0.264 ulvophyceae 0.002 11 0.132 desmids eustigmatophyceae 0.002 14 0.168 zygnematophyceae 0.004 21 0.252 diatoma bacillariophyceae 0.22 1098 13.192 coscinodiscophyceae 0.051 179 2.151 mediophyceae 0.032 174 2.091 undetermined h l k unidentified phytoplankton 0.034 314 3.773 total phytoplankton 0.6255 1765 38.568 zooplankton branchiopoda cladocerans 0.014 3 0.036 copepods 0.032 4 0.048 rotifera brachionidae 0.038 2 0.024 total zooplankton 0.083 9 0.108 worms nematoda 3.138 433 5.202 annelides oligochaetes 1.571 20 0.240 glossiphoniidae 0.026 2 0.024 total worms 4.735 455 5.467 insects ephemeroptera leptohyphidae 0.189 3 0.036 ephemerellidae 0.039 4 0.048 heptageniidae 0.124 3 0.036 baetidae 0.131 4 0.048 hydroptilidae 0.112 2 0.024 siphlonuridae 0.112 2 0.024 plecoptera pachygronthidae 0.004 2 0.024 leuctra geniculata 0.025 1 0.012 odonata libellulidae 1.128 53 0.637 lestidae 0.175 4 0.048 coenagrionidae 0.25 10 0.120 calopterygidae 0.112 3 0.036 aeshnidae 0.1 2 0.024 heteroptera tettigoniidae 0.05 1 0.012 notonectidae 0.037 1 0.012 pleidae 0.05 2 0.024 coleoptera aphodidae 0.006 1 0.012 copridae 0.149 1 0.012 coenagrionidae 0.093 2 0.024 curculionidae 0.301 13 0.156 dytiscidae 0.137 4 0.048 elmidae 0.398 7 0.084 elminthidae 0.121 3 0.036 ecnomidae 0.003 2 0.024 hydraenidae 0.243 10 0.120 hydrochidae 0.065 7 0.084 hydrophilidae 6.731 250 3.004 hydroporinae 0.162 4 0.048 noteridae 0.1 1 0.012 pleidae 0.1 1 0.012 psephenidae 0.025 1 0.012 tricoptera agupetidae 0.056 1 0.012 philopotamidae 0.137 5 0.060 sericostomatidae 0.028 5 0.060 helicopsychidae 0.025 1 0.012 46 arame et al./ food habits of synodontis schall from niger river in northern benin recorded during the wet period. also, the highest volumetric percentage of phytoplankton (0.73%) and zooplankton (0.14%) consumed were recorded during the dry season. nevertheless, there were no significant (p>0.05) seasonal dietary variations for mollusks (f2,1502=0.055, p=0.946), rice hulls (f2,1502=0.510, p=0.477), worms (f2,1502=0.275, p=0.760), crustaceans (f2,1502=2.613, p=0.074), sand particles (f2,1502=0.963, p=0.382) and fish scales (f2,1502=2.527, p=0.080) (fig. 2). frequency of occurrence in diet: the analysis of the occurrence frequencies (of) of the food resources revealed that phytoplankton was ingested by all 1505 individuals collected with an occurrence frequency of=100% indicating that, though of reduced volumetric percentage (0.6255%), phytoplankton appeared to be the preferential prey. also, insects, sand particles and detritus with a huge aggregated volumetric percentage (vp=58.42%), displayed a high of estimated at 73.53, 59.19 and 59.11%, respectively, that classified them as preferential preys (table 2). the secondary preys consumed were worms, roots, zooplankton, seeds and macrophytes that were common in some stomachs with moderate of ranging between 12.04 and 35.74% (table 2). the accidental preys, rice hulls, fish scales, mollusks, and crustaceans ingested by s. schall occurred just in few stomachs with reduced of varying between 0.55 and 6.66%. diet according to life stages: the ontogenetic analysis of the diet indicated that preys such as roots (59.63%), insects (16.75%) and sand particles (15.30%) dominated the stomach of juveniles. in addition, macrophytes (15.97%) and rice hulls (9.23%) consistently occurred in the stomach of subadults, and insects (38.56%) relatively dominated the diet of adults (table 3). overall, the three life stages tended to consume more roots, insects and sand table 1. continued. prey category prey / family/genus / species volumetric percentage (%v) number (n) numeric percentage (%n) insects hydroptidae 0.003 3 0.036 hydropsychidae 4.051 310 3.725 lepidostomatidae 0.152 6 0.072 limnephilidae 0.025 2 0.024 glossosomatidae 0.03 8 0.096 polycentropodidae 0.165 2 0.024 diptera chironomidae 9.918 621 7.461 ceratopogonidae 1.388 60 0.721 dasyheleinae 1.196 52 0.625 chaoboridae 0.065 3 0.036 psychodidae 0.006 3 0.036 insects parts 3.812 179 2.151 indetermine insects unidentified insects 1.994 204 2.451 total insects 34.32 1869 22.456 total mollusks mollusks sphaeriidae 1.204 41 0.493 crustacean branchipodidae 0.001 2 0.024 platyischnopidae 0.008 2 0.024 candonidae 0.106 2 0.024 gammaridae 0.047 1 0.012 total crustaceans 0.161 7 0.577 fish scales fish scales 0.796 59 0.709 roots roots 8.319 361 4.337 seeds seeds 8.549 170 2.043 machrophytes machrophytes 13.415 172 2.067 rice hull rice hull 3.681 84 1.009 detritus detritus 5.344 748 8.987 sand particles sand 18.768 1138 13.673 total 100% 8323 100% 47 int. j. aquat. biol. (2021) 9(1): 41-54 particles. the presence of sand in a high volumetric percentage indicated that s. schall is a benthic feeder. macrophytes occurred only in the diet of sub-adults and adults probably because their digestive tracts were more developed than those of juveniles. the food items consumed by different life stages of s. schall showed significant (p<0.001) variations for insects (f2,1502=26.943, p=0.001), roots (f2,1502=4.607, p=0.001), rice hulls (f2,1502=2.5393, p=0.001), worms (f2,1502=4.055, p=0.001), macrophytes (f2,1502=23.24, p=0.001), seeds (f2,1502=21.370, p=0.001), detritus (f2,1502=47.711, p=0.001). however, there were no significant (p>0.05) ontogenetic variations in the consumption of mollusks (f2,1502=1.147, p=0.273), crustaceans (f2,1502=0.114, p=0.758) and fish scales (f2,1502=0.29363, p=0.932), phytoplankton (f2,1502=0.00127, p=0.129), zooplankton (f2,1502=0.708, p=0.136) and sand particles (f2,1502=9.482, p=0.29). empty stomachs: for this study, 1505 stomachs of s. schall were examined and 243 of them were empty (table 4). in general, the coefficient of emptiness varied with seasons and life stages and ranged between 5.88 and 23.47%. empty stomachs were higher in adults and averaged 19.08±4.57% whereas relatively lower percentages were recorded among sub-adults (mean: 11.22±4.65%). in contrast, almost all juveniles exhibited full stomachs and only one individual was empty. also, significant seasonal variations of empty stomachs were recorded during the study. indeed, higher values were recorded during the wet and flood periods where the coefficient of table 2. occurrence frequencies of prey consumed by synodontis schall from niger river in northern benin. prey categories occurrence frequency (%) food importance phytoplankton 100.00 preferential prey insects 73.53 preferential prey detritus 59.11 preferential prey sand particules 59.19 preferential prey zooplankton 17.51 secondary prey worms 35.74 secondary prey seeds 13.15 secondary prey machrophytes 12.04 secondary prey roots 28.92 secondary prey crustaceans 0.55 accidental prey mollusks 3.25 accidental prey fish scales 4.68 accidental prey rice hulls 6.66 accidental prey table 3. ontogenetic variations of preys consumed (volumetric percentage) by synodontis schall from niger river in northern benin. volumetric percentage (%v) prey categories juveniles (tl < 5) sub-adults (5 ≤ tl < 8) adults (tl ≥ 8) phytoplankton 0.5504 0.6008 0.6332 detritus 0.0612 3.2259 5.6091 roots 59.633 11.937 7.4895 seeds 6.8155 8.9774 macrophytes 15.9743 12.912 rice hulls 6.1162 9.2315 2.4342 worms 1.5291 3.4191 3.1153 mollusks 1.3559 crustaceans 0.1976 zooplankton 0.0738 0.0032 0.0388 insects 16.7458 25.1464 38.5602 fish scales 0.9225 0.7717 sand particles 15.2905 22.7238 17.9051 total 100% 100% 100% 48 arame et al./ food habits of synodontis schall from niger river in northern benin emptiness reached 22.07 and 19.20%, respectively, whereas that of dry season was relatively low with a value of 14.16% (table 4). pianka’s diet overlaps and ontogenetic diet shifts: overall, diet overlaps between size classes of s. schall ranged from øjk=0.54 (pairing "juvenile x subadults") to øjk=0.93 (pairing "sub-adult x adults") and averaged øjk=0.71±0.20 indicating relatively high diet similarities among different life stage categories. nevertheless, the reduced øjk=0.54 and øjk =0.66 recorded respectively between "juvenile" and "subadults" and between "juvenile" and "adults" indicated an ontogenetic diet shift (table 5). diet breadth: in niger river in benin, s. schall consumed a wide range of food resources reaching 221 food items classified in 13 foods categories (phytoplankton, detritus, roots, seeds, macrophytes, rice hulls, worms, mollusks, crustacean, zooplankton, insects, fish scales, and sand particles). consequently, a high diet breadth (db=4.93) was recorded for the table 4. seasonal variations of empty stomachs of synodontis schall from niger river in northern benin. life stage flood dry wet total nt ne ec (%) nt ne ec (%) nt ne ec (%) nt ne ec (%) juveniles<5 mm 5 1 20 5 1 20 sub-adults [5-8 mm] 14 2 14.3 252 34 13.49 17 1 5.88 283 37 13.07 adults≥8 mm 247 48 19.4 774 111 14.34 196 46 23.47 1217 205 16.84 total 261 50 19.2 1031 146 14.16 213 47 22.07 1505 243 16.15 nt=total number of stomachs, ne= number of empty stomachs, ec (%) = coefficient of emptiness table 5. matrix of diet overlaps (øjk) by life stage category of synodontis schall from niger river in northern benin. length classes juveniles (tl<5) sub-adults (5≤tl<8) adults (tl≥8) juveniles (tl<5) 1 0.54 0.66 sub-adults (5≤ tl< 8) 1 0.93 adults (tl≥8) 1 table 6. diet breadth (db) variations by life stage of synodontis schall from niger river in northern benin. length classes (ts, cm) seasons juveniles (tl<5) sub-adults (5≤tl<8) adults (tl≥8) total wet 4.44 5.22 5.32 flood 1.86 4.42 4.22 dry 2.46 5.74 4.34 4.84 total 2.46 5 4.45 4.93 table 7. spearman correlation coefficients between standard length (sl)/gut length (gl) and the volumetric percentages of food resources consumed by synodontis schall from niger river in northern benin. prey categories sl gl r a (slope) b p-value r a (slope) b p-value phytoplankton 0.3162 23.1 10.30 0.842 0.3162 191 17.2091 0.374 detritus 0.4472 3.506 10.2936 0.066 0.3317** 14.628 17.3040 0.0001 roots 0.3162 0.88 10.3166 0.645 0.5477 7.437 17.3239 0.036 seeds 0.4472 1.52 10.2873 0.090 0.3464** 7.150 17.3174 0.0001 macrophytes 0.8944** 2.9363 10.2479 0.001 0.6325 4.104 17.3472 0.01 rice hulls -0.8367** -8.63 10.4 0.001 -0.4899** -28.732 17.6907 <0.001 worms -0.3873 -0.32 10.3353 0.876 -0.3162 -1.001 1.4751 0.793 mollusks 0.3162 5.684 10.318 0.141 0.3162 3.610 17.4564 0.615 crustaceans -0.3162 -3.12 10.3 0.799 -0.3162 -11.08 17.4687 0.626 zooplankton 0.3162 6.667 10.3025 0.175 0.4000** 44.790 17.2657 0.0001 insects 0.5000** 3.8133 10.0679 <0.0001 0.5916** 10.621 16.7272 <0.0001 fish scales 0.3464 1.627 10.3293 0.831 0.3162 4.81 17.4567 0.735 sand particles 0.4472* 2.569 102291 0.050 -0.3606 -1.280 17.5163 0.608 * : correlation is significant at p=0.05 level, **: correlation is significant at p=0.01 level. 49 int. j. aquat. biol. (2021) 9(1): 41-54 whole population. ontogenetically, the db increased with fish sizes and ranged between db=2.46 (juveniles and db=5 (sub-adults) (table 6). also, the results showed seasonal variations in the diet breadth with a higher value (db=5.32) recorded during the wet season whereas a relatively lower value (db=4.22) was recorded during the flood period (table 6). eco-morphological relationships: the diet of s. schall was evaluated by plotting the volumetric proportion of the food categories (phytoplankton, insects, crustaceans, fish scales, mollusks, zooplankton, worms, roots, seeds, detritus, macrophytes, rice hulls and sand particles) against standard length (sl) and gut length (gl) to explore ecomorphological correlates of food habits. the matrix of spearman correlation coefficients recorded indicated that sl was positively correlated with the volumetric proportions of insects (r=0.5000, p<0.01), macrophytes (r=0.8944, p<0.01), sand particles (r=0.4472, p≤0.05) and negatively correlated with rice hulls (r=-0.8367, p<0.01). also, gl was positively correlated with detritus (r=0.3317, p<0.01), insects (r=0.5916, p<0.01), seeds (r=0.3464, p<0.01) and zooplankton (r=0.4000, p<0.01) and negatively correlated with rice hulls (r=-0.4899, p<0.01) (table 7). in addition, the regressions between sl-gl and between w (weight)-gl were established to evaluate the ecomorphological trends. the regression equations were as follow: log(gl)=1.0878log(sl) +0.1146, r=0.6007 (fig. 4); and log (gl)=0.4223log (w)+0.5981, r=0.6731 (fig. 3). both equations indicated that gl increased with sl and weight with significant (p<0.05) correlation coefficients. the ratio (gl/sl) was also computed as a measure of relative gut length and compared to published reference ratio (kramer and bryant, 1995). gl/sl varied from 0.12 (sl=5 cm) to 5 (sl=9 cm) with a mean of 1.69±0.56. discussions food and feeding patterns: the dietary analysis indicated that s. schall consumed a wide range of food resources (221 food items) dominated by eight prey categories such as insects (34.32%), sand particles (18.768%), macrophytes (13.415%), seeds (8.549%), roots (8.319%), detritus (5.344%), worms (4.735%) and rice hulls (3.681%) aggregating 97.131% of the stomach contents. this large food spectrum resulted from the presence of numerous developed mandibular teeth ranging between 24-39 and numerous gill rakers figure 3. relationship between log (gut length) and log (body weight) of synodontis schall (n=1505) from niger river in north-benin. 50 arame et al./ food habits of synodontis schall from niger river in northern benin varying between 40-44 (personal records) on the first branchial arch that could have favored this trophic flexibility (paugy and roberts, 2003). minor food items were fish scales (0.80%), crustacean (0.16%) and zooplankton (0.08%) and none of them had a volumetric proportion more than 0.80%. the presence of fish scales may indicate that this species had tendency to lepidophag. this feeding pattern suggested that in niger river in benin, s. schall displayed an omnivorous feeding habit that was confirmed by the presence of balanced animal and plant matters in the stomach of s. schall (dadebo et al., 2014; admassu et al., 2015). these findings agreed with those reported by willoughby (1974) in lake kanji in nigeria, by yatabary (1983) in the central delta of niger river and by diomande et al. (2009) in the river-lacustrine hydrosystem of bia in ivory coast where s. schall foraged mainly on insect nymphs and larvae, fish eggs, detritus, zooplankton, benthos, dipteran larvae, animal fragments, fish scales, macrophytes and sediment. likewise, in oueme river in benin, laleye et al. (2006) reported identical food habits for s. schall that foraged mainly on macrophytes, algae, insect larvae, aquatic insects, crustacea, rotifera, molluscks, nematoda, fish eggs, fish scales and sand particles. ecomorphological patterns and food habits: the wide spectrum of food resources (221 foods items recorded) ingested by s. shall in niger river suggested that this dominant mochokid is an omnivore. this food trend depicted is consistent with the ecomorphology patterns of the species. indeed, the mean relative gut length (ratio: gl/sl) fall in most omnivore ranges. these findings agreed with those reported by al-hussaini (1947) and kapoor et al. (1975) where the relative gut length varied between 0.8 and 5 for omnivores while herbivore showed higher ratio ranging between 2 and 21. as reported by dadebo et al. (2012), omnivore fishes forage both on plant and animal items and tend to have a moderate to short intestine with reduced relative gut length (gl/sl). in contrast, herbivore fishes exhibit long intestines with greater relative gut length (alhussaini, 1947; fryer and iles, 1972; kapoor et al., 1975). niche breadth, diet shift and trophic plasticity: this opportunistic feeding habit displayed by s. schall is the result of the high diet breadth (db) varying between 1.86 and 5.74 that were computed from the 221 food items identified. the body morphology, the figure 4. relationship between log (gut length) and log (standard length) of synodontis schall (n=1505) from niger river in north-benin. 51 int. j. aquat. biol. (2021) 9(1): 41-54 feeding behavior and the various ecological niches explored, greatly accounted for this wide spectrum of food ingested. indeed, the ventrally positioned mouth of s. schall is adapted for benthic feeding. also, the fact that synodontis is able to swim in upside down position enables this genus to switch from benthic feeding to surface/pelagic feeding depending on the availability and emergence of some food items (bishai and gideiri, 1965; sanyanga, 1998). nevertheless, as s. schall grows and move to pelagic waters, this mochokid is limited in foraging large prey because of its small mouth. however, its specialized teeth are suited to pick scales from other fishes in pelagic ecological niches (fryer et al.,1955). although the proportional consumptions of the food items were not equally represented in the diet, the wide choice of foods available to s. schall suggested that when one prey was in reduced supply, the species could forage on another abundant and available prey. as reported by mbadu (2011), this can be, not only an adaptation to reduce intraspecific competition among individuals in different classes of size, but also an indicator of trophic plasticity in s. schall which may shift its feeding structure according to prey availability (adite et al., 2013; gbaguidi et al., 2016). the current study revealed ontogenetic diet shift of s. schall. indeed, juvenile foraged preferentially on aquatic larvae (chironomidae larva) while sub-adults and adults ingested mainly macrophytes in proportional consumptions of 15.97 and 12.912%, respectively. in contrast, in oueme river, laleye et al. (2006) reported s. schall juvenile foraging mainly on macrophytes that accounted for 59.65% of the stomach content while seeds, sand, roots, insects (diptera and coleoptera) were the dominant food items of sub-adults and adults. in lake chamo in ethiopia, dadebo et al. (2012) reported the same diet shift according to life stages. the differential development of the digestive tract and the trophic flexibility could have caused this ontogenetic diet shift in s. schall. also, the study consistently showed high diet similarities between life stages indicated by a relatively high diet overlaps ranging between øjk=0.54-0.93. however, the reduced diet overlaps between juveniles (tl<5 cm) and sub-adults (5≤tl<8 cm) confirmed the ontogenetic diet shift depicted (adite et al., 2005). these results are consistent with those reported by gbaguidi et al. (2016) with sarotherodon galilaeus from a man-made lake of southern benin. conclusion this study documents the feeding ecology of s. shall, the dominant mochokid in niger river in benin. synodontis schall is an omnivore foraging both in benthic and pelagic habitats and consuming mainly aquatic insects, macrophytes, sand particles, seeds, roots, detritus, worms, rice hulls, mollusks and phytoplankton that resulted in a large niche breadth and a high trophic plasticity. the omnivorous food habit was also shown by the ecomorphological analysis. the species exhibited an ontogenetic diet shift that was also indicated by pianka’s diet overlap. the reinforcement of fishing regulation, the protection of habitats and the permanent ecological follow-up of the river are required for the conservation and the sustainable exploitation of s. schall. acknowledgement we are grateful to the laboratory of ecology and aquatic ecosystem management (lemea) of the department of zoology, faculty of sciences and technics of the university of abomey-calavi for providing logistics and assistances. many thanks to m. razack, b.g. ikililou, a. germard, l. medard, a.d. didier, k. bernard and the numerous fishermen for their help in fish collections. references adite a. 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(2018) 6(3): 126-137 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article effects of pirimicarb carbamate insecticide alone and in combination with lead (pb) on biochemical parameters of soft tissues in freshwater snail, galba truncatula marziyeh raisi1, hamid reza pourkhabbaz*1, mahdi banaee2, ali reza pourkhabbaz3, saeideh javanmardi1 1department of environment, behbahan khatam alanbia university of technology, behbahan, iran. 2department of aquaculture, behbahan khatam alanbia university of technology, behbahan, iran. 3department of environment, university of birjand, birjand, iran. article history: received 19 april 2018 accepted 12 june 2018 available online 2 5 june 2018 keywords: pirimicarb insecticide lead biochemical parameters freshwater snail abstract: in this study, potential effects of pirimicarb and lead (pb) were investigated on biochemical parameters in tissues of freshwater snails, galba truncatula. during an 8-day experiment, snails were exposed to sub-lethal concentrations of pirimicarb (0.5 and 1 mg/l) and/or lead acetate (0.1 and 0.2 mg/l). biochemical analyses of tissues to photometric method in snails indicate that snails treated with pirimicarb, pb, or both pirimicarb and pb increased malondialdehyde (mda) and catalase (cat) and decreased gamma-glutamyl transferase (ggt) levels, compared to the control group. alanine transferase (alt), lactate dehydrogenase (ldh), and alkaline phosphatase (alp) activity were increased in combined treatments of pirimicarb and pb. total antioxidant (tao) level increased in snails exposed to both pirimicarb and pb, while it decreased in snails treated with either pb or pirimicarb. cholesterol level increased in most experimental groups. aspartate aminotransferase (ast) was showed no significant changes in groups treated with 0.1 and 0.2 mg/l of pb compared to the control; however, ast enhanced in other treatments. in groups exposed to 0.5 and 1 mg/l of pirimicarb, the inhibition of acetylcholinesterase (ache) was not significant, although a significant reduction was found in ache level in other treatments. the results indicated that cytotoxicity of pirimicarb alone and in combination with pb depended on their concentrations. higher concentrations of pb induced significant changes in some biochemical parameters. moreover, increased pb level in water intensifies toxic effects of pirimicarb in snails. pirimicarb or/and pb, in sub-lethal concentrations, induced oxidative damages in soft tissue of snails. finally, these data support the hypothesis that changes in biochemical parameters were induced by exposure to pirimicarb or/and pb. introduction carbamate pesticides are among the most common pesticides used worldwide to control pests in grains, fruit trees and ornamental plants (casafe, 2009). pirimicarb is a carbamate and selective pesticide with chemical formula 2-dimethylamino-5,6dimethylpyrimidin-4-yl-n-dimethylcarbamate that acts by preventing the activity of acetylcholinesterase (ache). lipid peroxidation and oxidative stress are among other mechanisms causing pirimicarb toxicity (vera-candioti et al., 2010). this insecticide is toxic for different species of aquatic organisms, and especially for aquatic invertebrates (walker et al., 2007; vera-candioti et al., 2010). an increase in production and widespread application of agrochemicals, such as chemical pesticides can cause *corresponding author: hamid reza pourkhabbaz doi: https://doi.org/10.22034/ijab.v6i3.459 e-mail address: pourkhabbaz@yahoo.com varied environmental and health problems for humans and other organisms (bravo et al., 2011). some environmental pollutants have increasing, synergistic or antagonistic effects on bioavailability and toxicity of each other (nematdoost haghi and banaee, 2017; hamidipoor et al., 2015). therefore, it is expected that pesticides influence the bioavailability and toxicity of other environmental pollutants, including heavy metals (banaee et al., 2015a; banaee et al., 2015b). these compounds and their interactions can affect the physiological response and detoxification system of organisms and turn into a serious threat for organisms inhabiting near agricultural farms or industrial areas. heavy metals are hazardous due to their bioaccumulation capacity (adedeji and okocha, 2011). 127 int. j. aquat. biol. (2018) 6(3): 126-137 these elements are really sustainable in aquatic environments, bio-concentrate in tissues and bones of organisms due to lack of biological disposal, and thus their concentration increases in food chains. this increase is more remarkable on top of the food pyramid and causes toxic effects and diseases for organisms consuming the top of the food pyramid, including human beings (abbas zadeh, 1995). lead (pb) is one the heavy metals that pollutes the environment more than any other elements. human activities are the main source of introducing pb into the environment. pb gradually accumulates in the body and may replace calcium in bones as divalent lead (jarup, 2003). the production of agricultural products and expansion of different industries is of a significant importance in iran; therefore, a wide range of pesticides and other environmental pollutants, including heavy metals enter surface and underground waters through wastewater of farm lands and industrial sites. in fact, the entrance of these pollutants to surface and heavy metals can have a significant effect on water pollution and health of aquatic organisms. in order to determine the combined effect of contaminants and their source and concentration in aquatic ecosystems, the environment should be evaluated and monitored. the pollutants are troublesome when they are absorbed by living organisms (wright and welbourn, 2002). therefore, the rate of pollutants being biologically available should be evaluated (ruelas-inzuna and paez-osuna, 2000). that is why evaluating the combined effect of sub-lethal concentrations of pirimicarb and pb on biochemical parameters of aquatic organisms seems essential. in the marun river (khuzestan province, iran), different species are found, including gastropods. because of their lifestyle and nutrition, gastropods are used as a bio-indicator to evaluate the pollution of aquatic systems (macías-mayorga et al., 2015). alterations in physiological indices, growth (coeurdassier et al., 2001), behavioral changes in bellamya aeruginosa (zheng et al., 2012), oxidative stress, damage to tissues and alterations in biochemical parameters in freshwater snail, pila globosa (bhattacharya et al., 2016), in asiatic hard clam, meretrix meretrix (wan et al., 2015), grooved carpet shell, ruditapes decussatus (kamel et al., 2012), lanistes carinatus (khalil, 2015), green garden snail, cantareus apertus (leomanni et al., 2015), biomphalaria alexandrina (ibrahim et al., 2018; barky et al., 2012), lymnaea luteola (ali et al., 2012), helix aspersa (abdel-halim et al., 2013), theba pisana (radwan et al., 2010), and genetic damages in p. aeruginosa (zheng et al., 2012) are reported following exposure to environmental pollutants, such as pesticides and heavy metals. also, accumulation of cd, zn, pb and cu in snails, t. pisana (radwan et al., 2010) and h. aspersa (abdel-halim et al., 2013) are studied. snails are highly prone to accumulate heavy metals in their tissues (silva et al., 2017). lymnaea, a genus of snails, are found in abundance in freshwater ecosystems, including the marun river. moreover, they are found among plants grown along the edge of rivers and dams. galba truncatula is a sedentary species of this genus that inhabits freshwater ecosystems. it can be easily collected, identified, and kept under laboratory conditions without much challenge. therefore, the present study uses g. truncatula as a lab model to study the synergistic and antagonistic effects of sub-lethal concentrations of pirimicarb and lead (pb) on biochemical parameters of freshwater snail, g. truncatula. the results of this study can be useful in environmental monitoring of aquatic ecosystems and a better evaluation of type and rate of pesticides in agricultural lands. materials and methods chemical materials: pirimicarb (2-dimethylamino5,6-dimethylpyrimidin-4-yl-ndimethylcarbamate) (produced by golsam chemicals company, with 50% active material) was bought from shiraz, iran, and lead acetate was provided by merck company (germany, 99% pure). biochemical kits were bought from pars azmoon co. and other chemical materials were bought from merck, germany. snail: the freshwater snails, g. truncatula, 128 raisi et al/ effects of pirimicarb and lead (pb) on snail considering the national ethical framework for animal research in iran (mobasher et al., 2008) were randomly collected from 30-40 cm depth of the vegetation by the marun river near the city of behbahan, khuzestan province, iran. snails were immediately sent to the laboratory of reproduction and cultivation of ornamental species at faculty of natural resources at behbahan khatam alanbia university of technology for an adaptation period prior to the beginning of the experiment. before the experiment, snails were kept in plastic buckets under certain conditions (22±2°c, ph: 7.4±0.2, 16/8 light/dark) to adapt to laboratory conditions. in order to do the experiment, snails were kept in tanks containing 2 liters of distilled water and fed with lettuce extract. sub-acute toxicity test: the snails were divided into nine treatment groups and each group was triplicate. each aquarium contained thirty freshwater snail g. truncatula initially. group 1 was the control group, while groups 2 and 3 were respectively exposed to 0.1 and 0.2 mg/l of lead acetate. groups 4 and 5 were respectively treated with pirimicarb 0.5 and 1 mg/l. in group 6, snails were exposed to pirimicarb (0.5 mg/l) and lead acetate (0.1 mg/l). group 7 was treated with 1 and 0.1 mg/l of lead acetate. finally, groups 8 and 9 were respectively exposed to pirimicarb 0.5 mg/l plus lead acetate 0.2 mg/l and pirimicarb 1 mg/l plus lead acetate 0.2 mg/l. each treatment had three replications with a totally randomized design. pirimicarb pesticide (laznik et al., 2010) and lead (grosell et al., 2006) are chosen according to their subacute concentrations for mollusks. during the experiment, 50% of the water was exchanged daily to reduce excessive metabolites. moreover, the pesticide and lead (pb) was added to keep concentrations of pirimicarb and pb constant. sampling and analysis of biochemical parameters of tissue: to evaluate enzymes, soft tissue of snails was removed from the hard part by cold physiologic saline in a porcelain mortar. then, tissue samples were homogenized by a mechanical glass homogenizer with a mixture of phosphate-buffered saline (ph 5.6) for 2 minutes and then centrifuged for 8 minutes in a refrigerated centrifuge at 12000 rpm at 4°c so that it turned into a complete solution. finally, the supernatant was extracted with a sampler from the centrifuged soft tissue. it was kept in a freezer at -20°c till analyses of biochemical parameters. biochemical parameters were measured by a uvvis spectrophotometer (biochrom libra s22). the activity of aspartate aminotransferase (ast) and alanine aminotransferase (alt) was measured according to nadph consumption and its conversion to nad+ at the wavelength of 340 nm (moss and henderson, 1999). the activity of lactate dehydrogenase (ldh) was evaluated based on pyruvate reaction with nadh and hydrogen in presence of ldh and the production of lactate and nad+ at 340 nm (moss and henderson, 1999). the activity of alkaline phosphatase (alp) in plasma was measured according to the conversion of nitrophenyl phosphate to nitrophenol and phosphate at 405 nm (moss and henderson, 1999). the activity of glucose6-phosphate dehydrogenase (g6pdh) was measured based on the reaction rate of glucose-6-phosphate to 6-phosphogluconate and restoration of nadp to nadph in presence of g6pd at 340 nm (burtis and ashwood, 1999). gamma-glutamyl transferase (ggt) level was estimated based on glutamic acid transfer to glycylglycine and 5-amino-2-nitrobenzoate release at 405 nm (moss and henderson, 1999). the activity of acetylcholinesterase (ache) was measured according to butyrylthiocholine hydrolysis at 405 nm (knedel and boetteger, 1967). cholesterol level was measured based on chod-pap enzymatic colorimetric analysis at 510 nm (rifai et al., 1999). the activity of catalase (cat) was measured based on degradation of hydrogen peroxide and formation of a stable complex with ammonium molybdate at 405 nm (goth, 1991). malondialdehyde (mda) level was measured by thiobarbituric acid at 532 nm. in this experiment, tetraethoxy propanol and absolute ethanol were used as the standard mda (placer et al., 1966). total antioxidant capacity (tao) of the cell was measured by the reducing ability of ferric iron to ferrous, i.e. frap method, and with tptz or 2,4,6-tripyridyl-s-triazine 129 int. j. aquat. biol. (2018) 6(3): 126-137 as the substrate at 593 nm. to plot a standard curve, ferrous sulfate heptahydrate solution at 100-1000 mol/l was used (benzie and strain, 1996). statistical analysis: ibm spss software (version 19) was used for statistical analysis with p<0.01 normality of data was assessed with kolmogorovsmirnov test. one-way analysis of variance (anova) was used to compare treatments and when data were significant, duncan method at 99% confidence interval was used. the results are shown in a diagram in excel and presented as mean ± standard deviation (sd). results during the experiment, no mortality was found in snails. alterations in biochemical parameters in snail tissues exposed to sublethal concentrations of pirimicarb and/or lead acetate (separately or in combination) and compared to control are depicted in diagrams. malondialdehyde: there was a significant increase in mda level in all experimental treatments compared to the control group. the highest increase was in snails treated with 0.5 mg/l of pirimicarb (fig. 1). total antioxidant: levels of total antioxidant (tao) concentration in snails treated with pb 0.1 mg/l + pirimicarb 0.5 mg/l, pb 0.1 mg/l + pirimicarb 1 mg/l, pb 0.2 mg/l + pirimicarb 0.5 mg/l, and pb 0.2 mg/l + pirimicarb 1 mg/l were significantly lower than tao concentration levels in the control group. however, antioxidant level in snails exposed to 0.1 and 0.2 mg/l of pb, and 0.5 and 1 mg/l of pirimicarb was significantly higher than its level in the control group (fig. 2). catalase: according to the results, catalase (cat) level was significantly increased in all experimental treatments compared to its level in the control group (fig. 3). glucose-6-phosphate dehydrogenase: the rate of glucose-6-phosphate dehydrogenase (g6pdh) in snails treated with 0.5 mg/l of pirimicarb was significantly reduced. however, no significant figure 1. alterations of mda level in different treatments. identical letters express the absence of a significant difference. figure 2. alterations of antioxidant level in different treatments. identical letters express the absence of a significant difference. 130 raisi et al/ effects of pirimicarb and lead (pb) on snail difference was found in the activity level of g6pdh in other treatments when compared to the control group (fig. 4). cholesterol: no significant difference was found in cholesterol level in groups treated with pb 0.2 mg/l and pirimicarb 0.5 mg/l. however, other treatments indicated a significant difference in cholesterol level compared to the control group (fig. 5). lactate dehydrogenase: levels of lactate dehydrogenase (ldh) in snails treated with the combined treatments of pb 0.1 mg/l + pirimicarb 1 mg/l, pb 0.2 mg/l + pirimicarb 0.5 mg/l, and pb 0.2 mg/l + pirimicarb 1 mg/l were significantly increased. no significant increase was found in ldh activity in other treatments compared to the control group (fig. 6). acetylcholinesterase: no significant difference was found in acetylcholinesterase (ache) level in the groups treated with 0.5 and 1 mg/l of pirimicarb, compared to the control. however, a significant difference was recorded in ache activity of other experimental groups, compared to the control group. figure 3. alterations of cat level in different treatments. identical letters express the absence of a significant difference. figure 4. alterations of g6pdh in different treatments. identical letters express the absence of a significant difference. figure 5. alterations of cholesterol level in different treatments. identical letters express the absence of a significant difference. 131 int. j. aquat. biol. (2018) 6(3): 126-137 the biggest reduction in ache activity was observed in the combined treatments of pb 0.1 mg/l + pirimicarb 0.5 mg/l, pb 0.1 + pirimicarb 1 mg/l, pb 0.2 mg/l + pirimicarb 0.5 mg/l, and pb 0.2 mg/l + pirimicarb 1 mg/l (fig. 7). gamma-glutamyl transferase: the activity level of gamma-glutamyl transferase (ggt) was significantly reduced in all experimental treatments and the biggest increase was found in snails treated with pirimicarb 1 mg/l (fig. 8). alkaline phosphatase: the activity of alkaline phosphatase (alp) in snails inhabiting water containing both pb and pirimicarb (0.1 mg/l + 1 mg/l; 0.2 mg/l + 0.5 mg/l; and 0.2 mg/l + 1 mg/l, respectively) was significantly increased. however, no significant difference was observed in alp activity of other groups when compared to the control (fig. 9). aspartate aminotransferase: no significant difference was found in aspartate aminotransferase (ast) levels in groups treated with 0.1 and 0.2 mg/l of pb, figure 6. alterations of ldh level in different treatments. identical letters express the absence of a significant difference. figure 7. alterations of ache activity in different treatments. identical letters express the absence of a significant difference. figure 8. alterations of ggt in different treatments. identical letters express the absence of a significant difference. 132 raisi et al/ effects of pirimicarb and lead (pb) on snail compared to the control group. however, a significant difference was observed in ast level in snails of other treatments compared to the control group (fig. 10). alanine aminotransferase: levels of alanine aminotransferase (alt) significantly increased in snails treated with pb 0.1 mg/l + pirimicarb 1 mg/l, pb 0.2 mg/l + pirimicarb 0.5 mg/l, and pb 0.2 mg/l + pirimicarb 1 mg/l. in other groups, no significant difference was found in alt activity compared to the control group (fig. 11). discussion the objective of the present study was to evaluate biochemical parameters in soft tissue of g. truncatula treated with sublethal concentrations of lead acetate and pirimicarb. an increase in mda level, as the final metabolite of lipid peroxidation, in tissues of snails in all groups treated with pb and/or pirimicarb can be attributed to an increase in reactive oxygen species (ros) and enhancement of lipid peroxidation rate. moreover, an increase in mda indicates an imbalance figure 9. alterations of alp level in different treatments. identical letters express the absence of a significant difference. figure 11. alterations of alp level in different treatments. identical letters express the absence of a significant difference. figure 10. alterations of ast level in different treatments. identical letters express the absence of a significant difference. 133 int. j. aquat. biol. (2018) 6(3): 126-137 between ros level and the cellular antioxidant defense system in snails treated with pb and pirimicarb. an increase in mda level is reported in snails, t. pisana and h. aspersa, exposed to lead, zinc, copper and cadmium (radwan, 2010; atailia et al., 2016). similar results are reported in fish treated with pb (nourian et al., 2015), and in snails (l. carinatus) treated with chlorpyrifos (m.khalil, 2014). the mda produced during peroxidation of fatty acids can make a covalent bond due to having a double bond with other elements of cell membrane, and therefore affect the physiologic activity of the cell membrane. the cellular antioxidant defense system, including antioxidant enzymes and non-enzymatic antioxidants has an important role in removing free radicals. in the present study, a significant increase in total antioxidant (tao) level in snails treated with pb and pirimicarb demonstrates a cellular response to an increase in the production rate of ros. however, a significant reduction in tao in snails treated with a combination of pb and pirimicarb can indicate oxidative stress and an imbalance between the antioxidant defense system and production level of free radicals. therefore, snails’ exposure to pb and pirimicarb can reduce the activity of antioxidant enzymes, decrease non-enzymatic antioxidant levels, and diminish the organism’s ability in restoring the antioxidant defense system in cells. catalase (cat) is one of the major antioxidant enzymes that is responsible in degrading hydrogen peroxide in cells under normal conditions. therefore, a significant increase in cat activity in tissues of snails treated with different concentrations of pirimicarb and/or pb can be a physiologic response to increased levels of hydrogen peroxide in cells. our results are in accordance with those of el-shenawy (2012) on snails, eobania vermiculata, treated with lead, cadmium, iron, and copper, and khalil’s research (2015) on snails, l. carinatus, exposed to chlorpyrifos. in addition, basopo and ngabaza’s work (2015) on snails (l. carinatus) treated with pb and chlorpyrifos, and mleiki’s research (2015) on snails, c. apertus, exposed to pb and cd announced similar results. g6pdh is an important cytoplasmic enzyme in glucose metabolism in the pentose phosphate pathway. this is an important source of producing nicotinamide adenine dinucleotide phosphate (nadph), essential for dna and rna bond, carbon or phosphate sugars (kletzien et al., 1994). nadph is used for restoration of glutathione and so important in regulating the intracellular redox state (leopold and loscalzo, 2000). therefore, g6pdh activity is highly important to maintain the cytosolic reserve and cellular redox balance. that is why g6pdh is known as an index of oxidative stress (mehrpak et al., 2016). a reduction or disturbance in the activity of g6pdh can increase the chance of oxidative stress-induced cell death (lin et al., 2013). a decrease in g6pdh in snail tissue treated with pirimicarb (0.5 mg/l) may reduce the cellular nadph level and disturb the balance between oxidant and antioxidant level in cells. ast, alt, ldh, ggt, alp are found in different tissues such as liver, heart, the nervous system, skeletal muscles, kidney, pancreas, spleen, blood cells, intestine and gills of aquatic organisms (banaee, 2013; azubuike, 2012). a significant increase in alt and ast levels in snails treated with pirimicarb and/or lead can indicate a severe damage to the cell membranes, and especially hepatocytes. due to the effective role of aminotransferase enzymes in cellular nitrogen metabolism, oxidation of amino acids, or glucogenesis (banaee, 2013; rao, 2006), it can be used as an appropriate clinical marker to diagnose liver damages. an increase in ast and alt is reported in blood plasma of fish exposed to pb (nourian et al., 2015), quails treated with deltamethrin and pb (hamidipour, 2015), and snails, l. carinatus, treated with methylmalonat (khalil, 2015). alp is a cell membrane enzyme that catalyzes the dephosphorylation of many molecules, including nucleotides, proteins, and alkaloids in an alkaline ph. a significant increase in alp in snails treated with a combination of pirimicarb and pb can be the result of degeneration and necrosis of hepatocytes, and damage to cell membranes. an increase in alp activity is found in blood plasma of fish exposed to pb (nourian et al., 2015), in snails (l. carinatus) treated with 134 raisi et al/ effects of pirimicarb and lead (pb) on snail methymalonat (khalil, 2015) and in fish exposed to paraquat (banaee et al., 2016). ldh is an anaerobic enzyme that catalyzes lactate to pyruvate. an increase in ldh in snails treated with a combination of pirimicarb and lead acetate might be due to hepatocytes necrosis, heart diseases, kidney failure, kidney cells necrosis, muscular dystrophy and anemia (banaee et al., 2016). enhanced levels of ldh in fish exposed to paraquat and microplastics is reported by banaee et al. (2016). a significant decrease in ggt activity in snails exposed to pirimicarb and pb in all experimental treatments compared to the control can be due to the direct effect of pirimicarb and pb on ggt and preventing its biosynthesis in damaged hepatocytes. ggt has a key role in glutamyl cycle and glutathione homeostasis. therefore, a reduction in ggt activity in tissues of snails exposed to pirimicarb and pb of all treatments may suggest a disturbance in glutathione homeostasis or an imbalance between oxidants and antioxidants. a reduction in ggt activity is reported in plasma of fish exposed to pb (ziyadlou et al., 2011) and in plasma of common carp that were treated with paraquat (sharifinasab et al., 2016; banaee et al., 2016). cholesterol is a precursor of steroid hormones and under stressful conditions its available concentration increases to provide cortisol precursor (hoseini and ghelichpour, 2011). moreover, changes in cholesterol concentration, as the main marker of animals’ health status, indicate liver metabolism (seyit et al., 2000). an increase in cholesterol level in tissues of snails that were treated with pirimicarb and/or pb might be due to a change in snails’ metabolism and a damage to kidney (zhou et al., 2009; gul et al., 2011). an increase in cholesterol activity is reported in quails treated with both deltamethrin and pb (hamidipour, 2015). ache plays an important role in breakdown of acetylcholine and is known as a behavioral marker in organisms exposed to environmental pollutants (banaee, 2013). in the present study, a significant decrease in ache activity in snails treated with only 0.1 or 0.2 mg/l of pb and in groups treated with both pirimicarb and pb can negatively affect the snails’ behavior. pb and pirimicarb influence cysteine in the active site of acetylcholinesterase and thus irreversibly inhibit ache activity. therefore, the enzyme cannot sit on substrate, nor hydrolyze acetylcholine. the inhibition of ache is reported in snails, c. apertus, exposed to pb and cd (mleiki et al., 2015). conclusion the imbalance between the reactive oxygen species and the antioxidant capacity in the snail exposed to the pirimicarb pesticide and lead metal alone and in combination is one of the important reasons that cause undesirable changes in the biochemical parameters of the tissue. consequently in this study exposure to sublethal concentrations of pirimicarb and/or pb (alone or combined) in snails causes remarkable biochemical changes that these changes were greater in snails treated with both pirimicarb and pb (pb 0.1 mg/l + pirimicarb 0.5 mg/l, pb 0.1 mg/l + pirimicarb 1 mg/l, pb 0.2 mg/l + pirimicarb 0.5 mg/l, and pb 0.2 mg/l + pirimicarb 1 mg/l), because an increase in pb concentration in aquatic ecosystems can due to the imbalance between the reactive oxygen species and the antioxidant capacity intensify the toxicity and bioavailability of pesticides. furthermore, biochemical parameters’ response in the soft tissue of snails (g. truncatula) that were exposed to different concentrations of pirimicarb and pb indicate that this species can be used as a suitable bioindicator to assess and monitor aquatic ecosystems that are contaminated with heavy metals and pesticides. references abbas zadeh k. 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(2009). comparison of hematology and serum biochemistry of cultured and wild dojo loach misgurnus anguillicaudatus. fish physiology and biochemistry, 35(3): 435-41. http://go.mining.com/apr08-a3 int. j. aquat. biol. (2018) 6(3): 126-137 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی یاییبیوشیم پارامترهای بر سرب فلز در ترکیب با و به تنهایی پریمیکارب کشآفت کشندهتحت هایغلظت اثرات (galba truncatula) شیرین آب حلزون بافت 1جوانمردی سعیده ،3پورخباز علیرضا ،2بنایی مهدی ،1*پورخباز حمیدرضا ،1رئیسی مرضیه ایران. بهبهان، بهبهان،( ص) االنبیاء خاتم صنعتی دانشگاه زیست، محیط و طبیعی منابع دانشکده زیست، محیط گروه1 ایران. بهبهان، بهبهان،( ص) االنبیاء خاتم صنعتی دانشگاه زیست، محیط و طبیعی منابع دانشکده شیالت، گروه2 ایران. بیرجند، بیرجند، دانشگاه زیست، محیط و طبیعی منابع دانشکده زیست، محیط گروه3 چکیده: ( بررسی شد galba truncatulaتأثیرات احتمالی پریمیکارب و فلز سرب بر روی پارامترهای بیوشیمیایی بافت حلزون آب شیرین ) در این مطالعه گرم بر لیتر( میلی 2/0و 1/0گرم بر لیتر( و استات سرب )میلی 1و 5/0های تحت کشنده پریمیکارب )روز در معرض غلظت 8به مدت هاو حلزون و های پریمیکاربهای در معرض غلظتحلزونصورت توأم قرار داده شدند. آنالیز بیوشیمیایی بافت به روش فتومتریک نشان داد که به تنهایی و به در مقایسه با گروه کنترل شدند. سطح ggtو کاهش سطح آنزیم catو mdaهای صورت توأم باعث افزایش سطح آنزیمتنهایی و به سرب به های تحت تیمار ترکیبی سرب در حلزون taoدر تیمارهای ترکیبی پریمیکارب و سرب افزایش یافت. سطح آنزیم alpو alt ،ldhهای آنزیم تنهایی افزایش یافت. سطح کلسترول در اکثر تیمارهای در معرض پریمیکاب های تحت تیمار سرب و پریمیکارب بهکاهش و در حلزون و پریمیکارب داری را نسبت به گروه میلی گرم بر لیتر سرب تغییر معنی 2/0و 1/0های های تحت تیمار غلظتدر حلزون astو سرب افزایش یافت. سطح آنزیم های تحت کشنده های در معرض غلظتدر حلزون acheداری در سطح آنزیم در سایر تیمارها افزایش یافت. مهار معنی astداد اما کنترل نشان ن در سایر تیمارها مشاهده شد. acheداری در سطح گرم بر لیتر در مقایسه با گروه کنترل مشاهده نشد اما کاهش معنیمیلی 1و 5/0پریمیکارب تغییرات های باالتر سرب سبب ایجادها دارد. غلظتدهد که سمیت سلولی پریمیکارب به تنهایی و یا توام با سرب بستگی به غلظت آننتایج نشان می ها را تشدیدداری در برخی از پارامترهای بیوشیمیایی شده است. عالوه براین، افزایش سطح سرب در آب اثرات سمی پریمیکارب در حلزونمعنی رم حلزون های نهای استرس اکسیداتیو در بافتهای زیرکشنده سبب ایجاد آسیبصورت توام در غلظتیمیکارب و سرب به تنهایی و یا بهکند. پرمی و ها فرضیه مطرح شده مبنی بر ایجاد تغییرات در پارامترهای بیوشیمیایی ناشی از قرار گرفتن در معرض پریمیکاربشده است. در نهایت، این داده کند.سرب را اثبات می .شیرین آب حلزون بیوشیمیایی، پارامترهای سرب، پریمیکارب، کشحشره کلمات کلیدی: int. j. aquat. biol. (2021) 9(6): 383-387 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology short communication effect of salinity and ph on the absorption of cadmium in lemna minor rajaa abdul-kadhim hanaf*1 college of marine sciences, department of natural marine science, university of basrah, basrah, iraq. article history: received 7 september 2021 accepted 14 november 2021 available online 2 5 december 2021 keywords: heavy elements, aquatic plants, pollution, bioaccumulation. abstract: this work was conducted to study the effect of salinity and ph on the absorption and accumulation of cadmium by the aquatic plant lemna minor. different concentrations of salinity (0, 1, 2 and 3 ppt) and ph (6.2, 7 and 8.4) were used. the results showed a decrease in the absorption of cadmium with increasing salinity, and the highest concentration of cadmium absorption at ph 6.2. introduction aquatic plants are an important part of aquatic ecosystems due to providing an important source of organic matter and maintaining biological balance in the circulation and organization of nutrients. in addition, they provide ground for living, reproduction and nutrition for fish and other aquatic organisms (caraco and cole, 2002). in recent years, global interest has increased in using aquatic plants as a biological index for heavy metal pollution due to their ability to accumulate these elements in their tissues (coleman et al., 2001). they have also been used as a biological monitor for heavy metal levels in the aquatic environment (mal et al., 2001). furthermore, aquatic plants are used as phytoremediation of heavy metals (al-edani et al., 2019). the process of heavy elements absorption by plants may be affected by several factors, including ph, temperature, salinity and the concentration of heavy elements in the surrounding water (pettersson, 1999). salinity is one of the main limiting factors of the growth and productivity of aquatic plants. the effect of high salinity on plants can be observed through decreased productivity or plant death or photosynthesis, protein synthesis, energy conversion and lipid metabolism (agastian et al., 2000), and salinity also has a significant role in the abundance and *correspondence: rajaa abdul-kadhim hanaf doi: https://doi.org/10.22034/ijab.v9i6.1489 e-mail: rajaa.hanif@uobasrah.edu.iq distribution of plant species (littles, 2005). the effect of salinity comes through its impact on the growth rate of plants as well as their ability to absorb heavy elements through the toxic effect of sodium ion and chloride ion (parida and das, 2005), as the sodium ion releases cadmium from sediments into the water, which leads to an increase in cadmium concentration (greger et al., 1995). it was found that the aquatic plant potamogeton pectinatus grows with high concentrations of salinity, contains low concentrations of cadmium, as increased salinity led to a decrease in cadmium absorption by the plant (noraho and gaur, 1995). munda and hudink (1988) pointed out that the ability of aquatic plants to absorb copper decreases with increasing salinity. leblebici et al. (2009) found that the ability of aquatic plants to absorb heavy metals decreases when salinity levels increase in water. it is known that iraqi waters are alkaline (richardson and hussain, 2006), i.e. the ph values of the southern marshes range between 7-8.5. the ph can decrease during the summer and autumn seasons due to the decomposition of organic matter, which increases by rising temperature (lateef et al., 2020). the dense plant growth leads to carbon dioxide consumption during the winter and spring seasons, which leads to high ph values (neghamish and ali, 2005). the ph is a limiting factor for the growth of 384 hanaf / effect of salinity and ph on the absorption of cadmium in lemna minor plant communities, as its value is less than 6, which can limit the growth of aquatic plants (hutchinson, 1995). it was found that the ph in rivers in which there are species of the family lemnaceae was between 5.68.5, and they die when the ph reached 1.2 (hicks, 1933). cadmium is one of the important toxic trace elements in the environment due to its high mobility and toxicity at low concentrations for plants (ye et al., 2003) and its ability to transfer through the food chain (hiroyuki et al., 2002). cadmium enters the air, soil and water from industrial works, burning coal, household waste, phosphate fertilizers containing cadmium, sewage and pesticides. (wu et al., 2004). cd accumulates in fish and plants from the environment (atsdr, 2012). the aquatic plant lemna minor is one of the efficient plants used for water purification, and it grows at a ph ranging between 5-8.5 (littles, 2005). experiments have shown that this plant can consume many elements, including boron, aluminium, manganese, iron and copper, and it can absorb lead, in addition to removing phosphorous and nitrogen from water (miranda and liaugovan, 2004). it also tolerates salt up to 2.5% (haller, 1974). based on the above-mentioned background, the purpose of this study is to investigate the effect of salinity concentrations and ph on the ability of l. minor to absorb cadmium for the possible application of this plant as a biological monitor. materials and methods samples of the l. minor were collected from the ponds near the basrah university, karmat ali, iraq. they were washed with pond water to remove suspended matters and placed in clean bags until reaching the laboratory. aquaria with a capacity of 20 liters (25x25x29 cm) were used for the experiments. a fluorescent light source was provided with a luminous intensity of 130-150 microenstein m2/sec with a period of 14:10 hours light: dark. for aeration, an air pump was added to each of the aquarium. the aquaria were filled with distilled water of 10 liters; then the collected l. minor plants were placed into them at a rate of 100 g. to prepare the cadmium nitrate solution, 2 mg/liter of cadmium was added to each experimental aquarium. nacl was used to make different salt concentrations, including 0, 1, 2 and 3 ppt and other aquaria for the experiment used for ph (6.2, 7 and 8.4) experiments. for this purpose, the acidity was adjusted using sodium hydroxide and hydrochloric acid. the experiment was performed for 40 days, and the concentration of cd was measured in the treatments every ten days. for this measurement, the samples were dried in an electric oven at 70°c for 24 hours, crushed and passed through a laboratory sieve with a mesh of 40 µ. then, the samples were prepared for the extraction and measurement of the heavy metals according to apha (2005). one g of the crushed aquatic plant was put in glass flasks with 25 ml capacity and added a certain volume of nitric and pyrochloric acids in a ratio of 3: 1. the samples were left for 24 hours under vacuum, and the flasks were placed in a water bath for 1 hour to speed up digestion. the flasks were taken out, and 2-3 ml of the distilled water were added and placed on a hot plate at a temperature of 70°c until the volume reached 2 ml. after centrifuge filtration and completion of the filtered sample to 50 ml of distilled water, the samples are measured with a flame atomic absorption spectrometer (pye unicam sp9 air acetyline). the result is expressed as µg/l dry weight. results and discussions based on the results, the plant's ability to absorb cd at high salinity levels decreases or almost stops, in agreement with the findings of witham et al. (1971). the rise of salinity leads to an increase in the available forms of cd and its uptake by the wheat crop (norvell et al., 2000; khoshgoftarmensh et al., 2002). the study also showed that the cd in salinity of 0 and 1 ppt were higher absorption than those with higher salinity i.e. confirming that the increase in salinity caused a decrease in accumulation of the heavy metals in aquatic plants (fritioff et al., 2005). mahmoud (2008) and hanaf (2016) found that heavy metals accumulate with increasing salinity. as for l. minor, the increase 385 int. j. aquat. biol. (2021) 9(6): 387-387 in the salinity led to a decrease in the accumulation of cd, and this was in agreement with the results of leblebici et al. (2009). table 1 shows the concentration of cd in l. minor at different salinities during 40 days’ experiment. the reason for this may be due to the complex structure between the chloride ion and metals (forstner, 1979), or increased competition with the sodium ion for the adsorption sites in both the cell and plasma walls due to the high salinity, which led to a decrease in the absorption of elements by the plant (noraho and gaur, 1995). salinity may increase the release of heavy metals from the sediments into the water column (yureki et al. 2001). the ph plays a fundamental role in the transfer of the heavy metals between the liquid and solid phases, as the low ph increases the solubility of some heavy elements in water, which causes an increase in their spread and availability to living organisms in the water (addy et al., 2004). the results of the current study (table 2) showed that the highest concentration of cadmium in l. minor was at ph 6.2 compared to the concentration of cadmium in the same plant at ph 8.4, this was also noted by puranik and paknikar (1999) in their study on the use of some aquatic plants in the removal of heavy elements (lead, copper, zinc and cadmium) by the influence of some factors, including ph. while jafari and akhavan (2011) found an increase in the concentration of some heavy metals with increasing ph, the reason for this may be because l. minor grows in this range of ph (littles, 2005), as the ability to take up and absorb increases during the plant growth period, which leads to an increase in binding and thus an increase in accumulation, and this was confirmed by ekval and greger (2002) in a study they conducted on the influence of biomass production factors in two types of the aquatic environment on the ability to take some elements. this was also found by (hanaf, 2016), as she noticed the correlation of the productivity of phytoplankton and aquatic plants with the concentration of some heavy metals. several studies have indicated that the aquatic plant l. minor can accumulate heavy metals and remove them from polluted water with high efficiency. this was also found by the current study, which agreed with athbi et al. 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(2016) 4(2): 108-116; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article distribution, body size, and eggs of ovigerous swimming crab (portunus pelagicus linnaeus 1758) at various habitats in lasongko bay, central buton, indonesia abdul hamid1, yusli wardiatno*2,1djamar tumpal floranthus lumban batu2, etty riani2 1department of aquatic resources management, faculty of fisheries and marine sciences, halu oleo university, kampus hijau bumi tridharma anduonohu kendari 93232 indonesia. 2department of aquatic resources management, faculty of fisheries and marine science, bogor agricultural university (ipb), kampus ipb darmaga, bogor 16680, indonesia. article history: received 9 february 2016 accepted 12 april 2016 available online 2 5 april 2016 keywords: embryonic development habitat type portunidae reproduction sulawesi island abstract: the distribution, body size, and egg diameter and volume of ovigerous blue swimming crabs, portunus pelagicus, were examined according to habitat type and egg color in lasongko bay, central buton, indonesia between april 2013 and march 2014. ovigerous female crabs were sampled using gillnets and traps at seven stations. based on the results, the ovigerous female crabs with yellow to dark gray eggs were found over a range of depths from 0.35 to 31.0 m, on sandy to muddy substrate, and in sea bottom covered by seagrass and in bare areas. the carapace widths of the ovigerous female crabs varied significantly between the types of habitat, egg color, and season. wet weight, diameter, and volume of eggs increased by 36.35%, 25.16%, and 91.76%, respectively, with embryonic development, while the dry weight of eggs decreased by 1.86%. ovigerous blue swimming crabs with different levels of embryonic development showed a wide habitat distribution from shallow to deeper waters. introduction the blue swimming crab, portunus pelagicus linnaeus 1758, distributes at diverse habitats inhabiting in coastal waters of the continental shelf to a depth of 50 m (edgar, 1990), and a maximum depth of over 65 m (juwana, 1997). in coastal waters, this crab can be found in the intertidal regions, mouths of small rivers (creeks), sublittoral zones, shallow bays, and coastal waters (edgar, 1990; chande and mgaya, 2003; de lestang et al., 2003) with seagrass and algal beds with mud, clay, and sand substrates (chande and mgaya, 2003; de lestang et al., 2003; dineshbabu et al., 2008). this species is also found in mangrove areas in brackish water ponds adjacent to the sea (juwana, 1997). this crab is found from the head to the mouth of lasongko bay, central buton, indonesia in varying habitat conditions, depth, substrate type, and presence/absence of seagrass. in lasongko bay, this * corresponding author: yusli wardiatno e-mail address: yusli@ipb.ac.id crabs caught in shallow waters that are smaller than those from deeper locations (hamid, 2011). early development of the ovigerous female crabs usually occurs in estuaries and in shallow waters, and they migrate to deeper waters for spawning (sumpton et al., 1994; kangas, 2000). the blue swimming crab is usually found in clear water with sandy substrate, with high levels of oxygen and salinity to support the development and survival of newly hatched larvae (sumpton et al., 1994; kangas, 2000; xiao and kumar, 2004). inhabiting ovigerous female crabs in the estuary are smaller or younger than those of deeper parts of the bay (kangas, 2000). the distribution of ovigerous female crabs varies spatially and is usually influenced by water depth, sediment fraction, and seagrass condition (nitiratsuwan et al., 2010, 2013; zairion et al., 2014). nitiratsuwan et al. (2013) studied the distribution of ovigerous female crabs according to 109 int. j. aquat. biol. (2016) 4(2): 108-116 habitat type, but not based on the color of their eggs. therefore, the distributions of ovigerous female crabs based on egg color and in relation to the type of habitat have not been reported. crab eggs change color during embryonic development, starting from yellow, and progressing to orange, brown, and finally gray or black, and also change in shape, diameter, and volume (arshad et al., 2006; samuel and soundarapandian, 2009; soundarapandian and tamizhazhagan, 2009; liao et al., 2011; ravi and manisseri, 2013). as the eggs grow in diameter, volume, and wet weight, the largest size is obtained on the second day before the end of the embryonic development (ravi and manisseri, 2013). there have been relatively few studies on the diameter of eggs related to color change in ovigerous female swimming crabs (potter et al., 2001; arshad et al., 2006; soundarapandian and tamizhazhagan, 2009; ikhwanuddin et al., 2011, 2012; ravi and manisseri, 2013; safaie et al., 2013). in a recent study, soundarapandian and tamizhazhagan (2009) determined the diameters of eggs from yellow to dark gray, but they did not record the volume of eggs for each color. the present study was performed to elucidate the distribution, body size, and egg size (diameter and volume) of ovigerous female crabs in relation to habitat type and egg color in lasongko bay, central buton, indonesia. materials and methods study area and sampling: this study was conducted between april 2013 and march 2014 in lasongko bay (latitudes: 05°15's and 05°27's and longitudes: of 122°27'e and 122°33'e). crabs were collected at seven stations (fig. 1). sampling stations were characterized with regard to seagrass condition, substrate type, and water depth (table 1). the ovigerous female crabs were caught monthly using bottom gillnets with mesh sizes of 3.8, 5.8, and 8.9 cm and also with traps. the collected crabs were kept in an icebox and stored in a freezer prior to examination in the laboratory. a total of 168 ovigerous female crabs were caught, consisting of 57 with yellow eggs, 48 with orange eggs, 31 with brown eggs, and 32 with dark gray eggs. all samples were weighed using a digital scale with a precision of 0.01 g. carapace width was measured to a precision of 0.05 mm. the eggs on the abdomen were gently removed from pleopods, weighed, and dried at 65°c for 24 hrs. for each egg color, three crabs were used to determine the number and diameter of the eggs. all measurements were conducted with the assumption that the eggs were spherical (figueiredo and narcisoi, 2008). data analysis: body and egg sizes of the ovigerous females according to station and egg color were analyzed using one-way anova followed by tukey’s test. the sizes in different seasons were analyzed by t-test assuming unequal variance. the size and egg’s data were tested for normality using the kolmogorov-smirnov test (steel and torrie, 1992), and if significantly different, the data were transformed to log 10 before one-way anova and examined by t-test. linear regression analysis was used to determine the relationship between fresh weight and dry weight of crab eggs. in all analyses, p<0.05 was taken to indicate statistical significance. results distribution of ovigerous female blue swimming crabs: distribution of the ovigerous female crabs was determined based on the color of eggs and habitat type along with data from catches by fishermen in the study area (table 2). the ovigerous female crabs figure 1. map of study sites and sampling stations in lasongko bay, central buton, indonesia. 110 hamid et al./ distribution, body size, and eggs of ovigerous blue swimming crab with eggs of four different colors (yellow, orange, brown, and dark gray) in lasongko bay were found in several types of habitat, i.e., at depths of 0.35 m (low tide) to 31.0 m (high tide), on various substrate types from sand to clay, as well as under substrate conditions of sand to overgrown seagrass (table 2). the proportion of ovigerous female crabs caught at each station ranged from 6.72% to 35.00%, with station characteristics of habitat type depth (m) substrate type bottom condition seagraass bead condition 1 0.35-6.10 sand, sand clay covered seagrass, bare sand less–dense, enhalus acoroides dominated 2 0.35-6.60 sand, usually rock covered seagrass, bare sand less–dense, thalassia hemprichii lessdense 3 0.62-8.85 sand, sand clay covered seagrass, bare sand moderat–dense, t. hemprichi dominated 4 0.40-13.02 sand, sand clay covered seagrass, bare sand moderat–dense, t. hemprichi dominated 5 0.35-9.50 sand, sand clay covered seagrass, bare sand moderat–dense, t. hemprichi dominated 6 3.65 9.55 sand, loam clay sand bare sand no seagrass 7 14.00-31.00 clay bare sand no seagrass table 1. characteristics of crab habitat type at each station in lasongko bay, central buton, indonesia. station habitat type distribution of ovigerous female crabs based on eggs color depth (m) substrate type bottom condition turbidity (ntu) yellow orange brown dark gray 1 0.35-2.35 sand seagrass ++ 2.79 √ √ √ 0 1.60-3.65 sand seagrass + 2.14 √ √ √ √ 2.65-6.10 sc bare sand 2.18 √ √ 0 √ 2 0.35-2.15 sand seagrass ++ 1.99 √ √ 0 0 1.70-3.30 sand seagrass + 2.27 √ √ √ √ 3.70-6.60 sand bare sand 1.60 √ 0 √ √ 3 0.62-2.25 sand seagrass ++ 1.36 √ √ √ 0 2.00-3.71 sand seagrass + 0.99 0 √ √ √ 4.00-8.85 sandy clay bare sand 1.55 √ √ √ √ 4 0.40-2.50 sand seagrass ++ 1.53 √ √ √ √ 2.00-3.30 sand seagrass + 1.06 0 0 √ 0 8.85-13.02 sandy clay bare sand 1.04 √ 0 √ √ 5 0.35-2.60 sand seagrass ++ 1.44 √ √ 0 √ 2.10-3.77 sand seagrass + 1.39 0 √ √ √ 4.70-9.50 sandy clay bare sand 1.17 0 0 0 0 6 3.65-9.55 sand, loam clay sandy bare sand 1.18 √ √ √ √ 7 14.0-31.0 clay bare sand 1.18 √ √ √ √ ++: densely; +: less dense; bs: bare sand; √: found; 0: not found table 2. distribution of the ovigerous female of blue swimming crabs (portunus pelagicus) according to habitat type in lasongko bay, central buton, indonesia. 111 int. j. aquat. biol. (2016) 4(2): 108-116 the highest found at station 7 and the lowest at station 2. the proportion of ovigerous female crabs during the study period found at depths ranged 1.5-2.5 m on sandy substrate and dense seagrass coverage was 11.95%, and that at depths ranging from 2.5 to 3.5 m on sandy substrate and seagrass coverage was less than 13.33%. the proportion of ovigerous female crabs at depths ranging from 5 to 12 m on sandy-clay type substrate with a bare sand bottom was 10.14%. the rates at which ovigerous female crabs were caught at stations 6 and 7 were higher than other stations, and those with eggs of all four colors were found at stations 6 and 7. stations 6 and 7 were located far from intertidal areas. characteristic habitat types of the ovigerous female crabs at station 6, which is located in the central part of the bay at depths ranging 3.65-9.55 m, were sand and sandyclay loam substrates, with relatively clear water and bare sandy bottom. station 7, which is located at the mouth of the bay at depths ranging from 14.0 to 31.0 m, had a clay type substrate with clear water and fine bare sand on the bottom. at the other five stations, all of which were directly related to intertidal zones at water depths ranging from 0.35 m (low tide) to 13.5 m (high tide), the substrates were generally sandy with overgrown seagrass, and stations 1 and 2 had relatively high turbidity levels (table 2). body size of ovigerous female crabs: the ovigerous female crabs caught in the lasongko bay ranged 86.6-162.3 mm in carapace width and 42.75-314.22 g in total weight. the mean carapace width at each station (habitat type) ranged 104.4-125.0 mm with body weight ranging 80.60-150.72 g. the largest crab was found at station 7 and the smallest one at station 2. there were significant differences in body size of the ovigerous female between stations (p<0.05), but not with regard to egg color (p>0.05) (table 3). variable specimen number carapace width (mm) body weight (g) based on the station 1 15 113.2±10.0bc 103.22±27.27b 2 8 104.4± 6.0c 80.60±10.44b 3 27 118.3±15.0ab 137.42±50.35ac 4 48 122.5±9.2ab 139.14±35.82ac 5 20 116.7±10.7ab 118.98±34.75ac 6 29 115.4±10.8b 113.51±31.17b 7 21 125.0±13.3a 150.72±54.57a based on the color of egg yellow 57 119.1±9.9a 123.62±33.39a orange 48 119.2±10.2a 129.69±33.84a brown 31 117.6±7.3a 119.76±25.52a dark grey 32 121.6±8.7a 142.16±33.83a column and the same item and followed the same letter show no significantly different (p>0.05) table 3. sizes of ovigerous female in blue swimming crabs (portunus pelagicus) based on station, egg color, and season. eggs color number egg wet weight (g) egg dry weight (g) egg diameter (µm) egg volume (µm3) yellow 30 17.14±8.08a 4.31±2.01a 249.2±26.5a 8.5 x 106 a orange 29 18.39±8.30a 4.18±1.61a 291.6±19.4b 13.2 x 106 b brown 26 20.00±7.55a 4.65±1.92a 310.3±17.8c 15.9 x 106 c dark grey 25 23.37±0.56a 4.23±1.78a 311.9±21.4c 16.3 x 106 c changes (%) +36.35 -1.86 +25.16 +91.76 + = increase; = decrease; column of the same followed the same letter show no significantly different (p>0.05) table 4. weight, diameter, and volume of eggs from ovigerous female blue swimming crab (portunus pelagicus) according to egg color. 112 hamid et al./ distribution, body size, and eggs of ovigerous blue swimming crab egg weight, diameter, and volume: the wet weight of eggs caught during the study period ranged from 1.50 to 46.47 g, while the dry weight of eggs ranged from 0.17 to 10.55 g. the wet weight of ovigerous female crab eggs increased by 36.35%, whereas the dry weight decreased by 1.86% with changes in egg color from yellow to dark gray. however, differences in wet and dry weights of crab eggs according to color were not significant (p>0.05) (table 4). the wet and dry weights of eggs for each of the four crab egg colors showed a highly significant, strong, and positive correlation (p<0.01) (fig. 2). these observations indicate that dry weight increased with increasing wet weight of eggs with changing color. the greatest weight gain for dried eggs was for those of yellow and the lowest was for eggs with dark gray colour (fig. 2), indicating that the water content of yellow crab eggs is lower than others. the eggs ranged 180-400 μm in diameter as color changed from yellow to dark gray, with an average of 249.2-311.9 μm. the egg diameter increased 25.16% with changing its color from yellow to dark gray. the volume of crab eggs ranged from 3.1×106 to 33.5×106 μm3, with an average of 8.5×10616.3×106 μm3, and increased by 91.76% from yellow to dark gray (table 4). both the diameter and volume of eggs differed significantly between ovigerous crab eggs of different colors (p<0.05). discussion distributions of ovigerous female crabs by habitat type: ovigerous female crabs in lasongko bay with varying egg colors were found in various habitat types and occurred in all months during the study period, with the highest proportion in november and the lowest in september (hamid et al., 2015). the distribution of ovigerous female crabs in lasongko bay according to the color of their eggs showed no clear pattern in relation to the different habitat types as characterized by water depth, type of substrate, and bottom water and turbidity conditions. this is in contrast to previous studies (kangas, 2000; potter and de lestang, 2000; potter et al., 2001; de lestang et al., 2003; nitiratsuwan et al., 2013) in which the distribution of ovigerous female crabs was influenced by depth, substrate type, and water figure 2. relationship between wet weight (bb) and dry weight (bk) of (a) yellow, (b) orange, (c) brown, and (d) dark gray ovigerous blue swimming crab (portunus pelagicus) eggs. 113 int. j. aquat. biol. (2016) 4(2): 108-116 turbidity. the four different egg colors of ovigerous females in lasongko bay were found at almost equivalent ratios in a variety of habitat types. some previous studies (potter et al., 1983; sumpton et al., 1994; kangas, 2000; xiao and kumar, 2004) indicated that ovigerous female crabs migrated to clear water with sandy substrates for spawning and hatching of eggs. the ovigerous females with dark gray eggs were found in all types of habitat, but stations 1, 2, and 3 at depths ranging from 0.35 to 2.35 m and overgrown with dense seagrass, and also at station 4 with a depth ranging from 2.0 to 3.30 m and with less dense seagrass. these observations indicates that the spawning and release of crab larvae in lasongko bay did not always occur in deep, and clear water. thus, spawning and larval release occurred in various types of habitat, from shallow seagrass areas at a depth of approximately 0.35 m (low tide) with sandy substrate to locations 31 m (high tide) deep with fine sand (dominated by clay) and in relatively high turbidity (2.27 ntu) to relatively clear water (0.99 ntu). the basis for determining larval release location was based on the fact that the dark gray eggs of ovigerous female crabs hatch within a period of 12 to 24 hours (arshad et al., 2006). this was also confirmed by the observations of crab fishermen in lasongko bay, who temporarily store ovigerous female crabs with dark gray eggs in cages for 16 hours, after which the crabs no longer have eggs on their abdomens (i.e., they have released their eggs). some researchers (sumpton et al., 1994; potter and de lestang, 2000; de lestang et al., 2003) mentioned that crabs in bay ecosystems often do not migrate when spawning, and under certain conditions when there is a change in salinity, crabs spawn in the waters of bays with high salinity. the average salinity at the study site was relatively high, ranging from 29.5 to 33.4 psu, which supported spawning and hatching of crab eggs. ovigerous female crabs spawn, and their eggs hatch, at salinities of 26-32 psu (juwana, 2006), and the optimum salinity for growth of larvae is 30 psu (ikhwanuddin et al., 2012b). further tagging studies are needed to determine the migration patterns of ovigerous female crabs and the distribution of their larvae in lasongko bay. body size and egg volume and diameter: body size location (source) eggs color diameter (µm) volume (µm3) parangipettai, tamil nadu, india (soundarapandian and tamizhazhagan, 2009) yellow 350-380 orange 390-400 brown 400-410 dark grey 410-420 palk bay, mandapam, india (ravi and manisseri, 2013) yellow 324 15 x 103 blackish 21 x 103 cockburn sound, australia (poter et al., 2001) orange clear 235-324 brown 320 sematan coast, serawak, malaysia (ikhwanuddin et al., 2011) yellow-orange 373.4 9.3 x 106* brown 383.2 black 390.3 port dickson, johor coast, malaysia (ikhwanuddin et al., 2012) yellow 307 yellow 386 orange 396 persia gulf and oman sea, iran (safaie et al., 2013) yellow 300 yellow grey 300 grey 310 lasongko bay, indonesia (this study) yellow 249.2 8.5 x 106 orange 291.6 13.2 x106 brown 310.3 15.9 x 106 dark grey 311.9 16.3 x 106 = data not available; * = the average of the three color eggs table 5. mean egg diameter and volume of blue swimming crab (portunus pelagicus) according to egg color at several locations. 114 hamid et al./ distribution, body size, and eggs of ovigerous blue swimming crab of ovigerous females showed greater variation between seasons than stations or egg colors. the ovigerous females caught at station 7 were larger than those caught at other stations, especially stations 1 and 2. station 7 is located at the mouth of the bay with deeper water (14-31 m), while stations 1 and 2 are located on the inner side of the lasongko bay near the coastline with muddy and shallow habitats (1-4 m) and a low density of seagrass beds. crab carapace width of ovigerous females in this study was in the range reported works (32-193 mm) (batoy et al., 1987; de lestang et al., 2003; arshad et al., 2006; johnson et al., 2010; kamrani et al., 2010; ikhwanuddin et al., 2011, 2012a; josileen, 2013; safaie et al., 2013; zairion et al., 2014). egg diameter was found ranged 300-420 μm, showing smaller size than previous reports (soundarapandian and tamizhazhagan, 2009; ikhwanuddin et al., 2011, 2012a; safaie et al., 2013). the volume of egg was also smaller than that reported by ravi and manisseri (2013). however, the pattern of change in the size and color of the eggs during embryo development was identical to the previous studies (arshad et al., 2006; soundarapandian and tamizhazhagan, 2009; ikhwanuddin et al., 2011, 2012a; ravi and manisseri, 2013; safaie et al., 2013). the diameter and volume of eggs during embryonic development characterized by changing from yellow to dark gray increased 25.16% and 91.76%, respectively. the increase in the egg volume, ranged from 46.52% to 75%, was higher than previous reports (ravi and manisseri, 2013), whereas the increase in its wet weight in the present study (36.35%) was lower. the increase in size of the crab eggs is probably due to an increase in size of the embryo, the absorption of water through the membrane of the egg, and water retention resulting from respiration and fat metabolism (figueiredo and narciso, 2008; ravi and manisseri, 2013), which are not uncommon phenomena in the brachyura (figueiredo and narciso, 2008). it was also reported that significant increase in wet weight, diameter, and volume of crab eggs is occurred on the seventh and eighth day during incubation period or two days before the end of embryonic development (ravi and manisseri, 2013). eggs of the blood-spotted swimming crab, portunus sanguinolentus, were also reported to change in diameter during embryonic development, i.e., from 260.16 μm at stage 1 to 290.2 μm and 340.32 μm at stages 2 and 3, respectively (soundarapandian et al., 2013). as conclusions, ovigerous blue swimming crabs, portunus pelagicus, with yellow to dark gray eggs showed a wide habitat distribution from shallow to deeper water. a large degree of variation in body size was detected in ovigerous female crabs collected from various types of habitat with different levels of embryonic development. the wet weight, diameter, and volume of the eggs increased with color change as an indicator of embryonic development, but the dry weight decreased. acknowledgments thanks go to kaharudin, umi kalsum and la mpiri for help during sampling in the field and laboratory as well as crab fishermen in lasongko bay for providing samples of ovigerous female crabs. agus alim hakim prepared the figures of the paper. references arshad a., efrizal, kamarudin m.s., saad c.r. 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(2019) 7(6): 351-356 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article modelling the spatial distribution of the yellowfin tuna, thunnus albacares in the persian gulf using a fuzzy rule-based classification mona ghaitaranpour1, hadi poorbagher*1, soheil eagderi1, jahangir feghhi2 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2department of forestry and forest economics, faculty of natural resources, university of tehran, karaj, iran. s article history: received 30 october 2019 accepted 20 december 2019 available online 2 5 december 2019 keywords: predictor distribution pattern yellowfin tuna environmental variables satellite data abstract: yellowfin tuna, thunnus albacares, are the most important ecological and economic fishes in the persian gulf. in recent decades, their populations have faced overfishing, environmental problems and climate change. in this study, using some environmental variables affecting the habitat of tuna fish, i.e. sea surface temperature at night and day, reflection of 645 nm wavelength as a water turbidity, angstrom view of aerosol 443 to 965 nm, aerosol optic thickness at 869 nm, organic and inorganic particle carbon, photosynthetic active radiation, absorption by phytoplankton at 443 nm and chlorophyll-a concentration from 2002 to 2018, on the spatial distribution of yellow-fin tuna has been modelled by fuzzy rule-based classification. over the years, the variables had different degrees of importance in the models. there was a great variation in the spatial distribution of the species from year to year. introduction aquatics are the largest natural wealth of the persian gulf, if we do not consider oil reserves in this region. in fact, fisheries is the most widespread profession in the area and the economy of the region is dependent on the persian gulf ecosystem after oil (alavian petroody et al., 2013). the tuna fish population, as the commercially important species, has declined in the persian gulf (sadeghi, 2001). overfishing (worm et al., 2009), climate change (cheung et al., 2008; klein et al., 2017; cheung and oyinlola, 2018) and pollution (moran et al., 2011) are amongst the factors that decreased their stocks especially in closed and semiclosed ecosystems. management of aquatic resources needs information about behaviour of species and environmental factors affecting distribution of aquatics such as temperature (guillen et al., 2014), salinity, turbidity (pekcan-hekim., 2007), sea surface temperature and chlorophyll-a (putri et al., 2018). often, comprehensive information is needed for management purposes. thus, access to such information is possible through satellite remotely *correspondence: hadi poorbagher doi: https://doi.org/10.22034/ijab.v7i6.751 e-mail: poorbagher@ut.ac.ir sensed data (chassot et al., 2011). this information helps to make predictive models to forecast future status of species. such models are beneficial in assessing the vulnerability of endangered species (miguel and santos, 2000; amiri et al., 2017). there are many numerical ways to predict the future status of ecosystems, including time series (coro et al., 2016; amiri et al., 2018), neural networks (kim et al., 2012) and fuzzy logic (nishidaa et al., 2007). among them, nonlinear methods can deal with uncertainty problems. what distinguishes fuzzy logic from other predictive methods is the knowledge of human experts that can be set in some rules to solve complex real-world problems (riza et al., 2015). this study investigated the distribution pattern of the yellowfin tuna, thunnus albacares, in the persian gulf using a fuzzy rule-based classification. this species is pelagic, distributing in tropical and subtropical seas except the mediterranean sea. it feeds on fishes, crustaceans and squids. its maximum length and weight reach to 150 cm and 200 kg, respectively (eagderi et al., 2019). yellowfin tuna is an ecologically and commercially important species and 352 ghaitaranpour et al./ distribution of thunnus albacares in the persian gulf one of the near-threatened species in the persian gulf and the world (eagderi et al., 2019). materials and methods data sets: the coordinates of the presence points of yellowfin tuna were downloaded from the gbif site (global biodiversity information facility, 2017). the environmental data of all oceans were obtained from the website of the modis project (modis.gsfc. nasa.gov) of nasa. the environmental data were reflection in 645 nm (r645, sr-1) as a surrogate of turbidity (chen et al., 2007), angstrom view of aerosol (965-443 nm, a443), aerosol optics thickness in 869 nm (a869), organic and inorganic carbon particles (pic, poc, mol m-3), photosynthetically active radiation (par, einstein m-2 day-1), the absorption of light by phytoplankton in 443 nm (phy, m-1), sea surface temperature at day and night (sstd, sstn, °c) and chlorophyll-a concentration (chl, mg m3). the annually-averaged data was in network common data form (.nc) format. data were transformed to raster format using the package raster in r3.5.1 and were stacked. statistical analysis: we used ishibuchi’s method based on hybridization of gccl and pittsburgh (riza et al., 2015) to model effects of environmental parameters on the presence or absence of yellowfin tuna. all fuzzy methods were performed using the package frbs in r. to avoid overfitting, a monte carlo crossvalidation was used (kuhn and johnson, 2013). seventy five percent of the data were randomly selected as the training data and the rest as the testing data. the selected 75% of the training data was used for modelling for 25 times. the performance of the model was examined using the area underneath of the roc chart, accuracy and the cohen’s kappa statistic (kuhn and johnson, 2013). the importance of each environmental factor, as a predictor in the model, was examined using the function varimp() of the package caret in r. to examine the relationship between independent variables, a pearson’s correlation coefficient was used. the plots and the maps were drawn using the dismo packages in r3.5.1. results the average of the importance of different environmental parameters during 2002-2018 indicated that among the 10 variables, par was the most important factor influencing the presence of this species. at the second and third degrees, water turbidity and angstrom view of aerosol were the important factors influencing the distribution of yellowfin tuna (table 1). par was the most important factor in prediction of spatial distribution of yellowfin tuna in 2002, 2003, 2004, 2006 and 2007 (table 1). the maximum correlation among the independent variables were belonged to those of par and sea surface temperature at night. in 2013, par was also the most important predictor in the model with the highest correlation was found between par and sstd. in the years 2005 and 2008, sstd was of the highest importance in the model. the greatest correlation was found between sstd and pic, and table 1. the importance of each environmental parameter in the studied years. refer to the text for the abbreviated terms of the table. no model was made in 2017 and 2018 due to the lack of enough data. 353 int. j. aquat. biol. (2019) 7(6): 351-356 between sstn and aerosol optics thickness in 869 nm in those years, respectively. in 2009 and 2011, the sstn the most important predictor of the model. in those years, the greatest correlations were found between sstn and poc, and between sstn and par, respectively. in 2010, a869 was of the highest importance in the model. the greatest correlation was found between poc and phy. in 2012 and 2014, a443 was of the highest importance in the model, and a869 had the highest correlation with turbidity. in 2015, water turbidity was the most important parameter of the model with the greatest correlation was found between poc and par. in 2016, phy had the highest importance in model. the most probable presence of tuna fish in the persian gulf was found in 2010, 2014, 2015 and 2016. no model was made for the persian gulf in 2017 and 2018 due to lack of enough data (table 1 and fig.1). the kappa's statistic in most years was >0.8 and in one case (2005) was <0.8. the kappa's statistic was zero in 2015 and 2016 (table 1). accuracy was >0.8 in all studied years (table 2). the fuzzy logic model predicted high variation in probability of presence of t. albacares. no clear trend was discernable in spatial distribution of the species over time. from 2000 to 2010, the middle part of the persian gulf had the highest probability of presence of this species. after 2014, an increase in probability of presence of yellowfin tuna was found in the region with all parts of the persian gulf was predicted have to highly probable of presence of this species. discussions fishes are poikilothermic animals and their life history follow the variation of several environmental parameters controlling their biology including seasonal migration and presence in any given place (gunarso, 1985). environmental parameters interact to each other causing positive or negative synergistic effects that are finally being reflected in behaviour (rezagholinejad et al., 2016). the present study, predicted spatial distribution of an economically important fish in the persian gulf using the distributional data of the species in other parts of the world. the present study found great variation in the probability of presence of yellowfin tuna. although table 2. the performance of the fuzzy model in 2002-2018. year accuracy kappa year accuracy kappa 2002 0.9438 0.8875 2010 0.9208 0.8417 2003 0.9609 0.9216 2011 0.9692 0.9366 2004 0.9732 0.9464 2012 0.9655 0.931 2005 0.8235 0.6483 2013 0.9568 0.9139 2006 0.9723 0.9444 2014 1 1 2007 0.9938 0.9876 2015 0.8000 0 2008 0.9885 0.977 2016 0.8571 0 2009 0.9733 0.9467 2017 table 3. the parameters used in the model. the data source of the all parameters was modis.gsfc.nasa.gov. temporal resolution of the data was annual with a 9-km spatial footprint. environmental variables abbre. unit reflection is 645 nm r645 sr-1 sea surface temperature at day sstd °c sea surface temperature at night sstn °c angstrom view of aerosol 443-965nm a443 nm aerosol optics thickness in 869nm a869 nm particles organic carbon poc mol/m3 particles inorganic carbon pic mol/m3 photosynthesis active radiation par einstein/m2/day absorption of light by phytoplankton in 443nm phy m-1 chlorophyll-a concentration chl mg/m3 354 ghaitaranpour et al./ distribution of thunnus albacares in the persian gulf the middle area of the persian gulf were predicted to had the greatest probability of presence before 2010, an increase was found in the area of the gulf that had a high probability of presence of this species after 2010. in later years, aerosol and turbidity were the environmental parameters that had the greatest importance in the models. this may suggest that those parameters may have had correlation with dust storm and enrichment of the persian gulf with the minerals carried by the storm (poorbagher and eagderi, 2017). in years 2003, 2004, 2006, 2007 and 2013, par had a high correlation with sea surface temperature at day and night indicating its increasing effect on the sea surface temperature. a study shows that there is an inverse relationship between sea surface temperature and chlorophyll-a (nurdina et al., 2013). therefore, this may explain decrease or limited presence of this fish in the persian gulf. our study (nishidaa et al., 2007) indicated that fuzzy rule-based modelling can successfully predict spatial distribution of species where the dependent variable is binary. there are many fuzzy rule-based models that are implemented in r. the package frbs provides various method that enable modelling both categorical and continuous data. references alavian petroody s, ashrafi s., eagderi s., khazaee m. (2013). investigation of body size effect on bioaccumulation pattern of cd, pb and ni in the soft tissue of rock oyster saccostrea cucullata from laft figure 1. the predicted probability of presence of yellowfin tuna in the persian gulf in 2002-2018. 355 int. j. aquat. biol. (2019) 7(6): 351-356 port. journal of the persian gulf, 4(14): 39-45. amiri k., shabanipour n., eagderi s. (2017). using kriging and co-kriging to predict distributional areas of kilka species (clupeonella spp.) in the southern caspian sea. international journal of aquatic biology, 5(2): 108-113. amiri k., shabanipour n., eagderi s. (2018). forecasting the catch of kilka species (clupeonella spp.) using time series sarima models in the southern caspian sea. caspian journal of environmental sciences, 16(4): 349358. chassot e., bonhommeau s., bonhommeau g., nieto k., polovina j.j., huret m., dulvy n.k., demarcq h. 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(2013). the relationship between sea surface temperature and chlorophyll-a concentration in fisheries aggregation area in the archipelagic waters of spermonde using satellite images. aip conference proceedings, 1571: 466-472. pekcan-hekim z. (2007). effects of turbidity on feeding and distribution of fish. ph.d. thesis, university of helsinki, department of biological and environmental sciences. poorbagher h., eagderi s. (2017). modeling the effects of sea surface temperature and aerosols on presence of epinephelus (perciformes: serranidae) in the persian gulf using remotely sensed data. the fifth iranian conference of ichthyology, islamic azad university of babol, 13-14 december 2017. pp: 71-721. putri a.r.s., zainuddin m., putri r.s. (2018). effect of climate change on the distribution of skipjack tuna katsuwonus pelamis catch in the bone gulf, indonesia, during the southeast monsoon. aacl bioflux, 11(2): 439-451. rezagholinejad s., arshad a., nurul amin s.m., ehteshami f. (2016). the influence of environmental parameters on fish larval distribution and abundance in the mangrove estuarine area of marudu bay, sabah, malaysia. survey in fisheries sciences, 2(2): 67-78. riza s.l., bergmeir ch., herrera f., benıtez m. (2015). frbs: fuzzy rule-based systems for classification and regression in r. statistical software. journal of 356 ghaitaranpour et al./ distribution of thunnus albacares in the persian gulf statistical software, 65(6): 1-30. sadeghi n. (2001). biological and morphological characteristics of southern iranian fish. naghsh mehr, tehran. 432 p. worm b., hilborn r., baum j.k., branch t.a., collie j.s., costello c., fogarty m.j., fulton e.a., hutchings j.a., jennings s., jensen o.p. (2009). rebuilding global fisheries. science, 578-585. int. j. aquat. biol. (2022) 10(4): 280-284 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology short communication culturable microflora of artemia franciscana reared under laboratory conditions haruka kurihara, tomoya akagi, hitomi nimura, shiro itoi, haruo sugita*1 department of marine science and resources, nihon university, kameino 1866, fujisawa, kanagawa 252-0880, japan. s article history: received 26 june 2022 accepted 1 august 2022 available online 2 5 august 2022 keywords: microflora vibrionaceae opportunistic pathogens 16s rrna gene abstract: artemia is widely used as an initial food for larval and juvenile fish in aquaculture facilities around the world. however, several lines of research have strongly suggested that artemia larvae may carry opportunistic pathogens such as listonella anguillarum, thereby serving as a source of infection of fish. in the present study, we investigated the dynamics of the culturable microflora of artemia reared under laboratory conditions, with the goal of understanding the risk of opportunistic infection mediated by this animal. after hatching decapsulated cysts of a. franciscana, the larvae were reared for an additional 27 days to examine, using the culture-dependent method, the culturable microflora of the rearing water and of washed artemia. the results showed that vibrionaceae, flavobacteriaceae, pseudoalteromonadaceae, alteromonadaceae and rhodobacteriaceae accounted for 8.3-35.8% of the rearing water isolates. in contrast, vibrionaceae dominated in artemia isolates, accounting for 79.2% of the flora. however, vibrionaceae were not detected in either decapsulated or undecapsulated cysts, or in the algal concentrates used as feed, suggesting that vibrionaceae is not indigenous to artemia cysts and instead is derived primarily from natural seawater. these results strongly suggest that hatching and rearing live diets such as artemia under sanitary conditions may reduce the risk of opportunistic infection. introduction artemia is one of the most important foods for marine fish in aquaculture facilities worldwide, as its cysts can be incubated in seawater or salt water for one to two days to produce nauplii of appropriate size. in addition, artemia can be enriched with special nutrients such as eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) for the use of artemia in the diets of captive animals that require these substances (lavens and sorgeloos, 1996). however, kurosaki et al. (2021), in an examination of the gut microbiota of red seabream, pagrus major during early development, found that vibrio spp. in larval guts are derived primarily from rotifers brachionus plicatilis sp. complex s-type and artemia sp. nauplii used as the diet. previously, mizuki et al. (2006), in an investigation of the dynamics of the fish pathogen, listonella anguillarum in a hatchery of japanese flounder, paralichthys olivaceus, suggested that l. anguillarum was being transmitted from rotifers, *correspondence: haruo sugita doi: https://doi.org/10.22034/ijab.v10i4.1637 e-mail: sugita.haruo@nihon-u.ac.jp b. plicatilis and artemia nauplii. similar results have been reported by many researchers (grisez et al., 1977; tanasomwang and muroga, 1988; lópeztorres et al., 2001; eddy and jones, 2002). however, to our knowledge, there are no reports on the dynamics of the microflora of artemia during the long-term rearing of this animal. in marine fish farming, vibrio spp. often is detected in the fish gut, and the rearing environment contains a number of opportunistic pathogens; the presence of these pathogens is an extremely important risk factor in the aquaculture industry. therefore, in this study, we investigated the dynamics of the culturable microflora of artemia reared for relatively long periods of time, specifically by employing a culturedependent approach in combination with 16s rrna gene (16s rdna) sequencing. materials and methods experiments were performed with a. franciscana 281 int. j. aquat. biol. (2022) 10(4): 280-284 cysts obtained from the great salt lake, utah, usa, as supplied by osi marine lab (snowville, ut, usa). artemia cysts were decapsulated as described by lavens and sorgeloos (1996). for hatching and rearing artemia, a glass medium bottle with a capacity of 1,200 ml was used; two glass tubes with cotton filters attached were inserted into the lid of the bottle, and the air was delivered from an air pump into one of the tubes. the tip of another glass tube was placed above the water surface, permitting exhaust of spent air into the atmosphere. after the apparatus without an air pump was autoclaved at 121°c for 15 min and allowed to cool to room temperature, 1,000 ml of unsterilized natural seawater and 0.01 g of decapsulated cysts were placed in the bottle; the culture then was incubated at 25°c with aeration at a flow rate of 2.9 ml/sec and exposed to constant incandescent light. the natural seawater collected on enoshima island, fujisawa, kanagawa was used. artemia larvae were fed an algal concentrate (chlorella industry co., tokyo, japan) once every two days for an additional 27 days. surface bacteria loosely attached to the artemia body were removed according to the method of austin and allen (1982). specifically, sterile seawater was added to ten washed artemia larvae to make a total volume 100-500 μl depending on artemia size, which then was homogenized to generate a washed sample. artemia homogenates and rearing water sampled on days 0, 1, 7, 14, 21, and 28 were diluted serially with sterile seawater and inoculated onto 1/20 pybg agar medium, which contains the following (per 1000 ml of aged seawater): trypticase peptone (beckton dickinson, franklin lakes, nj, usa), 0.5 g; phytone peptone (beckton dickinson), 0.25 g; bacto-yeast extract (beckton dickinson), 0.1 g; lab lemco powder (thermo fisher scientific, waltham, ma, usa), 0.1 g; glucose, 0.1 g; and agar, 15 g; the resulting solution is adjusted to ph 7.5 (sugita et al., 2005). the inoculated agar plates were incubated at 25°c for 7 days under aerobic conditions. additionally, homogenates of both decapsulated and undecapsulated cysts, as well as natural seawater and an algal concentrate, were processed similarly. after incubation, the viable counts (colony-forming units (cfus) per ml or nauplius) were determined, and 1920 colonies per sample were chosen and purified by streaking to a fresh solid medium. 16s rdna of each isolate was amplified and sequenced according to hiraishi (1992), with some modifications. briefly, 16s rdna was amplified by polymerase chain reaction (pcr) using a primer set consisting of 20f (5'-aga gtt tga tcc tgg ctc ag-3') and r2l (5'-catcgtttacggcgtggac3'), and the resulting fragments were partially sequenced using the bigdye terminator v3.1 cycle sequencing ready reaction kit with a model abi 3130xl automated sequencer (applied biosystems, foster city, ca, usa). the final sequence (approximately 690 bp) was determined from overlapping sequence data using the autoassembler ver. 2.1 computer program (applied biosystems). the isolates were identified using ezbiocloud (yoon et al., 2017) on the basis of their 16s rdna sequences. representative sequences from this study have been deposited into the ddbj/genbank/embl databases (accession numbers: lc13330-lc13337). results and discussions a total of 299 bacteria were isolated, and all were classified into 22 families based on 16s rdna sequences. vibrionaceae (140 isolates), pseudoalteromonas (34), flavobacteriaceae (26), moraxellaceae (16), alteromonadaceae (15), stappiaceae (12), and rhodobacteriaceae (12) were the major families of isolates obtained in this study. notably, no bacteria were detected in the decapsulated cysts. table 1 shows the composition of 16 families in bacterial isolates from the undecaspulated cysts, from an algal concentrate, and from natural seawater, which exhibited viable counts of 1.2×106 cfu/g, 6.5×105 cfu/ml, and 6.6×103 cfu/ml, respectively. moraxellaceae and planococcaceae accounted for 25.0-60.0% of the isolates from undecapsulated cysts; microbacteriaceae, bacilli, and moraxellaceae accounted for 20.0-30.0% of the isolates from the algal concentrate. however, bacteria belonging to vibrionaceae were not isolated from either of those 282 kurihara et al./ microflora of artemia reared under laboratory conditions samples. nine families were isolated from the natural seawater, with microbulbiferaceae and stappiaceae accounting for 21.1-36.8% and vibrionaceae accounting for 10.5% of those isolates. ten families were isolated from the artemia rearing water, with viable counts of 5.0×104-2.6×107 cfu/ml (table 2). vibrionaceae, flavobacteriaceae, pseudoalteromonadaceae, alteromonadaceae, and rhodobacteriaceae accounted for 8.3-35.8% of the rearing water isolates. bacteria isolated from washed artemia ranged in viable counts from 1.4×104-1.1×107 cfu/nauplius and consisted of six families that vibrionaceae and pseudoalteromonadaceae were the top-two-most-dominant bacteria, accounting for 79.2 and 14.2%, respectively. viable counts of bacteria in rearing water and washed artemia during the rearing period varied by as much as three orders of magnitude, and the families of bacteria detected varied widely, indicating that the densities of the dominant culturable bacteria in each sample also varied greatly. table 3 shows the species composition of vibrionaceae in isolates from the natural seawater, rearing water, and washed a. franciscana. photobacterium consisted of one species, and vibrio of seven species (table 3). among the vibrio, v. parahaemolyticus (bbqd01000032) and v. tubiashii (cp009354) are known to act as pathogenic bacteria in marine animals. v. azureus (batl01000140), v. hyugaensis (lc004912), and v. neocaledonicus (jq934828) were detected in 50.066.7% of the rearing water samples and 83.3-100% of the washed artemia samples. these results suggested that bacteria of the genus vibrio have a high affinity for artemia because these bacteria are easily taken up into the gut of artemia or adhere to these animals’ body surfaces. vibrio spp. are predominantly found in the gut and on the body surface of copepods, and v. cholerae and v. parahaemolyticus adhere to copepod shells, using this matrix as a nutrient source and as a cryoprotective environment (kaneko and colwell, 1975; sochard et al., 1979; shimodori et al., 1989). however, the differences among the vibrio species in their frequencies and percentage of the bacteria may reflect the compatibility of each bacterial species with artemia and the rearing environment. the large fluctuations in vibrio representation in closest family undecapsulated cysts algal concentrate natural seawater bacillaceae 1 6 0 cellulomonadaceae 0 1 0 erythrobacteraceae 0 0 1 flavobacteriaceae 0 0 1 gordoniaceae 0 1 0 halomonadaceae 2 0 1 intrasporangiaceae 0 1 0 maricaulaceae 0 0 1 microbacteriaceae 0 6 0 microbulbiferaceae 0 0 7 moraxellaceae 12 4 0 nocardiaceae 0 1 0 planococcaceae 5 0 0 rhodobacteraceae 0 0 1 salinisphaeraceae 0 0 1 stappiaceae 0 0 4 vibrionaceae 0 0 2 viable counts (cfu/g, ml) 1.2×106 6.5×105 6.6×103 table 1. composition of bacterial families in isolates derived from the undecapsulated cysts, an algal concentrate, and natural seawater. 283 int. j. aquat. biol. (2022) 10(4): 280-284 rearing water and among artemia-associated bacteria during the artemia rearing period may be related to the effects of artemia shell molting and other factors; further research will be needed to clarify these patterns. since no bacteria were detected in decapsulated cysts, the vibrio spp. detected in artemia and its rearing water presumably existed at low densities in natural seawater and/or in the algal concentrate, subsequently multiplying and becoming dominant in the artemia-rearing environment. we hypothesize, by extension, that many opportunistic pathogens, including l. anguillarum, are not cyst-derived, but instead derive from the live diets and/or the seawater environments in which cysts are hatched and reared for a period of time (mizuki et al., 2006). lópez-torres et al. (2001) reported that artemia larvae are vectors of vibrio spp. in aquaculture activities. however, the present work suggests that these vibrio spp.. instead, result from the manipulation of artemia hatchery tanks and are not derived from the cysts themselves. in other words, vibrio spp. are ubiquitous in aquaculture facilities, but keeping artemia in an environment free of opportunistic pathogens may be an effective way to prevent opportunistic infections in the early developmental stages of marine fish being fed artemia. these previous results, viewed in the context of the present study, lead us to recommend that artemia cysts be hatched in seawater and containers that both have been disinfected, and be reared on foods and food additives that are confirmed to be free of opportunistic pathogens. we propose that similar studies also should be performed on rotifers to facilitate further effective control of opportunistic infections in seed production for aquaculture. acknowledgment this study was supported, in part, by the japan society for the promotion of science (jsps) grant-in-aid for scientific research (c) (25450263). references austin b., allen d.a. (1982). microbiology of laboratorytable 2. composition of bacterial families in isolates derived from rearing water and washed artemia. closest family no. of isolates from rearing water no. of isolates from washed artemia day 0 day 1 day 7 day 14 day 21 day 28 day 0 day 1 day 7 day 14 day 21 day 28 alteromonadaceae 0 1 0 9 0 2 0 0 0 3 0 0 bacillaceae 0 0 0 0 0 1 0 0 0 0 0 0 cyclobacteriaceae 0 0 0 0 0 2 0 0 0 0 0 0 enterobacteriaceae 0 0 0 1 0 0 0 0 0 0 0 0 erythrobacteraceae 0 0 0 1 0 1 0 0 0 0 0 0 flavobacteriaceae 0 0 13 0 4 8 0 0 0 0 0 0 oceanospirillaceae 0 0 0 0 0 0 3 0 0 0 0 0 pseudoalteromonadaceae 6 10 1 0 0 0 10 0 0 5 2 0 rhodobacteraceae 0 0 1 3 2 4 0 0 0 1 0 0 stappiaceae 0 0 0 5 0 2 0 0 0 1 0 0 vibrionaceae 14 9 5 1 14 0 7 20 20 10 18 20 viable counts (cfu/ml, nauplius) 1.8× 106 2.6× 107 1.2x 106 4.3× 105 5.0× 104 1.3× 106 1.4× 104 7.5× 105 4.9× 105 2.4× 105 1.4× 106 1.1× 107 284 kurihara et al./ microflora of artemia reared under laboratory conditions hatched brine shrimp (artemia). aquaculture, 26: 369383. eddy s.d., jones s.h. (2002). microbiology of summer flounder paralichthys dentatus fingerling production at a marine fish hatchery. aquaculture, 211: 9-28. grisez l., reyniers j., verdonck l., swings j., ollevier f. (1977). dominant intestinal microflora of sea bream and sea bass larvae, from two hatcheries, during larval development. aquaculture, 155: 387-399. hiraishi a. (1992). direct automated sequencing of 16s rdna amplified by polymerase chain reaction from bacterial cultures without dna purification. letters in applied microbiology, 15: 210-213. kaneko t., colwell r.r. (1975). adsorption of vibrio parahaemolyticus onto chitin and copepods. applied microbiology, 29: 269-274. kurosaki m., kunimoto m., akiyama n., itoi s., sugita h. (2021). predominant gut microbiota in the early life stages of red seabream (pagrus major) raised in indoor tanks. international aquatic research, 13: 219-226. lavens p., sorgeloos p. (1996) manual on the production and use of live food for aquaculture. fao fisheries technical paper no. 361. fao. 295 p. lópez-torres m.a., lizárrage-partida m.l. (2001) bacteria isolated on tcbs media associated with hatched artemia cysts of commercial brands. aquaculture, 194: 11-20. mizuki h., washio s., morita t., itoi s., sugita h. (2006). distribution of a fish pathogen listonella anguillarum in the japanese flounder paralichthys olivaceus hatchery. aquaculture, 261: 26-32. shimodori s., moriya t., kohashi o., faming d., amako k. (1989). extraction from prawn shells of substances cryoprotective for vibrio cholerae. applied and environmental microbiology, 55: 2726-2728. sochard m.r., wilson d.f., austin b., colwell r.r. (1979). bacteria associated with the surface and gut of marine copepods. applied and environmental microbiology, 37: 750-759. sugita h., kurosaki m., okamura t., yamamoto s., tsuchiya c. (2005). the culturability of intestinal bacteria of japanese coastal fish. fisheries science, 71: 956-958. tanasomwang v., muroga k. (1988). intestinal microflora of larval and juvenile stages in japanese flounder (paralichthys olivaceus). fish pathology, 23: 77-83. yoon s.h., ha s.m., kwon s., lim j., kim y., seo h., chun j. (2017). introducing ezbiocloud: a taxonomically united database of 16s rrna and whole genome assemblies. international journal of systematic and evolutionary microbiology, 67: 1613-1617. table 3. composition of bacterial species belonging to vibrionaceae among isolates derived from natural seawater, rearing water, and washed artemia. closest species (accession no.; identity, %) natural seawater no. of isolates from rearing water no. of isolates from washed artemia day 0 day 1 day 7 day 14 day 21 day 28 day 0 day 1 day 7 day 14 day 21 day 28 photobacterium salinisoli (kp054474; 97.7) 0 0 0 0 0 0 0 0 1 0 0 0 0 vibrio azureus (batl01000140; 98.8-100) 1 4 4 0 1 5 0 2 9 7 7 15 12 vibrio hyugaensis (lc004912; 98.8-99.9) 0 1 2 0 0 8 0 2 9 9 0 2 6 vibrio japonicus (lc143378; 99.4-99.6) 0 0 0 0 0 0 0 2 1 0 0 0 0 vibrio natriegens (atwu01000093; 100) 0 1 0 0 0 0 0 0 0 0 0 0 0 vibrio neocaledonicus (jq934828; 98.6-100) 0 7 3 5 0 1 0 1 0 4 3 1 1 vibrio parahaemolyticus (bbqd01000032; 98.4-99.3) 0 1 0 0 0 0 0 0 0 0 0 0 1 vibrio tubiashii (cp009354; 99.2) 1 0 0 0 0 0 0 0 0 0 0 0 0 int. j. aquat. biol. (2015) 3(1): 42-51 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article effect of water quality on the composition of fish communities in three coastal rivers of karnataka, india arunkumar shetty1, mididodi venkateshwarlu1, murugan muralidharan*21 1department of p.g. studies and research in applied zoology, kuvempu university, shankaraghatta, 577 451, karnataka, india. 2sri paramakalyani centre for environmental sciences, manonmaniam sundaranar university, alwarkurichi, 627 412, tamilnadu, india. article history: received 2 march 2014 accepted 14 april 2014 available online 2 5 february 2015 keywords: fish diversity karnataka sita river swarna river varahi river abstract: the fish assemblage and diversity in relation to water quality of three coastal rivers sita, swarna and varahi of udupi district, karnataka, india was studied. 71 species representing 7 orders, 20 families and 41 genera were recorded from 21 sites along the three rivers. species composition varied longitudinally in relation to the environmental factors of the habitat. the downstream change in the three rivers indicates that fish assemblage changed with increasing loss of riparian canopy cover and increasing agricultural land-use. the richness and abundance of fishes were correlated with land-use type, canopy cover, ph and turbidity. diversion of water, discharge of domestic sewage and agricultural runoff were prominent among the disturbances that alter the habitat quality. introduction faunal composition is distinct to geographic regions because the diversity and distribution of animals across a landscape can be interpreted in terms of their responses to the habitat characteristics (belanger and rodriguez, 2002). tropical streams with their diverse habitats and the faunal components are commonly used to denote the unique features of pristine landscapes. under mounting pressure from urbanization, this aquatic systems and their existence is being threatened in a number of ways. fish communities despite their high degree of natural variability, are indicators of ecosystem health (moyle, 1994) and hence the occurrence and abundance of fish species can be associated to water chemistry, physical habitat, and land-use activities to provide a more complete picture of quality of water and habitat across a river basin (deacon and mize, 1997). further, freshwater fishes are poorly studied group since information regarding distribution, population dynamics and threats is incomplete, and * corresponding author: murugan muralidharan e-mail address: muralistream@gmail.com most of the information is available from a few locations. modifications in the water and habitat quality are mostly due to the forest removal, urbanization, embankments and diversions for irrigation and hydroelectric power stations (may and brown, 2002). the deterioration of water quality has been recognized as a potential challenge which directly impacts the aquatic organisms leading to decline in diversity. given the increasing pressure on aquatic systems, documenting the available richness and establishing accurate estimates of the magnitude of biodiversity loss resulting from common human disturbances, such as land-use change and habitat loss, species invasions, and climate change is of particular importance (murphy and romanuk, 2014). physico-chemical characteristics are important determinants reflecting the condition of freshwater fish assemblages. it has been established that habitat variables such as water temperature, velocity, substrate, conductivity, depth and width, altitude and distance from the source influence river 43 shetty et al./ water quality impact on coastal river fishes fish composition (li et al., 2012). extensive work has been done during the last few decades on freshwater fishes of karnataka (arunachalam et al., 1997, arunachalam, 2000; daniel, 2002; bhat, 2003, 2004; ashashree et al., 2008; vijaykumar et al., 2009; heda, 2009; shahnawaz et al., 2010; shivashankar and venkataramana, 2012; ahmad et al., 2013; anandi et al., 2013). the database on freshwater fishes of karnataka based on various works lists about 201 freshwater fish species belonging to 9 orders, 27 families and 84 genera. though details on the distribution, habitat use and preferred substrate, tropic category and niche occupied by species may be available in literature, the ecology of many species vary throughout the range with respect to the changes in that region. in spite of such rich diversity, literature concerning ecology and fish community structure in karnataka is scarce and little information is available on the coastal rivers. this study summarizes the composition and structure of fish assemblages from three coastal rivers sita, swarna and varahi in southern part of karnataka and the relation of physico-chemical characteristics on the fish communities of the respective rivers. materials and methods the ichthyofaunal survey in three coastal rivers sita, swarna and varahi of udupi district, karnataka state in india was carried out in 21 study sites (fig. 1) during the period from september 2007 to september 2012. selection of sites was randomly made such that each river comprised 7 stretches with varied environmental settings from upstream to the downstream. these rivers are mostly rain-fed and the magnitude of flow is related to rainfall. the river sita originates from narasimha parvatha (13°29'7''n; 75°0'36''e) and flows for about 56.5 km in udupi district and drains into arabian sea near mabukala. the river swarna takes its origin at kuduremukh, (13°21'48''n; 74°49'54''e) and drains into arabian sea near kalyanapura. the varahi river has its origin at hebbagilu (13°41'30''n; 74°59'35''e) near agumbe in shimoga district at an altitude of 730 m. there is a dam across varahi river constructed near yadur of hosanagar taluk. further a hydro-electric power project is located at siddapura, udupi district. varahi river flows about 65 km from its origin to drain in the arabian sea near kundapura. sampling of fishes were made in different habitats such as pools, riffles, runs and cascades using gill nets (mesh sizes 10-34 mm), drag nets, scoop nets and cast nets. most samples were released back immediately after capture, however few specimens (5) of unidentified species were preserved in buffered formalin (10%). fish identification was done using standard literature by day (1875), talwar and jhingran (1991) and jayaram (2010). water samples were collected (in 2 liter pvc container) from each site between 8 am to 11 am. important water parameters such as water temperature, ph, conductivity, dissolved oxygen (do), biological oxygen demand for a five-day period (bod) and total dissolved solids of the stream water at each preselected sampling sites were determined using standard methods (apha, 1998). figure 1. sampling sites along the stretches of the three coastal rivers. 44 int. j. aquat. biol. (2015) 3(1): 42-51 sl. no fish species river sita river swarna river varahi no sp ab no sp ab no sp ab 1 hyporhamphus xanthopterus 1 0.05 8 0.73 0 0.00 2 xenentodon cancila 31 1.52 20 1.81 9 1.00 3 devario malabaricus 36 1.77 0 0.00 0 0.00 4 devario aequipinnatus 39 1.91 20 1.81 16 1.79 5 hypselobarbus curmuca 13 0.64 0 0.00 0 0.00 6 hypselobarbus jerdoni 32 1.57 31 2.81 31 3.46 7 hypselobarbus kolus 20 0.98 17 1.54 18 2.00 8 hypselobarbus kurali 18 0.88 18 1.63 28 3.13 9 labeo calbasu 12 0.59 6 0.54 6 0.67 10 oreichythys cosuatis 36 1.77 12 1.09 18 2.00 11 cirrhinus reba 52 2.55 20 1.81 11 1.23 12 dawkinsa arulius 42 2.06 0 0.00 0 0.00 13 dawkinsa filamentosa 83 4.07 63 5.72 11 1.23 14 haludaria fasciata 35 1.71 12 1.09 8 0.89 15 haludaria pradhani 17 0.83 0 0.00 0 0.00 16 puntius amphibius 51 2.50 16 1.45 18 2.00 17 puntius chola 44 2.15 18 1.63 24 2.68 18 puntius dorsalis 58 2.84 44 4.00 9 1.00 19 puntius sahyadriensis 10 0.49 35 3.18 0 0.00 20 puntius parrah 55 2.70 55 5.00 55 6.14 21 puntius vittatus 30 1.47 17 1.54 17 1.90 22 pethia ticto 30 1.47 0 0.00 0 0.00 23 systomus subnasutus 63 3.09 18 1.63 18 2.00 24 salmophasia acinaces 57 2.80 20 1.81 20 2.23 25 salmophasia boopis 30 1.47 0 0.00 9 1.00 26 barilius barna 58 2.85 20 1.81 20 2.23 27 barilius bakeri 39 1.91 20 1.81 0 0.00 28 barilius canarensis 37 1.81 18 1.63 6 0.67 29 rasbora daniconius 18 0.88 31 2.81 23 2.57 30 garra gotyla stenorhynchus 69 3.39 18 1.63 18 2.00 31 garra mullya 28 1.37 0 0.00 0 0.00 32 balitora mysorensis 13 0.64 28 2.54 8 0.89 33 bhavania australis 12 0.59 10 0.91 9 1.00 34 schistura denisoni 16 0.79 6 0.54 5 0.56 35 schistura nagodiensis 4 0.20 0 0.00 0 0.00 36 aplocheilus lineatus 12 0.59 11 1.00 2 0.22 37 microphis cuncalus 11 0.54 0 0.00 0 0.00 38 megalops cyprinoides 16 0.79 9 0.82 0 0.00 39 pseudophromenus cupanus 15 0.74 10 0.91 11 1.23 40 glossogobius biocellatus 4 0.20 0 0.00 0 0.00 41 glossogobius giuris giuris 23 1.13 18 1.63 16 1.79 42 sicyopterus grisseus 18 0.88 16 1.45 9 1.00 table 1. fish species abundance and distribution in rivers sita, swarna and varahi. 45 shetty et al./ water quality impact on coastal river fishes the length of each study site was about 100 m, a length sufficient to characterize the fish assemblage and to encompass all habitat units (lyons, 1992). at each site, habitat variables such as stream width, depth, water velocity, canopy cover, and land use were measured or visually estimated along transects. spherical densiometer was used to measure riparian canopy cover. stream discharge was measured with an electronic flow meter, water transparency was measured with a secchi disk. stream width was measured with measuring ropes at transect where flows were recorded and mean depth was calculated from the depths recorded during flow measurements. land-use category was analyzed for a 50-m-wide region along each side of the stream upstream from the site. the categories were forested area, agricultural sites, grazing area and habitation expressed as % of the total area. the relationship between the water quality parameters and the fish abundance and species richness were determined by principal component analysis (pca) and canonical correspondence analysis (cca) using past (1.89) freeware (hammer et al., 2001). results fishes collected during the study comprised of 71 species representing 7 orders, 20 families and 41 genera from the 21 study sites of the three rivers (table 1). cypriniformes was the species rich group 43 chanda nama 17 0.83 23 2.09 11 1.23 44 parambassis thomassi 39 1.91 13 1.18 9 1.00 45 channa marulius 18 0.88 16 1.45 13 1.45 46 channa orientalis 20 0.98 9 0.82 16 1.79 47 channa striatus 18 0.88 0 0.00 13 1.45 48 pristolepis marginata 0 0.00 9 0.82 0 0.00 49 horabagrus brachisoma 16 0.79 10 0.91 11 1.23 50 mystus armatus 29 1.38 8 0.73 17 1.90 51 mystus cavasius 12 0.59 5 0.45 9 1.00 52 mystus gulio 20 0.98 18 1.63 12 1.34 53 mystus malabaricus 12 0.59 12 1.09 20 2.23 54 mystus montanus 0 0.00 8 0.73 0 0.00 55 mystus oculatus 20 0.98 18 1.63 16 1.79 56 clarias batrachus 6 0.29 17 1.54 12 1.34 57 heteropneustes fossilis 8 0.39 0 0.00 0 0.00 58 pseudeutropius mitchelli 22 1.07 9 0.82 10 1.12 59 ompok malabaricus 24 1.18 12 1.09 16 1.79 60 ompok pabda 0 0.00 12 1.09 0 0.00 61 ompok pabo 20 0.64 10 0.91 10 1.12 62 wallago attu 0 0.00 6 0.54 0 0.00 63 mastacembalus armatus 13 0.98 5 0.45 11 1.23 64 etroplus canarensis 24 1.17 22 2.00 18 2.00 65 etroplus maculatus 48 2.36 0 0.00 12 1.34 66 etroplus suratensis 27 1.32 18 1.63 17 1.90 67 oreochromis mossambicus 38 1.86 12 1.09 12 1.34 68 carinotetraodon travancoricus 22 1.07 0 0.00 0 0.00 69 gerres erythrourus 0 0.00 0 0.00 8 0.89 70 gerres limbatus 0 0.00 0 0.00 9 1.00 71 gerres filamentosus 0 0.00 0 0.00 13 1.45 table 1. fish species abundance and distribution in rivers sita, swarna and varahi (continued). 46 int. j. aquat. biol. (2015) 3(1): 42-51 among the assemblage composition followed by other orders and this was consistent throughout the survey in all the three river samplings. species representing the family cyprinidae were dominant with higher abundance, followed by bagridae (catfishes) and balitoridae (loaches). as for river wise species richness, sita river recorded 64 species (7 orders, 18 families and 38 genera), swarna river 54 species (6 orders, 14 families and 36 genera) and varahi river 52 species (5 orders, 14 families and 33 genera). dawkinsa filamentosa and garra mullya were prevalent in all the study sites of sita river. only represented exotic species was oreochromis mossambicus which was predominant in the lower reaches of the river. pristolepis marginata, mystus montanus, ompok pabda and wallago attu were unique to river swarna. gerres erythrourus, g. limbatus and g. filamentosus were recorded only from the lower reaches of river varahi. species evenness was higher in the upstream sites than the other sampling locations in all the three river systems. water quality variables of the sampling stations of the three rivers were provided in table 2. environmental variables were subjected to principal component analysis (pca) and sites were sita river swarna river varahi river parameter mean (±sd) range mean (±sd) range mean (±sd) range air temp (°c) 29.28 ± 1.79 27-32 29.42 ± 1.71 27-32 29.14 ± 1.06 28-31 water temp (°c) 25.0 ± 1.41 23-27 25.57 ± 1.27 24-27 25 ± 1.15 24-27 ph 6.95 ± 0.35 6.3-7.4 6.97 ± 0.31 6.5-7.4 6.87 ± 0.26 6.5-7.3 do (mg.l-1) 5.24 ± 1.02 3.6-6.1 6.75 ± 0.82 5.1-7.5 5.37 ± 1.33 3.2-6.9 total hardness (mg.l-1) 14.7 ± 1.11 13-16 13.71 ± 1.49 12-16 14.42 ± 0.97 13-16 alkalinity (mg.l-1) 0.1 ± 0.01 0.03-0.08 0.05 ± 0.01 0.04-0.07 0.05 ± 0.01 0.04-0.06 tds (mg.l-1) 32.27 ± 1.84 29.4-34.5 31.0 ± 1.48 27.2-34.8 33.3 ± 2.02 30.3-35.8 turbidity (ntu) 3.8 ± 1.8 1-6 3.7 ± 2.13 1-7 4 ± 2.16 1-7 conductivity (µs.cm-1) 53 ± 4.24 48-59 71.71 ± 7.12 50-82 48.14 ± 6.38 42-58 table 2. the mean values (±sd) and ranges of different physico-chemical parameters of sita, swarna and varahi rivers of udupi district, karnataka, india. table 3. principal component analysis of water quality parameters as studied from rivers sita, swarna and varahi. variables code axis 1 axis 2 axis 3 water temperature wt -0.0883 -0.2257 0.848 ph ph 0.9099 -0.1559 -0.1907 dissolved oxygen do 0.2181 -0.4889 0.5561 conductivity c -0.8831 0.4079 0.05089 turbidity t -0.8963 0.2029 0.1376 total dissolved solids ds 0.3371 -0.6974 0.1849 mean width wid -0.5599 -0.6198 -0.2821 velocity v 0.5495 0.5113 0.3674 stream depth dep -0.1409 0.4415 0.4109 land-use type lu 0.6984 0.312 -0.2036 canopy cover co 0.7006 0.3169 0.1047 eigen value 4.193 2.046 1.55 % variance explained 38.12 18.60 14.15 values bold are significant. 47 shetty et al./ water quality impact on coastal river fishes distinguished in relation to their influence on the system (table 3). the pca yielded five principal components (eigen values>1) which altogether explained 80% of the variance in the data. the first two components alone explained 56.7% of the variance (fig. 2). conductance, turbidity and ph are significant and showed higher loadings than other variables. factors land-use and canopy cover contributed to discriminating between sites of the three rivers. water temperature had highest loadings on the pc3. ph values varied between sites, however the trend decreased from upstream sites towards downstream gradually. canopy cover (%) was higher and land-use category was unaltered evergreen forests in sites close to the source of the river systems. turbidity and conductivity values were higher in the downstream in all the three rivers. pca results also indicate that the patterns of variations in physico-chemical variables among the three rivers and between the sampling sites are represented by patterns of variation in the habitat characteristics governing the respective sites. species richness was influenced by water quality variables such as turbidity, conductivity and alkalinity. fish diversity was inversely proportional to the level of turbidity, lower the turbidity higher the diversity. species also differed with respect to level of turbid conditions of the sites. dawkinsa filamentosa, xenentodon cancila, devario aequipinnatus, glossogobius giuris and etroplus suratensis were abundant and distributed widely. cyprinids in general occupied clear and shallow habitats. oreochromis mossambica was the nonnative species that has naturalized in most of the sites. heteropnuestus fossilis occurred only in limited sites of the three rivers. the first and second canonical axes of cca explained roughly 36% and 20% of the general variance, respectively (fig. 3). the cca axis 1 was influenced much by the habitat morphological features land-use category and percent canopy cover rather than the water quality parameters. factors velocity, ph and depth were correlated but were however unrelated to temperature, hardness, conductivity and width. the ordination plane, on the basis of environmental factors, divided the fish figure 2. principal component analysis biplot for environmental variables and sampling sites. 48 int. j. aquat. biol. (2015) 3(1): 42-51 assemblage into groups irrespective of the family they represent. the group defined by land-use category preferred the sites close to undisturbed evergreen forests the species composed of rare and specialized forms such as bhavanaia australis (ba), sicyopterus griseus (sg), mastacembeles armatus (ma), horabagrus brachysoma (hb) and garra gotyla stenorhyncus (ggs). the same group was also positively influenced by the factors percent canopy cover, velocity and ph. the second group in the ordination segregated based on the habitat depth, species that preferred shallow to moderate depth xenentodon cancila (xc), d. filmentosa (df), puntius amphibius (pa), pristolepis marginata (pm), parambassis thomassi (pt) and hypselobarbus kurali (hk) formed a cluster. the other group that occurred in deeper stretches of the river systems was predominately of bagrid cat fishes clarias batrachus (cb), mystus gulio (mg), m. oculates (mo) and a few cyprinids rasbora daniconius (rd) and hypselobarbus kolus (hko). other species not found in the above groups, notably channa orientalis (co) occurred in turbid waters influenced by low flow and high alkaline conditions most of these sites were adjacent to agricultural land. the study reveals that the environment and habitat variables influence fish assemblage structuring in the rivers sita, swarna and varahi. discussion differences in fish communities in the three river systems reflect the water quality, habitat structure and the land-use pattern they flow through. cyprinids dominated the species list comprising most of the endemic species as has been reported previously by other studies (pethiyagoda, 2006; senthilmurugan and prabahar, 2012) in south asian streams. the dominance of cyprinids in the tropical indian rivers is due to their high adaptive variability (johnson and arunachalam, 2009) and the availability of extensive heterogeneous habitat structure (bhat, 2004). our observation that the catfishes and loaches are dominant next to cyprinids and they co-occur with cyprinids in appropriate habitats was similar to the study in river systems of the central western ghats (bhat, 2003) and the bhadra river (shahnawaz et al., 2010) of karnataka. gerres erythrourus, g. limbatus and g. filamentosus are estuarine fishes able to withstand the salinity variations and move between the river and the sea through the river mouth. insectivore species were dominant in streams with good canopy cover and figure 3. canonical correspondence analysis biplot for environmental variables and fishes. 49 shetty et al./ water quality impact on coastal river fishes substrates due to the availability of terrestrial and aquatic insects that constitute major part of their diet. as for the distribution of fishes based on trophic category the omnivore species increased in a downward direction towards lowland. this was because the nature of food depends to a great extent upon the nature of environment (bhuiyan et al., 2006) and species diversity in a given area is the result of stochastic ecological and evolutionary factors that interact on both local and regional scales (leibold and mcpeek, 2006). physico-chemical variables of lotic systems are considered as important factors in structuring fish assemblage (marchetti and moyle, 2001; may and brown 2002). turbidity, ph and conductivity influenced the fish diversity and distribution in the present study. increase in turbidity showed a decline in fish species however the species count was adjusted by few carnivore species known to occur in turbid and deep habitats. the species richness and abundance were higher in clear steams as penetration of sunlight into the water favored the algal growth supporting benthic feeders and algal scrapers like g. mullya. the higher turbidity levels could be due to higher suspended solids by human activity. increasing trend in total dissolved solids may probably be due to stagnation at least at some stretches (campos et al., 1992). the high conductivity may be due to greater ionic concentration of inlet flow (oliveira, 2004). it was related to total dissolved solids content and its value becomes higher with proportional increase of the degree of pollution (prabhakar et al., 2012), which consequently impacts fish diversity, as evident in some sites during this study. dissolved oxygen (do) fluctuate between sites and is dependent on flow and the organic matter content at the particular site, thus could vary between sites and rivers (bhalla et al, 2007). variation in water temperature is regarded as one of the most important factors of environmental variability in rivers as it influences the chemical, biochemical and biological characteristics of the water body and the fish production (rashleigh, 2004). width and depth varied between sites irrespective of its position from its origin, flow regulation due to various activities in its course could be the reason. variations in ph, conductivity and turbidity could be the effect of municipal sewage and the pollution especially due to discharge of effluents from small industries along the stretches of these rivers. transformation of the surrounding landscape, removal of riparian vegetation agricultural activities adjacent to river lead to increasing nutrient concentrations, mainly of inorganic nitrogen (pizarro et al., 2010). moreover, water flow and habitat modifications associated with urbanization and deforestation also influence the habitat quality (vyas et al., 2012; ahmad et al., 2013). a variety of factors like water quality, habitat availability, flow variability and nutrient supplies from riparian habitats control the abundance and distribution of river fishes. such environmental variables are easier to predict than other biotic variables like predation and competition. the present results corroborate with the established fact that environmental factors have great impact on both species richness and the trophic structure of fish assemblages (pouilly et al., 2006). the diversity and distribution of fishes of any aquatic system depends on the certain features like type of the ecosystem, water level fluctuations, morphometric features and bottom that have great implications. the unique distribution and variation in fish species observed during our study among the three rivers is likely because of variation in natural environmental features like geographic and geological conditions (matthews and robinson, 1998). this suggests that habitat quality and species diversity are related. among the activities that influence the habitats as observed during the study channelization of water, discharge of industrial effluents and mixing of agricultural runoff are management priorities. effective measures in controlling these activities rely on the local bodies that govern the stretches of water. conservation priorities that may yield promising results include strengthening and directing resources to organizations and institutions meant for resource 50 int. j. aquat. biol. 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(2012). fish biodiversity of betwa river in madhya pradesh, india with special reference to a sacred ghat. international journal of biodiversity and conservation, 4(2): 203-216. international journal of aquatic biology (2013) 1(5): 228-232 issn: 2322-5270 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article efficacy of fish oiland linseed oil-enriched artemia nauplii on growth performance and stress resistance of tiger barb larvae (puntius tetrazona) mohammad hadi abolhasani, seyed abbas hosseini, rasool ghorbani, mohammad sudagar, seyyed morteza hoseini*1 department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 23 july 2013 accepted 14 august 2013 available online 2 5 october 2013 keywords: tiger barb fish oil linseed oil enrichment abstract: fish oil is the important fat source in fish nutrition. high demand for fish oil and low global supply arise a need of alternative oils in fish culture. plant oils are a good candidate in this case. the aim of the present study was to compare the efficacy of fish oil and linseed oil on growth and stress resistance of tiger barb (puntius tetrazona) larvae. artemia nauplii were enriched by 2.5, 5 and 7.5 of each oil. the enriched nauplii were offered to larvae for 14 d. thereafter, fish were fed non-enriched nauplii for another 14 d. at the end of the trial, larvae were subjected to osmotic stress and their survival was recorded. there was no significant difference in final weight, sgr and weight gain among the treatments at day 14. however, oil type and oil levels significantly affected these parameters after 28 d. fish of 2.5-lo and 5-fo groups showed the best and worst performance, respectively. there was no significant difference in survival rate among the treatments, after 14 and 28 d; however, oil type significantly affected survival of the larvae after osmotic stress. survival of larvae fed on linseed oilenriched nauplii was significantly higher than that of those fed on fish oil-enriched nauplii. linseed oil showed significantly better results in growth performance and stress resistance compared to fish oil. it is concluded that linseed oil is more suitable than fish oil for artemia enrichment to feed tiger barb larvae. the potential reasons for the better performance of larvae fed on linseed oil-enriched artemia were discussed. introduction lipids are the main energy sources for fish, especially at early life stage. docosahexaenoic acid (dha, 22:6n-3), eicosapentaenoic acid (epa, 20:5n-3), and arachidonic acid (ara, 20:4n-6) are the important fatty acids in fish nutrition (sargent et al., 2002). these fatty acids support suitable growth and survival of fish as well as required energy for reproduction (sargent et al., 1999). live foods, particularly artemia, are used in freshwater and saltwater aquaculture. artemia nauplii contains 50-60 % proteins (all amino acids at the sufficient level) and 5-20 % lipid (rich in long chain fatty acids), which is of particular importance * corresponding author: seyyed morteza hoseini e-mail address: seyyedmorteza.hoseini@gmail.com tel: +989112750713 in fish nutrition (sorgeloos et al., 1986). artemia is capable of transferring different materials such as vitamins, amino acids and drugs to fish larvae. essential fatty acids, particularly epa and dha, are necessary for growth, survival and resistance to diseases in fish larvae. feeding on enriched artemia nauplii improves in non-specific immune mechanisms, resistance to diseases and environmental stresses (gapasin et al., 1998; lim et al., 2002). on the other hand, there is diffidence among the biochemical composition of different artemia strains, which this difference can be minimized by enrichment (leger et al., 1986). mailto:seyyedmorteza.hoseini@gmail.com 229 abolhasani et al/ international journal of aquatic biology (2013) 1(5): 228-232 fish oil has been extensively used in fish nutrition; because it contains sufficient level of long chain unsaturated fatty acids (epa and dha). however, global fish oil supply is not sufficient for aquaculture, because of decrease in marine catchment (wassef et al., 2007). accordingly, alternative oils are needed for fish nutrition. plant oils are a good candidate for fish nutrition, which can be used sustainably. one of these oils is linseed oil. linseed oil contains high levels of linolenic and linolenic acid that are precursor epa, dha and ara. freshwater fish can utilize these precursors (sargent et al., 2002), so that, linseed oil seems to be a good alternative for fish oil in freshwater fish nutrition. tiger barb (puntius tetrazona) is a freshwater species, which naturally found in sumatra, brunei, thailand and malaysia (welcomme, 1988). this species is native to southwest asia, however, was reintroduced to the habitats from which they have been eliminated (ng and tan, 1997). in iran, this species is of particular interest due to its beauty and low price. tiger barb needs live food in early life stages, such as many fish species. it is not cleared that whether fish oil and linseed oil leave different effect on growth performance and stress resistance of tiger barb. thus, the aim of this study was to compare the efficacy of fish oiland linseed oilenriched artemia nauplii on growth performance and stress resistance of tiger barb larvae. materials and methods cysts of artemia franciscana were used for this study. to hatch the cysts, 1 g of cysts were dispersed in 1 l water in a conical jar supplied with aeration from bottom. water salinity and temperature was 33 ppt and 28 ºc, respectively (dhont and van stappen, 2003). hatching occurred 18 h after incubation. hatching rate was determined by counting three 0.1ml-samples of the jar water. to prepare oils emulsions, 0.5 g lecithin was mixed with 100 ml distilled water in a blender for 5 min. 100 ml of this emulsion was used for each concentration (2.5, 5 and 7.5 %) of either fish oil or linseed oil. to enrich the nauplii, 2 ml of each concentration was added to 1 l water containing 200000 nauplii for 6 h. after 6 h, inoculation was repeated and nauplii was enriched for another 6 h. then the nauplii were transferred to the tanks of larvae. tiger barb larvae (2.3±0.09 mg) were purchased from a commercial hatchery. 2160 larvae were randomly stocked in 18 glass aquariums supplied with continuous aeration. to acclimatize the larvae, they were fed non-enriched artemia for a couple of days. then aquariums were divided into 6 groups with three replicates: 2.5 % fish oil (2.5-fo), 5% fish oil (5-fo), 7.5% fish oil (7.5-fo), 2.5 % linseed oil (2.5-lo), 5% linseed oil (5-lo) and 7.5% linseed oil (7.5-lo). fish were fed ad libitum, 4 times a day, for a 14-d period. thereafter, all treatments were fed by non-enriched artemia nauplii for another 14-d period. growth performance was recorded at the end of each 14-d period. water temperature, ph and dissolved oxygen were 28±0.7 ºc, 7.2±0.12 and 6.4±0.34 ppm, during the feeding trials. at the end of the experiment, fish were subjected to osmotic stress (exposure to 13 ppt water) and survival of each treatment was recorded. data were examined for normality. data were subjected to two way anova to find the effect of oil type and oil concentration on tested parameters. duncan test was used to delineate significant difference among the treatments. data were presented as mean ± sd. results growth performance of different treatments is presented in table 1. there were no significant difference in final weight, sgr and weight gain among the treatments at day 14. however, oil type and oil levels significantly affected these parameters after 28 d. generally, fish of 2.5-lo and 5-fo groups showed the best and worst performance, respectively (table 1). there was no significant difference in survival rate among the treatments, after 14 and 28 d; however, oil type significantly affected survival of the larvae 229 230 abolhasani et al/ international journal of aquatic biology (2013) 1(5): 228-232 after osmotic stress (table 2). survival of larvae fed on linseed oil-enriched nauplii was significantly higher than that of those fed on fish oil-enriched nauplii (table 2). figure 1 shows efficacy of fish oil and linseed oil to improve growth performance and stress resistance of tiger barb larvae. accordingly, linseed oil showed significantly better results in all cases compared to fish oil. discussion fish oil has been the first choice oil for fish diet formulation, for a long time. however, great demand and tiny supply of fish oil caused it not to be a sustainable oil source for aquaculture. thus, alternative oils, such as plant oils, are needed to ensure sustainable fish production. although there are many studies focused on the use of plant oils as the alternative for fish oil in the fish feeds (alexis, 1997; lupatsch et al., 1997; olivateles, 2000; izquierdo et al., 2003; montero et al., 2003; wassef et al., 2007), few study can be found on the effect of linseed oil on turbot (psetta maxima) and atlantic salmon (salmo salar) nutrition and performance (regost et al., 2003; menoyo et al., 2007). replacement of fish oil by linseed oil resulted decrease in growth performance turbot, which is not in agreement with the present study. it is because that turbot is a marine species, which needs epa and dha (sargent et al., 2002) that are abundantly found in fish oil not linseed oil. however, menoyo et al. (2007) showed that linseed oil could be an alternative for fish oil in the atlantic salmon diet, without detrimental effects, which is in agreement table 1. growth performance of tiger barb larvae fed on artemia nauplii enriched with either fish oil or linseed oil, over 14 and 28 days. different letters in each column show significant difference. n = 3. 14-d 28-d final weight sgr weight gain final weight sgr weight gain 2.5-lo 24.1±0.7 16.5±0.5 914±91 145.1±5.3 c 14.7±0.3 c 6126±525 c 5-lo 27.6±0.4 17.0±0.3 985±51 130.1±2.3 b 14.4±0.1 b 5524±248 b 7.5-lo 23.7±0.8 16.4±0.4 895±81 132.4±1.1 b 14.3±0.2 b 5454±323 b 2.5-fo 24.1±0.8 16.7±0.3 932±50 123.9±5.8 ab 14.1±0.1 b 5105±269 b 5-fo 24.2±0.7 16.8±0.4 948±58 115.8±2.7 a 13.6±0.2 a 4439±181 a 7.5-fo 24.7±0.9 16.7±0.8 936±90 124.0±4.0 ab 14.2±0.1 b 5134±115 b p value oil type 0.40 0.82 0.86 0.0001> 0.0001> 0.0001> oil level 0.19 0.61 0.59 0.02 0.009 0.01 type × level 0.13 0.79 0.74 0.21 0.07 0.1 table 2. survival of tiger barb larvae fed on artemia nauplii enriched with either fish oil or linseed oil, over 14 and 28 days. different letters in each column show significant difference. n = 3. 14 d 28 d (before stress) 28 d (after stress) 2.5-lo 91.6±0.7 94.4±2.1 75.5±3.8 b 5-lo 90.5±1.7 93.6±0.6 70.0±6.6 b 7.5-lo 91.4±1.3 93.6±0.9 80.6±6.6 b 2.5-fo 93.3±3.1 94.1±4.3 51.1±7.6 a 5-fo 93.3±1.3 95.0±1.6 62.2±10.1 a 7.5-fo 94.1±0.9 95.1±2.1 51.1±7.6 a p value oil type 0.11 0.22 0.0001> oil level 0.81 0.83 0.21 type × level 0.83 0.56 0.002 231 abolhasani et al/ international journal of aquatic biology (2013) 1(5): 228-232 with the present results. atlantic salmon, like tiger barb, is a freshwater species that needs linolenic and linolenic acids which are richly found in linseed oil. this explains the better performance of larvae fed on linseed oil-enriched nauplii compared to those fed on fish oil-enriched nauplii. there was no difference in growth and survival among the treatments, after 14 d. previous study showed there was no significant difference in survival and growth performance of dolphin (coryphaena hippurus) larvae fed by rotifer enriched by squid (rich in dha and epa) and plant oils (ostrowski and divakaran, 1990). however, in the present study, larvae showed significantly different performance at day 28. this showed that feeding on enriched nauplii could affect the larvae performance in the future, when they did not feed on enriched nauplii. the underlying mechanism of this phenomenon is not clear and needs further research. linseed oil led to better resistance to stress compared to fish oil, in the present study. it is believed to be related to better nutritional status of larvae fed on linseed oil-enriched nauplii compared to those fed on fish oil-enriched nauplii. it is demonstrated that freshwater species need both n-3 and n-6 fatty acids (sargent et al., 2002), which are richly available in linseed oil. on the other hand, fish oil is more unsaturated than linseed oil, and in turn, is more under the risk of lipid oxidation (jalali et al., 2010). since no antioxidant was used in the present study, lower survival of larvae fed on fish oil-enriched nauplii could be related to more oxidative stress compared to those fed on linseed oil-enriched nauplii. similar results were reported by jalali et al. (2010). in conclusion results showed that linseed oil is more beneficial than fish oil for tiger barb larvae. using linseed oil in artemia enrichment could enhance growth and stress resistance of tiger barb. these effects seem to be related to high concentration of linolenic and linolenic acid concentration in linseed oil that are necessary for freshwater species. references alexis m.n., karanikolas k.k., richards r.h. (1997). pathological findings owing to the lack of ascorbic acid in cultured gilthead bream (sparus aurata l.). aquaculture, 151: 209-218. dhot j., van stappen g. (2003). biology, tank production and nutritional value of artemia. in: j.g. støttrup, l.a. mcevoy (ed.). live feeds in marine aquaculture. wiley-blackwell, pp. 65-112. gapasin r.s.j., bombeo r., lavens p., sorgeloos p., nelis h.j. 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(2020) 8(1): 73-82 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 20209 iranian society of ichthyology original article induced breeding, embryonic and larval development of macrognathus pancalus (hamilton, 1822) under captive condition rimle borah*,1jyotirmoy sonowal, nipen nayak, akash kachari, shyama prasad biswas freshwater biology research laboratory, department of life sciences, dibrugarh university, assam, india. s article history: received 24 november 2019 accepted 14 february 2020 available online 2 5 february 2020 keywords: induced breeding breeding performance spawning fertilization abstract: the present study was carried out to enumerate induced breeding technique and larval development of macrognathus pancalus (hamilton, 1822) reared under captivity. five different doses of ovasis hormone (t1, t2, t3, t4, and t5) with 3 replicas each were administered to the matured brooders to standardize the breeding performance of the target species. the results indicated variation in fertilization rate, latency period, egg output and hatching rate in response to different treatments. spawning was occurred between 20-24 hrs of injection in all the experiments at 26.33±0.88°c water temperature. among all the experimental trials, the highest fertilization rate was observed in t3 (96.15±0.60) of e2 and the highest hatching rate was observed in t3 (92.49±1.00) of e2. the present work elucidated the viability of seed production of m. pancalus reared under confined condition which will useful for aquaculture and conservation. introduction macrognathus pancalus (hamilton, 1822) or barred spiny eel, a member of the family mastacembelidae, is well-distributed in the asian countries (talwar and jhingran, 1991; froese and pauly, 2006). this fish due to their sturdy nature, inhabits in a variety of habitats, including beels, small rivers, streams, canals, inundated fields, river plains and estuaries (bhuiyan, 1964, rahman, 1989; talwar and jhingran, 1991; galib et al., 2009). this species commands good market value when sold alive as food and ornamental fish in domestic and international markets, including northeastern region of india (serajuddin and ali, 2005; suresh et al., 2006; abujam and biswas, 2011; raghavan et al., 2013). in recent years, impact of anthropogenic activities such as habitat modification, dam construction, introduction of invasive species, overexploitation etc. has had profound impact on population dynamics in freshwater fishes around the world including m. pancalus (maitland, 1995; suresh et al., 2006; abujam and biswas, 2011). mitigation of such man-made adversities on these ichthyofaunal resources necessitates time bound action to stop *correspondence: rimle borah doi: https://doi.org/10.22034/ijab.v8i1.746 e-mail: borahrimlee@gmail.com further decline in their natural stocks. in this regard, aquaculture which is the fastest growing production globally represents a viable alternative to boost productivity of fishes (fao, 2011). despite its contentious implications on fish biodiversity, aquaculture has made significant contribution in management and conservation of many extant fishes by reducing pressure on the depleted stocks and sustainable utilization of aquatic resources (diana, 2009; de silva, 2012). captive breeding and reintroduction of different species into the wild have shown positive outcomes in different regions of the world (philippart, 1995). successful propagation of fishes through aquaculture requires thorough studies on their taxonomy, distribution, feeding and reproductive dynamics of brood stocks etc. (meffe, 1990; maitland, 1995). many authors have carried out studies pertaining to sexual dimorphism, reproductive strategies, feeding habits etc. upto some extent in m. pancalus (swarup et al., 1972; karim and hossain, 1972; serajuddin and ali, 2005). similar studies have also been carried out in other members of mastacembelidae family (karim and hossain, 1972; 74 borah et al./ induced breeding of macrognathus pancalus under captive condition umezawa et al., 1991; serajuddin and mustafa, 1994; das and kalita, 2003; suresh et al., 2006; oliveira and hable, 2010; rahman et al., 2011). notwithstanding, limited information on breeding performance of m. pancalus still persists that merits immediate interventions to bridge the existing shortcomings. proper supervision of natural resource or to ventilate artificial propagation or culture is obligatory to conserve the species. with this background information, the present investigation aimed to enumerate information on breeding and rearing feasibility of m. pancalus reared under captivity that might provide vital inputs in management and conservation in near future. standardization of captive breeding would help to diminish the spawning interval and intensify the production of more seeds within a short duration for commercial enslavement and thereby conserve the natural population. materials and methods collection and rearing of broodstock: samples of m. pancalus (n=162) were collected from different natural habitats (beels, oxbow lake, rivers etc.) of dibrugarh district, assam during august 2016 to august 2019. the wild collected specimens were acclimatized in laboratory conditions and maintained in facilities at department of life sciences, dibrugarh university, assam, india. fishes were fed ad libitum with formulated feed containing all necessary ingredients at 5% of total body weight on daily basis and were kept in fiber tanks of 500 l capacity and aquariums (120 × 40 × 40 cm) with flow through systems. due to their burrowing and bottom feeding habits, sand substratum was provided in the experimental set ups to simulate their natural environmental condition. different aquatic plants such as eichhornia crassipes, ceratophyllum demersum, sagitlaria guayayensis, ludwigia repens etc. were also grown to mimic the natural environment. water parameters such as ph, water temperature, dissolved oxygen (do), free carbon dioxide (fco2) and hardness were regularly monitored following apha (1998). brood fish selection: the healthy and matured male and female brood fish were selected by visual and physical examination for secondary sexual characters. the females were comparatively larger in size with dark greenish brown colour on the dorsal side and light whitish coloration ventrally. they possessed a soft and swollen abdomen with fleshy translucent protruding genital papilla with oviduct. the males were comparatively smaller in size with greenish brown dorsal and yellowish ventral side with smaller genital papilla. mature males were observed to release milts through genital papilla on applying subtle pressure on the posterior abdomen region. experimental design: for induced breeding of m. pancalus, the brooders were collected from rearing tanks and separated and released in plastic containers for acclimatization prior to hormone administration. to standardize the hormonal doses and breeding performances, three experimental trials were employed: (e1) 1:1 male and female ratio (e2) 2:1 male and female ratio (e3) 3:1 male and female ratio. five different doses of ovasis hormone treatments (t1, t2, t3, t4 and t5) were used with three replications of each during experimentation (table 1). hormone administration: synthetic ovasis hormone doses were administered near the base of the dorsal and pectoral fin at 45° with the body. prior to hormone administration, doses of hormone were calculated and prepared according to the body weight of the brooders. table 1. doses of hormone (ovasis) applied to both the sexes of macrognathus pancalus brood. treatments hormonal doses (ml/kg) male female (control) no inducement no inducement t1 0.2 0.4 t2 0.4 0.6 t3 0.6 0.8 t4 0.8 1.0 t5 1.0 1.2 75 int. j. aquat. biol. (2020) 8(1): 73-82 after hormone administration, the fishes were released at the sex ratio of 1:1, 1:2 and 1:3 in separate fiber tanks with provision of continuous air and water flow system. water hyacinths were placed as a substratum to hold the sticky eggs after ovulation. breeding performance: after ovulation, the eggs of m. pancalus were found attached to the roots of water hyacinth. microscopic observation revealed that the fertilized eggs were transparent with intact nucleus whereas the unfertilized were dark brownish. effective fecundity of each female after spawning was determined through random sampling of released eggs. the total eggs count and fertilization rate was determined following behera et al. (2010). developmental stages of fertilized eggs were monitored and documented under leica dm 750 microscope. after hatching, the hatchlings were maintained in circular fiber reinforced plastic (frp) tanks. fertilization rate and hatching rate was counted visually following the formula: fertilization rate: number of fertilized eggs total number of eggs × 100 hatching rate: number of hatchlings total number of fertilized eggs × 100 statistical analysis: the results were calculated as mean±standard error (se) in all the experiments. the experimental calculations were statistically analyzed using spss software (version 17.0 for windows). one-way analysis of variance (anova) was used to compare significant level of differences between the experimental observations. significant results (p<0.05) were additionally tested using duncan’s new multiple range test (dmrt) to evaluate significance differences among means. results a total 162 fish specimens (male=108; female=54) were used for captive rearing and induced breeding of m. pancalus. the fishes attained maturity during june to august. fertilization rate, latency period, egg output and hatching rate in response to different treatments and sex ratios have been summarized in tables 2, 3 and 4. no fertilization took place in the control (without hormonal inducement) set of experiment. table 2. (e1): captive breeding experiment of macrognathus pancalus (m: f=1:1). size of males size of females hormonal doses (ml/kg body weight) latency period (hrs) fertilization rate (%) hatching rate (%) w (g) tl (cm) w (g) tl (cm) m f 8.21±0.29 12.38±0.59 13.87±0.39 15.27±0.53 control control --- 7.58±0.14 12.06±0.84 12.21±0.82 14.14±0.58 0.2 0.4 --- 6.41±0.09 10.65±0.58 11.68±1.12 13.23±0.64 0.4 0.6 --- 6.13±0.21 11.97±0.46 13.56±0.80 14.48±0.67 0.6 0.8 24 69.86±1.41 74.25±3.50 7.81±0.08 12.1±0.34 12.59±0.56 14.32±0.52 0.8 1.0 --- 6.70±0.25 12.25±0.13 12.92±1.01 14.81±0.38 1.0 1.2 --- w, fish weight in gram; tl, total length in cm, data expressed as mean±se; n=3. table 3. (e2): captive breeding experiment of macrognathus pancalus (m: f=2:1). size of males size of females hormonal doses (ml/kg body weight) latency period (hrs) fertilization rate (%) hatching rate (%) w (g) tl (cm) w (g) tl (cm) m f 8.54±0.29 12.65±0.62 12.33±0.77 14.94±0.13 control control --- 6.91±0.07 11.39±0.44 11.87±1.08 14.13±0.52 0.2 0.4 24 84.31±2.19 87.92±3.59 6.08±0.05 10.98±0.58 12.02±0.78 13.57±0.49 0.4 0.6 22 88.56±1.03 86.67±1.83 6.46±0.14 11.30±0.32 14.56±0.32 14.48±0.10 0.6 0.8 22 96.15±0.60 92.49±1.00 7.47±0.09 12.43±0.17 12.26±0.86 13.98±0.18 0.8 1.0 20 92.94±1.72 88.96±2.63 6.33±0.21 11.92±0.46 12.25±1.06 13.81±0.38 1.0 1.2 --- w, fish weight in gram; tl, total length in cm, data expressed as mean±se; n=3. 76 borah et al./ induced breeding of macrognathus pancalus under captive condition breeding behaviour: after hormonal administration, the brooders showed varied mating behaviour in all the treatments (except control) after 10-14 hrs of injection. each female was paired with male and the mating was preceded by courtship behaviour. latency period varied significantly in different hormonal doses and in different experimental set up. spawning took place between 20 to 24 hrs of injection in different experiments as shown in tables 2, 3 and 4. fertilization rate: fertilization rate in e1 was 69.86±1.41 in t3 and no fertilization took place in all the other doses of e1 (table 2). in e2, a significantly higher fertilization rate was observed in t3 (96.15±0.60) and lowest was in t1 (84.31±2.19) and no fertilization took place in control and t5 (table 3). in e3, the fertilization was observed only in t2, t3 and t4 where in t3 fertilization rate was highest (77.22±1.47) (table 4). in all the experiments, the highest fertilization rate was observed in t3 (96.15±0.60) of e2 (p<0.05). the fertilization rate of e2 is significantly higher (p<0.05) in all the treatments compared to e1 and e3. hatching rate: a contorted movement of the embryos was observed within 14-16 hrs of spawning and hatching within 20-22 hrs of fertilization. calculated hatching rate was significantly higher (p<0.05) in e2 compared to e1 and e3 and the highest hatching rate was observed in t3 (92.49±1.00) with male and female ratio of 2:1. water parameters: different water parameters such as ph, water temperature, free co2, do, total alkalinity in experimental tanks and aquarium under different treatments of m. pancalus were monitored and are presented in table 5. the means values of water parameters were not significantly (p<0.05) different among the different experimental treatments. embryonic development: the development of embryo of m. pancalus were categorized into eight stages viz. zygote, cleavage, blastula, gastrula, segmentation, pharyngula, hatching and larval stages were described as follows (figs. 1, 2): zygote stage: the fertilized eggs were adhesive, dark greenish brown in colour and the diameter measured was 1.3-1.4 mm. cytoplasmic movements were observed after fertilization. cleavage stage: the first cleavage observed 30-40 min after fertilization i.e. formation of blastomere was occurred. after the first cleavage simultaneously the blastomeres divided at an interval of 20-30 min and completed 64 cell divisional stages within 2-3 hrs. blastula stage: with the entire formation of 128 cell blastula period initiate. at this stage, the cell table 4. (e3): captive breeding experiment of macrognathus pancalus (m: f=3:1). size of males size of females hormonal doses (ml/kg body weight) latency period (hrs) fertilization rate (%) hatching rate (%) w (g) tl (cm) w (g) tl (cm) m f 8.87±0.57 12.71±0.64 14.20±0.38 15.93±0.46 control control --- 8.24±0.35 12.06±0.26 12.54±0.63 14.14±0.58 0.2 0.4 --- 7.08±0.28 10.98±0.58 12.35±0.45 13.56±0.49 0.4 0.6 24 74.02±3.86 76.47±1.70 7.12±0.15 12.30±0.77 12.56±0.32 13.81±0.31 0.6 0.8 22 77.22±1.47 83.17±2.48 7.47±0.09 12.76±0.41 12.59±0.02 13.98±0.74 0.8 1.0 22 75.31±2.60 86.17±1.15 6.99±0.25 11.58±0.38 13.58±0.56 14.48±0.65 1.0 1.2 --- w, fish weight in gram; tl, total length in cm, data expressed as mean±se; n=3. table 5. different physicochemical parameters of breeding and rearing tanks. parameters breeding and hatching tank rearing aquarium ph 7.30±0.05 7.43±0.02 water temperature (0c) 25.33±0.88 26±0.47 free co2 (mg/l) 4.15±0.22 3.30±0.23 do (mg/l) 11.13±0.41 9.30±0.49 total alkalinity (mg/l) 55.74±0.91 54.33±0.72 (mean ± se); values of the parameters in each column differ significantly (p<0.05). 77 int. j. aquat. biol. (2020) 8(1): 73-82 rearrangement took place and this period is observed to be 2-5 hrs and ended with the beginning of the gastrulation. at the end of this phase, the embryo has reached 50% epiboly stage. gastrula stage: in this period, substantial cell movements were observed simultaneously with convergence, involution and extension producing three germs layers and embryonic axis. gastrula period ranged 5-10 hrs. segmentation stage: the first morphological cells differentiated and first body movements become visible in segmentation period. sequential formation of the somites was also observed in this stage and this period continued prior to hatching. segmentation period was observed to be 10-18 hrs. pharyngula stage: the embryo was bilaterally organized in this period observing in 18-24 hrs. the notochord was developed gradually and complete set of the somites was formed extending to the end of a post anal tail. hatching stage: after 20-24 of fertilization, the larva emerged from the egg membrane and the newly hatched larva was 2.8±0.37 mm in length. the hatchling was appeared slender and transparent with a voluminous yolk with large oil globules and the yolk sac is measured to be 1.63±0.03 mm. half of the body of the hatchling was covered by the yolk sac. newly hatched larva was not very active and remained in resting condition within the aquatic plants and the wall and bottom of the aquarium. after 10-12 hrs, they became very active and came to the surface of water after a while continuously for gulping air. the heart is not prominent but heartbeat was observed as 108-113 beats/ min. after 24 hrs, the hatchlings were characterized by large pigmented eyes and olfactory pits and the yolk sac was decreasing gradually. two-day old (dph) hatchlings were characterized by prominent heart and was seen pulsating located between the head and yolk sac, ventral to the eyes. 3 dph hatchlings were observed with reduced yolk mass and the size of the yolk sac reduced to 0.83±0.03. the forehead region which attached to the yolk sac, observed free from the yolk mass. four-day old hatchlings were characterized by large and highly pigmented eyes. during this period, the mouth cleft appeared although not with any marked movement. five-day old hatchlings were characterized by figure 1. fertilized eggs with different developmental stages. (a) fertilized eggs, (b) zygote stage, (c) cleavage stage, (d) blastula stage, (e) gastrula stage, and (f) segmentation stage. 78 borah et al./ induced breeding of macrognathus pancalus under captive condition spherical heart appeared pulsating and the yolk sac became reduced to half. six-day old larva was observed with two chambered heart located ventrally anterior to the globular yolk and the streaming larval blood circulation perceptible ventrally. conspicuous melanophore was observed on the body. dorsal and caudal fins developed with continuous fin rays. gill movements and jaws movements were also visible. pectoral fins were developed. 7 dph hatchlings were characterized with completely absorbed yolk and it began wondering in search of food. the fully developed larva was measured to be 6.8±0.23. pigmentation on head and trunk region became remarkable. well-developed upper and lower jaw with movements was noticed. clear and functional fins and fin rays were observed. after one week of hatching, the spawns were released into another tank providing with continuous feed for growth. discussions the present investigation demonstrated the captive feasibility and artificial propagation of barred spiny eel, m. pancalus. it was hypothesized that simulation of natural environmental conditions in confined aquarium may trigger spawning. the present observation partially supported the theory as the fish successfully matured under captive conditions. however, it failed to naturally spawn under artificial stimulant. das and kalita (2003) described spawning success in peacock eel, m. aculeatus under captive environmental conditions, where spawning was triggered using synthetic hormone (ovaprim). contemporary investigation pertaining to the use of ovasis (synthetic spawning hormone) as a spawning inducer in captive breeding of m. pancalus, showed significant variation in breeding performance. the variation in their spawning regime might be attributed due to the different hormonal doses and respective sex ratios. the basic success of induced breeding is governed by the appropriate administration of specific hormone, hormonal doses, condition of the brood fish as well as the environmental conditions (miah et al., 2008; afroz et al., 2014). in m. pancalus, the hormonal doses of 0.6 and 0.8 ml/kg in males and females, respectively were found to be effective for inducing the species with optimum spawning success. das and kalita (2003) and alam et al. (2009) did a comprehensive evaluation on the efficacy of different figure 2. different developmental stages of newly hatched larva (a) 1-day old larva, (b) 2-day old larva, (c) 3-day old larva, (d) 4-day old larva, (e) 5-day old larva, (f) 6-day old larva, and (g) 7-day old larva. 79 int. j. aquat. biol. (2020) 8(1): 73-82 hormonal doses to trigger spawning activity in peacock and spiny eel, respectively. their findings highlighted differential hormonal doses in inducing spawning which are in conformity to the present study. in this regard, atz and pickford (1959) reported that females generally require higher doses as compared to males and further suggested the use of small multiple doses giving better results than the single dose. among all the experimental set up, the highest fertilization and hatching rate was observed with ovasis hormone at the sex ratio of m: f=2:1. the sex ratio m: f=2:1 was found effective in induced breeding experiment compared to other ratio maintained which was strongly supported by islam et al. (2011) and nagarajan (2012). latency period ranged 20-24 hrs in m. pancalus with different doses of ovasis in different experimental set up. contrary to the present findings, das and kalita (2003) reported relatively low latency period whereas rahman et al. (2011) found much higher latency period in some members of the mastacembelidae family. in m. pancalus, the lowest latency period (20 hrs) was observed at the experiment e2 where the hormonal dose was 0.8 and 1.0 ml/kg of male and female, respectively. the highest latency period (24 hrs) was observed at the hormonal doses of 0.2 and 0.4 ml/kg at 2:1 as well as 0.4 and 0.6 ml/kg of 3:1 male and female, respectively. variations observed in latency period might be due to use of different hormonal doses and their mode of action in that particular fishes. peter et al. (1986) also established similar result whereby differential level of dopamine activity in fishes is influenced by the dose concentration of a particular hormone. physico-chemical parameters of water in the experimental tank might also contribute to the increased fertilization rate, successful hatching etc. the mean water temperature (25.33±0.88ºc), ph (7.30±0.05) and do (11.13±0.41 mg/l) during the breeding period in the experimental tank coincided with the successful spawning activity. according to behera et al. (2007), labeo bata administered with ovaprim and ovatide showed high hatching rate induced under optimum environmental conditions. similar inferences have also been opined by alam et al. (2009) on the influence of water parameters for successful fertilization and quality hatching rate. in this experiment, hormonal doses administration apparently affected the fertilization and hatching rate. low dosing of the inducing hormone caused late inducement in species, whereas overdosing caused early milting. the results are in conformity to the findings of routray et al. (2007) and pandey et al. (2002b) regarding efficacy of different synthetic hormone doses in triggering successful fertilization and hatching rate. the fertilized eggs observed were transparent whereas unfertilized ones were opaque and dark in colouration. kimmel et al. (1995) in danio rerio, udit et al. (2014) in puntius sarana, dey et al. (2014) in devario aequipinnatus, and malla and banik, (2015) in d. aequipinnatus reported similar morphological development/changes in oocyte structure after fertilization. the first cleavage i.e. formation of blastomere occurred 30-40 min after fertilization. udit et al. (2014) reported similar findings where cleavage occurred 30 min after fertilization in p. sarana; kimmel et al. (1995) reported 40 min in d. rerio whereas dey et al. (2014) reported 45 min in d. aequipinnatus. following successful fertilization, the incubation period reportedly varied from 20-24 hrs in this particular species. earlier reports on incubation period by kimmel et al. (1995), udit et al. (2014), and dey et al. (2014) revealed 48 hrs of incubation in d. rerio, 15-17 hrs in p. sarana, 36 hrs in d. aequipinnatus, respectively. in the present work, the newly hatched larva measured 2.8±0.37 mm with voluminous yolk sac after 20-24 hrs of fertilization. the yolk sac and mass of the hatchlings showed gradual decreasing trend coupled with organ development till the 7th day. it was observed that the yolk sac was completely dissolved after 1 week following which the larva started self -feeding in the rearing tank. similar studies were reported by alam et al. (2006) and arockiaraj et al. (2003). conclusion maturation and seed production of m. pancalus 80 borah et al./ induced breeding of macrognathus pancalus under captive condition through captive breeding technique can be consummated through the administration of inducing hormone (ovasis) at variegated doses. however, it was observed that different factors viz. rate of stimulation, sex ratio, as well as optimum environmental conditions, acts synergistically for the success of viable seed production. post spawning period is a critical phase, which requires scrupulous monitoring and care as it determine the reproductive success of a particular species. this study gives an elaborative overview of the reproductive dynamics, feeding preference, environmental congeniality of the potential ornamental spiny eel m. pancalus in captive condition. a stepwise optimization of the involved techniques will offer an impetus to the aquaculturist and conservationist to comprehend proper management and further framing conservational strategies for this valued species. acknowledgments first author acknowledges university grants commission (ugc/net-jrf fellowship), new delhi for providing financial assistance during the study tenure. the second and last author also acknowledges department of biotechnology, government of india for providing financial assistance under dbt twinning project (ner). references abujam s.s., biswas s.p. 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(2020) 8(4): 296-299 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology short communication first stranding event of a minke whale calf, balaenoptera acutorostrata lacépède, 1804, in the syrian coast, eastern mediterranean amir ibrahim1, chirine hussein1, nahla ibrahim2, moena badran1, firas alshawy*1, alaa alcheikh ahmad3 1marine biology department, high institute of marine research, tishreen university, lattakia, syria. 2faculty of sciences, tishreen university, lattakia, syria. 3general commission of fisheries resources, coastal area branch, tartous, syria. article history: received 30 june 2020 accepted 23 august 2020 available online 2 5 august 2020 keywords: marine mammals, atlantic ocean, cetaceans, syrian waters. abstract: minke whale, a member of the sub order mysticeti, was sighted stranded in the northern, and southern parts of eastern mediterranean. this paper report stranding of a minke whale in banyas coast of syria, and fills the gap of the species distribution between the north and south of the eastern mediterranean. introduction the abundance of cetaceans has been reduced by human activities, especially entanglement in fishing gears and pollution (inniss et al., 2016). minke whale, balaenoptera acutorostrata (lacépède, 1804), a member of the sub order mysticeti, is regarded as the smallest great whales or rorquals (carpenter and niem, 2001; cooke, 2018). according to the iucn red list, minke whale is a species of least concern (lc) (cook, 2018). it is found in summer as far north as baffin bay in the canadian arctic, denmark, strait, franz josef land, and novaya zemlya; while the wintering grounds are poorly known but extend at least to the caribbean in the west and the straits of gibraltar in the east. very little is known about its distribution in the mediterranean where it is considered merely a visitor, even though it was sighted stranded in the northern (öztürk et al., 2015) and in the southern (kerem et al., 2012) parts of eastern mediterranean; it had not been seen in the syrian marine water before (maio et al., 2016). here we report for the first time in syria stranding of a minke whale in banyas coast. this record fills the gap of the species distribution between the north and south of the eastern mediterranean. *correspondence: firas alshawy e-mail: falshawy@gmail.com materials and methods on 8 march 2020, a stranded dead minke whale calf was found on the coast south of banyas, syria, (35°05´45.1˝n, 35°53´17.8˝e). it was identified according to carpenter and niem (2001), perrin et al. (2009) and jefferson et al. (2015). its measurements were recorded (to the nearest cm; table 1) and 80 photographs of various parts of the body were taken for future records and data sharing with the scientific community. the young whale’s corpse was then buried for later recovery of the skeleton and future display at the biodiversity museum of the high institute of marine research in lattakia. results and discussions the stranded individual was a female calf of the minke whale (fig. 1a, b). its carcass was in a good condition (category 2; mazzariol et al., 2015). it had a streamlined body with short and pointed head (fig. 1c), slightly protruding lower jaw and creamy baleen plates with white bristles. a conspicuous white patch is located on the middle of the upper surface of the flippers (fig. 1d). the fluke was slightly concave with a well-marked median notch (fig. 1e) and the dorsal fin having a sickle shape (fig. 1f). the morphometric 297 int. j. aquat. biol. (2020) 8(4): 296-299 measurements of this individual indicate that it is a calf, as the total body length was only 3.5 m (van waerebeek et al., 1999), while reportedly adults generally reach just over 9 m (öztürk et al., 2011). the death of this calf was very recent as it was still bleeding on the time of sighting. this was considered as an indication that this calf was present in the syrian marine waters and had not been brought by water currents from other nearby areas of the mediterranean. this was later confirmed by a local fisherman who reported that this whale calf was entangled in his trammel net (8 m height, 300 m length) which was setup at ~15 m water depth and ~300 m off the shoreline. balaenoptera acutorostrata had been recorded earlier at the southern part (kerem et al., 2012), and the northern part (öztürk et al., 2015) of the eastern coast of the mediterranean, and this new record fills the gap in the species distribution along this coast. the minke whale exists in all latitudes of both hemispheres and inhabits the polar and sub polar waters. individuals of the north atlantic populations occasionally visit the mediterranean, mostly during winter, to benefit from the warmer waters (van waerebeek et al., 1999, fraija-fernández et al., 2015, öztürk et al., 2015, maio et al., 2016). recording this stranded calf in the syrian waters supports the idea that the eastern mediterranean may be a potential calving and/or nursery ground for the minke whale. even though this species is regarded as least concern (cooke, 2018), national and regional initiatives should be taken (gonzalvo and bearzi, 2008) to protect habitats of this species because it is already facing various threats. acknowledgements the authors thank the high institute of marine research-tishreen university, the higher commission for scientific research (damascus), and figure 1. a female minke whale calf, stranded at banyas coast, syria: (a) general body shape, (b) female genital area, (c) short and pointed head, (d) white patch on the flippers, (e) concave fluke with median notch, and (f) sickle shaped dorsal fin. 298 ibrahim et al./ first stranding event of a minke whale in the syrian waters the general commission of fisheries resources (coastal area branch, tartous) who provided the financial and logistic supports to this work. our sincere thanks go to dr. joan gonzalvo for his valuable comments on this manuscript. references carpenter k.e., niem v.h. (2001). fao species identification guide for fishery purposes. the living marine resources of the western central pacific volume 6 bony fishes part 4 (labridae to latimeriidae), estuarine crocodiles, sea turtles, sea snakes and marine mammals.fao library. 866 p. cooke j.g. (2018). balaenoptera acutorostrata. the iucn red list of threatened species 2018: e.t2474a50348265 the iucn red list of threatened species downloaded on 16 may 2020. fraija-fernández n., crespo-picazo j.l., domènech f., míguez-lozano r., palacios-abella j.f., rodríguezgonzález a., villar-torres m., gozalbes p. (2015). first stranding event of a common minke whale calf, balaenoptera acutorostrata lacépède, 1804, reported in spanish mediterranean waters. mammal study, 2: 95100. gonzalvo j., bearzi g. (2008). action plan for the conservation of cetaceans in syria. regional activity centre for specially protected areas contract 39/2007_rac/spa. 45 p. inniss l., simcock a., ajawin a.y., alcala a.c., bernal p., calumpong h.p., araghi p.e., green s.o., harris p., kamara o.k. (2016). the first global integrated marine assessment.united nations. 1752 p. jefferson t.a., webber m.a., pitman r.l. (2015). marine mammals of the world, 2ed edition: a comprehensive guide to their identification.academic press. 616 p. kerem d., hadar n., goffman o., scheinin a., kent r., boisseau o., schattner u. (2012). update on the cetacean fauna of the mediterranean levantine basin the open marine biology journal, 6: 6-27 maio n., giovannotti m., caputo barucchi v., petraccioli a., pollaro f., guarino f.m., splendiani a., de stasio r., odierna g. (2016). haplotype characterization of a stranded common minke whale calf (balaenoptera acutorostrata lacepede, 1804): is the mediterranean sea a potential calving or nursery ground for the species? hystrix-italian journal of mammalogy, 2: 1-4. mazzariol s., cozzi b., centelleghe c. (2015). handbook for cetaceans’ strandings. massimo valdina the coffee house art & adv. 240 p. öztürk a.a., dede a., tonay a.m., danyer e., aytemiz i. (2015). stranding of a minke whale on the eastern mediterranean coast of turkey, april 2015. journal of the black sea/mediterranean environment, 2: 232-237. öztürk a.a., tonay a.m., dede a. (2011). strandings of the beaked whales, risso’s dolphins, and a minke whale on the turkish coast of the eastern mediterranean sea. journal of black sea/mediterranean environment, 17(3): 269-274. perrin w.f., würsig b., thewissen j. (2009). encyclopedia of marine mammals.academic press. 1355 p. table 1. measurements of the minke whale specimen stranded on the coast south of banyas, syria. measurement (cm) factors 350 length from tip of lower jaw to fluke notch 295 from tip of lower jaw to anus 290 from tip of lower jaw to genital opening 250 from tip of lower jaw to anterior margin of dorsal fin 103 from tip of lower jaw to anterior margin of pectoral fin 35 from tip of lower jaw to posterior corner of mouth gape 65 from tip of lower jaw to eye 35 length of snout 28 length of caudal fin 77 span (maximum width) of caudal fins 15 height of dorsal fin 52 length of pectoral fin along anterior margin 13 width of pectoral fin base 299 int. j. aquat. biol. (2020) 8(4): 296-299 van waerebeek k., andré m., sequeira m., vidal m., daniel r., anne c. (1999). spatial and temporal distribution of the minke whale, balaenoptera acutorostrata (lacépède, 1804), in the southern northeast atlantic and the mediterranean sea, with comments on stock identity. journal of cetacean research and management, 3: 223-237. int. j. aquat. biol. (2014) 2(4): 184-187 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article histopathological effects of cypermethrin on liver of aphanius sophiae (heckel, 1849) using rank-based estimation for linear models maryam nasrolah pourmoghadam, hadi poorbagher*,1soheil eagderi department of fisheries, faculty of natural resources, university of tehran, karaj po box 4111, iran. article history: received 2 may 2014 accepted 22 july 2014 available online 2 5 august 2014 keywords: sophiae toothed carp cypermethrin diet frequency liver salinity abstract: ecotoxicological studies that use histological techniques enjoy descriptive approach in explaining the damages to tissues. in the present study, a rank-based estimation for linear models was used to examine significant difference between levels of cypermethrin on histological changes of the liver in aphanius sophiae. the toxicity of cypermethrin associated with certain environmental factors such as salinity and feeding frequency was studied in the sophiae toothed carp (a. sophiae). specimens were exposed to concentration of 0.02 μg l-1 cypermethrin, two levels of salinity (0 and 14 ppt) and feeding frequencies (two times a day and one time every three days) under laboratory conditions. after the end of 14 days period of the experiment, the liver tissues were removed and histological sections prepared. the results revealed that liver tissues alter significantly with the changes in salinity and diet frequency. the results also showed that sensitivity to cypermethrin increased with decreasing salinity and increasing feeding frequency. the results suggested using a. sophiae as an indicator for cypermethrin assessment in aquatic ecosystems and appropriateness of rank-based estimation for linear models, to evaluate the effect of toxins on histopathological alternations. introduction cypermethrin is one of widely used pesticides of pyrethroid (jin et al., 2011). this toxin enters in natural water bodies mainly via runoff enters to watershed (marino and ronco, 2005). cypermethrin in vertebrates and invertebrates mostly affects the nervous system, making increases the nerve impulses in sense organs (vijverberg and bercken, 1990(. it is also shown that cypermethrin is atp-ase enzyme’s inhibitor, functioning especially in fishes and aquatic insects and regarded as an inherently toxic in aquatic organisms (siegfried, 1993). in toxicological studies the control groups are usually in optimum conditions (holmstrup et al., 2009), whereas the organisms in their natural environments are rarely experienced under optimal conditions. interactions between a natural and toxic stressor can sometimes be greater than the impact of a stressor * corresponding author: hadi poorbagher e-mail address: poorbagher@ut.ac.ir alone (holmstrup et al., 2009). hence, this study was conducted to study the interactions between natural stressors including salinity and feeding frequency with cypermethrin insecticide in aphanius sophiae with emphasize on liver histopathological changes using rank-based estimation for linear models. material and methods a total of 100 specimens of a. sophiae were captured from the eshtehard river and transferred to the laboratory. after adoption to laboratory conditions, 80 healthy specimens were used for experiment. two salinity levels (0 and 14 ppt), and two levels of feeding using biomar food (two times a day and one time every three days) were considered as main treatments. cypermethrin was prepared in 0.02 μg/l concentration and then introduced to four aquariums with the capacity of 10 liters, while the 185 nasrolah pourmoghadam et al./ effects of cypermethrin on liver of aphanius sophiae four others were without cypermethrin exposure. after the end of 14 days period of experiment, fishes were anesthetized in clove solution and then their liver were removed and fixed into buin solution. the histological sections were prepared based on banaee et al. (2013) in 5 μm thickness. then the prepared sections were stained with hematoxylin-eosin and investigated using light microscope (banaee et al., 2013). a rank-based estimation for linear models (kloke and mckean, 2012) in the rfit package in r software 3.0.1 (r core team, 2013) was used to examine the effects of cypermethrin on histological changes of the liver. in each interaction the difference between the two levels of each factors in a combination of other factors levels were examined (underwood, 1997). finally, in order to show levels making significant differences, mann-whitney analysis was used and α error was adjusted using bonferroni adjustment (quinn and keough, 2002). results liver in the absence of toxin, feeding twice a day and 14 ppt salinity had a normal mode, as hepatocytes and sinusoids did not show any alternations (fig. 1i). after the end of 14 days, the normal structure of liver tissue in the absence of toxin, feeding twice a day and zero salinity showed minor changes in some parts, where the most important alternations included atrophy (fig. 1-iv), pyknosis (fig. 1-iv) and fat degeneration (fig. 1-iv). in absence of poison, feeding one time every three days and 14 ppt salinity, the overlapped hepatocytes (fig. 1-ii), nucleous irregularity (fig. 1-ii) and cloudy swelling (fig. 1ii) were observed. the atrophy (fig. 1-iv), fat degeneration (fig. 1-iv) and pyknosis (fig. 1-iv) were the most observed changes in the liver with the conditions included lack of toxin, feeding one time every three days and zero salinity. most observed alternations in the presence of toxin, feeding twice a day and 14 ppt salinity were tissue level damage histopathological changes 1 normal liver 2 cloudy swelling, nucleous irregularity and overlapped hepatocytes 3 pyknosis , fat degeneration and atrophy, 4 accumulation of kupffer cell and karyolysis 5 necrosis and bile stagnation table 1. ranking histopathological changes were observed in the liver of different treatments. figure 1. histological sections showing alternations in liver tissue of aphanius sophiae after exposure to different treatment for 14 days. i. a: hepatocyte, b: sinusoid; ii. a: cloudy swelling, b: overlapped hepatocytes, c: nucleous irregularity; iii. a: karyolysis, b: accumulation of kupffer cell; iv. a: atrophy, b: fat degeneration, c: pyknosis; v. a: necrosis and vi. a: bile stagnation. 186 int. j. aquat. biol. (2014) 2(4): 184-187 karyolysis (fig. 1-iii) and accumulation of kupffer cell (fig. 1-iii) in the liver. while the presence of toxin, feeding twice a day and zero salinity treatment showed greater alternations including necrosis (fig. 1-v) and bile stagnation (fig. 1-vi). changes in the toxic condition, feeding one time every three days after the end of 14 days included atrophy (fig. 1-iv), karyolysis (fig. 1-iii) and accumulation of kupffer cell (fig. 1-iii). most observed changes in presence of toxin, feeding one time every three days and zero salinity involved necrosis (fig. 1-v), karyolysis (fig. 1-iii) and accumulation of kupffer cell (fig. 1iii). in this study, changes in liver tissues were ranked in five categories that each of them was involved specific injuries. the first status indicates little changes than normal condition and with increasing intensity of tissue damages, the higher ranking was accounted (table 1). the results showed that poison (df=1, f=150.681, p<0.001), feeding frequency (df=1, f=9.719, p=0.003) and salinity (df=1, f=37.768, p<0.001) factors had a significant effect on extension of tissues damage. among the two factors including toxin concentration and diet regime, the significant interactions were observed (df=1, f=11.620, p=0.001). therefore, due to these significant interactions four analysis were carried out. the results revealed that between the two levels of poison (presence and absence of the toxin) in one time feeding every 3 days condition as well as twice feeding in a day condition, there were statistically significant differences. discussion findings indicate that increase of salinity levels significantly affects the toxicity of cypermethrin, as the fishes in 14 ppt of salinity less suffered. this results are in agreements with findings about the effects of environmental factors such as hardness, temperature, salinity and ph on the toxicity of cypermethrin in poecilia reticulate (gautam and gupa, 2007). however, evelyn et al. (2002) reported that the toxicity of organophosphate pesticides increases when salinity levels rise. furthermore, wang et al. (2001) showed that by increasing salinity, the toxicity of aldicarb pesticide clearly increases in oncorhynchus mykiss. the results from present study indicate that the toxicity of cypermethrin in a. sophiae decreases with increasing the salinity levels. in presence of toxin, feeding twice a day was caused higher cypermethrin toxicity. it seems increasing of feeding frequency and food accumulation in the environment, the dissolved oxygen decreases in ecosystem. therefore, the reduction of dissolved oxygen, the toxicity of toxin increases (koskela, 2002). hence, the eutrophic aquatic ecosystems can increase the toxicity of the cypermethrin for a. sophiae. finally, the results revealed that presence of cypermethrin, decreasing salinity and increasing feeding frequency can impose dangerous and risky condition on a. sophiae. hence, due to no even functional responses in different environmental conditions, sophiae toothed carp (a. sophiae) could not be considered as a suitable candidate to be an indicator in monitoring scheme. nevertheless, due to observed responses of a. sophiae to lower levels of cypermethrin pollutant, it is suggested that a. sophiae could be regarded as a suitable biological indicator. therefore, this species can be a promising candidate for assessment of cypermethrin effects in aquatic ecosystems. in conclusion, the results revealed the appropriateness of applied method i.e. rank-based estimation for linear models, to evaluation the effect of toxins on histopathological alternations in a. sophiae in difference environmental condition and therefore this method is proposed for histopathological studies. refrences banaee m., sureda a., mirvagefei a.r., ahmadi k. (2013). biochemical and histological changes in the liver tissue of rainbow trout (oncorhynchus mykiss) exposed to sub-lethal concentrations of diazinon. fish physiology and biochemistry, 39(3): 489-501 evelyn h., heugens w., hendricks a.j., dekkor t., straalen n.m., admiraal w. (2002). a review of the effects of multiple stressors on aquatic organisms and 187 nasrolah pourmoghadam et al./ effects of cypermethrin on liver of aphanius sophiae analysis of uncertainty factors for use in risk assessment. critical reviews in toxicology, 31: 247284. gautam p.p., gupta a.k. (2007). toxicity of cypermethrin to the juveniles of freshwater fish poecilia reticulata (peters) in relation to selected environmental variables. natural product radiance, 7(4): 314-319. holmstrup m., bindesbol a.m., oostingh g.j., duschi a., scheil v., kohler h.r., loureiro s., soares a.m.v.m., ferreira a.l.g., kienle c., gerhardt a., laskowski r., kramarz p.e., bayley m., svendsen c., spurgeon d.j. 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(2002). experimental design and data analysis for biologists. cambridge university press, cambridge 537 p. r core team. (2013). r: a language and environment for statistical computing. r foundation for statistical computing, vienna, austria. url http://www.rproject.org. siegfried b.d. (1993). comparative toxicity of pyrethroid insecticides to terrestrial and aquatic insects. environmental toxicology and chemistry, 12: 16831689. underwood a.j. (1997). experiments in ecology: their logical design and interpretation using analysis of variance. cambridge university press, cambridge, 504 p. vijverberg h.p.m., bercken j. (1990). neurotoxicological effects and the mode of action of pyrethroid insecticides. critical reviews in toxicology, 21(2): 105-126. wang j., girsle s., schlenk d. (2001). effect of a salinity on aldicarb toxicity in juvenile rainbow trout (oncorhynchus mykiss) and striped bass (morone saxatilisx chrysops). science, 64: 200-207. int. j. aquat. biol. (2020) 8(1): 18-34 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article zooplankton diversity of three floodplain lakes of the dibru-saikhowa biosphere reserve, upper assam, northeast india bhushan kumar sharma*,1nogen noroh department of zoology, north-eastern hill university, shillong -793022, meghalaya, india. s article history: received 23 october 2019 accepted 4 january 2019 available online 2 5 february 2020 keywords: brahmaputra floodplains diversity temporal variations zooplankton paradox abstract: limnological survey (october 2013–september 2015) of maghuri, khamti guali and no.11 floodplain lakes (beels) of the dibru-saikhowa biosphere reserve (dsbr), upper assam, northeast india (nei) revealed 210 (184±4) species of zooplankton, belonging 78 genera and 32 families, with several species of global and regional distribution interest. the biodiverse zooplankton assemblage and interesting taxa are hypothesized to habitat diversity and environmental heterogeneity of these wetlands located in the assam-gateway’an important biogeographic corridor of india. the monthly richness and community similarities depicted heterogeneity of zooplankton composition of the individual beels. low abundance is attributed to soft–moderately hard waters with low ionic concentrations. zooplankton richness and abundance followed no definite pattern of monthly or annual variations; rotifera > cladocera influenced overall diversity in the three beels. high species diversity affirmed habitat heterogeneity, while high evenness and low dominance without quantitative importance of any species affirmed ‘generalist nature’ of zooplankton of the different beels. our results indicated limited influence of individual abiotic factors but cca registered moderately high cumulative importance of seventeen abiotic factors on zooplankton assemblages of dsbr beels. introduction zooplankton, an integral component of freshwater metazoans, has been studied from different parts of india since more than one century. the indian literature depicts proliferation of ‘routine’ reports from varied ecosystems loaded with incomplete species lists and even unidentified species and thus provides limited information for biodiversity and ecology considerations (sharma and sharma, 2008). nevertheless, some detailed works on zooplankton diversity from this country are limited to the floodplain lakes (beels) of assam (sharma, 2011a; sharma and sharma, 2011a, 2012; sharma and hatimuria, 2017) and pats of manipur (sharma, 2011b) states of nei. in addition, sharma and sharma (2008, 2017a, b, 2019) emphasized these floodplains to be one of the globally interesting habitats for zooplankton taxocoenosis. realizing paucity of meaningful information on *correspondence: bhushan kumar sharma doi: https://doi.org/10.22034/ijab.v8i1.730 e-mail: profbksharma@gmail.com freshwater zooplankton diversity of india in general and nei in particular, we undertook limnological survey of three beels of the upper brahmaputra river basin, and located in the assam-gateway and the indoburma biodiversity hot-spot. our observations deal with monthly variations in richness, species composition, abundance, species diversity, equitability and dominance, and analysis of individual and cumulative influence of abiotic factors on zooplankton and the constituent groups the sampled beels. the salient features of faunal diversity, abundance and ecology noted vide this study are highlighted and discussed vis-a-vis importance for zooplankton diversity of india as well as of the tropical and subtropical floodplain lakes. materials and methods the observations were undertaken in three floodplain lakes namely maghuri (altitude: 96.1 m asl, area: 19 int. j. aquat. biol. (2020) 8(1): 18-34 119 ha), khamti guali (altitude: 97.4 m asl, area: 11 ha) and no. 11 (altitude: 94.7 m asl, area: 12 ha) beels of the dibru-saikhowa biosphere reserve (dsbr), tinsukia district, upper assam, nei (fig. 1). the sampled beels are invariably refereed as ‘dsbr beels’ in this text. water samples as well as qualitative and quantitative plankton and semi-plankton samples were collected monthly from the three beels during october 2013-september 2015. water samples were examined for 17 abiotic parameters. water temperature was recorded using a centigrade thermometer; ph and specific conductivity were recorded with the field probes; and the rainfall data was obtained from the citrus research station, tinsukia, assam. dissolved oxygen was estimated by the modified winkler’s method, and other abiotic factors namely free carbondioxide, total alkalinity, total hardness, calcium, magnesium, chloride, dissolved organic matter, total dissolved solids, phosphate, nitrate, sulphate and silicate were analyzed following apha (1992). the qualitative plankton and semi-plankton samples collected each, by towing nylobolt plankton net (no. #50 µm), were preserved in 5% formalin. these samples were screened with a wild stereoscopic binocular microscope for isolation of various taxa which were mounted in polyvinyl alcohol–lactophenol mixture. various zooplankton were observed with leica (dm 1000) stereoscopic phase contrast microscope fitted with an image analyzer and species were identified following koste (1978), michael and sharma (1988), korovchinsky (1992), ranga reddy (1994, 2001), segers (1995), sharma (1998), sharma and sharma (1999a, b, 2000, 2008, 2013), orlova-bienkowskaja (2001), jersabek and leitner (2013), and sharma et al., (2017). the community similarities were calculated vide sørensen’s index and the hierarchical cluster analysis figure 1. map of india showing assam state indicating location of tinsukia district and satellite map showing the sampled beels (after sharma et al., 2017). 20 sharma and noroh / zooplankton diversity of floodplain lakes of upper assam was consequently done using spss (version 20). monthly quantitative plankton samples from dsbr beels were obtained by filtering 25 l of water each through nylobolt plankton net and were preserved in 5% formalin. quantitative enumeration of zooplankton and their constituent groups was done using sedgewick-rafter counting cell. the abundance of various taxa was expressed as n/l as well as ranges and means ±s.d. species diversity (shannon’s index), dominance (berger-parker’s index) and evenness (e1 index) were calculated following ludwig and reynolds (1988), and magurran (1988). two-way analysis of variance (anova) was applied to ascertain significance of variations of the biotic assemblages. pearson correlation coefficients for maghuri, khamti guali and no.11 beels (r1, r2 and r3, respectively) were calculated between abiotic and biotic parameters; p-values were calculated vide http://vassarstats.net/tabs.html and their significance were ascertained after applying bonferroni corrections. the canonical correspondence analysis (xlstat 2015) was done to observe cumulative influence of seventeen abiotic parameters namely water temperature, rainfall, ph, specific conductivity, dissolved oxygen, free carbon-dioxide, total alkalinity, total hardness, calcium, magnesium, chloride, dissolved organic matter, total dissolved solids, phosphate, nitrate, sulphate and silicate on zooplankton assemblages of the three beels. results the temporal variations (mean±sd) of the different abiotic parameters of maghuri, khamti guali and no.11 beels, during october 2013-september 2015, are presented in table 1. plankton and semi-plankton samples examined from dsbr beels revealed total 210 zooplankton species, spread over 78 genera and 32 families (table 2), with individual richness of 190 > 182 > 180 species in no.11 > khamti guali > maghuri beels, respectively (table 3). the monthly zooplankton richness (table 4) varied between 34-90, 39-99 and 30-105 species during the study period (figs. 2-3), and recorded 39.1-71.7, 37.8–73.0 and 37.0-76.2% community similarities table 1. abiotic parameters (range, mean±sd) of dsbr beels (after sharma et al., 2017). maghuri beel khamti guali beel no.11 beel parameters↓ range mean±sd range mean±sd range mean±sd water temperature (oc) 15.0 -30.8 24.7±4.6 14.0-32.6 25.4±4.8 15.5-30.7 25.4±4.6 rainfall (mm) 0.0-615.0 188.4 -±193.6 0.0-615.0 14.0±32.6 0.0-615.0 188.4±193.6 ph 6.51-8.26 7.38±0.50 6.84-8.71 7.51±0.54 6.39-8.72 7.42±0.54 specific conductivity (µs/cm) 69.0-140.0 100.0±19.4 65.0-150.0 103.1±24.6 46.0-139.0 84.7±22.3 dissolved oxygen (mg/l) 4.0-8.0 6.0 ±1.4 4.0-8.0 5.9±1.3 4.0-8.0 5.6±1.2 free carbon-dioxide (mg/l) 10.0-28.0 15.8±5.0 8.0-24.0 15.3±4.5 10.0-24.0 16.1±3.8 total alkalinity m(g/l) 40.0-80.0 58.9 ±12.9 40.0-84.0 54.6±12.6 38.0-80.0 52.4±10.0 total hardness (mg/l) 54.0-96.0 72.6 ±10.5 50.0-100.0 71.3±12.5 50.0-100.0 69.2±10.7 calcium (mg/l) 14.7-25.2 20.1±2.8 14.7-27.3 20.0±3.5 12.6-25.2 18.8± 3.7 magnesium (mg/l) 7.00-17.71 12.75 ±2.60 7.99-19.20 12.47±3.00 8.07-18.69 12.24±2.44 chloride (mg/l) 7.99 -20.97 13.23 ±3.43 9.90-24.98 14.46±4.19 10.98-24.98 16.52±3.67 dissolved organic matter (mg/l) 0.041 -0.131 0.101±0.027 0.048-0.150 0.100± 0.028 0.045-0.131 0.097±0.022 total dissolved solids (mg/l) 0.080 -0.320 0.160±0.075 0.040-0.280 0.157±0.070 0.040-0.320 0.155±0.077 phosphate (mg/l) 0.134-0.322 0.189±0.054 0.135-0.351 0.195 ±0.060 0.136-0.371 0.194±0.062 nitrate (mg/l) 0.352-1.881 0.733±0.352 0.440-1.702 0.728±0.293 0.369-1.550 0.720±0.293 sulphate (mg/l) 6.143-25.047 11.020 ±5.584 6.720-23.986 12.357±5.733 5.767-22.907 11.482±5.213 silicate (mg/l) 0.657-1.089 0.877 ±0.188 0.654-1.212 0.871±0.195 0.661-1.167 0.900±0.192 table 2. biodiversity of zooplankton of dsbr beels. groups↓ taxa → species genera families rotifera 141 31 17 cladocera 49 35 7 rhizopoda 11 5 5 copepoda 7 5 2 ostracoda 2 2 1 zooplankton species 210 78 32 21 int. j. aquat. biol. (2020) 8(1): 18-34 in maghuri, khamti guali and no.11, respectively (table 4). of the important components, rotifera richness ranged between 16-59, 15-70 and 14-74 species, while cladocera richness varied between 1026, 10-31 and 12-31 species maghuri beel, in khamti guali beel and no.11 beel, respectively (table 4). the hierarchical cluster of zooplankton assemblages of the three beels are indicated in figures 4-6. in maghuri beel (fig. 4), peak zooplankton similarity was observed between april, 2014 and june, 2014, during the first year and between june, 2015 and july, 2015 in the following year. in all 35, 26, 3 and 2 instances indicated 51-60, 41-50, 61-70, and 31-40% similarities, while 36, 15, 14 and 1 instance indicated between 51-60, 41-50, 61-70, and 71-80% similarities during two years, respectively. in khamti guali beel (fig. 5) peak similarity was observed between october, 2013 and november, 2013 during first year; 36, 16, 11 and 3 instances indicated 51-60, 61-70, 41-50, and 31-40% similarities, respectively. during the second year, peak similarity was observed between october, 2014 and november, 2014 and 27, 25, 10, 2 and 2 instances indicated 5160, 41-50, 61-70, 71-80 and 31-40% similarities, respectively. zooplankton similarities of no.11 beel (fig. 6) recorded peak between april, 2014 and may, table 3. zooplankton species richness of the three dsbr beels. groups↓ beels→ maghuri khamti guali no. 11 mean ± sd rotifera 112 119 122 118±4 cladocera 49 45 48 47±2 rhizopoda 10 9 11 10±1 copepoda 7 7 7 7±0 ostracoda 2 2 2 2±0 zooplankton 180 182 190 184±4 table 4. zooplankton richness variations of dsbr beels. groups↓ beels→ maghuri beel khamti guali beel no.11 beel richness (total) zooplankton (210 species) 180 species 182 species 190 species community similarity 39.1-71.7 % 37.8-73.0 % 37.0-76.2 % richness (monthly) zooplankton 34-90 56±13 39-99 62± 15 30-105 65±20 rotifera 16-59 30±9 15-70 34±15 14-76 37±17 cladocera 10-26 18±5 10-31 19±5 12-31 20±5 figure 2. monthly variations of richness of zooplankton of dsbr beels (2013-2014). 22 sharma and noroh / zooplankton diversity of floodplain lakes of upper assam 2014 during the first year and 34, 23, 7 and 2 instances indicated similarities between 51-60, 41-50, 61-70, and 31-40%, respectively. peak similarity was noted between july, 2015 and august, 2015 during second year and 27, 25, 10, 2 and 2 instances indicated similarities between 51-60, 41-50, 61-70, 71-80 and 31-40%, respectively. zooplankton density ranged between 139-286, 150-261 and 99-268 n/l in maghuri, khamti guali and no. 11 beels, respectively (table 5) during the study period (figs. 7-8); it comprised between 59.0±9.9 to 61.9±10.0% of net plankton abundance. rotifera recorded abundance between 56-152, 56-155 and 37152 n/l; cladocera abundance ranged between 24-101, figure 3. monthly variations of richness of zooplankton of dsbr beels (2014-2015). figure 4. hierarchical cluster analysis of zooplankton of maghuri beel. 23 int. j. aquat. biol. (2020) 8(1): 18-34 41-121 and 37-113 n/l; rhizopoda recorded density variations of 0-34, 0-34 and 12-37 n/l; and copepoda density ranged between 10-29, 4-24 and 0-21 n/l; and ostracoda density ranged between 0-6, 1-6 and 0-4 n/l in maghuri beel, khamti guali beel and no. 11 beel, respectively (table 4). lecanidae and chydoridae recorded quantitative importance; lepadellidae, brachionidae, daphniidae and macrothricidae figure 5. hierarchical cluster analysis of zooplankton of khamti guali beel. figure 6. hierarchical cluster analysis of zooplankton of no. 11 beel. 24 sharma and noroh / zooplankton diversity of floodplain lakes of upper assam indicated limited role, while no individual zooplankton species indicated importance in the three beels (table 4). the species diversity, dominance and evenness varied (table 4) between 3.372-4.206, 3.401-4.324 and 3.257-4.454; 0.041-0.074, 0.0360.100 and 0.027-0.120; 0.935-0.969, 0.921-0.963 and 0.930-0.972 in maghuri, khamti guali and no. 11 beels, respectively (table 5). zooplankton richness is positively correlated with total dissolved solids (r3=0.575, p=0.0033) and rotifera richness showed positive correlation with rainfall (r3=0.692, p=0.0002) only in no.11. zooplankton abundance positively correlated with rainfall (r2=0.746, p=0.0001) and water temperature (r2 = 0.603, p=0.0018); rotifera abundance showed positive correlation with rainfall (r2=0.596, p= table 5. quantitative variations of zooplankton of dsbr beels (ranges, mean ± sd) (october 2013september 2015). maghuri beel khamti guali beel no.11 beel abundance net plankton n/l 214-950 359±150 250-545 336±56 230-438 337±52 zooplankton n/l 139-286 198±36 150-261 205±31 99-268 199±46 % composition 14.6-83.0 60.4±15.8 41.3-78.5 61.9±10.0 33.3-73.3 59.0±9.9 species diversity 3.372-4.206 3.827±0.197 3.401-4.324 3.880±0.234 3.257-4.454 3.921±0.289 dominance 0.041-0.074 0.058±0.010 0.036-0.100 0.057±0.017 0.027-0.120 0.059±0.019 evenness 0.935-0.969 0.955±0.008 0.921-0.963 0.947±0.010 0.930-0.972 0.953±0.012 different groups rotifera (n/l) 56-152 99±24 56-155 103 ±30 37-152 90±32 % composition 35.0-61.7 50.0±6.5 28.9-62.4 49.6±9.5 31.7-58.0 44.6±8.6 cladocera 24-101 58±18 41-121 66±17 37-113 72±21 % composition 14.6-37.9 29.0±5.7 19.0-52.2 32.9±9.1 25.2-51.6 36.5±8.2 rhizopoda 0-34 20±8 0-34 20±7 12-37 23±7 % composition 0.0-19.9 10.2±4.7 0.0-17.8 9.6±3.8 5.6-18.1 11.6±3.3 copepoda 10-29 18±5 4-24 14±5 0-21 13±5 % composition 3.8-15.2 9.2±3.3 2.0-10.9 7.0±2.2 0.0-14.2 6.4±2.9 ostracoda 0-6 3±2 1-6 2±1 0-4 2±1 % composition 0.0-3.5 1.6±0.9 0.4-2.4 1.0±0.7 0.0-1.8 0.9±0.6 important families (n/l) lecanidae 32-81 48±12 24-84 48±16 14-77 42±17 lepadellidae 3-23 10±5 0-24 11±7 0-22 9±6 brachionidae 0-26 10±6 3-20 12±4 2-30 11±6 chydoridae 15-55 39±13 17-86 41±13 14-70 42±15 daphniidae 0-17 7±4 5-23 13±5 2-24 11±5 macrothricidae 1-23 9±5 2-17 8±4 4-25 12±5 figure 7. monthly variations in zooplankton abundance of dsbr beels (2013-2014). 25 int. j. aquat. biol. (2020) 8(1): 18-34 0.0023); lecanidae showed positive correlation with rainfall (r2=0.597, p=0.0021); chydoridae showed positive correlation with rainfall (r2=0.575, p= 0.0023) in khamti guali beel. zooplankton and its constituent groups showed no significant relationship with any abiotic factor in maghuri beel. the canonical correspondence analysis (cca) with 17 abiotic factors (figs. 9-11) registered cumulative influence figure 8. monthly variations in zooplankton abundance of dsbr beels (2014-2015). figure 9. cca coordination biplot of zooplankton and abiotic factors of maghuri beel. abbreviations: abiotic: ca (calcium), cl (chloride), dom (dissolved organic matter), do (dissolved oxygen), fco2 (free carbon dioxide), rain (rainfall), no3 (nitrate), po4 (phosphate), sio2 (silicate), sc (specific conductivity), so4 (sulphate), ta (total alkalinity), tds (total dissolved solids), th (total hardness), ph (hydrogen-ion concentration), wt (water temperature). biotic: bra (brachionidae), chy (chydoridae), cld (cladocera), clr (cladocera richness), cop (copepoda), dap (daphniidae), lec (lecanidae), lep (lepadellidae), lb (lecane bulla), ll (lecane leontina), mac (macrothricidae), m (macrothrix triserialis), np (net plankton), ost (ostracoda), rot (rotifera), rr (rotifera richness), rhz (rhizopoda), tri (trichocercidae), zoo (zooplankton), zr (zooplankton richness). 26 sharma and noroh / zooplankton diversity of floodplain lakes of upper assam figure 10. cca coordination biplot of zooplankton and abiotic factors of khamti guali beel. abbreviations: abiotic: ca (calcium), cl (chloride), dom (dissolved organic matter), do (dissolved oxygen), fco2 (free carbon dioxide), rain (rainfall), no3 (nitrate), po4 (phosphate), sio2 (silicate), sc (specific conductivity), so4 (sulphate), ta (total alkalinity), tds (total dissolved solids), th (total hardness), ph (hydrogen-ion concentration), wt (water temperature). biotic: bra (brachionidae), chy (chydoridae), cld (cladocera), clr (cladocera richness), cop (copepoda), dap (daphniidae), lec (lecanidae), lep (lepadellidae), lb (lecane bulla), ll (lecane leontina), mac (macrothricidae), m (macrothrix triserialis), np (net plankton), ost (ostracoda), rot (rotifera), rr (rotifera richness), rhz (rhizopoda), pp (plationus patulus), tri (trichocercidae), zoo (zooplankton), zr (zooplankton richness). figure 11. cca coordination biplot of zooplankton and abiotic factors of no. 11 beel. abbreviations: abiotic: ca (calcium), cl (chloride), dom (dissolved organic matter), do (dissolved oxygen), fco2 (free carbon dioxide), rain (rainfall), no3 (nitrate), po4 (phosphate), sio2 (silicate), sc (specific conductivity), so4 (sulphate), ta (total alkalinity), tds (total dissolved solids), th (total hardness), ph (hydrogen-ion concentration), wt (water temperature). biotic: bra (brachionidae), chy (chydoridae), cld (cladocera), clr (cladocera richness), cop (copepoda), cs (chydorus sphaericus), dap (daphniidae), lec (lecanidae), lep (lepadellidae), ll (lecane leontina), mac (macrothricidae), m (macrothrix triserialis), np (net plankton), ost (ostracoda), rot (rotifera), rr (rotifera richness), rhz (rhizopoda), pp (plationus patulus), tri (trichocercidae), zoo (zooplankton), zr (zooplankton richness). 27 int. j. aquat. biol. (2020) 8(1): 18-34 of 73.65, 61.42 and 63.56% on zooplankton assemblages, along first two axes, in maghuri, khamti guali and no.11 beels, respectively. discussions low ionic concentrations warranted their inclusion under class i category of trophic classification vide talling and talling (1965), while water temperature concurred with geographical location of the three beels. besides, other notable attributes included soft to marginally hard, circumneutral-alkaline, and moderately oxygenated calcium poor waters; total alkalinity attributed to bicarbonate ions; and low chloride, lower nutrients and occurrence of free carbon dioxide throughout the study period (sharma et al., 2017). total zooplankton richness (210 species) of dsbr beels compared with the reports of 209 species from 15 beels of assam (sharma and sharma, 2008), and 206 species from 15 pats of manipur (sharma, 2005a); it is more biodiverse than 141 species known from three beels (sharma and hatimuria, 2017) and is marginally species-rich than 197 species observed from 12 beels (bks, unpublished) of the majuli river island of upper assam. dsbr beels thus depicted one of the biodiverse zooplankton assemblages known from india which is hypothesized to habitat diversity and environmental heterogeneity of these low ionic concentration wetlands located in the assam-gateway, an important biogeographic corridor of india. this generalization supported hypothesis of sharma and sharma (2008, 2014, 2019) on biodiversity importance of the floodplains of nei and that of the tropics and subtropics (segers et al., 1993). individual dsbr beels recorded higher mean richness (184±4 species) with no. 11 > khamti guali > maghuri beels and registered 88.4-90.9% community similarities (vide sorenson index). the latter affirmed high homogeneity of zooplankton composition due to common occurrence of 157 species (~75%) amongst the three beels. our results recorded high mean richness individually with lower inter-beel variations as compared with the reports of 109±20 species known from 15 pats of manipur (sharma, 2005a), 102-118 species from > 30 beels of the brahmaputra river basin of assam (sharma and sharma, 2008), 118±8 species from three beels of the majuli river island (sharma and hatimuria, 2017), and 178±13 species from four beels of lower assam (bks, unpublished). further, zooplankton of individual dsbr beels are distinctly speciose than the reports of 110 species from various kashmir wetlands (wanganeo and wanganeo, 2006); 110 and 103 species from waithou and utra pats of manipur (sharma, 2011b); 76 species (khan, 2002) and 89 species (khan, 2003) from the floodplain lakes of southeastern west bengal; 76 species from two floodplain wetlands (datta, 2011) of north bengal; and 51 species from two floodplain lakes (khan, 1987) of kashmir. rotifera > cladocera mainly contributed to zooplankton richness of the three beels, while rhizopoda > copepoda > ostracoda indicated limited role. rotifera revealed total 141 species (sharma et al., 2017) comprising ~34.0 and ~50.0% of species of the phylum known from india and nei, respectively with one species new to the oriental region, two new records to the indian rotifera, three new to assam, 21 species (~15.0%) of global biogeographic interest and ~10% species restricted to nei (sharma et al., 2017). rotifera richness of dsbr beels corresponded with 140 species from the floodplains of the kashmir himalayas (sharma and sharma, 2018). the species predominance of this phylum concurred with the reports of sharma (2005a, b, 2009a, b, 2011a, b), sharma and sharma (2001, 2005, 2012, 2014, 2019) and sharma et al. (2015, 2016, 2018) from the floodplains of nei and west bengal (khan, 2002, 2003). it also concurred the reports from rao tapajos (koste, 1974), lago camaleao (koste and robertson, 1983), lake guarana (bonecker et al., 1994) of brazil, lake iyi-efi and lake oguta from niger delta (segers et al., 1993), thale-noi lake, thailand (segers and pholpunthin, 1997), laguana bufeos, bolivia (segers et al., 1998), and the rio pilcomayo national park, argentina (jose de paggi, 2001). cladocera richness (49, 47±2 species) deserved importance in light of a conservative estimate of 28 sharma and noroh / zooplankton diversity of floodplain lakes of upper assam occurrence of up to 60-65 species of the taxon from tropic and subtropics waters of the indian subcontinent (sharma and michael, 1987; michael and sharma, 1988; sharma and sharma, 2017b). with ~90.0% of the species observed from all dsbr beels and higher community similarities (94.6-98.9% vide sørensen’s index), our results affirmed high homogeneity in species composition of these microcrustaceans amongst the beels concurrent with the reports from certain floodplains of assam (sharma and sharma, 2008, 2013) and manipur (sharma and sharma, 2009, 2010). the mean richness is marginally higher than the report of 44±3 species from four beels of lower assam (bks, unpublished) while it is distinctly higher than 26±6 species known from 12 majuli beels (sharma et al., 2015). this group is represented by ~20.0% biogeographically interesting elements, including two australasian, three indochinese, three oriental endemics, the palearctic kurzia latissima; and the paleotropical dunhevedia serrata. our results depicted the diverse nature of chydoridae (~63%) and common occurrence of macrothricidae and thus depicted the littoral periphytic character to the cladoceran assemblages (sharma and sharma, 2017b). this study is notable for paucity of the bosminidae and moinidae and lack of daphnia concurrent with the report from the majuli floodplains, upper assam (sharma et al., 2015). dsbr cladocera recorded ten cosmotropical species and several tropical and sub-tropical taxa and thus depicted a broadly ‘tropical character’ concurrent with the reports on tropical assemblages (sharma and michael, 1987; sharma and sharma, 2008). rhizopoda indicated (11, 10±1 species) importance of species of lobosea than that of filosea concurrent with the remark of sharma and sharma (2011b). the rhizopod richness is lower than the reports of 21 species from deepor beel (sharma and sharma, 2011b) but broadly compared with the reports of 12 species from tripura (das et al., 2000), 10 species from melghat wildlife sanctuary, maharashtra (bindu, 2010); and 13 species from pench national park, maharashtra (bindu and das, 2010). amongst copepoda species, mesocyclops aspericornis, m. isabellae and tropodiaptomus signatus are new records from nei, while m. varicans and thermocyclops crassus are new records from assam. in general, zooplankton of dsbr beels depicted the littoral-periphytic assemblages and a broadly ‘tropical character’ following the remarks on the composition of several tropical zooplankton assemblages vide fernando (1980), dussart et al. (1984), and sharma and sharma (2008, 2013). zooplankton indicated wider monthly richness variations and registered 39.1-71.7, 37.8-73.0 and 37.0-76.2% community similarities in maghuri, khamti guali and no.11 beels, respectively with majority of instances in the similarity matrices indicating lower ranges of 41-60%. the stated features along with the differences in the hierarchical cluster groupings affirmed heterogeneity in monthly composition of zooplankton of the three beels which is hypothesized to habitat heterogeneity. our results highlight interesting zooplankton consortia per samples of 99 (april, 2015) and 105 (may, 2014) species in khamti guali and no. 11 beels, respectively. we categorize these as ‘zooplankton paradox’ following analogy to the classical ‘paradox of the plankton’ reported by hutchinson (1961). such instances are hypothesized to the intriguing possibility of the co-existence of a number of species in the floodplain ecotones due to high amount of niche overlap (macarthur 1965). rotifera monthly richness significantly influenced zooplankton richness variations in maghuri (r1=0.940, p<0.0001), khamti guali (r2=0.942, p=0.0001) and no.11 (r3=0.959, p<0.0001) beels, respectively. the report of maximum 76 species per single sample in no. 11 beel is interesting in light of ‘rotifera paradox’ of 80+ species advanced by sharma and sharma (2019) for certain floodplain lakes of nei. cladocera contributed significantly to zooplankton richness only in maghuri beel (r1=0.773, p<0.0001) although the record richness of 31 species per sample each in khamti guali beel and in no.11 beels is noteworthy. low zooplankton abundance of reported from maghuri, khamti guali and no. 11 beels, respectively is attributed to low ionic concentrations’ and ‘soft – 29 int. j. aquat. biol. (2020) 8(1): 18-34 moderately hard waters. this generalization concurred with the reports from loktak lake (sharma and sharma, 2009) and two floodplain lakes (sharma, 2011a) of manipur, ghorajan beel (sharma and sharma, 2012) of assam, and from a reservoir of mizoram (sharma and pachuau, 2013). anova indicated significant monthly density variations amongst the three beels (f23, 71=2.5061, p=0.0039). the zooplankton did not follow any definite pattern of quantitative variations during the study period; peak densities recorded during pre-monsoon (june, 2015), monsoon (july, 2015) and autumn (november, 2014) in maghuri, khamti guali and no. 11 beels, respectively differed from the winter maxima reported by sharma (2011a, b), and sharma and sharma (2011a). this study lacked quantitative importance of any individual zooplankton species and thus suggested their ‘generalist-nature’ (sharma and sharma, 2014; sharma and hatimuria. 2017) in contrast to importance of certain species reported by sharma and sharma (2008, 2011b, 2012). rotifera, the dominant component (50.0±6.5, 49.6±9.5 and 44.6±8.6%), significantly contributed to zooplankton density variations in maghuri, khamti guali and no. 11 beels, respectively (r1=0.871, p<0.0001; r2=0.818, p=<0.0001; r3=0.848, p<0.0001). the concurrence of peak densities of rotifera with zooplankton peaks in the three beels reaffirmed this generalization. the stated importance of rotifera agreed with the reports of khan (1987), sanjer and sharma (1995), sharma (2005a, b, 2011a, b), and sharma and sharma (2001, 2008, 2011a, 2012) while it differed from sub-dominant role of this taxon in holmari beel (sharma and hatimuria, 2017) as well as vide the reports of yadava et al. (1987), sharma (2000), sharma and hussain (2001) and khan (2002). the rotifer abundance of dsbr beels concurred with the reports from loktak lake (sharma, 2009a) and two floodplain lakes (sharma, 2011b) of manipur, and a reservoir of mizoram (sharma and pachuau, 2013) while it is relatively lower than the reports from deepor beel (sharma, 2011a) and from ghorajan beel of assam (sharma and sharma, 2012). peak rotifer densities were observed during monsoon (july, 2015) in maghuri beel, post-monsoon (october, 2014) in khamti guali beel and in summer (may, 2014) in no.11 beel. the post-monsoon peak in khamti guali beel concurred with the report from the floodplains of the kashmir valley (khan, 1987) while summer peak in holmari beel concurred with the reports of yadava et al. (1987) and sanjer and sharma (1995). the reported maxima differed from winter peaks known from certain floodplain lakes of nei (sharma and hussain, 2001; sharma, 2009a, 2011a; sharma and sharma, 2011a, 2012). lecanidae > brachionidae > lepadellidae influenced the rotifer abundance in maghuri and khamti guali beels, and lecanidae > brachionidae recorded importance in no.11 beel. lecanidae contributed significantly to zooplankton abundance in maghuri beel (r1=0.605, p<0.0017), khamti guali (r2=0.750, p<0.0001), and no.11 beel (r3=0.798, p<0.0001). the importance of these families concurred with the reports from the floodplains of nei (sharma and hussain 2001; sharma, 2005a, 2009a, b, 2011a; sharma and sharma 2001, 2008, 2014) but differed from lack of such trend from west bengal (khan, 2002) and assam (sharma and sharma, 2012). cladocera comprised between 29.0±5.7, 32.9±9.1 and 36.5±8.2% of zooplankton abundance in the three beels, respectively and contributed significantly to the latter in maghuri beel (r1=0.836, p<0.0001) and no.11 beel (r3=0.636, p=0.0008). cladocera abundance is higher than the results of khan (1987), yadava et al. (1987), sharma and hussain (2001) and sharma and hatimuria (2017) from certain floodplain wetlands of india. the cladocerans recorded peak densities during pre-monsoon (june, 2015) in maghuri, summer (may, 2015) in khamti guali and pre-monsoon (june, 2015) in no.11 beels. this group recorded importance of chydoridae in the three beels concurrent with the reports of sharma (2011a) and, sharma and sharma (2008, 2011a, 2012). rhizopoda formed 10.2±4.7, 9.6±3.8, and 11.6±3.3% of zooplankton abundance in maghuri, khamti guali and no. 11 beels, respectively. it recorded peak in february and november, 2014 and in july, 2015 in the three beels, respectively. the rhizopod abundance is relatively 30 sharma and noroh / zooplankton diversity of floodplain lakes of upper assam lower than the reports of sharma and sharma (2008) while it is higher than the results of sharma and pachuau (2013) and sharma and hatimuria (2017). copepoda density comprised 9.2±3.3, 7.0±2.2 and 6.4±2.9% of zooplankton in maghuri, khamti guali and no. 11 beels, respectively. anova registered significant monthly variations of copepods density amongst the three beels (f2, 71=10.486, p=0.0001). the sub-dominance of copepods in dsbr beels concurred with the reports from deepor beel (sharma and sharma, 2011a) and ghorajan (sharma and sharma, 2012) beels of assam, and from loktak lake (sharma and sharma, 2009b) and two floodplain lakes (sharma, 2011b) of manipur but was lower than the report from ghorajan beel (sharma and sharma, 2012) of assam. nauplii recorded throughout the study period showed an active continuous reproductive phase of the cyclopoids concurrent with the reports of sharma (2011a, b), sharma and sharma (2011a) and sharma and pachuau (2013). ostracoda contributed insignificant fraction of zooplankton of the three dsbr beels. high zooplankton species diversity with h´ values > 4.0 during 3, 9 and 10 months in maghuri, khamti guali and no. 11 beels, respectively, coupled with lower densities of majority of species, is attributed to fine niche portioning in combination with microand macro-scale habitat heterogeneity as hypothesized by segers (2008) and endorsed by sharma (2011a, b), sharma and sharma (2011a, 2012) and, sharma and pachuau (2013). the species diversity was directly influenced by richness of zooplankton, rotifera and cladocera; and abundance of zooplankton, rotifera, cladocera, lecanidae and brachionidae in maghuri beel. it was influenced by richness of zooplankton, and rotifera; and abundance of rotifers, lecanidae, brachionidae and trichocercidae in khamti guali beel. it was influenced richness of zooplankton and rotifera; and abundance of rotifera, lecanidae, lepadellidae in no. 11 beel. low zooplankton dominance in maghuri, khamti guali and no. 11 beels and lack of quantitatively important species is hypothesized to the fact that the habitat of the sampled beels has resources for utilization by majority of species and thus providing high amount of niche overlap (macarthur, 1965). zooplankton depicted higher evenness in maghuri, khamti guali and no. 11 beels, respectively which affirmed low densities and equitable abundance of various species and thus reiterated that the majority of zooplankton are ‘generalists’ vis-à-vis their general environment. the present results concurred with the reports from the floodplain lakes of nei (sharma 2011a, b; sharma and sharma, 2008, 2011a, 2012; sharma and hatimuria, 2017). our study depicted limited significance of individual abiotic parameters on richness and abundance of zooplankton in dsbr beels. the former concurred with the results of sharma (2005b) and sharma and sharma (2012), while the latter corresponded with the reports of yadava and dey (1990), sharma and hussain (2001), sharma (2011a), and sharma and sharma (2011a). these generalizations are affirmed by the fact that the richness of zooplankton and rotifera is positively correlated with total dissolved solids and rainfall only in no.11 beel. abundance of zooplankton and rotifera is negatively correlated with nitrate and sulphate in khamti guali beel; zooplankton abundance is positively correlated with rainfall and water temperature and rotifera abundance is positively correlated with rainfall in no.11 beel. on the other hand, the canonical correspondence analysis (cca) with 17 abiotic factors registered moderate cumulative influence of 73.65, 61.42 and 63.56 zooplankton assemblages, along first two axes, in maghuri, khamti guali and no.11 beels, respectively. cca coordination biplot indicated influence of water temperature, dissolved oxygen and chloride and total hardness on zooplankton abundance; water temperature on rotifera richness; rainfall on rotifera abundance; water temperature on lecanidae; rainfall on daphnidae; and dissolved organic matter on macrothricidae in maghuri beel. cca recorded influence of total alkalinity on macrothricidae and plationus patulus; rainfall on zooplankton abundance and brachionidae; dissolved organic matter on zooplankton richness and lecanidae; chloride on 31 int. j. aquat. biol. (2020) 8(1): 18-34 rotifera abundance and richness; ph, calcium, total dissolved solids and silicate on copepoda abundance; and water temperature on lecane bulla in khamti guali beel. zooplankton abundance was influenced by rainfall; magnesium influenced abundance of cladocera, daphnidae and l. leontina; ph influenced rhizopoda abundance; lecanidae was influenced by total dissolved solids; and p. patulus was influenced by nitrate in no. 11 beel. the present study thus indicated limited influence of individual abiotic factors, while cca registered moderately high cumulative importance of 17 abiotic factors vis-à-vis richness and abundance of zooplankton assemblages of dsbr beels. the former affirmed that zooplankton species are largely ‘generalist’ in terms of individual abiotic factors; the results, hence, suggested the importance of analysis of factors associated with habitat variations in the sampled wetlands. to sum up, the biodiverse zooplankton with high total richness in the three dsbr beels, new records and various species of biogeographic interest; the littoral-periphytic nature with broadly tropical character; species homogeneity amongst the three beels and heterogeneity in individual beels; and ‘zooplankton paradox’ are hypothesized to habitat diversity and environmental heterogeneity of these floodplain wetlands located in `the assam-gateway’. low abundance of zooplankton is attributed to ‘soft – moderately hard waters’ with ‘low ionic concentrations’. our results did not depict any pattern of monthly or annual richness and density variations, while rotifera > cladocera influenced diversity in the three beels. our results are characterized by high species diversity, high evenness, low dominance, and lack of quantitative importance of any individual zooplankton species; the last aspect indicated ‘generalist nature’ of various taxa. this study highlights limited influence of individual abiotic factors on richness and abundance of zooplankton but cca registered moderately high cumulative importance of seventeen abiotic factors on zooplankton assemblages of dsbr beels. this study marks an important contribution on diversity of freshwater zooplankton of india in general and that of the floodplain lakes of nei as well as the tropics and subtropics in particular. acknowledgments the senior author (bks) is thankful to the ministry of environment and forests (govt. of india) for sanction of a research grant no. 22018-09/2010-cs (tax) under which this study was initiated. we thank the head, department of zoology, north-eastern hill university, shillong for laboratory facilities. the authors have no conflict of research interests. references a.p.h.a. 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(1987). limnology and productivity in dighali beel (assam). tropical ecology, 28: 137-146. international journal of aquatic biology (2015) 3(3): 172-176 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article length-weight relationship and condition factor of seven fish species of totkabon river (southern caspian sea basin), guilan, iran mazaher zamani faradonbeh1, soheil eagderi*1, fariborz ghojoghi2 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2department of fisheries, faculty of natural resources, islamic azad university, azadshahr branch. article history: received 15 february 2015 accepted 24 april 2015 available online 2 5 june 2015 keywords: river freshwater fishes lwrs allometry condition factor abstract: length-weight relationship and condition factor were investigated in seven fish species, including barbus cyri, capoeta gracilis, alburnoides eichwaldi, acanthalbarnus microlepis, ponticola cyrius, cobitis keyvani and oxynemacheilus bergianus from totkabon river. a total of 570 specimens ranging from 25.90 to 146.97 mm in total length and 0.1 to 260.3 g in total weight were collected. based on the results, growth coefficient values “b” ranged from 2.429 (a. microlpis) to 3.71 (c. keyvani). all length-weight relationships were significant (p<0.05), with r2 greater than 0.856. the four species viz. a. microlepis, c. gracilis, p. cyrius and c. keyvani showed allometric (b<3<b) and three species viz. b. cyri, a. eichwaldi and o. bergianus isometric (b = 3) growth patterns. condition factor ranged from 0.544 (o. bergianus) to 0.940 (p. cyrius). all linear regressions were highly significant (p<0.05). the result of this study may be useful for biologists and fishery managers. introduction length-weight relationships (lwrs) data of fishes are useful tools for biologists (martin-smith, 1996) in fishery assessment and proper management of fish population. lwr permits assessment of the biomass from length observations and conversion of growthin-length equations to growth-in-weight (gurkan and taskavak, 2007) and also is applied to assess well-being of individuals and to compare life histories of separated populations of the same species in different regions (martin-smith, 1996; ak et al., 2009). in addition, lwrs is used to obtain information about the condition of fishes to determine whether somatic growth is isometric or allometric (gurkan and taskavak, 2007; ujjania et al., 2012). condition factor (k) is an important biological parameter, which indicates the suitability of a specific water body for growth of fish and an index of species average size (alam et al., 2014). the * corresponding author: soheil eagderi e-mail address: soheil.eagderi@ut.ac.ir values of this factor depend on physiological features of fish especially maturity, spawning, life cycle, environmental factors and food availability in a water body (ujjania et al., 2012; dan-kishiya, 2013). the present study aimed to find out the present status of length-weight relationship and condition factor of seven fish species viz. barbus cyri, capoeta gracilis, alburnoides eichwaldi, acanthalbarnus microlepis, ponticola cyrius, cobitis keyvani and oxynemacheilus bergianus inhabiting totkabon river, a tributary of sefidrud river in the caspian sea basin (guilan province, north of iran). findings of this work will help better understanding of their biology and play an important role for management of these species in the protection program of their natural stock. materials and methods totkabon river (36°53'48"n, 49°30'19"e) is a 173 zamani faradonbeh et al/ lwrs and condition factor of seven fish species of totkabon river shallow stream with the elevation of 129-208 meters above sea level and surrounded by a dense forest. the samplings were conducted during the daytime in autumn 2013 by a backpack electrofishing (samus mp750, 45 cm diameter, aluminum ring anode). a total of 570 fish specimens belonging to seven species were collected (table 1). the captured fish specimens were anesthetized in 1% clove oil solution at the field and then weighted by a digital balance and photographed by a digital camera (canon, 510 is, 12 mp). finally, they were returned to the river after identification, and unidentified specimens were fixed in 10% formalin and transferred to the laboratory for identification based on abdoli (2000) and coad (2015). a scale was put beside the photographed fishes for extracting morphometric measurements using imagej software (version: 1.47). for each individual, morphometric data, including total length (tl), fork length (fl) and standard length (sl) was measured from its pictures to the nearest 0.01 mm. the body weight (bw) of the specimens were measured using an electronic balance to the nearest 0.01 g after drying by a clean towel. the length-weight relationship was determined by the equation of w= 𝛼lb (le cren, 1951; kahraman et al., 2014; wang et al., 2012), where w is total weight (expressed in g), l is total length (expressed in cm), 𝛼 is intercept i.e. coefficient related to the body, and b is slope. the growth pattern is isometric when the value of b = 3 and allometric when significantly different from 3 (alam et al., 2014). the logarithm transformation of the equation was expressed as: log w= 𝛼 + b logl (kahraman et al., 2014; wang et al., 2012) to estimate the parameters 𝛼 and b. the degree of correlation between the variables was computed by the determination coefficient “r2”. the significance level of r2 was estimated by anova. the student’s t-test (ts) was used to determine whether the parameter b is significantly different from the expected or theoretical value of 3 (i.e. b = 3, p<0.05). the condition factor (k) was estimated according to le cren (1951) and froese (2006) using k =100 w/l3, where, k is condition factor, w is weight of fish (gr) and l is length (total, fork and standard) of fish (cm). here, factor 100 is used to bring k close unity. in the present study, we calculated condition factor (k) for all measured length groups, i.e. tl, fl and sl. all the statistical analyses were considered at significance level of 5%. the statistical package spss (version 22) and microsoft office excel software (version 2013) were used to analysis data. results and discussion the ranges of the length and weight parameters, estimated lwr parameters, coefficients of the correlation of seven studied fish species are presented in table 1. the specimens ranged from 25.90 mm for c. gracilis to 146.97 mm for b. cyri in total length and 0.1 to 260.3 g in total weight for c. gracilis and b. cyri, respectively. all relationships were highly significant (p<0.05) with r2 values species n total length (mm) body weight (gr) growth coefficient min max mean ± se min max mean ± se 𝛼 b r2 ts tg b. cyri 70 50.70 146.97 93.47±2.59 1.1 26.30 7.95±0.67 0.0006 3.0078 0.927 0.02 i c. gracilis 240 25.90 145.90 74.54±1.27 0.10 16.48 4.45±0.20 0.000006 3.116 0.902 0.574 +a a. eichwaldi 62 48.764 101.37 74.24±1.73 1.00 8.70 4.02±0.28 0.000006 3.056 0.933 0.142 i a. microlepis 22 30.92 83.26 50.58±2.89 0.30 2.88 1.04±0.17 0.0000005 2.429 0.856 -1.05 -a p. cyrius 46 49.18 110.32 81.31±2.08 0.70 10.59 5.54±0.44 0.00006 3.214 0.861 0.417 +a c. keyvani 15 40.67 65.97 53.74±2.04 0.30 1.9 1.08±0.14 0.0000004 3.71 0.964 0.686 +a o. bergianus 15 40.67 75.17 63.72±0.15 0.47 2.24 1.42±0.15 0.000007 2.98 0.93 0.023 i n: sample size, min: minimum, max: maximum, mm: millimeter, gr: gram, 𝛼: intercept, b: slope, r2: correlation coefficient, se: standard error of the slope, t: students t-test, tg: types of growth, i: isometric growth, +a: positive allometric growth, -a: negative allometric growth. table 1. summary of length-weight relationships for seven fish species from totkabon river. 174 international journal of aquatic biology (2015) 3(3): 172-176 greater than 0.90 in five species and r2 = 0.80-0.90 in two others (table 1). the values of b of the length-weight equations among studied species ranged 2.429 for a. microlepis to 3.71 for c. keyvani. the b values of other species were obtained 3.116 for c. gracilis, 3.056 for a. eichwaldi, 2.429 for a. microlpis, 3.214 for p. cyrius and 2.98 for o. bergianus. the parameter “b” of all studied species was within the expected range of 2.5-3.5 except for a. microlepis and c. keyvani with b values of 2.429 and 3.71, respectively. the results of student’s t-test showed that b value of a. microlepis (b = 2.429; t-test: t = -1.05; p<0.05) was significantly lower than the theoretical value of 3 indicating a negative allometric growth, whereas the results of student t-test for c. gracilis, p. cyrius and c. keyvani were significantly higher (b = 3.1163.214; t-test: t = 0.417-0.686; p<0.05) indicating a positive allometric growth. barbus cyri, a. eichwaldi and o. bergianus showed an isometric growth pattern (b = 2.98-3.056; t-test: t = 0.020-0.142; p>0.05), with no significant difference from the theoretical value of 3 (table 2). tahmasebi et al. (2014) reported lwrs of b. cyri from sefidrood river (caspian basin) with b value of 2.5134, showing a negative allometric growth. boron et al. (2008) observed that parameter b in standard length-weight equation for specimens of the spined loach, cobitis taenia (linnaeus, 1758), for males and females are 3.83779 and 3.1683, respectively, indicating allometric growth in both sexes, though more in males than females (boron et al., 2008). the b value of o. bergianus in the kordan river was 2.83 and close to that of the totkabon (tabatabae et al., 2015). for other studied species, no lwrs data were available from iranian inland waters to compare with the results of the present study. the result showed that the condition factor values (k, tl-k) range between 0.544-0.94, with minimum value for o. bergianus (k = 0.544 ± 0.016) and maximum value for p. ciryus (k = 0.94 ± 0.0045) (table 2). the maximum value of the condition factor was observed based on the tl group in p. cyrius (0.94), based on the fl group in a. eichwaldi (1.203) and based on the sl group in a. eichwaldi (1.608) (table 2). t-test analysis showed that k values calculated based on the three length groups i.e. total and fork and standard lengths, in the studied species are significantly different (p>0.05). the condition factor is an index reflecting interaction between biotic and abiotic factors in the physiological conditions of fishes. therefore, the condition factor may vary among fish species in different locations (blackwell et al., 2000). condition factor is based on the hypothesis that heavier fishes of a given length are in better condition. this factor is also used as an index of growth and feeding intensity (seher and suleyman, 2012). condition factors of ≥1 indicate a good level of feeding and proper environmental condition (ujjania et al., 2012). based on the results, it was <1 family species condition factor total length fork length standard length k sd k sd k sd cyprinidae b. cyri 0.847 0.153 1.08 0.207 1.41 0.259 c. gracilis 0.911 0.268 1.203 0.340 1.608 0.454 a. eichwaldi 0.890 0.137 1.217 0.189 1.642 0.252 a. microlepis 0.73 0.21 1.007 0.26 1.323 0.344 gobiidae p. cyrius 0.94 0.207 1.55 0.339 cobitidae c. keyvani 0.632 0.093 0.944 0.149 nemachilidae o. bergianus 0.544 0.070 0.63 0.093 0.83 0.167 k = condition factor coefficient and sd= standard deviation of condition factor coefficient. table 2. correlation of standard body length with body weight of studied fish from totkabon river at different length groups. 175 zamani faradonbeh et al/ lwrs and condition factor of seven fish species of totkabon river for o. bergianus and c. keyvani showing no proper environmental conditions of habitat for these species in totkabon river. whereas, k values of the rest of species were >1 showing perfect condition of this river for them. lwr parameters (𝛼 and b) and the “k” value of the fish have been reported to be affected by many factors such as feeding intensity, availability of food, fish size, age, sex, season, stage of maturation, fullness of the gut, degree of muscular development, the amount of reserved fat and life history (bagenal and tesch, 1978; ujjania et al., 2012; gupta and banerjee, 2015). none of the above mentioned effective factors on lwr and k in the studied fishes have been considered in the present study. therefore, for using the results of this study, it should be borne in mind that the samples were taken seasonally and the number of fish examined was limited. finally, the length-weight relationships and condition factor presented here will provide useful information for fisheries management and fish population dynamic studies. to the best of our knowledge, lwr and k of these fishes were presented in totkabon river for the first time. therefore, the results of the present study can serve as baseline data for these species and for comparisons with future studies. references abdoli a. 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(2015) 3(1): 28-34 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article comparative histological and histochemical studies on the pancreas of labeo rohita (hamilton, 1822), mystus vittatus (bloch, 1790) and notopterus notopterus (pallas, 1769) padmanabha chakrabarti*,1saroj kumar ghosh fisheries laboratory, department of zoology, the university of burdwan, burdwan 713104, west bengal, india. article history: received 10 november 2014 accepted 24 december 2014 available online 2 5 february 2015 keywords: histoarchitecture histochemistry pancreas labeo rohita mystus vittatus notopterus notopterus abstract: the histological analysis, disposition and histochemical localization of tryptophan were investigated in the pancreas to compare the cellular organization and histochemical characterization in the pancreas of labeo rohita (hamilton, 1822), mystus vittatus (bloch, 1790) and notopterus notopterus (pallas, 1769) having different feeding habits. histological analysis demonstrated that the exocrine pancreatic tissues were dispersed within the hepatic parenchyma and spleen in l. rohita. thin septa of connective tissue separated parenchyma of liver and also the spleen from exocrine pancreatic cells. however, in m. vittatus, the discrete pancreatic tissue formed distinct oval or elongated acini interspersed with small area of islet of langerhans and blood vessels. in n. notopterus, the rhomboidal acinar cells of discrete pancreatic tissue intercalated with comparatively clear and large islet of langerhans. the exocrine acinar cells in all the three species were provided with prominent nuclei and densely packed zymogen granules. histochemical localization revealed that the zymogen granules of exocrine acinar cells of all species exhibited varied intensities of tryptophan reaction, the precursor of various pancreatic enzymes which may be related to the food and feeding habits of the fishes under study. introduction one of the most important digestive glands in teleosts is pancreas consisting of exocrine and endocrine tissue with various degrees of morphoanatomical and cellular variations (eurell and haensly, 1982; youson et al., 2006; el-said et al., 2010; chakrabarti and ghosh, 2012). pancreas in teleosts shows numerous species variations with respect to their cytological structures as well as functional aspects (sinha and moitra, 1975; dwivedi and pandey, 1982; vicentini et al., 2005; petcoff et al., 2006). a hepatopancreas has been reported in the iridencent shark catfish pangasius hypophythalmus (seyrafi et al., 2009) and a spleenopancreas in barbus pectoralis (khaksar mahabdy et al., 2012). the formation of glandular acinuses is a regular part of the exocrine pancreas; their cytological details and chemical nature have been investigated in fishes * corresponding author: nahid gheisvandi e-mail address: dr.pchakrabarti@yahoo.in having different feeding habits (shafi, 1973; bremer, 1980). studies relating to precise cellular details, including the precise histochemical nature and functional aspects of the pancreas in indian freshwater teleosts are limited. the aim of our research was to explore the presence of pancreas in discrete or diffused pattern and to examine the structural and functional status of pancreatic tissues in labeo rohita, mystus vittatus and notopterus notopterus having different feeding habits by histological analysis and histochemical localization. these species are of great interest to fish culture, including their considerable growth and excellent quality. materials and methods mature specimens of l. rohita (28-30 cm in total length), m. vittatus (10-12 cm in total length) and 29 chakrabarti and ghosh/ comparative microscopical analysis of pancreas in l. rohita, m. vittatus and n. notopterus n. notopterus (23-25 cm in total length) were collected from local freshwater fish farm of burdwan (23.2333° n, 87.8667° e), west bengal, india during april-october, 2013. fishes were anesthetized with an overdose of chloral hydrate following the guidelines given by institutional ethical committee. histological preparation: the body cavity was cut open through abdominal incision and small pieces of the discrete pancreas, pancreatic tissues associated with liver and spleen were removed and fixed in aqueous bouin’s fluid for 18 hrs. the tissues were then placed in 70% ethanol and subsequently dehydrated through ascending series of ethanol, followed by acetone and cleared with xylene, impregnated and embedded in paraffin wax (5658°c). serial sections of 4 µm thick were obtained with leitz microtome. deparaffinised sections were brought to distilled water through descending series of ethanol and were stained with delafield’s haematoxylin-eosin (he) and mallory’s triple (mt) stain (mallory, 1936). histochemical preparation: for histochemical study, the pieces of pancreas, hepatopancreas and spleenopancreas were removed and fixed in 10% neutral formalin for 16 hrs. the tissues were passed through graded series of ethanol, cleared in xylene and embedded in paraffin wax at 52-54°c in vacuum embedding bath. serial sections of the tissues were cut at 8-10 µm in thickness. deparaffinised sections were brought to distilled water and then subjected to histochemical demonstration of tryptophan by dimethylaminobenzaldehyde (dmab) nitrate method (adams, 1957). staining slides were dehydrated with graded series figure 1. photomicrographs of section of pancreas in l. rohita showing histological architecture stained with delafield’s haematoxylin-eosin (he) and mallory’s triple (mt) stain. (a). scattered round or triangle shaped pancreatic tissue (pt) distributed in between the hepatic tissue (ht). note the presence of blood vessels (bv) (solid arrows) encircled by ac. broken arrow indicates central sinusoid with erythrocytes (mt x100), (b). higher magnification of hepatopancreas showing pyramidal or triangular acinar cells (ac) of pt arranged in several rows. ac are provided with basally located nucleus (n) with conspicuous nucleolus (arrow heads) and numerous zymogen granules (zg). note the presence of thin septa of connective tissue (broken arrows) which separates pt from ht. solid arrow indicates blood vessels (bv) encircled by ac (he x400), (c). spleenic pancreas showing orientation of pancreatic ac surrounding bv. ac are distinguished from spleenic tissue (st) by thin septa of connective tissue (solid arrows) (mt x200) and (d). higher magnification of spleenopancreas showing ac around bv contains prominent nucleolus in nucleus (n) (arrow heads) and acidophilic zg. pt is distinguished from ht by thin septa of connective tissue (solid arrows). broken arrows indicate bv adjacent to ac (mt x400). 30 int. j. aquat. biol. (2015) 3(1): 28-34 of ethanol, cleared in xylene and mounted with dpx. the slides were examined and photographed under an olympus-tokyo pm-6 compound microscope. results labeo rohita: the pancreas in l. rohita is distributed within the liver parenchyma forming hepatopancreas and within the spleenic tissues. the triangle oval or elongated patches of pancreatic tissues are randomly distributed within the hepatic parenchyma. the hepatocytes are arranged in cords around central sinusoids, which contained mainly erythrocytes (fig. 1a). the hepatic cells contain granular cytoplasm with prominent nuclei (figs. 1a, 1b). the hepatopancreas in this fish is separated from the hepatic parenchyma by a thin layer of connective tissue (fig. 1b). the pancreatic patched are made up of exocrine acini, which are bounded by connective tissue, existed around hepatocytes (figs. 1a, 1b) and the blood vessels and spleen (figs. 1c, 1d). the pyramidal or triangular acinar cells are large and arranged in two or more rows. a typical exocrine cell contains a broad basal region and a narrow apical part. the basal region contains a large spherical nucleus with a distinct nucleolus. the apical parts of the exocrine pancreatic cells contain acidophilic zymogen granules, which are relatively larger in size (figs. 1b, 1d). in the present study, the patches of the islet of langerhans cannot be detected in the hepatopancreas and spleenopancreas. mystus vittatus: the pancreatic tissue consists of the clusters of acinar cells observed adjacent to stomach or attached to the wall of the stomach (fig. 2a). in m. vittatus, the pancreas is relatively more organized and composed of a large number of acinar cells (figs. 2b, 2c, 2d). each acinus cell contains zymogen granules, relatively smaller in size than those of l. rohita and a spherical nucleus with a distinct nucleolus (fig. 2c). limited patches of islet of langerhans and blood vessels are present in figure 2. photomicrographs of section of pancreas in m. vittatus showing histological architecture stained with delafield’s haematoxylin-eosin (he) and mallory’s triple (mt) stain. (a). showing pancreatic tissue (pt) having clusters of acinar cells (ac) adjacent to the stomach wall (sw). broken arrow indicates small islet of langerhans (il) (mt x100), (b). pt attached with the wall of stomach (s) having dense organization of ac around blood vessels (bv) (arrow) and scattered il (he x200), (c). higher magnification of ac with prominent zymogen granules (zg) and conspicuous nucleolus in the nucleus (arrow heads). note the presence of bv (solid arrows) and il encircled by ac (he x400) and (d). presence of il (broken arrow) in between ac (solid arrows) (mt x400). 31 chakrabarti and ghosh/ comparative microscopical analysis of pancreas in l. rohita, m. vittatus and n. notopterus between the exocrine cells (figs. 2b, 2c). pancreatic duct is also observed, encircled by acinar cells (figs. 2a, 2d). notopterus notopterus: in n. notopterus, the pancreatic tissues are sandwitched in between pyloric caeca and the wall of the stomach in distal region. the exocrine pancreatic tissue consists of clusters of rhomboidal type of cells mostly organized in acini. the acinar cells are provided with basophilic cytoplasm, distinct nuclei and many eosinophilic or aniline blue positive zymogen granules (figs. 3b, 3c, 3d). blood vessels and pancreatic ducts are present here and there with in the ground pancreatic tissue (figs. 3c, 3d). the zymogen granules are diffused and/or relatively smaller in size than those of l. rohita in the pancreatic acinar cells (figs. 3b, 3d). moreover, the pancreas of n. notopterus also presents the endocrine tissues as islet organ in between the exocrine cells (figs. 3a, 3b, 3d). langerhan’s islets are surrounded by a delicate connective tissue. in the islet, cells are arranged in scattered way, between which were capillaries. the α and β cells are clearly differentiated in the islets. the α cells are bigger with the prominent nucleus while β cells are smaller, spherical with rounded nuclei (figs. 3b and 3d). histochemical demonstration of tryptophan: the zymogen granules of cytoplasm in pancreatic acinar cells of l. rohita exhibit maximum intensity of tryptophan reaction while hepatocytes display weak reaction for this histochemical test (figs. 4a, 4b). the blood cells of hepatic sinusoids also show intense tryptophan reaction (fig. 4a). the acinar cells in the spleenic pancreas exhibit intense reaction for tryptophan, though comparatively less than that of hepatopancreas. the spleen tissue itself shows a weak tryptophan reaction (fig. 4b). the zymogen granules of the acinar cells of the pancreas in figure 3. photomicrographs of section of pancreas in n. notopterus showing histological architecture stained with delafield’s haematoxylin-eosin (he) and mallory’s triple (mt) stain. (a). showing pancreatic tissue (pt) adjacent to stomach wall (sw) having bunch of acinar cells (ac) encircling islet of langerhans (il). note the presence of blood vessel (bv) (solid arrow) and pancreatic ducts (pd) (broken arrows) in the pt (mt x100), (b). higher magnification showing il separated from ac by connective tissue septa (broken arrows). il showing α cells (solid arrows) and β cells (broken arrows) in between bv (mt x400), (c). showing exocrine pt consisted of cluster of ac (solid arrows) attached to sw. note the presence of bv among ac and attached to the sw. broken arrows indicate scattered bv in between ac. pd indicates pancreatic duct (he x200) and (d). rhomboidal shaped ac (arrow heads) provided with diffused zymogen granules. note the presence of il and scattered blood cells (solid arrow) among ac (mt × 400). 32 int. j. aquat. biol. (2015) 3(1): 28-34 m. vittatus display a strong tryptophan reaction while the islet of langerhans cells exhibit very weak reaction (fig. 4c). in n. notopterus, the large number of acinar cells in the pancreatic tissue shows maximum intensity of reaction for tryptophan. the islet of langerhan cells in between the acinar cells exhibit weak reaction for tryptophan (fig. 4d). discussion the diffused form of the pancreas as an organ in teleosts has been studied by different authors (chen et al., 2006; tocher et al., 2008). gonzalez et al. (1993) reported the presence of diffuse exocrine pancreas, which expanded through the mesentry, around some digestive organs of disseminates within the intraperitoneal adipose tissue in serranus cabrilla. el-said et al. (2010) also described the acinar arrangement of hepatopancreas in mugil cephalus and sparus aurata. in the present investigation, in l. rohita the pancreas exists as intrahepatic as well as in the spleenic tissues. on the other hand, in carnivorous m. vittatus and n. notopterus, the pancreas is a compact gland extended over the surface of the stomach and anterior part of the intestine. khanna (1961; 1963) also observed compact nature of the pancreas in clarias batrachus, mystus aor, silonia silondia and heteropneustes fossilis. in the present study, the exocrine acinar cells in the hepatopancreas and spleenopancreas of l. rohita are arranged in two or more rows surrounding a central blood vessel. the zymogen granules in the acinar cells are numerous and large in size than those in m. vittatus and n. notopterus. in herbivorous, l. rohita the intestine is enormously coiled, an adaptation for retention of food materials for a large period of time and the only source and copious amount of digestive enzymes are needed for effective of enzyme is the hepatopancreas digestion of food materials. the pancreatic juices along with figure 4. photomicrographs of section of pancreas in l. rohita, m. vittatus and n. notopterus showing histochemical localization of trypotophan (dmab). (a). hepatopancreas of l. rohita showing intense tryptophan reaction in acinar cells (ac) of pancreatic tissue and weak reaction in hepatic tissue (ht). solid arrows indicate positive tryptophan reaction in blood vessels (bv) (dmab x200), (b). spleenopancreas of l. rohita showing intense tryptophan reaction in acinar cells (ac) of pancreatic tissue and weak reaction in spleenic tissue (st). note moderate reaction of tryptophan in bv (dmab x400), (c). strong intensity of reaction for tryptophan in zymogen granules of ac and weak reaction in islet of langerhans (il) of m. vittatus (dmab x400) and (d). pancreas of n. notopterus showing acute tryptophan reaction in the ac (solid arrows) and weak reaction in il (dmab x400). 33 chakrabarti and ghosh/ comparative microscopical analysis of pancreas in l. rohita, m. vittatus and n. notopterus bile juice after being secreted from the hepatopancreas is collected in the gall bladder and ultimately emptied in the alimentary canal. yamane (1973) gave an account of the presence of amylase in the cytoplasm of the acinar cells in diffuse pancreas and confirmed that these cells were the centre of amylase production in the carp. ray (1988) observed that gall bladder of three species viz., colisa fasciata, ambassis nama and a. ranga with hepatopancreas, exhibited higher enzymatic activities between ph 7.5 and 8.5. further, in the present study the concentration of the zymogen granules in the pancreatic acini of hepatopancreas in l. rohita appears to be higher than that of the spleenopancreas. therefore, it may be assumed that the acinar cells of hepatopancreas are physiologically more active in respect to secretion of digestive enzymes. alboghobeish and khaksar mahabdy (2005) emphasized that pancreatic tissue in ctenopharyngodon idella gradually invaded the liver along the branches of the portal vein. the combined hepatic and pancreatic tissue are collectively called hepatopancreas. based on the results, in l. rohita the scattered spleenic lobules along with pancreatic acinar cells enclose with the wall of the intestine may have similar digestive function like that of hepatopancreas. in barbus pectoralis, khaksar mahabdy et al. (2012) recorded that the spleenopancreas performed many functions such as lymphatic cell production and digestion of food materials. in m. vittatus and n. notopterus the basophilic zymogen containing cells of the exocrine pancreas is for the production and storage of pancreatic enzymes that are delivered to the digestive tract through a network of ducts for effective digestion of protein-rich food materials. field et al. (2003) reported that the exocrine pancreatic tissue produces digestive enzymes, such as trypsin, amylase and carboxypeptidase a for effective digestion of food in zebra fish (danio rerio). the results showed the prominent α and β like cells in the endocrine component of n. notopterus and m. vittatus may have some role in carbohydrate metabolism. ikpegbu et al. (2012) observed islet of langerhans interspersed in the majority of exocrine cells in clarias gariepinus responsible for hormone production like insulin. in the present study, the distribution and localization of tryptophan content was observed in the exocrine acinar cells at varied intensities in l. rohita, m. vittatus and n. notopterus. tryptophan, the precursor of pancreatic enzymes mainly associated with the zymogen granules of acinar cells. the varied intensities of tryptophan reaction in the acinar cells of the aforesaid fishes may be related with the synthesis and secretion of pancreatic enzymes in different proportions according to their requirement relating to their feeding habits. acknowledgement the authors are grateful to dr. a. basu, head of the department of zoology, the university of burdwan, burdwan for providing necessary laboratory facilities. references adams c.w.m. 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(2015) 3(1): 28-34 aquaculture, 256: 489-501. dwivedi a.k., pandey d.n. (1982). observation on the pancreas of a freshwater fish, mystus vittatus (bloch). reading in zoology, 1: 48-49. el-said n., el-bakary r., el-gammal h.l. (2010). comparative histological, histochemical and ultrastructural studies on the liver of flathead grey mullet (mugil cephalus) and sea bream (sparus aurata). global veterinaria, 4: 548-553. eurell j.a., haensly w.e. (1982). the histology and ultrastructure of the liver of atlantic croacker micropogon undulatua. journal of fish biology, 21: 113-125. field h.a., si dong p.d., beis d., stainier d.y. r. (2003). formation of the digestive system in zebra fish. ii pancreas morphogenesis. developmental biology, 261: 197-208. gonzalez g., crespo s., brusle j. (1993). histocytological study of the liver of the cabrilla sea bass, serranus cabrilla (teleosti, serranidae), an available model for marine fish experimental studies. journal of fish biology, 43: 363-373. ikpegbu e., nlebedum u.c., nnadozie o., agbakwuru i.o. (2012). histological structures of the accessory glands of the digestive system in adult farmed african catfish (clarias gariepinus). iosr journal of agricultural and veterinary science, 1: 41-46. khaksar mahabdy m., morovvati h., arefi a., karamifar m. (2012). anatomical and histomorphological study of spleen and pancreas in berzem (barbus pectoralis). world journal of fish and marine sciences, 4: 263267. khanna s.s. (1961). alimentary canal in some teleost fishes. journal of the zoological society of india, 13: 206-219. khanna s.s. (1963). on the pancreas of some freshwater teleosts. journal of the zoological society of india, 15: 53-58. petcoff g.m., diaz a.o., escalante a.h., goldemberg a.l. 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(2020) 8(1): 50-55 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article the olfactory mucosa of river catfish, eutropiichthys vacha (hamilton, 1822): a histochemical study saroj kumar ghosh*1, surajit das2 1department of zoology, bejoy narayan mahavidyalaya, itachuna, hooghly-712 147, west bengal, india. 2department of zoology, kabi nazrul college, murarai, birbhum-731 219, west bengal, india. s article history: received 19 december 2019 accepted 31 january 2020 available online 2 5 february 2020 keywords: olfactory epithelium cell types histochemical characteristics chemoreception abstract: miscellany in histochemical peculiarity of the olfactory mucosa was studied for localization of axons, mucopolysaccharides, glycogen, protein and lipid in schilbid catfish, eutropiichthys vacha (hamilton, 1822). silver deposition was detected in the abundance of receptor cells in the olfactosensory epithelium and well as marked in the knob and dendrite process of primary receptor cells. the mucous cells with various stages of maturity containing different degrees of mucopolysaccharides were identified by employing the periodic acid schiff's (pas) reaction in combination with alcian blue (ab) test. this combined test imparted purple colour due to pas for neutral mucin and blue colour for ab reaction due to the presence of acid mucin exclusively. the results of best’s carmine test indicated considerable amount of glycogen present in the receptor cells, basal cells and supporting cells in the olfactory mucosa. basic protein and bound lipid were ascertained in the various cells of the epithelial lining and in blood cells of the central core were discussed with behavioural activities of the fish interested. introduction olfaction is one of the most notable senses driving the basic types of behaviour in teleosts for communication with the aquatic environment. it is extremely important in fish participating in survival linked activities such as feeding, migrations, nest finding, reproductive strategies, parental behaviour and fright reaction to alarm substance (camacho et al., 2010). olfactory cues are ascertained by a pair of olfactory organs connected to the olfactory lobes of the brain and pertinent attitudes are released in any given species (mana and kawamura, 2002). in recent years, a widespread attraction has been exhibited by researchers on morphology, structural organization and function of the olfactory organ to understand the mechanism of olfaction in teleosts. cellular elements and their chemical constituents conceiving of the olfactory system are indispensable to annotation of olfactory function. scanty studies have been made on histochemistry of the olfactory epithelium of fish (singh and singh, 1987; pastor et *correspondence: saroj kumar ghosh doi: https://doi.org/10.22034/ijab.v8i1.773 e-mail: saroj.fisherylab@gmail.com al., 1991; saito et al., 2004; bettini et al., 2009; ghosh and chakrabarti, 2012, 2015; kim et al., 2019) to localize the chemical peculiarity in different cell types. in view of the paucity of knowledge regarding the chemical nature and functional significance of diverse cells lining the olfactory mucosa of schilbid catfishes in relation to mode of life and living, the present work has been undertaken on eutropiichthys vacha (hamilton, 1822) (actinopterygii: siluriformes: schilbeidae), a carnivore surface feeder which feeds on insects, crustaceans, rotifers, mollusks and small fishes (talwar and jhingran, 1991; nath, 1994). materials and methods sample collection: a total of 24 adult specimens of both sexes e. vacha (measured 22±2.07 cm in total length) were captured from the river ganga around kalyani, west bengal throughout the year 2018. the specimens were deeply anaesthetized with benzocaine (4 mg/l) and the olfactory organs were carefully 51 int. j. aquat. biol. (2020) 8(1): 50-55 excised from the floor of the nasal cavity under a zeiss stemi 2000-c stereoscopic binocular microscope. histochemical analysis: olfactory tissues were fixed in 10% neutral formalin for 16-18 h. after fixation, the tissues were washed well in 70% ethanol and dehydrated in graded ethanol series. the tissues were cleared in methyl benzoate and processed for paraffin embedding. sections were cut as 8-10 m thick using a rotary microtome (weswox mt-1090a) and proceed for silver impregnation method (sim) for detection of axons (marsland et al., 1954), periodic acid schiff's (pas) reaction in combination with alcian blue (ab) (pas-ab) for detection of neutral and acid mucins (mowry, 1956), best's carmine (bc) method for detection of glycogen (best, 1906), mercury-bromphenol blue (mbb) method for detection of basic protein (mazia et al., 1953) and acetone sudan black (asb) method for detection of bound lipid (berenbaum, 1958). all the slides were observed and photographed using a leica ec3 light microscope at different magnifications. results silver reaction: silver reaction furnishes dark brown colour and ascertains various kinds of receptor cells in the apical surface of epithelium (figs. 1a-b). positive reaction of silver staining is observed in the axonal processes of ciliated and rod receptor cells and nerve fascicles in the central core (fig. 1c). the axon of scanty primary receptor cells consequence with synaptic connection to the dendrite ends of secondary receptor cells. the basement membrane in between olfactory epithelium and central core and knob-like structure of receptor cells on the free epithelial surface shows positive silver reaction (fig. 1d). periodic acid schiff's reaction in combination with alcian blue: histochemical detection of neutral and acid mucopolysaccharides is denoted by pas reaction figure 1. photomicrographs of transverse sections of olfactory epithelium of eutropiichthys vacha showing silver deposition in the neurons by silver impregnation method (sim). (a) olfactory lamellae (ol) showing silver deposition in olfactory epithelium (oep) and central core (cc) (arrows) radiated from raphe (r) (40x). (b) showing silver reaction in the axonal processes of different receptor cells (d) lining the epithelium of ol. note positive reaction in nerve fascicles of cc (100x). (c) showing intense silver reaction in axonal processes of ciliated receptor cells (rc) and rod cells (arrows) of oep. note strong reaction in nerve fibres (n) and blood vessels (bv) in cc (400x). (d) maximum localization of silver staining in primary rc with knob like structure (k) and synaptic contact in between primary and secondary rc (arrows) of oep. note intense silver staining in basement membrane (bm) and bv in cc (sim) (1000x). 52 ghosh and das / histochemistry of the olfactory organ in eutropiichthys vacha in combination with ab (pas-ab) test. the positive reaction is due to the presence of 1-2 glycol groups in the mucous cells. this combined test furnishes a red colour due to pas reaction for neutral mucin and blue colour for ab reaction confirms the presence of acid mucin (fig. 2a). the secretory mucous cells at the surface of the epithelium and peripheral non-secretory mucous cells show intense blue and red colour assuring acid and neutral mucins, respectively. the secretory mucous cells are vacuolated and roughly reticulated. loosely disposed connective tissues and elastic fibres in the central core exhibit moderate reaction (fig. 2b). the secreted mucin also positively rebounds with ab test. negligible activity is observed in the basal cells, supporting cells and receptor cells. best's carmine reaction: the accrue of best’s carmine assay mark presence of glycogen in the receptor cells, basal cells, ciliated supporting cells and non-ciliated supporting cells of the epithelium as well as in the epithelial border (figs. 3a-b). diffuse granules are observed in the cytoplasm of basal cells and supporting cells. moderate glycogen content is present figure 2. photomicrographs of the olfactory lamella of eutropiichthys vacha showing periodic acid schiff's (pas) reaction in combination with alcian blue (ab) (pas-ab). (a) localization of acid mucin in the secretory mucous cells (mc) of olfactory epithelium (oep). central core (cc) displays moderate reaction. r marks raphe (100x). (b) intense acid mucin in the secretory mucous cells (mc) and neutral mucin in non-secretory mucous cells (mmc) of oep. cc shows moderate reaction. arrows indicate luminal secretion (400x). figure 3. part of horizontal section of olfactory epithelium of eutropiichthys vacha showing best’s carmine (bc) reaction for glycogen. (a) intense localization of glycogen in the epithelial border (arrows) of olfactory lamella (ol). central core (cc) and raphe (r) displays moderate to weak reaction (100x). (b) olfactory epithelium (oep) displays glycogen content in receptor cells (solid arrows), ciliated supporting cells (csc), non-ciliated supporting cells (broken arrows) and basal cells (bc). cc exhibits moderate reaction (400x). 53 int. j. aquat. biol. (2020) 8(1): 50-55 in the central core of the lamella. mercury-bromphenol blue reaction: the basal cells, labyrinth cells and receptor cells having cylindrical process up to the free epithelial surface are positive to mercury-bromphenol blue reaction furnishing a dignified concentration of proteins (figs. 4a-b). the nuclei of basal cells are darkly stained. the mucopolysaccharide filing of mucous cells in the olfactory epithelium show proteinaceous nature (fig. 4b). the puissant reaction of protein is also consorted with the blood cells of the central core. acetone sudan black resaction: this histochemical test has been employed for the detection of bound lipid associated with various cells of the olfactory lamella. the colours procured are generally black or grey. the basal cells, receptor cells along with sensory hairs at the epithelial surface showing lipoid nature. this method gives deep colouration of blood vessels in central core (figs. 5a-b). discussions the olfactory organ is the only organ of fish in which receptor neurons are directly exposed to aquatic figure 4. photomicrographs of transverse sections of olfactory lamella of eutropiichthys vacha showing mercury-bromphenol blue (mbb) reaction for basic protein. (a) distribution of protein in various layers of olfactory lamellae (ol) based on raphe (r) (100x). (b) showing intense localization of protein in receptor cells (solid arrows) and basal cells (bc) of olfactory epithelium (oep) and blood vessels (bv) of central core (cc). note moderate reaction in labyrinth cells (lc) and weak reaction in mucous cells (broken arrows) (400x). figure 5. photomicrographs of different regions of olfactory epithelium of eutropiichthys vacha showing acetone sudan black (asb) reaction for bound lipid. (a) distribution of lipids in the epithelial (oep) lining (arrows) and central core (cc) of olfactory lamella radiated from raphe (r) (100x). (b) showing sudanophilic materials in blood vessels (bv) of cc, basal cells and receptor cells (broken arrows) with sensory hairs (solid arrows) at surface of oep (400x). 54 ghosh and das / histochemistry of the olfactory organ in eutropiichthys vacha environment and vulnerable to water contaminants. in e. vacha, the acute localization of silver stain in the axons of the primary receptor cells, make synaptic contacts with the dendrites of the secondary neurons. the axons of the secondary neurons which enter into the central core are also intensely stained with silver reaction. this intimates that the impulses received by the terminal dendrites of primary receptor cells ultimately convey impulses to the central core for final transduction of impulses to the brain. this finding is accordance with that of olfactory epithelium of etroplus suratensis (chakrabarti and ghosh, 2013) and catla catla (ghosh and chakrabarti, 2015). the histochemical investigations advocate that the olfactory mucosa of e. vacha comprehends mucopolysaccharides, glycogen, protein and lipid. pas-ab positive materials are detected in the mucous cells. the presence of mucous cells in the present species makes their olfactory epithelium secretory in nature. in the present observation, the predominance of acid mucopolysaccharides in the epithelial lining prevents the friction against foreign particles, which enter into the olfactory chamber through water. the secreted mucin which covers the surface of the olfactory epithelium provides protection to delicate sensory hairs against osmotic effects of water (hopkins, 1926) and forms a favourable environment for ionic and molecular diffusion (chakrabarti, 2005). pas-ab positive material is a complex mucopolysaccharides or glycoprotein in nature (fullmer, 1965). it has been noticed that diffuse glycogen occurs in the cytoplasm of supporting and basal cells of e. vacha. the movement of cilia of the supporting cells maintains a continuous directional flow along the surface epithelium, which is an active process, requires energy and presence of glycogen particles is the main source of such energy. the presence of glycogen in the receptor cells facilitating the conduction of the sense of smell and glycogen probably acts as a substrate for the source of energy required for the activities. the presence of glycogen also been reported by ojha and kapoor (1972) in channa punctatus and chakrabarti (2005) in mugil parsia. glycogen in the basal cells in the fishes under study may help in maintaining their metabolic status as well as provide energy so essential for sustaining their motility. the mucous cells, receptor cells, labyrinth cells and basal cells are rich in protein. the contents of the mucous cells exhibit positive mercury-bromphenol blue reaction, confirming the proteinaceous nature which synthesis neutral glycoproteins. the basal and labyrinth cells in the olfactory epithelium exhibit intense to moderate reaction for protein probably for various metabolic as well as physiological activities (ghosh and chakrabarti, 2015). the presence of protein has reported in the elements of olfactory epithelium of hillstream teleosts (singh and singh, 1987). it has been observed that the most lipids found in the epithelial border of the present species. the acetone sudan black method demonstrated that the presence of lipids mostly concentrated in the basal cells and moderately distributed along dendrites of receptor cells may be a sign of myalinated sheath in the axons of receptor cells and probably help in the impulse transduction process. evans and hara (1977) reported that phospholipids occur in the dendrite process of receptor cells of the olfactory epithelium in fishes. mostly concentrated lipid material in blood cells of central core is required as a source of endogenous energy including its involvement for the physiological activities. acknowledgments the authors acknowledge the financial support of the university grants commission, eastern regional office, salt lake, kolkata-700 098 [no.: f.psw016/15-16 (ero) id no. wb1-014 dated 15-nov16]. references bernenbaum m.c. (1958). the histochemistry of bound lipids. quarterly journal of microscopical science, 99: 231-242. best f. (1906). uber carmine far bug des glycogens and derkerne. zeitschrift für wissenschaftliche mikroskopie und mikroskopische technik, 3: 319-322. bettini s., lazzari m., ciani f., franceschini v. (2009). 55 int. j. aquat. biol. (2020) 8(1): 50-55 immunohistochemical and histochemical characteristics of the olfactory system of the guppy, poecilia reticulata (teleostei, poecilidae). the anatomical record, 10: 1569-1576. camacho s., ostos-garrido m.v., domezain a., carmona r. (2010). study of the olfactory epithelium in the developing sturgeon characterization of the crypt cells. chemical senses, 35: 147-156. chakarbarti p. (2005). histological and histochemical studies on the olfactory rosette of mugil parsia (hamilton). folia morphologica, 64: 41-46. chakrabarti p., ghosh s.k. (2013). histochemical studies of the olfactory epithelium of brackish-water cichlid fish, etroplus suratensis (bloch). archives of polish fisheries, 21: 315-321. evans r.e., hara t.j. (1977). histochemical localization of phospholipids in the olfactory epithelium of fish. canadian journal of zoology, 55: 776-781. fullmer h.m. (1965). histochemistry of the connective tissue. international review of connective tissue research, 3: 1-65. ghosh s.k., chakrabarti p. (2012). histochemical study of the olfactory rosette of cyprinus carpio (linnaeus, 1758). iranian journal of fisheries sciences, 11: 305314. ghosh s.k., chakrabarti p. (2015). histochemical characterization of the olfactory epithelium of an indian major carp, catla catla (hamilton, 1822). iranian journal of ichthyology, 2: 43-52. hopkins a.e. (1926). olfactory receptors in vertebrates. journal of comparative neurology, 41: 253-289. kim h.t., yun s.w., park j.y. (2019). anatomy, histology and histochemistry of the olfactory organ of the korean shuttles mudskipper periophthalmus modestus. journal of morphology, 280: 1485-1491. mana r.r., kawamura g. (2002). olfactory organs of two pelagic teleost fish-opah (lampris guttatus) and dolphin fish (coryphaena hippurus). south pacific study, 22: 53-64. marsland t.a., glees p., erikson l.b. (1954). modification of the glees silver impregnation for paraffin sections. journal of neuropathology and experimental neurology, 13: 587-591. mazia d., brewer p.a., alfert m. (1953). the cytochemical staining and measurement of protein with mercuric bromphenol blue. biology bulletin, 104: 57. mowry r.w. (1956). alcian blue technique for the histochemical study of acidic carbohydrates. journal of histochemistry and cytochemistry, 4: 403. nath s. (1994). recent advances in fish ecology, limnology and eco-conservation. volume 3. daya publishing house. new delhi. 131 p. ojha p.p., kapoor a.s. (1972). histochemistry of the olfactory epithelium of the fish, channa punctatus bloch. acta anatomica, s3: 540-553. pastor l.m., graña l., frutos m.j., villaverde r., ramos d. (1991). lectin histochemistry of the olfactory surface in two teleostean fishes. acta histochemica, 90: 173-180. satio s., yamamoto y., mori m., amano m., yamanome t., taniguchi k., yamamori k., taniguchi k. (2004). variety in histochemical characteristics of the olfactory receptor cells in a flatfish, barfin flounder (verasper moseri). the journal of veterinary medical science, 11: 1409-1412. singh w., singh h.r. (1987). histochemical studies on the olfactory epithelium of some hillstream teleosts. journal of the indian fisheries association, 17: 25-30. talwar p.k., jhingran a.g. (1991). inland fishes of india and adjacent countries, vol. 2, oxford & ibh publishing co. pvt. ltd. new delhi-calcutta. 1158 p. int. j. aquat. biol. (2014) 2(3): 124-128 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article the acute effect of copper exposure on serum biochemical characteristics of common carp (cyprinus carpio l.) melika ghelichpour*1 department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 25 march 2014 accepted 28 april 2014 available online 2 5 june 2014 keywords: copper sulfate cyprinus carpio serum biochemistry stress toxicity abstract: effects of ambient copper was investigated on serum stress markers, sodium and enzyme levels in common carp (cyprinus carpio l.) over a 14-d exposure period. fish were exposed to 0, 25 and 100 μg l-1 copper (as copper sulfate) and blood was sampled at 0, 3, 7 and 14 d after exposure. serum profile was significantly affected by copper concentration, sampling time and their interaction. increase in serum levels of cortisol, glucose, alanine aminotransferase and aspartate aminotransferase and decrease in serum sodium levels were observed in both copper-exposed groups, 3 d after copper exposure, which lasted until the end of the experiment. it is concluded that copper exposure causes stress response and sodium loss in common carp. likewise alanine aminotransferase and aspartate aminotransferase increase after exposure which might be as results of either tissue damage or stress. introduction aquatic organisms are threatened by heavy metal pollution, since aquatic habitats are the final destination of effluents and wastes. fish may be exposed to waterborne metals including copper. copper resources in the water bodies are industrial waste or copper sulfate as a therapeutic or algicide agent. thus, fish may experience copper toxicity both in their natural habitat and rearing systems. copper sulfate is highly soluble in water producing cu2+ which is an important xenobiotic in aquatic ecosystems and a non-degradable and cumulative pollutant. the toxicity of cu2+ to teleosts has been extensively studied, as summarized by sorensen (1991). generally, copper attacks mainly the gill, an important organ in gas exchange and hydromineral balance in fish. therefore, waterborne copper exposure causes stress response in fishes (flik et al., 2002; fırat et al., 2011). common carp (cyprinus carpio l.) is an important pond-aquaculture species reared in iran. it is an * corresponding author: melika ghelichpour e-mail address: ml.ghelichpour@gmail.com important species of the caspian sea and plays an important role in producing food supply for people. this species may expose to ambient copper as result of copper sulfate, used as algicide or therapeutic. therefore, the water source of the common carp ponds might be contaminated by copper due to waste water introduction. also, this species might face copper toxicity in the caspian sea, as de mora et al. (2004) reported the occurrence of copper in the southern caspian coasts. the present study aimed to investigate the acute effect of copper (as copper sulfate) on serum characteristics of common carp (c. carpio l.) over a 14-day period. materials and methods fish maintenance and condition: two hundred and forty common carp (~ 100 g in weight) were randomly distributed in 12 fiberglass tanks, filled with 350 l decholorinated tap water. the fish were fed (2% of biomass) with an artificial diet (35.5% 125 ghelichpour/ effect of copper exposure on common carp protein, 10.1% lipid) twice a day. water of the tanks was exchanged 80%, daily. water quality was as following: temperature = 25-27˚c, dissolved oxygen > 5.5 mg l-1, ph = 7.03-7.1, total hardness = 210 mg l-1 (caco3), alkalinity = 200 mg l -1 (caco3), magnesium = 1 mg l-1, calcium = 84 mg l-1, iron = 0.01 mg l-1, potassium = 8.5 mg l-1, sulfate = 7.2 mg l-1 and free copper = undetectable. continuous aeration was supplied for all tanks. the fish remained under these conditions over a 2-week period. toxicity trail: after 2 weeks of acclimation period, the tanks were assigned as 3 treatments (4 tanks per treatment). one of the treatments served as control, which received freshwater (as acclimation period), while the two others received same water with 25 and 100 μg l-1 copper, as copper sulfate. feeding and the other conditions were similar to those of the acclimation period. the specimens remained under these conditions for 2 weeks. sampling and analyses: specimens from all treatments were blood-sampled before exposing to copper as well as at 3, 7 and 14 day after exposure. at each sampling point, two specimens were caught using a dip net from each tank (4 fish per treatment) and anesthetized with clove solution bath (3000 ppm) in less than 1 min. then blood samples were collected using caudal severance. all samples were kept at room temperature for 2 h prior to centrifugation (3000 rpm, 5 min). serum samples were stored at -80°c until further analyses. serum levels of cortisol were determined by elisa method using commercial kit (ibl, gesellschaft für immunchemieund immunbiologie, germany). serum glucose, alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were measured spectrophotometerically using commercial kits (pars azmun co. ltd, tehran, iran). sodium levels were determined using a flame photometer (seac, florence, italy). dissolved oxygen and ph were determined by portable multiparameter meter (sension 156, usa). water total hardness, alkalinity, magnesium, calcium, iron, potassium, sulfate and free copper were determined using a portable photometer with commercial kits provided by the manufacturer (wagtecch portable photometer 7100, berkshire, uk). statistical analyses: all data were tested for normality and homogeneity of variances using the shapiro-wilk's and the levene test, respectively. all data were examined using a two-way anova with copper concentration and sampling time as factors, and significant difference among the means was delineated by lsmeans' test. all analyses performed using the statistical software of sas version 9. data are presented as mean ± sd. the values of p<0.05 were considered significantly different. results the results showed that all tested parameters were significantly affected by copper concentration, sampling point and their interactions (table 1). there were no significant differences in cortisol, glucose, sodium, alt and ast levels over the time, in the control group (table 2). in addition, there were no significant differences among the treatments before exposing to copper (table 2). the levels of cortisol increased significantly at the days 3 and 7 and showed further increase at the day 14 compared to that of 0 d, in 25 μg l-1 treatment. in also, its levels in 100 μg l-1 treatment showed a significant increase at the day 3 and remained elevated until the day 14. pvalue cortisol glucose sodium alt ast dose < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 time < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 dose × time 0.014 < 0.0001 0.0014 < 0.0001 < 0.0001 two-way anova, n = 4. table 1. significance of copper concentration (dose), period of exposure (time) and their interaction on serum biochemical characteristics of common carp c. carpio 126 int. j. aquat. biol. (2014) 2(3): 124-128 the glucose increased over the time in both 25 and 100 μg l-1 treatments, however, the fish exposed to 100 μg l-1 copper showed higher glucose compared to that of 25 μg l-1 at the days 7 and 14. the levels of sodium decreased at the day 3 d in both copper-exposed groups and remained low until the day 14. alt and ast increased over the time in both copper-exposed treatments. discussion the present study showed that copper exposure alters serum characteristics of common carp. since, the experimental condition and sampling procedure was constant over the experiment, no significant change was observed in the measured parameters over the trial time, in the control group. in addition, there were no significant differences among the treatments at 0 d suggesting no tank effects before the experiment initiation. thus, any change in serum profile of the copper-exposed groups, assumed to be related to ambient copper. the levels of cortisol increased significantly in both copper-exposed treatments. cortisol is an important indicator of stress in fish (wendelaar bonga, 1997; barton, 2002). its elevation after stress and how long it remains elevated are the means for evaluating degree of stress. accordingly, both copper-exposed groups experienced stress after exposure and did not recover until the end of the experiment. fırat et al. (2011) showed increase in cortisol levels after copper exposure in nile tilapia, (oreochromis niloticus l.). the glucose also increased over the time in both groups with higher rate in 100 μg l-1 treatment. glucose is a secondary stress response which increased parallel to increase in cortisol with a certain delay (barton, 2002). glucose elevation supplies demanded energy to cope stress condition (wendelaar bonga, 1997; barton, 2002). stress caused by copper exposure found to be as a result of respiration stress (grosell et al., 2007) and oxidative stress (roméo et al., 2000). the levels of glucose found to remain elevated during the stress and even during recovery in common carp (ruane et al., 2002; hoseini, 2010). it is suggested that 100 μg l-1 treatmrnt experienced more severe stress than that of 25 μg l-1 which led to further glucose elevation at 7 and 14 d. previous studies (abdel-tawwab et al., cu exposure period (day) (μg l-1) 0 3 7 14 cortisol 0 38.7 ± 2.5 a † 41.3 ± 4.3 a † 40.3 ± 4.3 a † 43.3 ± 4.4 a † 25 37.5 ± 2.4 a † 47.8 ± 2.5 b † ‡ 50.5 ± 4.5 bc ‡ 55.8 ± 7.3 c ‡ 100 36.7 ± 4.3 a † 52.3 ± 2.5 b ‡ 52.5 ± 6 b ‡ 58.2 ± 6.2 b ‡ glucose 0 42.1 ± 2.2 a † 43.8 ± 3.9 a † 43.1 ± 4.1 a † 43.3 ± 3.9 a † 25 44.5 ± 3.2 a † 88.3 ± 6.2 b ‡ 85.8 ± 6.5 b ‡ 104 ± 4.4 c ‡ 100 43.2 ± 4.8 a † 92.2 ± 6.7 b ‡ 100 ± 3.6 c § 115 ± 4.5 d § sodium 0 140 ± 1.3 a † 140 ± 1.8 a † 140 ± 1.7 a † 139 ± 1.7 a † 25 140 ± 2.8 a † 132 ± 2.2 b ‡ 133 ± 2.2 b ‡ 132 ± 1.9 b ‡ 100 139 ± 1.8 a † 132 ± 1.3 b ‡ 131 ± 1.8 b ‡ 131 ± 2.5 b ‡ alt 0 13.8 ± 1.7 a † 15.5 ± 2.1 a † 13.5 ± 3.4 a † 14.3 ± 1.7 a † 25 14.8 ± 2.5 a † 28.8 ± 2.9 b ‡ 33.2 ± 2.2 c ‡ 40.5 ± 3.1 d ‡ 100 15.8 ± 2.2 a † 35.3 ± 2.6 b § 44.5 ± 2.1 c § 45.3 ± 2.9 c § ast 0 118 ± 6.2 a † 121 ± 2.8 a † 121 ± 2.6 a † 120 ± 4.3 a † 25 118 ± 1.7 a † 146 ± 5.1 b ‡ 175 ± 5.3 c ‡ 203 ± 5.4 d ‡ 100 120 ± 4.6 a † 150 ± 3.6 b ‡ 184 ± 4.1 c § 208 ± 5.4 d ‡ table 2. effect of copper concentration and period of exposure on common carp c. carpio serum levels (mean ± sd) of cortisol (ng ml-1), glucose (mg dl-1), sodium (meq l-1), alt (iu l-1) and ast (iu l-1). different lowercase letters show significant difference over the time in each copper concentration. different symbols show significant difference among the treatments at each sampling point 127 ghelichpour/ effect of copper exposure on common carp 2007; fırat et al., 2011) showed hyperglycemia after copper exposure. the levels of sodium decreased in both copperexposed groups and remained low until 14 d. grosell et al. (2007) found the main toxic effect of copper was due to loss of body sodium as a result of impact on gill function. hence, the constant sodium levels of both copper-exposed groups suggests that fish were partially capable to counteract the adverse effects of copper on sodium balance. mc donald and milligan (1997) suggested ion loss might be stressful for fish and caused increase in cortisol and glucose levels. this is, at least in part, the reason of higher levels of cortisol and glucose in the copper-exposed fish. similarly, previous studies (grosell et al., 2007; öner et al., 2008; fırat et al., 2011) showed loss of sodium after copper exposure. alt and ast increased over the time in both copper-exposed groups. alt and ast are cytosolic enzyme found in cell cytoplasm in high concentration. higher levels of these enzymes in circulation might be as a result of both leakage from cells (due to high concentrations) as well as cell damage which is caused enzyme release to peripheral fluids. copper is caused oxidative stress which in turn leads to lipid proxidation, mainly cell membrane lipids. damage to cell membrane fails its normal function and causes the cytosolic enzyme to escape from the cell into peripheral fluids and causes increase in enzyme activity levels in blood. on the other hand, these enzymes are involved in gluconeogenesis and increase during stress to supply amino acids for gluconeogenesis (tejpal et al., 2009). in the present study, the cortisol and glucose levels suggest that fish experienced stress until the end of the experiment, in both copper-exposed groups. thus, increase in enzymes' levels might be as a result of activation of gluconeogenesis. increase in alt and ast after copper exposure in this study is in agreement with previous studies (abdeltawwab et al., 2007; fırat et al., 2011). it is concluded that sublethal copper exposure caused stress response and sodium loss in common carp which lasted until 14 d. moreover, copper causes alt and ast elevation which might be as a result of tissue damage or stress. references abdel-tawwab m., mousa m.a.a., ahmad m.h., sakr s.f.m. (2007). the use of calcium pre-exposure as a protective agent against environmental copper toxicity for juvenile nile tilapia, oreochromis niloticus (l.). aquaculture, 264: 236-246. barton b.a. (2002). stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids. integrated comparative biology, 42: 517-525. de mora s., sheikholeslami m.r., wyse e., azemard s., cassi r. (2004). an assessment of metal contamination in coastal sediments of the caspian sea. marine pollution bulletin, 48: 61-77. fırat ö., cogun h., yüzereroğlu t., gök g., fırat ö., kargin f., kötemen y. (2011). a comparative study on the effects of a pesticide (cypermethrin) and two metals (copper, lead) to serum biochemistry of nile tilapia, oreochromis niloticus. fish physiology and biochemistry, 37: 657-666. flik g., stouthart x.j.h.x., spanings f.a.t., lock r.a.c., fenwick j.c., wendelaar bonga s.e. (2002). stress response to waterborne cu during early life stages of carp, cyprinus carpio. aquatic toxicology, 56: 167-176. grosell m., blanchard j., brix k.v., gerdes, r. (2007). physiology is pivotal for interactions between salinity and acute copper toxicity to fish and invertebrates. aquatic toxicology, 84: 162-172 hoseini s.m. (2010). efficacy of clove powder solution on stress mitigation in juvenile common carps, cyprinus carpio (linnaeus). comparative clinical pathology, 20: 359-362. mc donald, d.g., milligan, c.l. (1997). ionic, osmotic and acid-base regulation in stress. fish stress and health in aquaculture, (ed. iwama, g.k., pickering, a.d., sumpter, j.p and schreck, c.b.), pp 119-144. cambridge university press, cambridge, uk. öner m., atli g., canli m. (2008). changes in serum biochemical parameters of freshwater fish oreochromis niloticus following prolonged metal (ag, cd, cr, cu, zn) exposures. environmental toxicology and chemistry, 27: 360-366. roméo m., bennani n., gnassia-barelli m., lafaurie m., girard j.p. (2000). cadmium and copper display 128 int. j. aquat. biol. (2014) 2(3): 124-128 different responses towards oxidative stress in the kidney of the sea bass dicentrarchus labrax. aquatic toxicology, 48: 185-194. ruane n.m., huisman e.a., komen j. (2002). the influence of feeding history on the acute stress response of common carp (cyprinus carpio). aquaculture, 210: 245-257. sorensen e.m. (1991). cadmium. metal poisoning in fish. pp 175–234. crc press, boca raton, florida. tejpal c.s., pal a.k., sahu n.p., ashish kumar j., muthappa n.a., vidya s., rajan m.g. (2009). dietary supplementation of l-tryptophan mitigates crowding stress and augments the growth in cirrhinus mrigala fingerlings. aquaculture, 293: 272-277 wendelaar bonga s.e. (1997). the stress response in fish. physiological reviews, 77: 591-625. int. j. aquat. biol. (2015) 3(1): 35-41 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article age, growth and some biological characteristics of silver bream (blicca bjoerkna l., 1758) (cyprinidae) from aras dam lake in northwest of iran hadi jamali*1, soheil eagderi2, esmaeil esmaeilzadegan1, rahman patimar3 1department of fishery, urmia university, urmia, iran. 2department of fishery, faculty of natural resources, university of tehran, karaj, iran. 3department of fishery, faculty of natural resources, gonbad kavous university, gonbad kavous, iran. article history: received 1 march 2014 accepted 14 december 2014 available online 2 5 february 2015 keywords: blicca bjoerkna length-weight relationship condition factor aras dam lake abstract: we collected silver bream blicca bjoerkna from march to july 2013 from aras dam lake (north-west of iran) and investigated its age, size, and some biological characteristics. the maximum age was 5+ years. the total length and weight of specimens ranged 137-278 mm and 26-247 g for male and 134-282 mm and 26-289 g for female, respectively. length-weight relationship was estimated as w = 1e-06tl3.44 for females, w = 1e-06tl3.45 for males and w = 1e-06tl3.44 for the sexes combined. sex ratio was 1:1.42 in favor of males. the growth model was positive allometric for males, females and sexes combined. the calculated maximum condition factor was 0.34 in male and 0.37 in female. the most frequent age classes in the samples were 2+ years for males and females. introduction silver bream, blicca bjoerkna, is a cyprinid fish species and widespread in stagnant waters of lakes and reservoirs, calm rivers and canals throughout central and northern europe, from the east of england to the caspian sea basin (spillmann, 1961; wheeler, 1969; lelek, 1980; kottelat, 1997). this species has been reported from the southern caspian sea basin, including aras, atrak, gorgan, tajan, babol, haraz, sardab, tonekabon and safidrud rivers as well as the anzali and the boojagh wetlands (derzhavin, 1934; holčík and oláh, 1992; nejatsanatee, 1994; kiabi et al., 1999; abdoli, 2000; abdoli and naderi, 2009; khara et al., 2011). silver bream is strictly a phytophilous species (balon, 1975, 1984; philippart and vranken, 1983) reproducing from may to july (kottelat and freyhof, 2007). since, this species spawns at shallow depths, it is highly sensitive to changes in water level; in the bays of the kakhovka reservoir, spivak (1987) observed that this fish lays eggs along the shoreline * corresponding author: hadi jamali e-mail address: saeed.jamali11@gmail.com at depths to 40 cm on willow roots, floating residues of the previous year’s plants and stems of growing reed. males of this species become partially mature in the 2nd year and females in the 3rd-5th year. silver bream can live up to 16 years (pecl, 1990). despite being an abundant species, silver bream is of little importance as its meat is of poor quality. nevertheless, this species is an important component in the diet of predatory fishes (okgerman et al., 2012). in iran, studies on the biology and ecology of b. bjoerkna is rare, but out of iran several works on its feeding strategy and growth (specziár et al.,1997), growth and reproduction aspects (balık et al., 1999; hamalosmanoğlu, 2003), maturity and fecundity (gürsoy, 2001) and length-weight relationship (tarkan et al., 2006;yılmaz et al., 2012) have been published. although there is no economic value of silver bream in general, this species caught for consumption in aras dam lake due to the decrease of commercial 36 int. j. aquat. biol. (2015) 3(1): 35-41 fish as a result of overfishing. aras river is one of the largest rivers of the caspian sea basin. aras dam with max capacity of 1350 × 106 m3, max area of 153 km2, mean depth of 20 m and height of 36 m, was constructed in 1972 on this river. this study aimed to describe the age structure, growth pattern, condition factor (ka) and lengthweight relationship (lwr) of b. bjoerkna inhabiting aras dam lake (western-azerbaijan province, iran). these biological data of fishes are imperative for both fishery biology and taxonomic studies and give information on the condition and growth patterns of fish that are necessary for proper management and conservation of the wild population (oscoz et al., 2005). materials and methods a total of 392 specimens were caught using gillnets (20 m × 2 m) of various mesh sizes (16, 18, 20, 22, 24, 28, 32, 36, 40, 45, 50, and 55 mm, knot to knot) monthly during march to july 2013 (in the last week of each month) from aras dam lake (46°54'52.05''e, 39°09'42.11''n). in the field, all specimens were anaesthetized by 1% clove solution and then preserved in 10% formaldehyde solution. in the laboratory, all specimens were measured for total length (tl) and total body weight (tw) to the nearest 0.01 mm (using a digital caliper) and 0.01 g (using a digital balance), respectively. age was determined from both left and right opercula based on banding patterns, being reviewed three times (each time by a different person) using a 20-40x binocular microscope under reflected light. the relationship between tl and tw was determined using the equation: tw = atlb; where a is the intercept and b is the slope (coefficient of allometry) (pauly, 1984). sex was determined by visual examination of the gonadal tissue. condition factor (ka) was calculated using the equation ka = w/lb (ricker, 1975), where w is the body weight (bw) in g; l is the total length (tl) in cm; b is the parameters of the tl-bw relationship. according to froese (2006), the factor 100 is used to bring ka close to unity. analysis of co-variance (ancova) was performed to test for significant differences in weight-length relationships between sexes. any significant difference in the overall sex ratio was assessed using the chi-square test (zar, 1984). all statistical analyses were performed with a significance level of p<0.05 using the spss 21 software package. results the total length and weight of males range 137-278 mm and 26-247 g, respectively, while those of females range from 134-282 mm and 26-289 g, respectively. the majority of specimens were belonged to age group 2+, with 5+ being the oldest age recorded for both sexes. the observed length-atage group total length ± sd min–max total weight ± sd min–max n male 1+ 185.8 ± 26.17 154-226 79.6 ± 45.49 33-149 17 2+ 203.8 ± 34.6 137-262 112.7 ± 56.4 26-216 131 3+ 207.1 ± 33.1 138-278 114.2 ± 55.7 27-248 63 4+ 221.1 ± 13.5 200-247 131.7 ± 39.4 89-248 18 5+ 270 270 230.9 230.9 1 female 1+ 170.7 ± 22.2 146-206 58.7 ± 32.3 31-130 12 2+ 201.4 ± 38.4 134-266 112.2 ± 63.7 26-223 91 3+ 211.8 ± 34.2 143-276 126.1 ± 67.2 27-289 48 4+ 234.9 ± 21.87 209-282 157.9 ± 45.3 98-241 9 5+ 236.5 ± 13.4 227-246 161.7 ± 12.6 152-171 2 table 1. average total length (mm) and weight (g)-at-age of b. bjoerkna in aras dam lake 37 jamali et al./ some biological characteristics of b. bjoerkna age in the population was different between sexes, females being longer and heavier than males (table 1). length and weight frequency distribution of the fish (figs. 1 and 2)indicate that the most frequent size classes in the samples were 208-222.8 mm and 26-52.3 g for males and148.8-163.6 mm and 26-52.3 g for females, respectively. females were rare in length classes greater than 267.2-282 mm. the growth model was positive allometric for males, females and sexes combined because the b value was significantly different from 3 (pauly’s test, tmale = 9.5, tmale = 9.49, tsexes combined = 12.6, tpooled = 1.96, p>0.05). the overall ratio of females to males was 1: 1.42 and chi-square analysis indicated a significant difference from an expected ratio of 1: 1 (χ2 = 113.27, p<0.05). an unequal sex ratio was observed among length classes (fig. 1). the total length-weight relationships were evaluated for males, females and sexes combined. a significant relationship with the high regression coefficient (r>0.98) was found between the length and weight of the silver bream. the length-weight relationships were found as w = 1e-06tl3.45 for males, w = 1e06tl3.44 for females, and w = 1e-06tl3.44 for sexes combined (fig. 3). the calculated condition factor (ka) values are shown in figure 4. there was no significant difference in mean ka between sexes (t-test, p>0.05). the calculated minimum and maximum figure 1. total length (mm) frequency of male and female b. bjoerkna in aras dam lake. figure 2. total weight (g) frequency of male and female b. bjoerkna in aras dam lake. 38 int. j. aquat. biol. (2015) 3(1): 35-41 ka was 0.27 and 0.34 for 5+ and 2+ in male and 0.30 and 0.37 for 5+ and 2+ in female, respectively. age frequency distribution of the fish (fig. 5) indicated that the most frequent age classes in the samples were 2 year for males and females. discussion the total lengths and weights of examined silver bream specimens ranged 13.4-28.2 cm and 26-289 g, respectively. the standard lengths of this species ranged from 3.8 to 32.9 cm in lake balaton (specziár et al., 1997). balık et al. (1999) reported that distributions of the fork lengths and weights of kuş lake population of silver bream were 3.5-17.2 cm and 0.5-134.6 g, respectively. these ranges were determined as 9.6-49.2 cm and 11-269 g for the silver bream of the lake sapanca (hamalosmanoğlu, 2003). tarkan et al. (2006) studied with silver bream specimens were 12-21.2 cm of total lengths. differences of weight and length distributions can be attributed to sampling time and method, type of length measured, population density and ecological figure 3. relative growth curves (total length versus total weight) for males, females, and sexes combined of b. bjoerkna in aras dam lake. 39 jamali et al./ some biological characteristics of b. bjoerkna features of studied lakes (yılmaz et al., 2012). the growth model was positive allometric for males, females and sexes combined. specziár et al. (1997) reported that growth of silver bream population in balaton lake, hungary, was positive allometric with b value of 3.267. in turkey, b values of lwr for females, males and total individuals of b. bjoerkna inhabiting lake kuş were 3.24, 3.27 and 3.31, respectively (balık et al., 1999). these parameters were determined as 3.09 for females, 2.96 for males and 3.04 for entire population of b. bjoerkna in lake sapanca by hamalosmanoğlu (2003), and 3.18 for all specimens in the same lake by tarkan et al. (2006). our results were in accordance with the previous works. the variation in the b exponent which is interpreted as variation in fish condition or fitness could be attributed to the different environmental conditions that vary between rivers and between years in a river, and act as a local selective pressure on the fish. therefore, variation in the b exponent can be attributable to a species response to different habitat conditions (weatherley, 1972). the sex ratio (female: male) was 1:1.42 in favor of males. this ratio was estimated 1:0.35 in lake kuş population of b. bjoerkna (balık et al.,1999), 1:1.07 and 1:0.53 in sapanca lake (gürsoy, 2001; hamalosmanoğlu, 2003) and 1:0.98 in lake ladik (turkey) (yılmaz et al., 2012) populations of b. bjoerkna. nikolsky (1980) reported that sex ratio varied considerably from species to species; however, in the majority of species, it is close to one. however, subsequent changes in this ratio may be explained by a number of hypotheses, including differences in habitat preference according to the season or sex, sampling errors and selective mortality (fernandez and rossomanno, 1997). the maximum age of silver bream in this study was figure 4. condition factor of male and female b. bjoerkna in aras dam lake. figure 5. age (year) frequency of male and female b. bjoerkna in aras dam lake. 40 int. j. aquat. biol. (2015) 3(1): 35-41 5+ years that is less than that of reported by okgerman et al.(2012) in sapanca lake (9+ years), kirilyuk (1991) in kremenchug reservoir (11+ years) and sasi and berber (2012) in uluabat lake (7+ years). the calculated maximum ka was 0.34 in male and 0.37 in female for 2+ years of silver bream. there is no published information about the condition factor of silver bream to compare with the present results. ka reflects, through its variations, information on the physiological state of the fish in relation to its welfare. from nutritional and reproductive points of view, higher ka values show the accumulation of fat and gonadal development (lecren, 1951; angelescu et al., 1958). ka also gives information when comparing two populations living in certain feeding, density, climate, and other conditions in terms of the period of gonad maturation and degree of feeding activity of a species to verify whether it is making good use of its feeding source (weatherley, 1972). the length, weight and age characteristics of silver bream from the western iran basin indicate a short life span (5+ years in both sexes), and a moderate body weight (weight-length relationship: b>3). these findings provide important new data with respect to the biology of this species. it is suggested assessment of the potential impacts of habitat degradation on populations of this species. reference abdoli a. 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(2022) 10(2): 181-186 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article the removal efficiency of natural nano-coagulant produced from phragmites communis, schanginia aegyptiaca and portulaca oleracea in wastewater treatment maytham m. alabassi1, muthik a. guda1, manal a. muhammed2 1department of ecology, faculty of science, university of kufa, najaf, iraq. 2department of civil, faculty of engineering, university of wasit, wasit, iraq. s article history: received 1 february 2022 accepted 11 april 2022 available online 2 5 april 2022 keywords: normal coagulation turbidity cod toc abstract: sewage water contains suspended and dissolved solids, hydrocarbons, many types of organic matter and heavy metals. the reuse of wastewater faces the challenge of removing organic pollutant compounds before discharging them into any natural stream. therefore, in this study, natural nano-coagulant materials were used and their efficiency in removing turbidity, total organic carbon (toc), and chemical oxygen demand (cod) from wastewater were investigated. the natural nano-coagulant were synthesized using leaves of phragmites communis, schanginia aegyptiaca and portulaca oleracea. the results showed that 0.5 mg/l of p. oleracea nano-coagulant can remove 96.3% of cod, 86.2% of toc and 89.5% of turbidity. also, s. aegyptiaca leaves’ nano-coagulant in the 1 and 0.5 mg/l concentration was removed turbidity, cod and toc content of the wastewater by 93.1, 89.42, and 81.8%, respectively. moreover, p. communis leaves removed toc, cod, and turbidity up to 78.2, 83.48, and 86.18%, respectively. the results showed the efficiency of natural nano-coagulant materials in treating and depositing wastewater pollutants. introduction water contamination is growing at an alarming rate, and the most important cause of pollution is the discharge of wastes resulting from human activities. most of these wastes are rich in nutrients, causing eutrophication (guda et al., 2019). although maintainable water administration techniques are recognized worldwide, water resources face a serious threat from receiving a wide range of pollutants. this required more attention to wastewater treatment and ensuring that this is suitable to meet local conditions for pollution sources and wastewater treatment frameworks. the evacuation of major poisons such as suspended solids, biological oxygen demand (bod), supplements (organic and inorganic) and coliform microbes are the main objectives of wastewater filtration (fakhrul-razi et al., 2009). colloidal materials can be eliminated from treated water using numerous methods, such as coagulation, flocculation, adsorption, particle trade, buoyancy, film, correspondence: muthik a. guda e-mail: meethakha.almithhachi@uokufa.edu.iq sedimentation, dissolvable extraction, natural filtration, and electrolysis strategies (guda et al., 2018). coagulation is among the accessible water treatment strategies and is straightforward, dependable, and low-cost (oladoja, 2015). it is an effective way to treat water pollutants and has been widely used to treat different types of wastewater such as water from mills, sanitary waste from hospitals, and domestic wastewater (tatsi et al., 2003; yue et al., 2008) since it does not require complex machines, many manpower, and no need for energy consumption. this method can remove colloidal, soluble and suspended pollutants and other pollutants such as natural compounds, color, micro-pollutants, fats, and oils through the collection of fine particles until large particles of size are formed that can be deposited (oladoja, 2015). in the nano-coagulation method, small particles are formed into large aggregates (lumps) and the brokenup natural matter will be retained. this is often taken after expelling bigger particles by simple filtration or 182 guda et al./ the removal efficiency of natural nano-coagulant in wastewater treatment sedimentation, reducing natural matter and turbidity (yin, 2010). there is a particular definition for coagulation and flocculation, but it is worthy to refer to both forms as coagulation in wastewater treatment (jiang, 2007). coagulation incorporates the taking after several stages, including thrombus formation, particle instability and particle aggregation (jiang and graham, 1998). natural coagulants collect particles and masses by adsorption on their surfaces. four coagulation instruments occur within the molecule to prepare twofold layer compression, viz. clear flocculation, adsorption, charge neutralization, and adsorption and molecule fascination (yin, 2010). aluminum and iron salts are the most common nanomaterial used to treat wastewater (guda et al., 2017). however, they cause negative effects on human health, such as losing memory when aluminum is used as a coagulant in treated water treatment. the current trend is to use environmentally friendly coagulated nanomaterial (muthik et al., 2019; hussein et al., 2019). plants, animals, and minerals are natural sources for producing nanomaterial and the common natural coagulants are made from plants due to their suitability for mass production and applicability, and being non-toxic, renewable, producing less degradable sludge, relatively cost-effective and do not change the ph value of the treated water (fakhrulrazi et al., 2009; muthik et al., 2018; al-bayati, 2019; kumar et al., 2019). therefore, the present study aims to produce nanomaterial from the leaves of phragmites communis, schanginia aegyptiaca, and portulaca oleracea as natural nano-coagulants in wastewater treatment. materials and methods treatment of plant: leaves of p. communis, s. aegyptiaca and p. oleracea were collected and washed with deionized water several times to remove the impurities. 100 g of dried leaves were taken and put in an oven at 60°c until their weight stability, then placed in an airtight carton box, away from moisture. the dried leaves were ground into a fine powder and sieved using a 600 μm sieve (yue et al., 2008). 25 ml of 70% ethanol were added to a separating funnel, and after stirring for two hours, the sediment was left, and the supernatant was taken and dried. purified water was included in the powder to get a suspension of 1%, and shaken for 45 min to extract the coagulation proteins and then passed through the whatman filter paper. this was used as a coagulant stock for the experiment. the stock should be prepared daily and kept in the fridge to eliminate any impact, such as changing the thickness, coagulation movement, and ph. it must be shaken well before use. the nuclear constrain magnifying (afm) microscope was used to examine the structural arrangement of the nanomaterials, normal distance, and disseminations. the afm features images of the prepared nanomaterials in 2d. collecting water samples: samples of the untreated water were collected from the wastewater in barakia, najaf governorate, iraq, on 15 october 2020, as three replications from the main sedimentation tank using clean containers. physical and chemical parameters of samples: the ph and ec were measured using a multi photometer and turbidity by a turbo meter. all the devices were laboratory calibrated before starting the measurements. total dissolved solids (tds), total organic carbon (toc), biochemical oxygen demand (bod5) and chemical oxygen demand (cod) were measured based on wong et al. (2006). jar test: the jar test was performed to study the potency of nano-coagulants. the treated water characteristics in the experiments are shown in table root mean square (nm) roughness average (nm) particles size distributions (nm) 50% diameter (nm) average diameter (nm) nanoparticle 1.09 0.74 40-180 80 112.7 p. oleracea 0.68 0.38 65-135 85 98.7 s. aegyptiaca 1.04 1.17 70-155 60 76 p. communis table 1. characteristics of natural nano-coagulants by (afm) microscope. 183 int. j. aquat. biol. (2022) 10(2): 181-186 1. natural nano-coagulants were used at room temperature and the sample were mixed well before use. one liter of water was taken, the experiment substance was added, mixed at 150 rpm for 1 min, then mixed slowly at 50 rpm for 20 min, and finally leaves the sample to settle for 15 min. coagulants were added at different concentrations of 0.5-3 ppm to determine the best dose based on the minimum contaminant concentration. experiments were performed in the ph range of 3-10 to determine the best ph value. statistical analysis: data were analyzed using anova in spss (ver.17) software. lsd was used to calculate the significance of the means. results and discussions diameter and size are shown in the two-dimensional images of the prepared natural nano-coagulants nanomaterials (table 1, figs. 1, 2, 3). determination of optimum doses of coagulants: the required optimal dose for different natural nanocoagulants to reduce turbidity is shown in figure 4. the increasing natural nano-coagulants concentration increases the efficiency until it reaches an optimum point. the optimum concentration for p. oleracea was 0.5 mg/l, for s. aegyptiaca 1 mg/l, and 1.5 mg/l for p. communis. the highest removal rate was recorded in p. oleracea at 89.5%, s. aegyptiaca at 89.42%, and p. communis at 86.18%. the optimum dose for toc removal: the optimum dose for p. oleracea, s. aegyptiaca and p. communis was 1 mg/l. based on the results, exceeding the optimum dose leads to destabilization, which weakens the attraction between the organic materials. this causes a decrease in the stability velocity of the particles according to the stock law, causing a decrease in the removal efficiency (jiang, 2007). the highest removal rate was in p. oleracea with a toc removal rate of 86.2 %, then s. aegyptiaca (81.8%), and p. communis (78.2%) (fig. 5). the optimum dose for the removal of cod: the optimum dose for p. oleracea and s. aegyptiaca was 0.5 mg/l, and for p. communis was 1.5 mg/l. the use of natural coagulant nanomaterials increases sludge formation, and by exceeding the optimal limits, colloids were not stabilized in the treated water and thus increased (jiang and graham, 1998). the highest removal rate was found in p. oleracea with a rate of figure 1. 2d image of p. oleracea as natural nano-coagulants by (afm) microscope. figure 2. 2d image of s. aegyptiaca as natural nano-coagulants. figure 3. 2d image of p. communis as natural nano-coagulants. 184 guda et al./ the removal efficiency of natural nano-coagulant in wastewater treatment 96.3 %, then s. aegyptiaca (93.1%) and p. communis (83.48%) (fig. 6). comparing the optimal results of nano-coagulation dose of the three experimental plants showed that p. oleracea had the best removal rate for turbidity, toc, and cod. schanginia aegyptiaca had almost similar results with p. oleracea in removal percentage (fig. 7). the use of leaves of three examined plants in this work can be efficient in manufacturing nanocoagulate materials that can be applied to treat wastewater to remove turbidity, toc and cod. the highest removal percentage of toc, cod and turbidity were recorded 86.2, 96.3, and 89.5%, respectively, when using p. oleracea. parameters range cod (mg/l) 1457-28609 bod5 (mg/l) 260-308 tds (mg/l) 99800 toc (mg/l) 60-9000 turbidity (ntu) 441 ph 7-7.3 electrical conductivity (ec) (ds/m) 6.55 table 2. initial characteristics of the treated water that was used in the experiment. 86 88 90 92 94 96 98 100 0.5 1 1.5 2 2.5 3 tu rb id it y r e m o v a l (n t u ) dose (mg/l) p. oleracea s. aegyptiaca p. communis figure 4. removal of turbidity from treated water by the influence of natural nano-coagulants particles. 30 40 50 60 70 80 90 100 0.5 1 1.5 2 2.5 3 t o c r e m o v a l (m g /l ) dose (mg/l) p. oleracea s. aegyptiaca p. communis figure 5. the removal of toc from treated water by the influence of natural nano-coagulants particles. 185 int. j. aquat. biol. (2022) 10(2): 181-186 acknowledgments this work was supported by the department of environmental science, faculty of science, kufa university. references ahmad a.l., ismail s., bhatia s. (2005). optimization of coagulation–flocculation process for palm oil mill effluent using response surface methodology. environmental science and technology, 39(8): 28282834. al-bayati h.j.m. (2019). growth and yield of cauliflower as affected by boron and fertilizer type. international journal of agricultural and statistical sciences, 15(2): 595-599. fakhrul-razi a., pendashteh a., abdullah l.c., biak d.r.a., madaeni s.s., abidin z.z. (2009). review of technologies for oil and gas produced water treatment. journal of hazardous materials, 170(2-3): 530-551. guda m.a., nasir a.s., younus a.s., altamimi a.j. (2018). antioxidant enzyme responses of juncus aschers (etbuch.) adoms to some of environmental stresses and use it as indicators. indian journal of public health research and development, 9(12): 1-9. guda m.a., hakeem g.l., alabassi m.m., hamad m.o., naji h.a. (2019). investigation use irradiation to treat west water and their effects on plants. journal of alhussein bin talal university for research, 5(5): 35673578. guda m.a., hakeem j.i., alabassi m.m., almayahi b.a. (2017). effects of environmental stress on nutrients of typha domingensis pers. plant in najaf, iraq. annual research and review in biology, 1-6. hussein a.m., alshukri a.y., mohammed a.e. (2019). 75 77 79 81 83 85 87 89 91 93 0.5 1 1.5 2 2.5 3 c o d r e m o v a l (m g /l ) dose (mg/l) p. oleracea s. aegyptiaca p. communis figure 6. removal of cod from treated water by the influence of natural nano-coagulants particles. 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00% 100.00% turbidity toc cod r e m o v a l ra te % p. oleracea s. aegyptiaca p. communis figure 6. comparison between plants used in syntheses natural nano-coagulants under optimal conditions 186 guda et al./ the removal efficiency of natural nano-coagulant in wastewater treatment susceptibility of pleurotus ostreatusto growth on wheat, water hyacinth, barley straw and biodegrade its residues. international journal of agricultural and statistical sciences, 15(2): 585-589. jiang j.g. (2007). development of coagulation theory and prepolymerized coagulants for water treatment. separation and purification methods, 30(1): 127-141. jiang j.q., graham n.j.d. (1998). pre-polymerised inorganic coagulants and phosphorus removal by coagulation-a review. water sa, 24 (3): 237-244. kumar a., dutt b.d., kumar v. (2019). groundwater quality assessment in river hindon catchment of saharanpur, uttar pradesh, india. international journal of agricultural and statistical sciences, 15(1): 415-421. oladoja n.a. (2015). headway on natural polymeric coagulants in water and wastewater treatment operations. journal water process engineering, 6: 174192. tatsi a.a., zouboulis a.i., matis k.a., samaras p. (2003). coagulation–flocculation pretreatment of sanitary landfill leachates. chemosphere, 53(7): 737-744. wong s.s., teng t.t., ahmad a.l., zuhairi a., najafpour g. (2006). treatment of pulp and paper mill wastewater by polyacrylamide (pam) in polymer induced flocculation. journal of hazardous materials, 135: 378388. yin c.y. (2010). emerging usage of plant-based coagulants for water and wastewater treatment. process biochemistry, 45(9): 1437-1444. yue q.y., gao b.y., wang y., zhang h., sun x., wang s.g., gu r.r. (2008). synthesis of polyamine flocculants and their potential use in treating dye wastewater. journal of hazardous materials, 152: 221227. banoon s.r., ghasemian a. (2021). the characters of graphene oxide nanoparticles and doxorubicin against hct-116 colorectal cancer cells in vitro. journal of gastrointestinal cancer, 1-5. int. j. aquat. biol. (2014) 2(4): 188-192 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article does physical production of nanoparticles reduce their ecotoxicity? a case of lower toxicity of agnps produced by laser ablation to zebrafish (danio rerio) seyed ali johari1*,1 iman sourinejad2, niko bärsch3, somayye saed moocheshi4, andishe kaseb5, nina nazdar6 1fisheries department, natural resources faculty, university of kurdistan, sanandaj, kurdistan, iran. 2fisheries department, faculty of oceanic and atmospheric sciences and technologies, university of hormozgan, bandar abbas, iran. 3ceo, particular gmbh, langenhagen, germany. 4gorgan university of agricultural sciences and natural resources, gorgan, iran. 5marine sciences faculty, tarbiat modares university, noor, iran. 6natural resources faculty, urmia university, urmia, iran. article history: received 2 july 2014 accepted 14 july 2014 available online 2 5 august 2014 keywords: aquatic nanotoxicology silver nanoparticles bottom-up method top-down method. abstract: use of nano-materials has increased in various aspects of human life. however, possible outbreak of nano-materials toxicity in humans and other organisms is one of the future challenges. different chemical precursors which are used in chemical approaches for production of nano-materials may have secondary and sometimes toxic effects in living organisms. these secondary effects may be reduced in physical approaches due to not use of chemicals. to test this hypothesis, acute toxic effects of two types of silver nanoparticles (agnps) which were produced by physical (top-down) and chemical (bottom-up) methods on survival rate of zebrafish (danio rerio) were compared. according to the results, agnps produced by physical method were 38 times less toxic than ones generated by chemical method and therefore, the hypothesis was approved. the estimated 96 hr lc50 values of agnps produced by physical and chemical methods for zebrafish were 0.540 ± 0.032 and 0.014 ± 0.001 mg/l, respectively. according to these values and regarding the rules of european union, both types of agnps are considered as highly toxic chemicals to aquatic organisms. generally, agnps seems to have toxic effects on aquatic organisms regardless of the method used for their production, and so, their accidental or intentional entrance into the aquatic ecosystems should be inhibited. introduction nanotechnology is a highly promising technology that has extensive applications in many areas of science and industry. a nano-material is a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate and where, 50% or more of the particles in the number size distribution and one or more external dimensions is in the size range of 1-100 nm (european commission, 2011). in specific cases and where warranted by concerns for the environment, health, safety or competitiveness, the number size distribution threshold of 50% may be replaced by a threshold between 1 and 50%. * corresponding author: seyed ali johari e-mail address: a.johari@uok.ac.ir the use of nano-materials is growing and number of nano-products has increased from 54 in 2005 to 1628 in 2013 (woodrow wilson database, 2014). the silver, titanium, carbon, silicon, zinc and gold are respectively the more frequent nano-materials used in consumer products (woodrow wilson database, 2014). with rapid development of nanotechnology and the extension of nano-material applications in human life, concerns are rising on probable threats caused by releasing them to environment. as aquatic ecosystems are one of the final destinations of the man-made chemicals, the study of these materials effects on the aquatic organisms (aquatic toxicology) is of great importance (hosseini et al., 2013; 189 int. j. aquat. biol. (2014) 2(4): 188-192 mansouri chorehi et al., 2013; roy et al., 2013; banaee et al., 2014; ghelichpour, 2014). in this respect, "aquatic nanotoxicology" can be considered as a suitable indicator for prediction of possible impacts of nano-materials release into aquatic ecosystems. hence, this study was conducted to evaluate the toxicity of silver nanoparticles (agnps) on adult zebrafish (danio rerio). this species is a valuable model for investigating the impacts of chemical contaminants (hill et al., 2005) and suitable vertebrate for nanotoxicology studies and the prediction of safety and hazard level of nanomaterials on human is much simpler and less expensive compared with rodents (kim and tanguay, 2013). nano-materials can be manufactured by bottom-up and top-down approaches. in bottom-up approaches, molecules are joined together during special processes to generate larger structures with nano dimensions, whereas in top-down approaches, large dimension materials are changed into nano dimension structures by physical methods (rodgers, 2006). in top-down approach, a large volume of silver metal is first ablated by mechanical method and then the manufactured agnps are fixed by adding colloids protectants (gaffet et al., 1996; amulyavichus et al., 1998). bottom-up approaches include chemical reduction of silver ions, electrochemical methods and sonochemical processes (prabhu and poulose, 2012). different chemical precursors which are used in bottom-up approaches for reducing silver ion to agnps, may cause secondary effects and sometimes have toxic impacts on organisms. in contrast, no use of chemicals in top-down approaches may reduce these secondary effects. therefore, to test this hypothesis in this study, acute toxicity effects of two types of agnps produced by physical and chemical methods were investigated and compared based on the survival rate of zebrafish. materials and methods nanoparticles and characterization: two types of colloidal silver nanoparticles (agnps) were used in the present study. the first type of colloidal agnps produced by physical method (top-down approach), manufactured by particular gmbh (germany) through laser ablation of silver metal in water (bärsch et al., 2009). according to the information provided by the manufacturer, this product is a ligand-free colloidal agnps with concentration of 500 mg/l in pure distilled water. for enhancing the resistance and preventing the nanoparticles from settling, polyvinylpyrrolidone (pvp) has been added as 0.01% wt. the second type of colloidal agnps produced by chemical method (bottom-up approach), manufactured by nano nasb pars company (tehran, iran) through photochemical reduction of silver nitrate solution in presence of hydrazine solution and linear alkyl benzene sulfonate (rahman nia, 2009). according to the information provided by the manufacturer, this product is a citrate capped colloidal agnps with concentration of 4000 mg/l. the detailed specifications of this colloid have been analyzed and reported previously (asghari et al., 2012; johari et al., 2013). also, prior to using the above products in the present study, dynamic light scattering (dls), was performed using a zetasizer (malvern instruments, nano zs), to determine the hydrodynamic diameter of particles in colloids. experimental animals and conditions: a population of 1000 zebrafish with average weight of 1580 ± 282 mg was used in this research. for adaptation to experimental conditions, fish were kept for one week in a 1000 l tank with aeration and under 12l: 12d photoperiod. during this period, mean water temperature was 23 ± 1°c and fish were daily fed to 1% of body weight with aquarium fish food (biomar, germany). the water used during adaptation and toxicology experiments was dechlorinated tap water. the properties of water including ammonia, chloride, calcium and ph were 0.00 mg/l, 0.00 mg/l, 100 mg/l and 8.5, respectively. also during the experiments, mean 190 johari et al./ physical production reduces toxicity of agnps to zebrafis water temperature was 24 ± 1°c and dissolved oxygen was always more than 8 mg/l. ecotoxicity tests: toxicology experiments were conducted according to organisation for economic co-operation and development (oecd) guidelines for the testing of chemicals (oecd, 1992) in aquariums containing ten liters of aerated water. according to the oecd guidelines, feeding of fish was stopped 24 hours before and during the experiments. first, several range finding tests were conducted to determine the range of lethal concentration of each chemical. for this purpose, 200 zebrafish were exposed to concentrations of 5, 3, 1, 0.5, 0.3 0.1, 0.05, 0.03, 0.01, and 0 (control) mg/l of each type of the agnps for 96 hours. each concentration was in two replicates and five fish were exposed to chemicals in each replicate. the mortalities were recorded at 24, 48, 72 and 96 hours post-exposure. based on the data acquired from the preliminary experiments, lethal range of chemically produced agnps was 0.01-0.03 mg/l, while this range was 0.50-1.00 mg/l for agnps produced by physical method. for conducting the main experiments, a number of 600 fish were exposed to ten concentrations of agnps produced by physical method (including 0.98, 0.90, 0.82, 0.74, 0.66, 0.60, 0.58, 0.56, 0.54 and 0.52 mg/l) and nine concentrations of chemically produced agnps (including 0.030, 0.028, 0.025, 0.023, 0.020, 0.018, 0.015, 0.013 and 0.010 mg/l) along with control groups (aerated water without nanoparticles) for 96 hours with three replicates (10 fish per replicate). the mortalities were recorded at 24, 48, 72 and 96 hours post-exposure. the results obtained from the mortalities of fish during the main experiments were assessed using the epa probit analysis program (version 1.5) to calculate the lethal concentrations (lcs). results based on the results of dynamic light scattering, the hydrodynamic diameter of the physically produced agnps ranged from 5-20 nm in colloid, where particle sizes of 9.244 nm were dominant and polydispersity index (pdi) of these particles was 0.396. in the same way, the hydrodynamic diameter of the chemically produced agnps in colloid was in the range of 5 to 220 nm, where particle sizes of 63.45 nm were dominant and pdi of these particles was 0.544 (pdi <20, showing that particles are monodisperse, and their diameter distribution is uniform in the colloid). the number of mortalities in the control groups was always less than 5%. in the case of fish exposed to higher concentrations of both types of agnps, increase of gill mucus secretion and unbalanced swimming were observable that were finally resulted in the death of fish. based on the results, no observed effect concentration (noec) and lowest observed effect figure 1. magnitudes of 48 hours lc50, matc, loec, and noec of silver nanoparticles (agnps) produced by top-down approach (laser ablation method) compared to bottom-up approach (chemical reduction) in zebrafish. 191 int. j. aquat. biol. (2014) 2(4): 188-192 concentration (loec) of agnps produced by chemical method were 0.01 and 0.0125 mg/l, respectively. furthermore, according to the probit analysis, maximum acceptable toxicant concentration (matc) of this type of agnps was calculated 0.011 ± 0.0015 mg/l. median lethal concentration (lc50) of chemically produced agnps colloids was estimated 0.014 ± 0.001 mg/l during 96 hours of exposure (fig. 1). in the case of physically produced agnps, the noec, loec and matc were 0.50, 0.52, 0.427 ± 0.0597, respectively. lc50 of physically produced agnps colloids in zebrafish was estimated 0.540 ± 0.032 mg/l during 96 hours of exposure (fig.1). discussion the aim of this research was to test this hypothesis that chemically produced agnps are more toxic than agnps produced by physical method due to different chemical precursors which are used in bottom-up approaches for reducing silver ions to agnps, and may cause secondary effects and sometimes have toxic impacts on organisms. based on the results, no observed effect concentration, lowest observed effect concentration, maximum acceptable toxicant concentration and median lethal concentration of agnps produced by chemical method were respectively 50.00, 41.60, 38.81 and 38.57 times less than ones produced by physical method, therefore our hypothesis is accepted. in the acute aquatic toxicity tests, the median lethal concentration is usually the most important end point. with regard to the results, 96 h lc50 of physically and chemically produced agnps colloids in zebrafish were estimated to be 0.540 ± 0.032 and 0.014 ± 0.001, respectively. as both of these values are less than 1 mg/l, and based on the rules of the european union (european commission, 2008) and recommendation no. 67/548/eec of this union (european commission, 1999), both types of agnps colloids are classified as "highly toxic chemicals" to this fish species and therefore if these nano-materials were released into the aquatic ecosystems, they cause unfavorable effects on aquatic organisms. similar results on the acute toxicity of agnps in oncorhynchus mykiss (johari et al., 2013) and daphnia magna (asghari et al., 2012) have been reported. also sublethal toxicity of agnps has been shown by other works (sharifian et al., 2013). based on the findings of this research and also the results of other published studies, it seems that agnps regardless of the method used for their production (chemical or physical), have toxic effects on aquatic organisms, therefore more attention should be paid to preventing from their accidental or intentional entrance into aquatic ecosystems. references amulyavichus a., daugvila a., davidonis r., sipavichus c. (1998). study of chemical composition of nanostructural materials prepared by laser cutting of metals. the physics of metals and metallography, 85: 111-117. asghari s., johari s.a., lee j.h., kim y.s., jeon y.b., choi h.j., moon m.c., yu i.j. (2012). toxicity of various silver nanoparticles compared to silver ions in daphnia magna. journal of nanobiotechnology, 10:14. doi: 10.1186/1477-3155-10-14. banaee m., sureda a., zohiery f., nematdoust hagi b., garanzini d.s. (2014). alterations in biochemical parameters of the freshwater fish, alburnus mossulensis, exposed to sub-lethal concentrations of fenpropathrin. international journal of aquatic biology, 2(2): 53-57. bärsch n., jakobi j., weiler s., barcikowski s. (2009). pure colloidal metal and ceramic nanoparticles from high-power picosecond laser ablation in water and acetone. nanotechnology, 20: 44. doi:10.1088/09574484/20/44/445603. european commission. 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(2021) 9(3): 134-147 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article ameliorative and protective effects of prebiotic, microbial levan in common carp, (cyprinus carpio) fry under experimental exposure to fipronil sanjay kumar gupta 1 icar-indian institute of agricultural biotechnology, namkum ranchi, jharkhand-834010, india. s article history: received 1 february 2021 accepted 1 april 2021 available online 2 5 april 2021 keywords: microbial levan fipronil lipoprotein lpo hsp abstract: this study investigated the immuno-toxicological effect of the insecticide fipronil at sublethal concentration (10% of lc50) and the potential ameliorative effects of dietary microbial levan in cyprinus carpio fry. fish were randomly distributed into five treatments in triplicate for 60 days. five different treatment groups were: levan control l0f0 (basal feed + 0% levan without exposure to fipronil), pesticide control l0f (basal feed + 0% levan with exposure to fipronil), other three dietary supplemented groups exposed to fipronil with different inclusion levels of levan at 0.25% (l0.25f), 0.50% (l0.50f) and 0.75% (l0.75f), respectively. the results revealed that feeding common carp with 0.75% dietary levan significantly reduced (p<0.05) glutathione-s-transferase and glutathione peroxidase levels in various tissues. lipid peroxidation and heat shock protein level was significantly (p<0.05) reduced with supplementation of levan at 0.75% compared to other groups. higher glycogen content was observed in high levan fed groups. although fipronil exposure had no signficiant effect on lipid profile levels, dietary levan supplementation decreased lipid profile level in the fish exposed to fipronil stress. total immunoglobulin and myeloperoxidase content of common carp showed an increasing trend with the concomitant increase in the level of levan administration in the diet. overall, results revlealed that microbial levan at 0.75% in the fipronil induced c. carpio fry mitigated the stress due to its potent nutraceutical properties, thus presenting a promising immunoadditive for aquaculture. introduction insecticides employed in the agricultural practices for the primary aim of eradicating harmful insect’s pests and enhancing food production, eventually reach to freshwater bodies through run off (ghelichpour et al., 2020). these insecticides from agricultural field’s overflow pollute the aquatic environment resulting into serious distress to certain physiological and biochemical processes in non-target species even at sub-lethal concentrations (prusty et al., 2011; kumar et al., 2016). fipronil, a broad-spectrum, phenylpyrazole insecticides, and its application particularly in the asian countries is gaining momentum, due to emerging outlaw on the use of organochlorines and organophosphates as agricultural insecticides. agricultural runoff or drift from aerial or ground based spraying applications of fipronil pose threat to non-target cultured fishes (fenet et al., 2001; schlenk *correspondence: sanjay kumar gupta doi: https://doi.org/10.22034/ijab.v9i2.866 e-mail: sanfish111@gmail.com et al., 2001; walse et al., 2004). once entranced to fish body, marked alterations in many of the key physiobiochemical and haeamatoimmunological processes (prusty et al., 2011; akhtar et al., 2012; gupta et al., 2014a; kumar et al., 2016) are inevitable. in this context, gupta et al. (2014b) indicated perturbations of heamato-biochemical responses of c. carpio fry as a result of short term exposure of sublethal concentration of fipronil. further, long term sublethal exposure of fipronil resulted into immunesuppression, delayed adverse health effects, and death of cultivated fishes thus posing heavy economical losses to the fish farmers (gupta et al., 2013). hence, there is an utmost need to develop eco-friendly and sustainable methods to control the fish mortality mediated through the sublethal fipronil concentration. application of herbal products such as extract, phytochemical and prebiotics through dietary 135 int. j. aquat. biol. (2021) 9(2): 134-147 interventions in aqua-feed seems to be very promising and viable approach to combat pollutant stress (taheri mirghaed et al., 2019; rajabiesterabadi et al., 2020a, b; fazelan et al., 2020; ghelichpour et al., 2021). prebiotic is nothing but a form of health supplements which selectively stimulate the growth, and/or activity of intestinal microflora associated with health and wellbeing (gibson and roberfroid, 1995). application of fructans as prebiotic has gained attention in aqua feed industry as these are intended to manufacture a mixture of functional prebiotics (ávilafernández et al., 2011). levan is a unique polymer of carbohydrate containing fructose as monomer in its structure of backbone. apart from the various useful properties such as bio-compatibility, biodegradability, renewability, flexibility, and eco friendliness, levan also offers some multi-functional biomedical properties such as anti-oxidant, antiinflammatory, anti-carcinogenic, anti-aids, immunestimulating, growth augmenting, hyperglycaemic inhibitor etc. (gupta et al., 2010; dahech et al., 2011; huang et al., 2015). levan is distinctly distributed in plants, yeasts, fungi and bacteria (jang et al., 2003) and its biosynthesis is usually carried out by an enzyme levansucrase. several bacterial strains, including zymomonas mobilis, bacillus subtilis, b. polymyxa and acetobacter xylinum produce extracellular levan of high molecular weight which is serologically active (clarke et al., 1997). augmentation of haemato-immunological and physio-biochemical responses and protection against bacterial challenge have been reported in c. carpio (rairakhwada et al., 2007; gupta et al., 2014a), l. rohita (gupta et al., 2008, 2015) and epinephelus coioides (huang et al., 2015) owing to dietary microbial levan supplementation. of late, present authors reported stress amelioration efficacy of levan by modulation of cytokine expression in the pathogen aggravated rohu, l. rohita (gupta et al., 2018, 2020). despite the potential health benefits of prebiotics in fish, very limited information is available about the possible ameliorative effects of prebiotics when fish exposed to pesticides. thus, the present study was conducted to elucidate possible ameliorating efficacy of dietary microbial levan on the immuno-biochemical responses of cyprinus carpio fry as a result of fipronil exposure. materials and methods experimental animals: cyprinus carpio (average weight of 3.31±0.52 g) were procured from palghar fish farm, maharashtra, india, transported, stocked in cement tank (1000 l capacity) and left unobstructed for the whole night. on following day, fish were given a mild salt treatment (5%) to ameliorate the handling stress, if any. the stock was acclimatized under aerated condition for 15 days. during acclimation, fish were fed to satiation with control diet having 30% crude protein (cp). chemicals: fipronil (c12h4cl2f6n4os) (99.1% pure, technical grade) was procured from malti enterprises, mumbai, india and was kept in an airtight container in the refrigerated condition. a stock solution of fipronil was prepared using analytical grade acetone (solubility of fipronil in acetone is 545.9 g1-1 at water ph 9.0). required quantity of fipronil was drawn from this stock solution for later experimental use. the sublethal fipronil dose of 0.0428 mg l-1 (1/10th lc50 for 96 h) was calculated as described by gupta et al. (2013) and selected for this study. experimental site, design and feeding: the experimental setup was maintained in the wet laboratory of the aquaculture division of central institute of fisheries education (cife) mumbai, and the laboratory analysis was carried out at the fish nutrition, biochemistry and physiology division. following a completely randomized design, two hundred and twenty-five fry of c. carpio were distributed in five treatment groups in triplicates. the experimental system comprised of 15 uniform size circular fiber reinforced plastic tanks (150 l). four experimental diets were prepared (table 1) for feeding the fishes till 60 days and divided into five different treatment groups as: levan control l0f0 (basal feed+ 0% levan without exposure to fipronil); pesticide control l0f (basal feed+0% levan with exposure to fipronil); l0.25f (basal feed+0.25% levan with exposure to fipronil); l0.50f (basal feed+0.50% levan 136 gupta / protective role of levan in common carp with exposure to fipronil) and l0.75f (basal feed+0.75% with exposure to fipronil). purified microbial levan was procured from department of microbiology, bhavans college, mumbai, india. except l0f0, all the groups were exposed to sublethal dose of fipronil. the drinking tap water was used during the experimental trail to make sure that it is not contaminated with any other pollutants. this tap water was used purposefully as the facilities to measure the exact fipronil concentration was unavailable. aeration was provided through compressed air pump round the clock. feeding of the fry was done to satiation level (at 4% of their body) and changed accordingly to their biomass gain over a period of 15 days. daily ration was divided into two parts on the basis of biomass, about 2/3rd of total ration was given at 09:30 h and the rest 1/3rd at 18:30 h. the left over feed and faecal debris were manually siphoned out employing water exchange of approx. 50% of the tank volume on every second days with taking care to provide any discomfort to the animals. to maintain the sublethal effect of the fipronil, test solution was reintroduced with firponil treated water arranged from the stock solution. we followed the methodology as previously described by das and mukherjee (2003) and prusty et al. (2011) to maintain sublethal concentration. water quality parameters from each replicate tank monitored on daily basis, were dissolved oxygen (do), temperature, and ph, while alkanlity and hardness table 1. composition of experimental diets (g %), and its proximate analysis (% dry matter basis). ingredients experimental diets (% inclusion) l0 f0 l0.25f l0.50f l0.75f casein fat free* 35 35 35 35 gelatin* 9.5 9.5 9.5 9.5 dextrin* 10 10 10 10 starch soluble* 25 25 25 25 cellulose powder* 9.0 8.75 8.5 8.25 cod liver oil† 4 4 4 4 sun flower oil‡ 4 4 4 4 vitamin mineral mix§ 2.88 2.88 2.88 2.88 vit-c‼ 0.10 0.10 0.10 0.10 microbial levan -0.25 0.5 0.75 cmc* 0.48 0.48 0.48 0.48 betain hydrochloride* 0.02 0.02 0.02 0.02 bht* 0.02 0.02 0.02 0.02 total 100 100 100 100 proximate composition of diets organic matter (om) 96.63 96.64 96.67 96.59 crude protein (cp) 35.10 35.04 35.01 35.33 ether extract (ee) 7.56 7.50 7.48 7.61 ash 3.37 3.20 3.38 3.39 total carbohydrate (tc) 53.94 54.25 54.13 53.66 digestible energy♯ (de) 424.31 424.67 423.88 424.51 *himedia laboratories, mumbai, india, †densa pharma, mumbai, india, ‡ruchi soya industries, raigad, india,§composition of vitamin mineral mix (emix plus) (quantity 2.5kg -1), vitamin a 55,00,000 iu; vitamin d3 11,00,000 iu; vitamin b2 2,000 mg; vitamin e 750 mg; vitamin k 1,000 mg; vitamin b6 1,000 mg; vitamin b12 6 µg; calcium pantothenate 2,500 mg; nicotinamide 10 g; choline chloride 150 g; mn 27,000 mg; i 1,000 mg; fe 7,500 mg; zn 5,000 mg; cu 2,000 mg; co 450 mg; ca 500 g; p 300g; llysine 10 g; dlmethionine 10 g; selenium 50 mgl-1; selenium 50 mgl-1; satwari 250 mgl-1; (lactobacillus 120 million units and yeast culture 3000 crore units). ‼stay c (hoffman la roche, nutley, nj, usa) 15% ascorbic acid activity, ♯digestible energy (k cal/100g) = (%cp x 4)+(% ee x 9)+(tc x 4). 137 int. j. aquat. biol. (2021) 9(2): 134-147 were monitored on weekly basis. all water quality parameters (dissolved oxygen: 6.48–7.2 mg l-1; ph: 7.35–7.60; temperature: 26.8-27.2oc; alkalinity 51-59 mg l-1 and hardness 56-66 mg l-1) were found to be within the normal range for rearing of c. carpio (gupta et al., 2013). proximate analysis of feed: as per the standard methods of aoac (1990), proximate composition of the experimental diets was determined (table 1). the moisture content was determined by drying the samples at 105oc to a constant weight. nitrogen content was estimated by automated kjeldahl apparatus (2200 kjeltec auto distillation, foss tecator, sweden) and cp was estimated by multiplying nitrogen percentage by 6.25. ether extract (ee) was measured using a soxtec system (1045 soxtec extraction unit, tecator, sweden) using diethyl ether (boiling point, 40-60oc) as a solvent and ash content was determined by incinerating the samples in a muffle furnace at 600oc for 6 h. total carbohydrate was calculated by difference i.e., total carbohydrate % = 100−(cp%+ee%+ash%). the digestible energy (de) values of experimental diets and tissues were calculated as described by halver (1976). sample preparation: at the end of the experiment, two fish from each replicate with a total of six from each treatment were anaesthetized with cifecalm (cife, mumbai, india) at 50 μl l−1. whole intestine was dissected out aseptically and collected for enzyme assay after removing the intestinal contents and immediately homogenized (5%) in chilled sucrose solution 0.25 m using a teflon coated mechanical tissue homogenizer. during homogenization, care was taken by keeping the sample over ice to avoid heating. in a cooling centrifuge, homogenized samples were centrifuged (6000×g for 10 min) and supernatants were collected in 2 ml eppendorf tube and stored at – 20ºc for subsequent enzyme assays. enzyme assays: protease activity in the intestinal tissue was determined by the casein digestion method of drapeau (1974). lipase activity in the intestinal tissue was determined based on cherry and crandell (1932). alp activity in the intestinal tissue was determined by the method of garen and levinthal (1960). phosphate liberated was estimated at od of 660 nm (fiske et al., 1925). acp activity was estimated using the same method as alp, except that acetate buffer (0.2 m, ph 5) was used in place of bicarbonate buffer. protein estimation in the tissue was carried out using method of lowry et al. (1951). antioxidant assay: gluthathione-s-transferase (gst) activity was measured in the liver, gill, brain and kidney tissues following the method of habing et al. (1974) using s-2, 4-dinitrophenyl glutathione (cdnb) as substrate. the method was based on the principle of formation of adduct of cdnb, s-2, 4dinitrophenyl glutathione and was monitored by measuring the increase in absorbance at 340 nm against blank. glutathion peroxidase (gpx) was measured in the liver, gill and kidney tissues following the method of paglia and valentine (1967). gpx activity was analysed based on conversion of glutathione into glutathione disulphide, and decomposition of 1 µm of the substrate to the product. gsh levels were measured based on the formation of 2-nitro 5-thiobenzoic acid. tissue glycogen: liver and muscle glycogen content (mg g-1) was estimated colorimetrically by the method described by hassid and abraham (1957). the tissue was placed in a pre-weighed centrifuge tube containing 3 ml of 30% koh. after the weight of the tissue had been recorded, the tubes were placed in a boiling water bath for 20 min. after cooling, 5 ml of 95% ethanol was added to precipitate the glycogen. the precipitate obtained was dissolved in 1 ml of distilled water and again precipitated with 95% ethanol and centrifuged. the glycogen precipitate was then dissolved in distilled water, and this solution was used to estimate the quantity of glycogen. in 0.1 ml of aliquot, 5 ml of anthrone reagent was added and mixed by swirling the tube. the tubes were covered with glass marble and heated for 10 min in boiling water, followed by cooling, and the absorbance was recorded at 590 nm. the reading was compared with that of standard glycogen. collection of blood serum: for serum, two fish from each replicate and a total of 6 from each treatment were anesthetized as above; the blood was collected 138 gupta / protective role of levan in common carp without anticoagulant and allowed to clot for 2 h, centrifuged (3000 g for 5 min) and then kept at _ 80°c until use. serum cholesterol, triglyceride, ldl (low density lipoprotein) and (vldl) (very low density lipoprotein) cholesterol: serum cholesterol was measured by method of (palkonen et al., 1957) by using commercially available kit from sigma aldrich, saint louis, usa. in brief, 20 µl of serum was mixed with 2 ml of reaction solution (enzyme solution with colour reagent). the absorbance of samples was measured at 540 nm against the reagent blank value. serum triglycerides were measured by the method described (kaplan 1985) using commercially available kit from sigma aldrich, saint louis, usa. in brief, 10 μl of serum was mixed with 1 ml of reaction solution. the absorbance of sample was measured against the reaction solution. the increase in absorbance, measured at 540 nm, due to the formation of the quinoneimine dye. the increase in absorbance was directly proportional to the glycerol concentration in the sample. true serum triglycerides were calculated by subtracting the free glycerol concentration in the sample from total triglycerides. serum ldl and hdl contents were measured using the kit of daiichi pure chemicals co., ltd. chuo-ku, tokyo, japan. initially, 3 μl of sample was mixed with 300 μl of reagent 1 and kept for 5 min and then a700/600 nm was measured. this was followed by the addition of 100 μl of reagent 2 in the same microtitre plate. again the a700/600nm was measured for the same sample. the absorbance was measured by using the instrument chem well of awareness technology inc. palm city, florida, usa. estimation of vldl was calculated by (sattyanarayanan 2001). the value of vldl was calculated by density gradient centrifugation method based on the following formula: vldl = total cholesterol− (hdl+ldl) the estimated values of vldl were expressed in mg dl-1 lipid peroxidation (lpo) and hsp70: lpo was determined by the procedure of uchiyama and mihara (1978) in the liver, gill, kidney and brain tissues. briefly, 0.25 ml of homogenate was mixed with 25 ml of 10 mm butylated hydroxytoluene (bht). 3 ml phosphoric acid (1%) and 1 ml of 0.67% thiobarbituric acid (tba) were added and mixture was incubated at 90oc for 45 min. the absorbance was measured at 535 nm. the rate of lpo was expressed as nanomoles of thiobarbituric acid reactive substance (tbars) formed/h/mg of protein using a molar extinction coefficient of 1.56 × 105 m-1 cm-1. hsp70 levels (eia kit, catalog no. eks-700b) in the liver, muscle and gill was determined following the manufacturer's instructions (bioguenix/enzo life science, mumbai, india). the absorbance was read on the elisa plate reader (biotek india pvt. ltd.). myeloperoxidase content and total immunoglobulin: total myeloperoxidase content present in serum was measured according to quade and roth (1997) with some modifications. about 15 μl of the serum was diluted with 135 μl of hank's balanced salt solution (hbss) without ca2+ or mg2+ in 96-well plates. the wells were added with 25 μl of 20 mm 3,3′ 5, 5′tetramethyl benzidine hydrochloride (tmb) (hi-media) and 25 μl of 5 mm h2o2 (qualigens) (both substrates of mpo prepared freshly). the reaction was terminated after 2 min by adding 50 μl of 4 m sulphuric acid (h2so4). the plate was centrifuged (400 g) for 10 min, and 150 μl of the supernatant from each well was transferred into new 96-well plates. the absorbance was measured at 450 nm in a microplate reader (biotek india pvt. ltd.). total immunoglobulin level was measured and calculated as per the protocol of siwicki et al. (1994) with minor modification. od measurement was performed at 595 nm. the serum total immunoglobulin concentration was calculated by subtracting the concentration of proteins in the supernatant from the total protein concentration in the serum before precipitation with peg. the total immunoglobulin level was expressed as unit ng dl-1. statistical analysis: mean value of all parameters were subjected to one-way analysis of variance (anova) to study the treatment effect and duncan’s multiple range tests (dmrt) were used to determine the significant differences between the 139 int. j. aquat. biol. (2021) 9(2): 134-147 mean value. comparisons were made at 5% probability level. all the data were analyzed using statistical package spss (version 16) (spss inc., chicago, il, usa). results enzyme assays: dietary levan supplementation significantly (p<0.05) affected protease, lipase, alp and acp activity in the intestine of c. carpio fry in different experimental groups (table 2). highest (p<0.05) protease activity among levan fed group was observed in the (l0.75f) group fed with 0.75% levan supplementation and exposed to fipronil which was comparable to l0f0 group fed without levan supplementation and reared without exposure of fipronil stress. lipase activity was recorded highest (p<0.05) with highest level of dietary levan fed l0.75f group and varied significantly from all other groups except l0f0. lowest value of both protease and lipase activity was observed in the l0f group fed without levan supplemented diet and exposed to fipronil. fish fed 0.75% levan and exposed to fipronil had significantly (p<0.05) lowest intestinal alp activity than the other treatment group except l0f0 group. a decreasing trend in acp activity of intestine was observed with increasing concentration of levan and lowest (p<0.05) activity was observed in the group exposed to fipronil and fed with 0.75% dietary levan which was similar to the group fed without levan and reared without fipronil stress. higher value of alp and acp activities (p<0.05) were observed in the order of l0f > l0.25f > l0.50f groups (table 2). antioxidant assay: the oxidative stress in terms of gst and gpx in the liver, gill, brain and kidney of c. carpio exposed to fipronil stress for 60 days are presented in table 5. the cellular stress indicators such as gst and gpx were noticeably elevated (p<0.05) in fipronil exposed group with compared to all other treatment groups. we found that supplementation of levan at 0.75% diet significantly amended the oxidative stress levels in all the tissues which were reflected in terms of reduced gst and gpx levels. tissue glycogen content: dietary levan supplementation had significant (p<0.05) impact on the glycogen content in both liver and muscle of experimental groups (table 3). glycogen content was recorded higher in the liver than the muscle. lowest table 2. effect of dietary microbial levan on specific enzyme activities in the digestive tract of cyprinus carpio fry exposed to sublethal fipronil stress for 60 days. treatments protease lipase alp acp l0f0 12.78a±0.73 1.44a±0.06 27.33c±1.22 45.44c±1.95 l0f 7.16b±0.36 0.66b±0.03 40.09b±1.92 87.85a±2. 31 l0.25f 8.73b±0.55 0.80b±0.09 45.72b±1.89 83.94a±3. 07 l0.50f 8.91b±0.70 0.87b±0.12 39.18b±1.13 67.22b±1.89 l0.75f 12.48a±0.41 1.33a±0.12 28.79c±2.58 40.61c±2.41 activities are expressed as follows: protease as micromole of tyrosine released per min per mg protein; lipase activity as units per hour per mg protein; alp and acp as nanomoles p-nitrophenol released per min per mg protein at 37°c; values in the same column with different superscript (a, b, c) differ significantly (p<0.05). data are expressed as mean ±se (n=6) table 3. effect of dietary microbial levan on glycogen content in liver and muscle tissues of cyprinus carpio fry exposed to sublethal fipronil stress for 60 days. treatments glycogen content (mg g wet tissue-1) muscle liver l0f0 1.39ab±0.06 21.31c±0.79 l0f 1.21c±0.01 18.36d±0.99 l0.25f 1.28b±0.03 19.68bc±0.98 l0.50f 1.34ab±0.04 19.83ab±0.84 l0.75f 1.37a±0.02 21.09a±0.86 values in the same column with different superscript (a, b, c, d) differ significantly (p<0.05). data are expressed as mean ±se (n=6). 140 gupta / protective role of levan in common carp (p<0.05) value of glycogen level in both liver and muscle was observed in the l0f group fed without levan supplemented diet and exposed to fipronil. higher value among levan fed groups was observed in the l0.75f > l0.50f > l0.25f groups (table 3). lipid profile of serum: the lipid profile of serum of the experimental groups at the end of the feeding trial is represented in table 4. supplementation of dietary levan significantly (p<0.05) affected cholesterol, triglycerides, ldl and vldl of the experimental groups. as the incorporation of levan increased in the diet, the value of serum cholesterol decreased consequently in the experimental groups. similar trend was observed for triglyceride and vldl contents and the lower value was observed in the l0.75f group fed with 0.75% levan and exposed to fipronil stress. the lower value of ldl cholesterol was observed in the group fed with higher levan and exposed to fipronil stress. higher value of serum cholesterol, triglycerides and ldl were observed in the l0f group fed without levan supplementation and exposed to fipronil. lpo and hsp-70: the lpo levels were significantly higher (p<0.05) in the liver, gill, kidney and brain tissues of fipronil exposed group compared to all other groups. further, the level of lpo was significantly (p<0.05) reduced with supplementation at 0.75% diet compared to other treatment groups in all the tissues except lpo level in the brain which was similar (p>0.05) to the control l0f0 and l0.50f levan fed groups. the stress biomarkers hsp-70 of c. carpio exposed to fipronil and fed with different doses of levan for 60 days are presented in table 6. the hsp 70 in gill, liver and muscle were noticeably (p<0.05) elevated in fipronil exposed group (l0f), whereas higher supplementation of levan led to consequential reduction in the levels of hsp-70 and hence lowest (p<0.05) was found in the group fed at at 0.75% diet compared to other treatment groups in all the tissues. myeloperoxidase content and total immunoglobulin (ig): total immunoglobulin content of c. carpio showed a significant increasing trend with the concomitant increase in the level of dietary levan concentration. the content of total immuneglobulin was observed highest (p<0.05) in the group fed at 0.75% levan compared to other treatment table 4. impact of dietary levan on serum lipid profile (mg%) of cyprinus carpio fry exposed to sublethal fipronil stress for 60 days. treatments cholesterol triglyceride ldl1 vldl2 l0f0 154.67bc±6.38 246.33c±8.81 54.64b±0.90 32.76b±5.48 l0f 162.33c±2.40 253.33c±3.17 55.30b±3.56 31.10b±5.38 l0.25f 149.00abc±4.16 230.33b±2.90 43.56a±4.24 22.08ab±0.18 l0.50f 144.00ab±4.16 226.33ab±1.45 43.33a±2.69 18.38a±3.25 l0.75f 35.67a±5.36 214.33a±2.40 38.91a±2.06 15.27a±0.92 1ldllow-density lipoprotein, 2vldl-very low-density lipoprotein. values in the same column with different superscript (a,b,c,d) differ significantly (p<0.05). data are expressed as mean ±se (n=6). table 5. impact of dietary levan on on glutathione-s-transferase (gst) and glutathione peroxidase (gpx) in liver, gill, brain and kidney tissues of cyprinus carpio fry reared under fipronil stress for 60 days. treatments l0f0 l0f l0.25f l0.50f l0.75f gst-liver 0.18b±0.07 0.27c±0.02 0.17b±0.01 0.18b±0.03 0.11a±0.08 gst-gill 0.19b±0.01 0.37d±0.02 0.28c±0.05 0.23c±0.02 0.14a±0.02 gst-brain 0.32b±0.04 0.51d±0.04 0.43c±0.03 0.33b±0.06 0.24a±0.03 gst-kidney 0.26c±0.02 0.49d±0.06 0.23bc±0.01 0.25c±0.04 0.14a±0.01 gpx-liver 4.54b±0.60 8.56c±0.42 2.21a±0.18 2.11a±0.27 2.23a±0.06 gpx-gill 4.66b±0.35 10.43e±1.07 7.87d±1.04 5.67c±0.77 3.01a±0.22 gpx-brain 3.98b±0.32 11.02d±0.70 4.20b±0.53 7.11c±0.41 1.69a±0.14 gpx-kidney 3.97b±0.41 7.50e±0.65 5.48c±0.39 6.25d±0.42 2.01a±0.24 values in the same row with different superscript (a, b, c, d) differ significantly (p<0.05). data expressed as mean ±se (n=6). gst and gpx: units mg protein-1. 141 int. j. aquat. biol. (2021) 9(2): 134-147 groups (fig. 1). consistent to the total immuneglobulin results, the increasing trend of myeloperoxidase content was observed among the treatment groups and the higher value was observed in the group fed at 0.75% dietary levan which was similar to the group fed with 0. 50% diet (fig. 2). discussions microbial levan is a potent functional feed, and its efficacy as enhancer of immune response, heat tolerance, resistance to pathogen, growth performance and nutrient utilization in various species of fishes have been well documented (gupta et al., 2010, 2013, 2014a, 2015, 2020; rairakhwada et al., 2007; huang et al., 2015). in this present investigation, an attempt has been made to probe the protective role of dietary prebiotic levan on immuno-biochemical responses of c. carpio fry exposed to sublethal concentration of fipronil. the activity of digestive enzymes is an important indicator of digestive physiology which determines the capacity of digestion and absorption of nutrients, and eventually reflected in better growth. improved growth is also dependent on the progress of prebiotic fermentation by endogenous gut microbes (scheppach, 1994; dimitroglou et al., 2010; hoseinifar et al., 2011, 2015). the positive influence of prebiotics on growth performance may be associated with improved nutrient digestibility, as a result of enhanced digestive enzyme activity which allow the host to degrade more nutrients (huang et al., 2015). in the present study, dietary inclusion of prebiotic levan significantly increased digestive enzyme activities of c. carpio fry. fish fed 7.5 g kg–1 levan and exposed to fipronil had significantly higher protease and lipase activities compare to other treatment groups except l0f0, which might be due to alteration of local gut microbial communities by table 6. impact of dietary levan on lipid peroxidation (lpo) in liver, gill, brain and kidney tissues and hsp-70 in the gill, liver and muscle tissues of cyprinus carpio fry reared under fipronil stress for 60 days. treatments l0f0 l0f l0.25f l0.50f l0.75f lpo-liver 16.41b±0.25 31.23d±3.12 16.98b±1.54 18.98c±1.51 12.92a±1.44 lpo-gill 12.86b±0.52 27.29d±0.83 19.23c±1.90 19.40c±1.54 10.49a±0.89 lpo-kidney 17.62b±0.43 46.51e±0.78 34.34d±0.85 32.09c±1.85 12.97a±0.88 lpo-brain 10.60a±0.73 31.04d±1.78 18.86c±1.44 12.28ab±0.98 10.55a±0.95 hsp-70-gill 12.93b±0.37 32.37d±0.43 18.07bc±1.57 17.73b±1.46 9.67a±0.19 hsp-70-liver 13.47b±0.24 35.23c±0.51 23.27b±0.36 22.93b±0.63 9.35a±0.34 hsp-70-muscle 12.57b±0.21 38.23d±0.42 25.57c±0.22 23.40c±0.65 10.57a±0.43 values in the same column with different superscript (a, b, c, d) differ significantly (p<0.05). data expressed as mean ±se (n=6). lpo: n mole tbars formed/h/mg protein, hsp-70: ng ml-1. figure 1. total immunoglobulin of cyprinus carpio fry fed diteray levan and reared under fipronil stress for 60 day. data expressed as mean ± e (n=6). statistically significant (p<0.05) denoted as superscript letter a, b, c, d, on top of error bars. figure 2. myeloperoxdiase of cyprinus carpio fry fed diteray levan and reared under fipronil stress for 60 day. data expressed as mean ±se (n=6). statistically significant (p<0.05) denoted as superscript letter a, b, c, d, on top of error bars. 142 gupta / protective role of levan in common carp enhancing the proliferation of probiotics such as lactobacillus plantarum and bifidobacterium pseudocatenulatum (wang et al., 2016). our results are supported by findings of akhtar et al. (2012) in l. rohita juveniles fed with highest level of pyridoxine incorporation and exposed to endosulfan toxicity. protease is one of the digestive enzymes responsible for the protein digestion in animal, therefore highest activity in the maximum levan fed group indicates better protein digestion (swanson et al., 2002; teitelbaum and walker, 2002). to corroborate our findings, xu et al. (2009) reported parallel results of increased protease activity after feeding fructooligosacchsride in poultry. dimitroglou et al. (2010) revealed that dietary mos (mannan oligosaccharides) supplementation increased the absorptive surface by promoting longer mucosal folding and increasing the microvilli density and microvilli length in the anterior and posterior gut regions of atlantic salmon. the results of present investigation are also reflected with our previous finding gupta et al. (2013) where higher weight gain% and sgr, highest protein efficiency ratio and better food conversion ratio (fcr) were recorded in the l0.75f group fed with (0.75%) levan and exposed to fipronil stress. huang et al. (2015) suggested that the positive influence of levan on growth performance of fish may be associated with improvements in digestive enzyme activity or the gut morphology, thereby resulting in better degradation or absorption of nutrients in the gut. increase in enzyme activity correlated with improved growth of c. carpio which might be attributable due to increased number of benign bacteria facilitating in the production of organic acids (formic acid, acetic acid, lactic acid), hydrogen peroxide, and several other growth stimulating compounds such as bacteriocins, siderophores and lysozyme (hoseinifar et al., 2015). however, till to date, no reports are available on the mechanistic approaches on effect of dietary levan on digestive enzymes and microvilli structures in the gut of fish to substantiate our findings. alkaline phosphatase, an important regulative enzyme in bio-metabolic processes, plays a vital role in transition and absorption of nutrient in the enterocytes (harpaz and uni, 1999; gawlicka et al., 2000) whereas acp activity is highly correlated with growth in fish (debnath et al., 2007). lowest value of alp and acp in the intestine of l0f group fed diet without levan and exposed to fipronil is in agreement with the previous results (verma et al., 2007) in c. carpio exposed to endosulfan toxicity. significant decrease in alp and acp activity in the l0.75f group might be due to hydrolysis of high-energy phosphate bonds to release phosphate ions to fight stressful situation leading to improved fcr and better growth (gupta et al., 2013). our results are supported with the findings of (sarma et al., 2009) in channa punctatus who fed high protein and high vitamin c diet and kumar et al. (2012, 2014) in l. rohita fed with diet containing lecithin and betaine and exposed to combined effect of endosulfan and temperature. antioxidant defense plays a vital part in the response of an organism to pollutants. numerous processes excite the generation of hydrogen peroxide and production of free radicals or deplete the antioxidant defense, which may cause severe oxidative damage in the organisms if not eliminated effectively (martínez-álvarez et al., 2005). in this direction, gst and gpx are the most important antioxidant enzymes, that serves as the first line of defence in the organisms against oxidative stress (hoseini et al., 2019; paray et al., 2020; zargar et al., 2020). gpx and gst are involved in hydrogen peroxide degradation and is responsible for detoxification of harmful xenobiotics. the oxidative stress enhanced after exposure to fipronil in the present study, might be attributable due to the generation of hydrogen peroxide and production of oxygen free radicals. therefore, to counteract activities of oxygen free radicals, collective action of both enzymatic and non-enzymatic antioxidant is valuable (pérez-campo et al., 1993). to corroborate our findings of increased gpx and gst activities in different tissues of fipronil exposed group fed without levan, previous studies also reported enhanced oxidative stress in l. rohita, c. chanos and o. mossambicus exposed to various contaminants (kumar et al., 2012, 2016, 2017). significant 143 int. j. aquat. biol. (2021) 9(2): 134-147 reduction in the level of oxidative stress might be correlated with the stress ameliorative efficacy of dietary levan supplementation (gupta et al., 2014a). our results are also substantiated with the reduced level of lpo levels observed in the higher levan fed group. tissue glycogen is common measured parameter of stress response (manush et al., 2005). in the present study, the glycogen content of both liver and muscle of c. carpio fry decreased significantly in the group exposed to fipronil and fed diet without levan. our result is in agreement with the findings of akhtar et al. (2012) who observed decreased glycogen content in l. rohita fingerlings fed without pyridoxine and exposed to endosulfan toxicity. similarly, rawat et al. (2002) found decreased glycogen content in heteropneustes fossilis when reared under endosulfan stress. nevertheless, supplementation of levan at 0.75% diet restored the glycogen level to normal in muscle as compared to control (l0f0) group. to the best of our knowledge, no literature is available on the effect of levan on glycogen levels in fish to correlate our findings. lipoproteins transport the majority of plasma lipids, including cholesterol and triglycerides. ldl and vldl are the lipoproteins responsible for the vast majority of cholesterol transport in the blood. supplementation of dietary levan significantly led to reduction in cholesterol, triglycerides, ldl and vldl of the experimental groups. as the incorporation level of levan increased in the diet, serum cholesterol, triglycerides, ldl and vldl decreased consequently in the experimental groups. to corroborate our finding, daubioul et al., (2002) reported decrease in the triglyceride accumulation in the liver of rats fed diet supplemented with dietary fructans. this might be attributed due to high fermentation of fructans which selectively decreased the incorporation of acetate into total lipids that could contribute to lesser triglyceride accumulation. decreases of serum triglycerides in animals are shown to result from the reduction of very low-density lipoprotein-triglyceride secretion and the inhibition of hepatic lipogenesis by the reduced activity and down regulation of gene expression of lipogenic enzymes (kok et al., 1996; delzenne et al., 2001). feeding of high-molecular weight levan (1-5% w/v) decreased serum total cholesterol in a dose dependent manner and the oral administration of 2% levan reduced adiposity as well as serum lipids in rats (kang et al., 2002a). the possible mechanism of reduced serum lipid parameters might be attributed to peroxisome proliferation-activated receptors (ppars) that have important effects on lipid homeostasis, by regulating the expression of genes involved in lipid metabolism (escher and wahli, 2000). the supplementation of levan decreased adiposity and postprandial lipidaemia in rats, through the enhancement of uncoupling protein gene expression (kang et al., 2002b, 2004). the mrna expressions of hepatic fatty acid synthase and acetyl coa carboxylase, which are the key enzymes in fatty acid synthesis, are also downregulated by levan (kang, 2009). hsp-70 levels and lipid peroxidation (lpo) are sensitive biomarkers, and found to be highly elevated upon exposure to pollutants like heavy metals and pesticide (kumar and singh, 2019). the significant increase in hsp-70 level might be associated due to denatured protein leading to generation and accumulation of reactive oxygen species in response to different kinds of stressors in aquatic animal (zhang et al., 2009). in the current investigation, hsp-70 has been improved by supplementation of dietary levan which may be due to the stimulation of nonspecific defence mechanism by hsp, which is in accordance to the findings of sung et al. (2008) and(gupta et al. (2010). lipid peroxidation (lpo) is a process under which oxidants such as free radicals attack lipids containing carbon-carbon double bond(s), especially biological membranes that leads to impaired membrane function, structural integrity as well as inactivation of several membranes bound enzymes (goel et al., 2005). thus, lpo is an indicator of principal disorders in structural, functional and enzymatic organization of the biological membranes. in this study, level of lpo has been significantly reduced in levan supplemented groups, hence, it may be inferred that dietary levan 144 gupta / protective role of levan in common carp holds this mechanistic property for inhibition of lipid peroxidation and mitigation of stress in c. carpio. to supports our results, studies reported that use of other dietary nutraceuticals such as methyl donors (kumar et al., 2014) and pyridoxine (kumar et al., 2016) reduced the oxidative stress and maintained their level in the different tissues. total immunoglobulin (ig) and myeloperoxidase content of serum in levan fed common carp were significantly enhanced with concomitant increase in the level of supplementation. the major functions of immunoglobulins in defense-related activities such as destroying the pathogen and amelioration of stress are well recognized (gupta et al., 2020). similar to levan, inulin an another prebiotic, has shown to exhibit enhanced igm level and myeloperoxidase activity in leopard grouper, mycteroperca rosacea, (reyesbecerril et al., 2014). the enhanced level of total immunoglobulin and myeloperoxidase content in higher levan fed group at 0.75% suggested that levan incorporation in common carp might have triggered the activation of immune cells which might have led to ameliorate the stress mediated through fipronil exposure. furthermore, our results are in accordance to studies performed by ahmadifar et al. (2019) which showed that dietary ginger administration significantly increased total ig levels in zebrafish. besides, hoseini and yousefi (2019) demonstrated that dietary supplementation of thyme extract significantly improved plasma total ig in rainbow trout (oncorhynchus mykiss) against oxytetracyclineinduced stress, indicating the protective role of prebiotic levan in augmenting the immune status of common carp against pesticide stress. conclusion to summarize the above findings, dietary microbial leavn at 0.75% of incorporation level led to enhancement of the intestinal protease and lipase activities whereas as alkaline and acid phosphatase activity was declined. the ameliorating effect of microbial levan was confirmed by marked improvement in glycogen level, serum cholesterol, triglyceride, lpo, hsp-70, myeloperoxidase content as well as total immunoglobulin level of the fish fed with 0.75% of levan. overall results showed protective and ameliorative role of dietary microbial levan at 0.75% incorporation level and could be used as potent immuno-additive prebiotic supplements to mitigate the fipronil induced stress in aquaculture. further investigation using different concentrations of levan should be accomplished to unravel defensive mechanism against fipronil induced immune-toxic impact. acknowledgements the financial support and necessary facilities provided by indian council of agricultural research (icar), new delhi to the first author is duly acknowledged. references ahmadifar e., sheikhzadeh n., roshanaei k., dargahi n., faggio c. 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(2009). identification of two hsp90 genes from the marine crab, portunus trituberculatus and their specific expression profiles under different environmental conditions. comparative biochemistry and physiology part c: toxicology and pharmacology, 150: 465-473. international journal of aquatic biology (2015) 3(3): 149-154 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article length-length and length-weight relationships and condition factor of nine freshwater fish species of nageshwari, bangladesh most habiba ferdaushy, mohammad manjurul alam*1 department of fisheries, university of rajshahi, rajshahi-6205, bangladesh. article history: received 1 september 2014 accepted 1 april 2015 available online 2 5 june 2015 keywords: length-weight condition factor morphometrics indigenous species abstract: the research carried out the length-length relationships (llr), length-weight relationship (lwr) and condition factor of nine freshwater small indigenous species viz. g. giuris, e. danricus, m. pancalus, l. guntea, c. fasciatus, p. sophore, h. fossilis, c. punctatus and m. cavasius from the water bodies of nageshwari, bangladesh. relationships among different body length parameters of each species were found highly significant with all “r” values being >0.900. lwrs were obtained on the form of tw = 𝛼tlb. the “b” values range from 2.65-3.03. the parameter “b” of the length-weight relationship equation showed higher value (>3) only for g. giuris and c. fasciatus. rest of the specimens showed lower value (<3) but close to the value of 3 and relationships were found highly significant with all “r” values being >9.00. the fulton’s condition factors showed positive growth tendency for five specimens viz. g. giuris, c. fasciatus, p. sophore, c. punctatus, m. cavasius; and negative tendency for four specimens viz. e. danricus, m. pancalus, l. guntea, and h. fossilis. introduction morphometric measurements and statistical relationships of fishes are imperative for both fishery biology (sparre et al., 1989; mustafa and brooks, 2008) and taxonomic studies (tandon et al., 1993; simon et al., 2010; alam et al., 2014). the application of length-weight relationship in fishery biology solves various problems concerned with the life history of fishes. length-weight relationship also gives information about the condition and growth patterns of fish (bagenal and tesch, 1978; oscoz et al., 2005; alam et al., 2014). in addition, condition factors may be used to detect seasonal variations in the growth of fish, which may vary with food abundance and average reproductive stage of the stock (king, 1995; alam et al., 2014). for proper management and conservation of the wild population of fish species, morphometric study is necessary. in bangladesh, a total of 260 indigenous freshwater fish species were reported (rahman, 2005) of which * corresponding author: mohammad manjurul alam e-mail address: mamillat@yahoo.com small indigenous fishes species are important target species for the small-scale fishermen (criag et al., 2004; mustofa and brooks, 2008); and serve as a major source of protein and vitamin for the rural community (rubbi et al., 1978). the present study aimed to find out the present status of length-length and length-weight relationship; and condition factor of nine fish species viz. glossogobius giuris, esomus danricus, mastacembelus pancalus, lepidocephalus guntea, colisa faciatus, puntius sophore, heteropneustes fossilis, channa punctatus, and mystus cavasius. findings of the work will play an important role for the successful management of these species to conserve from probable depletion of their wild stock in the future. materials and methods the specimens were collected fortnightly from the fishers of nageshwari (25.9792°n 89.7083°e) water 150 international journal of aquatic biology (2015) 3(3): 149-154 bodies during day time from october, 2011 to january, 2012. during the period a total of 540 specimens (each of 60) were collected and confirmed to the species level and preserved by date in plastic jars with 5% formalin. for each individual different body lengths tl, pl, pvl, sl, hl, dl, al were measured with the help of a digital slide caliper and whole body weight (tw) was taken with the help of a digital balance model: kd-300kc with 0.01 g accuracy (simon and mazlan, 2008; alam et al., 2012). the relationships among all body length parameters were determined by the method of least squares to fit a linear regression as: y = 𝛼 + bx. where, y = various body lengths, x = total length, 𝛼 = proportionality constant and b = regression coefficient (alam et al., 2014). the length-weight relationships were determined by the general equation of tw = 𝛼tlb (le ceen, 1951). where, tw is the total weight (expressed in g), tl is the total length (expressed in cm), “𝛼” is a coefficient related to body form and “b” is an exponent indicating isometric growth when equal to 3 and indicating allometric growth when significantly different from 3 (simon et al., 2009; alam et al., 2014). the parameters “𝛼” and “b” of the exponential curve were estimated by linear regression analysis over log-transformed data expressed as: logtw = log𝛼 + blog tl. the values of the constant “a” and “b” of the linear regression was determined by following rounsefell and everhart (1953) and lagler (1966). the fulton’s condition factor, k was calculated by using the following formula: k = (tw/tl3) × 100. where, k = fulton’s condition factor, tw = total body weight, tl = total body length. here, factor 100 is used to bring k close unity. all the data were analyzed by using computer software spss version 15.0. results and discussion the length-length relationships with total length among standard length, dorsal length, pectoral length, pelvic length, anal length, head length and the coefficient of correlation of 9 fish species were presented in the table 1. the relationships were found highly significant with all “r” values being >0.900. the body lengths were gradually increased with the increase of tl. the obtained regression equations clearly revealed that the lengths of the body parts are proportional to the total length. such findings were also observed by tandon et al. (1993) while working with the morphometry of cirhinus reba kanjli wetland of india. these relationships were also observed in puntius chola (bhuiyan and biswas, 1982), mystus vittatus (hoque and hossin, 1992; hossain et al., 2006), parastromateus niger, (dadzie et al., 2008), and p. sophore (alam, et al., 2012). the findings are more similar to the findings of hossain et al. (2009) while worked on lengthweight and length-length relationship of 10 small fish species from the ganges, bangladesh; alam et al. (2013) and alam et al. (2014) on length-length relationship, length-weight relationship and condition factors of some freshwater fish species of bangladesh. the range of length and weight parameters of the length-weight equations and values of fulton’s condition factors were shown in table 2. the slope “b” values of the length-weight equations were obtained 3.011 for g. giuris, 2.812 for e. danricus, 2.852 for m. pancalus, for l. guntea, 3.030 for c. faciatus, 2.990 for p. sophore, 2.680 for h. fossilis, 2.840 for c. punctatus and 2.650 for m. cavasius. the length-weight relationships were found highly significant with all “r” values being >0.900 where the parameter “b” remained mostly within the expected range of 2.5-3.5. therefore, all the species seemed to be followed the cube law. the reason behind may be the observed specimens were the inhabitants of quite good environment and gravid females were more in the samples (le cren, 1951). the equations are therefore applicable for the total population as a whole. while working with different morphometric characters of other fish species by bagenal and tesch (1978), hoque and hossain (1992), kiran et al. (2004), oscoz et al. (2005), froese (2006), britton and devies (2007), aguirre et al. (2008), arshad et al. (2008), hossain et al. (2009), alam et al. (2013) and alam et al. (2014) observed 151 ferdaushy and alam/ morphometric relationship and condition of nine freshwater fishes of bangladesh similar results. the values of fulton’s condition factor were for g. giuris, c. faciatus, p. sophore, c. punctatus, and m. cavasius were being >1 indicate that growth of these species were perfect condition whereas the rest species were being <1 but very close to 1 that may species (ordinate tl) abscissa mean  se of abscissa regression equation r g. giuris (tl = 6.68  0.13) sl 5.18±0.10 sl = 0.3675 + 0.7211tl 0.992** dl 2.96±0.06 dl = 0.1298 + 0.4239tl 0.975** pl 1.46±0.03 pl = 0.0073 + 0.2198tl 0.965** pvl 1.63±0.03 pvl = 0.0873 + 0.2565tl 0.971** al 3.08±0.06 al = 0.0775 + 0.4498tl 0.985** hl 1.46±0.04 hl = 0.6664 + 0.3182tl 0.957** e. danricus (tl = 5.14 ± 0.09) sl 4.07±0.07 sl = 0.1544 + 0.7488tl 0.986** dl 2.69±0.05 dl = 0.2761 + 0.4690tl 0.960** pl 1.05±0.01 pl = 0.6211 + 0.0829tl 0.909** pvl 2.06±0.03 pvl = 0.4480 + 0.3131tl 0.954** al 2.93±0.05 al = 0.1824 + 0.5345tl 0.983** hl 0.79±0.02 hl = 0.2468 + 0.1054tl 0.921** m. pancalus (tl = 9.53 ± 0.15) sl 8.87±0.14 sl = 0.0425 + 0.9345tl 0.990** dl 1.95±0.04 dl = 0.1427 + 0.2199tl 0.916** pl 1.68±0.02 pl = 0.2289 + 0.1517tl 0.939** pvl al 5.40±0.08 al = 0.1262 + 0.5535tl 0.981** hl 1.58±0.03 hl = 0.0587 + 0.1592tl 0.950** l. guntea (tl = 7.17 ± 0.09) sl 5.95±0.09 sl = 0.5553 + 0.9074tl 0.993** dl 3.00±0.05 dl = 0.3927 + 0.4729tl 0.961** pl 1.04±0.01 pl = 0.4795 + 0.0784tl 0.914** pvl 3.03±0.05 pvl = 0.2697 + 0.4602tl 0.975** al 4.67±0.07 al = 0.3368 + 0.6981tl 0.990** hl 0.90±0.02 hl = 0.2040 + 0.1539tl 0.919** c. fasciatus (tl = 4.97 ± 0.18) sl 3.74±0.13 sl = 0.1033 + 0.7301tl 0.997** dl 1.42±0.05 dl = 0.1767 + 0.2500tl 0.962** pl 1.22±0.04 pl = 0.1229 + 0.2201tl 0.982** pvl 0.98±0.03 pvl = 0.1912 + 0.1597tl 0.972** al 1.41±0.05 al = 0.0767 + 0.2685tl 0.979** hl 1.12±0.04 hl = 0.1357 + 0.1982tl 0.962** p. sophore (tl = 6.90 ± 0.12) sl 5.33±0.10 sl = 0.1640 + 0.7964tl 0.979** dl 2.70±0.05 dl = 0.1470 + 0.4123tl 0.982** pl 1.32±0.02 pl = 0.1379 + 0.1715tl 0.957** pvl 2.60±0.05 pvl = 0.0903 + 0.3890tl 0.962** al 3.86±0.07 al = 0.0841 + 0.5706tl 0.985** hl 1.32±0.03 hl = 0.0414 + 0.1979tl 0.932** h. fossilis (tl = 9.09±0.19) sl 7.96±0.17 sl = 0.0434 + 0.8894tl 0.998** dl 2.63±0.06 dl = 0.1111 + 0.2803tl 0.951** pl 1.24±0.03 pl = 0.1048 + 0.1264tl 0.920** pvl 2.68±0.06 pvl = 0.1957 + 0.2764tl 0.972** al 3.23±0.07 al = 0.1560 + 0.3423tl 0.984** hl 1.27±0.03 hl = 0.0169 + 0.1427tl 0.943** c. punctatus (tl = 8.02 ± 0.18) sl 6.43±0.15 sl = 0.1446 + 0.8194tl 0.988** dl 2.29±0.08 dl = 0.969 + 0.4064tl 0.942** pl 2.21±0.05 pl = 0.1243 + 0.2910tl 0.976** pvl 2.44±0.06 pvl = 0.2148 + 0.3308tl 0.976** al 3.64±0.08 al = 0.0544 + 0.4465tl 0.984** hl 2.38±0.05 hl = 0.1536 + 0.2773tl 0.950** m. cavasius (tl = 6.65 ± 0.08) sl 5.08±0.05 sl = 0.4699 + 0.6937tl 0.984** dl 1.98±0.02 dl = 0.3547 + 0.2440tl 0.910** pl 1.20±0.02 pl = 0.3208 + 0.2289tl 0.953** pvl 2.59±0.03 pvl = 0.0910 + 0.4028tl 0.947** al 3.78±0.04 al = 0.7141 + 0.4611tl 0.923** hl 1.24±0.01 hl = 0.4591 + 0.1172tl 0.921** table 1. relationships with total length among different body lengths of nine species. 152 international journal of aquatic biology (2015) 3(3): 149-154 occur due to age and stage of maturity of the species as well as environmental conditions of habitat such as temperature, salinity and seasonality. such findings were also observed in puntius chola (bhuiyan and biswas, 1982), heteropneustes fossilis (mia, 1984), amblypharyngodon mola (afroze et al., 1992), mystus vittaus (hoque and hossain, 1992), puntius stigma (islam & hossain, 1992), ailia coila (alam et al., 1994), barbus canis (mir, 1996), chand ranga (iqbal et al., 1995-1996), botia lohachata (mortuza and mokarrama, 2000), trachurus mediterraneus (santic et al., 2006), parastromateus niger (dadzie et al., 2008) and p. ticto (hossain et al., 2012). the outcome of the present study has provided some new and updated information on the morphometric characters of nine freshwater fish species of nageshwari, bangladesh. though the fishes are small, they have very good consumer demand with relatively high nutritional value. findings of the present investigation may play an important role in country economy being key factors for the management and conservation of these species as well as other fish species of bangladesh. acknowledgement we are grateful to the chairman, department of fisheries, university of rajshahi for providing all sorts of lab facilities during the study period. references afroze s., hossain m.a., parween s. 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(2008). length-weight and length-length relationships of five fish species collected from seagrass beds of the sungai pulai estuary, peninsular malaysia. journal of applied ichthyology, 24: 328species total length characteristics total weight characteristics parameters of the relationship fulton’s condition factor range (cm) mean±se range (cm) mean±se 𝛼 b r (k) g. giuris 4.70-9.10 6.68 ± 0.13 1.10-5.85 2.77 ± 0.15 0.017466 3.011 0.980** 1.50 e. danricus 3.20-6.80 5.14 ± 0.09 1.00-1.30 1.14 ± 0.01 0.050511 2.812 0.904** 0.93 m. pancalus 7.00-13.70 9.53 ± 0.15 2.60-4.20 3.25 ± 0.04 0.059340 2.852 0.917** 0.99 l. guntea 4.30-8.30 7.17 ± 0.10 1.50-4.71 3.58 ± 0.10 0.008330 2.840 0.913** 0.98 c. fasciatus 3.10-7.80 4.97 ± 0.18 0.65-4.06 1.99 ± 0.16 0.004818 3.030 0.990** 1.15 p. sophore 5.20-9.50 6.90 ± 0.12 2.42-7.65 4.34 ± 0.29 0.001380 2.990 0.988** 1.32 h. fossilis 5.50-15.50 9.00 ± 0.19 1.50-9.50 4.26 ± 0.19 0.047410 2.680 0.942** 0.96 c. punctatus 5.20-11.20 8.02 ± 0.18 2.50-11.19 6.21 ± 0.32 0.068620 2.840 0.959** 1.19 m. cavasius 5.90-8.10 6.65 ± 0.08 2.40-3.60 2.93 ± 0.04 0.033610 2.650 0.904** 1.02 table 2. length-weight relationships and fulton’s condition factors of nine species. 153 ferdaushy and alam/ morphometric relationship and condition of nine freshwater fishes of bangladesh 329. bagenal t., tesch f.w. 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(2004). lengthweight relationship of feather back, notopterus notopterus (pallas) from jannapura pond, karnataka, india. zoos’ print journal. 19(4): 1449-1450. lagler k.f. (1966). freshwater fishery biology (2nd edn.). co. dubuque, iowa, 421 pp. le-cern e.d. (1951). length-weight relationship and seasonal cycle in gonad weight and condition in the perch (perca fluviatilis) journal of animal ecology, 20: 201-219. mia g.k. (1984). length-weight relationship and condition factor in the air-breathing catfish, heteropneustes fossilis (bloch). bangladesh journal of zoology, 12(1): 49-52. mir s. (1996). length-weight relation of a carp, barbus canis (cuvier and valenciennes). bangladesh journal of zoology, 24 (1): 89-90. mortuza m.g., mokarrama n.t. (2000). notes on the length-weight relationship and condition factor of mud loach botia lohachata (chaudhuri) (cypriniformes: cobitidae). university journal of zoology rajshahi university, 19: 113-114. mustafa m.g., brooks a.c. 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(2010). size, growth and age of two congeneric archer fishes (toxotes jaculatrix pallas, 1767 and toxotes chatareus hamilton, 1822) inhabiting malaysian coastal waters. sains malaysiana, 39(5): 697-704. sparre p., ursin e., venema s.c. (1989). introduction to tropical fish stock assessment. part 1. manual. fao fisheries technical paper no. 306, rome, 429 pp. tandon k.k., johal m.s., bala s. (1993). morphometry of cirrhinus reba (hamilton) from kanjli wetland, punjab, india. research bulletin of the punjab university, science. 43(1-4): 73-78. tandon k.k., johal m.s., bala s. (1993). morphometry of cirrhinus reba (hamilton) from kanjli wetland, punjab, india. research bulletin of the punjab university, science, 43 (1-4): 73-78. international journal of aquatic biology (2014) 2(5): 246-252 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2013 npajournals. all rights reserved original article effects of short-term starvation and re-feeding on antioxidant defense status in mesopotamichthys sharpeyi (günther, 1874) fingerlings asieh najafi1, amir parviz salati*1, vahid yavari1, farzad asadi2 1department of fisheries, faculty of marine natural resources, khorramshahr university of marine science and technology, khorramshahr, iran. 2department of biochemistry, faculty of veterinary medicine, university of tehran, tehran, iran. article history: received 4 august 2014 accepted 7 september 2014 available online 2 5 october 2014 keywords: starvation re-feeding oxidative stress mesopotamichthys sharpeyi enzymes abstract: the objective of this study was to evaluate the impact of short-term starvation and re-feeding on oxidative stress in mesopotamichthys sharpeyi (günther, 1874). after two weeks adaptation to new conditions, a total number of 270 fingerlings were distributed into nine 300-l fiberglass tanks, equipped with aeration system in three treatments including 4, 8 and 16 days starvation (each in 3 replicates). after starvation, all groups were fed for 32 days. at the beginning of trial and at the end of starvation and re-feeding periods, blood samples were collected, plasma was separated and activity of catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gpx) and malondialdehyde (mda) content were assayed. based on the results, no significant difference was observed in sod activity at the end of starvation and also after re-feeding between the different treatments. at the end of starvation the activity of the cat and gpx increased significantly in 8 and 16 days of starvation groups compared to the base. moreover, a significant increase in mda levels of plasma was found during food deprivation. these findings showed that short-time starvation increased antioxidant enzymes activity in plasma of m. sharpeyi but short-term food deprivation and re-feeding periods in this species does not cause disturbances in the antioxidant defense status. introduction study of oxidative stress indices can show the physiological and health status of fish in starvation and re-feeding. thus, the complete information provides regarding the optimal duration of these changes and return to the initial situation for any species (furne et al., 2009). most of investigations on oxidative stress in fish has been done on the aspects of toxicology, such as the influence of various xenobiotics and heavy metals on antioxidant defense system (bayir et al., 2011), the comparison of biological transformation processes, on the intensity of lipid peroxidation and other biomarkers of oxidative stress (trenzado et al., 2006). also, other studies have been evaluated the effects of season or another stimulus on the activity of antioxidant enzymes (trenzado et al., 2006). on the * corresponding author: amir parviz salati e-mail address: salatia@gmail.com other hand, most of researches on fasting and refeeding in fish are also concentrated on the muscular growth and metabolic and hormonal responses. newly, the issues related to the impact of food restriction on hematological, biochemical, immune parameters and indices of antioxidant defenses system has been investigated (pascual et al., 2003; morales et al., 2004; rios et al., 2005; feng et al., 2009; caruso et al., 2011). starvation as a stressor activates the axis of hypothalamus-pituitaryinterrenal, leading to increased release of glucocorticoid hormones and subsequently cause changes in fluid balance, function of cardiovascular and respiratory system and balance of peroxidation– antioxidant (vázquez-medina et al., 2010). furthermore, nutritional restriction augments poisoning of chemicals and disease outbreaks in 247 najafi et al/ effects of starvation and re-feeding in mesopotamichthys sharpeyi certain pathological conditions. these effects could be essentially related to the increment of ros that are created by food shortage (pascual et al., 2003). oxidative stress occurs as a result of increased rate of ros generation or decreased level of antioxidant defense, or both situations (langseth, 1995). the poor food supply and starvation could cause the enhancement of oxidative damage of lipids, oxidative stress (welker and congleton, 2005) and activation of antioxidant defense in fish (bayir et al., 2011). many of fish increase antioxidant enzymes activity, when encountered with unendurable starvation for adaptation to new circumstances (wilhelm filho, 2007). it is well known that most important antioxidant enzymes include superoxide dismutase (sod) which converts superoxide radical (o2•−) to hydrogen peroxide (h2o2); catalase (cat), which detoxifies h2o2, glutathione peroxidase (gpx) which detoxifies both h2o2 and organic hydroperoxides and glutathione reductase (gr), which accelerate the transformation of oxidized glutathione (gssg) to reduced glutathione (gsh) (morales et al., 2004). these enzymes destroy ros or convert it into metabolites that have less toxicity (romero et al., 2011). while the evidence suggests that oxidative stress may be involved in causing various diseases in fish, and since few studies have been conducted in the field of the effects of short-term periods starvation and refeeding on the antioxidant defense in fish. in this study the influence of the short-term food deprivation and re-feeding on oxidative stress indices in mesopotamichthys sharpeyi (günther, 1874) has been studied. material and methods fish and rearing condition: the present experiment was carried out on m. sharpeyi fingerlings with mean weight of 6.13 ± 0.40 g. before the beginning of the experiment, the fish were disinfected with brackish water, and were maintained for 2 weeks in 300-l tanks for acclimatization to laboratory conditions. during this period fish were fed with a commercial diet (bayza, shiraz, iran) containing 41.04% crude protein, 5.83% crude fat and 11.45% ash, two times per day (9:00 am and 17:00 pm). following the adaptation period, fish were weighed individually and distributed randomly to nine 300-l fiberglass tanks (40 fish/per tank) that equipped to aeration system. experimental design: to investigate the effects of starvation and re-feeding on blood parameters, three treatments including 4, 8 and 16 days starvation were considered with 3 replications. after starvation all groups were fed for 32 days. the duration of experiment was 48 days. the experimental design was performed such that re-feeding period was started in all treatments at same time. tanks were placed in an indoor system to protect from the direct solar radiation. during re-feeding period, fish were fed to apparent satiation twice a day. water temperature (25.6 ± 0.1ºc), dissolved oxygen (7.61 ± 0.05 mg/l) and ph (8.05 ± 0.03) were measured and recorded daily during experiment. sampling: fish were deprived one day before sampling procedure. fifteen specimens from each treatment (5 fish from each replicate) were randomly captured and were anesthetized with 2-phenoxyethanol (2%). the blood samples were taken from caudal vein at the beginning of the trial (as basal sample) and at the end of the starvation and refeeding periods. blood samples were centrifuged immediately at room temperature, plasma was separated and stored at -80ºc until analysis. analysis: sod activity was measured by a modified method of iodophenyl nitrophenol phenyltetrazolium chloride (ransod kit, randox com, united kingdom). this method employs xanthine and xanthine oxidase to generate superoxide radicals which react with 2-(4iodophenyl)-3-(4nitrophenol)-5-phenyltetrazolium chloride (int) to form a red formazan dye. the superoxide dismutase activity was then measured by the degree of inhibition of this reaction. one of int under the condition of the assay unit of sod was considered a 50% inhibition of reduction. gpx was measured based on paglia and valentine (1967) (ransel kit, randox com, united 248 international journal of aquatic biology (2014) 2(5): 246-252 kingdom). gpx catalyzes the oxidation of glutathione (gsh) by cumene hydroperoxide. in the presence of glutathione reductase (gr) and nadph, the oxidized glutathione (gssg) is immediately converted to the reduced form with a concomitant oxidation of nadph to nadp+. the decrease in absorbance was measured at 340 nm. catalase activity was measured according to aebi (1984). briefly, tissue sections were homogenized in triton x-100 1% (merck, darmstadt, germany) and the homogenates were diluted with phosphate buffer (ph 7.0). the reaction was initiated by addition of hydrogen peroxide to the reaction mixture and the level of enzyme activity was quantitated according to the ability of the tissue catalase to decompensate hydrogen peroxide by monitoring the decrease in absorbance at 240 nm against a blank contains phosphate buffer instead of substrate. the value of log a1/a2 for a measured interval was used for unit definition owing to the first-order reaction of enzyme. one unit of cat is the amount of enzyme that decomposes 1.0 mmole of hydrogen peroxide per minute at ph 7.0 and 25ºc. the total amount of plasma lipid peroxidation was indicated by the content of mda as described by buege and aust (1978). briefly, one volume of plasma was mixed with two volumes of a stock solution of 15% w/v trichloroacetic acid, 0.375% w/v thiobarbituric acid and 0.25 mol) hydrochloric acid thoroughly. the solution heated for 15 min in boiling water bath. after cooling, the precipitate is removed by centrifugation at 1000 g for 10 min. the absorbance of the clear supernatant determined at 535 nm. data were expressed as mean ± se (standard error). means were analyzed by one way analysis of variance (anova) test using spss 16, and where significant difference were indicated, means were tested using tukey’s post hoc to compare the means of treated groups against that of the corresponding control. p<0.05 was the accepted significance level. results the specific activity of the antioxidant enzymes is figure 1. effects of starvation and re-feeding on sod activity (a), cat activity. (b) and gpx activity (c) in mesopotamichthys sharpeyi (günther, 1874). similar capital letters represents no significant differences between the base sample and other experimental groups at the end of starvation and after re-feeding (p>0.05). small letters indicate changes in the activities of these enzymes in each treatment. data are presented as mean ± se. differences were considered statistically significant at the 0.05 probability level 249 najafi et al/ effects of starvation and re-feeding in mesopotamichthys sharpeyi shown in figure 1(a-c). no significant difference was found in the activity sod at the end of the fasting and re-feeding period compare to base (p>0.05) (fig. 1a). the comparison of sod activity in each treatment represents a significant decline only in the 16 days starvation after re-feeding (p<0.05). the cat activity significantly increased in 8 and 16 days starvation groups compared to the base and returned to initial values after re-feeding (p<0.05) (figs. 1b and 1c, respectively). moreover, comparison of variations in the activity of cat in each treatment, indicates significant differences in the food-deprived groups of 8 and 16 days, although in 4-days starvation group no significant difference were recorded at the end of fasting and re-feeding period (p>0.05). similar results were recorded for gpx, as its activity increased in 8 and 16 days starvation groups compare to base. gpx activity was decreased in these groups after re-feeding period. as presented in figure 2, the plasma content of mda significantly increased as starvation time increased (p<0.05). whereas, no significant differences were detected between the different starved groups and the base level after re-feeding (p>0.05).the statistical analysis of changes of plasma mda in each treatment represents significant decrement of this index in the three experimental groups (p<0.05). discussion in the present study, the gpx and cat activities increased along with the extension of the starvation period. increased activity of cat and gpx simultaneously, represents increased production of h2o2 throughout the cell (ritola et al., 2002). bayir et al. (2011) reported that total or partial food deprivation enhances the production of ros in liver and gills of brown trout (salmo trutta). moreover, enhanced ros rates have been reported as a consequence of starvation in sparus macrocephalus (zhang et al., 2007) and yellow croaker (pseudosciaena crocea) (zhang et al., 2008). like m. sharpeyi, limited food availability was caused an augmented production of h2o2 in wistar and gotokakizaki (gk) rats (santos et al., 2009). parihar et al. (1997) concluded that oxidative agents in cells may lead to enhancement of antioxidant enzymes as a defense mechanism. stimulation of antioxidant enzymes have been recognized as an important defensive line against oxidative stress in biological systems (velisek et al., 2011). in fact, when an organism is exposed to conditions that enhances the ros generation rates, will produce and activate more antioxidant enzymes to cope with additional reactive oxygen species. when the aerobic organisms were faced with energetic restrictions such as food shortage and/or severe hypoxia, maintain relatively high levels of their antioxidant components to deal with oxidative stress (ansaldo et al., 2007). pascual et al. (2003) observed a significant increase in the sod and gpx activities after food deprivation in gilthead seabream (sparus aurata). in common dentex (dentex dentex), morales et al. (2004) found a marked increase in activities of sod, cat and gpx after 5 weeks food restriction. a similar increase in the activity of sod, cat and gpx was found in the liver and gills of brown trout (salmo trutta) (bayir et al., 2011). figure 2. the content of plasma mda in mesopotamichthys sharpeyi (günther, 1874) fingerlings during the short-time starvation and re-feeding. similar capital letters represents no significant differences between the base sample and other experimental groups at the end of starvation and after re-feeding (p>0.05). small letters indicate changes of the plasma mda in each treatment. data are presented as mean ± se. differences were considered statistically significant at the 0.05 probability level. 250 international journal of aquatic biology (2014) 2(5): 246-252 guderley et al. (2003) also detected that fasting leads to increase gpx and gst activities in the liver of the starved atlantic cod (gadus morhua). increased gpx activity had been reported in wistar and gotokakizaki (gk) rats following food deprivation, whereas no observed any significant in the mn-sod activity in the both strains. sod, cat, gpx and gr are key enzymes in the antioxidant defense system (furné et al., 2009). sod converts superoxide anion (o2.-) to water (h2o) and hydrogen peroxide (h2o2). hence, increased sod activity in the starved fish represents the production the high levels of o2.-.on the other hand, increased sod activity is led to an incensement in the production of h2o2, which could be the reason for the increase in cat and gpx activities (pascual et al., 2003; morales et al., 2004; zhang et al., 2007, 2008; bayir et al., 2011). cat is a key antioxidant enzyme to remove h2o2 and also a basic mechanism to restrict the formation of hydroxyl radicals highly reactive (regoli et al., 2000b). increment in cat and gpx activities in the current study may be not only due to increased h2o2, but also caused by diminish in lipid peroxidation that has led to oxidative stress. inal et al. (2001) expressed that the increase in gpx activity could be related to lipid peroxidation. in addition, the high activities of cat and gpx have been reported to reduce lipid peroxidation in mammals and fish (barja de quiroga et al., 1989; rodriguez-ariza et al., 1993; regoli et al., 2005). valko et al. (2006) reported that peroxisomal fatty acid oxidation is a potentially important source of h2o2 production during prolonged starvation. hence, according to the results, the increase in cat activity and gpx, and no significant changes in the activity of sod could be due to the formation the excess h2o2 by the metabolism of peroxisomal long chain fatty acids. oxidative damage often assessed through the determination of lipid peroxidation in the form of mda (huang et al., 2010). mda is one of the compounds resulting from lipid peroxidation that is formed as a result of the decomposition of lipid peroxides in the presence of iron or copper during the fenton reaction (rumely and paterson, 1998). based on the results, the plasma mda content increased in parallel with food deprivation. increased plasma mda content in the present study represents increased lipid peroxidation and oxidative stress which correspond with the observed increase in cat activity and gpx. similar findings previously have been obtained in rainbow trout (o. mykiss) and sea bream (s. aurata) deprived of food that starvation caused an increase in mda levels in the liver (hidalgo et al., 2002; pascual et al., 2003). also, morales et al. (2004) found that long-term food deprivation increases the mda level in the liver of starved d. dentex compared to the control group. furthermore, bayir et al. (2011) in a study on brown trout (s. trutta) observed that mda level in the gill and liver rises with increasing time of starvation. a remarkable increase in the lipid peroxidation indices can show high sensitivity of lipid molecules into ros attack and determine the amount of oxidative damage imposed to these molecules (velisek et al., 2011). in the present study, a positive relationship was recorded between enzyme activities and time of starvation. increase in the activity of these enzymes could be due to increased use of lipid reserves that followed by augmented production of ros. activity of evaluated enzymes returned to normal after refeeding showing compensatory capacity of m. sharpeyi. references aebi h. (1984). catalase in vitro. methods in enzymology, 105: 121-126. ansaldo m., sacristán h., wider e. 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(2016) 4(1): 57-68 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article genetic characterization of garra rufa (heckel, 1843) populations in tigris basin, iran using microsatellite markers hamed kolangi miandare1, ghasem askari*1, ali shabany1, hamid reza rezaei2 1department of fisheries, gorgan university of agricultural sciences and natural resources, gorgan, iran. 2department of environmental science, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 7 november 2015 accepted 29 january 2016 available online 2 8 february 2016 abstract: the isolation-by-distance theory states that the genetic differentiation between individuals raised by increasing geographic distance. therefore, this study tested this hypothesis for garra rufa, a freshwater fish species of iranian inland waters, from six rivers located at the different distances in tirgis basin. for this purpose, eight variable microsatellite loci were applied to identify geographic-based population structure of g. rufa. from 240 fish of six populations, 102 alleles were found with a mean number of 11.625 to 13.250 alleles. heterozygosity was ranged 0.567-0.638 in six studied populations. moreover, a significant deviations from hardy-weinberg were found in the studied populations. unweight pair group analysis indicated that the six studied populations could be divided into four major clusters. the results revealed a fairly high level of genetic variation in the microsatellite loci within six studied populations. wright’s fixation index (fst) ranged between 0.013-0.044 indicating little genetic differentiation between populations. within this range, however, we found a strong positive relation between fst and geographical distance lending support to the isolation-by-distance theory. keywords: genetic diversity microsatellite doctor fish tigris basin iran introduction genetic diversity is one of the important indicators of ecological condition in aquatic ecosystems and has been considered as a useful and powerful tool for evaluation and management of biological communities (avise, 2000). it is one of three levels of biodiversity, proposed by the global conservation organization for the stocks conservation program (lucentini et al., 2009). since higher genetic diversity may lead to an increase in the survival rate of natural populations (zoller et al., 1999), maintaining genetic diversity in these populations is crucial for conservation biologists. of the common dna markers used to study genetic diversity at the molecular levels, microsatellite markers are especially informative (chen et al., 2008). because of unique features such as high polymorphism, high scope in genome, and high mutation rates, the microsatellite markers have * corresponding author: ghasem askari doi: http://dx.doi.org/10.7508/ijab.2016.01.008 e-mail address: askarighasem82@gmail.com been widely applied in population genetic studies (li et al., 2009). microsatellite markers have been identified in the genomes of many species, and widely used in relation to species with an economic value (wang et al., 2012). the application of these markers includes aspects of evolutionary biology, population genetics, ecology and pedigree identification in populations (cui et al., 2005; cruz et al., 2005; maremi et al., 2005). there are 257 fish species in the inland waters of iran (joulade-roudbar et al., 2015), which mostly belong to the family cyprinidae. garra rufa is the member of cyprinidae and occurs in the river basins of the northern and central middle east (keivany et al., 2015; mousavi-sabet and eagderi, 2016). this species has been utilized in psoriasis treatment (ündar et al., 1990) and are preyed by piscivorous fishes such as anguilla anguilla and clarias gariepinus in their habitats (yalçin-özdilek, 2007). 58 kolangi miandare et al./ genetic characterization of g. rufa populations in tigris basin, iran despite the ecological importance of g. rufa, the studies on its genetic structure and demography remain rudimentary. isolation by distance (ibd) model has been extensively used to study spatial patterns of genetic variation in natural populations (e.g., crispo and hendry, 2005; storfer et al., 2010). according to the ibd theory, the genetic distance between individuals or populations will increase with decreasing in gene flow as a result of increasing geographic distance between them (wright, 1943; rousset, 1997). this restriction of gene flow can finally result in speciation events (coyne and orr, 2004). although ibd has been detected in a range of species groups, including fishes, plants, oysters, beetles, and mammals across both small and large geographical scales (angers and bernatchez, 1998; launey et al., 2002; bockelmann et al., 2003; peakall et al., 2003; buonaccorsi et al., 2004; oleksa et al., 2012), some studies have not supported this theory (e.g., peterson, 1995; peter and slatkin, 2013). here, we tested ibd hypothesis by comparing genetic structure of six populations of g. rufa from six rivers located in different distances from each other in the tirgis basin of iran. materials and methods during october and november 2011, a total of 240 samples (40 specimens per population) were collected using cast net from six rivers belonging to three river drainages, including kabkiyan river (30°51'n, 51°19'e; population 1), berim river (30°19'n, 51°15'e; population 2), fahlyan river (30°11'n, 51°31'e; population 3), beshar river (30°26'n, 51°46'e; population 4); sarab bahram river (30°05'n, 51°26'e; population 5), and shahpour river (29°45'n, 51°33'e; population 6) (fig. 1). the average distance (± sd) between sampling sites was 98±44 km (range 30-180) (table 1). a small piece of the pelvic and pectoral fins of the specimens were removed and fixed in 95% ethanol and the fish were released. total genomic dna was extracted and stored at -20°c from tissues using the traditional proteinase-k digestion and standard phenol/chloroform techniques based on hillis et al. (1996). the extracted dna was analyzed by electrophoreses via a 0.6% agarose gel containing 5 μg ml-1 ethidium bromide. according to crooijmans et al. (1997) and matura et al. (2012), eight microsatellite loci, comprising mfw17, ggm002, ggm007, ggm023, ggm024, ggm027, ggm034 and ggm045 were used (table 2). pcr amplification was performed in total reaction volume of 25 ml containing 2.0 mm mgcl2, 0.2 mm dntp mix, 0.2 mm each primer, 1 u taq dna polymerase, 1 x pcr buffer, approximately 100 ng template dna and deionized water. initial denaturation was achieved at 94°c for 3 min followed by 30 cycles of denaturation in 30 seconds at 94°c, 30 seconds at the respective annealing temperatures, and extension to 72°c for 1 min. the final step was extended for 3 min at 72°c. pcr products were separated using 8% polyacrylamide gels stained with silver nitrate (bassam et al., 1991). the observed number of alleles (na), the effective number of alleles (ne), observed heterozygosity figure 1. sampling sites of garr rufa from tirgis basin in iran (1kabkiyan river, 2-berim river, 3-fahlyan river, 4-beshar river, 5-sarab bahram river, and 6-shahpour river). pop2 pop3 pop4 pop5 pop6 pop1 130 125 30 150 180 pop2 80 100 90 100 pop3 90 30 90 pop4 90 150 pop5 40 pop6 table 1. sampling locations and distance between sampled rivers (km). 59 int. j. aquat. biol. (2016) 4(1): 57-68 (ho), expected heterozygosity (he), number of migrant (nm), nei’s genetic distance, wright’s fixation index (fst), genetic identity, inbreeding coefficient (fis) and hardy–weinberg equilibrium (hwe) were calculated by genealex (ver.6.5) software (peakall and smouse, 2012). the unweighted pair group method with arithmetic mean (upgma) diagram based on the matrix of genetic distances between populations was produced by ntsys (ver.2.2) (smýkal et al., 2008). the null allele frequency was estimated using microchecker (ver.2.2.3) software (van-oosterhout et al., 2004). the hardy-weinberg equilibrium tests were adjusted using the sequential bonferroni correction (rice, 1989). analysis of molecular variance (amova) was calculated using arlequin (ver. 3.1) to evaluate genetic diversity (excoffier et al., 2005). amova is a suitable approach to determine the population structure and genetic differentiation between populations (grassi et al., 2004). effective population size reductions were evaluated using bottleneck (ver. 1.2.02) (cornuet and luikart, 1996). the distance matrix was then used to construct a upgma dendrogram using the software popgene (ver.1.31) (yeh et al., 1999). this program examines discrepancy between the observed heterozygosity and expected heterozygosity based on the observed number of alleles using twotails model (s.m.m.). results in 240 individuals of the six studied populations, 102 alleles were observed. the average number of alleles per locus was 12.750. the highest (0.638) and lowest (0.567) average heterozygosity was found in the populations 1 and 2, respectively, whereas the highest (0.876) and lowest (0.840) expected heterozygosity (he) were found in the populations 2 and 6, respectively (table 3). thirty-seven out of 48 loci showed consistent significant deviations from hardy-weinberg equilibrium expectations in populations after the probability level (p<0.05) (table 3). locus ggm002 and ggm024 in the populations 1, 3, 5 and 6; ggm002 and ggm045 in the population 2, and ggm024 in the population 4 were within hwe. fixation index (fis), a measure of heterozygote deficiency or over-plus (inbreeding co-efficient), was often larger than zero, showing a deficiency of heterozygotes in most of loci in all populations (table 3). according to fis values, there were no significant differences between regions. there were microsatellite loci gene bank accession no. primer sequence n size (bps) anneal (°c) mfw17 mfw17 f: ctcaactacagagaaatttcatc r: gaaatggtacatgacctcaag 9 112 232 46 ggm002 hq288485 f:cactttgtccttgccattga r:ctcaacaccgtggactctca 25 200 344 55 ggm007 hq288490 f:gctgtgctgactggcactt r:caaaccaacatttcatcaaaaa 11 232 300 52 ggm023 hq288506 f:tcaccatccactgaagacca r:gaaatatgtaacgtcattaattgtgtg 9 96 136 53 ggm024 hq288507 f:tccctctttttgctctcagg r:taggtgaacaaatggcatgg 14 128 208 52 ggm027 hq288510 f:tcggtgcacccctagtaaac r:ccaagtgtgtgtttggatgg 12 188 252 54 ggm034 hq288517 f:cgcgcaagtttctttcagtt r:gctgtgagacaagcctaaacc 11 128 184 56 ggm045 jf268662 f:tctcatgggtctctgggttc r:tgtgcagaaaggctgttgag 11 152 200 53 n: number of alleles table 2. characteristics of the used microsatellite loci in this study. 60 kolangi miandare et al./ genetic characterization of g. rufa populations in tigris basin, iran no significant indications of a recent reduction in the effective population size according to the s.m.m test in any of the populations and there was no evidence of any genetic bottlenecks (table 4). examination of genotyping errors revealed no evidence for large allelic dropout or stutter-band scoring at any of the eight loci. at the loci ggm007, ggm023, ggm027 and mfw17, null alleles might have appeared. for removing possible bias in results we repeated our analysis, excluding loci that showed null alleles in all populations. since the results remained the same, therefore we retained all loci for the analysis. population number mfw17 ggm002 ggm007 ggm023 ggm024 ggm027 ggm034 ggm045 na 10 23 12 8 13 13 10 9 pop 1 ne 5.939 17.231 9.011 6.644 9.446 8.711 4.795 5.407 ho 0.393 1.000 0.357 0.750 0.929 0.321 0.643 0.714 he 0.832 0.942 0.889 0.849 0.894 0.885 0.791 0.815 fis 0.528 0.062 0.598 0.117 0.039 0.637 0.188 0.124 phw *** ns *** *** ns *** *** ** na 7 24 9 8 15 8 11 11 pop 2 ne 4.159 18.892 5.851 5.091 12.346 4.709 7.502 5.244 ho 0.250 0.964 0.107 0.500 0.857 0.429 0.714 0.714 he 0.760 0.947 0.829 0.804 0.919 0.788 0.867 0.809 fis 0.671 0.018 0.871 0.378 0.067 0.456 0.176 0.117 phw *** ns *** *** ** *** *** ns na 11 22 13 8 15 15 10 9 pop 3 ne 9.064 16.860 7.127 3.655 10.116 9.924 7.362 5.580 ho 0.714 1.000 0.250 0.357 0.929 0.357 0.857 0.464 he 0.890 0.941 0.860 0.726 0.901 0.899 0.864 0.821 fis 0.197 0.063 0.709 0.508 0.030 0.603 0.008 0.434 phw *** ns *** *** ns *** *** ** na 7 25 10 9 11 11 13 11 pop 4 ne 4.780 18.667 6.701 4.284 8.760 5.521 9.503 8.859 ho 0.000 1.000 0.143 0.714 0.857 0.393 0.643 0.786 he 0.791 0.946 0.851 0.767 0.886 0.819 0.895 0.887 fis 1.000 0.057 0.832 0.068 0.032 0.520 0.282 0.114 phw *** ** *** *** ns *** *** *** na 10 30 12 8 14 10 10 12 pop 5 ne 5.297 24.889 7.362 5.209 11.281 6.426 6.348 5.620 ho 0.321 1.000 0.393 0.571 0.964 0.197 0.750 0.464 he 0.811 0.960 0.864 0.808 0.911 0.844 0.842 0.822 fis 0.604 0.042 0.545 0.293 0.058 0.789 0.110 0.435 phw *** ns *** *** ns *** *** *** na 9 23 11 10 17 15 10 11 pop 6 ne 6.426 16.505 7.575 6.759 11.701 9.800 5.502 7.840 ho 0.393 1.000 0.250 0.714 0.857 0.357 0.429 0.929 he 0.844 0.939 0.868 0.852 0.915 0.898 0.818 0.872 fis 0.535 0.064 0.712 0.162 0.063 0.602 0.476 0.064 phw *** ns *** *** ns *** *** *** na, number of observed alleles; ne, effective number of alleles; ho, observed heterozygosity; he, expected heterozygosity; fis, fixation indices; phw, hardy-weinberg probability test (*p< 0.05, **p<0.01,***p<0.001, n.s., non-significant). table 3. genetic variability of eight microsatellite loci in six studied populations for garra rufa. wilcoxon test s.m.m mode-shift pop 1 0.8432 no pop 2 0.7421 no pop 3 0.5468 no pop 4 0.7421 no pop 5 0.1953 no pop 6 0.7426 no table 4. analysis of the possibility of a recent bottleneck under two tails for h excess or deficiency. 61 int. j. aquat. biol. (2016) 4(1): 57-68 the average level of genetic differentiation between populations, as indicated by fst, was 0.039. there was a significant relationship between genetic divergence (pair-wise fst) and geographical distance (r2 = 0.93, p<0.001, fig. 2). pair-wise fst estimates between population pairs differed significantly (p<0.01) from zero for all the pairs in populations (table 5). analysis of molecular variance (amova) showed that 97% of the observed variation was found within populations and only 3% of the variation was observed between populations. the results revealed high levels of gene flow (nm) between populations (table 6). on the basis of nei's (1978) genetic distance values (table 7), an upgma dendrogram was created displaying four major clusters (fig. 3). most of the similarity was observed between populations 1 and 4 in cluster d and between populations 3 and pop 1 pop 2 pop 3 pop 4 pop 5 pop 6 pop 1 0.035** 0.033** 0.013** 0.039** 0.044** pop 2 0.023** 0.031** 0.025** 0.027** pop 3 0.027** 0.013** 0.030** pop 4 0.025** 0.040** pop 5 0.023** pop 6 **p<0.01 table 5. pairwise fst between six studied populations of garra rufa based on eight microsatellite loci. pop 1 pop 2 pop 3 pop 4 pop 5 pop 6 pop 1 0.000 pop 2 12.386 0.000 pop 3 9.831 13.888 0.000 pop 4 6.192 7.422 5.949 0.000 pop 5 9.157 9.748 10.460 6.802 0.000 pop 6 19.106 7.691 10.788 5.385 9.043 0.000 table 6. pairwise population nm values based on fst values between six studied populations. pop 1 pop 2 pop 3 pop 4 pop 5 pop 6 pop 1 0.272 0.316 0.404 0.354 0.208 pop 2 0.762 0.260 0.355 0.348 0.374 pop 3 0.729 0.771 0.426 0.330 0.293 pop 4 0.668 0.701 0.653 0.402 0.452 pop 5 0.702 0.706 0.719 0.669 0.351 pop 6 0.812 0.688 0.746 0.636 0.704 table 7. nei genetic distance (above diagonal) and genetic identity (below diagonal) on the studied garra rufa populations figure 6. the relationship between geographical distance and genetic differentiation between six studied populations of garra rufa figure 2. upgma dendrogram based on nei’s genetic distance, summarizing the data on differentiation between six studied populations of garra rufa, according to microsatellite dna marker analysis. 62 kolangi miandare et al./ genetic characterization of g. rufa populations in tigris basin, iran 5 in cluster c. the upgma revealed a distinct population structure for sixth population of g. rufa in tigris basin (fig. 3). discussion structure and genetic diversity of g. rufa has been studied using eight microsatellite loci in six populations that showed an average fst of 0.039 (range 0.013-0.044) indicating little genetic differences between studied populations (wright, 1987). in general, the fixation index can range from 0 to 1, where values close to 0 indicate that the populations are sharing their genetic material through high level of breeding and values close to 1 indicate that population do not share any alleles with one another (dewoody and avise, 2000). the population structure of freshwater organisms depends on the distribution of the river systems (nagarajan et al., 2006). the previous studies showed that genetic structuring can happen even across short geographical distances (e.g., angers and bernatchez, 1998; koskinen et al., 2001, 2002; primmer et al., 2006) as similar to the results of the present study. the positive significant relationship between genetic variation and geographical distance revealed that the genetic differentiation between studied populations is raised by increasing geographic distance. in addition, upgma dendrogram revealed that the six studied populations can be divided into four clusters. the populations 1 and 4 were in one group and were not significantly different (cluster d). similarly, populations 3 and 5 were categorized in cluster c and the population 6 (cluster a) was a distinct population from others. these findings indicated that the majority of migration occurred between populations being located at about 30 km from each other. thus, our study lends support to ibd theory because garra populations that lived in close spatial proximity were genetically similar. similarly, using 17 microsatellites, primmer et al. (2006) identified the relatively high level of genetic structuring and significant isolation-by-distance signal between atlantic salmon, salmo salar sampled from the tributaries and main stream of the varzuga river system. in this study, the average waterway distance between sampling site was 60 km (range 5-165 km) and the level of genetic differentiation between sampling locations (fst values) ranged 0.006-0.07, with the global fst being 0.014. our results revealed a fairly high level of genetic variation in the microsatellite loci within six studied populations. although no information is available on genetic diversity of g. rufa using microsatellite markers, durna et al. (2010) applied rflp markers in this fish species. here, the mean observed and expected heterozygosity, and number of alleles per locus were 0.46, 0.56 and 9.1, respectively. the values obtained in this study are higher than those reported by durna et al. (2010) and accordingly represent high genetic diversity of this species. the average observed heterozygosity among six regions was 0.567-0.638, higher than those generally reported in freshwater fish (dewoody and avise, 2000). the average observed heterozygosity across all populations was less than the expected average heterozygosity. the microsatellite data showed that allele diversity and heterozygosity levels are high in studied populations. in genetic diversity investigations, allelic richness is higher than heterozygosity values and high allelic richness represents effective population size (diz and persa, 2009). according to amova analysis, the mean observed number of alleles in the populations 1 to 6 was 12.250, 11.625, 12.875, 12.125, 13.250 and 13.250, respectively. the primer of ggm002 revealed the highest number of allele (25) compared to other primers. mean number of alleles per locus was 12.75 across the 8 microsatellite loci which is generally higher than rates reported for freshwater fish (dewoody and avise, 2000). however, the finding is similar to those of kanapen et al. (2006) in gobio gobio as the average number of alleles per locus ranged from 2 to 13. furthermore, kim et al. (2007) reported a positive linear relationship between microsatellite length and number of alleles as well as the average number of 63 int. j. aquat. biol. (2016) 4(1): 57-68 alleles 11.7 per locus in hemibarbus mylodon. many of the variation in polymorphism at microsatellite loci that exist between species can be ascribed to differences in their population biology and life history traits (neff and gross, 2001). this may be the reason for the observed differences in the number of alleles in the studied populations. in wild populations, fish are often seen deviating from the hardy-weinberg equilibrium (hwe) (lucentini et al., 2006; yue et al., 2004). based on the results, the studied populations deviated significantly from hwe at most of the microsatellite loci (37 out of 48 tests). zhuo et al. (2012) and quan et al. (2007) reported similar results for channa argus and silurus soldatovi, respectively. factors such as inbreeding, intra-population structure (wahlund effect), non-random sampling, fishing pressure, and fish migration have been reasons for deviation from hwe (bergh and getz, 1989; garcia deleon et al., 1995; castric et al., 2002; shao et al., 2002; ruzafa et al., 2006; gopalakrishnan et al., 2009; abbas et al., 2010). non-significant deviation from hwe was reported by israel et al. (2004) as a result of the presence of several stocks of green sturgeon that, in turn, showed the existence of one or more of the ovipositor population. heterozygote deficits could have resulted in null alleles and real biological phenomena including mixing of differentiated wild populations (mandal et al., 2012). the results indicated a null allele in all studied garra populations. examination of the genotyping errors revealed no evidence for large allelic dropout or stutter-band scoring at any of the eight loci. deviation from hardy-weinberg expectations observed in the present study might be a result of non-specific primers usage, mistakes in reading alleles (borrell et al., 2008), presence of migration, genetic drift (bhassu et al., 2004), and the occurrence of null alleles in the populations. as conclusion, this study revealed that the populations have a high genetic diversity and thus its ecological value requires adequate protection. the protection of g. rufa populations in iran, as an important economic and ecological species, requires genetic monitoring to track changes in their genetic diversity. therefore, the results of the present study could be useful as a reference to monitor any future genetic change created by environmental or anthropogenic factors. however, nature of stream habitats and susceptibility to barrier may lead to hierarchical genetic structure in freshwaters organisms. hence, further studies are needed to separate the potential confounding impact that hierarchical structure and ibd have on one another for full understanding of genetic structure in g. rufa populations. acknowledgements we are grateful to r. nadafi for helpful comments on the manuscript. we also thank h.r. rezaei for his advice during the project and z. ghodsi for her assistance in laboratory. this study was funded by gorgan university of agricultural sciences and natural resources. references abbas k., zhou x.y., li y., gao z.x., wang w.m. 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(2016) 4(1): 57-68 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی ریزماهواره نشانگرهای از استفاده با دجله حوضه در( garra rufa) چراغ گل ماهی هایجمعیت ژنتیکی خصوصیات 2، حمیدرضا رضایی1، علی شعبانی*1قاسم عسکری، 1میاندره کلنگی حامد .ایران ،گرگان گرگان، طبیعی منابع و کشاورزی علوم دانشگاه ،آبزیان پرورش و تکثیر گروه1 .ایران ،گرگان گرگان، طبیعی منابع و کشاورزی علوم دانشگاه ،محیط زیست گروه2 چکیده: ماهی عنوانهب چراغگل ماهی برای فرضیه این. یابدمی افزایش جغرافیای فاصله افزایش با افراد بین ژنتیکی تمایز ،فاصله با جدایی تئوری اساس بر ساختار مطالعه جهت ماهواره ریز جایگاه هشت تعداد منظور این برای. گرفت قرار ارزیابی مورد حوضه از مختلف رودخانه شش در ،ایران داخلی آبهای تا 635/11 بین للیآ میانگین گردید، مشاهده نمونه 240 در للآ 102 تعداد. گردید استفاده جغرافیای پراکنش پایه بر چراغگل ماهی ژنتیکی هایجمعیت که گردید مشخص عالوههب. گشت محاسبه 638/0-567/0 حدود در مطالعه مورد جمعیت شش در هتروزیگوسیتی میزان. بود 250/13 جمعیت شش که داد نشان( upgma) وزن بدون جفت گروه تحلیل و تجزیه. دارند واینبرگ-هاردی تعادل از داریمعنی انحراف مطالعه مورد نشان یقتحق این در استفاده مورد هایجایگاه در را ژنتیکی تنوع از باالیی سطح نتایج. گیرند قرار جداگانه کالستر چهار در توانندمی شده مطالعه از لحاص نتایج اساس بر. باشدمی مطالعه مورد هایجمعیت بین پایین ژنتیکی تماییز بیانگر که بود 044/0-013/0 دامنه در fst میزان. دهدمی .نمایدمی تقویت را فاصله با جدایی نظریه که دارد وجود جغرافیای فاصله و fst بین قوی بسیار ای رابطه که گردید مشخص تحقیق این .ایران دجله، حوضه ،ماهی دکتر ماهواره، ریز ژنتیکی، تنوع :کلمات کلیدی int. j. aquat. biol. (2016) 4(2): 80-86; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article heavy metal concentrations in gill and liver tissues of rutilus kutum and chelon aurata in the coast of babolsar, southern caspian sea fatemeh kardel*,1farzaneh mirzapour, shila omidzahir, maryam akhoundian faculty of marine science, university of mazandaran, babolsar, iran. article history: received 22 december 2015 accepted 15 april 2016 available online 2 5 april 2016 keywords: fish tissues zinc cadmium lead caspian sea abstract: heavy metal accumulation in the aquatic ecosystems is a main concern which threats human health. in this study two commercial fish species, rutilus kutum and chelon aurata were selected for assessing heavy metal (cd, pb, zn) concentrations in gill and liver tissues at babolsar’s coast, the southern caspian sea, iran. babolsar is one of the important fishery stations in the southern caspian sea. the results showed that liver tissue of c. aurata significantly accumulated higher concentration of cd, pb and zn compared to that of r. kutum, but these results were not significant for gill tissue. liver tissue accumulated higher concentration of cd and pb compared to gill tissue in c. aurata, but these results were not significant for r. kutum. it is concluded that the liver tissue of c. aurata has higher potential to accumulate heavy metal pollution compared to liver tissue of r. kutum. introduction the caspian sea is a valuable ecosystem, continuously suffering from various sources of pollution, mainly due to the domestic, industrial and agricultural wastewater. among the different pollutants entering the aquatic environment, heavy metals are known as the main important pollutants because of their toxic effects and potential of accumulation in many aquatic species, including fishes (blevins and pancorbo, 1986). metals act differently in aquatic organisms; they can be absorbed, accumulated or excreted via the organs of aquatic organisms. among different heavy metals, lead and cadmium as non-essential elements are highly toxic for living organisms (wood et al., 2012). fishes are very sensitive to the toxic effects of these metals, especially during early life stages (plumlee, 2001). some heavy metals, like zinc is essential element for living organisms. zinc is required for basic biological processes, including proteins, nucleic * corresponding author: fatemeh kardel doi: http://dx.doi.org/10.7508/ijab.2016.02.002 e-mail address: f.kardel@umz.ac.ir acids, carbohydrates and lipids metabolisms and also contributes in the functions of immunity, neurotransmission and cell signaling (murakami and hirano, 2008). however, the high amount of this metal can be toxic for fishes and other aquatic organisms (bodar et al., 2005). the liver, kidney and gill as the most sensitive tissues to toxic elements that absorb the high amounts of metals and transmit them to the other tissues through blood circulation (karaytug et al., 2007). due to direct contact of the large surface of gill tissues with aquatic environment, they are susceptible to absorb high amount of metals and can be used as suitable tissue for surveying the impact of pollutants in fish (heath, 1995; simonato et al., 2008). liver as high sensitive tissue to pollutants is used as a suitable bio-indicator of environmental pollutions in organisms (thophon et al., 2003; camargo and martinez, 2007). accumulation of metals in living organisms depends on different factors such as organism size, diet, metabolic 81 int. j. aquat. biol. (2016) 4(2): 80-86 activities, different life stages, concentration of heavy metals in water and sediment, fishing season (canli and atli, 2003). they may also depend on different chemical and physical characteristics of water and sediment such as ph, alkalinity, hardness, organic and inorganic materials (wood et al., 2012). according to various sources of pollutions, mainly due to untreated domestic wastewater and agricultural activity in the southern caspian sea, high amount of absorption and accumulation of heavy metals by aquatic organisms is possible. therefore, this study aimed to evaluate the accumulation of pb, cd, and zn in gill and liver tissues of two commercially important fishes, including rutilus kutum and chelon aurata at the babolsar’s coast, the southern caspian sea. materials and methods sampling and sample preparation: twenty male fishes of r. kutum and c. aurata were collected from the southern caspian sea (babolsar᾿s coast) in march 2015. the selected fish species are the most important commercial fishes in the southern caspian sea. the collected fishes were immediately transferred to a laboratory where their total length, fork length, standard length (in cm) and the weight (in gram) were measured using a biometric ruler, and an electronic digital balance with an accuracy of 0.01, respectively. the liver and gill tissues were carefully dissected and washed with distilled water. the washed tissues were put into a petri dish and dried in the oven at 70°c for 5-6 days to obtain the constant weight. chemical analysis: the dried tissues were homogenously powdered by a mortar and stored in a polyethylene container. for the digestion of tissue samples, 8 ml of hno3 (merck, darmstadt germany) was added to 1 g of each powdered sample tissue, and this solution was placed in an oil bath at 100°c for one hour. the solution was removed from the oil bath and after temperature reduction, 2 ml of h2so4 was added to the solution and placed in an oil bath at 100°c for one hour to complete dissolving the samples. the solution was passed through the watman filter paper with a pore size of 42 µm, then its volume increased to 25 ml with distilled water. it was injected into the atomic absorption spectrophotometer (analytik jena, model novaa 400, germeny) for the determination of metals concentrations. the metals concentrations were presented as mg.kg-1 dry weight. statistical analysis: the normality of the data was tested by kolmogorov-smirnov tests, and the data that did not meet normality, were normalised by logarithmic transformations. the differences between the two studied fish species i.e. r. kutum and c. aurata and their tissues (liver and gill) were analyzed using a two-way analysis of variance (anova) procedure and a duncan's test. the correlation between all considered parameters was performed through pearson’s correlation matrix. all analyses were performed in r 2.15.3 software (r development core team, 2013). results the statistical analysis of cadmium (cd), lead (pb), and zinc (zn) concentrations in the liver and gill tissues of the r. kutum and c. aurata are presented in table 1. in both species, the concentrations of metals were in order: zn> pb> cd in the liver and fish tissue cd pb zn rutilus kutum liver 8.07±0.44a 30.13±2.23b 154.40±18.78c gill 7.52±3.88a 25.08±3.87b 182.96±19.19c chelon aurata liver 11.02±2.47a 36.44±4.64b 199.3±39.48c gill 6.39±0.91a 23.15±6.09b 150.93±17.58c different letters indicate significant difference (p≤0.05) between different metal concentrations separately for each tissue of fish (p<0.05). table 1. mean concentrations (mg.kg-1 d.w.) and standard deviations of cadmium (cd), lead (pb), and zinc (zn) in liver and gill tissues of rutilus kutum and chelon aurata from the south caspian sea. 82 kardel et al./ cd, pb and zn in rutilus kutum and chelon aurata in the southern caspian sea gill tissues. cadmium (cd) concentration in the tissues of fish species: the results showed that the liver of c. aurata has a significantly higher concentration of cd (37.5%) compared to that of r. kutum, but the difference was not significant for the gill tissue (fig. 1a). although the liver cd concentration in c. aurata was significantly higher compared to its gill tissue (fig. 1a). also, in r. kutum, the liver tissue accumulated a higher concentration of this metal compared to gill tissue, but this result was not significant (fig. 1a). lead (pb) concentration in the tissues of fish species: the liver concentration of pb in c. aurata was significantly higher (24.7%) than that of r. kutum (fig. 1b). according to the results, the liver tissue of c. aurata accumulated a significantly higher concentration of pb compared to gill tissue, but this result was not significant for r. kutum (fig. 1b). zinc (zn) concentration in the tissues of fish species: the results revealed that only the liver tissue of c. aurata had a significantly higher concentration of zn (35.1 %) than that of r. kutum (fig. 1c). for both studied species, there was no significant differences between liver and gill tissue in regard to zn accumulation (fig. 1c). correlation between considered parameters: the results showed a positive and significant correlation between weight, standard length, fork length and total length of both considered fish species (table 2). a significant positive correlation was observed between cd and pb concentration in both the gill and liver tissues in c. aurata (table 2). there was no significant relationship between the fish biometric parameters (length and weight) and the concentration of the measured metals in the gill and liver of both species. discussion comparison of zinc, lead and cadmium concentration: zn had the highest concentration in the gill and liver tissues of both fish species (c. aurata and r. kutum) followed by pb and cd. the finding of the present study confirms the results of other works from different aquatic ecosystems (canli and atli, 2003; anan et al., 2005; ali et al., 2011). for instance, a higher concentration of zn figure 1. mean concentrations (mg.kg-1 d.w.) and standard deviations of (a) cd, (b) pb, and c) zn in liver and gill tissues of rutilus kutum and chelon aurata from the south caspian sea. different letters indicate significant difference (p≤0.05) between the fishes and their tissues. 83 int. j. aquat. biol. (2016) 4(2): 80-86 (114.83 mg.kg-1 dry weight) compared to pb (18.52 mg.kg-1 dry weight) and cd (4.06 mg.kg-1 dry weight) was observed for sturgeon species in the south caspian sea (mashroofeh et al., 2013). it is not surprising that the concentration of zn is higher than pb and cd in this stud, since this metal is an essential element for aquatic organisms and its content is more than other non-essential elements on the earth's crust, but its level can be increased as a result of industrial, agricultural and mining activities (saghali et al., 2012; wood et al., 2012). therefore, it is released increasingly into the aquatic environments, and increase its absorption and bioaccumulation in the tissues and organs of aquatics (canli and atli, 2003; wood et al., 2012). it is characterized by a higher solubility and a slower absorption relative to the amount of bioaccumulation (wood et al., 2012). pb and cd are considered as toxic and nonessential elements that have no known role in biological systems (wood et al., 2012) and even low concentrations of these metals threat aquatic organisms’ health (olmedo et al., 2013). these heavy metals transfer through water and sediments in different parts of aquatic organisms, including fish (rashed, 2001). high concentrations of heavy metals in water, sediments and organisms have reported in different areas of the caspian sea (de mora et al., 2004; parizanganeh et al., 2006; karbassi et al., 2008; bastami et al., 2015). the concentrations of cd, pb and zn in liver and gill of the both fish species i.e. c. aurata and r. kutum were higher than the limits of international standards viz. who (world health organization), fda (food and drug administration) and maff (ministry of agriculture, fisheries and food, united kingdom) (table 3). high concentration of heavy metals in the aquatic environments also is reported by other studies e.g. canli and atli (2003), ali et al. (2011) and mashroofeh et al. (2013). comparison of heavy metal accumulation in the fish species: based on the results, the accumulation of metals in the fish tissues were species specific. this can be attributed to the different diets and feeding a) sl fl tl cdgil cdliv pbgil pbliv zngil znliv w 0.90** 0.79* 0.89** 0.41 -0.15 0.31 -0.42 0.04 -0.57 sl 0.98*** 0.98*** 0.04 -0.20 0.01 -0.22 0.02 -0.61 fl 0.97** -0.14 -0.31 -0.05 -0.13 0.00 -0.63 tl 0.02 -0.38 0.14 -0.26 -0.03 -0.69 cdgil 0.15 0.65 -0.48 -0.13 -0.27 cdliv -0.58 0.08 0.11 0.55 pbgil -0.54 -0.23 -0.44 pbliv 0.74* 0.40 zngil 0.49 b) sl fl tl cdgil cdliv pbgil pbliv zngil znliv w 0.84** 0.88** 0.86** -0.39 0.35 -0.19 0.05 -0.44 -0.39 sl 0.99*** 0.99*** -0.32 0.43 -0.27 -0.04 -0.35 0.02 fl 0.99*** -0.30 0.42 -0.22 0.00 -0.40 -0.05 tl -0.32 0.42 -0.24 -0.02 -0.35 -0.01 cdgil 0.06 0.79** 0.05 0.15 0.43 cdliv -0.20 0.75** -0.13 0.54 pbgil -0.11 -0.10 0.04 pbliv -0.04 0.34 zngil 0.42 *: significant at p= 0.05, ** significant at p= 0.01, ***: significant at p=0.001 table 2. pearson's correlation matrix for different considered parameters (w= weight, sl= standard length, fl= fork length, tl= total length, cdgil= cd in gill, cdliv= cd in liver, pbgil= pb in gill, pbliv= pb in liver, zngil= zn in gill, znliv= zn in liver) in a) rutilus kutum and b) chelon aurata. 84 kardel et al./ cd, pb and zn in rutilus kutum and chelon aurata in the southern caspian sea behaviors of the two studied fishes. sediments are considered as an important source of heavy metals and play a key role in metal transition through the food chain (bastami et al., 2015). heavy metals are accumulated in the sea bed due to their low mobility in sediments, and can be easily available in large quantities for aquatic organisms, particularly benthic dwellers or eater (gnandi et al., 2006). studies have shown that fish species accumulate high levels of heavy metals such as zn, pb and cd in their tissues, depending on their feeding behaviors (canli and atli, 2003; voigt, 2004; evans et al., 2005; ben salem et al., 2014). thus, the higher values observed for c. aurata can be due to its feeding behavior which causes it to spending more time close to sediments relative to r. kutum. however, many factors may effect on the absorption or accumulation of metals by an organism such as biological and physiological factors, environmental parameters, and fish habitats (evans et al., 2005). canli and atli (2003) observed the highest zn (110.03 mg.kg-1 dry weight), pb (41.24 mg.kg-1 dry weight) and cd (4.5 mg.kg-1 dry weight) in the livers of mugil cephalus, atherina hepsetus, and trigla cuculus, respectively. comparison of heavy metal accumulation in liver and gill tissues: the results showed that the accumulation of heavy metals in liver tissue was significantly higher than gill tissue for both fish species i.e. c. aurata and r. kutum, except for zn in the gill of r. kutum. most studies confirm that the liver is a tissue which accumulates the highest concentration of heavy metals compared to gill tissue (canli and atli, 2003; ozturk et al., 2009; paulino et al., 2014; beg et al., 2015). the higher concentrations of metals in the liver compared to the gill can be due to the high capacity of liver tissue to induce the metal-binding protein metallothionein (canli and atli, 2003). these metallothionein can bind to certain metals and leads to higher concentrations of those metals in the liver (beg et al., 2015). most elements tend to react with carboxylate oxygen, amino groups, and nitrogen or mercapto group sulfur in metallothionein protein (cheung et al., 2004; scudiero et al., 2005). thus, the highest concentration of metals can be seen in the liver and this causes the liver to be the major tissue for metal accumulation (okocha and adedeji, 2011). relationship between all studied parameters: the results revealed that there is a significant correlation between cd and pb in the liver and gill of c. aurata which indicates a common contamination source of these metals. moreover, these results indicated that c. aurata is potentially a better bioindicator of heavy metal pollution in aquatic environments than r. kutum, because of its feeding behavior. the results also showed that the relationship of zn with cd and pb was positive and not significant. these results might be due to two reasons: (i) zn has a higher solubility and slower absorption and plays an important role in fish metabolism relative to cd and pb, and (ii) there are more various sources for zn compared to cd and pb in an aquatic environment. as conclusion, the present study revealed the liver tissue of c. aurata accumulated significantly higher concentration of cd, pb and zn compared to that of r. kutum, but these results were not significant for gill tissue. moreover, liver tissue accumulated significantly higher concentration of cd and pb compared to gill tissue in c. aurata, but these results were not significant for r. kutum. from the results of this study we can conclude that the liver tissue of c. aurata has higher potential to accumulate heavy metal pollution compared to liver tissue of r. kutum. references ali a.a., elazein e.m., alian m.a. 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(2016) 4(2): 80-86 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی chelon) طالیی کفال ماهی و( rutilus kutum) سفید ماهی کبد و آبشش هایبافت در سنگین فلزات غلظت aurata )خزر دریای جنوب بابلسر، ساحل در امیدزهیر، مریم آخوندیانپور، شیال ، فرزانه میرزا*فاطمه کاردل .ایران ،دانشکده علوم دریایی، دانشگاه مازندران، بابلسر چکیده: سفید ماهی تجاری، گونه دو مطالعه این در. کندمی تهدید را هاانسان سالمت که است نگرانی مهمترین آبی هایاکوسیستم در سنگین فلزات تجمع (rutilus kutum )طالیی کفال ماهی و (chelon aurata )سنگین فلزات بررسی جهت (cd, pb, zn )خزر دریای جنوب بابلسر، ساحل در بافت در zn وcd ، pb فلزات غلظت که داد نشان نتایج. است خزر دریای جنوب در ماهیگیری هایایستگاه مهمترین از یکی بابلسر. گردید انتخاب غلظت. نبودند اردمعنی آبشش بافت برای نتایج این اما باشد،می بیشتر داریمعنی طور به سفید ماهی کبد بافت با مقایسه در طالیی کفال ماهی کبد . نبود دارنیمع سفید ماهی برای نتایج این اما بود، بیشتر طالیی کفال ماهی در آبشش بافت با مقایسه در کبد بافت در کادمیوم و سرب فلز تجمع .دارد فلزات تجمع در سفید ماهی کبد به نسبت بیشتری توانایی طالیی کفال ماهی کبد بافت که گرفت نتیجه توانمی .خزر دریای سرب، کادمیوم، روی، ماهی، هایبافت :کلمات کلیدی int. j. aquat. biol. (2020) 8(2): 83-90 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article the effect of ripe papaya, carica papaya, as natural carotenoids meal on body pigmentation and growth performance in banded gourami, trichogaster fasciata anurag protim das*,1shyama prasad biswas department of life sciences, dibrugarh university, 786004, dibrugarh, india. s article history: received 28 january 2020 accepted 9 march 2020 available online 2 5 april 2020 keywords: carotenoids feeding regime nutrition skin pigmentation abstract: the present investigation elucidates the synergistic effects of improvised ecological parameters and ripe papaya (carica papaya) meal on skin pigmentation, growth performance and survival of banded gourami, trichogaster fasciata, under confined environment. a feeding trial of 60 days was done with initial length groups from 6.6 to 9.7 cm using five isonitrogenous experimental diets formulated by supplementing graded levels of carotenoids at 1 to 5% and a control without carotenoids. two ecological parameters temperature and light intensity were elevated using artificial modulators. at the onset of the feeding trial, the total carotenoid concentration in fish muscle in both male (2.94±0.07 μg.g-1) and female (2.54±0.05 μg.g-1), respectively, which significantly increased, highest being in male (6.86±0.12 μg.g-1) and female (5.96±0.07 μg.g-1) fishes fed with 4% papaya meal. positive correlation, (0.98) in male and (0.97) female was observed between elevated levels of dietary carotenoids and body pigmentation which revealed that incorporation of dietary carotenoids resulted in a significant increase in total carotenoid concentration in chromatophores. congenial effects were observed on body indices was revealed by positive correlation of weight (0.79) to elevated levels of carotenoid and 100% survival rate of the fishes. the feeding regimes showed ripe papaya meal as cheap natural colour enhancer source can be safely supplemented at 4% levels in the diets to increase the skin pigmentation and optimum conditioning in optimized captive environment having temperature range of 26-28oc and light intensity 344.0-346 lux, without any xenobiotic effect on banded gourami. introduction globally ornamental fish trade is estimated to be more than us$ 15 billion with trading of more than 2 billion live ornamental fishes, is emerging as a much lucrative industry (katia, 2001; das and biswas, 2016). vibrant body pigmentation is one of the major quality attributes for market acceptability of the ornamental fish (saxena, 1994; das and biswas, 2016). colouration of fish skin is mainly attributed to the presence of chromatophores that contain pigments, including melanins, pteridines, purines and carotenoids (chatzifotis et al., 2005). like other vertebrates, fish do not possess the ability of de novo synthesis of carotenoids (goodwin, 1951). the carotenoid pigmentation of fish is attributed to the pigment present in the diet (hata and hata, 1973). hence, a direct relationship between dietary *correspondence: anurag protim das doi: https://doi.org/10.22034/ijab.v8i2.782 e-mail: anuragprotim.99@gmail.com carotenoids and pigmentation in fish exists (halten et al., 1995). carotenoids are a group of naturally occurring lipid soluble organic pigments that are responsible for the red, orange and yellow colour in the skin, flesh, shell and exoskeleton of aquatic animal (pailan et al., 2012). ornamental fishes, when kept under captivity for long duration have been found to lose their natural skin coloration; henceforth the loss of pigments can be overcome by the addition of carotenoids to the artificial diet. hence, the diet for aquarium fish should be nutritionally balanced, palatable, and resistant to crumbling, water stable, and buoyant but it should also enhance body pigmentation in the fish in captivity (das and biswas, 2016). from commercial point of view, maintenance of the natural skin pigmentation is of great importance, as it is directly associated with acceptance or rejection by the 84 das and biswas / effect of papaya on body pigmentation of trichogaster fasciata consumers (shahidi et al., 1998) and its market demand. the prominence of the carotenoid based colouration is reflected as an authentic indicator influencing the foraging ability for carotenoid rich foods (endler, 1980). aside from colouring muscle and skin, there is evidence of the beneficial effects of dietary carotenoid supplementation, such as growth enhancement through increased metabolism of fish (tacon, 1981) and better nutrient utilization (amar et al., 2001). ecological factors such as water temperature is a significant abiotic driver of aquatic ecosystems which influence attributes of an organism including feeding, metabolic and growth rates, reproductive performance, behaviour and ultimate survival (caissie, 2006; dallas, 2008; webb et al., 2008). moreover, daily endogenous rhythms in fish as well as growth, metabolic rates, locomotory activities, body pigmentation, sexual maturation and reproduction is profoundly influenced by photoperiod since it acts as a synchronizer (duston and saunders, 1990; gross et al., 1995; silva-garcia, 1996; boeuf and le bail, 1999; trippel and neil, 2002; biswas and takeuchi, 2002; biswas et al., 2002; biswas et al., 2005). researches indicates that ornamental fish require between 50 and 400 mg/l of synthetic or natural carotenoids (e.g., red pepper and marigold extracts) in their diet to develop coloration identical to those of fish consuming live foods (boonyaratpalin and lovell, 1977; fey and meyers, 1980; lovell, 1992). in spite of significance of body pigmentation in depth studies on colouration enrichment in native ornamental fish are lacking (das and biswas, 2016). plant sources have been extensively harnessed for inducing pigmentation in ornamental fish. for example, arthrospira platensis (spirulina) have been used as a source of carotenoid pigments for rainbow trout and fancy carp (choubert, 1979; boonyaratpalin and phromkunthong, 1986; alagappan et al., 2004). gouveia et al. (2003) worked with microalgal biomass supplementation have shown that chlorella vulgaris is as efficient as synthetic pigments in the pigmentation of cyprinus carpio and carassius auratus. alagappan et al. (2004) studied the utilization of algae spirulina sp. as a source of carotenoid pigment for trichogaster trichopterus. marigold petal meal was used for the tiger barb (puntius tetrazona), red swordtail (xiphophorus helleri) and gold fish (c. auratus) to enhance body pigmentation (boonyarapatin and lovell, 1977; ezhil et al., 2008; alma et al., 2013). in fruits, such as papaya, with the progression of ripening, the content of carotenoids increases (wall, 2006). the main carotenoids that have been identified in papaya are lycopene, β-cryptoxanthin, and βcarotene (marelli de souza et al., 2008). the banded or giant gourami, trichogaster fasciata (bloch and schneider 1801) has high nutritional and ornamental value in india and global market. the present study was carried out to evaluate the efficacy of supplementation of graded levels of ripe papaya (carica papaya) meal on the skin pigmentation and body indices of the banded gourami, one of the beautiful and hardy native gourami species for tropical aquarium culture. materials and methods experimental feeding design: the experimental feeding trial was conducted in the department of life sciences, dibrugarh university from december 2018 to january 2019. banded gourami of size group with initial length groups from 6.6 to 9.7 cm was collected from a wetland of dibrugarh district. following collection, the fishes were transferred to aquaria of 50 litre with continuous aeration and acclimatized for 8 days in laboratory conditions. the experiment was conducted for 60 days and carried out in 15 aquaria of 50 litre. these 15 aquaria were grouped into five sets and each set consists of 3 replicates. the fishes were stocked at the rate of 15 per aquarium in which 10 were males and 5 were females in each trough. the length and weight of fishes were recorded weekly. the fishes were fed twice daily viz. morning and evening at the rate of 2% of body weight per day. the feed was adjusted after every 10 days according to the body weight of the fish. aquaria were cleaned properly by siphoning off feed particles and metabolic wastes daily from the bottom. water exchange was carried out weekly for the sufficient supply of oxygen to the 85 int. j. aquat. biol. (2020) 8(2): 83-90 fish (table 1). diet formulation: for the feeding trail, dry pelleted feed was formulated following pearson square method and trial and error method of hardy (1980). the control feed was prepared using the basic ingredients like fish meal 25%, soya bean meal 22%, groundnut oil cake 15%, rice bran 20% and wheat flour 12%. the selected ingredients were grounded, thoroughly mixed and dough was prepared by adding required amount of luke warm water. this dough was steamed in water bath at 70°c for 40 min. after this, the dough was cool for a while and added other ingredients like soya oil 2%, starch powder 3%, vitamins and minerals premixes 1% were added and mixed thoroughly. ripe papaya was dried in lypholizer until complete moisture was removed, finely powdered and subsequently added in respective concentration of 1, 2, 3, 4 and 5 g/100 g of basal diet by replacing same quantity of rice bran for the preparation of treatment diets (c1 to c5). the pelleted feed was air dried and kept in airtight containers until further use. analysis of proximate composition of feed, carotenoid concentration, feed conversion ratio (fcr), protein energy ratio (per) and survival: proximate compositions of the experimental diets were analyzed for moisture, crude protein, ether extract and total ash based on aoac (2005). quantitative estimation of crude fat was done following the protocol developed by das and biswas (2019). estimation of carotenoids in fish feed was carried out following britton (1995) with improvisations. feed conversion ratio (fcr) was calculated by relating the feed consumption to gain in wet weight of fish following vasudhevan et al. (2013), protein energy ratio (per) was calculated by relating wet weight gain to protein fed and the survival rate was estimated following francis (1995). fish skin color determination: total carotenoid concentration (tcc) in the muscle tissue from dorsal and abdominal skin of both male and female fishes was analyzed at an interval of 15 days and after the completion of experiment following olson (1979). all procedures were performed in low light and temperature as carotenoids are very sensitive to light, temperature and oxygen. improvisation and monitoring of water quality parameters: water parameters viz. water temperature was elevated to 26-28 using water heater and light intensity using candescent fluorescent light (cfl). parameters such as ph, dissolved oxygen, total hardness, and alkalinity from all the aquaria’s were analyzed at weekly intervals as per standard methods of apha (2005) and light intensity was measured using software lux meter of microsoft lumia. results water quality parameters: physico-chemical parameters of water in the experimental tanks were estimated weekly during the experimental period are presented in table 2. the water quality parameters were improvised to maintain within the normal range tolerable for tropical fishes viz., temperature 24 to 30ºc, dissolved oxygen > 5 mg and ph 7 to 8.5 (santhosh and singh, 2007) throughout the experimental period. proximate composition analysis, growth and survival: analyzing the proximate composition of the experimental diets was almost similar (table 3), which can be traced to the inclusion of feed ingredients, which were only differentiated by the level of carotenoids. the protein content of the diets varied from 30.39 to 31.47%. the survival and growth rates of the fishes at end of the experimental period are shown in table 4. there were significant effects of papaya meal supplementation on growth parameters table 1. rearing conditions for experimental trichogaster fasciata during acclimatization. days diet feeding frequency/amount water exchange rate (per day) density 0-3 none na twice 15 4-6 rice bran ad libitum twice 15 7-10 rice bran 2% of body weight once 15 86 das and biswas / effect of papaya on body pigmentation of trichogaster fasciata of banded gourami. fishes fed with cf4 (4%) papaya supplemented diet showed considerable increase in values in net weight gain (1.9±0.08 g) in comparison to the other treatments and control group. feed conversion ratio (fcr) in control group was 1.74±0.05 and the corresponding values in different experimental groups ranged from 1.75±0.06 to 1.79±0.07, highest being in cf4 and lowest being in cf1 (p<0.05) (table 4). feed conversion ratio and feed efficiency ratio are considered as indices for feed utilization. the results showed that increased level of supplementation of carotenoids in diets increased fcr and a significant affect was observed on the survival rate of the banded gourami with treatment diets and control diet ranged from 90 to 100% (table 3). the results may be attributed to lower densities and/or the hardiness of species, since banded gouramies are very hardy species and survive under extreme conditions. effects of the experimental diets on total carotenoid concentration (tcc) of banded gourami skin: at the beginning of the experiment, the total carotenoids concentration in the muscle and skin of banded gourami was 2.37 μg/g wet weights. enhancement of body pigmentation was witnessed with the brightening from 3rd week of feeding trial. total carotenoids concentration in the muscle and skin of banded gourami after 60 days of experimental feeding trial showed that the total carotenoids concentration increased with the supplementation of papaya meal in the diet, highest being in 4% levels (6.82±0.10 μg/g table 2. ecological parameters recorded in optimized experimental tanks. parameter minimum maximum improvised temperature (°c) 16±1.5 18±1 26±2 ph 7.2±0.3 7.4±0.1 dissolved oxygen (ppm) 5.3±0.9 6.1±0.2 light intensity (lux) 50.6 54.0 344.0±2.4 *temperature and light intensity was elevated using seibo water heater and using cfl bulb in the aquaria. table 3. feed composition and proximate analyses of diets. ingredients (%) control 1 % cf 2% cf 3% cf 4% cf 5% cf fish meal 15 15 15 15 15 15 soybean meal 20 22 22 22 22 22 groundnut cake 23 23 23 23 23 23 rice bran 20 19 18 17 16 15 wheat flour 14 14 14 14 14 14 starch powder 3 3 3 3 3 3 soybean oil 2 2 2 2 2 2 vitamin mineral premix 2 2 2 2 2 2 carotenoids (dried papaya) 1 2 3 4 5 vitamin c 1 1 1 1 1 1 chemical composition (% dry wt.) diet moisture crude fat crude protein crude fibre carotenoids (mg/gm) nfe total ash control 7.56±0.02 8.3±0.01 30.67±0.02 6.28±0.02 0.15±0.01 35.77 11.42±0.02 cf1 7.54±0.03 8.1±0.03 30.63±0.01 6.26±0.01 1.27±0.03 36.02 11.45±0.01 cf2 7.63±0.01 8.3±0.01 30.60±0.03 6.24±0.02 2.13±0.02 35.80 11.43±0.02 cf3 7.59±0.02 8.1±0.02 30.59±0.02 6.26±0.01 2.72±0.01 35.80 11.41±0.01 cf4 7.56±0.02 8.3±0.01 30.67±0.02 6.28±0.01 3.21±0.01 35.77 11.42±0.02 cf5 7.58±0.01 8.2±0.01 30.61±0.03 6.26±0.02 3.46±0.03 35.91 11.44±0.01 cfcarotenoids feed with numbers referring to percentage of carotenoids incorporated per 100 gm of basal ingredients, fcrfeed conversion ratio, nitrogen free extract = 100 (% moisture + % crude protein + % crude lipid + % crude fibre + % ash) 87 int. j. aquat. biol. (2020) 8(2): 83-90 wet weights) in male and (5.92±0.09 μg/g wet weights) in case of female, and beyond that no further significant increase in carotenoids content was found in both the sexes (fig. 1). positive correlation is observed between increasing dietary carotenoids level to increasing skin and muscle carotenoids concentration in both male (0.96) and female (0.97) fishes. discussions high adaptability of ornamental fishes to culture conditions enhances their capability of living in environments having wide ranges (chapman, 2000). successful ornamental fish culture necessities a continuous supply of nutritionally balanced, costeffective feed. the results showed that different level of supplementation of papaya meal in the treatment diets has notable effect on survival of banded gourami. lower survival of atlantic salmon (salmo salar) fry (christiansen et al., 1995) and juveniles (christiansen and torrissen, 1996) was found when fed with diets without asthaxanthin supplementation in comparison to the groups that were fed diets containing asthaxanthin. in the present work, increased fcr in the fish was observed with increasing level of carotenoids supplementation in diets. optimum fcr was obtained by christiansen and torrissen (1996) in atlantic salmon juveniles fed with astaxanthin figure 1. total carotenoid concentration (μg/gm) of male and female trichogaster fasciata fed with diets containing graded levels of papaya meal (means with different superscript indicate significant difference (p<0.05)). table 4. mean survival, weight, length, and fcr of trichogaster fasciata after 60 days of feeding trial. diet increase in weight (gm) increase in length (cm) feed intake (g) fcr survival % carotenoids% (μg/gm wet weight) male female control 1.6 ±0.07 1.2±0.01 97.2 1.74±0.05 90 3.07±0.08 2.64±0.09 cf 1 1.4±0.04 1.2±0.01 92.4 1.75±0.06 100 3.46±0.04 3.21±0.06 cf 2 1.7±0.07 1.4±0.01 95.4 1.77±0.03 100 4.34±0.10 4.04±0.03 cf 3 1.7±0.05 1.3±0.01 99.6 1.78±0.04 100 5.74±0.07 4.88±0.06 cf 4 1.9±0.08 1.6±0.01 96.3 1.79±0.07 100 6.82±0.10 5.92±0.09 cf 5 1.7±0.05 1.5±0.01 95.2 1.78±0.04 100 6.73±0.04 5.81±0.07 the means with different superscript in each row indicate a significant difference (p<0.05). 88 das and biswas / effect of papaya on body pigmentation of trichogaster fasciata supplemented diet. similarly, in penaeus monodon, better fcr was observed with prawn fed on the diet containing spirulina (arthrospira platensis). the onset of enhancement of body pigmentation from the 3rd week of feeding trial is due to time intake for breaking of dietary carotenoids and subsequent transformation and increase of carotenoids in pigment cells (chromatophore). the stabilization of body pigmentation during the progression of feeding trial is due to the consequent supply of dietary carotenoids for maintaining the quantity of carotenoids in chromatophores. the above result coincides to the results of sitorus et al. (2015). kalinowski et al. (2005) observed positive effect on growth and skin colour in pagrus pagrus when fed with different carotenoid sources with their dietary levels. ezhil et al. (2008) observed enhancement of pigmentation in x. helleri when fed with formulated feed containing calendula officinalis concluding that this lutein can be used as pigmenting source of natural origin. the carotenoid source and its effectiveness on deposition of pigments in fish body is species specific (ha et al., 1993). sexual dichromatism is often common in several fish species. in general males are more brightly coloured than their female counterparts (gogoi et al., 2013). in the present feeding trial, even female fish fed with carotenoid diets also exhibit the remarkable pigmentation due to increased total carotenoid concentration in their body. alma et al. (2013) reported that 200 mg of carotenoids from marigold meal is optimum to increase the pigmentation in skin of goldfish and over that level they have not found any additional accumulation of carotenoids. similar studies on etroplus maculatus fed with diet having marigold oleoresin contained the highest total carotenoid concentration (jagadeesh et al., 2014). the rate of colour development observed to depend on the amount and nature of carotenoid present on the pigment source/ingredients (boonyaratpalin and unprasert, 1989). the present results confirm that percentage of colour development was higher in carotenoids fed fishes and directly attributed to the higher level of carotenoid content in the particular ingredients. the study enunciates formulated diets incorporated with carotenogenic biomass revealed stabilization and enhancement of body pigmentation during the maximum study period. the most successful period in inducing the skin colouration was after 3 week of feeding papaya meal at 4% concentration and retained further. therefore, the supplementation of ripe papaya meal at 4% concentration in the feeds of banded gourami significantly improved its colouration without any adverse effect on growth, feed conversion efficiency and survival in aquarium conditions. due to the adverse effects of synthetic carotenoids on aquatic environment, many natural plant sources can be harnessed and incorporated in formulated feeds for colour retention or enhancement of ornamental fishes in captive environment. it will aid in creation of avenues for promotion of the ornamental fish industry as well as colour enhancer feed industry and employment generation (das and biswas, 2016). acknowledgement authors are thankful to department of life sciences, dibrugarh university for providing the laboratory facilities and ugc for providing the national fellowship to anurag protim das. references alagappan m., vijula k., sinha a. 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(2015) 3(6): 425-432 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article toxic effects of cadmium on antioxidant defense systems and lipid peroxidation in acipenser persicus (borodin, 1897) roghieh safari*1 faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 6 september 2015 accepted 1 december 2015 available online 2 5 december 2015 keywords: oxidative stress superoxide dismutase catalase malondialdehyde persian sturgeon abstract: cadmium is considered as a common residue in water and sediments which could readily enter aquatic organisms. the aim of this study was to evaluate the timeand concentration-dependent changes in antioxidant enzymes (sod and cat) activities as well as concentration of mda as a bi product of lipid peroxidation in the liver of persian sturgeon, acipenser persicus, following cdcl2 exposure at sub-lethal concentrations for 14 days. based on the results, activity of sod and cat showed a significant increase in the fish exposed to different concentrations of cdcl2 up to the day 7, and then their activity decreased in the fish of all treatments on the 14th day. in all treatments, mda content significantly increased after exposure at first day until the end of the experiment. the levels of sod, cat and mda followed a concentration-dependent manner and its increase was higher in 800 µgl-1 than those of 200 µgl-1. the results suggested that antioxidant enzymes could be used as an effective index to monitor ecotoxicological changes. introduction cadmium is considered as a highly toxic metal pollutant which easily enters the environment via industrial and agricultural activities such as mining, ore refining, electroplating processes, chemical fertilizers and pesticides (stohs and baghchi, 1995; duribe et al., 2007). it is a serious threat to the environment due to its persistent nature, long distance transport and toxicity to aquatic organisms (huang et al., 2005). even at low concentrations, this metal may accumulate in aquatic animal’s body and resulting in several toxic effects, including tissue damage, physiological and biochemical alterations, respiratory changes, and ultimately death (reynders et al., 2006; oner et al., 2008; banaee et al., 2015). the intensity and duration of these responses are influenced by several factors such as concentration of the toxicant, time of exposure and the fish species (dabas et al., 2012). alterations in biochemical levels are usually the first detectable responses to environmental perturbation * corresponding author: roghieh safari e-mail address: rsafari@gau.ac.ir which can provide information on sub-lethal cellular effects of stress factors in a particular species and have potential to be applied as sensitive biomarkers in field studies to monitor fish health (kim et al., 2008). among the most commonly used biochemical biomarkers, those related to oxidative stress are considered to have an important role, being frequently used both in environmental monitoring and laboratory assays (pandey et al., 2003). in fact, the metabolism of xenobiotics is a two-phase process. the reactions of the first phase include oxidation, reduction and hydrolysis in which the most important ones are oxidation enzymes involved in metabolism of the majority of xenobiotics. in the second phase of the reactions, endogenous enzymatic and non-enzymatic antioxidants converse the reactive oxygen species (ros) to harmless types and also protect and restore normal cellular metabolisms and functions as well (ballesteros et al., 2009). elevated levels of ros cause oxidative damage in biomolecules such as lipids, proteins and 426 int. j. aquat. biol. (2015) 3(6): 425-432 dna (livingstone, 2003; sevcikova et al., 2011). the key enzymes for detoxifying ros in all organisms are superoxide dismutase, glutathion-stransferase and catalase (lonidis and yu, 1995; wang et al., 2008; ballesteros et al., 2009; dazy et al. 2009; rastgoo and alemzadeh, 2011). estimation of lipid peroxidation has been also found in various studies to have predictive importance as a biomarker of oxidative stress (lackner et al., 1998). persian sturgeon, acipenser pesicus, has been listed as an endangered species, existing in the caspian sea, the largest continental water body on the earth containing more than 85% of the world’s sturgeon population (pourang et al., 2005). besides overfishing, contamination by industrial, domestic effluents and other anthropological activities (kaplin, 1995; lenhardt et al., 2006) result in declining its population to non-economic levels over the past decades. induction of oxidative stress has been reported in fish exposed to pollutants (dorts et al., 2009; almeida et al., 2009; sharma et al., 2013; sharma et al., 2014). considering these effects, studies on the effect of cd on acipenser species are limited to some studies including lc50 estimates (mirzaee et al., 2003), stress indices (glocose, cortisol, alt, and ast) (zahedi et al., 2013), cytotoxicity (shariati et al., 2011) and genotoxicity (safari et al., 2014). therefore, this study was designed to evaluate the timeand concentration-dependent changes in antioxidant enzymes (sod and cat) activities as well as the concentration of mda as a bi-product of lipid peroxidation in liver of a. persicus following cdcl2 exposure at sub lethal concentrations for 14 days. materials and methods fingerlings of the persian sturgeon (3-5 g) were obtained from shahid marjani breeding and rearing center (golestan province, iran). they were acclimated to the experimental conditions for two weeks. then the fish were randomly distributed into 9 tanks of 300 l at a density of 30 fish per tank and submitted to 200 and 800 µgl-1 cdcl2 for 14 days (lc50 was previously determined as 4000 μgl -1, shariati et al., 2011). three tanks (each for one replication) were considered for each treatment. during the exposure, the fish were fed with artemia biomass twice a day and the water was continuously monitored for temperature, dissolved oxygen, ph and conductivity (mean ± sd., t ~ 24 ± 1°c; do ~ 7 ± 0.2 mg l-1; ph ~ 7.6 ± 0.2; 1412 ± 167.9 μs/cm). oxidative stress analysis: nine fish per treatment were rapidly anesthetized by clove powder (0.5 gl1) solution; their liver were removed and then immediately deep-frozen in liquid nitrogen and stored at -80°c for further analysis. sample preparation: the sampled livers were homogenized (1:5) in ice-cold 50 mm phosphate buffer ph 7.5 containing protease inhibitor cocktail (sigma p2714). the homogenate was centrifuged at 10000 g for 10 min at 4°c and supernatant was kept at -20°c for antioxidant enzyme activities assays. protein content was assayed based on lowery et al. (1951). activity of antioxidant enzymes superoxide dismutase (sod): the sod activity was measured according to its ability to inhibit the photochemical reduction of nitroblue tetrazolium (nbt) as described by dhindsa et al. (1981). the activity of sod was expressed as unit per milligram protein. catalase (cat): activity of cat was assayed using spectrophotometric method by determining h2o2 decomposition in time at 240 nm based on aebi (1984). the enzyme activity was expressed as unit per milligram protein. malondialdehyde (mda): lpo which results in production of malondialdehyde (mda) and consequently to free radicals, was assessed by thiobarbituric acid reactive substances (tbars) (fatima et al., 2000). mda reacts with thiobarbituric acid (tba) and the product is read by spectrophotometric method at 535 nm. the homogenate was added at the ratio of 1:1 (v:v) to 5% trichloroacetic acid (tca), and incubated on ice for 15 min. the solution was then mixed at the ratio of 2:1 with 0.67% tba, and centrifuged at 2200×g at 427 safari/ effects of cadmium on antioxidant defense systems in a. persicus 4°c for 10 min. the whole supernatant was boiled for 10 min, and then refreshed at room temperature before recording the absorbency. a calibration curve with increasing mda concentrations allowed the calculation of lpo expressed as nmol g-1 per tissue. statistical analysis: for each index, the data were tested for normality and homogeneity. normalized data passed levens test for homogeneity of variance. statistics data were subjected to one way anova (α = 0.05). comparisons within each analysis day and within a treatment at different sampling days were made by duncan's test. data were reported as mean ± standard deviation. the spss software, version 16, was utilized for data analysis (spss, richmond, virginia, usa). results after exposure to the selected concentrations of cadmium chloride, the toxic stress on fish was manifested in the form of restlessness and jerky and erratic swimming movements. instantaneous secretion of excessive mucus all over the body surface of the exposed fish also was notable especially in higher concentration. the exposed fish rejected feeding in the earlier stages (up to the 4th day) of exposure, then, they started to consume the food (with hesitation) gradually and resumed feeding nearly as normal. throughout the experiment, no significant macroscopic behavioral changes were observed in the control group. no death was recorded either in the control or experimental group during the experiment. enzymes assay: activity of both antioxidant enzymes (sod and cat) in liver of cd (200 and 800 µgl-1) exposed fish (a. persicus) generally increased compared to that of the control group until the 7th day of exposure (p<0.05) followed by non-significant decrease on the day 14. the activity of enzymes significantly increased in the fish treated with 800 more than 200 µgl-1 cdcl2 (tables 1 and 2). lipid peroxidation: in the exposed fish, tbars levels increased compared to that of the control group. in both experimental concentrations, mda significantly increased at first day (p<0.05). mda exposure day/ concentration 1 4 7 14 control 22.63±0.3aa 22.4±0.28 c a 22.4±0.24 c a 22.4±0.1 b a 200 µgl-1 22.7±0.12ac 23.45±0.24 b bc 31.26±0.6 a a 27.3±0.4 a ab 800 µgl-1 22.96±0.2ab 27.65±0.2 a a 28.11±0.3 b a 27.76±0.0.3 a a the results are expressed as means with standard deviation (n=9). different superscript and subscript letters denote significant (p<0.05) difference in each column (a-c) and each row (a-d), respectively. table 1. alternation in sod (u/mg) activity in the liver of acipenser persicus exposed to 200 and 800 µgl-1 of cd for 14 days. exposure day/ concentration 1 4 7 14 control 2.76±0.1aa 2.9±0.1 b a 3±0.1 b a 2.88±0.16 c a 200 µgl-1 2.9±0.4ac 3.27±0.15 b c 7.8±0.3 a a 5.8±0.28 a b 800 µgl-1 3±0.3ac 5.8±0.3 a ab 6.72±0.4 a a 4.92±0.22 b b the results are expressed as means with standard deviation (n=9). different superscript and subscript letters denote significant (p<0.05) difference in each column (a-c) and each row (a-d), respectively. table 2. alternation in cat (u/mg) activity in the liver of acipenser persicus exposed to 200 and 800 µgl-1 of cd for 14 days. exposure day/ concentration 1 4 7 14 control 6.5±0.6aa 6.2±0.9 b a 6.8±0.2 c a 6.4±0.7 b a 200 µgl-1 6.5±0.5ad 9±0.34 b c 10.5±0.25 a b 13.5±0.66 a a 800 µgl-1 6.7±0.4ad 12.2±0.4 a c 14±0.9 b b 17±0.59 a a the results are expressed as means with standard deviation (n=9). different superscript and subscript letters denote significant (p<0.05) difference in each column (a-c) and each row (a-d), respectively. table 3. alternation in mda (nmol g−1 tissue) in the liver of acipenser persicus exposed to 200 and 800 µgl-1 of endosulfan for 14 days. 428 int. j. aquat. biol. (2015) 3(6): 425-432 content followed a concentration-dependent trend and grew in the fish treated with 800 more than 200 µgl-1 cdcl2 (table 3). discussion application of pollutant biomarkers has been the subject of various studies, especially because of the fact that distinct kinds of pollutants may interfere with animal physiology and behavioral processes, which is also of ecological importance (scott and solman, 2004). cadmium dose not generate ros directly, but can alter antioxidant enzymes, especially sod, cat and gsh and it is able to displace copper and iron in various proteins, and then freeing these metals to participate in the fenton reaction (ercal et al., 2001; remeo et al., 2000; sevcikova et al., 2011). based on the results, cd exposure increased sod and cat in the liver of persian sturgeon in comparison with that of the control indicating that the antioxidant system response to cadmium stress. a significant increase in sod and cat enzymes until the 7th day of exposure could be attributed to superoxide radical accumulation. similar results were reported in oreochromis mossambicus (basha and rani, 2003), channa punctatus (dabas et al., 2012), and o. niloticus (saglam et al., 2014) which were attributed to superoxide radical accumulation, denovo synthesis of enzymatic proteins and inducing expression of genes encoding sod and cat to detoxify ros (rastgoo and alemzadeh, 2011). the depressed catalytic activity of cat and sod on the 14th day of exposure may be linked to the binding of cd to –sh groups of these enzymes. the present findings are in agreement with the results obtained in cadmium exposed danio rario (ghazie et al., 2013) and arius arius (mani et al., 2014) in which it was reported that binding of non-essential heavy metals to the active center of enzyme resulted in overproduction of h2o2 that consequently caused decline in antioxidant enzymes activity. falling trend of antioxidant enzymes activity was attributed to the flux of superoxide radicals by filek et al. (2008) and modesto and martinez (2010). these bi-phasic responses have been reported in several studies (garcia sampaio et al., 2008; saglam et al., 2014). the antioxidant enzyme responses to pollutant could be species-specific in addition to the factors such as dosage and exposure duration (almeida et al., 2009; dorts et al., 2009; jia et al., 2011; wang et al., 2013). in this study, the activity of antioxidant enzymes increased in higher concentration treatments and followed a concentration-dependent trend. a contrary result was observed in lutjanus argentimaculatus after exposure to high levels of cd compared to low dosages and suggested that low dosages of cd might enhance the protective mechanism, while high dosages lead gradually to cytotoxicity and inhibit the antioxidant enzymes (wang et al., 2013). lipid peroxidation (lpo) is also one of the key manifestations of oxidative damage induced by various compounds, including metals (ercal et al., 2001; dabas et al., 2012). in this study, mda increased up to the 14th day of exposure to cd. the determined values of lpo were in agreement with those reported for other fish species (pandey et al., 2008). cd produces inhibitory effects on mitochondria electron transport (belyaeva et al., 2012), and as a result, the respiratory chain becomes highly reduced and the electrons are transferred directly to available oxygen, leading to an enhancement in ros formation (miyamoto et al., 2003), which cause peroxidative damage in liver. increasingn mda has been reported after exposure to cd in o. niloticus (almedia et al., 2009), c. puntatus (dabas et al., 2012), clarias gariepinus (asagba et al., 2008), and l. argentimaculatus (wang et al., 2013). in this study, tbars levels followed a concentration-dependent manner. in addition, the elevated lpo levels are supported by the results reported in c. gariepinus (asagba et al., 2008) and c. puntatus (dabas et al., 2012). contrary to these results, wang et al. (2013) showed higher content of mda at low concentrations of cd due to inhabitation of the antioxidant enzymes. in conclusion, exposure to sub-lethal dosage of 429 safari/ effects of cadmium on antioxidant defense systems in a. persicus cdcl2 showed a biphasic trend in antioxidant enzymes. antioxidant enzymes showed a rise until the 7th day and mda levels increased up to the end of experiment. the present results suggested that antioxidant enzymes could be used as an effective index to monitor ecotoxicological changes. acknowledgements the author is grateful to gorgan university of agricultural sciences and natural resources for providing facilities to accomplish this study. references aebi h. (1984). catalase in vitro. method enzymology, 105: 121-126. almeida j.a., barreto r.e., novelli e.l.b., castro f.j., moron s.e. 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(2015) 3(6): 425-432 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی ایرانی ماهیتاس در هاچربی پراکسیداسیون و اکسیدانتی آنتی دفاع سیستم بر کادمیوم تاثیر (acipenser persicus (borodin, 1897)) صفری رقیه .ایران گرگان، گرگان، طبیعی منابع و کشاورزی علوم دانشگاه ،زیست محیط و شیالت دانشکده چکیده: میزان تغییرات ارزیابی مطالعه این هدف. شودمی انآبزیبدن وارد آسانیبه که است رسوبات و آبی هایمحیط در معمول پسماندهای از یکی کادمیوم کشنده تحت هایغلظت با مواجه در ایرانی ماهیتاس کبد در چربی اکسیداسیون جانبی محصول عنوانبه آلدئیددیمالون و اکسیدانتیآنتی هایآنزیم تا کادمیوم مختلف هایغلظت با مواجه در را داریمعنی افزایش cat و sod فعالیت نتایج اساس بر. باشدمی روز 41 مدت برای کادمیوم کلرید تا اول روز از آلدئیددیمالون میزان بررسی مورد تیمارهای در. یافت کاهش تیمارها همه در ام41 روز تا هاآن فعالیت سپس و داد نشان ام7 روز لیتر در میکروگرم 088 تیمار در و کرده تبعیت دوزی به وابسته روند آلدئیددیمالون و sod ، catمیزان. یافت افزایش آزمایش دوره انتهای مؤثری شاخص عنوانبه تواندمی آلدئیددیمالون و اکسیدانتی آنتی هایآنزیم گیریاندازه که داد نشان نتایج. بود لیتر در میکروگرم 088تیمار از باالتر . باشد محیطی شناسیسم تغییرات پایش در .ایرانی ماهیتاس ،آلدئیددیمالونکاتاالز، ،دیسموتاز سوپراکسید ،اکسیداتیو استرس :کلمات کلیدی int. j. aquat. biol. (2016) 4(1): 43-50 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article evaluation of soybean meal replacement with sesame seed meal using activated charcoal as an additive in the diet of african catfish juveniles, clarias gariepinus muyideen owonire lawal*,1ademola zaid aderolu, oluwaseun olasunkanmi aarode, bolanle moriamo seriki, timothy o. alonge deparment of marine sciences, university of lagos, akoka, yaba, nigeria. article history: received 8 november 2015 accepted 27 january 2016 available online 2 5 february 2016 keywords: soybean meal growth performance activated charcoal african catfish abstract: this study evaluated the effects of 0.25% activated charcoal added to sesame seed meal (ssm) in partial replacement of soybean meal (sbm) of juvenile clarias gariepinus diet on growth and haematological parameters for 70 day experimental period. six experimental diets were formulated as: control diet 1 (100% ssm without activated charcoal), control diet 2 (100% ssm plus 0.25% activated charcoal), diet 3 (50% smb+50% ssm), diet 4 (50% sbm+50% ssm+0.25% ac), diet 5 (30% sbm+70% ssm) and diet 6 (30% sbm+70% ssm+0.25ac). the inclusion of activated charcoal recorded significant improvement in mean weight gain, mean feed intake, specific growth rate (sgr), protein intake (pi) and protein efficiency ratio (per). the control diet 2 recorded the highest values for growth and nutrient utilization parameters while diet 5 recorded the least values for these parameters. the best values for mean weight gain (65.33±5.57 g), specific growth rate (2.56±0.12% / day) and feed conversion ratio (0.71±0.05) were recorded in fish fed control diet 2 while the worst values (41.30±3.82 g, 1.92±0.11% / day, and 1.07±0.07, respectively) for these parameters were observed with diet 5. however, haematological parameters did not differ significantly across the diets. thus, from this study 0.25% activated charcoal could favorably be added to the feed of c. gariepinus for optimum performance without any adverse effect on the health status of the fish. introduction plant protein could be an alternative to expensive fishmeal; however, the inclusion of plant ingredients like soybean meal, groundnut seed meal and cotton seed meal in fish feed has been reported to reduce fish growth rate in comparison to fish meal (imoroutoko et al., 2008; li et al., 2010). this reduced growth rate of fish fed with plant proteins inclusion could be due to several factors such as poor digestibility (lech and reigh, 2012), imbalanced amino acid composition (xie et al., 2001) and inherent anti nutritional factors (anf) in most plant ingredients (francis et al., 2001). soybean has been the major plant ingredient used in livestock and fish feeds (shipton and hecht, 2005), but a sharp price pressure in recent times ensued by increasing utilization by livestock feed production and human being nutrition (hardy, * corresponding author: muyideen owonire lawal doi: http://dx.doi.org/10.7508/ijab.2016.01.006 e-mail address:mlawal@unilag.edu.ng 2010). the search for alternatives to soybean is thus important for sustainable aquaculture industry and its profitability. sesame (sesamum indicum l.) plant is a xerophyte adapted to many soil types (ram et al., 1990). according to ahmed (2005), there are about 335,000 hectares of land under sesame cultivation in nigeria with yields of between 1.5-2.0 tonnes/hectare. sesame seed has nutrient composition similar to other oilseed proteins including soybean meal and other conventional legumes (sintayehu et al., 1996) and its potential as dietary protein source is well-recognized (olveranovoa et al., 2002). full-fat sesame seed contains 22% crude protein and the meal after oil extraction about 44% crude proteins (mamputu and buhr, 1995). the seed contains 50-60% oil compared to 20% in soybean 44 lawal et al./ activated charcoal in sesame seed diet for catfish (kato et al., 1998; ahmed, 2005). hossain and jauncey (1989) found that sesame oilseed meal can be included up to 25% in common carp, cyprinus carpio l, however, tacon (1993) suggested that maximum level of its inclusion is 35% in both omnivorous and herbivorous fish species. whereas, mukhopadhyay and ray (1999) suggested that fermented sesame seed meal protein can replace up to 50% of fish meal protein in the diets for rohu fingerling. limitation to the effective utilization of sesame seed include high phytic acid up to 50 μg g-1, tannin and oxalic acid which reduces the biological availability of zinc, calcium, magnesium and iron (nahm, 2007), the following treatment methods already suggested; adequate heat treatment, soaking in water, fermented with lactic acid bacteria and extraction of oil, could reduce seed toxicants (tanin, oxalic and phytic acids) and crude proteins and lipids level improvement (mukhopadhyay and ray, 1999; diarra and usman (2008). effective approach to the reduction of anti-nutritional factors in sesame seed could be the use of non-nutritive and inert adsorbents additives which could bind anf and reduce the absorption of anf in the gastrointestinal tract. activated charcoal (ac) is a form of carbon with increased surface area for absorption. adsorption therapy with activated charcoal as a non-digestible carrier is one of the important methods of preventing the ingested toxicants or noxious substances formed in the gastrointestinal tract (van et al., 2006). the major advantages of ac include safety, easy administration and control of pathogenic bacteria through addition to the animal feed (dalvi and ademoyero, 1984; nikoleavia et al., 1994). the present study therefore, looked at the possibility of using bamboo activated charcoal to enhance sesame seed meal utilization on growth performance and haematology of african catfish, clarias gariepinus, juveniles. materials and methods the experimental catfish, c. gariepnus were purchased from a fish farm at cele, ikotun-egbe, lagos state. the fish were transported in a 25 litres plastic container with water to the nutritional unit of the department of marine sciences, university of lagos. the fish were kept in plastic tank to acclimatize for two weeks. during the acclimatization period, they were fed with 2 mm coppens fish feed (coppens international bv, the netherlands), thereafter 10 juveniles of an average weight 20.9 g were allocated to each of the twentyfour plastic containers making a total of two hundred and forty c. gariepinus juveniles. all the ingredients, including soybean meal (sbm) and activated charcoal (ac) used for the experiment except sesame seed were purchased at soleace nigeria limited, oko-oba, agege, lagos, nigeria. the sesame seed (sesamum indicum) was bought from bariga market, lagos, nigeria. ingredients were separately milled, screened to fine particle size (< 250 μm mesh size) and stored for future use. the sesame seeds were screened, winnowed and cleaned to remove dirt, sand, stones and other undesirable particles. using fire wood as a source of heat, the sesame seeds were placed in a wide open aluminum pan and toasted for about 15 minutes by stirring continuously to prevent the burning of the seed coat and enhance even distribution of heat. the toasting was stopped when pop sound was produced and the seeds slightly turned brown to produce sesame seed meal (ssm) according to yakubu and alfred (2014). six experimental feeds were formulated (table 1), including: the positive control (ctr) diet 1 (100% ssm without activated charcoal); the negative control (ctr) diet 2 (100% ssm plus 0.25% activated charcoal), diet 3 (50% smb + 50% ssm), diet 4 (50% sbm + 50% ssm + 0.25% ac), diet 5 (30% sbm + 70% ssm) and diet 6 (30% sbm + 70% ssm + 0.25ac). ingredients were carefully weighed, mixed and water was added to form dough. the dough was pelletized with 3 mm die using locally fabricated pelletizing machine and sundried. the study, which lasted for a period of 10 weeks (70 days), was conducted in twenty-four (55 x 33 x 45 int. j. aquat. biol. (2016) 4(1): 43-50 31 cm3) rectangular plastic tanks. the tanks were covered with nets to prevent fish from jumping out. the experiment was run in triplicate, making a total of 24 tanks. the fishes were hand fed to satiation three times a day (9:00 am, 1:00 pm and 5:00 pm). the water was changed every other day to ensure the fish were kept under standard condition; temperature (27.5-29.5ºc), dissolved oxygen (4.5-4.8 mg/l), and ph (7.3-8.0) as described by aderolu and akpabio (2009). the weekly weight gain of the fish was measured by bulk-weighing the fish and the weekly feed intake also recorded. after acclimatization, initial weight of fish was taken using an electronic scale (ohaus cs5000) and recorded. fish weighing was done every week. growth and nutrient utilization parameters: these parameters were calculated using following formulas: (1) mean weight gain (mwg) (g) = final mean weight (fmw) – initial mean weight (imw) (2) feed conversion ratio (fcr) = (feed intake (g)) / (weight gain (g)) (3) specific growth rate (sgr) (%/day) = (loge w2 – loge w1x 100) / (t2 – t1 (day)) where, e is natural logarithm, t2 – t1= experimental period, w1 = initial weight, and w2 = final weight. (4) protein efficiency ratio (per) = (mean weight gain) / (protein intake) (5) mean feed intake (mfi) = feed intake for experimental period) / (number of days in the period) (6) protein intake (pi) = (total feed intake) / (protein content of feed) haematological analysis: the blood samples were ingredients 100% ssm (ctr 1) 100% ssm + ac (ctr 2) 50% ssm (diet 3) 50% ssm + ac (diet 4) 70% ssm (diet 5) 70% ssm + ac (diet 6) fish meal 20.0 20.0 21.0 21.0 24.0 24.0 groundnut cake 22.0 22.0 25.0 25.0 24.0 24.0 noodle waste 25.0 25.0 21.0 21.0 19.0 19.0 soybean meal nil nil 15.0 15.0 9.0 9.0 ssm 30.0 30.0 15.0 15.0 21.0 21.0 oil 1.0 1.0 1.0 1.0 1.0 1.0 premix 0.25 0.25 0.25 0.25 0.25 0.25 calcium monohydrogen phosphate. 1.0 1.0 1.0 1.0 1.0 1.0 salt 0.25 0.25 0.25 0.25 0.25 0.25 vitamin c 0.25 0.25 0.25 0.25 0.25 0.25 ac nil 0.25 nil 0.25 nil 0.25 crude protein (%) 41 41 40 40 42 42 crude energy 3046 3046 3189 3189 3117 3117 table 1. composition of experimental diets. diets ctr diet 1 ctr diet 2 diet 3 diet 4 diet 5 diet 6 imw (g/fish) 21.30±0.02 21.00±0.20 21.30±0.17 21.17±0.21 21.33±0.06 19.93±2.45 fmw (g/fish) 86.63±5.57b 112.57±3.58a 64.17±4.66c 68.87±4.18c 62.63±3.76c 66.87±9.43c mwg (g/fish) 65.33±5.57b 91.47±3.62a 42.87±4.62c 47.70±3.99c 41.30±3.82c 46.93±7.50c mfi (g) 52.93±7.73ab 64.44±4.14a 42.19±14.02b 44.11±7.46b 44.01±1.54b 44.89±6.55b sgr (%/day) 2.56±0.12b 2.99±0.06a 1.97±0.13cd 2.10±0.09cd 1.92±0.11d 2.16±0.13cd fcr 0.81±0.07bc 0.71±0.05c 0.97±0.23ab 0.92±0.09ab 1.07±0.07a 0.96±0.01ab pi 44.75±3.63ab 51.21±3.38a 38.37±8.60bc 39.32±3.75bc 33.66±1.99c 37.59±0.53bc per 1.47±0.21ab 1.79±0.12a 1.17±0.39b 1.23±0.21b 1.15±0.04b 1.25±0.18b table 2. growth and nutrient utilization parameters of clarias gariepinus fed experimental diets. 46 lawal et al./ activated charcoal in sesame seed diet for catfish collected from the caudal peduncle as described by joshi et al. (2002) in a 2 ml syringe ad mixed with ethylene-diamine-tetra-acetic acid (edta) for haematological analysis at bioassay diagnostic laboratory, cele-egbe, ikotun, lagos. haemoglobin (hb), red blood cells (rbc), white blood cells (wbc), packed cell volume (pcv), mean cell haemoglobin concentration (mchc) and mean cell volume (mcv) were analyzed using the methods described by svobodova et al. (1991). data analysis: all the data were subjected to analysis of variance (anova) and duncan’s multiple range tests were used to compare means among the treatments.the computations were performed using the package spss 18.0 (spss inc., chicago, il, usa). results the results of the growth and nutrient utilization parameters are shown in table 2. the final mean weights (fmw) were higher in the groups with the inclusion of 0.25% activated charcoal (ac) across the experimental diets. the highest fmw (112.57±3.58 g) was recorded for fish fed control 2 diet (100% ssm plus 0.25% ac) while the lowest value (62.63±3.76 g) was recorded in diet 5 (70% ssm). the partial substitution of soybean meal with roasted sesame seed meal actually resulted in inferior (p<0.05) mean weight gain; however, the inclusion of ac led to significant (p<0.05) improvement in mean weigh gain (mwg), mean feed intake (mfi) and specific growth rate (sgr). the ctr diet 2 recorded highest values for mwg (91.47±3.62 g), mfi (64.44±4.14 g) and sgr (2.99±0.06% day-1). the lowest values for these parameters mwg (41.30±3.82 g), mfi (44.01±1.54 g) and sgr (1.92±0.11% day-1) were recorded in fish fed diet 5. furthermore, the nutrient utilization parameters tested recorded significant differences (p<0.05) across diets, the inclusion of ac positively (p<0.05) affected feed conversion ratio (fcr), protein intake (pi) and protein efficiency ratio (per). the best value (0.71±0.05) for fcr was recorded in fish fed ctr diet 2 while the worst value (1.07±0.07) was recorded by the group fed diet 5. similar results were observed with the protein intake (pi) and protein efficiency ratio (per). the ctr diet 2 recorded highest values for pi (51.21±3.38) and per (1.79±0.12) while diet 5 recorded least values (33.66±1.99, 1.15±0.04, respectively) for these parameters. no significant differences (p>0.05) were recorded in fmw, mwg, mfi and per among diets 3, 4, 5 and 6. also, significant differences (p<0.05) were not recorded between the graded levels (50 and 70% substitutions) of sesame seed meal with activated charcoal. amongst the experimental diets, the diets with activated charcoal inclusion had better growth and nutrient utilization performances than the other dietary groups (table 2). the results of the haematological parameters showed no significant differences (p>0.05), in hb, parameters ctr diet 1 ctr diet 2 + ac diet 3 diet 4 diet 5 diet 6 rbc (1012/l) 10166666 ±6048415.79 5300000 ±1137610.0 7050000 ±1060660.1 6950000 ±2050609.6 6300000 ±141421.35 6000000 ±565685.42 pvc (%) 35.33±16.92 18 ±0 28 ±2.83 26 ±8.49 25.5 ±0.71 24 ±2.83 wbc (109/l) 12000±4000 11000 ±0 12000±2828.43 9500 ±2121.32 13500±2121.32 11000±5656.85 hb (g/l) 11.53 ±5.56 5.9 ±0 9.05 ±92 8.4 ±2.69 8.25 ±0.21 7.85 ±0.78 mchc (g/l) 32.63 ±0.33 32.78 ±0 32.32 ±0.02 32.34 ±0.22 32.35 ±0.07 32.75 ±0.62 mcv (fl) 0.000036 ±0.000004 0.000034 ±0 0.00004 ±0.00002 0.000037 ±0.000012 0.00040 ±0.0000002 0.00004 ±0.0000009 table 3. haematological indices of clarias gariepinus fed experimental diets. 47 int. j. aquat. biol. (2016) 4(1): 43-50 rbc, wbc, pcv, mchc and mcv across the experimental diets despite the inclusion of 0.25% activated charcoal (table 3). discussion the reduction in growth parameters recorded at the inclusion of toasted sesame seed meal without activated charcoal in the diet of c. gariepinus could be due to dietary amino acid profile imbalance and the presence of anti-nutritional factors in test ingredient (sesame seed). sesame seed is reported to contain high amount of oxalate and phytic acid (narasinga rao, 1985). oxalate acid reduces the physiological availability of calcium from the seed (salunkhe et al., 1991). also, phytic acid reduces bio-availability of minerals, impairs protein digestibility by formation of phytic acid-protein complexes and depresses absorption of nutrients due to damage to pyloric caeca region of the intestine (francis et al., 2001). the above suggested reasons could then account for the resultant poor nutrient utilization and growth rate on the substitution of soybean meal for sesame seed meal in c. gariepinus (jimoh and aroyehun, 2011). however, the inclusion of activated charcoal along with the sesame recorded better growth performance and improved nutrients utilization, especially in the negative control diet. this result is corroborated by the works of yoo et al. (2005) on juvenile japanese flounder and thu et al. (2009) when they fed tiger fish, takifugu rubripes, with graded levels of bamboo charcoal. they reported improved growth and nutrient utilization which was attributed to increase availability of certain macronutrients, particularly proteins, leading to a better nutrient accumulation. also, jahan et al. (2014) attributed the enhanced growth and nutrient utilization of fish fed activated charcoal feed to the presence of nutrient enhancing microbes along the gastrointestinal tract and possibly the thinner and lighter surface area of the intestinal villi of animal. the increased weight gain might also be due to the fact that the activated charcoal has the capacity to improve apparent nutrient digestibility, reduce toxins in diets by adsorbing it and thereby preventing its absorption from the intestine and selectively inhibit the harmful coliforms (anjaneyulu et al., 1993; choi et al., 2009). the improved fcr, sgr and per were similar to previous studies when activated charcoal was included in the feed of chicken as reported by moe et al. (2009) and jahan et al. (2014) on pangasius hypophthalmus catfish, respectively. the result was attributed to the absorbent effect of activated charcoal which has the potential to condition the intestinal cell membranes to reduce surface tension by eliminating noxious substances along the intestine and the utilization and absorption of nutrients across the cell membranes. blood is an important index of physiological, pathological and nutritional status in the organism (olorode et al., 2007). it is an index and a reflection of the effects of dietary treatments on the animal in terms of the type, quality and amounts of the feed ingested and was available for the animal to meet its physiological, biochemical and metabolic necessities (ewuola et al., 2004). the haematological parameters measured did not show any significant difference across all experimental diets, despite the inclusion of activated charcoal. this result is supported by the findings of thu et al. (2009), when they recorded no significant difference in the values of heamatocrit and heamoglobin concentration across the different diets after feeding dietary bamboo charcoal to tiger puffer fish. probable implication of these results is that the experimental fish heamatology parameters were by no means affected by the dietary inclusion of activated charcoal. in conclusion, from the present study 0.25% ac could favorably be added as natural substance to the feed of c. gariepinusfor optimum performance without any adverse effect on the health status of the fish. references aderolu a.z., akpabio v.m. (2009). growth and economic performance of clarias gariepinus juvenile 48 lawal et al./ activated charcoal in sesame seed diet for catfish fed diets containing velvet bean, mucuna pruriens seed meal. african journal of aquatic science, 34(2): 1-5. ahmed m.f. 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(2014). nutritional evaluation of toasted white sesame seed meal sesamum indicum as a source of methionine on growth performance, carcass characteristics, haematological and biochemical indices of finisher broiler chickens. iosr journal of agriculture and veterinary science, 7(1): 46-52. yoo j.h., ji s.c., jeong g.s. (2005). effect of dietary charcoal and wood vinegar mixture (cv82) on body composition of olive flounder, paralichthys olivaceus. journal world aquaculture society, 36: 203-208. int. j. aquat. biol. (2016) 4(1): 43-50 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology چکیده فارسی عنوان یک افزودنی در خوراک گربه ماهی ارزیابی جایگزینی پودر سویا با پودر کنجد با استفاده از زغال فعال به clarias gariepinus نوجوان آفریقایی، آدرولو، اولوواسئوم آرود، بوالنل موریامو سریکی، تیموتی او. آلونگآدموال زید ، *موییدین اوونیره .علوم دریایی، دانشگاه الگوس، آکوکا، یابا، نیجریه وهگر چکیده: در خوراک ( sbm)عنوان جایگزینی بخشی از پودر سویا به( ssm) به پودر دانه کنجد( ac) درصد زغال فعال 52/0این مطالعه اثرات افزودن روزه مورد ارزیابی قرار داد. شش 00در یک دوره آزمایش را شناسیبر رشد و پارامترهای خون clarias gariepinus آفریقایی، نوجوان ماهیگربه ac( ،3 ) 52/0% به عالوه ssm %100حاوی 5( تیمار شاهد 5، )acبدون ssm %100حاوی 1( تیمار شاهد 1شامل ) خوراک آزمایشی smb %30شامل 2خوراک smb +20% ssm +%52/0 ac ،(2 ) %20 شامل 4خوراک smb +20% ssm( ،4 ) %20شامل 3خوراک +00% ssm 30شامل 6خوراک ( 6)و% smb +00% ssm +%52/0 ac .میانگین جذب بهبود افزایش وزن، افزودن زغال فعال فرموله شدند ی هامترار رشد و پاریداباالترین مق 5( را ثبت کرد. خوراک شاهد per( و نسبت پروتیئن موثر )piجذب پروتئین )(، sgrنرخ رشد ویژه )غذا، gهترین مقادیر میانگین افزایش وزن )را داشت. ب ایتغذیهمترهای اپار اینکمترین مقادیر 2که خوارک تیمار ای را نشان داد در حالیوری تغذیهبهره مد، بدست آ 5( در ماهیان تغذیه شده با خوراک شاهد 01/0±02/0درصد در روز( و نرخ تبدیل غذا ) 26/5±15/0(، نرخ رشد ویژه ) 20/2±33/62 مشاهده 2( در خوراک تیمار 00/1±00/0ر روز و ددرصد g 25/3±30/41 ،11/0±00/0ترتیب بهکه بدترین مقادیر برای پارامترهای فوق )در حالی به غذای درصد زغال فعال 52/0 داری را نشان نداد. بنابراین براساس نتایج این مطالعهشناختی در بین تیمارها تفاوت معنیپارامترهای خونشد. ماهیان اضافه شود. سالمت وضعیت بدون هیچ گونه اثر زیانباری برای تغذیهب سمنابرای کارایی دتوانمی ماهی آفریقاییگربه ماهی آفریقایی.کارایی رشد، زغال فعال، گربه سویا، پودر :کلمات کلیدی int. j. aquat. biol. (2021) 9(13: 177-186 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article influence of micro-habitats on the distribution of macroinvertebrates in the ziga reservoir, burkina faso, west africa victor bancé*1, adama ouéda1, idrissa kaboré1, idrissa ouédraogo1, 2, komandan mano1, 3, peter d.m. weesie4, gustave b. kabré1 1laboratory of biology and animal ecology (lbea), ufr/svt, university joseph ki-zerbo, burkina faso. 2dori university center, university thomas sankara, burkina faso. 3 university of dédougou, burkina faso. 4science and society group, faculty of science and engineering, university of groningen, groningen, netherlands. s article history: received 23 august 2020 accepted 7 may 2021 available online 2 5 june 2021 keywords: lake macroinvertebrate micro-habitat diversity distribution abstract: in this work, we assessed micro-habitat's influence on the distribution of macroinvertebrates in lake ziga in burkina faso from july to december 2016. the water quality variables were measured in situ and the macroinvertebrates were collected with a hand net. the organisms were identified to the lower taxonomic resolution as possible. the results show that the temperature is globally warm, characteristic of tropical area, with a good oxygen content and ph close to neutral. we found five micro-habitats, mainly dominated by fine substrates (32.5%) and aquatic plants (25.83%). the stone, roots and dead woods represented less than 20%. in total, 3,773 individuals of macroinvertebrates were collected. these individuals belong to 33 taxa and three classes. the insects class is the most abundant (88.22%) and the most diversified (24 taxa, 72.72%). the highest taxonomic richness is observed in aquatic plants and root zones. the diversity and density of the macroinvertebrate community varies according to micro-habitats but not according to the size of their surface area. the results showed that coleopterans and hemipterans were strongly and positively correlated to transparency and conductivity (adjusted r>60%, p<0.05). in the local area, the results showed that macroinvertebrates' diversity and distribution are more linked to habitat availability. our findings reveal a good habitat condition of the lake, and can be served as reference site and hostpot of aquatic biodiversity in the area. introduction the effects of anthropogenic activities and climate change are increasing threats to water resources (younes-baraillé et al., 2005; atique et al., 2020a; hara et al., 2020). these pressures negatively affect water quality and decrease aquatic organism biodiversity (younes-baraillé et al., 2005; agbohessi et al., 2012; atique and an, 2020). the preservation of aquatic ecosystems necessarily depends on their excellent management. traditionally, water quality assessment actions have been focused on the measurement of physicochemical parameters (atique et al., 2020b). nowadays, the use of biological indicators is a key element of the water resources management policies in many countries (moog et al., 1999; haque et al., 2020; tampo et al., 2020). a *correspondence: victor bancé doi: https://doi.org/10.22034/ijab.v9i3.976 e-mail: bance605@gmail.com biological description provides a holistic view of the state of an ecosystem, as biological communities reflect environmental conditions over time and in space (reyjol et al., 2012). macroinvertebrate communities are the most frequently used to assess the integrity of aquatic ecosystems (barbour et al., 1999; clarke et al., 2002; kaboré et al., 2016a). the use of macroinvertebrates in bioindication has several advantages. macroinvertebrate communities reflect in their structure any modifications, even temporary of the environment (kaboré et al., 2016a). they are an essential link in the aquatic ecosystem's trophic chain and have a long-life cycle (moisan and pelletier, 2008). a significant change in their community structure will inevitably impact the aquatic fauna 178 bancé et al./ influence of micro-habitats on distribution of macroinvertebrates (tachet et al., 2006; touzin, 2008). in burkina faso, several studies have examined the diversity and ecology of macroinvertebrates (guenda, 1986; kabré et al., 2002; sanogo et al., 2014; kaboré et al., 2016a). these authors have demonstrated that the freshwater ecosystem in burkina faso harbors the highest diversity of macroinvertebrates composed of insects, the most dominant, crustaceans, annelids and molluscs (ouédraogo et al., 2015; kaboré et al., 2016b, c). to analyze changes in the macroinvertebrate population due to human disturbance, all micro-habitats must be taken into account when sampling (metzeling et al., 2003, touzin, 2008). in burkina faso, few studies have been focused on the impact of micro-habitats in the distribution of macroinvertebrates (kaboré et al., 2016c). therefore, this study aims to analyze microhabitat diversity's influence on the spatial distribution of macroinvertebrates downstream of the ziga reservoir in burkina faso. the specific objectives are to (1) determine the disversity of macroinvertebrates in ziga reservoir, (2) analyze the impact of habitats on the macroinvertebrates distribution’s, and (3) explore the relationship between the physicochemical variables and the macroinvertebrate taxa’s. materials and methods study area: burkina faso is a dry sahelian country with several reservoirs as the central stocking waters. with a surface of 8 872.5 ha and a volume of 208 million m3 in the flood period, the reservoir of ziga (fig. 1) is one of the biggest in burkina faso. it is located between longitude 0 °49'w and latitude 12°37'n, in the middle course of nakambé basin, which is heavily impaired by human activities (melcher et al., 2012). it was built in 1998 on the "volta blanche" river to provide drinking water to the local population and the country's capital ouagadougou. with an increasing scarcity of surface water due to the lower rainfall in recent years, ziga dam lake is subjected by activities such as agriculture using fertilizers and pesticides, livestock, washing and bathing, mainly downstream. environmental data collection: the micro-habitats were characterized according to moog (2007) and kaboré et al. (2016a). for this study, five microhabitats were determined: dead wood (d-w) habitats dominated by dead leaves and woods; aquatic plants (a-p), the portion of habitats dominated by aquatic plants such as macrophytes (e.g. nymphaea, eichhornia, reeds); fine substrates (f-s) refers to bottom sediments; root zones (r-z), habitats dominated by tree roots, and stones area (s-a), the habitats where rocks and/or big pebbles are dominants. in each micro-habitat, the following keys physicochemical variables, including temperature, figure 1. study site with framed sampling area. 179 int. j. aquat. biol. (2021) 9(3): 177-186 ph, electrical conductivity and dissolved oxygen were measured in-situ between 9 and 11 am using a portable multi-parameter probe (hanna instrument, hi9829). the transparency and water depth were measured using a secchi disk and tri-weighted measuring tape. each parameter was taken monthly for six months, from july to december 2016. the physicochemical variables were measured before the macroinvertebrates sampling. macroinvertebrates sampling: the macroinvertebrates were sampled using a hand net (25*25 cm2 of aperture and a mesh size of 500 μm). per month, a total of 20 sample units were taken in each micro-habitat. for sampling in practice, aquatic plants and roots are well shaken inside the hand net to collect any hanging organisms. the surface of the rocks was scraped with a brush and then dragged into the hand net. where possible, stones were moved to capture any organisms that might be underneath. in the fine substrates, the bottom sediment was stirred with the hand or foot to dislodge the organisms and capture them with the net. the dead woods were taken with the hand and their surface was scraped with a brush and dragged into the hand net. after the collection, the content of the net is spilled in a bucket, then thoroughly washed and stripped of large debris such as pieces of wood, large pebbles and leaves. the samples were kept per micro-habitat with ethanol (70%) in plastic pots, then transported in an icebox to the laboratory. before sorting out the organisms, samples were sieved and the animals were sorted with the naked eye and under to a binocular microscope. finally, all organisms were identified to the lowest taxonomic level as possible using taxonomic manuals and keys (durand and lévêque, 1981; tachet et al., 2010) and enumerated. data analysis: to visualize the variation of physicochemical variables in micro-habitats, box plots were used. the richness and abundance of macroinvertebrate taxa were calculated. then, the common diversity indices, which provide more information about community composition, rarity and commonness of species in a community were used. thus, the shannon-wiener index (h’) (shannon and wiener, 1949) and equitability (piélou, 1969) were calculated in each micro-habitat using respectively formulas (1) and (2). 𝐻𝐻′ = −∑((𝑁𝑁𝑁𝑁 / 𝑁𝑁) 𝑥𝑥 𝑙𝑙𝑙𝑙 (𝑁𝑁𝑁𝑁 / 𝑁𝑁)) (1) where ni is the number of individuals of a given species and n: total number of individuals. 𝐸𝐸 = 𝐻𝐻 ′/ 𝐻𝐻𝐻𝐻𝐻𝐻𝑥𝑥 = 𝐻𝐻′ / 𝐿𝐿𝐿𝐿𝐿𝐿2 𝑆𝑆 (2) where s is a number of taxa observed. for the density calculation, we used a log transform following the formula. �𝑫𝑫� 𝒊𝒊𝒊𝒊𝒊𝒊. 𝒎𝒎𝟐𝟐 �� = 𝐥𝐥𝐥𝐥𝐥𝐥𝟏𝟏𝟏𝟏( 𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻𝑻 𝑵𝑵𝑵𝑵𝒎𝒎𝑵𝑵𝑵𝑵𝑵𝑵 𝑻𝑻𝒐𝒐 𝑻𝑻𝒊𝒊𝒊𝒊𝒎𝒎𝑻𝑻𝑻𝑻𝒂𝒂 𝑨𝑨𝑵𝑵𝑵𝑵𝑻𝑻 𝑻𝑻𝒐𝒐 𝒂𝒂𝑻𝑻𝒎𝒎𝑻𝑻𝒊𝒊𝒊𝒊𝒔𝒔 𝑵𝑵𝒊𝒊𝒊𝒊𝑻𝑻𝒂𝒂 ) (3) the non-parametric kruskal-wallis test was used to test the significances of physicochemical variables and macroinvertebrate diversity in micro-habitats. to explore the influence of environmental variables on macroinvertebrates, the redundancy analysis (rda) (van den wollenberg, 1977) were performed with software canoco for window (version 4.5). results variation in physicochemical variables: in some micro-habitats, the temperature is globally warm (general mean >25°c), characteristic of tropical area, with a good oxygen content and ph close to neutral (fig. 2). in the figure 2b, the minimum value of temperature (19.6°c) was found in dead woods (average = 20.3±0.7, p = 0.003) and roots (average=25.54±3.43), and the maximum value (32.4°c) was recorded in fine substrates (average = 26.93±4.06). the minimum and maximum values (2.8-8.09 mg/l) of the dissolved oxygen were reported in aquatic plants (average = 5.66±1.82, fig. 2d). the minimum value (7 cm) of water transparency and the maximum value (34 cm) were also reported in aquatic plants while the high mean was recorded in the coarse substrate habitats (average = 12.06 cm, p = 0.005, fig. 2e). the minimum value (6.08) of the ph were found in fine substrates and root habitats, and the maximum value (8.07) in roots (fig. 2a). the lowest mean (6.22±0.10, p = 0.002) was recorded in the dead woods. the minimum value (41 µs/cm) of the conductivity is recorded in roots (average = 80.12±25.02, p = 0.007) while the maximum value 180 bancé et al./ influence of micro-habitats on distribution of macroinvertebrates (230 µs/cm) was reported in aquatic plants and coarse substrates (averages =113.66±70.35; 109.23±67.87, respectively; fig. 2c). the aquatic plants have the widest ranges for ph, conductivity and dissolved oxygen. taxonomic composition and abundance of macroinvertebrates: in total, 3,773 individuals of macroinvertebrate were collected and all belong to 33 taxa, 10 orders and five classes (table 1). we found that the insects (72.72% of the total taxonomic diversity) are the most dominant group, with 7 orders and 24 taxa. they are follows by the molluscs (12%) and the annelids and crustceans are the less represented (<10%). influence of micro-habitats on the macroinvertebrate’s distribution: in the study area, the five micro-habitats selected were mainly dominated by fine substrates (32.5%), aquatic plants (25.83%), followed by coarse substrates (19.58%), roots (16.67%) and dead wood habitats (fig. 3). the macroinvertebrates structures are mainly influenced by the micro-habitats types. indeed, the highest figure 2. physico-chemical variables in the micro-habitats. (a) ph, (b) temperature, (c) conductivity, (d) dissolved oxygen; (e) transparency and (f) depth. legend: the number of stars indicates the difference; a _ h = all habitat; a-p= aquatic plants; cond=electrical conductivity; d-w = dead woods; f-s= fine substrates; o.d= dissolved oxygen; temp=temperature; trans=transparency; r-z= root zones; s-a =stone area. median value is shown in each box; vertical bars correspond to the minimum and maximum values; the ends of the box indicate the 25th and the 75th quartiles. 181 int. j. aquat. biol. (2021) 9(3): 177-186 taxonomic richness (more than 25 taxa) was found in plants, root and coarse substrates habitats (fig. 3a), while the lowest taxonomic diversity (8) was reported in the dead woods (p<0.05). we identified 8 common taxa (dysticidae, hydrophilidae, chironomidae, baetidae, caenidae (caenomedea), belostomatidae, gomphidae, libellulidae) in the five micro-habitats. gerridae and psychodidae were found only in aquatic plants and fine substrates, respectively (table 1). the results showed that the shannon diversity index follows the same trend as taxonomic diversity (fig. 3). in contrast, the low value (0.64 bits) of piélou equitability was recorded in the fine substrates (fig. 3d). the highest density was found in the aquatic table 1. occurrence of macroinvertebrates in the micro-habitats (note: *= presence). phylum class order taxa d-w a-p f-s r-z s-a arthropoda insects coleoptera dytiscidae * * * * * gyrinidae * * hydrophilidae * * * * * diptera bezzia (kieffer, 1924) * * chaoborus (lichtenstein,1800) * * * chironomidae * * * * * culicidae * * * psychodidae * simuliidae * * * * chrysops (meigen, 1803) * * * * ephemeroptera baetidae * * * * * caenomedea (thew, 1960) * * * * * hemiptera belostomatidae * * * * * micronecta (kirkadly, 1897) * * * * gerridae * nepidae * * * notonecta (linnaeus, 1758) * * * * veliidae * * * odonata anax (selys, 1872) * * * * coenagrionidae * * * * gomphidae * * * * * libellulidae * * * * * trichoptera hydropsychidae * * * leptoceridae * * * crustaceans decapoda caridina africana (kingsley, 1882) * * * gecarcinicidae * * * * macrobranchium dux (lenz, 1910) * * molluscs gasteropoda mesogasteropoda ampullaridae * * bellamya unicolor (olivier, 1804) * * * basommatophora biomphalaria (preston, 1910) * * bivalva unionoida irridinidae annelids clitellata arynchobdellida hirudinae * * haplotaxidae * * * * total 33 10 27 25 27 21 182 bancé et al./ influence of micro-habitats on distribution of macroinvertebrates figure 3. influence of micro-habitat on the macroinvertebrates distribution. (a) number of taxa, (b) log10 of the mean density, (c) shannon index and (d) equitability index; the histogram indicates the percentage of micro-habitat available and the curve indicates the index. figure 4. redundancy analysis (rda) of macroinvertebrates taxa and environmental variables. legend : [aesh = aeshnidae ; aty = atyidae ; baet = baetidae ; cerato= ceratopogonidae ; culi = culicidae ; coena = coenagrionidae ; gyr = gyrinidae ; chloro = chlorolestidae ; lepto = leptoceridae ; hydrops = hydropsychidae ; iridi = iridinidae ; veli = veliidae ; noto = notonectidae ; calopt = calopterigidae ; ger = gerridae ; caeni = caenidae ; simuli = simuliidae ; hydrom = hydrometridae ; chiro = chironomidae ; libel = lebellulidae ; gomph = gomphidae ; taba = tabanidae ; chaobo = chaoboridae ; nepi = nepidae ; gecar = gecarcinicidae ; belos = belostomatidae ; hiru = hirudinidae ; oligo = oligochaetae ; corix = corixidae ; dytis = dytiscidae ; ph = potentiel-hydrogen ; transp = transparence ; o. d = dissolved oxygen ; cond = conductivity ; temp. = temperature ; prof = depht]” 183 int. j. aquat. biol. (2021) 9(3): 177-186 plants (363.87 =±21.58 ind/m2), followed by roots (348±3.22 ind/m2), dead woods (188.30±4.41 ind/m2), fine substrates (1746.61 ind/m2) and stones (168.51 ind/m2), respectively (p<0.05) (fig. 3b). our findings revealed that macroinvertebrates community are linked to the aquatic plants, the coarse substrates and roots. influence of physicochemical variables on macroinvertebrates: the representativeness of all axes, given by the monte carlo test, is significant (p = 0.02; f-ratio = 5.131). axis i is very significant (p = 0.004; f-ratio = 6.887) and expresses 62% of the information, axis ii, 23.5%, i.e. a total of 85.5% for both axes. importantly, the polluo-sensitivity of the taxa is notably remarkable in figure 4. the taxa such as dytiscidae, nepidae, corixidae are positively and strongly correlated to the transparency and conductivity (r = 0.76) that oppose them to those taxa (iridinae, gomphidae and culicidae) which are positively correlated to ph and temperature (r = 0.63). in opposite, simulidae, caenidae and baetidae are positively and strongly correlated to the dissolve oxygen (r = 0.58). discussions the local taxa assemblages are strongly influenced by habitats quality and complexicity (allan, 2004; kaboré et al., 2016c). in this study, we recored diverses micro-habitats mostly dominated by fine sediments, aquatic plants and coarse substrates. the diversity and frequency of micro-habitats can be explained by the sampling period that corresponds to the rainy season. with rainfall, the floods increase and enlarge the ecological niche, which contributes to expanding micro-habitats for macroinvertebrates (ouéda et al., 2007; kaboré et al., 2016a). the similar results were obtained by kaboré et al. (2016a) who have proven a strong relationship between macroinvertebrates and physicochemical variables. the poor water conditions observed in some microhabitats could be attributed to man-induced activities, such as vegetable farms using fertilizers and pesticides, bathing, washing, and animal pasture. we found evidence of those activities near the study sites. this study reveals that benthic invertebrates are dominated by the arthropods community followed by molluscs and annelids typical of tropical freshwater ecosystems (barbour et al., 1999; edia et al., 2013; tampo et al., 2015; kaboré et al., 2016a; tanon et al., 2020; bancé et al., 2021). most macroinvertebrates taxa recorded here are similar to those encountered in previous studies in burkina faso (guenda, 1986; sanogo et al., 2014; ouédraogo et al., 2015; kaboré et al., 2016a, b) that have demonstrated that the lakes ecosystems harbor high diversity of insects. this can be explained by insects' remarkable capacity to adapt to different biotopes even those of extreme conditions (tachet et al., 2003; mereta et al., 2011). the results are similar to those reported by several authors in tropical regions (diomandé et al., 2009; sanogo et al., 2014; kaboré et al., 2016; bancé et al., 2021). our finfings revealed a high number of taxa (33) compared to those of kabré et al. (2000) who have idenfied four taxa in bagré lake. this could be justified by the sampling technique using multi-habitat approche (camara et al., 2012; kaboré et al., 2016a). in other hand, the number of taxa found here is lower compared to those of kaboré et al. (2016c), who have reported 60 taxa. that can be explained by the fact: (1) one site was sampled, and (2) the organisms were mostly identified to families and genera taxonomic resolution. the diversity of macroinvertebrates increases with habitat conditions and food resources availability. according to these authors (ouéda et al., 2007; kaboré et al., 2016a), organic matters contribute to the proliferation of phytoplankton, zooplankton and detritus which improve the food resources and enhance habitat quality for benthic macroinvertebrates. the high number of individuals found in plants and root reinforce our arguments that those habitats enclose an important amount of organic matters (bournaud and cogerino, 1986). the abundance of aquatic plants enhances environmental heterogeneity, protects from predators, and reduces competition between species (gong et al., 2000; uwadiae, 2013; kaboré et al., 2016a). for example, dipterans such as chironomidae deposit their eggs in 184 bancé et al./ influence of micro-habitats on distribution of macroinvertebrates the plants, which could explain their high density in this micro-habitat (dejoux, 1977). as well, dead wood by decaying organic matter can serve as supplementary food resources for aquatic organisms. the aquatic vegetations offer protection to rheophilic taxa such as hydropsychidae, simuliidae, and filterfeeding larvae that take advantage of organic matter inputs from water. cummins and lauff (1969) also demonstrated that the availability of food resources can influence the distribution of macroinvertebrates locally. the sampling area is located in the most threaned basin of burkina faso. this could explain the dominance of the tolerant taxa. according to melcher et al. (2012), nakambé bassin is the most populated and urbanized area with intense anthropogenic activities (e.g. intense agriculture using pesticides and fertilizers, vegetables farming). conclusion this study we assessed the biotic and abiotic factors that influence the distribution of macroinvertebrates in the lake. the results showed that the physico-chemical variables and habitat types determine strongly the structure of macroinvertebrates community. in our findings, coarse substrares and aquatic plants harbour the highest diversity of macroinvertebrates. the results also showed that the study area is impacted by anthropogenic activities as proven by the dominant of tollerants taxa recorded. the protection of habitats is a fundamental for aquatic biodiversity conservation in west africa, especially in burkina faso were the aquatic ecosystems are ongoing pressures. and the environmental restoration measures may help to mitigate the aquatic habitats degradation and biodiversity declining in the nakambé basin. acknowledgements we thank the national office of water and sanitation (onea) for allowing us to work on the reservoir of ziga. we are grateful to project susfish (sustainable management of fisheries and water in burkina faso) funded by appear (austrian partnership program for higher education and research for development) for considerable support for field activities. we thank the european union program erasmus + for the mobility scholarship, which allowed us to finish writing. we thank s. djidama, s.r. pierre, k. boukaré and s. mahamadi for field and laboratory work support. references agbohessi p.t., imorou t.i., attakpa e.y., kestemont p. 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(2021) 9(4): 234-243 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article influence of lunar cycle, moon driven tides and water physicochemical factors on the gonadal maturation of green mussel, perna viridis, in the inner malampaya sound, taytay, palawan, philippines adzel adrian guillen baldevieso1,2, fiona l. pedroso*2, 3,1karen grace andrino-felarca2, maria shirley m. golez4, mary jane apines-amar2 1college of fisheries technology, surigao del sur state university, lianga campus, lianga surigao del sur, philippines. 2institute of aquaculture, college of fisheries and ocean sciences, university of the philippines visayas, miagao, iloilo, philippines. 3school of fisheries and marine technology, mindanao state university at naawan, naawan, miasamis oriental, philippines. 4institute of marine fisheries and oceanology, college of fisheries and ocean sciences, university of the philippines visayas, miagao, iloilo, philippines. s article history: received 19 may 2020 accepted 31 july 2021 available online 2 5 august 2021 keywords: gonadal maturity body indices lunar cycle tidal fluctuations abstract: this study determined the influence of the lunar cycle, tidal fluctuation, as well as water physicochemical factors on the gonadal maturation of green mussel, perna viridis, in malampaya sound, taytay, palawan, philippines. monitoring and sampling were conducted for the 6 months, from october 2017 to march 2018. it was observed that the lunar cycle has a direct influence on gonadal maturity. gonadal changes in both male and female p. viridis were observed histologically and both exhibit the characteristic of lunar-synchronous pattern. it was observed that a high number of green mussels with spawning stages usually occur during the full moon and new moon. furthermore, gonadal maturity was also affected by the tidal fluctuation. a higher number of p. viridis with spawning stages were recorded during spring tide, whereas the percentage of spent individuals were higher during neap tides. furthermore, it was observed in the present study that salinity fluctuation could influence spawning of green mussel, wherein the number of spent individuals increased after the sudden drop in water salinity from 30 to 13 ppt after the heavy rainfall, an indication that spawning had occurred. the presence of individuals with spawning stages was also observed throughout of period of sampling which indicates that mussels were continuously breeding in the area. the highest percentage of individuals having spawning stages were recorded in october and november. the information on the factors that affect gonadal maturation and spawning schedule can help mussel growers predict the schedule of spatfall. introduction green mussel, perna viridis is one of the commercially important bivalve species and a cheaper source of protein (korringa, 1972; tortell et al., 1978). its cultivation is extensively carried out in tropical countries (rajagopal et al., 2006; soon and ransangan, 2014) in the intertidal and subtidal areas. in the philippines, green mussel culture started in 1962 in the cavite province (aypa, 1990) and became the most widely cultured bivalve species in the country with estimated production of 26.30 thousand metric tons in 2018 (philippine statistics authority 2018). currently, the philippine government is making an effort to improve and expand the culture of this mussel. in 2014, the government initiated to put *correspondence: fiona l. pedroso doi: https://doi.org/10.22034/ijab.v9i4.859 e-mail: fionapedroso@gmail.com up the first mussel hatchery in the country for a sustainable spat production (mero et al., 2019). furthermore, research efforts to increase the larval production has been conducted (maquirang et al., 2020) and improvement of the grow-out production is on-going. one of the areas in the country with high potential for grow-out culture of green mussel is inner malampaya sound, taytay, palawan, philippines. the mussel sizes produced in this area were larger compared to other mussels cultured in other locations of the country, thus inner malampaya sound can be an ideal source for breeders. one of the important considerations of successful cultivation of green mussel is to identify the factors, influencing their reproduction and spawning. 235 int. j. aquat. biol. (2021) 9(4): 234-243 knowledge on the reproductive biology of the green mussel can help farmers to predict their spawning and spatfall schedule. on the other hand, the reproductive development of mussels depends on the quality of their environment that varies in different areas. main factors that affect gonadal development of the green mussel in the different area includes, temperature (lee, 1988; shafee, 1989; rajagopal et al., 1998; alfaro et al., 2001 rajagopal et al., 2006), density and composition of food supply (rajagopal et al., 1998; lopes-lima et al., 2014; soon and ransangan, 2014), and light intensity, and wave action (wong and cheung, 2003). there is limited evidence about the relationship between the different lunar phases and the spawning activities of green mussel as reported in other mollusk species. in abalone, haliotis asinina, natural spawning usually occurs during new moon and full moon (capinpin, 1995; counihan, 2001). similarly, giant clams, tridacna spp. spawns during mid to late afternoon that coincides with the full moon and new moon periods (elis, 2009). detailed information regarding the lunar cycle in gonadal maturity can enhance the farming efficiency of green mussel particularly in the malampaya sound, taytay palawan, philippines. thus, this study aimed to investigate the gonadal maturity and spawning of green mussel in relationship to some uncontrolled onsite factors such as the lunar cycle, moon driven tides, and physicochemical parameters. materials and methods study area and sampling: the study was conducted at barangay new guinlo, taytay, palawan, philippines (fig. 1) located on the inner malampaya sound, taytay, palawan, philippines (10.78272 latitude, 119.437218 longitude). the mussel farms in this area supply most of the green mussel in the markets of palawan, philippines. the study was conducted during six months from october 2017 to march 2018. samplings were done weekly representing the different lunar phases (full moon, new moon, first lunar quarter and last lunar quarter). the daily tidal ranges and lunar cycle were identified using the book of philippine tide and current tables (2017 and 2018 editions) (namria, 2017, 2018). samples were collected regularly from 9 am to 12 noon. about 50 green mussels (40-60 mm) were randomly collected from three different random points at the mussel farm. samples were taken from the middle part of the bamboo stakes and then mixed and randomly analyzed for gonadal maturity and gonad index. reproductive biology: for every sampling schedule, twenty samples of adult mussel (10 males and 10 females) with shell length of 50-60 mm were sectioned dorsoventrally through the mid-body and gonads were removed from the mantle lobes. the gonads were individually stored in a screw cap container and immediately preserved in 70% ethanol. samples were sent to the microtechnique laboratory, institute of marine fisheries and oceanology, university of the philippines visayas for further histological analysis. samples were fixed using bouin’s solution, subsequently dehydrated with the series (80-100%) of ethanol and xylene, embedded in paraffin wax, sectioned (7 µm) and stained with hematoxylin and eosin based on bancroft and stevens figure 1. map of palawan and malampaya sound, taytay palawan pointing the location ( ) of mussel farms as sampling area. 236 baldevieso et al./ influence of lunar cycle on gonadal maturation of green mussel (1996). gonad sections were examined under a light microscope, classified, and scored based on the classification scheme based on shafee (1989) (figs. 2, 3). after classification of gonads, the gonad index (𝐺𝐺𝐺𝐺) was computed using the formula below (king et al., 1989): gi = ((no, in each stage) (rank of stage) / total sample size) x 100 water physico-chemical parameters: water parameters in the study area such as salinity (using rocker sa10t and rhsn-10atc refractometer), ph (using oakton eco testr ph1 and eutech phtestr20), temperature (using digital thermometers), and dissolved oxygen (kit) and turbidity (using secchi disk) were measured around 9-12 am during the sampling period. these monitoring covered three strategic points in the area at different depth (surface and bottom (2-3 m below depending on tidal height)). bottom waters were collected using niskin water sampler. data analyses: data were subjected to a normality test using the shapiro-wilk test and homogeneity test using levene’s test before further analysis was done. the gonad index and different water parameters were compared monthly, every lunar week, and tidal schedule (spring tide and neap tide), using anova, brown-forsythe, and t-test based on the homogeneity of each data set. results influence of lunar cycle on gonad index and gonadal maturation: observation of the gonad index (gi) and gonadal maturation was conducted at different lunar phases from october 2017 to march figure 2. light microscopic image of redeveloping (a-d) and mature (e-h) green mussel gonads. female = a, b, e, f; male = c, d, g, h (h&e). 237 int. j. aquat. biol. (2021) 9(4): 234-243 2018. although no significant no significant difference was observed, the highest percentage of individuals with spawning stages were recorded during full moon, while the fewest number was during the first quarter of the lunar cycle i.e. the redeveloping stage was high in almost all the lunar phases (table 1). furthermore, the females with spawning stages were observed to have a lunar synchronous pattern during the full moon and new moon which can be noted clearly after the reproductive/breeding/spawning peak (october) (fig. 4). influence of moon driven tides on gonad index and gonadal maturation: gonad index was found to be significantly higher during the neap tides. however, figure 3. light microscopic image of spawning (a-d) and spent/resting (ef) green mussel gonads. female = a, b; male c, d (h&e). figure 4. female green mussels at spawning stage during different lunar phases at the inner malampaya sound, taytay palawan. 238 baldevieso et al./ influence of lunar cycle on gonadal maturation of green mussel the number of individuals with spawning stages were observed to be high during spring tides (table 2). whereas, individuals having spent stages were high during neap tides. thus, these observations indicate that tidal fluctuation at different lunar phases can influence spawning activities of green mussels. influence of physicochemical parameters on gonad stages: the physicochemical parameters of the water in the study area were within the range required for mussel culture. salinity, ph, and temperature levels table 1. gonad maturity stages and gonad index of green mussel in relationship to the lunar phases. lunar phase gonad maturity stages (average % of occurrence) gonad index ripe spawning spent resting redeveloping full moon 4.93±3.60 22.97±7.77 3.54±1.76 10.79±8.44 57.77±10.30 184.88±14.96 last quarter 4.76±4.76 17.51±6.82 14.47±8.56 2.04±2.04 61.21±13.32 189.29±11.41 new moon 2.86±2.86 18.36±6.22 14.64±9.19 3.85±2.41 60.30±14.15 181.43±14.50 first quarter 8.73±8.73 3.17±3.17 11.04±9.34 8.51±5.40 68.54±15.32 184.17±21.61 table 2. body indices and gonad index, and gonad stages of green mussel in relationship to tides. tide gonad maturity stages (average % of occurrence) gonad index full/ ripe spawning spent resting redeveloping neap 6.56±4.59 10.89±4.33 12.88±6.06 5.03±2.76 64.60±9.70 186.92±11.02 spring 3.89±2.23 20.66±4.83 9.09±4.75 7.32±4.32 59.04±2.76 183.92±9.231 table 3. summary of means of physicochemical parameters and their significant values among different comparisons. parameter range mean ± se monthly lunar week tides neap vs spring salinity (ppt) 13-37 29.47±0.31 0.015* 0.002** 0.806 do (ppm) 4.8-11.2 7.8±0.18 0.976 0.233 0.816 ph 4.5-8.6 8.00±0.05 0.000** 0.000** 0.732 temperature (oc) 26-33.2 28.20±0.08 0.001** 0.166 0.0.816 turbidity 0.8-2.5 1.40±0.71 0.257 0.633 0.530 *= significant different, ** = high significant different figure 5. spawning and spent stages of green mussel in relationship with salinity fluctuations. 239 int. j. aquat. biol. (2021) 9(4): 234-243 have significant variations per month. significant changes in salinity and ph values were noted during weekly sampling (table 3). drastic salinity fluctuations were observed from the third week of october until the third week of november due to constant rain. a high percentage of the spent individuals were observed after the occurrence of a huge drop in salinity in the area. this may indicate that majority of the mature individuals have released their gametes as influenced by sudden changes in environmental factors (fig. 5). monthly trend of the gonadal maturity of green mussel: mussels with spawning stages were observed throughout the period of sampling (october 2017 to march 2018) as shown in table 4. an indication that mussel is continuously breeding in the sampling area. however, the highest percentage of spawning stages were recorded during october and november 2017. whereas the highest percentage of individuals with spent gonads were observed in november. high percentage of mussel having redeveloping stages were observed from december to march (table 4). discussions regulation of reproduction in an organism is genetically controlled in response to its environment (barber and blake, 2006). one of the abiotic factors that affect gametogenesis and spawning in marine organisms is lunar periodicity. several studies have shown that reproductive cycles of marine invertebrates are often linked to the lunar phase (counihan et al., 2001; collin et al., 2016; gupta 2017). in the present study, weekly gonadal changes in both male and female p. viridis were observed histologically and both exhibit the characteristic of lunar-synchronous pattern. it was observed that a high number of green mussel with spawning stages usually occur during the full moon and new moon and spent gonads were observed thereafter an indication that spawning had happened. the occurrence of spawning during or a few days before the new moon and full moon were also observed in other mollusk species (castanos, 1997; capinpin, 1995; counihan et al., 2001; elis, 2009). likewise, some marine teleost showed group synchronous gonadal development in response to the lunar phase. for instance, in rabbitfish, their gonads mature synchronously and showed spawning peaks between full and last quarter moon, after which the gonads return to spent condition (takemura at al., 2004). thus, lunar cues play a significant factor in allowing synchrony in the gonadal maturation of the marine organisms to ensure reproductive success (takemura et al., 2004). the rhythms of the moon influence the marine environment in the form of tidal rhythm (gupta, 2017). the tidal cycle or periodic fluctuation of tides can also influence the biological rhythms of the marine organisms (tran et al., 2011). those organisms that live in the intertidal environment are greatly affected by tidal variations (nishida et al., 2006). thus, these organisms need to synchronize their physiological activities especially their reproduction timing in response to the tidal pattern to ensure better survival of their offspring. majority of the intertidal animals favored broadcast spawning during nighttime and spring tide (korringa, 1957; naylor, 1976). this allows better transport and dispersal of gametes, as well as safety of their offspring from predation, which is greater at night time (berry, 1986). however, lugworm (duncan, 1960), spirorbid polychaetes table 4. monthly body indices, gonadal maturity stages and gonad index of green mussel in the inner malampaya sound, taytay, palawan. month gonad maturity stages % gonad index full/ ripe spawning spent resting redeveloping october 33.75 31.25 1.25 0.00 33.75 232.50±8.54a november 2.02 31.31 31.31 10.10 25.25 150.00±15.17b december 0.00 8.00 13.00 14.00 65.00 159.00±24.10ab january 0.00 8.08 4.04 1.01 86.87 193.84±3.77ab february 0.00 3.33 5.00 5.00 86.67 185.00±10.41ab march 0.00 12.00 1.00 0.00 87.00 199.00±1.00ab gonad index with similar superscript = no significant different 240 baldevieso et al./ influence of lunar cycle on gonadal maturation of green mussel (rothlisberg, 1974), and larviparous oysters (knightjones, 1952) spawn during neap tides (berry, 1986). in the present study, it was observed that a higher number of p. viridis with spawning stages were recorded during spring tide intervals. whereas the percentage of spent individuals was higher during neap tides, an indication that tides have a direct influence on the reproduction of green mussel. moreover, prolonged exposure of green mussel to air during low tide can possibly stimulate mature individuals to spawn. several reports have shown that bivalves and other invertebrates can be induced to spawn through desiccation (velasco et al., 2007; abidin et al., 2016; arendse et al., 2018). gonadal maturation and spawning of p. viridis were also influenced by salinity, temperature and food availability (walter, 1982; nagabhushanan and mane, 1991; rajagopal et al., 1998). temperature may have direct influence on the development of gametes. the optimal range for p. viridis reproduction is 26 to 32oc, however, an increase in temperature which ranges from 30 to 32oc accelerates the ripening of gametes (sreedevi et al., 2014). the temperature level recorded during the duration of the present study is within the optimum requirement for gonadal development. therefore, the temperature in the inner malampaya sound, palawan, philippines provides a suitable temperature requirement for the reproduction of p. viridis. however, temperature fluctuation throughout the day is not very evident in the present study since the monitoring of temperature was done only in the morning. therefore, it is inconclusive if the temperature has a direct effect on the spawning activities of mussels. however, based on the observation of walter (1982) temperature fluctuation had no obvious relationship with the spawning of mussel in the philippines. further, stephen (1980) also noted that changes in temperature have little importance in determining spawning in indian oyster. on the other hand, it was observed in the present study that the number of spent individuals increased after a sudden drop in salinity from 30 to 13 ppt after the occurrence of heavy rainfall. the sudden change in salinity may have influence mature individuals to release their gametes and the presence of spent individuals is an indication that spawning has occurred. the observation was in accordance with the study of stephen and shetty (1981) that rapid salinity change stimulates the spawning of bivalves in indian coastal water. thus, salinity is one of the key factors in influencing spawning activities of marine invertebrates in the coastal and estuarine environment. the p. viridis in the philippines is continuously breeding throughout the year as observed by walter (1982). the continuous gonadal development and spawning of p. viridis in the philippines is a likely occurrence since the country is situated in the tropical region thus p. viridis are exposed to a stable warm condition. the presence of individuals with redeveloping stages was high throughout the study an indication that mussel in the area is continuously undergoing gonadal maturation. the occurrence of individuals with spawning stages was also evident throughout the period of sampling (october to march). furthermore, the highest percentage of individuals having spawning stages were recorded in october and november. although green mussels in the country are continuously breeding, they have also shown reproductive or spawning peaks. these were confirmed by the occurrence of spat fall in some areas in the country. for instance, the peaks of spat fall were observed in cañas bay, iloilo during may and november; december to may for sapian bay, capiz; october to december and april to june in maqueda bay, samar; december to january and march to april for silanga bay, samar; and november to december and april to may for cambautatay bay, samar (baylon et al., 2016). furthermore, pcarr (1977) reported that the settlement and spat fall of green mussel in bacoor bay, cavite occurred in april to may and october to november. moreover, spawning peaks of green mussel may also vary in other countries depending on the season. for instance, spawning occurs during june and october in kalpakkam, east cost of india (rajagopal et al., 1998) and april and november in eastern johore strait water, singapore (low et al., 1991; wong and cheung, 2003; soon et al., 2016). on the other hand, lee (1988) reported a 241 int. j. aquat. biol. (2021) 9(4): 234-243 single breeding period that extended from june to september in the victoria harbor, hong kong. while, yoshiyasu et al. (2004) reported spawning from summer to early autumn in the sagami bay, japan. conclusion and recommendations detailed reproductive pattern is an important aspect in biology, conservation, and aquaculture of green mussels. information obtained in the present study showed that the lunar cycle, tidal fluctuation, and changes in physicochemical parameters in water can affect the gonadal maturation and spawning of green mussels in inner malampaya sound, taytay, palawan, philippines. further investigation on the effect of these factors on condition index, meat yield on the period not covered by this study is recommended. acknowledgment the study was funded by the philippine department of science and technology, science education institute, accelerated science and technology human resource development program (dost-seiasthrdp), philippine department of science and technology, philippine council for agriculture, aquatic and natural resources, graduate research and education assistantship for technology program (dost-pcaarrd-great program) under the project pilot testing of longline method for green mussel culture in traditional area, and the university of the philippines visayas – office of the vicechancellor for research and extension (upv ovcre). the study was also made possible through the permission of the palawan council for sustainable development and the department of environment (pcsd), and the department of environment and natural resources community environment and natural resources office (denr-cenro), of the municipality of taytay, palawan, philippines. the author also acknowledged the help and support extended by western philippines university puerto princesa campus, upv mussel team, upv-cfos laboratory personnel and staff, puerto princesa city water district laboratory and the malampaya live fish culturer's association. references abidin n.a., s.r.m.s., ching f., othman r., manjajimatsumoto m., senoo s., mustafa s. 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(crustacea: anostraca) population in al wathba lake in the abu dhabi emirate, uae anitha saji*,1shaikha al dhaheri, junid n. shah, pritpal s. soorae terrestrial and marine biodiversity sector, assessment and conservation, environment agency abu dhabi, p.o. box 45553, abu dhabi, united arab emirates. article history: received 9 february 2016 accepted 12 april 2016 available online 2 5 april 2016 keywords: artemia population physico-chemical parameters biomass cyst counts abstract: long term monitoring programme on brine shrimp (artemia sp.) is being carried out by the environment agency, abu dhabi, united arab emirates (ead) with the prime purpose of understanding the population dynamics, ecology and habitat requirements of artemia at al wathba lake, situated within al wathba wetland reserve, which is an artificial wetland near abu dhabi city. the present study, being a component of this programme, intends to understand the influence of chemical parameters such as dissolved oxygen, nitrate, nitrite, phosphate, ammonia and total organic carbon on artemia biomass and cyst production at different sites of the al wathba lake. the study was carried out by sampling lake water quarterly for a period of 5 years from 2010 to 2014. the artemia population was found to have direct impact of the above mentioned parameters on its abundance. the abundance was highest during the year 2010. further, temperature, dissolved oxygen, and nitrate were found to be the most crucial parameters for production of artemia. furthermore, this study aimed to determine the significant relationship between physico-chemical parameters and artemia sp. population dynamics and cyst production. introduction the brine shrimp, artemia which is a microcrustacean (crustacea: anostraca), are typical inhabitants of hypersaline habitats and are welladapted to thrive in the extreme environments. lenz (1987) observed that zooplankton dynamics are influenced by various biotic and abiotic factors of their habitat (e.g. food availability, temperature, salinity, etc.) as well as the predictability and seasonality of their environment. a monitoring study conducted to assess the influence of physicochemical parameters on the distribution and cyst production of artemia franciscana revealed a significant interaction among salinity, dissolved oxygen and nitrates on cyst production (krishnakumar et al., 2014). contrary to this, some studies have found no significant relationships between physico-chemical variables and artemia population structure and density (e.g. ben naceur et * corresponding author: anitha saji e-mail address: asaji@ead.ae al., 2011). the aim of this study was to identify physicochemical variables that influence the artemia biomass and cyst production across the sampling sites in the al wathba lake. therefore, an attempt was made to evaluate the impact of various physicochemical variables on the distribution and cyst production potential of artemia, confined to al wathba lake. previous study showed that artemia population inhabiting al wathba lake is mostly affected by water temperature, salinity and ph (al dhaheri, 2004). also, in a further study (al dhaheri and saji, 2013), it was found that salinity is correlated with artemia occurrences in sampling locations. in the wild, artemia generally is found in salinities from 10-300 ppt (sorgeloos, 1980), but are more rarely found in waters lower than 45 ppt and above 300 ppt. salinity is one of the crucial factors that control growth and survival of artemia (van 88 saji et al./ influence of chemical parameters on artemia sp. population stappen, 2002). in its natural environment, temperature, feeding and salinity are the most important factors influencing artemia populations (wear and huslett, 1987). materials and methods sampling site and sampling period: the present study was conducted in the al wathba lake at al wathba wetland reserve (awwr) (n: 24.25812, e: 54.60282) which is located 40 km east of abu dhabi city (fig. 1). the water body extends for approximately 1.5 km in length and 0.5 km in width a maximum depth of almost two metres (brook et al., 2004). the water in al wathba lake is supplemented by brackish water from the nearby al wathba camel race track as well as discharge of treated sewage effluent from the adjacent mafraq wastewater treatment plant. a set of water samples from all sites was collected for analysis of the following chemical parameters: ph (test method apha 4500 h+), salinity (test method apha2520 (b), dissolved o2, nitrate (no3), nitrite (no2), phosphate (po4), ammonia (nh3), total organic carbon (toc), copper (cu), cadmium (cd), and iron (fe). the chemical analysis was conducted at the laboratories of arab center for engineering studies, abu dhabi, uae (aces). sampling techniques: extensive field study was carried out for five years from 2010 to 2014. sample collection was carried out on quarterly (q1, q2, q3 and q4) basis at day time. the sampling sites for artemia monitoring were chosen on a 100 m grid overlaid on a geo-corrected digital satellite image of the awwr (al dhaheri, 2004). altogether, nine sampling locations were selected on the basis of their representation of the physical-chemical characteristics of al wathba waters. in addition to these nine sampling sites of the lake, water samples from two fresh water inlet valves was also included to understand the water quality of tertiary treated water from al mafraq sewage treatment plant (mstp). water samples were collected using a horizontal sampling device (van-dorn water sampler) deployed for surface sample collection. temperature and ph (in situ) was measured using wtw handheld multi-parameter instrument figure 1. map of al wathba wetland reserve with locations of sampling points 89 int. j. aquat. biol. (2016) 4(2): 87-95 (multi/350i/set). the time of sampling and depth of each sampling site was also documented. counting of artemia: artemia were counted on a grid under stereo-microscope after filtration of the water samples with plankton mesh size 20 µm and euthanized with sparkling water (artemia were killed by the co2 contained in the sparkling water). cyst density calculated in 1 litre of the sample (total cyst/1 litre = ((total cyst/6 ml) x 1000) (al dhaheri, 2004). data analysis: descriptive statistics such as the mean, standard error, standard deviation, minimum and maximum values of each physico-chemical factor were calculated. one-factor analysis of variance (anova) used for each parameters (temperature, salinity, dissolved oxygen, nitrate (no3), nitrite (no2), phosphate (po4), ammonia (nh3), total organic carbon (toc), copper (cu), cadmium (cd), and iron (fe)). pearson’s correlation coefficient (r) was calculated for the statistical interpretation of the influence of the physicochemical variables on artemia abundance. all the above mentioned statistical analyses were performed using ibm spss statistical software version 21.0. (ibm corp. released 2012). limits of significance for all critical ranges were set at p<0.05 two-tailed. results the salinity varied during the study period and ranged between 173.61-219.47 ppt and was found to be increased with the increase of temperature. over the whole period of the study, the lowest recorded value for salinity was in the month of june (q2) 2012 and the highest value in the month of september (q3) 2014, 61.79 and 357 ppt, respectively (table 1). the overall temperature varied between 22.8235.89°c across the years. the lowest temperature value recorded was in the month of december (q4) temperature (°c) ph salinity(mg l -1 ) dissolved oxygen (mg l -1 ) 2010 march 26.83 ± 1.65 8.13 ± 0.08 121.18 ± 001.19 6.73 ± 0.39 june 34.31 ± 1.03 7.01 ± 2.64 180.88 ± 073.25 7.92 ± 0.41 september 35.47 ± 2.66 7.81 ± 0.49 249.08 ± 119.15 4.64 ± 2.49 december 23.72 ± 1.34 7.62 ± 0.18 106.13 ± 002.31 4.94 ± 0.86 2011 march 25.10 ± 1.64 8.07 ± 0.26 137.28 ± 027.04 4.84 ± 2.23 june 33.84 ± 1.69 7.86 ± 0.22 213.86 ± 054.09 4.18 ± 1.76 september 35.75 ± 2.39 7.17 ± 0.48 295.68 ± 115.09 5.20 ± 2.24 december 22.82 ± 1.95 7.50 ± 0.18 126.78 ± 025.28 4.87 ± 1.51 2012 march 24.02 ± 1.00 7.64 ± 0.27 194.82 ± 050.49 3.67 ± 1.27 june 27.69 ± 1.38 7.53 ± 0.24 061.79 ± 073.78 6.67 ± 1.67 september 35.36 ± 3.08 7.83 ± 0.25 135.08 ± 114.14 6.82 ± 1.22 december 24.91 ± 0.41 8.05 ± 0.15 127.09 ± 022.87 7.05 ± 0.89 2013 march 24.64 ± 0.64 8.64 ± 0.19 213.66 ± 021.23 5.34 ± 1.79 june 33.37 ± 1.76 7.99 ± 0.49 313.26 ± 029.52 4.75 ± 1.68 september 34.96 ± 1.44 7.83 ± 0.45 272.04 ± 050.91 6.88 ± 0.84 december 25.68 ± 2.14 7.72 ± 0.32 152.45 ± 024.14 6.27 ± 0.79 2014 march 25.68 ± 0.91 7.92 ± 0.35 124.63 ± 034.86 7.54 ± 0.34 june 33.57 ± 1.69 8.00 ± 0.66 297.61 ± 091.37 6.56 ± 1.47 september 35.89 ± 1.53 7.45 ± 0.37 357.06 ± 017.07 7.64 ± 0.61 december 24.17 ± 1.99 8.14 ± 0.27 085.22 ± 019.75 7.39 ± 0.31 table 1. physico-chemical parameters (mean ± sd) for different sites at al wathba lake. 90 saji et al./ influence of chemical parameters on artemia sp. population 2011 and highest value recorded in the month of september (q3) 2014. the surface water ph ranged between 7.0 (june (q2)) 2010) and 8.6 (march (q1) 2013). high seasonal variations of dissolved oxygen were observed during the month of june (q2) 2010 and in the month of march (q1) 2012. the average values of oxygen concentration varied from 3.6-7.9 mg l-1. the results of the nutrient analysis (table 1 and figs. 2 and 3) showed that the mean values of nitrate (no3) ranged between 2.24 mg l -1 in september (q3) 2014 and 45.13 in june (q2) 2012. the mean value of nitrite (no2) ranged between 0.19 mg l -1 in june (q2) 2010 and 4.28 in december (q4) 2014. the minimum ammonia concentration (0.21 mg l-1) was recorded in march (q1), 2013 and the maximum (4.43 mg l-1) in march (q1) 2014. the minimum phosphate concentration (1.31 mg l-1) recorded was in june (q2) 2010 and the maximum concentration (6.24 mg l-1) in june (q2) 2012. the mean values of total organic carbon (toc) ranged figure 2. mean (± sd) concentration (mg l-1) values of physico-chemical parameters recorded across different months at nine sampling sites at al wathba lake. figure 3. mean (± sd) concentration (mg l-1) values of heavy metals (a-copper; b-cadmium; ciron) recorded across different months at nine sampling sites at al wathba lake. 91 int. j. aquat. biol. (2016) 4(2): 87-95 between 15.85 mg l-1 in june (q2) 2012 and 85.85 mg l-1 in september (q3) 2014. toc was the most frequently abundant nutrient and no3, no2, po4 and nh4 were moderately recorded during the study period. the inorganic heavy metals had generally very low concentration values except for a couple times (e.g. in 2011 and 2012). cd showed presence at a very high concentration only during march 2010, which was unexplainable as to how and why it had such high concentration value. based on test results, cu and cd concentrations are not exceeding the standard limits (ehsms, 2003). we conclude that brine shrimps are not currently at risk from heavy metals (cu and cd) and iron (fe) as the concentrations of heavy metals and iron were recorded below the critical limit of lake water quality standards. in al wathba lake, the average artemia adult density was measured between 0-64 individuals l-1 and juveniles between 0-78 individuals l-1. the maximum artemia adult and juvenile densities were observed in march (q1) 2010. the average number of individuals collected during the study period was lowest in the months of june (q2), september (q3) temperature ph salinit y do nitrates nitrites ammoni a phosphate s total organic carbon cu cd fe cysts -0.293** -0.025 -0.063 -0.187* -0.099 -0.002 0.064 -0.106 -0.080 0.028 -0.025 -0.007 juveniles -0.324** 0.098 -0.040 -0.100 -0.185* -0.048 0.012 0.007 0.072 -0.097 0.382** 0.021 adults -0.288** 0.036 -0.063 -0.161* -0.199** -0.102 0.091 0.055 -0.141 -0.052 0.433** 0.115 total -0.311** -0.018 -0.065 -0.193** -0.112 -0.008 0.067 -0.102 -0.080 0.021 0.008 -0.002 correlations is significant at the 0.05 level (2-tailed). **. correlation is significant at the 0.01 level (2-tailed). table 2. correlation between artemia and cyst density and water physico-chemical parameters. figure 4. fluctuation in artemia cyst, juvenile and adult density during the period 2010 to 2014 at al wathba lake. 92 saji et al./ influence of chemical parameters on artemia sp. population and december (q4) 2012, 2013 and 2014. whereas the maximum cyst density was observed in the year 2011 (335 individuals l-1) and the minimum density was observed in the year 2010 (251 individuals l-1). cyst density was highest when temperature, ph, salinity, dissolved oxygen, nitrate, nitrite, ammonia, phosphate and total organic carbon contents recorded were 29.3°c, ph 7.65, 4.77 mg l-1, 16.63 mg l-1, 1.51 mg l-1, 1.42 mg l-1, 2.73 mg l-1 and 38.09 mg l-1, respectively. for the population composition, cysts made up a very high fraction of the samples during the month of december (q4) 2010 (table 2 and fig. 4). overall, temperature was found to have a significantly negative effect (p<0.01; table 2) on all the artemia reproductive stages alike, while salinity was not found to play a significant effect on the artemia stages. similarly, dissolved oxygen also had a negative effect on the artemia populations except for the juveniles, are better able to cope with fluctuations in oxygen levels due to physiological adaptions in their life cycle stage, suggesting it survives in sites where dissolved oxygen is least in concentration. the present study showed that hatching and survival of cysts occur in two periods march (q1) and december (q4) and maximum adult density occurred in march (q1) in almost all sampling periods. amongst chemicals, nitrates were the only group that affects the juveniles and adults alike negatively. similarly, cd was the only hard metal affecting negatively the juveniles and adults alike in the al wathba lake, however, throughout the sampling years cd concentrations were negligible but its very high concentration at the start of the sampling cannot be explained. discussion artemia population was abundant when the average concentration of salinity, temperature and ph, dissolved oxygen, nitrite, nitrate, ammonia, phosphate and total organic carbon were 121 ppt, 26.83°c, 8.13, 6.73 mg l-1, 4.96 mg l-1, 0.55 mg l-1, 1.27 mg l-1, 4.68 mg l-1, 21.76 mg l-1, respectively. previous study on the influence of physico-chemical variables on the distribution of the invader artemia franciscana in the salterns of kelambakkam, southeast coast of india showed that artemia population was abundant when salinity, temperature and dissolved oxygen were from 70 to 200 ppt, 24.5 to 35.4°c and 0.8 to 6.1 mg l-1, respectively (krishnakumar et al., 2014). in the current study, it is evident that temperature, dissolved oxygen, nitrate are the most crucial parameters for production of artemia at al wathba lake. in natural environments, temperature, feeding conditions, and salinity are important factors influencing artemia populations (wear and haslett, 1987; van stappen et al., 2001; torrentera and dodson, 2004). in the current study, the maximum cysts were observed during the month of december (q4) 2010 when the temperature recorded low for the whole study period. also in a study of the correlations between environmental parameters and artemia populations in sabkhet el adhibet in tunisia (ben naceur et al., 2011), it was found that physico-chemical parameters present significant connections with the reproductive mode and offspring output. conversely, camargo et al. (2004) state that the variation in physico-chemical conditions of some thalassohaline (marine) sites in the colombian caribbean did not influence by environmental factors, principally oxygen, salinity, and temperature. torrentera and dodson (2004), who studied the ecology of the brine shrimp artemia in the yucatan (mexico) salterns, showed that the artemia population dynamics and abundance are highly influenced by environmental factors, principally oxygen, salinity, and temperature. in addition, camargo et al. (2004) reported that the reproductive experiment (mean cyst production per female) does not entirely agree with the estimated cyst production potential but may be due to a combination of certain parameters (i.e. salinity, oxygen concentration, low nitrate, and starvation of the adult artemia population after reaching a high density). in the current study, the maximum artemia adult and juvenile densities were observed in march 93 int. j. aquat. biol. (2016) 4(2): 87-95 (q1) 2010 and during this period maximum level of dissolved oxygen recorded. on the other hand, lenz (1987) observed that zooplankton population dynamics are influenced by abiotic factors (salinity, temperature, and nutrient concentration) and by biological interactions (predation, competition, and grazers). sorgeloos et al. (1986) reported that the best conditions for artemia biomass production are at the lower salinity levels (100 ppt) and under conditions of very regular food availability. according to a previous study (gaevskaya, 1916; gilchrist, 1960; amat, 1980; litvinenko et al., 2007), salinity and salt composition are the most important ecological characteristics affecting morphological and biometrical artemia parameters. similar study showed that different schedules in shrimp abundance and reproductive strategy correlated with habitat differences, principally oxygen, salinity and temperature (torrentera and dodson, 2004). the field data clearly showed lower abundance in all the populations at the highest salinities and highest peak of cyst production at the highest salinities during the dry season (torrentera and dodson, 2004). studies on the combined effect of temperature and salinity on the survival of artemia of various geographical origins (with two artemia salina populations from spain and cyprus) showed that temperature is the most important factor that affected artemia survival, and that the a. salina and a. parthenogenetica strains did not survive at temperature exceeding 30°c and a. franciscana at 34°c (ben naceur et al., 2009). in our study, the variation in water temperature showed a difference during the study period; the maximum surface water temperature in the lake recorded during the year 2010 was 29.9°c and the minimum salinity recorded during the year 2011 with a temperature value 27.9°c. vanheacke and sorgeloos (1989) reported that the maximum growth and biomass production occur in the range of 20-27°c. but the impact of temperature on growth varies from one strain to another, but for most strains, growth is limited below 15°c temperature (ben naceur et al., 2009). this is evident to artemia population at al wathba lake, the season for optimal density is confined to december-march. triantaphyllidis et al. (1995) showed that the salinity effects on survival, maturity, growth, biometrics, and reproductive and lifespan characteristics of bisexual and a parthenogenetic population of artemia. vos (1979) found nauplii growth decreases and the overall appearance of adults deteriorates with ph values below 7.0 and they concluded that the optimum ph for artemia growth ranges from 8.0 to 8.5. stao (1967) determined that cysts hatching efficiency was greatly compromised at ph below 8. in this study, the maximum artemia adult and juvenile densities were observed in march (q1) 2010 when the ph value observed 8.6 which is the maximum during the study period. however, several studies have been performed to estimate the various environmental and genetic components on the life span and reproductive traits of artemia (vanhaecke et al., 1984; wear et al., 1986; triantaphyllidis et al., 1995, browne et al., 2002; abatzopoulos et al., 2003; ben naceur et al., 2009). most of these researchers have focused on temperature and salinity as the most important physical parameters affecting the life history of artemia. the average number of individuals collected during the study period was lowest in the months of june (q2), september (q3) and december (q4) 2012, 2013 and 2014 and during these period highest salinity, temperature and also the levels of other parameters like nitrate, nitrite, ammonia, phosphate and total organic carbon remained high. the cyst production showed a decreasing trend along with increase of temperature. generally, brine shrimp (artemia spp.) have not been considered particularly sensitive to metals. numerous researchers have demonstrated that relative to other aquatic organisms, brine shrimp range from moderately sensitive to insensitive to a wide range of metals (brix et al., 2006). only cd at concentrations between 1.0 and 33 mg l-1 significantly suppressed growth rate and reproduction. cadmium was recorded highest in mach 2010 which is unusual phenomena with respect to its value as compared to rest of years. 94 saji et al./ influence of chemical parameters on artemia sp. population the present study clearly indicated that fe and cu are not influencing the artemia population due to its lower concentrations in the al wathba lake (ehsms, 2003). however, high levels of cd, cu, and pb, and fe can act as ecological toxins in aquatic and terrestrial ecosystems (balsberg-pahlsson, 1989; guilizzoni, 1991; joshi et al., 2002). to summarize, the results indicate a strong correlation between some of the physico-chemical parameters of water and artemia life cycle stages in the al wathba lake. the artemia population was found to be significantly negatively affected by physico-chemical parameters like temperature, dissolved oxygen and nitrate, which are the most crucial parameters for production of artemia. the concentration of dissolved oxygen is not influencing the survival of juveniles but affected cyst production as its number decreases with lower dissolved oxygen. acknowledgments the first author would like to acknowledge dr. m. al qassimi, former director, terrestrial biodiversity sector, environment agency abu dhabi, for his support and encouragement. financial support from the environment agency abu dhabi is greatly acknowledged. references abatzopoulos t.j., el-bermawi n., vasdekis c., baxevanis a.d., sorgeloos p. 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(2022) 10(6): 515-524 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2022 iranian society of ichthyology original article morphological plasticity of nemoura cinerea (arthropoda, nemouridae) as a biological indicator for aquatic systems esraa mohammad alnoiji1, fatemeh tabatabaei yazdi2, omid safari3 1department of biology, faculty of sciences, ferdowsi university of mashhad, iran. 2department of environment, faculty of natural resources and environment, ferdowsi university of mashhad, iran. 3department of fisheries, faculty of natural resources and environment, ferdowsi university of mashhad, iran. s article history: received 19 september 2021 accepted 4 october 2022 available online 2 5 december 2022 keywords: macrobenthos eco-morphology biodiversity fish farm wastewater geometric morphometric abstract: macrobenthos are indicators of the physical and chemical changes in aquatic ecosystems. in this research, the diversity of macrobenthos communities in the ortkand river, iran, was investigated during four consecutive seasons using the shannon-weiner, ept richness indices, and the hilsenhoff index for water quality at four sampling stations. in addition, sampling of nemoura cinerea (arthropoda, nemouridae) was done to compare morphological differences between specimens from two upstream and downstream sites using a geometric morphometric approach. the physicochemical parameters of water were also recorded. the entry of fish farm wastewater significantly affects biotic and abiotic environmental factors according to the shannon-weiner and hilsenhoff indices. the results showed a significant positive correlation between do and shannon-wiener index, tds and the hilsenhoff index, and do and ept richness index. a significant negative correlation was observed between bod and the shannon-wiener index, do and hilsenhoff index, and between bod and ept richness index. geometric morphometrics analyses revealed that the two groups differed mainly in pronotum and metanotum morphology. according to the results, monitoring of macrobenthos can help assess rivers’ water quality, and n. cinerea can be a proper bioindicator. introduction benthic communities play important roles in the food chains of aquatic ecosystems (fries and bowles, 2002). the quantification of their changes using indices of diversity provides crucial information. the study of macrobenthos communities is also one of the common methods for evaluating stresses on aquatic ecosystems and water quality monitoring (lydy et al., 2000). in addition, the physical and chemical status of the aquatic ecosystem can be recognized through the plasticity of the community structure of the benthic organisms, which is why benthic communities make the typical subjects for the biological assessment of water quality (young et al., 2014a). correspondence: fatemeh tabatabaei yazdi doi: https://doi.org/10.22034/ijab.v10i6.1354 e-mail: f.tabatabaei@um.ac.ir dor: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.8.1 although evaluating water quality includes measuring physicochemical and biological parameters, these methods are insufficient and often costly and time-consuming. the evaluation of diversity in the macrobenthos communities using biodiversity indices such as the shannon-wiener, margalef, and ept species richness (for ephemeroptera, plecoptera, and trichoptera), and the assessment of water quality by indicators such as the hilsenhoff biotic index (hbi), have already been recommended for the biological assessment of an ecosystem (borja et al., 2003). now becomes apparent that macrobenthos species play an effective role in improving and preserving water quality through the mineralization and recycling of organic 516 alnoiji et al./ morphological plasticity of nemoura cinerea materials (venkateswarlu, 1986). environmental factors are potent forces in the development of organisms during evolutionary processes (costa and cataudella, 2006). morphological plasticity in aquatic organisms such as fish and macrobenthos may reflect the physicochemical conditions of their habitats and food habits (ostrand et al., 2001). in other words, morphotype reflects genotype and can also reflect an organism's habitat type (sarker md et al., 2016). similarly, macrobenthos can be adapted to different environmental conditions and show morphological plasticity (nacua et al., 2010). shape analysis is crucial in many biological studies (zelditch et al., 2004). geometric morphometrics is a relatively new and efficient method for detecting similarities and differences between morphological structures and has been applied in biological sciences, e.g. evolutionary morphology investigations of fish and mammals (tabatabaei yazdi and adriaens, 2013; yazdi et al., 2015). this technique allows visualization of shape variations and quantitative comparison of morphological differences (sheets et al., 2004; zelditch et al., 2004). this type of morphometry can statistically represent the shape differences among species or populations of a species (sheets et al., 2004). aquatic organisms are used as water quality indicators to determine the quality of streams and rivers (eklöv and jonsson, 2007). aquatic organisms, especially fish and macrobenthos, are good indicators of changes in aquatic habitats because their body shape and size are susceptible to physical changes (hammer et al., 2001). nemoura cinerea (retzius) (nemouridae) plays a vital role in the food chain but is sensitive to the environment (franken et al., 2008). thus, it can be used as a bioindicator. the main objective of the present study is to investigate the effect of fish farm effluent on some of the biodiversity indices (shannon-wiener and ept richness) for macrobenthos communities and the hilsenhoff index of ortkand river. we also evaluate the morphological plasticity of n. cinerea in response to the conditions of its environment in ortkand river as a potential bio-indicator. material and methods study area and sampling stations: samples were collected during four seasons from four sampling stations along the ortkand river, which is located 110 km north of mashhad in the kalat, khorasan razavi province (fig. 1). the first station was located in the upstream area about two kilometers away from the point source of pollution (station a). since no pollution entered this station, it was considered a control station. the second station is located at 1348 meters above sea level (36˚48′10. n, 59˚48′39.41˝e), and it was considered the point source of pollution’s load station (fig. 1b). this station is located just after the main source of pollution discharged into the river. the third and fourth stations are located two and four kilometers downstream from the point source, respectively (fig. 1a-d). the type of substrate at the upstream station (station a) was rocky. the substrate was sandy at the fish farm wastewater entrance (station b), sandstone at the first downstream station (station c), and rocky at the second downstream station (station d). sampling of the macrobenthos communities was done randomly along a hypothetical line perpendicular to the flow of water, using a 40x40 cm-wide sorber with two repetitions. the sampling was done in august, february, october, and april (in four seasons). the sampled materials were separated, washed, and finally fixed in 4% formalin in the fishery laboratory of ferdowsi university of mashhad, iran. the specimens were identified using identification keys at the level of order, family, and genus (holland, 1972; edington and hildrew, 1995). the specimens were saved in containers containing ethyl alcohol based on armitage (1995). biological indicators: the shannon-wiener index, the ept richness index, and the hilsenhoff family biotic index (hfbi) were calculated using r ver. 3.3.0. the shannon index is one of the common indicators of species diversity that has applications in ecological assessment for measuring the pollution 517 int. j. aquat. biol. (2022) 10(6): 515-524 and distribution of macrobenthos groups in the environment. if species are distributed equally among all ecosystem groups, the shannon index will be higher (yazdian et al., 2014). this index was calculated using the formula of h`= -∑pilnpi to characterize species diversity at the stations (shannon and weaver, 1964). to calculate the ept richness index, the number of all individuals belonging to ephemeroptera, plecoptera, and trichoptera was counted and using the formula of ept% = (ne+p+t/ns)×100 was calculated. in this formula, ns is the sum of all collected macroinvertebrates, and ne+p+t is the number of single-day families with hairy wings. increased levels of this index indicate higher water quality (fries and bowles, 2002; kenney et al., 2009). the hilsenhoff family biotic index (hfbi) (hilsenhoff, 1988), was used to assess the water quality at each station using the equation of hfbi = (xi×ti)/n, where, xi: the number of individuals for each species, ti: tolerance value, and n: the density of macroinvertebrates. according to hfbi, the water quality is classified into five levels: high (0.00-3.75), very good (3.76-4.25), good (4.26-5.00), fair (5.015.75), and poor (6.51-7.25). physical and chemical parameters: the concentration of dissolved oxygen, electrical conductivity, temperature, and ph were measured with a portable apparatus (hanna instrument, model: hi98193, romania). bod5 (5-day biochemical oxygen demand) and cod (chemical oxygen demand) of the water samples from each sampling station were measured at the laboratory for each sampling station. the mean depth of the river, speed, and discharge were also measured at each station. also, the reynolds number (re) was calculated according to the following formulas: re = udv-1, where u: velocity proximal to the substrate, d: depth of water column and v: kinematic viscosity. the following formula was used to calculate discharge: q = s×v, where s: surface area (m2), v: average flow velocity (m/s) and q: water discharge (m3/s). morphological analyses: to study the morphological differences between specimens from stations a to c (away from the point source of pollution and just after entry of wastewater from the fish farm), the photographs were taken with a nikon d70 digital reflex camera (using a sigma 105 mm macro lens at five megapixels) using a standardized protocol. the camera was placed on a tripod). leftright symmetry on the ventral and dorsal sides was the criteria used to allow a standardized positioning of the specimens. the homolog points of the species’ body parts were digitized, with landmark points using tpsdig2 software. a total of 18 landmark points were digitized on the dorsal side (fig. 2), and 16 landmark points on their ventral side (fig. 3). figure 1. location of the sampling stations along the ortkand river, khorasan razavi province, iran. 518 alnoiji et al./ morphological plasticity of nemoura cinerea nine numerical attributes were determined for the ventral side (table 1), and 11 numerical attributes for the dorsal side (table 2). a generalized procrustes analysis (gpa) was performed using past (paleontological statistics) ver. 3.22 (hammer et al., 2001) to eliminate the effect of non-shape data, including size, rotation, and displacement (rohlf and marcus, 1993). one-way anova was used for comparing the mean sizes (centroid size) between the groups, using statistica (statsoft, version 12.0, www.statsoft.com). to investigate morphological variation and determine those morphological variables that discriminate the studied groups, principal component analysis (pca) was done using past, and discriminant function analysis (dfa) in morphoj ver. 1.02c (klingenberg, 2011), on the shape weight matrix of each data set separately for the dorsal and ventral sides. the scatter plots that illustrate the results of pca analysis on the dorsal side (a more informative plot compared to the ventral side) were generated to visualize how figure 2. landmarks on the ventral and dorsal side of nemoura cinerea. landmark’s number anatomical definition 1 anterior point of mouthpart 2-3 groove length of inferior lip 4-5 prosternum length 5-6 mesosternum length 7-8 head width at mouthpart 9-10 postgena width just behind the compound eye 11-12 post occiput width 13-14 distance between the coxa of forelegs 15-16 distance between the coxa of middle legs table 1. landmark points definitions on the ventral side of specimens. landmark’s numbers anatomical definition 1-2 pronotum length 2-3 mesonotum length 3-4 metanotum length 5-6 length of the upper lip 7-8 scape distance at pedicle base 9-10 head width behind the compound eyes 11-12 width of anterior pronotum 13-14 width of posterior pronotum 15-16 width of posterior metanotum 17-18 posterior tips of metanotum table 2. landmark points definitions on the dorsal side of specimens. 519 int. j. aquat. biol. (2022) 10(6): 515-524 the specimens are distributed in the morphospace. thin-plate spline deformation grids were used to visualize shape variation expressed by the first two pcs (rws axes). illustrating the shape difference between the groups was made using morphoj ver. 1.02c. cluster analysis (upgma) was done in past by combining the partial warp scores of specimens’ dorsal and ventral sides. statistical analyses: except for geometric morphometric analyses, the statistical analyses were performed using r ver. 3.3.0. results the highest shannon index was observed at stations a and d during summer at 3.14, and 3.01, respectively. station b showed the lowest shannon index. in the summer, the shannon-weiner index increased at station a, compared to winter, autumn, and spring. the difference in this index between summer and winter was significant (p<0.05). the highest value of the hilsenhoff index was observed in summer at stations b (8.99, at the entry point of farm wastewater to the river) and station c (7.75). the lowest hilsenhoff index was recorded at station a in spring. the average hilsenhoff index was 2.51 the highest ept index at the 95 percent confidence level (0.90) was observed in summer at station a, and the lowest value of this indicator was observed at the entry point of farm wastewater in summer (0.03) (table 3). the ept index increases in summer compared to winter, autumn, and spring (table 4). calculating pearson correlation between biological indices and physicochemical variables showed a significant correlation between shannonwiener and ept richness indices with do and bod (table 5). the hilsenhoff index showed a positive correlation with tds, bod, and cod and a negative correlation with do (table 5). geometric morphometric analysis: the correlation between procrustes distances and their corresponding euclidean distances after projection in shape space was tested using tpssmall (viscosi and cardini, 2011). the correlation value of 0.999 between the euclidean and tangent distances for the dorsal side indicates that the shape data matrix can be used for further analyses. an anova on the mean sizes on ventral and dorsal sides showed a significant difference (p<0.01) between the two studied groups (a vs. c). pca showed considerable variation in body shape i.e. the populations occupy different spaces in the scatter plot. however, there was some overlap between the two populations, and pca was unable to distinguish the two populations of n. cinere i.e. the populations of station a from the upstream station and the population of the first downstream station (station c). the first two axes (pcs) could explain a total of 76.02% of variations (48.05 and 27.97% for pc1 and pc2, respectively) (fig. 3). the results of the pca analysis for the dorsal side winter autumn summer spring station shannonwiener hfbi shannonwiener hfbi shannonwiener hfbi shannonwiener hfbi 1.99±0.09a,a 3.50±0.05a,a 2.04±0.07a,a 3.03±0.05a,a 3.14±0.09b,c 1.38±0.06a,a 3.12±0.02b,c 2.33±0.02b,b upstream (a) 1.33±0.01c,c 6.630.05c,b 1.61±0.05c,c 5.16±0.20b,b 1.40±0.04a,a 8.99±0.03a,c 1.38±0.06a,a 8.01±0.02a,c the entry point (b) 1.38±0.07c,b 5.51±0.01c,a 1.48±0.09c,b 5.14±0.02b,a 1.97±0.08a,a 7.75±0.1c,c 1.86±0.05a,a 7.63±0.01c,c downstream1 )c ( 1.82±0.06a,b 4.26±0.02a,a 2.01±0.04a,b 4.10±0.08a,a 3.01±0.05b,c 2.74±0.03b,b 2.58±0.05bc,c 2.45±0.02b,b downstream2 (d ( table 3. the mean (±standard deviation) hilsenhoff and shannon index of macrobenthos in the ortkand river at the studied stations (n=2). winter autumn summer spring stations 0.81±0.016b,a 0.86±0.085c,a 0.90±0.011b,a 0.78±0.015c,a upstream (a) 0.15±0.035a,b 0.16±0.00a,b 0.03±0.0a,a 0.04±0.00 a, a the entry point (b) 0.24 ±0.099a,b 0.32±0.04a, b 0.22±0.04a,a 0.22±0.046ab,a downstream 1(c) 0.35±0.017ab,a 0.67±0.028c, a 0.85±0.07b,b 0.77±0.05c, b downstream 2 (d) table 4. the mean (±standard deviation) ept richness index of macrobenthos in the ortkand river at the studied stations for every season (n=2). 520 alnoiji et al./ morphological plasticity of nemoura cinerea showed that considerable morphological variations along pc1 occur across the head and chest (mainly at the pronotum), but the highest variations along pc2 were observed along with the components of the mouth and body length (fig. 3). the specimens captured away from the point source of pollution (station a) have higher values on the pc1 axis and are characterizes by broader chests. dfa analysis of the dorsal side of the specimens (with 10,000 replicas) showed no overlap between do )1-(mg.l tds )1-(mg.l bod )1-(mg.l cod )1-mg.l) ec .s)1-(μmohs.cm re q )1-.s3(m index **0.5 **0.5**0.7*0.6**0.6 -0.9 0.3′h **0.7 -0.4 **0.7*0.5**0.6 -0.8 -0.4 ept **0.6**0.5 **0.8 **0.8 **0.5 0.6 **0.5 hfbi table 5. pearson correlation between biological indices and physicochemical variables. figure 3. scatter plot of pc1 versus pc2 of the dorsal side. deformation grids represent shape differences along each axis and correspond to pcs’ highest values. the specimens from station a are represented by the black solid polygon, and those from station b are represented by a grey dashed polygon. for the numbering of the landmarks, see figure 2. figure 4. histogram of dfa analysis and the deformation grids, including the arrows (3x magnified), which show shape differences between the groups, on the dorsal (above) and ventral (below) sides. 521 int. j. aquat. biol. (2022) 10(6): 515-524 the two populations (fig. 4). this analysis of the ventral side’s data (with 10,000 replicas) shows a clear separation of the two groups (fig. 4). the differences between groups are mainly in the metanotum, head width (fig. 4, the distance of landmarks 16-18 on the dorsal side), and chest (fig. 2. distances between 13-14 and 15-16 landmarks). a upgma cluster analysis based on euclidean distance showed that specimens belonging to different groups (sampled from stations a and c) are relatively well-clustered (fig. 5). discussion the environmental problems of the ortkand river will increase with the expansion of anthropological activities such as trout farms and direct release of their effluents into the river. the results of the diversity index showed that the number of macrobenthos decreased in stations b and c because of the wastewater entry from fish farms, which led to the elimination of macrobenthos such as ephemeroptera, followed by an increase in the number of resistant species such as chironomidae. yokoyama et al. (2007), in a study of macrobenthos diversity at the point of wastewater entry, pointed out that the highest self-purification occurs in summer. based on the results, wastewater entry could not affect the richness of macrobenthos at the second downstream station since pollutants were decomposed before reaching the station. in this station, the shannon diversity index was 1.4-3.14 in summer, showing poor to moderate water quality. the results also showed that with increased production in fish farm and the reduction of dissolved oxygen, especially in the summer, species diversity is decreased at station b, which consequently lead to an increase in the hilsenhoff index (classifying as “poor” level), and a considerable decrease in the number of pollutantsensitive groups such as ephemeroptera. the hilsenhoff index increased in stations downstream from the fish farm, indicating the accumulation of organic matter from fish farms and reduced riverbed quality at these stations. station d had considerably lower levels of hfbi due to its distance from the fish farms. this fact indicates that the water quality at the upstream and the second downstream station (about four kilometers from the last fish farm) is considerably different and demonstrates the river's ability to self-purify. additionally, improvement of the environmental conditions at the second downstream station shows that the pollution load could be noticeably lower after passing through an almost short distance. these findings are consistent figure 5. a upgma cluster analysis on the combined ventral and dorsal data. 522 alnoiji et al./ morphological plasticity of nemoura cinerea with the results of young et al. (2014), which showed that water quality is improved by increasing the distance from the pollution sources. the results showed a reduction in the ept richness index at the stations affected by aquaculture. these results agree with kani and murugesan (2014), who pointed out that pollution reduced the diversity of sensitive species while the density of resistant species is increased. releasing treated wastewater did not have much effect on tds (noroozrajabi et al., 2013). environmental conditions worsen as the dissolved solids increase, and consequently, sensitive organisms were significantly reduced (sarker, 2016). our results showed that the shannon-wiener index has a positive correlation with do, but a negative correlation with tds, which agrees with the previous studies (yazdian et al., 2014). in the present study, physicochemical parameters (tds and ec) and discharge showed a positive correlation with the hilsenhoff index and a negative correlation with do. the turbulence increased at the first and second downstream stations, which could reflect the substrate conditions. during the self-purification process, the dissolves organic matter, e.g. ammonia and phosphorus, convert to soluble salts, which cause an increase in ec. water quality gradually decreases, and electrical conductivity gradually increases down the river at the first and second downstream stations due to entry. since ec increased down the river, it can be concluded that changes in ec are mainly influenced by geological factors. regarding the morphological differences, the shape differences between the studied groups could be in response to environmental conditions and adaptation to different habitats (haas et al., 2010). as shown by the deformation grids, the most differences were in the width of the head, which was observed in the group affected by pollutants. the reason for this can be a morphological adaptation of macrobenthos to prevent being washed away. also, the chest width was smaller at the wastewater entry point, which can be a morphological adaptation and biological advantage for living in a polluted environment. this fact was previously reported in the perlidae and nemouridae families (briers, 2009). the differences in morphology are affected by various factors, such as environmental stressors, pollutants (taylor and kennedy, 2006), or even by access to food (johnson, 1987). using benthic organisms, especially macrobenthos indicators greatly enhances states’ ability to identify and subsequently improve water quality. acknowledgments this work was financially supported by ferdowsi university of mashhad, iran (project #41985). thanks to the anonymous reviewers in advance. references armitage p.d. 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(2015) 3(6): 409-413 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology short communication length-weight and length-length relationships in populations of garra rufa from different rivers and basins of iran yazdan keivany*1, ali nezamoleslami1, salar dorafshan1, soheil eagderi2 1department of natural resources (fisheries division), isfahan university of technology, isfahan, 84156-83111, iran. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 24 august 2015 accepted 7 november 2015 available online 2 5 december 2015 keywords: cyprinidae, doctor fish, growth, life history. abstract: this study describes the length-weight and length-length relationships for 28 populations of garra ruffa in different basins of iran, including tigris, karkheh, karun, persis and hormuz. the length-weight relationships from most localities are reported here for the first time. for most populations the b value was not significantly different from 3, indicating an isometric growth, in a few, it was significantly bigger than 3, indicating a positive allometric growth, and in some, it was significantly smaller than 3, indicating a negative allometric growth. in the whole samples, it was not significantly different from 3, indicating an isometric growth. introduction the length-weight (lwr) and length-length (llr) relationships are useful for the prediction of weight from length, assessment of fish stocks and give information on the condition and growth patterns of fish (radkhah and eagderi, 2015), and are widely used in fish biology. herein, we provide lwrs and llrs of doctor fish, garra rufa, for populations from 28 rivers and 5 basins of iran. there is some biological information available on this species. esmaeili et al. (2005), abedi el al. (2011) and geremew et al. (2015) studied the reproductive biology of this fish in central and southwestern iran. gozukara and cavas (2004) and gorshkova et al. (2012) studied the karyology of g. rufa in eastern meditereanian of turkey and jordan, respectively. patimar et al. (2010) studied some life history aspects of this species in western iran. shabani et al. (2013) studied the microsatellite loci of g. rufa to determine its population structure in khuzestan province, iran. stiassny and getahun (2007) studied the phylogenetic placement of the afro‐asian genus * corresponding author: yazdan keivany e-mail address: keivany@cc.iut.ac.ir garra. teimori et al. (2011) studied the microstructure of the adhesive organ in g. rufa from bushehr basin. vazirzadeh et al. (2015) studied the spawning induction of g. rufa using ovaprim and captive rearing of the obtained larvae. also, esmaeili and ebrahimi (2005), bibak et al. (2013), gerami et al. (2013), hamidan and britton (2013) and hashemzadeh et al. (2015) studied the lwr of this fish in southern iran. however, the length-weight and length-length relationships from many localities are reported here for the first time. materials and methods the specimens were collected during april 2010 to august 2011 by different types of fishing gears, including hand net, gill net with a mesh size of 5 mm and cast net with a mesh size of 15 mm. the samples were anesthetized in 1% clove oil solution and fixed in 10% buffered formalin and transferred to the ichthyology laboratory of isfahan university of technology for further examinations. for each specimen, total length (tl), fork length (fl) and 410 int. j. aquat. biol. (2015) 3(6): 409-413 standard length (sl) to the nearest 0.01 mm and whole body wet weight to the nearest 0.01 g was measured. the relationships between body length parameters were calculated by the method of least squares to fit a linear regression as: y = 𝛼 + bx, where, y is standard and fork lengths, x is total length, 𝛼 is proportionality constant and b is regression coefficient (le cren 1951; haddon, 2011).). the length-weight relationship was estimated with logtransformed equation: w=𝛼lb and log (w) = log(𝛼) + b log(l), where w is the whole body weight (g), l is the total length (cm), 𝛼 is intercept i.e. coefficient related to the body, and b is an exponential expressing relationship between length-weight. prior to regression analyses, log-log plots of the lengthweight pairs were performed to identify outliers (froese, 2006; froese et al., 2011). extreme outliers attributed to data error were excluded from the analyses. results and discussion length-weight and length-length relationships of the selected populations and related statistics are presented in tables 1 and 2. the exponent b in length-weight relationships should normally fall between 2.5 and 3.5 (froese, 2006). in this study, the exponent b for all the populations was within this total length (mm) weight (g) length-weight basin river counts min-max mean±sd mean±sd a b r2 tigris alvand 12 68.09-122.83 102.25±16.00 13.72±5.00 0.0126 2.98 0.99 chamgordalan 30 54.30-106.84 79.11±15.00 7.29±4.00 0.0120 3.05 0.99 doirej 26 58.37-97.85 70.36±10.16 3.99±1.80 0.0179 2.75 0.95 godarkhosh 7 40.89-89.55 55.37±18.00 2.63±2.80 0.0121 2.98 0.99 kangir 5 56.87-65.88 62.66±3.00 2.92±0.50 0.0110 3.04 0.97 little zab 6 50.45-77.25 61.74±9.00 2.92±1.00 0.0056 3.40* 0.98 mimeh 31 62.86-98.30 79.63±9.69 5.45±1.90 0.0116 2.95 0.94 sirvan 16 57.61-78.99 69.31±6.00 3.90±1.10 0.0115 2.99 0.96 zimakan 14 32.19-80.15 60.15±14.91 2.72±1.60 0.0061 3.30* 0.99 all basin 147 32.19-122.83 74.29±16.56 5.49±4.10 0.0093 3.10 0.97 karkheh chaghalvandi 24 43.17-131.30 87.89±29.69 11.84±9.60 0.0094 3.14 0.99 chardavol 7 19.82-104.31 43.07±35.10 2.92±4.00 0.0121 2.99 0.99 gamasiab 10 30.63-104.69 68.59±19.83 4.73±4.090 0.0094 3.14 0.99 kakareza 58 48.73-127.20 89.78±15.78 9.86±5.35 0.0121 3.01 0.98 kashkan 17 56.23-172.85 117.82±32.74 22.25±15.40 0.0140 2.91 0.97 saymareh 5 82.31±43.65 0.79-46.72 12.24±19.50 0.0076 3.19* 0.99 all basin 121 19.82-172.85 88.59±29.37 11.26±10.20 0.0115 3.02 0.99 karun beshar 33 36.94-113.13 79.74±20.74 6.80±4.70 0.0114 2.99 0.99 karun 10 81.61-130.93 106.69±18.00 15.44±6.90 0.0171 2.85* 0.97 katola 4 50.36-96.35 72.45±22.00 5.14±4.00 0.0094 3.08 0.99 marbor 15 55.40-127.58 80.98±21.77 7.53±7.00 0.0076 3.18* 0.98 all basin 62 36.94-130.93 83.92±22.64 8.26±6.00 0.0099 3.06 0.98 persis ahram 9 58.63-98.52 71.36±12.25 4.83±3.00 0.0142 2.92* 0.98 bahoosh 8 60.30-72.43 66.90±4.65 3.77±0.80 0.0099 3.11 0.78 darolmizan 11 29.25-65.24 49.22±10.64 1.55±0.80 0.0094 3.12 0.99 karzin 16 22.45-58.02 31.72±7.91 00.41±0.40 0.0132 2.83* 0.99 mond 17 61.95-106.09 74.07±10.96 4.55±2.50 0.0060 3.27* 0.93 safid 5 32.02-85.61 60.73±19.48 2.86±2.00 0.0097 3.04 0.99 shahpour 4 55.04-100.36 78.60±19.82 5.20±3.00 0.0209 2.63* 0.99 sheldan 33 39.49-68.47 49.05±5.96 1.30±0.50 0.0096 3.06 0.98 all basin 103 19.82-172.85 51.37±17.42 2.01±2.20 0.0095 3.06 0.98 hormuz axe rostam 19 33.48-75.74 54.57±12.00 2.00±1.00 0.0059 3.34* 0.99 total 452 19.82-172.85 72.05±25.80 6.19±7.10 0.0092 3.11 0.99 table 1. number, mean total length and weight of garra rufa from different rivers and basins of iran collected during april 2010 to august 2011. 411 keivany et al./ lwrs and llrs relationships in populations of garra rufa range and therefore, the parameters can be used within the referred length ranges. in most populations, the b value was not statistically different from 3, indicating an isometric growth. in some populations, it was significantly smaller than 3, indicating a negative allometric growth and in a few, it was significantly bigger than 3, indicating a positive allometric growth. in the whole samples, it was not significantly different from 3, indicating an isometric growth (fig. 1). however, length-weight relationships may vary due to changes in food availability, stage of sexual maturity and other factors such as sampling and preservations techniques (froese, 2006; alavi-yeganeh et al., 2011; daneshvar et al., 2013; hasankhani et al., 2013, 2014; ghanbarifardi et al., 2014), none of which were considered in this study. in conclusion, this study provides basic information on lwrs and llrs for this species that would be useful for fishery biologists and managers in iran. acknowledgments we would like to thank s. asadollah and m. nasri for their help in fish collection. this study was financially supported by isfahan university of technology and iran department of environment (grant no. 11023 to yk). references abedi m., shiva a.h., mohammadi h., malekpour r. (2011). reproductive biology and age determination of garra rufa heckel, 1843 (actinopterygii: cyprinidae) in central iran. turkish journal of zoology, 35(3): 317-323. alavi-yeganeh m.s., seifabadi s.j., keivany y., kazemi b., wallis g.p. (2011). comparison of length-weight relationships in different populations and sexes of iranian thoothcarps. journal of applied ichthyology, 27: 1401-1403. bibak m., hosseini s.a., izadpanahi g.r. (2013). length-weight relationship of barbus grypus (heckel, 1843) in dalaki river and garra rufa (heckel, 1843) in shahpur river in south of iran. world journal of fish and marine sciences, 5(2): 203-205. daneshvar e., keivany y., paknehad y. (2013). comparative biometry of the iranian cichlid, iranocichla hormuzensis, in different seasons and sexes. research in zoology, 3: 56-61. esmaeili h.r., ebrahimi m. (2006). length-weight relationships of some freshwater fishes of iran. journal of applied ichthyology, 22(4): 328-329. esmaeili h.r., yazdanpanah m., monsefi m. (2005). reproductive biology of doctor fish, garra rufa (cyprinidae: garrinae), in southwest of iran. journal of fish biology, 67: 282-282. esmaeili h.r., ebrahimi m. (2006). length-weight relationships of some freshwater fishes of iran. journal of applied ichthyology, 22: 328-329. froese r., pauly d. (2015). fishbase. available at: http:// www.fishbase.org, (last accessed: 5 june 2015). froese r. (2006). cube law, condition factor and weightlength relationships: history, meta-analysis and recommendations. journal of applied ichthyology, 22: 241-253. froese r., tsikliras a.c., stergiou k.i. (2011). editorial basin equation lwr parameters a b r2 tigris fl=a+b×tl 0.058 0.910 0.99 sl=a+b×tl 0.128 0.851 0.98 karkheh fl=a+b×tl 0.026 0.930 0.99 sl=a+b×tl 0.069 0.839 0.99 karun fl=a+b×tl 0.080 0.922 0.99 sl=a+b×tl 0.182 0.823 0.98 persis fl=a+b×tl 0.012 1.125 0.99 sl=a+b×tl 0.080 0.740 0.98 hormuz fl=a+b×tl 0.072 0.933 0.99 sl=a+b×tl 0.474 0.728 0.99 all fl=a+b×tl 0.933 0.074 0.99 sl=a+b×tl -0.114 0.850 0.99 table 1. length-length relationships for populations from different basins in iran. figure 1. length-length relationships for populations from different basins in iran. 412 int. j. aquat. biol. (2015) 3(6): 409-413 note on weight-length relations of fishes. acta ichthyologica et piscatoria, 41: 261-263. gerami m.h., abdollahi d., patimar r. (2013). lengthweight, length-length relationship and condition factor of garra rufa in cholvar river of iran. world journal of fish and marine sciences, 5(4): 358-361. geremew a., getahun a., dejen e. (2015). reproductive biology of garra regressus and garra tana (cypriniformes: cyprinidae) from lake tana, ethiopia. journal of threatened taxa, 7(6): 72237233. ghanbarifardi m., ghasemian s., aliabadian m., pehpuri a. (2014). length-weight relationships for three species of mudskippers (gobiiformes: gobionellidae) in the coastal areas of the persian gulf and gulf of oman, iran. iranian journal of ichthyology, 1: 29-31. gorshkova g., gorshkov s., abu-ras a., golani d. (2012). karyotypes of garra rufa and g. ghorensis (pisces, cyprinidae) inhabiting the inland water systems of the jordan basin. italian journal of zoology, 79(1): 9-12. gozukara s.e., cavas t. (2004). a karyological analysis of garra rufa (heckel, 1843)(pisces, cyprinidae) from the eastern mediterranean river basin in turkey. turkish journal of veterinary and animal sciences, 28: 497-500. haddon m. (2011). modelling and quantitative methods in fisheries. 2th edition. crc press, new york, usa. 449 p. hamidan n., britton j.r. (2013). length‐weight relationships for three fish species (capoeta damascina, garra rufa, and nemacheilus insignis) native to the mujib basin, jordan. journal of applied ichthyology, 29(2): 480-481. hasankhani h., keivany y., raeisi h., pouladi m., soofiani n.m. (2013). length-weight relationships of three cyprinid fishes from sirwan river, kurdistan and kermanshah provinces in western iran. journal of applied ichthyology, 29: 1170-1171. hasankhani m., keivany y., daliri m., pouladi m., soofiani n.m. (2014). length–weight and length– length relationships of four species (barbus lacerta, pseudorasbora parva, squalius lepidus and oxynoemacheilus angorae) from the sirwan river, western iran. journal of applied ichthyology, 30: 206207. hashemzadeh i., tabatabaei s.n., mansouri a., abdoli a., ghalenoei m., golzarianpour k. (2015). lengthweight relationships of garra rufa, in the tigris and persian gulf basins of iran. international journal of aquatic biology, 3(1): 25-27. patimar r., chalanchi m.g., chamanara v., naderi l. (2010). some life history aspects of garra rufa (heckel, 1843) in the kangir river, western iran: (osteichthyes: cyprinidae). zoology in the middle east, 51(1): 57-66. radkhah a., eagderi s. (2015). length-weight and length-length relationships and condition factor of six cyprinid fish species of zarrineh river (urmia lake basin, iran). iranian journal of ichthyology, 1: 61-64. shabani a., askari g. (2013). microsatellite loci to determine population structure of garra rufa (heckel, 1843) in the khuzestan province (iran). international journal of aquatic biology, 1(4): 188-194. shabani a., askari g., moradi a. (2013). genetic variation of garra rufa fish in kermanshah and bushehr provinces, iran, using ssr microsatellite markers. molecular biology research communications, 2(3): 81-88. stiassny m.l., getahun a. (2007). an overview of labeonin relationships and the phylogenetic placement of the afro‐asian genus garra hamilton, 1922 (teleostei: cyprinidae), with the description of five new species of garra from ethiopia, and a key to all african species. zoological journal of the linnean society, 150(1): 41-83. teimori a., esmaeili h.r., ansari t.h. (2011). microstructure consideration of the adhesive organ in doctor fish, garra rufa (teleostei; cyprinidae) from the persian gulf basin. turkish journal of fisheries and aquatic sciences, 11(3). vazirzadeh a., zahedinejad s., bahri a. (2015). spawning induction in doctor fish, garra rufa (heckel, 1843) by ovaprim and captive rearing of larvae. iranian journal of ichthyology, 1(4): 258-265. int. j. aquat. biol. (2015) 3(6): 409-413 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی های آبریز مختلف ایران ها و حوضهرودخانه در garra rufaهای طول جمعیت-وزن و طول -روابط طول 2، سهیل ایگدری1، ساالر درافشان1االسالمینظام لیع ،*1یزدان کیوانی .ایران ،65141-11111ستی پکد،اصفهان ،صنعتی اصفهان، دانشگاه گروه منابع طبیعی )بخش شیالت(1 .گروه شیالت، دانشکده منابع طبیعی، دانشگاه تهران، کرج، ایران2 چکیده: و هرمز را بوشهر، کرخه، کارون، دجلهدر حوضه های آبریز ایران شامل garra rufaجمعیت ماهی 21طول -وزن و طول-این مطالعه روابط طول ریب مقادیر ض ،هادر بیشتر جمعیت شود.زارش میگبرای اولین بار مطالعه برداری در اینهای نمونهوزن بیشتر مکان-روابط طول نماید.توصیف می b بود که 1تر از داری بزرگمعنی به طور باین ضری هایعنی الگوی ایزومتریک نشان نداد، در تعداد اندکی از جمعیت 1 عدد تفاوت معنی داری را از بود که بیانگر الگوی رشد 1داری کمتر از عدد نیز مقدار این ضریب به طور معنی هابود، و در برخی از جمعیت هاآن بیانگر رشد آلومتریک مثبت دهنده الگوی رشد ایزومتریک این گونه در نداشت که نشان 1داری از عدد ها، این ضریب تفاوت معنیباشد. در کل نمونهها میآلومتریک منفی آن های مورد بررسی است.حوضه .حیاتچرخه ،رشد ،دکتر ماهی ،کپورماهیان :کلمات کلیدی int. j. aquat. biol. (2019) 7(6): 322-331 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article germination and seedling development in pistia stratiotes l. gabriela costa barbosa¹, maria francilene souza silva*2, fátima de cássia evangelista de oliveira², maria da conceição prado de oliveira3, simon joseph mayo4, ivanilza moreira de andrade1 1herbário delta do parnaíba, campus parnaíba, universidade federal do piauí, parnaíba, brazil. 2laboratório de oncologia experimental, universidade federal do ceará, núcleo de desenvolvimento de medicamentos, fortaleza, ceará, brazil. 3departamento de biologia, universidade federal do piauí, teresina, piauí, brazil. 4royal botanic gardens, herbarium, kew, richmond, surrey, tw9 3ae, united kingdom. s article history: received 0 october 2019 accepted 18 december 2019 available online 2 5 december 2019 keywords: water-lettuce araceae macrophytes abstract: this study aimed to describe the post-seminal development, evaluate how the available water, light and temperature influence seed germination and to describe the vegetative propagation by selecting one of the collected plants of pistia stratiotes. the plant material was collected in a tributary of the rio igaraçu, parnaíba, piauí state, brazil. a botanical description of the species was made using individuals collected in the study area. for the germination tests, a total of 1400 seeds were harvested and three experiments were conducted, varying the amount of available water, light and temperature. the results showed that the highest percentage of germination is occurred with seeds pre-washed with distilled water, sown on filter paper with abundant water (enough to form a sheet of water) and exposed to alternating light at an average temperature of 37°c. germination began on the fourth day after sowing, marked by the appearance of the cataphyll. after 22 days, the seedling stage was completed. vegetative propagation of this species is efficient and fast. in 50 days, a total of 134 new vegetative shoots were recorded in just one individual. the results showed that the germination rate is related to the amount of light and temperature. introduction pistia stratiotes l., the only species of the genus pistia, is an aquatic macrophyte of the family araceae, known by the vernacular names of water lettuce (howard and harley, 1998). recent studies using molecular and fossil evidence (renner and zhang, 2004) concluded that pistia is part of an ancient clade originating in the cretaceous in the tethys sea region of the old world. its present-day pantropical distribution is probably the result of dispersion of seed and plant parts by birds (holm et al., 1977; cabi 2016), but in more recent times its foliage has made it attractive as an ornamental aquatic plant and this may also have contributed to its dispersal in modern times (cícero et al., 2007). the species is recorded in all five major regions of brazil (coelho et al., 2015). in piauí, where the present study was carried out, p. stratiotes occurs in annual or perennial ponds and eutrophicated lowland creeks and canals (igarapés). *correspondence: maria francilene souza silva doi: https://doi.org/10.22034/ijab.v7i6.713 e-mail: lenolysilva@hotmail.com pistia stratiotes is ecologically important in watersheds as it is a bioindicator of pollution and is characterized by its rapid vegetative multiplication. it is an invasive species whose proliferation may rapidly cover the whole surface of a water body, impeding the penetration of sunlight and thus affecting the efficiency of transmission and intensity of light within the water. the dense carpets of pistia thus formed result in the diminution of oxygen content in the water body and hence reduction of both animal and plant biodiversity (langeland and burks, 2008). studies of this species have focussed especially on the ecology of its control and proliferation (tucker, 1983; sommaruga et al., 1993; noelli, 1996; camargo and biudes, 2006; zimmels et al., 2006; lu et al., 2010; wang, 2010; sánchez-galván et al., 2013; khan et al., 2014; chen et al., 2014; das et al., 2014; yang et al., 2014; ugya et al., 2015). various studies of its germination and reproductive biology have been 323 int. j. aquat. biol. (2019) 7(6): 322-331 carried out since the 1980s, for example those of piertese et al. (1981), sharma and sridhar (1981), tucker (1983), dray-jr and center (1989), harley (1990), dewald and lounibos (1990), sajna et al. (2007), kan and song (2008), and eid et al. (2016). however only a few such studies have been published based on research in brazil, e.g. silva (1981), teixeira et al. (2007), henry-silva et al. (2008) and cancian et al. (2009). the identification of seedlings and study of their biology is fundamentally important for a better knowledge of plants in general, but also because of their practical importance for horticulture and agriculture, and for understanding how plants colonize and establish themselves in their natural habitat (guerra et al., 2006). morphological studies of seeds are also important for species identification both in the laboratory and the field, and as aids to ecological and developmental studies (oliveira and pereira, 1984). knowledge of the life cycle of species is in turn indispensable because basic knowledge of the morphology and germination of species makes possible understanding of the natural mechanisms existing in ecosystems, such as the renewal of their resources and how species behave at different stages of development (kuniyoshi, 1983). the aim of this study was to describe the morphology of p. stratiotes, investigate the effect on germination of available water content, light levels and temperature, and study the post-seminal development with the aim of understanding the population dynamics of this species in its natural habitat. materials and methods the materials were collected in a stretch of the rio igaraçu (02°55´39.73"s and 41°46'21.21"w) in the bebedouro district, parnaíba, piauí state, brazil. the altitude of sampling station was 5 m above sea level, with a mean annual minimum and maximum temperatures of 20 and 32ºc, respectively. mean annual rainfall is 1200 mm (in parnaíba), varying between 800 and 1600 mm, designated as an equatorial maritime regime, with five to six consecutive months of higher rainfall with a dry season for the rest of year (aguiar, 2004). the botanical description was made using randomly collected individuals. voucher specimens are deposited in the parnaíba delta herbarium (hdelta) of the federal university of piauí. seed germination was studied using a sample of 1400 seeds divided into three experiments assays to observe germination under different conditions of light, temperature and water availability. the petri dishes had a diameter of 12 cm and plastic trays with 46 x 29 cm comp and 6.5 cm height. the design of the experiments is described in table 1. the germination study was carried out in the plant cell and molecular laboratory of the federal university of piauí (ufpi) at parnaíba. the emergence of the cataphyll was used as the criterion to mark the initiation of germination. observations of the germination were done daily, the analysed variables were the percentage germination and germination rate index (ivg), which were calculated according to maguire (1962). the percentage germination follows standard norms for seed analysis (brasil, 1992). the data were analysed using analysis of variance (anova) followed by the tukey test for multiple comparisons. for the study of post-seminal development, seedlings from the third germination assay were used. the illustrations were prepared using an optical stereomicroscope equipped with a drawing tube and the terminology adopted for the description based on mayo et al. (1997). every day, the leaf number, leaf length and leaf width were recorded in the seedlings. the representative plants of each germination phase were illustrated and described in a chronological sequence from germination to the attainment of the young plant stage. observation of the vegetative propagation was made by selecting one of the collected plants, and placing it in a 30l plastic vessel with water collected from the plant's habitat. every day, the evaporated water was replaced from the tape water (agespisa). this experiment was carried out for 50 days. 324 barbosa et al./ germination and seedling development in pistia stratiotes results and discussion taxonomic description pistia stratiotes l. sp. pl. 963 (1753) (fig. 1a-e) floating aquatic macrophyte, free or fixed, 3.4-8.4 cm long, acaulous; stolons green, pilose, lacking foliar structures, 3-7.5 × 1-1.7 cm. roots adventitious, brown and white, pilose, 16-20 cm long. leaves simple, rosulate, subsessile to petiolate; petiole white, glabrous, 0.1-0.3 cm long; leaf blade green, spongiose, obovate-oblong, base attenuate, apex retuse, 2-9 × 1.54.5 cm; major veins parallel, 5-8, prominent on abaxial surface, pale green, with abundant trichomes on both surfaces. inflorescence solitary per leaf, 5-7 per plant, axillary, composed of unisexual flowers, 11.4 cm long; perigon absent; peduncle 0.2-0.4 cm long. spathe green at base, white at apex, adnate to ovary in basal portion, margins between stigma and androecium forming a lower, tubular feminine chamber, upper part more expanded forming an open male chamber with a blade, densely pilose externally, 0.2-0.5 × 0.2-0.3 cm. male flowers forming a verticel near or at spadix apex; stamens connate in pairs to form 5-7 yellow synandria in verticillate male zone. female flower at spadix base; pistil 5-7 mm long, ovary ovoid, oblique to spadix axis, trichomes present around ovary, 3-4 × 2-3 mm, unilocular, ovules numerous, placenta basal, stigma short but distinct. fruit a berry, green, pilose, ellipsoid, irregular, 5-7 × 3-3.1 cm. seed barrel-shaped, brown, 3-9 per fruit, 1.2-1.5 mm long, micropyle with an operculum, testa reticulate, thick. in the area of collection, the species occurs at the margins of creeks, in the polluted localities, and usually associated with other species such as eichhornia crassipes (c. mart.) solms, lemna sp., and wolffiella sp. germination of p. stratiotes first germination experiment: germination of the seeds in the first experiment took place four days after sowing, with rupture of the operculum. during this period, the germination index for treatment 1 was 18%; treatment 2, 19%; treatment 3, 2% (table 2). the results also showed a greater germination table 1. experimental design for seed germination in pistia stratiotes. experiment experiment conditions treatment wash no. seeds photoperiod exposure to light light intensity temperature 1 600 seeds 1 pre-wash with distilled water 2 plates, 100 seeds per plate 12h light/ 12h dark shade 50% 35ºc 2 12h light/ 12h dark sun 100% 37ºc 3 continuous light laboratory 100% 24ºc 2 800 seeds 1 (control) absence of prewash continuous light laboratory 100% 24ºc 2 pre-wash with distilled water 12h light/ 12h dark sun 100% 37ºc 3 12h light/ 12h dark shade 50% 35ºc 4 continuous light laboratory 100% 24 ºc 3 600 seeds * 2 group 1 pre-wash with distilled water and watered with collection water 100 seeds per plate 12h light/ 12h dark sun 100% 37ºc 2 group 2 pre-wash with distilled water ** 3 group 1 pre-wash with distilled water and watered with collection water shade 50% 35ºc 3 group 2 prewash with distilled water ** shade 50% 35ºc 4 group 1 continuous light laboratory 100% 24 ºc 4 group 2 * seeds were used which did not germinate in the second experiment (from treatments 2, 3 and 4). ** water from city supply network. 325 int. j. aquat. biol. (2019) 7(6): 322-331 percentage and germination rate index (ivg) in treatment 2. however, there was no significant difference between treatments 1 and 2 in the percentage germination. for ivg, a significant difference was found between treatment 3 (exposure to laboratory conditions), with a value of 0.7, and others, concluding that seeds of p. stratiotes germinate when pre-washed with distilled water and then sown in petri dishes lined with filter paper and kept wet with distilled water under continuous light in the laboratory, or in the open with light alternating between exposure to sunlight and shade. speed of the germination was also greater with the alternating sun/shade regime. second germination experiment: there was no germination in any treatments in this experiment using table 2. the results of the experiment 1. germination (%) and germination velocity index (gvi) of seeds of pistia stratiotes. pairs of values with the same letter indicate non-significant differences between means as tested by anova and tukey's multiple comparison post-test; those with different letters indicate significant difference. treatments germination gvi 1 76% 7.06 a 2 89% 8.18 a 3 64% 0.7 b figure 1. pistia stratiotes l. a. habit; b. detail of the inflorescence insertion; c. detail of the stolons; d. inflorescence; e. seed. 326 barbosa et al./ germination and seedling development in pistia stratiotes substrate irrigated with distilled water. the result shows that pistia seeds are sensitive to desiccation. this species is a floating of fixed aquatic plant (depending on the water level of the habitat) and it is therefore to be expected that its seeds germinate only in the presence of abundant water. according to teixeira et al. (2007), the species invests more in sexual reproduction in dry periods when the water level is lower; the seeds are able to resist the drier conditions through dormancy and later germinate in the rainy season. third germination experiment: in the third experiment, seeds which did not germinate in the second experiment were used. the seeds were watered abundantly so that they were covered by a layer of water derived either from their natural habitat, or from tape water. the results showed that water availability has an effect on seed germination. based on table 3, the percentage germination of seeds exposed to the sun (direct daylight, for 12h) was significantly different in three light regimes: direct sunlight for 12h, 50% sunlight (shade) for 12h, and laboratory artificial light for 24h. however, within each light regime, there was no significant difference in percentage germination or germination speed (ivg) between water treatments (habitat vs. mains water supply). the seeds germinate better with distilled water, sown on filter paper with sufficient water to form a water layer, and exposed to the sun for 12h at a mean temperature of 37ºc. cancian et al. (2009) showed that p. stratiotes is sensitive to low temperatures, showing less growth, leaf yellowing and leaf death at 15oc, whereas at 25oc with a 12h photoperiod higher growth rates were observed. kan and song (2008) studied the germination of p. stratiotes in relation to potential seed storage. they showed that there was no germination in darkness, or in far red light, but white light and red light and alternating 12h photoperiods of red light and darkness promoted germination. percentage germination of seeds that had been rapidly dehydrated was 73.3%, and in white light, seed germination changed from zero to 68%. addition of nitric oxide brought about germination even in darkness. water is the factor with the most influence on the process of germination. after inhibition, the tissues of the seed undergo rehydration and as a consequence, an intensification of respiration and other metabolic activities, which result in the provision of energy and necessary nutrients for the resumption of growth by the embryonic axis (kramer and kozlowski, 1972). the speed of water absorption varies among species with the number of pores in the tegument surface, the availability of water, temperature, hydrostatic pressure, area of contact between seed and water, intermolecular forces, chemical composition and physiological condition of the seed (nassif et al., 1998). the water layer can enhance germination in p. stratiotes. cassol et al. (2008) studied the effect of flood conditions (saturated soil at 5, 10 and 20 cm submersion depth) on the germination of seeds of sagittaria montevidensis, a weedy aquatic plant, and verified that a water layer favoured germination. rego et al. (2009) showed that there was an interaction between temperature and water quantity in the substrate in germination of seeds of blepharocalyx salicifolius. silva (1981) collected seeds of p. stratiotes in three different environments (lago castanho, rio solimões and lago baixio) and table 3. the results of the experiment 3. germination (%) and germination velocity index (gvi) of seeds of pistia stratiotes. pairs of values with the same letter indicate non-significant differences between means as tested by anova and tukey's multiple comparison post-test; those with different letters indicate significant difference. treatments germination gvi 2 group watered with collection water 98% a 98a 2 group 2 watered with agespisa tape water 92% a 92a 3 group 1 watered with collection water 55% b 47b 3 group 2 watered with agespisa water 64% b 48b 4 groups 1 and 2 watered with agespisa water 67.5% b 75.5c 327 int. j. aquat. biol. (2019) 7(6): 322-331 germinated them in beakers with water up to a level of 10 cm. with this method, this author obtained a low index of seed germination per fruit (24.2, 21.3 and 10.8%) in the three environments. when they were placed in the germination vessel, the seeds floated at first and then sank two to six days later, or sank as soon as they touched the water, and then in both cases germinated at the bottom and after about seven days rose to the surface as seedlings. silva (1981) carried out a second experiment testing the seed germination of p. stratiotes at depths of 10 and 30 cm (in beakers with water), obtaining 40 and 32% mean germination, respectively. at 10 cm, all seedlings floated to the top after germination but at 30 cm depth none did so. these results showed that depth could be a factor which influences propagation by seed (silva, 1981). the seeds of p. stratiotes are small, with an average length of 1.2-1.5 mm, and thus the water layer need be no more than 2 cm in depth to ensure that the water completely surrounds it to facilitat its penetration into the seed. in the present study, we suggest that the prewashing with distilled water provides a first contact between seed and water which predisposes it to further water penetration and thus initiates imbibition. in addition, the water washes the seed tegument and thus may remove any germination inhibitor substances figure 2. post-seminal development of pistia stratiotes. a. seed; b. turgid seed; c. emergence of the cataphyll (cat); d. first true leaf (fi), and the emergence of the radicle (r); e. first leaf begins to open; f. first leaf opens completely, showing trichomes and adventitious roots (ra); g. presence of the 3rd root, death of the radicle; h. appearance of the second true leaf; i. appearance of the third true leaf; j. roots now numerous; k. end of seedling phase. 328 barbosa et al./ germination and seedling development in pistia stratiotes which may be present. in addition to water, it is essential that other conditions are optimal for the seed to achieve maximum germination capacity (figliolia et al., 1993), among which are the light intensity and temperature to trigger germination. in the present study, the seeds of p. stratiotes were exposed to continuous light (in the laboratory) or alternating light and dark photoperiods of 12 hours, and either in shade (50% intensity) or in full sun (100% intensity). the results showed that exposure to full sun favoured germination. in the locality where the seeds were collected, the light intensity wais high from september to january. during this period, the temperature is also high with little variation from night to day. in their natural habitat the seeds probably germinate soon after dispersal. post-seminal development in p. stratiotes: seed production is high in its natural habitats and the collected seeds showed 98% viability of germination. at the beginning of germination, the seed becomes turgid by absorption of water. on the second day, the cataphyll pushes open the seed operculum (fig. 2b). on the third day, the cataphyll, ca. 2 × 1 mm in size, emerges completely and in some seeds the first leaf appeared (fig. 2c). the radicle, 0.5-1 mm long, and first leaf occur on the fourth day, making it possible for the seedling now to float. in this phase, the cataphyll measures 2 × 1 mm and first leaf 2×1 mm (fig. 2d). on the fifth day, the first leaf shows the presence of trichomes (fig. 2e). on the sixth day, the radicle measures 5.5 mm, the first leaf, now completely unfolded with 2 mm long, the trichomes are clearly evident and numerous, and a second root was appeared (fig. 2f). on the seventh day, the third root was appeared (fig. 2g) and on thirteenth day, the radicle was disappeared and the seedlings are now figure 3. vegetative propagation of pistia stratiotes l., days after the beginning of the experiment. (a) day 8, (b) day 12, (c) day 39, and (d) day 50. 329 int. j. aquat. biol. (2019) 7(6): 322-331 free-floating (figs. 2g-h). on the fourteenth day, other leaves appeared (fig. 2i) and on the eighteenth day, the roots were numerous (fig. 2j). on the twentieth day, the tegument falls off and seedling stage was completed (fig. 2k). vegetative propagation: in addition to an efficient reproduction by seed germination, p. stratiotes propagates itself vegetative. we observed the formation of approximately 28 stoloniferous shoots by eighth day of the experiment, 36 by twelfth day, and 134 by thirty ninth day. after 50 days, 146 new vegetative produced rosettes were observed (fig. 3ad). thus an intense asexual reproduction by means of stolons in a short time was observed, having an ivg of 4.4% individuals per day. after 50 days, we observed that 24 individuals were in flower, although we did not verify the presence of fruits. teixeira et al. (2007) studied the reproductive strategy of p. stratiotes and verified that 54% of individuals produced asexually did not flower in wet localities, and in flooded localities more than 85% failed to flower. they observed less than 34% of individuals with fruits. the vegetative multiplication of p. stratiotes is considered important for its propagation. normally an individual produces from 1-7 short stolons arising in the axils of inconspicuous prophylls that surround the base of each foliage leaf and they develop as the plant grows to its natural full size (lemon and posluszny, 2000). temperature, photoperiod and nutrient availability affect gaining biomass and production of shoots in p. stratiotes, although details of how this takes place remain little understood (cancian et al., 2009). the reproduction of aquatic plants consists in both sexual and asexual mechanisms (harper, 1977). although sexual reproduction introduces new genetic individuals into the population, vegetative propagation produces modules with the potential for physiological independence which contribute to the survival and reproductive success of the plant, as well as diminishing the risk of mortality of the species (cook, 1979), allowing it to explore wide areas and new localities, facilitating a greater dispersion of the propagules (lovettdoust, 1981). the present study shows that seed germination in p. stratiotes is influenced by temperature, abundance of water, and light levels. in the scarcity water, the seeds fail to germinate. knowledge of the germination of weedy species is of fundamental importance for the development of management practices. pistia stratiotes, a weedy aquatic species, can interfere with the life cycle of plant and animal species which share its environment. acknowledgments we are grateful for financial support provided to ima by the "edital mac-doubles fernandes do nascimento de apoio a ciência, tecnologia e inovação, prefeitura de parnaíba, piauí", as part of the project "flora de parnaíba, pi, brasil: diversidade, etnobotânica e conservação". references aguiar r.b (2004). diagnóstico do município de ilha grande. in: r.b. aguiar, j.r.c. gomes (eds.), projeto cadastro de fontes de abastecimento por água subterrânea, estado do piauí. companhia de pesquisa de recursos minerais-serviço geológico do brasil, fortaleza. 7 p. brasil, ministério da agricultura e reforma agrária. 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(2021) 9(5): 268-278 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article age, growth and mortality of atlantic chub mackerel, scomber colias gmelin, 1789 in the mediterranean waters of morocco mohamed techetach* 1, hassnae kouali1, hafid achtak1, fatima rafiq1, ahmed lemhadri1, abdallah dahbi1, rabia ajana2, younes saoud2 1environment and health team, department of biology, polydisciplinary faculty of safi, cadi ayyad university, safi, morocco. 2applied biology and pathology laboratory, department of biology, faculty of sciences, abdelmalek essaadi university, tetouan, morocco. s article history: received 20 november 2020 accepted 12 september 2021 available online 2 5 october 2021 keywords: age growth mortality southwestern mediterranean sea abstract: this work is a study of growth aspects and mortality of the atlantic chub mackerel, scomber colia, from the mediterranean moroccan coast. a total of 845 specimens were collected from commercial catches in m’diq bay, whose total length ranged between 16.4 and 35.9 cm. the length-weight relationship was w = 0.0019 tl3.4527 (r = 0.97) for the whole population. the atlantic chub mackerel displays positive allometric growth. otolith edge analysis indicated that opaque zones were formed between april and september and translucent ones during the remaining months of the year. the oldest individuals in the sample were 5 years old for both sexes. von bertalanffy growth parameters estimated for this species were: l∞ = 37.30 cm, k = 0.26 year-1 and t0 = 2.19 year. the difference in growth between sexes is not significant. the estimated natural mortality was 0.59 per year. introduction small pelagic fishes (sardines, mackerel, horse mackerel and anchovies) represent the fishery potential of morocco, contributing to nearly 80% of national production and ensuring domestic fish consumption and supply of processing units. the second most important species landed in morocco in 2017 after the sardine was the atlantic chub mackerel, scomber colias gmelin, 1789 (242 749 tons), which represent 17% of the whole catch of small pelagic. the catches from the mediterranean waters were 2189 tones, contributing 1% to the overall atlantic chub mackerel landings. the moroccan fleet targeting small pelagic fish is composed by coastal purse seiners, which mainly target sardine and anchovy but also atlantic chub mackerel depending on its availability (inrh, 2017). according to zardoya et al. (2004), s. colias is considered to belong to one stock management policy in mediterranean sea. for assessment purposes, the gfcm (general fisheries commission for the mediterranean) defined the moroccan mediterranean coast as southern alboran *correspondence: mohamed techetach doi: https://doi.org/10.22034/ijab.v9i5.1033 e-mail: mtechetach@gmail.com sea, geographical subarea 3 (fao, 2018). scomber colias is a coastal pelagic species inhabiting warm and temperate waters of the atlantic, mediterranean and black sea (collette and nauen, 1983). based on significant mitochondrial and nuclear dna data and phenotypic characteristics, s. colias is a separate species from its indo-pacific congener, s. japonicus (infante et al., 2007). besides their fishery’s worldwide importance, the atlantic chub mackerel occupies an intermediate position in food web and plays an important role in connecting the lower and upper trophic levels (cury et al., 2000). growth is one of the most important life history processes influencing the dynamics of a fish population. hence, reliable estimation of growth is critical to the development of sustainable fisheries (king, 1995). in spite of the economic and ecological interest, biological information about the atlantic chub mackerel and its fishery in moroccan mediterranean coast are still lacking, and there are no published studies on age structure and growth, except a recent study about its reproduction (techetach et al., 269 int. j. aquat. biol. (2021) 9(5): 268-278 2019). by contrast, in other areas of the mediterranean sea, several aspects of the biology of this species has been discussed, such as length-weight relationship and mortality (petrakis and stergiou, 1995; moutopoulos and stergiou, 2002; sinovčić et al., 2004; karakulak et al., 2006; özaydın and taşkavak, 2006; bayhan, 2007; i̇şmen et al., 2007; cengiz, 2012; allaya et al., 2013; cikes-kec and zorica, 2013), age and growth (rizkalla, 1998; hattour, 2000; kiparissis et al., 2000; perrotta et al., 2005; velasco et al., 2011), reproduction (cikes-kec and zorica, 2012) and feeding habits (hattour, 2000; bayhan et al., 2007). age determination based on otoliths has been the most used method for the atlantic chub mackerel in different areas (castro and santana, 2000). otoliths grow continuously throughout ontogeny and they are not metabolically active providing an ideal structure to infer growth (campana, 2001). in addition to the age estimation, otoliths are also used in taxonomical and biological archives of species in stock discrimination analyses, and study of the diet from partially digested stomach contents (cottrell et al., 1996). accurate ages are mandatory to define lifehistory traits applicable to management, including rates of growth and age-at-maturity (beamish and mcfarlane, 1983). therefore, the knowledge about age structure and growth are fundamental to achieve certain stock assessments. based on above mentioned background, the objectives of this study were to provide length-weight relationship, growth parameterization, validation of age interpretation and natural mortality of s. colias in the sw mediterranean sea for fisheries management purposes. materials and methods samples of atlantic chub mackerel were collected in m’diq region (35ºn, 5ºw) from purse seine commercial catches (fig. 1). a total of 845 s. colias were monthly analyzed between january and december 2004; 54% (453) were females and 46% (392) males. in the laboratory, for each fish, total length (tl, to the nearest 1 mm) and total body weight (w, 0.1 g) were measured. sex was determined by visual examination of the gonads. the sagitta otoliths were removed, cleaned, weighted (ow, 0.1 mg) and stored dry (table 1). length-weight relationships were calculated by applying the equation of w = atlb, where w is the body weight (g), tl is the total length (cm), a is a scaling coefficient and b is the allometry coefficient which usually ranges from 2.5 to 4 (ricker, 1973). student’s t-test was used to ascertain whether the coefficient b was significantly different from 3, using the following formula (morey et al., 2003): ts = (b – 3)· ts = (b – 3)sb-1, where ts is student’s t-test, b is slope and sb is standard error of the slope. the association degree between variables was calculated by the correlation coefficient (r). analysis of variance (anova) was used to check the significance of relationships and to compare the regression lines between sexes. for age determination, whole otoliths were mounted on a black background, covered with water and examined with binocular microscope under reflected light. each otolith was read twice at different times to limit subjective interpretations. unreadable otoliths were excluded from the study (n = 226). the age determination criteria were (ices, 2016): (1) the figure 1. location of study area in m’diq bay (moroccan mediterranean coast) showing the fishing ground. 270 techetach et al./ growth pattern of scomber colias date of birth was considered to be 1 january according to the reproduction season (winter) (lorenzo, 1992), (2) a translucent and an opaque ring were deposited on the otolith every year (castro and santana, 2000; perrotta et al., 2005), and (3) the age of the fish depends on the month of capture: in otoliths taken from fish caught in the first half of the year, their age corresponds to the number of complete translucent bands, but for fish caught during the second half of the year, their age group corresponds to the number of translucent bands surrounded by opaque band (lorenzo, 1992). to validate the annual periodicity of band formation, monthly changes in proportions of opaque and translucent rings in the otolith edge were examined. in addition, to assess that otoliths continue to grow throughout the lives of the fish, weights of 147 otoliths were regressed on age and size of fish, given that a strong positive relationship suggests that the otoliths continue to grow at a measurable rate through the lifespan of the fish (fowler and doherty, 1992). to describe the growth of the atlantic chub mackerel, the observed mean length-at-age data were fitted to the von bertalanffy growth equation: lt = l∞[1 – e-k(t-t0)], where lt is the total length (cm) at age t, l∞ is the asymptotic total length (cm), k is the growth coefficient (year-1), and t0 (year) is the hypothetical age at which lt = 0. analysis of variance was applied to compare male and female mean lengths at age. to compare the growth parameters obtained with other studies, the growth performance index (φ’) was calculated using the following formula (pauly and munro, 1984): φ’ = log10 k + 2 log10 l∞. the condition factor (kn) was used for comparing the condition or well-being of fish and could be used as an indicator of seasonal energy investment, related to changes between somatic growth and reproductive processes. the kn was calculated using formula (fulton, 1904) of kn = w (g) tl-3 (cm) 100. natural mortality (m) was calculated by applying the pauly (1980) formula as log10m = – 0.0066 – 0.279 × log10l∞ + 0.6543 × log10k + 0.4634 × log10t, where l∞ and k are the parameters of the von bertalanffy growth equation and t is the mean annual sea water temperature, which was 19.8°c in m’diq bay (https:// www.seatemperature.org/africa/morocco/). averaged temperature was obtained for the area and the study time period. additionally, based on the results published in techetach et al. (2019), the age at first maturity (a50) was estimated using von bertalanffy growth equation. all the statistical analyses were conducted in statistica software v.6.0. results size composition: the length of fish ranged between 16.4 and 35.9 cm tl (mean length = 24.07, standard deviation (sd) = 3.25). males ranged 16.6-35.9 cm (23.97±3.26) and females varied from 16.4 to 35.8 cm (24.15±3.25). most of the fish sampled ranged 20-27 cm, representing 79.27% of the whole capture, and the modal was 22-23 cm (fig. 2). the anova showed table 1. statistic data from the sample of atlantic chub mackerel collected in this study (tl = total length; w = total body weight; ow = otolith weight; sd = standard deviation; now = number of otoliths weighted). month number of specimens tl range (cm) mean tl (cm) ± sd mean w (g) ± sd now mean ow (mg) ± sd males females total january 57 36 93 17.3-30.0 23.2±2.5 105.6±47.0 35 6.3±1.4 february 35 31 66 16.6-29.8 21.3±3.2 80.4±45.6 22 5.0±1.1 march 27 31 58 16.4-28.4 24.1±3.0 117.1±40.2 32 6.2±1.0 april 12 21 33 21.5-26.7 23.9±1.3 99.1±18.2 3 5.5±0.4 may 22 38 60 21.0-29.8 24.6±2.2 122.9±44.6 7 8.3±0.8 june 22 30 52 19.8-35.8 23.6±2.9 102.3±65.8 3 9.4±2.2 july 41 51 92 20.6-35.6 24.8±3.5 137.1±77.4 16 9.4±1.0 august 43 50 93 19.1-31.1 23.3±2.6 109.6±45.6 7 9.6±1.0 september 24 48 72 21.1-35.9 26.5±4.6 186.5±118.2 3 7.4±0.4 october 46 48 94 19.1-33.1 24.5±3.4 136.2±67.2 18 9.4±1.5 november 22 29 51 18.1-31.4 24.2±3.2 125.0±55.0 1 10.6 december 41 40 81 18.6-28.3 24.0±1.7 126.7±32.4 271 int. j. aquat. biol. (2021) 9(5): 268-278 that difference between mean tl by sex not significant (f = 0.69, p>0.05). length-weight relationship: the relationship between length and weight was highly significant for all fish combined and per sex (p<0.001). in addition, high value of the correlation coefficient (r>0.97) indicated that variables were strongly correlated. the b-values showed significance difference from isometric growth for both sexes and all fishes (table 2). therefore, the atlantic chub mackerel displays positive allometric growth (b>3). the slopes of the length-weight regressions lines were not significantly different between sexes (f = 1.6; p = 0.2). age validation: otolith analysis showed two types of edges (opaque and translucent). monthly changes in the frequency of opaque and translucent bands on the otolith edge seemed to show a seasonal variation in the formation of these bands during the study period (fig. 3). opaque edges were more frequent in spring and summer months (april-september), while translucent edges dominated during autumn and winter (octobermarch), thus confirming the expected annual pattern of deposition of two rings: one opaque and one translucent. the weights of 147 otoliths varied from 3.4 to 12.8 mg, with a mean value of 7.19±2.06 mg. a positive linear relationship was observed between otolith weight and age (r = 0.88) (fig. 4). a high linear correlation was also obtained between the total length of the fish and the otolith weight (r = 0.88) (fig. 5). condition factor: the kn ranged from 0.51 to 1.1 for males and from 0.46 to 1.072 for females. a similar monthly trend was observed for male and female fish (fig. 6). the highest values were recorded in table 2. parameters of length–weight relationship for atlantic chub mackerel (n = number of fish; a = scaling coefficient; b = allometry coefficient; r = correlation coefficient; ts = t-test). sex n a b r ts significance allometry males 392 0.0017 3.48 0.98 14.10 p<0.05 positive females 453 0.0020 3.42 0.97 11.33 p<0.05 positive all fish 845 0.0019 3.45 0.97 17.54 p<0.05 positive figure 2. size frequency distributions by sex for atlantic chub mackerel caught in m’diq bay. 272 techetach et al./ growth pattern of scomber colias september and december, while the lowest ones were occurred in april and june. age and growth: the estimated ages of s. colias varied from 0 to 5 years both for males and females. age class 1 (37.16%) was dominant, followed by 2 (32.47%) and 3 (12.92%). juvenile specimens (age class 0) were poorly represented in the sample. the age-length key per sex including totals is shown in table 3. the von bertalanffy growth curve fitted well to the mean length at age data of atlantic chub mackerel (table 4). the growth performance index (φ’) was calculated as 2.56 for males, females and all samples. no significant differences were found in the mean length at age between males and females (p>0.05). there were no differences in the growth between both sexes, thus data can be pooled (fig. 7). the estimated natural mortality was 0.59 per year. age at first maturity: in m’diq bay, the size at first maturity has been estimated at 19.19 cm (techetach et al., 2019), which corresponds to an age at first figure 3. monthly changes in the occurrence frequency of edge type in otoliths of atlantic chub mackerel. figure 4. relationship between age and otolith weight (ow) for atlantic chub mackerel in m’diq bay (n = number of fish; r = correlation coefficient. figure 5. relationship between total length (tl) and otolith weight (ow) for atlantic chub mackerel in m’diq bay (n = number of fish; r = correlation coefficient). 273 int. j. aquat. biol. (2021) 9(5): 268-278 table 3. age-length key per sex and total of scomber colias from m’diq bay (n = number of fish; tl = total length; min = minimum; max = maximum; sd = standard deviation). length class (cm) age (year) 0 1 2 3 4 5 16-17 17-18 18-19 19-20 20-21 21-22 22-23 23-24 24-25 25-26 26-27 27-28 28-29 29-30 30-31 31-32 32-33 33-34 34-35 6 1 8 43 110 61 7 1 8 46 84 45 17 8 17 38 16 1 1 16 17 20 6 3 3 2 6 6 8 10 4 n mean tl min. tl max. tl sd tl % 6 16.68 16.40 16.90 0.18 0.97 230 22.51 19.60 24.40 0.82 37.16 201 24.51 21.70 26.90 0.99 32.47 80 26.32 24.30 28.10 0.85 12.92 66 28.93 26.80 32.40 1.34 10.66 36 32.16 29.80 34.50 1.41 5.82 table 4. parameters of the von bertalanffy growth equation for scomber colias (l∞ = asymptotic total length; k = growth coefficient; t0 = hypothetical age at which size fish is zero; r = correlation coefficient). sex l∞ (cm) k (year-1) t0 (year) r males 36.65 0.27 2.15 0.94 females 38.10 0.25 2.19 0.94 all fish 37.30 0.26 2.19 0.94 figure 6. relationship between total length (tl) and otolith weight (ow) for atlantic chub mackerel in m’diq bay (n = number of fish; r = correlation coefficient). 274 techetach et al./ growth pattern of scomber colias maturity (a50) of 0.61 year, based on the present study. discussions positive allometric growth of s. colias observed in this study is in agreement with results reported in other studies in mediterranean sea (kiparissis et al., 2000 in the hellenic seas; moutopoulos and stergiou, 2002 in the aegean sea; bayhan, 2007 in izmir bay; velasco et al., 2011 in alboran sea; cengiz, 2012 in saros bay). however, isometric growth was obtained by allaya et al. (2013) in tunisian coasts, cikes-kec and zorica (2013) in adriatic sea, and özaydın and taşkavak (2006) in izmir bay. there is no significant difference in length-weight relationship parameters between sexes, which indicated a similar relative weight growth between males and females. this result is in concordance with those revealed in the gulf of cadiz and the alboran sea (velasco et al., 2011), madeira island (vasconcelos et al., 2011), and canary islands (lorenzo and pajuelo, 1996). variation in parameters of length-weight relationship for atlantic chub mackerel might be related to sampling area, sampling strategy, fish feeding, sexual maturity, sex, size-range and health (tesch, 1971; ricker, 1975). age validation is critical process to accurately determine age. monthly evolution of the nature of the otolith edges presented a clear annual pattern of the annuli deposition with opaque and hyaline rings being laid down during warm and cold months, respectively. it matches with the results of velasco et al. (2011) in the same area. the same pattern has been also obtained in other areas (lorenzo, 1992; kiparissis et al., 2000). the formation of an opaque zone, which corresponds to rapid growth, was related to food resource abundance, increase of temperature, and best condition. in contrast, translucent zone formation coincides with the spawning period in this area (lorenzo and pajuelo, 1996). indeed, the otolith growth depends on local environmental factors, which influence the otolith deposition pattern (campana and neilson, 1985). the atlantic chub mackerel gets the energy required for gonad growth from food that explained the maximum values of kn recorded in summerautumn, whereas, the stored energy is devoted to somatic growth. accordingly, the feeding activity of s. colias does not change during the reproductive period. the phase of slimming will take place after the spawning, due to the release of genital products (kartas, 1981; aristizabal, 2007). similar results have been found in sardines, which do not stop feeding during the spawning period (somarakis et al., 2004). species that allocate ingested food directly to reproduction seem to be income breeder (stephens et al., 2009; mcbride et al., 2015). in addition, the condition of a species is the consequence of the figure 7. the von bertalanffy growth curve for the whole sample of atlantic chub mackerel. 275 int. j. aquat. biol. (2021) 9(5): 268-278 interaction of biotic and abiotic factors (vazzoler, 1981). the present study is the first work on age and growth pattern of atlantic chub mackerel, s. colias in the west mediterranean coast of morocco. the age of atlantic chub mackerel can be estimated from whole otoliths. this is supported by all results presented in this study, especially the positive relationship between otolith weight and length and age of fish, thus, suggesting the increase of otolith weight throughout the lifespan of fish. the otolith weight would be an accurate indicator of somatic growth, since it is sensitive to variations in growth rate and closely related to changes in fish metabolism (pawson, 1990; fletcher, 1995; zorica et al., 2010). as commented, the maximum age observed in the present study was 5 years. this longevity is similar to that obtained by kiparissis et al. (2000), and slightly lower than the 6 years recorded in alboran sea (velasco et al., 2011). however, cikes-kec and zorica (2013) reported the maximum age as 9 years in the adriatic sea. growth of atlantic chub mackerel is rapid during the first year of their life reaching the 60.35% of its maximum length, which seems to be closer to the value reported by velasco et al. (2011; 59%). the similar growth patterns between sexes noted in this study was in accordance with the results of vasconcelos et al. (2011) and velasco et al. (2011). growth parameters (l∞, k and t0) and growth performance index (φ’) from the present study and previously published studies are shown in table 5. our estimate of the growth parameters (k, l∞) was in agreement with previous studies. the growth performance indices ranged from 2.48 to 2.82 for chub mackerel in their area of distribution. the lowest one corresponds to saros bay and the highest in azorean waters. the obtained performance index was similar to the values in adriatic sea (cikes-kec and zorica, 2013) and in hellenic seas (kiparissis et al., 2000) although different from all the other studies (table 5). the geographic differences in growth parameters could be due to variations in environmental conditions such as water temperature, food quality and availability might lead to different results for growth parameters (weatherley, 1976; perrotta et al., 2005). the natural mortality in this study was similar to that found by jurado-ruzafa et al. (2017, m = 0.57 year-1) but different to those of cengiz (2012, m = 0.34 year-1) and cikes-kec and zorica (2013, m = 0.35 year-1). disparity of mortality rates between different localities could be explained by ecological conditions and fishing pressures (vetter, 1988), as well as on the growth estimation method, the sample size, the sampling strategy and the ageing methodology. the first maturity of the atlantic chub mackerel obtained in this work is early (0.61 year) compared to previous studies in other areas. in fact, this species achieved maturity at 27 cm and 3 years in portuguese coast (martins, 1996). in the azores, carvalho et al. (2002) estimated size at maturity at 27.78 cm and 2.23 years. the trend of reduction in age and size at first maturity could be explained by the result of genetic changes as an effect of intense and prolonged exploitation and also the effect of global climate table 5. comparison of growth parameters from different regions for atlantic chub mackerel (l∞=asymptotic total length; k=growth coefficient; t0=hypothetical age at which size fish is zero; φ’=growth performance index). area l∞ k t0 φ’ reference m’diq bay 37.30 0.26 -2.19 2.56 this study alboran sea 40 0.37 -0.10 2.77 velasco et al. (2011) adriatica sea 45.31 0.18 -1.65 2.57 cikes-kec and zorica (2013) hellenic seas 47.60 0.15 -2.18 2.53 kiparissis et al. (2000) saros bay (turkey) 39 0.20 -2.13 2.48 cengiz (2012) egypt 39.42 0.31 -1.39 2.68 rizkalla (1998) gulf of cadiz 43 0.27 -1.10 2.69 velasco et al. (2011) canary islands 49.20 0.21 -1.40 2.71 lorenzo et al. (1995) madeira island 50.08 0.25 -1.33 2.80 vasconcelos et al. 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(2018) 6(4): 242-247 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology short communication morphological comparison of western and eastern populations of caspian kutum, rutilus kutum (kamensky, 1901) (cyprinidae) in the southern caspian sea masoud sattari*1,3, javid imanpour namin1, mehdi bibak1, mohammad forouhar vajargah1, aliakbar hedayati2, arash khosravi1, mohammad hossein mazareiy1 1department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, iran. 2department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. 3department of marine biology, caspian basin research center, university of guilan, rasht, iran. article history: received 5 may 2018 accepted 2 august 2018 available online 2 5 august 2018 keywords: morphological variation length-weight relationship condition factor stock abstract: this study aimed to investigate morphological differences of western and eastern populations of caspian kutum rutilus kutum in the southern caspian sea and its providing lengthweight relationships and condition factor (cf). a 13-landmark based morphometric truss network system was used to investigate the hypothesis of population fragmentation of western and eastern populations of this species. the studied populations were differentmorphologically based on pre anal, body height, distance from pectoral fin to ventral fin, distance from pectoral fin to anal fin, caudal peduncle length, head length, pre orbital, pre ventral, and dorsal length. the results also revealed a negative allometry (b<3) of length-weight relationships for both sexes. maximum condition factors was found in march. the results suggest distinct stocks in the western and eastern of caspian sea for fisheries management. introduction caspian sea is unique ecosystem not only because of its size, but also due to its salinity which is brackish as well as those of freshwater fish species that adapted to live in such a salinity. caspian kutum, rutilus kutum, is a commercially important native fish species of the caspian sea basin representing over 50% of the total bony fish catch, and 60% of the fishermen’s income in this basin (abdolmaleki and ghaninejad, 2007). the iranian fisheries organization (ifo) has been releasing up to 200 million r. kutum fry (with average weight of 1 g) into its rivers annually since 1982, to rehabilitate their reduced populations in this basin (abdolmaleki and ghaninejad, 2007). caspian kutum attains a maximum fork length and weight of 71 cm and 4 kg, respectively. its spawning migration in the rivers and anzali wetland occurs from mid-march to mid-may, depending on water temperature, and ecological conditions (valipour and khanipour, 2006). the relationship between body weight and length is a simple but essential parameter in fishery *corresponding author: masoud sattari doi: https://doi.org/10.22034/ijab.v6i4.529 e-mail address: msattari@guilan.ac.ir management (chien-chung, 1999). length-weight relationships is used to convert growth-in-length equations to growth in weight in stock assessment models (bobori et al., 2010). to estimate growth rates, determining age structure is necessary to obtain the condition of fish and comparative growth studies (kolher, 1995). in addition, these relationships contribute to the comparison of life history and morphological aspects of populations between different regions. hence, the present study aimed to study the morphological differences of western and eastern populations of r. kutum in the southern caspian sea in terms of morphometric and providing length-weight relationships and condition factor (cf) this species in the southern caspian sea. materials and methods the sampling was carried out on a monthly basis between december 2017 and march of 2018 in five regions, including astara (38˚30'n, 48°88'e), kiyashahr (37˚42'n, 49˚98'e), anzali (37°46'e, 49°59'e) in the western part and sari (36°48'n, 243 int. j. aquat. biol. (2018) 6(4): 242-247 53°6'e) and bandar-e turkmen (36°33'n, 55°50'e) in the eastern part of the caspian sea. for morphological comparison, those of western and eastern were merged and considered as two populations. a total of 240 fish were collected and fixed into 10% buffered formalin and transferred to the laboratory of ichthyology, university of guilan, rasht, iran. total length (tl) and whole body wet weight (g) were recorded for each specimen. the length-weight relationship was estimated using following equation: w = a lb where w is the whole body weight (g), l is the total length (mm), a is the intercept of the regression and b is the regression coefficient (slope) (ricker, 1975). the parameters a and b of the length-weight relationship were estimated by the least-squares method based on logarithms (zar, 1999): log (w) = log (a)+b log (l) when b = 3, increase in weight is isometric and when the value of b is other than 3, weight increase is allometric (positive if b>3, negative if b<3). also a ttest was used to compare b value in the linear regression of males and females (zar, 1999). 𝑡 = 𝑏1 − 𝑏2 𝑆𝑏1−𝑏2 these parameters (a, b) are important in stock assessment studies (froese, 1998). data analysis was performed by excel and spss version 19 software. the condition factor (cf) was computed by the following equation: 𝑘 = 100𝑤 𝑙3 where w = the observed total weight for each fish, l = the observed standard length for each fish and k= the condition factor (cf). for morphological study, measurements of specimens were extracted by collecting x–y coordinate data for relevant morphological features, followed by the three-step process as described below (turan, 1999). the fish were placed on a white board with dorsal and anal fins held erect by pins. the right body profile of each fish was photographed using a 300-dpi, 32-bit color digital camera (cybershot dscf505; sony, japan). images were saved in jpg format and analyzed by tps dig2 (ver. 2.04) to digitize 13 landmark points (fig. 1). a box truss of 26 lines connecting landmark points was generated for each fish to extract the basic body shape (cardin and friedland, 1999). all data were transferred to a spreadsheet file (excel 2010), and the x–y coordinate data transformed into linear distances by computer for subsequent analysis (turan, 1999). size-dependent variation was corrected by adopting an allometric method based on elliott et al. (1995): madj = m (ls / l0) b where m is the original measurement, madj = size adjusted measurement, l0 the standard length of fish, ls = the overall mean of the standard length for all fish from all samples in each analysis, and b = estimated for each character from the observed data as the slope of the regression of log m on log l0 using all fish in figure 1. locations of the 13 landmark points for constructing the truss network on rutilus kutum. 1. tip of the snout; 2. center of the eye; 3. end of the frontal bone; 4. ventral end of the gill slit; 5. origin of the pectoral fin; 6. insertion of the dorsal fin; 7. origin of the pelvic fin; 8. insertion of the dorsal fin; 9. origin of the anal fin; 10. insertion of anal fin; 11. dorsal end side of caudal peduncle, at the nadir; 12. ventral end of the caudal peduncle, at the nadir; 13. end of body lateral line. 244 sattari et al./ morphological comparison of western and eastern populations of caspian kutum any group. the derived data from the allometric method by testing significance of the correlation between transformed variables and standard length were confirmed (turan, 1999). univariate anova was performed for each morphometric character to evaluate the significant differences between the populations (zar, 1984). the morphometric characters that showed significant difference (p<0.05) were used for further analysis. in addition, the recommended ratio of the number of organisms (n) measured to the parameters (p) were included in the analysis to be at least 3-3.5 (kocovsky et al., 2009) for obtaining the stable outcome from the multivariate analysis. linear discriminant function analysis (dfa) was used to calculate the percentage of correctly classified (pcc) fish. a cross-validation using pcc was carried out to estimate the expected actual error rates of the classification functions. to examine the suitability of the data for pca, bartlett’s test of sphericity and the kaiser–meyer–olkin (kmo) measures were performed. the bartlett’s test of sphericity can be used to determine if the value of the correlation matrix is equal to zero, while the kmo measures sampling adequacy tests whether the partial correlation among variables is sufficiently high (nimalathasan, 2009). the kmo statistics varies between 0 and 1. kaiser (1974) suggested that values greater than 0.5 are acceptable. statistical analyses for morphometric data were performed using the spss software (version 16). results and discussion length and weight parameters of the studied r. kutum are shown in table 1. the minimum and maximum length of females were 32 and 51 (cm), while those of weight were 330 and 1324 (g), respectively. the minimum and maximum length of males were 29.5 and 47 (cm), while those of weight were 260 and 1010 (g), respectively (table 1). figures 2 and 3 show length–weight relationships of male and female caspian kutum, respectively. figures 4 and 5 present length–length relationships in males and female caspian kutum, respectively. all the three relationships were positively correlated at 0.05% level (p<0.05%). a negative allometry (b<3) in length-weight relationship were found for r. kutum in the caspian sea. according to weatherley and gill (1987), the annual length-weight relationships could differ between seasons and years and also many table 1. length and weight parameters of the studied rutilus kutum. sex parameter f m 32-51 29.5-47 length range (cm) 43.7±0.8 38.71.2 mean length (cm)±se 330-1324 260-1010 weight range (g) 892.1±46.9 628.1±55.9 mean weight (g)±se figure 2. length-weight relationship of the studied rutilus kutum (male). figure 3. length-weight relationship of the studied rutilus kutum (female). 245 int. j. aquat. biol. (2018) 6(4): 242-247 factors could contribute to these differences such as maturity, temperature, salinity, food availability and size. length-weight relationship may vary seasonally according to the degree of sexual maturity, sex, diet, stomach fullness, sample preservation techniques (wootton, 1992), number of specimens examined, area/season effects and sampling duration (moutopoulos and stergiou, 2002). figure 6 shows the mean monthly cf of the caspian kutum from november 2017 till march 2018. the monthly cfs were higher in march reflecting better conditions of fish in this month. the highest value (1.63) was observed in march, and the lowest value (1.5) in january. significant differences were found between eastern and western populations of r. kutum in 42 morphometric characters out of 78 standardized traits (p≤0.05). in the present study, the value of kmo for overall matrix was 0.6 and the bartlett’s test of sphericity is significant (p<0.05). principal component analysis of 42 morphometric measurements extracted 12 factors with eigenvalues >1, explaining 96% of the variance. the first principal component (pc1) accounted for 26.84% of the variation, while the second one (pc2) for 16.33%. the most significant loadings on pc1 were 1-3, 1-4, 1-5, 1-8, 1-11, 1-12, 1-13, 2-3, 2-4, 27, 3-5, 3-6, 3-7, 3-9, 3-10, 4-6, 4-8, 4-9, 4-11, 4-12, 56, 5-8, 5-9, 5-11, 5-12, 6-12, 7-9, 7-10, 8-9, 8-10, 913, 10-11, 10-13, while on pc2 were 1-7, 1-10, 1-12, 2-6, 2-9, 2-10, 2-11, 3-8, 3-9, 3-10, 3-11, 4-7, 4-10, 4-12, 5-7, 5-8, 5-10, 6-10, 6-11, 7-8, 7-9, 7-11, 7-12, 8-11, 9-10, 9-11, 9-12, 9-13. in this analysis, the characteristics with an eigenvalues exceeding 1 were included and others discarded. also, in female visual examination of plots of pc1 and pc2 scores, specimens were grouped into two areas with moderate overlap between two stations (fig. 7). wilks’s lambda distribution of discriminant analysis indicated significant differences in 13 morphometric characters of the two populations. in this test, one function was highly significant (p<0.01). the linear discriminant analysis gave an average pcc (percentage of specimens classified) of 86.5% for morphometric characters indicating a high rate of correct classification of individuals into their original populations. figure 4. lengthlength relationship the studied rutilus kutum (male). figure 5. lengthlength relationship the studied rutilus kutum (female). figure 6. monthly changes of the condition factor in the studied rutilus kutum in the caspian sea. 246 sattari et al./ morphological comparison of western and eastern populations of caspian kutum the studied populations were distinguished from each other by morphologic differences especially in pre anal, body height, distance from pectoral fin to ventral fin, distance from pectoral fin to anal fin, caudal peduncle length, head length, pre orbital, preventral and predorsal length. geographical isolation can also affect growth pattern and reproductive strategy of fish species. the importance of such factors on producing morphological differentiation in fish species is well-known (yamamoto et al., 2006; pollar et al., 2007). the causes of morphological differences among populations are often quite difficult to explain (bookstein 1991). it was suggested that morphological characteristics of fishes are determined by genetic, environment and the interaction between them (kohestan-eskandari et al., 2014). the environmental factors prevailing during the early development stages, when the individual’s phenotype is more amenable to environmental influence is of particular importance (eschmeyer and fong, 2011; burgos et al., 2016). the phenotypic variability may not necessarily reflect population differentiation at the molecular level (bookstein 1991). apparently, different environmental conditions can lead to an enhancement of pre-existing genetic differences, providing a high inter-population structuring (eschmeyer and fong, 2011; burgos at al., 2018). these morphologically different populations can be considered as distinct stocks in the caspian sea for fisheries management. nevertheless, future studies on determination of population structure will be elucidated using biochemical and molecular genetic methods or not. acknowledgments this study was financially supported by the caspian basin research center, university of guilan, rasht, iran, file number 21195170 references abdolmaleki s., ghaninzhad d. 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(2014) 2(4): 206-214 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article phytoremediation efficiency of pondweed (potamogeton crispus) in removing heavy metals (cu, cr, pb, as and cd) from water of anzali wetland hajar norouznia1, amir hossein hamidian*21 1department of environment, faculty of natural resources and earth science, kashan university, p.o. box 8731751167, kashan, iran. 2department of environment, faculty of natural resources, university of tehran, p.o. box 31585-4314, karaj, iran. article history: received 1 june 2014 accepted 22 july 2014 available online 2 5 august 2014 keywords: phytoremediation heavy metal potamogeton crispus bioconcentration translocation abstract: plant-based remediation (i.e. phytoremediation) is one of the most significant ecosustainable techniques to cope with devastating consequences of pollutants. in the present study, the potential of a wetland macrophyt (i.e. potamogeton crispus) for the phytoremediation of heavy metals (i.e. cu, cr, pb, as and cd) in the anzali wetland was evaluated. the results showed that p. crispus tends to accumulate notable amounts of cu, cr, pb, as and cd according to their assayed concentrations as follows: 8.2 µg g-1 dw, 0.97 µg g-1 dw, 6.04 µg g-1 dw, 2.52 µg g-1 dw and 0.34 µg g-1 dw, respectively. further accurate perception of the phytoremediation efficiency were conducted using both bioconcentration factor and translocation factor. the average of the highest bioconcentration factors was presented in a descending order as: 2.9×103, 1.9×103, 1.17×103, 0.68×103 and 0.46×103 for the cu, cr, pb, cd and as, respectively. based on the results, p. crispus presents high potential to absorb all the alluded metals except for as and partly cd. correspondingly, the mean values of translocation factor were reported in the range of 0.41 to 2.24. eventually, relying on the observed findings, the results support the idea that p. crispus species would be employed as the prospective candidate for the phytoremediation processes in anzali wetland. introduction heavy metals pollution has been posed as a growing predicament worldwide. hence, the natural environment quality levels tends to be worsened due to poor problem-solving skills and inadequate ecosuited techniques to diminish the detrimental demeanor of pollutants. unlike organic pollutants, non-biodegradability property of heavy metals displays the immediate cause of bioaccumulation and its harmful subsequences on food chains (khan et al., 2010; hamidian et al., 2014). therefore an acute health risk occurs to the environment and its living organisms. thus, removing heavy metals from natural medium requires necessary action (ali et al., 2013). the heavy metals are divided broadly into essential and non-essential. essential heavy metals such as zn, * corresponding author: amir hossein hamidian e-mail address: a.hamidian@ut.ac.ir cu, mn, fe, cr and ni are necessary for functions in trace quantities in biological operations (cempel and nikel, 2006). in contrast, hg, cd, as and pb known as non-essential heavy metals which are not only unnecessary for organism, but also have toxic and deleterious effects (dabonne et al., 2010). major origin of contaminants such as heavy metals are anthropological activities including industrial and municipal effluents, mining, untreated wastewater and agrochemicals and also, natural-driven origins such as volcanic eruption, ore weathering and mineral deposition (kabata-pendias and pendias, 1989; rai et al., 2008). concern about marked release of heavy metal pollutants and cleaning up of contaminated areas has been increasing as the major controversial and disputed issues in the contemporary time. therefore, 207 norouznia and hamidian/ phytoremediation efficiency of pondweed in removing heavy metals many physical, chemical and biological approaches have been applied to reduce the impacts of pollutants especially heavy metals (sheoran et al., 2011). most of these methods appear to cost high and disruptive to the natural properties of environment and ended to soil erosion rapidly, and may also cause multiplied environmental problems (ali et al., 2013). the most innovative and effective alternative method to omit the heavy metals from environment is phytoremediation that is a green, safe, solar-oriented and cost effective method using the capability of plant species to absorb high levels of metals in specific tissues like roots and leaves especially in the aquatic substratum (carranza-άlvarez., 2008; sigh and prasade, 2011). the inception of phytoremediation idea was owing to chaney (1983) and came to apply eagerly during the recent two decades as a better way to solve contamination problems particularly by aquatic plants for removal of pollutants from contaminated surface water (i.e. phytofilteration) (mukhopadhyay and maiti, 2010). based on ali et al. (2013), proper plant species for phytoremediation needs some distinctive features such as to spread widely, easy to grow and mainly high potential to accumulate heavy metals. the aquatic macrophyts have innate ability to uptake heavy metals from polluted water medium and wastewater (hamidian et al., 2014). the aquatic plants can absorb more cu, as, br, cr, cd, sr, v and pb than terrestrial plants (sood et al., 2012). phytoremediation and metal removal using aquatic macrophyts can be enormously reinforced by preferring appropriate plants species (fritioff and greger, 2003; hamidian et al., 2014). identification of proper plants for phytoremediation in a contaminated area is a full-scale model to survey its mechanisms of heavy metals purification and accumulation (carranza-alvarez et al., 2008; hamidian et al., 2014). therefore, this study aimed to assess the phytoremediation potential of a native and dominant floating plant in anzali wetland, potamogeton crispus (pondweed), and evaluate its innate capability to accumulate heavy metals as hyper-accumulators to meet the phytoremediation purposes. potamogeton crispus is a widespread floating species in wetland ecosystems especially in anzali wetland (pajevic et al., 2008). this species is a floating plant and its metal-accumulation aspect is related to its surrounding water metal concentrations (favas and pratas, 2013). hence, this study intends to determine the extent to which the abovementioned species is well-suited for phytoremediation and whether could be used as a proper accumulator species to uptake specific heavy metals including cu, cr, pb, as and cd based on (1) to investigate the concentration of metals in water and plant body, (2) to determine the removal potential of cu, cr, pb, as and cd using p. crispus, (3) to estimate the metal transportability according to bioconcentration factor (bcf) for water and plant tissues; moreover, transfer factor (tf) for shoot and root and (4) to confirm the positive correlation figure 1. location of anzali wetland in iran 208 int. j. aquat. biol. (2014) 2(4): 206-214 between metal concentration in water and plant in the eastern part of anzali wetland in iran. material and methods description of study area: anzali wetland is an outstanding coastal lagoon that is located in the southwest of caspian sea including an area of about 200 km2 located between 37°28´n and 49°25´e. it is an excellent natural aquatic ecosystems that enhances a widely heterogeneous floristic composition and wildlife refuges. the wetland is covering 1% of bird wintering immigrant community of middle-east region (jafari, 2009). anzali wetland similar to other coastal ecosystems is likely to be affected by anthropocentric activities. hence, the contaminants deriving from industrial and urban effluents, agrochemicals and untreated wastewater can cause irretrievable adverse effects on this natural ecosystem (fig. 1). preparation of plant samples: sampling was performed in october 2013. the water samples were removed from five stations and prepared by filtration and addition of 2% hno3 subsequently. also, twenty plant samples were collected from the same site. the body samples were washed with tap and distilled water to wipe out any adhering substances. the root and body of plants were homogenized and then dried in oven (60°c for 24 hrs). then, the dried samples were pulverized and obtained powder was sieved (0.15 mm) (kalra, 1998). according to the digestion protocol, 0.5 gram samples of plant tissues put into the digestion tube and then 10 ml of nitric acid was added and stored overnight. furthermore, samples were heated (120°c) for 4 hrs. finally, the samples were transferred into 25 ml volumetric flasks and after addition of 3 ml diluted hydrochloric acid, filled with distilled water. inductivity coupled plasma-optical emission spectrometry (icp-oes) (perkinelmer, usa) was used to measure the concentration of cu, cr, pb and cd. the detection limits for the analytical instrument were 2.5, 0.5, 0.5, 5 ppb for cu, cd, cr and pb, respectively. as concentrations were assayed by hg-faas (hydrogen generation flame atomic absorption spectroscopy) with detection limit of 1 ppb. the analysis procedure was confirmed by analyzing 2 blanks and 2 spiked specimens for each twenty plant samples considering all laboratory conditions being equal. statistical analysis: all statistical analysis were performed using origin 8.0 software. each metal was analyzed individually. data was analyzed for normalization using kolmogorov-smirnov test. anova and duncan tests were applied to parallel the mean values of metals in different sampling sites. pearson correlation coefficients were used for estimating the correlation among metal concentrations in the water and plant tissues. also, comparisons between metal concentrations in water and plant tissues were performed using t-test. when concentrations were not definite (e.g. below the detection limit), they were considered as half of the element sample (mean ± sd, n=20) bcf tf water (µg l-1) shoot (µg g-1) root (µg g-1) cu 2.82 ± 0.14 4.35 ± 0.12 8.2 ± 0.17 2900 0.530 cr 0.51 ± 0.03 0.471 ± 0.024 0.97 ± 0.05 1900 0.485 pb 5.14 ± 1.43 6.04 ± 0.2 2.686 ± 0.134 1170 2.25 as 5.42 ± 0.65 1.037 ± 0.052 2.525 ± 0.127 460 0.411 cd 0.50 ± 0.01 0.235 ± 0.012 0.344 ± 0.02 688 0.683 bioconcentration factor (bcf) = concentration in plant/concentration in water translocation factor (tf) = concentration in shoot / concentration in root table 1. heavy metals in potamogeton crispus species tissues (µg-1, dry weight) and in fresh water (μg lit-1). 209 norouznia and hamidian/ phytoremediation efficiency of pondweed in removing heavy metals detection limit. data were reported based on mean values of at least triplicates with standard deviation (sd). result and discussion variation of heavy metals in water and plant tissues: no significant difference was observed in heavy metal concentrations of different sampling sites, in the same time (table 1). thus, it can be inferred that the metal ions were equally distributed throughout the wetland. however, the concentration of heavy metals in station 1 (pirbazar river outfall to the wetland) and station 3 (hendekhaleh river outfall to the wetland), which are located in the east and southeast of anzali wetland, respectively, were little higher than those of other stations. meanwhile, based on geographical interpretation of the sampling sites, it was obvious that heavy metal effluents outfall were participated in the contaminating of the water substratum (pajevic et al., 2008; pratas et al., 2012). the results showed a significant differences between the heavy metals concentration in plant tissues and water (p<0.05). the concentrations of heavy metals in water are shown in figure 2. the heavy metals concentration in plant tissues are depicted in figure 3. based on the results, a range between 0.12 µg g-1 dw and 14.05 µg g-1 dw in plant and a range between 0.5 µg lit-1 and 6.91 µg lit-1 in water were recorded. this results show a significant differences in the different tissues of plant and water. copper: cu not only is a necessary nutrient for plants, but also a toxic element at extra concentration. it seems having high level of potency to accumulate in the lower tissues of plants (kabatapendias and pendias, 2001). the results revealed that the concentration of cu in the water, p. crispus roots and shoots tissues were 2.82 µg lit-1, 8.20 µg g-1 dw and 4.01 µg g-1 dw, respectively. the highest registered bcf was calculated in p. crispus with the average of 2.9×103. similarly, the tf were calculated in the range of 0.17 and 0.56. moreover a significant (p<0.001) positive correlation between the cu concentration in the plant and water was found. this result shows the positive aspects of p. crispus as a reliable accumulator species to omit cu-oriented contaminants loaded to anzali wetland. chromium: contents of cr in plants have appealed warning notice not only due to its main function as an essential metal, but also because of its carcinogenic impacts. chromium is slightly available to plants, thus it is accumulated eminently in roots. chromium concentration vary between 0.4 and 3.2 mg kg-1 in rooted emergent plants species (kabata-pendias, 2011). nonetheless, chromium concentrations more than 10 mg kg-1 have phytotoxic (pais and jones, 2000). in the current investigation, the mean values of cr in the water and p. crispus roots and shoots were calculated to be 0.51 µg lit-1, 0.97 µg g-1 dw and 0.47 µg g-1 dw, respectively. the cr values in the p. crispus tissues were measured in a descending order from root to shoot. although, cr concentration of p. crispus was shown positive correlation with its concentration in water. low transport of cr from root to aerial parts in figure 2. concentration of heavy metals in water (µg.litˉ1). figure 3. concentration of heavy metals in potamogeton crispus tissues (root (first column) and shoot (second column)) (µg g-1 dw). 210 int. j. aquat. biol. (2014) 2(4): 206-214 potamogeton sp. can be described due to being as a non-essential element for plant growth (shewry and peterson, 1974). on the other hand, the high accumulation of cr in some wetland plants depends on the plant ability to decrease toxic cr(vi) to the non-toxic cr(iii) form in roots and then trasmittancy of cr(iii) to the shoots (lytle et al., 1998). the highest bcf was calculated with the average of 1.9×103. also, the translocation factor values ranged between 0.47 and 0.6 in p. crispus. based on tfs, cr transfer ability of p. crispus from root to shoot, implying low transfer rate of chromium due to incompetence in plant transfer system. according to the results a significant (<0.05) positive correlation among cr concentrations in tissues and water was detected. owing to the results, p. crispus seems to act as a proper alternative to reduce negative effects of pollution especially in the cr-based contaminated aquatic environments. arsenic: as is a prevalent metalloid that found in water, atmosphere, minerals and living organisms (adriano, 2001). concentration of as in unpolluted surface water differs from 1 to 10 μg lit-1. in freshwater, the as concentrations were reported in the range of 0.15–0.45 and 2 μg lit-1 (sharma and sohn, 2009; smedley and kinniburgh, 2002). although, aquatic macrophyts have a significant effect on the as uptake, but the presence of as in plants is calculated more than 1 mg kg-1 (sasmaz and obek, 2009). garcía et al. (2010) noticed that as can hardly is removed via direct absorption by plants. in addition, heung lee (2013) mentioned that 0.5–1% of the total as input was accumulated in plant tissues. in the current investigation, the mean concentration of as in water, p. crispus roots and shoots tissues were measured as 5.4 µg lit-1, 2.52 µg g-1 dw and 1.03 µg g-1 dw, respectively. besides, the assayed metal concentrations in plant were more considerable than those of water. the highest bcf was 0.46×103 and tfs values were varied between 0.28 and 0.42. tfs quantities pronounced that as were not transferred from roots to shoots efficiently. moreover, a significant (p<0.001) positive correlation between as concentrations in the p. crispus and water was recognized. according to results, p. crispus does not appear to be a proper aquatic species to omit as. lead: pb is a major pollutant of the environment and absorbed by aerial tissues of plant passively and tightly is bound in root (kabatapendias, 2011). the pb transfer from roots to aerial parts is limited, whereas raskin (1996) described, only 3% of the pb in roots was transferred to the upper tissues. pb is a gradually phytoavailable metal thus, hard to be phytofilter (kabata-pendias, 2011). in spite of above mentioned reports, in present study, the mean concentration of lead in water, p. crispus roots and shoots tissues were detected as 5.13 µg lit-1, 2.68 µg g-1 dw and 6.04 µg g-1 dw, respectively. the most concentration of pb was found in the shoots of p. crispus (6.04 µg g-1 dw). this value were correlated with the pb value of water. as a result, p. crispus was able to meet strong pb concentrations overstepped that of the surrounding substratum. the highest bcfs of pb in the p. crispus was 1.17×103. also, the tfs were in the range of 1.77-2.24. based on the results, the concentration of pb in aerial parts is higher than that of roots, therefore pb transferring from root to aerial part is feasible and furthermore, a well-developed root-rhizome form was not detected in plant species (demirzen and aksoy, 2004; aksoy et al., 2005). a highly significant (p<0.001) positive correlation between pb in p. crispus and water was found. based on the findings, p. crispus seems to be an appropriate species to omit pb from wastewater (favas et al., 2012). cadmium: cadmium is noted as one of the main ecotoxic metals that reveals catastrophic effects on the plants and entire physiological processes of living organisms (kabata-pendias, 2011). although cd is a non-essential element for metabolic processes, it is easily absorbed by both root and leaf. there are proofs that acceptable values of cd is absorbed passively by root and leaf (kabata-pendias, 2011). hence, the linear relationship between cd in plant and water medium was reported (kabata-pendias, 2011). cd values in natural water are normally lower 211 norouznia and hamidian/ phytoremediation efficiency of pondweed in removing heavy metals than 0.001 μg ml-1 and can be reached up to 1.9 µg g-1 in stems and leaves of aquatic plants (friberg et al., 1986; kabata-pendias, 2004). the growth of plants in higher concentrations of cd usually is stopped (wang and zhou, 2005). according to the results, the mean values of cd in the water and p. crispus roots and shoots were recorded as 0.50 µg lit-1, 0.34 µg g-1 dw and 0.23 µg g-1 dw, respectively. the highest cd concentrations in the roots of p. crispus (0.34 µg g-1 dw) indicates that this species is capable to accumulate a high cd concentration. therefore, the highest bcfs were estimated 0.68×103 for the root of p. crispus, indicating its moderate ability to accumulate cd. similarly, the tf recorded between 0.56 and 1.52. tfs reveal that cd was not transferred from roots to leaves efficiently but was absorbed greater than cu, cr and as. a significant (p<0.001) positive correlation between cd concentration and plant and water was observed, therefore p. crispus seems to be an appropriate species to remove cd-derived pollutants from surrounding water effectively. quantification of phytoremediation possibility: evaluation of phytoremediation efficiency is determined using bioconcentration factor (bcfs) and translocation factor (tfs) (pratas et al., 2012; ali et al., 2013). both factors (bcfs and tfs) would help trial to establish a greater degree of accuracy on phytoremediation efficiency. the both factor values are presented in table 1. bioconcentration factor: bcfs demonstrate the capability of plant to uptake heavy metals from surrounding medium, indicating that plants are able to accumulate heavy metals and therefore more appropriate for phytoremediation (pratas et al., 2012). the bio-concentration factor is defined as metal concentration in dry mass in relation to its concentration in external substratum (favas and pratas, 2012). it is calculated as follows (zhuang et al., 2007). bioconcentration factor (bcf) = 𝐶 ℎ𝑎𝑟𝑣𝑒𝑠𝑡𝑒𝑑 𝑡𝑖𝑠𝑠𝑢𝑒𝑠 𝐶 𝑤𝑎𝑡𝑒𝑟(𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑢𝑚) where c harvested tissues is the concentration of the metal in the plant harvested tissue and c water(substratum) is the concentration of the same metal in water (substratum). a bcf value of > 1000 indicates a considerable hyper-accumulation potency of plant (boonyapookana et al., 2002). in present study, the highest bcf value was calculated in p. crispus for copper (2.9×103), showing that p. crispus exhibits acceptable efficiency to decline cu in water. similarly, the highest chromium bcfs with the average of 1.9×103 was reported in this plant species. also, the highest bcfs for pb, cd and as were as 1.17×103, 0.68×103 and 0.46×103, respectively. consequently, p. crispus appears to be a noticeable accumulator in anzali wetland for all mentioned metals except for as. translocation factor: translocation factor explains the capacity of plant in transporting the concentrated metals from root to aerial parts (ali et al., 2013). this factor is defined as the metal concentration in plant shoot in relation to its concentration in plant root (pratas et al., 2012). it is calculated as follows (padmavathiamma and li, 2007): translocation factor (tf) = 𝐶 𝑠ℎ𝑜𝑜𝑡 𝐶 𝑟𝑜𝑜𝑡 where c shoot is the concentration of metal in plant shoots and c root is its concentration in plant roots. the tf with higher valuse of 1, imply the high potency of plant metal transport systems (zhao, 2002). also, kabata-pendias and pendias (2000) reported that the translocation factor in the range of 0.01-1 indicates the moderate bioavailiblity and accumulation of metals in aerial tissues of plants. in the present study, the mean values of tf in the p. crispus were calculated lower than 1 for cu, as, cr due to dysfunction in metal transmittancy operations (sasmaz et al., 2008). the mean tfs in the plant evaluated higher than1 for pb and relatively cd. the highest tfs refer to p. crispus (2.24) for pb, due to high transmittancy of pb from roots to leaves actively. the mean tfs were presented in a descending order as pb, cd, cu, cr and as. this results confirmed that above-mentioned heavy metals were not transferred from roots to aerial parts efficaciously except for pb and partly cd. general conception: many works have been 212 int. j. aquat. biol. (2014) 2(4): 206-214 conducted to assess the bioaccumulation of heavy metals using aquatic macrophyts (ye et al., 1997; robinson et al., 2003; pajevic et al., 2008; carranzaalvarez et al., 2008; sasmaz et al., 2008; alonsocastro et al., 2009; bonanno and giudice, 2010; pratas et al., 2012; favas et al., 2012; hamidian et al., 2014). also, rai (2008) has been emphasized to introduce p. crispus as proper metal accumulator. ali et al. (1999) noticed the prominence of p. crispus to phytoremediate cu, cr, pb and zn. demirzen and aksoy (2004) investigated remediation potential of potamogeton sp. to accumulate cd, pb, cr, ni, zn and cu in wetlands. furthermore, fritioff and greger (2006) recognized p. crispus ability to participate on the heavy metals phytoremediation process. moreover, pajevic et al. (2008) pronounced that potamogeton sp. can be used as reliable accumulators for heavy metals (fe, mn and cd) pollution. numerous factors may influence on the concentration-dependent variation of heavy metals in the plant species such as total concentration of metals in aquatic environment, the wide scope of metal species, various metal mechanisms and movability, and also plant–water interface. in addition, several physiochemical factors and physiological features such as water depth, water overflow, natural attributes of heavy metals, water ph, organic compound volume and biological characteristic of each plant species are used to determine whether a particular heavy metal is likely to be accumulated (caranz-alvarez et al., 2008). conclusion macrophyts are the key elements of wetland ecosystems. they not only uptake heavy metals, but also exhibit inherent strength to clean up surrounding water by absorbing of increasing pollutants particularly heavy metals (jenssen et al., 1993). in addition, the highlighted functions of wetland macrophyts on phytoremediation and bioaccumulation assist to achieve a profound binary role (rai et al., 2008). eventually, this study showed a concentration-bound accumulation of cu, cr, pb, as and cd in p. crispus tissues. namely, the metals concentrations in plant species was increased in a linear model along with increase in the water. the inspected positive correlation between metal concentrations in p. crispus and water, revealed its valuable role to remove metallic pollutants. the root of p. crispus is more adapted to concentrate metals than aerial parts, due to confined metal transporting system of plant. by returning to the objectives, it is now possible to state that p. crispus is an important qualified representative to meet the phytoremediation needs in the polluted substratum of anzali wetland by cu, cr, cd, pb and as, respectively. acknowledgment the authors are sincerely grateful to the anonymous reviewers for their profitable comments on the manuscript. references adriano d. 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(2015) 3(2): 78-88 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article ecosystem diversity of cladocera (crustacea: branchiopoda) of the floodplain lakes of majuli river island, the brahmaputra river basin, northeast india bhushan kumar sharma*,1mrinal kumar hatimuria, sumita sharma freshwater biology laboratory, department of zoology, north-eastern hill university, shillong 793 022, meghalaya, india. article history: received 12 january 2015 accepted 18 february 2015 available online 2 5 april 2015 keywords: beels abiotic factors composition interesting taxa richness similarities abstract: plankton and semi-plankton samples collected from twelve floodplain lakes (beels) of majuli river island of the brahmaputra river basin, upper assam reveal rich cladocera assemblage of 48 species belonging to 32 genera and 7 families. this report assumes biodiversity value as ~65.0% and ~37.0% of the species, and ~78.0% and ~72.0 of genera of the taxon known from assam state of northeast india (nei) and india, respectively. picripleuroxus quasidenticulatus (smirnov) is a new record from the indian sub-region. biogeographically important elements include one australasian, three indo-chinese and two oriental species. total cladoceran richness in individual beels ranged between 16-38 (26 ± 6) species while monthly and seasonal richness in six beels each varied between 8 ± 3-13 ± 3 species and 11 ± 4-17 ± 3 species, respectively and showed lack of any pattern of temporal variations. the community similarities (40.1-86.5% vide sørensen’s index) and the hierarchical cluster analysis affirm heterogeneity in cladocera composition in different beels. individual abiotic factors indicated insignificant influence on richness except for significant positive correlation with alkalinity only in khorkhoria beel. introduction freshwater cladocera have been documented from distant parts of india since the initial study by baird (1860) but little is yet known about their ecosystem diversity in inland environs of this country (sharma, 2010). some notable studies on the latter aspect (sharma and sharma, 2008, 2009, 2010, 2014) are limited to the floodplain lakes (beels or pats) of northeast india (nei) while fewer other ad-hoc studies elsewhere from india are riddled with incomplete species inventories for meaningful analysis. the present study on cladocera assemblages of twelve beels of majuli, the largest river island of the world situated in the upper reaches of the river brahmaputra in upper assam, thus deserves ecology value. we provide an inventory of the examined cladoceran species from different beels of this * corresponding author: bhushan kumar sharma e-mail address: profbksharma@gmail.com geographically interesting landform of fluvial geomorphology faced with extinction due to alarming rate of erosion by the brahmaputra flood waters. comments are made on their composition and richness, interesting elements, community similarities and influence of abiotic parameters. the present report is of biodiversity interest and for following meta-analyses on the cladoceran fauna of majuli studied earlier vides sharma and sharma (2014). materials and methods this study is based on water and plankton samples collected, during september, 2010-august, 2012, from twelve floodplain lakes (beels) of majuli river island (93°-95°e, 25°-27°n), upper assam (fig. 1, table 1). the sampled beels possessed different aquatic macrophytes’ namely eichhornia crassipes, 79 sharma et al./ cladocera of the floodplain lakes of majuli river island hydrilla verticellata, utricularia flexuosa, trapa natans, lemna major, l. minor, pistia striates, salvinia sp., nymphaea spp., nymphoides spp., potamageton spp., azolla pinnata, euryale ferox, and sagittaria sp. the collections were obtained monthly from six beels (marked with*) and seasonally from rest of the beels (table 1). water samples were examined for abiotic parameters, including water temperature, specific conductivity and ph were recorded by the field probes; do was estimated by winkler’s method while free co2, total alkalinity and total hardness were analyzed following apha (1992). the qualitative plankton samples were collected by figure 1. district map of assam state indicating location of majuli river island (insert map of india indicating assam state of northeast india). parameters↓ beels→ bhereki* ghotonga* holmari* chela* chakuli* khorkhoria* latitude 26°57′09.1″n 27°01′52.7″n 26°59′17.3″n 27°04′58.2″ n 26°56′40.3″n 26° 56′47.4″n longitude 94°12′23.0″e 94°15′28.7″e 94°12′30.6″e 94°17′51.9″ e 94°09′01.9″e 94°12′28.8″e altitude 67 m asl 73 m asl 75 m asl 89 m asl 69 m asl 74 m asl water temperature o c 23.7 ± 1.7 23.9 ± 1.7 23.6 ± 1.7 23.4 ± 1.9 23.6 ± 1.9 23.9 ± 1.8 ph 6.67 ± 0.23 6.51 ± 0.16 6.87 ± 0.13 7.04 ± 0.19 6.82 ± 0.18 6.80 ± 0.24 sp. conductivity µs/cm 140.7 ± 24.4 121.4 ± 26.8 173.6 ± 32.5 210.4 ± 41.3 180.8 ± 37.8 172.1 ± 44.4 dissolved oxygen mg/l 6.3 ± 0.9 6.2 ± 1.0 7.1 ± 0.8 7.8 ± 0.7 6.2 ± 0.8 6.4 ± 1.2 free co2 mg/l 13.6 ± 4.0 13.8 ± 3.4 10.2 ± 2.8 10.3 ± 2.4 14.8 ± 4.6 13.9 ± 5.0 total alkalinity mg/l 70.3 ± 20.7 62.2 ± 13.4 92.3 ± 14.2 113.5 ± 24.6 105.8 ± 29.0 90.2 ± 29.9 total hardness mg/l 69.8 ± 20.3 60.8 ± 13.6 89.3 ± 16.9 113.0 ± 23.8 104.0 ± 26.2 88.8 ± 27.2 parameters↓ beels→ doriya dubori tuni baatomaari jur chereki latitude 26°57′27.7″n 26°57′01.9″n 26° 58′35.3″n 26°59′25.9″n 26°59′45.3″n 26°58′25.4″n longitude 94°10′02.4″e 94°16′13.8″e 94°15′57.8″e 94°13′08.0″e 94°14′34.4″e 94°10′38.7″e altitude 70 m asl 70 m asl 67 m asl 71 m asl 71 m asl 67 m asl water temperature o c 24.2 ± 2.3 24.1 ± 1.9 23.9 ± 2.1 24.0 ± 1.9 23.9 ± 2.5 24.2 ± 1.7 ph 6.70 ± 0.32 6.61 ± 0.19 6.69 ± 0.14 6.87 ± 0.13 6.71 ± 0.14 6.62 ± 0.21 sp. conductivity µs/cm 110.2 ± 20.8 132.4 ± 18.6 123.6 ± 23.0 114.2 ± 20.5 130.8 ± 24.6 128.2 ± 33.2 dissolved oxygen mg/l 5.8 ± 1.2 7.0 ± 0.9 5.1 ± 1.8 6.2 ± 0.9 5.9 ± 1.1 6.1. ± 1.0 free co2 mg/l 12.0 ± 5.2 11.8 ± 4.2 12.2 ± 1.9 11.4 ± 1.9 12.7 ±3.5 12.1 ± 3.6 total alkalinity mg/l 67.3 ± 12.2 72.2 ± 11.4 82.3 ± 12.3 91.5 ± 16.2 88.9 ± 12.9 90.8 ± 16.6 total hardness mg/l 62.8 ± 12.6 70.8 ± 10.6 79.1 ± 15.6 89.0 ± 12.8 81.4 ± 12.0 86.9 ± 17.0 * sampled monthly; the rest sampled during winter (december/january), pre-monsoon (march-may), monsoon (juneaugust) and post-monsoon (september-october) table 1. the sampled floodplain lakes (beels) and their abiotic parameters (mean ± sd) (after sharma et al., 2015). 80 int. j. aquat. biol. (2015) 3(2): 78-88 towing a plankton net (#50 µm) from the littoral, limnetic/semi-limnetic regions of different beels, preserved in 5% formalin and were screened with a wild-stereoscopic binocular microscope. various cladocerans and their disarticulated appendages were mounted in polyvinyl alcohol-lactophenol mixture, and observed with a leica (dm 1000) stereoscopic phase contrast microscope fitted with an image analyzer. cladocera species were identified following smirnov (1971, 1976, 1992, 1996), michael and sharma (1988), korovchinsky (1992), sharma and sharma (1999), orlova-bienkowskaja (2001), and sharma and sharma (2008, 2013). the percentage similarities between cladoceran communities of different beels were calculated vide sørensen’s index (sørensen, 1948) and their hierarchical cluster analysis was performed using spss (version 20). ecological relationships between abiotic factors and rotifer richness were determined by pearson’s correlation coefficients (r); their p values were calculated vide http://faculty.vassar.edu/ lowry/tabs.html and significance was ascertained after use of bonferroni corrections. the reference collections were deposited in the holdings of freshwater biology laboratory, department of zoology, north-eastern hill university, shillong. results the details of the sampled beels and their basic abiotic parameters (mean ± sd) are indicated in table 1. water temperature ranged between 23.4 ± 1.9-24.2 ± 2.3°c, ph between 6.51 ± 0.16-7.04 ± 0.19, specific conductivity between 110.2 ± 20.8210.4 ± 41.3 µs/cm, do between 5.1 ± 1.8-7.8 ± 0.7 mg/l; free co2 between 10.3 ± 2.4-14.8 ± 4.6 mg/l; total alkalinity between 62.2 ± 13.4-113.5 ± 24.6 mg/l; and total hardness between 60.8 ± 13.6-113.0 ± 23.8 mg/l. a total of 48 cladocera species belonging to 32 genera and 7 families are documented. picripleuroxus quasidenticulatus (smirnov) is a new addition (fig. 2, a-c) to the indian cladocera. the species composition of the taxon of sampled beels is indicated in appendix i. total richness in individual beels ranged between 16-38 (26 ± 6) species and recorded 40.1-86.5% community similarities vide sørensen’s index (table 2). the monthly and seasonal richness in six beels each varied between 8 ± 3-13 ± 3 species and 11 ± 4-17 ± 3 species, respectively. the hierarchical analysis between cladocera assemblages in different beels is shown in figure 3. discussion the sampled beels are characterized by slightly acidic to circum-neutral, ‘moderately hard’ to ‘hard’ well-oxygenated waters with ‘bicarbonate alkalinity’ and occurrence of free co2, and are notable for their low specific conductivity (refer sharma et al., 2015). the last salient feature, indicating low ionic concentrations, warrants inclusion of these floodplain lakes under ‘class i’ category of trophic classification vide talling and talling (1965). forty-eight species of cladocera, spread over 32 genera and seven families, observed in our plankton and semi-plankton collections from twelve beels of majuli river island reveal rich and diverse figure 2. picripleuroxus quasidenticulatus (smirnov, 1996), parthenogenetic female, (b) head, parthenogenetic female, (c) postabdomen, parthenogenetic female. 81 sharma et al./ cladocera of the floodplain lakes of majuli river island assemblage of the taxon in south asia. total richness is of biodiversity value as ~65.0% and ~37.0% of the species, and ~78.0% and ~72.0 of genera of the taxon known from assam state (nei) and india, respectively. this report affirms the hypothesis of sharma and sharma (2008, 2013, 2014) on the floodplains of the brahmaputra basin to be cladocera rich habitats of the indian sub-region. though based on observations from much limited geographical location, these results merit biodiversity value in light of a conservative estimate of occurrence of up to 60-65 cladoceran species from tropical and subtropical parts of the india subcontinent (fernando and kanduru, 1984; sharma and michael, 1987). picripleuroxus quasidenticulatus (smirnov) is a new record from india; this erstwhile australasian species is now known (sinev and sanoamuang, beels 1 2 3 4 5 6 7 8 9 10 11 12 1 68.1 60.5 68.4 77.8 54.5 58.6 54.5 63.6 62.5 64.0 59.6 2 72.0 70.8 69.8 82.3 64.6 58.1 62.7 69.1 63.1 59.3 3 74.4 73.8 72.3 59.0 48.3 59.6 54.9 56.6 40.1 4 86.5 84.4 57.6 71.1 57.8 61.2 62.7 50.0 5 75.0 48.1 54.9 55.0 59.1 60.9 46.5 6 61.3 67.8 58.3 65.4 66.7 54.9 7 82.2 64.5 63.6 79.4 76.9 8 64.4 66.7 76.9 71.0 9 69.2 66.7 62.7 10 75.9 61.8 11 66.7 12 1-bhereki, 2-ghotonga, 3-holmari, 4-chakuli, 5-chela, 6-khorkhoria, 7-doriya, 8-dubori, 9tuni, 10-baatomari, 11-jur, 12-chereki table 1. percentage similarities between cladocera assemblages of majuli beels. figure 3. hierarchical cluster analysis of cladocera assemblages of majuli beels. 82 int. j. aquat. biol. (2015) 3(2): 78-88 2013) from thailand, vietnam and the far east of russia. the present study further extends its distribution to the indian sub-region within the oriental region. further, in consideration of this record, we suggest re-examination of all earlier reports of the congener p. denticulatus (birge) from india. the biogeographically important elements include the australasian disperalona caudata; three indo-chinese species namely alona cheni, a. kotovi and chydorus angustirostris; and two oriental endemics: celsinotum macronyx and kurzia (rostrokurzia) brevilabris. of these, d. caudata is an important link between the cladocera faunas of nei, southeast asia and australia while the rest endorse affinity of nei cladocera with se asia (sharma and sharma, 2007, 2014). of these, a. kotovi and c. angustirostris are recent additions (sharma and sharma, 2014) to the cladoceran faunas of india and nei, respectively; sinev and korovchinsky (2013) extended the distribution of the latter as well as erstwhile indian endemic to vietnam imparting it an indo-chinese character. total cladocera richness is distinctly higher than the reports of only 11 species from two floodplain lakes (khan, 1987) of kashmir; 9 species from 65 wetlands of 24-parganas district (nandi et al., 1993) of west bengal; 4 species (sinha et al., 1994) and 12 species (sanjer and sharma,1995) from the floodplains of bihar; 14 species from 37 floodplain lakes (sarma, 2000) of assam, 36 species from 20 wetlands from the floodplains of south-eastern west bengal (khan, 2003) and 30 species from 30 wetlands of keoladeo national park (venkataraman, 1992). we, however, caution against over-emphasis on comparisons with poor richness of certain reports because of inadequate sampling or incomplete species inventories. cladocera assemblages of individual beels recorded 40.1-86.5% community similarities (vide sørensen’s index) with ~67% instances indicating 51-70% similarities and <70% similarity only in ~24.0% instances in the matrix. this generalization suggests relatively lower similarity and more heterogeneity in species composition of these micro-crustaceans amongst different beels. the former is in contrast to higher similarities reported in certain floodplain lakes of assam (sharma and sharma, 2008; sharma and sharma, 2008, 2013) and manipur (sharma and sharma, 2009, 2010). peak similarity is observed between chakuli and chela beels while a lowest value is noticed between holmari and chereki beels. the hierarchical cluster analysis endorses closeness in cladoceran communities of the first two beels. on the contrary, bhereki reflected most divergence in its species composition and is followed by tuni, chereki and holmari in the stated order. total cladoceran richness in individual majuli beels ranged between 16-38 (26 ± 6) species; maximum richness in doriya beel is followed by 35 and 30 species in dubori and jur beels, respectively, while six beels in all recorded more than average number of species. in general, our report broadly concurs with total richness of 15 beels (21-29 species) of assam (sharma and sharma, 2008) and 14 pats (2131 species) of manipur but notwithstanding the report of 51 species from loktak lake a ramsar site (sharma and sharma, 2010). the monthly and seasonal richness in six beels each varied between 8 ± 3-13 ± 3 species and 11 ± 4-17 ± 3 species, respectively and showed lack of any pattern of temporal variations; the former is attributed to growth of eichhornia crassipes affirming the recent remarks on rotifera of these floodplains (sharma et al., 2015). referring to the influence of individual abiotic factors, our report of significant positive correlation with alkalinity only in khorkhoria beel (r = 0.561, p = 0.0022) is in contrast to their importance noticed earlier from assam (sharma and sharma, 2010; sharma and sharma, 2008) and manipur (sharma and sharma, 2010). the present observations affirm remarks of sharma and sharma (2014) on occurrence of weedassociated biota in general and member of the family chydoridae (62% of total richness), macrothricidae and sidiidae in particular, common occurrence of macrothrix spp., simocephalus mixtus and 83 sharma et al./ cladocera of the floodplain lakes of majuli river island guernella raphaelis, a relative paucity of members of the bosminidae and moinidae and lack of daphnia. the common occurrence of the members of the former three families is attributed to the prevalence of the littoral-periphytonic conditions; 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(1971). chydoridae of the world fauna. fauna sssr. rakoobraznie, 1(2): 1-531 (in russian). smirnov n.n. (1976). macrothricidae and moinidae of the world fauna. fauna sssr, novaya seriya. rakoobraznye, 1(3): 1-237 (in russian). smirnov n.n. (1992). the macrothricidae of the world. in: guides to the identification of the microinvertebrates of the continental waters of the world. 1: 1-143. spb academic publishers, the hague. smirnov n.n. (1996). cladocera: the chydorinae and sayciinae (chydoridae) of the world. in: guides to the identification of the microinvertebrates of the continental waters of the world, 11: 1-197. spb academic publishers, the hague. sørensen t. (1948). a method of establishing group of equal amplitude in plant sociology based on similarity of species content and its application to analyze the vegetation of danish commons. biologiske skrifter, 5: 1-34. talling j.f., talling i.b. (1965). the chemical composition of african lake waters. internationale revue der gesamten hydrobiologie, 50: 421-463. van damme k., maiphae s., sa-ardrit p. (2013). inland swamps in south east asia harbor hidden cladoceran diversities: species richness and the description of new paludal chydoridae (crustacea: branchiopoda: cladocera) from southern thailand. journal of limnology, 72 (2): 174-208. venkataraman k. (1992). i. cladocera of keoladeo national park, bharatpur and its environs. journal of bombay natural history society, 89 (1): 17-26. 85 sharma et al./ cladocera of the floodplain lakes of majuli river island order: ctenopoda family: sididae 1. diaphanosoma excisum sars, 1885 2. diaphanosoma sarsi richard, 1895 3. diaphanosoma senegal gauthier, 1951 4. pseudosida szalayi (daday, 1898) 5. sida crystallina (o. f. muller, 1776) order: anomopoda family: daphniidae 6. ceriodaphnia cornuta sars, 1885 7. scapholeberis kingi sars, 1901 8. simocephalus (echinocaudus) acutirostratus (king, 1853) 9. simocephalus (coronocephalus) serrulatus (koch, 1841) 10. simocephalus (simocephalus) mixtus sars, 1903 family: bosminidae 11. bosmina longirostris (o. f. muller, 1776) s. lato 12. bosminopsis deitersi richard, 1895 family: moinidae 13. moina micrura kurz, 1874 14. moinodaphnia macleayi (king, 1853) family: macrothricidae 15. macrothrix laticornis (fischer, 1857) 16. macrothrix triserialis (brady, 1886) 17. guernella raphaelis richard, 1892 18. grimaldina brazzai richard, 1892 family: ilyocryptidae 19. ilyocryptus spinifer herrick, 1882 family: chydoridae subfamily: chydorinae 20. alonella (alonella) clathratula sars, 1886 21. alonella (alonella) excisa (fischer, 1854) 22. chydorus angustirostris frey, 1987 23. chydorus sphaericus (o. f. muller, 1776) s.lat 24. chydorus ventricosus daday, 1898 25. dadaya macrops (daday) 26. disperalona caudata smirnov, 1996 27. dunhevedia crassa king, 1853 28. dunhevedia serrata daday, 1898 29. ephemeroporus barroisi (richard, 1894) 30. picripleuroxus quasidenticulatus (smirnov, 1996)* 31. picripleuroxus similis vavra, 1900 subfamily: aloninae 32. alona cheni sinev, 1999 33. alona guttata tuberculata kurz, 1875 34. alona kotovi sinev, 2012 35. anthalona harti van damme et al. 36. camptocercus uncinatus smirnov, 1973 37. celsinotum macronyx (daday, 1898) 38. celsinotum rectangula (sars, 1862) s.lat 39. euryalona orientalis (daday, 1898) 40. graptoleberis testudinaria (fischer, 1854) 41. karualona karua (king, 1853) 42. kurzia (kurzia) latissima kurz, 1874 appendix i: systematic list of examined cladocera taxa super-class: crustacea class: branchiopoda super-order: cladocera (sensu strictu) ----------------------------------------------------------------------------------------------------------------------------------------------- * new record from india 86 int. j. aquat. biol. (2015) 3(2): 78-88 43. kurzia (rostrokurzia) brevilabris rajapaksa & fernando,1986 44. kurzia (rostrokurzia) longirostris (daday, 1898) 45. leberis diphanus (king, 1853) 46. leydigia acanthocercoides (fischer, 1854) 47. notoalona globulosa (daday, 1898) 48. oxyurella singalensis (daday, 1898) 87 sharma et al./ cladocera of the floodplain lakes of majuli river island super-class: crustacea class: branchiopoda super-order: cladocera (sensu strictu) taxa↓ beels sr. no.→ 1 2 3 4 5 6 7 8 9 10 11 12 order: anomopoda family: bosminidae 1. bosmina longirostris (o. f. muller) + + + + + 2. bosminopsis deitersi richard + + family: chydoridae subfamily: aloninae 3. alona affinis (leydig) s.lat + + + + 4. alona cheni sinev + + + + + + + + 5. alona guttata tuberculata kurz + + + + + + + + 6. alona kotovi sinev + + + + 7. anthalona harti van damme et al. + + + 8. camptocercus uncinatus smirnov + + + + + + + + + 9. celsinotum macronyx (daday) + + + + 10. celsinotum rectangula (sars) s.lat + + + + + + 11. euryalona orientalis (daday) + + + + + + + + + + 12. graptoleberis testudinaria (fischer) + + + + 13. karualona karua (king) + + + + + + + + + + + + 14. kurzia brevilabris rajapaksa & fernando + + 15. kurzia latissima kurz + + + + 16. kurzia longirostris (daday) + + + + + + + + + + 17. leberis diphanus (king) + + 18. notoalona globulosa (daday) + + + + + + + + + + + + 19. oxyurella singalensis (daday + + + + + + subfamily: chydorinae 20. alonella clathratula sars + + + + + + + + 21. alonella excisa (fischer) + + + + + + + + + + + + 22. chydorus angustirostris frey + + + + + + + + + 23. chydorus sphaericus (o. f. muller) + + + + + + + + + + + + 24. chydorus ventricosus daday + + + + + + + 25. dadaya macrops (daday) + + + 26. disperalona caudata smirnov + + + + + + 27. dunhevedia crassa king + + + + + + 28. dunhevedia serrata daday + + + + + + 29. picripleuroxus quasidenticulatus (smirnov)* + + + 30. picripleuroxus similis + + + + + + + + + + + + family: daphniidae 31. ceriodaphnia cornuta sars + + + + + + + + 32. scapholeberis kingi sars + + + + + + 33. simocephalus acutirostratus (king) + + + 34. simocephalus serrulatus (koch) + + + + + + + + 35. simocephalus mixtus sars + + + + + + + + + + + appendix ii: species composition of cladocera of beels of majuli river island. 88 int. j. aquat. biol. (2015) 3(2): 78-88 appendix ii: species composition of cladocera of beels of majuli river island (contd.) taxa↓ beels sr. no.→ 1 2 3 4 5 6 7 8 9 10 11 12 family: ilyocryptidae 36. ilyocryptus spinifer herrick + + family: macrothricidae 37. grimaldina brazzai richard + + 38. guernella raphaelis richard + + + + + 39. macrothrix laticornis (fischer) + + + + + + + + + + + + 40. macrothrix spinosa king + + + + 41. macrothrix triserialis (brady) + + + + + + + + + + + + family: moinidae 42. moina micrura kurz + + + + + 43. moinodaphnia macleayi (king) + + + + + order: ctenopoda family: sididae 44. diaphanosoma excisum sars + + + + + + + + + + + + 45. diaphanosoma sarsi richard + + + + + + + 46. diaphanosoma senegal gauthier + + 47. pseudosida szalayi (daday) + + + + + 48. sida crystallina (o. f. muller) + + + + + total richness (species) 20 27 23 21 16 24 38 35 24 28 30 27 1-bhereki, 2-ghotonga, 3-holmari, 4-chakuli, 5-chela, 6-khorkhoria, 7-doriya, 8-dubori, 9-tuni, 10-baatomari, 11-jur, 12-chereki * new records from india int. j. aquat. biol. (2016) 4(2): 69-79; doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2016 iranian society of ichthyology original article loktak lake, manipur, northeast india: a ramsar site with rich rotifer (rotifera: eurotatoria) diversity and its meta-analysis bhushan kumar sharma*,1telsing paongam haokip, sumita sharma freshwater biology laboratory, department of zoology, north-eastern hill university, shillong-793 022, meghalaya, india. article history: received 9 november 2015 accepted 4 january 2016 available online 2 5 april 2016 keywords: composition distribution interesting species new records richness wetland abstract: a total of 162 species (s) of rotifera belonging to 40 genera and 20 families examined from loktak lake, an important floodplain lake of northeast india (nei) that is one of the richest assemblages of the taxon known from the indian sub-region. it merits biodiversity value as ~40.0% and ~62.0% of species recorded from india and nei, respectively. one species is new to india, 23 species are new to manipur and 14 species are new to loktak basin. biogeographically interesting elements included three australasian, five oriental, ten palaeotropical and one cosmo-subtropical species. lecanidae > lepadellidae > brachionidae > trichocercidae collectively comprised 65.4% of s; lecane > lepadella > trichocerca are diverse genera; and paucity of brachionus spp. is distinct. loktak rotifera indicated importance of cosmopolitan, the littoral-periphytonic and small-sized species, and ‘tropical character’. anova recorded significant variations of the rotifer richness amongst three sampling sites of loktak during june 2010–may 2012 survey. the richness followed osscillaring monthly variations and indicated lack of significant influence of any individual abiotic parameter at all three stations. introduction segers et al. (1993) hypothesized tropical and subtropical floodplain lakes to be globally rich habitats for the rotifer diversity. sharma and sharma (2014a, 2014b) extended this hypothesis to the floodplains lakes (beels) of the brahmaputra river basin of northeast india (nei) with deepor beel, a ramsar site and an important wetland of this basin, as one of the globally interesting rotifera habitat (sharma and sharma, 2015). realizing biodiversity value of the floodplain lakes of nei vis-a-vis the role of extensive sampling, we undertook meta-analysis of rotifera diversity of loktak lake, a ramsar site of india and another important floodplain lake of nei, based on recent collections and our earlier reports (sharma, 2007, 2009a). an inventory of 162 species recorded till date from this floodplain lake (pat) of manipur is presented and interesting species are illustrated. the * corresponding author: bhushan kumar sharma doi: http://dx.doi.org/10.7508/ijab.2016.02.001 e-mail address: profbksharma@gmail.com nature and composition of the rotifer diversity are discussed with remarks on richness, new records and species of global and regional distribution value. this study merits importance for ecosystem diversity, biogeography and for following metaanalysis of the rotifer diversity of this second well sampled freshwater ecosystem of india. materials and methods the present study is a part of limnological survey undertaken (june 2010 may 2012) and collections on several occasions during 2013-2015 from loktak lake (93°46'–93º55'e, 24º25'–24º42'n; area: 286 km2; max. depth: 4.58 m, mean depth: 2.07 m) located in bishnupur / imphal districts of manipur state (nei). this wetland is characterized by floating mats of vegetation called “phumdi” which are inhabited by an endangered brow-antlered deer (rucervus eldi eldi). the common aquatic plants of 70 sharma et al./ rotifer diversity of loktak lake, manipur, northeast india this ramsar site included ceratophyllum demersum, eichhornia crassipes, pistia stratiotes, salvinia cucullata, savinia natans, euryale ferox, hydrilla verticillata, nymphoides cristatum, trapa natans, potamogeton crispus, alternanthera philoxeroides, rumex nepalensis, ipomoea arguta, polygonum pulchrum, utricularia sp., valllissnaria sp., oryza rufipegon, spiranthus sinesis and nelumbo nucifera. water samples and qualitative plankton were collected, at regular monthly intervals during june 2010 may 2012 limnological survey, at three sampling sites namely loktak a (93°45'56.3''e; 24°32'13.5''n; alt. 726 m asl), loktak b (93°47'58.1''e; 24°30'39.1''n; alt. 714 m asl) and loktak barrage (93°45'43.5''e; 24°32'46.9''n; alt. 718 m asl). in addition, qualitative plankton samples were collected from different parts of loktak basin during the study period. water temperature, specific conductivity and ph were recorded with field probes; dissolved oxygen was estimated by winkler’s method; and total alkalinity, total hardness, calcium and chloride were analyzed following apha (1992). the plankton samples were collected by towing a nylobolt plankton net (#50 μm) and preserved in 5% formalin; aquatic vegetation was disturbed before each sampling to facilitate collection of planktonic and semi-planktonic rotifers. individual collections were screened with a wild stereoscopic binocular microscope; the rotifers were isolated and mounted in polyvinyl alcohol-lactophenol, and observed with leica (dm 1000) stereoscopic phase contrast microscope fitted with an image analyzer. the measurements were given in micrometers (μm). the rotifer taxa were identified following koste (1978), segers (1995), sharma (1983, 1998a), sharma and sharma (1997, 1999, 2000, 2008, 2013). the rotifer community similarities were calculated vide sørensen’s index (sørensen, 1948) and two-way anova was used to analyze the significance of temporal variations of richness. ecological relationships between abiotic factors and the rotifer richness were determined by pearson’s correlation coefficients (r); p values were calculated vide http://faculty.vassar.edu/ lowry/tabs.html and significance was ascertained after use of bonferroni corrections. results the variations (ranges, mean ±sd) of the recorded abiotic factors are presented in table 1 in comparison with the report of sharma (2009a). water temperature at three sampling stations of loktak lake varied between 22.6±3.2 – 23.4±3.4°c, ph ranged between 6.8±0.2 – 7.0±0.3 and specific conductivity varied between 80.9±21.7 – 113.1±24.1 µs cm-1 while dissolved oxygen and free carbon dioxide fluctuated between 4.6±2.0 – 6.0±2.7 mg l-1 and 8.8±3.1 13.5±5.6 mg l-1, respectively. total alkalinity, total hardness, calcium and chloride ranged between 65.2±16.5–72.8±24.7 mg l-1; 51.0± 12.8 73.1±17.9 mg l-1; 33.7±9.8 38.1±9.6 mg l-1 and 19.3±5.5 23.7±6.2 mg l-1 between three sampling stations of loktak, respectively. we report a total of 162 species spread over 40 genera and 20 families from loktak lake (appendix june 2010 – may 2012 (present study) sharma (2009a) parameters↓ stations→ loktak a loktak b loktak barrage nov. 2002-oct. 03 mean ± sd mean ± sd mean ± sd mean ± sd water temperature (°c) 23.1±3.0 22.6±3.2 23.4±3.4 21.4 ± 4.0 ph 6.9±0.3 6.8±0.2 7.0±0.3 6.38 ± 0.23 specific conductivity (µs cm-1) 80.9±21.7 86.1±21.7 113.1±24.1 98.9 ± 19.7 free carbon dioxide (mg l -1) 10.6±4.3 13.5±5.6 8.8±3.1 9.5 ± 2.1 dissolved oxygen (mg l-1) 5.2±1.6 4.6±2.0 6.0±2.7 6.2 ± 1.1 total alkalinity (mg l-1) 65.2±16.5 72.8±24.7 72.6±12.3 16.0 ± 4.4 total hardness (mg l-1) 51.0±12.8 59.2±18.4 73.1±17.9 38.1 ± 8.2 calcium (mg l-1) 33.7±9.8 37.4±18.9 38.1±9.6 8.9 ± 3.0 chloride (mg l-1) 23.7±6.2 23.4±7.8 19.3±5.5 14.9 ± 3.1 table 1. variations in certain abiotic factors of loktak lake. 71 int. j. aquat. biol. (2016) 4(2): 69-79 1) and interesting taxa are illustrated; of these, 152 species are observed in 2010-2015 collections. further, 135 species are observed at three sampling stations namely loktak a, loktak b and loktak barrage during june 2010 may 2012 survey. the rotifer richness (figs. 1, 2) ranged between 44-79 (60±8), 46-72 (58±7) and 39-67 (50±8) species at three stations, respectively and recorded 73.2-78.5% community similarities vide sørensen’s index. peak richness is observed during december 2011, november 2011 and january 2012 at loktak a, loktak b and loktak barrage, respectively. mytilina lobata (fig. 3a) is a new record to the indian rotifera. brachionus caudatus, b. durgae, b. kostei (fig. 3b), e. meneta, keratella tecta, lecane aspasia (fig. 3c), l. bifurca, l. rhenana (fig. 3d), l. rhytida (fig. 3e), l. undulata, lepadella minuta, l. quadricarinata (fig. 3f), l. quinquecostata (fig. 3g), mytilina acanthophora (fig. 3h), m. michelangellii (fig. 3i), trichocerca edmondsoni (fig. 3j), t. hollaerti (fig. 3k), t. maior (fig. 3l), t. scipio, t. weberi (fig. 3m), sinantherina figure 1. species richness of loktak rotifera (2010-2011). figure 2. species richness of loktak rotifera (2011-2012). 72 sharma et al./ rotifer diversity of loktak lake, manipur, northeast india semibullata, testudinella amphora (fig. 3n) and wolga spinifera (fig. 3o) are new records from manipur. brachionus bidentatus, epiphanes brachionus, e. incisa, k. lenzi, lecane aeganea, l. arcula, l. bulla diabolica, l. haliclysta, l. pusilla, l. thienemanni, lepadella desmeti, l. triba, platyias leloupi and trochosphaera aequatorialis are new records from loktak lake. euchlanis semicarinata, figure 3. (a) mytilina lobata pourriot (lateral view), (b) brachionus bennini leissling (dorsal view), (c) lecane aspasia myers (dorsal view), (d) lecane rhenana hauer (dorsal view), (e) lecene rhytida harring & myers (dorsal view), (f) lepadella quadricarinata (stenroos) (ventral view), (g) lepadella quinquecostata (lucks) (dorsal view), (h) mytilina acanthophora hauer (lateral view), (i) mytilina michelangellii reid & turner (lateral view), (j) trichocerca edmondsoni (myers) (ventral view), (k) trichocerca hollaerti de smet (lateral view), (l) trichocerca maior hauer (lateral view), (m) trichocerca weberi (jennings) (lateral view); (n) testudinella amphora hauer (dorsal view), and (o) wolga spinifera (western) (dorsal view) . 73 int. j. aquat. biol. (2016) 4(2): 69-79 filinia brachiata, floscularia ringens, habrotrocha angusticollis, lecane acanthinula and l. solfatara, macrochaetus danneelae, rotaria macroceros and r. tardigrada are not observed in 2010-2015 collections. discussion water temperature affirmed sub-tropical nature of loktak lake concurrent with its location. the slightly acidic-slightly alkaline, marginally hard and calcium-poor waters are characterized by low ionic concentration as indicated by specific conductivity at all three sampling stations; the latter warranted their inclusion under ‘class i’ category of trophic classification vide talling and talling (1965). this study indicated well-oxygenated waters, low free co2 and low chloride content. our report differed in ph, alkalinity, hardness and marginal increase in calcium and chloride values than an earlier report (sharma, 2009a) based on sampling at one station (sendra). the present collections revealed a total of 152 rotifer species, including one species new to india, 23 species new to manipur and 14 species new to loktak basin. this is in contrast to an earlier report of 120 species from this wetland (sharma, 2009a) with ~82% community similarity between two lists. our report raised total richness (s) known to date from this ramsar site to 162 species which, in turn, is one of the richest rotifera assemblage known from any freshwater ecosystem of the indian sub-region following the report of 171 species from deepor beel (sharma and sharma, 2015) another ramsar site of india. the rotifer fauna of loktak reflected 78.7% community similarity with the latter because of common occurrence of several cosmopolitan, cosmotropical and pantropical species. total richness (s) merits biodiversity value as ~ 40.0% and ~ 62.0% of species of rotifera known from india and nei, respectively. the report of 40 genera and 20 families from loktak lake reflected rich higher diversity as compared with 50 genera and 23 families as well as 65 genera and 25 families of this phylum known from nei (sharma and sharma, 2014a) and india (bks, unpublished), respectively while it broadly concurred with 38 genera and 20 families known from deepor beel (sharma and sharma, 2015). the rich and diverse assemblage of loktak rotifera, affirming biodiversity value of this ‘hotspot’, is hypothesized to habitat diversity and environmental heterogeneity of this ramsar site. this generalization supported hypothesis of segers et al. (1993) indicating (sub) tropical floodplains to be the world’s rotifer rich habitats and also concurred with reports from the floodplains of argentina (jose de paggi, 1993, 2001), brazil (bonecker et al., 1998), australia (shiel et al., 1998), and the brahmaputra river basin of india (sharma and sharma, 2014b). mytilina lobata is a new addition to the indian rotifera. known from neotropical region (segers, 2007), the present report extended its distributional limit to the oriental region. twenty-three species are new records from manipur. besides, this study extended distribution of fourteen species to loktak lake basin (appendix 1). a total of nineteen globally interesting elements (~12.0% of s) now known from loktak lake is of biogeography value than twelve species listed by sharma (2009a). these included the australasian brachionus kostei, macrochaetus danneelae and notommata spinata; five oriental endemics, namely filinia camasecla, lecane acanthinula, lecane bulla diabolica and l. niwati and l. solfatara; the palaeotropical euchlanis semicarinata, lecane lateralis, l. simonneae, l. unguitata, lepadella bicornis, l. discoidea, l. vandenbrandei, testudinella brevicaudata, trichocerca abilioi and t. hollaerti are palaeotropical species; and brachionus durgae is a cosmo (sub) tropical species. of these, brachionus kostei, euchlanis semicarinata, notommata spinata, lecane niwati, l. solfatara lepadella vandenbrandei, notommata spinata, testudinella brevicaudata, trichocerca abilioi and t. hollaerti are interestingly restricted in their distribution in india exclusively to nei. ascomorpha ecaudis, brachionus mirabilis, filinia brachiata, f. saltator, keratella lenzi, lecane 74 sharma et al./ rotifer diversity of loktak lake, manipur, northeast india aeganea, l. aspasia, l. bifurca, l. doryssa, l. elegans, l. haliclysta, l. rhenana, l. rhytida, l. simonneae, l. pusilla, l. tenuiseta, l. thienemanni, lepadella benjamini, l. bicornis, l. costatoides, l. dactyliseta, l. desmeti, l. elongata, l. quadricarinata, l. quinquecostata, lophocharis salpina, mytilina acanthophora, m. bisulcata, m. michelangellii, macrochaetus longipes, platyias leloupi, testudinella amphora, t. parva, t. emarginula, t. tridentata, trichocerca bicristata, t. edmondsoni, t. insignis, t. flagellata, t. maior, t. scipio, t. tenuior, t. weberi, tripleuchlanis plicata and trochosphaera aequatorialis are examples of regional distributional importance in the indian sub-continent. of these, testudinella amphora is a recently reported from india from assam (sharma et al. 2015); lecane aeganea, trichocerca hollaerti and t. maior are new additions to the indian rotifera from mizoram state of nei (sharma and sharma 2015). our present tally of 152 species of loktak rotifera reflected its diverse nature than the reports of 111 species from the floodplains of argentina (jose de paggi, 1993); 124 species (lake) and 136 species from oguta and iyi-efi lakes, respectively of the niger delta (segers et al., 1993); 130 species from lake guarana, brazil (bonecker et al., 1994); 106 taxa from thale-noi lake, a ramsar site in thailand (segers and pholpunthin, 1997); 104 species from laguana bufeos, bolivia (segers et al., 1998); and 114 taxa examined from the rio pilcomayo national park (a ramsar site), argentina (jose de paggi, 2001). the richness is higher than 67-103 species (sharma, 2005), 69-93 species (sharma and sharma, 2008) and 60-100 species (sharma et al., 2015) examined from various beels of the brahmaputra river basin, assam; and 62-73 species reported from 14 floodplain lakes (pats) of manipur (sharma, 2009b). we caution on overemphasis on comparisons of richness as it was likely to be influenced by sampling intensity (dumont and segers, 1996). we attribute high richness of rotifera of loktak to the rotiferologist effect following fontaneto et al. (2012) though our biodiversity update is also the result of the sampling intensity. loktak rotifera is characterized by diverse lecanidae (47 species) > lepadellidae (27 species) > brachionidae (17 species) > trichocercidae (15 species) which collectively formed 65.4% of s. it differed from the importance of lecanidae > lepadellidae > trichocercidae > brachionidae listed earlier from this ramsar site (sharma, 2009a) with distinct increase in the richness of the first two families in our recent collections. nevertheless, marginal increase in the brachionid diversity deserved caution due to their yet restricted occurrence and of brachionus spp. in particular. this feature differed from high brachionidae richness (28 species) reported from deepor beel (sharma and sharma, 2015). notommatidae = euchlanidae > testudinellidae = trochosphaeridae = mytilinidae, other diverse families (~20.0% of s), deserved attention in loktak lake. the littoral-periphytonic lecane (47 species) > lepadella (22 species) > trichocerca (17 species), together, included ~52.0% of s of loktak rotifera and thus supported hypothesis of green (2003) on the possibility of assemblage rules for the periphytic community. interestingly, their significance broadly concurred with the reports from the floodplains of niger delta (segers et al., 1993), broa reservoir, brazil (segers and dumont, 1995), river nan, thailand (sanoamuang, 1998), bolivia (segers et al., 1998) and okavango delta of south africa (green, 2003); it also concurred with the report from deepor beel (sharma and sharma, 2015). the rich diversity of ‘tropic-centered’ lecane, more cosmopolitan species (~60% of s), and collective importance (~24% of s) of cosmotropical and pantropical species assigned ‘tropical character’ to loktak rotifera. this conclusion is concurrent with several tropical faunas (green, 1972; pejler, 1977; fernando, 1980; dussart et al., 1984; segers, 1996, 2001) globally as well as from india (sharma, 1996, 1998b, 2005; sharma and sharma, 2008; 2014a, 2014b, 2015). these remarks are supported by relative paucity of ‘temperate-centered’ keratella in our collections. 75 int. j. aquat. biol. (2016) 4(2): 69-79 high richness of the littoral-periphytonic and fewer euplanktonic species in loktak are hypothesized to paucity of definite limnetic habitats (de manuel, 1994). the occurrence of both nonplanktonic species and planktonic taxa in the littoral weedy margin of this ramsar site affirmed occupation of different niches as hypothesized by bonecker et al. (1998). these features concurred with the reports from deepor beel (sharma and sharma, 2013, 2015) and other floodplain lakes of the brahmaputra river basin (sharma and sharma, 2014b). loktak rotifera is notable for large number of small-sized species particularly of lecane, lepadella, trichocerca and colurella. this feature is hypothesized to predation by juvenile fish and invertebrates (baumgartner et al., 1997) but specific observations are desired to confirm predation effect. our june 2010 may 2012 limnological survey registered significant variations of the rotifer richness amongst three loktak stations during the study period (f2, 46=11.473, p=9.07e-05) as well as during two successive years (f 2, 22=4.327, p=0.026; f2, 22=7.051, p=0.004) as against insignificant variations reported earlier (sharma, 2009a). further, it recorded significant monthly variations at two sampling sites: loktak b (f1, 11=3.536, p=0.023) and loktak barrage (f1, 11=6.618, p=0.002) and followed osscillaring variations with maxima during december 2011, november 2011 and january 2012 at loktak a, loktak b and loktak barrage, respectively. this study indicated lack of significant influence of any individual abiotic factor on the richness in contrast to inverse correlation with rainfall, ph, hardness, nitrate, chloride and total dissolved solids and positive correlation with dissolved oxygen recorded vide sharma (2009a). the differences suggested that the rotifers are generalists in terms of abiotic factors vs. their occurrence with factors associated with microhabitat being more important. to sum up, the specious and diverse rotifer assemblage of loktak lake, with various new records and species of global and regional interest, imparted biodiversity and biogeography merit to our meta-analysis. with our bias towards inclusion of monogonont plankton and semi-plankton taxa, this study suggested scope for further up-date of the rotifer inventory of this ‘hot-spot’ based on specific sampling of periphytic, sessile, colonial and benthic communities. we estimate the report of 220+ species from this ramsar site while analysis of the ‘rotifermacrophytes associations’ merits biodiversity interest due to lack of such studies in india. acknowledgements we thank the head, department of zoology, northeastern hill university, shillong for laboratory facilities. the samples for this study were collected by tph. the authors have no conflict of interests. references a.p.h.a. 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(1965). the chemical composition of african lake waters. internationale revue der gesamten hydrobiologie, 50: 421-463. 78 sharma et al./ rotifer diversity of loktak lake, manipur, northeast india class: monogononta order: ploimida family: brachionidae 1. anuraeopsis fissa gosse, 1851 2. brachionus angularis gosse, 1851 3. b. bidentatus anderson, 1889 *** 4. b. calyciflorus pallas, 1766 5. b. caudatus barrois & daday, 1894 ** 6. b. falcatus zacharias, 1898 7. b. durgae dhanapathi, 1974 ** 8. b. kostei shiel, 1983 ** 9. b. mirabilis daday, 1897 10. b. quadridentatus hermann, 1783 11. keratella cochlearis (gosse, 1851) 12. k. lenzi hauer, 1953 *** 13. k. tropica (apstein, 1907) 14. k. tecta (gosse, 1851) ** 15. platyias leloupi (gillard, 1967) *** 16. p. quadricornis (ehrenberg, 1832) 17. plationus patulus (o.f. müller, 1786) family: epiphanidae 18. epiphanes brachionus (ehrenberg, 1837) *** family: euchlanidae 19. beauchampiella eudactylota (gosse, 1886) 20. dipleuchlanis propatula (gosse, 1886) 21. euchlanis dilatata ehrenberg, 1832 22. e. incisa carlin, 1939 *** 23. e. meneta myers, 1930 ** 24. e. semicarinata segers, 1993 # 25. e. triquetra ehrenberg, 1838 26. tripleuchlanis plicata (levande, 1894) family: mytilinidae 27. lophocharis salpina (ehrenberg, 1834) 28. mytilina acanthophora hauer, 1938 ** 29. m. bisulcata (lucks, 1912) 30. m. lobata pourriot, 1996 * 31. m. michelangellii reid & turner, 1988 ** 32. m. ventralis (ehrenberg, 1830) family: trichotriidae 33. macrochaetus danneelae koste & shiel, 1983 # 34. m. longipes myers, 1934) 35. m. sericus (thorpe, 1893) 36. trichotria tetractis (ehrenberg, 1830) 37. wolga spinifera (western, 1894) ** family: lepadellidae 38. colurella adriatica (ehrenberg, 1837) 39. c. obtusa (gosse, 1886) 40. c. sulcata (stenroos, 1898) 41. c. uncinata (o. f. müller, 1773) 42. lepadella acuminata (ehrenberg, 1834) 43. l. apsicora myers, 1934 44. l. apsida harring, 1916 45. l. bicornis vasisht & battish, 1971 46. l. benjamini harring, 1916 47. l. costatoides segers, 1992 48. l. dactyliseta (stenroos, 1898) 49. l. desmeti segers & chittapun, 2001 *** 50. l. discoidea segers, 1993 51. l. eurysterna myers, 1942 52. l. ehrenbergi (perty, 1850) 53. l. heterostyla (murray, 1913) 54. l. lindaui koste, 1981 55. l. minuta (weber & montet, 1918) ** 56. l. ovalis (o.f. müller, 1786) 57. l. patella (o.f. müller, 1773) 58. l. quadricarinata (stenroos, 1898) ** 59. l. quinquecostata (lucks, 1912) ** 60. l. rhomboides (gosse, 1886) 61. l. triba myers, 1934 *** 62. l. triptera ehrenberg, 1832 63. l. vandenbrandei gillard, 1952 64. squatinella mutica (ehrenberg, 1832) family: lecanidae 65. lecane acanthinula (hauer, 1938) # 66. l. aculeata (jakubski, 1912) 67. l. aeganea harring, 1914 *** 68. l. arcula harring, 1914 *** 69. l. aspasia myers, 1917 ** 70. l. bifurca (bryce, 1892) ** 71. l blachei berzins, 1973 72. l. bulla (gosse, 1851) l. bulla diabolica (hauer, 1936) *** 73. l. closterocerca (schmarda, 1859) 74. l. crepida harring, 1914 75. l. curvicornis (murray, 1913) 76. l. decipiens (murray, 1913) 77. l. doryssa harring, 1914 78. l. elegans harring, 1914 79. l. flexilis (gosse, 1886) 80. l. furcata (murray, 1913) 81. l. haliclysta harring & myers, 1926 *** 82. l. hamata (stokes, 1896) 83. l. hornemanni (ehrenberg, 1834) 84. l. inermis (bryce, 1892) 85. l. inopinata harring & myers, 1926 86. l. lateralis sharma, 1978 87. l. leontina (turner, 1892) 88. l. ludwigii (eckstein, 1883) 89. l. luna (o.f. müller, 1776) 90. l. lunaris (ehrenberg, 1832) 91. l. monostyla (daday, 1897) appendix 1: systematic list of rotifera recorded from loktak phylum: rotifera super-class: eurotatoria 79 int. j. aquat. biol. (2016) 4(2): 69-79 92. l. nitida (murray, 1913) 93. l. niwati segers, kothetip & sanoamuang, 2004 94. l. obtusa (murray, 1913) 95. l. ohioensis (herrick, 1885) 96. l. papuana (murray, 1913) 97. l. ploenensis (voigt, 1902) 98. l. pusilla harring, 1914 *** 99. l. quadridentata (ehrenberg, 1830) 100. l. rhenana hauer, 1929 ** 101. l. rhytida harring & myers, 1926 ** 102. l. ruttneri hauer, 1938 103. l. signifera (jennings, 1896) 104. l. simonneae segers, 1993 105. l. solfatara (hauer, 1938) # 106. l. stenroosi (meissner, 1908) 107. l. tenuiseta harring, 1914 108. l. thienemanni (hauer, 1938) *** 109. l. undulata hauer, 1938 ** 110. l. unguitata (fadeev, 1925) 111. l. ungulata (gosse, 1887) family: notommatidae 112. cephalodella forficula (ehrenberg, 1830) 113. c. gibba (ehrenberg, 1830) 114. c. mucronata myers, 1924 115. monommata longiseta (o.f. müller, 1786) 116. m. maculata harring & myers, 1930 117. monommata sp. 118. notommata spinata koste & shiel, 1991 family: scaridiidae 119. scaridium longicaudum (o.f. müller, 1786) family: gastropodidae 120. ascomorpha ecaudis perty, 1850 family: trichocercidae 121. trichocerca abilioi segers & sarma, 1993 # 122. t. bicristata (gosse, 1887) 123. t. cylindrica (imhof, 1891) 124. t. edmondsoni (myers, 1936) ** 125. t. elongata (gosse, 1886) 126. t. flagellata hauer, 1938 127. t. hollaerti de smet, 1990 ** 128. t. insignis (herrick, 1885) 129. t. longiseta (schrank, 1802) 130. t. maior hauer, 1936 ** 131. t. rattus (o.f. müller, 1776) 132. t. scipio (gosse, 1886) ** 133. t. similis (wierzejski, 1893) 134. t. tenuior (gosse, 1886) 135. t. weberi (jennings, 1903) ** family: asplanchnidae 136. asplanchna priodonta gosse, 1850 family: synchaetidae 136. ploesoma lenticulare herrick, 1855 137. polyarthra vulgaris carlin, 1943 138. synchaeta pectinata ehrenberg, 1832 family: dicranophoridae 139. dicranophoroides caudatus (ehrenberg, 1834) 140. dicranophorus forcipatus (o.f. müller, 1786) order: flosculariaceae family: flosculariidae 141. floscularia ringens (linnaeus, 1758) # 142. s. semibullata (thorpe, 1893) ** 143. sinantherina spinosa (thorpe, 1893) 144. s. socialis (linnaeus, 1758) family: conochilidae 145. conochilus unicornis rousselet, 1892 family: trochosphaeridae 146. filinia brachiata (rousselet, 1901) # 147. f. camasecla myers, 1938 148. f. longiseta (ehrenberg, 1834) 149. f. opoliensis (zacharias, 1898) 150. f. saltator (gosse, 1886) 151. trochosphaera aequatorialis semper, 1872 *** family: testudinellidae 152. testudinella amphora hauer, 1938 ** 153. t. brevicaudata yamamoto, 1951 154. t. emarginula (stenroos, 1898) 155. t. parva (ternetz, 1892) 156. t. patina (hermann, 1783) 157. t. tridentata smirnov, 1931 class: bdelloidea order: philodinida family: philodinidae 158. philodina citrina ehrenberg, 1832 159. rotaria macroceros (gosse, 1851) # 160. r. neptunia (ehrenberg, 1830) 161. r. tardigrada (ehrenberg, 1832) # family: habrotrochidae 162. habrotrocha angusticollis (murray, 1905) # * new record from india; **new record from manipur state; *** new record from loktak; # not observed in present collections int. j. aquat. biol. (2019) 7(1): 35-37 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology short communication a new record of potamanthellus caenoides ulmer, 1939 (ephemeroptera: neoephemereidae) from the chalakudi river, southern western ghats of india ollappilly abdulkadher banazair*,1guna christopher advanced centre of environmental studies and sustainable development (acessd), mahatma gandhi university, kottayam, india. article history: received 4 january 2019 accepted 23 february 2019 available online 2 5 february 2019 keywords: ephemeroptera, new record, chalakudy river, southern western ghats. abstract: as part of an ongoing study on the benthic macro-invertebrates of chalakudi river basin, of the southern western ghats of india, we establish a new record of a mayfly larva (potamanthellus caenoides). larva of potamanthellus caenoides ulmer, 1939 is recorded for the first time from the upstream of chalakudy river. the specific location, sample and brief ecological notes are appended. introduction ephemeroptera is a biogeographically significant archaic order of aquatic insects abounding in several enigmatic families in the pantropical region, especially in the oriental realm. neoephemeridae is a small group of mayflies presently confined to holartic and oriental regions. bae (1998) recognized only three genera of mayflies viz., potamanthellus lestage, 1930 (seven species), neoephemera mcdunnough, 1925 (five species) and ochernova bae, 1998 (one species), synonymizing leucorhoenanthus lestage, 1930 (one species) with neoephemera. this is not accepted by kluge (2004), and bauernfeind and soldán (2012). larvae of neoephemeridae have unique operculate gills on the second abdominal segment that are fused medially. the larvae of potamanthellus are distinguished from those of neoephemera and ochernova by their densely setate mouthparts, lack of well-developed lateral expansions of the pronotum and mesonotum, and possession of rows of long setae on the caudal filaments (bae, 1998). however, the larvae of potamanthellus cannot be differentiated from leucorhoenanthus by any plesiomorphic or apomorphic traits. based on our larval collections of *corresponding author: ollappilly abdulkadher banazair doi: https://doi.org/10.22034/ijab.v7i1.555 e-mail address: benazeeroa@gmail.com ephemeroptera from the chalakudy river of the western ghats, and subsequent taxonomical examinations, we establish a new record of potamanthellus caenoides (ulmer, 1939), which is a significant range extension to south asia from its known range viz., sumatra island located in southeast asia. differential diagnosis of p. caenoides is verified based on the larval descriptions given (ulmer, 1939) and subsequent revisionary studies (bae, 1998). materials and methods the material was collected from the up-streams of chalakudy river, near valpparai and sholayar in the south-western region of the western ghats of peninsular india, by kick/dip net methods and by direct hand picking (needham and needham, 1969) and (pennak, 1989). the specimens were preserved in 90% ethanol. some specimens were mounted on slides to enable detailed microscopic observations. results potamanthellus caenoides ulmer, 1939 taxonomic status: accepted; kingdom: animalia; phylum: arthropoda; class: insecta; order: ephemeroptera; family: neoephemeridae; taxon rank: 36 banazair and christopher / new record of potamanthellus caenoides from chalakudi river species; genus: potamanthellus; specific epithet: caenoides country: india; state province: kerala & tamil nadu; locality: valparai; verbatim latitude: 10°18'38.8''n; verbatim longitude: 076°56'45.1''e; sampling protocol: hand picking; year: 2018; month: april; day: 18; habitat: cascade; individual count: 4; sex: male & female; life stage: larva; identified by o.a. banazair & g. christopher. diagnosis: potamanthellus caenoides is distinguished from other species of potamanthellus by the following combination of characters in larvae: (i) a distinct diagonal ridge on operculate gills, (ii) distinct tubercles on abdominal terga 6-8, (iii) dorsal forefemora with transverse row of setae (iv) relatively small body size (<8 mm), and (v) relatively short caudal filaments that possess strongly developed lateral setae. potamanthellus caenoides is distinguished from closely related species p. ganges by the following characters: (i) posteromedian tubercle on abdominal terga 1-2 and 6-8 distinct, (ii) rows of hair like setae strongly developed and mature body ca. 6-8 mm, and (iii) dorsal fore-femora with transverse row of setae (fig. 1). distribution: indonesia (sumatra (ulmer, 1939), java, bali, lombok and flores), malaysia (malay peninsula, sabah and sarawak), philippines (mindanao), thailand (bae, 1998), vietnam (nguyen and bae, 2003) and india (southern western ghats). ecology: the larvae of p. caenoides occur in moderately fast flowing mountain streams and rivers ranging 850-935 m in altitude. the substrates consist of relatively coarse particles (boulder 30%, cobble 20%, pebble 20% and gravel and sand 30%), fallen leaves and detritus. the water temperature in april ranges 18-23°c. taxon discussion: presently this genus consists of seven species viz. p. amabilis (eaton, 1892), p. caenoides (ulmer, 1939), p. chinensis (hsu, 1936), p. edmundsi (bae, 1998), p. ganges (bae, 1998), p. shaowuensis (gui et al., 1999) and p. unicutibius (nguyen and bae, 2003). however, two species viz. p. ganges is known from india from the tributary of ganges (bae, 1998). potamanthellus caenoides (ulmer, 1939) is from the southern western ghats and p. caenoides (ulmer, 1939) is a new record from chalakudi river. acknowledgments the authors express their gratitude to dr. a.p. thomas and dr. s. prasanth narayan for their valuable suggestions. thanks are also to the kerala forests and wildlife department for permitting to do field work in vazhachal forest. the research work was financially supported by ugc maulana azad national felloship (manf). conflict of interest the authors declare that they have no conflict of interest. references bae y.j. (1998). phylogenetic systematics and biogeography of the neoephemeridae (ephemeroptera: pannota). aquatic insects, 20: 35-68. bauernfeind e., soldán t. (2012). the mayflies of europe (ephemeroptera). apolla books, ollerup. 781 p. eaton a.e. (1892). new species of ephemeridae from the tenasserim valley. transactions of the entomological society of london, 185-190. gui h., zhou c.f., sc c. (1999). ephemeroptera. in: b.k. huang (ed.). fujian insect fauna.1. fujian science and technology press, fuzhou. hsu y. (1936). new chinese mayflies from kiangsi province (ephemeroptera). pekingnatural history bulletin, 10: 119-126. kluge n. (2004). the phylogenetic system of ephemeroptera. kluwer academic publishers, figure 1. potamanthellus ceanoids, collected from the chalakudi river. 37 int. j. aquat. biol. (2019) 7(1): 35-37 dordrecht. 442 p. lestage j. (1930). contribution à l'étude des larves des éphéméroptères. vi. les larves dites fouisseurses. le regime des larves. les larves et les poisons. bulletin et annales de la société entomologique de belgique, 70: 79-89. mcdunnough j. (1925). new canadian ephemeridae with notes iii. canadian entomology, 57: 168-176. needham j.g., needham p.r. (1969).a guide to the study of freshwater biology holden-day inc. san francisco. 108 p. nguyen v.v., bae y.j. (2003). taxonomic review of the vietnamese neoephemeridae (ephemeroptera) with description of potamanthellus unicutibius, new species. pan-pacific entomologist, 79: 230-236. pennak r.w. (1989).freshwater invertebrates of the united states, 2ndedn. john wiley and sons, new york. 810 p. ulmer g. (1939). eintagsfliegen (ephemeropteren) von den sunda-inseln. archiv für hydrobiologie, 16: 443692. int. j. aquat. biol. (2020) 8(2): 126-131 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article does length-weight equation fit clupeid fishes? an evaluation of lwrs for six clupeids from iran (teleostei: clupeiformes) leyli purrafee dizaj1, hamid reza esmaeili*1, keyvan abbasi2, tooraj valinassab3, ali salarpouri4 1ichthyology and molecular systematics research laboratory, zoology section, department of biology, college of sciences, shiraz university, shiraz, iran. 2inland waters aquaculture research center, iranian fisheries sciences research institute, agricultural research, education and extension organization, bandar anzali, iran. 3iranian fisheries science research institute, agricultural research, education and extension organization, tehran, iran. 4iranian fisheries science research institute, agricultural research, education and extension organization, persian gulf and oman sea ecological research center, bandar abbas, iran. s article history: received 9 january 2020 accepted 18 april 2020 available online 2 5 april 2020 keywords: length-weight parameters brackish water marine waters morphology abstract: this study investigates length–weight relationships of six clupeid species (alosa braschnikowi, alosa caspia, dussumieria acuta, nematalosa nasus, sardinella albella and tenualosa ilisha) captured from three main water bodies of iran (persian gulf, oman sea and caspian sea), to evaluate if the lwr parameters fit for these fishes having specific morphological characteristics. based on the obtained results, i) the b value was influenced by recorded length (tl, sl, fl) and body shape, ii) it was within the expected range of 2.27–3.48, iii) length–weight relationships were highly correlated and significant (r2>0.82-0.98, p<0.001), and hence length-weight equations fit well with six clupeid species in the iranian water bodies. the results presented here, would be useful for fishery biologists and fisheries stake-holders in the study area. introduction length-weight relationships (lwrs) have been implemented to assess status of fish populations in fisheries since the beginning of the 20th century (froese, 2006; jellyman et al., 2013). lwr presents how fish weight changes as a function of length, and it provides essential information for fisheries scientists trying to infer age structure (jellyman, 1997), to estimate growth rates (hansen and closs, 2009), to model bioenergetics (hayes et al., 2000; booker et al., 2004) or to quantify some other aspects of fish population dynamics (safran, 1992). it also provides suitable data to determine breeding season, feeding state, fatness, the suitability of environment and gives knowledge of species ecology (al-jebory et al., 2018). moreover, lwrs are required for assessing biomass of a fish community using only length and species data (greig et al., 2010), estimating fish condition factor (hiddink et al., 2011) and constructing comparisons of life history characteristics of fish (fonseca and cabral, *correspondence: hamid reza esmaeili doi: https://doi.org/10.22034/ijab.v8i2.847 e-mail: hresmaeili22@gmail.com 2007). length and weight of fishes are related to each other and can be estimated by having one in hand. therefore, these equations are useful during field studies to convert length into weight (esmaeili and ebrahimi, 2006; valinassab et al., 2012; esmaeili et al., 2014, 2015; sayyadzadeh and esmaeili, 2016; jafari-patcan et al., 2018) or any measured length to another (e.g. tl to sl) (zareian et al., 2018; mouludisaleh et al., 2019). fish length is often more quickly and reliably measured than fish weight (le cren, 1951). some previous studies have provided lwrs for some clupeid species (see samsun, 1995; tarkan et al., 2006; erguden et al., 2011; yılmaz and polat, 2011; saç, 2012; aydoğan and özuluğ, 2020). the family clupeidae with about 198 species (fricke et al., 2020), with many important food fishes in the world, are commonly caught for production of fish oil and fish meal. here, the objective of this paper is to provide lwrs of six species of this family from the 127 int. j. aquat. biol. (2020) 8(2): 126-131 persian gulf, oman sea and caspian sea to evaluate if the lwr parameters fit for these fishes with specific morphological characteristics. materials and methods fish specimens were caught from 2016 to 2019 from three main water bodies, including the persian gulf, oman sea (both in the south) and the caspian sea in the north of iran (fig. 1). specimens washed with freshwater and dried with paper. total length (tl), fork length (fl), and standard length (sl) were measured to the nearest 0.05 mm, and weight (w) was measured to the nearest 0.001 g. then they were preserved in 5% formaldehyde solution and deposited in zm–cbsu, zoological museum, collection of biology department, shiraz university. the parameters of the length–weight relationship w= alb were estimated by linear regression of the logtransformed weight and length, where w is weight and l is length (tl, fl or sl), a the intercept, and b the regression slope (koutrakis and tsikliras, 2003). firstly, log-log plots of length and weight values were conducted for visual inspection of outliers (froese, 2006). in addition, 95% confidence intervals (ci) for a and b were estimated. based on the slope (b) of the lwr, one can estimate whether fish growth is isometric (b=3, all fish dimensions increase at the same rate), hypoallometric (b<3) or hyperallometric (b>3). exploring which growth (i.e., isometric or allometric) is presented by a provided species gives assumption on how fish body proportions may differ at a given geographic zone or throughout a specific season (froese, 2006). materials used for lwr: alosa braschnikowi zmcbsux013-1 to 23, x029-1 to 14, x032-1 to 5, x034-1 to 3, x061-1 to 8; 3.83-394.5 g, 7.91-33.70 cm (fig. 1a); alosa caspia zm-cbsux015-1 to 8, x035-1 to 9, x063-1 to 2; 12.16-385.70g, 11.9634.70cm (fig. 1b); dussumieria acuta zmcbsux047-1 to 9, x042-1 to 31; 10.41-36.75 g, 11.75-17.75 cm (fig. 1c); nematalosa nasus zmcbsux010-1 to 19, x011-1 to 17, x016-1 to 8, x046-1 to 7; 35.30-91.50 g, 15.24-21 cm (fig. 1d); sardinella albella zm-cbsux001-1 to 9, x018-1 to 12; 5.82-18.79 g, 9.12-13.70 cm (fig. 1e); tenualosa ilisha zm-cbsux003-1 to 10, x012-1 to 13; 66.20205.20 g, 19.90-28.80 cm (fig. 1f). results a total of 194 fishes with tl ranging from 7.91 to34.8 cm (19.26±6.56) and w from 3.83 to 394.50 g (82.96±78.18 se) were studied for lwrs (table 1). principal estimated parameters of lwrs between fish figure 1. six studied clupeid species from the persian gulf, oman sea and caspian sea. 128 purrafee dizaj et al./ does length-weight equation fit clupeid fishes? lengths (tl, fl and sl) and wet weight for the 6 clupeids are presented in table 1. the results indicated that the length-weight relationships for all studied species were highly correlated and significant (r2>0.82-0.98, p<0.001). in the present study, the mean b value (based on tl) varied from a minimum of 2.68 for d. acuta to 3.47 for t. ilisha. in all fishes, except d. acuta the mean b values (based on fl) are greater than those for sl and tl (table 1). discussion this work provides lwr for 6 clupeid fishes of the genera alosa, dussumieria, nematalosa, sardinella and tenualosa from iran. for all species investigated during this study, the coefficient (b) of lwr is within the expected range of 2.27-3.48 as supposed by froese (2006). the slope (b) of the length-weight relationships for all 6 species fell within the expected range of 2.68–3.47 for tl, varying from a minimum of 2.68 for d. acuta to a maximum of 3.47 for t. ilisha; 2.36–3.48 for fl, varying from a minimum of 2.36 for d. acuta to a maximum of 3.48 for t. ilisha; 2.27–3.11 for sl, varying from a minimum of 2.27 for d. acuta to a maximum of 3.11 for a. caspia. the b parameter of the length-weight relationships of fishes are influenced by a number of factors, including health, nutrition, habitat, area, environmental conditions (such as salinity and temperature), season, sex, gonad development, level of stomach fullness, variations in the length range of the caught specimen and fishing table 1. principal parameters and linear relations (y = a + bx) between total length and whole wet weight for the 6 clupeids, from iran. (tl = total length; fl= fork length; sl= standard length; w = fish wet weight, a = intercept value; b = regression slope; r2 = coefficient of determination; n = number of fish sampled; min = minimum; max = maximum, 95% ci= 95% confidence limit). species locality n w (g), (min – max) length (min-max) regression parameters 95% cl of b 95% cl of a a b (se) r2 alosa braschnikowi (borodin, 1904) caspian sea, anzali 51 128.95 (3.83-394) sl 18.10 (6.42-28.10) 0.01148 3.08 (0.09) 0.95 2.90-3.27 0.006-0.019 tl 22.55 )7.91-33.70) 0.00549 3.11 (0.06) 0.98 2.98-3.23 0.003-0.007 fl 20.02 (7.15-30.40) 0.00691 3.15 (0.06) 0.98 3.02-3.28 0.004-0.010 alosa caspia (eichwald, 1838) caspian sea, anzali 18 109.37 (12.16-385.7) sl 18.28 (9.37-27.7) 0.01047 3.11 (0.12) 0.97 2.84-3.38 0.004-0.022 tl 22.51 (11.69-34.8) 0.00467 3.16 (0.14) 0.96 2.85-3.47 0.001-0.012 fl 19.90 (10.48-30.6) 0.00537 3.25 (0.14) 0.96 2.93-3.56 0.002-0.013 dussumieria acuta valenciennes, 1847 persian gulf, bandar abbas 32 19.03 (10.41-36.75) sl 11.44 (9.20-14.64) 0.07079 2.27 (0.11) 0.93 2.04-2.51 0.040-0.125 tl 14.31 (11.7517.75) 0.01479 2.68 (0.10) 0.95 2.46-2.90 0.008-0.026 fl 12.49 (10.10-15.60) 0.04786 2.36 (0.11) 0.92 2.11-2.60 0.025-0.087 nematalosa nasus (bloch 1795) persian gulf, bandar abbas and bushehr 49 64.82 (35.3-91.50) sl 14.22 (11.0716.31) 0.10715 2.41 (0.0.16) 0.82 1.772.62 0.043-0.254 tl 18.40 (14.1621) 0.02137 2.75 (0.12) 0.91 2.703.27 0.0100.043 fl 15.74 (13.32-18.20) 0.01548 3.01 (0.19) 0.83 2.62-3.41 0.005-0.046 sardinella albella (valen ciennes 1847) persian gulf, bandar abbas 21 12.37 (5.82-18.89) sl 8.86 (6.88-10.81) 0.01949 2.93 (0.12) 0.96 2.67-3.18 0.011-0.033 tl 11.56 (9.1213.70) 0.00660 3.05 (0.12) 0.97 2.803.30 0.003-0.011 fl 9.67 (7.42-11.36) 0.01096 3.07 (0.13) 0.96 2.78-3.35 0.005-0.021 tenualosa ilisha (hamilt on 1822) persian gulf, abadan 23 152.35 (66.2-199.70) sl 20.05 (15.04-23.80) 0.03801 2.75 (0.18) 0.91 2.12-3.19 0.011-0.12 tl 25.16 (19.90-28.80) 0.00194 3.47 (0.18) 0.94 2.94-3.67 0.001-0.0067 fl 21.91 (17.42-24.90) 0.00316 3.48 (0.19) 0.95 3.07-3.87 0.009-0.010 129 int. j. aquat. biol. (2020) 8(2): 126-131 gear (tesch, 1971; froese, 2006; moradinasab et al., 2012; keivany et al., 2016). lwr is also related to fish body shape. there is great variation in body shape and depth (round-bodied to strongly compressed and deep) of clupeid fishes. as a general rule, eel-like fishes show smaller b value. according to jellyman et al. (2013) based on 285,124 fish records, anguillidae and geotriidae were classified as ‘eel-like’, pleuronectidae as ‘compressed’ body shape, cyprinidae and poeciliidae as ‘short and deep’ and the remaining studied families were considered to have ‘fusiform’ shape. in study of lwr for the spiny eel, macrognathus pancalus, the pool data for the co-efficient of regression (b) was recorded as 1.408 in juveniles; 2.977 in males and 3.034 in females (abujam and biswas, 2016). in the present study, minimum mean b value (based on tl) belongs to d. acuta (2.68) with fusiform body shape, and maximum belongs to t. ilisha (3.47) with short, deep and almost laterally compressed body form (fig. 1) revealing relationship of b value and body shape. according to erguden et al. (2011), in some other clupeid fishes, the coefficient b ranged from a minimum 2.97 for females of alosa immaculata to a maximum 3.75 for both sexes a. c. caspia. the mean b values for two other clupeid fishes were also in the expected range of 2.4-3.5 (varied from 3.177 to 3.496, for different populations of c. cultriventris and 3.258 for c. muhlisi) (see aydoğan and özuluğ, 2020). for all species, r2 values were high (r2>0.82-0.98, p<0.001). it has been reported that r2 values less than 0.8 are associated with either low numbers of individuals or a limited size range (see jellyman et al., 2013) which was not observed for the studied species here. based on the results of present study, i) the b value is influence by recorded length (tl, sl, fl), ii) the b value is influence body shape, iii) it is within the expected range of 2.27–3.48 as supposed by froese (2006), iv) length–weight relationships were highly correlated and significant (r2>0.82-0.98, p<0.001), and hence length-weight equations fit with for six clupeid species in the iranian water bodies. the results presented here would be useful for fishery biologists and managers in the studied area. acknowledgment the research was funded by shiraz university and was approved by the ethics committee of the biology department (su-9430246). references abujam s.k.s., biswas s.p. 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(2019) 7(5): 315-321 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article the protective effect of damask rose, rosa damascena extract on the liver of cyprinus carpio following zinc exposure milad taleghani1, seyyed mohammad hoseini*1, shila omidzahir2 1department of pathology, babol branch, islamic azad university, babol, iran. 2faculty of marine and oceanic sciences, university of mazandaran, babolsar, iran. s article history: received 25 february 2019 accepted 21 october 2019 available online 2 5 october 2019 keywords: common carp bioaccumulation histopathology biochemical parameters abstract: this study aimed to examine two concentrations of rosa damascene extract on toxic effect of zn on the liver of common carp. for this purpose, four treatments with three replicates were designed as following: treatment one as control, treatment 2 exposed to 3 mg/l znso4, and treatments 3 and 4 supplemented with 2.5 and 5 g of r. damascene extract along with exposure to 3 mg/l znso4. after 45 days, the bioaccumulation of zn and histopathological alternation in the liver, as well as aspartate aminotransferase (ast), alanine transaminase (alt), triglyceride, cholesterol and albumin were measured. the results showed the highest concentration of zn in treatment, and its decreasing trend in treatments 3 and 4. the histopathological examinations showed the most alternations in treatment 2, but lower changes in treatment 3 and 4 due to application of r. damascene extract. in addition, those measured biochemical parameters had increased in treatment 2 following exposure to znso4, whereas they had decrease in treatments 3 and 4. these results revealed positive effect of r. damascene extract to decrease toxicity of zn on the liver of common carp. introduction damask rose, rosa damascene, is one of important species of the rosaceae family (kaul et al., 2000; cai et al., 2005) and its main components are anthocyanin, cyanidin, 3-5 diglucoside, kaempferol, quercetin, galactoside, arabinoside, citronellol, linalool, geraniol and terpenes (velioglu and mazza, 1991), having antibacterial and antioxidant activities (ozkan et al., 2004). among these components, quercetin can prevent many types of cancers e.g. intestine cancer (shoskes et al., 1999) and reduce blood pressure (edwards et al., 2007). in addition, kaempferol extracted from damask rose reported to prevent the activity of the hiv virus’s proteases (mahmood et al., 1996). zinc is an essential and second trace element in vertebrates’ body (pagiannisa et al., 2004), necessary for physiological processes such as cell division, metabolism, wounds healing, defense and reproduction systems, and taste and sight *correspondence: seyyed mohammad hoseini doi: https://doi.org/10.22034/ijab.v7i5.749 e-mail: dr_hosseini2323@yahoo.com performances (aggett and comerford, 1995). its concentrations in aquatic environments vary due to anthropological activities, and natural geological events (janssen et al., 2000; luoma and rainbow, 2008; wood et al., 2012). total zn concentrations has been reported ranging 0.02 µg/l in rivers to more than 1000 µg/l in freshwater water bodies near mining and metal industrial activities (luoma and rainbow, 2008; wood et al., 2012). despite the essentiality of zn, it can be toxic and considered as a serious threat for health, if organisms exposed to a high concentrations (mazrouh and mahmoud 2009; murakami and hirano, 2008). zinc can be toxic for fish, causing disturbances in ionoregulation and hypoxia (murugan et al., 2008), and histological damages e.g. edema, epithelial cell destruction, hyperemia, inflammatory cell infiltration, leydig cell hyperplasia, severe fibrosis, loss of natural structure, and reduction of spermatids and spermatozoa in the lumen of the seminiferous tubules have been reported following 316 taleghani et al./ protective effect of damask rose on liver of cyprinus carpio zinc poisoning in carassius auratus (hoseinifard et al., 2018). the liver is an important organ for zn storage in fishes, and increasing the levels of zn will lead different degrees of abnormalities effecting fish performance (gregorović et al., 2008), therefore liver is a proper biomarker for evaluation of environmental pollution (chavan and muley, 2014). hence, this study aimed to examine two concentrations of r. damascene extract on toxic effect of zn on the liver of common carp (cyprinus carpio). materials and methods a total of 120 healthy fish with an average weight and length of 50±2.14 g and 13±0.25 cm, respectively, were purchased from a local fish farm and transferred to the laboratory. fish were acclimated in an aerated freshwater 1000l tank (24±1°c; ph, 7.2±0.2) for two weeks. during the acclimatization period, fish were fed with commercial pelleted feed including 41% crude protein, 6% crude fat, 5% crude fiber and 12% moisture (chineh co.). fish were fed as 1% of body weight twice a day during the acclimatization period. they were also maintained at 24±1°c with continuously aeration, 6.5 mg/l do, and ph 7.2±0.2. for experiment, fish were randomly distributed into 12 circular 100 l tanks as 4 treatments with three replications. treatments were fed with commercial pelleted feed. treatment 1 was control one, treatment 2 exposed to 3 mg/l znso4 (merck, germany) without adding r. damascene extract, and treatments 3 and 4 were exposed to 3 mg/l znso4 along with 2.5 and 5 g r. damascene extract supplementation in 100 gr commercial feed. each supplemented diet was mixed in a mixer for 30 min and then homogenized into a paste by adding fish oil and distilled water into the food mixer. the amount of distilled water required for pelleting (40% of feed weight) was then added to the mixture and further homogenized. this mixture was passed through a meat grinder, producing string shapes, dried and then broken to produce pellets. the control diet was prepared by the same process, although no supplement was added. the fish were subjected to artificial light of 12h d: 12h l. after 45 days, fishes were taken out randomly, anesthetized with clove oil and sacrificed for further analysis after blood sampling. the water conditions were similar to those of the acclimatization period. damask rose extract analysis: the extract component of r. damascena was analyzed based on yassa et al. (2015). determination of total amount of phenol was measured using folin-ciocaltue colorimetric method (slinkard and singleton, 1977) and total flavonoid by aluminum chloride colorimetric method (kim et al., 2002). histopathological analysis: the liver samples were removed and fixed into 10% buffered formalin. histological sections were prepared based on eagderi et al. (2013) using tissue processor device of did sabz2080/h (iran), and microtome of leitez 1512 (germany) with a diameter of 5 µm. the tissue sections were stained with haematoxylin and eosin (h&e) (bancroft and gamble, 2008; moëzzi et al., 2013). the histological sections were examined with light microscope (olympus cx31, japan) equipped to a digital camera (tucsen truechrome metrics). the histopathological alternations were graded as , +, ++ and +++ showing no change, mild, moderate, and severe lesions, respectively (thophon et al., 2003; nasrolah pourmoghadam et al., 2014). biochemical parameters analysis: the blood samples were taken and their serum were separated by 3000 r/m centrifuging for 10 min (ahmadivand et al., 2014, 2015). the biochemical parameters, including aspartate aminotransferase (ast), alanine transaminase (alt), triglyceride, cholesterol and albumin were measured by pars azmoon kit (iran) using autoanalyzer (hitachi -911, japan). bioaccumulation analysis: the samples of liver tissue were removed from fish and dried in the oven (105°c) for 96 hours and powdered, then 1 g of this powder was mixed with 5 ml of nitric acid (merck, germany) in the polyethylene tubes and then 2 ml of perchloric acid (merck, germany) were added. the samples were kept overnight at room temperature, and afterward transferred to the water bath for 2 hours at 100°c. after digestion process, they filtered using filter paper, and their volume increased to 25 ml by adding 317 int. j. aquat. biol. (2019) 7(5): 315-321 1% nitric acid (al-weher, 2008). zn concentration measured using atomic absorption spectrophotometer (analytic jenaaa-400, germany), and the following formula was used to calculate the concentration (al-weher, 2008): zn concentration in sample (µg/g) = aas reading of digest (µg/ml) × volume of digest used (ml) ⁄ weight of sample digested (g) statistical analysis: normality of the data were checked using kolmogorov-smirnov. one-way analysis of variance (anova) was used for analysis of significant difference among treatments. means were compared by duncan’s test and a p<0.05 was considered statistically significant. statistical analysis was performed using software spss ver. 22. data are presented as mean±se. results damask rose extract components: the total phenol and flavonoid in r. damascena extract were 18.6 and 1.54 mg/g, respectively. histopathology finding: in treatment 1, the prepared histological sections showed normal conditions. those of treatment 2 had altered as moderate hyperaemia, hepatocytes necrosis, bile duct hyperplasia and inflammatory cells infiltration and sever vacuolar degeneration. in treatment 3, mild hyperaemia, inflammatory cells infiltration and bile duct hyperplasia, moderate vacuolar degeneration and hepatocytes necrosis were observed. in treatment 4, the histopathological alternations significantly were healed, and only mild necrosis and vacuolar degeneration were observed (table 1, fig. 1). bioaccumulation of zn in the liver tissue: the highest zn of the liver was measured in treatment 2, significant than the others (p<0.05). in those treated with r. damascene, zn concentration in the liver were lower, and no significant difference found between treatments 4 and control one (p>0.05) (fig. 2). biochemical parameters: the measured biochemical parameters showed an increase in treatment 2, whereas, those of 3 and 4 had decreased due to application of using r. damascene extract. the results are shown in table 2. discussions the present study examined the toxic effect of zn and therapeutic properties of r. damascene in liver tissue of common carp. liver is a biological indictor for determination of environmental pollution (sadauskas et al., 2007; chavan and muley, 2014) and the main organ for heavy metals bioaccumulation (wood et al., 2012; chavan and muley, 2014; georgieva et al., 2016). histopathological changes such as hepatocyte necrosis, degenerative vacuolation and hyperemia in liver were reported subsequent zn poisoning in common carp (georgieva et al., 2016). the highest zn concentration was observed in table 1. histopathological alternations of the liver of cyprinus carpio in different treatments (-, +, ++ and +++ indicate no change, mild, moderate, and severe lesions, respectively). hyperaemia hepatocytes necrosis bile duct hyperplasia inflammatory cells infiltration vacuolar degeneration treatment 1 treatment 2 ++ ++ ++ ++ +++ treatment 3 + ++ + + ++ treatment 4 + + table 2. biochemical parameters (mean±se) of cyprinus carpio in different treatments (letters above the column indicate significant differences; p<0.05). ast (u/lit) alt (u/lit) triglyceride (mg/dl) albumin (g/dl) cholesterol (mg/dl) treatment 1 85±3.16b 8.50±0.67c 174±4.60a,b 1.05±.02a 70±1.78b treatment 2 130±9.48a 20.25±1.77a 225.50±27.05a 1.25±.11a 125.5±18.64a treatment 3 89.50±2.50b 12±0.89b 166±16.09b 1.15±.08a 125±9.83a treatment 4 86.50±4.94b 9±0.70b,c 130±14.75b 1.02±.05a 98±3.13a,b 318 taleghani et al./ protective effect of damask rose on liver of cyprinus carpio treatment 2 i.e. the group only exposed to zn, whereas this metal was low in treatments 3 and 4 due to application of r. damascene extract. the results also revealed that zn has been caused sever histopathological lesions in treatment 2, whereas in the supplemented treatments with r. damascene i.e. groups 3 and 4, the histopathological alternation were less. these results revealed the positive effect of r. damascene extract to decrease toxicity of zn on the liver of common carp. damask rose possesses the flavonoids, phenol and phenolic compounds with valuable antioxidant and anti-aging effects (kalim et al., 2010). the other works revealed higher antioxidant effect of r. damascene extract in compared to usual antioxidants such as ascorbic acid (alam et al., 2008( and in this regard, quercetin, an important flavonoids, is a main compounds of damask rose extract (velioglu et al., 1991; arts et al., 2004). previous studies indicated that quercetin can reduce necrosis and apoptosis in the injured liver of rats figure 1. (a, b) treatment 1, normal liver tissue, (c, d, e) treatment 2, (1) necrosis, (2) vacuolar degeneration, (3) congestion, (4) bile duct hyperplasia, (5) inflammatory cells infiltration, (f, g) treatment 3, (1) necrosis, (2) vacuolar degeneration, (3) congestion, (4) bile duct hyperplasia, and (h) treatment 4, necrosis (1), vacuolar degeneration (2) (x40 h&e). 319 int. j. aquat. biol. (2019) 7(5): 315-321 (velioglu et al., 1991; khaki, 2010). in addition, quercetin has shown protective effect on liver damage induced by biliary obstruction in rats (kanter, 2010). antioxidant and hepatoprotective effect of the ethanolic extract of the r. damascene have been studied by alam et al. (2008), showing that this ethanolic extract reduces some hepatic injuries such as hepatocyte necrosis, degenerative vacuolation, inflammatory cells infiltration and congestion in central vein and sinusoids. moreover a dose dependent decrease of ast, alt, alp enzymes in rat liver that damaged by paracetamol was reported (alam et al., 2008). in line with the above-mentioned work, in the current study, an increase of the biochemical parameters was observed in treatment 2, whereas in the treatments 3 and 4 they were lower. shalaby (2000) showe the increase of alt and ast in liver of common carp following exposure to zno. in addition, application of quercetin have improved the biochemical parameters, including activities of cholinesterase, ast, alt, alp, bilirubin, total lipids, cholesterol and triglycerides in the rat liver, damaged due to exposure to ammonium fluoride (czerny et al., 2000). as conclusion, the result of the present study showed r. damascene can decrease the toxic effect of zn in liver tissue of common carp. references aggett p.j., comerford j.g. 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(2019) 7(1): 9-13 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology short communication first report of the golden mantis shrimp, lysiosquilla tredecimdentata holthuis, 1941 (crustacea: stomatopoda) from chennai coastal waters, southeast india krishnan silambarasan*1, palaniswamy senthilkumaar2 1fishery survey of india, visakhapatnam, andhra pradesh, india. 2post graduate and research department of zoology, sir theagaraya college, chennai, india. article history: received 9 january 2019 accepted 22 february 2019 available online 2 5 february 2019 keywords: new record, golden mantis shrimp, tamil nadu. abstract: the golden mantis shrimp, lysiosquilla tredecimdentata was reported for the first time from chennai coastal waters, southeast india. one adult male specimen was found in the trawl by-catch near kasimedu fishing harbour on september, 2014. the specimen was identified, described, illustrated and morphometrically measured. introduction stomatopods, also called mantis shrimps, are elongate, flattened, shrimp-like or lobster-like crustaceans (carpenter and neim, 1998). these species are among the most aggressive and behaviourally complex crustaceans. all species are active predators and mark one of the few radiations of obligate carnivores within the crustacea. stomatopod crustaceans are common members of benthic ecosystems in tropical and subtropical marine and brackish waters throughout the world. few species are known from temperate seas (hans-georgmuller, 1994). characteristic features of stomatopods are the large powerful raptorial appendages. prey is captured by ‘spearing’ or ‘smashing’; depending on the dactyl is extended or held folded during the strike. the two methods of prey capture distinguish two broad functional groups; the “smashers” and the “spearers”. these two groups comprise the order ‘stomatopoda’, the only living representatives of the subclass hoplocarida (calman, 1900). worldwide 485 species, 115 genera and 17 families of mantis shrimp are currently described (worms december 25, 2018). the indian stomatopod fauna comprises 66 species, of which 17 species are *corresponding author: krishnan silambarasan doi: https://doi.org/10.22034/ijab.v7i1.542 e-mail address: silambuplankton@hotmail.com recorded from chennai coastal waters (divibala and thirumilu, 2013). the pantropical stomatopod genus lysiosquilla, which includes the largest known stomatopods, comprises 12 species, five of which are reported in the indo-west pacific region. our knowledge of indian stomatopod fauna from the seas around india is fairly good, as many extensive studies were made. the monograph on kemp (1913) is an extensive work in indo-pacific region. subsequently, the contributions by kemp and chopra (1921), chopra (1934, 1939), alikunhi (1967) and shanbhogue (1970) have added further information on the taxonomy of this group. recently pillai and thirumilu (2006) have reported l. tredecimdendata from cuddalore fishing harbour, tamil nadu coast of india. the present study reports the first occurrence of golden mantis shrimp, l. tredecimdentata, from kasimedu fishing harbour, chennai coast, tamil nadu, india. materials and methods a single specimen of l. tredecimdentata was collected from trawl by-catch in the kasimedu fish landing centre (fig. 1) from a depth of around 80-120 m during the early hours of september 25, 2014. the specimen 10 silambarasan and senthilkumaar / first report of lysiosquilla tredeimdentata from chennai coast was collected by hand picking and its identification was carried out using standard guidelines (manning, 1978; ahyong et al., 2008). therefore, confirmation of the species, the specimen was deposited in the department of zoology (voucher number: zoological museum specimen-234), sir theagaraya college (stc), chennai. terminology and size are based on manning (1978) and ahyong (2001). all measurements are in millimeters. total length (tl) is measured along the midline from the anterior margin of the rostral plate to the posterior median spines of the telson. carapace length (cl) is measured along the midline and exclusive of rostrum. corneal index, given as 100 * cl divided by cornea width (cw) (table 1). results and discussion taxonomy phylum arthropoda von siebold, 1848 sub phylum crustacea brunnich, 1772 class malacostraca laterille, 1802 sub class hoplocarida calman, 1904 order stomatopoda laterille, 1817 super family lysiosquilloidea giesbrecht, 1910 family lysiosquillidae giesbrecht, 1910 genus lysiosquilla dana, 1852 species lysiosquilla tredecimdentata holthuis, 1941 materials examined: material examined india, tamil nadu, chennai, kasimedu fishing harbour, trawl catch, 24.ix.2014, coll. k. silambarasan, 1 male (tl: 129 mm), zomusp 234 (fig. 2). family characteristics: carapace devoid of carinae; telson lacking distinct median carina; eye t-shaped, cornea bilobed. marginal teeth or spines of telson in conspicuous, rostral claw slender and elongate, adapted for spearing prey, with toothed edge of dactylus bearing numerous, large, serrated teeth or spines. lysiosquillids are usually are clearly banded with alternate light and darkly pigmented bands. species description: anterior part of carapace depicting rostrum and antennal scale is given in figure 3a; rostrum-dome shaped, broader at the base and pointed at the distal end (fig. 3b), antennal scale long, leaf-like with rounded border at both basal and distal borders, dactlus of raptorial claw possessing eight teeth (fig. 3c), spination on postero-lateral and posterior border 4 + 4, the uppermost 2 pairs the sharpest (fig. 3d). color: dorsal base color dark brown or black and yellow horizontal bands alternate all over the body; table 1. morphometry of the only collected specimen of lysiosquilla tredecimdentata holthuis, 1941 male. morphometric characters measurement (mm) total length carapace length corneal index rostral plate length rostral plate width antennal scale length antennal scale width telson length telson width 129.0 22.1 32.5 4.5 6.0 13.5 4.0 18.0 26.0 figure 1. map showing the location of the study area on the kasimedu fishing harbour, chennai coast. 11 int. j. aquat. biol. (2019) 7(1): 9-13 uropodal exopod with distal half of proximal segment and proximal two-thirds of distal segment black; scale with brown outline. pereiopods with distal pink setae. habitat: the species inhabits deep burrows on intertidal sand and mud flat areas (ahyong, 2001). distribution: western indian ocean from aden (yemen), india, pakistan, madagascar, south africa, to thailand, taiwan, australia and central pacific waters (shanbhogue, 1986; ahyong, 2001). remarks: the specimen collected is in perfect agreement with the original description given by holthuis (1941). shanbhogue (1970) on comparing the original specimens of the two species remarked that: the tip of the rostrum reached either to base or up middle or beyond tip of dorsal process and the raptorial dactylus was with 10-11 teeth. in holthuis specimens, the tip of the rostrum did not surpassed the dorsal process of ophthalmic segment and the raptorial dactylus showed 13 teeth. while re-describing the species; manning (1968) observed that the antennal peduncle did not surpassed the eyes. in the present specimen, the antennal peducle is extended slightly beyond the eye. according to manning (1978) l. tredecimdentatais a distinctive species that resembles l. capensis hansen, 1895 and l. sulcirostris kemp, 1913 and differs from l. sulcata, in having an elongated oval shaped antennal scale with a dark pigmented border, antennal protopod, and a posterior spine on the ventral keel of the eighth thoracic somite. it differs from l. capensisin a broad triangular rostrum with a distinct dorsal carina and 10-13 rather than 15-17 teeth on the figure 2. lysiosquilla tredecimdentata holthuis, 1941, male collected from kasimdeu fishing harbour, chennai coast. (a) dorsal view, and (b) ventral view. 12 silambarasan and senthilkumaar / first report of lysiosquilla tredeimdentata from chennai coast claw. lysiosquilla tredecimdentata differs from l. sulcirostris in having a broader rostral plate that lacks deep grooves flanking the median carina and in having 10-13 rather than 7-8 teeth on the dactylus of the claw. the colouration of present specimen resembles with the observations reported by ahyong et al. (2008) in having yellow and black bands alternatively on carapace, abdomen and telson. acknowledgments the authors are grateful to dr. m. kathirvel, formerly principal scientist, central institute of brackish water aquaculture (icar) for critical evaluation of the manuscript and offering valuable suggestions. references ahyong s.t. (2001). revision of the australian stomatopod crustacea. records of the australian museum supplement, 26: 1-326. ahyong s.t., chan t.y., liao y.c. 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(2020) 8(2): 98-108 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article toasted jatropha curcas seed meal in nile tilapia (oreochromis niloticus) diet: effect on growth, economic performance, haematology, serum biochemistry and liver histology wasiu adeyemi jimoh*1, ahmed ayodeji ayeloja1, musa idi-ogede abubakar1, yusuf olatunji yusuf1, mohammed olayemi shittu2, somrat adeola abdulsalami3 1department of aquaculture and fisheries, university of ilorin, pmb 1515, ilorin, kwara state, nigeria. 2department of fisheries technology, federal college of animal health and production technology, pmb 5029, ibadan, nigeria. 3fisheries and aquaculture unit, department of biological sciences, crescent university, abeokuta, ogun state, nigeria. s article history: received 1 january 2020 accepted 9 april 2020 available online 2 5 april 2020 keywords: tilapia profit margin total cholesterol jatropha feed conversion ratio abstract: the effect of dietary inclusion of differently timed dry heat-treated jatropha curcas on the growth and economic performance of nile tilapia, oreochromis niloticus, was evaluated in a 56-day feeding trial. five isonitrogenous and isolipidic dietary treatments (35% crude protein and 10% crude lipid) were made consisting of soybean meal (control) which was replaced by j. curcas seed meal toasted either 5 min or 10 min at 20 and 40% to make other four test diets. a total of 225 juveniles of o. niloticus were acclimatized for a week, weighed and allotted into five dietary treatments. each treatment was replicated three times with fifteen fish per replicate. fish were fed 5% body weight on two equal proportions per day for 56 days. growth data were collected at two-week intervals. the results from the study indicated that there was significant difference (p<0.05) in the growth and economic performance parameters among the fish exposed to different dietary treatments. however, there was no significant variations (p>0.05) in the different growth and economic performance parameters of fish fed ctr and fish fed d520t (5 min toasted, 20%). there was significant reduction in haematological and biochemical parameters of the blood of o. niloticus fed the different dietary treatments containing j. curcas seed meal. based on economic and physiologic performance, soybean meal in nile tilapia diet could be replaced up to 40% by 5-minute toasted j. curcass seed meal. introduction protein feedstuffs such as fish meal used in fish feed formulation in developing countries are scarce, competitive and their cost is usually beyond the reach of fish farmers and producers of aqua feed (fasakin and balogun, 1996). legume seeds and oilseed cake are known to be good alternative dietary protein sources for fish feed and are available in sub-saharan africa on a large scale (fagbenro et al., 2003). soybean meal is a conventional plant protein source in fish feeds that has the capability to replace up to 50% of the fish meal (storebakken, 2000). however, its sustainable use as fishmeal replacer is threatened because of the various uses to which soybean is put as feed for livestock and also consumed by human. this consequently led to its hike in price (azaza et al., 2009). there is, therefore, need to look for cheaper plant protein source that can replace soybean meal in *correspondence: wasiu adeyemi jimoh doi: https://doi.org/10.22034/ijab.v8i2.808 e-mail: jimoh.wa@unilorin.edu.ng fish diets (yue and zhou, 2008) so that sustainable aquaculture development could be witnessed from constant supply of cheaper, lesser used and available plant protein feed ingredients with nutritional profile similar to or better than soybean meal especially at village farm level where farmers can harness natural gift to the maximal. more so that secondary metabolites in plant sources such as tannins, alkaloids, polypeptides, saponin, volatile oils have been reported to have antimicrobial, anti-stress and disease resistant generally initiating immunomodulatory activity in fish (hoseinifar et al., 2020). the review of hoseinifar et al. (2020) detailed mechanisms and strategies of boosting immunity in fish using plant derived natural products. the natural products derived from plants, are capable of stimulating or suppressing the components of both adaptive and innate immune systems in such a way to provide prophylactic 99 int. j. aquat. biol. (2020) 8(2): 98-108 measures rather than therapeutic measures against diseases (hoseinifar et al., 2018). jatropha curcas grows very well in the tropical regions and it is well-distributed in nigeria. its seed meal has nutrient density that is comparable to any other protein sources. it has a good amino acid profile with its essential amino acids level (except lysine) higher than soybean meal (kumar et al., 2011). studies on the use of jatropha seed meal are found in kumar et al. (2008) on rainbow trout diets; kumar et al. (2011) on carp diet; fakunle et al. (2013) and alatise et al. (2014) on clarias gariepinus diet. there is paucity of information on the use of the seed meal as dietary protein source in nile tilapia diets, especially reports on the use of dry heat-treatment to reduce anti-nutritional factor in the seed meal and economic analysis of feeding the diets containing j. curcas seed meal in nile tilapia diets are scarce. thus, this study examined the effect of dietary inclusion of graded level of differently timed dry heattreated j. curcas seed meal as protein source on the growth, haematology, serum biochemistry, liver histology and economic performance of nile tilapia (oreochromis niloticus) juveniles. materials and methods collection and processing of j. curcas seed: three kilograms of mature j. curcas seed were sourced from neighboring village within ilora, oyo town, nigeria. before the commencement of the experiment, the j. curcas were decoated to obtain the seeds. the seeds were divided into two parts, the first part was roasted for 5 min and the second part was roasted for 10 min to eliminate the possible effect of anti-nutritional factor that could be present in the kernel. these seed meal and some other basic feed ingredients were then taken to the laboratory for proximate composition analyses following the procedure of (aoac, 2010). diet formulation and preparation: based on the proximate composition of the basic feed ingredients in table 1, five experimental diets were formulated consisting of a control diet which is made up of soybean meal and 4 test diets replacing soybean meal at a rate of 20 and 40% by 5-min and 10-min toasted j. curcas seed meal. the ingredients were mixed together in the laboratory using hand after which vitamin premix and fish oil were added to the dry ingredient and mixed thoroughly. warm water was added to these ingredients and mixed until a doughlike paste was formed. the dough was passed through an improvised pelleting machine to produce 2 mm pellet size. the pellets were sun-dried and preserved in air tight containers. the gross composition of the experimental diets is given in table 2. table 2 reveals the proximate composition, calculated amino acid and fatty acid profile of the experimental diets. the optimal dietary protein and lipid requirement of tilapia were met by the prepared diets (luquet, 2017). experimental design: a completely randomized design was adopted with three replicates per treatment. 225 juveniles of o. niloticus, obtained from masopa fish farm along alakia road ibadan oyo state, nigeria, were equally randomly distributed into fish tank and grouped fed. the fish were allowed to acclimatize for 15 days while being fed on commercial diet. prior to the commencement of the feeding trial, all fish were starved for 24 hours. this practice was to prepare the gastrointestinal tract for the experimental diet while at the same time to increase the appetite of the fish. feeding trial: the feeding trial was conducted in the wet laboratory of federal college of animal health and production technology, ibadan. the experimental system contained a set of 15 rectangular table 1. proximate composition of some feed ingredients. parameter fish meal soybean meal 5-min toasted jcm 10-min toasted jcm** maize moisture 9.75 10.70 3.80 3.80 10.48 crude protein 72.4 38.74 33.96 34.72 9.87 crude lipid 10.45 30.68 39.03 39.43 4.28 crude fibre 5.10 13.11 10.16 5.78 ash 8.32 4.48 6.09 4.25 6.73 *nfe 10.30 4.01 7.64 62.35 *nfe = nitrogen free extract; **jcm = jatropha curcas meal 100 jimoh et al./ toasted jatropha curcas seed meal in nile tilapia diet plastic tanks each with a capacity of 55 liters of water. 225 o. niloticus juveniles with initial weight of 15.19±0.02 g was used for the experiment. all fish were fed twice daily at a fixed feeding rate of 5% of their body weight per day. feeding was generally done in the morning at 09:00 a.m. and 05:00 p.m. in the evening. periodic weighing was done at two weeks interval and the feed adjusted as required. the procedures of jimoh and aroyehun (2011) was used in calculating growth performance indices. a table 2. nutrient composition (g/100 g) of experimental diets containing differently timed dry heat-treated jatropha curcass seed meal fed to oreochromis niloticus. ingredients ctr d520t d540t d1020t d1040t fishmeal 20.83 20.83 20.83 20.83 20.83 soybean meal 42.10 33.68 25.62 33.68 25.62 5-min toasted 9.42 18.85 10-min toasted 9.21 18.43 corn 10.00 10.00 10.00 10.00 10.00 fish premix 5.00 5.00 5.00 5.00 5.00 fish oil 2.50 2.50 2.50 2.50 2.50 starch 19.57 18.59 17.20 18.78 17.62 proximate composition moisture 9.95±0.17a 10.06±0.12a 9.70±0.17a 10.16±0.16a 9.94±0.25a crude protein 35.17±0.05a 35.18±0.08a 35.18±0.01a 35.17±0.01a 35.16±0.01a crude lipid 10.49±0.24a 10.66±0.31a 10.38±0.06a 10.38±0.01a 10.38±0.01a crude fibre 11.92±0.14c 12.40±0.49bc 12.60±0.11b 13.16±0.16a 13.27±0.40a ash 6.44±0.01b 6.50±0.03ab 6.52±0.03a 6.37±0.06c 6.19±0.02d nfe 26.04±0.1a 25.20±0.39bc 25.62±0.25ab 24.75±0.35c 25.06±0.33bc energy 4.00±0.03a 4.02±0.03ab 4.01±0.01ab 3.97±0.02b 3.99±0.01bc calculated amino acid profile arginine% 2.5 2.3 2.1 2.3 2.1 histidine% 0.8 0.8 0.7 0.8 0.7 isoleucine% 1.6 1.4 1.3 1.4 1.3 leucine% 2.6 2.3 2.1 2.3 2.1 lysine% 2.4 2.2 2.0 2.2 2.0 methionine% 0.7 0.7 0.6 0.7 0.6 m+c% 1.2 1.1 1.0 1.1 1.0 phenylalanine% 1.5 1.3 1.2 1.3 1.2 p+t% 2.6 2.3 2.1 2.3 2.1 threonine% 1.5 1.4 1.3 1.4 1.3 tryptophan% 0.4 0.4 0.3 0.4 0.3 valine% 1.7 1.5 1.4 1.5 1.4 calculated fatty acid profile loa (18:2n-6)% 5.1 4.2 3.3 4.2 3.3 lna (18:3n-3)% 0.8 0.6 0.5 0.6 0.5 ara (20:4n-6)% 0.1 0.1 0.1 0.1 0.1 epa (20:5n-3)% 0.3 0.3 0.3 0.3 0.3 dha (22:6n-3)% 0.7 0.7 0.7 0.7 0.7 total n-3% 1.8 1.7 1.6 1.7 1.6 total n-6% 5.2 4.2 3.4 4.2 3.4 n3:n6 0.4 0.4 0.5 0.4 0.5 loa: linoleic acid; lna: linolenic acid; ara: arachidonic acid; epa: eicosapentanoic acid; dha docosahexanoic acid; fatty acid and amino acid profiles of the experimental diets were calculated using a program (excel) developed by naca (2008) specification: each kg contains: vitamin a , 4,000,000 iu; vitamin b, 800,000 iu; vitamin e, 40,000 iu, vitamin k3, 1,600 mg; vitamin b1, 4,000 mg; vitamin b2, 3,000 mg; vitamin b6, 3,800 mg, vitamin b12 3 mcg; nicotinic acid 18000 mg. pantothenic acid 8,000 mg; folic acid 800 mg biotin, 100 mcg choline chloride 120,000 mg; iron 8,000 mg; copper 800 mg; manganese,6,000 mg; zinc 8,000 mg; iodine 400 mg; selenium, 40 mcg, vit c coated 60,000 mg, inositol 10,000 mg; cobalt,150 m, lysine 10,000 mg; methionine 10,000 mg, antioxidant 25,000 mg manufactured by bi-mix brrand, corporate head office/factory: 1, odo-olowu street, ijesatedo, lagos, nigeria. 101 int. j. aquat. biol. (2020) 8(2): 98-108 combined digital dissolved oxygen meter (ysi model 57, yellow spring ohio) was used to measure water temperature and dissolved oxygen; ph meter (mettler toledo – 320, jenway uk) was used to monitor ph weekly. bioeconomic analysis was done following the methods explained in faturoti (1989), abu et al. (2010), boateng et al. (2013) and jimoh et al. (2015b); straight line method of depreciation was used to evaluate the cost of aquaria tanks with the following properties. cost of plastic tanks n57,000 no of years (life span) 5 yrs. savage value 10% of cost price histology, haematological, and serum biochemistry analysis were done following the methods explained in jimoh et al. (2015c), fagbenro et al. (2013) and jimoh et al. (2015a), respectively. statistical analysis: data obtained from the experiment was expressed in mean±sd and it was subjected to one-way analysis of variance (anova) using spss 16.0 version. where the anova reveals significant difference (p<0.05). duncan multiple range test was used to compare differences among individual treatment means. results growth performance: table 3 reveals the growth performance of o. niloticus fed diets containing graded levels of differently timed dry heat-treated j. curcas seed meal. the weight gain of fish fed ctr was the highest which was significantly different (p<0.05) from the weight gain of fish fed other dietary treatments except the fish fed d520t. the lowest weight gain was observed among fish fed d1040t which was not significantly different (p>0.05) from the weight gain of fish fed d1020t. no significant variation (p>0.05) existed among the weight gain of fish fed d520t, d540t and d1020t. similarly, trends of the results as observed for weight gain was also as observed for final weight, table 3. growth performance of oreochromis niloticus fed diets containing graded levels of differently timed dry heat-treated jatropha curcass seed meal. parameters ctr d520t d540t d1020t d1040t initial weight (g) 15.19±0.02a 15.14±0.01a 15.17±0.01a 15.15±0.01a 15.19±0.01a final weight (g) 21.71±0.45a 21.08±0.17ab 20.77±0.65b 20.39±0.54bc 19.75±0.37c 1weight gain (g) 6.52±0.45a 5.94±0.16ab 5.60±0.65b 5.24±0.54bc 4.56±0.37c 2%wg 42.92±2.97a 39.23±1.01ab 36.92±4.30b 34.59±3.44bc 30.02±2.44c 3sgr (%/day) 0.64±0.36a 0.59±0.02ab 0.56±0.06b 0.53±0.05bc 0.47±0.03c food fed 7.71±0.54 7.05±0.13 6.77±0.67 6.52±0.02 5.87±0.53 4fcr 1.18±0.25c 1.19±0.02c 1.21±0.0bc 1.24±0.02b 1.29±0.02a protein fed 2.70±0.19 1.21±0.02 2.37±0.24 2.28±0.26 2.05±0.19 5per 2.41±0.05a 2.41±0.05a 2.36±0.04ab 2.30±0.04b 2.22±0.04c 6survival (%) 88.00±1.73 91.00±10.15 93.33±6.51 82.33±4.04 83.33±15.28 row means with different superscripts are significantly different (p<0.05) from each other. 1mean weight gain= final mean weight – initial mean weight; 2 percentage weight gain= [final weight-initial weight /initial weight] x 100; 3 specific growth rate= [in final weightin initial weight] x 100; 4 feed conversion ratio=dry weight of feed fed /weight gain (g); 5 protein efficiency ratio=fish body weight (g)/ protein fed; 6 percentage survival = {(total number of fishmortality)/ total number of fish] x 100. table 4. cost of production (n) of the experimental diets containing differently timed dry heat-treated jatropha curcas seed meal fed to oreochromis niloticus. price/kg ctr d520t d540t d1020t d1040t fishmeal 680 141.64 141.64 141.64 141.64 141.64 soybean 155 65.26 52.20 39.71 52.20 39.71 5-min toasted 68 6.41 12.82 10-min toasted 68 6.26 12.53 cornmeal 72 7.20 7.20 7.20 7.20 7.20 fish premix 700 35.00 35.00 35.00 35.00 35.00 fish oil 600 15.00 15.00 15.00 15.00 15.00 starch 50 9.78 9.29 8.60 9.39 8.81 total 273.88 266.74 259.97 266.70 259.90 102 jimoh et al./ toasted jatropha curcas seed meal in nile tilapia diet %weight gain and sgr, and a reverse trend was also observed for fcr. the per of fish exposed to diet ctr was the highest which was not significantly different (p>0.05) from the per of fish fed d520t and d540t. the per of fish exposed to diet d1040t was significantly the lowest among other treatments. there was no significant difference (p>0.05) in the per of fish fed diet d540t and diet d1020t. economic analysis cost of production of 1 kg diet: the cost (n) of producing 1 kg of each experimental diet containing differently timed dry heat-treated j. curcas fed to o. niloticus is presented in table 4. there was reduction trend in the cost of producing the diet with increasing inclusion of j. curcas when compared with control. table 5 shows the economic analysis of producing o. niloticus with diets containing differently timed dry heat-treated j. curcas seed meal. the incidence of cost, which is the cost of producing 1 kg tilapia fish, was highest using diet d1040t and lowest using diet d520t. there existed significant variation (p<0.05) in the incidence of the cost of the fish produced using different dietary treatments. however, there was no significant difference (p>0.05) in the incidence of the cost of producing 1 kg tilapia fish with diet d1040t, d1020t, and ctr. the profit index and gross profit of the produced with d540t was the highest which is not significantly different (p>0.05) from profit index and gross profit of fish produced by using diet ctr and d520t. table 6 shows the profitability analysis of producing o. niloticus with diets containing differently timed dry heat-treated j. curcas seed meal. benefit cost ratio (bcr) of producing fish with d540t was the highest which is not significantly different (p>0.05) from the bcr of fish produced with other dietary treatments except d520t and d1020t. bcr of fish produced by using diet d1040t was the table 5. economic analysis of producing oreochromis niloticus with diets containing differently timed dry heat-treated jatropha curcas seed meal. parameters ctr d520t d540t d1020t d1040t output biomass (kg) 21.71±0.45a 20.39±0.57ab 19.75±0.37b 21.08±0.17bc 20.77±0.65c cost of feeding (x103 ₦) 2.11±0.15a 1.88±0.0.36ab 1.76±0.17bc 1.74±0.0.20bc 1.53±0.00c incidence of cost (₦) 324.10±6.89ab 316.53±4.08b 314.57±5.20b 331.60±5.55a 334.40±5.41a value of fish (₦) 3.91±0.27a 3.57±0.96ab 3.36±0.39b 3.14±3.15bc 2.73±0.22c profit index (₦) 1.85±0.40ab 1.89±0.25a 1.91±0.30a 1.81±0.26bc 1.79±0.31c gross profit (₦) 275.90±6.89ab 283.47±4.10a 285.43±5.20a 268.40±5.55b 265.60±5.41b total variable cost (x103 ₦) 2.34±0.15a 1.96±0.20ab 1.75±0.14b 2.10±0.34bc 1.98±0.18c *total fixed cost (x103 ₦) 1.71 1.71 1.71 1.71 1.71 total cost (x103 ₦) 4.05±0.15a 3.67±0.20ab 3.46±0.14bc 3.81±0.35bc 3.69±0.18c total revenue (x103 ₦) 13.03±0.27a 12.23±0.32ab 11.85±0.22b 12.65±0.10bc 11.33±0.58c gross margin (x103 ₦) 10.80±0.42 10.40±0.23 10.69±0.41 10.60±0.00 10.40±1.58 net return (x103 ₦) 8.98±0.13a 8.56±0.12a 8.39±0.09ab 8.83±0.07bc 8.77±0.21c cost of juveniles: ₦15 1kg tilapia=n600 1 usd= n365 cost of aquaria tank 57,000 less 10% savage value 5,700 depreciation 10,260 value of aquaria tank (2 months) 1,710 table 6. profitability analysis of producing oreochromis niloticus with diets containing differently timed dry heat-treated jatropha curcas seed meal. parameters ctr d520t d540t d1020t d1040t benefit cost ratio 3.22±0.60b 3.32±0.15ab 3.38±0.51a 3.33±0.95ab 3.43±0.70c gross ratio 0.31±0.10a 0.30±0.00ab 0.29±0.06b 0.30±0.10ab 0.29±0.06b expense structure ratio 0.42±0.15c 0.45±0.00bc 0.46±0.21ab 0.46±0.25ab 0.50±0.25a rate of return 2.22±0.60b 2.32±0.15ab 2.38±0.51a 2.33±0.95ab 2.43±0.70a row means with different superscripts are significantly different (p<0.05) from each other 103 int. j. aquat. biol. (2020) 8(2): 98-108 lowest. rate of return of producing nile tilapia diets with different dietary treatments follow the same trends of results as accounted for bcr. gross ratio (gr) of fish with ctr was the highest but it was not significantly different from the gr of fish produced by using diets d520t and d1020t. the expense structure ratio (esr) followed reverse trends of results. haematology and serum biochemistry: table 7 shows the haematological and serum biochemical changes in the blood of o. niloticus fed diets containing differently timed dry-heat-treated j. curcas seed meal. the haemoglobin content of the blood of fish fed ctr was the highest while the lowest haemoglobin content was found in d1040t. there was significant difference (p<0.05) in the haemoglobin content of the blood of fish fed various dietary treatments. however, there was no significant variation (p>0.05) in the haemoglobin content of the blood of fish fed diets ctr and d520t. there was significant difference (p<0.05) in the total cholesterol of the blood of fish exposed to different dietary treatments. fish fed diet table 7. haematological and biochemical response in oreochromis niloticus juveniles fed diets containing differently timed dry-heat-treated jatropha curcas seed meal. parameters ctr d5020t d5040t d1020t d1040t pcv (%) 17.33±0.58a 16.87±0.15ab 16.53±0.05b 15.73±0.44c 15.13±0.06c hb (g/dl) 9.60±0.26a 9.20±0.36ab 9.10±0.36ab 8.70±0.20bc 8.17±0.31c rbc (x1012 mm) 1.38±0.02a 1.36±0.04a 1.32±0.03ab 1.29±0.03b 1.26±0.06b wbc (x106mm) 86.0±4.58a 83.67±2.51a 84.67±3.80a 87.00±6.60a 88.67±9.02a mch(ρɡ) 69.40±1.31a 67.63±1.40a 68.90±1.30a 67.63±1.80a 64.70±0.60b mchc (g/dl) 5.53±0.15a 5.43±0.25a 5.50±0.30a 5.53±0.06a 5.40±0.20a mcv (fl) 125.30±2.90a 124.10±4.40a 125.23±3.80a 122.23±3.82a 119.93±5.60a total cholesterol 141.6±16.80a 125.67±7.77ab 113.00±3.00bc 108.33±8.51bc 100.00±9.85c total protein(g/dl) 7.53±0.31a 6.70±0.20b 5.80±0.36c 5.60±0.20c 4.90±0.20d albumin (mg/dl) 5.13±0.21a 4.37±0.06b 3.73±0.21c 3.73±0.15c 3.50±0.27c globulin (mg/dl) 2.40±0.17a 2.33±0.25ab 2.07±0.15bc 1.87±0.06c 1.40±0.10d glucose(mg/dl) 84.33±2.52d 88.67±2.08cd 91.67±2.52bc 95.33±3.79b 102.00±3.00a row means with different superscripts are significantly different (p<0.05). figure 1. photomicrographs (5 µm, x400, h&e stained) of the liver of oreochromis niloticus fed: (a) diet ctr: diffuse vacuolar degeneration and necrosis of hepatocytes (long arrows) and moderate central venous congestion (short arrows). melanomicrophage centre (star); (b) diet d520t: severe diffuse hepatic necrosis (+). portal congestion (single head arrow); prominent melanomicrophage centre (double head arrow); (c) diet d540t: there is mild sinusoidal congestion. mild to moderate hepatic vacuolation (single head arrow). the melanomicrophage centre is not very prominent(double head arrow); (d) diet d1020t: there is very mild vacuolation of hepatocyte (single head arrow) and the melanomicrophage centre is not very prominent(double head arrow); (e) diet d1040t: there is mild to moderate diffuse vacuolar degeneration and necrosis of hepatocytes (arrow). 104 jimoh et al./ toasted jatropha curcas seed meal in nile tilapia diet ctr had the highest blood total cholesterol while fish fed diet d1040t had the lowest. however, there was no significant difference (p>0.05) in the blood total cholesterol of fish fed diets ctr and d520t. histology: figure 1 shows the photomicrographs (5 µm, x400, h&e stained) of the liver of o. niloticus fed diets containing differently timed dry-heat-treated j. curcas seed meal. diffuse vacuolar degeneration, necrosis of hepatocytes and moderate central venous congestion were recorded in the liver of fish fed control diet. severe diffuse hepatic necrosis cum prominent melanomicrophage centre were observed in the liver of fish fed d520t. mild to moderate hepatic vacuolation were observed in the livers of fish fed diet d540t; d1020t and d1040t: there is mild to moderate diffuse vacuolar degeneration and necrosis of hepatocytes (arrow) discussions based on the results, processing time and levels of inclusion of j. curcas had impact on the growth performance of nile tilapia. the growth performance of tilapia fed diets containing lower timed heat-treated j. curcas were comparable to that of control even at higher replacement level with soybean using fcr and per as indices of assessment. these results are in consonance with the work of workagegn et al. (2013) for nile tilapia juveniles fed the same seed meal replacing soybean meal that the growth performance of heat-treated j. carcass kernel meal was comparable to that of control at 10% replacement level. the results, however, differ from what kumar et al. (2011), who reported that inclusion of cooked jatropha seed meal in the diet of carp was possible at levels higher than 50% in the diet of fish without compromising their growth performance. plausible reasons might be the different heat treatments applied more so that the seed meal was completely detoxified of the phorbol esters (pes) which is a toxic compound in the seed meal (kumar et al., 2011). makkar et al. (2012) reported that detoxified j. carcass seed meal could replace more than 50% of fishmeal in the fish diets. although davies and gouveia (2008) reported that thermal processing of raw pea seed meal, dry heat treatment in particular (180oc:30 min), led to a greatly improved feed utilization and consequent better growth performances; various factors could imply the relatively poor growth performance recorded at higher processing time and inclusion level recorded in this study, one of which could be incomplete inactivation of the anti-nutrients in the seed meal, the principal of it being pes by the heat treatment applied which consequently led to poor digestibility of protein (kumar et al., 2008). kumar et al. (2011) reported that antinutrients in j. curcass seed meal especially the major toxic components called phorbol esters restricts its use in fish feed. lower growth observed among the group fed test dietary treatments might not be unconnected to reduced feed consumption recorded among the test dietary treatment group relative to control group. similar observation to what was observed in this study was made by paray et al. (2020) when cyprinus carpio was fed oak (quercus castaneifolia) leaf extract. this incidence of cost analysis of feeding j. curcas seed meal to o. niloticus showed the impact of the cost of feed on the variable cost of fish production since maintenance and sustenance of aquaculture depends on its economic viability and relative profitability (adeparusi and balogun, 1999). a profit index above 1 showed that it is profitable to feed the fish with the diet (jimoh et al., 2013). gross profit remains the primary interest on most capital investment, the gross profit margin and loan repayments where applicable form the basis after operational evaluation (faturoti, 1989). gross margin was reported to be a good measure of profitability (olagunju et al., 2007). the experiment showed that it is profitable to replace soybean meal with differently timed dry heat-treated j. curcas seedmeal using gross profit as an index of assessment. this result agrees with the finding of fagbenro et al. (2001), abu et al. (2010) and jimoh et al. (2012) that feeding fish with cheaper and lesser known feed ingredients left some profit margin. although the economic implication of using the different dietary treatments might not be well appreciated since the margin might be too small, it will be much explicit when the magnitude of total cost and 105 int. j. aquat. biol. (2020) 8(2): 98-108 expected revenue of its large-scale operation is critically and objectively considered (faturoti, 1989). adeparusi and balogun (1999) reported profit margin as increasing when fish meal was replaced by roasted pigeon peal meal in a diet fed to c. gariepinus. jimoh (2004) also reported an increase in profit margin in the production of tilapia up to 30% of soybean meal with jackbean meal. the reduced gross profit on tilapia produced by diet d1040t was due to lower growth rate of growth of fish. this result is in agreement with the report of jimoh (2004) that profit margin reduced with jackbean replacement level beyond 30%. this implies that feeding fish with differently timed dry heat-treated j. curcas seed meal had minimum impact on variable cost of production hence the profit recorded. the results showed that the average total variable and total cost of producing o. niloticus with the diets was met or covered by the total revenue realized from the sale tilapia and left a positive gross margin and net returns in all the dietary treatment groups. positive gross margin and net returns indicate that it is profitable to feed o. niloticus differently timed dry heat-treated j. curcass seed meal. a result similar to what was reported in jimoh et al. (2015b) when soybean meal was replaced by watermelon seed meal. (boateng et al., 2013) reported a positive operating profit when all variable cost of production of all male tilapia was covered by the gross revenue as an indicator of profitability of all male tilapian aquaculture enterprise in ghana. jimoh et al. (2014) reported similar trends of results on incidence of cost analysis when c. gariepinus was fed diets containing chrysophyllum albidum seed meal. the esr value reported in this study is lower than the value reported by adebayo and daramola (2013). the rate of return recorded in this study is higher than what boateng et al. (2013) reported on return on investment for all male tilapia farming in ghana. adebayo and daramola (2013) reported ror value of 0.62 for catfish production. the values recorded for some haematological parameters of the blood of nile tilapia fed diets containing differently timed dry-heat-treated j. curcas seed meal fell within the range of normal haematology of a healthy fish (fagbenro et al., 1993). the observed reduction in haematological parameters of the blood of nile tilapia fed diets containing differently timed dry-heat-treated j. curcas seed meal in this study might not be unconnected to the presence of antinutrients present in the seed meal. increase in white blood cell as observed in the fish fed differently timed dry-heat-treated j. curcas seed-meal based diets is attributed to increase in the production of leucocyte in the haematopoietic tissue of the kidney and perhaps the spleen (akinwande et al., 2016). the increase in white blood cell among the test dietary treatment groups may plausibly be induced by the anti-oxidant effect of bioactive compounds in j. curcas seed-meal based diets thereby improving the immune system of fish (hoseinifar et al., 2020). decrease in the blood total protein levels with higher levels of plant protein based diets may be attributed to the destruction of cells and consequent impairment in protein synthesis (singh and singh, 2002) and may be due to mobilization of protein to meet energy requirements and to sustain increased physiological activity (arellano-martinez et al., 2004). a reduction in blood cholesterol may be related to its utilization in the manufacture of cortisol arising from stress created by consumption of diets containing higher concentration of anti-nutrients similar to the observation of kumar et al. (2011). cortisol elevation due to stress always aggravate blood glucose (paray et al., 2020) consistent with the observation recorded in this study; blood glucose increased among the test dietary treatment groups relative to control group. the trend of increase in blood glucose levels recorded in this study is in line with the results of previous studies on common carp during stress (hoseini et al., 2019; hosseini and hoseini, 2012; yousefi et al., 2019, 2020) which are plausibly due to adaptive responses to provide energy required by fish during the stress condition (barton, 2002). reduction in albumin and globulin among the test dietary treatment groups when compared with control group is consistent with the result obtained in liver histology of this study; from control to test dietary treatment groups liver condition improved 106 jimoh et al./ toasted jatropha curcas seed meal in nile tilapia diet from degraded liver (graded 3 for control and d520t fed group) to intermediate liver (grade 2 for d540t, d1020t and d1040t fed groups) condition, respectively using the assessment of martínez-llorens et al. (2012), indicating some forms of hepatoprotection. significantly higher concentration of serum total protein in the control group might have been as a result of liver damage (coz-rakovac et al., 2005; francesco et al., 2012) which consequently results into reduced deamination capacity of the liver by reducing its aminotransferace ability (kavadias et al., 2003). the higher serum protein in the control might be due to protein mobilization as a substrate for hepatic glyconeogenesis (barcellos et al., 2003). some forms of hepatoprotection was also conferred to jatropha fed fish. conclusion and recommendation it is evident from the present study that soybean meal could be replaced up to 40% by incompletely detoxified j. curcass seed meal at lower dry heat processing time without compromising the growth performance of tilapia fish but with a mild negative impact on the haematological and biochemical profiles of the blood of the fish. references abu o.m.g., sanni l.o., erondu e.s., akinrotimi o.o. 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(2020) 8(3): 166-177 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article which is a better chelating agent in beluga, huso huso (l.), coriander, coriandrum sativum, or active charcoal? masoumeh bahrekazemi*,1mahboubeh eslami, sadaf samadaii, jaber nikbakhsh department of fisheries, qaemshahr branch, islamic azad university, qaemshahr, iran. s article history: received 21 december 2019 accepted 4 february 2020 available online 2 5 june 2020 keywords: growth active charcoal beluga chelating effect coriander detoxification abstract: to compare the chelating effect of coriander, coriandrum sativum, and active charcoal on beluga, huso huso, a total of 270 beluga (260±2 g) were initially fed with a diet containing 0, 5, 10, 15 and 20 g of coriander and charcoal per kg of diet for 60 days. the fish were then subjected to 1 mg/l of heavy metals (lead, cadmium, and copper) for seven days. the best growth and feeding performances before and after challenging with heavy metals were observed in fish fed with 15 g/kg active charcoal. also, the best growth performance before challenging with heavy metals was observed in fish fed with 5 and 10 g/kg coriander followed by fish fed with 10 and 15 g/kg coriander after the challenging stage. the growth and nutritional efficiency were better in coriander than the charcoal treatments, especially in the pre-challenge stage. no mortalities were observed in charcoal treatments at all. while mortality occurred only in fish fed with 20 g/kg coriander in the first stage, a 100% survival rate was observed only in the second stage of fish fed with 10 g/kg coriander. in both stages of the experiment, the highest percentages of carcass protein and lipid were obtained in fish fed with 10 g/kg coriander and 15 g/kg of charcoal. also, the lowest ash and the highest moisture were related to the control group. the amounts of protein and ash were higher in carbon treatments while the amounts of lipid and moisture were higher in coriander treatments. minimum amounts of cadmium, copper, and lead were obtained in fish fed with 15 and 20 g/kg charcoal and coriander. the concentration of the heavy metals was significantly lower in fish fed with carbon (p<0.05). therefore, 5-10 g/kg coriander is a better additive in low heavy metal concentration. but in an environment with a higher concentration of heavy metals, adding 15 g/kg active charcoal to beluga diet is recommended. introduction due to the effect of heavy metal pollutions on the health of fishes and consequently humans, using dietary supplements called chelating agents, which can absorb heavy metals and accelerate their excretion from the body, is of great importance. chelation is a process by which a chemical or natural compound binds a metal ion and holds it firmly. substances with such property have been used in the treatment of metal poisoning (aaseth et al., 2016). various chelating agents identified so far such as ethylene diamine tetraacetic acid (edta), methyl sulfonylmethane (msm), active charcoal (ac), coriander, garlic, and chlorella algae (mirzavand et al., 2015; abdeltawwab et al., 2017a). active charcoal refers to a group of charcoalaceous substances with high porosity *correspondence: masoumeh bahrekazemi e-mail: bahr.kazemi@gmail.com and high internal surface area, which are of paramount importance due to their significant internal area, powdery and porous structure, high absorbability, surface reactivation ability, and low cost compared to other adsorbents (bradley et al., 1996; yoo et al., 2005). it should be noted that negative side effects in the use of chemical chelating agents have also been reported. even edta, with much lower toxicity than msm, can excrete some metal elements like zinc. therefore, using natural substances with lower side effects and costs have attracted researchers (aaseth et al., 2016). coriander, coriandrum sativum, or chinese parsley, from the family of umbelliferae/ apiaceae, is a glabrous aromatic, herbaceous annual plant, with effective compounds of metallothionein and 167 int. j. aquat. biol. (2020) 8(3): 166-177 glutathione compounds, for removing metals such as mercury, cadmium, lead, copper, and aluminum (abidhusen et al., 2012). since coriander leaves can attach to heavy metal and excrete these particles from the body. coriander can also excrete heavy metals from the bloodstream (abidhusen et al., 2012). various studies have been reported the detoxification of heavy metals by ac in fish. also, they indicated ac positive effects on the growth efficiency, and the body chemical composition, including nile tilapia, oreochromis niloticus, (abdeltawwab et al., 2017a), red tilapia, o. mossambicus × o. niloticus, (michael et al., 2017) and japanese flounder, paralichthys olivaceus, (thu et al., 2010). moreover, the chelating effect of coriander, itself or in combination with other substances, has been studied in fish and other animals. for example, coriander has led to the non-accumulation of aluminum and lead in mice (aga et al., 2001; kansal et al., 2011). the chelating effect of coriander in iron excrement is much higher than parsley (mirzaei and khatami, 2013). also, the use of coriander, garlic, and chlorella algae in carassius gibelio has shown a positive effect (2% individually) on kidney protection against 10 mg/l of cadmium (nicula et al., 2016). besides, using 2% coriander powder in rainbow trout, oncorhynchus mykiss, diet reduced cadmium amount by 20-30% in the liver and kidney of fish (ren et al., 2006). beluga is one of the most important sturgeon species of the caspian sea with high economic value for producing meat and caviar (chebanov and galich, 2013; hoseini et al., 2013; gisbertet al 2014; asgari et al., 2014). since the caspian sea is polluted with heavy metals due to wastewater influx from farms and industries (parizanganeh and lakhan, 2007; saeedi saravi and shokrzadeh, 2013; saghali et al., 2014 abadi et al., 2018), bioaccumulation of heavy metals in the tissues and muscles of sturgeons with a lengthy culture is highly important. hence, the present study aims to investigate and compare the effects of two available and cheap chelating agents, coriander as a natural substance and active charcoal as a chemical substance, on growth efficiency, fish nutritional value, and chelating of heavy metals in beluga. materials and methods fish and experimental design: this research was carried out at qaraboron sturgeon culture center (sari, iran). a total of 270 beluga (with the average weight and length of 260±2 g and 40±1 cm, respectively, was randomly selected and stocked in 27 concrete ponds (1.70×1.50 m) with a depth of 0.6 m and a watering depth of 0.3 m. this trial consisted of a control group (no coriander powder and active charcoal addition), four experimental treatments received coriander powder (qaemshahr, iran), and four treatments received active charcoal (tina chem., iran) orally at 5, 10, 15 and 20 g/kg of diet (abdel tawwab et al., 2017a, b). each treatment had three replications. the first stage of the experiment lasted for 60 days and 12 fish were sampled from each treatment for carcass and heavy metal analysis. the rest fish were challenged with heavy metals for seven days as the second stage of the experiment (abdel tawwab et al., 2017a, b). table 1 represents the caspian sea water analysis in the qaraboron center regarding the concentration of heavy metals in 12 months, conducted by the laboratory of alborz health test, iran. accordingly, the highest amount of the above three heavy metals was related to lead (0.9 mg/l). therefore, heavy metals as lead nitrate, copper sulfate, and cadmium chloride (merch, germany) were each challenged at the concentration of 1 mg/l. it should be noted that in the pre-challenging stage, the fish rearing was carried out in ponds with continuous water exchange, while in the second stage, flowing water was not used to maintain the concentration of the above heavy metals at a constant concentration (1 mg/ l). therefore, the ponds were first watering and in addition to using oxygen, one-third of the ponds water was siphoned from the bottom especially after feeding and freshwater was added to the ponds together with the same doses of heavy metals (abdeltawwab et al., 2017a, b). water quality parameters: during the experimental process, the qaraboron center water was supplied from a well that flowed into the system after aeration. the water flow rate was 15 l/s for all ponds in the 168 bahrekazemi et al./ which is a better chelating agent coriander or active charcoal? center (chebanov and galich, 2013). the chemical properties of water are given in table 1. during the experiment, factors such as temperature, oxygen, and ph were measured daily using a multimeter (hi 9828, multiparameter meter, hanna instruments, united states). diet preparation: the basal diet used in this study was a pellet (2 mm) made by qaraboron company, iran (table 2). for coriander diets preparation, the coriander was washed and dried in a cool environment and circulating air which was away from direct sunlight. the leaves were then powdered by grinding machine, dissolved in distilled water (5 g of coriander in 70 ml of water), and finally, added to per kg of diet. about active charcoal, the diets were prepared manually too, by dissolving the active charcoal powder in distilled water (5 g of charcoal in 70 ml of water) and adding to per kg of diet. the diets were then grounded in a meat grinder. after re-pelleting, the diets were dried in an oven for 48 hrs and stored at 4 °c. the diets were prepared once a week and fed to fish 5 times a day at 24, 5, 10, 14, and 20 hrs until its satiation (chebanove and galich, 2013). growth and feeding parameters: the growth and nutritional performances were calculated based on the following formulae (abdeltawwab et al., 2017a). body weight gain (bwg, %) = final weight (g) – initial weight (g)/ initial weight (g) × 100 specific growth rate (sgr, %/day) = [(ln w2 – ln w1)/t2 – t1] × 100 where, lnw1= natural logarithm of initial weight, lnw2= natural logarithm of final weight and t2 – t1= experimental period. survival rate (sr, %) = (nt/n0) × 100 where nt = number of fish at the end of experimental period and n0 = number of fish at the beginning of experimental period. feed conversion ratio (fcr) = weight gain (g)/feed eaten (g) protein efficiency ratio (per) = protein eaten (g)/weight gain (g) nutritional value of fish flesh: before and after challenging with heavy metals, six fish per treatment were randomly selected for carcass composition analysis. the skin of the fish was isolated and crushed. after removing viscera, they were placed in a meat grinder (pars khazar, mg 1400, iran). this was performed separately for all treatments. afterward, they placed into a plate and the total weight of plate and fish was measured by a digital scale. the specimens were then inserted into an oven of 55 °c for 24 hrs and re-weighed with the same scale to get moisture content. the important parameters calculated for approximate fish carcass analysis were crude protein, total lipid, and total ash which were estimated by kjeldahl device (kjeltec auto analyzer, 2300 tecator, sweden), soxhlet (dissolving fat in ether), and standard method of aoac (1990) (placing samples in an electric furnace (herius-germany) at 550°c for 4 hrs), respectively. heavy metals in fish flesh: at the end of both stages of the experiment, six fish per treatment were table 1. physico-chemical parameters and heavy metals concentrations of the caspian sea and well waters used in this study. parameter parameters caspian sea water well water temperature (ºc) 13 (winter) 25 (summer) 17.1±0.2 dissolved oxygen (mg/l) salinity (ppt) ph 9-14 10-13 8.3-8.9 8±0.11 0.3±0.05 7.51±0.03 total alkalinity (mg/l) 260 370±2.01 total hardness (mg/l) 5100 500 ±3.00 nh4(mg/l) 0.080.096 0.576±0.032 no3 (mg/l) 3.5-11.1 23.79±2.21 (mg/l) 2no copper (mg/l) cadmium (mg/l) lead (mg/l) 0.011-0.11 <0.1 <0.01 0.013-0.9 0.01±0001 <0.002 <0.001 <0.0005 169 int. j. aquat. biol. (2020) 8(3): 166-177 randomly selected in each stage and kept at −20°c in the laboratory for five days for heavy metal analysis. to digest frozen flesh, all fish were placed in a dryer for 48 hrs until they completely dried. thereafter, the dry tissues (0.2 g) were separately powdered and poured into test tubes. after adding concentrated nitric acid (5 ml), samples were placed in ambient temperature for 1 hr. and completely digested on a hot plate at 100 °c for 3 hours. after cooling down, the volume of samples reached 25 ml by adding distilled water (okoye, 1991). in order to estimate the values of heavy metals (copper, lead and cadmium), the samples were filtered with acetate cellulose (0.2 µ) and the concentrations of heavy metals were measured in all fish by an atomic absorption device (model icpoes) and the following formula: c= gs ×v/w where c = metal concentration in the solid sample (mg/ g dry weight), gs = metal concentration in solution from digestion (ppm), v = dilution volume (ml) and w= the dry weight of the sample (g). statistical analysis: this research was conducted in a factorial design including one control group, four groups with coriander treatments, and four groups with active charcoal treatments. all data were tested for normal distribution by the kolmogorov– smirnov test. moreover, data were analyzed by two-way analysis of variance (factorial two-way anova). besides, mean values were compared by tukey’s test through spss software (version 23) at a 95% confidence level. results growth and nutritional performances before and after challenging with heavy metals: the results of pre and postchallenging with heavy metals showed that the highest bwg, sgr, and per and the lowest fcr amounts were obtained in fish fed with 15 g/kg active charcoal without significant differences with fish fed 10 g/kg carbon in the first stage and the other treatments except for per in the second stage (p>0.05). the best growth and feed utilization results were obtained in fish fed with 5 g/kg coriander without significant differences with fish fed 10 g/kg coriander before challenging with heavy metals, while in the next stage, the best growth and feeding parameters were obtained in fish fed with 10 g/kg coriander with no significant differences with the fish fed 15 g/kg coriander. in the first phase, sr was 100% in all carbon treatments which was the same for fish in all table 2. ingredients and proximate analysis of the experimental diets used in this study (nrc, 1993). ingredients amount (%) fish meal (57.35% protein) 37.07 wheat gluten 10 wheat flour 10 canola oil 5.5 soybean meal 24 meat meal 10 mineral premix* 1.5 *vitamin premix 1.5 anti-fungi 0.1 stable vitamin c 0.13 binder (molasses) 0.2 20 15 10 5 0 20 15 10 5 0 parameter (%) 33.5±0.04 33.7±0.09 33.9±0.003 34±1.4 34±0.6 34.0±0.01 34.2±0.08 34.4±1.00 34.4±1.2 34.5±0.3 protein 14.6±0.24 14.7±0.01 14.7±0.13 14.7±0.50 14.8±0.30 14.8±0.07 14.8±0.16 14.8±0.50 14.8±0.03 14.8±0.11 lipid 11.2±2.01 10.7±1.90 10.2±1.31 9.7±0.46 9.2±0.008 9.4±0.72 9.3±0.11 9.3±0.25 9.2±0.006 9.2±0.30 ash 14.0±0.66 14.4±2.05 14.5±1.98 14.5±1.91 14.8±0.80 14.5±1.00 14.5±1.36 14.5±1.7 14.5±1.08 14.5±0.92 moisture *mineral premix, provided following amounts per gram of premix: mg: 70 mg, fe: 50 mg, cu: 3 mg, co: 0.01 mg, mn: 20 mg, zn: 30 mg, i: 0.1 mg, se: 0.1 mg. *vitamin premix, provided following amounts per gram of premix: a: 1000 iu, d3: 200 iu, e: 10 iu, k: 2 mg, b1: 4 mg, b2: 4 mg, b12: 4 mg, pantothenic acid: 10 mg, niacin: 20 mg, pyridoxine: 4 mg, biotin: 0.02 mg, folic acid: 1 mg, c: 44 mg, choline chloride: 98 mg. 170 bahrekazemi et al./ which is a better chelating agent coriander or active charcoal? coriander treatments except fish fed with 20 g/kg coriander. in the second phase, the 100% survival rate was also observed in all carbon treatments but it was seen only in fish fed with 10 g/kg coriander. the comparison of carbon and coriander treatments showed that except sr and per (only in the prechallenging stage), coriander yielded better results than carbon in growth and nutritional parameters. except for per, no significant difference was obtained in fish fed with 15 g/kg carbon or coriander, in the first stage (p>0.05; table 3). in the next stage, a significant difference was only obtained in fish fed with 10 g/kg of carbon and coriander (p<0.05; table 4). carcass composition before and after challenging with heavy metals: the highest percentages of carcass protein was measured in fish fed with 10 g/kg coriander which was not significantly different from other treatments (except fish fed with 20 g/kg coriander in the pre-challenging stage and fish fed with 15 and 20 g/kg coriander in the post-challenging stage). the highest amount of lipid was obtained in fish fed with 10 g/kg coriander which was not significantly different from other coriander treatments in both stages (p>0.05; table 5). in charcoal treatments, however, the highest percentages of carcass protein and lipids were measured in fish fed with 15 g/kg carbon in both stages. in the first stage, carcass ash percentage did not differ significantly between all treatments while in the next stage, it increased significantly in 10, 15, and 20 g/kg coriander compare to other treatments. in carbon treatments, the percentage of carcass ash increased significantly with increasing carbon content in both table 3. growth and feeding parameters of beluga fed with different levels of coriander and active charcoal before challenging with heavy metals. parameter concentration (g/kg) chelating agent 0 5 10 15 20 body weight gain (%) coriander a88.33±3.25 ba127.88±3.03 ba121.09±13.42 aba100.44±4.84 aa96.07±7.14 active charcoal a88.33±3.25 ab88.92±3.13 ab91.89±2.96 ba100.74±0.01 cb75.66±2.91 specific growth rate (%/day) coriander a0.453±0.02 ba0.597±0.01 ba0.573±0.04 ca0.503± 0.02 aca0.487±0.03 active charcoal ac0.453±0.02 acb0.462±0.01 abb0.470±0.01 ba0.500±0.00 cb0.410±0.01 survival rate (%) coriander a100.00±0.00 a100.00±0.00 a100.00±0.00 a100.00±0.00 ba93.33± 0.00 active charcoal a100.00±0.00 a100.00±0.00 a100.00±0.00 a100.00±0.00 ab100.00±0.00 feed conversion ratio coriander a2.13±0.044 ba1.39±0.02 ba1.49±0.16 ca1.77±0.06 aa1.97±0.11 active charcoal a2.13±0.044 ab2.12±0.05 abb2.01±0.05 ba1.85±0.01 ab2.34±0.07 protein efficiency ratio coriander a1.71±0.048 ba2.10±0.04 ba1.98±0.21 aca1.65±0.05 ca1.49±0.09 active charcoal ad1.71±0.048 bda2.06±0.05 bcb2.18±0.06 cb2.36±0.01 adb1.87±0.05 data are presented as means ± standard deviation. different lowercase letters show significant difference of each parameter at different levels of coriander and active charcoal treatments with the control group and each other; whereas, different uppercase letters show significant difference of each parameter in each level of coriander and active charcoal (factorial twoway anova, p<0.05). table 4. growth and feeding parameters of beluga fed with different levels of coriander and active charcoal after challenging with heavy metals. parameter concentration (g/kg) chelating agent 0 5 10 15 20 body weight gain (%) coriander a3.75±0.33 aa4.47±0.74 ba6.03±1.01 aba4.95±1.02 aba4.42±0.59 active charcoal a3.75±0.33 aa3.88±0.10 ab3.91±0.14 aa4.27±0.18 aa3.50±0.31 specific growth rate (%/day) coriander a0.227±0.03 aa0.270±0.04 ba0.363±0.06 aba0.300±0.06 aa0.267±0.03 active charcoal a0.227±0.03 ab0.232±0.01 ab0.237±0.01 aa0.260±0.01 aa0.213±0.02 survival rate (%) coriander a0.00±93.33 aa0.00±93.33 ba100.00±0.00 ca0.00±97.67 da83.33±0.00 active charcoal a0.00±93.33 bb100.00±0.00 ba100.00±0.00 bb100.00±0.00 bb100.00±0.00 feed conversion ratio coriander a2.42±0.12 bca1.79±0.27 ba1.42±0.15 bca1.86±0.38 bca2.21±0.27 active charcoal ab2.42±0.12 aba2.26±0.07 abb2.13±0.06 aa1.94±0.08 aba2.62±0.25 protein efficiency ratio coriander a1.23±0.097 ba1.65±0.26 ca2.07±0.23 bda1.61±0.32 ada1.33±0.18 active charcoal a1.23±0.097 aa1.29±0.04 ab1.37±0.04 ba1.50±0.06 aa1.12±0.10 data are presented as means±standard deviation. different lowercase letters show significant difference of each parameter at different levels of coriander and active charcoal treatments with the control group and each other; whereas, different uppercase letters show significant difference of each parameter in each level of coriander and active charcoal (factorial twoway anova, p<0.05). 171 int. j. aquat. biol. (2020) 8(3): 166-177 stages of the study. however, no significant differences were observed in fish fed with 10, 15, and 20 g/kg carbon (p>0.05; tables 5, 6). also, in both stages of the experiment, the highest moisture was related to the control group with significant differences with other treatments (tables 5, 6). the comparison of fish nutritional value in both carbon and coriander treatments, the amounts of protein and ash were higher in carbon treatments while the amounts of lipid and moisture were higher in coriander treatments (tables 5, 6). in the first stage, significant differences in protein, lipid and ash contents were obtained only in fish fed with 15 g/kg carbon and coriander (p<0.05). this result was obtained in fish fed with 10 g/kg carbon and coriander after challenging with heavy metals (table 6). heavy metal amounts in beluga before and after challenging with heavy metals: for both stages of the experiment, the amounts of carcass heavy metals significantly decreased compared to the control group by adding both chelating agents to the diet, (p<0.05; tables 7, 8). the lowest levels of the above metals were obtained in fish fed with 15 and 20 g/kg coriander with an only significant difference about the lead amount in fish fed with 10 g/kg coriander in the first stages. in the second stage, there was no significant difference in the amount of copper between the coriander treatments. the lowest amounts of the other two heavy metals were also obtained in fish fed with 15 and 20 g/kg coriander. in the case of carbon treatments, the lowest amounts of the heavy metals were observed in fish fed with 20 g/kg carbon, which was only significant in terms of copper content compared to fish fed with 15 g/kg carbon in both stages of the study (p<0.05; tables 7, 8). a comparison of the results of the two chelating agents showed that the amounts of cadmium, copper, and lead were lower in the fish fed with activate carbon than the fish fed coriander. in the pre-challenging stage, no significant difference was only obtained in table 5. carcass composition of beluga fed with different levels of coriander and active charcoal before challenging with heavy metals. parameter concentration (g/kg) chelating agent 0 5 10 15 20 total protein (%) coriander a15.65±0.250 aa15.77±0.252 aa16.37±0.153 aba15.53±0.153 ba14.93±0.115 active charcoal a15.65±0.250 bca16.57±0.378 ba17.00±0.200 bb17.23±0.305 aca15.93±0.902 total lipid (%) coriander a8.58±0.265 ba8.833±0.153 ba9.008±0.115 ba9.000±0.000 ba8.900±0.100 active charcoal a8.58±0.265 ab8.400±0.100 bcb8.667±0.208 cb8.800±0.1000 ab8.500±0.100 total ash (%) coriander a1.88±0.08 aa1.67±0.15 aa1.70±0.10 aa1.77±0.15 aa1.67±0.06 active charcoal a1.88±0.08 bb2.01±0.02 cb2.39±0.03 cb2.43±0.03 cb2.54±0.11 moisture (%) coriander a74.30±0.100 ba72.97±0.235 ba72.77±0.030 ba71.91±0.080 ba71.75±0.089 active charcoal a74.30±0.100 bb70.02±0.965 cb71.50±0.500 ca71.83±0.289 bb68.93±0.950 data are presented as means±standard deviation. different lowercase letters show significant difference of each parameter at different levels of coriander and active charcoal treatments with the control group and each other; whereas, different uppercase letters show significant difference of each parameter in each level of coriander and active charcoal (factorial twoway anova, p<0.05). table 6. carcass composition of beluga fed with different levels of coriander and active charcoal after challenging with heavy metals. parameter concentration (g/kg) chelating agent 0 5 10 15 20 total protein (%) coriander ab15.29±0.115 aba15.38±0.202 aa15.80±0.346 bca15.07±0.115 ca14.60±0.529 active charcoal a15.29±0.115 bb16.50±0.200 bb16.66±0.210 bb17.15±0.150 ca14.63±0.077 total lipid (%) coriander a8.167±0.153 ba8.800±0.000 ba8.900±0.173 ba8.833±0.153 ba8.767±0.252 active charcoal a8.167±0.153 ba8.600±0.100 ab8.067±0.115 ba8.800±0.100 ba8.667±1.53 total ash (%) coriander a1.80±0.10 aa1.80±0.00 ba2.04±0.00 ba2.12±0.00 ba2.10±0.10 active charcoal a1.80±0.10 bb2.13±0.15 cb2.39±0.00 cb2.47±0.15 cb2.67±0.15 moisture (%) coriander a76.48±0.040 ba74.47±0.351 ba73.74±0.151 ba72.50±0.500 ba72.00±0.500 active charcoal a76.48±0.040 bb73.23±0.586 ba73.00±1.000 ca72.16±0.110 db70.12±0.080 data are presented as means±standard deviation. different lowercase letters show significant difference of each parameter at different levels of coriander and active charcoal treatments with the control group and each other; whereas, different uppercase letters show significant difference of each parameter in each level of coriander and active charcoal (factorial twoway anova, p<0.05). 172 bahrekazemi et al./ which is a better chelating agent coriander or active charcoal? cadmium amounts in the fish fed coriander and carbon (p>0.05; table 7). in the post-challenging stage, significant differences were obtained in the amounts of all three metals in fish fed with 15 and 20 g/kg coriander and carbon (p<0.05; table 8). discussions based on the results, the best growth and feeding performances before challenging with heavy metals were observed in fish fed with 5 and 10 g/kg coriander followed by fish fed with 10 and 15 g/kg coriander after the challenging stage. in carbon treatments, the best results were obtained in fish fed 10 and 15 g/kg carbon in both stages of the study. the positive effect of carbon and coriander on growth and nutrition efficiency was not significant in the second stage due to the short period of rearing at this stage. but in the first phase, in particular, the results indicated a higher efficacy of coriander than carbon. considerable effect of coriander and carbon on growth and feeding parameters is related to their ability in enhancing the status of the digestive system and food digestion, along with absorbing gases and food toxicants (pish jang, 2011; abdel-tawwab et al., 2017a). active charcoal enhances the activity of intestinal function by increasing the length of intestinal villi and enterocytes, thereby raising food intake associated with increased growth rate (michael et al., 2017). but coriander with more nutritional value can be more effective on growth rate than carbon. the aromatic oil in coriander is a digestive stimulant. the oil contains linalool and other important compounds include flavonoids, phenolic acids and mucilage (a soluble fiber) (bhat et al., 2014). ramakrishna et al. (2003) reported that the effectiveness of pancreatic lipase and amylase were increased through the supplementation of essential oils by coriander. coriander also contains some substances with mild anti-bacterial activity (kubo et al., 2004). although, in the post-challenging phase, due to the better effect of carbon on the removal of heavy metals, the difference in growth rate in carbon and coriander treatments was not significant. the effect of coriander on animals such as vanaraja chicken (kumari et al., 2014), and mice (aga et al., 2001; ren et al., 2013) has been widely investigated with a proven positive effect on growth. moreover, 1% dietary coriander led to the highest growth rate of broilers (ali taneh et al., 2016). according to table 7. heavy metal amounts of beluga flesh fed with different levels of coriander and active charcoal before challenging with heavy metals. parameter concentration (g/kg) chelating agent 0 5 10 15 20 mg/g) 3-cadmium (×10 coriander a5.00±1.000 aba3.33±0.577 ba2.33±0.577 ba2.00±0.000 ba2.00±0.000 active charcoal a5.00±1.000 ba2.33±0.577 bca2.00±0.000 bca1.33±0.577 ca0.33±0.577 copper (mg/g) coriander a1.517±0.058 aa1.433±0.058 ba1.167±0.058 ba1.200±0.100 ba1.200±0.000 active charcoal a1.517±0.058 bb1.300±0.000 cb1.000±0.000 db0.500±0.000 eb0.200±0.000 mg/g) 3-lead (×10 coriander a260.80±0.72 ba207.33±6.43 ca198.67±1.15 da150.30±0.26 da148.17±3.55 active charcoal a260.80±0.72 bb191.53±0.50 bb189.43±0.51 cb135.00±1.00 cb129.00±3.60 data are presented as means±standard deviation. different lowercase letters show significant difference of each parameter at different levels of coriander and active charcoal treatments with the control group and each other; whereas, different uppercase letters show significant difference of each parameter in each level of coriander and active charcoal (factorial twoway anova, p<0.05). table 8. heavy metal amounts of beluga flesh fed with different levels of coriander and active charcoal after challenging with heavy metals. parameter concentration (g/kg) chelating agent 0 5 10 15 20 mg/g) 3-cadmium (×10 coriander a9.00±0.577 aba7.00±1.000 bca5.67±0.577 cda4.33±0.577 da3.67±1.15 active charcoal a9.00±0.577 ba6.33±0.577 ca4.67±0.577 db1.67±0.577 db1.00±0.000 copper (mg/g) coriander a6.167±0.045 ba5.200±0.436 ba5.027±0.025 ba5.017±0.015 ba5.013±0.042 active charcoal a6.167±0.153 bb4.500±0.100 cb2.100±0.100 db0.600±0.000 eb0.267±0.058 mg/g) 3-lead (×10 coriander a930.33±15.04 ba834.00±15.09 ca731.33±20.13 da680.00±10.0 da660.33±26.65 active charcoal a930.33±13.61 ba795.67±5.86 ba763.67±14.84 cb232.00±22.00 cb223.67±16.20 data are presented as means±standard deviation. different lowercase letters show significant difference of each parameter at different levels of coriander and active charcoal treatments with the control group and each other; whereas, different uppercase letters show significant difference of each parameter in each level of coriander and active charcoal (factorial twoway anova, p<0.05). 173 int. j. aquat. biol. (2020) 8(3): 166-177 hosseinzadeh et al. (2014), a treatment containing 2% coriander reduced growth rate and raised gut content viscosity due to increasing crude fiber in the diet along with changes in the diet taste and palatability. this might end to a decrease in growth rate in beluga fed with 20 g/kg coriander. there are limited reports on the effect of coriander on fish growth rate. jia et al. (2009) for example, showed that the addition of coriander to rainbow trout diet did not effect on fish growth which was attributed to the types of fish diet and digestive enzymes. in this study, although 5 to 15 g/kg dietary coriander powder increased the growth of beluga, more coriander amounts might be toxic. abdel-tawwab et al. (2017a) have reported an optimal active charcoal amount of 7 g/kg diet for nile tilapia. on the other hand, pirarat et al. (2015) reported a better growth rate in fish fed with 20 g/kg carbon in the same species. no significant difference in the growth performance of nile tilapia was reported by using the same amount of active charcoal (boonanuntanasarn et al, 2014). there are contrasting results about other species, too. michael et al. (2017), for instance, introduced 30 g/kg carbon as the optimum amount in red tilapia while thu et al. (2010) reported 5 g/kg carbon as an appropriate amount for japanese flounder. the differences in optimum concentrations could be attributed to differences in the charcoal production source, and fish feeding habits. also, the absorption capacity of active charcoal depends on the challenging duration so that slowfeeding fish needs less active charcoal for better growth, while in those that feed rapidly, active charcoal has a lower time to expose to toxic compounds and toxic gases, resulting in their increased in charcoal demands (thu et al., 2010). although there is no report about the effects of coriander on nutritional parameters in fish, an improvement in fcr was reported by using 1% coriander powder in broilers (ghazanfari and mohammadi, 2015; ali taneh et al., 2016). the best fcr and per values in red tilapia were obtained in fish fed with 30 and 40 g active charcoal (michael et al., 2017) while in nile tilapia, fcr amounts were not significantly different by increasing charcoal (abdel tawwab et al., 2017a). here, no mortalities were recorded in charcoal treatments. likewise, abdel-tawwab et al. (2017a) have noticed a survival rate of 96.7-100% in nile tilapia, so that no toxic effects were observed in dietary active charcoal. this result was not obtained in using coriander especially at high concentration in this study. coriander can be added to the diet at 10-15 g/kg without mortality. it should be noted that there was no mortality in the fish fed active carbon even during the heavy metal challenge. the result was only obtained in fish fed with 10 g/kg coriander. it can relate to interactions between the two chelating agents and the heavy metals, which made the active carbon a better chelating agent. adding coriander to beluga diet, increased the amount of lipid and decreased the amount of carcass moisture. the highest percentage of carcass protein was obtained in fish fed with 10 g/kg coriander in both stages. the highest percentages of carcass protein and lipids were obtained in fish fed with 15 g/kg charcoal. also, the lowest carcass ash and highest moisture content were related to the control group. the comparison of carcass composition in both carbon and coriander treatments, the amounts of protein and ash were higher in carbon treatments while the amounts of lipid and moisture were higher in coriander treatments. this result showed that charcoal was more suitable as an additive to beluga diet. the decrease of carcass moisture is related to the increase of other carcass constituents such as protein, lipid, and ash as well as a high absorption of water by active charcoal (abdel-tawwab et al., 2017a). the result of this study is similar to other researchs about increasing carcass protein amount by a rising amount of dietary charcoal in nile tilapia (boonanuntanasarn et al., 2014; abdel-tawwab et al., 2017a), red tilapia (michael et al., 2017), and japanese flounder (thu et al., 2010). the above studies suggested that the digestibility of dietary protein increases dramatically in charcoal-contained diets, giving rise to the protein content of fish fed with active charcoal compared to the control group. also, the stimulatory effects of coriander essential oils on gastrointestinal secretions https://www.tandfonline.com/author/hayashi%2c+tetsuhito https://www.tandfonline.com/author/hayashi%2c+tetsuhito 174 bahrekazemi et al./ which is a better chelating agent coriander or active charcoal? result in more digestion and absorption of lipids in the digestive tract and hence, improving carcass lipid (ali taneh et al., 2016). mandal and mandal (2015) showed that fish fed with coriander had an increase in concentrations of eicosapentaenoic acid and docosahexaenoic acid. there are also different reports focused on other carcass components such as moisture, total lipid, and ash. for example, boonanuntanasarn et al. (2014), thu et al. (2010) and michael et al. (2017) reported no significant differences in the above-mentioned components other than protein, while abdel-tawwab et al. (2017a) reported a significant difference in all parameters. based on the results, exposure to environmental heavy metals did not affect protein and lipid amounts considerably which may because of the protective role of coriander and carbon in reducing the toxicity of heavy metals. furthermore, the decrease of lipid and protein contents is because of changes in their synthesis or degradation in the body after challenging with heavy metals since they provide energy against ensuing stress, a process observed in the control group. however, the effects of metals were largely neutralized by increasing coriander and charcoal concentrations, especially in fish fed with 10 and 15 g/kg carbon or coriander. in this study the highest amounts of cadmium, copper, and lead were measured in the control group while the lowest amounts were obtained in fish fed with 15-20 g/kg of active charcoal and coriander, respectively. the amount of copper in fish carcass was higher compared to cadmium and lead in both stages of the experiment. also, the amount of lead in beluga was higher than cadmium. similar results have been reported in three fish species of the caspian sea (saeedi saravi and shokrzadeh, 2013). comparing two chelating agents in this study, the amounts of cadmium, copper, and lead were lower in fish fed with active carbon than the fish fed with coriander. at very low concentrations of metals, no considerable difference was found but in cadmium amounts in fish fed with coriander and carbon while at 1 mg/l concentration of each metal, differences were obtained in fish fed with 15 and 20 g/kg coriander and carbon. in fact, at higher concentrations, carbon was a better chelating agent than coriander. several researches have shown increasing accumulation of heavy metals, particularly in the gills, liver and muscle of fish by rising concentrations of metals in the environment or fish diet (boonanuntanasarn et al., 2014; al-asgah et al., 2015). the present study also confirmed this result in fish muscles. also, our results indicated that although both substances were able to reduce the accumulation of heavy metals in beluga, active charcoal was more effective. active charcoal can reduce heavy metals bioaccumulation through excretion and /or absorption because of its micro-porous structure through surface adsorption mechanism. similar results were reported in nile tilapia in which heavy metal accumulation reduced by increasing the amount of charcoal (abdeltawwab et al., 2017a). metallothionein and glutathione compounds of coriander are combined with heavy metals; besides, accelerating the urinary excretion of these metals reduces the bulk accumulation of them (kansal et al., 2011). aga et al. (2001) suggested that coriander removes toxins from the cell and it was recommended to be mixed with other chelating agents for better disposal. moreover, nicula et al. (2016) reported that using 2% garlic powder with 2% coriander powder had the most protective role against cadmium toxicity in c. gibelio. coriander was shown to combine with cadmium chloride, stabilize it, and remove the metal in rainbow trout (ren et al., 2009). ren et al. (2006) reported that inclusion of 2% dried coriander powder by freezedrying reduced the 20-30% cadmium accumulation in the liver of rainbow trout; in fact, the accumulation of toxic form of cadmium was greater in the control group but its effect on preventing the kidney accumulation of cadmium was less. coriander also protects the cells against free radicals produced by the lead (wangensteen et al., 2004; kansal et al., 2011). lead can promote the generation of reactive oxygen, and affect antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase (tellez-lopez et al., 2017). 175 int. j. aquat. biol. (2020) 8(3): 166-177 conclusion the heavy metals were chelated better in fish fed with 15 to 20 g/kg of active charcoal and coriander. but considering other parameters such as growth performance, survival rate, and carcass composition led to better results in fish fed with 10 g/kg coriander and 15g/kg active charcoal. however, coriander is a better additive in an environment with a low concentration of heavy metals. but in an environment with a higher concentration of heavy metals, adding 15 g/kg active charcoal to beluga diet is recommended. acknowledgments this research received a grant from islamic azad university, qaemshahr branch, number: 813109. also, the authors are grateful to qaraboron company for the kind provision of the fish. references aaseth j., crisponi g., andersen o. (2016). chelation therapy in the treatment of metal intoxication. academic press of elsevier. 388 p. abadi m., zamani a., parizanganeh a., khosravi y., badiee h. 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(2020) 8(3): 178-183 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology short communication small pelagic fisheries condition in north sulawesi: a case study on traditional purse seine practice in likupang village, indonesia silvester benny pratasik*,1,inggrid akerina, nego elvis bataragoa, lefrand manoppo fisheries resources management, faculty of fisheries and marine science, sam ratulangi university, jl. kampus bahu, manado-95115, north sulawesi, indonesia. s article history: received 13 january 2020 accepted 2 june 2020 available online 2 5 june 2020 keywords: size composition, d. macrosoma, d. macarellus, maturity. abstract: this study was conducted to know the impact of traditional purse seine fisheries in likupang village, north minahasa regency, north sulawesi, on pelagic fish stocks of scad decapterus spp. by size at first maturity estimation. samples were collected from the purse seine catch of likupang fishermen. they were individually measured and dissected for maturity level examinations. the results showed a wide size range in d. macarellus catch and high catch of small individuals of d. macrosoma reflecting that traditional purse seiners in likupang could become potential fishing gear to deplete the stocks of scad. this situation makes the purse seine fishermen have to adjust the mesh size to the fishing target. introduction overfishing can occur when many small individuals are caught, young individuals enter the fishing ground, and efforts are maximized for fishing (pauly, 1988). thus, knowledge on growth, length at maturity and spawning season is crucial to detect when and at which length the fish should be protected (hunter et al., 1992) or to explain the variation of the level of population, such as estimation of fishing mortality, population of cohorts, and population of spawning stock (karna and panda, 2011) as well as to make efforts to increase the amount of fish harvest (das et al., 1989). size composition of catches should represent mature individuals, the highest catch of a cohort, and reflects the conservation of big-sized mature individuals (froese, 2004). size at first maturity is important information for the proper management and conservation of fish stocks, and the information is very helpful to researchers and policymakers for the preparation of very effective sustainable management plans of fishery resources (jennings et al., 1998; waddy and aiken, 2005; karna and panda, 2011; nandikeswari, 2016; peixoto et al., 2018). size at first *correspondence: silvester benny pratasik e-mail: spjong07@yahoo.com maturity (lm) is the length at which 50% of the fish have reached maturity and has been taken as a reference point of minimum size and a useful index for determining the size of the exploitable stock (siegel and loeb, 1994; jirapunpipat, 2008; nadikeswari, 2016). it is related to the reproductive cycle of fishes that can be seen through the seasonal development changes in their gonads (karlou-riga and economidis, 1996; gomiero et al., 2008) and affected by the environmental changes particularly temperature, photoperiod and food supply (bagenal, 1978). estimation of size at first maturity has been used to let adult individuals spawn at least once or to protect young individuals (fontoura et al., 2009), and can be employed as stock availability indicator and applied for the determination of mesh size used in fishing operation (carlucci et al., 2006; omar et al., 2015). information on fish gonad maturity is required to know the mature and immature fish ratio of a stock, size or age at the first spawning, spawning time, duration, and pattern etc. change in fish gonad size is expressed with gonad maturity level (kordi, 2010). each fish species can reach first gonad maturity at 179 int. j. aquat. biol. (2020) 8(3): 178-183 different body size, and same species that are distributed in the different latitude more than 5 degrees, can have different size and age at first gonad maturity due to different environmental conditions of their habitats. therefore, knowledge on size at first maturity is crucial, since it allows us to examine mature and spawning stocks for management of their exploitation (jennings et al., 1998) and developing a successful management program (gupta and triphati, 2017; tesfahun, 2018). scads, decapterus spp. are small pelagic fishes that have good economic value consumed in indonesia. they belong to five different species, including d. kuroides, d. ruselli, d. macarellus, d. macrosoma, and d. maruadsi, with a maximum individual size range of 40-50 cm and common size of 25-30 cm (www.fishbase.org). scads are target fish in light fishing in multiple hook-hand lines and purse seine fisheries. fishermen in likupang village, north minahasa, indonesia, have run mini purse seine as income source. this fishing gear is operated by encircling the fish school with net. this study is intended to describe the impact of traditional purse seine fisheries in likupang village on pelagic fish stocks, especially decapterus spp. by observations on size maturity of the fish catches and size composition. materials and methods fish samples were obtained from small purse seiner’s fishing operations in likupang village. the fishing grounds locate 1°41’05.54”n and 124°13’50.61e 1°59’30.42”n and 125°22’32.86”e. all dimensions of the purse seine were measured. the fish were randomly taken from the boat and separated by species type. body length was recorded in the standard length, the distance from the tip of the snout to the posterior end of the last vertebra. the scads were separated into several different size classes. then, they were dissected for gonad maturity examination. the maturity level was determined following effendie (2002) (table 1). data grouping into size classes was done using struges (1926) as follows: k = 1 + 3.3 log n where k = number of classes and n = number of data. for class interval determination, the following formula was used: c = 𝑋𝑛 − 𝑋1 𝑘 where c = class interval, xn = the largest data value, x1= the smallest data value, and k = number of classes. the data were then arranged from the smallest to the largest and grouped in the class interval. all data were plotted in a graph to present the size distribution. size at first maturity was estimated based on gonad maturity level and size class following spearmankarber equation (udupa, 1986) as follows: m = xk+ 𝑥 2 – (xʃpi) where xk = log of last size in which 100% of the fish are fully mature, x = log of size increment = xi+1 xi, i = 1, 2,…k-1, and xo = log of last size in which table 1. fish gonad maturity level (effendie, 2002). maturity stage note female male i immature small ovary up to ½ the length of the body cavity. it is transculent. oocyte does not appear. the testis is small up to ½ the length of the body cavity. it is whitish. ii maturing the ovary is about half the length of the body cavity. it is orange, transculent, and oocyte cannot be seen by the naked eye. the testis is about ½ the length of the body cavity. it is white and about symmetrical. iii ripening the ovary is about 2/3 the length of the body cavity. ovary yellow-orange, oocyte appears. ovary with blood vessels on the surface. no transparent eggs or transculent, eggs are still dark. the testis is about 2/3 the length of the body cavity. iv ripe the ovary is about 2/3 up to full of the body cavity. the ovary is orange-pink with blood vessels on the surface, eggs are apparent. the testis is about 2/3 up to fulfilling the body cavity. it is white-soft cream. v spent ovary shrinks down to ½ the body cavity. wall is thick. there may be dark and mature eggs in the ovary that disintegrate from absorption, dark or transcluent. testis shrinks down to ½ the body cavity. wall is thick. the testis is soft. 180 pratasik et al./ small pelagic fisheries condition in north sulawesi there is no fully mature fish, ri = number of fully mature fish at size group i, pi = proportion of fully mature fish at size group i, pi = rl/ni, if ni ≠ ni+1 for i = 1, 2, …k-1 and pi = ri/n, if n = ni = ni+1 for i = 1, 2…..k-1. mean size at first maturity was obtained by antilog (m) = m. results and discussion decapterus malarellus (n = 89) and d. macrosoma, (n = 50) caught by traditional purse seiners in likupang, randomly collected from one of the fishing vessels. sex composition consisted of 24 males and 65 females for the former and 36 males and 14 females for the latter. gonad maturity of d. macarellus catches revealed that male size ranged from 117-166 mm dominated by gonad maturity i and ii, and one individual with maturity iii. females had a size range of 117-131 mm belonging to gonad maturity of i, while in the size range of 132-161 mm gonad maturity of i and ii dominated, but there were individuals with gonad maturity of iv. at the size range of 162-176 mm, a small number of females have reached gonad maturity of iv and v, but the larger size was dominated by figure 1. the size distribution of decapterus macarellus, catch: orange – male; grey – female. figure 2. the size distribution of decapterus macrosoma, catch: orange male; grey female 181 int. j. aquat. biol. (2020) 8(3): 178-183 individuals of gonad maturity, and then no fish were found in gonad maturity level. therefore, d. macarellus reaches the first gonad maturity at the size larger than 176 mm. estimation of size at first maturity calculation found that d. macarellus reached the first maturity at 177 mm long in the size range of 177-191 mm (fig. 1). in west sulawesi waters, nur et al. (2017) found that d. macarellus reached the first maturity at a larger size, 224 mm long for male and 188 mm long for female. size composition of d. macrosoma catch ranged 120-185 mm (fig. 2) with the highest catch at the class interval of 131-141 mm (n = 21) and the lowest catch at the class interval of 175-185 mm {n = 4). decapterus macrosoma reaches the first maturity at 163 mm in a range of 153-163 mm. other previous studies have found different size at first maturity for the same species, 143-149 mm (prihartini et al., 2006) with first spawning at 145-151 mm in jawa sea, and 195 mm for male and 210 cm for female in bone bay waters (dahlan et al., 2015), in which males reach gonad maturity earlier than females. these differences could be as a result of different geographic localities influencing the ecological conditions (blaxter, 1989). since the size at maturity may vary between populations in different geographic locations, detailed information of the reproductive biology of widely distributed species is critical for developing effective management approaches (herrera et al., 2016). the size composition of the catch reflects that traditional purse seiners in likupang catch also immature fish so that it could degrade the scad populations in north sulawesi waters. fingerlingsized scads are taken as well since they have a market value at both local and national levels. fisheries companies purchase the small sized-individuals for cheaper price to meet the national market demand. this condition shows that scad populations in this area get sufficiently high fishing pressures, even though fish maturity in earlier stage could occur as an adaptation to the fishing pressure because size at maturity could respond very quickly both to natural selection and to additional selective pressures such as those caused by fisheries. high fishing pressures could make mean length at sexual maturity of a population decrease in response to the removal of large individuals (motta et al., 2005), and recovery rate is related with mortality rate in which the closer the length at first maturity to the maximum length is, the lower the mortality rate or the population pressure (ectf, 2004). the present data show a wide size range in d. macarellus catch and high catch of small individuals of d. macrosoma reflect that traditional purse seiners in likupang could become potential fishing gear to deplete the decapterus spp. and other small pelagic fish groups, such as euthynnus spp., and large pelagic fish groups, such as tuna, that have similar schooling behavior. this situation makes the purse seine fishermen have to adjust the mesh size to the fishing target. therefore, the use of larger mesh-size at the pocket part of the traditional purse seine should be established to let the fish spawn at least once before caught and save the small individuals from exploitation for the small pelagic fish target. references bagenal t.b. 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(2018) 6(5): 274-280 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article how did dams affect length-weight and length-length relationships of capoeta razii (cyprinidae) in sefid river, the southern caspian sea basin? hamed mousavi-sabet*1,2, adeleh heidari1, meysam salehi3 1department of fisheries sciences, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. 2the caspian sea basin research center, university of guilan, rasht, iran. 3abzi-exir aquaculture co., agriculture section, kowsar economic organization, tehran, iran. s article history: received 11 july 2018 accepted 14 october 2018 available online 2 5 october 2018 keywords: sefid river caspian sea population dam abstract: fish populations are affected by dams in terms of morphology, reproduction, migration, growth rate and etc. to examine the hypothesis; how dams can affect the length-weight and lengthlength relationships in cyprinids, four capoeta razii segregated populations (by dams) were studied. the length-weight (lwr) and length-length (llrs) relationships were calculated for three populations from upstream, dam lakes and downstream of manjil and tarik dams in sefid river, in the southern caspian sea basin. also, one independent population from the damming impacts was considered as the control, to examine other possible annual effects on lwr. the b values in the lwr ranged from 2.893 to 3.586 in downstream and dam lakes populations, respectively. the r2 value ranged from 0.966 to 0.988. the averages of recorded length and weight in dam lake population were up to two and six times (respectively) more than the ranges in up and downstream populations. monthly lwr is presented for the control population. the sex and maturity were found as effective factors on lwr in the control population. no significant differences were observed in lwr by seasons. all llrs were highly significant (r2> 0.95). this study presents that the dams and the sex and maturity can be considered as effective non-biological and biological factors (respectively) affecting growth patterns as expressed by length and weight relationships in cyprinid (c. razii) populations. the results may be helpful in future fisheries studies and conservation programs. introduction knowledge of length-weight relationship (lwr) and length-length (llrs) relationships is an important tool for the adequate management of fish stocks and populations (king, 2007; ndome et al., 2012; qiang et al., 2013; mousavi-sabet et al., 2013, 2014, 2015a; zamani faradonbeh et al., 2015). the lwr is useful in local and interregional morphological and lifehistory comparisons across species and populations, also represent an important fishery management tool. llrs also have great importance for comparative growth studies (moutopoulos and stergiou, 2002; heidari et al., 2018, mousavi-sabet et al., 2015b, c). sefid river is one of the most important rivers in the southern caspian sea basin with 765 km in length. the manjil and tarik dams were constructed on sefid river in 1962 and 1968, respectively (najmaii, 2004), *correspondence: hamed mousavi-sabet doi: https://doi.org/10.22034/ijab.v6i5.534 e-mail: mousavi-sabet@guilan.ac.ir which have effectively fragmented the river into upstream and downstream and have probably blocked the migration of the fish among up and downstream. the manjil dam (also known as the sefidroud dam) is a buttress dam on the sefid river near manjil (guilan province, iran). it was constructed as a water reservoir for irrigation and produce hydroelectric power. it is 106 m high and forms a reservoir with a capacity of 1.82 km3. the tarik dam is located 35 km downstream from manjil dam and diverts releases from manjil dam for irrigation (najmaii, 2004). heidari et al. (2013, 2014) showed that the dams could affect morphological traits of fish populations in the river. the genus capoeta contains potamodromous cyprinid fishes, which capoeta razii being an important native species in sefid river (zareian et al., 275 int. j. aquat. biol. (2018) 6(5): 274-280 2016; esmaeili et al., 2018) and for this species lwr studies are still poorly known from the southern caspian sea basin. also, there is currently no report about the impact of dams on lwr and llrs for any fish species, inhabiting the caspian sea basin. according to litterateurs (jageret al., 2001; heidari et al., 2013, 2014; mousavi-sabet et al., 2015a, 2016a, b): (1) the lwr can be affected by biological and nonbiological factors, and (2) dam separate fish populations and make different habitats in upstream and downstream which impacts on fish morphology and growth parameters; therefore, the present study aimed to assess the impact of dams on length-weight and length-length relationships of c. razii as the case species here. materials and methods fish were collected from upstream, dam lake and downstream of dams and a population as control, specimen’s measurements, analysing and estimating possible relations of the studied parameters with different habitats caused by dam constructor. for this purpose, a total of 439 individuals of c. razii were collected by electrofishing (energy level: 200-300 v; distance between electrodes: 10-50 meters; area covered each time: 100 meters; water depth covered: 40-120 cm; substrate of the fishing areas: with diverse structures, including pools, riffles, gravel bed, sandy shore, etc.) from sefid river in the southern caspian sea basin, northern iran. of the total samples, 108 adult specimens (sex ratio 1:1) were caught in november 2014 from three sampling sites according to dams locations in the river, including upstream of manjil dam (37°18′29″n, 49°15′25″e; n = 44), dam lake (36°46′52.86″n, 50°1′18″e; n = 30) and downstream of tarik dam (37°39′15″n, 49°58′11″e; n = 33) (fig. 1). also, to estimate the effects of biological factors on lwr, 331 specimens were figure 1. location of sampling sites. upstream and downstream of manjil and tarik dams on the sefid river, southern caspian sea basin, iran 276 mousavi-sabet et al./ effect of dam on length-weight and length-length relationships of capoeta razii collected monthly (during september 2013 to august 2014) from tutkabon stream in the river drainage, as an independent and protected stream from the damming impacts (37°01′16.82″ to 36°50′44.71″n, 49°37′56.86″ to 49°35′01.23″e), which considered as control population. the sampled fish were fixed in 10% formaldehyde upon capture and transported to the laboratory for further analysis. in the laboratory for each individual, total length (tl), standard length (sl) and fork length (fl) (cm) were measured and whole-body weight (w) was taken using a digital balance to the nearest 0.1 g. the lwr of the fishes was calculated by using the formula w = a lb (le cren, 1951; froese, 2006; froese et al., 2011), where w is the total body weight in grams and l is the total length (tl) in cm. regression parameters a and b of the lwr were estimated by linear regression equation log tw = log a + b log tl. for lwr with r2< 0.95, the regression was repeated after removing outliers (froese, 2006). additionally, 95% confidence limits (cl) of b were estimated. the model fit to the data was measured by the coefficient of the pearson r-squared (r2) test. the null hypothesis that b = 3 was tested using two tailed t-test as described by zar (1999). tl vs fl, tl vs sl and sl vs fl relationships were also estimated by linear regression (zar, 1999). all statistical tests were at  = 0.05. results the mean (±sd) and range of total length, sample size, weight range, length-weight parameters a and b, the standard error of the slope, and the coefficient of determination (r2) for the studied populations of c. razii are given in table 1. monthly lwr for the control population is given in table 2. maximum recorded total length and body weight were 23.5 cm and 162.60 g respectively. also, llrs were found to be highly correlated (in all cases: r²>0.95, p<0.001) (table 3). the average recorded tl in dam lake population was 17.51±2.91 cm (vs. 9.99±3.41 and 9.24±2.02 in up and downstream populations) and average recorded weight was 79.66±43.64 g (vs. 13.76±11.93 and 11.50±9.88 in up and downstream populations). the values of coefficient of determination ‘r2’ calculated for all relationships viz., tl-wt, tl-sl, tl-sl and sl-fl for the studied populations of c. razii are shown in tables 1 and 3 which are highly significant (p<0.001). discussion this study presents new records of maximum total length for c. razii in sefid river from the southern caspian sea basin. maximum recorded total length for capoeta gracilis (now is c. razii in the southern caspian sea basin) was reported to be 41 cm from turkey in fishbase database (froese and pauly, 2015). the b values in the lwr ranged from 2.893 in downstream to 3.586 in dam lake populations, which table 1. descriptive statistics and estimated parameters of length-weight relationship for capoeta razii in up and downstream of manjil and tarik dams in sefid river, iran. locality n total length (cm) total weight (g) relationship parameters t value gro wth range mean ± sd range mean ± sd log a b r2 seb ±95% ci of b upstream 40 5.815.3 9.99±3.41 4.744.6 13.76±11.93 -1.9303 2.939 0.988 0.052 2.835-3.43 -1.1731 -a dam lake 30 11.321.7 17.51±2.91 15.4162.6 79.66±43.64 -2.6089 3.586 0.966 0.172 3.218-3.953 3.4011 +a downstream 34 6.916.2 9.24±2.02 4.157.3 11.50±9.88 -1.7937 2.893 0.983 0.067 2.758-3.029 -1.5970 -a tutkabon stream (control population) 33 1 3.923.5 11.26±3.30 2.3126.7 21.38±20.57 -4.8735 2.735 0.903 0.025 2.67-2.83 -1.5825 -a sd = standard deviation, n = sample size; a and b, the parameters of the length-weight relation, seb = standard errorof the slope, r2 = the coefficient of determination, t value (difference of b from 3), ci = confidence interval, +a = allometric (+), -a = allometric (-) 277 int. j. aquat. biol. (2018) 6(5): 274-280 table 2. descriptive statistics and estimated monthly lwrs for both sexes of capoeta razii in sefid river from the southern caspian sea basin (sep 2013 to aug 2014). month sex n total length (cm) weight (g) regression parameters min max min max a b r2 sep m 18 7.1 13.1 4.45 23.97 0.0170 2.827 0.975 f 12 7.5 14.2 5.11 32.10 0.0159 2.863 0.983 oct m 13 7.0 13.4 3.16 19.77 0.0127 2.874 0.986 f 5 6.7 9.3 2.74 8.010 0.0054 3.262 0.971 nov m 17 7.7 14.5 5.80 37.30 0.0161 2.864 0.990 f 14 6.2 17.5 3.03 53.07 0.0194 2.769 0.989 dec m 30 7.3 14.2 4.40 41.00 0.0087 3.121 0.982 f 16 5.6 15.3 2.30 36.50 0.0161 2.836 0.992 jan m 9 7.5 19.5 4.21 96.42 0.0080 3.141 0.996 f 10 6.0 14.8 2.77 32.42 0.0195 2.738 0.997 feb m 41 7.2 17.8 4.10 55.88 0.0114 2.978 0.968 f 8 9.2 20.0 8.13 109.93 0.0050 3.331 0.991 mar m 27 8.5 15.3 7.63 37.92 0.0255 2.694 0.974 f 4 14.5 20.7 34.65 99.00 0.0136 2.943 0.992 apr m 25 10.2 15.3 13.20 42.40 0.0202 2.800 0.968 f 5 14.7 21.13 43.30 126.00 0.0200 2.848 0.979 may m 4 13.0 18.5 21.90 65.00 0.0071 3.112 0.988 f 5 12.0 15.0 18.10 33.00 0.0403 2.478 0.970 jun m 12 9.5 15.0 11.39 35.29 0.0433 2.465 0.973 f 8 7.0 23.5 3.74 124.66 0.0124 2.929 0.998 jul m 34 7.5 16.5 6.20 62.70 0.0209 2.819 0.984 f 3 15.0 18.5 40.90 70.00 0.0308 2.644 0.981 aug m 14 7.0 16.0 4.24 44.12 0.0108 3.034 0.991 f 3 14.0 15.5 35.10 48.01 0.0085 3.158 0.985 over all m 244 7.0 19.5 3.16 96.42 0.0151 2.894 0.966 f 83 5.3 23.5 2.30 126 0.0105 3.036 0.990 immature 4 7.0 9.5 4.35 10.95 0.0139 2.946 0.989 table 2. length-length relationships between total length, fork length and standard length of capoeta razii in sefid river, iran. locality equation n a b r2 tl = a + bfl 0.057 1.010 0.994 upstream fl = a + bsl 44 0.014 0.057 0.994 sl = a + btl 0.968 0.119 0.994 tl = a + bfl 1.073 0.130 0.974 dam lake fl = a + bsl 30 1.090 0.146 0.990 sl = a + btl 0.820 0.285 0.957 tl = a + bfl 0.018 0.056 0.989 downstream fl = a + bsl 34 1.042 0.083 0.983 sl = a + btl 0.918 0.153 0.979 tutkabon stream (control population) tl = a + bfl 0.986 0.062 0.992 fl = a + bsl 331 0.972 0.099 0.989 sl = a + btl 1.029 0.137 0.991 n = sample size, a = intercept, b = slope, r2= coefficient of determination 278 mousavi-sabet et al./ effect of dam on length-weight and length-length relationships of capoeta razii was 2.735 in the control population. overall, the values of parameter b, which varies between 2 and 4, mostly remained within the expected range of 2.5-3.5 (froese, 2006), with extreme values of 2.893 in downstream and 2.939 in upstream populations. in terms of growth type, the results showed that upstream, downstream and control populations had negative allometric growth pattern while the dam lake population had positive allometric growth. the differences between growth patterns can be explained by the differences between the different habitats and food availability, which up and downstream habitats are shallow and free flowing river with low primary production in compare with the dam lake (fig. 2). in addition, water flow and quality in the up and downstream are affected by the damming, which are normally fast and muddy (fig. 2). the present study has revealed that the manjil and tarik dams on the sefid river have created differences in length and weight relationship of c. razii in upstream and downstream. the lwr in fishes can be affected by a number of factors, including season, habitat, gonad maturity, sex, diet, health and preservation techniques of the captured specimens (jager et al., 2001; mousavi-sabet et al., 2017a, b; alavi‐yeganeh et al., 2018), which were considered for the control population in the present study. thus, differences in lwr among different studied populations could be potentially attributed to differentiation of habitats caused by the dams construction. it was also previously suggested that these dams separated capoeta populations morphologically (heidariet al., 2013, 2014), which could be cause by the differences in growth parameters. dams influence on upward migration of fish especially that of the migratory species and prevent of migration them resulting in an ecological trap for fishes that ascend the fish passages (pelicice and agostinho, 2008). fragmentation of the river converts a free-flowing river into reservoir habitat, affecting the ecosystem and this in turn affects the migration patterns of fish populations (jageret al., 2001; mohadasi et al., 2013; jalili et al., 2015). in conclusion, the present study indicates that there exist different length-weight and length-length relationships in populations of c. razii in upstream, figure 2. habitats in the sampling sites: (a) upstream of manjil dam, (b) manjil dam lake, (c) downstream of tarik dam, (d) stream tutkabon. 279 int. j. aquat. biol. (2018) 6(5): 274-280 dam lake and downstream of the manjil and tarik dams on the sefid river, and the probable reason for the differentiation of these populations are the construction of the dams. this study provides basic information and suggests that biological variations observed in c. razii should be considered in fisheries management and commercial exploitation of this species and any stock enhancement program. acknowledgements the authors are grateful to h. bagherpour, a. habibi, m. mohammadi-darestani, m. mansuro-chorehi and k. ghasemzadeh for their help in fish collections. references alavi‐yeganeh m.s., sharifiniya m., ghasemzadeh j., amoeii m., mousavi‐sabet s.h. (2018). length–weight and length–length relationships of three blenny species from the persian gulf and the gulf of oman. journal of applied ichthyology, 34: 1238–1240. esmaeili h.r., sayyadzadeh g., eagderi s., abbasi k. (2018). checklist of freshwater fishes of iran. fishtaxa, 3(3): 1-95. froese r., pauly d., (2016). fishbase. world wide web electronic publication. available at: http://www.fishbase.org. (accessed on 29 february 2016). froese r. (2006). cube law, condition factor and weight– length relationships: history, meta-analysis and recommendations. journal of applied ichthyology, 22: 241-253. froese r., tsikliras a.c., stergiou k.i., (2011). editorial note on weight-length relations of fishes. acta ichthyologica et piscatoria, 41 (4): 261-263. heidari a., mousavi-sabet h., khoshkholgh m., esmaeili h.r., eagderi s. (2013). the impact of manjil and tarik dams (sefidroud river, southern caspian sea basin) on morphological traits of siah mahi capoeta gracilis (pisces: cyprinidae). international journal of aquatic biology, 1 (4): 195-201. heidari a., mousavi‐sabet h., sattari m., alavi‐yeganeh m.s., abbasi k. (2018). length–weight and length– length relationships for two gobiids from the anzali wetland, in the southern caspian sea basin. journal of applied ichthyology, 34: 1042-1044. heidari a., khoshkholgh m., mousavi-sabet h. (2014). tracing the effects of sefidroud dams on capoeta gracilis (cyprinidae) populations using truss distances in southern caspian sea basin. iranian journal of ichthyology, 1 (2): 106-113. jager h.i., chandler j.a., lepla k.b., winkle w.v. (2001). a theoretical study of river fragmentation by dams and its effect on white sturgeon populations. environmental biology of fishes, 60: 347-361. jalili p., eagderi s., keivany y. (2015). body shape comparison of kura bleak (alburnus filippii) in aras and ahar-chai rivers using geometric morphometric approach. research in zoology, 5(1): 20-24. king m. (2007). fisheries biology, assessment and management. wiley-blackwell, 400 pp. le cren e.d. (1951). the length-weight relationship and seasonal cycle in gonad weight and condition in the perch (perca fluviatilis). journal of animal ecology, 20 (2): 201-219. li q., x.u r., huang j. (2013). length-weight relations for 20 fish species from the pearl river, china. acta ichthyologica et piscatoria, 43 (1): 65-69. mohadasi m., shabanipour n., eagderi s. (2013). habitatassociated morphological divergence in four shemaya, alburnus chalcoides (actinopterygii: cyprinidae) populations in the southern caspian sea using geometric morphometrics analysis. international journal of aquatic biology, 1(2): 82-92 mousavi-sabet h., habibi a., bagherpur o. (2013). studies on length-weight and length-length relationships, relative condition factor and fulton’s condition factor of hemiculter leucisculus (pisces: cyprinidae) from the southwestern caspian sea basin. our nature, 11 (1): 25-30 mousavi-sabet h., heidari a., fekrandish h. (2015c). population structure, length-weight and length-length relationships of six populations of the bartail flathead platycephalus indicus (scorpaeniformes: platycephalidae) along the persian gulf coastal waters. journal of threatened taxa, 7(1): 6810-6814. mousavi‐sabet h., heidari a., paknejad s. (2015b). length‐weight and length‐length relationships of the genus alosa (clupeoidei: clupeiformes: clupeidae) along the southern caspian sea coast. journal of applied ichthyology, 32: 129-130. mousavi‐sabet h., heidari a., vatandoust s. (2017a). length–weight and length–length relationships for 11 species of the genus alburnoides jeiteles, 1861 (cyprinidae) from iran. journal of applied ichthyology, 33: 609-612. 280 mousavi-sabet et al./ effect of dam on length-weight and length-length relationships of capoeta razii mousavi‐sabet h., heidari a., vatandoust s. (2017b). length–weight and length–length relationships for nine species of the genus paracobits (nemacheiliidae) in iran. journal of applied ichthyology, 33(3): 617-619 mousavi‐sabet h., heidari a., mohamadi‐darestani m., mansouri‐chorehi m., ghasemzadeh k. (2016a). length‐weight relation‐ships and condition factors of two fish species from the southern caspian sea basin: alburnoides samiii mousavi‐sabet, vatandoust & doadrio, 2015 and ponticola iranicus vasil′eva, mousavi‐sabet & vasil′ev, 2015. journal of applied ichthyology, 32: 751-752. mousavi‐sabet h., heidari k., abbasi s., vatandoust s. (2016b). length‐weight relationships of six species of the genera cobits and sabanejewia (cobitdae) in iranian river systems. journal of applied ichthyology, 32: 1310-1312. mousavi‐sabet h., homayouni h., marjani m., heidari a. (2015a). length‐weight relationships for 5 clupeiformes species from the persian gulf and oman sea. journal of applied ichthyology, 32: 169-170. mousavi-sabet h., khataminejad s., vatandoust s. (2014). length–weight and length–length relations of the seven endemic alburnus species (actinopterygii: cypriniformes: cyprinidae) in iran. acta ichthyologica et piscatoria, 44(2): 157–158. moutopoulos d.k., stergiou k.i. (2002). length-weight and length-length relationships of fish species from the aegean sea (greece). journal of applied ichthyology, 18: 200-203. najmaii m. (2004). dam and environment. department of energy-iranian large dams national committee. 385 p. (in persian) ndome c.b., eteng a.o., ekanem a.p. (2012). lengthweight relationship and condition factor of the smoothmouth marine catfish (carlarius heudelotii) in the gulf of guinea, niger delta, nigeria. aacl bioflux, 5(3): 163-167. pelicice f.m., agostinho a.a. (2008). fish-passage facilities as ecological traps in large neotropical rivers. conservation biology, 22: 180-188. zar j.h, (1999). biostatistical analysis. fourth edition. pearson education india. 663 p. zareian h., esmaeili h.r., heidari a., khoshkholgh m., mousavi-sabet h. (2016). contribution to the molecular systematics of the genus capoeta from the south caspian sea basin using mitochondrial cytochrome b sequences (teleostei: cyprinidae). molecular biology research communications, 5(2): 65-75. zamani faradonbeh m., eagderi s., ghojoghi f. (2015). length-weight relationship and condition factor of seven fish species of totkabon river (southern caspian sea basin), guilan, iran. international journal of aquatic biology, 3(3): 172-176. int. j. aquat. biol. (2014) 2(4): 201-205 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology review article morphology, growth pattern, feeding and reproductive biology of mystus gulio (hamiltonbuchanan, 1822) (siluriformes: bagridae) sandipan gupta1 central inland fisheries research institute (icar), barrackpore, kolkata-700120, india. article history: received 2 july 2014 accepted 3 august 2014 available online 2 5 august 2014 keywords: long whiskered catfish age and growth food reproduction abstract: mystus gulio is a euryhaline fish, occurring mostly in freshwater; it has also been found to thrive in backwaters of low salinity. it is a popular food fish due to its good taste and recently it has also been reported to be exported as indigenous ornamental fish from india. number of workers earlier has studied morphology, age, growth pattern, food and feeding habit and reproductive biology of this fish species; but no such collective documentation on these aspects is available. with this view, the current review work has been made to document all available information along with noting down those which are lacking and will be beneficial for future fishery and management of this fish species. introduction mystus gulio (hamilton-buchanan, 1822), commonly known as long whiskered catfish, is a euryhaline fish, occurring mostly in freshwater and has also been found to thrive in backwaters of low salinity (pandian, 1966) (fig. 1). talwar and jhingran (1991) have reported that this fish primarily inhabits brackish water and also enters and lives in fresh water. the adults occur mainly in larger water bodies (rivers and streams) with mud or clay substrates, and rarely found in smaller streams (talwar and jhingran, 1991). shafi and quddus (2001) have documented its availability in canals, beels, haors, oxbow lakes, rivers and estuaries of bangladesh. mystus gulio has been documented to be distributed in india, bangladesh, sri lanka, indonesia, vietnam, pakistan, nepal, java, thailand, malaysia and myanmar (day, 1878; weber and de beaufort, 1913; smith and schultz, 1945; pethiyagoda, 1991; talwar and jhingran, 1991; roberts, 1993; jhingran, 1997; kottelat, 2001; senarathne and pathiratne, 2007; froese and pauly, 2014). recently it has also been reported from china * corresponding author: sandipan gupta e-mail address: sandipangupta2007@gmail.com (dong et al., 2012). this fish species is widely cultured in rice fields and brackish water areas of deltaic bengal, where it forms a valuable culture fishery (pantulu, 1961). it has good market demand as food fish due to its delicious taste (haniffa, 2009; begum et al., 2010) and recently has been documented to be exported as indigenous ornamental fish from india (gupta and banerjee, 2014). till date number of workers has documented information on its morphology, age and growth pattern, food and feeding habit and reproductive biology; but all are in scattered format. no consolidated review report is available on these aspects. therefore, the present review has been conducted to accumulate all those available information along with documentation of the lacunae of information which will be beneficial to explore its future fishery and management. morphology day (1878) and talwar and jhingran (1991) have documented the morphological characters of 202 int. j. aquat. biol. (2014) 2(4): 201-205 m. gulio which are as follows: body elongated and compressed; body depth 3.8 to 4.1 times the standard length. head depressed, its upper surface rough and granulated; occipital process triangular, rounded posteriorly, about 1.5 times longer than broad at its base, extending (in adults) to basal bone of dorsal fin; median longitudinal groove on head short, not very conspicuous, not reaching base of occipital process. snout broad and slightly depressed; mouth terminal, upper jaw somewhat longer; teeth villiform in bands on jaws, vomerine tooth patch narrow, continuous and crescentic. barbels four pairs; nasal shorter than the head, maxillary barbells extend posteriorly to end of pelvic fins, the external mandibular are longer than the head or than the internal ones. dorsal spine strong, serrated on its inner edge; adipose fin small, inserted considerably behind rayed dorsal fin, pectoral fin does not reach the ventral, its spine is as long as the head excluding the snout, strong and denticulated internally, ventral fin arises on the vertical behind the last dorsal ray and does not reach the anal; caudal fin forked, upper caudal lobe the longer, lower one sometimes rounded. colour bluish-brown on the head and back, becoming dull white beneath; fins usually black on their outer halves, maxillary barbells black, those from freshwater sometimes whitish or white tipped. age and growth pattern no other workers except pantulu (1961) have determined the age of m. gulio using pectoral spine. he has documented age wise variation in mean total length; mean total length of 47.42 mm, 87.66 mm, 109.33 mm, 130.00 mm and 158.67 mm, respectively, at the age of 1 to 5. no significant difference in mean total length at different ages between sexes has been reported. a proportional relationship between spine radius and total length has also been reported by pantulu (1961). kaliyamurthy (1981) has reported positive allometric growth in m. gulio, which has later been supported by dasgupta (1997); on contrary isometric growth pattern has been reported by pantulu (1961) and begum et al. (2008b). food and feeding habit pandian (1966) has documented that food of m. gulio consists mainly of crustaceans and insects; insect populations of chironomus and micronecta has been reported to constitute 40% of the total food items while crustaceans (copepods and cladocerans) have been reported to form 55% of the diet; rare occurrence of prawns and fishes has also been reported. on the other hand, pantulu (1961) has reported high predation of m. gulio on prawns and small fishes (about 57% of the total annual food requirement) in hooghly estuary. debris, zooplanktons, zoobenthos, other benthic organisms, fish eggs and larvae have been documented in stomach content of juveniles and adults by siddique (2007). yusuf and majumdar (1993) have reported m. gulio as omnivorous fish with inclination towards carnivore as animal foods cover 79% of the overall diet of this fish; among animal food, nauplius larvae and brachionus sp. have been reported as main food items with higher frequency of occurrence. they also have reported bottom feeding habit for this fish species. david (1963) has reported small crustaceans forming the dominant food item over the entire study period along with presence of fish and vegetable matters like algae, diatoms etc. in the gut content. kaliyamurthy and rao (1972) have reported this species as carnivorous with high preference for amphipods, copepods and other crustacean; they have reported it as benthos feeder. pasha (1964) earlier has also reported its bottom feeding habit. tripathi (1996) has reported its insectivorous feeding habit. pandian (1966) has reported monthly variation in the amount of different food items in the stomachs of m. gulio. he also has reported low feeding activity figure 1. long whiskered catfish, mystus gulio. 203 gupta/ review on morphology, growth pattern and biology of mystus gulio during pre-spawning period and marked rise of the same in the month following spawning. pantulu (1961) and begum et al. (2008a) have reported the same for m. gulio in hooghly estuary and in bangladesh, respectively. monthly variation in amount of different food items in the stomachs has also been reported by begum et al. (2008a). pandian (1966) has documented no remarkable change in the food progression with increasing size in m. gulio. kaliyamurthy and rao (1972) have reported variation in food preference in relation to size; small size fishes have been reported to prefer copepods, diatoms and amphipods while large size fishes prefer prawns, fish, hermit crab, polychaetes etc. begum et al. (2008a) have observed difference in food preference in adult and juveniles of m. gulio; adult fishes have been reported to prefer insects and crustaceans, whereas the immature and juvenile fishes have been reported to consume diatoms, copepods, cladocerans and rotifers mostly. pantulu (1961), david (1963), pandian (1966) and kaliyamurthy and rao (1972) have reported m. gulio as carnivorous fish whereas begum et al. (2008a) have reported this fish species as an omnivorous fish as the diet covers a wide spectrum of food ranging from various types of plankton to invertebrates and plants. begum et al. (2008a) stated that m. gulio exhibit overlapping in food and feeding habits to avoid inter and intra-specific competition for available food. yusuf and majumdar (1993) have pointed out its omnivorous diet with inclination towards carnivorous feeding habit. reproduction sexual dimorphism: mookherjee et al. (1941) have reported the presence of genital papilla in male and its absence in female of m. gulio; later david (1963) and begum et al. (2008b) have also supported this observation. sex ratio: kaliyamurthy (1981) has reported female dominance over male in m. gulio population; later it has been supported by islam et al. (2008); though pantulu (1961) has reported no such significant variation from 1:1 (male: female) ratio in his studied sample. minimum size at maturity: pantulu (1961) has reported 6.2 cm as minimum size at maturity for m. gulio in hooghly estuary while 7.9 cm, 8.2 cm and 5.4 cm has been documented as minimum size at maturity by david (1963), jhingran and natarajan (1969) and kaliyamurthy (1981), respectively. fecundity: the fecundity of m. gulio to be ranged from 1,285 to 24,768 with an average of 4,754 (kaliyamurthy, 1981). fecundity range of 12,07421,437 and 425-18,199 have been documented by sarker et al. (2002) and dasgupta (2002), respectively. islam et al. (2008) has reported the fecundity to be ranged from 3,891-1,68,358 with an average of 32,909.49. breeding periodicity: pantulu (1961) has reported july as the spawning month for m. gulio in hooghly estuary; though spawning of some fishes again in december has been reported by him. pandian (1966) has stated commencement of gonadal activity in january and spawning in late september and early october for m. gulio in cooum backwaters of madras (currently chennai); spawning of few fishes again in january has also been documented by him. in both these cases, spawning activity has been reported to be correlated with commencement of monsoon season. jhingran and natarajan (1969) have reported june to november as breeding season for this fish species. kaliyamurthy (1981) has reported august-october as the breeding season with september-october as spawning months for m. gulio in lake pulicat, india. sarker et al. (2002) have reported march-november as breeding season with july as the spawning month in bangladesh; and later islam et al. (2008) have reported the same duration (march to november) of breeding season with the same spawning month (july) in bangladesh. single spawning nature of m. gulio has been earlier reported by pantulu (1961) and later has been supported by david (1963), pandian (1966) and kaliyamurthy (1981). conclusion from the early literatures available on food and 204 int. j. aquat. biol. (2014) 2(4): 201-205 feeding habit of m. gulio, it is quite clear that maximum of the workers have concluded this fish as a carnivorous species; though omnivorous feeding habit has been reported by begum et al. (2008a). analysis of the mucosal surface of the alimentary canal is an effective tool to determine the feeding habit of a fish; yusuf and majumdar (1993) have studied the same in m. gulio and have reported its omnivorous feeding habit with tendency towards carnivore character. therefore, more detail study is needed in this aspect to firmly conclude something about its feeding habit. enzymatic analysis of the alimentary tract can also be a good technique to ascertain regarding its feeding habit. age wise and size wise variation in food preference which is unavailable for many of the mystus sp.; but the same has been documented in details for this fish species which is quite satisfactory. most of the workers have documented female dominance over male in this fish population along with high fecund nature of m. gulio females. there lies major lacking of information regarding length at first maturity (length at which 50% of the fish population is in mature condition) for this fish species as all the previous workers have documented minimum size at maturity (minimum length at which mature specimen has been observed); so detail work is needed in this aspect. all the previous workers have reported m. gulio as a single spawner; variation in spawning month and breeding periodicity has been reported earlier. some of the workers have correlated onset of monsoon season with spawning activity for this fish species and thus have concluded variation in breeding periodicity with change in time of monsoonal onset in different regions. but still detail work is needed to conclude firmly on this type of variation along with existing relationship of breeding periodicity and spawning with other hydrological and meteorological parameters if any for this fish species. there exists contradiction regarding growth pattern of m. gulio; kaliyamurthy (1981) and dasgupta (1997) have reported positive allometric growth while pantulu (1961) and begum et al. (2008b) have reported isometric growth pattern. this difference may be due variation in feeding, reproductive activities, habitat, season, health, environmental condition, sampling procedure etc. references begum m., alam m.j., islam m.a., pal h.k. (2008a). on the food and feeding habit of an estuarine catfish (mystus gulio hamilton) in the south-west coast of bangladesh. university journal of zoology, rajshahi university, 27: 91-94. begum m., mamun a.a., islam m.l., alam m.j. (2008b). morphometric characters and their relationship in estuarine catfish. journal of bangladesh agricultural university, 6(2): 349-353. begum m., pal h.k., islam m.a., alam m.j. (2010). length-weight relationship and growth condition of mystus gulio (ham.) in different months and sexes. university journal of zoology, rajshahi university, 28: 73-75. dasgupta m. (1997). biometry and length-weight relationship of the catfish mystus gulio from west bengal. uttar pradesh journal of zoology, 17(3): 241244. dasgupta m. (2002). fecundity of mystus gulio (hamilton) from west bengal. indian journal of fisheries, 49(4): 457-459. david a. (1963). sexual dimorphism, fecundity and food of the estuarine bagrid, mystus gulio (ham.). proceedings of the national academy of sciences, india, 33(b): 385-410. day f. (1878). the fishes of india; being a natural history of the fishes known to inhabit the seas and fresh waters of india, burma, and ceylon. william dowson and sons, london. 778 p. dong l., chun-guang z., wen-qiao t. (2012). first record of long whisker catfish mystus gulio from china with discussions on the taxonomic status of the genus mystus. acta zootaxonomica sinica, 37(3): 648-653. froese r., pauly d. (editors). (2014). fishbase. world wide web electronic publication. www.fishbase.org, version (04/2014). gupta s., banerjee s. (2014). indigenous ornamental fish trade of west bengal. narendra publishing house, new delhi. 63 p. haniffa m.a. (2009). native catfish culture-a technology 205 gupta/ review on morphology, growth pattern and biology of mystus gulio package for fish farmers. aquaculture asia magazine, 14(3): 22-24. islam m.a., begum m., pal h.k., alam m.j. (2008). studies on the gonado-somatic index and fecundity of mystus gulio (ham.). progressive agriculture, 19(2): 161-166. jhingran v.g. (1997). fish and fisheries of india. hindustan publishing corporation, delhi, india. xxiii, 727 p. jhingran v.g., natarajan a.v. (1969). a study of the fisheries and fish populations of the chilka lake during the period 1957-65. journal of inland fisheries society of india, 1: 49-126. kaliyamurthy m. (1981). spawning biology of mystus gulio in lake pulicat, india. indian journal of fisheries, 8(1&2): 36-40. kaliyamurthy m., rao k.j. (1972). preliminary studies on the food and feeding habits of some fishes of the pulicat lake. journal of inland fisheries society of india, 1: 115-121. kottelat m. (2001). freshwater fishes of northern vietnam. a preliminary check-list of the fishes known or expected to occur in northern vietnam with comments on systematics and nomenclature. the world bank, environment and social development unit, east asia and pacific region, washington d.c. 123 p. mookherjee h.k., mazumdar s.r., dasgupta b. (1941). sexual dimorphism in macrones gulio (ham.). indian journal of veterinary science, 10(3): 295. pandian t.j. (1966). feeding and reproductive cycles of the fish mystus gulio in the cooum backwaters, madras. indian journal of fisheries, 13(1 & 2): 320333. pantulu v.r. (1961). determination of age and growth of mystus gulio (ham.) by the use of pectoral spines, with observations on its biology and fishery in the hooghly estuary. proceedings of the national institute of sciences of india, 27: 198-225. pasha s.m.k. (1964). the anatomy and histology of the alimentary canal of an omnivorous fish mystus (=macrones) gulio (ham.). proceedings of the indian academy of sciences, b59 (4): 211-221. pethiyagoda r. (1991). freshwater fishes of sri lanka. the wildlife heritage trust of sri lanka, colombo. 362 p. roberts t.r. (1993). the freshwater fishes of java, as observed by kuhl and van hasselt in 1820-1823. zoologische verhandelingen, 285(1): 1-94. sarker p.k., pal h.k., rahman m.m., rahman m.m. (2002). observation on the fecundity and gonadosomatic index of mystus gulio in brackishwater of bangladesh. online journal of biological sciences, 2(4): 235-237. senarathne p., pathiratne k.a.s. (2007). accumulation of heavy metals in a food fish, mystus gulio inhabiting bolgoda lake, sri lanka. sri lanka journal of aquatic sciences, 12: 61-75. shafi m., quddus m.m.a. (2001). bangladesher matsho shampad. fisheries of bangladesh. kabir publication, dhaka, bangladesh. pp: 186-187. siddiqui k.u. (ed.) (2007). encyclopaedia of flora and fauna of bangladesh freshwater fishes vol. 23. asiatic society of bangladesh, dhaka, bangladesh. 300 p. smith h.m., schultz l.p. (1945). the freshwater fishes of siam, or thailand. washington united states government printing office. 622 p. talwar p.k., jhingran a.g. (1991). inland fishes of india and adjacent countries. vol-1 and vol-2. oxford and ibh publishing co. pvt. ltd. new delhi, bombay and calcutta. 1063 p. tripathi s.d. (1996). present status of breeding and culture of catfishes in south asia. in: m. legendre, j.p. proteau (ed.). the biology and culture of catfishes. aquatic living resources. 9, hors serie. pp: 219-228. weber m.c.w., de beaufort l.f. (1913). the fishes of the indo-australian archipelago. ii. malacopterygii, myctophoidea, ostariophysi: i siluroidea. e.j. brill ltd. leiden. 404 p. yusuf z.a., majumdar p.k. (1993). morphology of the alimentary canal and food habit of mystus gulio ham. from saline sewage-fed wetlands at minakhan. journal of inland fisheries society of india, 25(1): 58-61. int. j. aquat. biol. (2020) 8(3): 154-165 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article evaluation of kidney histopathological alterations in crucian carp, carassius carassius, from a pesticide and pcb-contaminated freshwater ecosystem, using light microscopy and organ index mathematical model eldores sula1, valbona aliko*2, 1elda marku3, aurel nuro3, caterina faggio4 1department of laboratory technician and imaging, faculty of technical medical sciences, aldent university, tirana, albania. 2department of biology, faculty of natural sciences, university of tirana, tirana, albania. 3department of chemistry, faculty of natural sciences, university of tirana, tirana, albania. 4department of chemical, biological, pharmaceutical and environmental sciences, university of messina, italy. s article history: received 2 march 2020 accepted 8 may 2020 available online 2 5 june 2020 keywords: organ index histopathology kidney organochlorine compounds abstract: this study aimed to evaluate the potential toxic effects of chronic sublethal pesticides and polychlorinated biphenyl (pcbs) exposure on feral crucian carp, carassius carassius, using histopathological alterations as an endpoint. besides, a mathematical model of organ index was used to evaluate the severity of tissue damages. circulatory disturbances, inflammation, regressive and progressive changes of tubules, glomerulus and interstitial tissue of kidneys were the most frequent damages observed. organ index calculation revealed moderate occurrence of damage in kidneys of fish compared to the reference site. findings highlight the effectiveness of organ index as a measuring kidney’s damage severity and health status of fish. the present work is the first study that determines the levels and effects of pesticides and pcbs in water and fish kidneys in seferani lake in albania. the results suggest that the observed changes in kidney structure of c. carassius, could possibly indicate a prolonged chemical stress caused by pesticides and pcbs suggesting continuous monitoring of the lake to protect human consumer’s health. introduction anthropogenic activities are led to stress on aquatic environments by a mixture of pollutants, which may cause molecular, biochemical, cellular and consequently physiological damages on aquatic organisms such as fishes (lópez-barea, 1995; rand, 1995; oheet al., 2004; faggio et al., 2014a, b, 2018; savorelli et al., 2017). a finest example is the feedback of wildlife communities under constant influence of pesticides and industrial chemicals, such as pcbs (jeremy, 2000). an ample number of pesticides are in use nowadays, belonging to a wide diversity of classes, with an extensive range of physicochemical characteristics (haygarth and jarvis, 2002; chromcova et al., 2015; fiorinoet al., 2018; plhalova et al., 2018; stara et al., 2019). organochlorine pesticides and pcbs are a group of toxic substances that can get into water bodies from different routes (carter and heather, 1995), and are *correspondence: valbona aliko e-mail: valbona.aliko@fshn.edu.al defined by their ability to have chemical and biological stability and persistence (jackson et al., 1994; ross and birnbaum, 2001; pfeuffer and rand, 2004). despite the presence of these hazardous compounds in aquatic biota, their chronic biological effects on aquatic life in the lake are still poorly studied. fishes as inhabitants of aquatic ecosystems are frequently exposed to contaminated water, particularly in those areas where contaminants are stable on the environment and has a high potential of accumulation and biological effects (bernet et al.,1999). thereby, fishes are good bio-indicators of environmental health status because of their position in the trophic chain and their responsiveness to low stimuli of pollutants (aliko et al., 2018; burgosaceves et al., 2018, 2019; gobi et al., 2018). biological reactions to contaminants measured in an organism are defined as biomarkers, including 155 int. j. aquat. biol. (2020) 8(3): 154-165 parameters such as enzymatic, hematological, immunological, genotoxic, endocrine, physiological, histological and morphological biomarkers (van der oost et al., 2003). histopathology of various fish tissues have been used as reliable biomarker in assessment of stressor’s effects both in laboratory and field studies (wester and canton, 1991; hinton et al., 1992; dutta, 1996; leonardi et al., 2009; marchand et al., 2009). histopathological changes are displayed as a medium response to chemical contaminants, and histology provides a rapid method to detect a number of lesions in various tissues and organs (bucke et al., 1996; miranda et al., 2008). liver (hinton and lauren, 1990; camargo and martinez, 2007; miranda et al., 2008), kidney (bucher and hofer, 1993; hinton, 1993; miranda et al., 2008), and gills (fernandes and mazon, 2003; oliveira et al., 2005) are suitable organs for histopathological investigation to determine the effects of various stressors. kidney is especially involved in the maintenance of water and body fluids homeostasis (hinton et al., 1992) and serves as an excretory organ for the metabolites of a group of xenobiotics to which fish are possibly exposed (who, 1993). in fish, kidney collects and filtrate a large amount of postbranchial blood and so renal lesions might be expected to be good indicators of environmental pollution (hinton and lauren, 1990; camargo and martinez, 2007). seferani lake, is one of three biggest among 85 karstic lakes found in dumrea region, central-north albania. during two last decades, this freshwater lake, is facing with an intense anthropogenic activity. local agriculture is mainly relied on cultivation of wheat, corn and tobacco. shifting the land use towards agriculture brought about several problems related to the extensive use of pesticides and herbicides, often favored by the lack of control by the authorities. the majority of dumrea karst lakes have been used for irrigation without any restriction or regards to the consequent detrimental effects on the biota, reducing it critically to values below biological minimum. hence, the natural lacustrine ecosystems are declining severely leading to the disappearance of many aquatic species, including fishes (qiriazi and sala, 2000). in addition, further developments in the area contributed to reduce of water quality and increase pollution due to the discharge of liquid waste and chemicals directly into the lakes, and intensive use of herbicides and pesticides for agricultural activity (selenica, 1998; unpublished data from sula, 2018, 2019). until now, few studies on the effects of pollution of seferani lake on its biota have been conducted, viz. its microbiological load (mali and shumka, 2011), and histopathological changes of fish liver tissue (sula and aliko, 2017). the present study aimed to investigate the potential toxic effects of organochlorine pesticides and pcbs found in seferani lake, on feral freshwater crucian carp using kidney histopathology as an endpoint. a reference site, dushku bulcarit lake, was used to allow comparison between a polluted and organochlorine and pcbs-free lake. to evaluate quantitatively tissue damages, an organ index mathematical model was applied. in addition, an identification of cellular alterations resulting from natural stressors other than contaminants, is also discussed. materials and methods fish collection: in total, thirty live adult male fish (six individuals at each station) with total body length of 18.7±2.4 cm, and weight of 183.9±24.5 g, were collected from the seferani lake during april 27 and may 11, 2019. since the water data analysis showed no variation between stations, all individuals were pooled regarding the histopathological analysis. before the experiments, fish were anesthetized with a 0.75% aminobenzoic acid ethyl ester (ms-222) solution and ph adjusted to 7.7 with nahco2. the body cavity was opened and the kidneys were immediately removed and fixed into 10% formaldehyde solution. a total of five reference crucian carp fish were caught in the same period from dushku bulcarit lake, gramshi district, north east of study area as control. dushku bulcarit lake was selected as a reference site due to its pristine natural conditions, its proximity to seferani lake, and absence of any anthropogenic influences. water analysis: water samples were taken at 5 stations 156 sula et al./ histopathological alterations in crucian carp from a pesticide and pcb-contaminated ecosystem at 1.5 m depth. one liter of water was analyzed for each sample for physico-chemical characteristics of lakes water and presence of organochlorine pesticides and pcbs. the physico-chemical characteristics of the water were determined according to apha (1998). the water quality parameters of seferani lake are shown in table 1, where ph = 7.8, temperature = 19.73±1.34°c, do = 3.91±1.38 mg/l, ammonium (nh4+) = 1.86±0.12 mg/l, sulphates = (so4 2-) 1.65±0.41 mg/l, nitrites (no2-) = 0.4±0.02 mg/l, total phosphate = 2.95±0.3 mg/l, and n-no3 = 3.26±1.42 mg/l. meanwhile, the reference site, dushku bulcarit lake, showed a normal situation with: ph 8.0, temperature 19.89±2.1°c, do 9.1±0.2 mg/l, ammonium (nh4+) 0.07±0.02 mg/l, nitrites (no2-) 0.012±0.01 mg/l, and sulphates (so4 2), total phosphate and n-no3 in non-available amounts. analysis of pesticide and pcbs was done using gas chromatography hp 6890 series ii with electron capture detector (gc/ecd). rtx-5 capillary column and detector ecd was used for the separation and detection of organochlorine and pcbs compounds. quality and quantity analysis of pesticides and pcbs was based on epa 8081b standard. table 1. histopathological assessment tool for fish kidney. an importance factor (w) ranging from 1 to 3 is assigned to every alteration: it is composed of the respective organ (org), the reaction pattern (rp), and the alteration (alt)*. # extracted from (bernet et al., 1999). reaction pattern functional unit of tissue alteration importance factor score value index circulatory disturbances hemorrhage/ hyperemia/aneurysm wkc1=1 akc1 ikc intercellular edema wkc2=1 akc2 regressive changes tubule architectural and structural alterations wkr1=1 akr1 ikr plasma alterations wkr2= 1 akr2 deposits wkr3= 1 akr3 nuclear alterations wkr4= 2 akr4 atrophy wkr5= 2 akr5 necrosis wkr6= 3 akr6 glomerulus architectural and structural alterations wkr7= 1 akr7 plasma alterations wkr8= 1 akr8 deposits wkr9= 1 akr9 nuclear alterations wkr10= 2 akr10 atrophy wkr11= 2 akr11 necrosis wkr12= 3 akr12 interstitial tissue architectural and structural alterations wkr13= 1 akr13 plasma alterations wkr14= 1 akr14 deposits wkr15= 1 akr15 nuclear alterations wkr16= 2 akr16 atrophy wkr17= 2 akr17 necrosis wkr18= 3 akr18 progressive changes tubule hypertrophy wkp1=1 akp1 ikp hyperplasia wkp2= 2 akp2 glomerulus hypertrophy wkp3=1 akp3 hyperplasia thickening of bowman’s capsular membrane wkp4= 2 akp4 interstitial tissue hypertrophy wkp5=1 akp5 hyperplasia wkp6= 2 akp6 inflammation exudates wki1=1 aki1 iki activation of res wki2=1 aki2 infiltration wki3= 2 aki3 tumor benign tumor wkt1= 2 akt1 ikt malignant tumor wkt2= 3 akt2 ik. 157 int. j. aquat. biol. (2020) 8(3): 154-165 histopathogical analysis: histopathology was conducted based on sula and aliko (2017). after fixation, samples of the kidney were dehydrated in an ethanol series, cleared in xylene and embedded in paraffin wax and sectioned at 5 μm. after dewaxing with xylene and hydration in ethanol series of decreasing concentration, sections were stained with hematoxylin and eosin (luna, 1968) and examined under a light microscope. cytological results were expressed as a prevalence of fishes with histopathological lesions, the kidney lesion index was determined according to bernet et al. (1999). this index is based on the sum of the score rankings (a) and importance factors (w) for each considered lesion in the tissue (table 1). the score ranking was based on the percentage of lesions ranging from 0 to 6 depending on the degree and extent of alteration: (0) unchanged; (2) mild; (4) moderate; and (6) severe. intermediate values were also considered. the importance factor (w) was determined for each lesion depending on the effects considered as minimal (1), moderate (2) and irreversible (3). mathematical calculation of lesion indices: because the lesions within one organ are studied, two indices are applicable (bernet et al., 1999). reaction index of an organ (i org rp) = ∑ (aorg rp alt xworg rp alt) / alt where: org = organ (constant); rp = reaction pattern; alt = alteration; a = score value; w = importance factor. the quality of the lesions in an organ is expressed by the reaction index. organ index (iorg): ∑ ∑ (aorg rp alt xworg rp alt) / rp alt where: org = organ (constant) (for abbreviations see reaction index formula). this index represents the degree of damage to an organ. statistical analysis: statistical analyses were refined by analysis of variance (anova) using spss software, version 15. one-way analysis of variance (anova) was used to test the level of significance at 0.05 level of probability for the pesticide and pcbs residue levels and kidney tissue alterations. a pearson correlation analysis (pca) was used to determine the relationship among the pcbs and pesticides and organ index coefficient. all data were expressed as mean ± sed. the significance was assigned for p<0.05. results water analysis: water quality data regarding pesticide and pcbs amounts measured in seferani lake, are showed in table 2. no pesticide residue was determined in the water samples of dushku bulcarit lake. among the pesticides, dieldrin (660.87±2.1 ng/l), 4.4”-ddd (755.31±2.34 ng/l) and endosulfan (546.92 ± 2.34 ng/l) were the components found at appreciably higher concentration. meanwhile, pcb 52 (145.14±3.1 ng/l), pcb153 (623.33±4.1 ng/l) and pcb209 (203.66±2.61 ng/l) were in the highest table 2. pesticides and pcbs measured concentrations in seferani and d. bulcarit lakes parameter seferani lake d. bulcarit lake pesticides alfa-hch 5.57±0.3 ng/l n/a beta-hch 14.6±1.02 ng/l n/a dieldrin 660.87±2.1 ng/l n/a 4.4'-ddd 755.31±2.34 ng/l n/a endosulfansulfat 546.92±2.34 ng/l n/a pcb-s pcb 28 48.75±1.03 ng/l n/a pcb 52 145.14±3.1 ng/l n/a pcb 101 19.62±1.23 ng/l n/a pcb 118 17.47±2.01 ng/l n/a pcb 153 623.33±4.1 ng/l n/a pcb 209 203.66±2.61 ng/l n/a 1 5 8 s u la e t al ./ h is to p at h o lo g ic al a lt er at io n s in c ru ci an c ar p f ro m a p es ti ci d e an d p c b -c o n ta m in at ed e co sy st em c a ra ss iu sc a ra ss iu ss e fe ra n i l a k e c a ra ss iu sc a ra ss iu s d . b u lc a ri t im p o rt a n c e fa c to rs / in d e x e s 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 1 2 3 4 5 w k c 1 = 1 2 /2 2 /2 2 /2 2 /2 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 w k c 2 = 1 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 i k c 2 2 2 4 2 0 2 4 4 2 2 0 4 2 2 2 4 2 4 0 2 2 4 0 2 0 4 4 2 2 0 0 2 0 0 w k r 1 = 1 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 2 /2 2 /2 4 /4 2 /2 2 /2 2 /2 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 w k r 2 = 1 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 2 /2 2 /2 2 /2 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 1 /1 w k r 4 = 2 6 /1 2 4 /8 2 /4 2 /4 4 /8 4 /8 2 /4 2 /4 6 /1 2 4 /8 6 /1 2 2 /4 2 /4 6 /1 2 2 /4 2 /4 4 /8 2 /4 6 /1 2 4 /8 4 /8 2 /4 2 /4 2 /4 2 /4 2 /4 4 /8 1 /2 1 /2 w k r 5 = 2 4 /8 2 /4 2 /4 4 /8 2 /4 2 /4 2 /4 2 /4 2 /4 4 /8 2 /4 2 /4 2 /4 2 /4 2 /4 4 /8 2 /4 2 /4 4 /8 2 /4 4 /8 2 /4 w k r 6 = 3 4 /1 2 2 /6 2 /6 2 /6 2 /6 4 /1 2 2 /6 4 /1 2 2 /6 4 /1 2 2 /6 4 /1 2 4 /1 2 2 /6 4 /1 2 2 /6 2 /6 2 /6 2 /6 w k r 7 = 1 4 /4 2 /2 2 /2 4 /4 4 /4 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 4 /4 4 /4 2 /2 4 /4 2 /2 4 /4 2 /2 4 /4 4 /4 2 /2 2 /2 4 /4 4 /4 4 /4 4 /4 2 /2 w k r 1 0 = 2 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 4 /8 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 4 /8 2 /4 2 /4 4 /8 2 /4 2 /4 2 /4 2 /4 w k r 1 1 = 2 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 w k r 1 2 = 3 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 2 /6 w k r 1 4 = 1 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 2 /2 2 /2 2 /2 2 /2 i k r 6 0 3 4 3 0 4 2 4 0 4 2 1 8 2 8 5 6 4 4 5 0 1 2 3 2 4 4 2 4 3 4 3 6 6 4 2 1 0 6 0 1 2 3 2 3 2 4 8 1 8 2 6 4 0 3 8 2 4 3 0 0 w k p 1 = 1 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 2 /2 4 /4 4 /4 4 /4 4 /4 2 /2 4 /4 4 /4 4 /4 4 /4 4 /4 2 /2 4 /4 1 /1 1 /1 w k p 2 = 2 2 /4 2 /4 4 /8 4 /8 2 /4 2 /4 4 /8 4 /8 4 /8 4 /8 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 4 /8 2 /4 2 /4 2 /4 2 /4 w k p 3 = 1 2 /2 2 /2 2 /2 4 /4 2 /2 4 /4 4 /4 4 /4 4 /4 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 2 /2 4 /4 2 /2 4 /4 2 /2 2 /2 w k p 4 = 2 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 4 /8 4 /8 4 /8 2 /4 2 /4 2 /4 2 /4 w k p 6 = 2 2 /4 2 /4 2 /4 2 /4 2 /4 2 /4 i k p 1 4 1 6 1 2 2 2 1 6 6 0 1 4 2 4 1 4 2 0 0 1 4 2 2 1 4 1 0 8 6 1 8 0 2 0 0 8 1 0 0 0 0 1 2 1 2 1 4 1 0 1 0 0 w k i3 = 2 4 /8 2 /4 2 /4 4 /8 2 /4 4 /8 2 /4 4 /8 4 /8 2 /4 4 /8 i k i 8 4 0 4 0 0 0 0 8 4 8 0 0 4 0 0 0 0 8 0 8 0 0 0 0 0 0 0 4 8 0 0 0 0 0 i k 8 4 5 6 4 4 7 2 5 8 4 8 2 0 4 6 9 2 6 4 8 0 1 2 5 0 7 2 4 0 4 6 4 8 1 4 7 2 1 0 9 0 1 4 4 4 4 2 6 8 2 2 4 2 5 8 6 2 3 4 6 0 0 m e a n i k 4 7 .2 2 .6 t ab le 3 . r ea ct io n i n d ex es a n d o rg an i n d ex o f c . ca ra ss iu s fi sh k id n ey s fr o m s ef er an i an d d . b u lc ar it l ak es 159 int. j. aquat. biol. (2020) 8(3): 154-165 concentrations among pcbs detected in the sites. all pesticide and pcbs values are considered higher than the reported values of their toxicity for cyprinid fish (epa, 2001; helfrich et al., 2009; jayraj et al., 2016). kidney tissue morphology: the histopathological observations of c. carassius are shown in figure 1, and calculated indexes in table 3. circulatory disturbances, inflammation, regressive and progressive changes of tubules, glomerulus and interstitial tissue were the important histopathological alternations found in the fish of the study site, compared to the reference lake. carassius carassius’ kidneys have displayed various alterations related to circulatory viz. hemorrhage and hyperemia, tubular figure 1. photomicrographs of kidneys of carassius carassius fishes caught in bulcarit and seferan lakes. all sections are stained with h&e. (a) normal kidney tissue showing glomerulus and bowman’s space well defined (arrow), hematopoietic tissue (asterisk), proximal tubules (double arrow), and distal tubule (arrow head), (b) rupture of an artery and hemorrhage (arrow), (c) blood congestion, hyperemia (arrow), (d) intercellular edema of hematopoietic tissues (double arrow), (e) tubule with architectural and structural cell alterations (arc), plasma alterations (arrow head), and nuclear alterations (double arrow), (f) tubules showing atrophy and necrosis (asterisk), (g) glomerulus with architectural and structural variations, alterations of nucleuses of epithelial endothelial cells and podocytes (arrow), (h) atrophy and necrosis of glomerular cells (arrow), (i) plasma alterations, hyaline degeneration of interstitial tissue (arc), (j) hypertrophy of tubular cells (asterisk), (k) increase in number of tubule cells known as hyperplasia (arrow head), (m) hypertrophy of glomerulus cells occupying all renal corpuscle (arrow), (n) thickening of bowman’s capsular membrane (hyperplasia), (o) hyperplasia of hematopoietic leukocyte cells (arc), and (p-q) infiltration of fibroblasts, neutrophils, monocytes and macrophages known as peritubular fibrosis (asterisks and double arrow). 160 sula et al./ histopathological alterations in crucian carp from a pesticide and pcb-contaminated ecosystem and glomerular alterations like nuclear and plasma alterations, atrophy, hypertrophy and hyperplasia of tissue cells, even tubular and glomerular focal necrosis. (fig. 1; b-q). the fish of dushku bulcarit i.e. reference lake, showed a normal kidney structure, consisting of normal glomerulus, a well-defined bowman’s capsule, and renal tubules (fig. 1). organ index for all crucian carp fish from seferani lake revealed significantly higher values ranging 6 to 92, with a mean of 47.2, indicating a moderate occurrence (table 3). meanwhile, that of dushku bulcarit i.e. reference site, had a mean organ index of 2.6 (fig. 2). a straightforward correlation of kidney hitopathological status and pesticide and pcbs concentrations analysed for seferani lake, established a weak correlation of r=0.0346 (p<0.05). discussions the present study revealed that crucian carp inhabiting the seferani lake is affected by combined toxic effects, such as moderate hypoxia and chronic sub-lethal pesticide and polychlorinated biphenyl (pcbs) exposure. intensive and long-term use of pesticides in agriculture, especially in developing countries, have increased due to their persistence in nature. additionally, the fast urbanization can contribute to increase influent of urban wastes to the lake, decreasing the water quality. chemical analyses are essential to evaluate water quality as they provide information for types of xenobiotics like organochlorines that influence water biota. it has been estimated that only a small amount, about 0.1% of pesticides reach the target organisms for which they are used; the remaining contaminates the surrounding environment (carriger et al., 2006). compounds such as pesticides and pcbs are among those substances which are capable to cause endocrine, genetic, or severe reproductive problems in fauna and also qualified for inclusion under the broad heading of substances which possess carcinogenic properties which all together lead to death of organisms (epa, 2001). in the present study, water analysis revealed presence of various toxic organochlorine pesticides and pcbs such as ddd, dieldrin, endosulfan-sulfat and pcb 153 in sub-lethal concentrations. the retention of pcbs in tissues, especially those with high lipid concentration is linked to the degree of chlorination, higher chlorinated compounds persist for long periods of time. pcbs induce interactive effects like increased kidney toxicity of trichloroethylene (epa, 1999). ddt and dieldrin are banned in united states for survival of fish and protection of water quality (helfrich et al., 2009). dieldrin has extreme nonpolar properties that result in a high affinity for organic matter with strong tendencies to adsorb sediments and accumulate in the tissue of aquatic biota, where they can persist for long time (atsdr, 1993; nowell et al., 2000). the results showed a great concentration of dieldrin, approximately 0.66 µg/l compared to hrl (health reference level) i.e. 0.002 µg/l (based on estimated excess lifetime cancer risks of 10-6) (epa, 2003). occurrence of dieldrin is high in fishes, which highlight the potential of bioaccumulation for this pesticide (kolpin et al., 1998). endosulfan sulfate is banned in many countries (wan et al., 2005); it is a derivate of endosulfan but more stable in environment (hose et al., 2003). according to helfrich (2009), endosulfan is classified at category “super” with lc50 <10 µg/l (lethal concentration) related to its toxicity. in the present study, endosulfan-sulphate value was 0.00055 mg/l figure 2. mean kidney index differences between seferani and d. bulcarit lake fishes. the values given are mean±std. significance * = p<0.05. 161 int. j. aquat. biol. (2020) 8(3): 154-165 that is half of 96-hour lc50 (0.001 mg/l) to for rainbow trout and bluegill (helfrich et al., 2009). endosulfan is extremely toxic to aquatic organisms, especially fish. it has a relatively high potential to bioaccumulate in fish tissues (unep and gef, 2002). the subacute and chronic toxicity studies of endosulfan in animals suggest that liver, kidneys and immune system are the main target organs (calepa, 2008). teleost kidney act as an excretory route for all xenobiotic metabolites which fish may be exposed and consequently are one of the first organs to be affected (hinton et al., 1992; thophon et al., 2003; fazio et al., 2015; moyson et al., 2016). the exposure of fish to sub-lethal concentration of organic compounds such as pesticides may result in different histopathological alterations in specific tissues (altinok and capkin, 2007; ghelichpour and mirghaed, 2019). important changes related to circulatory disturbances, inflammation, regressive and progressive changes of tubules, glomerulus and interstitial tissue are found on fish from the polluted site, compared to the reference one. in our case, c. carassius’ kidneys have displayed various alterations that corroborate with findings of pandey et al. (1993), who described histopathology of estuarine mullet liza parsia exposed to ddt kidney with eosinophilic material in cell plasma, edema, hypertrophy of epithelial cells, karyopyknosis, karyolysis and fibrosis, shrinkage of glomeruli and focal necrosis. mchugh et al. (2011) also showed hyaline degeneration, vacuolation of the renal tubule and dilation of the glomerulus capillaries in tiger fish, hydrocynus vittatus, kidneys exposed to ddt in a natural habitat. an interesting finding of this study was the presence of small granules in the cytoplasm, which later, can progress to hyaline degeneration. these granules may be formed inside of cells or by the reabsorption of plasma proteins lost in the urine by glomerular damage, which can lead necrosis (hinton and lauren, 1990; takashima and hibiya, 1995; boran et al., 2010). altinok and capkin (2007) studied rainbow trout exposed to endosulfan and described that kidney of fish had eosinophilic exudates on glomeruli, occluded glomerular capillaries and segregation of tubular epithelium from connective tissue. they observed also necrosis of hematopoietic, glomerular and tubular cells. capkin et al. (2006) showed that kidney was the most affected organ of rainbow trout treated with endosulfan. assessing the histopathology of c. carassius kidneys exposed to dieldrin, satyanarayan et al. (2012) described presence of vacuolated cells in glomeruli, atrophy of glomeruli, and hematopoietic and tubular necrosis. such feedback alterations might be due to variations in type, strength, and persistence of a stressful chemical factor to which the fish is exposed. the high incidence of histological alterations in the kidney of c. carassius living in seferani lake, indicates their chronic expose to toxic substances in their natural habitat. therefore, the renal lesions might be expected to be good bio-indicators of environmental pollution in this species. inevitably, all kidney damages reported here for c. carassius, are expected to impair the osmoregulatory function of the fish, since their kidney is not only an excretory organ but, also an osmoregulatory one. in addition, as a longterm effect, the impaired organ structure and function can directly affect survival, growth and reproduction of the organism (wahli, 2002). different studies support the fact that organ histopathological changes integrate the impact of a variety of stressors (schmidtposthaus et al., 2001; au, 2004). histopathological assessment is subjective and often it is related to proficiency of the researcher. it is a descriptive method and quantitation is rather challenging. usually, a histopathological assessment is done routinely using light microscopy (lm), and extend of a tissue alteration is categorized as either mild, moderate or a severe change (cengiz, 2006) or as a percentage of altered tissue (benli et al., 2008). for a better semi-quantitative evaluation of the severity of tissue damages, a mathematical approach of calculating organ index, named bernet’s system (1999) which sum up all detected changes in a tissue, being so a parameter that measures the importance factor and type and/or severity of the change. according to bernet’ scoring system, a definition of 162 sula et al./ histopathological alterations in crucian carp from a pesticide and pcb-contaminated ecosystem certain patterns of tissue response to pollutants, can be achieved. using this mathematical approach, we calculated that the mean organ index for all fishes was 47.2 indicating moderate occurrence. values of organ indexes ranged 6-92 indicating that in most cases kidney possess lesions of severe damages of tissues. in the current study, a weak positive correlation between nature and intensity of kidney damages and water concentrations of pesticides and pcbs, does not necessarily mean that these pollutants are not the direct responsible actors, but can support the idea that under the complex exposure situation in the field, with the presence of other co-stressors, establishing straightforward relationships between a single stressor and a biological response can be complicated (segner, 2005; zimmerli et al., 2007). in this context, the importance of taking into consideration of mixture effects for biological responses has to be emphasized, including combination of chemicals (silva et al., 2002) as well as combinations between chemicals and physical or biological stressors. in our previous studies on the same ecosystem, we found a significant increased level of cortisol, plasmatic glucose and oxidative stress enzymes accompanying liver histological alterations in this fish (morina et al., 2013; sula and aliko, 2017). the results of this study suggest that crucian carp at seferani lake show poor health status than those of dushku bulcarit i.e. reference site. the conclusion on a decreasing health status of fish in seferani lake is based mainly on the kidney histopathological findings. however, to get a full tableau of the complex interactions between pollution and histological changes, other parameters such as sediment chemistry and bioaccumulation must be assessed in conjunction with. as conclusion, the results of this study showed that the histopathology of kidney is a useful biomarker for monitoring fish health. histological changes appear as medium-term responses to sub-lethal stressors, considering their intermediate location with regard to the level of biological organization, and histology represents a rapid method to detect effects of stressors, especially chronic ones, in various tissues and organs. organ index mathematical model represents an integrative indicator of fish conditions and offers a better evaluation of the severity of tissue damages. as such, it can be successfully applied to better predict the severity of xenobiotic toxicity on fish tissue level, and in comparison, between two or more polluted sites, including different freshwater ecosystems, such as streams, rivers, lakes and fish farms. acknowledgments this study is part of phd research thesis and was supported partially by the faculty of natural sciences, university of tirana, project no. #2571/5. references aliko v., qirjo m., sula e., morina v., faggio c. 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(2019) 7(3): 146-154 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article assessment of habitat suitability index of capoeta species in the caspian sea and namak lake basins, iran maryam nasrolah pourmoghadam, hadi poorbagher*,1soheil eagderi, kamran rezaei tavabe department of fisheries, faculty of natural resources, university of tehran, karaj, iran. s article history: received 22 march 2019 accepted 16 june 2019 available online 2 5 june 2019 keywords: habitat suitability kordan taleghan jajrood abstract: habitat suitability index (hsi) models are usually used to forecast habitat quality and species distributions and are used to develop biological studies, management priorities and anticipate possible changes under different management or climate change situations. this study was conducted to identify the habitat suitability index of three species namely, capoeta buhsei, c. razii and c. alborzensis in the kordan, taleghan and jajrood rivers, respectively. at each station, environmental variables including temperature, dissolved oxygen, ph, ec, tds and hydrological parameters such as flow velocity, depth, width, average diameter of stones and amount of phosphate, nitrate and ammonium were measured. the results showed that suitable habitats for these species are those with a high stone diameter, high temperature, low flow velocity and in areas where the width of the river is low. with respect to the abundance of fishes sampled in this study, the central and lower regions of the jajrood and kordan rivers and the stations far from the dam on the taleghan river are favorable habitats for the studied capoeta species. introduction many habitats on the planet including rivers are seriously affected by human activities which strengthen the vitality of studies on these ecosystems especially those that investigating the relationship between habitat factors and their inhabitants (bovee, 1986). desirable habitats have significant impacts on species survival and reproduction, and are highly regarded in the wildlife management and conservation (mack et al., 1997). the fish species distribution undergoes by the environmental factors and meanwhile the structure of fish communities can be highly impacted by these factors both at spatial and temporal scales (hackradt et al., 2011). each fish species prefers a particular habitat and this habitat is where a fish encounter desirable properties, such as sufficient oxygen, tolerable temperature, adequate food and hiding places (thurow, 1997). the frequency of loss and fragmentation of these habitats due to the pollution, manipulation, and other human activities are common problems that threaten the *correspondence: hadi poorbagher doi: https://doi.org/10.22034/ijab.v7i3.622 e-mail: poorbagher@ut.ac.ir ecological functions (dong et al., 2013). loss of habitat, in terms of quantity and quality, is an important factor decreasing the abundance of aquatic organisms a n d a major threat to biodiversity. therefore, habitat is considered as one of the most important factors in species conservation (rashleigh et al., 2004). the quality of different habitats in a river for one species is defined by the relative abundance of its members. usually, organisms are more abundant in places where the quality of the habitat is high, and few individuals are found in poor habitats, with no species in completely unsuitable habitats (jowett et al., 2008). endangered species can be assisted before extinction by focusing on the conservation of specific habitats and identification of suitable habitat types for these species. the habitat suitability index is an analytical tool used to indicate the preference of different species for a combination of variables (armour and taylor, 1991; jowett et al., 2008). habitat suitability models (hsm) 147 int. j. aquat. biol. (2019) 7(3): 146-154 are used for a range of expected habitat conditions in a study area. in these models, a number of habitat stations are evaluated and habitat suitability index is calculated. the purpose of these studies can be to determine the high (0.7-1), medium (0.3-0.7) and low (0.3-0) quality stations, respectively (brooks, 1997) to find the availability of habitat for many freshwater, brackish and saline water species or determine the species' habitat preferences which is generally intended to assess the effects of human activities (brown et al., 2000). the genus capoeta consists of medium to large species that have a fusiform body, small scales and almost big and subterminal mouth (eagderi et al., 2015; coad, 2018; çiçek et al., 2018). capoeta species are important in terms of fishing in inland waters, aquaculture, sport fishing and animal geographical studies (sammaee et al., 2006). fish ecosystem conservation has a great importance, this study was thus conducted to identify the habitat suitability index of three species namely, capoeta buhsei, c. razii and c. alborzensis in the kordan, taleghan and jajrood rivers, respectively. capoeta buhsei has a wide distribution in the namak lake and dasht-e kavir basins (roudbar et al., 2017; esmaeili et al., 2018), which can be an appropriate candidate for the study of intraspecific compatibility. all three rivers have similar latitude and the amount of anthropogenic effects, and almost are in the same situation, therefore this study stands for answering the question whether the greater distribution of c. buhsei is due to its more resilience in habitat suitability index compared to the other two species? materials and methods sampling stations: sampling of capoeta species from the kordan, taleghan and jajrood rivers was carried out in december 2017. first of all, a field survey was conducted to determine the places of sampling stations and examine their approximate situation in terms of river morphology and the variables such as the width, depth and vegetation type of the area. then the exact coordinates of the stations were determined using google earth software and garmin gps device (etrex 30x) (fig. 1). five sampling stations in the kordan river, six stations in the taleghan river and four stations in the jajrood river were determined. fish sampling: the specimens were sampled using an electrofishing device (samus mp750). samples were anesthetized in 0.1% clove solution after collection and the number of each species at each station was counted. after taking the pictures of the specimens, they were returned to the river (price and peterson, 2010). physicochemical analysis of water: immediately after figure 1. map of study areas and location of sampling stations in, kordan, taleghan and jajrood rivers. 148 nasrolah pourmoghadam et al./ habitat suitability index of capoeta species sampling, environmental variables, including temperature (°c), dissolved oxygen (ppm), ph, ec (µs), tds (mg/l) and hydrological parameters such as flow velocity (cm/s), depth (cm), width (m) and average diameter of stones (cm) were measured at each station. then the water samples were transferred to the laboratory to measure concentration of phosphate, nitrate and ammonium as the most important nutrients, using the standard methods. the width of the river was measured using a tape meter at three points; downstream, middle and upstream of each station and the mean of them was considered as the mean width of the river at each station. the depth of the river at each station was measured at 20 points and the mean of these numbers was considered as the depth of the river (gorman and karr, 1978; pusey et al., 1993). using a floating object, the flow velocity was measured three times in each station, and the mean value after the multiplication in the correction factor (0.88), was considered as the flow velocity (hasanli, 2000). at each station, temperature, oxygen, ph, ec and tds were randomly measured at three points the portable electronic device (wtw) and their average were calculated. the average stone diameter was recorded randomly using a quadrat (50 × 50 cm) (platts et al., 1983). imaging and statistical analysis: to calculate the hsi, a nonparametric method namely kernel smoothing was used to examine the relationship between each environmental variable and the number of fish in each station. si graphs: first, to find the bandwidth of the kernel smoothing, a polynomial regression was calculated to model the predicted values. to find the si graphs, the predicted values were standardized using the following equation: min( ) max( ) min( ) i x x x x   where, xi= the environment variable, min (x) = the minimum value of each variable and max (x) = the maximum value of each variable. then, the graphs of these values were standardized and plotted for each environmental variables, including ph, temperature, ec, tds, depth, width, velocity, stones diameter, po4, no3 and nh4. calculating hsi: the mean of the arithmetic, the geometric, the minimum, and the maximum of the si values from the si graphs for each independent figure 2. si charts for environmental variables: ph, temperature, ec, tds, dissolved oxygen, river depth, river width, water velocity, stone diameter, po4, no3, nh4 in the kordan river. 149 int. j. aquat. biol. (2019) 7(3): 146-154 variable were calculated and considered as hsi at each sampling station. the relationship between these values and the number of fish at each station was examined using a linear regression. after fitting the linear regression between the number of fish and hsi, the aic value was calculated. then, to illustrate the relationship between the number of fish and hsi obtained from different stations, four graphs were plotted based on four hsi calculation methods. results si graphs: si graphs for the environmental variables in the kordan river are shown in figure 2. the number of fishes increased in ph = 8-8.2 and also an increase was found with rise o f temperature and ec values. as tds increased, the number of fish increased significantly and the highest number of fish was found in high do concentration and a depth of 20 cm. in the river width of 6-7 m, the number of fish increased while the number of fish had negative correlation with flow velocity. the larger the stone diameter, the more fish were found and w i t h i n c r e a s e o f po4 to a certain extent, the number of fish increased. also, increase of no3 and nh4 concentration had positive effects on the number of fishes. the si graphs for the environmental variables in the taleghan river are shown in figure 3. in the range of ph = 7.9-8 and in the high temperatures, the number of fish increased. when the ec and tds increased, the number of fish increased. in the stations with more do, the more fish were available. up to 15 cm depth, the number of fish increased, and then the number of fish decreased with increase of depth. the number of fish was more in the 2-5 m of the river width. at high velocity above 2 m/s, the number of fish decreased. more specimens were found by the stones with larger diameters. by increasing po4 to a certain extent, the number of fish increased and above that, the number of fish decreased. both no3 and nh4 had positive correlation with the number of fish. the si graphs from the jajrood river indicated that the number of fish increased in the range of ph = 7.5-8 and in high temperatures. when ec and tds increased, a significant increase was found in the number of fish. the highest number of fish was found in high do values. the maximum number of fish was found at the depth of 20 cm and declined by increase of depth. there was a large number of fish in the widths between 14 to 16 m while water velocity figure 3. si charts for environmental variables: ph, temperature, ec, tds, dissolved oxygen, river depth, river width, water velocity, stone diameter, po4, no3, nh4 in the taleghan river. 150 nasrolah pourmoghadam et al./ habitat suitability index of capoeta species showed a negative effect on fish number. more fish were found in stations with large stone diameter and also the fish abundance increased by rise of po4, no3 and nh4 (fig. 4). hsi results: the greatest hsi were found in the 5 th station of the kordan river, the 1st and 6 t h stations of the taleghan and the 4th station of the jajrood river (table 1, fig. 5). discussions rivers are amongst ecosystems which have been wellknown to highly be affected by human activities. fish communities are the organisms which reflect such effects through different ways, including theirs distribution and abundance (ferreira, 2007). as a result, conservation of aquatic animals requires the recognition of the natural requirements of species and the study of habitat priorities (garland et al., 2002). the results of si graphs for environmental variables, including ph, temperature, ec, tds, do, river depth, river width, velocity, stone diameter, po4, no3 and nh4 for assessing the habitat suitability of figure 4. si charts for environmental variables: ph, temperature, ec, tds, dissolved oxygen, river depth, river width, water velocity, stone diameter, po4, no3, nh4 in the jajrood river. table 1. hsi index calculated with four methods of arithmetic mean, geometric, minimum and maximum. hsi (maximal si( hsi (minimal si) hsi (geometric mean) hsi (arithmetic mean) station river 0.933 0.000 na 0.095 1 1.000 0.004 0.249 0.454 2 1.000 0.000 na 0.656 3 kordan 1.000 0.000 na 0.404 4 1.000 0.839 0.977 0.979 5 1.000 0.366 0.824 0.859 1 1.000 0.000 na 0.387 2 1.000 0.000 na 0.536 3 taleghan 0.661 0.000 na 0.106 4 0.545 0.000 na 0.257 5 1.000 0.152 0.736 0.808 6 1 0.103 0.000 na 0.009 2 jajrood 1.000 0.000 na 0.814 3 1.000 1.000 1.000 1.000 4 151 int. j. aquat. biol. (2019) 7(3): 146-154 three species, c. buhsei in the kordan river, c. razii in the taleghan river and c. alborzensis in the jajrood river showed that for all three species the suitable habitat was a habitat with neutral ph, high temperature and sufficient do. several studies have been conducted on the effect of temperature on fish life. nukazawa et al. (2010) used water temperature as an environmental index for plecoglossus altivelis altivel as an annual migratory species. smith and sklarew (2012) also showed that the water temperature had a negative effect on salvelinus fontinalis populations. water temperature is an important factor in fish life and in some cases, its variation is a natural stimulant and indicates the beginning of some processes such as spawning and migration. usually within optimum temperature ranges, an increase in temperature increases the digestibility rate of capoeta species. consequently, the digestion rate increases, the food intake also increases (nikolski, 1963; zamani faradonbe et al., 2017) that may be the point that high temperature is more appropriate for capoeta species. increasing the ec and tds increased the number of fish but above it the number of fish showed an inverse trend. in this study, ec and tds increased from upstream to downstream, which is consistent figure 5. relationship between number of fish and hsi in the a: kordan, b: taleghan and c: jajrood rivers. 152 nasrolah pourmoghadam et al./ habitat suitability index of capoeta species with the results of an et al. (2012). the electrical conductivity (ec) is the ability of a solution to carry electrical current, and associated with total dissolved solids (tds) and concentration of many materials (simonson et al., 1993). differences in water discharge across the river can be the most relevant parameter changing these two factors. according to our results, the suitable habitat for each of the three studied species is a habitat with lower velocity and depth. depth and water velocity are two influencing factors in distribution and abundance of fishes (baker and ross, 1981). for example, mccain (1992) found that chinook salmon (oncorhynchus tshawytscha) in early life stages uses low-velocity areas, as these areas can be a good protector against predators and flood. in the present study, the preferred points were the places with small width and large stone diameter. habitats with great river width may decrease the survival rate, therefore fish species prefer to live in points of rivers with small width (littlejohn et al., 1985). preference of fishes to substrates with large stones is probably due to the fact that large pieces of stone are used as a shelter against the flow. in addition, due to the creation of a large surface and more dead space, the surface of such stones has a high density of food. in all three rivers, the increase of water chemical factors such as po4, no3 and nh4 increased the number of fishes to a certain extent. nutrients in form of phosphorus and nitrogen increase the growth of phytoplankton and provide more natural food for fishes. in these cases, the increased amount of nutrients to a certain extent will result in an optimal increase in the number of fish and their size. similar to the present study, zamani faradonbe et al. (2015) evaluated the habitat suitability index of c. razii in the taleghan river. the results showed that the suitable habitat characteristics of this species were the height of 1550-1400 m, the depth of 40-55 cm, the river width of less than 5 m, the flow velocity between 0.6 to 0.3 m/s, the average stone diameter of 30-45 cm and the temperature between 1618°c. then, the optimal habitat for this fish is low altitudes, large stone diameter, high water temperature, low flow velocity and small river width. regarding the abundance of sampled fish in this study, it can be noted that the central and lower regions of the kordan and jajrood rivers and the far stations from the dam (1 and 6) in the taleghan river provide the most favorable habitats for these species. this suggests that the habitat of a species is influenced by various factors and any change in each of the environmental variables can change the living conditions of the fish. conclusion preliminary studies and pre-sampling showed that among these species, c. buhsei has a higher distribution in namak lake and dasht-e kavir basins, which can be an appropriate candidate for the interspecific adaptability study. all three rivers in terms of latitude and the amount of anthropogenic effects almost are in the same situation, and the results of this study rejected the hypothesis that the wide distribution of c. buhsei is due to its different habitat preferences compared with those of the other two species. therefore, the greater distribution of c. buhsei may be due to the ability of this species to adapt to environmental changes that requires further studies. acknowledgements the authors wish to thank the iranian national science foundation and the research deputy of the faculty of natural resources, university of tehran, for partially funding the thesis of m. nasrolah pourmoghadam, under the scheme of “supporting the ph.d. theses. references an k.g., park s.s., shin j.y. 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(2019) 7(4): 211-217 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article effects of florfenicol on skin mucus immune parameters and immune related genes expression in zebrafish (danio rerio) narmin ezatrahimi1, siyavash soltanian*1, mostafa akhlaghi1, seyed hossein hoseinifar2 1department of clinical sciences, school of veterinary medicine, shiraz university, shiraz, iran 2department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. s article history: received 3 june 2019 accepted 24 august 2019 available online 2 5 august 2019 keywords: florfenicol immune mucus gene expression zebrafish abstract: florfenicol (ff) is a common, inexpensive antibiotic with relatively low toxicity. the present study investigates the possible effects of florfenicol on cutaneous mucus immune parameters and immune related genes expression in danio rerio. after two weeks adaptation, fish were stocked in experimental units at density of 20 fish per aquaria and fed with experimental diets containing 0, 10, 30 and 50 mg/kg ff per for 11 days. evaluation of immune parameters at the end of trial showed that the control group had a significantly lower level of lysozyme activity when compared with those treated with antibiotic. however, the skin mucus total immunoglobulins level of fish fed diets containing antibiotics did not show any significant difference compared to the control group. the highest expression level of tnf-α, il1β and lysozyme was observed in fish treated with 10 mg/kg ff while the lowest expression was noticed in those fish treated with 30 and 50 mg/kg ff. the present results indicate the relatively positive effects of this antibiotic on the immune system of zebrafish, and it seems that the appropriate dosage of the drug can serve as an immunostimulant for zebrafish. introduction fishes are vulnerable to a vast variety of pathogenic and non-pathogenic micro-organisms. as aquaculture is developing and new methods of culture are developed, there is an increase in incidence rates of bacterial, viral, parasitic diseases as well as environmental problems. to deal with stressful conditions, fish develop adaptation mechanism. in fact, through development of such mechanism, natural or homeostatic status of body can be maintained (makvandi et al., 2011). a complex set of specific and non-specific immune parameters concerning stress response in fish have been identified (ellis, 2001). primary response to pathogens is formed by the primary defense system which include the skin mucus, skin, gills, gastrointestinal tract as well as blood components, including natural killer cells and phagocytes (anbarasu and chandran, 2001). the mucus layer consists of glycoprotein, proteoglycan and proteins that, besides providing physical *correspondence: siyavash soltanian doi: https://doi.org/10.22034/ijab.v7i4.631 e-mail: siyavashsoltanian@yahoo.com protection, contain secretory compounds such as agglutinins, lysines, lysozymes, non-specific precipitators, acute phase protein. also, the presence of natural anti-bodies provide defensive-chemical role against environmental micro-organisms (soltani, 2008). fish are commonly exposed to chemicals released into aquatic system which per se can disrupt innate and adaptive immune system (yousefi and hoseinifar, 2018). antibiotics have significant roles in controlling diseases. besides, in aquaculture they have formerly been used as growth stimulators, antibacterial agents, and for a variety of other purposes (masahiro et al., 1999; li et al., 2012). however, due to their residual effects on the fish and shrimp tissues and consumers' lack of interest, application of antibiotics is not recommended in aquaculture (khajeali, 2012). the interaction between medications and lymphoid tissues may affect the performance and balance of immune system causing unintentional negative effects such as 212 ezatrahimi et al./ florfenicol effect on zebrafish immunity immunosuppression, uncontrolled cell proliferation, changes in defensive mechanisms of host against pathogens, and even neoplasia (ren et al., 2014). several immunomodulatory effects of anti-bacterial and other medications have been reported (lunden et al., 1999). in recent years, several reports on antibiotic resistance in pathogens raised a concern about fisheries and public health, hence, emphasizing identification and application of new antibacterial compounds to deal with such bacterial diseases (park et al., 2008). florfenicol (ff) is a broad-spectrum antibiotic, uses for treatment of various infections, which has been confirmed in food-producing animals due to low price and relatively low toxicity (carty et al., 2008). in addition to merits of chloramphenicol such as wide spectrum effects and high tissue infiltration, ff has a longer elimination half-life. on the other hand, due to substitution of fluorine with a hydroxyl group in its chemical structure, florfenicol is more resistant to enzymatic inactivation, putting effects on a wider spectrum of chloramphenicol-resistant bacterial strains (anadon et al., 2008). despite an increase in use of ff, some studies document its toxicity (hu et al., 2014). therefore, it is essential to determine its effects on mucosal immune level of fishes as their primary defense mechanism against pathogens. zebrafish (danio rerio) has recently been introduced as a model for rapid analysis of gene performance (zon et al., 2005). it is widely used in different laboratories for genetic research (haffter et al., 1996; driever et al., 1996). hence, the present study aims to investigate the effects of florfenicol on the fish mucosal immunity as the primary defensive mechanism against pathogens. materials and methods fish and experimental design: a total of 240 zebrafish were purchased from a private farm. each experimental unit (84 l aquarium) contained 20 fish. prior to the study, a 2-week adaptation period with laboratory conditions was considered. they were fed a commercial food biomar (biomar sas, nersac, france). throughout the study, water temperature, ph, and dissolved oxygen were monitored daily and maintained at optimum rates. ff was purchased from the rooyan company and added to the basal diet. the experiment was designed as completely randomized with four treatments (three experimental and one control) repeated in triplicates. the feeding trial was lasted for 11 days, and during this period, fish fed 3% of body weight, twice a day. experimental diet preparation: in this study, commercial food, biomar (biomar sas, nersac, france) (table 1) was used as the basal diet and different doses (10, 30 and 50 mg/kg) of ff were added. skin mucus immune parameters: skin mucus samples were obtained based on ross et al. (2000). accordingly, three fish from each tank were randomly selected and anaesthetized. the mucus samples were kept in -80°c before determining the lysozyme activity and the total ig level. enzymatic assay of lysozyme was performed using turbidimetric method (subramanian et al., 2007). in this procedure, micrococcus luteus (atcc4698) was used as substrate. the mucus total ig levels was measured based on the method suggested by siwicki and anderson (1993). determination of immune genes expression: the intestinal tissue samples were obtained from samples and immediately transferred to liquid nitrogen tank. to extract total dna, 50-100 milligrams of the tissue was homogenized in 1 ml of rnx-plus for 15 minutes in room temperature according to company guidelines. after drying the product, it was dissolved in 50 ml of distilled water and then kept in the refrigerator at 80°c. prior transferring samples to refrigerator, the concentration enzyme activity of rna were checked using spectrophotometer in 260/280 nanometers. table 1. the proximate composition of the basal diet. proximate analysis (%) 93.2 dry matter 38.6 crude protein 15.1 crude lipid 11.0 ash 213 int. j. aquat. biol. (2019) 7(4): 211-217 also, the quality of rna was assessed using agarose gel electrophoresis as well as dyeing with ethidium bromide (miandare et al., 2013). one ml of total rna was used to build cdna using bio rad kit. copying process was conducted according to the manufacturer's instructions. the resulted cdna was kept in -20°c (gioacchini et al., 2010). the primers of qpcr were designed for tnf-∝, il-1 and lys genes in gene bank based on protected areas of dna sequences (table 2). then, the primers were added to the samples for analyzing gene expressions. copying process was conducted according to a specific protocol. cyber green method was used to investigate gene expression and the samples were put in real time pcr instrument to measure gene expression. statistical analysis: spss version 16.0 (spss inc., chicago, il, usa) was used for statistical analysis. the statistically significant differences (p<0.05) were determined using one-way analysis of variance (anova) followed by duncan's multiple range tests. results skin mucus immune parameters: the skin mucus lysozyme activity of fish in control group's was significantly lower than that of the experimental groups treated by various doses of antibiotic (p<0.05) (fig. 1). the results showed the total immunoglobulin in fish mucus was not significantly different between treatments (p>0.05) (fig. 2). immune related genes expression: gene expression studies revealed the highest expression of tnf-α gene in 10 mg of ff treatment (fig. 3), whereas the others did not have significant differences compared to control. in addition, in 10 mg of ff treatment the expression of il-1β gene was the highest (fig. 4), and those of 30 and 50 mg had the lowest level, and the control one showed an intermediate levels (p<0.05). the expression of lysozyme gene in 10 mg/g ff was table 2. sequences and accession numbers of primers used. primer name primer sequence application accession number i1b q-pcrf cgtctccacatctcgtactca immune ay340959.1 il1b q-pcrr gtgtctttcctgtccatctcc tnf-alpha q-pcrf ctgcttcacgctccataaga immune ay427649.1 tnf-alpha q-pcrr ctggtcctggtcatctctcc lyz q-pcrf ggcagtggtgtttttgtgtc immune nm_139180.1 lyz q-pcrr cgtagtccttccccgtatca β-actin q-pcrf agcagatgtggatcagcaag housekeeping gene nm_131031.1 β-actin q-pcrr tacctccctttgccagtttc figure 1. the effect of different doses of florfenicol on mucus lysozyme activity in zebrafish. values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. different letters above the bars denote a significant difference between treatments (p<0.05). figure 2. the effect of different doses of florfenicol on mucus total ig in zebrafish. values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. different letters above the bars denote a significant difference between treatments (p<0.05). 214 ezatrahimi et al./ florfenicol effect on zebrafish immunity significantly higher than others (fig. 5). however, no significant difference was noticed when compared with control group. discussions improvement and protection of fish health is one of the main factor in the aquaculture industry (mahghani et al., 2014). increasing the productivity of fish farming is associated with stressful conditions, which provides a sensitive environment for infectious diseases (dehghan et al., 2016). lysozyme is one of the most important parameters of the non-specific humoral immune system. it is a polypeptide with a molecular weight of 14 to 18 kda. it exists in a wide range of vertebrates, including freshwater and marine species, and is known as an antibacterial agent (itami et al., 1992). this enzyme is secreted from white blood cell granules (mostly by neutrophils, monocytes and less by macrophages) as well as leukocyte-rich tissues such as kidneys, mucus membranes, spleen, gills and digestive tract since these organs are highly susceptible to invasion by pathogenic bacteria (holloway et al., 1993). the enzyme can break down the glycosidic bond of peptidoglycan layer in the bacterial cell wall. lysozyme has direct effects on gram-positive bacteria; and can eliminate the gramnegative bacteria with the help of complement system (yano, 1996). in the present study, evaluation of skin mucus lysozyme activity revealed significant increases in all experimental groups compared to the control group. however, the treatment containing 30 mg ff showed more activity, which was not statistically significant. reda et al. (2013) reported that lysozyme activity is significantly increased in tilapia after ff administration, but had no significant effect on igm, phagocytosis and alt as seen in the present study. in contrast, administration of ff in hybrid tilapia diet at rate of 0.02 g kg-1 had no effect on serum lysozyme. it may be due to different conditions used in previous studies, fish species, temperature, antibiotic dose and figure 2. the effect of different doses of florfenicol on relative expression of tnf-α gene in zebrafish. values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. different letters above the bars denote a significant difference between treatments (p<0.05). figure 4. the effect of different doses of florfenicol on relative expression of il-1β gene in zebrafish. values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. different letters above the bars denote a significant difference between treatments (p<0.05). figure 5. the effect of different doses of florfenicol on relative expression of lysozyme gene in zebrafish. values (n=9) are presented as the mean ± standard deviation, obtained by dividing each sampling value by the mean control value at the same sampling time. different letters above the bars denote a significant difference between treatments (p<0.05). 215 int. j. aquat. biol. (2019) 7(4): 211-217 route of administration which can affects drug absorption (bjorklund and bylund, 1990). according to ren et al. (2014), juveniles of litopenaeus vannamei revealed no significant differences in antibacterial activities in plasma following administration of 100 mg kg-1 ff, whereas 200 mg kg1 ff treatment suppressed them, the total hemocyte count and phagocytic activity decreased significantly in both treatments. in the present study, the total ig level in the skin mucus of fish fed diet containing different antibiotic doses showed no significant difference compared to control. since the function of a gene at the mrna level varies from that of the protein after translation, the evaluation of the expression of lysozyme gene can be reliable in evaluation of the effect of immunostimulants on this gene. the level of lysozyme or its activity is viewed as an important indicator in the primary defense of fish. there are changes in levels of lysozyme activity or mrna expression in response to infectious diseases or stress. based on the results, the highest expression of lysozyme was in fish treated with 10 mg/g ff. however, there was no significant differences when compared with the control. il1β and tnf-α expression in the 10 mg/kg ff were highest; however, 30 and 50 mg/kg of ff had the lowest expression level, and the fish in control group showed an intermediate level. the tumor necrosis factor (tnf) is a signaling protein (cytokine) participating in systemic inflammation as one of the cytokines forming the reaction of the acute phase. tnf-α serves as intermediate proteins in immune cells and puts effects on them (gruss, 1996). tnf-α is the main mediator of acute inflammatory response to gram-negative bacteria and other bacterial microbes and accounts for most of the systemic disorders of acute infections. the results showed that the expression of this gene in fish treated with 10 mg/kg was highest. in contrast, suppressing effects of ff on rat immune responses showed that ff could suppress humoral and cellular immune responses in mice (guan et al., 2011). similarly, some antibiotics have a suppressive effect on increased levels of tnf-α (er et al., 2010; er and yazar, 2012). in this study, the expression of this gene decreased in a dose-dependent manner. in brown trout, serum levels of tnf-α in fish treated with ff were not suppressed following lps injection. this may be due to the dose of ff or differences in the species type (ayse and burak, 2014). according to caipang et al. (2009), oral administration of ff and oxolinic acid significantly increase the anti-inflammatory cytokines, including il-1β and il-8 in atlantic cod (gadus morhua). changes in the immune response and antioxidant defense of the atlantic cod have also been documented in relation to the oral administration of oxolinic acid and ff. these antibiotics could modulate some components of the innate humoral immune responses, bacterial pathogen proliferation in the sera, antioxidant defense genes and transcription of selected immune response when given at therapeutic concentration (caipang et al., 2009). based on the present results, it can be concluded that ff had the relatively positive effects on the immune system of zebrafish. it seems that the appropriate dose of drug in the diet can act as an immunostimulant which preventing stressors and disease occurrence. references anadon a., martinez m. a., martinez m., rios, a., caballero v., ares i., martinez-larranaga r. m. 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(2019) 7(2): 100-105 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article improvement of fish production in benin: which strain of tilapia oreochromis niloticus is recommended to fish farmers? gangbé luc1,2, achoh mardochée ephraim*3,1aboudou zouberou aboudou4, hounhoedo aimé5, agbohozo buenas5, aizonou romaric3, agadjihouèdé hyppolite1,3 1laboratory of hydrobiology and aquaculture (lha), faculty of agronomic sciences, university of abomey-calavi, 01 po box: 526 cotonou, benin. 2national institute of agricultural research of bénin, 01 po box 884 cotonou, benin. 3laboratory of research in aquaculture, biology and aquatic ecology (larabea), the valley’s school of aquaculture, national university of agriculture, po box 43 kétou, benin. 4polytechnic school of abomey-calavi, university of abomey-calavi, 01 po box 2009 cotonou, benin. 5department of aquaculture and fisheries management, university of ibadan, ibadan, nigeria. s article history: received 15 january 2019 accepted 6 april 2019 available online 2 5 april 2019 keywords: tilapia strain zootechnical performance local feed abstract: controversies on the performance of the tilapia strain oreochromis niloticus (s2 inrab, si), led us to be interested to the evaluating of that strain’s growth in comparison with the strain commonly used by the fish farmers named faizou’s strain (sf). fry of average weight 10.17±2.0663 g were stocked in concrete tank (density: 10 individuals/m2). the experiment lasted 60 days where the temperature, oxygen-dissolved and ph parameters were measured morning and evening every 48 hours. the feed used was made from local products at protein level of 45%. the results revealed that physicochemical parameters have no negative influenced on fish growth even though they were significantly different from one strain to another. the gain in weight obtained was significantly higher for the strain sf (38.12±5.65 g) than the si (37.15±4.99 g) despite it recorded 13.33% more mortality than the si strain. above of this, the average daily gain (adg) of the sf strain was almost linear over the entire study period, unlike the si strain whose has no linear adg. it shows that the sf strain is more productive despite the recorded mortality. this confirms the fish farmers’ claims on the lowest growth of the si strain, which however is to be considered in selection and breeding programmes. introduction in west africa especially in benin republic, agriculture is one of the essential components of the economy. it contributes 36% to benin's gross domestic product (insae, 2008). it uses 48% of the active population with 60% of male workers and 40% of female workers. among of the agricultural production sectors in benin, aquaculture is one on which benin is committed to increase its production of animal protein (psrsa, 2011). the new government orientations for the 2016-2020 aim reaching 20,000 tons against 5,000 tons in 2015 as aquaculture production. these new orientations are aimed to reduce as far as possible the deficit in proteins from animal origin but also to ensure good employment, around 18 000 created for young people *correspondence: mardochée ephraim achoh doi: https://doi.org/10.22034/ijab.v7i2.560 e-mail: mardoachoh12345@gmail.com in aquaculture sector by 2021. beninese fish farming mainly exploits two species, including tilapia oreochromis niloticus and african catfish, clarias gariepinus (imorou, 2007). oreochromis niloticus is widely used in aquaculture in benin with the support of several programmes and projects promoting industrial and family aquaculture (achoh et al., 2018). however, fish farmers are faced with a collapse in the genetic aptitude on o. niloticus (rurangwa et al. 2014). this is happened because, of a multitude strains which are present with uncontrolled crossings in aquaculture farms in benin. it has been necessary to look at breeding and genetic improvement issues to obtain a good performance strain leading to a good production yield. it was in this perspective, chikou et al. (2014) established and 101 int. j. aquat. biol. (2019) 7(2): 100-105 disseminated among important fish farmers’ strains of o. niloticus named s2 inrab strain (si). in a logic of fish famers’ appreciation on the performance of that strain, it is insisted that the strain s2 inrab is less efficient and does not have the expectations of yield. to better understand the perception of fish farmers, it is better to conduct a comparative study on growth performance and to indicate new orientations in terms of research on the genetic improvement of tilapia in benin republic. this is the main objective of this study which aims to confirm or not the controversies of fish farmers on the zootechnical performance of the s2 inrab strain under breeding conditions. materials and methods experimental procedure: the experimental set-up was composed of six (6) concrete tanks (1 m3) arranged by total randomization. each strain was arranged in 3 replicates. the tanks were mid-filled with drilling water 72 hours before fish stocking with measurement of physicochemical parameters to monitor the stabilization of the water before starting the experiment. the comparative study was carried out with the faizou’s strain (sf) (strain produced on the farm ‘’johan estève’’ in benin) which, according to the fish farmers, has proven zootechnical performance. the producer of this strain, faizou has a good system of strain safety (with the support of the west african agricultural productivity project waapp-benin/ procad), avoiding any kind of genetic pollution. the faizou’s strain was purchased and acclimatized for one week in the research station before the start of the experiment. the stocking density was 10 individuals per tank (10 individuals/m2). sixty (60) fingerlings of o. niloticus were used i.e. 30 s2 inrab strain and 30 of faizou’s strain with average weight 10.17±2.0663 g. conduct of experimentation: after stocking, the fish were fed twice daily (8 a.m and 6 p.m), at the rate of 12% body weight. this rate was revised gradually downwards according to the growth of the specimens. thus, the rate was revised firstly at a rate 10% body weight after the first control fishery, secondly at a rate of 6% body weight after the second control fishery and finally at a rate of 5% body weight after the third control fishery. the feed used contained a protein level of 45% and made from local ingredients whose incorporation rates are summarized in table 1. during the experiment, ph, temperature and dissolved oxygen were measured every 48 hours in the morning at 7 o'clock and in the evening at 17 o'clock. the control fishery took place every fortnight and covered 50% of the total population of each experimental unit. during the fishing, water of each concrete tank was renewed. the experiment lasted 60 days. data analysis: the data collected were analyzed statistically with the statistica software (2004, version 6). indeed, after verifications of the normality, the averages of the physicochemical parameters were compared between the plot using t student test with p-value = 0.05. the curve of weight with the time was presented and the coefficients of variation were calculated and compared between the plots using z bilateral test. the following zootechnical parameters were calculated and compared using t student test. average daily gain (adg) = (pf pi)/t survival rate (sr %)= (nfx100)/ni gain in weight (gw) = pf pi net production rate (%) (npr) = (pf pi) x 100 / pf specific growth rate (sgr) = (logpf-logpi) x 100/t yield of harvest: (t / ha) = (gw) / area unit where pi = initial weight, ni = initial number, pf = final weight, nf = final number; t = duration of the experiment, log = logarithm with base 10, si = strain s2 inrab, and sf = strain faizou. table 1. percentage of food composition. ingredients quantity of ingredients (%) bran 5 wheat flour 14 cotton oilcakes 5 soy oilcakes 10 fishmeal 60 cmv 1 oyster shell 4 iodized salt 0,5 red oil of palm 0,5 total 100% proteins 40% 102 gangbé et al./ improvement of fish production in benin results physico-chemical characterization of the water: the averages of the physico-chemical parameters for the duration of the trial are summarized in table 2. according to the results, the physicochemical parameters (temperature, dissolved oxygen and ph) have significant difference between the two strains areas (p<0.05). specifically, the temperature is significantly higher at the si strain (p=0.011) while the dissolved oxygen and ph are significantly higher at the sf strain with a p-value 0.013 and 0.0000, respectively. the value of the temperature varied between 29 and 30°c, the dissolved oxygen between 3 and 4.5 mg.l-1 and the ph between 7 and 8.5 at the two strains. this, despite the significant difference noted, the values are within the tolerance range of the species. growth in weight and length of fish: the evolution of the growth in weight of the specimens for the two strains is shown in figure 1. the trend of evolution of the growth is almost similar for the two strains except that the strain si seems to have presented a more accelerated growth at the first month before matching the same growth rate with the sf strain. in this way, the growth of the strain si has no linear evolution while the sf strain has linear evolution over the time. the table 3 shows the initial weights, final weights, the coefficient of variation, and the probabilities associated with the analysis. it appears that the averages obtained for the final weights are respectively 47.74±7.836 and 47.86±6.853 g against initial weights of 9.62±2.178 and 10.71±1.860 g for the sf and the si strains. at the end of trial, the growth in weight of that two strains is significant and the average values are significantly higher than the initial values with p<0.05. on the other hand, the final average weights are no significant different between the strains (p>0.05). thus, the two strains have the same weight gain after 60 days of experimentation. regarding the homogeneity of the batches, no significant difference was recorded between the two strains (p>0.05). evolution of average daily gain: the variation in daily average after control fishery for both strains is presented in figure 2. the average daily gain is roughly equal for both strains at the first control table 2. average of physicochemical parameters. temperature (°c) disolved oxygen (mg.l-1) ph mean sd mean sd mean sd sf 29.19 1.871 3.33 1.306 7.88 0.715 si 29.26 1.950 3.22 1.345 7.67 0.739 p-value 0.013 0.013 0.000 sd = standard deviation; p>0.05 = no significant difference; p<0.05 = significant difference table 3. average initial weight and final weight. initial weight final weight mean sd mean sd p-value cv sf 9.62 2.178 47.74 7.836 < 0.05 16.41 si 10.71 1.860 47.86 6.853 < 0.05 14.39 p-value >0.05 >0.05 >0.05 sd = standard deviation; p>0.05 = no significant difference; p<0.05 = significant difference figure 1. weight curve evolution for control fishery. 103 int. j. aquat. biol. (2019) 7(2): 100-105 fishery (0.51 g/day). growth acceleration is noted at the end of the second fortnight of the si strain (0.80 g/day); and felt down considerably in the third fortnight till recorded the lowest gain in fifteen days for the whole experimentation period (0.44 g/day). the average daily gain of the si strain evolved no linear during the experiment, while the sf strain showed an almost linear weight gain over the entire period. at the end, the means of daily gain obtained for the entire trial period are no significant different (p>0.05) and are 0.635±0.09 and 0.619±0.17 g/day for sf and si strains, respectively. however, strain sf recorded a numerically higher value than strain si. averages of total length: the body length gain of the two strains was evaluated and is summarized in table 4. the length growth was significant at the end of the experiment (p<0.05) for the both strains. the final averages obtained are 14.37±1.174 and 14.61±0.593 cm against 7.94±0.698 and 8.49±0.539 cm, respectively for the sf and si strains. the coefficient of variation revealed that the plot of si strain (4.06%) is significantly more homogeneous than sf strain (8.17%) (p<0.05). the table 5 shows the average zootechnical parameters (wg, sr, npr, sgr and yield) of two strains. the zootechnical parameters reveals that the weight gain is significantly higher at the sf strain than the si strain (p=0.4841). although it had the highest gain in weight, the sf strain had the lowest survival rate (86.67%) against 100% for the si strain (p<0.05). in the same way, the yield is significantly higher with the sf strain (12.71 g.m-2 or 127.1 tons. ha-1) against 12.38 g.m-2 or 123.8 tons.ha-1 for the strain si. it should be noted that no significant difference is recorded between the strains for the other parameters, especially the net production rate (npr) and the specific growth rate. discussions physicochemical parameters are the characteristics of the ecological conditions that determine aquatic life. table 4. total initial length and final length of fish. initial length final length mean sd mean sd p-value cv (%) sf 7.94 0.698 14.37 1.174 <0.05 8.17 si 8.49 0.539 14.61 0.593 <0.05 4.06 p-value >0.05 >0.05 <0.05 sd = standard deviation; p>0.05 = no significant difference; p<0.05 = significant difference table 5. evaluation of the zootechnical parameters. sf si mean sd mean sd wg (g) 38.12a 5.657 37.15b 4.992 sr (%) 86.67 c 5.7735 100.0 d 0.0 npr (%) 79.84e 72.198 79.65e 72.853 sgr 1.16f 0.927 1.15f 0.944 yield (g/m2) 12.71g 1.886 12.38h 1.664 sd= standard deviation; means bearing the same letters are not significantly different from other while those wearing the different letters are significantly different figure 2. curve evolution of the average daily gain according to control fisheries. 104 gangbé et al./ improvement of fish production in benin the values obtained for these parameters into the water of the two strains (29-30°c for the temperature, 3-4.5 mg.l-1 for the dissolved oxygen and 7 to 8.5 for ph) are within the range required for good growth of o. niloticus (lacroix, 2004; lazard, 2009; amoussou et al., 2016) for that, the values obtained for the parameters could not negatively influence the zootechnical performances during the trial. the both strains have final average weights that are no significant different. the weight gain compared to the initial weight of the two strains is significant and shows the quality of the feed, but also the physicochemical conditions recorded which did not influence the growth of the fish whatever the strain (toguyeni, 1996). the average daily gain is no significant different between the two strains during the entire study period, but remains numerically higher in the sf strain. however, the variations obtained for the average daily gain of the sf strain express an almost linear growth, unlike the si strain, which show the best quality of the sf strain. besides, considering the survival rate, it is obvious that the sf strain, although it recorded a mortality rate of 13.33%, achieved the highest gain in weight. mortalities recorded from sf strain relieve an adaptation to the environment since this strain was purchased from producers while the si strain is in stock at the fish station. this adaptive behavior is supported by the work of lan et al. (2008), which states that stoking, environmental and handling stress influence the survival and growth of fisheries resources. the weight gain, although different between the two strains, indicates the zootechnical quality of the two strains to express a good growth performance especially in sf strain. this corroborates the observations bamba et al. (2008), which, with a density of 10 individuals/m2, had obtained a similar result in four months of breeding on o. niloticus against two months of rearing in the present study. avit et al. (2012) reported having the weight gain of 11.04±0.05 g in four months of rearing. the average daily gains for sf and si strains (0.635±0.09 and 0.619±0.17 g/day) are lower than those obtained by chakrabortry and banerje (2010) and githukia et al. (2015) in fertilized pond that are respectively 1.74 and 0.6677 g/day (all sexes combined). on the other hand, the average daily gain obtained in this study for both strains is higher than that obtained by olufagla et al. (2017) in aquarium (0.013 g/day) for 24 weeks of rearing. bamba et al. (2008) obtained less, using the same stocking density as in this study. these results imply the both strains express some growth performance that should be considered in future genetic selection and breeding programmes. however, the sf strain, in terms of its almost linear growth throughout the study period, would result in a significantly higher average final weight, especially if the technical management favours a better survival rate than the one obtained in this study (86.67%). as conclusion, it should be noted that the sf strain is the one which has the most growth. this strain had an almost linear average daily gain and a higher weight gain than the si strain. nevertheless, the both strains showed reasonably acceptable growth in tilapia culture. it would be advisable to consider them in future selection and genetic improvement programmes and projects, especially to try to combine the growth capacity of the sf strain with the resistance capacity of the si strain for having expressed a survival rate of 100%. acknowledgements the authors are grateful to the fisheries research subprogram of the zootechnical, veterinary and fisheries research laboratory for providing the infrastructure and equipment to carry out this study. references achoh m.e., agadjihouèdé h., gangbé l., dougnon t.v., hounmanou y.m.g., baba-moussa l. (2018). diversity and abundance of tilapia exploited in benin and virus of tilv (tilapia lake virus): synthesis and risk of outbreak. afrique science, 14(2): 90-99. amoussou t.o., toguyeni a., imorou t.i., chikou a., issaka y.a.k. (2016). caractéristiques biologiques et zootechniques des tilapias africains oreochromis niloticus (linnaeus, 1758) et sarotherodon melanotheron rüppell, 1852: une revue. international. journal of. biological and chemical sciences, 10(4): 1869-1887. 105 int. j. aquat. biol. (2019) 7(2): 100-105 avit j-b.l.f., bony k.y., kouassi n.c., konan k.f., assemian o., allouko j.r. (2012). conditions écologiques de production de fingerlings d’oreochromis niloticus (linné, 1758) en association avec le riz wita 12 en étang. journal of applied biosciences, 59: 4271-4285. bamba y., ouattara a., da-costa k.s., gourene g. (2008). production d’oreochromis niloticus avec des aliments à base de sous-produits agricoles. sciences and nature, 5(1): 8-99. chakraborty s.b., banerjee s. (2010). effect of stocking density on monosex nile tilapia growth during pond culture in india. international journal of biological, biomolecular, agricultural, food and biotechnological engineering, 4(8): 2010. chikou a., senouvo p., sodjinou e. (2014). etude de faisabilité de l’introduction et vulgarisation du tilapia (oreochromis niloticus) souche volta en aquaculture dans les communes de soava, bopa, adjohoun et ouinhi. rapport pana1, projet n°:00074252. githukia c.m., ogello e.o., kembenya e.m., achieng a.o., obiero k.o., munguti j.m. (2015). comparative growth performance of male monosex and mixed sex nile tilapia (oreochromis niloticus l.). reared in earthen ponds. croatian journal of fisheries, 73: 20-25. imorou t.i. (2007). amélioration de la production halieutique des trous traditionnels à poissons (whedos) du delta de l’ouémé (sud bénin) par la promotion de l’élevage des poissons-chats clarias gariepinus et heterobranchus longifilis. phd thesis, université de namur, belgique. 186 p. insae. (2008). institut national de la statistique et de l’analyse economique. rapport annuel d’activité. 15 p. lacroix e. (2004). pisciculture en zone tropicale. gtz & gfa terra systems: hamburg. lan l.m., micha j.c., long d.n., yen p.t. (2008). effect of densities and culture systems on growth, survival, yield, and economic return of freshwater prawn, macrobrachium rosenbergii farming in the rice field in the mekong delta vietnam. journal of applied aquaculture, 18(1): 43-62. lazard j. (2009). la pisciculture des tilapias. cahiers agricultures, 18(2-3): 393-401. maep/provac. (2011). au bénin; doc technique de pilotage. 437 p. olufeagba s.o., okomoda v.t., adoga t. (2017). growth performance and nutrient utilization of hormonal sexreversed male and mixed sex oreochromis niloticus under outdoor rearing condition. international journal of aquaculture, 7(16): 106-110. psrsa. (2011). ministère de l’agriculture, de l’élevage et de la pêche (revue), bénin. 116 p. rurangwa e., van den berg j., lalèyè p.a., van duijn a.p., rothuis a. (2014). mission exploratoire pêche, pisciculture et aquaculture au bénin. un quick scan du secteur pour des possibilités d’interventions. embassade du royaume des pays-bas à cotonou / instut for marine ressources & ecosystem studies/ report c072/14 lei report 14-049. toguyeni a. (1996). la croissance différentielle liée au sexe chez le tilapia (pisces: cichlidae), oreochromis niloticus (linnaeus, 1758): contribution des facteurs génétiques, nutritionnels, comportementaux, et recherche d’un relais endocrinien. phd thesis, université de renne i, renne. 250 p. int. j. aquat. biol. (2013) 1(3): 132-137 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article age and sex specific variation in hematological and serum biochemical parameters of beluga (huso huso linnaeus, 1758) reza akrami1, ahmad gharaei*2,1roghayeh karami2 1department of fisheries, islamic azad university, azadshahr branch, p.o. box: 30, iran. 2department of fisheries, international hamoun wetland research institute, university of zabol, p.o. box: 98615-538, iran. article history: received 26 april 2013 accepted 28 may 2013 available online 2 0 june 2013 keywords: sturgeon biochemical parameters, hematology feeding rate abstract: in the present study, the ageand sex-specific changes of various haematological and blood serum biochemical parameters of beluga (huso huso) were investigated. blood samples were collected from 4-, 6-, 7-, and 8-year-old beluga (n = 7 for each sex and age). the specimens were fed at a rate of 0.5-3% body weight per day. ast and ldh levels in 7and 8-year-old fish of both sexes were significantly higher (p<0.05) than those in 4and 6-year-old individuals. the mean alt were significantly different (p<0.05) in both sexes of 4-, 6-, and 7-year-old sturgeon. however, the 6-, 7-, and 8-year-old female sturgeon had higher alp levels (p<0.05). additionally, mean rbc, pcv, and hb values were significantly higher (p<0.05) in 7and 8-year-old females and males than the others. two-tailed pearson’s correlation between the biochemical and haematological parameters obtained for beluga sturgeon indicated significant positive correlations between ast and alp, ast and ldh, alp and ldh, rbc and hb, rbc and pcv, hb and pcv, mch and mchc, and mcv and mch. however, significant negative correlations were found between rbc and mcv and mch. these results suggest that the blood parameters of beluga are influenced by ageand sex-specific factors. introduction analysis of serological and hematological parameters is one of the most informative methods to monitor physiological status because these parameters are specific among different species and sexes (celik, 2004; asadi et al., 2006). the physiological parameter provides a diagnostic tool to assess possible dysfunction and detect the metabolic disturbances, infectious and non-infectious etiologies (aldrin et al., 1982). there are many studies concerning the hematological and serum enzyme activities of different sturgeon species (slynko, 1976; shahsavani et al., 1999, 2001, 2010; falahatkar et al., 2005; asadi et al., 2006; gharaei et al., 2010; rajabipour et al., 2006, 2010; ahmadifar et al., * corresponding author: ahmad gharaei e-mail address: agharaei551@gmail.com tel: +985422232600 2011). however, fluctuations in serum component levels and hematological indices during the artificial conditions have not been investigated yet. sturgeons, the producers of valuable black caviar, live exclusively in the northern hemisphere. the caspian sea is the habitat of four commercial species of sturgeon (keyvanshokooh et al., 2009). the giant sturgeon (huso huso) is the largest species of acipenseriformes and one of the most important species of sturgeon in the caspian sea (jalali et al., 2008). this species is suitable for aquaculture because of its fast growth, ease of reproduction, high tolerance to adverse environmental conditions (keyvanshokooh et al., 2009). the aim of this study was to (1) determine the normal fluctuations of the serum biochemical properties of 133 akrami et al./ int. j. aquat. biol. (2013) 1(3): 132-137 beluga reared in ponds until maturity, (2) compare the parameters between sexes, and (3) provide a basis for future comparative investigations. materials and methods this experiment was carried out on 4-, 6-, 7-, and 8years old beluga. the fish were reared in the earthen ponds supplied with ground water (19 ± 3°c) from the larval stage at the shahid marjani breeding and rearing centre, iran, from may 2005 to may 2009. they were fed a commercial feed containing 39% protein, 12% lipid, 8% ash, 3% fiber, 0.8% phosphorus and 11% moisture (abizan™, iran). following sampling from each age group, total length and body weight were measured and recorded (table 1). to measure serum biochemical factors during the culture period, blood samples (n = 7 for each sex and age) were taken on 4-, 6-, 7-, and 8-years old fish. all blood samples were collected from behind the anal fin using a 5 ml plastic syringes with 23 gauge needles. prior to sampling, the fish were anesthetized with 200 mg l-1 ms 222. blood sera were obtained by centrifuging the sample at 3,000 rpm for 10 min (using a heraeus labofuge 400) and the sera were removed with a disposable transfer pipette (shakoori et al., 1996; wood et al., 1996). the sera samples were analyzed using an auto-analyzer (technicon ra1000, usa) and diagnostic kits (pars azmoon co., tehran, iran). to study the hematological parameters, blood samples (n = 7 for each sex and age) were collected in heparinized tube prior to anaesthesia of the fish to prevent haemolysis. the following parameters were subsequently determined according to akrami et al. (2009): red blood cell (rbc), white blood cell (wbc), hematocrit (pcv), hemoglobin (hb), mean corpuscular volume (mcv), mean corpuscular hemoglobin (mch) and mean corpuscular hemoglobin concentration (mchc). statistical analysis: data were tested for normality and homogeneity of variance before the analysis. the data were examined using a one-way analysis of variance (anova), followed by duncan’s multiple range test (duncan, 1995) to rank the groups using spss (version 10). the pearson’s correlation coefficient was used to examine the correlation between each hematological index and serum biochemical parameters. results the fluctuations in serum enzyme activity (i.e. ast, alp, alt, and ldh) in female and male beluga are listed in table 2. ast levels were significantly higher in 7and 8-years old males and females than in 4and 6-years old individuals (p<0.05). mean alt levels were significantly different (p<0.05) in both sexes of 4-, 6-, and 7-year-old fish. 6-8 years old females had higher alp levels than 4 years old females (p<0.05). however, 7and 8-year-old males and females exhibited significantly higher ldh levels than the others (p<0.05). there were significant positive correlations between ast, alp, and ldh (table 3). the hematological parameters of both sexes and all ages of the fish are listed in table 4. the mean values of rbc, pcv, and hb in 7and 8-year-old females and males different significantly from those of the other age group. age (year) sex weight (kg) (mean±sd) length (cm) (mean±sd) 4+ female 7.60 ± 0.84 101.20 ± 8.30 male 7.80 ± 0.59 102.20 ± 8.90 6+ female 12.43 ± 0.97 111.14 ± 12.86 male 13.12 ± 1.23 114.37 ± 15.92 7+ female 19 ± 1.11 139.75 ± 8.12 male 23.25 ± 2.21 140.05 ± 16.17 8+ female 25 ± 2.41 142.04 ± 12.65 male 26.50 ± 2.36 145.25 ± 9.71 table 1. the age, mean weight and total length of female and male (n = 7 for each sex and age) beluga. 133 134 akrami et al./ int. j. aquat. biol. (2013) 1(3): 132-137 there were significant positive correlation between rbc and hb, rbc and pcv, hb and pcv, mch and mchc, and mcv and mch. there were significant negative correlations between rbc, and mcv and mch (table 5). discussion many studies have documented changes in hematological and biochemical parameters in fish according to age or size (satheeshkumar et al., 2011). however, these investigations are limited to the comparisons between juvenile and adult fish or examination of only wild-caught fish, in which other factors may influence blood variables (terry et al., 2001). few studies have investigated blood values from sturgeon of known ages (gharaei et al., 2012; terry et al., 2001), which makes comparison of our results with those on other fish species difficult. the mean levels of ast and ldh in the sera of 7 and 8-years old beluga were significantly higher than those of 4and 6-year old. the activity of serum enzymes can be increased in two ways; leakage from damage, or induction that increases the amount of the enzyme and hence the amount that is also likely to leak. it is also important to mention that such an induction may occur when the organism mobilizes its energy sources, including amino acids, to manage stress and repair damage caused by metals (masola et al., 2008). however, asadi et al. (2006) reported that mature female a. persicus exhibited higher ast activity than immature females. ldh is very nonspecific and is located in the cytoplasm of the most cells. ldh catalysis the conversion of lactate to pyruvate and is therefore an important enzyme for generating energy in cells (shahsavani et al., 2010). in this study, the higher ast and ldh levels in the older beluga may have been due to the beginning of the sexual maturation. it should also be noted that ldh levels are directly correlated with growth rate (rajabipour et al., 2010). alt and ast are non-plasma-specific enzymes located within the cells of the liver, heart, gills, kidneys, muscles, and other organs (gharaei et al., 2010). in beluga, simultaneous increases of alt and ast enzyme activity with age may be related to changes in physiological metabolites due to sexual development, feeding management and differences in size (trivedi et al., 2001; cech et al., 2000). shahsavand et al. (2010) measured the activity of ast in the sera of mature beluga and stated that the parameter (ul-1) 4+ years 6+ years 7+ years 8+ years male female male female male female male female ast a 20 ±297.4 a 76.2 ±315.7 a 51.23 ±280.8 a 40.5 ±274.5 b 5 15 ±666.5 b 113 ±656.2 b 81.6 ±565.6 b 124 ±570.2 alt b 1.14 ±3.4 b 0.92 ±3.8 a 0.69 ±1.1 a 0.55 ±1.6 c46 3. ±10.3 c 4.1 ±10.3 bc 1.51 ±6.1 bc 1.1 ±6.3 alp a 28 ± 184.8 a 41 ±184 a 39.4 ±165.8 b 46.5 ±266 a6 51. ±184.7 b 32.6 ±208 a 37.4 ±157 b5 33. ±204.3 ldh a 103 ± 487.4 a 204 ±628.3 a 214 ±600 a 176.4 ±717.5 c 673 ±3392 c 712 ±4094 b 580.2 ±1852 b 634 ±2219 table 2. the values of serum enzymes activity of female (n = 7) and male (n = 7) beluga in difference ages. values followed by different superscript letters in each row are significantly different (p<0.05). parameter ast alt alp ldh ast p value 1 0 -0.06 0.939 *0.911 <0.001 *0.865 <0.001 alt p value 1 0 -0.033 0.842 -0.053 0.753 alp p value 1 0 *0.836 <0.001 ldh p value 1 0 table 3. pearson correlation and p value between serum enzymes activity of beluga. * showed significantly correlation between parameters. 135 akrami et al./ int. j. aquat. biol. (2013) 1(3): 132-137 significant increase in ast levels may be related to sexual maturation. alp exhibited increased serum activity in 6-, 7-, and 8-years old female beluga. alp induces activity transfer and is normally secreted in bile from the liver. this enzyme mainly leaks from the lining of the bile canaliculi and sinusoidal surfaces of hepatocytes (gottelli et al., 1985). based on our results, the increase of alp activity may be due to an increase in biliary canal or osteoblast activity, or feeding conditions (moraes et al., 2005; cech et al., 2000). the activities of enzymes that play roles in amino acid catabolism differ based on sex and stage of maturity. direct significant correlations were obtained between ast, alp, and ldh, suggesting that individuals with higher ast levels also tend to have higher alp and ldh levels. rbc, pcv, and hb values were significantly higher in both sexes of 7and 8-years old beluga, suggesting that these parameters are age-specific (table 4). the direct significant correlations found among some hematological parameters suggest that individuals with higher rbc and hb levels tended to have the parameters that have greater correlations with each other. rbc and hb concentration tended to increase with length and age of the fishes (satheeshkumar et al., 2011). however, the significant negative correlations between rbc, mcv and mch could be argumentative. * showed significantly correlation between parameters. parameters 4+ years 6+ years 7+ years 8+ years male female male female male female male female (fl) mcv 8.6± 314.3 10.4±317.9 9.65 ±288.6 11.32 ±295.7 12.9 ±283.2 27 ±300.8 19.14 ±294 16.11 ±293.8 mch(pg) 4.5 ± 103.8 11.17±101.3 8.1 ±92.2 12 ±94.8 8.5 ±92.3 13.7 ±98.5 14.3 ±95.9 8.3 ±94.56 mchc (%) 1.21 ±33 1.4±32 0.96± 32 1.3±32.1 0.95 ±32.6 1.44 ±32.5 0.91 ±32.6 1.63 ±32.1 rbc (106 mm) a 0.1± 0.82 a 0.14 ±0.79 a 0.2 ±0.85 a 0.13 ±0.84 b 0.19 ±1.13 b 0.13 ±0.97 b 0.17 ± 1.02 b 0.03 ±0.99 pcv (%) a 2.9 ±22.51 a 4.4 ±21.96 a 3.4 ±24.1 a 2.1 ±24.8 b 4.3 ±28.9 b 3.1 ±27.6 b 3.4 ±29.6 b 2.2 ±29.2 hb (g dl-1) a 0.87 ±7.3 a 1.24 ±7.52 a 1.2 ± 7.74 a 0.67 ±7.62 b 1.19 ±10.1 b 1.2 ±9.93 b 1.12 ±9.7 b 1.01 ±10.4 leukocyte (mm3) 1.8 ±24.01 1.5±24.3 2.3± 23.6 1.88 ±24.2 1.12 ±25.7 1.19 ±23 0.84 ±25.4 1.84 ±24.3 lymphocyte (%) 2.94 ±68.2 3.4±72.5 3.5 ±71 3.14 ±69.6 3.8 ±70.3 3.33 ±71 3.69 ±70.25 3.43 ±70.2 neutrophil (%) 1.8 ±18.2 1.16±19.1 2.14± 19.4 1.87 ±20.5 2.21 ±20.4 2.9 ±19.2 2.45 ±19 2.1 ±20.3 eosinophil (%) 1.2 ±5.8 1.3±5.4 1.58 ±6 1.4± 6.3 1.16 ±6 1.87± 6.2 1.12 ±6.5 1.11 ±5.5 monocyte (%) 0.86± 4.2 0.77±3.41 0.62± 3.6 0.46 ±3.3 0.57 ±3.75 1.01 ±3.66 0.94 ±4.01 0.34 ±4.00 values followed by different superscript letters in each row are significantly different (p<0.05). table 4. hematological parameters of female (n = 7) and male (n = 7) beluga in difference ages. parameter leukocyte )3(mm rbc mm) 6(10 hb )1-(g dl pcv (%) mcv (fl) mch (pg) mchc (%) )3leukocyte (mm p value 1 0 0.224 0.144 0.214 0.164 0.259 0.090 0.011 0.944 0.059 0.702 0.200 0.194 mm) 6rbc (10 p value 1 0 *0.832 <0.001 *0.873 <0.001 *0.489 0.002 *0.465 <0.001 0.77 0.619 )1-hb (g dl p value 1 0 *0.982 <0.001 0.026 0.869 0.096 0.535 0.183 0.236 pcv (%) p value 1 0 0.017 0.915 0.000 0.998 0.003 0.978 mcv (fl) p value 1 0 *0.949 <0.001 0.064 0.681 mch (pg) p value 1 0 *0.373 0.013 mchc (%) p value 1 0 table 5. pearson correlation and p value between hematological parameters of beluga. 135 136 akrami et al./ int. j. aquat. biol. (2013) 1(3): 132-137 in this study, we determined the ageand sexspecific changes in biochemical and hematological parameters of the serum in cultured beluga. a better understanding of relationships between changes in these parameters could have beneficial impact on commercial rearing of this species. these findings can assist environmental and aquaculture officials responsible for making decisions on the management and rearing of this fish. acknowledgements the authors would like to thank the university of zabol and shahid majani propagation and rearing center for providing the fish and necessary facilities for conducting this study. references ahmadifar e., akrami r., ghelichi a., mohammadi zarejabad a. (2011). effects of different dietary prebiotic insulin levels on blood serum enzymes, hematologic, and biochemical parameters of great sturgeon (huso huso) juveniles. comparative clinical pathology, 20: 447-451. aldrin j.f., messager j.l., laurencin f.b. 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(2020) 8(5): 327-336 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article the effects of hand-stripping on some epidermal mucus immune parameters in rainbow trout, oncorhynchus mykiss fatemeh hashemian, siyavash soltanian*,1mostafa akhlaghi, amin gholamhosseini department of clinical sciences, school of veterinary medicine, shiraz university, shiraz, iran. s article history: received 21 january 2020 accepted 4 september 2020 available online 2 5 o c t o b e r 2 0 2 0 2020 keywords: hand-stripping mucosal immunity skin mucus abstract: fish epidermis functions as the first defense barrier against physical, chemical, and biological stressors. however, the effects of hand-stripping on fish mucosal immune responses have been hardly investigated. the present study investigated the effects of stripping procedures on skin mucosal immune responses of rainbow trout (oncorhynchus mykiss) breeders. the skin mucus was sampled from six male and six female trout before and one week after the stripping handling. the results showed that stripping had significant effect on all parameters except protease activity, but gender had a significant effect only on the mucosal protease and alkaline phosphatase activity. the results did not show any effect of the interaction between stripping and gender on mucosal lysozyme activity. the data revealed that enzymatic activities of alkaline phosphatase, lysozyme, esterase, as well as the total immunoglobulin level and bactericidal activity were significantly reduced in the skin mucus of fish one week after the stripping. the reduction of immune parameters in the skin mucus could be related to immunosuppression caused by stripping stress which, in turn, might have made the fish more susceptible to microbial infections and diseases. therefore, care should be taken during stripping to minimize the manipulation stress. introduction fish are in continuous interaction with different challenges, such as variations in water quality (e.g. dissolved oxygen, temperature, salinity, etc.), culture conditions (e.g. feed supply and fish population density), and regular handling (e.g. transportation and grading) (guardiola et al., 2016). generally, breeders are highly sensitive to stress due to manipulation during breeding. fish catches, air exposure, transportation, hormone therapy, confinement, and hand-stripping can suppress the fish's immune system. several studies have been performed on the effects of stressors on mucosal parameters in different fish species (vatsos et al., 2010; tacchi et al., 2015; guardiola et al., 2015). the skin mucus of fish acts as the first line of defense against stressors (jung et al., 2012; guardiola et al., 2015; tacchi et al., 2015; parra et al., 2015; khansari et al., 2018). fish skin mucus is composed of several immune and antimicrobial molecules, such *correspondence: siyavash soltanian doi: https://doi.org/10.22034/ijab.v8i5.774 e-mail: siyavashsoltanian@yahoo.com as lysozymes, immunoglobulins, complements, lectins, and antimicrobial peptides (ingram, 1980; subramanian et al., 2007; esteban, 2012; guardiola et al., 2014; salinas, 2015). the structure and cellular composition of fish skin are affected by various stressors, such as environmental contaminants, pathogens, transportation, and crowding density (lindenstrøm et al., 2004; tacchi et al., 2015; guardiola et al., 2015; roosta and hosseinifar, 2016). moreover, the mucus composition and immune functions change with the fish species, alternation in the environment, and the fish’s physiology (guardiola et al., 2014). while the fish skin plays a vital role in mucosal immune reactions, there are no available data on the changes to epidermal immune parameters under stripping conditions. although most fish species show a generalized stress reaction in mucosal membranes, the pattern and scale of the response may be influenced not only by environmental factors (such as temperature and salinity) but also by the nature of 328 hashemian et al./ the effects of hand-stripping on epidermal mucus immune parameters in rainbow trout stressors and specific evolutionary life stories (khansari et al., 2017, 2018). therefore, the present study aimed to evaluate the effects of stripping manipulation on some immune parameters in the skin mucus of rainbow trout breeders. materials and methods experimental fish and mucus collection: the trial was conducted during the spawning season at a private rainbow trout hatchery farm in fars province, southwestern iran. during the sampling, water temperature, ph, and dissolved oxygen were monitored as 12ºc, 7.3, and 6 ppm, respectively. the broodstock (with average weight of 967.8±221.2 g) were held in raceway ponds under natural photoperiod and fed with a commercial trout diet (beyza technology co. ltd., iran) at 2% of their body weight once a day. after being kept 24 h without feeding, six male and six female fish with different weights (table 1) were randomly netted, individually placed in a bathtub tank, and anesthetized with clove powder (150 mg/l). different weights were selected for an easy and stress-free individual recognition because tagging or marking techniques could be invasive and potentially add stress (sharpe et al., 1998; carballo et al., 2018) to the normal stripping manipulation. in this study, a factorial design (2×2) was used to evaluate the effect of stripping (fish sampled before and after stripping), and gender (male and female) on some mucosal immune parameters in the rainbow trout breeders. the skin mucus was scraped from the dorso-lateral surface using a plastic spatula with enough caution to avoid contamination with blood and urino-genital and intestinal excretions (palaksha et al., 2008). the mucus samples were transferred to 15 ml sterile falcon tubes. after mucus collection, the fish were handstripped. the collection of semen and egg samples was carried out applying massage from the anterior portion of the belly (testis or ovary region) towards the genital papilla. after stripping, the fish were released back into the same ponds and fed for one week. mucus sampling was repeated of the same individuals one week after stripping based on the procedure applied in the first sampling. identical volumes of mucus samples were then homogenized using one volume of sterile normal saline (0.9%) following the method of kuppulakshmi et al. (2008). the samples were well shaken, and then centrifuged (2000 × g (equivalent to 4226 rpm) for 10 min at 4°c, following guardiola et al. (2014, 2016). the supernatant was kept at -80°c before use. skin mucus total immunoglobulin: the immuneglobulin concentration was measured based on siwicki and anderson (1993). immunoglobulins were precipitated with 10,000 kda polyethylene glycol (peg, sigma). mucus (100 µl) was mixed with an equal volume of 12% peg solution for 2 h at room temperature under constant shaking. after centrifugation at 5000 g for 15 min, the supernatant was collected and the concentration of proteins was determined based on bradford (1979). the difference in protein contents prior to and after the immunoglobulin molecules precipitation was considered as the ig content. lysozyme activity: lysozyme activity was measured based on a turbidimetric assay (demers and bayne, 1997). in brief, 50 μl suspension of bacterium micrococcus luteus (sigma, st louis, mo) (0.3 mg/ml of lyophilized cells dissolved in 40 mm sodium phosphate buffer, ph 6.5) was mixed with 50 μl of the mucus sample. the mixture was then incubated at 30°c and the decline in absorbance at 450 table 1. the weights of male and female rainbow trout brooders. brooder’s weight immediately after stripping (g) brooder’s weight one week after stripping (g) male female male female 673 695 714 734 782 806 826 846 864 886 912 934 976 997 1182 1203 1163 1185 1268 1291 1282 1305 1337 1358 329 int. j. aquat. biol. (2020) 8(5): 327-336 nm was detected after 0 and 15 min in a microplate reader (biotekelx 808 instrument, usa). a unit of lysozyme activity was defined as the quantity of enzyme that induced a reduction in the absorbance of 0.001 per minute. enzyme activity was expressed as u mg-1 of protein. alkaline phosphatase activity: alkaline phosphatase activity was measured over the incubation of mucus supernatants with 4 mm para-nitrophenyl phosphate (sigma) in 100 mm ammonium bicarbonate buffer containing 1 mm magnesium chloride, ph 7.8 at 30°c, based on the method described by palaksha et al. (2008). one unit of activity was defined as the amount of enzyme that released 1 mmol of paranitrophenyl product in 1 min. the activity unit was expressed per mg of protein (specific activity). protease activity: protease activity was determined using the azocasein hydrolysis assay as described by palaksha et al. (2008). azocasein hydrolysis was assayed by incubating 50 μl of the mucus sample re‐ suspended in 100 mm ammonium bicarbonate, ph 7.8, with 50 μl azocasein substrate 0.25% (w/v) in the same buffer for 19 h at 30°c. the reaction was stopped by adding 50 μl of 20% (w/v) trichloroacetic acid followed by a 5 min centrifugation at 15400 × g. equal volumes (100 μl) of the resultant supernatant and 0.5 m naoh were added to a 96‐well plate and the absorbance was measured at 405 nm. one unit of activity was defined as the amount of enzyme that caused a change in absorbance of 0.001/min. the activity unit was expressed per mg of protein (specific activity). esterase activity: esterase activity was detected following the incubation of mucus supernatants with 0.4 mm para-nitrophenyl myristate in 100 mm ammonium bicarbonate buffer containing 0.5% triton x-100, ph7.8 at 30°c, based on the method described by palaksha et al. (2008). the absorbance was recorded constantly for 2 h at 405 nm by a plate reader. the activity was defined as the quantity of enzyme needed to release 1 mmol of para-nitrophenyl product in1 min. enzyme activity was expressed as u mg−1 of protein. antibacterial assay: prior to analysis, the mucus samples were thawed in room temperature. preliminary screening for the antimicrobial activity of mucus samples was carried out against different bacterial species, including aeromonas hydrophila, yersinia ruckeri, and lactococcus garviae, which were obtained from the stock cultures maintained at the microbiology laboratory of the aquatic animal health and diseases department, school of veterinary medicine, shiraz university. these bacteria were selected based on their influence in causing diseases and mortalities in rainbow trout. the antimicrobial activity was studied following the slightly modified method offered by subramanian et al. (2008). all the bacterial species were grown in mueller-hinton (mh) broth medium for 24 h at 25ºc. one milliliter of mh broth and one milliliter of each bacterial culture (containing 1.5×106 cfuml-1) were added into a tube containing a different concentration of mucus samples. assays were carried out in triplicate for each mucus sample. the antimicrobial activity was confirmed by the visual inspection, absorbance at 595 nm using microplate reader (bmg labtech, germany). the antimicrobial activity was further confirmed by spread plating on mh agar plates. all the plates were performed in triplicate. the minimal bactericidal concentrations (mbcs) of mucus extracts were defined as the minimum mucus concentrations (μlml-1) that could cause complete inhibition of bacterial growth. statistical analysis: the normality of data was assessed by shapiro-wilk test. then the effects of the time of stripping and sexuality on skin mucus immune parameters were analyzed through two-way full factorial analysis of variance (anova). tukey’s multiple comparison tests were used for comparing the means of treatments following two-way anova. all statistical analyses were tested at the 0.05 level of probability (p<0.05), using spss 16.0 for windows. data are presented as mean ±sd. results mean values±sd of the skin mucosal immune parameters recorded in the experimental fish are shown in tables 2 and 3 and figures 1 and 2. no 330 hashemian et al./ the effects of hand-stripping on epidermal mucus immune parameters in rainbow trout significant difference was noted in the skin mucosal immune parameters between male and female fish either before or after stripping, except for lysozyme activity that showed significantly higher values (p<0.05) in female before and after stripping (fig. 2). however, the results did not show any effect of gender alone or any effect of the interaction between stripping and gender on mucosal lysozyme activity (table 4). the skin mucus lysozyme activity significantly decreased one week after the stripping in male and female fish. the results also showed that gender had a significant effect on the mucosal protease activity. however, there was no significant change in the activity of protease under stress induced by stripping. although the effects of stripping and gender alone on the activity of mucosal alkaline phosphatase were significant, the results showed that the interaction between these two factors had no significant effects on the activity of alp (table 4). the enzymatic activity of esterase, as well as the level of total ig, was significantly reduced in the skin mucus of fish one week after the stripping (p<0.05). similar results were found for skin mucus bactericidal table 2. enzymatic activities of protease, esterase and alkaline phosphatase (alp) (unit/mg protein) found in skin mucus of male and female rainbow trout brooders before and one week after stripping manipulation. (mean±sd, n=6). values in the same rows showing the same superscript letters are not significantly different (p>0.05). parameters before stripping after stripping male female male female protease activity (unit/mg protein) 20.5±2.5a 21.8±2.4a 19.5±1.5a 22.2±2.1a esterase activity (unit/mg protein) 3.00±0.35b 3.15±0.40b 2.23±0.34a 2.20±0.30a alp activity (unit/mg protein) 11.8±1.7b 13.6±1.7b 8.5±1.4a 9.9±1.3a table 3. minimum bactericidal concentration (mbc) (μl/ml) found in skin mucus of male and female rainbow trout brooders against selected bacterial pathogens before and one week after stripping manipulation (mean±sd, n=6). mbc was measured using broth dilution method. values in the same rows showing the same superscript letters are not significantly different (p>0.05). bacteria before stripping after stripping male female male female a. hydrophila 225±52a 200±45a 358±74b 325±52b y. ruckeri 208±38a 200±55a 317±52b 300±45b l. garviae 275±27a 250±45a 392±38b 375±69b figure 1. alterations in skin mucus total immunoglobulin of rainbow trout brooders before and one week after stripping manipulation. (mean ± sd, n=6). different letter notations indicate significant differences at p<0.05. 331 int. j. aquat. biol. (2020) 8(5): 327-336 activities against selected bacterial pathogens (table 3). the results revealed that neither gender nor the interaction between stripping and gender had a significant effect on total ig level, skin mucus esterase, and bactericidal activities (table 4). discussions there is some evidence that the mucus composition table 4. statistical significances of immune modulations; results of two-way anova performed on skin mucus immune parameters considering male and female rainbow trout brooders before and one week after stripping manipulation. differences were considered significant for p>0.05. parameters factors sig lysozyme activity (unit/mg) stripping <0.0001 sex 0.347 stripping × sex 0.949 total ig (mg/ml) stripping <0.0001 sex 0.224 stripping × sex 0.851 alp activity ( unit/mg protein ) stripping <0.0001 sex <0.05 stripping × sex 0.672 esterase activity (unit/mg protein ) stripping <0.0001 sex 0.691 stripping × sex 0.533 protease activity (unit/mg protein) stripping 0.711 sex <0.05 stripping × sex 0.462 mbc (µl/ml) (yersinia ruckeri) stripping <0.0001 sex 0.528 stripping × sex 0.833 mbc (µl/ml) (aeromonas hydrophila) stripping <0.0001 sex 0.223 stripping × sex 0.859 mbc (µl/ml) (lactococcus garviae) stripping <0.0001 sex 0.293 stripping × sex 0.831 figure 2. alterations in skin mucus lysozyme activity of rainbow trout brooders before and one week after stripping manipulation. (mean ± sd, n=6). different letter notations indicate significant differences at p<0.05. 332 hashemian et al./ the effects of hand-stripping on epidermal mucus immune parameters in rainbow trout varies with stress, disease, and parasite attack (schrock et al., 2001; mustafa, 2005; easy and ross, 2010; lü et al., 2012). the skin mucus comprises different biologically active molecules such as complement factors, hydrolytic enzymes (e.g., lysozyme, cathepsin b, proteases), immunoglobulins, lectins, interferons and antimicrobial peptides which plays a vital role in mucosal immune reactions (böckelmann et al., 2010; huang et al., 2011; esteban, 2012; khansari et al., 2018). different enzymes with recognized functions in the immune reactions have been identified in numerous fish species, a finding which was also noticed in our study. among them, alp in mucus has been reported to act as an antibacterial factor because of its hydrolytic activity (ross et al., 2000). also, it has been reported that alp has a protective role both in the initial stages of wound healing (iger and abraham, 1990, 1997; rai and mittal, 1991) and against stress (iger and abraham, 1990, 1997). our results revealed a significant reduction in the alp activity in the mucus of fish one week after exposure to stripping manipulation. in addition, a gender effect on alp activity was noticed that might be associated with different levels of sex hormones in male and female fish (thongprajukaew and kovitvadhi, 2013; dash et al., 2018; reverter et al., 2018). similar results were found in turbot (scophthalmus maximus) reared in high density for 120 days (jia et al., 2016). however, despite the results of the present study, it has been shown that some stressors, such as high stocking densities (roosta and hosseinifar, 2016), hypoxia (vatsos et al., 2010), and aluminum exposure at lower ph (ledy et al., 2003) could significantly enhance mucosal alp activity in tiger barbs (puntigrus tetrazona), sea bass (dicentrarchus labrax), and brown trout (salmo trutta fario), respectively. moreover, a significant variation in alp activity was reported in the skin mucus of gilthead sea bream (sparus aurata) depending on the waterborne metal tested and the exposure time (guardiola et al., 2015). such alteration in alp activity could be used as a stress indicator in some circumstances (ross et al., 2000). proteases and esterase were measured in the current work because both of them have been linked with skin immune response and defense against microbial infections (esteban, 2012). in skin mucus, proteases may play a defensive role against pathogens by direct way i.e. splitting their proteins (subramanian et al., 2007), and indirect ways i.e. hindering their colonization and invasion mechanisms (aranishi et al., 1998). furthermore, the proteases may activate other immune parameters such as complement, immunoglobulins or antibacterial peptides (hjelmeland et al., 1983; fernandes and smith, 2002; kennedy et al., 2009). some studies describing changes in mucus proteases following stress (aranishi and nakane, 1997; aranishi et al., 1999; easy and ross, 2010) and infection (ross et al., 2000; aranishi and mano, 2000) pointed out its importance in mucosal immunity. in the current study, no significant change was noticed on mucosal protease activity following the stripping, although a gender effect was detected. the effect of gender on mucosal protease activity may be due to the difference between the level of proteases and antiproteases in the mucus of male and female trout. hormonal changes, especially for sex hormones during the reproductive season, may have a significant effect on the activity of mucus protease (thongprajukaew and kovitvadhi, 2013; dash et al., 2018; reverter et al., 2018). likewise, jia et al. (2016) found no changes in the protease activity in epidermal mucus of turbot exposed to crowding stress. it seems that the protease level in the skin mucus differs depending on the specific stressor conditions (guardiola et al., 2016). although the function of esterase in fish mucus is not known, it could act separately or in collaboration with other immune components in the mucus in defending against pathogens (sheikhzadeh et al., 2012). previously, increased esterase activity was demonstrated in gilthead seabream following waterborne exposure to mercury, arsenic, and cadmium (guardiola et al., 2015). in the current work, the esterase activity reduced in the mucus of fish after exposure to stripping manipulation, which is consistent with a previous report in the epidermal mucus of turbot cultured in high-density conditions 333 int. j. aquat. biol. (2020) 8(5): 327-336 (jia et al., 2016). total ig remarkably decreased in the epidermal mucus of fish after stripping procedures. contrary to the usual expectation, the level of ig measured in the skin mucus of fish exposed to heavy metals was reduced on day 2 of exposure; however, it increased afterward (guardiola et al., 2015). studies conducted pertinent to fish immunity has primarily evaluated serum lysozyme activity (ellis, 2001), but, comparatively, less attention has been paid to the function of lysozyme in the mucosal surfaces (bergsson et al., 2005; nigam et al., 2012). in the current study, the female brooders exhibited higher lysozyme activity in their skin mucus. the results are in agreement with the findings of ghafoori et al. (2014), who reported a significant higher lysozyme level in the skin mucus of female caspian kutum (rutilus kutum). studnika et al. (1986) observed an increase in the lysozyme level in the spawning stage of the common carp during the development of oocyte. in another study, lysozyme activity in the serum of female tilapia increased with the development of oocyte, especially during the vitellogenesis (takemura et al., 1995). in the spawning stage, the lysozyme trend shows an exponential increase, particularly in the skin, due to its involvement in overcoming probable pathogenic factors (ghafoori et al., 2014). the mucus lysozyme activity diminished in turbot raised in a high-density condition (jia et al., 2016). similarly, in blackspot seabream (pagellus bogaraveo), a significant decrease in lysozyme values was found in the mucus subjected to a 31-day starvation (caruso et al., 2011). however, in the atlantic salmon (salmo salar), short-term stress did not cause any significant changes in the mucus lysozyme activity 3 and 24 h post-stress (easy and ross, 2010). it seems that the discrepancy in the mucus lysozyme activity could be connected to several factors such as season (schrock et al., 2001), species and genetic variations, sex, maturity, diet, stress handling (balfry and iwama, 2004; caruso et al., 2011), and even the thickness of the epidermis and the number of mucus cells (subramanian et al., 2007). regardless of the effector components and the mechanisms involved in bacterial killing, the assessment of the bactericidal activity of skin mucus could be more important in practice than the enzymatic activities alone (guardiola et al., 2014). several studies have shown that the skin mucus of some fish species presents a strong antibacterial and antifungal activity against a wide range of microbial pathogens (hellio et al., 2002; subramanian et al., 2008; dhanaraj et al., 2009). in the present study, a remarkable decrease in the bactericidal activity was detected in the mucus of fish subjected to stripping manipulation. our results conflict with those of roosta and hosseinifar (2016), who found the elevation of antibacterial activity in the skin mucus of tiger barbs subjected to crowding stress. moreover, a significant increase in the bactericidal activity of the skin mucus of seabream was observed following exposure to heavy metals (guardiola et al., 2015). the present study demonstrated a negative relationship between stripping handling and the immune response in the skin mucus of rainbow trout breeders. the enzymatic activities of lzm, alp, esterase, ig level and bactericidal activity were significantly reduced in the skin mucus one week after exposure to stripping manipulation. the reduction of the immune parameters in the mucus might suggest immunosuppression due to the stripping stress which, in turn, might have made the fish more susceptible to infections and diseases. overall, the dramatic variations observed between the results of the current study with those of others can be ascribed to factors such as differences in sex, species, maturity, diet, stressor types, and stress duration (guardiola et al., 2014). funding this study was funded by the aquatic animal health and diseases department, school of veterinary medicine, shiraz university, through a research grant to the second author. references aranishi f., mano n. 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(2021) 10(3): 262-272 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article life-history and population dynamic of the white treads fish, holothuria leucospilota in the iranian part of the oman sea with a note on its conservation and management seyed ahmad reza hashem*1, mastoorh doustdar2, teymour aminrad1, alberto de jesús navarrete3, sylvie m. gaudron4,5 1offshore fisheries research center, iranian fisheries science and research institute, agricultural research education and extension organization, chabahar, iran. 2iranian fisheries science and research institute, agricultural research education and extension organization, tehran, iran. 3el colegio de la frontera sur-unidad chetumal. av. centenario km 5.5, chetumal, quintana roo, mexico. 4lille university, littoral côte d’opale, cnrs, umr 8187 laboratoire d’océanologie et de géosciences, 59000 lille, france. 5sorbonne university, ufr 918 & ufr 927, 75005 paris, france. s article history: received 18 march 2022 accepted 24 june 2022 available online 2 5 june 2022 keywords: black sea cucumber exploitation coefficient growth mortality abstract: in the present study, the life-history and population dynamic characteristics of holothuria leucospilota were evaluated in the iranian part of the oman sea by sampling at nine sites, including ramin, kachoo, aliabady, beries, beries plane, pasabandar, tis, pozm and gurdium from march 2017 to march 2018. biometric data of 862 specimens were obtained, and the growth and mortality indices, including infinite length (l∞ = 50.5 cm), growth coefficient (k = 0.51 (yr-1)), growth performance index (ф = 3.11), natural mortality (m = 0.94(yr-1)), fishing mortality (f = 0.56 (yr-1)), total mortality (z = 1.50±0.12 (yr-1)) and exploitation coefficient (e = 0.36 (yr-1)) and time zero (-0.27) were calculated. mean of relative production per recruitment (y' / rp), relative biomass per recruitment (b' / rp) and exploitation rate (u) of the studied population of h. leucospilota were 0.02, 0.30, and 0.28, respectively. mean gsi and maturity stage indicated that the spawning seasons were june (spring) and december (autumn). the mean size at first sexual maturity (lm50) was 246 mm for males, 220 mm for females and 225 mm for both sexes. the results of the current work showed that the studied h. leucospilota stock had not yet reached to overfished’ status. introduction sea cucumbers conservation and management are necessary because these marine benthic species have an important ecological role, such as the recycling of nutrients (shiell and knott, 2010), and are a significant source of income for many coastal communities worldwide (purcell, 2010). the current status of sea cucumber stocks in many countries shows poor fishery management, and international efforts are made on their culture in different countries to minimize the depletion of their natural population. the unique life-history traits of holothurians e.g. low or infrequent recruitment and density-dependent reproductive success, make these species vulnerable to overfishing (purcell, 2010). population dynamics are driven by changes in abundance or biomass of a population through time by a series of life-history traits such as fecundity, *correspondence: seyed ahmad reza hashem doi: https://doi.org/10.22034/ijab.v10i3.1626 e-mail: seyedahmad91@gmail.com successful recruitment, growth, and mortality. estimates of population dynamics can provide great insight into a harvested marine population species. it can indicate how a population arrived at its current state and how it might change in the future (brown and guy, 2007). recruitment, growth, and mortality rates are the primary population dynamics parameters that explain the harvestable part of a fish population (brown and guy, 2007). holothuria leucospilota (brandt, 1835) (dendrochirotida; holothuriidae) is harvested commercially. this species lives in shallow habitats up to 10 m, mainly on outer and inner reef flats, back reefs, seagrass beds, microatoll detrital fringe, and shallow coastal lagoons but with higher densities on the inner reef slopes. it is common, with its distribution extending into warm-temperate zones. holothuria leucospilota is a deposit feeder with an 262 hashem et al./ life-history and population dynamic of the holothuria leucospilota in the oman sea important ecological role (purcell et al., 2012). studies on the biological characteristics of sea cucumber species in different parts of the world are done (herrero-perezrul et al., 1999; skewes et al., 2002; uthicke et al., 2004; conand, 2008; choo, 2008; purcell et al., 2009; dissanayake and stefansson, 2010; chávez et al., 2011; de jesús-navarrete et al., 2018). the oman sea is a region of the northern indian ocean that connects the arabian sea with the strait of hormuz, which then runs to the persian gulf. it borders iran and pakistan on the north, oman on the south, and the united arab emirates on the west (taghavimotlagh and shojaei, 2017). with its unique ecological conditions, the oman sea hosts a wide variety of marine species that provide livelihood, employment, and vast economic activities for the settlers. iran has more than 120,000 fishermen; therefore, fishing has significantly created employment in coastal areas and economic activities for post-harvest operations (taghavimotlagh and shojaei, 2017). recently, the illegal fishing of h. leucospilota in the northern waters of the oman sea has increased significantly, and they are caught for export. there is currently no specific management or restriction for this species in this area. despite the economic importance of this species, little is known about its stock population. hence, this study aimed to investigate some life-history traits and assessment measures of h. leucospilota from the oman sea to provide basic information. first, the structure of the population, spawning period, gonadosomatic index, size at first maturity, and growth rate of h. leucospilota will be studied. then, a series of fisheries assessment data, such as biomass, recruitment, and mortality, will be calculated to evaluate its population sustainability in the northern part of the oman sea. materials and methods nine sampling sites, including the ports of ramin figure 1. map of holothuria leucospilota sampling stations in the northern oman sea, iran. 263 int. j. aquat. biol. (2022) 10(3): 262-272 (60˚45´e, 25˚15´n), kachoo (60˚55´e, 25˚16´n), aliabady (61˚05´e, 25˚10´n), beris (61˚10´e, 25˚82´ n), beris plane (61˚15´e, 25˚72´n), pasabandar (61˚ 25´e, 25˚70´n), tis (60˚35´e, 25˚24´n), pozm (60˚ 16´e, 25˚23´n) and gurdim (59˚61´e, 25˚22´n) (fig. 1) were selected for sampling in the iranian part of northern oman sea. samplings were done monthly from march 2017 to march 2018. samples were collected with the help of divers from subtidal areas of sandy and pebbles shore at depths of less than 10 meters. additionally, they were captured using a linear transect 50 meters long and 2.0 meters wide using scuba diving. a total of 802 specimens of h. leucospilota were collected (table 1). the weight and length of the specimens were measured after placing them in a potassium chloride solution (10%) for half an hour. biometric measurements, including length and weight, were performed on sea cucumber after anesthesia. total length was measured using a biometric ruler with 1 mm precision (fig. 2) and wet weight by the nearest 1 g. the equation of wi = a × li b was used to calculate the relationship between the total length and wet weight, where wi is the sea cucumber weight (g), li is the sea cucumber length (mm), a is a constant coefficient, and b is an equation power. the equation of t = [(s.dx)/ (s.dy)] × [(lb-3l)/ (√ (l-r2)] × [√ (n-2)] was used to decipher significant differences between the calculated b. in this equation b = 3 for a sea cucumber of similar growth with s.dx = standard deviation of total length natural log, s.dy = standard deviation of weight natural log, b = slope, r2 = coefficient of determination and n = sample sizes (zar, 2010). life-history traits and population dynamic parameters structure of population and growth rates: the data was pooled monthly from different stations and subsequently grouped into classes with 3 cm intervals. two methods were used to analyze the data for growth rates viz. (1) shepherd, and (2) elefan (electronic length frequency analysis). in method 1, the data was analyzed using fisat ii (faoiclarm stock assessment tools, www.fao.org/fi) based on the shepherd method (gayanilo et al., 2003). in method 2, the estimation of l∞, the infinite length was obtained using the equation of log loo = 0.044 + 0.9841 * log (lmax) and maximum length of samples (lmax) based on froese and binohlan (2000). the growth rate was obtained by applying the elefan (optimization model) using the rstudio software with the tropfishr package (mildenberger et al., 2017). the optimum value of t0 (time that length is zero) was calculated by the pauly equation of log (-t0) = 0.3922 0.2752 log l∞ 1.038 log k, where k is the growth factor (froese and binohlan, 2000). a comparison of growth indices such as infinite length species (sex) method l∞ (cm) k ) 1-yr ) ot φ' m f z e h. leucospilota (male) shepherd method (fisat ii) 49 0.6 -0.28 3.09 0.95 0.64 1.59 0.4 h. leucospilota (female) shepherd method (fisat ii) 47 0.45 -0.28 3.09 0.95 0.64 1.59 0.4 h. leucospilota (total) shepherd method (fisat ii) 49 0.5 -0.28 3.09 0.95 0.64 1.59 0.4 h. leucospilota (male) elefan method (r) 56 0.54 -0.26 3.13 1 0.4 1.4 0.29 h. leucospilota (female) elefan method (r) 50 0.44 -0.31 3.15 0.84 0.71 1.55 0.46 h. leucospilota (total) elefan method (r) 52 0.54 -0.25 3.14 0.95 0.35 1.3 0.26 h. leucospilota (total) mean of both 50.5±3 0.51±0.06 0.27±0.02 3.11±0.02 0.94±0.05 0.56±0.14 1.5±0.12 0.36±0.07 table 1. comparison of population dynamics values of holothuria leucospilota with two methods (shepherd and elefan) (l∞ = infinite length, k = growth rate, to = time that length is zero, φ' = growth performance index, m = natural mortality, f = fishing mortality, z = total mortality, and e = exploitation rate). 264 hashem et al./ life-history and population dynamic of the holothuria leucospilota in the oman sea (l∞) and growth factor (k) was performed using the equation of ф ' = log (k) + 2 log (l∞). in addition, the maximum lifespan of this species was calculated based on the formula of tmax = t0 + 3 / k (froese and pauly, 2017). mortality: natural mortality (m) was calculated based on the pauly equation of ln (m) = 0.0152 0.297 ln (l∞) + 0.654 ln (k) + 0.642 ln (t), where, m is the annual natural mortality coefficient, l∞ is the infinite length of the sea cucumber (cm), k is the growth curve parameter of von bertalanffy growth equation and t (celsius) is the mean environmental temperature (sparre and venema, 1998). the mean annual temperature of the northern oman sea surface was estimated as 26°c (keymaram et al., 2009). total mortality (z) was calculated using the length-converted catch curves data. the fishing mortality was estimated using the eq f = z – m equation, where z = the total mortality, f= the fishing mortality, and m the natural mortality. the exploitation rate (e), which is the ratio of fishing mortality to total mortality, was calculated using the equation of e = f / z (sparre and venema, 1998). recruitment and biomass: the relative yield per recruitment (y/r) was estimated against the fishing mortality coefficient or exploitation rate. in the equation of y'/r = eum/k (-3um/ (1 + m) + 3 u2 / (1 + 2m) + u3 / (1 + 3m) with u = 1 (lc / l∞); m = (1 e) / ( m / k) = ( k / z); e = f / z, e, is the exploitation coefficient, u is the exploitation rate, m is the natural mortality coefficient, f is the fishing mortality coefficient and lc (length at first capture) is the same as lc50 (gayanilo et al., 2003). in addition, the relative biomass per recruitment (b' / r) was calculated using the equation of b' / r= y' / r / f. the gonadosomatic index (gsi): the maturity stage for males and females (fig. 6) were determined macroscopically using a 5-stage maturity key (gaudron et al., 2008), including (1) resting (i), immature stage (ii), growing stage (iii), maturation stage (iv), and post-spawning stage (v). the gsi was calculated by expressing the mean gonad weight as a proportion of the total body weight (biswas, 1993). the gi was calculated using the equation, gi = gw / ev, where gi is gonad index (%), gw is gonad weight and ew is eviscerated body weight (gaudron figure 2. holothuria leucospilota from the northern oman sea, iran. 265 int. j. aquat. biol. (2022) 10(3): 262-272 et al., 2008). the mean size at first sexual maturity (lm50): the mean size at first sexual maturity (lm50) was estimated for females by fitting the logistic function to the proportion of mature sea cucumbers in 4 cm (tl) size categories y = 1 / 1 + exp(-a-bx), where y is the proportion of the number of all mature males and females to all immature males and females in the same length class, x is the total length in cm and a and b are correlation constants (king, 2007). the mean size at first maturity was taken when 50% of individuals were mature. monthly sea temperature data (°c) were recorded in different stations (by multi-parameter device, hach model). statistical analyses: comparison of population dynamic values in two methods (shepherd and elefan methods) and between male and female lengths and weights were tested using student’s test (t-test) with paired t-test and independent t-test, respectively. the correlation between temperature and gsi was tested by a pearson correlation test. a chisquare test was used to assess the sex ratio difference between males and females. the normality of data was assessed by using the kolmogorov–smirnov test. data analyses were performed using fisat ii and r studio (1.1.46) with the tropfishr package. results length frequency distribution: the mean ± standard deviation of total length and total weight for male (624 specimens) and female (178 specimens) were 31±6 (18-45) and 31±5 (19-43) cm, and, 770±164 (3701220) and 762±155 (375-1125) g, respectively. the differences between total length and total weight in both sexes were not significant (t = 0.83, p>0.05; t = 0.58, p>0.05, respectively). the length (tl) data were categorized into 3-cm groups, which the highest frequency (195 sea cucumber), were belonging to individuals with 27 to 30 cm length (fig. 3a). length-weight relationship (lwr): in lwr, the figure 3. length and frequency (a) and length-weight relationship (b) of total holothuria leucospilota in the northern oman sea, iran. 266 hashem et al./ life-history and population dynamic of the holothuria leucospilota in the oman sea parameters were a = 25.76 and b = 0.97 (r2 = 0.61) for female, a = 30.79 and b = 0.92 (r2 = 0.93) for male, and a = 24.93 and b = 0.98 (r2 = 0.59) for both sexes. the results showed significant differences between estimated b from 3 (p<0.05) (fig. 3b), which means an allometric growth pattern for both sexes. growth parameters: the population dynamic parameters using the two methods of shepherd and elefan for male, female, and both sexes are presented in table 1. there were no significant differences between the two methods (p>0.05). growth parameters for both sexes were estimated as l∞ = 50.5 cm (w∞ = 1.211 kg), k = 0.51 (yr-1), and t 0= -0.27. the growth curve (fig. 4a). highlighted six cohorts and age groups and the growth performance index was estimated as ф = 3.11. there is recruitment throughout the year and the highest recruitment rate was observed in the winter and summer seasons (fig. 4b). based on the results, the maximum lifespan of this species was near 6 years. mortality estimate: the natural mortality (m), fishing mortality (f) and total mortality (z) were estimated 0.94 (yr-1), 0.56 (yr-1), and 1.5 (yr-1), respectively. the exploitation coefficient was estimated as 0.36 (yr-1) (fig. 5). based on the results, the von bertalanffy equation for this species in the northern oman sea of iran was lt = 50.5 (1 exp (0.51 (t + 0.27))) and wt = 1211 (1 exp (-0.51 (t + 0.27))) ^ 0.98. yield per recruit and biomass per recruit: based on figure 4. growth curve (a) and monthly recruitment in percent (b) derived from the structure of the population of holothuria leucospilota from the northern oman sea (iran). (a) the growth curve plot shows reconstructed frequencies, with negative and positive values as white and black colored histograms, respectively. the background shading shows runs of peaks, with positive peaks in blue, negative peaks in red, and values of zero in white. the different colour backgrounds were added in order to help visualize the sign and magnitude of the bin values. the sum of all positive peaks is called the “available sum of peaks” (asp), which represents a maximum possible score. the “estimated sum of peaks” (esp) is the sum of peak values crossed by the growth curves (pauly, 1985). 267 int. j. aquat. biol. (2022) 10(3): 262-272 length at first capture (lc = 27 cm), which is 50% the probability of catching sea cucumber, the relative production per recruitment and relative biomass per recruitment were estimated y' / rp = 0.02 and b' / rp = 0. 3, respectively. the results showed an exploitation rate (u) of 0.28 and fishing mortality at a maximum sustainable yield (fmsy) of 0.6 (fig. 5). sex ratio, gsi, and lm50: from all sea cucumbers sampled, 639 were male (75%) and only 223 were female (25%). the sex ratio was significantly biased towards males (1 female/ 3 males) (chi2 = 100.38, p<0.05). the mean value of gsi for both sexes was 5.59±1.75. the highest gsi value was observed in june (21±6) and december (8±2), and the lowest value in august (1±0.5) (fig. 6). moreover, there was no significant correlation between the gsi temporal evolution and the monthly temperature (pearson correlation = 0.30, p>0.05). mean gsi and maturity stage indicated that spawning occurred in june (spring) and december (autumn). the mean size at first sexual maturity (lm50) was 246 mm for males, 220 mm for females, and 225 mm for both sexes (fig. 7). discussions for the first time, we investigated some life-history traits and population dynamics of h. leucospilota in the study area. this species is economically valuable in the south and northern oman sea, iran. the sea cucumbers play a ‘key role in the structure of marine ecosystems mainly in organic matter processing. overfishing sea cucumbers on a tropical scale is likely to affect their structure in ecosystems (uthicke et al., 2009). life-history traits: the lwr of h. leucospilota in the current study showed an allometric growth pattern figure 5. exploitation coefficient curve (a, method 2) and relative yield per recruit (y' / r) and relative biomass per recruit (b' / r), fmsy (b) of holothuria leucospilota (total) in the northern oman sea (iran) (fmsy = fishing mortality rate of maximum sustainable yield, f 0.5= fishing mortality rate at which the slope of the yield-per-recruit curve is only half the slope of the curve at its origin, f 0.01= fishing mortality rate at which the slope of the yield-per-recruit curve is only one percent the slope of the curve at its origin). 268 hashem et al./ life-history and population dynamic of the holothuria leucospilota in the oman sea and the female was heavier than males in the same length group. the growth curve (length and weight) of h. leucospilota slows down after two years and an allometric growth pattern is common in sea cucumbers (al-rashdi et al., 2007; herrero-perezrul et al., 1999; chávez et al., 2011; dereli et al., 2016). the relationship between length and weight for h. tubulosa had reported w = 7.66 l1.06 (r2 = 0.52) (dereli et al., 2016), for parastichopus parvimensis w = 0.4 l1.83 (chávez et al., 2011), for h. scabra w = 0.0033 l2.17 (r2 = 0.80) (al-rashdi et al., 2007), and for isostichopus fuscus w = 1.14 l1.83 (herreroperezrul et al., 1999). according to marthin (1994) the range of "b" could be 2.5 to 4. the b-value shows the body form and is directly related to the weight affected by ecological factors such as temperature, food supply, spawning conditions, and other factors, such as sex, age, fishing time, and area and fishing vessels (ricker, 1973). in the present study, h. leucospilota was shown to spawn twice a year, with a major peak in june and a weaker one in december that was not correlated with temperature. the percentage of recruitment also emphasizes spawning in the winter and summer seasons. spawning time of h. leucospilota was reported from november to april in australia (franklin, 1980), november to march in the cook islands (drumm and loneragan, 2005), juneseptember in taiwan (choo, 2008), february and may in the indian ocean (conand, 2008), february and may in the western indian (gaudron et al., 2008), and from june to october in daya bay, china (huang et al., 2018). the difference in the spawning period of any species is affected by regional conditions, and in tropical areas, almost shallow-water holothurians have an annual 2to 3-month spawning period. most of the tropical shallow-water holothurians spawn during the warm months. the holothurians have different reproductive patterns and need to be studied in each region (chao, 1995). the sea cucumber reproduction cycle differs among species and even within a species distributed in different regions (huang et al., 2018). the size at first sexual maturity (lm50) for white threads fish was estimated at 246 mm for males and figure 6. monthly variation of gsi (male and female) of holothuria leucospilota (total) and temperature in the northern oman sea, iran. 269 int. j. aquat. biol. (2022) 10(3): 262-272 220 mm for females. females mature earlier, therefore, their growth is slower than males, resulting from the high energy they need in earlier years for their growth and reproduction. therefore, females h. leucospilota have a l∞ value smaller than males. the size at first maturity (lm50) of h. sanctori was reported as 201 to 210 mm in the eastern atlantic, spain (conand, 1993) and h. atra as 165 mm (males), 155 mm (females) in egypt (abdel razek et al., 2005). the size at first maturity is an important parameter for managing the stock, as it helps to limit capture sizes (conand, 2008) and tools for enhancing sustainable management of the fisheries (kohler et al., 2009). this parameter is needed for fisheries management, conservation of exploited sea cucumbers, and small immature individuals’ collection decreases for the sustainability of the population. population dynamic: comparisons of the population dynamic parameters of h. leucospilota with other studies in different parts of the world are presented in table 2. as seen in table 2, the l∞ and growth coefficient of females in different species are smaller than those of males. in addition, these characters in different species differ in various regions (table 2). differences in the l∞ and growth rate are influenced by the ecological differences of each region (king, 2007). the growth rate is expected to be higher in the tropical zones. higher k values for species are common in tropical waters due to their poikilothermic nature resulting in higher metabolic rates in high temperate (hashemi et al., 2015). the differences in the l∞ and growth rate might be due to the quantity and quality of food and climatic conditions (bartulovic et al., 2004). various factors can also affect holothurian growth including age, sex, season, year, type of feeding, physiological conditions, differences in food availability, and reproductive period (lalèyè, 2006). the mean growth performance index (ф ׳) was 3.11 in the current study. there is a correlation figure 7. the lm50 (male (a)) and (female (b)) of holohturia leucospilota (total) and temperature in the northern oman sea, iran. 270 hashem et al./ life-history and population dynamic of the holothuria leucospilota in the oman sea between l∞, and growth rate. the growth curve has a rate with constant changes at different times and sizes. differences in ecological conditions and latitude changes can affect the values of inf l∞ and growth rate (king, 2007). this study's natural mortality rate of h. leucospilota was higher than fishing mortality. the ratio of fishing mortality to maximum sustainable yield (f / fmsy) was less than one. the f / fmsy of more than 1 indicates overfishing (arrizabalaga et al., 2012). however, our work's exploitation coefficient was less than 0.5, indicating that the fisheries were not more than the optimum level. the exploitation coefficient and exploitation rate should not exceed 0.5, and the fishing mortality should not exceed natural mortality otherwise, they indicate overfishing (sparre and venema, 1998; king, 2007). the most important factors affecting the pressure on stocks are the amount of catch and the environmental factors that affect survival and access to the fishery resources (mateus and estupinan, 2002). management and conservation: it is recommended that appropriate instructions be established for the harvesting and management of h. leucospilota in the northern oman sea. this species should not harvest in lengths less than 250 mm and in winter and summer because of its spawning season. the size at first maturity is an important parameter for managing this stock, as it helps to limit capture sizes (conand, 2008). this parameter is needed for fisheries management, conservation of exploited sea cucumbers, and avoiding small immature individuals’ collection its sustainability of the population. the present work revealed that the studied stocks had not yet reached ‘overfished’ status. acknowledgments we would like to thank dr. bahmani, the manager of the iranian fisheries science research institute (ifsri), and the experts of the offshore fisheries research center (ofrc, chabahar) for their and holger weissenberger for drawing the map. references abdel razek f.a., abdel rahman s.h., el shimy n.a., omar h.a. 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(2019) 7(5): 254-259 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article study of the macroalgae and application of ecological evaluation index (eei-c) in the coastal waters of algeria benkhedda belhaouari *1, zoubeyda bezzina2 1higher agronomic school of mostaganem, algeria. 2university of chlef, hassiba benbouali, algeria. s article history: received 1 august 2019 accepted 21 october 2019 available online 2 5 october 2019 keywords: marine pollution monitoring algerian coastal macroalgae abstract: the diversity of macroalgae and evaluation of the algerian coastal waters using eei-c index were studied. macroalgae were sampled at seven stations in summer 2019. coverage data for the macroalgae at each site were analysed at species level. eleven species have been identified and the results showed the prevalence of cystoseira compressa and ulva sp.. the least abundant species were cladophora sp. and corallina elongata. classification of sites based on the cluster analysis shewed an agreement with the water degradation information. based on the results, the eei-c values were quite homogeneous over all the studied sites, and generally correspond to undisturbed states except for one site. introduction macroalgae are the main primary producer in aquatic water bodies (graham et al., 2007), and also habitat for microfauna and microflora (chabot and rossignol, 2003). the coastal environment that shelters algae is strongly influenced by anthropogenic activities, such as fishing, submarine hunting and leisure activities, urbanisation, industries and agriculture (mcmanus and polsenberg, 2004). therefore, algae are not adapted to these disturbances, declining their communities as seen in the mediterranean (thibaut et al., 2005; serio et al., 2006; ballesteros et al., 2007). in addition, macroalgae are used as bioindicators of the ecological status of coastal waters (cabioc’h et al., 2014; pereira and neto, 2015). the ecological status assessment is based on macroalgae, invertebrates and angiospermae (marques et al., 2009; abbasi and abbasi, 2012; badreddine et al., 2018; belhaouari, 2019). the eei-c (ecological evaluation index) is one of the ecological index based on macroalgae (orfanidis et al., 2011), which is used to assess the ecological status of coastal waters (gabriel et al., 2014; caldeira et al., 2017; amaral et al., 2018; caldeira and reis, *correspondence: benkhedda belhaouari doi: https://doi.org/10.22034/ijab.v7i5.695 e-mail: belhaouaribio@hotmail.fr 2019). algerian coast macroalgae are poorly studied and their exploitation is still marginal (chabane et al., 2018; traich et al., 2018), therefore, the present study aimed to examine the diversity of macroalgae and the evaluation of the algerian coastal waters using eei-c index. materials and methods sampling was carried out at six sites (as s1-s6) on the coast of the tenes in summer 2019 (fig. 1), selected based on a non-aligned block design, in which a sample is located randomly within a representative permanent cell of dimensions 10×10 m. each site was selected along the coastline at a depth range of 0.1-0.5 m (orfanidis et al., 2003). the samples were scrapped using a hammer and chisel in a quadrat of 25×25 cm from 25 squares with five replicates (boudouresque, 1971; orfanidis et al., 2001). characteristics of the sampling sites are presented in table 1. species were identified based on cabioc'h and floc'h (2014) and mangialajo et al. (2008). in each site, total average cover (tac) of each species was calculated using the formula of tac = σri/n (boudouresque, 1971), where ri is the percentage of 255 int. j. aquat. biol. (2019) 7(5): 254-259 area of the quadrat covered by species i, and n = number of quadrat. hierarchical cluster analysis (hca) was performed to establish a hierarchy of clusters (rokach et al., 2005). hca is used to classify species with similar behaviour according to a set of variables based on orfanidis et al. (2011). ecological status of the six sites were determined by eei-c (orfanidis et al., 2011). macroalgae were used as bio-indicators of the ecosystem shifts, from the pristine state with late-successional species (ecological state group i) to the degraded state with opportunistic species (ecological state group ii). e s g i comprises thick perennial (ia), thick plastic (ib) and shade-adapted plastic species (ic), and e s g i i comprises fleshy opportunistic (iia) and filamentous sheet-like opportunistic (iib) species (table 2). the absolute abundance (% coverage) of esg i = [(ia*1) + (ib*0.8) + (ic*0.6)] and esg ii = [(iia*0.8) + (iib*1)] the eei-c was applied using the formula: eei-c (esgi, esgii) = a + b * (esgi/100) + c * (esgi/100)2 + d * (esgii/100) + e * (esgii/100) 2 + f * (esgi/100) * (esgii/100). the coefficients of the hyperbola are a = 0.468, b = 1.2088, c = −0.3583, d = −1.1289 and = 0.5129, f = −0.1869 (orfanidis et al., 2011, 2014). the eei-c can be transformed in accordance to the ecological quality ratios as: eei-c eqr = 1.25 x (eei-c value/rcvalue)-0.25 rc = 10 table 1. characteristics of the sampling sites. sites ph temperature salinnity anthropogenic activities anglaise beach (s1) 8.365±0.17 25 ±2.0 38.173±0.12 swimming ain el kadi beach (s2) 7.62±0.63 24.5±1.0 37.974±0.42 domesticwaste waters charrir beach (s3) 8.346±0.15 24±1.0 37.490±0.21 swimming sonaric beach (s4) 8.317±0.02 25.5±2.0 37.940±0.32 touristic complex mainis beach (s5) 7.983±0.23 25±2.0 38.222±0.26 swimming and artisanal fishing cap kaf-kala beach (s6) 8.35±0.46 25.5±2.0 37.865±0.57 swimming table 2. ecological status class boundaries coastal waters based on the ecological evaluation index (eei-c) continuous formula. ecological status classes eei-c boundary values eei-c eqr boundary values high 9.72±0.46sd 0.97±0.06sd good-high 8.09±0.74 sd 0.76±0.09sd good-moderate 5.84±0.70 sd 0.48±0.09sd moderate-low 4.04±0.68 sd 0.25±0.08sd bad 2.34±0.78 sd 0.04±0.10sd figure 1. map of the sampling sites on the coast of the tenes. 256 belhaouari and bezzina / ecological evaluation index of the coastal waters of algeria results the species richness (sr) of the studied sites are shown in table 3. the total species richness of the study area was 11, including seven species of phaeophyceae, one species of rhodophyta and three species of chlorophyta (table 3). all species were observed in sites s1, s3 and s5 and the lowest species richness in the site 2 with four species. the average cover of each species in the studied sites is shown in figure 2. cystoseira compressa reached its highest percentage (94.4%) in s3 and s5. cystoseira compressa is the most abundant species in all sites, except s2, where it does not exist. this site is marked by the presence of c. mediterrania. the results showed an abundance of ulva sp. with an average cover of 24.8-78.4% between stations. hca identified three groups with 70% similarity that the first one consists of sites s1 and s3, and second s4, s5 and s6. the analysis highlights a third group represented by s2. the results of eei-c are shown in table 4 and it revealed that the studied sites are not disturbed, except sites 2 and 4 with a moderate-low and good-moderate status, respectively. the results showed that s1 has high status and s3, s5 and s6 are in good-high status. discussions the observed macroalgae in the present study are common in the mediterranean sea (cabioc’h et al., 2014; traiche et al., 2018; chabane et al., 2018). these eleven species have been already reported in the algerian coast (perret-boudouresque and séridi, 1989). biodiversity of the macroalgae is influenced by many biotic and abiotic parameters (arévalo et al., 2007; belhaouari et al., 2014, 2017), and the specific richness of algae reaches the highest level in summer (thibaut et al., 2005; ballesteroset al., 2007). based on the results, the members of the genus cystoseira were found in all the sites. it is one of the widely distributed genera of the fucales (ochrophyta, phaeophyceae) (amico, 1995; draisma et al., 2010), and the majority of taxa are found in the mediterranean sea and the adjacent atlantic ocean (oliveras and gomez, 1989; amico, 1995). table 3. specific richness of the sampling sites. species s1 s2 s3 s4 s5 s6 phaeophyceae cystoseira compressa cystoseira mediterrania halopteris scoparia sargassum sp. dictyopteris sp. dictyota fasciola padina pavonica rhodophyta corallina elongata chlorophyta ulva sp. ulva lactusa cladophora sp. + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + table 4. ecological evaluation index eei-c and eei-c eqrof the six sites. sites eei-c eei-c eqr ecological status s1 8.07 0.75 high s2 3.96 0.24 moderate-low s3 7.33 0.66 good-high s4 5.46 0.43 good-moderate s5 6.83 0.60 good-high s6 7.18 0.64 good-high 257 int. j. aquat. biol. (2019) 7(5): 254-259 the average cover results indicate that the dominance of macroalgae in the study area is shared by cystoseira and ulva. cystoseira species are slowgrowing and late-succession taxa, i.e. species with low growth rates and long life cycles (ecological state group i) and ulva sp. is a fast-growing opportunistic species i.e. species with high growth rates and short life cycles (ecological state group ii) (orfanidis et al., 2003). cohabitation between these two macroalgae was reported by traiche et al. (2018) in the coastal waters of the chlef. chabane et al. (2018) reported similar results in the coastal waters of algiers. the most abundant species i.e. c. compressa in the study area are not present in six sites. species abundance may be influenced by nutrient inputs, coastal hydrodynamics and climatic conditions (birje et al., 1996; pereira and neto, 2015; traiche et al., 2018). on the other hand, by the anthropogenic pressure (boudouresque et al., 2009; gihan, 2013; bermejo et al., 2016), the combination of these phenomena may explain the difference in overall average cover at the sampling sites. hca allowed the acquirement coherent and informative data sites 1 and 3, representing the same groups, this is due to the types of species that these beaches shelter. these two stations shelter all the species listed in this study. site 5 shelter 11 species, but it grouped with s4 and s6 which shelter 10 species. it appears that the abundance of species has a strong influence on their classification (orfanidis et al., 2011; caldeira et al., 2017; amaral et al., 2018). site 2 constitutes an isolated group, by having four species. the results of eei-c showed that the site 1 has high quality, explained by non-effecting from any anthropic pressure. the site 2 has low quality, and in accordance with our expectance because of receiving wastewater of the tenes. wastewater has a negative figure 2. total average cover of species in the six sites figure 3. the results of the ascending hierarchical classification of sampling sites. 258 belhaouari and bezzina / ecological evaluation index of the coastal waters of algeria impact on quality of the coastal waters (smith and shackley, 2006; boucetta et al., 2016). this result is in agreement with the classification of the sites in our study. many studies have shown that with a nutrient enrichment gradient, the most impacted sites are systematically characterized by low taxa richness (arévalo et al., 2007; pinedo et al., 2007). site 4 has a moderate quality; a hotel complex is located near this site, which can have a negative effect on the quality of the coastal waters and their ecological status. indeed, it is considered that tourist activities can cause multiple negative impacts on aquatic ecosystems (marchand, 2014; valavanidis, 2018). the results of eei-c confirm the reliability of eei-c to be adapted for evaluation of algerian coastal. conclusion this study has allowed us to better understand the biodiversity of the macroalgae and ecological status of algerian coastal waters. the eei-c results confirm that the coastal waters of our study area are generally qualified as a good ecological state. the distribution of macroalgae is influenced by anthropogenic activities. this study confirms the importance of preserving macroalgae to protect coastal ecosystems and indicated macroalgae as good indicators of the algerian coastal waters. we suggest the use of the eei-c index as part of the biomonitoring and sustainable management of the algerian coastal. references abbasi t., abbasi s.a. 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(2013) 1(4): 188-194 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article microsatellite loci to determine population structure of garra rufa (heckel, 1843) in the khuzestan province (iran) ali shabani, ghasem askari*1 1 department of fisheries, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 23 may 2013 accepted 4 august 2013 available online 2 1 august 2013 keywords: microsatellites genetic variation polymorphism garra rufa khuzestan province abstract: genetic diversity of garra rufa was studied using 6 polymorphic microsatellite dna loci. the specimens of g. rufa were collected from the kheirabad and maroon rivers. despite high importance of this species, there is no genetic information about its population structure. a total of 133 alleles were detected at the 6 loci across the two populations. the kheirabad population exhibited a lower genetic variation (ho=0.429 and he=0.850) than the maroon one. the average numbers of observed alleles in the kheirabad and maroon populations were 11.8 and 10.3, respectively. the genetic similarity and distance between the two populations were 0.721 and 0.326, respectively. it seems that maroon population live under better conditions in contrast to the kheirabad one. diminution of genetic variation within examined populations decreases its adaptation to environmental alterations. based on the results of this study, we can identify two different garra rufa populations in the khuzestan province. introduction there are about 200 fish species in the inland waters of iran, which generally belong to three families: cyprinidae, balitoridae, and cobitidae (abedi et al., 2011). the cyprinid species exhibit a wide range of geographical distribution, life histories, and reproductive styles (winfield and nelson, 1991). the family cyprinidae, with about 220 genera and about 2420 species, is the largest family of freshwater fishes and, with the possible exception of gobiidae, the largest family of vertebrates (nelson, 2006). the members of the genus garra hamiltonbuchanan, 1822 are found throughout the southwest asia and from africa to southeast asia, and are predominantly adapted to live in swift-flowing waters, streams, and lakes (krupp and schneider, 1989). among the iranian inland fishes, garra rufa is one of the important biological species that is native to the tigris basin. it has a small size and no * corresponding author: ghasem askari e-mail address: askarighasem82@gmail.com economic importance. some of the common names of this fish used in iran are gel-cheragh, gel-khorak, mahi-e-sang lis, and shirbot. garra rufa has a wide dispersion, but there is little information on its biology in iran. microsatellite dna markers are utilized in the assessment of genetic variation and population differentiation studies for a variety of vertebrates, including aquatic organisms (o’connell and wright, 1997; neff and gross, 2001; askari et al., 2013). their high level of polymorphism and co-dominant inheritance pattern makes them markers of choice for population genetic studies. microsatellites which occur in non-coding parts of dna have conserved flanking sequences (ellegren, 2004), and there exists the potential for using microsatellite pcr primers developed for one species to characterize loci in other related species (moore et al., 1991; zardoya et 189 shabani and askari/ int. j. aquat. biol. (2013) 1(4): 188-194 al., 1996; galbusera et al., 2007) or unrelated species across families. microsatellite and mitochondrial dna haplotype markers became the methods of choice for many fish studies. taxonomy and systematic have undoubtedly benefited from dna sequencing technology (hillis et al., 1996). therefore, the principal objectives of this study were to assess the intraand interpopulation genetic variations and genetic differentiation in two populations of garra rufa inhabiting the khuzestan province, using the microsatellite dna markers developed by matura et al. (2012). materials and methods the tissue samples for dna extraction were collected using the fin clipping procedure of lourie et al. (1999 a, b). the specimens used in this study, were collected from the kheirabad and maroon rivers of iran between september and october 2012. fin tissues were collected from 80 fish from kheirabad and maroon rivers (fig. 1), and then stored in 96% ethanol for subsequent dna extraction and amplification. genomic dna was extracted from fin clips using the phenolchloroform procedure described by hillis and moritz (1990). the quality and concentration of dna from samples were assessed through 1% agarose gel electrophoresis. pcr reactions were carried out in a thermal cycler (ptc 200 gradient; m.j. research, watertown, ma, usa). pcr amplifications were done using six microsatellite loci analyzed: ggm014, ggm015, ggm021, ggm024, ggm034, ggm044 (matura et al., 2012) (table 1). the polymerase chain reaction (pcr) conditions, especially the annealing temperatures, were optimized for the 6 microsatellite primers as necessary to produce amplification products. amplification was performed in pcr system (gradient eppendorf) using a 25 μl total volume containing 5 μl of 10x reaction buffer, dntps 10 mm, mgcl2 50 mm, primer 20 pmol of each (foward and reverse) (table 1), genomic dna 100 ng and 1.5-2 unit of taq polymerase. initial denaturation was achieved at 94°c for 3 min figure 1. map showing the sampling localities in the kheirabad and maroon rivers microsatellite loci primer sequence n size (bps) anneal (°c) ggm014 f:tgatgcattatgggaacagg r:tcatcaatacttcagaaacgaaat 7 100-132 54 ggm015 f:tgcagttctgacctgaatgag r: ttgtgggacctaatcgattttt 11 220-292 55 ggm021 f:tcctaagaatttttggcataaaaga r:aaatggaactttcagcataataaac 11 184-248 54 ggm024 f:tccctctttttgctctcagg r:taggtgaacaaatggcatgg 14 128-212 54 ggm034 f:cgcgcaagtttctttcagtt r:gctgtgagacaagcctaaacc 10 160-208 56 ggm044 f:ggacgacgttcacagcagta r:caagccaacagcaaattcaa 16 144-220 52 n: number of allele table 1. characteristics of garra rufa microsatellite loci used in this study. 189 190 shabani and askari/ int. j. aquat. biol. (2013) 1(4): 188-194 followed by 30 cycles of denaturation at 94°c for 30 seconds, 30 seconds at the respective annealing temperatures, and extension at 72°c for 1 minute, and an extension for 5 minutes at 72°c. pcr products were separated using 8% polyacrylamide gels stained with silver nitrate. the presence of null alleles was tested using microchecker version 2.2.3 (van oosterhout et al., 2004). the recorded microsatellite genotypes were applied as input data for the genealex software version 6 (peakall and smouse, 2012) to calculate allelic and genotypic frequencies, observed (ho) and (he), expected heterozygosity and to test for deviations from hardy-weinberg equilibrium (hwe). for each marker allelic variation was estimated by the polymorphic information content (pic) value first described by botstein et al. (1980) and modified by anderson et al. (1993). results primer sequences and specific annealing temperature (ta°c) of the resource species (matura et al., 2012) and studied garra rufa species are given in table 1. the optimal annealing temperatures to get scorable bands in samples differed from that reported for the resource species. all primer pairs tested yielded successful amplification. a total of 133 alleles were detected at the 6 loci and across two populations (the kheirabad and maroon rivers) (table 2). in the kheirabad population, a total of 71 alleles were produced in microsatellite analysis across all samples. the number of allele per locus ranged from 5 to 17. in the maroon population, a total of 62 alleles were detected in microsatellite analysis across all samples. the number of alleles at different microsatellite loci in maroon population varied from 8 to 14 with an average value of 10.3. primer ggm044 exhibited maximum allele number (14) compared to other primers. considerable differences among 2 populations in the number of alleles were found at some of these loci (table 2). the number of alleles in ggm024 ranged from 12 to 15 and ggm015 from 8 to 13 with a tendency to a reduction in the maroon population. allele sizes ranged from 100 to 292 bp across the microsatellite loci. the effective number of alleles varied from 3.58 for ggm015 to 10.02 for ggm024. in all populations, the effective number of alleles was lower than the observed number of alleles, except ggm014 loci in the kheirabad population. the average of observed and expected heterozygosity ranged from 0.286 to 0.857 and from 0.721 to 0.902, respectively. the maximum and minimum numbers of the unique alleles were found at loci ggm044 (17) and loci ggm014 (5), respectively. in the kheirabad population, the mean observed heterozygosity (ho) and expected heterozygosity (he) values were 0.429 and 0.850. in the maroon population, these values were 0.532 and 0.859, respectively. location ggm014 ggm015 ggm021 ggm024 ggm034 ggm044 na 5 13 11 15 10 17 ne 7.17 3.58 7.87 10.02 6.89 9.00 kheirabad ho 0.381 0.381 0.333 0.810 0.286 0.381 he 0.721 0.861 0.873 0.900 0.855 0.889 fis 0.472 0.557 0.618 0.101 0.666 0.571 phw *** *** *** *** *** *** na 8 8 11 12 9 14 ne 6.48 6.48 7.41 7.73 5.65 10.25 maroon ho 0.429 0.381 0.524 0.857 0.286 0.714 he 0.846 0.846 0.865 0.871 0.823 0.902 fis 0.493 0.550 0.394 0.016 0.653 0.209 phw *** *** ns ns *** ns na, number of observed alleles; ne, number of effective alleles; ho, observed heterozygosity; he, expected heterozygosity; fis, fixation indices; phw, hardy-weinberg probability test (*p<0.05, **p<0.01,***p<0.001, n.s, non-significant). table 2. genetic variability of six microsatellite loci in two populations for garra rufa. 191 shabani and askari/ int. j. aquat. biol. (2013) 1(4): 188-194 significant deviations from hardy-weinberg equilibrium (hwe) at the locus level are shown in table 2. all six loci used in this study were tested for departure from hwe. nine out of 12 (6 loci × 2 populations) possible tests for hwe were statistically significant (p<0.05). the population differentiation (fst) was modest whit fst value between the kheirabad and maroon rivers population was 0.022 and no significant. rst value between the two populations was high (0.108) and significant. the estimated gene flow (nm) value between the kheirabad and maroon rivers’ populations across all the studied loci was 11.243 (table 3). genetic distances among the respective populations were small. the genetic distances and genetic similarity, as computed by saitou and nei (1987) between the kheirabad and maroon rivers’ populations were 0.326 and 0.721, respectively and the unweight pair group method with arithmetic mean (upgma) dendrogram, based on the genetic distance, showed that these two populations are distinctly two different branches. the fis values ranged from 0.016 for the locus ggm024 to 0.666 for the locus ggm034 between two populations. discussion genetic diversity is important for ecological and evolutionary processes ranging from individual fitness to ecosystem function. heterozygosity serves as an indicator of evolutionary potential and is important in determining population dynamics as well as population viability (reed, 2009). the result is consistent with earlier reports, suggesting the possibility of using primers interspecifically among teleosts (gopalakrishnan et al., 2004). all of the loci were polymorphic and the genotypic distribution frequencies across all the loci were significantly different, suggesting genetic structuring among these two populations. frequencies of alleles in the kheirabad samples are higher from the maroon samples, except at one locus (ggm014). it is likely that the maroon population had originated from the kheirabad, and that it had lost some alleles during the course of fisheries and environmental management. the losses of alleles and heterozygosity in the garra rufa stocks may be intensified by bottlenecks and inbreeding. heterozygosity is an important measurement of population diversity at the genetic level and has drawn much attention from ecologists and aquaculturists (xu et al., 2001). the results of the study indicated that the average number of alleles per locus and the observed heterozygosity in kheirabad (0.429 and 0.850) and the average number of alleles per locus and the observed heterozygosity in the maroon river were 0.532 and 0.839, respectively. in the current study, the observed heterozygosity for 6 microsatellite loci was lower than the expected heterozygosity in the two populations. however, the kheirabad population showed the lowest genetic diversity among the two populations in terms of the average number of alleles and genotypes per locus, the number of unique alleles, and low-frequency alleles. the results of this study indicated that considerable heterozygosity excess was observed in intra-population based on allelic and genotypic frequencies. significant deviations from hardyweinberg expectations (hwe) were observed in the two populations. genetic drift, inbreeding and divergent evolution are likely to be the causes for deviation from the h–w disequilibrium (zolgharnein et al., 2011). several hypotheses have been mentioned to explain the deviation from hwe, including inbreeding, intra-population structure (wahlund effect), non-random sampling, selection against heterozygote, and fishing pressure (abbas et al., 2010; bergh and getz, 1989; castric et al., 2002; ruzafa et al., 2006). these results are compatible loci ggm014 ggm015 ggm021 ggm024 ggm034 ggm044 nm 6.009 12.754 10.646 13.702 10.725 13.621 fst 0.04 0.019 0.023 0.018 0.023 0.018 table 3. number of migrant and fst index of six microsatellite loci in two populations for garra rufa. 191 192 shabani and askari/ int. j. aquat. biol. (2013) 1(4): 188-194 with a previous study that had been conducted for paraschistura bampurensis (askari and shabani, 2013), and with studies on other types of fish (salari aliabadi et al., 2009; bradshaw et al., 2007; alam and islam, 2005; hansen and mensberg, 1998). the partitioning of variability of populations observed after f-statistics comparisons with total types of markers showed that most of the genetic variation is within populations. there was a low level of genetic differentiation among the two populations and significant fst value of 0.022 (p<0.01). based on analysis of molecular variance (amova), fst (0.022) was observed between the kheirabad and maroon rivers’ populations (nm=11.243). this issue represents the low differentiation between the two populations. according to wright (1987), fst value less than 0.05 indicates the low differentiation among communities. li et al. (2007) noted that when nm > 1 and nm < 1, then genetic differentiation occurred due to number of migrant and gene flow, respectively; hence the results of this study revealed that number of migrant fish was the main reason for low genetics differentiation between the studied populations. it was demonstrated using upgma dendrogram, that there were two separate population groups in these rivers. genetic structure of garra rufa in these rivers was probably due to number of migrates which occurred during decades. the results obtained from the present study show that at least 2 different populations of garra rufa are found in the two rivers, which include the kheirabad and maroon rivers’ populations. this information should be taken into account for any genetic conservation and stock improvement plan. however further study involving low numbers of populations covering all parts of the country with additional microsatellite loci is recommended to reveal detailed genetic structure of this important fish species in iran. references abbas k., zhou x.y., li y., gao z.x., wang w.m. 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(2018) 6(4): 179-188 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology review article review of the danionids of iran (family danionidae) brian w. coad1 canadian museum of nature, ottawa, ontario, k1p 6p4 canada. article history: received 4 july 2018 accepted 16 october 2018 available online 2 5 august 2018 keywords: biology, morphology, barilius, cabdio. abstract: the systematics, morphology, distribution, biology and economic importance of the danionids of iran are described, the species are illustrated, and a bibliography on these fishes in iran is provided. there are two native species in the genera barilius and cabdio found in southwestern and southeastern iran, respectively. introduction the freshwater ichthyofauna of iran comprises a diverse set of about 297 species in 109 genera, 30 families, 24 orders and 3 classes (esmaeili et al., 2018). these form important elements of the aquatic ecosystem and a number of species are of commercial or other significance. the literature on these fishes is widely scattered, both in time and place. summaries of the morphology and biology of these species were given in a website (www.briancoad.com) which is updated here for one family, while the relevant section of that website is now closed down. other families will also be addressed in a similar fashion. family danionidae bleeker, 1863 the danionids, or danios, rasboras and allies, are found from africa to indonesia and comprise about 347 species (catalog of fishes, downloaded 15 october 2018). the danionids of iran belong to the subfamily chedrinae bleeker, 1863, or troutbarbs, with two species. the family was formerly placed as a subfamily within the family cyprinidae but is distinguished on the basis of osteological and molecular data (e.g., tang et al., 2010; stout et al., 2016; betancur-r et al., 2017; tan and armbruster, 2018). * corresponding author: brian w. coad doi: https://doi.org/10.22034/ijab.v6i4.516 e-mail address: bcoad@mus-nature.ca genus barilius hamilton, 1822 the members of this genus are found from pakistan to thailand with one species in the tigris-euphrates and an adjacent basin. their systematics is still poorly understood and there may be about 32 species. this genus is characterised by a compressed but slender and small body, having small to moderate sized scales, a decurved lateral line, running for example on the lower part of the caudal peduncle, lateral line complete, incomplete or absent, a short dorsal fin and a long anal fin, no fin spines, a moderate and terminal mouth, barbels absent or in one or two pairs, short gill rakers, pharyngeal teeth in three rows, and usually with dark bands or spots on the flank. these fishes are found mostly in mountain streams although some are lowland species. barilius mesopotamicus berg, 1932 (figs. 1-2) common names: sebil mahi (= moustache or barbelled fish) [sboura iraqia (from subura and shabil meaning shad after mikaili and shayegh (2011), in arabic; mesopotamian barilius, mesopotamian minnow]. systematics: the holotype, 44.0 mm total length and 180 coad/ danionids of iran 35.4 mm standard length, is in the zoological institute, st. petersburg (zisp 23955) and is decoloured. the collection date was given by berg (1949) as 16.iv.1914, as 3.iv.1914 in the zisp catalogue and 5.iv.1914 in the jar. the first two dates are probably correct, one old style and one new style. the type locality is "stromgebiete des tigris, in (siaret) seid-hassan, an der persisch-türkischen grenze, unter 33°20'n. br., 46°20'ö. l. seid-hassan liegt am flusse gawi, welcher sich mit dem kundschian (gundschian)-tschai vereinigt; der letztere mündet in den tigris". seyyed hasan (33°06'n, 46°11'e) lies on a tributary of the kanjan cham river near the iranian town of mehran on the iran-iraq border. the tributary is presumably the gavi river. howes (1980) stated that this species has apomorph characters shared with species assigned to leucaspius heckel and kner, 1857 but this seems unlikely on general morphological grounds. coad (1982), bianco and banarescu (1982) and liao et al. (2011) concurred, the latter also incorporating molecular evidence. it resembles other barilius in having barbels (none in leucaspius), a lateral line low on the body (short and mid-body), broad suborbital bones, and flank bars (none), while leucaspius is unique in females having a fold of skin in the shape of two, large, rounded papillae around the genital opening. bianco and banarescu (1982) stated that this species may be generically distinct from south asian barilius but do not diagnose a new genus. bănărescu and coad (1991) and bănărescu (1992) stated that its position and biogeographical affinities are uncertain. berg (1949) considered it closer to indian species of the genus barilius than to african ones. jouladeh roudbar and vatandoust (2015) and jouladeh roudbar et al. (2017) examined fish from the seimarreh (= simareh), changoleh and siahgave rivers for 29 morphometric and seven meristic characters, finding 10 and four of these respectively showing population differences. the siahgave population could be separated from the other two. the populations were not genetically differentiated figure 1. barilus mesopotamicus, line drawing by s. laurie-bourque. figure 2. barilus mesopotamicus, khabour river, syria, f. krupp. 181 int. j. aquat. biol. (2018) 6(4): 179-188 using cyt b gene sequences and morphological differences were attributed to environmental factors. keivany and ghorbani-ranjbari (2017) examined 460 fish morphometrically from the bushehr, diala, jarrahi, karkheh and karun rivers and found no differences between populations although some differences in head size and shape, body depth and caudal peduncle length were observed. ghorbaniranjbari et al. (2018) examined 99 fish from the changooleh, doyrej, kangir and meymeh rivers and found morphometrics did not fully differentiate populations although the kangir river fish were shown as a separate group. ghorbani-ranjbari and keivany (2018) examined 140 fish from six rivers in the bushehr (= persis) basin morphometrically and found significant differences between them with only two overlapping. body shape, head size and body depth were the main separating characters. key characters: the only member of its genus in iran, this species is easily identified by the pigment pattern, low lateral line, broad suborbital bones and the barbels. morphology: the lower jaw bears a small symphysial knob. the mouth is slightly subterminal, oblique and elongate with the mouth corner under the anterior half of the eye. a well-developed barbel has its origin just anterior to the level of the nostril above the upper lip and lies in a groove between the upper lip and the beginning of the suborbital bone series. this barbel can be absent or minute in some fish (females from habbaniyah, iraq) (coad and krupp, 1983). in addition to these maxillary barbels, a second pair of barbels has their origin slightly above the posterior edge of the mouth in eight out of 259 fish examined. they are usually rudimentary but may reach 10.7% of head length. barbels are difficult to see in smaller fish without magnification. dorsal fin unbranched rays 2-3, usually 3, branched rays 7-9, usually 8, anal fin unbranched rays 2-3, usually 3, branched rays 10-14, pectoral fin branched rays 11-15, and pelvic fin branched rays 6-8, usually 7. lateral line scales 42-58. the lateral line is incomplete or complete, rarely terminating at the pectoral fin level. lateral line decurved and parallel to the ventral body profile from the pelvic fin origin to the caudal peduncle, being 2-3 scales above this profile. on the caudal peduncle, the lateral line is below the mid-line while scales on the caudal fin posterior to the hypural plate are perforated in the mid-line. pectoral and pelvic axillary scales present. scales are regularly arranged over the whole body but are not strongly imbricate, particularly on the belly and back anterior to the dorsal fin. anterior flank scales are oval with subcentral anterior focus and a moderate number of circuli. radii are found principally on the posterior and lateral fields. anterior field radii are usually absent although 1-2 radii may occasionally be found. scale radii based on five anterior flank scales from five fish (40.7-50.7 mm standard length) number 5-11 primary radii, 013 secondary radii and 5-23 total radii. total gill rakers 7-14. gill rakers are short and rounded, reaching to or part way to the raker below when appressed. total vertebrae 38-41. pharyngeal teeth usually 4,5-5,4, often 4,5-5,3, or more rarely in three rows 1,3,5-5,3,1 or 1,4,5-5,4,1. teeth are hooked at the tip, slender and have a concave grinding surface below the tip. the gut is a simple s-shape. meristic values for iranian specimens are: dorsal fin branched rays 7(2), 8(32) or 9(2), anal fin branched rays 10(2), 11(19), 12(13) 13(1) or 14(1), pectoral fin branched rays 11(1), 12(8), 13(23), 14(2) or 15(1), pelvic fin branched rays 6(1), 7(33) or 8(2), scales in lateral series 42(1), 43(3), 44(3), 45(2), 46(6), 47(6), 48(1), 49(3), 50(4), 51(4), 52(2) or 54(1), total gill rakers 7(4), 8(6), 9(6), 10(7), 11(6), 12(1), 13(1) or 14(1), pharyngeal teeth 4,5-5,4(8), 4,5-5,3(3) or 1,3,5-5,3,1(1), and total vertebrae 39(8), 40(20) or 41(5). sexual dimorphism: unknown. colour: overall colour is brilliant silver with a golden-yellow glimmer, with the back darker and having a thin median stripe. scales are highly deciduous and leave a silvery smear on the hand. the upper flanks have 6-11 roundish dark, grey-green spots, not always clearly apparent in live fish. in preserved fish the spots are brown. a median dorsal stripe is variably developed. barbels can be pale. 182 coad/ danionids of iran fins are lightly pigmented, most melanophores being on the rays rather than the membranes. the anal and paired fins are almost entirely hyaline. the caudal fin may show one or two irregular bars running parallel to the posterior margin. the peritoneum is light to silvery but bears scattered melanophores which give a greyish tinge in preserved fish. some fish from iraq (habbaniyah stream) lacked, or had weakly expressed, flank spots. size: reaches 69.8 mm total length (esmaeili et al., 2014). distribution: this species is found in the tigriseuphrates basin, including its iranian part, and the adjacent persis basin. found in the tigris river basin in the ab-e shur, a'la, arvand, bala, bushehr, changoleh, chikhab, dez, diala, doveyrich, doyrej, gavi, jarrahi, kahnak, kangir, karkheh, karun, meymeh, qeski, shur, siahgave, simareh and zard rivers; and in the persis basin in the ahram, dalaki, darolmizan, helleh, karzin, kheyrabad, mond, shapur, shirin and zohreh rivers (berg, 1932; 1949; bianco and banarescu, 1982; abdoli, 2000; pazira et al., 2013; esmaeili et al., 2015; jouladeh roudbar and vatandoust, 2015; jouladeh roudbar et al., 2017; keivany and ghorbani-ranjbari, 2017; keivany and zamani-faradonbe, 2017a, b; ghorbani-ranjbari et al., 2018; ghorbani-ranjbari and keivany, 2018). zoogeography: found in the tigris-euphrates basin of turkey, syria, iraq and iran. it does not appear to be common in turkey, at least in the upper reaches of this basin there, nor in upper reaches of iranian rivers. the distribution in the persis basin of iran is outside the modern tigris-euphrates basin. it is presumably a relict of the late pleistocene when the tigris-euphrates flowed down a drained persian gulf receiving tributaries now isolated by the postpleistocene rise in sea level (coad and krupp, 1983). kosswig (1951, 1952, 1955a, 1955b) noted the similarity at the generic level between indian and african fishes, e.g., the cyprinoids barilius, garra and labeo, indicating that these fishes arrived in africa from india after the desiccation of the syrianiranian sea in the pliocene. barilius, it should be noted, appears to prefer, in asia and the tigris-euphrates basin, large lowland rivers and its dispersal across iran is difficult to envisage by headwater capture (other genera can be found in small streams at higher altitudes as well as lowland rivers). however, berg (1940) suggested that fish dispersal across this region was facilitated by the coastal rivers of iranian and pakistani baluchestan being part of a single river system in the pliocene, since submerged by subsidence. the presence of the spiny eel mastacembelus and barilius in western iranian basins is attributed to headwater capture and/or colonisation from the tigris-euphrates basin when persian gulf rivers were tributary to an expanded tigris-euphrates basin during lowered sea levels in glacial times. this distribution of these genera is not, therefore, a remnant of the dispersal across iran from asia. habitat: it is present in both running and still water, from small streams only 1.0 m wide and irrigation ditches to major rivers more than 200 m across. current is slow to fast but generally an obvious flow is apparent. however, one specimen was collected in a fish pond near ahvaz (zsm 25701). the collection localities in iran are all at low altitudes and no fish were taken in high zagros mountain streams and rivers. collections were made over mud and pebble substrates in shallow streams or at river margins. the species may also occur at the surface in mid-river but no collections confirm this supposition. capture temperatures were 12-24°c and conductivity 0.4510.5 ms. salinity in drying pools of 20 cm depth in syria where this species was caught in march had cl1 = 390 mg/l and a salinity of 1.5‰ (fig. 3) (coad and krupp, 1983). age and growth: esmaeili et al. (2014) gave a b value for 13 fish from the tigris river basin, 4.266.98 cm total length, as 3.22. keivany and zamanifaradonbe (2017a) gave a b value of 3.33 for 21 zohreh river fish (3.01-5.45 cm total length). keivany and zamani-faradonbe (2017b) examined 94 fish, 24.3-57.2 mm total length, from the jarrahi river and found a b value of 3.61. food: gut contents include winged insects 183 int. j. aquat. biol. (2018) 6(4): 179-188 (coleoptera, heteroptera, thysanoptera and diptera) and spiders, suggestive of surface feeding (coad and krupp, 1983). abdoli (2000) also reported hymenoptera, brachycera and culicidae. reproduction: most fish were collected in january when eggs were small but developing suggestive of spring spawning. al-rudainy (2008) gave an absolute fecundity of about 200 eggs for iraq. parasites and predators: none reported from iran. economic importance: none. experimental studies: none. conservation: this fish is found in suitable habitats of large rivers and in small ditches and does not appear to be in need of conservation. vulnerable in turkey (fricke et al., 2007). listed as of least concern by the iucn (2015, https://newredlist. iucnredlist.org/, downloaded 15 october 2018). sources: type material: barilius mesopotamicus (zisp 23955). iranian material: cmnfi, 1979-0120, 3, 19.3-50.7 mm standard length, bushehr, dalaki river near konar takhteh (29º28'n, 51º21'e); cmnfi 19790357, 1, 27.6 mm standard length, khuzestan, ditch in karkheh river drainage (31º34'n, 48º12'e); cmnfi 1979-0363, 11, 21.4-30.2 mm standard length, khuzestan, karkheh river (31º52'n, 48º20'e); cmnfi 1979-0365, 7, 20.0-34.4 mm standard length, khuzestan, stream in doveyrich river drainage (32º25'n, 47º36'30"e); cmnfi 1979-0367, 1, 34.2 mm standard length, khuzestan, meymeh river 11 km north of dehloran (32º44'30"n, 47º09'30"e); cmnfi 1979-0368, 29, 21.6-41.9 mm standard length, khuzestan, karkheh river (32º24'30"n, 48º09'e); cmnfi 1979-0372, 2, 30.7-33.1 mm standard length, khuzestan, dez river near chogha zanbil (ca. 32º02'n, ca. 48º30'e); cmnfi 1979-0377, 3, 28.0-39.4 mm standard length, khuzestan, karkheh river (ca. 32º57'n, ca. 47º50'e); cmnfi 1979-0378, 7, 31.9-42.4 mm standard length, khuzestan, stream tributary to karkheh river (ca. 32º48'n, ca. 48º04'e); cmnfi 1979-0379, 21, 23.8-45.6 mm standard length, khuzestan, dez river (32º12'n, 48º27'e); cmnfi 1979-0380, 10, 25.3-41.0 mm standard length, khuzestan, stream tributary to dez river (ca. 32º10'n, ca. 48º35'e); cmnfi 1979-0381, 7, 24.331.2 mm standard length, khuzestan, stream west of shushtar (ca. 32º10'n, ca. 48º35'e); cmnfi 19790382, 4, 25.9-30.8 mm standard length, khuzestan, karun river at shushtar (32º03'n, 48º51'e); figure 3. habitat of barilius mesopotamicus, khuzestan, a'la river at pol-e tighen (31º23'30"n, 49º53'e), 20 september 1995, b.w. coad. 184 coad/ danionids of iran cmnfi 1979-0383, 8, 28.6-34.8 mm standard length, khuzestan, stream in ab-e shur drainage (31º59'30"n, 49º06'e); cmnfi 1979-0384, 3, 26.8-40.8mm standard length, khuzestan, river in ab-e shur drainage (32º00'n, 49º07'e); cmnfi 1979-0392, 3, 35.0-39.3 mm standard length, khuzestan, zard river (ca. 31º32'n, ca. 49º48'e); cmnfi 1979-0396, 35, 25.1-48.8 mm standard length, khuzestan, kheyrabad river 20 km from behbehan (30º32'n, 50º23'30"e); cmnfi 19950009a, 1, 35.2 mm standard length, khuzestan, a'la river at pol-e tighen (31º23'30"n, 49º53'e); cmnfi 2008-0165, not kept, khuzestan, dez river near shush (32º14’40”n, 48º20’07”e); cmnfi 2008-0171, not kept, khuzestan, a'la river at pol-e tighen (31º23'20"n, 49º52'44”e); zsm 25701, 1, 36.5 mm standard length, khuzestan, fishpond near ahvaz (no other locality data); issb uncatalogued, 1, 48.7 mm standard length, bushehr, helleh river (ca. 29º20'n, ca. 51º15'e) (coad and krupp, 1983). comparative material: bm(nh) 1974.2.22:12561267, 11, 33.7-46.2 mm standard length, iraq, stream between lake habanniyah and euphrates river (ca. 33º22'n, 43º34'e); bm(nh) 1968.12.13:217-220, 4, 18.5-47.4 mm standard length, syria, euphrates river at mayadine (35º01'n, 40º27'e); bm(nh) 1968.12.13:221-236, 16, 30.8-42.4 mm standard length, syria, tigris river at ain diwar (37º17'n, 42º11'e); smf 16442, 5, 28.2-35.9 mm standard length, syria, nahr balikh at jisr shanine (36º03'n, 39º06'e); smf 16443, 63, 17.0-34.9 mm standard length, syria, nahr balikh at jisr shanine (36º03'n, 39º06'e); issb uncatalogued, 4, 32.8-34.4 mm standard length, turkey, batman suyu (ca. 37º55'n, ca. 40º15'e). genus cabdio hamilton, 1822 this oriental genus has only three species, one of which enters southeastern iran. aspidoparia heckel, 1847 is a synonym (tang et al. 2010). mirza (2000) proposed that the members of the genus aspidoparia be placed in a new subfamily, aspidoparinae. it is characterised by an elongate and almost cylindrical body with a rounded abdomen, the head has a broad ring of suborbital bones, the mouth is small and inferior, the roof of the mouth has a papillose nodule, the lower jaw has a sharp, crescentic bony edge, there are no barbels, pharyngeal teeth are in 2-3 rows, the dorsal fin is short, anal fins are short to moderate, scales are moderate in size, the lateral line is decurved and runs on the lower half of the caudal peduncle, and the gut is long and coiled. cabdio morar (hamilton, 1822) (fig. 4) common names: zanboor-mahi (= bee or wasp fish, presumably a mis-reading of the name in pakistan) [common chilwa or waspi in pakistan; morar, morari]. systematics: no relevant synonyms. this species was originally described from the yamuna and tista figure 4. cabdio morar, line drawing by s. laurie-bourque. 185 int. j. aquat. biol. (2018) 6(4): 179-188 rivers, india. no types are known (eschmeyer et al., 1996). there are some minor differences in characters with literature reports, particularly in pharyngeal tooth count, but sample sizes do not permit an adequate comparison for this wide-ranging species. jalili et al. (2015) found osteological differences between mashkid and sarbaz river fish although zamani faradonbeh et al. (2015) found no meristic differences but some morphometric ones, attributed to phenotypic plasticity in different river conditions. key characters: the suborbital ring of bones is large and distinctive, being almost as deep as the eye, and this feature is unique in southeastern iranian cyprinids. morphology: the snout is short and rounded and overlaps the upper lip. the mouth is small, ventral and transverse. the lower jaw is straight with a slightly horny cutting edge and no lip. the dorsal fin origin is over or slightly behind the pelvic fin origin. the dorsal fin margin is straight to somewhat emarginate and the anal fin is emarginate. dorsal fin unbranched rays 2-3 (the first unbranched ray is very small, usually 3 rays are present but not discernible) and branched rays 6-8, anal fin unbranched rays 2 and branched rays 8-10, pectoral fin branched rays 9-16, usually 12 or more, and pelvic fin branched rays 7-8. lateral line scales 36-45. scales have few anterior and more numerous but not many posterior radii. there is a pelvic axillary scale and several elongate and overlapping scales in the pectoral axil. gill rakers are very short, not touching the adjacent one when appressed, difficult to count at the fleshy ends of each arch, and numbering about 17-25. pharyngeal teeth 2,4,5-5,4,2 in the literature but the main row count of 4 teeth observed here differs. the main row teeth have large, oval to oblong flattened crowns. the gut is a very elongate s-shape with a small anterior loop. total vertebrae 36-37. the chromosome number is 2n= 48-50 (klinkhardt et al., 1995; arai, 2011). meristic values for iranian specimens are: dorsal fin branched rays 6(1) or 7(18), anal fin branched rays 8(4) or 9(15), pectoral fin branched rays 9(1), 12(5), 13(11) or 14(2), pelvic fin branched rays 7(19), lateral line scales 36(1), 37(4), 38(1), 39(5), 40(4), 41(2), 42(1) or 45(1), scales above the lateral line 7(10) or 8(9), scales below the lateral line to the anal fin 3(1), 4(12) or 5(6), scales between the lateral line and the pelvic fin 4(13), 5(5) or 6(1), predorsal scales 17(1), 18(3), 19(2), 20(2), 21(3), 22(3), 23(2) or 24(2), caudal peduncle scales 15(1), 16(5), 17(6), 18(5), 19(1), total gill rakers 17(1), 18(2), 19(2), 21(2), 22(4), 23(2) or 25(1), pharyngeal teeth 2,4,44,4,2(5), 2,4,4-4,4,1(1) or 2,3,4-4,4,2(1), and total vertebrae 36(3) or 37(9). sexual dimorphism: unknown. colour: back light brown to brown-green with the flanks very silvery to silvery-yellow and the belly lighter. there is a golden stripe along the flank. fins are a distinct dark yellow. the caudal fin may be yellow to orange and paired fins a very light orangeyellow. preserved fish have immaculate fins except for the caudal fin which has some melanophores lining the rays, a broad stripe along the midline of the back, and fine melanophores on the back and upper flank. some fish have small, dark dots on the back and upper flank. the peritoneum is black. size: attains 20.0 cm (malhotra and munshi, 1985). distribution: this species is reported from the makran and mashkid river basins in pakistan (mirza, 1992) and eastwards to thailand. the iranian distribution encompasses the hamun-e mashkid (= mashkel) basin in the mashkid river, and the makran basin westwards from the pakistan border to the straits of hormuz. in the makran, it is found in the bahu kalat, gabrik, jaghin, mazaei, rask gando, sarbaz and validad rivers (kiabi and abdoli, 2000; malekzehi et al., 2014; zamani faradonbeh et al., 2015). zoogeography: the species and genus reaches its westernmost limit of distribution in southeastern iran. barriers to further dispersal are unknown but it may be limited by temperature, habitat availability and poor recent connections between streams in the makran and the southern deserts of iran. habitat: this species favours streams and rivers with slow current. it is also recorded from lakes but not in 186 coad/ danionids of iran iran. mashkid river fish were caught in pools isolated by the drying river. capture temperatures in december 1977 were 15-21ºc. in pakistan, it prefers comparatively high water temperatures and avoids waters with greater water depths, high stream orders and sandy beds (rafique, 2007). age and growth: a female, 9.8 cm total length, from iran had mature eggs (berg, 1949). esmaeili et al. (2014) gave a b value for 55 fish from the makran, 6.08-12.0 cm total length, as 3.31. food: this minnow is a carni-omnivore and a voracious feeder (bhattacharjee and dasgupta, 1988). iranian specimens contained no discernible food items in their guts. reproduction: spawning occurs from february to april in india (malhotra and munshi, 1985). iranian specimens caught in december were not mature suggesting a later spawning season. parasites and predators: jalali et al. (2000) described two new species of monogeneans, dactylogyrus yousefpouri and d. mobedii, from this species in the bahu kalat river of baluchestan. malekzehi et al. (2014) recorded lernaea sp. from fish in the mashkel river basin. economic importance: not of any economic importance in iran but it is eaten in india. experimental studies: none. conservation: although known from only a few localities in southeastern iran, this species may not be threatened other than by water abstraction and pollution. listed as of least concern by the iucn (2015, https://newredlist.iucnredlist.org, downloaded 15 october 2018). sources: iranian material: cmnfi 1979-0316, 1, 22.1 mm standard length, baluchestan, stream in sarbaz river drainage (26º48'n, 61º02'e); cmnfi 1979-0322, 7, 42.3-86.3 mm standard length, baluchestan, bahu kalat river (ca. 25º45'n, ca. 61º26'e); cmnfi 1979-0333, 7, 17.7-69.5 mm standard length, baluchestan, mashkid river west of kuhak (ca. 27º05'n, ca. 63º12'e); cmnfi 19790334, 10, 22.8-62.0 mm standard length, baluchestan, mashkid river 5 km east of esfandak (27º04'n, 62º54'e); osu 8123, 5, 45.7-50.6 mm standard length, baluchestan, sarbaz river (no other locality data). comparative material: cmnfi 2008-0048a, 2, 43.9-67.8 mm standard length, afghanistan, probably kabul river basin (no other locality data); cmnfi 2010-0002, 1, 89.8 mm standard length, afghanistan, daruntah lake, kabul river drainage (34°30’n, 70°22'e); bc 55-61, 2, 67.0-68.2 mm standard length, afghanistan, india, barakar river near tillya dam (no other locality data). acknowledgments i am indebted to the department of biology, shiraz university and the canadian museum of nature, ottawa for funding of research. numerous colleagues and co-authors assisted in developing the website on iranian fishes, providing specimens, data and photographs and are listed at www.briancoad.com. references abdoli a. 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(2019) 7(6): 368-373 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article physiological, biochemical and neurochemical responses of cirrhinus mrigala upon short term exposure to cerium oxide sharath chandra*1, sandya sukumaran2 1department of of biochemistry, government science college, hassan, karnataka, india. 2department of inorganic and physical chemistry, indian institute of science, bangalore, india. s article history: received 10 september 2019 accepted 21 december 2019 available online 2 5 december 2019 keywords: biochemistry cirrhinus mrigala neurochemical response physiology abstract: the current study was performed to ascertain the biochemical and physiological impact of cerium oxide (ceo2) on freshwater fish cirrhinus mrigala, which are widely consumed. biochemical, neurochemical and physiological modifications were evaluated and lc50 of ceo2 was found to be 22 ppm, observed for 24 h. further 1/10th of the lc50 concentration of ceo2 (2.2 ppm) was used for short term investigation at 96 h. the results demonstrate an increase in physiological levels of serum lactate dehydrogenase (ldh), aspartate transaminase (ast) and alanine transaminase (alt) compared to control groups. studies revealed variations in oxidative stress markers with a significant reduction in the serum superoxide dismutase (sod) and catalase (cat) activities and an increase in malondialdehyde (mda). the study reported the increase in brain glutamate concentrations indicating possible brain tissue damage. the above analysis highlights the potential toxicological impact of ceo2 on freshwater fish and their ecosystem. introduction the existence of xenobiotics, chemicals and their derivatives in the environment and ecosystem due to their agricultural and industrial applications are welldocumented. the majority of these chemicals in the form of heavy metals and their derivatives are not degradable and have the possibility of endogenous binding molecules as they enter the biological system may lead to unfavorable toxic effects (amdur et al., 1991). the existence of these metals in a biological system can bring about undesirable modifications in a cell which may be circulated to the tissue or the organ (bernet et al., 1991). the eventuality of these metals to accumulate in the aquatic ecosystem, particularly fish, has a detrimental impact on the food chain. human populations who consume aquatic products as a primary source of food are also part of the food chain, thus generating a cause of concern in public health (di giulio and hinton, 2008). the impact and bioaccumulation of metals and their derivatives in the organs of aquatic animals like fish depend on a variety of factors like proximity of the animals from the *correspondence: sharath chandra doi: https://doi.org/10.22034/ijab.v7i6.657 e-mail: biosharath123@gmail.com pollutants, metabolic activity and membrane transport potential, and climatic conditions (abhijith et al., 2016). cerium oxide is extensively used as diesel fuel additive exploiting their potential of oxygen storing, and their applications can also be found in television tubes, uv absorbents, and glass industries (khan et al., 2011), besides their uses also found in biomedical industry like to retinal neurodegeneration and protection against radiation caused injury (celardo et al., 2011). in this study, we used freshwater fish cirrhinus mrigala for the measurement of acute toxicity of cerium oxide. it is bottom-dwelling fish that is widely consumed in the indian subcontinent. lc50 concentration is a vital indicator of the extent of resistance of an organism to metal and its derivatives (reda et al., 2010). biochemical and physiological changes of exposed fish give the precise extent of damage caused by these metals (banaee et al., 2019b). toxic stress leads to oxidative damage by the production of ros, consequently with the exhaustion of natural antioxidants (banaee et al., 2019a). 369 int. j. aquat. biol. (2019) 7(6): 368-373 quantification of oxidative stress markers such as sod, catalase and lipid peroxidation (lpo) demonstrates the biochemical variations due to toxicity. alterations in brain neurotransmitter glutamate is assessed given that neurons are susceptible to ros, ensuing in variations of neurotransmitter and causing neurodegenerative disorders due to oxidative damage (li et al., 2010). thus in the current research, we evaluated the biochemical, neurochemical (glutamate) and oxidative stress imparted to freshwater fish c. mrigala upon short term exposure (96 h) to cerium oxide. materials and methods cerium oxide (ceo2) sample preparation and inoculation: 22 g/l of the solution was prepared by dissolving ceo2 in double-distilled water. the groups of fishes were exposed to ceo2 for a short period of 96 h (acute toxicity). concentrations of sample 2.2 mg/l based on 1/10th of lc 50concentration (22 mg/l) were introduced into 100 l aquariums containing 50 fishes each. the fishes with similar size and weight were kept in the groups. every 24 h, the water was changed and freshly prepared solution of ceo2 was introduced to the aquariums. the control group of 50 fishes was also maintained under the comparable physical conditions like the treated group fishes. food was withdrawn 24 h before sacrifice to circumvent postprandial variations. control and treated fish (2.2 mg/l) were taken for further investigation. zero mortality was observed during 96 h of exposure time. sample collection: the cardiac puncture was adopted to draw blood from the fish. the blood drawing syringes were precoated with heparin, an anticoagulant. the samples (abhijith et al., 2016) for biochemical evaluation were transferred to edta vials and centrifuged at 9392 g for 20 min at 4ºc. the brain tissue (raju et al., 2004) sample for estimation of neurotransmitter (glutamate) was immediately dissected and placed in ice. the brain tissue during analysis was homogenized with 8 volumes of cold tris buffer saline (10 mm tris-hcl, 10 mm sucrose, 0.7% nacl, 0.1 mm edta of ph 7.2 at 4ºc (raju et al., 2004). the homogenate was then centrifuged at 3000 rpm for 12 min at 4ºc, and the supernatant was obtained for further evaluation. biochemical variable measurement: blood glucose level was measured by preparing a reaction mixture of 0.1 ml of plasma, 4 ml of o-toluidine (cooper and mcdaniel, 1970), which was then mixed and incubated for 10 min on a boiling water bath. the samples were cooled and the absorbance was measured at 630 nm in a uv spectrophotometer. the glucose concentration was expressed in mg/dl. plasma protein concentration was assessed by lowry’s method (lowry et al., 1951). 100 µl of plasma, 900 µl of double distilled water and 4 ml of lowry reagent were mixed well and incubated for 10 min at room temperature, to the reaction mixture, 0.5 ml of fc reagent was added and incubated for 20 min at room temperature. the sample was then measured at 720 nm and expressed in µg/ml. analysis of serum transaminases: serum l aspartate aminotransferase (l-ast) and l-alanine aminotransferase (l-alt) activities were demonstrated at 37oc by a colorimetric method (reitman and franckel, 1957) and enzyme activity was expressed as iu/l. lactate dehydrogenase (ldh) was analyzed by the method developed by anon (1984) and activity was expressed in iu/l. oxidative stress markers and glutamate analysis: superoxide dismutase (sod) was measured (das et al., 2000) by the inhibition of superoxide led nitrite formation from hydroxylamine hydrochloride and the absorbance was measured at 540 nm and activity was expressed as u/mg of protein. catalase activity was determined based on the formation of a stable complex of hydrogen peroxide with ammonium molybdate and absorbance was read at 405 nm and expressed as u/mg (goth, 1991). lipid peroxidation was determined by taking malondialdehyde (mda) as standard (buege and aust, 1978). the reaction mixture of tca-tba hcl reagent and the sample was boiled for 10 min, cooled and then centrifuged at 10000 g and the supernatant was used for quantification at 535 nm and expressed as nmol/mg. glutamate levels were demonstrated by multiple development of paper chromatography (raju et al., 2004). the supernatant 370 çiçek et al./ the effect of ceo2 on cirrhinus mrigala was evaporated at 70ºc and mixed with 100 ml double distilled water. the two mm glutamate standard solution with the sample was spotted on whatman no.1 chromatography paper which is then allowed to develop on the mobile phase (butanol: acetic acid: water 12:3:5 v/v). the chromatogram is developed again, following which the papers are dried and sprayed with ninhydrin and incubated at 100ºc for 4 min. the bands which reveal glutamate corresponding to the standard is cut and eluted in 75% ethanol with 0.005% cuso4. the absorbance is measured against the blank at 515 nm in a spectrophotometer. the concentration is expressed as µmol/g of glutamate. statistical analysis: all data were statistically analyzed and represented as mean±se. in all experiments, the level of statistical significance was set for p<0.05. the significance was calculated by using an independent sample t-test by ms-excel. results the fish were divided into treated (2.2 ppm of ceo2) and control groups and studied for 24, 48, 72 and 96 h. the blood analysis of ceo2 treated c. mrigala demonstrates a remarkable raise in glucose levels (table 1) throughout the 4 days of experiment, however major difference of 5.81±0.05 / 26.55±1.43 was found to be on the 4th day (96 h). the lowering in plasma protein level (table 1) was significantly (p<0.05) demonstrated in treated groups (2.2 ppm of ceo2) on the 4 th day (8.58±0.03 / 6.33±0.24) compared to control group. in the current analysis, there was a marked rise in both ast and alt concentration of treated groups on all the four days in comparison to the control groups. lactate dehydrogenase (ldh) is an isoenzyme playing a vital task in glycolysis, which is also considered as important biomarker of organ and tissue damage. table 1 reports the increased activity of ldh on treatment of c. mrigala fish to ceo2 (2.2 ppm). figure 1 shows the increase in brain glutamate concentration levels in treated fish (8.35±0.16) when compared to the control groups (14.53±0.82) on the 4th day. the results revealed decrease in sod and cat in the treated groups compared to the control one on all the days of sample inoculation. the lpo analysis in c. mrigala exposed to 2.2 ppm of ceo2, suggested a remarkable raise in mda levels in treated fish compared to the control fish (table 2). discussions in the current study, there was a considerable increase in glucose concentration of treated fish compared to the untreated fish. this can be credited to stimulation of stress resulting in hyperglycemia (simakani et al., table 1. alterations in the biochemical variables of cirrhinus mrigala exposed to ceo2 during short term exposure. biochemical variables exposure duration (in h) 24 48 72 96 c e c e c e c e glucose (mmol/l) 5.42±0.51 8.37±0.45 5.53±0.82 20.11±1.33* 5.72±0.63 24.72±1.54* 5.81±0.05 26.55±1.43* protein (g/l) 8.66±0.47 14.24±0.31* 8.63±0.13 13.12±0.18 8.78±0.75 11.57±0.92* 8.58±0.03 6.33±0.24* ldh (iu/l) 4.07±0.51 8.46±0.27* 4.28±0.15 8.07±1.37* 4.92±038 7.86±0.95* 4.88±0.41 8.17±1.06* ast (iu/l) 14.91±1.05 17.62±1.09* 14.53±0.92 18.43±0.39* 14.34±0.44 19.53±1.05* 15.01±0.48 20.17±0.96* alt (iu/l) 18.25±0.91 22.58±1.15* 18.91±1.16 22.93±0.24* 19.07±0.86 23.41±0.87* 18.95±0.36 23.58±1.15* all values are expressed as mean± se of three individual samples,* p<0.05 is significant (control: c. experimental: e) table 2. alterations in the ros variables of cirrhinus mrigala exposed to cerium oxide during short term exposure. ros variables exposure duration (in h) 24 48 72 96 c e c e c e c e sod (u/ml protein) 5.11±0.43 3.37±0.26* 5.23±0.12 4.04±0.35* 5.01±0.27 4.17±0.33* 5.93±0.65 3.53±0.41 cat (µmol/ml protein/min) 5.54±0.12 3.44±0.26* 5.98±0.11 3.97±0.25* 5.43±0.33 2.68±0.42* 4.95±0.73 3.74±0.32* nmol of mda/ mg protein) 6.28±0.25 7.34±0.15* 6.77±0.27 7.08±0.58 6.14±0.15 7.34±1.12* 5.99±0.43 7.93±0.68* all values are expressed as mean± se of three individual samples,* p<0.05 is significant. 371 int. j. aquat. biol. (2019) 7(6): 368-373 2018). similar results were reported which are ascribed to the changes in carbohydrate metabolism, on account of glycogenolysis in freshwater fish (ghelichpour and mirghaed, 2019). boost in glucose levels during metals exposure have also been reported due to synthesis of glucose from extra hepatic tissue amino acids (almeida et al. 2001; banaee et al., 2019c;). many reports also related increase in plasma glucose levels to discharge of glucocorticoids and catecholamine`s from adrenal tissues of fish during stress incidents (klaper et al., 2010). the protein concentration decreased in the treated fish as duration of exposure was increased. the reduced protein levels are correlated to metal binding to blood and tissue proteins culminating in tissue injury and oxidative stress. it also causes changes in physicochemical properties which are evident in protein structure conformational change (chen et al., 2011), and this might also be the basis of decline in protein concentration in this investigation. toxicity of metals might also be the rationale for declined protein as it inhibits transcription and translational processes. the detection of transaminases like aspartate transaminase (ast/got) and alanine transaminase (alt/gpt) in blood have been clinically employed to identify with any tissue damage. presence of these biomarkers can also be coupled to the enzyme inhibition in metabolic pathways upon exertion of stress. there was a notable increase in both alt/ast concentrations in treated fishes. these can be attributed to the tissue and organ damage caused by exposure to ceo2 and other toxicants (abhijith et al., 2016). accordingly, indicating the potential role of transaminases as chief biomarkers during exposure to toxicants and ensuing in metabolic stress. ast and alt are indicators in liver function tests, as ast is synthesized by liver hepatocyte and typically found in liver and heart, while alt primarily is present in liver and kidney. subsequently, higher activity of these enzymes has been reported in fish exposed to pesticides (rao, 2006). the raise in ldh can be ascribed to amplified glycolysis upon metabolic stress. furthermore, the onset of anoxia is also an important reason for increased ldh activity. the prevalence of anaerobic conditions due to metal toxicity is also reported to rise in ldh activity (min and ju-chan, 2008). the result represents an increase in brain glutamate in treated fish. this might be coupled with excitotoxicity and excessive production of glutamate, which results in damage and death of nerve cells. the increased glutamate concentration makes alterations in the brain physicochemical environment which activates the glutamate receptors by allowing a high concentration of calcium ions to enter the cell (manev et al., 1989). figure 1. alterations in the brain glutamate level of cirrhinus mrigala exposed to ceo2 upon short term exposure (all values are expressed as mean±se of three individual samples,* p<0.05 is significant) 372 çiçek et al./ the effect of ceo2 on cirrhinus mrigala the generation of ros is the reason for oxidative stress, which agitates the biological processes by disconcerting homeostasis (banaee et al., 2019a, b). this outcome is imbalance of equilibrium of the detoxification mechanism of ros (manke et al., 2013). cells supply the range of enzymatic and nonenzymatic responses to scavenge the excessive generation of ros. the pro-oxidant uniqueness of metals causes ros generation during mitochondrial respiration and thus activating nadph-like enzymes. enzymes like sod and cat play a principal role in scavenging ros formed during modifications of metabolic (raisi et al., 2018) and physiological processes (mirghaed et al., 2018). sod has been reported to be the primary and instantaneous response to oxidative stress in a biological system (winston and di giulio, 1991). this is owing to the generation o2 and their conversion to h2o2, which may additionally lead to oxidation of cysteine in the antioxidant enzyme, thus reassuring ceo2 toxicity. lipid peroxidation (lpo) is the primary culprit in the interruption of cell structure and function due to unwarranted generation of reactive oxygen species (huang et al., 2003; safari, 2016). the lipid peroxidation mechanism is assessed by measuring the malondialdehyde (mda) concentration, which is one of the end products of the breakdown of lipids due to peroxidation. the observation suggests the existing cellular defense mechanism was not competent to ward off the oxidative damage. all the above mentioned analysis suggests the bioaccumulation of ceo2. the study proposes that the release of cerium oxide to the aquatic environment may lead to detrimental effects on aquatic animals and the human population who are reliant on aquatic products as source of diet. ethical approval: all experiments performed involving animals were following the ethical standards of the institution and fish from which biological samples were collected and held in the laboratory via approval iaec committee (permit caf/ethics/648/2018). this article does not contain any studies with human participants performed by any of the authors. conflict of interest: the authors declare that they have no conflict of interest. references abhijith b.d., ramesh m., poopal r.k. 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(2020) 8(2): 143-147 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology review article alkaline phosphatase activity as a biochemical biomarker in aqua-toxicological studies mahdi banaee*1 aquaculture department, faculty of natural resources and the environment, behbahan khatam alanbia university of technology, behbahan, iran. s article history: received 16 january 2020 accepted 20 april 2020 available online 2 5 april 2020 keywords: detoxification, alkaline phosphatase, interfering factors, aquatic organisms. abstract: alkaline phosphatase (alp) is a glycoprotein with a metallophosphatase structure that catalyzes the hydrolysis of monophosphate esters of biomolecule esters at alkaline ph. alp activity is a useful bioindicator to assess the physiological health of cellular membranes, cell growth, apoptosis and cell migration, cellular metabolic status, hepatocyte function, and detoxification activity in hepatocytes. alp activity is detected in a colorimetric method using the para-nitrophenyl phosphate substrate (p-npp) at a wavelength of 405 nm in biological samples. cell hemolysis, especially erythrocytes; increased levels of sex hormones and corticosteroids, biological infections, and poor nutrition can adversely affect alp activity. introduction alkaline phosphatase (alp) is a metalloenzyme that is anchored to the cell membrane by a glycosylphosphatidylinositol (gpi) (atkins et al., 2011). alp is a glycoprotein with an external domain and a catalytic domain with a serine base. the activity of alp depends on the binding of two zn+ 2 ions and one mg+2 ion to the active site of the enzyme. the serine base can inhibit the activity of the alp enzyme (banaee and taheri, 2019). therefore, alp activity depends on the concentration of these ions in cells and intercellular fluids (banaee et al., 2011; nafisi bahabadi et al., 2014; soleimany et al., 2016). therefore, gender, health status, ontogeny (kim et al., 2001), oral supplements administration (taheri et al., 2017; gholizadeh zare tavana et al., 2018), or exposure to pollutants, and environmental factors can have a significant effect on alp activity (woo et al., 2018). alkaline phosphatase plays a vital role in phosphate hydrolysis and membrane transfer (derikvandy et al., 2020), and is also involved in transphosphorylation of many biomolecules, such as nucleotides, proteins, and alkaloids in alkaline ph *correspondence: mahdi banaee doi: https://doi.org/10.22034/ijab.v8i2.880 e-mail: mahdibanaee@yahoo.com (nematdoost haghi and banaee, 2017). the activity of this enzyme is essential for cell growth, planned cell death (apoptosis), and cell migration (sharma et al., 2014; banaee et al., 2019c). alp activity often increases during differentiation of b lymphocytes to plasmocytes (akcakaya et al., 2007). alkaline phosphatase is often synthesized in the liver, bones, and to a lesser extent, in the intestine and kidneys (telega, 2018). additionally, the alp gene is expressed in the endothelial blood vessels and the brain tissue of fish (goishi and klagsbrun, 2004). alp activity is essential for bone mineralization (atkins et al., 2011). so far, 12 isoenzymes from alp have been identified that may have different origins and synthesis sites. alkaline phosphatase plays a vital role in glycogen metabolism (kim et al., 2001). the activity of this enzyme in hepatocytes can inactivate phosphorylase and increase the rate of glycogen breakdown in the liver (raisi et al., 2018; rezaei shadegan and banaee, 2018). increased levels of synthesis and alp activity can play an essential role in providing energy for stressed cells (agrahari et al., 2007; saha and kaviraj, 2009). 144 banaee / alkaline phosphatase activity as a biochemical biomarker purpose of measuring alp activity: alkaline phosphatase is a non-specific indicator for the diagnosis of liver and bone damage. furthermore, alp is an excellent biological indicator for assessing cellular stress (banday et al., 2019). it plays a vital role in the xenobiotic detoxification process in hepatocytes (vaziryan et al., 2017; hatami et al., 2019). alp activity measurement method: alkaline phosphatase activity is measured by a quantitative colorimetric method using the para-nitrophenyl phosphate substrate (p-npp) at a wavelength of 405 nm (moss and henderson, 1999). reasons for increased alp activity: tumor formation in the liver and bone tissue, hyperthyroidism, bile duct obstruction, liver nodules, liver cysts, liver failure, and increased alp biosynthesis rates in hepatocytes, and intestinal dysfunction can lead to increased alp activity in the serum or plasma. for example, alp activity in rainbow trout, oncorhynchus mykiss, and cyprinus carpio fish plasma increased significantly after exposure to diazinon (banaee et al. 2011, 2012). increased alp activity in the body tissues of freshwater fish of alburnus sellal exposed to fenpropathrin indicates damage to cell membranes (banaee et al., 2014). additionally, damage to the cell membrane led to the release of alp into the fish plasma exposed to titanium dioxide nanoparticles (banaee et al., 2019b). increased alp activity in fish hepatocytes exposed to dimethoate and bacillar fertilizer may also be a physiological response to increased glycogen breakdown in the liver (rezaei shadegan and banaee, 2018). nematdoost haghi and banaee (2017) and wan do et al. (2019) stated that damage to the hepatocyte membrane, gallbladder ducts, intestinal epithelial cells, and renal ducts increased alp in fish plasma exposed to microplastics and paraquat. banaee et al. (2019b) reported an increase in alp activity in plasma of fish exposed to paraquat and nano-tio2. research shows that sometimes pollutants may increase alp activity in cells by affecting the process of transphosphorylation in cells (banaee et al., 2019a). increased alp activity in cells of c. carpio following oral exposure to zinc oxide nanoparticles may be related to the dysfunction of cells (taheri et al., 2017). furthermore, an increase in the production rate of reactive oxygen species (ros) may elevate alp activity (banday et al., 2020; banaee et al., 2017). woo et al. (2018) observed elevated activity of alp in plasma of c. carpio exposed to trichlorfon (woo et al., 2018). an increase in alp activity was observed after exposure of clarias gariepinus to deltamethrin (amin and hashem, 2012; eni et al., 2019), cypermethrin (eni et al., 2019), heavy metals (banday et al., 2019). similar results have been reported after exposing oreochromis niloticus to deltamethrin (abdel-daim et al., 2015; abdelkhalek et al., 2015), diazinon (abdelkhalek et al., 2017). an increase in alp activity was observed in the blood of channa punctatus exposed to monocrotophos (agrahari et al., 2007). reasons for decreased alp activity: alp activity may be reduced by malnutrition, especially protein, zinc, magnesium and vitamin c deficiency, and increased vitamin d levels in the diet. anemia and hypothyroidism can lead to decreased alp activity. severe cellular necrosis can be another reason for decreased alp activity in fish plasma (banaee et al., 2016). moreover, disturbance in the transport of zinc, magnesium, and phosphorus ions from cell membranes can lead to decreased alp activity (banaee et al., 2019c; banaee et al., 2020). decreased absorption of essential ions for alp biosynthesis can lead to decreased enzyme activity (veerappan et al., 2012). besides, the interaction of environmental pollutants with alp can reduce its activity (banaee et al., 2019a; b). for example, decreased alp activity in gill cells of the rainbow trout exposed to cadmium chloride may be due to the effect of cadmium on cell membranes (evaz-zadeh samani et al., 2017). exposure of c. carpio to cd reduced alp activity in muscle tissue (banaee et al., 2015). decreased plasma alp activity may be due to damage to erythrocyte membranes and their hemolysis (farah et al., 2012). vaziryan et al. (2017) suggested that reduced alp activity may be associated with impaired alp biosynthesis, hepatic cell necrosis, and damage to the intestinal epithelial cells of fish exposed to oral 145 int. j. aquat. biol. (2020) 8(2): 143-147 aflatoxins. decreased alp activity may affect bone marrow cell activity (prins et al., 2014). decrease in alp activity was reported in serum of rhamdia quelen fed with a feed artificially contaminated with aflatoxin b1 (anater et al., 2020). interventional factors in measuring alp activity: sex hormones (ahmad et al., 2002), viral and bacterial infections (banaee et al., 2017; xia et al., 2017), parasites infection, administration of antibiotics (gora et al., 2018), dietary supplements, hemolysis of blood cells (farah et al., 2012) and feeding fish with oxidized and fatty foods can interfere with alp activity in blood or tissue samples. conclusion measurement of the alp activity in the intercellular and extracellular fluids can be indirectly useful for evaluating the membrane function of cells exposed to xenobiotics. interaction of environmental pollutants with the biochemical structure of alp or lipid peroxidation of cell membranes can cause fluctuations in alp activity in cells or extracellular fluids. therefore, each change in alp activity outside the normal range can indicate stress on the cells. references balon e.k. 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(2014) 2(3): 147-154 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article structural characterization of the olfactory epithelium of freshwater olive barb, puntius sarana (hamilton, 1822) saroj kumar ghosh, padmanabha chakrabarti*1 fisheries laboratory, department of zoology, the university of burdwan, burdwan-713 104, west bengal, india. article history: received 30 may 2014 accepted 17 june 2014 available online 2 5 june 2014 keywords: olfactory epithelium function histoarchitecture surface architecture abstract: the cellular organization of the olfactory epithelium of puntius sarana (hamilton, 1822) was explored by means of optical and scanning electron microscopy. the oval shaped olfactory rosette was composed of 26-28 primary lamellae distributed from both sides of the central raphe. the sensory epithelium confined chiefly on the linguiform processes of the lamella and rest of the portion consisted of non-sensory epithelium. the sensory epithelium was embossed with morphologically distinct three types of sensory cells: ciliated, rod and microvillus receptor cells. the non-sensory epithelium was made up of mainly stratified epithelial cells and mucous cells. different cells lining the olfactory epithelium were discussed in relation to mode of life and living of the fish concerned. introduction the olfactory organs are of special attention in fish biology because they are basically chemoreceptors, efficient to ascertain water soluble compounds and afford the fish with information about the nearby environment. in fish, water with dissolved chemicals flow from anterior to posterior nostril over the olfactory organ, covered with olfactosensory epithelium which performs an important role in olfaction (hara, 1971). olfactory information is extremely important in the lives of fishes including feeding, prey detection, predator avoidance, species and sex recognition, and migration (camacho et al., 2010). teleostean olfactory organs exhibit a respectable diversity, reflecting different developmental strategies and ecological habitats (zeiske et al., 1992). the microstructure and function of olfactory organ in several fishes have been widely described by several workers (mandal et al., 2005; sawad et al., 2006; bhute and baile, 2007; kumari, 2008; ma and wang, 2010; chakrabarti and ghosh, 2011; ghosh and * corresponding author: padmanabha chakrabarti e-mail address: dr.pchakrabarti@yahoo.in chakrabarti, 2012; 2013; 2014). however, no attempt has been made to correlate the functional significance of various cells lining the olfactory epithelium in relation to the species feeding adaptations. puntius sarana (cypriniformes, cyprinidae) is freshwater, omnivorous, bottom dwelling teleost and distributed throughout the indian subcontinent (chetia borah, 2012). its food primarily consists of algae, unicellular and single celled animals, larvae of aquatic insects and sands (shafi and quddus, 2001). the aim of the present work is to examine the distribution of different cell types and their functional aspects in the olfactory epithelium of important food fish, p. sarana by histological and ultrastructural examination. materials and methods living mature specimens of p. sarana (14 to 16 cm in total length) were obtained from the local freshwater body of burdwan, west bengal, india. the specimens were anaesthetized with tricane 148 ghosh and chakrabarti/ structure of olfactory epithelium in puntius sarana methone-sulphonate (ms 222; sigma chemical co.) solution (100 mg/l) and sacrificed following the guidelines given by the institutional ethical committee. the olfactory organs were carefully dissected out from the floor of the nasal cavity under a stereo microscope and immediately processed for the histological and scanning electron microscopic studies. histological study: the olfactory tissues were fixed in aqueous bouin’s fluid for 16-18 hour. after fixation the tissues were washed repeatedly in 70% ethanol and dehydrated properly through ascending series of ethanol. then they were cleaned with xylene and embedded in paraffin wax of 56-58°c under a thermostat vacuum paraffin-embedding bath for a period of 1 hour and 30 minute. serial sections were cut at 4 µm thick using a rotary microtome (weswox). after routine histological procedure deparaffinized sections were stained with delafield’s haematoxylin-eosin (he) and mallory’s triple (mt) stain. the prepared histological sections were examined and photographed by olympus-tokyo pm-6 compound microscope. scanning electron microscopic (sem) study: the olfactory rosettes were perfused in vivo with 2.5% glutaraldehyde solution in 0.1 m na-cacodylate buffer (ph 7.4) for 15 minute. the entire olfactory rosettes were dissected out and rinsed with heparinised saline (heparin sodium salt 10,000 iu dissolved in 0.67% nacl solution) and 1% tween 40 mixture to remove the adhering mucus. after rinsing in 0.1 m na-cacodylate buffer, the tissues were again fixed with 2.5% glutaraldehyde in 0.1 m nacacodylate buffer (ph 7.4) for 24 hour at 4°c. after primary fixation the tissues were rinsed in the same buffer and post-fixed in 1% osmium tetroxide in 0.1 m na-cacodylate buffer (ph 7.4) for 2 hour. the tissues were washed thoroughly in the same buffer and dehydrated through graded series of ethanol followed by acetone and isoamyl acetate. the samples were dried with critical point drier (hitachi 8cp2), mounted on metal stubs, coated with gold palladium by vacuum gold coater. the tissues were examined under a hitachi s-530 scanning electron microscope. results according to sem observations, the olfactory rosette of p. sarana is nearly oval in outline with a convex ventral and a concave dorsal appearance. the olfactory rosette consists of a series of 26 to 28 more or less flattened lamellae, distributed in a feather like style on both sides of the central raphe and occupies the entire cavity of the olfactory chamber (fig. 1). the outer edges of the lamellae are adhered to the olfactory chamber, while the inner edges are attached to the median raphe. the lamellae are capacious in the middle portion of the rosette and diminish in sizes towards the preceding and succeeding ends. the middle dorsal part of the lamella is characterized by free well-developed linguiform process, grows from the distal edge (fig. 1). the sensory epithelium is restricted mainly on the linguiform processes of the lamella, while rest of the portion is consisted of non-sensory epithelium. histologically, the olfactory lamellae are radiated from raphe and composed of two layers of thick olfactory epithelium separated from the narrow central lamellar space, central core by a welldeveloped basement membrane (fig. 2). the central core is packed with connective tissue fibers, nerve fibers and blood vessels (figs. 3, 4, 8). the sensory figure 1. photomicrographs of the olfactory epithelium of p. sarana. oval shaped olfactory rosette exhibits olfactory lamellae (ol) radiating from central median raphe (r). arrows indicate linguiform processes of ol (sem × 50). 149 int. j. aquat.biol. (2014) 2(3): 147-154 epithelium is typified with mainly receptor cells having long dendrite reaching up to the free epithelial surface and few rod cells and microvillous cells (fig. 3). rod cells are ovoid in appearance, extend as a spike like texture and characterized with basally situated deeply stained nuclei. microvillous cells are situated in more superficial layer of the epithelium, possess a round nucleus and without cilia (fig. 3). receptor cells are the sensory elements of the olfactory epithelium and differentiated into primary and secondary receptor cells. the primary receptor cells are highly basophilic with almost rounded and deeply stained nuclei in the distal portion of the cell. the dendrite process of each receptor cell runs to the superficies of the lamellae as a spare cylindrical process (fig. 4). the secondary receptor cells are distributed below the primary receptor cells and marked by their oval and elongated nuclei. the axonal ends of primary receptor cells synapse with the dendrite tips of secondary receptor cells (fig. 4). under sem observations, the linguiform process of olfactory lamella has a spongy structure due to the appearance of compact ciliated receptor cells (fig. 5). the figure 2. histological sections of the olfactory epithelium of p. sarana. olfactory lamellae (ol) based on raphe (r) shows olfactory epithelium (oep) separated from central core (cc) by basement membrane (mt × 100). figure 3. histological sections of the olfactory epithelium of p. sarana. sensory olfactory epithelium (oep) lined with receptor cells (rc) (solid arrows), rod cells (arrow heads) and microvillous cells (broken arrows). note the presence of blood vessels (bv) in cc (he × 400). figure 5. photomicrographs of the olfactory epithelium of p. sarana. sensory epithelium exhibits bunch of ciliated receptor cells (rc) (sem × 3500). figure 4. higher magnification of sensory olfactory epithelium (oep) of p. sarana shows the arrangement of primary receptor cells (solid arrows) and secondary receptor cells (broken arrows). note presence of blood vessels (bv) in the central core (cc) (mt × 1000). 150 ghosh and chakrabarti/ structure of olfactory epithelium in puntius sarana receptor cells are distinguished by the distal free end of the dendrites and categorized into ciliated, rod and microvillous receptor cells. ciliated receptor cells are furnished by tuft of long cilia, rise to the epithelial surface and dominant over both rod cells and microvillous cells (fig. 6). the rod cells are few in number and their apical end protrude as a simple rod like structure, distributed randomly in the epithelial surface. the microvillous cells are embossed with tuft of microvilli and dipped into the thickness of the ciliated receptor cells (fig. 6). the transitional zone of sensory and non-sensory figure 6. photomicrographs of the olfactory epithelium of p. sarana. sensory epithelium displays dendrite processes of receptor cells (rc), rod cells (solid arrows) and microvillous cells (arrow heads) (sem × 4000). figure 7. photomicrographs of the olfactory epithelium of p. sarana. transitional zone of sensory and non-sensory epithelium shows ciliated receptor cells (rc), microvillous cells (broken arrows), stratified epithelial cells (sec) with complex microridges and mucous cells (solid arrows). note mucin droplets over the sec (sem × 4500). figure 9. photomicrographs of the olfactory epithelium of p. sarana. surface epithelium of raphe characterized with stratified epithelial cells (sec) having labyrinth pattern microridges. note the presence of opening of mucous cells (solid arrows) in between sec and mucin mass (arrow heads) over the sec (sem × 4000). figure 8. histological sections of the olfactory epithelium of p. sarana. non-sensory olfactory epithelium (oep) shows mucous cells (solid arrows) and stratified epithelial cells (arrow heads). note presence of blood vessels (bv) in the central core (cc) which is detached from oep by basement membrane (bm) (he × 400). 151 int. j. aquat.biol. (2014) 2(3): 147-154 epithelium is provided with stratified epithelial cells intercalated with the opening of mucous cells leaving microvillous cells adjacent to the ciliated receptor cells (fig. 7). histologically, the non-sensory epithelium is lined with chiefly stratified epithelial cells with prominent basophilic nuclei and mucous cells (fig. 8). under sem study, the non-sensory epithelium and raphe is represented by tightly packed stratified epithelial cells having labyrinth pattern microridges and opening of mucous cells. secreted mucin masses are placed over the microridges of stratified epithelial cells (fig. 9). discussion olfactory mucosa comprising the sensory neurons is typically placed on the floor of the olfactory chamber, which is frequently folded, forming olfactory lamellae (hara, 1975). the feeding habits of fishes are reflected on the structure and cellular organization of the olfactory organ (hara, 1994). the oval shaped olfactory rosette of p. sarana with two rows of olfactory lamellae present on both side of the median raphe belongs to burne’s (1909) rosette column i or bateson’s (1889) rosette type iii. teichmann (1954) classified this oval type of olfactory organ under the category of eye-nose fish, which means that this kind of fish possess similarly developed optic and olfactory senses. the olfactory organ of p. sarana holds 26-28 lamellae arranged on both left and right side of the rosette, adapted maximum to the space availability and is similar to other cyprinid olfactory organ can be placed to the type vi by yamamoto and ueda (1979). the multilamellar arrangement increases the sensitivity and efficiency of the olfactory organ (zeiske et al., 1976). the distribution of sensory and non-sensory areas in the olfactory epithelium is variable among teleosts (yamamoto, 1982). in p. sarana the sensory epithelium is bounded in the free linguiform processes, while the rest portion of lamellae is comprised of non-sensory epithelium. this disposition may be due to the fact that the linguiform process with sensory cells faces the flow of incoming water current and the receptor cells interact with the water soluble chemicals during olfaction. similar findings were also reported by ojha and kapoor (1973) in the olfactory apparatus of labeo rohita. the most interesting characteristics of the olfactory epithelium of p. sarana is the histological existence of secondary receptor cells in addition to primary receptor cells and the presence of synaptic connection between these two kinds of receptor cells. this is in conformity with the findings of goel (1978) in the olfactory epithelium of notopterus notopterus. buck and axel (1991) reported that olfaction commences at the apical tip of the receptor cells. in the present observation, the sensory epithelium chiefly consists of three morphologically distinct types of receptor cells: ciliated, rod and microvillous cells; intermingled in different proportions. the ciliated receptor cells correspond to the type i cells of yamamoto and ueda (1978), the rod cells similar to the type iv cells of ichikawa and ueda (1977), whereas the microvillous cells are harmonized with the type ii cells of muller and marc (1984). the present study reveals that the ciliated receptor cells dominating over the rod cells and microvillous cells. the cilia are the site of transduction process, stimulated by odour bearing substances and also enable the fish to detect food. sparsely distributed rod cells in the olfactory epithelium of p. sarana are considered as a sensory receptor cells. yamamoto (1982) opined the rod cells as a sign of aging of ciliated receptor cells and terminate with single cilium at the free surface of the epithelium. reite and evensen (2006) believed that these are non-specific immune cells, rolled up in immunity as their number proliferates during parasitic infection. however, on the basis of experimental work and developmental studies, zielinski and hara (1988) and moran et al. (1992) have established that the rod shaped process of these cells represent the dendritic apical processes of olfactory receptor cells. in p. sarana the microvillous receptor cells probably form a different olfactory transduction mechanism for pheromones in the regulation of reproductive activities. this is in compliance with the findings of biju et al. (2003) in 152 ghosh and chakrabarti/ structure of olfactory epithelium in puntius sarana the olfactory epithelium of cirrhinus mrigala. bhute and baile (2007) also advocated that the microvillous receptor neurons perceive and process signals of pheromone, which is an important step of breeding in labeo rohita. on contrary bakhtin (1977) and bannister (1965) considered that microvillous cells in the olfactory surface of squalus acanthias and teleostean fishes are predecessors of ciliated receptor cells. in the transitional zone of sensory and non-sensory epithelium few ciliated receptor cells in between stratified epithelial cells are responsible for better monitoring of the water quality even up to this zone. in p. sarana, the non-sensory cells lining the olfactory epithelium may help in mechanical assist of sensory cells. furthermore, the non-sensory epithelium and the raphe consist of stratified epithelial cells provided with labyrinth pattern microridges on their apical surface. such microridges may enhance the epithelial surface and serve as holding the mucus film over the epithelial membrane and protect the epithelium from mechanical abrasion. the mucin secreted by mucous cells over the lamellae probably forms a suitable medium for diffusion of odorants. the mucus layer may also help in ion trap, which obstructs the penetration of salts and heavy metals to underlying organs (banerjee, 1993). in addition the mucin secreted from the mucous cells of raphe probably helps the smooth flow of water through the olfactory chamber by binding microscopic debris which is ejected through the posterior nostril. this is in confirmed with the findings of bandyopaghyay and datta (1998) in the olfactory organ of heteropneustes fossilis. acknowledgement the authors are grateful to dr. a. basu, head of the department of zoology, the university of burdwan for providing laboratory facilities and thankful to dr. s. chakraborty, the scientist-in-charge of the university science instrumentation centre, the university of burdwan, for his technical support in sem analysis. references bakhtin e.k. 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(2014) 2(4): 193-200 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article the relationship between morphological and reproductive status in suitable russian sturgeon, acipenser gueldenstaedtii (brandt and ratzeburg, 1833) broodstocks bahram falahatkar*,1javid imanpour fisheries department, faculty of natural resources, university of guilan, sowmeh sara, p.o. box 1144, guilan, iran. article history: received 8 july 2014 accepted 11 august 2014 available online 2 5 august 2014 keywords: russian sturgeon artificial breeding morphometric parameters broodstock selection abstract: owing to reduction of sturgeon stocks in various water bodies, artificial propagation has become significantly important for stock restoration and appropriate broodstock selection is vital in this process. selection of suitable broodstocks may influence the quality and quantity of obtained eggs and larva. the present study aimed to examine correlation between some morphometric and reproductive parameters to find suitable brood fish for artificial reproduction in russian sturgeon, acipenser gueldenstaedtii. forty fish free of any external disease symptom and abnormality were selected for the study. following biometric measurements including weight, total length, abdominal girth, pv (distance between pectoral and pelvic fins), lx (distance between anal and caudal fins), polarization index (pi), gonadosomatic index, absolute fecundity, and fertilization rate, correlations between these morphometric and biological characteristics with emphasis on breeding were calculated. there was higher correlation between weight-absolute fecundity and length-pv in fish responded to artificial breeding, while correlation between age-pv, length-pv and weight-abdominal girth were higher in those fish not responded to artificial breeding. the results suggests that it is quite possible to select suitable russian sturgeon spawners for artificial propagation based on combination of body weight, lx, pv, age, abdominal girth and total length, however the most useful criteria for the selection seems to be precise measurement of the polarization index. introduction the caspian sea is the most important habitat and home for five sturgeon species including beluga (huso huso), ship (acipenser nudiventris), stellate (a. stellatus), persian (a. persicus) and russian sturgeon (a. gueldenstaedtii). over-fishing, habitat and spawning ground degradation along with environmental pollution have caused these species to be included in the iucn red list of endangered and consequently in cites appendices (iucn, 2012). several governments and organizations have considered conservation and rehabilitation of sturgeon stocks and various restoration programs, e.g., allocation of catch quota on the base of stock assessment programs, restoration of natural habitats and spawning grounds and developing artificial * corresponding author: bahram falahatkar e-mail address: falahatkar@guilan.ac.ir breeding strategies. artificial propagation is a regular practice to produce and release sturgeon fingerlings into the caspian sea (falahatkar et al., 2011). although russian sturgeon is important for meat and caviar production, nevertheless its artificial propagation in the southern caspian sea basin has dramatically been hampered by lack of suitable spawners. in addition, large numbers of spawners do not respond efficiently to artificial induction which leads to failure of propagation processes. ovulation in fish sometimes is unpredictable and failed because of state of gonad development and handling stress during transportation and maintenance in hatchery (schreck, 2010; williot et al., 2011). due to high fecundity and survival rate of the obtained larvae in 194 int. j. aquat. biol. (2014) 2(4): 193-200 russian sturgeon, precise determination of gonad developmental stage will lead to an increase in induced ovulation and ultimate enhancement in hatchery production (hurvitz et al., 2008). several studies have revealed that sex steroids are appropriate components for determination of maturity stages of brood fish (ceapa et al., 2002; webb et al., 2002; alberto et al., 2008; davailcuisset et al., 2008; petochi et al., 2011). since blood sampling is considered as an invasive technique to determine sex and maturity in sturgeon (wildhaber et al., 2007), introduction of a method to enables us effectively monitor gonad development in spawners is crucial for field and hatchery practices (falahatkar et al., 2011). morphometric parameters of ovarian follicles have been used for selection of suitable broodstock in artificial breeding (see goncharov et al., 1999). although this method is not as precise as physiological methods, however it might be very useful where laboratory facilities are not available. the most potent way to assess the reproductive potential of female sturgeon prior to hormonal injection is determining the polarization index and in vitro maturation competence of ovarian follicles (lutes et al., 1987; williot et al., 1991). in present study, several morphometrical and sexual indexes were examined to highlight correlations between those parameters. this helps us to determine the reliable parameters to predict ovulation to select suitable spawners in final stage of maturation. materials and methods fish: forty russian sturgeons (27 females and 13 males) were caught at sturgeon fishing stations (turkmen and miankaleh bay) by gill-net in the southern caspian sea basin during the winter season and transported (1.5 h) by a truck equipped with aeration to the shahid mardjani sturgeon hatchery center in golestan province, iran. the weight, length and age range of females were 10-39 kg, 112.5-167 cm, and 11-18 years old, respectively and were in range of 10-22 kg, 122-153.5 cm, and 10-15 years old, respectively, for males. maintenance and artificial breeding: fish were kept under natural fluctuations over winter temperature and photoperiod. the collected fish were kept in broodstocks maintenance facilities in concrete ponds prior to oocyte examination from 2-6 days according to their fishing date. during the appropriate water temperature (march) for artificial breeding of russian sturgeon, oocyte samples were taken by a special probe (chebanov and galich, 2011) to determine polarity index. approximately, 10-15 eggs were sampled and put into 4% formaldehyde solution for later examination. for this purpose, eggs were put in boiled water for 2-3 minutes. delicate sections were made across the animal to vegetal poles and polarization index (pi) was determined according to dettlaff et al. (1993). the core idea is to estimate the stage of germinal vesicle migration under a dissecting microscope. based on the obtained pi, 10 broodstocks were suitable for hormonal induction and underwent hormonal treatment using common carp pituitary extract (males 40-60 mg; females 50-70 mg). females were received intramuscular injection of two separate doses (10% as priming and 90% as resolving doses) in 12 hours interval (pourasadi et al., 2009; falahatkar and efatpanah, 2011) at 14.515.5°c. ovulation occurred within 22-26 h of resolving injection. all 13 caught males were received a single injection and spermiation occurred within 18-20 h. sperm from 5 males were used for fertilization. oocytes were collected traditionally by opening the abdomen (pourasadi et al., 2008). eggs were fertilized using 10 ml of milt diluted to 1:100 with addition of hatchery water. following 5 min of fertilization, eggs were mixed with 1:10 clay suspension and softly stirred for 40 min until they were no longer sticky (pourasadi et al., 2008). eggs were then washed and transferred to youshchenko incubator. morphometric and sex parameters: fish weight (kg ± 50 g), total length (cm ± 1 mm), condition factor (cf = weight/total length3 × 100), abdominal girth (cm ± 1 mm), pv (the distance between pectoral fin to pelvic fin; cm ± 1 mm), lx (the distance between 195 falahatkar and imanpour/ russian sturgeon broodstocks selection according to morphological features anal fin to caudal fin; cm ± 1 mm), ovary weight (kg ± 10 g), gonadosomatic index (gsi = gonad weight/body weight × 100) and age were determined for all broodstocks. weight of 100 oocytes and absolute fecundity (number of oocyte/fish) were determined in broodstocks responded to hormonal injection. for estimation of fish age, the first pectoral fin ray was sampled. a thin slide of 0.5-1 mm thickness was prepared and after putting into the glycerin solution for 24 h, the cross-section detail was studied using stereoscope (hughes et al., 2005). absolute fecundity was determined by below formula: absolute fecundity = number of eggs per gram × total weight (g) of ovaries (eggs) after transferring the fertilized eggs to the incubators, the overall fertilization rate at blastula stage was recorded with collecting 200-300 eggs and counting live and dead eggs. statistical analyses: data were analyzed using spss (version 12; chicago, il) to examine correlation between parameters and success of artificial reproduction. normality of data and homogeneity of variances were tested by kolmogorov-smirnov and levene’s tests, respectively. the independent samples t-test was used to compare fish responded to hormonal treatment and those indifferent to hormonal treatment at 95% confidence level. results correlations of measured parameters in responsive and non-responsive broodstocks to hormonal treatment are presented in tables 1 and 2. there were no significant correlation between fertilization rate and any measured indexes in responsive females. regarding pi, a significant correlation was found only with age (r = 0.634, p = 0.049). significant correlation was also observed between weight and absolute fecundity (r = 0.874, p = 0.001), length and pi (r = 0.740, p = 0.014), egg weight and fish length (r = -0.782, p = 0.07), egg weight and age (r = -0.685, p = 0.029), pv and lx with age (r = 0.721, p = 0.019; r = 0.680, p = 0.031, respectively) in broodstocks responded to artificial breeding. fertilization rate pi absolute fecundity gsi abdominal girth lx pv age cf total length weight -0.188 0.409 0.874** -0.205 0.223 0.656* 0.619 0.415 0.623 0.647* total length 0.536 0.740* 0.555 -0.477 -0.123 0.830** 0.907** 0.803** -0.186 cf 0.168 -0.160 0.584 0.279 0.442 -0.018 -0.119 -0.288 age -0.222 0.634* 0.229 -0.612 -0.364 0.680* 0.721* pv -0.187 0.605 0.441 -0.447 0.112 0.840** lx -0.249 0.511 0.497 -0.503 -0.129 abdominal girth -0.170 -0.135 0.313 0.546 gsi 0.007 0.024 0.200 absolute fecundity -0.375 0.573 pi -0.299 pi: polarization index; gsi: gonadosomatic index; lx: the distance anal fin to caudal fin; pv: the distance between pectoral fin up to the pelvic fin; cf: condition factor. * correlation is significant at the 0.05 level; ** correlation is significant at the 0.01 level. table 1. correlation (r) of measured parameters in russian sturgeon broodstocks responded to hormonal injection (n = 10). pi abdominal girth lx pv age cf total length weight 0.298 0.933** 0.697** 0.885** 0.905** 0.501* 0.846** total length 0.111 0.665** 0.774** 0.953** 0.918** -0.013 cf 0.539 0.696** 0.023 0.114 0.190 age 0.121 0.783** 0.780** 0.939** pv 0.124 0.732** 0.726** lx 0.029 0.542* abdominal girth 0.473 pi: polarization index; lx: the distance between the anal fin the caudal fin; pv: the distance the pectoral fin to pelvic fin; cf: condition factor. * correlation is significant at the 0.05 level; ** correlation is significant at the 0.01 level. table 2. correlation (r) of measured parameters of russian sturgeon broodstocks not responded to hormonal injection (n = 17). 196 int. j. aquat. biol. (2014) 2(4): 193-200 in non-responsive females to hormonal treatment no correlation was observed between pi and any measured parameters, while significant correlations were observed between age and pv (r = 0.939, p<0.000), length and pv (r = 0.953, p<0.000), age and lx (r = 0.780, p<0.000) and weight and abdomen girth (r = 0.933, p<0.000). mean weight of responsive spawners (10 specimens) to artificial breeding was 22 ± 4.6 kg and those nonresponsive (17 specimens) was 22.1 ± 7.5 kg. there were no significant differences between these two groups (p>0.05). no significant difference was figure 1. regression of fertilization rate with weight (a), total length (b), condition factor (c), age (d), abdomen girth (e), gonadosomatic index (f) and polarization index (g) of russian sturgeon broodstocks (n = 10) in southern basin of the caspian sea which responded to hormone injection. 197 falahatkar and imanpour/ russian sturgeon broodstocks selection according to morphological features noticed between total length, cf, pv, lx, abdomen girth and age (p>0.05), while pi and gsi were significantly different in two groups (p<0.05; table 3). figure 1 (a-g) shows correlation of some parameters in fish responded to artificial breeding with fertilization rate. there was no positive correlation in any measured parameters except for fertilization rate and cf (fig. 1a). negative correlation also was found between pv (fig. 2a; r = -0.447) and lx (fig. 2b; r = -0.503) with gsi. discussion this is important to find a simple approach to select sturgeon breeders for artificial propagation and present study showed some relations but not powerful to examine the suitable broodstocks. it is clear that the age and body size of fish are closely related and when the effect of size is eliminated, the effect of age on fecundity may be small or inconsistent (wootton, 1991), which is in correlation with our findings. colombo et al. (2007) and tripp (2007) reported a significant positive relationship between fecundity and weight of the shovelnose sturgeon (scaphirhynchus platorynchus) harvested from middle of mississippi river. fecundity and both fork length and wet weight in upper wabash river female shovelnose sturgeon (kennedy et al., 2006) demonstrated positive relationships > 69%. positive relationships between fecundity and fork length were observed in atlantic sturgeon (a. oxyrinchus) in more than 70% and in the lower mississippi river over 40% of female sturgeons (van eenennaam et al., 1996; van eenennaam and doroshov, 1998; wildhaber et al., 2006). also, some positive relationships were observed in siberian sturgeon (a. baeri) breeders for selection of suitable fish (williot et al., 1991). žikov and petrova (1993) reported a significant relationship between absolute fecundity with body length, age and weight in pikeperch (stizostedion lucioperca). similar results were observed on absolute fecundity and body weight of russian sturgeon in present study. these results show that fecundity is influenced by the egg size, species, season and environmental conditions. fecundity varies among populations of the same species and also differs from one year to another. a key feature with fecundity is that it increases (with certain exceptions) in accordance with fish growth. a large fish lays more eggs than a small one and the correlation between fecundity and weight is higher than the correlation between length and age weight (kg) total length (cm) condition factor pv (cm) lx (cm) age (year) pi gsi (%) abdomen girth (cm) responsive 22.3 ± 4.6 148.5 ± 8.1 0.7 ± 0.1 58.5 ± 3.2 28.2 ± 2.2 14.8 ± 0.8 8.1 ± 1.0* 15.7 ± 2.8* 59.3 ± 2.9 nonresponsive 22.1 ± 7.5 144.3 ± 14.3 0.7 ± 0.1 56.4 ± 6.4 27.5 ± 3.0 14.5 ± 1.7 12.5 ± 6.2 22.1 ± 6.5 60.9 ± 8.0 pv: the distance between pectoral fin to pelvic fin; lx: the distance between the anal fin up to the caudal fin; pi: polarization index; gsi: gonadosomatic index. table 3. some measured indices (mean ± sd) in broodstocks responded (n = 10) and not responded (n = 17) to hormonal injection. asterisks show significant differences between the two means (p<0.05). figure 2. regression of pv (a) and lx (b) with gonadosomatic index (%) of russian sturgeon broodstocks (n = 10) in southern basin of the caspian sea which responded to hormone injection. 198 int. j. aquat. biol. (2014) 2(4): 193-200 (wootton, 1991). information on gonadal development in relation to fish weight and age is essential tool to manage sturgeon culture activities and caviar production (hurvitz et al., 2008). van eenennaam and doroshov (1998) demonstrated a relationship between individual fecundity with fork length and age in mature females of atlantic sturgeon (a. oxyrinchus). in present study, these correlations were r = 0.555 and r = 0.229, respectively. the results showed a very high correlation between absolute fecundity and fish weight (r = 0.874). smith and baker (2005) also demonstrated a relationship between weight and length for female lake sturgeon (a. fulvescens) spawners (r = 0.776). there was no significant relationship between body size of white sturgeon (acipenser transmontanus) broodstock and ovulation success in study conducted by lutes et al. (1987). we observed a positive correlation between weight and total length and also between condition factor and fish age. this could be interpreted that increase in length does not necessarily coincide with increase in weight (fawole, 2002). in this study, we found a small negative regression of polarization index with fertilization rate, while the value was significantly different between responded and not-responded females. it seems that germinal vesicle (gv) position is one of the best parameters for selection of brood fish in spawning season and even transferring fish from wild to hatcheries. former studies showed that gv position is a key morphological parameter for general discrimination of potentially responsive females as suggested by lutes et al. (1987) and williot et al. (1991). in white sturgeon, gv position and in vitro oocyte maturation showed high relationships with ovulation. there were also significant correlations between ovulation success and germinal vesicle breakdown, oocyte diameter and plasma concentration of 17α, 20βdihydoxy progesterone (lutes et al., 1987). it seems that the best way to test the ovarian follicles near to spawning is in vitro study (mojazi amiri et al., 2001; williot et al., 2009). also, a study on persian sturgeon (a. persicus) revealed that the calcium content of the oocytes are positively correlated with the polarization index (rafiee and hajrezaee, 2011). therefore, it is appropriate that to identify the possibility of some methods for pre-selection or for first selection of genitors for the reproduction based on morphometrical parameters; final selection (fish with hormonal treatment) being based on microscopical observation of oocytes (pi, oocyte size) and bio-tests. owing to deterioration of suitable spawning grounds for sturgeons in the rivers of the southern caspian sea, most of the broodstocks used for artificial breeding are caught at the sea. since these fish are at earlier stages of maturity, or even between 2 spawning seasons, they do not respond duly to hormonal treatments. some 33-55% of persian sturgeons caught and transported to the hatcheries does not respond efficiently to hormone injection and discarded from artificial breeding cycle (azari takami et al., 1997). a major problem with artificial breeding of sturgeons in comparison to bony fishes is that the bony fishes have annual reproduction cycle, while this cycle in sturgeons depending upon the slow gonads development, environmental conditions, feed availability and some other parameters occurs in 2-5 year intervals. therefore, one may not guarantee the success of artificial breeding based on age and other parameters since fish may appear mature with appropriate length and weight in fact in the period between two consecutive spawning. consequently, majority of the results obtained in teleost species are not compatible with sturgeons. in conclusion, considering the facts mentioned above, selection of spawners based on morphometric parameters e.g., tracking the position of nucleus and other measurements and indices are practical methods to select suitable broodstocks for artificial propagation and discard unsuitable ones for caviar production. our study demonstrated that it is quite possible to select suitable broodstocks for artificial propagation based on combination of weight, lx, pv, age, abdomen girth and length, but the most practical tools for this selection which can be 199 falahatkar and imanpour/ russian sturgeon broodstocks selection according to morphological features consider is polarization index. undoubtedly physiological parameters like sex steroids and corticosteroids measurements should also be given special priority for selection of sturgeon spawners with regards to their fragile stock condition which leaves no place for try and error. based on previous findings, our results confirmed that examination of gv position and some in vitro studies on oocyte maturation response may be adequate tools for selection of russian sturgeon females in artificial propagation. detailed studies are required to combine practical approaches and sophisticated physiological and biochemical methods for better selection of broodstock to secure successful breeding programs. acknowledgments we would like to extend our thanks to staff of the international sturgeon research institute, especially dr. m. pourkazemi, dr. k. amini, and staff of the shahid mardjani sturgeon hatchery. references alberto a., williot p., vizziano d. 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(2019) 7(6): 342-350 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article biological features of chanda nama (ambassidae) in the old brahmaputra river, bangladesh zoarder faruque ahmed1, ferdous ahamed*2,1mst. kaniz fatema1 1department of fisheries management, bangladesh agricultural university, mymensingh 2202, bangladesh. 2department of fisheries management, patuakhali science and technology university, patuakhali 8602, bangladesh. s article history: received 15 september 2019 accepted 21 december 2019 available online 2 5 december 2019 keywords: sex ratio sexual maturity spawning season growth pattern abstract: biological features including sex ratio, length-frequency distributions (lfds), size at sexual maturity, spawning season, length-weight relationships (lwrs) and condition factor of chanda nama were studied in the old brahmaputra river, bangladesh. there was no significant difference in sex ratio. lfds indicated no significant differences in size between the sexes. size at sexual maturity was estimated at ~3.0 cm standard length. monthly variations in gonadosomatic index indicate that the main spawning season is from july to august. the lwrs showed isometric growth in males and positive allometric growth in females. fulton’s condition factor varied in both sexes and was attributed to variations in gsi with maturity. the findings of this study will be helpful to formulate conservation and management strategies of c. nama population in the old brahmaputra river and surrounding ecosystems. introduction chanda nama (hamilton, 1822), a member of the family ambassidae, is commonly known as elongate glass-perchlet (talwar and jhingran, 1991). it is a nocturnal or crepuscular fish famous for lepidophagy or scale eating (grubh and winemiller, 2004) and one of the important small indigenous fish species and popular food fish having good consumer preference. chanda nama is found in running and standing fresh and brackish water throughout the indian subcontinent including pakistan, india, nepal, bangladesh, and myanmar. in bangladesh, the consumer’s interest is vigorously growing towards small indigenous fish species because of their good taste and high nutritional value essential to prevent malnutrition of the rural communities, particularly of vulnerable groups such as poor women and children (thilsted et al., 1997; thilsted, 2003). it is also a popular aquarium fish and has high market demand among the ornamental fish hobbyists (gupta and banerjee, 2012). previous studies on this species have only been concentrated to length-weight relationships and *correspondence: ferdous ahamed doi: https://doi.org/10.22034/ijab.v7i6.683 e-mail: ferdous@pstu.ac.bd condition factor (hossain et al., 2012a; sarkar et al., 2013; alam et al., 2014; jahid et al., 2017; khoso et al., 2018; bhuvaneswari and serfoji, 2018), sexual maturity (grubh and winemiller, 2004) and spawning season (jones, 1946; gupta, 1984; parween et al., 2000; grubh and winemiller, 2004) using seasonal samples. however, the study of biological parameters, including sex ratio, length-frequency distributions (lfds), size at sexual maturity, spawning season, length-weight relationships (lwrs) and condition factor of this species using year-long large data sets is evidently lacking. therefore, the present study was conducted to broaden the knowledge of important biological features of c. nama in the old brahmaputra river, bangladesh over a year-long study period. materials and methods study site and sampling: the present study was conducted in the old brahmaputra river of bangladesh, which is comprised of two channels: the main channel flowing through bangladesh is known as the jamuna and the old channel commonly known as the old brahmaputra river running through 343 int. j. aquat. biol. (2019) 7(6): 342-350 mymensingh, a northeastern district of bangladesh. monthly samples were collected from the old brahmaputra river near bangladesh agricultural university, mymensingh (24º75´n, 90º43´e) for a period of one year. the samples were collected using a combination of fine-meshed (< 2 mm) cast and seine nets by the help of local fishermen. all specimens at each sampling were preserved with 10% formalin and transferred to the laboratory for further analysis. fish measurement: standard length (sl) for all individuals were measured to the nearest 0.1 cm using a measuring ruler, while the body weight (bw) was recorded using a digital balance to 0.01 g accuracy. sex was determined by abdominal incision of each individual and visual inspection of the gonad. in case of females, the gonads were weighed to the nearest 0.001 g. sex ratio and length-frequency distributions (lfds): sex ratio (male/ female) was calculated on a monthly basis and by size (sl) class, and the results were analyzed to compare whether the sex ratio departed from the expected 1:1 using a chi-square test at 95% significance level. length-frequency distributions were constructed using 0.5 cm sl intervals for both sexes. the normal distributions were fitted to the pooled length-frequency data for each sex, using a computer analysis based on hasselblad’s maximumlikelihood method (hasselblad, 1966). each normal distribution was presumed to represent an age group in the population. in cases where two normal distributions representing two different age groups overlapped, individuals were separated into two age groups using a discriminate function: zi = (lm×σn+ln×σm) / (σm+σn) − li, where lm is the mean sl and σm is the standard deviation at age m; ln is the mean sl and σn is the standard deviation at age n; and li is the sl of individual i. if zi > 0, i belonged to the m age group; if zi < 0, i belonged to the n age group. size at sexual maturity and spawning season: the gonadosomatic index (gsi) was calculated as gsi (%) = (gw/ bw) ×100. size at sexual maturity was estimated by the relationship between sl and gsi. spawning season was estimated based on the monthly variations of gsi (kamal et al., 2009). length-weight relationships (lwrs) and condition factor: lwrs were calculated according to the equation: bw = a × slb, where bw is the body weight (g) and sl is the standard length (cm) (zamani faradonbeh et al., 2015). the parameters a and b were estimated by linear regression analysis based on natural logarithms: ln (bw) = ln (a) + b ln (sl). extreme outliers were excluded from the analyses (froese, 2006). significant deviation of b-value from the theoretical isometric value (b = 3) indicates either positive (b > 3) or negative (b < 3) allometric growth (tesch, 1971; zamani-faradonbe et al., 2015), which was verified with student’s t-tests (sokal and rohlf, 1981). analysis of covariance (ancova) (zar, 1984) was used to test for significant differences in slopes and intercepts between sexes. fulton’s condition factor (k) was estimated using the following equation: k = (bw/sl3) × 100 both monthly and by size (sl cm) class (mouludi-saleh and eagderi, 2019). results sex ratio and length-frequency distributions (lfds): a total of 1169 specimens of c. nama were collected during this study, among them 598 (51.2%) were male and 571 (48.8%) female (table 1). the sl ranged from 1.9 to 8.1 cm in males and from 2.0 to 8.1 cm in females, whereas bw ranged from 0.2 to 11.5 g and 0.2 to 12.9 g for male and female, respectively. sex ratios were 1:1 throughout the year (fig. 1) except in february and november when males significantly outnumbered females and in july when females dominated significantly (χ2 test, p<0.001). sex ratios by size classes and overall sex ratio was even between the sexes. the overall lfds showed two age groups with similar pattern in both sexes (fig. 2). the mean sl of smaller size groups were similar (3.4±0.5 cm) in both sexes and therefore, no significant difference was observed between the sexes (t-test, p>0.05). the mean sl of larger size group was 4.9 (±1.2) cm in males and 5.4 (±1.4) cm in females. the mean sl of larger size group was 0.5 cm longer in females than males; 344 ahmed et al./ biological features of chanda nama however, the difference was non-significant (t-test, p>0.05). in addition, lfds showed that the 3.0–4.0 cm sl size group was dominant in both sexes, constituting 70 and 53% of its population, respectively. size at sexual maturity: the relationship between sl and gsi of female is shown in figure 3. the lowest and highest gsi recorded during this study were 0.03 and 14.64, respectively. the gsi of <3.0 cm sl was low. however, the gsi rose sharply at ~3.0 cm sl. therefore, the size at sexual maturity was considered to be 3.0 cm sl. spawning season: the monthly mean gsi with minimum and maximum values of female were table 1. collection records of chanda nama from the old brahmaputra river, bangladesh. sampling month total fish no. of males size range no. of females size range sl (cm) bw (g) sl (cm) bw (g) january 99 55 2.5-8.1 0.3-11.3 44 2.6-8.1 0.3-10.7 february 100 61 2.4-6.8 0.3-7.2 39 2.9-7.8 0.4-8.9 march 100 45 1.9-5.9 0.3-4.6 55 2.5-6.8 0.3-8.1 april 100 43 2.6-7.6 0.3-9.7 57 2.6-8.1 0.3-12.1 may 81 39 3.1-7.9 0.4-11.5 42 3.7-8.0 1.1-12.9 june 100 47 2.2-5.3 0.2-5.1 53 2.4-7.5 0.3-12.1 july 89 32 2.7-6.6 0.3-6.2 57 2.7-7.8 0.3-8.6 august 100 50 2.7-5.4 0.3-2.7 50 2.8-7.4 0.3-6.9 september 100 56 2.1-5.4 0.2-3.5 44 2.1-6.9 0.2-6.4 october 100 50 2.3-5.8 0.2-3.8 50 2.5-6.6 0.3-7.5 november 100 66 2.3-6.5 0.2-5.4 34 2.6-6.9 0.3-6.8 december 100 54 2.2-5.2 0.2-2.7 46 2.0-6.2 0.2-5.8 overall 1169 598 1.9-8.1 0.2-11.5 571 2.0-8.1 0.2-12.9 sl, standard length; bw, body weight. figure 1. sex ratio with regard to month and size class (sl, cm) of chanda nama ( statistically significant difference from 1:1 ratio,  not significant) in the old brahmaputra river, bangladesh. figure 2. length-frequency distribution of pooled males and females chanda nama in the old brahmaputra river, bangladesh. 345 int. j. aquat. biol. (2019) 7(6): 342-350 plotted in figure 4. the mean gsi varied from 1.17 in january to 5.14 in july. the mean gsi value was around 2 in majority of the months; however, the gsi value was higher during july to august with a peak in july. therefore, the spawning season of c. nama was estimated to be from july to august. table 2. descriptive statistics of lwrs of chanda nama collected from the old brahmaputra river, bangladesh. month sex n parameters of the lwr r2 a (95% cl) b (95% cl) gt january m 55 0.0062 (0.0052-0.0074) 3.20 (3.09-3.31) 0.984 a+ f 44 0.0054 (0.0042-0.0069) 3.29 (3.14-3.45) 0.978 a+ february m 61 0.0058 (0.0038-0.0089) 3.27 (3.00-3.53) 0.911 a+ f 39 0.0057 (0.0046-0.0071) 3.26 (3.13-3.39) 0.986 a+ march m 45 0.0110 (0.0060-0.0203) 2.87 (2.48-3.26) 0.835 i f 55 0.0141 (0.0072-0.0276) 2.74 (2.33-3.15) 0.773 i april m 43 0.0066 (0.0037-0.0119) 3.15 (2.79-3.51) 0.883 i f 57 0.0066 (0.0051-0.0087) 3.16 (3.00-3.33) 0.964 i may m 39 0.0049 (0.0035-0.0068) 3.36 (3.17-3.55) 0.971 a+ f 42 0.0072 (0.0051-0.0101) 3.15 (2.96-3.34) 0.964 i june m 47 0.0095 (0.0057-0.0159) 2.97 (2.63-3.32) 0.871 i f 53 0.0062 (0.0052-0.0074) 3.27 (3.16-3.38) 0.986 a+ july m 32 0.0057 (0.0044-0.0075) 3.26 (3.09-3.44) 0.980 a+ f 57 0.0062 (0.0052-0.0073) 3.22 (3.11-3.32) 0.985 a+ august m 50 0.0099 (0.0055-0.0177) 2.89 (2.51-3.27) 0.829 i f 50 0.0069 (0.0053-0.0089) 3.12 (2.97-3.28) 0.971 i september m 56 0.0121 (0.0076-0.0193) 2.81 (2.50-3.11) 0.863 i f 44 0.0099 (0.0075-0.0131) 2.96 (2.78-3.13) 0.964 i october m 50 0.0107 (0.0057-0.0200) 2.85 (2.44-3.25) 0.809 i f 50 0.0066 (0.0050-0.0086) 3.19 (3.02-3.36) 0.968 a+ november m 66 0.0078 (0.0060-0.0101) 3.04 (2.87-3.21) 0.952 i f 34 0.0057 (0.0043-0.0075) 3.25 (3.06-3.43) 0.975 a+ december m 54 0.0098 (0.0067-0.0145) 2.93 (2.67-3.19) 0.907 i f 46 0.0078 (0.0058-0.0104) 3.08 (2.89-3.28) 0.959 i overall m 598 0.0080 (0.0071-0.0089) 3.06 (2.99-3.13) 0.924 i f 571 0.0068 (0.0063-0.0074) 3.16 (3.11-3.21) 0.964 a+ n, sample size; m, male; f, female; cl, confidence limit of mean; gt, growth type; i, isometric; a+, positive allometric. figure 3. relationship between gonadosomatic index and standard length (cm) for female chanda nama in the old brahmaputra river, bangladesh. 346 ahmed et al./ biological features of chanda nama length-weight relationships (lwrs): the detailed statistics of lwrs of c. nama are given in table 2. the overall lwrs indicated isometric growth in males (t-test, p>0.05), while positive allometric growth was observed in females as the allometric coefficient b values were significantly larger than the expected isometric value of 3 (t-test, p<0.05). significant difference in both slope (b) and intercept (a) were observed between sexes (ancova, p<0.05). monthly variations in lwrs were observed with the calculated b-values ranged from 2.81 in september to 3.36 in may for males, and from 2.74 in march to 3.29 in january for females. all lwrs were highly significant with r2 exceeding 0.800. condition factor: the variations of fulton’s condition factor by month and size class for both sexes are presented in figure 5. the monthly k-values varied for both sexes, ranging from 1.63 to 2.09 in males and from 1.63 to 2.21 in females. the lowest k was found in november, whereas the highest was in june for both sexes. by size class, males showed minimum k-value at 3.5 cm sl and maximum at 7.5 cm sl, whereas females showed minimum value at 3.5 cm sl and maximum at 8.5 cm sl. k tended to be lower at ~3.5 cm sl, thereafter started to increase in the subsequent size class for both sexes. k in females was always higher than males. discussions the present study investigated some basic biological features of c. nama necessary to develop the management and conservation strategies. there was no significant difference in overall sex ratio in the present study, even no temporal (except february, july and november) and size class variations in sex ratio were observed. usually deviation from 1:1 sex ratio is not expected for most fish species, although some fish populations may present a strong bias in this ratio. such variation in sex ratio might be due to (a) different growth rate and longevity between sexes or (b) sex change (oh et al., 2002; chilari et al., 2005; ahamed et al., 2014, 2018). analysis of lfds is important to know ecological and life-history traits of a fish population (ranjan et al., 2005). in the present study, the lfds revealed two size groups in both sexes; however, no significant differences in size were observed between the sexes. determination of size at sexual maturity is very crucial for fisheries management as it indicates the minimum permissible capture size (lucifora et al., 1999; ahamed and ohtomi, 2011, 2014; hossain et al., 2013; ahamed et al., 2014, 2015). in the present figure 4. monthly changes of mean gonadosomatic index (gsi) with minimum and maximum values, for female chanda nama in the old brahmaputra river, bangladesh. 347 int. j. aquat. biol. (2019) 7(6): 342-350 study, the size at sexual maturity of c. nama was estimated at ~3.0 cm sl. on the other hand, grubh and winemiller (2004) reported the size at sexual maturity of this species as 2.5 cm sl from a wetland, tamilnadu, india. the differences in size at sexual maturity might be attributed to variations in environmental factors, particularly water temperature, population densities and food availability (king, 2007). monthly variations of gsi indicated the spawning season of c. nama from july to august. a number of studies have also reported the spawning season of this species, e.g. parween et al. (2000) reported the spawning season as july-november and februaryaugust respectively in bangladesh, while jones (1946), gupta (1984), and grubh and winemiller (2004) have documented june-august, april-may and march-july respectively for the same in india. several studies (ahamed and ohtomi, 2012; ahamed et al., 2014; allen, 1966; bauer, 1992; kikuchi, 1962) figure 5. changes of fulton’s condition factor by month and size class (sl, cm) for both genders of chanda nama in the old brahmaputra river, bangladesh. 348 ahmed et al./ biological features of chanda nama reported the temperature and rainfall as important factors controlling the spawning of fish. however, in the present study, the factor/s controlling spawning season could not be deciphered due to lacking of such environmental data. the calculated b-values of the lwrs were within the expected range of 2.5 to 3.5 (froese, 2006) with the overall b indicating isometric growth in males and positive allometric growth in females. however, numerous studies (alam et al., 2014; hossain et al., 2012a; jahid et al., 2017; sarkar et al., 2013; khoso et al., 2018; bhuvaneswari and serfoji, 2018) reported negative allometric growth for combined sex of this species using only seasonal samples from different habitats. we also observed some monthly variations in growth types with positive allometric growth recorded during january-february, may and july in males and january-february, june-july and october-november in females. while isometric growth was observed during march-april, june and august-december in males and march-may, august-september and december in females. the differences in growth types among different populations of the species can be attributed to the seasonal variation in sample collection, maturity status, and size range of the specimens observed (tesch, 1968). the condition factor is an index reflecting interactions between biotic and abiotic factors on the physiological condition of the fishes; therefore, it can also be used as an index to assess the status of the aquatic ecosystem in which fish live (anene, 2005). k is generally correlated with the temporal changes of fish gsi (ahamed et al., 2014; hossain et al., 2012b, 2013) although we found no definite trends between these two variables. however, k across size classes showed a noticeable decrease at 3.5 cm sl for both sexes, which may indicate the start of sexual maturation at 3.0 cm sl for c. nama. decreasing kvalue after sexual maturity due to reproductive activity is a common phenomenon in fishes. in conclusion, the present study provides some important baseline information on the population biology of c. nama, which will be helpful to formulate conservation and management strategies of this species. acknowledgements we acknowledge the support of the department of fisheries management, bangladesh agricultural university for providing the laboratory facilities. we would like to thank the local fisher for helping in sampling. references ahamed f., saha n., nishat m.a., biswas m.k., sultana m., khatun m.s., ahmed z.f., hossain m.y., ohtomi j. 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(2020) 8(3): 194-208 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article evaluation of probiotic adequacy, immunomodulatory effects and dosage application of bacillus coagulans in formulated feeds for catla catla (hamilton 1822) anita bhatnagar*,1shashi raparia department of zoology, kurukshetra university, kurukshetra -136119, india. s article history: received 19 january2020 accepted 2 june 2020 available online 2 5 june 2020 keywords: hydrophobicity probiotic properties phagocytic ratio indian carp abstract: the present study was conducted to study the probiotic properties, antagonistic effect against pathogenic aeromonas hydrophila of bacillus coagulans isolated from intestine of healthy catla catla hamilton, 1822; and its optimum dosage for growth promotion and immunostimulation. the isolated b. coagulans from the gastrointestinal tract of c. catla was first assessed for its probiotic properties viz., antagonism towards pathogen and cell surface adhesion. a feeding trial of 90 days was conducted to optimize the inclusion level of b. coagulans in diets and c. catla fingerlings (avg. wt. 0.30±0.03g) were fed on feed supplemented with 1x103 (diet d1), 2x103 (diet d2), 3x103 (diet d3) and 5x103 (diet d4) b. coagulans cfu g-1 of feed in triplicate treatments. the growth and digestibility parameters, intestinal enzyme activities were significantly higher in group of fish fed on feed d3 (3x103 cfu g-1) in comparison to other dietary treatments except for food conversion ratio which was significantly higher in control group. significantly higher value of carcass protein level, lower excretion of metabolites (ammonia and phosphates), enhancement of non-specific immune response and increase of total erythrocyte count (tec) and total leucocyte count (tlc) were observed in fish fed with probiotics supplemented diets. the results obtained in the present study support the use of b. coagulans for better growth and proper nutrient utilization. the broken line analysis was carried out and polynomial fit curve further suggest that the optimum concentrations of b. coagulans as high as 3000 (3x103) cfu g-1 of feed is required for improving the overall physiological performance and enhancement of defense mechanisms in the fingerlings of c. catla. introduction aquaculture is most promising, viable and fastgrowing sector to provide nutritional security and its intensification is required to keep pace with surging need of animal protein. intensification increases stress level in the animal as well as the environment. disease outbreak is considered as most important constraint to its continued expansion. the application of antibiotics and chemotherapeutics to control diseases has led to serious problems such as the evolution of drug resistant pathogens, suppression of the aquatic animal's immune system, significant risk to human health and environmental hazards (brogden et al., 2014; allameh et al., 2015). an alternate approach to enhance disease resistance, immune responses and other health benefits is the administration of probiotics *correspondence: anita bhatnagar e-mail: anitabhatnagar@gmail.com which play an important role in improving health of fish (bandyopadhyay et al., 2015; sivagami and ronald, 2018). merrifield et al. (2010) defined probiotics “as any microbial cell provided via the diet or rearing water that benefits the host fish, fish farmer or fish consumer, which is achieved, in part at least, by improving the microbial balance of the fish”. many studies have reported that probiotic supplemented diets have a major impact on growth performance of fish (gao et al., 2016; bhatnagar and lamba, 2017; liu et al., 2018; gobi et al., 2018; sivagami and ronald, 2018; ullah et al., 2018). probiotic are also known to improve intestinal enzymatic activities in fishes (sivani et al., 2016; makled et al., 2019). zhang et al. (2018) reported that supplementation of lactobacillus delbrueckii as 195 int. j. aquat. biol. (2020) 8(3): 194-208 probiotic enhanced growth performance and intestinal enzymatic activities as well as whole body composition of common carp. selection of probiotics demands that is should be isolated from the gastrointestinal tract of host species intended to study (patel et al., 2010; makled et al., 2019) as commercially available probiotics are mainly from non-fish sources, which are believed to be unable to survive and/or remain viable at optimum concentrations in the fish intestine (ghosh et al., 2008). probiotics isolated from mature animals are well accepted in feeds of immature animals of same species (gomez-gil et al., 2000; ghosh et al., 2003). bacillus has been evaluated as probiotics in fish due to its antagonistic property, ability to enhance growth and immune response and is environment friendly to use (shelby et al., 2006; sumathi et al., 2014; bhatnagar and lamba, 2015, 2017; bhatnagar and saluja, 2019; bhatnagar and dhillon, 2019; bhatnagar and rathi, 2020). bacillus circulans and bacillus sp. have been isolated from the gut of catla catla and cirrihinus mrigala and their effect on growth, nutritional quality and immunity have been studied when incorporated in formulated diets (bandyopadhyay and patra, 2004). studies were undertaken to isolate gut adherent potential probiotic bacterium to improve fish growth and digestibility in c. catla (bhatnagar et al., 2012; bhatnagar and raparia, 2014; bhatnagar and saluja, 2019), and it was observed that significantly high growth performance can be achieved in the group of fishes fed on diet containing b. coagulans. in our earlier studies, three doses 200, 2000 and 20000 cfu per gram were used (bhatnagar and raparia, 2014), and it was found that optimum dose is somewhere near 2000 cfu g-1, therefore to evaluate the proper dose four doses 1000, 2000, 3000 and 5000 cfu g-1 were used in the present study. however, it was felt that there is a need to assess the probiotic properties of this bacterium and its impact on growth performance, nutritional physiology and hematological parameters. therefore, the present studies were conducted to evaluate the antagonistic properties of this probiotic species against aeromonas hydrophila (major diseasecausing agent in water), its influence on growth performance, blood parameters and immunity of c. catla and its optimum inclusion level in formulated diets. materials and methods experimental animals: fingerlings of c. catla were obtained from “sultan fish seed farm” in butana, nilokheri, district karnal (haryana, india). fingerlings were kept in glass aquarium of 30l capacity in laboratory conditions where temperature was maintained at 25±1°c. fishes were acclimatized for 10 days prior to experiment start. each aquarium was filled with de-chlorinated tap water and then stocked with 20 fingerlings with average body weight (bw) 0.30±0.03g. during the experiment, the water samples from all the aquaria were collected fortnightly and temperature, dissolved oxygen (do), ph, electrical conductivity, calcium, chlorides and total alkalinity were measured following american public health association (2017) to investigate the influence of supplemented feeds on quality of holding water. at the end of feeding trials, water samples from each tub were collected at two-hour intervals for the estimation of excretory levels of total ammonia (n-nh4+) and reactive orthophosphate following apha (2017), and calculated following sumagaysay-chavoso (2003). mass culture of b. coagulans and feed preparation: bacillus coagulans, isolated from gut of c. catla (bhatnagar et al., 2012) was used in the present studies. it was kept in nutrient agar slant at 4oc for further use. bacillus coagulans was inoculated into conical flask (500 ml) containing nutrient broth and incubated at 30oc for 24 h in shaker incubator. the culture was centrifuged at 10000 rpm for 20 minutes at 4°c and supernatant was discarded, while the pellets were resuspended in phosphate buffer saline (pbs; ph 7.2). the suspension was similarly washed and recentrifuged four times and then quantified by spread plate technique (nutrient agar), incubated at 30oc for 24 h to determine the number of colonies forming units (cfu) (bhatnagar and raparia, 2014). 196 bhatnagar and raparia / bacillus coagulans as probiotic bacterium and its dosage for catla feed was prepared by thoroughly mixing the ingredients (table 1) followed by steaming for 20 minutes, cooled mixed thoroughly and then pellets were made by a hand pelletizer. feed were air dried, probiotic were sprayed in respective concentrations and finally stored in vacuum sealed plastic containers at 4°c. in all the treatments, fishes were fed with respective diets daily at 4% bw in two instalments at 8:00 and 16:30 hours for 90 days. growth parameters and enzymatic analysis was done using standard methods. biochemical analysis of feed and fish carcass was carried out following aoac (2019). antibiotic resistance study: bacillus coagulans was examined for its inhibitory effects against the pathogenic a. hydrophila (imtech, chandigarh) using well diffusion assay (lyon and glatz, 1993). aeromonas hydrophila was spread by a sterile swab, evenly, over the face of a sterile agar plate. a microbial suspension of each intestinal bacterial strain was applied in well at the centre of the agar plate (in a fashion such that the antimicrobial doesn't spread out from the centre) and incubated for 24 hours at 30º c to check the prevention of aeromonas growth by the antibiotic activity. the strains that showed halos larger than 20 mm were considered positive. hydrophobicity assay: the cell surface hydrophobicity was evaluated according to the ability of the microorganisms to partition into hydrocarbon from phosphate buffer solution using the method of savage (1992). bacterial isolates were grown in nutrient broth (merck, germany) at 37°c for 24 h. after being centrifuged at 5000 rpm for 15 min, the pellets (bacterial precipitates) were washed twice with phosphate buffer solution and optical density (od450) of the bacteria at 450 nm adjusted to 0.5 a. about1 ml of bacterial suspension was added with 60 μl of a hydrocarbon viz., xylene (fisher, england) and toluene (merck, germany), and vortexed for 1 min followed by determination of optical density of the water phase. hydrophobicity was calculated according to the equation: [(od450 before – od450 after)/od450 before] ×100 = % hydrophobicity. growth experiment experimental setup: the experiment was conducted table 1. ingredient and proximate composition (% dry weight basis) of experimental diets. dietary treatments dc (control) d1 (1000 cfu g-1) d2 (2000 cfu g-1) d3 (3000 cfu g-1) d4 (5000 cfu g-1) ingredient composition groundnut oil cake 650.0 650.0 650.0 650.0 650.0 rice bran 42.0 42.0 42.0 42.0 42.0 processed soybean* 266.0 266.0 266.0 266.0 266.0 wheat flour 32.0 32.0 32.0 32.0 32.0 chromic oxide (cr2o3) 10.0 10.0 10.0 10.0 10.0 mineral mixture** 10.0 10.0 10.0 10.0 10.0 probiotic bacterium (cells g-1) bacillus coagulans 0.0 1000 2000 3000 5000 *soybean was hydrothermally processed in an autoclave at 121°c (15 lbs for 15 minutes) to eliminate antinutrient factors (garg et al., 2002). **each kg has nutritional value: copper 312 mg, cobalt 35 mg, magnesium 2.114g, iron 979 mg, zinc 2 mg, iodine 15 mg, dl-methionine 1.920 g, l-lysine monohydrochloride 4.4 g, calcium 30%, phosphorous 8.25%. proximate composition crude protein (%) 39.80±1.36 a 38.24±0.98a 39.51±0.86 a 39.86±0.79 a 39.35±0.79 a crude fat (%) 9.10±0.26 b 9.21±0.24 b 9.7±0.31 a 9.29±1.26 b 9.18±1.26 b crude fiber (%) 6.23±0.06 a 6.13±0.07 a 6.28±0.08 a 6.37±0.06 a 6.14±0.06 a total ash (%) 6.64±0.39 a 6.66±0.34 a 6.51±0.26 a 6.42±0.47 a 6.45±0.47 a moisture (%) 7.41±0.20 a 7.37±0.28 a 7.39±0.19 a 7.22±0.37 a 7.38±0.37 a nitrogen free extract (%) 30.82±1.42 b 32.38±1.49 a 31.84±1.11 ab 30.8±1.07 ab 31.5±1.07 ab gross energy (kj g-1) 17.90±0.09 b 18.53±0.18 a 18.63±0.08 a 18.37±0.09 a 18.33±0.09 a feed phosphorus (%) 1.48±0.11 c 1.42±0.08 c 1.54±0.20 b 1.62±0.07 a 1.47±0.07 c all values are mean±s.e of mean. means with different letters in the same row are significantly (p<0.05) different. (duncan’s multiple range test). 197 int. j. aquat. biol. (2020) 8(3): 194-208 under laboratory conditions (25±1°c) in glass aquarium (30 l capacity) at aqaculture research unit of department of zoology, kurukshetra university, kurukshetra. each aquarium was filled with dechlorinated tap water and then stocked with 20 fish fingerlings with average bw 0.3±0.03 g. five dietary treatments (dc, d1, d2, d3 and d4) were performed with three replicates of each treatment. in treatment 1 (dc), fishes were fed on artificial diet without probiotic bacteria (i.e., control diet). ingredient composition in g kg-1: ground nut oil cake, 650; rice bran 42; hydrothermically processed soybean 266; wheat flour 32; mineral mixture 10. in treatment 2 (d1), fishes were fed on diet containing b. coagulans suspension in proportion 1000 (1x103) cfu g-1 of feed. in treatment 3 (d2), fishes were fed on artificial diet containing b. coagulans in proportion of 2000 (2x103) cfu g-1 of feed. in treatment 4 (d3), fishes were fed on artificial diet containing b. coagulans in proportion of 3000 (3x103) cfu g-1 of feed and in treatment 5 (d4), with proportion of 5000 (5x103) cfu g-1 of feed. all these diets were isocaloric and isoproteic with approximately 40% protein content. after spraying, the feed was air dried at room temperature and the bacterial concentration of feed (cfug-1) was calculated. finally, the feed was stored in vacuumed plastic container at 4oc. all groups of fish were fed daily at 4% bw in 2 installments at 08:00 and 16:30 hours for 90 days. growth parameters and enzymatic analysis was done using standard methods. biochemical analysis of feed and fish carcass was carried out following aoac (2019). blood parameters study: after the end of feeding trial, blood samples were collected from fingerlings of each dietary treatment for hematological diagnosis by using a heparinized syringe from caudal vein or heart by cardiac puncture (lavanya et al., 2011). blood samples of five fishes were pooled for analysis. edta (1 mg edta ml-1) was used as anticoagulant in blood. total erythrocyte count (tec) and total leucocyte count (tlc) were estimated by analyzing the blood samples from each treatment with the help of hemocytometer using a neubauer’s counting chamber. nonspecific immune response: blood samples were heparinsed and immediately used for phagocytic assay (park and jeong, 1996). phagocytic assay: for phagocytic assay cells of freshly grown pathogenic bacteria a. hydrophila in 0.1 ml of pbs were added to 0.1 ml of blood sample (pooled samples of blood of five fishes mixed with edta as anticoagulant) of fishes of each treatment in sterile microplate. blood was then incubated for 30 min at 25°c after thorough mixing in the well. the plate was removed and fifty ml of each suspension was transferred on glass slides to make smears. after air drying, smear was fixed in 95% ethanol, re-dried and stained with may grunwald giemsa. the phagocytic cells and phagocytosed bacteria were enumerated. phagocytic ratio (pr) and phagocytic index (pi) were determined by enumerating 100 phagocytes per slide under a microscope. the average of three slides was calculated depending on the formula which is given below. phagocytic ratio (pr; i.e. percentage of cell with engulfed bacteria) = (no. of phagocytic cells with engulfed bacteria/no. of phagocytic cells) × 100. phagocytic index (pi; i.e. number of engulfed bacteria per cell) = no. of engulfed bacteria/no. of phagocytic cells. nitroblue tetrazolium (nbt) assay: the oxygen radical production by blood phagocytes during respiratory burst activity was measured through nitroblue tetrazolium (nbt) assay as described by anderson and siwicki (1995). briefly, 0.1 ml of edta mixed blood from each treatment group was taken in eppendorf to which 0.1 ml of 0.2% nbt solution was added. the mixture was incubated for 30 minutes at 25°c. from the suspension, 50 µl was taken, added to 1.0 ml n, n-dimethyl formamide in a glass tube and centrifuged at 3000g for 5 minutes. the optical density (od) of the supernatant was measured at 540 nm in the spectrophotometer. challenge trial: after feeding for 90 days, 10 fishes from each treatment were challenged with a. hydrohila which has been cultured and maintained 198 bhatnagar and raparia / bacillus coagulans as probiotic bacterium and its dosage for catla in the selective medium. fishes from each replicate were immersed in a suspension of a. hydrophila ~ 105 cfu ml-1 followed by a second immersion ~107 cfu ml-1 after 7 days (austin et al., 1995). per cent survival was measured for 10 days based on observation that mortality reached its plateau after one week (sahoo et al., 1998) and relative percentage survival was calculated by the following formula (ellis, 1998) below: rps = 1(percent mortality in treated group/ percent mortality in control group) × 100 statistical analysis: data were examined by one-way analysis of variance (anova). when anova identified differences among groups, a multiple comparison, duncan’s test was conducted to examine significant differences among treatments using statistical package for social science (spss-11.5) and significant differences were declared at p≤0.05. data of experiments were further subjected to orthogonal polymonials (broken line regression analysis) for trend analysis (zeitoum et al., 1976). results antagonistic activity of b. coagulans/antibiotic resistance assay: the size of zone of inhibition was found to be 19.0±1.6 and ranged between 15 to 21 mm (fig. 1). hydrophobicity assay: the use of xylene, and toluene to evaluate the hydrophobic cell surface properties of the tested b. coagulans showed consistent positive results. the hydrophobicity of b. coagulans was 30.49±0.84% in xylene, and 22.79±3.96% in toluene. proximate composition: the average proximate composition of formulated diet revealed that the diets were isonitrogenous. values of moisture, crude protein, crude fat, total ash, crude fiber and nfe are shown in table 1 as % dry weight basis. fish growth, survival, digestibility and nutrient retention: the growth responses of test fish fed on experimental diets (dc to d4) are shown in table 2. fish satisfactorily accepted the experimental diet from the beginning of the experiment and maintained normal behavior throughout the experimental period. survival rate (%) was high in all dietary treatments and slight mortality occurred only during the initial days of experiment. statistical analysis revealed that the growth of fish in terms of weight gain (g), growth per cent gain (%) in body weight, growth per day (%) in bw and specific growth rate (sgr) were significantly (p<0.05) higher in treatment d3 in comparison to dietary treatments dc, d1, d2 and d4. also, significantly (p<0.05) higher values of digestibility parameters viz., apparent protein digestibility (apd), gross conversion efficiency table 2. growth performances and intestinal enzyme activities of catla catla fed on soybean based diets containing varying proportions of probiotic bacterium bacillus coagulans. growth parameters dietary treatments dc (control) d1 (1000cfug-1) d2 (2000cfug-1) d3 (3000cfug-1) d4 (5000cfug-1) initial length (cm) 1.95±0.05a 2.0±0.04a 1.90±0.06a 1.95±0.04a 2.05±0.02a initial weight (g) 0.32±0.02a 0.29±0.01a 0.31±0.02a 0.32±0.03a 0.31±0.02a final weight (g) 1.40 ±0.03e 1.87±0.04c 2.08±0.05b 2.30±0.03a 1.69±0.02d live weight gain (g) 1.09±0.02e 1.57±0.04c 1.77±0.04b 1.98±0.03a 1.4±0.02d survival rate (%) 100a 100a 100 a 100 a 98.3±1.36a growth (%) gain in bw 349.3±17.09e 524.3±28.4c 572.7±17.7b 635.6±28.5a 445.9±31.5d growth/day (%) in bw 1.35±0.03c 1.56±0.03b 1.63±0.02ab 1.77±0.02a 1.5±0.04 bc specific growth rate (sgr) (%bw d-1) 0.72±0.02c 0.85±0.02ab 0.88±0.02a b 0.99±0.01a 0.81±.03b feed conversion ratio (fcr) 2.6±0.09 a 1.96±0.04b 1.74±0.02c 1.56±0.03d 1.99±0.04b gross conversion efficiency (gce) 0.41±0.02d 0.53±0.01c 0.65±0.01b 0.80±0.02a 0.59±0.01c protein efficiency ratio (per) 1.18±0.05e 1.34±0.03c 1.45±0.01b 1.67±0.02a 1.2±0.03d apparent protein digestibility (apd) (%) 73.3±0.64e 79.8±0.42c 81.5±0.57b 86.4±0.46a 77.7±0.72d all values are mean±s.e of mean. means with different letters in the same row are significantly (p<0.05) different. (duncan’s multiple range test) 199 int. j. aquat. biol. (2020) 8(3): 194-208 (gce) and protein efficiency ratio (per) and significantly (p<0.05) lower fcr (1.56±0.03) were observed in the dietary treatment d3. the highest fcr was found in the control group (2.6±0.09) (table 2). the data on weight gain revealed that initially up to 15 days not much variations were observed in the weight gain of group of fishes fed on varying dietary treatments. however, growth rate increased significantly (p<0.05) in the fishes fed on diet d3 after 30 till 90 days (fig. 2). diet containing b. coagulans at 3000 cfu g-1 of diet depicted 81.65% increase in weight gain in comparison to control diet (fig. 3). data of experiments were further subjected to orthogonal polymonials (broken line regression analysis) for trend analysis, also showed a clear dose dependent trend line curve. polynomial curve fitting to the data of weight gain in the fingerlings of c. catla is shown in figure 4. intestinal digestive enzyme activities: intestinal digestive enzyme activities for protease, amylase and table 3. proximate carcass composition of catla catla fed on soybean based diets containing varying proportions of probiotics bacterium bacillus coagulans. proximate composition initial value dietary treatments dc (control) d1 (1000 cfug-1) d2 (2000 cfug-1) d3 (3000 cfug-1) d4 (5000 cfug-1) moisture (%) 73.07±0.36 70.65±0.51a 69.5±0.34ab 68.62±0.32b 66.10±0.34c 68.5±0.34b crude protein (%) 8.90±0.06 11.93±0.21d 14.41±0.07c 16.34±0.19b 17.04±0.21a 13.99±0.07c crude fat (%) 2.2±0.04 5.77±0.07a 3.97±.06 bc 4.45±0.15b 3.75±0.06c 4.07±0.07bc total ash (%) 3.6±0.06 4.27±0.18a 4.15±0.10a 3.95±0.09b 3.93±0.04b 4.29±0.10a nitrogen free extract (%) 12.2±0.40 7.4±0.15c 8.60±0.54b 8.51±0.45b 8.82±0.20a 8.40±0.36b gross energy (kj/g) 5.06±.06 6.36±0.08d 6.3±0.05d 6.74±0.04b 7.07±0.05a 6.5±0.05c phosphorus (%) 0.53±0.02 0.59±0.03d 0.71±0.02ab 0.67±0.03c 0.69±0.03b 0.73±0.02a all values are mean±s.e of mean. means with different letters in the same row are significantly (p<0.05) different. (duncan’s multiple range test). figure 1. antagonistic activity shown by probiotic bacterium bacillus coagulans for pathogenic aeromonas hydrophila. 200 bhatnagar and raparia / bacillus coagulans as probiotic bacterium and its dosage for catla cellulase were determined. it was found that specific activity of digestive enzymes was significantly (p<0.05) higher in all the dietary treatments in comparison to control group. the values showed an increasing trend from treatment dc to d3. thereafter, with further increase in the inclusion level of probiotic bacteria (diet-d4), containing b. coagulans in proportion of 5000 cfu g-1 of feed, the values decreased (fig. 5). fish carcass composition: initial and final carcass composition with respect to proximate nutrients of test fish on the basis of feeding trial is shown in table 3. crude protein (%) and gross energy (kjg-1) were found to be significantly (p<0.05) higher in the carcass of fish fed on diet d3. moisture (%) and crude fat (%) figure 2. increase in mean fish weight (g) ±s.e of mean of catla catla fingerlings fed on diets supplemented with varying proportions of probiotics bacillus coagulans (dc=control, d1=1000 cells g-1, d2=2000 cells g-1, d3=3000 cells g-1 and d4=5000 cells g-1 of diet) from day 15 to 90. figure 3. polynomial fit curve using broken line analysis to show effect of bacillus coagulans supplementation (dc=control, d1=1000 cells g-1, d2=2000 cells g-1, d3=3000 cells g-1 and d4=5000 cells g-1 of diet) fitting to the data of weight gain in the fingerlings of catla catla. figure 4. per cent increase in growth of catla catla fed on varying dietary treatments containing varying proportion of bacillus coagulans (dc=control, d1=1000 cells g-1, d2=2000 cells g-1, d3=3000 cells g-1 and d4=5000 cells g-1 of diet). figure 5. intestinal enzyme activities of catla catla fed on varying dietary treatments containing varying proportion of bacillus coagulans (dc=control, d1=1000 cells g-1, d2=2000 cells g-1, d3=3000 cells g-1 and d4=5000 cells g-1 of diet). 201 int. j. aquat. biol. (2020) 8(3): 194-208 was found to be significantly (p<0.05) higher in dietary treatment dc. nitrogen free extract (nfe) was found to be higher in diet d3. however, no significant (p<0.05) variations were observed in total ash (%) of carcass of fishes fed on different diets. effect of experimental diets on water quality characteristics: the data on water quality characteristics pertaining to five dietary treatments is presented in table 4. in general, significant low values in total ammonia excretion and reactive phosphate production (mg kg-1 bw d-1) were recorded in fish fed on diet d3 supplemented with 3000 cfu g-1 of feed. haematological parameters: the rbc was significantly higher (p<0.05) in fishes fed on diet d3 (2.4±0.08) than in the control treatment dc (1.33±.03). in the present study, significant increase (p<0.05) in wbc count was observed in fishes of treatment d3 (50.5 ± 2.1) when compared to control treatment dc (20.7± 0.82). among the post-challenge groups, dc showed significantly (p<0.05) lower rbc than the others. the post-challenge data showed increase in leukocyte count irrespective of the b. coagulans inclusion signify a possible increased infection and inflammatory response mediated by leukocyte against bacteria (table 5; fig. 6). phagocytic responses: phagocytic ratios and phagocytic indices in the fish fed with varying proportion of b. coagulans were significantly (p<.05) higher than in control fish during the assay period. the highest values of phagocytic ratio (79.01±1.72) and phagocytic index (2.61±0.05) were observed in dietary treatment d3 and the lowest in fish fed on the control diet (59.58±1.19 and 1.75±.02, respectively). (table 6; fig. 7). table 4. effect of fish fed on soybean-based diets with different proportion of probiotic bacterium bacillus coagulans supplementation on water quality characteristics. physico-chemical parameters dietary treatments dc (control) d1 (1000 cfug-1) d2 (2000 cfug-1) d3 (3000 cfug-1) d4 (5000 cfug-1) dissolved oxygen (do) mgl-1 6.4±0.07a 6.1±0.02c 6.4 ±0.08a 6.3±0.10a 6.1±0.10a ph 7.80±0.01a 7.79±0.02a 7.82±0.01a 7.84±0.01a 7.78±0.01a conductivity (µ mho cm-1) 624.66±3.32b 629±3.42b 687.33±3.61a 685.83±2.68a 644.83±2.68ab alkalinity(carbonates) 21.33±0.48b 22.61±0.79ab 24.76±0.33a 24.80±0.58a 24.2±0.58b alkalinity(bicarbonates) 128.63±3.87c 144.54±4.17ab 149±5.33a 143.5±4.20b 144.5±4.20 ab chloride (mg l-1) 24.36±0.76a 20.87±0.57b 25.3±1.05a 24.98±0.87a 25.08±0.87a calcium (mgl-1) 25.17±1.08ab 24.19±0.96b 22.73±0.53c 26.41±0.98a 19.41±1.92d total dissolved solids 575.5±16.77a 539.51±8.53b 458.33±14.53e 479.30±19.27d 486.30±18.27c total nh3-nexcretion (mg kg -1 bw day-1) 1890.2±32.74a 1287.31±19.4c 752.8±16.36d 619.3±13.4e 1326.3±19.9b total o-po4 production (mg kg -1 bw day-1) 766.02±11.3a 472.62±7.55c 335.16±8.07d 278.55±13.1e 473.15±13.6b means with different letters in the same row are significantly (p<0.05) different. (duncan’s multiple range test). table 5. hematological values of catla catla fed on soybean based diets containing varying proportions of probiotics bacterium bacillus coagulans. treatments haematological parameters rbc (106mm3) wbc (103mm3) pre-challenge post challenge pre challenge post challenge dc (control) 1.33±0.03e 1.01±0.04 d 20.7±0.82 e 22.58±0.97d d1 (1000 cfug-1) 1.64±0.06c 1.51±0.03c 32.3±1.2c 36.52±1.4c d2 (2000 cfug-1) 1.96±0.04b 1.84±0.06b 39.3±1.6b 43.67±1.9b d3 (3000 cfug-1) 2.4±0.08a 2.15±0.03a 50.5±2.1a 53.58±2.5a d4 (5000 cfug-1) 1.44±0.04d 1.08±0.02d 25.3±0.7d 34.76±1.8c all values are mean±s.e of mean. means with different letters in the same column are significantly (p<0.05) different. (duncan’s multiple range test). 202 bhatnagar and raparia / bacillus coagulans as probiotic bacterium and its dosage for catla nbt assay: respiratory burst activity of phagocytes was measured by reduction of nitro blue tetrazolium (nbt) by intracellular superoxide radicals produced by leukocytes. the production of superoxide radicals was significantly influenced by the probiotic diets. maximum increase in the nbt reduction value was observed in treatment d3 (fig. 8). survival rate with challenge test: after challenge with a. hydrophila, the first mortality was recorded after 24 h. mortality was recorded up to 10 days after challenge. significantly (p<0.05) higher mortality (73.3%) was recorded in fishes of control group. the data on relative per cent survival is presented in the form of survivorship curve (fig. 9). treatment d2 and d3 fed groups showed significantly (p<0.05) higher relative percent survival, 86.36 and 90.9% respectively. clinical signs observed after challenge: the fish were sluggish and gradually lost their equilibrium 24-48 h after challenge with a. hydrophila. the clinical signs were characterized by hyperemic condition on the ventral side of the body, a visibly swollen abdomen and a slightly protruding reddish vent. the eyes of the infected fish were opaque and during the terminal stages the animals were seen floating dorsal side down at the water surface. the abdomen was distended due to accumulation of fluid in the peritoneal cavity. these changes were not evident in d3 group. mortality table 6. effect of bacillus coagulans supplementation on phagocytic ratio and phagocytic index of catla catla. peripheral blood monocytes fish group phagocytic index phagocytic ratio (%) bacteria cells within phagocytes no. of ingesting phagocytes total no. of phagocytes 1.75±0.02d 59.58±1.19e 72.67±3.18 41.33±1.76 69.33±2.18 dc (control) 2.06±0.07c 68.39±1.74c 95±2.30 46.4±2.4 67.6±2.02 d1 (1000 cfug-1) 2.31±0.03b 73.17±1.09b 135.8±6.17 58.3±1.91 79.7±2.12 d2 (2000 cfug-1) 2.61±0.05a 79.01±1.72a 173.6±3.67 66.3±1.83 84.2±2.41 d3 (3000 cfug-1) 1.95±0.04c 63.5±2.41d 86±2.88 44±1.52 69.4±0.98 d4 (5000 cfug-1) all values are mean±s.e of mean. means with different letters in the same column are significantly (p<0.05) different. (duncan’s multiple range test). figure 6. (a) erythrocytes (400x), (b) thrombocytes (a), basophils (b), and lymphocytes (c) and (c) macrophages (a) and early phagocytic cell (b), of catla catla in d3 treatment after 90 days of feeding trial (1000x). 203 int. j. aquat. biol. (2020) 8(3): 194-208 percentage was as low as 10.0±5.7 and 6.6±3.3 in treatment d2 and d3, respectively. discussions in the present study, the novel strain of b.coagulans isolated from the gut of c. catla (bhatnagar et al., 2012) was tested for antagonistic effect on the growth of common indicator fish pathogen a. hydrophila by the appearance of clear inhibition zone by well diffusion assay. the results revealed a clear zone of inhibition ranging from 15 to 21 mm with a mean value of 19.0±0.9 mm (plate-6) clearly, indicating that this strain of b. coagulans can limit the growth of fish pathogen a. hydrophila by producing antimicrobials. urdaci and pinchuk (2004), bhatnagar and lamba (2015) and bhatnagar and dhillon (2019) have also reported that bacillus species could produce a large number of antimicrobials. the potential of probiotics is further inferred through the ability to adhere and to colonize in the intestinal tract. the hydrophobicity of this strain of b. coagulans was 30.49±0.84% in xylene and figure 7. (a) early phagocytic cell, (b) phagocytic cells and(c) mature phagocytic cells, of catla catla in d3 treatment after 90 days of feeding trial (1000x). figure 8. nitro blue tetrazolium (nbt) activity of catla catla fed on diet containing various proportions of probiotic bacterium bacillus coagulans. figure 9. survivorship curve of catla catla in different dietary treatments containing varying proportion of bacillus coagulans (dc=control, d1=1000 cells g-1, d2=2000 cells g-1, d3=3000 cells g-1 and d4=5000 cells g-1 of diet) challenged with aeromonas hydrophila. 204 bhatnagar and raparia / bacillus coagulans as probiotic bacterium and its dosage for catla 22.79±3.96% in toluene, clearly revealing that this strain can colonize the gut of c. catla and has properties of successful probiotic. mahdhi et al. (2011) have also advocated the ability of b. subtilus and b. coagulans to adhere to the intestinal surface and reported hydrophobicity with toluene 30.3±9.40 and 31.3±3.70 and with xylene 32.2±5.60 and 36.10, respectively. bhatnagar and lamba (2015) and bhatnagar and dhillon (2019) have also characterized properties of probiotics on the basis of hydrophobicity. these findings of the present study suggested that the isolated b. coagulans has potential to be the probiotic bacterium for c. catla. in the present study, attempt has also been made to evaluate the optimum dose of probiotic supplementation in the formulated feed for c. catla. the optimum probiotic level which resulted in highest growth in c. catla fingerlings in terms of live weight gain (g), growth per cent gain, sgr (specific growth rate) and nutrient retention (per, gce and apd) was found to be around 3000 cfu g-1 of feed (treatment d3). the polynomial fit curve (broken line regression analysis) of weight gain also represented the optimum dose at dietary treatment d3 (b. coagulans @ 3000 cfu g-1) with high r2 values (0.9637, y=-0.0425x3 + 0.2318x2+0.0043x+0.912). fcr (feed conversion ratio) values decreased with each increase in the dietary probiotic content upto 3000 cfu g-1 of feed. thereafter, further increase in dietary probiotic level resulted in increase in fcr and growth depression. the findings of the present study showed similarity with the study of sivani et al. (2016) in which inclusion rate of probiotic bacterium increased after a certain level, a decrease in growth performance was observed. although, all the feeds were isonitrogenous but the concentration of probiotics in dietary treatment d3 might have been helpful for proper nutrient utilization. high carcass crude protein and lesser nitrogen and phosphate excretion were also observed in dietary treatment d3 which can be attributed to proper probiotic concentration, whereas lesser carcass protein and greater nitrogen and phosphate excretion were observed in dietary treatment d4 which could have been due to the overall low feed utilization level. the high apd (apparent protein digestibility) values for the diet containing b. coagulans at 3000 cfu g-1 of diet may be attributed to high dietary utilization. ghosh et al. (2003) using b. circulans as probiotic in labeo rohita fingerlings; rengpipat et al. (1998) using bacillus sp. as probiotics in paneus monodon, bhatnagar and lamba (2015) using b. cereus in c. mrigala, bhatnagar and dhillon (2019) using aneurinibacillus aneurinilyticus for l. calbasu also reported high values of apd values at doses coinciding with high growth performance. the enhanced enzyme activity level in the gut because of extracellular enzyme production by b. coagulans might have helped in increasing the food absorption and thus resulted in high growth in treatment d3. rani et al. (2004), bhatnagar and khandelwal (2009) and makled et al. (2019) have reported extracellular enzyme production in significant amounts because of presence of suitable gut adherent enzyme producing microflora. the specific enzyme activities were also found highest in treatment d3 and lowest in control dc which may be due to better dietary protein utilization or due to colonization of probiotics bacteria and its exogenous enzyme production. when probiotics supplementation exceeds the optimum level, no further improvement in growth performance and nutritive physiology of the fish was observed, rather these parameters decreased. this might be due to the fact that probiotics bacteria incorporated in the feed might have competed amongst themselves and their colonization was not proper, resulting in the decline in exogenous/ extracellular enzyme production and thus low digestibility, low growth and high feed conversion ratio. these findings could be attributed to the specific feature of probiotic bacterium which stimulate the digestive system of host to increase the intestinal enzymatic activities (eslamloo et al., 2012; bhatnagar and saluja, 2019) and inhibition of other harmful flora along fish gut (makled et al., 2019) thus resulting in better growth performance of fish. in aquaculture, water quality deteriorates mainly 205 int. j. aquat. biol. (2020) 8(3): 194-208 due to accumulation of metabolic wastes such as ammonia and orthophosphate excretion in the holding water. bacillus sp. reduces the quantity of ammonia and nitrite in the water as it degrades the organic matter and facilitates nutrients recycling (skjermo and vadstein, 1999; sanders et al., 2003). the findings of raparia and bhatnagar (2016) and bhatnagar and lamba (2017) showed that dietary supplementation of b. coagulans and b. cereus, respectively, lowered the excretion of total ammonia (n-nh4) and orthophosphate (o-po4), respectively. similarly, in the present study, b. coagulans supplementation at 3000 cfu g-1 improved the water quality parameters and also reduced pathogenic bacteria load to significant levels. rbc and wbc increased in yellowtail infected with n. kampachi (ikeda et al., 1976). the result of the present experiment also revealed an increase in tlc and tec counts in groups d2 and d3 compared to the control (dc). this indicated the heightened immune response in the fish fed on feed containing b. coagulans, probably due to its immunostimulatory effect. similar findings were reported by bandyopadhyay et al. (2015), makled et al. (2017) and bhatnagar and dhillon (2019) where hematological parameters i.e. tlc and tec count showed enhancement when fish were fed on probiotic supplemented diet. it has been shown that bacillus strains supplementation in diet could increase disease resistance in fish through the stimulation of cellular immune function, such as phagocyitc activity (merrifield et al., 2009). phagocytosis is responsible for early activation of the inflammatory response and is mediated by phagocytic cells such as neutrophils, monocytes and macrophages in fish (kwak et al., 2003). significant increases of phagocytic activity (pa) and phagocytic index (pi) was recorded in e. coioides fed b. pumilus or b. clausii containing diets for 60 days compared with those fed the control diet (sun et al., 2010). sumathi et al. (2014) reported that diets with b. megaterium and pontibacter inclusion induced highest phagocytic ratio and phagocytic index in l. rohita. in present study also, significant increase in pa and pi were found in treatment d3 compared with those fed on control diet. in line with our finding, bandyopadhyay and patra (2004) found that isolated bacterium b. circulans pb7 could significantly improve the phagocytic ratio and phagocytic index of c. catla (ham.). bhatnagar and dhillon (2019) in l. calbasu and bhatnagar and saluja (2019) in c. catla have also reported high pi and pa with high growth performance. zhou et al. (2010) confirmed the isolated probiotics b. coagulans 16 from the gut of oreochromis niloticus enhances the immune and health status, thereby improving growth performance which supports the results of present studies for c. catla. however, they used culture of probiotics as water additives where as in present study probiotic bacterium was used as dietary supplement. in l. rohita fed with feed containing b. subtilis, the survival rate after challenge with a. hydrophila was significantly higher in the treatment group compared to the control. administration of yeast glucan enhances the survival of carp infected with a. hydrophila (selvaraj et al., 2005). the per cent mortality during challenge trial with a. hydrophila was low in the groups fed with probiotic bacterial strain b. coagulans @ 3000 cfu g-1. the survivorship plot indicated that there is a significant difference between the survivorship curves in each treatment; similar plot has been reported by bhatnagar and lamba (2017) and bhatnagar and dhillon (2019) in their studies on c. mrigala and l. calbasu, respectively. the high rates of establishment of bacterium in the gastro-intestinal tract of fish treated with b. coagulans have suppressed the a. hydrophila infection, which ultimately resulted in the higher survival in treatment d2 and d3 in present investigation. acknowledgements we are grateful to university grants commission, new delhi, india for sanctioning support under special assistance programme at drs-i. 206 bhatnagar and raparia / bacillus coagulans as probiotic bacterium and its dosage for catla references alameh s.k., yusoff f.m., ringø e., daud h.m., saad c. r., ideris a. 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(2020) 8(3): 209-215 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article the trophic status of the zayandeh river dam lake in the spring and summer, 2017 nasim asadian1, atefeh chamani*1,2, mohammad hadi abolhassani1,2 1environmental science department, islamic azad university, isfahan (khorasgan) branch, isfahan, iran. 2waste research center, islamic azad university, isfahan (khorasgan) branch, isfahan, iran. s article history: received 25 august 2019 accepted 15 february 2020 available online 2 5 june 2020 keywords: tsi index trophy chlorophyll-a oligotroph abstract: the zayandeh river dam lake, supplies freshwater for municipal, agricultural and industrial activities of three central iranian provinces. monthly water sampling was conducted in the spring and summer 2017 at four stations in the lake to determine trophic state. electro-conductivity, temperature, ph, turbidity, total suspended solids, total dissolved solids, nitrate, phosphate, dissolved oxygen, biological oxygen demand also chlorophyll-a levels were measured in the samples. the maximum value of trophy state index (tsi) was recorded in may and the minimum value in september. based on tsi, the lake was oligotrophic in the spring and summer. however, in the may, the lake was in mesotrophic state, probably due to floods, runoff and drainage of farmlands. due to high temperatures and biological activity in the summer, nitrate and phosphate decomposition increased. on the other hand, agricultural activities decreased compared to the spring, resulted in decreases in the concentration of nutrients, especially nitrate. therefore, the lake is in oligotrophic state from june to september. introduction eutrophication is a harmful environmental phenomenon in inland waters such as lakes, reservoirs, rivers, estuaries and other habitats (fernández et al., 2009; rigosi et al., 2014), which occurs due to increase in the concentration of nutrients, especially nitrogen and phosphorus (wilkinson, 2017). increase in application of nitrogenous and phosphorous fertilizers has increased agricultural crops production, but led to serious problems, such as algae bloom, change in diversity and abundance of aquatic species and water quality (huang et al., 2017). these impacts threaten the trophic state and ecological sustainability in aquatic ecosystems. eutrophication also causes water turbidity and replacement of macrophytes by phytoplanktons (portielje and van der molen, 1999). a range of water quality parameters, such as physical, chemical and biological parameters and primary production are evaluated to monitoring trophic state (hollister et al., 2016). trophic state in freshwaters is *correspondence: atefeh chamani e-mail: atefehchamani@yahoo.com determined based on total phosphorous, total nitrogen and chlorophyll-a concentrations. resident time, depth, geology and morphology of lakes, industrial wastewater, aquaculture and fertilizers are effective in trophic state of the lakes (brönmark and hansson, 2005). eutrophication is directly linked to primary production by phytoplankton. these organisms play an important role in the environment. however, high concentrations of certain species may lead to health problems for humans and aquatic organisms. cyanobacteria create toxins that can cause serious liver, nervous system diseases and death at certain concentrations. phytoplankton has photoactive pigments that may be used to identify these toxins (watanabe et al., 2015). trophic state in freshwaters has been the subject of several researches in the world (rigosi et al., 2014; yang et al., 2016; boucek et al., 2017; lee and liu, 2018; kiersztyn et al., 2018) as well as iran (saghi et al., 2015; ghorbani et al., 2016; taheri tizro et al., 2016; esfandi et al., 2018). 210 asadian et al./ trophic status of the zayandeh river dam lake chlorophyll-a (chl-a) presents in all phytoplankton species (watanabe et al., 2015) and is an efficient indicator of water eutrophication (malek et al., 2011). there are several linear models for chl-a concentration and environmental variables. to date, several trophic models have been presented by researchers. one of the most important and common models is trophic state index (tsi) that was introduced by carlson (1977). the zayandeh river with more than 350 km length (nabinejad, 2018), is the most important river in central iran with semi-arid region (safavi et al., 2014). the zayandeh river drainage has water supply from the central zagros mountains, covers 41,500 km2 and finally enters the gavkhuni international wetland (babaei et al., 2013; sanayei et al., 2009). the zayandeh river dam lake, locating 110 km west of isfahan city (32°44′ 06.51″n, 50°44′15.75″ e), covers an area of 54 km2 and has capacity to hold 150 × 106 m3 water (shams et al., 2012). the lake supplies freshwater for municipal, agricultural and industrial activities of three central iranian provinces (hajian and rahsepar, 2010). according to the importance of the lake (as mentioned above), it is important to monitor its water quality; therefore, this study aimed to assess the trophic state of the zayandeh river dam lake in the spring and summer of 2017. materials and methods sampling: the randomized complete block design (rcbd) (anderson and mclean, 2018) was used to choose 4 sampling sites in the north (a: chadegan), west (b: mashhad kaveh), east (c: hojjatabad) and south (d: yancheshmeh) of the lake (fig. 1). monthly sampling of surface water was done in the spring and summer, 2017. all samples were collected at 10 am to 2 pm. from each station, three 1.5-liter dark bottles of water were collected and transferred to the laboratory under controlled conditions. the water temperature was measured in the sampling site; whereas, electroconductivity (ec), total dissolved solids (tds), total suspended solids (tss), ph, turbidity, dissolved oxygen (do), biological oxygen demand (bod5), nitrate and phosphate were determined according to apha (2005). the samples’ chl-a content was measured according to arnon (1967). chl-a extraction: all samples were transferred to the laboratory at 4°c. in order to determine the chlorophyll contents, the samples were extracted, using 80% acetone. the supernatant was used to measure the absorbance with a spectrophotometer (arnon, 1967). finally, chl-a content was determined at 663 and 645 nm. table 1. carlson classification for trophic status (oecd, 1977). tsitrophic state 0-40oligotrophic 40-60mesotrophic 60-100eutrophic figure 1. geographical map of sampling stations. 211 int. j. aquat. biol. (2020) 8(3): 209-215 chl-a (mg/ g tissue) = )12.7 (a663) – 2.69 (a645) (× v/1000 × w where v is the final volume of chl-a extract in 80% acetone, a= absorbance of specific wavelength and w= fresh weight of tissue extract. tsi: tsi (carlson, 1977) was developed based on transparency as relative indicators of algae biomass, that is the most suitable and acceptable method for evaluating inland lake’s eutrophication (duan et al., 2007). but several studies have claimed that transparency is influenced by various factors (aizaki et al., 1981). therefore, modified carlson index was proposed based on chl-a concentration (table 1), with 0–100 continuous numerical classes of lakes trophic states (oecd, 1982). statistical analyses: all data were tested in terms of normality and homogeneity of variance before conducting parametric statistical analysis. variability among sampling sites was analyzed for each water parameter by one-way anova. to detect differences among individual mean, we used duncan multiple range test. the relationships among the tested parameters were evaluated by pearson correlation (p<0.05) using spss software (van belle et al. 2004; thode, 2002). results analysis of variance between the sampling stations and times are presented in table 3. the results of comparison between the stations and times are reported in tables 4 and 5, respectively. based on the results, turbidity showed significantly different between the stations and the highest value was observed in the station b (mashhad kaveh). according to the results (table 5, fig. 2), the lowest and highest ec and tds were found in august and june, respectively. the highest water tds, bod5 and po4 were recorded to june (spring), august (summer) and may (spring), respectively. the lowest tds was recorded in the summer and the lowest bod5 in may and september. there was no significant difference in po4 levels between the spring and summer, except in may that had the highest value. the highest water and ambient temperature table 2. modified carlson classification for trophic status (oecd, 1982). oligotrophic mesotrophic eutrophic total po4(µg/l) mean 8 26.7 84.4 range 3.7-17 9.6-10.95 16-386 total no3(µg/l) mean 661 753 1857 range 307-1630 361-1387 393-6100 chlorophyll-a(µg/l) mean 1.7 4.7 14.3 range 0.4-3.5 3-11 3-87 table 3. analysis of variance between the stations. parameters stations months f-value pvalue f-value pvalue ph 0.372 0.77 2.911 0.058 ec (μmhos/cm) 0.462 0.713 3.842 0.024* turbidity (ntu) 7.43 0.002 0.404 0.803 tds (mg/l) 0.459 0.715 3.805 0.025* tss (mg/l) 0.822 0.501 1.60 0.225 do (mg/l) 0.919 0.454 3.023 0.052 bod5 (mg/l) 0.037 0.990 6.22 0.004* po4 (mg/l) 0.932 0.448 2.43 0.008* no3 (mg/l) 0.4 0.755 1.31 0.093 chlorophyll-a (µg/l) 0.280 0.839 0.842 0.52 ambient temperature (°c) 0.016 0.997 4.47 0.014* water temperature (°c) 1.45 0.265 7.83 0.001* *significant difference at 0.05; ** significant difference at 0.01 212 asadian et al./ trophic status of the zayandeh river dam lake table 4. mean comparison (duncan) between the stations. parameters station a station b station c station d ph 7.58±0.41a 7.59±0.28a 7.71±0.13a 7.72±0.17a ec (μmhos/cm) 288.7±39.11a 318.46±37.78a 310.22±40.25a 299.36±51.73a turbidity (ntu) 2.68±0.87b 16.28±11.45a 2.26±1.65b 1.48±0.68b tds (mg/l) 152.00±21.20a 168.00±20.28a 164.40±21.76a 158.00±27.28a tss (mg/l) 7.60±4.04a 5.60±0.89a 5.60±1.52a 5.40±2.61a do (mg/l) 6.28±0.13a 6.50±0.47a 6.20±0.52a 6.58±0.43a bod5 (mg/l) 6.70±0.52a 6.64±0.47a 6.68±0.50a 6.74±0.43a po4 (mg/l) 0.82±0.045a 1.14±0.078a 0.62±0.027a 0.6±0.022a no3 (mg/l) 6.26±1.68a 7.18±0.87a 6.54±1.53a 6.88±1.34a chlo-a (µg /l) 2.9±0.16 a 5.02±0. 24a 4.06±0. 24a 3.86±0. 27a ambient t(°c) 22.60±1.95a 23.00±4.30a 22.60±3.21a 23.00±5.96a water t(°c) 17.80±2.49a 14.20±3.56a 14.20±3.27a 14.60±3.51a similar letters mean non-significant difference among the stations. table 5. mean comparison (duncan) between the sampling time and the results of tsi index. parameters may june july august september ph 7.87±0.13a 7.74±0.22a 7.59±0.22ab 7.37±0.31b 7.68±0.17ab ec (μmhos/cm) 337.38±23.85ab 339.50±14.93a 287.88±20.08bc 281.45±61.09c 275.05±16.70c turbidity (ntu) 8.35±2.48a 4.01±1.38a 9.00±4.03a 2.77±0.75a 4.22±1.64a tds (mg/l) 178.00±12.73ab 179.25±7.80a 151.75±10.84bc 147.75±32.82c 145.00±8.76c tss (mg/l) 8.50±3.42a 6.50±3.79a 5.00±0.82a 5.00±0.82a 5.25±0.96a do (mg/l) 6.00±0.16a 6.83±0.42a 6.45±0.21a 6.43±0.55a 6.25±0.24a bod5 (mg/l) 6.28±0.10 c 7.05±0.51ab 6.60±0.18bc 7.13±0.32a 6.40±0.26c po4 (mg/l) 1.6±0.08a 0.5±0.01b 0.5±0.01b 0.5±0.01b 0.87±0.01b no3 (mg/l) 7.92±0.85a 7.13±0.22a 6.78±0.34a 6.24±1.86a 5.53±1.54a chlo-a (µg/l) 5.37±0.34a 2.37±0.22a 3.15±0.23a 2.80±0.28a 2.20±0.22a ambient t (°c) 19.00±2.58b 20.50±1.00b 25.75±4.99a 23.00±2.73a 25.75±0.96a water t (°c) 12.25±1.26c 12.75±2.87c 15.25±3.30b 16.00±1.41b 19.75±0.50a tsi (chlorophyll-a) 44.6 31.75 37.8 35.1 35.9 similar letters mean non-significant difference between the sampling times. figure 1. variations of water quality parameters among the sampling time. 213 int. j. aquat. biol. (2020) 8(3): 209-215 were observed in the summer (july, august and september) and the lowest in the spring (may and june). there was no significant difference in chl-a content between the sampling stations. according to tsi (table 5), chl-a was higher in may compared to the other times. discussions based on the results, ph, tss, turbidity, do, no3 and chl-a were relatively stable in the spring and summer, as there were no significant differences. the highest phosphate concentrations were measured in may, which might be as a result of raining and input of agricultural drainage to the dam lake. the highest and lowest water temperatures were measured in august and may, respectively and the highest water turbidity in the station b (mashhad kaveh), which might be due to water turbulence near the station. based on tsi (chl), the mean trophy level of the zayandeh river dam lake in the spring and summer is oligotrophic. however, the lake is in mesotrophic state in the spring (may), probably due to floods, runoff and drainage of farmlands. due to high temperatures and biological activity in the summer, nitrate and phosphate decomposition increases. on the other hand, the concentration of the nutrients especially nitrate decreased compared to the spring, probably due to decreases in agricultural activities. therefore, the lake was oligotrophic from june to september. shams et al. (2012) reported that zayandeh river dam lake is oligo-mesotrophic based on the seasonal variations in phytoplankton communities. based on chl-a measurment, movahhedinasab (2013), in the spring and summer, and rajae (2013), in the autumn and winter, categorized the lake as oligotrophic. according to tsi(sd) and tsi(chl), malekzadeh (2014) reported the lake as mesotrophic. based on physical and chemicals parameters and phytoplankton communities, hamidi et al. (2014) classified the lake as oligo-mesotrophic. khalaji et al. (2017) estimated that the water quality of the zayandeh river's dam lake is good (50-100) based on wqi. the main notable point is that the eutrophic is often equal to poor water quality. the quality of water depends on the water applications and the local attitude of the people. the concept of trophic status and its index should be merely a framework for assessing water quality and should remain neutral to such subjective judgments. references aizaki m., otsuki a., fukushima t., hosomi m., muraoka, k. (1981). application of carlson's trophic state index to japanese lakes and relationships between the index and other parameters. internationale vereinigung für theoretische und angewandte limnologie: verhandlungen, 21: 675-681. anderson v.l., mclean r.a. (2018). design of table 6. pearson correlations between the tested parameters. p h e c t u rb id i ty t d s t s s d o b o d 5 p o 4 n o 3 c o l. a a m b ie n t t w a te r t ph 1 0.47 10.0** 0.48 0.28 -0.29 -0.61 0.40 0.31 60.1* -0.29 -0.13* ec 0.47 1 0.00* 1 0.34 90.0* -0.10* 20.1* 0.77 40.1* -0.53 -0.57 turbidity 0.01** 0.00** 1 0.00** -0.10* 50.0** -0.13* 0.46 10.0** 0.37 80.0** -0.26 tds 0.48 1 0.00** 1 0.34 90.0* -0.11* 30.1* 0.77 50.1* -0.52 -0.57 tss 0.28 0.34 -0.10* 0.34 1 -0.34 -0.47 30.1* 0.44 0.38 -0.25 -0.13* do -0.29 0.09* 50.0** 90.0* -0.34 1 0.65 -0.42 40.0** -0.14* 0.02** -0.29 bod5 -0.61 -0.10* -0.13* -0.11* -0.47 0.65 1 -0.50 -0.23 -0.34 0.00** -0.20* po40.40 0.12* 0.46 30.1* 30.1* -0.42 -0.50 1 0.11 50.1* -0.31 -0.15* no30.31 0.77 10.0** 0.77 0.44 40.0** -0.23 10.1* 1 0.33 -0.51 -0.51 chlo.a 60.1* 0.14* 70.3* 0.15 80.3* -0.14 -0.34* 0.15 30.3* 1 -0.25 -0.32 ambient t -0.29 -0.53 80.0** -0.52 -0.25 -0.02** 0.00** -0.31 -0.51 -0.25 1 0.51 water t -0.13* -0.57 -0.26 -0.57 -0.31* -0.29 -0.20* -0.15* -0.51 -0.32 0.51 1 * significant difference at 0.05 ** significant difference at 0.01 214 asadian et al./ trophic status of the zayandeh river dam lake experiments: a realistic approach: routledge. crc press. 440 p. apha (2005). standard methods for the examination of water and wastewater. american public health association (apha): washington, dc, usa. arnon a. 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(2021) 9(1): 1-10 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article genetic diversity and population structure of barilius barna (hamilton, 1822) in the subhimalayan dooars region of west bengal, india through mitochondrial cytochrome oxidase i sequence analyses ajoy paul1,2, tanmay mukhopadhyay1,3, soumen bhattacharjee*1 1cell and molecular biology laboratory, department of zoology, university of north bengal, p.o. north bengal university, raja rammohunpur, siliguri, india. 2protein engineering and neurobiology laboratory, department of biosciences and bioengineering, indian institute of technology bombay, powai, mumbai, india. 3department of zoology, north bengal st. xavier’s college, rajganj, india. s article history: received 8 june 2020 accepted 22 january 2021 available online 2 5 february 2021 keywords: teesta coi haplotype phylogeny abstract: the genetic diversity and the population structure of barilius barna (hamilton, 1822) wild population from the teesta river were assessed through mtdna cytochrome oxidase i (coi) sequence analyses. the haplotype and nucleotide diversity analyses revealed low level of genetic diversity in the b. barna wild populations, especially in the lower reaches of teesta (bholarhat). the genetic differentiation and gene flow between the two study sites were 0.08434 and 2.71, respectively. tajima’s d, fu and li’s d and fu and li’s f analyses were used to assess population differentiation in the two study sites. haplotype networking and phylogenetic analyses clearly distinguished the two populations from each other, as well as from other populations from other parts of the country. nature and implications of the genetic and haplotype diversities among the populations are discussed. phylogenetic analyses also indicated that the gajoldoba population is genetically closer to north indian river populations, than that to bholarhat population. introduction barilius barna (hamilton, 1822) (cyprinidae) is an economically important tropical fresh water fish found in the teesta river and adjoining rivers of north bengal, india. barilius barna has a “ne” or “not evaluated” status according to the iucn but has noteworthy ornamental and market values. the conservation status of this species is considered as “lower risk near threatened” (lrnt) according to camp (conservation assessment and management plan) report for freshwater fishes of india (molur and walker, 1998). however, during the past few years, its population has dwindled substantially and the fish has become increasingly rare in rivers of north bengal, india. the dwindling population of b. barna may be ascribed to over-exploitation, dam/hydroelectric power plant constructions, urban effluents and/or pesticide run-offs from the nearby tea gardens. the loss of genetic diversity of any economically valuable species in the hotspot region is a great threat to *correspondence: soumen bhattacharjee doi: https://doi.org/10.22034/ijab.v9i1.901 e-mail: soumenb123@rediffmail.com biodiversity locally, as well as globally. the study region is situated at the north-eastern sub-himalayan region of india which is known for its proximity to himalayan biodiversity hotspot. previously we have studied the genetic architecture of b. barna by rapd and issr markers (paul et al., 2018) and have found that the genetic diversity of this fish has dwindled. owing to the inherent nature of those dominant and arbitrary markers, we set out to ascertain the present status of genetic diversity through mitochondrial dna-based assessment of the local wild populations of b. barna in the teesta river of north bengal, india. application of molecular data in conservation of endangered species has been practiced in various organism groups at the national and international level for a long time. mitochondrial dna (mtdna) has been widely adopted for population genetic studies and it being almost exclusively maternally inherited has some advantages over the nuclear dna 2 paul et al./ genetic diversity of barilius barna (billington, 2003). mitochondrial dna is highly variable in natural populations because of its elevated mutation rate, which can generate some information about population history over short time frames. being involved in basic metabolic functions (cellular respiration), mitochondrial genes have been considered as less likely than other genes to be involved in adaptive processes (galtier et al., 2009). one consequence of maternal transmission is that the effective population size for mtdna is smaller than that of nuclear dna, therefore, mtdna variation is a more sensitive indicator of population phenomena (avise, 1994). among the most frequently used mitochondrial genes for detecting genetic data, the one coding for cytochrome oxidase subunit i (coi) is amplified using polymerase chain reaction methods using conserved primers (folmer et al., 1994). in this regard, the primary goal of the present study was to ascertain the genetic diversity available at mitochondrial coi and also to investigate the phylogenetic relationships of the isolates with other b. barna populations, based on publicly available sequences. materials and methods a detailed survey has been carried out in two spots (at two different altitudes) of the teesta river of the subhimalayan, dooars region of west bengal, india. barilius barna were collected from the river during october, 2015 and march, 2017. geographic coordinates were recorded using handheld gps (etrex vista hcx, garmin, usa). the fishes were carried to the laboratory immediately after collection in ice bucket and identified (shaw and shebbeare, 1934; talwar and jhingran, 1991). the collection spots were as follows: gbb (gajoldoba b. barna) and bbb (bholarhat b. barna). the location and geographical co-ordinates of the collection spots are mentioned in figure 1. isolation of genomic dna and quantification: genomic dna (gdna) was isolated from small amount of tissue samples (10-15 mg of fin clips) using commercial dna isolation kit (dneasy blood and tissue kit, qiagen). the isolated gdna samples were stored in 1.5 ml microcentrifuge tubes at -20ºc. the gdna samples were quantified using spectrophotometer (rayleigh uv-2601 spectrophotometer, beijing, china). the concentration of the extracted gdna was adjusted to 50 ng/µl for subsequent pcr amplifications. the used primer sequences are follows: f1: 5′tcaaccaac cacaaagacat tggcac-3′ (gc content 42.30%); r1: 5′taga c ttctgggtggccaaa gaatca-3′ (gc content 46.15%) (ward et al., 2005). they were synthesized by imperial life science (gurgaon, india). pcr amplification and documentation of amplified products: mitochondrial coi gene amplification was performed in a 96 wells eppendorf® (germany) peltier thermal cycler. the final reaction volumes were 30 µl, each containing a final concentration of ~100-150 ng of isolated gdna, 1 pm figure 1. map showing two collection spots of barilius barna from the teesta river of dooars region of west bengal, india (gbb= gajoldoba (n 26°44´584, e 88°35´314 elev 354 amsl) and bbb= bholarhat (n 26°21´423, e 88°50´485 elev 193 amsl). 3 int. j. aquat. biol. (2021) 9(1): 1-10 of each oligonucleotide primers (forward and reverse), 1x standard taq polymerase buffer (10 mm tris-hcl, ph 8.3, 50 mmkcl, 1.5 mm mgcl2) (neb, usa), 200 µm of each dntps (datp, dttp, dctp, dgtp) (neb, usa), and one unit of taq dna polymerase (neb, usa). pcr cycling programs were as follows: initial denaturation at 94ºc for 5 min followed by 40 cycles of 94ºc, 1 min for denaturation; 50ºc, 45 sec for annealing; 72ºc, 1 min for elongation and finally an extension at 72ºc for 10 min. the amplified products were electrophoresed in an ethidium bromide (0.5 µg/ml) pre-stained 1.5% (w/v) agarose gel (lonza, switzerland) at a constant voltage 100 v and current 100 ma in tae buffer (40 mm tris-hcl, ph 8.0; 20 mm acetic acid; 1 mm edta, ph 8.0) using benchtop labsystems bt-ms-300 (taiwan) electrophoretic apparatus. molecular weight of each band was estimated using a standard 100 base pair ladder (neb, usa). the gels were visualized on the uv-transilluminator (spectroline bi-ovision®ny, usa) and photographed using a nikon d3100 camera (fig. 2). purification of pcr product and sequencing of coi: the amplified products were purified using invitrogen purelinktm pcr purification kit (thermofisher scientific, india), following manufacturer’s protocol. the amplified products were confirmed as coi partial coding gene by restriction digestion using hind iii restriction enzyme (hind iii cutting point between 252th-253th bp). each pcr product was sequenced at least twice by dye-dideoxy automated chain termination method (mwg-biotech pvt. ltd., bangalore india). all the coi partial cds were used separately to search the gb/embl/ddbj combined nr database in ncbi blast search through the implementation of blastn and blastx, optimized for highly similar sequences (megablast), using stringent expect threshold (0.01) and excluding low complexity regions within the combined database. all the sequences identified b. barna and b. bendelisis had high scores and low expect values (data not shown). the curated sequences (approximately 606 bp) were submitted to genbank and accession numbers were obtained (table 1). genetic diversity and population structure: levels of genetic variation within the gajoldoba and bholarhat populations were estimated in terms of the number of variable sites (s), total number of mutations (eta), number of haplotypes (h), haplotype diversity (h), nucleotide diversity (πn) and average number of nucleotide differences by dnasp ver. 5.1 software (librado and rozas, 2009). the genetic differentiation (fst) and gene flow (nm) were calculated following the method of wright (1978), govindaraju (1989) and low et al. (2014). tajima’s d (tajima, 1989), fu and li’s d and fu and li’s f (fu, 1997) were calculated to verify selective neutrality in relation to mtdna sequences, which would help to accumulate information regarding the demographic history of the population and used to deduce whether a population has undergone a sudden population expansion or construction. figure 2. representative gel photograph of the mtdna co1 pcr amplicons. m=100 bp dna size ladder, lane no. 1-10= individuals from gbb and lane no. 11-20= individuals from bbb. 4 paul et al./ genetic diversity of barilius barna preparation of sequence dataset and phylogenetic analyses: twenty raw sequence reads, each pcr product sequenced at least twice, were curated in bioedit ver. 7.0.9 (hall, 1999) for character uncertainty and then assembled in geneious r7 ver 7.0.6 (trial) (rozen and skaletsky, 2000) using pro features and retrieved the final sequences as fasta files. multiple sequence alignment was implemented in clustalx ver 2.0.3 (thompson et al., 1997) using high gap opening (50) and gap extension (50) penalties. the best model of dna substitution selected was gtr or general time reversal (tavare, 1986) plus i+γ based on the akaike information criterion (aic) implemented in jmodeltest v0.1.1 (guindon and gascuel, 2003; posada, 2008). publicly available twenty-eight b. barna coi partial cds corresponding to isolates submitted from northern india were retrieved from genbank before august 21, 2017. the isolate names and their accession numbers are presented in table 1. the final dataset contained 655 positions which included twenty-eight 606 to 655 nucleotides long genbank sequences and twenty 605 to 606 nucleotide long b. barna coi sequences from the teesta river. phylogenetic analyses: the evolutionary relationship between forty-eight b. barna mitochondrial coi sequences was estimated using bayesian coupled with markov chain monte carlo (bmcmc) methods of phylogenetic inference and maximum likelihood (ml). bmcmc searches (huelsenbeck, 2001) were performed in mrbayes v3.1 (huelsenbeck, 2001; ronquist, 2003) using the following model parameters: base frequencies f(a), 0.2220; f(c), 0.2506; f(g), 0.2164; f(t), 0.3110; substitution rates r[ct], 3.3482; r[cg], 0.9238; r[at], 2.1907; r[ag], 4.7431; r[ac], 1.1675; proportion of invariant sites pinv, 0.0780; and shape parameter of the gamma distribution α, 1.3620. four markov chains (4x2x106 cycles; ngen = 1000000) were run simultaneously, which were started from random trees and sampled every 1,000th cycle (samplefreq = 1000). tracer v1.2 (rambaut, 2003) was used to check whether stationarity in the fluctuating values of the likelihood and all the phylogenetic parameters had been reached. each simulation was repeated four times (nchain = 4) starting from different random trees (startingtree = random). all sample points prior to reaching stationarity were discarded as burn in (burninfrac = 0.25). the posterior probabilities for individual clades obtained from separate analyses were compared for congruence and then combined and summarized in a table 1. list of species used for molecular analysis and their genbank accession number and deposited as result of the present study. accession number species reference for sequences kx649920 barilius barna paul et al. 2016 kx649921 barilius barna paul et al. 2016 kx649922 barilius barna paul et al. 2016 kx649923 barilius barna paul et al. 2016 kx649924 barilius barna paul et al. 2016 kx649925 barilius barna paul et al. 2016 kx649926 barilius barna paul et al. 2016 kx649927 barilius barna paul et al. 2016 kx649928 barilius barna paul et al. 2016 kx649929 barilius barna paul et al. 2016 kx649930 barilius barna mukhopadhyay et al. 2016 kx649931 barilius barna mukhopadhyay et al. 2016 kx649932 barilius barna mukhopadhyay et al. 2016 kx649933 barilius barna mukhopadhyay et al. 2016 kx649934 barilius barna mukhopadhyay et al. 2016 kx649935 barilius barna mukhopadhyay et al. 2016 kx649936 barilius barna mukhopadhyay et al. 2016 kx649937 barilius barna mukhopadhyay et al. 2016 kx649938 barilius barna mukhopadhyay et al. 2016 kx649939 barilius barna mukhopadhyay et al. 2016 hm042158 barilius barna mishra et al. 2010 hm042159 barilius barna mishra et al. 2010 hm042160 barilius barna mishra et al. 2010 hm042161 barilius barna mishra et al. 2010 hm042162 barilius barna mishra et al. 2010 hm042163 barilius barna mishra et al. 2010 hm042170 barilius barna mishra et al. 2010 hm042171 barilius barna mishra et al. 2010 hm042172 barilius barna mishra et al. 2010 hm042173 barilius barna mishra et al. 2010 hm042174 barilius barna mishra et al. 2010 hm042175 barilius barna mishra et al. 2010 hm042164 barilius barna mishra et al. 2010 hm042165 barilius barna mishra et al. 2010 hm042166 barilius barna mishra et al. 2010 hm042167 barilius barna mishra et al. 2010 hm042168 barilius barna mishra et al. 2010 hm042169 barilius barna mishra et al. 2010 hm042176 barilius barna mishra et al. 2010 hm042177 barilius barna mishra et al. 2010 hm042178 barilius barna mishra et al. 2010 hm042179 barilius barna mishra et al. 2010 hm042180 barilius barna mishra et al. 2010 hm042181 barilius barna mishra et al. 2010 eu417797 barilius barna lakra et al. 2008 eu417798 barilius barna lakra et al. 2008 eu417799 barilius barna lakra et al. 2008 jn965190 barilius barna kalyankar et al. 2011 5 int. j. aquat. biol. (2021) 9(1): 1-10 majority rule consensus tree. ml searches (felsenstein, 1981) of sequence data set were performed in mega v6 (tamura et al., 2013) based on software suggested model (k2+g) parameters: equal base frequencies; substitution rates r(at), 0.032; r(ac), 0.032; r(ag), 0.187; r(ta), 0.032; r(tc), 0.187; r(tg), 0.032; r(ca), 0.032; r(ct), 0.187; r(cg), 0.032; r(ga), 0.187; r(gt), 0.032; r(gc), 0.032; and shape parameter of the gamma distribution α, 0.3845. ml heuristic search was implemented using 100 bootstrapping option by a slow but accurate subtree pruning and regrafting (spr level 5) method with starting bionj trees. all the trees were built as midpoint-rooted consensus trees using figtree v1.3.1 (http://tree.bio.ed.ac.uk /software/figtree/). haplotype network analysis: intraspecific gene genealogies were inferred using haplotype network construction method, implemented in freely available software package network ver 5 (www.fluxusengineering.com/sharenet.htm). the algorithm for constructing the minimum spanning trees (msts) from a matrix of pairwise distances (absolute number of differences) among haplotypes (prim, 1957; rohlf, 1973) has been modified to include all possible msts within a single graph as the minimum spanning network (msn) (excoffier and smouse, 1994). all 48 taxa were aligned by clustalw ver. 2 (thompson et al., 1994) and were concatenated to yield a total length of 605 bp. in the median-joining network approach (bandelt et al., 1999), all msts are first combined within a single network (msn) following an algorithm analogous to that proposed by excoffier and smouse (1994). then, using the parsimony criterion, inferred intermediate haplotypes were added to the network to reduce overall tree length. using the parsimony criterion, inferred intermediate haplotypes were added to the network to reduce overall tree length. in addition, by setting a parameter nucleotide character weight = 10 and epsilon (ε) value = 0, less parsimonious pathways were excluded in the inferred network (bandelt et al., 1999). results diversity and population structure: we have found a total number of variable sites were 106 and 102 in bholarhat (bbb) and gajoldoba (gbb), respectively, and 108 when two populations were considered together (table 2). total numbers of mutations were 107 and 102 in bbb and gbb populations, respectively (table 2). total 13 haplotypes were found in the teesta river population, where 5 were for bbb population and 8 for gbb (table 2). the haplotype diversity and nucleotide diversity of bbb population were 0.756±0.01678sd and 0.06329 ±0.0000684sd, respectively; and of gbb population were 0.933±0.00597sd and 0.03607±0.0006035sd, respectively (table 2). the genetic differentiation (fst) and gene flow (nm) between gbb and bbb populations were 0.08434 and 2.71, respectively (table 2). the observed values of tajima’s d, fu and li’s d and fu and li’s f analyses of bholarhat population were 0.10843 (not significant; p>0.10), 1.61711 (significant; p<0.02) and 1.40063 (not significant; p>0.10), respectively; and in gajoldoba population were 1.95643 (significant; p<0.05), 2.35348 (significant; p<0.02) and -2.54752 (significant; p<0.02), respectively. table 2. population genetic diversity parameters based on barilius barna mtdna coi partial coding sequences. popu lations no. of variable sites (s) total no. of mutatio ns (eta) no. of haplotype s (h) haplotype (gene) diversity (hd±sd) nucleotide diversity (pi±sd) average number of nucleotide differences (k) genetic differentiation, (fst) between gbb and bbb population gene flow (nm) between gbb and bbb population bholarhat (bbb) 106 107 5 0.756 ±0.01678 0.06329 ±0.0000684 38.28889 0.08434 2.71 gajoldoba (gbb) 102 102 8 0.933 ±0.00597 0.03607 ±0.0006035 36.05555 whole population 108 109 13 0.926 ±0.00185 0.08996 ±0.0000487 54.42632 *coi, cytochrome oxidase subunit i; sd, standard deviation 6 paul et al./ genetic diversity of barilius barna bmcmc and ml analyses: the dichotomy of the two main lineages was strongly supported by high posterior probabilities (pp) (1.0) and bootstrap (100% represented as 1.0) in the bmcmc and ml midpoint rooted phylogenetic trees, respectively (fig. 3). the phylogenetic trees suggested that eight tbbrn (bholarhat population), one tgbrn (gajoldoba population) and one north indian isolate (kr04) are closely related to each other than to other b. barna coi sequences. while nine tgbrn (gajoldoba population) and two tbbrn (bholarhat population) formed a highly supported sister clade along with all the other north indian b. barna sequences in bmcmc analysis (fig. 3). almost identical topology and phylogenetic relationships were produced in the ml analysis as well as found in bmcmc analyses implemented in mega ver. 6 (fig. 3). network analysis of haplotypes: total 48 taxa were used to construct the haplotype network (fig. 4). haplotype network of b. barna coi were in agreement with the phylogenetic reconstruction (fig. 3). the haplotype network showed that the segregation of mitochondrial haplotypes was in accordance with the locality (figs. 3, 4). a total of 21 haplotypes were identified in the b. barna, distributed in three main geographic groups. among them five (hap 01-05) haplotypes were distributed in bholarhat and eight (hap06-13) were distributed in gajoldoba (fig. 4). remaining eight (hap14-21) haplotypes were distributed in the northern region of india. a unique haplotype (hap21 i.e., kr04) connects with the hap 04 and hap 05 haplotypes in bholarhat population. there was no sharing of haplotypes within these three groups. the network shows variations in nucleotide character positions 601 (hap7-hap8, hap9-mv10 and hap1-hap2, hap11-hap12, mv1-mv2), 148 (hap7mv10, hap8-hap9, mv1-hap11, mv2-hap12), 589 (hap1-mv1, hap2-mv2, mv2-mv3, mv4-mv5) and 271 (mv3-mv 4, mv2-mv5) which indicate parallel mutations or homoplasy (fig. 4). figure 3. bmcmc and ml phylogenetic analyses showing the evolutionary relationship of forty-eight barilius barna mitochondrial coi inferred by using the gtr and kimura 2 models, respectively. the posterior probability (bmcmc) and bootstrap values (ml) in which the associated taxa clustered together are shown next to the major branches (gajoldoba isolates are in green and bholarhat isolates are in blue. posterior probabilities (pp) (1.0) and bootstrap (100% represented as 1.0) values are represented as pp/bootstrap). 7 int. j. aquat. biol. (2021) 9(1): 1-10 number of haplotypes name of haplotypes taxon names source 1 hap 01 tbbrn10 (teesta river) bholarhat 2 hap 02 tbbrn6 3 hap 03 tbbrn2, tbbrn4, tbbrn7, tbbrn8, tbbrn9 4 hap 04 tbbrn5 5 hap 05 tbbrn3, tbbrn13 6 hap 06 tgbrn15 (teesta river) gajoldoba 7 hap 07 tgbrn2, tgbrn11, tgbrn20 8 hap 08 tgbrn4 9 hap 09 tgbrn5 10 hap 10 tgbrn9 11 hap 11 tgbrn6 12 hap 12 tgbrn14 13 hap 13 tgbrn13 14 hap 14 wl-f53, wl-f55, wl-f56 northern india 15 hap 15 brn46, brn49, brn50, brn52, brn53, brn54 16 hap 16 brn15, brn17, brn20 17 hap 17 brn30, brn33, brn35, brn36, brn39, brn40 18 hap 18 brn23, brn25, brn29 19 hap19 brn12 20 hap 20 brn1, brn3, brn5, brn6, brn10 21 hap 21 kr04 figure 4. median-joining network for the coi haplotypes in barilius barna populations from teesta river of north bengal, india and northern region of india. each circle represents a unique haplotype, with the area being proportional to the frequency of the haplotypes. the network showed the mutational changes. the black and red boxes indicate the nucleotide position of mutational changes. m1-4=contain >5 number of mutations, m5=583,586,589; m6=37, 220, 268, 313, 454; m7=34, 35, 133; m8=214, 336; m9=474, 558; m10=306, 474; m11=72, 132. 8 paul et al./ genetic diversity of barilius barna discussions the results revealed low level of genetic diversity in the dwindling wild population of b. barna from two different locations of the teesta river of subhimalayan northern west bengal, india. the mitochondrial dna coi gene sequences are highly polymorphic in nature, as reported in many genetic studies on geographically isolated populations of different organisms. similar study had carried out on snakehead fish from thailand, a total 33 haplotypes among 60 individuals was found and; haplotype and nucleotide diversity ranged from 0.75-1.0 and 0.00442-0.2672, respectively (boonkusol et al., 2016). in the present study, we found that the total numbers of haplotypes were 13 for 20 individuals of b. barna distributed in two different populations of teesta river of north bengal, india. these reveal that the genetic diversity (number of haplotypes, haplotype diversity and nucleotide diversity) is low in b. barna population as evident from the present mitochondrial coi study. the present findings are also in accordance with our previous study on b. barna of the same river, where we have found that the overall genetic diversity (polymorphism, nei’s genetic diversity and shannon’s information index) was low as revealed by rapd and issr marker (paul et al., 2018). the study carried out by low et al. (2014) categorized the levels of genetic differentiation as fst>0.25 (great differentiation), 0.15 to 0.25 (moderate differentiation), and fst<0.05 (negligible differentiation). the levels of gene flow can be categorized as nm>1 (high gene flow), 0.25 to 0.99 (intermediate gene flow), and nm<0.25 (low gene flow). the genetic differentiation (fst) observed in our study was 0.08434 which is comparatively low than the study carried by boonkusol et al. (2016) on snakehead fish from thailand (fst ranged from 0.27891 to 0.40627). the low level of genetic divergence between the study regions may be attributed to the migratory behaviour and gene flow (nm=2.71), as evident in our present study. these findings were also supported by our previous study on b. barna where we have detected sufficient level gene flow between gajoldoba and bholarhat population (paul et al., 2018). anthropogenic could be the possible reasons behind the dwindling population structure of this species in the study region. the bholarhat population gave positive values for tajima’s d, fu and li’s d and fu and li’s f test, which indicated that the population showed balancing selection and sudden population contraction; whereas, the gajoldoba population gave negative values for all the test, which indicated a selective sweep and a population expansion after a recent bottleneck (tajima, 1989; fu and li, 1993). the study of thirumaraiselvi and thangaraj (2015) on six eleutheronema tetradactylum populations from south asian countries also found negative values for the tests which indicated sudden population expansion of the e. tetradactylum population. in our study, we have found a total 13 haplotypes from twenty different individuals of b. barna from teesta river of north bengal india and 9 haplotypes from 28 individuals from northern india. the haplotypes network showed possible ancestral connections within the network along with differentiation of haplotypes with respect to its connecting haplotypes. our study also revealed that some of the haplotypes showed shared parallel mutation at specific nucleotides or characters. this homoplasic condition decreases the genetic diversity within the population (wake, 1991; wake et al., 2011) and this observation is also in accordance with the haplotype diversity in our study. our mitochondrial cytochrome oxidase i based bmcmc and ml trees showed the phylogenetic relatedness between the two populations of the teesta river system of the dooars region of north-eastern west bengal, india. the clubbing of tgbrn9 (gajodoba) with the bholarhat population (blue taxa) and that of tbbrn6 and tbbrn10 (bholarhat) with the gajoldoba population (green taxa) also indicate a significant level of gene flow between the sites. with the unavailability of any data from the region we resorted to comparing our data with that of other b. barna coi sequences from the uttar pradesh state of northern india. our result indicated that the gajoldoba isolates are genetically closer, than that of 9 int. j. aquat. biol. 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(2018) 6(5): 294-295 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology short communication length-weight relationship for four fish species from the oman sea, iran asghar jafari-patcan, soheil eagderi*,1atta mouludi-saleh department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 28 september 2018 accepted 22 october 2018 available online 2 5 october 2018 keywords: oman sea, lwr, trawling, parameter b. abstract: the present study reports length-weight relationship (lwr) of four fish species viz. pennahia macrophtalmus, epinephelus bleekeri, acropoma joponicum and trichiurus lepturus from the oman sea, iran. a total of 880 specimens were collected from december 2016 to august 2017 by trawling. the lwr parameter b for the studied species was 2.68 to 3.07. introduction the length-weight relationship of fishes is used to estimate the weight of a specimen from its length and vice versa, evaluation of fish stocks, estimating biomass, ontogenetic changes, growth rate studies and understanding the life cycle (froese, 2006; sarkar et al., 2008; kumolu-johnson and ndimele 2010). such information is scarce for fish species of the oman sea, hence feels its necessity for fisheries management (oscoz et al., 2005). this contribution presents the parameters of lwrs for four species viz. pennahia macrophtalmus, (acanthuriformes: sciaenidae), epinephelus bleekeri (perciformes: serranidae), acropoma joponicum (perciformes: acropomatidae) and trichiurus lepturus (scombriformes: trichiuridae) from the oman sea. materials and methods the sampling was carried out on a monthly basis. a total of 880 specimens of four species, including 219 p. macrophtalmus, 81 e. bleekeri, 241 a. joponicum and 339 t. lepturus were collected in the depths of 168-276 m by trawling (30 mm mesh size) from the oman sea during december 2016 to august 2017. the collected fishes were preserved in the buffered 10% formalin at the field and transported to the *corresponding author: soheil eagderi doi: https://doi.org/10.22034/ijab.v6i5.562 e-mail address: soheil.eagderi@ut.ac.ir fisheries laboratory of university of tehran. the total length (tl) and total weigh of each individual were measured using dial calipers and digital scale to the nearest 0.1 mm and 0.1 g, respectively. the length-weight relationship was estimated by w=alb, and the logarithmic length‐weight equation as follows: log(w) = log(a)+blog(l). where w is the whole-body weight (g); l = the total length (mm), a = the intercept and b = the slope. prior to regression analyses, log-log plots of the length-weight pairs were performed to identify outliers (froese et al., 2011; radkhah and eagderi 2015). outliers perceive in the log-log plots of all species were evacuated from the regression. all statistical analyses were performed in excel 2016. results the specimens were ranged from 64.1-870.2 mm in total length and 48.1-5400 g in total weight. the number of samples, minimum and maximum of total length (mm), minimum and maximum of weight (g), length-weight relationships parameters (a, 95%ci-a, b and 95%ci-b) and the coefficient of determination (r2) of four studied species, including macrophtalmus, e. bleekeri, a. joponicum and t. lepturus are presented in table 1. lwr ranged 2.68 for a. joponicum to 3.07 for e. bleekeri. all 295 int. j. aquat. biol. (2018) 6(6): 294-295 relationships were highly significant (p<0.05) with r2 values greater than 0.75 in four species (r2 = 0.750.95). discussion the values of the b in lwrs falls between 2.5 and 3.5 (froese, 2006) or 2-4 (tesch, 1971). the value of b for p. macrophtalmus (2.72), e. bleekeri (3.07), a. joponicum (2.68) and trichiurus lepturus (2.71) are in the expected range (tesch, 1971; froese, 2006; zamani-faradonbe et al., 2015a, b). this study provided some basic information that will be useful for their fishery management and fish population dynamic studies. acknowledgments we are pleased to thank university of tehran for financial support. references froese r. (2006). cube law, condition factor and weightlength relationships: history, meta-analysis and recommendations. journal of applied ichthyology, 22: 241-253. froese r., tsikliras a.c., stergiou k.i. (2011). editorial note on weight–length relations of fishes. acta ichthyologica et piscatoria, 41(4): 261-263. kumolu-johnson c.a., ndimele p.e. (2010). lengthweight relationships and condition factors of twentyone fish species in ologe lagoon, lagos, nigeria. asian journal of agricultural science, 2: 174-179. oscoz j., campos f., escala m.c. (2005). weight–length relationships of some fish species of the iberian peninsula. journal of applied ichthyology, 21: 73-74. radkhah a., eagderi s. (2015). length-weight and lengthlength relationships and condition factor of six cyprinid fish species of zarrineh river (urmia lake basin, iran). iranian journal of ichthyology, 2(1): 61-64. sarkar u.k., negi r.s., deepak p.k., lakra w.s., paul s.k. (2008). biological parameters of the endangered fish chitala chitala (osteoglossiformes: notopteridae) from some indian rivers. fisheries research, 90(1-3): 170-177. tesch f.w. (1971). age and growth. in: w.e. ricker (ed.). methods for assessment of fish production in fresh waters. blackwell scientific publications, oxford. pp. 98-130. zamani faradonbeh m., eagderi s., ghojoghi f. (2015a). length-weight relationship and condition factor of seven fish species of totkabon river (southern caspian sea basin), guilan, iran. international journal of aquatic biology, 3(3): 172-176. zamani faradonbe m., eagderi s., naserabad s.s. (2015b). length-weight relationships and condition factor of three fish species from taleghan river (alborz province, iran). journal of advance botany and zoology, 2: 1-3. table 1. descriptive statistics and estimated parameters of length-weight relationships for four species from oman sea, iran. species total length (mm) weight (g) regression parameters n min max min max a b r2 95% cl of b 95% cl of a p. macrophtalmus (bleeker,1549) 219 166.3 438.2 48.1 1004.1 0.0005 2.72 0.85 2.32-3.07 0.0001-0.0009 e. bleekeri (vaillant,1878) 81 240.2 870.2 203.0 5400.3 0.0001 3.07 0.95 2.47-3.10 0.0001-0.0005 a. joponicum (gunther,1859) 241 64.1 271.3 5.3 65.5 0.0008 2.68 0.88 2.57-3.13 0.0005-0.0009 t. lepturus (linnaeus,1758) 339 191.6 545.6 100.0 1601.0 0.0006 2.71 0.75 2.64-3.24 0.0003-0.0009 n, number of individuals; a, intercept; b, slope; ci, confidence intervals; r2, coefficient of determination. int. j. aquat. biol. (2015) 3(3): 102-107 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article gillnets selection of alburnus chalcoides (güldenstädt, 1772) in almus dam lake (tokat, turkey) mehmet karataş1, erdoğan çiçek*21 1department of biology, faculty of science, karamanoglu mehmetbey university, karaman, turkey. 2department of biology, faculty of arts and sciences, nevsehir hacı bektas veli university, nevsehir, turkey. article history: received 12 december 2014 accepted 25 february 2015 available online 2 5 april 2015 keywords: alburnus chalcoides caspian shemaya gill-net selectivity almus dam lake abstract: in this study gill-net selectivity for alburnus chalcoides inhabiting the almus dam lake was estimated. experimental gill-net fishing was conducted using gill-net with 15 mm and 20 mm mesh-size, during october 2002 and september 2003. a total of 254 and 198 specimens of a. chalcoides were caught by gill-net with 15 mm and 198 with the 20 mm mesh-size, respectively. the total length of the fish ranged from 10.5 cm to 21.5 cm for 15 mm mesh-size and 13.6 cm to 25.6 cm for 20 mm mesh-size. mean total lengths were calculated as 14.34 ± 1.52 cm and 17.85 ± 1.97 cm for 15 mm and 20 mm mesh-size, respectively. selectivity analysis indicated an optimum length (100% probability of retention) of 14.77 cm for the 15 mm mesh size and 19.69 cm for the 20 mm mesh size gill-nets. selectivity factor was calculated as 0.9846. the vast majority (81%) of fish caught by 15 mm mesh size and 20 mm mesh size were mature. therefore, both the 15 mm and 20 mm mesh size were suitable for the sustainable fisheries of a. chalcoides in the almus dam lake. introduction the caspian shemaya (danube bleak), alburnus chalcoides inhabit lower reaches of rives, coastal lakes, estuaries, and brackish areas of sea. this species is widely distributed in the black, caspian and aral seas and the rivers connected them and reported from the western and southern parts of anatolia and probably has been spread toward further west turkey by translocations (bogustkaya, 1997; innal and erk’akan, 2006). alburnus chalcoides is a pelagic species occurring close to surface and adults predominantly prey on planktonic crustaceans, terrestrial insects and small fish while its larvae and young juveniles feed on zooplankton, algae and insect larvae (kottelat and freyhof, 2007). this species spawns in small rivers or streams with heavy current water on the gravel bottom. furthermore, the land-locked populations of a. chalcoides spawn in reservoir’s tributaries by * corresponding author: erdoğan çiçek e-mail address: n.ghysvandi@yahoo.com depositing their sticky eggs on pebbles or stones of river beds (kottelat and freyhof, 2007). spawning period of a. chalcoides in anatolia has been reported may till june (ünver and yıldırım, 2011; balık and sarı, 1994). the caspian shemaya is considered as an endangered species and threatened by habitat loss, eutrophication/pollution and construction of dams (fricke et al., 2007). in addition, this species is locally caught and utilized as a food resource in turkey, but does not consider as a commercial fish (geldiay and balik, 2007). therefore, there is no catch statistics available about this species (anonymous, 2014). the almus dam lake was constructed on yeşilırmak river in 1966 (almus, tokat). the total area of the lake is 3130 km2 with depth of 43 m. the mean water surface temperatures were reported as 17.4 ± 6.47°c (with range of 3.00-25.53°c) (kayım, 2002) and 14.8 ± 6.7°c (with range of 5.6-22.8°c) 103 karataş and çiçek/ effect of water temperature on food transit in caspian kutum (buhan et al., 2010). commercial fishing in the lake is conducted by almus fisheries cooperative mostly using gill-nets. water quality of the lake appropriate for rainbow trout cage culture (kayım, 2002; buhan et al., 2010) that have been started in 1999 (zengin and buhan, 2007). several studies have been conducted on the almus dam lake, e.g. water quality (pabucçu, 2000; kayım, 2002; buhan et al., 2010), fish fauna (akyurt and karataş, 1994; zengin and buhan, 2007), biology of inhabiting fish (cengizler, 1991; karataş, 1995; karataş and akyurt, 1997; yılmaz and suiçmez, 2010; yılmaz et al., 2011; suiçmez et al., 2011), parasitic fauna of fish (cengizler, 1993) and hydrographical characteristics (pabucçu, 2000). however, there is no information available regarding the gill-net selectivity of fish species inhabiting the almus dam lake. therefore, this study was aimed to identify the gill-net selectivity parameters of a. chalcoides inhabiting the almus dam lake. material and methods this study was carried out between october 2002 and september 2003 in the almus dam lake (40°24'28''n; 36°54'13''e). the specimens were monthly caught using gill-nets. the gill-nets with 15, 20, 25 and 30 mm mesh sizes were used for fishing, but most of a. chalcoides specimens were caught by gill-nets with 15 and 20 mm mesh sizes. the used monofilament gill-nets had a length of 100 m and its hanging ratio was 0.5 m. fishing operations were perform at night and it duration was 12 hrs. fish samples were transferred to the laboratory immediately, and their total length (tl), fork length (fl) and standard length (sl) were measured to the nearest 1 mm. in order to compare the results of this study with previous works (ünver and erk’akan, 2009; ünver and yıldırım, 2011), the relationships between tlfl, tl-sl, and fl-sl were estimated using linear regression method by microsoft excel 2010. the selectivity parameters were estimated using holt’s model (sparre and venema, 1998) by the following formula:         2 2 *2 )( exp s lml s l where, lm is optimum length for being caught, sl, fraction (ranged 0 to 1) and s, the common standard deviation. input data for this model were the numbers of caught fish by length group for each gear, i.e. ca and cb, and the two used mesh sizes, including ma and mb. the calculation of selectivity parameters was performed as following: step 1: calculation of log ratios for each length group using y = ln(cb/ca). only the lengths where the frequencies overlap, were used. in this formula, ca is length group caught using the smaller meshed net (15 mm) and cb is length group caught using the larger meshed net (20 mm). step 2: performing a regression analysis of the calculated log ratios [y = ln(cb/ca)] versus the interval midpoint for fish length (x = l), to determine α and b coefficients as following: y = ln(cb/ca)= α + b*l step 3: the obtained values of α and b along with ma and mb were used to estimate the selection factor (sf) in following formula: )(* *2 mbmab a sf    the optimum fish lengths for the small and large mesh size were calculated as following: masflma * and mbsflmb * where, lma id optimum length for the smaller meshed net and lmb is optimum length for the larger meshed net. the common standard deviation (s) is determined as following: b mamb sf mbmab mamba s      * )(* )(**2 2 2 step 4: points on the selection curves are found by inserting values of l:         2 2 *2 )( exp)( s lmal lsa         2 2 *2 )( exp)( s lmbl lsb where, sα is selection curve for gill-net with 15 mm 104 int. j. aquat. biol. (2015) 3(2): 102-107 mesh size and sb, selection curve for gill-net with 20 mm mesh size. step 5: from obtained data of step 4 and the catches ca(l) and cb(l), an index of the number in the population is estimated for each mesh size as following: )(/)()( lsalcalna  )(/)()( lsblcblnb  results a total of 254 and 198 specimens of a. chalcoides were caught with 15 mm and 20 mm mesh-size gillnet, respectively. total length of the fish ranged from 10.5 to 21.5 cm for 15 mm mesh-size and 13.6 to 25.6 cm for 20 mm mesh-size. mean total lengths were calculated as 14.34 ± 1.52 cm and 17.85 ± 1.97 cm for 15 mm and 20 mm mesh-sizes, respectively. the distribution at the points designated by ln(cal/cbl) for successive pairs of gill-nets are presented in figure 1. the regression constants α and b were estimated as -14.262 and 0.8277, respectively (fig. 1). for the calculation of selectivity parameters using holt’s method, the data and value of the selectivity parameters are presented in table 1. the values of the selectivity factor (sf) and standard deviation figure 1. the regression of ln(cb/cα) on fish length for alburnus chalcoides for gill nets of 15 mm and 20 mm mesh size from almus dam lake. figure 2. selection curves for alburnus chalcoides for gill nets of 15 mm and 20 mm mesh size from almus dam lake. 105 karataş and çiçek/ effect of water temperature on food transit in caspian kutum (sd) were obtained 0.9846 and 5.95, respectively. selectivity curve is shown in figure 2. the optimum catch lengths of the monofilament gill-nets with 15 and 20 mm mesh sizes were calculated as 14.77 cm (tl) and 19.69 cm (tl), respectively. in order to compare the results of this study with previous works, the relationships among tl-fl, tlsl and fl-sl were described as follows; fl = 0.886*tl+0.4644 (r2 = 0.9171) sl = 0.7933*tl+0.7555 (r2 = 0.8299) sl = 0.9105*fl+0.0948 (r2 = 0.8914) discussion proper estimation of age and length at first maturity is very crucial for fish stock management. yılmaz and suiçmez (2010) reported the ages of a. chalcoides ranged i-iii year old with mean total lengths of 12.85, 14.70 and 16.50 cm, respectively in almus dam lake. in other works on a. chalcoides, the mean total lengths for each age given as i-10.28 length interval midpoint l (x) number caught )ln( cal cbl (y) selection population estimates ma=15 ca(l) mb=20 cb(l) sa(l) sb(l) na(l) nb(l) 10.5 1 0 0.2161 0.0008 4.6284 11.5 3 0 0.4072 0.0035 7.3677 12.5 13 0 0.6486 0.0129 20.0424 13.5 55 0 0.8733 0.0398 62.9769 14.5 96 1 -4.56434 0.9939 0.1037 96.5870 9.6448 15.5 49 9 -1.69459 0.9561 0.2282 51.2492 39.4380 16.5 21 52 0.906721 0.7774 0.4246 27.0130 122.482 17.5 4 51 2.545531 0.5343 0.6676 7.4867 76.3924 18.5 6 23 1.343735 0.3104 0.8873 19.3319 25.9200 19.5 2 14 1.945910 0.1524 0.9969 13.1238 14.0436 20.5 2 25 2.525729 0.0632 0.9466 31.6217 26.4090 21.5 2 16 2.079442 0.0222 0.7598 90.1418 21.0575 22.5 0 5 0.0066 0.5155 9.6995 23.5 0 0 0.0016 0.2956 0 24.5 0 0 0.0003 0.1433 0 25.5 0 1 0.0001 0.0587 17.0353 26.5 0 1 0 0.0203 49.1931 lba cal cbl *)ln(  a=-14.262; b=0.8277 9846.0 )2015(*8277.0 )262.14(*2 )(* *2        mbmab a sf lma=sf*ma=14.77 lma=sf*ma=19.69 95.5 8277.0 1520 9846.0* 2      b mamb sfs 44.2 2  ss table 1. estimation of net selectivity curves for alburnus chalcoides from almus dam lake. 106 int. j. aquat. biol. (2015) 3(2): 102-107 cm, ii-16.04 cm, iii-21.26 cm, iv-24.17 cm and v28.00 cm for ömerli dam lake (tarkan et al., 2005); i-11.59 cm, ii-15.93 cm, iii-18.04 cm and iv-20.23 cm for bird lake (bandırma) (balık et al., 1996). in the previous studies, the length and age at first maturity of a. chalcoides was reported 13.30 cm (ünver and yıldırım, 2011) at age of i-iii (ünver and yıldırım, 2011; balık and sarı, 1994). based above mentioned information, the lengths at first maturity was estimated as 15.93 cm, 16.04 cm and 16.50 cm for a. chalcoides of the almus dam lake, bird lake and ömerli dam lake, respectively. the fishery can greatly influence the population structure and reproductive potential of the species. therefore, adult organism should reproduce at least once in their lifetime before to be exploited for the sustainable fisheries. fishing gear selectivity studies are important for fisheries management due to regulating the mesh size of gill-net and recognizing the approximate minimum catch sizes of the target species (spare et al., 1989). the results showed that, the optimum catch lengths of gill-nets with 15 and 20 mm mesh sizes were estimated as 14.77 cm (tl) and 19.69 cm (tl) of length, respectively. the results given above indicate 15 mm mesh size gill-net for fishing operations should be discouraged for a. chalcoides in the almus dam lake. however, the use of gill-net with 20 mm mesh size is appropriate due to enhancing sustainability of the fisheries resources of the almus dam lake. ünver and yıldırım (2011) suggested the specimen size allowed for commercial fishing must be over 15.8 cm according to length at first maturity. in conclusion, selectivity parameter of a. chalcoides was estimated and allowable gillnet mesh size was determines as 20 mm for sustainable fisheries in the almus dam lake. references akyurt i., karataş m. 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(2018) 6(3): 170-178 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article developmental morphology and growth patterns of laboratory-reared giraffe cichlid, nimbochromis venustus boulenger, 1908 maryam saemi-komsari1, meysam salehi2, mohammad mansouri-chorehi1, soheil eagderi3, hamed mousavi-sabet*1,4 1department of department of fisheries sciences, faculty of natural resources, university of guilan, sowmeh sara, guilan, iran. 2abzi-exir aquaculture co., agriculture section, kowsar economic organization, tehran, iran. 3department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 4the caspian sea basin research center, university of guilan, rasht, iran. article history: received 7 march 2018 accepted 22 june 2018 available online 2 5 june 2018 keywords: ontogeny growth embryo juvenile abstract: the giraffe cichlid nimbochromis venustus boulenger, 1908 is a well-known species in aquarium trade. the laboratory-reared electric giraffe cichlid was studied in terms of early morphological development and allometric growth pattern from hatching until the juvenile stage i.e. 51 dph. total length increased linearly from hatching until 51 dph, with a high regression coefficient. the yolk sac exhaustion completed throughout 15 days. significant morphological and morphometric variations occurred before the yolk sac absorption and early juvenile stage by evolving the anterior and posterior body section which improve swimming ability and food capturing. positive growth coefficient of the body and trunk lengths were occurred after inflexion point coinciding with development of digestive system function. eye diameter showed negative allometry at the inflexion point of 9.3 mm, continuing with positive growth rate until day 51. body shape variation in early life history revealed consequent development in anterior and posterior body section in preflexion phase and development of body and trunk length in post flexion phase to support high survival of larvae. the present study emphasizes ontogenic study of different species to interpret biology and ecology of fish in association with evolutionary biology. introduction the giraffe cichlid, nimbochromis venustus (boulenger, 1908), is a large cichlid endemic to lake malawi in africa (bangerter, 2007). juveniles of this species are found in shallow water near rocks while adults forage in deeper regions with sandy substrate. they usually fed small fish and invertebrates. halfgrown individuals have been observed to remain motionless, partially buried in the sand, waiting for small fishes to come within reach. nimbochromis venustus, as a mouth brooding fish (konings, 1990; snoeks and manuel, 2004), prefers waters with ph, depth and temperature 7.2-8.8, 6-23 m and 25-27°c (riehl and baensch, 1996). fish morphological transformation from larvae to a juvenile or young adult form is occurred during early developmental stages in a relatively short period of time with completion of their functional systems as a *corresponding author: hamed mousavi-sabet doi: https://doi.org/10.22034/ijab.v6i3.525 e-mail address: mousavi-sabet@guilan.ac.ir survival strategy (osse and van de boogart, 2004). in addition, study of the allometric growth patterns during early developmental stages of different teleost species helps to corroborate the importance of morphological development and growth patterns of young fish, by providing a better understanding of early life events (fuiman, 1983; khemis et al., 2013), their priorities during the early growth, and sizerelated behavior and ecology (gisbert, 1999). in this regard, this study aimed to describe early morphological development and allometric growth pattern of n. venustus. materials and methods the broodstocks of n. venustus were obtained from a local ornamental fish farm in february 2015 and transferred to a rearing glass aquarium at the fisheries laboratory of university of guilan, guilan province, 171 int. j. aquat. biol. (2018) 6(3): 170-178 iran. a total of 20 breeding pairs were transferred to breeding aquaria for spawning. the eggs were kept in glass aquaria with water temperature of 26-28°c and after hatching, larvae were transferred to new aquaria for rearing. rearing conditions were 27±0.8°c, 6.49±0.15 mg/l dissolved oxygen, 12l:12d artificial photoperiod and light intensity of 500 lux at the water surface (mousavi-sabet, 2011). newly hatched larvae were fed with artemia nauplii and micro-worms from 1-5 dph, then with a mixture of nauplii and commercial food pellets (biomar a/s; 58% protein, 15% lipid) twice a day from 6-51 dph. daily sampling were randomly carried out from 15 dph (day post hatching), followed every other day till 51 dph from the same larval batch. larvae were sacrificed with an overdose of ms222 (35 mg ml-1) and fixed in 5% buffered formalin solution. then, their left sides were photographed using a stereomicroscope equipped with a cannon camera with a 5 mp resolution. the following morphological characteristics were measured from the digital images to the nearest 0.01 mm using image-j software (version 1.240): body length (bl), head length (hl), head depth (hd), trunk length (trl), tail length (tal), maximum body depth (bd), eye diameter (ed) and snout length (snl). measurement method followed leis and trnski (1989). ontogenic development followed criteria as described by balon (1977, 1986, 1999). specimens were examined for general morphology, pigmentation, and fin development under a leica mc5 stereozoom microscope. the allometric growth patterns were calculated as a power function of total length using nontransformed data: y=axb, where (y) was the dependent variable, (x) the independent variable, (a) the intercept and (b) the growth coefficient. isometric, positive and negative allometric growth patterns are indicated by b=1, b>1, b<1, respectively. the inflexion points of growth curves were determined according to fuiman (1983) and van snik et al. (1997). drawing graphs was performed in ms-excel 2013 (microsoft corporation) and data analysis in past (ver 2.17). results general morphology: total length of n. venustus increased linearly from hatching up to 51 dph (fig. 1). bl of day 0 larvae ranged 8.23-8.32 (mean±sd: 8.28±0.04) mm (n=5) and reached 27.3930.92 (mean±sd: 28.93±1.08) mm on day 51 (fig. 2, table 1). the mouth and anus were closed at hatching. newly hatched larvae had a large oval yolk sac (with horizontal length of 3.05±0.10 mm). the head initially was separated from the yolk sac (fig. 2a) at hatching, upper and lower jaws formed in day 1, mouth opened, and anus hardly seen on day 2. formation of the anal-fin with visible rays and starting y = 0.3709x + 8.2629 r² = 0.96 0 10 20 30 40 0 10 20 30 40 50 60 t o ta l l e n g th ( m m ) day after hatching (dah) figure 1. linear regression between total length (mm) and days after hatching of laboratory-reared nimbochromis venustus from days 0 to 51 after hatching. 172 saemi-komsari et al/ early developmental morphology of nimbochromis venustus notochord flexion were observed on day 3 (fig. 2c). operculum appeared on day 4 (fig. 2d), myomeres were visible from hatching to day 11. lateral line was appeared on day 15, yolk moved dorsally to above abdominal cavity beside upper base of the pectoral fin (fig. 2h), completely absorbed by day 15. head length calculated 22-25 %bl in day 1 and increased with growth, reaching 28–30 %bl on day 51 (fig. 3a). the proportion of body depth, regardless of the yolk sac depth, increased continuously to over 27 %bl on day 51 from 12 %bl at hatching (fig.3b). trunk length was nearly constant at all stages (40–44 %bl) (fig. 3c). eye diameter slightly decreased by day11 (118 %bl) and thereafter reached to 11%bl on day 51 (fig. 3d). snout length increased continuously up to day 51 from 2 to 7 %bl (fig. 3e). tail length showed increasing trend as 24-32% by day 9 then decreased around 29% on day 51. the soft rays of the caudal fin formed from hatching (with about 16 rays) (fig. 2a), and finalized with 26 soft rays on day 51(fig. 2i). the other fins not observed on day 1. dorsal-fin soft rays appeared on figure 2. early developmental stages of giraffe cichlid nimbochromis venustusa. (a) newly hatched embryo 1 day after hatching, (b) two day after hatching (dah), (c) 3 dah, (d) 4 dah, (e) 5 dah, (f) 7 dah, (g) 11 dah, (h) 15 dah, and (i) 51 dah; completion of fin rays and juvenile formation after day 15. scale bar=1 mm. 173 int. j. aquat. biol. (2018) 6(3): 170-178 day 2 (fig. 2b), attained 13 spines in anterior part on day 11 (entirely 26 rays) (fig. 2g), and it was constant thereafter. anal-fin soft rays appeared on day 3 (fig. 2c) attaining 3 spines in anterior part on day 9 (entirely 12 rays) and was constant thereafter. pelvic and pectoral find soft rays appeared on day 11 with 13 and 10 rays, respectively (fig. 2g). allometric growth: the head length allometric growth pattern was negative (b= 0.84, r2=0.68) up to 9 dph, then turned to positive (b=1.125, r2=0.96) up to day table 1. linear growth of larvae of the nimbochromis venustus in the experiment. above line: the mean value and standard deviation (in parentheses); under the line: limit of variations of the parameter, (tl: total length n: the number of specimens). age, days tl±sd/min-max (mm) n 1 8.28 (0.04) 8.23 − 8.32 5 11 13.31 (0.56) 12.74 − 13.78 5 21 15.78 (0.64) 15.10 − 16.40 5 31 18.50 (1.70) 16.78 − 20.19 5 41 22.39 (0.53) 21.77 − 22.73 5 51 28.93 (1.80) 27.39 − 30.92 5 figure 3. proportions of (a) head length (hl), (b) body depth (bl), (c) trunk length (trl), (d) eye diameter (ed), (e) snout length (snl), and (f) tail length (tal) to total length (tl) in young laboratory-reared nimbochromis venustusa. 174 saemi-komsari et al/ early developmental morphology of nimbochromis venustus 51 (fig. 4a). the body depth growth coefficient was positive throughout the entire study period with decreasing trend, separated with an inflexion point on day 9 (fig. 4b). the growth pattern of the trunk length (trl) was allometrically negative ((b=0.78, r2=0.77) until the ninth day at the 10.8 mm of total length (fig. 3b). the growth pattern of trl positively increased until day 51 (fig. 4c). the growth of eye diameter (ed) showed negative allometric growth pattern (b=0.27 r2=0.13) by day 11 followed positive allometric growth pattern (b=1.16 r2=0.8) by day 51 (fig. 4d). snout length biphasic pattern of the growth pattern showed a strong positive allometry in relation to total length (b=2.37, r2=0.58), following days displayed positive growth with decreasing gradient to the previous phase (b= 1.31, r2=0.83) (fig. 4e). tail length grew negatively by day 9 and positively until day 51 (b=1.37, r2=0.93; b=0.77, r2=0.89, respectively) (fig. 4f). pigmentation: the newly hatched fry are transparent, grayish in color and slender with melanophores on head. melanophore deposition in eyes was observed on 1 dph (fig. 2a). the yolk sac in the anterior part, operculum, dorsal part of the body and upper surface figure 4. allometric growth equations and relationship between different measured body proportions with total length in nimbochromis venustusa during early stages of development (from hatching up to day 51). (a) head length, (b) body depth (bl), (c) trunk length (trl), (d) eye diameter (ed), (e) snout length (snl), (f) tail length (tal). the dashed line represents the inflexion point of growth. power regression equations are displayed (p<0.05 for all regression equations; exception was the first phase of eye diameter r2=0.138; p>0.05). 175 int. j. aquat. biol. (2018) 6(3): 170-178 of eyes had a bit dispersed punctate pigment on day 1 (fig. 2a). melanophres emerged some dispersed melanophores on lateral line (on myomeres) with low density on day 2 (fig. 2b). melanophores initially were few and increased in number with growth in the following days. melanophores were seen as colony in some area on lateral line and appeared around anal fin on day 7 (fig. 2f). few melanophores observed on snout on day 11(fig. 2g) and then increased for the following days. moreover, the concentration of melanophores increased on operculum and lateral body on day 13. discussion in the present study, the newly-hatched free embryos of n. venustus were transparent, grayish and slender thereafter with increasing chromatophores. the pigmentation provides camouflage against predators or distracting flickering effect during swimming (moser, 1981). also, there has been shown in many species such as pleuronectes platessa larvae and atlantic halibut, hippoglossus hippoglossus that pigmentation could be positively related to metabolism, some hormones, and growth factor (christensen and korsgaard, 1999; solbakken et al., 1999), genetic and environmental factors (urho, 2002). pigmentation pattern in n. venustus like other cichlidae showed differentiation compared to other species (maan and sefc, 2013; ahmadi et al., 2013). furthermore, it is assumed that identical pigmentation can imply similar function while body coloration is inextricably referred to diversification as well as responding to both natural and sexual selection (meyer, 1993; kocher, 2004; maan and sefc, 2013). yolk sac depletion was completed on 15 dph in the present study. it is assumed that n. venustus has 15day preparatory period for shifting from endogenous to exogenous energy dependent periods. the previous studies have showed longer period for shifting from endogenous to exogenous feeding as observed in clarias gariepinus (matsumoto et al., 2001), climbing perch, anabas testudineus (morioka et al., 2009a) and snakeskin gourami, trichogaster pectoralis (morioka et al., 2009b). in the current study, complete exhaustion of yolk sac on the day 15 coincided with transformation of free embryo to juvenile supporting its survival in environment conducting the importance of evolutionary ecology during early life history (kamler and keckeis, 2000). based on the results, the lateral line was appeared on day 15 coincident with yolk depletion and exogenous feeding. it is supposed that lateral line plays a critical role in the prey/ predator detection (coombs and montgomery, 1994; coombs et al., 1996) and recognition of the low frequency movement of water (harries and van burgeijk, 1962) in fish during early life history especially with the beginning of exogenous feeding which need further tools for survival against predators to interact better with their environment. in addition, as the yolk sac depletion increases, external morphological efforts manage to optimize the localization and uptake of prey in favor of fish survival in the early life stage (comabella et al., 2013). food capturing and predator avoidance are critical issues contributed to essentially related organogenesis for feeding (porter and theilacker, 1999; makrakis et al., 2005) and swimming (murphy et al., 2007; huysentruyt et al., 2009). development of organs associated with these functions must occur in a mutual balance (osse et al., 1997; rodríguez-mendoza et al., 2011; saemi-komsari et al., 2018). nimbochromis venustus larvae exhibited positive growth in tail length by day 9 followed by negative allometric growth pattern later on. the positive allometry of tail has been reported in sturgeon (gisbert and doroshov, 2006), croaker (shan and dou, 2009) and catfish (huysentruyt et al., 2009). tail length positive growth coefficient in the early life history could enhance swimming capacity considering the changes in swimming style from anguilliform to subcarangiform (van snik et al., 1997; osse and boogaart, 1999; osse, 1990). in our study, the pelvic and pectoral fins developed after unpaired fins. in more developed fishes, including teleost, the pelvic fin with trimming function decreases the pitching and up-ward body displacement in fish effort of braking (murata et al., 2010). in many other fish species, the maneuverability 176 saemi-komsari et al/ early developmental morphology of nimbochromis venustus function of the effective swimming in the favor of feeding has been reported (comabella et al., 2013). the trunk and body allometric growth pattern of n. venustus was negative by day 9 and positive till 51 dph. the negative growth pattern of the trunk during the first stages of development in n. venustus has been reported for most fish species to develop primary functions (feeding, respiration and locomotion) related to the anterior and posterior body sections prior to the abdominal region (osse and van den boogart, 1995). trunk allometric growth increases after the development of the head and tail (osse et al., 1997; van snik et al., 1997; gozlan et al., 1999) that is considered as the priority of digestive system development. the head growth showed negative pattern by day 9 in n. venustus. thereafter, when the larvae were less dependent on endogenous feeding, positive growth coefficient was observed. it is assumed that positive growth of head can describe the development of nerve, sensory and respiratory organs as well as feeding systems essential for exogenous feeding (gisbert and doroshov, 2006). negative growth pattern (b=0.2) of eye diameter by day 11 followed by positive growth pattern afterward. the morphological variation toward positive growth of eye diameter could explain visionary essence after shifting to exogenous feeding as vision provide better spatial orientation in swimming (gisbert, 1999; petereit et al., 2008). in conclusion, the present study highlights the preference of morphogenesis as energy demanding transformation to improve the strategy of survival considering the respective importance of functionality. acknowledgments we would like to thank the financial support of university of guilan. references ahmadi s., khodadadi m., salehi-farsani a., samadikuchaksaraei b., mousavi-sabet h. 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(2014) 2(4): 180-183 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology short communication digestive tube contents of blood cockle (anadara granosa) in a tropical mangrove estuary in malaysia tatsuya yurimoto*1, faizul mohd kassim2, alias man3 1japan international research center for agricultural sciences, tsukuba, ibaraki, japan 2penang office of japan international research center for agricultural sciences, penang, malaysia. 3fisheries research institute, department of fisheries, penang, malaysia. article history: received 13 may 2014 accepted 30 july 2014 available online 2 5 august 2014 keywords: digestive tube contents mangrove estuary blood cockle abstract: this study was carried out to clarify the feeding biology of the blood cockle (anadara granosa). we collected blood cockles from 8 stations in the matang mangrove estuary of malaysia in july and august 2010. the digestive tube contents of the specimens were stained with congo red and observed under a light microscope. the results showed blood cockles take in particles containing cellulose as well as phytoplankton such as diatoms. as blood cockles in estuaries are known to exhibit cellulolytic enzyme activity in their digestive gland, the present results indicate blood cockles in estuaries feed on litter supplied from mangrove forests and terrestrial plants. introduction blood cockles, anadara granosa, inhabit the coastal areas of the middle east, south asia, southeast asia, east asia, and northern australia (faulkner, 2010). they mainly live in the silt clay muddy bottoms of estuaries or the inner parts of bays from the tidal zone to shallow areas (narashimham et al., 1984; nakao et al., 1989). the blood cockle is an important species for bivalve aquaculture industry due to being popular food in southeast asia, including thailand and malaysia (watanabe, 2009; yurimoto et al., 2014). the matang mangrove is approximately 40,000 ha and located on the west coast of peninsular malaysia. moreover, there is a huge muddy tidal flat suitable for blood cockle culture that contains wide sowing aquaculture grounds (pathansali and song, 1958; fontalvo-herazo et al., 2011). blood cockles occupy a large proportion of the biomass of the ecosystem of the matang mangrove (man et al., 2012). in addition to clarifying their ecological position, understanding the feeding behaviors of the blood cockle is * corresponding author: tatsuya yurimoto e-mail address: yurimoto@outlook.com necessary for stable production in sowing aquaculture in mangrove estuary ecosystems. many benthic invertebrate organisms including clams such as ruditapes philippinarum and corbicula japonica in brackish waters contain cellulolytic enzymes (sakamoto and toyohara, 2009; niiyama and toyohara, 2011; sakami and higano, 2012). moreover, the presence of cellulolytic enzymes was recently revealed in the digestive gland of the blood cockle, suggesting they can feed and digest organic matter including cellulose supplied from terrestrial plants including those from mangrove forests (niiyama et al., 2012). therefore, this study aimed to clarify the feeding biology of the blood cockle in the matang mangrove estuary (peninsular malaysia). therefore, we collected the blood cockles from 8 stations in the matang mangrove estuary and analyzed their digestive tube contents for terrestrial plant particles using congo red staining, which adsorbs to cellulose (samiey and dargahi, 2010). in addition, the composition of digestive tube contents of the blood 181 yurimoto et al./ digestive tube contents of blood cockle cockle was analyzed and compared among stations. materials and methods a total of 79 individual blood cockles with shell lengths ranging from 14-35 mm were collected from a total of 8 stations (st.) in matang mangrove estuary in peninsular malaysia (fig. 1). sampling at st. 1-5 and 6-8 was carried out in july and august 2010, respectively. the digestive tube contents of specimens were analyzed. in brief, each specimen was fixed in formalin and dissected, and the digestive tube contents were aspirated from the lip with a micropipette. the contents were subsequently stained with congo red solution to visualize cellulose and observed under a light microscope. in detail, the digestive tube contents were immersed with 1% (w/v) congo red solution for at least 1 hour, and the extra solution after staining was washed with an alkaline alcohol solution [1 ml 1% (w/v) sodium hydroxide + 100 ml 50% (v/v) alcohol] and ionexchanged water. the contents in the microtube were washed repeatedly with bleaching in alkaline alcohol solution followed by purified water. the contents were then flash centrifuged at 3,000 rpm to exchange the solution. finally, the sample was diluted with a predetermined amount (500 μl) of purified water, dropped to a glass slide, and covered with a cover glass before observation under a light microscope. in addition, particles > 10 μm stained positive (i.e., land plant-derived particles) and negative (i.e., phytoplankton, etc.) with congo red were counted. results and discussion the numbers of collected specimens at each station are shown in table 1; 5-14 individuals were collected from each station. however, only 3-7 individuals from each station were successfully analyzed. thus, the success rate at each station ranged from 21-80%. in particular, the success rate was higher at stations from the inner part of the mangrove area than the estuaries in both july (7080% at st. 1-3) and august (36% at st. 6). these results indicate that food consumption is higher in the inner part of the mangrove area than estuaries. photomicrographs of the gut contents of blood cockles are shown in figure 2. the particles positively and negatively stained with congo red are figure 1. sampling locations of the blood cockle, a. granosa, in the matang mangrove estuary, malaysia. ●: sampled in july 2010, ★: sampled in august 2010. figure 2. photomicrographs of the digestive tube contents of blood cockle, a. granosa, stained with congo red solution. positive staining: asterisk and red arrows show terrestrial plant fragments, and red arrow pl shows phytoplankton cell-like particle. negative staining: blue arrows m and p show mineral particle and phytoplankton such as diatom, respectively. 182 int. j. aquat. biol. (2014) 2(4): 180-183 shown in figure 2a; particles containing cellulose components from mangrove forests or terrestrial plants stained strongly with red (fig. 2a, asterisk and red arrows). in addition, the silicate cell walls of diatoms (fig. 2a, b, blue arrows p) (hecky et al., 1973) and mineral particles (fig. 2a, blue arrow m) as well as many other unidentified particles were not stained. phytoplankton-like particles were stained strongly (fig. 2b, red arrow pl) could be green algae cells, which contain cellulose in their cell walls (sugiyama et al., 1991). the composition of the digestive tube contents (particles > 10 μm) from blood cockles collected from each sampling station is shown in figure 3. the ratios of components differed somewhat among sites. mineral and terrestrial plant particles and phytoplankton, which is a common food source for bivalves (nakamura and shinotsuka, 2007), were observed. regarding staining, positively and negatively stained particles accounted for 11-57% and 43-89% of all particles at each sampling station, respectively. among the positively stained particles, phytoplankton-like particles accounted for approximately 13% of all particles in samples from st. 4. diatoms, and minerals and unidentified particles accounted for 950% and 30-63% of the negatively stained particles, respectively. in addition, the composition of digestive tube contents differed between survey months. there was a greater ratio of positively stained particles in july (st. 1-5) than august (st. 68). on the other hand, the ratio of negatively stained particles (e.g., diatoms) was higher in august than july 2010 august 2010 st. 1 st. 2 st. 3 st. 4 st. 5 st. 6 st. 7 st. 8 number of specimens 10 5 7 10 10 11 14 12 number of successful samples 7 4 5 4 3 4 3 3 success rate (%) 70 80 71 40 30 36 21 25 table 1. number of the blood cockle specimens collected at each sampling station and success rate of the digestive tube contents sampling. figure 3. composition of the digestive tube contents (>10 μm in diameter) of blood cockles, a. granosa, at each sampling station. the numbers of total counted particles and observed samples at each station are as follows: st. 1: n = 391 (7), st. 2: n = 204 (4), st. 3: n = 405 (5), st. 4: n = 319 (4), st. 5: n = 116 (3), st. 6: n = 112 (4), st. 7: n = 229 (3) and st. 8: n = 31 (3). positive (plankton like) indicates positively stained particles of phytoplankton-like cells (fig. 2b, red arrow pl), positive (others) indicates positively stained terrestrial plant-derived particles (fig. 2a, asterisk and red arrows), negative (plankton) indicates unstained particles identified as phytoplankton such as diatom (fig. 2a, b, blue arrows p), and negative (others) indicates an unstained mineral particle (fig. 2a, blue arrow m) or unidentified. 183 yurimoto et al./ digestive tube contents of blood cockle july. in addition, the ratio of negatively stained particles was higher in mangrove areas (st. 1, 2, 3, 6 and 7) than estuaries (st. 4, 5, and 8), which had a higher ratio of positive to negative particles. these results indicate that the digestive tube contents of blood cockles vary with respect to environmental differences and changes in their habitat. as mentioned above, the presence of cellulolytic enzyme activity has been confirmed in the mid-gut glands of blood cockles living in estuaries (niiyama et al., 2012). accordingly, the present results indicate that blood cockles in the matang mangrove estuary take in terrestrial plant particles containing cellulose as well as phytoplankton. thus, blood cockles digest particles containing cellulose in their digestive tube with cellulolytic enzymes. acknowledgements this study was supported in part by the japan international research center for agricultural sciences. references faulkner p. (2010). morphometric and taphonomic analysis of granular ark (anadara granosa) dominated shell deposits of blue mud bay, northern australia. journal of archaeological science, 37: 1942-1952. fontalvo-herazo m. l., piou c., vogt j., saint-paul u., berger u. (2011). simulating harvesting scenarios towards the sustainable use of mangrove forest plantations. wetlands ecology and management, 19: 397-407. hecky r.e., mopper k., kilham p., degens e.t. (1973). the amino acid and sugar composition of diatom cellwalls. marine biology, 19: 323-331. nakamura y., shinotsuka y. (2007). suspension feeding and growth of ark shell anadara granosa: comparison with ubiquitous species scapharca subcrenata. fisheries science, 73: 889-896. nakao s., nomura h., statar m.k.b.a. (1989). macrobenthos and sedimentary environments in a malaysian intertidal mudflat of the cockle bed. bulletin of the faculty of fisheries, hokkaido university, 40: 203-213. narashimham k.a., selvaraj g.s.d., lalitha devi s. (1984). the molluscan resources and ecology of kakinada bay. marine fisheries information series: technical and extension series, 59: 1-16. niiyama t., toyohara h. (2011). widespread distribution of cellulase and hemicellulase activities among aquatic invertebrates. fisheries science, 77: 649-655. niiyama t., toyohara h., tanaka k. (2012). cellulase activity in blood cockle (anadara granosa) in the matang mangrove forest reserve, malaysia. japan agricultural research quarterly: jarq, 46: 355-359. man a., watari s., tanaka k., hanamura y., chong v.c., mohd kassim f. (2012). ecopath trophic model for the matang mangrove estuary, malaysia. japan international research center for agricultural sciences (jircas) working report, 75: 69-79. pathansali d., song m.k. (1958). some aspects of cockle (anadara grunosa l.) culture in malaya. proceedings of the indo-pacific fisheries, pp. 26-31. sakami t., higano j. (2012). an attempt to assess the feeding activity of short-neck clam ruditapes philippinarum juveniles by a digestive enzyme cellobiosidase activity. journal of fisheries technology, 5: 49-55. sakamoto k., toyohara h. (2009). a comparative study of cellulase and hemicellulase activities of brackish water clam corbicula japonica with those of other marine veneroida bivalves. journal of experimental biology, 212: 2812-2818. samiey b., dargahi m.r. (2010). kinetics and thermodynamics of adsorption of congo red on cellulose. central european journal of chemistry, 8: 906-912. sugiyama j., vuong r., chanzy h. (1991). electron diffraction study on the two crystalline phases occurring in native cellulose from an algal cell wall. macromolecules, 24: 4168-4175. watanabe k. (2009). coastal-zone use of bandon bay: area study in surat thani province, south thailand. kyoto working papers on area studies: g-coe series, 68: 1-12. yurimoto t., mohd kassim f., man a. (2014). sexual maturation of the blood cockle, anadara granosa, in matang mangrove estuary, peninsular malaysia. international journal of aquatic biology, 2(3): 115123. int. j. aquat. biol. (2018) 6(5): 281-287 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article histological characterization of the olfactory organ in schilbid catfish, clupisoma garua (hamilton, 1822) saroj kumar ghosh1 department of zoology, bejoy narayan mahavidyalaya, itachuna, hooghly-712147, west bengal, india. s article history: received 10 september 2018 accepted 24 october 2018 available online 2 5 october 2018 keywords: morphology histoarchitecture olfactory mucosa clupisoma garua olfaction abstract: fishes have a good sense of smell and are able to ascertain odour with the help of a pair of olfactory organs connected to the olfactory lobes of the forebrain by means of olfactory tracts. the functional anatomy and structural characterization of olfactory organ in the freshwater indian catfish clupisoma garua (hamilton, 1822) was investigated by light microscopy. the paired well-developed olfactory organs were located in nasal cavity having two exterior apertures: incurrent and excurrent nares. the olfactory rosettes were elongated structure, possessed 40±02 lamellae on each side of the narrow median raphe. histologically, each lamella consisted of two principal layers: an epithelium consisted of sensory and non-sensory cells and a central core, which was composed of connective tissues, nerve fibers and blood vessels. the sensory epithelium was composed of three types of receptor cells: two described as classical bearing cilia or microvilli and third bearing rod like dendritic terminal. synapses between primary and secondary neurons were formed. the indifferent epithelium comprised the greater surface area of the olfactory lamella, was typified with ciliated non-sensory cells, secretory mucous cells, mast cells and supporting cells. undifferentiated basal cells were scattered in the deeper part of the epithelium above the basement membrane. organization of various cells on the olfactory epithelium was correlated with essential life process of the fish concerned. introduction olfactory organ in fishes is of primary importance because it is innately a chemoreceptor and performs an indispensable role in detection and location of food, recognition of sex, exposure of predators, parental behaviour and migration. olfaction results from stimulation of the sensory receptor cells lining the olfactory mucosa, which is innervated by the olfactory nerve. a large number of researchers investigated diverse views of the olfactory organ of teleosts (fishelson, 1995; hansen and zielinski, 2005; sinha, 2008; chakrabarti and ghosh, 2009; 2010; waryani et al., 2013, kim and park, 2016; ghosh, 2018). variation in the morphology of the olfactory organ correlates with the enormous diversity of life-styles among fish species, the long, divergent evolutionary history of primary aquatic vertebrates and their inferred actual ecological adaptations (zeiske et al., 2009). the knowledge of the cellular organization of *correspondence: saroj kumar ghosh doi: https://doi.org/10.22034/ijab.v6i5.543 e-mail: saroj.fisherylab@gmail.com the olfactory organs in schilbid catfish is almost unknown. considering the dearth of information, the present study was undertaken to describe the morphology and histoarchitecture of the olfactory epithelium of clupisoma garua (siluriformes; schilbeidae); a bottom dweller river catfish which feeds on mollusks, insects, small fishes and decaying matter (talwar and jhingran, 1991). materials and methods this study used 16 samples of adult c. garua (17±1.57 cm in total length), caught from the river ganga at kalyani, nadia, west bengal. the fishes were deeply anaesthetized with benzocaine (4 mg/l) and decapitated following the guidelines of the institutional animal ethics committee. the olfactory apparatus with the brain was dissected out under a zeiss stemi 2000-c stereoscopic binocular microscope and further processed for respective 282 ghosh / histological characterization of the olfactory organ in clupisoma garua studies. for histological preparation, the olfactory tissues were immediate fixed in aqueous bouin’s fluid for about 24 h. fixated tissues were thoroughly washed in 70% ethanol, dehydrated through ascending grades of ethanol, cleared in xylene and then embedded in paraffin wax of 56-58°c during 1 h 30 min. serial transverse sections were cut in 4 m thick using a rotary microtome (weswox mt-1090a). sections were firmed on glass slides, deparaffinized and stained with romies azan (ra) and mallory’s triple (mt) stain (mallory, 1936). the staining slides were observed and photographed using a leica ec3 light microscope at different magnifications. for scanning electron microscopy, the rosettes were taken out and fixed in glutaraldehyde 2.5% in 0.1m sodium phosphate buffer (ph 7.4) for 4 h, postfixed in 1% osmium tetroxide in phosphate buffer. the rosettes were then repeatedly washed in same buffer, dehydrated in a growing acetone series and subsequently processed through critical point drying method. the dried samples were mounted on aluminium stubs, coated with gold palladium and documented photographically with a zeiss evo 18 scanning electron microscope. results general anatomy: paired olfactory organ of c. garua (fig. 1a) are located on the nasal cavity on the dorsolateral side of the head and at a distance from the eyes, near to the rise of maxillary barbels (fig. 1b). each nasal cavity contains two separate openings, the anterior inlet and a posterior outlet. a nasal bridge distinguishes these openings. the anterior nostril is roughy rounded, whereas posterior nostril is crescentic and occupied a transverse position. the olfactory apparatus consists of nasal cavity, anterior and posterior nostrils, olfactory rosette, olfactory bulb and olfactory nerve terminated to the telencephalon (fig. 1c). the deck of the nasal cavity is lined by mucosa, which is aroused from the bottom into a series of lamellae to construct olfactory rosette. a very small figure 1. photographs showing the position of nostrils, olfactory rosette and connection of olfactory organ with brain in clupisoma garua. (a) complete structure of clupisoma garua, (b) dorsolateral view of the head showing the anterior nostril (solid arrow), posterior nostril (arrow head) and nasal bridge (broken arrow), (c) dorsal view of dissected olfactory rosette (or) showing the relationship of the olfactory bulb (ob), olfactory nerve (on), cerebral hemisphere (ch), optic lobe (opl), cerebellum (c) and medulla oblongata (mo), and (d) olfactory rosette within olfactory chamber (oc) holds a series of olfactory lamellae (ol) radiated from central raphe (r). note the presence of linguiform processes (arrows) on ol. 283 int. j. aquat. biol. (2018) 6(5): 281-287 amount of fibrous connective tissue is observed, which engrossed the olfactory organ within the nasal cavity. the olfactory rosette is an elongated structure having concave dorsal and convex ventral appearance inhabited the major space of the corresponding chamber (fig. 1d). it consists of 40±02 leaf lets, the olfactory lamellae, arranged on each side of the median raphe. the number of lamellae on the either side of the rosette exhibits bilateral symmetry. the lamellae are affixed to the wall of olfactory chamber by their ventral borders and to the raphe by their proximal ends. the dorsal portion of the lamellae is characterized with linguiform processes at their distal ends. each lamella is broad anteriorly and narrow posteriorly. histology: the lamella of the olfactory organ consists of olfactory epithelium, which is pseudostratified in nature, enclosing a connective tissue layer, central core (fig. 2a). the central core is distinguished from the epithelium by a basement membrane, made up of loose fibres, connective tissues and blood vessels (fig. 1b-e). the epithelium is dissociated into two regions: sensory and indifferent, which are regularly interspaced. the sensory epithelium is embossed with morphologically distinct ciliated, microvillous and rod figure 2. photomicrographs of the transverse section of the olfactory organ in clupisoma garuaa stained with romies azan (ra) and mallory’s triple (mt) stain. (a) section of olfactory lamellae (ol) showing olfactory epithelium (oep) (arrow heads) and central core (cc) (solid arrows), radiated from raphe (r) (ra 40x), (b) sensory oep lined with ciliated receptor cells (rc) and microvillous cells (mv). cc contains blood vessels (bv) distinguished from oep by basement membrane (bm) (ra 400x), (c) oep typified with rc, rod cells (solid arrows), mucous cells (mc), mast cells (m) and basal cells (bc). note the presence of connective tissue (ct) and bv in cc. r marks raphe (mt 400x), (d) magnifying sensory epithelium (se) shows rod cells (asterisks), mv, synaptic contact (s) in between primary (solid arrows) and secondary rc (broken arrows). note knob like structure of ciliated rc on the epithelial surface and presence of bv and ct in cc, separated from oep by bm. (ra 1000x), and (e) non-sensory epithelium (nse) shows mc, bc, mast cells (arrow heads), supporting cells (broken arrows) and ciliated non-sensory cells (solid arrows). note cc with copious bv (mt 1000x). 284 ghosh / histological characterization of the olfactory organ in clupisoma garua receptor cells. ciliated receptor cells are columnar in shape with a basally located cell body and a thin long dendrite, which formed a swelling, the olfactory knob at the free surface of the epithelium. in some areas, the axonal end of primary neurons is synapsed with the dendrite tips of the secondary neurons (fig. 2d). microvillous receptor cells are of small size and have broad apical surface but without cilia. the cell body and light stained nucleus are located in more superficial layer in the epithelium (fig. 2b). the rod receptor cells are scattered along the surface zone of the mucosa, consisted of basally placed vesicular nuclei and narrow protruded dendrons (fig. 2c). the indifferent epithelium is typified with supporting cells, ciliated non-sensory cells, mast cells and mucous cells. the supporting cells give the basic structure of the olfactory epithelium. they are elliptical in shape with intensely basophilic nuclei and faintly stained less granular cytoplasm (fig. 2e). ciliated non-sensory cells are columnar and composed of numerous long cilia at their surface. deeply stained round nucleus is placed in the middle of the cell. the mast cells are rounded having relatively smaller amount of cytoplasm and centrally placed nuclei. (fig. 2c, e). mucous cells are fairly larger and oval, distributed at the margin of the olfactory epithelium in between other non-sensory cells. nucleus is present towards the basal ends. the basal cells appeared to be stem cells, are grouped in the deeper part of the epithelium just above the basement membrane. they are small, round in shape with an enormous central nucleus. discussion olfactory mucosa containing the sensory neurons is typically located on the floor of the nasal chamber, which is often folded, forming olfactory lamellae (hara, 1975). the present study reveals that olfactory rosette of c. garua is elongated texture which can be placed under bateson’s (1889) rosette type 2, burne’s (1909) rosette column ii and teichmann’s (1954) 1st group. according to teichmann (1954) the elongated olfactory organ belongs to the category of “nose” fishes, which means that, c. garua having a predominantly developed sense of olfaction. the presence of 40±02 lamellae around a central raphe designated as macrosmatic, furnishes their bottom dwelling habit. the capacious surface area furnished by the olfactory lamella increases and sensitivity and efficacy of the olfactory system (zeiske et al., 1976). in c. garua, the paired olfactory chambers are communicated to surrounding aquatic environment by means of two opening, through which water enters and leaves. adequate ventilation is needful to bring the odorants in the nasal chamber for perceiving the chemical signals (belanger et al., 2003). hara (1993) reported that ventilation of the nasal chamber occurs by either forward movement of fish or synchronous beating of ciliated non-sensory cells. in the present study, the sensory epithelium of c. garua is consisted of ciliated, microvillus and rod receptor cells. the ciliated receptor cells correspond to type i cell of yamamoto and ueda (1978), whereas microvillus to those of type ii cells of muller and marc (1984) and rod cells of those type iv cells of ichikawa and ueda (1977). the ciliated receptor cells dominate over the microvillous and rod receptor cells. the ciliated receptor cells with knobs are of special interest because they are able to detect chemical changes in the surrounding environment. the long and welldeveloped dendrite process of the receptor cell enables the fish to smell its food even in muddy surrounding. bhute and baile (2007) advocated that the microvillous receptor neurons perceive and process signals of pheromone, which is an important step of breeding in labeo rohita. on the other hand, bakhtin (1977) and bannister (1965) reported that microvillous cells in the olfactory surface of squalus acanthias and teleostean fishes are predecessors of ciliated receptor cells. zielinski and hara (1992) and moran et al. (1992) have mentioned that rod shaped processes represent the dendrite apical processes of olfactory receptor cells. hernadi (1993) proposed that the occurrence of the rod-shaped olfactory neuron has been observed in the presence of a new physiological condition. the epithelial surface is covered with non-sensory cilia, which propel streams of incoming water and/or 285 int. j. aquat. biol. (2018) 6(5): 281-287 dissolved chemicals between the lamellae and act as guides enabling chemicals to reach olfactory terminals containing receptor sites (singh and singh, 1989). the supporting cells have been suggested to perform several functions: secretory, absorbing and glial (yamamoto and ueda, 1978; hernádi, 1993; theisen, 1972; hansen and zeiske, 1998). mast cells in the olfactory mucosa are believed to play an important role in reproduction of baltic trout (bertmer, 1982) and indian major carp, l. rohita (bhute and baile, 2007). mast cells can change metabolic activity of receptors and thereby the sensitivity of olfactory epithelium. the mast cells are thought to cause fluctuations in the production of mucus over the olfactory epithelium. as the terminal mucus film is believed to be an important factor in the olfactory process this may also influence the variations in the olfactory sensitivity (moulton and beidler, 1967). the basal cells are located in the deeper layers of the epithelium. graziadei and metcalf (1971) advocated that the basal cells are able to differentiate into receptor cells while ojha and kapoor (1973) reported their transformation into supporting cells. mucous cells secret mucin to shield the epithelium from mechanical abrasion. the secreted mucin from the mucous cells probably helps for coagulating microscopic debris and thereby keeps the receptor ready for new stimuli. zeni and stagni (2002) opined that the mucus covering the olfactory lamellae constitutes an important medium in which the odorants are diffused like that of other olfactory systems of vertebrates. in conclusion, elongated olfactory rosette bounded by 40±02 lamellae associated with a predominantly developed sense of olfaction for exploring the surrounding in which c. garua live. it is a macrosmatic species, can be classified as “nose fishes” whose olfaction is better developed than vision. during forward rhythm, unidirectional water flows into anterior opening and passes out through the posterior one after bathing the olfactory epithelium. ascendant receptor cells in the epithelial lining manifest the olfactory stimuli and assess the surrounding habitat. acknowledgements this research was supported by the grants from the department of higher education, science & technology and biotechnology, government of west bengal [memo number: 275 (sanc.)/st/p/s&t/1g37/2017 dt. 27/03/2018]. references abai e.a., ombolo a., ngassoum m.b., mbawala a. 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(2019) 7(5): 280-290 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article biosorption of nickel by halobacillus sp. kn57 isolated from the miankaleh wetland, iran nafiseh torabia1, fatemeh kardel*21 1department of marine biology, faculty of marine science, university of mazandaran, babolsar, mazandaran, iran 2department of environmental sciences, faculty of sciences, university of mazandaran, babolsar, mazandaran, iran. s article history: received 10 july 2019 accepted 22 october 2019 available online 2 5 august 2019 keywords: biosorption heavy metal wetland kinetic isotherm abstract: heavy metals are generally discharged from untreated wastewater in the aquatic ecosystem. this study was aimed to investigating the effective parameters in the sorption of nickel metal by a native halophile bacterium isolated from sediments of the miankaleh wetland in the north of iran. halobacillus sp. kn57 as the most resistant gram-positive strain was selected and identified by phenotypic and phylogenetic properties (16s rrna). the optimum contact time, ph, temperature, salinity, and initial biosorbent concentration of nickel for this strain were 100 min, 7, 30°c, 10%, 1 g.l-1, and 150 mg.l-1, respectively. the nickel biosorption was 111.11 mg/g by the selected strain under the laboratory conditions, followed langmuir isotherm with a correlation coefficient of more than 0.98 and the maximum single layer sorption. in addition, the kinetics of nickel biosorption of this strain correspond to a pseudo second order kinetic model with a correlation coefficient of more than 0.99. scanning electron microscopy was applied to confirm the biosorption of ni2+ by halobacillus sp. kn57. finally, the ft-ir spectrum identified that the amide, carbonyl, and amine functional groups were participated in binding to nickel ions. the results showed that the native bacterial strain (halobacillus sp. kn57) isolated from miankaleh wetland, is potentially a promising biosorbent for sorption of nickel metal. introduction heavy metals are one of the most important threats to the wetlands (bai et al., 2011). nowadays, various human activities have been led to entrance of a worrying amount of the heavy metals into aquatic ecosystems, including wetlands. these pollutants can impact the ecosystems, and ultimately, human beings due to their persistence and accumulation in the food chain (pascoe et al., 1996). nickel is found in industrial wastewater such as stainless steel, nonferrous alloys and super alloys, mineral processes, color formulation, electrotyping, glazing, pumice, copper sulfate production, electronics products, welding products, textile, nickel-cadmium batteries, and fertilizers (coman et al., 2013). since the wastewaters enter aquatic ecosystems, if they are not properly cleaned, hence, removal of the heavy metals from industrial effluent and aquatic ecosystems need to be done using physicochemical and biological *correspondence: fatemeh kardel doi: https://doi.org/10.22034/ijab.v7i5.704 e-mail: f.kardel@umz.ac.ir methods (regine et al., 2000). in this regard, biological methods can preferred than physicochemical methods due to their costeffectiveness, eco-friendly and less sludge production (regine et al., 2000; masoumi et al., 2017). bacteria, fungi, algae, yeasts, agricultural waste and other polysaccharide materials are of the most potent to extract and remove heavy metals (volesky, 1986; padmavathy et al., 2003; ozturk, 2007; özer et al., 2008; abdel-ghani and el-chaghaby, 2014). among the biological adsorbents, bacteria are good adsorbent because of their high surface-to-volume ratio, and having surfaces for the absorption of chemicals like teichoic acid (ozturk, 2007; masoumi et al., 2017). in addition, bacteria, due to their small size, growth ability under controlled conditions, and resistance to a wide range of environmental conditions can be considered as a good bioadsorbent (ansari et al., 2011; kulkarini et al., 2014). halophile bacteria https://www.sciencedirect.com/science/article/pii/s0921344913000281#! 281 int. j. aquat. biol. (2019) 7(5): 280-290 are used in this research due to their importance in biotechnology, and their industrial application to produce enzymes, polymers, proteins and so on (kushner and kamekura, 1998). in such a way that their growth in high salt concentrations can minimize the risk of contamination of the culture medium, and their simple nutritional needs help to use different combinations of carbon and energy sources (ventosa et al., 1998). according to our knowledge, there is no information on nickel-resistant native halophile bacteria species from the miankaleh wetland and biosorption capability of nickel metal by halobacillus sp. are available. hence, this study aimed to evaluate kinetics, isotherms and effective parameters in biosorption of nickel by a native halophile bacterium (halobacillus sp.) from the miankaleh wetland. materials and methods study area and sampling: sediment samples were taken from the miankaleh wetland at latitudes of 36°49'24'' and 36°56'45'', and longitude of 53°24'50'' and 541'20'' in the golestan province, iran during fall 2015. sediments were sampled from the top 5 cm, and stored in a plastic bag and transferred to the laboratory. isolation and identification of bacteria: the isolation of gram-positive strains was carried out by culturing of the samples on moderate halophilic media (mh) (nacl 1.1 g.l-1, mgcl2. 6h2o 7 g.l -1, mgso4. 7h2o 9.6 g.l-1, cacl2. 2h2o 0.36 g.l -1, kcl 2 g.l-1, nahco3 0.06 g.l -1, nabr 0.026 g.l-1, yeast extract 10 g.l-1 , peptone 5 g.l-1, glucose 1 g.l-1, and agar 15 g.l-1) (gaballa et al., 2003), and gram staining for 48 h at 30°c. the adjusted ph of the growth media was 7 which was made using 0.1 n h2so4 and/or naoh. further steps for preparation of samples were the same. a strain with the maximum growth at the highest concentration of nickel was selected as the most resistant gram-positive strain to nickel metal. morphological characteristics (color and form of colony), gram staining method, and biochemical tests (simon citrate, triple sugar iron, motion, indole, sulfide production, methyl red and voguesproskauer) were done using bergey's manual by sequencing of 16s rrna gene to identify halobacillus sp. kn57 (bergey et al., 1930; krieg et al., 1984). biosorption time and kinetic study: a solution containing 150 mg nickel nitrate and 1000 ml deionized water was prepared in an erlenmeyer flask. a sample of zero time in kinetics (control) was isolated from this solution. then, time was recorded by adding 1 g.l-1 of bacterial biomass to the solution and placing flask in an incubator shaker at 30°c and 150 rpm. samples were removed from the solution and filtered at different time periods (5, 10, 20, 30, 40, 50, 60, 75, 90, 120, 150, 180 and 240 min). concentration of the residual nickel ion in the solutions (ce) were measured by a flame atomic absorption spectroscopy (agilent varian aa240 fs) with three replications. then, the biosorption was calculated at different times. the sorption rate was calculated using following equation: qe = (ci − ce) v m eq. (1) where qe is the metal uptake or biosorption (mg.g1), ci=initial concentration of metal (mg.l-1), ce=metal concentration in solution (mg.l-1), v=solution volume (l), and m=starting sorbent weight (g). the data of this stage were compared with the pseudo-first and second kinetic models (ho and mckay, 1999; ho, 2006). pseudo-first kinetic model (equation 2) and its linear form (equation 3) are as follow (lagergren, 1898; ertugay and bayhan, 2008): dq dt = k1(qe − qt) eq. (2) ln (qe−q) qe = −k1 t eq. (3) pseudo-second kinetic model (equation 4) and its linear form (equation 5) also were (chojnacka, 2010): dq dt = k2(qe − q) 2 eq. (4) t qt = 1 k2qe 2 + 1 qe t eq. (5) where qe and qt are the amount of metal biosorbed per unit weight (mg.g-1 dry weight) of biosorbent at equilibrium at any time, t time (min),and k1 and k2 the rate constant of pseudo-first-order sorption (min-1) and rate constant of pseudo-second-order sorption (mg.g-1 min-1), respectively. 282 torabia and kardel/ biosorption of nickel by halobacillus sp. initial metal concentration and biosorption isotherms: to evaluate the isotherm of equilibrium sorption of nickel, amount of the nickel biosorption at various initial concentrations of metal ions (25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 and 300 mg.l-1) were investigated under constant conditions (ph=7, temperature=30°c, adsorbent concentration=1 g.l-1, and salinity=10%). at last, the compatibility of the findings was investigated which resulted in the effect of the initial concentration of metal on biosorption with langmuir and freundlich isotherm models. based on langmuir isotherm model, biosorbent surface assumes homogeneous, no interaction between adsorbed molecules, and once a site is occupied by a species, no more sorption take place at that site, while the freundlich isotherm model assumes adsorption on a heterogeneous surface, and there is interaction between adsorbed molecules in this model. langmuir equations (equation 6) and freundlich (equation 7) are as follows (langmuir, 1918; freundlich, 1906): 𝑞𝑒 = 𝑏𝑞𝑚𝐶𝑒 1+𝑏𝐶𝑒 eq. (6) 𝑞𝑒 = 𝑘𝐹𝐶𝑒 1 𝑛⁄ eq. (7) where qe is the amount of metal absorbed per unit weight of biosorbent (mg.g-1), ce = equilibrium metal concentration (mg.l-1), qm = maximum metal uptake of biosorbent (mg.g-1), b = langmuir constant (l.mg1), kf = distribution coefficient, and n = correction factor (n>1). optimum ph, temperature, salinity, and adsorbent concentration: after obtaining the best initial concentration for optimal sorption (150 mg.l-1), the experiments were performed to find the effect of ph (4-9), temperature (5-40°c), salinity (4-16% sodium chloride), and adsorbent concentration (0.5-2 g.l-1) on the nickel biosorption in three replication. unless otherwise stated, for the experiments of nickel sorption, ph, temperature, adsorbent concentration, and salinity were 7, 30°c, 1 g.l-1, and 10%, respectively. biosorption by live and dead bacteria: in an erlenmeyer flask, the nickel solution was prepared by solving 150 g of the nickel nitrate in 1000 ml deionized water (150 g.l-1). next, a reference sample was taken from the prepared solution, then it was shared between two 250 ml erlenmeyer flask. the dead biomass, due to autoclaving of the living bacteria biomass, and living bacteria were added to solutions with the same concentration (1 g.l-1) at temperature of 30°c for 10 min. at the end of experiment, the samples were taken by syringe and filtered, then concentration of the nickel in a solution was measured using aas. surface morphology of the selected strain using scanning electron microscope: the liquid culture medium containing bacteria was centrifuged at 10000 rpm for 10 min, after filtering a wet biomass of bacteria was obtained. this biomass was divided into two parts, one part was exposed to the nickel metal and another one not. then, both portions were dried in an oven for 48 h at 40°c. scanning electron microscopy (vega-lmu model manufactured by tescan usa) was applied to observe the bacteria surface without and with present of the nickel (kulkarini et al., 2014). fourier transforms infrared (ft-ir) spectroscopy: to identify the functional groups for sorption of ni by bacteria, a ft-ir spectrophotometer was used. in order to prepare solid samples, the bacterial biomass without and with present of the nickel were placed in an oven at 40°c for 48 h and dried. the dried samples, which were completely powdered, mixed with potassium bromide (kbr) and converted to pillshaped under a high pressure, and finally pills were examined by the ft-ir spectroscopy (verma et al., 2017). results and discussions isolation and purification: the bacterial strains (n=14) were isolated and purified from the sediment of the miankaleh wetland. after gram staining, culturing of 9 isolated gram-positive halophile bacteria strains on salt-free nutrient agar culture medium resulted in the separation of halophile strains from salt tolerant, and 7 gram-positive strains were selected to continue the experiment. then, kn57 strain was selected as the most resistant strain to nickel by testing the minimum 283 int. j. aquat. biol. (2019) 7(5): 280-290 concentration inhibiting bacterial growth. identification: according to the morphological characteristics of bacteria, the strain of halobacillus sp. was identified by gram staining and biochemical tests. the 16s rrna gene sequence of the kn57 strain with access number of ky490583 has shown the highest match (98%) with halobacillus sp. strains (supplementaries 1-2). optimum biosorption time and kinetics: biological sorption of bacteria occurs in most cases with two different mechanisms of biosorption (which is a fast, inactive, and independent of metabolism) and bioaccumulation (which is a slow, active, and dependent on cellular metabolism) (goyal et al., 2003). study of sorption time showed that biosorption of the nickel by kn57 strain had two stages. at the first, the absorption rate reached 54.52 mg.g-1 over 5 min. in fact, at an initial 5 min of the experiment, 36.14% of the nickel metal was absorbed by the selected strain indicating metal binding to the bacteria surface, due to a large available surface area at the initial times, which accrues very fast. up to 240 min, the absorption rate reached 76.75 mg.g-1 of nickel, i.e. an average of 50.87% of the nickel was absorbed (fig. 1). the biosorption over time showed no significant increase in sorption of metal after 100 min, due to the competition between metal ions for binding to active sites on bacteria surface in solution. therefore, 100 min is selected as an optimum biosorption time. in order to understand the mechanism of biosorption process, the experimental data of biosorption at different times (qt) with first and second order kinetic models, diagrams of log (qe-qt) and t/qt versus t (time) were plotted, respectively. the results figure 1. biosorption of nickel metal by halobacillus sp. (kn57) at different contact time. figure 2. pseudo first (a) and pseudo second order model (b) for the biosorption kinetics of ni (ii) by halobacillus sp. (kn57). 284 torabia and kardel/ biosorption of nickel by halobacillus sp. showed a higher value for correlation coefficient (r2) of the pseudo second order model (r2=0.999) than to the pseudo first order model (r2=0.719) (table 1). moreover, the qt values derived from second order kinetic model were more fitted to experimental qt values at different times than first order kinetic model (table 1, fig. 2). thus, our findings follow a second order kinetic model which this model explains chemisorption of ni2+ by halobacillus sp. (kn57) biomass as rate limiting step (fig. 3). some former studies on biosorption of metal by bacteria also reported that the kinetics of metal biosorption follow a pseudo-second order kinetics (kulkarini et al., 2014; masoumi et al., 2016; tabaraki et al., 2013). optimum concentration of metal and biosorption isotherm: the initial concentration of metal is playing an important role for the metal absorption by a biosorbent in the solution (abdel-ghani and elchaghaby, 2014). in this study, biosorption of the nickel at various concentrations (25-300 mg.l-1) showed that by increasing the nickel up to 150 mg.l1, biosorption increased under constant conditions (fig. 4). at lower concentrations of nickel, an increase in concentration resulted in an increase of biosorption per unit weight of the sorbent which could be related to the greatest surface area of bacteria being available table 1. parameters for pseudo first and second order models for ni (ii) biosorption kinetic by halobacillus sp. (kn57). biosorption pseudo first order kinetic model pseudo second order kinetic model 𝑞e(mg.g -1) 𝑘1 r 2 𝑞e (mg.g -1) 𝑘2 r 2 kn57 strain 549.540 0.101 0.719 83.330 0.003 0.999 table 2. parameters of langmuir and freundlich model for adsorption of ni (ii) on halobacillus sp. (kn57). biosorption langmuir frendlich 𝑞max (mg.g -1) 𝑏 (l.mg-1) r2 𝐾𝐹 (l.g -1) 𝑛 r 2 kn57 strain 111.110 0.009 0.987 4.139 1.865 0.872 figure 3. comparing the experimental data with the non-linear form of pseudo-first and pseudo second order model for the biosorption of ni (ii) by halobacillus sp. (kn57). figure 4. effect of initial concentration of ni (ii) on biosorption of this metal by halobacillus sp. kn57 (error bars represent ± standard deviations). 285 int. j. aquat. biol. (2019) 7(5): 280-290 (kulkarini et al., 2014). at higher levels of initial concentration of the metal (> 150 mg.l-1), this increase did not affect the biosorption rate, which could be due to the reduction of existing sites on the adsorbent surface comparing to the existing nickel ions in the solution. the similar results also reported in other studies (kulkarini et al., 2014; masoumi et al., 2016; tabaraki et al., 2013). biosorption isotherms was investigated using langmuir and freundlich model at different concentration of the nickel metal to describe the relationship between the amount of metal adsorbed by bacterial biomass and concentration of the metal in the solution. the obtained experimental data was plotted the 1/qe versus 1/ce based on langmuir model, and also was plotted the log qe versus log ce based on freundlich model (fig. 5a, b). the r2 value obtained from langmuir (r2=0.987) and freundlich model (r2=0.872) (table 2) showed that the experimental data were more consistent with the langmuir model than the freundlich model (fig. 6). thus based on the langmuir model, halobacillus sp. (kn57) surface is homogeneous and no more sorption take place at the saturated sites. this results can be applied for further design of adsorption species by halobacillus sp. (kn57). effect of ph: ph is an important parameter for absorption of metal in aquatic solution by a bacterium (cayllahua et al., 2009; fan et al., 2014; sarada et al., 2014; masoumi et al., 2016). the effect of ph on the biosorption of nickel by kn57 strain is shown in figure 7. the biosorption rate was small in low ph due to the presence of proton ions (h+) and their competition with nickel ions. with increasing ph and decreasing h+, the biosorption rate increased so that the maximum biosorption was observed at neutral ph (ph=7) (huang and liu, 2013). the absorption rate decreased with increasing ph from 7 to 9, due to low solubility of ions at high ph, and consequently nickel could be deposited in the form of hydroxide (kulkarini figure 5. plots for langmuir (a) and freundlich model (b) for adsorption of ni (ii) on halobacillus sp. (kn57). figure 6. the non-linear form of langmuir and freundlich biosorption isotherms of ni (ii) by halobacillus sp. (kn57). 286 torabia and kardel/ biosorption of nickel by halobacillus sp. et al., 2014; masoumi et al., 2016). our finding was in line with those of kulkarini et al. (2014). effect of temperature: effect of temperature on biosorption of metal by microorganisms is species specific (fan et al., 2014; kulkarini et al., 2014; masoumi et al., 2016). it is well-known that temperature is an important parameter which affects microbial growth and metabolism (fan et al., 2014). in this study, nickel biosorption at different temperature under constant conditions showed that biosorption increased by increasing temperature up to 30°c and after that, the biosorption was decreased (fig. 8). increasing biosorption by temperature rise can be due to increasing the size of pores on the adsorbent surface, creating new active sites, increase the speed of some slow sorption steps or increase mobility of metal ions. however, excessive increase in this factor can lead to the destruction of the physical structure (tabaraki et al., 2013). biosorbent concentration: for, absorption of the nickel at different concentrations of biosorbent (0.5, 1, 1.5 and 2 g.l-1) under constant conditions, it was observed that absorption increased by raising the concentration of biosorbent up to 1 g.l-1, but when the figure 7. effect of ph on biosorption of ni2+ by halobacillus sp. kn57 (error bars represent ± standard deviations). figure 8. effect of temperature on biosorption of ni2+ by halobacillus sp. kn57 (error bars represent ± standard deviations). figure 9. effect of ni concentration on biosorption of metal by halobacillus sp. kn57 (error bars represent ± standard deviations). figure 10. effect of salinity on biosorption of ni2+ by halobacillus sp. kn57 (error bars represent ± standard deviations). figure 11. biosorption of ni2+ by live and dead biomass of halobacillus sp. kn57 (error bars represent ± standard deviations). 287 int. j. aquat. biol. (2019) 7(5): 280-290 biosorbent was more than 1 g.l-1, the adsorption was reduced (fig. 9). the increase of biosorbent concentration led to the increase of metal absorption attributed to the availability of the more surface area, and the biosorption reduction at a high concentration of biosorbent could be related to the complex interaction of different factors in the solution (masoumi et al., 2016). effect of salinity: the effect of salinity on adsorption under constant conditions showed that the absorption rate decrease with increasing salt content (fig. 10). the increase in na+ ion resulted in declining absorption due to the competition between na+ ions and heavy metal cations in absorption to the bacterial binding sites (functional groups with negative charge) (deschatre et al., 2013). biosorption potential of live and dead bacteria: the nickel sorption by live and dead mass under constant conditions revealed that the absorption rate was 69.76 mg.g-1 in living and 44.08 mg.g-1 in the dead (autoclaved) bacteria (fig. 11). therefore, the highest rate of biosorption was inactive and independent of metabolism that occurred quickly. the cell metabolism depended active biosorption was carried out at a lower rate with a lesser extent which obtained by the difference between the biosorption of live and dead mass (i.e. 25.68 mg.g-1). therefore, the live mass of the selected bacteria as native bacterium has a high capacity for removal of metal from the miankaleh wetland, and dead mass has a high potential to be produced on large scale in industries due to its metabolic independent sorption process. scanning electron microscopy (sem): sem examination revealed the adsorption of ni2+ by the surface of halobacillus sp. kn57 (fig. 12). fourier transform infrared (ftir) analysis: the results of the fourier transform infrared spectroscopy figure 12. morphology of the surface of halobacillus sp. kn57 before (a) and after (b) adsorption of nickel with scanning electron microscopy (sem). figure 13. ft-ir spectrum for halobacillus sp. kn57 before (a) and after (b) adsorption of ni2+. 288 torabia and kardel/ biosorption of nickel by halobacillus sp. in the absence and presence of nickel showed a shifts of the bands and change in the intensity of biosorption peaks (fig. 13). the change in the biosorption peak intensity at wavelengths of 3773 cm-1 and 3857 cm-1 indicates interaction of the nickel metal with amide groups. at a wavelength of 3435 cm-1, the stretching band of hydroxyl (o-h) was observed, which after exposure to nickel does not differ significantly in terms of the biosorption and displacement. the stretching band at the wavelength of 12362 cm-1 is related to the cyanide functional group (≡ nc), which does not have an effect on biosorption as this functional group does not form a complex with heavy metals. biosorption peaks in the regions of 1653 cm-1 and 1547 cm-1 are related to the carbonyl (c-o) and amine (c-n) groups that considering the change in sorption peak intensity and peak displacement of 100 nm. then it is concluded that the functional groups of carbonyl and amine are highly effective in biosorption. in the wavelength of 1057 cm-1, the c-h bending band can be observed, which has no effect on biosorption. conclusion this study demonstrated that biosorption of the nickel by halobacillus sp. kn57 is highly dependent on ph, temperature, biosorbent concentration, salinity, and contact time. the live biomass of the selected bacteria had a higher biosorption of metal compared to dead biomass which suggests both dead and live bacteria from the miankaleh wetland can play an important role for sorption of metals in wastewater treatment and aqueous ecosystems. the sem examination confirmed the biosorption of metal on the surface area of halobacillus sp. kn57. the ft-ir analysis identified functional groups involved for metal absorption by halobacillus sp. kn57. the biosorption of nickel ions by halobacillus sp. kn57 follows pseudo-second order kinetic model indicating chemisorption as rate limiting step. based on langmuir model, the maximum biosorption capacity of nickel ions by halobacillus sp. kn57 was 111.11 mg.g-1. in conclusion, the kinetic and isotherm models allowed to gain insight into the biosorption mechanism of nickel ions by halobacillus sp. kn57. this study based on kinetics study, langmuir and frendlich model, effective factors, biosorption capacity of dead and live bacteria, sem examination, and ft-ir analysis suggests that halobacillus sp. kn57 is a promising biosorbent for removal of metal specially nickel from contaminated sites. acknowledgements the authors thank l. satari faghihi, j. abbaszadeh and s. abbaskhani davanloo for their help in the isolation and identification of the selected bacteria in the lab. this work was granted by university of mazandaran for a master student research program. references abdel-ghani n., ei-chaghaby a. 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(2009). evaluation of equilibrium, kinetic and thermodynamic parameters for biosorption of nickel (ii) ions onto bacteria strain, rhodococcus opacus, minerals engineering, 22: 1318-1325. chojnacka k. (2010). biosorption and bioaccumulation the prospects for practical applications, environment international, 36: 299-307. coman v., robotin b., ilea p. (2013). nickel recovery/removal from industrial wastes: a review. resources, conservation and recycling, 73: 229-238. goyal n., jain s.c., banerjee u.c. (2003). comparative 289 int. j. aquat. biol. (2019) 7(5): 280-290 studies on the microbial adsorption of heavy metals. advances in environmental research, 7: 311-319. deschatre m., ghillebaert f., guezennec j., colin c.s. (2013). sorption of copper(ii) and silver(i) by four bacterial exopolysaccharides. applied biochemistry and biotechnology, 171: 1313-1327. ertugay n., bayhan y.k. (2008). biosorption of cr (vi) from aqueous solutions by biomass of agaricus bisporus, journal of hazardous materials, 154432. fan j., okyay t.o., rodrigues d.f. (2014). the synergism of temperature, ph and growth phases on heavy metal biosorption by two environmental isolates, journal of hazardous materials, 279: 236-243. freundlich h. (1906). adsorption in solutions. zeitschrift fur physikalische chemie (germany), 57: 385-470. gaballa a., amer r., hussein h., moawad h., sabry s. (2003). heavy metal resistance pattern of moderately halophytic bacteria. arab journal of biotechnology, 6(2): 267-278. ho y.s., mckay g. (1999). pseudo-second order model for sorption processes, process biochemistry, 34, 451-465. ho y.s. (2006). review of second-order models for adsorption systems. journal of hazardous materials, 136: 681-689. huang w., liu z. (2013). biosorption of cd(ii)/pb(ii) from aqueous solution by biosurfactant-producing bacteria: isotherm kinetic characteristic and mechanism studies. colloids and surfaces b: biointerfaces, 105: 113-119. krieg n.r., holt j.g. (1984). bergey’s manual of systematic bacteriology, vol. 1. pp: 343-352. kulkarni r.m., vidyashetty k., srinikethan g. (2014). cadmium (ii) and nickel (ii) biosorption by bacillus laterosporus (mtcc 1628). journal of the taiwan institute of chemical engineers, 45(4): 1628-1635. kushner d.j., kamekura m. (1998). physiology of halophilic eubacteria, in: f. rodriguez-valera (ed.). halophilic bacteria, 1sted boca raton, crc press. pp: 109-140. lagergren s. (1898). about the theory of so called adsorption of soluble substances. kungliga sevenska vetenskapsakademiens hanlingar, 24: 1-6. langmuir i. (1918). the adsorption of gases on plane surfaces of glass, mica and platinum. journal of the american chemical society, 403-1361. masoumi f., khadivinia e., alidoust l., mansourinejad z., shahryari s., safaei m., mousavi a., salmanian a.h., zahiri h.s., hojatollah vali h., akbari noghabi k. (2016). nickel and lead biosorption by curtobacterium sp. fm01, an indigenous bacterium isolated from farmland soils of northeast iran. journal of environmental chemical engineering, 4(1): 950-957. özer a., gürbüz g., çalimli a., körbahti b. (2008). investigation of nickel (ii) biosorption on enteromorpha prolifera: optimization using response surface analysis. journal hazardous materials, 2: 778788. ozturk a. (2007). removal of nickel from aqueous solution by the bacterium bacillus thuringiensis. journal of hazardous materials, 147: 518-523. padmavathy v., vasudevan p., dhingra s.c. (2003). biosorption of nickel (ii) ions on baker’s yeast, process biochemistry, 38: 1389-1395. pascoe g.a., blanchet r.j., linder g. (1996). food chain analysis of exposures and risks to wildlife at a metalscontaminated wetland. archives of environmental contamination and toxicology, 30(3): 306-318. regine h.s., vieira f., volesky b. (2000). biosorption: a solution to pollution? international microbiology, 3(1): 17-24. sarada b., krishna prasad m., kishore kumar k., ramachandra murthy ch.v. (2014). cadmium removal by macro algae caulerpa fastigiata: characterization, kinetic, isotherm and thermodynamic studies. journal of environmental chemical engineering, 2: 1533-1542. ventosa a., nieto j.j., oren a. (1998). biology of moderately halophilic aerobic bacteria. microbiology and molecular biology review, 62: 504-544. verma a., kumar s., kumar s. (2017). statistical modeling, equilibrium and kinetic studies of cadmium ions biosorption from aqueous solution using s. filipendula. journal of environmental chemical engineering, 5(3): 2290-2304. 290 torabia and kardel/ biosorption of nickel by halobacillus sp. supplementary 1: the results of morphological investigation and biochemical test for kn57 strain. dark cream colonies color spherical colonies shape positive gram staining bacillus bacterial cell shape negative motility negative sulfide negative indole negative triple sugar iron negative simmons citrate negative methyl-red pink ring negative vogesproskaure negative gelatinase halobacillus sp. identification supplementary 2: phylogenetic tree of kn57 using neighborhood-joining and the bootstrap based on thousand replications. int. j. aquat. biol. (2014) 2(3): 115-123 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article sexual maturation of the blood cockle, anadara granosa, in matang mangrove estuary, peninsular malaysia tatsuya yurimoto*1, faizul mohd kassim2, alias man3 1japan international research center for agricultural sciences, tsukuba, ibaraki, japan. 2penang office of japan international research center for agricultural sciences, penang, malaysia. 3fisheries research institute, department of fisheries, penang, malaysia. article history: received 29 march 2014 accepted 22 april 2014 available online 2 5 june 2014 keywords: anadara granosa, blood cockle, sexual maturation, redox potential, malaysia. abstract: the sexual maturation and spawning period of blood cockle in matang mangrove estuary was studied by naked eye and histological observations of the gonads from july 2010 to april 2011. the high spawning period was from november to february. however, at one station where bottom sediment exhibited a severe reduction in potential of −100 mv lower, immature individuals were common. these results suggest the redacting environment inhibits the sexual maturation of blood cockle. introduction the blood cockle, anadara granosa, is an ark shell species bearing hemoglobin. the valve of the adult blood cockle forms a thick oval shape, and the surface has about 18–21 ribs and many small nodules (narashimham, 1988b). this species lives on the surface of muddy bottoms from the intertidal zone to the subtidal zone at depths less than about 5 m distributing mainly in the coastal waters of india, china, west japan and north australia (narashimham et al., 1984; nakao et al., 1989; faulkner, 2010). in southeast asia, this cockle species is an important species for aquaculture, and natural blood cockle spats are used for seeds to sow cultures (watanabe, 2009). sowing cultures of blood cockle is very popular in the west coast of the malay peninsula, particularly the coastal areas of penang, perak, and selangor districts (pathansali and song, 1958). however, sites with natural high density have been limited to the coastal waters of johor and selangor in recent years. therefore, it is important to clarify the current status * corresponding author: tatsuya yurimoto e-mail address: yurimoto@outlook.com of blood cockle reproduction in the penang and perak districts to ensure the sustainable production of blood cockle seed in those areas. however, little is known about the sexual maturation or main spawning period of the blood cockle in these districts. therefore, unstable factors of the occurrence of the natural spat fall area are unclear. thus, the natural spat area depends on factors such as defective maturation of adult blood cockles, depletion of the planktonic larvae, and high mortality of the spat in the early life stage. in this study, 3 survey stations were set up in the cockle culture grounds of matang mangrove estuary on the west coast of peninsular malaysia. the sexual maturation and spawning period of blood cockle were studied by naked eye observations at the visceral site as well as histological observations of the gonads. materials and methods survey and environmental measurement: three sampling stations (st. 1-3) were set in matang mangrove estuary in peninsular malaysia from july 116 yurimoto et al./ sexual maturation of the blood cockle, a. granosa 2010 to april 2011 (fig. 1). temperature, salinity, and dissolved oxygen in bottom-layer water (i.e., 20 cm above the seabed) at each sampling station were monitored by a water quality sensor (aaq-rinko, jfe advantech inc., japan). in addition, adult blood cockles were sampled to study their sexual maturation. also, surface sediments (i.e., 0–1 cm) of the 3 stations were monitored for the redox potential by an oxidation reduction potential (orp) sensor (rm-20, toa-ddk co. ltd., japan) in november 2011. the blood cockles collected from each station were transported to the laboratory of the fisheries research institute in penang. the cockles were dissected, their visceral sites were observed, and their gonads were fixed in 10% seawater–formalin solution. naked eye observations: a total of 246 blood cockles with shell lengths ranging from 16.8–39.1 mm were used for naked eye observations of the visceral site. the shells were observed, and the thickness of the visceral site was assessed; specimens were classified into 3 groups according to thickness. class i (immature stage): thickening is not observed at the visceral site; the midgut gland tissue is visible through the membrane of the organ; however, the status is not visible on gonadal tissue (fig. 2i). class ii (growth or regression stages): thickening on the visceral site is slightly observed, and gonadal tissue is visible through the membrane tissue of the visceral site (fig. 2ii). class iii (mature stage): marked thickening is observed at the visceral site, and gonadal tissue is filled at the site. sex was determined according to appearance (fig. 2iii). as a measure of thickness, individuals in the classes i, ii, and iii were scored 0, 1, and 2, respectively. the points were subsequently averaged for each survey, and plotted with standard errors on a graph. histological observations: the same samples were reused for histological observations. after dehydration in 70–100% alcohol, fixed tissue was embedded in paraffin by replacing the lemosol. tissues were cut from paraffin blocks into 7–10 μm sections, which were subsequently double-stained with meyer’s hematoxylin and eosin and observed under an optical microscope. in addition, the gonads were classified into 5 stages: the immature, developing, mature, spawning, and spent stages. the stages are described in detail below. immature stage: sex is indistinguishable in the gonads. it is difficult to observe germ cells in the gonads, and the site is mainly occupied by connective tissue or an empty genital tube (fig. 3). figure 1. locations of the 3 sampling stations in matang mangrove estuary, peninsular malaysia. figure 2. naked eye observations of the visceral site (arrow in photo i) were used to classify specimens into 3 categories: (i) immature, (ii) developing, and (iii) mature. figure 3. photomicrograph of immature stage. no traces of gametes are observed, so the sex is indistinguishable. h&e staining. scale bar = 100 μm. 117 int. j. aquat. biol. (2014) 2(3): 115-123 developing stage: sex discrimination is possible starting from this stage. in males, the genital tube is enlarged and the spermatogonia appear along the wall (fig. 4a). in the late stage, spermatocytes move toward the center of the genital tube and a small number of sperm cells appear. in females, the genital tube is enlarged and oogonia appear along the wall (fig. 4e). in addition, the ovary becomes a capsulelike tube structure, and oocytes and oogonia are attached to the tube wall. mature stage: in males, sperm and sperm cells occupy a large part of the genital tube; the sperm are arranged radially in center of the tube (fig. 4b). in females, there are many mature or late developing oocytes in the ovary; the oocytes transform into irregular or polygonal shapes (fig. 4f). figure 4. photomicrographs of testes (a–d) and ovaries (e–h) at each reproductive stage. (a, e) developing, (b, f) mature, (c, g) spawning, and (d, h) spent stages. h&e staining. scale bar = 100 μm. 117 118 yurimoto et al./ sexual maturation of the blood cockle, a. granosa spawning stage: in males, some sperm have been released, and many sperm and sperm cells remain in the genital tube (fig. 4c). in addition, some of the genital tube is contracted and partially collapsed after spawning. in females, a tube in the ovary is observed, because mature eggs have partially spawned (fig. 4g). oocytes in the late developing or mature stages remain in much of the genital tube. in addition, mature eggs usually exhibit nuclear disappearance because of germinal vesicle breakdown. spent stage: in males, the genital tube is almost devoid of sperm, which have completely spawned, although a few sperm remain (fig. 4d). in females, the genital tube is deformed and folded, and much of the tube is completely empty (fig. 4h). results naked eye observations: the thicknesses of the viscera of blood cockle specimens collected from st. 2 in september were observed. the thickness increased in november (fig. 5), continuing until january. the average score decreased in february, and the thickness had completely contracted by april. changes in the thickness of blood cockles at st. 3 exhibited similar changes as those of st. 2. the thickness increased from september to november, peaking from november to january, regressing in february, and completely regressed by april. on the other hand, at st. 1, thickening was almost 0 points in july, which continued until the last survey in april. histological observations: the histological changes in the gonads of both sexes from st. 2 and st. 3 were almost the same. specimens were mainly classified as being in the developing and mature stages in september, the mature and spawning stages in november and january, the spawning and spent stages in february, and the spent and immature stages in april (table 1). individuals in the spawning stage were first observed in males from st. 3 in october; individuals in this stage at st. 3 were observed until april. in addition, males in the spawning stage at st. 2 were observed from january to april; thus, individuals in the spawning stage were observed later than at st. 3. females in the spawning stage were first observed at st. 3 in november until february. in addition, individuals in the spawning stage at st. 2 were observed from january to figure 5. changes in the thickness of the visceral site at the 3 sampling stations. values represent mean ± se. numbers in parentheses are the numbers of samples. station 1: ●, station 2: △, and station 3: ○. table 1. occurrence of each developmental stage of anadara granosa in matang mangrove estuary from july 2010 to april 2011. 119 int. j. aquat. biol. (2014) 2(3): 115-123 february. on the other hand, all 18 individuals at st. 1 in july were immature; the percentage of immature individuals exceeded 70% in each survey throughout the study period. environment: the water temperature at the bottom of 3 stations ranged from 28.4–32.1°c (fig. 6a). the maximum temperature at st. 1–3 was recorded 30.3°c in april, 32.1°c in september, and 31.1°c in september, respectively. meanwhile, the minimum temperatures ranged from 28.4–28.5°c in late january (fig.6), the average temperature was not significant difference during these stations (fig. 6b). the minimum salinity at st. 1–3 was 19.3 psu in late january, 22.3 psu in late january, and 18.7 psu in april, respectively (fig. 6c). regarding the average salinity at each station during the study period, the average salinity at st. 2 was significantly higher than that of st. 3 (p<0.05) (fig. 6d). the minimum dissolved oxygen concentrations at st. 1 and st. 2 were 1.4 and 3.5 mg/l in february, respectively, and 2.2 mg/l in april and october at st. 3. the average dissolved oxygen concentration at st. 1 was significantly lower than that of st. 2 (p<0.05) but not significantly different from that of st. 3 (fig. 6f). finally, the redox potential in the bottom sediment at st. 1 was −100 mv lower in november and was significantly different from those of other stations (p<0.01) (fig. 7). discussion environment: matang mangrove is about 40,000 ha and has a long coastline of about 50 km from north figure 6. changes in and averages of water temperature, salinity, and dissolved oxygen on the bottom layer at the 3 stations in matang mangrove estuary during the survey period. (a) changes in water temperature. (b) average water temperature. (c) changes in salinity. (d) average salinity. (e) changes in dissolved oxygen. (f) average dissolved oxygen. large bars (means) and small bars (sd) without common letters indicate significant differences in each graph in b, d, and f (p<0.05; tukey test). station 1: ●, station 2: △, and station 3: ○. 119 120 yurimoto et al./ sexual maturation of the blood cockle, a. granosa to south (fontalvo-herazo et al., 2011). in addition, this region has a typical tropical rainforest climate; annual precipitation is 2,000–3,000 mm (ashton et al., 1999), and average temperature is 22°c at night and 33°c in the daytime with little annual variation (aldrie amir, 2012). therefore, water temperature is stable even in shallow water, which is easily affected by changing temperature. in the present study, the water temperature of the bottom layer was almost constant at the 3 stations throughout the study period. on the other hand, salinity exhibited clear changes among surveys, decreasing at 3 stations in september, february, and april. this is considered to be due to freshwater inflow from rivers as a result of heavy rainfall at the time. in addition, salinity remained low during the study period at st. 1 and st. 3, which are located in estuaries; these results indicate both stations are affected by inland water. in addition, dissolved oxygen concentrations were high at st. 2 throughout the study period, while concentrations were often low at st. 3 and particularly low at st. 1. okamura et al., (2010) surveyed the distributions of dissolved oxygen in the waters of matang mangrove estuary and found hypoxic waters (dissolved oxygen < 2 mg/l) were distributed in tributary rivers and the inner part of the mangrove during neap tide; furthermore, the hypoxic water mass extended to estuaries and coastal areas during spring tide. therefore, estuaries affected by land waters, such as st. 1 and st. 3, were considered susceptible to hypoxic water distributing in the inner part of the mangrove. in addition, okamura et al., (2010) surveyed redox potential in the surface of bottom mud in the same area; their results indicate bottom mud from the coastal to offshore areas is oxidative. however, in the present study, redox potential measurement of bottom mud in november showed oxidative values at st. 2 and st. 3., and a significant reduction of −100 mv lower at st. 1. it is necessary to consider the effect of specific organic loading on the oxidative reduction of sediment around st. 1., because this area has been recently exposed to increasing numbers of net cages for finfish culture. therefore, remaining feed from these cages may be one of the causes for the observed severe reduction at this site. spawning season: the spawning season of the blood cockle in tropical regions is very long. the spawning seasons of both sexes in the rusamilae waters of pattani bay, thailand off the east coast of peninsular malaysia are observed in september and from december to june (suwanjarat et al., 2009). the appearance of mature blood cockles in selangor estuary off the west coast of peninsular malaysia are observed from september to april (broom, 1983). in addition, in kakinada bay off the west coast of india, the spawning season of the blood cockle differs each year but spawning-stage individuals appear almost year round (narashimham, 1988a). on the other hand, from the naked eye observations of the visceral site of blood cockles collected at st. 2 and st. 3 in the present study, the thickness index peaked in november, continuing until january and regressing from january to april. in addition, from gonadal tissue observations of the same specimens, males and females in the spawning stage were observed from november to february. therefore, the peak of spawning season of the blood cockle in matang mangrove estuary is likely from november to february. the sexual maturation of bivalves is usually closely associated with water temperature and food availability (mackie, 1984). the figure 7. comparison of redox potential in the surface sediment at the 3 stations in matang mangrove estuary on november 2010. large bars (means) and small bars (se) without common letters indicate significant differences (p<0.01; tukey test). 121 int. j. aquat. biol. (2014) 2(3): 115-123 importance of these factors has been confirmed experimentally in manila clam, ruditapes philippinarum (mann, 1979; toba, 1989; toba and miyama, 1995). however, in tropical areas such the present survey sites, the water temperature of the bottom layer is stable almost year round. therefore, it is unlikely water temperature is a primary factor confining the spawning season. on the other hand, according to the average monthly precipitation in sitiawan, which is about 60 km south of matang mangrove estuary, referenced from the malaysian meteorological agency website (http://www.met.gov.my/), a rainy season of more than 200 mm/month occurs from october to december. in addition, it is well known phytoplankton in matang mangrove estuary increases from november to january; in one case, the peak phytoplankton volume was more than 5 times the normal volume (tarutani et al., 2005). therefore, the above mentioned studies suggest changing food availability during the rainy season is likely one of the major factors defining the spawning season of the blood cockle in matang mangrove estuary. immaturity: in this study, the visceral sites of blood cockles from st. 2 and st. 3 were remarkably thick; these tissues were confirmed to be developing germ cells by histological observation. meanwhile, the blood cockles collected in st. 1 did not exhibit the same thickness at the visceral sites; moreover, the gonads were completely immature or some cells in these gonads were slightly developing upon histological observation. therefore, this behavior observed in blood cockles from st. 1 is considered to be a unique phenomenon, which will be discussed below. comparing the environments at st. 1 and st. 3, which are located at the river mouth, both stations have almost the same temperature and salinity. however, regarding the average dissolved oxygen concentrations, the average concentration at st. 1 was significantly lower than those at st. 2 and st. 3. in addition, in november, when the blood cockles at st. 2 and st. 3 were in high spawning season, the redox potential at the surface layer of bottom mud was measured at each station; oxidative values were detected at st. 2 and st. 3, whereas a severe reduction below −100 mv was detected at st. 1. hata (1965) examined air-dried fine seabed mud containing 1% each of minced fish meat and powdered cellulose, which was filled with seawater and incubated at 30°c, and observed the environmental changes. the results show changes in redox potential: the mud was 340 mv on the first day, decreasing to below −100 mv after about 5 days, and to about −250 mv, stabilizing for about 20 days until the end of the experiment. in addition, when the same experiment was set up at different temperatures, the decrease in potential was significant at temperatures exceeding 22°c. furthermore, the results of that study demonstrate potential is closely associated with bacterial activity. on the other hand, the monitoring of bottom mud in an inner bay of the danish coast by jorgensen (1977) revealed seasonal variation in the redox potential; the results show the reduction of surface mud and hypoxic water just above the seabed is likely to occur in high temperatures in summer and disappear in winter. considering these findings and the fact that water temperature in tropical areas is stable at about 30°c year round, it is possible, mud with reduced redox potential remains. furthermore, it is well known that hypoxic water is likely to occur at the boundary layer immediately above the water on sediment (gundersen and jorgensen, 1990). therefore, it is very likely blood cockles, which commonly exist in the boundary layer, are exposed to the hypoxic environment detected in the present study or harsher environments. on the other hand, bivalves in remarkably low-oxygen environments are well known to have anaerobic metabolism including decreased metabolic and filtration rates (sobral and widdows, 1997). however, corresponding knowledge in the blood cockle is currently insufficient. nevertheless, it is quite possible the blood cockle is restricted by food availability when exposed to hypoxic environments. thus, the specimens collected from st. 1 suggest blood cockles cannot maintain good nutritional conditions for sexual maturation. 121 122 yurimoto et al./ sexual maturation of the blood cockle, a. granosa conclusion the sexual maturation and spawning of blood cockles from 3 stations of matang mangrove estuary in peninsular malaysia were estimated by naked eye and histological observations of visceral and gonadal tissues. the results indicate a high period of spawning from november to february during rainy season at 2 stations. however, at one other station, where redox potential was −100 mv lower at the surface bottom in november, maturation was not clearly observed during the study period. therefore, the results suggest the redacting environment inhibits the sexual maturation of the blood cockle. references aldrie amir a. 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(2014) 2(3): 164-171 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article allometric body shape changes and morphological differentiation of shemaya, alburnus chalcoides (guldenstadf, 1772), populations in the southern part of caspian sea using elliptic fourier analysis mohammad mohadasi1, soheil eagderi*2,1nader shabanipour1, mahboube sadat hosseinzadeh3, hossein anvarifar4, roozbahan khaefi5 1department of biology, faculty of science, guilan university, rasht, iran. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 3department of biology, faculty of science, ferdowsi university, mashhad, iran. 4department of fisheries, university applied science and technology, gorgan, iran. 5department of biology, faculty of science, shiraz university, shiraz, iran. article history: received 27 august 2013 accepted 21 february 2014 available online 2 5 june 2014 keywords: allometric changes caspian sea shemaya efa abstract: study of phenotypic diversity among populations can help better understanding of diversification of species within ecosystems and intraspecific diversification in fishes. a geometric morphometric study was carried out using the elliptic fourier analysis to demonstrate the effect of habitat type on morphological features of shemaya (alburnus chalcoides) populations. populations were sampled from three rivers and one lagoon, from the southern part of caspian sea. significant differences in body shape were found among the populations. differences in shapes of the riverine populations were minute compared to those of lagoon one in terms of size and shape. shemaya is an anaderemus fish and its populations have a common origin, therefore, observed differences could be as result of environmental factors. in addition, this study suggest that the amount of curvature i.e. fusiform body shape of this species could be independent form environmental condition. introduction the general body shape of an organism is determined not only by its genetics, but also by its ecology and environment (sara et al., 1999). different selective environmental pressures can generate and maintain different phenotypes. however, some geographical variations such as morphology, reproductive patterns, growth rates and mortality, are not always consistent with genetic variation and then can be related to phenotypic plasticity as a result of different environmental conditions (orensanz et al., 1991; cadrin, 2000). fish are among the most diverse aquatic organism in terms of morphology and ecology (helfman et al., 2009) and their intra-specific diversification is welldocumented (robinson and wilson, 1994; jonsson * corresponding author: soheil eagderi e-mail address: soheil.eagderi@ut.ac.ir and jonsson, 2001; robinson and wilson, 1994). fishes’ morphological characters provide information about their ecological niches (winemiller, 1991), allow more efficient use of available resources and improving fitness and performance (pianka, 1994). morphological characters are essential for identifying discrete phenotypic stocks (booke, 1999), and the historical development of stock identification methods has paralleled the advancement of morphometric techniques (cadrin, 2000). morphometric variation has been used as a method of stock identification for many fishery resources (palmer et al., 2004; cadrin and friedland, 2005). therewith, significant advances in morphometric analysis have occurred in the last two decades, offering more efficient and powerful techniques, such as image analysis (cadrin and 165 mohadasi et al./ morphological differentiation of shemaya populations friedland, 1999) and geometric morphometrics methods (rohlf and marcus, 1993) for detecting differences among groups. traditional morphometrics apply a variety of lengths, widths, angles, and ratios to obtain information about shape, whereas geometric morphometric approaches focus on complete, uniform measurement of shape, retaining all geometric information throughout its analysis. within this context, measurement of curves or outlines poses some challenges, since mathematically curves are infinite sets of points. in this way, fish outline data is collected automatically from images (ruff et al., 1994; russ, 1995) and analyzed using elliptic fourier analysis (efa) (kuhl and giardina, 1982; rohlf and archie, 1984; lestrel, 1997) to compute growth trajectories and visualizing shape changes. shemaya, alburnus chalcoides (guldenstadf, 1772), is widely distributed in the river systems of black, caspian and aral seas (bogutskaya, 1997). this species is benthoplagic and anadromous and found in fresh and brackish water of the southern caspian sea basin (slastenenko, 1959). therefore, this study aimed to study allometric body shape changes and morphological differentiation of shemaya populations in the southern part of caspian sea using elliptic fourier analysis. material and methods sampling: a total 120 individuals of the shemaya from lisar, shiroud and babolroud rivers and anzali lagoon were sampled during april and may, 2008 (table 1). the specimens were caught by handy net, cast net, and electrofishing and then fixed into 10% formalin solution. the specimens were photographed (canon g12, 2,304×1,704 pixel dimensions) and digitization was performed to obtain the body shape data for elliptic fourier analysis (efa) analysis. outline data were automatically collected as 150 profile point coordinates by tpsdig2 software version 2.16 (rohlf, 2004), excluding fins (fig. 1). elliptical fourier analysis: the applied method in this study was explained by kuhl and giardinia (1982) and were derived as a parametric formulation from conventional fourier analysis, i.e. as a pair of equations that represent the variation in x and y coordinates as a function of a third variable t, along the body outline (kuhl and giardina, 1982; lestrel, 1989). the efa includes description of the outline of a specific shape with several components (harmonics) with an ellipse as the first approximation step. each harmonic is characterized by four coefficients (fds), come from the sine and cosine part of the variation in the x and y coordinates (lestrel, 1997). for this study, first thirteen harmonics were selected as statistical variables and were forwarded to efw software (rohlf and ferson, 1992) using the gmtp software (taravati, 2010) for further analysis. before subsequence analysis, data were normalized and invariant to size, location, and rotation. size and multivariate analysis: after transformation, the centroid size (cs) was calculated (by gmtp software) for each specimen. in addition, the body length and width were measured using tpsdig2. for visualizing purpose, the size variation among groups, a 95% confidence interval error bar graph was plotted. to determine significant differences among groups multivariate analysis of variance (manova) was performed. for analyzing size, three variables including centroid size, area, and perimeter were used. these variables were obtained from gmtp software. for variables with normal distribution and similar variances, one-way anova was employed, and for others, with past program, the kruskal-wallis test was used. canonical variate analysis (cva) was performed to analyze the data and for classification functions and to assign individual specimens to putative populations, the stepwise discriminant function analysis (dfa) was performed. the classification success rate was evaluated based on percentage of individuals correctly assigned into figure 1. manual curve tracing of the fish (a. chalcoides). 166 int. j. aquat. biol. (2014) 2(3): 164-171 original sample. as a complement to discriminant analysis, morphometric distances among the individuals of four groups were inferred to cluster analysis (veasey et al., 2001) by adopting the euclidean square distance as a measure of dissimilarity and the upgma (unweighted pair group method with arithmetical average) method as clustering algorithm (sneath and sokal, 1973). allometry: the relationship between shape variables and cs was evaluated by multivariate regression analysis (rohlf and marcus, 1993), to investigate the allometric patterns associated with size. therefore, a principal component analysis (pca) was performed for each new set of variables. the correlation test was used between cs and pca scores using the pearson product-moment correlation coefficient and the pca with the highest correlation which was plotted against cs. results anova assumes data normality and homogeneity of variances. the centroid size (cs) and perimeter data were checked for normality (p>0.05) for each population, and the homogeneity of variances were tested by levene's test (p>0.05). the p value for anova of cs was 1.586e-21 (< 0.05) and 4.75e-21 for perimeter, showing cs and perimeter of populations are significantly different (fig. 2). the manova (wilks’ lambda) indicated a significant difference for mean vectors among four populations (ʌ= 0.031, f= 2.78, p= 3.082e-15). tukey's test was also used to determine which populations were different from each another (table 2). table 3 shows the bonferroni corrected mann– whitney pairwise comparison for all groups. confidence interval is another way of visualizing the difference among means of three or more populations. means and 95% confidence intervals for cs and body length are presented in figure 2. for assessing the power of multivariate analysis, the length/width ratio was calculated for each specimen, and its confidence interval was obtained for comparing the results of multi-width univariate analysis (fig. 2). 76.6% of individuals were correctly classified into their respective groups by discriminant function analysis (table 4), indicating a high differentiation between the studied populations. the cva scatter plot of elliptic fourier coefficients locality brevity gps sample size lisar river lr n: 37°58', e:48°56' 33 anzali lagoon al n: 37°28', e: 49°27' 38 shiroud river shr n: 36°49', e: 50°52' 36 babolroud river br n: 36°42', e: 52°39' 35 table 1. brevity of sampling site and sample size of shemaya (a. chalcoides) from southern of the caspian sea. lisar anzali shiroud babolroud lisar --7.72e-06* 7.72e-06* 1.179e-5* anzali 7.72e-06* --0.01664* 0.03918* shiroud 0.8926 7.721e-06* --7.814e-06* babolroud 0.0865 7.721e-06* 0.01174* -- table 2. tukey's pairwise comparisons for centroid size (over diagonal) and perimeter (under diagonal) showing the p (same) value. asterisks (*) indicate significant differences. lisar anzali shiroud babolroud lisar --1.47e-09* 5.91e-13* 1.24e-07* anzali 8.81e-09* --0.002723* 0.09694 shiroud 3.55e-12* 0.01634* --0.01634* babolroud 7.44e-07* 0.5816 0.000141* -- table 3. mann-whitney pairwise comparisons (bonferroni corrected) of body area. asterisks (*) indicate significant differences. 167 mohadasi et al./ morphological differentiation of shemaya populations showed differences among studied populations. in cva analysis, the two first components were responsible more than 80% of all variation (cv1= 53.3 and cv2= 32). figure 3 shows the projection of the specimens on the first two canonical functions and the changes in shape associated with them showing lisar anzali shiroud babolroud total original (%) lisar 70.7 2.8 2.8 5.6 100 anzali .0 94.9 .0 2.8 100 shiroud 2.9 2.9 67.5 2.9 100 babolroud 11.1 11.1 5.6 72.5 100 table 4. classification matrix showing the percentage of individuals that were correctly classified. (bold values indicate correct classifications). figure 3. canonical variate analysis scatter plot of four samples of a. chalcoides. species are shown with different shape. figure 2. four size variables: a: centroid size, b: body length, c: area, d: perimeter. (lr: lisar river, al: anzali lagoon: shr: shiroud river, br: babolroud river). 168 int. j. aquat. biol. (2014) 2(3): 164-171 that lisar and anzali populations are separated and shiroud and babolroud groups have overlapped to some extent. the lisar population with positive scores of cv1, was characterized by a stout-shaped body, whereas the anzali population, with negative scores on cv2, presented a slender-shaped body. the upmga graph shows two major distinct clads that first one includes anzali population and the rest positioned in the second one. the second clad is divided to lizar and shiroud-babolroud branchs (fig. 5). for distinguishing correlation between size and shape, the pearson product-moment correlation was used to find the correlation between the first three pc axes scores and cs. the scores of pc1 had the highest correlation (r = 0.72; p<0.001). the growth trajectory related in pc1 clarifies high shape variability in small specimens followed by a better defined pattern of shape change in larger specimens. figure 4 shows the plot of pc1 versus cs and shapes related in the extreme values of the axis, and it appears as a saturating curve. gradually, the shape of larger fish is more fusiform, the anterior region sharpens and the caudal peduncle is longer and slimmer as they are growing. discussion the results showed a significant difference in size of studied groups. the population of anzali lagoon has a larger and more curved body shape than others and their body curvature increases with increase of fish size. it is known that physical conditions of lagoons differ from rivers/streams systems (e.g. hendry et al., 2000; brinsmead and fox, 2002). lagoons have lentic physical conditions, lower water transparency, higher figure 4. pc1 versus centroid size (cs). graphical outline representations are reported for extreme values of principal components. figure 6. the 95% confidence intervals of body length/width ratio of four a. chalcoides populations (lr: lisar river, al: anzali lagoon: shr: shiroud river, br: babolroud river). figure 5. dendogram resulting from cluster analysis based on euclidean distances among the mean shapes of the species. 169 mohadasi et al./ morphological differentiation of shemaya populations water temperature, and greater leaf litter accumulation on the substratum (haas et al., 2011). it is commonly known that growth of lagoon fish is greater than that of riverine populations (warburton, 1979; marian et al., 2002). coban et al. (2008) also reported that there is no significant shape variation between cultured fish (that are always fed well) and lagoon caught. lisar is a river and characterized due to less depth, muddy bottom and high turbidity, fast-running water and less nutritious. meanwhile, the shiroud and babolroud rivers characterized due to more water clarity (particularly the shiroud river has a sandy bottom), lower turbidity, higher depth and more nutritious having a better environmental conditions than lisar river. therefore, it seems that hard environmental conditions of the lizar specimens could be led a smaller size (boily and magnan, 2002). the cva showed that the shape of four groups are significantly different. bagherian and rahmani (2007) reported a morphometric separation of shemaya populations from two geographical regions of the southern caspian sea. the reasons of morphological differences between populations are often quite difficult to explain (poulet et al., 2004; anvarifar et al., 2011), but it is well-known that morphometric characters can show high degree of the plasticity in response to environmental conditions (wimberger, 1994), such as food availability, water depth and flow, temperature and turbidity (allendorf, 1988; wimberger, 1994). it is generally considered that variation in size between populations depends largely on environmental conditions, whereas a variation in shape reflects in the genetic constitution (adams and funk 1997; orr and smith, 1998; schluter, 2000). since, the studied shemaya population are anadromous and have a common origin population. therefore, the difference in shape can therefore be considered to be a result of environmental affection. many fish species are famous to show distinct morphologies between lotic and lentic habitats (robinson and wilson, 1994; taylor et al., 1997; hendry et al., 2000; pakkasmaa and piironen, 2000; brinsmead and fox, 2002). hydrodynamic theory proves that a fusiform body shape decrease drag, and hence reduces energy consumption to maintain position in flowing water (keast and webb, 1966; blake, 1983; webb, 1984; videler, 1993; vogel, 1994), therefore, it is expected that in the same ages, river samples (particularly lizar specimens) show more fusiform body shape, but our results showed that the size of body is more effective, and river water current does not have any important role forming a more fusiform body shape (mohadasi et al., 2013). the anzali population (having better food condition with relatively bigger size) that lives in a lentic environment, shows more fusiform body shape than other populations. therefore, this study suggest that the amount of curvature could be as result of genetic and independent form water current. references adams d.c., funk d.j. 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(2019) 7(2): 112-116 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology book review fishes of guilan, by abbasi ranjbar k. 2017. 206 p. iliya culture publication, rasht, isbn: 978-964-190-517-2. ali reza radkhah, soheil eagderi*,1hadi poorbagher department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 7 february 2019 accepted 1 march 2019 available online 2 5 april 2019 keywords: caspian sea, diversity, guilan, inland waters. abstract: the caspian sea basin is one of the most important inland water basin of iran having the most diverse inland water fishes. the present study aimed to review the book entitled "fish of guilan'' by keyvan abbasi ranjbar published in 2017. this book provides general and biological information of the reported fishes in guilan province that can be used as a reference and identification key by fishermen and researchers who are involved in aquaculture, fisheries and biology. introduction iran, with 19 inland water basins, possesses a high fish diversity with 297 species mostly belonging to the cyprinidae followed by gobiidae and nemacheilidae (esmaeili et al., 2018). the caspian sea is one of the most important inland water basin of iran locating in the palaearctic region (coad, 1998; esmaeili et al., 2014). this basin has the highest diversity of fishes among the iranian inland waters (esmaeili et al., 2010, 2017) with 119 species belonging to 63 genera and 18 families, which 19 of them are exotic species (esmaeili et al., 2014). cyprinids are the most diversified family (35 species), at the next levels, gobiidae (19), and clupeidae (8) have the greatest diversity in the caspian sea basin. due to the high diversity of fishes in this basin, it is necessary to provide a comprehensive work regarding their taxonomic statues, morphological and biological features. although several studies have been carried out over recent years, e.g. esmaeili et al. (2014) included a checklist of the caspian sea basins, information regarding these fishes is still scanty. guilan is one of the northern provinces of iran with great diversity of fish species due to having a long coast of the caspian sea and major aquatic ecosystems of iran such as the sefid river and anzali wetland, *correspondence: soheil eagderi doi: https://doi.org/10.22034/ijab.v7i2.628 e-mail: soheil.eagderi@ut.ac.ir which provide rich and proper habitats for fishes (radkhah et al., 2015, 2016; asadi et al., 2016). abbasi ranjbar et al. (1998) published a book entitled “atlas of iranian fishes, inland waters of the guilan province” being updated recently by abbasi ranjbar as “fishes of guilan” published by iliya culture publication (rasht, guilan province). considering the importance of fish species in guilan province, this study aimed to review this book providing a brief overview of it different sections. the introduction of the book has presented a brief overview of the caspian sea basin plus pervious published works. the author has addressed some researches performed on this basin in the recent years. then, it reviewed previous studies regarding the status of some economically important fishes such as sturgeon, kilka and bony fishes. in addition, the book has provided some terms and concepts of fish taxonomy and morphology. at the end of this section, an identification keys of the families has been provided. the next section introduces fish families and the species that are found in the guilan province, i.e. the species belong to the families of petromyzontidae, acipenseridae, anguillidae, clupeidae, cyprinidae, cobitidae, nemacheilidae, siluridae, esocidae, salmonidae, gadidae, 113 int. j. aquat. biol. (2019) 7(2): 112-115 table 1. a list of the reported fish species in the guilan province (e: endemic, n: native, ex: exotic, t: translocated). family species/subspecies status petromyzontidae petromyzon wagneri n acipenseridae acipenser baerii acipenser gueldenstaedtii acipenser nudiventris acipenser persicus acipenser ruthenus acipenser stellatus huso huso ex n ex n n n n anguillidae anguilla anguilla ex clupeidae alosa braschnikowi alosa caspia caspia alosa kessleri alosa saposhnikovi alosa sphaerocephala alosa volgensis clupeonella caspia clupeonella engrauliformis clupeonella grimmi ex n n n n n n n n cyprinidae abramis brama acanthobrama microlepis alburnoides samiii alburnus chalcoides alburnus filippi alburnus hohenackeri ballerus sapa barbus cyri blicca bjoerkna capoeta razii carassius gibelio ctenopharyngodon idella cyprinus carpio hemiculter leucisculus hypophthalmichthys molitrix hypophthalmichthys nobilis leucaspius delineatus leuciscus aspius leusiscus sp. luciobarbus brachycephalus luciobarbus capito luciobarbus mursa mylopharyngodon piceus pelecus cultratus pseudorasbora parva rhodeus amarus rutilus caspicus rutilus kutum scardinius erythrophthalmus squalius turcicus tinca tinca vimba persa n n e n n n n n n e ex ex n, ex ex ex ex n n ? n n n ex n ex n n n n n n n cobitidae cobitis saniae cobitis aurata sabanejewia caspia n n n 114 radkhah et al. / fishes of guilan atherinidae, poeciliidae, gasterosteidae, syngnathidae, percidae, mugilidae and gobiidae. in addition, extra information regarding each species are presented, including local name, english name, external characteristics, feeding, reproduction, economical value and how to consume. some parts of the book need a major review. the taxonomic status of some species such as alosa caspia table 1. continued. family species/subspecies status nemacheilidae oxynoemacheilus bergianus e siluridae silurus glanis n pangasiidae pangasius cf. sanitwongsei ex loricariidae hypostomus plecostomus ex esocidae esox lucius n salmonidae oncorhynchus keta oncorhynchus mykiss salmo caspius salmo trutta stenodus leucichthys ex ex n n n gadidae lota lota n atherinidae atherina caspia n poeciliidae gambusia holbrooki poecilia reticulata ex ex gasterosteidae gasterosteus aculeatus pungitius platygaster ex n syngnathidae syngnathus caspius n percidae perca fluviatilis sander lucioperca sander marinus n n n mugilidae chelon auratus chelon saliens ex ex gobiidae anatirostrum profundorum benthophilus abdurahmanovi benthophilus baeri benthophilus ctenolepidus benthophilus granulosus benthophilus leobergius benthophilus macrocephalus benthophilus pinchuk hyrcanogobius bergi knipowitschia caucasica knipowitschia iljini knipowitschia longecaudata mesogobius nonultimus neogobius caspius neogobius melanostomus neogobius pallasi ponticola bathybius ponticola gorlap ponticola iranicus ponticola goebelii ponticola syrman proterorhinus nasalis rhinogobius lindbergi n n n n n n n n n n n n n n n n n n e n n n ex serrasalmidae piaractus brachypomus e channidae channa micropeltes e notopteridae chitala chitala e lepisosteidae genus atractosteus or lepisosteus e 115 int. j. aquat. biol. (2019) 7(2): 112-115 volgensis (p. 68; promoted to species level as a. volgensis in esmaeili et al. (2018)), acanthalburnus microlepis (p. 75; synonym of acanthobrama microlepis), rutilus caspicus (p. 104; synonym of r. lacustris), ponticola ratan (p. 172; synonym of p. goebelii), rhinogobius sp. (p. 176; named as r. lindbergii in sadeghi et al. (2019)) and cobitis keyvani (p. 112; synonym of c. saniae) are not correct (neilson and stepien, 2009; eagderi et al., 2017; jouladeh-roudbar et al., 2017; esmaeili et al., 2018; levin et al., 2016). in addition, capoeta gracilis (p. 85) does not exist in the caspian sea basin, hence, the reported c. gracilis has probably been mistaken by c. razii (esmaeili et al., 2018). cobitis faridpaki (p. 111) does not exist in guilan province, and probably this species has incorrectly been included in the book (jalili et al., 2015, 2016), and finally an unknown species has been presented in the book as leuciscus sp. (p. 95), however, its picture does not confirm the claimed species and further investigation is necessary. in the final section, some exotic ornamental fishes have been reported from the natural water bodies of guilan province. the author has discussed the negative impacts of these species. these ornamental fishes, including channa micropeltes, chitala chitala, pangasius cf. sanitwongsei and hypostomus plecostomus (pp: 183-181) are first records in iranian inland waters, therefore, they are needed to be listed in an updated checklist of iranian freshwater fishes. alligator gar, atractosteus spatula (lepisosteidae) has also been reported in this province from the anzali coast, however, it has already been reported from marivan (zarivar) lake (tigris basin) (esmaeili et al., 2017). despite having many positive features, there are still some shortages which we will briefly review them here and suggest them to be addressed in the next edition. there is no information regarding systematic status of the listed species. providing such data can help to understand the evolutionary processes of fish species in the caspian sea basin. there are many exotic fish species reported in the caspian sea basin (esmaeili et al., 2014) but a list of figure 1. cover page of the book. 116 radkhah et al. / fishes of guilan them has not been presented in the book as a table or graph. information such as richness of the families and their distribution could be useful even for general readers. for this purpose, we have provided a table of fishes of guilan (table 1). providing a list of fishes in some provinces of iran (such as fars and guilan provinces) is a useful trend that has been carried out in recent years by the iranian researches. this book has presented information similar to those provided for other provinces, making better understanding about identification, distribution and conservation of the iranian freshwater fishes. references abbasi k., valipour a.r., talebi-haghighi d., sarpanah a.n., nezami s. (1998). atlas of fishes of iran. inland water of guilan province. guilan fisheries research center, bandar anzali, iran. 126 p. (in farsi) asadi h., sattari m., radkhah a.r. (2016). distribution pattern of alburnoides eicwaldii in the tootkabon river (caspian sea basin, iran). journal of entomology and zoology studies, 4(4): 92-96. coad b.w. 1998. systematic biodiversity in the freshwater fishes of iran. italian journal of zoology. 65 (supplement): 101-108. eagderi s., joulade-roudbar a., jalili p., sayyadzadeh g., esmaeili h.r. (2017). taxonomic statue of the genus cobitis linnaeus, 1758 (teleostei: cobitidae) in the southern caspian sea basin, iran with description of a new species. fishtaxa, 2(1): 48-61. esmaeili h.r., coad b.w., gholamifard a., nazari n., teimory a. (2010). annotated checklist of the freshwater fishes of iran. zoosystematica rossica, 19: 361-386. esmaeili h.r., coad b.w., mehraban h.r., masoudi m., khaefi r., abbasi k., mostafavi h., vatandoust s. (2014). an updated checklist of fishes of the caspian sea basin of iran with a note on their zoogeography. iranian journal of ichthyology, (september 2014), 1(3): 152-184. esmaeili h.r., masoudi m., amini chermahini m., esmaeili a.h., zarei f., ebrahimi m. (2017). invasion of the neotropical and nearctic fishes to iran. fishtaxa, 2(3): 126-133. esmaeili h.r., sayyadzadeh g., eagderi s., abbasi k. (2018). checklist of freshwater fishes of iran. fishtaxa, 3(3): 1-95. jalili p., eagderi s., mousavi-sabet h., mafakheri p. (2015). descriptive osteology of faridpaki spined loach, cobitis faridpaki (mousavi-sabet et al., 2007) (cypriniformes: cobitidae) from southern caspian sea basin. journal of marine biology, 24: 37-70. (in farsi) jalili p., eagderi s., pourbagher h. (2016). phylogeny of the genus cobitis (linnaeus, 1758) in the southern caspian sea basin using osteological characters. the forth iranian conference of ichthyology, ferdowsi university of mashhad, 20-21 july 2016. pp: 90-96. (in farsi) jouladeh-roudbar a., eagderi s., sayyadzadeh g., esmaeili h.r. (2017). cobitis keyvani, a junior synonym of cobitis faridpaki (teleostei: cobitidae). zootaxa, 4244(1): 118-126. levin b.a., simonov e.p., ermakov o.a., levina m.a., interesova e.a., kovalchuk o.m., malinina y.a., mamilov n.s., mustafayev n.j., pilin d.v., pozdeev i.v., prostakov n.i., roubenyan h.r., titov s.v., vekhov d.a. (2016). phylogeny and phylogeography of the roaches, genus rutilus (cyprinidae), at the eastern part of its range as inferred from mtdna analysis. hydrobiologia, 788(1): 33-46. radkhah a.r., eagderi s., asadi h. (2016). length–weight relationship and condition factor for five fish species in anzali wetland and talar river of the caspian sea basin of iran. journal of entomology and zoology studies, 4(3): 122-124. radkhah a.r., eagderi s., poorbagher h., nowferesti h. (2015) morphological differences in populations of sawbelly, hemiculter leucisculus (basilewsky, 1855) in zarineh river and anzali wetland using geometric morphometric technique, journal of aquatic exploitation and aquaculture, gorgan university of agricultural sciences and natural resources, 5(1): 7382. (in farsi) sadeghi r., esmaeili h.r., zarei f., esmaeili a.h., abbasi k. (2019). the taxonomic status of an introduced freshwater goby of the genus rhinogobius to iran (teleostei: gobiidae). zoology in the middle east, 65(1): 51-58. int. j. aquat. biol. (2018) 6(4): 198-201 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology case report the curious case of the missing face: death of california sea lion by dungeness crab trap rif s. el-mallakh*1, michael hartman2 1department of psychiatry and behavioral sciences, university of louisville school of medicine, louisville, kentucky, usa. 2eugene, oregon, usa. article history: received 5 august 2018 accepted 25 august 2018 available online 2 5 august 2018 keywords: california sea lion, zalophus californianus, dungeness crab, metacarcinus magister, crab traps. abstract: marine mammals frequently interact with human detritus due to their proximity to shores and shared target foods. sea lions have been known to attempt to obtain bait inside crab traps. a case is described in which the pattern of decomposition of a california sea lion, zalophus californianus, suggests that it likely became entangled and drowned while attempting to get dungeness crab trap bait. this represents the first case of california sea lion death related to interaction with a crab trap. introduction marine mammals not infrequently wash up on beaches. routine examinations of these corpses can be instructive as to what stressors these animals experience and the circumstances that can kill them (stroud and roffe, 1979). deaths related to interactions with human detritus are not uncommon (carretta et al., 2017). pinnipeds are frequently victims of fishing nets and only rarely are associated with shellfish traps (woodley and lavigne, 1991). california sea lion injury and death have not been previously associated with the expanding dungeness crab (metacarcinus magister) fishery despite anecdotal observations that sea lions attempt to get crab trap baits (https://www.youtube.com/watch ?v=wrxwqxrohsw or https://www.youtube.com/ watch?v=nktydouj1g4). case report on 22 july 2017, after one of the highest tides of the year, the carcass of a juvenile male california sea lion (zalophus californianus) was noted to have washed up on gleneden beach, on the central oregon coast (figs. 1-3). the animal was examined within 12 hours of being washed up on the beach, but autopsy was not performed because the authors were not authorized to *corresponding author: rif s. el-mallakh doi: https://doi.org/10.22034/ijab.v6i4.517 e-mail address: rselma01@louisville.edu remove the carcass and the oregon marine mammal stranding network declined to investigate. the carcass was about 2 m long and mostly in a moderate degree of decomposition (level 3) (fig. 1), but with a skeletonized head with a clear demarcation (level 5) (fig. 2) (coding according to moore, 2013). discussion the differential level of degradation of the body suggests that the head and the remainder of the body were exposed to different environments. crabbing activities were notable off of gleneden beach around that time. we deduce that the sea lion attempted to obtain the bait inside of the trap (since it is unlikely that the sea lion was attempting to eat the crabs (lowry et al., 1991), as they have been observed to do (https://www.youtube.com/watch?v=wrxwqxrohsw and https://www.youtube.com/watch?v=nktydouj1 g4), but became trapped and drowned. juvenile sea lions, as the one described here, are much more likely to be victims of entanglement (stewart and yochem, 1987). we suspect his struggles resulted in disconnection of the buoy, so that the trap was lost. about 10% of all dungeness traps are lost by the fishermen, and they generally continue to catch crabs (breen, 1987). with a population of hungry crabs 199 int. j. aquat. biol. (2018) 6(4): 198-201 trapped in the trap, the sea lion’s flesh within their reach was totally removed. other parts of the body may have been in close enough proximity to the crab trap to also be skeletonized, such as one of the front flipper (fig. 3). the rest of the body decomposed at a slower rate, and experienced typical scars, such as a figure 1. the carcass of a california sea lion, zalophus californianus, within 12 hours of washing up on gleneden beach, oregon. the overall state of decomposition is moderate (level 3). the skeletonized head (level 5) can be seen at right. figure 2. a close up showing a skeletonized skull with a missing jaw bone and fractured zygomatic arches. the level of decomposition of the skull is 5, with a fairly clear demarcation between the two states of decomposition. 200 el-mallakh and hartman/ crab trap drowning bite from a cookie-cutter shark (fig. 1) (hayashi et al., 2015; ebert et al., 2015). ultimately, the body drifted away from the trap and was brought to the beach by one of the highest tides of the year. obviously, these are just deductions. we do not actually have direct evidence for this scenario. however, it is quite difficult to find an alternate explanation for this pattern of differential tissue degradation. this interpretation strongly suggests that even large animals, such as this juvenile sea lion, can potentially drown due to relatively small traps, such as dungeness crab traps. references breen p.a. (1987). mortality of dungeness crabs caused by lost traps in the fraser river estuary, british columbia. north american journal of fisheries management, 7(3): 429-435. carretta, j.v., muto, m.m., greenman, j., wilkinson, k., lawson d., viezbicke j., jannot j. (2017). sources of human-related injury and mortality for u.s. pacific west coast marine mammal stock assessment, 2011-2015. https://swfsc.noaa.gov/publications/tm/swfsc/noa a-tm-nmfs-swfsc-579.pdf ebert d.a., pien c., kamikawa d.j. (2015). confirmation of the cookiecutter shark, isistius brasiliensis, from the eastern north pacific ocean (squaliformes: dalatiidae). marine biodiversity record, 8:e118. hayashi t., higo e., orito h., ago k., ogata m. (2015). postmortem wounds caused by cookie-cutter sharks (isistius species): an autopsy case of a drowning victim. forensic science and medical pathology, 11(1): 119121. lowry m.s., stewart b.s., heath c.b., yochem p.k., francis j.m. (1991). seasonal and annual variability in the diet of california sea lions zalophus californianus at san nicolas island, california, 1981-86. fishery bulletin, 89(2): 331-336. moore m.j., van der hoop j., barco s.g., costidis a.m., gulland f.m., jepson p.d., moore k.t., raverty s., mclellan w.a. (2013). criteria and case definitions for serious injury and death of pinnipeds and cetaceans caused by anthropogenic trauma. diseases of aquatic organisms, 103: 229-264. stewart b.s., yochem p.k. (1987). entanglement of pinnipeds in synthetic debris and fishing net and line fragments at san nicolas and san miguel islands, california, 1978-1986. marine pollution bulletin, 18 (6, suppl b): 336-339. figure 3. the left front flipper, showing the different levels of decomposition. the rostral edge of the flipper is skeletonized (level 5), while the remainder of the flipper is in an early state of breakdown (level 2). 201 int. j. aquat. biol. (2018) 6(4): 198-201 stroud r.k., roffe t.j. (1979). causes of death in marine mammals stranded along the oregon coast. journal of wildlife disease, 15(1): 91-97. woodley t.h., lavigne d.m. (1991). incidental capture of pinnipeds in commercial fishing gear. international marine mammal association, inc., technical report no. 91-01. http://citeseerx.ist.psu.edu/viewdoc/down load?doi=10.1.1.9.1659&rep=rep1&type=pdf int. j. aquat. biol. (2020) 8(6): 424-433 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article abundance and characteristics of microplastics in commercially sold fishes from cebu island, philippines bianca sofia f. abiñon1, boniver s. camporedondo1, esther mae b. mercadal1, kathryn marie r. olegario1, evan marie h. palapar1, chrisian wilfred r. ypil1, antonio e. tambuli2, christine anna lou m. lomboy1, jake joshua chi garces*3 1 1biology department, college of arts and sciences, velez college, f. ramos street, cebu city, philippines. 2biology department, college of arts and sciences, university of san carlos, talamban, cebu city, philippines. 3biology department, college of arts and sciences, cebu normal university, osmeña boulevard, cebu city, philippines. s article history: received 5 may 2020 accepted 18 september 2020 available online 2 5 d e c e m b e r 2020 keywords: mps wet public market commercially sold fishes plastic pollution abstract: this study documents microplastics (mps) in the top three commercially sold fishes viz. auxis rochei, rastrelliger kanagurta and chanos chanos in major public markets of cebu island, philippines. mps were found in the gastrointestinal tracts (fgit) and quantified and characterized according to size, type, and color. in general, nine (97.3%) of 81 fgit samples contained 635 total pieces of mps with size ranging 0.01 to 0.50 and 1.00 to 2.00 mm. transparent microfibers (91%) were the most predominant mps, with blue (48%) as the most common mp color observed, followed by red (39%), black (8%) and white (5%). chanos chanos proved to be the most susceptible fish to mp ingestion with a mean average of 11.6 pieces per individual fish, followed by a. rochei with 6.6 pieces, and r. kanagurta with 5.3 pieces. the results indicated that mps were ubiquitous and high in commercially sold fishes in major public wet markets of cebu island, philippines. the ingestion of fishes is of primary concern as a route of human exposure to mps because they filter a large volume of seawater and are typically eaten whole without gut removal. further study is needed on the potential consequences of mps to aquatic populations to assess comprehensive exposure integrating multiple sources and routes. introduction since the 1930s, plastics are essential raw materials in the global plastic industry due to various applications (lusher et al., 2013; setala et al., 2014). this resulted in widespread occurrence of the plastics in the marine environments and has drawn global attention, because its pollution may give a considerable impact on marine organisms (bendell, 2018). however, the information available on the abundance of plastics in aquatic organisms in the philippine waters, mainly fishes, remain to be scarce. as plastics are usually broken down by uv light, various environmental conditions often result in the formation of secondary microplastics (mps hereafter, both singular and plural, nematdoost haghi and banaee, 2017). due to their small size, various fragmented forms, and a wide range of potential sources, mps are proven difficult to pinpoint and separate from bodies of water (farel et al., 2018). as *correspondence: jake joshua chi garces doi: https://doi.org/10.22034/ijab.v8i4.874 e-mail: garcesjj@cnu.edu.ph most plastic wastes end up accumulating in the oceans and degraded slowly by sunlight, microbes or mechanical abrasion, it is noteworthy to study the abundance of mps in fishes as well as their characteristics (e.g. type, size and composition). the tendency of fishes to accumulate mps and their unhealthy physiological effects (nematdoost haghi and banaee, 2017; banaee et al., 2019) reveal importance of such mps studies in fishes. understanding how mps affect marine organisms has not been only the interest of marine biologists but also the policy makers and environmental managers. many studies have documented that mps are ingested by various marine organisms such as bivalves, zooplankton, copepods, fish, etc. (browne et al., 2008; lusher et al., 2013; chua et al., 2013; rochman et al., 2014; watts et al., 2014) and transported up through high trophic levels in the food chain (cedervall et al., 2012; setala et al., 2014). therefore, the widespread 425 int. j. aquat. biol. (2020) 8(6): 424-433 use of mps is expected to lead its accumulation in the environment. thus, greater exposure risk of wild organisms and human populations occurs over time. marine fisheries in the philippines contribute to fish production as the primary source of protein for most of the population (macusi et al., 2011). however, the possible effects of mps on human compels need for research on mp contamination in major public wet markets in cebu island, the philippines. considering the importance of marine resources to the livelihood of cebuano people, as well as the lack of information regarding the levels of mps in the fishes, this study aims to: (1) investigate the abundance of mps in the top three commercially sold fishes in a wet public market in cebu island, philippines and (2) characterize mps based on type, size, color and composition. this study will shed light on the risks of consuming seafood, particularly on fishes sold in major public markets of cebu island, philippines. this study was carried out in three major public wet markets of cebu, philippines during 2017-2018. materials and methods description of the study area and context: the study was conducted in three public markets in cebu island, philippines, namely (1) consolacion public market, consolacion, (2) pasil fish market, barangay pasil, and (3) lapu-lapu public market, barangay opon, lapu-lapu city. these sites were selected to covers whole distribution of the cebu island, philippines (fig. 1). pasil fish market is located in barangay pasil downtown cebu city. it is the largest seafood market in the visayas. it is located near the pasil fish port where fisher folks arrive with their fish mainly caught in the nearby open cebu strait. lapulapu public market in the island of mactan, is a large public market in the district where most of the population purchase their commodities, including fish which are mainly caught in their jurisdiction, the olango group of islands. consolacion public market is the only public market in the municipality of consolacion where dry and wet goods are sold. one of the primary commodities that are usually bought by the consumer in the area is fish which are mainly caught in the seas of carmen. ethical considerations: the researchers were granted clearance from the ethics review committee of velez college. a transmittal letter was given to the head of the marine biology section, dr. danilo t. dy, in the university of san carlos (usc) – talamban campus figure 1. geographic location of the study areas (major public markets) in cebu island, philippines. 426 garces / microplastics in fishes from cebu island, philippines cebu, philippines as permission to utilize their marine research station in mactan island as a research locale. then, a certification for exemption from the institutional animal care and use committee (iacuc) was obtained. sample collection: mps were assessed in fish samples, following the protocol of rochman et al. (2015). for the identification of the top three commercially sold fishes, the researchers utilized an open-ended interview with 30 local fish vendors which were selected randomly from the public wet markets. in gathering fish samples, a selective method was utilized based on the interview result. samples were processed in marine research station, maribago, mactan island facilitated by uscdepartment of biology. all fish samples were securely wrapped with aluminium foil and zip lock bags and stored in the freezer to avoid possible contamination. then the sampling site and fishing data were also recorded. commercial species: three representative commercial fish species viz. auxis rochei (scrombridae), rastrelliger kanagurta (scrombridae) and chanos chanos (chanidae) were collected from major public wet markets in metro cebu, philippines. only those fish species with age ranging between 1 and 2 years were included for mp analysis. auxis rochei is highly commercialized and found in marine brackish waters. it is endemic in pelagic-neritic zones and distributed in the atlantic, indian, and pacific oceans. this species is carnivorous feeding on small fishes, crustacean, and squid. rastrelliger kanagurta is found in the pelagic-neritic area and distributed from east africa to indonesia. it is an omnivorous fish, feeding on phytoplankton, zooplankton, shrimps, and small fish. chanos chanos is a benthopelagic organism found in marine, fresh, and brackish waters. it is distributed mostly in the indo-pacific waters. this species is omnivorous feeding on cyanobacteria, soft algae, small benthic invertebrates, and pelagic fish eggs. microplastic identification and analysis: samplings were performed once every week for three weeks, gathering a total of 81 fishes. the samples have roughly the same in length with size ranging from 33 36 cm. each fish was placed in an aluminium foil which was then brought and processed in marine research station on the same day. the mps in the fish gut was extracted and inspected based on budimir et al. (2018). dissection was done under the laminar flow cabinet with controlled airflow to prevent contamination with the use of synthetic-free gloves (de witte et al., 2014). all beakers and apparatuses used in the study were also rinsed several times with distilled water to avoid potential contamination with mps. the gastrointestinal (gi) tract of the fishes was isolated by cutting lengthwise in the belly from head to anus. the gi tract was separated from the animal and weighed in a large tared jar covered with aluminium foil to prevent any contamination. reagents were prepared by separately dissolving technical grade powdered naoh and sds (sodium dodecyl sulphate) (5g/l) in a distilled water. digestion method was done by adding a 10 ml naoh and 5 ml sds 0.5% to the jar per 1 g of fish tissue. it was incubated in the oven at 50℃ for 24 h, then incubated for another 24 h after the jars are gently shaken. after the incubation process, the contents were vacuum filter using a 0.45 μm filter membrane and the jars were rinsed with 95% ethanol and milli-q water three times and filtered separately. then 10 ml of technical 2m hcl was added and rinsed with distilled water after 5 min. if there are still organic materials left, 30% hydrogen peroxide was added in the same way as hcl. all mps were placed in a filter paper and inspected under the microscope. mps were placed in a petri dish covered with filter papers; identified and confirmed figure 2. (a) before and (b) after result of the hot needle test on mps identification under the stereomicroscope. 427 int. j. aquat. biol. (2020) 8(6): 424-433 using the stereomicroscope and a hot needle technique, respectively. hot needle procedure was done by heating the tip of a thin needle and poking a suspected mps under the stereomicroscope (de witte et al., 2014). plastic tends to shrink or warp when exposed to heat (fig. 2). amscope™ (digital microscope camera) used for documentation. precautionary methods were made such as closing the petri dish always and opening it only under the laminar flow cabinet to prevent from any contamination during the whole procedure. the researchers also wore proper laboratory gown and sterile, non-synthetic gloves. all authors made sure that all mps present in the gi tracts of fishes sampled have homogenous color, and undeformed feature before undergoing further analysis (lusher et al., 2014). for microfibers, there should be consistency in terms of thickness throughout lengths without debris found on both ends of the fibers sampled in all fishes. throughout the mp extraction process, all containers were appropriately covered with aluminium foil. to reduce contamination, some of the members who did the experiment wore protective gears such as mask, gloves and goggles. data analysis: the normality and homoscedasticity of the gathered data were tested using shapiro-wilk and levene’s tests, respectively. all data (mean average and %) were statistically analyzed using kruskal–wallis test to investigate differences on the amount of the ingested mps between fish species with subsequent post hoc dunn's test analysis at 5% level of significance was performed using statistical package for the social sciences (spss) software (ibm co. ltd, usa). results mps were detected in all the fishes (fig. 3) and out of 81 fishes (fig. 4), 79 (97%) had the ingested mps in their digestive tract. measured densities of mps showed a statistically significant difference in all fishes (kruskal-wallis test, χ2(2) = 13.75, df=3, p=0.001, <0.05). chanos chanos (with mean rank of 52.89) had higher mp density, followed by a. rochei (mean rank = 40.89) and r. kanagurta (mean rank = 29.22). a significant difference in mps density was found between r. kanagurta and c. chanos (dunn’s test, t = -23.67, sd = 6.38, p = 0.001) while there was no significant difference in mps density between r. kanagurta and a. rochei (dunn’s test, t = -11.67, sd = 6.38, p = 0.203) and a. rochei and c. chanos (dunn’s test, t = 12.00, sd = 6.38, p = 0.180) (fig. 3). microplastic debris characteristics: four types of mps, including microfibers, microfragments, micropellets and microfilms were recovered in all fishes (fig. 5f). a total of 635 pieces of mps were detected and categorized according to size, type, and figure 3. average amount of ingested mps per individual fish. 428 garces / microplastics in fishes from cebu island, philippines color. microfibers were the most common type of mps found in all fishes sampled, with a proportion of 80 to 91%. microfragments were the second most abundant mps type, making an average of 7% of each figure 4. identification of top 3 commercially sold fishes in three large public markets in cebu island, philippines. figure 5. mps types found in fish samples: (a) microfragment, (b) microfiber, (c) microfilm, and (d) micropellet along with the (e) distribution of mps types between fish samples and (f) its total proportion in all samples. 429 int. j. aquat. biol. (2020) 8(6): 424-433 sample. the other two types, micropellets and microfilms were only found in a few fish samples at 1% for each mps type (fig. 5). both microfragments and microfibers were present in a. rochei while in r. kanagurta, micropellet, microfragment, microfiber and microfilm were the most common mp types. in c. chanos, microfragment, microfiber and microfilm were present. only microfoam was not present in all fishes (fig. 7). most detected mps in fishes have sizes ranging 0.015-3.800 mm, with 1.001-2.000 mm as the most common size class (fig. 7). moreover, the classification of mp by size showed that the most abundant mps were in the range of 0.01-0.5 to 1.001figure 6. size distribution of mps in three study sites figure 7. mp distribution according to color and type. 430 garces / microplastics in fishes from cebu island, philippines 2.000 mm (fig. 6). the dominant color group of mps was the colored one particularly blue (48%) color is the most prevalent in all fishes (fig. 7) followed by red (39%), black (8%) and white (5%). discussions the results indicated that a. rochei, r. kanagurta and c. chanos were vulnerable to mp contamination, of which c. chanos accumulated most mps (±11.6 pieces per individual fish) following a. rochei (±6.6) and r. kanagurta (±5.3). this result was in congruence with the study of ory et al. (2017). mp abundance in fishes can be explained series of environmental factors (i.e., wave action and water currents) as well as human-mediated activities (i.e. disposal of waste materials and wastage produced from industrial and municipal facilities). to cite, the total population in metro cebu (798,634), lapu-lapu city (550,467) and mandaue city (331,320) when combined (1,680,421) was higher than those of any other cities in the philippines higher population in these cities could contribute to mp contamination in marine ecosystems. the higher degree of development in metro cebu indeed, is caused the higher mp pollution in marine environment. dominance of the microfibers in this study indicated high change of ingestion events. chanos chanos had ingested the most amounts of mps as a bentho-pelagic species. it lives near the bottom as well as in midwaters or near the surface and feeds on free swimming organisms. philips and bonner (2015) showed high plastic ingestion from lower to higher trophic level. both a. rochei and r. kanagurta are pelagic-neritic species found in coastal waters and mid-waters. microfibers come from industrial and municipal waterways which indirectly and directly contaminate freshwater and marine ecosystems (lusher et al., 2014). also, sewage treatment facilities produce voluminous amounts of microfibers through the production of textiles and become part of the waterways, which can be hardly determined when studied on-site (bessa et al., 2014). other industries probably generate a considerable number of microfibers from the fishing nets and ropes which contribute to high microfiber accumulation in the studied fishes. our result is pivotal for management purposes in studied markets in metro cebu since successful management of mp pollution can be attained based on the source of mp origin. in recent studies, ingestion of mps among fishes could be attributed to their accidental identification of mps as their prey (boerger et al., 2010) since these mps have similar color, shapes and texture with that of food (foekema et al, 2013; lusher et al., 2013, 2015; ory et al., 2017). the highest value of microfibers is found in high volume and number (>85%) on coastal shorelines and even on the surface of oceans globally. uncertain with its main source, most research suggests that these microfibers are debris produced from syntheticallymade fibers (with dimensions <5 mm) which can easily pass through microfiber materials (lusher et al., 2015; bessa et al., 2018). other domestic materials such as clothing and plastic materials from laundry are also considered as a major source of microfibers (bagchia et al., 2016; carr et al., 2016). moreover, the color of the dominant mps found was consistent with previous studies (bagchia et al., 2016; carr et al., 2016; ory et al., 2017). their color selectivity may potentially contribute to the likelihood of floating mps to their natural food for aquatic organisms (andrady, 2011). blue particles were preferentially ingested by c. chanos and a. rochei (ismail et al., 2019), and r. kanagurta (martin et al., 2017). the results were in concordance with the mp colors observed in many previous studies (ogata et al., 2009; rochman, 2015; tekman et al., 2017) where blue color is more abundant. mp ingestion among fish species is a major concern to human systems since these mps can be engulfed in bulk and can be left in their gut for a long period. left unnoticed, these fishes with high mps in their guts are commonly bought by market-goers daily. moreover, wide-scale assessment on the most commonlypurchased fishes is also recommended as this will identify the possible route of mp contamination to humans. suggested guidelines to minimize mp exposure include correct assessment of mps during 431 int. j. aquat. biol. 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(2014) 2(3): 129-137 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article effect of copper on the characterization of proteins in the spiny lobster, panulirus homarus homarus (linnaeus, 1758) maharajan athisuyambulingam*1, rajalakshmi sundar rao2, vijayakumaran manambrakat3 1post graduate and research department of zoology, khadir mohideen college, adirampattinam, thanjavur dist, tamil nadu, india. 2department of zoology, kanchi mamunivar centre for post graduate studies, puducherry, india. 3national institute of ocean technology, niot campus, narayanapuram, pallikaranai, chennai, tamil nadu, india. article history: received 9 december 2013 accepted 3 june 2014 available online 2 5 june 2014 keywords: panulirus homarus homarus copper sds page protein abstract: copper is most toxic metal in marine organisms. characterization of protein occurring in the metabolically active tissues of muscle (mu), hepatopancreas (hp) and gills (gl) of the spiny lobster, panulirus homarus homarus on exposure to two sub-lethal doses (9.55 and 19.1 µg/l) of copper were studied for 28 days of exposure (doe). the electrophoretic pattern of muscle, hepatopancreas and gill proteins revealed 12, 8 and 8 slow moving bands (control). the number of bands decreased to 8 and 7, 6 and 5, 6 and 4 after 7 days of exposure to 9.55 µg/l and 19.1 µg/l concentrations of copper, respectively. after 28 days, the protein bands decreased to 7 and 6, 5 and 4, 4 and 4 at 9.55 µg/l and 19.1 µg/l concentrations of copper, respectively. present study to indicate that to avoid the cupronickel coil in lobster holding centers in chiller plants used for cooling of water was found to be responsible for the mortality of lobsters during live transportation. introduction the electrophoretic techniques are promising tools for identifying the protein profile in response to stressful and sublethal level of heavy metals. heavy metal binding proteins have been found to be associated with copper and the lower molecular weight protein in the lake fauna (dutta et al., 1984). chang et al. (1987) isolated and purified the aminoacids from the lobster, homarus americanus exposed to copper. similarly, canli et al. (1997) analyzed the effect of copper in the gill and hepatopancreas tissues of the norway lobster, nephrops norvegicus. he also suggested presence of copper metallothionein in the gill and hepatopancreas. brouwer et al. (1989) studied the structural and functional diversity of copper metallothioneins from the american lobster, homarus americanus and sequenced 56 aminoacids of copper binding proteins. brouwer et al. (1991) * corresponding author: maharajan athisuyambulingam e-mail address: athimaha@yahoo.co.in studied a crucial role of copper binding proteins in the regulation of reactive ions in the same species. lobster metallothioneins share a number of similarities with mammalian metallothioneins with respect to the presence of copper and cadmium, apparent molecular weights and amino acids composition, but differ substantially in their electrophoretic behaviour (chou et.al., 1991). copper toxicity of spiny lobster, panulirus homarus homarus in muscle conductivity, bioaccumulation, chromosomal aberrations and histopathological changes also has been reported (maharajan et al., 2010a; maharajan et al., 2010b; maharajan et al., 2011; maharajan et al., 2012). krishnamoorthy and subramanian (1997) reported intensities of major polypeptide bands in the freshwater prawn, macrobrachium lamerrei lamerrei, exposed to copper. similarly, the reduction in the number of protein fraction in scylla serrata treated with copper 130 maharajan et al./ effect of copper on proteins of the spiny lobster was reported by ramanibai (1986). the present study intended to evaluate the lethal and sublethal effect of copper in the spiny lobster, p. homarus homarus, the major spiny lobster exported live from india. since mortalities due to copper toxicity have been reported in lobster holding centers. it is hoped that the study will provide a useful blueprint for live transportation and packing agencies in designing an effective programme for avoiding toxicity of copper during live transport of lobsters. materials and methods experimental animals and acclimation: actively moving juveniles of spiny lobster, p. homarus homarus (weight 150-200 g) with no visible signs of disease or morbidity were collected from kovalam using bottom set gill net at a depth of 5-8 meters located in tamil nadu. immediately after the collection, juvenile lobsters were transferred to the laboratory conditions in two large frp tanks (cap. 200 l) for more than 2 weeks before the initiation of experiments. the seawater used in acclimation tanks was treated by rapid sand filtration, bio-filtration and passed through ultraviolet radiation. adequate aeration was provided using air blowers, and optimum water quality parameters were maintained during the acclimation period: temperature, (29 ± 1°c), salinity (33 ± 1 ppt), dissolved oxygen (6.4 ± 0.8 mg l-1), ph (7.9 ± 0.4), no2-n (< 0.02 mg l -1) and nh3/nh4 (0 mg l -1). the seawater used for acclimation and exposure experiments was free from residues of copper. a photoperiod of 12 l (0700 h1900 h):12 d (1900 h-0700 h) was maintained. lobsters were fed ad libitum twice a day (0800 h and 1600 h) with live marine clam donax cuneatus. faeces and uneaten feed was siphoned out twice a day (1000 h and 1730 h) and 50% of water exchanged daily (07.30 h). in order to reduce the amount of excreted products in the test tanks, feeding was stopped 48 h prior to the commencement of acute bioassay tests. test chemical: stock solution of copper were prepared by dissolving 3.963 g copper sulphate pentahydrate (cuso4 5 h2o, merck, germany) in 100 ml of 2% sulphuric acid solution and making upto 1000 ml with double deionised water. it was stored in a clean standard flask at room temperature in the laboratory. acute bioassay tests: acute toxicity test, 10 active animals each were exposed to various concentrations of the copper (80, 100, 120, 140, 180 and 200 μg/l) using filtered sea water as control. experimental animals were starved for one week. the experiments were conducted in three replicates at room temperature. no feed was given during the test period. mortality of lobsters was recorded continuously for 12, 24, 36, 48, 72 and 96 hrs. percent mortality was calculated and the values were transformed into the probit scale. probit analysis was carried out based on finney (1971). test solutions and sub-acute tests: it has been hypothesised that sublethal concentrations of copper offer an excellent scope for observing the behavioural and physiological changes in animals. two sublethal doses corresponding to 10% and 20% of 96-h lc50 were selected for sub-acute toxicity experiments. concentrations of active ingredient of copper present in two sublethal concentrations computed from commercial-grade composition were found to be 9.55 and 19.1 μg/l, respectively. the exact amount of active ingredient of copper present in each sublethal solution however, was not quantified. for evaluation of effects of sublethal concentrations, 90 randomly sampled lobsters of similar size (weight 150-200 g; n=10) were divided into 3 groups, each group comprising of 30 intermoult juveniles (stage.c) of p. homarus homarus. one group served as control, while two other groups were exposed to two sublethal doses of copper (one group to one sublethal dose). a total of three replicate aquaria (500 l capacity) were maintained for each dose and the control group (10 lobsters per concentration per replicate). during the exposure period, a mild aeration was provided using air pumps (boyu, japan) in order to maintain do levels not less than 5 mg l-1. juveniles were fed with live marine clam donax cuneatus, ad libitum and 131 int. j. aquat. biol. (2014) 2(3): 129-137 were deprived of feed 24 h before exposure experiments. in toxicological studies, chronic tests of shorter duration (~28 days) have been recommended as an alternative to longer chronic tests (maki, 1979). the experiment was run for a period of 28 doe as the estimated intermoult period of p. homarus homarus under the laboratory conditions was estimated to be 21±1 days (personal observation). in order to maintain constant concentration of copper in test solutions, the entire toxic medium in each aquarium was gently siphoned out daily (09.00 h) and renewed with freshly prepared solution of respective sublethal concentrations of copper. aeration was suspended temporarily during water exchange and feeding, and care was taken that the disturbance caused to the lobsters was minimal. characterization of protein: the lobsters were exposed to 9.55 μg/l and 19.1 μg/l concentrations of copper for 28 days. after 0, 7 and 28 days, the lobsters were sacrificed. muscle, gills and hepatopancreas were excised out and analyzed for characterization of protein. sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sdspage) was performed following the protocol of weber and osborn (1969). the gel was immersed in 5 ml of staining solution (200 mg coomassie brilliant blue r250+50 ml meoh (methyl hydroxide) + 7 ml acetic acid + 43 ml distilled water and was allowed to stain for 4 hours at room temperature. the stain was removed and the gel destained with acetic acid and methyl hydroxide solution (7 ml acetic acid + 30 ml methyl hydroxide + 63 ml distilled water). the gel was stored in 7% acetic acid, the bands were visualized under uv trans-illuminator. results protein pattern in muscle: twelve distinct protein fractions, eight with high molecular weights and four with low molecular weights, have been detected in the muscle tissue of control lobster (p. homarus homarus) after 7 days of experiment using standard marker proteins (fig. 1). the high molecular weight fractions ranged from 190 kd to 77 kd. the low molecular weight fractions ranged from 42 kd to 18 kd. the high molecular weight polypeptide constituted 20.642 mg/g protein at 77 kd at an area of 10.646% and 2.25 seconds retention time. the low molecular weight polypeptide constituted 41.103 mg/g protein at 38 kd at an area of 21.198% and 3.38 seconds retention time (fig. 2a). after 7 days of exposure to 9.55 µg/l concentration of copper, the number of protein fractions declined and only eight numbers of distinct protein fractions were observed. seven were with high molecular weights and one with low molecular weight. the high molecular weight fractions ranged from 198 kd to 73 kd. the low molecular weight fraction was found to be at 29 kd. the high molecular weight polypeptide constituted 39.323 mg/g protein at 73 kd at an area of 20.502% and 2.11 seconds retention time. the low molecular weight polypeptide constituted 32.788 mg/g protein at 29 kd at an area of 17.098% and 3.42 seconds retention time (fig. 2b). similarly lobsters exposed to 19.1 µg/l concentration of copper had seven distinct fractions six with high molecular weights and one with low molecular weight. the high molecular weight proteins ranged from 167 kd to 88 kd. the low molecular weight protein was of 32 kd. the high molecular weight protein was maximum at 88 kd (24.556 mg/g) at an area of 13.353% and 2.32 seconds retention time. the low molecular weight polypeptide constituted 20.233 figure 1. electrophorogram and densitometric scan image of marker protein. 132 maharajan et al./ effect of copper on proteins of the spiny lobster mg/g protein at 32 kd at an area of 11.002% and 3.48 seconds retention time (fig. 2c). muscle tissue of control lobster after 28 days (closure of the experiment) revealed a broad spectrum of polypeptides with twelve fractions. among them ten were with high molecular weights and two with low molecular weights. the high molecular weight fractions ranged from 198 kd to 74 kd and the low molecular weight ranged fractions from 27 kd to 19 kd. the high molecular weight protein fraction with maximum quantity was at 92 kd (24.656 mg/g) at an area of 13.032% and 2.18 seconds retention time. the low molecular weight protein with maximum quantity was observed at 27 kd (30.397 mg/g) at an area of 16.066% and 3.49 seconds retention time (fig. 3a). after 28 days of exposure to 9.55 µg/l concentration of copper the number of protein fractions reduced to seven, five with high molecular weights and 2 with low molecular weights. the high molecular weight proteins ranged from 88 kd to 192 kd and the low molecular weight protein ranged from 27 kd to 20 kd. among the high molecular weight fractions, the maximum was found to be 24.492 mg/g protein at 88 kd at an area of 14.484% and 2.34 seconds retention time. the maximum value for low molecular weight polypeptide was found to be 17.191 mg/g protein at 27 kd at an area of 10.1665 and 3.49 seconds retention time (fig. 3b). similarly lobsters exposed to 19.1 µg/l concentration of copper revealed less number of protein fractions and thicker bands. however, the effect was found to be severe with many interfering bands between high and low molecular weight peptides. six protein fractions were observed and among them, five had high molecular weights and one, low molecular weight. the high molecular weight protein ranged from 168 kd to 62 kd. the low molecular weight protein was of 28 kd. among the high molecular weight fractions the maximum was observed at 142 kd figure 2. electrophorogram and densitometric scan image of total proteins in the muscle of p. homarus homarus after 7 days of the experiment (a) control, (b) exposed to 9.55 µg/l and (c) exposed to 19.1 µg/l concentration of copper. figure 3. electrophorogram and densitometric scan image of total proteins in the muscle of p. homarus homarus after 28 days of the experiment (a) control, (b) exposed to 9.55 µg/l and (c) exposed to 19.1 µg/l concentration of copper. 133 int. j. aquat. biol. (2014) 2(3): 129-137 (30.077 mg/g protein) at an area of 18.362% and 1.22 seconds retention time. the low molecular weight protein at 28 kd contained 22.331 mg/g protein at an area of 13.633% and 3.43 seconds retention time (fig. 3c). protein pattern in hepatopancreas: the protein profile of hepatopancreas of control group after 7 days revealed eight distinct protein fractions seven with high molecular weights and one with low molecular weight. the high molecular weight proteins ranged from 142 kd to 56 kd and the low molecular weight fraction was of 18 kd. among the high molecular weight fractions, maximum was observed at 64 kd (20.521 mg/g protein) at an area of 12.675% and 2.32 seconds retention time. the low molecular weight fractions at 18 kd had 15.170 mg/g protein at an area of 9.370% and 2.95 seconds retention time (fig. 4a). after 7 days of exposure to 9.55 µg/l concentration of copper, the zymogram pattern revealed six protein fractions, four with high molecular weights and two with low molecular weights. the high molecular weight proteins ranged from to 122 kd to 71 kd. the low molecular weight proteins were found to be between 28 kd and 17 kd. maximum high molecular protein (17.378 mg/g) was found at 125 kd at an area of 11.9275 and 0.93 seconds retention time. the low molecular weight fraction at 17 kd contained 16.092 mg/g protein at 2.14 seconds retention time and extending to an area of 11.045% (fig. 4b). similarly, after 7 days of exposure to 19.1 µg/l concentration of copper, the hepatopancreas revealed five fractions, three with high molecular weights and two with low molecular weights. the high molecular weight proteins ranged from 140 kd to 60 kd and the low molecular weight protein range was upto 25 kd (fig. 4c). the high molecular protein having maximum quantity was found at 125 kd (9.808 mg/g) at 0.86 seconds figure 4. electrophorogram and densitometric scan image of total proteins in the hepatopancreas of p. homarus homarus after 7 days of the experiment (a) control, (b) exposed to 9.55 µg/l and (c) exposed to 19.1 µg/l concentration of copper. figure 5. electrophorogram and densitometric scan image of total proteins in the hepatopancreas of p. homarus homarus after 28 days of the experiment (a) control, (b) exposed to 9.55µg/l and (c) exposed to 19.1µg/l concentration of copper. 134 maharajan et al./ effect of copper on proteins of the spiny lobster retention time having an area of 6.636%. low molecular protein was maximum 12.140 mg/g at 25 kd at 2.10 seconds retention time, in 8.214% of area. besides, several minor insignificant fractions were also exhibited. the zymogram pattern of hepatopancreas in control lobster at the end of the 28 days revealed eight distinct protein fractions. six distinct bands with high molecular weight proteins ranged from 148 kd to 73 kd (fig. 5a). the 81 kd protein had the maximum quantity of 21.890 mg/g (1.95 seconds retention time and an area of 13.571%). similarly, two low molecular weight proteins ranged from 28 kd to 22 kd. maximum protein of low molecular weight was observed at 22 kd (12.280 mg/g) at an area of 7.613% and 3.50 seconds retention time. the effect of copper on hepatopancreas was found to be severe with indistinct protein profiles after 28 days of exposure to 9.55 µg/l concentration of copper. five protein fractions were observed all with high molecular weights ranging from 120 kd to 62 kd (fig. 5b). the 62 kd protein had the maximum quantity of 15.059 mg/g (2.08 seconds retention time and an area of 13.291%). after 28 days of exposure to 19.1 µg/l concentration of copper, the characterization of protein revealed only four distinct fractions of high molecular weight ranging from 132 kd to 69 kd. few minor fractions of protein were also recorded. among the high molecular weight fractions, maximum was recorded at 132 kd (16.221 mg/g) at an area of 13.395% and 2.08 seconds retention time (fig. 5c). protein pattern in gills: the zymogram pattern of gills in control lobster after 7 days revealed eight distinct bands, six with high molecular weight proteins ranging from 180 kd to 80 kd and two with low molecular weights ranging from 34 kd to 27 kd (fig. 6a). the 180 kd protein had maximum quantity 14.953 mg/g (0.85 seconds retention time and an area of 36.741%). maximum low molecular weight protein was observed at 34 kd (3.647 mg/g) at an area of 3.458% and 3.72 second retention time. after 7 days of exposure to 9.55 µg/l concentration of copper, the gills revealed six distinct bands, five with high molecular weights ranging from 180 kd to 60 kd and one low molecular fraction at 28 kd (fig. 6b). the 180 kd protein had the maximum quantity of 16.691 mg/g (1.06 seconds retention time and an area of 60.258%). the low molecular weight fraction at 28 kd constituted 2.018 mg/g protein with 7.287% coverage of area and 3.82 seconds retention time. after 7 days of exposure to 19.1 µg/l concentration of copper, four distinct protein fractions three with high molecular weights and one low molecular weight were observed. the high molecular weight proteins ranged from 150 kd to 54 kd. the 150 kd protein had the maximum quantity of 13.821mg/g (0.87 seconds retention time and an area of 36.372%). the low molecular weight protein at 22 kd had 2.597 mg/g protein covering an area of 6.834% with a retention time of 3.76 seconds (fig. 6c). the zymogram pattern of gills in control lobster after 28 days revealed eight fractions, seven with high figure 6. electrophorogram and densitometric scan image of total proteins in the gills of p. homarus homarus after 7 days of the exposure (a) control, (b) exposed to 9.55µg/l and (c) exposed to 19.1µg/l concentration of copper. 135 int. j. aquat. biol. (2014) 2(3): 129-137 molecular weights and one with low molecular weight. the high molecular weight proteins ranged from 180 kd to 62 kd (fig. 7a). the 180 kd protein had the maximum quantity of 15.128 mg/g (0.81seconds retention time and an area of 45.431%). similarly, the low molecular weight protein at 28 kd had 1.147 mg/g protein with a coverage of 3.446% and 3.80 seconds retention time. the zymogram pattern of gills in lobster exposed to 9.55 µg/l concentration of copper for 28 days revealed four fractions two with high molecular weights and two with low molecular weights. the high molecular weight proteins ranged from 145 kd to 80 kd with the 145 kd fraction having maximum quantity of 16.850 mg/g (0.78seconds retention time and an area of 43.204%). the low molecular weight protein ranged from 18 kd to 24 kd and the 24 kd protein had the maximum quantity of 7.323 mg/g (3.50 seconds retention time and an area of 18.777%) (fig. 7b). after 28 days of exposure to 19.1 µg/l concentration of copper, the gills revealed four distinct fractions, three with 145 kd to 72 kd and one with low molecular weight (17 kd). the 145 kd protein had maximum quantity of 20.731 mg/g (1.00 seconds retention time and an area of 47.877%). the low molecular weight protein at 17 kd had 2.939 mg/g covering an area of 6.787% and a retention time 3.79 seconds (fig. 7c). discussion the electrophoretic technique remains a promising tool for identifying the protein profile in response to stressful and sublethal level of heavy metals (dutta et al., 1983; costa et al., 2002). heavy metal binding proteins are associated with copper and the lower molecular weight protein in particular is found to have a significant percentage of copper contained in the muscle, hepatopancreas and gills. the lower molecular weight protein probably plays a significant role in the metabolism of the copper. the hepatopancreas of decapod crustaceans has been implicated as being important in the metabolism of heavy metal, for it is the site of absorption of products derived from digested food and also it appears to act as a store for metal taken up from solution over body surface such as the gills (bryan, 1968; jennings and rainbow, 1979 jacqueline et al., 2010). the disappearance of bands in the muscle, hepatopancreas and gills of p. homarus homarus on exposure to copper may be due to the interference of copper in the protein synthesis process and the reduction in the number of banding pattern as reported in freshwater prawn, macrobrachium lamerrei lamerrei (krishnamoorthy and subramanian, 1997). in the present study, 12 distinct bands are accounted in the muscle tissue of p. homarus homarus. extensive disruption in the number of banding is well-documented at 19.1 μg/l of concentration with seven and six polypeptide fractions during 7 and 28 days of copper exposed lobster. low molecular myofibrillar proteins (troponin) hydrolyzed by specific proteinase are of interest, because these proteins are hampered by the figure 7. electrophorogram and densitometric scan image of total proteins in the gills of p. homarus homarus after 28 days of the exposure (a) control, (b) exposed to 9.55µg/l and (c) exposed to 19.1µg/l concentration of copper. 136 maharajan et al./ effect of copper on proteins of the spiny lobster fragments produced from bigger molecule proteins such as myosin heavy chain (pangkey et al., 2000). lim and lee (1970) working on prawn muscle myogen reported species specificity and used the electrophoretic technique to trace the toxicity levels between species. in addition, the muscle myogen are found to vary considerably between different concentrations and tissues. the present investigation confirms the above findings on copper toxicity. the muscle myogens are generally more in number than in the gills and hepatopancreas indicating the number of glycoproteins is more in muscle than in other tissues in p. homarus homarus. in hepatopancreas, the number of portein bands reduced from 8 (control) to 6 and 5 at 9.55 μg/l concentration during 7 and 28 days, respectively. similarly, at 19.1 μg/l of concentration, the total number of bands reduced from 8 (control) to 4 at either days of exposure (7 and 28). it is also evident that exposure to copper disturbed the banding pattern under stress conditions. krishnamoorthy and subramanian (1997) reported that the intensities of the major polypeptide bands in the gills of prawns when treated with heavy metal were less than that of the control. the result is in accordance with the current observation in p. homarus homarus in which the 8 distinct bands (control) decreased to 6 and 4 at 9.55 μg/l during 7 and 28 days, respectively. similarly, the bands reduced to four at 28 days of exposure to 19.1 μg/l concentration of copper. similar reduction in the numbers of protein fractions was found in scylla serrata when treated with copper (ramanibai 1986). panulirus homarus homarus has minimum protein residue in muscle, hepatopancreas and gills due to copper toxicity which interfered with the banding pattern of proteins as reported in other aquatic invertebrates (wright, 1978). it is also evident that exposure to copper disturbed the banding pattern of protein under stress condition in p. homarus homarus. similar alterations in the banding pattern of protein due to trace metal interaction has been reported in fishes salmo gairdnerii due to arsenic (kothary and candido, 1982) and in oncorhynchus tshawtsche due to zinc and cadmium (heikkila et al., 1982). references bryan g.w. 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(2020) 8(6): 434-446 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article macrophytes as indicators of the ecological status of a tropical rehabilitated wetland ecosystem: application of multivariate statistics and ecological state macrophyte index (esmi) dimuthu wijeyaratne, aravinda bellanthudawa*1 department of zoology and environmental management, university of kelaniya, sri lanka. s article history: received 18 november 2019 accepted 7 december 2020 available online 2 5 december 2020 keywords: anthropo-pressure macrophyte settlement rate sri lanka tropical wetlands abstract: the present study used the ecological state macrophyte index (esmi) and the multivariate statistical methods to assess the ecological status and the variation of macrophytes in a tropical wetland system. six sites were selected from rehabilitated and non-rehabilitated areas of an urban tropical wetland and the water quality parameters (water ph, temperature, conductivity, total dissolved solids (tds), dissolved oxygen (do), visibility, biological oxygen demand 5 days after incubation (bod5), chemical oxygen demand (cod), nitrate, chlorophyll-a and total phosphorus concentrations), sediment quality parameters (ph, organic matter content, percentage sand, silt and clay content) and abundance of aquatic macrophytes were measured. shannon weiner diversity index, percentage vegetation under anthropo-pressure, macrophyte settlement rate and esmi were calculated. significant variations in the water and sediment quality parameters were observed and ten species of aquatic macrophytes were recorded. salvinia melosta and cypreus iria were recorded only from the non-rehabilitated sites. although there was no significant difference in the percentage anthropo-pressure among study sites, the rehabilitated sites were displayed low anthropo-pressure. the sites in the non-rehabilitated area showed a significantly lower macrophyte settlement rate. esmi and macrophyte abundance showed significant correlations with water quality parameters. based on the results, it can be recommended that applications based on esmi and multivariate statistics can be used to assess the ecological status of tropical wetlands. introduction aquatic macrophytes are important components of wetland ecosystems. they can grow as rooted emergent, rooted submerged or floating vegetation in wetlands and play a major role in wetland communities by performing direct and indirect ecological functions. aquatic macrophytes are important in nutrient cycling, maintenance of water quality, prevention of sediment re-suspension and providing food and habitats for many other wetlands associated organisms (gidudu et al., 2011). a healthy macrophyte community is an indicator of a healthy wetland ecosystem. as the rooted macrophytes are rooted in the soft muddy bottoms of wetland ecosystems, they are able to integrate long term changes in water and sediment quality, making them ideal indicators of assessing the changes in wetland *correspondence: dimuthu wijeyaratne doi: https://doi.org/10.22034/ijab.v8i6.675 e-mail: dimuthu.wijeyaratne@kln.ac.lk environments for several seasons or several years (murphy et al., 2003; lee and mcnaughton, 2004). several studies have been conducted to investigate effect of environmental characteristics on the changes in macrophyte community in various types of wetland ecosystems. these studies have shown that, the physical and chemical properties of the water and sediments can determine the composition of the aquatic macrophyte community, thereby influencing the health of the ecosystems (lee and mcnaughton, 2004; lacoul and freedman, 2006; henry-silva et al., 2008; fu et al., 2014). concentration of nutrients in both water and sediments and light penetration were recorded to be the strongest predictors of macrophyte distribution (bini et al., 1999; henry-silva et al., 2008). in addition to these major predictors, variation of conductivity, mg, ca and na concentrations, 435 int. j. aquat. biol. (2020) 8(6): 434-446 alkalinity, altitude, ph and depth can also have strong effects on abundance and distribution of macrophytes (kunii, 1991). in addition to the physical and chemical factors, biological factors also play a key role in determining the abundance and distribution of macrophytes in the wetlands. many studies have identified that primary and secondary succession, competitive interactions among plants, patterns of herbivory by invertebrates and vertebrates as major biological factors that control the composition and distribution of aquatic macrophytes in wetlands (gidudu et al., 2011; dar et al., 2014). biological monitoring is very important in predicting the stressors associated with wetland communities, as the biological community can have both direct and indirect effects due to natural and human induced changes in an ecosystem. therefore, there should be reliable, predictable and cost-effective comprehensive studies on wetland biological communities (birk et al., 2012; lyche solheim et al., 2013). however, the common biological indicators of wetland health assessment involve macrobenthos (braccia and vosell, 2006; dahanayaka and wijeyaratne, 2006; brraich and kaur, 2017; wijeyaratne and bellanthudawe, 2017; wijeyaratne and kalaotuwawe, 2017; basu et al., 2018) and fish (karr, 1981; brousseau et al., 2011; priyatharsini et al., 2018). comparatively less priority is given to studies using aquatic macrophytes as bioindicators. several studies on macrophyte indices in wetland health monitoring programmes have been used in europe, but applications are rare in other regions of the world (ciecierska, 2006; birk et al., 2012; lyche solheim et al., 2013). aquatic macrophyte indices are also used as indicators in wetland health assessment in wetland management and rehabilitation programmes. these methodologies involve analysis of the spatial and structural distribution of aquatic macrophytes in a wetland ecosystem for a predetermined time period and their spatial and temporal arrangements are modeled to predict the wetland health (ciecierska, 2006). ecological state macrophyte index (esmi) is a biological monitoring method to assess the ecological status of wetlands based on macrophyte abundance and diversity characteristics. this method involves development of a macrophyte index based on ratio of redundancy index and colonization index of aquatic macrophytes in wetland ecosystems (ciecierska and kolada, 2013). this method is applied in several studies in the europian union to study the ecological status of small lakes and wetland ecosystems (willby et al., 2009; søndergaard et al., 2010; ciecierska and kolada, 2013). however, applications of esmi is very limited in the other parts of the world. therefore, the present study was designed to apply esmi to assess the ecological status of a tropical urban wetland system that is associated with wide array of diverse land-uses. the present study was conducted in an urban wetland system located in the commercial capital of sri lanka, diyawanna wetland system. this wetland is identified as an important marshland in the area and iucn sri lanka and central environment authority of sri lanka have recognized the diyawanna oya wetland, as the colombo flood retention area and as a wetland system that is at a high level of risk. this wetland system contains both rehabilitated and nonrehabilitated areas. the rehabilitated areas are restored by wetland rehabilitation programme initiated by the sri lanka department of land reclamation and highly contribute to the social well-being of the surrounding urban and sub-urban communities by facilitating income generation activities such as fishing and cattle grazing, and collecting reeds, rushes and fuel wood and serving as recreational area for family outings and water sports. the unmanaged (non-rehabilitated) areas are left as pristine habitats and are rich in indigenous fauna and flora. however, clearance of land, illegal reclamation and construction, dumping of garbage, and encroachments are taking place at some areas of the ecosystem and these environmental changes are affecting the health of the wetland. the present study aims to use esmi and mulitivariate analysis techniques to study the abundance and distribution of macrophytes in different parts of the diyawanna wetland ecosystem and to thereby characterize the ecological status of this 436 wijeyaratne and bellanthudawa / application of ecological state macrophyte index wetland system, which will be very useful in planning and management of wetland development activities in future wetland restoration activities. materials and methods study sites: six study sites were selected from both rehabilitated and non-rehabilitated areas of the diyawanna wetland system. a map showing the locations of the study sites is given in figure 1. site a (06°54'585''n, 079°54'722''e), site b (06°54'664''n, 079°54'633''e) and site c (06°54'609''n, 079°54'604'') are located in non-rehabilitated areas. but sites b and c were in close proximity to the rehabilitated area compared to site a (fig. 1). site d (06°54'68''n, 079°54'610''e), site e (06°54'751''n, 079°54'735''e) and site f (06°54'741''n,079°54'525''e) are located in the rehabilitated area (fig. 1). water and sediment quality parameters: from each site, water samples and shallow sediments samples (00.5 m depth) were collected in seven replicates for water and sediment quality analysis. sampling was carried out once in 6 weeks for a period of 7 months from april to december in 2016. at each sampling site, water ph, temperature, conductivity, total dissolved solids (tds) and salinity were measured insitu using a calibrated digital multi parameter (ysi environmental model-556 mps). dissolved oxygen concentration (do) was measured using do meter (hq 40b model-hach). visibility was recorded using a secchi disk. the biological oxygen demand 5 days after incubation (bod5), chemical oxygen demand (cod), nitrate concentration, chlorophyll-a concentration and total phosphorus concentrations were measured using the methods described by apha (1992). in addition, sediment ph was measured in-situ using the calibrated digital multiparameter (ysi environmental model-556 mps). in the laboratory, sediment organic matter content was measured by the loss on ignition method and the percentage sand, silt and clay content of the sediments were measured using the sedimentation jar. macrophyte abundance and percentage coverage: line-transect sampling described by southwood and henderson (2000) was followed to sample the macrophytes. a measuring tape was taken to mark approximately 5 m distance from bank to the middle of the wetland. intervals of 0.5 m distance was marked off using colored tags. each interval was treated as a separate unit of 5 m line transects. at each site, individual macrophytes were counted along the line transect at 0.5 m intervals started from bank to middle of wetland. seven replicate transects were used at each site for macrophyte sampling. identification of macrophytes to the lowest taxonomic level as possible was done using the photographic guide of aquatic plants prepared by the national aquaculture development authority (naqda) and the flora of ceylon (dassanayake and fosberg, 1980-1991; dassanayake et al., 1994-1995; dassanayake and clayton, 1996-2000). the identified samples were verified by comparison with the specimens from the specimen collection of department of botany, university of kelaniya, sri lanka. the abundance of figure 1. the study sites in the diyawannawa wetland, sri lanka. the study sites a, b and c are located in the non – rehabilitated area and the sites d, e and f are located in the rehabilitated area. 437 int. j. aquat. biol. (2020) 8(6): 434-446 each species at each site were recorded. the percentage cover of macrophytes at each study site was determined by determining the proportion of locations where a particular species is present compared to the total number of sampled locations as described by southwood and henderson (2000). the macrophyte abundance data were used to calculate ecological state macrophyte index (esmi) for each site in the rehabilitated and non-rehabilitated areas of the wetland. determination of esmi: esmi was determined using the phytocoenotic diversification index (h), maximum phytocoenotic diversification index (hmax) and vegetation under anthropo-pressure (j). phytocoenotic diversification index (h) was calculated from the shannon-wiener index (panek, 2001) as following: 𝐻𝐻 = − � 𝑛𝑛𝑖𝑖 n ∗ ln 𝑛𝑛𝑖𝑖 𝑁𝑁 𝑖𝑖=100 𝑖𝑖 where, h = phytocenotic diversification index, 𝑛𝑛𝑖𝑖= area of specific plant community in the percentage of the total phytolittoral area and n = total area of phytolittoral area (100%). the maximum phytocoenotic diversification index (h max) was calculated as described by ciecierska and dynowska (2013) using the equation of 𝐻𝐻𝑚𝑚𝑚𝑚𝑚𝑚 = 𝑙𝑙𝑛𝑛 𝑆𝑆 , where s is the total number of communities in the sampling site. the vegetation under anthropo-pressure (j) was calculated as described by pielou (1966) using equation of j = h/hmax. the settlement rate of macrophytes was determined considering the relationship between the area actually occupied by the macrophytes (phytolittoral surface) and the surface potentially available to them, considered as the area of littoral zone where the water is shallower than 2.5 m (ciecierska and dynowska, 2013). for each site, the settlement rate of macrophytes was calculated using following equation (ciecierska and dynowska, 2013): 𝑍𝑍 = 𝑁𝑁 𝑃𝑃 − 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖. 2.5 where z = settlement rate of macrophytes, n = total area of phytolittoral zone in the site (m2), p = the total surface area of the site (m2) and isob.2.5 = area where the water is shallower than 2.5 m (m2). using the values calculated in above equations, esmi for each site was calculated using following equation (kolada and soszka, 2004; ciecierska 2008; ciecierska et al., 2010): 𝐸𝐸𝑆𝑆𝐸𝐸𝐸𝐸 = 1 − exp [ − 𝐻𝐻 𝐻𝐻𝑚𝑚𝑚𝑚𝑚𝑚 ∗ 𝑍𝑍 ∗ exp � 𝑁𝑁 𝑃𝑃 �] where, h = phytocenotic diversification index (shannon weiner diversity index), hmax = the maximum phytocoenotic diversification index, z = settlement rate of macrophytes, n-total area of phytolittoral zone in the site (ha), and p = the total surface area of the site (ha). the calculated esmi values were compared with the water quality classes established by ciecierska and kolada (2014). statistical analysis: minitab 14 statistical software package was used in the statistical analysis. the data were tested for normality using anderson darling test. if the data were not normally distributed, data were log 10 transformed before further analysis. however, the portioned variables, such as the percentage sand, silt, clay, toc and percentage abundance were arcsine transformed before analysis. one-way anova followed by tukey’s pairwise comparison was used to assess the spatial variation of water and sediment quality parameters and the abundance of macrophytes. the shannon weiner diversity index, maximum phytocenotic diversification index, the vegetation under anthropopressure, settlement rate of macrophytes and emsi among the study sites were compared by one-way anova followed by tukey’s pairwise comparison. principal component analysis (pca) was used to determine water and sediment quality parameters and diversity and biotic indices that describes the distribution and abundance of macrophytes in this wetland system. regression relationship of water and sediment quality parameters with the diversity of macrophytes were used to identify the influence of these parameters on the distribution of aquatic macrophytes. results the spatial variation of mean±standard deviation of 438 wijeyaratne and bellanthudawa / application of ecological state macrophyte index water quality parameters of the study sites are given in table 1. water ph, visibility, temperature, dissolved oxygen concentration (do), salinity, total phosphate concentration (tp) did not show significant spatial variations (p>0.05). site a of the non-rehabilitated area showed significantly high conductivity, tds and significantly low bod5 and cod compared to other sites (p<0.05). significantly high water depth, nitrate concentration, tds and bod5 were recorded in sites e and f of the rehabilitated area, and significantly high cod and chlorophyll-a concentration were in all the rehabilitated sites compared (p<0.05) (table 1). the spatial variation of mean±standard deviation of sediment quality parameters of the study sites is given are table 2. total organic matter content and sediment ph did not show significant spatial variations between the studied sites (p>0.05). the sites in the rehabilitated area showed significantly high sand, clay and silt contents compared to the sites table 1. spatial variation of mean±standard deviation of water quality parameters at each sampling site. for each parameter, mean values indicated by different superscript letters at each row are significantly different from each other (anova, tukey’s pairwise comparison; n = 7). parameter non-rehabilitated area rehabilitated area site a site b site c site d site e site f ph 6.98 ±0.3a 7.54 ±0.2a 7.63±0.3a 7.78±0.2a 7.38±0.2a 8.05±0.3a visibility (cm) 42.7±4.0a 40.2±4.4a 37.7±1.5a 43.3±2.6a 40.8±1.7a 43.76±1.2a temperature (oc) 30.74±0.4a 31.34± 0.3a 31.44±0.3a 31.54±0.4a 31.75±0.5a 32.14±0.3a conductivity (µs/cm) 345.5±10.5a 253.7± 9.7b 271.6±14.6b 252.4±8.3b 248.69±4.8b 245.23±7.4b water depth (cm) 85.5±3.2a 118.6±9.5a 98.9±5.9a 119.4±2.5a 85.7±6.2b 62.7±6.8b tds (mg/l) 166.25±5.0a 121.68±4.7b 129.98±7.1b 120.47±4.0b 109.90±5.3b 116.84±3.6b do (mg/l) 2.82±0.09a 6.84±0.4b 7.68±0.4b 7.81±0.4b 10.61±0.2c 10.28±1.0c salinity (o/oo) 0.16±0.004a 0.12±0.004a 0.12±0.006a 0.12±0.004a 0.11±0.006a 0.12±0.003a bod5 (mg/l) 1.20±0.5a 3.13±0.5b 3.85±0.3b 3.79±0.3b 6.56±0.3c 5.13±0.3c total phosphate (mg/l) 0.02±0.004a 0.02±0.003a 0.02±0.004a 0.03±0.005a 0.03±0.006a 0.04±0.008a nitrate (mg/l) 0.01±0.001a 0.02±0.001a 0.02±0.002a 0.02±0.003a 0.037±0.004b 0.04± .002b chlorophyll-a (mg/dm3) 2.05±0.2a 2.45±1.7b 2.40± 0.6a 11.61±0.5b 12.13±2.6b 12.42±0.9b cod (mg/l) 173.1 ± 36.4a 305.3±44.7b 285.5±46.9b 384.9±38.8c 387.5±38.2c 454.8±0.1c table 2. spatial variation of mean±standard deviation of sediment quality parameters at each sampling site. for each parameter, mean values indicated by different superscript letters at each row are significantly different from each other (anova, tukey’s pairwise comparison; n = 7). parameter non-rehabiliated area rehabiliated area site a site b site c site d site e site f sand content (%) 44.6±9.7a 54.3±11.9a 48.7±10.6a 7.7±9.4b 7.3±1.6b 5.5±1.2b silt content (%) 9.9±2.1a 3.9±1.2a 8.1±1.7a 13.6±3.1b 11.9±2.6b 13.1±2.8b clay content (%) 45.5±11.9a 41.8±12.7a 43.3±12.4a 83.8±12.3b 80.76±4.20b 81.50±4.04b total organic carbon (%) 12.42±0.01a 12.34±0.02a 12.36±0.03a 12.35±0.02a 12.48±0.03a 12.48±0.03a conductivity (µs/cm) 47.98±0.6a 43.80±0.7a 70.87±2.5b 74.49±2.4b 77.05±1.5b 91.44±1.2c ph 6.19±0.08a 5.76±0.24a 6.17±0.11a 6.12±0.12a 6.23±0.13a 6.31±0.10a table 3. spatial variation of mean±standard deviation of percentage coverage of macrophytes at each sampling site. for each species, mean values indicated by different superscript letters at each row are significantly different from each other (anova, tukey’s pairwise comparison; n = 7). species non-rehabilitated area rehabilitated area site a site b site c site d site e site f nymphaea ampla 63.3±3.39a 12.5±3.67b 18.33±5.03b 8.33±0.83c cryptocoryne wendtii 10.0±1.75a 1.67±0.11b 11.1±0.1a 6.67±0.14c annona glabra 10.8±1.94 a 4.17±0.15b 1.67±0.14b eichhornia crassipes 3.1±0.25a 21.67±3.06b 9.17±3.63a 4.17±1.8a 21.67±4.7b 15.83±3.89ab pistia stratiotes 2.2±1.08a 15.83±2.63b 9.17±2.30bc 12.5±1.81b 29.17±5.75c 11.67±2.44b hydrilla verticillata 6.3±035 6.67±2.26 a 7.5±3.08a 7.67±0.17a 7.5±0.2a ceratophyllum demersum 5.9±1.2c 11.67±2.64a 10.15±3.89a 8.33±0.83b nymphaea rubra 2.1±0.5c 29.17±0.62b 52.5±3.59a salvinia melosta 1.1±0.1 1.67±0.11a 0.8±0.02 cypreus iria 5.02±0.18a 2.5±0.13a 5.01±0.15a 439 int. j. aquat. biol. (2020) 8(6): 434-446 in the non-rehabilitated area (p<0.05). significantly high sediment conductivity was recorded at site f of the rehabilitated area and significantly lower sediment conductivity was recorded at the sites a and b of the non-rehabilitated area (p<0.05) (table 2). the percentage cover of aquatic macrophytes at each study site is given in table 3. ten species of aquatic macrophytes namely, nymphaea ampla, cryptocoryne wendtii, annona glabra, eichhornia crassipes, pistia stratiotes, hydrilla verticillata, ceratophyllum demersum, nymphaea rubra, salvinia melosta and cypreus iria were recorded from the study sites. salvinia melosta and c. iria were recorded only from the sites located in the non-rehabilitated area. significantly higher percentage coverage of n. ampla (63.3%) and a. glabra (10.8%) were recorded from site a of the non-rehabilitated area (p<0.05). sites b and e showed significantly higher percentage coverage of e. crassipes (21.67%) and site e showed significantly higher percentage coverage of p. stratiotes (29.17%) (p<0.05). sites d (11.67%) and e (10.15%) of the rehabilitated area showed significantly high percentage coverage of c. demersum and site f showed significantly high percentage coverage of n. rubra (52.5%) (p<0.05). hydrilla verticillata was recorded from all the study sites except site b, and there was no significant difference of the percentage coverage between the studied sites (p>0.05) (table 3). the shannon wiener diversity index, maximum phytocenotic diversification index, the vegetation under anthropo-pressure, settlement rate of macrophytes and ecological state macrophyte index of the study sites are given in table 4. the shannon wiener diversity index of the study sites ranged from 1.09 to 2.01 and esmi ranged from 0.29 to 0.8. comparatively high esmi were recorded from the sites located in the rehabilitated area. the highest shannon wiener diversity index was recorded from site c (2.01) and the lowest was from site a (1.09) of the non-rehabilitated area (table 4). maximum phytocenotic diversification index ranged from 1.09 to 2.20 and the variation was similar to that of the shannon wiener diversity index (table 4). the percentage vegetation under anthropo-pressure ranged from 83.1 to 100%. site a was having the highest anthropo-pressure (100%) and site e of the rehabilitated area was having the lowest anthropopressure (83.1%). although there was no significant difference in the percentage anthropo-pressure among study sites, the rehabilitated sites were displaying comparatively low anthropo-pressure compared to the non-rehabilitated sites (table 4). the sites in the nonrehabilitated area recorded a significantly lower macrophyte settlement rate compared to the sites in the rehabilitated area (p<0.05) (table 3). pca score plot for variation of water and sediment quality parameters among the study sites in the diyawannawa wetland is given in figure 2. the eigenvalues of the first two pcs, eigenvectors of the water and sediment quality variables and the principal component scores for the study sites are given in table 5. two pcs displaying a cumulative variance of 87.4% were obtained after applying pca for 5 principal components. according to the pca on water and sediment quality parameters, the sites c and b of table 4. the mean±standard deviation values of shannon-weiner diversity index (h`), maximum phytocenotic diversification index, percentage vegetation under anthropo-pressure, settlement rate of macrophytes and ecological state macrophyte index (esmi)in the study sites (n = 7). different superscripts in each column indicate statistically significant differences (one-way anova, tukey’s pairwise test; p<0.05). sites a, b and c; non-rehabilitated area and sites d, e, and f; rehabilitated area. site shannon wiener diversity index maximum phytocenotic diversification index vegetation under anthropo-pressure (%) settlement rate of macrophytes (per m2) (esmi) a 1.09±0.02a 1.09±0.02 a 100±1.2 a 3.50±0.1 a 0.30±0.01 a b 1.72±0.01b 1.8 ±0.01b 95±3.6 a 3.25±0.2 a 0.29±0.01 a c 2.01±0.01c 2.2±0.01 c 91±1.6 a 3.75±0.2 a 0.32±0.02 a d 1.61±0.02b 1.8±0.02 b 89.4±2.6 a 4.5±0.5 b 0.72±0.02 b e 1.33±0.02ab 1.6±0.02 ab 83.1±4.6 a 4.5±0.3 b 0.80±0.02 b f 1.54±0.02b 1.8±0.02 b 85.5±1.6 a 4.5±0.3 b 0.76±0.1 b 440 wijeyaratne and bellanthudawa / application of ecological state macrophyte index the non-rehabilitated area and site d of the rehabilitated area grouped together. sites e and f of the rehabilitated area were grouped together characterizing by high visibility, depth and percentage sand content. site a of the non-rehabilitated area was separated from the other groups characterizing by high total phosphorous concentration, total organic carbon and percentage clay content of sediments (fig. 2, table 5). pca score plot based on abundance of macrophytes among the study sites in the diyawannawa wetland is given in figure 3. the eigenvalues of the first two pcs, eigenvectors and the principal component scores for the study sites are given in table 6. two pcs display a cumulative variance of 68.2%. the results of the pca on macrophyte abundance indicated that site a was grouped separately from other sites and was categorized by high abundance of n. ampla. sites b and d were grouped together and they were characterized by high abundance of e. crassipes, p. stratiotes and s. melosta. sites e and f were characterized by h. verticillata and c. iria, and site c was characterized by a. glabra (fig. 3, table 6). table 5. summary of the pca of physico-chemical parameters of water and shallow sediments of the study sites at the diyawannawa wetland. cumulative % variation of only the pc1 and pc2 are shown. a high cumulative percentage as high as 87.4 % of the total variation among physico-chemical parameters are explained by pc1 and pc2 axis. sites a, b and c; non-rehabilitated area and sites d, e, and f; rehabilitated area. eigenvalues pc eigenvalues %variation cum.%variation 1 12.2 64.2 62.8 2 4.41 23.2 87.4 3 1.47 7.8 95.1 4 0.52 2.7 97.9 5 0.40 2.1 100.0 eigenvectors (coefficients in the linear combinations of variables making up pc's) variable pc1 pc2 pc3 pc4 pc5 water ph 0.218 -0.251 -0.182 -0.339 0.281 temperature 0.280 -0.058 -0.041 -0.175 0.170 ec -0.234 0.272 0.039 -0.029 -0.049 tds -0.245 0.232 0.013 -0.173 0.199 do 0.263 -0.165 -0.089 -0.089 -0.232 bod5 0.264 -0.025 -0.202 0.018 -0.466 cod 0.266 -0.148 -0.109 0.094 0.221 nitrate 0.262 -0.100 0.267 -0.177 -0.023 chlorophyll a 0.215 -0.171 0.348 0.451 0.246 total phosphorous 0.251 0.149 -0.080 0.208 0.495 visibility -0.245 -0.157 -0.248 0.361 -0.085 depth -0.142 -0.367 -0.236 0.369 -0.153 %toc 0.172 0.346 0.238 0.165 -0.199 sediment ph 0.122 0.389 -0.304 -0.113 -0.140 %sand -0.247 -0.204 -0.199 -0.136 0.090 % silt 0.130 0.305 -0.447 0.388 0.156 %clay 0.239 0.213 0.224 0.169 -0.203 sediment conductivity 0.240 0.123 -0.383 -0.155 -0.003 principal component scores sample pc1 pc2 pc3 pc4 pc5 site a 5.093 -2.819 -0.411 -0.218 -0.111 site b 1.702 3.153 -1.532 0.009 -0.180 site c 1.013 0.515 1.064 1.125 0.575 site d -0.047 1.171 1.811 -0.712 -0.514 site e -3.176 -0.529 -0.402 -0.717 0.938 site f -4.586 -1.492 -0.530 -0.513 0.707 table 6. summary of the pca of the abundance of macrophytes in the study sites at the diyawannawa wetland. cumulative % variation of only the pc1 and pc2 are shown. a high cumulative percentage as high as 68.2 % of the total variation among macrophyte abundance are explained by pc1 and pc2 axis. sites a, b and c; non-rehabilitated area and sites d, e, and f; rehabilitated area. eigenvalues pc eigenvalues %variation cum.%variation 1 4.13 41.3 41.3 2 2.69 26.9 68.2 3 1.63 16.3 84.5 4 1.24 12.4 96.9 5 0.31 3.1 100.0 eigenvectors (coefficients in the linear combinations of variables making up pc's) variable pc1 pc2 pc3 pc4 pc5 nymphaea ampla 0.489 -0.034 -0.043 0.086 -0.016 cryptocoryne wendtii 0.342 -0.317 0.310 -0.239 -0.236 annona glabra 0.478 0.121 0.048 -0.072 -0.168 eichhornia crassipes -0.338 -0.405 -0.114 0.159 -0.337 pistia stratiotes -0.378 -0.285 0.189 -0.235 -0.459 hydrilla verticillata -0.183 0.548 0.034 -0.167 -0.239 ceratophyllum demersum -0.299 0.076 0.542 -0.168 0.573 nymphaea rubra -0.112 0.191 0.067 0.822 -0.120 salvinia melosta -0.078 -0.318 -0.629 -0.086 0.388 cypreus iria -0.131 0.441 -0.395 -0.333 -0.201 principal component scores sample pc 1 pc 2 pc 3 pc 4 pc 5 site a 4.025 -0.367 0.438 -0.124 -0.137 site b -0.656 -1.749 -2.098 -0.219 0.243 site c 0.065 1.832 -0.861 0.376 -0.849 site d -1.063 1.960 0.320 -1.283 0.575 site e -1.354 -1.751 1.491 -0.713 -0.499 site f -1.017 0.076 0.710 1.963 0.393 441 int. j. aquat. biol. (2020) 8(6): 434-446 the results of the linear regression analysis showed the coefficients of determination (r2) being greater than 0.5 at 95 % level of significance indicated strong negative relationship of esmi with pc1 score of water and sediment quality parameters (r2 = 71.4, p = 0.034). pc1 score of the macrophyte abundance showed a strong negative correlation with the pc1 of water and sediment quality parameters (r2 = 64.4, p = 0.025, fig 4). however, shannon weiner diversity index (h) did not show a significant relationship with figure 2. ordination of the study sites based on pc1 and pc2 scores of pca of the physico-chemical parameters of over lying water and sediments of the study sites in rehabilitated and non – rehabilitated areas in the diyawannawa wetland. the study sites a, b and c are located in the non – rehabilitated area and the sites d, e and f are located in the rehabilitated area. figure 3. ordination of the study sites based on pc1 and pc2 scores of pca of the abundance of macrophytes in the study sites in rehabilitated and non – rehabilitated areas in the diyawannawa wetland. the study sites a, b and c are located in the non – rehabilitated area and the sites d, e and f are located in the rehabilitated area. 442 wijeyaratne and bellanthudawa / application of ecological state macrophyte index the water and sediment quality parameters (fig. 4). discussions macrophytes are important part of the wetland ecosystems as they serve as major primary producers, sediment stabilizers and habitat providers (schaumburg et al., 2004). the results of the present study indicate that there is a significant variation in the abundance of macrophytes in the rehabilitated and non-rehabilitated areas in the wetland. further, significantly high abundance of invasive alien species is recorded in the non-rehabilitated area compared to the rehabilitated area. the rehabilitated area is managed under the wetland management programmes and the management actions involve dredging to increase the depth of the wetland and continuously monitoring for the water quality, detecting occurrence of invasive alien species and removing them accordingly. this may have resulted in a significantly low number of invasive alien plants in the rehabilitated area. however, high chlorophyll-a concentration, biochemical oxygen demand, chemical oxygen demand, nitrate and do were recorded in some sites of the rehabilitated area. the increased water quality parameters in the rehabilitated area may be due to the presence of high concentrations of phytoplankton. when macrophytes are abundant, they can serve as nutrient sinks, utilizing much of the available phosphorus and nitrates (jasser, 1995; zimmer et al., 2011; hilt, 2015). therefore, in macrophyte abundant environments, less potential for high growth of phytoplankton can be expected as the nutrient availability for phytoplankton is low (zimmer et al., 2011; hilt, 2015). in the rehabilitated area, continuous removal of invasive macrophytes may have caused availability of nutrients to phytoplankton and resulted in high growth of phytoplankton, increasing chlorophyll-a concentration, do, bod5 and cod. application of univariate to assess the variation of abundance of the biological communities are a commonly practiced methodology in ecological assessments. however, multivariate statistical techniques are more sensitive and accurate in studying environmental disturbance associated community changes in ecosystems (warwick and clarke, 1993). in sri lanka, few studies have been conducted using multivariate statistics to assess the variation of biological communities in relation to water and sediment quality parameters (dahanayake and wijeyaratne, 2006; idroos and manage, 2012; wijeyaratne and bellanthudawe, 2017). these studies have focused on variation of benthic macrofigure 4. linear regression against the pc1 score for physicochemical parameters of the sediments and overlying water in the study sites. (a) ecological state macrophyte index, (b) shannon wiener diversity index, (c) pc1 score for abundance of macrophytes in study sites. 443 int. j. aquat. biol. (2020) 8(6): 434-446 invertebrate communities in wetlands in relation to the water and sediment quality parameters. in the present study, the pca was used to categorize the study sites based on water and sediment quality parameters and abundance of macrophytes. based on the results, the sites a and b of the non-rehabilitated area and site d of the rehabilitated area were grouped together characterizing high visibility and high percentage sand content in the sediments. the sites a, b and d were located in close proximity to each other and this may have caused these sites to share common physical parameters. in the pca on the abundance of macrophytes, sites b and d were grouped together and site a was separated from others. the results revealed that site a is characterized by n. ampla and high percentage of total phosphorous, high total organic carbon and high percentage clay content of sediments. nymphaea ampla is considered as an invasive species that has originated in caribbean and central america and grows as a dense patch which is covering the water surface like a mat preventing light penetration and blocks the interface between air and water, decreasing do in water (maddy, 2009). the results of the present study also agree with maddy (2009) regarding the site a with highest abundance of n. ampla has significantly lower do. maddy (2009) indicates that high abundance of n. ampla can cause nutrient release from the degenerating mats increasing the phosphorous and organic matter composition of the water and sediments. the results also agree with these findings regarding site a with highest abundance of n. ampla characterized by high total phosphorous in water and high total organic carbon in sediments. sites e and f of the rehabilitated area were grouped together characterizing by high visibility, depth and percentage of the sand content, and aquatic macrophytes of h. verticillata and c. iria. hydrilla is identified as an important plant used in constructed wetlands to remove nutrients and to trap suspended solids (langeland, 1996; barko and james 1998; knight et al., 2003; tanaka et al., 2007). the results support the water purification ability of hydrilla as the significantly high visibility recorded from two sites where hydrilla is abundant. macrophyte based ecological quality assessment methods provide important information regarding the ecological status of the wetlands. however, absolute numbers such as the number of species recorded are less informative compared to the quantitative ratios of abundance of dominant species with relevance to the area of the wetland (ciecierska and kolada, 2014). in esmi, a taxonomic composition is quantified using the phytocenotic diversity index (h) and the maximum theoretically possible hmax. if the anthropogenic or natural influences disturb the phytocenotic balance, vegetation patterns are simplified and extinction of some communities and dominance of other communities can result (rejewski, 1981; ciecierska et al., 2010). in the present study, the esmi ranged from 0.29 to 0.80 with significantly lower values in the nonrehabilitated sites. however, there was no significant different of the shannon weiner diversity index (phytocenotic diversity index (h)) among the study sites. the percentage anthropo-pressure in the non– rehabilitated sites were comparatively higher. the rehabilitated sites of this wetland are carefully monitored and managed by the land reclamation department of sri lanka, which in turn provided less opportunities for people to engage in activities that disturbs the ecological functions of the wetland. however, in the non-rehabilitated area, it was observed that wetland associated animal farm management activities and waste deposition is prominently carried out. this may have resulted in the comparatively increased anthropo-pressure in the sites of the non-rehabilitated area. further, the increased anthropo-pressure may have significant effects on the macrophyte resettlement rate. in the present study, the macrophyte resettlement rate in the rehabilitated sites were significantly higher than that of the non-rehabilitated sites. the weed removal, dredging and water quality monitoring activities conducted by the land reclamation department of sri lanka may be impose positive effects on the macrophyte resettlement rate in the rehabilitated portion of the diyawanna wetland. 444 wijeyaratne and bellanthudawa / application of ecological state macrophyte index according to boundary values of the esmi index for classifying the ecological status in wetlands introduced by ciecierska and kolada (2014), esmi values between 0.205-0.409 indicate moderate ecological status, 0.410-0.679 indicate good ecological status and values at or above 680 indicate high ecological status. in the present study, all the sites in the non-rehabilitated area were categorized into the moderate ecological status and all other sites are categorized as the high ecological status. therefore, the present study proves that wetland rehabilitation programmes are successful in improving the ecological status of wetlands. further, the regression analysis between esmi and the pc1 score based on water and sediment quality parameters indicated a significant positive association indicating that 71.5% of the variation of esmi can be accounted due to variations in the water and sediment quality parameters. therefore, the present study provides evidence of the suitability of adopting esmi in ecological status classifications in the tropical wetland ecosystems. these macrophyte indices, together with multivariate statistical applications provide important information on the relationships between water quality, sediment quality and macrophyte indices which in turn can be used in long term wetland restoration and wetland management programmes. funding statement this research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. references barko j.w., james w.f. 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(2013) 1(4): 143-149 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article an investigation on morphology, age and growth of the caspian sea kilka (clupeonella cultriventris) in babolsar, southern caspian sea zohreh mazaheri kohanestani *1, rasoul ghorbani1, saeid yelghi2, abdolazim fazel3, mahmood zoghi1 1 department of fisheries, gorgan university of agricultural sciences and natural resources, gorgan, iran. 2gorgan research center of inland water fishes, gorgan, iran. 3department of fisheries, university of guilan, guilan, iran. article history: received 8 may 2013 accepted 27 june 2013 available online 2 0 august 2013 keywords: morphology growth natural and fishing mortality caspian sea abstract: in this study, 160 fishes were randomly collected from commercial catch by a cone net in babolsar port from january to october 2010. the biological features of specimens were measured. 2+ years old fishes made the dominant age group with 33.75% and 1+ and 5+ years old had the least frequency (8.75%). relationship between length and weight indicated negative allometric growth pattern (b=2.581). the von-bertalanffy growth parameters were calculated as l=131.57 mm, k=0.26 and t0=-1.02. growth performance index was 1.66 and the total mortality (z), natural (m) and fishing (f) mortality coefficients were 0.9 year, 0.43 and 0.47, respectively. the exploitation ratio (e) was calculated as 0.52. introduction identification of fishes is the first stage to manage the aquatic ecosystem and conservation of the stocks (yaoungs and robson, 1978). kilka belongs to the species clupeonella and clupeidae or herring family. there are three genus of kilka in the caspian sea including common kilka (clupeonella cultriventris), anchovy (clupeonella engrauliformis) and big-eye kilka (clupeonella grimmi) that are identified by morphometric parameters (anonymous, 1978). kilka is fished by cone nets using under water light to attract fishes at nights (yermalchev and sedov, 1990; fazli and rouhi, 2002). kilka has an important role in the caspian sea ecosystem and costal countries economic. they are considered as an important part of the trophic chains (mamedov, 2006) and also a health indicator of the caspian sea environment (fazli, 1990; razavi sayyad, 1993; pourgholam et al., 1996). their stocks are needed to be protected (pourgholam et al., 1996). * corresponding author: zohreh mazaheri kohanestani e-mail address: zohremazaheri_65@yahoo.com since 1998, kilka populations have decreased due to invasion of mnemiopsis leidyi as a new trophic rival, chronic poisoning with oil, phenol and heavy metal pollutants, temperature variation of the caspian sea and over-fishing (paritskii et al., 2001). mnemiopsis leidyi has already damaged the pelagic ecosystem of the central and southern caspian sea, directly or indirectly impacting all trophic levels. previous studies confirm decline of kilka biomass in some neighbor countries as mamedov (2006) reported that exploitable biomass of kilka varied from 18500t to 5100t between 2000-2004 in azerbaijan. since kilka is an important part of diet of some valuable fishes like sturgeon (acipenser spp. and huso huso) and seal (phoca caspica), decline of its biomass can threat their stocks. there are some studies on distribution (besharat and khatib, 1993; razavi sayyad, 1993), stock assessment (pourgholam et al., 1996; fazli et al., 2002) and biology (karimzadeh et al., 2010; fatemi 144 kohanestani et al./ int. j. aquat. biol. (2013) 1(4): 143-149 et al., 2009; parafkandeh haghighi, 2009; fazli et al., 2002, 2004, 2005, 2007; sayyad bourani, 1997) in kilka of southern part of the caspian sea. the present study aimed to provide a renew of information on biological characteristics of clupeonella cultriventris in southern part of the caspian sea. such information may help to manage kilka stocks. materials and methods this study was carried out from january to october 2010. 160 samples were randomly collected from the commercial catches, which taken by fishing boat and cone nets with a 1500 kw light to attract the fish in babolsar port, the southern part of caspian sea. samples were preserved in 10% formaldehyde and transported to laboratory. weight and standard length were measured by a digital scale (0.01 g) and vernier calipers (0.01 mm), respectively. age was determined using annuli of 10 scales. in order to increase precision of age determination, scales were examined by 3 experts and, in some cases, compared with otolith. relationship between length and age estimated using regression model. the standard von bertalanffy growth equations and ford-walford method were used to estimate growth in each age (bagenal and tesch, 1978; erdogan, 2002): lt = l∞ (1-e -k (t-t0)) where lt is the length-at-age t, l∞ is the maximum theoretical length, k is growth coefficient and t0 is equal to age of fish when the length is zero. the growth performance index was calculated using the following equation (pauly and munro, 1984): φ´= log k +2 log l∞ the condition factor (cf) and instantaneous growth rate (g) was calculated, respectively, as: cf = w/l3 and g = (lnw2-lnw1)/(t2-t1) where w1 = initial wet weight of fish, w2 = final weight of fish, t1 = age at stocking and t2 = age at end of the period (usually one year). total morality (z) was calculated based on beverton and holt (1956) method follow as: z= k[ ] where lc, is average length of captured fishes and lc is length at first capture. also, natural (m) mortality was estimated by pauly’s empirical equation (1984): ln m=0.0066 -0.279 ln (l∞) + 0.6543 ln (k) + 0.4634 ln (t) according to total (z), natural (m) mortality and bellow relationship, fishery mortality (f) was calculated as: z= m+f the exploitation ratio (e) was calculated using the equation: fm f e   for the morphological study, 10 features (7 morphometric and 3 meristic features) described by berg (1949), were measured and illustrated in tables 2 and 3. before analysis, data were examined for normality by kolmogorov-smirnov test. morphological features were standardized before the analysis and were compared in different ages by oneway analysis of variance (for metric parameters) and the kruskal-wallis test (for meristic parameters). result age of samples ranged between 1+ and 5+. the 2+ years old fishes were the dominant age group (33.75%) and also 5+ years old with 8.75% had the lowest frequent age groups (table 1). the specimens had an average age of 2.9 years. standard length ranged from 63 to 115 mm. mean values and standard deviations of characteristics are shown in table 2. morphometric and meristic characteristics did not vary significantly with increasing age (table 2). according to length-age relationship, length increased with increasing age logarithmically (fig. 1, p<0.001) indicating that young specimens grow faster than older ones. )( )( lclc lcl   145 kohanestani et al./ int. j. aquat. biol. (2013) 1(4): 143-149 according to pauly test (p<0.05), weight and length had negative allometric relation (b=2.58), showing that the length increased faster than the weight (fig. 2). standard von-bertalanffy growth equation was estimated as follow: lt=123.7 (1-e -0.356(t+1.4)) according to this formula, coefficient growth, infinity length (basically standard length) and age of fish at zero length calculated as 0.3585 per years, 120.71 mm and -1.4 years, respectively. growth performance index was 1.66. the total mortality (z), natural (m) and fishing (f) mortality coefficients were 0.9 year, 0.43 and 0.47, age number frequency (%) standard length (mm) weight (g) condition factor growth rate 1+ 16 10 67.4 ± 3.2 3.64± 0.26 1.14 ± 0.08 ----- 2+ 54 33.75 80.2 ± 6 5.37 ± 1.16 1.03 ± 0.1 0.17 3+ 40 25 95.9 ± 2.1 8.65 ± 1.73 0.98 ± 0.18 0.18 4+ 36 22.5 102.1 ± 1.9 9.32 ± 1.16 0.88 ± 0.12 0.06 5+ 14 8.75 109.1 ±1.8 10.95 ± 1.15 0.88 ± 0.12 0.07 total 160 100 89.3 ± 13.2 7.38 ± 2.64 0.98 ± 0.15 0.48 table 1. mean value of standard length (±sd), weight (±sd) and condition factor of common kilka. character/age 1+(n= 16) 2+(n= 54) 3+(n= 40) 4+(n=36) 5+(n=14) mean body depth 15±1.41 18.13±2.67 20.82±2.72 22.21±1.93 23.24±2.82 20.23 caudal depth 5.62±0.75 7.19±1.71 8.10±1.58 8.05±1.02 8.08±1.05 7.71 caudal fin length 13±0.82 16±1.83 18.15±1.86 18.83±2.04 19.88±2.18 17.58 distance between eyes 2.17±0.15 2.81±0.80 3.29±0.76 3.52±0.62 3.43±0.48 3.17 eyes diameter 3.5±0.27 4.12±0.55 4.51±0.54 4.80±0.38 5.03±0.49 4.45 head and dorsal fin d 35.50±1.29 40.98±3.18 46.71±4.61 50.29±1.60 51.88±2.87 45.56 dorsal and caudal fin d* 28±0.82 32.82±3.29 36.62±3.90 39.88±2.52 41.65±1.77 36.17 dorsal fin rays 14.25±0.50 14.84±0.89 14.80±0.83 14.71±0.86 14.88±0.78 14.79 pelvic fin rays 14.75±0.50 15.13±2.07 15.09±2.30 14.71±0.81 14.82±0.64 15 caudal fin rays 28.25±0.96 26.71±3.73 27.30±4.09 27.46±2.08 28.12±1.65 27.24 *d= distance table 2. mean (±sd) of morphometric-meristic characteristics of common kilka. parameter fork length (mm) body weight (g) b age (year) dominant ages (year) year mean s.d min max mean s.d min max 1997 92.8 9.92 67.5 112.5 6.24 1.55 3.1 9.5 2.6 0-5 1 (0-3) 1998 87.3 12.38 57.5 122.5 4.89 1.77 1.4 10.2 1999 82.5 8.48 47.5 107.5 4.17 1.17 0.8 8 2000 81.5 6.76 57.5 107.5 3.82 0.84 1.5 7.5 2.512 0-5 2 (0-3) 2001 88.3 5.89 67.5 107.5 5.24 0.83 1.7 10.9 2.455 0-5 3 (2-4) 2002 85.12 7.84 52 121 4.446 1.35 1.37 14.02 2.28 1-6 3 (2-4) 2004 84.35 --65 105 4.28 --1.7 7.19 ------ 2008 93.8 11.8 50 127 7.1 2.1 1.1 16.5 2.77 1-6 4 (4-6) 2010 90.3 13.2 63 111 7.42 2.72 3 13.03 2.581 1-5 2 (2-4) table 3. some biological parameters of c. cultriventris were taken from previous studies (abtahi et al., 2002; abtahi et al., 2005; fazli et al., 2006 and janbaz and abdolmaleki, 2009) and present study. 145 146 kohanestani et al./ int. j. aquat. biol. (2013) 1(4): 143-149 respectively. the exploitation ratio (e) was calculated as 0.52. discussion among the clupeonella species, common kilka (c. cultriventris) has a distinctive distribution depth and lives in pelagic area at depth less than 50 m in the iranian water. recent study has shown that kilka stocks are over-exploited (karimzadeh et al., 2010). also, comparison of the caught rates of three clupeonella species indicates that, cone net is not proper fishing device for kilka, so it can be exploited by independent catching methods (abtahi, 2001; paritskii et al., 2001; razavi sayyad, 1993). some biological parameters of c. cultriventris which were reported in previous studies are showed in table 3. according to table 3, similar changes have happened in some parameters such as length, weight compositions and regression coefficient (b) since 1997. these changes show that common kilka stock has been changed. for example the mean fork length was 92.8 mm in 1997 and it decreased to 81.5 during 1997-2000 (fazli et al., 2006). after 2000, it increased to 93.8 (in 2008) again. in present study it was 90.3 ± 13.2 mm. this variation can be seen in weight, too. mean body weight of kilka is reported 6.24 g in 1997, 3.84 g in 2000 and 7.1 g in 2008. based on the previous and present studies it seems that kilka population has become younger during 1997-2000 and then older in 2000-2010. fazli et al. (2006) suggested that after mnemiopsis sp invasion in 1998, the kilka stock migrated to deeper waters because of food competition with mnemiopsis sp. migration of common kilka to deeper waters has resulted in over-exploitation and decrease of c. engrauliformisas as the main species in commercial catch (fazli et al., 2002). kilka had negative allometric growth which varied during these years. variation of the coefficient b depends on species, habitat, sex, age, feeding, season, etc. (bagenal and tesch, 1978). age composition data help us to understand the effect of environment factors on growth parameters and fishery recruitment (stevensen and campana, 1992). at the present study five age groups were found. age structure varied like other parameters described before. the dominant age has increased from 1+ to 4+ since 1999, and decreases to 2+ (because the mean age is 2.9 and the 3+ years old have a high frequency (25%), we can considered it equal 3). a decrease in the mean and maximum length, number of age groups and dominant age in present study, indicate that kilka population is under pressure and also it can be a sign that other species of kilka who lived in deeper water may be in risk. at the present study fishing (f) mortality coefficient was 0.47, whereas janbaz and abdolmaleki (2009) reported this value as 0.44. also the exploitation ratio of common kilka reported as 0.294 – 0.411 and 0.51 by chilton et al. (1982) and janbaz and figure 2. relationship between standard length and weight, in common kilka figure 1. the relationship between standard length and age in common kilka 147 kohanestani et al./ int. j. aquat. biol. (2013) 1(4): 143-149 abdolmaleki (2009). at the present study, it was 0.51. all of these data confirm that the kilka population is under pressure (fazli et al., 2002). the analysis of mean values of characteristics did not reveal significant differences between age groups especially in meristic characteristics because, most of specimens were adult (kilka matured in 2 years old), so there was not a lot of variation in morphological characteristics. the average of body and caudal depths, caudal fin length, distance between eyes, eyes diameter and caudal fin rays of c. engrauliformis were 19.3±1.33, 6.96±0.7, 3.61±1.24, 5.59±0.88, 5.02±0.72 and 14.64±2.5, respectively (rahimi bashar and alipour, 2009). except the caudal fin length and distances between eyes which are higher in common kilka, other parameters are similar to each other. this is maybe explained by species-specific characteristics’. in conclusion, it is important to avoid overand unmanaged fishing in spawning period of clupeonella sp to recruit and preserve their stocks. we suggest continuation of kilka’s biological features to have a good view on status of stocks for better management and conservation. also we suggest design of special fishing method for common kilka. acknowledgment we are grateful to alireza kalantari for providing samples from babolsar commercial catch site, to masoud mollaei, expert of fishery laboratory and to fishery department of gorgan university of agricultural sciences and natural resources for his support. references abtahi b. 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(2020) 8(1): 1-8 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article morphohistological characteristic of digestive tract of an endemic cichlid fish, iranocichla hormuzensis coad, 1982 (teleostei: cichlidae) mina hojat ansari1, mehregan ebrahimi*2,3,1hamid reza esmaeili2 1gastroenterohepatology research center, shiraz university of medical sciences, shiraz. 2department of biology, college of sciences, shiraz university, shiraz, iran. 3school of biological sciences, flinders university, australia. s article history: received 27 november 2019 accepted 4 january 2020 available online 2 5 february 2020 keywords: cichlids morphohistology digestive tube omnivore abstract: objective of the current study was to investigate morphohistology of digestive tract of iranocichla hormuzensis, one of two native cichlid fishes in iran. the species present omnivorous feeding habits and restricted in shallow, high temperatures and high salinity water in the mehran river and its drainages. this species has a terminal mouth with several rows of serrated incisive teeth, which covered the upper and lower lips. esophagus is a short tubular between pharynx and stomach, stomach is relatively small and sac-shape, and intestine is long. gastric wall, through the digestive tube, consists of four layers of mucosa, submucosa, muscularis, and serosa with some characters specialized for each organ, such as a well-developed mucosa layer and numerous goblet cell in the ventral section of stomach. morphohistology of digestive tract of i. hormuzensis supports its generalist diet. introduction histological and anatomical study of digestive tract in fish is essential to understand feeding physiology, structure of digestive tract, and adaptation in response to feeding habits. there is an increasing number of studies focus on characterising morphohistology of digestive system in fish (e.g. canan et al., 2012; rønnestad et al., 2013; purushothaman et al., 2016; arman and ucuncu, 2017; kalhoro et al., 2018). however, anatomical and histological aspects of digestive system are poorly characterised in many fishes, hindering an understanding of feeding dynamics and habitat occupation of the species. the cichlids (family cichlidae) particularly remain underrepresented despite their high species richness among vertebrates. the iranian cichlid fish iranocichla hormuzensis coad, 1982, is an endemic fish restricted to a small river tributary in the persian gulf basin (esmaeili et al., 2016; schwarzer et al., 2017). this fish is distributed in shallow, high temperature and high salinity water of the mehran river and its drainages *correspondence: mehregan ebrahimi doi: https://doi.org/10.22034/ijab.v8i1.779 e-mail: m.ebrahimi@shirazu.ac.ir (esmaeili et al., 2016; schwarzer et al., 2017). diet of i. hormuzensis is mainly detritus, cyanobacterium, diatoms and green algae, but zooplankton and small fishes have also been observed (unpublished data). this trophic plasticity attributed to seasonal variation, breeding strategies and a higher protein needs for reproduction and potential growth in adults (unpublished data). iranocichla hormuzensis, beside of its natural beauty, interesting mouth-brooder behavior and importance as nutritional food for local people, is the only cichlid fished in iran. this endemic species link to trophic dynamics of the mehran river ecosystems makes it as an important species that its morphohistological data is missing. therefore, the present study aimed to provide data on morphohistology of i. hormuzensis digestive tract. materials and methods four specimens of i. hormuzensis (two males and two females) were collected from the mehran river near the city of bastak (27.04889n, 54.28360e). these specimens were collected under the appropriate 2 hojat ansari et al./ morphohistological characteristic of digestive tract of iranocichla hormuzensis permits (subc-840388) and transferred alive to the laboratory. in the laboratory, the structures of the digestive tract, position, and size of the mouth, dentition, and shape of the gill rakers were morphologically described in both sexes. the pharyngeal jaw, position, and size of mouth and structure of skull have been drawn using camera lucida coupled with a stereomicroscope (zeiss sv 11). the teeth structure examined using the scanning electron microscope (sem) following monsefi et al. (2010). briefly, samples were dehydrated in series of 30, 50, 70, 90 and 100% ethanol, fixed, mounted on metallic stubs, coated with a layer of gold (sc7640 sputter coater fisons) and photographed (leica cambridge). for histological description, a fragment of the pharynx, esophagus, stomach, and intestine (anterior, intermediate, and posterior portions) from two specimens (one male and one female) were examined through transverse and longitudinal sections (fig. 1). in summary, the tissue samples initially were fixed in bouin’s fluid and then following the routine histological methods described in bancroft and stevens (1990), the tissue blocks were obtained. these tissue blocks were submitted to a microtome (microtome spencer 820, u.s.a) to obtain micro section at 5-7 μm. then micro sections were stained with hematoxylin-eosin (he) based on gurr (1962) and finally examined using a zeiss microscope coupled to a canon eos digital camera. results the structure of the digestive tract in i. hormuzensis is given in figure 1. this fish has a terminal mouth that in female being smaller in size, although they have a bigger mouth cavity than males. the upper and lower lips include a stratified epithelium, with several rows of the serrated teeth sets on jaws. the upper jaw has three to four rows of tricuspid teethes, and the lower one often is observed with one more row of tricuspid teethes (fig. 2a, b). the individual jaws are also including bicuspid teeth forms but just in the outer line (fig. 2a). pharyngeal jaw is covered with papilliform teethes in both upper and lower parts (fig. 2c, d). the upper pharyngeal jaw consists of two oval shape bones and the lower jaw (lpj) is triangle in shape (fig. 2e, f). there is no external structure at the ventral lpj when the two halves of the lpj meet. the esophagus is a short tubular drive from the posterior end of the pharynx extending towards the anterior part of the stomach. the esophagus consists of several concentric tissue layers of tunica; mucosa, submucosa, and muscularis (which formed by a thin inner longitudinal and an outer circular) (fig. 3a). the mucosal epithelium has numerous goblet cells near the surface. the mucosa consists of many longitudinal folds along with stratified epithelium, which includes a large number of mucous cells (fig. 3b). it then follows by lamina propria, which is formed by loose connective tissue with many fibers of the striated muscles (fig. 3c). the submucosa, a thin layer of the dense connective tissue, support the mucosa and connects to the muscular. the stomach is sac-shape and relatively small with lateral position to the intestine. the transitional region between esophagus and stomach simply detected when the cubic epithelium of the esophagus reduced and replaced by the cylindrical epithelium characterized for stomach. in addition, the strained muscular seen in the transitional region is replaced with smooth muscle fibers (a thick inner circular and an outer thin longitudinal layer) later in the stomach (fig. 4a). at the transition region (base of the stomach), a confluence of the striated muscle and collagen fibers exist, which is similar to a sphincter figure 1. the structure of the digestive tract in iranocichla hormuzensis. a fragment of the eight highlighted positions was used for preparing transverse and longitudinal sections. 1= esophagus; 2, 3, and 4= stomach; 5= transition region; 6= anterior intestine; 7= intermediate intestine; 8= posterior intestine. 3 int. j. aquat. biol. (2020) 8(1): 1-9 delimiting operating as a control mechanism (fig. 4b). presence of the fibromuscular folds of wall, including both striated and smooth muscle, that facing towards the intestinal lumen provided stronger support to existence of a true pyloric sphincter in the base of stomach. the stomach includes a thin layer of smooth muscle, which is approximately one in third of its epithelium layer. the thickness of muscular layer reduced towards the end (bottom of the sack) and instead of that, the elastic fibers often are observed. in the stomach, the gastric glands predominated in lamina properia and often presents in tubular forms towards the end. the cell composition is often varied in dorsal and ventral sections, and across the wall figure 2. shape and the structure of teethes in iranocichla hormuzensis. a, b, c, d, e, and f. figure a represents the scanning electron microscope (sem) of the jaw showing the outer line including both bicuspid (a) and tricuspid (b) teethes. figure b showing outer and inner lines of teethes that bicuspid (a) just seen in outer line and tricuspid (b) in all lines. figure c demonstrates the sem scan of the pharyngeal jaw and their papilliform teethes. figure d showing the papilliform teethes cover all over the upper and lower pharyngeal jaw. figure e represents a schematic view of the lower pharyngeal jaw. figure f showing a schematic view of half of the upper pharyngeal jaw. 4 hojat ansari et al./ morphohistological characteristic of digestive tract of iranocichla hormuzensis extensions of stomach (fig. 4c, d, e). the beginning part of the stomach exhibits average and branched folds mucosa, which then replaces with uniform and tall mucosa through the middle and finally reduced in number and size at the fundus. in particular, the dorsal mucosa exhibits patulous, short and uniform folds, with single goblet cell and presence of a single layer of simple cylindrical epithelium. the mucosa in the ventral section shows tall and branched folds that are completely glandular and presents a much thicker layer of simple cylindrical epithelium compare with the dorsal section (fig. 4d). the goblet cell seen in the beginning part of the dorsal section and often disappears towards fundus whereas in ventral section they increased by number (fig. 4f). the intestine is a long and thin tube, which forms several spiral loops joined by adipose tissue. this adipose tissue is significantly thicker in female, especially during the reproductive seasons. average length of intestine tube measured at 45.73 mm approximately half (58.25%) of total body length. anterior intestine has tall, thin and numerous folds, but they reduced in number and height posteriorly (fig. 5a, b). the epithelium of the intestine is cylindrical with nucleus positioned in basal region forming a border facing lumen. goblet cells exhibit across entire range of intestine (fig. 5c), although their frequency is decreased at posterior part. after lamina propria is smooth muscular fibers, which arranged internally with a circular tunica and an outer longitudinal tunica, and overall muscular layer is ticker in the posterior part of the intestine (fig. 5d). the tunica of the intestine wall lastly followed by serous. discussions principal objective of this study was to provide a description of the digestive tract of i. hormuzensis. current findings indicated that histomorphological and anatomical structure of digestive tract of i. hormuzensis is well-adapted to its trophic plasticity (unpublished data). iranocichla hormuzensis has a terminal mouth with serrated incisive teeth. we observed a smaller mouth size but a bigger mouth cavity in female. digestive tract of teleost fishes often well-adapted to the feeding behavior exhibits certain characterised variation in morphological and functional features (banan khojasteh, 2012; ghosh and chakrabarti, 2015). consequently, shape and position of mouth, dentition, and form and type of gill rakers can present feeding behavior of species (motta, 1984; albrecht et al., 2001; monsefi et al., 2010). in figure 3. the esophagus of iranocichla hormuzensis: a, b, and c. figure a shows a transverse section of esophagus showing folds (f) and mucosa layer, (ebar) epithelial layer and (m-bar) muscle layer. figure b represents esophagus fold consisting of (mg) mucous glands and (ec) epithelium cells. figure c demonstrates the muscle layer of esophagus highlighted the (ms) striated muscle fibers. 5 int. j. aquat. biol. (2020) 8(1): 1-9 this context, mouth size or gap is stated to be a proportion to the prey size consumed by the species (canan et al., 2012). however, this condition may not totally be supported in i. hormuzensis, because more than 80% overlap in food items have been recorded in male and female (unpublished data). on the other hand, it was indicated a larger size prey (a small size fish) consumed by male that might indicate the ability of male to swallow bigger prey. however, having a bigger mouth cavity observed in female of i. hormuzensis potentially is a specific adaptation related to the mouth brooder behavior of female figure 4. the stomach of iranocichla hormuzensis: a, b, c, d, e, and f. figure a represents a transition region that highlights the strained muscle (ms-bar). figure b demonstrates a transverse section of the first part of the stomach (close to transition region), showing cell variation between dorsal and ventral (curly bracket), including epithelium (e), submucosa (s), mascularis layers (m-bar), and gastric glands in lamina propria (arrows). figure c show dorsal folds in the stomach showing the epithelium cells (ec) and mucous glands (mg). figure d represents an intermediate part of the stomach that showing clear variation in the dorsal epithelium and epithelium cells compare with the first part. figure e shows folds in the ventral section of the stomach. figure f represents the epithelial and epithelial cells of the stomach from the dorsal region from intermediate towards the end of the stomach. 6 hojat ansari et al./ morphohistological characteristic of digestive tract of iranocichla hormuzensis (esmaeili et al., 2009). several unicuspid and serrated rows of teeth observed respectively in pharyngeal and oral jaws of i. hormozensis. the dentition of both oral and pharyngeal jaws are highly correlated with the foraging behavior of cichlid fishes (kocher, 2004; streelman and albertson, 2006). for example, the papilliforms pharyngeal teeth in herichthys minckleyi are specialized for herbivores to shred plants (darrin hulsey, 2006). in overall, morphology of oral jaw tooth ranged from unicuspid [observed in poscivorous, planktivorous and insectivorous species (e.g. afra cichlid, cynotilapia afra)] to tricuspid [stated for algal scrapers (e.g. blue mbuna, labeotrophenus fuelleborni)] in cichlid fishes (kocher, 2004; streelman and albertson, 2006). likewise, the morphology of teeth in i. hormuzensis with papilliforms pharyngeal teeth and bicuspid and tricuspid forms of teeth in the oral jaw is the norm for cichlid fishes. iranocichla hormuzensis have an outer raw of bicuspid and tricuspid teeth followed by several tricuspid rows as similar as described for zebra mbuna, metriaclima zebra (streelman and albertson, 2006) with exception of missing third cusp on the outer row of teeth in m. zebra. the morphology of teethes noticed in i. hormuzensis from both oral and pharyngeal jaw potentially justified by the fact that these parts assist in its generalist feeding habits. in esophagus, it is generally assumed that striated fibers predominate in anterior part, which then replaces with smooth muscle in posterior region (canan et al., 2012). nevertheless, this structure seems to be evolved with certain variation in some species (see canan et al., 2012), particularly in cichlid fishes, that esophagus consists of striated muscles across the entire region of its full length (e.g. gargiulo et al., 1996; morrison and wright, 1999; da silva et al., 2012). in support of our finding, a similar pattern figure 5. intestine of iranocichla hormuzensis: a, b, c, and d. figure a represent anterior intestine with mascularis externa (me) and tall folds (f) that are very close to each other. figure b shows anterior intestinal folds formed by a simple cylindrical epithelium including goblet cells (g). figure c represents an intermediate section of the intestine with short folds and a thick muscularis externa (me), goblet cells (g), serosa (s), mucosa (mu) and submucosa (sm). figure d demonstrates posterior intestine contains very short folds and smooth muscular fibers, arranged internally with a circular tunica (mc) and an outer longitudinal tunica (mi). 7 int. j. aquat. biol. (2020) 8(1): 1-9 previously described for esophagus morphology in other cichlid fishes (gargiulo et al., 1996; morrison and wright, 1999; da silva et al., 2012). position and histological structure (that the strained muscle predominated at the externa tunica) of the sphincter in i. hormuzensis suggests that this region potentially prevents direct food passes into intestine before chemical digestion in the stomach. in general, stomach in fish with huge variation in shape and structure is an organ that serves chemical food digestion (canan et al., 2012). the fact that i. hormuzensis has a small size stomach probably associated with two scenarios: first this organ provides a brief passage of food for chemical digestion (see da silva et al., 2012), and second presence of pharyngeal plates (as described by hahn and cunha, 2005). wall of stomach in i. hormuzensis showed specific structure that is the most interesting variation compare with previous studies, which perceived either three histologically distinct areas [cardiac, intermediary and pyloric (e.g. osman and caceci, 1991; morrison and wright, 1999) or a unique structure across the whole organ (see da silva et al., 2012). the presence of welldeveloped folds from the mucosa layer as well as their structural variation across dorsal and ventral sections, observed in the present study, may suggest an exclusive evolution towards adaptation in this species. as described by osman and caceci (1991) the mucosal folds would slow down the food passage and therefore provide sufficient time for acid digestion. the length of intestine in i. hormuzensis seems to be in the intermediate range and possibly related to its feeding habits. it is in accordance with other studies that highlighted a correlation between relative length of intestine and diet of species, which is in order of carnivores < omnivores < herbivores and detritivores (al-hussaini, 1949; fryer and iles, 1972; fugi et al., 2001; rodrigues and menin, 2008). the quantitative feature of digestion and food absorption are an expression of length of intestine as well as advanced structure of intestinal mucosa (manjakasy et al., 2009; canan et al., 2012). the complex and developed structure of intestinal mucosa with long and numerous folds, observed especially in the anterior part of i. hormuzensis intestine, probably involved in final digestion and also increase absorption processes in line with previous studies (manjakasy et al., 2009; canan et al., 2012; da silva et al., 2012). in addition, our finding that characterised a higher proportion of goblet cells in the first part of intestine (especially approximate part), support description by takashima and hibiya (1995). high variation in histological structure of digestive tract wall in fish compared with other vertebrates, illustrate their dynamic food habits (santos et al., 2011) constant environment changes (da silva et al., 2012) and different behavior and anatomical changes related to the feeding mechanism (kullander, 2003). consequently, different species of fish adapted to their habitat in response to environmental variation to increase their ability to take available food, thus optimize their fitness regarding environmental changes in their habitat. acknowledgments supports received from the members of kokherd village, especially e. divjam and h. dehdar, are gratefully acknowledged. references al-hussaini a.h. 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(2015) 3(2): 89-101 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article improvement of nutritive value of sesame oil cake in formulated diets for rohu, labeo rohita (hamilton) after bio-processing through solid state fermentation by a phytase-producing fish gut bacterium paramita das, koushik ghosh*1 aquaculture laboratory, department of zoology, the university of burdwan, golapbag, burdwan 713 104, west bengal, india. article history: received 24 september 2014 accepted 19 january 2015 available online 2 5 april 2015 keywords: sesame oilcake bacillus subtilis subsp. subtilis solid state fermentation labeo rohita fingerlings abstract: sesame oil cake (ssc) was bio-processed through solid state fermentation (ssf) under optimized conditions by a phytase-producing fish gut bacterium, bacillus subtilis subsp. subtilis (jx292128). ssf significantly reduced anti-nutritional factors (e.g., phytic acid, tannins and trypsin inhibitor) and crude fibre, while enhanced free amino acids, fatty acids and different minerals. phytase production (39.72 ± 1.06 u/g) during ssf was also recorded. along with a fish meal based reference diet (rd), 8 isonitrogenous (36% crude protein) and isocaloric (4.60 kcal g-1) experimental diets incorporating raw (r1-r4) and ssf processed (f1-f4) ssc(20%, 30%, 40% and 50%, w/w) were fed to rohu, labeo rohita fingerlings (mean weight 3.28 ± 0.15 g) in triplicate treatments for 70 days. in general, growth and feed utilization efficiencies in fish fed diets containing ssf-processed ssc were superior to the groups fed diets containing raw ssc. the diet f3 (40% fermented ssc) showed significantly (p<0.05) better result in terms of weight gain, feed conversion ratio, protein efficiency ratio, apparent net protein utilization, activities of digestive enzymes, carcass composition and apparent digestibility of protein, lipid and phosphorus. faecal phosphorus discharge reduced significantly (p<0.05) in fish fed fermented diets. the results indicated that incorporation of ssf-processed ssc might be practiced as a function to replace fish meal in the diets of l. rohita fingerlings. introduction sustainability of the growing aquaculture industry depends on the progressive reduction in wild fish inputs into fish feed (naylor et al., 2000). oil seed by-products might be the most promising alternative sources of protein and energy for formulating economic and environment-friendly aqua-feed (hardy, 2000). however, apart from deficiencies in the essential amino acids, the use of oil cakes has been restricted by the presence of some antinutritional factors (anfs), majority of which are polyphenols, trypsin inhibitors, non-starch polysaccharides and phytic acid (mandal and ghosh, 2009). phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakis dihydrogen phosphate) represents approximately 70-80% of the total phosphorus in plant seeds (lott et al., 2000). due to high density of * corresponding author: koushik ghosh e-mail address: kghoshbu@gmail.com negative charge, it can bind with mineral cations (na, k, mg, ca, zn, fe, cu, mn etc.) forming phytates, and also forms insoluble complexes with proteins and amino acids, thereby appears as a major anf diminishing the bioavailability and digestibility of the essential nutrients (kumar et al., 2011). fish cannot digest phytate compounds as they lack the intestinal phytase (pointillart et al., 1987). moreover, poor degradation of phytates leads to increased faecal phosphorus release and exerts detrimental effects on the aquatic environment like eutrophication (persson, 1991). therefore, endogenous phytate compounds reduce feed value of the protein rich oil cakes unless destroyed or inactivated. as evidenced by an upsurge in research reports, the influence of microbial phytase supplementation on 90 int. j. aquat. biol. (2015) 3(2): 89-101 protein digestion and utilization is a topic of recent interest. in contrast to the success achieved in farmed animals, dietary supplementation of microbial phytase in fish diets produced contradictory and inconsistent results. different authors have reported an increase (vielma et al., 1998; debnath et al., 2005a), no change (lanari et al., 1998) or even decrease (teskeredzic et al., 1995) in protein digestibility owing to phytase supplementation in the fish diets. the phytases have highest activity at two ph optima, i.e., 5.0-5.5 and 2.5 (simons et al., 1990). unlike the farmed animals (pig, poultry and swine, that do have an acidic ph within their gi tract), agastric (stomach less, e.g., carps) or even monogastric fishes do not have such ph ranges within their gut making the supplemented phytase either ineffective or less effective. alternately, pretreatment/processing of plant derived feed ingredients have been indicated to ameliorate feed utilization through deactivation of some anfs (ramachandran and ray, 2007). solid state fermentation (ssf) has been shown to reduce the phytate content in plant ingredients by phytases produced by the bacteria (khan and ghosh, 2013). therefore, microbial deactivation (through ssf) has been considered in the present study for removal of plant derived phytate and other anfs. after oil extraction, processing of the oil cakes through ssf might pretend great economic feasibility to the agro-based oil production sectors providing an eco-friendly way of nutrient recycling. efficacy of the fermented oil seed meals for partial or complete substitution of fishmeal has been suggested by several authors (ramachandran et al., 2005). as likely incorporation of harmful metabolites during the ssf process cannot be ruled out, the use of autochthonus fish gut microorganisms might be reasonable in processing of plant feed stuffs for likely use in fish feed (mandal and ghosh, 2013). in this context, the major aim of the presently reported study was reclamation of plant ingredients into value added products. de-oiled sesame oil cake (ssc) is rich source of protein and minerals, such as calcium and phosphorus (salunkhe et al., 1991). the seeds were reported to contain 25% protein that is rich in methionine and tryptophan (godin and spensley, 1971). the specific objectives of the present study were value addition of ssc through bio-processing with a phytase-producing fish gut bacterium, bacillus subtilis subsp. subtilis (jx292128) and to appraise nutritive value of the bio-processed ssc with partial replacement of fish meal and other conventional ingredients in formulated diets for rohu, labeo rohita fingerlings. materials and methods bacteria culture and optimization of solid-state fermentation parameters: the extracellular phytaseproducing bacterium bacillus subtilis subsp. subtilis (genbank accession no: jx292128) was isolated from the gut of a freshwater carp, cirrhinus mrigala (das and ghosh, 2013). the culture was grown and maintained on selective modified phytase screening media (mpsm) with minor modifications (howson and davis, 1983). inoculum was prepared from a freshly raised 5-d-old slant culture in mpsm broth grown at 35°c for 48 hrs. the inoculant thus obtained contained 4.8 × 107 cells ml-1. optimization of ssf parameters influencing phytate degradation in the ssc was done following khan and ghosh (2013) and das and ghosh (2014) to detect processing conditions of the ssc. the parameters studied were: initial moisture content of the substrate (10%-90%), initial ph of the moistening media (3-8), incubation temperature (25°c-50°c), inoculum volume (1-5%, v/w), different surfactants (tween20, tween40, tween 80, dmso, sds; 1%, v/w) and nacl supplementation (1-5%, w/w). further, the medium was supplemented with different carbon sources (1%, w/v) (glucose, sucrose, lactose, maltose and starch) and different organic and inorganic nitrogen sources (1%, w/v) (peptone, tyrosine, tryptophane, ammonium sulfate, ammonium nitrate and yeast extract). impact of additional carbon and nitrogen sources were further optimized by incorporating the best source at varying levels (1-5%, w/w). finally, a time course study was conducted to optimize the 91 das and ghosh/ use of bio-processed sesame oil cake in carp diets duration of fermentation (2-12 days) incorporating the optimized physico-chemical parameters. to determine extracellular phytase production by the bacteria during ssf enzyme extraction from the fermented material was carried out following khan and ghosh (2013) and quantitative phytase assay of the crude enzyme was done after yanke et al. (1999) using sodium phytate as the substrate. one phytase unit (u) was defined as the amount of enzyme per ml of supernatant that released 1 µg of inorganic phosphorus per minute. enzyme yield was expressed as u/g (gram dry substrate). analysis of proximate composition, minerals and antinutrients: proximate composition of the raw and fermented ssc were analysed following the standard methods of association of official analytical chemists (aoac, 1990): crude protein (n% × 6.25) by micro kjeldahl digestion and distillation, lipid was determined by extracting the residue with 4060°c petroleum ether in a soxhlet apparatus, crude fiber was determined as loss on ignition of dried lipid free residue after digestion with 1.25% h2so4 and 1.25% naoh and ash was determined by ignition at 550ºc in a muffle furnace to constant weight. total free amino acids and fatty acids were measured according to moore and stein (1948) and cox and pearson (1962), respectively. the mineral elements were analysed by atomic absorption spectrophotometer (perkin elmer aanalyst 700) using standard reference chemicals. na and k were analysed by flame photometry. calcium and phosphorus were estimated by biochemical methods as described by oser (1971). among the anfs, tannin content in both fermented and raw ssc was determined using folin-denis reagent (schanderi, 1970) and phytic acid content determined according to vaintraub and lapteva (1988). trypsin inhibitor activity was determined according to smith et al. (1980). formulation and processing of experimental diets: eight isonitrogenous (36% crude protein) and isocaloric (4.60 kcal g-1) experimental diets were formulated using raw (r1-r4) and fermented (f1f4) ssc at 20%, 30%, 40% and 50% levels (w/w) replacing fish meal and other conventional ingredients. a diet with fish meal as the main protein source was used as the reference diet (rd). each feed was formulated separately using winfeed 2.8 software. composition of the experimental diets has been presented in table 2. diets were prepared as described by saha and ghosh (2013). experimental design: the feeding trial was conducted under laboratory condition, in 27 glass aquaria, each containing 90-l of water, for 70 days, with continuous aeration. rohu, l. rohita fingerlings were obtained from a local fish farm and acclimatized for 15 days. the fingerlings (mean individual weight of the 405 fingerlings: 3.28 ± 0.15 g) were randomly distributed in the glass aquaria at a stocking density of 15 fish per aquarium with three replicates for each experimental diet. the fish were fed twice daily: at 07.00 h and 13.00 h, at a feeding rate of 3% of the total body weight per day. the daily ration was adjusted every tenth day after weighing the fish from each replicate. the uneaten feed was siphoned off 6 hrs after each feeding, and oven dried at 100°c for 24 hrs to calculate the feed conversion ratio. the uneaten feeds remained almost intact due to the binder used (carboxy-methyl-cellulose, cmc) during preparation of experimental diets. the faecal samples released by the fish were collected daily from each aquarium by pipetting (spyridakis et al., 1989). the oven dried (60°c) faecal samples were analysed for digestibility estimation. the water quality parameters, viz., temperature (°c), ph and dissolved oxygen content (mg l-1) from each experimental set were monitored at regular intervals following the standard methods of american public health association (apha, 1998). the ranges of water quality parameters were 29-31°c, ph 7-7.5, dissolved oxygen 6.3-6.8 mg l-1 and alkalinity 148153 mg l-1 (n=10). chemical analysis: the proximate composition of the feed ingredients, experimental diets, faecal samples and fish carcass were analysed both prior to commencement, and on termination of experiment by following the standard methods of aoac (1990) as described previously. five fish from each 92 int. j. aquat. biol. (2015) 3(2): 89-101 aquarium were sampled at the termination of the feeding experiment; they were homogenised and analysed for whole body (carcass) composition (on wet weight basis). chromic oxide in diets and faecal samples were estimated following the method of bolin et al. (1952). apparent dry matter or total and nutrient digestibility values, apparent protein digestibility (apd%), apparent lipid digestibility (ald%) and apparent phosphate digestibility (aphd%) were calculated after de silva and anderson (1995). specific growth rate (sgr, % day1), feed conversion ratio (fcr), protein efficiency ratio (per) and apparent net protein utilization (anpu%) were calculated using standard methods (steffens, 1989). assay of digestive enzymes: α-amylase activity was determined following the dinitro-salicylic-acid (dnsa) method described by bernfeld (1955). amylase activity was expressed as mg maltose liberated h-1 mg protein-1. protease activity was determined by the casein digestion method of walter (1984). one unit of enzyme activity was defined as µg of tyrosine liberated h-1 mg protein-1. lipase activity was measured following the method described by bier (1955). lipase activity was expressed as µ mole of fatty acid liberated h-1 mg protein-1. statistical analysis: all experiments were performed in triplicate and the mean values were reported along with standard error (mean ± se, n=3). statistical analysis of the data was performed by analysis of variance (anova). mean difference between treatments were tested for significance at p<0.05 and comparisons were made by tukey’s test following zar (1999) to find out which treatment differed significantly from the other in respect of growth, carcass composition, digestibility, profiles of digestive enzymes and general performance of the fish. all the statistical analyses were done using spss ver11 (kinear and gray, 2000) software. results the results revealed that phytate content in the ssc decreased from 2.58 ± 0.05 to 1.02 ± 0.04 g 100 g-1 dry weight (75.25% reduction) after 8 days through ssf under optimized conditions, i.e., 60% initial moisture content, ph 6, 35°c temperature, 3.5% (v/w) inoculum volume, and supplementation of tween 80 (1%, v/w), nacl (4%, w/w), starch (4%, w/w) and ammonium sulphate (3%, w/w). maximum phytase production (39.72 ± 1.06 u/g) was also recorded after 8 days (fig. 1). data pertaining proximate compositions of nutrients and anfs (tannin, phytate and trypsin inhibitor) in raw and processed ssc are summarized in the table 1. there were marginal increase (t-value significant at p<0.05) in the contents of protein, lipid, and minerals (na, k, ca, mg, zn, fe, cu, p and mn) in the ssc after fermentation at optimal conditions by the fish gut isolate b. subtilis subsp. subtilis (jx292128). ingredient composition and proximate analysis of the experimental diets are presented in the table 2. in comparison to the diets containing raw ssc, the contents of the anfs (tannin, phytic acid and trypsin inhibitor) were lower in the diets containing fermented ssc. although, average final weight of the fish increased considerably from the initial value in all the dietary treatments, the results clearly established that inclusion of the bio-processed oil cake in diets improved overall growth performance and nutrient utilization in l. rohita fingerlings (table figure 1. phytate degradation in sesame oil cake and phytase production during solid state fermentation by b. subtilis subsp. subtilis. 93 das and ghosh/ use of bio-processed sesame oil cake in carp diets 3). the performance of fish in terms of average live weight gain (%), specific growth rate (sgr, % day1) and protein efficiency ratio (per) increased significantly (p<0.05) up to 40% incorporation (diet f3) of the ssf processed ssc and thereafter decreased. values for anpu and fcr were the best for fish fed the diet f3, and worst for the diet r4 containing 50% raw ssc. figure 2 depicts apparent digestibility of dry matter, protein, lipid and phosphate in l. rohita fed experimental diets. a progressive decline in the digestibility parameters with increasing level of raw ssc was observed in the present study. in comparison to all of the experimental diets, significantly (p<0.05) higher values for the digestibility parameters were noticed with the fish fed diet f3, while, apparent lipid digestibility (ald) values did not differ significantly (p<0.05) between the diets f3 and f4. faecal p concentrations in fish fed different experimental diets are presented in figure 3. the highest faecal p concentration was associated in the fish fed diet r4, whereas, the lowest value was noticed in the fish fed diet f3. higher phosphate digestibility was allied with significantly lower faecal p output in the groups of fish fed fermented ssc incorporated diets than in fish fed raw ssc incorporated diets. parameters raw ssc ssf processed ssc % increase (↑) / reduction (↓) nutrients crude protein 41.75 ± 0.03 46.57 ± 0.04 11.54 ↑ crude lipid 7.2 ± 0.04 8.9 ± 0.02 23.61 ↑ crude fibre 4.23 ± 0.04 2.23 ± 0.04 47.28↓ crude ash 5.47 ± 0.03 6.8 ± 0.04 24.31↑ total free fatty acid 1.02 ± 0.03 1.5 ± 0.02 47.05↑ total free amino acid 0.78 ± 0.05 1.51 ± 0.04 93.58↑ antinutrional factors tannin (mg/g) 2.8 ± 0.03 0.61 ± 0.03 68.21↓ phytate (g %) 2.58 ± 0.05 1.02 ± 0.04 75.25↓ trypsin inhibitor (mg/g) 9.21 ± 0.04 2.61 ± 0.03 71.66↓ minerals na (mg /g) 0.98 ± 0.51 1.14 ± 0.43 16.32↑ k (mg /g) 10.25 ± 0.43 12.45 ± 0.23 21.46↑ ca (ppm) 0.78 ± 0.31 0.95 ± 0.42 21.79↑ mg (mg /g) 3.11 ± 0.53 4.51 ± 0.21 44.61↑ zn (mg /g) 1.22 ± 0.41 1.47 ± 0.55 20.49↑ fe (ppm) 9.51 ± 0.22 11.35 ± 0.31 19.31↑ cu (mg/g) 11.71± 0.9 14.38 ± 0.63 22.80↑ p (mg/g) 4.05 ± 0.51 4.33 ± 0.26 6.91 ↑ mn (ppm) 11.86 ± 0.11 13.45 ± 0.9 13.40 ↑ values are mean ± s.e of five determinations. figures in parentheses indicate the percent increase (↑)/decrease (↓) over the values of the corresponding raw oil cakes. table 1. proximate composition of nutrients, anti-nutritional factors and minerals (% dry matter) in raw and fermented sesame oil cake. figure 2. apparent digestibility of dry matter and nutrients (%) of labeo rohita fingerlings fed experimental diets for 70 days (error bars show deviation among three replicates). 9 4 in t. j . a q u at . b io l. ( 2 0 1 5 ) 3 (2 ): 8 9 -1 0 1 p a r a m e te r s r d d ie ts w it h r a w s s c d ie ts w it h b io -p r o c e ss e d s s c r 1 r 2 r 3 r 4 f 1 f 2 f 3 f 4 in g r e d ie n t c o m p o si ti o n f is h m e a l 3 5 3 0 2 6 2 3 2 0 3 0 2 6 2 3 2 0 m u st a rd o il c a k e 3 0 1 7 1 3 1 2 7 1 7 1 3 1 2 7 r ic e b ra n 3 2 3 0 .0 2 8 .0 2 2 .0 2 0 .0 3 0 .0 2 8 .0 2 2 .0 2 0 .0 s e sa m e o il c a k e 2 0 3 0 4 0 5 0 2 0 3 0 4 0 5 0 c o d l iv e r o il 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 p re m ix 1 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 c h ro m ic -o x id e 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 1 .0 p r o x im a te c o m p o si ti o n d ry m a tt e r 9 4 .5 7 ± 0 .5 8 9 4 .3 2 ± 0 .6 4 9 3 .9 4 ± 0 .6 6 9 3 .6 1 ± 0 .7 8 9 3 .8 5 ± 0 .4 6 9 4 .3 2 ± 0 .6 4 9 3 .9 4 ± 0 .6 6 9 3 .6 1 ± 0 .7 8 9 3 .8 5 ± 0 .4 6 c ru d e p ro te in 3 6 .5 7 ± 1 .0 8 3 6 .2 3 ± 1 .0 5 3 5 .2 5 ± 1 .0 2 3 5 .1 8 ± 1 .0 3 3 6 .4 1 ± 0 .9 5 3 6 .5 3 ± 0 .8 9 3 5 .8 5 ± 0 .7 5 3 6 .8 7 ± 0 .9 6 3 6 .2 1 ± 0 .8 4 c ru d e l ip id 7 .4 5 ± 0 .2 9 7 .2 9 ± 0 .2 4 7 .4 5 ± 0 .3 3 7 .4 1 ± 0 .1 8 7 .4 3 ± 0 .2 7 7 .4 9 ± 0 .3 8 7 .2 7 ± 0 .2 7 6 .9 5 ± 0 .3 5 7 .3 9 ± 0 .4 1 a sh 1 1 .8 5 ± 0 .6 2 1 1 .3 4 ± 0 .6 1 1 0 .6 2 ± 0 .4 3 1 0 .2 4 ± 0 .5 5 1 0 .1 1 ± 0 .5 4 1 1 .7 5 ± 0 .3 2 1 1 .2 4 ± 0 .2 4 1 0 .4 9 ± 0 .4 3 1 0 .5 5 ± 0 .3 2 c ru d e f ib re 6 .7 5 ± 0 .4 1 6 .9 4 ± 0 .5 5 6 .4 1 ± 0 .2 8 6 .1 5 ± 0 .3 5 5 .8 1 ± 0 .3 3 5 .9 5 ± 0 .6 3 6 .3 9 ± 0 .2 5 6 .8 1 ± 0 .4 5 6 .9 1 ± 0 .3 5 n f e 2 2 7 .7 9 ± 0 .4 3 2 6 .5 7 ± 0 .4 1 2 8 .3 6 ± 0 .3 5 2 8 .9 1 ± 0 .3 5 2 7 .4 1 ± 0 .4 2 6 .5 7 ± 0 .4 1 2 8 .3 6 ± 0 .3 5 2 8 .9 1 ± 0 .3 5 2 7 .4 1 ± 0 .4 g ro ss e n e rg y ( k c a l g -1 ) 4 .6 5 ± 0 .3 5 4 .4 7 ± 0 .3 1 4 .4 5 ± 0 .2 9 4 .3 7 ± 0 .2 5 4 .4 8 ± 0 .2 8 4 .6 1 ± 0 .2 3 4 .6 7 ± 0 .3 1 4 .8 2 ± 0 .3 6 4 .7 5 ± 0 .3 5 t a n n in 0 .7 1 ± 0 .0 3 0 .8 9 ± 0 .0 5 1 .2 1 ± 0 .0 3 1 .3 8 ± 0 .0 5 0 .4 5 ± 0 .0 2 0 .5 2 ± 0 .0 3 0 .6 2 ± 0 .0 1 0 .5 7 ± 0 .0 2 p h y ti c a c id 0 .6 7 ± 0 .0 3 0 .8 4 ± 0 .0 1 1 .1 8 ± 0 .0 3 1 .4 1 ± 0 .0 5 0 .0 7 ± 0 .0 1 0 .0 6 ± 0 .0 3 0 .0 9 ± 0 .0 2 0 .0 8 ± 0 .0 2 t ry p si n i n h ib it o r (m g /g ) 2 .7 6 ± 0 .0 5 3 .7 1 ± 0 .0 3 5 .1 2 ± 0 .0 4 6 .3 1 ± 0 .0 6 0 .8 8 ± 0 .0 4 1 .2 5 ± 0 .0 3 1 .4 7 ± 0 .0 2 2 .8 1 ± 0 .0 3 p h o sp h a te 9 4 .5 7 ± 0 .5 8 9 4 .3 2 ± 0 .6 4 9 3 .9 4 ± 0 .6 6 9 3 .6 1 ± 0 .7 8 9 3 .8 5 ± 0 .4 6 9 4 .3 2 ± 0 .6 4 9 3 .9 4 ± 0 .6 6 9 3 .6 1 ± 0 .7 8 9 3 .8 5 ± 0 .4 6 v al u es a re m ea n s ± s e o f th re e d et er m in at io n . 1 v it am in a n d m in er al m ix tu re ( s u p ra d y n , b a y er c o n su m er c ar e a g , b as el , s w it ze rl an d ). 2 n it ro g en -f re e ex tr ac t; r d = r e fe re n ce d ie t t ab le 2 . in g re d ie n t co m p o si ti o n a n d p ro x im at e co m p o si ti o n ( o n % d ry m at te r b as is ) o f th e ex p er im e n ta l d ie ts . 9 5 d as a n d g h o sh / u se o f b io -p ro ce ss ed s es am e o il c ak e in c ar p d ie ts p a ra m e te rs r d d ie ts w it h r a w s s c d ie ts w it h b io -p ro c e ss e d s s c r 1 r 2 r 3 r 4 f 1 f 2 f 3 f 4 in it ia l w t (g ) 3 .0 5 ± 0 .0 6 3 .0 3 ± 0 .0 5 3 .0 5 ± 0 .0 4 3 .0 4 ± 0 .0 6 3 .0 2 ± 0 .0 3 3 .0 3 ± 0 .0 4 3 .0 5 ± 0 .0 6 3 .0 2 ± 0 .0 7 3 .0 3 ± 0 .0 5 f in a l w t (g ) 6 .3 5 ± 0 .0 7 e 5 .9 8 ± 0 .0 5 d 5 .6 1 ± 0 .0 4 c 5 .3 2 ± 0 .0 5 b 5 .1 1 ± 0 .0 6 a 6 .0 5 ± 0 .0 5 d 6 .2 9 ± 0 .0 4 e 6 .5 3 ± 0 .0 8 f 6 .3 8 ± 0 .0 6 e f w e ig h t g a in ( % ) 1 0 8 .1 9 ± 3 .5 1 e 9 7 .3 5 ± 3 .2 2 d 8 3 .9 3 ± 2 .6 7 c 7 5 .0 1 ± 3 .1 1 b 6 9 .2 0 ± 2 .8 5 a 9 9 .7 6 ± 2 .7 5 d 1 0 6 .2 2 ± 3 .4 7 e 1 1 6 .2 2 ± 3 .6 2 f 1 1 0 .5 7 ± 3 .4 3 e f e e d i n ta k e # 1 .9 1 ± 0 .0 8 a 1 .9 3 ± 0 .0 6 a 1 .9 6 ± 0 .0 5 b 1 .9 9 ± 0 .0 6 b 2 .0 1 ± 0 .0 4 c 2 .0 3 ± 0 .0 3 c 2 .0 6 ± 0 .0 7 d 1 .9 8 ± 0 .0 9 b 2 .0 5 ± 0 .0 6 c s g r ( % d a y -1 ) 4 .7 1 ± 0 .0 6 f 4 .2 1 ± 0 .0 5 d 3 .6 5 ± 0 .0 4 c 3 .2 5 ± 0 .0 3 b 2 .9 8 ± 0 .0 5 a 4 .3 1 ± 0 .0 4 c 4 .6 2 ± 0 .0 3 e 5 .0 1 ± 0 .0 6 g 4 .7 8 ± 0 .0 5 f f c r 2 .4 1 ± 0 .0 6 b 3 .2 9 ± 0 .0 4 e 3 .5 ± 0 .0 3 f 3 .8 8 ± 0 .0 2 g 4 .3 1 ± 0 .0 4 h 3 .1 1 ± 0 .0 5 d 2 .5 6 ± 0 .0 3 c 2 .3 2 ± 0 .0 8 a 2 .4 3 ± 0 .0 5 b p e r 1 .3 0 ± 0 .0 5 e 1 .1 7 ± 0 .0 3 c 1 .0 2 ± 0 .0 2 b 0 .9 6 ± 0 .0 5 b 0 .6 5 ± 0 .0 3 a 0 .9 8 ± 0 .0 4 b 1 .2 5 ± 0 .0 2 d 1 .4 6 ± 0 .0 7 f 1 .3 3 ± 0 .0 6 e a n p u ( % )$ 2 0 .5 1 ± 0 .5 8 d 1 5 .9 5 ± 0 .6 1 b 1 5 .2 7 ± 0 .5 2 b 1 2 .6 3 ± 0 .4 9 a 1 0 .7 1 ± 0 .4 8 a 1 7 .4 7 ± 0 .4 7 c 1 8 .6 9 ± 0 .6 3 c 2 2 .6 5 ± 0 .6 5 e 2 0 .5 7 ± 0 .5 6 d v al u es a re m ea n s ± s e o f th re e d et er m in at io n s. m ea n v al u e w it h s am e su p er sc ri p ts i n t h e sa m e ro w a re n o t si g n if ic an tl y d if fe re n t (p < 0 .0 5 ). # g 1 0 0 g -1 b o d y w ei g h t o f fi sh d ay -1 . $ a n p u = ( n et i n cr ea se i n c ar ca ss p ro te in /a m o u n t o f p ro te in c o n su m e d ) × 1 0 0 d ie ts p r o te a se a c ti v it y * a m y la se a c ti v it y # l ip a se a c ti v it y $ in it ia l 1 2 .4 5 ± 0 .2 1 a 7 .1 4 ± 0 .2 7 a 9 .7 4 ± 0 .1 6 a r d 1 7 .6 1 ± 0 .1 9 e 1 2 .3 5 ± 0 .2 5 e 1 4 .3 5 ± 0 .1 9 d r 1 1 6 .3 1 ± 0 .1 8 d 1 1 .2 6 ± 0 .2 9 d 1 3 .3 9 ± 0 .2 1 c r 2 1 5 .5 3 ± 0 .2 3 c 1 0 .6 3 ± 0 .2 3 c 1 3 .1 2 ± 0 .2 5 c r 3 1 5 .1 9 ± 0 .1 7 c 1 0 .4 1 ± 0 .2 1 c 1 2 .3 1 ± 0 .2 7 b r 4 1 3 .4 7 ± 0 .2 5 b 9 .3 5 ± 0 .1 8 b 1 1 .3 9 ± 0 .2 2 a f 1 1 6 .5 5 ± 0 .1 7 d 1 1 .3 6 ± 0 .2 2 d 1 4 .5 1 ± 0 .1 8 d f 2 1 7 .4 8 ± 0 .2 2 e 1 2 .6 1 ± 0 .1 7 e 1 5 .2 3 ± 0 .1 6 d f 3 1 8 .3 3 ± 0 .1 6 f 1 4 .2 6 ± 0 .1 9 f 1 6 .8 2 ± 0 .1 9 e f 4 1 7 .8 5 ± 0 .2 1 e f 1 3 .4 7 ± 0 .1 8 f 1 6 .4 1 ± 0 .2 1 e v al u es w it h t h e sa m e su p er sc ri p t in t h e sa m e co lu m n a re n o t si g n if ic a n tl y d if fe re n t (p < 0 .0 5 ); * µ g o f ty ro si n e li b er at e d h -1 m g p ro te in -1 ; # m g m al to se l ib er at ed h -1 m g p ro te in -1 ; $ µ m o le o f fa tt y a ci d l ib er at e d h -1 m g p ro te in -1 t ab le 3 . g ro w th p er fo rm a n ce s an d f ee d u ti li za ti o n e ff ic ie n ci es i n l ab eo r o h it a fi n g er li n g s fe d e x p er im en ta l d ie ts f o r 7 0 d a y s. t ab le 4 . a ct iv it ie s o f p ro te as e, a m y la se a n d l ip as e in t h e g u t o f l ab eo r o h it a fi n g er li n g s fe d e x p er im en ta l d ie ts f o r 7 0 d a y s. 96 int. j. aquat. biol. (2015) 3(2): 89-101 proximate compositions of the carcass in fish fed various experimental diets are presented in figure 4. although all the fish were fed isonitrogenous diets, the deposition of carcass protein and lipid was significantly higher in fish fed bio-processed ssc incorporated diets than the reference diet, and an increasing level of raw ssc was associated with a decrease in carcass protein and lipid contents. the highest values for protein gain and lipid accumulation in the carcass was recorded in the group of fish reared on diet f3. carcass ash content was also revealed the highest value in fish fed diet f3. activities of intestinal protease, amylase and lipase in l. rohita fingerlings fed experimental diets are presented in table 4. in general, activities of all the three enzymes were significantly (p<0.05) higher in the fish fed diets containing fermented ssc as compared to the fish fed diets containing raw ssc. maximum protease, amylase and lipase activities were noticed in the fish fed diet f3, though it was not significantly (p<0.05) different from the diet f4. discussion the present investigation was intended to assess the effectiveness of a phytase-producing fish gut bacterium, b. subtilis subsp. subtilis (jx292128) in improving the nutritive value of sesame (sesamum indicum) oil cake (ssc) under ssf. the results showed that fermentation was effective in reducing the crude fibre content and the anfs, such as tannins, phytic acid and trypsin inhibitor, and enhancing available free amino acids and fatty acids. in the present study, increased level of crude protein, free amino acids and free fatty acids in fermented ssc in comparison to the raw oil cake is consistent to the findings of roy et al. (2013). in vitro processing by autochthonus microorganisms might be assumed as an effective strategy as the organism itself and their metabolites would not cause harm to the fish providing the basis for mutual relationship (khan and ghosh, 2013). suitability of the ssf processed ssc as an alternate plant derived feed ingredient has been evaluated in the formulated diets for rohu, l. rohita fingerlings. the results revealed that inclusion of the bioprocessed oil cake in diets improved significantly overall growth performance and nutrient utilization in l. rohita fingerlings in terms of average live weight gain, sgr and per up to 40% incorporation of the ssf processed ssc and thereafter decreased. the results might indicate that inclusion of the 40% fermented oil cake in the diet was optimal for augmenting the bioavailability of nutrients in l. rohita fingerlings. reports on the effectiveness of dietary microbial phytase and/or phytase pretreatment were contradictory, as some authors could not detect significant effect in diverse fish species figure 4. proximate carcass composition (% wet weight) of experimental fish at the end of the 70 days feeding trial (initial values: moisture, 83.48 ± 0.51; crude protein, 10.35 ± 0.35; crude lipid, 3.3 ± 0.07 and ash, 3.1 ± 0.06). figure 3. faecal phosphorus concentration of labeo rohita fingerlings fed experimental diets for 70 days (error bars show deviation among three replicates). 97 das and ghosh/ use of bio-processed sesame oil cake in carp diets fed plant-based diets (cain and garling, 1995; sajjadi and carter, 2004). diet composition, methods of phytase pre-treatment/application and rearing conditions may be closely associated with the inconsistency of the experimental results. however, the present study evidenced positive effect of microbial phytase on the growth performance and nutrient utilization in rohu fingerlings, which were consistent with the results of other researchers (vielma et al., 2002; debnath et al., 2005a; liebert and portz, 2005; sardar et al., 2007; roy et al., 2013). the treatment of fish feed with phytase has been reported to result in improvement of protein digestibility and retention in fish (cheng and hardy, 2002; debnath et al., 2005a, b; baruah et al., 2005). a declining trend in apparent protein digestibility (apd) values had also been reported previously with higher levels of raw plant ingredient inclusions in carp diets (ramachandran and ray, 2007; saha and ghosh, 2013; roy et al., 2013). in the present study, fermentation of ssc by phytase-producing fish gut bacteria resulted in increased phytate hydrolysis enhancing availability of protein and minerals that are chelated by phytate. not only protein, the apparent lipid digestibility also increased significantly in fish fed fermented ssc incorporated diets in comparison with the diets with raw ssc. indeed, sesame proteins are amphiphilic globulins whose functional property of absorbing fat may reduce lipid digestibility in case of raw ssc incorporated diets (johnson et al., 1979). an essential mineral nutrient for fish is phosphorus (p), a vital component of the skeletal system. it plays an important role in energy and cell metabolism, including synthesis of nucleic acids, phospholipids and some major enzymes (luo et al., 2010). apparent phosphate digestibility is one of the most sensitive criteria for assessing the influence of phytase on minerals utilization in fish (sajjadi and carter, 2004). in the present study, apparent phosphate digestibility increased with incorporation of fermented ssc, confirming the established properties of phytase with respect to dietary phosphorus availabilities. the increase in phosphate digestibility is in accordance with other studies carried out in common carp (nwanna et al., 2008) and rohu juveniles (baruah et al., 2005). in the present study, increased incorporation of fermented oil cake was associated with decreased faecal p output, which was in accordance with the observations made by vielma et al. (2002) and sugiura et al. (2001), who opined that addition of phytase in rainbow trout, oncorhynchus mykiss diets reduced the faecal p excretions up to 95-98% compared with the fish fed diets without phytase. addition of microbial phytase has been reported to be effective in improving bioavailability of phytate phosphorus due to hydrolysis of phytate to orthophosphate by phytase (reddy et al., 1982) making the chelated phosphorus available to fish resulting in less faecal excretion (baruah et al., 2004). the results of the presently reported study indicated that bacterial phytase was effective in enhancing the bioavailability of p considerably, thereby reducing the p output in the faeces. in the present study, the proximate carcass composition, i.e., moisture, crude protein, ash and crude lipid, of the experimental fish was significantly influenced by the level of incorporation of raw and fermented ssc in the diets. phytate forms compounds with a large number of minerals and also forms complexes with proteins and amino acids, thereby reduces bioavailability of minerals and decrease digestibility of proteins as phytate-protein and protein-mineral complexes are resistance to proteolytic digestion (kumar et al., 2011). this results in lowering the gastrointestinal absorption of protein and other nutrients (debnath et al., 2005a, b). sesame seed α-globulin and sodium phytate use to form complex through a bi-phasic reaction (rajendran and prakash, 1993). at the first step phytase binds protein through strong electrostatic attractions, which is followed at the next step by slower protein-protein interactions ensuing precipitation of the protein-phytate complex. consequently, protein utilization in fish has been reported to be reduced by phytate (nang thu et al., 98 int. j. aquat. biol. (2015) 3(2): 89-101 2011). it was apparent in the present study that the bacterial phytase could prevent the formation of protein-phytate complexes to some extent by prior hydrolysis of the phytate complex through the ssf process making nutrients and minerals bio-available for growth. therefore, improvement in growth performance and carcass composition of rohu fingerlings could be attributed to enhanced release of the nutrients. improved ash contents in the fish carcass indicated better mineral deposition in the fish fed ssf processed ssc incorporated diets. it has been reported that the addition of microbial phytase enhances the availability of various minerals from the plant oilseed meals and improves their absorption (debnath et al., 2005b; cao et al., 2008). the results indicated that the fish was able to digest the nutrients from the diets containing fermented seed meal more efficiently. decreased protease activities with increased raw ssc in the diets might correspond to decrease in protein availability from ssc. similar results have been documented by krogdahl et al. (1994), who concluded that proteases might be highly sensitive to plant anfs. the decrease in protease activity at higher inclusion levels of raw ssc might be caused by the presence of the anfs like tannin and phytic acid. moreover, activity of the digestive enzymes in fermented sscfed groups comparable with the rd-fed group might correlate with improved nutrient availability and decreased anfs in the fermented ssc. conclusions the present study demonstrated an inclusion level of fermented ssc (up to 40%) in the practical diet for l. rohita fingerlings without any adverse effect on growth and feed utilization efficiencies. on the other hand, the study highlighted that dietary incorporation of bio-processed ssc improved carcass composition and apparent phosphate digestibility in l. rohita fingerlings. thus, preparation of fish feed incorporating ssc after processing through ssf by phytase-producing fish gut bacteria might be expected to provide both economic and environmental benefits through decreased expenditures on supplemental minerals and mineral outputs to the aquatic ecosystem. however, further experimentation in the field condition with large number of fish and replication are essential prior to recommend it to the aquaculture industry. acknowledgements this research was supported by the university grants commission (ugc), new delhi, india [major research project f. no. 37–383/2009(sr)]. research facilities provided by the department of zoology, the university of burdwan, west bengal, india, the department of science and technology (fist programme) and the university grants commission (ugc-sap-drs programme) are also gratefully acknowledged. references aoac (1990). official methods of analysis. v.a. arlington (16th ed), association of official analytical chemist, new york, 1134 p. apha (1998). standard methods for the examination of water and wastewater, 20th edition. in: l.s. clesceri, a.e. greenberg, a.d. eaton (eds.). american public health association, american water works association, water environment federation. washington. dc, usa, 1220 p. baruah k., pal a.k., sahu n.p., jain k.k., mukherjee s.c., debnath d. 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(2021) 9(3): 187-199 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article strategies to control invasion of sailfin armoured catfish, pterygoplichthys spp. in wastewater-fed aquaculture bheries of east kolkata wetland, india with suggestion of a modified barrier based on the biological and behavioural characteristics ajmal hussan* 1, rathindra nath mandal1, farhana hoque1, jitendra kumar sundaray2, arabinda das1, partha pratim chakrabarti1, subhendu adhikari1, uday kumar udit2, gourab choudhury1, bindu raman pillai2 1regional research centre, icar-central institute of freshwater aquaculture, rahara, kolkata700118, west bengal, india. 2icar-central institute of freshwater aquaculture, kausalyaganga, bhubaneswar-751002, odisha, india. s article history: received 7 june 2020 accepted 21 august 2020 available online 2 5 june 2021 keywords: alien species control eradication wetland catfish abstract: sailfin armoured catfish (pterygoplichthys spp.), an alien invasive species of family loricariidae has invaded extensively in wastewater-fed large aquaculture ponds (locally called ‘bheries’) of east kolkata wetlands (ekw), west bengal, india. as there is no viable controlling method at present, commonly these fishes are removed by different physical methods and discarded. in the present study, we investigated the effectiveness and suitability of different in-practice pterygoplichthys spp. control methods, based on on-field sampling, biological and behavioural study of the fish and also response analysis of the stakeholder’s of ekw. the results indicate that inpractice eradication efforts, like ‘repeated seine netting’ with or without removal of eichhornia sheath of the pond periphery and ‘dewatering of pond’ aiming to reduce or eradicate pterygoplichthys spp., are not fully effective, because of the capture avoidance ability and burrowing habit of these fishes. we found deep and branching burrows of pterygoplichthys spp. in aquaculture ponds of ekw, with maximum burrow depth of 58 cm, and water in that burrows even after 12 days of dewatering. hence, it is suggested stakeholders to keep dewatered pond exposed to sunlight for at least four weeks or above to ensure complete water-out from the burrows in which pterygoplichthys spp. take shelter or lay their eggs. ‘multilayer bamboo fencing’ or ‘combination of bamboo fencing and net barrier’ use by the stakeholders of ekw to prevent intrusion or re-intrusion of pterygoplichthys spp. were found only partially effective, because of the capability of these fishes to damage net-blocking through their hard dorsal and pectoral spines or entry through the holes dug across the barrier in beneath or banks of the sewage intake channel. based on learning on the biological and behavioural characteristics of pterygoplichthys spp., we then suggested a modified version of barrier to the stakeholder’s of ekw, incorporating a sewage feeder pipeline, a concrete collection chamber with size separation arrangement made of hard materials like wire mesh and a dam of specific dimensions across the channel, for effective prevention of intrusion of these fishes in their aquaculture bheries. introduction convention on biological diversity (2014) defined invasive species as “species that their introduction and/or spread outside their natural past or present distribution threaten biological diversity”. freshwater fishes form a key component of invasive alien fauna in many countries around the world, and non-native species contribute high proportions among them in several regions (leprieur et al., 2008). high impact invaders when established in new habitats cause more chronic negative impact on native biota in terms of *correspondence: ajmal hussan doi: https://doi.org/10.22034/ijab.v9i3.897 e-mail: ajmal.hussan@icar.gov.in diversity loss, change in disturbance regime and retarding ecosystem succession (simberloff et al., 2012; paolucci et al., 2013; hussan and sundaray, 2020). pterygoplichthys spp., a non-native armoured ‘sailfin catfish’, having good capability of modifying their life history patterns to take advantage of new habitat, has successfully invaded in aquatic systems around the world including india and silently expanding its range (chaichana et al., 2011; ruedajasso et al., 2013; hussan et al., 2019, 2020). the 188 hussan et al./ control of invasive pterygoplichthys spp. in east kolkata wetland, india basket of biological traits like herbi-detrivorous feeding habit, high fecundity coupled with prolonged reproductive period, batch spawning and active parental care, broad physiological tolerance (e.g. salinity, ph, pollution), toleration of poor oxygen content in polluted water due to accessory respiration, capacity to down regulate metabolism during periods of food scarcity and rapid growth (liang et al., 2005; german et al., 2010), coupled with lack of natural predators and non-use as food, are most likely contributing to its range expansion and aggressive invasion in different parts of the world including east kolkata wetlands (ekw) in india (hoover et al., 2004; hussan et al., 2019). moreover, due to large amount of yolk, fishes of the genus pterygoplichthys directly develop into a definitive adult phenotype (juvenile stage) without undergoing a true larval metamorphosis between the free-living embryo and the juvenile stage, which improves its competitiveness even in its early stages (hoover et al., 2014). in ekw, these fishes were most likely introduced via aquarium hobbyist releases, and at present at least two species and also several intermediary forms of unknown identity, of the genus pterygoplichthys co-exist in this ecosystem (hussan et al., 2019; hussan et al., 2020). negative impacts of pterygoplichthys spp. and other loricariids on recipient ecosystems and aquatic communities have been reported widely (chaichana et al., 2011; nico et al., 2012; hussan et al., 2019). these fishes often grow exponentially and reach high densities and alter aquatic systems through direct consumption of organic matter, algae and benthicdwelling invertebrates (chaichana et al., 2011), and thus can compete with natural and culture production of native small indigenous and economically important fishes (hussan et al., 2019). pterygoplichthys spp. can also cause decline in native fish abundance by consuming or destroying the eggs of native fishes (hoover et al., 2014), and/or displacing them because of their aggressive territorial behavior (wakida-kusunoki et al., 2007). in ekw, significant decline in populations of native small indigenous fishes like puntius sp. and chanda sp. and depletion of productivity of commercial carp culture ponds due to pterygoplichthys spp. invasion were reported (kumar et al., 2018; hussan et al., 2019). hussan et al. (2019) also reported economic losses due to injuries/scratches on economically important fishes due to presence of a large quantity of armoured catfishes in the net during harvest, excavation in pond dykes and pond bottoms, and payment of incentives to the fishermen for catching/handling and discarding these fishes. but no systematic effort for suppressing its population and restricting its spread into new habitat has been reported so far, not only from ekw, but also from any other parts of the world. only known eradication of an introduced loricariid catfish by direct human intervention was reported by hill and sowards (2015), who reported complete eradication of p. disjunctivus from lower rainbow river of florida by hand and fish spear with an effort of two years. chaichana and jongphadungkiet (2012) and sumanasinghe and amarasinghe (2013) also suggested physical effort like ‘intensive fishing’ as one of the effective and feasible means of controlling population growth of armoured catfishes. as an effort to restrict entry of pterygoplichthys spp. in their aquaculture ponds and suppress its population, fish farmers of ekw are also practicing different physical methods like net-blocking, repeated netting and even dewatering of their aquaculture ponds (hussan et al., 2019). but evaluation of effectiveness of these efforts on suppression of pterygoplichthys spp. population has not been undertaken yet. therefore, in this study we attempted to describe and evaluate the effectiveness and drawbacks of current management strategies associated with pterygoplichthys spp. control in ekw, and suggest improvements for better prevention/elimination of the species from commercial carp culture bheries of the ecosystem. in the present study, we used the terms, ‘control’ to refer to all the efforts aimed to the prevention of new introductions and re-introductions, ‘eradication’ to refer to all efforts aimed at maintaining/reducing the population or eliminating the species from the system and ‘management’ to refer to all efforts aimed to control and eradicate the species. 189 int. j. aquat. biol. (2021) 9(3): 187-199 materials and methods study site: the east kolkata wetlands (ekw), one of the ramsar sites in west bengal (ramsar, 2019), located between 22°25’-22°40’n and 88°20’-88°35’e is the world’s largest wastewater ecosystem created to sustain successive resource recovery systems in the form of vegetable farms, fish ponds and paddy fields. an estimated 30-50% of the sewage generated by the kolkata city is treated and reused by the fishponds of the ekw and produces over 15,000 mt fish per annum from its 264 functioning aquaculture ponds, locally called bheries (edwards, 2008; hussan, 2016). pterygoplichthys spp. got entry into this system through deliberate introduction and invaded widely in ekw water bodies through the sewage feeder channels (hussan et al., 2019). we selected two sites, north-west site [bidhanagar area (bid)] and south site [anandapur area (ana)], of ekw for the present study based on the abundance of the pterygoplichthys spp. data collection: between may to december, 2017 randomly 40 big farms (n1) [20 farms from each site] having water area >10 ha were surveyed. to get qualitative information and also to evaluate the usefulness of the in-practice management efforts related to the eradication, control and containment of pterygoplichthys spp., a total of 80 respondents (n2) (one fisherman and farm manager/farm owner from each farm) were interviewed using a semi-structured interview schedule. the respondents were asked about the pros and cons of each method and also asked to give a score to each of the management practice against each factors (effectiveness, cheapness and ease of implementation), on a five-point scale (1, 2, 3, 4 or 5) following the concept detailed in table 1. to cross-check the views of the key informants (interviewed respondents), we visited the selected farms, done sampling and recorded the number and biomass of pterygoplichthys spp. removed by different methods. in-practice eradication efforts include repeated seine netting (rs), repeated seine netting after removing eichhornia sheath of the pond periphery (rse) and dewatering of water bodies (dw). in rs method, intensive, continuous seine netting covering portions of the water bodies are done throughout the year by the stakeholders of ekw for localized population reduction of invasive pterygoplichthys spp. for on-field sampling, we had done 32 numbers of removal treatments covering a total of 15.36 ha water area of eight selected bheries using seine net of size 48x5.4 m and mesh size of 15 mm. as pterygoplichthys spp. have tendency of hiding in burrows and in sheltered areas, rse method include removal of eichhornia sheath of the pond periphery first, followed to repeated seine net hauling. physical sample of rse were collected from three ponds using same seine net as rs. dewatering of water bodies (dw) refer to removal of fishes after complete draining of water of the bheries. to prevent entry of invasive pterygoplichthys spp. stakeholders of ekw are using two types of physical barriers, viz. layers of bamboo fencing (mb) or combination of both bamboo and net fencing (mbn). in case of mb, split bamboos are tied together side-by-side to form a mat, and then 2-3 layers (at a distance of 5-10 m) of such mats having different finger space are placed across in the sewage feeder channel. in mbn method along with 12 layers of bamboo fence, an additional layer of fine mesh net fence is placed either across the channel or in the outlet end (towards aquaculture pond) of the sewage intake pipe fixed by creating a dam across the channel. table 1. analysis of factors for scoring against a management practice. factors analysis of factors for scoring effectiveness higher the effectiveness, higher the score and vice versa (very effective=5; effective=4; keeps population in control / moderately effective=3; not so effective/little bit effective=2; not at all effective=1) cheapness cheaper the cost, higher the score and vice versa (very cheap=5; cheap=4; not cheap nor costly=3; costly=2; very costly=1) ease of implementation lower the difficulty in implementation, higher the score and vice versa (very easy=5; easy=4; little bit difficult=3; difficult=2; very difficult=1) 190 hussan et al./ control of invasive pterygoplichthys spp. in east kolkata wetland, india during the study, we also located burrows of pterygoplichthys spp. on the pond dykes (exposed after dewatering of the ponds) as well as banks of sewage intake channels. the burrows were identified based on nico et al. (2009) who reported most of the pterygoplichthys spp. burrows as close to triangular. data including burrow tunnel length/depth, burrow heightfloor to roof at the entrance, and burrow width at entrance were collected. measurements were taken using a combination of meter sticks, surveying rods, and tape measures. we also thoroughly studied the structural build up and installation practice of mb and mbn, to identify the drawbacks of these methods, in correlation to biological and behavioural characteristics of pterygoplichthys spp. we also collected secondary information on biological and behavioural characteristics of pterygoplichthys spp. from stakeholders of ekw and from literature search. integrating this information, we then suggested a modified version of barrier, for effective prevention of intrusion of pterygoplichthys spp. in the aquaculture bheries of ekw. data analysis: length-weight relationship of the fishes removed was estimated using the equation, w=alb given by le cren (1951); where l is the body length and w is the body weight of fish. average number and biomass of fishes removed per unit effort were expressed simply as mean±sd and fishes removed per hectare efforts were calculated, individually, as follows and expressed as mean±sd: fishes removed (as per hectare effort) = number or biomass of fishes removed/area covered during sampling. to analyse the perception of the respondents, ‘score’ against each factor against each management practices given by each respondent were converted to percentage, to simplify the understanding, using following formula (modified after paul et al., 2018): score in % = (score obtained against a factor/ maximum attainable score against the factor) × 100. then overall perception of the stakeholders towards in-practice control and eradication methods was calculated using ‘usefulness index (individual) (uii)’, using the formula: uii = cumulative score against the management practice against all factors/maximum attainable score against all factors) × 100 after that, overall efficiency of each management practices was calculated using the formula of usefulness index (overall) (uio) = ∑ uii/no. of respondent. uio value was used as an indicator of the overall efficiency of the management practices with minimum and maximum attainable value of 20 and 100, respectively. greater the uio value, greater the overall efficiency and vice versa. descriptive statistics of frequency tables, simple percentages and averages were used for the generation of conclusion. results large numbers of pterygoplichthys spp. with a wide range of the body sizes were recorded during present study (table 2), which indicates that a selfmaintaining population of sailfin armoured catfish has established in ekw. the total length of the fishes collected was found in the range of 114 to 478 mm with a weight of 27 to 810 g. although these fishes get slimmer with increasing length, as suggested by bvalue, which is less than ‘3’; r2 value of about 0.95 indicate a strong linear relationship between total length and total weight. as these fishes are not considered as important commercial fish because of their hard body armour and very little meat, farmers of table 2. the weight and total length (mean±sd), maximum and minimum values recorded, and the calculated values for the total length-weight relationship for pterygoplichthys spp. removed by different eradication methods at ekw, west bengal, india. eradication method n total length (mm) total weight (g) w =alb max min mean± sd max min mean± sd a b r2 rs 264 483 145 322.87±87.72a 810 46 317.39±207.62a 0.019 2.74 0.948 rse 210 452 132 298.69±86.61b 690 39 272.13±167.51b 0.016 2.82 0.913 dw 328 478 114 277.54±114.35c 785 27 266.83±219.24b 0.022 2.68 0.937 values in the same column having different superscript letters are significantly different (p<0.05) among eradication methods. rs = repeated seine netting, rse = repeated seine netting after removing eichhornia sheath, dw = dewatering of pond. 191 int. j. aquat. biol. (2021) 9(3): 187-199 ekw are generally practicing ‘culling after capture’ as a measure to limit pterygoplichthys spp. population (fig. 1). a total of 7831 specimens were removed during present study by different eradication efforts, summary of which is presented in table 3. the number and biomass of fishes removed by repeated seine netting (rs) or even by repeated seine netting after removing eichhornia sheath of the pond periphery (rse) were found indifferent statistically. analysis of stakeholder’s perception also indicates that, there is no significant difference in the effectiveness of rs and rse (table 4). whereas, 381.41±112.49 numbers and 118.52±45.62 kg of fishes removed by per-ha effort of dewatering of ponds/bheries (dw) is about 4-5 times higher than rs or rse. though dw was found advantageous both in terms of effectiveness (table 4) and feasibility (uio = 74.75±7.77) (fig. 2), in many cases, even after dewatering and despite leaving ponds empty for 15-20 days, pterygoplichthys spp. appeared once again when the ponds were filled with water. this is due to the refuge these fishes take in deep and branching table 3. summary of the on-field eradication efforts in the ponds of east kolkata wetlands (ekw). sampling site eradication method no. of efforts area covered (ha) number of fishes removed per effort* biomass of fishes removed per effort (in kg)* number of fishes removed per-ha effort* biomass of fishes removed per-ha effort (in kg)* ekw rs 32 15.36 39.71±16.22a 14.69±5.96a 84.16±27.09a 29.74±9.72a rse 11 6.15 49.45±21.98a 15.58±6.81a 87.34±32.97a 25.69±10.57a dw 03 14.30 1724±114.51b 529.47±63.55b 381.41±112.49b 118.52±45.62b *mean±sd. values in the same column having different superscript letters are significantly different (p<0.05) among eradication methods. rs = repeated seine netting, rse = repeated seine netting after removing eichhornia sheath, dw = dewatering of pond. table 4. pterygoplichthys spp. eradication methods: analysis of stakeholder’s perception on “five point scale” and in percentage (n = n2=80). factors eradication methods rs rse dw effectiveness 2.69±0.70a (53.75±14.08) 2.81±0.69a (56.25±13.90) 4.56±0.57b (91.25±11.40) cheapness 2.22±0.83a (44.50±16.52) 2.63±0.60b (52.50±12.06) 3.04±0.74c (60.75±14.74) ease of implementation 2.38±0.79a (47.50±15.71) 2.12±0.56b (42.50±11.19) 3.61±0.61c (72.25±12.11) values in parenthesis represent the score value in %. data in the same raw having different letters are significantly different (p<0.05) among eradication methods. rs = repeated seine netting, rse = repeated seine netting after removing eichhornia sheath, dw = dewatering of pond. table 5. measurement of pterygoplichthys spp. burrows found in water bodies of ekw, west bengal. parameters n min max mean±sd burrow height – floor to roof at entrance (cm) 13 8 16.7 11.99±2.67 burrow width at entrance (cm) 13 9.8 20.6 15.22±2.82 burrow tunnel length/depth (cm) 12 17 58 37.75±11.51 burrow volume (cm3) 12 1281 15751 7246±4076 *burrows identified based on nico et al. (2009) were only considered for the study. figure 1. proportion of farms practicing different pterygoplichthys spp. eradication methods (n=n1=40) [*c: combinations of rs, rse or dw). rs = repeated seine netting, rse = repeated seine netting after removing eichhornia sheath, dw = dewatering of pond. 192 hussan et al./ control of invasive pterygoplichthys spp. in east kolkata wetland, india burrows, large number of which were recorded during the study (table 5). maximum burrow depth of 58 cm, and water in burrows even after 12 days of dewatering was recorded during present study. sewage intake channels are the major pathway of pterygoplichthys spp. intrusion into the bheries of ekw, and hence farmers are using different types of physical barrier viz. layers of bamboo fencing (mb) or combination of both bamboo and net fencing (mbn) (fig. 3), to avert its intrusion into their pisciculture bheries. although these methods are cost-effective and also easier to implement, their effectiveness were found in the range of 40-60% only (table 6) and overall usefulness were found almost indifferent (fig. 2). major drawbacks of mb and mbn were identified as frail materials and improper installation with respect to biological and behavioural characteristics of pterygoplichthys spp. discussions as the invasion of pterygoplichthys spp. can results in serious ecological and economic consequences (nico et al., 2012), by directly interacting with native animals and physically altering the invaded aquatic habitats (chaichana et al., 2011; wei et al., 2017), adequate management efforts are needed urgently for effective control of their population growth and range expansion in india, including ekw (hussan et al., 2016, 2019). eradication efforts aimed at eliminating an invasive species from a given system through ‘early detection and rapid response’, are considered as the second most cost‐effective method to deal with invasive species, after prevention (qdpi, 2001). physical eradication methods like rs has been table 6. pterygoplichthys spp. control methods: analysis of stakeholder’s response on “five point scale” and in percentage (n = n2=80). factors control methods mb mbn effectiveness 1.97±0.39a (39.50±7.78) 2.97±0.55b (59.50±11.01) cheapness 3.91±0.62a (78.22±12.48) 3.46±0.55b (69.25±10.99) ease of implementation 3.78±0.56a (75.50±11.19) 3.64±0.48a (72.75±9.67) values in parenthesis represent the score value in %. data in the same raw having different letters are significantly different (p<0.05) among eradication methods. mb = layers of bamboo fencing, mbn = combination of both bamboo and net fencing. figure 2. overall usefulness (uio values) of different eradication and invasion control methods (n=n2=80). rs = repeated seine netting, rse = repeated seine netting after removing eichhornia sheath, dw = dewatering of pond, mb = layers of bamboo fencing, mbn = combination of both bamboo and net fencing. 193 int. j. aquat. biol. (2021) 9(3): 187-199 reported effective in controlling pond and lake populations of invasive fishes, such as topmouth gudgeon, pseudorasbora parva in the united kingdom (britton et al., 2010). but in ekw, though most practiced, the effectiveness of rs to eradicate pterygoplichthys spp. in-terms of per-ha effort was found only about 22% compare to dw. thus, complete eradication of pterygoplichthys spp. by rs from ekw is unlikely, because of the large sizes of the water bodies and heavy vegetation coverage along the pond periphery making it difficult to net whole water body at a time, and locate individuals within vegetation coverage (eichhornia shed). even after removing eichhornia sheath effectiveness of repeated seine netting (i.e. rse) did not improve significantly (only 23% effective compare to dw in-terms of per hectare effort). this is mainly because of the burrowing habits and capture avoidance ability of these fishes. generally, males of pterygoplichthys spp. excavate deep burrows in the banks and sides of the water bodies, which these fishes use as spawning and nesting sites, and as hide-outs, particularly in early life stages (nico et al., 2009). the greater number of younger individuals hauled per unit effort of rse also indicates their preference for hide-outs in the early stages. while the number of fishes removed per-ha effort by rse (87.34±32.97) was higher than the number of fishes removed by rs (84.16±27.09), biomass removed by rse (25.69±10.57 kg) was found lesser than rs (29.74±9.72 kg). taking advantage of this behavior, target removal of the colonized young one’s from hide-outs and/or egg masses from male-guarded burrows during the spawning season, thus can offer an option for localized control of pterygoplichthys spp. population by restricting recruitment (orfinger and goodding, 2018). such a policy was proven successful in restricting the population growth of invasive red lionfish (pterois volitans) locally in the united states (barbour et al., 2011). habitat manipulation, such as removing protective cover of vegetation, armouring of pond dyke walls, thus preventing egg deposition can offer a useful technique for altering the abundance of pterygoplichthys spp. within ekw. while possible, these strategies can be expensive and would likely impact aquatic ecosystems unfavourably (holdich et al., 1999; simberloff, 2001). therefore, rs or rse can be effectuated regularly, as a suppression tool, to keep population of pterygoplichthys spp. under control, which has very high recruitment potential. dewatering, though considered as the most feasible tool for complete eradication of invasive species from figure 3. traditional physical barrier in use at east kolkata wetland. 194 hussan et al./ control of invasive pterygoplichthys spp. in east kolkata wetland, india controlled environment (copp et al., 2007), in many circumstances dewatering may not be fully effective and also often need an increased investment of money and time (collier and grainger, 2015). hence in ekw practice of this method is largely limited to small water bodies, having water area upto 10-15 ha. in the case of pterygoplichthys spp. achieving 100% removal by simple dw is most unlikely, as they take shelter in deep branching burrows and also lay eggs there (zworykin and budaev, 2013). moreover, these fishes are resilient to hypoxia, anoxia, and brief aerial exposure due to their ability of aerial respiration through the modified gastrointestinal tract (armbruster, 1998; cook-hildreth, 2009). gibbs and groff (2014) reported that these fishes can survive out of water even more than 30 hours. eggs of these fishes are also very resistant to the environmental conditions and can develop normally even at low water levels, the only condition being that they should be covered with water (hoover et al., 2014). all these adaptations enable pterygoplichthys spp. to withstand potentially lethal events and stressors such as droughts and polluted water. therefore, to ensure 100% eradication of pterygoplichthys spp. even after draining, ponds of ekw needs to be sun-dried for at least four weeks or above to ensure complete water-out from the burrows. kozak and policar (2003) concluded that dewatering method may yield greater efficacy in controlling invasive fishes, when used alongside another eradication technique such as the application of chemicals. eradication of an established population of nonnative species is considered as a less biologically and economically feasible option as the species occupies more area and most of the detection methods are not completely reliable (pluess et al., 2012; tobin et al., 2014). hence, the best and most cost-effective way to reduce total impacts from non-native invasive species is to prevent their arrival and establishment (iucn, 2000; lodge et al., 2006; keller et al., 2007). prevention strategies include regulation, border protection, public engagement, and public‐private partnerships to restrict introduction (mack et al., 2000). prevention of re-introduction is also critical for any successful eradication (bomford and o’brien, 1995). physical control using fish barriers or screens are sometimes very effective in preventing new fish entry or excluding eradicated fish following removal to promote restoration of the degraded habitat (collier and grainger, 2015). brammeier et al. (2008) reported ‘physical barrier’ method as 95-100% effective to prevent the transfer of aquatic invasive species on a small scale. but in ekw, in-practice physical barrier methods, like use of layers of bamboo fencing (mb) or combination of both bamboo and net fencing (mbn) were found only partially effective in preventing pterygoplichthys spp. intrusion. lower effectiveness of mb or mbn in ekw were found related with the shortcoming of these structures and their implementation, in correlation to biological and behavioural characteristics of pterygoplichthys spp. these fishes have tendency to excavate burrows in the banks and peripheral area of aquatic bodies. generally, males of these fishes dig deep and branching burrows, length of which can extend even upto 1.2-1.5 m and are often horizontal in direction (nico et al., 2009; capps et al., 2011). during present study, we also recorded number of burrows in dykes of aquaculture bheries and also in the banks of sewage feeder channels, average length of which was 37.75±11.51 cm, with maximum of 58 cm. in addition, these fishes have tendency of digging burrows in the steep and exposed portion of the banks, just above the water level (nico et al., 2009), and hence sufficient dyke height to be maintained above the maximum water level for effective control of these fishes. pterygoplichthys spp. also can damage netblocking and even hard structures, like cages, thorough their hard dorsal and pectoral spines (wakida-kusunoki et al., 2007; zworykin and budaev, 2013). these biological and behavioural oddities of pterygoplichthys spp. were not taken into consideration in use of mb and mbn in ekw, and hence these fishes got their way in the aquaculture bheries, by passing through the holes dug across the barrier in beneath or banks of the channel or by damaging the net blocking or through the finger spaces of the bamboo fencing. therefore, a modified 195 int. j. aquat. biol. (2021) 9(3): 187-199 version of barrier, named hereby as ‘modified vertical barrier (mvb), was suggested taking into consideration these biological and behavioural characteristics of pterygoplichthys spp. to minimise their intrusion in the bheries of ekw (figs. 4, 5). mbv suggested includes a sewage feeder pipeline, a concrete collection chamber with size separation arrangement and a dam of specific dimensions across the channel, to cope with the biological and behavioural oddities of these fishes. juvenile and adult pterygoplichthys spp., which have capability to damage plastic net by their hard dorsal and pectoral spines can be retained by pvc coated wire mesh (square mesh 3x3 cm and 1.5x1.5 cm size) and finemesh fishing net as third layer will optimise retention of smaller ones having softer and thinner armour (e.g. p. pardalis stretch out their body fins at size >10 cm) (chaichana and jongphadungkiet, 2012; gibbs et al., figure 4. photographic image of the suggested ‘modified vertical barrier (mvb). figure 5. cross section of the suggested ‘modified vertical barrier (mvb). 196 hussan et al./ control of invasive pterygoplichthys spp. in east kolkata wetland, india 2013). moreover, debris blockage issues that discourage many ekw stakeholders to use fine mesh net as barrier can be minimised through this separation technique. considering the burrowing characteristics of pterygoplichthys spp. recorded during present study and also described by nico et al. (2009) and capps et al. (2011), a dam width of 2.5-3.0 m across sewage feeder channel and height of at least one meter above maximum water level was suggested for mbv. ruebush et al. (2012) found that, barrier designed considering the behavioural characteristics of fish (named as ‘bubble barrier’) was successful in preventing hypophthalmichthys nobilis and h. molitrix from moving upstream in the illinois river of united states. whereas, an attempt to prevent migration of pacifastacus leniusculus in the river buaa at the border between sweden and norway, using a simple barrier was reported unsuccessful, as behavioural aspects of the fish like ability to crawl a height and travel distances over land were not addressed in application of the barrier (johnsen et al., 2008). the present study concurs the remark of hill and sowards (2015), who stated pterygoplichthys spp. eradication as difficult, potentially time consuming and not economically feasible. hence, efforts need to explicitly focus towards containing the current established populations and preventing future expansions of pterygoplichthys spp. (lawson et al., 2015). exploiting these fishes as human food or as an animal feed ingredient, may be an option towards limiting its population. but though these fishes have good nutritional quality and potential for uses as human food fish in the form of fresh fillets, processed product like surimi (rueda-jasso et al., 2013); fishermen, as well as general public in and around ekw, are averse to eating these catfishes. educating the public, especially fishers and other stakeholders not to release unwanted pterygoplichthys spp. into water bodies or water channels, also has paramount importance, as most transfer between catchments are human-assisted (lintermans, 2004; maceda-veiga et al., 2016). acknowledgement the authors wish to thank the director, icar-central institute of freshwater aquaculture, bhubaneswar for all the logistic supports and scientific guidance. the authors are thankful to d. biswas and r. mandal for helping in drawing engineering impression, and to r. nath and r. lal for helping in sample collection. also heartfelt thanks to the stakeholder’s of ekw for their wholehearted support and co-operation. references armbruster j.w. 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(2020) 8(2): 148-153 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article methylation levels of lysozyme gene in rainbow trout (oncorhynchus mykiss) fed by commercial immunogen probiotic ali choubkar1, hamid farahmand*1, alireza mirvaghefi1, houshang alizadeh2, amirreza abed-elmdoust1 1department of fisheries sciences, faculty of natural resources, university of tehran, karaj, iran. 2department of agronomy and plant breeding, faculty of agricultural sciences and engineering university of tehran, karaj, iran. article history: received 16 february 2020 accepted 21 april 2020 available online 2 5 april 2020 keywords: lysozyme dna methylation epigenetics immunity abstract: in the present study, we evaluated the effect of commercial immunogenic prebiotic on the rate of methylation of type-c gene. a total of 120 rainbow trout were divided into two treatments, including a control group and another one with 2.0% commercial prebiotic immunogen each in three replicates. on the first, 15 and 45 days, dna of adrenal tissue was extracted and treated with bisulfite. samples were amplified by polymerase chain reaction (pcr) and sequenced. based on the results, there was no significant difference (p<0.05) between the 1st day (r), 15th day of control (c), 15th day of immunogen (i) and 40th day of control (cs). however, there was a significant difference (p<0.05) between the 45th day immunogenicity (is) and other samples, i.e. the cpg islet methylation rate in the is samples was lower leading to increase in the expression of the lysozyme gene. introduction development of intensive aquaculture systems have been caused problems such as disease and physiological disturbances following the increase of stress. the most common way to treat fish diseases is application of chemicals and antibiotics, which are led many health and social problems, e.g. the antibiotic bioaccumulation and producing antibiotic-resistant strains. therefore, application of these substances for treatment should be seriously monitored (austin and austin, 2016). in this regard, many researches have focused to maximize aquaculture productivity in the short time, with minimal cost and side effects using additives with no health and environmental consequences (khurana, 2005). hence, the use of various dietary supplements, including probiotics, prebiotics and their combinations (synbiotics) has received much attention and interest to elicit the immune response and enhance immunity of aquatic species (huynh et al., 2017). prebiotics are indigestible nutrients (carbohydrates) that have beneficial effects on the host and improve their health by growing or activating one *correspondence: hamid farahmand doi: https://doi.org/10.22034/ijab.v8i2.881 e-mail: hfarahmand@ut.ac.ir or a limited number of bacterial species in the gut. therefore, they improve and balance the intestinal microflora enhancing the host defense mechanism (fooks and gibson, 2002). in recent years, nutrition has been shown to exert drastic changes in gene expression by affecting transcription mechanisms and subsequent steps (waterland el al., 2004). nutrition also has a permanent effect on the gene expression pathway by epigenetic mechanisms, which can be inherited (waterland and michels, 2007). immunogen is a commercial prebiotic consisting several stimulants, such as beta-glucan and mannaligosaccharide (salze et al., 2008). it can prevent diarrhea, increase immunogenicity, stimulate growth and absorb mycotoxin. this dietary compound also prevents the accumulation of pathogenic bacteria such as salmonella, clostridium, and escherichia coli in the intestine and increases the secretion of iga, igg, and igm immunoglobulins, which impedes the attachment of bacteria or toxins to the intestinal epithelium (sado et al., 2008). supplementation of immunogen as a prebiotic to rainbow trout increases the expression of lysozyme type-c gene resulting in 149 int. j. aquat. biol. (2020) 8(2): 148-153 increased resistance to environmental stress and disease (hashim, 2016). epigenetic changes are reversible in gene expression, which can be inherited through mitosis, meiosis, or both, which does not include changes in dna sequences (novik et al., 2002). epigenetic changes can be classified into three major categories, including changes in covalent histones, non-coding rnas and dna methylation (zargar and rabbani, 2000, 2002; herceg, 2007). in dna methylation process, a methyl group, in a covalent form, binds to a cytosine base (at carbon number 5) by the action of dna methyl transferase. dna methylation usually occurs at or near the cpg islands located within or near the gene promoter (lętowska-andrzejewicz et al., 2011) which highly effects the gene expression level. bisulfite sequencing is a method of dna methylation analysis in which bisulfite treatment of dna is used before routine sequencing to determine the pattern of methylation (li and tollefsbol, 2011). changes in global methylation rate in different nutritional and environmental circumstances have been investigated in some aquatic species (blouin et al., 2010; morán and pérez-figueroa, 2011; xiao et al., 2013; anastasiadi et al., 2017; fan et al., 2019). the present study aimed to investigate the relationship between increased specific gene expression and methylation rate of the lysozyme gene in oncorhynchus mykiss treated with commercial immunogenic prebiotic is investigated. materials and methods a total of 120 rainbow trout (with mean weight of 81±0.05 g) were obtained from karaj fish farm and transported to the aquaculture lab of fisheries department, university of tehran. for adaptation, fish were divided into two 1000-liter tanks equipped with a central aeration system and kept for 48 hours without feeding. the water replacement was 50% daily. they were adapted to lab conditions for 10 days prior to the experiment. the experiment was conducted as a completely randomized design in two treatments and each with three replications. treatments were a group fed with the commercial immunogenic prebiotic supplement (2 g/kg) and control group with no dietary supplement. the group were randomly assigned to six tanks of 1000 fiberglass tanks (20 fish per tank). food preparation and feeding: the commercial immunogenic prebiotic product was obtained from icc company, usa, and added with a ratio of 0.2% to rainbow trout commercial diet from behparvar company at mixing step before pelleting. during the experiment period, feeding was performed manually based on 2% of live biomass weights in the tank twice a day. biomass was measured at 14 days intervals. tissue sampling: the methylation rate of the lysozyme gene was measured from the anterior part of the kidney obtained from three fish from each treatment at the first, 15th and 45th days of the experiment. the sampling was performed in a sterile condition and the fish were anesthetized with clove powder prior to the sampling. the samples were kept in -80°c before dna extraction. dna extraction, bisulfite treatment and amplification: express primer methyl software (v 1.0) was used for primer design, and the primers were manufactured and shipped by macrogen company, korea (table 1). dna extraction was performed by modifying the protocol of tierling et al. (2007). nanodrop model 7.v3.1000 was used to evaluate the quantity and quality of the extracted dna. bisulfite treatment was performed by modifying the protocol of fernandez et al. (2007). for pcr amplification, topaz pcr mix master with 1x final concentration, primers with 0.4μm and dna template with 50-150 ng concentration were used. the pcr amplification was performed with 33 cycles with 94°c for 3 min for initialization step, 95°c for 45s for denaturation step, table 1. forward and reverse primers designed by express primer methyl software for lysozyme type-c gene. forward primer 5’ gtatgtttttgagttttaaaaatatttt 3’ reverse primer 5’ cacatttcttatcaaaaataatactcta 3’ 150 choubkar et al./ methylation levels of lysozyme gene in rainbow trout 55°c for 40s for annealing step, 72°c for 25s for extension/elongation step and 72°c for 10 min final elongation step (table 1). after amplification of dna fragments, nanodrap model 7.v3.1000 was used to evaluate the quantity and quality of the fragments and bands obtained from 1.5% agar electrophoresis were prepared according to the protocol from macrogen company, korea and sent for sequencing. flanking bases at the two ends of each sequence with low quality were removed by ugene unipro software and sequences were aligned. statistical analysis: changes in methylation levels of cpg islets in each treatment were measured by biq analyzer software. data were analyzed using lollipop diagrams and bars. in order to compare the mean methylation rate in different groups, spss software (v. 17) was used at a significant level of 5%. results methylation levels in day 1 (r), control treatment on day 15 (c), immunogen treatment on day 15 (i), control treatment on day 45 (cs) and immunogen treatment on day 45 (is) were compared with the original gene version. there was no significant difference (p<0.05) between control on day 15 and 45 and immunogen treatments on day 15 and first-day samples, while there was a significant difference (p<0.05) between the methylation percentage of the immunogen treated samples on day 45 to all other samples (fig. 1). based on the results, no significant relationship was observed between methylated and unmethylated cpg dinucleotides, and each sample exhibited a different response in different locations of the cpg dinucleotides (fig. 2), but the regions with two or three cpgs were more susceptible to methylation. discussions in bisulfite sequencing technique, the number of treated bases should be in the range of 300 to 400 bp so that all the bases can be exposed to bisulfite during treatment, which in this way the rate of cleavage and breakage of dna would be minimal (tierling et al., 2007). in the present study, the primers were designed to amplify 309 bp of the lysozyme gene close to the promoter and 18 cg sites were measured. more investigations on other regions of the lysozyme gene can highly elucidate the relation between methylation and gene expression. our findings showed that methylation in cgs increases in regions closer to the promotor and incidentally the cgs near the end of the 5' gene play an important role in the expression of the gene. in the is group, cg dinucleotides that were closer to the 5' end of the gene were more demethylated. 45 days of commercial immunogenic prebiotic use was caused partial dimethylation of the type-c lysozyme gene, which increased the expression of this gene. epigenetic traits, e.g. genetic traits, are transmitted to the next generation known as figure 1. methylation percentage in each group in the experiment. 151 int. j. aquat. biol. (2020) 8(2): 148-153 vertical transmission. therefore, the positive changes in the epigenome will be permanent and transferable organisms (portela and esteller, 2010). reinforcing the innate immune system is critical to improving disease resistance in aquatic species (liu et al., 2012). most epigenetic studies are based on global methylation in a single generation and investigated the percentage of methylated cpg in the whole genome of an organism. there are also few works on global cpg methylation after consuming a dietary treatment and its comparison with a control treatment without aiming at a specific gene or trait. although there have been many studies of dna methylation in mammals in recent years, there are very few studies on aquatic species to compare with our findings. global methylation levels in mature rats, which were fed by prebiotics, were significantly decreased (zheng et al., 2014), which is consistent with our results. in another work, feeding mice with vitamin b12 had significantly increased dimethylation rates in the gr1 and ppar genes, which are genes related to resistant to stress (lee, 2015). as immunogen figure 2. (a) bar graphs immunogen treated samples and (b) control group on day 45th. 152 choubkar et al./ methylation levels of lysozyme gene in rainbow trout prebiotic is rich in vitamins and antioxidants that are not digestible, the fish takes advantage of these substances with the help of bacterial flora in their intestine. furthermore, a study on daphnia showed that exposing it to high levels of nacl is led higher overall methylation rate, with the greater amount of methylation being returned to immunity and stress control and the overall methylation status. the downside is the function of the body's immune system, which was inconsistent with our results (asselman et al., 2015). however, in this study, no dietary supplements were used. based on our results, it can be concluded that commercial immunogenic prebiotic can affect the methylation rate. the current work examined one gene that has been proven to increase expression by commercial immunogenic prebiotics. references anastasiadi d., díaz n., piferrer f. 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(2014) 2(3): 155-163 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article acute toxicity of alkali and alkaline earth metals on rohu, labeo rohita (hamilton) egg and hatchlings anusaya mallick*1, 2, bikash chandra mohapatra2, niranjan sarangi2 1department of environmental science, university of kalyani, kalyani, west bengal -741235, india. 2central institute of freshwater aquaculture, (indian council of agricultural research), bhubaneswar751 002, orissa, india. article history: received 14 november 2013 accepted 18 april 2014 available online 2 5 june 2014 keywords: alkali and alkaline earth metals bioassay lc50 rohu abstract: the acute toxicity of salts of alkali and alkaline earth metals, such as sodium (na), potassium (k), calcium (ca) and magnesium (mg) were studied on the egg and larval stages of indian major carp labeo rohita (hamilton). the acute toxicity experiments were conducted followed by the range finding bioassay tests. the experiments were conducted in triplicates. the cumulative percentage of dead or damaged eggs at the end of 6, 12, 18, 24, 36, 48, 60, 72 and 96 hours was recorded for the calculation of lc50. the increase in salt concentrations in water increased their toxicity and reduced the duration to damage 50% of the eggs. the eggs became smaller than their normal size and whitish before being damaged in the test solutions. most of the exposed eggs and hatchlings tended to lay on the floor of the tank. the toxicity of the metals was in the order of k>na>mg>ca. the 96 hours lc50 values were 3.25, 2.73, 28.9 and 20.52 ppm for sodium, potassium, calcium and magnesium, respectively. introduction indian major carps (imc) are the most important groups of fishes cultured in indian subcontinent. rohu (labeo rohita) belongs to the family cyprinidae and is found commonly in rivers and freshwater lakes, mainly in south-east asia (gupta et al., 1997). in order to bridge the gap between the everincreasing demand and supply of the seed of the species, induced breeding plays a major role. alkali and alkaline earth metals affect fish breeding, water hardening of eggs, growth and survival of hatchlings (mallick et al., 2010). the embryological stages of an organism have important considerations, when examining the effect of heavy metals and concentration of these in the body tissues. it can vary with the age or size of the organism (bennett and dooley, 1982; newman and mitz, 1988). the health of fish may be affected, either directly through uptake from the water or indirectly through their diet of vegetation, * corresponding author: anusaya mallick e-mail address: anusaya.cifa@gmail.com invertebrates or smaller fish (kime et al., 1996). metals released into aquatic ecosystems are responsible for several fish physiological irregularities (sehgal and saxena, 1986). these can also disturb the ion regulatory mechanism in aquatic organisms (hansen et al., 1996). in an aquatic environment, metal toxicity can be influenced by various abiotic factors such as oxygen, calcium/water hardness (skidmore, 1964; cairns and mount, 1990; ghillebaert et al., 1995), ph and temperature (cairns and mount, 1990; kotze et al., 1999). according to rose et al. (1993) abiotic factors are defined as variables that exert a direct effect on individuals in the population. the sodium level in water bodies is quite variable. wide ranges of seasonal fluctuations of sodium in freshwater bodies have been reported by khan and siddqui (1974), goel et al. (1986) and khatavkar et al. (1990). high concentrations of sodium chloride have strong local effects in fish (metelev et al., 156 mallick et al./ acute toxicity of alkali and alkaline earth metals on rohu international journal of aquatic biology (2014) 2(3): 138-146 1983). potassium is a naturally occurring element, which remains in lesser concentration than calcium, magnesium and sodium in freshwater. the potassium level in aquatic habitats is quite variable from 0.2-35.0 ppm. mohan and zafar (1986) demonstrated that the potassium is fast accumulated with the passage of time and doubling up in every fifteen years in the aquatic habitat. according to metelev et al. (1983) and mohapatra (1999) the symptoms of poisoning due to potassium compounds are analogous to those of poisoning with sodium compounds. calcium is a very important cation for the animal body because not only takes part in the structural formation, but also in the various metabolic and physiological activities in the body. the increase in environmental calcium rises the tolerance of aquatic animals to ammonia toxicity (tomasso et al., 1980). calcium is a primary structural component of hard tissues such as bones, exoskeleton, scales and teeth of aquatic organisms. freshwater fish obtain magnesium ions through active uptake from the environment or from dietary sources. fish eggs contain a significant amount of magnesium associating with the yolk (hayes et al., 1946). therefore, the yolk may serve as a magnesium source for the developing embryo during the early developmental stage. magnesium is essential for cellular respiration and neuromuscular transmission (lall, 1989). egg hatchability and hatching time are more sensitive indicators of toxicity than “standard” end point, like mortality and growth (pyle et al., 2002). except one report of mohapatra (1999) on the toxicity studies on fingerlings of catla catla, no work has been done on the determination of tolerance limits of alkali and alkaline earth metals on egg and larvae of indian major carps viz., catla catla, labeo rohita and cirrihinus mrigala. therefore, the present study was planned to investigate the comparative sensitivity of rohu egg and hatchlings to alkali and alkaline earth metals through toxicity tests. materials and methods test containers: the glass jar tanks with 20 l capacity were used as test containers after with laboratory detergents, then with 100% acetone and tap water. after each test, the containers were washed appropriately with acid to remove metals, bases and organic compounds. each of the test containers was provided with facilities of continuous aeration. test solutions: the test solutions were prepared by dissolving the calculated amount of salts, such as sodium chloride, potassium chloride, magnesium chloride and calcium chloride as per the experiments in the dilution water of the jars. the solutions were prepared immediately prior to the initiation of the experiments. de-chlorinated tap water was used as dilution water for control tests and for making concentrations of the test substances. the dilution water was clean, uncontaminated and of constant quality. test concentrations: selection of test concentrations was made as per the protocol of apha (1998). the concentrations of the metals ware expressed as parts per million (ppm). the test organisms were exposed to a range of salt concentrations in logarithmic scale, such as 0.0001, 0.001, 0.01, 0.1, 1.0, 10, 100, 1000 and 10,000 ppm. based on the results of the range finding bioassay, the concentrations 0.5-10,000; 0.05-1000; 1-10,000 and 1-5000 ppm for the salts of sodium, potassium, magnesium and calcium, respectively were selected as the test concentrations for definite bioassay experiment. to avoid contamination, the controls were maintained little away from bioassay tanks. bioassay of egg: the rohu eggs of appropriate quality were selected for the experiment. eggs were received from cifa carp hatchery at bhubaneswar and were transferred to the experimental tanks with test media on arrival at laboratory, and the containers were calibrated to determine the approximate number of eggs for experimentation. the eggs were measured volumetrically before release into the test containers including control tanks. the percentage of dead/ damaged eggs at the end of every 6, 12, 18, 24, 48, 72 and 96 hours were recorded and tabulated. to avoid contamination, the dead eggs were removed 157 int. j. aquat. biol. (2014) 2(3): 155-163 from the experimental tanks immediately. data analysis: the data obtained from the experiments were processed by probit analysis (finney, 1971; reish and oshida, 1987; mohapatra and rengarajan, 1995) for determination of lc50 values in computer using spss software. the lethal concentrations were plotted against time in hours to get "toxicity curve"(seegertet al., 1979). determination of toxicity of elementary form of metals: grams of compound containing 1.0 g element= molecular weight of compound/molecular weight of element i.e., 1.0 g of nacl, kcl, cacl2 2h2o and mgcl2 6h2o contain 0.39, 0.524, 0.273 and 0.12 g of na+, k+, ca++ and mg++, respectively. results range finding bioassay: in range finding bioassay a wide range of concentrations i.e. 0.0001, 0.001, 0.01, 0.1, 1.0, 10, 100, 1000 and 10,000 ppm were selected for each salt solution. no mortality was observed in table 1. percentage death of eggs or hatchlings of rohu exposed to different concentrations of sodium chloride (nacl) salt solution conc. (ppm) % death of eggs and hatchlings of rohu in different exposure time (hours) 6 12 18 24 36 48 60 72 96 0.5 30 35 40 40 40 40 40 40 40 1 45 50 50 50 50 50 50 50 50 10 50 50 50 50 55 55 55 55 55 100 50 50 50 50 55 55 60 70 70 1000 60 75 80 90 100 100 100 100 100 10000 65 85 100 100 100 100 100 100 100 control 10 15 15 20 20 25 30 35 40 table 2. percentage death of eggs or hatchlings of rohu exposed to different concentrations of potassium chloride (kcl) salt solution conc. (ppm) % death of eggs and hatchlings of rohu in different exposure time (hours) 6 12 18 24 36 48 60 72 96 0.05 20 25 30 30 45 45 45 45 50 0.1 20 30 35 45 45 45 45 45 45 1 30 30 40 50 50 50 50 50 50 10 35 45 50 50 60 60 65 65 75 100 40 55 70 90 100 100 100 100 100 1000 45 60 80 100 100 100 100 100 100 control 10 20 20 30 30 30 30 30 35 table 3. percentage death of eggs or hatchlings of rohu exposed to different concentrations of magnesium chloride (mgcl2) salt solution conc. (ppm) % death of eggs and hatchlings of rohu in different exposure time (hours) 6 12 18 24 36 48 60 72 96 1 30 35 40 40 45 45 45 45 45 10 40 40 45 50 50 50 50 50 50 100 45 45 50 50 50 60 60 60 65 1000 50 50 55 70 90 100 100 100 100 10000 50 55 65 100 100 100 100 100 100 control 20 20 20 30 30 30 40 40 40 table 4. percentage death of eggs or hatchlings of rohu exposed to different concentrations of calcium chloride (cacl2) salt solution conc. (ppm) % death of eggs and hatchlings of rohu in different exposure time (hours) 6 12 18 24 36 48 60 72 96 1 30 35 45 40 40 40 40 40 40 10 40 45 50 50 50 50 50 50 50 100 40 45 50 50 60 60 65 65 70 1000 50 60 80 90 100 100 100 100 100 5000 55 70 80 100 100 100 100 100 100 control 20 20 20 20 20 30 40 40 40 158 mallick et al./ acute toxicity of alkali and alkaline earth metals on rohu international journal of aquatic biology (2014) 2(3): 138-146 0.0001-0.01 ppm for all the tested salts i.e. sodium chloride, potassium chloride, calcium chloride and magnesium chloride, but 75% mortality occurred at 10,000 ppm in case of salts of sodium and magnesium, 1000 ppm for potassium and 5000 ppm for calcium in 96 hour of exposure. hence, the concentrations of 0.5, 1, 10, 100, 1000 and 10,000; 0.05, 0.1, 1, 10, 100 and 1000; 1, 10, 100, 1000 and 10,000; and 1, 10, 100, 1000 and 5000 ppm were selected for the salts of sodium, potassium, magnesium and calcium, respectively for conducting definitive bioassay. bioassay results: the percentage of damaged or dead eggs was not similar in all experimental concentrations. in higher concentrations, the percentage of damaged eggs was high. egg became smaller, whitish just before death and coelomic content turned opaque or white. most of the exposed eggs and hatchlings after death were settled on the aquarium floor. the cumulative percentage of dead or damaged eggs; or hatchlings after hatching out from eggs at 6, 12, 18, 24, 36, 48, 60, 72 and 96 hours of exposure was recorded for the calculation of lc50 (tables 1-4). the results of toxicity studies expressed in terms of lc50 values obtained from probit analysis for different salts are given in table 5. the median lethal concentration (lc50) decreased gradually with the increase in exposure time from 6 to 96 hours. the rank order of toxicity of metal salts for rohu egg and hatchlings was found to be potassium chloride > sodium chloride > magnesium chloride > calcium chloride. the toxicity of inorganic solids such as sodium, potassium, calcium and magnesium were calculated from the results of bioassay studies with their salts and presented in table 6. based on 96 h lc50 values, potassium was found to be more toxic to rohu larval stages and the least was the calcium. the toxicity order was k>na>mg>ca (table 6). toxicity curve: the toxicity curves were obtained for different salts by plotting the log time against the log lc50 values (fig. 1a-d). the toxicity curve may not pass through all the lc50 points on the graph at all times. it gives the overall picture of the progress table 5. lc50 values of salts of alkali and alkaline earth metals on eggs and hatchlings of rohu, labeo rohita exposure period (hours) lc50 (ppm)a nacl kcl mgcl2 cacl2 6 5561 1170 9122.5 3743.8 12 2471.1 560.6 6890.1 2326.4 18 345.7 188.8 4413.8 1075.1 24 353.1 17 387.8 256.1 36 93.2 7.2 193.4 64.5 48 21.6 6 58.8 59.1 60 13.9 6 38.8 45.4 72 4.1 4.6 36.2 42.7 96 3.3 2.7 20.5 28.9 a after 18 hours of exposure the eggs hatched to hatchlings table 6. lc50 values of elementary forms of alkali and alkaline earth metals on eggs and hatchlings of rohu, labeo rohita exposure period (hours) lc50 (ppm) na+ k+ mg++ ca++ 6 2168.8 613.1 1094.7 1022.1 12 963.7 293.7 826.8 635.1 18 134.8 98.9 529.6 293.5 24 137.7 8.9 46.5 69.9 36 36.3 3.8 23.2 17.6 48 8.4 3.2 7.1 16.13 60 5.4 3.2 4.6 12.4 72 1.6 2.4 4.3 11.6 96 1.3 1.4 2.5 7.9 159 int. j. aquat. biol. (2014) 2(3): 155-163 figure 1. toxicity curve for rohu eggs hatchlings exposed to different lethal concentrations of nacl, kcl, mgcl2, and cacl2 up to 96 hours. table 7. lc50 values of elementary forms of alkali and alkaline earth metals on eggs and hatchlings of rohu, labeo rohita salts species value (ppm) toxicity source nacl roach and tench 10,000-11,000 not toxic within 24 hrs metelev et al.,1983 13,000 mortality after 1 day carp (100-150g) 15,000 toxic trout (fry and fingerlings) 10,000 non-toxic for several hrs catla catla 15,600 6 hr lc50 mohapatra, 1999 13,700 12 hr lc50 10,900 24 hr lc50 10,800 48 hr lc50 10,100 72 hr lc50 9,000 96 hr lc50 sarotherodon mossambicus 31,300 24 hr lc50 30,300 48 hr lc50 29,700 72 hr lc50 27,600 96 hr lc50 labeo rohita (egg) 5561 6 hr lc50 present study 2472.1 12 hr lc50 labeo rohita (hatchling) 345.7 18 hr lc50 353.1 24 hr lc50 93.2 36 hr lc50 21.6 48 hr lc50 13.9 60 hr lc50 4.1 72 hr lc50 3.3 96 hr lc50 kcl perch and white salmon 10,000 toxic after 18 hrs metelev et al.,1983 fish 1000 toxic 500 non toxic emerald shiner, feathead minnow, golden shiner, white bass, carp 10,000 no mortality up to 24 hrs snyder et al., 1991 catla catla 3350 12 hr lc50 mohapatra, 1999 1830 24 hr lc50 1270 48 hr lc50 800 72 hr lc50 3400 96 hr lc50 160 mallick et al./ acute toxicity of alkali and alkaline earth metals on rohu international journal of aquatic biology (2014) 2(3): 138-146 table 7. continued. sarotherodon mossambicus 3,400 12 hr lc50 1,750 24 hr lc50 920 48 hr lc50 900 72 hr lc50 810 96 hr lc50 labeo rohita (egg) 1170 6 hr lc50 present study 560.6 12 hr lc50 labeo rohita (hatchling) 188.8 18 hr lc50 17 24 hr lc50 7.2 36 hr lc50 6 48 hr lc50 6 60 hr lc50 4.6 72 hr lc50 2.7 96 hr lc50 mgcl2 6h2o three-spined stickle back 2,300 can withstand up to 6 months without damage metelev et al.,1983 perch and white salmon 10,000 can tolerate up to 24 hours carp (100g) 6000 can tolerate for 3-4 days without damage brown trout fry 4900 can live up to 8 days barbel 10,000 can withstand for 4 weeks 15,000 a percentage die after 5 days 20,000 die after 1hrs carp 1000-15,000 can tolerate for 4 weeks 20,000 a percentage die after 5 days 30,000 die after 4 hours eel 10,000 live for 4 weeks 15-20,000 die after 14 days catla catla 18,500 12 hr lc50 mohapatra, 1999 18,300 24 hr lc50 18,200 48 hr lc50 18,000 72 hr lc50 17,900 96 hr lc50 sarotherodon mossambicus 38,000 12 hr lc50 36,500 24 hr lc50 36,100 48 hr lc50 35,600 72 hr lc50 35,100 96 hr lc50 labeo rohita (egg) 9122.5 6 hr lc50 present study 6890.1 12 hr lc50 labeo rohita (hatchling) 4413.8 18 hr lc50 387.8 24 hr lc50 193.4 36 hr lc50 58.8 48 hr lc50 38.8 60 hr lc50 36.2 72 hr lc50 20.5 96 hr lc50 cacl2 6h2o white salmon, carp and perch 10,000 toxic after 16-29 hours metelev et al.,1983 juvenile brown trout 13,900 toxic after 10 days carp, rainbow trout and barbel 5,000 not toxic in 4 weeks fishes 15,000 toxic in 1 hr to several days eel 27,000 can tolerate catla catla 7,500 12 hr lc50 mohapatra, 1999 6,350 24 hr lc50 4,950 48 hr lc50 4,100 72 hr lc50 3,950 96 hr lc50 161 int. j. aquat. biol. (2014) 2(3): 155-163 of the test and also indicates when acute lethality has stopped. with longer exposure times, the curve tends to be parallel to the time axis. discussion acute toxicity test is necessary in water pollution control to determine whether a potential toxicant is dangerous to aquatic life and if so, to find the relationship between the toxicant concentration and its effect on aquatic animals. bioassay is necessary to determine the concentration of a toxicant, which may be allowed in receiving waters without adverse effects on the living resources (standing committee of analysts, 1981; ward and parrish, 1982; reish and oshida, 1987). bioassay technique has been the cornerstone of programmes on environmental health and chemical safety (ward and parrish, 1982; mohapatra and saha, 2000). the median lethal concentration (lc50) of the metals sodium, potassium, calcium and magnesium on rohu eggs and hatchlings decreased gradually with the increase in exposure time from 6 to 96 h. the toxicities of the salts were seen in the order of potassium chloride > sodium chloride > magnesium chloride > calcium chloride. potassium was found more toxic and calcium was the least. these toxicity values cannot be are compared directly with the available results of other workers, but, a comparative statement is given in table 7. because of eggs and larval stages, the toxicity values in the present study for rohu were found less than the values reported by mohapatra (1999). metelev et al. 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(2019) 7(6): 332-341 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article demographic parameters and exploitation rate of five key fishes of okpara stream, oueme river, benin, west africa rachad sidi imorou1, alphonse adite*1, edmond sossoukpe2, kayode nambil adjibade1, hamidou arame1, stanislas pejanos sonon1 1laboratory of ecology and aquatic ecosystem management (lemea), department of zoology, faculty of sciences and technics, university of abomey-calavi, bp 526 cotonou, benin. 2laboratory of wetland researches (lrzh), department of zoology, faculty of sciences and technics, university of abomey-calavi, bp 526 cotonou, benin. s article history: received 22 september 2019 accepted 8 december 2019 available online 2 5 december 2019 keywords: growth parameters exploitation rate length at first capture management recruitment abstract: the current study was carried out on okpara stream in northern benin to evaluate the demographic factors with inferences on fisheries status of five dominant fish species, which were sampled monthly intervals for 18 months. asymptotic length (l∞) ranged from 36.23 cm (brycinus macrolepidotus) to 18.8 cm (hemichromis fasciatus). growth rates (k) varied between 1.6 yr-1 and 0.66 yr-1 with growth performance index (φ’) ranging from 2.4 to 3.1. theoretical age at length zero (t0) varied -0.95 year for marcusenius senegalensis to -0.59 year for shilbe intermedius. except for b. macolepidotus, total mortalities were higher than 2 year-1 and length at first capture (l50) ranged 7.72-12.09 cm. marcusenius senegalensis and s. intermedius showed one annual peak of recruitment, whereas h. fasciatus, oreochromis niloticus and b. macrolepidotus displayed two peaks. the results indicated that the stocks of these five species were underexploited. however, the low length at first capture (l50) recorded for some species, requires the implementation of early fisheries management scheme to assure a sustainable exploitation of these fishes. introduction in most regions of the world, fishing is one of the foremost activity for human subsistence because of generating substantial incomes for grassroots (ekouala, 2013). as reported by fao (2018), in year 2016, global world fish production reached 171 million metric tons that was estimated at about 362 billion us dollars. in benin, fisheries appeared to be a key component of rural activities and the fishery’s sector contributed substantially to the national economy. indeed, total production of inland and marine fisheries reached 42400 tons estimated at about 42,400,000,000 fcfa or 80,761,904 usd (fao, 2019). moreover, the increase of the african population in general and that of benin in particular, has led to a high fishing pressure and to the overexploitation of inland and marine fish resources (welcomme, 2001). thus, in addition to the drastic decrease of the fish production in most water bodies, exploitable stocks were dominated by small-sized *correspondence: alphonse adite doi: https://doi.org/10.22034/ijab.v7i6.642 e-mail: alphonseadite@gmail.com individuals (dulvy et al., 2003; mullon et al., 2005; chikou, 2006; fao, 2010; sossoukpe et al., 2013). therefore, a rational management scheme of the fish stocks is needed to assure the conservation and the sustainable exploitation of the fish resources. in fish population stock, estimating parameters such as asymptotic length (l∞), growth coefficient (k), fishing/natural mortalities (f/m) and exploitation rate (e), are means of conservation and management (abohwere and falaye, 2008; tah et al., 2010). these fish stock management tools are powerful instruments for development policies and decision making in fisheries sector. data on age and growth are particularly important for describing the status of a fish population and for predicting the potential yield of the fishery. they also facilitate estimation of production, stock size, recruitment and mortality (lowe-mcconnel, 1987). though several aspects of the fish population dynamic and community structure have been 333 int. j. aquat. biol. (2019) 7(6): 332-341 investigated in many aquatic ecosystems in benin, little is known about the demographic parameters and exploitation rate of marine and inland fish species. such studies have been restricted to few species such as galeoides decadactylus, sardinela madarensis and chloroscombrus chrysurus from the marine environment (sossoukpe et al., 2016a, b; 2017), sarotherodon melanotheron from lac toho (lederoun et al., 2015), s. galileus from lac doukon et lac togbadji (lederoun et al., 2016) and tilapia guineensis, hemichromis fasciatus, eleotris vittata, clarias gariepinus, chrysichthys auratus, hepsetus odoe, parachanna obscura and heterotis niloticus from sô river (hazoume, 2017). okpara stream is the main tributary of the oueme river, and in spite of its fisheries importance and degradation pressures, little is known about the demographic parameters and exploitation rates of its fish stocks. as reported by sidi imorou et al. (2019a), okpara stream dwelled 53 species belonging to 29 genera and 14 families. dominant species are hemichromis fasciatus (29.49%), marcusenius senegalensis (16.43%), shilbe intermedius (10.44%), oreochromis niloticus (8.90%) and brycinus macrolepidotus (9.23%) aggregating numerically 74.49% of the fish community total relative abundance (sidi imorou et al., 2019b). these species display a high commercial, economic and nutritional importance for the grassroots. therefore, the current study aims to investigate growth rates, mortality coefficients and exploitation rates of the five key fish species, namely h. fasciatus, m. senegalensis, s. intermedius, o. niloticus and b. macrolepidotus of okpara stream to gather fisheries documentation that will contribute to ensure successful management of these fishes. materials and methods study area: this study was carried out on okpara stream, a tributary of oueme river (200 km), located between 8°14'-9°45'n and 2°35'-3°25'e. this riverine water belongs to the northern hydrographic system and traverses the borgou province of northern benin and that of zou in central benin. annual ambient temperature around the stream averaged 26.6°c and the low temperatures were recorded in december and january. the climate is tropical and comprised two seasons: the dry season in november to april and wet season in may to october. the annual pluviometry averaged 1200 mm, peaked at about 1300 mm in july, august and september. the soil is covered by a wooded savanna characterized by the presence of parkia biglobosa, khaya senegalensis and vitellaria paradoxa. also, the plant community comprised marshy meadows, bamboo and fallow bushes. the okpara stream is the main source of fish resources for the grassroots and provides water for irrigated agriculture. in addition, a dam was built on the stream to supply the surrounding populations with drinking water. sampling sites: five sampling sites were selected (fig. figure 1. location of okpara’s stream and sampling sites. site 1= perere township, site 2 = gadela village (parakou township), site 3= kpassa village (tchaourou township), site 4= yarimarou village (tchaourou township), and site 5 = sui village (tchaourou township). 334 sidi imorou et al./ demographic parameters of five fishes from okpara stream 1) based on localities, accessibility, fisheries importance and level of site degradations (sidi imorou et al., 2019a). site 1 is situated in perere town at okpara up stream and site 2 is located in parakou town at gadela village (okpara up stream) about 2 km from soneb dam. site 3 is located at kpassa village where a dam was built to serve as a source of drinking water for the populations of tchaourou and parakou towns and surrounding villages. site 4 is situated around okpara downstream at yarimarou village (tchaourou town) where the dam withdraws its water. site 5 is also located around okpara downstream at sui village of tchaourou. at these five sites, samplings were done in the “aquatic vegetation habitat” at the edge of the stream and in the “open water habitat” exempt of vegetation and characterized by a high depth. fish collection and identification: fish samplings were done for 18 months by monthly intervals in all habitats at the five sampling sites with experimental gillnet and seine (sidi imorou et al., 2019a, b). in addition, fish samplings were directly made in fishermen artisanal captures by taking one third of each fisherman catches. all rare and uncommon species were included in the sample (okpeicha, 2011). fishing gears such as gillnets, seines, cast nets, hooks, and traps were used by the fishermen to collect the fishes. after collection, the fish samples were first identified in situ using fish identification references such as van thielen et al. (1987), skelton (1993), and lévêque and paugy (2006). the fish assemblages were preserved in a cooler and then transported to the laboratory of ecology and management of aquatic ecosystem to confirm the identifications. the valid scientific names of the fish species have been confirmed on fishbase (froese and pauly, 2018). in the laboratory, length of each specimens was measured to the nearest 1 mm with a measuring board, weighted to the nearest 1 g with an electronic scale (camry) and preserved in 10% formalin and latter in 70% ethanol to make easier other biological observations such stomach content analysis and aspects of reproductive biology (schreck and moyle, 1990; murphy and willis, 1996). data analysis: basic parameters are length-weight relationship (lwr), age-based growth, mortality (z), exploitation rate (e), and condition factor (k) (ricker, 1975). length data were combined monthly and converted into length frequencies with a constant class interval of 2 cm. the mean lengths and weights of classes were used for data analysis using accepted fisat’s format (gayanilo and pauly, 1997). growth parameters (l∞, k and t0) and growth performance index (’): growth parameters are important to determine the stock state. the model of von bertalanffy (1938) of elefan i program in fisat ii is the most used in studies of fish’s growth: tl = l∞{1 − exp[−k(t − t0)]} (king, 1995) where, tl (cm) is the total length of the fish, l∞ (cm) = asymptotic length of fish, l∞ = maximum length that a fish could reach if it lived many years, k (yr-1) = growth coefficient, and t0 (yr) = theoretical age of the fish individual when its length is zero. t0 is determined by the following pauly’s equation: log10(−t0) = −0.392– 0.275 log10l∞ − 1.038 log10k (pauly, 1979). where t0 (yr) is the age of the fish at various lengths and calculated by the inverse of von bertalanffy growth equation. k and l estimates were used to determine the growth performance index (') of the species (munro and pauly, 1983; pauly and munro, 1984) according to the formula: ’ = log10k+ 2log10 l∞ where ’ is growth performance index. this index is an indicator of fish’s well-being. mortality parameters: the instantaneous annual rate of total mortality (z) was evaluated by the construction of linearized curve of length converted into catch (sparre and venema, 1992). instantaneous natural mortality rates (m) were determined by pauly’s (1980) empirical equation: log10m = -0.0066 – 0.279 log10l∞ + 0.6543 log10k + 0.463 log10t with t = annual temperature of okpara stream waters. the fishing mortality (f) rate was determined by the formula: f = z-m probability of capture (l50), longevity (tmax) and epxloitation rate (e): estimates of length at first 335 int. j. aquat. biol. (2019) 7(6): 332-341 capture (l50) were derived from capture probabilities generated by fisat’s capture curve analysis. the estimate of the average growth coefficient k was used to generate longevity as follows: tmax = 3/k (anato, 1999). the exploitation rate (e) was calculated by fisat from the linearized curve of length converted to capture curve of each species: e = f/z, with f = fishing mortality and z = annual total mortality. this value gives a rough estimate of whether the stock is overexploited or not (pauly, 1983). when e>0.5, the stock is overexploited. when e is approximately equal to 0.5, the yield is optimized and f is approximately equal to m (gulland, 1971). results growth parameters: growth parameters are shown in table 1. the asymptotic length, l∞ ranged 18.8 (h. fasciatus) to 36.23 cm (b. macrolepidotus). the values of growth rates (k) varied between 0.66 yr-1 for s. intermedius and 1.6 yr-1 for m. senegalensis. growth performance index (') ranged 2.4 for h. fasciatus and 3.1 for o. niloticus. the theoretical age at length zero (t0) varied -0.95 (m. senegalensis) to -0.59 year (s. intermedius). longevity, instantaneous mortality and exploitation rates: the length converted catch curves (fig. 2) generated the value of total mortality (z), natural mortality (m), fishing mortality (f) and exploitation rate that are presented in table 2. for most species (4/5), z were higher than 2 year-1 excepted for b. macolepidotus (1.79 yr-1) and the higher value of z is 3.65 yr-1 for m. senegalensis. the natural mortality ranged from 2.786 (m. senegalensis) to 1.441 yr-1 (s. intermedius), and the fishing mortality (f) ranged 1.50 yr-1 for h. fasciatus and 0.36 yr-1 for o. niloticus. the exploitation rates ranged 0.44 for h. fasciatus to 0.16 for o. niloticus. these rates were weak compared to the optimum (e = 0.5) when fishing mortality is equal to natural mortality (f = m) (pauly and munro, 1984). the values of longevity were relatively high table 1. growth parameters of von bertalanffy function output by fisat of exploited fishes in okpara stream (oueme river, north-benin) in comparison to populations from other african waters. species locality country l∞ (cm) k (yr-1) t0 (yr) ' references b. macrolepidotus okpara stream benin 36.23 0.73 -0.68 2.98 current study lake ayame i cote d’ivoire 32 0.46 2.67 tah et al. (2010) h. fasciatus okpara stream benin 18.8 0.88 -0.67 2.40 current study lake nokoue benin 16.75 1 2.45 niyonkuru (2007) so stream benin 18.38 0.96 -0.48 2.51 hazoume (2018) lake ayame i cote d’ivoire 27 0.57 2.62 tah et al. (2010) ebrie cote d’ivoire 25 0.85 villanueva (2004) m. senegalensis okpara stream benin 18.90 1.6 -0.95 2.76 current study o. niloticus okpara stream benin 35.18 1.2 -0.90 3.1 current study lake ayame i cote d’ivoire 35.5 0.48 2.78 tah et al. (2010) lake victoria kenya 61.3 0.35 getabu (1992) tanguiga reservoir burkina faso 17.6 0.46 2.15 bajot and moreau (1997) s. intermedius okpara stream benin 25.2 0.66 -0.59 2.62 current study lake nokoue benin 26 0.7 2.67 niyonkuru (2007) table 2. longevity, mortality and exploitation rate of the five key fishes of okpara stream (oueme river, north-benin). species tmax (yr) z (yr-1) m (yr-1) f (yr-1) e z/k l50 (cm) l50/l∞ b. macrolepidotus 4.11 1.79 1.39 0.4 0.22 2.45 7.90 0.22 h. fasciatus 3.41 3.45 1.95 1.50 0.44 3.92 8.78 0.47 m. senegalensis 1.88 3.65 2.786 0.86 0.24 2.28 10.54 0.56 o. niloticus 2.50 2.30 1.941 0.36 0.16 1.92 7.72 0.22 s. intermedius 4.55 2.13 1.441 0.69 0.32 3.23 12.09 0.48 tmax = longevity, z= total mortality, m=natural mortality, f=fishing mortality, e=exploitation rates, l50=length at first capture, k=growth coefficient, l∞= asymptotic length. 336 sidi imorou et al./ demographic parameters of five fishes from okpara stream and the highest was found for s. intermedius (tmax = 4.55 year) and the lowest value was found for m. senegalensis (tmax = 1.88 year). the ratio between z and k was superior to 1 for the five species and ranged from 1.92 (o. niloticus) to 3.92 (h. fasciatus) (table 2). with regards to the ratio between l50 and l∞, the lowest value was recorded for b. macrolepidotus and o. niloticus (l50/l∞ = 0.22) and the highest value was recorded for m. senegalensis (l50/l∞ = 0.56) (table 2). length at first capture and recruitment: the length at first capture (l50) is the estimate length of fishes at 50% probability of capture (fig. 3). in this study, the highest l50 was recorded for s. intermedius (12.09 cm) and the lowest one for o. niloticus (7.72 cm) (table 2). the recruitment structure (fig. 4) of fishes showed one annual peak of recruitment for m. senegalensis and s. intermedius. species like h. fasciatus, o. niloticus and b. macrolepidotus showed two peaks of recruitment in a year (fig. 4). discussions the asymptotic length (l∞ = 18.8 cm) value recorded for h. fasciatus, the dominant species, agreed with that reported by hazoume (2017) in the sô river (18.38 cm) in benin. however, these values of l∞ were lower than those reported by villanueva (2004) in ebrie lagoon (25 cm) and by tah et al. (2010) in lake ayame i (27 cm) in ivory coast. also, the value recorded for o. niloticus (35.18 cm) in this study agreed with that reported by tah et al. (2010) in lake ayame i (35.5 cm) of ivory coast. in contrast, o. niloticus showed higher l∞ = 64.6 cm in lake victoria in kenya (getabu, 1992) while s. intermedius exhibited an asymptotic length (25.2 cm) nearly identical to that reported by niyonkuru (2007) in lake nokoue (26 cm) in benin. in this study, the growth rates (k) between 1.6 and 0.66 yr-1 recorded for these key species were higher than those reported by tah et al. (2010) in lake ayame, by villanueva (2004) in ebrie lagoon, by getabu (1992) in lake victoria and by bajot and moreau (1997) in tanguiga reservoir of burkina faso. nevertheless, in the current study, h. fasciatus displayed a lower growth rate (0.88) compared to those of lake nokoue (1.0) and sô river (0.96). this a b c d e figure 2. length converted catch curves of dominant fishes of okpara stream (oueme river, north-benin): a = hemichromis fasciatus; b =marcusenisius senegalensis; c = schilbe intermedius; d = oreochromis niloticus; e = brycinus macrolepidotus. 337 int. j. aquat. biol. (2019) 7(6): 332-341 could be explained by the fact that in transitory ecosystems such as coastal lagoons (lake nokoue, brackish sites of sô river), fishes quickly reach their maximum size with shorter life span than those living in freshwaters (longhurst and pauly, 1987; tah et al., 2010). in addition, the high abundance of h. fasciatus in okpara river could cause intraspecific food competition, reducing the growth rate of this topa b c d e figure 3. probability of capture and length of first capture of fishes in okpara stream (oueme river, north-benin): a = hemichromis fasciatus; b =marcusenisius senegalensis; c = schilbe intermedius; d = oreochromis niloticus; e = brycinus macrolepidotus. a b c d e figure 4. recruitment patterns of dominant fishes in okpara stream (oueme river, north-benin): a = hemichromis fasciatus; b =marcusenisius senegalensis; c = schilbe intermedius; d = oreochromis niloticus; e = brycinus macrolepidotus. 338 sidi imorou et al./ demographic parameters of five fishes from okpara stream carnivorous species. the growth performance index (') ranged between 2.40 (h. fasciatus) and 3.1 (o. niloticus). the ' fall within 2.65<'<3.32, the range of ' values reported by baijot and moreau (1997) for slow-growing fish species. the ecosystem disturbances factors could have negatively affected the well-being of the fishes and leading to reduced growth performances. most of these degradation factors of okpara stream were the dumping of domestic garbage, the withdrawal of water by soneb, a benin water company, the withdrawal of water for irrigation, the use of chemical fertilizers/pesticides for agriculture, the introduction of invasive exotic fishes such the o. niloticus and the proliferation of water hyacinth, an invasive floating plant. the average value of ' recorded in this study agreed with to those reported by hazoume (2017) in sô river in benin and by adeyemi et al. (2009) in gbedikere lake in nigeria exhibiting average growth performances ' = 2.75 ± 0.41 and ' = 2.79 ± 0.19, respectively. however, the current value of ' in okpara stream was lower than those reported by ahouansou montcho et al. (2011) in pendjari river in benin and by du feu (2003) in kainji lake where ' values averaged 3.22 ± 0.31 and 3.19 ± 0.31, respectively. inversely, niyonkuru (2007) found an average value of ' = 2.58 ± 0.19, lower than that recorded in this study. these differences could be mainly attributed to environmental conditions and food availability. overall, fish longevity (tmax) ranged between 1.88 yr (m. senegalensis) and 4.55 yr (s. intermedius). these values were lower than those reported by hazoume (2017) on sô river where tmax was higher and reached 11.66 yr, probably because the sô river floodplains stand as breeding and nursery/growing grounds for several fish species. in okpara stream, natural mortality (m) of fishes was higher than those of the sô river by hazoume (2017). these results imply that as natural mortalities (m) of fishes increase, longevities (tmax) decrease. in the current investigation, for all species, natural mortalities (m) were greater than fishing mortalities (f) and the exploitation rates (e) were lower than 0.5. as results, stocks of b. macrolepidotus, h. fasciatus, m. senegalensis, o. niloticus and s. intermedius were underexploited in okpara stream (tah et al., 2010). villanueva (2004) and tah et al. (2010) came up with the same observation and trends in some west african lagoons/estuaries such as lake ayame i of ivory coast. nevertheless, in the current study, for each species, the ratio of total mortality (z) to k is greater than 1, leading to a situation where mortalities dominated growth (barry and tegner, 1989). with regards to length at first capture (l50), species such as b. macrolepidotus and o. niloticus had l50 of 7.9 and 7.72 cm, respectively. these values were lower than those reported by tah et al. (2010) in lake ayame i, l50 = 11.57 and l50 = 14.03 cm for b. macrolepidotus and o. niloticus, respectively. recruitment are occurred throughout the year with two peaks per year for the majority of species (h. fasciatus, o. niloticus and b. macrolepidotus), indicating two main periods of spawning. this observation agreed with the findings of many african fisheries biologists (pauly, 1982; welcomme and de merona 1988; ahouansou montcho et al., 2011) who found that tropical fish species reproduced twice a year, and mainly during the wet season. in the current study, all the five fish species displayed some exploitation rates (e = f/z) ranging between 0.160.44, less than 0.5, suggesting that the stocks of these fishes in okpara stream were underexploited (pauly and moreau, 1997). conclusion the relatively high growth rates (k) and growth performance index (') displayed by b. macrolepidotus, m. senegalensis, h. fasciatus, o. niloticus and s. intermedius indicated that these fish species showed a good adaptation to ecological condition of okpara stream. also, the low exploitation rates (k) indicated that the stocks of these fishes in okpara stream were underexploited. nevertheless, the low length at first capture (l50) of some fishes such o. niloticus, b. macrolepidotus and h. fasciatus is harmful for the survival, the conservation and the 339 int. j. aquat. biol. 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(2019) 7(4): 180-194 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article updated list of alien macrozoobenthic species along the syrian coast izdihar ammar*1 department of marine biology, high institute of marine research, tishreen university, lattakia, syria. s article history: received 5 february 2019 accepted 27 july 2019 available online 2 5 august 2019 keywords: marine biodiversity macrozoobenthos invasive alien species eastern mediterranean sea abstract: marine biodiversity along the syrian coast is affected by climate change-related temperature increase leading to the migration and entrance of alien species, especially from the suez canal (biological invasion). the syrian coast is chracterized by many different types of habitats, including marine caves, midilittoral bioconstructions (lithophyllum byssoides rim and vermetid tarraces), coraligenous communities, sandy dunes, rocky coast and seagrass meadows. the present study summarizes early and most recent results on the presence, distribution, abundance and conservation status of marine alien species along the syrian coast and provides an updated list of alien macrozoobenthic species. a total of 79 alien species belonging to mollusca, crustacea, tunicata, polychaeta, spongia, echinodermata and chaetognatha are reported; among them gastropoda is being the most abundant and successful taxon. in the light of these results, some recommendations on future research directions are provided. in particular, more effort is needed to monitor and record the entrance of alien species by adopting a comprehensive national plan to study marine biodiversity and to protect local resources in the syrian marine environment. introduction marine biodiversity in the mediterranean sea is undertaking major changes due to climate changerelated increase in water temperature. local species from temperate waters disperse throughout the mediterranean sea, while phytoand zoobenthic species from the warmer red sea enter the mediterranean sea through the suez canal. the introduction of alien species (biological invasions) is considered to be a major threat to marine biodiversity along with overfishing, pollution and habitat destruction, causing serious environmental and economic consequences (ruiz et al., 1997; carlton, 2000; bax et al., 2003). there are many examples of disastrous invasions by these species that caused the loss of native species, changes in community structure and their functions, besides damages to local fishery and aquaculture. the alien species found in the mediterranean sea is approaching 1,000 (zenetos et al., 2012; katsanevakin et al., 2016), with an introduction rate of one species *correspondence: izdihar ammar doi: https://doi.org/10.22034/ijab.v7i4.556 e-mail: izdiammar@gmail.com every 1.5 week (zenetos, 2010; zenetos et al., 2012). in the mediterranean sea, the distribution and spread of alien species varies from country to another. they are more preponderant in the eastern basin (775 alien species) than in the western part, most of them being lessepsian species that have entered the mediterranean through the suez canal. these species include 13 taxonomic groups dominated by mollusca (215 species), followed by crustacea (159 species) and polychaeta (132 species). among these alien species, over 600 species are well-established in the mediterranean sea (zenetos et al., 2017). some alien invertebrates have also become dominant components of the region and greatly altered the benthic community structures (çinar et al., 2011). these species can drastically alter the food web in the area, compete with native species for food and space, and transmit new diseases to native species (por, 1978; çinar et al., 2005). the syrian coast (183 km in length) is located in the middle part of the eastern mediterranean sea, extending geographically from 181 int. j. aquat. biol. (2019) 7(4): 180-194 al-hamedia in the south to ras al-bassit in the north. the mediterranean coast forms an integrated unit in terms of geological structure and topography. it is exposed to several environmental problems as a result of anthropological activities, which dispose untreated wastes directly into the marine environment. hence, water pollution causes deterioration of coastal ecosystems, which influences the public health, fishing and tourism. furthermore, the suez canal which is 650 km away from the syrian coast represents a critical passage for indian and pacific species migrating through the red sea to the mediterranean sea, some of which settle and become highly invasive. for these reasons, the geographical distribution, dispersion and reproduction of those species along the syrian coast has to be considered a fundamental issue to study. benthic habitats along the coast of syria have been densely colonized by lessepsian species. in addition, tartous in the south and lattakia in the north have large international harbours and jetties for crude oil and coal transfers. therefore, the region is also a recipient area for ship-transferred species into the mediterranean sea. over the last several years zoobenthic communities and their distributional patterns in syria have been studied within scientific projects and various studies at littoral, sublittoral and deep sea areas. some papers reported quantitative and qualitative attributes of zoobenthic communities (saker and ammar, 1996; kucheruk et al., 1998; saker et al., 1999, 2000) in the north sector of the syrian coast, such as lattakia and al-bassit (torchia et al., 2004; ammar, 2004, 2010; ammar et al., 2008, 2011). the objective of the present study was to review, update and present a unique and updated list of alien macrozooobenthic species and taxa which have been recorded until 2018 along the syrian coast. materials and methods we conducted a review of the alien species found between 1994 and 2018 at various depths and sites of the syrian coast (fig. 1). available information was gathered from both published and unpublished works. samples were collected either by hand or using benthic sampling devices (e.g. trawl, trammel net and grab). in addition, new soft bottom benthic samples were collected in 2015 and 2016 in the marine protected area of ibn-hani, north of lattakia, at depths ranging between 15-40 m. more recent samples were collected from different areas of the syrian coast during the year of 2018. nomenclature follows the world register of marine species (worms, 2018). results and discussion alien species along the syrian coast: the rise of water temperatures due to climate change in the eastern shores of the mediterranean sea overlaps with the entry and settlement of new species in the region in several ways. these include a direct impact on individuals and populations due to changes in the local physical and chemical conditions and an indirect impact related to changes in species distribution and reproduction, and competition between local and alien species often resulting in an expansion of former. during previous researches, which were conducted along the syrian coast, more than 600 species belonging to 16 macro-taxa, including porifera, cnidaria, bryozoa, sipunculida, polychaeta, gastropoda, bivalvia, cephalopoda, scaphopoda, figure 1. map of syrian coast. 182 ammar / alien macrozoobenthic species along the syrian coast crustacea, echinodermata, brachiopoda, ascidiacea and other minor taxa, were recorded at a depth up to 600 m in both hard and soft bottom (ammar et al., 2013; ammar, 2016). most of these species are atlantic and mediterranean fauna, 79 of them being alien. the largest number of alien species in syria belongs to mollusca (bitar et al., 2003; ammar, 2004; the present study). this phylum also contributes the most of alien species in the eastern mediterranean and european seas (katsanevakis et al., 2013; nunes et al., 2014). an updated list of alien zoobenthic species found along the syrian coast, including the time of first record, the type of habitat and depth was explained in the table 1. early studies on lessepsian migration took place along the coast of lattakia and banias, where 16 gastropod species, 9 bivalve species and 11 crustaceans were found (saker and ammar, 1994a, b; saker and farah, 1994). subsequent research recorded new species belonging to several benthic taxa, including mollusca, crustacea, polychaeta, spongia and chaetognata (hassan et al., 2008; arabia, 2011; ammar et al., 2013; ammar, 2016a, b). currently, alien species of zoobenthos represent almost 11.55% of the total number of zoobenthos in the syrian coast. the great majority of the alien species belong to 3 taxa, including gastropoda (33 species, 13.41% of the total), bivalvia (12 species, 9.02%) and crustacea (24, species 23.76%), followed by tunicata (3 species 3.79%) and annelida (2 species, 2.27%) and 5 species other minor taxa (table 2). figure 3 shows the percentage of alien species for each main taxonomic group represented by mollusca (57%) followed by crustacea (30%), tunicata (4%) and the rest form (9%). a total of 53 alien zoobenthic species were already described along the syrian coast in previous local studies (ammar, 1995, 2002, 2010, 2014, 2016a, b). of these, 22 species belong to mollusca (acteocina mucronata, brachidontes pharaonis, cerithium (thericium) scabridum, chama pacifica, pyrgulina figure 2. number of native, alien and cryptogenic species for each taxonomic group. figure 3. percentage of alien species for each taxonomic group. 183 int. j. aquat. biol. (2019) 7(4): 180-194 table 1. updated list of the 79 alien and cryptogenic species zoobenthic found along the syrian coast. species first record habitat depth abundance status reference gastropoda diodora ruppellii (sowerby, 1835) lattakia, 1994 hard bottom, littoral littoral 10 m common established ammar (1995) smaragdia souverbiana (montrouzier in souverbie & montrouzier, 1863) al-bassit, 2003 mud-gravel 5 -35 m few established katsanevakis et al. (2014) trochus erithreus brocchi, 1821 lattakia, 1992 hard bottom, littoral littoral available established ammar (1995), bitar et al. (2003) pseudominolia nedyma (melvill, 1897) lattakia albassit, 2005 mud-gravel 5-140 m 8-912 ind./m2 established ibrahim et al. (2005), arabia (2011), ammar et al. (2013) cerithium scabridum (philippi,1848) lattakia, 1938, 1994 hard bottom, littoral, sandgravel < 25 m dominant established pallary (1938) rhinoclavis kochi (philippi, 1848) lattakia, 1994 sandy < 17 m dominant established ammar (1995) finella pupoides adams a., 1860 al-bassit, 2007 mixed bottom 25-40, 120140 m 8-432 ind./m2 established arabia (2011), ammar et al. (2013) clathrofenella ferruginea (adams, 1860) al-bassit, 2007 mixed bottom 40 m 12-24 ind./m2 established arabia (2011), ammar et al. (2013) cerithiopsis pulvis (issel, 1869) al-bassit, alhamedeia, 2007 mixed bottom 25-40 m 24-40 ind./m2 established arabia (2011) cerithiopsis tenthrenois (melvill, 1896) al-hamedeia, 2007 mixed bottom 17-25 m 24-56 ind./m2 established arabia (2011) rissoina bertholleti issel, 1869 lattakia, 1994 sand gravel 5-12 m few established ammar (1995) alvania dorbignyi (audouin, 1826) al-bassit, banias and alhamedeia, 2007 mixed bottom 17-40, 120160 m 8-368 ind/m2 cryptogenic arabia (2011), ammar et al. (2013) conomurex persicus (swainson, 1821) lattakia, 1985, 1994 hard bottom; soft bottom littoral 70 m dominant established gosselck et al. (1986), ammar (1995, 2002) erosaria turdus (lamarck, 1810) al-bassit, 2007 mixed bottom 25, 160 m 8 ind/m2 established arabia (2011), ammar et al. (2013) sticteulima cf. lentiginosa (adams a., 1861) al-hamedeia, 2007 detritus 17-25 m 16 ind./m2 established arabia (2011) ergalatax junionae (houart, 2008) al-bassit, 2004 hard bottom, littoral littoral dominant established ammar (1995, 2002, 2010), ibrahim et al. (2005) indothais lacera (born, 1778) lattakia, 1994 hard bottom, littoral littoral few established ammar (1995) murex forskoehlii (röding, 1798) lattakia, 2005 mud-sand >30 m few established katsanevakis et al. (2014) fusinus verrucosus (gmelin, 1791) lattakia, 2004 mud 70 m available established katsanevakis et al. (2014) zafra selasphora (melvill and standen, 1901) al-bassit and al-hamedeia, 2007 mixed bottom 15-40 m 8-78 ind./m2 established arabia (2011) conus fumigatus hwass in bruguière, 1792 al-bassit and tartous, 2007 mixed bottom 17-40, 120140 m 3264 ind./m2 established arabia (2011), ammar et al. (2013) pyrgulina maiae hornung & mermod, 1924 lattakia, 1992 muddy-detritus 5-140 m 8-72 established arabia (2011), ammar et al. (2013, 2015) styloptygma beatrix melvill, 1910 ibn-hani, 2015 mixed bottom 15-40 few established present study (new records) syrnola cinctella a. adams, 1860 ibn-hani, 2015 mixed bottom 15-40 m few established present study (new records) syrnola fasciata (jickeli, 1882) al-bassit, 2007 mixed bottom 30, 120-140 m 8-232 ind./m2 established arabia (2011), ammar et al. (2013) odostomia lorioli (hornung mermod, 1924) al-bassit, 2007 mixed bottom 30-40, 110160 m 8-72 ind./m2 established arabia (2011), ammar et al. (2013) 184 ammar / alien macrozoobenthic species along the syrian coast table 1. continued. species first record habitat depth abundance status reference acteocina mucronata (philippi, 1849) al-bassit and al-hadmedia, 2003 mixed bottom 5, 20-40 m 16-24m established ibrahim et al. (2005), arabia (2011) ventomnestia girardi (audouin, 1826) ibn-hani, 2015 mixed bottom 15-40 few established present study (new records) pyrunculus fourierii (audouin, 1826) al-bassit and banias 2007 mixed bottom 30-40, 120140 m 8-168 ind./m2 established arabia (2011), ammar et al. (2013) bulla ampulla linnaeus, 1758 al-bassit, 2007 mixed bottom 40 m 8-16 ind./m2 established arabia (2011) thecacera pennigera (montagu, 1813) lattakia, 2013 muddy 100 m 1 ind. cryptogeni present study (new records) aplysia dactylomela (rang, 1828) lattakia, 2013 rocky littoral 2 specimens established katsanevakis et al. (2014) goniobranchus annulatus (eliot, 1904) tartous, 2018 rocky 10 m 1 ind. established ammar and solaiman (unpublished) bivalvia brachidontes pharaonis (krauss, 1962) lattakia, 1931, 1994 hard bottom littoral 5 m dominant established gruvel and moazzo (1931), ammar (1995) septifer cumingii récluz, 1848 lattakia, 2015 sandy 15 m dominant established present study (new records) crassostrea gigas (thunberg, 1793) lattakia, 1993 rocky littoral dominant established ammar (1995, 2002, 2010), ibrahim et al.(2005) saccostrea cucullata (born, 1778) lattakia, 1993 hard bottom littoralsublittoral – 100 m dominant established ammar (1995, 2002, 2010), ibrahim et al. (2005) pinctada imbricata radiata (leach, 1814) lattakia, 1994 (since 1975) hard bottom littoral-8 m dominant established kinzelbach (1985) malleus regula (forsskål in niebuhr, 1775) 1931 (collecting place not given) hard bottom 5 m dominant established gruvel and moazzo (1931) spondylus spinosus (schreibers, 1793) banias, 1999 hard bottom 3-15 m dominant established bitar et al. (2003) chama pacifica broderip, 1834 lattakia, 1993 hard bottom littoral-8 m dominant established bitar et al. (2003) afrocardium richardi (audouin, 1826) lattakia, 2009 soft bottom 15-120 m 8 ind/m2 established ammar et al. (2013), pressent study gafrarium savignyi (jonas, 1846) lattakia, 1993 rocky and sand littoral -50 m dominant established ammar (1995, 2002), ammar (2010) circenita callipyga (born, 1778) lattakia and tartous, 2005 mud 25 m rare established ibrahim et al. (2005) paratapes textilis (gmelin, 1791) lattakia, 19921993 shelf bottoms offshore common established kucheruk and basin (1999) cephalopoda ommastrephase bartramii (lesueur, 1821) ras al-basset and jableh, 2016 mud 40-130 m common established ammar and maaroof (2016c) crustacea penaeus pulchricaudatus stebbing, 1914 lattakia, 1928 soft bottom > 70 m dominant established gruvel (1928) metapenaeopsis mogiensis consobrina (nobili, 1904) lattakia, 2012 muddy 64-100 single specimen established ammar et al. (2013) metapenaeus monoceros (fabricius, 1798) 1980 (collecting place not given) soft bottom >60 m abundant established holthuis (1980), ammar, (2016b) metapenaeus stebbingi nobili, 1904 lattakia, 1996 muddy sand 50-90 m rare established saker and farah (1994) penaeus semisulcatus de haan, 1844 (in de haan, 1833-1850) lattakia, 1928 soft bottom 500-600 m abundant established gruvel (1928) trachypenaeus curvirostris malaiana balss, 1933 banias, 1999 soft bottom > 70 m rare established saker and farah (1994), hasan et al. (2008) 185 int. j. aquat. biol. (2019) 7(4): 180-194 table 1. continued. species first record habitat depth abundance status reference leptochela (leptochela) aculeocaudata paul'son, 1875 banias, 1999 hard bottom and soft bottom 10, 14 m few established saker and farah (1994), ammar (2002) leptochela (leptochela) pugnax de man, 1916 lattakia, 1994 muddy 110160 m 8 ind./m2 established saker and farah (1994), hasan et al. (2008), ammar et al. (2013) alpheus inopinatus holthuis & gottlieb, 1958 lattakia, 2006 in cystoseira 1 m single specimen established hassan et al. (2008) alpheus migrans lewinsohn & holthuis, 1978 lattakia, 2009 rocky 64-100 m single specimen established ammar et al. (2013) saron marmoratus (olivier, 1811) latakia, 2018 rocky 8-10m 4 individuals established ammar and raea (2019) ixa monodi holthuis & gottlieb, 1956 lattakia, 1995 among macroalgae 2 m 2 specimens established hassan et al. (2008) coleusia signata (paul'son, 1875) lattakia, 1994 sand, mud up to 100 m rare established saker (2002), hasan et al. (2008) myra subgranulata kossman, 1877 lattakia, 1996 sand, mud 14 m, 500 600 dominant established saker and farah (1994), hassan et al. (2008), ammar et al. (2013), ammar (2016b) micippa thalia (herbst, 1803) lattakia, 1993 hard bottom 2 m one specimen established hassan et al. (2008) ashtoret lunaris (forskål, 1775) latakia, 2017 sand littoral 2 specimen established ammar and arabia (2018) charybdis (charybdis) hellerii (a. milne-edwards, 1867) lattakia, 1992 soft bottom 5 >70 m abundant established kuznetsov et al. (1993), saker and farah (1994, 1997), saker (2002), ibrahim et al. (2005), hassan et al. (2008), ammar (2010) charybdis (goniohellenus) longicollis leene, 1938 lattakia, 2003 soft bottom 69-100, 500-600 m dominant established ibrahim et al. (2005), ammar et al. (2013), ammar (2016b) portunus (portunus) segnis (forskål, 1775) lattakia, 1930 soft bottom 5-70 m dominant established gruvel, 1928), monod 1931), saker and farah (1994), ammar (2002), ibrahim et al. (2005) hassan et al. (2008) ammar et al. (2013), ammar (2016b) thalamita poissonii (audouin, 1826) lattakia, 2005 soft bottom 50 60 m rare established ibrahim et al. (2005), hasan et al. (2008) glabropilumnus laevis (dana, 1852) banias, 1996, 2002 hard bottom and soft bottom 10, 14 m few established ammar (2002), hassan et al. (2008) atergatis roseus (rüppell, 1830) lattakia, 2002 rocky sandy infralittoral abundant established saker (2002), ammar (2010) macrophthalmus (macrophthalmus) indicus davie, 2012 lattakia, 2003 hard bottom 13-120 m 24 ind./m2 established bitar et al. (2003), brahim, et al. (2005), ammar et al. (2013) erugosquilla massavensis (kossmann, 1880) banias, 2002 mud > 70 m abundant established ammar (1995), hassan et al. (2008) tunicata phallusia nigra savigny, 1816 banias, 1999 hard bottom littoral dominant established bitar et al. (2003) herdmania momus (savigny, 1816) ibn hani, 2002 rocky 26-31 m dominant established bitar et al. (2003) ascidiella aspersa (müller, 1776) himr, 1993 rocky littoral dominant established present study (new records) annelida spirobranchus tetraceros (schmarda, 1861) al bassit, 2007 sandy-muddy 40-50 m 8-16 ind./m2 established bitar et al. (2003), ammar et al. (2011) spirorbis (spirorbis) marioni caullery, mesnil, 1897 lattakia, 1995 rocky littoral dominant established ammar, 1995), zibrowius and bitar (2003) https://en.wikipedia.org/wiki/guillaume-antoine_olivier https://en.wikipedia.org/wiki/peter_forssk%c3%a5l 186 ammar / alien macrozoobenthic species along the syrian coast maiae, diodora ruppellii, ergalatax junionae, fusinus verrucosus, gafrarium savignyi , malleus (malvufundus) regulus, paratapes textilis, pinctada imbricate radiata, pseudominolia nedyma, rhinoclavis kochi, rissoina bertholleti, saccostrea cucullata, smaragdia souverbiana, spondylus spinosus, conomurex persicus, indothais lacera and trochus erithreus) and 24 species belong to crustacean (alpheus inopinatus, a. migrans, atergatis roseus, balanus trigonus, charybdis (charybdis) hellerii, c. (goniohellenus) longicollis, erugosquilla massavensis, heteropanope laevis, ixa monodi, leptochela aculeocaudata, l. pugnax, coleusia signata , macrophthalmus graffei, penaeus pulchricaudatus, p. semisulcatus, metapenaeopsis mogiensis consobrina, metapenaeus monoceros, m. stebbingi, micippa thalia var. caledonica, myra subgranulata, portunus (portunus) segnis, thalamita indistincta, t. poissonii and trachysalambria palaestinensis. other species already reported include two species of echinodermata (ophiactis parva and synaptula reciprocans), two species of annelida (spirobranchus tetraceros and spirorbis marioni), one species of cnidaria (macrorhynchia philippina) and two species of chordate (herdmania momus and phallusia nigra). other species known as aliens in the mediterranean were already described in local studies on the syrian coast, but not reported in the international database as present in syria. these include 13 species of gastropoda collected during the period 2007-2014 from different sites, including alvania dorbignyi, bulla ampulla, cerithiopsis pulvis, c. tenthrenois, clathrofenella ferruginea, conus fumigatus, erosaria turdus, finella pupoides, odostomia lorioli, pyrunculus fourierii, sticteulima cf. lentiginosa, syrnola fasciata and zafra selasphora (arabia, 2011; ammar, 2016a). furthermore, 2 species of bivalvia viz. afrocardium richardi and circenita callipyga were reported (ammar, 2002; ibrahim et al., 2005; ammar et al., 2013). as research continued, one species of cephalopods i.e. the flying squids, ommastrephase bartramii (lesueur, 1821) has recorded for the first time locally, three individuals table 1. continued. species first record habitat depth abundance status reference echinodermata ophiactis macrolepidota marktanner-turneretscher, 1887 lattakia, 2003 rocky 7-11 m few established zibrowius and bitar (2003) synaptula reciprocans (forsskål, 1775) ras samra, ibnhani, 2003 cymodocea nodosa meadow 12-32 m few established bitar et al. (2003) cnidaria macrorhynchia philippina kirchenpauer, 1872 north of lattakia, 1999, rocky 7m few established zibrowius and bitar (2003) spongia agelas linnaei (de voogd et al. 2008) latakia, 2014 rocky 15-30 m one specimen established ammar and fadel (2017) table 2. number and percentage composition of native and alien species along the syrian coast. total number of species native species alien and cryptogenic species percentage of alien species on the total gastropoda 246 213 33 13.41 bivalvia 133 121 12 9.02 polychaeta 88 86 2 2.27 crustacea 101 77 24 23.76 spongia 29 28 1 3.45 others 87 82 7 8.04 total 684 607 79 11.55 187 int. j. aquat. biol. (2019) 7(4): 180-194 figure 4. new recorded alien species of the syrian coastal water. 188 ammar / alien macrozoobenthic species along the syrian coast were collected in march and april 2016, two individuals from the ras al-basset coast and one from the jableh coast, at depths ranging from 40-130 m (ammar and maaroof, 2016). in addition, one species of sea slugs, goniobranchus annulatus (eliot, 1904) has recently collected (in october 2018) from tartous in the south of syria. this lessepsian migrant was invaded the mediterranean sea, and recorded since 2007 (daskos and zenetos, 2007). new recorded alien crustaceans are four individuals of saron marmoratus (olivier, 1811) collected from the rocky bottom near lattakia port in 2018, (ammar and raya, 2019) and two individuals of ashtoret lunaris (forskål, 1775) from the sandy beach in south of latakia in 2017 (ammar and arabia, 2018), seven individuals of metapenaeopsis aegyptia (galil and golani, 1990) from the depth of 60 m in 2012 from the latakia coast (ammar, 2016). furthermore, new record of exotic species of demospongia agelas linnaei (de voogd et al., 2008) was reported for the first time in the syrian coast from sublittoral area at 15-30 m of ibn hani region in aprilmay 2014. this species is indo-pacific origin (ammar and fadel, 2017). the present study also reports new records of alien species in the marine protected area of ibn hani, north of lattakia, at depths ranging between 1-40 m, collected during 2015-2016. these include two species of gastropoda, including s. cinctella and ventomnestia girardi, and one species of bivalvia, septifer cumingii (table 1). these species are indopacific origin, introduced to the mediterranean sea via the suez canal (zenetos et al., 2004). trends and distribution: the increase in the number of alien species in the syrian waters is due to the continuing introduction of new species from the red sea and neighboring areas as well as the increase in research effort which is conducted in new areas and depths. regarding mollusca, 31% of the total number of individuals are found in the soft bottom at depth 90160 m (ammar et al., 2013). in the littoral region, the invasive species of b. pharaonis, e. junionae, t. erithareus and c. scabridum are dominate in the coastal region, where they have been found for more than twenty years, similar to b. pharaonis (saker and ammar, 1994b) and c. scabridum and e. junionae (saker and ammar, 1994a). on the hard bottom of the sublittoral zone, the most important alien species in terms of distribution and abundance are p. nedyma, c. scabridum, f. pupoides, s. fasciata, r. kochi, c. persicus (gastropoda), and b. pharaonis, g. savignyi (jonas, 1846), p. imbricata radiata, m. regula, c. pacifica and s. spinosus (bivalves). in the soft bottom, septifer forskali is dominant at depth 15-40 m and s. cucullata attached to s. tetraceros at depth of 60 m. all new alien species are of indopacific and/or red sea origin, introduced to the mediterranean via the suez canal. the majority of new alien gastropods are still spreading in few numbers, which did not exceed 56 individual/m2 in the benthic habitats at the littoral and sublittoral zone, lower than a depth of 160 m. many other species known as invasive are dominant or common in the littoral and/or sublittoral habitats such as c. scabridum, and r. kochi. conomurex persicus, e. junionae, d. ruipplli and t. erithraeus. rhinoclavis kochi, c. scabridum, and murex forskoehli were found at deep syrian water, although both r. kochi and c. scabridum are the most important invasive species in the shallow area. abundance of g. savignyi is increasing and ruditapes decussatus appears at deeper sites. concerning crustacean, 90 species known from different areas and found between 1992 and 2016 (saker and farah, 1994, 1997; ammar, 2002; arabia, 2011; hassan et al., 2008; ammar, et al., 2013). 24 species are indo-pacific origin (23.76%). most of these species are brawns, lobsters and crabs that economically important. lessepsian migrant shrimps, p. pulchricaudatus and p. semisulcatus are commercially important and more abundant at the depth (50-100 m) on the muddy and sandy bottoms as well as presence of m. monoceros in the same medium. portunus segnis is common at depths of 5070 m. erugsquilla massawenses (migrant lobster) is found at a depth over 50 m. two crab species viz. m. subgranulatais and c. helleri are dominant on https://en.wikipedia.org/wiki/mediterranean_sea https://en.wikipedia.org/wiki/guillaume-antoine_olivier 189 int. j. aquat. biol. (2019) 7(4): 180-194 muddy bottom at depth of >50 m. leptochela aculeocaudata and glabropilumnus laevis are found on the rocky bottom in sea grass habitats. recent studies showed occurrence of two species of crabs, m. subgranulata and c. longicollis in the muddy bottom at depth of 500-600 m (ammar, 2016). the number of alien crabs on the syrian coast, which is 14, does not seem to be significant in comparison to total number of exotic crabs recorded in the mediterranean sea that reported to be 39 until 2013 (zaouali et al., 2012; karhan et al., 2013). two cryptogenic alien species viz. thecacera pennigera and a. d’orbigny are found in 2013 from a muddy bottom at depth of 100 m and in 2011 at depths ranged 17-160 m in the mixed bottom, respectively. concerning annelida, the total number is 89 species (ammar 1995, 2002; ammar et al., 2011) that two alien species reported in previous studies (ammar, 1995; bitar, 2003), s. (spirorbis) marioni being dominant on the rocky substrate in the littoral zone and s. tetraceros is found in the soft bottoms in the sublittoral zone. in conclusion, the expansion of alien species in the syrian coast could be explained in the different behaviour or dynamic of these species with time (van aartsen, 2006) and their ability to tolerate the disturbances in the marine environment (çinar et al.,2006; occhipinti-ambrogi and savini, 2003), especially regarding the increase in water temperature in deep sea associated with climate changes, with a sharp change in the hydrological characteristics of the eastern basin (lejeusne et al., 2010; raitsos et al., 2010; lelieveld et al., 2012). many alien species were found at depth >100 m, few were dominant, this observations refer to expand some of alien species from the shallow water to the deep sea some of them dominating since more than 20 years. establishment success of invasive species: most of 79 reported alien species found along the syrian coast are established, none of them is questionable, casual or cryptogenic (table 1). some of them are dominant in the marine habitats and compete with native species for food and habitat. these include c. scabridum, p. maiae, f. pupoides, r. kochi, c. persicus, p. textilis. eleven species are listed in the black list of marine invasive species and some of these species are harmful since they affect local species or biodiversity, especially f. pupoides, r. kochi, c. persicus, c. pacifica, s. tetraceros, l. pugnax and c. longicollis. in addition, species such as p. imbricata radiata, crassostrea gigas and s. cucullata have a negative impact on the fish resources and environmental health as well as on underwater constructions in sublittoral zone. chama pacifica and s. spinosus replaced the two native species c. phoides and s. gaederopus. the pharaonic mussel b. pharaonis displaced the native mytilid, mytilaster minimus completely, with a great impact on the local benthic populations. cerithium scabridum has replaced c. vulgatum, and c. rupestra. c. persicus, a. drobigni and c. kochi are present in high density and biomass. the economic impact of the most of invasive species in syria is little explored. some of them, such as c. pacifica and s. spinosus, are valuable species for seashell collectors or embellishment with a small trading market. pinctada imbricate radiata is sold for food in the markets and restaurants p. pulchricaudatus, p. semesolcatus and m. monocerus are also commercially important and valuable in the market. the number of invasive species increases with the number of species entering the eastern mediterranean. according to zenetos (2010, 2012), presently, more than 30 species of zoobenthos have been described as invasive species in the syrian coast. it is also noticeable the spread of some invasive species into deeper areas. many species of crustacean viz. p. pulchricaudatus and p. semisulcatus are abundant along the syrian coast in the sublittoral area down to depth 100 m. in addition, m. monoceros, m. stebbingi, p. segnis and e. massavensis are also abundant. the negative impact of alien species in the mediterranean sea is documented in many works (occhipinti ambrogi, 2000; streftaris and zenetos, 2006; eea, 2012). success of the most established alien zoobenthic species in syrian coast can be explained with the hydrological characteristics of syrian marine waters, showing an increase in salinity higher than 39 psu, and a temperature ranging 13-32°c. taking in 190 ammar / alien macrozoobenthic species along the syrian coast consideration that all of alien species are thermophilic species, the syrian marine environment confirm to be a favorite environment to establish these alien species. comparison with neighbouring countries: a comparison of the total number of alien species of zoobenthos in syria with those of some neighbouring countries of the eastern mediterranean revealed a relatively low number of the recorded species. a total of 239 marine nis and cryptogenic species are reported from greece (zenetos et al., 2015b), most of them i.e. 214 species are aliens and 62 cryptogenic (zenetos et al., 2018). out of these, 146 are zoobenthos as follows: one porifera, two ctenophora, one platyhelminthes, five cnidaria, 44 mollusca, 35 annelida, eight bryozoa, 42 crustacea, and three echinodermata and seven tunicata (see http://elnais.hcmr.gr/elnaisdatabase-2), while the rest are plants, foraminifera and fishes, etc.. in italy, 35 alien molluscan species recorded (crocetta et al., 2013a). in turkey, a total of 111 molluscan species (öztürk et al., 2014), including six echinodermata (öztoprak et al., 2014) from the levantine coast, 50 crustacean (suat ate et al., 2013), and 57 polychaeta have been reported (çinar et al., 2014). a recent study revealed that the contribution of alien mollusca account for approximately 30% of the molluscan fauna i.e. 32 out of 106 (guarnieri et al., 2017). in cyprus 42 species of mollusca, 19 polychaeta, 12 crustacean, and 10 species from other taxa have been reported up to july 2009 (katsanevakis et al., 2009). the updated checklist of mollusca of lebanon is 32 species, including 13 opisthobranches (12 alien and one cryptogenic) (crocetta et al., 2013b) and 18 bivalvia (crocetta et al., 2013c). conclusion the present study indicate that the benthic fauna along the syrian coast has suffered from an increase in the number of alien species, with the record of new species, their extension in the northern part of the syrian coast and disappearance of many native species. alien species consist 11.55% of the total number of species of zoobenthos, with dominance of mollusca and crustacea. many of the established species have become an invader and dominant in the marine environment of syria. their proliferation is accelerating and also their negative impact on the native species on the socio-economic activities seems to be clear. what is required today of the concerned scientific community is increasing the effort to monitor the environmental situation in the syrian coast and help to predict future changes and find ways to address them or managed. rapid assessment surveys are one approach to quickly characterize the native, nonnative and cryptogenic species present in the syrian marine environment. the study of the relation between native and alien species will be the aims of our future research based on the knowledge gathered in the present study. acknowledgements i would like to thank everyone who helped get the samples and i am grateful to s. eagderi the managing editor of ijab who comments substantially improved the quality of the manuscript. sincere thanks to a. zenetos and p. magni for commenting on the article. references ammar i. 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(2019) 7(5): 291-300 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article the effects of starvation on some epidermal mucus immune parameters in rainbow trout, oncorhynchus mykiss siyavash soltanian*,1amin gholamhosseini aquatic animal health and diseases department, school of veterinary medicine, shiraz university, shiraz, iran. s article history: received 2 july 2019 accepted 25 august 2019 available online 2 5 august 2019 keywords: starvation skin mucus mucosal immunity rainbow trout abstract: the skin of a fish acts as the primary protective agent against biological, physical, and chemical stress. however, the effects of such stressors on fish mucosal immune responses have been hardly investigated. fasting or feed deprivation commonly is occurred in aquaculture due to season, production policies, or disease. this research was aimed to investigate the impacts of 20-day starvation on skin mucosal immune responses of rainbow trout. the results revealed that the enzymatic activities of lysozyme (lzm) and alkaline phosphatase (alp), as well as the total immunoglobulins (ig) level and bactericidal activities were significantly reduced in the skin mucus of fasted fish. no significant changes were observed in the esterase and protease activities. bactericidal activity in the mucus of starved fish was significantly lower than control group after 20 days. therefore, it could be strongly suggested that this species should not remain under starvation stress as this kind of stress impairs mucosal immune barriers which, in turn, could make the fish more susceptible to infections or harmful agents. introduction fasting or feed deprivation is a normal occurrence that many fish species may experience in their natural environments during migration, reproduction, and due to some adverse conditions in the environment, like red tides or cold-water masses in the summer (cho et al., 2006; sridee and boonanuntanasarn, 2012; park et al., 2012). furthermore, feed deprivation can be used as a managing approach to decrease mortality due to disease occurrence and overproduction (shoemaker et al., 2003) or to reduce water quality complications and decrease stress management (davis and gaylord, 2011). nevertheless, in several previous researches, starvation was perceived as a stressor (furné et al., 2009; langer et al., 2014; varga et al., 2014; piccinetti et al., 2014; arslan et al., 2015). unlike mammals, fish could tolerate long periods of fasting. however, extended starvation exhausts the energy reserves of the fish (rundles, 2008) and depress their immune system. as a result, the fish become predisposed to the infectious diseases (raina and sachar, 2014). in fact, study of the immune *correspondence: siyavash soltanian doi: https://doi.org/10.22034/ijab.v7i5.634 e-mail: siyavashsoltanian@yahoo.com defense mechanisms is of main importance for assessing fish health since a direct correlation between the functioning of the immune system and the ability to prevent disease outbreaks has been recognized (hoseini and ghelichpour, 2013; hoseini et al., 2014; hoseini et al., 2019; de souza e silva et al., 2019; gholizadeh zare tavana et al., 2019; foysal et al., 2020;). the skin mucus of fish acts as the first line of defense against stressors (jung et al., 2012; guardiola et al., 2015; tacchi et al., 2015; khansari et al., 2018). little is known regarding how the skin mucosal immune system operates in different species of fish. fish skin mucus is composed of water, mucin, complement, c-reactive proteins, lectins, cathepsins, defensins, haemolysins, immunoglobulin m (igm), agglutinin, proteolytic enzymes, antimicrobial peptides, acid and alkaline phosphatases, superoxide dismutase, and lysozyme, act as inhibitory agents against various infections (ingram, 1980; subramanian et al., 2007; esteban, 2012; guardiola et al., 2014). 292 soltanian and gholamhosseini/ effects of starvation on epidermal mucus immune in rainbow trout recently, numerous studies have revealed that some factors that cause stress, such as infectious agents, environmental pollutants, prolonged transport stress, and over-crowding stress can change the complete structural and cellular composition in skin of oncorhynchus mykiss, sparus aurata, and puntius tetrazona (lindenstrøm et al., 2004; tacchi et al., 2015; guardiola et al., 2015; roosta and hosseinifar, 2016). furthermore, different fish species undergo some changes in their mucus structure and immune responses, following any environmental and physiological changes (guardiola et al., 2014). hoseini et al. (2014) revealed the serum levels of t4, t3, cortisol, glucose, lactate, triglyceride and cholesterol were significantly affected by fasting period in rainbow trout; however, there were no significant changes in serum total protein, albumin, globulin or a:g among the fish fasted 0-72 h (hoseini et al., 2014). the same results showed by hoseini et al. (2019) on common carp. three days of fasting led to a significant decrease in serum levels of lactate and thyroid hormones. fourteen days of fasting led to initiation of physiological process to maintain serum glucose by increase in serum cortisol level and lactate utilization. these fish had lower levels of thyroid hormones suggesting suppression of basal metabolism. the fish also had lower wbc and serum lysozyme and total ig levels indicating immunosuppression. therefore, this research was aimed to study some immune system alteration in the skin mucus of rainbow trout following a short-term feed deprivation. materials and methods in accordance with the national ethical framework for animal research in iran, rainbow trout was chosen for the experimentation in this study (mobasher et al., 2008). specimens with a body weight of 22.6±4.6 g, were selected from a local fish farm, shiraz, iran. in order to adaptation, fish kept in 250 l fiberglass tanks for three weeks. during this period, the water was recirculated, fish were exposed to natural photoperiod, and water temperature, dissolved oxygen, and ph were 13.2±1.8ºc, 6.2±0.9 ppm and 7.5±0.2, respectively. fish fed twice a day with commercial pellets (beiza co, iran) as 2% of their body weight. after acclimation, fish were divided into two groups in triplicates, one maintained at starvation and the other was fed daily with the same diet to apparent satiation twice daily, as a control. the experiment lasted 20 days (tran et al., 2018), during which 6 specimens from both the control and fasted groups were sampled at time zero (before starvation) and on day 20 after the start of the starvation for the measurement of selected skin immune parameters. in both treatments (control and starved fish), before sampling, the fish were first anesthetized by 0.1 ppm ms222 (argent laboratories inc., redmond, wa, usa). then, a sterile plastic spatula, with enough caution to prevent contamination with blood, intestinal and urogenital secretions, was used to scratch the skin mucus from the dorso-lateral surfaces (palaksha et al., 2008). once the mucus was collected, an overdose (100 ppm) of ms222 was used to euthanize the fish. mucus samples were homogenized using one volume of sterile normal saline (9‰). afterward, the samples were centrifuged at 2000 g for 10 min at 4°c. the supernatant was stored at -80°c for further analysis. skin mucus total immunoglobulin: the method described by siwiki and anderson (1993) was used to measure the total immunoglobulins (total ig). first, ig was precipitated in serum using a 12% polyethylene glycol solution and remaining protein in the supernatant was assayed by bradford method (bradford, 1979) and it was subtracted from the total protein to give total ig content. lysozyme activity: the turbidimetric assay was applied to measure the lysozyme activity (demers and bayne, 1997). briefly, 50 μl suspension of bacterium micrococcus luteus (sigma) (0.3 mg/ml of lyophilized cells dissolved in 40 mm sodium phosphate buffer, ph 6.5) and 50 μl of the mucus sample were mixed together. the incubation of the mixture was done at 30°c; then, after 0 and 15 min, the reduction in the absorbance at 450 nm was measured in a microplate reader (biotek elx 808 293 int. j. aquat. biol. (2019) 7(5): 291-300 instrument, usa). the quantity of enzyme that caused a decline in the absorbance of 0.001 per minute was defined as a unit of lysozyme activity. alkaline phosphatase activity: using the method of palaksha et al. (2008), the measurement of the activity of alkaline phosphatase was done after the incubation of the mucus supernatants with 4 mm paranitrophenyl phosphate (sigma) in 100 mm ammonium bicarbonate buffer which contained 1 mm magnesium chloride, ph 7.8 at 30°c. the quantity of enzyme that freed 1 mmol of para-nitrophenyl product in 1 min was defined as one unit of activity. protease activity: utilizing the method offered by palaksha et al. (2008), the activity of protease was determined by azocasein hydrolysis assay. 50 μl of the mucus sample re-suspended in 100 mm ammonium bicarbonate, ph 7.8, with 50 μl azocasein substrate 0.25% (w/v) in the same buffer for 19 h at 30°c were incubated for assaying azocasein hydrolysis. fifty μl of 20% (w/v) trichloroacetic acid was added to the process to bring the reaction to a halt. this was followed by a 5 min centrifugation at 15400 g (equal volumes of supernatant (100 μl)). then, 0.5 m naoh was added to a 96 well plate and the absorbance was evaluated at 405 nm. the quantity of the enzyme that triggered a variation in the absorbance of 0.001/min was defined as one unit of activity. each unit of activity was determined per mg of protein (specific activity). esterase activity: based on palaksha et al. (2008), when mucus supernatants were incubated with 0.4 mm para-nitrophenyl myristate in 100 mm ammonium bicarbonate buffer containing 0.5% triton x-100, ph7.8 at 30°c; then, the detection of esterase activity was processed. a plate reader was used to keep a constant record of the absorbance for 2 h at 405 nm. the amount of enzyme needed to set free 1 mmol of para-nitrophenyl product in1 min was defined as the activity. antibacterial assay: before the samples of mucus were analyzed, they were defrosted at room temperature. in vitro bactericidal activity of mucus samples were examined against aeromonas hydrophila, yersinia ruckeri, and lactococcus garviae, which had been taken from the stock culture kept at the microbiology laboratory of the aquatic animal health and diseases department, school of veterinary medicine, shiraz university. the standard disc diffusion method was used to detect the antibacterial activity of the mucus samples (bauer et al., 1966). at first, different species of bacteria of interest to the present study were cultured in nutrient broth medium for 24 h at 25ºc; then, 0.1 ml of broth culture medium (contains 1.5×108 cfu/ ml) was cultivated on mueller hinton agar. aseptic paper discs with a 6 mm diameter were soaked with 200 µl of mucus sample and put on the medium. the incubation was performed at 25ºc for 24 h. following that, the discs were checked and a ruler was used to calculate the growth inhibition area diameter in mm (minus the diameter of paper discs). the clear part which surrounded the discs was considered as an indication of the antibacterial activity. statistical analysis: immunological data and antibacterial activity were analyzed using a twosample t-test. each data set was checked for normality using a shapiro-wilk test. non-normal data were transformed for statistical comparison. all the statistical analyses were performed using spss software (version 16.0, chicago, il) and the probability of p<0.05 was considered statistically significant. data are presented as mean±sd. results all biomarkers were measured before the final experiment to ensure that the individual conditions of the fish tested were uniform (at 0 days). the results showed that there was no significant difference between the fish assigned to the different experimental groups (table 1). there was no case of fish death throughout the experiment in spite of the feed deprivation. mean±sd of the epidermal immune factors observed in the sample fish are tabulated in figures 1. a significant reduction in the enzymatic activities of lzm (fig. 1) and alp (fig. 2), as well as total ig level (fig. 3), was observed in the skin mucus of the starved fish compared to those measured in the fed ones (p<0.05). similar results were found for skin 294 soltanian and gholamhosseini/ effects of starvation on epidermal mucus immune in rainbow trout mucus bactericidal activities against selected bacterial pathogens (table 2). however, the protease and esterase activities did not show any differences after exposure to starvation (figs. 4, 5). discussions although it is clear that nutritional disturbance can interfere with the resistance to pathogens in table 1. immunological and antibacterial results before the start of the final experiment. table 2. antibacterial activity of epidermal mucus in rainbow trout 20 days after subjection to starvation. biomarkers control starved fish lysozyme 29.67±1.86 30.17±3.31 alkaline phosphatase 8.17±1.60 9.67±1.03 esterase 2.22±0.34 2.23±0.28 protease 20.67±2.73 21.33±2.66 total immunoglobulins 5.87±0.37 6.25±0.41 yersinia ruckeri 5.17±1.72 7.17±1.32 aeromonas hydrophila 9.83±2.14 11±2.10 lactococcus garviae 7.00±1.79 5.83±2.14 20 days bacteria control starved fish a. hydrophila 8.8±2.0b 4.0±2.2a y. ruckeri 6.0±1.4bc 2.5±1.6a l. garviae 7.7±1.6b 3.7±2.0a figure 1. alterations in skin mucus lysozyme activities of rainbow trout 20 days after subjection to starvation. (mean±sd, n=6). different letter notations indicate significant differences at p<0.05. figure 2. alterations in skin mucus alkaline phosphatase activities of rainbow trout 20 days after subjection to starvation. (mean±sd, n=6). different letter notations indicate significant differences at p<0.05. figure 3. alterations in skin mucus total immunoglobulin levels of rainbow trout 20 days after subjection to starvation. (mean±sd, n=6). different letter notations indicate significant differences at p<0.05. figure 4. alterations in skin mucus protease activities of rainbow trout 20 days after subjection to starvation. (mean±sd, n=6). different letter notations indicate significant differences at p<0.05. 295 int. j. aquat. biol. (2019) 7(5): 291-300 domesticated animals (klasing, 1998), the mechanisms by which nutritional variations modulate the fish immune system are largely unidentified. on the other hand, the significance of the mucosal surfaces, as the first defending position and the initial target in an attack by infectious agents, has been increasingly highlighted (rombout et al., 2011; esteban, 2012). nevertheless, the vulnerability of the mucosal defensive system to alterations in nutrients has just recently been noticed in fish species (vieira et al., 2011; landeira-dabarca et al., 2013). different enzymes with recognized functions in the immune reactions have been shown in numerous species of fish as they were noticed in the present research. of these enzymes, alp in mucus has been reported to function as a factor with antibacterial properties because it is an enzyme involved in hydrolysis (ross et al., 2000). also, it has been reported that alp plays a pivotal role in the primary phases of wound healing (rai and mittal, 1991; iger and abraham, 1990, 1997) and against stress (iger and abraham, 1990, 1997). no available data were found regarding the effects of starvation on mucosal alp activity in fish species. our findings pointed to a significant reduction in the mucosal activity of alp in the fasted fish. similar results were found in turbot (scophthalmus maximus) reared in high density for 120 days (jia et al., 2016). however, despite to the results of the present work, it has been shown that some stressors namely high stocking densities (roosta and hosseinifar., 2016), aluminum acidity (ledy et al., 2003), and hypoxia (vatsos et al., 2010), significantly enhanced the alp activity in the mucus of the tiger barbs (pentius tetrazona), brown trouts (salmo truta fario), and sea bass (dicentrarchus labrax), respectively. such alteration in the activity of alp could be used as an indication of stress in some circumstances (ross et al., 2000), for instance in the conditions tested in the current work. however, further studies are required to approve this hypothesis. since alp activity is dependent on maintaining the homeostasis of ions, starvation may affect alp activity by reducing the level of uptake of ions such as calcium. proteases and esterase were also measured in the current work because both of them have been linked with skin immune responses and defense against microbial infections (esteban, 2012). in skin mucus, proteases can act as a defense means against infectious agents both directly, via splitting their proteins (subramanian et al., 2007), and indirectly, via hindering their colonization and invasion mechanisms (aranishi et al., 1998). furthermore, proteases may activate other immune parameters such as complement, immunoglobulins or antibacterial peptides (hjelmeland et al., 1983; fernandes and smith., 2002; kennedy et al., 2009). some studies have documented the alterations that take place in the mucus proteases in the face of stress (aranishi and nakane., 1997; aranishi et al., 1999; easy and ross., 2010) and infection (ross et al., 2000; aranishi and mano, 2000) and pointed their importance in mucosal immunity. however, our data showed no changes in the fish epidermal proteases activities following 20 days of starvation. in a similar way, jia et al. (2016) found no changes in protease activity in the epidermal mucus of turbots (scophthalmus maximus) exposed to crowding stress. furthermore, in the current research, the effects of feed deprivation on esterase level in the skin mucus were examined. even though the function of the esterase in fish mucus is not known, it is known that it can function separately or in collaboration with other mucosal immune components defending against the figure 5. alterations in skin mucus esterase activities of rainbow trout before and 20 days after subjection to starvation. (mean±sd, n=6). different letter notations indicate significant differences at p<0.05. 296 soltanian and gholamhosseini/ effects of starvation on epidermal mucus immune in rainbow trout infectious agents (sheikhzadeh et al., 2012). previously, increased esterase activity was demonstrated in gilthead sea bream fish (sparus aurata) following waterborne exposure to some heavy metals (guardiola et al., 2015). conversely, a significant reduction in the esterase activity was reported in the skin mucus of turbots reared in highdensity conditions (jia et al., 2016). however, in the current work, no change in the esterase activity was found in the mucus of the starved fish. in addition, the total ig was measured in the present research. immunoglobulins have crucial roles to play in defending the fish from serious infectious agents and conserving the mucosal homeostasis (esteban, 2012). igm and teleost polymeric immunoglobulin receptor (pigr) found locally in the skin play an essential role in the mucosal immunity of fish (esteban, 2012). our data revealed that total ig took a remarkable reduction in the epidermal mucus of the fasted fish. hormonal changes during starvation may affect the production of immunoglobulins. furthermore, starvation can affect the stimulation of b cells and the secretion of t cell-derived cytokines (ota et al., 2016). interestingly, the value of the ig measured in the skin mucus of the sea bream (sparus aurata) which had been exposed to heavy metals reduced on the second day of exposure; however, it elevated later (guardiola et al., 2015). nevertheless, no change in the serum igm values was recorded after starvation in either european sea bass (dicentrarchus labrax) (caruso et al., 2011) or mesopotamichthys sharpeyi (najafi et al., 2015). in fish immunity studies, serum lysozyme activity has been mainly evaluated (ellis, 2001), yet the lysozyme functions in the mucosal surfaces have received less attention comparatively (bergsson et al., 2005; nigam et al., 2012). for supporting the importance of lysozyme for disease resistance, in a study carried out by yazawa et al. (2006), they generated a transgenic zebrafish (danio rerio) strain expressing a chicken lysozyme gene and the results showed that the transgenic strain survived 65% when confronted with flexibacter columnare while the wildtype fish survived 0% when challenged with the same bacterial agent. we revealed that lysozyme content in the skin mucus significantly decreased after starvation. likewise, the mucus lysozyme activity obviously decreased in turbots raised in high-density condition (jia et al., 2016). similarly, in blackspot sea bream (pagellus bogaraveo), which had been starved for 31 days, a significant reduction was found in the lysozyme activity of their skin mucus. however, the activity of lysozyme was doubled in the skin mucus of european seabass, dicentrarchus labrax reared at the same condition (caruso et al., 2011). it seems that the difference in the mucosal activity of lysozyme could be associated with many factors such as season (schrock et al., 2001), type of species, stress management, gene mutation, diet, maturity, sex (balfry and iwama., 2004; caruso et al., 2011), and even epidermis thickness and the number of the mucous cells (subramanian et al., 2007). regardless the effector components and the mechanisms participating in the bacterial killing, the assessment of bactericidal activity of skin mucus could be more important in practice than single enzymatic activities (guardiola et al., 2014). several studies have shown that the skin mucus of some fish species presented a strong antibacterial and antifungal activity against a wide range of microbial pathogens (hellio et al., 2002; subramanian et al., 2008; dhanaraj et al., 2009). in a study, one-week starvation in channel catfish significantly increased the mortality to f. columnare which normally penetrates through surface mucosa (liu et al., 2013). the results of the present study showed that when the sample fish were exposed to starvation, some antimicrobial activities started to operate against specific infectious agents and a remarkable reduction was observed in the antibacterial activity. our results conflict with those obtained by roosta and hoseinifar (2016) who found the elevation of antibacterial activity in the skin mucus of the tiger barbs (pentius tetrazona) subjected to crowding stress. in addition, the exposure of sea bream to heavy metals caused a significant increase in their skin mucus bactericidal activity (guardiola et al., 2015). 297 int. j. aquat. biol. (2019) 7(5): 291-300 in conclusion, the current work demonstrated a converse relationship between starvation and immune response in the skin mucus of rainbow trout. the enzymatic activities of lzm, alp as well as ig level and bactericidal activity were significantly reduced in the skin mucus of fish after 20 days of starvation. moreover, the dramatic variations observed between the findings of the present study and those of others could possibly be attributed to factors such as season, length of feed deprivation, evolutionary adaptation to starvation, and pathogen dynamics (prevalence, routes of transmission, and so on). the present findings suggest the important role of nutrition in maintaining the immunological functioning of the skin mucus in the rainbow trout. thus, it is strongly recommended that this fish species should not remain under starvation stress since this kind of stress can impair the mucosal immune barriers which, in turn, could make the fish more susceptible to infections or harmful agents. acknowledgments the authors would like to thank the aquatic animal health and diseases department, school of veterinary medicine, shiraz university, for its support of the present research by providing a research grant to the first author. references aranishi f., mano n. 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(2019) 7(5): 245-253 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article detection and mode of action of retinoids on ovarian development in the mud crab, scylla serrata kaduru venkaiah, daveedu thathapudi, sri bhashyam sainath*1 department of biotechnology, vikrama simhapuri university, nellore-524 320, ap, india. s article history: received 17 july 2019 accepted 13 august 2019 available online 2 5 october 2019 keywords: ecdysteroids hplc ovaries oocyte diameter retinoic acid abstract: in the current study, the retinoic acid isomers such as 9-cis retinoic acid and all-trans retinoic acid were detected in the mature ovaries of mud crabs, scylla serrata using hplc analysis. given the detection of retinoids in the ovaries, an attempt has been made to elucidate the possible role of retinoic acid in the regulation of reproduction in mud crabs. injection of 9-cis retinoic acid induced ovarian maturation in intact mud crabs as evidenced by a significance elevation in the ovarian index (226.76%; p<0.001), and oocyte diameter (150.61%; p<0.001) accompanied by accumulation of yolk globules in the oocytes as compared to the untreated crabs. further, a significant increase (258.63%; p<0.0001) in the circulatory ecdysteroid levels were also observed in 9-cis retinoic acid injected mud crabs over vehicle injected crabs. from the results, it can be postulated that retinoic acid-induced stimulation of ovarian maturation at least in part mediates ecdysteroids in the mud crabs. introduction it is wellknown that the ovarian development in decapods is primarily under the control of two antagonistic hormones viz., gonad/vitellogenin inhibiting hormone (gih or vih) of the major neuro-endocrine system located in the eyestalks, xorgan-sinus gland complex (xo-sg) and gonad/vitellogenin stimulating hormone (gsh or vsh) secretion from the brain and the thoracic ganglia, respectively (subramoniam, 2017). in addition, ecdysteroids of y-organs and methyl farnesaote of mandibular organs also play a crucial role in the regulation of crustacean ovarian development (swetha et al., 2011). however, their precise crosstalk during ovarian maturation is not well-defined (rotllant et al., 2018). therefore, understanding the molecules that coordinate the crosstalk between the endogenous hormones are instrumental to gain insights into the regulatory effects of peptides, steroids and the terpenoids during the ovarian development of decapod crustaceans (sainath et al., 2013). *correspondence: sri bhashyam sainath with all authors equal contribution doi: https://doi.org/10.22034/ijab.v7i5.787 e-mail: drsainath@simhapuriuniv.ac.in vitamin a (retinol: rol) is a multifaceted molecule with wide array of functions in vertebrates. the biologically active metabolite of vitamin a, retinoic acid (ra) acts as one of the signaling molecules in the coordination of reproductive regulators in vertebrates (andre et al., 2014). in addition, the role of ra in the regulation of a range of physiological processes such as embryonic development and organogenesis, tissue homeostasis, cell proliferation, differentiation, embryonic growth and development and vision is well acknowledged (theodosiou et al., 2010; clagget-dame and knutson, 2011; macejova et al., 2016). most notably, the biological functions of ra are operated via genomic actions where ra through the ligation of its cognate nuclear receptors [wherein 9-cis retinoic acid (9cra) exerts its genomic actions via retinoic acid x receptors (rxrs) and retinoic acid receptor (rar), whereas all-trans retinoic acid (atra) exerts its genomic action via rars]. after ligand bounded, the rar/rxr or rxr/rxr specifically binds the retinoid response elements on the dna and 246 venkaiah et al./ role of retinoids on ovarian growth in mud crabs regulate the transcriptional expression of ra target genes (andre et al., 2014). although complex, it has been shown that ra through autocrine and paracrine activities and via its retinoid receptors regulate the ovarian functions, including the formation and development of postnatal follicles and a complex differentiation process during the ovarian development in vertebrates (macejova et al., 2016). the information with respect to vitamin a metabolism and its signaling components are still at its infancy in decapods (andre et al., 2014). however, traces of information is available with regard to the crustacean retinoid system which includes the occurrence of retinoids, including retinoic acid, retinoid transport proteins: cellular retinoic acid binding protein and the retinoid signaling proteins: retinoid x receptors (gu et al., 2002; asazuma et al., 2007; hopkins et al., 2008; techa et al., 2013; tang et al., 2014). surprisingly, the role of rxr in the crustacean reproduction is well acknowledged (nagaraju et al., 2011; cui et al., 2013; gong et al., 2016). given that the genomic actions of retinoic acid are crucial for vertebrate reproduction and the retinoic acid x receptors and the retinoic acid isomers occurs endogenously in crustaceans, the current study was investigate to address the probable role of retinoic acid on the ovarian development in mud crabs, scylla serrata. scylla serrata is widely consumed all over the world and has a huge demand at the international markets. however, the mud crab culture is not expanding as expected due to the lack of sufficient seed supply. although, esx is common practice to induce seed in hatchery industry, because of its side effects such as mortality in the brood stock and inferior quality and quantity of seed, there is a need for the development of reliable strategies. currently, the seed requirements for mud crab culture is sustained from the wild-caught seedlings and the gravid female crabs, consequently a reduction on their population in the wild. therefore, an effective way of inducing ovarian maturation thereby seed in the mud crabs has long been sought, which in turn minimizes the intensive fishing of gravid female mud crabs in the wild. materials and methods collection and maintenance of animals: uninjured intermolt stage (c4) mud crabs, s. serrata used in this study were collected from the nellore coast, andhra pradesh, india. female crabs (body weight: 140±10 g; carapace width: 10-12 cm) were brought to the laboratory (approximately 7.5 km from the collection point) and separately maintained in large aquaria (3.0 x 1.0 x 1.0 m). the bottom of the aquaria was covered by sand (4 cm). all the crabs were acclimatized (10 days) to the laboratory conditions (constant salinity=30±1 ppt, ph=7.3±0.1, temperature= 25±3ºc) and continuous aeration with moderate intensity) before being used in the experiments. during their acclimatization, they were fed with fish flesh ad libitum (between 8 am to 9 am) and the ambient medium was removed during the next feeding day to reduce contamination. experimental design: in the present study, two experiments were designed wherein the first experiment was aimed to investigate the occurrence of retinoic acid (9-cis retinoic acid: 9cra and alltrans retinoic acid: atra) in the ovaries of mud crabs and the second experiment was to evaluate the probable effect of ra on ovarian maturation in the mud crabs, s. serrata. experiment 1: the procedure followed in the identification of retinoid isomers (9cra and atra) in mature ovaries of the crabs was based on the previously published reports (hopkins et al., 2008; kane and napoli, 2010; kim and quadro, 2013). briefly, the crabs were dissected under yellow light and the ovaries (mature stage or iv: colour dark orange and oocytes are completely occupied by yolk globules), weighed, and immediately processed for the detection of ra isomers (9cra and atra) under yellow light. to one aliquot (1 ml) of homogenate (obtained by homogenizing the ovarian tissue (30 mg) in 2 ml of phosphate buffered saline (ph=7.4) using hand homogenization with a ground glass homogenizer placed on ice) 3 ml of 0.025 m koh in ethanol was added followed by vortexing for at least 10 s. after vortex, 10 ml of hexane was added to the mixture and again followed by vortexing (at least for 247 int. j. aquat. biol. (2019) 7(5): 245-253 10 s). the mixture was centrifuged at 1000 x g and the resultant supernatant was taken into a disposable fresh glass tube followed by evaporation step under liquid nitrogen to get a yellow colour crystalline compound. methanol (100 µl) was added to the resultant residue and subjected to hplc analysis using the conditions similar to the detection of standards (see below). the hplc chromatograms were interpreted using the computer software provided by the manufacturer. standard solutions of atra and 9cra were prepared by dissolution in methanol (1 mg/ 1000 µl) followed by brief vortex. the following were the column conditions: zodiac c18 column: 250 mm x 4.6 mm and i.d, 5 µm, total analysis time per sample: 20 min and the flow rate: 1 ml/min. the standard solutions of selected retinoids (20 µl) were injected into the column using the mobile phase (75% acetonitrile and 25% methanol). the corresponding absorbance (280 nm) and retention times for 9cra and atra were recorded via agilent hplc system (1100 series, waldbronn, germany). this served as identification parameters for 9cra and atra from the mature ovaries of the crabs. experiment 2: uninjured intact female crabs (c4) were randomly divided into three groups. crabs which did not receive any treatment served as initial control group (group 1; n=12) and sacrificed on day 1 of the experiment, whereas the crabs in group 2 (concurrent controls; n=12) received injections (100 µl) of crustacean saline containing ethanol (1 µl of ethanol was mixed in 99 µl of crustacean saline). on the other hand, crabs in group 3 (n=12) were independently injected (100 µl) with 10-7 mole/crab 9cra (cayman chemicals, inc., ann arbor, mi). the required concentration of 9cra was prepared freshly on the day of experimentation by dissolving in ethanol and diluted accordingly in crustacean saline (pantin, 1934). the injections were given on days 1, 7, 14, and 21 and all the animals were sacrificed on 28th day to analyze the selected ovarian maturation endpoints. the dose selection was based previous studies (cui et al., 2013) and also our preliminary studies (data not shown). the total duration of the experiment was 28 days. before the start of the experimentation (one day) feeding was stopped. the vehicle and/or test chemicals were administered into the crabs through the arthrodial membrane at the base of one of the walking legs. all the crabs were ice anesthetized before handling. after completion of the experimental period, the crabs from the control and the experimental groups were sacrificed to analyze ovarian index, oocyte diameter, and histology of the ovary. the crabs from control, concurrent and 9crainjected groups were weighed and the ovaries were excised, cleaned in crustacean saline, blotted on filter paper, and weighed wet to the nearest milligram. the ovarian index was determined using the following formula: ovarian index = [wet weight of the ovary (g)/ weight of the crab (g)] x 100. the diameter of 20 oocytes from each ovary was measured using an ocular micrometer under a compound microscope (olympus, model-bx41tf hb, japan). the measurements were made on the longest and shortest axes of each oocyte, both dimensions were added, and the mean was taken as mean oocyte diameter. the ovarian sections were prepared according to the method described by bancraft and stevens (1982) and they were visualized using the phase contrast microscope (olympus, model -bx41tf, japan) and the hematoxylin and eosin stained ovarian sections were photographed. hemolymph collection and ecdysteroid assay: ecdysteroids in the hemolymph was analyzed in crabs at different reproductive stages (quinitio et al., 2007) and also during the experimental conditions using enzyme-linked immunosorbent technique (kingan, 1989; abuhagr et al., 2014). the primary antibody and hrp-conjugated ecdysone was purchased from dr. timothy kingan (timkingan@gamil.com) and the secondary goat anti-rabbit igg antibody was purchased from merck, india. for ecdysteroid analysis, crab hemolymph samples (100 µl) were collected at the base of the 3rd walking leg using 1 ml syringe (27-guage needle). they were mixed with 300 248 venkaiah et al./ role of retinoids on ovarian growth in mud crabs µl of methanol and the mixture was centrifuged for 10 min at 20,000 g at 4°c to remove precipitated proteins. the supernatants (10 μl) were evaporated to dryness using an evaporator (thermo scientific, savant) and the samples were dissolved in 150 μl assay buffer containing the components [sodium phosphate (25 mmol; ph 7.5), nacl (150 mmol), edta disodium dehydrate (1 mmol) and 0.1% bovine serum albumin] in one litre of autoclaved water. to the overnight incubated pre-coated elisa plates (96wells) with secondary antibody [0.5 µg in 90 µl of phosphate-buffered saline (ph 7.5) per well] assay buffer was added. this step was performed to block the unspecific sites. the plates were incubated for 2 h at room temperature followed by washing step three times with the assay buffer containing 0.05% tween-20. after washing steps, the diluted hemolymph samples (50 µl) and standards of 20hydroxyecdysone were incubated with 50 µl of primary antibody (1: 100,000) and 50 µl of 20e/hrp conjugate (1:10,000) were added to the wells and incubated at 4ºc overnight. the plates were washed thrice with pbs containing 0.05% tween 20 and 100 µl of tmb substrate (tetramethylbenzidine solution and peroxidase solution: thermo scientifics) was added to the wells. the plates were incubated in dark for 20 min for colour development and the reaction was stopped by the addition of 100 µl of 1m phosphoric acid. the colour development was read at 450 nm using elisa plate reader (bio-rad, usa). figure 1. hplc chromatogram of retinoic acid isomers (atra: all-trans retinoic acid; 9cra: 9-cis retinoic acid); a: standard mixture b: analysis in mature ovaries of mud crabs, scylla serrata. 249 int. j. aquat. biol. (2019) 7(5): 245-253 the intraand inter-assay variations for this elisa were found to be < 11 % and < 15 %, respectively. statistical analysis: the data were expressed as mean ±s.d. statistical analysis was performed using one way analysis of variance (anova) followed by tukey’s post-hoc test (spss: student version 7.5, spss inc, cherstsey., uk). differences was considered significant at p<0.05. results detection of retinoic acid isomers (9cra and atra) in mature ovaries of the mud crabs, s. serrata: a total of three hplc separations were run using mature ovarian extracts of the crabs, where in two of the separations were done on the same mature ovarian extracts. figure 1a and b shows the hplc chromatogram of the standard and the test samples, respectively. the hplc retention times of the standard retinoic acid isomers viz., 9cra and atra were 6.891 minutes and 7.549 minutes, respectively. in the test samples, two peaks were noticeable with the hplc retention times of 6.841 minutes and 7.513 minutes, which correspond to the standard hplc retention times of 9cra and atra, respectively suggesting the occurrence of endogenous retinoic acid isomers in mature ovaries of the mud crabs (fig. 1). effect of 9cra on ovarian index, oocyte diameter, and histology of the ovary in intact mud crabs, s. serrata during 28-day experimental period: the ovarian index and oocyte diameter in initial control and concurrent controls were 0.68±0.064 (ww%), 49.13±2.73 µm and 0.71±0.061 (ww%), and 52.12±3.82 µm, respectively in a 28 day experimental period (table 1), suggesting no significant changes in the ovarian index and oocyte diameter in the concurrent controls as compared to initial controls. injection of 9cra significantly enhanced the ovarian index (226.76%) and oocyte diameter (150.61%) as compared to concurrent controls in a 28 day experimental period (table 1). histological observations of the ovaries in concurrent control (fig. 2a) crabs indicated that the ovary was at the immature stage as evidenced by ovarian lobes completely occupied by oogonia surrounded by follicular cells. whereas, the histological analysis of figure 2. transverse sections of ovary of vehicle (a), and 9cra injected crabs (b) in a 28-day experimental period in the crab, scylla serrata. pvo: pre-vitellogenic oocyte; vo: vitellogenic oocyte. scale line = 100 μm. table 1. effect of in vivo injection of 9-cis retinoic acid (9cra) on ovarian index (ww%) and oocyte diameter (µm) in intact mud crabs. groups ovarian index oocyte diameter control 0.68a ± 0.064 49.13a ± 2.73 vehicle-injected 0.71a ± 0.061 (4.411) 52.12a ± 3.68 (6.085) 9cra-injected 2.32b ± 0.053 (226.760) 130.62b ± 7.92 (150.61) values are mean ±s.d. of 12 individual crabs; values in the parentheses are percent change from that of control; for evaluation of statistical analysis and percent change, for vehicle injected crabs, crabs in initial control group served as controls and for 9cra injected group crabs, crabs in vehicle injected group served as controls; values with same superscript in a column do not differ significantly from each other at p<0.001. 250 venkaiah et al./ role of retinoids on ovarian growth in mud crabs ovaries in 9cra injected crabs indicated that the ovaries were at mature stage as evidenced by the accumulation of yolk globules in the oocytes (fig. 2b). effect of 9cra on haemolymph ecdysteroid levels in the intact mud crabs during 28-day experimental period: significant increase in the hemolymph ecdysteroid levels was noticed during ovarian development as the crabs shift from immature stage (stage i) to late mature stage (stage iii) (fig. 3). however, no significant changes were observed in circulatory levels of ecdysteroids in crabs at stage iii as compared to the crabs at stage iv (complete mature stage). no significant changes were observed in the hemolymph ecdysteroid levels in vehicle alone injected crabs as compared to initial controls. whereas, injection of 9cra showed a significant elevation (258.63%; p<0.0001) in the circulatory levels of ecdysteroids in crabs as compared to concurrent control crabs over a period of 28-days (fig. 3). discussions previously, retinoids such as all-trans ra, ral, 13-cis-ral and retinoic acid isomers such as all transra and 9-cisra using hplc coupled with gc/ms in the blastemas of regenerating limbs in the fiddler crab, u. pugilator has been reported (hopkins et al., 2008). moreover, studies of liñán-cabello et al. (2003) reported the ral in the ovary of shrimp, litopenaeus vannamei using diode array spectrophotometer. liñán-cabello and paniagua (2004) also discovered the retinoids such as 13cisral, all-transral and 13-cisrol in the ovaries at maturation stage iv and all-transral in the eyestalks of the shrimp, l. vannamei. in this study, we detected the ra isomers such as 9cra and atra in mature ovaries (stage iv) of mud crabs, s. serrata using hplc analysis. retinoic acid is the active metabolite of vitamin a. thus, identification of retinoic acid isomers in mature ovaries might suggest the occurrence of vitamin a metabolism in crabs. interestingly, enzymes that convert retinaldehydes to ra have been identified in the extracts of uca pugilator blastemas (hopkins, 2001) and, carotene 15 -15 monooxygenase cdnas have been identified in an est library of crab blastema tissues (durica et al., 2006). vitellogenesis is a key step through which the developing oocytes are nourished via yolk globules and is under the control of vitellogenin gene in crustaceans (warrier and subramoniam, 2002; jia et al., 2013). in this study, we found a significant increase in the ovarian index and oocyte diameter associated with the accumulation of the oocytes in ovary of mud crabs injected with 9cra. this may suggest the stimulatory effect of 9cra on figure 3. changes in the ecdysteroid levels of hemolymph in mud crabs: at different reproductive stages (a) and injected with 9cra (b). bars are mean ±s.d. of 6 individual crabs (a) and 12 individual crabs (b); values in the parentheses are percent change from that of control; for evaluation of statistical analysis and percent change, for crabs at stage ii, iii and iv, crabs at stage i served as controls (a) and for evaluation of statistical analysis and percent change, for vehicle injected crabs, crabs in initial control group served as controls and for 9cra injected group crabs, crabs in vehicle injected group served as controls (b). bars with same superscript in a column do not differ significantly from each other at p<0.001. 251 int. j. aquat. biol. (2019) 7(5): 245-253 vitellogenesis. recently, it has been shown that the exogenous injection of 13cra could lead to concurrent up-regulation of vitellogenin gene expression from the hepatopancreas of crabs, o. senex senex (girish et al., 2018). the results also indicated that the injection of 9cra also enhanced the circulatory levels of ecdysteroids in intact mud crabs, suggesting that the 9cra at least in part mediates ecdysone signaling (girish et al., 2018). it is wellknown that the process of vitellogenesis is regulated by different hormones, including the ecdysteroids (subramoniam, 2017). the ecdysteroid levels were also significantly elevated as the crabs shift from the immature stage to the mature stage (gong et al., 2015). it has been shown that the genomic actions of ecdysone signaling occur via ecdysteroid receptors (ecr), a member that belongs to the nuclear receptor family in crustaceans. it was reported that the promoter region of vitellogenin gene contains several transcription binding sites, including the dimer partners, ecr and the ultra spiracle that are required to execute the genomic actions of ecdysteroids (martin et al., 2001). interestingly, injection of 13cra also enhanced the expression levels of ecr and rxr mrna in the hepatopancreas (the site for vitellogenesis) in freshwater crabs, o. senex senex. thus, ligand bound ecr combines with the rxr, which is also known as ultra-spiracle in insects to form a heterodimer complex. this complex (ligand bounded ecr/rxr) attaches to its respective response elements on the promoter region of vitellogenin thereby facilitates its transcription (tiu et al., 2010). to summarize, accumulation of yolk globules in the oocytes of 9cra-injected mud crabs may be attributed to the stimulation of vitellogenesis mediating ecdysone signaling. the second plausible reason could be attributed to the ability of 9cra to inhibit the release of gih from the xo-sg complex which eventually leads to the release of gsh from the brain and thoracic ganglia thereby ovarian maturation. nevertheless, the detection of retinoic acid in the mature ovaries of crabs and stimulation of ovarian growth after 9cra injection suggests, at least in part, a selective mode of action of retinoic acid. further, studies to elucidate the mechanism(s) of action of retinoic acid and its crosstalk with other endogenous hormones in the regulation of ovarian maturation might be helpful to develop reliable strategies for captive breeding in crustacean aquaculture. references abuhagr a., malea k., chang s., donald m. 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(2021) 9(2): 97-104 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article optimized exploitation of pharaoh cuttlefish (sepia pharaonis ehrenberg, 1831) stocks in the iranian part of persian gulf and oman sea seyed ahmadreza hashemi* 1, mastooreh doustdar2 1offshore fisheries research center, iranian fisheries science research institute, agricultural research, education and extension organization, chabahar, iran. 2iranian fisheries science research institute, agricultural research, education and extension organization, tehran, iran. s article history: received 29 february 2020 accepted 22 march 2021 available online 2 5 april 2021 keywords: maximum sustainable yield monte carlo simulation method arima model abstract: the purpose of the present study was to investigate the trends in pharaoh cuttlefish (sepia pharaonis) capture fisheries and determine the suitable range for optimized exploitation of s. pharaonis resources in the iranian part of persian gulf and oman sea using catch data. the data on pharaoh cuttlefish capture fisheries in iranian southern waters for the twenty-three years was collected and the suitable range for optimized exploitation of s. pharaonis was estimated using the r software. the average values (95% confidence interval) using the monte carlo simulation method for intrinsic population growth rate (r), maximum sustainable yield (msy), the biomass of maximum sustainable yield (bmsy) and maximum fishing mortality rate of maximum sustainable yield (fmsy) were 0.92 (0.73-1.17) per year, 5100 (4200-6200) tons, 1100 (8670-13900) tons, 0.46 (0.36-0.58) per year, respectively. the results showed that the annual catch of s. pharaonis exceeded the maximum sustainable yields and measures should be taken to reduce the number of capture fisheries and fishing effort. with results of the prediction model was observed moving average analysis (mape=2.85, mad=0.10, msd=0.02) and arima (0, 0, 1) (aic=9.79, bic=6.38), are better than other models for a period of five years for modeling annual this species landing. it seems that reducing fishing permits and fishing effort will put the s. pharaonis stock situation in a more favorable condition in the long term and will further benefit the exploiters and the fishing community. introduction global total capture fisheries production was about 91 million tons in 2016, of which 87% were in seawater (79.3 million tons) and 13% from inland waters (11.6 million tons) (fao, 2018). in recent years, there are remarkable signs of over exploitation of major fish stocks and other aquatic resources. the proportion of stocks fished at biologically sustainable levels (bsls) to biologically unsustainable levels (buls) in 1974 was about 90%, whereas this proportion in 2016 was about 67%. thus, buls has increased and requires immediate management measures (fao, 2018). the entire amount of fisheries production in iran was near 800,000 tons, of which 720,000 tons (more than 90 percent) were fisheries production within the seawaters of southern iran (fisheries statistical yearbook, iran area, 2020). *correspondence: seyed ahmadreza hashemi doi: https://doi.org/10.22034/ijab.v9i2.736 e-mail: seyedahmad91@gmail.com the quantity and quality of knowledge from many of the world’s fisheries are inadequate to enable traditional methods of assessment to be applied. the management of "data-rich" stock approaches is usually focused on complex models of stock evaluation and involves variety of knowledge sources. stock management is currently faced with numerous fish stocks that have little knowledge that do not help these with data-rich approaches (dick and maccall, 2011; ghaitaranpour et al., 2019). today, length-based models, like length based spawning potential ratio (lbspr), length-based integrated mixed effects (lime), and length-based bayesian (lbb), and also as catch-based methods, like catch-maximum sustainable yield (catch-msy), depletion based stock reduction analysis (dbsra), simple stock synthesis (sss), and catch-msy (cmsy) in many fishery scenarios and 97 int. j. aquat. biol. (2021) 9(1): 97-104 in several countries are developed (known as “datapoor” or “data-limited” fisheries) (wetzel and punt, 2015). with their unique ecological conditions, the persian gulf and oman sea host a good range of aquatic species that provide settlers with livelihoods, jobs and various economic activities (rajaei et al., 2014; taghavimotlagh and shojaei, 2017; eagderi et al., 2019). iran has quite 120,000 fishermen, mainly engaged in fishing, and fishing has played a serious role in generating jobs in coastal areas and in post-harvest economic activities (taghavimotlagh and shojaei, 2017). mollusks are the largest and most diverse group of invertebrates after arthropods with more than 80,000 extant species (bruska et al., 2016). among the economically important mollusks, pharaoh cuttlefish (sepia pharaonis) is a neritic demersal species inhabiting coastal waters to the depth of 130 meters. it is more likely to be found at the depth of 10 to 40 m (jereb and roper, 2005). this species is a predominant species in the persian gulf and oman sea (valinasab, 1993, 1997; roper et al., 1984). in addition, s. pharaonis is found in the indo-pacific region, the red sea, the arabian sea to the south china sea, the east china sea and the northern australian waters (jereb and roper, 2005). estimation of optimal fishing based on the time series of various aquatic species fishery has been carried out in some fish species (martell and froese, 2013; froese et al., 2016; zhou et al., 2017). hence, the present study aimed to investigated the trends in pharaoh cuttlefish capture fisheries in the iranian part of persian gulf and oman sea with the aim of determining the suitable range for its optimized exploitation and proper and principled exploitation management. materials and methods the data on pharaoh cuttlefish capture fisheries landings (based on metric ton) in the iranian part of persian gulf and oman sea (fig. 1) for the twentythree years was collected from the iran fisheries organization (from 1997 to 2019). catch-msy (cmsy): the catch-msy model has the same characteristics as the graham-shaefer surplus production model. these models rely on only a catch time series dataset and prior ranges of r and k and possible ranges of stock sizes in the first and final years of the time series. the cmsy is a method for estimating maximum sustainable yield (msy) and related fisheries reference points (bmsy, fmsy) from catch data and information on resilience (froese et al., 2017). this model requires a prior distribution on r and k as well as priors on the relative proportion of biomass at the beginning (martell and froese, 2013). the biomass in subsequent years was then generated figure 1. map of the study area and main iranian fishing ports (green color denoted the fishing ports). 98 hashemi and doustdar / optimized exploitation of pharaoh cuttlefish stocks in the persian gulf and oman sea from a schaefer model according to equation of by+1=by+rby (1-by/k) es1-ct es2, where by is biomass in the year y+1, r = population instantaneous growth rate, k = carrying capacity, cy = catch in the time series. in this method, the values of population instantaneous growth rate and carrying capacity are calculated with depletion formula (d) and storage saturation (s): d=1-s=1-by/ky. the maximum steady-state mortality rate with the formula of fmsy=r/2 and the maximum sustainable yield is calculated from msy=rk/4 and bmsy=k/2 (zhou et al., 2017). a prior range was set for r based on the resilience of the stock as proposed by martell and froese (2013), where stocks with a high resiliency were allocated a r value from 0.6–1.5 (zhou et al., 2017). the intrinsic population growth rate was calculated based on the inverse range factor and the formula of irf=3/r high–r low (froese et al., 2016). the es1 and es2 remove the bias in the equation and simulation. the es1 is related to the process error, whereas the es2 is related to the observation error. the desired pattern of the model is achieved when the standard deviation of the process error is set to 0.2 and the observation error is set to 0.1 (froese et al., 2016). forecasting methods: univariate time series models: moving average (ma) method is the arithmetic mean of observations of the full data set and uses the arithmetic mean as the predictor of the future period (karmaker et al., 2017). exponential smoothing (es) method is a kind of weighted averaging method which estimates the future value based on previous forecast plus a percentage of the forecasted error. trend analysis (td) may be a general model to multiple statistic data having trend pattern and provides idea to traders about what is going to happen within the future supported historical data (karmaker et al., 2017). winters method (wm) is employed to smooth data employing a level component, a trend component, and a seasonal component at each period and provides short to medium range forecasting. decomposition method (dm) is employed to separate the statistic into linear trend and seasonal components and error (karmaker et al., 2017). the autoregressive integrated moving average (arima) models demonstrated a good performance in terms of explained variability and predicting power. the autocorrelation (acf) and partial autocorrelation functions (pacf) were estimated, which led to the identification and construction of arima models (tsitsika et al., 2007). the arima were implemented on the data and the best model was chosen using the test of akaike coefficient data autocorrelation functions (lawer, 2016). the general form of the arima models is referred to as arima (p, d, q), where p is the order of the autoregressive term (ar term), d is the degree of differencing involved to achieve stationarity, and q is the order of the moving average term (ma term). the best forecasting model: the values of mean absolute percentage error (mape), mean absolute deviation (mad) and mean square deviation (msd) are used to identify the best model (karmaker et al., 2017). 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 = � �𝑒𝑒𝑒𝑒𝐷𝐷𝑒𝑒� 𝑛𝑛 ∗ 100 mad=∑ |𝐷𝐷𝐷𝐷−𝐹𝐹𝐷𝐷| 𝑛𝑛 mse=∑(𝐷𝐷𝑒𝑒 − 𝐹𝐹𝑒𝑒)2/ 𝑛𝑛 − 1 where dt is actual demand for time period t, ft is forecast demand for time period t, n is specified number of time periods, and et is forecast error = (dt − ft). akaike information criterion (aic), bayesian information criterion (bic)) were estimated as follows: aic = -2 ln (maximum likelihood) + 2m bic = -2 ln (maximum likelihood) + m ln(n) where m is that the number of the estimated parameters and n is that the number of the observations. statistical analyses were performed with r studio (1.1.45), spss (22) and minitab (16) software package and a significance level of 0.05 was adopted. results the average catch (yi) of the studied period was 3536 tones with 95% confidence intervals of 4284-2919 tones and the average catch was significantly 99 int. j. aquat. biol. (2021) 9(1): 97-104 decreased for the twenty-three years (r=-0.14, p<0.05) (fig. 2). the amounts of trend catch of this species showed a decreasing trend (fig. 2). the cmsy model: based on the annual catch catches and initial pharaoh cuttlefish information (initial growth 0.6-1.5 per year), the software used the initial value to start of modeling based on the cmsy and the monte carlo simulation method. the initial relative biomass as 0.5-0.9 and the final relative biomass as 0.2-0.6 were considered. the output values table 1. average values (95% confidence interval) of cmsy model parameters for pharaoh cuttlefish in the persian gulf and oman sea (iranian coastal waters). indices / models cmsy average (maximum-minimum) biomass (1000 tonnes) 11.9 ( 7.1-13.2) msy (1000 tonnes) 5.1 (4.2-6.2) bmsy (1000 tonnes) 11 (8.67-13.9) fmsy 0.46 (0.36-0.58) f 0.46 (0.41-0.77) b/bmsy 1.08 (0.64-1.2) f/fmsy 1 (0.93-1.32) k (1000 tonnes) 22 (17.3-27.9) r 0.92 (0.73-1.17) bt/k 0.54 bmsy/k 0.50 figure 2. the changes trend in the annual (yr) and catch (total) (a) and trend catch of this period with 95% confidence intervals (b) for sepia pharaonis in the persian gulf and oman sea (iranian coastal waters). 100 hashemi and doustdar / optimized exploitation of pharaoh cuttlefish stocks in the persian gulf and oman sea of the monte carlo simulation method with 30,000 times the modeling repeat result the average values (95% confidence interval) of state-space surplus production model for intrinsic population growth rate (r), carrying capacity (k), maximum sustainable yield (msy), biomass of maximum sustainable yield (bmsy), current or existing biomass (b), maximum fishing mortality rate of maximum sustainable yield (fmsy) and current or existing fishing mortality (f) that are shown in table 1. the ratios of the current biomass to the biomass of maximum sustainable yield (b/bmsy) and the ratio of the current fishing mortality to the maximum fishing mortality rate of maximum sustainable yield (f/fmsy) were 1.08 (0.64-1.2) and 1 (0.91-1.2), respectively (fig. 3). according to the kobe plot, which represents the graphic of the values of b/bmsy and f/fmsy, it can be concluded that the increase in the fishing table 2. forecasting and error calculations of different methods (mean absolute percentage error (mape), mean absolute deviation (mad), mean square deviation (msd) for pharaoh cuttlefish. method mape mad msd multiplicative decomposition (yt=3.431+0.0375*t) 4.43 0.15 0.035 additive decomposition (yt=3.429+0.0377*t) 4.43 0.15 0.035 moving average 2.85 0.1 0.02 single exponential smoothing 3.88 0.13 0.025 double exponential smoothing 3.87 0.13 0.027 trend analysis (linear) (yt=3.56+0.0017*t) 4.43 0.15 0.035 trend analysis (exponential) (yt=3.56*(0.99*t) 4.43 0.15 0.035 trend analysis (quadratic) (yt=3.90-0.083*t+0.0033*t2) 3.03 0.10 0.016 winters multiplicative 4.13 0.14 0.036 winters additive 4.13 0.14 0.036 figure 3. changes in biomass of maximum sustainable yield, intrinsic population growth rate and carrying capacity of pharaoh cuttlefish in the persian gulf and oman sea (iran) based on 30,000 times repeat of modeling using the monte carlo simulation method for cmsy model. 101 int. j. aquat. biol. (2021) 9(1): 97-104 mortality and the reduction of the current biomass has begun and is still ongoing. the mortality fishing (f) to mortality fishing of the maximum sustainable yield (fmsy) ratio (f/fmsy), the biomass (b) to biomass of the maximum sustainable yield (bmsy) ratio (b/bmsy) in the cmsy model were 1 (0.93-1.32) and 1.08 (0.64-1.20) (in 2019 year), respectively. forecasting methods: performance of sixteen methods was evaluated based on forecasting accuracy. the ten different forecasting techniques, including moving average and using of six methods identified orders of arima (p, d, q) based on the aic and bic. with results of the prediction model was observed moving average analysis (mape=2.85, mad=0.10, msd=0.02) and arima (0, 0, 1) (aic=9.79, bic=6.38), are better than other models for a period of five years for modeling annual this species landing (fig. 4). various models were then fitted and compared as presented in tables 2 and 3, using identified orders of arima (p, d, q) based on the aic and bic. however, arima (0, 0, 1) with drift was suitable for modeling annual s. pharaonis landings table 3. coefficients and summary statistics of multivariate arima modeling for pharaoh cuttlefish in the persian gulf and oman sea (iran) in the persian gulf and oman sea (iran). method b (coefficient) constant standard error of b log likelihood aic bic ar1 ma1 ar1 ma1 arima(1,0,0) 0.58 3.58 0.18 9.52 13.03 9.62 arima(0,1,0) 7.07 12.14 11.05 arima(0,0,1) 0.37 3.55 0.16 7.78 9.79 6.38 arima(1,0,1) 0.77 -0.29 3.59 0.19 0.27 9.95 11.90 7.35 arima(0,1,1) -0.38 0.19 8.59 13.05 10.86 arima(1,1,1) -0.16 -0.24 0.60 0.59 8.56 11.13 7.85 figure 4. the moving average trend fitted models (a), the arima (0,0,1) trend fitted (b) models (forecast plot is blue line) for of sepia pharaonis in the persian gulf and oman sea (iranian coastal waters) 102 hashemi and doustdar / optimized exploitation of pharaoh cuttlefish stocks in the persian gulf and oman sea (fig. 4) based on the selection criteria (aic=9.79, bic=6.38). discussions in recent years, the catching of pharaoh cuttlefish in the waters of southern iran has been decreased. over the past two decades, its catch rate in the waters of southern iran has dropped from about 9,000 tons in 1997 to about 5,000 tons in 2019, indicating a sharp decline (fisheries statistical yearbook, iran, 2020). in this regard, the sistan and baluchestan province had the highest percentage of pharaoh cuttlefish capture fisheries (63%), followed by bushehr (19%) and hormozgan (10%) provinces. khuzestan province had the smallest percentage of pharaoh cuttlefish capture fisheries (8%) and the hormozgan province showed the highest reduction. the intrinsic population growth rate (r) value of this species is high flexibility (0.6-1.5) indicating its high potential to stand up to fishing pressure and recovery i.e. it demonstrates the ability to withstand fishing pressure and the recovery of declining fish stocks (martell and froese, 2013; froese et al., 2016; zhou et al., 2017). the earlier work showed s. pharaonis as the most flexible species (froese and pauly, 2015). a strong correlation exists between the intrinsic population growth rate (r) and other parameters of life history, especially natural mortality (m). the best model is r=1.73 m for teleosts and r=0.76 m for elasmobranches (zhou et al., 2017). froese and pauly (2015) confirmed that the population intrinsic growth rate (r) is approximate twice the maximum fishing mortality rate of maximum sustainable yield (fmsy), 2 times the natural mortality (m), 3 times the growth rate coefficient of the von bertalanffy curve (k), 3 divided by the generation time (tgen) and 9 divided through the most age (tmax) (r ≈ 2fmsy ≈ 2 m ≈ 3 k ≈ 3 / tgen ≈ 9 / tmax). in the present study, the decreasing trend in the amount of current biomass to biomass of maximum sustainable yield (b/bmsy) showed that pharaoh cuttlefish had been in full exploitation condition for catching fisheries in the iranian part of persian gulf and oman sea. in addition, the level of current fishing mortality at the fishing mortality rate to be the maximum sustainable yield (f/fmsy), is shown with an increasing trend and toward overfishing pattern (arrizabalaga et al., 2012). the fishery status is usually evaluated based on b/bmsy and is divided into three general categories: b/bmsy≥1/5 means stocks under exploited, 0.5<b/bmsy<1.5 shows stocks fully exploited, 0.2<b/bmsy<0.5 means stocks overexploited and b/bmsy<0.2 shows collapsed stocks (branch et al., 2011; anderson et al., 2012). the biomass ratio of maximum sustainable yield to carrying capacity (bmsy/k) is one of the most relevant indicators for biological reference points. this ratio was 50% for this species in the persian gulf and oman sea (iran), suggesting average resource depletion (palomares and froese, 2017). the optimum value of this ratio typically varies from species to species and usually ranges 30-60%. there is a small amount of this index for species with high intrinsic population growth rates, while species with low intrinsic population growth rates have a high value. the minimum value for this index is 20-30%, and a sharp decrease in capital is less than this (gabriel and mace, 1999). the rate of exploitation and population biomass undoubtedly affects the rate of population growth and impacts the ratio of maximum sustainable yield biomass to carrying capacity (zhou et al., 2017). according to the results, the annual catch of pharaoh cuttlefish in the persian gulf and oman sea (iran), exceeded the maximum sustainable yield of pharaoh cuttlefish (approximately 5000 tons in 2019) and steps should be taken to minimize the amount of catch and fishing effort. it seems that reducing fishing permits and fishing effort, especially by fishing vessels, will put the s. pharaonis stock situation in a more favorable condition in the long term and will further benefit the exploiters and the fishing community. restoring fishery stocks that have been overexploited is unlikely to occur in a short time, because the rebuilding of a resource usually requires 2-3 times of its life span (fao, 2018). in this study, we illustrate linear forecasting methods that not capable of accurately forecasting the 103 int. j. aquat. biol. (2021) 9(1): 97-104 fishery landing time series, due to the fact that the series which is often highly nonstationary, and nonlinearity (shabri and samsudin, 2015). nonlinear forecasting approach better than linear forecasting and the best models of the arima scheme are also arima (0, 0, 1). time series forecasting using arima models have been widely applied in fisheries and univariate and multivariate arima models are useful tools for predicting the total fish production in different local. the major objective of this work was to develop a composite forecasting model to improve the prediction of this species in the persian gulf and oman sea (iranian coastal waters). despite all the uncertainty of the catch data, we need to use them in their work. fisheries managers for better and more stable management in the persian gulf and oman sea should concentrate on the available trends (rosenberg et al., 2005). further research can be check to compare the forecast ability of the model with other time series models like sarima, parima, etc. acknowledgments we are going to like to thank m. bahmani, the head of the iranian fisheries science research institute (ifsri) and the offshore fisheries research center (chabahar) experts are also very grateful for helping the project work. references anderson s.c., branch t.a., ricard d., lotze h.k. 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(2019) 7(6): 374-382 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article feeding habits of bigeye houndshark, iago omanensis (elasmobranchii; triakidae); a typical deep water shark from the gulf of oman ali reza rastgoo*1, eelia etemadi-deylami2, mohammad reza mirzaei3 1persian gulf and oman sea ecological center, iranian fisheries science research institute (ifsri), agricultural research, education and extension organization (areeo), bandar abbas, iran 2department of marine biology, school of marine science and technology, hormozgan university, iran. 3offshore fisheries research center, iranian fisheries science research institute, agricultural research education and extension organization (areeo), chabahar, iran. s article history: received 29 august 2019 accepted 24 december 2019 available online 2 5 december 2019 keywords: shark foraging ecology diet ecological role abstract: in this study, the feeding habits of bigeye houndshark, iago omanensis, a typical deep water shark, were examined in the gulf of oman by analyzing of stomach contents. in addition, the effects of sex and seasons (spring and summer) on its feeding habits were evaluated. bigeye houndshark diet consists of mostly teleost fishes, and to a lower extent on crustaneans, molluscs and sea snakes. the great importance of teleost in the diet of bigeye houndshark may be due to the fact that teleosts are the dominant in terms of biomass and abundance in the area where bigeye houndshark exist, allowing them to exploit food resources available in the environment. no significant differences were found between sexes and seasons. this species occupy high trophic position within the food webs. these results present new data that will allow us to understand the role of bigeye houndshark in the deep water of gulf of oman to effect of fishing activity on its population dynamics in the future. introduction marine predators have remarkable effects on the food web and are interesting case in understanding the factors that control their ecological role within the aquatic ecosystem (navarro et al., 2013). sharks classified as those predators which have wide effects on various potential prey within food web. teleost fishes, sea turtles, sea snakes, sea birds, crustaceans, other elasmobranchs, cephalopods and marine mammals have reported as potential prey groups for sharks (simpfendorfer et al., 2001). beside the vast range of favorite prey, the degree of foraging specialization in sharks is an important aspect which shows that they utilize only a subset of prey in their environment (davies et al., 2012; navarro et al., 2013) or they select a wider range of trophic resources (davies et al., 2012). this trait can be changed between different groups of population for example between sex (o’shea et al., 2013), maturity stages or/and size classes (simpfendorfer et al., 2001; *correspondence: ali reza rastgoo doi: https://doi.org/10.22034/ijab.v7i6.668 e-mail: rastgoo.alireza@yahoo.com rastgoo et al., 2018a) and also geographic location (simpfendorfer et al., 2001). knowledge of top predators’ diet is important for understanding ecosystem dynamics (baremore et al., 2010), providing the foundation for ecosystem-based management and a multi-species approach to fisheries management (brodziak et al., 2004) and for understanding food web dynamics (espinoza et al., 2015). stomach content analysis is traditionally a standard method for determining the diet and trophic ecology of sharks (cortés, 1997; hyslop, 1980). analysis of diet can represent many factors of ecological interactions, including the feeding competition (o’shea et al., 2013; rastgoo et al., 2018b), prey preference (baremore et al., 2008), movement of predators and preys, and also reveal that how an animal might respond to changes in its ecosystem and prey assemblage (hambright, 1994; juanes et al., 2001), even the role in effects on commercially 375 int. j. aquat. biol. (2019) 7(6): 374-382 important species (navia et al., 2007). furthermore, quantification of diet through stomach content analysis (to determine the food habits and feeding behavior) is essential for recognizing the roles of predators in an ecosystem (baremore et al., 2010) and providing high taxonomic details on the diet specially for what species has recently consumed (hussey et al., 2011). it is also pivotal in the management of shark fisheries from both natural processes as well as anthropogenic influences (wetherbee and cortés, 2004; jabado et al., 2015). despite many studies defined the relation between ontogeny of sharks and differences in selected prey and size of species consumed (see cortés, 1997; ebert, bizzarro, 2007; hyslop, 1980), the number of studies in the gulf of oman and related regions are very rare (e.g. jabado et al., 2015). gulf of oman is related to arabian sea on southward and the persian gulf on westward. it is environmentally unique with an unusual faunal assemblage (valinassab et al., 2006). every year, thousands tons of various fishes are caught in this area, which sharks are the remarkable portion of total catch quantity. according to increase in commercial fishing efforts, sharks are also over exploited next to other fishes. the bigeye houndshark, iago omanensis (norman, 1939) is found on continental shelves and slopes at depths of 110-1000 m, and possibly to as deep as 2.195 m (eagderi et al., 2019). jabado et al. (2017) recorded this species in the red sea and along the coast from oman to india with the exception of the persian gulf. total biomass of i. omanensis, in one of the annual stock assessment survey through iranian related waters in the gulf of oman, has estimated around 59 tons (unpublished data). sharks as agile predators can form the marine ecosystems especially in deep waters. in this study, we aimed to analyze food content consumed by i. omanensis in the gulf of oman, where there is a lack of data, to answer how this species connect to ecosystem through food webs. materials and methods study area and sampling procedure: the study area was the northern part of oman gulf, with coordinates of 2426 and 5855 west and 2413 and 6125 east. the gulf of oman is a marginal sea with a narrow continental shelf that 3/4 of its body is deeper than 1,000 m (reynolds, 1993). it has a rapid floristic turnover, which could be one of the sharpest biotic transition represented in marine biogeography (schils and wilson, 2006). the distinct partitions from the persian gulf and gulf of oman have significant differentiations from those of the arabian sea based on their species richness, species composition, average distribution range per species, general temperature, affinity of the composing species, and seasonal temperature data of the coastal waters (schils and wilson, 2006) and remarkable faunal assemblages (valinassab et al., 2006). the main atmospheric phenomenon in the area is indian monsoon, which makes important upwelling systems in this area and affects the structure of ecosystem communities (schils and wilson, 2006). specimens were collected as by-catch from commercial bottom trawlers during two cruises with the r/v ferdows-1 between may and july 2017. the mesh size of cod end net was 80 mm and the headline net mesh was 72 mm. specimens were collected at bottom depths between 50 and 110 m from 27 hauls. the duration of each haul varied from 150 to 180 min, depending on the sampling station. specimens were identified on board and the sex, body weight (to the nearest 10 g), and the total body length for each individual were recorded. stomach content analyses: we weighed the stomachs of the i. omanensis, and then recovered the stomach contents during dissections in the laboratory. all prey parts recovered were separated, identified to the lowest possible taxon, counted, and weighed to the nearest 0.1 g. the number of individuals of each prey was determined as the least number that these fragments could have originated from to avoid overestimation of the occurrence of a particular prey item. we combined the data from the stomach contents into four functional groups (teleosts, crustaceans, mollusca, chordata). in order to examine effect of sample size in estimating the diet of species, we constructed 376 rastgoo et al./ feeding habits of iago omanensis cumulative prey curves (cortés, 1997) using the shannon-weiner method to evaluate if the number of sampled stomachs was enough to describe the diversity of the diet of each group of the species or not. it was randomized the samples 50 times with the computer routine “sample-based rarefaction” using estimates 9.1 software (colwell, 2005; bornatowski et al., 2014). the sample size was considered to be sufficient if the curves visually reached an asymptote (magurran, 2013). a combined index of relative importance (pinkas et al., 1971) used to estimate the relative importance of each prey group in the diet of each group as: irii = (ni + wi )· foi (eq. 1) where foi is the frequency of occurrence of a particular functional prey group (i) in relation to the total number of stomachs, ni is the contribution by number of a type of prey group (i) in relation to the whole content of the stomach, and wi is the weight of a prey group (i) in relation to the whole content of the stomach. all calculations were based on the number of non-empty stomachs. iri values were expressed as a percentage to allow comparisons between prey groups (cortés, 1997): %irii=100· irii ∑ irii n i=1⁄ (eq. 2) the diet diversity for each group of the species were estimated with using of shannon-weiner diversity index (h) (ludwig and reynolds, 1988) as: h = ∑ 𝑝𝑖𝑛𝑖=1 log pi (eq. 3) where pi is the proportion of the i prey group in the diet. the pielou’s index (j) was also used to estimate evenness of the prey distribution in the stomach contents of predator as: 𝐽 = 𝐻 𝐿𝑜𝑔 𝑆 (eq. 4) where j is the pielou’s index, h is the shannon wiener index and s is the number of species in the diet of predator. margalef’s index also was estimated to calculate the species richness of different prey taxa in the diet of predator as: 𝑑 = 𝑆−1 𝐿𝑜𝑔 𝑁 (eq. 5) where s is the number of species recorded in the diet and n is the total of individuals present in the diet. we also categorically estimated the trophic level (routley et al., 2002) that the bigeye houndshark preyed at using the w% with the trophlab software (pauly et al., 2000). trophlab estimates tl considering the diet composition and the trophic level of the different prey present in the diet, according to w% (pauly et al., 2000) as: tli = 1 + ∑ dcij ∗ tlj 𝐺 𝑗=1 (eq. 6) where dcij is the fraction of prey (j) in the diet of consumer i, tlj is the trophic level of prey (j), and g is the number of prey categories. the trophic level of each prey category was extracted from the fishbase dataset (froese and pauly, 2000). statistical analysis: we tested for differences among the sex and seasons (spring and summer) in their stomach contents (based on %w) with the semiparametric permutation multivariate analyses of variance tests (permanova test) on the braycurtis distance matrix. permanova allows for the analysis of complex designs (multiple factors and their interactions) without the constraints of multivariate normality, homoscedasticity, and when there are a greater number of variables than in traditional anova tests. the method calculates a pseudo-f statistic analogous to the traditional f-statistic for multifactorial univariate anova models, using permutation procedures to obtain p-values for each term in the model. when results were significant, we then conducted pair-wise tests. we evaluated similarities in diets using the bray–curtis similarity coefficient and then we applied non-metric multidimensional scaling analysis (nmds). all statistical tests were performed using primer v.6 software (clarke and gorley, 2006). results a total of 63 specimens were collected and examined. the sample was composed of 40 females and 23 males ranging 35 to 66 cm. all individuals categorized among 8 separated length class. the less length classes were belonged to 30-35, 35-40 and 65-70, whereas the highest collected specimens was categorized in 50-55 and 55-60 length classes (fig. 1). specimens were collected in spring (29 individuals) 377 int. j. aquat. biol. (2019) 7(6): 374-382 and summer (34 individuals). in the examined specimens, 61 individuals (96%) contained food in their stomachs. the cumulative prey curves based on the diversity of prey for each group indicated that sample sizes were adequate to suggest their feeding habits (fig. 2). diet composition including percentages number (%n), percentage by weight (%w), frequency of occurrence (%fo), and index of relative importance (%iri) of prey items are shown in table 1. in terms of importance of prey in the overall diet (%iri), the bigeye houndshark mainly fed on teleosts (%iriteleosts = 89.94 %), followed by crustaceans (%iricrustaceans = 9.21 %), mollusca (%irimollusca = 0.83 %) and chordata (%irichordate ~0.01 %) (table 2). the overall biodiversity indices, including shannon-wiener index, pielou’s index and margalef table 1. diet composition of iago omanensis from the gulf of oman expressed as percentages number (%n), percentage by weight (%w), frequency of occurrence (%fo), and index of relative importance (%iri) of prey items food items %n %w %fo %iri teleosts 67.45 87.98 90.16 89.94 trichiuridae trichiurus lepturus 3.30 6.70 9.83 1.82 nemipteridae nemipterus japonicus 1.88 9.82 4.91 1.06 acropomatidae acropoma sp. 26.88 3.39 37.70 21.13 synodontidae saurida tumbil 3.30 6.18 8.19 1.43 platycephalidae grammoplites suppositus 0.94 2.14 3.27 0.18 carangidae selar crumenophthalmus 0.47 4.34 1.63 0.14 other carangids 1.88 4.92 4.91 0.61 mullidae upeneus sulphureus 2.35 8.2 6.55 1.28 sphyraenidae sphyraena putnamae 0.94 9.05 3.27 0.60 unidentified fishes 25.47 33.16 54.09 58.73 crustaceans 28.30 4.12 44.26 9.21 penaeidae 2.35 0.93 6.55 0.39 squillidae 6.60 0.81 14.75 2.02 portunidae 0.94 0.96 3.27 0.11 isopod 4.24 0.11 8.19 0.66 amphipod 1.41 0.03 3.27 0.08 unidentified crustaceans 12.73 1.26 27.86 7.22 mollusca 3.77 7.57 11.4 0.83 sepiidae sepia sp. 3.77 7.57 11.47 2.41 chordata 0.47 0.31 1.63 0.00 hydrophiidae hydrophis sp. 0.47 0.31 1.63 0.02 figure 1. size distribution of iago omanensis sampled for stomach content analyses 378 rastgoo et al./ feeding habits of iago omanensis table 2. percentage of the index of relative importance (%iri) of the main taxonomic groups in the diet of iago omanensis in function of a) the sex (females and males) and b) the season (spring and summer). number of individuals is indicated between branches. teleosts crustaceans mollusca chordata a) sex female (40) 94.23 4.88 0.85 0.02 male (23) 79.48 19.15 1.36 0.00 b) season spring (29) 93.68 6.58 0.73 0.00 summer (34) 86.89 12.14 0.92 0.03 table 3. biodiversity indices for the prey items of iago omanensis in the gulf of oman. n s h j d a) sex female 40 54 3.80 0.95 11.28 male 23 36 3.32 0.92 7.56 b) season spring 29 36 3.35 0.93 7.70 summer 34 49 3.66 0.94 10.23 s: total species; n: total individuals; h: shannon wiener index; j: pielou’s index of evenness and d: margalef’s index of richness figure 2. cumulative average and standard deviation of shannon–wiener diversity index for iago omanensis. 379 int. j. aquat. biol. (2019) 7(6): 374-382 index for each group are shown in table 3. however, except the margalef’s index of richness, no variation of indices was observed for prey items among groups. diet differences, based on %w were not found between males and females or between seasons (sex comparisons, f =0.41, df =1, p-value = 0.91 and seasons differences, f=0.30, df=1, p-value=0.97). in addition, nmds confirmed an overlap between groups (fig. 3). overally, the bigeye houndshark has occupied a high trophic level (tl = 4.40), placing this species in top predator levels. discussions current study revealed new information on the diet of i. omanensis, a deep water shark, in the northern gulf of oman. although the stomach contents analyses have restricted to consumed preys within a few days before (rastgoo et al., 2018b), but it can provide fundamental information for rare species, like i. omanensis that occur in the deeper water. previous studies on bottom-dwelling sharks and skates revealed that the proportion of empty stomachs is generally low (kamura and hashimoto, 2004; scenna et al., 2006; yick et al., 2011), as we observed in this study that 96% of stomachs of i. omanensis were full. based on the results, teleosts were the most preferred preys of i. omanensis. in addition, crustaceans, cephalopods and sea snakes were recorded in several stomachs. nair and appukkuttan (1973) reported the similar diet for this species at the southeast coasts of india. furthermore, waller and baranes (1994) pointed out that deep water cephalopods and benthopelagic fishes are important prey group in the diet of i. omanensis in the red sea. the importance of teleosts and cephalopods in the diet of triakids has been recorded in costa rica (espinoza et al., 2015). also, cortés (1999) mentioned that teleosts, crustaceans and cephalopods are the most important food for several species of mustelus (more than 70%). mesopelagic aquatics such as teleosts and cephalopods are able to form an important dietary contribution to the deep sea ecosystem (valls et al., 2014). crustaceans also have been documented as preferred prey group in mustelus sp. in north-east atlantic (ellis et al., 1996) and coastal waters of colombia (navia et al., 2007). therefore, i. omanensis shows a similar diet like other members of the family triakidae. focusing on possible variations based on sex to found any ontogenetic shifts in diets as well as what occurs based on habitat variation, should be studied (jabado et al., 2015). in the present study, there was no markedly dietary preference seen by males or females of i. omanensis, indicating that the foraging figure 3. nmds scaling of the stomach content of iago omanensis sampled from the gulf of oman. 380 rastgoo et al./ feeding habits of iago omanensis habitats and dietary requirements are similar for both sexes, which caused a high degree of dietary overlap (simpfendorfer et al., 2001). but, based on the results, the prey diversity was slightly higher in female (h:3.80) than male (h:3.32), while the richness of prey items had significant differences in female (d:11.28) than male (d:7.56). the most proportion of diet in both sexes was teleosts which followed by crustaceans. but there was a clear difference between the volumes of consumed crustaceans in male (19.15) compare with female (4.88), which revealed that the crustaceans are more noteworthy by males of i. omanensis. these differences can be explained with the fact that females reach to a greater size than males, where maximum size for female and male are 84 and 54 cm from oman (henderson et al., 2009), 67 and 43 cm from red sea (waller and baranes, 1994), 83 and 59 cm in india (barnes et al., 2018), respectively. the results also indicate that i. omanensis is probably an important predator of teleosts and crustaceans along the deep waters of the gulf of oman. interestingly, in one hand the dominated diet by teleosts shows the role of agility of this predator, followed by crustaceans which need bottom-dwelling behavior, on the other hand. the high trophic level of bigeye houndshark (more than tl = 4) indicates that this species occupied high trophic level, a position shared by other sharks and some batoids (rastgoo and navarro, 2017) and more than other genera in this family, such as mustelus (cortés, 1999). however, this species have an important potentially predator in the deep water food web of the gulf of oman (rastgoo and navarro, 2017). due to the likely current abundance of bigeye houndshark in the gulf of oman’s ecosystem, its ecological role may be potentially high and effective. in conclusion, here we presented new information on feeding habits of bigeye houndshark in the northern part of gulf of oman. although, the sampling period did not cover through the year, we reported first evidence of the diets of i. omanensis in this area. however, increasing the fleets and fishing effort maintain intensive pressure on the gulf of oman marine resources (valinassab et al., 2006). indeed, the outspread of fishing technology to exploitation of deep sea resources is similar with the depth that this species exists. it can be predicted that in the future, the stocks or habitat of this species may be seriously damaged. thus, our results present important data that will allow an exploration of the role of bigeye houndshark in the gulf of oman and how to connect to the food web. acknowledgements we thank the captain h. mohammad zadeh and the crew of ferdows 1 for sampling. references baremore i.e., murie d.j., carlson j.k. 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(2018) 6(6): 303-306 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology short communication phagotrophic algae in a wastewater stabilization pond emmanuel bezerra d’alessandro*1, ina de souza nogueira2, nora katia saavedra del aguila hoffmann3 1institute of chemical-1, federal university of goiás. avenida esperança s/n, bloco iq-1, câmpus samambaia cep 74690-900. goiânia – goiás, brasil. 2department of biology, federal university of goiás. avenida esperança s/n, câmpus samambaia cep 74690-900. goiânia – goiás, brasil. 3school of civil and environmental engineering, federal university of goiás. av. universitária, no. 1488. setor universitário. cep 74605-220. goiânia – goiás, brasil. article history: received 5 december 2018 accepted 25 december 2018 available online 2 5 december 2018 keywords: euglenophyta peranema phagotrophic pheophytin abstract: in the course of evolution, phagotrophic euglenoids developed before the phototrophic species. most phagotrophic euglenoids have a robust feeding apparatus. members of the algal genus peranema are able to eat a wide variety of living organisms that have little or no mobility, such as other unicellular algae and live yeasts. stabilization ponds are artificial environments rich in organic matter, nutrients and microorganisms, and are therefore suitable environments for growing species of euglenophyta. this contribution records, through photographs and videos, the operation of the feeding apparatus of peranema collected in a wastewater stabilization pond. the mean pheophytin content of the water was higher than the mean chlorophyll a content, which indicates a non-ideal environment for the growth of microalgae. thus, peranema can be used as a bioindicator of the quality of wastewater stabilization pond. the operation of the feeding apparatus of peranema sp. and the strategy for phagocytizing plastids of lepocinclis sp. are described. introduction the euglenoids represent an ancient and diverse eukaryotic lineage. huber-pestalozzi (1955) described over 800 species belonging to about 40 genera. about two-thirds of the species are non-photosynthetic. in the course of evolution, phagotrophic euglenoids developed before the phototrophic species. the phototrophic species evolved from a single secondary endosymbiotic event involving a chloroplast from a green alga, a member of prasinophyceae (marin, 2004). the transition from a phagotrophic to a phototrophic lifestyle occurred several times independently in the evolution of eukaryotes. one of these events occurred within the euglenophyta, a moderately large group of flagellates with diverse modes of nutrition. phototrophic euglenoids constitute a clade that is nested within lineages of phagotrophic euglenoids, and originated through a secondary endosymbiosis with green algae (yamaguchi et al., 2012). most phagotrophic euglenoids have a robust feeding apparatus comprised of two main bundles of *corresponding author: emmanuel bezerra d’alessandro doi: https://doi.org/10.22034/ijab.v6i6.557 e-mail address: dalessandro.e.b@gmail.com microtubules termed rods, and four interior vanes (leander et al., 2007). members of the euglenoid genus peranema are able to consume a wide variety of living organisms that lack or have low mobility, such as live yeasts and other unicellular algae. smaller organisms are swallowed whole. larger organisms are also swallowed, but when this is not possible peranema uses its rod-organ to make a cut in the periplast of the prey and ingests the contents, including starch grains, oil droplets and protein particles (chen, 1950). marin (2004) noted that algae may be colorless not only through the loss of chloroplasts, but also from having undergone a mutation that results in the formation of leucoplasts rather than chloroplasts. leucoplasts are non-pigmented plastids that function to store reserve substances. stabilization ponds are artificial environments rich in organic matter and nutrients, and are therefore suitable environments for growing euglenophyta. for instance, soldatelli and schwarzbold (2010) found that euglenophytes comprised 27% of the total number 304 d’alessandro et al./ phagotrophic algae in a wastewater stabilization pond of taxa in the phytoplankton community.this contribution records, based on microphotography, the operation of the feeding apparatus of a species of peranema collected in a stabilization pond. materials and methods the samples were collected in september 2011, from a facultative pond of the trindade wastewaterstabilization pond system, goiás, brazil. the identifications of the organisms in living samples were based on morphological and morphometric characteristics, analyzed in an inverted microscope equipped with an olympus ckx41 camera connected to the software cell d®. the reference used for identification was huber-pestalozzi (1955). physical and chemical analyses of the wastewater, including temperature, electrical conductivity, orthophosphate, ammonia, biochemical oxygen demand, ph and transparency, according to apha (1998). the transparency of the stabilization ponds was measured with a secchi disk, and the depth of the euphotic zone was calculated as three times the transparency value (cole, 1975). results and discussion as seen in table 1, the dissolved-oxygen content was low (1.9 mg l-1), which is common in stabilization ponds. the mean pheophytin content was higher than the mean chlorophyll content, which indicates a nonideal environment for the growth of microalgae, i.e., they were dying. in such a situation, the phagotrophic euglenoids exploit the weakness of other algae. the pond depth was approximately 1.70 m and the euphotic zone was 0.45 m. figure 1 shows an individual peranema sp. swallowing plastids of a weakened lepocinclis sp. to access the plastids, the peranema used its flagellum to make a hole in the anterior part of the pellicle of lepocinclis sp. and, when it had pierced the cell, projected its feeding apparatus to phagocytize the plastids (fig. 1a, b and c). triemer (1997) showed that peranema can ingest living cells, paramylon grains, and whole plastic beads, and has a strong preference for nonmotile or injured cells. alternatively, peranema can “drill” or tear a hole in the prey cell and then suck out the contents. the projected rod-organ seems to help to push food into the body of the cell. after phagocytizing approximately 7 plastids, peranema sp. used its flagellum to exit from inside the lepocinclis sp. cell (fig. 1d, e, f). upon exiting, peranema sp. showed cell contractions for more than 1 minute (fig. 1g, h). figure 1g shows the hole in the anterior part of lepocinclis sp. left by peranema sp. flagellated algae in stabilization ponds help to stabilize the organic-matter content (madoni, 1994). in general, the protist community is directly related to the depuration state and the final effluent quality (salvado et al., 1995). akpor and momba (2010) studied phosphate and nitrate removal in activated sludge, and found that using peranema and acetate as a carbon source, the phosphate and nitrate concentration decreased dramatically after 96 hours. in conclusion, peranema is a good microorganism for use in sewage treatment, because it can survive in environments with high organic matter and low dissolved oxygen. thus, peranema can be used as a bioindicator of the quality of wastewater stabilization pond. acknowledgments the author expresses his sincere thanks to the company saneago s.a., especially to operator irmon, for allowing the collection of peranema in the stabilization pond. table 1. mean and standard deviation (sd) of physicochemical and biological variables analyzed in facultative pond. variables mean sd temperature (°c) 25.7 1.6 ph 7.7 0.1 dissolved oxygen (mg l-1) 1.9 0.4 electrical conductivity (µs cm-1) 1,343 7 orthophosphate (mg l-1) 0.9 0.2 ammonia (mg l-1) 13.2 2.0 chemical oxygen demand (mg l-1) 390 39 chlorophyll a (µg l-1) 691 475 pheophytin(µg l-1) 1008 875 transparency (m) 0.15 0.01 euphotic zone (m) 0.45 0.03 305 int. j. aquat. biol. (2018) 6(6): 303-306 figure 1. sequential movements of peranema sp. phagocytizing plastids of a lepocinclis sp. (a b and c) opening the pellicle and phagocytizing the plastids, (d, e and f) using the flagellum to remove matter from inside a cell of lepocinclis sp., and (g and h) contractions of the cell after phagocytizing the plastids (scale = 20 µm). 306 d’alessandro et al./ phagotrophic algae in a wastewater stabilization pond references akpor, o.b., momba, m.n.b. (2010). relationship of protozoan biomass to phosphate and nitrate removal from activated sludge mixed liquor. biotechnology journal, 5: 304-313. apha. (1998). standard methods for examination of water and wastewater. 20th ed. ediciones diaz de santos s.a/american public health association; american water works association/water pollution control federation, madrid. chen y.t. (1950). investigations of the biology of peranema trichophorum (euglenineae). quarterly journal of microscopical science, 3-91: 279-308. cole g.a. (1975). textbook of limnology. mosby company, st. louis. huber-pestalozzi g. (1955). das phytoplankton des süßwassers. systematik und biologie: euglenophyceen. e. schwiezerbat’sche verlagsbuchhandlung, stuttgart. leander b.s., esson h.j., breglia s.a. (2007). macroevolution of complex cytoskeletal systems in euglenids. bioessays, 29: 987-1000. madoni p. (1994). microfauna biomass in activated sludge and biofilm. water science and technology, 29: 63-66. marin b. (2004). origin and fate of chloroplasts in the euglenoida. protist, 155: 13-4. salvado h., gracia m.p., amigó j.m. (1995). capability of ciliated protozoa as indicators of effluent quality in activated sludge plants. water research, 29: 10411050. soldatelli v.f., schwarzbold a. (2010). comunidade fitoplanctônica em lagoas de maturação. iheringia. série botânica, 65: 75-86. triemer r.e. (1997). feeding in peranema trichophorum revisited (euglenophyta)1. journal of phycology, 33: 649-654. yamaguchi a., yubuki n., leander b.s. (2012). morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy: description of rapaza viridis n. gen. et sp. (euglenozoa, euglenida). bmc evolutionary biology, 12: 29. int. j. aquat. biol. (2014) 2(3): 111-114 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article first record of redneck goby schismatogobius deraniyagalai (teleostei: gobiidae) from seethanathi river, karnataka, southern india muthukumarasamy arunachalam1, murugan muralidharan*1, karunakaran remadevi2 1sri paramakalyani centre for environmental sciences, manonmaniam sundaranar university, alwarkurichi 627 412, tamil nadu, india. 2southern regional station, zoological survey of india, 130, santhome high road, chennai 600 028, tamil nadu, india. article history: received 2 march 2013 accepted 14 april 2013 available online 2 5 june 2014 keywords: gobiidae, new record, seethanathi, redneck goby, india abstract: schismatogobius deraniyagalai is recorded from the seethanathi river of karnataka state in the southern part of india. previous records of these species were from the streams in kerala of india and from freshwater habitats of sri lanka. herein we report the occurrence of this species in seethanathi river showing its distribution extended further north along the west coast of peninsular india. introduction schismatogobius is a genus of small freshwater gobies with a naked and elongated body with a characteristic marmorated color pattern, living among pebbles in the freshwaters above tidal influence (chen et al., 2001). distribution of the genus is chiefly confined to indo-pacific islands from sri lanka to fiji. the 10 nominal species known till date include schismatogobius marmoratus (peters, 1868), s. bruynisi (de beaufort, 1912), s. insignus (herre, 1927), s. pallidus (herre, 1934), s. roxasi (herre, 1936), s. deraniyagalai (kottelat and pethiyagoda, 1989), s. ampluvinculus (chen, fang and shao, 1995), s. fuligimentus (chen, seret, pollabauer and shao, 2001), s. vanuatuensis (keith, marquet and watson, 2004) and s. vitiensis (jenkins and boseto, 2005). the single species of schismatogobius recorded from the indian subcontinent is s. deraniyagalai described from sri lanka (kottelat and pethiyagoda, 1989; pethiyagoda, 1991). the discovery of this species from sri lankan water bodies is significant in terms of biogeography and distribution as it was for the first time the genus has * corresponding author: murugan muralidharan e-mail address: muralistream@gmail.com been documented from an indian ocean island as all other known species have been collected only form islands of the pacific ocean, southern japan, indonesia, philippines and australia. as for the indian subcontinent schismatogobius deraniyagalai was first reported from the upper reaches of chaliyar river system inside the nilgiri biosphere reserve of kerala (easa and basha, 1995; easa and shaji, 1997). this region comprises part of the western ghats known for its rich species diversity and high levels of endemism, also regarded as one of the world’s biodiversity hotspots (myers et al., 2000; sajan et al., 2014). that was the only site record for this species after which there are no reports showing its occurrence in any of the freshwater bodies in southern india to date. this species has been now found in the seethanathi river of karnataka showing the extension of distribution of the species further north along the west coast of india. materials and methods the specimens were collected using cast nets and preserved in 10 % formalin. measurements were 112 arunachalam et al./ first record of redneck goby made with dial calipers with an accuracy of 0.01 mm. methods for taking counts and measurements follow hubbs and lagler (1947). the specimens are deposited at the zoological survey of india/southern regional station (zsi/srs), chennai, india. abbreviations of fins: d1 and d2, 1st and 2nd dorsal fins; a, anal fin; p, pelvic fin; v, ventral fin; c, caudal fin. results eight specimens (2 males and 6 females) of s. deraniyagalai range 26.0-38.5 mm of standard length (table 1, fig. 1) were collected from the upstream of seethanathi river at thuthinjet (13º 24’ n, 71º 01’ e), karnataka state, india during 2002– 2004. the specimens were deposited in the ichthyological section of the zoological survey of india, southern regional station, chennai, india and the accession register number is f.7149 srs/zsi. schismatogobius deraniyagalai is distinguished from all its congeners by its very slender body (6.5–7.9 times sl) 7.9–8.3 times as per original description by kottelat and pethiyagoda (1989). however the specimens collected from indian waters differ in being stouter than the sri lankan forms. preopercular canal and associated pores absent in the cephalic sensory pore system. fin ray counts d1: vi; d2: i,9; a:i,9; p:14-15;v:i,9-10, c:12-13. 1 2 3 4 5 6 7 8 mean ♀ ♀ ♀ ♂ ♂ ♀ ♀ ♀ total length 44 44.5 40 40 36 35 32 31 standard length 37 38.5 33 33 30 29 27 26 (% sl) body depth 18.2 15.4 16.4 17.0 15. 7 15.9 15.9 15.0 16.2 body width 16.2 14.0 14.2 14.2 13.3 13.8 13.3 11.9 13. 9 head length 25.1 26.8 25.8 29.7 28.0 26.6 27.0 25.0 26.7 length of snout 5.1 5.2 4.9 5.8 5. 7 4.8 4.8 4.6 5.1 length of maxilla 10.0 9.6 8.5 18.8 15. 7 9.0 8.2 6.9 10.8 eye diameter 5.4 5.9 4.9 6.4 6.0 5.2 5. 6 5.4 5.6 interorbital width 3.7 2.6 3.0 4.2 3.3 3.5 3.3 3.5 3.4 predorsal distance 34.5 34.8 34.9 36.4 33.3 32.4 33.3 35.0 34.3 pre ventral 30.0 30.1 30.9 32.7 32.0 30.0 30.0 28.9 30.6 pre anal 60.5 61.0 61.2 58.8 59.0 59.3 56. 7 57.7 59.3 length of pectoral 24.3 20. 8 25.8 21.8 25.3 24.8 21.9 23.1 23.5 height of dorsal i 14.0 11.7 9.7 12.1 11.3 12.1 8. 9 10.4 11.3 height of dorsal ii 9.7 10.4 11.2 11.2 10.0 12.7 10.0 11.5 10.9 height of anal 5.4 10.4 10.3 11.2 10.0 10.3 9.6 9.6 9.6 length of pelvic 22.1 22.9 22.4 22.1 21.7 23.1 23.7 21.2 22.4 width of head 16.2 15.3 15.2 15.8 14.3 13.8 14.1 13.5 14.7 length of caudal peduncle 15.9 15.3 17.9 18.8 17. 7 17.6 19.3 17.3 17.5 height of caudal peduncle 7.8 7.5 7.88 8.2 8.0 8.6 7.4 7.7 7.9 length of caudal fin 19.7 20.0 20.6 20.7 21.7 20.4 20.4 20.5 distance between pelvic tip and anal 10.0 10.4 9.7 6.4 7.7 7.6 6.3 3.9 7.7 distance between pectoral origin pelvic 5.9 5.5 6.36 5.8 6.3 6.2 5.9 6.2 6.0 pelvic to anal base 33.2 33.0 33.0 29.1 29.7 32.4 29.3 28.9 31.1 length of body opercular border to anus 39.2 35.8 37.9 31.0 30.0 35.9 33.7 31.9 34.4 (% hl) length of snout 20.4 19.4 18.8 19.4 20.2 18.2 17.8 18.5 19.1 length of maxilla 39.8 35.9 32.9 63.3 56 33.8 30.1 27.7 40.0 eye diameter 21.5 22.3 18.8 21.4 21.4 19.5 20.5 21.5 20. 9 interorbital width 15.1 9.71 11.8 14.3 11.9 13 12.3 13.8 12.7 width of head 64.5 57.3 58.8 53.1 51.2 51.9 52.1 53.8 55.3 table 1. morphometry of schismatogobius deraniyagalai from seethanathi river of south india (f.7149 srs/zsi) 113 int. j. aquat. biol. (2014) 2(3): 111-114 discussion variations are observed between the type specimens as noted from the original description and the samples collected during this study. body shape of specimens in the present collection are slender but stouter than the sri lankan forms, body depth in percentage sl in the former is 16.2 (15.0-18.2) vs. 12.4 (12.0-12.7) in the latter; width of body 13.9 (11.9-16.2) vs. 10.4 (10.0-10.8). slight variations in snout length are also observed in that the percentage snout length in sl is 5.1 (4.6-5.8) vs. 6.3 (5.4-7.3). further difference observed was in predorsal distance 34.3 (32.4-36.4) vs. 38.7 (38.3-39.3) and depth of caudal peduncle 7.9 (7.4-8.6) vs. 7.2 (7.0-7.3). in addition the teeth in jaws are reported to be in three rows whereas in the specimens from india the rows are more in number and irregularly arranged. all other morphometric and meristic characters overlap between the species. however the colour pattern is similar in both original types and the present samples. the differences observed are only in the girth of the species and also there is much overlap in many of the characters these specimens hence are reported as s. deraniyagalai. this species was not recorded during the survey undertaken in the tributaries of chaliyar river of the new amarambalam reserve forest (narf), which forms the core area of the nilgiri biosphere reserve from where the fish was recorded earlier (baby et al., 2010). anthropogenic interventions pose pressure and threat to the survival of this species and hence their distribution is restricted only to isolated streams/rivers in dense forests. acknowledgements first author is thankful for the financial support from national bureau of fish genetic resources of indian council of agricultural research (icar), government of india through the national agricultural technology project. references baby f., tharian j., ali a., raghavan r. (2010). a checklist of freshwater fishes of the new amarambalam reserve forest (narf), kerala, india. journal of threatened taxa, 2(12): 1330-1333. chen i.s., seret b., pollabauer c., shao k. t. (2001). schismatogobius fuligimentus, a new species of freshwater goby (teleostei: gobiidae) from new caledonia. zoological studies, 40 (2): 141-146. easa p.s., basha s.c. (1995). a survey on the habitat and distribution of stream fishes in the kerala part of nilgiri biosphere reserve. 86 pp. kfri research report 86. kerala forest research institute, thrissur, india. easa p.s., shaji c.p. (1997). freshwater fish diversity in kerala part of the nilgiri biosphere reserve. current science, 73 (2): 180-182. hubbs c.l., lagler k.f. (1947). fishes of great lakes region. bulletin of cranbrook institute of sciences, 26: 1-213. kottelat m., pethiyagoda r. (1989). schismatogobius deraniyagalai, a new goby from srilanka description and field observation. spixiana, 12: 315-320. myers n.r.a., mittermier c.g., da fonseca g.a.b., kent j. (2000). biodiversity hotspots for conservation priorities. nature, 403: 853-858. pethiyagoda r. (1991). freshwater fishes of sri lanka. the wildlife heritage trust of sri lanka, colombo. p.362. figure 1. schismatogobius deraniyagalai collected from seethanathi river, karnataka 114 arunachalam et al./ first record of redneck goby sajan s., anna mercy t v., malika v (2014). biometric parameters of the redline torpedo fish puntius denisonii day 1865, an endemic barb in the western ghats hotspots of southern india. international journal of aquatic biology, 2(2): 75-84. shaji c.p., easa p.s., gopalakrishnan a. (2000). freshwater fish diversity of western ghats. in: a.g. ponniah, a. gopalakrishnan (eds.). endemic fish diversity of western ghats. nbfgr-natp publication volume 1 national bureau of fish genetic resources, lucknow, india. pp: 33-35. international journal of aquatic biology (2015) 3(3): 129-134 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article hydrocortisone treatment may enhance survival and stocking of beluga sturgeon (huso huso linnaeus, 1758) in estuaries of the caspian sea nastaran mazloumi*1, bagher mojazi amiri2, mohammadali nematollahi2, gholamreza rafiee2 1southern seas ecology laboratories, school of biological sciences, university of adelaide, south australia 5005 australia. 2department of fisheries, faculty of natural resources, university of tehran, po box 4314-31583, karaj, iran. article history: received 30 october 2014 accepted 10 april 2015 available online 2 5 june 2015 keywords: cortisol chloride cell salinity acipenseridae great beluga abstract: beluga sturgeon (huso huso linnaeus, 1758) fingerlings are released into the caspian sea for recruiting and enhancing commercial and recreational fishing purposes. these fingerlings are reared in fresh water, but released to the estuaries that may be caused mortalities due to acute osmotic stress. in this study, the fingerlings in whole (in vivo) or their gill tissue (in vitro) were exposed to three different levels of ‘the stress hormone’ cortisol (3, 5, 7 mg l-1 hydrocortisone sodium phosphate) for 24 hrs. the effects of treatments on blood cortisol levels and the size and numbers of gill chloride cells were monitored. in each case, hormonal treatment significantly increased blood cortisol levels and also the number but decreased the size of the chloride cells. we conclude that bathing in hydrocortisone could promote the survival rate of the fingerlings in brackish water and may be have a positive effect on their osmoregulation potentiality. introduction the beluga sturgeon, huso huso is an anadromous acipenserid species in the caspian sea basin, and subjected to intensive fishing as a source of caviar. the stock of this species has been declined in recent years (fakharzadeh et al., 2011), and therefore, listed as ‘critically endangered’ (iucn, 2012). in marchjune of each year, the belugas migrate into inflowing rivers, e.g. the volga (russia) and sefidrood (iran), where they spawn (vosoghi and mostajir, 2002). in early march, the broodstocks are captured from the sea and transferred to the hatchery for artificial breeding, and then produced juveniles (with 2-3 g) released to the estuaries, where the water salinity is about 12 ppt. this releasing may be caused mortality because of insufficient fish osmoregulation capabilities (abdolhay and tahori, 2006). strategies to minimise osmotic stress and promote juvenile survival, therefore, are likely to be commercially and ecologically beneficial. * corresponding author: nastaran mazloumi e-mail address: nastaran.mazloumi@adelaide.edu.au the steroid hydrocortisone (cortisol) hormone is produced in response to stress, and acts on receptors in the gills, kidneys and intestines of fishes (van der salm et al., 2002). this hormone increases salinity tolerance by increasing the size and the number of gill chloride cells, and promotes chloride ion transport through increased na+/k+/atp–ase enzyme activity (khodabandeh et al., 2009). as blood cortisol levels are known to increase in sturgeons, salmon, lampreys and other fishes during downstream, post-spawning migrations from fresh to saline water (tipsmark and madsen, 2001), it is possible that artificially increase of cortisol levels could help to offset osmotic stress. therefore, the present study aimed to describe the effects of direct (fingerlings in whole, in vitro) and indirect (gill tissue culture, in vivo) exposure of beluga sturgeon to cortisol. we anticipated that hormonal treatment will increase the osmoregulation potency through the blood cortisol level and increase the number and 130 international journal of aquatic biology (2015) 3(3): 129-134 reduce the size of gill chloride cells, preparing the fingerlings for osmotic shock associated with their release to the brackish water. materials and methods a total of 500 beluga fingerlings (3.00 ± 0.5 g) were obtained from the hyrkan sturgeon culture farm (ramsar, mazandaran province, northern iran), and transferred to the aquaculture laboratory at the university of tehran. fish were acclimatized for seven days to laboratory conditions in circular 150l tanks with continuously aerated, filtered and recycled tap water (water temperature 20 ± 2°c, dissolved oxygen >5 mg l-1, ph 7, 12/12h photoperiod). throughout the acclimatization and experimental periods, the fish were fed twice daily with a dry commercial pellet diet (45% protein, 12% lipid, 3.5% carbohydrate, 10% ash; behparvar co., iran), at 1.5% of tank biomass. uneaten food and faeces were removed from the tanks twice a day. water quality parameters were checked daily, and salinity was adjusted, if necessary, using dechlorinated tap water. all dissecting equipment (scalpels, scissors, and forceps) was autoclaved for 24 hrs before use. in the in vivo experiment, three treatments (each of 20 fingerlings) were exposed to 3, 5 and 7 mg l-1 of the hydrocortisone sodium phosphate, respectively, along with a control group without hormonal exposure, each with three replicates. after 24 hrs, fish were anaesthetized using clove oil solution (150 ppm) and killed by spinal section. the blood samples were randomly taken from 20 specimens in eppendorf® tubes with a drop of edta anticoagulant, and transferred to a glass jar containing ice (without direct contact with the ice). the blood cortisol levels were determined in the blood serum, using the eliza method (diagnostics biochemistry canada, ontario; sensitivity 0.4 μg dl1) (lucía et al., 2011). in addition, the second left gill arch of the specimens were removed in sterile conditions, and fixed in 4% formalin for 24 hrs (merck®), maintained in 70% alcohol for one week (eagderi et al., 2013). the tissues were processed and 5 µm histological section prepared and stained with haematoxylin and eosin based on eagderi et al. (2013). furthermore, the chloride cells on the prepared gill sections were counted, and their sizes measured along the bases of five gill lamella according to khatooni et al. (2011) using an optical micrometer. in the in vitro experiment, the second left gill arch of the fingerlings were removed, divided by scalpel into 20 mm pieces and washed three times for 15 min in phosphate-buffered saline solution (pbs: 80 g nacl, 2 g kcl, 26.8 g na2hpo4.7h2o, 7.4 g k2hpo4 and 1 l distilled water) (mccormick, 1995). then, the samples were transferred to the 24-chamber cell culture-plate (bd falcon®, cat. 3043) with each chamber containing 1 ml ringer’s solution (mojazi amiri et al., 1999). furthermore, three concentrations of hydrocortisone, i.e. 3, 5 and 7 mg l-1, were added to three treatments with three replicates. meanwhile, a control group was left without adding hormone with three replicates. the plates were kept over ice before transferring to an incubator with 15°c (wood and pärt, 1997). after 24 hrs, the samples were fixed in bowen’s solution (75 ml picric acid, 25 ml formalin, 5 ml acetic acid), and 5 µm histological section prepared and stained based on eagderi et al. (2013). the chloride cells were counted, and their sizes measured as mentioned for the in vivo experiment. statistical analyses were performed by the packages spss 19 and sas 9. one-way anova was applied to compare treatment means (α = 0.05); the lsd test was applied to compare the treatments with controls and duncan’s test to pairwise combinations of treatments. results the results showed significant differences in blood cortisol between the controls and treatments (p<0.01). based on the results, the blood cortisol levels increased by increasing the levels of the hormonal exposure (fig. 1). this correlation is proportional with high sensitivity (fig. 2). the results revealed that the in vivo hormonal treatment 131 mazloumi et al/ hydrocortisone treatment of beluga have significantly affected the numbers and size of the gill chloride cells in 7 mg l-1 treatments (figs. 3 and 4). the similar results obtained in the in vitro experiment (figs. 5 and 6). the effects of in vivo and in vitro exposures are presented in table 1. in general, the exposure to the hydrocortisone increased the number of chloride cells and decreased their size. the changes were slightly greater in vitro, that can be due to the effect of the ringer’s solution or pbs buffer on tissues. discussion we exposed beluga fingerlings to three levels of the hydrocortisone, anticipating increase of the osmoregulation potency through the changes of their physiological indices. the results support this hypothesis and the levels of the cortisol in the blood plasma increased and also, the number of the gill figure 1. effects of three levels of hydrocortisone on blood cortisol levels in beluga fingerlings, data are mean ± sd, n = 20. figure 2. relationship between blood cortisol levels and exposure to hydrocortisone treatments in beluga fingerlings. figure 3. effects of three levels of the hydrocortisone on the number (a) and size (b) of gill chloride cells in beluga fingerlings in the in vivo experiment. data are mean ± sd, n = 20. table 1. the effect of hydrocortisone treatment (mg l-1) on numbers and sizes (µm2) of gill chloride cells of beluga fingerlings in vivo and in vitro experiments. each cell shows the mean ± sd for 3 replicates. the trend lines estimate the effect (y) from the treatment (x); r2 is the coefficient of determination. treatment in vivo in vitro number size, µm2 number size, µm2 control* 9 ± 1 68.37 ± 0.93 12 ± 2 80.37 ± 0.75 3 mg l-1 14 ± 3.21 38.21 ± 1.10 20 ± 5 51.21 ± 3.5 5 mg l-1 21 ± 4.04 26.04 ± 2.9 35.3 ± 6.4 35.42 ± 0.32 7 mg l-1 45 ± 5 19.69 ± 1.03 45 ± 5.1 24.69 ± 1.8 trend, y = 11.833x–6.833 –18.645x+94.447 13.333x–4.667 –17.376x+75.808 r2 0.912 0.965 0.993 0.991 132 international journal of aquatic biology (2015) 3(3): 129-134 chloride cells increased, whereas their size reduced. plasma cortisol levels in fish are known to increase as an adaptive response to osmotic stress, implying that an artificial increase in plasma cortisol could increase the tolerance of fingerlings to saline water (swallow and fleming, 1970). khodabandeh et al. (2009) pointed out that the blood cortisol level of persian sturgeon fingerlings, acipenser persicus, is increased by exposing to hydrocortisone. the present study showed similar results in beluga fingerlings by increasing the blood cortisol, up to about 40 µg dl-1, with those of khodabandeh et al. (2009). in the present study, the numbers of gill chloride cells in beluga fingerlings increased with increasing the concentration of the hydrocortisone, so that, the 7 mg l-1 treatment showed a fourfold increase in compare to those of control group, in both in vitro and in vivo experiments. the in vitro results suggest that this was due to mitotic divisions of the chloride cells. furthermore, the sizes of the cells decreased more than threefold, both in vitro and in vivo. this response may be different if the hormonal treatment was extended for more than 24 hrs (cf. kazemi et al., 2003). an increase in the plasma cortisol generally is accompanied by changes in gill chloride cells, implicated in osmoregulation (e.g. adams, 1990), figure 4. gill chloride cells (arrows) of a beluga fingerling exposed to 7 mg l-1 hydrocortisone in the in vivo experiment (h&e, 100x). figure 5. effects of three levels of the hydrocortisone on the number (a) and size (b) of gill chloride cells of beluga fingerlings in vitro. data are mean ± sd, n = 20. figure 6. gill chloride cells (arrows) of the beluga fingerlings in the control group (a) and after exposure to 7 mg l 1 hydrocortisone in the vitro experiment (b) (h&e, 100x). 133 mazloumi et al/ hydrocortisone treatment of beluga although the nature of the response differs between bony and cartilaginous fish. in bony fish like rainbow trout (oncorhynchus mykiss: van der salm et al., 2002) and atlantic salmon (salmo salar: madsen et al., 1997), cortisol increases the size of the chloride cells, but in cartilaginous species the opposite is true (mccormick, 2001). in persian sturgeon fingerlings, khoshnood et al. (2010) showed that treatment with 5 mg l-1 hydrocortisone increased the number of gill chloride cells. carmona et al. (2004) and farabi et al. (2009) demonstrated similar responses in adriatic sturgeon (a. naccarii) and ship sturgeon (a. nudiventris). although 7 mg l-1 hydrocortisone treatment was most effective in juvenile beluga, this could be lethal for other fish, including persian sturgeon (cf. fakharzadeh et al., 2011), and the optimal dose should be established for each species. for beluga fingerlings, we conclude that 24 hrs exposures to 7 mg l-1 hydrocortisone could be a practical measure to forestall mortality after transfer from fresh to brackish water. we suggest that our observations warrant further testing under field conditions. acknowledgements we are grateful for assistance from nourallah ghorbani, director of the hyrkankesht sturgeon culture farm, mehdi hedayati, institute of endocrinology, shahid beheshti university, and mohammad ebrahimi, bs fisheries laboratory and faculty of veterinary histology, university of tehran. our thanks also to keith walker, school of earth and environmental sciences, the university of adelaide, for help in preparing this work for publication. references abdolhay h.a., tahori h.b. (2006). fingerling production and release for stock enhancement of sturgeon in the southern caspian sea: an overview. journal of applied ichthyology, 22: 125-131. adams s.m. (1990). status and use of biological indicators for evaluating the effects of stress on fish. fisheries symposium 8, american fisheries society, maryland, usa. 191 p. carmona r., garcía-gallego m., sanz a., domezaín a., ostos-garrido m.v. (2004). chloride cells and pavement cells in gill epithelia of acipenser naccarii: ultrastructural modifications in seawater-acclimated specimens. journal of fish biology, 64: 553-566. eagderi s., mojazi amiri b., adriaens d. (2013). description of the ovarian follicle maturation of the migratory adult female bulatmai barbel (luciobarbus capito, güldenstädt 1772) in captivity. iranian journal of fisheries sciences, 12(3): 550-560 fakharzadeh s.m.e., farhangi m., mojazi amiri b., ahmadi m., mazloumi n. (2011). the effect of hydrocortisone treatment by bathing and daphnia enrichment on the osmotic stress in persian sturgeon acipenser persicus juvenile. international aquatic research, 4: 39 44. farabi s., najafpour s., najafpour g. (2009). aspect of osmotic-ions regulation in juvenile ship, acipenser nudiventris (lovetsky, 1828) in the southeast of caspian sea. world applied sciences journal, 7: 1090-1096. lucía g.a., lehotay s.j., fortis l.l., paoli g., wijey c., heinzen h. (2011). development and validation of a rapid method for microcystins in fish and comparing lc-ms/ms results with elisa. analytical and bioanalytical chemistry, 401(8): 2617-2630. iucn (2012). red list of threatened species, v. 2012.2. at: http://www.iucnredlist.org, november 2012. kazemi r., bahmani m., krayushkina l.s., pourkazemi m., ogorzalek a. (2003). changes in blood serum osmolarity and ultrastructure of gill chloride cells in young persian sturgeon acipenser persicus (borodin) of different sizes during adaptation to sea water. zoologica poloniae, 48: 5 30. khodabandeh s., mosafer s., khoshnood z. (2009). effects of cortisol and salinity acclimation on na+/k+/2cl--co-transporter gene expression and na+, k+, -atpase activity in the gill of persian sturgeon, acipenser persicus, fry. scientia marina, 73(s1): 111116. khoshnood z., khodabandeh s., mosafer s., khoshnood r. (2010). effects of cortisol on gill chloride cells in persian sturgeon, acipenser persicus, fry. yakhteh medical journal, 11: 424-431. madsen s., mathiesen a., korsgaard b. (1997). effects of 17β-estradiol and 4-nonylphenol on smoltification and vitellogenesis in atlantic salmon (salmo salar). fish physiology and biochemistry, 17: 303-312. 134 international journal of aquatic biology (2015) 3(3): 129-134 mccormick s.d. (1995). 11 hormonal control of gill na+/k+/–atpase and chloride cell function. fish physiology, 14: 285 315. mccormick s.d. (2001). endocrine control of osmoregulation in teleost fish. american zoologist, 41: 781-794. mojazi amiri b., maebayashi m., adachi s., moberg g.p., doroshov s.i., yamauchi k. (1999). in vitro steroidogenesis by testicular fragments and ovarian follicles in a hybrid sturgeon, bester. fish physiology and biochemistry, 21: 1-14. schreck c.b, moyle p.b. (1990). methods for fish biology. american fisheries society, maryland, usa. swallow r.l., fleming w. (1970). the effect of oxaloacetate acth and cortisol on the liver glycogen levels of tilapia mossambica. comparative biochemistry and physiology part a, 36: 93-98. tipsmark c.k., madsen s.s. (2001). rapid modulation of na+/k+/–atpase activity in osmoregulatory tissues of a salmonid fish. journal of experimental biology, 20: 701-709. van der salm a., nolan d., wendelaar bonga s. (2002). in vitro evidence that cortisol directly modulates stress-related responses in the skin epidermis of the rainbow trout (oncorhynchus mykiss walbaum). fish physiology and biochemistry, 27: 9-18. vosoghi g., mostajir b. (2002). freshwater fishes. tehran university press, tehran. wood c., pärt p. (1997). cultured branchial epithelia from freshwater fish gills. journal of experimental biology, 200: 1047-1059. int. j. aquat. biol. (2019) 7(6): 383-386 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology short communication stonefish synanceia verrucosa bloch & schneider, 1801 (actinopterygii: synanceiidae): the first record in the syrian coast and the fourth in the mediterranean amir ibrahim, firas alshawy*,1chirine hussein department of marine biology, high institute of marine research, tishreen university, lattakia, syria. article history: received 27 november 2019 accepted 24 december 2019 available online 2 5 december 2019 keywords: synanceiidae, synanceia verrucosa, mediterranean, syrian marine waters. abstract: fish species introduction into the mediterranean sea is constantly increasing, and this is what worries biologists especially after the arrival of poisonous species. in this paper, synanceia verrucosa is recorded for the first time in the syrian marine waters, filling the gap of its distributions between north and south of the eastern mediterranean. introduction synanceiidae family (stonefishes) has nine genera and 36 species (carpenter and niem, 1999; carpenter and de angelis, 2016) characterizing by its warty skin and frightening shape. the stonefish of synanceia verrucosa has a deadly poison in the bases of the fin spines and capable of burying itself in the sand to camouflage and grab prey. before 2010, no individual was recorded in the mediterranean sea (froese and pauly, 2019) but, it was recorded from palmakhim (edelist et al., 2011), then from iskenderun bay in 2012 at (bilecenoğlu, 2012) and for third time from tyr coast in 2014 (crocetta et al., 2015). it has not been recorded yet in the european and african coasts of the mediterranean sea (bearez et al., 2017; el sayed haroun and karachle, 2017; froese and pauly, 2019). in this paper, s.verrucosa is recorded for the first time in the syrian marine waters, filling the gap of its distributions between north and south of the eastern mediterranean. materials and methods on 12 october 2019, during a field trip in the marine waters facing lattakia city, syria (35°31'5.97"n, 35°42'48.57"e), a specimen of s. verrucosa was collected using longline, with assistance of fishing *correspondence: firas alshawy doi: https://doi.org/10.22034/ijab.v7i6.750 e-mail: falshawy@gmail.com boat (9.5 m, 19hp). the specimen was identified according to carpenter and niem (1999), and the morphometric measurements (length to the nearest mm, weight to the nearest g) and meristic counts were recorded. the specimen was then photographed, preserved into 7% formaldehyde, and placed at the biological laboratory of the high institute of marine research (tishreen university lattakia, syria) as a reference sample (unnumbered yet). results and discussions a single specimen of stonefish, s, verrucosa bloch & schneider, 1801 was caught at ~15 m depth off lattakia coast (fig. 1). it has a large body and a warty skin. the mouth is upward with a deep pit behind and a smaller one below of eye. the pectoral, ventral and caudal fins are large and strong, with severe sharp spines, allowing them to move the sand and deposit the body. the body is completely covered with spots of various colours, especially light orange and brown. the meristic characters are d, xiii-7; a, iii-6; p, 18; v, 6; c 11. the morphometric measurements are shown in table 1. synanceia verrucosa is a tropical fish, spreads from the red sea and east africa to french polynesia, north to the ryukyu and from ogasawara islands, south to 384 ibrahim et al./ first record of synanceia verrucosa in the syrian marine waters. queensland of australia (carpenter and niem, 1999; froese and pauly, 2019; eagderi et al., 2019). it had been recorded in the eastern part of the mediterranean sea, as a lessepsian fish species (edelist et al., 2011; bilecenoğlu, 2012). the specimen recorded in this study provides an evidence of changes in the mediterranean environment (ibrahim, 2009; alshawy et al., 2019g, a; hussein et al., 2019; ibrahim et al., 2019a). this species had not been recorded in the syrian marine waters (alshawy et al., 2019c). synanceia verrucosa possesses the poisonous spines that have a deadly effect on humans since it inhabits the swimming areas and thus imposing a threat to human life. in addition, presence of such species in these new environments is hazardous because of exploiting the available food and habitats (katsanevakis et al., 2014; alshawy et al., 2019e). the mediterranean sea had received a number of similar species in the recent years (hallom et al., 2014; alshawy et al., 2019e, b; ibrahim et al., 2019b), and requires further studies to determine the resulting environmental, economic and health impacts. this necessitates building regional and international capacities for mutual cooperation (vallerga et al., 2003; drago et al., 2004; hussein et al., 2011; alshawy et al., 2019d) to develop plans of actions to mitigate the negative effects of these species in the eastern mediterranean (hussein et al., 2011a, b, alshawy et al., 2019f, hussein et al., 2019). acknowledgements the authors thank the higher commission for scientific research (damascus), tishreen university and the high institute of marine research (lattakia) who provided the financial and logistic supports to this work. references alshawy f., ibrahim a., hussein c., lahlah m. (2019a). first record of arrow bulleye, priacanthus sagittarius starnes, 1988 from the syrian marine waters (eastern mediterranean). fishtaxa, 2: 21-24. alshawy f., ibrahim a., hussein c., lahlah m. (2019b). first record of the blacktip cardinalfish apogon atradorsatus heller & snodgrass, 1903 from syrian marine waters (eastern mediterranean). international journal of advanced research in science, engineering and technology, 3: 8299-8302. alshawy f., ibrahim a., hussein c., lahlah m. (2019c). first record of the broadbanded cardinalfish ostorhinchus fasciatus white, (1790) from the syrian marine waters (eastern mediterranean). international journal of agriculture and environmental science, 6: 14-16. alshawy f., ibrahim a., hussein c., lahlah m. (2019d). first record of the flat needlefish ablennes hians figure 1. synanceia verrucosa, caught on 12 october 2019 from the syrian coast. 385 int. j. aquat. biol. (2019) 7(6): 383-386 (valenciennes, 1846) from syrian marine waters (eastern mediterranean). marine biodiversity records, 12(15): 1-4 alshawy f., ibrahim a., hussein c., lahlah m. (2019e). first record of the oceanic puffer lagocephalus lagocephalus (linnaeus, 1758) from the syrian marine waters (eastern mediterranean) marine biodiversity records, 12(11): 1-4: alshawy f., ibrahim a., hussein c., lahlah m. (2019f). first record of the spotfin cardinal fish jaydia queketti (gilchrist, 1903) (teleostei: apogonidae) from the syrian marine waters (eastern mediterranean). iranian journal of ichthyology, 2: 138-142. alshawy f., ibrahim a., hussein c., lahlah m. (2019g). new distribution of the serpent eel ophisurus serpens (linnaeus, 1758) in eastern mediterranean: first record from the syrian marine waters. international journal of agriculture and environmental science, 3: 50-52. bearez p., pruvost p., feunteun e., iglesias s., francour p., causse r., de mazieres j., tercerie s., bailly n. (2017). checklist of the marine fishes from metropolitan france. cybium, 4: 351-371. bilecenoğlu m. (2012). first sighting of the red sea originated stonefish (synanceia verrucosa) from turkey. coral reefs, 1: 76-82. carpenter k.e., de angelis n. (2016). the living marine resources of the eastern central atlantic. volume 4: part 2, bony fishes (tetradontiformes to perciformes) and sea turtles. fao. 1509 p. carpenter k.e., niem v.h. (1999). the living marine resources of the western central pacific volume 4 bony fishes part 2 (mugilidae to carangidae). fao. 736 p. crocetta f., agius d., balistreri p., bariche m., bayhan y., çakir m., ciriaco s., corsini-foka m., deidun a., el zrelli r. (2015). new mediterranean biodiversity records (october 2015). mediterranean marine science, 3: 682-702. drago a., aarup t., abdelbaki a., abuissa a., awad h., awad m.b., beken c., besiktepe s., boargob a.f, brundrit g., capari m., carlier a., cermelj b., casazza g., civili f.s., cohen y., christos t., dahlin h., dalla costa m., drakopoulos p., flemming n.c., font j.f., gertman i., harzallah a., herrouin g., ibrahim a., kabbara n. (2004). medgoos-building a strong regional partnership for operational oceanography in the mediterranean rapports et proces verbaux des réunions-commission internationale pour l'exploration scientifique de la mer méditerranée.158 p. eagderi s., fricke r., esmaeili h.r., jalili p. (2019). annotated checklist of the fishes of the persian gulf: diversity and conservation status. iranian jornal of ichthyology, 6(suppl. 1): 1-171 edelist d., spanier e., golani d. (2011). evidence for the occurrence of the indo-pacific stonefish, synanceia verrucosa (actinopterygii: scorpaeniformes: synanceiidae), in the mediterranean sea. acta ichthyologica et piscatoria, 2: 129. el sayed haroun k.a., karachle p.k. (2017). the marine ichthyofauna of egypt. egyptian journal of aquatic biology and fisheries, 3: 81-116. froese r., pauly d. (2019). fishbase available from: www.fishbase.de. retrieved 15/10/ 2019. hallom n., ibrahim a., galiya m. (2014). first record of the hen-like blenny aidablennius sphynx (blenniidae) from syrian marine waters (eastern mediterranean). marine biodiversity records, 73: 2. hussein c., ibrahim a., alshawy f. (2019). first record of red cornetfish, fistularia petimba lacepède, 1803 (actinopterygii: fistulariidae) from the syrian coast. international journal of aquatic biology, 3: 175-179. hussein c., verdoit-jarraya m., pastor j., ibrahim a., saragoni g., pelletier d, mahévas s., lenfant p. (2011a). assessing the impact of artisanal and recreational fishing and protection on a white seabream (diplodus sargus sargus) population in the northwestern mediterranean sea using a simulation model. part 1: parameterization and simulations. fisheries research, 1: 163-173. hussein c., verdoit-jarraya m., pastor j., ibrahim a., saragoni g., pelletier d., mahévas s., lenfant p. (2011b). assessing the impact of artisanal and recreational fishing and protection on a white seabream (diplodus sargus sargus) population in the northwestern mediterranean sea, using a simulation model. part 2: sensitivity analysis and management measures. fisheries research, 1: 174-183. hussein m., courp t., ibrahim a., benkhelil j. (2011). seasonal variability of hydrographical properties of the syrian marine water. journal of marine systems, 1: 3044. ibrahim a. (2009). national overview on vulnerability and impacts of climate change on marine and coastal biodiversity in syria unep/map-rac/spa. ibrahim a., alshawy f., hussein c., lahlah m. (2019a). confirmation record of the spot fin cardinal fish jaydia queketti (gilchrist, 1903) (teleostei: apogonidae) in 386 ibrahim et al./ first record of synanceia verrucosa in the syrian marine waters. the syrian marine waters. ssrg international journal of agriculture and environmental science, 4: 48-50. ibrahim a., hussein c., alshawy f. (2019b). new distribution of pteragogus trispilus randall, 2013 (actinopterygii: labridae) in the syrian marine waters (eastern mediterranean). ssrg international journal of agriculture and environmental science, 5: 24-26. katsanevakis s., wallentinus i., zenetos a., leppäkoski e., çinar m.e., oztürk b., grabowski m., golani d., cardoso a.c. (2014). impacts of invasive alien marine species on ecosystem services and biodiversity: a paneuropean review. aquatic invasions, 4: 391-423. vallerga s., drago a., aarup t., abdelbaki a., abuissa a., awad h, awad m., beken c., besiktepe s., boargob a, 2003. mama—towards a new paradigm for ocean monitoring in the mediterranean. elsevier oceanography series. athens: elsevier. int. j. aquat. biol. (2013) 1(3): 125-131 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article feeding and reproductive biology of amblypharyngodon mola (cyrpiniformes: cyprinidae) from two floodplain lakes of india debjit kumar mondal, anilava kaviraj*1 department of zoology, university of kalyani, kalyani – 741235, w.b. india. article history: received 10 may 2013 accepted 22 may 2013 available online 2 0 june 2013 keywords: freshwater fish diet gill and gut morphology spawning abstract: pattern of feeding and reproduction of amblypharyngodon mola (hamilton, 1822) was studied in two floodplain lakes of north 24 parganas districts of west bengal in india during 2011. results indicated that the fish exhibited a predominantly herbivorous diet with algae as the main content of gut. a long, thin walled, coiled gut and closely packed filamentous gill racker suited this feeding habit. feeding intensity of the fish fluctuated throughout the year, males showing maximum intensity in june and females showing maximum intensity in february and november. females outnumbered males (1.9:1) and were found as highly fecund with a calculated fecundity factor of 1445 ova g-1 of body weight. from the peak of gonado-somatic index and ova diameter it was revealed that a. mola apparently spawned only once during july in these floodplain lakes. it was concluded that improvement of ecosystem health of these two lakes were necessary to ensure spawning of the fish. introduction amblypharyngodon mola (hamilton, 1822) is a conspicuous member of the small indigenous freshwater fish (siff) species that inhabit ponds, rivers, floodplain lakes, canals, paddy fields and many other small water bodies in the indian subcontinent. siff species are defined as fish which grow to the size of 25-30 cm at mature or adult stage of their life cycle (sarkar and lakra, 2010). amblypharyngodon mola has drawn special interest among more than 200 siff available from north east india because of its high nutritional value (alam et al., 2004) and demand in the markets of indian subcontinent (azadi and mamun, 2004). although various aspects of culture and farming of the species are known (kohinoor, 2000; kohinoor et al., 2001; roy et al., 2002), most farmers of the subcontinent do not include this species in freshwater aquacultures, because it is an auto-breeder in * corresponding author: anilava kaviraj e-mail address: akaviraj@gmail.com tel: +913325828477 confined water and compete with major carps for space, natural food as well as supplementary feed (data, 2010). however, roos et al. (1999) observed that a. mola could be cultured successfully in small seasonal ponds in polyculture with carps subject to availability of good seeds and minor modification of the culture techniques. the floodplain lakes, particularly those which are opened and maintain a connection with the river, remained as the unique habitat for a large number of small freshwater selfrecruiting riverine species of fish like a. mola. in a recent study on two floodplain lakes of icchamati river basin a. mola has been found as one of the most abundant species in spite of a general trend of decline of finfish species in these two floodplain lakes (mondal and kaviraj, 2009) and many other floodplain lakes of india (panigrahi et al., 2003; kar et al., 2006). suresh et al. (2007) observed that a. mola once formed a major component of the 126 mondal and kaviraj/ int. j. aquat. biol. (2013) 1(3): 125-131 fisheries from floodplain lakes of west bengal, but its availability had been drastically reduced during the last few years. the floodplain lakes in west bengal and other north eastern states of india are gradually shrinking due to several allocthonous and autocthonous factors (das, 2002; deka et al., 2005; mondal and kaviraj, 2008). therefore, it has become very pertinent to study the biology of a. mola inhabiting these water bodies for a sustainable supply of the fish. comprehensive information on the biology of a species is an essential prerequisite to plan its stock rehabilitation programme in a water body. reproduction of the freshwater fish particularly the siff is greatly influenced by the environmental condition of the habitat occupied by them. at present, only limited information is available on the reproduction capacity of a. mola in floodplain lakes (piska et al., 1991). the objectives of the present study were to make a detailed study on the biology of a. mola with emphasis on its reproduction capacity to protect the natural population of the species in the floodplain lakes of north 24 parganas district of west bengal in india. materials and methods study sites and sampling: studies were made in two floodplain lakes, gopalnagar and dumar, situated on ichhamati river basin in the bongaon sub-division (23.07º n; 88.82º e) of west bengal in india. a detailed account of these two lakes, locally called as baur, has been described previously (mondal and kaviraj, 2009). the study period covered from january to december 2011. random samples of a. mola were collected in the morning from different sites of each baur using fine mesh cast net and gill net and pooled together to make a composite sample for study at every month. feeding habit: detailed feeding habit was studied every month on random samples of 15 specimens captured from these two lakes. the sampled fish were dissected out by a clean scissor and the viscera were cut open. length of the gut and length of the body cavity were recorded. structure of gut, gill and gill racker of five sampled fish were recorded. the gut was cut open and emptiness or fullness of the gut was recorded in all the sampled specimens. the period of intense feeding was determined as monthly feeding index (fi) using the following formula fi = (nf/n) x 100, where nf is the number of fishes with food in stomach and n is the number of fish examined. gut contents of the sampled fish were taken out from the anterior portion of the gut in a watch glass. necessary dilution of the gut content was made with normal saline and it was observed under light microscope using a sedgwick-rafter cell. food items were identified up to major taxonomic group. determination of reproductive pattern: the numbers of male and female fish in a sample of fifty specimens was recorded and reproductive pattern was studied separately for each sex. however, length and weight of only ovary could be recorded, because testis of this species was too small to be weighed. the selected mature ovaries were preserved in 5% formalin, which helped in separating the eggs from the ovarian wall (shafi and quddus, 1974) and make further studies. following parameters of reproductive behaviors were determined: sex ratio: the sex ratio was analyzed on total samples of fish by chi-square test following the equation of fisher (1970), assuming that the ratio of male and female in the population to be 1:1. gonado-somatic index (gsi): the gonado-somatic index (gsi) was determined using the formula gsi = (gw / bw) × 100, where gw = gonad weight, bw = body weight of fish. fecundity, maturity and ova diameter: fecundity was measured on random samples of 50 mature female fishes (length 4.70 to 7.7 cm and weight 2.50 to 7.0 g). samples taken from the anterior, middle and posterior region of both ovaries were weighed separately and number of mature ova present within each sample was counted. absolute fecundity was estimated on the basis of total weight of the ovaries using the following formula f = (wo × n) / w, where f is fecundity, wo is total weight of ovary, w is the weight of the sub-samples of ovary and n is 127 mondal and kaviraj/ int. j. aquat. biol. (2013) 1(3): 125-131 the number of mature ova counted in the sub sample. ova diameter was calculated every month on ovaries collected at random from 10 mature fishes with the help of ocular and stage micrometer. diameters of 10 ova were measured at random from anterior, middle and posterior region of each ovary and then the mean diameter was calculated. results anatomical features related with feeding: specimens of a. mola collected from these two baurs showed terminal mouth. gills were found with long filaments and minute but closely packed filamentous gill rackers (fig. 1). alimentary canal was characterized by a long, thin walled and highly coiled gut. there was practically no stomach. instead, the anterior part of the intestine was slightly swollen forming an intestinal bulb behind the oesophagus. feeding behavior and gut contents: feeding intensity fluctuated throughout the year showing three peaks both in male and female. maximum feeding activity, as revealed from the feeding index, was recorded in june in case of male and february and november in case of female. females showed less feeding activity for the months of may to july than rest of the months, while males showed less feeding activity for the months of april, july and august than the rest of the months (fig. 2). the fish showed a preference of herbivorous diet (fig. 3). algae were the main component of the gut table 1. monthly variation in sex ratio of amblypharyngodon mola. months total samples male female sex ratio chi-square j 50 16 34 1:2.1 6.48* f 50 14 36 1:2.6 9.68** m 50 21 29 1:1.4 1.28 a 50 15 35 1:2.3 8.00** m 50 14 36 1:2.6 9.68** j 50 15 35 1:2.3 8.00** j 50 12 38 1:3.2 13.52** a 50 17 33 1:1.9 5.12** s 50 22 28 1:1.3 0.72 o 50 20 30 1:1.5 2.00 n 50 22 28 1:1.3 0.72 d 50 22 28 1:1.3 0.72 overall 600 210 390 1:1.9 54.00** significant at *p < 0.05 and **p < 0.01 figure 1. structure of gill and alimentary canal of amblypharyngodon mola. figure 2. monthly variation of feeding index of amblypharyngodon mola. 127 128 mondal and kaviraj/ int. j. aquat. biol. (2013) 1(3): 125-131 content followed by plant materials, debris and mud, crustacean, rotifera and rhizopoda. among different groups of algae observed in the gut cholorophyceae was the dominant group followed by mixophycae and bacillariophyceae. the rests included cyanophyceae and euglenophyceae. there was a slight variation in food preference among different weight class. lower weight class preferred more algae than higher weight class. the higher weight class though preferred algae consumed high amount of other plant materials and debris and mud than the lower weight class. the ratio of total length to gut length was 1: 3.56. reproductive pattern: the ratio of male and female varied between 1:1.3 to 1:3.2, which has been mentioned in table 1. the ratio significantly departed from the expected 1:1 ratio in the months of january (p<0.05), february, april, may, june, july, august, october (p<0.01). during these months the females significantly outnumbered the males. the overall sex ratio for the whole sample over a period of twelve months also varied significantly from the expected ratio (p<0.01) with 1.9 female for every male. the gsi of the female ranged from a minimum value of 1.39 ± 0.45 in december to a maximum value of 12.75 ± 3.75 in july. the diameter of ova ranged from 0.05 to 0.62 mm. it showed a single peak, which coincided, with the peak of gsi (fig. 4). the fecundity of mature female a. mola ranged from 3785 (fish with body length 4.90 cm, body weight 2.70 g and ovary weight 0.2 g) to 12590 (fish with body length 7.20 cm, body weight 6.50 g and ovary weight 0.92 g) and the calculated fecundity factor was 1445 ova g-1 of body weight. discussion feeding habit: gut content analysis of a. mola shows that fish is predominantly herbivorous in feeding habit. algae are the main food item of this fish. very little amount of zooplankton in the gut content indicates that a. mola feeds zooplankton occasionally. this is also supported from the observations of gut content of a. mola captured from reservoir of andhra pradesh (piska et al., 1991), of bangladesh (mamun et al., 2004) and floodplain lakes of west bengal (suresh et al., 2007). however, piska et al. (1991) found higher aquatic plants as the second most important food items next to algae in the gut content and found no animal food contents in the gut. in the present study, chlorophycae was recorded as the most dominant group in the gut content of mola. mamun et al. (2004) also found chlorophycae as the dominant group of algae in the gut content of a. mola. but suresh et al. (2007) observed myxophyceae as the dominant group of algae in the gut content of a. mola. gut length more than three times longer than the body length strongly support the herbivorous nature of the fish (mamun et figure 3. food preference of different weight class of amblypharyngodon mola. figure 4. gonado-somatic index and ova diameter of female amblypharyngodon mola. 129 mondal and kaviraj/ int. j. aquat. biol. (2013) 1(3): 125-131 al., 2004). present data indicate that the feeding habit of a. mola changes with the increase in body weight. piska et al., (1991) also showed that feeding habit of a. mola changed with the increase in length of the fish. the present study indicates that intensity of feeding of mola in the two floodplain lakes is high (less number of empty stomach and high feeding index) during september to february. piska et al. (1991) observed september to january as the period of intense feeding for this fish. the lowest feeding intensity (high number of empty stomach and low feeding index) of mola in these two floodplain lakes during july coincided with the active spawning month. piska and waghray (1986) also reported that high incidence of empty guts coincided with the spawning season. reproductive pattern: sex ratio of a. mola showed a predominance of females over males (1 male: 1.9 female) in the floodplain lakes under study. this is also supported by the observation of piska and waghray (1986), afroze et al. (1991), azadi and mamun (2004), suresh et al. (2007). the single peak of gsi and ova diameter indicate that a. mola breeds once in a year (july) in the studied floodplain lakes. however, breeding time varies with habitat. suresh et al. (2007) observed april to october as the breading season for this species in another floodplain lake of west bengal. piska and waghray (1986) observed that the breeding season of a. mola from himayatsagar, andhra pradesh extended from february to july. but in kaptai reservoir of bangladesh a. mola was found as a multiple breeder and spawned during july, august, october and march (azadi and mamun, 2004). kohinoor et al. (2003) obtained highest value of gsi during july and concluded that a. mola breeds twice in a year once during may-july and again during septemberoctober from samples collected from the pond of bangladesh agricultural university. hoque and rahaman (2008) also concluded that a. mola breeds twice in a year once in may and another in september in ponds and beels of bangladesh. result of the present study indicates that a. mola is a highly fecund fish and capable of breeding twice. fecundity of a. mola in the floodplain lakes under study increased with length and weight of the fish and weight of the gonad. similar observation had been recorded by azadi and mamun (2004) in a reservoir of bangladesh. it is concluded from the present study that a. mola is predominantly planktophagus and predominantly herbivorous in feeding habit. it is necessary to maintain ecosystem health of the two floodplains so that there is adequate supply of plankton in the lakes as food for the fish. the fish breeds only once i.e. during july in the floodplain lakes under study and it is also necessary to keep the environment of the lakes free of any stress during this period. acknowledgements authors acknowledge the help provided by the fishermen cooperative society associated with gopalnagar and dumar baur and partial financial assistance provided by the dst-purse programme of the university of kalyani. references afroze s., hossain m.a., parween s. 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(2003). reproductive biology of three indigenous small fish, mola (amblypharyngodon mola), chela (chela cachius) and punti (puntius sophore). in: m.a. wahab, s.h. thilsted, m.e. hoq (ed). small indigenous species of fish in bangladesh: proceedings of bauenreca/danida workshop on potentials of small indigenous species of fish (sis) in aquaculture and rice-field stocking of improved food and nutrition security in bangladesh. 30-31 october 2002, bangladesh agricultural university, mymensing, bangladesh & enrec/danida. pp. 3-22. mamun a., tareq k.m.a., azadi m.a. (2004) food and feeding habits of amblypharyngodon mola (hamilton) from kapti reservoir, bangladesh. pakistan journal of biological sciences, 7: 584588. mondal d.k., kaviraj a. (2008). ecotoxicological effects of jute retting on the survival of two freshwater fish and two invertebrates. ecotoxicology, 17: 207-211. mondal d.k., kaviraj a. 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(2020) 8(1): 66-72 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article dna barcoding of aphanius vladykovi from different habitats in chaharmahal va bakhtiari province, iran farideh abolhasani koupaie1, iraj hashemzadeh segherloo*2, 1mohamad saieed heydarnejad1, seyedeh narjes tabatabaei3 1department of biology, faculty of basic sciences, shahrekord university, shahrekord, iran. 2department of fisheries and environmental sciences, faculty of natural resources and earth sciences, shahrekord university, shahrekord, iran. 3department of biodiversity and ecosystem management, environmental sciences research center, shahid beheshti university, tehran, iran. s article history: received 4 june 2019 accepted 6 january 2020 available online 2 5 february 2020 keywords: cytochrome oxidase i differentiation genetic barcodes haplotypes phylogeny abstract: this study was aimed to reveal the possible cryptic diversity of the aphanius vladykovi populations in the chaharmahal va bakhtiari province, iran using mitochondrial cytochrome-c oxidase subunit i (coi). a total of 30 specimens from the beheshtabad river, choghakhor and gandoman wetlands, and brovi, shalamzar, and balagholi springs from the chaharmahal va bakhtiari province were collected. the maximum within-population genetic distance based on k2p was 0.28% and this distance was 0.22% between populations of gandoman and brovi with shalmazar, whereas the least genetic distance was observed between choghakhor and beheshtabad (0.09%). a total of six haplotypes were observed between the studied specimens. maximum likelihood (ml) and neighbor-joining (nj) trees reconstructed and all haplotypes from a. vladykovi specimens collected from non-type localities nested in one group with a. vladykovi from choghakhor wetland i.e. type locality. the results of this study detected no cryptic diversity in a. vladykovi inhabiting different habitats in the studied region. hence, it is proposed to consider all the studied populations in conservation measures related to a. vladycovi. introduction the family aphaniidae is composed of the genus aphanius with 15 reported species from iranian inland waters (esmaeili et al., 2018). the greatest diversity of this genus is found in the near east, particularly iran and turkey (teimori et al., 2018; cicek at al., 2018). the members of this genus can adapt to a wide range of environmental parameters (cavraro et al., 2017), inhabiting rivers, wetlands, pools, springs, and qanats in semiarid and arid regions. therefore, this ability makes it possible for them to form different isolated populations causing diversification and radiation of different taxa. hence, there may also be more unknown diversity in this genus that should be explored using molecular tools. aphanius vladykovi coad, 1988, described from choghakhor wetland (31°57'n, 51°01'e) in zagros mountains of iran (coad, 2017), is found in the karoun river drainage in the chaharmahal va bakhtiari province, iran (coad, 2017; esmaeili et al., *correspondence: iraj hashemzadeh segherloo doi: https://doi.org/10.22034/ijab.v8i1.612 e-mail: ihashem@sku.ac.ir 2018). male a. vladykovi is characterized by a light transverse strip on the body, while female has dark spots on both sides of the body (coad, 2017). the populations of this species inhabit different isolated habitats in the chaharmahal va bakhtiari province. in previous studies, a. vladykovi from its type locality has been compared to different aphanius spp. (esmaeili et al., 2014; teimoori et al., 2012), but there is no molecular data of different populations of this species. mitochondrial dna (mtdna) that widely used for identifying cryptic fish species (rezvani gilkole, 1997; ivanova et al., 2007; asgharian et al., 2011), play an important role in taxonomic studies and phylogenetic inferences (dabert, 2006; yang et al., 2010). during the past decade a movement known as dna barcoding has started in which scientists from all over the world produce partial sequences of a standard gene region of around 650 base pairs using universal primers to identify different animal 67 int. j. aquat. biol. (2020) 7(8): 66-72 species (kerr et al., 2007). the purpose of dna barcoding is to improve the identification of species and to discover new species by studying patterns of sequence differentiation in a standard region in the genome (http://www.boldsystems.org/). the partial coi sequence is used to study different groups, especially at species and population levels (tala et al., 2011; asgharian et al., 2011; hashemzadeh segherloo et al., 2012b). dna barcodes have been used successfully for delimitation of species in more than 90% of animal species studied (ward et al., 2005; hajibabaei et al., 2006; hubert et al., 2008). in this study, the a. vladykovi populations in the chaharmahal va bakhtiari province, iran was investigated in different localities to explore possible cryptic diversity. materials and methods fish were collected using a scope net from different localities, including beheshtabad river, choghakhor and gandoman wetlands, and shalamzar, berovi and balaghololi springs (table 1). the right pectoral-fin of five specimens from each locality were clipped and subsequently fixed in 96% ethanol. dna extraction was performed using salt extraction method (aljanabi and martinez, 1997). for amplification of the coi gene, the primers fishf1-5′tcaaccaaccacaaa gacattggcac3′ and fishr15′tagacttctg ggtggccaaagaatca3′ were used (hubert et al., 2008). the pcr reaction (25 μl per reaction) contained 2.5 μl of 10× buffer, 0.5 μl of (50 mm) mgcl2, 0.5 μl of (25 mm) deoxynucleotide triphosphate (dntp), 0.5 μl (10 μm) of each primer, 0.5 μl of taq polymerase (5 u μl−1), 2 μl of total dna, and 18 μl of h2o. amplification cycles were: denaturation for 2 min at 94°c; 30 cycles at 94°c for 1 min, 59°c for 0.5 min, 72°c for 0.5 min, and a final extension for 2 min at 72°c. the 5’ end of coi with an approximate length of 652 nucleotides was amplified. the 5’ end of the coi gene was sequenced on an abi-3130xl sequencer using forward primer. the sequences were checked and edited visually based on chromatograms, compared to sequences in each population, and genbank. the sequences were translated to their respective amino acid sequences with mega7 (kumar et al., 2016) to see whether the confirmed mutation had led to any change in protein sequences compared to standard amino acid sequence of coi or not. the sequences were compared to the published aphanius sequences using blast search in genbank (altschul et al., 1997) to find similar sequences (table 2). a common 584 bp length of the selected coi segment was used for further analyses. the gene diversity indices, including number of polymorphic sites, number of haplotypes (h), haplotype diversity (hd), and nucleotide diversity (pi) were calculated with dnasp v6 (rozas et al., 2017). to provide a quantitative measure of the sequence divergence, the divergence among studied populations, genetic distances between sequences were calculated based on the kimura two-parameter (k2p) model of sequence divergence with mega7. phylogenic trees were reconstructed based on maximum likelihood (ml) and neighbor joining (nj) methods with mega7 and raxml (silvestro and michalak, 2012). the best fit model of sequence evolution to be used in maximum likelihood approach was selected with the modeltest table 1. details of the sampling stations. locality sampling date coordinates lon lat brovi spring 2017-01-02 32°16'48.3"n 50°59'25.1"e balagholi spring 2017-06-03 32°05'02.8"n 50°42'47.2"e behesht-abad river 2017-01-03 32°05'09.3"n 50°39'41.9"e choghkhor wetland 2016-11-28 31°56'05.1"n 50°54'29.7"e gandoman wetland 2017-06-03 31°49'40.2"n 51°06'01.5"e shalamzar spring 2016-11-28 32°01'27.4"n 50°49'18.8"e 68 abolhasani koupaie et al./ dna barcoding of aphanius vladykovi utility implemented in mega7. to check for the robustness of each branch on phylogenetic trees a bootstrap test with 1000 replicates was used for both nj and ml approaches. to visualize the mutational relationships among the haplotypes identified in this study and the haplotypes retrieved from genbank a rooted tcs network was reconstructed with popart1.7 (http://popart.otago.ac.nz). to root the phylogenetic tree bathygobius sp., canthigaster rivulata, canthigaster smithae, parupeneus spilurus, and pseudamia gelatinosa were used as out-group species (table 2). results in this study six haplotypes were resolved, which differ from one another by 1-3 mutations (fig. 1). haplotype diversity (hd) and nucleotide diversity (pi) were 0.680 and 0.001, respectively. the haplotypes a. vla2 was the most frequent one observed in all populations except beheshtabad river. the second table 2. list of sequences used from ncbi-genbank. species accession distribution aphanius alexandri kj552647 middle east. aphanius almiriensis kj552360 europe: greece. aphanius anatoliae kj552467 asia: endemic to turkey. aphanius asquamatus kj834543 asia: lake hazar, eastern anatolia and iran. aphanius baeticus kj552418 europe: spain along atlantic coast between huelva and gulf of cadiz. aphanius danfordii kj834531 asia: eastern turkey. aphanius fasciatus kj552516 europe: france, italy, slovenia, croatia, albania, greece and montenegro. asia: turkey. mediterranean basin: north africa from egypt to eastern algeria. aphanius fontinalis kj552742 asia: endemic to turkey. aphanius fontinalis kj552560. aphanius iconii kj552688 asia: endemic to turkey. aphanius iconii kj552481 aphanius maeandricus kj552515 asia: endemic to turkey. aphanius mento kj552511 middle east. aphanius mentoides kj552397 middle east. aphanius orontis kj552683 middle east. aphanius saldae kj552398 asia: central turkey. aphanius saourensis kj552623 africa: algeria. aphanius similis kj552500 middle east. aphanius similis kj552367 middle east. aphanius sureyanus kj834526 asia: central turkey. aphanius transgrediens kj552368 asia: central turkey. bathygobius sp. kt357897 southwest pacific: australia, including the lord howe and norfolk islands. canthigaster rivulata jf952693 indo-pacific: east africa south to natal, south africa and east to hawaii, north to southern japan, south to northwestern australia. canthigaster smithae jf493017 western indian ocean: agalega islands, mauritius to durban, south africa. also maldives. parupeneus spilurus dq107798 western pacific: japan to western australia, new caledonia and northern new zealand. recently reported from tonga. pseudamia gelatinosa fj346820 indo-pacific: red sea and east africa to french polynesia, north to ryukyu islands, south to sydney harbor, new south wales (australia). table 3. k2p sequence divergence of the studied populations. group within group between group shalamzar (1) 0.28 gandoman (2) 0.2 0.22 choghakhor (3) 0.17 0.21 0.17 brovi (4) 0.08 0.22 0.17 0.21 beheshtabad (5) 0 0.17 0.13 0.09 0.21 balagholi (6) 0 0.17 0.13 0.17 0.14 0.17 69 int. j. aquat. biol. (2020) 7(8): 66-72 frequent haplotype was a. vla1 observed in all populations except in balagholi and berovi springs. other haplotypes were population specific. the maximum within group k2p sequence differentiation was calculated in the shalamzar population (0.28%). the maximum between group genetic distance was calculated between the gondoman and berovi populations and shalamazar population (0.22%) and the minimum genetic distance was found between the choghakhor and beheshtabad populations (0.9%; table 3). the maximum likelihood and neighbor-joining phylogenic trees were similar in topology (fig. 2). aphanius spp. used for reconstruction of phylograms were nested in three monophyletic groups including a. vladykovi (bs= 100), a. orontis, a.mento, a. mentoides, a. alexondri, and a. similis (middle eastern group; group ii; bs=100), and a. baeticus, a. saourensis, a. almiriensis, a. facsiatus, a. asquamatus, a. danfordii, a. anatoliae, a. maeandricus, a. iconii, a. transgrediens, a. saldae, a. fontinalis, and a. sureyanus (european-anatolian group; group iii; bs=66-67). all a. vladykovi specimens collected from different localities, nested in a monophyletic group with absolute bootstrap support on both nj and ml phylograms (bs=100). aphanius spp. from europe, north africa, and the middle east nested in the third group (iii). discussions the intra-species sequence differentiation in freshwater fishes had been reported to be 0.27% and the intra-genus divergence among different freshwater fishes falls in a range of 0-19.3%, with an average of 8.37% (hubert et al., 2008). the divergence values observed between the studied populations of a. figure 1. the coi haplotype network showing mutational relationship of sequences produced in this study and sequences from genbank. the hatch lines along the connection lines denote the number of mutational steps between each pair of haplotypes. black circles are probable haplotypes from which different mutational paths radiate. abbreviations: a. vla: a. vladykovi. 70 abolhasani koupaie et al./ dna barcoding of aphanius vladykovi vladykovi were in the ranges of intra-specific levels. hence, all studied aphanius specimens in the current study are a. vladykovi. the distribution of the most frequent haplotypes in the different localities is expectable (hashemzadeh segherloo et al., 2012 a), since these localities are in close proximity to one another in the same river drainage, hence population interchange among them during different climatic events like flooding or even via anthropogenic activities is likely. as indicated in previous studies a sample size of 5-7 individuals can trap a good representative haplotype diversity of each population (hubert et al., 2008), accepting this notion we can infer that shalamzar, balagholi, and beheshtabad populations with more haplotypes have higher genetic diversity compared to other populations studied here. among the haplotypes identified here, haplotypes a. vla3 (choghakhor) and a. vla5 (shalamzar) are probably younger haplotypes, since they are peripheral on the network with no haplotypes radiating from them. this inference should be treated cautiously, since in this study we have not included other iranian aphanius species, which are probably phylogenetically closer to a. vladykovi compared to species used from genbank. because it is possible that inclusion of other aphanius spp. from iran or nearby regions would change the connection patterns in haplotype network and the noted peripheral haplotypes may be intermediate to other species. based on what is seen on the haplotype network figure 2. phylogentic relationships of aphanius vladykovi populations with other aphanius spp. based on maximum likelihood and neighbor joining methods. numbers along the branches are maximum likelihood and neighbor joining bootstraps values, respectively. the bootstrap support values of smaller than 50% are not shown. the blue bars denote the resolved groups. 71 int. j. aquat. biol. (2020) 7(8): 66-72 a. vladykovi is highly diverged from other aphanius spp. included here. so far, conservation activities regarding a. vladykovi have been focused only on the choghakhor wetland (the type locality of this species). based on the results, there are more than one population of the species in the studied region. these populations exist mostly in small springs with different extents of anthropogenic effects like habitat changes, pollution, and water exploitation. each of these isolated populations can be considered as a gene pool for conservation of a. vladycovi. hence, we propose to consider all these populations and their related habitats in any conservation-oriented plans. acknowledgments this project was supported with a grant (no.94grd1m688) from shahrekord university. references aljanabi s.m., martinez i. 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(2020) 8(1): 35-49 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article the effect of oral administration of lactic acid bacteria isolated from kefir on intestinal microbiota, growth performance and survival in juvenile rainbow trout, oncorhynchus mykiss mohsen ali1, siyavash soltanian*1, ali taheri mirghaed*2, mostafa akhlaghi1, seyed hossein hoseinifar3, atefeh esmailnejad4 1division of aquatic animal health, department of clinical sciences, school of veterinary medicine, shiraz university, shiraz, iran. 2department of aquatic animal health and diseases, faculty of veterinary medicine, university of tehran, tehran, iran. 3department of fisheries, gorgan university of agricultural sciences and natural resources, gorgan, iran. 4department of pathobiology, school of veterinary medicine, shiraz university, shiraz, iran. s article history: received 10 october2019 accepted 5 january 2020 available online 2 5 february 2020 keywords: lactic acid bacteria kefir probiotic growth lactobacillus enterococcus abstract: lactic acid bacteria (lab) are used in the aquaculture industry to improve growth indices and nutrition efficiency of farmed fish. kefir is a natural probiotic that largely consists of these microorganisms. this study aimed to isolate lab strains from kefir grains and investigate their effects on the intestinal microbiota and growth indices of juvenile rainbow trout. based on the results, one isolate was identified as lactobacillus faraginis (lf) and other one as enterococcus durans (ed), both were gram positive, non-hemolytic, catalase and oxidase negative. both strains showed resistance to acidic environments and gastric juice and were able to grow against bile salts i.e. the hydrophobicity potential of both strains was evaluated above 50%. the results showed that both strains had appropriate and acceptable probiotic properties. a total of 480 juvenile fish with were randomly divided into eight groups viz. control (basal diet), lf1 and lf2 receiving diets supplemented with l. faraginis at 107 and 108 cfu/g, respectively, ed1 and ed2 diets supplemented with e. durans at 107 and 108 cfu/g, respectively, bactocell group receiving a diet supplemented with commercial probiotic bactocell pa10 (1010 cfu/g), combined treatments lf1+ed1 (5×106 cfu/g) and lf2+ed2 (5×107 cfu/g). the strains were prepared in the form of lyophilized powder and added to the basal diet as supplements. sampling was performed after 0, 30, 15, and 45 days feeding with the diets. in the in vivo investigation, the highest lab colony counts were obtained for the ed2, bactocell, lf2+ed2 and lf2 groups, respectively. the highest bwi, sgr, dwg, per, and cf, and the lowest fcr were observed in the lf2+ed2, bactocell, ed2, and lf2 groups, respectively. on the other hand, the lf2+ed2, bactocell and lf2 treatments also had the highest survival rate. apparently, the use of the tested lab strains as probiotic in high doses (lf2, ed2) and in combination (lf2+ed2) could improve growth indices, the intestinal lab population and survival rate in juvenile rainbow trout. introduction lactic acid bacteria (lab) and bifidobacteria are among the microorganisms most commonly used as probiotics, although other bacteria and some yeast strains are also used for this purpose (didari et al., 2014). an important branch of the family of lab is the genus lactobacillus, which is an anaerobic or microaerophilic gram-positive bacterium. other notable genera of this family include lactococcus, enterococcus, pediococcus, streptococcus, and leuconostoc (makarova et al., 2006). some strains of the genus enterococcus with probiotic properties are *correspondence: siyavash soltanian and ali taheri mirghaed doi: https://doi.org/10.22034/ijab.v8i1.697 e-mail: siyavashsoltanian@yahoo.com, mirghaed@ut.ac.ir also used in the pig and poultry feed industry as well as pharmaceutical products (liu et al., 2014). so far, many lactobacillus bacteria have been categorized as gras (generally recognized as safe) (soccol et al., 2010). although some strains of the enterococci are known to have probiotic properties and are used as animal feed supplements for improving growth and preventing gastrointestinal disorders (franz et al., 2011), they are yet to be cauterized as gras (huys et al., 2013). lab are resistant to bile salts, gastric acid, and pancreatic enzymes and adhere to the mucosal 36 ali et al./ oral administration of lactic acid strains kefir in oncorhynchus mykiss membrane of the gastrointestinal tract (fioramonti et al., 2003). research in the field of aquaculture has shown that the use of some probiotics in the form of live and dead microorganisms may have positive effects on the host and its environment (lazado and caipang, 2014; hoseinifar et al., 2017, 2019; gobi et al., 2018). the notable applications of lab in the aquaculture industry include improving the nutrition efficiency and growth performance, increasing the survival rate, preventing digestive and intestinal disorders by neutralizing anti-nutritional and toxic substances in the diet, controlling and monitoring the growth of microorganisms in intestines, improving immune responses and preventing the growth of pathogenic organisms in fishes (panigrahi et al., 2005; suzer et al., 2008; mohapatra et al., 2012a, b). also, lab have been reported to have beneficial effects on growth parameters of juvenile fish by improving the digestion of nutritional compounds of the diet and enhancing digestive enzymes and intestinal microbiota (nayak, 2010). moreover, the multiple strains of lab can increase the growth and protein efficiency, reduce the feed conversion ratio, and improve digestion and food absorption of fish, which they have attributed to their role in reducing stressors (gomez et al., 2000; lara-flores et al., 2003). under stressful situations in artificial and intensive fish culture systems, which disrupt the balance of the bacterial flora in the water and in the digestive system of the fish, the use of probiotic strains can be beneficial way of restoring microbial balance both in water and in the fish and increase the chance of proper growth (ringo, 1998; lara-flores et al., 2013). according to previous studies, the use of lab probiotics in aquaculture can increase survival, enhance disease resistance, and improve growth parameters in various species of fish (nayak, 2010; estebanet al., 2014; muñoz-atienza et al., 2014; hai, 2015). kefir is a traditional fermented milk product originating from the caucasus mountains, which is widely used in eastern europe, southwest asia, and many other parts of the world (rodrigues et al., 2005). since the onset of industrial-scale kefir production in eastern and central europe, the microbiology of kefir grains has attracted the attention of many researchers (atalan et al., 2003). kefir grains contain complex probiotic microorganism produced following milk fermentation (atalan et al., 2003; rodrigues et al., 2005). the fermentation agents of kefir grains include the species of lactobacillus, streptococcus, saccharomyces, and some other bacterial genera, which coexist in the form of gelatinous colonies (mukai et al.,1988,1991). it has been shown that the microorganisms that exist in kefir grains include lab and yeasts that are used as leaven in its preparation (rodrigues et al., 2005). lab species of kefir can be both homofermentative and heterofermentative (gao and li, 2016). in kefir grains, lab and yeast are present in a matrix of proteins, fats, and sugars, which also contains important species of beneficial bacteria such as bifidobacterium spp. and l. acidophilus (guzel-seydim et al., 2011; uluköy et al., 2015). there is evidence suggesting that some of the microorganisms found in kefir can potentially exhibit probiotic properties (zheng et al., 2013; bolla et al., 2013). studies carried out on the probiotic properties of lactobacillus strains isolated from kefir grains has shown their high resistance to gastric acid and bile salts, their ability to attach and adhere to the gut epithelial cells in in-vitro conditions, and lack of their adverse enzymatic or hemolytic activity (zheng et al., 2013; leite et al., 2015). also, some studies have reported the beneficial effects of the addition of kefir grains to the fish diet on the growth performance (uluköy et al., 2016; van doan et al., 2017). rainbow trout is an important aquaculture species in iran, with total annual production of more than 160000 tons (hoseini et al., 2014; hoseini et al., 2019a). it is cultured under semi-intensive and intensive conditions in tanks, concrete ponds and earthen ponds. although previous studies have explored the impacts of direct use of kefir grains in the fish diet, they have not investigated the probiotic potential of specific lab strains isolated from kefir when used as a fish diet supplement. in the present work, after isolating and identifying two strains of lab from kefir grains, they were added to the diet of 37 int. j. aquat. biol. (2020) 8(1): 35-49 juvenile trout to investigate the effects on growth parameters, the intestinal lactic acid bacteria population and survival rate. materials and methods preparation of kefir: imported kefir grains originating from the russian caucasus mountains were purchased from an official sales representative in iran (oxan company). according to unal and arslanoglu (2013), 20 g of kefir in 200 ml of fresh cow milk was incubated for 24 hours at 25-28°c (wszolek et al., 2006). then, kefir grains were filtered from the fermented milk and stored at 4°c (uluköy et al., 2016). isolation and purification of kefir bacteria: to isolate the bacteria, 1 g of kefir grains was homogenized with t18 basic ultra-turrax® homogenizer (ika, germany) and subsequently, serial dilution was made with sterile saline solution (0.9%). strains were isolated and purified using direct and indirect methods. in the indirect method, primary enrichment of kefir with mrs broth and the steps were continued as in the direct method. in both cases (krieg and padgett, 2011), the serially diluted sample was cultured on mrs agar with ph=6.2 at 37°c under 5% co2 atmospheres for 48 hours in the nuaire dhd autoflow (nu-5510) incubator. then, purification of lab colonies was performed by streaking bacteria on mrs agar in three replications (dworkin et al., 2006). strain identification: gram staining method was used to identify the purified strains and investigate their morphological characteristics. staining results were confirmed with the test of solubility in 3% koh solution based on suslow et al. (1982). catalase test was performed with 3% hydrogen peroxide (h2o2) solution (macfaddin, 2000) and oxidase test was performed using 1% oxidase (n, n, n', n' tetramethyl-p-phenylenediamine) reagent (harrigan and mccance, 1990). of the four types of colony purified by these tests, the two that were positive in gram-positive staining and negative in koh, catalase, and oxidase tests were chosen for molecular identification. this identification was carried out by 16srrna gene sequencing. whole dna extraction from the strains was performed with cinnagen kits (cinnagen, iran) based on marmur (1961) with some modifications. the polymerase chain reaction (pcr) was then performed using the universal primers (27f: 5'-agagtttgatcmtggctcag-3') and (1492r: 5'-ggttaccttgttacgactt-3') and confirmed on 1% agarose gel by electrophoresis (powerpac™) and observation with a gel doc (syngene gbox ef). the product was sequenced with a macrogen sequencer (macrogen, korea) and the results were analyzed using the chromas software (v.2.01, technelysium pty ltd). finally, the software blastn was used to compare the results with the sequences of the ncbi gene database and the isolate with the highest similarity in terms of srna16 gene sequence was identified. in-vitro evaluation of the probiotic potential of the strains isolated from kefir: hemolytic activity of the isolated lab strains was studied in columbia agar (merck, 110455) (leite et al., 2015). ph tolerance test was carried out using the method of klayraung et al. (2008). resistance of strains to gastric juice was evaluated based on yanyan et al. (2010). also, resistance to bile salts was measured according to walker and gilliland (1993), briefly the suspension of the strains was added to the mrs broth with different levels of bile salts (0%, 0.15%, and 0.3%), and the optical density (od) of the media was measured during 0, 2, 4, 8 hours of incubation with a novaspec ii visible spectrophotometer at 620 nm, then the coefficient of inhibition of the strains for bile oxalate was calculated by the equation of gopal et al. (1996). hydrophobicity was evaluated by the method of goldberg et al. (1990) based on adhesion to hexadecane. to measure the antagonistic activity of the strains, as instructed by balcazar et al. (2008), the strains were cultured in mrs broth, then a suspension with a concentration of 0.5 mcfarland of lactobacillus garvieae ir-lgt-ms-1 (kf918779), streptococcus iniae (gq850377), yersinia ruckeri (kc291153), and aeromonas hydrophila (ah04) freshly grown on tsa medium was added to muellerhinton agar plates and cultured. multiple wells were created on each culture medium and filled with the 38 ali et al./ oral administration of lactic acid strains kefir in oncorhynchus mykiss bacterial filtrates, and finally, the inhibition zones around the wells were measured in mm (balouiri et al., 2016). preparation of the lyophilized form of the strains for diet supplementation: lyophilized powder of the strains was prepared according to john et al. (2007). in summary, a cold-trap was used to prepare a suspension of the strains from skim milk (protective lyophilization medium) and after freeze-drying, the lyophilized form of the strains was stored at 4°c until use. to determine the exact amount of powder that should be added to the diet, colonies in one gram of lyophilized powder were counted according to li et al. (2012). design of experiment and treatments (in vivo): a total of 480 healthy rainbow trout with mean weight and length of 41.9±2.4 gr and 14.5±0.5 cm were obtained from a private fish farm, haraz, tehran province. the fish were stocked in 24 tanks (20 fish per tank) and allowed to acclimatize for 10 days, during which, the fish were monitored for health problems. the water physicochemical conditions during the experiment were as follows: temperature = 18±1.5°c, ph = 7.5±0.1, do = 8.5±0.5 mg/l, hardness = 180±15 caco3 mg/l, total ammonia ≤ 0.01 mg/l and nitrite ≤ 10 mg/l. during the treatment period, water was changed on a daily basis and aeration was carried continuously so that the physicochemical conditions of water remained optimal for growth. a daily ration of 3% of biomass was divided into four meals. after acclimation, the tanks were divided into eight treatments as follow: (1) control: basal diet without any supplement (2) lf1: basal diet + 107 cfu/g l. ferraginis (3) lf2: base diet + 108 cfu/g l. ferraginis (4) bactocell: basal diet + commercial probiotic bactocell pa 10 (lallemand animal nutrition s.a., blagnac, france) containing 1010 cfu/g pediococcus acidilactici (according to manufacturer) (5) ed1: basal diet + 107 cfu /g e. durans (6) ed2: basal diet + 108 cfu/g e. durans (7) lf1 + ed1: basal diet + 5×106cfu/g l. farraginis + 5×106 cfu/g e. durans (8) lf2 + ed2: basal diet + 5×107 cfu/g l. farraginis + 5×107 cfu/g e. durans. basal diet specifications: commercial extruded feed for the juvenile rainbow trout (ff2-extruded) was purchased from faradane co. (iran). the type, shape, size, and composition of the basal diet were decided according to the average weight and length of the fish (table 1). addition of bacterial strains to the basal diet and preparation of daily feed: first, the daily feed requirements of the treatments were determined according to water temperature and weight of the fish. then, the lyophilized strains (as defined for the treatments) were added to the diet based on panigrahi et al. (2004) with minor modifications. for this purpose, the exact amount of lyophilized powder needed for each diet was calculated according to the number of colonies counted in one gram of that powder (l. farraginis: 1014cfu/g; e. durans: 1012cfu/g). these amounts of the lyophilized powder were dissolved in edible oil and sprayed uniformly on the commercial feed pellets and mixed with a drum mixer. for the bactocell treatment, bectocell powder was added to the diet in the same way. for the control diet, all of the above steps were performed without adding the strains. for control one, number of the bacteria added to the diet, the colonies in one gram of table 1. specifications and chemical analysis of extruded rainbow trout feed (ff2-extruded). proximate composition content (%) crude protein 40 crude lipid 12 crude fiber 2 ash 7 moisture 5 phosphorus 1 feed form floating feed size (mm) 4±0.3 39 int. j. aquat. biol. (2020) 8(1): 35-49 diet were counted basedon swanson et al. (1992). for this purpose, a suspension of the diet in buffered peptone water was prepared and then serially diluted with physiological serum (0.85%). solutions of different dilutions were cultured in mrs agar and counting was performed after 48 hours of incubation at 37oc (table 2). growth parameters and survival: the measurement of growth parameters was taken at days 0, 15, 30, 45 after the start of the experiment. from each treatment, a total of 15 fish (5 from each replicate) were randomly collected. growth indices were calculated by following equations (yousefi et al., 2016; hoseini and mirghaed et al., 2018): specific growth rate = log (wf-wi)/ n * 100 feed conversion ratio = fg(g) / wg (g) condition factor = wf (g) / tl3 (cm) *100 protein efficiency ratio = wg (g) / tp (g) daily growth rate = wf-wi / n percentage weight gain = (wf-wi) / wi * 100 survival rate = (nf / ni) * 100 where wf is mean final weight, wi = mean initial weight, n = number of treatment days, tl = final total length, wg = weight gain, fg = feed intake, tp = total protein received, ni = initial number of fish and nf = number of fish survive. evaluation of intestinal lab: after sampling, the external surfaces of fish were first disinfected with benzalkonium chloride and then a portion of the intestinal tract was sampled 0, 15, 30, and 45 days after start feeding trial. the samples were homogenized with normal saline, serially diluted, and immediately cultured in mrs agar. finally, the media were incubated at 25°c for 5 days and then numbers of the colonies were counted (mahious et al., 2006). the number of colonies of each sample was calculated by the following equation: cfu/g intestine = number of colonies × inverse of the dilution factor statistical methods and data analysis: to analyze the data, first, all data were compiled in excel 2007. normality of data was checked by kolmogorovsmirnov test and homogeneity of variance by levene test. analyses of the collected data were performed with one-way anova. in the cases where there was a significant difference between treatments, the tukey test was performed at 95% confidence level and p≤0.05 significance level. all tests were performed using the statistical analysis software spss 19 (chicago, il, usa). results isolation and purification of lab strains from kefir: four bacterial strains were isolated and purified from kefir grains. one strain, lf, was bacillus-shaped and observed often in single units and occasionally in pairs. another strain, ed, was cocci-shaped with slight elongation and observed often in clusters and sometimes in pairs and short chains. both of these strains were gram positive (confirmed by the koh test). the catalase and oxidase tests of these strains were negative. in terms of characteristics, these strains were very similar to the family of lactic acid bacteria. the other two strains were gram negative (confirmed by the koh test) and did not belong to the lab family. molecular identification of the isolated strains: le strain had 99.7% phylogenetic affinity with table 2. the average number of colonies of the strains per gram of diet. treatments lactic acid bacteria (cfu/g) control lf1 1.1×107 lf2 1.9×108 bactocell 1.01× 1010 ed1 1.2×107 ed2 1.7 ×108 lf1+ed1 2.1×107 lf2+ed2 2.65×108 40 ali et al./ oral administration of lactic acid strains kefir in oncorhynchus mykiss l. farraginis jcm 14108 (t) and the ed showed 99.9% affinity with e. durans nbrc 100479 (t). probiotic characteristics of the strains: investigation of hemolytic activity of the strains lf and ed in blood agar did not show any type of hemolytic activity (α, β, or γ) around the colonies of these strains. both strains showed resistance to acidic environments and gastric juice and were able to grow against bile salts. the hydrophobicity of the lf and ed strains was measured to 72.47% and 51.42%, respectively. the strains exhibited good in vitro antagonistic activity against lactobacillus garvieae and aeromonas hydrophilia, but their activity against streptococcus iniae was extremely weak. results of analysis of intestinal labs: according to the results (fig. 1), comparison of the number of lab in the midgut of fish at the first sampling time and the beginning of the treatment period (initial: 0) showed no significant difference (p>0.05) between the treatments and control groups. analysis of the samples collected at three time points (days 15, 30 and 45) showed a significant figure 1. total count of lab colonies in the intestine of rainbow trout fed with different levels of the lab strains isolated from kefir at days 0, 15, 30, 45 (mismatching letters (a, b, c) signify a significant difference (p≤0.05) between means). figure 2. comparison of percentage of survival rate of rainbow trout fed with different levels of the lab strains isolated from kefir at the end of the treatment period (day 45) (mismatching letters (a, b, c) signify a significant difference (p≤0.05) between means). 41 int. j. aquat. biol. (2020) 8(1): 35-49 increase (p<0.05) in the total number of intestinal labs in ed2, bactocell, lf2+ed2 and lf2 treatments compared to others. although no significant difference was observed between the intestinal lab colony counts of lf1, ed1, and lf1+ed1 in any of the sampling times, the number of colonies in all of these treatments was significantly higher (p<0.05) than in the control. in addition, in all treatment groups except the control, the number of lab colonies increased as the experiment progressed (fig. 1). growth parameters and survival rate: according to growth indices (table 3), the parameters bwi, sgr, dwg, per, and cf were highest in the treatments lf2+ed2, bectocell, ed2, and lf2, respectively (p<0.05). in these treatments, the least significant change compared to the control group was observed in fcr. the parameters length gain and total length in the treatments lf2+ed2, bectocell, and lf2 showed the greatest increase in comparison with other groups (table 3). inaddition, the survival rate was found to be significantly (p<0.05) higher in lf2+ed2, bactocell and lf2 than in other treatments (fig. 2(. discussions probiotics can contribute to aquaculture as beneficial and eco-friendly microorganisms with multiple mechanisms of positive effect on the host and environment (lazado and caipang, 2014). indeed, some of the probiotic microorganisms are able to provide the essential nutrients for fish to grow more favorably (balcazar et al., 2006; panigrahi et al., 2007). kefir grains, as a probiotic product, consist of many members of the family of lactic acid bacteria (rodrigues et al., 2005) and can serve as a useful source for the extraction of new probiotic strains (kumura et al., 2004). however, after molecular identification of lab strains, they should be subjected to vigorous in vitro examinations to study their probiotic and health characteristics (saarela et al., 2000; hammes and hertel, 2002; mercenier et al., 2008). in the current study, two strains of the lab family were isolated and purified in the anaerobic and microaerophilic conditions from the fermentation of kefir grains in the milk. colonies of the strain l. ferraginis (lf) were showed 99.7% phylogenetic similarity with the l. farraginis strain jcm 14108 (t) in the ncbi database. another study that isolated l. farraginis and l. para ferraginis strains from a traditional beverage called shochu produced by fermentation of rice extract (endo and okada, 2007b). the incubation of kefir can take place not only in cow milk but also in soy milk, rice milk, and coconut milk (semih and cagindi, 2003). according to the taxonomic classification, lf strain belongs to the phylum firmicutes, the class bacilli, the order table 3. comparison of growth parameters and survival rate of rainbow trout fed with different levels of the lab strains isolated from kefir at the end of the treatment period (day 45). lf2+ed2 lf1+ed1 ed2 ed1 bactocell lf2 lf1 control parameters 42.16±0.83a 41.78±0.91a 41.98±1.51a 42.40±1.26a 42.48±1.08a 42.16±0.82a 41.82±1.55a 41.42±1.21a initial weight (g) 99.99±2.50d 92.01±1.32b 95.75±2.72c 94.67±2.46bc 99.79±2.69d 92.15±2.71b 86.56±1.72a 84±3.60 a final weight(g) 58.19±2.49d 50.21±1.32b 53.95±2.72c 52.87±2.46bc 57.99±2.68d 50.35±2.71b 44.76±1.71a 42.2±2.60a weight gain(g) 14.56±0.32a 14.78±0.52a 14.64±0.50a 14.54±0.53a 14.62±0.46a 14.70±0.31a 14.66±0.50a 14.60±0.51a initial length (cm) 21.5±0.4c 21.31±0.18bc 21.28±0.35abc 21.25±0.36abc 21.5±0.27c 21.3±0.38bc 20.95±0.4ab 20.9±0.37a total length (cm) 4.77±0.40c 4.58±0.18bc 4.55±0.35abc 4.52±0.36abc 4.77±0.26c 4.57±0.37bc 4.22±0.40ab 4.17±0.36a length gain (cm) 139.21±5.98d 120.1±3.17b 129.08±6.51c 126.49±5.89bc 138.73±6.43d 120.45±6.50b 107.08±4.11a 100.95±8.6a bwi (%) 1.01±0.06b 0.95±0.03ab 0.99±0.05b 0.98±0.06b 1.01±0.05b 0.95±0.05ab 0.94±0.06ab 0.92±0.06a cf 1.94±0.06d 1.75±0.03b 1.84±0.06c 1.82±0.05bc 1.93±0.06d 1.76±0.07b 1.62±0.04a 1.55±0.10a sgr (% day-1) 2.60±0.11d 2.25±0.06b 2.41±0.12c 2.37±0.11bc 2.59±0.12d 2.25±0.12b 2.00±0.08a 1.89±0.16a per 1.29±0.06d 1.11±0.03b 1.19±0.06c 1.17±0.05bc 1.28±0.06d 1.11±0.06b 0.99±0.04a 0.93±0.08a dwg (gr) 0.91±0.04d 1.05±0.03b 0.98±0.05bcd 1±0.05bc 0.91±0.04d 0.96±0.12cd 1.06±0.05b 1.46±0.11a (fcr) * mismatching letters (a, b, c) in the rows signify a significant difference (p≤0.05) between means. 42 ali et al./ oral administration of lactic acid strains kefir in oncorhynchus mykiss lactobacillales, the family lactobacillaceae, and the genus lactobacillus, and has a phylogenetic affinity with l. hilgardii, l. buchneri, l. diolivoran, l. parakefi, l. kefiri and l. parabuchneri species (felis and dellaglio, 2007; endo and okada, 2007a). the second strain isolated in the present study from kefir grains was e. duran (ed), which has 99.9% phylogenetic similarity with the strain e. durans nbrc 100479 (t). the strain e. durans was first isolated from kefir grains by rosi (1978), who reported it as streptobacterium durans and later reported by yuksekdag et al. (2004) as streptococcus durans, which is one of the strains of the lab family (rosi and rossi, 1978; yuksekdag et al., 2004). in a study similar to the present work, three strains of e. durans were isolated from turkish kefir (yuksekdag et al., 2004), and recently, e. durans was isolated from the kefir incubated in goat milk and reported using api-50 biochemical methods (aloklah et al., 2017). according to previous studies, e. durans is homologous to e. faecium, e. hirae, and e. lactis in terms of molecular structure (farrow, 1983; yu et al., 2011; techo et al., 2018). in the present study, the two isolates of kefir lacked any hemolytic activity in blood agar and they also presented no catalase activity. in a similar study, the hemolytic activity of l. kefiri isolated from kefir grains, which has a genetic affinity with l. farraginis, was also negative (carasi et al., 2014). on the contrary, some e. durans strains have been reported to have α and β hemolytic activity in blood agar (collins et al., 1984), and meanwhile, there are other studies indicating that e. durans strains have no hemolytic activity (foulquie et al., 2006; pieniz et al., 2014). these contradictory results are probably due to the large variety of strains of this species and the difference in the sources from which they have been isolated, because e. durans is one of the most prevalent of enterococcus species (morandi et al., 2006). many studies have reported negative catalase activity for l. farraginis and e. duran (devriese et al., 2002; endo and okada, 2007b). in general, it can be concluded that the two strains isolated from kefir belong to the family of labs, because the members of this family are all gram-positive, negative catalase, and anaerobic or microaerophilic (orla-jensen, 1919), and also because these strains were isolated from kefir grains in milk and, according to 16srrna gene sequencing, they both belong to the phylum firmicutes, which are all characteristics of the lab family (kandler and weiss, 1986). another finding of this study was the resistance of the isolated strains to acidic conditions (ph = 2.5-4) of the gastrointestinal tract and pepsin and trypsin enzymes of gastric juice (ph = 2) for 1-3 hours. also, the ed strain showed generally higher resistance levels than the lf strain. the strains were able to grow in the presence of bile salts with a concentration of 0.15%, but are not able to grow when bile salt concentration increases to 0.3%. however, there was no significant difference between the growth rates of the strains in these conditions. in a similar study, the strains isolated from kefir grains, including l. acidophilus, l. plantarum, and l. kefiri exhibited high resistance to gastric acid and bile salts (zheng et al., 2013). a study on the strain l. kefiranofaciens isolated from kefir found it to be resistant to acidic conditions but unable to grow in the presence of bile salts at 0.20% level (xing et al., 2017). in addition, l. paracasei isolated from kefir showed high resistance to acidic conditions, pepsin enzyme, and 0.15% bile salts (bengoa et al., 2017). however, e. durans isolated from dairy products (fermented milk) can survive and grow under acidic conditions (ph = 2-3), gastric juice, and 1.5 and 0.3% bile salts (pieniz et al., 2014; guo et al., 2016). in general, one of the interesting characteristics of labs is their resistance to acid, gastric juice, and bile salts (fioramonti et al., 2003). the findings of this study showed the in-vitro antagonistic activity of the lf and ed strains against l. garvieae and a. hydrophila, which are two important pathogens of fish farms, but they had very weak activity against s. iniae. the lf strain showed stronger antagonistic activity against y. ruckeri than the ed strain. in a study of lactobacillus strains isolated from kefir, they were able to inhibit the growth of salmonella enterica, escherichia coli, 43 int. j. aquat. biol. (2020) 8(1): 35-49 listeria monocytogenes, and staphylococcus aureus (zanirati et al., 2014). a study reported that e. durans species isolated from kefir can inhibit the growth of gram-positive and negative bacterial pathogens (carasi et al., 2014). the strains isolated from cheese showed broad antimicrobial activity against a. hydrophila, e. coli, l. monocytogens, and p. aeruginosa (pieniz et al., 2014). the antagonistic activity of ed and lf is probably caused by the production of antibacterial compounds by these strains, because the metabolites resulting from the function of lab strains of kefir including acetic acid, lactic acid, ethanol, diacetyl, peptides, and bacteriocins, have sufficient antibacterial effect to act against pathogenic bacteria (jamuna and jeevaratnam, 2004). another important characteristic of probiotic strains that is highly associated with their stability and survival in the gastrointestinal tract is hydrophobicity (nikoskelainen et al., 2001). the in-vitro investigation of hydrophobicity of the isolated strains showed that they both have a hydrophobicity level of more than 50%, with the lf strain (72.47%) being significantly more hydrophobic than the ed (51.42%). these results are consistent with the findings of a study conducted on l. faraignis, l. plantarum kefiri, and l. acidophilus, which showed a high level of adhesion to digestive tract epithelium in the culture medium (zheng et al., 2013; thamacharoensuk et al., 2017). the strain of l. kefiranofaciens xl10 isolated from tibetan kefir was also reported to have 79.9% hydrophobicity (xing et al., 2017). in contrast, a study on e. durans isolated from milk products showed low levels of hydrophobicity in the medium (guo et al., 2016). in general, it can be argued that the hydrophobicity of the ed and lf strains signifies their ability to adhere to the intestinal mucus and avoid excretion with feces. the analysis of lab colony counts in the intestines of the fish fed with different doses of ed and lf showed that all treatment groups had significantly more lab colonies in the intestine than the control group. among the treatment groups, ed2, bactocell, and lf2+ed2 had the highest colony counts. this also revealed that the number of lab colonies in the intestines increased with increasing the dose of the strains in the diet. also, the combined use of ed + lf in the diet increased the lab colony count in the intestines. similar to the results of the present study, the addition of l. sakei (clfp 202) isolated from the intestines of salmonids to the diet of salmo trutta significantly increased the number of lab colonies in the intestine, which was attributed to their high potential for adhesion and survival in the intestinal tract of the fish (balcazar et al., 2007). other studies have also reported that the addition of probiotics e. faecium and e. casseliflavus to the diet of common carp and rainbow trout, respectively, increased the lab population in their intestine (dehaghani et al., 2015; safari et al., 2016). a study of oral administration of l. kefiranofaciens strain isolated from kefir to mice reported an increase in the lab colony count in the intestine and a decrease in the number of pathogenic bacteria (xing et al., 2017). since the presence of labs in the intestines of most fish species has been proved (ringo and gatesoupe, 1998), the increased number of colonies in the sampled intestines may be attributed to the introduction of probiotic strains into the diet, which significantly increases the share of labs in the intestinal flora and limit the growth of other bacteria, including the harmful ones (ziaei-nejad et al., 2006). both of the isolated strains seem to have a good survival in the gastrointestinal environments, as the conditions for adherence and colonization in the intestinal wall is favorable and this increases the population of lab. a significant increase in bwi, sgr, per, cf, dwg, and tl indices of the fish fed with lf2 + ed2, bectocell, ed2, and lf2 diets indicate that using higher doses of the strains (lf + ed) (108) and a combination of the two strains (lf + ed) improves growth parameters more successfully. in contrast, fcr ratio of these treatments was the lowest in comparison with the control group. the addition of l. acidophilus to diet of clarias gariepinus improved the growth and fcr, sgr, and bwi (al-dohail et al., 2009). adding a mixture of b. subtilis, e. faecium and 44 ali et al./ oral administration of lactic acid strains kefir in oncorhynchus mykiss p. acidilactici to the diet of rainbow trout has shown significant effects on growth performance and nutrition efficiency (giannenas et al., 2015). supplementing of 2.5 × 106 cfu/g of e. faecium and e. durans isolated from the gastrointestinal tract of oreochromis niloticus has been shown to improve the nutrition efficiency and growth parameters fish (laraflores et al., 2013). probiotics can increase fish growth by positively affecting digestive functions such as digestion and absorption of nutrients, intestinal microbiota, appetite, and fcr (ringo and cratesoup, 1998; panigrahi et al., 2005; nayak, 2010). the combined use of probiotic strains in fish diets has also shown to increase sgr and per (mohapatra et al., 2012a, b). in the present study, the treatments lf2 + ed2, bactocell and lf2 had the highest rate of survival. adding probiotic bacteria has been shown to increase the survival of fry and larvae of the spotted seatrout (cynoscion nebulosus), turbot (scophthalmus maximus), goldfish (carassius auratus), swordtail (xiphophorus helleri( (gatesoup, 1994; kennedy et al., 1998; abraham et al., 2007) and catla catla (mohanty et al., 1996). several studies suggest that lab species increase the survival rate by preventing digestive system impairment, neutralizing antinutritional substances and toxins, regulating the growth of beneficial intestinal microorganisms, improving immune responses, and limiting the activity of pathogenic microorganisms (ringo and gatesoupe, 1998; panigrahi et al., 2005; suzer et al., 2008). in contrast, some researchers have not observed any significant difference in the survival rate after using probiotics (farzanfar et al., 2006). this discrepancy is probably due to differences in the type of probiotic strains, growth conditions, and fish species. in general, lf and ed strains are likely to increase the resistance and survival rate of fish by improving their health condition and growth performance and stimulating the immune system. in conclusion, the two lab strains isolated from kefir showed suitable probiotic properties, including resistance to acidic conditions, gastric juice enzymes, and low concentrations of bile salts, as well as high hydrophobicity. they showed in-vitro antagonistic activity against some pathogens that are common in fish farms. the addition of lf and ed strains to the trout diet in higher doses (lf2, ed2) and in combined form (lf2 + ed2) increased the number of lab colonies in the intestine, enhanced and improving the biometric indicator and growth performance and survival of the fish. although both lf and ed appear to be good candidates for addition to the diet of juvenile rainbow trout, further studies with more invitro and in-vivo investigations seem necessary. acknowledgments this study is a product of project no. 96011993, financially supported by the iran national science foundation and also funded by a research grant from shiraz university. this project was performed using the laboratory facilities generously provided by the aquatic animal health department of the faculty of veterinary medicine, university of tehran. references abraham t.j., babu c.s., mondal s., banerjee t. 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(2020) 8(5): 311-316 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article effects of apple cider vinegar on growth performance and non-specific immune parameters of skin mucus in common carp (cyprinus carpio) fingerlings hamed nekoubin*,1abdolmajid hajimoradloo, seyyed hosein hoseinifar department of fisheries, gorgan university of agricultural sciences and natural resources, gorgan, iran. s article history: received 7 november 2019 accepted 14 march 2020 available online 2 5 october 2020 keywords: common carp apple cider vinegar non-specific immune mucus abstract: this study was undertaken to evaluate the effects of apple cider vinegar on growth performance and non-specific immune parameters of skin mucus (alkaline phosphatase, lysozyme and total protein) in common carp fingerlings. for this purpose, a total of 240 fish were stocked in twelve tanks for four treatment with three replications and fed by diets supplemented with 0, 1, 2 and 4 % apple cider vinegar for 60 days. the result showed that there was no significant difference in body weight increase (bwi), feed conversion ratio (fcr), specific growth rate (sgr) and condition factor (cf) among the treatments (p>0.05). the results revealed that there is no significant difference among skin mucus alkaline phosphatase of fish in all treatments. although skin mucus lysozyme activity and total protein significantly increased by apple cider vinegar in comparison with the control group. the highest and lowest skin mucus lysozyme activity and total protein in common carp fingerlings were observed at 2% apple cider vinegar and control group, respectively. therefore, apple cider vinegar improves skin mucus lysozyme activity and total protein and can be a good candidate for antibiotic replacement in common carp fingerlings. introduction according to the un food and agriculture organization, aquaculture is growing more rapidly than all other animal food-production sectors (www.fao.org). aquaculture is becoming a more concentrated industry, with fewer, but much larger, farms (yousefi et al., 2019). infectious diseases are always a hazard and may cause significant economic losses and problems with animal welfare. intensive aquaculture led to growing problems with bacterial diseases (taheri mirghaed et al., 2018; hoseini et al., 2020a, b). to solve this problem, farmers frequently use antibiotic compounds to treat bacterial diseases (alderman and hastings, 1998); therefore it has resulted in the development and spread of antibiotic resistance bacteria (yonar, 2012; hoseini and yousefi, 2019), so there is a need to use alternative therapies for bacterial pathogens in fish (chakraborty and hancz, 2011; chakraborty et al., 2014). in this case, various immunestimulants, including certain feed additives are being used to increase the resistance *correspondence: hamed nekoubin doi: https://doi.org/10.22034/ijab.v8i5.818 e-mail: nekoubin.hs@gmail.com of aquatic organisms to infectious agents (lee et al., 2015). recently, some scientists proved the positive effects of medicinal plants or herbs as feed additives. these herbs improved the growth and feed utilization of the fish and also reduced diseases by regulating pathogens in gastrointestinal tract (fazelan et al., 2020; rajabiesterabadi et al., 2020; yousefi et al., 2020). one of feed additives is organic acid components which are known to have stimulatory potential effect on the immune system and have been proven efficient for disease prevention (taheri mirghaed et al., 2019). various types of organic acids, such as acetic acid, butyric acid, citric acid, lactic acid, malic acid, sorbic acid, propionic acid, as well as their salts, are used to improve the health of fish (hossain et al. 2007; ng et al., 2009; safari et al., 2017) and shrimp (pourmozaffar et al. 2017). apple cider vinegar is an acidic solution, which contains organic acids such as acetic acid and malic acid, vitamin b and c, and minerals (iman et al., 2015). previous study 312 nekoubin et al./ apple cider vinegar and carp immunity demonstrated that using organic acid in diet resulted in improve blood indexes and healthy in rainbow trout fingerling (taheri mirghaed et al., 2019). also, in another studies positive effect of apple cider vinegar in fish (beheshti et al., 2012; safari et al., 2017) and shrimp (pourmozaffar et al., 2017) healthy and immunology are demonstrated. therefore, in this study we used apple cider vinegar to determine whether this solution has an effect on growth performance including and skin mucus nonspecific immune parameters in common carp fingerlings. materials and methods two hundred forty common carp, cyprinus carpio, fingerlings (10 ±0.56 g) were purchased from a private sector farm and transferred to the aquaculture laboratory of gorgan university of agricultural sciences and natural resources (iran). in this study, we used 12 glass tanks (40×60×40 cm), and each tank (40-l) was aerated with an air pump. after 7 days acclimation, fish were distributed into three experimental groups and one control group, each with 3 replicates. preparing the experimental diet: apple cider vinegar (5% acetic acid) was supplied from 1&1 co. (fars, iran). 1, 2 and 4 percentage of apple cider vinegar were mixed with basal diet (8.8% moisture, 11.36% ash, 34.5% crud protein and 10.7% fat), and then pelleted by a meat grinder (hoseinifar et al., 2017). experimental diets were kept at 4°c until used. we chose the doses based on the literature (safari et al., 2017). fish were fed on experimental diet at 5% of body weight and 3 times per day for 60 days (nrc, 2011). sampling: at the first and the end of experiment, 10 specimens were randomly selected from each treatment and were anesthetized in clove oil solution (150 ppm) for 40-50 s (yousefi et al., 2018), dried with tissue paper, and then weight and length were measured. in addition, skin mucus was obtained following the method described before (ross et al., 2000, subramanian et al., 2007) for non-specific immune parameters assay, at the end of the experiment. the mucus samples were transferred to 15 ml sterile tube, centrifuged at 1500 g (4ºc) for 10 min and the supernatants were poured in 2-ml tubes and stored at -80 ºc until analysis. growth performance: in this study growth indices were calculated by following formula (hoseini et al., 2016): bwi% = (wtw0)/w0×100 fcr (feed conversion ratio) = f/(bt – b0) sgr % (specific growth rate) = (ln wt -ln w0) ×100/t cf = 100 × (wt/tl3) where wt and w0 are final and initial body weight (g), respectively, f is relative food intake (g), bt and b0 are final and initial fish biomass (g), respectively, t = time of rearing (days) and tl = total length (cm). non-specific immune parameters of skin mucus: alkaline phosphatase in skin mucus was determined using commercial competitive kits (bio-chemistry kit, iran). the skin mucus lysozyme activity was measured based on lysis of micrococcus luteus using turbidimetric assay following (hoseini et al., 2018). total protein content of samples was determined according to lowry et al. (1951). data analysis: all data are shown as mean±sem. the spss 16 software was used for statistical analyses. one-way analysis of variance (anova) followed by lsd tests was used to assess the significant effects of apple cider vinegar concentrations on growth performance indices and skin mucus non-specific immune parameters. a value of p<0.05 was considered statistically significant for all statistical tests. results the result showed that there was no significant difference in bwi, fcr, sgr and cf among the treatments (p> 0.05) (table 1). skin mucus alkaline phosphatase, lysozyme activity and total protein: the effects of apple cider vinegar on non-specific immune parameters of common carp skin mucus are showed in figures 1 to 3. the results revealed that there is no significant difference among skin mucus alkaline phosphatase activity of fish in all 313 int. j. aquat. biol. (2020) 8(5): 311-316 treatments and control group (p>0.05) (fig. 1). common carp fingerlings showed a dose-dependent increase in lysozyme activity with apple cider vinegar. lysozyme activity was significantly enhanced by apple cider vinegar relative to controls (fig. 2) (p<0.05). according to figure 3, there was significant difference detected among different treatment groups in skin mucus total protein (p<0.05). the highest skin mucus total protein in common carp fingerlings was observed in 2% apple cider vinegar and lowest skin mucus total protein was detected in the control group. discussions in this study, apple cider vinegar used as dietary supplements to assess the potential growth rates and immunological effects on common carp fingerlings. our results showed that using 1, 2 and 4% apple cider vinegar as a supplementary in diet had no effect on bwi, fcr, sgr and cf after 60 days in fish. similarly, using of organic acid in diet had not significant effect on growth performance in oreochromis sp. (ng et al., 2009) and pagrus majo (hossein et al., 2007). also, this study assessed the ability of the apple table 1. effect of different concentrations of apple cider vinegar on growth performance in common carp fingerlings after 60 days. growth indices concentrations of apple cider vinegar control 1% 2% 4% bwi (%) 98.89 ± 48.83 101.68 ± 21.45 99.02± 59.42 90.47± 42.17 fcr 2.53± 0.24 2.84± 0.44 3.12± 0.09 2.4± 0.18 sgr (%) 1.64± 0.81 1.75± 0.35 1.65± 0.99 1.50± 0.70 cf (%) 1.24± 0.38 1.23± 0.08 1.30± 0.09 1.23± 0.95 figure 1. effect of different concentrations of apple cider vinegar on skin mucus alkaline phosphatase in common carp fingerlings after 60 days. data are presented as relative gene expression and mean±sem. different lowercase letter indicates significant differences among treatments. figure 2. effect of different concentrations of apple cider vinegar on skin mucus lysozyme activity in common carp fingerlings after 60 days. data are presented as relative gene expression and mean±sem. different lowercase letter indicates significant differences among treatments. figure 3. effect of different concentrations of apple cider vinegar on skin mucus total protein in common carp fingerlings after 60 days. data are presented as relative gene expression and mean±sem. different lowercase letter indicates significant differences among treatments. 314 nekoubin et al./ apple cider vinegar and carp immunity cider vinegar in skin mucus non-specific immune parameters of common carp fingerlings. mucus is an important barrier in fish, because it provides the substrate in which antibacterial mechanisms may act, and in most fish species the mucus covers most of the external surfaces, and mainly the skin (tort et al., 2003). fish skin mucus contains many humoral nonspecific defense factors for example, lysozyme, complement, interferon, c-reactive protein, and lectin, transferrin (saurabh and sahoo, 2008). these innate immune molecules are especially important for each species, because each live in a water medium rich in pathogens (ingram, 1980) and thus play a key role in maintaining homoeostasis in the animal (saurabh and sahoo, 2008). we demonstrated that 60 days of feeding with apple cider vinegar altered the skin mucus levels of lysozyme activity and total protein in common carp fingerlings that are related to non-specific immunity in fish. previous researches demonstrated that some immune-stimulant such as organic acids can enhance some of specific and non-specific immune responses (hossain et al. 2007; ng et al., 2009; beheshti et al., 2012; safari et al., 2017), and can be a good candidate as an alternative to the drugs, chemicals and antibiotics currently being used to control fish diseases in fish culture (bairwa et al., 2012). in addition, lysozyme activity were significantly higher in fish japanese flounder (paralichthys olivaceus) fed a fermented vegetable product supplemented diet (ashida and okimasu, 2005). it has been reported that apple cider vinegar has multiple antimicrobial properties on different microbial species (yagnik et al., 2018). aapple cider vinegar consists of acetic acid, flavonoids such as gallic acid, tyrosol catechin, epicatechin, benzoic acid, vaninilin, caftaric acid, coutaric acid, caffeic acid, acid and ferrulic acid that can effect on immune defense and oxidative responses (budak et al., 2011; nazıroğlu et al. 2014). in general, the results of this study showed that the addition of apple cider vinegar in diet of common carp has beneficial effects on the activity of lysozyme and mucus protein that related to non-specific immune. therefore, our data suggest that feeding with apple cider vinegar may positively affect non-specific immune in fish. also, 2% vinegar in the diet showed better results than other levels. therefore, apple vinegar can be used as immune-stimulant in diet of common carp fingerlings. acknowledgments the present work was supported by gorgan university of agricultural sciences and natural resources. references alderman d., hastings t. 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(2019) 7(3): 175-179 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology short communication first record of red cornetfish, fistularia petimba lacepède, 1803 (actinopterygii: fistulariidae) from the syrian coast chirine hussein, amir ibrahim, firas alshawy*1 department of marine biology, high institute of marine research, tishreen university, lattakia, syria. article history: received 22 july 2019 accepted 9 september 2019 available online f o r ( v 7 , n 3 ) 2 5 june 2019 keywords: mediterranean, lessepsian, fistularia petimba, lattakia. abstract: fistulariidae species (cornetfishes) exist in tropical and subtropical waters having four species; two of them are found in the mediterranean sea. this study records fistularia petimba from the syrian marine waters for the first time, filling the gap in its distribution along the eastern mediterranean. introduction for decades, marine species are moving from tropical to subtropical waters, especially to the mediterranean, which is under natural and anthropogenic stressors (mavruk and avsar, 2008; alshawy et al., 2019 b, c) and accommodates more than 100 new species (zenetos et al., 2012). some of these species exploit the resources and succeeded in colonizing and threatening the native populations and human health (zenetos et al., 2004; ibrahim, 2008; plan, 2009). fistulariid species (cornetfishes) exist in tropical and subtropical waters having four species that two are found in the mediterranean sea (froese and pauly, 2019). fistularia commersonii is known to be distributed along the mediterranean sea, and was record for the first time in the syrian coast in 2002 (galyia, 2003). the second one, f. petimba lacepède, 1803, was recorded at the spanish mediterranean coast (west mediterranean) in 1997 (cárdenas et al., 1997), and in 2016 was recorded in the eastern mediterranean, at ashdod coast and mersin bay (stern et al., 2017; ünlüoğlu et al., 2018), but never in the syrian coasts (ali, 2018). this paper report the first record of f. petimba in the syrian marine waters, filling the gap in its distribution along the eastern mediterranean between ashdod coast and mersin *correspondence: firas alshawy doi: https://doi.org/10.22034/ijab.v7i3.660 e-mail: firas.ahmad.alshawy@tishreen.edu.sy bay. materials and methods on 29 july 2019, a field trip was performed in the marine waters facing lattakia city, syria (35°31'5.97"n, 35°42'48.57"e; fig. 1) to collect fish samples using fixed gillnet (18 mm mesh size, 3 m height, 200 m length: with duplicates), with fishing boat (9.5 m, 19hp). the collected f. petimba was identified according to carpenter and de angelis (2016) and morphometric measurements (length to the nearest mm, weight to the nearest g) and meristic counts were recorded. the specimen was then photographed, preserved in 7% formaldehyde, and placed at the biological laboratory of the high institute of marine research (tishreen university, lattakia, syria) (unnumbered yet). results and discussions a single specimen of f. petimba (fig. 2) was caught at ~45 m water depth off lattakia coast. it has a long and lightly compressed body, with a long tubular snout and small mouth. the dorsal fin locates approximately at the end of the body, caudal fin is clearly forked and has one elongated filament (fig. 2a). the dorsal side of the body has a row of bony http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatget.asp?genid=141 http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatget.asp?spid=14692 176 hussein et al./ first record of fistularia petimba from the syrian coast plates (fig. 2b) stretching along to the end. the posterior part of the body has a series of backwardpointing spines on each side (fig. 2c). the body is colored orange-brown and the abdominal side is pearly white. the margins of dorsal, anal and caudal fins are bright orange. the meristic formula are d: 15, a: 15; p: 15, v: 6, c: 7. these features of f. petimba are in agreement with carpenter and de angelis (2016) and ünlüoğlu et al. (2018). the morphometric measurements are shown in table 1. the red cornetfish exists in the atlantic and indopacific oceans (carpenter and de angelis, 2016) and red sea (bogorodsky et al., 2014). it was passed from atlantic to the spanish mediterranean coast for the first time via gibraltar (cárdenas et al., 1997). then, it was recorded in ashdod and mersin bay, presumably interring through the suez canal (çiftçi et al., 2019). this species is metaphorically regarded as lessepsian one, especially that it was not recorded in the mediterranean coasts of africa or europe, except spain (bearez et al., 2017; el sayed haroun and karachle, 2017). fistularia petimba was not recorded from the syrian marine before (alshawy et al., 2019a, d, e), may be because of misidentification with the other similar species e.g. f. commersonii. in fact, despite the large similarity, many morphological differences occur between these two species. fistularia commersonii does not have any bony plates or backward-pointing spines that are the distinctive features of f. petimba. in addition, f. commersonii is green or bluish green, while f. petimba is red to orange-brown. the sagittal body of f. petimba may enable easy escape through the mesh of fishing nets, which may lower its landings and accelerates its population enlargement and possible establishment in this area. fistularia petimba feeds on small fish and shrimp (carpenter and niem, 1999), which may threatens the native fish population through the competition for food and for space. this record adds an additional species to the fish checklist of the syrian marine waters and confirms that human activates support the species introduction to the mediterranean sea (ibrahim, 2009; alshawy et al., 2019g, f). in addition, climatic changes make the seawaters feasible to accommodate the tropical species (ibrahim et al., 2010; alshawy et al., 2016; alshawy et al., 2017). however, further investigations should be carried out table 1. morphometric and biometric characteristics of fistularia petimba caught from the syria marine water. features measurement (mm or g) total length 642 standard length 448 body depth 13 (2.9% sl) head length 157 (35.04% sl) eye diameter 15 (3.35% sl) snout length 128 (28.57% sl) dorsal fin length 17 (3.79% sl) dorsal fin height 28 (6.25% sl) pectoral fin length 9 (2.01% sl) pectoral fin height 20 (4.46% sl) pelvic fin length 4 (0.89% sl) pelvic fin height 10 (2.23% sl) caudal fin length 26 (5.80% sl) anal fin length 17 (3.79% sl) anal fin height 27 (6.03% sl) pre-dorsal length 372 (83.04% sl) pre-pectoral length 175 (39.06% sl) pre-pelvic length 258 (57.59% sl) pre-anal length 372 (83.04% sl) total weight 54 figure 1. collection site of fistularia petimba from the syrian marine waters. http://researcharchive.calacademy.org/research/ichthyology/catalog/fishcatget.asp?genid=141 177 int. j. aquat. biol. (2019) 7(3): 175-179 to reveal the economic and environmental impacts of this fish on the native fish populations (unep-map rac/spa, 2009; ibrahim et al., 2019). this necessitates the need for international and regional cooperation for fisheries management to ensure proper marine biodiversity conservation (vallerga et al., 2003; drago et al., 2004) and native fish stocks protection (hussein et al., 2011a, b). acknowledgements the authors thank tishreen university and the high institute of marine research, lattakia who provided the financial and logistic supports to this work. references ali m.f. (2018). an updated checklist of the marine fishes from syria with emphasis on alien species. mediterranean marine science, 2: 388-393. alshawy f., ibrahim a., hussein c., lahlah m. (2019a). first record of arrow bulleye, priacanthus sagittarius starnes, 1988 from the syrian marine waters (eastern mediterranean). fishtaxa, 2: 21-24. alshawy f., ibrahim a., hussein c., lahlah m. 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(2019) 7(3): 117-122 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article cocos frillgoby, bathygobius cocosensis (bleeker, 1854): an additional fish element for the iranian marine waters (teleostei: gobiidae) reza sadeghi, hamid reza esmaeili*1 ichthyology and molecular systematics research laboratory, zoology section, department of biology, college of sciences, shiraz university, shiraz, iran. article history: received 2 april 2019 accepted 5 june 2019 available online 2 5 june 2019 keywords: gobies, taxonomy, diversity, morphology, distribution, indo-pacific region. abstract: members of the pantropical/subtropical genus bathygobius are small and common gobies in sheltered and exposed shallow rocky or sandy shorelines, reef crests, mangroves, seagrass beds, rock jetties and seawalls in the atlantic and indo-pacific regions. this report documents a significant range extension of cocos frillgoby, bathygobius cocosensis into the western parts of indo-pacific regions, in the iranian intertidal coast of makran sea. the morphological description of collected individuals and its comparison with morphologically similar related goby species, b. meggitti is given and discussed. introduction gobiidae is one of the most diverse and species-rich family of marine fishes with more than 1904 valid species (chakrabarty et al., 2012; fricke et al., 2019). members of this family, are found worldwide in freshwater, brackish, and marine, with the majority of these species being associated with shallow tropical and subtropical environments (agorreta et al., 2013). they are generally small benthic fishes whose paired fins are modified into an adhesive disk that enables them to rest on the bottom (gill and mooi, 2012). some gobies spend most of their time hiding among the cracks, crevices and rocks of coral reefs (walker and wood, 2005). due to their cryptic nature, small size and lack of immediate economic importance, gobies are poorly understood. gobies of the genus bathygobius bleeker, 1878 are a circumtropical fishes that normally found intertidally around coral, rocks, or mangroves that currently comprises about 29 valid species (smith and heemstra, 1987; miller and stefanni, 2001; fricke et al., 2019). due to high diversity and distribution of bathygobius, it has been used as an excellent group to study evolution patterns within the atlantic *correspondence: hamid reza esmaeili doi: https://doi.org/10.22034/ijab.v7i3.613 e-mail: hresmaeili@shirazu.ac.ir (rodríguez-rey et al., 2017) and indo-pasific regions. bathygobius cocosensis (bleeker, 1854), commonly referred to as cocos frillgoby is known from indo-pacific, east africa to johnston, marquesas and tuamoto islands, north to southern japan, south to the southern great barrier reef and rapa islands; marianas and marshall islands in micronesia (myers, 1999). although b. cocosensis has been reported from several regions of the indo-pacific ocean, but there has been no record of it from the iranian marine waters. hence, in this study, (i) presence of b. cocosensis in the iranian waters is documented, (ii) its detailed morphological characteristics are provided, and (iii) its significant range extension into the western indo-pacific ocean are discussed. materials and methods the specimens of b. cocosensis (fig. 1) were collected by hand net with mesh size of 1.30 mm in seven trips from october 2016 to may 2018 (figs. 2, 3) from 10 cm depth of the intertidal rocky pools in two localities of the iranian intertidal coast of makran sea, including bahal (25°41'n, 57°53'e) and chabahar (25°16'n, 60°39'e) (figs. 2-4). after 118 sadeghi and esmaeili / first record of bathygobius cocosensis from makran sea anesthesia with quinaldine sulphate, the specimens were preserved in 10% neutralized formalin and catalogued in the zoological museum of shiraz university, collection of biology department, shiraz (zm-cbsu). the specimens were identified following smith and heemstra (1987) and randall (1995). all morphometric measurements were taken point to point by calipers to an accuracy of 0.1 mm under the stereomicroscope (zeiss stemi sv6). morphometric characters were given as % standard length (sl) and % head length (hl) in table 1. meristic and morphometric methods follow miller (1988), chen and fang (2006) and chen and miller (2008). meristic abbreviations are as follows: d1 = first dorsal fin; d2 = second dorsal fin; v = ventral fin; a = anal fin; p = pectoral fin; psd = predorsal figure 1. bathygobius cocosensis. (a) bahal, female, 39.3 mm sl, and (b) chabahar, female, 45.4 mm sl. figure 2. distribution map of bathygobius cocosensis. the solid red circle are the new record localities. (a) bahal and (b) chabahar. http://onlinelibrary.wiley.com/doi/10.1111/j.1095-8649.1977.tb04048.x/full 119 int. j. aquat. biol. (2019) 7(3): 117-122 scales; lss= longitudinal scales series; and tss = transverse scales series. results bathygobius bleeker, 1878 bathygobius bleeker [p.] 1878:54 [archives néerlandaises des sciences exactes et naturelles v. 13 3; masc. gobius nebulopunctatus valenciennes 1837. according to miller and stefanni (2001) and the genus bathygobius can be diagnosed by the presence of postorbital blotches, body width greater than body length, uppermost pectoral fin rays free from membrane near tips, no barbels on underside of head, no spines on preopercle, chin with curved mental frenum, a small bump below anterior nostril bordered below by a longitudinal groove, cheek papillae pattern longitudinal, gill opening equals pectoral fin base, scales cycloid or ctenoid; no curved canine tooth in each side of lower jaw (carpenter and niem, 2001). remarks: bathygobius is easily confused with drombus and palutrus, which lack free pectoral fin rays, and monishia and caffrogobius, which have a transverse cheek papillae pattern. bathygobius cocosensis (bleeker, 1854) (fig. 1) gobius cocosensis, bleeker [p.] 1854:47 [natuurkundig tijdschrift voor nederlandsch indië v. 7 (no. 1); indonesia [original locality was nova selma, cocos island/cocos-keeling islands, eastern indian ocean]. morphological description: d1: vi; d2: i, 9; a: i, 8; p: 17-20; psd: 12-15; lss: 33-38; tss: 17-20. some morphometric data of the examined specimens of this species and the similar species, b. meggitti are given in table 1. bathygobius cocosensis is characterized by body width greater than length, body depth 16.53% sl, body width 22.08% sl, head length 28.25 in sl, upper 3-4 pectoral rays branched to base and free from membrane, rounded table 1. measurements (in percentage sl) of bathygobius meggitti and b. cocosensis from two localities in the iranian coast of makran sea. locality mean of all localities bahal chabahar species b. meggitti b. cocosensis b. cocosensis b. cocosensis number of specimens mean of all specimens mean of all specimens mean of 4 male mean of 3 female mean mean of 3 male mean of 5 female mean standard length (mm) 40.36 34.92 34.43 30.39 32.41 37.79 37.07 37.43 body depth 17.22 16.53 16.95 16.29 16.61 16.42 16.45 16.42 body width 23.32 22.08 21.55 22.27 21.93 22.99 21.6 22.27 head length 30.3 28.25 29.15 28.57 28.9 27.86 27.32 27.62 snout length 8.55 7.55 8.25 8.25 8.25 6.8 7.11 6.95 eye diameter 7.53 7.32 7.07 7.07 7.07 7.67 7.48 7.58 upper jaw length 10.63 10.11 10.04 10.04 10.04 10.72 9.7 10.18 caudal peduncle depth 11.05 11.21 11.42 11.42 11.42 11.66 10.45 11.01 caudal peduncle length 23.14 23.75 22.73 22.73 22.73 24.75 24.94 24.88 predorsal length 36.37 34.25 35.09 35.09 35.09 32.47 34.13 33.33 preanal length 59.51 57.14 56.82 56.82 56.82 55.25 59.52 57.14 prepelvic length 28.89 27.55 27.62 27.62 27.62 28.33 26.6 27.47 first dorsal base length 18.19 18.59 18.08 18.08 18.08 18.87 19.42 19.16 anal base length 15.93 16.67 16.64 16.64 16.64 20.37 14.14 16.69 caudal fin length 23.98 24.63 23.04 23.64 23.31 25.97 26.25 26.11 pectoral fin length 26.66 27.1 24.75 24.75 24.75 29.76 30.21 29.94 pelvic fin length 23.09 24.88 23.64 23.64 23.64 26.6 26.04 26.32 120 sadeghi and esmaeili / first record of bathygobius cocosensis from makran sea caudal fin, predorsal scales nearly reaching to above rear margin of preopercle, scales absent on cheek and operculum, prepelvic area scaled, body scales ctenoid, becoming cycloid on abdomen, breast and nape. color: the principal color characters distinguishing this species are: body with mottled brown overall color, with five alternating irregular whitish and brown blotches or saddles dorsally on body, lower half of side with 5-7 rectangular brown blotches, white spots and blotches on cheek and operculum, midsides with about 6-8 elongate black spots, males with numerous longitudinal lines, females mottled, caudal fin spots small, horizontal dark line near base of 1st dorsal fin (smith and heemstra, 1987). habitat. marine waters. it is usually found in the rockpools which are a common feature in the rocky intertidal zone (fig. 3). distribution: east and south africa, seychelles, madagascar and mascarenes east to hawaiian islands and pitcairn, north to southern japan, south to western australia, queensland (australia), new caledonia and rapa and now in the makran sea (fig. 4). discussions recent field investigations in the persian gulf, strait of hormuz and makran/oman sea have resulted in the discovery or new records of different fish groups (randall, 1995; béarez et al., 2008; uiblein and heemstra, 2011; sadeghi et al., 2017; mehraban and esmaeili, 2018) including gobies. nevertheless, work still remains to be done on fish taxonomy and ecology in the area. the persian gulf and oman sea are included in the arabian sea ecosystem whose southern limit is defined by the line between cape figure 3. collecting sites of bathygobius cocosensis. (a) bahal and (b) chabahar. (b) (a) figure 4. world distribution map of bathygobius cocosensis, including new record (blue solid square) and previous other countries/islands (red solid circle). 121 int. j. aquat. biol. (2019) 7(3): 117-122 guardafui, the horn of africa (the north-east point of somalia) and the cape comorin (the southernmost tip of india). briggs (1974) considers the oman sea as a zoogeographic boundary between the western indian ocean and the indo-polynesian provinces. this situation is important from biogeography point of view as it receives fish elements from indo-pacific ocean as presented here by record of cocos frillgoby, b. cocosensis. till date, b. cocosensis was known from east and south africa, seychelles, madagascar and mascarenes east to hawaiian islands and pitcairn, north to southern japan, south to western australia, queensland (australia), new caledonia and rapa, marianas and marshall islands in micronesia (randall et al., 1993; myers, 1999; fricke et al., 2019). however, it seems that this goby has wider distribution range and now it is found in the makran sea. the specimens of this species in two localities were taken from tide pools with some gravel and sand at depths of 5-20 cm. as, daryanavard et al. (2015) reported b. meggitti (hora and mukerji, 1936) and bathygobius sp. from the kandaloo (in qeshm island in the persian gulf), it is possible that bathygobius sp. be b. cocosensis and therefore, coastal area of the persian gulf is also to be considered as suitable habitat for this species. bathygobius cocosensis is apparently very similar to b. meggitti (fig. 5), but it can be distinguished from b. meggitti by certain characteristics: 33-38 longitudinal scales rows (vs. 38-40 longitudinal scales rows in b. meggitti), 12-15 predorsal scales (vs. 14-22 predorsal scales in b. meggitti), 17-20 pectoral fin rays with the upper 3 or 4 with rays free of membranes, each with two branches (vs. 20-22 pectoral fin rays with the upper 5 or 6 with rays free of membranes, each with two to four branches in b. meggitti), no flaps on nostrils (vs. a small dorsoposterior flap on anterior nostril), no large black spot in the upper base of pectoral fin (vs. present in b. meggitti). also in b. cocosensis, predorsal scales are extending forward to above rear preopercle margin and mental frenum distinctly curved with long free lateral lobes. acknowledgements we thank h. mehraban and h. hashemi for their help and support in the fieldworks. this study was supported by shiraz university and was approved by the ethics committee of biology department (su9330207). conflict of interest the authors declare that they have no conflict of interest. references agorreta a., san mauro, d., schliewen u., van tassell j.l., kovačić m., zardoya r., rüber l. 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(2020) 8(2): 132-142 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article a redescription of tenagomysis species and gastrosaccus australis from estuarine environments (crustacea: mysida) in auckland region, new zealand neetha nandanie punchihewa*1 the open university of sri lanka, nawala, nugegoda, sri lanka. s article history: received 16 february 2020 accepted 26 march 2020 available online 2 5 april 2020 keywords: estuarine mysida north island tenagomysis abstract: investigation of mysid habitats in estuarine waters were conducted at 59 sites throughout auckland region from may 2006 to january 2009. this paper provides the taxonomic descriptions of the three species, collected during this survey. among the 59 sites mysids were collected only at 26 sites: tenagomysis chiltoni collected from 15 sites, t. novaezealandiae from 21 sites and gastrosaccus australis from four sites. ontogenetic variation observed in the size of the antennal scale, and counts of lateral spines and cleft spines of telson and uropod for both t. chiltoni and t. novaezealandiae. such apparent characteristics should not be used alone to differentiate species. it is important to use a combination of characteristics such as shape of the rostrum, anterolateral margin of the carapace, number of articulations of carpo-propodus of thoracic limbs and shape and size of the antennal scale. several aspects of the description of t. chiltoni provided by tattersall (1923) and hodge (1964), do not agree with the present specimens, this is due to size variations. introduction seventeen mysid species have been so far described from new zealand waters, presently accommodated within six genera: tenagomysis thomson, 1900 (10 species), siriella dana, 1850 (2 species), paralophogaster hansen, 1910 (1 species), euchaetomera sars, 1883 (2 species), boreomysis sars, 1869 (1 species) and gastrosaccus norman, 1868 (1 species). several species have not been reported subsequent to original descriptions, but other species have proven to be more widespread, and more regularly reported: tenagomysis chiltoni tattersall, 1923, t. macropsis tattersall, 1923, and t. novaezealandiae thomson, 1900. the spatial distribution of mysids validated from many locations in the south island and few locations in the north island. the most diverse genus tenagomysis described by thomson (1900) in new zealand which currently comprises 15 species from australia and new zealand. this paper provides the taxonomic descriptions of three species, t. chiltoni tattersall, 1923, t. novaezalandiae thomson, 1900 and gastrosaccus australis tattersall, 1923 which were collected during *correspondence: neetha nandanie punchihewa doi: https://doi.org/10.22034/ijab.v8i2.834 e-mail: neetha_punchihewa@yahoo.co.nz the ecological survey throughout the auckland estuaries. materials and methods the material examined was collected as a part of reconnaissance surveys and monthly surveys to investigate the distribution of mysids throughout the greater auckland region. reconnaissance surveys for mysids in estuarine habitat were conducted at 59 sites extending from mathesons bay, north of auckland, to miranda in the firth of thames, along the east coast of the auckland region, and from bethells beach, north of manukau heads, to waiuku, manukau harbour, on the west coast of the auckland region. mysids were collected using a hand held dip net with a mouth area of 25×20 cm and with 500 µm mesh size, and drag along the stream bed. all mysids retained in the net along each transect were immediately collected into separate bottles containing 70% ethyl alcohol. selected mysid specimens were photographed using scanning electron microscopy (sem). further, morphology of mysid specimens was investigated under light microscope and the illustrations were 133 int. j. aquat. biol. (2020) 8(2): 132-142 made. total body length (tl) was measured from the tip of the rostrum to the posterior end of the telson excluding the spines. results order: mysida boas, 1883 family: mysidae haworth, 1825 subfamily: leptomysinae czerniavsky, 1882 genus: tenagomysis thomson, 1900 tenagomysis thomson, 1900: 483–484; tattersall, 1918: 9–10; 1923: 289–290; fenton, 1991: 325–326. tenagomysis chiltoni tattersall, 1923 (figs. 1-4) tenagomysis chiltoni tattersall, 1923: 292–293, plate ii, figs. 5–8; hodge, 1964: 387–394, figs. 2-3; chapman and lewis, 1976: 149–152, figs. 9.1–9.4 & 9.5a–b. material examined: 2270♀, 5.7–18.52 mm (tl), 1067♂, 5.9–16.00 mm (tl). distribution: east coast; mathesons bay (6♀, 3♂), matakana (10♀6♂), clevedon-wairoa river (16♀3♂), west coast; paturoa bay (6♀5♂), waitangi falls (6♀), figure 1. scanning electron micrographs (sem), tenagomysis chiltoni: (a–b) anterior part of the body (♀, 13.08 mm); (c) thoracic region of the body (♀,16.00 mm); (d) thoracic appendages (♀, 13.08 mm). legend: alp, antennular peduncle; ap, antennal peduncle; al, antennule; an, antennae; as, antennal scale; rm, rostrum; ce, cornea; en, endopod; ex, exopod; mr, marsupium. 134 punchihewa / a systematic study on estuarine mysids in new zealand orua bay (1♀), kakamatua (1386♀601♂), cornwallis (663♀340♂), mill bay (112♀71♂), karekare stream i (13♀10♂0), karekare stream ii (16♀4♂), piha stream i (6♀4♂), piha stream ii (12♀8♂), piha stream iii (11♀6♂), huia bay (6♀6♂). distribution-elsewhere around new zealand: tidal inlet, parakai (tattersall, 1923); lake oturi, waverley (hodge, 1964); stream at piha (chapman and lewis, 1976); coopers lagoon, forsyth and the avonheathcote estuary (waite, 1980); lower waikato river (kirk, 1983); avon-heathcote estuary (roper et al., 1983; jones et al., 1989); estuarine systems figure 2. scanning electron micrographs (sem), tenagomysis chiltoni: (a) uropod and telson, lateral view (♂, 13.54 mm); (b) uropods, ventral view (♀, 13.08 mm); (c–d) pleopods (♂, 13.54 mm). legend: ou, outer uropod (exopod); in, inner uropod (endopod); es, endopod spines; ts, telson spines. 135 int. j. aquat. biol. (2020) 8(2): 132-142 along the east otago coastline from the clutha river to oamaru and kaikorai lagoon (lill, 2011); taieri river estuary (bierschenk et al., 2008). habitat: recorded from both estuarine and fresh waters, new zealand. diagnosis: antennal scale elongate; mandibles with a prominent spine; carpo-propodus of endopod of thoracic limbs 3–7 with four short subjoints, 8 with five subjoints, dactylus with distinct claw; anterolateral angles of carapace produced into acute spines; telson with deeply cleft, with combinations of armature of the telson and uropodal endopod. description: carapace leaving last 1.5–2.5 thoracic somites exposed. front median line of the carapace bluntly pointed and sloping into concave shape rostrum. antero-lateral angles of carapace produced into acute spines (figs. 1a, 3a). eyes large, about 1.1–1.8 times longer than broad, with 3/8 of eye occupied by black-pigmented cornea (figs. 1a, 3a). antennal scale lanceolate, with setose margins; distal joint short and distinct; with prominent spine on outer distal corner of sympod from which scale arises. antennal scale 7–10 times as long as broad. antennal peduncle length approximately half that of antennal scale; peduncle extends to mid of distal segment of antennular peduncle. antennular peduncle longer than antennal peduncle; length nearly 1.4 times that of antennal peduncle with prominent spine on (upper mid line) distal joint of antennular peduncle (figs. 1a, b, 3a). mandibles welldeveloped, with prominent spine and large lacinia. (fig. 3e). endopods of thoracic appendages well-developed. carpo-propodus of thoracic limbs 3–7 with four short articulations, and distinct nail. carpo-propodus of thoracic limb 8 with five articulations, and distinct nail. outer distal corner of the basal segment of exopod produced into acute spine (figs. 4 a–d). telson about as long as last abdominal somite, 14– 17 times longer than terminal spine. cleft deep, basal third of cleft without armature, cleft armed with pair of plumose setae and a dense row of spines on distal 2/3; cleft spine number varies ontogenetically, for individuals 3.5–17.5 mm it ranges 10–52, and those mature individuals (10.0–18.9 mm) have 32–52 spines. telson with ontogenetically variable number of lateral spines; on individuals 10.0–18.9 mm, spines number 16–22 each side, and 10–18 spines on smaller individuals; spine number also subject to variation on an individual, with one side occasionally with 1 or 2 more spines than its opposite; terminal spine either side of telson longer than others. endopod of uropod 1.2–1.5 times longer than telson, with 40–64 spines on inner margin; exopod of uropod 1.2–1.4 times longer figure 3. tenagomysis chiltoni. (a) ♂, 13.64 mm; (b–e) ♀, 13.35 mm): (a) anterior part of the body; (b) maxilla; (c) uropod; (d) telson; (e) mandible. figure 4. tenagomysis chiltoni. (♀, 13.35 mm): thoracic appendages: (a) 1st leg; (b) endopod of 2nd leg; (c) endopod of 8th leg; (d) endopod of thoracic legs (3rd -7th). 136 punchihewa / a systematic study on estuarine mysids in new zealand than endopod, and 1.6–1.9 times longer than telson (figs. 2a, b, d, 3c, d). tenagomysis novaezealandiae thomson, 1900 (figs. 5-7) tenagomysis novaezealandiae thomson, 1900: 484, pl 33, figs. 6–8, pl 34, figs 9–17; chilton, 1906:703; tattersall, 1923: 291–292; chapman and lewis, 1976: 149–152, pl 30 & figs. 9.5 c, d. material examined: 2435♀, 3.01–10.32 mm (tl), 842♂, 3.25–8.50 mm (tl). distribution: east coast: orewa-nukumea stream figure 5. scanning electron micrographs (sem), tenagomysis novaezealandiae (♀, 7.5 mm); (a) anterior part of the body; (b) pleopod; (c) uropods and telson, ventral view. legend: ap, antennal peduncle; an, antennae; as, antennal scale; mn, mandible; rm, rostrum; ce, cornea; en, endopod; ex, exopod; mr, marsupium; tl, telson; in, inner uropod (endopod); ou, outer uropod (exopod); es, endopod spines. 137 int. j. aquat. biol. (2020) 8(2): 132-142 (358♀106♂), red beach (6♀), big manly bay (923♀310♂), okoromai bay (405♀150♂), murrays bay (taiorahi creek) (4♀21♂), hobson bay (2♀6♂), wenderholm beach (10♀5♂), waiwera river (estuary) (16♀4♂), mathesons bay (7♀6♂); west coast: kakamatua (506♀127♂), cornwallis (26♀15♂), mill bay (3♀), lower nihotopu reservoir (spillway) (10♀6♂), karekare stream i (15♀6♂), karekare stream ii (21♀6♂), piha stream i (58♀15♂), piha stream ii (35♀18♂), piha stream iii (35♀18♂), orua bay (4♀), paturoa bay (5♀3♂), armour bay (18♀12♂). distribution-elsewhere around new zealand: kaikorai lagoon, estuary of the waikouaiti river, and rock pools at brighton, all near dunedin (thomson, 1900); lake waikare (chilton, 1906); stream at piha (chapman and lewis, 1976); avon-heathcote estuary (roper et al., 1983; jones et al., 1989); raglan harbour and the mouth of the waikato river (nipper and williams, 1997); taieri river (sutherland and closs, 2001; bierschenk et al., 2008, unpubl. data); estuarine systems along the east otago coast line from clutha river to oamaru and kaikorai lagoon (lill, 2011). habitat: estuarine, capable of living in fresh waters, new zealand. diagnosis: a small tenagomysis species with carpopropodus of thoracic limbs 3–8 with three articulations; mandibles without lateral spine; anterolateral angles of carapace produced into acute spines with combinations of armature of telson and uropodal endopod description: carapace leaving last three thoracic somites exposed. front margin of carapace evenly rounded and obtusely pointed in median line between eyes. antero-lateral angles of carapace produced into acute spines (fig. 5a). eyes large, about 1.3 times as long as broad, with distal half of eye occupied by black-pigmented cornea (figs. 5a, 6a). figure 6. tenagomysis novaezealandiae. (♀, 9.42 mm): (a) anterior part of the body; (b) antenna and antennal scale; (c) antennule (antennuler peduncle and antennal peduncle); (d) uropod (endopod and exopod); (e) telson. figure 7. tenagomysis novaezealandiae. (♀ 8.46 mm): thoracic appendages: (a) 1st leg; (b) mandible with mandibular palp; (c) endopod of 3rd leg; (d) 2nd leg; (e) maxilla; (f) endopod of 6th leg. 138 punchihewa / a systematic study on estuarine mysids in new zealand antennal scale lanceolate, with setose margins; distal joint short and distinct; with prominent spine on outer distal corner of sympod from which scale arises. antennal scale nearly 4.6-6.3 times as long as broad. antennal peduncle length, approximately half that of antennal scale; antennal peduncle extends to proximal end of third segment of antennular peduncle, with prominent spine on (upper mid line) distal joint of antennular peduncle (figs. 6a, c). mandibles welldeveloped, with larger lacinia (fig. 7b). endopods of thoracic appendages well-developed (figs. 7a, c, d, f). tarsal joint of thoracic limbs 3–8 with three articulations and a distinct nail. outer corner of basal segment of exopod rounded. telson shorter than last abdominal somite, 9–12 times longer than terminal spine, cleft shallow, armed with a pair of plumose setae and 13–32 spines; lateral margins of telson armed with 12–15 spines on entire length, terminal spines largest (fig. 6e). endopod of uropod 1.2–1.6 times longer than telson, with 20–26 spines; exopod 1.5–2 times longer than endopod (fig. 6d). subfamily: gastrosaccinae, norman, 1892 genus: gastrosaccus norman, 1868 gastrosaccus norman, 1868: 153–15 diagnosis: carapace emarginate dorsally behind; eyes small, cylindrical; antennal scale small, shorter than peduncle, outer edge naked with a terminating spine; peduncle of antennules long and strong, outer filament swollen at base. thoracic legs with multiarticulate tarsus; spines and setae at each articulation, no nail; first pleopods well-developed with elongated curved cylindrical peduncle and two minute one-jointed branches, other pleopods simple. first segment of pleon with a large epimeral process which acts in figure 8. scanning electron micrographs (sem), gastrosaccus australis (♀, 11.00 mm): a, anterior part of the body; b, thoracic legs; c, fifth abdominal somite including small lobe; d, uropods (exopod and endopod) and telson. 139 int. j. aquat. biol. (2020) 8(2): 132-142 support of marsupial pouch; marsupium pouch with two pairs of marsupial lamellae. telson quadrangular, elongated, with larger marginal spines, short cleft armed with serrations which are larger distally. outer edge of outer uropod with series of strong spines. male pleopods biramous; peduncle of first pair margined with long setae, peduncle of remaining pairs naked; inner branches of first, fourth and fifth pairs very small, outer branch of fourth pair very long with nearly seven articulations without setae, gradually becoming more slender distally. gastrosaccus australis tattersall, 1923 (figs. 8-10) gastrosaccus australis tattersall, 1923: 282–283, pl 1, figs 7–9, pl ii figs. 1–4. material examined: 23♀, 9.6-12.6 mm (tl) (all 23, mature♀ with broods). distribution: east coast: big manly bay, 7♀, orewanukumea stream 2♀ and awanohi bridge, 2♀; west coast: kakamatua 12♀. distribution-elsewhere around new zealand: sprits bay near north cape (tattrsall, 1923), avon heathcote estuary (roper et al., 1983; jones et al., 1989); estuarine systems along the east otago coast line from clutha river to oamaru and kaikorai lagoon (lill, 2006); taieri river (bierschenk et al., 2008). habitat: estuarine, capable of living in fresh waters. diagnosis: a gastrosacus species with posterior margin of fifth abdominal somite with a short median process (lobe), six spines on inner margin of endopod of uropod; endopod slightly longer than exopod and figure 10. gastrosaccus australis. (♀, 8.9 mm): (a) 1st leg; (b) 2nd leg; (c) 8th leg; (d) endopod of 4th leg. figure 9. gastrosaccus australis. (♀, 9.6 mm): (a) anterior part of the body; (b) maxilla; (c) uropod (endopod and exopod); (d) mandibular palp with mandible; (e) telson. 140 punchihewa / a systematic study on estuarine mysids in new zealand six large lateral spines on telson (a species of the spinifer group). description: carapace large, leaving half of last thoracic somite exposed. front margin of carapace produced into short obtusely rounded rostral plate with prominent pseudo-rostral process below rostral plate, being triangular and acute in dorsal view (fig. 9a). eyes small, about 1.6 times as long as broad, with approximately 1/3 of eye occupied by black pigmented cornea (figs. 8a, 9a). antennal scale small, rounded apex, with setose margins (except outer margin), anterior-lateral margin of antennal scale extends as a strong spine; about three times as long as broad, extends up to margin of first segment of antennular peduncle. antennal scale length approximately two third that of antennal peduncle; antennal peduncle extending beyond distal joint of antennular peduncle. antennular peduncle with two prominent spines on outer margin of second segment; two spines and small process on (upper mid line) distal joint of antennular peduncle (fig. 8a). mandibles well-developed, with large lacinia (fig. 8d). posterior median margin of fifth abdominal somite produced into a small process (fig. 8c). endopods of thoracic appendages well-developed. carpo-propodus of thoracic limbs 4–8 (endopod) with 8–10 joints; third with seven joints (fig. 10a–d). telson about 1.1 times long as last abdominal somite, 5–6 times longer than terminal spine, and more than twice as long as broad at base. cleft armed with 11– 12 spines on each side; lateral margins of telson armed with four spines arranged their entire length and another two terminal spines on apical lob. endopod of uropod slightly longer than exopod of uropod. exopod with 13–15 spines; endopod with six spines spread on inner margin (figs. 9c, e). discussions of particular note is the ontogenetic variation observed in the size of the antennal scale, and counts of lateral spines and cleft spines of telson and uropod (endopod spines). a small mysid may differ considerably from a larger specimen. this has been found true for t. chiltoni. therefore, several aspects of the description of t. chiltoni provided by tattersall (1923) and hodge (1964) do not agree with the present specimens. the antennal scale of t. chiltoni was described by tattersall (1923) as being 10 times as long as was broad; hodge (1964) described it as nine times long as it was broad; and the present specimens have a antennal scale about 8-10 times as long as broad (24% of current specimens agree with hodge, 1964), and 21% tattersall,1923). additionally, tattersall (1923) described this species with 16–18 lateral spines on the telson, and hodge (1964) recorded 16–20 spines, whereas the number of spines on specimens reported herein is in the range 10–22, although it is subject to table 1. major diagnostic criteria of each species. character t. chiltoni t. novaezealandiae g. australis length of adult females (mm) 10.00–18.5 6.00–10.31 9.60–12.60 length of males (mm) 5.90–15.64 3.25–8.48 not found rostrum obtuse with a blunt apex rounded short obtusely rounded rostrum with pseudorostral process antennal scale long, lanceolate in shape short, lanceolate in shape short & stout, rounded apex with anteriorlateral margin extend as a strong spine telson cleft deep shallow shallow number of cleft spines of telson (juvenile to adult) 10–52 (body size 3.5–17.5 mm); 32–52 (body size 10.0–18.9 mm) 13–32 6 number of lateral spines of telson (juvenile to adult) 10–22 12–15 11–12 carpo-propodus of thoracic limbs (endopod) 3–7 with 4 articulations and 8 with 5 3–8 with 3 articulations 4–8 with 8–10 articulations and 3 with 5 special features a prominent acute spine on the body of the mandible immediately outside the attachment of the palp. 141 int. j. aquat. biol. (2020) 8(2): 132-142 ontogenetic variation; unfortunately tattersall (1923) did not report the number of cleft spines for this species, although hodge (1964) recorded 32–36 on each border, and in this current study 32–52 were counted on mature animals. a second difference concerns the number of spines on the uropods, according to hodge (1964) being 55–60 spines, whereas these number 40–64 on adult auckland region specimens. some of this variation could be attributable to the different ontogenetic stages reported by these earlier two authors, as tattersall’s t. chiltoni specimens ranged 8–10 mm and the average length of hodge’s specimens was 12.5 mm, while specimens of 5.5–18 mm were examined in this study. examination of extant type material of this species is required to determine whether all forms attributed to it truly are conspecific. certain characters of t. novaezealandiae show similar ontogenetic variations to t. chiltoni. such apparent characteristics as lateral and cleft spines and proportions of different body regions should not be used alone to differentiate species. the number of carpo-propodus joints on the 8th leg for t. chiltoni described by tattersall (1923) was four, in addition to a distinct nail, whereas present specimens referred to this species have five short joints, as did those referred to this taxon by hodge (1964). tattersall (1923) described this species with only the last thoracic somite being exposed; whereas those specimens referred to this species from the auckland region have the last 1.5–2.5 thoracic somites exposed, again similar to hodge (1964). tattersall (1923) described only five female specimens (8–10 mm), so that a complete description is impossible and the variations may be related to size and the preservation condition of the animal. thus, in identification of mysid species, it should be important to use combination of characters. the size of the species, with their significant ontogenetic variations also important in combination with other major characters (table 1). therefore, the present study considered combination of all the characters to differentiate tenagomysis species. these included, adult size of the species (both male and female), shape of the rostrum, anterolateral margin of the carapace, the carpo-propodus of thoracic limbs, shape and size of the antennal scale, and counts of lateral spines and cleft spines of telson and uropod (endopod spines). acknowledgment this project was funded by the auckland university of technology, auckland, new zealand. dr. steve o’ shea is hereby acknowledged for his support given to me during my study. references chapman m.a., lewis m.h. (1976). an introduction to the freshwater crustacea of new zealand. auckland and london: collins. fenton g.e. (1991). three new species of tenagomysis from the coastal waters of south-eastern tasmania (crustacea: mysidae: mysinae: leptomysini). memoirs of museum victoria, 52(2): 325-335. hodge g. (1964). a redescription of tenagomysis chiltoni (crustacea: mysidacea) from a freshwater coastal lake in new zealand. new zealand journal of science, 7: 387-395. jones m.b., greenwood j.g., greenwood j. (1989). distribution, body size, and brood characteristics of four species of mysids (crustacea: peracarida) in the avon-heathcote estuary, new zealand. new zealand journal of marine and freshwater research, 23: 195199. lill a.w.t., closs g.p., savage c., schallenberg m. (2011). annual secondary production of two estuarine mysid species (mysidacea: mysidae) inhabiting an intermittently closed estuary, south-eastern new zealand. new zealand journal of marine and freshwater research, 62(7): 823-834. melland k., willassen e. (2007). the disunity of “mysidacea” (crustacea). mollecular phylogenetics and evolution, 44(3): 1083-1104. nipper m.g., williams e.k. (1997). culturing and toxicity testing with the new zealand mysid species. australian journal of ecotoxicology, 3(2): 117-129. roper d.s., simons m.j., jones m.b. (1983). distribution of zooplankton in the avon heathcote estuary, christchurch. new zealand journal of marine and freshwater research, 17: 267-278. sutherland d.l., closs g.p. (2001). diel patterns of mysid 142 punchihewa / a systematic study on estuarine mysids in new zealand drift (crustacea: mysidacea) in the taieri river estuary, new zealand. new zealand journal of marine and freshwater research, 35: 197-200. tattersall w.m. (1918). euphausiacea and mysidacea. australian antarctic expedition, 1911– 1914. scientific reports. series c zoology and botany, 5(5): 1-15. tattersall w.m. (1923). crustacea. part vii: mysidacea. british antarctic (terra nova) expedition, 1910. natural history report (zoology), 3(10): 273-304. thomson g.m. (1900). on some new zealand schizopoda. journal of the linnean society of london, zoology, 27(178): 482-486. int. j. aquat. biol. (2013) 1(4): 195-201 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article the impact of manjil and tarik dams (sefidroud river, southern caspian sea basin) on morphological traits of siah mahi capoeta gracilis (pisces: cyprinidae) adeleh heidari1, hamed mousavi-sabet*1, majidreza khoshkholgh1, hamid reza esmaeili2, soheil eagderi3 1department of fisheries, faculty of natural resources, university of guilan, sowmeh sara, p.o. box: 1144, guilan, iran. 2department of biology, college of sciences, shiraz university, iran. 3department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 2 june 2013 accepted 4 august 2013 available online 2 1 august 2013 keywords: capoeta gracilis dam geometric morphometrics shape variation abstract: it has been postulated that the building of the manjil and tarik dams on sefidroud river has led to the body shape variation of capoeta gracilis in upand downstream populations due to the isolation. in this study, geometric morphometric approach was used to explore body shape variations of capoeta gracilis populations in upand downstream manjil and tarik dams in sefidroud river from south of the caspian sea basin. the shape of 90 individuals from three sampling sites were extracted by recording the 2-d coordinates of 13 landmark points. pca, cva, dfa and ca analysis were used to examine shape differences among the populations. the significant differences were found among the shape of the populations and these differences were observed in the snout, the caudal peduncle and the head. the present study indicated the body shape differences in the populations of capoeta gracilis in the sefidroud river across the manjil and tarik dams, probably due to the dam construction showing anthropogenic transformation of rivers influences body shape in an aquatic organism. introduction many freshwater fish species are typically threatened by effects of human activities, such as habitat demolition and fragmentation (yamamoto et al., 2006). biodiversity is based on genetic diversity, which is likely to be influenced by habitat fragmentation (hanski and gaggiotti, 2004), one of the most prevalent negative man-induced processes for nature conservation (heywood, 1995). construction of dams affects fish movements, which may conduct to differentiation of populations (meldgaard et al., 2003). it has been suggested that fragmentation of river ecosystems may result in the conversion of migration patterns between fish populations (horvath and municio, 1998; craig, 2001; jager et al., 2001), ‘producing genetic stocks’ that are reproductively isolated units and are genetically different from other stocks (carvalho and * corresponding author: hamed mousavi-sabet e-mail address: mousavi-sabet@guilan.ac.ir hauser, 1994). water impoundment also changes the natural landscapes of a river by its transformation into reservoirs (haas et al., 2010). this can cause novel ecological and evolutionary challenges (baxter, 1977) for fishes that need to be responded for adaptation to new condition of ecosystem. the genus capoeta contains potamodromous cyprinid fishes with about 10 species, of which seven occur in iran and it is one of the most taxonomically complex genera of cyprinidae (coad, 2013; abdoli et al., 2008; samaee et al., 2009). species and subspecies of capoeta occur sympatrically and allopatrically in all iranian freshwater basins (saadati, 1997). this genus inhabiting both lotic and lentic habitats (samaee et al., 2006). siah mahi, capoeta gracilis, is a predominant fish of the sefidroud river. this species matures within 2–4 years of age and at a length of 15–20 cm, some mailto:mousavi-sabet@guilan.ac.ir 196 heidari et al./ int. j. aquat. biol. (2013) 1(4): 195-201 populations attains maturity as early as their first year and at a size of 10 cm (coad, 2013). in addition to its ecological significance, c. gracilis is an important species being harvested for sport and inland water fishing (kiabi et al., 1999). two sexes of c. gracilis have similar morphometrical characteristics (anvarifar et al., 2011). geometric morphometric is a modern approach to analyze the shape of body (bookstein, 1991). application of this method in fish body shape has been reported (loy et al., 1999) and has been considered as a useful tool to assess fish populations. this method describes organisms’ body shape in terms of x and y (and also z) coordinates, obtained from a set of landmark points (loy et al., 1999). landmark points are defined as homologous points that bear information on the geometry of biological forms (bookstein, 1991). this method quantifies changes in shape, and patterns of morphometric variations within and between groups if each individual deviates from an average shape, i.e. the consensus configuration (cadrin, 1999). the sefidroud river, with 765 km length is one of the important rivers in the southern caspian sea basin (aghili et al., 1999). the manjil and tarik dams were constructed on the sefidroud river in 1962 and 1968, respectively (najmaii, 2004). since there is no information on the impact of the manjil and tarik dams on morphological characteristics of siah mahi as an endemic fish of sefidroud river, this study was conducted to assess the impact of the manjil and tarik dams on the body shape of c. gracilis using geometric morphometric method. the results of this study can help to understand better the response of this species to alterations in natural flow regimes as a result of dam construction. materials and methods a total of 90 individuals of c. gracilis were collected from three sampling sites, one each from upstream of the manjil dam (36° 77′ 89″ n, 49° 15′ 25″ e; 40 individuals: station 1), downstream of the manjil dam which is upstream of the tarik dam (36° 46′ 52.86″ n, 49° 61′ 18″ e; 17 individuals: station 2) and downstream of the tarik dam (36° 99′ 15″ n, 49° 57′ 71″ e; 33 individuals: station 3), in november 2013 by electrofishing (fig. 1). care was taken to collect specimens of similar size to remove any allometric difference. the specimens were anaesthized, then fixed in 10% formaldehyde at the sampling site and transferred to the laboratory. the left side of the specimens (with dorsal and anal fins were held erected using pins) were photographed using digital camera (cybershot dscf505; sony, japan), thirteen landmark points were defined and digitized on two-dimensional images using tpsdig2 (rohlf, 2005; fig. 2). the landmark data after generalized procrustes analysis (gpa) to remove non-shape data, analyzed using principal components analysis (pca) to explore patterns of variation in their body shape. in addition, to assess the variation among the three studied populations, canonical variate analysis (cva) and manova were used (rohlf, 1993). a discriminant function analysis (dfa) was also performed to compare the body shape of the populations using consensus configuration and deformation grids. as a complement to discriminant analysis, morphometric distances among the body shape consensus of three studied populations were measured using a cluster analysis (veasey et al., 2001) by adopting the euclidean square distance as a measure of dissimilarity and the upgma method as the clustering algorithm (sneath and sokal, 1973). all analyses for morphometric data were performed using the morphoj software package and past. figure 1. sampling sites: (1) upstream and (2) downstream of the manjil dam and (3) downstream of the tarik dam on sefidroud river (north of iran). 197 heidari et al./ int. j. aquat. biol. (2013) 1(4): 195-201 results pca for all specimens explained 70.2% of shape variations by the first two pc axes extracted from the variance-covariance matrix (pc1 = 45.38%, pc2 = 15.67% and pc3 = 9.16). separation of the three studied populations showed along the first and second axis, respectively (fig. 3) revealed that the populations were clearly distinct from each other. pc1 scores were related to elongation of the head area and increase of caudal peduncle width, whereas pc2 scores were related to decrease of the head size and body depth. the manova/cva analysis showed significant differences between the populations in terms of body shape (p < 0.0001; fig. 4). the cva plot indicated that body shapes in the three studied groups were completely separated from each other. dfa on relative warps classified specimens into the correct groups at the rate of 97.6% in origin data and 93.9% in cross-validation (table 1) indicating a high differentiation among the populations of c. gracilis. the body shape differences (mean body shape) from the stations to other ones have shown in figure 5. the deformation grids were extracted from the dfa for pairwise groups. based on observed differences, the specimens of station 3 (upstream of the manjil dam) had larger heads, deeper caudal peduncles and dorsal position of the snout relative to those from the second station. also, comparison of the populations in upand downstream manjil dam showed that the upstream population had smaller heads, ventral position of the snout and deeper body. the downstream tarik dam population showed a little deeper body than those of the upstream manjil dam. figure 2. defined landmark-points to extract the body shape data of capoeta gracilis. 1. tip of snout; 2. center of eye; 3. posterior end of forehead (end of frontal bone); 4. posterior edge of operculum; 5. dorsal origin of pectoral fin; 6. anterior of dorsal fin base; 7. origin of pelvic fin; 8. posterior end of dorsal fin base; 9. anterior of anal fin base; 10. posterior of anal fin base; 11. posterodorsal end of caudal peduncle, at the nadir; 12. posteroventral end of caudal peduncle, at the nadir and 13. posterior most of body lateral line. figure 3. scatter plot of individual scores from the first two principal components based on 13 landmarks, distinguishing c. gracilis. vectors indicating the changes in the relative position of landmarks for each pc. figure 4. canonical variates analysis (cva) based on 13 landmarks, distinguishing capoeta gracilis along two canonical axes. 197 198 heidari et al./ int. j. aquat. biol. (2013) 1(4): 195-201 the upmga analysis divided the studied populations into two major distinct groups. the first branch was the upstream manjil dam (station 1) and downstream tarik dam (station 3) and the second branch was comprised of downstream manjil dam (station 2; fig. 6). the mahalanobis distance among the three studied groups of c. gracilic is shown in table 2. discussion morphometric analysis has been significantly enhanced using image processing techniques and in this regard, the geometric morphometrics (gm) approach is a powerful tool that can complement other methods for stock identification (cadrin, 1999). in the present study, landmark-based gm technique was applied to study the body shape changes among c. gracilis populations as a result of the manjil and tarik dams in the sefidrud river. the present study detected morphological differences in body shape of the studied populations. effects of dams on fish populations have extensively been documented in the recent years (yamamoto et al., 2006; dakin et al., 2007). dakin et al. (2007) showed that the morgan-falls dam on the chattahoochee river caused morphological differences between two populations of shoal bass, micropterus cataractae, especially in the upstream population compared to the downstream one. yamamoto et al. (2006) also noted habitat fragmentation caused by damming resulted in different body shape in whitespotted charr, salvelinus leucomaenis populations 20 years after the construction of the dam. anvarifar et al. (2011) found two distinct populations of figure 5. comparison of the body shape between three studied populations (deformation grids show differences of body shape from consensus shape of a station to another one). figure 6. the upgma graph for the three studied populations of c. gracilis. 199 heidari et al./ int. j. aquat. biol. (2013) 1(4): 195-201 capoeta capoeta gracilis at the upstream and downstream of the shahid-rajaei dam on the tajan river. the long-term isolation of populations and interbreeding may lead to morphometric variations between populations, and provides a basis for population differentiation. the body shape variations of c. gracilis populations in upstream and downstream of the dam may be caused by limitation of downstream dispersal of fish, and elimination of upstream migration (dakin et al., 2007). the dams obstruct the upward migration of fish, especially the migratory species resulting in an ecological trap for migratory fish that ascend the fish passages (pelicice and agostinho, 2008). many fish species shows morphological differences between different habitats (robinson and wilson, 1994; smith and skulason, 1996; taylor, 1999; jonsson and jonsson, 2001) and intraspecific polymorphism is commonly believed to arise from divergent selection pressures between various environments (robinson and wilson, 1994; smith and skulason, 1996; schluter, 2000). we have found almost similar body shape pattern in the specimens of stations 1 and 3 i.e. they differ significantly from the specimens which were collected between these two dams. differentiation between the samples from adjacent stations may be due to the geographic isolation of stations by artificial obstacles that allow morphological differentiation to proceed independently at each station (samaee et al., 2006). different body shape pattern of the second station suggests that this population may have a smaller population size (founder effect) or procured these morphological traits in responses to different environmental conditions (compared to two others). our results suggest that characteristics of created habitats (as result of dam construction) may determine evolutionary and ecological conditions driving alternations in the body shape of its resident fishes (langerhans et al., 2003). the main differences among the studied populations were found in the head region, the caudal peduncle and the body depth. it is common that morphological characteristics can show high plasticity in response to different environmental conditions (wimberger, 1992). the new environmental conditions of the upstream and downstream regions as result of the anthropogenic transformation of river may underline the shape differentiation among these three sites (haas et al., 2011). hence, the differences in body shape may be related to morphological adaptation to new habitats. for example, deeper body and caudal peduncle of the third station’s specimens can be area upstream manjil dam dawnstream manjil dam (upstream tarik dam) dawnstreamtarik dam original upstream manjil dam 100 0 0 dawnstream manjil dam (upstream tarik dam) 0 93.3 6.7 dawnstream tarik dam 3.3 0 96.7 cross-validated upstream manjil dam 94.6 2.7 2.7 dawnstream manjil dam (upstream tarik dam) 0 93.3 6.7 dawnstream tarik dam 6.7 0 93.3 table 1. classification matrix showing the number and percentage of individuals that were correctly classified. (bold values indicate correct classifications). station 1 station 2 station 2 5.1774 station 3 3.8369 4.3906 table 2. mahalanobis distances among the three studied groups of capoeta gracilic. 199 200 heidari et al./ int. j. aquat. biol. (2013) 1(4): 195-201 translated to be an adaptation for the rapid acceleration and maneuverability, whereas, the more fusiform body (a more streamlined body) of the population of second station may lower the drag force (nacua et al., 2010). also, differences in the head region may indirectly be related to changes in food seeking habits reflecting the difference between feeding habits and availability of food resources (langerhans et al., 2003). dams can alter the feeding habits, availability of food items, growth pattern and reproductive strategy of fish species living upstream and downstream of a river which these factors can influence the morphological differentiation in fishes (akbarzadeh et al., 2009). in conclusion, the present study found differences in the body shape of c. gracilis populations fragmented by the manjil and tarik dams on the sefidroud river probably as a result of dam construction showing that anthropogenic transformation of rivers influences the body shape in an aquatic organism. in addition, this study indicated that dam play an effective role in body shape of fishes and can be considered as main evolutionary drivers acting on aquatic biodiversity. references abdoli a., rasooli p., mostafavi h. 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(2018) 6(6): 321-329 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article ovarian maturatıon stages in arabian carpet shark, chiloscyllium arabicum gubanov, 1980 (orectolobiformes, hemiscylliidae) during spring and autumn in the persian gulf farideh porforugh1, negin salamat*1, abdolali movahedinia2 1department of marine biology, faculty of marine sciences, khorramshahr university of marine science and technology, khorramshahr, iran. 2department of marine biology, faculty of marine sciences, university of mazandaran, babolsar, iran. article history: received 8 october 2018 accepted 20 december 2018 available online 2 5 december 2018 keywords: carpet shark ovary 17-β estradiol progesterone gonadotropin abstract: the present study aimed to assess the ovarian tissue structure and plasma levels of the hypophysial-gonadal hormones, including 17-β estradiol, progesterone, gth-i and gth-ii of the arabian carpet shark, chiloscyllium arabicum inhabiting the persian gulf in spring (april to july) and autumn (august to december). in this regards, a total of 60 c. arabicum female specimens were collected from the bahrakan creek, in the north of the persian gulf. fish were bleed after euthanization and biometrical characters and levels of 17-β estradiol, progesterone, gth-i and gthii were measured. fish were then dissected and samples were taken from the ovary and fixed in bruin’s solution for 48 hrs. histological sections were prepared using routine histological techniques and stained with hematoxylin and eosin. based on the results, four different developmental stages were observed in the ovary in spring, including stage i, stage ii (primary oogenesis), stage iii (mediate oogenesis), and stage iv (final oogenesis). however, only three first stages were detected in autumn samples. the plasma levels of the studied hormones were higher in spring. based on the results, spring (especially mid-april to mid-july) is the reproduction season of c. arabicum in the persian gulf. introduction the reproductive system of female sharks includes a pair of ovaries and passageways for eggs. similar to other vertebrates, ovaries have three major functions viz. producing sex cells, accumulation of yolk, and synthesis and secretion of steroid hormones (koob and callard, 1999). in sharks, ovaries are positioned in the anterior part of the body cavity and hung by mesovarium from the abdomen roof (koob and callard, 1999). like teleost, the hypothalamic– pituitary-gonadal (hpg) axis is the most important axis of controlling reproduction in the sharks. hypothalamus secretes a variety of hormones and neurotransmitters (such as gonadotropin-releasing (gnrh) and dopamine-releasing (da)) and then regulates the production and secretion of the pituitary gonadotropic hormones, which in turn, affect gonads to release the steroid hormones (tostivint, 2011). two types of gonadotropin hormones are secreted from *corresponding author: negin salamat doi: https://doi.org/10.22034/ijab.v6i6.540 e-mail address: salamatnegin@yahoo.com pituitary anterior lobe of several elasmobranchs which are similar structurally to follicle-stimulating hormone (fsh) and luteinizing hormone (lh) (querat et al., 2001). similar to other vertebrate, gonadal steroids such as progesterone, androgen and estrogen play important role in the reproductive process of elasmobranchs (lutton et al., 2005). in female elasmobranchs, androgen and estrogen are primarily produced by granulose and theca cells of the ovarian follicles; however the granulosa cells can also produce progesterone (lutton et al., 2005). there are 26 shark species in the persian gulf (rima et al., 2014). arabian carpetshark, chiloscyllium arabicum is a member of carpet sharks inhabiting the coral reefs, wetlands, rocky shores, mangrove forests and river mouth. it can be found in the depth of 3-100 m, in the persian gulf to western india ocean, oman sea, and india ocean. they have been reported to be abundant in the spring and summer 322 porforugh et al./ ovarian maturatıon stages in arabian carpet shark in the persian gulf, oman sea and india ocean (compagno, 2002). there is a little information available regarding various biological aspects of sharks such as their reproductive system. however, c. arabicum is a proper model for laboratory studies because of its small size and tame and non-aggressive mood. therefore, the present study aimed to investigate the reproduction features of c. arabicum such as the reproductive time, morphological and histological structure of ovary, levels of steroid (17 beta-estradiol (e2) and progesterone (p4)) and gonadotropin hormones (gthi and gthii) during its reproduction and rest seasons. materials and methods a total of 60 mature females of c. arabicum were caught from the bahrakan creek using trawl net in autumn (september to december 2015) and spring (april to july 2016) (30 fish/season) and transported alive to the laboratory (fig. 1). blood and tissue sampling: the fish were euthanized by 2-phenoxy ethanol (0.35 ml-l) and blood samples were collected from the caudal vein after measurement of their weight and total length. then, the blood samples were centrifuged at 6000 rpm for 10 min, plasma was separated and frozen at -80°c for further analysis. fish were dissected after bleeding and ovaries were detached and weighted. the samples were taken from the ovaries and fixed in bouin's solution for 48 hours before histological processing. histological and histometrical study: tissue samples were washed in 70% ethanol for 24 hours and then dehydrated in ethanol series and embedded in paraffin using tissue processor apparatus (tissue tek-rotary, rx 11b, japan). histological sections of 5-6 µm thickness were prepared using a rotary microtome (leica, rm2245, germany) and stained by hematoxylin and eosin (eagderi et al., 2013). stained sections were studied under the light microscopy, and digital images were taken using dino capture software (fdp2, new taipei city, taiwan). for histometric analysis of the ovaries in both seasons, 10 tissue sections from each sample were studied and number of the ovarian follicles in different developmental stages was counted. the thickness of follicular wall and follicular diameter also were measured. ganado-somatic index: the fish weight (g) and gonad weight (mg) were measured in both seasons to calculate the gonado-somatic index (gsi) according to roff (1983) as following: gsi = (weight of ovaries/total body weight) × 100 steroid hormone assay: plasma levels of e2 and progesterone were measured using the radioimmunoassay (ria) method according to rinchard et al. (1993). briefly, 50-100 μl of standards, controls and plasma samples were moved into microtubes coated by polyclonal rabbit antibodies. 500 μl of 125ilabelled e2 (radioactivity 170 kbq, immunotech, france), and 1 ml of 125i-labelled progesterone (radioactivity 185 kbq, immunotech, france) tracer were added to microtubes and incubated in a water bath. the tubes were washed in phosphate buffer saline three times and radioactivity was counted using a gamma counter (lkb gamma counter/france). gonadotropin hormone assay: the plasma levels of gthi and gthii hormones were measured by commercial kits (immunotech, france) using the radioimmunoassay (ria) method described by van der kraak et al. (1983). statistical analysis: all data were represented as means ± standard error (se). the normality of data was controlled using shapiro-wilk test. then, figure 1. sampling area in the bahrakan creek at the north west of the persian gulf. 323 int. j. aquat. biol. (2018) 6(6): 321-329 independent-sample student t-test was used to determine the significant difference among data obtained in spring and autumn. the significant level of p<0.05 was accepted. all analyses were performed in spss 16.0 software. results the average weight and total length of female were significantly smaller during autumn season (table 1). chiloscyllium arabicum possesses a pair of large yellow oval ovaries located in the ventral part of the epigonal organ and hung by mesentery in the anterior part of the abdominal cavity (fig. 2). follicles at various developmental stages were observed in the ovaries in spring and autumn confiming the asynchronous ovary in the arabian carpetshark (fig. 2). in spring, the ovary occupies a great volume of the abdominal cavity. follicles were recognizable separately with naked eye (fig. 3), although follicles were significantly larger in spring (p<0.05, fig. 6). the structure of ovary in c. arabicum consists of the stromal connective tissue with many blood vessels and ovarian follicles at different stages of growth (fig. 4). epigonal organ was a vessels-abundant lymphomyeloid tissue, consisting various blood cells (leukocytes especially granulocytes were dominant). this organ is positioned around the ovarian follicles (fig. 4). the ovary is covered by a cell layer i.e. germinal epithelium and a fibrous and dense connective tissue so-called tunica albuginea (fig. 4). the ovarian follicles include one oocyte and surrounding membranes in both spring and autumn, at various developmental stages. meanwhile, tubular glands i.e. oviducal glands were detected among ovarian follicles (fig. 4). oviduct secretes egg shell to cover the released oocytes. in spring, sperm was found inside the follicles (fig. 4). the wall of ovarian follicles was thicker in spring (p<0.05, figs. 4, 6). follicles had also larger diameter in spring; then, they might be torn during preparation, therefore full image of mature follicle could not be prepared (fig. 4). developmental stages of the ovarian follicles were as following in spring: stage i: no ovarian follicles were formed and a group of the primordial germ cells (oogonia) were observed. oogonia were spherical or ovoid in shape (409.04519.83 μm diameters). a nucleus with dense chromatin was seen at the center of oogonia. stage ii (early oogenesis): the primary follicles (631.23-784.06 μm diameters) include oocytes (nucleus with 106.08-175.36 μm diameters in center or between center and perimeter of oocyte) covered by zona radiata and a layer of the follicular cells. inner and outer theca layers consisted of wide cells and collagen fiber, respectively were observed around the follicles. table 1. biometrical data of chiloscyllium arabicum collected from the bahrakan creek in spring and autumn. spring autumn weight (gr) 986.7±133.88 672.5±87.67 length (cm) 73.7±5.18 55.6±4.5 figure 2. the position and morphology of the ovary in chiloscyllium arabicum; ovarian follicle (white arrowhead), epigonal organ (black arrowhead), and mesentery (black star). 324 porforugh et al./ ovarian maturatıon stages in arabian carpet shark stage iii (middle oogenesis): the growing follicles with 1027.32-2914.64 μm diameters were the most follicles detected in the ovary. the increase in size of these follicles was mainly because of the vitellogenesis (yolk deposition). many yolk granules in the oocyte cytoplasm, a nucleus in the periphery of the oocyte, thicker zona radiata and thicker follicle membrane (comprising of the granulosa cells) were characteristics of the ovarian follicles in this stage. stage iv (late oogenesis): the growing follicles (3147.45-3566.62 μm diameters) with many yolk granules were observed. meanwhile, lipid vacuoles were appeared inside the oocyte and they become large. zona radiata was very thick. granulosa cells were cylindrical in shape around oocyte and sperm was observed in some follicles (fig. 4). a large amount of follicles in stages i (85.21120.18 μm diameter) and ii (160.28-236.64 μm diameter) and a little number of the ovarian follicle in stage iii (213.33-660.72 μm diameter) were found in the ovary of c. arabicum in autumn. no ovarian follicle at stage iv was detected in this season (fig. 5). the ovarian follicles in autumn were primarily immature (previtelloginic stages) while those observed in spring were mainly mature (fig. 6). the mean of diameter and wall thickness of the ovarian follicles was significantly higher in spring (p<0.05, fig. 6). the weight of the ovaries was significantly higher in spring and it had close correlation with the fish’s weight. the weight of the ovaries and percentage of the gonadosomatic index (gsi) was higher in spring (p<0.05, fig. 6). the results showed a significant difference between the amounts of steroid (17 beta-estradiol (e2) and progesterone (p4)) and gonadotropin hormones in plasma of female sharks in autumn and spring. the plasma level of these hormones were significantly higher in spring (p<0.05, fig. 7). discussion since the fishes have scheduled reproductive behavior, morphohistological study of the gonads may precede the study of maturity procedure. thus, structural and morphological changes in the oocyte figure 3. the anatomical structure of the ovary in chiloscyllium arabicum in spring (a, c) and autumn (b, d). ovary (white star), ovarian follicles (black arrowhead), and epigonal organ (black star). 325 int. j. aquat. biol. (2018) 6(6): 321-329 figure 4. representative photomicrograph of the ovarian tissue structure in chiloscyllium arabicum in spring. (a) germinal epithelium (black arrowhead), tunica albuginea (ta), ovarian follicle (black star); (b) epigonal organ (eo), oogonium (white arrowhead); (c) oogonium (white star), oogonial nucleus (n), oogonial squamous epithelial cell (white arrowhead); (d) ovarian follicle at stage i (black star), nucleus (n), ovarian follicle at stage ii (white star), epigonal organ (eo); (e) ovarian follicle at stage iii: yolk granules (y), zona radiata (z), granolosa cells (g), inner theca layer (it), outer theca layer (et); f. ovarian follicle at stage iv (black star), oviducal gland (og), sperm (s); g. oviducal gland (og); a,b,d,g (h&e;×725), c, e (h&e;×2900), f (h&e;×290). 326 porforugh et al./ ovarian maturatıon stages in arabian carpet shark and ovary could be used to determine the various stages of maturity (khalatbari, 2013). follicular cells are appeared around oocytes and change in shape during follicular development. in the present study, these cells were in cuboidal shape in the previtellogenic follicles and cylindrical at the end of vitellogenic stage. the theca layer was observed in both previtellogenic and vitellogenic follicles in the ovary of c. arabicum. sperm was found inside a number of ovarian follicles in c. arabicum. in this regard, chen and liu (2006) reported that a delay is occurred between the maturation and fertilization in c. plagiosum is mainly due to the sperm storage in the ovarian follicles of females. conde-moreno and galvan-magana (2006) reported that only one ovary of shortfin mako shark, isurus oxyrinchus is active and ovarian follicles are presented at the four developmental stages: in the first stage a group of oogonia were observed; in the early oogenesis, the primary follicles with zona radiate and follicular cells are detected; in the middle oogenesis, the diameter of follicles increase due to yolk disposition; and in the late oogenesis, the diameter of follicles and thickness of zona radiate are more than figure 5. representative photomicrograph of the ovarian tissue structure in chiloscyllium arabicum in autumn. (a) germinal epithelium (ge), tunica albuginea (ta), ovarian follicle at stage ii (ii), nucleus (n), ovarian follicle at stage i (i); (b) epigonal organ (eo); (c) nucleus (n), ovarian follicle at stage i (i); (d) ovarian follicle at stage ii (ii), tunica albuginea (ta); (e) the wall of ovarian follicles at stage i and ii, zona radiata (z), granolosa cells (g), inner theca layer (it); a,c,d (h&e, 290x), b, e (h&e, 2900x). 327 int. j. aquat. biol. (2018) 6(6): 321-329 previous stages and sperm present inside some follicles. in the present study, similar developmental stages of the follicles were observed. the gonadosomatic index (gsi) is considered as figure 6. the percent of ovarian follicles at different developmental stages, diameter and wall thickness of the ovarian follicles and gsi amount in chiloscyllium arabicum in spring and autumn. data are represented as mean±se. the letters indicate the significant difference between seasons (p<0.05). figure 7. the plasma level of e2, p4, gthi and gthii hormones in chiloscyllium arabicum in spring and autumn. data are represented as mean±se. the letters indicate the significant difference between seasons (p<0.05). 328 porforugh et al./ ovarian maturatıon stages in arabian carpet shark the most conventional method to determine the growth of gonads and spawning season (biswas, 1993). according to the results, the spring is the reproduction season of c. arabicum. in spring, the body weight and weight of sharks’ ovaries were higher. increasing the gonadosomatic index of c. arabicum may be as the result of increased number of the vitellogenic and/or watered follicles in the final stage (lee and yang, 2002). watered follicles absorb the water during the developmental process and increase the weight of ovary two to four times (lee and yang, 2002). abasi et al. (2003) stated that changes in gsi are closely related to the number of eggs and their size. in the present study, increase in the size of the follicles was the most salient sign of follicle growth. barone et al. (2007) found that the ovary was asynchronous in raja asterias, in which, the developing follicles with various sizes were detected in the ovary and the largest follicles were observed in the vitellogenic stage. the results of the present study were in agreement with these findings. the gradual increase in thickness and complexity of zona radiate represent its function in transferring the necessary materials. vitellogenesis, egg maturity and hatching are complex processes that need the transfer of raw materials to oocyte and active synthesis. mackie (2000) and samira et al. (2008) pointed out that diameter of the follicles increases in the reproduction season. erfani majd et al. (2015) also reported that diameter of the zona radiata reaches its maximum in the mature follicles in hypophthalmichthys molitrix. it has been well-documented that gnrh secretion from the hypothalamus stimulates the gth secretion form the pituitary and consequently secretion of steroid hormones from the gonads (omoto et al., 2005). gth-i controls the estradiol synthesis by ovarian follicular cells and vitellogenin absorption by follicles (omoto et al., 2005). meanwhile, the oocyte maturation is directly started under gth-ii control by means of a progestin so-called induced maturation steroid made in the follicular cells (jalabert, 2005). based on the studies on salmonids that have reproductive stages with a specific time difference, it was reported that gth-i is the mediator of vitellogenesis, and gth-ii regulates the final maturation of oocytes (tyler et al., 1991). seasonal changes of the gonadotropin levels in carassius auratus have indicated that synthesis and secretion of gth-i and gth-ii occur simultaneously for fishes with asynchronous ovaries (sohn et al., 1999). mateos et al. (2003) reported that the level of gth-i in pituitary of striped bass increase as the gsi increased. these results were in agreement with the results of the present research. gomez et al. (1999), sohn et al. (1999) and melamed et al. (2000) reported that synthesis and secretion of gth-ii increased during gametogenesis and spawning. similar results were found in c. arabicum. conflict of interest statement the authors report no conflict of interest. references abasi f., orian sh., matinfar a. (2003). histology and morphology of ovary in orange spotted grouper, epinephelus coioides, from the persian gulf. journal of pajouhesh and sazandegi, 66: 68-74. (in farsi) barone m., de ranieri s., fabiani o., pirone a., serena f. (2007). gametogenesis and maturity stages scale of raja asterias delaroche, 1809 (chondrichthyes, raijdae) from the south ligurian sea. hydrobiologia, 580: 245-254. biswas s. (1993). manual of methods in fish biology. south asian publishers. pp: 123-125. chen w.k., liu k.m. (2006). reproductive biology of whitespotted bamboo shark chiloscyllium plagiosum in northern waters off taiwan. fisheries science, 72: 12151224. compagno l.j.v. (2002). sharks of the world: an annotated and illustrated catalogue of shark species known to date (volume 2). rome: food and agriculture organization of the united nations. pp: 167-168. conde-moreno m., galvan-magana f. (2006). reproductive biology of the mako shark isurus oxyrinchus on the south-western coast of baja california, mexico. cybium, 30(4): 75-83. eagderi s., mojazi amiri b., adriaens d. (2013). description of the ovarian follicle maturation of the migratory adult female bulatmai barbel (luciobarbus 329 int. j. aquat. biol. (2018) 6(6): 321-329 capito, güldenstädt 1772) in captivity. iranian journal of fisheries sciences, 12(3): 550-560. erfani majd n., mesbah m., rahimi s. (2015). histometrical study of ovary in silver carp, hypophthalmichthys molitrix, in two age groups. journal of veterinary research, 70: 203-211. jalabert b. (2005). particularities of reproduction and oogenesis in teleost fish compared to mammals. reproduction, nutrition, development, 45: 261-279. khalatbari n. (2013). determination of gonadal structure and development in female yellowfin seabream. msc thesis. khorramshahr university of marine science and technology, pp: 64-67. koob t.j., callard i.p. (1999). reproductive endocrinology of female elasmobranchs: lessons from the little skate (leucoraja erinacea) and spiny dogfish (squalus acanthias). journal of experimental zoology, 284: 557-574. lutton b.v., george j.s., murrin c.r., fileti l.a., callard i.p. (2005). the elasmobranch ovary, in: w. hamlett (ed.). reproductive biology and phylogeny of chondrichthyes: sharks, batoids, and chimaeras. science publisher inc, einfeld, nh. pp: 237-281. mackie m. (2000). reproductive biology of the halfmoon grouper, epinephelus rivulatus at ningaloo reef, western australia environmental. journal of fish biology, 57: 363-376. mateos j., mananos e., martinez-rodrguez g., carrillo m., querat b., zanuya s. (2003). molecular characterization of sea bass gonadotropin subunits (a, fshb, and lhbþ and their expression during the reproductive cycle. general and comparative endocrinology, 133: 216-32. melamed p., gur g., rosenfeld h., elizur a., shulz w.r., yaron z. (2000). reproductive development of male and female tilapia hybrids (oreochromis niloticus x o. aureus) and changes in mrna levels of gonadotropin (gth) ib and iib subunits. journal of experimental zoology, 286: 64-75. omoto n., maebayashi m., adachi s., arai k., yamauchi k. (2005). the influence of oocyte maturational stage on hatching and triploidy rates in hybrid (bester) sturgeon huso huso× acipneser ruthenus. aquaculture, 245: 287-294. querat b., tonnerre-doncarli c., genies f., salmon c. (2001). duality of gonadotropins in gnathostomes. general and comparative endocrinology, 124: 308314. rima w., jabado r.w., saif m., ghais a.l., waleed h., mahmood s., shivji m.s., aaron c., henderson a.c. (2014). shark diversity in the persian gulf higher than previously thought: insights based on species composition of shark landings in the united arab emirates. marine biodiversity, 27-29. rinchard j., kestemont p., kühn e., fostier a. (1993). seasonal changes in plasma levels of steroid hormones in an asynchronous fish the gudgeon gobio gobio l. (teleostei, cyprinidae). general and comparative endocrinology, 92: 168-178. roff d.a. (1983). an allocation model of growth and reproduction in fish. canadian journal of fisheries and aquatic science, 40: 1395-1404. samira s., assem a., mourad mona m. (2008). reproductive biology (histological & ultrastructure) and biochemical studies in ovaries of mugil cephalus from mediterranean water. journal of arabian aquaculture society, 3: 33-58. sohn c.y., yoshiura y., kobayashi m., aida k. (1999). seasonal changes in mrna levels of gonadotropin and thyrotropin subunits in the goldfish, carassius auratus. general and comparative endocrinology, 113: 436-444. tostivint h. (2011). evolution of the gonadotropinreleasing hormone (gnrh) gene family in relation to vertebrate tetraploidizations. general and comparative endocrinology, 170: 575-581. tyler c.r., sumpter j.p., kawauchi h., swanson p. (1991). involvement of gonadotropin in the uptake of vitellogenic into vitellogenic oocytes of the rainbow trout, oncorhynchus mykiss. general and comparative endocrinology, 84: 291-299. van der kraak g., lin h.r., donaldson e.m., dye h.m., hunter g.a. (1983). effects of lh-rh and desgly10[d-ala6] lh-rh ethylamide on plasma gonadotropin levels and oocyte maturation in adult female coho salmon (oncoskynchus kisutch). general and comparative endocrinology, 49: 470-4196. int. j. aquat. biol. (2018) 6(6): 321-329 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology چکیده فارسی chiloscyllium arabicum gubanov, 1980 عربی کوسه گربه ماهی در تخمدانی بلوغ مراحل orectolobiformes, hemiscylliidae) )فارس خلیج در پاییز و بهار طی 2نیاموحدی عبدالعلی ،1*سالمات نگین ،1پرفروغ فریده .ایران ،خرمشهر ،خرمشهر دریایی فنون و علوم دانشگاه ،اقیانوسی و دریایی علوم دانشکده ،دریا زیست وهگر1 .ایران ،، بابلسرمازندران دانشگاه ،دریایی علوم دانشکده ،دریا زیست گروه2 چکیده: ، بتا استرادیول، پروژسترون-17گنادی شامل -های هیپوفیزیمطالعه حاضر با هدف ارزیابی ساختار بافتی تخمدان و سطوح پالسمایی هورمون در طی بهار )انتهای اسفند تا خرداد( و پاییز chiloscyllium arabicumدر ماهی گربه کوسه عربی خلیج فارس iiو گنادوتروپین iگنادوتروپین شد. پس از آوری از خور بحرکان واقع در شمال خلیج فارس جمع c. arabicumقطعه ماهی 60)شهریور تا آذر( صورت پذیرفت. در این راستا، بتا استرادیول، -17های های خونی سانتریفوژ شده و سطوح هورمونان انجام شد. نمونهسنجی، خونگیری از ماهیزیستبیهوش نمودن و انجام و برداشتها های بافتی از تخمدان آنگیری شد. پس از خونگیری، ماهیان تشریح شده و نمونهاندازه iiو گنادوتروپین i، گنادوتروپین پروژسترون و ائوزین هماتوکسیلینشناسی تهیه و با استفاده های معمول بافت. مقاطع بافتی با استفاده از روشندساعت تثبیت گردید 48مدت بوئن بهدر محلول iii)اووژنز اولیه(، مرحله ii، مرحله iصید شده در فصل بهار مشاهده شد: مرحله چهار مرحله تکاملی مختلف در تخمدان ماهیان آمیزی شدند. زنگ )اووژنز نهایی(. تنها سه مرحله اول تکاملی در تخمدان ماهیان صید شده در پاییز مشاهده شدند. سطوح پالسمایی iv)اووژنز میانی( و مرحله ویژه اواخر اسفند تا اوایل خرداد( فصل فصل بهار )بهبه براساس نتایجبود. ترهای مورد مطالعه حاضر در ماهیان صید شده در فصل بهار بیشهورمون باشد.گونه در خلیج فارس میتخمریزی این گنادوتروپین. پروژسترون، استرادیول، بتا-17 تخمدان، ،عربی کوسه گربه :کلمات کلیدی int. j. aquat. biol. (2015) 3(2): 119-128 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article population dynamic parameters of the highly endemic fish, alburnoides qanati coad and bogustkaya 2009, (teleostei: cyprinidae) in the kor river basin, iran mohadeseh sadat tahami1, hamid reza esmaeili*1, mohsen safaie2 1 1ichthyology lab., department of biology, college of science, shiraz university, shiraz, iran, 71454. 2fisheries department, hormozgan university, bandar abbas, iran. article history: received 25 january 2015 accepted 17 march 2015 available online 2 5 april 2015 keywords: asymptotic length growth coefficient growth performance index management conservation abstract: the present study provides the data on the population parameters of an endemic qanat spirlin, alburnoides qanati from the endorheic kor river basin of iran to assess the stock status of this cyprinid fish species, which is highly important from management and conservation points of views. fish specimens (387) were collected from moshkan stream, kor river basin, iran in 2011-2012. asymptotic length (l∞) and growth coefficient (k) were estimated at 123.9 mm and 0.31/year for females and 93 mm and 0.49/year for males, respectively. growth performance index (ø') was calculated as 8.47 and 8.35 for female and male specimens, respectively. total mortality (z) of females (1.56/year) was higher than males (1.14/year) whereas natural mortality (m) of female specimens (0.44/year) was lower than male specimens (0.65/year). data on growth and mortality parameters and also length-weight relationship of a. qanati revealed significant differences with corresponding data from other alburnoides species from iran and other countries, which could be attributed to habitat’s differences and specific species characteristics. introduction alburnoides bipunctatus (bloch, 1782) was the name applied to most populations of spirlins (riffle minnows or tailor fish) across europe and the middle east from france north of the alps eastwards to the black, caspian and aral sea basins (coad and bogutskaya, 2012) and reportedly to be one of the abundant and widespread groups in iranian endorheic and exoreic basins (esmaeili et al., 2010). however, ongoing research was revealed a greater diversity and presence of at least seven species in different basins of iran, including the caspian sea, lake orumiyeh (urmia), tedzhen river, kavir, namak lake, tigris river, persian gulf drainage and kor river basins (bogutskaya and coad 2009, coad and bogutskaya, 2009, 2012; esmaeili et al., 2010, 2014a) of which the kor river populations have been assigned to alburnoides qanati, qanat * corresponding author: hamid reza esmaeili e-mail address: esmaeili@susc.ac.ir tailor fish by coad and bogustkaya (2009). members of the genus alburnoides are lithophilic and rheophilic fishes, which inhabit in barbell and grayling zones and spawn on gravel and rubble (breitenstein and kirchhofer, 2000). the spirlins are very sensitive to human activities and levels of dissolved oxygen. having low tolerance to water polluted by industrial, agriculture or urban wastes makes these cyprinid fishes a good biological indicator of the environment quality (čihăr, 1999). in european waters, spirlins are extremely threatened and nearly close to extinction because of this sensitivity (kirchhofer, 1997; lusk et al., 1998). the freshwater fishes of iran are also faced to recent severe droughts, climate change, pollutions, introduction of exotic fishes and anthropogenic impacts, and as a consequence, many fish populations have been intensively affected 120 int. j. aquat. biol. (2015) 3(2): 119-128 especially sensitive fishes, like the spirlins (esmaeili et al., 2014a, b). hence, there is a constant need for an increase in knowledge on different aspects of biology of these fishes, including estimating growth parameters as an indicator of fish health and habitat quality to implant effective management and conservation measures. however, many of these aspects on a. qanati have not been documented in iran, partly because of narrow endemic range and no commercial importance due to its slow growth rate and small size. therefore, due to the information scarcity on the population dynamics of a. qanati, coupled with the need to provide much-needed scientific data for the management and rational exploitation of this valuable resource, the present study was undertaken to evaluate and to estimate the growth characteristics (growth parameters) of this species to assess the stock status of this endemic fish species from a spring-stream system in kor river basin of iran for the first time. material and methods the samples were collected from moshkan springstream system in kor river basin located at n 30˚36'16.9'' and e 52˚56'40.1''. sampling was performed monthly from march 2011 to april 2012 by electrofishing device. samples were fixed in 10% formalin solution and transported to the laboratory. some morphological characters, including: total length (tl), fork length (fl), standard length (sl) and maximum and minimum body width were measured to the nearest 0.01 mm and fish weight (gr) to the nearest 0.1 gr. sexes were determined by investigation of the gonads. the relationship between the length and weight were determined by fitting the data to a potential relationship in the form of: w = 𝛼lb, where w is the fish weight; l, fish length; and 𝛼 and b are the parameters to be estimated, with b being the coefficient of allometry based on the test given by pauly (1980). prior to regression analyses, log–log plots of length and weight values were performed for visual inspection of outliers (froese, 2006). one-way analysis of variance (anova) was used to test significant relationship between length and weight. to calculate growth performance and measure the back-calculation of growth in length, data were put in to the fisatii software. the von bertalanffy growth function was used to study the overall growth performance using the values of growth in length and phi-prime (ø') (bertalanffy, 1934; sparre and venema, 1992), as follows: 𝐿𝑡 = 𝐿∞(1-𝑒 −𝑘(𝑡−𝑡0)) ∅′ = 𝑙𝑛𝐾 + 𝑙𝑛𝐿∞ where lt is total length at age t, l∞ is the ultimate total length that an average fish should achieve if it continues to live and growth, k is the growth coefficient that determines how fast the fish approaches to l∞, t0 is hypothetical age lt=0, and ø' is overall growth performance. estimations of the mean total mortality rate (z) was obtained from using length converted catch curve analysis. natural mortality (m) was calculated using the equation of pauly (1980): 𝐿𝑜𝑔10𝑀 = 0.0066 − 0.279𝐿𝑜𝑔10𝐿∞ + 0.6543𝐿𝑜𝑔10𝐾 + 0.4634𝐿𝑜𝑔10𝑇 where, m is the natural mortality; l∞ is the asymptotic length; k is the growth co-efficient of the von bertalanffy growth function (vbgf) and t is the mean annual water temperature (°c) of sampling site. mortality rate was calculated by length converted catch curve method (pauly, 1980): 𝐹 = 𝑍 − 𝑀 , 𝐸 = 𝐹 𝑍⁄ = 𝐹 𝐹 + 𝑀⁄ where z is total mortality, f is fishing mortality, e is exploitation coefficient and m is natural mortality. results the descriptive statistics on a. qanati specimens, collected from moshkan spring-stream, kor river basin are presented in table 1. minimum and maximum total length (tl) for female and male specimens were 24.76 and 118.66 and 24.88 and 87.07, respectively (table 1). one-way analysis of variance (anova, p<0.05) showed a high significant relationship between length and weight of a. qanati in both sexes and the estimates of the 121 tahami et al./ population dynamics of alburnoides qanati parameter b, varied between 3.30 and 3.45 for females and 3.32 and 3.44 for males with high r2 values of 0.97 to 0.98 for both sexes (table 1, figs. 1a, b). the parameters of growth including growth coefficient (k), asymptotic length (l∞), overall growth performance (ø'), and total, fishing, natural and exploitation mortality (z, f, m, e) for females and males of a. qanati are given in table 2 and von bertalanffy growth curves are shown in figure 2. it can be realized that infinity length (l∞) of females is greater than males (123.9 vs. 93 mm, respectively) and value of k parameter for females is lower than length parameter (mm) sex min max b a r2 n tl female 24.76 118.66 3.45 0.0033 0.98 209 male 24.88 87.07 3.44 0.0033 0.98 155 fl female 23.00 110.1 3.35 0.0044 0.97 196 male 23.42 81.86 3.32 0.0047 0.97 141 sl female 21.02 102.96 3.30 0.0055 0.98 219 male 20.76 75.06 3.35 0.0047 0.98 168 tl, total length; fl, fork length; sl, standard length; b, regression slope; α, intercept; r2, coefficient of determination; n, number of specimens. table 1. descriptive statistics and estimated parameters of the length-weight relationships (lwrs) for females and males of alburnoides qanati collected from kor river basin, during 2011-2012. figure 1. lengthweight relationship of alburnoides qanati from kor river basin, iran. a c b 122 int. j. aquat. biol. (2015) 3(2): 119-128 males (0.31 vs. 0.49, respectively) (table 2). discussion the ranges of a. qanati length obtained in this study is in the rages reported for other alburnoides species from different regions. it indicates that females are almost larger than males (table 3). this attribute could be interpreted as an inter-population pattern related to nature of water body (river, stream, spring, qanat), different habitat quality, growth rate and some intrinsic factors. length-weight relationships (lwrs) showed highly significant positive relationships for a. qanati as it can be seen in other reports of this genus (table 4) in the range of 2.5-3.6 (treer et al., 2000; patimar et al., 2012; tabatabaei et al., 2014). the estimation of b parameter is remained within the expected range of 2.5–4 reported by tesch (1971). the upper limit showing deep body form and that fish getting more weight as its length increases. the reasons for the variation of b in the different regions are reported to be due to seasonal fluctuations in environmental parameters, growth parameters l∞(mm) k ø' z m f e female 123.9 0.31 8.47 1.56 0.44 1.12 0.72 male 93 0.49 8.35 1.14 0.65 0.49 0.43 von bertalanffy growth parameters (l∞, k,), overall growth performance (ø'), total mortality (z), fishing mortality (f), natural mortality (m) and exploitation mortality (e). table 2. growth parameters for females and males of alburnoides qanati from kor river basin, iran. figure 2. von bertalanffy growth curves of alburnoides qanati (a., female; b. male). 123 tahami et al./ population dynamics of alburnoides qanati physiological conditions of the fish at the time of collection, sex, gonad development, food availability and quality in habitat, diet, stomach fullness, health and the preservation techniques of the samples (tesch, 1971; esmaeili, 2001; esmaeili and ebrahimi, 2006; esmaeili et al., 2014c). fitting the von bertalanffy growth formula (vbgf) to back-calculated lengths resulted in the estimation of higher values of l-infinity than the maximum observed total lengths for both sexes. this length is seen as a capacity for growth (bagenal and tesch, 1978) and inter-sex differences in this parameter of the vbgf correspond to different growth rates of the sexes. on the other hand, a different vbgf can be a result of resource allocation between growth and reproduction. therefore, differences between sexes in the vbgf parameters could also reflect differences in the reproductive effort of the fish (see bagenal and tesch, 1978; patimar et al., 2012). it is theoretically admitted that growth parameters l∞ and k are negatively correlated (gaertner et al., 2008). the parameters of von bertalanffy’s equation obtained for linear growth of a. qanati confirm the theoretical assumption explained in raikova-petrova et al. (2011) that the smaller (l∞), shows a faster growth rate (k) which is remarkably exhibited in male and female populations of a. qanati. the k parameter of male specimens is higher than females (0.49 vs. 0.31, respectively) revealing that the male grows rapidly initially and approaches its asymptotic length (l∞=93 mm) earlier in life. the same as other species of the family cyprinidae, the females of a. qanati were found to have reasonably larger l∞ (123.9 vs. 93 mm, respectively) and smaller kvalues than males (0.31 vs.0.49, respectively). a comparison between estimates of von bertalanffy species sex tl age country reference a.b. f 10.3 6+ velika morava river, former yugoslavia soric and ilic (1985) a.b. f 12.53 6+ oltu stream, coruh basin, turkey yıldırım et al. (1999) a.b. u 11.72 --rudava river, slovakia siryova (2004) a.b. f 11.0 4+ sava river, croatia treer et al. (2006) a.b. u 13.0 (sl) --european freshwater systems kottelat and freyhof (2007) a.b. u 16.0 3+ azerbaijan abdurahmanov (1962) a.b. u 12.5 --madarsu stream, national park of golestan, northern iran akbaripasand (1999) a.e. u 15.0 --sardabroud river, northern iran abdoli (2000) a.e. m 9.5 4+ zarrin-gol river, northern iran patimar and dowlati (2007) a.e. f 11.0 4+ zarrin-gol river, northern iran patimar and dowlati (2007) a.e. u 14.0 6+ rivers of northern iran abdoli and naderi (2009) a.e. m 11.0 4+ qanat of uzineh, northern iran patimar et al. (2012) a.e. f 11.1 4+ qanat of uzineh, northern iran patimar et al. (2012) a.n. a.n. a.n. a.n. a.n. a.n. a.e. a.e. m f m f m f m f 6.06* 7.45* 6.49* 6.91* 8.41* 8.62* 8.01* 8.70* gharachai, namk basin, iran gharachai, namk basin, iran jajrud, namak basin, iran jajrud, namak basin, iran cheshme ali, kavir basin, iran cheshme ali, kavir basin, iran tajan river, caspian basin, iran tajan river, caspian basin, iran tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) a.q. a.q. m f 8.71 11.87 kor river basin, iran kor river basin, iran present study present study table 3. maximum observed length (tl, cm) and age (years) for different alburnoides species in the european region and iran. a.b., a. bipunctatus; a.e., a. eichwaldii; a.n., a. namaki; a.q, a. qanati; f, female; m, male; u, unsexed; * mean tl. 124 int. j. aquat. biol. (2015) 3(2): 119-128 length-at-age growth parameters for different species and populations of spirlins (skorva, 1972; breitenstein and kirchhofer, 2000; treer et al., 2000; treer et al., 2006; raikova-petrova et al., 2011; seifali et al., 2012; patimar et al., 2012; tabatabaei et al., 2014 and present study) reveals that these parameters vary for different populations, species at different water bodies (e.g. qanat, stream, river) and different distribution ranges (table 5). the l∞ and k-values ranges from 93 to 123.9 and 0.31 to 0.49, respectively. many studies have revealed that under different environmental conditions, biological features such as life span (abdoli et al., 2007; mann et al., 1984; ricker, 1975), age at maturity (lobón‐ cerviá et al., 1996), age structure and growth rate (abdoli et al., 2007; kamal et al., 2009; patimar et al., 2012b; basilone et al., 2004; naddafi et al., 2005; tabatabaei et al., 2014), the maximum length (l∞) (basilone et al., 2004; kamal et al., 2009; naddafi et al., 2005; tabatabaei et al., 2014) can change. for example, according to saifali et al. (2012), population parameters for south caspian spirlin (a. eichwaldii) are as follows: asymptotic length (l∞) =104.48 mm; growth co-efficient (k/year)= 1.19; natural mortality (m/year)= 0.97; fishing mortality (f/year)= 2.43; total mortality (z/year) =3.4 and exploitation level (e)= 0.71. in the present work, ø' was estimated to be 3.86 and 3.75 for female and male (based on the l∞ of 9.3 cm and 12.39 cm) specimens of a. qanati, respectively, which is less than other spirlins (table 6). according to pauly (1979), phi prime values are very similar within related taxa and have narrow normal distributions. however, similarity of this index in species area sex length α b reference a.b. dobra river, croatia c tl 0.0059 3.2245 treer et al. (2000) a.b. bednja river, croatia c tl 0.0150 2.7970 treer et al. (2000) a.b. middle korana river, croatia c tl 0.0088 3.1043 treer et al. (2000) a.b. lower korana river, croatia c tl 0.0030 3.5567 treer et al. (2000) a.b. sava river, croatia c tl 0.0044 3.4032 treer et al. (2000) a.b. a.b. a.b. ҁoruh river, turkey ҁoruh river, turkey ҁoruh river, turkey c f m fl fl fl 0.0249 0.0375 0.0166 2.79 2.62 2.95 torcu-koҁ et al. (2006) torcu-koҁ et al. (2006) torcu-koҁ et al. (2006) a.b. sava river, croatia c tl 0.0083 3.025 treer et al. (2006) a.b. seyhan dam lake, turkey c tl 0.0028 2.72 ergu ̈den and goksu (2009) a.b. emajõgi river basin, estonia c sl 0.0103 3.251 www.fishbase.org a.e. a.e. zarrin-gol river, northern iran zarrin-gol river, northern iran m f tl tl 0.0054 0.0088 2.59 2.52 patimar and dowlati (2007) patimar and dowlati (2007) a.e. a.e. a.e. qanat of uzineh, northern iran qanat of uzineh, northern iran qanat of uzineh, northern iran m f p tl tl tl 0.0068 0.0079 0.0072 3.2559 3.2067 3.2387 patimar et al. (2012) patimar et al. (2012) patimar et al. (2012) a.q. a.q. a.q. a.q. a.q. a.q. a.e. a.e. gharachai, namk basin, iran gharachai, namk basin, iran jajrud, namak basin, iran jajrud, namak basin, iran cheshme ali, kavir basin, iran cheshme ali, kavir basin, iran tajan river, caspian basin, iran tajan river, caspian basin, iran f m f m f m f m tl tl tl tl tl tl tl tl 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 2.87 3.05 3.26 3.20 2.77 3.05 3.26 3.30 tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) a.q. a.q. kor river basin, iran kor river basin, iran f m tl tl 0.0033 0.0033 3.45 3.44 present study present study * small letters indicate comparison of column and large letters indicate comparison of row. table 4. parameters of the length-weight relationship of spirlins, alburnoides species. a.b., a. bipunctatus; a.e., a. eichwaldii; a.n., a. namaki; a.q., a. qanati; f, female; m, male; c, combined sex; p, population. 125 tahami et al./ population dynamics of alburnoides qanati northern populations of alburnoides (ex. south caspian sea spirlin and the european populations) and its difference with a. qanati may reflect the distinctiveness of northern and southern populations of alburnoides. mortality parameters are higher for caspian spirlin than a. qanati from kor basin; it can be due to more sympatric predator species and over-fishing activities in caspian basin. being small spring, moshkan has no predatory fish, and a. qanati and species location sex l∞ (mm) k (year-1) t0 (year) reference a.b. dunajec river, czechoslovakia u 20.1 0.15 --skora (1972) a.b. turiec river, former czechoslovakia u 15.6 0.28 --bastl et al. (1975) a.b. radimna river, romania u 14.4 0.30 --papadopol and cristofor (1980) a.b. a.b. a.b. a.b. a.b. a.b. dobra river, croatia bednja river, croatia middle korana river, croatia lower korana river, croatia sava river, croatia sava river, croatia u u u u u u 20.5 15.5 15.1 17.7 11.5 12.0 0.16 0.33 0.28 0.19 0.59 0.59 –1.38 –0.42 –0.86 –1.47 –0.47 –0.14 treer et al. (2000) treer et al. (2000) treer et al. (2000) treer et al. (2000) treer et al. (2000) treer et al. (2006) a.e. a.e. zarrin-gol river, northern iran zarrin-gol river, northern iran m f 99.64 107.23 0.51 0.55 –0.715 –0.548 patimar and dowlati (2007) patimar and dowlati (2007) a.e. a.e. a.e. qanat of uzineh, northern iran qanat of uzineh, northern iran qanat of uzineh, northern iran m f u 140.7 153.7 148.3 0.27 0.23 0.24 –0.92 –1.08 –1.04 patimar et al. (2012) patimar et al. (2012) patimar et al. (2012) a.e. kesselian stream, caspian sea, iran u 104.5 1.19 --seifali et al. (2012) a.n. a.n. a.n. a.e. gharachai, namk basin, iran jajrud, namak basin, iran cheshme ali, kavir basin, iran tajan river, caspian basin, iran u u u u 122.43 112.66 120 123.01 0.27 0.54 0.29 0.29 –0.76 0.18 –1 –1 tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) tabatabaei et al. (2014) a.q. a.q. kor river basin, iran kor river basin, iran f m 123.9 93 0.31 0.49 -- -- present study present study table 5. estimates of von bertalanffy length-at-age growth parameters for populations of different alburnoides species in the distribution area (iran and european regions). a.b., a. bipunctatus; a.e., a. eichwaldii; a.n., a. namaki; a.q, a. qanati;, f, female; m, male; u, unsexed. locality species ø′ reference turiec ,slovenian a. bipunctatus 4.22 bastl et al. (1975) radimna, slovenian a. bipunctatus 4.13 papadopol and cristofor (1980) croatianslovenian a. bipunctatus 4.17 treer et al. (2000) caspian sea, iran alburnoides sp. 4.87 saifali et al. (2012) kor river, iran a. qanati (f) 3.86 present study kor river, iran a. qanati (m) 3.75 present study table 6. phi prime (ø′) values of different species of alburnoides from europe and iran. 126 int. j. aquat. biol. (2015) 3(2): 119-128 oxynemacheilus persa are the only fish species inhabit in this stream. it also has been reported that the spirlin population reflects the changes in habitats (jurajda et al., 1996); therefore, water dam construction can critically endanger this species (lusk, 1995) which can be considered for all species of genus alburnoides including a. qanati species (kirchhofer, 1997; lusk et al., 1998; treer et al., 2006). based on the available data, it can be concluded that many factors affect growth parameters of fish populations. the commonly proposed explanations include the effects of changes in environment conditions, (most notably temperature and food availability), density-dependent effects, eutrophication, habitat disruption and specific species characteristics. these factors can operate singly or in combination and have positive or negative effects on growth parameters. according to ma et al. (2010), the slow-growing and long-lived fish tend to be, particularly, vulnerable to excessive mortality and rapid stock collapse and population turnover may be lower than expected recovery. study of these parameters for the endemic fish, a. qanati is highly recommended. acknowledgments we wish to thank r. khaefi, b. parsi, g. sayadzadeh and s. mirghiasi for their valuable helps with the fish collections and shiraz university for the financial support. references abdoli a., pont d., sagnes p. (2007). intrabasin variations in age and growth of bullhead: the effects of temperature. journal of fish biology, 70: 1224-1238. abdurahmanov y.a. (1962). ryby presnykh vod azerbidjana. baku. 224 pp. in azerbaijani. akbaripasand a. (1999). ecological investigation of fishes of gorganroud river in the national park of golestan. msc thesis, tarbiat modarres university, tehran, pp: 114. basilone g., guisande c., patti b., mazzola s., cuttitta a., bonanno a., kallianiotis a. 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(2013) 1(4): 150-157 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article sialic acid specific lectins from episesarma tetragonum (decapoda, grapsidae): isolation, purification and characterization r. viswambari devi*,1m. r. basil rose, p. d. mercy department of zoology, holy cross college, nagercoil-629 004, tamil nadu, india. article history: received 18 may 2013 accepted 11 july 2013 available online 2 0 august 2013 keywords: sialic acid glycoproteins affinity purification hemagglutination abstract: two sialic acid specific lectins episesarma tetragonum agglutinin–1 and 2 were purified from the hemolymph of the mangrove crab, episesarma tetragonum. the major lectin was purified using cnbr-activated sepharose 4b conjugated to fetuin. n-acetyl glucosamine containing buffer was used for elution. the hemagglutination activity of purified lectin was inhibited by glycoproteins containing siaα, 2-3galβ, 1-4 glcnac linkages. on sds-page, the molecular weight of calcium dependent lectin was observed to be 70 kda. lectin had the maximum activity at a wide range of ph (6.5 – 9.5) and temperature (0 40 °c). the physicochemical characteristics of the minor agglutinin showed that its hemagglutinating activity was calcium dependent, optimum at ph 8 – 9.5 and temperature 0 – 37 °c. the only potent inhibitor of minor lectin was bovine submaxillary mucin. an attempt was also made to purify minor lectin by affinity chromatography using bovine submaxillary mucin coupled to cnbr-activated sepharose 4b column. the lectin was eluted with elution buffer containing ethylene diamine tetra acetate. strong inhibition of purified minor lectin by bovine submaxillary mucin and non-inhibitory action of de-o-acetylated bovine submaxillary mucin suggested that the lectin was o-acetyl sialic acid specific. introduction invertebrates lack an adaptive immune system, but have developed efficient innate immune systems to defend themselves against foreign materials. molecular structures and functions of various defense components that participate in immune processes are being discovered. new molecules such as fibrinogen-related proteins (freps) are being uncovered that might have the potential to recognize and attack specific pathogens, while the roles of better studied molecules continue to expand. this challenges the idea that invertebrates are adequately served by broad-spectrum pathogen recognition proteins. lectins are one such protein that is involved in defense and various biological phenomena. lectins may recognize a part of a sugar, a whole * corresponding author: r. viswambari devi e-mail address: viswambaridevi@gmail.com tel: +919941635133 sugar, their glycosidic linkages or a sequence of sugars. this property of lectins has moved their efficacy to humans in various biological applications (devi et al., 2010). among invertebrates, arthropods are the major source of such sialic acid specific lectins. it is interesting to note that some of the agglutinins of arthropods are capable of binding specifically to sialic acids, the family of sugars not synthesized by protostomian invertebrates (warren, 1963). sialic acids discriminating against agglutinins of arthropods will definitely be of immense value in identifying sialylated tumor-associated antigens. careful search for these may provide lectins with unique specificity for different kinds of sialic acids and their glycosidic linkages. therefore in the present study, our aim is to isolate and purify 151 devi et al./ int. j. aquat. biol. (2013) 1(4): 150-157 significant sialic acid specific lectins from the hemolymph of episesarma tetragonum, which may be added to the library of potential diagnostic tools for identifying sialyl epitopes in pathogenic bacteria, and malignant tumor cells. materials and methods collection of samples: the mangrove crab, episesarma tetragonum was collected for the study from the mangrove and fresh water regions of manakudy, kanyakumari district, tamil nadu. hemolymph was collected from fresh, uninjured non-autotomized crabs. the hemolymph of small crabs (5-15 g) was drawn using a sterile 1.0 ml syringe and 22 gauge needles through the arthrodial membrane at the base of third walking leg. for large crabs (20-60 g), after cutting the dactylus, the hemolymph was allowed to bleed directly into centrifuge tubes placed on ice and, was allowed to clot for 15 minutes. the hemolymph was then centrifuged to separate the serum which was stored at -20°c. hemagglutination assay: the mammalian blood samples were collected directly in sterile modified alsevier’s medium. before use, the erythrocyte types were suspended and washed three times with tris buffered saline and resuspended in the same buffer to give 1.5% v/v cell suspension for agglutination assays. hemagglutination titer is defined as the reciprocal of the highest dilution of the test sample showing visual agglutination of the test erythrocytes. cross adsorption assay: the adsorption assays carried out using dog, horse and rat erythrocytes in this study were essentially the same as those carried out by mercy and ravindranath (1992). hemagglutination inhibition (hai) assay was performed to test the ability of various glycoproteins and sugars (mono and oligosaccharides) to inhibit agglutination. hemagglutination inhibition titer was reported as the reciprocal of the lowest dilution of inhibitors giving complete inhibition of agglutination after 1 hour. ha assays to determine physicochemical parameters: the physicochemical properties were determined by hemagglutination assays with serum samples under conditions of varying ph, temperature, bivalent cation of diverse concentration, edta and some chemical agents. affinity purification of episesarma tetragonum agglutinin–1 (eta-1): clarified serum (70 ml) was applied to 3.5 ml of fetuin coupled to cyanogen bromide activated sepharose 4b in an econo column (bio-rad) previously equilibrated with tbs at 4°c. the elution of lectin was done with elution buffer that contained 100 mm glcnac and collected 1 ml fractions on ice in polypropylene tubes containing 10 μl of 100 mm calcium chloride at a rate of 0.3 ml/minute. the fractions were vortexed immediately after collection and kept on ice. fractions containing lectin were pooled on the same day and dialyzed against 1 mm cacl2 , at 4°c for 3 hours and the dialysate was then aliquoted, lyophilized (speed-vac, sawant), and stored at -20°c. sialidase treatment of dog erythrocytes: a reaction mixture (total 1.0 ml) containing 10% washed dog erythrocytes in pbs-bsa (ph 7.0) and 140 milliunits of neurminidase of clostridium perfringens (sigma type x) was incubated at 37°c for 4 hours. neuraminidase treated cells were washed with pbs-bsa three times and pelleted by low speed centrifugation. ha assays were performed against the desialylated erythrocytes using purified lectin, eta-1. sialidase treatment of sialoglycoprotein: asialo fetuin was prepared by incubating 2 mg of glycoprotein (fetuin) with 0.1 unit of clostridium perfringens sialidase (sigma type x) in 400 μl of 5 mm acetate buffer, ph 5.5 for 2 hours at 37°c. as a control, fetuin was treated similarly without sialidase. hai assay was performed with purified lectin for sialidase treated and untreated fetuin against 1.5% dog erythrocyte suspension. polyacrylamide gel electrophoresis: sdspolyacrylamide 12.5% slab gel electrophoresis was performed according to laemmli (1970). purification of eta-2: clarified serum (50 ml) was applied to 1.5 ml of bsm coupled to cyanogen bromide activated sepharose 4b in an econo column 151 152 devi et al./ int. j. aquat. biol. (2013) 1(4): 150-157 (bio-rad) previously equilibrated with tbs at 4°c. the elution of lectin was done with elution buffer that contained 10 mm edta and collected 1 ml fractions on ice in polypropylene tubes containing 10 μl of 100 mm calcium chloride at a rate of 0.3 ml/minute. the fractions were vortexed, dialyzed, lyophilized and stored at -20°c. de-o-acetylated preparation of glycoproteins: deo-acetylation of sialic acids was performed following the procedures of sarris and palade (1979) and schauer (1982). results identification of agglutinins of different specificity: the presence of naturally occurring agglutinins in the serum of e. tetragonum was detected using a panel of 15 mammalian erythrocyte types. ha titer with the different species of erythrocytes can be graded as follows: dog > albino rat = mice = horse > buffalo = cat > human b = o > a = rabbit = goat = donkey. the ha titer was high (ha = 64) with dog erythrocytes, was moderate (ha = 16) with albino rat, mice and horse erythrocytes followed by cat and buffalo (ha = 8). results of cross adsorption studies showed that serum adsorbed with dog and rat erythrocytes completely lost the agglutinating activity towards all the three erythrocytes tested. but the serum adsorbed with horse erythrocytes retained the agglutinating activity with dog erythrocytes even after four adsorptions and thus indicating the presence of heteroagglutinins, namely e. tetragonum agglutinin-1 (eta-1) specific for dog erythrocytes and e. tetragonum agglutinin-2 (eta-2) specific for horse erythrocytes. purification of eta-1: the specific activity of the purified lectin increased about 1141 folds from 673 to 7.68 x 105 ha unit/mg of protein (table 1, fig. 1). an analysis of purified eta-1 on sds-page in step sample volume (ml) protein (mg) total activity (ha units) specific activity (ha units/mg) purification fold 1 crude 70 530 1.792 x 105 338 1 2 clarified sample 30 57 3.84 x 104 673 2 3 purified using formalinized dog rbc 10 1.2 5.12 x 104 4.26 x 104 63 4 purified using fetuin – agarose affinity column 6 0.04 3 x 104 7.68 x 105 1141 table 1. purification of eta-1 from the native hemolymph of e. tetragonum. glycoproteins (n=5) nature of sialic acid hai minimum concentration for inhibition µg/ml relative inhibitory potency% fetuin transferrin porcine thyroglobulin -acid glycoprotein psm apotransferrin bovine thyroglobulin bsm lactoferrin neugc neugc neugc neugc neugc neugc neuac/neugc neuac 256 128 128 16 8 4 4 0 0 19.531 39.062 39.062 312.5 625 1250 1250 0 0 100 50 50 6.25 3.125 1.5625 1.5625 0 0 table 2. hai assay of eta-1 by sialoglycoproteins. figure 1. fetuin affinity elution profile-purification of eta-1. 153 devi et al./ int. j. aquat. biol. (2013) 1(4): 150-157 the presence of 2-mercaptoethanol revealed a major band at molecular weight of 70 kda (fig. 2). physicochemical properties of eta-1: the ha activity of the agglutinin was sensitive to ph and temperature. the ha was stable between ph 6.5 9.5 and at temperature ranging from 10 – 40°c. among the cations tested, calcium ions did not have any effect on ha titer, however magnesium at 10 – 0.1 mm concentration reduced the ha titer to 32 while at 100 mm concentration gave the normal ha titer. though calcium ions were not required for ha, the metal ion chelator – edta showed a different action towards the ha activity. at very low concentrations (1 – 0.01 mm) the ha activity decreased (ha = 32). concentrations ranging from 1 – 5 mm, the ha activity increased one fold from the normal ha (128) and at concentrations from 10 20 mm there was sudden decrease in ha activity, after which the ha activity was completely lost. the agglutinating activity was completely inhibited by chloroform while the activity was greatly inhibited by incubation with denaturing agents such as hcl and naoh. binding specificity of purified eta-1: with a view to ascertain the nature of the binding specificity of purified lectin, hemagglutination assays were table 3. effect of neuraminidase treatment of glycoprotein on hai of eta-1. sugars (n=5) hai minimum concentration for inhibition µg/ml relative inhibitory potency% glcnac galactose neugc maltose lactose mannac galnac neuac trehalose 64 64 32 16 16 8 0 0 0 1.5625 1.5625 3.125 6.25 6.25 12.5 0 0 0 100 100 50 25 25 12.5 0 0 0 table 4. inhibition of eta-1 hemagglutination by various sugars. step sample volume (ml) protein (mg) total activity (ha units) specific activity (ha units/mg) purification fold 1 2 3 crude clarified sample purified 125 50 2 3125 250 0.07 1.6 x 105 4 x103 160 51.2 16 2000 1 3 125 table 5. purification of eta-2 from the native hemolymph of e. tetragonum. glycoprotein hai titer minimum conc. required for inhibition (µg /ml) relative inhibitory potency (%) bsm crude 512 9.7 100 purified 8192 0.6103 100 table 6. hemagglutination inhibition titer of crude and purified eta-2 by bsm. glycoprotein treatment hai titer with dog erythrocytes fetuin (without sialidase) fetuin + sialidase (3 hours) fetuin + sialidase (6 hours) fetuin + sialidase (12 hours) fetuin + sialidase (18 hours) fetuin + sialidase (20 hours) 256 128 8 8 8 8 153 154 devi et al./ int. j. aquat. biol. (2013) 1(4): 150-157 performed with a variety of sialoglycoproteins. the inhibitory potency of eta-1 was as follows: fetuin > porcine thyroglobulin = transferrin > α-acid glycoprotein > psm > apotransferrin = bovine thyroglobulin (table 2). the purified eta-1 showed a remarkable inhibitory potency with fetuin containing sialic acids with α, 2-3 linkages. on the other hand, bsm and lactoferrin failed to inhibit the ha activity of eta-1. to further define the possible role of sialic acids as potent inhibitor of lectin, the sialoglycoprotein, fetuin was enzymatically modified and its derivative was examined for hai. sialidase treatment of fetuin reduced its inhibitory properties at 20 hours (table 3). in order to find out if eta-1 was neuac or neugc specific, free sialic acids neuac and neugc were tested as inhibitors of hemagglutination. neuac did not inhibit hemagglutination whereas neugc inhibited (table 4). however neugc linked glycoproteins were inhibitorier than free neugc. galactose that showed very low inhibitory potency with crude agglutinin was a potent inhibitor of purified eta-1. purification of eta-2: both the clarified serum, which had passed through the affinity matrix and the effluent collected during subsequent washing of this matrix with high salt buffer and low salt buffer, did s.no glycoprotein treatment hai titer 1 de-o-acetylation bsm untreated bsm + 0.04 n naoh, 4° c, 45 minute bsm + 0.4 n naoh, 4° c, 45 minute 8192 0 0 2 desialylation bsm (without sialidase) bsm + sialidase (3 hour) bsm + sialidase (6 hour) bsm + sialidase (12 hour) bsm + sialidase (18 hour) bsm + sialidase (20 hour 8192 2048 1024 512 512 128 table 7. hai of purified eta-2 of e. tetragonum by bsm before and after de-o-acetylation and desialylation. figure 2. sds-page of purified lectin eta-1. lane a, fetuinagarose purified lectin; lane b, formalinised dog erythrocyte adsorbed purified lectin; lane c, protein from crude serum; lane d, protein from crude serum after ultracentrifugation; lane e, standard of known molecular weight (prestained, broad range marker, bio-rad laboratories). figure 3. bsm – affinity elution profile-purification of eta-2. 155 devi et al./ int. j. aquat. biol. (2013) 1(4): 150-157 not show hemagglutination activity against horse erythrocytes. this indicated that lectin in the serum was adsorbed by the affinity matrix. when elution buffer with 10 mm edta was passed through the column, peaks at 280 nm absorbance coincided with agglutinating activity against horse erythrocytes emerged from the affinity matrix (table 5, fig. 3). physicochemical properties of eta-2: the ha was stable between ph 8 – 9.5 and at temperature ranging from 10 35°c. among the cations tested calcium ions did not have any effect on ha titer, however magnesium at 0.1 – 10 mm concentration reduced the ha titer to 16 while at 100 mm concentration gave the normal ha titer. though calcium ions were not required for ha, the metal ion chelator – edta showed a different action towards the ha activity similar to eta-1. binding specificity of eta-2: it was interesting to note that only bsm was the potent inhibitor of purified eta-2. it gave a hemagglutination inhibition (hai) titer of 8192, when compared to crude agglutinin that gave only 512 hai titer (table 6). no inhibitors that inhibited eta-1 were capable of inhibiting eta-2. base treatment, specific for hydrolysis of the o-acetyl groups of sialic acids without cleavage of peptide bonds (schauer, 1982), completely reduced the ability of bsm to inhibit hemagglutination. sialidase treatment of bsm reduced its inhibitory properties at 20 hours (table 7). discussion the serum of mangrove crab, e. tetragonum possess two naturally occurring agglutinins, the major agglutinin, eta-1 specific for dog erythrocytes and the minor agglutinin, eta-2 specific for horse erythrocytes. the heterogeneity of agglutinin in these crustaceans gives important support to the fact that, agglutinins as defense molecules for recognition of “non-self” are the part of an invertebrate immune system. eta-1 shared some common properties of other crustacean agglutinins namely ph and thermal sensitivity and calcium dependency. purified agglutinin might yield precise information on its sugar specificity and would be of great interest for biomedical applications. glycoprotein inhibition experiments provide valuable information pertaining to the sialyl oligosaccharides preferred by eta-1. the potent inhibitor of eta-1 was fetuin. fetuin, the major glycoprotein in calf serum contain carbohydrates distributed between an equal numbers of nand oglycosidically linked units. fetuin contains sialic acid α, 2-3 and sialic acid α, 2-6 in a 2:1 ratio. the second potent inhibitors were porcine thyroglobulin and bovine transferrin. porcine thyroglobulin is a major glycoprotein synthesized in the thyroid gland. the carbohydrate moiety of porcine thyroglobulin was shown to consist of a complex type (unit b-type) oligosaccharides being linked to asparagine residues (arima et al., 1972). unit b-type oligosaccharides of the glycoprotein were of triantennary and biantennary complex type. in triantennary oligosaccharides, the sialic acid residues were not localized on certain specific branches but distributed on all three branches. α, 2-3 linked sialic acid residues were exclusively located on the terminal of the branch arising from c-4 of the branching α mannose residue, whereas α, 2-6 residues occupied terminals of the other branches. the natural position of α, 2-3 linked sialic acid residue of triantennary complex type sugar chain was similar to that observed to exist in glycoprotein fetuin (nilsson et al., 1979; baenziger and fiete, 1979). but the ratio of α, 2-3 linked sialic acid to α, 2-6 linked sialic acid in triantennary complex type of thyroglobulin was 1:2 and this explained the higher reactivity of fetuin to eta-1 than thyroglobulin. clear information pertaining to sialyl linkages of bovine transferrin and α-acid glycoprotein are not available. they are also known to possess sialic acid α, 2-3 gal β1-4 glcnac β1 residues (vliegenthart et al., 1982). this corroborates the specificity of eta-1 towards sialic acid α, 2-3 gal β1-4 glcnac β1 residues. this can be further assured by the non-inhibitory potency of bsm which contains a terminal sialic acid sequence of neuac α, 2-6 galnac (gottschalk and graham, 1959; graham and gottschalk, 1960). the affinity of 155 156 devi et al./ int. j. aquat. biol. (2013) 1(4): 150-157 eta-1 to neugc was also reflected in the glycoprotein inhibition study. these investigations obviously demonstrate that eta-1 exhibits high affinity towards sialoglycoconjugates possessing terminal neugc α, 2-3 linked to penultimate galactose residues. to the best of our knowledge, eta-1 is the first known arthropodan lectin with sialic acid α, 2-3 gal β1-4 glunac-r specificity. bsm was the only potent inhibitor of eta-2. all other glycoproteins which are found to possess neugc or neuac were non inhibitory. the most common type of glycosidic linkages involving sialic acid in bsm is α-2, 6 galnac. although the terminal oligosaccharide sequence of bsm is neuac α-2, 6 galnac, a major fraction (>50%) of the sialic acid is o-acetylated (graham, 1966). since eta-2 is not inhibited by any other sugars and glycoproteins, it is of clear evidence that eta-2 has unique specificity to o-acetyl sialic acid and not to neuac α-2, 6 galnac fraction of bsm. hemagglutination inhibition tests thus revealed bsm, which contains mainly 9-o-acetyl and 8, 9-di-o-acetyl-n-acetyl neuac (schauer, 1982), as the most potent inhibitor of the agglutinin. de-o-acetylation of bsm by base treatment specifically hydrolyses o-acetyl groups of sialic acids without cleavage of peptide bonds (sarris and palade, 1979; schauer, 1982). in the present study, the inhibitory potency of bsm was completely abolished, after de-o-acetylation. this observation further reveals the specificity of eta-2 for o-acetyl sialic acid. from our studies the most important prerequisite for eta-1 binding to glycoconjugate seems to be sia(neugc)α, 2-3galβ, 1-4 glunac linkages rather than neugc per se. considering the importance of sialic acids in cell sociology, lectins which specifically recognize terminal sialic acid residues are potentially useful as analytical tools in studying the biological functions of sialoglycoconjugates. these lectins, along with monoclonal antibodies raised against sialoglycoconjugates, have been used in the detection, affinity purification, cytochemical localization and quantification of such glycoconjugates (varki, 1997). lectins that recognize linkages or modifications of sialic acid are thus indispensable as reagents in biochemical research and diagnostic analysis. carbohydrate residues of the membrane glycoproteins can be detected using lectins due to their binding specificity to carbohydrates. lectins, therefore have gained an importance in the field of cancer research (sherwani et al., 2003). it is believed that an elevated level of α, 2-3-linked sialylation increases the metastatic potentials of tumor cells (dennis et al., 1986). the increase of α, 2-3-linked sialic acids with increased branching of glycans is considered to accompany hepatocarcinoma (montreuil et al., 1997). tumor associated antigen sialyl lewis x (slx) 1 contains a sialic acid α, (2-3) galactose moiety and has been implicated in inflammation and cancer metastasis (tyrrell et al., 1991; kannagi et al., 2004). hence eta-1 that can recognize sialic acid α, 2-3 linkages can be used in detection and quantification of those glycoconjugates accompanying hepatocarcinoma, inflammations and cancer metastasis. thus there is no doubt that eta-1 is a valuable diagnostic tool for identifying the sialoconjugates in normal and malignant tissues. acknowledgements the authors express their thanks to jawaharlal nehru memorial fund, new delhi for the financial assistance aided. we also thank k. thanalakshmi, m. jayalakshmi, vinoliya josephine mary and josephine priyadarshini for their overall support to complete my research work successfully. references arima t., spiro m.j., spiro r.g. 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(2014) 2(3): 138-146 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article the effect of water temperature on food transit time and digestive enzymes activity in caspian kutum (rutilus kutum) larvae nahid gheisvandi*,1abdolmajid hajimoradloo, seyed hossein hoseinifar department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 23 december 2013 accepted 4 june 2014 available online 2 5 june 2014 keywords: digestive enzyme food transit time rutilus kutum water temperature abstract: the present study investigates the effects of water temperature on digestive enzymes activity and food transit time in caspian kutum (rutilus kutum) larvae. caspian kutum larvae (532 ± 0.05 and 543 ± 0.02 mg) were divided into two groups with three replicates and reared at different water temperature i.e. 25.6 ± 0.4°c (t1) and 18.4 ± 0.1°c (t2). at the end of the experiment, sampling of intestine was performed at 0, 1, 3, 5, 8, 16, 24 and 30 h after feeding from each treatment. in t2, food was observed until 24 h after feeding and the intestine was empty 29 h after feeding, while in t1 19 h after feeding the intestine was empty. digestive enzymes activities were higher in t2 treatment. the peaks of trypsin and alkaline phosphatase enzymes activity were found 8 h after feeding in t1, while occurred 16 h after feeding in t2. the highest chymotrypsin and alpha-amylase enzymes activity were observed 5 and 8h after feeding in t1 and t2, respectively. these results confirmed remarkable effects of temperature on food transit time and digestive enzymes activity of caspian kutum. introduction the rutilus kutum is an important commercial fish species in the caspian sea that faced a sharp decline of natural population due to overfishing, water pollution, and loss of habitat and spawning grounds (hoseinifar et al., 2014). the severe decline in natural population motivated the iranian government to launch its restocking project in 1984 by releasing the fingerlings obtained from artificial propagation to the rivers of southeastern region of caspian sea. at present, around 200 million of fry (~ 1 g) are annually released to southern rivers for stocks enhancement purposes (caspian environment. org, 2007). the passage of food in fish gut is depend on a number of factors including species, fish age/size, water temperature, food quality, and feeding frequency (temming and herrmann, 2001; specziár, 2002; wuenschel and werner, 2004). in particular, water temperature has a significant effect on food * corresponding author: nahid gheisvandi e-mail address: n.ghysvandi@yahoo.com transit time that as it can affects digestive enzymes activity and feed intake (temming and herrmann, 2001; kofuji et al., 2005). nakada (2000) reported remarkable increase of food transit time in japanese yellowtail (seriola quinqueradiata) following decrease of water temperatures. also decreased digestion rate in the same species during the cold months has been reported (kofuji et al., 2005) which results in lower apparent protein digestibility (kofuji et al., 2005). type and function of the digestive enzymes clearly affect digestive process and is a very important issue in the study of the digestive physiology (gisbert et al., 2009). moreover, efficiency of the enzyme function and digestive process is related to important factors such as sufficient time for hydrolysis of food and sufficient amount of enzymes in the gut (olsson and holmgren, 2001; uscanga et al., 2010). the first parameter is determined by measurement of the food transit time. 139 gheisvandi et al./ effect of water temperature on food transit in caspian kutum due to seasonal variation, the caspian kutum (r. kutum) is exposed to different water temperatures throughout the year, which may affects food transit time, food intake and digestive enzyme activity. therefore, the purpose of this study was investigation of the effects of water temperature on food passage time and digestive enzymes activity in caspian kutum. materials and methods experimental design and management: the present study was performed in august and december 2012 at shahid fazli aquaculture research station of gorgon university (golestan province, iran). in august and december, average water temperature of the tanks were 25.6 ± 0.4°c and 18.4 ± 0.1°c, respectively. the caspian kutum larvae were supplied from the sijaval fish farm (golestan province, iran) and acclimated to laboratory conditions for 2 weeks. during acclimation, fish were fed twice a day with a commercial formulated diet (biomar, 0.8 mm). after acclimation, a total of 360 caspian kutum including 180 specimens (532 ± 0.05 mg) in 25.6± 0.4°c (t1) and 180 specimens (543 ± 0.02 mg) in 18.4±0.1°c (t2) were used, respectively in three replicates fiberglass tanks (420 l capacity with 300 l water volume with 60 fish per tank). there were no significant differences between weights of fishes in both treatments (p>0.05). the photoperiod condition was similar in both treatments. also water quality parameters such as ph, dissolved oxygen, ammonia, nitrate and nitrite had no significant difference in two treatments (p>0.05). determination of gut transit time: food transit time was determined according to miegel et al. (2010) by visual observations of the digestive tract contents at 0, 1, 3, 5, 8, 16, 24 and 30 h post feeding. this was performed by monitoring the passage of feed and digesta through the entire digestive tract in dissected intestines. sampling and preparation of crude enzyme extract: digesta, whole gut and intestinal tissue samples were collected by dissection at different times post feeding as mentioned above. half-hour after feeding, the rearing tanks were cleaned to remove remaining food. three fish were randomly selected from each tank. the entire digestive tract (from esophagus to the anus) was removed and adipose tissues were cautiously cleaned. afterward samples were rinsed with chilled refined water and stored at −80°c for further analysis (harpaz et al., 2005; perez-jimenez et al., 2009). prior to assessment, the stored samples were partially thawed, weighed and homogenized in five volumes of 0.2 m nacl solution (w/v) using an electric homogenizer (ika t25 digital, ultra turrax model). the tip of the homogenizer was cleaned for each dissimilar homogenizing process with distilled water and wiped with tissue paper (gawlicka et al., 2000). the suspensions were centrifuged for 30 min at 10,000 g, 4°c using a refrigerated centrifuge (eppendorf 5810r) (klomklao et al., 2007). thereafter, the supernatants were softly pipetted into separate sterile centrifuge vials and frozen at −80°c until enzyme activity or protein content analysis. sample preparations were conducted at low temperature by working on ice. enzyme assays: the trypsin (ec 3.4.21.4) activity was estimated using benzoyl-dlarginine-pnitroanilide (bapna) as substrate according to erlanger et al.(1961) and absorbance was read at 410 nm using spectrophotometer (lightwave-s2000 uv/vis). chymotrypsin (ec 3.4.21.1) activity was determined after hummel (1959) with btee (nbenzoyl-l-tyrosine ethyl ester) as substrate and absorbance was read at 256 nm using spectrophotometer (lightwave-s2000 uv/vis). the activity of α-amylase (ec 3.2.1.1) was calculated using starch as the substrate according to bernfeld (1951) and absorbance was read at 540 nm using spectrophotometer (lightwave-s2000 uv/vis). alkaline phosphatase (ec 3.1.3.1) activity was assayed as previously described (bessey et al., 1946) at 37°c using 4-nitrophenyl phosphate (pnpp) as 140 int. j. aquat. biol. (2014) 2(3): 138-146 substrate and absorbance was read at 407 nm using spectrophotometer (lightwave-s2000 uv/vis). bovine serum albumin solution (bsa) was used as the standard in the determination of soluble protein (bradford, 1976). all enzymes activities were expressed as specific activity (u mg protein−1). all enzymatic assays were performed at 25 °c (except alkaline phosphatase at 37°c) in triplicate. statistical analysis: the study of food transit was performed by split-plot completely randomized design in time (5 levels: 0, 1, 3, 5, 8 h) with two treatments. also for the food location changes in the gut, the regression analysis was used. the study of digestive enzymes activity and protein was performed by split-plot completely randomized design in time (7 levels: 0, 1, 3, 5, 8, 16 and 24 h) and (8 levels: 0, 1, 3, 5, 8, 16, 24 and 30 h) in t1 and t2, respectively. when f values were significant (p<0.05), means in a treatment in different times were compared with lsd test and in two treatment in a time were compared with t-test. statistical analyses were done using spss software. results are expressed as mean ±se. results food transit time: there results of water temperature effects on food transit time are presented in figures 1-3. the results showed that the changes in water temperature significantly affected food transit time (p<0.05). the influence of the treatment, time and their interaction on food passage in the gut was significant (p<0.05). the results of the lsd test showed there was no significant difference between the food relative location in two treatments up to 3 h post feeding (p>0.05). however, at 5 and 8 h post feeding sampling significant difference was noticed (p<0.05) (fig. 1). according to the results of the figure 1. the relative location of food after feeding in the gut (t1: summer and t2: winter). bars assigned with the same letter are significantly differed (p< 0.05); values are presented as the mean ± s.e. figure 2. the passage percent of initial section of food in the intestine of caspian kutum reared in different water temperature (t1: summer and t2: winter). 141 gheisvandi et al./ effect of water temperature on food transit in caspian kutum lsd test, food reached the intestinal distal in t1 and t2, after 11.69 and 23.57 h, respectively (fig. 2). while the distal part of food, reached after 19.81 and 29.28 h (fig. 3). intestinal protein: according to table 1, soluble protein concentrations in caspian kutum in t2 was lower than that of t1 (p<0.05). the soluble protein levels increased from 0 to 1 h post feeding, decreased from 3 to 5 h post feeding and increased again from 8 to 24 h after feeding in t1. while decreased 0 to 1 h after feeding, increased from 1to 3 h after feeding, then decreased from 5 to 8 h after feeding and increased again from 16 to 30 h after feeding in t2. proteolytic enzymes: the influence of the treatment factor, time factor and their interaction on the specific activity of trypsin was significant (p<0.05). the specific trypsin activity in the experimental period is shown in table 2. although no significant difference was noticed, the enzymatic activity in t1 was lower compared to t2 (p>0.05). the peak of enzyme activity occurred at 8 and 16 h post feeding in t1 and t2, respectively and declined thereafter. the enzyme activity decreased from 0 to 1 and 0 to 3 h post feeding in t1 and t2, respectively. the effects of treatment, time and their interaction on the chymotrypsin specific activity were significant (p<0.05). table 3 represents the specific activity of chymotrypsin during the experimental period. the activity of chymotrypsin in t1 was lower than that of t2. the peak of enzyme activity occurred at 5 and 8 h post feeding in t1 and t2, respectively and declined thereafter. enzyme activity decreased from 0 to 1 and 0 to 3 h post feeding in t1 and t2, respectively. α-amylase: the analysis for amylase specific activity showed significant effect of treatment, time and their interaction (p<0.05). the specific activity of α-amylase during the experimental period is shown in table 4. the enzymatic activity of αamylase int1 was lower thant2.the peak of enzyme activity occurred at 5 and 8 h after feeding and figure 3. the passage percent of last section of food in the intestine of caspian kutum reared in different water temperature (t1: summer and t2: winter). time/protein treatment 1 treatment 2 0 a d1.74±0.09 a c1.65±0.14 1 ac 3.07±0.31 bc1.48±0.11 3 a a4.77±0.6 b ab2.49±0.25 5 a ab3.17±0.4 a bc2.04±0. 5 8 a ab4.26±0.31 b c1.89±0.4 16 a a4.36±0.08 b b2.42±0.01 24 a a4.72±0.15 b a2.857±0.06 30 a3.012±0.4 * small letters indicate comparison of column and large letters indicate comparison of row. table 1. changes in soluble protein concentration (mean ± se, n=3) post feeding in caspian kutum reared in different water temperature (t1: summer and t2: winter). 142 int. j. aquat. biol. (2014) 2(3): 138-146 declined thereafter in t1 and t2, respectively. alkaline phosphatase: the treatment factor, time factor and their interaction had significant effect on the specific activity of alkaline phosphatase (p<0.05). table 5 shows the alkaline phosphatase specific activity during the experiment. the enzymatic activity of alkaline phosphatase in t1 was lower than that of t2. the peak of enzyme activity occurred at 8 and 16 h after feeding and declined thereafter in t1 and t2, respectively. enzyme activity decreased from 0 to 1 and 0 to 3 h after feeding in t1 and t2, respectively. comparison of digestive enzymes activity: the lowest alkaline phosphatase, amylase, chymotrypsin and trypsin specific activities were observed in in t1. the amylase had the maximum enzymatic activity between all enzymes analyzed (p<0.05). in addition, among both proteases, the activity of trypsin was time/trypsin treatment 1 treatment 2 0 a b0.039±0.003 a b0.0416±0.004 1 a d0.021±0.005 a bc0.0356±0.004 3 a cd0.024±0.006 a c0.0271±0.007 5 a bc0.031±0.002 a bc0.0391±0.008 8 a a0.054±0.002 a a0.0573±0.006 16 b b0.036±0.01 a a0.0597±0.003 24 b b0.019±0.004x a b0.0419±0.003 30 b0.038±0.005 * small letters indicate comparison of column and large letters indicate comparison of row. table 2. changes in specific trypsin activity (mean ± se, n=3) post feeding in caspian kutum reared in different water temperature (t1: summer and t2: winter). time/cymotrypsin treatment 1 treatment 2 0 a bc0.81±0.05 a b0.9±0.04 1 b d0.42±0.06 a d0.65±0.02 3 a bcd0.68±0.05 a d0.62±0.05 5 a a1.21±0.49 a a1.23±0.05 8 b ab1.02±0.051 a a1.30±0.02 16 b cd 0.52±0.0 a b0.96±0.03 24 b d0.43±0.02 a c0.83±0.03 30 d0.65±0.04 * small letters indicate comparison of column and large letters indicate comparison of row. table 3. changes in specific cymotrypsin activity (mean ± se, n=3) post feeding in caspian kutum reared in different water temperature (t1: summer and t2: winter). time/ amylase treatment 1 treatment 2 0 5.75±0.767c a 8.418±0.396c a 1 7.557±2.206b a 7.187±0.2de a 3 7.925±2.814b a 7.629±0.195d a 5 10.799±0.485a a 10.631±0.309b a 8 10.271±0.931a a 11.913±0.734a a 16 7.268±0.625c a 11.718±0.242a b 24 6.792±0.402c a 7.634±0.51d a 30 6.59±0.401e * small letters indicate comparison of column and large letters indicate comparison of row. table 4. changes in specific amylase activity (mean ± se, n=3) post feeding in caspian kutum reared in different water temperature (t1: summer and t2: winter). 143 gheisvandi et al./ effect of water temperature on food transit in caspian kutum lower than that of chymotrypsin. discussion food transit time: this study demonstrates that water temperature has a significant effect on the food transit time in r. kutum. factors that affect food transit time may also affect appetite and feed intake (vinagr et al., 2007; imsland et al., 2001). in this study higher evacuation rate of the gastrointestinal tract was observed int1. this is in agreement with the results of previous studies (shimeno et al., 1993; nakada, 2000; watanabe et al., 2001), which water temperature has been shown to influence feed passage time and digestion rates. also miegel et al. (2010) reported the stomachs were almost empty in seriol alalandi at the 20 h and 36 h sampling time in summer and winter, respectively. in addition, study on japanese yellowtail (seriola quinqueradiata) showed that the stomach evacuation time was less than 12 h after feeding at water temperatures between 20 and 28°c, whereas the stomach was empty less than 48 h after feeding in winter, when water temperatures was 13 and 18°c (nakada, 2000; watanabe et al., 2001). the gut evacuation time in r. kutum increased during in lower temperature due to the slower gut motility (miegel et al., 2010). intestinal protein activities: in the present study, soluble proteins changes trend was different in the two treatments. the amount of soluble protein in t2 was lower than that of t1. this could be support the hypothesis suggested by raoofinia (2011) that reduction of soluble proteins intestinal and tissue occurs due to stress. stress causes an immediate mobilization of protein resources from the intestine, apparently for systemic use. also it has been reported in other fish species that reductions of endogenous protein before the lipid reserves are completely utilized (greene, 1926; templeman and andrews, 1956; love, 1958). therefore, the reduction of soluble protein in r. kutum in t2 due to stress-induced, decrease food intake, under nutritional stress and lack of nutrients and therefore it begins to break down protein. digestive enzyme activities: the results revealed that tendency for protease, amylase and alkaline phosphatase activities is higher in t2 due to slower gut motility at colder water temperatures or slower movement of digesta through the gastrointestinal tract, which allow enzymes within the digesta to accumulate (miegel et al., 2010). a study on the japanese yellowtail showed that the activity of stomach enzymes, trypsin and chymotrypsin, within the pyloric caeca decreased by 10% and 30%, respectively when water temperature dropped from 25°c to 16°c (kofuji et al., 2005). enzyme activity could also be higher during winter due to the lower feed intake. in winter, when fish are under nutritional stress due to the reduction in feed intake, enzymes activity may increase to compensate and maximize the nutrient digestion and absorption (miegel et al., 2010). in marine fishes, increase of digestive enzyme time/ alkaline phosphatase treatment 1 treatment 2 0 a ab1.87± 0.4 a b2.254±0.432 1 a b1.43 ±0.35 a c1.746±0.185 3 a ab2.22 ±0.21 a cd1.459±0.103 5 a ab1.84 0.36 a b2.496±0.462 8 a a2.6±0.85 a a3.453±0.176 16 b b1.73 ±0.05 a a3.504±0.182 24 b b1.58 ±0.07 a b2.679±0.315 30 d1.055±0.077 * small letters indicate comparison of column and large letters indicate comparison of row. table 5. changes in specific alkaline phosphatase activity (mean ± se, n=3) post feeding in caspian kutum reared in different water temperature (t1: summer and t2: winter). 144 int. j. aquat. biol. (2014) 2(3): 138-146 activity has been reported due to food deprivation (lamarre et al., 2004). also cara et al. (2007) reported increase of trypsin and chymotrypsin activities in sea bass (dicentrarchus labrax), following food deprivation. indeed reduced feed intake in lower temperature would be compensated by increase of enzyme activity and protein digestion capacity. the trypsin activity was also higher at lower temperatures in anarhichas minor that probably such a positive recompense mechanism renders available protein energy from food sources or internal reserves more efficiently harnessed and available for growth and physiological maintenance (savoie et al., 2008). digestive enzyme activities after feeding: food consumption is caused changes in digestive enzymes activity (onishi et al., 1973). the study of digestive enzyme activities changes in chrias guriepinus after feeding revealed gradual decrease of activity after the 4 h post feeding (uys et al., 1987). the present study showed that peak of enzymes activities were 5 and 8 hour after feeding and after the 15 and 18 h, the activities gradually decreased. the decrease of the protease and alkaline phosphatase specific activities of rutilus kutum during 0 to 1 and 0 to 3 h after feeding in t1 and t2, respectively was probably due to dilution by the food and an inadequate rate of secretion (uys et al., 1987). previous works showed that maximum trypsin activity in oreochromis niloticus (uscanga et al., 2010) and cyprinus carpio (onishi et al., 1973) occur 6 and 5 h post feeding. the peak of enzyme activity of rutilus kutum was in line with those obtained previously on other cyprinids in conclusion, this study showed that temperature had significant impact on the food transit time and digestive enzyme activity in caspian kutum. thus the feeding amount and frequency should be determined considering temperature. acknowledgments we express our gratefulness to fish farm sijaval (turkmen seaport, golestan province, iran) for supplying fish and mr. a. jafar and a. naimi (gorgan university of agricultural sciences and natural resources fisheries laboratory personnels, golestan province, iran) for their assistance during the experiments. references bernfeld p. 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(2021) 9(2): 124-133 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2021 iranian society of ichthyology original article dietary conjugated linoleic acid (cla) and lecithin affects levels of serum cholesterol, triglyceride, lipoprotein and hypoxic stress resistance in rainbow trout (oncorhynchus mykiss) mohammad reza maleki moghaddam1, naser agh*2, 1 kourosh sarvi moghanlou1, farzaneh noori2, akbar taghizadeh3, enric gisbert4 1department of fisheries, faculty of natural resources, urmia university, urmia, iran. 2artemia and aquaculture research institute, urmia university, urmia, iran. 3department of animal science, faculty of agriculture, tabriz university, tabriz, iran. 4institut de recerca i tecnologia agroalimentàries (irta), centre de sant carles de la ràpita, unitat de cultius aqüícoles, crta. poble nou km 5.5, 43540 sant carles de la rapita, spain. s article history: received 11 december 2019 accepted 7 february 2020 available online 2 5 april 2021 keywords: linoleic acid lecithin hypoxia stress cortisol lipoprotein abstract: considering the role of conjugated linoleic acid (cla) and soybean lecithin (sbl) in fat digestion, absorption and metabolism as well as stress resistance in aquatic organisms, the current study was a 74-day attempt to investigate the synergistic effects of these two compounds on stress resistance and blood lipoprotein levels of rainbow trout (oncorhynchus mykiss) weighing 120±5 g. nine isonitrogenous and isocaloric experimental diets containing 1, 2 and 3% cla and 2, 3 and 4% soybean lecithin on the basis of the basal control diet (free from cla and lecithin) were formulated. the fish were exposed to three ascending periods of hypoxic stress (7.5, 15 and 30 min) at the end of culture period. the dissolved oxygen was decreased to 1.96 ppm by injecting nitrogen gas. significantly higher levels of cortisol and glucose were detected in fish fed on diets having higher levels of sbl (2%) and cla (3%) at higher stress time improving the resistance to hypoxia stress (p≤0.05). moreover, levels of triglyceride (trg), cholesterol (col), low density lipoprotein (ldl), very low-density lipoprotein (vldl) and high density lipoprotein (hdl) also increased significantly compared to the control group (p≤0.05). this study reveals that using 2% sbl and 3% cla in rainbow trout feed can promote resistance to hypoxic stress. introduction conjugated linoleic acid (cla) is a member of specific group of geometric isomers of linoleic acid that their double bonds are separated from each other by a single bond. cla is produced as a result of isomerization and bio-hydrogenation of microbes in the rumen of ruminant (wahle et al., 2008). in aquaculture nutrition, the two bioactive isomers of cla viz. sis-9 trans-11 and sis-10 trans-12 are used (zanqui et al., 2013). these two isomers affect lipid metabolism in aquatic’s body affecting enzymes such as acyl-coa oxidase, carnitine acyl transferase i, malic enzyme, desaturases, elongase and lipoprotein lipase (valente et al., 2005; leaver et al., 2006). cla can influence the ability of aquatic organisms in converting fatty acids to each other (bioconversion) *correspondence: naser agh doi: https://doi.org/10.22034/ijab.v9i2.544 e-mail: n.agh@urmia.ac.ir and change the ratio of omega-3 to omega-6 fatty acids in muscle and liver (leaver et al., 2006) and creates a tendency to increase the growth performance (berge et al., 2004). the above-mentioned enzymatic effects can influence the level of blood lipoproteins of the fish fed diets containing cla supplement. for example, it has been proven that cla increases insulin resistance and decreased serum cholesterol and high-density lipoprotein (hdl) levels (yang et al., 2014). the positive effect of cla on lipolysis in comparison to proteolysis and glycolysis can be attributed to the low amount of co2 produced in fish fed diets containing cla supplement. therefore, cla is expected to affect rq and consequently the amount of co2 produced in intensive fish culture systems and as a result increase fish resistance to 125 int. j. aquat. biol. (2021) 9(2): 124-133 hypoxia stress. cla not only increased insulin resistance and decreased lipogenesis and lipoprotein lipase activity in mice, but also increased mice adaptation to hypoxic stress in vivo (luo et al., 2011). the role of dha when being exposed to stress as well as its role in cortisol secretion in fish has been proven by kanazawa (1979) and tago et al. (1999). on the other hand, cla increases dha level in fish (berge et al., 2004; makol et al., 2009), therefore, it indirectly influences the production of dha affecting hypoxic stress resistance in fish. according to rohmah et al. (2016), cla can increase insulin resistance and affect blood glucose level of the fish under hypoxic stress. to maximize intestinal absorption of dietary cla, new methods should be taken into account when formulating diets. cla has the capacity to bond with phospholipid compounds (choi et al., 1999). therefore, it seems that phospholipid compounds such as lecithin have specific emulsifier roles (atar et al., 2009) and are capable of increasing the enterocyte absorption rate of cla. lecithin consists of phosphoric acid and choline, fatty acids, glycerol, glycolipid, triglycerides and phospholipids such as phosphatidylcholine, phosphatidylcholine ethanol amine, phosphatidyl serine and phosphatidylinositol. lecithin has an interactive role in intestinal absorption of cholesterol and can improve the survival of aquatic species (adm specialty ingredient, 2003). since fish and crustaceans cannot produce enough phospholipid to maximize their growth, phospholipids should be added to their diets (ketola, 1976; hung and lutes, 1988; hung, 1989; poston, 1991a, b). the main role of lecithin seems to be its synergic property in lipid metabolism. the increased resistance to stress has been reported by supplementing of phospholipids to the diets (salhi et al., 1999; tocher et al., 2008). under the hypoxic stress, i.e. salinity and temperature, phosphatidylcholine containing dha has increased the resistance to the stress in flounder (paralichthys olivaceus) more efficiently than triglycerides containing dha (tago et al., 1999). therefore, since lecithin is a rich source of phosphatidylcholine, it can play a role in creating resistance to stress in fish. moreover, kanazawa (1979) stated that dha and soy lecithin increases the stress tolerance of oxygen, temperature and salinity in sea bream. liu et al. (2002) also found lecithin and eicosapentaenoic acid (epa) effective in producing cortisol or stress hormone in gilthead sea bream (sparus aurata). additionally, researchers pointed to the role of lecithin as an emulsifier in increasing the enterocyte absorption rate of lipids and increasing blood lipoproteins. several studies on marine fishes have shown the synergistic effects of phospholipids in fat metabolism and these studies attributed the increase in digestion and absorption efficiency of fat in intestinal cells (enterocytes) by phospholipids to the emulsification role of phospholipids in increasing the intestinal absorption of fat (fontagne et al., 1998; olsen et al., 1999; salhi et al., 1999; liu et al., 2002; azarm et al., 2012). hence considering the important role of cla and lecithin in reducing environmental stress and digestive processes, absorption, transfer and metabolism of lipids, the present study aimed to investigate the synergistic effects of these two dietary supplements on coping with hypoxia stress and their effects on the concentration of lipoproteins in experimental rainbow trout, oncorhynchus mykiss, under stress. materials and methods experimental diets and fish: during a 74-day experiment, nine isonitrogenous (45% crude protein) and isocaloric (3.65 mega calories per kg) experimental diets with different levels of cla and lecithin were formulated according to the requirements of cold-water fishes (table 1). experimental diets were proximately analyzed according to aoac (1995). the control diet was free from cla and lecithin. the required cla with 58% purity was provided by salamat pakhsh-e-yalda company, iran pharmaceutical products technology incubator. the used lecithin was soybean lecithin, the product of vicentin company, argentina. the pellets were produced industrially and the amounts of cla and lecithin were added to experimental diets (table 126 maleki moghaddam et al./ cla and lecithin affecting stress resistance in rainbow trout 1). thirty 300-l tanks were used for this experiment, each containing 20 rainbow trout (with average weight of 120±5 gr). the fish were fed the control diet (2% of body weight) for two weeks (three times a day) during the adaptation period. length and weight measurements were done every 14 days. to reduce the biometry stress, the experimental fish were not fed 24 hours before the biometry. hypoxic stress: all experimental fish were exposed to hypoxic stress for 7.5, 15 and 30 min by injecting nitrogen gas into the culture tanks (bloom et al., 1993; dabrowskia et al., 2004). after each stress period, the fish were recovered and feeding continued until second and third phases of stress. the concentration of oxygen gas during hypoxic stress fell to 1.96 ppm. sampling and measuring parameters: at the end of each hypoxic stress stage, four fish were caught from each tank and were anesthetized using clove powder (eugenia caryophyllata) with a concentration of 200 ppm. blood samples were taken from the caudal peduncle using 5 ml syringes without heparin. blood samples were centrifuged at 3000 rpm for 10 min (burel, 2001). sera were separated by pipette and poured into 0.5 ml micro tubes and were frozen and kept at a temperature of -20oc till the determination of their biochemical parameters. cortisol was measured by elisa method using accubind kits made by monobind company. to measure cholesterol and glucose, autoanalyzer (alpha-6 model) and the kits of the man company were used. samples were read at a wavelength of 450 nm. triglyceride was determined by enzymatically glycerophosphate dehydrogenase (god-pap) method (fukuda, 1958) and to measure the serum lipoproteins, the kits of pishtazteb company were utilized and all measurements were repeated three times. statistical analysis: the experiment was completely randomized in a factorial design of 10 treatments with 3 replications. at the end of the study, the data from the effect of experimental diets on biochemical parameters of hypoxic stress (cortisol levels, glucose) as well as cholesterol, triglyceride and blood table 1. formulation of experimental diets containing different levels of cla & soy bean lecithin (%). d9 d8 d7 d6 d5 d4 d3 d2 d1 control ingredients 36 36 36 36 36 36 36 36 36 36 fish meal1 6 6 6 6 6 6 6 6 6 6 fish oil2 7 7 7 7 7 7 7 7 7 7 safflower oil 15 15 15 15 15 15 15 15 15 15 soybean meal 15.5 15.5 15.5 15.5 15.5 15.5 15.5 15.5 15.5 15.5 wheat gluten 7 7 7 7 7 7 7 7 7 7 corn gluten 4 4 4 4 4 4 4 4 4 4 canola meal 3 2 1 3 2 1 3 2 1 0 cla3 4 4 4 3 3 3 2 2 2 0 soybean lecithin4 0 1 2 1 2 3 2 3 4 7 starch 1 1 1 1 1 1 1 1 1 1 vitamin supplements5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 binder6 1 1 1 1 1 1 1 1 1 1 mineral supplements7 analyzed proximate composition (percent in dry matter) 40.1 40.12 40.12 39.97 40 40 40.15 40.23 40.31 40.42 crude protein (n*6.25) 15.36 15.1 15.08 15.26 15.18 15.12 15.25 15 15.01 15 crude lipid 12.03 13.13 12 12.3 12 12.28 12.13 13.10 13 3.13 ash 3.37 3.40 3.42 3.36 3.42 3.40 3.44 3.40 3.36 3.40 crude fiber 94 94.15 93.97 94 93.98 94 94.1 94.21 94 94.2 dry matter 3.63 3.61 3.60 3.62 3.60 3.60 3.60 3.63 3.60 3.60 digestible energy (mcal kg-1 diet) 1, kilka meal. iran; 2 kilka oil. iran; 3, conjugated linoleic acid. salamat pakhsh yalda co, iran purity 58%; 4 soybean lecithin, vicentin co, argentina; 5, vitamin mixture (unit kg-1 of diet): vitamin a, 1200 000 iu; d3, 400 000 iu; e, 3000 mg; k3, 1200 mg; c, 5400 mg; h2; 200 mg; b1, 200 mg; b2, 3600 mg; b3, 7200 mg; b5, 9000 mg; b6, 2400 mg; b9, 600 mg; b12, 4 mg; antioxidant 500 mg career up to 1 kg; 6, binder: amet binder (component: crude protein: 71.98%, crude fibre: 0.9%, ash: 17.8%, moisture: 9.55%) and 7, minerals mixture (mg kg-1 of diet): fe, 4500 mg; cu, 500 mg; co, 50 mg; se, 50 mg; zn, 6000 mg; mn, 5000 mg; i, 150 mg; choline chloride, 150 000 mg; career up to 1 kg. 127 int. j. aquat. biol. (2021) 9(2): 124-133 lipoproteins were analyzed using sas (9.1) software, glm procedure. mean comparisons were performed by duncan test. presence or absence of statistical differences between treatments was set at 0.05. results the results of the blood analysis at the end of rearing period before application of stress showed significantly lower cortisol and glucose in treatments received 3% cla and 2-4% sbl, but cholesterol, triglyceride, hdl, ldl and vldl were significantly higher in most fish fed experimental diets compared to the control (table 2). cortisol level in fish exposed to hypoxic stress increased gradually with increasing the stress period but was significant only in the third phase (30 min stress) compared to others (fig. 1, table 3, table 2. effect of different feeding treatments on the levels of glucose, cortisol, cholesterol, triglyceride, high-density, low-density and very lowdensity lipoproteins at the end of the experiment before stress (μg/dl). treatments cort glu chol try hdl ldl vldl 0 4.38±0.62a 152.92±9.87a 268.93±19.97d 262.4±31.65d 47.45±2.94 c 26.26±5.82d 58.68±6.96 d 1 3.98±0.66ab 150.1±9.23ab 294.73±21.96dc 317.19±36.2cd 53.73±3.55bc 29.41±6.33d 63.24±7.02 cd 2 3.14±0.44abc 142.48±5.98abc 326.95±21.02bcd 325.69±38.2bcd 61.61±3.74ab 37.32±6.42cd 70.47±9.23abcd 3 2.56±0.43abc 118.12±10.98cd 346.67±11.46abc 423.87±52.3abc 62.84±2.85ab 50.61±6.66abc 89.4±9.86a 4 4.13±0.7ab 140.58±11.04abc 314.42±20.87cd 323.7±35.76cd 56.81±4.01bc 44.34±8.5bcd 62.95±6.29d 5 2.78±0.56abc 144.8±8.95ab 346.08±17.24abc 354.98±21.5bcd 60±2.53ab 43.9±6.16bcd 74.6±5.83abcd 6 2.2±0.37bc 113.83±5.22d 385.9±19.06ab 518.1±84.25a 69.59±3.9a 64.62±7.21a 82.2±7.01abc 7 3.64±0.5ab 145.53±9.8ab 309.8±18.14cd 338.6±26.6bcd 60.93±2.57ab 39.34±6.7cd 67.38±5.27bcd 8 2.53±0.65abc 139.2±8.54abc 336.57±17.28abc 389.13±31.7bc 63.59±2.94ab 45.55±6.18abcd 72.36±6.9abcd 9 1.51±0.24c 126.75±9.92bcd 389.07±22.27a 453.7±50.3ab 69.96±4.25a 60.9±8.08ab 85.7±5.25ab lack of common letters indicates significant differences between treatments (p<0.05). figure 1. effect of hypoxic stress on cortizol level (a); glucose; cholesterol; triglycerides; high, low and very low density lipoprotein (b, c, d) at different stress timings. 128 maleki moghaddam et al./ cla and lecithin affecting stress resistance in rainbow trout p≤0.05). the lowest level of cortisol was observed in fish fed experimental diets containing highest level of cla and 4-2% sbl, respectively. significant differences in cortisol level were observed between treatment 9 containing 3% cla and 4% sbl with control group and with treatments 1, 4 and 7 containing lowest levels of cla (table 2, p≤0.05). higher stress period resulted in a significant increase in blood glucose level (p≤0.05) (fig. 1). in addition, blood glucose in fish under 7.5 min stress showed significant differences between treatments 6, 8 and 9 with the control group exposed to 30 min stress table 3. the effect of duration of hypoxic stress (7.5, 15 and 30 min) on changes in levels of cortisol, glucose, cholesterol, triglyceride, highdensity, low-density and very low-density lipoproteins (μg/dl). cort glu chol try hdl ldl vldl time1_t0 3.26±1.75abc 123.75±9.6cdefgh 255.25±44.1c 323.8±84.4bcdef 55.9±3.94bc 34.78±8.9bcde 83.37±12.1bcde time1_t1 2.66±1.5abc 121.75±10.8cdefgh 311±53.22abc 401.7±88.7bcdef 59.6±5.07bc 33.18±5.7bcde 81.5±18.4bcde time1_t2 2.57±1.3abc 134±1.73abcdefgh 358±14.98abc 414±115.2bcdef 66.33±8.04ab 50.85±8.9abcde 90.13±27.9bc time1_t3 2.13±1.08bc 94±31.96h 359.75±27.8abc 549±143.1b 66.53±7.4ab 58.2±8.45abcd 125.23±17.6a time1_t4 2.74±1.07abc 122.5±30.7cdefgh 352.75±43.2abc 432.8±69.1bcde 61.38±6.15ab 45.8±15.9abcde 82.2±12.5bcde time1_t5 2.06±1.03c 116.8±17.03defgh 355.75±19.9abc 433.3±26.8bcde 60.88±6.25ab 56.25±8.66abcd 89.45±8.22bc time1_t6 1.36±0.68c 104.75±7.18gh 374±29.17abc 782±223.25 a 77.7±6.4a 73.38±15.7a 89.88±16.64bc time1_t7 2.92±1.2abc 109.5±3.1efgh 344.5±44.46abc 428.3±9.7bcdef 63.93±5.3ab 49.5±8.25abcde 84.6±3.36bcde time1_t8 1.48±1.03c 117.5±19.1defgh 361.75±31.4abc 470.5±50.1bcd 64.45±5.57ab 44.63±7.1abcde 89.1±16.45bcd time1_t9 1.19±0.58c 106±5.6fgh 369.75±21.4abc 493.25±114bcd 79.55±8.78a 72.05±5.27ab 94.75±9.28ab time2_t0 3.9±1.21abc 155±10.95abcde 295.75±16bc 272.25±42.2def 48.23±5.64bc 32.6±11.9bcde 54.4±8.4cdeg time2_t1 3.33±1.45abc 153.8±18.8abcdef 317±26.47abc 297.5±50.7cdef 53.93±8.72bc 36.6±17.3abcde 57.8±4.4bcdeg time2_t2 2.83±0.7abc 145.2±17.3abcdefg 330±30.4abc 289 ±22.1cdef 61.53±4.58ab 38.9±13.5abcde 66.5±7.2bcdeg time2_t3 2.44±1.1abc 117.7±3.8cdefgh 351.25±14.6abc 348±69.3bcdef 61.8±2.89ab 61.03±11.1abc 73±15.6bcdeg time2_t4 3.71±1.97abc 140.2±6.98abcdefgh 304.5±37abc 332.75±36bcdef 56.35±10.55bc 53.3±16.3abcd 61.5±4.8bcdeg time2_t5 2.39±1.7abc 151.2±10.7abcdefg 331.5±53.8abc 307.5±23.9cdef 60±3.1ab 42.4±8.4abcde 69.5±13.8bcdeg time2_t6 1.69±0.38c 118.75±7.9cdefgh 420.75±20.5a 392.5±15.1bcdef 68.35±3.24ab 66.88±4.7ab 78.48±3bcde time2_t7 3.5±1.22abc 149.5±10.7abcdefg 305.5±22.1abc 331.8±34.9bcdef 59.45±2.16ab 49.5±12.9abcde 66.4±6.95bcdeg time2_t8 2.04±1.71c 140.5±11.4abcdefgh 321.75±24.8abc 354.5±67.9bcdef 64.03±6.03ab 32.9±14.5bcde 59.5±10.2bcdeg time2_t9 1.48±0.41c 130.25±18bcdefgh 418.25±57.7a 518.3±112bc 67.8±3.8ab 67.4±12.2ab 86.08±9.7bcde time3_t0 5.98±0.44a 180±16.77a 255.8±39.5c 191±21.37f 38.21±1.84c 11.4±7.37e 38.26±4.26g time3_t1 5.96±0.11a 174.8±8.2ab 256.2±33.5c 252±37.7def 47.67±4.65bc 18.4±8.39de 50.4±7.55eg time3_t2 4.03±0.65abc 148±8.5abcdefg 292.6±47.7bc 274±25.6def 56.98±6.92bc 22.2±8.4cde 54.78±5.1cdeg time3_t3 3.1±0.69abc 142.6±7.4abcdefg 329±17.8abc 374.6±35bcdef 60.2±4.55ab 45.48±8abcde 70±4.7bcdeg time3_t4 5.94±0.9ab 159±14.3abcd 286±30.56bc 205.6±22.4ef 52.7±5.26bc 19.78±8.2de 45.12±6.9g time3_t5 3.88±0.68abc 166.4±10.1abc 351±16.7abc 324±30.3bcdef 59.12±4.51ab 33.02±12bcde 64.8±6.1bcdeg time3_t6 3.55±0.58abc 118±10.9cdefgh 363±40.87abc 380±59.4bcdef 62.72±7.93ab 53.62±14abcd 78.32±13.9bcde time3_t7 4.49±0.9abc 177.6±12.55ab 279.4±24.8bc 255.8±37.6def 59.42±5.38ab 19.02±8.16de 51.14±7.5bcdeg time3_t8 4.06±1.2abc 159.6±8.68abcd 326.2±34.2abc 342±38bcdef 62.28±5.13ab 46.2±14.5abcde 68.5±7.6bcdeg time3_t9 1.86±0.64c 144±19.7abcdefg 379.2±36.4ab 349.6±23.5bcdef 62.52±7.3ab 57.4±17.14abcd 76.3±8.3bcde *the lack of common letters indicates significant differences between treatments (p<0.05) # the abbreviations used in diagrams (1) and (2) and tables (2), (3) and (4) are as follows: t1=7.5 min, t2=15 min, t3=30 min cort=cortisol, glu=glucose, chol=cholesterol, try=triglyceride, hdl=high-density lipoprotein, ldl=low-density lipoprotein, vldl=very low-density lipoprotein. 129 int. j. aquat. biol. (2021) 9(2): 124-133 (p≤0.05) (table 3). comparing the effect of different treatments on the levels of cholesterol, triglyceride and serum lipoproteins (table 2) showed an increase in these parameters with increasing the amount of dietary cla (p≤0.05). comparison of the means revealed significant differences between the levels of triglyceride, vldl and hdl in fish exposed to 30 and 15 min hypoxic stress compared to those under 7.5 min stress (p≤0.05). considering the combined effect of feeding treatments and duration of stress on the serum triglyceride level (table 3), significant differences were observed between the fish of treatment 6 and 9 under 7.5 and 15 minutes compared to the control group (p≤0.05), but no significant difference was observed at 30 min stress among the treatments. cholesterol level was significantly higher in treatment 6 and 9 stressed for 15 min and treatment 9 stressed for 30 min compared to the control fish (p≤0.05) (table 3). ldl and hdl were significantly higher in treatments received highest levels of cla and sbl stressed for 7.5 and 30 min compared to control. similarly, vldl also was significantly higher in treatments 6 and 9 containing highest amount of cla and sbl stressed for 30 min in comparison to control (p≤0.05, table 3). discussions one of the indicators of stress is cortisol (hydrocortisone) which is secreted via the renal tissue as a result of the release of adrenocorticotropic hormone (acth) (pierson et al., 2004; ramsay et al., 2006; martinez-porchasi et al., 2009). based on the results, the lowest levels of cortisol in all stress phases was observed in fish fed experimental diets with the highest levels of cla (3, 6 and 9 treatments), indicating the effect of dietary cla on reducing stress. cla will increase the beta-fat oxidation rate in the body of experimental fish (leaver et al., 2006) as seen in the current study. since fat catabolism generates less respiratory fraction than carbohydrates and proteins, therefore in exchange for taking a unit of fat in the body, small amounts of co2 is released (mcdonald et al., 1979) and this reduces the need of experimental fish to absorb dissolved oxygen and increases the ability of the fish to tolerate hypoxia stress i.e. cla increases the production of phospholipids such as dha which affect resistance to hypoxic stress (berg et al., 2004, makol et al., 2009). the treatments 3, 6 and 9 have the highest ratio of cla to lecithin. considering lecithin’s role as an emulsifier in the transmission of intestinal fatty acids (atar et al., 2009; azarm et al., 2012), lecithin also indirectly effects increase of stress resistance in the fish due to increased intestinal absorption of cla. lecithin as a source of phospholipids such as phosphatidylcholine can have an indirect impact on generation, transmission and absorption of epa and dha (tago, 1999; liu et al., 2002), that increase fish resistance to hypoxic stress. increased blood glucose level with increasing the duration of hypoxia stress was evident from the first to third phase. by increasing stress time, increase of both blood glucose and cortisol level was observed. pickering et al. (1989) and martinez (2009) pointed out the regulatory relationship between cortisol and blood glucose levels through the process of gluconeogenesis i.e. cortisol increases blood glucose level (hyperglycemia) and stimulates gluconeogenesis of proteins and lipids. after applying stress, cortisol ranges from 40 to 200 ng/ml (pickering et al., 1989) and in some species it can exceed 1000 ng/ml (barton et al., 1998). according to pickering et al. (1989) and tocher et al. (2008), the cortisol secretion is a hormonal response of the body of the fish under stress that increases the resistance of the aquatic body to stress conditions i.e. the level of plasma cortisol and changes in carbohydrate metabolism, such as glucose can be used as a general indicator of stress. our results were in line with the findings of pickering et al. (1989) and martinez et al. (2009). the lowest amount of blood glucose was observed in treatments of 3, 6 and 9. the reason for the significant difference in the level of glucose in treatments 3, 6 and 9 (treatments that received the highest levels of cla) is the physiological effect of cla on reducing the oxygen demand of the fish. the undeniable role of cla in lipid metabolism (leaver et al., 2006) and the reduction of oxygen 130 maleki moghaddam et al./ cla and lecithin affecting stress resistance in rainbow trout demand during hypoxia stress (berge et al., 2004), as well as the role that lecithin plays in the absorption of cla and the production, absorption and transfer of phospholipids, such as dha and epa (kanazawa 1999; liu et al., 2002) can be reasons for interpreting the synergistic effects of these two compounds in reducing stress and subsequently reducing cortisol and glucose levels in fish fed diets of 3, 6 and 9. furthermore, these are reasons for explaining the effects of treatments on changes in blood glucose levels. cla also increases polyunsaturated fatty acids (pufas) by increasing the enzyme gene transcription rate of aceyl coenzyme a oxidase. therefore, in both cases, cla provides a substrate for the pathway of gluconeogenesis. given the fact that the experimental diets 3, 6 and 9 had the highest levels of cla, it was expected that the highest levels of glucose would be obtained for fish fed these diets, but the results showed the reverse. as cla increases the activity of carnitine palaeomethyl transferase 1 and beta-oxidation rate, the consumption of free fatty acids, which are substrates for the process of gluconeogenesis, increases and its result can be the reduction of blood glucose concentration in fish fed diets containing cla. in addition to increasing the activity of carnitine palaeomethyl transferase 1 and increasing the betaoxidation of fatty acids in the aquatic body, cla decreases the activity of ∆ 9-desaturase (pariza et al., 2001; berg et al., 2004; leaver et al., 2006). lipoprotein lipase is an enzyme which hydrolyzes the core of chylomicrons and vldl and changes them to di-glycerides and mono-glycerides and finally free fatty acids. it seems that the effect of cla on serum lipoprotein levels occurs by inhibition of lipoprotein lipase (lpl) (pariza et al., 2001, luo et al., 2011). an increase in the rate of gluconeogenesis requires substrates such as fatty acids (pickering et al., 1989) which can be a feed-back to increase blood lipoproteins. an increase in the rate of gluconeogenesis requires providing substrates such as fatty acids (pickering et al., 1989), the consumption of which can be a feedback to produce blood lipoproteins. lecithin plays an interactive role in the absorption of cholesterol (adm specialty ingredient, 2003). cholesterol is a lipoprotein precursor and, the lecithin has an emulsifier role, therefore, lecithin increases fat absorption rate and forms chylomicrons, increasing in the concentration of lipoproteins. based on the results, levels higher than 2% lecithin and 3% cla increased lipoprotein accumulation rate and by increasing the stress time from 7.5 to 30 min, the blood lipoprotein levels decreased. this process showed that with increasing stress time, the consumption of fatty acids (as gluconeogenesis substrate) increased and as a result cholesterol, triglyceride and lipoprotein concentrations are decreased. as conclusion, it can be noted that the greatest synergistic effect of lecithin and cla on increasing rainbow trout resistance was observed against oxygen deficit stress when cla was 3% and lecithin was 2, 3 and 4% in the diet. therefore, it is recommended that in intensive culturing systems which are under hypoxia stress, the above-mentioned levels of these two supplements are used in formulation of rainbow trout feed in growing phase. regarding the influence of 3% cla and 2, 3 and 4% lecithin on levels of serum cholesterol, triglyceride and lipoproteins, it is suggested that these levels be periodically used in formulating diets for rainbow trout fed fat-rich diets. references adm specialty ingredients (2003). lecithin in aquaculture. p.o. box 2, 1540 aa koog aan de zaan, netherlands, feedingredients@admworld.com. aoac (1995). official methods of analysis of aoac international. 2 vols. 16th edition. arlington, va, usa, association of analytical communities. atar h.h., bekcan s., olmez, m. 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(2019) 7(1): 14-26 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article effect of leucine enkephalin administration on ovarian maturation in the freshwater crab travancoriana schirnerae navya gopal, arath raghavan sudha devi*1 department of zoology, mary matha arts and science college, wayanad, kerala 670 645, india. s article history: received 15 december 2018 accepted 22 february 2019 available online 2 5 february 2019 keywords: leucine encephalin ovarian maturation histological parameters crab abstract: the current study focused on the effect of administration of the neurotransmitter leucine enkephalin on ovarian maturation in the freshwater crab travancoriana schirnerae. the crabs were administered with leucine enkephalin in multiple doses (10 µl/injection) and their dissected ovaries were processed for histomorphological analyses. ovarian maturation was assessed by both macroscopic and microscopic observations such as ovarian index, mean oocyte diameter, oocyte proportion values and histological examinations of the ovaries of control and treated crabs. our observations revealed significantly higher ovarian index, oocyte diameter and oocyte proportion values in treatments over controls. leucine enkephalin treatment induced previtellogenic ovary to grow into primary vitellogenic, primary vitellogenic to secondary vitellogenic 1 secondary vitellogenic 1 and 2 to secondary vitellogenic 3 stage as evinced by the presence of a large number of primary previtellogenic ovaries in previtellogenic oocytes, larger proportion of secondary vitellogenic oocytes in primary vitellogenic ovaries and secondary vitellogenic stage 3 oocytes in secondary vitellogenic stage 1 ovaries of treated crabs over the controls. the conversion of previtellogenic ovary to early vitellogenic, early to middle and middle to late vitellogenic ovaries in treated crabs probably indicate the stimulatory effect of leucine enkephalin on ovaries either by triggering the release of the gonad stimulating hormone synthesized and released from the brain or thoracic ganglion or by blocking the release of the gonad inhibiting hormone synthesized and secreted by the x organ-sinus gland complex of the eyestalks or both. the results of the present study clearly indicate that leucine enkephalin has a stimulatory effect on ovarian maturation in t. schirnerae, thus shortening the period of maturation of ovary, which can be utilized in large-scale production of the species concerned. further studies are needed to check the efficacy of this neurotransmitter as a supplement in diet to induce ovarian maturation of this species. introduction enkephalins are endogenous opioid pentapeptides occurring naturally in the brain of both invertebrates and vertebrates, including crustaceans where they act as neurotransmitters or neuromodulators (kream et al., 1980; duve and thorpe, 1990; salzet et al., 1997). the presence of enkephalins was reported in retinular cells of the ommatidia (mancillas et al., 1981; jaros, 1986), sinus gland, eyestalk ganglia and brain of many crustaceans (jaros and keller, 1983). two structurally different forms of enkephalins were found, namely leucine enkephalin (leu-enk) (simantov and synder, 1976) and methionine enkephalin (met-enk) (hughes et al., 1975), products of the proenkephalin gene *correspondence: arath raghavan sudha devi doi: https://doi.org/10.22034/ijab.v7i1.526 e-mail: arsudhadevi@gmail.com (gubler et al., 1982; undenfriend and kilpatrick, 1983). leucine enkephalin has the amino acid sequence tyr-gly-gly-phe-leu and met-enk has the sequence tyr-gly-gly-phe-met (lazarus and guillemin, 1976). these two pentapeptides bind to morphine receptors in the central nervous system and have opioid properties of relatively short duration (takahashi, 2016). enkephalins enhance the release of neurohormones that control the reproductive activities in both invertebrates (fingerman, 1987) and vertebrates (crim et al., 1984). leucine enkephalin and met-enk have antagonistic effects on reproductive indices in crustaceans with leu-enk stimulatory in action while 15 int. j. aquat. biol. (2019) 7(1): 14-26 met-enk has an inhibitory role (kumar et al., 2012). leucine enkephalin induces the development of gonads either by triggering the production of gonad stimulating hormone (gsh) from the brain or thoracic ganglion or by inhibiting gonad inhibiting hormone (gih) release from the sinus gland or both (kulkarni et al., 1981; reddy, 1991; sarojini et al., 1995). methionine enkephalin stimulates the production of gih or inhibits the production of gsh or both (fingerman, 1997; nagaraju, 2011; swetha et al., 2011). apart from reproduction, enkephalins are also involved in regulating nociception (noda et al., 1982; titus et al., 1989), euphoria, susceptibility to seizures (bergola et al., 2002), decrease in gastrointestinal motility, cardiovascular regulation and food consumption behaviour through opiate receptors (micheal et al., 1989; froehlich, 1997). among invertebrates, the role of exogenous enkephalins in reproduction has been studied extensively in decapod crustaceans (schoofs et al., 1998). sarojini et al. (1995) observed stimulatory and inhibitory effects of leu-enk and met-enk in the mud crab, uca pugilator. in the indian white prawn, penaeus indicus, reddy et al. (2000) revealed an increase in ovarian index following leu-enk administration and a decrease following met-enk administration. influence of leu-enk on moulting and vitellogenesis in the freshwater field crab oziotelphusa senex senex was demonstrated by kishori and reddy (2003). stimulation of ovarian growth and vitellogenesis was reported by kishori et al. (2012) in o. senex senex following leu-enk administration. one major problem faced by aquaculture industry is the shortage or non-availability of quality seed as several commercially important species are unable to spawn spontaneously under artificial conditions. eyestalk ablation (esa) has been used as the most successful procedure by hatchery industries for induction of moult, gonadal maturation and spawning which often leads to the production of poor quality seed, juveniles and huge mortality of the broodstock. alternative modes to uphold sustainable aquaculture are the administration of hormones: gsh, methyl farnesoate, ecdysteroids, vertebrate type steroids and stimulatory neurotransmitters either as injections or as feed supplements. recently, administration of anti gih antibody which blocks the gih has been practiced by aquaculture industries for inducing gonadal maturation in captivity. of these methods, the hormonal manipulation technique is very expensive and thus not cost effective. moreover, the use of hormones can have potential health hazards to man and the environment. eating foods contaminated with hormones especially steroid hormones may cause endocrine disorders and development of cancer (kandarakis et al., 2009). for most crustaceans, the best alternative to hormone injection is the use of neurotransmitters as they have the same effectiveness of hormone injection, cost effective and cause no harmful consequences. literature is scanty regarding the role of neurotransmitters on ovarian growth and maturation in freshwater crabs. further, so far no efforts have been made to study the stimulatory effects of the neurotransmitter leu-enk on ovarian growth and maturation in the edible freshwater crab travancoriana schirnerae. in this scenario, the present investigation on the effect of exogenous administration of a stimulatory neurotransmitter on ovarian maturation in this species is attempted. the neurotransmitter, leu-enk has been shown to accelerate ovarian maturation by reducing the overall maturation time (kishori and reddy, 2003; kishori et al., 2012), which can be utilized in large-scale production of the species concerned. materials and methods adult intermoult females in various phases of oogenesis (previtellogenic, early, middle and late vitellogenic phases) were collected from the paddy fields near mary matha arts and science college campus, mananthavady, wayanad (kerala, india) during june 2017 to june 2018. they were acclimatized to the conditions of the laboratory for 3 to 4 days. they were fed with pulses, boiled egg and sliced beef liver once in two days. the carapace width, wet weights and moult stages of animals were 16 gopal and sudha devi / effect of leucine enkephalin on ovarian maturation recorded. leucine enkephalin purchased from sigma (sigma aldrich, st. louis, mo, usa) was used for injection (1 mg/1 ml distilled water). the crabs were divided into two groups of 10 each. group i formed the controls and group ii which received leucine enkephalin (10 µl) injections on 1st, 7th, 14th and 21st day through the arthrodial membrane at the base of the coxa of the third walking leg formed the experimentals. both control and experimental crabs were sacrificed on 30th day. their ovaries were dissected out; the body weight and wet weight of gonads were recorded to calculate the gonadosomatic index (gsi). one half of the ovary was fixed in bouin’s fluid for histological analysis and the other half used for the measurement of oocyte diameter to determine the stage of development of the ovary. colour of ovary, oocyte diameter and ovarian index were the criteria used to determine the stage of development of the ovary. sections were stained with hematoxylin-eosin and observed under a leica dm 500 research microscope. photomicrographs were taken with a dg 330 /210 camera using biowizard software. mean ovarian index, mean oocyte diameter, oocyte proportion values and histological characteristics of the ovaries of control and treated crabs were the parameters used to evaluate the impact of leucine enkephalin administration on ovarian maturation. the results were calculated as mean±sd and the data obtained for controls and experimentals were subjected to one-way analysis of variance (anova). a value of p<0.05 was considered statistically significant. results morphology of the female reproductive system: the female reproductive system of t. schirnerae consists of paired ovaries, oviducts, gonopores and spermathecae. the ovary is h-shaped with anterior and posterior lobes, connected by a transverse bridge of ovarian tissue, located mid-dorsally in the cephalothorax. an oviduct which arises laterally from the posterior part of each ovary extends ventrally and opens out through the coxa of the third walking leg. a pear shaped spermatheca which stores spermatozoa received during copulation was found proximally attached to each oviduct. travancoriana schirnerae breeds once in a year, accommodating a single ovarian cycle during the prolonged intermoult phase (sudha devi and smija, 2013). based on the size and degree of yolk deposition, ten distinct stages were identified in the development of the oocyte: oogonia, chromatin nucleolus (cn) stages 1, 2, 3, perinuclear (pn) stage, primary vitellogenic (pv) stage, secondary vitellogenic (sv) stages 1, 2, 3 and tertiary vitellogenic (tv) stage (smija and sudha devi, 2015). the whole process of oogenesis was divided into six phases: proliferation, previtellogenic, primary vitellogenic, secondary vitellogenic, tertiary vitellogenic and oosorption phases based on morphological and histological features of the ovary. morphology and histology of ovary of control and treated crabs during previtellogenic phase (juneseptember): morphologically, no remarkable change was noticed in the ovary of treated crabs over controls. they were opaque and cream in colour. on the other hand, injection of leucine enkephalin significantly increased the ovarian index (p<0.001) and mean oocyte diameter values (p<0.001) of treated females compared to the controls (table 1). the ovary of experimental crabs showed a predominance of pv oocytes (49%) followed by pn oocytes (30%), cn3 (16%), cn2 (4%) and cn1 (1%) oocytes whereas the control ovary demonstrated larger proportion of pn oocytes (56%) followed by cn3 (29%), cn2 (11%) and cn1 oocytes (4%) (fig. 1a). histological observations of the ovary of control and experimental crabs revealed substantial differences with regard to the pattern of oocyte development. the pn oocytes of injected ovaries were mostly developed into pv oocytes while the control ovaries remained at pn stage. the treated ovaries which reached primary vitellogenic phase contained mainly pv oocytes that occupied towards the periphery of the ovary. the pv oocytes of treated crabs were characterized by the presence of numerous 17 int. j. aquat. biol. (2019) 7(1): 14-26 large vacuolated globules (19.69±5.16 µm) and yolk globules (15.16±2.18 µm). in addition, some pv oocytes (3%) in treated ovaries showed accumulation of highly basophilic yolk globules in the peripheral ooplasm, which is characteristic of sv1 oocytes. the size of nuclei (55.21±11.10 µm in diameter) and the number (2-4) and size of nucleoli (8.56±2.60 µm in diameter) of pn oocytes were increased considerably in experimental ovaries than the controls (50.32±8.74 µm, 1-2 and 6.14±1.84 µm, respectively). ovaries injected with leucine enkephalin evinced an increase in the width of perinuclear zone (65.37±16.07 µm) and table 1. mean ovarian index and oocyte diameter values of control and treated crabs during different phases of oogenesis. phases of oogenesis ovarian index oocyte diameter control (µm) experimental (µm) control (µm) experimental (µm) previtellogenic phase 0.31±0.04 0.37±0.03** 296.48±0.12 356.77±8.86** primary vitellogenic phase 0.53±0.02 0.65±0.10* 436.34±0.41 481.33±4.52** secondary vitellogenic phase 2.72±0.38 3.32±0.33* 787.19±9.46 1097.96±15.77** tertiary vitellogenic phase 4.48±0.42 4.50±0.38# 1363.60±11.58 1384.60±10.96# the values are represented as mean±sd; * p<0.01; ** p<0.001; # not significant figure 1. graph showing the effect of leucine enkephalin on proportion of oocytes during different phases of oogenesis. 18 gopal and sudha devi / effect of leucine enkephalin on ovarian maturation number and size of vacuolated globules (13.74±4.26 µm diameter) in the pn oocytes over controls (45.43±18.09 and 11.74±3.26 µm, respectively). histological examination also revealed that in control crabs, follicle cells were few in number surrounding the cn and pn oocytes whereas treated ovaries indicated the accumulation of more number of follicle cells surrounding the cn and pn oocytes and formation of epithelium around the pv oocytes. the treated ovaries also displayed increased proliferation of oogonia and follicle cells (fig. 2a-d, fig. 3a-d). morphology and histology of ovary of control and treated crabs during primary vitellogenic phase (october-november): the ovaries of both control and treated crabs were light yellowish in hue. the average ovarian index (p<0.05) and the mean oocyte diameter (p<0.001) values were significantly high in experimental females compared to the controls (table 1). the ovaries of treated crabs were dominated by sv oocytes (sv1 64% and sv2 26%) followed by pv oocytes (10%). on the other hand, pv oocytes predominated (62%) the control ovaries with less number of pn (27%) and cn oocytes (11%) (fig. 1b). histological analyses of treated ovaries showed enhanced vitellogenic activities in as much as their ovaries attained secondary vitellogenic phase. the cortical ooplasm of sv1 oocytes was profoundly laden with dense large yolk globules (41.51±9.63 µm in diameter) and vacuolated globules (37.97±8.27 µm diameter). in some sv1 oocytes, the yolk globules were fused to form mildly basophilic yolk platelets, which is characteristic of sv2 oocytes. the sv2 oocytes contained a large store of yolk platelets and vacuolated globules, organized in the entire ooplasm figure 2. photomicrograph of control and leucine enkephalin treated ovaries during previtellogenic phase in travancoriana schirnerae. (a) control ovary dominated by pn and cn oocytes; (b) treated ovary with a predominance of primary vitellogenic oocytes; (c) control ovary with pn oocyte at higher magnification; (d) treated ovary showing pn oocytes with increased perinuclear zone width and large vacuolated globules (ca: cortical alveoli; cn1: chromatin nucleolus stage 1 oocyte; cn2: chromatin nucleolus stage 2 oocyte; cn3: chromatin nucleolus stage 3 oocyte; dg: dispersed yolk globule; n: nucleus; nu: nucleolus; pn: perinuclear stage oocyte; pv: primary vitellogenic stage oocyte; pz: perinuclear zone; sf: shrunken follicle; vg: vacuolated globule). 19 int. j. aquat. biol. (2019) 7(1): 14-26 except the perinuclear zone. in contrast, the ovaries of control crabs showed normal development with large proportion of pv oocytes occupied by small to large yolk globules (14.08±4.28 µm diameter) and vacuolated globules (20.99±4.22 µm diameter) in the peripheral ooplasm. stimulation of ovarian growth was also evidenced by the proliferation of oogonia and follicle cells in the germinal zone of treated ovaries (fig. 4 a-d). morphology and histology of ovary of control and treated crabs during secondary vitellogenic phase (december-january): leucine enkephalin treatment did not cause any appreciable change in the ovarian morphology. both control and treated ovaries were bright yellow in coloration. administration of leucine enkephalin induced a significant rise in the ovarian index (p<0.05) as compared to the control crabs. likewise, the treated groups had higher mean oocyte diameter values which were statistically significant (p<0.001) than the controls (table 1). leucine enkephalin treatment during secondary vitellogenic phase indicated a greater proportion of sv3 oocytes (81%) with minor proportions of sv2 (17%) and sv1 (2%) oocytes. in contrast, the control ovaries contained mostly sv2 (52%) and svi (41%) with only a few sv3 (7%) oocytes (fig. 1c). the sv3 oocytes contained copious amounts of eosinophilic polygonal yolk platelets (56.14±4.28), narrowing the perinuclear zone to a thin strip. in such oocytes, the nucleus has a shrunken appearance, loses its rounded or spherical form with the advancement of yolk platelets towards the nucleus. there was an increase in the number and size of yolk globules (51.51±7.52 µm diameter) and vacuolated globules (43.97±9.21 µm in diameter) in sv1 oocytes of treated ovaries over controls (41.51±9.63 µm and 37.97±8.27 µm in diameter, respectively). moreover, the ovaries of injected crabs displayed signs of follicle cell proliferation and growth of younger oocytes (fig. 5 ad). morphology and histology of ovary of control and treated crabs during tertiary vitellogenic phase figure 3. treated previtellogenic ovary exhibiting follicle and oogonial proliferation and accumulation of yolk globules and vacuolated globules characteristic of sv1 oocytes (a) primary vitellogenic oocyte displaying accumulation of highly basophilic large yolk globules and vacuolated globules characteristic of sv1 oocytes; (b) follicle cell proliferation; (c) oogonial cell proliferation (fc: follicle cell; fn: follicle nucleus; n: nucleus; nu: nucleolus; oo: oogonia; vg: vacuolated globule; yg: yolk globule). 20 gopal and sudha devi / effect of leucine enkephalin on ovarian maturation (february-march): the administration of leucine enkephalin during this phase of vitellogenesis did not result in any significant change neither in the morphology nor in the histology of the ovary. the ovary appeared bright orange in both control and experimental crabs. the mean ovarian index (4.502±0.38) and oocyte diameter values (1384.60±10.96 µm) of the treated crabs were slightly but not significantly, larger than the corresponding values of the control crabs (4.48±0.42 and 1363.44±11.58 µm, respectively) (table 1). the ovaries of both control and experimental crabs were compactly filled with a larger proportion of tv oocytes (95 and 97%, respectively) followed by a few cn oocytes (5 and 3%, respectively) (fig. 1d). the ooplasm of tv oocytes was abundant with eosinophilic yolk platelets which form a homogeneous matrix. the nuclei and nucleoli were not apparent. the follicle cells were no longer visible around the tv oocytes (fig. 6 a-d). discussions the current study focused on the effect of administration of the neurotransmitter leucine enkephalin on ovarian maturation in the freshwater crab t. schirnerae. ovarian maturation was assessed by both macroscopic and microscopic observations such as ovarian index, mean oocyte diameter, oocyte proportion values and histological examinations of the ovaries of control and treated crabs. our observations in t. schirnerae revealed no remarkable changes in the ovarian morphology with leu-enk treatment when compared to the control ovaries. similar observations were made by reddy et al. (2013) in the giant freshwater prawn macrobrachium rosenbergii treated with serotonin. contrary results were obtained by kishori and reddy (2003) and kishori et al. (2012) in o. senex senex and figure 4. ovarian histology of control and experimental crabs during primary vitellogenic phase. (a) control ovary dominated by primary oocytes; (b) experimental ovary dominated by sv1 and sv2 oocytes; (c) injected ovary showing oogonial proliferation; (d) treated sv1 oocytes with yolk platelets, characteristic of sv2 oocytes (cn2: chromatin nucleolus stage i oocyte; cn3: chromatin nucleolus stage 3 oocyte; fn: follicle nucleus; n: nucleus; nu: nucleolus; oo: oogonia; pn: perinuclear stage oocyte; pv: primary vitellogenic stage oocyte; pz: perinuclear zone; sv1: secondary vitellogenic stage 1 oocyte; sv2: secondary vitellogenic stage 2 oocyte; vg: vacuolated globule; yg: yolk globule; yp: yolk platelet). 21 int. j. aquat. biol. (2019) 7(1): 14-26 reddy (2000) in p. indicus. the present study observed significantly higher ovarian indices in the experimental females than the control crabs. several authors described increased ovarian index as an outcome of leu-enk administration. kishori and reddy (2003), kishori et al. (2012) and reddy (2000) observed increased ovarian index values in o. senex senex and p. indicus injected with leu-enk. increased ovarian indices were also reported with other stimulatory neurotransmitters like serotonin, nalaxone, spiperone and octopamine. treatment of serotonin drastically enhanced the ovarian index value in u. pugilator (richardson et al., 1991), the freshwater crayfish procamburus clarkii (kulkarni et al., 1992), the freshwater field crab paratelphusa hydrodromous (ragunathan and arivazhagan, 1999), white shrimps litopenaeus vannamei and l. stylirostris (vaca and alfaro, 2000; alfaro et al., 2004), p. semisculatus (kumulu, 2005), m. rosenbergii (meeratana et al., 2006; aprajitha et al., 2014), m. nipponense (forsatkar et al., 2013) and the mole crab emerita emeritus (akhila et al., 2016). prasad et al. (2014) noticed elevated gonadosomatic index values in the freshwater crab barytelphusa guerini, administered with serotonin and nalaxone. food containing spiperone and spiperone treatment boosted up the gonadosomatic indices in p. clarkii (rodriguez et al., 2002), charybdis granulata (zapata et al., 2003), aegla platensis (cahansky et al., 2008), a. uruguayana (castiglioni et al., 2009) and cherax quadricarinatus (cahansky et al., 2011). tinikul et al. (2009) suggested a rise in the ovarian index in m. rosenbergii with octopamine treatment. ovarian index is often used to evaluate ovarian growth and larger ovarian indices are the results of greater number of oocytes developing in response to leu-enk injection. the results of this study clearly showed that leuenk was capable of increasing the mean oocyte diameter values in t. schirnerae. our observations figure 5. ovaries of control and experimental crabs during secondary vitellogenic phase. (a) control ovary with greater proportions of sv1 and sv2 oocytes; (b) treated ovary dominated by sv3 oocytes; (c) injected ovary populated by younger oocytes; (d) treated ovary displaying signs of follicle cell proliferation (cn1: chromatin nucleolus stage 1 oocyte; cn2: chromatin nucleolus stage 2 oocyte; cn3: chromatin nucleolus stage 3 oocyte; fc: follicle cell; fn: follicle nucleus; n: nucleus; nu: nucleolus; pz: perinuclear zone; sv1: secondary vitellogenic stage 1 oocyte; sv2: secondary vitellogenic stage 2 oocyte; sv3: secondary vitellogenic stage 3 oocyte; vg: vacuolated globule; yp: yolk platelet). 22 gopal and sudha devi / effect of leucine enkephalin on ovarian maturation were consistent with the findings of kishori et al. (2012) wherein a significant enhancement in the mean oocyte diameter value was reported for female o. senex senex administered with leu-enk. kulkarni et al. (1992), meeratana et al. (2006) and akhila et al. (2016) have recorded augmented oocyte diameter values in p. clarkii, m. rosenbergii and e. emeritus treated with serotonin. in b. guerini, the diameter of oocytes was significantly increased with serotonin and nalaxone injections (prasad et al., 2014). cahansky et al. (2011) observed a significant increase in the oocyte diameter of c. quadricarinatus treated with spiperone. by contrast, kishori and reddy (2003) demonstrated insignificant mean oocyte diameter values in leu-enk treated females of o. senex senex. the elevated oocyte diameter value of oocytes in experimental ovaries of the present investigation is suggestive of increased accumulation of lipoproteins and yolk granules in response to leu-enk injection. leucine enkephalin administration in t. schirnerae showed considerable changes in histology of the ovary compared to the controls. leucine enkephalin treatment induced previtellogenic ovary to grow into primary vitellogenic, primary vitellogenic to sv1 and sv2 and sv1 to sv3 stage as evidenced by the presence of a large number of pv oocytes with yolk globules and vacuolated globules in previtellogenic oocytes, increased width of perinuclear zone in pn oocytes, larger proportion of sv1 and sv2 oocytes in pv ovaries and sv3 oocytes in sv1 ovaries of treated crabs than the controls. this observation was wellsupported by the experimental evidence provided by kishori et al. (2012) in o. senex senex that the ovaries of leu-enk injected crabs were in vitellogenic stages, confirmed by the accumulation of yolk globules. reddy et al. (2013) observed that the ovary of serotonin injected m. rosenbergii possessed large number of mature oocytes whereas the control crabs exhibited more oogonia and early vitellogenic oocytes. likewise in p. hydrodromous, ragunathan and arivazhagan (1999) noticed increased ooplasmic volume with much reduced nucleus in oocytes treated with serotonin. meeratana et al. (2006) observed a rise in the number of oocytes developing into later stages figure 6. photomicrograph of ovaries of control and treated crabs during tertiary vitellogenic phase. (a and c) control and treated ovaries with compactly packed tv oocytes; (b and d) enlarged view of tv oocytes of control and treated crabs with eosinophilic yolk platelets (cn3: chromatin nucleolus stage 3 oocyte; tv: tertiary vitellogenic stage oocyte; yp: yolk platelet). 23 int. j. aquat. biol. (2019) 7(1): 14-26 of maturation in the ovaries of m. rosenbergii injected with serotonin. babu and reddy (2014) documented that the ovaries of p. monodon treated with serotonin were in the late maturation phase with majority of the oocytes laden with yolk granules. aprajitha et al. (2014) and akhila et al. (2016) made observations on the accumulation of large number of yolk globules in ovaries of m. rosenbergii and e. emeritus treated with serotonin. chen et al. (2003), santhoshi et al. (2008) and reddy et al. (2013) demonstrated increased hemolymph vitellogenin levels in m. rosenbergii, fenneropenaeus indicus and o. senex senex as a consequence of serotonin injection. the increased accumulation of yolk globules and vacuolated globules, a typical feature of vitellogenesis, in experimental over controls in the current investigation may possibly suggest a positive role for leu-enk in the regulation of vitellogenesis. in t. schirnerae, the conversion of previtellogenic ovary to early vitellogenic, early to middle and middle to late vitellogenic ovaries in the treated crabs compared to the controls probably indicate the stimulatory effect of leu-enk on the ovaries either by triggering the release of the gonad stimulating hormone (gsh) synthesized and released from the brain or thoracic ganglion or by blocking the release of the gonad inhibiting hormone (gih) synthesized and secreted by the xo-sg complex of the eyestalks, or both. in decapod crustaceans, ovarian growth and maturation are known to be regulated, directly or indirectly by an array of factors: neurohormones like gsh (gomez, 1965; eastman-reks and fingerman, 1984) and gih (otsu, 1963; bomirski et al., 1981), terpenoid hormone methyl farnesoate (mf) synthesized and released by the mandibular organ (laufer et al., 1998), ecdysteroids, synthesized and secreted by the y organ, vertebrate type steroids (estradiol and progesterone) from the ovary or hepatopancreas (subramoniam, 2000) and neurotransmitters (both stimulatory and inhibitory). of these endocrine modulators, the neurotransmitters indirectly influence ovarian growth and maturation by modulating the release of gsh or gih (luschen et al., 1993). stimulatory neuropeptides like leu-enk, serotonin, spiperone, nalaxone and octopamine stimulate the release of gsh (richardson et al., 1991; kulkarni et al., 1991) and thus stimulate vitellgenesis by acting directly on vitellogenin synthesizing sites (kulkarni et al., 1981; sarojini et al., 1995) while inhibitory neurotransmitters like dopamine and methenk stimulate gih which in turn act directly on oocytes by blocking vitellogenin uptake and thus yolk protein synthesis (nagaraju, 2011; swetha et al., 2011). conclusion the results of the present study clearly indicate that leu-enk has a stimulatory effect on ovarian maturation in t. schirnerae, thus shortening the period of maturation of ovary. leucine enkephalin seems to be a cheap and effective alternative to eyestalk ablation or to the use of other endocrine modulators to induce ovarian maturation in species of commercial importance. further studies are needed to check the efficacy of this neurotransmitter as a supplement in diet to induce ovarian maturation in this species. acknowledgements the financial assistance in the form of inspire fellowship from the department of science & technology, new delhi, india (dst/inspire fellowship/2015/if150375) is gratefully acknowledged. references akhila n., sangeetha s., deepa rani s., munuswamy n. 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(2020) 8(1): 9-17 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article diversity of anoxygenic phototrophic bacteria in anaerobic lagoons and facultative stabilization pond used in treatment of sewage nora katia saavedra del aguila hoffmann*1, iolanda cristina silveira duarte2, maria bernadete amâncio varesche silva3 1school of civil and environmental engineering, federal university of goiás ufg. av. universitária, no. 1488. setor universitário. cep 74605-220, goiânia – goiás, brazil. 2department of biology, federal university of são carlos ufscar, joão leme dos santos highway, km 101, cep 18052-780, sorocaba, são paulo, brazil. 3laboratory of biological processes, department of hydraulics and sanitation, engineering school of são carlos – university of são paulo (eesc – usp) campus ii, são carlos, sp, cep 13563-120, brazil. s article history: received 20 september 2019 accepted 25 december 2019 available online 2 5 february 2020 keywords: blooms sulfur pcr/dgge pufm abstract: the objective of this study was to identify the anoxygenic phototrophic bacteria in the anaerobic lagoons and facultative stabilization pond of the vale do ribeira, cajati, são paulo, brazil, and their correlation with physical and chemical parameters of the ponds’ water. the samples were collected seasonally (spring, summer, autumn and winter) in the sub-surface, intermediate layer and sediment-water interface. we used the pcr/dgge with pufm 557fgc and pufm 750r primers specific to the reaction center of the photosynthetic phototrophic bacteria for their identification. the amplification products were separated by electrophoresis on denaturing gradient gel. from the bands cut out and sequenced from dgge, the identified bacteria were rhodopseudomonas palustris (99% similarity), chromatium sp. (92%), thiocapsa sp. (90%), rhodospirillum sp. (95%), roseobacter sp. (93%) and other uncultured bacteria. introduction the anoxygenic phototrophic bacteria are part of a metabolically diverse group of microorganisms, grouped according to the use of sulfate and specific pigmentation. they play an important role in anaerobic degradation of organic matter, as primary producers or consumers of the reduced organic compounds (madigan et al., 2006). stagnant water exposed to sunlight facilitates sulfate production by decomposing organic matter, allowing the growth of anoxygenic phototrophic bacteria. in such environments, these bacteria often occur in large numbers and are visible because they produce pink, red, brown and green blooms. in general, blooms of the anoxygenic phototrophic bacteria contain mostly purple and green sulfur bacteria (hiraishi et al., 1995). phototrophic bacteria species are generally found in concentrations of 105 cells.ml-1 in environments with high amounts of the organic matter. moreover, they coexist with heterotrophic anaerobic microorganisms in hypolimnion, or in aquatic systems surface such as *correspondence: nora katia saavedra del aguila hoffmann doi: https://doi.org/10.22034/ijab.v8i1.688 e-mail: kasaavedra@ufg.br eutrophic lagoons or wastewater treatment plants. in wastewater treatment processes, anoxygenic phototrophic bacteria inhabit symbiosis with other heterotrophs and photoautotrophs (ng et al., 1989). anoxygenic phototrophic bacteria often bloom in waste stabilisation ponds used to treat sanitary sewage and form a purple layer on their surface. these blooms can cause malfunctions and toxicity in these lagoons, because under anoxic and/or facultative conditions, color of the wastewater appears purple due to the predominance of purple anoxygenic phototrophic bacteria, and consequently the presence of sulfate. as a result of this massive growth, the physicochemical quality of pond effluents can be seriously affected, such as color, odor and concentration of suspended solids (belila et al., 2013a, b). therefore, study of the conditions that provide these blooms, the microbial diversity, potential for organic matter removal and establishment of relations between such knowledge, allow the comprehension of the system metabolism, and contributes to the efficiency maintenance of the 10 hoffmann et al./ anoxygenic phototrophic bacteria in anaerobic lagoons and facultative stabilization pond stabilisation ponds, which are particularly sensitive to environmental changes such as temperature, nutrients and organic load instability (belila et al., 2011). in addition to visual verification of the phototrophic bacteria’s blooms, it is possible to use molecular methods to characterize a microbial community and identify microorganisms. the analysis of nucleic substances extracted from the full community member of an environment allows the phylogenetic detection and identification of most organisms, including those are not cultivated. the denaturing gradient gel electrophoresis (dgge) is widely used to describe a genetic diversity of microorganisms in different environments. depending on the objective of the study, the total number of bands visualised in the dgge gel indicates a spatial and temporal diversity of variations in the determined environment. through this technique, it is possible to identify microorganisms by sequencing selected bands in the dgge. the combination of these molecular methods with those related to microbiology allows the detailed analysis of the microorganisms’ diversity in the system. achenbach et al. (2001) reported the application of specific primers to evaluate green sulfur bacteria, non-sulfur green bacteria and heliobacteria. in addition, specific photosynthetic primers, known as pufm, were used for anoxygenic phototrophic abnormalities detection, that encode a m subunit of the photosynthetic reaction center, which is universally distributed for purple photosynthetic anoxygenic bacteria. pufm primers amplify, not only purple and non-sulfur anoxygenic phototrophic bacteria dna, but also that of chloroflexus sp. (nonsulfur green bacteria). though the structure of the purple bacterium reaction center is similar to the photosystem ii of green plants, pufm primers do not amplify cyanobacterial dna, which indicates its specificity for anoxygenic phototrophic bacteria. this study aimed to identify the anoxygenic phototrophic bacteria in anaerobic lagoon and facultative stabilization ponds of the vale do ribeira (cajati, são paulo, brazil) and their correlations with physical and chemical parameters of water from the ponds verified at different times of the year and different collection times. identification is performed using a partial sequencing technique of pufm photosynthetic reaction center of the purple anoxygenic phototrophic bacteria. materials and methods the selected ecosystem for this study was the cajati microbasin (são paulo brasil), in the baixo ribeira de iguape region, located at 23º30’-25º30’ south latitude and 46º50’-50º00’ west longitude, in an area of 24.980 km2. samples were taken from anaerobic and facultative ponds of cajati, at subsurface (s) and intermediate layer (ci), and water-sediment interface (as) twice a day (14:00 h and 02:00 h), as well as the affluent raw sewage and final effluent were sampled from the stabilisation ponds. in the anaerobic lagoon, the intermediate layer and water-sediment interface were collected at 1.50 and 3.00 m below the surface, respectively. in the facultative lagoon, the collections were made at 0.70 and 1.20 m from the surface. the specific collection point in the lagoons present circulation and mixing due to the wind at different times of year. microbial diversity analysis: samples were collected using van dorn sampler, transferred to flasks and subjected to a temperature of 4°c. for measuring biomass concentration, 200 ml of the pond water was centrifuged at 6000 rpm for 10 min at 4°c. the created pellet was washed three times with 1x pbs phosphate (137.0 mm nacl, 2.6 mm kcl, 1.7 mm kh2po4, 10.0 mm na2hpo4, ph=7.4). dna extraction was performed using glass beads, phenol and chloroform according to the griffiths protocol et al. (2000). in polymerase chain reaction (pcr) for the amplification of the photosynthetic reaction center of the purple anoxygenic phototrophic bacteria, pufm.557f (5'-cgcacctggactggac3') with gc clamp and pufm.750r (5'-cccatggtc cagcgccagaa-3') primers were used (achenbach et al., 2001). pcr conditions were as follows: 1.5 μl mgcl2 (50 mm), 5 μl 10x pcr buffer, 5 μl dntps (2 mm each), 0.5 μl each primer (100 μmol, invitrogen), 0.5 u amplitaq dna polymerase (5 u.ml-1), containing 2 μl template dna (50-100 ng) 11 int. j. aquat. biol. (2020) 8(1): 9-17 completing at an ultrafurified rate up to 50 μl. amplification cycles were as follows: denaturation at 94°c for 3 min, followed by 30 cycles of 1 min denaturation at 94°c, 1 min annealing at 55°c, and 1 min extension at 72°c; and final extension at 72°c for 10 min. the amplified product was analyzed on 1% agarose gel for use in dgge (muyzer et al., 1993). the samples were transferred to 10% acrylamide/ bis-acrylamide gel prepared from 40% acrylamide/ bis-acrylamide (38.93 g acrylamide and 1.07 g bisacrylamide dissolved in 100 ml ultrapure water) with 20 to 80% denaturants (urea and formamide) for the electrophoresis gel in dgge. the electrophoretic run time of 16 hrs was used in tae 1x buffer, with temperature of 65°c and constant voltage of 75 v. the dgge gel was stained in ethidium bromide for 20 min and then visualised in uv transilluminator. the resulting predominant bands were cut out of the gel and eluted separately in 20 μl of ultrapure water and stored at 4°c overnight. the eluted dna was reamplified using the respective primer set (without gc clamp), purified and applied to ge healthcare's megabace 1000 sequencer. the identification of organisms was made using a set of sequencing reagents based on fluorochromelabeled dideoxynucleotides (dye terminators). the concentrations of dna template and primers followed the manufacturer’s instructions. sequencing reactions were purified on sephadex g50 column and applied to ge healthcare's megabace 1000 sequencer. the analysis was performed by the sequence analyzer 3.0 software with cimarron 3.12 basecaller. the sequences were checked using dna star program (seqman) and then compared with other sequences previously in the genbank using the basic local aligment search tool (blastn). aligned nucleotides were used to make the consensus phylogenetic tree using the neighbor-joining algorithm (kimura et al., 1980) in mega 3.1 software (kumar et al., 2004). the known sequences of r. palustris (cp000301), r. rubrum (j03731) and chromatium vinosum (aj544223) were retrieved from the genbank to identify the sampled microorganisms. physicochemical analysis: the dissolved oxygen, ph and temperature were measured using membrane electrode (yellow springer, 556 mps). solar radiation was measured with a quanta-meter ly-cor radiometer with a sensitivity of 400-700 nm. chemical oxygen demand (cod), and total and dissolved nutrients determinations were measure based on apha (2012). samples for analysis of volatile suspended solids were filtered on 0.45 μm pore diameter glass fiber membranes. results and discussions physical and chemical characteristics of the stabilisation ponds: the raw tributary organic matter concentration was below the recommended values for strong sewage. according to tchobanoglous (2016), domestic sewage is considered strong when it presents cod of 1,000 mg.l-1. in the present study, the affluent cod ranged from 50 mg.l-1 in spring to 290 mg.l-1 in summer. total phosphorus concentration ranged 0.6 mg.l-1 in summer to 7.0 mg.l-1 in spring; total nitrogen kjeldahl (ntk) ranged 13.1 mg.l-1 in spring to 39.9 mg.l-1 in autumn; ammonia nitrogen ranged 1.8 mg.l-1 in summer to 36.8 mg.l-1 in spring. all these values were below the values considered strong for sanitary sewage and equal to 15.0 mg.l-1; 85.0 mg.l-1 and 50.0 mg.l-1, respectively. nitrate remained above the recommended (0.0 mg.l-1), as values ranged 1.3 mg.l-1 in spring to 3.7 mg.l-1 in summer. according to andrade neto (1997), in the stabilisation ponds without final effluents, cod range values should be between 200-300 mg.l-1, ntk 28.045.0 mg.l-1 and total phosphorus 4.5-6.9 mg.l-1. in the present study, the organic matter was below this range, except in winter reaching 300.4 mg.l-1. ntk values were below this range, except in spring with 28.7 mg.l-1. total phosphorus values were less than the recommended one by andrade neto (1997). during four seasons, ph, dissolved oxygen and solar radiation fluctuated in the stabilisation lagoons. in the anaerobic lagoon, the solar radiation were similar in summer and autumn at 14:00 h, with 245.5 and 264.6 μem-2s-1, respectively, which were possibly propitious for the development of anoxygenic 12 hoffmann et al./ anoxygenic phototrophic bacteria in anaerobic lagoons and facultative stabilization pond phototrophic bacteria, where a larger number of populations of these microorganisms were reported. in spring (545.8 μem-2s-1) and winter (574.8 μem-2s-1), the solar radiation was not favorable for the proper development of anoxygenic phototrophic bacteria. during summer, in the facultative lagoon, a higher light intensity was registered at 14:00 h (1344 μem-2s1) and a smaller number of populations of the anoxygenic phototrophic bacteria, compared to the anaerobic lagoons. the solar radiation during fall was 409.9 μem-2s-1, which determined a lower number of anoxygenic phototrophic bacteria compared to those of the anaerobic lagoon at the same period. according to dgge standard band profiles, anoxygenic phototrophic bacteria prefer a certain light intensity for their optimal development. in the this work, this range was 245.50-264.61 μem-2s-1, for the 14:00 h. collection time, where 6, 10 and 10 bands were observed in the subsurface, intermediate layer and sediment water interface in summer, and 14, 17 and 18 bands in fall, respectively. according to belila et al. (2013), the quality of the light spectrum stimulates different distribution of the photosynthetic bacteria, occupying different ecological niches along the visible spectrum, encouraging their coexistence. the minimum and maximum ph in the anaerobic lagoon in both collection times were 6.81 and 10.91, respectively. during fall, the ph were lower i.e. 6.81 and 9.08, respectively. in this ph range, a greater diversity of the anoxygenic phototrophic bacteria was found. in the facultative lagoon, the minimum ph value was 6.98 recorded in fall, and maximum of 12.05 in summer. also, in the facultative lagoon, the lowest ph along with greater diversity of these microorganisms were observed in autumn. this range of ph in both lagoon system possibly allow a better development of the anoxygenic phototrophic bacteria. temperature ranged 19.6 to 35.1°c during the seasons at both collection times. the highest temperature was recorded in autumn, when a greater diversity of anoxygenic phototrophic bacteria was verified. above-mentioned temperature range is probably favored for development of these microorganisms. according to von sperling et al. (2003), optimum removal efficiency ranges may serve as a reference for most brazilian regions, mentioned as 20-25ºc, in the coldest month of the year. the minimum temperature of 18.4°c was found in winter and a maximum of 34.3°c in autumn. greater temperature values were recorded in the facultative lagoon in fall collection, where the lower number of the anoxygenic phototrophic bacteria was detected in relation to the anaerobic lagoon at the same time. the lowest dissolved oxygen (do) was close to zero, while the maximum one was 8.22 mg.l-1. do on the subsurface during collections at different times ranged 0.14 to 8.22 mg.l-1. in fall, in the intermediate layer and water-sediment interface, no dissolved oxygen was detected. during this period, a greater diversity of anoxygenic phototrophic bacteria was verified at the sampling points, possibly favored for this facultative environment. minimum do of 0.02 mg.l-1 and maximum one of 9.54 mg.l-1 were recorded in summer. do was variable during four seasons, different sampling points and collection times. its values in autumn were higher than those of the anaerobic lagoon, where a greater diversity of anoxygenic phototrophic bacteria was observed. possibly, the lower do in the anaerobic lagoon favored the development of these microorganisms. a higher removal of organic material (cod) was verified in the facultative lagoon, with 25.6%. the removal of 62.4% of volatile suspended solids was verified, in spring, with a temperature of 22.9˚c for affluent and 24.3˚c for effluent. removal of 60.2% of ntk was obtained in fall (affluent temperature of 27.2˚c and effluent temperature of 32.4˚c) and 58.8% for total phosphorus in fall. higher nitrate removal was observed in spring, with 14.6%, and 73.0% of ammonia nitrogen in fall. higher removal of total dissolved phosphorus and 95.8% for orthophosphate were verified in spring. in this work, the nutritional conditions were verified in different collections and sample locations probably favored the development of anaerobic, facultative anaerobic, microaerophilic and aerobic micro-organisms. when considering the temperature, development of the psychrophilic microorganisms in 13 int. j. aquat. biol. (2020) 8(1): 9-17 winter and mesophilic during other times were favored. the variation of ph noted in the ponds suggests presence of the neutrophils and alkaliphiles microorganisms. evaluating the nutrients i.e. ammonia nitrogen, nitrite and nitrate, the highest temperature was verified in fall, which favored a major metabolic activity, as well as a faster ammonia nitrogen oxidation. almost every ammonia nitrogen was oxidized to nitrate in the facultative pond. do was not limited by nitrifying activity associated to the highest temperatures registered during summer and fall. in the anaerobic pond, the ammonia nitrogen concentration was higher than the facultative pond during the study, associated to the greatest organic charge and ammonification process in anaerobic environment. on the other side, there was a major nitrifying activity in the facultative pond, mainly during summer and fall, possibly due to the temperature and algal biomass (primary production) increase, consequently, a greater oxygenation of the environment. during this period, it was noticed that almost all ammonia nitrogen was oxidized to nitrate in the facultative pond, contrary to the anaerobic pond. hence, there was a bacteria population raise during summer and fall, both autotrophs (including nitrifying) and heterotrophs confirmed by the major diversity bacteria domain in the dgge standard band profile. anoxygenic phototrophic bacteria identification: sampling from the stabilisation ponds were done two times (14:00 h and 02:00 h) to verify presence of different anoxygenic phototropic bacteria in three sampling locations (subsurface: s; intermediate layer: ci; and water-sediment interface: as), as well as different times of year (spring: p; summer: v; fall: o, and winter: i). table 1 shows the cut-off dgge bands and phylogenetic approximation with the sequences of anoxygenic phototrophic bacteria deposited in genebank, samples from anaerobic and facultative lagoons at different times of year and different collection points. approximately 229 base pairs (bp) were sequenced. in the anaerobic pond, the bands 3, 4, 6, 7 and 9 (uncultivated bacteria), 8 (rhodospirillum sp. ay390602), and 11 (rhodopseudomonas palustris, ab015977) were verified. the sequence for band 8 was similar to rhodospirillum sp., it is a nonsulphurous, gram negative, anoxygenic phototrophic purple bacterium with flagella. such bacteria grow preferentially under anaerobic photoheterotrophic conditions, in the presence of various organic compounds, or under dark microaerobic conditions (beja et al., 2002). rhodospirillum and rhodoferax are known for their key roles in carbon and nitrogen metabolism, and often found in sewage, activated sludge and wastewater treatment plants (wan et al., 2011; belila et al., 2013; hulsen et al., 2014). the sequence of band 11 was similar to r. palustris, a purple non-sulfur phototrophic, gram negative bacterium. its photoautotrophic growth is possible using hydrogen, sulfite and thiosulfate as electron donors in the presence of yeast extract and carbon dioxide. photoheterotrophic growth is occurred in the presence of organic substrates such as benzoate, acetate, pyruvate, malate, fumarate, lactate and succinate. such bacteria may grow under dark microaerobic and aerobic conditions, or under anaerobic conditions in the presence of some organic substrates. sulphate can be used as a source of sulfur, while ammonia, dinitrogen, and some amino acids are used as nitrogen sources (madigan et al., 2006). these microorganisms represented part of the autochthonous bacterial community of the anaerobic lagoon, probably responsible for the blooms in the stabilisation lagoons, causing purple color in the surface. in general, purple non-sulphurous phototrophic bacteria are characterised by their metabolic versatility, including photoautotrophic, chemoheterotrophic, and photoheterotrophic organisms, using sugars and a variety of organic acids as electron donors and carbon sources (kim et al., 2004; hulsen et al., 2014). in the facultative pond, the bacteria similar to thiocapsa sp. (aj544223, band 5) were found. thiocapsa sp. is a purple phototrophic sulfur, and gram negative bacteria. under anaerobic conditions, it has phototrophic growth and under aerobic or 14 hoffmann et al./ anoxygenic phototrophic bacteria in anaerobic lagoons and facultative stabilization pond microaerophilic conditions, it has chemoautotrophic or chemoorganotrophic growth in the darkness. according to wieland et al. (2003), the purple sulfur bacteria release sulfate, which can be used as an electron acceptor in anaerobic respiration by sulfate reducing bacteria, producing sulfide. the latter compound may have been used by phototrophic bacteria as an electron donor in anoxygenic photosynthesis. belila et al. (2013) evaluated red-coloured blooms in stabilisation ponds for domestic sewage treatment (anaerobic pond, facultative pond and two serial maturation ponds), and identified anoxygenic phototrophic bacteria using the pcr/dgge technique (with pufm 557fgc and pufm 750r) from the selected and sequenced dgge bands. molecular analysis revealed that 59.3% of pufm sequences corresponded to non-sulfur purple bacteria (rhodospirillum, rhodoferax, rhodobaca, rhodobacter, rhodoplanes and phaeospirillum); 27.8% to purple sulfur bacteria (thiocapsa, roseopersicin and chromatium weissei), and 12.9% to purple anoxygenic phototrophic bacteria (roseobacter sp. and erythrobacter sp.). in both ponds, band 1 (chromatium sp., abo11811), 2 (roseobacter sp., ay675565), and 10 (chromatium sp., d50647) were verified. band 1 was similar to chromatium sp., gram-negative purple table 1. sequence information obtained from dgge cropped bands with pufm primers from anaerobic and facultative lagoons. band microorganis m anaerobic pond facultative pond #access similarity % reference 14:00 h 02:00 h 14:00 h 02:00 h band 1 chromatium sp. sp, cip, asp, sv, civ, asv, so, cio, aso, si, cii, asi sp, cip, asp, sv, civ, asv, so, cio, aso, si, cii, asi sp, cip, asp, sv, civ, asv, so, cio, aso sp, cip, asp, sv, civ, asv, so, cio, aso abo11811 92 nagashima (1998) band 2 roseobacter sp. sp, cip, asp, sv, civ, asv, so, cio, aso, si, cii, asi sp, cip, asp, sv, civ, asv, so, cio, aso, si, cii, asi sp, cip, asp, sv, civ, asv, so, cio, aso sp, cip, asp, sv, civ, asv, so, cio, aso ay675565 91 oz et al. (2005) band 3 uncultivated bacteria so, cio, aso am162697 99 ranchou-peyruse et al. (2006) band 4 uncultivated bacteria so, cio, aso ay044247 89 beja et al. (2002) band 5 thiocapsa sp. so, cio, aso, si, cii, asi aj544223 90 wieland et al. (2003) band 6 uncultivated bacteria civ, asv, aso am162737 88 ranchou-peyruse et al. (2005) band 7 uncultivated bacteria civ, asv dq080988 89 yutin et al. (2000). not published (genbank) band 8 rhodospirillu m sp. so, cio so, cio ay390602 95 lee et al. (2003) band 9 uncultivated bacteria aso ay044246 90 beja et al. (2002) band 10 chromatium sp. sp, sv, so, cio, si, cii, asi sv, so, cio, si so, cio, aso sp, cip, asp, so, cio, aso d50647 91 nagashima et al. (1997) band 11 rhodopseudo monas palustris civ, asv, cio, aso, civ, asv, cio, aso, ab015977 99 beja et al. (2002) 15 int. j. aquat. biol. (2020) 8(1): 9-17 sulfur phototrophic bacteria. chromatium species present flagella with no gas vacuoles and intracellular sulfur granules in photoautotrophic growth in the presence of hydrogen sulfide. under microaerophilic conditions, they grow under chemoautotrophic and mixotrophic conditions. in addition, they may grow under photoheterotrophic conditions in the presence of ammonium acetate (madigan, 2006). this microorganism, which survives in microaerophilic conditions, possibly uses sulfur compounds and organic acids as electron donors in anoxygenic photosynthesis. when using sulfide in photoautotrophic growth, intracellular sulfur accumulates, which will be oxidized culminating with increased sulfate. in general, such bacteria grow close to sulfatereducing bacteria and both actively participate through syntrophic relations in the establishment of the sulfur cycle. the sequence for band 2 was similar to roseobacter sp., gram-negative anoxygenic phototrophic bacterium with one or two flagella, of the α-proteobacterium group. however, it performs aerobic anoxygenic photosynthesis, i.e. requiring oxygen for growth and bacteriochlorophylla (bchla) syntheses. these microorganisms are not able to grow under strict photoautotrophic conditions as they require organic carbon supplementation (oz et al., 2005). at 14:00 h, in both lagoons, bands 3, 4, 6, 7 and 9 (uncultivated bacteria) and 5 (thiocapsa sp.) were figure 1. consensus phylogenetic tree based on cut-off dgge sequences with primers from the pufm photosynthetic center, obtained from anaerobic and facultative lagoons. values present in tree nodes indicate percentages that branch repeated (500 bootstrap resamples). 16 hoffmann et al./ anoxygenic phototrophic bacteria in anaerobic lagoons and facultative stabilization pond verified. the other sequences at both times indicate the presence of anoxygenic phototrophic bacteria day and night, contributing in the degradation of the organic matter in the stabilisation ponds. the primer sets of pufm gene not only detected sulphurous and non-sulphurous purple bacteria, but also aerobic anoxygenic phototrophic bacteria (roseobacter sp.), as verified by ranchou-peyruse et al. (2006). however, such microorganisms are also capable to grow in an anaerobic environment. figure 1 shows the result of phylogenetic tree based on the sequence of the organisms from the stabilisation lagoons. similarity ranges 88-99%, indicating phylogenetic diversity of microorganisms, possibly due to the vertical transfer of genes from the photosynthetic center, which occurs between photosynthetic bacteria (karr et al., 2003). conclusions although bacteria blooms were not found during this study, it was possible to verify their presence in anaerobic and facultative stabilising ponds. in both lagoons, it was detected a major number of bacteria during the fall. in spring, it was ascertained a lower number of bands in dgge profile. a greater number of dgge bands of bacteria was found in the anaerobic lagoon compared to the facultative lagoon, this was probably facilitated by the lower concentrations of do, observed in the first lagoon. it is very important to keep track of these communities to maintain the operation and guarantee the efficiency of stabilising lagoons. researches on evaluations of seasonal and temporal diversity of bacteria community in stabilizing ponds are rare. thereby, there is not much knowledge concerning the variability of water column and its connections to the system nutritional conditions. acknowledgement the authors thank cnpq, for providiing scholarship, and fapesp for the financial support of this research. references achenbach l.a., carey j, madigan m.t. 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(2015) 3(2): 114-118 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology short communication length-weight relationship of mystus tengara (ham.-buch., 1822), a freshwater catfish of indian subcontinent sandipan gupta*,1samir banerjee aquaculture research unit, department of zoology, university of calcutta, 35, ballygunge circular road, kolkata700019, west bengal, india. article history: received 19 january 2015 accepted 3 march 2015 available online 2 5 april 2015 keywords: tengara mystus growth pattern morphometrics bagridae abstract: length-weight relationship is the most commonly used analysis which has been used for several purposes in fisheries field among which estimation of weight from length is the most popular one. the present study has been performed to analyze the length-weight relationship of mystus tengara, a freshwater catfish of indian subcontinent. total length and body weight of the studied specimens have been observed to vary from 7.2-11.3 cm (male), 7.3-11.7 cm (female) and 3.43-13.63 g (male), 2.83-14.88 g (female). the calculated regression coefficient (b) values are 2.941, 3.119 and 3.071 for male, female and combined sex, respectively; thus depicting negative allometric growth for male; while positive allometric growth for female and combined sex of this fish species. the correlation coefficient values (0.94, 0.95 and 0.95 for male, female and combined sex, respectively) are suggesting a significant relationship between length and weight of the studied fish. the present study provides the first baseline information on the length weight relationship of m. tengara which will be beneficial for future management of this fish species. introduction length-weight relationship (lwr) is one of the most commonly used analyses of fisheries data (mendes et al., 2004). it has been widely used in fish biology with several purposes: to predict weight from length measure for yield assessment, to calculate the standing crop biomass, to estimate weight at age, stock assessment, to evaluate index of well-being of fish population, to assess age structure and function of fish populations, growth studies, to assess fish population dynamics and growth, to make morphometric comparisons between species and populations and life history comparisons between regions (pauly, 1993; petrakis and stergiou, 1995; goncalves et al., 1997; haimovici and velasco, 2000; morato et al., 2001; stergiou and moutopoulos, 2001; moutopoulos and stergiou, 2002; ozaydin et al., 2007). thus this parameter is of great importance in fishery assessments, more * corresponding author: sandipan gupta e-mail address: sandipangupta2007@gmail.com importantly for proper exploitation and management of fish population (haimovici and velasco, 2000). mystus tengara is a freshwater species which is distributed throughout the indian subcontinent including india, bangladesh, pakistan, nepal and afghanistan (talwar and jhingran, 1991; petr, 1999). this fish species is a popular food fish due to its high nutritional value with good amount of protein and mineral content (siddiqui et al., 2010; gupta and banerjee, 2013). recently it has also made its entry in domestic ornamental fish markets of india (gupta and banerjee, 2012) and has been reported to have moderate export price too (gupta and banerjee, 2014). earlier, some works have been carried out on lwr of different species of the genus mystus in india, bangladesh and pakistan (hossain et al., 2006, 2009, 2012; krishna rao, 2007; begum et al., 2010; venkateshwarlu et al., 2007; sani et al., 2010; karna and panda, 2012; naeem et al., 2012; 115 gupta and banerjee/ length-weight relationship of mystus tengara srivastava et al., 2013; victor raj et al., 2014), but no such work so far has been reported on lwr of mystus tengara. therefore, to put the first basic information on lwr of mystus tengara the present study has been performed. materials and methods data collection: specimens of m. tengara have been collected on monthly basis from an undisturbed wetland at baruipur, south 24 paraganas, west bengal (latitude n 22°34', longitude e 88°43') for one year, from february, 2011 to january, 2012. samplings have been carried out during early morning in between 6.00 am to 8.00 am. in total 400 specimens of m. tengara have been collected during the entire study period to study the lwr. after collection, fish specimens have been transferred to ice-box and morphometric study has been performed after reaching the laboratory. total length (tl) in cm and total body weight (tbw) in gram have been measured to the nearest of 0.1 cm and 0.01 g, respectively. data analysis: lwr has been calculated for male, female and combined sex using the conventional formula described by le cren (1951) as follows: w = alb, where, w is the total body weight (g), l is the total length (cm), a is the intercept of the regression and b is the regression coefficient (slope or better to say growth rate). the parameters a and b have been estimated using the least-square linear regression method (zar, 1999) after log-transforming the above equation as follows: logw = a + b logl the significance of the regression has been assessed by anova. the determination coefficient (r2) has been used as an indicator of the quality of the linear regression (zar, 1999). in order to check if the calculated value of b is significantly different from 3 (isometric value), student’s t-test has been used. the value of b gives information on the kind of growth of fish; growth is isometric (no change of body shape as the fish grows) if b = 3 and the growth is allometric if b ≠ 3; negative allometric (fish becomes more slender as it grows) if b<3 and positive allometric (fish becomes relatively stouter or deeperbodied as it grows) if b>3 (bagenal and tesch, 1978). all statistical analysis have been considered at significance level of 1% (p<0.01). statistical analysis was performed using statistical software spss version 10.0 for windows (spss inc. chicago, usa). results and discussion sample size, weight and length range and calculated lwr parameters a, b and r2 has been represented in table 1. the linear relationships calculated for female, male and combined sex are as follows: logw = -2.159 + 3.119 logl, logw = -1.992 + 2.941 logl and logw = -2.115 + 3.071 logl. the values of regression coefficient b are 2.941, 3.119 and 3.071 for male, female and combined sex, respectively, which are suggesting negative allometric growth for male, and positive allometric growth for female and combined sex. as till now no such information is available on lwr of m. tengara, it is not possible to compare the current result with previous data. however, they have been compared with results of the earlier studies on the same aspect in different species of the genus mystus as follows: hossain et al. (2006) have reported 2.96, 3.13 and 3.05 as “b” values for male, female and combined sex no. body weight (gm) total length (cm) regression parameters 95% cl of a 95% cl of b r2 max. min. max. min. a b female 197 14.88 2.83 11.7 7.3 2.159 3.119 2.016-2.301 2.974-3.264 0.95 male 203 13.63 3.43 11.3 7.2 1.992 2.941 1.844-2.141 2.788-3.094 0.94 combined 400 14.88 2.83 11.7 7.2 2.115 3.071 2.014-2.214 2.968-3.173 0.95 table 1. descriptive statistics and estimated parameters of length-weight relationships for male, female and combined sex of mystus tengara. 116 int. j. aquat. biol. (2015) 3(2): 114-118 sex of m. vittatus while victor raj et al. (2014) have reported values of 2.405, 2.873 and 2.732 for the same. hossain et al. (2009) and srivastava et al. (2013) have documented “b” values of 3.27 and 2.88 for combined sex of m. vittatus, respectively. krishna rao (2007), sani et al. (2010), hossain et al. (2012) and karna and panda (2012) have documented “b” values of 2.83, 2.91, 3.21 and 3.009, respectively, for combined sex of m. cavasius while venkateshwarlu et al. (2007) have reported “b” values of 2.493 and 2.7402 for male and female of the same species. karna and panda (2012) have reported “b” value of 3.032 for combined sex of m. gulio while “b” values of 1.388 and 1.468 for male and female specimen have been reported by begum et al. (2010) for the same species. naeem et al. (2012) have reported “b” values of 2.64, 2.70 and 2.62 for male, female and combined sex of m. bleekeri, respectively. among the earlier documented information on “b” value of different species of mystus other than mystus tengara, only values reported by hossain et al. (2006) on m. vittatus are somehow close to the value of the current study. lwr parameters (a and b) of the fish have been reported to be affected by a number of factors like season, habitat, gonad maturity, sex, diet, stomach fullness, health, annual differences in environmental conditions, differences in the length range of the caught specimens, sampling procedure etc. (bagenal and tesch, 1978; froese, 2006). even though the change of “b” values depends primarily on the shape and fatness of the species, such differences in values “b” can be due to one or combination of most of the above listed factors including differences in the number of specimens examined, area/season effects and duration of sample collection etc. (moutopoulos and stergiou, 2002). according to bagenal and tesch (1978), goncalves et al. (1997) and ozaydin et al. (2007), the parameter b, unlike the parameter a, may vary seasonally, and even daily, and between habitats. muchlisin et al. (2010) have assumed that the b values are mostly affected by the availability of food and environmental conditions such as temperature, ph and dissolved oxygen. thus, lwr in fish can be affected by a number of factors discussed above; none of which has been considered in the present study. therefore, the present study provides first ever baseline information on the lwr of m. tengara which will be beneficial for future management of this fish species. acknowledgements authors are thankful to head, department of zoology, university of calcutta for providing the laboratory facility for the research work. financial assistance of university grants commission (ugc), india is gratefully acknowledged. references bagenal t.b., tesch f.w. (1978). age and growth. in: t.b. bagenal (ed.). methods of assessment of fish production in fresh waters, oxford blackwell scientific publication. pp. 101-136. begum m., pal h.k., islam m.a., alam m.j. 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(2019) 7(6): 357-367 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article antimicrobial properties of thymus vulgaris, origanum majorana and ziziphora clinopodioides combined essential oils and their effects on growth behavior of aeromonas hydrophila zein talal barakat1, ashkan zargar*1, ali taheri mirghaed1, alireza khosravi2, hamed paknejad3 1department of aquatic animal health, faculty of veterinary medicine, university of tehran, tehran, iran. 2department of mycology, faculty of veterinary medicine, university of tehran, tehran, iran. 3department of fisheries, faculty of fisheries and environmental sciences, gorgan university of agricultural sciences and natural resources, gorgan, iran. ss article history: received 20 july 2019 accepted 21 november 2019 available online 2 5 december 2019 keywords: herbal essential oils chemical composition growth curves antimicrobial assays abstract: this study aimed to determine the chemical composition of thymus vulgaris, origanum majorana and ziziphora clinopodioides and evaluate the antimicrobial properties of their combined essential oils i.e. t.o.z 50% t.v, 25% o.m and 25% z.c; t.o.z = 25% t.v, 50% o.m and 25% z.c and t.o.z = 25% t.v, 25% o.m and 50% z.c, against aeromonas hydrophila (in vitro). the compositions of the herbal essential oils were determined using gas chromatography–mass spectrometry (gc-ms) and the antimicrobial effects was conducted using agar-disc diffusion method, determination of mic and mbc, and bacterial growth curves determination based on od at 600 nm. the main compounds were thymol (40.60%) and limonene (15.98%) for t. vulgaris, carvacrol (57.86%) and thymol (13.54%) as the major compounds in o. magorana. regarding z. clinopodiodes, α-pinene (22.6%) and carvacrol (21.1%) represented the major constituents. base on the disc-diffusion results, t.o.z showed the best inhibition zone (26 mm). the inhibitory activity and bactericidal effect of t.o.z, unveiled by the mic and mbc values, was clearly the highest between all combined herbal oils. regarding the bacterial growth curves, the combined essential oils exhibited significant differences in all tested concentrations. the t.o.z was the most effective between the three tested combined oils at different temperature that cannot affect herbal oil performance. as conclusion, we suggested that the mixing of herbal oils with growth media was delaying exponential phase starting in the bacterial growth curves. introduction application of antibiotics has been led to increase in bacterial resistance, and therefore, scientists has recently focused on herbal extracts as a natural antioxidants and antibacterial substances (dhull et al., 2016). in addition, herbal extracts have been reported to promote various functions such as growth, appetite stimulation, stress resistance, immune functions, skin coloration, egg hatching rates, hematological and biochemical status as well as increasing disease resistance in aquaculture due to having different active components (yılmaz et al., 2010). the antimicrobial properties of medicinal plant’s essential oils are due to their hydrophobic characteristic that act over the lipids of the cell membrane, modifying its structure and turning it more permeable, and allowing the passage of ions and or other substances (millezi et al., 2012). *correspondence: ashkan zargar doi: https://doi.org/10.22034/ijab.v7i6.645 e-mail: azargar@ut.ac.ir according to ouedrhiri et al. (2016) essential oils are also accumulated in the cytoplasmic membrane, causing damages such as loss of function of selective barrier. thyme, thymus vulgaris, an aromatic plant of the lamiaceae family, is typical of the mediterranean area and extensively used as a culinary herb. its essential oil is utilized as flavor ingredients in a wide variety of food, beverage and confectionery products, as well as in perfumery for the scenting of soaps and lotions. because of its antiseptic, antispasmodic and antimicrobial properties, it is also used for medicinal purposes (cosentino et al., 1999). thyme has a strong antimicrobial and antioxidant activity due to its very high contents of thymol, p-cymene, carvacrol, eugenol, and 4-allylphenol (gültepe et al., 2014). the antioxidative effect of thyme is based on polyphenolic 358 barakat et al./ antimicrobial properties of t. vulgaris, o. majorana and z. clinopodioides compounds as flavonoids, while thymol and carvacrol have lower activity (selmi and sadok, 2008). marjoram, origanum majorana, belongs to the lamiaceae family with interesting pharmacological effects (dantas et al., 2016). it is an aromatic plant rich in essential oils and commercially grown in southern europe and in the mediterranean region (el-ashmawy et al., 2007; ramadan et al., 2012). marjoram has an extensive range of biological activity, such as antioxidant, antimicrobial, anti-inflammatory, antitumorigenic and hepatoprotective activities (abdel-massih et al., 2010; al dhaheri et al., 2013). ziziphora clinopodioides, a member of the family lamiaceae, grows in several regions throughout the world, especially middle east ( tian et al., 2011; shahbazi, 2017). its antibacterial, antioxidant, antifungal, anti-inflammatory properties is already reported (tian et al., 2010; kheirkhah et al., 2015). moreover, its essential oil has been proved to possess insecticidal activity (lolestani and shayesteh, 2009). aeromonas hydrophila is a motile, facultatively anaerobic gram-negative rod, oxidase and catalasepositive bacterium, participating with other members of this genus in motile aeromonad infection (mai) which is probably the most common bacterial disease of freshwater fishes (cipriano et al., 1984). this motile aeromonad may also inhabits brackish water but decrease in prevalence with increasing salinity (thune et al., 1993). motile aeromonads are relatively weak pathogens but isolate widely in pathogenicity. an slayer on the cell wall and more elastase production are present in more pathogenic strains. while both endotoxin and exotoxins (proteases, hemolysins) are produced, their precise relationship with pathogenicity is unclear (peatman et al., 2018). some references consider motile aeromonads as a part of the normal intestinal microflora of healthy fish and disease occurrence are usually secondary to infection by a primary pathogen (cipriano et al., 1984). the in-vitro conditions in which microorganisms can be able to grow such as temperature and ph are particularly simulated those in certain parts of the host body (for instance, respiratory system, gastrointestinal and urogenital tracts) (vukanti et al., 2012). therefore, this research aimed to evaluate the antibacterial combined effects of essential oils extracted from thymus vulgaris, o. majorana and z. clinopodioides on optimal inhibitory effect against a. hydrophila in different temperatures. materials and methods microorganisms: aeromonas hydrophila was obtained from a bacterial bank in department of aquatic animal health and disease university of tehran. bacterial samples were cultured on bloodagar media and overnight-cultures were used in the conducted experiments. essential oils: thymus vulgaris, o. majorana and z. clinopodioides essential oils were provided from a commercial firm (pars-imen daroo, iran). three combination of the essential oils (ceo) were prepared as follows: t.o.z contains 50% t. vulgaris, 25% o. majorana and 25% z. clinopodioides; t.o.z = 25% t. vulgaris, 50% o. majorana and 25% z. clinopodioides and t.o.z = 25% t. vulgaris, 25% o. majorana and 50% z. clinopodioides. the compositions of the herbal essential oils in this study were determined by gas chromatography–mass spectrometry (gc-ms). the analyses were performed using shimadzu gc2010 system with an auto injector aoc-20i and plus mass detector qp2110, equipped with an hp5-ms fused silica capillary column (30 m × 0.25 mm × 0.25 µm). the chromatographic conditions were as follows: carrier gas, helium at a flow rate of 1.02 ml min–1; oven temperature programmed initially at 60°c and increased to 310°c at a ramp of 3°c min–1; injector temperature, 220°c; injector mode in split ratio of 1:20 with 2 ml min–1 purge; ms interface temperature, 280°c; ion source temperature, 260°c; and ionization energy, 70 ev. the oil samples (15 mg) were dissolved in 1.5 ml of purified ethyl acetate and 1 µl this solution was injected for analysis. the isolated compounds were identified by their respective kováts retention indices determined in reference to a series of n-alkanes, and 359 int. j. aquat. biol. (2019) 7(6): 357-367 verified by a comparison of mass spectral data with those obtained using pure standards and those reported in the literature, 10 and eventually by comparing their mass spectra with the gc–ms spectral library (wiley 8 and ffnsc 1.2 libraries). antibacterial assays agar-disc diffusion method: the essential oils were first screened for their antibacterial activity against a. hydrophila using agar-disc diffusion method according to clsi (2012) with some modifications. briefly, fresh bacterial suspension was prepared in physiological solution (sodium chloride 0.9%) and adjusted to (1) mcfarland used to inoculate on agar plates (tsa-merck, germany). then, the sterile paper discs (6.4 mm in diameter) were applied on the surface of each plate and impregnated with 10 µl of essential oil. the plates were incubated at 25ºc for 18-24 h. the diameters of inhibition zones were measured in mm. florfenicol, oxytetracycline, trimethoprimsulfamethoxazole and enrofloxacin were used as positive samples and all the experiments were carried out in triplicate. micro-well determination of mic: the minimum inhibition concentration (mic) values were determined for the three ceos as described by hajlaoui et al. (2016). the inoculums of a. hydrophila strain were prepared from 24 h broth cultures, and suspensions were adjusted to (0.5) mcfarland standard turbidity. t.o.z, t.o.z and t.o.z were dissolved in 10% dimethyl sulfoxide dmso (sigmaaldrich) and diluted to the highest concentrations (12.8 µl /ml). then serial two-fold dilutions were prepared i.e. concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 µl /ml from each ceo into 5 ml sterile test tubes containing nutrient broth. the 96well plates were prepared by dispensing 95 µl of nutrient broth and 5 µl of the bacterial inoculum into each well. a 100 µl aliquot from the stock solutions of the combined essential oil was added into the first wells. then, 100 µl from the serial dilutions were transferred into the consecutive wells. the last well containing 95 µl of nutrient broth without ceo and 5 µl of the bacterial inoculum on each strip was used as the negative control. the final volume in each well was 100 µl. the plates were incubated at 25°c for 1824 h. the experiments were carried out in triplicate for each ceo concentration. after 18-24 h, the results were taken by elisa microplate reader (od at 600 nm) and the mic value was defined as the lowest concentration (the highest dilution) of the compounds to inhibit the growth of the microorganisms. determination of (mbc): the minimum bactericidal concentrations were determined by spreading 5 µl from negative wells in the previous stage (i.e. mic determination) on blood agar plates. the mbc value corresponded to the lowest concentration of the combined essential oil yielding negative subculture after incubation at 25ºc for 24 h (ouedrhiri et al., 2016). bacterial growth curves: bacterial growth intensities were determined in 96-well plates with assay similar to that one in mic determination. for every combined essential oil, four concentrations (lower than mbc concentration) were used to investigate their effects on a. hydrophila growth curves. this trail was conducted in different temperature of 20, 25 and 30°c, ph=7 which was monitored by titration with 5% h2so4 or 5% naoh. 96-well plates were incubated in a shaker cold incubator (fg-iran). growth curves were obtained by measuring the optical density (od) at 600 nm (by elisa microplate reader) at regular time intervals (1, 3, 6, 9, 12, 24, 48, 96 h). all experiments were conducted for 5 replicates and resulting od data over time for each replicate-sample was analyzed for growth yield (maximum absorbance at 600 nm) (vukanti et al., 2012). statistical analyses: data for each variable at each time point were compared using anova method and duncan test in spss var.17, and significance was determined if p-value is less than 0.05 (n = 5). results chemical composition of essential oils: a total of 18 components were determined accounting for 99.42% of the total amount of t. vulgaris essential oil (table 1). the main compounds were thymol (40.60%), 360 barakat et al./ antimicrobial properties of t. vulgaris, o. majorana and z. clinopodioides limonene (15.98%), cymene (12.17%) and αterpinolene (8.90%). other compounds were found in small quantities ranging between 0.16% (d-fenchyl alcohol) to 5.82% (γ-terpinene). in the o. majorana table 1. chemical composition of thymus vulgaris, origanum majorana and ziziphora clinopodioides essential oils. t. vulgaris o. majorana z. clinopodioides compound rt (min) area (%) rt (min) area (%) rt (min) area (%) α-pinene 6.708 0.46 6.158 0.17 6.631 22.6 camphene 7.077 1.6 β-pinene 8.255 0.96 8.078 0.8 β-myrcene 8.836 0.66 8.191 0.10 8.654 0.3 camphane 9.131 0.53 8.935 0.2 δ.3-carene 9.588 0.67 9.391 0.5 isocineole 9.811 1.93 9.599 3.6 1-phellandrene 9.375 2.88 α-terpinene 9.899 3.22 9.177 0.09 cymene 10.309 12.17 9.578 6.78 γ-terpinene 11.752 5.82 10.99 3.11 dihydrocarvone 18.108 0.18 trans-caryophyllene 27.971 0.32 27.366 11.52 cymol 10.035 8.2 limonene 10.548 15.98 9.696 0.67 10.216 8.3 1.8-cineole 10.315 9.1 α-terpinolene 13.08 8.90 12.753 0.8 linalool l. 13.314 0.5 d-fenchyl alcohol 14.133 0.16 13.853 0.3 terpinene 1-ol 14.818 0.3 cyclohexanone, 5methyl-2 (1-methylethyl) 15.695 3.0 ethanone, 1bicyclo[2.2.1]hept2-yl-, exo 16.214 1.1 menthol 16.619 3.4 α-thujene 5.942 0.08 2-δ-pinene 7.616 0.61 α-cubebene 24.547 0.10 3-allyl-6methoxyphenol 25.097 1.98 copaene 25.579 0.43 α-humulene 28.481 1.57 δ-cadinene 30.9 0.17 4-terpineol 16.775 0.3 para-cymen-8-ol 17.184 0.9 α-terpineol 17.408 0.8 pulegone 19.867 0.6 carvone 20.666 3.03 20.132 3.3 anethole 22.529 0.94 22.249 0.5 thymol 23.468 40.60 22.56 13.54 22.778 7.0 carvacrol (or isothymol) 23.556 57.86 23.292 21.1 piperitenone 24.666 0.6 caryophyllene oxide 32.758 0.27 33.233 0.4 total 99.42 99.06 100.0 361 int. j. aquat. biol. (2019) 7(6): 357-367 essential oil, 17 compounds were identified accounting for 99.06% of the total oil. carvacrol (or isothymol) (57.86%), thymol (13.54%), transcaryophyllene (11.52%) and cymene (6.78%) were major compounds (table 1) and the minimum quantity was 0.08% for α-thujene. for z. clinopodioides, α-pinene (22.6%), carvacrol (21.1%), 1.8-cineole (9.1%), limonene (8.3%), cymol (8.2%) and thymol (7.0%) were major compounds out of 27 (table 1). a remarkable richness in monoterpenes compared to sesquiterpenes was observed in the studied essential oils. antibacterial effects: all the tested ceos exhibited an antibacterial effect by disc-diffusion test (fig. 1). among them, t.o.z (with 50% o. majorana) presented the best inhibition zone (26 mm), followed by t.o.z (25.33 mm) and t.o.z (24.66 mm). but no significant differences were found between them (p<0.05). indeed, when the effects of coes were compared to the synthetic antibiotics (i.e. four antibiotics), oxytetracycline had the lowest efficacy, whereas enrofloxacin (inhibition zone= 32.66 mm) showed priority compared to other ceos (p<0.05). the results of mic and mbc are shown in table 2. the t.o.z and t.o.z exhibited the same inhibitory activity (mic=0.8 µl /ml), while that of the t.o.z was the lowest (mic=0.4 µl /ml). regarding the bactericidal effect, all ceos showed similar results (mbc=3.2 µl /ml). bacterial growth curves: the growth curves of a. hydrophila under t.o.z treatment were significantly uneven in both treated with ceos and control group at all tested temperatures (fig. 2 a, b, c). significant differences were found in the exponential phase of growth curves, but in the lag duration i.e. lag phase, a. hydrophila growth in either essential oils tsb (tryptic soy agar) or blank tsb table 2. inhibitory and bactericidal effects against aeromonas hydrophila (values= µl /ml). concentration combined oil 0.1 0.2 0.4 0.8 1.6 3.2 6.4 12.8 t.o.z mic mbc t.o.z mic mbc t.o.z mic mbc figure 1. disc diffusion results against aeromonas hydrophila (disc diameter = 6 mmincluded) (ff = florfenicol, t = oxytetracycline, t.s = trimethoprim sulfamethoxazole and en.r= enrofloxacin). 362 barakat et al./ antimicrobial properties of t. vulgaris, o. majorana and z. clinopodioides (b) did not affect by temperature, suggesting that different temperatures neither stimulated nor suppressed the duration of the lag phase. in addition, the differences in the growth curves were apparent figure 2. bacterial growth curves (based on od at 600 nm) under various concentrations of t.o.z (50% t. vulgaris) essential oil at temperatures of 20 (a), 25 (b) and 30°c (c) for aeromonas hydrophila. growth yields in the previous conditions (d). (approved concentrations of combined essential oil were less than mbc and ranged between 0.1 to 0.8 µl /ml and b (blank) = no essential oil; values are means (n=5). a, b, bc, and c = standard deviation). figure 3. bacterial growth curves (based on od at 600 nm) under various concentrations of t.o.z (50% o. majorana) essential oil at temperatures of 20 (a), 25 (b) and 30°c (c) for aeromonas hydrophila. growth yields in the previous conditions (d). (approved concentrations of combined essential oil were less than mbc and ranged between 0.1 to 0.8 µl /ml and b (blank) = no essential oil; values are means (n=5). a, b, bc, and c = standard deviation). 363 int. j. aquat. biol. (2019) 7(6): 357-367 even in the lowest concentrations of t.o.z , confirmed by the highest points of the growth curves and further bacterial growth yields i.e. a significant differences between t.o.z treatments and blank one (b=tsb with no essential oil) were observed (fig. 2d). od-value reached 0.960 nm at 48 h as the final growth yield in blank group i.e. without essential oil at 20°c, whereas it was 0.363 nm at 96 h in the lowest t.o.z concentration (=0.1 µl /ml). in the bacterial growth curves at 25°c with the previous conditions, the final growth yield was 0.700 nm for blank at 24 h reaching 0.360 nm in 0.1 µl /ml of t.o.z at 96 h. in the final growth yield at 30°c, od was 0.496 nm at 12 h for blank, reaching 0.375 nm at 96 h for the lowest concentration of ceo. the bacterial growth curves for a. hydrophila in t.o.z treatments i.e. 50% o. majorana, essential oil and control were similar to those of t.o.z (fig. 3a, b, c) where, od reached 0.960 nm at 48 h as the final growth yield in the blank situation at 20°c. it was 0.360 nm at 96 h in the lowest t.o.z concentration (=0.1 µl /ml). in the bacterial growth curves at 25°c with the previous conditions, the final growth yield measured 0.700 nm for blank at 24 h reaching 0.368 nm in 0.1 µl /ml of t.o.z at 96 h. regarding the final growth yield at 30°c, od was 0.496 nm at 12 h for blank, while it reached 0.352 nm at 96 h for the lowest concentration of ceo. at all blank (b) situations, a significant differences (p<0.05) were found comparing to various concentrations of essential oil (fig. 3d). the t.o.z showed the weakest effect at 30°c (fig. 4c). od-value at 30°c reached 0.496 nm at 12 h as the final growth yield for a. hydrophila with no essential oil (blank), whereas it was 0.420 nm at 96 h in the lowest t.o.z concentration (=0.1 µl /ml). in contrast, these two values were almost similar, but significantly different (p<0.05) (fig. 4d). the bacterial growth curves at 20 and 25°c were similar to those of t.o.z and t.o.z (fig. 4a, b). discussions phytochemicals is considered as secondary metabolites of low-molecular weight occurring naturally in plants. these biologically active molecules have a main role in the interaction between the plant and its environment, i.e. they serve as a defense against insects, fungi, and other figure 4. bacterial growth curves (based on od at 600 nm) under different concentrations of t.o.z (50% z. clinopodioides) essential oil at temperatures of 20 (a), 25 (b) and 30°c (c) for aeromonas hydrophila. growth yields in the previous conditions (d). (approved concentrations of combined essential oil were less than mbc and ranged between 0.1 to 0.8 µl /ml and b (blank) = no essential oil; values are means (n=5). a, b, bc, and c = standard deviation). 364 barakat et al./ antimicrobial properties of t. vulgaris, o. majorana and z. clinopodioides microorganisms, as growth regulators, pigments, and flavors (leitzmann, 2016). with the discovering of the medical properties of the herbal extracts, most recent research focused to investigate their effects on pathogenic organisms such as bacteria and fungi. based on the results, the major components of t. vulgaris were thymol, limonene, cymene and αterpinolene. this finding was in agreement with those reported by al-asmari et al. (2017), cosentino et al. (1999), divband et al. (2017) and yılmaz et al. (2010). however in other reports, the borneol and α-terpineol have been found as major compounds of t. vulgaris (radaelli et al., 2016). this difference shows that its compound concentrations affected by several factors, including geographic areas, climatic conditions, season of the plant collection, species, growth stages, origin of herb, drying conditions and distillation conditions (nhu-trang et al., 2006). in addition, every component in essential oil composition has different effects. for instance, the antioxidative effect of thyme is based on polyphenolic compounds as flavonoids (luteolin), while thymol and carvacrol have high antimicrobial activity (justesen and knuthsen, 2001). in the o. majorana essential oil, the most abundant compounds were carvacrol, thymol, transcaryophyllene and cymene. this result is contrast to previous reports (tabanca et al., 2004; mossa and nawwar, 2011; dantas et al., 2016) that terpinene, terpineol and sabinene were major compounds. although they were conducted in different geographic regions, but their data were similar regarding the reported components, suggesting that differences in geographic areas cannot always affect herbal oil composition. in other study, the thymol and/or carvacrol were found to be the predominant compounds as same as of the present study (banchio et al., 2008). the α-pinene, carvacrol, 1.8-cineole, limonene, cymol and thymol were respectively exhibited as the major constituents in the z. clinopodioides essential oil and this is consistent with the rsults of shahbazi (2017), who investigated the composition of z. clinopodioides in the three geographical region of iran showing that thymol was the highest compound in some areas, while carvacrol in others. however in previous studies, the pulegone reported as the most abundant component (ding et al., 2014; kheirkhah et al., 2015). the studied essential oils have a remarkable richness in polyphenols (such as phenolic acids and flavonoids) and monoterpenes (for instance menthol, carvone and limonene). the therapeutic agents as antibiotics and various chemicals have been used against a. hydrophila that have been led to bacterial resistance for antibiotics. application of the plant products such as thymus and origanum sp. essential oil, as therapeutic agents has been well-known (aligiannis et al., 2001; baranauskienė et al., 2003; baydar et al., 2004). the results showed that t.o.z exhibited the best inhibition zone (26 mm) but had no significant differences with other ceos. its superiority can be explained due to having a higher concentration of the carvacrol and thymol. the hydroxyl group of thymol and carvacrol and the presence of a system of delocalized electrons in their chemical structure play a major role in their antibacterial effects (nazzaro et al., 2013). thymol interacts with cell membrane affecting its permeability leading to the loss of membrane potential, cellular uptake of ethidium bromide and leakage of potassium ions, atp and carboxyfluorescein. the antimicrobial effect of the carvacrol is expected to be similar to the thymol, causing functional and structural damages to cell membrane (ouedrhiri et al., 2016). according to mic and mbc results, t.o.z has the superiority to other coes. this can be explained by more carvacrol content in t.o.z, which has been previously reported having high inhibitory and bactericidal effects. in addition, the cymene in o. majoran potentiates the antibacterial effect of the carvacrol (ultee et al., 2002). this shows that combination of the herbal essential oils leads to synergistic effect between their (not at all situations), saving essential oil consumption. recently, there is a high interest in bacterial growth laws (bren et al., 2013). bacteria respond to 365 int. j. aquat. biol. (2019) 7(6): 357-367 environmental changes by reprogramming their metabolism (baev et al., 2006). the presence of the herbal oils in growth media and alternation in temperature can be considered as environmental changes. the bacterial growth curve consists of three phases, including lag (initial), log (exponential) and stationary (deceleration) (bren et al., 2013). bren et al. (2013) showed that deceleration phase exist and the main reason of its presence is nutrient limitation in growth media. this is in accordance with our results especially for bacterial growth curves in blanks (with no essential oils). in particular, deceleration phase was occurring in different cases after 24 or 48 h. furthermore, in the present study significant differences was found between a. hydrophila growth curves with the presence of combined herbal oils and its absence particularly, in exponential phase at all conditions. based on the results, we can point out that in most cases (under ceos treatment), a. hydrophila exhibits a significant growth comparing to blanks. moreover, increasing the growth curves in some situation e.g. in t.o.z at 30°c is occurred after 24-48 h. hence, it is suggested that the mixing the herbal oils with growth media was delaying exponential phase starting in the bacterial growth curves. acknowledgements the authors appreciate pars-imen daroo commercial firm, iran for providing the herbal essential oils. references abdel-massih r., abdou e., baydoun e., daoud, z. 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(2015) 3(6): 414-424 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article parasitic worms and their histopathological effects in four sturgeon species from the southwest shores of the caspian sea mohammad reza noei1, shaig ibrahimov1, masoud sattari*21 1institute of zoology, the azerbaijan national academy of sciences, baku, azerbaijan. 2fisheries department, faculty of natural resources, university of guilan, sowmeh sara, iran. article history: received 17 august 2015 accepted 2 n o v e m b e r 2015 available online 2 5 d e c e m b e r 2015 keywords: trematode cestode acanthocephalans huso huso acipenser stellatus acipenser nudiventris acipenser gueldenstaedtii abstract: this study conducted to provide the status of parasite communities of four sturgeon species viz. acipenser stellatus, a. gueldenstaedtii, a. nudiventris, and huso huso in the southwest of the caspian sea and their histopathological effects on the examined fishes. for this purpose a total of 93 individuals of four sturgeon species were caught in two fisheries regions from the southwest of the caspian sea (guilan province, iran) from march 2010 to may 2011. the histological slides of the infested tissues of the examined fishes were prepared for study of the histopathological effects of the parasites. classical epidemiological variables, including mean intensity, prevalence, abundance and dominance were calculated for overall samples, grouped by season, geographical region, and sex. five worm species, including two nematodes (cucullanus sphaerocephalus and eustrongylides excisus larvae), one cestode (bothrimonus fallax), one acanthocephalans (leptorhynchoide plagicephalus) and one digenean trematode (skrjabinopsolus semiarmatus) were found in examined sturgeons and their histopathological effects on the fish tissues were assessed. based on the results, the diversity of the parasites (including freshwater ones) in the southern part of the caspian sea have decreased since the time of the first study in 1972. this may be related to unfavorable conditions in freshwater ecosystems. introduction sturgeons are evolutionary relicts with a wide distribution in the northern hemisphere (holcik, 1989). their status as basal actinopterygian fish, unique benthic specializations, and variation in their basic diadromous life history make sturgeons interesting biological and biogeographical subjects (holcik, 1989). extensive studies on eurasian sturgeons indicate that they are also unique among fishes in possessing a markedly diverse assemblage of host-specific parasites. the parasites of sturgeons have been studied in several works (dogiel and bykhovskiy, 1939; dubinin, 1952; shulmann, 1954; nechaeva, 1964; mokhayer, 1972; skryabina, 1974; sattari and mokhayer, 2006; sattari et al., 2007, 2008, 2009; khara et al., 2009: mousavi sabet and sattari, 2013; daghigh roohi et al., 2014a, b; khara * corresponding author: masoud sattari e-mail address: msattari@guilan.ac.ir and sattari, 2014). however, there are only a few reports about their parasites in the southern part of the caspian sea. mokhayer (1972) studied the parasites of three sturgeon species, namely a. stellatus, a. gueldenstaedtii, and huso huso from the caspian sea, and reported 17 parasite species, including trichodina reticulata, polypodium hydriforme, skrjabinopsolus skrjabini, s. acipenseris, amphilina foliacea, bothrimonus fallax, eubothrium acipenserinum, ascarophis ovotrichuria, cyclozone acipenserina, cucullanus sphaerocephalus, contracaecum squalii, anisakis schupakowi, eustrongylides excisus, leptorhynchoides plagicephalus, pomphorhynchus laevis, corynosoma capsicum and pseudotracheliastes stellatus. gorogi (1996a) studied the parasites of a. persicus and reported three parasite species, 415 noei et al./parasitic worms and histopathologic effects in sturgeons including c. sphaerocephalus, s. semiarmatus and l. plagicephalus. in another study, gorogi (1996b) reported five parasite species viz. c. sphaerocephalus, anisakis schupakovi, s. semiarmatus, corynosoma strumosum and e. acipenserinum from h. huso. there are also other reports regarding the parasites of the sturgeons from the caspian sea (sattari and mokhayer, 2006). therefore, this study attempts to provide the status of parasite communities (prevalence, mean intensity of infection, abundance and dominance) of four sturgeon species viz. stellate sturgeon, acipenser stellatus pallas, 1771, russian sturgeon, a. gueldenstaedtii brandt, 1833, ship sturgeon, a. nudiventris lovetsky, 1828, and great sturgeon, huso huso in the southwestern of the caspian sea and their histopathological effects on the examined fishes. materials and methods a total of 93 individuals of four sturgeon species, namely a. stellatus (n=60), a. gueldenstaedtii (n=12), a. nudiventris (n=9), and huso huso (n=12) were collected from march 2010 to may 2011. the samples includes sturgeons caught in fisheries regions 1 (region 1: western coast of guilan province) and 2 (region 1: eastern coast of guilan province) along a shore line of more than 200 km. the stellate sturgeons (60 fish) had a mean weight of 8.725 kg (±2.735 kg, range=4-15 kg) and fork length of 129.57 cm (±18.11 cm, range=83-170 cm). the russian sturgeons (12 fish) had a mean weight of 19.417 kg (±6.007 kg, range=12-32 kg) and fork length of 135.33 cm (±15.20 cm, range=110-155 cm). the ship sturgeons (9 fish) had a mean weight of 28.444 kg (±11.304 kg, range=11-41 kg) and fork length of 156.78 cm (±29.28 cm, range=112-190 cm). great sturgeons (12 fish) had a mean weight of 119.500 kg (±71.008 kg, range=22-271 kg) and fork length of 207.5 cm (±41.097 cm, range=144-274 cm). since the samplings of this study were restricted by the governmental fishing program (i.e. for artificial propagation and then exporting their flesh), therefore, age determination was not possible. after recording biometric characteristics of fishes, common necropsy and parasitology methods (bykhovskaya-pavlovskaya, 1985; stoskopf, 1993) were used for finding parasites. live trematodes and acanthocephalans were relaxed in distilled water at 4ºc for 1 hrs and fixed in 10% hot buffered formalin. live nematodes were fixed in hot 70% ethanol and cleared in hot lactophenol. frozen specimens were thawed in water, and then fixed with 10% formalin (trematodes and acanthocephalans) or 70% ethanol (nematodes). all fixed specimens in 10% formalin were stained with aqueous acetocarmine, dehydrated and mounted in permount. after fixation of the infested tissues of the examined fishes for 24 hrs, they were processed based on eagderi et al. (2013), embedded in paraffin and subsequently cut (with a thickness of 5 μm) with a microtome (model 1130 rotary microtome, reichert-jung) for preparing histological slides. then, they were stained with haematoxylin-eosin for study of the histopathological effects of the parasites. the worms were identified using parasite identification keys (yamaguti, 1961; bykhovskaya-pavlovskaya et al., 1962; avdeyev, 1987; moravec, 1994) and then were deposited at the laboratory of fish diseases, faculty of natural resources, university of guilan, iran. statistical analysis: classical epidemiological variables (prevalence, intensity and abundance) were calculated according to bush et al. (1997). mean intensity of infection was determined by dividing the total number of recovered parasites by the number of infected fish samples, while calculating abundance was carried out by dividing the total number of recovered parasites by the number of (infected and uninfected) fish samples. prevalence was also calculated by dividing the number of infected fish samples by the total number of the examined fishes and expressed as a percentage. the dominance of a parasite species was calculated as n/n sum (where n=abundance of a parasite species and n sum=sum of the abundance of all parasite species found) and expressed as a percentage (modified after leong and holmes, 1981). mean 416 int. j. aquat. biol. (2015) 3(6): 414-424 intensity of infection and abundances of parasite species (with prevalences >10%) among seasons, age classes and sexes were tested by the kruskalwallis test (kw, multiple comparisons) and mannwhitney u test (mw, pairwise comparisons). the results were considered significant at the 95% level (p<0.05). data analysis were performed using spss software (ibm spss statistics, ibm corporation). results a total of 762 parasite belonging to five species, including two nematodes viz. cucullanus sphaerocephalus rudolphi, 1809 and eustrongylides excisus jagerskiold, 1909, one cestode species, namely bothrimonus fallax luhe, 1900, one acanthocephalans, namely leptorhynchoides plagicephalus westrumb, 1821, and one digenean trematode, namely skrjabinopsolus semiarmatus molin, 1858 were found in the examined sturgeons, including a. stellatus, a. gueldenstaedtii, a. nudiventris and h. huso. the prevalence, mean intensity, range, abundance and dominance of the collected parasites are presented in tables 1 and 5-7. a total of 631 worms belonging to five species were found in a. stellatus. leptorhynchoides plagicephalus had the highest prevalence (45%), abundance (5.22) and dominance (49.6%), while s. semiarmatus had the highest mean intensity of infection (24.45) (table 1). these two parasites constituted up to 92.23% of parasite communities in a. stellatus. the prevalence of c. sphaerocephalus and s. semiarmatus in a. stellaus was higher in spring (16.22 and 27.02) than that of winter and autumn, respectively, whereas the prevalence of l. plagicephalus was higher in winter than that of spring and autumn, respectively (table 2). the mean intensity of infection of these three parasites was higher in spring than winter and autumn, respectively, but the differences between seasons were not significant (kw, χ2=0.778, df=2, p=0.678 for c. sphaerocephalus; kw, χ2=0.00, df=1, p=1.00 for parasites prevalence (%) mean intensitysd range abundancesd dominance (%) s. semiarmatus n=269 18.33 24.4562.41 1-212 4.4827.4 42.63 l. plagicephalus n=313 45 11.5917.06 1-71 5.2212.7 49.6 c. sphaerocephalus n=22 17.26 2.442.30 1-8 0.371.22 3.49 b. fallax n=12 1.66 12 0 12 0.21.55 1.9 e. excisus larvae n=15 5 5.03.61 1-8 0.251.28 2.4 table 1. the prevalence, mean intensity, range, abundance and dominance of some parasites in a. stellatus. parasite season c. sphaerocephalus prevalence (%) meansd range s. semiarmatus prevalence (%) meansd range l. plagicephalus prevalence (%) meansd range e.exisus prevalence (%) meansd range b. fallax prevalence (%) meansd range spring n=37 16.22 2.82.7 (1-8) 27.02 26.465.4 (1-212) 45.95 14.2920.58 (1-71) 2.70 1 8 2.70 12 12 autumn n=16 12.5 21.4 (1-3) 0 37.5 5.57.23 (1-20) 0 0 winter n=7 14.29 1 1 14.29 5 5 57.14 9.257.27 (2-19) 28.57 3.53.53 (1-6) 0 table 2. the prevalence, mean intensity and range of some parasites of a. stellatus in various seasons. 417 noei et al./parasitic worms and histopathologic effects in sturgeons s. semiarmatus; and kw, χ2=1.606, df=2, p=0.448 for l. plagicephalus). the same results were found regarding the abundance of these three parasites in various seasons (kw, χ2=0.098, df=2, p=0.952 for c. sphaerocephalus; kw, χ2=5.374, df=2, p=0.068 for s. semiarmatus; and kw, χ2=1.524, df=2, p=0.467 for l. plagicephalus). the prevalence of c. sphaerocephalus and s. semiarmatus in a. stellatus in region 1 was higher, but the prevalence of l. plagicephalus in region 2 was higher (table 3). the mean intensity of infection of c. sphaerocephalus in region 1 was significantly higher (kw, χ2=4.8, df=1, p<0.05, but no significant differences were found between these two regions concerning to s. semiarmatus and l. plagicephalus (kw, χ2=1.457, df=1, p=0.224 for s. semiarmatus; and kw, χ2=0.223, df=1, p=0.637 for l. plagicephalus). also, no significant differences were found regarding the abundance of these three parasites between regions (kw, χ2=0.685, df=1, p=0.408 for c. sphaerocephalus; kw, χ2=0.031, df=1, p=0.806 for s. semiarmatus; and kw, χ2=0.166, df=1, p=0.684 for l. plagicephalus). the prevalence of c. sphaerocephalus in females of a. stellatus (16.66) was higher than males (12.5), but the prevalence of s. semiarmatus and l. plagicephalus in males (20.83 and 45.83, respectively) was higher (table 4). the mean intensity of the infection of these three parasites in females was higher than males, although the differences between males and females were not significant (kw, χ2=0.333, df=1, p=0.564 for c. sphaerocephalus; kw, χ2=0.009, df=1, p=0.926 for s. semiarmatus; and kw, χ2=4.054, df=1, p=0.054 for l. plagicephalus). the same results were found regarding the abundance of these three parasites in males and females (kw, χ2=0.179, df=1, p=0.673 for c. sphaerocephalus; kw, χ2=0.153, df=1, p=0.695 for s. semiarmatus; and kw, χ2=0.326, df=1, p=0.568 for l. plagicephalus) (table 4). the prevalence of c. sphaerocephalus, s. semiarmatus and l. plagicephalus in higher length groups of a. stellatus were higher than in lower length groups. the mean intensity of the infection of these three parasites in higher length groups were higher than lower length groups, but the differences were parasite locality c. sphaerocephalus prevalence (%) mean sd range s. semiarmatus prevalence (%) meansd range l. plagicephalus prevalence (%) meansd range e. excisus prevalence (%) meansd range b. fallax prevalence (%) meansd range location 1 n=27 18.52 18.52 40.74 3.70 3.70 3.62.61 8.86.1 10.7315.25 8 12 (1-8) (2-17) (1-54) 8 12 location 2 n=33 12.12 18.18 48.48 6.06 1.00 37.585.5 12.1918.67 3.53.54 0 1 (1-212) (1-71) 1-6 table 3. the prevalence, mean intensity and range of some parasites of a. stellatus in two sampling regions. parasite sex c. sphaerocephalus prevalence (%) meansd range s. semiarmatus prevalence (%) meansd range l. plagicephalus prevalence (%) meansd range e. excisus prevalence (%) meansd range b. fallax prevalence (%) meansd range female n=36 16.66 16.66 44.44 5.56 2.82.7 39.3  84.7 13.818.03 3.53.53 0 (1-8) (1-212) (1 71) (1-6) male n=24 12.5 20.83 45.83 4.17 4.17 1.671.15 6.66.35 8.3615.8 8 12 (1-3) (1-17) (1-54) 8 12 table 4. the prevalence, mean intensity and range of some parasites of a. stellatus in males and females. 418 int. j. aquat. biol. (2015) 3(6): 414-424 not significant (kw, χ2=6.667, df=7, p=0.464 for c. sphaerocephalus; kw, χ2=9.119, df=8, p=0.332 for s. semiarmatus; and kw, χ2=15.750, df=15, p=0.399 for l. plagicephalus). the same results were found regarding the abundance of these three parasites in various length groups (kw, χ2=18.789, df=27, p=0.878 for c. sphaerocephalus; kw, χ2=25.993, df=27, p=0.519 for s. semiarmatus; and kw, χ2=20.508, df=27, p=0.809 for l. plagicephalus). in addition, the prevalence of c. sphaerocephalus and s. semiarmatus in higher weight groups of a. stellatus was more than lower weight groups. the mean intensity of infection of these two parasites in higher weight groups was also more than lower weight groups, but the differences were not significant ((kw, χ2=6.667, df=5, p=0.247 for c. sphaerocephalus; kw, χ2=6.103, df=5, p=0.296 for s. semiarmatus). the same results were found regarding the mean intensity of infection of this parasite, but the differences were not significant (kw, χ2=4.769, df=5, p=0.782). no significant differences was found concerning to the abundance of these three parasites in various weight groups of a. stellatus (kw, χ2=12.681, df=11, p=0.315 for c. sphaerocephalus; kw, χ2=14.596, df=11, p=0.202 for s. semiarmatus; and kw, χ2=13.111, df=11, p=0.767 for l. plagicephalus). a total of 43 worms belonging to three species were found in a. gueldenstaedtii (table 5). in this species, c. sphaerocephalus had the highest prevalence (50%), mean intensity of infection (6.17), abundance (3.08), and dominance (86.04%). eustrongylides excisus larvae had the second highest prevalence (16.66), mean intensity of infection (2.5), abundance (0.41,) and dominance (11.63%). these two parasites were constituted up to 97.67% of parasite communities in the examined specimens of a. gueldenstaedtii (table 5). a total of 48 worms belonging to four species were found in the specimens of a. nudiventris (table 6). based on the results, c. sphaerocephalus had the highest prevalence (55.5%), abundance (3) and parasites prevalence (%) mean intensity  sd range abundance sd dominance (%) l. plagicephalus n=37 8.33 1 1 0.080.01 2.32 c. sphaerocephalus n=1 50 6.174.02 1-12 3.080.13 86.04 e. excisus larvae n=5 16.66 2.52.12 1-4 0.410.02 11.63 table 5. the prevalence, mean intensity, range, abundance and dominance of some parasites in a. gueldenstaedtii. parasites prevalence (%) mean intensity  sd range abundance sd dominance (%) l. plagicephalus n=37 8.33 1 1 0.080.01 2.32 c. sphaerocephalus n=1 50 6.174.02 1-12 3.080.13 86.04 e. excisus larvae n=5 16.66 2.52.12 1-4 0.410.02 11.63 table 6. the prevalence, mean intensity, range, abundance and dominance of some parasites in a. nudiventris. parasites prevalence (%) mean intensity  sd range abundance sd dominance (%) s. semiarmatus n=4 8.33 4 4 0.330.11 10 c. sphaerocephalus n=35 41.67 77.65 1-19 2.920.1 87.5 e. excisus larvae n=1 8.33 1 1 0.080.01 2.5 table 7. the prevalence, mean intensity, range, abundance and dominance of some parasites in huso huso. 419 noei et al./parasitic worms and histopathologic effects in sturgeons dominance (56.25%). skrjabinopsolus semiarmatus had the second highest prevalence (22.2), abundance (1.56) and dominance (29.17%). the mean intensity of the infection of s. semiarmatus (7) was higher than c. sphaerocephalus (5.4). these two parasites constituted up to 85.42% of the parasite communities in a. nudiventris. a total of 40 worms belonging to three species were found in the samples of h. huso (table 7). cucullanus sphaerocephalus had the highest prevalence (41.67%), mean intensity of infection (7) abundance (2.92) and dominance (87.5%). skrjabinopsolus semiarmatus had the second prevalence (8.33), mean intensity of infection (4), abundance (0.33) and dominance (10%). these two parasites constituted up to 97.5% of parasite communities in h. huso. the histopathological effects of some parasites on the gut of the sturgeons are as follows: eustrongylides excisus larvae: the parasites were encapsulated in the wall of the intestinal mucosa. there was chronic inflammation around the parasites figure 1. histopathologic section of the infested tissues by e. excisus larvae in the gut of h. huso (h&e, 40x). figure 2. histopathologic section of the infested tissues by e. excisus in the gut of h. huso (h&e, 400x). figure 3. histopathologic section of the infested tissues by l. plagicephalus in the gut of a. nudiventris (h&e, 40x). figure 4. histopathologic section of the infested tissues by l. plagicephalus in the gut of a. nudiventris (h&e, 400x). figure 5. histopathologic section of the infested tissues by c. sphaerocephalus in the gut of a. gueldenstaedtii (h&e, 40x). 420 int. j. aquat. biol. (2015) 3(6): 414-424 with infiltration of the macrophages, lymphocytes, fibroblasts, and multiple eosinophils (figs. 1, 2). leptorhynchoides plagicephalus: the head of the parasite with horny proboscis was attached in a pitted zone of intestinal wall of the sturgeon. the spines of proboscis formed severe necrosis in the epithelium with chronic inflammation. the mononuclear cells with multiple lymphocytes were infiltrated and also some lymphoid follicles were present (figs. 3, 4) cucullanus sphaerocephalus: this parasite is caused the focal necrosis and acute to sub-acute inflammation with multiple lymphocytes (figs. 5, 6). bothrimonus fallax: necrosis and destruction of the mucosa were observed in the site of attachment this parasite. mononuclear cells such as lymphocytes and a few number of eosinophils were also observed (figs. 7, 8). discussion there are few reports about the parasites of sturgeons in iran. mokhayer (1972) studied the parasites of three sturgeon species, namely a. stellatus, a. gueldenstaedtii and h. huso, and reported 17 parasite species. gorogi (1996a, b) also studied the parasites of two sturgeon species, including a. persicus and h. huso with reporting 3 and 5 parasite species, respectively. sattari and mokhayer (2006) studied the parasites of 206 individuals of a. persicus and reported nine parasite species. however, in the present study, trichodina reticulata, polypodium hydriforme, ascarophis ovotrichuria, cyclozone acipenserina, contracaecum squalii and pomphorhynchus laevis were not recovered from the samples. skryabina (1974) found that a. stellatus is parasitized by more worm species than a. gueldenstaedtii, particularly in the caspian sea basin. skryabina (1974) also found that in the azov and caspian seas, the prevalence of c. sphaerocephalus in a. stellatus is less than other sturgeon species from the same localities. similarly, in the present study, it was found that the diversity of helminth fauna in a. stellatus was more than other examined sturgeons. it was also found that the prevalence and mean intensity of c. sphaerocephalus in a. stellatus were less than in the other examined figure 6. histopathologic section of the infested tissues by c. sphaerocephalus in the gut of a. gueldenstaedtii (h&e, 400x). figure 7. histopathologic section of the infested tissues by b. fallax in the gut of a. stellatus (h&e, 40x). figure 8. histopathologic section of the infested tissues by b. fallax in the gut of a. stellatus (h&e, 400x). 421 noei et al./parasitic worms and histopathologic effects in sturgeons sturgeons. markov et al. (1967) found that the most important parasites of a. stellatus from the lower course of the volga river are cestodes and acanthocephalans. they also found that along the coast of dagestan, the most important species are e. acipenserinum, b. fallax, l. plagicephalus and s. semiarmatus. similarly, in the present study, the prevalence and mean intensity of l. plagicephalus in a. stellatus were higher than other examined sturgeons. bothrimonus fallax was only found in a. stellatus. of all acipenserid species, the russian sturgeon has the best-known parasite fauna (holcik, 1989). the complete list of the parasites found in a. gueldenstaedtii includes 46 species; of these, parasitic worms are the largest group (dogiel and bykhovskii, 1939; shulman, 1954; nechaeva, 1964; skryabina, 1974). skryabina (1974) reported that the parasite fauna of a. gueldenstaedtii is similar to that of a. stellatus, while in this study and also in sattari and mokhayer (2006), the parasite fauna of a. gueldenstaedtii was similar to that of a. nudiventris and h. huso than a. stellatus. the main parasites of a. nudiventris includes 32 parasites species and most of them are specific to acipenserids (dogiel and bykhovskii, 1939; shulman, 1954; skryabina, 1974). shulman (1954) pointed out that the adult ship sturgeons are infested primarily in the sea by helminthes such as s. semiarmatus, e. acipenserinum and c. sphaerocephalus. in juveniles, the predominant parasites are freshwater species such as t. acipenseri, amphilina foliacea, hysterothylacium bidentatum, piscicapillaria tuberculata, chilodonella cyprini, trichodina domerguei and argulus foliaceus. along the iranian shore of the caspian sea, however, the catching of juvenile sturgeons is forbidden by government. therefore, there is no report about their parasite fauna in this region. although some researchers have reported anisakis sp. larvae and cystoopsis acipenseris from dead juvenile sturgeons (a. hajimoradloo, personal communications). the parasite fauna of h. huso has been studied by many authors (dogiel and bykhovskii, 1939; shulman, 1954; nechaeva, 1964; skryabina, 1974; bauer et al., 1977) and 33 parasite species have been reported (holcik, 1989). with great probability, the local populations of the great sturgeon are infested by different aggregations of parasitic worms (dogiel and bykhovskii, 1939). dogiel and bykhovskii (1939) stated that the stocks inhabiting the northern caspian region are mostly infested to typical freshwater parasitic worms than those of the southern part of the caspian sea. similarly, in the present study and also in the previous studies (mokhayer, 1972; gorogi, 1996b; sattari and mokhayer, 2006), more marine typical worms such as e. acipenserinum, anisakis sp. (l.), e. excisus larvae, c. strumosum and c. sphaerocephalus were found in the great sturgeon. the sample of the sturgeons, particularly a. nudiventris and h. huso, were small in the present study due to decreasing numbers of these species in catching yields. however, with respect to the works of gorogi (1996a, b) and sattari and mokhayer (2006), it seems that expected helminthofauna in the sturgeons of the southern part of the caspian sea does not exceed 13-15 species. in this study, c. sphaerocephalus and s. semiarmatus were the most prevalent worms and their mean intensity, abundance and dominance were higher than the others parasites. in addition, e. excisus larvae were mostly found in more carnivorous sturgeons such as h. huso, a. gueldenstaedtii and a. nudiventris. this is likely because e. excisus larvae needs some benthophagous fishes (e.g., rutilus caspius and neogobius spp.) as obligatory second intermediate hosts. in the present study, amphilina foliacea and d. armatum (belonging to freshwater parasite fauna) were not found in the examined sturgeons that have already been reported by sattari and mokhayer (2006). this may be because of decreasing spawning migrations of the sturgeons into freshwater, which can be as result of unfavorable conditions of freshwater ecosystems caused by pollution, dam construction, etc. according to the results of this study and the results 422 int. j. aquat. biol. (2015) 3(6): 414-424 of mokhayer (1972), gorogi (1996a, b), and sattari and mokhayer (2006), the diversity of the sturgeons’ parasites in the southern part of the caspian sea is lower than that of the northern part. it should be noted that the maximum depth of the caspian sea in the northern part is about 12 m, while in the southern part it is about 980 m. furthermore, the salinity in the northern part of the caspian sea is about 5 ppt, while in the southern part, it is about 13 ppt and may reach to 20 ppt in the southeast region. in addition, the productivity and carbonate ions between the southern and northern parts are different. these factors may have some impacts on the parasite communities of the sturgeons. based on the results, the diversity of parasites (including freshwater ones) in the southern part of the caspian sea have decreased since the time of the first study by mokhayer (1972). this may be related to unfavorable conditions in freshwater ecosystems, such as pollution and dam construction. in these conditions, it is impossible for the sturgeons to migrate into the rivers for spawning. acknowledgments we would like to thank dr. m. pour kazemi for providing the laboratory equipment, and the international research institute of sturgeons in iran and the university of tehran for their financial supports. references avdeyev v.v. 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(2015) 3(6): 414-424 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology چکیده فارسی خزر دریای غربیجنوب سواحل در خاویاری ماهیان از چهارگونه روی بر هاآن شناسیآسیب اثرات و کرمی هایانگل *2ستاری مسعود ،1ابراهیموف شایق ،1نوعی محمدرضا .آذربایجان باکو، آذربایجان، علوم ملی آکادمی جانورشناسی، انستیتو1 .ایران سرا، صومعه گیالن، دانشگاه طبیعی، منابع دانشکده شیالت، گروه2 چکیده: هاآن شناسیآسیب اثرات و ماهیفیل و شیپ چالباش، برون،ازون شامل تاسماهیان از گونه چهار انگلی هایجمعیت وضعیت تعیین برای مطالعه این دریای غربیجنوب شیالتی ناحیه دو در گونه چهار این از ماهی عدد 39 تعداد منظور، این برای. گرفت صورت خزر دریای غربیجنوب سواحل در مطالعه برای آزمایش مورد ماهیان آلوده هایبافت شناسی بافت هایالم. شدند صید 2211 می ماه تا 2212 مارس ماه از( ایران گیالن، استان) خزر شاخص و وانیفرا شیوع، آلودگی، شدت میانگین شامل کالسیک شناختی گیریهمه متغیرهای. گرفتند قرار استفاده مورد هاانگل شناسیآسیب اثرات وسکوکوالن) نماتود دو شامل انگل گونه پنج. شدند محاسبه جنسیت و جغرافیایی ناحیه فصل، اساس بر همچنین و هانمونه تمام برای غالبیت یک و( سفالوسیپالژ لپتورینکوئیدس) آکانتوسفال یک ،(فاالکس بوتریمونوس) سستود یک ،(اکسیسوس یواسترونژیلیدس نوزاد و اسفروسفالوس مورد ماهیان هایبافت روی بر آنها شناسیآسیب اثرات و شدند یافت آزمایش مورد هاینمونه در( آرماتوسسمی اسکریابینوپسولوس) دیژن ترماتود کاهش 1392 سال در مطالعه اولین زمان از خزر دریای غربیجنوب در( شیرین آب هایگونه شامل) هاانگل تنوع نتایج، اساس بر. گرفت قرار ارزیابی . باشد ارتباط در شیرین آب هایاکوسیستم در نامطلوب شرایط با است ممکن امر این. است یافته .چالباش ،شیپ برون،ازونماهی، فیل ،آکانتوسفال ،سستود ،ترماتود :کلمات کلیدی int. j. aquat. biol. (2019) 7(2): 93-96 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article interpreting biomass and catch per unit area (cpua) to assess the status of demersal fishes in oman sea mohammad reza mirzaei*1, pedram hatami2, seyed abbas hosseini1 1offshore fisheries research center, iranian fisheries science research institute, agricultural research, education and extension organization (areeo), chabahar, iran. 2sistan and baluchistan fisheries organization, chabahar, iran. s article history: received 27 december 2018 accepted 28 march 2019 available online 2 5 april 2019 keywords: biomass demersal fish catch per unit area oman sea abstract: this study aimed to assess the biomass and catch per unit area (cpua) of demersal fish resources in the northern part of the oman sea based on trawl survey. the study area was stratified into five stratum (i, ii, iii, iv and v) covering the depth layers of 10-20, 20-30, 30-50 and 50-100 m. a total number of 68 stations were monitored during the study period. the highest values of biomass were belong to stratum v and iv in the east coast of the oman sea and the highest species biomass was belong to batoid fishes (8054.4 tons). the lowest cpua was recorded in the central region of the study area (stratum iii, approximately 8212 kg/nm2) and the highest cpua was on both sides of the study area (the east and west coast). moreover, the highest cpua (2031 kg/nm2) was observed in 20-30 m depth layer. the results showed the changes in biomass and cpua based on different depths and the most abundant species groups were trichiurus lepturus (50-100 meter), sphyraena jello (30-50 meters), caranx ignobilis (20-30 meters), and pomadasys kaakan (10-20 meters). introduction estimation of fish abundance is essential for fisheries management purposes. catch per unit area (cpua) and biomass as important indices of stock assessment are fundamental elements of knowledge for an effective fishery management (monjezi veysi et al., 2017). increase fishing pressure is threatening the sustainability of demersal resource in oman sea. currently, there are few effective management control in this area. due to vulnerability of aquatic systems, it is necessary to monitor the changes in aquatic stocks and trends. bottom research trawler as regional fishery survey in different populations is a method to achieve this goal. the first study of the bottom trawls as regional fishery in the persian gulf and oman sea emerged during 1976 to 1979 (kesteven et al., 1981). in recent years, valinassab et al. (2006) assessed the abundance of demersal fishes in the persian gulf and oman sea *correspondence: mohammad reza mirzaei doi: https://doi.org/10.22034/ijab.v7i2.531 e-mail: mirzaei.mr@gmail.com and total demersal fish biomass was estimated as 73,000 tonnes in persian gulf and approximately 39,000 tonnes in the oman sea. they reported the most abundant species groups were rays, catfish, grunts, nemipterids and carangids. furthermore, several studies have been conducted on catch per unit area and biomass of demersal fishes such as saurida tumbil (ghotbeddin et al., 2015), trichiurus lepturus (raeisi et al., 2012), some members of the families viz. haemulids, nemipterids and ariids (monjezi veysi et al., 2017) and grammoplites suppositus (mirzaei et al., 2017) in northern part of the persian gulf and oman sea. in this order, this study aimed to estimate the biomass and catch per unit area (cpua) of demersal fishes in northern part of the oman sea to provide basic information for better understanding of their biological parameters, population dynamics and stock enhancement. 94 mirzaei et al./ biomass and cpua in demersal fish in oman sea materials and methods this study was conducted in northern region of the oman sea from the meidani (58º55'e) to gwatre bay (61º30'e) using kavian trawler vessel equipped with fish bottom-trawl net (with headline of 72 m) during a period of 30 days in september 2015. the study area divided into five stratums (i, ii, iii, iv and v) and each stratum was divided into four substrata on the basis of depth i.e. 10-20, 20-30, 30-50, and 50-100 m (fig. 1, table 1). the study area, stratum area and different depth were calculated using a platometer. a total number of 66 stations were chosen based on the random stratified sampling method (valinassab et al., 2006). each trawl session lasted for 60 min at speed of 3 knots. furthermore, the sampling date, sampling time, towing distance, towing speed, water depth, and geographic location were recorded at each sampling station. the whole catch was transferred on board and all sizeable fishes were separated, counted and weighed. the remaining small fishes were placed in the same shape baskets and chosen some baskets and then using the number and weight of fishes in each basket to calculate an estimate of the entire population (fishes) in each station (smith and heemstra., 2012; bianchi., 1985). the following equation was used to calculate cpua in trawl survey (valinassab et al., 2006): cpua=cw/a. where the cw is catch weight (kg), and a is swept area (nm2) for each hauling that is calculated by a= d.h.x1, where d is the distance covered (nm), h= the headline height (m), and x1=the wing spread coefficient (is equal to 0.6). in addition, the biomass was calculated based on sparre and venema (1998) as follows: b=cpua / x2, where cpua is catch per unit area in each station at different stratum, and x2= equal to 0.5 as the catch coefficient. moreover, the total biomass is calculated by equation of b=cpua×a /x2, where a is the total area (nm2) (maunder et al., 2006). results sampling was carried out at 68 defined stations in different area in northern part of the oman sea by covering 10-100 m depths (20-10, 30-20, 50-30, and 100-50 m). a total of 135 species were identified during 30 days study period. total biomass of the demersal fish was estimated 33560.3 tones. the maximum and minimum biomass was 10034.4 and 3640.3 in stratum v and iii, respectively. furthermore, the highest and lowest biomass values were observed in 50-100 and 10-20 m depth, respectively (figs. 2, 3). among the catch species, batoid species had the highest biomass (8054.4 tons) in total catch (24% of total biomass). after batoids, the highest biomass in separate depth layers belongs to largehead hairtail (t. lepturus) (50100 meter), pickhandle barracuda (sphyraena jello) (30-50 meters), giant trevally (caranx ignobilis) 20table 1. number of trawl stations, stratum area, and location of each stratum in oman sea strata 10-20 m 20-30 m 30-50 m 50-100 m start end area (nm2 ) total stations i 3 3 4 5 58°55'e 59°25'e 116 15 ii 2 3 3 4 59°25'e 59°55'e 180.9 12 iii 2 3 3 3 59°55'e 60°25'e 235 11 iv 2 4 4 5 60°25'e 60°55'e 268.5 15 v 3 4 4 4 60°55'e 61°25'e 363.8 15 figure 1. map of the sampling area in northern part of the oman sea. 95 int. j. aquat. biol. (2019) 7(2): 93-96 30 meters, and javelin grunter (pomadasys kaakan) (10-20 meters). the mean cpua was estimated 75436 nm2 during study period. the highest mean cpua value observed in stratum v (19884 nm2) and lowest one in stratum iii (8212 nm2). the results showed that the abundance was higher in the western part of the studied area. the highest and lowest mean cpua values were 20311.4 and 5258.7 kg/ nm2 in 20-30 and 10-20 m depth layers, respectively (figs. 4, 5). discussions biomass and cpua are employed as stock indices for managing of the demersal fish species. the present study was conducted to evaluate the cpua and biomass of the demersal fishes in different stratums and depth layers in northern part of the oman sea and findings showed higher biomass of the stratums i, ii, iv, and v. in addition, the results of this study revealed a better condition regarding the abundance of marine resources in the western and eastern regions of the studied area which indicate an ecologically rich area with high primary production and appropriate habitats for aquatic marine species. comparison of biomass in different depth layers showed that maximum biomass was at the depth of 50 to 100 m. this result is contrary to that of valinassab et al. (2006) who found the highest biomass value at 10-20 meter depth layer. differences between biomass in these two layers during the last decade might be due to overexploitation, use of non-standard fishing equipment and gears, more catch per unit of effort (cpue) i.e. increasing number of fishermen, boats, ships, and fishing gears especially gillnets along the coastal waters (10-20 meter depth layer) leading lack of opportunity for regeneration of fish populations in this area. another explanation for differences between biomass in1020 meter depth layer and 50-100 meter depth layer is the difference in the extent of these two areas. figure 2. biomass (tone) in different stratum at the northern coast of the oman sea. figure 3. biomass (tone) in different depth layer at the northern coast of the oman sea. figure 4. catch per unit area (kg/ nm2) in different stratum at the northern coast of the oman sea. figure 5. catch per unit area (kg/ nm2) in different depth layer at the northern coast of the oman sea. 96 mirzaei et al./ biomass and cpua in demersal fish in oman sea differences in mean cpua values between different depth layers can be due to horizontal distribution of species. changing trends in mean cpua at different stratum showed that the stratums i and ii in the east coast and stratums iv and v in west coast of the oman sea represent more favorable fishing conditions. these results are in line with valinassab et al. (2006) who also found changes in mean cpua at different stratums (i (7524.9), ii (9975.5), iii (6365.6), iv (13943.4), and v (9001.9)) in the oman sea. furthermore, these results are in agreement with abbaspour et al. (2010) findings which showed mean cpua in west (i=13808, ii=11782) and east (iv=11482, v=9609) coast is more than central (iii=5703) part of oman sea. this results can be explained due to high fishing effort of artisanal (gillnet) and industrial (trawl vessels) fisheries in this region. the mean cpua in different depth layers of the oman sea during 2004 to 2008 showed that is reduced with increasing water depth; its value in 1020 m depth layer was 3.8 times higher than that of 50100 m (valinassab, 2011). however, according to the present study, the highest cpua was found in 20-30 m depth layer, which was 8.3 times higher than that of 10-20 m. our findings suggests that immediate management is required to optimal exploitation of the studied fishes, such as limited fishing season during spawning and limit the catch on heavily-fished waters. furthermore, imposed a ban on bottom trawls and other unselective fishing gear would be beneficial to avoid damage to the fishing industry and the marine environment by catching juvenile fish, damaging the seafloor, and leading overfishing. references abbaspour n.r., vosoughi g., valinasab t., jamili s. (2010). study of biomass rate, catch per unit area (cpua), distribution and abundance of demersal fishes in deep-sea layers of oman sea. journal of animal environment, 2: 29-40. bianchi g. (1985). field guide to the commercial marine and brackish-water species of pakistan. prepared with the support of pak/77/033 and fao (firm) regular programme. rome, fao. 200 p. ghotbeddin n., izadpanah z., valinassab t., azhir m. (2015). biomass and cpua estimation and distribution pattern of saurida tumbil from northwest of the persian gulf. journal of the persian gulf, 6: 2936. kesteven g.l., nakken o., strømme t. (1981). the smallpelagic and demersal fish resources of the north-west arabic sea surveys 1975-1976. institute of marine research, bergen. 110 p. maunder m.n., sibert j.r., fonteneau a., hampton j., kleiber p., harley s.j. (2006). interpreting catch per unit effort data to assess the status of individual stocks and communities. ices journal of marine science, 63: 1373-1385. mirzaei m.r., azhang b., kazemi s. (2017). evaluation of demersal trawl survey data for assessing the biomass and catch per unit area (cpua) of platycephalidae. research in marine sciences, 2: 59-64. monjezi veysi m., mahboobi soofiani n., valinassab t., daryanabard g. (2017). determination of cpua and distribution pattern of families haemulidae, nemipteridae and ariidae in the oman sea. iranian journal of fisheries sciences, 16: 1297-1311. raeisi h., hosseini s., paighambari s., davoodi r. (2012). abundance of cutlassfish trichiurus lepturus (linnaeus, 1758) in bushehr waters, persian gulf. annual review and research in biology, 2: 37-45. smith m.m., heemstra p.c (1986) smiths' sea fishes. macmillan south africa, johannesburg. 1047 p. sparre, p. (1998). introduction to tropical fish stock assessment. part 1. manual. fao fish. tech. paper., 306: 1-407. valinassab t. (2011). change in species combination of demersal fishes in persian gulf and oman sea a threat for environment. in: (ed.). conference for coordination of iranian maritime organizations. ramsar-iran. pp:115. valinassab t., daryanabard r., dehghani r., pierce g. (2006). abundance of demersal fish resources in the persian gulf and oman sea. journal of the marine biological association of the united kingdom, 86: 1455-1462. int. j. aquat. biol. (2019) 7(4): 195-201 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article effects of sublethal exposure to new pesticides lufenuron and flonicamid on common carp, cyprinus carpio, hydromineral balance to further saltwater exposure melika ghelichpour, ali taheri mirghaed *1 department of aquatic animal health, faculty of veterinary medicine, university of tehran, tehran, iran. s article history: received 7 april 2019 accepted 24 august 2019 available online 2 5 august 2019 keywords: lufenuron flonicamid adaptation stress biochemical assay abstract: the effects of 21 days exposure to lufenuron and flonicamid were investigated on common carp responses to saltwater exposure. fish were assigned in three groups: control (21 days in freshwater), lufenuron (exposed for 21 days) and flonicamid (exposed for 21 days). after 21 days, all fish were subjected to 15 g/l saltwater for further 4 days. plasma glucose, cortisol, sodium, potassium, chloride, calcium and phosphorus levels were monitored 0, 24, 72 and 120 h after saltwater exposure. type of pesticides and time of salt water exposure had interaction on plasma glucose and cortisol levels. these two stress indicators were increased significantly in all groups after saltwater exposure. the cortisol elevation continued until 72 h after salinity challenge and then decreased significantly after 120 h salinity exposure in all treatments. plasma glucose level showed fluctuation during saltwater exposure. it was increased after 24 h saltwater exposure, then decreased after 72 h and again it was increased after 120 h salinity challenge. increased plasma sodium showed significant elevation along with elongation of saltwater exposure. pesticide exposure significantly affected plasma chloride levels as the flonicamid group had significantly lower chloride compared to the control and lufenuron groups. plasma chloride showed significant elevation along with elongation of saltwater exposure. pesticide and time of salinity challenge had interaction on plasma calcium levels as 24 h after salinity challenge calcium level of pesticide groups increased significantly. along with elongation of saltwater exposure, calcium level of pesticides treatments decreased but it higher than the pre salinity challenge. plasma phosphorus level increased 24 h after saltwater exposure and decreased along with elongation of saltwater exposure. in conclusion, lufenuron and flonicamid induce stress and alter gill function and blood ionic homeostasis during saltwater exposure. introduction now a days, input and distribution of pollutants to ecosystems and their effects are of the main environmental concerns. industrial developments and population growth led to chemical pollutant accumulation in aquatic ecosystems (saghali et al., 2014). the pollutants behaviors may be assessed at three levels: water column, sediments and biomass of aquatic organisms (saghali et al., 2014). one of the serious threats for human is pollutant entry to waters, leading to accumulation in aquatic organisms’ body and moving towards higher levels of food chain (shaw and handy, 2011). agricultural pesticides are considered as one of the largest group of environmental pollutant, which are extensively *correspondence: ali taheri mirghaed doi: https://doi.org/10.22034/ijab.v7i4.662 e-mail: mirghaed@ut.ac.ir studied in aquatic toxicology (wang et al., 2015). pesticide exposure can affect fish responses to further stress. hpi is involved in this case and studies have shown pesticide exposure significantly affects cortisol responses (bonga and lock, 1991). the effects of the pesticides on fishes are of important environmental concerns. flonicamid (iki220; n‐ cyanomethyl‐4‐trifluoromethylnicotinamide), a pyridinecarboxamide compound, is a novel systemic insecticide with selective activity against hemipterous pests, such as aphids and whiteflies, and thysanopterous pests (staetz et al., 2006). lufenuron, a benzoylurea pesticide, inhibits the production of chitin in insects. without chitin, a larval flea will never develop a hard outer shell (exoskeleton). lufenuron is 196 ghelichpour and taheri mirghaed / effects of new pesticides lufenuron and flonicamid on common carp also sold as an agricultural pesticide for use against lepidopterans, eriophid mites, and western flower thrips. common carp, cyprinus carpio, is an economically important species rearing in many parts of iran (hosseini and hoseini, 2012). it is a popular fish in iran and its population in the caspian sea is supported by stock rehabilitation activities of iranian fisheries organization, because of declining its natural population. this species may face saltwater stress during stock rehabilitation. there are no data about lufenuron and flonicamid biochemical effects in common carp when exposed to saltwater. thus, the aim of the present study was to study the effects of 21 days exposure to lufenuron and flonicamid on biochemical responses to saltwater exposure in common carp. materials and methods fish and pesticides preparation: a total number of 600 juvenile common carp with an average weight of 48±4.25 g were stocked in 2000 l fiberglass tank and acclimatized under laboratory condition for two weeks (in research station of gharahsoo, bandar turkeman, iran). during adaptation period, water was aerated persistently and it was exchanged about 75 percent daily. the fish were fed (1.5% of body weight) twice a day with commercial carp feed (mazandaran animal and aquatic feed co., sari, mazandaran, iran). physico-chemical parameters of water monitored daily during the experiment by hach hq40d portable apparatus (loveland, colorado, usa); temperature (27.5±1.25ºc), dissolved oxygen (7.1±0.84 mg l-1), salinity (2.63±0.15 g l-1), and ph (8.5±0.25). total hardness 300±17.5 mg l-1 (as caco3), alkalinity 350±20.3 mg l-1 (as caco3), and calcium 110±11.7 mg l-1 were measured by a portable photometer (wagtech 7100, berkshire, uk). lc50-96-h determination: lethal concentration of lufenuron and flonicamid pesticides were determined using oecd (1992) procedure. according to pretreatment test, 5 different concentrations of each pesticide were considered. a total of 300 common carp were randomly distributed in 30 fiberglass tanks (15 tanks for each pesticide; 3 tanks per concentration and 5 concentrations per pesticide) with a volume of 160 l. the fish were exposed to concentrations of 0 (control), 5, 10, 15, 20 ppm lufenuron and 0 (control), 30, 40, 50 and 60 ppm flonicamid. water exchange was 75 percent daily and required amounts of pesticides were added to each tank to set concentrations. the other physico-chemical parameters of water and fish feeding were same as the acclimation period. during the experiment, the number of dead fish were recorded after 96 h exposure to lufenuron and flonicamid. chronic experiment and salinity challenge: in this part of the experiment, 12 fiberglass tanks were assigned for 3 different treatments (4 tanks per treatment); control (without any pesticides), 10% of lc50-96-h lufenuron (0.11 mg l-1), 10% of lc50-96-h flonicamid (4.3 mg l-1). each tanks stocked with 25 fish which exposed to sub-lethal concentrations of the pesticides for 21 days. at the end of the pesticides exposure period, feeding was ceased and all groups were subjected to saltwater (addition of 12 g nacl to one litter of water) over 120 h. this salinity was chosen according to a preliminary experiment to determine tolerable salinity for the experimental fish. biochemical analysis: blood samples of all groups were taken 0 (just before the salinity appending), 24, 72, and 120 h after the salinity challenge from caudal vein (six samples at each point). two fish per tank were sampled randomly with a dip net and immediately anesthetized with 100 mg l-1 eugenol (hoseini et al., 2015) within 1 min. blood samples were taken using heparinized syringes and plasma was separated after 10 min centrifugation (1000×g), and maintained at -80ºc until analysis. all the experiments were conducted under a protocol accepted by the committee of ethics of the faculty of sciences of the university of tehran (357; 8 november 2000). plasma levels of chloride, calcium, phosphorus and glucose were determined photometrically using pars azmun kits (hoseini et al., 2016) and zist shimi kits (tehran, iran) (hoseini and tarkhani, 2013). plasma samples were assayed for sodium and potassium using a flame photometer (seac, florence, italy) (hoseini 197 int. j. aquat. biol. (2019) 7(4): 195-201 and tarkhani, 2013). plasma cortisol was determined by elisa method based on competition principle using a commercial kit (ibl, gesellschaft für immunchemieund immunbiologie, hamburg, germany). statistical analysis: the mortality of the fish was subjected to probit analysis to calculate the lc50. the plasma data were subjected to two-way anova and duncan tests. mean values were considered significantly different when p<0.05. data are presented as mean ± standard error. all analysis were performed using spss software version 16.0 (spss inc., chicago, il, us). results in the present study, lc50-96h lufenuron and flonicamid were determined 1.1 mg/l and 43 mg/l, respectively. according to the results, the pesticide exposure significantly affected plasma glucose levels as the control group had significantly lower glucose compared to the pesticide groups. also, time of saltwater exposure significantly affected plasma glucose levels. plasma glucose level of the control group significantly increased 24 h after saltwater exposure. plasma glucose level of the control group decreased and again increased significantly in 72 and 120 h after salinity challenge, respectively. in the lufenuron treatment, the highest level of plasma glucose was after 24 h exposure to the saltwater and it was decreased over time. salt water exposure caused a significant elevation in flonicamid glucose level 24 h after salinity challenge and decreased along with elongation of saltwater exposure up to 120 h (fig. 1). the difference between plasma cortisol levels of pre challenge (0 h) treatments was similar to the plasma glucose levels. bar chart shows plasma cortisol of the control group and pesticide treatments significantly increased in exposure to 24 h salt water compared to the pre challenge and remained high within 72 h salinity challenge. plasma cortisol level of the control group and pesticide treatments decreased significantly after 120 h saltwater exposure in such a way there were no differences in plasma cortisol of each treatment compared to the pre challenge. also, 120 h after exposure to the salt water, there were no differences between plasma cortisol of the control group and pesticide treatments (fig. 2). figure 3 shows pesticides had no significant effects on plasma sodium, but time of saltwater exposure significantly affected plasma sodium levels. plasma sodium showed significant elevation along with elongation of saltwater exposure. plasma potassium level had a fluctuation along with elongation of saltwater exposure. flonicamid affected plasma figure 1. effects of pesticide exposure on plasma glucose response to a 120-h saltwater exposure in common carp. uppercase letters indicate significant difference among the treatments at each time. lowercase letters indicate significant difference among each treatment in all times (n=6, p<0.05). figure 2. effects of pesticide exposure on plasma cortisol response to a 120-h saltwater exposure in common carp. uppercase letters indicate significant differences among the treatments at each time. lowercase letters indicate significant differences among each treatment in all times (n=6, p<0.05). 198 ghelichpour and taheri mirghaed / effects of new pesticides lufenuron and flonicamid on common carp potassium, so that plasma potassium level of flonicamid treatment was significantly higher than the lufenuron and control groups (fig. 4). pesticide exposure significantly affected plasma chloride levels as the flonicamid group had significantly lower chloride compared to the control and lufenuron groups. time of saltwater exposure significantly affected plasma chloride levels. plasma chloride showed significant elevation along with elongation of saltwater exposure (fig. 5). the results show that the kind of pesticides and the time of exposure to the salinity challenge had interaction on plasma calcium levels. plasma calcium level of fish exposed to lufenuron and flonicamid increased significantly after 24 h exposure to the salt water. more elevation of plasma calcium level were observed in flonicamid fish exposure after 72 h salinity challenge. however, after 120 h exposure to the salt water calcium level were decreased in both pesticides groups (fig. 6). figure 3. effects of pesticide exposure on plasma sodium response to a 120-h saltwater exposure in common carp. different letters show significant differences among the time exposure (n=6, p<0.05). figure 4. effects of pesticide exposure on plasma potassium response to a 120-h saltwater exposure in common carp. different letters show significant differences among the time exposure and treatments (n=6, p<0.05). figure 5. effects of pesticide exposure on plasma chloride response to a 120-h saltwater exposure in common carp. different letters show significant differences among the time exposure and treatments (n=6, p<0.05). 199 int. j. aquat. biol. (2019) 7(4): 195-201 duration of exposure to salinity affected plasma phosphorus level, so that it is significantly increased after 24 h exposure to saltwater. along with elongation saltwater exposure (after 72 and 120 h) plasma phosphorus decreased significantly with no difference compared to the control group (fig. 7). discussions plasma glucose and cortisol are indicator of stress in fish (bonga, 1997; barton, 2002). stressors cause temporary increase in plasma cortisol levels to provide demanded energy to deal with stress during stressful conditions (bonga, 1997, 2002; aluru and vijayan, 2009). during salinity challenge, more level of cortisol needs to compensate the energy to osmoregulation and osmotic stress as increase chloride cell number and size (mccormick, 1990) and increase in na+,k+-atpase activity (madsen, 1990). in the present study, the cortisol elevation was occurred 24 and 72 h after saltwater exposure. this result is agree with ghelichpour et al. (2018) which common carp exposed to indoxacarb. glycogen utilization leads to glucose production in fish liver, thus, increased circulating glucose levels. as glycogen stores are limited, prolonged stress leads to gluconeogenesis from lactate and amino acids to maintain hyperglycemia (barton, 2002). in the present study, the pesticide groups had significantly higher glucose compared to the control, suggesting induction of stress by these pesticides. similarly, taheri mirghaed et al. (2018) found increase in plasma glucose due to indoxacarb exposure in common carp. simakani et al. (2018) found hyperglycemia in common carp after exposure to mancozeb. on the other hand, exposure to saltwater led to increase plasma glucose. similarly, ghelichpour et al. (2018) and hoseini and hosseini (2010) reported increased plasma glucose in common carp exposed to saltwater. sodium, potassium and chloride are the most abundant ions in fish blood with varieties of vital roles (bonga and lock, 1991). in the present study, saltwater exposure led to significant elevation in plasma sodium and chloride in common carp. it is in line with previous studies showing such elevations in plasma sodium and chloride in common carp exposed to saltwater (ghelichpour et al., 2018; taheri mirghaed et al., 2018). potassium level of plasma increased in pesticide groups and after saltwater exposure, it showed fluctuation. common carp is a stenohaline species and cannot tolerate wide range of water salinity, instead, it augment internal ions levels to mitigate osmotic gradient across body surface and prevention of water lose (van der linden et al., 2004). exposure to lufenuron and flonicamid significantly decreased plasma chloride. it has been previously reported that pesticides decrease chloride levels via two ways: 1) induction of stress and increase in kidney figure 6. effects of pesticide exposure on plasma calcium response to a 120-h saltwater exposure in common carp. uppercase letters indicate significant differences among the treatments at each time. lowercase letters indicate significant differences among each treatment in all times (n=6, p<0.05). figure 7. effects of pesticide exposure on plasma phosphorus response to a 120-h saltwater exposure in common carp. different letters show significant differences among the time exposure (n=6, p<0.05). 200 ghelichpour and taheri mirghaed / effects of new pesticides lufenuron and flonicamid on common carp chloride loss, and 2) effects on carbonic anhydrase activity in fish gill (taheri mirghaed et al., 2018). similarly, banaee et al. (2014) found decrease in plasma chloride in common carp exposed to chlorpyrifos. calcium is an important ion in fish circulation, which its levels is tightly controlled in healthy fish (kaneko and hirano, 1993). in this study, pesticide exposure modified calcium responses to saltwater exposure; the control group showed no changes in plasma calcium levels during saltwater exposure, but the pesticide groups had elevated calcium levels within this period. such changes might be due to stress induced by pesticides that leads to cortisol elevation and induction of calcium pump in fish (hoseini et al., 2018). on the other hand, it has been reported that saltwater exposure leads to increased calcium (jalali et al., 2010). higher changes in calcium in the pesticide groups might be due to gill damages, impairing osmoregulation and ionoregulation during saltwater exposure. phosphorus level increased 24 h after salinity challenge and then decreased which the same as pre salinity challenge. conclusion in conclusion, the results demonstrate that sub-lethal exposure to lufenuron and flonicamid impairs cortisol and glucose response to saltwater stress. it interferes glucose metabolism and hydromineral balance. all these effects threatens the fish life chance in response to saltwater stress. references aluru n., vijayan m.m. 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(2010). effect of dietary ltryptophan on osmotic stress tolerance in common carp, cyprinus carpio, juveniles. fish physiology and biochemistry, 36: 1061-1067. hoseini s.m., tarkhani r. (2013). effect of short‐term treatment with potassium permanganate on stress markers and blood biochemistry in goldfish carassius auratus. aquaculture research, 44: 869-875. hoseini s.m., rajabiesterabadi h., tarkhani r. (2015). anaesthetic efficacy of eugenol on iridescent shark, pangasius hypophthalmus (sauvage, 1878) in different size classes. aquaculture research, 46: 405-412. hoseini s.m., rajabiesterabadi h., kordrostami s. (2016). chronic exposure of rutilus rutilus caspicus fingerlings to ambient copper: effects on food intake, growth performance, biochemistry and stress resistance. toxicology and industrial health, 32: 375-383. hoseini s.m., tort l., abolhasani m.h., rajabiesterabadi h. 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(2013) 1(4): 175-184 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article studies on reproductive biology of mystus tengara (ham.-buch., 1822), a freshwater catfish of west bengal, india sandipan gupta*,1samir banerjee aquaculture research unit, department of zoology, university of calcutta 35, ballygunge circular road, kolkata700019, west bengal, india article history: received 25 july 2013 accepted 8 august 2013 available online 2 0 august 2013 keywords: mystus wetland gonad maturity spawning sex ratio abstract: studies on reproductive biology are essential to assess culture potential of a fish species. mystus tengara is a popular food fish as well as preferred as an ornamental fish in west bengal. till date detailed report on reproductive biology of this fish species in the agro-climatic context of west bengal is lacking. therefore, the present work was aimed to study the detailed reproductive biology of mystus tengara with an emphasis on sex ratio, length at first sexual maturity, cycle of gonadal maturation and spawning periodicity using standard methods. results of the study revealed female dominance of the species over male in the population. however, the males showed earlier maturation than females. five gonadal maturity stages namely immature, maturing, mature, ripe and spent were identified both for female and male fishes. monthly study of gonadosomatic index (gsi), condition factor and mean ova diameter revealed that the breeding season for this fish species extended from may to september with a single spawning month in july. total spawning behaviour along with synchronous oocytes development was also observed in this fish species. introduction mystus tengara is commonly known as tengara mystus which is a freshwater species, inhabits both flowing and standing waters. the species is distributed in india, nepal, bangladesh and pakistan (talwar and jhingran, 1991). in west bengal it is locally known as tengara and is a preferred food fish due to its good taste, high nutrient profile; and in recent times it has also got its importance as ornamental fish too (gupta and banerjee, 2012). studies on reproductive biology of any fish species are essential for assessing commercial potentialities of its stock, life history, culture practice and actual management of its fishery (doha and hye, 1970). reproductive potential of a population is one of the basic exigencies to designate the individuals of that population in respect to their gonadal conditions * corresponding author: sandipan gupta e-mail address: : sandipangupta2007@gmail.com tel: +919830082686 (jhingran and verma, 1972). in order to make success in fish culture, it is important to assess the yearly breeding cycle of culturable fishes (stoumboudi et al., 1993). spawning of fish occurs during a particular phase of reproductive cycle; some of them breed once annually while others at regular intervals throughout the year. knowledge of gonadal development and spawning season of a species allow subsequent studies on spawning frequency of its population, which is important for its management (chakraborty et al., 2007). study of sex-ratio, length at first sexual maturity, cycle of maturation and spawning periodicity etc. are essential part of reproductive biology investigation of fishes (reddy, 1979; vazzoler, 1996). a number of workers (qasim and qayyum, 1961; bhatt, 1971a, b; rao and sharma, 1984; roy and 176 gupta and banerjee / int. j. aquat. biol. (2013) 1(4): 175-184 hossain, 2006; musa and bhuiyan, 2007) have studied different aspects of reproductive biology of different species of mystus. rastogi and sexena (1968) and guraya et al. (1975) have studied seasonal changes in morphology and activity of mystus tengara ovary. however, till date there is no report on reproductive biology of mystus tengara in the agro-climatic context of west bengal. the objective of the present study was to study the reproductive biology of mystus tengara collected from a selected wetland in west bengal. materials and methods monthly samples of mystus tengara were collected from an undisturbed wetland baruipur, south-24paraganas district of west bengal (latitude n 22°34', longitude e 88°43') for a period of 12 months starting from september, 2008. in total, 721 specimens (never less than 50 in a month) were collected during the entire study period for evaluation of reproductive biology of the fish. sampled fish were brought to the laboratory, total length (to the nearest of 0.1 cm) was measured by a measuring scale, washed thoroughly with clean water, soaked by a blotting paper and total body weight was measured (to the nearest of 0.01gm) by an electronic balance (sartorius, model no. bt 223s). fish specimens were dissected out ventrally to remove gonads carefully. surface moisture of gonads was removed using blotting paper and the weight and length of the gonads were measured to the nearest of 0.01 gm and 0.1 cm, respectively. sexes of the sampled fish specimens were determined after examination of the gonads. monthly variation in sex ratio was determined from the total number of two sexes in monthly collected samples. chi-square test (zar, 1999) was performed to investigate the differences in sex-ratio (monthly value and over-all value) from the expected ratio of 1:1. fish specimens were grouped into different length classes with interval of 0.5 cm and the length class in which at least 50% of the fish specimens were observed to be mature was regarded as length at first sexual maturity (rao and sharma, 1984; suresh et al., 2006; mitra et al., 2007). cycle of maturation was studied by macroscopic and microscopic observation of the different maturation stages of gonad; male and female gonads were grouped into different gonadal stages of development according to nikolsky (1963). additional information for differentiation of gonadal maturity stages was gathered following the work of bhatt (bhatt, 1970, 1971b) who worked on two other species of mystus. spawning periodicity was determined by monthly evaluation of the gonadosomatic index (gsi), condition factor and mean ova diameter. gsi and condition factor (k) were measured using the following formulae (htun-han, 1978): gsi = gonad weight (gm) x 100 total body weight (gm) k = (tbw − gw)x 100 tl3 where tbw is total body weight, gw is gonad weight and tl is total length. size frequency distribution of the intra-ovarian oocytes was studied on monthly basis to determine the type of oocytes development. for measuring the size frequency distribution of the intraovarian oocytes, a small representative part from the middle portion of the right or left ovary was taken out separately and put into physiological saline solution (0.85% nacl) in a petridish. the ova present in the ovary samples were separated and spread on a glass slide to measure the diameter under a microscope fitted with micrometer following the method of lecren (1951). the ocular micrometer reading was standardized with that of the stage micrometer for measurement of ova diameter in micrometer (μm) and then the values were transformed to mm unit. ova were then grouped into four size classes; immature ova (0.10-0.30 mm), maturing ova (0.300.45 mm), mature ova (0.45-0.60 mm) and ripe ova (0.60-0.85 mm) depending on the maturity status of the ova and then monthly percentage frequency of 177 gupta and banerjee / int. j. aquat. biol. (2013) 1(4): 175-184 four size classes was calculated to get the information on the spawning type. results sex-ratio: among the 721 specimens studied, 451 and 270 were observed to be female and male, respectively (table 1). the average ratio of males to females was observed to be 1:1.67. overall females showed significant (p<0.01) dominance over males; though on monthly basis only from may to july (p<0.01) and also in april, october and november (p<0.05) significant dominance of females over males was observed. length at first sexual maturity: the smallest males with mature gonads were observed to appear in 7.58 cm size group. 50% of all males were observed to be mature in the 8.5-9 cm size group and all males above 10 cm were observed with mature testes during the spawning season. few females in 8-8.5 cm size group were observed with mature gonads while 50% of the females were found with mature gonads in the size group of 9-9.5 cm. all females male (observed value) female (observed value) ratio of male and female p 2 remark month no. of fish no. % no. % sept., 2008 52 24 46.15 28 53.85 1:1.17 0.579 0.31 ns oct., 2008 60 22 36.67 38 63.33 1:1.73 0.039 4.27 s* nov., 2008 62 22 35.48 40 64.52 1:1.82 0.022 5.23 s* dec., 2008 45 18 40.00 27 60.00 1:1.50 0.180 1.80 ns jan., 2009 56 22 39.29 34 60.71 1:1.54 0.109 2.57 ns feb., 2009 62 25 40.32 37 59.68 1:1.48 0.128 2.32 ns mar., 2009 75 31 41.33 44 58.67 1:1.42 0.133 2.25 ns apr., 2009 62 22 35.48 40 64.52 1:1.82 0.022 5.23 s* may, 2009 66 22 33.33 44 66.67 1:2.00 0.007 7.33 s** june, 2009 65 21 32.31 44 67.69 1:2.09 0.004 8.14 s** july, 2009 62 19 30.65 43 69.35 1:2.26 0.002 9.29 s** aug., 2009 54 22 40.74 32 59.26 1:1.45 0.174 1.85 ns p = probability; 𝟀2 = chi-square; ns = non significant; s** = significant at 1% level; s* = significant at 5% level table 1. monthly variation of sex ratio in mystus tengara. figure 1. percentage of mature fish in different length groups in mystus tengara. 177 178 gupta and banerjee / int. j. aquat. biol. (2013) 1(4): 175-184 above 10.5 cm were observed with mature ovaries during the spawning season (fig. 1). cycle of gonadal maturation: five stages of maturity of ovary and testes were recognized as follows: stage i (immature): ovaries translucent and colorless; ova not visible to naked eyes, but under microscope ova were irregular in shape, transparent, yolk not formed. testes whitish in color, very narrow, thread like, no testicular lobules were visible. stage ii (maturing): ovaries yellowish white in color; ova visible to naked eyes but not prominent; under microscope ova were spherical in shape, slightly opaque due to deposition of yolk at the central position. testes milky white in color, thread like, appearance of little testicular lobules. stage iii (mature): ovaries deep yellowish in color and enlarged in size; ova clearly visible to naked eyes; under microscope spherical in shape and completely opaque (except the periphery) in appearance due to presence of yolk. testes light yellowish in color, testis lobes increased in size and length; increased number of testicular lobules. stage iv (ripe): ovaries reddish yellow in color; with maximum size; ova clearly visible to naked eyes; under microscope were spherical in shape and opaque due to presence of huge amount of yolk. in this stage, ova were with their full size, came out on putting light pressure on the abdomen. testes yellowish white in color, testes lobes extended much and two lobes were nearly touching each other. testicular lobules increased in number and length. milt came out while putting slight pressure on abdomen. stage v (spent): ovaries very much reduced in size, shrunken and reddish in appearance. under microscope, few ripe ova along with irregular shaped small translucent ova were visible. testes translucent, thread like and flaccid in appearance; no testicular lobules were visible. females with immature gonads were observed from october to april; highest percentage being observed from october to december while lowest percentage was observed in april. maturing females first were observed in january and available till may; highest and lowest percentage being observed in april and january, respectively. mature females were observed from april to july; highest percentage was observed in may while lowest percentage in july. ripe females were observed from may to august; highest percentage being observed in july and lowest percentage in may. spent females were observed from july to september; highest percentage being observed in september and lowest percentage in july (fig. 2). males with immature gonads were observed from october to may; highest percentage being observed in november and december while lowest percentage was observed during may. maturing males first were observed in january and available till june; highest figure 2. monthly percentage of different gonadal maturation stages in female mystus tengara. 179 gupta and banerjee / int. j. aquat. biol. (2013) 1(4): 175-184 percentage being observed in april and lowest percentage in january. mature males were observed from may to july; highest percentage was observed in june while lowest percentage in july. ripe males were observed from june to september with highest percentage being observed in july and lowest percentage in september. spent males were observed from july to october; highest percentage being observed in september and lowest percentage in july (fig. 3). gonado somatic index (gsi): in both female and male, gsi was observed to reach peak once a year during the month of july. the lowest value of gsi was observed in the month of december; then it started to increase from january onwards and reached the peak in july; then dropped down in august to reach the lowest value again in december (fig. 4). in respect to both sexes, gsi showed significant (p<0.01) positive relationship with total body weight (tbw), total length (tl), gonad weight (gw) and gonad length (gl) as follows: gsi = -3.38 + 0.73 tbw (r = 0.39, p<0.01, se = 4.37) gsi = -7.59 + 1.06 tl (r = 0.21, p<0.01, se = 4.65) gsi = 0.31 + 8.85 gw (r = 0.95, p<0.01, se = 1.55) gsi = -8.87 + 6.42 gl (r = 0.63, p<0.01, se = 3.68) condition factor: in both female and male, condition factor was observed to reach peak once a year during june. the lowest value of condition factor was observed in the month of december; then it started to increase from january onwards and reached the peak in june; then dropped down in july to reach the lowest value again in december (fig. 5). figure 4. monthly variation of gonado-somatic-index (gsi) of male and female mystus tengara. figure 3. monthly percentage of different gonadal maturation stages in male mystus tengara. 179 180 gupta and banerjee / int. j. aquat. biol. (2013) 1(4): 175-184 size frequency distribution of intra-ovarian oocytes: the frequency of occurrence of ova of different diameter plotted against months showed that immature ova were observed from august to april; the percentage occurrence of immature ova was observed to be highest from october to december and lowest percentage being observed in april. maturing ova were observed from january to may; highest percentage being observed in april and lowest percentage in january. mature ova were observed from april to july; highest percentage being observed in may and lowest percentage in july. ripe ova were observed from may to september; highest percentage was observed in july while lowest percentage in may (fig. 6). the mean monthly ova diameter was observed to reach peak once in a year, in the month of july. the lowest value of mean monthly ova diameter was observed in october; then it started to increase gradually from november onwards to reach the peak in july; then dropped down in august to reach the lowest value again in october (fig. 7). discussion monthly record of sex-ratio of m. tengara indicated a female dominance over male in the population and deviation from the expected ratio of 1:1. bhatt (1971b), rao and sharma (1984), roy and hossain (2006), musa and bhuiyan (2007) etc. also reported female dominance over male in different species of mystus. similar observations on the deviation of sex ratio from the expected value and female dominance over male in the population of other fish species have been reported earlier by number of workers (suresh figure 5. monthly variation of condition factor of male and female mystus tengara. figure 6. monthly percentage frequency of four size groups of intra-ovarian ova in mystus tengara. 181 gupta and banerjee / int. j. aquat. biol. (2013) 1(4): 175-184 et al., 2006; mondal and kaviraj, 2010; olurin and savage, 2011; parvin et al., 2011; banik et al., 2012) supporting the results of the present study. the reason behind the female dominance in this fish population is not clear; but may be it is a mechanism of population regulation as earlier described by fagade et al. (1984). on the other hand, some authors (ursin, 1963; cooper, 1983) concluded that the metabolic strain of spawning was generally greater in older males than in older females rendering more mortality amongst males than females specifically during the spawning period in this studied fish population. early maturation of males over females also could account for greater strain in males. bhatt (1971b) and rao and sharma (1984) also reported earlier maturation of males than females in different species of mystus. same trend of early maturation of males in respect to females have also been reported by many workers (suresh et al., 2006; banik et al., 2012; rahman and tachihara, 2005) for other fish species and thus supporting the observation of the present study. study of monthly gsi values showed only one peak in the month of july for both the sexes and high gsi values were observed from may to september. the relationship between gsi and spawning periodicity has been stated earlier; gsi tend to increase with maturation of gonad (initiation of breeding season), become maximum during the period of peak maturity and decline abruptly thereafter, when the fish becomes spent after gamete extrusion and/or reabsorption (le cren, 1951; nikolsky, 1963; olurin and savage, 2011). therefore, during the monthly study of gsi value in any fish species; the month(s) at which gsi value(s) reach at peak(s) depict the spawning period for that particular fish species and the months with high gsi values represent the breeding periodicity of that particular fish species. results of the present study indicate that mystus tengara is a single-spawner with july as the spawning month and the breeding season expanding from may to september. throughout maturation, the gsi values of females were observed to be much higher than males implying that a greater proportion in body reserves was allocated to the gonads in females (chatzifotis, 2004). gsi showed highly significant (p<0.01) positive relationship with total body weight, total length, gonad weight and gonad length; but comparison of correlation coefficients of gsi-total body weight (r=0.39), gsi-total length (r=0.21), gsi-gonad weight (r=0.95) and gsi-gonad length (r=0.63) have indicated that variation in gsi can be explained better in terms of gonad weight than in terms of total body weight, total length and gonad length for this fish species. monthly condition factor (k) values showed only one peak during june and dropped down in july. the correlation between condition factor and gonad weight indicated that k value increased with figure 7. monthly variation of mean intra-ovarian ova diameter in mystus tengara. 181 182 gupta and banerjee / int. j. aquat. biol. (2013) 1(4): 175-184 increasing gonad weight and reached maximum just before the spawning period and then dropped during the spawning month due to loss of gonadal products (marammazei et al., 2000; hernandez et al., 2003; kiran and puttaiah, 2003) confirming july as the spawning month for this fish species. guraya et al. (1975) also reported july as its spawning month, though rastogi and saxena (1968) reported june as the spawning month. the small duration of breeding season is very common in mystus; qasim and qayyum (1961) reported june-september as the breeding season for mystus vittatus; while bhatt (1971a, b) found mystus vittatus and mystus cavasius to breed during august-september in aligarh. mean monthly ova also showed maximum value during july, the spawning month due to highest percentage of ripe ova available during this period in the gonad further confirming july as the spawning month for mystus tengara. the changing trend of mean monthly ova-diameter corresponded with the frequency of occurrence of different size-classes of intra-ovarian oocytes depicting a synchronous development of oocytes in mystus tengara (qasim and qayyum, 1961; bhatt, 1971b; rao and sharma, 1984). ripe ova were present from may to september in the gonads indicating this period as the breeding season for mystus tengara. absence of maturing and mature ova during august is an indication of the completion of the spawning period for this fish species (chakraborty et al., 2007). till now, fishery of mystus tengara is capture-based. it is now essential to start its captive culture. since mystus tengara is available with a sex ratio of almost 1:2 (male: female) two females for one male can be stocked for captive breeding of mystus tengara to get success. the present study reveals that the right time to collect the brooders from nature is may-june for mystus tengara. however, proper strategies to conserve the fish species in its natural habitat are required. in this respect length at first sexual maturity is of special interest in fisheries management and is widely used as an indicator of minimum-permissible capture size (lucifora et al., 1999). the data of the present study have revealed that the minimum capture size for mystus tengara in nature is 10-10.5 cm. acknowledgements authors are thankful to the head, department of zoology, university of calcutta for providing the lab facilities for the research work. the financial support of the university grants commission (ugc) for this research work is gratefully acknowledged. references banik s., goswami p., acharjee t., malla s. 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(2020) 8(2): 91-97 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article effects of dietary olive leaf extract on intestinal immune-related gene expressions in common carp, cyprinus carpio hamid rajabiesterabadi1, afshin ghelichi*1, sarah jorjani1, seyyed morteza hoseini2, reza akrami1 1department of fisheries, azadshahr branch, islamic azad university, azadshahr, iran. 2inland waters aquatics resources research center, iranian fisheries sciences research institute, agricultural research, education and extension organization, gorgan, iran. s article history: received 31 january 2020 accepted 17 march 2020 available online 2 5 april 2020 keywords: common carp olive leaf intestine health abstract: this study aimed to investigate the effects of dietary olive leaf extract (ole) on intestinal immune-related genes expression of tumor necrosis factor alpha (tnfa), interleukin 1 beta (il1b), lysozyme (lys), and mucin2 (muc2). for this purpose, common carp (~15 g) were fed with 0 (control), 0.1 (ole-0.1), 0.5 (ole-0.5) and 1 (ole-1) % ole diets for eight weeks. the fish were sampled after one and eight weeks to study intestinal tnfa, il1b, lys, and muc2 gene expressions. the results showed that dietary ole administration significantly up-regulated intestinal tnfa gene expression after one (all ole-treated groups) and eight (ole-0.5) weeks. moreover, ole-0.1 and ole-1 groups showed up-regulated intestinal il1b expression, after one week, all the ole-treated fish had significantly higher intestinal il1b expression, after eight weeks. ole had no significant effects on lys gene expression after one week, but ole-0.1 and ole-0.5 had significantly higher gene expressions after eight weeks. ole-0.1 and ole-1 had significantly lower muc2 gene expression after one week, but all ole-treated fish had significantly higher muc2 gene expression after eight weeks. in conclusion, dietary 0.1-0.5% ole supplementation is suitable to support common carp intestinal health. introduction aquaculture is an important industry supplying human foods and common carp, cyprinus carpio, is one of the most common aquaculture reared species (yousefi et al., 2019). one of the critical issues in aquaculture is fish health deterioration under artificial conditions, which leads to fish susceptibility to diseases. therefore, several researches have been conducted to find methods for boosting fish health. in this case, dietary supplementation with herbal materials have gained great attention, because the herbal materials are natural antioxidant and most of them stimulate fish immune system (chakraborty and hancz, 2011; chakraborty et al., 2014). there are several studies on common carp, showing dietary herbal material supplementation, including basil, ocimum basilicum extract, palm fruit extract and ginger, significantly increased fish immune and health (amirkhani and firouzbakhsh, 2015; *correspondence: afshin ghelichi doi: https://doi.org/10.22034/ijab.v8i2.839 e-mail: afshin.ghelichi@yahoo.com hoseinifar et al., 2015, 2017; fazelan et al., 2020). among the herbal extracts, olive leaf extract (ole) has recently gained attention as feed additives in aquaculture. administration of ole to fish diet significantly up-regulated tumor necrosis factor alpha (tnfa) and interleukin 1 beta (il1b) along with higher resistance against bacterial challenge (baba et al., 2018; zemheri-navruz et al., 2019). therefore, it might be used as feed supplements in common carp to boost the fish health. fish intestine is an important organ, involving in both nutrient absorption and fish health. fish intestine is directly connected to ambient water, thus is under the threat of ambient pathogens (jutfelt, 2011). therefore, fish must have healthy and high-immunity intestine. there are several studies showing herbal materials are capable to augment fish intestinal health. for example, dietary supplementation with guava, psidium guajava, leaf extract (giri et al., 2015), 92 rajabiesterabadi et al./ effects of dietary olive leaf extract on cyprinus carpio jujube, ziziphus jujube, extract (hoseinifar et al., 2018a), raffinose (karimi et al., 2020), beta-glucan (van der marel et al., 2012) and pectin (edirisinghe et al., 2019) were found to augment intestinal tnfa, il1b, lysozyme (lys), and mucin (muc) gene expression. accordingly, the aim of the present study was to investigate the effects of dietary ole administration on intestinal gene expression in common carp. for this, the expression of tnfa, il1b, lys, and muc2 were studied, as they are important genes in immune responses in fish (karimi et al., 2020). materials and methods ole preparation: ole extraction was performed according to sarhadi et al. (2020) with some modifications. olive leaves were dried against a fan blow for one week. after pulverizing, the leaves were mixed with 70% ethanol with 1:5 proportion and remained at room temperature for a week. then, the mixture was passed through a filter and filtrates were concentrated by a freeze-drier (beta ldpluse, martin christ gefriertrocknungsanlagen gmbh, germany) 72 h (-50ºc). experimental protocol: four diets were used in this experiment, namely control, ole-0.1, ole-0.5 and ole-1 according to fazelan et al. (2020) (table 1). common carp (~15 g) were stocked in 12 tanks (100 l) at a density of 10 fish per tank. the fish were fed the control diet for one week to acclimate with the experimental conditions. then the fish were fed control, ole-0.1, ole-0.5 or ole-1 diets for eight weeks based on 2% of biomass per day. the fish intestine samples were collected after one and eight weeks of rearing for intestinal gene expression analysis. water temperature, dissolved oxygen, ph and ammonia were measured by hach hq40d (loveland, colorado, usa) and wagtech photometer (7100, berkshire, uk), being 22.9±0.55°c, 6.21±0.78 mg/l, 7.15±0.24 and 0.28±0.03 mg/l, respectively. for intestine sampling, two fish were caught from each tank and anesthetized by eugenol (100 mg/l) (yousefi et al., 2018). then the fish were euthanized by spinal cord cutting, followed by cutting the posterior part of the intestine. the samples were immediately frozen in liquid nitrogen and transferred to -70ºc freezer. the rnx-plus extraction kit (sinagene, iran) was used for total rna extraction from the kidney samples according to the kit instructions. the primer sets for quantification of mrna levels of the selected genes were designed based on the common carp sequences found in gen bank (table 2). the oligo 7 program was used for designing the primers. the sybr green table 1. composition of diets (%) used in this study. control ole0.1 ole0.5 ole1 soybean meal 17 17 17 17 fish meal 16 16 16 16 poultry meal 15 15 15 15 wheat meal 38.1 38 37.6 37.1 wheat gluten 10 10 10 10 fish oil 1 1 1 1 soybean oil 1 1 1 1 phytase 0.5 0.5 0.5 0.5 lysine 0.6 0.6 0.6 0.6 methionine 0.3 0.3 0.3 0.3 mineral mix 0.25 0.25 0.25 0.25 vitamin mix 0.25 0.25 0.25 0.25 ole 0 0.1 0.5 1 dry matter 90.8 91 90.6 91.1 protein 39.3 39.2 39.1 39.2 lipid 8.87 8.81 8.78 8.91 ash 6.21 6.22 6.18 6.20 energy (kcal/kg) 3831 3831 3831 3831 93 int. j. aquat. biol. (2020) 8(2): 91-97 method was followed for determination of relative expression of the selected genes using real-time pcr analysis as described by karimi et al. (2020). statistical analysis: data normal distribution and variance homogeneity were confirmed by shapirowilk and levene tests (abtahi et al., 2013). then, the data were analyzed by two-way anova (sampling time and ole levels as factors); as there were interaction effects of the factors on tested parameters, the data were reanalyzed by one-way anova and tukey tests. all data were analyzed in spss v.22 and expressed as mean±sd. results all ole-treated fish showed up-regulated tnfa expression after one week, with the highest value in ole-0.5 treatment. however, after eight weeks, only ole-0.5 treatment had significantly higher gene expression compared to the control treatment (fig. 1). compared to the control group, the ole-0.1 and ole-1 showed up-regulated il1b expression after one week. all ole-treated fish showed up-regulated il1b expression after eight weeks, and the highest value was recorded in the ole-0.5 (fig. 2). there was no significant difference in lys gene expression among the treatments after one week. whereas ole-0.1 and ole-0.5 treatments had a significantly higher lys gene expression compared to the control treatment after eight weeks (fig. 3). those of ole-0.1 and ole-1 had significantly lower muc2 expression compared to the control treatment after one week. the ole-treated fish had significantly higher table 2. sequences of the used primers in this study. gene name primer sequences accession number/reference beta-actin f: cctgtatgccaacaccgtgctg jq619774.1 r: cttcatggtggagggagcaagg il1b f: accagctggatttgtcagaag ab010701.1 r: acatactgaattgaactttg tnfa f: ggtgatggtgtcgaggaggaa aj311800.1 r: tggaaagacacctggctgta lys f: gtgtctgatgtggctgtgct ab027305 r: ttccccaggtatcccatgat muc2 f: tgactgccaaagcctcattc van der marel et al. (2012) r: ccattgactacgacctgtttctc figure 1. effects of different levels of dietary ole supplementation on intestinal tnfa gene expression, after one (gray bars) and eight (white bars) weeks. different letters above the bars show significant difference among the treatments at each sampling time. 94 rajabiesterabadi et al./ effects of dietary olive leaf extract on cyprinus carpio muc2 expressions after eight weeks (fig. 4). discussions tnfa is a pro-inflammatory cytokine with important role in cell proliferation and differentiation (hoseinifar et al., 2019). it stimulates il1b production, which acts as cell proliferator and apoptotic (yarahmadi et al., 2016). these two cytokines have remarkable role in immune response signaling. up-regulation of these genes indicates boosted immune strength in the present study. the present results are in line with previous studies on ole administration to fish. baba et al. (2018) reported elevated tnfa and il1b gene expressions in rainbow trout spleen, along with higher resistance against yersiniosis. moreover, intestinal transcriptome modulation by other herbal materials has also been reported. giri et al. (2015) reported that dietary figure 2. effects of different levels of dietary ole supplementation on intestinal il1b gene expression, after one (gray bars) and eight (white bars) weeks. different letters above the bars show significant difference among the treatments at each sampling time. figure 3. effects of different levels of dietary ole supplementation on intestinal lys gene expression, after one (gray bars) and eight (white bars) weeks. different letters above the bars show significant difference among the treatments at each sampling time. asterisks show significant difference between the sampling times. 95 int. j. aquat. biol. (2020) 8(2): 91-97 supplementation with guava leaf extract significantly up-regulated tnfa and il1b expression in intestine of rohu carp, labeo rohita. hoseinifar et al. (2018a) found that dietary supplementation with jujube fruit extract significantly up-regulated the cytokines gene expression in common carp intestine. lys is a bactericidal enzyme, effective against gram-positive bacteria (saurabh and sahoo, 2008). elevation of lys benefits the host to better react to bacterial infections. the present results indicated that intestinal lys is not sensitive to short-tern-ole administration; however, long-term administration improves lys gene expression. there are several studies reporting up-regulation of intestinal lys gene expression by dietary herbal material administration. karimi et al. (2020) reported that dietary raffinose administration significantly up-regulated intestinal lys gene in common carp. similar results were observed in common carp, treated with jujube fruit extract (hoseinifar et al., 2018a). intestinal mucus is a complex fluid and muc is its main component. muc is filamentous and highlyglycosylated glycoprotein, with high adherence capacity that play important roles in intestinal defense (edirisinghe et al., 2019). the short-term ole treatments significantly down-regulated intestinal muc2 gene expression, which needs further studies to find the exact reasons. however, long-term ole administration clearly up-regulated muc2 gene expression, which is indication of higher immune strength of the fish intestine. previous study on common carp showed that dietary beta-glucan administration significantly up-regulated muc5, but not muc2, gene expression in the fish gill (van der marel et al., 2012). moreover, dietary pectin administration significantly up-regulated whole body muc5, but not muc2, gene expression in zebrafish, danio rerio (edirisinghe et al., 2019). such differences among the studies might be due to different studies tissues, which need further studies for clarification. references abtahi b., yousefi m., kenari a.a. 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(2014) 2(4): 172-179 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2014 iranian society of ichthyology original article fluoxetine and diclofenac interaction on food intake in goldfish, carassius auratus mohammad navid forsatkar1, maryam hedayatirad1, kazem kookaram1, mohammad ali nematollahi*1, wen-bin huang2 1faculty of natural resources, department of fisheries, university of tehran, karaj, iran. 2department of natural resources and environmental studies, national dong hwa university, hualien, taiwan. article history: received 2 march 2014 accepted 14 april 2014 available online 2 5 august 2014 keywords: carassius auratus food intake weight gain fluoxetine diclofenac abstract: this study was carried out to investigate the interaction of simultaneous use of fluoxetine (flx), a selective serotonin reuptake inhibitor, and diclofenac (dcf), a non-steroidal anti-inflammatory drug, on food intake in goldfish, carassius auratus. treatments with different dosage of flx including control, 0, 1, and 10 µg/g body weight (bw) were injected in the fish with mean weight of 30.16 ± 8.57 g every other day in total of 5 times. then fish were exposed to 3 different levels of dcf including 0, 10, and 100 mg/l for 5 days. injection of fluoxetine significantly decreased food intake and consequently body weight. after 5 days exposure to dcf, the amount of food intake in the dcf receiving treatments of 1 mg/l and 10 mg/l was significantly larger than that of 0 mg/l dcf receiving treatment in both the flx dosage groups of 1 μg/g bw and 10 μg/g bw. our results indicated that dcf inhibits behavioral change effects of flx showing the complex effects of pharmaceuticals on fish. introduction study on the antagonistic relationship between nonsteroidal anti-inflammatory drugs (nsaids) and antidepressant pharmaceuticals indicated that the nsaid significantly reduced antidepressant-like effects of ssris but had less effect on other types of drugs, and concluded that reduced use of nsaids in patients with depressive disorders can increase the efficiency of treatment with ssris (warner-schmidt et al., 2011). in addition, simultaneous use of nsaids would increase risk of gastrointestinal bleeding associated with ssris intake. furthermore, the antinociceptive action of nsaids is modulated by the serotonergic system (miranda et al., 2003). therefore, more study is needed to assess the complex relationship between nsaids and ssris and their interactions. flx is one of the most common ssri antidepressants (mennigen et al., 2010), which is metabolized in the liver by the cytochrome p450 enzyme (especially cyp2d6) (hiemke and hartter, 2000). levels of * corresponding author: mohammad ali nematollahi e-mail address: malahi@ut.ac.ir ingested drug are excreted in urine as two forms of parent compound and its main metabolite, norfluoxetine (de vane, 2000). these compounds pass through biological treatment in wastewater treatment plants (gaworecki and klaine, 2008) and the reported environmental concentration range of flx is 12 ng/l to 540 ng/l (calisto and esteves, 2009). flx is a lipophilic drug indicating its ability to accumulate in tissues (hiemke and härtter, 2000). the brain and the liver are the main sites for accumulation of flx in non-target organisms, such as fish (brooks et al. 2005), that have similar actions in both fish and mammalian brain (mennigen et al., 2009). dcf has special pharmacological properties. the activation of cyclooxygenases (cox) is inhibited by dcf, leading to prevent prostaglandin synthesis and subsequently pain is abated (miranda et al., 2003). consumption of 85.80 tons of dcf in germany only in 2001 (huschek et al., 2004) represents the environmental occurrence of this drug in aquatic 173 forsatkar et al./ goldfish nutrition affected by an ssri and nsaids contrast systems. concentration ranges of dcf in german rivers has been reported at 0.15-1.2 µg/l (ternes, 1998). like other drugs, dcf has adverse side effects which relate to inhibition of prostaglandin synthesis (hoeger et al., 2005). pathogenic alterations in gill and kidney of rainbow trout has been reported after dcf exposure (schwaiger et al., 2004). twenty-one days of dcf exposure to brown trout (salmo trutta) resulted in the breakdown of gill lamella and trunk kidney; lysozyme activity increased significantly after 21 days in a 5 µg/l exposure group; however haematocrit value was not significantly different (hoeger et al., 2005). it is well known that alteration in behavior and physiological functions are related to occurrence of disrupting chemicals. in mammals, physiological mechanisms of food intake in relation to changes in serotonin levels have been well investigated (leibowitz, 1985). it has been shown that any process associated with increases in the serotonergic system may induce satiety and reduce appetite (simansky et al., 1992). flx, which inhibits reuptake of serotonin from synaptic junctions, promotes the extracellular serotonin levels within the brain (hemeryck and belpaire, 2002). in fish, de pedro et al. (1998) reported that intracerebroventricular injection of serotonin in goldfish, carassius auratus, significantly reduced food intake 2 hours post injection, however, intraperitoneal administration of serotonin had low effect. they concluded that serotonin acts as a potential factor to control appetite in goldfish. there are many studies on the effects of sub-lethal concentrations of pharmaceutical agents alone in aquatic organisms, especially fish. however, influence of multiple drugs together and interactions between them on fish physiology and behavior has been given less attention. given the complexity of pharmaceutical contaminants in the environment, special attention to this part of ecotoxicological knowledge is needed. therefore, the aim of the present study was to investigate the effects of flx injection and dcf exposure on food intake and weight gain of goldfish. this species is an excellent model to evaluate food intake (mennigen et al., 2009), and has been the subject of many growth studies. material and methods animals: one hundred and fifty goldfish, c. auratus, with an average weight of 30.16 ± 8.57 g were purchased from a commercial fish farm in rasht, iran. they were divided equally and kept in five 100liter fiberglass tanks with constant aeration, water temperature of 21-23 °с, and a photoperiod of 12l: 12d. half of the water in the tanks was replaced with dechlorinated tap water every 2 days from the beginning of a 3-week acclimation period until the beginning of the dcf exposures. fish were fed daily at 11:00-11:30 with commercial pellets at a rate of 2% of body weight (bw). this amount of food is enough for optimal growth in goldfish (volkoff et al., 1999). chemicals and procedures: there were 2 experiments in this study, including experiment-i, flx injection, and experiment-ii, connected dcf exposure. in experiment-i, an 8 mg/ml flx solution was prepared with 8 mg of fluoxetine hcl dissolved in 1 ml of 0.9% physiological saline. concentration of the flx solution was diluted with saline for intraperitoneal injections. nine fish were randomly selected from the five 100-liter fiberglass tanks, weighed, and put into a 25-liter experimental tank (9 individuals per tank). there were 12 experimental tanks that were randomly divided into 4 flx injection dosage treatments (3 replicate tanks per treatment), including no injection (control, flx c), 0.9% saline only (flx 0), 1 µg/g bw (flx 1), and 10 µg/g bw (flx 10). fish were anesthetized with extract of clove powder (20 mg/l) and injected intraperitoneally with the flx dosage of its treatment group every other day in the 9 days of the experiment-i. a total of 5 injections were performed. a food intake test was done 15 minutes after each injection for each tank. in experiment-ii, a 400 mg/ml dcf solution was prepared with 2000 mg of diclofenac sodium dissolved in 5 ml of ethanol for dcf exposures. three dcf exposure concentration treatments, no dcf 174 int. j. aquat. biol. (2014) 2(4): 172-179 addition (dcf 0), 1 mg/l dcf (dcf 1), and 10 mg/l dcf (dcf 10), were used for 5-day dcf exposures in experiment-ii which began continually on the 4th day after accomplishment of experiment-i. the 3 tanks in each flx dosage injection treatment of experiment-i were randomly assigned to the 3 dcf exposure concentration treatments. in each tank, the 9 fish were randomly separated into 3 sub-tanks (3 individuals per sub-tanks) which formed 3 replicates per dcf exposure treatment. therefore, 3 dcf concentration exposure treatments crossed with 4 flx dosage injection treatments with 3 replicates (sub-tanks) were tested. after 5 days of dcf exposures, the food intake tests, 36 in total, were done. execution of the food intake test followed mennigen et al. (2009) and de pedro et al. (1998). fish were not fed for one day before the flx injections as well as one day before the dcf exposures. fifteen minutes after the injection in the first 9 days as well as the day after the 5-day dcf exposures, fish were given excess food with commercial pellets at a rate of 4% of bw. one hour after feeding, residual food was slowly siphoned out and dried in an oven at 60°c for 2 hours, then weighed. food intake (fi) was calculated with the formula: fi= wi (initial dry food weight) – [wf (residual dry food weight) × f (correction factor)], which f, determined by residual ratio of the dry weights of commercial pellets kept in water alone for 1 hour, was 0.88 ± 0.02 (n = 3). to calculate weight gain, fish weight was measured 3 times: (1) before flx injections, after flx injections, and at the end of dcf exposure. until end of injections, 9 fish in each tank, were weighed individually and weight gain calculated by subtracting the initial weight from the final. for weight gain calculation during dcf exposure, 9 fish in each tank were divided into 3 groups of 3 individuals as one replicate and weighed. at the end of exposure, those 3 fish were weighed again and weight gain calculated as before. statistical analysis: two-way and one-way anovas followed by the duncan’s multiple range tests (p<0.05) were used to determine significance source of variation ss df ms f p flx injection 9.846 3 3.282 21.648 0.000*** dcf exposure 1.343 2 0.671 4.429 0.023* interaction 6.171 6 1.028 6.784 0.000*** error 3.639 24 0.152 total 20.998 35 source of variation ss df ms f p dcf exposure effects in the flx group of control 0.815 2 0.408 3.051 0.122 0 μg/g flx 0.634 2 0.317 2.703 0.146 1 μg/g flx 2.366 2 1.183 5.525 0.044* 10 μg/g flx 3.699 2 1.849 13.077 0.006** flx injection effects in the dcf group of 0 mg/i dcf 11.706 3 3.902 19.937 0.000*** 1 mg/i dcf 3.502 3 1.167 6.100 0.018* 10 mg/i dcf 0.808 3 0.269 3.978 0.053 table 1. two-way anova table for the food intake affected by the flx injection treatments crossed with the dcf exposure treatments. * p<0.05; ** p<0.01; *** p<0.001. 175 forsatkar et al./ goldfish nutrition affected by an ssri and nsaids contrast differences of the food intake among the 4 flx dosage treatments crossed with the 5 injection times and the weight gain among the 4 flx dosage treatments in experiment-i, respectively. the twoway anova was also used to determine significance differences of the food intake and the weight gain among the 3 dcf exposure treatments crossed with the 4 flx dosage treatments in experiment-ii. statistics were performed using spss 19.0 software. results in experiment-i of the flx treatment, results of the two-way anova showed that the food intake was significantly affected by both the main factors, the flx dosage (f3,40=66.820, p=0.000) and the injection time (f4,40 =5.222, p=0.002) but not by the interaction (f12,40=1.342, p=0.234). the food intake was significantly the lowest in the flx 10 treatment, followed by the flx 1 treatment and the flx 0 treatment, and the largest in the flx c treatment (fig. 1). the food intake was the lowest after the first injection, significantly increased to the largest after the second injection, and decreased moderately from the third injection to the fifth injection (fig. 2). after the 5 time injections, a dramatic standard deviation of the weight gain in the flx 1 treatment was found while means of the weight gains were insignificantly different among the 4 flx treatments (f3,8=3.041, p =0.093) even though the means in the flx 10 and flx a b c d 0 2 4 6 8 10 control 0 μg/g 1 μg/g 10 μg/g f o o d i n ta k e ( g ) flx injection treatment figure 1. means ± standard deviations of the food intake (g per fish per day) of goldfish (carassius auratus) in the 4 flx injection treatments. n = 15 (3 replicate tanks with 5 injection times); letters that differ indicate significance differences at p<0.05. a b bc bc ac 0 2 4 6 8 10 1st 2nd 3rd 4th 5th f o o d i n ta k e ( g ) injection time figure 2. means ± standard deviations of the food intake (g per fish per day) of goldfish (carassius auratus) in the 5 injection times. n = 12 (3 replicate tanks with 4 flx injection treatments); letters that differ indicate significance differences at p<0.05. figure 3. means ± standard deviations of the weight gain (g per fish per day) of goldfish (carassius auratus) after the 5 time injections in the 4 flx injection treatments. n = 3; letters that differ indicate significance differences at p<0.05. a a a a a a b ba a b b 0 2 4 6 8 10 control 0 μg/g 1 μg/g 10 μg/g f o o d i n ta k e ( g ) flx injection treatment 0 mg/l 1 mg/l 10 mg/l dcf treatment figure 4. means ± standard deviations of the food intake (g per fish per day) of goldfish (carassius auratus) in the 4 flx injection treatments crossed with the 3 dcf exposure treatments. n = 3; letters that differ indicate significance differences at p<0.05. a a a a 0 1 2 3 4 5 6 control 0 μg/g 1 μg/g 10 μg/g w e ig h t g a in ( g ) flx injection treatment 176 int. j. aquat. biol. (2014) 2(4): 172-179 1 treatments were smaller than those in the flx 0 and flx c treatments (fig. 3). in experiment-ii of the flx injection crossed with the dcf exposure, results of the twoway anova showed that the food intake was significantly affected by both the main factors, the flx dosage (f3,24=21.648, p=0.000) and the dcf exposure (f2,24=4.429, p=0.023), and also by the interaction (f6,24=6.784, p=0.000). the effect of the dcf exposure treatments on the food intake was not significant in both the flx c group (f2,24=3.051, p= 0.122) and the flx 0 group (f2,24=2.703, p=0.146) but was significant in both the flx 1 group (f2,24= 5.525, p=0.044) and the flx 10 group (f2,24=13.077, p=0.006) (table 1). in both the flx 1 and flx 10 groups, the food intakes in the dcf 1 and dcf 10 treatments were significantly larger than that those in the dcf 0 treatment (fig. 4, right 2 panels). the effect of the flx injection treatments on the food intake was not significant in the dcf 10 group (f3,24=3.978, p=0.053) but was significant in both the dcf 0 group (f3,24=29.937, p=0.000) and the dcf 1 group (f3,24=6.100, p=0.018) (table 1). in the dcf 0 group, the food intake was significantly lower in the flx 1 and 10 treatments than those in the flx 0 and flx c treatments (fig. 5, left panel). in the dcf 1 group, the food intake was the lowest in the flx 0 and flx 10 treatments, followed by the flx 1 treatment, and the largest in the flx c treatment (fig. 5, middle panel). the results of two-way anova in experiment-ii showed that the weight gain was significantly affected only by the flx injection treatment (f3,24=52.886, p=0.000) but not by the dcf exposure treatment (f2,24=0.064, p=0.938) and the interaction (f6,24=0.265, p=0.948). the weight gain was significantly the lowest in the flx 10 treatment, followed by the flx 1 treatment, and the largest in the flx 0 and control treatments (fig. 6). discussion there are many parameters regulating food intake in fish, including metabolic, neuro-physiological, and hormonal mechanisms. also, environmental conditions indirectly affect the appetite. in mammalian species, lam and heisler (2007) showed that the serotonergic pathway is involved in appetite regulation and energy homeostasis. some evidence in humans suggests an inhibitory role of flx on appetite and weight gain (halford et al., 2007). in our study, the amount of food intake significantly decreased after flx injection treatments of 1 µg/g bw and 10 µg/g bw. this indicates that the flx had a negative effect on appetite of the goldfish. after the second injection, food intake of all treatment groups increased in comparison to the first injection. this increase may be related to manipulation stress in fish. the first food intake test performed immediately after the acclimation period where the fish were divided from 100-liter tanks to smaller 50liter ones; while in the second test, fish were adapted a a aa b ab ab a b b a 0 2 4 6 8 10 0 mg/l 1 mg/l 10 mg/l f o o d i n ta k e ( g ) dcf exposure treatment control 0 μg/g 1 μg/g 10 μg/g flx treatment figure 5. means ± standard deviations of the food intake (g per fish per day) of goldfish (carassius auratus) in the 3 dcf exposure treatments crossed with the 4 flx injection treatments. n = 3; letters that differ indicate significance differences at p<0.05. a a b c 0 1 2 3 4 5 6 7 control 0 μg/g 1 μg/g 10 μg/g w e ig h t g a in ( g ) flx injection treatment figure 6. means ± standard deviations of the weight gain (g per fish per day) of goldfish (carassius auratus) in the 4 flx injection treatments. n = 9 (3 replicate tanks with 3 dcf exposure treatments); letters that differ indicate significance differences at p<0.05. 177 forsatkar et al./ goldfish nutrition affected by an ssri and nsaids contrast to the new situation. the results show that flx affects weight gain of goldfish, although it was not significant in our study. the effect of flx on human nutrition is contradictory. some cases reported a repressive action (pijl et al., 1991); for this, higher doses of flx are needed i.e., several times greater than the dose used for treatment of depression. fogelson (1991) showed that flx has no effect or even additive action on weight gain. in this study, after five injections of flx, the lowest weight gain was observed in flx 10 group. this is because food intake in this group was less than other treatment groups. with lower food consumption, fish remained under starvation condition and did not have enough energy to devote to growth and weight gain. also, the potential impacts of flx on appetite parameters such as increased secretion of crf (de pedro et al., 1998) and reducing npy (lopez-patino et al., 1999), causes negative weight gain after administration of flx. similar results are observed in rats in which flx administration after 44 days prevented weight gain (cantor et al., 1999). mennigen et al. (2010) reported that flx exposure caused 7-fold reduction in goldfish weight after a 28 day experiment. study on fat mice showed the main effect of flx on lipid tissues and protein content has slight reduction (gutierrez et al., 2002). therefore, only 9 days duration in our study might be too short to bring out the entire effect of the flx treatments, which come out insignificantly in the results. in addition, the small sample size (n=3) and high variance among the 3 replicates (dramatic standard deviation) could be one of the other reasons why the effect of the flx was masked in our study. after the dcf exposure, the food intake was significantly larger in the dcf exposure treatments of 1 mg/l and 10 mg/l than that in the 0 mg/l dcf exposure treatment in both the flx dosage groups of 1 μg/g bw and 10 μg/g bw. these results show the inhibitory role of dcf on the flx side effect of appetite reduction. de la garza and asnis (2003) reported that dcf sodium reduces the serotonin turnover in the prefrontal cortex of rat brains. concentration of serotonin in goldfish tissue was not measured in our study, but it is known the serotonergic pathways are affected by flx (mennigen et al., 2010) and/or nsaids (warnerschmidt et al., 2011). there is a central anorectic action of serotonin in teleost fish (de pedro et al., 1998), as a result, flx can reduce food intake by increasing serotonin levels and promoting the amount of food intake is related to the antagonistic relationship of dcf exposure on flx. after 5 days dcf exposure, insignificant results of the dcf effects on the weight gain were found in this study, although the food intake recovered due to the dcf exposure effects in the flx dosage groups of 1 μg/g bw and 10 μg/g bw. cleuvers (2003) indicated that models of toxicity mixture between flx and dcf is synergistic because the combined effects of these two compounds are more than the sum of the effects of each alone. therefore, only 5 days dcf exposure duration in our study might has been too short for the entire effect of the dcf treatments to emerge. this could be one of the main reasons why the effect of the dcf was insignificantly in the results. in conclusion, flx injection can significantly affect food intake and weight gain in goldfish. in the relationship between flx, a potential augmentor of extracellular serotonin levels, and dcf, a cyclooxygenase (cox) enzyme inhibitor, it was found that dcf exposure regulated flx impacts on appetite and growth conditions. molecular and physiological studies are needed to further understand the effects of multiple compounds interaction on aquatic organisms. references brooks b.w., chambliss c.k., stanley j.k., ramirez a., banks k.e., johnson r.d. and lewis r.j. 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(2013) 1(3): 119-124 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article antibacterial effects of medicinal plant extracts against lactococcus garvieae, the etiological agent of rainbow trout lactococcosis mohammad saeid fereidouni*1, mostafa akhlaghi1, aliasghar khadem alhosseini1 1aquatic animal health unit, school of veterinary medicine, shiraz university, shiraz, iran, po box 71345-173 article history: received 4 may 2013 accepted 21 may 2013 available online 2 0 june 2013 keywords: antibacterial effect medicinal plants lactococcus garvieae rainbow trout abstract: eight medicinal plants were assessed for antimicrobial activity against lactococcus garvieae isolate obtained from diseased oncorhynchus mykiss collected from rainbow trout fish farms in iran. lactococcus garvieae is among the major pathogens of a large number of fish species cultured in fresh and marine recirculating and net pen production systems. the antibacterial activity of the medicinal plants against l. garvieae was evaluated using disc diffusion, well diffusion and minimum inhibitory concentration. results showed that the extracts and essential oils had a relatively high antibacterial activity against l. garvieae. of the plants studied, the most active extracts were those from the methanol extract of peganum harmala, the essential oil of satureja bachtiarica, the ethanol extract of juglans regia and trachyspermum copticum with minimum inhibitory concentration (mic) of 105, 126, 510 and 453 μg/ml, respectively. conversely, some of the extracts such as quercus branti lindley and glycyrrhiza glabra l. had lower activity against l. garvieae with mic values of 978 and 920 μg/ml, respectively. plant extracts as natural and environment-friendly compounds can be an important source of antibacterial agents against l. garvieae. they may be used for disinfection of instruments and rainbow trout raceways or treatment of the fish. introduction lactococcus garvieae is a fish pathogen causing lactococcosis that has become a major problem in fish culture. lactococcosis is a serious septicemic disease in freshwater culture of salmonid fish and marine culture species, especially when water temperature is over 15°c (austin and austin, 2007). lactococcosis is a kind of streptococcosis caused by l. garvieae. lactococcosis has spread to many countries causing significant economic loss to the rainbow trout industry as in turkey (diler et al., 2002), portugal (pereira et al., 2004), france and the balkans (eyngor et al., 2004). the iranian l. garvieae isolate was diagnosed using a specific pcr assay based on 16s rdna gene by producing a single band of 1107 bp. partial analysis of 16s rdna * corresponding author: mohammad saeid fereidouni e-mail address: saeid.fereidouni@gmail.com of the bacterium was in close genetic relationship with those previously reported for mullet in taiwan (af352166) and yellowtail in japan (ab267897) based on genbank data antibiogram tests on l. garvieae isolates showed a high susceptibility to erythromycin, enrofloxacin and chloramphenicol (sharifiyazdi et al., 2010). many bacterial diseases in aquaculture, are controlled by antibiotics. however, continuous use of antibiotics leads to drug resistance and thereby to a reduced efficacy of the drugs. antibiotics accumulate in the environment and fish, pose a potential risk to consumers and to the environment. antibiotics (such as oxytetracycline, erythromycin, tetracycline and etc.) and other chemical disinfectants are widely used to prevent bacterial 120 fereidouni et al./ int. j. aquat. biol. (2013) 1(3): 119-124 disease in fish. the rapidly expanding aquaculture industry in iran has suffered from heavy economic losses due to bacterial pathogens, particularly streptococcus iniae and l. garvieae, which are the major agents of streptococcosis in rainbow trout (akhlaghi and keshavarzi, 2002; akhlaghi and mahjoor, 2004). increased public awareness of the negative effects caused by overexposure to synthetic chemicals has led to the search for ‘‘green solutions’’, such as organic and synthetic chemical-free food products (abutbul et al., 2004). for organic fish production it is essential to develop antibacterial treatments that are made from materials with natural sources. medicinal herbs contain physiologically active gradients that over the years have been exploited in traditional medicine for the treatment of various ailments because of having anti-microbial properties (kelmanson et al., 2000; srinivasan et al., 2001; ghasemi pirbalouti et al., 2011; negi et al., 2011). in spite of tremendous efforts to provide an alternative to medicinal plants with minimum side effects, easy accessibility and excellent compatibility, future clinical trials and standardization of medicinal plants are still required as an important steps in drug discovery (sarwar et al., 2011). this paper describes the in vitro use of eight iranian medicinal plants as antibacterial against lactococcosis caused by l. garvieae in rainbow trout. materials and methods plant materials: eight medicinal plants were collected from herbal medicine shop and their identity was confirmed using monographs by mozaffarian (1996; table 1). extract preparation: dried plant material was pulverised (200 g) and subjected to hydro-distillation (2000 ml distillated water) for 4 h using a clevengertype apparatus. the leaves and seeds of some of the plants were shade dried and ground into a powder (100 g), macerated in 200 ml of methanol or ethanol, filtered and dried at 35 °c using a rotary vacuum. the extract of samples were stored in universal bottles and refrigerated at 4 °c prior to further analyses. bacterial strain: lactococcus garvieae was isolated from the infected rainbow trout (oncorhynchus mykiss) from a commercial aquaculture farm in fars province, iran. the isolate was identified as l. garvieae using conventional morphological as well as biochemical tests. it was then confirmed by molecular methods (sharifiyazdi et al., 2010). the bacteria were kept frozen in 15% glycerol, 85% saline solution or brain heart infusion (bhi) broth, in aliquots, at -70 °c until used. for infection trials, 100 ml of bhi broth was inoculated with 50 μl of the frozen isolate. the cultures were shaken (100 rpm) at 27 °c for 24 h. absorbance (at 600 nm) of known bacterial densities was determined to obtain a standard calibration curve. an initial bacterial suspension containing 107 cfu/ml was made from the flask broth culture. subsequent dilutions were made from the above suspension, which were then used in tests. disc diffusion assay: the disc diffusion methods of lennette (1985) were used with some modification parts used habit local name family name scientific name 1 seed herb esfand nitrariaceae peganum harmala 2 root herb shirin bayan fabaceae glycyrrhiza glabra l. 3 seed herb zenian umbelliferae trachyspermum copticum 4 leaves tree mort myrtaceae myrtus communis l. 5 leaves tree barge gerdo juglandaceae guglans regia 6 seed tree balout fagaceae quercus branti lindley 7 leaves herb baboneh gavi asteraceae tanacetum parthenium l. 8 leaves herb marzeh koohi lamiaceae satureja bachtiarica bung table 1. characteristics of eight medicinal plants used in this study. 121 fereidouni et al./ int. j. aquat. biol. (2013) 1(3): 119-124 to determine the growth inhibition of plant extracts and essential oils on the bacterium. bhi agar was used to prepare the culture medium and autoclaved at 121 °c for 15 min. briefly, plates (8-cm diameter) were prepared with 10 ml agar inoculated with 1 ml of bacterial suspension. sterile paper discs (6 mm in diameter) were impregnated with 60 μl of dilutions of known extract concentrations (100 μg/disc) and incubated at 35°c for 18 h. the extracts were dissolved in dimethyl sulfoxide (dmso, 15 μl) before being tested for antimicrobial activity. discs (6 mm diameter) of ampicillin, amkcacin, penicillin, cephalexin, cefazolin and cefixime (10 μg) were used as positive controls. bacterial growth inhibition was determined using the diameter of the inhibition zones around the discs (mm). the growth inhibition diameter was calculated as an average of three measurements, from three different directions. all tests had three replicates. well diffusion assays: four equidistant holes were made in the agar using sterile cork borers (q=6 mm). 20 μl of each extract and essential oil was added to the holes using a micropipette (sagdic et al., 2007). minimal inhibitory concentration: the minimal inhibitory concentration (mic) value was determined by serial dilution assay. the mic was defined as the lowest concentration of the compound to inhibit the growth of the microorganism to 50%. all extracts were initially tested at 10000 μg/ml and serially diluted to 10 μg/ml. each tube was inoculated with 5 ml of bacterial suspension at a density of 107 cfu/ml and incubated at 37 ° c for 48 h. the growth of microorganisms was observed as turbidity determined by measuring the optical density at 600 nm with a spectrophotometer. erythromycin was included as a positive control in each assay. extract-free solution was used as a negative control. control tubes were incubated under the same condition. all assays were carried out in triplicate. the inhibition demonstrated by the extracts is expressed by the following equation (zampini et al., 2005): inhibition % = [(od c –od t) / od c] ×100 where odc is the od600 for the negative control (containing no extract) and odt is the od600 for the sample treated with the antimicrobial compounds. results the growth inhibition value of the extracts and essential oils on the bacterium strain is shown in table 2. the extracts from the different plant species studied showed antibacterial activities, with the diameters of the inhibition zone. there were significant differences in the antibacterial activities of plant extracts (p≤0.01). among the plants tested, the essential oil of satureja bachtiarica leaves, the plant species extraction disc diffusion (mm)* well diffusion (mm)* mics values (μg/ml) disc content (µg) 1 peganum harmala methanol extract 28±2.30 39±3.43 105 2 glycyrrhiza glabra l. ethanol extract 14±2.15 13±1.20 920 3 trachyspermum copticum ethanol extract 18±3.13 21±3.14 453 4 myrtus communis l. essential oil 16±2.53 20±1.23 672 5 juglans regia ethanol extract 19±4.12 22±3.15 510 6 quercus branti lindley ethanol extract 12±3.80 15±1.93 978 7 tanacetum parthenium l. essential oil 15±1.96 17±3.15 824 8 satureja bachtiarica bung essential oil 25±4.70 29±5.12 126 9 erythromycin 32±1.54 35±2.61 15 10 ampicillin 22±3.87 24±3.49 25 11 flofenicol 29±2.82 33±2.67 30 12 enrofolxacin 27±1.42 30±2.11 5 13 trimethoprim 28±2.30 28±2.30 20 table 2. inhibition zones and mic values produced by antibacterial activity of the medicinal plants against l. garvieae. 121 122 fereidouni et al./ int. j. aquat. biol. (2013) 1(3): 119-124 methanol extract of peganum harmala seeds and the ethanol extract of juglans regia leaves and trachyspermum copticum seeds showed the best antibacterial activity that could effectively inhibit the growth of l. garvieae (table 2). subsequent experiments were conducted to determine the minimal inhibitory concentration (mic) of all the selected plant extracts and essential oils. the essential oil of satureja bachtiarica and the methanol extract of peganum harmala showed the best antibacterial activities against l. garvieae in rainbow trout (table 2). the mic values for the active extracts and essential oils ranged 105-978 μg/ml. the highest level of antibacterial activity against l. garvieae was demonstrated by the methanol extract prepared from the seeds of peganum harmala and ethanol extract of trachyspermum copticum seeds when compared with the results of five antibiotic discs. these preparations showed mic values from 105 to 453 μg/ml. other extracts used in this study only showed a slight inhibition of the tested microorganism. discussion plant extracts as natural and environmentally friendly compounds could be an important source of antibacterial agents against l. garvieae. lactococcosis could be controlled by a health management protocol using disinfectants such as natural antibacterial compound besides employing vaccination of fish against the etiological agent. such antibacterial with natural sources are not expensive and could be prepared and ordered by registered agencies around the world. there has been no large scale systematic investigation into the relationship between bacterial inhibition and total phenolic content of spices and herbs. previous studies (shan et al., 2005; pritam and purushottam, 2007) showed that a highly positive linear relationship exists between the antioxidant activity, cytotoxic activity and total phenolic content in some spices and herbs. some studies claim that the phenolic compounds present in spices and herbs might also play a major role in their antimicrobial effects (hara-kudo et al., 2004). the essential oil and extract of some aromatic plants with a higher percentage of carvacrol and thymol (e.g. the mint family, lamiaceae), have a higher efficacy against the bacterial strains. also, the antimicrobial activity of the essential oils of some thymus spp. that possess large quantities of phenolic monoterpenes, have shown activity against viruses, bacteria, food-derived microbial strains and fungi (rasooli et al., 2006). previous works showed that the essential oil of satureja bachtiarica exhibited antifungal activities against saprolegnia parasitica from cutaneous lesions of oncorhynchus mykiss eggs (ghasemi pirbalouti et al., 2009). the essential oils of satureja bachtiarica and thymus daenensis exhibited antibacterial activities against staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and klebsiella pneumonia (ghasemi pirbalouti et al., 2010b). essential oils of myrtus communis, thymus daenensis and satureja bachtiarica exhibited antimicrobial activities against escherichia coli o157:h7, bacillus cereus, listeria monocytogenes and candida albicans (ghasemi pirbalouti et al., 2010a) and streptococcus iniae (ghasemi pirbalouti et al., 2011). results of study by sonboli et al., 2004 indicated the essential oil of satureja laxiflora c. koch contained a high concentration of oxygenated monoterpenes (76.3%) of which thymol (63.9%) was the major compound followed by carvacrol (4.8%) and geraniol (3.2%) and it exhibited antimicrobial activities against candida albicans, aspergillus niger, saccharomyces cerevisiae, klebsiella pneumonia and enterococcus faecalis. they suggested that a major portion of this antimicrobial activity is due to the thymol present in the oil. the compounds from the essential oil of satureja bachtiarica included 20% carvacrol and 19% thymol before flowering and 26% carvacrol and 5% thymol at full flowering stage, as the main components (sefidkon and jamzad, 2000; sefidkon et al., 2007). sefidkon et al. (2007) reported that the anti-bacterial effect (five bacteria including: bacillus subtilis, bacillus cereus, micrococcus luteus, staphylococcus 123 fereidouni et al./ int. j. aquat. biol. (2013) 1(3): 119-124 sp. and staphylococcus aureus; three gram negative bacteria including: klebsiella pneumonia, klebsiella oxytoca and pseudomonas aeruginosa) of satureja bachtiarica oil was stronger before the flowering stage, because of a higher percentage of phenolic compounds (thymol and carvacrol). the essential oil of tanacetum parthenium l. containing camphor showed minimum antibacterial effect on s. aureus and p. aeuroginosa (saharkhiz et al., 2008). similarly this essential oil had a low antibacterial effect on l. gavieae in this study. the influence of three medicinal plants, ocimum bascilicum, adathoda vasica and calendula officinalis on the biochemical parameters of normal and aeromonas hydrophila-infected fish (labeo rohita) was assessed using feeds supplemented with 3% concentrations of the three plants extracts. at the culmination of feeding experiment, biochemical analysis of serum for enzymes was undertaken and the highest level of serum glutamate oxaloacetate transferase, serum glutamate phosphor transferase, alkaline phosphatase, were recorded 60.0, 54.1, 26.3 mg dl-1 respectively in ocimum basilicum supplemented group. serum protein content was also higher in calendula officinalis supplemented group (6.0 mg dl-1). this study revealed that feed supplementation of three medicinal plant extracts alters biochemical parameters to overcome disease induced stress in fishes (john et al., 2011). in three methods used for extraction of the eight medicinal plants in this study, the highest level of antibacterial activity was demonstrated by the essential oil of the leaves of satureja bachtiarica, the methanol extract of peganum harmala, the ethanol extracts of juglans regia and trachyspermum copticum. thus they are potential source of natural antibacterial against l. garvieae isolated from rainbow trout. they might be used for disinfection of instruments and rainbow trout raceways. further work should be performed to describe their in vivo antibacterial activities in more detail in fish. references abutbul a., golan-goldhirsh a., barazani o., zilberg d. 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(2020) 8(2): 109-125 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article distribution and bioaccumulation of heavy metal in water, sediment and fish tissue from the river mahananda in seemanchal zone, north bihar, india arbind kumar1, anil kumar2, suman kumar jha3 1p.g. department of chemistry, darshan sah college, katihar, purnea university, purnia, bihar, india. 2department of zoology, l.s.t. gramin mahavidylaya aungaridham, nalanda, patliputra university, patna, bihar india. 3p.g. department of zoology, darshan sah college, katihar, purnea university, purina, bihar, india. s article history: received 12 september 2019 accepted 16 march 2020 available online 2 5 april 2020 keywords: bioaccumulation heavy metals water sediment fish tissue abstract: in the present study, distribution, and bioaccumulation of cu, zn, cd, and pb were analysed in water, sediment and freshwater fish tissues of catla catla and mystus seenghala which were seasonally collected from river mahananda in seemanchal zone. the results showed that except pb, level of cu, zn and cd in water were below than allowance limit of who (2008), while level of cu, zn and cd in sediment was higher than a toxicity reference value (trv) recommended by usepa (1999), except pb. heavy metal concentration in water and sediment were recorded in the order: cu>pb>zn>cd and zn>cu>pb>cd, respectively. the seasonal variation of this metal in water was in the sequence: rainy>winter>summer, and in sediment summer>winter>rainy. bioaccumulation of studied metal by different tissues of m. seenghala was maximum than c. catla, and found that following order of magnitude: liver>gill>muscle. the order of studied metal concentration in liver zn>pb>cu>cd, in gills zn>pb>cu>cd, in muscle zn>pb>cu. pb was only metal whose concentration was higher than fao, fao/who and who standards in all examined tissues of both species. bioaccumulation of pb in different tissues of both fishes was observed maximum in summer followed by winter and rainy (monsoon) seasons. bioaccumulation factor (baf) of all four metals in organs of m. seenghala was higher than c. catla. metal index value (mi) > 1 for cd and pb in water suggests that worse quality of water. the levels of heavy metals accumulated in two fishes might be due to the increase in agricultural influx water, domestic wastes, poultry farm, municipal and some other anthropogenic activities. this study shows that river water in the studied zone is a serious concern of human health and selected fishes do not feed directly without proper treatment of the riverine ecosystem, and potential danger may occur in the future. introduction heavy metal pollution in the aquatic system has attracted global attention due to their environment toxicity, persistence, bioaccumulation and biomagnifications in the food chain, which can pose adverse effect on living beings and the entire ecosystem (kumar et al., 2015; kumar and seema, 2016; kumar et al., 2017; xu et al., 2018; ali et al., 2019). anthropogenic activities continuously increase the amount of heavy metal in the aquatic ecosystem (xu et al., 2018; kumar et.al, 2019). as heavy metals cannot be degraded, they are deposited, assimilated or incorporated in water, sediment and aquatic organisms and thus, causing heavy metal pollution in an aquatic ecosystem (kumar and kumar, 2018; farsani, et al., 2019). in the light of the extreme human activity, *correspondence: arbind kumar doi: https://doi.org/10.22034/ijab.v8i2.676 e-mail: drarbindktr@gmail.com natural sources of heavy metals are usually of a little importance (xu et al., 2016a; patel et al., 2018; yan et al., 2018). anthropogenic activities such as industrial effluent, sewage sludge, domestic wastes (guatam et al., 2013; mozumdar et al., 2015; banaee et al., 2015; arbind and seema, 2016; kumar et al., 2017; farsani et al., 2019; banaee and tahari, 2019), atmospheric pollutants (arbind and seema, 2017) and agricultural runoff (zhang et al., 2014; singh et al., 2016, 2017) entering the water bodies are one of the prime sources of heavy metal toxicity, which deteriorates water quality and danger to human health and aquatic organisms (xu et al., 2016a, b). in water bodies, metals can be in dissolved and particulate form (farsani, et al., 2019), and their levels in water depend on some physiochemical parameter such as redox 110 kumar et al./ distribution and bioaccumulation of heavy metal in water, sediment and fish tissue potential, ph, temperature, dissolve oxygen, salinity, conductivity and ionic strength (hassan et al., 2015; sim et al., 2016). sediments often act as carries potential sources for metals in the aquatic environment (hassan et al., 2015; sim et al., 2016). about 99% of the heavy metals entering into river from different sources have been found to ultimately precipitate onto the sediment (sim et al., 2016; singh et al., 2017; xu et al., 2018). their quality can indicate the states of water pollution (zahra et al., 2014). the distribution of heavy metals in the sediments is affected by the chemical composition of the sediments, grain size and content of total carbon matter (zhao et al., 2014; azidi et al., 2018). the release of heavy metals from sediment to water bodies is affected by the overlying water conditions, ph, alkalinity, salinity, dissolved oxygen concentration and suspended solid (simpon et al., 2004; li et al., 2013) and found that physical disturbance of sediments released metals more rapidly than biological disturbance (atkinson et al., 2007). the ph is an important factor of metal bioavailability in sediment (li et al., 2013). decreasing the ph, increases the competition between metal ion and h+ for binding sites such as oh, co3 2-, so4 2-, cl-, s2 and po4 3in sediments, therefore absorption abilities and bioavailability of the metals in the sediments subsequently decrease and then increase the mobility of metal ions and may result in dissolution of metal complexes, thereby releasing free metal ions into the water system (nowrouzi et al., 2014; rajeskumar and li, 2018). contaminated sediments do not always remain at the bottom of the water body, anything that stirs up the water, such as dredging, can resuspend sediments. resuspension may mean that all of the animals in the water, and not just the bottom-dwelling organism, will be directly exposed to the toxic contaminants (begum et al., 2009). thus, sediments can be a sensitive indicator to detect the quality of the aquatic system, and it may act not only as a sink but also as sources of contamination in aquatic bodies (li et al., 2013; rajeskumar et al., 2018; ali et al., 2019). in recent years, the consumption of fish has increased rapidly with an awareness of its nutritional and therapeutic benefits (sioen et al., 2007; bawuro et al., 2018). fish contains high level of unsaturated acids and low levels of cholesterol and also has a high level of many essential nutrients (el-mosclhy 2000; kris-etherton et al., 2002; afshan et al., 2014; bawuro et al., 2018; rajeskumar and li, 2018). various researches have shown the adverse effect of heavy metals on human health, such as renal failure, cardiovascular diseases, liver damage and even death (castro-gonzalez and armenta, 2008; al-bussaidi et al., 2011; rahman et al., 2012). bioaccumulation of heavy metal in freshwater fish depends upon the various factor such as fish characteristics and external environmental factors. the retention of heavy metal in the body of an organism depends on many factors such as the speciation of the metal concerned and the physical mechanism developed by the organism for the regulation, homeostasis, and detoxification of the heavy metal. the degree of bioaccumulation in different tissues of fish is generally different depending on the active tissue like liver, gills, and kidney have a higher accumulation of the heavy metal than other tissues such as skin and muscles (maurya and mallik, 2019; ezekiel et al., 2019). thus the presence of heavy metals in water and sediment would be primary source for the distribution and bioaccumulation of metals in the fish and cause adverse effect to those who consume the contaminated fish (yu et al., 2012; bao et al., 2015; bawuro et al., 2018; maurya and mallik, 2019; ezekiel et al., 2019). therefore, this research aimed to evaluate the distribution, bioaccumulation and seasonal variation of cu, zn, cd and pb in water, sediment and freshwater fishes from river mahananda in three districts of seemanchal zone and monitoring the health risk of heavy metal in fish and people within the environment. materials and methods study area: the river mahananda originates 6 kms north of kurseong in the darjeeling district of west bengal in the himalayan range, at the elevation of 2062 m. it has a powerful role in regulating overall economy of the catchment area, such as darjeeling, 111 int. j. aquat. biol. (2020) 8(2): 109-125 utter dinajpur and maldah districts of west bengal and kishanganj, purnea and katihar districts of bihar. the river mahananda starts its 360 kms long journey to the ganga out of which 324 kms are in india and 36 kms are in bangladesh. the total drainage area of this river is 24,753 sq kms, of which 5,293 sq kms are located in nepal, 6,677 sq kms in west bengal, 7,975 sq kms in bihar and rest is located in bangladesh, where it finally joins the ganga (padma) near godagarighat in nawabganj district. the river mechi and kankai flow through nepal and form the boundary between india and nepal and then flow through the indian state of bihar to join the mahananda in kishanganj district (fig. 1). collection of water, sediment and fish samples: a total of 66 samples (water and sediment) were collected from mahananda river during year 20172018 in summer, rainy (monsoon) and winter season, including 22 samples from each season and at 8 sites of each kishanganj and katihar and 6 sites of purnia district of seemanchal zone, bihar (table 1). about 500 ml water samples were collected at 0.5 meter below the water surface, filtered in pre cleaned bottle and preserved by adding 5 ml of 65% concentrated hno3 to it and then packed in ice bath (4 °c) and brought to the laboratory for further digestion (apha, 2005). about 500 g sediments samples were collected at a depth of 0-10 cm using a portable ekman grab sampler by applying the method of us epa (2001) and immediately transferred into polyethylene bags, which were already washed with 10% hno3 solution and successively rinsed with distilled water. at each site, three samples were collected and were subsequently well-mixed to get a composite mixture (kumar et al., 2019). five specimens of each species (c. catla and m. seenghala) were also collected seasonally at each sampling sites, using a multifilament, nylon gillnet with help of local fisherman. these fish species represent different biotopes (table 2) that were immediately preserved on ice in an ice chest and then transferred to the laboratory where they were weighed and their total length recorded, and kept frozen at -20°c. after identifying, all fishes were dissected into separate organs such as liver, gills and muscles using stainless steel instrument by applying the method of voegborlo et al. (2012) and al-busaidi et al. (2011) and put in to petri plate to dry at 120°c until reaching a constant weight. digestion of water, sediment, and fish samples: 100 ml water sample was taken in a conical flask, and 5 ml of concentrated hcl acid was added to it and then heated on the hot plate for two hours at 105°c to 25 ml. the concentrated water sample was then transferred into 100 ml volumetric flask, and distilled water was added to fill up to the mark and then analysed for cu, zn, cd and pb using atomic absorption spectrophotometer. each sediment sample was air dried and homogenized by grinding, using an agate mortar and pestle to pass through 63 µ mesh nylon sieve at room temperature. to estimate the heavy metal content, 2 gram of sediment sample figure 1. map showing sampling location of river mahananda in seemanchal zone. 112 kumar et al./ distribution and bioaccumulation of heavy metal in water, sediment and fish tissue collected from each site was digested separately with 25 ml of tri-acid mixture of hno3, h2so4 and hclo4 in the proportion of 5:1:1 respectively in teflon measuring beaker at about 80°c for 4-5 h based on allen et al. (1986) modified by singh et al. (2017). after this digested, solution was filtered using whatman no. 42 filter paper in pre-cleaned 100 ml measuring flask and volumes were made up to mark and then subjected to atomic absorption spectrophotometer for analysis of cu, zn, cd and pb. the dried fish tissues were digested by the method described by voegborlo et al. (2012), modified by bawuro et al. (2018). in this method one gram of each sample was digested separately with hclo4 and hno3 in the ratio 1:1 followed by sulphuric acid, and the mixture was heated at 200°c for 30 min. after complete digestion, each digested mixture was cooled at room temperature and then transferred in to 50 ml volumetric flask. distilled water was added to it to fill up to the mark and analysed for cu, zn, cd, and pb using atomic absorption spectrophotometer. metal index (mi): for assessing the water quality, table 1. sampling sites, geographic coordinates and description of sampling sites. geographic coordinates description sampling site latitude longitude kishanganj district upstream thakurganj (kj-1) 26.42720 n 88.12540 e few factories too have been established in this region in the recent decades. the water body receives a lot of water of wastes ranging from industrial, agricultural and domestic sources, which apart from adversely affecting the normal hydrochemistry of the river, also decreases its channel capacity at various points, and this has been largely responsible for flood disasters in the river. the main rivers flowing through this district are mahananda, kankai, mechi, donk, rauta, sudhani and ramzan. arrabari (kj-2) 26.27030 n 88.03630 e halamla (kj-3) 26.37070 n 88.23470 e palkoaikund (kj-4) 24.21130 n 87.83530 e balubari (kj-5) 24.14900 n 87.91260 e gobondpur (kj-6) 26.11770 n 87.89670 e chakandra (kj-7) 26.13350 n 87.88030 e kishanganj town (kj-8) 26.079440 n 87.937220 e purnea district midstream talbari (pr-1) 26.00930 n 87.75420 e the district is one of the fastest growing districts in the state and this can be seen in the increasing infrastructural facilities in the city. the rivers also receives effluents from many small industries, textile battery producing unit and also agricultural runoff from its catchment area. kankai river is major tributary of mahananda river, when enters in purnea district. there are four important rivers which traverse the city and also divide the district into four distinct zones. the rivers which traverse the city are kosi in the west, panar in the northeast, mahananda in the east and ganga in the south. surjapur (pr-2) 25.96800 n 87.76400 e amor (pr-3) 25.956940 n 87.725000 e khari (pr-4 25.93850 n 87.76400 e chanargaon (pr-5) 25.1335 n 87.8193 e bhasia (pr-6) 25.86730 n 87.74430 e katihar district downstream taiyabpur (kr-1) 25.71170 n 87.82060 e the main rivers of the district are ganga, kosi and mahananda. this district shares boundary with two states i.e. jharkhand at the southern side and west bengal at the eastern side. the bangladesh lies around 80 km east of katihar town and nepal lies around 100 km north of katihar town. the river receives allochtonous input of organic matter from the surrounding vegetation derived through runoff from the surface of soil. solid wastes are produced daily from domestic uses in katihar city and about 65% of them are dumped in the river. the right portion of river mahananda at bagdob known as jhawa branch, when enters in katihar district of seemanchal zone, majhok (kr-2 25.69700 n 87.83800 e jhawa r.s.(kr-4) 25.61340 n 87.77550 e meena r.s. (5) 250 35'00" n 870 51'00" e mukuria (kr-5) 25.62690 n 87.88690 e lava (kr-6) 26.1037 n 87.8457 e singha (kr-7) 26.1655 n 87.5634 e gobindpur (kr-8) 26.49760 n 87.65190 e table 2. fish species ecological characteristic. species common name habitant feeding behavior no. of samples length (cm) weight (gr) catla catla catla surface and mid-water feeders mainly omnivorous 5 14-25 225-450 mystus seenghala dariai tengara middle bottom feeder carnivorous 5 20-30 205-420 113 int. j. aquat. biol. (2020) 8(2): 109-125 metal index can be applied. it was calculated by the following equation, defined by tamasi and cini (2004) and described by yehia and sebaee (2012): 𝑀𝐼 = ∑ 𝐶𝑖 (𝑀𝐴𝐶)𝑖 where 𝐶𝑖 is the metal concentration in water sample and 𝑀𝐴𝐶 is the maximum allowed concentration for each element in drinking water and subscript i is the ith sample. bioaccumulation factor (baf): baf is inductive of the degree of accumulation of heavy metal in an organism relative to that in its habitant (water and sediment). it was calculated by the following equation described by kalvins et al. (1998) and modified by ali et al. (2019): 𝐵𝐴𝐹 = 𝐶𝑓𝑖𝑠ℎ 𝑡𝑖𝑠𝑠𝑢𝑒 𝐶 𝑠𝑒𝑑𝑖𝑚𝑒𝑛𝑡 where c fish tissue is the metal concentration in fish tissue and c sediment is the metal concentration in the sediment. statistical analysis: all statistical analysis was performed on using the microsoft excel 2007. similarly, the significance of differences between the concentrations of heavy metals in water, sediments and selected tissues were calculated using casio calculator (made in china) fx-991 ms. a probability of p<0.05 was considered statistically significant. results the seasonally variation of averages mean concentration with standard deviation of cu, zn, cd, and pb in water, sediment and selected organs viz. liver, gills and muscles of two c. catla and m. seenghala of river mahananda in seemanchal zone are presented in tables 3 and 4, and 5-8, respectively. water: the average maximum mean concentration of cu, zn and pb in water sample was found metal location summer (m±sd) rainy (m±sd) winter (m±sd) total (m±sd) metal index who (2008) cu (mg/l) kishanganj, n=8 0.36±0.02 0.41±0.01 0.40±0.011 0.39±0.026 0.195 1-3 mg/l purnia, n=6 0.38±0.01 0.49±0.14 0.45±0.05 0.44±0.056 0.22 katihar, n=8 0.43±0.13 0.47±0.02 0.47±0.03 0.456±0.023 0.225 zn (mg/l) kishanganj, n=8 0.12±0.01 0.17±0.05 0.15±0.11 0.147±0.025 0.049 10-15 mg/l purnia, dt., n=6 0.15±0.15 0.21±0.01 0.17±0.01 0.176±0.030 0.051 katihar, n=8 0.18±0.02 0.25±0.02 0.22±0.06 0.217±0.035 0.072 cd (mg/l) kishanganj, n=8 0.058±0.01 0.068±0.03 0.061±0.01 0.062±0.005 20.44 2 mgl purnia, n=6 0.068±0.02 0.078±0.04 0.072±0.04 0.073±0.005 23.33 katihar, n=8 0.065±0.02 0.075±0.01 0.065±0.02 0.068±0.006 22.78 pb (mg/l) kishanganj, n=8 0. 15±0.08 0.20±0.05 0.17±0.03 0.173±0.025 17.33 0.1-0.2 mg/l purnia, n=6 0.25±0.02 0.35±0.02 0.31±0.04 0.303±0.05 30.33 katihar, n=8 0.31±0.33 0.38±0.01 0.34±0.11 0.343±0.035 34.33 table 3. seasonal variation of heavy metal in water of river mahananda in seemanchal zone. table 4. seasonal variation of heavy metal in sediment of river mahananda in seemanchal zone. metal location summer (m±sd) rainy (m±sd) winter (m±sd) total (m±sd) trv (usepa) cu (mg/l) kishanganj, n=8 65.32±7.32 58.72±7.63 60.38±8.61 61.47±3.433 16 mg/kg purnia, n=6 65.73±9.24 58.92±9.28 60.09±21.9 61.58±6.641 katihar, n=8 71.5±7.81 68.12±8.24 69.32±9.41 69.65±1.713 zn (mg/l) kishanganj, n=8 108.5±8.47 102.41±9.51 104.8±10.2 105.24±3.068 110 mg/kg purnia, n=6 117.34±20.27 113.62±19.21 115.46±21.9 115.47±1.860 katihar, n=8 119.43±9.81 116.71±9.55 117.35±10.08 117.83±1.422 cd (mg/l) kishanganj, n=8 0.921±0.11 0.82±0.101 0.864±0.118 0.868±0.0501 0.6 mg/kg purnia, n=6 0.73±0.15 0.657±0.21 0.68±0.109 0.689±0.037 katihar, n=8 0.79±0.12 0.78±0.12 0.712±0.103 0.755±0.053 pb (mg/l) kishanganj, n =8 23.23±4.12 21.46±3.71 22.95±4.34 22.55±0.951 31 mg/kg purnia, n=6 24.47±4.43 22.67±2.81 23.95±3.62 23.67±0.926 katihar, n=8 25.81±3.72 23.76±3.21 24.41±3.66 24.66±1.048 114 kumar et al./ distribution and bioaccumulation of heavy metal in water, sediment and fish tissue 0.456±0.023, 0.217±0.035 and 0.343±0.035 mg/l, respectively at study point of katihar (downstream), whereas cd was 0.073±0.005 mg/l at location of purnea district (midstream). similarly, average lowest mean concentration of cu, zn, cd and pb were found 0.39±0.026, 0.147±0.025, 0.062±0.005 and 0.173± 0.025 mg/l, respectively at location of kishanganj, upstream of river (table 3). the order of heavy metal in the water of river was cu>pb>zn>cd. the results indicated that cu was maximally and cd was least accumulated in the water of the river. the results also indicated the greater values of heavy metals in water were found at downstream of river and seasonally higher concentration of these metals were detected in the rainy season followed by winter and summer season. the maximum mi of cu, zn and pb in water samples was found 0.225, 0.072 and 34.33, respectively at the study point of katihar and for cd 23.33 at purnea district of midstream of river mahananda (table 3). sediment: the average maximum mean concentration of cu, zn, and pb in sediment of river was 69.65± 1.713, 117.83±1.422 and 24.66±1.048 mg/kg, respectively in downstream of river at katihar, whereas cd was 0.868±0.0501 mg/ kg at location of kishanganj. similarly, the average minimum mean concentration of cu, zn and pb was 61.47±3.433, 105.24±3.068 and 22.55±0.951mg/kg, respectively at upstream of river at the location of kishanganj, whereas cd was 0.689±0.037 mg/kg at midstream of the river at purnea zone. the order of heavy metal accumulation in sediment of river was zn>cu>pb >cd. the data also indicated that zn was maximally accumulated and cd got the least concentration in the sediment of each location of mahananda river. the maximum concentrations of these metals in sediment were detected in summer, followed by winter and rainy season (table 4). heavy metal content and baf in fish tissues: copper: in the liver of c. catla, cu recorded its highest mean concentration 14.5±0.755 mg/kg and in m. seenghala was 19.93±1.09 mg/kg at the same location of downstream of river at katihar, whereas lowest mean concentration in c. catla was 2.51± 0.344 mg/kg at location of kishanganj, and in m. seennghala was 4.7±0.556 mg/kg at location of purnea (table 5). similarly, in gills, maximum mean concentration of cu in c. catla was recorded 4.35±0.5408 mg/kg and in m. seenghala was 3.3±0.356 mg/kg at locations of river at katihar and purnia areas, respectively. minimum concentration was 1.32±0.036 and 1.97±0.113 mg/kg, respectively, at the same study points of upstream of river at kishanganj (table 5). in muscles of c. catla and m. seenghala, the highest table 5. seasonal variation of cu concentration (mg/kg dry wt) in different organs of catla catla and mystus seenghala in river mahananda. location organ catla catla mystus seenghala summer rainy winter total summer rainy winter total (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) kishanganj liver 2.9±0.05 2.4±0.34 2.2 4± 0.26 2.51±0.344 4.8±0.21 5.1±0.11 4.7±0.06 4.83±0.152 baf 0.0444 0.0408 0.037 0.041±0.003 0.0734 0.0851 0.0778 0.0787±0.006 gills 1.33±0.02 1.35±0.012 1.28± 0.31 1.32±0.036 1.91±0.21 2.1±0.01 1.9±0.02 1.97±0.113 baf 0.0203 0.0229 0.0211 0.0214±0.001 0.0292 0.0357 0.0314 0.0321±0.003 muscles 0.92±0.23 0.85±0.15 0.82±0.31 0.853±0.051 1.21±0.06 1.09±0.11 1.06±0.21 1.12±0.079 baf 0.014 0.0144 0.0101 0.013±0.002 0.0185 0.0185 0.0175 0.018±0.0005 purnea liver 5.5±0.41 5.1±0.03 4.1±0.34 5.133±0.351 5.2±0.32 4.8±0.21 4.1±0.08 4.70±0.556 baf 0.0836 0.0868 0.0798 0.0834±0.0035 0.0791 0.0814 0.0682 0.076±0.007 gills 3.2±0.131 2.9±0.31 2.6±0.13 2.9±0.32 3.7 ±0.41 3.2±0.05 3.01±0.02 3.3±0.356 baf 0.0499 0.0492 0.0432 0.0474±0.0037 0.0456 0.0543 0.0499 0.05±0.004 muscles 1.46±0.05 1.24±0.12 1.2±0.31 1.3±0.14 1.76±0.23 1.54±0.13 1.52±0.34 1.607±0.133 baf 0.0222 0.021 0.0199 0.021±0.0031 0.0267 0.0261 0.0252 0.026±0.0007 katihar liver 15.3±0.34 14.4± 13.8±0.43 14.5±0.755 20.6±0.50 20.5±0.45 18.7±0.38 19.93±1.069 baf 0.2139 0.2113 0.199 0.208±0.008 0.2881 0.3009 0.2677 0.286±0.017 gills 4.5±0.01 2.8±0.01 3.75±0.03 4.35±0.5408 3.7±0.31 2.8±0.23 2.6±0.04 3.03±0.586 baf 0.0629 0.0704 0.054 0.0624±0.008 0.0517 0.0411 0.375 0.0434±0.007 muscles 2.65±0.34 2.25±0.02 2.04±0.41 2.313±0.309 1.78±0.07 1.52±0.21 1.48±0.25 1.593±0.162 baf 0.037 0.033 0.0294 0.033±0.004 0.0248 0.0223 0.0213 0.023±0.002 115 int. j. aquat. biol. (2020) 8(2): 109-125 mean concentration of cu was 2.313±0.309 and 1.607±0.133 mg/kg at the station of katihar and purnia, respectively, whereas lowest mean concentration was 0.853±0.051 and 1.12±0.079 mg/kg at same station of kishanganj (table 5). the maximum baf of cu in the liver of c. catla and m. seenghala was 0.208±0.008 and 0.286±0.017, respectively at the same study point of river at katihar, whereas minimum mean baf value was 0.041±0.003 and 0.076±0.007 at the location of kishanganj and purnia district, respectively. in gills of c. catla and m. seenghala the maximum mean baf value obtained was 0.0624±0.008 and 0.05±0.004 at the station of katihar and purnia, respectively, and minimum baf was 0.0214±0.001 and 0.0321±0.003 at the same study point of kishanganj. in muscle of c. catla and m. seenghala highest baf value of cu was 0.033±0.004 and 0.023±0.002, respectively at same location katihar and lowest value was 0.013±0.002 and 0.018±0.0005, respectively at same location kishanganj. zinc: the highest concentration of zn in liver of c. catla and m. seenghala was 63.9±1.708 and 71.93±3.028 mg/kg at location of katihar and purnea, respectively, whereas lowest mean concentration was recorded 27.53±1.20 and 35.07±0.3214 mg/kg, respectively at same station of river at kishanganj (table 6). also, at location of downstream of river at katihar the gills of c. catla and m. seenghala, recorded the maximum mean concentration of zn was 36.43±0.850 and 46.5±1.509 mg/kg, respectively and minimum mean concentration was 22.3±0.01 and 36.33±0.5507 mg/kg, respectively at upstream of river at kishanganj (table 6). in muscles of c. catla and m. seenghala the highest mean concentration of zn was recorded 17.93±1.115 and 23.86±1.656 at the same location of katihar, whereas minimum concentration was recorded 12.13±0.351 and 11.87±0.378 mg/kg at the stations purnea and kishanganj (table 6). the maximum mean value of baf for zn in liver and gills of c. catla was 0.5422±0.01 and 0.309±0.005, respectively at same location of katihar and in muscles was 0.1532±0.005 at location of kishanganj, whereas in gills and muscles of m. seenghala highest baf value for zn was 0.4016±0.020 and 0.2058±0.0111, respectively at the same station of katihar and in liver was 0.6243±0.0185 at purnea location. the lowest value of baf for zn in liver and gills of c. catla was 0.2615±0.004 and 0.212±0.007and of m. seenghala was 0.3333±0.009 and 0.3484±0.008, respectively at upstream of river at kishanganj. in muscle of c. catla table 6. seasonal variation of zn concentration (mg/kg dry wt) in different organs of catla catla and mystus seenghala in river mahananda. location organ catla catla mystus seenghala summer rainy winter total summer rainy winter total (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) kishanganj liver 28.7±2.65 26.3±1.32 27.6 ±2.21 27.53±1.20 35.3±2.76 35.2±5.31 34.7± 6.01 35.07±0.3214 baf 0.2645 0.2568 0.2633 0.2615±0.004 0.3253 0.3437 0.3311 0.3333±0.009 gills 22.2±2.31 22.4±4.12 22.3± 1.42 22.3±0.01 36.7±3.21 36.6±3.25 35.7±3.54 36.33±0.5507 baf 0.2046 0.2187 0.2127 0.212±0.007 0.3474 0.3573 0.3406 0.3484±0.008 muscles 16.9±2.01 15.1±3.2 16.1±2.43 16.03±0.902 12.3±0.56 11.6±1.02 11.7±1.05 11.87±0.378 baf 0.1587 0.1474 0.1536 0.1532±0.005 0.1133 0.1132 0.1116 0.1127±0.009 purnea liver 41.7±0.76 38.5±0.54 37.8±6.01 39.33±2.079 75.4±8.3 70.6±7.8 69.8±5.2 71.93±3.028 baf 0.3553 0.3388 0.3273 0.3405±0.014 0.6435 0.6231 0.6065 0.6243±0.0185 gills 24.7±4.32 22.5±3.54 22.7±2.12 23.3±1.216 40.1±3.23 39.2±5/42 39.4±2.03 39.9±1.044 baf 0.2104 0.198 0.1966 0.2016±0.007 0.3417 0.345 0.3369 03512±0.0040 muscles 12.5±0.42 12.1±0.6 11.8±1.01 12.13±0.351 16.6±2.1 17.3±3.32 17.4±2.11 17.1±0.4358 baf 0.1065 0.1064 0.1021 0.109±0.0025 0.1414 0.1522 0.1507 0.1481±0.0058 katihar liver 65.7±5.98 63.7±8.32 62.3±5.48 63.9±1.708 45.2±6.30 46.6±4.32 47.8±1.23 46.53±1.301 baf 0.5501 0.5457 0.5308 0.5422±0.01 0.3984 0.3992 0.4073 0.4016±0.005 gills 37.3±4.31 36.4±3.03 35.6±2.01 36.43±0.850 48.1±3.21 46.3±1.95 45.1±2.01 46.5±1.509 baf 0.3123 0.3118 0.3033 0.309±0.005 0.424 0.3967 0.3843 0.4016±0.0203 muscles 19.2±0.98 17.5±1.06 17.1±1.83 17.93±1.115 22.3±2.56 23.7±2.13 25.6±4.10 23.86±1.6563 baf 0.1607 0.1499 0.1457 0.1521±0.007 0.1965 0.203 0.2181 0.2058±0.0111 116 kumar et al./ distribution and bioaccumulation of heavy metal in water, sediment and fish tissue lowest baf was recorded 0.109±0.0.0025 and of m. seenghala was 0.1127±0.009 at study point of purnea and kishanganj district, respectively. cadmium: the highest mean concentration of cd in liver of c. catla and m.seenghala was 0.924±0.004 and 0.979±0.019 mg/kg, respectively at the same location of kishanganj (upstream) and whereas lowest mean concentration 0.471±0.0065 and 0.534±0.0239 mg/kg was recorded at the same location of downstream of katihar (table 7). in gills maximum mean concentration in c. catla and m. seenghala was 0.4576±0.0124 and 0.870±0.0181 mg/kg at study point of purnea and kishanganj, respectively but minimum mean concentration was found 0.269±0.0359 and 0.576±0.0062 mg/kg at study point of kishanganj and purnia respectively (table 7). cd table 7. seasonal variation of cd concentration (mg/kg dry wt) in different organs of catla catla and mystus seenghala in river mahananda. location organ catla catla mystus seenghala summer rainy winter total summer rainy winter total (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) kishanganj liver 0.928±0.11 0.925±0.04 0.92±0.01 0.924±0.004 0.996±0.06 0.985±0.06 0.958±0.04 0.979±0.019 baf 1.007 1.004 1.0033 1.005±0.002 1.081 1.069 1.0401 1.063±0.021 gills 0.256±0.01 0.242±0.03 0.31±0.12 0.269±0.035 0.891±0.04 0.856±0.03 0.865±0.06 0.870±0.018 baf 0.2779 0.2951 0.2905 0.2878±0.009 0.9674 1.0439 1.001 1.004±0.038 muscles nd nd nd nd nd nd nd nd baf nd nd nd nd nd nd nd nd purnea liver 0.524±0.12 0.501±0.01 0.511±0.05 0.512±0.01 0.548±0.01 0.545±0.03 0.54±0.11 0.5443±0.004 baf 0.7178 0.7625 0.7514 0.7439±0.23 0.7506 0.8298 0.7941 0.7915±0.396 gills 0.472±0.02 0.451±0.02 0.45±0.03 0.4576±0.012 0.581±0.01 0.578±0.05 0.569±0.03 0.576±0.0062 baf 0.6465 0.6864 0.6617 0.6648±0.02 0.7958 0.8797 0.8367 0.8374±0.419 muscles nd nd nd nd nd nd nd nd baf nd nd nd nd nd nd nd nd katihar liver 0.478±0.05 0.47±0.06 0.465±0.03 0.471±0.0065 0.562±0.04 0.521±0.03 0.52±0.01 0.534±0.0239 baf 0.605 0.6025 0.6568 0.6214±0.03 0.7113 0.6679 0.7303 0.7031±0.031 gills 0.46±0.021 0.392±0.03 0.402±0.04 0.418±0.0367 0.751±0.04 0.72±0.02 0.71±0.02 0.727±0.0213 baf 0.5822 0.5025 0.5646 0.5497±0.041 0.9506 0.923 0.9971 0.9569±0.037 muscles nd nd nd nd nd nd nd nd baf nd nd nd nd nd nd nd nd nd = not detected table 8. seasonal variation of pb concentration (mg/kg dry wt) in different organs of catla catla and mystus seenghala in river mahananda. location organ catla catla mystus seenghala summer rainy winter total summer rainy winter total (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) (m±sd) kishanganj liver 14.2±1.2 13.1±2.2 12.7±2.31 13.33±0.776 14.78±3.32 15.4±2.12 15.1±5.2 15.09±0.310 baf 0.6612 0.6104 0.5523 0.6079±0.054 0.6362 0.7176 0.6579 0.6705±0.042 gills 6.4±0.45 6.1±1.34 5.7±0.47 6.066±0.351 9.3±2.38 8.7±1.61 8.6±1.23 8.866±0.378 baf 0.2755 0.2842 0.2483 0.2693±0.018 0.4003 0.4054 0.3747 0.3937±0.016 muscles 3.88±0.23 2.79±0.04 2.58±2.1 3.083±0.698 1.78±0.21 1.79±0.41 1.76±0.22 1.776±0.015 baf 0.167 0.13 0.1124 0.1364±0.02 0.0766 0.0834 0..0766 0.0788±0.003 purnea liver 14.3±2.1 13.5±4.2 13.1±4.21 13.63±0.611 18.4±2.41 16.7±5.12 16.2±3.25 17.1±1.153 baf 0.5843 0.5955 0.5469 0.5755±0.02 0.7519 0.7366 0.6764 0.7216±0.04 gills 9.57±0.12 8.32±1.36 7.88±1.1 8.59±0.8767 1.25±0.32 11.7±0.45 11.81±0.27 12.0±0.4336 baf 0.591 0.367 0.302 0.42±0.1516 0.5108 0.5161 0.4931 0.5066±0.012 muscles 2.7±0.56 2.6±0.56 2.2±0.31 2.5±0.2645 3.7±0.33 3.6±0.51 3.1±1.1 3.466±0.321 baf 0.1103 0.1146 0.0918 0.1055±0.012 0.1512 0.1588 0.1294 0.1464±0.015 katihar liver 30.5±5.61 30.1±7.1 28.2±4.27 29.2±1.229 32.51±7.13 31.4±4.34 32.2±5.41 32.03±0.572 baf 1.181 1.2668 1.155 1.201±0.058 1.2592 1.3215 1.3191 1.299±0.035 gills 16.5±3.81 15.3±2.4 15.4±2.36 15.73±0.665 24.3±2.23 24.6±4.21 24.4±3.01 24.43±0.152 baf 0.6392 0.6439 0.6308 0.638±0.006 0.9414 1.0353 0.7995 0.9254±0.11 muscles 8.5±1.31 8.2±0.37 8.01±2.41 8.236±0.247 7.6±0.56 7.1±0.392 7.2±0.51 7.3±0.264 baf 0.3293 0.3451 0.3277 0.334±0.009 0.2944 0.2988 0.2949 0.296±0.002 117 int. j. aquat. biol. (2020) 8(2): 109-125 was not detected in any concentration in the muscles of c. catla and m. seenghala at any location of river in seemanchal zone. the maximum value of baf for cd in the liver of c. catla and m. seenghala was recorded 1.005±0.002 and 1.063±0.0211 at the same location of river (upstream), whereas minimum baf value 0.6214±0.0306 and 0.7031±0.031 were found at same study point, downstream of river in seemanchal zone. like concentration of cd in gills, maximum baf value in galls of c. catla and m. seenghala was obtained 0.6648±0.0201 and 1.004±0.0383 at study point of purnea and kishanganj, respectively and minimum baf value was obtained 0.2878±0.009 and 0.8374 ±0.419 at study point of kishanganj and purnia, respectively. lead: the highest mean concentration of pb in liver, gills and muscles of c. catla was 29.2±1.229, 15.73±0.663 and 8.265±0.247 mg/kg respectively and of m. seenghala was 32.03±0.572, 24.43±0.152 and 7.3±0.264 mg/kg, respectively at the same location, downstream of river at katihar (table 8). the lowest mean concentration of pb in the liver, gills and muscles of c. catla was 13.33±0.776, 6.066±0331 and 3.083±0.698 mg/kg and of m. seenghala was 15.09±0.310, 8.866±0.378 and1.776±0.0015 mg/kg respectively at the same location, upstream of river at kishanganj (table 8). the maximum mean value of baf for pb in liver of c. catla was 1.201±0.0581 at katihar area and of m. seenghala was 1.299±0.035 at same location, whereas the minimum value of baf for pb in the liver of c. catla was 0.5755±0.02 at station purnea and in m. seenghala was 0.6705±0.042 at kishanganj district. in gills of c. catla and m. seenghala, the maximum mean value of baf for pb was 0.638±0.006 and 0.9257±0.11 at the same station of katihar and the minimum value was 0.2693±0.018 and 0.3937±0.016 at same location of kishanganj. in muscle of c. catla and m. seenghala, the highest value of baf of pb was 0.334±0.009, and 0.296±0.002 at same station of katihar area, whilst the lowest baf value was 0.1055±0.01 and 0.0788±0.003 at station purnea and kishanganj for the two types of fish’s c. catla and m. seenghala, respectively. discussions the results show that except pb, the heavy metal load (cu, zn and cd) in water was below than toxicity threshold level recommended by who (2008), whereas the concentration of cu, zn and cd in sediment samples was higher and of pb was lower than trv recommended by usepa (1999). the concentrations of cu, zn, cd and pb in the water system were found to be maximum during the rainy season, which may be due to extremely low alkalinity and ph of the aquatic phase (battacharya et al., 2008). during the summer season, the concentration of cu, zn, cd and pb in water system attained their minimum value with high surface water temperature. the parameter like ph, alkalinity, tds and turbidity value also reached their highest values during the summer season (battacharya et al., 2008). the effect was also observed in the sediment phase with the highest heavy metal concentration in summer followed by winter and rainy season. the opposite trend of seasonal variation of heavy metals in water and sediment might be also due to as the decreased river flow in summer, the rate of sedimentation and consequently the concentration increase. on the other hand, in rainy season, increased river flow causes a dilution effect, and consequently, metal level in sediment decreases. though at the one set of rainy season, the first flush effect may enhance the level, the dilution effect predominates as the season progresses. similar observations were also recorded by pandey et al. (2017). similarly, higher levels of metal in winter than rainy season could be linked to decreased river flow table 9. heavy metals in fish’s tissues (mg/kg dry wt) and maximum permissible limits (mpl) international standard. metal c. catla m. seenghala fao (1983) fao/who (1989) who (1995) cu 0.853± 0.051-14.5±0.755 1.12±0.079-19.93±1.069 30 30 30 zn 12.13±0.351-63.9±1.708 11.87±0.378-71.93±3.028 30 40 100 cd 0.269±0.036-0.924±0.004 0.534 ±0.024-0.979 ±0.02 0.05 0.5 1 pb 2.5±0.264-29.2 ±1.2288 1.78±0.01532.03±0.572 0.5 0.5 2 118 kumar et al./ distribution and bioaccumulation of heavy metal in water, sediment and fish tissue during the winter season. similar seasonal patterns have been reported in another research (kumar et al., 2013). a high concentration of heavy metal in water and sediment at katihar zone (downstream) may be due to urban release of sewage and industrial effluents together with agricultural runoff and atmospherically deposited substances also reach the river directly or indirectly through land surface runoff (pandey et al., 2013; pandey et al., 2017; kumar et al., 2019). a high level of heavy metal in water and sediment in a downstream could be downward flow of water resuspension of deposited sediments under high flow rate of water tend to carry heavy metal in downstream. the elevated level of heavy metals, especially in the sediment can be a good indication of pollution and often can be attributed to anthropogenic influences, rather than a natural process, are also supported by work of mustafa et al. (2007) and karabassi et al. (2008). higher concentrations of heavy metals in riverside sediments may pose an ecological risk to bottom-dwelling organisms (decan et al., 2018). the maximum mean concentration of cu was recorded in liver (19.93±1.069 mg/kg), followed by gills (4.35±0.5408 mg/kg) and in muscle (2.313±0.309 mg/kg) and the level of cu in the different tissue samples of the fishes were varied between 0.92±0.23-20.6±0.50 mg/kg in summer, 0.85±0.15-20.5±0.45 mg/kg in rainy and 0.82±0.3118.7±0.38 mg/kg in winter, respectively. the mean concentration of cu present in this study was exceeded the several folds than the literature (ambedkar and muniyan, 2012; dhanakumar et al., 2014; rajeshkumar and li, 2018) and also exceeded the several folds than the permissible limit (3 mg/kg) recommended by who (2008), but lower the maximum permissible limit (mpl) recommended by fao, fao/who for human consumption. the highest levels of cu in the different tissues of selected fish species may be due to domestic waste, agricultural and industrial wastes and also due to increased boating activities, recurrent usage of antifouling paint, oil dropping from boats, and commercial fishing in the study area. several researchers have also observed the level of cu in the liver and other fish tissues (storelli et al., 2006; frang et al., 2007; uysal et al., 2009; leung et al., 2014; karunanidhi et al., 2017). pyle et al. (2005) reported that in the liver, cu concentration are usually regulated by homeostatic control below50 μm/g/drywt and can exceed this threshold only if the control mechanisms are overloaded. cu is an essential element that serves as a cofactor in some enzymes system and necessary for the synthesis of haemoglobin (sivaperumal et al., 2007), but any high intake of cu can cause adverse health effect problems for most living organisms. in the present study, zn was observed the highest amount in both fishes’ tissues in the order of magnitude as liver> gills>muscle and seasonal variation of zn in the fish tissues were observed in the order of summer>winter≈rainy season. similar trends were reported by some researches (yehia and sebaee, 2012; maurya and malik, 2016; singh and kumar, 2017). in these finding, zn level was within the range of permissible limit (10-75 mg/kg) as recommended by who but lower than mpl, recommended by fao, fao/who for human consumption and approximately same as 13.08±0.30-78.15±2.04 and 58.44±3.67-26.67±1.37 mg/kg in liver, gills and muscle of different fishes as reported by yehia and sebaee (2012) and maurya and malik (2016), respectively. the sources of zn in the study area may be geological rock weathering or human activities such as industrial and domestic wastes water discharges. zn is an essential element as more than one hindered specific enzyme require for their catalytic function (kayrak and terkin, 2018). however, at higher levels, zn produced adverse effects in fish by structural damage, which affects the improvement, growth, and survival of fish (kori et al., 2008). zn is a potential toxicant to fish (vosylien et al., 2006), which causes ion regulation, disturbances, disruption of gill tissue and hypoxia (murugan et al., 2008). in human beings, significant levels of zn can cause prominent health problems, and high dose of zn damage the pancreas and disturb the protein metabolism and cause arteriosclerosis (afshan et al., 2014). 119 int. j. aquat. biol. (2020) 8(2): 109-125 among the studied metal, cd was observed minimum amount in both types of fish tissues; it was also detected as lowest in water and sediment. the highest cd concentration was found in liver followed by gills and not detected in muscle in both fishes. the same distribution pattern of cd was reported by jaric et al. (2011). the level of cd was highest in c. catla and m. seenghala in the summer followed by winter and rainy season. the level of cd found in liver and gill of c. catla and m. seenghala was exceeded than the mpl, recommended by fao, fao/who for human consumption, but were lower than 34.44±0.79, 1.6±0.068 mg/kg in liver and 30.89±.21, 1.35±0.061 mg/kg in gill of other fishes as reported by ambedkar and muniyan (2012) and maurya and malik (2016), respectively. in the study points, cd enter into the fresh water by disposal of industrial, municipal and household waste and also agricultural runoff. cd is the non-essential and most toxic heavy metal which is widely distributed in aquatic environment and earth's crust. the nutritive need of different tissues of fishes depends on their biochemical configuration of mineral contents, amino acids, protein and vitamins, etc. (afshan et al., 2014). the distribution of pb in different tissues of c. catla and m. seenghala was in the order of liver>gill> muscle. the level of pb found in liver, gill and muscle of c. catla and m. seenghala was approximately same as available in literature yehia and sebaee (2012) and maurya and malik (2016) and was exceeded several folds than the mpl, recommended by fao, fao/who for human consumption and also higher than 0.40±0.011-0.68±0.32 mg/kg as reported by ambedkar and muniyan (2012). among the four metals, pb was observed as the second major amount in both types of fishes in summer, followed by the rainy and winter season. similar results have been reported in different fishes by yehia and sebaee (2012). pb enter in water system through runoff, industrial and sewage waste streams. the high concentration of pb in studied areas may be due to extended agriculture, textile poultry form, industrial and other activates near to study points. the sediments could be the primary sources of pb contamination, and bottom-dwelling organisms may be directly affected by this deposited element (rajeshkumar and li, 2018). pb is a nonessential element for a living organism, and also it possesses a severe adverse effect on a living organism. fish and humans are primarily exposed to pb by food ingestion and breathing. an increasing level of pb in the water can cause generative damage in some aquatic life and cause blood and nervous changes in animals and fish. pb accumulates in muscles, bones, blood, and fat. newborns and young children are especially delicate to even low levels of pb (afshan et al., 2014). the concentration of heavy metal in two type fishes was different, as distribution and bioaccumulation heavy metal have a direct link with the feeding habit of fish and fish niche in the water system (shrivastava et al., 2001; oguzie, 2003). moreover, many factors such as age, sex, size, reproductive cycle, summing patterns, geographical location, as well as other factors like salinity, temperature and interacting agents can influence metal uptake (mustafa et al., 2003; yilmaz, 2005; zhao et al., 2012). a higher level of metal in m. seenghala compared to c. catla is that m. seenghala is a carnivorous and bottom feeder, while c. catla is omnivorous, herbivorous and surface, mid water feeder (maurya and malik, 2016; adebayo, 2017; rajeshkumar and li, 2018). seasonal variation of metal in fish may be due to varying seasonal growth rate, reproductive cycle, water salinity and temperature may be the cause of high metal accumulation of metal mainly during summer in comparison of winter and rainy season. a similar pattern was reported by ebinger et al. (2015), singh and kumar (2017), rajeshkumar and li (2018) and rajeshkumar et al. (2018). in this work, accumulation of metal in different tissues of c. catla and m. seenghala was liver>gill>muscle. mormede and devies (2001) have reported that the liver was the target organ, showing the detoxification and accumulation role of the liver. the muscle is generally considered to have weak accumulating potential (erdogrul and erbilir, 2007; uysal et al., 2009). the liver is the preferred organ for metal accumulating, as could be deduced from the present study. a similar 120 kumar et al./ distribution and bioaccumulation of heavy metal in water, sediment and fish tissue pattern has been observed in some other researches (stprelli et al., 2006; dural et al., 2007; ploetz et al., 2007; agah et al., 2009). the difference in the level of accumulation of metal in different organs of a fish can be attributed to the differences in the physiological role of each organ (rajeshkumar and li, 2018; rajeshkumar et al., 2018). regular ability, behaviour and feeding habits are other factors that affect the bioaccumulation differences in the different organs. the liver of the c. catla and m. seenghala obtained the highest level of all studied metals, while muscles appeared to be least. this finding is an agreement with those of other studies regarding fish tissue (kir et al., 2006; karaded and unlu, 2007; karaded-akin, 2009; mohamadi et al., 2011; ebrahimpour et al., 2011; liu et al., 2012; rajeshkumar and li, 2018). the liver is a vital organ in vertebrates and has a significant role in metabolism (liu et al., 2012). the high accumulation of metals in the liver is due to the greater tendency of the element to react with the oxygen carboxylate, amino group, nitrogen, sulphur of mercapto group in the metallothionein protein, whose level is highest in the liver as supported by al-yousuf et al. (2000). gill is an essential site for the entry of heavy metals (vohodhani and narayanan, 2008; rajeshkumar and li, 2018). in the present work, higher metal concentration in the gill is due to element complexation with the mucus, which is difficult to be obliterated from the tissue before analysis (khalil and faragallah, 2008). thus, level of metals in the gill reflects the level of the metals in the water system where the fish lives, whereas the concentration in liver and kidney storage of metals (vohodhani and narayanan, 2009). thus, the gills in fish are more often recommended as environment indicator organs of water pollution than any other fish organ. level of metals was lower in muscle compared to liver and gill because at being inactive tissue in accumulating heavy metals (karaded et al., 2004; stprelli et al., 2006; dural et al., 2007; ploetz et al., 2007; agah et al., 2009). health risk assessments for fish consumption: to assess the public health risk of mahananda river fish consumption, metal concentrations in liver, gills, and muscles of the fishes in this study were compared with the maximum permissible limits (mpl) for human consumption as set by various organizations. the concentrations of metals in the different tissues of c. catla and m. seenghala collected from river mahananda in seemanchal zone was found to be below the mpl for human use recommended by fao (1993), fao/who (1989) and who (1995) with few exceptions. the essential metals zn and cu were clearly below the mpl, for human consumption, whereas the nonessential metal cd was lower and pb was higher than mpl. both fishes were contaminated by pb and a threat to public health. this could be likely due to anthropogenic sources. conclusions based on experimental findings, it was concluded that metal index value (mi) for cd and pb in water was more significant than one; therefore, river water cannot be used for drinking purposes. in the sediment concentration of cu, zn and cd exceeded the trv suggest that adverse effects on sediment-dwelling organisms, and different fish species as well as the impact on human health, who consumed fish from the study area. the results also showed that metal accumulation in the fish varied between organs and species depending on species-specific factors like feeding behaviour, swimming patterns, and a genetic tendency or other factors like geographical distribution, age, and ambient concentration of metals in the water system. metals accumulations were higher in the liver, followed by gills and muscles. the high level of cu, zn, cd and pb was observed in liver and gill and even though fish liver and gill are rarely consumed. the low-level of heavy metal in muscle is particularly important because muscle is the main part of the fish and directly influences human health. however, pb in both types of fishes during all three seasons exceeded mpl, hence unsafe for consumption and therefore they pose a threat to public health. references al-busaidi m., yesudhason p., al-mughairi s. 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(2019) 7(5): 271-279 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article microbial load and diversity in the gastro-intestinal tract of cultured nile tilapia (oreochromis niloticus) and hybrid catfish (clarias gariepinus ♀ x heterobranchus bidorsalis ♂) in ilorin metropolis, nigeria wasiu adeyemi jimoh*,1taofeek sulyman, ayomide damilare taiwo department of aquaculture and fisheries, faculty of agriculture, university of ilorin, pmb 1515, ilorin, kwara state, nigeria. s article history: received 11 june 2019 accepted 29 september 2019 available online 2 5 october 2019 keywords: yeast load bacteria load fish gut shannon-weiner index simpson dominance abstract: this study investigated microbial load and diversity of gastro-intestinal tract of the cultured nile tilapia (oreochromis niloticus) and hybrid catfish (clarias gariepinus ♀ x heterobranchus bidorsalis ♂) in ilorin metropolis, nigeria. a set of apparently healthy nile tilapia and hybrid catfish were obtained from a fish farm in ilorin metropolis. after dissecting the fish sample aseptically, the entire alimentary canals of the specimens were divided into foregut, midgut and hindgut. then bacterial isolates were characterised, following standard operating procedures for gram reaction, morphology, motility, catalase and oxidase reactions, citrate utilization, coagulase production, starch hydrolysis, sugar fermentation, and eventual identification of the resultant colonies. the moulds were examined based on their micro-morphology as well as the colour and micro-morphology of their sporulating structures and conidia. the results of the study revealed that microbes were present in the entire gastro-intestinal tract of cultured hybrid catfish and nile tilapia with highest microbial load found in the hindgut of the two fish species under study. also, larger number of bacteria diversity indices were found in the hindgut of cultured nile tilapia, while the hindgut of cultured clariid catfish had higher fungi diversity indices. introduction gut microbes perform a variety of nutritionally important functions. they improve nutritional intake and utilization. it is well-known that dietary fiber cannot be digested in the small intestine of fish as there are no endogenous enzyme which can do so. dietary fiber and other complex polysaccharides are broken down by gut microbes producing short-chain fatty acid (scfas) and lower intestinal ph inhibits the growth of harmful bacteria producing toxins (scott et al., 2008) and conversely, scfas serve as precursors to develop certain beneficial gut bacteria that could offer probiotic effects (swennen et al., 2006; ogueke et al., 2010). they enhance the growth of lactic acid bacteria and bifidobacterium (gibson et al., 2004; nugent, 2005), which improve a host’s health (gibson and roberfroid, 1995) by promoting beneficial effects on glucose and lipid metabolism (clements and choat, 1995; gray, 2006; scott et al., 2008). fish gut *correspondence: jimoh wasiu adeyemi doi: https://doi.org/10.22034/ijab.v7i5.672 e-mail: jimoh.wa@unilorin.edu.ng microflora varies with the nature, and complexity of the digestive tract (cahill, 1990) and they could be autochthonous or allochthonous. in addition, digestion and utilization of feed are enhanced by presence of gut-associated microbiota (ghosh et al., 2002; saha et al., 2006; nayak, 2010). these microbes are known to play a complementary role of breaking down feed in fishes by producing exogenous enzymes that can degrade starch or cellulose (bairagi et al., 2002; saha et al., 2006; banerjee et al., 2016) and chitin (banerjee et al., 2015). fish gut microflora have the capacity of using complex polysaccharides such as mannose, xylose, raffinose, and cellulose; but ordinarily endogenous enzymes lack this capacity (kar and ghosh, 2008; ray et al., 2010). the microbes break down organic matter in complex polysaccharides for their survival. they can also denature anti-metabolite in feed, thereby improving feed utilization (ghosh and ray, 2017). in 272 jimoh et al./ microbial load and diversity in the gastro-intestinal tract of cultured nile tilapia and hybrid catfish the same vein, gut microbes have been reported to have the capacity to degrade phytate; das and ghosh (2014) and khan and ghosh (2012) reported that phytase is present in yeasts isolated from the gastrointestinal tracts of four major carps. similarly, tannase‐producing microbiota have been isolated from the gastro-intestinal tracts of some freshwater fish (mandal and ghosh, 2013a, b). mondal et al. (2008) reported that fish gut provides a conducive environment for establishment of microbes owing to the large amount of nutrients therein. bacteria have been reported to constitute the majority of gut colonizing microbiota (ray et al., 2012). moreover, banerjee and ghosh (2014) and das and ghosh (2014) reported that yeasts are present in fish gut, and yeasts have commonly been isolated from fish gut (gatesoupe, 2007; moffitt and mobin, 2006). gatesoupe (2007) has reported that the presence of these gut yeasts stimulates the immune system of freshwater fish. also, yeasts produce several compounds that have enormous organic values such as enzymes and immunostimulants (chi et al., 2009). hence, their presence in fish gut has a great value for the host (romero et al., 2014). gatesoupe et al. (2005a, b) isolated some fungi from freshwater fish, oncorhynchus tschawytscha and o. mykiss, respectively. genus candida is prominent in the gut of rainbow trout (gatesoupe et al., 2005a). similarly, jimoh et al. (2009a, b) isolated bacteria and fungi, respectively from different gut sections of the cultured and captured clarias gariepinus sampled in abeokuta, nigeria. also, microflora was isolated from different parts of the gastro-intestinal tract of tilapia and c. gariepinus obtained from the river dandaru, oyo state, nigeria by jimoh et al. (2013, 2014), respectively. paucity of information still exists in exploring gut microbes of different cultured fish species. an attempt is made in this study to investigate the microbial load and diversity in the gut sections of two principally cultured fish species in ilorin metropolis, nile tilapia (oreochromis niloticus) and hybrid catfish (clarias gariepinus ♀ x heterobranchus bidorsalis ♂) materials and methods a set of apparently healthy nile tilapia and hybrid catfish were obtained from a fish farm in ilorin metropolis. the entire alimentary canals of each of the specimens were divided into foregut, midgut and hindgut after the fish sample had been dissected aseptically. prior to this, the working table was sterilized with absolute alcohol and an oven at 160°c was used to sterilize the glassware for 90 min. isolation and characterization of microflora: the different gut sections of each fish in a sterile bottle containing 5 ml sterile distilled water were shaken vigorously to allow the content separate into water. for fungi isolation, the method of onions et al. (1981) was followed. using freshly prepared sabouraud dextrose agar medium (sda), 0.1 ml of each suspension was pour plated, covered and gently swirled to evenly mix up, and it was allowed to gel after which the plates were put in the inoculating chamber for 3 to 4 days. the representative colonies developing from the plates were assembled according to their cultural characteristics, purified by recurrent sub-culturing and maintained on appropriate agar slants as stock culture. the moulds under study were examined on the basis of their micro-morphology as well as the colour and micro-morphology of their sporulating structures and conidia according to onions et al. (1981). for bacteria isolation, the gastrointestinal tracts (gits) of each sample in sterile bottles containing 5 ml sterile distilled water were vigorously shaken to allow the content to detach in water. using nutrient agar, 1 ml of the suspension was taken and serially diluted to 10-6. using serial dilution and pour plate method, microbial load, isolation and identification of microorganisms were done. after incubation at 37°c for 24 hours, representative colonies emerging from the plates were grouped according to their cultural characteristics, purified by recurrent sub-culturing and maintained on appropriate agar slants as stock cultures. characterization of bacterial isolates followed standard operating procedures for gram reaction, morphology, motility, catalase and oxidase reactions, citrate utilization, coagulase production, starch hydrolysis and sugar 273 int. j. aquat. biol. (2019) 7(5): 271-279 fermentation (claus, 1992; harrigan and mccance, 1976; seeley jr and vandemark, 1962). the criteria of holt et al. (1994) were used in identification of the resultant colonies. microbial count: counting of the bacteria colonies, which evolved after incubation, was expressed in colony forming unit (cfu)/g. the total fungal counts were expressed as spore/g. diversity study: the following diversity indices were employed for diversity study of the different sections and the entire gastro-intestinal tract of cultured nile tilapia and hybrid catfish sampled from farms in ilorin metropolis shannon weiner index (h) = − ∑ plnp s i=1 simpson dominance index (1 − d) = 1 − d where d = ∑ p2si=1 . margalef richness index = (s − 1) ln n ⁄ where s = total number of species and n = total number of items in the sample. statistical analysis: data obtained from microbial count were transformed using logarithmic transformation (log cfu), expressed as mean ± sd before subjecting them to one-way analysis of variance using spss version 17.0. duncan multiple range test was used to separate the means where significant difference (p<0.05) existed among the treatment means. results microbial load analysis: table 1 shows the microbial load in the different sections of the fish sampled. there were significant variations (p<0.05) in the microbial load of the gastro-intestinal tract of nile tilapia in ilorin metropolis with hindgut having significantly (p<0.05) higher bacteria and fungi load than other sections of the gastro-intestinal tract. the fungi and bacteria load of foregut and midgut of the sampled nile tilapia were not significantly different (p>0.05). table 2 shows the microbial load (logcfu/g) of different gut sections of sampled hybrid catfish cultured in ilorin metropolis. the hindgut had the highest bacteria load which was not significantly different (p>0.05) from other sections of the gastrointestinal tract of hybrid catfish. microbial diversity: microbial occurrence, distribution, and diversity of the gastro-intestinal tract of nile tilapia is presented in table 3. the hindgut of nile tilapia was colonized by a variety of microorganisms. it had the highest value of diversity indices, while the midgut had the lowest diversity. table 4 shows the occurrence, distribution and diversity indices of the different gut sections and the entire gastro-intestinal tract of cultured hybrid catfish. the hindgut harbored a larger number of microorganisms. the diversity indices increased from the foregut to hindgut table 5 reveals the bacteria and fungi diversity indices of different sections and the entire gastrointestinal tract of nile tilapia. comparatively, the bacteria were more diverse than the fungi in the foregut, midgut, and hindgut. using margalef's richness as index of assessment, bacteria were more table 1. microbial load (log-cfu/g) of the different sections of gastro-intestinal tract of nile tilapia (oreochromis niloticus) in ilorin metropolis. microbial load bacteria fungi foregut 5.89±1.29b 3.90±0.19b midgut 6.06±0.08b 4.15±0.22b hindgut 7.21±0.08a 5.00±0.28a column means with different superscripts were significantly different (p<0.05) from each other table 2. microbial load (log-cfu/g) of different gut sections of the sampled hybrid catfish (clarias gariepinus ♀ x heterobranchus bidorsalis♂) cultured in ilorin metropolis. bacteria fungi foregut 5.28±0.30a 3.31±0.15a midgut 5.46±0.28a 3.33±0.17a hindgut 5.48±0.23a 3.38±0.10a column means with different superscripts were significantly different (p<0.05) from each other 274 jimoh et al./ microbial load and diversity in the gastro-intestinal tract of cultured nile tilapia and hybrid catfish diverse than fungi in the entire gastro-intestinal tract of nile tilapia. bacteria and fungi were equally distributed in the midgut, using simpson’s dominance and shannon-weiner (h) as indices of assessment. table 6 shows the taxa differential diversity indices of the microbes in different sections and the entire gastro-intestinal tract of cultured hybrid catfish. comparatively, fungi were more diverse than bacteria in the mid and hind guts, and the entire gastrointestinal tract of the fish, using simpson dominance, shannon-weiner (h) and maargalef’s richness as indices. discussions the microbial community in the gastro-intestinal tract of fish provides a synergistic role in a host’s health, nutrition and development (romero et al., 2014). the microbial load in cultured nile tilapia and hybrid catfish in ilorin metropolis revealed a differential table 3. microbial occurrence, distribution and diversity of the different sections and the entire gastro-intestinal tract of nile tilapia (oreochromis niloticus) in ilorin metropolis microbial isolates foregut mid gut hindgut git enterococcus faecalis 1(0.20) 0(0.00) 1(0.11) 2(0.11) kleibsiella oxytoca 0(0.00) 0(0.00) 1(0.11) 1(0.06) citrobacter freundii 0(0.00) 0(0.00) 1(0.11) 1(0.06) escherichia coli 1(0.20) 1(0.25) 1(0.11) 3(0.17) shigella flexneri 1(0.20) 1(0.25) 1(0.11) 3(0.17) aspergillus flavus 0(0.00) 1(0.25) 1(0.11) 2(0.11) aspergillus niger 0(0.00) 1(0.25) 1(0.11) 2(0.11) penicillum notatum 1(0.20) 0(0.00) 1(0.11) 2(0.11) aspergillus fumigatus 1(0.20) 0(0.00) 1(0.11) 2(0.11) diversity indices simpson dominance index (1-d) 0.80 0.75 0.89 0.88 shannon-weiner index (h) 1.61 1.39 2.20 2.14 margalef's richness 1.82 1.37 8.54 7.74 values in parenthesis are proportion. table 4. microbial occurence, distribution and diversity indices of the different sections and the entire git of cultured hybrid catfish (clarias gariepinus ♀ x heterobranchus bidorsalis ♂) from ilorin metropolis microbial isolates foregut midgut hindgut git proteus vulgaris 0(0.00) 0(0.00) 1(0.14) 1(0.06) bacillus substilis 0(0.00) 0(0.00) 1(0.14) 1(0.06) streptococcus faecalis 1(0.25) 1(0.20) 0(0.00) 2(0.13) enterobacter aerogenes 1(0.25) 1(0.20) 1(0.14) 3(0.19) aspergillus flavus 1(0.25) 1(0.20) 1(0.14) 3(0.19) aspergillus niger 0(0.00) 1(0.20) 1(0.14) 2(0.13) saccharomyces cerevisae 1(0.25) 1(0.20) 1(0.14) 3(0.19) penicillium citrinum 0(0.00) 0(0.00) 1(0.14) 1(0.06) diversity indices simpson dominance (1-d) 0.75 0.80 0.86 0.85 shannon-weiner index(h) 1.39 1.61 1.95 1.98 margalef's richness 2.16 2.49 3.08 5.41 values in parenthesis are proportion. table 5. taxa differential diversity indices of the gastro-intestinal tract of nile tilapia (oreochromis niloticus) in ilorin metropolis. foregut midgut hindgut git diversity indices b f b f b f b f simpson dominance (1-d) 0.67 0.50 0.50 0.50 0.50 0.50 0.50 0.75 shannon-weiner index 1.10 1.39 0.69 0.69 0.69 0.69 0.72 1.39 margalef's richness 1.24 0.72 1.38 1.28 4.38 2.16 9.38 7.28 git: gastro-intestinal tract b: bacteria f: fungi 275 int. j. aquat. biol. (2019) 7(5): 271-279 increment from foregut to hindgut. comparative assessment of the microbial loads of the two fishes under studies showed that cultured nile tilapia had higher microbial load than hybrid catfish. our findings are in tandem with huber et al. (2004) who observed that bacteria loads vary from fish to fish and across locations. the highest microbial load was found in the hindgut of both fishes in this study, which supports the report of mountfort et al. (2002) that hindgut harbors larger numbers of microbial community that contribute a great to the energy need of the host fish. short-chain fatty acid (scfa) produced by the activities of these microbes have beneficial effect on lipid and glucose metabolism that serve as a source of energy to the host (gray, 2006; scott et al., 2008). the isolated gut microbes of both fish types in this study closely related to what was reported by jimoh et al. (2009a, b) from captured and cultured c. gariepinus sampled in abeokuta north local government, nigeria. similarly, jimoh et al. (2013) isolated aspergillus flavus and a. niger from nile tilapia caught from river dandaru, ibadan and some bacteria isolates similar to what is observed in the present study. enterobacter and bacillus spp. observed in this study are in consonance with the report of jimoh et al. (2014) on microbial flora of the gastrointestinal tract of c. gariepinus caught from river dandaru ibadan, nigeria. the results of the present study also reveal that most of the isolates were found in the hindgut sections of git as evidenced in the measurement of diversity indices. the foregut and the midgut having lower diversity indices could plausibly be because of pancreatic, bile and acid secretion into the stomach, which could inhibit colonization of these sections by microflora (guarner and malagelada, 2003; jimoh et al, 2014). anaerobes and facultative anaerobes were found in large number in git of fish (bairagi et al., 2002; saha et al, 2006). bacillus spp., citrobacter spp. and entrobacter spp. were isolated from the gut sections of three indian major carp (ray et al., 2010), which are known to be enzyme-producing bacteria in fish gut. kar et al. (2008) isolated b. subtilis similar to what was observed in this study. anerobic bacteria such as escherichia coli and klebsiella isolated in the fish gut regions have been reported to contribute a lot to fish nutrition (clements et al., 2009); they are known to produce amylase (ray et al., 2007), lipase and glycosidase (ramirez and dixon, 2003). the gram negative type among the isolated bacteria, such as proteus vulgaris, e. aerogenes, shigella and e. coli, in this study have enzymes that can digest complex carbohydrates (ray et al., 2012). they are known to produce short chain fatty acids (scfas) mainly acetate, propionate and butyrate (clements and choat, 1995; seeto et al., 1996; clements, 1997; stevens and hume, 2004). mountfort et al. (2002) reported that an important energy source for fish is the acetate produced by microbial fermentation to fish. bacillus subtilis and enterobacter spp. observed in this study, isolated from git of atlantic salmon and gray mullet respectively, have been reported to produce chitinase (hamid et al., 1979; askarian et al., 2012). bacteria were more diverse in git of nile tilapia, which is in line with the report of pond et al. (2006) and nayak (2010). however, the concentration of bacteria was lower than that of yeast in the git of cultured hybrid catfish in this study. this variation might be attributed to the food habit of the two fishes which is known to vary with nature and complexity of gastro-intestinal tract (cahill, 1990). higher concentration of the yeast in the gut was observed by yoshimizu (1980), who found larger number of yeasts in fish intestinal microflora. andlid et al. (1995) table 6. taxa differential diversity indices of the different sections and the entire gastro-intestinal tract of cultured hybrid catfish (clarias gariepinus ♀ x heterobranchus bidorsalis ♂). foregut midgut hindgut git diversity indices b f b f b f b f simpson dominance (1-d) 0.50 0.50 0.50 0.67 0.67 0.75 0.69 0.72 shannon-weiner index(h) 0.69 0.69 0.69 1.10 1.10 1.39 1.28 1.31 margalef's richness 1.44 1.44 1.44 2.09 1.82 2.16 3.08 3.64 git: gastro-intestinal tract b: bacteria f: fungi 276 jimoh et al./ microbial load and diversity in the gastro-intestinal tract of cultured nile tilapia and hybrid catfish reported that rainbow trout git could contain up to 3x106 yeast cell per gram of intestinal tissue. the values of yeast count recorded in this study was much lower. andlid et al. (1995) and sakata et al. (1993) have similar reports to the yeast load found in this study (3.31-5.00 log-cfu/g) for the two fish species under study. a range of 2-4 and 5-7 log-cfu/g was reported by andlid et al. (1995) and sakata et al. (1993) as naturally occurring yeast load in the gastrointestinal tract of freshwater farmed o. mykiss. 0-2 log-cfu/g of yeast load was reported by gatesoupe et al. (2005a) for freshwater farmed o. mykiss. other researchers have reported 0-4 log-cfu/g of yeast load in the same freshwater farmed o. mykiss (gatesoupe et al., 2005b; waché et al, 2006). moffitt and mobin (2006) reported the same value of 0-4 log-cfu/g of yeast load as naturally occurring in freshwater farmed o. tschawytscha. higher yeast concentration observed in this study might be as a result of lower concentration of yeast used as additive in commercial fish feed (tovar et al., 2002). tovar-ramırez et al. (2004) reported that dietary yeast improved gut maturation in sea bass (dicentrachus labrax) larvae. it has immunostimulatory and immunomodulatory effect (waché et al., 2006; kutty and philip, 2008; song et al., 2010). tannase activity of candida spp. was reported by mandal and ghosh (2013a). saccharomyces cerevisae in fish gut have been identified to stimulate enzyme activities (waché et al., 2006). conclusion it is evident from the report above that microbes can be found in the entire gastro-intestinal tract of cultured hybrid catfish and nile tilapia, with the highest microbial load found in the hindgut of the two fish species under study. larger number of bacteria diversity indices were found in the hindgut of cultured nile tilapia, while the hindgut of cultured hybrid catfish had higher fungi diversity indices. references andlid t., juárez r.-v., gustafsson l. 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(2002). effect of live yeast incorporation in compound diet on digestive enzyme activity in sea bass (dicentrarchus labrax) larvae. aquaculture, 204(1-2): 113-123. waché y., auffray f., gatesoupe f.-j., zambonino j., gayet v., labbé l., quentel c. (2006). cross effects of the strain of dietary saccharomyces cerevisiae and rearing conditions on the onset of intestinal microbiota and digestive enzymes in rainbow trout, onchorhynchus mykiss, fry. aquaculture, 258(1-4): 470-478. yoshimizu m. (1980). microflora of the embryo and the fry of salmonids. bulletin of the japanese society of scientific fisheries, 46: 967-975. international journal of aquatic biology (2015) 3(3): 135-148 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.npajournals.com © 2015 npajournals. all rights reserved original article metal bioaccumulation levels in different organs of three edible fish species from the river ravi, pakistan hafiz abdullah shakir1, javed iqbal qazi*1, abdul shakoor chaudhry2, shaukat ali3 1department of zoology, university of the punjab, lahore-54590, pakistan. 2school of agriculture, food and rural development, newcastle university, newcastle upon tyne, ne1 7ru, uk. 3department of zoology, the university of azad jammu and kashmir, muzaffarabad. article history: received 2 february 2015 accepted 21 march 2015 available online 2 5 june 2015 keywords: river ravi lahore urban and industrial effluents major carps metals contents of fish abstract: metals bioaccumulation in five organs of cirrhinus mrigala, labeo rohita and catla catla captured from three industrial and sewage polluted downstream sites (shahdera = b, sunder = c and balloki = d) were compared with a non-industrial upstream site (siphon = a) during high (post monsoon) and low (winter) flow seasons of river ravi. mean concentrations of metals were significantly higher in low flow than the high flow season. pattern of metal accumulation in the studied organs was: zn > fe > mn > cu > cr > pb > ni > hg > cd. kidneys showed mostly greater metal bioaccumulation than intestines, hearts, eyes and gills. among fish species, the highest concentrations (µg/g dry weight) of cr (3.77), zn (56.22), mn (8.95), ni (1.70) and hg (1.60) and lowest of pb (2.53) were detected in c. mrigala whereas cu (7.19), fe (62.11) and pb (2.64) appeared higher while zn (52.69), mn (7.82) and ni (1.41) with lowest concentrations in c. catla. in contrast, lower concentrations of cd (0.15), cr (3.16), cu (7.06) and fe (54.18) were recorded in l. rohita. accumulation of the metals was significantly different in organs among the different sampling sites. based on metals accumulation pattern, second downstream site (sunder) identified as the most polluted site due to untreated industrial and municipal discharges. measured elevated levels of metals concentrations in fish organs indicated potential health risks for the fish and the food chain. introduction like many other developing countries, disposal of untreated municipal and industrial effluents into natural waters is known to cause a serious damage to the water quality in pakistan. due to unmanaged and large-scale addition of wastewaters, water quality of rivers in densely populated cities and towns, especially during low-flow months of the year remains highly degraded (bhatti and latif, 2011; shakir et al., 2013). lahore being the second-largest city of pakistan, comprises rapidly expanding population and industrial zones. the city represents one of the few major agricultural, industrial, and urbanization centers of pakistan. consequently, a large number of toxic chemicals and effluentproducing industries are located in and around * corresponding author: javed iqbal qazi e-mail address: qazi.zool@pu.edu.pk lahore. the ravi river receives large quantities of untreated domestic sewage and industrial chemical pollutants from seven major municipal sewage outfalls, and two drains called hudaira and degnullah when the river passes through lahore (shakir and qazi, 2013; shakir et al., 2013). hudiara drain enters in pakistan loaded with effluents of around 100 industries situated alongside the 55 km indian side and more than 112 industries alongside 63 km of the punjab, pakistan. deg-nullah carries effluents from kala shah kaku industrial complex, which has more than 149 industrial units. some industries located along-sides lahore-sheikhupura road, also discharge their wastewater into this drain. the load of hazardous and untreated waste going into ravi river from lahore is about 728.75 tons per day. 136 international journal of aquatic biology (2015) 3(3): 135-148 also, about 1810 sources of domestic and industrial effluents are dumped in this river through pumping stations and urban drains (saeed and bahzad, 2006; yasar et al., 2010). while the toxic effects of heavy metals on freshwater animals are known for a long time, using biological methods has been recognized as great economic and sensitive ways to monitor metals in fresh water environment. fish are widely used to evaluate the health of aquatic ecosystems because pollutants that build-up in the food chain can exert adverse effects and even death of the biotic components of the aquatic systems (yousuf and el-shahawi, 1999; farkas et al., 2002). studies carried out on various fish have shown that pollutants alter the physiological activities of the animals and biochemical parameters of their tissues (sanchez et al., 2011; yousafzai and shakoori, 2011). previous studies on the river ravi inhabitant fauna were restricted to one catch site, single flow season, single fish species or non-edible fish varieties, specific tissues or limited metal types (javed and hayat, 1998; javaid and mahmood, 2000; javed, 2005; nawaz et al., 2010; jabeen et al., 2012). this study was therefore extended to investigate site-wise seasonal variations of various metal bioaccumulation in eyes, gills, heart, intestine and kidneys of three nutritionally and economically important carp fish species; catla catla (thaila), labeo rohita (rohu), cirrhinus mrigala (mori) comprehensively. the bioaccumulation of metals in muscles and liver samples of same species already reported (shakir et al., 2013; shakir et al., 2014). therefore, it was not included in this paper. these three fresh water carp species, are regarded as important fish species in aquaculture and these are the most common inhabitants of the river ravi of pakistan and other water sources of sub-continent, south east asia and china. these fish represent different parts of their habitat where c. catla is a surface feeder, l. rohita is a column feeder and c. mrigala is a bottom feeder. these fish are the most preferred species by the pakistani consumers due to their size, flavour and taste. however, their food quality, safety, and market value can be affected by the level of pollutants in their inhabiting waters. therefore, the aim of this study to monitor different organs of these fish species for metal bioaccumulation by capturing representative samples of fish from different sites during different flow seasons of the river. materials and methods sampling of fish species and dissection: the selected stretch of the river receives direct discharge of untreated urban and industrial effluents from the lahore city. the upstream site, siphon (31°41΄n and 74°25΄e) was characterized least polluted site as no point source of pollution was identified at or above this site. the first downstream site shahdera (31°36΄n and 74°18΄e) receives discharges from three untreated municipal pumping stations. solid waste damping on the banks of river with blackish water colouration associated with urbanized overcrowded towns were noted for this site. the second downstream site sunder (31°21΄n and 74°3΄e) receives untreated effluents from hudiara and deg-nullah loaded with pollutants of more than 212 and 112 industries, respectively as reported by saeed and bahzad (2006) and four municipal pumping stations. inflows of polluted upstream domestic sewage water plus effluents of drains carrying industrial effluent together make the river segment a highly polluted site. qadirabad link canal join river ravi before head balloki, last downstream sampling site (31°13΄n and 73°52΄e). the industrial and urban effluent not discharge at or before this site. brief descriptions of the study sites have been reported by shakir and qazi (2013) and shakir et al. (2013). all fish samples were netted from three polluted sites (shahdera = b, sunder = c and balloki = d) and an upstream site a (siphon) of the river ravi. during low flow season, in winter (novemberdecember 2009) and high flow season after monsoon (septemberoctober, 2010). the metal accumulation was studied in eyes, gills, heart, intestine and kidneys of replicated samples of three 137 shakir et al/ metal bioaccumulation levels in river ravi fish species comprising c. mrigala, l. rohita and c. catla. nine fish specimens for each of the three species of comparable size from each collection site during each flow period were saved from triplicate netting per site. the detailed fish size and sampling procedure have been described earlier by shakir and qazi (2013). each fish specimen was washed with water before their transfer to separate polythene bags being placed in an ice box that was immediately transported to the laboratory for analysis. each fish specimen was then identified based on mirza (2003). in total two hundred and sixteen specimens of the three fish species were dissected under aseptic condition by using sterilized forceps, scissors and scalpel to incise and remove kidneys, heart, eyes, intestine, and gills. the removed organs were carefully washed with distilled water and stored in marked polythene bags at -20ºc until their laboratory analysis. acid digestion of the fish tissues: the frozen fish organs were thawed, rinsed in distilled water and dried on blotting paper. then whole kidneys and heart, both eyes and homogenized portions of gills and intestines were shifted into individually labeled and pre-weighed dried glass vials. a known weight of each dried fish organ was acid digested according to du preez and steyn (1992) with a slight modification made by yousafzai and shakooki (2008) and shakir et al. (2013). to each sample, about 5 ml of nitric acid (55%) and 1 ml of perchloric acid (65%) were added as a first dose while working in a fume hood and the samples were then kept for overnight at room temperature. next day, 5 ml of nitric acid and 4 ml of perchloric acid were added as a second dose to each flask containing a few glass beads to prevent bumping during heating of flasks on a hot plate in fume hood at 200-250ºc. turning of dense brown fumes into white fumes escaping from the flask indicated completion of the digestion process. however, the mixture was evaporated until the mixture approached about 0.5 ml of volume. the sample within each flask was then cooled and diluted up to 20 ml with distilled water while rinsing the digestion flask. the diluted sample was filtered through whatman filter paper no. 541 filter paper. the filtrate was stored in properly washed labeled vials at 4ºc until the metal concentrations analyses. standard solutions and metal analysis by atomic absorption spectrophotometer (aas): the diluted samples were analyzed for cd, cr, cu, pb and ni using fast sequential atomic absorption spectrometer (varian spectra aa-240). mn and fe concentrations were determined with a pye unicam atomic absorption spectrophotometer whereas hg and zn were measured with a varian atomic absorption spectrophotometer (varian aas-1275). different instruments were used in this study due to limitation of lamps. single standard solutions of cd, cr, cu, fe, hg, mn, ni, pb and zn (1000 μg/ml) were purchased from bdh (england). different diluted working standard solutions were prepared stepwise from the stock solutions. standard curves were prepared for different metals between working standard solutions concentrations verses their corresponding absorbances. the accuracy of the aas was checked after each 10 samples by feeding diluted working standard solution of the respective element as a reference sample. samples that were over calibrated were further diluted. the absorbance of samples were calibrated against their relevant standard curves to find out the concentration of metals present in the analyzed samples. metal concentrations were expressed in µg/g of dried fish organs. statistical analysis: basic descriptive statistics of metal concentrations in organs of different fish species was performed using microsoft excel. the general linear model in minitab-16 software was used to the statistically compare the sampling sites, flow seasons, fish species, fish tissues and 2 or 3 way interactions for each metal concentration. the effect of these factors were declared highly significant at p<0.001, very significant at p<0.01, significant at p<0.05 and non-significant at p>0.05. results mean cd, cr, cu, fe, pb, zn, mn, ni and hg concentrations and their respective standard deviations of dried samples of eyes, gills, heart, inte 1 3 8 in te rn at io n al j o u rn al o f a q u at ic b io lo g y ( 2 0 1 5 ) 3 (3 ): 1 3 5 -1 4 8 f is h s p e c ie s m e ta ls e y e s g il ls h e a r t in te st in e k id n e y s l o w h ig h l o w h ig h l o w h ig h l o w h ig h l o w h ig h cirrhinus mrigala c d 0 .0 9 ± 0 .0 1 0 .0 3 ± 0 .0 0 0 .0 6 ± 0 .0 0 0 .0 4 ± 0 .0 0 0 .0 7 ± 0 .0 0 0 .0 5 ± 0 .0 0 0 .0 8 ± 0 .0 0 0 .0 4 ± 0 .0 0 0 .1 3 ± 0 .0 4 0 .0 8 ± 0 .0 3 c r 1 .4 ± 0 .0 1 1 .1 2 ± 0 .0 4 1 .7 4 ± 0 .0 4 0 .9 8 ± 0 .0 1 1 .5 8 ± 0 .0 4 1 .3 9 ± 0 .0 6 1 .8 2 ± 0 .0 2 1 .4 6 ± 0 .0 7 1 .9 8 ± 0 .0 2 1 .5 8 ± 0 .0 5 c u 5 .2 4 ± 0 .1 0 4 .4 2 ± 0 .0 6 4 .3 8 ± 0 .0 8 3 .7 6 ± 0 .0 5 6 .1 3 ± 0 .1 1 5 .6 9 ± 0 .0 8 5 .8 6 ± 0 .1 1 6 .0 9 ± 0 .0 8 6 .9 3 ± 0 .1 3 6 .2 1 ± 0 .1 3 f e 3 6 .5 5 ± 1 .3 6 3 2 .0 4 ± 1 .7 9 2 9 .7 6 ± 1 .1 1 2 4 .3 5 ± 1 .3 6 3 5 .2 4 ± 1 .3 1 3 9 .5 1 ± 2 .2 1 5 1 .9 5 ± 2 .2 9 4 5 .9 2 ± 2 .5 7 5 6 .9 9 ± 2 .5 6 4 8 .4 5 ± 2 .7 1 p b 0 .3 1 ± 0 .0 3 0 .2 4 ± 0 .0 5 0 .2 6 ± 0 .0 2 0 .1 8 ± 0 .0 4 0 .4 4 ± 0 .0 5 0 .3 7 ± 0 .0 8 0 .4 7 ± 0 .0 5 0 .2 7 ± 0 .0 6 0 .4 1 ± 0 .0 3 0 .2 8 ± 0 .0 6 z n 3 5 .0 8 ± 2 .2 3 2 9 .9 4 ± 3 .5 4 3 1 .9 2 ± 2 .0 3 2 5 .6 6 ± 3 .0 4 4 1 .4 1 ± 2 .6 3 3 5 .3 4 ± 4 .1 8 4 4 .8 4 ± 2 .8 5 3 8 .2 7 ± 4 .5 3 5 1 .7 0 ± 3 .2 8 4 1 .6 5 ± 4 .9 3 m n 3 .3 9 ± 0 .0 3 3 .3 1 ± 0 .0 3 3 .0 8 ± 0 .0 3 3 .0 1 ± 0 .0 3 4 .0 0 ± 0 .0 4 3 .9 0 ± 0 .0 4 4 .3 3 ± 0 .0 4 4 .2 3 ± 0 .0 4 4 .9 9 ± 0 .0 5 4 .8 7 ± 0 .0 5 n i 0 .5 3 ± 0 .0 3 0 .4 2 ± 0 .0 3 0 .4 8 ± 0 .0 3 0 .3 6 ± 0 .0 2 0 .6 1 ± 0 .0 3 0 .4 6 ± 0 .0 3 0 .6 5 ± 0 .0 4 0 .5 4 ± 0 .0 3 0 .6 9 ± 0 .0 4 0 .6 2 ± 0 .0 4 h g 0 .1 5 ± 0 .0 5 0 .1 0 ± 0 .0 2 0 .1 3 ± 0 .0 4 0 .1 4 ± 0 .0 2 0 .1 7 ± 0 .0 5 0 .1 4 ± 0 .0 2 0 .2 2 ± 0 .0 7 0 .2 2 ± 0 .0 3 0 .1 9 ± 0 .0 6 0 .1 6 ± 0 .0 2 labeo rohita c d 0 .0 7 ± 0 .0 1 0 .0 4 ± 0 .0 1 0 .0 9 ± 0 .0 1 0 .0 5 ± 0 .0 2 0 .0 6 ± 0 .0 0 0 .0 4 ± 0 .0 0 0 .0 7 ± 0 .0 0 0 .0 3 ± 0 .0 1 0 .0 8 ± 0 .0 0 0 .0 5 ± 0 .0 1 c r 1 .8 3 ± 0 .1 1 1 .6 9 ± 0 .1 2 1 .3 3 ± 0 .1 2 1 .2 9 ± 0 .1 7 1 .7 9 ± 0 .0 6 1 .2 ± 0 .0 7 1 .8 3 ± 0 .0 6 1 .1 5 ± 0 .0 6 1 .9 9 ± 0 .0 6 1 .2 7 ± 0 .0 7 c u 5 .0 9 ± 0 .0 7 4 .1 7 ± 0 .1 0 4 .2 5 ± 0 .0 6 3 .5 5 ± 0 .0 9 5 .9 6 ± 0 .0 9 5 .3 6 ± 0 .1 3 5 .9 9 ± 0 .3 5 5 .7 4 ± 0 .1 4 6 .7 4 ± 0 .1 0 6 .0 4 ± 0 .1 5 f e 3 7 .5 2 ± 1 .0 0 3 5 .5 ± 1 .4 9 3 0 .5 5 ± 0 .8 1 2 6 .9 8 ± 1 .1 3 4 6 .1 8 ± 0 .9 6 4 1 .7 8 ± 0 .8 3 5 3 .3 3 ± 1 .4 2 5 0 .8 8 ± 2 .1 4 5 8 .2 4 ± 4 .3 2 5 3 .6 8 ± 2 .2 6 p b 0 .3 5 ± 0 .0 7 0 .2 7 ± 0 .0 6 0 .3 0 ± 0 .0 6 0 .1 9 ± 0 .0 4 0 .5 0 ± 0 .1 1 0 .4 0 ± 0 .0 8 0 .5 3 ± 0 .1 1 0 .3 0 ± 0 .0 6 0 .4 6 ± 0 .1 0 0 .3 1 ± 0 .0 6 z n 2 9 .4 8 ± 1 .0 3 2 7 .7 3 ± 1 .3 4 2 6 .8 2 ± 0 .9 4 2 3 .7 6 ± 1 .1 5 3 4 .8 0 ± 1 .2 1 3 2 .7 3 ± 1 .5 8 3 7 .6 8 ± 1 .3 1 3 5 .4 4 ± 1 .7 1 4 3 .4 4 ± 1 .5 2 3 8 .5 7 ± 1 .8 6 m n 3 .3 8 ± 0 .0 4 2 .5 0 ± 0 .1 1 3 .0 8 ± 0 .0 4 2 .2 7 ± 0 .1 0 3 .9 9 ± 0 .0 5 2 .9 5 ± 0 .1 4 4 .3 2 ± 0 .0 5 3 .1 9 ± 0 .1 5 4 .9 8 ± 0 .0 6 3 .6 8 ± 0 .1 7 n i 0 .5 6 ± 0 .0 5 0 .4 8 ± 0 .0 4 0 .5 1 ± 0 .0 4 0 .4 1 ± 0 .0 4 0 .6 4 ± 0 .0 5 0 .5 3 ± 0 .0 5 0 .6 9 ± 0 .0 6 0 .6 2 ± 0 .0 6 0 .7 3 ± 0 .0 6 0 .7 0 ± 0 .0 6 h g 0 .1 2 ± 0 .0 1 0 .1 1 ± 0 .0 1 0 .1 0 ± 0 .0 1 0 .1 4 ± 0 .0 1 0 .1 3 ± 0 .0 1 0 .1 4 ± 0 .0 1 0 .1 6 ± 0 .0 1 0 .2 2 ± 0 .0 1 0 .1 4 ± 0 .0 1 0 .1 6 ± 0 .0 1 catla catla c d 0 .0 5 ± 0 .0 1 0 .0 3 ± 0 .0 0 0 .0 8 ± 0 .0 2 0 .0 6 ± 0 .0 2 0 .0 9 ± 0 .0 2 0 .0 5 ± 0 .0 1 0 .1 2 ± 0 .0 2 0 .0 9 ± 0 .0 3 0 .1 5 ± 0 .0 4 0 .1 1 ± 0 .0 4 c r 1 .5 2 ± 0 .0 6 1 .2 3 ± 0 .0 7 1 .5 7 ± 0 .0 5 1 .1 2 ± 0 .0 5 1 .8 1 ± 0 .0 6 1 .5 ± 0 .5 0 1 .8 5 ± 0 .0 6 1 .4 3 ± 0 .4 8 2 .0 1 ± 0 .0 7 1 .5 8 ± 0 .5 3 c u 5 .4 2 ± 0 .1 7 4 .3 8 ± 0 .0 5 4 .5 3 ± 0 .1 4 3 .7 3 ± 0 .0 4 6 .3 5 ± 0 .2 0 5 .6 4 ± 0 .0 6 6 .0 6 ± 0 .1 9 6 .0 3 ± 0 .0 7 7 .1 8 ± 0 .2 3 6 .3 5 ± 0 .0 7 f e 3 8 .2 8 ± 2 .1 8 3 7 .1 2 ± 1 .7 3 3 1 .1 7 ± 1 .7 7 2 8 .2 1 ± 1 .3 1 5 6 .9 1 ± 2 .3 7 4 5 .7 8 ± 2 .1 3 5 4 .4 1 ± 3 .1 0 5 3 .2 ± 2 .4 8 6 9 .2 1 ± 3 .0 5 5 6 .1 4 ± 2 .6 2 p b 0 .3 3 ± 0 .0 7 0 .3 2 ± 0 .0 6 0 .2 8 ± 0 .0 6 0 .2 3 ± 0 .0 5 0 .4 8 ± 0 .1 0 0 .4 7 ± 0 .1 2 0 .5 0 ± 0 .1 3 0 .3 5 ± 0 .0 7 0 .4 4 ± 0 .1 0 0 .3 6 ± 0 .0 7 z n 2 8 .3 8 ± 1 .8 7 2 2 .8 3 ± 1 .5 7 2 5 .8 2 ± 1 .7 0 1 9 .5 7 ± 1 .3 5 3 3 .5 0 ± 2 .2 1 2 6 .9 5 ± 1 .8 5 3 6 .2 8 ± 2 .4 0 2 9 .1 8 ± 2 .0 1 4 1 .8 2 ± 2 .7 6 3 1 .7 6 ± 2 .1 8 m n 3 .5 0 ± 0 .1 5 1 .9 2 ± 0 .1 1 3 .1 8 ± 0 .1 3 1 .7 5 ± 0 .1 0 4 .1 3 ± 0 .1 7 2 .2 7 ± 0 .1 4 4 .4 7 ± 0 .1 9 2 .4 6 ± 0 .1 5 4 .8 9 ± 0 .2 0 2 .8 4 ± 0 .1 7 n i 0 .5 7 ± 0 .0 2 0 .4 2 ± 0 .0 2 0 .5 1 ± 0 .0 2 0 .3 5 ± 0 .0 2 0 .6 5 ± 0 .0 2 0 .4 6 ± 0 .0 3 0 .6 9 ± 0 .0 2 0 .5 4 ± 0 .0 3 0 .7 4 ± 0 .0 3 0 .6 1 ± 0 .0 3 h g 0 .1 3 ± 0 .0 1 0 .1 0 ± 0 .0 1 0 .1 1 ± 0 .0 1 0 .1 3 ± 0 .0 1 0 .1 4 ± 0 .0 1 0 .1 4 ± 0 .0 1 0 .1 8 ± 0 .0 1 0 .2 2 ± 0 .0 1 0 .1 6 ± 0 .0 1 0 .1 5 ± 0 .0 1 t ab le 1 . m ea n s ± s d ( µ g /g d ry w ei g h t) o f m et al s co n ce n tr at io n s in d if fe re n t o rg a n s o f th e sa m p le d f is h s p e ci es s am p le d f ro m s it e a ( s ip h o n ) d u ri n g l o w a n d h ig h f lo w s ea so n . 1 3 9 s h ak ir e t al / m et al b io ac cu m u la ti o n l ev el s in r iv er r av i f is h s p e c ie s m e ta ls e y e s g il ls h e a r t in te st in e k id n e y s l o w h ig h l o w h ig h l o w h ig h l o w h ig h l o w h ig h cirrhinus mrigala c d 0 .1 8 ± 0 .0 2 0 .0 5 ± 0 .0 2 0 .1 4 ± 0 .0 2 0 .1 ± 0 .0 1 0 .1 5 ± 0 .0 1 0 .1 2 ± 0 .0 1 0 .1 4 ± 0 .0 1 0 .1 1 ± 0 .0 1 0 .1 9 ± 0 .0 1 0 .1 4 ± 0 .0 1 c r 2 .4 3 ± 0 .0 5 2 .0 3 ± 0 .0 5 2 .9 9 ± 0 .0 9 2 .4 ± 0 .0 6 3 .2 6 ± 0 .0 7 2 .8 9 ± 0 .0 7 3 .3 4 ± 0 .0 7 2 .1 ± 0 .1 4 4 .1 8 ± 0 .0 9 3 .2 9 ± 0 .1 2 c u 6 .4 8 ± 0 .1 3 5 .1 5 ± 0 .3 5 6 .3 1 ± 0 .4 6 5 .3 ± 0 .3 3 7 .8 3 ± 0 .1 6 6 .2 2 ± 0 .3 2 8 .5 1 ± 0 .1 7 7 .6 9 ± 0 .0 8 1 0 .2 6 ± 0 .2 1 9 .0 8 ± 0 .3 9 f e 4 2 .7 8 ± 3 .3 3 3 6 .7 ± 2 .0 7 4 9 .3 7 ± 3 .8 0 4 1 .0 7 ± 2 .3 1 5 7 .8 7 ± 3 .9 2 5 1 .2 6 ± 3 .6 5 6 4 .2 6 ± 3 .1 4 5 7 .9 6 ± 3 .2 7 7 5 .7 7 ± 2 .5 6 6 9 .8 2 ± 3 .9 4 p b 2 .1 9 ± 0 .1 6 1 .7 5 ± 0 .0 9 1 .8 3 ± 0 .1 3 1 .3 6 ± 0 .0 7 2 .8 8 ± 0 .2 1 1 .8 6 ± 0 .0 9 2 .0 2 ± 0 .1 4 1 .7 9 ± 0 .0 9 2 .1 6 ± 0 .1 1 2 .0 5 ± 0 .1 5 z n 4 3 .4 9 ± 2 .0 7 3 9 .3 5 ± 2 .2 7 3 7 .2 7 ± 1 .7 8 3 3 .7 3 ± 1 .9 5 5 7 .7 6 ± 2 .7 5 5 0 .2 9 ± 2 .9 0 5 5 .5 8 ± 2 .6 5 4 3 .1 9 ± 2 .4 9 7 7 .5 4 ± 3 .7 0 5 7 .9 9 ± 3 .3 5 m n 4 .8 7 ± 0 .0 3 4 .3 1 ± 0 .0 8 5 .1 7 ± 0 .0 3 3 .9 2 ± 0 .0 7 6 .3 7 ± 0 .0 3 5 .0 9 ± 0 .0 9 7 .9 1 ± 0 .0 4 6 .0 0 ± 0 .1 1 8 .3 8 ± 0 .0 4 6 .3 6 ± 0 .1 2 n i 0 .6 1 ± 0 .0 3 0 .6 5 ± 0 .0 3 0 .6 5 ± 0 .0 3 0 .6 0 ± 0 .0 3 0 .8 0 ± 0 .0 4 0 .8 3 ± 0 .0 4 0 .8 5 ± 0 .0 4 0 .7 8 ± 0 .0 3 0 .9 2 ± 0 .0 4 0 .9 5 ± 0 .0 4 h g 0 .3 4 ± 0 .0 6 0 .2 8 ± 0 .0 8 0 .3 2 ± 0 .0 6 0 .1 9 ± 0 .0 5 0 .5 0 ± 0 .0 9 0 .3 4 ± 0 .0 9 0 .6 9 ± 0 .1 2 0 .3 3 ± 0 .0 9 0 .5 4 ± 0 .0 9 0 .4 2 ± 0 .1 1 labeo rohita c d 0 .1 2 ± 0 .0 2 0 .0 8 ± 0 .0 1 0 .1 ± 0 .0 0 0 .0 9 ± 0 .0 1 0 .1 2 ± 0 .0 0 0 .1 1 ± 0 .0 1 0 .1 1 ± 0 .0 0 0 .1 ± 0 .0 1 0 .1 5 ± 0 .0 0 0 .1 3 ± 0 .0 1 c r 2 .6 5 ± 0 .0 5 1 .8 9 ± 0 .0 3 3 .0 4 ± 0 .0 5 2 .1 3 ± 0 .0 4 3 .3 4 ± 0 .0 6 2 .4 6 ± 0 .0 3 3 .6 4 ± 0 .0 6 2 .6 4 ± 0 .0 4 4 .5 5 ± 0 .0 8 3 .2 2 ± 0 .0 4 c u 6 .0 5 ± 0 .0 6 4 .8 2 ± 0 .1 0 5 .6 2 ± 0 .2 1 5 .5 1 ± 0 .1 2 7 .3 1 ± 0 .0 7 5 .8 3 ± 0 .1 2 7 .9 4 ± 0 .0 8 7 .5 8 ± 0 .1 6 9 .5 8 ± 0 .0 9 9 .2 4 ± 0 .2 0 f e 4 6 .1 3 ± 2 .1 4 3 9 .8 4 ± 2 .7 3 4 1 .0 9 ± 1 .8 2 3 8 .1 1 ± 3 .2 3 5 1 .2 8 ± 2 .2 7 4 7 .0 7 ± 4 .1 4 6 8 .0 8 ± 4 .0 7 5 6 .6 1 ± 1 .9 3 6 8 .2 8 ± 4 .7 3 5 9 .1 9 ± 2 .3 2 p b 2 .2 8 ± 0 .1 3 1 .8 4 ± 0 .1 7 1 .9 1 ± 0 .1 1 1 .4 3 ± 0 .1 3 3 .0 0 ± 0 .1 8 1 .9 6 ± 0 .1 8 2 .1 1 ± 0 .1 2 1 .8 9 ± 0 .1 7 2 .2 8 ± 0 .2 1 2 .1 4 ± 0 .1 3 z n 4 2 .2 2 ± 1 .0 6 3 8 .3 4 ± 1 .1 6 3 6 .1 9 ± 0 .9 1 3 2 .8 6 ± 0 .9 9 5 6 .0 8 ± 1 .4 1 4 9 .0 0 ± 1 .4 8 5 3 .9 7 ± 1 .3 5 4 2 .0 8 ± 1 .2 7 7 5 .2 8 ± 1 .8 9 5 6 .5 0 ± 1 .7 1 m n 4 .8 8 ± 0 .0 4 5 .5 3 ± 0 .1 0 5 .1 8 ± 0 .0 4 5 .0 3 ± 0 .0 9 6 .3 8 ± 0 .0 6 6 .5 3 ± 0 .1 2 7 .9 3 ± 0 .0 7 7 .6 9 ± 0 .1 4 8 .4 0 ± 0 .0 7 8 .1 5 ± 0 .1 5 n i 0 .6 5 ± 0 .0 7 0 .6 4 ± 0 .0 7 0 .7 0 ± 0 .0 7 0 .6 0 ± 0 .0 7 0 .8 5 ± 0 .0 9 0 .8 3 ± 0 .0 9 0 .9 1 ± 0 .1 0 0 .7 8 ± 0 .0 9 0 .9 8 ± 0 .1 0 0 .9 4 ± 0 .1 0 h g 0 .3 1 ± 0 .0 2 0 .2 6 ± 0 .0 3 0 .2 9 ± 0 .0 2 0 .1 8 ± 0 .0 2 0 .4 6 ± 0 .0 3 0 .3 1 ± 0 .0 3 0 .6 2 ± 0 .0 4 0 .3 0 ± 0 .0 3 0 .4 9 ± 0 .0 3 0 .3 9 ± 0 .0 4 catla catla c d 0 .0 9 ± 0 .0 1 0 .0 7 ± 0 .0 1 0 .1 2 ± 0 .0 1 0 .0 8 ± 0 .0 1 0 .1 3 ± 0 .0 2 0 .0 9 ± 0 .0 1 0 .1 6 ± 0 .0 2 0 .1 2 ± 0 .0 3 0 .2 1 ± 0 .0 3 0 .1 5 ± 0 .0 3 c r 2 .5 7 ± 0 .0 5 2 .1 9 ± 0 .0 7 2 .9 5 ± 0 .0 5 2 .4 6 ± 0 .0 8 3 .2 4 ± 0 .0 6 2 .8 4 ± 0 .0 9 3 .5 3 ± 0 .0 6 3 .0 5 ± 0 .1 0 4 .4 1 ± 0 .0 8 3 .7 3 ± 0 .1 2 c u 6 .0 5 ± 0 .0 5 4 .8 5 ± 0 .0 5 5 .7 2 ± 0 .2 0 5 .5 4 ± 0 .0 6 7 .3 1 ± 0 .5 1 5 .8 6 ± 0 .5 0 7 .9 4 ± 0 .5 1 7 .6 2 ± 0 .4 7 9 .5 8 ± 0 .4 6 9 .2 8 ± 0 .5 5 f e 4 7 .6 ± 3 .7 6 3 9 .5 6 ± 1 .7 3 5 2 .7 6 ± 1 .5 9 4 4 .2 7 ± 1 .9 4 5 8 .3 7 ± 2 .2 1 5 3 .2 5 ± 2 .7 1 6 2 .0 3 ± 4 .5 9 5 0 .4 7 ± 4 .4 1 7 5 .6 5 ± 4 .3 3 6 3 .2 5 ± 5 .8 8 p b 2 .2 8 ± 0 .2 0 1 .7 3 ± 0 .2 2 1 .9 1 ± 0 .1 7 1 .3 5 ± 0 .1 7 3 .0 1 ± 0 .2 7 1 .8 4 ± 0 .2 3 2 .1 1 ± 0 .1 9 1 .7 8 ± 0 .2 2 2 .1 4 ± 0 .1 9 2 .1 4 ± 0 .2 7 z n 4 3 .9 1 ± 1 .6 0 3 5 .9 7 ± 1 .4 5 3 7 .6 4 ± 1 .3 7 3 0 .8 3 ± 1 .2 4 5 8 .3 3 ± 2 .1 2 4 5 .9 7 ± 1 .8 5 5 6 .1 3 ± 2 .0 4 3 9 .4 8 ± 1 .5 9 7 2 .3 1 ± 2 .6 3 5 3 .0 0 ± 2 .1 3 m n 3 .8 5 ± 0 .0 4 4 .0 6 ± 0 .0 9 4 .0 9 ± 0 .0 5 3 .6 9 ± 0 .0 8 5 .0 3 ± 0 .0 6 4 .7 9 ± 0 .1 0 6 .2 5 ± 0 .0 7 5 .6 5 ± 0 .1 2 6 .6 2 ± 0 .0 7 5 .9 8 ± 0 .1 3 n i 0 .7 9 ± 0 .0 2 0 .6 8 ± 0 .0 2 0 .8 5 ± 0 .0 2 0 .6 4 ± 0 .0 2 1 .0 3 ± 0 .0 3 0 .8 8 ± 0 .0 3 1 .1 0 ± 0 .0 3 0 .8 2 ± 0 .0 3 1 .1 9 ± 0 .0 3 1 .0 0 ± 0 .0 3 h g 0 .3 0 ± 0 .0 3 0 .3 0 ± 0 .0 3 0 .2 8 ± 0 .0 2 0 .2 0 ± 0 .0 2 0 .4 4 ± 0 .0 4 0 .3 5 ± 0 .0 3 0 .6 0 ± 0 .0 5 0 .3 4 ± 0 .0 3 0 .4 7 ± 0 .0 4 0 .4 4 ± 0 .0 4 t ab le 2 . m ea n s ± s d ( µ g /g d ry w ei g h t) o f m et al s co n ce n tr at io n s in d if fe re n t o rg a n s o f th e sa m p le d f is h s p e ci es s am p le d f ro m s it e b ( s h ah d er a) d u ri n g l o w a n d h ig h f lo w s ea so n . 1 4 0 in te rn at io n al j o u rn al o f a q u at ic b io lo g y ( 2 0 1 5 ) 3 (3 ): 1 3 5 -1 4 8 f is h s p e c ie s m e ta ls e y e s g il ls h e a r t in te st in e k id n e y s l o w h ig h l o w h ig h l o w h ig h l o w h ig h l o w h ig h cirrhinus mrigala c d 0 .3 6 ± 0 .0 3 0 .2 ± 0 .0 1 8 0 .2 9 ± 0 .0 3 0 .1 8 ± 0 .0 2 0 .3 8 ± 0 .0 4 0 .2 5 ± 0 .0 2 0 .4 ± 0 .0 4 0 .2 8 ± 0 .0 3 0 .4 9 ± 0 .0 5 0 .3 4 ± 0 .0 3 c r 7 .6 7 ± 0 .1 6 3 .2 8 ± 0 .1 6 6 .8 6 ± 0 .1 4 3 .9 1 ± 0 .4 7 8 .6 2 ± 0 .1 8 5 .6 4 ± 0 .1 7 1 0 ± 0 .2 1 4 .2 7 ± 0 .2 0 1 0 .3 8 ± 0 .2 2 4 .9 5 ± 0 .2 3 c u 7 .4 6 ± 0 .0 5 5 .8 2 ± 0 .0 9 7 .9 9 ± 0 .6 3 6 .6 7 ± 0 .1 1 8 .4 1 ± 0 .4 9 7 .7 ± 0 .1 2 1 2 .7 9 ± 0 .0 8 1 1 .0 3 ± 0 .1 8 1 1 .8 5 ± 0 .0 7 9 .6 4 ± 0 .9 0 f e 7 3 .1 3 ± 2 .1 6 6 1 .4 4 ± 2 .0 6 6 6 .2 4 ± 1 .9 5 5 4 .8 ± 2 .7 2 7 0 .2 2 ± 2 .9 7 6 4 .2 5 ± 2 .1 5 8 5 .6 ± 2 .5 3 7 7 .5 ± 2 .5 9 9 7 .4 3 ± 5 .0 6 8 0 .3 2 ± 2 .6 9 p b 4 .0 8 ± 0 .0 7 3 .9 7 ± 0 .2 1 4 .7 1 ± 0 .0 8 3 .5 9 ± 0 .1 9 4 .9 2 ± 0 .2 6 4 .4 3 ± 0 .0 7 6 .2 3 ± 0 .1 0 5 .1 1 ± 0 .2 7 6 .2 8 ± 0 .4 0 5 .9 4 ± 0 .3 1 z n 8 2 .0 4 ± 8 .0 5 5 6 .2 7 ± 2 .6 5 7 4 .6 3 ± 7 .3 2 5 4 .2 6 ± 2 .5 6 9 6 .8 4 ± 9 .5 0 6 7 .9 0 ± 3 .2 0 1 0 4 .8 6 ± 1 0 .2 9 6 9 .3 5 ± 3 .2 7 1 2 0 .8 9 ± 1 1 .8 6 7 9 .9 6 ± 3 .7 7 m n 2 1 .6 7 ± 1 .3 2 1 0 .9 6 ± 1 .7 3 1 7 .5 8 ± 1 .0 7 9 .9 7 ± 1 .5 8 2 6 .9 8 ± 1 .6 4 1 2 .9 3 ± 2 .0 5 2 2 .7 3 ± 1 .3 9 1 5 .2 4 ± 2 .4 1 2 8 .4 9 ± 1 .7 4 1 6 .1 5 ± 2 .5 5 n i 3 .7 8 ± 0 .0 3 2 .8 9 ± 0 .1 4 4 .0 9 ± 0 .0 3 2 .6 5 ± 0 .1 3 5 .1 1 ± 0 .0 3 3 .2 8 ± 0 .1 6 5 .3 6 ± 0 .0 4 3 .5 1 ± 0 .1 7 5 .8 8 ± 0 .0 4 4 .2 9 ± 0 .2 1 h g 3 .7 7 ± 0 .1 4 2 .7 8 ± 0 .1 1 1 .8 3 ± 0 .0 7 1 .2 8 ± 0 .0 5 4 .0 7 ± 0 .1 5 3 .0 0 ± 0 .1 2 3 .9 5 ± 0 .1 5 3 .0 0 ± 0 .1 2 5 .7 0 ± 0 .2 2 4 .1 4 ± 0 .1 6 labeo rohita c d 0 .2 6 ± 0 .0 0 0 .1 9 ± 0 .0 1 0 .2 1 ± 0 .0 0 0 .1 7 ± 0 .0 1 0 .2 7 ± 0 .0 0 0 .2 4 ± 0 .0 1 0 .2 9 ± 0 .0 0 0 .2 ± 0 .0 6 0 .3 8 ± 0 .0 4 0 .2 9 ± 0 .0 5 c r 6 .6 3 ± 0 .6 5 3 .3 7 ± 0 .1 9 4 .1 3 ± 0 .1 4 2 .9 9 ± 0 .1 7 5 .1 9 ± 0 .1 8 3 .7 5 ± 0 .2 2 6 .0 3 ± 0 .2 1 4 .4 ± 0 .2 5 6 .2 6 ± 0 .2 1 5 .1 ± 0 .2 9 c u 7 .0 8 ± 0 .0 4 5 .8 7 ± 0 .1 2 6 .9 3 ± 0 .4 6 6 .7 2 ± 0 .1 4 8 .0 3 ± 0 .8 8 7 .7 7 ± 0 .1 6 1 2 .1 3 ± 0 .0 7 1 1 .1 3 ± 0 .2 2 1 1 .2 4 ± 0 .0 7 8 .7 1 ± 0 .1 8 f e 6 5 .8 ± 2 .4 2 5 6 .4 5 ± 0 .8 8 5 9 .6 1 ± 2 .1 9 4 7 .6 ± 0 .7 4 6 4 .1 9 ± 6 .0 8 5 9 .0 3 ± 0 .9 2 7 7 .0 3 ± 2 .8 3 7 1 .2 1 ± 1 .1 1 7 9 .6 7 ± 4 .5 4 7 3 .7 9 ± 1 .1 5 p b 4 .0 0 ± 0 .2 3 3 .3 7 ± 0 .2 1 4 .6 2 ± 0 .2 7 3 .0 5 ± 0 .1 9 4 .3 4 ± 0 .2 5 4 .1 8 ± 0 .2 6 6 .1 1 ± 0 .3 5 4 .3 4 ± 0 .2 7 5 .9 2 ± 0 .4 0 5 .0 4 ± 0 .3 2 z n 6 2 .6 1 ± 1 .4 5 5 9 .7 4 ± 1 .2 1 5 6 .9 6 ± 1 .3 2 5 7 .6 0 ± 1 .1 6 7 3 .9 1 ± 1 .7 2 7 2 .0 8 ± 1 .4 6 8 0 .0 3 ± 1 .8 6 7 3 .6 2 ± 1 .4 9 9 2 .2 7 ± 2 .1 4 8 4 .8 8 ± 1 .7 2 m n 1 7 .7 0 ± 1 .1 5 1 2 .0 3 ± 1 .3 5 1 4 .3 6 ± 0 .9 3 1 0 .9 5 ± 1 .2 3 2 2 .0 3 ± 1 .4 3 1 4 .2 0 ± 1 .5 9 1 8 .5 6 ± 1 .2 0 1 6 .7 3 ± 1 .8 8 2 3 .2 7 ± 1 .5 1 1 7 .7 3 ± 1 .9 9 n i 3 .1 8 ± 0 .1 0 2 .1 3 ± 0 .1 4 3 .4 4 ± 0 .1 1 1 .9 6 ± 0 .1 3 4 .3 0 ± 0 .1 3 2 .4 2 ± 0 .1 6 4 .5 1 ± 0 .1 4 2 .5 9 ± 0 .1 7 4 .9 5 ± 0 .1 5 3 .1 7 ± 0 .2 1 h g 3 .5 6 ± 0 .1 0 2 .7 3 ± 0 .1 1 1 .7 3 ± 0 .0 5 1 .2 6 ± 0 .0 5 3 .8 5 ± 0 .1 0 2 .9 4 ± 0 .1 2 3 .7 4 ± 0 .1 0 2 .9 4 ± 0 .1 2 5 .3 9 ± 0 .1 4 4 .0 6 ± 0 .1 4 catla catla c d 0 .3 3 ± 0 .0 3 0 .2 3 ± 0 .0 4 0 .2 9 ± 0 .0 2 0 .1 8 ± 0 .0 2 0 .3 5 ± 0 .0 3 0 .2 5 ± 0 .0 3 0 .3 7 ± 0 .0 3 0 .2 7 ± 0 .0 3 0 .5 5 ± 0 .0 4 0 .3 9 ± 0 .0 4 c r 6 .5 4 ± 0 .0 7 3 .6 8 ± 0 .1 4 5 .9 2 ± 0 .0 7 3 .2 6 ± 0 .1 2 7 .2 2 ± 0 .0 8 4 .0 9 ± 0 .1 6 8 .4 7 ± 0 .8 2 4 .7 9 ± 0 .1 8 8 .8 2 ± 0 .1 0 5 .5 6 ± 0 .2 1 c u 7 .9 9 ± 0 .5 0 6 .0 1 ± 0 .2 3 7 .4 9 ± 0 .5 7 6 .8 9 ± 0 .2 7 8 .1 3 ± 0 .4 2 7 .9 6 ± 0 .3 1 1 3 .6 9 ± 1 .0 1 1 .4 ± 0 .6 4 1 2 .6 8 ± 1 .0 0 1 0 .9 3 ± 1 .3 4 f e 8 3 .5 6 ± 2 .2 1 6 6 .7 8 ± 2 .7 2 7 5 .6 9 ± 2 .0 2 5 6 .3 1 ± 2 .3 0 7 8 .8 1 ± 1 .8 2 6 9 .8 4 ± 2 .8 4 9 7 .8 1 ± 2 .5 9 8 4 .2 4 ± 3 .4 3 8 8 .4 7 ± 2 .3 4 8 7 .3 ± 3 .5 5 p b 4 .3 6 ± 0 .2 3 4 .2 3 ± 0 .4 3 4 .8 9 ± 0 .2 6 3 .8 3 ± 0 .3 9 5 .2 5 ± 0 .5 3 4 .5 9 ± 0 .2 1 5 .4 6 ± 0 .3 0 5 .4 5 ± 0 .5 5 6 .4 3 ± 0 .3 1 6 .3 3 ± 0 .6 4 z n 6 5 .5 6 ± 1 .6 1 6 1 .6 7 ± 1 .5 3 5 9 .6 5 ± 1 .4 6 5 9 .4 6 ± 1 .4 7 7 7 .3 9 ± 1 .9 0 7 4 .4 1 ± 1 .8 5 8 3 .8 0 ± 2 .0 5 7 6 .0 0 ± 1 .8 9 9 6 .6 2 ± 2 .3 7 8 7 .6 2 ± 2 .1 7 m n 1 6 .4 8 ± 1 .3 7 1 1 .7 3 ± 0 .9 3 1 3 .3 7 ± 1 .1 1 1 0 .6 7 ± 0 .8 5 2 0 .5 1 ± 1 .7 0 1 3 .8 4 ± 1 .1 0 1 7 .2 8 ± 1 .4 4 1 6 .3 1 ± 1 .3 0 2 1 .6 6 ± 1 .8 0 1 7 .2 8 ± 1 .3 8 n i 2 .1 9 ± 0 .0 4 2 .6 2 ± 0 .0 7 2 .3 7 ± 0 .0 5 2 .4 6 ± 0 .0 6 2 .9 6 ± 0 .0 6 3 .1 3 ± 0 .0 8 3 .1 0 ± 0 .0 6 3 .2 6 ± 0 .0 9 3 .4 1 ± 0 .0 7 3 .9 7 ± 0 .1 0 h g 3 .6 7 ± 0 .1 1 2 .8 1 ± 0 .0 6 1 .7 9 ± 0 .0 5 1 .3 0 ± 0 .0 3 3 .9 7 ± 0 .1 2 3 .0 3 ± 0 .0 6 3 .8 5 ± 0 .1 2 3 .0 3 ± 0 .0 6 5 .5 5 ± 0 .1 7 4 .1 8 ± 0 .0 9 t ab le 3 . m ea n s ± s d ( µ g /g d ry w ei g h t) o f m et al s co n ce n tr at io n s in d if fe re n t o rg a n s o f th e sa m p le d f is h s p e ci es s am p le d f ro m s it e c ( s u n d er ) d u ri n g l o w a n d h ig h f lo w s ea so n . 1 4 1 s h ak ir e t al / m et al b io ac cu m u la ti o n l ev el s in r iv er r av i f is h s p e c ie s m e ta ls e y e s g il ls h e a r t in te st in e k id n e y s l o w h ig h l o w h ig h l o w h ig h l o w h ig h l o w h ig h cirrhinus mrigala c d 0 .2 4 ± 0 .0 3 0 .1 3 ± 0 .0 3 0 .1 7 ± 0 .0 3 0 .1 3 ± 0 .0 3 0 .2 4 ± 0 .0 3 0 .1 6 ± 0 .0 2 0 .3 1 ± 0 .0 3 0 .2 ± 0 .0 3 0 .3 6 ± 0 .0 5 0 .2 5 ± 0 .0 4 c r 4 .0 5 ± 0 .6 1 2 .7 5 ± 0 .0 9 4 .3 6 ± 0 .1 7 2 .8 5 ± 0 .3 4 5 .3 0 ± 0 .3 5 3 .1 6 ± 0 .4 8 6 .4 6 ± 0 .2 6 3 .0 3 ± 3 .0 3 5 .9 5 ± 0 .5 6 3 .4 3 ± 0 .1 3 c u 5 .5 7 ± 0 .6 8 4 .9 5 ± 0 .8 7 6 .5 1 ± 0 .7 4 5 .7 4 ± 0 .6 9 8 .0 2 ± 0 .4 7 6 .4 9 ± 0 .1 4 6 .9 3 ± 0 .7 0 6 .3 2 ± 0 .7 3 9 .3 2 ± 0 .7 4 8 .6 6 ± 1 .0 8 f e 5 9 .4 6 ± 3 .2 4 0 .5 5 ± 3 .1 4 5 6 .7 5 ± 4 .5 8 4 4 .4 ± 5 .3 2 6 7 .9 ± 3 .6 9 5 1 .4 8 ± 5 .8 2 7 0 .6 ± 4 .6 7 5 2 .1 3 ± 3 .0 2 8 3 .4 1 ± 5 .1 5 7 6 .0 2 ± 4 .4 2 p b 3 .4 0 ± 0 .1 8 2 .0 2 ± 0 .0 9 2 .7 0 ± 0 .1 5 1 .6 8 ± 0 .0 8 2 .8 6 ± 0 .1 3 2 .8 0 ± 0 .1 5 3 .8 4 ± 0 .2 1 2 .6 0 ± 0 .1 2 4 .0 6 ± 0 .2 2 3 .0 2 ± 0 .1 4 z n 5 7 .3 0 ± 2 .3 2 3 9 .4 6 ± 2 .4 1 4 8 .5 4 ± 1 .9 7 4 3 .3 8 ± 2 .6 5 6 9 .3 3 ± 2 .8 1 6 0 .3 4 ± 3 .6 8 6 7 .5 2 ± 2 .7 3 4 8 .6 0 ± 2 .9 7 7 1 .5 3 ± 2 .9 0 6 3 .9 2 ± 3 .9 0 m n 6 .4 2 ± 1 .1 4 6 .3 2 ± 0 .9 6 6 .0 5 ± 1 .0 8 5 .7 5 ± 0 .8 7 9 .8 2 ± 1 .7 5 7 .4 6 ± 1 .1 3 8 .3 3 ± 1 .4 8 8 .0 7 ± 1 .2 3 1 0 .4 0 ± 1 .8 5 9 .3 1 ± 1 .4 1 n i 1 .3 9 ± 0 .0 5 1 .1 4 ± 0 .0 4 1 .2 8 ± 0 .0 4 0 .9 7 ± 0 .0 4 1 .4 1 ± 0 .0 1 1 .4 3 ± 0 .0 5 1 .5 2 ± 0 .0 5 1 .4 7 ± 0 .0 5 1 .7 4 ± 0 .0 5 8 1 .6 8 ± 0 .0 6 h g 1 .9 4 ± 0 .0 7 1 .5 4 ± 0 .0 8 1 .3 9 ± 0 .0 5 1 .1 1 ± 0 .0 6 3 .2 5 ± 0 .1 2 2 .5 8 ± 0 .1 3 4 .0 3 ± 0 .1 5 3 .2 0 ± 0 .1 6 3 .2 5 ± 0 .1 2 2 .5 8 ± 0 .1 3 labeo rohita c d 0 .1 9 ± 0 .0 8 0 .1 2 ± 0 .0 4 0 .1 5 ± 0 .0 3 0 .0 9 ± 0 .0 2 0 .1 8 ± 0 .0 3 0 .1 2 ± 0 .0 2 0 .2 3 ± 0 .0 5 0 .1 6 ± 0 .0 3 0 .2 7 ± 0 .0 7 0 .2 1 ± 0 .0 5 c r 3 .6 9 ± 0 .4 8 2 .5 5 ± 0 .6 7 3 .3 4 ± 0 .5 2 2 .4 7 ± 0 .4 7 3 .8 9 ± 0 .4 0 2 .6 5 ± 0 .2 0 3 .0 3 ± 0 .1 5 2 .9 9 ± 0 .3 9 5 .4 3 ± 0 .4 9 3 .4 6 ± 0 .4 3 c u 6 .7 3 ± 0 .5 4 5 .3 3 ± 0 .4 0 5 .8 7 ± 0 .3 0 5 .7 2 ± 0 .4 0 7 .8 5 ± 0 .3 6 6 .4 7 ± 0 .2 6 8 .4 3 ± 0 .4 4 7 .8 ± 0 .4 7 1 0 .5 3 ± 0 .3 5 9 .6 4 ± 0 .5 0 f e 5 1 .4 2 ± 3 .7 3 4 7 .2 5 ± 3 .4 8 4 3 .3 5 ± 4 .0 3 3 9 .0 7 ± 4 .1 3 5 5 .8 8 ± 5 .8 7 5 1 .7 9 ± 4 .9 7 6 4 .1 4 ± 6 .2 6 6 0 .4 6 ± 4 .8 3 7 8 .7 9 ± 4 .6 4 7 0 .2 7 ± 4 .8 6 p b 3 .3 9 ± 0 .1 9 2 .5 5 ± 0 .1 8 2 .6 9 ± 0 .1 5 2 .1 2 ± 0 .1 5 3 .6 1 ± 0 .2 5 2 .7 9 ± 0 .1 5 3 .8 3 ± 0 .2 2 3 .2 9 ± 0 .2 3 4 .0 5 ± 0 .2 4 3 .8 2 ± 0 .2 7 z n 6 8 .8 4 ± 1 .7 7 4 2 .5 3 ± 1 .6 8 5 8 .3 1 ± 1 .5 0 4 6 .7 5 ± 1 .8 5 8 3 .2 9 ± 2 .1 4 6 5 .0 2 ± 2 .5 7 8 1 .1 1 ± 2 .0 8 5 2 .3 7 ± 2 .0 7 8 5 .9 4 ± 2 .2 0 6 8 .8 9 ± 2 .7 2 m n 6 .4 1 ± 0 .1 2 6 .9 4 ± 0 .1 1 6 .0 3 ± 0 .1 1 6 .3 1 ± 0 .1 0 9 .7 9 ± 0 .1 8 8 .1 9 ± 0 .1 4 8 .3 1 ± 0 .1 5 8 .8 7 ± 0 .1 5 1 0 .3 8 ± 0 .1 9 1 0 .2 3 ± 0 .1 7 n i 1 .2 7 ± 0 .0 8 0 .9 2 ± 0 .4 1 1 .1 7 ± 0 .0 7 0 .7 8 ± 0 .3 4 1 .2 8 ± 0 .0 8 1 .1 5 ± 0 .5 1 1 .3 9 ± 0 .0 8 1 .1 9 ± 0 .5 2 1 .5 8 ± 0 .1 0 1 .3 5 ± 0 .5 9 9 h g 1 .9 6 ± 0 .0 3 1 .5 1 ± 0 .0 4 1 .4 1 ± 0 .0 2 1 .0 9 ± 0 .0 3 3 .2 9 ± 0 .0 5 2 .5 4 ± 0 .0 6 4 .0 7 ± 0 .0 6 3 .1 5 ± 0 .0 7 3 .2 9 ± 0 .0 5 2 .5 4 ± 0 .0 6 catla catla c d 0 .2 8 ± 0 .0 4 0 .1 9 ± 0 .0 3 0 .1 3 ± 0 .0 3 0 .1 ± 0 .0 2 0 .2 2 ± 0 .0 3 0 .1 7 ± 0 .0 3 0 .3 4 ± 0 .0 4 0 .2 6 ± 0 .0 3 0 .2 9 ± 0 .0 3 0 .2 4 ± 0 .0 3 c r 3 .7 7 ± 0 .3 6 2 .5 3 ± 0 .3 4 3 .0 4 ± 0 .5 2 2 .7 5 ± 0 .4 4 3 .9 8 ± 0 .5 9 3 .6 3 ± 0 .5 2 3 .1 3 ± 0 .4 4 2 .9 6 ± 0 .0 9 3 .5 5 ± 0 .3 9 3 .1 3 ± 0 .3 5 c u 6 .3 5 ± 0 .4 5 5 .0 6 ± 0 .5 5 6 .2 ± 0 .6 8 5 .8 1 ± 0 .3 4 7 .3 3 ± 0 .1 3 6 .0 6 ± 0 .1 0 7 .6 6 ± 0 .4 5 6 .8 9 ± 0 .4 9 9 .0 3 ± 0 .8 6 8 .7 2 ± 0 .5 3 f e 6 6 .3 5 ± 2 .9 2 6 0 .6 9 ± 2 .8 8 5 8 .5 3 ± 5 .3 0 5 3 .6 1 ± 2 .5 4 7 6 .1 2 ± 3 .6 3 7 0 .8 ± 3 .3 6 6 5 .5 6 ± 3 .9 4 6 3 .3 8 ± 0 .0 1 8 4 .5 ± 3 .0 1 7 8 .5 5 ± 3 .7 3 p b 3 .4 9 ± 0 .2 4 2 .2 4 ± 0 .5 6 2 .7 7 ± 0 .1 9 1 .8 6 ± 0 .4 7 3 .1 7 ± 0 .8 0 2 .8 7 ± 0 .2 0 3 .9 4 ± 0 .2 7 2 .8 8 ± 0 .7 2 4 .1 7 ± 0 .2 8 3 .3 5 ± 0 .8 4 z n 5 8 .2 1 ± 1 .9 9 4 2 .6 8 ± 1 .1 4 4 9 .3 1 ± 1 .6 9 4 6 .9 2 ± 1 .2 5 7 0 .4 3 ± 2 .4 1 6 5 .2 6 ± 1 .7 4 6 8 .5 9 ± 2 .3 5 5 2 .5 6 ± 1 .4 0 7 2 .6 6 ± 2 .4 9 6 9 .1 4 ± 1 .8 4 m n 6 .3 0 ± 0 .0 3 5 .4 7 ± 0 .7 4 5 .9 4 ± 0 .0 3 4 .9 8 ± 0 .6 7 9 .6 4 ± 0 .0 4 6 .4 6 ± 0 .8 7 8 .1 8 ± 0 .0 3 8 7 .0 0 ± 0 .9 5 1 0 .2 1 ± 0 .0 5 8 .0 7 ± 1 .0 9 n i 1 .1 5 ± 0 .0 3 1 .1 1 ± 0 .0 3 1 .0 6 ± 0 .0 3 0 .9 4 ± 0 .0 2 1 .1 6 ± 0 .0 3 1 .3 8 ± 0 .0 3 1 .2 6 ± 0 .0 3 3 1 .4 3 ± 0 .0 3 1 .4 3 ± 0 .0 4 1 .6 3 ± 0 .0 4 h g 1 .8 7 ± 0 .0 2 1 .5 5 ± 0 .0 4 1 .3 5 ± 0 .0 2 1 .1 2 ± 0 .0 3 3 .1 4 ± 0 .0 4 2 .6 1 ± 0 .0 7 3 .8 9 ± 0 .0 4 9 3 .2 3 ± 0 .0 8 3 .1 4 ± 0 .0 4 2 .6 1 ± 0 .0 7 t ab le 4 . m ea n s ± s d ( µ g /g d ry w ei g h t) o f m et al s co n ce n tr at io n s in d if fe re n t o rg a n s o f th e sa m p le d f is h s p e ci es s am p le d f ro m s it e c ( s u n d er ) d u ri n g l o w a n d h ig h f lo w s ea so n . 1 4 2 in te rn at io n al j o u rn al o f a q u at ic b io lo g y ( 2 0 1 5 ) 3 (3 ): 1 3 5 -1 4 8 m e ta ls c d c r c u f e p b z n m n n i h g s a m p li n g s it e s s it e a : s ip h o n ( c o n tr o l) 0 .0 7 1 .5 3 5 .4 4 4 3 .5 3 0 .3 5 3 3 .4 1 3 .5 0 0 .5 6 0 .1 5 s it e b : s h a h d e ra 0 .1 2 2 .9 8 7 .0 7 5 3 .1 5 2 .0 3 4 8 .4 1 5 .8 0 0 .8 2 0 .3 8 s it e c : s u n d e r 0 .2 9 5 .7 3 8 .9 4 7 2 .4 7 4 .8 3 7 5 .4 3 1 6 .9 8 3 .4 3 3 .3 0 s it e d : h e a d b a ll o k i 0 .2 1 3 .5 8 7 .0 7 6 1 .4 7 3 .0 6 6 0 .6 2 7 .7 2 1 .2 9 2 .4 7 s e m 0 .0 0 2 0 .0 1 5 0 .0 2 1 0 .1 9 5 0 .0 1 5 0 .1 7 8 0 .0 5 4 0 .0 0 7 0 .0 0 5 f lo w s e a so n s h ig h 0 .1 4 2 .7 8 6 .6 8 5 3 .8 4 2 .3 1 4 9 .1 8 7 .4 0 1 .3 7 1 .3 7 l o w 0 .2 0 4 .1 3 7 .5 8 6 1 .4 7 2 .8 3 5 9 .7 6 9 .6 0 1 .6 8 1 .7 8 s e m 0 .0 0 1 0 .0 1 1 0 .0 1 5 0 .1 3 8 0 .0 1 1 0 .1 2 6 0 .0 3 8 0 .0 0 5 0 .0 0 3 f is h s p e c ie s c ir rh in u s m ri g a la 0 .1 8 3 .7 7 7 .1 4 5 6 .6 7 2 .5 3 5 6 .2 2 8 .9 5 1 .7 0 1 .6 0 l a b e o r o h it a 0 .1 5 3 .1 6 7 .0 6 5 4 .1 8 2 .5 4 5 4 .4 9 8 .7 3 1 .4 6 1 .5 5 c a tl a c a tl a 0 .1 8 3 .4 4 7 .1 9 6 2 .1 1 2 .6 4 5 2 .6 9 7 .8 2 1 .4 1 1 .5 7 s e m 0 .0 0 1 0 .0 1 3 0 .0 1 8 0 .1 6 9 0 .0 1 3 0 .1 5 4 0 .0 4 7 0 .0 0 6 0 .0 0 4 f is h e s o r g a n s e y e s 0 .1 5 3 .0 4 5 .6 8 5 0 .1 0 2 .2 9 4 6 .4 0 7 .2 5 1 .2 8 1 .3 4 g il ls 0 .1 3 2 .9 1 5 .7 0 4 5 .5 7 2 .0 7 4 2 .4 1 6 .4 8 1 .2 4 0 .7 9 h e a rt 0 .1 6 3 .5 2 6 .9 0 5 6 .2 7 2 .6 3 5 8 .2 7 9 .0 5 1 .5 7 1 .7 3 in te st in e 0 .1 9 3 .6 4 8 .3 0 6 4 .1 2 2 .8 0 5 7 .1 1 9 .1 7 1 .6 5 1 .9 2 k id n e y s 0 .2 3 4 .1 6 9 .0 6 7 2 .2 2 3 .0 7 6 8 .1 6 1 0 .5 5 1 .8 8 2 .0 9 s e m 0 .0 0 2 0 .0 1 7 0 .0 2 4 0 .2 1 8 0 .0 1 7 0 .1 9 9 0 .0 6 0 0 .0 0 8 0 .0 0 5 t ab le 5 . m ea n s o f m et al s’ c o n c en tr at io n s (µ g /g d ry w ei g h t) f o r th e sa m p li n g s it es , fl o w s e as o n s, f is h s p ec ie s an d f is h es ’ o rg a n s w it h s ta n d ar d e rr o r o f m e an s (s e m ). t ab le 6 . m ea n s o f m et al s’ c o n c en tr at io n s (µ g /g d ry w ei g h t) f o r th e sa m p li n g s it es , fl o w s e as o n s, f is h s p ec ie s an d f is h es ’ o rg a n s w it h s ta n d ar d e rr o r o f m e an s (s e m ). m e ta ls s a m p li n g s it e s (d f = 3 ) f lo w s e a so n s (d f = 1 ) f is h s p e c ie s (d f = 2 ) f is h o r g a n s (d f = 4 ) s it e x s e a so n x s p e c ie s x ti ss u e s (d f = 2 4 ) f v a lu e s ig n if ic a n c e le v e l f v a lu e s ig n if ic a n c e le v e l f v a lu e s ig n if ic a n c e le v e l f v a lu e s ig n if ic a n c e le v e l f v a lu e s ig n if ic a n c e le v e l c d 3 7 6 1 p < 0 .0 0 1 1 4 8 1 p < 0 .0 0 1 2 3 8 p < 0 .0 0 1 4 8 3 p < 0 .0 0 1 2 .9 1 p < 0 .0 0 1 c r 1 2 8 3 9 p < 0 .0 0 1 7 7 6 5 p < 0 .0 0 1 5 3 6 p < 0 .0 0 1 8 4 6 p < 0 .0 0 1 2 2 .3 p < 0 .0 0 1 c u 4 5 2 7 p < 0 .0 0 1 1 8 0 7 p < 0 .0 0 1 1 3 .5 p < 0 .0 0 1 4 1 2 1 p < 0 .0 0 1 2 .5 p < 0 .0 0 1 f e 3 9 7 0 p < 0 .0 0 1 1 5 2 9 p < 0 .0 0 1 5 7 5 .8 p < 0 .0 0 1 2 4 0 7 p < 0 .0 0 1 9 .1 p < 0 .0 0 1 p b 1 5 6 2 4 p < 0 .0 0 1 1 1 7 7 p < 0 .0 0 1 2 1 .3 p < 0 .0 0 1 5 5 5 p < 0 .0 0 1 6 .3 p < 0 .0 0 1 z n 1 0 0 5 6 p < 0 .0 0 1 3 5 3 0 p < 0 .0 0 1 1 3 2 p < 0 .0 0 1 2 6 4 3 p < 0 .0 0 1 1 .3 p > 0 .0 5 m n 1 2 0 8 2 p < 0 .0 0 1 1 6 6 9 p < 0 .0 0 1 1 6 6 p < 0 .0 0 1 7 3 6 p < 0 .0 0 1 0 .7 1 p > 0 .0 5 n i 3 1 0 1 8 p < 0 .0 0 1 1 7 8 5 p < 0 .0 0 1 5 5 6 p < 0 .0 0 1 1 0 2 8 p < 0 .0 0 1 3 .5 p < 0 .0 0 1 h g 1 1 2 6 8 4 p < 0 .0 0 1 7 6 3 7 p < 0 .0 0 1 3 5 .7 p < 0 .0 0 1 1 0 1 4 9 p < 0 .0 0 1 0 .3 7 p > 0 .0 5 d f = d e g re e o f fr e e d o m 143 shakir et al/ metal bioaccumulation levels in river ravi stine and kidneys of each fish species are presented in tables 1-4. mean metals accumulation appeared to be the highest for site c and lowest for the site a whereas the sites b and d had higher metal contents than the upstream site a. the trend of the metal concentrations appeared to be significantly higher during the low than the high flow season. highest contents of zn followed by fe, mn, cu, cr, pb, ni, hg and cd appeared for the studied fish organs (table 5). metals bioaccumulation was significantly different (p<0.001) among sampling sites, flow seasons and fish organs (table 6). cadmium (cd): highest mean cd bioaccumulation was found at site c followed by the sites d, b and a, respectively. the cd contents of the fish organs were found higher during low than the high flow periods of the river (table 2). among the fish species, lowest cd bioaccumulation was recorded in l. rohita than c. catla and c. mrigala. mean cd accumulations pattern in the fish organs were in the order of: kidneys > intestine > heart > eyes > gills (table 5). the highest cd concentration of 0.55 ± 0.039 µg/g was found in kidneys of c. catla from site c during low flow season (table 3) whereas the eyes of c. mrigala sampled from site a during high flow season showed the lowest cd accumulation of 0.03 ± 0.003 µg/g (table 1). chromium (cr): highest mean chromium bioaccumulation was recorded at site c than the sites d, b and a (table 4). effects of seasons appeared more during the low flow than high-flow season. the highest chromium accumulation was recorded in the organs of c. mrigala than c. catla and l. rohita. the accumulation pattern in fish organs was in the order of: kidneys > intestine > heart > eyes > gills (table 5). the cr accumulation ranged from 1.4 ± 0.012 µg/g to 10.38 ± 0.216 µg/g in c. mrigala. while in l. rohita the metal bioaccumulation ranged from 1.83 ± 0.109 µg/g to 6.26 ± 0.214 µg/g, in c. catla, it ranged from 1.23 ± 0.012 µg/g to 8.82 ± 0.100 µg/g. copper (cu): the site c had the highest mean cu bioacuumulation than the sites b, d and a. the mean cu accumulation differed significantly between low and high-flow seasons (p<0.001). the species also showed significant variations where c. catla contained the highest cu bioaccumulation than c. mrigala and l. rohita. the metal accumulation pattern in the fish organs was in the order of kidneys > intestine > heart > gills > eyes (table 5). mean cu bioaccumulation in different organs of c. mrigala ranged from 4.38 to 6.93 µg/g and 3.76 to 6.21 µg/g at site a during low and high flow seasons, respectively. in contrast for sites b, c and d, the cu concentration ranged from 6.31 to 11.16 and 5.15 to 9.08 µg/g; 7.42 to 12.79 and 5.82 to 11.03 µg/g; and 5.56 to 9.98 and 4.95 to 8.66 µg/g during low and high-flow seasons, respectively. iron (fe): the sites differed for the mean fe concentrations where the site c had the highest and upstream site a the lowest fe accumulation. also, fe concentration was significantly greater during the low than high-flow season. the fish species also differed for the mean fe concentration which was highest in c. catla followed by c. mrigala and l. rohita. the fe bioaccumulation pattern in the fish organs was in the order of: kidneys > intestine > heart > eyes > gills (table 5). the highest fe concentration of 97.43 ± 5.060 µg/g was found in kidneys of c. mrigala that were caught from site c during the low-flow season whereas the gills of c. mrigala from site a showed the lowest fe concentration of 24.35 ± 1.362 µg/g during the highflow season (table 1). lead (pb): similar to the trends for previous metals, the pb contents differed between sites with highest pb at site c followed by the sites d, b and a. the flow seasons also differed for the pb content which was higher during the low than high-flow season. the metal bioaccumulation pattern in fish organs was in the order of kidneys > intestine > heart > eyes > gills. the fish species also differed for the pb contents which were highest in c. catla and lowest in c. mrigala (table 5). higher pb accumulation in the organs of c. mrigala was measured at site c where the pb ranged from 4.08 ± 0.066 to 6.28 ± 0.401 µg/g and from 3.59 ± 0.189 to 5.94 ± 0.313 µg/g during the low and high-flow season, respectively (table 3). in contrast, the lowest pb accumulation ranging from 144 international journal of aquatic biology (2015) 3(3): 135-148 0.26 ± 0.022 to 0.47 ± 0.053 µg/g and from 0.18 ± 0.037 to 0.37 ± 0.077 µg/g during the low and highflow seasons, respectively, were recorded in c. mrigala from site a (table 1). the pb contents in the organs of c. catla ranged from 0.28 ± 0.064 to 0.50 ± 0.126 µg/g and 0.23 ± 0.046 to 0.47 ± 0.117 µg/g at site a the low and high-flow seasons, respectively. the corresponding pb accumulation in the fish sampled from site c ranged from 4.36 ± 0.229 to 6.43 ± 0.310 µg/g and 3.83 ± 0.385 to 6.33 ± 0.636 µg/g during low and high flow season, respectively. the mean pb accumulation in organs of l. rohita ranged from 0.30 ± 0.061 to 6.11 ± 0.351 µg/g and from 0.19 ± 0.040 to 5.04 ± 0.318 µg/g during the low and high-flow season, respectively. zinc (zn): the order of mean zn bioaccumulation for sites was c > d > b > a where mean zn contents differed significantly (p<0.001) for low and highflow seasons (table 6). the fish species differed significantly for zn which was highest in c. mrigala and lowest in c. catla. the zn accumulation pattern in fish organs was in the order of: kidneys > heart > intestine > eyes > gills (table 5). highest zn concentration of 104.86 ± 10.287 µg/g was found in the intestines of c. mrigala than the intestines of c. catla (83.80 ± 2.052 µg/g) and l. rohita (80.03 ± 1.858 µg/g) during low flow season at site c (table 3). manganese (mn): mean highest maganese accumulation was measured at site c. then the metal content appeared in descending order at the sites d, b and a. the mean metal contents of the fishes’ organs representing low flow to high-flow season differed significantly (p<0.001) from each other (table 6). among the three fish species, the c. mrigala had highest maganese accumulation. while l. rohita and c. catla showed the metal levels in descending order. the metal bioaccumulation pattern in fish organs was in order of: kidneys > intestine > heart > eyes > gills. the gills of c. catla showed lowest mn accumulation (1.75 ± 0.104 µg/g) at site a during high flow season (table 1). nickel (ni): among sampling sites, highest mean nickel bioaccumulation occurred at site c (3.43 µg/g). while up to 1.29, 0.82 and 0.56 µg/g of fishes’ organ/tissues of ni appeared for the sites d, b and a, respectively. higher metal accumulation was recorded during low than high flow season, respectively (table 5). among the fish species, c. mrgiala showed highest bioaccumulation of ni. while c. catla showed lowest concentration of the metal (table 5). the ni bioaccumulation pattern in the fishes’ organs was in descending order: kidneys > intestine > heart > eyes > gills. mercury (hg): mean mercury (hg) bioaccumulation measured highest at site c than d, b and a during low as well as high-flow seasons. hg bioaccumulation pattern in the fishes’ organs was the same observed by ni. highest mercury accumulation was recorded in c. mrigala than c. catla and l. rohita (table 5). mean hg bioaccumulation in different organs of c. mrigala caught from different sites ranged from 0.13 ± 0.039 to 5.70 ± 0.216 µg/g and from 0.10 ± 0.015 to 4.14 ± 0.163 µg/g during low and high flow seasons, respectively. hg accumulation in organs of l. rohita corresponding to low and high flow seasons ranged from 0.10 ± 0.005 to 5.39 ± 0.143 µg/g and from 0.11 ± 0.005 to 4.06 ± 0.143 µg/g. whereas hg bioaccumulation in organs of c. catla were recorded higher during low flow and ranged from 0.11 ± 0.007 to 5.55 ± 0.168 µg/g than high flows seasons when metal concentrations ranged from 0.10 ± 0.005 to 4.18 ± 0.087 µg/g. discussion in the present study, metals’ concentrations in fishes’ organs varied significantly (p<0.001) among the selected sampling sites and flow seasons of the river ravi. it is worth mentioning that means of total length and total wet body weight of same sampled specimen of each species (c. mrigala, l. rohita, c. catla) did not differ significantly (p>0.05) among sampling sites and flow seasons as already reported by shakir and qazi (2013). site specific metals’ accumulations in the fishes’ organs sampled from the river ravi have been reported by several workers (javed, 2003; nawaz et al., 2010; jabeen et al., 2012; shakir et al., 2013). metals’ bioaccumulation in 145 shakir et al/ metal bioaccumulation levels in river ravi tissues are related to change in feeding behaviour and physiological activities of fish species during different seasons (farkas et al., 2000; tekin-ozan and kir, 2007). seasonal variation in metals’ accumulation may be influenced by stream conditions, toxicants load, water chemistry and other environmental factors which affect the availability of different metals differently (heiny and tate, 1997). physico-chemical and ecological factors do influence the intensity of heavy metals uptake in animals. avenant-oldewage and marx (2000) reported that physico-chemical parameters such as temperature, ph and total dissolved solids influence the availability of heavy metals. for the present studied sites, shakir et al. (2013) reported alongstream increasing trend of ambient temperature with negative correction of dissolved oxygen and higher values of total dissolved solids, especially during low flow season of the river ravi. increasing temperature at downstream sampling sites leads to increase in metabolic rate. thus, increased diffusion or active transport associated with higher rates of water movement across the gills, might have led greater amounts of metals uptake by the fish (prosi, 1979). the results showed that metals’ accumulation in different organs of the fishes increased progressively at the downstream locations. pattern of metals’ bioaccumulation in fishes’ organs with respect to sampling sites provided evidences of exposure to contaminated aquatic environment. it was documented that fish can absorb and bioaccumulate available metals directly from their surrounding environment via skin and gills or through the ingestion of contaminated water and food (holliset al., 1999; kotze et al., 1999; qadir and malik, 2011). elevated level of metals in different fish tissues mainly originates from abiotic and biotic components of aquatic resources polluted by municipal sewage and industrial effluents (mansour and sidky, 2002; van aadt and erdmann, 2004; altindag and yigit, 2005; javed, 2006). therefore, metals’ bioaccumulation in different fish species of different trophic levels can be considered as an index of metal pollution in the aquatic bodies (tawarifufeyin and ekaye, 2007; karadede-akin and unlu, 2007). in the present study, cd, cr, cu fe, pb, zn, mn, ni and hg bioaccumulations in different organs appeared significantly (p<0.001) different among selected fish species. highest level of zn and fe, while lowest of cd were recorded in the fishes’ organs. the present study results are in line with jabeen et al. (2012) that reported higher zn content than as, ba, cr, ni in different tissues/organs (gills, liver, kidneys, intestine, reproductive organs, skin, muscle, fins, scales, bones and fats) of c. catla, c. mrigala and, l. rohita caught from different sampling sites (shahdara bridge, balloki head works and sidhnai barrage) of river ravi, pakistan. higher fe has been reported in various organs of fishes sampled from river chenab, pakistan as compared to pb, cd, cr, ni, cu and zn (qadir and malik, 2011). trace amount of cd can cause anomalies such as reduction in development and growth rates (hollis et al., 1999). during the present study, lowest concentration of cd was detected in all organs as compared to the other metals. such variations have been correlated by various workers with difference in uptake, absorption, storage, regulation, animals’ age, geographical location, season and excretion abilities of given fish species (al-yousuf et al., 2000; scerbo et al.,2005; solhaug jenssen et al., 2010). the metals’ contents in different organs of the investigated fish species appeared several folds higher than their corresponding values in the waters (shakir et al., 2013) as well as the level of water quality guidelines and proposed standards (neqs,2000; who,2004; wwf, 2007; nsdwq, 2008; usepa, 2009). there is fairly high amount of hg in different organs of fish species particularly sampled from site c. the authors’ best knowledge, no specific source of mercury contamination in river ravi reported. however, this might be related to untreated industrial effluents discharged through hudiara and deg-nullah into river ravi before this site. hudiara drain received effluents from around 100 industries before leaving from india and then 146 international journal of aquatic biology (2015) 3(3): 135-148 loaded with 112 industrial effluents from pakistan side before joining with river ravi. the deg-nullah also carries effluents of more than 149 industrial units (saeed and bahzad, 2006). the metals’ accumulation also significantly varied among different organs of the same fish. highest metals’ accumulation in kidneys than the other organs of sampled fish species might be associated to the fact that kidneys play a vital role in excretion. the higher zn and other metal accumulation in studied fishes’ kidneys might indicate maximum deloading capability of the sampled fishes under the prevailing condition. murugan et al. (2008) reported that fish have a tendency to push zinc burden from muscles to other tissues like kidney, during metallic stress and this deloading is beneficial to consumers who use fish muscle for food. varying levels of the metals’ bioaccumulations in different organs of the fishes are attributed to differences in their physiological functions (karuppasamy, 2004). metals uptake from blood at tissue level is a biphasic process, which involves rapid adsorption or binding to the surface, followed by a slower transport into cell interior. transport of different metals ions in to intracellular compartment may be facilitated by either diffusion of the metals ions across the cell membrane or by active transport of metals ions through binding with different specific carrier proteins. presence of different metal binding proteins is an indication of toxic metal pollution in an aquatic environment (hennig, 1986; crist et al., 1988). fish regulate metal ions through excretion via kidney and gills, however, such capacity of a tissue is directly related to the total amount of metal’s accumulation in that specific tissue. fish’s ability to synthesize metal binding proteins is limited. when metabolic capabilities for excretion and binding the pollutants are exceeded from threshold limit, toxic effects results, unless the fish has an alternate way of detoxification (kojima and kagi, 1978; cosson, 1994). the present study highlights the metal accumulation greater in fish species dwelling downstream sites, indicating impairment of ambient water due to continuous discharges of untreated industrial and municipal effluents into the studied segment of the river. the studied organs of fish species sampled from site c (industrial area) showed highest bioaccumulation levels which are directly associated with the pollution level of this site due to discharge of untreated industrial effluents and urban sewage in comparison with non-industrial upstream site a. the water and sediment samples also showed highest concentration of metals for this site (shakir et al., 2013). measured concentrations of the metals in fish organs indicated potential health risks for the fish and the food chain. the accumulation intensity of studied metals in different organs of the economically important fishes as a consequence of municipal sewage and industrial effluents. these discharges are not only threatening the ecological integrity of aquatic resources but also putting the health of local population at risk. therefore, contaminations due to heavy metals should be considered a priority concern and needs to be addressed urgently. acknowledgement first author acknowledged to higher education commission (hec), pakistan for funding under scholarship scheme “indigenous ph.d. 5000 fellowship program” (pin no. 074-1921-bm4-146) and “irsip” to support this research at university of the punjab, pakistan and newcastle university, uk. many thanks of dr. diky ramdani from newcastle university uk for his help in the statistical analysis. references al-yousuf m.h., el-shahawi m.s., al-ghais s.m. 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(2018) 6(4): 202-207 doi: issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2018 iranian society of ichthyology original article first record of the amur goby rhinogobius lindbergi berg 1933 (gobiidae) from the tigris river drainage, iran soheil eagderi*1, manoochehr nasri2, erdoğan çiçek3 1department of fisheries, faculty of natural resources, university of tehran, karaj, iran. 2department of fisheries science and engineering, faculty of agriculture and natural resources, lorestan university, khorramabad, iran. 3department of biology, faculty of arts and science, nevsehir haci bektas veli university, nevsehir, turkey. article history: received 22 may 2018 accepted 17 july 2018 available online 2 5 august 2018 keywords: sirvan river eivashan river zoogeography exotic abstract: iran harbors a great faunistic diversity especially in freshwater fishes from different endorheic and exorheic basins with about 32 reported exotic fishes. fishes are widely introduced and translocated aquatics due to anthropological activities. the present study reports first occurrence of the amur goby, rhinogobius lindbergi berg 1933 (gobiidae), from the gaveh river, a tributary of sirvan river drainage and eivashan river, a tributary of kashkan river system. in addition, we present meristic and morphometric data of the specimens herein examined. the collected specimens probably introduced to the rivers along with other exotic species as accidental introduction. therefore, an effective management strategy required to minimize their negative impacts. introduction fishes are widely introduced and translocated aquatics (esmaeili et al., 2014) by wide ranges of mechanisms such as aquaculture, sport fishing, control of malaria, research and accidental introductions (coad, 1996; mooney and cleland, 2001; esmaeili et al., 2007, 2011). iran harbors a great faunistic diversity especially in freshwater fishes with 297 species in 109 genera, 30 families and 24 orders reported from different endorheic and exorheic basins (esmaeili et al., 2018). about 32 species in 12 families have been introduced or translocated and some of them are wellestablished in different iranian water bodies (khaefi et al., 2014; esmaeili et al., 2014; mousavi-sabet and eagderi, 2014, 2015; radkhah et al., 2016). members of the genus rhinogobius (gill, 1859) with 66 species are distributed in japan, korea, taiwan, hainan, philippines, china, russia, vietnam, laos, cambodia, thailand and the amur river basin of eastern asia (chen et al., 2008; suzuki et al., 2016, coad 2018; froese and pauly; 2016). they are mainly amphidromous, non-diadromous, land-locked, and *corresponding author: soheil eagderi doi: https://doi.org/10.22034/ijab.v6i4.528 e-mail address: soheil.eagderi@ut.ac.ir fluvial species (mizuno and goto, 1987; iguchi and mizuno, 1991; akihito et al., 2002), as well as lake– river migratory and lentic species in lake biwa, japan (takahashi and okazaki, 2002). the lake goby, r. similis, has been recorded from different iranian inland waters, including the kashaf and hari rivers, hari river basin (coad and abdoli, 2000), anzali wetland, caspian sea basin (coad, 2018), zarineh river, urmia lake basin (eagderi and moradi, 2017) and jajrud river, namak lake basin (eagderi et al., 2017). shakirova and sukhanova (1994), sal'nikov (1995) and aliev et al. (1988) reported r. similis from some water bodies of turkmenistan on the east-north borders of iran. vasil'eva and vasil'ev (1995) and vasil'eva and kugac (2008) pointed out that the rhinogobius introduced to central asia is r. cheni (nichols, 1931), a chinese species of the yangtze river, and this could be the species found in iran. rhinogobius lindbergi berg 1933 is described species from amur and ussuri rivers, russia and the iranian specimens probably have been originated from the amur river basin via 203 int. j. aquat. biol. (2018) 6(4): 202-207 hari river and all other reported r. similis from iran could be r. lindbergi. based on our recent survey, amur goby, r. lindbergi, was found in the gaveh and eivashan rivers, tigris river system and probably can be found in iraq and turkish inland waters as well. therefore, this paper is aimed to report the first record of r. lindbergi from iranian part of the tigris river drainage by providing meristic and morphometric data of the specimens herein examined. materials and methods a total of 15 r. lindbergi were collected from the gaveh river (34°55'53.6"n 47°13'14.0"e), kermanshah province and 8 specimens from the eivashan river (33°27'43.06"n 48°50'44.21"e), lorestan province of iran by electrofishing device in 15 september 2016 and 1 october 2018, respectively (fig. 1). the collected specimens were preserved in 5% buffered formaldehyde after anesthetizing and transferred to laboratory for further study. the specimens were identified based on coad (2016), chen et al. (2008), suzuki et al. (2016), and sakai et al. (2000). nine meristic characteristics were counted by a stereomicroscope and 31 morphometric features were measured using a caliper to the nearest 0.1 mm (tables 1, 2). the morphometric data are presented as standard and head lengths. methods for taking measurements and counts follow hubbs and lagler (1958). the specimens were deposited in the ichthyological figure 1. observed distribution of rhinogobius lindbergi in kermanshah and lorestan provinces. figure 2. lateral view of rhinogobius lindbergi from the eivashan river, tigris river drainage, iran. 204 eagderi et al./ first record of the amur goby rhinogobius lindbergi from iran museum of natural resources faculty, university of tehran (imnrf-ut1024). results the collected specimens ranged 29.5-39.7 mm in standard length and the general body shape of the collected amur goby is displayed in figure 2. the morphometric and meristic data of the specimens are presented in tables 1 and 2. three other exotic fish species, including hemiculter leucisculus, pseudorasbora parva and carassius gibelio along with 10 native fishes, including alburnus sellal, barbus lacerta, capoeta damascina, c. trutta, cyprinion macrostomum, garra rufa, squalius berak, s. lepidus, oxynoemacheilus sp. and turcinoemacheilus kosswigi were collected table 1. morphometric characteristics of rhinogobius lindbergi from the gaveh and eyvashan rivers, tigris basin (min=minimum; max=maximum; sd=standard deviation). gaveh river eyvashan river characters min max mean ±s d min max mean ±sd standard length (mm) 29.5 39.7 30 38 33.4 3.1 % in standard length body depth maximal 16.2 20.7 17.9 1.3 16.6 17.4 17 0.4 caudal peduncle depth 9.3 11.2 10.5 0.5 9.1 11.2 10.2 1.1 predorsal 1 length 34.9 40.5 37.9 1.3 35.9 36.9 36.5 0.5 postdorsal 1 length 59.2 65.1 62.2 1.7 63.1 64.5 64 0.8 predorsal 2 length 53.9 58.6 56.6 1.2 56.3 58.6 57.6 1.2 postdorsal 2 length 40.8 45.1 42.9 1.5 45.3 48.7 47.6 2 preanal length 60.1 64.5 61.9 1.3 56.8 59 58 1.1 caudal peduncle length 23.0 27.0 24.8 1.3 26.9 28.6 27.5 1 dorsal-fin1 base length 10.8 16.5 13.3 1.7 7.3 12 10.1 2.5 dorsal-fin 1 depth 6.0 13.5 9.8 2.5 11 12.4 11.9 0.8 dorsal-fin 2 base length 14.6 19.7 16.8 1.9 13.1 18.5 15.3 2.8 dorsal-fin 2 depth 10.6 15.2 13.1 1.7 11.7 15.7 13.8 2 anal-fin base length 10.2 17.6 14.6 2.1 10.6 18.4 16.9 1.4 anal-fin depth 7.2 12.3 10.0 1.9 10.5 13.9 12.4 0.8 pectoral fin length 14.1 23.0 18.2 2.8 15.4 19.7 17.5 2.1 pectoral – anal-fin origin distance 28.7 37.7 32.5 2.0 30.1 32.6 31.4 1.3 caudal-fin length 19.6 26.3 22.1 1.7 19.1 20.7 20.1 0.9 body width 12.9 16.2 14.9 0.8 15.9 17.6 16.4 1 caudal width 3.0 5.4 3.9 0.6 4.2 4.7 4.5 0.3 pelvic fin width 9.8 17.1 13.9 2.3 9.1 14.7 12.3 2.1 pelvic fin length 16.6 20.4 18.6 1.2 16 17.9 16.8 1 head length (hl) 23.2 29.9 27.0 2.2 24 26.1 25.2 1.8 % in head length snout length 22.5 32.5 27.6 2.6 29.7 31.9 30.4 1.3 eye horizontal diameter 12.2 22.5 17.8 3.1 12.4 18.3 16.7 3.1 postorbital distance 45.1 64.8 56.9 4.6 55.5 59.1 57.1 1.8 head depth at nape 61.2 75.3 67.7 4.9 59.4 80.1 66.6 11.7 head depth through eye 48.1 62.2 53.5 4.0 48.5 52.9 50.4 2.3 mouth width 35.2 62.0 45.6 7.8 32.6 61.6 43.2 8.2 interorbital 7.6 15.5 9.9 2.2 8.1 11.6 10.3 2.7 head width 56.3 85.0 69.5 9.8 66.3 69.5 67.8 1.6 205 int. j. aquat. biol. (2018) 6(4): 202-207 during sampling in gaveh river. in addition, two other exotic fish species viz. cyprinus carpio and oncorhynchus mykiss along with 6 native species including a. sellal, c. shajariani, c. aculeate, c. macrostomum, g. rufa and chondrostoma regium were sampled from the eivashan river. discussion rhinogobius lindbergi is native to the lower and middle portion of the amur river (novomodny et al., 2004), and was previously known to extend upstream only to the vicinity of the mouth of the bidzhan river, russia (neely et al., 2008). rhinogobius lindbergi is distinguished by having 27 vertebrae, two preopercular canal pores, 19-20 pectoral-fin rays and i, 9 anal-fin rays (sakai et al., 2000; li et al., 2018). the collected specimens have similar morphological characters to those reported for r. lindbergi (sakai et al., 2000; li et al., 2018). new record of r. lindbergi from the gaveh river, a tributary of sirvan river and eivashan river, a tributary of kashkan river, both from tigris river drainage shows the range extension of this species further to west and southwest from its previous records. the sirvan river originates near the hamadan, in the zagros mountains of iran and then runs west mainly through eastern iraq and finally feeds into the tigris below baghdad but the eivashan river origins from zaghe near khoramabad city, also in zagros mountains of iran and then runs westward to the karkhe river, tigris river drainage (coad, 2018). in total of 14 species were collected from gave river, four of them are exotic to the river. also, hypophthalmichthys molitrix, h. nobilis, and, c. carpio are other exotic species recorded from this river that have been released into the lakes of the soleimanshah and gavshan dams, reconstructed on the gaveh river, by iranian fisheries organization (alizadeh et al., 2016). in the eyvashan river, three exotic species, including r. lindbergi, c. carpio and o. mykiss were collected. therefore, r. lindbergi probably introduced to this river along with commercially important cyprinids as accidental introduction. introductions are always led to risks for the native biota if species is able to integrate itself successfully into the ecosystem (gozlan et al., 2009), and also is resulted in possible detrimental interactions with native species (gozlan et al., 2010; esmaeili et al., 2014). therefore, an effective management strategy needs to minimize their negative impacts. acknowledgments this study was financially supported by the university of tehran and lorestan university. references abdoli a., coad b., naderi m. 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(2013) 1(3): 138-142 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article acute toxicity of two pesticides diazinon and deltamethrin on tench (tinca tinca) larvae and fingerling mohammad forouhar vajargah*1, aliakbar hedayati1, sayede amene hossaini1, elaheh hassan nataje niazie1, mohammad javad vesaghi1 1 department of fisheries, faculty of fisheries and environment, gorgan university of agricultural sciences and natural resources, gorgan, iran. article history: received 4 may 2013 accepted 27 may 2013 available online 2 0 june 2013 keywords: fish lethal toxicity pesticide poison abstract: diazinon and deltamethrin are common useful agricultural pesticides in the world. the present research compared the acute toxicity of diazinon and deltamethrin to tench larvae and fingerling. lc50 of 24 h, 48-h, 72 h and 96 h were determined using a probit. fish samples (21 fish in each test group) were exposed to different concentrations of diazinon and deltamethrin (diazinon: for fingerling between1-20 ppm and larvae 0.25-2 ppm, deltamethrin: for fingerling between 0.01-0.2 ppm and larvae 0.0025-0.02) for 96 h and mortality were recorded. the lc50 96 h of diazinon for fingerlings and larvae were 6.77 and 0.63, respectively. the lc50 96 h of deltamethrin for fingerlings and larvae were 0.07 and 0.005 ppm, respectively. according to the results, larvae are more sensitive than fingerlings, lc50 values indicated that deltamethrin is more toxic than diazinon to tinca tinca, so we suggest to use diazinon instead of deltamethrin in agriculture. introduction tench (tinca tinca) is a freshwater and brackish water cyprinid. it normally inhabits slow-moving freshwater habitats, particularly lakes and lowland rivers (whitton, 1982). typically, this species lives in shallow, densely vegetated lakes and backwaters. it often overwinters in mud and spawns among dense vegetation in still water. t. tinca has economic and ornamental values. tench is distributed throughout the europe for centuries and thus has blurred the original distribution pattern. hypothesized to be native in most of europe, naturally absent only in ireland, scandinavia north of 61°30' n, eastern adriatic basin and western and southern greece where it is now introduced. in asia, native eastward to western yenisei drainage south of 60° n. introduced to north and south africa, tasmania, australia, * corresponding author: mohammad forouhar vajargah e-mail address: forouhar.eco89@yahoo.com tel: +981712220320 new zealand, india, north america, chile and probably elsewhere (iucn, 2012). the present study was performed to determine toxicity of diazinon and deltamethrin as potential dangerous organic pesticides to assess mortality effects of these chemicals to the tench, these tow pesticides are common to control herbal pests in many agriculture fields that are located in the vicinity of fresh water resources. deltamethrin is a synthetic pyrethroid insecticide and acaricide. it is used topically for the control of ectoparasites in cattle, sheep and poultry. it is also widely used as a pesticide on crops (emea, 2000). diazinon is one of the most functional organophosphate known pesticides commonly enters through the agricultural fields drain into surface water and groundwater (sohrabi et al., 2011). in recent studies of surface water, coastal and even 139 forouhar vajargah et al./ int. j. aquat. biol. (2013) 1(3): 138-142 wastewater treatment systems in urban areas in different parts of the world, including iran, significant amounts of these toxins have been reported (shayeghi et al., 2001; u.s. epa, 2005). fish is one of the important aquatic organism because of the economic value and the sensitivity against contaminants, thus they are used in a wide range for biological assays (ola, 1369). today major threat of tench populations is river engineering (iucn, 2012) and given that it is a potamodromous species (riede, 2004), investigate the effects of agricultural pesticides on its life seems to be essential. materials and methods fishes and concentration treatments: this research aimed to determine the toxicity of diazinon and deltamethrin on tinca tinca larvae and fingerlings. lethal experiments were conducted using 125 larvae and 125 fingerling for each of the tested pesticides. physicochemical properties of water were measured: temperature 23±1, ph 6/5-8, total hardness 220 mg/l and dissolved oxygen 7-9/5 mg/l. during the experiment, water was not exchanged. before the test, fish were feed twice daily with biomar with the amount of 2% of body weight. applied concentrations of diazinon were 1, 5, 10, and 20 for fingerlings and 0.25, 0.5, 1 and 2 mg/l for larvae. these concentrations for deltamethrin were 0.01, 0.05, 0.1 and 0.2 for fingerling and 0.0025, 0.005, 0.01 and 0.02. for each experiment, fingerlings and larvae were divided into four groups i.e. there were four group including fingerlings exposed to diazinon, larvae exposed to diazinon, fingerling exposed to deltamethrin and larvae exposed to deltamethrin. in each group, there were four treatments and one control. mortality rates were recorded at 0, 24, 48, 72 and 96 h. statistical analysis: experiments were performed according to the o.e.c.d standard method (1998), to find the lc50 96 h of tench larvae and fingerlings. the numbers of dead fish were recorded at 24, 48, 72, 96 h. acute toxicity tests was carried out according to hotos and vlahos (gooley et al., 2000). the nominal concentration of diazinon and deltamethrin estimated to result in 50% mortality of tinca tinca within 24 h (24 h lc50), 48 h, 72 h and 96 h was attained by probit analysis by software spss version 16. results during the study number of dead larvae and fingerlings for deltamethrin and diazonin doses were examined depending on duration (24, 48, 72 and 96 h) of exposure in tench larvae and fingerlings. with increasing deltamethrin concentrations, both larvae and fingerling exposed duration 24-96 h had significantly increased number of dead fish. the highest diazinon and deltamethrin concentration showed the highest larvae and fingerling mortality (tables 1 and 2). no fish died during the acclimation period before exposure, no control fish also died during acute toxicity tests. median lethal concentrations of 10%, 30%, 50%, 70%, 90% and 99% test are presented in tables 3 and 4. the results of this study indicated group concentration (ppm) no. of dead fish 24h 48h 72h 96h f in g e rlin g 1 0 1 2 5 5 0 1 3 9 10 1 3 8 14 20 3 7 14 21 control 0 0 0 0 l a rv a e 0.25 0 1 2 8 0.5 1 3 6 10 1 2 4 9 14 2 3 7 14 21 control 0 0 0 0 table 1. the mortality rate of tinca tinca exposed to acute commercial diazinon (21 fish for each concentration). 139 140 forouhar vajargah et al./ int. j. aquat. biol. (2013) 1(3): 138-142 lc50 96 h of diazinon was 6.77 ppm for fingerling and 0.63 ppm for larvae. thus mac value was equal to 0.677 and 0.063 ppm, respectively (table 3). for deltamethrin, mac value for fingerling and larvae were 0.007 and 0.0005 for fingerlings and larvae, respectively (table 4). discussion contamination of aquatic environment with pesticides via rainfall runoff is very common (willis and mcdowell, 1982). the toxicity of deltamethrin and diazinon on tench increased with increasing concentration and exposure time. previous studies indicated the high toxicity of deltamethrin to fish species, which is in agreement with our results (boateng et al., 2006). it has been shown that a low level of deltamethrin (0.005 µg/l) in the aquatic environment may have a significant effect on carp populations. as toxicant for aquatic life, it should be used with great attention in agriculture to protect natural waters (kӧprϋcϋ and aydın, 2004). although synthetic pyrethroids are less persistent and less toxic to mammals and birds (sayeed et al., 2003), they are highly toxic to a number of non-target organisms such as bees, freshwater fishes and other aquatic organisms even at very low concentrations (oudou et al., 2004). for this reason, these organisms are extremely sensitive to neurotoxic effects of pyrethroids when they reach surface water-courses (bradbury and coats 1989, haya, 1989; mittal et al., 1994). deltamethrin alters the hepatic metabolism and the normal ionic flux in some species; bálint et al. (1995) observed 20% decreases in acetyl cholinesterase activity of the brain, heart, blood, liver and skeletal muscle of carp after a three-day exposure to deltamethrin. (cristina da silva de assis et al., 2009) group concentration(ppm) no. of dead fish 24h 48h 72h 96h f in g e rlin g 0.01 0 0 1 4 0.05 0 2 5 8 0.1 1 4 8 14 0.2 2 7 13 21 control 0 0 0 0 l a rv a e 0.0025 0 1 3 6 0.005 0 3 7 11 0.01 2 6 11 16 0.02 4 11 17 21 control 0 0 0 0 concentration (ppm) group point 24 h 48 h 72 h 96 h f in g e rlin g 1lc 6.24 10lc 17.2 8.88 2.93 30lc 25.14 18.34 9.99 3.99 50lc 30.64 24.9 14.88 6.77 70lc 36.13 31.46 19.77 9.56 90lc 44.07 40.92 26.83 13.58 99lc 55.03 53.98 36.57 19.12 l a rv a e 1lc 10lc 1.48 0.62 0.12 30lc 2.68 1.72 0.87 0.35 50lc 3.51 2.47 1.39 0.63 70lc 4.33 3.23 1.91 0.91 90lc 5.53 4.32 2.65 1.31 99lc 7.18 5.83 3.68 1.87 table 3. lethal concentrations of diazinon. table 2. the mortality rate of tinca tinca exposed to acute commercial deltamethrin (21 fish for each concentration). 141 forouhar vajargah et al./ int. j. aquat. biol. (2013) 1(3): 138-142 diazinon is a very highly toxic organophosphate compound. organophosphates are long known and widely applied active ingredients of different insecticides. in the living organism, organophosphates inhibit the enzyme acetyl cholinesterase, accumulate acetylcholine, a neurotransmitter involved in impulse transmission, and lead to an over-stimulation of the parasympathetic nerves (farage-elawar, 1989). poisoned animals show salivation, lachrymation, diarrhea and convulsions followed by depression, prostration, ataxia and cyanosis, and then death usually within a short time (brown et al., 2003). the results of researches on carp and other fish indicate diazinon causes anemia, reduction blood factors and weakening of the immune system of fish (shamooshaki et al., 1390) and the results of this study agree with many studies on different fish species of cyprinidae family such as effect of diazinon on rutilus frisii kutum, abramis brama, hypophthalmichthys molitrix (nasri tajan, 1997), ctenopharyngodon idella (pourgholam et al., 2001), cyprinus carpio embryos and larvae (rahmi and kenan, 2005), pimephales promelas (sibel et al., 2006) and deltamethrin on cyprinus carpio (mestres and mestres, 1992). our finding indicate that deltamethrin is more toxic than diazinon to this species and larvae are more sensitive than fingerlings, so we suggest to use diazinon instead of deltamethrin in agricultural applications. reference: bálint t., szegletes t., szegletes z., halasy k., nemcsók j. (1995). biochemical and subcellular changes in carp exposed to the organophosphorous metidation and the pyrethroid deltamethrin. aquatic toxicology, 33: 279-295. boateng j.o., nunoo f.k., dankwa e.h.r., ocran m.h. (2006). acute toxic effects of deltamethrin on tilapia, oreochromis niloticus (linnaeus, 1758). west africa journal applied ecology, 9: 1-5. bradbury s.p., coats j.r. (1989). comparative toxicology of the pyrethroid insecticides. reviews of environmental contamination and toxicology, 108: 133-177. brown t.p., julian r.j., saif j.m. (2003). diseases of poultry. iowa state press, a blackwell publishing company. iowa, pp: 1133-1159. cristina da silva de assis h., nicareta l., salvo l.m., klemz c., truppel j.h., calegari r. (2009). biochemical biomarkers of exposure to deltamethrin in freshwater fish, ancistrus multispinis. brazilian archives of biology and technology, 52: 1401-1407. emea. (2000). committee for veterinary medicinal products, deltamethrin (extension to fin fish). the europen agency for the evaluation of medicinal products veterinary medicines and information technology. canary wharf, london, e14 4hb, uk. mrl/ 731/00-final report. 4 p. farage-elawar m. (1989). enzyme and behavioral changes in young chickens as a result of carbaryl treatment. journal of toxicology and environmental health, 26: 19-31. gooley g.j., gavine f.m., dalton w., de silva ss b.m., concentration (ppm) group point 24 h 48 h 72 h 96 h f in g e rlin g 1lc 0.04 10lc 0.2 0.08 0.02 0.006 30lc 0.31 0.17 0.1 0.04 50lc 0.39 0.23 0.15 0.07 70lc 0.47 0.29 0.2 0.09 90lc 0.58 0.38 0.28 0.13 99lc 0.74 0.51 0.38 0.18 l a rv a e 1lc 0.005 10lc 0.015 0.0007 0.0003 30lc 0.023 0.08 0.006 0.003 50lc 0.028 0.18 0.011 0.005 70lc 0.033 0.29 0.015 0.008 90lc 0.041 0.44 0.021 0.011 99lc 0.051 0.65 0.029 0.015 table 4. lethal concentrations of deltamethrin. 141 142 forouhar vajargah et al./ int. j. aquat. biol. (2013) 1(3): 138-142 samblebe m. (2000). feasibility of aquaculture in dairy manufacturing wastewater to enhance environmental performance and offset costs. final report drdc project no. maf001. marine and freshwater research institute. snobs creek. 84 p. haya k. (1989). toxicity of pyrethroid insecticide to fish. environmental toxicology and chemistry, 8: 381391. iucn. (2012). iucn red list of threatened species. from: www. fishbase. org. retrieved 4/05/2013. kӧprϋcϋ k., aydın r. (2004). the toxic effects of pyrethroid deltamethrin on the common carp (cyprinus carpio l.) embryos and larvae. pesticide biochemistry and physiology journal, 80: 47–53. mestres r., mestres g. (1992). deltamethrin: uses and environmental safety. reviews of environmental contamination and toxicology, 124: 1-18. mittal k., adak t., sharma v.p. (1994). comparative toxicity of certain mosquitocidal compounds to larvivorous fish, poecilia reticulate. indian journal of malariology, 31: 43-47. nasri tajan m. (1997). determination of lethal concentration of toxin (organophosphate insecticide), diazinon granule 5 percent and emulsion 60 percent on the abramis brama population in anzali lagoon. msc thesis. lahijan islamic azad university, pp: 7-31. ola y. (1369). pollution from domestic waste, municipal, agricultural, industrial and natural, structure and function of anzali lagoon in front of them. guilan fisheries reserche center. doc. 2: 38 p. oudou h.c., alonso r.m., bruun hansen h.c. (2004). voltammetric behavior of the synthetic pyrethroid lambda-cyhalothrin and its determination in soil and well water. analytica chimica acta, 523: 69-74. pourgholam r., soltani m., hassan d.m., esmaeili f., farhoomand h., usefi p. (2001). evaluation of blood characteristics of grass carp (ctenopharyngodon idella) after exposure to organophosphate, diazinon. iranian journal of fisheries sciences, 3:1-18. int. j. aquat. biol. (2019) 7(5): 301-314 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2019 iranian society of ichthyology original article the use of zingiber officinale extract against yersinia ruckeri and its effects on the antioxidant status and immune response in oncorhynchus mykiss mehrdad soltanian1, hamid faghani langrodi*1, majid mohammad nejad2 1department of fisheries, tonekabon branch, islamic azad university, tonekabon, iran. 2department of fisheries, bandar gaz branch, islamic azad university, bandar gaz, iran. s article history: received 15 july 2019 accepted 15 august 2019 available online 2 5 august 2019 keywords: rainbow trout non-specific immune response alt ldh ast abstract: the present work aimed to explore the effects of nutritional ginger on rainbow trout. the fish were fed various concentrations of ginger extract i.e. 1, 3, 6, and 10 g/kg; the control was fed no ginger. having fed for 56 days, the fish were exposed to yersinia ruckeri and any case of death was documented for two weeks. the diet with the extract of ginger could check the experimental infection in rainbow trout. the highest survival rate, i.e. 91.4%, was attained in the group nourished with ginger at 10 g/kg while the survival rate for the control was 41.6%. ginger diet of 6 and 10 g/kg affected the biochemical parameters and the immune response. total serum protein and total serum immunoglobulin, together with lysozyme, phagocytic, bactericidal and antioxidant enzymes activities, significantly improved in some groups, other than the control, in a dose-reliant mode (p<0.05). no variations were detected in the levels of alanine aminotransferase, lactate dehydrogenase, and aspartate aminotransferase activities in the ginger-fed groups, indicating no liver toxicity. the findings of the current research revealed that some doses of nutritional ginger (6 and 10 g/kg) could reinforce the non-specific immunity and diminish the vulnerability of rainbow trout to y. ruckeri. introduction as one of the most extensively introduced fish species in the world, rainbow trout has been introduced into at least 99 countries (stanković et al., 2015). the production of rainbow trout in freshwater amounted to 576.902 metric tons (mt) globally in 2014 (fao, 2016). iran, turkey, and italy stood as the top three producers and produced 126.515, 107.533, and 34.400 mt, respectively, comprising almost 47% of the worldwide culture of rainbow trout (fao, 2016). diseases are still the main limit for the development of aquaculture. enteric red mouth disease (ermd), also known as yersiniosis, caused by yersinia ruckeri, is an important pathogen in salmonid fish (gregory et al., 2010). as a gram-negative enterobacterium, this pathogen is isolated from different fish species (haig et al., 2011; chettri et al., 2013; soltani et al., 2014). chemotherapies are extensively used to control and prevent bacterial diseases in intensive aquaculture (austin and austin, 2007); however, the broad usage *correspondence: hamid faghani langrodi doi: https://doi.org/10.22034/ijab.v7i5.630 e-mail: hamid_faghani1@yahoo.com of antibiotics may cause environmental risks, lead to the development of resistant pathogens, and threaten human health (esiobu et al., 2002; jones et al., 2004; rao et al., 2006). immune enhancers augment resistance to stress and disease by enhancing the immune responses in aquatic animals (harikrishnan et al., 2011). herbal immunostimulants consist of numerous terpenoid, polyphenolic, polypeptide, phenolic, quinone, lectine, and alkaloid complexes, most of which are very adequate replacements to chemical drugs (talpur et al., 2013). they also support growth, are anti-stress and antimicrobial in aquaculture (citarasu et al., 2001; maqsood et al., 2011). currently, much attention has been paid to herbal immunostimulants in aquaculture because they are inexpensive and environmentally friendly (abdeltawwab et al., 2018; hoseini and yousefi, 2018; adeshina et al., 2018; yousefi et al., 2019). zingiber officinale roscoe, commonly known as ginger, comprises flavonoids, alkaloids, steroids, gingerols, shogaols, polyphenols, carotenoids, 302 soltanian et al./ zingiber officinale extract against yersinia ruckeri in oncorhynchus mykiss zingeron, vitamins, and minerals (iheanacho et al., 2017). ginger is a significant immunostimulant and also has antibacterial, antiviral, antifungal, antioxidant, and anti-inflammatory effects on various living organisms (nya and austin, 2009a; otunola et al., 2010). to the best of our knowledge, no data is available regarding the anti-infectious potential of ginger against y. ruckeri in fish species. therefore, the objective of the current study was to determine the impact of nutritional ginger on immunity response and disease resistance against y. ruckeri in rainbow trout. materials and methods preparation of ginger extracts and diets: ginger fresh rhizomes were bought from a local shop in karaj, iran. clean water was used to wash the ginger. then, it was skinned, chopped, and shade-dried at room temperature. then, it was dried in an oven at 60°c and powdered using an electrical blender. samples were then homogenized in ethanol (75%) for three days. then, the mixture was filtered through whatman filter paper (no. 1) and became concentrated at 40°c by a rotary evaporator. the dried extract was put in closed bags and kept at 4°c for further use. the pellet feed (faradaneh company, shahrekord, iran) was used as the basal diet. table 1 presents how the basal diet was formed. the extract was homogenized in 75% ethanol and sprayed gently over the feed pellets in five dissimilar levels (0, 1, 3, 6, and 10 g/kg feed). for drying out the pellets, they were put in an oven at 35°c for 24 h. the feed pellets were covered with 4% aqueous gelatin dissolved in distilled water at a ratio of 5:40 (v/w) for keeping the plant extract intact (wu et al., 2013). the pellets were kept at room temperature (25°c) for 6 h to be air-dried and were then conserved in airtight bags at 4°c for further use. the approximate ingredients (crude lipid, fiber, crude protein, dry matter, and ash) of the feeds used in the study were analyzed in triplicate based on the association of official analytical chemists (aoac, 2005). the diets were examined for the presence of nitrogen-free extract (nfe) and the amount of this extract was estimated based on the following formula (shaluei et al., 2016): nfe = dry matter – (crude lipid + crude protein + fibre +ash). table 2 shows the approximate constitutions of test diets. experimental design: the research tests were performed at a private trout farm at the hashtgerd, karaj, iran. a total of 750 juvenile rainbow trout (45.8±3.6 g), were arbitrarily distributed into five different groups with three repeats for each. the fish (50 per replicate) were held in 15 circular tanks (3000 l), supplied with well water (constant temperature 15±0.5°c; ph=7.6; oxygen>7.5 mg/ l; nh3<0.01 mg/l; no2<0.1 mg/l and total hardness 278 mg/l), table 1. formulation and element constitution of basal diet. ingredients approximate constitution of additives (g/kg) (g/kg) crude protein crude fat fiber ash moisture sardine fish meal 350 650 80 10 190 70 anchovy fish meal 250 700 90 10 140 60 soybean meal 75 400 20 60 70 120 wheat flour 184 100 10 25 10 180 soybean oil 60 0 990 0 0 10 fish oil 30 0 990 0 0 10 wheat gluten 25 750 20 20 15 75 gelatin 10 750 0 0 130 110 vitamin and mineral mix 16 mineral and vitamin mixture (mg/ kg or iu/kg): vitamin: retinol acetate (a), 1200000 iu; thiamin mononitrate (b1), 200 mg; riboflavin (b2), 3600 mg; calcium d-pantothenate (b3), 7200 mg; niacinamide (b5), 9000 mg; pyridoxine hydrochloride (b6), 2400 mg; folic acid (b9) 600 mg; cyanocobalamin (b12), 4 mg; l-ascorbic acid (c), 5400 mg;cholecalciferol (d3), 400000; d-biotin (h2), 200 mg; dl-a-tocopheryl acetate (e), 30 iu;menadione sodium bisulfite (k3), 1200 mg;antioxidant500 mg: mineral: cu, 500 mg; se, 50 mg; co, 50 mg; zn, 6000 mg; fe, 4500 mg; i, 150 mg; choline chloride, 150 000 mg; mn,5000 mg; carrier up to 1 kg. 303 int. j. aquat. biol. (2019) 7(5): 301-314 with a 10:14 h light: dark natural photoperiod. water parameters were measured every two weeks. oxygen and total hardness were measured based on standard methods (clesceri et al., 1999), and ph using ph meter (model: hanna instrument model no. h1 8915 atc). ammonia, nitrite and nitrate were monitored using a commercial testing kit (fish farming test kit, model ff1a; hach co., loveland, co, usa) and maintained in standard ranges. the basal diet was used to nourish the control while the experimental groups were supplied with the basal diet supplemented with four concentrations of ginger extract (1, 3, 6, and 10 g/kg). as proposed elsewhere, all the groups were fed to proximate the estimated levels (2% biomass per day) at a frequency of thrice a day for 8 weeks (56 days) (merrifield et al., 2010). following every 30 min of feeding interval, siphoning was used to remove uneaten feed and feces for all the groups (li et al., 2015). blood and serum collection: after every 2 weeks, the fish were first fasted for 24 h and then anesthetized with eugenol (25 mg/ml) before blood sampling was done. in each group, 15 fish were randomly selected for blood collection (five fish per group replicate), and a 2-ml syringe was used to collect blood from their caudal vein. for each blood sample, one part of it was transferred into pre-heparinized plastic eppendorf tubes, whereas the other part was transferred into eppendorf tubes without anticoagulant and left to clot there for 2 h at room temperature. the tubes were kept at 4ºc during the night and then centrifuged at 2500g for 15 min. next, the collection of the supernatant serum was done and kept at -20ºc in screw cap glass vials to be used later. immunoglobulin assay: the total serum immunoglobulin (ig) was measured based on the method suggested by siwicki and anderson (1993). immunoglobulins were separated from serum by precipitation with 10000 kda polyethylene glycol (peg, sigma). serum (100 µl) and an equal volume of 12% peg solution were mixed together and the mixture was shaken for 2 h at room temperature. at first, the supernatant was centrifuged at 5000 g for 15 min; then, it was removed and the proteins concentration was detected based on biuret method (goldenfarb et al., 1971). to determine the total immunoglobulin content, this value was subtracted from the total serum concentration of the protein. lysozyme assay: jian and wu’s (2003) method was used to evaluate serum lysozyme activity. concisely, a suspension of micrococcus luteus, which had been grown during the night, was produced by dissolving 20 mg of m. luteus into 100 ml of 0.067 mol/l sodium phosphate buffer, ph 6.4. one hundred μl of fish serum was added to a 3 ml suspension of m. luteus at 22°c and absorbance at 540 nm was read after 0.5 and 4.5 min. one unit of lysozyme activity was considered as the quantity of lysozyme, inducing a reduction in absorbance of 0.001 per minute. phagocytic activity ex vivo: phagocytic activity was examined according to zhou et al. (2002), and the commercial baker’s yeast, saccharomyces cerevisiae, was used as an indicator. dried live yeast was mixed in 2% sucrose solution (ph 3-4) for 2 h at 30°c and boiled for 30 min. then, the yeast was centrifuged (800 g for 10 min); the pellet was washed two times table 2. approximate constitution of diets containing different concentrations of ginger extract. nutritional ginger extract inclusion levels (g/kg) approximate analysis (g/kg dry weight) 0 1 3 6 10 dry matter 918.24±8.48a 918.62±8.38 a 920.26±6.14 a 934.63±9.45 a 944.64±10.26 a crude protein 492.36±8.26 a 503.34±7.16 a 496.34±10.12 a 502.23±9.14 a 494.26±10.14 a crude lipid 164.42±8.66 a 165.36±6.82 a 172.26±7.45 a 170.56±8.45 a 167.26±7.86 a fiber 19.24±4.28 a 20.12±3.68 a 21.14±4.22 a 22.12±3.86 a 24.18±4.48 a ash 108.36±6.42 a 110.42±5.57 a 117.34±6.23 a 120.14±8.24 a 122.35±6.89 a nfe 135.36±18.3 a 116.34±20.46 a 118.54±15.42 a 121.26±17.32 a 137.42±12.8 a gross energy (mj/kg) 22.26±0.12 a 22.58±0.16 a 23.14±0.17 a 20.18±0.08 a 19.76±0.13 a data in rows assigned with different letters are significantly different at 5% level of significance. 304 soltanian et al./ zingiber officinale extract against yersinia ruckeri in oncorhynchus mykiss and it was re-suspended in 0.85% saline (at 2×108 cell/ml). after this, an aliquot [20 μl] of suspension, together with 40 μl heparinized whole blood, was added to a 0.1 ml eppendorf tube. the mixture was incubated at 22°c for 30 min and shaken mildly. afterward, the preparation of glass smears took place and wright-giemsa was used to stain the slides which had been air-dried earlier. phagocytic activity (pa) was estimated by assessing 100 phagocytes per slide under a light microscope. the evaluation was done on three slides/fish. the mean pa was determined as 100 × number of phagocytic cells and the engulfed yeast cells/number of phagocytes were enumerated. complement activity: the assessment of the components of the complement (c3 and c4) was done using laboratory kits (pars azmoon co., tehran, iran) based on immunoturbidimetry (sun et al., 2010). total serum protein was determined as described in the biuret method (goldenfarb et al., 1971). serum enzymes activities: lactate dehydrogenase (ldh), aspartate aminotransferase (ast), alanine aminotransferase (alt), and alkaline phosphatase (akp) activities were detected in serum colorimetrically using the kits offered by ziest chem diagnostics, tehran, iran (fazlolahzadeh et al., 2011). serum bactericidal activity: bactericidal activity of serum was detected according to rao et al. (2006). the centrifugation of the fresh nutrient broth culture of y. ruckeri was done for 3000 g for 15 min; also, the washing and suspension of the pellet was done in pbs. the suspension optical density was accustomed to 0.5 at 546 nm. the dilution of the bacterial suspension was done serially (1:10) with pbs and it was repeated for five times. two ml of this diluted y. ruckeri suspension was incubated with 20 μl of serum in an eppendorf tube for 1 h at 22ºc to evaluate the bactericidal activity of serum. pbs substituted the serum in the bacterial control group. once the incubation finished, the counting of the viable bacteria was started by enumerating the colonies grown on a nutrient agar plate for 24 h at 22ºc. antioxidant-related parameters assay: total antioxidant capacity (tac) and the activities of catalase (cat), superoxide dismutase (sod), and glutathione peroxidase (gsh-px) in serum were measured using a spectrophotometer (uv-2100, shanghai jinhua technology instrument co., ltd., shanghai, china) at 520, 550, 412, 405, and 520 nm, respectively (deng et al., 2013). the antioxidantrelated parameter detection kits (tac, cat, gsh-px, and sod) were ordered from nanjing jiancheng bioengineering institute (nanjing, china). one unit of tac was considered as a 0.01 rise of the absorbance of the reaction induced by serum per ml reacting at 37˚c for 1 min. one unit of cat activity was interpreted as the quantity of the enzyme that accelerated the breakdown of 1 mmol of h2o2 per min. one unit of gsh-px activity was defined as the quantity of the enzyme that decreased the gsh level in the reaction system at 1 mmol/ l per minute. one unit of sod activity was considered as the quantity of the enzyme needed to induce a 50% inhibition of the nitroblue tetrazolium reduction rate determined at 550 nm. challenge experiment: a virulent isolate of y. ruckeri (fj870985) was cultured in tryptic soy broth (tsb, germany) for 48 h at 22°c. the feeding continued for 8 weeks. at the end of the 8 week feeding trial, the intraperitoneal injection of all the groups (6 fish from each tank in triplicate, n=18) was done with 0.1 ml of y. ruckeri suspension, grown in one day, in pbs containing 106 cells/ml. formerly, the challenge dose was established to cause 50% mortality (ld50) in non-supplemented fish. to form a control, some fish were injected with 0.1 ml pbs. after the bacterial challenge, all the fish (ginger supplemented and nonsupplemented) were monitored for two weeks to document any strange behavior, clinical signs, and daily fish death. eventually, the following equation was used to measure the values of the relative percent survival (rps) (amend, 1981): rps = 1 – [(treatment mortality/control mortality) × 100]. statistical analysis: the data are presented as mean±sd of the number of fish per treatment. one way analysis of variance (anova) and tukey’s multiple comparison test were performed for the analysis of the data. statistical analysis was performed 305 int. j. aquat. biol. (2019) 7(5): 301-314 using spss software (version 16.0, chicago, il) and the probability of p<0.05 was considered statistically significant. results no abnormal behavior or mortality was noticed throughout the experimental period. the effects of z. officinale as feed additive on total protein and total ig can be observed in figure 1 and table 3. after 4 and 8 weeks of feeding, the total protein and total ig increased significantly in the 10 g ginger-fed group compared to those of the other groups (p<0.05). table 3. average serum biochemical parameters for rainbow trout juveniles fed with 0, 1, 3, 6 and10 g ginger per 1000 g of feed for 8 weeks. parameters g ginger per 1000g of feed experimental period 2th week 4th week 6th week 8th week alt (u/l) 0 1 3 6 10 9.38±0.18 9.49±0.23 9.54±0.24 9.56±0.21 9.41±0.16 9.56±0.18 9.61±0.16 9.46±0.18 9.65±0.24 9.28±0.22 9.23±0.19 9.31±0.17 9.29±0.22 9.33±0.18 9.52±0.19 9.40±0.25 9.36±0.18 9.70±0.05 9.36±0.14 9.40±0.05 ast (u/l) 0 1 3 6 10 224.3±9.8 238.4±8.6 246.8±9.3 233.6±7.4 246.3±8.6 236.6±10.2 242.8±7.9 238.6±8.5 227.8±8.4 248.3±9.2 241.5±9.7 224.9±8.4 247.3±8.3 251.4±10.3 250.4±9.8 234.8±9.8 239.1±10.3 241.3±9.4 245.9±8.8 252.6±7.2 ldh (u/l) 0 1 3 6 10 729.6±33.4 736.5±34.2 764.8±27.2 775.8±28.3 786.4±33.6 732.4±28.7 728.3±26.9 783.7±34.8 796.7±35.8 813.8±26.7 724.8±32.6 738.8±34.1 792.6±31.9 804.8±28.6 812.8±29.1 744.6±27.2 755.9±34.2 773.7±28.4 809.6±29.2 810.7±32.8 akp (u/ l) 0 22.36±0.42 23.64±0.53 22.17±0.64 23.38± 0.67 1 21.14±0.51 24.18±0.49 22.46±0.43 19.95±0.53 3 22.43±0.62 23.91±0.57 24.17±0.68 25.74±0.86 6 23.19±0.48 25.16±0.64 26.11±0.72* 28.45±0.97* 10 25.47±0.73 27.34±0.86* 29.27±1.08* 31.54±2.79* total protein (g/dl) 0 1 3 6 10 3.68±0.25 3.52±0.27 3.65±0.18 3.89±0.22 4.08±0.27 3.49±0.18 3.59±0.26 3.82±0.23 4.17±0.26 4.48±0.19 3.56±0.24 3.73±0.17 3.93±0.18 4.59±0.26 5.17±0.24* 3.75±0.19 3.89±0.26 4.12±0.21 5.14±0.24* 5.86±0.23* data in a column assigned with asterisk indicates significant difference from the control (p<0.05) (n=15). figure 1. alterations in serum total ig of rainbow trout juveniles fed with 0, 1, 3, 6 and10 g ginger per 1000 g of feed for 2, 4, 6 and 8 weeks. bars assigned with asterisk indicate significant difference from the control (p<0.05) (n=15). 306 soltanian et al./ zingiber officinale extract against yersinia ruckeri in oncorhynchus mykiss lysozyme activity enhanced significantly at the end of weeks 6 and 8 in the fish fed with 10 g extract compared to that of the other groups (p<0.05) (fig. 2). furthermore, a significant enhancement, starting when the second week of the feeding was about to finish, was observed in the phagocytic activity of the fish supplemented with 10 g plant additive compared to other groups (fig. 3). this value was maximized when the feeding trial reached the end of the 8th week. a significant increase was observed in the complement components (c3 and c4) in the 10 g ginger-fed group, compared to the control groups, only by the end of the 8-week feeding trial (figs. 4, 5). in the present research, the activities of ast, alt, and ldh did not show any significant change when the fish diet was supplemented with ginger (p>0.05) (table 3). as regards the serum bactericidal activity, the number of viable bacterial colonies showed a dosereliant reducing trend in fish supplemented with ginger. a significant increase was observed in the serum bactericidal activity of the fish which were fed with both 6 and 10 g plant additive, compared to their table 4. effects of dietary ginger supplementation on antioxidant capacity parameters in serum of rainbow trout. parameters g ginger per 1000g of feed experimental period 2th week 4th week 6th week 8th week sod (u/ mg protein) 0 1 3 6 10 5.63±0.46 8.24±0.56 8.86±0.67 12.29±1.08 15.43±1.59* 7.11±0.83 7.79±0.61 9.27±0.84 14.48±1.37 18.49±2.07* 6.44±0.58 9.33±0.68 11.66±0.96 18.26±1.93* 23.18±2.56* 7.18 ±0.72 10.25±0.73 13.81±1.27 22.36±2.17* 29.62±3.24* cat (u/ mg protein) 0 1 3 6 10 6.12±0.42 4.28±0.46 7.52±0.43 9.47±0.59 12.67±0.73 5.86±0.54 5.88±0.51 8.26±0.53 11.27±0.64 16.48±0.94* 6.43±0.59 6.83±0.43 9.12±0.47 14.52±0.82* 23.4±1.26* 6.94±0.46 8.85±0.47 10.24±0.51* 18.21±1.13* 29.36±2.47* gsh-px (u/ mg protein) 0 1 3 6 10 0.08±0.01 0.11±0.01 0.09±0.01 0.11±0.01 0.11±0.01 0.09±0.01 0.09±0.01 0.10±0.01 0.12±0.01 0.13±0.01 0.11±0.01 0.12±0.01 0.11±0.01 0.11±0.01 0.12±0.01 0.08±0.01 0.11± 0.01 0.12±0.00 0.13±0.01* 0.14±0.01* tac (u/ mg protein) 0 1 3 6 10 3.61±0.24 3.74±0.29 4.42±0.23 4.75±0.28 5.37±0.31 3.76±0.28 3.88±0.23 4.84±0.27 5.07±0.23 6.24±0.26* 3.54±0.22 4.31±0.27 5.11±0.36 5.87±0.36 6.88±0.38* 3.78± 0.20a 5.22±0.21ab 5.81±0.31ab 6.12±0.28b* 7.52±0.42b* data in a column assigned with asterisk indicates significant difference from the control (p<0.05) (n =15). figure 2. alterations in serum lysozyme activity of rainbow trout juveniles fed with 0, 1, 3,6 and10 g ginger per 1000 g of feed for 2, 4, 6 and 8 weeks. bars assigned with asterisk indicate significant difference from the control (p<0.05) (n=15). 307 int. j. aquat. biol. (2019) 7(5): 301-314 respective control groups, at the end of weeks 2, 4, 6, and 8 (fig. 6). dietary ginger supplementation largely increased the activities of serum antioxidant enzymes in a dosedependent manner (table 4). the serum sod activity elevated significantly in the fish fed ginger at 10 g level compared to the control group; this elevation started at the end of week 2 and peaked by the end of the 8-week feeding trial. besides, a significant increase in tac, cat, akp and gsh-px activities was first noticed at the end of week 4 in fish fed ginger at 1% level which continued by both 6 and 10 g plant additive and peaked by the end of feeding trial. the fish mortality trends during the two weeks after the presentation of y. ruckeri challenge are tabulated in table 5. the incorporation of ginger extract in the table 5. effects of dietary ginger supplementation on relative percentage survival (rps) against ld50 concentration of yersinia ruckeri in rainbow trout. g ginger per 1000g of feed survival (%) mortality (%) rps (%) 0 (control) 41.6d 58.4 1 48.4d 51.6 11.6 3 55.4c 44.6 23.6 6 65.2b 34.8 40.5 10 91.4a 8.6 85.3 values with the same superscript in a column do not differ significantly (p<0.05) (n =15). figure 3. alterations in phagocytic activity of peripheral blood leukocytes of rainbow trout juveniles fed with 0, 1, 3, 6 and10 g ginger per 1000 g of feed for 2, 4, 6 and 8 weeks. bars assigned with asterisk indicate significant difference from the control (p<0.05) (n=15). figure 4. alterations in serumc3 component of complement of rainbow trout juveniles fed with 0, 1, 3,6 and10 g ginger per 1000 g of feed for 2, 4, 6 and 8 weeks. bars assigned with asterisk indicate significant difference from the control (p<0.05) (n=15). 308 soltanian et al./ zingiber officinale extract against yersinia ruckeri in oncorhynchus mykiss diet of rainbow trout led to a significant decrease in the cases of fish death after the bacterial challenge was presented. the control group showed 58.4% mortality whereas the groups supplemented with different concentrations of the ginger extract had mortalities ranging 8.6–51.6% in a dose-reliant fashion with the lowermost mortality rate (i.e. highest resistance) noticed in fish fed with 10 g plant extract. furthermore, no clinical signs of the disease were observed in the survived fish belonging to the group fed with ginger at the end of the bacterial challenge. discussions over the past two decades, significant attention has been paid to the use of herbal immune enhancers in aqua farming. the present research aimed to evaluate the biochemical and immunological responses of young rainbow trout fed with experimental diets supplemented with various levels (1, 3, 6, and 10 g per kg feed) of z. officinale. some researchers believe that the serum total protein is the most important representative of the nutritional, biochemical, and health condition in fish species (patriche et al., 2009). the highest serum protein level was found in the group fed with 10 g ginger per 1000 g of feed, a finding which was in parallel with what nya and austin (2009a) reported in their study. similarly, ginger powder as a feed additive could increase the total protein in l. calcarifer (talpur et al., 2013) and juvenile h. huso (gholipour kanani et al., 2014). likewise, increased values of total serum protein in beluga (h. huso) and rainbow trout after figure 6. alterations in serum bactericidal activity of rainbow trout juveniles fed with 0, 1, 3,6and10 g ginger per 1000 g of feed for 2, 4, 6 and 8 weeks. bars assigned with asterisk indicate significant difference from the control (p<0.05) (n=15). figure 5. alterations in serum c4 component of complement of rainbow trout juveniles fed with 0, 1, 3, 6 and10 g ginger per 1000 g of feed for 2, 4, 6 and 8 weeks. bars assigned with asterisk indicate significant difference from the control (p<0.05) (n=15). 309 int. j. aquat. biol. (2019) 7(5): 301-314 dietary supplementation with stinging nettle (urtica dioica) have been reported by binaii et al. (2014) and bohlouli and sadeghi (2014). the increment in total serum protein value in fish treated with ginger supplemented diet might be accompanied by the increased levels of immune parameters which have a protein structure, such as total complements and immunoglobulins. fish innate immunity is the first barrier against microbial invaders and more critical for fish than mammals (uribe et al., 2011). immunoglobulin (igm) is a main element of the teleost humoral immunity and contributes in the recognition and nullification of exogenic agents such as viruses and bacteria which are harmful to fish health (magnadóttir, 2006). in the present work, the total immunoglobulins values elevated following nutritional inclusion of z. officinale. similar results were found in beluga (h. huso) and lates calcarifer following dietary supplementation of ginger (vahedi et al., 2017; talpur et al., 2013). several fish species also presented elevated levels in serum immunoglobulins upon dietary supplementation of the herbal ingredients (nya and austin, 2009b; binaii et al., 2014; bohlouli and sadeghi, 2014; akrami et al., 2015; ngugi et al., 2015; bohlouli and sadeghi, 2016). this might be due to an augmentation effect of herbal extract on lymphocyte activity and their proliferation resulting in provoking immunoglobulin production by b lymphocytes. phagocytosis by macrophages and neutrophils is a critical defense activity against pathogenic bacteria (rao et al., 2006). the increase observed in the phagocytic activity of the fish belonging to the gingersupplemented groups in this study is believed to have been caused by the bioactive elements of ginger (tan and vanitha, 2004), elements which allowed the fish to be less affected by the infection. the findings of the present study are comparable and similar to those of dügenci et al. (2003), nya and austin (2009a), haghighi and rohani (2013), and talpur et al. (2013 a, b), who all reported an enhanced phagocytic activity in rainbow trout and asia sea bass after they had been fed with ginger. the serum lysozyme activity is considered as an effective antibacterial parameter against bacterial pathogens (misra et al., 2006; zhou et al., 2013). in the current work, the lysozyme activity presented significant increment in the group fed with 10 g ginger. these findings are similar to those of haghighi and sharif rohani (2013) and talpur et al. (2013a, b), who reported that the dietary administration of ginger could significantly enhance the serum lysozyme activity in o. mykiss and l. calcarifer, respectively. ginger supplementation at 2% concentration in epinephelus fuscoguttatus, resulted in an increased seric lysozymeactivity (apines-amar et al., 2013). nevertheless, and despite the findings of the present research, some studies have reported no change in the lysozyme activity of young belugas which were fed with ginger (gholipour kanani et al., 2013). such conflicting results might be due to differences in fish species, fish size, and environmental fluctuation. the complement system can quickly recognize and opsonize bacteria for phagocytosis or break them up directly by inducing cell membrane disorder (ahmadi et al., 2012). complement components (c3 and c4) determined in the present study were significantly improved with 10 g of ginger extract indicating that ginger could influence the activity of the complement system in rainbow trout. this is in agreement with saeidi-asl et al. (2017) who recorded elevated levels of complement components c3 and c4 in rainbow trout following nutritional addition of stinging nettle extract. moreover, similar results were found in rainbow trout supplemented with ferulago angulata extract and persian oak (quercus brantii) fruit extract, respectively (bohlouli and sadeghi, 2016; bohlouli et al., 2016). various studies stated that ginger is an effective antioxidant which has cellular protecting and reformative properties induced by destroying free radicals (mallikarjuna et al., 2008; lebda et al., 2012). live organisms encountered to pathogens and various stressors are exhibited to free radicals generated by oxidative stress (tovar-ramırez et al., 2010). to protect themselves against free radicals, the living organisms rely on their enzymatic defense mechanism, and the first line of this mechanism is 310 soltanian et al./ zingiber officinale extract against yersinia ruckeri in oncorhynchus mykiss formed by antioxidant enzymes, such as sod, cat, and gsh-px (abdel-tawwab and hamed heba, 2018). moreover, total antioxidant capacity, which is considered a reliable disease biomarker, could be used to estimate efficiently the antioxidant capability of antioxidants (kusano and ferrari, 2008). our findings reveal that antioxidant enzymes (sod, cat, and gsh-px) activities and total antioxidant capacity increased in the experimental groups with increased ginger concentration. it was stressed that this may be related with several bioactive ingredients (shagol, gingerols, zingeron, etc.) which exist in the composition of ginger and are responsible for anti-oxidative property of ginger (şahan et al., 2018). in a similar study conducted in tilapia, a significant increment in the activity of cat and sod following intensive feeding with garlic has been reported (metwally, 2009). likewise, dietary supplementation of cholesterol considerably improved the serum and hepatic gsh-px, sod, cat and tac activities in rainbow trout (denget al., 2013). noteworthy, sukumaran et al. (2016) reported that nutritional incorporation of ginger at 0.8 and 1% level remarkably upregulated the expression of antioxidant genes (zinc/copper superoxide dismutase [sod1] and glutathione peroxidase [gpx]) in the intestine, kidney, and hepatopancreas of labeo rohita fingerlings. alanine aminotransferase, ast, and ldh enzymes are functional indicators of liver cells impairment. increased values may point to cell necrosis, degeneration, and damage to the liver caused by cellular destruction (bhardwaj et al., 2010). in the current study, no changes were detected in ast, alt and ldh levels in the experimental treatments, representing no liver toxicity in fish following dietary supplementation of ginger. these findings are consistent with those of gholipour et al. (2014) and binaii et al. (2014), who reported no notable changes in the level of alt and ast in beluga supplemented with dietary ginger and nettle. moreover, ast and alt levels indicated even a considerable reduction in sobaity sea bream (sparidentex hasta) fry following dietary supplementation of ginger (jahanjoo et al., 2018). the serum bactericidal activity has an important task in the killing of the pathogens in fish (ellis, 2001). our results showed that serum bactericidal activity increased in the experimental groups with increased ginger concentration. these results are in concord with the findings of nya and austin (2009a, b) and talpur and ikhwanuddin (2012, 2013), who reported an elevated serum bactericidal activity in rainbow trout and l. calcarifer upon diet supplementation with ginger, garlic, and neem leaf (azadirachta indica), respectively. the microbial challenge is an essential stage to assess the influence of immune enhancers on the immune response of the host, as some immunerelevant indicators do not definitely represent the real health condition of the fish (adel et al., 2016a). the enhanced disease resistance observed in the current work is due to improvement in immunological responses. the increase in the immune indices tested here well-connected with the enhanced resistance to an infectious pathogen (y. ruckeri). the findings of the current research obviously indicated that when the rainbow trout’s diet was supplemented with ginger, the rainbow trout resistances to y. ruckeri increased to some extent. the maximum protection was detected in the group fed with 10 g of the extract. likewise, earlier studies found that nutritional addition of various herbal ingredients decreased the mortality against bacterial pathogens (adel et al., 2016b; awad and austin, 2010; talpur, 2014; ngugi et al., 2015). conclusion in conclusion, the current research brings some pieces of evidence that nutritional supplementation of ginger at 10 g for 56 days has improved the antioxidant status and stimulated non-specific immune responses in rainbow trout. evidently, dietary application of ginger has a considerable effect in the control of y. ruckeri infection. moreover, the incorporation of ginger in the fish diet as a substitute to chemotherapies has a great value in disease control in aquaculture. these findings propose that nutritional doses of ginger should be noticed when durable field trials are carried out. 311 int. j. aquat. biol. 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(2013) 1(4): 167-174 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article length-weight and length-length relationships, condition factors and optimal length of some fish species from the persian gulf and oman sea seyed hesam kazemi*1, seyed yousef paighambari1, moslem daliri2, reza abaspour naderi3 1 department of fisheries, gorgan university of agricultural sciences and natural resources, gorgan, iran. 2faculty of marine science and technology, hormozgan university, bandar abbas, iran. 3iranian fisheries organization, tehran, iran. article history: received 19 may 2013 accepted 30 june 2013 available online 2 0 august 2013 keywords: length-weight relationship marine fish condition factor optimum length persian gulf abstract: length-weight relationships (lwrs), relative condition factor (krel), relative weight (wr) and optimal length (lopt) were calculated for five important commercial fishes from iranian waters of the persian gulf and oman sea. samples were collected from 2011 and december 2012 in 11 stations in three iranian provinces (hormuzgan, khozestan and sistan and baluchestan) using trawl and gillnets. also, length-length relationships (llrs) for pampus argenteus and scomberomorus commerson were computed. the values of the exponent b in the length-weight relationship ranged from 2.593 for s. commerson to 2.995 for p. argenteus. krel varied between 1.01 ± 0.08 for parastromateus niger and 1.06 ± 0.41 for p. argenteus. also, wr ranged from 59.12 ± 47.74% for s. commerson to 107.78 ± 107.29% for eleutheronema tetradactylum. the lopt were calculated for all five species. a negative allometric growth was found in s. commerson, while other four species (e. tetradactylum, otolithes ruber, p. niger and p. argenteus) had isometric growth. the length-weight and length-length relationships presented here are for the first time in the iranian coastal waters of the persian gulf and oman sea, which can provide a basis for fisheries management. introduction the persian gulf is lying in sub-tropical climate and located between latitudes 24° 30’ n and longitudes 49° 61’ e (kampf and sadrinasab, 2006; valinassab et al., 2006). at the end of eastern side of the persian gulf, the hormuz strait restricts the water exchange between the persian gulf and the northwestern indian ocean. a wide variety of marine biota are found in the persian gulf, including sea turtles, marine birds, dugongs, whales, dolphins and many (over 500) fish species. many of the fishes are endemic and heavily dependent on the gulf environment (unep, 1999). in the last decade, fish landing has been decreased from 110,000 tonnes to 87,240 tonnes in the persian * corresponding author: seyed hesam kazemi e-mail address: hesam_pep@yahoo.com gulf (valinassab et al., 2006; planning and development department, 2003). there are few studies on fishing management and biological information on fish resources in the region (hosseini, 2002; shokri et al., 2005; raeisi et al., 2011; daliri et al., 2012). length-weight relationships (lwrs) are often used to estimate biomass of the standing stock (martin, 1996), condition indices, ontogenetic changes (sarafan, 1992) and growth studies (garcia et al., 1989; haimovici and velasco, 2000; moutopoulos and stergion, 2002). a lwrs study for a species can provides important insights into the ecology of the species (froes, 2006). 168 kazemi et al./ int. j. aquat. biol. (2013) 1(4): 167-174 length-length relationships (llrs) are useful for standardization of length type when data are summarized (froes, 1998) and also functional for comparative growth studies (moutopoulos and stergiou, 2002). this study reports lwrs, llrs, condition factors and optimum size (lopt) of eleutheronema tetradactylum (shaw, 1804), otolithes ruber (bloch and schneider, 1801), parastromateus niger (bloch, 1795), pampus argenteus (euphrasen, 1788) and scomberomorus commerson (lacepede, 1800) from the persian gulf and oman sea. lwrs and llrs data are available for most fishes of the world, while these data are unavailable in tropical fish species. all these species are of the most commercial importance in the area and this study is the first report of its type on these species from the persian gulf (froes and pauly, 2012). material and method sampling was carried out from may 2011 to june 2012 in 11 stations (10 stations using gillnet and 1 by trawl) in the iranian coastal waters of the persian gulf and oman sea (fig. 1). nominal mesh sizes of gillnets for pampus argenteus, scomberomorus commerson and parastromateus niger, were 135 mm and 95 mm for otolithes ruber and eleutheronema tetradactylum. a shrimp trawl net with 25 mm and 40 mm nominal mesh size of cod-end and panel, respectively, was used for pampus argenteus and scomberomorus commerson, where we measured length-length relationships data. after each catch operation subsamples were collected from 5-10% of the total catch and fork length (fl) or total length (tl) were measured to the nearest 0.1 cm using a measuring board. total weight was measured to the nearest 0.1 g using an electronic balance. the relationship between fl or tl (l) and total weight (w) usually expressed by the equation w= a lb, which w is weight in gram and l is length in centimeters (ricker, 1975). lwr parameters (a and b) have been calculated from the logarithmic equivalent as log w= log a + b log l (zar, 1984). llrs were determined by the same method: fl = a tl where fl = fork length and tl = total length. the 95% confidence limits of b-value were calculated using the equation 95% cl = b±t0.05, n-2.sb, where n is the number of specimens and sb is the standard error of the slope (b). a t-test was used to compare the b-values, which obtained in the linear regression, with isometric values for each species (sokal and rohlf, 1987): ts = (b-3)/sb, where ts is the t test value, b is the lwr parameter and sb the standard error of the b-value. the relative weight (wr) and relative condition factor (krel) were computed using the equations, wr = 100 (w/ws), where wr is the relative weight, w is the total weight of a specimens and ws is a standard weight (75 percentile of observed weights at that length; wege and anderson, 1978), krel = w/(a l b), where w is the body weight in g, l is total length in cm, a and b are lwr parameters (le cren, 1951). the optimal length (lopt) was computed by the following equations (froese and binohlan, 2000; froese 2006); lopt = 10 1.0421 log linf 0.2742, linf = 10 0.044 + 0.9841 log lmax, where linf is the asymptotic length and lmax is the mean length of the three largest specimens caught in last 20 years (from data in this study; carpenter et al., 1997; asadi et al., 1996). results a total of 3089 specimens of five important commercial fish species of the persian gulf belonging to five families were collected from 11 stations. length and weight characteristics and parameters of lwrs of all species, confidence limit of b, standard error of a, r2 and correlation coefficient (r) are presented in table 1. the r2 for all five species were over 0.847 and all regressions (lwrs) are significantly different from 0 (p<0.05). the estimates of the parameter b ranged from 2.593 for scomberomorus commerson to 2.995 for pampus argenteus. the length-length relationship parameters for pampus argenteus and f a m ily s p e c ie s l e n g th ra tio (t l /f l ) c h a ra c te ristic w e ig h t c h a ra c te ristic l w r p a ra m e te rs n t l /f l m e a n s d m in m a x m e a n s d m in m a x a s e b c l r 2 r s tro m a te id a e p a m p u s a rg e n te u s 1 0 8 9 f l 1 8 .9 2 5 .2 8 6 .5 3 7 .5 2 1 0 .5 2 3 3 .0 9 1 0 1 5 0 0 0 .0 2 3 0 .0 0 3 2 .9 9 5 0 .6 0 3 7 0 .8 4 7 0 .9 2 s c o m b rid a e s c o m b e ro m o ru s c o m m e rso n 5 8 2 f l 8 9 .7 2 3 3 .2 7 1 2 1 4 8 6 7 2 8 .4 3 5 4 3 3 .2 7 1 1 0 2 4 1 0 0 0 .0 4 4 0 .0 1 3 2 .5 9 3 0 .2 4 5 4 0 .9 8 6 0 .9 9 c a ra n g id a e p a ra stro m a te u s n ig e r 8 5 8 f l 3 6 .2 3 4 .7 4 2 6 4 9 1 2 4 2 .6 4 4 8 6 .9 9 5 0 0 2 7 0 0 0 .0 3 3 0 .0 0 2 2 .9 1 9 0 .1 2 9 9 0 .9 5 9 0 .9 8 s c ia e n id a e o to lith e s ru b e r 3 9 8 t l 3 5 .9 6 7 .0 8 2 2 .5 5 8 5 8 4 .0 2 3 5 4 .6 7 1 0 3 2 1 0 5 0 .0 3 2 0 .0 0 6 2 .7 0 6 0 .3 5 1 2 0 .8 6 1 0 .9 3 p o ly n e m id a e e le u th e ro n e m a te tra d a c ty lu m 1 6 2 f l 3 9 .6 1 1 1 .9 3 1 9 7 6 1 1 4 4 .7 2 1 1 3 9 .4 7 1 0 9 6 1 5 7 0 .0 1 6 0 .0 0 3 2 .9 6 1 0 .3 7 0 7 0 .9 4 1 0 .9 7 t ab le 1 . t h e le n g th a n d w eig h t d ata an d p ara m eters o f len g th -w eig h t relatio n sh ip (l w r ) o f fiv e c o m m ercial fish sp e cies in th e p ersian g u lf (m ay 2 0 1 1 to ju n e 2 0 1 2 ). kazemi et al./ int. j. aquat. biol. (2013) 1(4): 167-174 s p e c ie s t l c h a ra c te ristic s f l c h a ra c te ristic s l l r p a ra m e te rs n m e a n s d m in m a x m e a n s d m in m a x a s e b c l r 2 p a m p u s a rg e n te u s 3 9 0 1 8 .5 3 3 .8 9 9 .5 2 9 1 5 .4 6 5 .3 2 6 .5 2 4 .5 0 .4 7 6 0 .0 1 2 1 .1 8 6 0 .0 6 2 5 1 0 .9 8 s c o m b e ro m o ru s c o m m e rso n 7 6 3 3 .4 2 5 .7 4 1 5 .5 4 3 2 9 .5 5 5 .5 1 1 2 3 9 0 .6 1 1 0 .0 2 9 1 .1 0 5 0 .0 3 7 8 8 0 .9 8 9 t ab le 1 . t h e le n g th d ata a n d le n g th -len g th relatio n sh ip (l l r ) o f tw o m arin e fish sp ecies in th e p ersian g u lf. d ata are ta k e n fro m tra w l statio n . n : th e n u m b er o f sam p les, s d : stan d ard d ev iatio n , s .e : stan d ard erro r an d c l: 9 5 % c o n fid en ce lim its, f l : fo rk len g th (cm ), t l : to tal len g th (cm ), to tal w eig h t is in g . n : th e n u m b er o f sam p les, s d : stan d ard d ev iatio n , s .e : stan d ard erro r an d c l : 9 5 % c o n fid en c e lim its, f l : fo rk len g th (cm ), t l : to tal len g th (c m ). 169 kazemi et al./ int. j. aquat. biol. (2013) 1(4): 167-174 species ws 𝑊𝑟 mean sd min max 95% cl pampus argenteus 235 89.57 99.18 4.25 638.29 4.94 scomberomorus commerson 11380 59.12 47.74 0.96 211.77 3.25 parastromateus niger 1500 82.84 32.46 33.33 180 1.82 otolithes ruber 785 74.39 45.11 13.12 168.15 3.73 eleutheronema tetradactylum 1062 107.78 107.29 10.26 579.75 13.95 table 3. table 3. relative weight (wr) of five fish species of persian gulf during may 2011 to june 2012. pampus argenteus scomberomorus commerson parastromateus niger otolithes ruber eleutheronema tetradactylum lopt 34.33 109.45 34.66 54.64 88.38 table 4. the optimum length (lopt in cm) of five different fish species in persian gulf. table 5. the parameters of the length-weight relationship (lwr) of selected species in the region and other location of the world. species location length sex a b authors eleutheronema tetradactylum chilka lake 20-840 tl unsexed 1.57 × 10-6 3.0405 patnalk (1969) parastromateus niger bushehr (northwest of persian gulf) 13.5-43 fl unsexed 0.0342 2.9477 daliri et al. (2012) bangladesh, bay of bangal fl unsexed 0.0211 3.012 mustafa (1999) indonesia, western region 5-38 tl unsexed 0.0073 3.319 pauly et al. (1996) bangladesh, bay of bangal sl unsexed 0.0138 2.5411 pati (1981) otolithes ruber kuwait 14.2-45.5 sl unsexed 0.0203 2.916 hussain and abdullah (1971) pampus argenteus bangladesh, bay of bangal sl male 0.0134 2.5307 pati (1981) bangladesh, bay of bangal sl female 0.009523 2.692 pati (1981) korea rep, east china and southern korean waters fl unsexed 0.0345 3.000 lee et al. (1992) persian gulf, coastal waters of iran fl male 0.0187 2.91 sadeghi et al. (2009) scomberomorus commerson persian gulf, coastal waters of iran fl female 0.0194 2.89 sadeghi et al. (2009) queens land, east coast stock 47-155 fl unsexed 0.0099 2.95 mcpherson (1992) 170 169 kazemi et al./ int. j. aquat. biol. (2013) 1(4): 167-174 scomberomorus commerson are shown in table 2. the r2 values for both species were greater than 0.98. the t-test for lwrs showed that eleutheronema tetradactylum (ts = 0.174), otolithes ruber (ts = 1.38), parastromateus niger (ts = 1.02) and pampus argenteus (ts = 0.013) were isometric in growth while, scomberomorus commerson (ts = 2.731) showed negative allometric growth (all computations were considered at α = 5%). the mean relative condition factors (krel) was 1.0299 (± 0.021) and it ranged from 1.01 (± 0.08) for parastromateus niger to 1.06 (± 0.41) for pampus argenteus (fig. 2). relative weight (wr) of all species are tabulated in table 3. values were ranged between 59.12 ± 47.74% for scomberomorus commerson to 107.78 ± 107.29% for eleutheronema tetradactylum. the optimal length (lopt) of all species are presented in table 4. discussion the length-weight relationship is a very important tool in fisheries assessment (garcia et al., 1989; haimovidici and velasco, 2000; arslan et al., 2004) and also standing crop biomass can be estimated based on this value (morey et al., 2003). froese (2006) expressed that the exponent b should normally lies between 2.5 to 3.5. the b-value ranged from 2.593 (for scomberomorus commerson) to 2.995 (for pampus argenteus) in this study, so the parameters can be used safely within the indicated length ranges. previous studies on the parameters of the lwr of selected species in persian gulf and other location of the world presented in table5. in general, the variations in parameters (table 5) may occur according to sex, gonad maturity, season, habitat type, health, food availability, environmental condition (such as temperature and salinity), degree of stomach fullness, differences in the length range of the caught specimens, sampling procedure and fishing gear (bagenal and tesch, 1978; avşar, 1988; wootton, 1992; froese, 2006). also, field measurement can be fluctuating according to differences in fish surface wetness; boat movements and other adverse environmental condition (gutreuter and krzoslen, 1994). isometric growth (b = 3) for eleutheronema tetradactylum, otolithes ruber, parastromateus niger and pampus argenteus indicated that length increases with body weight (gayanilo and pauly, 1997) and small specimens have the same form and condition of large specimens (froese, 2006). however, scomberomorus commerson shows negative allometric growth (b < 3), the condition decreases with increase of age or elongation of form and increase of length (hile, 1936). condition factors are used to compare the condition, fatness, or well-being (tesch, 1968) of fishes, based on the assumption that heavier fish of a given length are in better condition (froese, 2006). lecren (1951) provided an equation for calculating relative figure 1. the map of the study area with trawl () and gillnet sampling points (⬤). figure 2. t relative condition factor (krel) (± sd) of five fish species from iranian waters of the persian gulf. 171 170 kazemi et al./ int. j. aquat. biol. (2013) 1(4): 167-174 condition factor (krel) which compares the observed weight of an individual with the mean weight of the same length. the relative weight (wr) that indicates the percentage of the weight of an individual fish in comparison to the standard weight at the same length (wege and anderson, 1978), which can be used as a management tool for fisheries managers. in this study, eleutheronema tetradactylum (wr = 107.7 ± 107.29%) had the largest relative weight showing the best performance, whereas scomberomorus commerson (wr = 59.1 ± 47.74%) was not a proper representative of the environmental condition. froese and binohlan (2000) represented the equation of the optimal length (lopt) which indicates the fit size of fish species for catch. “for most iteroparous fishes (lopt) lies between the first and second spawning, thus, making overfishing is theoretically impossible” (mayers and mertz, 1998; froese, 2006), so these data can be useful for fisheries management in the region. here, the sex of specimens and seasonal variations were not determined, so it can be considerable for future studies. references arslan m., yildirim a., bekta s. 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(2015) 3(2): 108-113 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2015 iranian society of ichthyology original article effects of grape pomace extract on the quality and shelf life of silver carp (hypophthalmicthys molitrix) fillets during chill storage shirin hasani1, ebrahim alizadeh*1, mostafa yousef elahi2 1department of fisheries, faculty of natural resources, university of zabol, zabol 98615-538, iran. 2department of animal science, faculty of agriculture, university of zabol, zabol 98615-538, iran. article history: received 11 june 2014 accepted 24 september 2014 available online 2 5 april 2015 keywords: grape pomace tvb-n sensory analysis antimicrobial abstract: the effects of grape pomace extract (0, 2 and 4%) on quality and shelf life of silver carp (hypophthalmicthys molitrix) fillets during chill storage (4°c) were investigated. the control and the treated fillets were analyzed periodically for microbiological (tvc and ptc), chemical (tvb-n), and sensory characteristics. the results showed that grape pomace-treated samples have lower tvb-n (24.2 and 21.2 mg n/100 g, respectively), tvc (7.33 and 7.09 log cfu/g, respectively) and ptc (7.26 and 7.03 log cfu/g, respectively) at the end of the storage period. the results revealed that the addition of grape pomace extract has a positive effect on the sensory quality of silver carp fillets by retaining proper quality characteristics for a longer time and extends their shelf life during chill storage. introduction the smell, taste, freshness, absence of specific microorganism, size and composition are the most important factors for determining the quality of marine products (connell, 2002). seafood is spoiled more than other foods containing high percentage of the protein (huss, 1988). the psychrophilic bacteria spoilage is occurred in cold storage in seafood (gram and huss, 1996) and has serious health risk to consumers. therefore, additions of the antioxidant and antibacterial substances to seafood products are useful to improve their quality, increase the shelf life and prevent economic losses (yin and cheng, 2003). grape pomace is a waste product of the grape juice (özkan et al., 2004) having high phenolic compounds (arvanitoyannis and van houwelingenkoukaliaroglou, 2005); therefore, it has drawn attention of researches to use it in seafood products (caillet et al., 2006; bozan et al., 2008). as free radical, the phenolic compounds can potentially interact with biological systems and prevent the human neurodegenerative diseases, * corresponding author: ebrahim alizadeh e-mail address: ebi_alizadeh2003@yahoo.com cardiovascular disorders and cancer (poudel et al., 2008). phenolic compounds are found in grape seeds, skins and stem extracts (jayaprakasha et al., 2003). flavanols are the most abundant phenolic compounds in grape skins with grape seeds being rich in monomeric phenolic compounds, such as (+)catechins, (-)-epicatechin and (-)-epicatechin-3ogallate, and dimeric, trimeric and tetrameric procyanidins (kammerer et al., 2004). these compounds act as antimutagenic and antiviral agents and can also be used to preserve food because of their protective effects against microorganisms (vattem et al., 2004). tesaki et al. (1999) confirmed that phenolic compounds are the important compounds acting against bacteria. therefore, the present study aimed to determine the effect of the grape pomace extract on the quality and shelf life of silver carp (hypophthalmichthys molitrix) fillets during chill storage. materials and methods sample preparation: experiments were carried out 109 hasani et al./ antimicrobial effects of grape pomace on the quality of silver carp fillets with fresh silver carp purchased from market (zabol, southeast iran) that had been delivered 5 hrs after slaughtering. the fresh silver carp with weight of 1.5 kg were selected and transported in isothermal iceboxes to the fish product processing laboratory at university of zabol in 2011. the fishes were cleaned and filleted and then 0 (as control group), 2% and 4% total phenolic (tp) of red grape pomace extract (g extract/100 g flesh) were added to the same weighed fillets (about 100 g). the samples were placed in moisture-impermeable plastic bags, stored in chill room (4°c) and used for analysis on 0, 3, 6, 9, 12 and 15 days. the experiments were performed in three replicates. extraction: fresh red grapes (vitis vinifera) were prepared from shahryar city (iran) and transferred to the laboratory. they were stored at -20°c until they were made into pomace. grape pomaces were dried at 45°c for 72 hrs and milled to a particle size less than 0.5 mm. the dried grape pomace (200 mg) was placed in a test tube, then 20 ml diethyl ether containing 1% acetic acid was added to remove pigments and fat. the solution was thoroughly shaken at room temperature for 20 min and centrifuged at 3000 g and 4°c for 10 min, after which the supernatant was recovered. ten milliliters of acetone 70% (v/v) was added to the residue, and shaking and centrifugation were repeated. extractions were performed to calculate the total phenolic content (makkar, 2000). bacteriological analysis: the total viable counts (tvc) and psychrophilic total counts (ptc) of fish fillets were estimated. fish muscle (10 g) of each treatment was aseptically weighed and homogenized with 90 ml sterile 0.85% normal saline for 1 min at room temperature. furthermore, the decimal dilutions were prepared and then 0.1 ml of each dilution was pipetted onto the surface of tripthic soye agar (tsa). they were incubated for 48 hrs at 35°c for tvc according to egan et al. (1997) and 10 days at 4°c for ptc based on mcmeekin et al. (1993). microbial loads were expressed as log10 cfu/g. chemical analysis: the tvb-n was estimated using steam distillation method (aoac, 2002). the steam distillation was carried out by distillation after the addition of mgo to the homogenized fish samples. the distillate was collected in a flask containing a 2% aqueous solution of boric acid, and a mixed indicator produced from dissolution of 0.1 g of methyl red to 100 ml of ethanol. then, the boric acid solution was titrated with a 0.1 n sulphuric acid solution. the tvb-n value (mg n/100 g of flesh fish) was determined based on the consumption of sulphuric acid. sensory evaluation: sensory quality of fish sample was evaluated by twenty trained panelists. the panelists have scored for colour, odour, flavour, overall acceptability and texture, using a seven-point hedonic scale (0, dislike extremely to 7, like extremely) (astm, 1969). a sensory score of two was taken as the borderline of acceptability. for this evaluation, the fillets were fried (sunflower oil, bahar, iran) for approximately 3 minutes at 180°c. statistical analysis: data were analyzed using twoway anova by sas version 9.1 software. when differences were significant (p<0.05), the mean values were evaluated by the least significant differences (lsd). kruskal-wallis test was used to sensory evaluation. the mann-whitney u-test is used to paired comparisons test. results microbiological analysis: figure 1 and 2 show total viable counts (tvc) and psychrophilic total counts (ptc) of silver carp fillets during chill storage. the lowest and highest of tvc and ptc were found on the first and 15 days of storage between 3.96 to 7.91 and 3.87 to 7.89 log cfu/g for the 4% total phenolic and control, respectively. the tvc and ptc values were increased significantly (p<0.05) during chill storage. there were significant differences between all treatments for all days of storage. chemical analysis: table 1 shows the results of the chemical analysis. the results show that the tvb-n values increase significantly (p<0.05) during chill storage. the total phenolic also had a significant effect on all the tvb-n values (p<0.005). the 110 int. j. aquat. biol. (2015) 3(2): 108-113 lowest values were found on the first day of storage for the 2 and 4% total phenolic and the highest were found on the 15 days of storage for control group. the significant differences were observed between all treatments for all days except first day of storage. sensory evaluation: the results of the sensory evolution are given in table 2. the results indicate that sensory scores showed a significant decline in all samples with increasing storage period, and the results also indicate that the sensory evolution values decreased significantly with the increasing chemical and microbial spoilage (p<0.05). the overall scores of 4% total phenolic were higher than the 2% total phenolic and control. discussion microbiological analysis: it has been estimated that about one-third of the world’s food production is lost annually on account of microbial spoilage (tingting et al., 2012). in the present study, the alternations in tvc during 15 days storage showed that microbial growth had been significantly (p<0.05) influenced by the addition of grape pomace extract. the control group attained a tvc value of 7.13 log cfu/g on day 9, which was close to the microbial acceptability limit of 7 log cfu/g for raw fish (ojagh et al., 2010), indicating a microbiological shelf-life about 9 days for the control samples. in other works, a shelf-life of less than 10 days has been reported for pacific salmon (onchorhynchus nerka) stored at 1ºc (sallam, 2007) and crucian carp (carassius auratus) stored at 4ºc (tingting et al., 2012). the results showed that grape pomace extract delayed microbial growth and extended the shelf-life of the silver carp fillets. in the present study, the treated samples with grape pomace extract did not reach the microbiological acceptability limit during the 12 (2% tp) and 15 (4% tp) days storage showing significantly lower tvc values than that of the control one (p<0.05). phenolics, with effective 3 4 5 6 7 8 9 0 3 6 9 12 15 storage time (days) t v c ( lo g 1 0 c fu /g ) control 2% 4% figure 1. changes in total viable counts of silver carp fillets during chill storage. 3 4 5 6 7 8 9 0 3 6 9 12 15 storage time (days) p t c ( lo g 1 0 c fu /g ) control 2% 4% figure 2. changes in psychrophilic total counts of silver carp fillets during chill storage. table 1. changes in tvb-n (mg n/100 g fish flesh) values of silver carp fillets during chill storage. storage time (days) control 2% 4% 0 4.71 ± 0.45 af 4.2 ± 0.00af 4.2 ± 0.00af 3 9.8 ± 0.00ae 7.23 ± 0.40be 5.37 ± 0.40ce 6 13.53 ± 0.40ad 11.67 ± 0.40bd 10.4 ± 0.56cd 9 21.28 ± 0.37ac 16.8 ± 1.40bc 13.67 ± 0.35cc 12 27.4 ± 0.70ab 20.83 ± 1.10bb 19.37 ± 0.50bb 15 30.50 ± 0.82aa 24.2 ± 0.30ba 21.2 ± 0.78ca values are mean ± standard deviation of three determinations. capital letters (a-c) in the same line indicate significant differences (p<0.05) of treatment. small letters (a-f) in the same column indicate significant differences (p<0.05) of storage. 111 hasani et al./ antimicrobial effects of grape pomace on the quality of silver carp fillets antimicrobial and antioxidant activities, could significantly delay microbial growth and have been widely applied as a natural food preservative (fan et al., 2008). tingting et al. (2012) confirmed that shelf-life of crucian carp could be prolonged for an additional 6 days during 4ºc storage by adding natural preservative (tea polyphenols and rosemary extract). based on the results, ptc increased significantly (p<0.05) throughout chill storage in all treatments, especially in control one. the significant reduction of ptc was observed in 2% and 4% treatments showing inhibitory effect of total phenolic on spoilage bacteria (fan et al., 2008). based on the microbiological acceptability limit of 7 log10 cfu/g for fresh water and marine species (icmsf, 1986), the results indicated that treatments with 2% and 4% tp have equal effect in inhibiting spoilage bacterial growth and extending the chill storage life in silver carp fillets to 12 and 15 days compared to 9 days in control group. chemical analysis: the results of alternation in tvb-n values in silver carp fillets during chill storage showed that the values increased gradually in all treatments during the 15-day storage. the tvbn value is one of the most widely used indicators of seafood deterioration. it is mainly composed of ammonia and primary, secondary and tertiary amines (beatty, 1938), resulted from degradation of the proteins and non-protein nitrogenous compounds. these compounds are chiefly produced by microbial activity (ruiz-capillas and moral, 2005; yilmaz et al., 2009) and proteolytic enzymes (yasin and aboutaleb, 2007). this increase can be related to microbial activity during cold storage (yilmaz et al., 2009; pacheco-aguilar et al., 2000). tvb-n values increased from an initial value (mg n per 100 g of fish flesh) of 4.71 ± 0.45–30.50 ± 0.82 in control group, to 4.2 ± 0.00–24.20 ± 0.30 in treatment with 2% total phenolic, and to 4.2 ± 0.00– 21.20 ± 0.78 in treatment with 4% total phenolic at the end of storage (day 15). the two treated groups maintained a significantly (p<0.05) lower tvb-n value than that of control one. this can be attributed to either a more rapidly reduced bacterial population or decreased capacity of bacteria for oxidative deamination of non-protein nitrogen compounds or both (fan et al., 2008; tingting et al., 2012), which was due to the effect of the grape pomace extract on silver carp fillets. by considering a tvb-n value of 25 mg n/100 g fresh fish as the acceptable limit proposed by kilincceker et al. (2009), the shelf-life table 2. changes in sensory scores of silver carp fillets during chill storage. storage time (days) flavour odour texture colour overall acceptability 0 control 7aa 7aa 7aa 7aa 7aa 2% 7aa 7aa 7aa 7aa 7aa 4% 7aa 7aa 7aa 7aa 7aa 3 control 5ab 5bb 5bb 5bb 5ab 2% 5ab 7aa 5bb 7aa 5ab 4% 5ab 7aa 7aa 7aa 5ab 6 control 1.5bc 1.5bc 1.5bc 1.5bc 1.5bc 2% 3ac 5ab 5ab 5ab 5ab 4% 4ac 5ab 5ab 5ab 5ab 9 control 2% 1.5bd 2ac 2ac 2ac 1.5bc 4% 3ad 3ac 3ac 3ac 3ac 12 control 2% 4% 1.5ae 1ad 1.5ad 1ad 1.5ad capital letters (a-b) in the same day indicate significant differences (p<0.05) of treatment. small letters (a-e) in the same column indicate significant differences (p<0.05) of storage. 112 int. j. aquat. biol. (2015) 3(2): 108-113 of the control and the treated groups were about 12 and 15 days, respectively. sensory evaluation: spoilage and quality deterioration can be assessed by chemical and physical methods and sensory evaluation (connell, 1990). not all chemical assessments give good correlation to quality changes, hence sensory evaluation is a necessity (hardy, 1979). the results indicated a significant decline of sensory scores in all groups with increasing storage time (özogul et al., 2004). the sensory properties of the control group was received a lower score than that of 2% and 4% groups. thus, the control group was acceptable up to 6 days and 2% and 4% samples were in acceptable conditions up to 9 and 12 days of chill storage. this results showed that the acceptable limits of microbial and chemical parameters is not confirmed the quality changes, thus the sensory evaluation is requirement. fan et al. (2008) reported that sensory scores of silver carp is reduced with increasing storage period in compared to those of treated with tea polyphenols. based on the results, 4% treatment could be retaining their good quality in terms of sensory evolution compared to others. these results are agreed with the results of microbial and chemical analyses. as conclusion, the present study showed that treatment with 2% and 4% total phenolic of red grape pomace extract could effectively retard microbial growth, delay chemical deterioration, maintain or improve sensory attributes and extend the shelf-life of silver carp fillets for 3-6 days during chill storage. natural preservatives such as grape pomace extract can be used as a safe method for storage of silver carp fillets, which is quite promising for food industry. references aoac. (2002). official methods of analysis (17th ed.). washington, dc: association of official analytical chemists. arvanitoyannis i.s., van houwelingen-koukaliaroglou m. 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(2020) 8(3): 184-193 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article antioxidant and antibacterial properties of protein hydrolysate from rocky shore crab, grapsus albolineathus, as affected by progress of hydrolysis mina emadi shaibani1, behrooz heidari*1,2, saber khodabandeh3, shirin shahangian1, saeed mirdamadi⁕4, mahta mirzaei5 1department of biology, faculty of science, university of guilan, rasht, iran. 2department of marine sciences, caspian sea basin research center, university of guilan, rasht, iran. 3department of marine biology, faculty of marine science, tarbiat modares university, noor, iran. 4department of biotechnology, iranian research organization for science and technology (irost), tehran, iran. 5department of food science and technology, shahr-e-qods branch, islamic azad university, tehran, iran. s a rticle history : received 18 julay 2019 accepted 31 may 2020 available online 2 5 june 2020 k e y w o rd s : crab protein hydrolysis antioxidant antibacterial abstract: antibacterial and antioxidant activity of the rocky shore crab, grapsus albolineathus, protein hydrolysate (cph), with different degree of hydrolysis (dh) prepared using alcalase was investigated. the results showed that by increasing dh with reaction time up to 90 min, the dpph radical scavenging activity of the hydrolysates raise, followed by a decrease in the next stages from 90 to 180 min. interestingly, abts radical scavenging of the hydrolysates increase up to 120 min, and cph120 show the highest activity with no significant difference with cph90 and cph180. the degree of hydrolysis applied a significant influence on the antibacterial activity of crab hydrolysates against gram-positive bacteria, with a significant increase up to 90 min. the maximum zone of inhibitions was recorded against listeria monocytogene for cph90:14.55 mm. the results suggest that the alcalase hydrolysis of rocky shore crab can produce bioactive peptides with potent antioxidant and antibacterial activities as affected by the degree of hydrolysis up to a certain level. introduction marine by-products comprise fishery wastes or low commercial species. enzymatic hydrolysis is one of the best methods for protein recovery from such a marine protein sources (petrova et al., 2018). the primary purpose of a hydrolysis process is to obtain high-quality protein with functional properties. an enzymatic hydrolysis process under optimized conditions could improve the functional properties and bioactivities of protein hydrolysates. several studies have been proved alcalase 2.4l (from bacillus licheniformis) as an effective enzyme for controlled protein hydrolysis due to high degree of hydrolysis in a short duration under moderate ph condition (kristinsson and rasco, 2000; rajapakse et al., 2005; ovissipour et al., 2009). bioactivity of protein and peptides from fish and shellfish by-products are of great interest for pharmaceutical and sea food industries. the antioxidant activity of threadfin bream surimi wastes (wiriyaphan et al., 2012) and unicorn *correspondence: behrooz heidari; saeed mirdamadi e-mail: bheidari@guilan.ac.ir; mirdamadi@irost.ir leatherjacket skin (karnjanapratum and benjakul, 2015), ace inhibitory and antihypertensive activities of tilapia by-product and mackerel skin gelatin (khiari et al., 2013), anticancer activity of shrimp shell waste (kannan et al., 2011) and gelatin hydrolysate of unicorn leatherjacket skin (karnjanapratum et al., 2016), and antimicrobial activity of american lobster (battison et al., 2008) and atlantic mackerel (ennaas et al., 2015) are some of works in this regard. natural compounds with high reactive oxygen species (ros) reduction activity have attracted a great consideration since ros are associated in cancer development. the great advantageous of the antioxidant peptides isolated from marine sources in reactive oxygen species (ros) and free radicals scavenging and terminating the radical chain reaction were reported (ngo and kim, 2013). in addition, during the past decades, the increment of antibiotic-resistant bacteria has been required for natural antimicrobial agents. therefore, natural 185 int. j. aquat. biol. (2020) 8(3): 184-193 antimicrobial agents have attracted increasing attentions due to their potential role as biopreservative or therapeutics (sperstad et al., 2011). antimicrobial peptides (amps) are part of the innate immune systems of some animal species, as a first line of defense against infections and have been recently isolated and characterized from different organisms (sila et al., 2014b). to date, several antimicrobial peptides (amps) with low molecular weights and cationic nature which are active against diverse group of bacteria have been identified and isolated from marine organisms by enzymatic hydrolysis (doyen et al., 2012; ghanbari et al., 2012; beaulieu et al., 2013; sila et al., 2014a). however, the information regarding the functional properties of bioactive peptides from crabs as protein-rich by-products is limited. grapsus albolineatus is a relatively large-size crab species in the persian gulf, where it has a large population on the rocky intertidal shores. therefore, purpose of the present study was to produce protein hydrolysate from g. albolineatus, as a noncommercial species, in the controlled hydrolysis conditions of the alcalase and to determine the effect of degree of hydrolysis (dh) on the antioxidant and antibacterial activity of hydrolysates produced. materials and methods summarizing the experimental design is shown in figure 1. in the current study, alcalase was provided from novozymes (denmark), and methanolwas from merck (darmstadt, germany) companies. abts 2,2azino-bis(3-ethylbenzothiazoline6-sulfonic acid) diammonium salt, dpph (2,2-diphenyl-1-picryl hydrazyl), trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and tca (trichloroacetic acid) were purchased from sigma (st. louis, mo, usa). bhi (brain–heart infusion broth, life, milan, italy) and mrs (de man, rogosa and sharpe, containing: peptone 10 g l-1; yeast extract 4 g l-1; glucose 20 g l-1; beef extract 8 g l-1; k2hpo4 2 g l1; sodium acetate trihydrate 5 g l-1; ammonium citrate 2 g l-1; mgso4, merck, germany) used as culture media. bacterial strains in the study, including listeria monocytogenes (ptcc 1306), staphylococcus aureus (ptcc 1112), escherichia coli (ptcc 1330) and lactobacillus sakei (ptcc 1712) were preserved strains from the persian type culture collection, irost, iran. samples preparation: live specimens of g. albolineatus (n=10, 55±2.63 g) were collected off rocky shore of the qeshm island, the persian gulf (fig. 2). the samples were frozen at −20°c, and then stored in a sealed styrofoam box containing ice and transported to the laboratory within 2 hrs. enzymatic hydrolysis: the total body of g. albolineatus was first minced using a grinder meat mincer (parskhazar, tehran, iran) blades, then for inactivating the endogenous enzymes, the sample heat-treated at 85°c for 20 min. afterwards, it was mixed with deionized water in a ratio of 1:2 and homogenized. enzymatic hydrolysis was performed with alcalase (2% v/w) under the optimal condition of the enzyme (ph 8.5; 55°c). crab protein hydrolysate (cph) were taken at 30 (cph30), 60 (cph60), 90 (cph90), 120 (cph120) and 180 (cph180) min of hydrolysis and inactivated at 95°c (15 min) followed by 10 min centrifugation at 8000 g. estimation of soluble protein (sp): soluble protein concentration during different stages of the hydrolysis was determined based on lowry et al. (1951). bsa (bovine serum albumin) was used as the protein standard. absorbance was measured at 540 nm using a uv-1601 spectrophotometer (shimadzu, kyoto, japan). change in the amount of soluble protein was calculated as the ratio of soluble protein in the hydrolysate relative to the initial amount of soluble protein in non-hydrolyzed mixture by following equation: soluble protein ratio = [soluble protein in a hydrolyzed crab-soluble protein in non-hydrolyzed crab / soluble protein in nonhydrolyzed crab] × 100. the degree of hydrolysis (dh)l: the dh of all hydrolysates (cph30, cph60, cph90, cph120, and cph180) were measured according to the method described by hoyle and merritt (1994), using tca (trichloroacetic acid) protein precipitation. briefly, an equal volume of 20% tca was added to the 186 emadi shaibani et al./ antioxidant and antibactrial properties of grapsus albolineathus protein hydrolysate hydrolysates followed by centrifugation at 6700×g for 20 min. the following equation was applied to calculate the dh: dh (%) = [soluble protein in tca-sample mixture / soluble protein in initial sample] × 100 peptide chain length (pcl): the average peptide chain length (pcl) introduced by adler-nissen (1986) was calculated from the degree of hydrolysis in the following equation. pcl = 100/ dh antioxidant activity dpph assay: the dpph radical scavenging activity was assayed according to the methods described by binsan et al. (2008) with a slight modification. briefly, 1.5 ml of samples (cphs) were added to 1.5 ml of 0.15 mm methanolic solution of dpph. the mixture was vigorously shaken and then incubated at 25°c in the dark for 30 min. the absorbance was read at 517 nm by a uv-1601 spectrophotometer (shimadzu, kyoto, japan). the dpph radical scavenging capacity was estimated according to the following equation: radical-scavenging activity (%) = [(acontrol _ ahydrolysate / acontrol)] x 100 the trolox standard curve was used to determine the antioxidant capacity as μmol trolox/mg protein sample. abts assay: abts radical scavenging activity was determined by the slightly modified method of re et al. (1999). for the present study, the test samples and abts solution were produced by reacting 7 mm abts solution with 2.45 mm potassium persulphate and leaving the mixture in the dark for 12-16 hrs. afterward, the solution was diluted with phosphate buffer (5 mm, ph 7.4) to an absorbance of 0.700±0.05 at 734 nm. then 25 µl of sample was added to 1 ml of reagent and incubated at 25°c. the calculation of scavenging percentage is as described for the dpph assay. the activity was calculated as μmol trolox/mg protein sample using trolox standard curve. antibacterial activity bacterial strains and growth media: the antibacterial activity of hydrolysate samples was studied against five gram-positive and gram-negative bacterial strains, including l. monocytogenes (ptcc 1306), s. aureus (ptcc 1112) and e. coli (ptcc 1330) grown in bhi and l. sakei (ptcc 1712) in mrs broth. antibacterial assay: antibacterial activity was determined by the agar well-diffusion assay method and expressed by measuring the diameter of the inhibition zone (iz) (sreeramulu et al., 2001). iz was measured in mm, and expressed as mean±sd. the culture medium (20 ml) was poured into petri dishes (90 mm in diameter). a 20 μl of 24 h cultured suspensions of reference strains were spread on the plates uniformly, and wells of 6 mm in diameter were made. sterile protein hydrolysate samples (40 μl) obtained from hydrolysis of the protein sample (1 mg dry weight /ml) during different times of hydrolysis, were transferred into the wells. the plates were incubated at 37°c for 24 hrs. figure 1. flow diagram of the crab hydrolysis process and investigation of antioxidant and antibacterial activities. 187 int. j. aquat. biol. (2020) 8(3): 184-193 bacteriocidal (mic) studies: the minimum concentration (mic) of the sample that prevents bacterial growth was determined for the protein hydrolysate with the most inhibition zone through the broth microdilution method in the 96-well microplates (portella et al., 2009). the turbidometric method was used to determine the antibacterial activity of each concentration of the sample (tafreshi et al., 2010). the indicator suspension was adjusted to a mcfarland standard of 0.5. then 50 µl of indicator strains (5×106) were added into 96-well plates containing 50 µl supernatant (serial dilution concentration of hydrolysate) and 100 µl nutrient broth. bioactivity was calculated by determining the turbidity of the cell suspension after 24 hrs of incubation at 600 nm by a spectrophotometer (turcotte et al., 2004). the lowest concentration of the samples showing no turbidity was recorded as mic. statistical analysis: the data were analyzed using spss (statistical package for the social sciences version) 19.0 (chicago, illinois, usa). analysis of variance (anova) and duncan’s multiple range test was used to evaluate the differences between samples at p≤0.05. figure 2. rock crabs grapsus albolineathus, from rocky shores of the qeshm island and nearby intertidal zones. 188 emadi shaibani et al./ antioxidant and antibactrial properties of grapsus albolineathus protein hydrolysate results degree of hydrolysis (dh), peptide chain length (pcl) and soluble protein (sp): the dh at the end of hydrolysis was 38.12% with the pcl of about 2.62, and maximum protein soluble ratio of the hydrolysates was 45.83% (fig. 3a, b). as described in figure 3a, the dh values increased rapidly with increasing the hydrolysis duration from 15.11 to 32.2% and the pcl of the hydrolysates decreased correspondingly during the first 90 min (p≤0.05). this positive dh trend followed by a slight increase until 120 min (cph120), and then the enzymatic reaction reached the steadystate phase up to 180 min at dh 38.12 (cph180) (p≥0.05). the pcl trend followed by a sequential decrease in the rate up to 180 min (p≥0.05) (fig. 3a). figure 3b indicates that during the hydrolysis of rocky shore crab, sp increased with increasing of dh up to a certain level (at 90 min) (p≤0.05). by increasing time up to 180 min, the ratio was slightly stopped in the late phases of hydrolysis (p≥0.05). antioxidant activity: the dpph radical-scavenging capacity of cphs improved with increasing dh (p≤0.05), except for an increase from 32.2 (cph90) to 36.8% dh (cph120) (p≥0.05) (fig. 4). the results showed that when dh increased up to 32.2%, the cph3 exhibited the strongest dpph radical scavenging (p≤0.05). in the abts assay, the abts radical scavenging activity increased interestingly with increasing dh and reached a maximal value at dh 36.8% for cph120 (p≤0.05), with no noteworthy difference with cph90 and cph180 (p≥0.05) (fig. 5). antibacterial activity: the antibacterial effects of the five types of rocky shore crab hydrolysate with different dh were determined by the appearance of zone of growth inhibition around the well containing the hydrolysates (table 1). the results showed that the antibacterial activity of the rocky shore crab hydrolysates significantly increased by the progress of hydrolysis. the strongest antibacterial activity was obtained against l. monocytogenes with izs of 14.50 mm by cph90 followed by s. aureus with izs of 11.16 mm by cph180 and l. sakei with izs of 11.00 mm by cph180. there was no significant difference with izs of cph90, cph120, and cph180 against these three bacteria (p≥0.05). the mic value of cph90 as the table 1. antibacterial activity of rocky shore crab protein hydrolysate with different dh. cph180 cph120 cph90 cph60 cph30 bacteria strains 11.00±0.44a 10.83±0.68a 10.68±0.51a 9.16±0.25b 8.70 ±0.00b staphylococcus aureus 14.30±0.55a 14.16±0.51a 14.50±0.44a 12.50±0.00b 11.66±0.55c listeria monocytogene 11.16±0.25a 11.00±0.00a 11.00±0.68a 11.00±0.00a lactobacillus sakei escherichia coli inhibition zones: (mm), (-): no activity. different letters indicate significant differences between groups. mean ± sd, n = 3. figure 3. effect of hydrolysis time on the degree of hydrolysis (dh) and peptide chain length (pcl) (a) effect of hydrolysis time on the soluble protein ratio (sp) (b). different letters indicate significant differences between groups, (mean ± sd, n = 3). 189 int. j. aquat. biol. (2020) 8(3): 184-193 most potent hydrolysate against l. monocytogenes was evaluated to be about 2.75 mg/ml. the minimum activity was observed against s. aureus for cph30 with izs of 8.70 mm. there was no activity against e. coli. an increase in dh value up to 32.2% within the first 90 min was caused an improvement in the antibacterial activity value (for cph30 to cph90) (p≤0.05) while in the next phases, with the slight increase in dh value up to 38% (for cph90 to cph180) antibacterial activity did not change significantly (p≥0.05) (table 1). discussions progress of enzymatic hydrolysis: one of the most imperative outcomes of hydrolysis is the capacity to part the soluble and insoluble proteins from each other in enzymatic processes (guerard et al., 2002). enzymatic hydrolysis is a process in which enzymes facilitate the cleavage of the peptide bonds and releasing the bioactive peptides that are inactive within the sequences of the parent proteins with the usually 2-20 amino acid residues in length (ryan et al., 2011). therefore, monitoring the progress of hydrolysis by the degree of hydrolysis (dh), the peptide chain length (pcl) and the amount of soluble protein (sp) could describe the hydrolysis process. according to the results, the fast reaction within first 90 min of the hydrolysis implied that alcalase cleaved the most susceptible peptide bonds in the early phase of hydrolysis and the most available cleavage sites for alcalase to act were at this time. a sequential decrease in the progress of hydrolysis in the steadystate phase can be attributed to the competition between the substrate and the hydrolysates achieved, the reduction in peptide bonds with the capacity to being cleaved, and enzyme denaturation that lessens its activity (adler-nissen, 1986). similar dh trends have been reported for other crustaceans alcalase hydrolysates (dey and dora, 2014; gunasekaran et al., 2015; antunes-valcareggi et al., 2017). the increased soluble protein in the initial phase of the hydrolysis could be because of an increase in the concentrations of soluble peptides released by the alcalase digestion of the crab crude protein. reduction in the number of peptide bonds available for hydrolysis could be attributed to the reduction in the rate of hydrolysis and stability in the percentage of sp during the later stages of hydrolysis. antioxidant activity: the wide specificity of proteases to peptide bonds could generate a vast range of smaller peptides and free amino acids with diverse activities. the results of radical scavenging activity assays (dpph and abts) showed that the alcalase hydrolysates of rocky shore crab with different dh are enriched with antioxidant peptides, which can act as an electron donor and could convert the free radicals to stabilized products. in several cases, alcalase represented a good candidate for producing figure 4. dpph radical scavenging activity of rocky shore crab protein hydrolysate with different dh. different letters indicate significant differences between groups, (mean ± sd, n = 3). figure 5. abts radical scavenging activity of rocky shore crab protein hydrolysate with different dh. different letters indicate significant differences between groups, (mean ± sd, n = 3). 190 emadi shaibani et al./ antioxidant and antibactrial properties of grapsus albolineathus protein hydrolysate antioxidant peptides based on the ability to cleave the peptide bonds from the interior of the peptide chain and releasing short or medium chain peptides containing hydrophobic amino acids such as leu, phe, ile, val, trp, met and tyr. it was hypothesized that the presence of these hydrophobic amino acids in the protein sequence of hydrolysate could enhance the radical scavenging activity (rajapakse et al., 2005; song et al., 2008; dryáková et al., 2010; gallegos‐ tintoré et al., 2011). the results also demonstrated that an increase in dh and reduction in pcl resulted in more antioxidant activity of crab hydrolysates up to a certain level. several studies preferred hydrolysates with the highest dh to warrant the existence of small peptides (jeon et al., 1999; kim et al., 2001; garcía-moreno et al., 2014). this could be because of small size of the peptides obtained by progress of the hydrolysis may facilitate electron reduction and results in more antioxidant capacity. zhang et al. (2015) pointed out that the dpph radical scavenging capacity of oyster hydrolysates increased with the progress of protein hydrolysis by alcalase (dh: 30.12 to 39.89%) due to producing small and medium-sized peptides with active amino acid residues. in contrast, you et al. (2009) reported that although the dpph and abts radical scavenging ability of papain hydrolysate of loach hydrolysis increased when the dh increased from 18 to 23%; this value decreased when the dh was increased from 23 to 33%. prolonged hydrolysis and excessive increase in dh may result in the formation of highly soluble peptides or free amino acids, completely lacking the functional properties of the native proteins (kristinsson and rasco, 2000). hence, the lack of a direct relationship between the hydrolysates radical scavenging activities and dh at the final stages of hydrolysis (90-180 min), could be attributed to the presence of free amino acids or peptides with different size, solubility and sequence compared to peptides in the first stages of hydrolysis, resulting in lower antioxidative activity. antibacterial activity: the antimicrobial activities of amp are contributed to the charge, size, hydrophobicity, and solubility of their structure. while the peptide length required for spanning bacterial membrane is approximately under 22 amino acid residues, the adequate peptide solubility is critical for the antimicrobial peptides performance to suppress bacterial growth (bahar and ren, 2013). the results showed the interesting antibacterial potential of all alcalase hydrolysates of rocky shore crab against all gram-positive bacteria and no activity against gram-negative bacterium e. coli. this antibacterial activity is probably due to the release of some small and medium-sized peptides consisting of the hydrophobic amino acids upon alcalase hydrolysis. the hydrophobic characters of antibacterial peptides have been found as the main factor in peptide interactions with bacterial membranes, while they can directly kill bacteria or inhibit bacterial growth (sperstad et al., 2011). the reason for no antibacterial effect of cphs against e. coli can be due to the lack of active amino acid sequences with inhibitory effect towards the bacterial membrane composition. the results also indicated that the antibacterial activity of the rocky shore crab hydrolysates significantly enhanced by the progress of hydrolysis up to certain level. this result suggested that cleavage of the peptide bonds alongside with subsequent decrease of pcl and sp in the initial stage of hydrolysis, exert a considerable influence on the antibacterial activity, but the slight progress of hydrolysis in the last phases could not improve this activity. similar to our result, the oyster alcalase hydrolysates showed inhibitory effect against the microorganisms viz. l. monocytogenes, e. coli, s. aureus, p. aeruginosa, v. harveyi and v. parahaemolyticus, whereas the highest dh hydrolysate (39.89%) showed a higher antimicrobial activity than lower dh hydrolysates with the maximum izs of 13.44 mm against v. parahaemolyticus (zhang et al., 2015). sila et al. (2014b) indicated that five alcalase hydrolysates (dh: 2.8, 5.1, 6.6, 10.45 and 13%) from barbel muscle protein that show antibacterial activity, no antibacterial activity was detected with its hydrolysates with the highest dh (14.58 and 16.2% 191 int. j. aquat. biol. (2020) 8(3): 184-193 after 2 hrs). in this case, the fii-3 sub-fractions, which obtained from purification and identification of (6.6% dh) hydrolysate showed the hydrophobic characteristics with a protective effect against pathogenic bacteria (l. monocytogenes, e. coli, p. aeruginosa, k. pneumonia., s. aureus and m. luteus). according to the results, the progress of alcalase hydrolysis of rocky shore crab is led to increase the number of the soluble peptides with small and medium size and higher availability of hydrophobic regions. these structural changes of the protein improve the antibacterial activity of hydrolysates up to a certain level. as conclusion, it can be mentioned that the hydrolysis of rocky shore crab enhances its antioxidant and antibacterial activities. we demonstrated that the progress of hydrolysis degree with time could modulate the biological activity of hydrolysate at a certain level, due to the presence of small and medium-sized peptides with specific amino acid composition, revealing the most antioxidant and antibacterial activity. hence, the careful monitoring of the degree of hydrolysis based on enzyme specificity is crucial due to generating peptides with different molecular size and amino acid contents and resultant differences in bioactivity. thus, alcalase hydrolysates of rocky shore crab may potentially serve as a suitable natural antioxidant and antibacterial source for future purification and identification. references adler-nissen j. 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(2020) 8(1): 56-65 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article growth and mortality parameters of caspian kutum, rutilus kutum, in southern caspian sea reza shahifar1, rahman patimar*1, hasan fazli2, hadi raeisi1, mohammad gholizadeh1, hojjatallah jafaryan1 1department of fisheries, faculty of agriculture and natural resources, gonbad kavoos university, gonbad kavoos, iran. 2caspian sea ecology research center, sari, iran. s article history: received 7 july 2019 accepted 11 august 2019 available online 2 5 february 2020 keywords: kutum l-infinity total mortality natural mortality caspian sea abstract: the caspian kutum, rutilus frisii, is one of the endemic and most important commercial cyprinid species in the southern caspian sea. a detailed study on growth and mortality parameters of this species was conducted based on 700 samples collected from commercial catches of beach seining in guilan and mazanderan provinces during fishing season 2017-2018. females were dominated in both studied populations. size frequency distributions showed significant variation among same sexes and between different sexes. the different wlrs were observed, positive allometric in mazanderan, and negative allometric in guilan. there were significant differences in growth parameters between sexes, females were of much greater asymptotic length than males, while the male fish had a higher growth rate and attained a smaller theoretical l∞ size than females. the theoretical maximum length (l∞) was larger than the maximum one recorded during sampling. based on the bhattacharya method, the caspian kutum from guilan fishing grounds was more diverse, and included nine cohorts, while the population from mazanderan province showed only six cohorts. the linearized catch curve based on age composition data showed that total mortality rates (z) are 1.32 year-1 and 0.63 year-1 for males and females of guilan, respectively, that of males in mazanderan is 1.04 year-1 and of females 0.86 year-1. the natural mortality rates (m) were 0.48 year-1 for males and 0.26 year-1 females in guilan, and was found to be as 0.26 year-1 and 0.45 year-1 for males and females of caspian kutum caught in mazanderan. the exploitation ratio (e) was found to be higher than 0.5 for both sexes from guilan, and to be lower than the expected optimum level of exploitation in caspian kutum males and females caught in mazanderan. introduction the ichthyofauna of the southern caspian sea basin includes 119 species belong to 63 genera (esmaeili et al., 2014, 2018). caspian kutum, rutilus kutum, is endemic to this basin occurring mainly in the southern coast of the caspian sea with a distribution extending from the terek river in the north to the gorgan bay in the southeastern region. this species is among of the most important commercial species for fisheries and stock enhancement programs in the iranian caspian coast (kiabi et al., 1999; farhang and eagderi, 2019). despite intensive investigations on caspian kutum in the context of aquaculture and ecotoxicology, few publications are available on its growth parameters (abdolmaleki et al., 2007; hoseini *correspondence: rahman patimar doi: https://doi.org/10.22034/ijab.v8i1.677 e-mail: rpatimar@yahoo.com et al., 2010; aghili and mohamadi, 2011; fazli et al., 2013). however, no paper has so far considered the variation in growth of different populations of this species on a relatively broad regional scale. fish growth is an indeterminate plastic process that can change considerably among populations of a species in its distribution areas. furthermore, growth parameters are not only required input to several stock assessment methods, but also allow tests of lifehistory hypothesis (stergiou, 2000). therefore, verification of local values and growth variability within species (considered as inter-population variations) has great importance in fisheries ecology and fish population dynamics. hence, this study aimed to investigate in greater detail growth aspects of 57 int. j. aquat. biol. (2020) 8(1): 56-65 caspian kutum from two main fishery areas, to increase our knowledge on the variability of its growth parameters. the results could be used for better and effective management practices of exploiting the species stock in this basin. materials and methods the samples were collected from commercial catches in fishing grounds of guilan and mazanderan provinces during 2017-2018 fishing season i.e. fall and winter. however, the size range did not include individuals smaller than the size at first capture because they were not selected by the commercial fishing gears. the fishing operations in these areas are conducted using huge beach seine with mesh sizes of 26–32 mm. then, the collected specimens were transported to the laboratory, where their fork length (fl; ±0.1 mm) and total weight (w; ±0.1 g) were recorded. all specimens were dissected to determine their sex by direct observation of the gonads. in addition, 5-10 scales of each individual were removed from the area just above the lateral line and mounted between two glass slides for age estimation. two readers interpreted the growth marks on scales without prior knowledge of length, weight or sex of the fish, its date of capture or the previous reading to avoid reading bias. only coincident readings were accepted. to describe size structures of the populations studied, histograms of size-frequency distributions of the caspian kutums were made, using 4 cm fl intervals. two-sample kolmogorov–smirnov (k–s) tests (α=0.05) were used to compare size frequency distributions between sexes (sokal and rohlf, 1981). estimation of the weight and length relationship was made by adjustment of an exponential curve to the data (ricker, 1975): w = alb, where w is the total weight (g), l the fork length (cm), a the intercept (initial growth coefficient or condition factor) and b the slope (allometric exponent). the relationship coefficients were calculated using non-linear least squares estimation. student’s t-test was applied to determine the significance of differences between the isometric growth and the estimated b-value of the equation. additionally, an analysis of covariance was used to compare fl–w relationships between sexes (keivany et al., 2016). estimates of theoretical growth in length were obtained by fitting the von bertalanffy growth function (vgbf) to the mean length at age data separately for males and females by non-linear regression: lt= l∞[1-e-k(t-t0)] using the method of gulland-holt (everhart and youngs, 1975), where lt is the length at age t, l∞ is the asymptotic length, k is the growth coefficient, and t0 is the hypothetical time when the fish total length is zero. the overall growth performance index (øʹ) was calculated based on the growth parameter estimates by the equation of munro and pauly (1983): øʹ= log10k+ 2 log10 l∞. the index was used to compare growth parameters obtained in this study with those reported by other authors. in addition, the weight-based of vbgf calculated as wt=w∞[1-e-k(t-t0)]b, where wt is the weight of the fish in g at age t, w∞ is the asymptotic weight of the fish in g and b is the constant in the length–weight relationship (ricker, 1975; sparre and venema, 1992). monthly length-frequency data was analysed using the package fisat model progression analysis, and bhattacharya’s (1967) method subroutine (sparre et al., 1989; gayanilo et al., 1994) for to identify the modes in the polymodal length-frequency distributions of caspian kutum cohorts. the total mortality rate (z) for each sex of population was estimated using powell weatherall plot, where the regression equation has the form ln(n) = a + bt′, where n is the number of fish in cohorts by means of successive growth curves, t′ is the relative age of the fish in that cohort, and b with the sign changed provides an estimate of z (powell, 1979; wetherall et al., 1987). to obtain an independent estimate of natural mortality rate (m), the pauly’s empirical equation (1980) log(m) = -0.0066-0.279 log(l∞) + 0.6543 log(k) + 0.4634 log(t) was employed. here, t is mean annual habitat temperature in southern caspian sea and l∞ is expressed in cm. results a total of 700 caspian kutum were collected from 58 shahifar et al./ growth and mortality parameters of caspian kutum commercial catches in the guilan (n=310) and mazanderan (n=390) provinces. the number of females sampled was statistically more than the number of males sampled (χ2=46.27, p<0.05). in both sampling areas, the sex ratio was almost the same (1:1.7) in favoure of females (guilan: χ2=19.6, mazanderan: χ2=26.7, p<0.05). size frequency distributions showed significant variation among same sexes and between different sexes (k–s test, p<0.05) (fig. 1). the length–weight relationships (wlrs) of males and females in both populations are given in figure 2. the wlrs for females and males were significantly different (p<0.05). therefore, the female and male data cannot be pooled. different types of growth were observed: positive allometric in males and females from mazanderan, and negative allometric in both sexes of guilan (t-test, p<0.05). table 1 summarizes the von bertalanffy growth parameters and mortality rates estimated from our data set. there were significant differences in growth parameters between sexes, females were of much greater asymptotic length than males, while the male fish had a higher growth rate and attained a smaller theoretical l∞ size than females. the theoretical maximum length (l∞) was larger than the maximum one recorded during sampling and no samples reached or were longer than the theoretical value of calculated for each sex. the plots of von bertalanffy growth functions for males and females fitted to the observed length and weight-at-age data of each sex and year separately show that the growth patterns of females figure 1. relative proportion (%) of caspian kutum males and females from southern caspian sea (2017-2018). table 1. growth parameters of the caspian kutum from southern caspian sea (2017-2018). location sex year w∞(gr) l∞(fl,cm) k(year-1) t0(year) z m guilan male 2017-2018 2053.41 60.74 0.20 -0.68 1.32 0.48 guilan female 2017-2018 4356.91 69.12 0.14 -0.99 0.63 0.26 mazanderan male 2017-2018 1610.10 52.77 0.36 -0.39 1.04 0.26 mazanderan female 2017-2018 2396.10 69.12 0.14 -0.99 0.86 0.45 59 int. j. aquat. biol. (2020) 8(1): 56-65 and males are similar in younger ages, thereafter; females grew to be larger than males (figs. 3, 4). the bhattacharya method showed different cohorts in commercial catch of caspian kutum in the southern area (fig. 5). the caspian kutum from guilan fishing grounds was more diverse, and included nine cohorts, while the population from mazanderan province showed only six cohorts. the linearized catch curve figure 2. fork length–weight relationship for combined sexes, as well as male and female caspian kutum, southern caspian sea (2017-2018). figure 3. length-age based von bertalanffy growth model for the caspian kutum sampled in southern caspian sea (2017-2018). 60 shahifar et al./ growth and mortality parameters of caspian kutum based on age composition data, corresponding to the slope of the descending limb of the catch curve, is presented in figure 6. estimated instantaneous total mortality rates (z) were 1.32 year-1 and 0.63 year-1 for males and females of guilan, respectively, that of males in mazanderan was 1.04 year-1 and of females 0.86 year-1. instantaneous natural mortality rates (m) using the equation of pauly (1980) was 0.48 year-1 for males and 0.26 year-1 females in guilan. the m parameter was found to be as 0.54 year-1 and 0.45 year-1 for males and females caught in fishing ground of mazanderan. the exploitation ratio (e) was found figure 4. weight-age based von bertalanffy growth model for the caspian kutum sampled in southern caspian sea (2017-2018). figure 5. the sequences of operation in the use of the bhattacharya method of separating a length–frequency distribution into normal components for caspian kutum from southern caspian sea (2017-2018). 61 int. j. aquat. biol. (2020) 8(1): 56-65 to be higher than 0.5 for both sexes of caspian kutum from guilan, and to be lower than the expected optimum level of exploitation (e=0.50) in caspian kutum males and females caught in mazanderan, indicating that caspian kutum suffered great fishing pressure in guilan sea areas, and fishing pressures on the caspian kutum in mazanderan sea areas have been relatively low. discussions as a comprehensive study on growth parameters of caspian kutum in the southern caspian sea can still be considered as limited to date, the present study provides a more detailed insight into the information on growth of this species. estimates of demographic growth parameters are important in a broader context as they provide a fundamental description of the life history characteristics. the determination of age and growth is of great importance to both fisheries biology and management as it forms the basic knowledge required for the estimation of mortality, recruitment and yield (mehanna, 1996). the size frequency used in the present study, on which growth and mortality estimates depended, were not based on a scientific sampling schedule but rather on random subsamples taken from commercial catches. fishing gear selectivity is an important consideration in studies determining demographic characteristics of fish populations. here, the nets used in the beach‐seine fishery were selective for the caspian kutum. furthermore, the catching this species in the study area was seasonal, which was not caught year-round (hasanpour et al., 2016). therefore, this study lacks data some lengths and ages. irrespective of this selectivity, this study was able to develop growth protocols, provide representative population parameters and mortality rates of this important exploited species. in this study, specimens were successfully aged using scales, shown to be problematic for some caspian cyprinids in previous studies. in general, scales were hard to read due to the existence of numerous annuli. this is relatively often an important source of error in ageing cyprinids. it is unlikely that our scale ages represent the true age of the caspian kutum, despite the fairly precise age estimates achieved by multiple readers using scales. the two data sets used to assess the caspian kutum populations’ growth variability in the present study were collected approximately within the same time period, and we assume that the pool specimens’ data figure 6. linearized catch curve of the caspian kutum and the regression equation obtainedsouthern caspian sea (2017-2018). 62 shahifar et al./ growth and mortality parameters of caspian kutum represents the average stock composition. although it is difficult to compare size and length data among the various studies because of seasonal differences and methods of collection, length frequency analysis applied to the caspian kutum from the study areas revealed that the stocks of the species consist of different sizes groups. this might be explained by the fact probably stemming from fishery selections on the populations. length-weight relationships provided a good fit to data for the caspian kutum, while there was a great deal of individual variability for the populations. this variability could be attributed to greater differences in size selectivity and/or condition factors among individuals of this species. the length–weight relationship equations had b-values significantly differ from “3” reflecting an allometric mode of growth for the caspian kutum. the allometry coefficient of the length-weight relationship shows positive allometric growth for the population from mazanderan (>3) and negative mode for the guilan (<3) populations. however, the b-values lie still between the expected range of 2.5–3.5 (bagenal and tesch, 1978). the estimated exponent value for female population from guilan and male population from mazanderan shows that the fish body is close to the isometric growth model, i.e. body weight increases approximately three times faster than body length. according to froese (2006), if b=3, then small specimens in the sample under consideration have the same form and condition as the large specimens. patimar et al. (2009) noted that a variation in b-values among populations can be affected by the geographic location as well as the environmental conditions. the difference in the exponents of caspian kutum could be attributed to the different sampling areas as well as the differences in number of specimens and length ranges of the populations belong to distinct regions (patimar et al., 2009). the present b-value of caspian kutum is more or less than those reported by previous studies; abdolmaleki et al. (2007) gave b=3.04, while in aghili and mohammadi (2011), 2.96 in the southeastern caspian sea and fazli et al. (2012) reported 3.12 for this species, which all differ from the present ones. these differences in b-values could probably be explained by the catches being through several different seasons or they could be attributed to different size distribution, as well as to differences in age composition in different years. estimates of von bertalanffy (vbgf) growth parameters indicated that females reach a larger asymptotic size than males. the larger asymptotic length for females could be attributed to their relatively longer longevity and greater absolute growth rate. the longer lifespan and larger size of females could be considered a life history strategy for supporting increasing egg production (roff, 1983). the values of l∞ which are lower than the maximum fl should be accepted considering that no male and female longer than these asymptotic sizes were caught. the estimated vbgf parameters show significant differences between sexes and areas as well, which can be attributed to interand intrapopulation variation of caspian kutum. growth parameters (l∞, k and t0) are the basic input data into various models used for managing and assessment the status of the exploited fish stocks, these parameters facilitate the comparison between growth of populations belonging to the same species at different times and different localities. comparison of vbgf parameters from different populations in the southern caspian sea, despite differences in methodology and data quality, showed that the growth patterns were different. in the previous studies, growth parameters estimated for combined genders of caspian kutum were l∞=70.1 cm fl, k=0.38 per year, and t0=-1.56 years (ghaninezhad et al., 2004), l∞=60.70 cm fl, k=0.15 per year, and t0=-1.75 years (abdolmaleki et al., 2007), and l∞=58.00 cm fl, k=0.24 per year, and t0=-0.16 years (hosseini et al., 2010). the difference is may be due to the difference in the ecological parameters in different localities, or the maximum observed lengths in the catch or the methods used in calculations by different authors. we did not find any studies describing weight-based growth parameters of von bertalanffy for the caspian kutum. because of the lack of this kind of data, the parameter w∞ might not 63 int. j. aquat. biol. (2020) 8(1): 56-65 be well-discussed in this study. for fishes that grow according to the von bertalanffy function, the vbgf parameters cannot be directly used to compare growth rates among populations, there is practical problem that none of its parameters have the dimensions of growth. the growth performance index (øʹ) emerges as an alternative (munro and pauly, 1983; pauly and munro, 1984). the relation between (ln)k and (ln)l∞ turns out to be a consequence of the so called beverton and holt dimensionless life-history invariants (beverton and holt, 1959; beverton, 1992; charnov, 1993), which allows the comparison of growth performance as represented by the øʹ index (munro and pauly, 1983) of stocks of the same species. it also represents a potential check for the accuracy of growth parameter estimates. comparison of øʹ calculated using reported vbgf parameters revealed that small difference is in this index among populations, ranging from 6.3 to 7.5. this confirms the accuracy of the calculations, and different vbgf parameters seem to indicate differences in dynamics status of the caspian kutum populations. higher total mortality (z) and natural mortality (m) for males suggest that the higher survival rate of females may be due to mechanisms developed for perpetuation of the species. due to direct fishing interest, caspian kutum is an important species in terms of fisheries management in southern caspian sea. therefore, large difference between the estimates of mortality rates for different populations in the basin indicates a different and variable natural and fishing mortalities. the total and natural mortality rates of caspian kutum estimated in the present study were larger than those estimated previously in the southern caspian sea (z=0.83 per year and m=0.31 per year for sexes combined population) (abdolmaleki et al., 2007). this could potentially be due to a combination of factors including (i) the previous estimates were made potentially prior to any significant fisheryassociated impacts, and (ii) the effects of sustained levels of harvesting of the species in our study area. nevertheless, the values of m seem reasonable, considering the biological features of caspian kutum. the same species may have different natural mortality rates in different areas (sparre et al., 1989). the estimate of the exploitation rate (e) indicates a relatively low fishing pressure in mazanderan fishing areas; this should be treated with caution as mortality rates were estimated from a single sampling season and may be biased due to annual differences in yearclass strength. it is generally recommended that assessment studies incorporate a range of mortality estimation methods as a precautionary approach and to improve certainty, particularly in data-poor fisheries as studied here. in guilan fishing grounds, at the same time, the exploitation rate (e) of the caspian kutum exceeded an optimally exploited value (gulland, 1971), indicating that the stocks had been overfishing. therefore, an urgent fisheries management response is required o prevent the collapse of the caspian kutum stocks in guilan fishing areas and to achieve sustainability. in addition, these results could be underestimated because of the lack of illegal catch information and other gear in current use, which may include a significant portion of undersized caspian kutum. in conclusion, the present study provides new information about mortality and growth parameters of caspian kutum, needed for stock assessment and management. however, further research is necessary to elucidate if the relatively high degree of variability found in some of growth parameters could represent either adaptations to local selective pressures (both fishery and environmental) or ecophenotypic variations. the estimated caspian kutum population parameters indicated a trend toward its being a k‐ strategist species based on high maximum theoretical length, low growth rate, long lifespan and low natural mortality. this indicates that the regulation strategies developed for the fishery management should be based on the caspian kutum’s different growth patterns. references abdolmaleki s., hashemi a., nahrvar r. 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(2020) 8(4): 262-271 issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2020 iranian society of ichthyology original article ageing nile tilapia (oreochromis niloticus): a comparative study between scales and otoliths khaled y. abouelfadl1, walid aly*2,1alaa g.m. osman3 1aquatic ecology department, faculty of fish and fisheries technology, aswan university, egypt. 2fisheries biology lab., fisheries division, national institute of oceanography and fisheries, egypt. 3department of zoology, faculty of science, al-azhar university (assiut branch), assiut, egypt. s article history: received 7 june 2020 accepted 21 august 2020 available online 2 5 august 2020 keywords: relative precision percent agreement age groups longevity abstract: this study is the first to compare age estimates based on scales and otoliths of the nile tilapia, oreochromis niloticus, from lake nasser, egypt. ageing precision between readers was estimated by calculating the percent agreement between three independent readers, and the coefficient of variation (cv) ages estimated from otoliths and scales. the relation between the total length of fish and the radius of its scale and otolith was determined and appeared to be linear. the estimated age composition of the o. niloticus included six age groups estimated using scales and five age groups estimated by otoliths. the relative precision (cv and sd) of ages estimated from otolith was higher than that from scales. higher percentages of agreement of overall annuli identification and age assignment between readers were noted in otoliths comparing to that in scales. scale showed some inaccurate estimation in the age of fish older than 4. the precision and bias information in this study will be beneficial to fisheries professionals in assessing the age of nile tilapia and other cichlids in the future. introduction growth and age studies investigate the essential demographic characteristics to examine and assess the structure of fish populations (maceina and sammons, 2006). estimating the age of fish is a common technique used to model its population dynamics and productivity by understanding fish longevity, growth and mortality rates i.e. an accurate understanding of these metrics needs precise age information (campana, 2001). if a population that is or will come under considerable fishing pressure is to be studied, it is imperative that ageing should be done accurately as it is the most important parameters upon which recommendations can be based for the rational exploitation of a fish species. many structures have been used for age estimation of fishes, including scales, various fin rays, fin spines, vertebrae, opercle, and otoliths. ageing of fishes from subtropical and tropical regions have been reported via annual increments in such calcified structures, for example, scales (ilieş et al., 2014), dorsal and pectoral *correspondence: walid aly e-mail: walid.soton@gmail.com spines (metcalf and swearer, 2005), vertebral centra (branstetter and stiles, 1987; bahuguna, 2013), opercular bones (gómez-márquez et al., 2008), and otoliths (pilling et al., 2003; fowler, 2009). a comparison of these various calcified structures has been performed in many species to obtain valuable information on the accuracy and bias of such age estimating structures (kruse et al., 1993; abecasis et al., 2008; lozano et al., 2014). studying the variation in age estimations using different calcified structures provide collateral evidence for the validity of the ageing method (campana, 2001). significant variation in age estimates based on different calcified structures indicates inaccurate ageing methodologies, imprecise ageing structures, or problems associated with interpretation (muir et al., 2008). balancing the accuracy and precision of the ageing method with sample size limitation is the main criteria to select the proper method for determination of age and growth in fishes (zymonas and mcmahon, 2009). tilapias are the main species produced in egypt 263 int. j. aquat. biol. (2020) 8(4): 262-271 and contributed about 31% of the total wild fish catch in 2017 (gafrd, 2017). nile tilapia (oreochromis niloticus) is widely distributed in all fresh and brackish water bodies in egypt (el-sawy, 2006). while various aspects of the biology of o. niloticus and other commercially important tilapias have been thoroughly studied in egypt (el-sawy, 2006; elbokhty et al., 2013; hassan and el-kasheif, 2013; elkasheif et al., 2015; hussian et al., 2019; shalloof et al., 2020), there were no comparative studies to validate used o. niloticus ageing methods based on calcified structures. the present work aims to compare scales and otoliths as reliable calcified materials for estimating the age of o. niloticus from lake nasser by obtaining information on the accuracy and bias of such age estimating structures which can provide collateral evidence for the validity of these ageing methods. materials and methods sampling and data collection: a total of 266 nile tilapia were collected from lake nasser during 2018 from local fishermen. they were caught using trammel and gill nets which are the main commercial fishing gears used to catch this species in the lake. for each fish specimen, total length (tl) was measured to the nearest 0.1 cm and total body weight (wt)) to the nearest 0.1 g. fish samples were transported to the laboratory and stored at -20ºc. in the laboratory, several scales (8–10) were removed from each fish sample from the area below the pectoral fin (abecasis et al., 2008) and stored in individually labelled envelopes. scales were then cleaned in dilute aqueous ammonia solution, rinsed, and dried. all scales showing signs of regeneration were eliminated. cleaned scales were kept between two clean slides. digital images of at least three scales for each fish sample were obtained using a camera (abbot dec2000) mounted on a binocular stereomicroscope (zeitess 530). the radius of the scale was measured on the digital image. both sagittas were recovered intact from each fish sample by exposing the otoliths capsules by applying an incision on the dorsal side of the head. otoliths then washed in water and cleaned from all extraneous tissue, and dried, labeled, then stored in plastic vials. digital images were obtained using a camera (abbot dec2000) mounted on a binocular stereomicroscope (zeitess 530) for each pair of otoliths, submerged in 50% glycerol and illuminated with oblique reflected light. the clearest image was chosen for interpretation. the radius of the otolith was measured on the digital image. age determination: each scale and otolith were read twice by three different readers separately (no previous knowledge of count, or length of the sample). each reader assigned each fish to an annuli class based on the number of opaque zones of the scale or the otolith (murie and parkyn, 2005). aging precision between readers was estimated by calculating: (1) the percent agreement between three independent readers and (2) the coefficient of variation (cv) (kimura and lyons, 1991). results out of 266 examined fish, 136 were females (51.1%), and 130 males (48.9%). the total length (tl) of the samples ranged 13.5-48.0 cm with an average of 20.75±8.61 cm. the total weight (tw) varied 46.6table 1. coefficient of variation (cv), percentage of agreement, and standard deviation of age group estimation by different readers based on scales and otoliths reading. age group scales otoliths cv agreement sd cv agreement sd i 34.2% 68.7% 0.56 16.2% 88.5% 0.38 ii 30.2% 58.4% 0.68 11.1% 86.1% 0.39 iii 17.2% 58.5% 0.59 12.2% 80.4% 0.50 iv 16.1% 61.0% 0.67 5.8% 82.7% 0.37 v 10.2% 70.1% 0.62 0.0% 100.0% 0.00 vi 10.4% 71.3% 0.62 total 24.8% 64.7% 0.62 11.6% 87.5% 0.33 264 abouelfadl et al./ scale and otolith-based ageing of nile tilapia 2301.7 g with an average of 652.96±537.2 g. the total length of males ranged between 13.5 and 48.0 cm and their total weight between 46.6 and 2301.7 g while females tl ranged between 14.0 and 45.5 cm tw between 56.3 and 2055.3 g. the tl for females and males did not differ significantly (t-test t=0.66, p<0.05) (fig. 1). the relationship between total length and the radius of its scales and otolith was determined and appeared to be linear. the correlation coefficient of the linear relationship was lower in scales (r2=0.93) than that of otolith (r2= 0.98) (fig. 2). the otoliths and scales of o. niloticus showed annual rings, each ring composed of a faint broader growth zone and a dark opaque line (fig. 3). the estimated age composition of the o. niloticus represented by the samples, including six age groups estimated using scales and five age groups estimated by otoliths (table 1). the relative precision (cv and sd) of ages estimated from otolith was higher than figure 1. length frequency of oreochromis niloticus from lake nasser during 2018. figure 2. the relation between scales and otoliths radius and the total body length of oreochromis niloticus from lake nasser during 2018. 265 int. j. aquat. biol. (2020) 8(4): 262-271 that of scales (fig. 4). a higher percentage of agreement of overall rings identification and age assignment between readers was noted in otoliths (87.5%) comparing to that of scales (64.7%) (table 1). calculating the agreement percentage between readers in the identification of each ring and assignment of each age group in both otolith and scales showed that higher percentages were recorded in otoliths reading reaching to 100% in the determination of age group v while the same group in scale had 71.1% agreement and the lowest percentage of agreement was recorded in the age group iii with 84.4% in otoliths and 58.5% in scales (table 1). the relationship between the assigned age groups and the total body length was determined and appeared to be linear. the correlation coefficient of this linear relationship for of scales (r2=0.91) was lower than that of otolith (r2= 0.93) (fig. 5). the proportion of fish in each age group increased from the group i to group ii, then it decreased gradually with increasing age groups where the second year of the age (age ii) had the highest frequency percentage of 37.6 and 43.6% for age estimation by scales and otoliths, respectively (fig. 6). fifty-six samples were assigned to age group i using both scales and otoliths and they had mean tl of 17.5 cm while group ii had some differences where the number of assigned samples were less when scales were used instead of otoliths (100 and 110, respectively) and the mean tl were 25.2 and 25.9, respectively, but these differences were not significant. furthermore, significant differences (p<0.05) was noted in samples assigned to age group table 2. mean and standard deviation of total body weight, total body length, hard structure radii, and number and frequency (%) of fishes of oreochromis niloticus assigned to each age group based on scales and otoliths reading. age scales otolith total weight (g) total length (cm) radius (mm) no % total weight total length radius (mm) no % (min-max) mean±sd (min-max) mean±sd (min-max) mean±sd (min-max) mean±sd (min-max) mean±sd (min-max) mean±sd i (46.6-336.0) 116.8±35.3 (13.5-20.0) 17.5±2.4 (0.116-0.157) 0.138±0.012 56 21.1 (46.6-336.0) 116.8±35.3 (13.5-20.0) 17.5±2.5 (0.091-0.139) 0.120±0.016 56 21.1 ii (157.4551.7) 332.5±118.3 (20.5-29.5) 25.2±3.1 (0.156-0.372) 0.242±0.059 100 37.6 (157.4639.0) 366.1±139.2* (20.5-30.8) 25.9±3.3 (0.139-0.230) 0.188±0.027 116 43.6 iii (480.7-891.2) 680.9±59.4 (30.0-35.0) 32.1±1.6 (0.286-0.397) 0.357±0.038 54 20.3 (555.8-947.5) 749.5±95.4* (31.0-36.0) 33.4±5.5* (0.185-0.289) 0.253±0.037 46 17.3 iv (803.0-1236.0) 995.8±135.7 (35.3-39.5) 36.9±3.2 (0.366-0.540) 0.436±0.045 24 9.02 (888.0-1904.0) 1240±406.2** (36.5-42.5) 39.2±7.5* (0.258-0.361) 0.326±0.031 28 10.5 v (1082.2-2059.4) 1649.4±304.5 (40.0-45.0) 42.8±4.1 (0.401-0.592) 0.486±0.062 22 8.27 (1582.0-2301.7) 1985.7±223.7* (44.0-48.0) 45.6±10.8* (0.3610.437) 0.395±0.0.24 20 7.5 vi (1939.5-2301.7) 2118.4±170.7 (45.5-48.0) 46.8±6.8 (0.543-0.630) 0.591±0.036 10 3.76 significant compared with scales * p<0.05, **p<0.01 figure 3. a scale (a) and an otolith (b) of oreochromis niloticus with annual rings, each ring composed of a faint broader growth zone and a dark opaque line. 266 abouelfadl et al./ scale and otolith-based ageing of nile tilapia iii and higher (table 2) where the frequency of samples and mean tl of each age group varied significantly between scale and otolith-based age estimation (table 2). this variation is noted in the last assigned age group which was the group v in scalesbased estimation while it was group vi in otolithbased estimation. discussions in the present study, the otoliths and scales of o. niloticus showed rings, each ring composed of a faint broader growth zone and a dark opaque line. many factors were reported to be correlated with ring formation in fish’s hard structures, including changes in temperature, total dissolved solids, food quantity, and changes, associated with the cycle of wet-dry seasons (karakiri and von westernhagen, 1989; lecomte et al., 1989; gauldie et al., 1990; yosef and casselman, 1995; admassu and casselman, 2000). indeed, seasonal variations are less intensive in the subtropics and tropics than in the temperate regions. however, regular climatic fluctuation occurs in many tropical freshwaters (oppenheimer, 1989). this fluctuation may follow either a uniannual or multiannual cycle, lead to the formation of one or more macrozones or checks in the calcified structures of the fish (admassu and casselman, 2000). in addition, fluctuation in body condition associated with spawning activity has also been considered an important factor especially in tropical and subtropical areas, where ring formation has often been attributed to the reproduction, particularly in cichlid fishes (garrod, 1959; pannella, 1974; hecht, 1980). the results of both scales and otoliths-based age estimation in the present study showed that the growth of o. niloticus in lake nasser followed the pattern shown by cichlids in other systems (el-sawy, 2006; el-bokhty et al., 2013; hassan and el-kasheif, 2013; figure 4. the coefficient of variation (cv%), percent agreement, and the standard deviation (stdev) in age estimation by different readers based on scales (a) and otoliths (b). figure 5. the relation between total body length and the assigned age group using scales and otoliths of oreochromis niloticus from lake nasser during 2018. figure 6. age composition of oreochromis niloticus from lake nasser based on scales and otolith reading. 267 int. j. aquat. biol. (2020) 8(4): 262-271 el-kasheif et al., 2015). growth during the first year is extremely rapid, reaching almost one third (17.5 cm) of its maximum length. the advantage of this growth pattern is the ability of juveniles to avoid the intense predation by rapidly attaining a size large enough (hecht, 1980). after sexual maturation, the growth rate decreases with an asymptotic length being approached early on in life (booth et al., 1995). this study is the first to use two different tools simultaneously to assess the quality of ageing information of o. niloticus. all age estimation of this species in different water bodies in egypt used scale method except anonymous (1997) and khalifa (2000) who used otoliths for ageing o. niloticus from qarun and wadi el-raiyan lakes. generally, most of the earlier studies of fishes age and growth used scales for age estimation due to ease of collection, process, and avoids sacrificing the specimens. emphasis on proper animal care in the fisheries profession would justify use of the scale in areas where other tools provide similar results, or even in areas where scales are slightly less precise (kruse et al., 1993). in this study, the comparison between age estimates using scales and otoliths showed that otoliths were more accurate for age determination of o. niloticus. it was found that otolith-based age estimates would be more precise than scale-based estimates as the average precision estimates for scales (cv=24.8%) is twice more than that of otoliths (11.6%). our results are in accordance with the growing evidences that the scale as a tool of age estimation may be unreliable under certain growth conditions. many studies have suspected on scale ageing due to difficulties in reading annuli, low precision (lowerre-barbieri et al., 1994), and that scale ages may become inaccurate when growth becomes asymptotic. as scale growth is proportional to body growth, in older fish annuli become crowded on the scale edges making scale interpretation difficult. as a result, true age can be misestimated, especially in species that growth is concentrated in early life history like o. niloticus (beamish and mcfarlane, 1987; shepherd, 1988). moreover, several studies have reported that scales can provide unreliable estimates of fish age because of the lack of a distinct cold season, as annuli are formed during cold months when growth is slow, likely results in scale annuli that are indistinguishable for some fish populations in subtropical and tropical areas (huish, 1954; schramm and doerzbacher, 1985). several studies reported that otoliths are the most reliable ageing structure in several temperate as well as tropical fish species (beamish, 1979; brothers, 1979; kalish, 1989; cailliet et al., 2001; phelps et al., 2007; gunn et al., 2008; fowler, 2009). age estimation of o. niloticus from tropical and subtropical lakes using calcified structures are more challenging when compared to temperate species, where, in many cases, the between-reader agreement is above 90% (bwanika et al., 2007). nevertheless, the ageing precision (cv=11.6 %, the agreement between readers=87.5%, and sd=0.33) for o. niloticus using otoliths in this study indicated comparable to that of the majority of studies ageing fish using otoliths (campana, 2001). this precision estimate is reflected by 100% agreement obtained between readers for age 5 years. the ageing precision results for scales and otoliths in the present study were similar to those of different species (hecht, 1980; boxrucker, 1986; welch et al., 1993; booth et al., 1995; abecasis et al., 2008) which reported that the percent of accurate agreement for scale readers is always less than that of otolith readers. the age composition of the catch is used in agestructured stock assessments, which can be used to estimate exploitable biomass. the results of this study showed that the proportion of fish in each age group increased from the group i to group ii, then it decreased gradually with increasing age groups. this pattern is following the classical dome shaped catch curve which is an indicator of two oppositely directed drives: the process of recruitment and the influence of mortality. the process of recruitment results in the formation of the left (rising from group i to group ii) region on the catch curve. at a sufficiently mature age, the age group to which recruits belong almost completely transits into the harvested stock. approximately at this age, the catch curve goes past 268 abouelfadl et al./ scale and otolith-based ageing of nile tilapia the maximum and starts to decrease monotonously (sukhanov, 2016). age-length keys for o. niloticus developed in this study showed high variability between tools in assigning ages to fish >30 cm tl. great variation in the length-at-age key of o. niloticus from lake nasser is noted in the previous longevity and age estimation studies as they all were solely based on scale (table 3). this variation may have resulted from the inaccuracy of the estimation method and /or a combination of both extrinsic factors such as the variable abiotic conditions experienced by the fishes in different areas of the lake and intrinsic factors such as genetic variation between the individuals themselves. much of the extrinsic intra-year variation in the growth of oreochromis spp. was linked to flood intensity and duration (booth et al., 1995). in the present study, age frequency distributions of o. niloticus based on scales were different from those based on otoliths, nevertheless, scale ages can fulfil a manager's need for information about population age composition if the oldest age-groups (>4) are combined. however, the scale is not sufficiently precise to assess the ages of individuals in a population, and does not, for the most part, accurately recognize older o. niloticus in lake nasser. conclusion this study is the first to compare age estimates based on scales and otoliths of the nile tilapia. otoliths are recommended for the best age estimates due to its higher precision of age estimates compared to scales. if nonlethal techniques for estimating age are required, scales can provide close age estimates for fish in comparison to otolith age estimates, but they may still inaccurately estimate the age of fish older than age 4. the precision and bias information in this study will be beneficial to fisheries professionals in assessing age of nile tilapia and other cichlids in the future. references abecasis d., bentes l., coelho r., correia c., lino p. g., monteiro p., gonçalves j. m. s., ribeiro j., erzini k. 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(2013) 1(4): 158-166 e-issn: 2322-5270; p-issn: 2383-0956 journal homepage: www.ij-aquaticbiology.com © 2013 iranian society of ichthyology original article optimizing the co-feeding strategy of persian sturgeon (acipenser persicus) larvae using artemia nauplii and formulated diet naser agh *1, reza asgari1, 2, farzaneh noori1 1artemia and aquatic animals research institute, urmia university, dr. beheshty avenue, urmia, 57153, iran. 2department of fisheries, faculty of natural resources, university of tehran, karaj, iran. article history: received 18 june 2013 accepted 28 july 2013 available online 2 0 august 2013 keywords: acipenser persicus artemia nauplii formulated diet co-feeding abstract: high mortality and labor costs are associated with first-feeding sturgeon culture, particularly during the period of dietary transition from live to formulated feed. therefore we investigated the effects of various feeding treatments on the survival and growth of the persian sturgeon (acipenser persicus) larvae during a 20-day culture period. three replicate groups (250 fish/replicate) of firstfeeding larvae were fed according to four main feeding regimes: (1) live food (live nauplii of brine shrimp artemia urmiana); (2) indirect transition (5 days live food followed by gradual transition to formulated diet); (3) direct transition (using different combinations of live and formulated diet from the start feeding onwards); (4) formulated feed (fd) from the start feeding. results indicated that growth and survival were higher in the indirect transition feeding regime than in other regimes. based on our study, co-feeding of a. persicus should start five days after prior feeding with live food. introduction sturgeon fish are mainly cultured for the production of caviar, as a result of the sharp decrease in production capacity of caviar from natural resources such as the caspian sea. additionally they are also an important source of commercially valuable fish meat. however, the feeding patterns of these species on natural food have only been studied on a small scale. this is especially true for the larval and juvenile stages, which are the most critical stages in the commercial production of these species. the persian sturgeon (acipenser persicus) as a sturgeon fish is a migratory species which are especially adaptable to changes in their environment and in food supply; thus, they can occur and attain satisfactory growth in various climatic zones. these fish species have been the focus of much attention in iran over the last decade because they are * corresponding author: naser agh e-mail address: n.agh@urmia.ac.ir tel: +984413440295 particularly interesting species in terms of rearing value. at the onset of exogenous feeding, different sturgeon species possess an anatomically complete digestive tract with a marked specialization at different segments (buddington and christofferson, 1985; gawlicka et al., 1995; gisbert et al., 1998; asgari et al., 2013). artificial larval diets have been used for intensive commercial culture of several acipenserid species from the onset of exogenous feeding onwards (charlon and bergot, 1991; giovannini et al., 1991; hung, 1991; gisbert and williot, 1997). however, the end of the lecithotrophic stage and transition to exogenous feeding are still characterized by considerable larval mortality (buddington and christofferson, 1985; giovannini et al., 1991; gisbert and williot, 1997; bardi et al., 1998). this observation suggests that nutritional 159 agh et al./ int. j. aquat. biol. (2013) 1(4): 158-166 problems are associated with the digestion and assimilation of artificial diets, which are normally formulated for salmonids or marine fish species (hung, 1991; gisbert and williot, 1997). combined feeding of live and manufactured diets (referred to as co-feeding) from the start of exogenous feeding or from an early larval age, could be considered as an alternative strategy to reduce larval mortality (rosenlund et al., 1997). mohler et al. (2000) indicated high survival in atlantic sturgeon acipenser oxyrinchus oxyrinchus using artemia nauplii and a commercial feed. they reported a complete transition to formulated feed with less than 25% mortality in a 20–26 day feeding trial. dilauro et al. (1998) offered five different formulated diets in combination with live brine shrimp artemia sp., to larvae of the lake sturgeon acipenser fulvescens. they reported no dietary effect on mean survival, but significantly higher growth in fish fed only brine shrimp. ware et al. (2006) investigated the effects of six feeding regimes on the survival and growth of shortnose sturgeon, acipenser brevirostrum fry over 30 days using formulated diets and co-feeding. they reported significantly higher survival and growth in groups co-fed with artemia compared to live food and commercial feed as sole diets. bardi et al. (1998) reported more than 95% survival in gulf of mexico sturgeon acipenser oxyrinchus desotoi larvae fed brine shrimp, but nearly complete mortality (>99%) when a formulated feed was used during a three week feeding trial. according to their findings survival and growth rate of first-feeding larvae increased if they were fed brine shrimp for one week and then switched to an experimental microdiet. in iranian sturgeon fish hatcheries, the sturgeon larvae are mainly fed on artemia nauplii and daphnia during early stages of growth (table 1). the fry are then released into fertilized earthen ponds containing different zooplankton population, mostly daphnia and chironomidae, when their average weight reaches to about 150 mg. introduction to the caspian sea or concrete culture ponds takes place at an average weight of 10 g. none of the hatcheries use formulated diet during early stages of growth. this imposes huge expenditure for purchasing/production of live food and involvement of expert personnel. moreover, huge mortality has been reported on sudden transition from live food feeding in earthen ponds into formulated diet in concrete ponds. the aim of the present work was to contribute to reduce the massive mortality in the culture of a. persicus, associated with the dietary transition from live food to formulated feed. more specifically, we wanted to investigate under controlled laboratory conditions how different feeding regimes, using live weight of larvae at different stages until transfer into earthen ponds feeding strategy at hatching (mg) at start feeding (mg) at transfer to pond (mg) artemia nauplii (days) artemia nauplii + daphnia (days) daphnia (days) huso huso 20-22 60-65 100-120 1 1 8-9 acipenser persicus 16-17 40-42 80-100 2 2 5-6 acipenser gueldenstaetii 16-17 40-42 80-100 2 2 5-6 acipenser nudiventris 10-14 32-38 150 6 4 8-10 acipenser stellatus 7-9 28-30 150 6 4 8-10 table 1. weight of sturgeon larvae at different stages from hatching to introduction into earthen pond and the normal feeding strategy in iranian sturgeon fish hatcheries. 159 160 agh et al./ int. j. aquat. biol. (2013) 1(4): 158-166 food as a sole diet or co-fed with an inert diet through various weaning regimes, influence the survival and growth in a. persicus larvae. we also aimed at replacing the long term live food regimen with combination of live food and formulated diet in shortest period helping to reduce the expenses and excessive involvement of expert staff. materials and methods larval fish culture conditions: polyethylene larval culture tanks (43 cm length, 30 cm wide, 35 cm height, 45 l total volume) contained 25 l uv treated fresh water obtained in a flow through system from a well with a flow rate of approximately 1 l min-1. dissolved oxygen was maintained above 7 mg l-1 using constant aeration. fish larvae were exposed to a natural photoperiod of approximately 12:12 l:d. tanks were siphoned daily in the morning to remove trapped feces. water temperature, do, ph, tan and nitrite were 20 ± 1°c, 7.7 ± 0.5, 7.4 ± 0.1, 0.23 ± 0.08 and 0.01 ± 0.01 mg l-1 respectively, throughout the experiment. temperature, ph and dissolved oxygen were monitored once or twice daily, but other parameters were measured once a week due to the constant quality of the well water and the low water retention time in the tanks. yolk sac stage larvae were collected from shahid marjani sturgeon hatchery (gorgan city, ne iran). the larvae were transferred to the lab in oxygenated plastic bags. after acclimatization to the new environment they were randomly distributed over the 45-l larviculture tanks (300 larvae/tank) and the feeding experiment started after absorption of the yolk sac at the time of mouth opening and commencement of external feeding, 14 and 10 days post-hatch for beluga and persian sturgeon larvae, respectively. each feeding treatment was run in three replicate tanks. two feeding strategies were adopted for both fish species. in the first strategy, the fish larvae were fed with newly hatched artemia urmiana nauplii (n) for 5 days followed by gradual replacement with a commercial formulated starter diet (fd) provided by joosen-luyckx, turnhout, belgium. in the second strategy, the fish larvae were fed different combinations of newly hatched artemia nauplii and fd from the first day of exogenous feeding. feeding rations were based on wet body weight: initially 35% of body weight (first 5 days), followed by 25% (days 6-10), 15% (days 11-15) and 10% of body weight (days 16-20) (agh, unpublished data). to determine the daily feeding rations, actively swimming newly hatched artemia nauplii were transferred into a big beaker, aerated and 15 sub-samples (250 µl each) were collected, weighed, dried and weighed again in order to calculate their wet and dry weight. daily rations were divided into six equal portions fed at intervals of four hours. the feeding rates were adjusted according to the daily mortalities in each tank. each feeding strategy included several feeding treatments which varied in the rate of transition from a live food to a formulated diet. additionally, two controls were included in the experimental set-up, one fed live food, and the other fed formulated diet throughout the culture period. all feeding treatments were continued for 20 days until transition to fd were completed for all treatments. experimental feeding regimes used in this study were as follows: 1. artemia nauplii (n) throughout the experiment. 2. fd throughout the experiment. 3. n for first 5 days + 10% daily replacement of n with fd from day 6 (total conversion to fd occurring on day 15). 4. n for first 5 days + 20% replacement of n with fd on day 6 and 10% daily additional replacement feed crude protein (% dw) crude lipid (% dw) carbohydrate (% dw) ash (% dw) energy (cal g⁻¹) formulated diet 50 ± 2 12 ± 1.5 12.5 ± 1 13.5 ± 1 4000 ± 31 nauplii 61.6 ± 0.8 11.6 ± 2.1 20 ± 2.9 6.8 ± 2 5013.1 ± 88.3 table 2. the chemical composition of artemia nauplii and formulated diet. (the values indicate the averages values of replicates with standard deviations). 161 agh et al./ int. j. aquat. biol. (2013) 1(4): 158-166 with fd from day 7 (total conversion to fd occurring on day 14). 5. n for first 5 days + 30% replacement of n with fd on day 6 and 10% daily additional replacement with fd from day 7 (total conversion to fd occurring on day 13). 6. n for first 5 days + 40% replacement of n with fd on day 6 and 10% daily additional replacement with fd from day 7 (total conversion to fd occurring on day 12). 7. n for first 5 days + 50% replacement of n with fd on day 6 and 10% daily additional replacement with fd from day 7 (total conversion to fd occurring on day 11). 8. n (90% feed weight) and fd (10% feed weight) on day 1 + 10% daily replacement of n with fd from day 2 (total conversion to fd occurring on day 10) 9. n (80% feed weight) and formulated feed (20% feed weight) on day 1 + 10% daily replacement of n with fd from day 2 (total conversion to fd occurring on day 9). 10. n (70% feed weight) and formulated feed (30% feed weight) on day 1 + 10% daily replacement of n with fd from day 2 (total conversion to fd occurring on day 8). 11. n (60% feed weight) and formulated feed (40% feed weight) on day 1 + 10% daily replacement of n with fd from day 2 (total conversion to fd occurring on day 8). table 3. final length, wet weight (ww) and dry weight (dw) (mean ± standard deviation) of a. persicus larvae fed on different combinations of live food and commercial feed (initial total length, wet weight and dry weight were 20.7 mm, 39.1 mg and 4.5 mg respectively). different superscripts in each column indicate significant difference among treatments (p < 0.05). treatments: (1) n; (2) fd; (3, 4, 5, 6, 7) indirect transition; (8, 9, 10, 11, 12) direct transition. 161 162 agh et al./ int. j. aquat. biol. (2013) 1(4): 158-166 12. n (50% feed weight) and formulated feed (50% feed weight) on day 1 + 10% daily replacement of n with fd from day 2 (total conversion to fd occurring on day 6). the approximate chemical composition of the formulated food and artemia nauplii used in this study is shown in table 2. survival: the dead larvae were counted daily and removed from each culture tank. based on these figures the mean survival in each treatment was calculated. growth: at the beginning of the experiment ten randomly collected fish larvae from each species were weighed. the initial feed requirement for each feeding regime was based on this wet weight value. every alternate day six individuals were collected from each culture tank to measure the total length of the larvae with the help of a stereomicroscope figure 1. final length (a), final wet weight (b), final dry weight (c), sgr (d), fcr (e) and % survival (f) of a. persicus larvae fed on different combinations of live food and commercial feed (initial total length, wet weight and dry weight were 20.7 mm, 39.1 mg and 4.5 mg, respectively). different superscripts in each column indicate significant difference among treatments (p<0.05). treatments: (1) n; (2) fd; (3, 4, 5, 6, 7) indirect transition; (8, 9, 10, 11, 12) direct transition. 163 agh et al./ int. j. aquat. biol. (2013) 1(4): 158-166 equipped with a drawing tube and micrometer (zhiss, germany). drawings were later digitized using a digitizer (graphica, japan) connected to a computer. the same specimens were also used for determination of wet and dry weight (dried at 60°c for 24 h). based on the increase of wet weight of the fish larvae in each tank, the amount of feed needed for the next two days was calculated. specific growth rate (sgr) and food conversion rate (fcr) were calculated at the end of the experiment. statistical analysis: data were examined using analysis of variance (anova) followed by the tukey test. results results obtained from the experiments are presented in table 3 and figure 1. significantly higher survival was observed in larvae fed live food (85 ± 4%) compared to other feeding treatments except the indirect co-feeding regimes 3 and 4 with 80 ± 4 and 78 ± 3% survival respectively (p<0.05). significantly lower survival was observed in larvae when 40-50% live food was replaced with fd at start feeding. highest growth was observed in fish of cofed groups (indirect transition treatments 3 and 4) followed by larvae fed on artemia nauplii, which was significantly higher compared to fd and direct transition groups (p<0.05). direct transition treatments resulted in higher mortality of a. persicus larvae compared to indirect transition regimes. growth and survival gradually and often significantly decreased in treatments receiving higher percentages of fd in both weaning strategies. significantly lower growth and survival was observed in larvae fed on the inert diet solely (p<0.05). discussion in most marine fish species, compound diets fed alone have a poor ability to sustain larval growth and development (cañavate and fernández-díaz, 1999; robin and vincent, 2003; curnow et al., 2006a). the low performance usually observed when feeding an inert diet to marine fish larvae from mouth opening onwards may be due to sub-optimal diet composition and the larva’s poor ability to modulate its digestive enzymes (cahu and zambonino infante, 2001). therefore, the co-feeding strategy has been proposed for farmed species, such as dourado (vega-orellana et al., 2006), asian sea bass (curnow et al., 2006b), pikeperch (hamza et al., 2007), and cod (rosenlund and halldórsson, 2007). artemia nauplii are used world-wide as live food for the larval stages of commercially important fresh water and marine fish species. the costs of infrastructure, labor and energy to culture this zooplankton organism are considerable and the supply and nutritional quality of brine shrimp are variable (sorgeloos, 1980; watanabe et al., 1983). furthermore, it seems that acceptable growth rates in a number of fish species cannot be maintained using exclusively live food due to its low nutrient content and restricted food intake (olsen et al., 1992). this has prompted a great deal of interest in the development of an artificial larval microdiet (md) as an economic alternative to live food. however, a lower performance is commonly reported when inert diets are fed to larvae from the onset of exogenous feeding. this may be due to the composition, palatability, or physical characteristics of the dry feed (person le ruyet et al., 1993), to the larva’s inability to properly digest the feed (holt, 1993; kolkovski et al., 1993; walford and lam, 1993; zambonino infante and cahu, 1994), or to the low attractiveness of the non-mobile particle for the fish larva. however, the performance of md’s for a variety of fish species was considerably enhanced when co-fed with live zooplankton (kanazawa et al., 1982; szlaminska and przybyl, 1986; ehrlich et al., 1989; fermin and bolivar, 1991; marte and duray, 1991; tandler and kolkovski, 1991; walford et al., 1991; person le ruyet et al., 1993; lavens et al., 1995). the successful use of artemia nauplii alone or co-fed with a commercial diet at the start feeding or during early development of different sturgeon species has been reported by a number of researchers (dilauro et al., 1998; bardi et al., 1998; mohler et al., 2000; volkman et al., 2004). 163 164 agh et al./ int. j. aquat. biol. (2013) 1(4): 158-166 results obtained in the present study indicated that a carefully programmed diet of live food co-fed with a commercial diet could be successfully used in first and early larval feeding of persian sturgeon (acipenser persicus). the results obtained with different co-feeding regimes showed that maximum survival is obtained with live food but it was not significantly different with treatments 3 and 4 (indirect transition starting with 10 and 20% replacements of live food with fd from day 6). maximum growth was obtained in the same indirect transition treatments, significantly higher than all other treatments except those fed solely on live food. this means that in a. persicus, the best results are obtained with a gradual transition process where the larvae are fed on artemia nauplii for five days, and then slowly weaned to fd. this finding is in congruence with the data of dilauro et al. (1998) for acipenser fulvescens and of bardi et al. (1998) for acipenser oxyrinchus desotoi. it has been proved that co-feeding enhances larval performance beyond the level achieved by feeding either type of feed alone (kanazawa et al., 1989; holt, 1993; leu et al., 1991; abi-ayad and kestemont, 1994), and that it permits weaning in a shorter time (person le ruyet et al., 1993). this study proved that this feeding strategy is applicable for a. persicus too, but different transition regimes need to be adopted. an increased supply of more suitable nutrients may be the main reason for better performance of fish larvae accepting fd along with live food. however, the larvae suffered significantly higher mortality caused by starvation when they were offered only formulated diet from first feeding. the empty digestive tract suggested low attractiveness of the formulated diet for the larvae. apparently there is a specific period during development, related to feeding behavior and physiological capacity, when sturgeon fish larvae accept manufactured diets. successful co-feeding thus depends on the ability of the fish larvae to eat dry feed when live food is also present. we may conclude that a. persicus prefers an early feeding with live food followed by gradual transition to fd from day 6 onwards. co-feeding resulted in a considerable reduction of costs needed for consumables (including artemia cysts), infrastructure, labor and space needed for feed preparation and for the feeding process. acknowledgements this study was carried out with the financial support of the artemia and aquatic animals research institute, urmia university, iran. we thank the iranian fishery organization and the staff of shahid marjani and shahid beheshty sturgeon hatcheries for technical assistance and providing the fish larvae. references abi-ayad a., kestemont p. 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